Synthesis, Biological Evaluation and Molecular Docking of Deferasirox and Substituted 1,2,4-Triazole Derivatives as Novel Potent Urease Inhibitors: Proposing Repositioning Candidate

Article abstract of DOI:10.1002/cbdv.201900710

A series of new deferasirox derivatives were synthesized through the reaction of monosubstituted hydrazides with 2-(2-hydroxyphenyl)-4H-benzo[e][1,3]oxazin-4-one. For the first time, deferasirox and some of its derivatives were evaluated for their in vitro inhibitory activity against Jack bean urease. The potencies of the members of this class of compounds are higher than that of acetohydroxamic acid. Two compounds, bearing tetrazole and hydrazine derivatives (bioisoester of carboxylate group), represented the most potent urease inhibitory activity with IC₅₀ values of 1.268 and 3.254 μm, respectively. In silico docking studies were performed to delineate possible binding modes of the compounds with the enzyme, urease. Docking analysis suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket and thus restrict the mobility of the flap by interacting with its crucial amino acid residues, CME592 and His593. The overall results of urease inhibition have shown that these target compounds can be further optimized and developed as a lead skeleton for the discovery of novel urease inhibitors.

Full text of DOI:10.1002/cbdv.201900710

Title: Synthesis, Biological Evaluation and Molecular Docking of
Deferasirox and Substituted 1,2,4-Triazole Derivatives as Novel
Potent Urease Inhibitors: Proposing Repositioning Candidate
Authors: Saeed Balalaie, Razieh Salehi Ashani, Homa Azizian, Nahid
Sadeghi Alavijeh, Vaezeh Fathi Vavsari, Shabnam Mahernia,
Niloofar Sheysi, Mahmood Biglar, and Massoud Amanlou
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201900710
Link to VoR: http://dx.doi.org/10.1002/cbdv.201900710

10.1002/cbdv.201900710
Chemistry & Biodiversity
Chem. Biodiversity
Synthesis, Biological Evaluation and Molecular Docking of
Deferasirox and Substituted 1,2,4-Triazole Derivatives as Novel
Potent Urease Inhibitors: Proposing Repositioning Candidate
Razieh Salehi Ashani1, Homa Azizian2, Nahid Sadeghi Alavijeh1, Vaezeh Fathi Vavsari1,
Shabnam Mahernia3, Niloofar Sheysi4, Mahmood Biglar3, Massoud Amanlou3, and Saeed
Balalaie*1,5
1Peptide Chemistry Research Center, K. N. Toosi University of Technology, P.O. Box 15875-4416, Tehran, Iran,
balalaie@kntu.ac.ir, Tel: +98-21-23064226, Fax: +98-21-22889403
2Department of Medicinal Chemistry, School of Pharmacy, International Campus, Iran University of Medical Sciences, P.O Box:
14665-354, Tehran, Iran
3Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences (TIPS), Tehran University of
Medical Sciences, P.O. Box. 14155-6451, Tehran, Iran
4Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, P. O. Box 14155-6451,
Tehran, Iran
5Medical Biology Research Center, Kermanshah University of Medical Sciences, P. O. Box 67155-1616, Kermanshah, Iran.
Abstract. A series of new deferasirox derivatives were synthesized through the reaction of monosubstituted hydrazides with 2-(2-
hydroxyphenyl)-4H-benzo[e][1,3]oxazin-4-one. For the first time, deferasirox and some of its derivatives were evaluated for their
in vitro inhibitory activity against Jack bean urease. The potencies of the members of this class of compounds are higher than that
of acetohydroxamic acid. Compounds 7d and 7e, analogs bearing tetrazole and hydrazine derivatives (bioisoester of carboxylate
group), represented the most potent urease inhibitory activity with IC50 values of 1.268 and 3.254 µM, respectively. In silico docking
studies were performed to delineate possible binding modes of the compounds with the enzyme, urease. Docking analysis
suggests that the synthesized compounds were anchored well in the catalytic site and extending to the entrance of binding pocket
and thus restrict the mobility of the flap by interacting with its crucial amino acid residues, CME592 and His593. The overall results
of urease inhibition have shown that these target compounds can be further optimized and developed as a lead skeleton for the
discovery of novel urease inhibitors
Keywords: Heterocycles • Inhibitors • Deferasirox analogs • Cyclization • Repositioning candidate
Introduction
Urease (urea amidohydrolase: EC 3.5.1.5), is a nickel-dependent metalloenzyme. Followed by the spontaneous hydrolysis of the
carbamate to ammonia and carbonic acid, it catalyzes the hydrolysis of urea to yield ammonia and carbamate.[1-4] Urease is
broadly distributed in nature among prokaryotes, as well as in eukaryotes, including fungi and plants.[5]
Although there are differences in organization and number of whole enzyme subunits in nature, the structure of the active site
around the nickel(II) ions is conserved and has a consequence of the similar catalytic activity.[6]
Ammonia production leads to an increase in the local pH. Thus urease activity plays an essential role in the pathogenesis of
human and animal diseases, including urinary stones, peptic ulcers, hepatic encephalopathy, and, more importantly, gastric
cancer, which is the second leading cause of cancer-related deaths worldwide.[7] The most substantial known risk factor for
developing peptic ulcers and gastric cancer is the urease activity of Helicobacter pylori (H. pylori), which helps to neutralize the
stomach acid, making a more hospitable environment for this pathogen.[8, 9] Consequently, besides other targets for controlling H.
pylori infection,[10] urease inhibitors have recently drawn significant attention as potential drugs for gastric and urinary tract
infections.[11, 12]
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During current years, several structural classes of urease inhibitors have been reported including hydroxamic acids,[13-15]
18]
20]
phosphoramidates,[16] urea and thiourea derivatives,[17,
oxoindoline and isoindolin-1-one derivatives,[19,
polyphenols,
isoniazids,[21] thiosemicarbazones,[22-24] benzimidazoles,[25] benzophenone sulfonamides,[26] catechol-based inhibitors,[27] diflunisal
derivatives,[28] aryl urea-triazole-based derivatives,[29] 1,3,4-oxadiazoles,[30] cinnamate-based phosphonic acids,[31] coumarin-
hybrids,[32] and metal complexes.[33]
The mode of action of the above inhibitors is based on two main mechanisms. The first one is the non-competitive inhibition
mechanism. These types of inhibition can be obtained by compounds containing groups that are reactive to thiol groups, which
can interact with Cys592 forming an enzyme-inhibitor complex. It was hypothesized that the active site flap in these enzyme-
inhibitors complex is unable to completely close, a position that is essential for the correct accommodation of the catalytic residues
such as His593.[34]
The other mechanism is based on competitive inhibition. Most of these inhibitors, like hydroxamic acid and imidazole [35], are
suitable metal chelators, and their mechanism of inhibition involves binding to the metal ions of the enzyme active site.
[1, 36]
Nevertheless, regardless of the chemical class of the compounds, it is reported
that “only a few functional groups with
electronegative atoms such as oxygen, nitrogen and sulfur act either as bidentate, tridentate or as ligand chelator to form
octahedral complexes with two slightly distorted octahedral nickel atoms of the enzyme.” Deferasirox (DFX) is a new and essential
oral tridentate chelator that binds to the metallic center with a ratio of 1:2. It is used to remove the toxic metals in the case of iron
overload disease and was approved for treating iron overload diseases such as ß-thalassemia.[37] Farkas et al. showed that like
Fe ion, iron chelators could coordinate with other metals such as Ni(II), Zn(II), and Cu(II) ions. Additionally, this study indicated
that the formation of nickel and iron chelation starts above pH ca. 5.[38]
DFX has a 1,2,4-triazole ring and two phenolic groups that, as electron donor groups, are considered to be useful metal chelators
[37, 39]
with various affinity
(Figure 1). Furthermore, the heterocyclic 1,2,4-triazole moiety has attracted considerable attention in
medicinal chemistry due to its wide range of pharmacological activities, low toxicity, and high bioavailability.[40, 41]
Among different classes of heterocyclic compounds, 1,3,4-oxadiazole, 1,3,4-thiadiazole, and 1,2,4-triazole have been found able
43]
to interact with the urease nickel metallocenter, which results in urease inhibitory activity.[42,
The high potency of these
compounds over all other inhibitors of ureases can be attributed to the fact that Ni ions of urease have a higher affinity for nitrogen-
based ligands, thereby stabilizing the binding of N substituents of the inhibitors’ pharmacophores.[44]
Figure 1. The structure of DFX and its complexation with iron.
These observations stimulated our interest to study the urease inhibitory activity of the DFX and some of its new derivatives for
the first time and investigate the role of –OH and various substituted 1,2,4-triazole groups in the inhibition of urease activity. In
this regard and continuation of our research interest in the synthesis of heterocyclic skeletons [45-50], we report an efficient
procedure for the synthesis of some DFX analogs, (N-substituted bis-hydroxyphenyl-1,2,4-triazoles), containing triazole backbone
(Scheme 1). The present article, therefore, describes urease inhibitory properties of newly synthesized compounds and
investigates their mode of molecular interaction to reveal the probable mechanism class of these compounds, including
complexation of nickel ions, blocking the active channel site, or the possibility of covalent interaction with a cysteine residue.
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Scheme 1. Synthesis of deferasirox analogs containing triazole moiety.
Results and Discussion
In the beginning, 2-hydroxybenzoic acid 1 was reacted with thionyl chloride 2 to obtain benzoyl chloride, which was then reacted
with 2-hydroxy benzamide 3 to yield compound 4 in 55% yield. Spectroscopic data confirmed the structure of 4. The IR spectrum
of 4 showed a band of -NH stretching located at 3250 cm-1, and the carbonyl C=O stretching band was observed at 1692 cm-1. 1H
NMR spectrum of compound 4 exhibited a singlet at δ 10.98 ppm for –NH amide. Thus, these data confirm the successful synthesis
of compound 4. In the next step, the cyclization of compound 4 was investigated using p-toluenesufonic acid (p-PTSA) to access
the benzoxazinone 5. Spectroscopic data confirmed its structure, and its purity was established based on the pharmacopeia. The
schematic representation of the acid-catalyzed cyclization reaction is shown in Scheme 2.
Scheme 2. Synthesis of benzoxazinone 5.
The proposed mechanism for the synthesis of benzoxazinone 5 through cyclization reaction is shown in Scheme 3.
Scheme 3. Proposed mechanism for the acid-catalyzed synthesis of benzoxazinone 5.
In the final step, the cyclization reaction of compound 5 was achieved using 4-hydrazinobenzoic acid in DMF at 80 ºC to afford
the final DFX.
With the optimized reaction conditions in hand (Table 1), we next studied the scope and limitations of reaction by employing five
types of hydrazines and hydrazides, which resulted in the formation of compounds 7a-e in good to excellent yields in DMF at 80
ºC (Scheme 4).
Table 1. The optimization of reaction condition in the synthesis of 1-(3,5-bis (2-hydroxyphenyl)-1H-1,2,4-triazol-yl) ethan-1-one 7b.
Entry
Solvent
Time (h)
Yield (%)[a]
1
2
3
4
EtOH
EtOH/H2O (1:1)
CH3CN
3.5
4
30
35
30
55
4.5
2
DMF
[a] Isolated yields.
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Scheme 4. Synthesis of DFX derivatives.
The absence of the amidic hydrogen in all 1H NMR spectra confirmed the cyclization reaction and access to the target products
7a-e, which was further supported by the distinguished peak at 151.5-159.0 ppm in 13C NMR spectra related to =CH in triazole
ring. Furthermore, the HR-MS data confirmed the molecular formula of the synthesized compounds. 2-(1H-Tetrazol-5-yl)
acetohydrazide as one of the starting materials in the cyclization reaction was synthesized from the reaction of ethyl cyanoacetate
with sodium azide and then reaction with hydrazine hydrate. It was selected as one of the members of this library because of
more nitrogen atoms in its structure that result in higher activity.
The most probable mechanism for the synthesis of compounds 7a-e is shown in Scheme 5. Benzoxazinone ring-opening occurred
followed by protonation of benzoxazinone (formation of intermediate A), and the nucleophilic addition of hydrazine or hydrazide
led to intermediate B. Then, after ring-opening and proton transfer, intermediate C is formed. Finally, in the acidic media, triazole
ring formation was carried out through intermediate D (Scheme 5).
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Scheme 5. The proposed reaction mechanism for the synthesis of DFX and its analogs 7a-e.
Urease inhibitory activity of the synthesized compounds was evaluated according to the Berthelot urease assay method[51, 52] and
compared with acetohydroxamic acid (AHA) as the positive control (Table 2). Interestingly, all compounds showed significantly
higher urease inhibition (in a range of 48.35± 0.23 to 1.27± 2.03 µM) in comparison to AHA (IC50 = 100 ± 2.49 µM). Among the
investigated compounds, compounds 7d and 7e having tetrazol-5-yl and benzohydrazide moieties in their structure were set up
to be the most effective compounds with IC50 values of 3.25 ±1.80 and 1.27 ± 2.03 µM, respectively. On the other hand, compound
7a with no substituent and 7c with acetoxy substituent showed reduced activities (IC50 value of 48.35± 0.23 and 7.34± 2.19 µM),
respectively. These findings reveal that 3,5-bis(2-hydroxyphenyl)-1H-1,2,4-triazol-1-yl) scaffold that has substituents present in
the 1,2,4-triazol-1-yl part, can be considered as a potential lead for new H. pylori urease inhibitors.
Molecular modeling
The dependability of the applied docking protocol was measured using re-docking of acetohydroxamic acid (AHA) into the active
site of the JBU. The essential characteristic of a good docking program is its ability to reproduce the experimental or
crystallographic binding modes of ligands. Evaluating this issue, a ligand is taken out of the X-ray structure of its protein-ligand
complex and re-docked into its binding site. The docked binding mode is then compared with the experimental binding mode, and
the RMSD is calculated; a prediction of a binding mode is considered successful if the RMSD is below a specific value (usually<2.0
Å). The urease active site which located in (αβ)8 TIM barrel domain and the surrounded residue composition is shown in Figure
2a. Figure 2b shows the superimposed structure between the docked and the experimental binding mode for AHA over JBU in
which the RMSD value was within the cutoff limit of 1.02 Å. Therefore, the performed docking procedure was applied to evaluate
the interaction between newly synthesized compounds 7a-e and DFX over the JBU active site in comparison to the AHA reference
urease inhibitor. The top-scoring pose of compounds was analyzed inside the binding site of JBU.
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Figure 2. The location of JBU active site over C-terminal (αβ)8 TIM barrel domain (a), Close-up representation of the active site, the AHA co-
crystallized, and the corresponding re-docked form are represented in green and cyan color, respectively (b).
In the binding model, all compounds were successfully occupied in the bi-nickel active site cavity. The scores of the docking
energy of the synthesized compounds varied from −4.28 to −7.78 Kcal mol-1, proposing that the binding interaction differs as the
functional group is varied (Table 2).
Table 2. Docking energy and IC50 values of compounds 7a-e over JBU
Docking energy (Kcal
Compound
7a
R
IC50 ± SD (µMol)
mol-1)
-4.28 (1H)
-3.10 (4H)
H
48.35 ± 0.23
7b
-
-5.25
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7c
7d
7e
7.34± 2.19
1.27 ± 2.03
3.25 ±1.80
-6.34
-7.78
-7.66
DFX
AHA
16.64 ±1.13
100 ± 2.1
-8.27
-4.74
-
Based on docking results, the synthesized compounds oriented in two distinct poses relative to the bi-nickel center over the active
site. Figure 3a exhibits the first pose, including compounds 7d, 7e, and DFX, in which the (3,5-bis (2-hydroxyphenyl) moiety
(shown in red color) adapts flexible conformation in the large hydrophobic opening of the active site pocket. In contrast, the N1
substitution on the 1,2,4-triazole ring (shown in yellow color) tends to orient toward the two nickel atoms. The next pose includes
compounds 7a, 7b, and 7c, which adapted in a manner that one of the 2-hydroxyphenyl moieties pointed toward the metals at the
center and the remaining part of the molecule occupied the surrounding hydrophobic pocket (Figure 3b).
Figure 3. Representation of two different docking poses of compounds relative to bi-nickel center over JBU active site: Compounds with 1,2,4-
triazole-N1 substituted oriented toward the metal center (a), and compounds with 2-hydroxyphenyl pointed one (b) (3,5-bis(2-hydroxyphenyl)
moiety, related N1 substitution and 1,2,4-triazole ring are shown in red, yellow, and navy, respectively.
According to the docking binding energy calculations, compounds 7d and 7e have the lowest free energy of docking (−7.78 and -
7.66 Kcal mol-1, respectively), which is inconsistent with the result of the highest inhibitory activity over JBU active site.
Figure 4 gives an explanation and understanding of molecular interactions of AHA and DFX into the binding site of JBU. As shown
in Figure 4a, AHA forms one H-bond with Asp633 by donating hydrogen and two ionic bonds with two Ni2+ ions. Also, the binding
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mode of DFX was depicted in Figure 4b, which revealed that like AHA carboxylate group pointed to the bi-nickel ion center.
Besides, DFX formed a π-π stacking with His593 and two H-bond interactions from its 2-hydroxyl group with Ala440 (C=O) and
CME592 (-OH). The binding modes of DFX suggested that it positioned more stably into the urease binding pocket by interacting
with Ni2+ ions (mimicked the binding mode of the reference drug AHA) and key residues His593 and CME 592. Thus, these
interactions supported lower docking energy (-8.27 vs. -4.74 Kcal mol-1) and higher anti-urease activity than the AHA (16.64 ±1.13
vs. 100 ± 2.1 µMol).
Figure 4. Molecular docking representation of AHA (a), and DFX (b) over JBU active site. H-bond and π – π staking are shown in yellow and
navy, respectively.
Figure 5a shows the 1-(1H-tetrazol-5-yl)propan-2-one moiety of compound 7d oriented along the bi-nickel center through salt
bridge interaction and formed π-π stacking with His492 and His519. The other part of compound 7d consists of 3,5-bis(2-
hydroxyphenyl)-1H-1,2,4-triazole moiety in which 2-hydroxyphenyl groups formed H-bond interaction with Asp494 and Ala636.
Also, the phenol ring center interacted with His593 through π-π interaction. In the docked conformation of compound 7e (Figure
5b), the benzohydrazide moiety pointed to the Ni+2 ions center. In addition, the NH2 group of hydrazide interacted with H-bonding
to the backbone carbonyl (C=O) of Gly550 and carboxylate side chain of Asp633 (1.9 ꢀ and 2.3 ꢀ). The proximal 2-hydroxyphenyl
ring interacted with Arg439 through H-bond and charge-cation interactions. Furthermore, the other 2-hydroxyphenyl group and
the 1,2,4-triazole moiety contacted with H-bonding interaction Ala436 (C=O) and CME592, respectively.
As mentioned above, like the carboxylate group of DFX, the tetrazol-5-yl moiety of compound 7d and benzohydrazide group of
compound 7e pointed toward the existed positive field around two Ni2+ ions. Both of these groups have the potential to produce
ionized form as a result of weak acidic character. The resulting carbanion is stabilized to a considerable degree due to the
additional aromatic delocalization of negative charge (Figure 6). In other words, this resonance stabilized con base may mimic
the behavior of the hydroxyl oxygen in AHA, which acts to stabilize the tetrazol-5-yl moiety and the benzohydrazide group with
the surrounded positive field of the Ni2+ ions.
Moreover, based on the ability of hydrazide and tetrazole functional groups to bridge transition metals [53, 54], one of the nitrogen
atoms in both of the mentioned groups may coordinate with the nickel ion, which is reminiscent of the behavior of the carbonyl
oxygens in AHA.
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Figure 5. Molecular docking representation of tetrazol-5-yl derivative, 7d (a), and benzohydrazide derivative, 7e (b) over JBU active site. H-bond,
π – π staking, and π –cation interactions are shown in yellow, navy, and green, respectively.
Figure 6. Representation of the aromatic stabilization and tautomerization of tetrazol-5-yl (a) and benzohydrazid (b)
Unlike the above structures, compound 7a with no substitution on the 1,2,4-triazole ring, and compounds 7b and 7c with acetoxy
and 1-(furan-2-yl)ethan-1-one moieties, respectively, which cannot be ionized or being metal coordinated, resulted in interacting
with different manner over JBU active site pocket.
The interaction patterns shown in Figure 7 represent different poses rather than those of compounds 7d and 7e. In all compounds
7a-c, beside chelation of the oxygen atom of one of the proximal 2-hydroxyphenyl groups with the bi-nickel center in the catalytic
site, the related phenyl group forms π – π stacking with His545. Further stability was due to the formation of hydrophobic π–π
stacking interactions of the distal 2-hydroxyphenyl group of compounds 7b and 7c with the active site flap His593, hence resulting
in urease active site inhibition (Figures 7c and 7d).
Furthermore, as compound 7a could be in two distinct tautomers (1H and 4H-1,2,4 triazol), it can lay in two slightly different poses
over the active site. Docking result showed although they contributed to H-bonding and hydrophobic interactions with the active
site residues, there are some differences in the way both tautomers coordinated with the center of bi-nickel. The alteration in the
interaction pattern is shown in Figures 7a and 7b.
In the case of 4H tautomer, the 1,2,4-triazole ring was stabilized by charge-transfer interaction with Arg633 and contributed H-
bond interaction through its –NH group to Ala636 (C=O). Consequently, it went closer to the helix-turn-helix motif over the active-
site cavity so it could interact with His593, while the 1,2,4-triazolo ring of the 1H tautomer just contributed in π -π and H-bonding
with His519 and Ala440, respectively and it could not interact with any of the flap residues at the entrance of the active site channel.
The difference in interaction mode between 7a 4H and 1H tautomers was reflected in different docking binding energies of -4.28
and -3.10 Kcal mol-1, respectively.
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Figure 7. Docking mode of compounds 7a (4H tautomer) (a), 7a (1H tautomer) (b), 7b (c), and 7c (d) over JBU active site. H-bond, π – π
stacking, and π –cation interactions are shown in yellow, navy and green, and cyan, respectively.
Conclusions
We have developed an efficient method for the synthesis of DFX derivatives using the reaction of benzoxazinone and hydrazide
derivatives to obtain 1,2,4-triazoles through cyclization reaction with good to high yields efficiency.
Using this approach, a novel class of urease inhibitors was identified with significant biological activities. This is the first study to
investigate the inhibition activity of deferasirox and some of its derivatives over jack bean urease. The potencies of this class of
compounds are higher than those of acetohydroxamic acid. Among the series compounds 7d and 7e, the analogs bearing
tetrazole and hydrazine derivatives (bioisoester of carboxylate group) provided the highest urease inhibition activity. These
compounds can be in the form of conjugated base orienting toward the positive field around the bi-nickel center.
According to the docking analysis, DFX and the synthesized compounds were harbored well in the catalytic site. They interacted
like ligand chelator (substrate-like inhibitor) and, at the same time, took advantage of interacting with the entry of active site
channels, accordingly, limited the flexibility of the helix-turn-helix motif which known as flap by interacting with His593 or CME592
as the critical amino acid residues.
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Generally, although the exact mechanism of urease inhibition of our test compounds is not known, deferasirox repositioning can
be proposed by our investigation in which this structure can be further optimized and developed as the lead for urease inhibition
activity, and it is intriguing to investigate detailed kinetics of such interaction.
Experimental Section
Urease inhibition assay
The enzyme assay was performed by the Berthelot alkaline phenol-hypochlorite method. This method is based on the release of
ammonia (NH3), which reacts with hypochlorite (OCl-) to form a monochloramine.[51, 52] This product then reacts with phenol to
form blue-colored indophenols that its absorbance is measured at 625 nm. In brief, 10 μl of enzyme solution was incubated with
140 μl of urea and 5 μl of inhibitor at a final concentration of 25 mM in phosphate buffer solution (pH 7.6, 100 mM) for 15 min at
37°C. The ammonia liberated was estimated using 500 μl of solution A (containing 5.0 g phenol and 25 mg of sodium nitroprusside)
and 500 μl of solution B [containing 2.5 g sodium hydroxide and 4.2 ml of sodium hypochlorite (5% chlorine) in 500 ml of distilled
water] at 37°C for 30 min. The absorbance was measured at 625 nm against the control. For all analyzed compounds, the values
of IC50 were calculated using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA).
Molecular docking procedure
To find out the interactions mode of designed molecules over the urease enzyme, the flexible docking study was performed on
Autodock 4.2.6 software.[55] The X-ray crystallographic structure of Jack bean urease (JBU) (in complex with acetohydroxamic
acid, AHA) was downloaded from the Protein Data Bank (PDB ID; 4h9m) (www.rcsb.org). The urease are reported to be
functionally active in a monomeric state, then all the docking studies were performed on a single monomer. Also, the prosthetic
group and co-factors are not directly involved in urease inhibition, so they removed before docking investigation. Water molecules
and co-crystallized ligands were removed from the enzyme's crystallographic structures. The pKa values of the residues in the
enzyme were calculated to determine if any of them were likely to adopt nonstandard ionization states, using PROPKA 2.0.[56] The
side chains of the lysine, arginine, and histidine residues were protonated, while the carboxylic groups of glutamic acid and aspartic
acid were deprotonated. After adding polar hydrogens and partial atomic charges, the enzyme structures saved in PDBQT. A grid
box of 60× 60× 60 Å (x, y, and z) created at the center of the enzyme's active pocket with the spacing of 0.375 nm in each
dimension to evaluate the ligand-protein interactions. The center of the grid box set to the center average coordinates of the
crystallographic AHA over JBU structure. The 2D structures of all synthesized compounds were drawn in Marvin 15.10.12.0
program (http://www.chemaxon.com)[57] and converted into PDB. Gasteiger charges assigned and saved in PDBQT file format.
Of the three different search algorithms offered by AutoDock 4.2.6, the Lamarckian genetic algorithm (LGA) consisting of 100
runs, 25×106 energy evaluations, and 27,000 generations was applied. Other docking parameters were set to default. Cluster
analysis was performed on the docked results by means of a root mean square (RMS) tolerance of 2.0 Å.
Description of the active site of JBU
The JBU consists of two domains; (αβ)8 TIM barrel domain andβ domain. The Ni2+–Ni2+ distance is 3.4 Å. These two Ni2+ ions are
bridged by a carbamylated lysine (KC490) through its O atoms. One of the Ni2+ ions is coordinated by His407 (NE2), His409 (ND1),
Asp633 (NE2), and KCX490 (OQ1), and the other is coordinated by His519 (NE2), His545 (NE2), KCX490 (OQ2), and Gly550
(OD2). Also, Arg439, CME592, and His593 located at the mouth of the active site channel. CME592 (modified Cys592), located
on the mobile flap, is an essential residue and highly conserved in many ureases. Other pocket residues are Ala440, CME592,
Gly550, Ala636, Met637, His492, Thr441, and Arg609.[58]
Supplementary Material
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/MS-number, which contains the
detailed procedure for synthesis and characterization of all compounds.
Acknowledgments
We thank the National Institute for Medical Research Development (NIMAD, Project No. 963388) and Research Council of
Tehran University of Medical Sciences for financial supports for financial support.
11
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Author Contribution Statement
RSA, NSA, VFV, and SB performed the experiments, analyzed the data, and wrote the paper. HA and MA contributed to docking,
the analysis of the data, and the writing of the article. SM and NS contributed samples/reagents/materials/analysis tools analyzed
the data. MA and MB conceived and designed the experiments. All authors read and approved the final manuscript.
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Twitter Text
For the first time, deferasirox and some of its derivatives were evaluated for their in vitro inhibitory activity against Jack bean
urease.
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