4-Hydroxybenzoic Acid-Based Hydrazide–Hydrazones as Potent Growth Inhibition Agents of Laccase-Producing Phytopathogenic Fungi That Are Useful in the Protection of Oilseed Crops

The research on new compounds against plant pathogens is still socially and economically important. It results from the increasing resistance of pests to plant protection products and the need to maintain high yields of crops, particularly oilseed crops used to manufacture edible and industrial oils and biofuels. We tested thirty-five semi-synthetic hydrazide–hydrazones with aromatic fragments of natural origin against phytopathogenic laccase-producing fungi such as Botrytis cinerea, Sclerotinia sclerotiorum, and Cerrena unicolor. Among the investigated molecules previously identified as potent laccase inhibitors were also strong antifungal agents against the fungal species tested. The highest antifungal activity showed derivatives of 4-hydroxybenzoic acid and salicylic aldehydes with 3-tert-butyl, phenyl, or isopropyl substituents. S. sclerotiorum appeared to be the most susceptible to the tested compounds, with the lowest IC50 values between 0.5 and 1.8 µg/mL. We applied two variants of phytotoxicity tests for representative crop seeds and selected hydrazide–hydrazones. Most tested molecules show no or low phytotoxic effect for flax and sunflower seeds. Moreover, a positive impact on seed germination infected with fungi was observed. With the potential for application, the cytotoxicity of the hydrazide–hydrazones of choice toward MCF-10A and BALB/3T3 cell lines was lower than that of the azoxystrobin fungicide tested.


Introduction
In recent years, the number of active substances used as plant protection products has decreased as the ones with the highest toxicity and harmfulness have been withdrawn from the environment [1].Furthermore, the fungicide resistance of phytopathogens is an increasingly severe problem worldwide [2].Improper application of available preparations, which often work according to similar mechanisms of action, makes the pathogens immune to them.Therefore, plant protection strategies have evolved and changed in more complex activities, including nonchemical and chemical control methods [3].Both approaches, when applied simultaneously, may decrease pathogen growth rates and the use of fungicides, minimizing selection for resistant strains.Nevertheless, chemical treatment is still a valuable and required tool for plant infection control and also reduces selection for pathogen strains with increased virulence against resistant cultivars [3,4].Biologically active compounds for fungal disease control of plants could be divided into several classes as multisite mode-of-action fungicides, single site-of-action fungicides, plant activators, natural products and natural product-derived fungicides, biopesticides, chemical fumigants, and biofumigants [4].Most have fungicidal or fungistatic effects, while others do not influence pathogens as they induce plant resistance to their attack.
In mild climates, many pathogens of plants can cause significant economic losses during crops' cultivation and storage.One of the most difficult to limit is polyphagous fungi, such as Botrytis cinerea Pers.and Sclerotinia sclerotiorum (Lib.) de Bary, due to there being many host plants and the ability to create resting bodies (sclerotia) in plant debris in the soil that can last for many years.Botrytis cinerea, which causes gray mold disease, is a significant plant threat in the growing season.It is a cosmopolitan fungus noted throughout the world.It infects more than 240 different plant species [5].The most significant losses result in temperate and tropical climate regions.It is a facultative parasite that can live a saprotrophic lifestyle in the form of mycelium and sclerotia on plant debris in the soil.Still, very often, it becomes a parasitic lifestyle under favorable conditions for its development.During the growing season, large amounts of conidial spores spread the disease on the plantation.The pathogen contributes to significant economic losses in horticultural crops (mainly strawberries and berry bushes), vegetable crops (e.g., cucumber), and ornamental plants [6,7].The fungus produces toxins, which are derivatives of botrydial and botcinins, which are undesirable in strawberry fruit [8].The result of infestation may be gangrene of seedlings, gangrene of flowers, and gray rot of shoots and fruit.Sclerotinia sclerotiorum causes white mold disease.The fungus is a polyphag that infects about 400 species of plants [9] and occurs on all continents.In mild climates, it has an adverse effect on the cultivation of rapeseed, sunflower, and vegetables.It causes yield losses of up to 60% in oilseed rape, as infected plants often result in massive seed drop.White mold causes considerable losses in the storage of root vegetables, especially carrots.The pathogen does not produce conidial spores, only ascospores.They form in fruiting bodies (apothecia).Under unfavorable conditions, the fungus produces sclerotia, in the form of which it overwinters in plant debris or stored roots.In the spring, sclerotia grows cupped apothecia on long legs, promoted by moist soil and higher temperatures [10,11].
Cerrena unicolor (Bull.)Murrill is a different type from the above-characterized mold fungi, as it is a decaying arboreal fungus belonging to the Basidiomycetes and the family Polyporaceae, classified as a wound parasite and saprotroph [12].It is found in deciduous and mixed woodland and in parks and gardens.It attacks and colonizes living and weakened broadleaf trees, most commonly chestnut trees (Aesculus sp.), maples (Acer sp.), beech trees (Fagus sp.), and oaks (Quercus sp.).The fungus is widespread throughout the year in Europe, Africa, and South America but is rare.It is also commonly known as the mossy maze polypore but presents high similarity to the genus Trametes fungi [13].It is among the fungi that cause white wood rot, characterized by the decomposition of all the components of plant biomass: cellulose, hemicellulose, and lignin.The fungi described above have the common trait of producing laccases-copper-dependent oxidoreductases.The enzymes from B. cinerea [14,15], S. sclerotiorum [16], and C. unicolor [17] have been isolated and described in the literature.
Laccases are catalytic proteins with particular substrate specificity toward a broad group of electron-rich arenes, mostly phenols, that are oxidized through a one-electron mechanism directly with molecular oxygen or in the presence of mediators.The laccase active center comprises four copper atoms (type Cu1, type Cu2, and two type Cu3s), in which Cu1 interacts with an electron-rich substrate, and the trinuclear-Cu cluster binds oxygen molecules.In nature, laccase conducts the polymerization and depolymerization reactions, which are related to the natural role of lignification and delignification processes in plants and fungi, respectively [14].Laccases may be produced in different isoforms by fungi, usually in the presence of polyphenols and copper ions [18][19][20][21].
This research focuses on compounds containing fragments of naturally occurring molecules that may act as antifungal agents or plant resistance activators.In this context, phenolic compounds are an interesting group of low-molecular-weight species, including simple phenols, aldehydes, polyphenolic acids, flavonoids, coumarins, stilbenes, and tannins that are widely distributed in the plant kingdom [22].Phenolic acids have essential physiological functions, such as defense against herbivores and pathogens, antioxidation, protection of cell structures from harmful solar radiation or free radicals, and other substructural functions [23].Of the various phenolic compounds found in plants, hydroxybenzoic acids are among the simplest phenolic acids that act in response to environmental stresses.For instance, in cucumber leaves infected with Pseudomonas syringae van Hall, the synthesis and accumulation of both salicylic and 4-hydroxybenzoic acid were observed in the stems and petioles in response to a mobile signal from the inoculated leaf as part of systemic acquired resistance [24].Furthermore, it was tested when applied exogenously to wheat cultivars to test crop tolerance to short-term drought and freezing, resulting in increased stress tolerance [25].In other studies, 4-hydroxybenzoic acid was found to be synthesized de novo and covalently linked to cell wall polysaccharides in the carrot protoplast system upon activating the phenylpropanoid pathway induced through fungal elicitor from plant pathogen Pythium aphanidermatum (Edson) Fitzp.[26,27].Plant material contains, among others, thymol, salicylic acid, and salicylic aldehyde, which were tested separately and showed total fungal inhibition growth of Fusarium, Penicillium, and Aspergillus species at a high concentration of 1 g/L [28].Interestingly, thymol was effective against non-plant and plant pathogens such as Cryptococcus neoformans Vuill.and Rhizopus sp., respectively, and its isomer carvacrol against Candida albicans (CP Robin) Berkhout [29].Among various phenolic compounds, 4-hydroxybenzoic acid is one of the simpler polyphenolic laccase mediators [30], and it is still within our area of interest.
Hydrazide-hydrazones, derivatives of 4-trifluoromethylhydrazide, showed inhibition of two important enzymes involved in the breakdown of choline neurotransmitters, important in the treatment of neurological diseases, including Alzheimer's disease.As a result, the salicylaldehyde derivative IX was found to be a highly selective acetylcholinesterase (AChE) inhibitor in relation to butyrylacetylcholinesterase (BuChE) with a BuChE/AChE selectivity of 18.6.This was a promising result for the non-selective drug galantamine (BuChE/AChE = 1.8) [45].Furthermore, studies have shown that this derivative is non-toxic in tests on liver cancer cell HepG2 (human hepatoblastoma cell line).In the article dealing with the same issue, derivatives of isoniazid with salicylic aldehydes or 4-hydroxybenzaldehydes were inactive against AChE, but the gentisaldehyde derivative X had activity comparable to isoniazid with an IC 50 of 4.8 µM on MPO (myeloperoxidase), and the free radical scavenging efficiency was at the level of quercetin control [44].The research [38] was conducted on a series of diflunisal (2 ′ ,4 ′ -difluoro-4-hydroxybiphenyl-3-carboxylic acid) hydrazidehydrazones to determine antiviral activity against hepatitis C virus (HCV) and cytotoxicity toward liver cancer cell lines (Huh7, HepG2, HepB3, Mahlavu).From the results of these authors, the imine derivative of diflunisal hydrazide with 2-pyridylaldehyde (XI) was identified as the most active against HCV with an EC 50 of 3.9 µM and sequential on all launched lines, with the lowest EC 50 of 4.74 µM for the Mahlavu line.In the work [49] on α-glucosidase inhibitors used in the treatment of type-2 diabetes, a series of chromones aldehydes derivatives with various substituted hydrazides were obtained.In general, hydroxylated derivatives of hydrazide-hydrazones and chromene aldehydes XIIa-c showed much higher activity compared to the typically used αglucosidase inhibitor, acarbose drug.
The above examples demonstrate a broad spectrum of hydrazone activity in different biological areas.Interest in this class of compounds is still growing in plant fungicide development [53][54][55][56].
The hydrazide-hydrazones were tested as a part of our ongoing scientific program to discover low-molecular-weight compounds that are antimicrobial agents [57], the inhibitors of essential enzymes that are overproduced by microorganisms during disease development in plants [47,48] and humans [58][59][60], as well as those that act directly toward cell lines [61,62].In our previous work, we discovered potent laccase inhibitors as a target, as this enzyme is secreted by phytopathogenic fungi, contributing to various plant diseases.Therefore, these potent laccase inhibitors can prevent or weaken pathogen attacks through chemical protection.Continuing our previous research on laccase inhibitors [47,48], we have addressed the effect of changing the structure of the acyl and phenylidene units in hydrazide-hydrazones, inspired by plant secondary metabolites such as 4-hydroxybenzoic acid and salicylic aldehydes, on the direct mycelium growth inhibition of selected phytopathogenic fungi.

Syntheses and Characterization
In general, hydrazide-hydrazones, which are the subjects of our studies, comprise aromatic aldehydes and benzoic acid fragments linked by hydrazine (Ar-CH=N-NH-(C=O)-Ar ′ ).These are usually crystalline compounds synthesized in a condensation reaction of an equimolar amount of hydrazide, formed from benzoic acid esters and hydrazine hydrate with aldehyde molecules.The reactions are carried out in the classic conditions or with a mechano-chemical procedure.The first procedure is conducted in an organic solvent with hydrazine hydrate in the first step and an acetic acid additive in the second step to form a crystalline pure product directly isolated from the reaction mixture [47].The second procedure covers the step of grinding substrates, esters with hydrazine hydrate, and later forming hydrazide and aldehyde with a few drops of acetic acid, followed by a recrystallization step from organic solvent [39].Both classical and mechanical approaches generally provide pure products from good to quantitative yields.However, the first approach does not require the additional purification step and offers safer conditions for working with hydrazine hydrate and acetic acid.
In the synthesis of the nifuroxazide drug (12), the 4-hydroxybenzoic acid hydrazide (36) reacted with 5-nitro-2-furaldehyde diacetate (69) in the absence of the catalyst in agreement with the nature of the diacetate substrate 69 (Scheme 2).
In the synthesis of the nifuroxazide drug (12), the 4-hydroxybenzoic acid hydrazide (36) reacted with 5-nitro-2-furaldehyde diacetate (69) in the absence of the catalyst in agreement with the nature of the diacetate substrate 69 (Scheme 2).
The results of spectroscopic analyses of new hydrazide-hydrazones and their precursors have typical characteristics for these groups of compounds [47,48,72].Interestingly, on the HRMS analysis of both known and new hydrazide 25 and 26 derivatives of 5-methyl-and 5-methoxy-3-hydroxymethyl-salicylaldehydes (62 and 63), respectively, additional mono-dehydration ions were observed.On the contrary, only mono-dehydration ions were detected in the case of the salicylic aldehyde 62 and 63 substrates.
The results of spectroscopic analyses of new hydrazide-hydrazones and their precursors have typical characteristics for these groups of compounds [47,48,72].Interestingly, on the HRMS analysis of both known and new hydrazide 25 and 26 derivatives of 5-methyland 5-methoxy-3-hydroxymethyl-salicylaldehydes (62 and 63), respectively, additional mono-dehydration ions were observed.On the contrary, only mono-dehydration ions were detected in the case of the salicylic aldehyde 62 and 63 substrates.

Biological Studies
All prepared hydrazide-hydrazones were used in a test for determination of their antifungal activity against Botrytis cinerea, Sclerotinia sclerotiorum, and Cerrena unicolor at basal 50 µg/mL concentration.Detailed antifungal tests were conducted for the most active compounds 18, 19, 27, 28, and 30 to determine the IC 50 value in the 0-50 µg/mL range.We also investigated the phytotoxicity of seven representative compounds of choice 13, 17, 18, 19, 25, 27, and 30 using three oilseed dicot plant representatives as flax (Linum usitatissimum L.), sunflower (Helianthus annuus L.), and rapeseed (Brassica napus L. var.napus).We chose the representatives of dicots as they are the most frequently attacked by the B. cinerea and S. sclerotiorum, preferentially growing on aerial plant tissues rich in pectin [73].For flax and sunflower, we also performed further experiments on the properties of tested compounds to induce the resistance for B. cinerea and S. sclerotiorum attack, respectively.Finally, the representative compounds of choice were evaluated on their cytotoxicity on the human and mouse cell lines.

Antifungal Activities
Results of the screening activities of the hydrazide-hydrazones 1-35 at a concentration of 50 µg/mL are presented in Tables 1-3.The magnitude of mycelial growth inhibition (%) caused by the selected compound was related to a control sample with a fungus growing on a medium free of the tested compound.A value of 100% refers to complete inhibition of mycelial growth, and 0% refers to no effect on growth.The discussed thirty-five hydrazidehydrazones were divided into three groups depending on the structure of the aldehyde fragment used.Twenty-nine compounds are derivatives of 4-hydroxybenzoic acid.Results of the screening activities of the hydrazide-hydrazones 1-35 at a concentration of 50 µg/mL are presented in Tables 1-3.The magnitude of mycelial growth inhibition (%) caused by the selected compound was related to a control sample with a fungus growing on a medium free of the tested compound.A value of 100% refers to complete inhibition of mycelial growth, and 0% refers to no effect on growth.The discussed thirty-five hydrazide-hydrazones were divided into three groups depending on the structure of the aldehyde fragment used.Twenty-nine compounds are derivatives of 4-hydroxybenzoic acid.
The determined direct antimicrobial activities were compared between groups 1 and 3. Further comparison concerned antifungal activity and laccase inhibition constants previously reported for compounds 1-3, 6-10, 17-19, 21, 27, 30-31, and 33 [47,48] and determined in this study for phloroglucinol derivative 32.For the preliminary studies, the concentration of 50 µg/mL was admitted to the test.Results of the screening activities of the hydrazide-hydrazones 1-35 at a concentration of 50 µg/mL are presented in Tables 1-3.The magnitude of mycelial growth inhibition (%) caused by the selected compound was related to a control sample with a fungus growing on a medium free of the tested compound.A value of 100% refers to complete inhibition of mycelial growth, and 0% refers to no effect on growth.The discussed thirty-five hydrazide-hydrazones were divided into three groups depending on the structure of the aldehyde fragment used.Twenty-nine compounds are derivatives of 4-hydroxybenzoic acid.
The determined direct antimicrobial activities were compared between groups 1 and 3. Further comparison concerned antifungal activity and laccase inhibition constants previously reported for compounds 1-3, 6-10, 17-19, 21, 27, 30-31, and 33 [47,48] and determined in this study for phloroglucinol derivative 32.For the preliminary studies, the concentration of 50 µg/mL was admitted to the test.Results of the screening activities of the hydrazide-hydrazones 1-35 at a concentration of 50 µg/mL are presented in Tables 1-3.The magnitude of mycelial growth inhibition (%) caused by the selected compound was related to a control sample with a fungus growing on a medium free of the tested compound.A value of 100% refers to complete inhibition of mycelial growth, and 0% refers to no effect on growth.The discussed thirty-five hydrazide-hydrazones were divided into three groups depending on the structure of the aldehyde fragment used.Twenty-nine compounds are derivatives of 4-hydroxybenzoic acid.
The determined direct antimicrobial activities were compared between groups 1 and 3. Further comparison concerned antifungal activity and laccase inhibition constants previously reported for compounds 1-3, 6-10, 17-19, 21, 27, 30-31, and 33 [47,48] and determined in this study for phloroglucinol derivative 32.For the preliminary studies, the concentration of 50 µg/mL was admitted to the test.toxicity on the human and mouse cell lines.

Antifungal Activities
Results of the screening activities of the hydrazide-hydrazones 1-35 at a concentration of 50 µg/mL are presented in Tables 1-3.The magnitude of mycelial growth inhibition (%) caused by the selected compound was related to a control sample with a fungus growing on a medium free of the tested compound.A value of 100% refers to complete inhibition of mycelial growth, and 0% refers to no effect on growth.The discussed thirty-five hydrazide-hydrazones were divided into three groups depending on the structure of the aldehyde fragment used.Twenty-nine compounds are derivatives of 4-hydroxybenzoic acid.
The determined direct antimicrobial activities were compared between groups 1 and 3. Further comparison concerned antifungal activity and laccase inhibition constants previously reported for compounds 1-3, 6-10, 17-19, 21, 27, 30-31, and 33 [47,48] and determined in this study for phloroglucinol derivative 32.For the preliminary studies, the concentration of 50 µg/mL was admitted to the test.toxicity on the human and mouse cell lines.

Antifungal Activities
Results of the screening activities of the hydrazide-hydrazones 1-35 at a concentration of 50 µg/mL are presented in Tables 1-3.The magnitude of mycelial growth inhibition (%) caused by the selected compound was related to a control sample with a fungus growing on a medium free of the tested compound.A value of 100% refers to complete inhibition of mycelial growth, and 0% refers to no effect on growth.The discussed thirty-five hydrazide-hydrazones were divided into three groups depending on the structure of the aldehyde fragment used.Twenty-nine compounds are derivatives of 4-hydroxybenzoic acid.
The determined direct antimicrobial activities were compared between groups 1 and 3. Further comparison concerned antifungal activity and laccase inhibition constants previously reported for compounds 1-3, 6-10, 17-19, 21, 27, 30-31, and 33 [47,48] and determined in this study for phloroglucinol derivative 32.For the preliminary studies, the concentration of 50 µg/mL was admitted to the test.Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.
The hydrazide-hydrazones from the first group are inactive toward tested microorganisms (Table 1).However, some residual activities against white-rot fungus C. unicolor were observed in the case of molecules 4, 5, and 9, which inhibited mycelium growth in the range of 20.9-23.6% at the tested concentration.Generally, these molecules have larger iPr or SO3Na and OCH3 substituents in benzylidene units localized at opposite or near imine linkers, respectively.In our test, nifuroxazide (12) showed no activity toward the molds, and to a small extent, it reduced the growth of C. unicolor by 19.5%.Nifuroxazide is typically used to cure bacterial infections against bacteria that cause travelers' diarrhea and colitis [74].The results of antimicrobial activity correlate with the results of laccase inhibition tested in our previous study [48] on the enzyme from white-rot fungi Trametes versicolor.Generally, these simple, unsubstituted, monosubstituted phenylidene fragments in hydrazide-hydrazones in the first group are weak inhibitors and antifungal agents.
In the second group of tested hydrazide-hydrazones, two molecules numbered 18 and 19 derived from 4-hydroxybenzoic acid showed the highest activity against tested microorganisms (Table 2).These have salicylaldehyde units having hindered tert-butyl or bulky phenyl substituents near the hydroxy group.Both compounds showed relatively strong antifungal activity against B. cinerea (82.1 and 73.5%) and C. unicolor (85.9 and 71.4%).Furthermore, the tert-butyl-salicylic aldehyde derivative was the most fungicidal and almost completely inhibited the growth of S. sclerotiorum.Such a significant reduction in the growth of tested microorganisms makes this compound a highly nonselective antifungal agent.In contrast, in the same group, the compound numbered 22 presented high activity toward solely C. unicolor.Interestingly, this compound is a derivative of 2,4- Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.
The hydrazide-hydrazones from the first group are inactive toward tested microorganisms (Table 1).However, some residual activities against white-rot fungus C. unicolor were observed in the case of molecules 4, 5, and 9, which inhibited mycelium growth in the range of 20.9-23.6% at the tested concentration.Generally, these molecules have larger iPr or SO3Na and OCH3 substituents in benzylidene units localized at opposite or near imine linkers, respectively.In our test, nifuroxazide (12) showed no activity toward the molds, and to a small extent, it reduced the growth of C. unicolor by 19.5%.Nifuroxazide is typically used to cure bacterial infections against bacteria that cause travelers' diarrhea and colitis [74].The results of antimicrobial activity correlate with the results of laccase inhibition tested in our previous study [48] on the enzyme from white-rot fungi Trametes versicolor.Generally, these simple, unsubstituted, monosubstituted phenylidene fragments in hydrazide-hydrazones in the first group are weak inhibitors and antifungal agents.
In the second group of tested hydrazide-hydrazones, two molecules numbered 18 and 19 derived from 4-hydroxybenzoic acid showed the highest activity against tested microorganisms (Table 2).These have salicylaldehyde units having hindered tert-butyl or bulky phenyl substituents near the hydroxy group.Both compounds showed relatively strong antifungal activity against B. cinerea (82.1 and 73.5%) and C. unicolor (85.9 and 71.4%).Furthermore, the tert-butyl-salicylic aldehyde derivative was the most fungicidal and almost completely inhibited the growth of S. sclerotiorum.Such a significant reduction in the growth of tested microorganisms makes this compound a highly nonselective antifungal agent.In contrast, in the same group, the compound numbered 22 presented high activity toward solely C. unicolor.Interestingly, this compound is a derivative of 2,4- Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.
The hydrazide-hydrazones from the first group are inactive toward tested microorganisms (Table 1).However, some residual activities against white-rot fungus C. unicolor were observed in the case of molecules 4, 5, and 9, which inhibited mycelium growth in the range of 20.9-23.6% at the tested concentration.Generally, these molecules have larger iPr or SO3Na and OCH3 substituents in benzylidene units localized at opposite or near imine linkers, respectively.In our test, nifuroxazide (12) showed no activity toward the molds, and to a small extent, it reduced the growth of C. unicolor by 19.5%.Nifuroxazide is typically used to cure bacterial infections against bacteria that cause travelers' diarrhea and colitis [74].The results of antimicrobial activity correlate with the results of laccase inhibition tested in our previous study [48] on the enzyme from white-rot fungi Trametes versicolor.Generally, these simple, unsubstituted, monosubstituted phenylidene fragments in hydrazide-hydrazones in the first group are weak inhibitors and antifungal agents.
In the second group of tested hydrazide-hydrazones, two molecules numbered 18 and 19 derived from 4-hydroxybenzoic acid showed the highest activity against tested microorganisms (Table 2).These have salicylaldehyde units having hindered tert-butyl or bulky phenyl substituents near the hydroxy group.Both compounds showed relatively strong antifungal activity against B. cinerea (82.1 and 73.5%) and C. unicolor (85.9 and 71.4%).Furthermore, the tert-butyl-salicylic aldehyde derivative was the most fungicidal and almost completely inhibited the growth of S. sclerotiorum.Such a significant reduction in the growth of tested microorganisms makes this compound a highly nonselective antifungal agent.In contrast, in the same group, the compound numbered 22 presented high activity toward solely C. unicolor.Interestingly, this compound is a derivative of 2,4- Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.
The hydrazide-hydrazones from the first group are inactive toward tested microorganisms (Table 1).However, some residual activities against white-rot fungus C. unicolor were observed in the case of molecules 4, 5, and 9, which inhibited mycelium growth in the range of 20.9-23.6% at the tested concentration.Generally, these molecules have larger iPr or SO3Na and OCH3 substituents in benzylidene units localized at opposite or near imine linkers, respectively.In our test, nifuroxazide (12) showed no activity toward the molds, and to a small extent, it reduced the growth of C. unicolor by 19.5%.Nifuroxazide is typically used to cure bacterial infections against bacteria that cause travelers' diarrhea and colitis [74].The results of antimicrobial activity correlate with the results of laccase inhibition tested in our previous study [48] on the enzyme from white-rot fungi Trametes versicolor.Generally, these simple, unsubstituted, monosubstituted phenylidene fragments in hydrazide-hydrazones in the first group are weak inhibitors and antifungal agents.
In the second group of tested hydrazide-hydrazones, two molecules numbered 18 and 19 derived from 4-hydroxybenzoic acid showed the highest activity against tested microorganisms (Table 2).These have salicylaldehyde units having hindered tert-butyl or bulky phenyl substituents near the hydroxy group.Both compounds showed relatively strong antifungal activity against B. cinerea (82.1 and 73.5%) and C. unicolor (85.9 and 71.4%).Furthermore, the tert-butyl-salicylic aldehyde derivative was the most fungicidal and almost completely inhibited the growth of S. sclerotiorum.Such a significant reduction in the growth of tested microorganisms makes this compound a highly nonselective antifungal agent.In contrast, in the same group, the compound numbered 22 presented high activity toward solely C. unicolor.Interestingly, this compound is a derivative of 2,4- Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.
The hydrazide-hydrazones from the first group are inactive toward tested microorganisms (Table 1).However, some residual activities against white-rot fungus C. unicolor were observed in the case of molecules 4, 5, and 9, which inhibited mycelium growth in the range of 20.9-23.6% at the tested concentration.Generally, these molecules have larger iPr or SO3Na and OCH3 substituents in benzylidene units localized at opposite or near imine linkers, respectively.In our test, nifuroxazide (12) showed no activity toward the molds, and to a small extent, it reduced the growth of C. unicolor by 19.5%.Nifuroxazide is typically used to cure bacterial infections against bacteria that cause travelers' diarrhea and colitis [74].The results of antimicrobial activity correlate with the results of laccase inhibition tested in our previous study [48] on the enzyme from white-rot fungi Trametes versicolor.Generally, these simple, unsubstituted, monosubstituted phenylidene fragments in hydrazide-hydrazones in the first group are weak inhibitors and antifungal agents.
In the second group of tested hydrazide-hydrazones, two molecules numbered 18 and 19 derived from 4-hydroxybenzoic acid showed the highest activity against tested microorganisms (Table 2).These have salicylaldehyde units having hindered tert-butyl or bulky phenyl substituents near the hydroxy group.Both compounds showed relatively strong antifungal activity against B. cinerea (82.1 and 73.5%) and C. unicolor (85.9 and 71.4%).Furthermore, the tert-butyl-salicylic aldehyde derivative was the most fungicidal and almost completely inhibited the growth of S. sclerotiorum.Such a significant reduction in the growth of tested microorganisms makes this compound a highly nonselective antifungal agent.In contrast, in the same group, the compound numbered 22 presented high activity toward solely C. unicolor.Interestingly, this compound is a derivative of 2,4-99.9± 0.4 100 ± 0.3 39.9 ± 0.7 -b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.dihydroxybenzaldehyde and benzoic acid hydrazide and showed better selective activity when compared to 4-hydroxybenzoic acid derivative 21.Furthermore, in this group, the hydrazide-hydrazones 14 and 15 derivatives of a salicylic aldehyde with bulky bromine opposite to the hydroxy group and methoxy group near imine linker, respectively, present a selectivity against Basidiomycetes fungus as C. unicolor acting as moderate antifungal agents.Among the tested compounds in the more complex second group, a high correlation between antifungal activity and the potency of inhibition of laccase activity is apparent.The most potent inhibitors with hindered 3-tert-butyl and bulky 3-phenyl groups, 18 and 19, respectively, are also strong antifungal agents, and those that did not inhibit the enzyme are also weak or moderate antifungal compounds.dihydroxybenzaldehyde and benzoic acid hydrazide and showed better selective activity when compared to 4-hydroxybenzoic acid derivative 21.Furthermore, in this group, the hydrazide-hydrazones 14 and 15 derivatives of a salicylic aldehyde with bulky bromine opposite to the hydroxy group and methoxy group near imine linker, respectively, present a selectivity against Basidiomycetes fungus as C. unicolor acting as moderate antifungal agents.Among the tested compounds in the more complex second group, a high correlation between antifungal activity and the potency of inhibition of laccase activity is apparent.The most potent inhibitors with hindered 3-tert-butyl and bulky 3-phenyl groups, 18 and 19, respectively, are also strong antifungal agents, and those that did not inhibit the enzyme are also weak or moderate antifungal compounds.dihydroxybenzaldehyde and benzoic acid hydrazide and showed better selective activity when compared to 4-hydroxybenzoic acid derivative 21.Furthermore, in this group, the hydrazide-hydrazones 14 and 15 derivatives of a salicylic aldehyde with bulky bromine opposite to the hydroxy group and methoxy group near imine linker, respectively, present a selectivity against Basidiomycetes fungus as C. unicolor acting as moderate antifungal agents.Among the tested compounds in the more complex second group, a high correlation between antifungal activity and the potency of inhibition of laccase activity is apparent.The most potent inhibitors with hindered 3-tert-butyl and bulky 3-phenyl groups, 18 and 19, respectively, are also strong antifungal agents, and those that did not inhibit the enzyme are also weak or moderate antifungal compounds.dihydroxybenzaldehyde and benzoic acid hydrazide and showed better selective activity when compared to 4-hydroxybenzoic acid derivative 21.Furthermore, in this group, the hydrazide-hydrazones 14 and 15 derivatives of a salicylic aldehyde with bulky bromine opposite to the hydroxy group and methoxy group near imine linker, respectively, present a selectivity against Basidiomycetes fungus as C. unicolor acting as moderate antifungal agents.Among the tested compounds in the more complex second group, a high correlation between antifungal activity and the potency of inhibition of laccase activity is apparent.The most potent inhibitors with hindered 3-tert-butyl and bulky 3-phenyl groups, 18 and 19, respectively, are also strong antifungal agents, and those that did not inhibit the enzyme are also weak or moderate antifungal compounds.dihydroxybenzaldehyde and benzoic acid hydrazide and showed better selective activity when compared to 4-hydroxybenzoic acid derivative 21.Furthermore, in this group, the hydrazide-hydrazones 14 and 15 derivatives of a salicylic aldehyde with bulky bromine opposite to the hydroxy group and methoxy group near imine linker, respectively, present a selectivity against Basidiomycetes fungus as C. unicolor acting as moderate antifungal agents.Among the tested compounds in the more complex second group, a high correlation between antifungal activity and the potency of inhibition of laccase activity is apparent.The most potent inhibitors with hindered 3-tert-butyl and bulky 3-phenyl groups, 18 and 19, respectively, are also strong antifungal agents, and those that did not inhibit the enzyme are also weak or moderate antifungal compounds.a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
In the third group, the most active and simultaneously non-selective is compound 30-a derivative of 3-tert-butylsalicylidene 18 with the presence of methyl, the smallest alkyl group in the 5th position of aldehyde fragment.Similarly, it almost completely inhibited the growth of S. sclerotiorum (96.8%), and it is the most promising antifungal agent against C. unicolor.Similar compounds derived from 4-methoxybenzoic acid 31, benzoic acid 33, and nicotinic acid 34 showed no antifungal activity against molds and residual activity against white-rot fungi.Interestingly, the total replacement of the acyl unit in 33 and 34 on the acetyl unit in 35 slightly improved activity against C. unicolor.Compounds 24-26, having differently localized hydroxymethyl group and methyl or methoxy groups at the salicylidene unit, showed moderate and reasonable activity toward C. unicolor.Replacing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor [48].a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
In the third group, the most active and simultaneously non-selective is compound 30-a derivative of 3-tert-butylsalicylidene 18 with the presence of methyl, the smallest alkyl group in the 5th position of aldehyde fragment.Similarly, it almost completely inhibited the growth of S. sclerotiorum (96.8%), and it is the most promising antifungal agent against C. unicolor.Similar compounds derived from 4-methoxybenzoic acid 31, benzoic acid 33, and nicotinic acid 34 showed no antifungal activity against molds and residual activity against white-rot fungi.Interestingly, the total replacement of the acyl unit in 33 and 34 on the acetyl unit in 35 slightly improved activity against C. unicolor.Compounds 24-26, having differently localized hydroxymethyl group and methyl or methoxy groups at the salicylidene unit, showed moderate and reasonable activity toward C. unicolor.Replacing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor [48].a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
In the third group, the most active and simultaneously non-selective is compound 30-a derivative of 3-tert-butylsalicylidene 18 with the presence of methyl, the smallest alkyl group in the 5th position of aldehyde fragment.Similarly, it almost completely inhibited the growth of S. sclerotiorum (96.8%), and it is the most promising antifungal agent against C. unicolor.Similar compounds derived from 4-methoxybenzoic acid 31, benzoic acid 33, and nicotinic acid 34 showed no antifungal activity against molds and residual activity against white-rot fungi.Interestingly, the total replacement of the acyl unit in 33 and 34 on the acetyl unit in 35 slightly improved activity against C. unicolor.Compounds 24-26, having differently localized hydroxymethyl group and methyl or methoxy groups at the salicylidene unit, showed moderate and reasonable activity toward C. unicolor.Replacing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor [48].99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 -b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined.Bold values refer to compounds selected for detailed IC 50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
The determined direct antimicrobial activities were compared between groups 1 and 3. Further comparison concerned antifungal activity and laccase inhibition constants previously reported for compounds 1-3, 6-10, 17-19, 21, 27, 30-31, and 33 [47,48] and determined in this study for phloroglucinol derivative 32.For the preliminary studies, the concentration of 50 µg/mL was admitted to the test.
The hydrazide-hydrazones from the first group are inactive toward tested microorganisms (Table 1).However, some residual activities against white-rot fungus C. unicolor were observed in the case of molecules 4, 5, and 9, which inhibited mycelium growth in the range of 20.9-23.6% at the tested concentration.Generally, these molecules have larger iPr or SO 3 Na and OCH 3 substituents in benzylidene units localized at opposite or near imine linkers, respectively.In our test, nifuroxazide (12) showed no activity toward the molds, and to a small extent, it reduced the growth of C. unicolor by 19.5%.Nifuroxazide is typically used to cure bacterial infections against bacteria that cause travelers' diarrhea and colitis [74].The results of antimicrobial activity correlate with the results of laccase inhibition tested in our previous study [48] on the enzyme from white-rot fungi Trametes versicolor.Generally, these simple, unsubstituted, monosubstituted phenylidene fragments in hydrazide-hydrazones in the first group are weak inhibitors and antifungal agents.
In the second group of tested hydrazide-hydrazones, two molecules numbered 18 and 19 derived from 4-hydroxybenzoic acid showed the highest activity against tested microorganisms (Table 2).These have salicylaldehyde units having hindered tert-butyl or bulky phenyl substituents near the hydroxy group.Both compounds showed relatively strong antifungal activity against B. cinerea (82.1 and 73.5%) and C. unicolor (85.9 and 71.4%).Furthermore, the tert-butyl-salicylic aldehyde derivative was the most fungicidal and almost completely inhibited the growth of S. sclerotiorum.Such a significant reduction in the growth of tested microorganisms makes this compound a highly nonselective antifungal agent.In contrast, in the same group, the compound numbered 22 presented high activity toward solely C. unicolor.Interestingly, this compound is a derivative of 2,4dihydroxybenzaldehyde and benzoic acid hydrazide and showed better selective activity when compared to 4-hydroxybenzoic acid derivative 21.Furthermore, in this group, the hydrazide-hydrazones 14 and 15 derivatives of a salicylic aldehyde with bulky bromine opposite to the hydroxy group and methoxy group near imine linker, respectively, present a selectivity against Basidiomycetes fungus as C. unicolor acting as moderate antifungal agents.Among the tested compounds in the more complex second group, a high correlation between antifungal activity and the potency of inhibition of laccase activity is apparent.The most potent inhibitors with hindered 3-tert-butyl and bulky 3-phenyl groups, 18 and 19, respectively, are also strong antifungal agents, and those that did not inhibit the enzyme are also weak or moderate antifungal compounds.
In the third group, the most active and simultaneously non-selective is compound 30-a derivative of 3-tert-butylsalicylidene 18 with the presence of methyl, the smallest alkyl group in the 5th position of aldehyde fragment.Similarly, it almost completely inhibited the growth of S. sclerotiorum (96.8%), and it is the most promising antifungal agent against C. unicolor.Similar compounds derived from 4-methoxybenzoic acid 31, benzoic acid 33, and nicotinic acid 34 showed no antifungal activity against molds and residual activity against white-rot fungi.Interestingly, the total replacement of the acyl unit in 33 and 34 on the acetyl unit in 35 slightly improved activity against C. unicolor.Compounds 24-26, having differently localized hydroxymethyl group and methyl or methoxy groups at the salicylidene unit, showed moderate and reasonable activity toward C. unicolor.Replacing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor [48].
We selected compounds for further testing based on the preliminary results of antifungal activity at a concentration of 50 µg/mL, with more than 40% growth inhibition for at least two microorganisms (see bolded values in Tables 2 and 3).The IC 50 values were determined by estimating parameter values of the log-logistic model along with goodness of fit (R 2 and RMSE), as shown in Table 4.The IC 50 's values were calculated only when the model predicted a decrease in growth rate by more than 50%.Of the hydrazide-hydrazones tested, five hydroxybenzoic acid derivatives, 18, 19, 27, 28, and 30, were selected, and the results of their antifungal activity were compared with those obtained for the reference fenhexamid (fungicide).The most active compounds are those in the second and third groups, which include derivatives of 4-hydroxybenzoic acid and functionalized salicylic aldehydes, as presented in Figure 2. The benzoyl unit from benzoic acid hydrazides and salicylidene unit from salicylic aldehydes, used in the synthesis of the potent hydrazide-hydrazones toward fungi, were indicated in the green and violet frame, respectively.Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined; c SD = 5.75 (%) for a test of laccase from Trametes versicolor inhibition on 2,4-diformylphloroglucinol derivative 32 Ki determination [75].Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
We selected compounds for further testing based on the preliminary results of antifungal activity at a concentration of 50 µg/mL, with more than 40% growth inhibition for Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined; c SD = 5.75 (%) for a test of laccase from Trametes versicolor inhibition on 2,4-diformylphloroglucinol derivative 32 Ki determination [75].Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
We selected compounds for further testing based on the preliminary results of antifungal activity at a concentration of 50 µg/mL, with more than 40% growth inhibition for Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined; c SD = 5.75 (%) for a test of laccase from Trametes versicolor inhibition on 2,4-diformylphloroglucinol derivative 32 Ki determination [75].Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
We selected compounds for further testing based on the preliminary results of antifungal activity at a concentration of 50 µg/mL, with more than 40% growth inhibition for Fenhexamid 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined; c SD = 5.75 (%) for a test of laccase from Trametes versicolor inhibition on 2,4-diformylphloroglucinol derivative 32 Ki determination [75].Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
We selected compounds for further testing based on the preliminary results of antifungal activity at a concentration of 50 µg/mL, with more than 40% growth inhibition for 99.9 ± 0.4 100 ± 0.3 39.9 ± 0.7 -b a Results were calculated as the percent of microorganism growth inhibition in the presence of 50 µg/mL of tested compounds in potato dextrose agar (PDA) medium compared to 0.5% (v/v) methanol-treated control; the result is expressed as the mean of three determinations.b not determined; c SD = 5.75 (%) for a test of laccase from Trametes versicolor inhibition on 2,4-diformylphloroglucinol derivative 32 K i determination [75].Bold values refer to compounds selected for detailed IC50 testing-the criteria are at least 40% inhibition of mycelial growth and such activity against at least two pathogens.
The salicylic aldehyde fragment, in active molecules, always contains a larger substituent, such as isopropyl, tert-butyl, and phenyl, at position 3 (near the hydroxy group).Molecules 18, 19, 28, and 30 have IC 50 values in the range of 13.9-62.5µg/mL against B. cinerea, which are two orders of magnitude lower when compared to the reference compound.Compounds 18, 19, and 30 show similar 13.5-20.3µg/mL activity against C. unicolor.On the contrary, three of the five tested compounds, 18, 27, and 30 with bulky tert-butyl show the most promising 0.5-1.8µg/mL activity against S. sclerotiorum, which is at least twice as high as fenhexamid fungicide.Of particular interest is hydrazide-hydrazone 18 with simple 3-tert-butylsalicylidene unit, which is active against all plant pathogens tested, while more substituted compounds 27 and 28 show high activity against the white mold fungus (Table 4).
The profiles of anti-B.cinerea, -S.sclerotiorum, and -C.unicolor activity for tested hydrazide-hydrazones derivatives of 4-hydroxybenzoic acid and fenhexamid (control fungicide) were illustrated in Supplementary Materials (see Figure S129a-c).Herein, we provide some insight into the relationship between the hydrazide-hydrazones concentrations applied and the inhibition potency of fungi growth in comparison to fenhexamid.Four concentrations of hydrazide-hydrazones, such as 6.25, 12.5, 25.0, and 50.0 µg/mL, were subjected to the estimation of the fungicidal profiles for 18, 19, 27, 28, 30, and fenhexamid.After analyzing the data, it is apparent that, generally, the most susceptible microorganism for tested compounds was S. sclerotiorum (Tables 4 and S3 and Figure S129b).Even the lowest tested concentration (6.25 µg/mL) of the 18, 27, and 30 molecules brought relatively high antifungal activity between 70.6 and 87.6%, compared to 95.6% of fenhexamid.A two-fold increase in the concentration of the hydrazide-hydrazones 18 and 30 showed their activity similar to fenhexamid, 96.3, 98.7, and 96.3%, respectively, approaching almost total inhibition of S. sclerotiorum growth at 50 µg/mL.Although derivative 27 presented a high 82.9% of S. sclerotiorum inhibition at the lowest investigated concentration, it strengthened the further effect to 93.1% only at 50 µg/mL.All discussed compounds (18, 27, and 30) are derivatives of 3-tert-butyl-2-hydroxybenzaldehydes and 4-hydroxybenzoic acid hydrazide.In that context, the presence of the tert-butyl group in the 3 position seems important for anti-S.sclerotiorum activity.Far less susceptible on tested hydrazide-hydrazones was C. unicolor (Tables 4 and S3, Figure S129c).The smallest concentration inhibited the fungus in the range of 9.2-21.5%.A two-fold increase in the dose enhanced the effect to 21.7-42.2%.At 25 µg/mL, the molecules 18, 19, and 30 showed the highest activity, which reached 76.5, 70.5, and 90.5% of C. unicolor inhibition at maximal concentration.But again, the hydrazide-hydrazones with 3-tert-butyl-5-methyl-2-hydroxybenzaldehyde fragment showed the highest antifungal activity.The highly active mold fungicide, fenhexamid, was weakly active on the fungus, with 32.1% in mycelium inhibition at 50 µg/mL.The least susceptible to tested compounds was B. cinerea (see Tables 4 and S3, Figure S129b).None of the hydrazide-hydrazones reached the activity of fenhexamid, which presented constant very high activity in the whole range of tested concentrations (98.5% inhibition at 6.25% to 100% inhibition at 50 µg/mL).In contrast to previous fungi, the most active compound against B. cinerea was a derivative of 3-phenyl-salicylic aldehyde and 4-hydroxybenzoic acid hydrazide 19.In fact, for this compound, the antifungal activity profile covers the systematic increase of inhibition from 30.1 to 70.1% of fungal development from 6.25 to 25.0 µg/mL, but at 50 µg/mL, only 74.1% was observed.A similar course of the profile could be attributed to molecule 18 but with slightly weaker activity corresponding to 20.2, 56.4, and 64.7%.The remaining compounds showed rather low activity, less than 50% of B. cinerea inhibition.and the results of their antifungal activity were compared with those obtained for the reference fenhexamid (fungicide).The most active compounds are those in the second and third groups, which include derivatives of 4-hydroxybenzoic acid and functionalized salicylic aldehydes, as presented in Figure 2. The benzoyl unit from benzoic acid hydrazides and salicylidene unit from salicylic aldehydes, used in the synthesis of the potent hydrazide-hydrazones toward fungi, were indicated in the green and violet frame, respectively.The salicylic aldehyde fragment, in active molecules, always contains a larger substituent, such as isopropyl, tert-butyl, and phenyl, at position 3 (near the hydroxy group).Molecules 18, 19, 28, and 30 have IC50 values in the range of 13.9-62.5µg/mL against B. cinerea, which are two orders of magnitude lower when compared to the reference compound.Compounds 18, 19, and 30 show similar 13.5-20.3µg/mL activity against C. unicolor.On the contrary, three of the five tested compounds, 18, 27, and 30 with bulky tertbutyl show the most promising 0.5-1.8µg/mL activity against S. sclerotiorum, which is at least twice as high as fenhexamid fungicide.Of particular interest is hydrazide-hydrazone 18 with simple 3-tert-butylsalicylidene unit, which is active against all plant pathogens tested, while more substituted compounds 27 and 28 show high activity against the white mold fungus (Table 4).Comparing the discussed results of the antimicrobial activity on the laccase-producing strains-B.cinerea, S. sclerotiorum, and C. unicolor-presented in this work to the results of the enzymatic studies performed on laccase from Trametes versicolor published in our previous work ([48], Tables 1-3), it is shown that potent laccase inhibitors 18, 19, 27, and 30 are also potent fungicides to the microorganisms producing these oxidoreductases.The presented results also showed some exceptions that do not meet this rule.These concern compounds 31, 33, and 28.Compounds 31 and 33 inhibit laccase but do not inhibit the growth of the tested microorganisms.In contrast, the thymol derivative 28 does not inhibit laccase (K i > 1000 µM) but selectively inhibits the growth of S. sclerotiorum IC 50 = 9.8 µg/mL.Therefore, further research is needed to clarify these phenomena.These compounds will be the subject of future experimental studies to determine the mechanism of action of this class of compounds.Concerning the last hydrazide-hydrazone (28), it is worth focusing on the thymol molecule.So far, unsubstituted thymol has been the subject of research due to its advantageous properties used in the food industry and traditional medicine and its activity against bacteria and fungi [76].In studies of unsubstituted thymol on Fusarium, Penicillium, and Aspergillus species, it inhibits mycelium growth at only high concentration (1 mg/mL) [28].In later detailed studies, the determined antifungal activity (MIC-minimum inhibitory concentration, µg/mL) of thymol on plant pathogens were 300 µg/mL for B. cinerea and Fusarium oxysporum Schltdl., 400 µg/mL for Alternaria alternata (Fr.)Keissl, and 450 µg/mL for Rhizopus oryzae [77].Recent studies on the antifungal activity of thymol have shown that it is a promising chemosensing agent for various fungi, and combined treatment with commercial fungicide allowed for a lower dosage of chemical treatment [78].Thus, thymol derivatives represent an interesting and noteworthy research area for discovering new fungicides.

Dicotyledonous Plant Germination Tests
Among the tested hydrazide-hydrazones derived from 4-hydroxybenzoic acid, we chose seven derivatives of 2-and 4-hydroxybenzaldehydes 13, 17, 18, 19, 25, 27, and 30.Four of them, 18, 19, 27, and 30, with a salicylidene framework, are simultaneously the most potent laccase inhibitors and strong antifungal agents.They have the salicylic aldehyde fragment modified with hindered tert-butyl or large phenyl substituents in 3-position.The hydrazide-hydrazone 25 has a hydroxymethyl substituent next to the hydroxy group of salicylic aldehyde fragment.This molecule represented the potential against C. unicolor without activity toward the laccase enzyme.The last two molecules, 13 and 17, are derivatives of gentisaldehyde and vanillin aldehyde, respectively.These natural components of both molecules are ubiquitous in the plant kingdom.Therefore, we use them to compare their influence on seed germination and induction of pathogen resistance with the highly potent antimicrobial potential application of our compounds in agriculture and horticulture.The grey and white molds that were studied usually attacked dicotyledons [73].Thus, we have selected three representatives of temperate climate plants whose seeds produce valuable oils, both for food and industrial purposes.The crops of choice are Linum usitatissimum (flax), Helianthus annuus (sunflower), and Brassica napus var.napus (rapeseed) for the test of phytotoxicity (test 1).Moreover, the flax and sunflower seeds were used to evaluate their resistance to B. cinerea and S. sclerotiorum, respectively (test 2).The results of test 1 were presented in Figure 3 and discussed based on plant germination index (GI, %, for methodology, see Section 3.2.4).Both tests were performed by using 50 µg/mL hydrazide-hydrazones solutions.According to the adopted methodology [86,87], a germination index (GI, %) ran from 90 to 110% means no effect of the tested substance on germination and root gro below 90% means an adverse effect, and above 110% means a favorable effect on ge nation and root growth.The phytotoxic effect showed six out of seven compounds f least one kind of seed.Among them, derivative 27 has a GI of 83% and 82% for rape and sunflower, respectively, which are the single compounds that negatively influ two plants.In single cases, the germination index was reduced to 67%, the lowest GI v calculated for rapeseed and sunflower for hydrazide-hydrazone 18 and 30, respectiv having one tert-butyl group.The sensitivity of the dicotyledonous crop seeds tested to tested hydrazide-hydrazones can be presented as follows: sunflower > rape > flax, w the listed seeds were inhibited by four or three compounds, and for flax seeds, no in tion effect was observed.No general trend describes the influence of hydrazide-hy zones tested, but some rules could be applied for at least two kinds of seeds.Surprisin hydrazide-hydrazones 13 and 17 with a fragment of naturally occurring aldehydes ringaldehyde, and salicylaldehyde, respectively, had little negative effect on sunflo and rapeseed with GIs of 87 and 81% (Figure 3).The positive, stimulating effect was served for 18, 19, and 25 molecules.However, derivatives 18 and 19 showed differen fects on the seed tested, having no effect on germination and root growth for sunflo and stimulating germination and growth for flax and rape, respectively.Hydrazide drazone 19 is a derivative of 2-hydroxybiphenyl (ortho-phenylphenol).The orthonylphenol itself is usually used in harvesting fruits to prevent fungal growth.An u pected result was observed for hydrazide-hydrazone 25, with the aspect of C. uni grown inhibition, which was non-toxic for all seeds tested and stimulated the grow rapeseed and flax.
The second test (test 2) assessed the seeds' germination and root growth abilit fected with the mycelium of the pathogen on soil with a filter in which the correspon test compound was present.For the test, we selected pairs of plants-pathogens suc sunflower-S.sclerotiorum and flax-B.cinerea.The results were compared with a con sample that contained soil with a water-ethanol solution (0.5%, v/v) and seeds infe with a particular mold.The results of test 2 were also expressed in terms of the germ tion index (%), as shown in Figure 4.According to the adopted methodology [86,87], a germination index (GI, %) ranging from 90 to 110% means no effect of the tested substance on germination and root growth, below 90% means an adverse effect, and above 110% means a favorable effect on germination and root growth.The phytotoxic effect showed six out of seven compounds for at least one kind of seed.Among them, derivative 27 has a GI of 83% and 82% for rapeseed and sunflower, respectively, which are the single compounds that negatively influence two plants.In single cases, the germination index was reduced to 67%, the lowest GI value calculated for rapeseed and sunflower for hydrazide-hydrazone 18 and 30, respectively, having one tert-butyl group.The sensitivity of the dicotyledonous crop seeds tested to the tested hydrazide-hydrazones can be presented as follows: sunflower > rape > flax, where the listed seeds were inhibited by four or three compounds, and for flax seeds, no inhibition effect was observed.No general trend describes the influence of hydrazide-hydrazones tested, but some rules could be applied for at least two kinds of seeds.Surprisingly, hydrazide-hydrazones 13 and 17 with a fragment of naturally occurring aldehydes, syringaldehyde, and salicylaldehyde, respectively, had little negative effect on sunflower and rapeseed with GIs of 87 and 81% (Figure 3).The positive, stimulating effect was observed for 18, 19, and 25 molecules.However, derivatives 18 and 19 showed different effects on the seed tested, having no effect on germination and root growth for sunflower and stimulating germination and growth for flax and rape, respectively.Hydrazide-hydrazone 19 is a derivative of 2-hydroxybiphenyl (ortho-phenylphenol).The ortho-phenylphenol itself is usually used in harvesting fruits to prevent fungal growth.An unexpected result was observed for hydrazide-hydrazone 25, with the aspect of C. unicolor grown inhibition, which was non-toxic for all seeds tested and stimulated the growth of rapeseed and flax.
The second test (test 2) assessed the seeds' germination and root growth ability infected with the mycelium of the pathogen on soil with a filter in which the corresponding test compound was present.For the test, we selected pairs of plants-pathogens such as sunflower-S.sclerotiorum and flax-B.cinerea.The results were compared with a control sample that contained soil with a water-ethanol solution (0.5%, v/v) and seeds infected with a particular mold.The results of test 2 were also expressed in terms of the germination index (%), as shown in Figure 4. Almost all compounds showed positive effects on GI for sunflower and flax pared to the infected seeds control.The GI for sunflower was between 97 and 151%; m while, for flax, the effect was more pronounced, which is reflected in GI values bet 162 and 408%.Furthermore, to see the positive effect on GI for seeds infected with a f pathogen, we compare this parameter for uninfected and infected control samples infected samples treated with a particular hydrazide-hydrazone solution, as show Figure 5.In comparing the control sample uninfected with fungal pathogens to t fected control samples, a significant reduction in the germination index of 62 and 25% apparent for sunflower and flax, respectively.In all cases tested, hydrazide-hydra eliminated the negative effect of the pathogen.In the case of 3,5-di-tert-butylsalicyl derivative 27 and 5-methyl-3-tert-butylsalicylidene derivative 30 on sunflower and no negative effect of the pathogen was observed (compounds eliminated the negati fect of the pathogen).Almost all compounds showed positive effects on GI for sunflower and flax compared to the infected seeds control.The GI for sunflower was between 97 and 151%; meanwhile, for flax, the effect was more pronounced, which is reflected in GI values between 162 and 408%.Furthermore, to see the positive effect on GI for seeds infected with a fungal pathogen, we compare this parameter for uninfected and infected control samples with infected samples treated with a particular hydrazide-hydrazone solution, as shown in Figure 5.In comparing the control sample uninfected with fungal pathogens to the infected control samples, a significant reduction in the germination index of 62 and 25% was apparent for sunflower and flax, respectively.In all cases tested, hydrazide-hydrazones eliminated the negative effect of the pathogen.In the case of 3,5-di-tert-butylsalicylidene derivative 27 and 5-methyl-3-tert-butylsalicylidene derivative 30 on sunflower and flax, no negative effect of the pathogen was observed (compounds eliminated the negative effect of the pathogen).

Cytotoxicity Studies
The cytotoxicity studies were conducted on normal human breast epithelial MCF-10A and mouse fibroblasts Balb/3T3 cell lines using cis-platin and azoxystrobin as positive controls.The calculated IC 50 values are presented in Table 5, and the illustrations of cytotoxicity profiles are provided in the Supplementary Materials (see Table S4 and Figure S130).To our surprise, despite the presence of a hydrazine toxicophore in the molecules 13 and 17 consisting of unmodified naturally occurred gentisaldehyde (52) and vanillin aldehyde (56) units, respectively, they did not show cytotoxicity on tested lines.In addition to the salicylic aldehyde fragment, the CH 2 OH and CH 3 substituents at positions 3 and 5, respectively, in compound 25, lead to a 3-fold reduction in cytotoxicity compared to cis-platin.The naturally occurring gallic acid (3,4,5-hba), 4-hydroxybenzoic acid (4-hba), and its hydrazide 36 were non-toxic.All highly fungicidal molecules tested, i.e., 18, 19, 27, and 30, have the same cytotoxicity as cis-platin but at least one order of magnitude less cytotoxic on the mouse cell line than azoxystrobin, a commonly used fungicide in plant protection.Azoxystrobin is semisynthetic, a well-known, widely used pesticide of natural origin, characterized by a broad spectrum of action against plant fungal diseases, and has already been used for almost three decades [88].Therefore, we assume that all compounds with lower cytotoxicity could be classified for future studies concerning the determination of the mechanism of action and further research on plants in the context of future applications in agriculture.

Cytotoxicity Studies
The cytotoxicity studies were conducted on normal human breast epithelial MCF-10A and mouse fibroblasts Balb/3T3 cell lines using cis-platin and azoxystrobin as positive controls.The calculated IC50 values are presented in Table 5, and the illustrations of cytotoxicity profiles are provided in the Supplementary Materials (see Table S4 and Figure S130).To our surprise, despite the presence of a hydrazine toxicophore in the molecules 13 and 17 consisting of unmodified naturally occurred gentisaldehyde (52) and vanillin aldehyde (56) units, respectively, they did not show cytotoxicity on tested lines.In addition to the salicylic aldehyde fragment, the CH2OH and CH3 substituents at positions 3 and 5, respectively, in compound 25, lead to a 3-fold reduction in cytotoxicity compared to cis-platin.The naturally occurring gallic acid (3,4,5-hba), 4-hydroxybenzoic acid (4hba), and its hydrazide 36 were non-toxic.All highly fungicidal molecules tested, i.e., 18, 19, 27, and 30, have the same cytotoxicity as cis-platin but at least one order of magnitude less cytotoxic on the mouse cell line than azoxystrobin, a commonly used fungicide in plant protection.Azoxystrobin is semisynthetic, a well-known, widely used pesticide of natural origin, characterized by a broad spectrum of action against plant fungal diseases, and has already been used for almost three decades [88].Therefore, we assume that all compounds with lower cytotoxicity could be classified for future studies concerning the determination of the mechanism of action and further research on plants in the context of future applications in agriculture.Summarizing the biological studies, our results showed that a specific group of hydrazidehydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 µg/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for further research aimed at application in the agricultural sector, mainly in the context of protecting oilseed crops from which edible and industrial oils are currently obtained.It will be crucial to elucidate their mechanism of action and determine the effects of the tested compounds in dose-response assays on plants, as well as further modification of the substituents to improve antifungal activity and lower phyto-and cytotoxicity.Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we believe that compounds with the highest activity against S. sclerotiorum are candidates for  Summarizing the biological studies, our results showed that a specific group of hydrazide-hydrazones, derivatives of naturally occurring salicylic aldehyde and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, showed promising antifungal activity against phytopathogens.The most susceptible to the tested compounds was S. sclerotiorum, for which hydrazones 18 and 30 were shown to exhibit activity close to fenhexamid at 12.5 ug/mL.In phytotoxicity tests, derivative 18 showed no effect on the germination index (GI), while derivative 30 had a negative effect on GI.On the other hand, in a phytotoxicity test on seeds infected with S. sclerotiorum mycelium, a positive effect on GI was evident for all tested compounds, as well as the effect of eliminating the pathogen's negative effect on germination and root growth in the case of derivative 30.Moreover, all of the aforementioned compounds showed at least an order of magnitude lower cytotoxicity compared to the commonly used fungicide azoxystrobin.With future plans in mind, we 174.11 ± 14.33 163.17
The cultures of Botrytis cinerea strain FBc05 and Sclerotinia sclerotiorum strain FSc10 were obtained from the collection of the Division of Plant Pathology and Mycology at the Department of Plant Protection of Wroclaw University of Environmental and Life Sciences (Poland).Cerrena unicolor (Bull.ex.Fr.)Murr, strain no.139, originated from the culture

In Vitro Antifungal Activity
Prior to the fungicidal activity assay of hydrazide-hydrazones 1-35, we considered ethanol and DMSO as potential dissolution media for tested compounds.Most of the hydrazide-hydrazones have poor solubility in water.Furthermore, a very important aspect of biological testing is the careful selection of organic solvents.These are typically used, e.g., methanol, ethanol, acetone, and DMSO, and may be highly toxic for microorganisms in even very low concentrations, such as 1% (v/v) [100].Ethanol and DMSO with a final concentration between 0.1 and 1.0% (v/v) in a PDA medium were used and applied to evaluate the inhibition growth of B. cinerea, S. sclerotiorum, and C. unicolor (see Supplementary Materials).The following procedure was applied to the microorganism testing.The assays of fungi inhibition growth were performed at a basal concentration of 50 µg/mL for all compounds, and detailed experiments were extended for testing between 0 and 50 µg/mL (0, 6.25, 12.5, 25.0, and 50.0 µg/mL) for selected compounds.The tests were carried out on Petri dishes (diameter 60 mm) with PDA medium containing appropriate compound, co-solvent control, and hydrazide-hydrazone dissolved in co-solvent assay.A piece of mycelium with a 5 mm edge taken from the outer edge of the growing fungi on the PDA plate with co-solvent was transferred to a central part of the PDA plate with the examined compound.The plates thus inoculated were incubated at 22 • C in the dark for a time when the control plate containing only the co-solvent was covered entirely with the tested microorganism.For B. cinerea and S. sclerotiorum, the test took 3 days, and for C. unicolor, it took 7 days.Each assay was performed at least in triplicate.The dose-response relationship between the concentration of tested chemicals and the growth of three chosen fungi species was described by log-logistic Equation ( 1 4. The IC 50 values were calculated only in cases when the model predicted a decrease in growth rate by more than 50%.The low value of R 2 , e.g., 0.13 for 27 and S. sclerotiorum, indicated a small difference between the regression line and the simple average, which was noted when increasing concatenations of chemicals hardly influenced the toxic effect, and therefore, the data points were arranged close to the horizontal line reflecting their average value.The model parameters were not estimated for ethanol influence on S. sclerotiorum growth because no signs of inhibition were observed, even in the highest ethanol concentrations (see Supplementary Materials, Tables S2 and S3).

Kinetic Study for 32
The inhibition constant for 32 was determined according to procedures and calculations reported previously [47,48] using commercially available laccase from Trametes versicolor (Sigma-Aldrich).

Phytotests
In the experiments with plants, two aspects were taken under consideration: (1) phytotoxicity, which determined the toxicity of the hydrazide-hydrazones on seed germination and root growth, and (2) study on the behavior of seeds in the presence of the tested hydrazide-hydrazones and pathogens producing laccase (B.cinerea and S. sclerotiorum).The non-coated seeds of Linum usitatissimum, Helianthus annuus, and Brassica napus var.napus were provided by the Institute of Soil Science and Plant Cultivation, National Research Institute in Pulawy, Department of Weed Science and Tillage Systems, and Institute of Technology and Life Sciences-National Research Institute Falenty (Poland).Both phytotests were performed based on direct-contact microbiotests following the protocol recommended by the manufacturer (PhytotestkitTM, MicroBio Tests, Mariakerke, Belgium) with the authors' modifications.
The main phytotest equipment (Tigret, Warsaw, Poland) and general methods are described as follows.Test plates (21 × 15.5 × 0.8 cm) provide optimal moisture and enable observation of seed germination and data reading at the end of the test.A single plate is made of polyvinylchloride and composed of both a transparent bottom and a covering part that are stuck together by a set of press buttons.The bottom part is separated horizontally with a middle ridge into two equal shallow rectangular areas, in which the lower area was filled with 90 mL of roasted sand and 30 mL of the ethanol solution of the tested compound.In tests ( 1) and ( 2), the selected hydrazide-hydrazones were dissolved to 50 µM in the water-ethanol solution 0.5% (v/v) and distributed uniformly into the sand.Such prepared wet support was covered with a black filter paper that soaked the solution and was applied for further testing.
In the assay of phytotoxicity (1), the seeds of three dicota plants were used, namely ten seeds of flax or rapeseed and six seeds of sunflower.The seeds were transferred directly on the filter surface by placing them along the line at approximately 1 cm from the ridge at equal distances.Then, the seeds on the filter surface were covered with a covering part and placed in a holder.
In assay (2), the direct interaction between seed-microorganism-tested hydrazidehydrazone was tested.Before the seeds were transferred on the surface of wet support, they were covered with mycelium of B. cinerea or S. sclerotiorum for flax or sunflower, respectively.For that purpose, 100 or 50 seeds of flax or sunflower, respectively, were transferred to the Petri dish (90 mm diameter) with a 7-day-old culture and gently shaken.Then, the seeds were placed on the prepared soaked support with the black filter and covered with the upper part of the plate.The choice of type of fungi and seeds was arbitrary since both molds attack both types of plant.
All test plates were put vertically in a special incubator to allow free germination and growth of roots at 25 • C through 5 days in the dark.The test was performed in triplicate for each test plant.The results were expressed as the germination index values (GI, Equation ( 2)) concerning the plant growth controls in water-ethanol solution for (1).The results obtained for (2) were also related to the control obtained in (1) test.The GI was calculated from the formula and categorized as an inhibition for GI values < 90%, as stimulation > 110%, and between 90 and 110% as non-toxic/no effect as proposed in the literature [86,87].
where Gs is seed germination (%), Ls is root elongation (mm) for the test plate, and Gc and Lc are corresponding values for the control plates.Gc was calculated as the proportion of germinated seeds in the test in relation to the number of germinated seeds in the control.
The control was the representative number of seeds that germinated on a medium with 0.5% (v/v) of ethanol.

Cytotoxicity Cell Lines
Antiproliferative tests were performed on a normal human breast cell line, MCF-10A, and a mouse embryonic fibroblast: Balb/3T3.Both cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA).The cell lines were maintained in the Institute of Immunology and Experimental Therapy, Wroclaw, Poland.MCF-10A cells were cultured in the F-12 nutrient mixture (Gibco, Scotland, UK), supplemented with 5% horse serum (Gibco, Scotland, UK), 10 µg/mL of cholera toxin (Vibrio cholerae Pacini), 10 µg/mL of hydrocortisone and 20 ng/mL of human epidermal growth factor (all from Sigma-Aldrich, Chemie GmbH, Steinheim, Germany).Balb/3T3 cells were cultured in Dulbecco medium (Gibco, Darmstadt, Germany) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 2 mM l-glutamine (Sigma-Aldrich, St. Louis, MO, USA).All culture media contained antibiotics: 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA) and 100 µg/mL streptomycin (Polfa-Tarchomin, Warsaw, Poland).Both cell lines were cultured during the entire experiment in a humid atmosphere at 37 • C and 5% CO 2 .Amistar 250 SC (fungicide) with active compound azoxystrobin was provided by the Institute of Soil Science and Plant Cultivation, National Research Institute in Pulawy, Department of Weed Science and Tillage Systems in Wroclaw, Poland in a humid.

Antiproliferative Assay In Vitro
Twenty-four hours prior to the applications of the tested compounds, MCF-10A and Balb/3T3 cells were plated in 96-well plates at a density of 10 4 cells per well and cultured at 37 • C in a humid atmosphere saturated with 5% CO 2 for 24 h.Stock solutions of the compounds were prepared ex tempore for each test by dissolving in DMSO.The cells were also exposed to the reference drugs: cis-platin (Accord, London, UK) (10-0.01µg/mL) and DMSO (Sigma-Aldrich) (at the concentrations corresponding to those in the tested agent dilutions).The solutions were then diluted in culture medium RPMI 1640 + Opti-MEM (1:1) (both from Gibco, Scotland, UK), supplemented with 2 mM l-glutamine, 5% fetal bovine serum (all from Sigma-Aldrich Chemie GmbH, Steinheim, Germany).An assay was performed after 72 h of exposure to the tested compounds 13, 17, 18, 19, 25, 27, 30, 4-hba, 4-hbah, and 3,4,5-hba (0.5, 5.0, 50, and to 500 µM) and azoxystrobin fungicide (0.05, 0.5, 5.0 and 50 µM).The antiproliferative effect in vitro was determined by the SRB method [103] (sulforhodamine B colorimetric assay).The optical densities of the samples were measured on the Synergy H4 photometer (BioTek Instruments, Winooski, VT, USA) at 540 nm.The results were calculated as 50% inhibitory concentration (IC 50 ), namely the dose of the tested compound inhibiting proliferation of the tested cells by 50% as compared to the untreated control cells, in the Prolab-3 system based on Cheburator 0.4 software using the two-point method [104].The mean values of ID 50 for each experiment ± SD are presented in Table 5.Each test was repeated 3-5 times.

Conclusions
In the present article, we presented the results of the biological evaluation of hydrazidehydrazones, derivatives of carboxylic acid hydrazides, and aldehydes, which previously were tested as laccase inhibitors.We postulated that the laccase might be the target enzyme produced by the phytopathogenic fungi, and inhibition may prevent or slow pathogen development in infected plants.We performed screening tests of all hydrazidehydrazones at 50 µg/mL on laccase-producing fungi, Botrytis cinerea, Sclerotinia sclerotiorum, and Cerrena unicolor and further detailed tests to determine IC 50 parameters.Among the tested molecules, derivatives of 4-hydroxybenzoic acid and salicylic aldehydes, namely 3-tert-butyl-, 3-Ph-, 3,5-di-tert-butyl, and 3-tert-butyl-5-methylsalicylic aldehyde (18, 19, 27, and 30, respectively), showed high antifungal activity, which corresponded to their high laccase inhibition potency [48].The most susceptible phytopathogen among tested laccase-producing fungi was S. sclerotiorum, for which the determined IC 50 was up to 0.5 µg/mL.Good results were also obtained for the thymol derivative 28, which was not active against laccase but showed high potency on S. sclerotiorum with IC 50 9.8 µg/mL.We also performed tests of phytotoxicity and cytotoxicity on selected hydrazide-hydrazones to determine their potential future application in the plant protection sector.Most of the tested molecules showed low or no phytotoxicity for tested dicotyledonous plants, and for two compounds, we observed no pathogen effect on the germination index of sunflower and flax seeds.Cytotoxicity results show that our compounds are non-toxic or less toxic than the azoxystrobin fungicide commonly used in agriculture.Therefore, we assumed that the derivatives of salicylic aldehydes and 4-hydroxybenzoic acid, namely 18, 19, 27, 28, and 30, are promising antifungal agents that can be further optimized as fungicidal candidates for application in the agriculture of dicot plant produced of oilseed crops used in manufacturing edible and industrial oils, especially for biofuels.

Figure 1 .
Figure 1.Structures of the aryl OH-substituted hydrazide-hydrazones I-XII with significant biological activities.

Figure 1 .
Figure 1.Structures of the aryl OH-substituted hydrazide-hydrazones I-XII with significant biological activities.

Molecules 2024 , 31 Figure 5 .
Figure5.Germination index (GI, %) determined for the uncoated seeds of dicot plants: sunflower (green) and flax (blue); uninfected control-GI determined for uninfected seeds, assumed as 100%; infected control-GI determined for the sunflower and flax seeds infected with mycelium of S. sclerotiorum and B. cinerea, respectively, grew on water-ethanol solution 0.5% (v/v); tests performed for sunflower and flax seeds treated with 13, 17, 18, 19, 25, 27, and 30 with 50 µg/mL concentration and infected with S. sclerotiorum and B. cinerea, respectively.The results are presented as a mean of three independent repetitions.

Figure 5 .
Figure5.Germination index (GI, %) determined for the uncoated seeds of dicot plants: sunflower (green) and flax (blue); uninfected control-GI determined for uninfected seeds, assumed as 100%; infected control-GI determined for the sunflower and flax seeds infected with mycelium of S. sclerotiorum and B. cinerea, respectively, grew on water-ethanol solution 0.5% (v/v); tests performed for sunflower and flax seeds treated with 13, 17, 18, 19, 25, 27, and 30 with 50 µg/mL concentration and infected with S. sclerotiorum and B. cinerea, respectively.The results are presented as a mean of three independent repetitions.
[48]acing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor[48].
[48]acing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor[48].
[48]acing methyl, the smallest alkyl group in compound 30, on the second bulky tert-butyl group in compound 27 slightly decreased activity against S. sclerotiorum; nonetheless, it improved the selectivity among all tested fungi.Noteworthy, compound 28, a derivative of thymol formylated near the hydroxy group, has potent activity against S. sclerotiorum (also moderately active toward B. cinerea) with a complete lack of inhibitory activity toward laccase from Trametes versicolor[48].