Exploiting oxadiazole-sulfonamide hybrids as new structural leads to combat diabetic complications via aldose reductase inhibition

Article abstract of DOI:10.1016/j.bioorg.2020.103852

A series of oxadiazole-sulfonamide hybrids was synthesized through multistep reaction and for the formation of targeted thioethers 6(a-l), a much facile route was adopted through which S-alkylation was successfully carried out at room temperature. These novel thioethers 6(a-l) were later screened against aldehyde reductase (ALR1) and aldose reductase (ALR2). Beside the enzyme inhibition studies, the compounds were also tested against cervical cancer cell lines (HeLa). The results suggested the significant inhibition pattern towards ALR2, while few compounds were active against ALR1. The synthesized derivatives have shown weak to moderate cytotoxicity. The most potent inhibitors (6b, 6e, 6f and 6l) were selected for molecular docking studies and the binding interactions were reported.

Full text of DOI:10.1016/j.bioorg.2020.103852

Journal Pre-proofs
Exploiting oxadiazole-sulfonamide hybrids as new structural leads to combat
diabetic complications via aldose reductase inhibition
Noman Javid, Rubina Munir, Faryal Chaudhry, Aqeel Imran, Sumera Zaib,
Ayesha Muzaffar, Jamshed Iqbal
PII:
S0045-2068(19)30810-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103852
YBIOO 103852
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
20 May 2019
12 April 2020
13 April 2020
Please cite this article as: N. Javid, R. Munir, F. Chaudhry, A. Imran, S. Zaib, A. Muzaffar, J. Iqbal, Exploiting
oxadiazole-sulfonamide hybrids as new structural leads to combat diabetic complications via aldose reductase
inhibition, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103852
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier Inc.

Exploiting oxadiazole-sulfonamide hybrids as new structural leads to
combat diabetic complications via aldose reductase inhibition
Noman Javida, Rubina Munirb*, Faryal Chaudhryb, Aqeel Imranc, Sumera Zaibc,d, Ayesha
Muzaffarb, Jamshed Iqbalc*
a Institute of Chemistry, University of the Punjab, Lahore 54590, Pakistan
b Department of Chemistry, Kinnaird College for Women, Lahore 54000, Pakistan
c Centre for Advanced Drug Research, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
dDepartment of Biochemistry, Faculty of Life Sciences, University of Central Punjab, Lahore
54590, Pakistan
*Corresponding Authors:
Tel.: +92-992-383591-96; fax: +92-992-383441; e-mail: drjamshed@cuiatd.edu.pk (J. Iqbal)
Tel.: +92-99203781; e-mail: rubina.munir@kinnaird.edu.pk; organist94@gmail.com (R.
Munir)
Abstract:
A series of oxadiazole-sulfonamide hybrids was synthesized through multistep reaction and for
the formation of targeted thioethers 6(a-l), a much facile route was adopted through which S-
alkylation was successfully carried out at room temperature. These novel thioethers 6(a-l) were
later screened against aldehyde reductase (ALR1) and aldose reductase (ALR2). Beside the
enzyme inhibition studies, the compounds were also tested against cervical cancer cell lines
(HeLa). The results suggested the significant inhibition pattern towards ALR2, while few
compounds were active against ALR1. The synthesized derivatives have shown weak to
moderate cytotoxicity. The most potent inhibitors (6b, 6e, 6f and 6l) were selected for
molecular docking studies and the binding interactions were reported.
Keywords: Aldose reductase; oxadiazole-sulfonamides; cytotoxicity; docking studies.
Graphical Abstract:

N
O
N
S
O
HN
NH
O
CH3
S
O
OCH3
Compound 6e
Against ALR1: IC50 3.26 ± 0.23 M
Against ALR2: IC50 3.06 ± 0.48 M
Potent Dual Inhibitor
Introduction
Among the nitrogen/oxygen containing heterocycles, oxadiazole nucleus has become a
significant pharmacophore in the medicinal arena. Oxadiazole moiety is present in the
structures of many drugs such as nesapidil, furamizole, and raltegravir whereas some other
derivatives have also been reported as plant growth regulators [1]. Literature studies expose
that compounds bearing 1,3,4-oxadiazole moiety hold noteworthy biological and
pharmacological properties such as analgesic [2], anticancer [3], hypoglycemic [4], anti-HIV
[5] and antitubercular activities [6]. Oxadiazole derivatives have also been reported as
photosensitizers and a few of their derivatives possess liquid crystals properties [7]. On the
other hand, sulfonamides are an important class of compounds of paramount significance to
humanity. Since sulpha drugs were reported as the first anti-microbial agents, these active
molecules have turned into important class of heterocyclic compounds that find their
applications in pharmaceutical and agrochemical industries. Various types of pharmacological
agents carry sulfonamide moiety which exhibit antitumor [8], anti-carbonic anhydrase [9],
hypoglycemic [10], diuretic [11] and anti-HIV activities [12]. Most of the commercially
available antibacterial, antihypertensive and hypoglycemic drugs such as topiramate, mafenide,
and acetazolamide etc. are sulfonamides in nature [13]. The literature reveals that the
combination of oxadiazole and sulfonamide as hybrid molecules could exhibit diverse
pharmacological properties (Fig. 1) [14-17].
The hybrid structures of oxadiazole and sulfonamide were selected for aldose reductase
inhibition because both moieties had previously shown selective inhibition against ALR2 [18].
So oxadiazole and sulfonamide moieties might inhibit the enzyme synergistically. As in
previous studies, similar structure oxazolidine possess good inhibitory activities against ALR2
[19]. Other sulfonamide derivatives have also shown inhibitory potential for ALR2 [20-21].

H
O
N
N
N
N
N
S
O
N
O
S
S
H
O
O
O
O
N
N
NH2
O
N
N
N
O
Cl
N
O
O
Anticancer Drug
Antibacterial Agent
Carbonic Anhydrase Inhibitor
H3CO
N
N
H
O
N
N
N
O
O
O
Cl
S
S
O
O
N
H
O
Cl
F
Cl
Anti-inflammatory Agent
Anti-proliferative Agent
Figure 1. Biologically important oxadiazole-sulfonamide hybrids [14-17]
Aldo-keto reductases comprise an oxidoreductase superfamily having ubiquitous
distribution throughout the living organisms, most of them are NADPH dependent and are
monomeric proteins present in cytosol. The protein contains variable external loops and
conserved substrate binding region, which are responsible for the reduction of carbonyl groups
present in various compounds [22-23].
Aldose reductase AKR1B1 (ALR2; AR; EC 1.1.1.21) is responsible for the
determination of reaction rate in polyol pathway in which ALR2 converts glucose into sorbitol
alongside the parallel conversion of NADPH to NADP+ [24]. Sorbitol dehydrogenase is another
enzyme which further converts sorbitol to fructose (Fig. 2). The accumulation of sorbitol inside
the interior of the cell causes osmotic imbalance which ultimately leads to cell damage. While
passing through several intermediates, the end products of fructose metabolism are the
advanced glycation end products which lead to the production of reactive oxygen species.
Moreover, ALR2 causes NADPH depletion which results in increase in oxidative stress [25].
ALR2 is known as very critical in diabetic patients for their cardiovascular, renal, ocular system
and nervous neuropathies [25]. Moreover, AKR1B1 has been found in retina, nervous tissues,
kidney, aorta and the tissues in which diabetic complications occur. Aldehyde reductase
(ALR1, EC 1.1.1.2) is known for the detoxification of metabolites in liver [26]. The structures
of ALR1 and ALR2 have 65% sequence homology, with few differences in their respective
active sites [27].

Figure 2. Polyol pathway of glucose metabolism
ALR2 was initially reported to be involved in diabetic complications like neuropathy,
nephropathy, retinopathy and atherosclerotic plaque formation [27]. Several research studies
suggest that inhibition of ALR2 could be helpful in the prevention of complications associated
with diabetes including atherosclerosis, kidney damage, cataract, retinopathy and neuropathy.
But on the other hand, increased level of ALR2 may also induce the oxidative stress which
could lead to many inflammatory pathologies, tissues dysfunction, cell death and modified
intracellular signaling [27-29]. Several cardiac complications including myocardial ischemia,
congestive heart failure, cardiac hypertrophy, cardiomyopathy linked with increased
generation of reactive oxygen species (ROS) due to over expression of ALR2 are the reported
side effects [30].
There are several inhibitors of ALR2 identified in recent years but only few showed
enhanced therapeutic advancement and are in clinical trials, while epalrestat is the only ALR2
inhibitor available commercially in market till now. Most of the reported drugs and known
inhibitors either show toxicity in the body or have poor blood brain barrier and pharmacological
properties. Therefore, new drugs as ALR2 inhibitors with enhanced biological profile are
required. The targeted drugs to be used as enzyme inhibitors are good therapeutic design to
combat the associated diseases. The novel sulfonamide hybrids with 4-thiadiazoles and 1,3,4-
oxadiazoles act as potent antibacterial and antifungal agents [14]. Sulfonamides other than
potential carbonic anhydrase inhibitors [15] revealed antidiabetic, anti-inflammatory and
anticancer activities when substituted with 2,5-disubstituted-1,3,4-oxadiazole derivatives [16].
Moreover, antiproliferative activity of new 1,3,4-oxadiazole analogues having sulfonamide
moiety is also reported [17]. Therefore, these hybrid compounds (having oxadiazole and
sulfonamide moieties) were designed and synthesized to investigate their inhibition profile
against ALR2 and ALR1. Afterwards the toxicity of the compounds was explored against
cancer cell lines. Moreover, the selective inhibitors were docked inside the protein to justify

the experimental results so these inhibitors may be a good addition to the field of medicinal
chemistry.
Results and discussion
Chemistry
A series of oxadiazole-sulfonamide hybrids 6(a-l) have been synthesized by using the synthetic
route illustrated in Scheme 1. The designed route involved the initial synthesis of sulfonamide
(2) by the reaction of methyl anthranilate (1) and methanesulfonyl chloride. Later, hydrazide
(3) was prepared and further cyclized into corresponding oxadiazole (4). On the other side,
different chloroacylated anilines 5(a-l) were synthesized from substituted anilines and
chloroacetyl chloride. Finally, S-alkylation was carried out at room temperature in ethanol and
the new products 6(a-l) were obtained in good to excellent yields. The reaction was
successfully carried out under basic medium (KOH) through SN2 mechanistic route. The
washings with dil. HCl also helped in neutralizing KOH remains.
For structure elucidation, different spectroscopic methods were employed. In 1H NMR,
the singlet of methyl (-SO2-CH3) protons appeared around 3.10-3.20 ppm whereas another
significant singlet of methylene (-S-CH2)appeared around 4.20-4.45 ppm which confirmed the
formation of new S-CH2 linkage. Two important NH singlets also noted around 9.50-10.50
ppm in all compounds 6(a-l). Beside other 13C NMR signals, an important methyl carbon (-S-
CH3) has appeared around 37.00 ppm. Unfortunately, methylene carbon (-S-CH2) has been
masked by solvent signal, however, significant carbonyl carbon (-C=O) spotted near to 165.00
ppm in all samples. In addition, FTIR data and elemental analysis also confirmed the product
formation.

O
O
O
CH3
NH2
NH2NH2
CH3
CH3SO2Cl
O
N
O
H
NH
S
NH
S
NH2
O
O
O
O
CH3
CH3
(3)
(2)
(1)
CS2
KOH
N
N
N
N
EtOH, KOH, rt
S
O
HN
S
O
O
H
Cl
O
NH
S
NH
S
R
O
O
O
O
HN
CH3
CH3
R
(4)
6(a-l)
5(a-l)
6a. R= H
6g. R= 4-I
6b. R = 2-CH3
6c. R = 3-CH3
6d. R = 4-CH3
6e. R = 2-OCH3
6f. R = 4-OCH3
6h. R = 2-COOCH3
6i. R = 4-COOC2H5
6j. R = 2-COOH
6k. R = 4-COOH
6l. R = 4-NO2
Scheme 1. Synthesis of oxadiazole based thioethers.
Biological Activity
Enzyme Inhibition Studies
All the synthesized compounds (6a-l) were tested for their potential inhibition against two
enzymes of aldo-keto reductase family: ALR2 and partially purified ALR1. Due to the
sequence homology (65%) in the structures of both the enzymes, among the screened
compounds, some dual inhibitors have been identified. The main purpose of the study was to
identify the most potent and selective inhibitors of ALR2, as the enzyme was found to be
involved in diabetic complication, inflammatory pathologies, cardiac injuries and different
types of cancers. On the other side, the ALR1 is mainly responsible for the detoxification of
toxic aldehyde and lipids. Non-selective inhibitors of ALR2 bear some unwanted side effects.
The inhibition data obtained from the enzymatic analysis is presented in Table 1. Valproic acid
and sorbinil were taken as reference standards during the assay. The inhibitory concentration
obtained for the valproic acid was 57.4 ± 0.89 µM for the ALR1, whereas, IC50 value of sorbinil
against ALR2 was found as 3.14 ± 0.02 µM.

Table 1. The aldose and aldehyde reductase inhibition activity and percent scavenging
activity (%RSA) of synthetic derivatives 6(a-l)
Compounds
ALR1b
ALR2
%RSAc Selectivity Indexd
IC50 ± SEM (µM) / %Inhibition
6a
6b
6c
6d
6e
6f
5.84 ± 0.32
4.26 ± 0.84 18.9 ± 1.07
1.37
4.77 ± 0.47 6.38 ± 0.64% 17.5 ± 0.99
-
5.66 ± 0.87
5.13 ± 0.57
3.26 ± 0.23
3.94 ± 0.32 15.2 ± 1.21
3.69 ± 0.21 2.81 ± 0.58
3.06 ± 0.48 5.70 ± 0.43
1.43
1.39
1.06
14.3 ± 1.29% 2.21 ± 0.73 35.4 ± 1.26
-
6g
6h
6i
-
-
-
-
22.1 ± 1.98% 24.8 ± 1.87% 27.5 ± 2.11
-
-
-
-
-
6j
18.2 ± 1.05% 30.6 ± 2.33% 2.81 ± 0.22
13.4 ± 1.14% 8.60 ± 0.38% 21.2 ± 1.51
-
6k
6l
-
3.99 ± 0.45
3.56 ± 0.02 20.4 ± 1.06
1.12
Valproic acid 57.4 ± 0.89
-
-
-
-
-
Sorbinila
-
3.14 ± 0.02
-
Ascorbic Acid
81.4 ± 4.12
a: IC50 for sorbinil and valproic acid was taken as reference value; b: partially purified preparation containing
ALR1; c: %radical scavenging activity by DPPH assay; d: Defined as IC50 [ALR1]:IC50 [ALR2]. The compounds
were tested at 1 mM end concentration for ALR1, ALR2 and %RSA activity.
Structure-activity Relationship
Figure 3 represents the detailed structure-activity relationship of active scaffolds and
substituents attached. A series of thioether derivatives was synthesized and evaluated for their
ability to inhibit ALR1 and ALR2. These thioether derivatives contained phenyl ring attached
to the nitrogen atom of acetamide. Varying the nature and position of these substituents on this
phenyl ring lead to an altered inhibitory activity. Among all the synthesized compounds, ten
compounds were screened for their inhibitory potential for ALR1 and ALR2. In this regard, six
compounds were identified as potent inhibitors of these enzymes. Only one compound i.e. 6b
was identified as selective inhibitor of ALR1 with an IC50 value of 4.77 ± 0.47 µM, whereas,

compound 6f (IC50 = 2.21 ± 0.73 µM) inhibited ALR2, selectively. However, remaining
analogues showed dual inhibition of both enzymes. Valproic acid was used as standard
inhibitor for ALR1 while sorbinil was employed as standard inhibitor for ALR2
In case of ALR1, six compounds were identified as potent inhibitors with IC50 values ranging
from 3.26 ± 0.23 to 5.84 ± 0.32 µM. Whereas, % inhibition of four compounds (6f, 6h, 6j and
6k) was less than 50%. The highest activity was shown by compound 6e (IC50 = 3.26 ± 0.23
µM) containing a methoxy group at ortho position. Here position of substituent had an
important role since the introduction of this methoxy group at para position resulted in
decreased activity (<50%). However, in case of methyl substitution, change of position resulted
in the maintenance of activity. Thus, with respect to position of methyl substitution, following
order of activity was observed ortho-CH3 > meta-CH3 > para-CH3. Moreover, compound 6b
was also found to be selective inhibitor of ARL1. Likewise, incorporation of a nitro group at
para position (6l) lead to good inhibitory activity for ALR1. However, inhibitory activity of
compounds containing either carboxyl group or ester was <50%.
On the other hand, six compounds were found to be potent inhibitors of ALR2, with their IC50
values in micromolar range (2.21 ± 0.73-6.38 ± 0.64 µM). Only one compound i.e. 6f was
identified as selective inhibitor of ALR2. Here again same pattern of activity was observed as
seen in ALR1, although with slight changes. The most potent inhibitory activity was shown by
the compounds containing a methoxy group. Although para position was slightly more
favorable as compared to ortho position, as reflected by the IC50 values of 6f (IC50 = 2.21 ±
0.73 µM) and 6e (IC50 =3.06 ± 0.48 µM), respectively. Moreover, 6e and 6f are the only
compounds whose IC50 value is higher as compared to standard i.e. sorbinil (IC50 = 3.14 ± 0.02
µM). On the other hand, compounds incorporating a methyl group also had good inhibitory
activity. In this regard, meta and para position was favorable since the introduction of methyl
group at ortho position resulted in reduced activity i.e. <50%. Similarly, introduction of nitro
group also resulted in good inhibitory activity with an IC50 value of 3.56 ± 0.02 µM. The
compounds 6j and 6k did not show any promising inhibition of this enzyme.

N
O
N
S
O
N
O
N
O
N
N
HN
S
O
S
O
NH
O
CH3
S
HN
CH3
O
HN
NH
CH3
NH
O
S
O
O
H3C
S
O
CH3
CH3
6c
6d
N
O
N
6b
S
O
N
O
N
S
O
HN
NH
O
CH3
HN
NO2
H3CO
S
O
NH
O
S
O
N
O
CH3
N
6e
S
O
6l
N
N
HN
N
S
O
N
NH
O
S
O
O
O
HN
OCH3
S
O
NH
S
HN
COOH
CH3
O
CH3
NH
O
CH3
O
S
O
6a
6f
N
6k
N
S
O
N
O
N
S
O
HN
I
O
NH
HN
O
S
O
NH
CH3
O
HOOC
S
O
6g
CH3
N
O
N
N
N
S
O
S
O
O
6j
HN
COOCH3
HN
COOC2H5
NH
O
CH3
NH
S
O
O
S
O
CH3
6h
6i
Figure 3. Structure-activity relationship of synthetic compounds 6(a-l)
Surprisingly, switching to the electron-withdrawing -NO2 group, as in compound 6l, has dual
and potent inhibitory effects against both the enzymes. This compound showed significant
inhibition against ALR1 along with good inhibitory activity against ALR2. Compounds 6g
having 4-iodophenyl and 6i having 4-ethyl benzoate as substituents were not tested against any
of the enzyme. These results pointed out towards the influential effects of electron-donating
functionalities that remarkably improve the inhibition potential of molecules.

Docking studies
In order to justify the experimental results, the docking analysis was carried out against the
potent and selective inhibitors of ALR2 as well as ALR1. The in vitro results clarified that 6b
was potent and selective inhibitor of ALR1, whereas, 6f was selective as well as potent inhibitor
of ALR2. However, some dual inhibitors were found and docking analysis was carried out
against those dual inhibitors, 6e and 6l in the active site of both the receptors. To investigate
the binding poses of selected compounds, the protein structures were selected from protein
databank. In case of ALR2, the crystal structure of human ALR2 was available (1US0) and
was downloaded [31]. However, human ALR1 crystal structure was not available and porcine
ALR1 structure (3FX4) [32] was selected (as human and porcine show about 97% sequence
homology) [33-34]. Fig. 4(a,b) depicts the overlap of all the selected compounds docked inside
the active site of ALR1 and ALR2, respectively.
(b)
(a)
Figure 4. The overlap of all the docked inhibitors (6a-f and 6l) inside the active site of
aldehyde reductase (a) and aldose reductase (b)
To validate the docking studies, the co-crystallized ligands of ALR1 and ALR2 were docked
inside the active site after extraction from respective receptor. The re-docking of co-crystallized
ligands (FX4401 for 3FX4 and IDD594 for 1US0) were carried out by root mean square
deviation of 0.89 and 1.01 Å for ALR1 and ALR2, respectively. The active pockets were
selected after analysis of binding interactions of co-crystallized ligands within the active site.
The 3D interaction diagrams of co-crystallized ligand, selective inhibitor and dual inhibitors of
ALR1 were shown in Fig. 5, while the 3D interaction diagrams of co-crystallized ligand,
selective inhibitor and dual inhibitors of ALR2 were presented in Fig. 6.

The detailed analysis of ALR1 co-crystallized ligand and the inhibitors showed that Trp22 and
Tyr50 were the most important residues showing interaction with the inhibitors within the
active site. The cognate ligand presented the same interactions and our selective inhibitor, 6b
was making π-π interactions with Tyr50. In addition to π-π interactions, the hydrogen bonds
were noticed between active site amino acid residues and the compound. The dual inhibitors,
6e and 6l were making the hydrogen bonds and π-π interactions with important residues. The
interaction analysis suggested that the selected compounds showed best fit interaction inside
the pocket of ALR1. The bond distances for all the interactions presented in 3D in Fig. 5 were
shown in Fig. S1. The cognate ligand showed the bond distance of 2.62 Å with Tyr50.
However, the bond distance showed by Trp22 with the phenyl ring was 4.78 and 4.87 Å. The
selective inhibitor 6b, was found at a bond distance of 4.11 and 4.35 Å from Trp22 from 2-((5-
(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide group, while at a
distance of 2.85 Å from Tyr50. Similarly, the dual inhibitors showed the interactions with
active site residues. The 2-methoxyphenyl group of 6e was found at 5.44 Å from Tyr50 and
2.54 Å from Trp22. In the compound 6l, 4-nitrophenyl group was present at a bond distance of
5.74 Å from Try22 and 2.90 Å from Tyr50 inside the active pocket of ALR1. Moreover, the 2-
((5-(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)acetamide was found as
contributing group for 6b towards the inhibition, while, 2-methoxyphenyl group of 6e and 4-
nitrophenyl group of 6l were found to show interactions more than other atoms of the respective
compounds. The selected compounds showed the binding interactions necessary for the
inhibition of ALR1 within the active site.
(a)
(b)

(c)
(d)
Figure 5. 3D interaction diagrams of cognate ligand (FX4401) (a) and selective inhibitor (b;
6b) of ALR1 along with dual inhibitors (c: 6e; d; 6l) inside the 3FX4
When ALR2 active site residues and interaction analysis of selected compounds were
investigated, it was noted that the compounds exhibited the interactions which were reported
in the literature [35-36] and are responsible for the inhibitory behavior towards ALR2. In
addition to Typ111 and Trp20, His110 and Tyr48 are also important to show interactions and
are playing key role towards the inhibition of enzyme. The detailed bond distances of selected
compounds with all the active site residues have been provided in Fig. S2. However, the bond
distance of some of the important residues and their type of interactions are explained here.
The co-crystallized ligand showed the hydrogen bonding with Tyr48 at the bond distance of
2.73 Å. However, the selective inhibitor of ALR2, compound 6f showed the same interaction
with Tyr48 at bond distance of 3.47 Å. The dual inhibitors, 6e and 6l exhibited 4.36 and 4.03
Å, respectively, with Tyr48. When π-π interactions of Trp20 were considered, the cognate
ligand showed 4.68 and 5.26 Å bond distance, while 6e was 3.98 and 3.76 Å away from Trp20.
Similarly, compound 6l was at 4.53 and 4.80 Å from Trp20. The cognate ligand was at a bond
distance of 3.43 and 3.73 Å from Trp111 while selective compound 6f was at 2.86 Å from the
same residue in the active pocket of ALR2. The compound 6f showed 4.17 and 4.14 Å distance
from another important residue of active site, His110 from middle of the compound. The
anionic pocket/ catalytic pocket of ALR2 contain His110, Tyr48 and Trp111 [37], therefore,
upon binding of negatively charged group (thioacetamide in the case of compound 6f), flexible
adaptation has been noticed and as a result additional hydrophobic pocket was opened. In
addition to sulfonamide moiety, here the role of thioacetamide moiety was found which anchor
inside the anionic site and was responsible for selective behavior of 6f towards ALR2 as
compared to ALR1 and exhibited hydrogen bonds with the side chains. The interaction analysis

suggested that the compounds were found in best fitted poses inside the active site of ALR2
and therefore the investigated interactions are responsible for the inhibition profile of the
compounds.
(a)
(b)
(d)
(c)
Figure 6. 3D interaction diagrams of cognate ligand (IDD594) (a) and selective inhibitor (b;
6f) of ALR2 along with dual inhibitors (c: 6e; d; 6l) inside the 1US0
Molecular Dynamic Simulations
In order to justify the docking analysis, the molecular dynamic simulations were performed for
both the enzymes (ALR1 and ALR2). The structure of proteins (apo) were first subjected to
MD run of 30 ns and then docked poses of selected ligands were submitted to MD run. The
results showed that 3FX4 and the ligand attained stability shown by RMSD value for apo
(protein) and holo (Protein + ligand) structures (Figure 7). However, the RMSD plot of ALR2
showed that the trajectories of apo and holo forms showed less fluctuations after 4 ns and both
the plots were found stable afterwards (Figure 10). When root-mean-square fluctuation
(RMSF) of ALR1 and ALR2 were examined, it was observed that residues were found stable

in both the cases (Figure 8 and 11). When radius of gyration was taken into account for ALR1
and ALR2, it was observed that the range of ALR1 apo and holo protein was 1.8 to 1.9 nm
(Figure 9), however, the radius of gyration lies between 1.80 and 1.95 nm in ALR2 (Figure
12).
The results of docking studies revealed that the after binding of potent compounds inside ALR1
and ALR2, the tight complex formation made the complex stable and resulted in lowering of
the energy. Our results suggested that docked complex throughout MD trajectories exhibit
stable behavior and therefore, increasing the efficacy of docked poses and hence docking
results.
Figure 7. Root Mean Square Deviation (RMSD) of amino acid residues of 3FX4 and protein
plus selective compound (6b) during 30 ns MD-simulation run

Figure 8. Root Mean Square Fluctuation (RMSF) of amino acid residues of 3FX4 and
protein plus selective compound (6b) during 30 ns MD-simulation run
Figure 9. Radius of gyration (Rg) of amino acid residues of 3FX4 and protein plus selective
compound (6b) during 30 ns MD-simulation run
Figure 10. Root Mean Square Deviation (RMSD) of amino acid residues of 1US0 and
protein plus selective compound (6f) during 30 ns MD-simulation run

Figure 11. Root Mean Square Fluctuation (RMSF) of amino acid residues of 1US0 and
protein plus selective compound (6f) during 30 ns MD-simulation run
Figure 12. Radius of gyration (Rg) of amino acid residues of 1US0 and protein plus selective
compound (6f) during 30 ns MD-simulation run
Cytotoxic activity against HeLa cervical carcinoma cells
The cytotoxic activity of 6(a-l) against cervical carcinoma cells (HeLa) was evaluated using
MMT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [38-39]. The

percentage inhibition of all the synthetic derivatives was assessed at 100 µM (Table 2).
Cisplatin was used as a positive control and exhibited 89.3% growth inhibition at the same
concentration level. As cisplatin is known to exhibit highly cytotoxic behavior, most of the
derivatives revealed lower cytotoxic activity than cisplatin towards the HeLa cells. The
synthetic compounds exhibited %age inhibition against cancer cells in the range of 12.6 –
53.1%. Compounds 6j and 6k were found to have more cytotoxic activity than other derivatives
and both the derivatives contain carboxylic acid substituent at ortho and para positions,
respectively.
Table 2. Cytotoxic potential of oxadiazole–sulfonamide hybrids 6(a-l) against cervical
carcinoma cells HeLa at 100 µM
Codes
%age inhibition ± SEM
29.8 ± 2.31
26.4 ± 1.17
19.2 ± 3.09
21.0 ± 0.52
32.9 ± 3.26
35.8 ± 2.14
-
6a
6b
6c
6d
6e
6f
6g
6h
44.6 ± 0.98
-
6i
6j
6k
49.2 ± 3.35
53.1 ± 3.14
12.6 ± 1.05
89.3 ± 1.99
6l
Cisplatin
DPPH assay (Free radical scavenging assay)
The synthesized analogues were screened for their percent scavenging activity (%RSA)
following the previously used protocol with slight modifications [40]. The tested compounds
exhibited less percent scavenging activity (Table 1), however, the standard, ascorbic acid used

in the experiment showed 81.4% activity. All the compounds revealed less than 40%
scavenging activity. Although, the synthesized derivatives were identified as potent inhibitors
of ALR1 and ALR2, but unfortunately none of them showed antioxidant activity.
Conclusions
Aldose reductase belongs to aldo-keto super family which converts glucose into sorbitol along
the parallel conversion of NADPH to NADP+. The enzyme along with taking part in polyol
pathway is also responsible for the antioxidant role in the body. In this way, formation of free
radicals may also be targeted by inhibiting the above enzyme. Therefore, a series of compounds
was designed to synthesize different oxadiazole-sulfonamide hybrid molecules 6(a-l) through
simple and facile protocol. The synthesized products were tested against ALR1 and ALR2
enzymes. Most of the compounds exhibited good results and among them 6b, 6e, 6f and 6l
were the most prominent. Only one compound i.e. 6b was identified as selective inhibitor of
ALR1 with an IC50 value of 4.77 ± 0.47 µM, whereas, compound 6f (IC50 = 2.21 ± 0.73 µM)
inhibited ALR2, selectively. The common observation was the presence of electron-donating
groups which plausibly enhanced the activity. The docking results also helped in finding
ligand-protein interactions. Furthermore, these compounds have also shown weak to moderate
cytotoxic activity. Although sulfonamides have been reported to possess some other biological
activities, we currently have focused on the aldose reductase inhibition studies. Thus, off-target
effects of these compounds have not been evaluated and will be studied as a part of future
research.
Material & methods
Chemistry
Melting points were obtained on Gallenkamp melting point apparatus and were uncorrected.
1H NMR spectra were recorded in DMSO-d6 on Brücker Avance NMR (300 MHz) on a TMS
scale as internal standard. Chemical shifts were recorded in δ (ppm). IR spectra were recorded
on Thermo Nicolet FT-IR Spectrophotometer in KBr pellets. Elemental analyses were
performed by LECO 630-200-200 TruSpec CHNS micro analyzer and the values are within ±
0.4% of the calculated results. The initial precursors (2-5) were prepared by using the literature
reported methodologies [41-43].

General procedure for the synthesis of compounds 6(a-l)
To an ethanolic solution (10 mL) of substituted oxadiazole (4) (1 mmol, 0.27 g) was added
KOH (1 mmol, 0.05 g). The reaction mixture was stirred until KOH was dissolved followed
by the addition of acylated aniline (5a) (1 mmol, 0.17 g) at room temperature. After completion
of the reaction (monitored by TLC), the precipitates formed were filtered off, washed with
dilute HCl and then with distilled water and dried.
The similar procedure was followed to prepare other derivatives 6(b-l).
2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)-N-phenylacetamide (6a)
White crystalline solid; mp 168 ºC; Yield: 58%; IR (νmax/cm-1; neat): 3352 (N-H), 3143-2940
1
(C-H), 1691 (C=O), 1601 (C=N), 1546 (C=C), 1327, 1145 (S=O); H NMR (DMSO-d6, 300
MHz), δ: 3.20 (s, 3H; -CH3), 4.38 (s, 2H; -CH2), 7.08 (t, 1H, J = 7.4 Hz; Ar-1H), 7.26-7.35 (m,
3H; Ar-2H), 7.58-7.89 (m, 4H; Ar-4H), 7.90 (d, 1H, J = 7.3 Hz; Ar-1H), 9.95 (s, 1H; NH),
10.47 (s, 1H; NH); 13C NMR (DMSO-d6, 75 MHz), δ: 37.28, 112.34, 119.63, 119.96, 124.19,
124.42, 129.14, 129.35, 133.60, 137.00, 139.10, 163.86 (C-5 Oxd), 164.53 (C-2 Oxd), 165.25
(C=O); Anal. Calcd. For C17H16N4O4S2: 50.48; H, 3.99; N, 13.85; S, 15.86 %. Found: C, 50.18;
H, 3.78; N, 13.91; S, 15.95 %.
2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(o-tolyl)acetamide (6b)
White powder; mp 138-139 ºC; Yield: 70%; IR (νmax/cm-1; neat): 3369 (N-H), 3151-2926 (C-
1
H), 1662 (C=O), 1609 (C=N), 1555 (C=C), 1292, 1156 (S=O); H NMR (DMSO-d6, 300
MHz), δ: 2.20 (s, 3H; -CH3), 3.20 (s, 3H; -CH3), 4.40 (s, 2H; -CH2), 7.07-7.23 (m, 3H; Ar-3H),
7.28-7.35 (m, 1H; Ar-1H), 7.41 (d, 1H, J = 7.5 Hz; Ar-1H), 7.64-7.69 (m, 2H; Ar-2H), 7.93 (d,
1H, J = 7.5 Hz; Ar-1H), 9.75 (s, 1H; NH), 9.95 (s, 1H; NH); 13C NMR (DMSO-d6, 75 MHz),
δ: 18.22, 37.28, 112.42, 120.05, 124.46, 125.26, 126.01, 126.51, 129.18, 130.87, 132.12,
133.61, 136.24, 137.00, 163.87 (C-5 Oxd), 164.56 (C-2 Oxd), 165.45 (C=O); Anal. Calcd. For
C18H18N4O4S2: C, 51.66; H, 4.34; N, 13.39; S, 15.32 %. Found: C, 51.82; H, 4.31; N, 13.19; S,
15.51 %.
2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(m-tolyl)acetamide (6c)
White powder; mp 182 ºC; Yield: 66%; IR (νmax/cm-1; neat): 3353 (N-H), 3147-2927 (C-H),
1687 (C=O), 1610 (C=N), 1556 (C=C), 1343, 1148 (S=O); 1H NMR (DMSO-d6, 300 MHz), δ:
2.28 (s, 3H; -CH3), 3.20 (s, 3H; -CH3), 4.36 (s, 2H; -CH2), 6.90 (d, 1H, J = 7.5 Hz; Ar-1H),
7.20 (t, 1H, J = 7.8 Hz; Ar-1H), 7.29 (t, 1H, J = 7.3 Hz; Ar-1H), 7.37 (d, 1H, J = 8.1 Hz; Ar-
1H), 7.43 (s, 1H; Ar-1H), 7.60-7.69 (m, 2H; Ar-2H), 7.90 (d, 1H, J = 7.5 Hz; Ar-1H), 9.95 (s,

1H; NH), 10.38 (s, 1H; NH); 13C NMR (DMSO-d6, 75 MHz), δ: 21.64, 37.28, 112.31, 116.83,
119.94, 120.13, 124.41, 124.89, 129.14, 129.18, 133.61, 137.01, 138.57, 139.03, 163.86 (C-5
Oxd), 164.53 (C-2 Oxd), 165.18 (C=O); Anal. Calcd. For C18H18N4O4S2: C, 51.66; H, 4.34; N,
13.39; S, 15.32 %. Found: C, 51.41; H, 4.29; N, 13.13; S, 15.79 %.
2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(p-tolyl)acetamide (6d)
White powder; mp 210 ºC; Yield: 64%; IR (νmax/cm-1; neat): 3347 (N-H), 3126-2928 (C-H),
1657 (C=O), 1608 (C=N), 1546 (C=C), 1345, 1148 (S=O); 1H NMR (DMSO-d6, 300 MHz), δ:
2.25 (s, 3H; -CH3), 3.20 (s, 3H; -CH3), 4.37 (s, 2H; -CH2), 7.13 (d, 2H, J = 8.3 Hz; Ar-2H),
7.30 (t, 1H, J = 7.4 Hz, Ar-1H), 7.48 (d, 2H, J = 8.3 Hz; Ar-2H), 7.60-7.69 (m, 2H; Ar-2H),
7.90 (d, 1H, J = 7.8 Hz; Ar-1H), 9.95 (s, 1H; NH), 10.38 (s, 1H; NH); 13C NMR (DMSO-d6,
75 MHz), δ: 20.91, 37.27, 112.31, 119.63, 119.93, 124.41, 129.13, 129.71, 133.15, 133.59,
136.60, 137.00, 163.88 (C-5 Oxd), 164.52 (C-2 Oxd), 164.98 (C=O); Anal. Calcd. For
C18H18N4O4S2: C, 51.66; H, 4.34; N, 13.39; S, 15.32 %. Found: C, 51.61; H, 4.01; N, 13.55; S,
15.91 %.
N-(2-Methoxyphenyl)-2-((5-(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (6e)
Light brown powder; mp 211-212 ºC; Yield: 84%; IR (νmax/cm-1;neat): 3346 (N-H), 3086-2929
(C-H), 1655 (C=O), 1607 (C=N), 1550 (C=C), 1332 & 1144 (S=O), 1250, 1021 (C-O-C); 1H
NMR (DMSO-d6, 300 MHz), δ: 3.10 (s, 3H; -CH3), 3.79 (s, 3H; -OCH3), 4.26 (s, 2H; -CH2),
7.08 (t, 1H, J = 7.5 Hz; Ar-1H), 7.20-7.3 (m, 3H; Ar-3H), 7.47-7.55 (m, 3H; Ar-3H), 7.76 (d,
1H, J = 7.1 Hz; Ar-1H), 10.56 (s, 1H; NH), 11.18 (s, 1H; NH); 13C NMR (DMSO-d6, 75 MHz),
δ: 33.01, 56.37, 113.16, 119.93, 120.67, 121.20, 123.61, 129.09, 130.45, 131.26, 133.09,
138.95, 155.22, 164.70 (C-5 Oxd), 164.83 (C-2 Oxd), 171.50 (C=O); Anal. Calcd. For
C18H18N4O5S2: C, 49.76; H, 4.18; N, 12.89; S, 14.76 %. Found: C, 50.01; H, 4.03; N, 13.09; S,
14.35 %.
N-(4-Methoxyphenyl)-2-((5-(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (6f)
White powder; mp 157-158 ºC; Yield: 62%; IR (νmax/cm-1; neat): 3306 (N-H), 3043-2931 (C-
H), 1672 (C=O), 1608 (C=N), 1558 (C=C), 1323, 1110 (S=O), 1243 & 1058 (O-C-O); 1H NMR
(DMSO-d6, 300 MHz), δ: 3.11 (s, 3H; -CH3), 3.72 (s, 3H; -OCH3), 4.32 (s, 2H; -CH2), 6.89 (d,
2H, J = 8.9 Hz; Ar-2H), 7.04-7.28 (m, 2H; Ar-2H), 7.51 (d, 2H, J = 8.9 Hz; Ar-2H), 7.59-7.64
13
(m, 1H; Ar-1H), 7.83 (d, 1H, J = 8.2 Hz; Ar-1H), 9.98 (s, 1H; NH), 10.32 (s, 1H; NH); C
NMR (DMSO-d6, 75 MHz), δ: 37.20, 55.63 (-OCH3), 114.42, 119.96, 121.22, 127.20, 129.30,

132.24, 133.41, 155.96, 164.77 (C-5 Oxd); Anal. Calcd. For C18H18N4O5S2: C, 49.76; H, 4.18;
N, 12.89; S, 14.76 %. Found: C, 49.34; H, 4.55; N, 13.25; S, 14.73 %.
N-(4-Iodophenyl)-2-((5-(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamide (6g)
White powder; mp 116-118 ºC; Yield: 87%; IR (νmax/cm-1; neat): 3295 (N-H), 3040-2992 (C-
H), 1677 (C=O), 1629 (C=N), 1339, 1125 (S=O); 1H NMR (DMSO-d6, 300 MHz), δ: 3.18 (s,
3H; -CH3), 4.30 (s, 2H; -CH2), 7.25-7.31 (m, 1H; Ar-1H), 7.39-7.50 (m, 2H; Ar-2H), 7.56-7.72
(m, 4H; Ar-4H), 7.88 (d, 1H, J = 7.5 Hz; Ar-1H), 9.94 (s, 1H; NH), 10.59 (s, 1H; NH); Anal.
Calcd. For C17H15IN4O4S2: C, 38.50; H, 2.85; I, 23.93; N, 10.56; S, 12.09 %. Found: C, 38.69;
H, 2.95; N, 10.73; S, 12.26.
Methyl 2-(2-((5-(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamido)benzoate (6h)
White powder; mp 163-166 ºC; Yield: 57%; IR (νmax/cm-1; neat): 3350 (N-H), 3180-2916 (C-
H), 1709 (C=O), 1687 (C=O), 1607 (C=N), 1589 (C=C), 1320, 1157 (S=O); 1H NMR (DMSO-
d6, 300 MHz), δ: 3.20 (s, 3H; -CH3), 3.80 (s, 3H; -COOCH3), 4.42 (s, 2H; -CH2), 7.20-7.33 (m,
2H; Ar-2H), 7.61-7.69 (m, 3H; Ar-3H), 7.90-7.93 (m, 2H; Ar-2H), 8.23 (d, 1H, J = 8.1 Hz; Ar-
13
1H), 9.94 (s, 1H; NH), 11.08 (s, 1H; NH); C NMR (DMSO-d6, 75 MHz), δ: 37.12, 52.89,
112.31, 118.81, 119.98, 121.90, 124.41, 129.14, 131.07, 133.64, 134.47, 137.00, 139.26,
163.48 (C-5 Oxd), 164.69 (C-2 Oxd), 165.86 (C=O), 167.74 (C=O); Anal. Calcd. For
C19H18N4O6S2: C, 49.34; H, 3.92; N, 12.11; S, 13.87%. Found: C, 49.51; H, 3.99; N, 12.21; S,
13.65 %.
Ethyl 4-(2-((5-(2-(methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-
yl)thio)acetamido)benzoate (6i)
Light yellow powder; mp 258-260 ºC; Yield: 86%; IR (νmax/cm-1; neat): 3291 (N-H), 3050-
2991 (C-H), 1756 (C=O), 1672 (C=O), 1631 (C=N), 1342, 1117 (S=O); 1H NMR (DMSO-d6,
300 MHz), δ: 1.31 (t, 3H, J = 7.1 Hz; -CH3), 2.28 (s, 3H; -CH3), 4.22-4.39 (m, 4H; 2×CH2),
7.18-7.51 (m, 6H; Ar-6H), 7.60-7.65 (m, 2H; Ar-2H), 7.73-7.78 (m, 2H; Ar-2H), 7.90-7.95 (m,
2H; Ar-2H), 10.32 (s, 1H; NH), 10.80 (s, 1H; NH); Anal. Calcd. For C20H20N4O6S2: C, 50.41;
H, 4.23; N, 11.76; S, 13.46 %. Found: C, 50.63; H, 4.43; N, 11.89; S, 13.26 %.
2-(2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)acetamido)benzoic acid
(6j)

White powder; mp 216-217 ºC; Yield: 51%; IR (νmax/cm-1; neat): 3352-3116 (N-H & O-H),
3050-2929 (C-H), 1702 (C=O), 1672 (C=O), 1604 (C=N), 1587 (C=C), 1330, 1157 (S=O); 1H
NMR (DMSO-d6, 300 MHz), δ: 3.19 (s, 3H; -CH3), 4.39 (s, 2H; -CH2), 7.13 (t, 1H; J = 7.5 Hz;
Ar-1H), 7.28 (t, 1H; J = 7.8 Hz; Ar-1H), 7.50-7.67 (m, 3H; Ar-3H), 7.88 (d, 1H; J = 7.6 Hz;
Ar-1H), 7.98 (d, 1H; J = 7.0 Hz; Ar-1H), 8.44 (d, 1H; J = 8.3 Hz; Ar-1H), 10.06 (s, 1H; NH),
12.53 (s, 1H; NH); 13C NMR (DMSO-d6, 75 MHz), δ: 37.36, 112.45, 120.04, 120.10, 123.39,
124.39, 129.23, 131.60, 133.37, 133.56, 137.08, 140.53, 163.59 (C-5 Oxd), 164.64 (C-2 Oxd),
165.50 (C=O), 169.74 (C=O); Anal. Calcd. For C18H16N4O6S2: C, 48.21; H, 3.60; N, 12.49; S,
14.30 %. Found: C, 47.99; H, 3.81; N, 12.75; S, 14.51 %.
4-(2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)acetamido)benzoic acid
(6k)
White powder; mp 208 ºC; Yield: 75%; IR (νmax/cm-1; neat): 3486 (O-H), 3248 (N-H), 3114-
3004 (C-H), 1685 (C=O), 1665 (C=O), 1608 (C=N), 1588 (C=C), 1326, 1150 (S=O); 1H NMR
(DMSO-d6, 300 MHz), δ: 3.09 (s, 3H; -CH3), 4.41 (s, 2H; -CH2), 7.15 (t, 1H; J = 7.4 Hz; Ar-
1H), 7.52-7.62 (m, 2H; Ar-2H), 7.68 (d, 2H; J = 8.5 Hz; Ar-2H), 7.80-7.83 (m, 2H; Ar-1H,
13
NH),7.89 (d, 2H; J = 8.5 Hz; Ar-2H), 10.90 (s, 1H; NH) C NMR (DMSO-d6, 75 MHz), δ:
37.34, 112.82, 118.82, 119.99, 124.54, 129.42, 130.73, 133.28, 142.26, 163.41 (C-5 Oxd),
165.29 (C-2 Oxd), 165.84 (C=O), 167.83 (C=O); Anal. Calcd. For C18H16N4O6S2: C, 48.21; H,
3.60; N, 12.49; S, 14.30 %. Found: C, 48.56; H, 3.47; N, 12.73; S, 14.19 %.
2-((5-(2-(Methylsulfonamido)phenyl)-1,3,4-oxadiazol-2-yl)thio)-N-(4-
nitrophenyl)acetamide (6l)
White powder; mp 226 ºC; Yield: 89%; IR (νmax/cm-1; neat): 3290 (N-H), 3155-2921 (C-H),
1
1705 (C=O), 1611 (C=N), 1562 (C=C), 1335, 1115 (S=O), 1478 & 1350 (NO2); H NMR
(DMSO-d6, 300 MHz), δ: 3.19 (s, 3H; -CH3), 4.44 (s, 2H; -CH2), 7.27-7.32 (m, 1H; Ar-1H),
7.60-7.68 (m, 2H; Ar-2H), 7.84 (d, 2H, J = 9.1 Hz; Ar-2H), 7.89 (d, 1H, J = 7.9 Hz; Ar-2H),
8.25 (d, 2H, J = 9.1 Hz; Ar-2H), 9.92 (s, 1H; NH), 11.07 (s, 1H; NH); 13C NMR (DMSO-d6,
75 MHz), δ: 37.34, 112.37, 119.28, 119.43, 124.46, 125.59, 129.13, 133.62, 136.99, 143.01,
145.15, 163.71 (C-5 Oxd), 164.60 (C-2 Oxd), 166.40 (C=O); Anal. Calcd. For C17H15N5O6S2:
C, 45.43; H, 3.36; N, 15.58; S, 14.27 %. Found: C, 45.21; H, 3.63; N, 15.81; S, 14.43 %.
Biological Protocols
The details of biological protocols have been provided in the Supporting Information file.

Financial & competing interest disclosure
J.I. is thankful to the Higher Education Commission of Pakistan for the financial support
through Project No.Ph-V-MG-3/Peridot/R&D/HEC/2019. The authors have no other relevant
affiliations or financial involvement with any organization or entity with a financial interest in
or financial conflict with the subject matter or materials discussed in the manuscript apart from
those disclosed.
References
[1] G. Chawla, B. Naaz, A.A. Siddiqui, Mini-Rev. Med. Chem., 18 (2018) 216-233.
[2] A.G. Banerjee, N. Das, S.A. Shengule, R.S. Srivastava, S.K. Shrivastava, Eur. J Med.
Chem., 101 (2015) 81-95.
[3] W. Gu, X.Y. Jin, D.D. Li, S.F. Wang, X.B. Tao, H. Chen, Bioorg. Med. Chem. Lett., 27
(2017) 4128-4132.
[4] R. Bhutani, D.P. Pathak, G. Kapoor, A. Husain, R. Kant, M.A. Iqbal, Bioorg. Chem., 77
(2018) 6-15.
[5] S. Hameed, T. Akhtar, N.A. Al-Masoudi, W.A. Al-Masoudi, P.G. Jones, C. Pannecouque,
Med. Chem. Res., 25 (2016) 2399-2409.
[6] A.S. El-Azab, Y.S. Mary, A.A. Abdel-Aziz, P.B. Miniyar, S. Armaković, S.J. Armaković,
J. Mol. Struct., 1156 (2018) 657-674.
[7] A. Nas, H. Kantekin, M. Durmuş, N. Gümrükçüoğlu. J. Lumin., 154 (2014) 15-21.
[8] M. Majewsky, D. Wagner, M. Delay, S. Bräse, V. Yargeau, H. Horn, Chem. Res. Toxicol.,
27 (2014) 1821-1828.
[9] F. Niu, Y. Liu, Z. Jing, G. Han, L. Sun, L. Yan, L. Zhou, Y. Wu, Y. Xu, L. Hu, X. Zhao,
Cancer Lett., 420 (2018) 60-71.
[10] C. De Monte, S. Carradori, A. Gentili, A. Mollica, D. Trisciuoglio, C.T. Supuran, Curr.
Med. Chem., 22 (2015) 2812-2818.
[11] J.R. Diaz, G.E. Camí, M. Liu-González, D.R. Vega, D. Vullo, A. Juárez, J.C. Pedregosa,
C.T. Supuran, J. Enzyme Inhib. Med. Chem., 31 (2016) 1102-1110.
[12] S.T. Asundaria, C. Pannecouque, E. De Clercq, K.C. Patel. Pharm. Chem. J., 48 (2014)
260-268.

[13] a). B. Yulug, L. Hanoglu, T.A. Mithat, T. Cakir, O. Olmuscelik, B. Pakoz, Cent. Nerv.
Syst. Agents Med. Chem., 16 (2016) 227-230; b). C. Capasso, C.T. Supuran, J. Enzyme Inhib.
Med. Chem., 29 (2014) 379-387.
[14] P. Zoumpoulakis, C. Camoutsis, G. Pairas, M. Soković, J. Glamočlija, C. Potamitis, A.
Pitsas, Bioorg. Med. Chem., 20 (2012) 1569-1583.
[15] M. Krasavin, A. Shetnev, T. Sharonova, S. Baykov, T. Tuccinardi, S. Kalinin, A. Angeli,
C.T. Supuran, Bioorg. Chem., 76 (2018) 88-97.
[16] S. Kavitha, K. Kannan, S. Gnanavel, Saudi Pharm. J., 25 (2017) 337-345.
[17] M.M.G. El-Din, M.I. El-Gamal, M.S. Abdel-Maksoud, K.H. Yoo, C.H. Oh, Eur. J. Med.
Chem., 90 (2015) 45-52.
[18] R. Maccari, R. Ottana, Targeting Aldose Reductase for the Treatment of Diabetes
Complications and Inflammatory Diseases: New Insights and Future Directions. Journal of
Medicinal Chemistry 58 (2015) 2047−2067
[19] S. Rapposelli, F. Da Settimo, M. Digiacomo, C. La Motta, A. Lapucci, S. Sartini, M.
Vanni, Arch Pharm., 344 (2011) 372-385.
[20] P. Alexiou, V.J. Demopoulos, J. Med. Chem., 53 (2010) 7756-7766.
[21] S. Ali, A. Saeed, N. Abbas, M. Shahid, M. Bolte, J. Iqbal, Med. Chem. Comm., 3 (2012)
1428-1434.
[22] Y. Jin, T. M. Penning, Annu. Rev. Pharmacol. Toxicol., 47 (2007) 263-292.
[23] Y. Shen, L. Zhong, S. Johnson, D. Cao, Chem-Biol. Interact., 191 (2011) 192-198.
[24] I. Crespo, J. Giménez-Dejoz, S. Porté, A. Cousido-Siah, A. Mitschler, A. Podjarny, H.
Pratsinis, D. Kletsas, X. Parés, F.X. Ruiz, K. Metwally, Eur. J. Med. Chem., 152 (2018) 160-
174.
[25] P. Taslimi, H.E. Aslan, Y. Demir, N. Oztaskin, A. Maraş, İ. Gulçin, S. Beydemir, S.
Goksu, Int. J. Biol. Macromol., 119 (2018) 857-863.
[26] M.G. Salem, Y.M.A. Aziz, M. Elewa, H.A. Elshihawy, M.M. Said, Bioorg. Chem., 79
(2018) 131-144.
[27] F. Balestri, L. Quattrini, V. Coviello, S. Sartini, F. Da Settimo, M. Cappiello, R. Moschini,
A. Del Corso, U. Mura, C. La Motta, Cell Chem. Biol., 25 (2018) 1-5.
[28] A. Cousido-Siah, F.X. Ruiz, A. Mitschler, S. Porté, A.R. de Lera, M.J. Martín, S.
Manzanaro, J.A. de la Fuente, F. Terwesten, M. Betz, G. Klebe, Acta Crystallogr. D: Biol.
Crystallogr., 70 (2014) 889-903.
[29] R. Tammali, S.K. Srivastava, K.V. Ramana, Curr. Cancer Drug Targets, 11 (2011) 560-
571.

[30] B. Vyas, S. Choudhary, P.K. Singh, B. Singh, R. Bahadur, A.K. Malik, O. Silakari, Chem.
Biol. Drug Des., 92 (2018) 1981-1987.
[31] E.I. Howard, R. Sanishvili, R.E. Cachau, A. Mitschler, B. Chevrier, P. Barth, V. Lamour,
M. Van Zandt, E. Sibley, C. Bon, D. Moras, Proteins, 55 (2004) 792-804.
[32] V. Carbone, M. Giglio, R. Chung, T. Huyton, J. Adams, R. Maccari, R. Ottana, A. Hara,
O. El-Kabbani, Eur. J. Med. Chem., 45 (2010) 1140-1145.
[33] O.S.S.A.M.A. El‐Kabbani, N.C. Green, G. Lin, M. Carson, S.V.L. Narayana, K.M. Moore,
T.G. Flynn, L.J. DeLucas, Acta Crystallogr. D, 50 (1994) 859-868.
[34] V. Carbone, H.T. Zhao, R. Chung, S. Endo, A. Hara, O. El-Kabbani, Bioorg. Med. Chem.,
17 (2009) 1244-1250.
[35] A. Ibrar, Y. Tehseen, I. Khan, A. Hameed, A. Saeed, N. Furtmann, J. Bajorath, J. Iqbal,
Bioorg. Chem., 68 (2016) 177-186.
[36] A. Saeed, Y. Tehseen, H. Rafique, N. Furtmann, J. Bajorath, U. Flörke, J. Iqbal, Med.
Chem. Comm., 5 (2014) 1371-1380.
[37] S.I. Avram, L. Crisan, L.M. Pacureanu, A. Bora, E. Seclaman, M. Balint, L.G. Kurunczi.
Med Chem Res 22 (2013) 3589–3605.
[38] T. Mosmann, J. Immunol. Methods, 65 (1983) 55-63.
[39] M. Nikš, M. Otto, J. Immunol. Methods, 130 (1990) 1149-1151.
[40] T. Khan, M. Ahmad, M. Nisar, M. Ahmad, M.A. Lodhi, M.I. Choudhary, J. Enzyme Inhib.
Med. Chem., 2005, 20 (2005) 245-249.
[41] M. Shafiq, M. Zia-Ur-Rehman, I.U. Khan, M.N. Arshad, S.A. Khan, J. Chil. Chem. Soc.,
56 (2011) 527-531.
[42] S.D. Joshi, H.M. Vagdevi, V.P. Vaidya, G.S. Gadaginamath, Eur. J. Med. Chem., 2008,
43 (2008) 1989-1996.
[43] S. Xue, J. McKenna, W.C. Shieh, O. Repic, J. Org. Chem., 69 (2004) 6474-6477.