Design, synthesis and biological evaluation of novel glyoxalase I inhibitors possessing diazenylbenzenesulfonamide moiety as potential anticancer agents

Article abstract of DOI:10.1016/j.bmc.2020.115608

The enzyme glyoxalase-I (Glo-I) is an essential therapeutic target in cancer treatment. Significant efforts have been made to discover competitive inhibitors of Glo-I as potential anticancer agents. Herein, we report the synthesis of a series of diazenylb

Full text of DOI:10.1016/j.bmc.2020.115608

Journal Pre-proofs
Design, synthesis and biological evaluation of novel glyoxalase I inhibitors
possessing diazenylbenzenesulfonamide moiety as potential anticancer agents
Buthina A. Al-Oudat, Hana'a M. Jaradat, Qosay A. Al‑Balas, Nizar A. Al-
Shar'i, Amanda Bryant-Friedrich, Mel F. Bedi
PII:
DOI:
Reference:
S0968-0896(20)30438-7
https://doi.org/10.1016/j.bmc.2020.115608
BMC 115608
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Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
19 March 2020
19 June 2020
22 June 2020
Please cite this article as: Al-Oudat, B.A., Jaradat, H.M., Al‑Balas, Q.A., Al-Shar'i, N.A., Bryant-Friedrich, A.,
Bedi, M.F., Design, synthesis and biological evaluation of novel glyoxalase I inhibitors possessing
diazenylbenzenesulfonamide moiety as potential anticancer agents, Bioorganic & Medicinal Chemistry (2020),
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Graphical Abstract
Design, synthesis and biological evaluation of
novel glyoxalase I inhibitors possessing
Leave this area blank for abstract info.
diazenylbenzenesulfonamide
potential anticancer agents
moiety
as
Buthina A. Al-Oudata, , Hana’a M. Jaradata, Qosay A. Al‑Balasa, Nizar A. Al-Shar’ia, Amanda Bryant-
Friedrichb, Mel F. Bedib
aDepartment of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science
and Technology, P.O. Box 3030, Irbid 22110, Jordan
bDepartment of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences,
University of Toledo, Toledo, OH, 43606, USA
NH2
O
S O
H3C
N
N
N
Zn
SO2NH2
OH
OH
H3C
IC50 = 1.28 µM
N
Optimization
N
NH2
NH2
O
S O
IC50 = 3.65 µM
H3C
O
N
N
OH
OH
Zn
IC50 = 1.13 µM
1

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Design, synthesis and biological evaluation of novel glyoxalase I inhibitors
possessing diazenylbenzenesulfonamide moiety as potential anticancer agents
Buthina A. Al-Oudata, , Hana’a M. Jaradata, Qosay A. Al‑Balasa, Nizar A. Al-Shar’ia, Amanda Bryant-
Friedrichb, Mel F. Bedib
aDepartment of Medicinal Chemistry and Pharmacognosy, Faculty of Pharmacy, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110,
Jordan
bDepartment of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, OH, 43606, USA
———
ARTICLE INFO
ABSTRACT
 Corresponding author. Tel.: +962-775816841; fax: +962-27095123; e-mail: baoudat@just.edu.jo (Buthina A. Al-Oudat)
Article history:
Received
Received in revised form
Accepted
The enzyme glyoxalase-I (Glo-I) is an essential therapeutic target in cancer treatment.
Significant efforts have been made to discover competitive inhibitors of Glo-I as potential
anticancer agents. Herein, we report the synthesis of a series of diazenylbenzenesulfonamide
derivatives, their in vitro evaluation against Glo-I and the resulting structure-activity
relationships. Among the compounds tested, compounds 9h and 9j exhibited the highest activity
with IC50 1.28 µM and 1.13 µM, respectively. Docking studies to explore the binding mode of
the compounds identified key moieties that may contribute to the observed activities. The active
compounds will serve as suitable leads for further chemical optimization.
Available online
Keywords:
Anticancer agents
Glyoxalase-I
Diazenylbenzenesulfonamide
Zinc binding groups
Molecular docking
2009 Elsevier Ltd. All rights reserved.
7
Methylglyoxal is a cytotoxic by-product generated from the
glycolysis process during normal metabolism.8 Its cytotoxicity is
due to its ability to form adducts with cellular proteins, lipids,
and nucleic acids.9-12 The glyoxalase system consists of two
1. Introduction
Despite the enormous efforts put into cancer prevention and
treatment, cancer remains a major public health problem
worldwide.1 Cancer is a rebound system of uncontrolled growth
that originates in the human body from the accumulation of
genetic and epigenetic changes in normal cells.2 Cancer
chemotherapeutic treatment, where chemical substances are used
as anti-cancer agents, is often accompanied by toxic side effects.
This is usually due to the lack of selectivity for tumor cells over
normal cells. For that reason, there is a growing need for the
identification of suitable druggable macromolecules that can be
targeted selectively by anti-tumor agents in cancer cells. In an
effort to find unique targets for anticancer agents, the glyoxalase
system has been demonstrated as a plausible one.3-4 The
glyoxalase system has a detoxification function in living cells. It
mediates the catalysis process of the GSH-dependent conversion
of the α-oxoaldehydes such as methylglyoxal into D-lactic acid.5-
enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II) and a
catalytic amount of glutathione (GSH). In the detoxification
process, the methylglyoxal 1 is converted nonenzymatically into
thiohemiacetal 2; the substrate for the Glo-I. Through the
formation of ene-diolate intermediate 3, Glo-I catalyzes the
isomerization of thiohemiacetal
2 to the thioester S-D-
lactoylglutathione 4 which then undergoes a catalytic hydrolysis
to the non-toxic D-lactic acid 5 and reduced glutathione by Glo-II
13-14
(Figure 1).5,
It has been proposed that inhibition of the
glyoxalase pathway in cancer cells leads to accumulation of the
toxic methylglyoxal resulting in self-destruction of the cancer
cells.15
2

O
OH
O
O
GlxI
OH
SG
O
SG
H
GSH
H3C
+
H3C
H3C
H3C
HO
H
SG
O
Methylglyoxal
enediolate intermediate
Thiohemiacetal
1
3
2
OH
O
OH
H
H
GlxII
H2O
O
O
H
GSH
+
+
H3C
H3C
SG
D-lactate
S-D-lactoylglutathione
5
4
Figure 1. Normal pathway of glyoxalase enzyme system
Selective inhibition of the glyoxalase system in cancerous
cells is expected to be obtained due to the increasing activity and
overexpression of the enzyme in the metabolically active
cancerous cells.16-18 Among the two enzymes of the glyoxalase
detoxification pathway, Glo-I has gained special attention as a
promising target for the development of potential anti-cancer
agents for two reasons. First, Glo-I drives the rate- limiting step
of the detoxification biological process. Second, Glo-I
overexpression was associated with multidrug resistance of a
wide variety of tumors suchlike monocytic leukemia and erythro
leukemia.19-20 Potent and selective inhibition of Glo-I in
cancerous cells would therefore induce elevated concentration of
the cytotoxic methylglyoxal leading to apoptosis of tumor cells.
Structurally, the human Glo-I is a homodimeric mononuclear
zinc metalloenzyme with each monomer consisting of 184 amino
acids and a mass of 42 kDa.21 It has been determined that the
Glo-I active site can be divided into three main regions, namely a
positively charged entrance (Arg37, Arg122, and Lys156), a
central zinc atom which is involved in stabilizing the enediolate
intermediate, and a deep hydrophobic pocket (Phe62, Leu69,
Leu92, and Met179).22
inhibitors
of
Glo-I.
The
selected
hit
possesses
diazenylbenzenesulfonamide moiety, exhibited IC50 value equal
to 3.65 µM. The structure of the selected hit is shown in Figure
2. In the present study, structural analogues of the hit compound
6 were proposed based on their synthetic feasibility and the
pivotal chemical features that are required for optimal inhibitor
binding to the active site. Three chemical features can be utilized
to assure better inhibitor binding to the active site of the enzyme
including; a zinc binding group to chelate the zinc atom at the
center of the active site, a negatively ionizable group for binding
to the positively ionized active site mouth and a hydrophobic
group that can accommodate the deep hydrophobic pocket of the
enzyme. The proposed compounds were synthesized and
biologically evaluated in vitro against human Glo-I enzyme.
Structure-activity relationships were obtained through structural
manipulations of the synthesized analogues. Docking studies
were performed on the most active compounds to explore the
binding modes of these compounds at the active site.
SO2NH2
OH
In the past decades there have been extensive efforts to
identify and design potent Glo-I inhibitors with structural
diversity and a range of different inhibitory activities utilizing
different drug discovery methodologies.23 Substrate and
transition-state analogues were among the first class of
N
N
NH2
24-26
compounds designed as competitive Glo-I inhibitors.3,
6
Natural products and natural product derivatives were utilized as
another approach to obtain such inhibitors.27-30 Recent studies
have utilized different approaches to identify new Glo-I
Figure 2. Structure of obtained hit with IC50 equal to 3.65 µM.36
inhibitors including high-throughput screening31-32 and in silico
2. Results and discussion
33-35
computer aided drug design.29,
Recently, Al-Sha'er et al.
utilized a structure-based pharmacophore approach to obtain a
pharmacophore model for the Glo-I active site.36 Ninety two
pharmacophore models were generated utilizing eighteen co-
crystallized protein-ligand structural complexes. Subsequent
QSAR modeling followed by ROC evaluation identified a single
pharmacophore model called Hypo (3VW9) which was able to
predict the expected Glo-I inhibition. Hypo (3VW9) was used to
screen compounds in the National Cancer Institute (NCI)
database that resulted in the identification of several promising
hits. The hit compounds were purchased and subjected to
experimental biochemical assay that led to the identification of
seven hits with IC50 at the low micromolar range. Herein, one of
the hits was used as a lead compound to develop more potent
2.1. Chemistry
To discover and develop competitive inhibitors of Glo-I as
potential anticancer drugs, we report here the preparation of a
series of compounds (9a-x) that are structurally related to the
lead compound 6. The synthetic routes adopted for the synthesis
of all target compounds are outlined in Scheme 1. The syntheses
utilized a set of different structures of commercially available
starting materials containing aromatic amino moiety (7 or 10)
that were subjected to diazotization reactions yielding diazonium
salt intermediates (8 or 11) which then were coupled with
NH2
NH2
NH2
O
S
O
O
S
O
O
S
O
R2 = Substituted aromatic
carbocyclic or heterocyclic
compounds
R1
R1
R1
NaNO2(aq)
40% HCl, 0 °C
3
N
R1 = CH3 or H
Cl
N2
N
R2
NH2
AcONa(aq) or NaOH (aq) , 0 °C
9a-q
7
8

Scheme 1. Synthesis of the target compounds 9a-x.
(compound 9f) or a 7-hydroxy-2H-chromen-2-one (compound
different structures of substituted aromatic carbocyclic and
heterocyclic compounds.37 We have chosen various R moieties
(R1, R2 and R3) to be present in the derivatives 9a-x including:
polar moieties such as sulfonamide, hydroxyl and amino groups,
ionizable moieties such as carboxylic and sulfonic acid groups,
and hydrophobic moieties such as benzene and naphthalene
groups. Besides the above mentioned ionizable and polar groups,
9g) moieties led to moderate inhibitory activities. Interestingly,
inclusion of 8-hydroxyquinoline moiety provided compound 9h
with IC50 value of 1.28 µM. Selection of the 8-hydroxyquinoline
moiety was based on a reported Glo-I inhibitor (compound 12,
Figure 3) which was developed with a novel sulfonamide core
pharmacophore exhibiting IC50 0.46 µM.35 It has been
demonstrated that compound 12 binds the catalytic zinc ion of
Glo-I in a bidentate fashion through the endocyclic nitrogen and
exocyclic sulfonamide-nitrogen donor atoms (in red). Here, we
propose that similar interactions with the zinc ion were attained
by the 8-hydroxyquinoline moiety in compound 9h utilizing the
8-hydroxyquinoline,
2-hydroxybenzoic
acid
and
2-
hydroxynaphthoic acid moieties were included in the derivatives
as they were presumed to chelate with the zinc ion of the enzyme
and/or form hydrogen bond interactions with the target protein
which should be highly beneficial for the inhibitory activity of
the compounds. The identities of all the synthesized compounds
were confirmed by 1H-NMR, 13C NMR and high resolution (HR)-
MS. All the target compounds are listed in Table 1 and Table 2.
endocyclic
nitrogen
and
exocyclic
hydroxyl-oxygen
functionalities (in red). To investigate the binding modes of the
synthesized compounds, particularly compound 9h, within the
binding site of Glo-I, we performed molecular docking using
CDOCKER algorithm within Discovery Studio (DS) software.
The best predicted docked pose for compound 9h in addition to
its 2D interaction map with active site residues are shown in
Figure 4, which explains the good activity of the compound. The
docked pose showed that the 8-hydroxyquinoline moiety
coordinated with the Zn ion in a bidentate fashion through the
endocyclic nitrogen and exocyclic hydroxyl-oxygen donor atoms
of the tautomerized form of the compound. Moreover, the
sulfonamide group contributed to the compound potency through
providing hydrogen bond interactions with Lys156 and Met157,
whereas the aromatic rings enhanced the compound’s activity
through providing binding interactions with the hydrophobic
region of the enzyme including Met65, Met179 and Leu69.
On the other hand, it was reported that the Glo-I inhibitor
(compound 13, Figure 3) that possesses di-salicylic acid moieties
exhibits good Glo-I inhibitory activity with IC50 0.34 µM.38 It has
been shown that the activity of this compound is due to the
ability of the ionizable di-salicylic acid moiety to form strong
interactions with the zinc ion, Arg37 and Arg122 at the active
site of the enzyme. For that reason, 2-hydroxybenzoic acid
moiety was used to produce compound 9i (IC50 11.5 µM) that
was optimized through introducing another fused benzene ring
(hydroxynaphthoic acid) which gave compound 9j that was
found to be the most active among the synthesized compounds
with IC50 1.13 µM. The binding mode of compound 9j within
Glo-I is depicted in Figure 4. The docked pose showed the
importance of the ionizable carboxylic moiety to the activity of
the compound due to its ability to form a metal-acceptor
coordination and an electrostatic interaction with the zinc ion.
Moreover, the docked pose showed that the sulfonamide group
provided significant hydrogen bond interactions with Lys156 and
Met157. In addition, the hydrophobic aromatic benzene and
naphthalene moieties stabilized the compound in the active site
2.2. Glo-I Inhibition, SAR study and Molecular docking
The lead compound 6 has been previously reported as Glo-I
inhibitor with IC50 value equal to 3.65 µM.36 Herein, structure–
activity relationship (SAR) studies around this lead compound
resulted in the identification of a series of analogues that
demonstrated a broad range of inhibition (low μM to inactive) as
shown in Table 1 and Table 2. Structurally, compound 6 has the
diazenyl-2-methylbenzenesulfonamide moiety attached to
position 8 of the 2-hydroxy-5-aminonaphthalene moiety. The
SAR studies were started through investigating the importance of
the identity and position of the substituents on the naphthalene
moiety of compound 6 through the synthesis of compounds 9a-
9c. Moving the hydroxyl group of compound 6 from position 2 to
position 3 or 1 (compounds 9a or 9b) led to moderate or
significantly reduced inhibitory activity, respectively. Removing
the hydroxyl group from the naphthalene moiety produced a
totally inactive compound (compound 9c). On the other hand,
attaching the diazenyl-2-methylbenzenesulfonamide moiety to
position 1 of the 2-hydroxynaphthalene resulted in compound 9d
with weak inhibitory activity. Introducing an amino group to
position 8 in compound 9d led compound 9e with moderate
inhibitory activity. These results reveal that the hydroxyl group at
position 2 and the amino group at position 5 of the naphthalene
ring are important for the inhibitory activity of compound 6. At
this point, we decided to modify compound 6 through replacing
the 2-hydroxy-5-aminonaphthalene moiety with functional
groups that have the potential to chelate the zinc ion of the
enzyme and/or form hydrogen bond interactions with amino acid
residues within the active site in order to enhance the inhibitory
activity of the compounds. For that reason, compounds 9f-9k
were synthesized. Inclusion of a 2-hydroxy-1-naphthaldehyde
4

through providing interactions with the hydrophobic region of the
enzyme (Phe67 and Lys150). An attempt to further optimize
compound 9j through changing the position of the carboxyl and
hydroxyl groups, as shown in compound 9k, led to significant
reduction in activity producing totally inactive compound. This
result might be attributed to disruption of the orientation of these
key functional groups seen in compound 9j, which upon
changing their positions as seen in compound 9k had resulted in
detrimental steric clashes within the active site rendering the
compound completely inactive.
Table 1: The structures and inhibitory activities of the target compounds 9a-q
NH2
O
S
O
R1
N
N
R2
No.
6
R1
R2
OH
- CDOCKER Energy
(Kcal/mol)
% Inhibition at 25 µM
IC50a (µM)
CH3
27.59
76.43
3.65
NH2
OH
9a
9b
9c
9d
CH3
CH3
CH3
CH3
32.8617
28.4877
17.5752
18.675
57.2
ND
18.90 ± 0.52
NH2
HO
>100
NH2
24.74
34.45
95.70 ± 12.2
45.02 ± 1.95
NH2
OH
OH
9e
9f
24.7535
34.6445
35.3771
54
24.00 ± 2.41
18.57 ± 0.10
17.81 ± 0.08
CH3
CH3
CH3
H2N
OH
O
57.36
58.98
H
OH
9g
O
O
5

9h
9i
33.7361
51.033
92.14
67.72
100
1.28 ± 0.12
11.48 ± 0.45
1.13 ± 0.06
CH3
CH3
CH3
N
OH
O
OH
OH
O
9j
41.0963
OH
OH
O
OH
9k
39.7131
ND
>100
CH3
OH
OH
9l
H
H
H
41.3362
20.0072
20.0229
66.04
28.096
39.04
21.83 ± 0.24
85.81 ± 10.32
37.51 ± 0.42
NH2
9m
9n
NH2
OH
OH
9o
H
36.5897
64.36
18.95 ± 0.37
O
O
O
9p
9q
55.1658
48.4885
76.47
96.54
8.66 ± 0.12
1.22 ± 0.07
H
H
OH
OH
O
OH
OH
Myricetin
-
-
3.38 ± 0.41
a The IC50 values are represented as the mean ± standard error of the mean of three independent experiments performed in triplicate.
6

O
O
NH2
NH2
O
S O
O
S O
N
OH
OH
OH
O
NH
N
N
S
N
O
N
N
O
O
O
N
OH
HO
OH
OH
9j
IC50 = 1.13 µM
This work
9h
IC50 = 1.28 µM
This work
12
IC50 = 0.46 µM
13
IC50 = 0.34 µM
(
)
(
)
Figure 3. Molecular structures of previously reported Glo-I inhibitors with corresponding IC50 values. Metal binding atoms are highlighted in red.
Figure 4. A. A 3D representation of the docked pose of compound 9h within the active site of the Glo-I enzyme (PDB code 3W0T (resolution of 1.35 Å)). The
binding site is represented as a hydrophobic surface (blue is positively charged areas and brown is hydrophobic areas), docked compounds are shown in balls and
sticks with carbons colored green, and the Zn2+ ion as a grey sphere. B. A 2D interaction map showing the different interactions between compound 9h and the
active site of the enzyme. Residues are colored according to the type of their interactions with the ligand, the interactions are shown as dashed lines. C. A 3D
7

representation of the docked pose of compound 9j within the active site of the Glo-I enzyme (PDB code 3W0T (resolution of 1.35 Å)). D. A 2D interaction map
showing the different interactions between compound 9j and the active site of the enzyme.
8

Then, we investigated the importance of the methyl group at
position 2 of the benzenesulfonamide moiety through the
synthesis of compounds 9l-9q. The SAR study showed that the
analogues lacking the methyl group exhibited similar activities
as the methyl-containing analogues. This may be attributed to
the small size of the methyl moiety which may have no
influence on the binding modes of the inhibitors with the target
enzyme.
important binding groups that provide the critical binding
interactions with the active site of the enzyme. To this end,
additional synthetic efforts were made to obtain compounds 9r-
9x (Table 2). Moreover, molecular docking of these
compounds into the Glo-I active site was performed to give an
explanation and understanding of the activity observed
providing an insight for structural interpretation of SAR
(Figure
5
and
Figure
6).
Next, we decided to explore the SAR for one of the most
active analogues (compound 9h) in order to identify the
Table 2: Structural modifications on compound 9h
IC50a (µM)
- CDOCKER Energy
No.
9h
Structure
% Inhibition at 25 µM
(Kcal/mol)
NH2
O
S
O
33.7361
92.14
1.28 ± 0.12
N
N
N
OH
NH2
O
S
O
9r
9s
33.0826
26.1108
98.28
38.31
1.36 ± 0.09
52.19± 0.72
N
N
N
OH
NH2
O
S
O
N
N
OH
NH2
O
S
O
9t
35.5576
59.91
13.36 ± 0.53
N
N
OH
N
9u
9v
N
N
21.8737
38.8429
82.78
90.71
9.13 ± 0.044
4.37 ± 0.43
OH
O
OH
N
N
N
OH
O
9w
9x
HO
HO
38.0737
14.7322
79.87
74.99
4.45 ± 0.30
9.70 ± 0.06
N
N
N
OH
O
S
O
N
N
N
OH
a The IC50 values are represented as the mean ± standard error of the mean of three independent experiments performed in triplicate.
9

Compound 9r that lacks the methyl group at position 2 of the
benzenesulfonamide moiety exhibited similar activity to
compound 9h, which is supported by the 2D interaction map that
displayed similar binding interactions to compound 9h (Figure
5). This result confirms the insignificant role of the methyl group
in the binding mode with the target enzyme as previously
observed in compounds 9l-9q. To investigate the importance of
the pyridine-nitrogen atom to the activity of compound 9h,
compound 9s that lacks the nitrogen atom was synthesized which
was found to have weak inhibitory activity. The significant
reduction in the inhibitory activity of 9s may be attributed to the
mono-chelation of the zinc ion to the hydroxyl group in contrast
to the di-chelation to the endocyclic pyridine-nitrogen and
exocyclic hydroxyl-oxygen donor atoms of the 8-
hydroxyquinoline moiety, as shown by the 2D interaction maps
(Figure 5). These results suggest that the pyridine-nitrogen atom
greatly contributes to the Glo-I inhibitory activity of compound
9h. In contrast to compound 9s, total removal of the pyridine
moiety (compound 9t, IC50 = 13.36 µM) led only to small
decrease in activity, when compared to compound 9h, which can
be explained through the analysis of its binding mode within the
active site. The 2D interaction map of compound 9t shows that
the compound flips over in the enzyme active site allowing the
formation of several binding interactions with the amino acid
residues at the active site of the enzyme (Figure 5). The hydroxyl
group of the phenol moiety interacts with Lys156 while the
sulfonamide moiety interacts with the zinc ion, Glu172 and
His126. We propose that these binding interactions compensated
for the loss of binding interactions provided by the pyridine ring.
Compound 9h
Compound 9r
Compound 9s
Compound 9t
Figure 5. 2D interaction maps of compounds 9h, 9r-9t showing the types of different interactions they establish with key amino acids within Glo-I active site.
Next, we decided to investigate the importance of the
sulfonamide moiety to the activity of compound 9h through the
synthesis of compound 9u. As predicted, this modification
resulted in some loss in the inhibitory activity leading to
moderately active compound (IC50 = 9.13 µM). Keeping some of
the potency for compound 9u may be attributed to the binding
10

interactions exhibited by the 8-hydroxyquinoline moiety through
di-chelation of the Zn ion to the endocyclic pyridine-nitrogen and
exocyclic hydroxyl-oxygen donor as well as the extra
hydrophobic interactions by the benzene ring with Met65 and
Met157, as shown by the 2D interaction map (Figure 6).
Isosteric replacement of the sulfonamide moiety with a carboxyl
group, produced compound 9v with IC50 value of 4.37 µM. The
di-chelation interactions of the zinc ion to the 8-
hydroxyquinoline moiety as well as the ionic bonds with Lys150
and Lys156 and hydrogen bond interaction with Lys156 by the
carboxyl group contributed to the potency of compound 9v.
Shifting the carboxyl group in compound 9v to the para position
relative to the diazinyl group generated compound 9w (IC50 4.45
µM) with similar activity to compound 9v. Replacement of the
sulfonamide moiety with a sulfonate group produced moderately
active compound (9x) with IC50 value of 9.7 µM. Although this
highly acid functionality was anticipated to enhance the binding
affinity, hence the overall activity, the observed activity could be
attributed to solubility issues, since this compound was the least
soluble under the used assay conditions. The 2D interaction maps
of
compounds
9v-9x
are
shown
in
figure
6.
Compound 9u
Compound 9v
Compound 9w
Compound 9x
Figure 6. 2D interaction maps of compounds 9u-9x showing the types of different interactions they establish with key amino acids within Glo-I active site.
11

2.3. ADMET and toxicity predictions
performed on precoated silica gel plates (Merk, Kieselgel 60 F-
254, 0.2 mm). Merck silica gel 60 was used for column
chromatography. Compound spots were visualized by UV light
(254 nm). 1H/13C NMR spectra were recorded at ambient
temperature on a Bruker AVANCE 400 MHz or 600 MHz
instruments. The spectra were obtained in ppm using automatic
calibration to the residual proton peak of the solvent, DMSO-d6.
Given the high cost of drug discovery and development
processes, it is highly recommended to direct and focus efforts
towards lead compounds with favorable ADMET properties and
eliminate ones that could lead to later problems due to
unfavorable ADMET characteristics. Therefore, we calculated
the ADMET descriptors and Toxicity parameters for compounds
that showed promising Glo-I inhibitory activities in order to
guide future lead optimization. ADMET refers to the Absorption,
Distribution, Metabolism, Excretion, and Toxicity properties of a
molecule within an organism.
The ADMET Descriptors protocol in DS was used to calculate a
range of ADMET related properties including, aqueous solubility
(AS), blood brain barrier (BBB) penetration, CYP2D6 inhibition,
hepatotoxicity, human intestinal absorption (HIA), plasma
protein binding (PPB), AlogP, and polar surface area (PSA). In
addition, different toxicity parameters were also calculated using
the Toxicity Prediction (TOPKAT) protocol in DS. The calculated
toxicity parameters were rodent carcinogenicity (for both male
and female rats), Ames mutagenicity (AM), skin irritation (SI),
ocular irritancy (OI), aerobic biodegradability and developmental
toxicity potential (AB) (Table 3, Supplementary Material).
Based on the calculated values, particularly mutagenicity and
carcinogenicity, compounds 9j and 9q show promising ADMET
and toxicity profiles, and as we show above, these compounds
also showed the highest activity.
1
The H NMR data are presented as follows: chemical shift (δ
ppm), multiplicity (s = singlet, d = doublet, dd = doublet of
doublet, t = triplet, q = quartet, m = multiplet), coupling constants
(Hz), and integration. The 13C NMR analyses were reported in
terms of chemical shift. High resolution mass spectra were
acquired using Waters Synapt HDMS (ESI Q-TOF MS) equipped
with nanoAcquity UPLC system and traveling wave ion mobility
MS in the College of Arts and Sciences and Mathematics
Instrumentation Center at the University of Toledo, 2801 W
Bancroft drive, Toledo, OH, USA.
4.1.1 General procedure for the synthesis of target compounds
9a-9x.
The synthesis of the target compounds was achieved according to
published literature procedures with some modifications utilizing
the following two steps.37
Step 1: Preparation of diazonium chloride solutions of aromatic
amines: The aromatic amine-containing compound (0.20 g, 1.0
eq) was dissolved in a freshly prepared 40% solution of
concentrated hydrochloric acid in de-ionized water (3 ml) then
cooled down to -5 ºC in ice/sodium chloride path. To that was
added dropwise an aqueous solution of NaNO2 (1.2 eq, 2M).
Then the reaction mixture was kept stirring at the same
temperature for 15 min to produce the diazonium chloride
solution which is required for the next step. The solution was
freshly prepared prior to use.
3. Conclusions
In summary, a series of novel diazenylbenzenesulfonamide
derivatives were synthesized and evaluated in vitro against Glo-I
enzyme. Structural modification of compound 6 produced several
derivatives that exhibited inhibitory activity with IC50 values in
the low micromolar range. Replacing the 2-hydroxy-5-
aminonaphthalene moiety of compound
6
with 8-
hydroxyquinoline moiety provided compound 9h with IC50 1.28
µM. SAR analysis of compound 9h revealed the importance of
the nitrogen atom and hydroxyl group of the 8-hydroxyquinoline
moiety as well as the sulfonamide moiety in exerting the
inhibitory activity of the compound. Isosteric replacement of the
sulfonamide moiety with a carboxyl group produced active
compounds with comparable potency to compound 9h with IC50
values less than 5 µM. On the other hand, hydroxynaphthoic acid
moiety in compound 9j was proved to be an exceptional
replacement for the 2-hydroxy-5-aminonaphthalene moiety of
compound 6 as it was found that compound 9j (IC50 1.13 µM) is
the most active among the synthesized compounds. Docking
studies of these active compounds into Glo-I active site provided
the probable binding modes and thus highlighted the important
functional groups that have the potential to chelate the zinc ion of
the enzyme and/or form hydrogen bond interactions with the
polar amino acid residues within the active site as well as the
hydrophobic groups that can accommodate the deep hydrophobic
pocket of the enzyme. Lastly, the calculated toxicity profile of 9j
also supports this compound as a lead worthy of further
investigation
Step 2: Diazo coupling of the diazonium chloride solutions with
substituted aromatic carbocyclic and heterocyclic compounds:
Method A:
The solution of diazonium chloride (prepared in step 1) was
added dropwise to
a solution of a substituted aromatic
carbocyclic or heterocyclic compound (1.0 equiv.) in a saturated
aqueous solution of sodium acetate (3.0 ml) at -5 ºC. Upon
reaction completion, the mixture was warmed to room
temperature and the pH was adjusted to 7. The solid was
collected by filtration, washed with a minimum amount of
deionized H2O, air dried and purified by silica gel column
chromatography eluting with 5% MeOH in DCM to give the
desired
compounds
in
good
yields.
Method B: In this method, similar procedure as in method A was
used to synthesize the target compounds except in method B
sodium hydroxide was used as a base instead of sodium acetate.
4.1.1.1. (E)-5-((4-amino-6-hydroxynaphthalen-1-yl)diazenyl)-
2-methylbenzenesulfonamide (9a). The product was obtained as
1
a dark purple solid using method A. Yield (79%). H NMR (400
MHz, DMSO-d6): δ 8.60 (d, J = 9.0 Hz, 1H), 8.30 (d, J = 9.2 Hz,
1H), 8.25 (s, 1H), 7.90 (d, J = 8.2 Hz, 1H), 7.60-7.58 (m, 3H),
7.48 (d, J = 8.2 Hz, 1H), 7.39 (d, J = 9.0 Hz, 1H), 7.08 (d, J = 9.2
Hz, 1H), 2.61 (s, 3H). 13C NMR (DMSO-d6): δ 156.95, 143.66,
143.26, 133.95, 133.47, 128.08, 125.28, 123.36, 122.81, 120.83,
116.33, 107.32, 19.53. HRMS (ESI, m/z): calculated for
C17H17N4O3S [M+H]+ 357.1016; found 357.1013.
4. Experimental Section
4.1. Chemistry
All reagents and solvents were obtained from commercial
sources and used without further purification. Reaction’s progress
was qualitatively analyzed by thin-layer chromatography
12

4.1.1.2. (E)-5-((4-amino-8-hydroxynaphthalen-1-yl)diazenyl)-
4.1.1.7. (E)-5-((7-hydroxy-2-oxo-2H-chromen-6-yl)diazenyl)-
2-methylbenzenesulfonamide (9b). The product was obtained as
purple solid using method A. Yield (44%). H NMR (400 MHz,
2-methylbenzenesulfonamide (9g). The product was obtained as
brown solid using method B. Yield (79%). H NMR (400 MHz,
1
1
DMSO-d6): δ 12.38 (s, 1H), 8.19-8.15 (m, 2H), 7.71 (m, 2H),
7.56 (m, 4H), 7.35 (t, J = 8.0 Hz, 1H), 7.06 (d, J = 7.2 Hz, 1H),
6.82 (d, J = 8.8 Hz, 1H), 2.66 (s, 3H). 13C NMR (DMSO-d6): δ
155.75, 153.48, 150.10, 143.40, 138.38, 136.67, 133.72, 125.57,
123.47, 122.24, 120.25, 120.14, 117.09, 114.13, 113.88, 109.29,
19.76. HRMS (ESI, m/z): calculated for C17H17N4O3S [M+H]+
357.1016; found 357.1013.
DMSO-d6): δ 11.65 (s, 1H), 8.41 (s, 1H), 8.12-8.07 (m, 3H),
7.62-7.59 (m, 3H), 7.02 (s, 1H), 6.36 (d, J = 9.2 Hz, 1H), 2.69 (s,
3H).13C NMR (DMSO-d6): δ 159.68, 158.63, 157.17, 149.53,
144.67, 143.26, 139.41, 136.52, 133.49, 125.49, 121.44, 120.09,
113.33, 112.00, 104.49, 19.91. HRMS (ESI, m/z): calculated for
C16H14N3O5S [M+H]+ 360.0649; found 360.0659.
4.1.1.8. (E)-5-((8-hydroxyquinolin-5-yl)diazenyl)-2-
4.1.1.3. (E)-5-((4-aminonaphthalen-1-yl)diazenyl)-2-
methylbenzenesulfonamide (9h). The product was obtained as
brown solid using method B. Yield (92%). H NMR (400 MHz,
1
methylbenzenesulfonamide (9c). The product was obtained as
1
black solid using method B. Yield (53%). H NMR (400 MHz,
DMSO-d6): δ 9.64 (d, J = 8.4 Hz, 1H), 9.11 (m, 1H), 8.44 (s,
1H), 8.14-8.02 (m, 3H), 7.66-7.61 (m, 3H), 7.48 (d, J = 8.6 Hz,
1H), 2.69 (s, 3H). 13C NMR (DMSO-d6): δ 154.70, 150.03,
146.37, 143.40, 139.14, 138.87, 137.83, 133.49, 131.60, 128.08,
125.19, 123.63, 121.26, 116.78, 114.72, 19.92. HRMS (ESI,
m/z): calculated for C16H15N4O3S [M+H]+ 343.0859; found
343.0863.
DMSO-d6): δ 8.76 (d, J = 8.3 Hz, 1H), 8.46 (d, J = 8.0 Hz, 1H),
8.39 (d, J = 8.3 Hz, 1H), 8.32 (s, 1H), 7.97 (d, J = 8.0 Hz, 1H),
7.79 (t, J = 7.5 Hz, 1H), 7.63-7.48 (m, 4H), 7.07 (d, J = 9.2 Hz,
1H), 2.62 (s, 3H). 13C NMR (DMSO-d6): δ 151.44, 150.59,
143.18, 136.40, 136.26, 133.50, 133.28, 127.98, 124.96, 124.67,
122.84, 122.70, 121.13, 119.50, 116.20, 108.12, 19.75. HRMS
(ESI, m/z): calculated for C17H17N4O2S [M+H]+ 341.1067; found
341.1046.
4.1.1.9. (E)-2-hydroxy-5-((4-methyl-3-
sulfamoylphenyl)diazenyl)benzoic acid (9i). The product was
1
4.1.1.4.
(E)-5-((2-hydroxynaphthalen-1-yl)diazenyl)-2-
obtained as yellow solid using method B. Yield (43%). H NMR
methylbenzenesulfonamide (9d). The product was obtained as
orange solid using method B. Yield (41%). H NMR (400 MHz,
(400 MHz, DMSO-d6): δ 8.33 (d, J = 9.2 Hz, 2H), 8.10 (d, J =
8.8 Hz, 1H), 7.99 (d, J = 8.0 Hz, 1H), 7.59 (m, 3H), 7.17 (d, J =
9.2 Hz, 1H), 2.67 (s, 3H). 13C NMR (DMSO-d6): δ 171.37,
163.89, 149.69, 144.43, 143.32, 139.04, 133.56, 129.05, 126.08,
120.23, 118.59, 113.87, 19.92. HRMS (ESI, m/z): calculated for
C14H14N3O5S [M+H]+ 336.0649; found 336.0631.
1
DMSO-d6): δ 15.51 (s, 1H), 8.56 (d, J = 8.1 Hz, 1H), 8.34 (s,
1H), 7.99 (d, J = 8.9 Hz, 2H), 7.81 (d, J = 7.8 Hz, 1H), 7.66-7.63
(m, 3H), 7.55 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.3 Hz, 1H), 6.97
(d, J = 9.3 Hz, 1H), 2.65 (s, 3H). 13C NMR (DMSO-d6): δ
167.16, 143.74, 143.61, 139.89, 135.75, 133.74, 132.57, 129.38,
129.13, 128.95, 127.98, 125.96, 123.40, 122.55, 121.30, 117.13,
19.66. HRMS (ESI, m/z): calculated for C17H16N3O3S [M+H]+
342.0907; found 342.0910.
4.1.1.10. (E)-1-hydroxy-4-((4-methyl-3-
sulfamoylphenyl)diazenyl)-2-naphthoic acid (9j). The product
1
was obtained as dark red solid using method B. Yield (85%). H
NMR (400 MHz, DMSO-d6): δ 8.91 (d, J = 8.4 Hz, 1H), 8.46 (d,
J = 1.6 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.26 (s, 1H), 8.17 (d, J
= 8.0, 1.6 Hz, 1H), 7.91 (t, J = 7.6 Hz, 1H), 7.75 (t, J = 7.6 Hz,
1H), 7.63 (m, 3H), 2.70 (s, 3H). 13C NMR (DMSO-d6): δ 172.78,
163.44, 150.16, 143.39, 138.83, 138.60, 134.38, 133.50, 130.81,
126.90, 126.01, 124.53, 123.56, 122.78, 120.38, 111.95, 106.34,
19.86. HRMS (ESI, m/z): calculated for C18H16N3O5S [M+H]+
386.0805; found 386.0810.
4.1.1.5. (E)-5-((8-amino-2-hydroxynaphthalen-1-yl)diazenyl)-
2-methylbenzenesulfonamide (9e). The product was obtained as
1
dark purple solid using method B. Yield (23%). H NMR (600
MHz, DMSO-d6): δ 7.97 (s, 1H), 7.81 (dd, J = 9.6, 2.6 Hz, 1H),
7.57 (d, J = 8.1 Hz, 1H), 7.52 (s, 2H), 7.48 (dd, J = 8.2, 2.1 Hz,
1H), 7.22 (td, J = 7.9, 2.7 Hz, 1H), 7.16 (s, 2H), 7.09 (d, J = 8.1
Hz, 1H), 6.99 (d, J = 7.2 Hz, 1H), 6.65 (dd, J = 9.6, 2.6 Hz, 1H),
2.59 (s, 3H). 13C NMR (DMSO-d6): δ 181.66, 145.85, 145.01,
143.50, 140.17, 134.05, 133.83, 131.74, 129.48, 127.43, 125.50,
119.21, 118.72, 118.47, 113.88, 112.41, 19.30. HRMS (ESI,
m/z): calculated for C17H17N4O3S [M+H]+ 357.1016; found
357.1026.
4.1.1.11. (E)-2-hydroxy-3-((4-methyl-3-
sulfamoylphenyl)diazenyl)-1-naphthoic acid (9k). The product
was obtained as red solid using method B. Yield (81%). 1H NMR
(400 MHz, DMSO-d6): δ 15.51 (s, 1H), 8.58 (d, J = 8.4 Hz, 1H),
8.35 (s, 1H), 8.00 (d, J = 9.2 Hz, 2H), 7.83 (d, J = 7.6 Hz, 1H),
7.67 -7.47 (m, 6H), 6.99 (d, J = 9.2 Hz, 1H), 2.65 (s, 3H). 13C
NMR (DMSO-d6): δ 167.05, 143.72, 143.59, 139.82, 135.71,
133.67, 132.53, 129.33, 129.07, 128.90, 127.94, 125.89, 123.34,
122.53, 121.26, 117.08, 19.62. HRMS (ESI, m/z): calculated for
C18H16N3O5S [M+H]+ 386.0805; found 386.0833.
4.1.1.6. (E)-5-((4-formyl-3-hydroxynaphthalen-2-yl)diazenyl)-
2-methylbenzenesulfonamide (9f). The product was obtained as
1
red solid using method B. Yield (14%). H NMR (600 MHz,
DMSO-d6): δ 8.58 (d, J = 8.4 Hz, 1H), 8.35 (s, 1H), 8.01 (d, J =
9.0 Hz, 2H), 7.84 (d, J = 7.8 Hz, 1H), 7.67-7.64 (m, 3H), 7.56 (d,
J = 7.8 Hz, 1H), 7.50 (t, J = 7.8 Hz, 1H), 6.99 (d, J = 9.0 Hz,
1H), 2.65 (s, 3H). 13C NMR (DMSO-d6): δ 166.98, 143.75,
143.60, 139.85, 135.74, 133.69, 132.53, 129.33, 129.10, 128.94,
127.96, 125.92, 123.35, 122.62, 121.27, 117.04, 19.61. HRMS
(ESI, m/z): calculated for C18H16N3O4S [M+H]+ 370.0856; found
370.0918.
4.1.1.12. (E)-3-((4-amino-6-hydroxynaphthalen-1-
yl)diazenyl)benzenesulfonamide (9l). The product was obtained
as dark purple solid using method A. Yield (79%). 1H NMR (400
MHz, DMSO-d6): δ 8.66 (d, J = 8.8 Hz, 1H), 8.31 (m, 1H), 8.19
(s, 1H), 8.00 (d, J = 6.4 Hz, 1H), 7.70-7.66 (m, 2H), 7.60-7.54
13

(m, 3H), 7.41 (d, J = 9.2 Hz, 1H), 7.04 (d, J = 8.8 Hz, 1H). 13C
NMR (DMSO-d6): δ 156.90, 146.30, 145.48, 134.19, 130.25,
128.05, 125.30, 123.24, 122.73, 121.36, 118.16, 117.34, 116.10,
114.27, 107.74, 107.40. HRMS (ESI, m/z): calculated for
C16H15N4O3S [M+H]+ 343.0859; found 343.0896.
4.1.1.18. (E)-3-((8-hydroxyquinolin-5-
yl)diazenyl)benzenesulfonamide (9r). The product was obtained
as brown solid using method B. Yield (76%). H NMR (400
MHz, DMSO-d6): δ 9.76 (d, J = 7.6 Hz, 1H), 9.16 (m, 1H), 8.40
(s, 1H), 8.22-8.12 (m, 3H), 7.99 (m, 1H), 7.81 (m, 1H), 7.64-7.56
(m, 3H). 13C NMR (DMSO-d6): δ 155.17, 151.96, 146.31,
145.59, 139.17, 137.68, 131.50, 130.41, 128.29, 127.65, 126.23,
123.84, 118.81, 117.30, 114.98. HRMS (ESI, m/z): calculated for
C15H13N4O3S [M+H]+ 329.0703; found 329.0708.
1
4.1.1.13. (E)-3-((4-aminonaphthalen-1-
yl)diazenyl)benzenesulfonamide (9m). The product was obtained
as black solid using method B. Yield (30%). 1H NMR (400 MHz,
DMSO-d6): δ 8.82 (d, J = 8.4 Hz, 1H), 8.37 (m, 2H), 8.25 (s,
1H), 8.06 (d, J = 7.6 Hz, 1H), 7.82-7.68 (m, 3H), 7.64-7.55 (m,
3H), 7.04 (d, J = 8.8 Hz, 1H). 13C NMR (DMSO-d6): δ 159.76,
145.98, 145.49, 135.19, 133.20, 132.15, 130.24, 127.14, 125.45,
125.34, 124.48, 123.32, 122.88, 121.51, 115.99, 114.35. HRMS
(ESI, m/z): calculated for C16H15N4O2S [M+H]+ 327.0910; found
327.0904.
4.1.1.19. (E)-3-((4-hydroxynaphthalen-1-
yl)diazenyl)benzenesulfonamide (9s). The product was obtained
as orange solid using method B. Yield (33%). H NMR (400
MHz, DMSO-d6): δ 8.79 (s, 1H), 8.25- 8.18 (m, 2H), 8.03 (s,
2H), 7.81-7.73 (m, 3H), 7.62-7.56 (m, 3H), 6.95 (m, 1H). 13C
NMR (DMSO-d6): δ. 158.58, 152.64, 145.46, 139.44, 132.81,
130.33, 128.12, 126.90, 126.68, 125.65, 124.38, 122.51, 118.04,
114.63, 108.57. HRMS (ESI, m/z): calculated for C16H14N3O3S
[M+H]+ 328.0750; found 328.0744.
1
4.1.1.14. (E)-3-((2-hydroxynaphthalen-1-
yl)diazenyl)benzenesulfonamide (9n). The product was obtained
1
as orange solid using method B. Yield (40%). H NMR (400
MHz, DMSO-d6): δ 8.53 (d, J = 8.4 Hz, 1H), 8.28 (s, 1H), 8.03-
7.97 (m, 2H), 7.80-7.69 (m, 3H), 7.64 (t, J = 7.6 Hz, 1H), 7.58 (s,
2H), 7.49 (t, J = 7.6 Hz, 1H), 6.90 (d, J = 9.2 Hz, 1H). 13C NMR
(DMSO-d6): δ 171.77, 145.85, 145.08, 141.43, 132.77, 130.83,
129.95, 129.58, 129.29, 128.20, 126.67, 124.58, 124.14, 122.28,
121.67, 114.86. HRMS (ESI, m/z): calculated for C16H14N3O3S
[M+H]+ 328.0750; found 328.0757.
4.1.1.20. (E)-3-((4-
hydroxyphenyl)diazenyl)benzenesulfonamide (9t). The product
was obtained as yellowish brown solid using method B. Yield
(22%). 1H NMR (400 MHz, DMSO-d6): δ 10.46 (s, 1H), 8.21 (s,
1H), 8.05 (d, J = 7.7 Hz, 1H), 7.93 (d, J = 7.5 Hz, 1H), 7.86 (d, J
= 8.6 Hz, 2H), 7.77 (t, J = 7.8 Hz, 1H), 7.52 (s, 2H), 6.97 (d, J =
8.6 Hz, 2H). 13C NMR (DMSO-d6): δ 161.64, 152.02, 145.38,
145.13, 130.29, 127.04, 126.56, 125.28, 117.73, 116.13. HRMS
(ESI, m/z): calculated for C12H12N3O3S [M+H]+ 278.0594; found
278.0545.
4.1.1.15. (E)-3-((7-hydroxy-2-oxo-2H-chromen-6-
yl)diazenyl)benzenesulfonamide (9o). The product was obtained
1
as brown solid using method B. Yield (83%). H NMR (400
MHz, DMSO-d6): δ 11.67 (s, 1H), 8.36 (s, 1H), 8.22 (d, J = 8.0
Hz, 1H), 8.12-8.06 (m, 2H), 7.99 (d, J = 7.6 Hz, 1H), 7.81 (t, J =
8.0 Hz, 1H), 7.55 (s, 2H), 7.03 (s, 1H), 6.36 (d, J = 9.6 Hz, 1H).
13C NMR (DMSO-d6): δ 159.61, 158.90, 157.40, 151.63, 145.49,
144.65, 136.63, 130.39, 127.96, 126.78, 119.72, 118.74, 113.39,
111.97, 104.54. HRMS (ESI, m/z): calculated for C15H12N3O5S
[M+H]+ 346.0492; found 346.0506.
4.1.1.21. (E)-5-(phenyldiazenyl)quinolin-8-ol (9u). The product
was obtained as purple solid using method B. Yield (50%). H
1
NMR (400 MHz, DMSO-d6): δ 9.31 (d, J = 8.4 Hz, 1H), 8.99 (s,
1H), 8.0-7.98 (m, 3H), 7.78-7.75 (m, 1H), 7.63-7.54 (m, 3H),
7.24 (d, J = 8.4 Hz, 1H). 13C NMR (DMSO-d6): δ 157.62, 152.56,
149.01, 138.63, 137.94, 131.81, 130.77, 129.41, 127.52, 123.20,
122.48, 114.80, 111.66. HRMS (ESI, m/z): calculated for
C15H12N3O [M+H]+ 250.0975; found 250.0983.
4.1.1.16. (E)-2-hydroxy-5-((3-sulfamoylphenyl)diazenyl)benzoic
acid (9p). The product was obtained as yellow solid using
1
method B. Yield (39%). H NMR (400 MHz, DMSO-d6): δ 8.39
4.1.1.22. (E)-3-((8-hydroxyquinolin-5-yl)diazenyl)benzoic acid
(s, 1H), 8.26 (s, 1H), 8.14-8.11 (m, 2H), 7.97 (d, J = 7.2 Hz, 1H),
7.80 (t, J = 7.2 Hz, 1H), 7.54 (s, 2H), 7.19 (d, J = 8.8 Hz, 1H).
13C NMR (DMSO-d6): δ 171.24, 164.01, 151.76, 145.48, 144.39,
130.41, 129.08, 127.66, 126.78, 126.30, 118.58, 118.07, 113.86.
HRMS (ESI, m/z): calculated for C13H12N3O5S [M+H]+
322.0492; found 322.0496.
(9v). The product was obtained as orange solid using method B.
Yield (70%). H NMR (400 MHz, DMSO-d6): δ 9.29 (d, J = 8.0
1
Hz, 1H), 9.00 (s, 1H), 8.44 (s, 1H), 8.22 (d, J = 6.8 Hz, 1H),
8.10-8.03 (m, 2H), 7.81-7.71 (m, 2H), 7.25 (d, J = 8.4 Hz, 1H).
13C NMR (DMSO-d6): δ 166.85, 158.06, 152.54, 149.09, 138.61,
137.93, 132.19, 131.71, 131.11, 129.85, 127.57, 126.48, 123.41,
122.96, 115.47, 111.74. HRMS (ESI, m/z): calculated for
C16H12N3O3 [M+H]+ 294.0873; found 294.0880.
4.1.1.17. (E)-1-hydroxy-4-((3-sulfamoylphenyl)diazenyl)-2-
naphthoic acid (9q)
The product was obtained as dark red solid using method B.
Yield (80%). H NMR (400 MHz, DMSO-d6): δ 12.89 (s, 1H),
4.1.1.23. (E)-4-((8-hydroxyquinolin-5-yl)diazenyl)benzoic acid
(9w). The product was obtained as brown solid using method B.
Yield (100%). 1H NMR (400 MHz, DMSO-d6): δ 9.65 (d, J = 8.8
Hz, 1H), 9.09 (s, 1H), 8.17-8.2 (m, 6H), 7.42 (d, J = 8.8 Hz,
1H).13C NMR (DMSO-d6): δ. 166.99, 154.35, 147.20, 137.92,
137.58, 133.65, 132.15, 130.86, 128.70, 124.15, 122.38, 117.41,
115.02. HRMS (ESI, m/z): calculated for C16H12N3O3 [M+H]+
294.0873; found 294.0872.
1
8.84 (d, J = 8.8 Hz, 1H), 8.35 (d, J = 8.4 Hz, 1H), 8.23 (s, 1H),
8.10-8.0 (m, 5H), 7.85 (t, J = 7.6 Hz, 1H), 7.70 (t, J = 7.6 Hz,
1H), 7.30 (m, 1H), 6.88 (m, 1H). 13C NMR (DMSO-d6): δ
172.52, 169.08, 164.53, 153.74, 143.43, 138.40, 134.56, 130.92,
127.20, 126.91, 124.63, 123.62, 122.80, 112.70, 108.57, 106.85.
HRMS (ESI, m/z): calculated for C17H14N3O5S [M+H]+
372.0649; found 372.0261.
14

Authors are greatly thankful to the financial support from
Deanship of Research at the Jordan University of Science and
Technology (Grant No. 513/2019). The high-resolution mass
spectrometry data were acquired in the College of Arts and
Sciences and Mathematics Instrumentation Center at the
University of Toledo, 2801 W Bancroft drive, Toledo, OH, USA.
4.1.1.24. (E)-4-((8-hydroxyquinolin-5-
yl)diazenyl)benzenesulfonic acid (9x). The product was obtained
as dark red solid using method B. Yield (75%). H NMR (400
MHz, DMSO-d6): δ 9.66 (d, J = 6.4 Hz, 1H), 9.10 (s, 1H), 8.12
(d, J = 7.6 Hz, 1H), 7.99 (m, 3H), 7.82 (m, 2H), 7.40 (d, J = 7.6
Hz, 1H). HRMS (ESI, m/z): calculated for C15H12N3O4S [M+H]+
330.0543; found 330.0554.
1
4.2. In vitro enzyme inhibition assay
The Glo-I inhibitory activity of the synthesized compounds was
performed in vitro using the recombinant human Glo-I (R&D
Systems® Corporation, USA) as detailed elsewhere.29, 39 Using a
double-beam UV–Vis spectrophotometer (Biotech engineering
management Co. Ltd., UK), the percent of Glo-I inhibition for
each tested compound was measured through three independent
experiments performed in triplicate, and the average was
calculated. Calculation of the IC50 values of all compounds was
performed using GraphPad Prism 6 (2012).
4.3. In silico design
4.3.1. Compounds preparation
All synthesized compounds were sketched using ChemBioDraw
Ultra 12, imported into discovery studio (DS) and converted into
their corresponding 3D structures using "prepare ligand"
protocol. This protocol was utilized to perform many tasks
involving the assignment of proper bond orders, generation of
ionization states, as well as assignment of chemical isomers and
tautomers. Default parameters were utilized except for the pH-
based ionization, which was changed into 7.2-7.6 range.
4.3.2
Protein preparation
The crystal structure of Glo-I in complex with N-
hydroxypyridone derivative inhibitor (HPU) was retrieved from
the Protein Data Bank (PDB code 3W0T: resolution 1.35 Å) to
serve as docking template.40-41 The structural model was checked
for missing loops, alternate conformations, or incomplete
residues using Protein Report tool within DS. The Prepare
Protein protocol within DS was applied to clean the crystal
structure, correct connectivity and bond order, standardize atom
names, and protonate protein at pH of 7.4.42-43
4.3.3 Molecular Docking
Prior to molecular docking, the prepared crystal structure was
solvated and energy-minimized, using the Simulation Tools
within DS. Following minimization, water molecules were
deleted and the co-crystalized ligand (HPU) was used to define
the binding site using the Define and Edit Binding Site tool, then
it was deleted. The radius of the generated sphere was chosen to
be 10 Å radius. Subsequently, the Dock ligands (CDOCKER)
protocol was employed using default parameters.44
4.3.4 ADMET descriptors and Toxicity parameters
The ADMET descriptors were calculated using the ADMET
Descriptors protocol in DS. For toxicity parameters, the Toxicity
Prediction (TOPKAT) protocol in DS.
Acknowledgments
15

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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships that
could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as potential
competing interests:
17