Antiureolytic Activity of Substituted 2,5-Diaminobenzoquinones

Article abstract of DOI:10.1002/cbdv.201900503

A series of 2,5-bis(alkyl/arylamino)-1,4-benzoquinones (1–12) were investigated in vitro for their potential to inhibit the activity of jack bean urease. Compounds 1–6, 8, 9, 11 and 12 effectively inhibited the jack bean urease activity by 90.8 % when tes

Full text of DOI:10.1002/cbdv.201900503

Title: Anti-ureolytic activity of substituted 2,5-diaminobenzoquinones
Authors: Amalyn Nain-Perez, Luiz Claudio Almeida Barbosa, Diego
Rodríguez-Hernández, Yane C. C. Mota, Thamara F. S Ávila,
Teodorico C. Ramalho, and Luzia V. Modolo
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To be cited as: Chem. Biodiversity 10.1002/cbdv.201900503
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900503

10.1002/cbdv.201900503
Chemistry & Biodiversity
Chem. Biodiversity
Anti-ureolytic activity of substituted 2,5-diaminobenzoquinones
Amalyn Nain-Perez,a Luiz C. A. Barbosa,*,a,b Diego Rodríguez-Hernández,a Yane C. C. Mota,c Thamara F. Silva,c
,c
Teodorico C. Ramalho,d Luzia V. Modolo*
a Department of Chemistry, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, e-mail:
lcab@ufmg.br
b Department of Chemistry , Universidade Federal de Viçosa, Viçosa, MG, Brazil
c Departamento de Botânica, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil, e-mail:
lvmodolo@icb.ufmg.br
d Department of Chemistry, Universidade Federal de Lavras, Lavras, MG, Brazil
A series of 2,5-bis(alkyl/arylamino)-1,4-benzoquinones (1-12) were investigated in vitro for their potential to
inhibit the activity of jack bean urease. Compounds 1 6, 8, 9, 11 and 12 effectively inhibited the jack bean
urease activity by 90.8% when tested at 5 µM, whereas 7 and 10 had relatively little effect. The IC for most
50
compounds was in the nanomolar range (31.4 nM and 36.0 nM for 2 and 8, respectively). The mechanism of
enzyme inhibition shown by 2 and 8 is typical of mixed-type inhibitors, whose affinity for the active site is over
6- and 2-fold higher (Ki = 30.0 and 22.8 nM, for 2 and 8, respectively) than that of an allosteric site. Molecular
docking studies revealed that both 2 and 8 establish hydrogen bonds with the amino acids residues Asp494,
Met588, His593 and Ala636 in the active site of jack bean urease. These results indicate that such aminoquinones
are useful leads for the development of more efficient urease inhibitors of wider utility.
Keywords: Urease inhibitors; Jack bean urease; 2,5-diamino-1,4-benzoquinones; Molecular docking.
Introduction
Urease (E.C.:3.5.1.5) is a well-known nickel metalloenzyme that catalyzes the hydrolysis of urea to ammonia
and carbon dioxide.[1] The primary environmental role of ureases is to allow organisms to use urea as nitrogen
source. Moreover, urease participates in systemic nitrogen transport pathways in plants, fungi, algae, bacteria
and microorganisms.[2] However, the activity of soil ureases leads to nitrogen losses when urea is used as
fertilizer for crop production.[3-4] Additionally, urease is a known virulence factor for the bacteria Helicobacter
pylori and Staphylococcus saprophyticus, involved in gastrointestinal diseases and urinary stone formation.[5-6] It
is estimated that each human being produces 10 kg of urea per year. Spontaneous degradation of urea in cells
is believed to occur with a half-life of c.a. 3.6 years,[7] whilst this process becomes 100 trillion-fold faster in the
presence of ureases.[8] Since urea hydrolysis yields ammonia,[9] the ammonia-dependent increase of cell pH in
the media has medicinal implications, while excessive ammonia emission to atmosphere causes environmental
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issues and economic problems due to lower availability of nitrogen to crops.[2, 10] For this reason, the control of
urease activity is of considerable clinical and agricultural importance.
Among all the nutrients used in agriculture, the nitrogen fertilizers are the highest with an annual
consumption in 2018/2019 over 107 Mt, of which 55% is represented by urea. It is estimated that 14 40% of
nitrogen is lost as ammonia depending on both climate and the soil type.[11] The greater part of such losses is
due to the activity of urease found in soils. Consequently, finding urease inhibitors that can be so applied with
urea is a research field of great economical interest. Among the few urease inhibitors in commercial use is the
N-butyl thiophosphoric triamide (NBPT), whose agronomic efficiency has been recently reviewed.[11]
During the last two decades a large array of metals and organic substances have been found to inhibit
urease. Amongst synthetic organic compounds, the most actives include phosphoramidates, thioureas, dimeric
vanadium complexes, benzothiazoles, coumarinyl pyrazolinyl thiomides and dihydropyrimidin(thi)one.[11 15]
Several classes of plant-derived compounds, including terpenoids, phenolic acids, flavonoids, stilbenes,
xantones, coumarins alkaloids and quinones, among others, have preceding last years, special attention has
focused on quinones due to their wide array of biological properties, specially anti-ureolytic activity. Quinones
also play important roles in the electron transfer chain of photosynthesis[18 20] and cell respiration[19-20] . For the
latter, coenzymes Q is involved in centers of reduction and in the translocation of protons across the inner
mitochondrial membrane via quinone/quinol (Q/H2Q) redox couple turnover.[19] The abilityof 1,4-benzoquinone
related compounds to inhibit H. pylori or Canavalia ensiformis (jack bean) ureases is also important.[21 25] The
inhibition of urease by naphthoquinones and their mechanism of action have been elucidated.[23, 26]
Although the anti-ureolytic potency of 1,4-benzoquinones has been attributed to the presence of electron-
withdrawing chlorine groups,[21] quinones bearing electron-donating amino groups (2,5-bis(alkylamino)-1,4-
benzoquinones derivatives) were also shown to inhibit urease with IC50 values as low as 27.3 µM.[27] Furthermore,
our group has previously shown that substituted 1,4-benzoquinones are promising phytotoxic and algicidal
agents.[28 32] Therefore, considering the limited information on the anti-ureolytic activity of 2,5-diamino-1,4-
benzoquinones, this work reports the effect of a series of such compounds[29] on the activity of jack bean type
III urease and the interaction between these compounds and urease (PDB ID: 4H9M) by molecular docking
studies.
Results and Discussion
A series of twelve 2,5-bis(alkyl/arylamino)-1,4-benzoquinones (1-12) were prepared employing a protocol
developed and reported by our group.[29] In short, the compounds were obtained from the aza-Michael addition
of primary amines to 1,4-benzoquinone (1:3 equiv. ratio) in methanolic solution, followed by spontaneous
oxidation (1-12; Scheme 1). The synthesis of compound 12 required the addition of NaHCO3 to promote the
deprotonation of the zwitterion, even though the yield was only 46%, much lower in comparison with the yields
obtained for the aliphatic amines derivatives 66-93%. All compounds were fully characterized by their physical
and spectroscopic data, that were in full agreement with the literature.[29]
2
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Scheme 1. Structure of 2,5-bis(alky/arylamino)-1,4-benzoquinones used in this study.
Anti-ureolytic Activity
The benzoquinones 1-12 were subjected to an in vitro assay using a purified Canavalia ensiformis (jack
bean) type III urease to assess their anti-ureolytic potential. Hydroxyurea (HU), commonly used in in vitro
screening,[14] was employed as a reference urease inhibitor. The percentages of urease inhibition caused by all
compounds at 5 µM are displayed in Table 1.
All 2,5-bis(alkyl/arylamino)-1,4-benzoquinones tested, except for 7 and 10, inhibited the activity of jack
bean urease by 90.8 ± 0.8 % (on average) while the inhibitor-reference HU barely affected the urease activity
under the same experimental conditions (Table 1). Notably, the only 2,5-diamino-1,4-benzoquinones previously
reported as urease inhibitors were shown to inhibit Helicobacter pylori urease by up to 81.6% when tested at
100 µM – a concentration 20 times higher than the one used in the current work.[27]
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Table 1. Inhibition of Canavalia ensiformis (jack bean) type III urease by 2,5-bis(alkyl/arylamino)-1,4-
benzoquinones 1-12.
Compound
Urease inhibition (%)[a]
Compound
Urease inhibition
(%)
89.5 ± 0.8
89.7 ± 1.0
88.4 ± 0.6
89.1 ± 1.0
90.1 ± 0.7
91.4 ± 0.8
13.7 ± 6.7
91.6 ± 0.4
97.7 ± 0.1
18.9 ± 6.6
89.1 ± 0.8
91.6 ± 1.3
5.5 ± 0.8
1
2
3
4
5
6
7
8
9
10
11
12
HU
[a]Compounds were tested at 5 µM in the presence of 10 mM urea. HU, hydroxyurea (reference of urease
inhibitor). Data are the means (n = 4) ± SD.
Although the number of compounds and structural variations are somehow limited, the data clearly show
that quinones derived from aliphatic amines (1-6, 11, 12) are very active. In general, quinones 1 6, 8, 9, 11 and
12 were approximately 16.5-fold more potent than the inhibitor-reference HU. Indeed, the
aminobenzoquinones tested were very active, regardless of the nature of the N-alkyl or N-benzyl groups.
Amongst the products derived from substituted anilines (7-10), those bearing a substituent at an ortho position
are very potent (>90% inhibition) whilst those with substituents either at the para (7) or meta (10) positions
were ineffective at 5 µM (<20% inhibition). Although a detailed computational calculation has not been carried
out, the presence of a group at ortho position on the phenyl ring clearly interferes with the relative conformation
of the rings and with the conjugation degree of the nitrogen lone electron pair with the quinone core, probably
impacting the biological potency. Further investigation is required to fully understand the effect of ortho
substituents on the measured bioactivity.
Based on the screening results (Table 1), compounds 2 and 8, typical examples of N-alkyl and N-arylamino
derivatives, respectively, were randomly selected out of the 10 equally highly active aminobenzoquinones to
further explore the mechanism of urease inhibition. Various concentration of each compound (up to 100 nM)
were used in in vitro experiments carried out with 10 mM urea and 12.5 mU urease to determine the
corresponding concentrations necessary to inhibit the enzyme by 50% (IC ). Such data served as a guide for
50
choosing appropriate concentrations (< IC50) for the kinetics studies. The IC50 values for compounds 2 and 8
were found to be 31.4 nM and 36.0 nM, respectively. These IC50 values are approximately eighteen times lower
than those found for the most active 2,3,5,6-tetrachloro-1,4-benzoquinone (IC = 600 nM) evaluated against
50
the same urease.[21]
Based on the IC values found for 2 and 8, concentrations of 10, 20 and 30 nM were chosen for the kinetics
50
assay. As observed on Figures A and C, both aminobenzoquinones caused increments in the urea K and
M
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decreases in the urease V . The analyses of the Lineweaver-Burk plots for 2 and 8 (Figures 1B and 1D) show
max
that both compounds function as mixed-inhibitors as the lines intersect one with another in the second
quadrant. These results indicate that the aminobenzoquinones tested are eligible to bind to either free urease
to yield an EI complex or urease-urea complex to furnish an ESI complex. The equilibrium dissociation constants
for urease-compound 2 and urease-compound 8 complexes (K) and for urease-urea-compound 2 and urease-
i
urea-compound 8 complexes (K were 30.0 ± 0.9 nM, 22.8 ± 3.7 nM, 191.4 ± 96.1 nM and 61.6 ± 13.7, respectively.
i
This clearly demonstrates the greater affinity of 2 and 8 to the active site of jack bean urease than to an allosteric
site. Indeed, the affinity of 2 to the urease active site is 6.4-fold higher than that of other sites while the affinity
of 8 to an allosteric site is only 2.7-fold lower compared to that of the active site. Other quinones such as 1,4-
benzoquinone, 2,5-dimethyl-1,4-benzoquinone, tetrachloro-1,2-benzoquinone and tetrachloro-1,4-
benzoquinone act as competitive slow-binding inhibitors of jack bean urease, yielding K values of 45 nM, 1,200
i
-1
nM, 2.4 × 10-6 nM and 0,45 nM, respectively, in reactions (pH 7.0) containing 15 µg mL urease.[1, 33]
2.5
2.0
1.5
1.0
0.5
6
5
4
3
2
1
0
B
A
0 nM
20 nM
30 nM
30 nM
20 nM
0 nM
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
5
10
15
20
25
30
35
Urea [mM]
1/[Urea]
2.5
2.0
1.5
1.0
0.5
6
5
4
3
2
1
0
D
C
30 nM
20 nM
0 nM
20 nM
30 nM
0 nM
-0.2
0.0
0.2
0.4
0.6
0.8
1.0
1.2
0
5
10
15
20
25
30
35
Urea (mM)
1/[Urea]
Figure 1. Effects of aminobenzoquinones 2 and 8 on the kinetics of jack bean urease (type III). Increasing amounts of urea
(0 32 mM) were incubated for 5 min with jack bean urease in the presence of the indicated concentrations of inhibitors.
Plots are representative of independent experiments done in triplicates. Michaelis-Menten hyperbolas (A and C) and
Lineweaver-Burk plots (B and D) for the results originated from reactions performed with 2 and 8, respectively.
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Molecular Docking Analysis
Since kinetics studies have shown preferential interaction with the active urease site, we analyzed
compounds 2 and 8 through molecular docking using the jack bean enzyme to shed more light on the
mechanism of urease inhibition, revealing biophysical interactions that play a fundamental role in this process.
The jack bean urease PDB ID: 4H9M was chosen since the crystal that originated its X-ray
diffraction data was available with resolution of 1.5 Å. Acetohydroxamic acid (AHA) was used as a reference
ligand, showing a root-mean-square deviation of atomic positions (RMSD) of 0.57 with docking score of -4.1
kcal/mol. The docking scores for 2 and 8 were -5.1 and -8.1 kcal/mol, respectively, lower than that of AHA. The
lower docking score shows a stronger interaction between the aminoquinones and the enzyme active site,
compared with the standard AHA, in agreement with the experimental data, in which 2 and 8 are more potent
than HU. The binding mode of compounds 2 and 8 is shown in Figure 2, serving as a representative template
for the binding mode of the series of aminoquinones tested in this study.
A)
B)
C)
D)
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Figure 2. Docking of aminoquinone 2 (green dash) into the active site of Canavalia ensiformis urease (PDB 4H9M; A) and a
2-D diagram of 2 (B). Docking of aminoquinone 8 (red dash) into the active site of Canavalia ensiformis urease (PDB 4H9M;
C) and a 2-D diagram of 8 (D).
The NH group and oxygen from quinone moiety may interact with different amino acid residues in the
urease active site, probably through hydrogen bonding (Figure 2). For instance, 2 interacts with Asp494 and
Ala636 via hydrogen bonding through its amino group, while the carbonyl of quinone moiety makes a hydrogen
bond with Arg609 residue (Figure 2A). The van de Waals interactions also are observed in the representative
Figure 2B, mainly by the amino acids residues Arg439, His492, His593, Asp633, Met637 and the nickel ions Ni901
and Ni902. The amino groups of 8 makes hydrogen bonds with Met588 and His593 and the carbonyl of the
acetyl group makes a hydrogen bound with the modified cysteine residue Cme592 (Figure 2C). The aromatic
moiety and the benzoquinone core of 8
-
-alkyl interactions with Met588, Cme592
and Ala440, respectively (Figure 2D). Overall, the binding mode of the aminoquinones to the active site of jack
bean urease resulted in docking scores similar to those obtained for other aminoquinones and the active site of
urease from Helicobacter pylori.[27] Also, in contrast to the study of urease from H. pylori[27], in the current study
the aminoquinones revealed no direct interaction with the nickel atoms.Although the docking study has shed
some light on the type of interaction that such compounds can have with the amino acid residues at the enzyme
active site, a full understanding of their mode of action requires further investigation. Also, compounds 2 and 8
can also bind to allosteric sites of jack bean urease. In the case of a simple 1,4-benquinone (1,4-BQ), it has been
demonstrated that the inhibition of urease from Sporosarcina pasteurii involves a covalent bond formation
between the thiol group of Cys322 residue, a key residue found on the mobile flap regulating the substrate
access to the active site.[34] The covalent bond formation between 1,4-BQ and 2,3,5,6-tetrachloro-1,4-
benzoquinone (TC-1,4-BQ) and thiol groups of cysteine residues from the active site of jack bean urease has
also been proposed as a key interaction for their mode of action.[21] Although the aminoquinones herein
investigated herein are less electrophilic than a simple 1,4-BQ, the formation of covalent bonds with thiol
groups is not excluded and further investigation is necessary to refute or corroborate this hypothesis.
Conclusions
The synthetic aminoquinones 1-6, 8, 9, 11 and 12 are excelent urease inhibitors, showing great
performance at nanomolar concentrations. Compounds 2 and 8 exhibit a mixed-type mode of urease inhibition
with much more affinity to the enzyme active site. Molecular docking reveals that the oxygen atoms at quinone
core and amino groups in compounds 2 and 8 are likely to establish hydrogen bonds with amino acid residues
located at the active site of jack bean urease. These results suggest that compounds with an aminoquinone core
are excellent candidates for the development of novel urease inhibitors of several interests.
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Experimental Section
General Experimental Procedures
Reagents were procured from Sigma-Aldrich (Milwaukee, Wisconsin, USA) and solvents were purchased from
Fluka (Rio de Janeiro-Brazil). They were used without further purification. Analytical thin layer chromatography
(TLC) was performed on silica gel 60 F254 0.2 mm thick plates (Merck, Rio de Janeiro-Brazil). The plates were
visualised under an UV-lamp at a wavelength of 254 nm or by staining with phosphomolibdic acid solution. All
compounds were fully characterized by IR, EI-MS, 1H NMR and 13C NMR spectroscopy. Infrared spectra were
recorded on a Perkin-Elmer Paragon 1000 FTIR spectrophotometer, preparing the samples as potassium
1
13
bromide disks (1% w/w). The H and C-NMR spectra were recorded on a Varian Mercury 300 spectrometer at
300 and 75 MHz, respectively, using CDCl3 as solvent and TMS as internal reference, unless otherwise stated. The
melting points were measured with on a MQAPF-301 apparatus and are given uncorrected.
Procedure for the preparation of 2,5-amino-1,4-benzoquinone derivatives (1-12)
Compounds 1-12 were synthesized using methods previously published.[29] The physical and spectroscopic data
of all compounds were in agreement with the previously reported[22] and are not repeated here.
Urease inhibition assay
The screening for identifying potential urease inhibitors was done by incubating each 2,5-bis(alkyl/arylamino)-
1,4-benzoquinones (1-12) at 5 µM (5,000 nM) in reactions containing 50 mM phosphate buffer (pH 7.4), urea (10
mM), and 1.25 x 10-2 U Canavalia ensiformis (jack bean) type III urease (Sigma U-1500-100 kU). Each mixture was
incubated for 15 min at 25 °C, and the reactions were interrupted according to Weatherburn,[35] with
modifications reported by Brito et al.[36] The ammonium concentration was determined by the phenol
hypochloride assay (measured at 636 nm), and the inhibition percentage [INH(%)] was calculated using the
equation: INH(%) = 100 [(AINH/AB) x 100], where AINH and AB are ammonium concentration in the tubes with
and without inhibitor, respectively. The inhibitory potential of the compounds was compared with that of the
standard-inhibitor hydroxyurea (HU; 5 µM). Two compounds (2 and 8) were randomly selected among the most
potent aminobenzoquinones to perform kinetics assays, in which each inhibitor was added at a fixed
concentration (0, 10, 20 or 30 nM) to the reaction media (pH 7.4) containing 12.5 mU jack bean urease and
increasing concentrations of urea (1-32 mM). Reactions were stopped after 5 min incubation and analysed for
the production of ammonium.
8
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Docking simulations
Molecular docking of 2,5-bis(alkyl/arylamino)-1,4-benzoquinones (2 and 8) with the active site of jack bean
urease (PDB ID: 4H9M) was performed using the AutoDock Vina program suite.[37-38]
prepared for molecular docking simulation by removing water, ligands and cofactors. Hydrogen polar atoms
and gasteiger charges were added to each protein atom. The Ni initial parameters were set as r = 1.170 Å, q =
+2.0 and van der Waals well depth of 0.100 kcal/mol.[39] Auto-Dock Tools-1.5.6 (ADT) was used to prepare and
analyze the docking simulations for the AutoDock 4.2. Coordinates ligands were generated using Chemdraw
14.0 followed by MM2 energy minimization, and non-polar hydrogen atoms were merged using ADT package.
The interaction of protein and ligands in binding pocket for AutoDock 4 was defined by a Grid box. The grid box
was created using 30 x 35 x 30 points for each direction of x, y, and z, the maps were centered on the Ni atom in
the catalytic site of the protein. AutoGrid4 was used to produce grid maps for docking calculations where the
search space size utilized grid points of 1.0 Å. Each docking experiment was performed 50 times, yielding 50
docked conformations. The best docking pose was picked on the best binding energy. The docking procedure
of aminoquinones 2 and 8) with the active site of jack bean urease was performed as described.
Acknowledgements
Author Contribution Statement
Amalyn Nain-Perez carried out literature reviews, molecular docking, data analyses, interpretation of the results
as well as compilation and preparing the manuscript. Diego Rodríguez-Hernández contributed to the
manuscript writing, data analysis, and preparation of graphics. Yane C. C. Mota, and Thamara F. S. Ávila carried
out the experiments with urease, performed data analyses and discussed the results. Luiz C. A. Barbosa
conceived and coordinated the project
as urease inhibitors
and worked on
the manuscript preparation. Luzia V. Modolo supervised all biological assays, discussed the results and helped
writing the manuscript while Teodorico C. Ramalho contributed with molecular docking.
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Chem. Biodiversity
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