Synthesis and structure-activity relationship studies of hydrazide-hydrazones as inhibitors of laccase from trametes versicolor

Article abstract of DOI:10.3390/molecules25051255

A series of hydrazide-hydrazones 1-3, the imine derivatives of hydrazides and aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor. Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of the plant pathogens that produce it in various biochemical processes. The kinetic and molecular modeling studies were performed and for selected compounds, the docking results were discussed. Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24-674 μM with the predicted and desirable competitive type of inhibition. The structure-activity relationship (SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger 3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site of laccase and, interestingly, acted as non-competitive (Ki = 32.0 μM) or uncompetitive (Ki = 17.9 μM) inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to environment and non-specific, was over 6 times more active than the above compounds.

Full text of DOI:10.3390/molecules25051255

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molecules
Article
Synthesis and Structure-Activity Relationship Studies
of Hydrazide-Hydrazones as Inhibitors of Laccase
from Trametes versicolor
Halina Maniak 1,
* *
, Michał Talma 2 , Konrad Matyja 1, Anna Trusek 1 and Mirosław Giurg 3,
1
Department of Micro, Nano and Bioprocess Engineering, Faculty of Chemistry, Wroclaw University of
Science and Technology, Wybrzez˙e Wyspian´skiego 27, 50-370 Wrocław, Poland;
konrad.matyja@pwr.edu.pl (K.M.); anna.trusek@pwr.edu.pl (A.T.)
Department of Bioorganic Chemistry, Faculty of Chemistry, Wroclaw University of Science and Technology,
Wybrzez˙e Wyspian´skiego 27, 50-370 Wrocław, Poland; michal.talma@pwr.edu.pl
2
3
Department of Organic and Medicinal Chemistry, Faculty of Chemistry, Wroclaw University of Science and
Technology, Wybrzez˙e Wyspian´skiego 27, 50-370 Wrocław, Poland
*
Correspondence: halina.maniak@pwr.edu.pl (H.M.); miroslaw.giurg@pwr.edu.pl (M.G.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 19 February 2020; Accepted: 8 March 2020; Published: 10 March 2020
Abstract: A series of hydrazide-hydrazones 1–3, the imine derivatives of hydrazides and
aldehydes bearing benzene rings, were screened as inhibitors of laccase from Trametes versicolor.
Laccase is a copper-containing enzyme which inhibition might prevent or reduce the activity of
the plant pathogens that produce it in various biochemical processes. The kinetic and molecular
modeling studies were performed and for selected compounds, the docking results were discussed.
Seven 4-hydroxybenzhydrazide (4-HBAH) derivatives exhibited micromolar activity Ki = 24–674 µM
with the predicted and desirable competitive type of inhibition. The structure–activity relationship
(SAR) analysis revealed that a slim salicylic aldehyde framework had a pivotal role in stabilization of
the molecules near the substrate docking site. Furthermore, the presence of phenyl and bulky
tert-butyl substituents in position 3 in salicylic aldehyde fragment favored strong interaction
with the substrate-binding pocket in laccase. Both 3- and 4-HBAH derivatives containing larger
3-tert-butyl-5-methyl- or 3,5-di-tert-butyl-2-hydroxy-benzylidene unit, did not bind to the active site
of laccase and, interestingly, acted as non-competitive (Ki = 32.0 µM) or uncompetitive (Ki = 17.9 µM)
inhibitors, respectively. From the easily available laccase inhibitors only sodium azide, harmful to
environment and non-specific, was over 6 times more active than the above compounds.
Keywords: oxidoreductase; hydrazones; salicylaldehyde derivatives; 4-hydroxybenzhydrazide
1. Introduction
The discovery of new eco-friendly pesticides, having readily biodegradable natural units, is an
essential need of the agrochemical industry [1]. The global human population is constantly growing,
therefore, food production needs to be still enhanced. Synthetic pesticides, which usually act
indirectly or non-specifically, cause considerable contamination of the environment. Furthermore,
crop losses result from the rapid evolution of pest resistance to these chemicals. These problems
lead to a high demand for new cultivation approaches and less toxic plant protection products [2,3].
Commonly occurring and troublesome pathogens of crop plants are filamentous fungi that belong to
the Sclerotniaceae family, for instance, gray mold (Botrytis cinerea) which attacks flowers, green tissues
of crop plants [
fungi that attack young and healthy but also old vintage trees in forestry, fruit orchard culture,
horticulture, and in the old parks [ ]. Insects e.g., aphids, Drosophila species, termites, etc., also cause
4] and fruits such grapes [5]. The increasing problem is the abundancy of wood-decay
6
Molecules 2020, 25, 1255; doi:10.3390/molecules25051255
www.mdpi.com/journal/molecules

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Molecules 2020, 25, 1255
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plant damage which influences the crop production, vitality and aesthetic values of gardens and
domestic ornamental plants [ ]. Therefore, new natural and natural product-derived plant protection
agents with an easily biodegradable structure and specific unique mechanisms of action are highly
desirable [ ]. A common feature of the fungi and insects mentioned above is the production of laccase,
which is specifically involved in various survival and physiological processes [ 10]. White-root
fungi, for instance, use laccase to destroy the lignin that is the physical barrier in trees to prevent
the degradation of cellulose [ ]. Laccases of some molds neutralize phenolic antibiotics, such as
phytoanticipins or phytoalexins, produced by the plant immune system as a response to a pathogen
attack [11 12]. Laccase together with tyrosinase also play a crucial role in the sclerotia formation and
virulence of Sclerotinia sclerotiorum [13 14] as well as hardening cuticle in insects [15]. Thus, regulation
7
8
9
,
6
,
,
of the activity of this enzyme might weaken pathogen activity and provide time to strengthen the plant
defense system.
Laccase (EC 1.10.3.2) is a copper-containing polyphenol oxidase that is widely distributed in
nature. The enzyme catalyzes the radical reduction of atmospheric oxygen to water with simultaneous
oxidation of electron-rich aromatic compounds such as polyphenols and anilines [16]. The broad
spectrum of substrates that can be converted, mostly by laccases of fungal origin, is due to the high
redox potential of a specific copper atom in the enzyme active site [17]. Laccase from Trametes versicolor
is a typical “blue” oxidase which contains a cluster of four copper atoms categorized as type 1, 2 and
3 [18]. Type 1 is a paramagnetic blue copper and a site of a substrate molecule binding. One copper
atom of type 2 and two copper atoms of type 3, form a trinuclear center which binds and reduces
dioxygen. The electron transfer from oxidized substrate follows from type 1 Cu center through
His458–Cys453–His452 tripeptide to the trinuclear-copper cluster of 2 and 3 types where it is used
for the reduction of dioxygen to water molecules [19]. The catalytic cycle includes oxidation of four
single-center substrates to four radical products with simultaneous reduction of one molecule of O2 to
two molecules of H2O. The oxidation of a substrate results in the active radicals, which either could
be mediators in the radical oxidation reactions or could be non-enzymatically converted to the final
oxidation products (Scheme 1).
Scheme 1. Oxidation mechanism through the laccase and laccase-mediator system (modified from [20]).
Laccase mediators are low-molecular-weight compounds of great importance in the oxidation of
complex high-molecular-weight molecules such as lignin since accessibility to the substrate-binding
pocket in the enzyme is spatially limited [20,21]. At least one hundred of these mediators have
been described in the literature so far, including the synthetic phenothiazine, 1-hydroxybenzotriazole
(HOBt), 2,20-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), or the natural laccase substrates
such 3-hydroxyanthranilic acid, 4-formyl-2,6-dimethoxyphenol (syringaldehyde), 4-hydroxybenzyl
alcohol and 4-hydroxybenzoic acid (4-HBA) (Figure 1) [20–23].

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Figure 1. Representative synthetic (upper row) and natural (lower row) laccase mediators (based
on [22,24]).
Both 4-hydroxybenzyl alcohol and 4-hydroxybenzoic acid (4-HBA) are naturally occurring and
are recognized as useful mediators in laccase catalyzed reactions. These simple compounds are also
readily available. Moreover, the 4-HBA is only needed in 0.1 mM concentration to significantly increase
the oxidation efficacy of resistant compounds in laccase-catalyzed reactions [22]. It seems that such
high affinity of the 4-HBA for the substrate pocket in laccase and the presence of an easily chemically
modified free carboxyl group would enable us to prepare a collection of 4-HBA derivatives with better
fitting to the enzyme cavity and control of their inhibition potency.
Isolation of bioactive natural compounds from plant sources and microorganisms for agricultural
applications is usually a challenge due to the small amount of the active ingredient(s), availability of the
biomass and cost of the process. Therefore, in our studies, we focused on the use of commonly available
natural salicylic aldehydes and carboxylic acids combined with hydrazine linkers to form natural
product-derived compounds such hydrazide-hydrazones. Furthermore, we aimed at discovering a
new group of organic low-molecular-weight compounds which activity that weakens a pest’s activity
and consequently provides conditions for improving the plant defense system. The imines 1–3 were
synthesized as part of a program to discover low-molecular-weight compounds that were both the
inhibitors of essential enzymes overexpressed during disease development [25–27] and act directly
against pathogenic microorganisms [28 29]. A common polypore fungus Trametes versicolor, which is
,
an abundant tree parasite causing a white-rot of wood, was chosen as a model organism. Infection
by T. versicolor results in a degradation of lignin by the laccase mediator system, and ultimately to
decomposition of the wood structure.
Hydrazide-hydrazones are known as antitumor [30
as enzyme inhibitors [31 36 41]. Hydrazones are less recognized as inhibitors of metallo- enzymes and
only the rare examples can be found in the literature so far [38 40 43]. Therefore, this work provides
–32] and antimicrobial agents [33–35], as well
,
–
,
–
insight into a relationship between hydrazide-hydrazones and laccase.
2. Results and Discussion
2.1. Syntheses and Characterizations
The various twenty-three imine derivatives 1–3 were synthesized using 4-hydroxybenzoic acid
hydrazide (4a) and diverse aldehydes 5–7 having a benzene ring. In two cases, 4-methoxy- and
3-hydroxybenzoic acid hydrazide 4b and 4c were used for the preparation of hydrazide-hydrazones
2h and 3g, respectively. Among them, fourteen products are new. The hydrazide-hydrazones listed in
Table 1 were thus prepared as pure compounds in good to quantitative (76–100%) yields.

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Table 1. Characteristics of hydrazide-hydrazones 1a–j, 2a–h, and 3a–g.
No.
R
R1
R2 Rx time
Yield(%)
Mp (◦C)
Mp lit.(◦C) Lit.
1a
1b
1c
1d
1e
1f
1g
1h
1i
Ph
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OMe
OH
OH
OH
OH
OH
OH
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OH
2h
6h
2h
2h
2h
2h
2h
4h
5h
5h
6h
6h
2h
3h
2h
6h
3h
3h
3h
3h
6h
2h
6h
5h
2h
96
72
80
97
86
242.0–244.0
243.5–244.0
246.0–247.0
251 dec.
265.0–266.5
254 dec.
240–242
[44]
C6H5CH2CH2
4-MeC6H4
2-NaSO3C6H4
2-HOC6H4
3-HOC6H4
4-HOC6H4
2-MeOC6H4
3-MeOC6H4
–
[45]
[46]
–
[47]
[48]
[49]
[50]
[50]
[50]
–
252.6
–
257–258
>250
265
230–231
205–206
220–221
93 a
96 b
91
261 dec.
236.0–237.0
210.0–212.0
223.0–224.0
215.0–218.0
240.0–242.0
294 dec.
276.0–278.0
256.5–257.5
214.5–216.0
218.5–220.0
240.0–242.5
229.0–230.0
266 dec.
79
1j
4-MeOC6H4
82
2a
2b
2c
2d
2e
2f
2g
2h
3a
3b
3c
3d
3e
3f
2-HO-3-PhC6H3
2-HO-3-tBuC6H3
2,4-(HO)2C6H3
85 c
93
–
–
–
–
90
90
71
97
100
84
77
86
93.5
76
[48]
[49]
–
–
[51]
–
–
–
–
–
2-HO-5-BrC6H3
2-HO-6-MeOC6H3
2,6-(MeO)2C6H3
3-MeO-4-HOC6H3
2-HO-3-PhC6H3
2-HO-3-CH2OH-5-MeC6H2
2-HO-3-Me-5-CH2OHC6H2
2-HO-3,5-(tBu)2C6H2
2-HO-3-tBu-5-MeC6H2
2-HO-3-iPr-6-MeC6H2
2-HO-4,6-(MeO)2C6H2
2-HO-3-tBu-5-MeC6H2
279–280
–
–
226–227
–
–
–
–
–
–
–
–
274.5–275.5
261.0–262.0
258.5–260.5
231.5–234.5
240.0–242.5
94.5
99
94
–
–
–
3g
a
Monohydrate was isolated, b Crystallize with 1/3 molecule of CH3OH, c Crystallize with one molecule of CH3OH.
Generally, in their Fourier-transform infrared spectroscopy (FT-IR) spectra, the salicylaldehyde
derivatives 2a–c and 3a–b, which were functionalized in the neighborhood of the hydroxyl group,
had a very wide absorption band at 2500–3300 cm−1, caused by hydrogen bonds in the crystal lattice.
For all of the remaining hydrazide-hydrazones 1–3, the wavenumbers corresponding to the
stretching vibration of an amide single bond N–H was around 3016–3307 cm−1, the carbonyl group
(C=O) bands were at 1580–1644 cm−1, and the imine bond (C=N) at 1538–1589 cm−1
.
A sole exception was compound 1b, in which the aliphatic aldimine C=N group was bound
1
to an ethylene linker that shifted the band a lower wavelength (1507 cm−1). The H-NMR and
13C-NMR spectra were measured in DMSO-d6 except for the insoluble sodium 2-{(E)-[2-(4-hydroxy-
benzoyl)hydrazinylidene]methyl}benzenesulfonate (1d). For this compound, the spectra were
1
measured in fully deuterated CH3OH. In the H-NMR spectra measured in DMSO-d6, the amide
proton (NHCO), phenolic hydroxy group of the 4-HBAH fragment (4–OH) and conjugate imine
(CH=N) resonances were observed at ca.: 11.24–12.18, 9.83–10.80 and 8.32–8.93 ppm, respectively.
In the 13C-NMR spectra, the resonances of the conjugated carbonyl and imine C=O and C=N
carbond, and tertiary C-4 phenolic carbon atoms, and ArC-2 were observed at ca.: 161.93–162.80
and 142.19–150.36, 160.43–161.04 and 154.72–160.77 ppm, respectively. For the sulfonic acid
sodium salt derivative 1d, similar chemical shifts were observed in the 1H-NMR resonance.
The hydrazide-hydrazones which had phenyl or alkyl group near the salicylic hydroxy group
caused a weak bathochromic effect. The spectroscopic data were consistent with the data published
in the literature [46–48,50–53]. The geometry of the imine CH=N double bond of the aroyl
hydrazones has an E configuration, which was proved by crystallographic studies of the imine
derivatives of 4-hydroxybenzhydrazide with aldehydes such as 2,3-dimethoxybenzaldehyde [50],
5-bromosalicylaldehyde (2d) [53] and vanillic aldehyde (2g) [52].
Methods of preparation of the hydrazides 4b–c and aldehydes of choice 6a–b, 6d–f, 7a–b, and 7d–f
were adapted from the original literature [54 64]. 4-Methoxy- and 3-hydroxybenzoic acid hydrazides
–
4b and 4c were prepared in 67–85% yields from the appropriate benzoic acid methyl esters 8b and 8c
and hydrazine monohydrate following a literature procedure [63]. The suitable benzoic acid methyl