Inhibitory Potential of New Phenolic Hydrazide-Hydrazones with a Decoy Substrate Fragment towards Laccase from a Phytopathogenic Fungus: SAR and Molecular Docking Studies

Laccase from pathogenic fungi participates in both the delignification and neutralization of phytoantibiotics. Furthermore, it interferes with the hormone signaling in plants and catalyzes melanization. Infections of these pathogens contribute to loss in forestry, agriculture, and horticulture. As there is still a need to expand knowledge on efficient defense strategies against phytopathogenic fungi, the present study aimed to reveal more information on the molecular mechanisms of laccase inhibition with natural and natural-like carboxylic acid semi-synthetic derivatives. A set of hydrazide-hydrazones derived from carboxylic acids, generally including electron-rich arene units that serve as a decoy substrate, was synthesized and tested with laccase from Trametes versicolor. The classic synthesis of the title inhibitors proceeded with good to almost quantitative yield. Ninety percent of the tested molecules were active in the range of KI = 8–233 µM and showed different types of action. Such magnitude of inhibition constants qualified the hydrazide-hydrazones as strong laccase inhibitors. Molecular docking studies supporting the experimental data explained the selected derivatives’ interactions with the enzyme. The results are promising in developing new potential antifungal agents mitigating the damage scale in the plant cultivation, gardening, and horticulture sectors.


Introduction
Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) is a blue multi-copper oxidase specific to a broad group of electron-rich arenes such as anilines and phenols. It has the exceptional ability to one electron oxidize both low-and high-molecular-weight substrates via direct or mediated reactions applied in green chemistry [1]. This nonspecific property has been reflected in numerous industrial, medical, and environmental applications [2][3][4][5]. Prokaryotes [6] and eukaryotes such as insects [7], plants, and fungi [8] produce laccase. Among the fungi, Ascomycota and Basidiomycota are well-known producers of this enzyme. Laccase participates in fungal metabolic pathways such as developing fruit bodies, the pigmentation of spores, and sexual differentiation [9]. It is also known as the virulence factor involved in the pathogenicity of humans [10][11][12] and plants [13][14][15][16]. Among the most troublesome filamentous fungi that produce laccase are Botrytis cinerea [17,18], Sclerotinia sclerotiorum [19], and Rhizoctonia sp. [20,21]. Furthermore, fungi parasitizing on trees use this enzyme in the degradation of lignin. Fungi release laccase into the plant tissues. It catalyzes lignin decomposition and neutralization of the auxins and phytoalexins, a primary barrier against pathogen attack. Such an infection strategy contributes to white-root parasitizing on trees use this enzyme in the degradation of lignin. Fungi release laccase into the plant tissues. It catalyzes lignin decomposition and neutralization of the auxins and phytoalexins, a primary barrier against pathogen attack. Such an infection strategy contributes to white-root of wood, crop, and ornamental plants diseases resulting in an enormous ecological and economic loss in forestry and agriculture [22,23].
We aimed this enzyme as a target since it is secreted by pathogenic fungi, contributing to various plant diseases. We believe that laccase inhibition might prevent or weaken pathogen attacks by chemical protection. Continuing our previous study, in the present article, we have addressed the effect of changing the structure of the acyl unit in hydrazidehydrazones while retaining their salicylidene character on laccase activity, see Figure 2. ized in the benzene ring of the acyl unit. An example is a 3,4-dihydroxybenzoic acid a catechol, and a well-known laccase substrate [81][82][83]. The next examples are the atives of 3-or 4-monohydroxybenzoic acid, both described in our previous work. We tigated the role of the benzylidene unit of hydrazide-hydrazones in laccase inhibition We showed that the 4-hydroxybenzoyl fragment derived from 4-hydroxybenzoic (4-HBA) constitutes a specific decoy being the mediator of reactions catalyzed by se [84]. However, the units from salicylic aldehydes are also responsible for specific action with amino acids in the substrate cavity of the enzyme. These results showed among the slim-shaped molecules, the most active representatives act as competitive itors with the KI in the range of 24.0-26.4 μM. Interestingly, the sterically hindered e derivative with a 3,5-di-tert-butyl-salicylidene unit has the most promising KI = 17.9 nd acts as an uncompetitive inhibitor. Generally, the values of the constant inhibition ify these compounds as effective agents that inhibit laccase. We aimed this enzyme as a target since it is secreted by pathogenic fungi, contribto various plant diseases. We believe that laccase inhibition might prevent or en pathogen attacks by chemical protection. Continuing our previous study, in the nt article, we have addressed the effect of changing the structure of the acyl unit in azide-hydrazones while retaining their salicylidene character on laccase activity, see re 2. e 2. The general structure of investigated hydrazide-hydrazones. The fragment derived from lic aldehydes is a salicylidene unit, and the acyl unit originated from acid hydrazides.

yntheses and Characterizations
We synthesized and investigated the new phenolic hydrazide-hydrazones as a part r ongoing project to discover aromatic compounds as antifungal and antiviral agents ey enzyme inhibitors of pathogenic microorganisms and viruses [31,[85][86][87][88][89][90]. The tarroducts were synthesized in a three-step procedure starting from carboxylic acid 1 arboxylic acid methyl ester 2 [91] as the first intermediate product. The final hydrahydrazone products 3-5 [31] were obtained by the direct condensation reaction of lic aldehydes 6-9 [92,93] and carboxylic acid hydrazides 10-19 [94], using the literaprocedure [31], Scheme 1, Table 1. Products 3-5 were formed quantitatively and charized. The high purity samples for the enzymatic study were prepared by direct crysation from the reaction mixtures with a good to almost quantitative yield between 81 Figure 2. The general structure of investigated hydrazide-hydrazones. The fragment derived from salicylic aldehydes is a salicylidene unit, and the acyl unit originated from acid hydrazides.

Syntheses and Characterizations
We synthesized and investigated the new phenolic hydrazide-hydrazones as a part of our ongoing project to discover aromatic compounds as antifungal and antiviral agents and key enzyme inhibitors of pathogenic microorganisms and viruses [31,[85][86][87][88][89][90]. The target products were synthesized in a three-step procedure starting from carboxylic acid 1 via carboxylic acid methyl ester 2 [91] as the first intermediate product. The final hydrazidehydrazone products 3-5 [31] were obtained by the direct condensation reaction of salicylic aldehydes 6-9 [92,93] and carboxylic acid hydrazides 10-19 [94], using the literature procedure [31], Scheme 1, Table 1. Products 3-5 were formed quantitatively and characterized. The high purity samples for the enzymatic study were prepared by direct crystallization from the reaction mixtures with a good to almost quantitative yield between 81 and 99%.  Among the spectroscopic methods used, the infrared spectra are the most diagnostic in the analysis of product formation due to the vanishing of the band with a very strong intensity usually observed around 1700 cm -1 that corresponded to stretching CH=O group vibration. Furthermore, the IR method is also helpful in monitoring the reaction progress and determining the product purity. Generally, the ATR-IR spectra of the functionalized salicylaldehyde derivatives 3-5 had an extensive absorption band between 3300-2500 cm -1 . This absorption contained bands corresponding to stretching vibrations Scheme 1. Preparation of hydrazide-hydrazones 3a-b, 4a-o, and 5a-d scheme started from carboxylic acids 1 via carboxylic acid methyl esters 2 and carboxylic acid hydrazides 11-19. Among the spectroscopic methods used, the infrared spectra are the most diagnostic in the analysis of product formation due to the vanishing of the band with a very strong intensity usually observed around 1700 cm −1 that corresponded to stretching CH=O group vibration. Furthermore, the IR method is also helpful in monitoring the reaction progress and determining the product purity. Generally, the ATR-IR spectra of the functionalized salicylaldehyde derivatives 3-5 had an extensive absorption band between 3300-2500 cm −1 . This absorption contained bands corresponding to stretching vibrations of O-H, N-H, C Ar -H, and CH 2 or CH 3 of alkyls. The band from stretching vibration of O-H bond was not observed in most of the analyzed spectra as a characteristic one. It probably contributed to a mentioned broad absorption typical for crystalline compounds with strong H-bonding [97]. The vibrations of the hydrazide fragment are frequently described as amide vibrations, in which stretching vibration of C=O bond is named amide I and bending vibration of NH group-amide II. These are relatively strong and the medium absorptions found between 1700-1600 cm −1 and 1650-1550 cm −1 , respectively. The other characteristic bands expected in the FT-IR spectrum correspond to stretching vibrations of C=N and C-O bonds. For all of the hydrazide-hydrazones 3-5, the wavenumbers corresponding to the following vibrations were assigned as following 3248-3164 cm −1 to stretching N-H bond, 1655-1591 cm −1 the amide I, 1615-1580 cm −1 amide II, 1567-1527 cm −1 stretching imine bond (C=N), and 1265-1227 cm −1 to stretching C-O phenolic bond. In the cases of methoxylated compounds 4c and 4d, the following stretching O-CH 3 vibration was observed at 1044 cm −1 and 1029 cm −1 , respectively.
The structural similarities of the synthesized hydrazides 11-19 to the corresponding hydrazide-hydrazones 3-5 resulted in partial coverage of the wavelength values in their IR spectra. The broad absorption between 3300-2500 cm −1 contained stretching vibrations the bands of NH 2 (3251-3182 cm −1 ), and N-H (3329-3283 cm −1 ), and C-H alkyl groups vibration in hydrazides 11, 14, and 15 (2963-2809 cm −1 ). Band of O-H stretching vibration was also not observed in this region. Absorptions with medium and strong intensities of amide I and amide II vibrations were attributed to bands between 1673-1605 cm −1 and 1604-1547 cm −1 , respectively. The other characteristic bands corresponded to the stretching vibration of C-O in phenolic and O-CH 3 in alkyl substituents found between 1269-1249 cm −1 and 1034-1030 cm −1 , respectively. The stretching vibration of the N-N bond had a weak absorption around 1000-1200 cm −1 . Although one nitrogen atom in the N-N group is acylated, it remains highly symmetrical and is not a diagnostic band in infrared spectra [98].
All di-aryl hydrazide-hydrazones numbered 4 and 5 were isolated as pure geometric isomers E. These having acetyl units numbered 3a and 3b were isolated as E isomer predominantly with the ratio 79:21 and 87:13, respectively. We determined the ratio of isomers based on CH=N integration in 1 H-NMR spectra.

Kinetic Studies
In our previous work, a 4-hydroxybenzoic acid (4-HBA) fragment of choice played a pivotal role in laccase inhibition as a decoy similar to its substrate. We showed that the salicylic aldehyde fragment with a bulky group localized neighbor to the hydroxy group, presented in Figure 3, contributes to a strong stabilizing effect with the hydrophobic area in a substrate-binding pocket [31]. In the present work, we extended the structureactivity relationship (SAR) after the change of acyl fragment ( Figure 2) introduced from the carboxylic acid. We preserved the salicylidene unit in hydrazide-hydrazone molecules introduced directly from salicylic aldehyde substrates 6-9.
H bond was not observed in most of the analyzed spectra as a characteristic one. It probably contributed to a mentioned broad absorption typical for crystalline compounds with strong H-bonding [97]. The vibrations of the hydrazide fragment are frequently described as amide vibrations, in which stretching vibration of C=O bond is named amide I and bending vibration of NH group-amide II. These are relatively strong and the medium absorptions found between 1700-1600 cm -1 and 1650-1550 cm -1 , respectively. The other characteristic bands expected in the FT-IR spectrum correspond to stretching vibrations of C=N and C-O bonds. For all of the hydrazide-hydrazones 3-5, the wavenumbers corresponding to the following vibrations were assigned as following 3248-3164 cm -1 to stretching N-H bond, 1655-1591 cm -1 the amide I, 1615-1580 cm -1 amide II, 1567-1527 cm -1 stretching imine bond (C=N), and 1265-1227 cm -1 to stretching C-O phenolic bond. In the cases of methoxylated compounds 4c and 4d, the following stretching O-CH3 vibration was observed at 1044 cm −1 and 1029 cm −1 , respectively.
The structural similarities of the synthesized hydrazides 11-19 to the corresponding hydrazide-hydrazones 3-5 resulted in partial coverage of the wavelength values in their IR spectra. The broad absorption between 3300-2500 cm -1 contained stretching vibrations the bands of NH2 (3251-3182 cm -1 ), and N-H (3329-3283 cm -1 ), and C-H alkyl groups vibration in hydrazides 11, 14, and 15 (2963-2809 cm -1 ). Band of O-H stretching vibration was also not observed in this region. Absorptions with medium and strong intensities of amide I and amide II vibrations were attributed to bands between 1673-1605 cm -1 and 1604-1547 cm -1 , respectively. The other characteristic bands corresponded to the stretching vibration of C-O in phenolic and O-CH3 in alkyl substituents found between 1269-1249 cm -1 and 1034-1030 cm -1 , respectively. The stretching vibration of the N-N bond had a weak absorption around 1000-1200 cm -1 . Although one nitrogen atom in the N-N group is acylated, it remains highly symmetrical and is not a diagnostic band in infrared spectra [98].
All di-aryl hydrazide-hydrazones numbered 4 and 5 were isolated as pure geometric isomers E. These having acetyl units numbered 3a and 3b were isolated as E isomer predominantly with the ratio 79:21 and 87:13, respectively. We determined the ratio of isomers based on CH=N integration in 1 H-NMR spectra.

Kinetic Studies
In our previous work, a 4-hydroxybenzoic acid (4-HBA) fragment of choice played a pivotal role in laccase inhibition as a decoy similar to its substrate. We showed that the salicylic aldehyde fragment with a bulky group localized neighbor to the hydroxy group, presented in Figure 3, contributes to a strong stabilizing effect with the hydrophobic area in a substrate-binding pocket [31]. In the present work, we extended the structure-activity relationship (SAR) after the change of acyl fragment ( Figure 2) introduced from the carboxylic acid. We preserved the salicylidene unit in hydrazide-hydrazone molecules introduced directly from salicylic aldehyde substrates 6-9.
We synthesized a set of twenty-one hydrazide-hydrazones from four salicylic aldehydes of choice 6-9 ( Figure 3) and ten differently substituted acid hydrazides 10-19. We synthesized a set of twenty-one hydrazide-hydrazones from four salicylic aldehydes of choice 6-9 ( Figure 3) and ten differently substituted acid hydrazides 10-19.
Based on the character of the acyl fragment, we formally divided the tested hydrazidehydrazones numbered 3-5 into three groups. The first group contained two alkyl representatives, methyl and 4-hydroxybenzyl in 3a and 3b, respectively. The second group included a strong electron-deficient 3-pyridyl or phenyl rings in 4a and 4b, respectively, and an electron-rich phenyl ring having a strong electron-donating methoxy 4c, 4d, hydroxy 4e, 4g, 4h, 4j, 4k, 4m, 4n, and dihydroxy 4f, 4i, 4l, 4o substituents. The structures of the compounds 5a-d in the third group contained a bigger 1-hydroxy-2-naphthoyl ring that provides their slim-like character ( Table 2). To compare the inhibition potency of new compounds, we chose NaN 3 as a classic standard reference. Additionally, we used hydrazide-hydrazone derivatives of 4-HBA numbered 20-22 and 4-methoxybenoic acid derivative 23 negative standards described in our previous work (   [31].

No.
Ar The kinetic studies showed that the nineteen hydrazide-hydrazones exhibited an inhibition potency in the micromolar range of K I between 8.0-233 µM and different modes of interaction with the enzyme molecules. Nine compounds 3a-b, 4a-f, and 5a (Table 2) contain the 3-tert-butyl-5-methyl-salicylidene leading motif introduced from aldehyde 8 ( Figure 2). We used previously reported hydrazide-hydrazone 20 derived from leading aldehyde 8 and 4-hydroxybenzoic acid hydrazide to compare the kinetic results. It acted as the competitive inhibitor with a constant K I = 26.4 µM. Among the hydrazide-hydrazones, the acetyl derivative 3a, and nicotinoyl derivative 4a, were not active at the desirable micromolar range. The benzoyl derivative 4b had a moderate activity with K I = 82.0 µM. Comparing it with 3b derivative with para hydroxylated benzene ring with methylene linker, almost a two-fold improvement of inhibition activity was observed (K I = 49.1 µM). On the other hand, the alteration from standard 20 having the 4-hydroxyphenyl framework to above compound 3b resulted in an almost two-fold drop in the activity, which confirmed our predictions that the hydroxylated aromatic fragment serves as a decoy and is required on the benzoyl fragment. Despite the 3b seeming structurally like laccase substrate, the distance of the para-hydroxylated phenyl ring of the molecule core resulted in its higher affinity to the enzyme-substrate complex ( Figure 4). Similar to 3b, the more hindered salicylic acid derivatives, the 4e, 4g, 4j, and 4m, also did not act as competitive inhibitors. Among the discussed compounds, 4e molecule acted as an uncompetitive laccase inhibitor, which corresponded to dropped inhibition potency approximately six-fold compared to the control 20, both derivatives of our leading aldehyde. As it resulted from kinetic measurements, removing the smallest alkyl group in the 5-position of the aldehyde fragment increases the inhibition potency from 150 µM to 65.6 µM for 4e and 4j compounds, respectively, simultaneously preserving the uncompetitive type of action.
-NaN3 d 2.7 [31] non-competitive a Not active up to 1000 μM of concentration tested, b not determined, c insoluble in medium, KI-an inhibition constant, determined for a related type of inhibition according to reference [99], d controls [31].
The kinetic studies showed that the nineteen hydrazide-hydrazones exhibited an inhibition potency in the micromolar range of KI between 8.0-233 μM and different modes of interaction with the enzyme molecules. Nine compounds 3a-b, 4a-f, and 5a (Table 2) contain the 3-tert-butyl-5-methyl-salicylidene leading motif introduced from aldehyde 8 ( Figure 2). We used previously reported hydrazide-hydrazone 20 derived from leading aldehyde 8 and 4-hydroxybenzoic acid hydrazide to compare the kinetic results. It acted as the competitive inhibitor with a constant KI = 26.4 μM. Among the hydrazide-hydrazones, the acetyl derivative 3a, and nicotinoyl derivative 4a, were not active at the desirable micromolar range. The benzoyl derivative 4b had a moderate activity with KI = 82.0 μM. Comparing it with 3b derivative with para hydroxylated benzene ring with methylene linker, almost a two-fold improvement of inhibition activity was observed (KI = 49.1 μM). On the other hand, the alteration from standard 20 having the 4-hydroxyphenyl framework to above compound 3b resulted in an almost two-fold drop in the activity, which confirmed our predictions that the hydroxylated aromatic fragment serves as a decoy and is required on the benzoyl fragment. Despite the 3b seeming structurally like laccase substrate, the distance of the para-hydroxylated phenyl ring of the molecule core resulted in its higher affinity to the enzyme-substrate complex ( Figure 4). Similar to 3b, the more hindered salicylic acid derivatives, the 4e, 4g, 4j, and 4m, also did not act as competitive inhibitors. Among the discussed compounds, 4e molecule acted as an uncompetitive laccase inhibitor, which corresponded to dropped inhibition potency approximately six-fold compared to the control 20, both derivatives of our leading aldehyde. As it resulted from kinetic measurements, removing the smallest alkyl group in the 5-position of the aldehyde fragment increases the inhibition potency from 150 μM to 65.6 μM for 4e and 4j compounds, respectively, simultaneously preserving the uncompetitive type of action. On the other hand, replacing the 3-tert-butyl group in 4j on a phenyl ring in 4m resulted in a change of inhibition mechanism to a mixed type. Such structural modification in 4m improved the value of constant inhibition of competitive component to 26.4 µM, at the same value as for the control 20. Comparing the compounds having bulky 3,5-ditert-butyl-2-hydroxy-benzylidene unit showed that a change of 4-hydroxybenzoyl group in control 21 to 2-hydroxybenzoyl in 4g resulted in a three-fold decrease in inhibition activity. Interestingly, the only exception was the derivative 4m of 2-hydroxybenzoyl and 3-phenyl-salicylidene units, for which the value of the competitive component K I = 26.4 µM remained the same as for control 20.
Well-known facts supported the choice of the salicylic acid hydrazide as the substrate for synthesizing potent hydrazide-hydrazone laccase inhibitors. Salicylic acid (SA) is a natural signaling molecule that activates plant defense responses to a pathogen attack [100,101]. The SA functional analogs are applied in the crop protection sector [102], and the salicylic acid [103] and a sulphonyl salicylic acid [104] are not laccase substrates. Moreover, the SA is not a promotor of laccase production in white-rot fungi culture [105].
The remaining hydrazide-hydrazones of the second group are also benzoyl monoand di-hydroxylated in 3-position or 3,5-positions derivatives 4h, 4k, 4n or 4f, 4i, 4l, 4o, respectively. Five of seven compounds were pure competitive inhibitors with good inhibition potency between 18.9-35.8 µM and 32.3-55.6 µM for mono-and di-substituted, respectively. Comparison of appropriate pairs of 3-hydroxy and 3,5-dihydroxy (α-resorcinic acid unit) derivatives 22 and 4f, 4h and 4i, and 4k and 4l showed their inhibition potency and the beneficial effect of benzoyl fragments on laccase inhibition. Compounds 4k and 4l and 22 and 4f had comparable inhibition effects, and these pairs derived from aldehyde 7 and 8 differ the CH 3 group in 5-position, respectively, see Figure 3. We observed the most significant difference between the last pair of inhibitors 4h and 4i with a common backbone of di-tert-butyl-salicylic aldehyde (9). These compounds acted as competitive inhibitors. For example, see Figure 5 for mono-hydroxylated on acyl unit derivative 4h. This molecule was three-fold more effective than its disubstituted counterpart 4i, because the crowded phenolic rings seemed too large to effectively interact with the substrate cavity. In general, a hydroxy group in meta-position may offer preferential interactions with the substrate cavity in the enzyme. Changes in the positioning of the single 3-OH group may occur by flipping around the axis C1-C4 of the benzoyl ring. We expected a similar but strengthened interaction for disubstituted α-resorcinic acid hydrazide. In this case, interaction in symmetrically localized hydroxy groups was probably slightly weaker or unfavorable to the fit of the molecules in the active site.
Two remaining compounds, 4o and 4n, have a common 3-phenyl salicylaldehyde fragment. These present the lowest inhibition potency between 69.9 and 233 μM with the mixed or non-competitive type of inhibition. On the other hand, these derivatives showed improved activities compared to an inactive control 23 with the aldehyde 6 core but meth- In general, a hydroxy group in meta-position may offer preferential interactions with the substrate cavity in the enzyme. Changes in the positioning of the single 3-OH group may occur by flipping around the axis C1-C4 of the benzoyl ring. We expected a similar but strengthened interaction for disubstituted α-resorcinic acid hydrazide. In this case, interaction in symmetrically localized hydroxy groups was probably slightly weaker or unfavorable to the fit of the molecules in the active site.
Two remaining compounds, 4o and 4n, have a common 3-phenyl salicylaldehyde fragment. These present the lowest inhibition potency between 69.9 and 233 µM with the mixed or non-competitive type of inhibition. On the other hand, these derivatives showed improved activities compared to an inactive control 23 with the aldehyde 6 core but methoxy substituent in acyl fragment. It seems that the bulky electron-rich phenolic substituent represents adverse properties to bind to the substrate-binding cavity. Interestingly, the hydrazide-hydrazone 4c and 4d, which are methoxylated in metaand para position of benzoyl unit and containing the common aldehyde 8 core, are the most active in this group. The constants inhibition of 4c and 4d are 17.4 µM and 25.8 µM, respectively. The slim 4d hydrazide-hydrazone has a high affinity to substrate-binding pocket. A mobile methoxy group localized in meta position in 4c prevented the interaction with the active site of the enzyme (Figure 6). It was in agreement with the reactivity of para-methoxylated arenes such as para-anisic alcohol in the presence of laccase [106]. Further discussion of OCH 3  Finally, the third group numbered 5a-d contains a 2-hydroxynaphtalene hydrazide leading fragment (Table 2). Such substituent would require a large area in the enzymesubstrate cavity for hydrophobic interaction of such a bulk polycyclic arene and provide possible hydrogen bonding with hydrophilic amino acids. As it resulted from form experiments, three out of four compounds acted as mixed inhibitors. For 5a, 5b, and 5d, the non-competitive component KI had a consistently lower value than the competitive counterpart. Together with the 5b derivative, they acted as non-competitive inhibitors preferentially. Furthermore, the magnitude of inhibition constants indicates that the effect of mono-substitution of aldehyde fragment in 3-position (5b and 5d) is insufficient to obtain higher inhibition potency than 24.3 μM ( Table 2). The additional alkyl substituent in 5position improved inhibition potency. The small CH3 group in 5a preserved the mixed type of inhibition (Figure 7) with a very good value of non-competitive component KI = 8.0 μM and a good value of competitive constant equal to 19.0 μM. The additional bulk tert-butyl group in position 5 in compound 5c changed the mechanism to pure non-competitive with relatively good KI = 16.2 μM. The found hydrazide-hydrazones with the highest activity towards laccase will be the subject of further research on SAR and future Finally, the third group numbered 5a-d contains a 2-hydroxynaphtalene hydrazide leading fragment (Table 2). Such substituent would require a large area in the enzymesubstrate cavity for hydrophobic interaction of such a bulk polycyclic arene and provide possible hydrogen bonding with hydrophilic amino acids. As it resulted from form experiments, three out of four compounds acted as mixed inhibitors. For 5a, 5b, and 5d, the non-competitive component K I had a consistently lower value than the competitive counterpart. Together with the 5b derivative, they acted as non-competitive inhibitors preferentially. Furthermore, the magnitude of inhibition constants indicates that the effect of mono-substitution of aldehyde fragment in 3-position (5b and 5d) is insufficient to obtain higher inhibition potency than 24.3 µM ( Table 2). The additional alkyl substituent in 5position improved inhibition potency. The small CH 3 group in 5a preserved the mixed type of inhibition (Figure 7) with a very good value of non-competitive component K I = 8.0 µM and a good value of competitive constant equal to 19.0 µM. The additional bulk tert-butyl group in position 5 in compound 5c changed the mechanism to pure non-competitive with relatively good K I = 16.2 µM. The found hydrazide-hydrazones with the highest activity towards laccase will be the subject of further research on SAR and future application to protect cultivating plans against phytopathogenic fungi [107].

Docking Studies
The compounds used in the enzymatic assay were docked in the active center of the laccase (PDB 1GYC). Used procedures were the same as in the previous publication on our laccase research [25]. This time, however, we decided to put forward a hypothesis and check whether the changes in the inhibition type may depend only on the construction of the active center, previously called the substrate cavity. Especially, that several of the inhibitors we have tested showed a mixed mechanism of inhibition. The amino acids Asp206, Asn208, Asn264, and His258 played a key role in interacting with active compounds. They carry an electron from the substrate to the oxygen cavity. However, the other numerous amino acids building the active site ( Figure 8A) have a significant influence on the location of the substrate in the active site. The laccase active center can be divided into two hydrophobic parts and one creating numerous hydrogen bonds. Rather, the compounds we investigated fill the volume of one of the hydrophobic cavities and the area of hydrophilic interactions. The position of selected inhibitors is shown in Figure 8B: compounds with a competitive mechanism are shown as a green mesh, while the others are shown as an orange mesh. The inhibitors with the lowest values of the inhibition constants are shifted deeper into the active center.
The compounds used in the enzyme assay were docked to the active site of laccase (PDB 1GYC). The procedures used were the same as in the previous publication on our research on laccase [31]. This time, however, we decided to make a hypothesis and check whether the changes in the type of braking may depend only on the construction of the active center, previously called the ground cavity. Especially since several of the inhibitors we have tested exhibited a mixed inhibition mechanism. The amino acids Asp206, Asn208, Asn264, and His258 played a crucial role in interacting with active compounds. They transfer an electron from the substrate to the oxygen cavity. However, numerous other amino acids building the active site ( Figure 8A) have a significant influence on the location of the substrate in the active site. The active site of laccase can be divided into two hydro-

Docking Studies
The compounds used in the enzymatic assay were docked in the active center of the laccase (PDB 1GYC). Used procedures were the same as in the previous publication on our laccase research [25]. This time, however, we decided to put forward a hypothesis and check whether the changes in the inhibition type may depend only on the construction of the active center, previously called the substrate cavity. Especially, that several of the inhibitors we have tested showed a mixed mechanism of inhibition. The amino acids Asp206, Asn208, Asn264, and His258 played a key role in interacting with active compounds. They carry an electron from the substrate to the oxygen cavity. However, the other numerous amino acids building the active site ( Figure 8A) have a significant influence on the location of the substrate in the active site. The laccase active center can be divided into two hydrophobic parts and one creating numerous hydrogen bonds. Rather, the compounds we investigated fill the volume of one of the hydrophobic cavities and the area of hydrophilic interactions. The position of selected inhibitors is shown in Figure 8B: compounds with a competitive mechanism are shown as a green mesh, while the others are shown as an orange mesh. The inhibitors with the lowest values of the inhibition constants are shifted deeper into the active center.
investigated fill the volume of one of the hydrophobic cavities and the area of hydrophilic interactions. The position of selected inhibitors is shown in Figure 8B: compounds with a competition mechanism are shown as a green grid, while others are shown as an orange grid. Inhibitors with the lowest values of inhibition constants are shifted deeper into the active center. The pairs of the inhibitors like 4j and 4k, and 4g and 4h have a common backbone. The slide difference between them is the position of the hydroxyl group from the hydrazide fragment. That difference plays a key role in the type of binding and inhibition of the compounds. Derivative of salicylic acid 4j having one tert-butyl group ( Figure 9A) is placed like inhibitors from our earlier studies [31]. The hydrophobic ring with the tertbutyl substituent is directed toward the Phe265 with medium-range aromatic interactions (4.29 Å). Asp206 is involved in a hydrogen bond with its hydroxyl group (1.86 Å) and with the backbone nitrogen atom (2.22 Å). The other hydrogen positioning of the inhibitor is seen by the interactions with Asn208 (2.05 Å) and Asn264 (1.85 Å). Those close distance non-bond bindings are strengthened by Pro394 (3.60 Å, 5.17 Å) and Pro396 (4.93 Å). The derivative 4k ( Figure 9B) with a hydroxy group in meta position has a different inhibition mechanism, and it is slightly moved of the cavity depth. In this case, the hydrazide hydroxyl group interacts with Asn206 (2.20 Å) and Asn264 (2.04 Å). The position of the ring is stabilized by extra hydrophobic interactions with Leu164 (5.20 Å). The carbonyl oxygen atom and the hydrazide nitrogen create a hydrogen bond triangle with His458, and the position of the ring is stabilized by Phe162 (4.22 Å) and Pro391 (5.06 Å).
The larger molecules having two tert-butyl substituents in the salicylidene framework, such as 4g ( Figure 9C) and 4h ( Figure 9D) have less freedom in the binding. They are placed deep in the pocket engaging the same set of amino acids. The different position of a hydroxyl localized on benzoyl unit in ortho vs meta in 4g and 4h, respectively, causes the flip of the phenyl ring. Consequently, the extra hydrogen bond between 4h and the carbonyl oxygen of the Asp206 (1.67 Å) is created. The hydrogen interaction of the hydroxyl group with Asp206 is of the most significant importance in the inhibition mechanism by blocking the flow of electrons. In other instances, the inhibition is by blocking the substrate's access site for the amino acid. The presence of a hydrogen bond between the hydroxy group and Asn208 can also change the type of inhibition from uncompetitive to non-competitive (4j vs 4g). Compounds with methoxy substituents 4c and 4d are also The compounds used in the enzyme assay were docked to the active site of laccase (PDB 1GYC). The procedures used were the same as in the previous publication on our research on laccase [31]. This time, however, we decided to make a hypothesis and check whether the changes in the type of braking may depend only on the construction of the active center, previously called the ground cavity. Especially since several of the inhibitors we have tested exhibited a mixed inhibition mechanism. The amino acids Asp206, Asn208, Asn264, and His258 played a crucial role in interacting with active compounds. They transfer an electron from the substrate to the oxygen cavity. However, numerous other amino acids building the active site ( Figure 8A) have a significant influence on the location of the substrate in the active site. The active site of laccase can be divided into two hydrophobic parts and one forming numerous hydrogen bonds. Rather, the compounds we investigated fill the volume of one of the hydrophobic cavities and the area of hydrophilic interactions. The position of selected inhibitors is shown in Figure 8B: compounds with a competition mechanism are shown as a green grid, while others are shown as an orange grid. Inhibitors with the lowest values of inhibition constants are shifted deeper into the active center.
The pairs of the inhibitors like 4j and 4k, and 4g and 4h have a common backbone. The slide difference between them is the position of the hydroxyl group from the hydrazide fragment. That difference plays a key role in the type of binding and inhibition of the compounds. Derivative of salicylic acid 4j having one tert-butyl group ( Figure 9A) is placed like inhibitors from our earlier studies [31]. The hydrophobic ring with the tertbutyl substituent is directed toward the Phe265 with medium-range aromatic interactions (4.29 Å). Asp206 is involved in a hydrogen bond with its hydroxyl group (1.86 Å) and with the backbone nitrogen atom (2.22 Å). The other hydrogen positioning of the inhibitor is seen by the interactions with Asn208 (2.05 Å) and Asn264 (1.85 Å). Those close distance non-bond bindings are strengthened by Pro394 (3.60 Å, 5.17 Å) and Pro396 (4.93 Å). The derivative 4k ( Figure 9B) with a hydroxy group in meta position has a different inhibition mechanism, and it is slightly moved of the cavity depth. In this case, the hydrazide hydroxyl group interacts with Asn206 (2.20 Å) and Asn264 (2.04 Å). The position of the ring is stabilized by extra hydrophobic interactions with Leu164 (5.20 Å). The carbonyl oxygen atom and the hydrazide nitrogen create a hydrogen bond triangle with His458, and the position of the ring is stabilized by Phe162 (4.22 Å) and Pro391 (5.06 Å). pairs of inhibitors with different inhibition types. The meta position of the methoxy group 4c creates an unpreferred position in the cavity space. The freedom of the rotation is not appreciated in the deep binding of the inhibitor. That creates a phenomenon, the 4c is turned opposite to the projected mechanism. In contrast, the 4d binds properly to the inhibition types and interacts with the Asp206. The thesis is also confirmed by analyzing the interaction of more hydrophobic compounds having a naphthoic ring in their structure with amino acids in the active center. Compound 5a ( Figure 10A) had the lowest value of the inhibition constant, but it has a mixed mode of inhibition. The low value of the non-competitive inhibition constant for the binding method of this inhibitor is related to the fact that it is located deep but also in its upper part in the active center, blocking access to His458. Additionally, 5a forms a strong hydrogen bond with Asp206 (1.79 Å). The lack of interaction with the amino acids that make up the cavity's lower area is one of the reasons for the dual binding mechanism. However, 5c ( Figure 10B) is bound to amino acids of the lower part of the active center but does not interact with the Asp206, and it has the non-competitive type of inhibition. The larger molecules having two tert-butyl substituents in the salicylidene framework, such as 4g ( Figure 9C) and 4h ( Figure 9D) have less freedom in the binding. They are placed deep in the pocket engaging the same set of amino acids. The different position of a hydroxyl localized on benzoyl unit in ortho vs meta in 4g and 4h, respectively, causes the flip of the phenyl ring. Consequently, the extra hydrogen bond between 4h and the carbonyl oxygen of the Asp206 (1.67 Å) is created. The hydrogen interaction of the hydroxyl group with Asp206 is of the most significant importance in the inhibition mechanism by blocking the flow of electrons. In other instances, the inhibition is by blocking the substrate's access site for the amino acid. The presence of a hydrogen bond between the hydroxy group and Asn208 can also change the type of inhibition from uncompetitive to non-competitive (4j vs 4g). Compounds with methoxy substituents 4c and 4d are also pairs of inhibitors with different inhibition types. The meta position of the methoxy group 4c creates an unpreferred position in the cavity space. The freedom of the rotation is not appreciated in the deep binding of the inhibitor. That creates a phenomenon, the 4c is turned opposite to the projected mechanism. In contrast, the 4d binds properly to the inhibition types and interacts with the Asp206.
The thesis is also confirmed by analyzing the interaction of more hydrophobic compounds having a naphthoic ring in their structure with amino acids in the active center. Compound 5a ( Figure 10A) had the lowest value of the inhibition constant, but it has a mixed mode of inhibition. The low value of the non-competitive inhibition constant for the binding method of this inhibitor is related to the fact that it is located deep but also in its upper part in the active center, blocking access to His458. Additionally, 5a forms a strong hydrogen bond with Asp206 (1.79 Å). The lack of interaction with the amino acids that make up the cavity's lower area is one of the reasons for the dual binding mechanism. However, 5c ( Figure 10B) is bound to amino acids of the lower part of the active center but does not interact with the Asp206, and it has the non-competitive type of inhibition. This leads to the conclusion, the compound to inhibit laccase competitively must interact with amino acids from the depths and bottom of the active center, such as Asp206, Asn264, Leu 164, or Phe265. Another criterion required for this type of inhibition is the formation of a strong hydrogen bond between Asp206 and the inhibitor using heteroatoms that do not build the backbone of the inhibitor, blocking the flow of electrons.
Methanol (CH3OH) was distilled prior to reactions from Mg element shavings in the presence of I2. The following substrates such as 3-phenylsalicylicaldehyde (6), 3-tert-butyl-5-methylsalicylic aldehyde (8)  This leads to the conclusion, the compound to inhibit laccase competitively must interact with amino acids from the depths and bottom of the active center, such as Asp206, Asn264, Leu 164, or Phe265. Another criterion required for this type of inhibition is the formation of a strong hydrogen bond between Asp206 and the inhibitor using heteroatoms that do not build the backbone of the inhibitor, blocking the flow of electrons.

Synthesis of Aroylhydrazones 3-5
To a mixture of salicylaldehyde 6-9 (2.0 mmol), and carboxylic acid hydrazide 10-19 (2.0 mmol) in CH 3 OH (5.0 mL), AcOH (200 µL) was added at RT and then the resulting solution was gently refluxed during stirring. The reaction progress was monitored by TLC. Then the reaction was finished. After slow cooling to room temperature (RT) and cooled to ca. +4 • C, the reaction mixture was left in a refrigerator (−24 • C) overnight [31]. The crystals formed were collected by filtration to give pure products 3-5, fully characterized by determination melting point, analysis NMR, IR, and HRMS spectra. The known compound was identified by comparing its melting points and FT-IR and/or NMR spectra with literature data and/or by HRMS determination. The error of molar mass determination by HRMS method was ≤0.0005%.

Reaction Condition and Kinetic Calculations
Reaction conditions and kinetic assays were performed as reported previously [25]. Briefly, the kinetic measurements were conducted in pH 5.3 at 25°C. A reaction was carried out in a plastique cuvette with a total capacity of 2.0 mL. It contained 1.5 mL of a diluted in McIlvain buffer enzyme (10-50 nM), 0.1 mL of inhibitor (1 × 10 3 µM), and 0.1 mL of syringaldazine (0.5-15 µM). Hydrazide-hydrazones, sodium azide, and syringaldazine were dissolved in methanol as co-solvent before use. Before reaction, the enzyme was preincubated with an inhibitor for 30 min. The progress of the reaction was measured at 525 nm (Shimadzu, UV-VIS 1800, Tokyo, Japan) using UVProbe 2.70 software. Each reaction was performed at least in triplicate. The velocity of the syringaldazine conversion was expressed as a change in product concentration in time (µM/min) and calculated from the region of a first-order kinetic using an extinction coefficient ε 0 = 65,000 cm −1 [110]. The kinetic parameters for all tested compounds were calculated by using Lineweaver-Burk transformation and the linear least square method in Matlab (The Mathworks, R2019R Version 9.6, academic license) using steady-state kinetics. The goodness of fit was assessed using the coefficient of determination (R 2 ), the sum of square errors (SSE), and root mean squared error (RMSE). The following equations were applied for the determination of the competitive Equation (1), non-competitive Equation (2), uncompetitive Equation (3), and mixed Equation (4) types of inhibition [99]. Mixed type of inhibition Equation (4) was determined based the Segel's mathematical model where K I component corresponds to the competitive component, whereas α·K I is a non-competitive or uncompetitive component for α < 1 or α > 1, respectively. The resulted from inhibition changes in V max [µM·min −1 ] and K M [µM] parameters were expressed as their apparent counterparts V max,app [µM·min −1 ] and K M,app [µM]. The constant inhibition values (K I ) were determined by replotting fixed apparent values versus inhibitor concentrations into a linear function (Figures 2 and 3).

Conclusions
In this work, we aimed to investigate the inhibition activity of hydrazide-hydrazones towards laccase from Trametes versicolor. The hydrazide-hydrazones were synthesized using four salicylic aldehydes of choice and eleven hydrazides to obtain 21 derivatives, of which 20 have not been described in the literature so far. The most active compounds have the constant inhibition in the range of 8 and 25.8 µM and present diversified types of inhibition, but none of them interact with the enzyme-substrate complex. Kinetic results revealed that the presence of an arene electron-rich benzoyl fragment containing hydroxy and methoxy group in 3 and 4 positions of the benzene ring is necessary to inhibit laccase activity effectively. Performed for selected derivatives, the structure-activity relationship analysis showed that the slice differences in the built of the inhibitors might reflect the inhibition type. We evaluated the contribution of the selected amino acids from the substrate center with the possibility of changing the type of inhibition. The deep and substrate-like binding is preferred to interact competitively, but the mixed systems with uncommon interactions resulted in lower K I values. The found hydrazide-hydrazones with the highest activity towards laccase will be the subject of further research on SAR and future application to protect cultivating plans against phytopathogenic fungi [107].