Design, synthesis and biological evaluation of selective hybrid coumarin-thiazolidinedione aldose reductase-II inhibitors as potential antidiabetics

Article abstract of DOI:10.1016/j.bioorg.2021.104970

Thiazolidinediones (TZD), benzopyrans are the proven scaffolds for inhibiting Aldose reductase (ALR2) activity and their structural confluence with the retention of necessary fragments helped in designing a series of hybrid compounds 2-(5-cycloalkylidene-2,4-dioxothiazolidin-3-yl)-N-(2-oxo-2H-chromen-3-yl)acetamide (10a-n) for better ALR2 inhibition. The compounds were synthesized by treating substituted 3-(N-bromoacetyl amino)coumarins (9a-d) with potassium salt of 5-cyclo alkylidene-1,3-thiazolidine-2,4-diones (4a-d). The inhibition activity against ALR2 with IC₅₀ values range from 0.012 ± 0.001 to 0.056 ± 0.007 μM. N-[(6-Bromo-3-coumarinyl)-2-(5-cyclopentylidene-2,4-dioxothiazolidin-3-yl)] acetamide (10c) with cyclopentylidene group on one end and the 6-bromo group on the other end showed better inhibitory property (IC₅₀ = 0.012 μM) and selectivity index (324.166) against the ALR2, a forty fold superiority over sorbinil, a better molecule over epalrestat and rest of the analogues exhibited a far superior response over sorbinil and slightly better as compared with epalrestat. It was further confirmed by the insilico studies that compound 10c showed best inhibition activity among the synthesized compounds with a high selectivity index against the ALR2. In invivo experiments, supplementation of compound 10c to STZ induced rats delayed the progression of cataract in a dose-dependent manner warranting its further development as a potential agent to treat the diabetic secondary complications especially cataract.

Full text of DOI:10.1016/j.bioorg.2021.104970

Bioorganic Chemistry 114 (2021) 104970
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and biological evaluation of selective hybrid
coumarin-thiazolidinedione aldose reductase-II inhibitors as
potential antidiabetics
,
Vijay Kumar Pasalaa *, Gopinath Gudipudia, Venu Sankeshib, Manohar Basudea,
Rambabu Gundlac, Surendar singh Jadavd, Burra Srinivasa, E. Yadaiah Gouda,
Devasani Nareshkumara
a Department of Chemistry, Osmania University, Hyderabad (T.S) 500 007, India
b Department of Biophysics, Centre for Cellular and Molecular Biology, Hyderabad (T.S) 500 007, India
c Department of Chemistry, School of Technology, GITAM University, Hyderabad (T.S) 502 102, India
d Centre for Molecular Cancer Research, Vishnu Institute of Pharmaceutical Education and Research (VIPER), Narsapur, Medak 502313, India
A R T I C L E I N F O
A B S T R A C T
Keywords:
Thiazolidinediones (TZD), benzopyrans are the proven scaffolds for inhibiting Aldose reductase (ALR2) activity
and their structural confluence with the retention of necessary fragments helped in designing a series of hybrid
compounds 2-(5-cycloalkylidene-2,4-dioxothiazolidin-3-yl)-N-(2-oxo-2H-chromen-3-yl)acetamide (10a-n) for
better ALR2 inhibition. The compounds were synthesized by treating substituted 3-(N-bromoacetyl amino)
coumarins (9a-d) with potassium salt of 5-cyclo alkylidene-1,3-thiazolidine-2,4-diones (4a-d). The inhibition
Benzopyrans
1,3-Thiazolidine-2,4-diones
Diabetic complications
Aldose reductase inhibitors
Molecular modeling
activity against ALR2 with IC50 values range from 0.012 ± 0.001 to 0.056 ± 0.007
marinyl)-2-(5-cyclopentylidene-2,4-dioxothiazolidin-3-yl)] acetamide (10c) with cyclopentylidene group on one
end and the 6-bromo group on the other end showed better inhibitory property (IC50 = 0.012 M) and selectivity
μM. N-[(6-Bromo-3-cou-
μ
index (324.166) against the ALR2, a forty fold superiority over sorbinil, a better molecule over epalrestat and rest
of the analogues exhibited a far superior response over sorbinil and slightly better as compared with epalrestat. It
was further confirmed by the insilico studies that compound 10c showed best inhibition activity among the
synthesized compounds with a high selectivity index against the ALR2. In invivo experiments, supplementation of
compound 10c to STZ induced rats delayed the progression of cataract in a dose-dependent manner warranting
its further development as a potential agent to treat the diabetic secondary complications especially cataract.
1. Introduction
composed of approximately 315–330 residues, generally form mono-
meric proteins with a molecular weight of 36 kDa [10,11] adopting an
Diabetes mellitus (DM) is one of the leading metabolic disorders with
an estimated 200 million casualities of morbidity and mortality. Under
hyperglycemic conditions more than 30% of the blood glucose is con-
verted by the aldose reductase (AR) [1] to the sorbitol causing diabetes
and secondary complications such as retinopathy, neuropathy, ne-
phropathy and cataracts [2–5]. Then sorbitol is converted to fructose by
sorbitol dehydrogenase in the polyol pathway. Hence, a better strategy
to treat the long term complications of diabetes is to inhibit the first step
of polyol pathway (AR) with molecules like sorbinil, epalrestat, fidera-
stat but these too suffer from serious side effects [6–9].
α/β-TIM barrel structure [12–14] located in the cytosol of most of the
cells but not equally distributed. The major physiological role of AR
appears to be the removal of potentially toxic aldehydes generated
during lipid peroxidation, such as 4-hydroxy-trans-2-nonenal (HNE) and
their glutathione adducts (GS-HNE), which are reduced more efficiently
than glucose by AR [14–16]. The pathogenesis of diabetic complications
in polyol pathway can be due to various reasons like sorbitol-osmotic
effects, depletion of myoinositol [17], subsequent perturbations in
Na+/K + ATPase activity [18], disturbances in cellular redox and free
radical defense, increased oxidative and glycation stress [19], activation
of PKC [20], nitric oxide (NO) mediated vascular tone [21] and
AR is a member of the AKR (Aldo Keto Reductase) super family,
* Corresponding author at: Chemistry, Natural Product Laboratory, Department of Chemistry, Osmania University, Hyderabad, Telangana 500 007, India.
E-mail address: kumar004vijay@gmail.com (V. Kumar Pasala).
https://doi.org/10.1016/j.bioorg.2021.104970
Received 23 December 2020; Received in revised form 21 March 2021; Accepted 4 May 2021
Available online 4 June 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
induction of hyperglycemic pseudo hypoxia [22]. These alterations due
to AR mediation cause vascular damage and decreased nerve conduction
velocity resulting in microvascular and macrovascular pathologies as
well as neuropathy. Under hyperglycemic condition, platelet aggrega-
tion leads to myocardial infarction, which is due to the excessive flow of
glucose through the polyol pathway and this can be arrested by using AR
inhibitors like epalrestat. (Fig. 1).
that would find enhanced binding interactions with enzyme active site.
The findings of Maccari.et.al [26,28] suggest that a free N-acetic acid
fragment on 1,3-thiazolidine-2,4-dione fragment was found as the
important moiety in enhancing the inhibition property. With this pre-
occupation, in the present work sorbinil (a closed model) and epalrestat
(an open model with N-acetic acid group) were used as templates for the
development of thought-conceived molecules. Opening the spi-
roimidazolidine of the sorbinil and the bioisosteric replacement of N-
CH2-COOH fragment of epalrestat with an amide resulted in the target
compounds 10a-n with more hydrophobic ends than the controls lead-
ing to theoretically safer agents.
Eventhough sorbinil was a failed molecule [23,24] due to its inad-
equate efficacy, the higher availability of it at the target tissue [25,26]
against diabetic retinopathy suggested that benzopyran skeleton acts as
a better carrier and hence in the present work, coumarin and 3-acet-
amide fragments were derived from benzopyran and spiro 1,3-diazole
fragments of sorbinil respectively. 3-Acetamide part is isosteric to urea
fragment of sorbinil and the strategies - simplification of the structure
and change of position - were employed to open the spiro ring at 4th
position of sorbinil to get the open chain 3-acetamide fragment and
epalrestat - a noncompetitive, reversible aldose reductase inhibitor -
available in the market [27] with 5-alkylidiene-1,3-thiazolidine-2-thi-
oxo-4-one, gave N-acetic acid fragments representing the other half of
the target scaffold (Fig. 2).
The thiazolidine-2,4-dione derivatives isosterically related to rho-
danine skeleton of epalrestat are better ALR2 inhibitors over spi-
rohydantoins like sorbinil due to the absence of hypersensitivity found
in hydantoin systems [29,30]. Introduction of an acetic acid chain on N-
3 of the thiazolidinedione moiety led to a marked increase in inhibitory
activity [31]. With free N-acetic acid fragment the corresponding mol-
ecules have very poor absorption in target tissues, while cyclic imides
and their related derivatives appear to adequately reach target tissues
but suffer from several side effects [25,26]. In spite of such liabilities
associated with these compounds, they helped in understanding the
binding interactions and accordingly developing new molecules. Indeed,
studies in animal models and in humans showed that AR inhibitors
(ARIs) can prevent or slow down the progression of certain DM- asso-
ciated disorders [32–34]. In the ALR2 the substrate-binding site is
located in a large, deep elliptical pocket at the COOH-terminus of the
beta barrel [35] with a bound NADPH in an extended conformation. The
pyrophosphate bridge of NADPH is tied down by loop B (residues
214–230) fastened by the interaction of Asp 217 with lysine residues at
23 and 263 [36] to give a binary complex which then coordinates with
the aldehyde to a ternary complex [37]. The highly hydrophobic nature
of the active site pocket greatly favors aromatic and apolar substrates
over highly polar monosaccharides and the largest measurable in-
teractions were observed between the apolar residues of the ALR2 active
site and inhibitor [35]. Hence the designed molecules (10a-n) too are
anticipated to act by the formation of ternary complexes and exhibit the
ALR2 inhibitory activity.
In the enzyme active site, the ligands bind as a ternary complex with
NADPH in the extended form. Hence an elongated molecule was
contemplated. Thus, the spiro fragment of sorbinil was opened and
retained at 3rd position of benzopyran to get the extended molecule, as a
probe for the extra binding interactions in active site along with NADPH
that helped in maintaining the geometry of the enzyme.
With this background and as part of our continued interest in the
exploration of scaffolds against ALR2 [19], hybrid molecules (10a-n)
were synthesized and tested for inhibition property. The synthesized
compounds (10a-n) were evaluated by invitro activity studies before
proceeding for the invivo studies with the most potent compound 10c of
invitro results, with further corroboration from docking studies of these
compounds (10a-n) against rat ALR2 enzyme to evaluate their affinity
and binding interaction in the active site.
2. Results and discussion
2.1. Chemistry
2.1.2. Synthesis
2.1.1. Design
The starting materials for the synthesis of acetamide conjugates of
1,3-thiazolidine-2,4-dione-3-substituted coumarins (10a-n) were po-
tassium salt of 5-cycloalkylidene-1,3-thiazolidine-2,4-diones (4a-d) and
In designing these compounds (10a-n) (Fig. 2), the intension was to
explore the selective ALR2 inhibitors with more hydrophobic groups
Fig. 1. Known Aldose reductase inhibitors.
2

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
O
NH
O
O
S
H
N
HN
O
F
N
N
X
HO
R
S
O
O
O
O
O
S
O
Sorbinil
Epalrestst
Designed analogues (10a-n)
I
N
H
I
B
Step 1
I
Sorbitol
Glucose
T
I
O
N
AR
Better treatment for diabetes
Fig. 2. Design and development of AR inhibitors.
3-N-bromoacetylaminocoumarins (9a-d). The former compounds (4a-d)
(Scheme 1) were obtained as solids in good yields via Knoevenagel
condensation of 1,3-thiazolidine-2,4-dione (1) with cycloalkanones,
namely cyclopentanone (2a), cyclohexanone (2b), 4-methyl cyclohex-
anone (2c) and 4-tetrahydrothiopyran-4-one (2d) followed by the
treatment of ethanolic solution of 5-cycloalkylidine-1,3-thiazolidine-
2,4-diones (3a-d) with ethanolic KOH at room temperature [38–40]
(Scheme 1). 3-N-Bromo acetylaminocoumarins [41] (9a-d) were syn-
thesized from 3-amino coumarins [42–43] (8a-d) which were in turn
obtained from 3-acetamido coumarins(6a-d) via N-Boc-3-amino-
coumarins (7a-d). Specifically, salicylaldehydes (5a-d) and N-acetyl
glycine were refluxed in acetic anhydride with sodium acetate to give 3-
acetamido coumarins (6a-d). Subsequently, these compounds (6a-d)
were converted to N-Boc-3-amino coumarins (7a-d) by refluxing with
Boc-anhydride in the presence of DMAP in tetrahydrofuran (Scheme 2).
N-Boc-deprotection of compounds (7a-d) with trifloroacetic acid in
dichloromethane at room temperature resulted in the 3-aminocoumar-
ins (8a-d) which reacted with bromoacetylbromide to furnish 3-N-bro-
moacetylaminocoumarins (9a-d) (Scheme 2). In the final step of the
synthesis, potassium salt of 5-cycloalkylidene-1,3-thiazolidine-2,4-dio-
nes (4a-d) were refluxed at 80 ◦C with 3-N-bromoacetylaminocoumar-
ins (9a-d) in dry.DMF in the presence of triethylamine to give 2-(5-
cycloalkylidiene-2,4-dioxothiazolidin-3-yl)-N-(3-coumarinyl)acet-
amides (10a-n) (Scheme 3).
compounds screened under invitro studies, 10c was identified as the
potent molecule whose potential was further evaluated under invivo AR
inhibition studies.
2.2.1. Invitro inhibition of ALR1 and ALR2 activity
All the synthesized compounds were tested for invitro inhibition of
ALR2 and ALR1 with Sorbinil, Epalrestat [44] and Valproic acid [45]
respectively as positive controls. Both ALR2 and ALR1 enzymes share
65% similarity of amino acid residues with few variations in their
respective active site and binding sites, which makes it difficult in
designing specific inhibitors with selectivity and no side effects [46].
The entire series of compounds (10a-n) tested against AR were found
to be good inhibitors with IC50 values between 0.012
μ
M to 0.056
μM,
falling closer to epalrestat (0.016 ± 0.002
μM) (Table 1). The compar-
ison of IC50 values for these compounds against ALR1 and ALR2 enzymes
provide a selectivity index ranging in between 324.166 and 19.285
(Table 1) with 10c (SI = 324.166) emerging as the most potent
compound.
2.2.2. Structure-Activity relationship (SAR) studies of compounds against
ALR2 activity
Among the compounds 10a-d with cyclopentylidene ring on the 5th
position of 1,3-thiazolidin-2,4-dione moiety, change of substituents at
the 6th position over benzopyran with decreasing electro negativity and
increasing + M.effect enhances the electron density in the aromatic ring
enabling them to get involved in vander walls interaction with the
phenyl ring of the tyrosine (210) residue in the enzyme active site. In
this light, 10c with 6- bromo substituent has emerged as the best com-
pound with selectivity index value 324.166. This is followed by 10b with
6-chloro substituent and 10a (unsubstituted). The poor IC50 value for
10a is due to the lack of intermolecular H bonding with the tyrosine OH
group. Then 10d with highly electronegative oxygen as OMe group at
2.2. Biological studies
All newly synthesized derivatives of 2-(5-cycloalkylidene-2,4-diox-
othiazolidin-3-yl)-N-(3-coumarinyl) acetamides (10a-n) were screened
for their inhibitory potential against purified ALR1 (aldehyde reductase)
and ALR2 (aldose reductase) enzymes extracted from rat kidney and
lenses respectively to determine their selectivity. Of the fourteen
Scheme 1. Synthesis of potassium salt of 5-cycloalkylidene-1,3-thiazolidine-2,4-diones (4a-d).
3

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
Scheme 2. Synthesis of 3-N-bromoacetylaminocoumarins (9a-d).
Scheme 3. Synthesis of 2-(5-cycloalkylidiene-2,4-dioxothiazolidin- 3-yl)-N-(3-coumarinyl) acetamides (10a-n).
8th position is poorly active due to its presence on the other side of the
molecule, far off from tyrosine OH group. In addition, the docking pose
(see the docking pose) of the 10c suggests that at the 6th position the C-X
among 10i to 10 k the IC50 values point out that a 4-methyl substitution
over cyclohexylidene ring pushes the groups at 6th position further
away from phenolic OH and demonstrates the reduced activity. Hence it
was concluded that as the size of the cycloalkylidene ring changed from
six to five with respect to a substituent on the 6th/8th position on
coumarin part, a gradual enhancement in the inhibition strength was
observed across the groups, starting from Group III → IV → II → I
(Table 1).
–
bond length gradually increases from C H to C-Cl to C-Br bond, keeping
the X group closer to phenolic OH of tyrosine allowing a possible
hydrogen bonding. Then on moving from 10e to 10 h the similar trend is
followed with slightly increased IC50 values which may be attributed to
the presence of six membered tetrahydrothiopyranylidene ring on the
5th position of 1,3-thiazolidin-2,4-dione part. This size variation from
five to six membered ring drifts the other end of the molecule
(substituted benzopyran) little further reducing the vander walls and
inter molecular H bonding interactions. Hence IC50 values from 10e to
10 h are relatively higher as compared to 10a-10d. In this series, best
compound is 10 g with 6-bromo substituent similar to 10c with a two
2.2.3. Delayed cataract progression in STZ induced rats
In order to evaluate the invivo inhibitory effect of compound 10c,
experiments were performed in rats by following earlier established
protocols [22]. Rats were divided into six groups of 12 rats each, Group-
I: normal control rats, Group-II: diabetic control rats (diabetes was
induced by intraperitoneal injection of Streptozotocin (STZ), 32 mg/kg
body weight), Group-III and Group-IV: diabetic rats were fed with 10
and 100 µM/kg body weight of compound-10c respectively. Group-V:
diabetic rats were fed with known AR inhibitor (sorbinil 100 µM/kg).
All rats (Group I-IV) were fed ad libitum and it was noticed that there was
an increase in food intake in all diabetic rats (Group II-V). Despite
increased food intake, the body weight of diabetic rats was found to be
lower (Group-II 186.01 ± 6.91 g), in relation to the control (Group-I
fold reduction in activity (IC 50 value 0.029 μM of 10 g Vs 0.012 μM of
10c). Both 10d and 10 h have 8-OMe group on the other side of the
benzopyran skeleton and increased electro negativity of oxygen reduces
the electron density in the benzopyran part resulting in the reduction in
activity. Surprisingly, from 10i to 10n, 10 l looks to be good but rela-
tively poor in comparison with 10c (three fold) and 10 g. From group II
to group IV, either doubled or significantly increasing IC50 values sug-
gest the lack of sulfur interaction of 4-thio-1-pyranylidene moiety. But
4

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
Table 1
Inhibitory Effect of Compounds 10a-n on Rat lens Aldose reductase (ALR2) and Aldehyde reductase (ALR1).
Compound
R
R’
IC50
μ
M
IC50
μ
M ALR1a
Selectivity Index (ALR1 IC50 ± SD/ALR2 IC50 ± SD)b
Basic structure Compound
ALR2a
10a
10b
10c
10d
H
H
0.028 ± 0.003
0.020 ± 0.009
0.012 ± 0.001
0.039 ± 0.002
1.06 ± 0.05
1.92 ± 0.09
3.89 ± 0.55
2.66 ± 0.19
37.857
96.0
Cl
Br
H
H
Group-I
H
324.166
68.205
OMe
10e
10f
H
H
0.023 ± 0.005
0.037 ± 0.007
0.029 ± 0.002
0.035 ± 0.003
5.80 ± 0.81
2.51 ± 0.016
1.17 ± 0.22
2.43 ± 0.075
252.173
67.837
40.344
69.428
Cl
Br
H
H
10 g
10 h
H
Group-II
Group-III
Group-IV
OMe
10i
H
H
0.043 ± 0.005
0.055 ± 0.006
0.041 ± 0.003
0.038 ± 0.007
4.08 ± 0.48
94.883
36.545
72.439
33.684
10j
Cl
Br
H
H
2.01 ± 0.064
2.97 ± 0.099
1.28 ± 0.0171
10 k
10 l
H
OMe
10 m
10n
H
H
H
0.056 ± 0.007
0.048 ± 0.001
1.08 ± 0.222
1.85 ± 0.083
19.285
38.541
Cl
Epalrestat
0.016 ± 0.002
0.47 ± 0.046
21.9 ± 0.011
0.037 ± 0.061
50.8 ± 0.38
Sorbinil C
–
ValproicacidD
–
c Reported IC50value of 0.43
μM by [35].
d Reported IC50value of 53.1 ± 0.55
μ
M [36].
a
n = 5.
b
Defined as IC50 [ALR1]&IC50[ALR2].
363.41 ± 0.33 g) and compound 10c fed rats (Group-III 302.12 ± 1.1 g
and Group-IV, 337.97 ± 0.43 g, Fig. 4D). Elevated blood glucose levels
have been attenuated by feeding with compound 10c (Group-III
136.477 ± 4.80 mg/dl, Group-IV 116.22 ± 3.56 mg/dl vs Group-II
214.022 ± 5.3 mg/dl, Fig. 4C). At the end of 8 weeks, a decrease in
AR activity in Group-III and IV rats in a dose dependent manner
(Table 2.; Fig. 4A) was observed. On extended feeding of compound 10c
to diabetic rats, a delay in the progression of cataract (Fig. 3) was
noticed due to decreased levels of sorbitol (Table 2; Fig. 4B) in STZ
induced diabetic rat lens, and it was comparable to known ARI control-
sorbinil (Group-V). The amelioration property of 10c against cataract is
evidenced by the betterment of antioxidant parametrs like Superoxide
dismutase (SOD), Catalase (CAT), Glutathione peroxidase (GPx)
(Table 2), TBARS, GSH and Protein carbonyl levels (Fig. 4E, 4F & 4G)
from the different groups of STZ induced diabetic rats.
2.3. Molecular docking studies
In order to rationalize the inhibitory concentration (IC50) and bind-
ing mode of the compounds 10a-n, molecular docking studies were
performed. These studies were carried out using as reference the known
ALR1 and ALR2 ligands [5-(3-carboxymethoxy-4-methoxybenzylidene)-
2,4-dioxothiazolidin-3-yl]acetic acid and [2-(4-Bromo-2-fluoro-ben-
zylthiocarbamoyl)-5-fluoro-phenoxy]-acetic acid, respectively. It was
observed that the invitro activity values of ligands and the docking scores
were in agreement and the data to this regard is shown in Table 3.
Table 2
Effect of compound 10c on polyol pathway and oxdative stress markers in STZ induced rats. AR activity (
μ
moles of NADPH oxidized/h/100 mg of protein), Catalase
moles of NADPH oxidized/h/100 mg of protein) and Sorbitol (nmoles/g of lens) in rat lens
analysed at the end of the experimentation.The data are the mean ± SD (n = 3).
(Units/100 mg of protein/min), SOD (Units/100 mg of protein/min), GPx (
μ
Parameter
Normal Control
Diabetic Control
Diabetic + 10c (10 µM)
Diabetic + 10c (100 µM)
Diabetic + Sorbinil(100 µM)
(Group-I, n = 12)
(Group-II, n = 12)
(Group-III, n = 12)
(Group-IV, n = 12)
(Group-V, n = 12)
Aldose reductase(ALR2)
Sorbitol level (nmoles/g lens)
Glucose level(mg/dl)
HbA1c
32.20 ± 1.32
42.19 ± 0.11
89.73 ± 0.044
4.7%(4.9 mmol/L)
9.12 ± 0.65
47.39 ± 0.03*
38.89 ± 0.06#
36.81 ± 0.06#
34.13 ± 0.027
48.32 ± 0.097
128.019 ± 1.84
6.1%(7.1 mmol/L)
8.66 ± 0.55
87.76 ± 0.013*
214.022 ± 5.3*
9.1%*(11.8 mmol/L)
5.96 ± 0.02*
54.26 ± 0.15#
52.66 ± 0.01#
136.477 ± 4.80
6.4%##(7.5 mmol/L)
7.15 ± 0.73##
116.22 ± 3.56##
5.7%#(6.4 mmol/L)
7.57 ± 0.12##
Catalase (Units/100 mg of
protein/min)
SOD (Units/100 mg of protein/
min)
5.15 ± 0.022
14.39 ± 0.43
2.79 ± 0.039*
11.55 ± 0.63*
3.21 ± 0.024##
12.03 ± 0.60##
3.73 ± 0.043##
12.66 ± 0.19##
4.08 ± 0.098
13.77 ± 0.04
GPx (μmoles of NADPH
oxidized/h/100 mg of protein)
n = numbe of live animals used in each individual group
*Statistically significant from Group-I to Group II (analyzed by ANOVA; p ≤ 0.05).
#
Statistically significant from Group-II to Group III & IV (analyzed by ANOVA; p ≤ 0.05).
##
Statistically significant from Group-V to Group III & IV (analyzed by ANOVA; p ≤ 0.05).
5

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
accommodated near to the nicotinamide nucleus of NADPH (Fig-S7-8),
where in, the alicyclic portion of 10a-n was hosted near to nicotinamide
nucleus at the ALR1 active site (Fig-S6). Further, the positioning of
coumarin aromatic ring (10a-n) in deep cavity of ALR2 active site might
be favored, responsible for making very crucial π-π interactions and
aliphatic carbonyl group in hydrogen bonding with Trp111 (Fig-S7-8).
However, this order was not present for compounds 10a-n in ALR1
active site. Eventhough other hydrogen bond interactions with Arg218,
Val300, Lys23 were present, the absence of π-π interactions might be a
major rate limiting cause for losing their selectivity. Among the ALR2
selective potent compounds, the halogenated coumarin analogues were
displayed an additional salt bridge interaction with Thr113.The chloro
substituted compound exhibited hydrogen bond with thiazolidinedione
nucleus.The bromo aryl groups linked to coumarin ring were reported to
have π-π interactions with the lipophilic cavity of the ALR2 active site
residues (25). On the other hand, the presence of supplementary linker
between the coumarin ring and arylated thiazolidinone enabled 10a-n
to accommodate in the deep cavity of ALR2 lipophilic active site.
Furthermore, the halogen or methoxy substituents on coumarin basic
ring has not affected the binding orientation of 10a-n in ALR2 pocket
(Fig. S7-8). Additionally, the predicted docking scores were in agree-
ment with experimental selectivity. Among the tested compounds, most
active ones were halogenated compounds and this might be due to salt
bridge interactions with Thr113. Compounds that bind to specificity
pocket possess much higher selectivity for ALR2 as compared with
ALR1. The selected docked conformations of compounds into the ALR1
and ALR2 binding sites are shown in Figs-S6-8. The overlap of 10c with
co-crystal ligand IDD594 displayed that the halogenated core is rela-
tively mapping with halogenated coumarin by making π-π interactions
with Trp111 (Figs. 5-9). The docking poses of 10a-n were additionally
provided in Figs- S6-9 (See supporting information).
2.3.1. Drug-like and pharmacokinetic studies
The pharmacokinetic profiling of 10a to 10n is done by insilico
techniques and presented in Table 4. In brief, the ADMET properties
(rule of five), solubility, oral absorption, serum albumin binding affin-
ities and permeability through gut blood barrier of 10a to 10n are pre-
dicted by QikProp. The pharmacokinetic predictive theoretical results
clearly stated 10a to 10n as following the rule of five which is an
essential parameter for most of drug-like compounds. Later, the phar-
macokinetic parameters of potential candidate 10c is compared with
existing ALR inhibitors, comparative analysis is made and results are
presented in Figure S10. The parameters presented in the Table 4 and
Figure S10 are obtained based on reference values mentioned in Qik-
Fig. 3. Delay in the progression of cataract in STZ induced rats. Fig- 3A & 3B:
Delayed onset progresstion of diabetic cataract by compound10c. (A) The STZ
induced rat eye lenses morphology from each group at the end of 8 weeks and
stages of cataract progression was performed using slit-lamp examination on a
weekly basis. In Group - I normal control rats, Groups-II to V all were STZ
induced rats, Group - II diabetic rats were not supplemented with any inhibitory
compounds (Diabetic control), Groups III & IV were supplemented with com-
pound 10c at a concentration of 10 µM &100 µM per kg body weight respec-
tively. In Group-V supplemented with Sorbinilat a concentration of 100 µM per
kg body weight. (B) Stage of cataract formation was scored every week and (C)
Classification cataract followed by: clear normal lens (0), peripheral vesicles
(1), peripheral vesicles and cortical opacities (2), diffuse central opacities (3)
and mature cataract (4). The data are the mean ± SD (n = 3). Cataract devel-
opment in rats was observed between 4th and 8th of the study and the number
of lenses that developed opacity against the total number of lenses was
considered as 100%, for calculating the percentage of incidence of cataract in
each group.
¨
Prop (QikProp, Schrodinger, LLC, New York, NY, 2021.). The gut perme-
ability and serum albumin binding affinity of 10c is found to be
equivalent to that of existing ALR inhibitors.
2.3.2. Docking studies protocols
¨
We conducted docking analysis using the Maestro13 Schrodinger
molecular modeling suite to verify the binding mode of the compounds
in the active site of aldose reductase (AR). The crystal structure of the
ternary complex which includes cofactor NADPH was used in this study
(PDB ID: 3FX4) [45]. The energy-minimized structure of 10c, Glucose,
Sorbinil, Epalrestat and crystal ligand (3FX4) were preliminarily docked
into ALR2 active site to examine how closely the Glide algorithm can
reproduce the experimentally determined binding conformation of
3FX4at the active site of AR. An overlay of docked 3FX4 onto the crys-
tallographic geometry resulted a root-mean-square (rms) deviation of
0.65 Å and revealed that Glide program algorithm was successful in
reproducing the binding conformation of 3FX4 to evaluate the rela-
tionship between corresponding ALR2 inhibition activities and binding
energy scores obtained from Glide docking. They fit well into the active
site pocket of enzyme occupied by 3FX4 in the X-ray crystal structure.
All the compounds primarily contain polar functional group. Those
groups anchor into the anionic binding site by forming numerous
The docking studies explained the interaction between the docked
conformations of the compounds with surrounding aminoacids of ALR1
and ALR2 (Fig-S6-9, (S = Supporting information)). In ALR2, the active
site consists of two major binding pockets, namely, anionic binding site
and specificity pocket [22]. The active site residues Tyr48, Lys77, and
His110 involved in catalytic role with the nicotinamide moiety of the
cofactor NADPH and surrounded by Trp111, Phe122, Trp219, Cys298,
Ala299, Leu300, Ser302 and Cys303 (Fig-S7-8). The validated molecular
docking protocol against both the targets ALR1 and ALR2 has been
conceded to get essential variation and selectivity with respect to bio-
logical evaluator test results.The significant binding orientation of
compounds 10a-n was obtained and upon visual inspection of docking
poses, it was noticed that the accommodation of coumarin nucleus was
entirely different in both ALR1/ALR2 active sites.The presence of aro-
matic ring of coumarin compounds 10a-n in ALR2 was observed to be
6

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
Fig. 4. Analysis of AR activity and its by products such as Sorbitol accumulation in different groups of diabetic rat lenses. Due to the delayed or inhibition of AR
activity by Compound 10c subsequently decreased the over accumulation of Sorbitol and increased the body weight in different groups of diabetic rat lens (A, B, & C).
Increasing GSH level (E), decreased TBARS (D) and Protein carbonyls levels (F) compound 10c showed significant effect on polyol pathway. Eight weeks old rat lens
was dissected and used to estimate the Sorbitol and levels in different groups. In Group I normal control rats, GroupsII to V all were STZ induced rats, Group II
diabetic rats were injected with only STZ, Groups III &IV were supplemented with compound 10c at a concentration of 10 μM &100 μM, respectively. Group V were
supplemented with known ARI, Sorbinil100 μM.
Table 3
Docking scores of compounds 10a-n against ALR1 & ALR2.
Compound
R
R’
Dock score
ALR2
Compound
R
R’
Dock score
ALR1
ALR2
ALR1
10a
H
H
ꢀ 9.177
ꢀ 8.536
ꢀ 8.262
ꢀ 8.438
ꢀ 8.693
ꢀ 8.410
ꢀ 9.301
ꢀ 7.809
ꢀ 6.809
ꢀ 8.732
ꢀ 6.000
ꢀ 5.709
ꢀ 5.641
ꢀ 5.565
ꢀ 5.736
ꢀ 6.276
ꢀ 4.718
ꢀ 6.263
ꢀ 6.343
ꢀ 6.568
10 h
10i
H
OMe
H
ꢀ 8.262
ꢀ 9.137
ꢀ 8.572
ꢀ 8.874
ꢀ 8.291
ꢀ 8.878
ꢀ 9.009
ꢀ 5.955
ꢀ 7.875
ꢀ 6.896
ꢀ 7.224
ꢀ 5.624
ꢀ 6.361
ꢀ 4.488
10b
Cl
Br
H
H
H
10c
H
10j
Cl
Br
H
H
10d
OMe
H
10 k
10 l
10 m
10n
H
10e
H
OMe
H
10f
Cl
Br
H
H
10 g
H
Cl
H
Sorbinil
Valproic acid
Epalrestat
hydrogen bonds with NADP + or the side chain of Arg312, Trp114 and
Arg309. Glide scoring function performs finest, and the produced
ranking showed good relative results with the ALR2 inhibitory activities
of the compounds. Based on the in-silico analysis, we finalized the high
scoring conformer of each above ligand based on the Glide scores
[47–50], and positioned onto the active site of enzyme.
the substituent on the coumarin part either at 6th or 8th position. Invitro
aldose reductase inhibition assay of these compounds suggested that
compound 10c with cyclopentyl ring on the 1,3-thiazolidine-2,4-dione
ring at 5th position and a bromine substituent on the 6th position of
coumarin exhibited the most potent inhibitory activity. As the size of the
cycloalkyl group increased to six, there was a steep fall in the inhibition
strength of the molecules. Molecular docking studies have further vali-
dated compound 10c as a promising, potent and selective ALR2
inhibitor.
2.4. Conclusions
In this paper we report the synthesis, aldose reductase inhibition and
docking based molecular modeling studies of a new series of acetamide
conjugates of 1, 3-thiazolidine-2,4- dione-3-substituted coumarins. The
newly synthesized derivatives differ in the size of cycloalkyl group on
1,3-thiazolidine-2,4-dione ring at 5thposition and also in the nature of
2.5. Experimental section
2.5.1. General experimental methods
Unless otherwise specified, all solvents and reagents were obtained
7

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
Fig. 5. Surface view of ALR2 (PDB:1US0) and ligand site is colored by green; (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
Fig. 6. Ribbon pose of ALR2, both NAD and 10c were represented in yellow boxes; (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)
O
O
O
O
N
ii
i
HN
K
X
HN
X
+
S
S
S
X
O
O
(4a-d)
O
(1)
(2a-d)
(3a-d)
X = nil, Cyclopentyl (a) ; -CH2,Cyclohexyl (b) ; -CH-CH3 , 4-methylcyclohexyl (c); -S-, 4-tetrahydro
thiopyran (d).
Reagents & Conditions: i) KF, dry.DMF, 80 °C, 14hrs; ii) Ethanolic KOH, RT.
Fig. 7. Overlap of 10c with co-crystal ligand (green thick sticks) and their interactions with active site residues (thin sticks);
from commercial suppliers. Solvents were purified as per the procedures
given in the ‘’Text book of practical organic chemistry’’ by Vogel (6th
Edition). All reactions were performed under nitrogen atmosphere un-
less otherwise noted. Column chromatography was performed using
Merck silica gel 60–120 mesh. Melting points were recorded on a Kofler
hot-stage apparatus and are uncorrected. TLC controls were carried out
on protecting silica gel plates (F 254 Merck). 1H NMR spectra were
recorded on Bruker spectrometer at 400 MHz spectrometer, 13C NMR
spectra were acquired on 100.6 MHz with tetramethylsilane as an in-
ternal standard, chemical shift (δ) are reported in ppm. Mass spectral
analysis was accomplished using electro spray ionization (ESI)
techniques.
acetyl glycine(11mmoles,12.8 g,1.1 eq) and sodium acetate (NaOAc,
40mmoles, i.e; 4 equivalents of NaOAc for 1 equivalent of salicylalde-
hyde) was refluxed at 110–120 ◦C for 3.5 to 4 h under nitrogen atmo-
sphere. At the end, the reaction mixture was poured into crushed ice and
stirred vigorously to give a gummy material. Extraction into ethyl ace-
tate and column chromatography with 20% ethyl acetate: 80% petro-
leum ether gave 3-N-acetyl amino coumarin.
2.5.2.1. 3-N-acetylaminocoumarin (6a). Brown colour, 65% yield,
ꢀ 1
melting point 199–200 ◦C, I.R (KBr) (cm ):1708 (C O str, NH-CO),
–
–
1
–
–
1683(C O str, C = OO, lactone), H NMR (CDCl ) (400 MHz): δ ppm:
3
8.68 (s,H-4, 1H), 8.07 (Broad peak, singlet, –NH, 1H), 7.52 (d, H-5, 1H),
7.45 (dd, H-7, 1H), 7.30 (m, H-6, H-8, 2H), 2.25 (s, –CH3, 3H), 13C NMR
(DMSO‑d6) (100.6 MHz): δ ppm: 170.6 (–NH-CO-), 157.9 (C-2), 150.0
(C-8a), 129.9 (C-3), 128.2 (C-7), 125.3 (C-5), 125.0 (C-6), 123.9 (C-4a),
2.5.2. General procedure for the synthesis of 3-acetamidocoumarins (6a-d)
A mixture of appropriate salicylaldehyde (10mmoles, 9.6 g, 1 eq), N-
8

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
O
O
O
O
OH
ii a, b
O
O
O
i
H
N
H
N
H
R
R
R
O
(7a-d)
(6a-d )
iv
(5a-d)
O
O
O
O
O
iii a, b
Br
N
H
NH2
R
R
(9a-d)
(8a-d)
(a) R = H; (b) R = 5-Cl; (c) R = 5-Br; (d) R = 3-OMe.
Reagents &Conditions: i) N-acetylglycine, NaOAc, Ac2O, 110 -120 °C, 4hrs; ii) a) (BOC)2O, DMAP, THF,
reflux, 4hrs; b) NH2NH2.H2O, CH3OH. iii) a). TFA, DCM, overnight stirring ; b). H2O ; iv) Bromoacetyl
bromide, dry.DCM, stirring, rt, 24hrs
Fig. 8. 10c showed in deep binding cavity of ALR2;
Fig. 9. 10c interactions with Trp111(thick sticks = ligands, green sticks = NADPH, thin sticks = active residues and blue dotted line =
π-
π
interactions).
QPlogKhsa
Table 4
ADMET and Pharmacokinetic properties of 10a-n.
SNo
Mol wt
HBD
HBA
QPlogPo/w
% Oral Absorption
QPlogS
QPPCaco
10a
10b
10c
10d
10e
10f
384.406
418.851
463.302
414.432
416.466
450.911
495.362
446.492
412.459
446.904
491.355
442.486
398.433
432.878
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
0.25
7.25
7.25
7.25
8
2.503
2.999
3.076
2.574
2.754
3.254
3.331
2.826
3.15
82.518
85.425
85.87
ꢀ 4.908
ꢀ 5.655
ꢀ 5.771
ꢀ 5.108
ꢀ 5.283
ꢀ 6.008
ꢀ 6.124
ꢀ 5.46
193.238
193.274
193.131
204.815
199.986
207.301
207.277
204.971
195.579
195.738
197.498
418.437
199.124
198.751
0.048
0.167
0.191
0.024
0.057
0.174
0.198
0.039
0.327
0.445
0.47
83.384
84.252
87.459
87.91
7.75
7.75
7.75
8.5
10 g
10 h
10i
84.865
86.398
89.277
89.824
93.513
84.5
7.25
7.25
7.25
8
ꢀ 5.823
ꢀ 6.567
ꢀ 6.678
ꢀ 5.467
ꢀ 5.333
ꢀ 6.081
10j
3.64
10 k
10 l
10 m
10n
3.722
3.355
2.802
3.298
0.27
7.25
7.25
0.175
0.295
87.392
HBD: Hydrogen bond donor & HBA: Hydrogen bond acceptor
120.0 (C-8), 116.2 (C-4), 24.3 δ (–CH3), DIP Mass : Molecular ion peak at
m/z 227 [M + H + Na]+.
68% yield, melting point 248–249 ◦C, I.R (KBr) (cmꢀ 1): 1730–1680
1
–
(C O str, NHC = O, OC = O), H NMR (CDCl ) (400 MHz): δ ppm: 9.82
–
3
–
(s, N H, 1H), 8.57 δ (s, H-4, 1H), 7.969–7.964 (d, H-5, 1H, J = 2.0 Hz),
7.62–7.59 (dd, H-7, 1H, J = 8.8 Hz, 2.0 Hz), 7.34–7.32 (d, H-8, 1H, J =
2.5.2.2. 6-Bromo-3-N-acetylaminocoumarin (6b). Light brown colour,
9

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
ꢀ 1
8.8 Hz), 2.17 (S, –CH3, 1H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm:
170.7 (–NH-CO), 157.5 (C-2), 149.0 (C-8a), 132.1 (C-3), 130.2 (C-7),
125.9 (C-5), 122.2 (C-4a), 118.4 (C-8), 117.1 (C-4), 24.4 (CH3), DIP
Mass: Molecular ion peak at m/z 307 [M + H + Na]+.
yield, melting point 111–112 ◦C I.R (KBr) (cm ): 3404 (N H str), 1703
–
1
–
–
–
–
(C O str, OC = O,lactone), 1633 (C O str, NHC = O), H NMR (CDCl )
3
(400 MHz): δ ppm: 8.18 (s,–NH, 1H), 7.42 (d, H-5, 1H), 7.34 (dd, H-7,
1H), 7.15 (d, H-8, 1H), 7.22 (s, H-4, 1H), 1.54 (s, 3xCH3 groups of Boc,
9H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 157.4 (C-2), 152.8 (C-
8a), 148.4 (–NH-CO-), 129.2 (C-3), 129.1 (C-6), 127.0 (C-7), 126.1 (C-
2.5.2.3. 6-Chloro-3-N-acetylaminocoumarin (6c). Brown colour, 72%
◦
ꢀ 1
yield, melting point 236–237 ◦C, I.R (KBr) (cm ): 1710 (C O str, NHC
–
–
– –
C
5), 121.7 (C-4a), 120.8 (C-8), 118.1 (C-4), 81.1 (-O
3 -carbon),
1
–
–
28.3 (3xCH3 of Boc), DIP Mass: Molecular ion peak atm/z 318 [M +
= O), 1695 (C O str, OC = O, lactone), H NMR (CDCl ) (400 MHz): δ
ppm: 9.53 (s, –NH, 1H), 8.62 (s, H-4, 1H), 7.55 (s, H-5,31H), 7.38 (dis-
torted doublet, H-7, 1H), 7.25 (distorted doublet, H-8, 1H), 2.52 (s,
–CH3, 3H, acetyl methyl), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm:
170.7 (–CO-NH), 157.5 (C-2), 148.6 (C-8a), 129.4 (C-3), 129.2 (C-6),
127.2 (C-7), 125.9 (C-5), 122.3(C-4a), 121.7 (C-8), 118.1(C-4), 24.4
(CH3 of acetyl), DIP Mass : Molecular ion peak at m/z 261 [M + H +
Na]+.
Na]+, M + 2 peak at m/z 320 [M + Na + 2]+.
2.5.3.4. 8-Methoxy-N-Boc-3-aminocoumarin (7d). Cream colour, 82%
ꢀ 1
yield, melting point 142–143 ◦C, I.R (KBr) (cm ): 3325 (N H str),
–
1
–
–
–
–
1687 (C O str, OC = O, lactone), 1631 (C O str, NHC = O), H NMR
(CDCl3) (400 MHz): δ ppm: 8.21 (s, H-4, 1H), 7.39 (s, broad peak,–NH,
1H), 7.15 (distorted triplet, H-6, 1H), 7.07 (distorted doublet, H-5, 1H),
6.91 (distorted doublet, H-7, 1H), 3.96 (s, -OMe group , 3H), 1.58 (s, Boc
group , 3xCH3 group , 9H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm:
157.6 (C-2), 152.9 (C-8), 146.7 (–NH-CO-), 139.0 (C-8a), 125.3 (C-3),
2.5.2.4. 8-Methoxy-3-N-acetylaminocoumarin (6d). Brown colour, 76%
ꢀ 1
yield, melting point 216–217 ◦C, I.R (KBr) (cm ): 1712 (C O str, NHC
–
–
1
–
–
125.31 (C-6), 122.6 (C-4a), 120.6 (C-5), 119.3 (C-4), 112.2 (C-7), 80.9
= O), 1683 (C O str, OC = O, lactone), H NMR (CDCl ) (400 MHz): δ
3
◦
– –
C
(-O
3 -carbon), 56.4 (-O-CH3 groups), 28.3 (3xCH3 groups), DIP
ppm: 8.51 (s, H-4, 1H), 8.08 (s, broad peak, –NH, 1H), 7.21 (t, H-6, 1H),
7.08 (d, H-5, 1H), 7.00 (d, H-7, 1H), 3.95 (s, -OMe, 3H), 2.25 (s, –CO-
CH3, 3H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 170.7 (–NH-CO),
157.6 (C-2), 146.7 (C-8), 139.2 (C-8a), 125.3 (C-3), 125.2 (C-6), 124.0
(C-4a), 120.6 (C-5), 119.5 (C-4), 112.4 (C-7), 56.4 (–OCH3), 24.4
(–CH3), DIP Mass : Molecular ion peak at m/z 257 [M + H + Na]+.
Mass: Molecular ion peak at m/z 314 [M + Na]+.
2.5.4. General procedure for the synthesis of 3-aminocoumarins (8a-d)
To a solution of N-Boc-3-aminocoumarin (7a-d) (10mmoles, 2.61
gm, 1 eq) in dichloromethane was added trifluroacetic acid (TFA)
(20mmoles, 15.2 ml, 2 eq) dissolved in dichloromethane drop wise at
room temperature and left it overnight for stirring. At the end, TFA was
removed under vaccum to give TFA salt to which water was added and
then extracted into ethyl acetate, combined extracts were dried,
concentrated under reduced pressure and the reaction mixture was then
column chromatographed using 38% ethylacetate: 62% petroleum ether
to give 3-aminocoumarin (8a-d).
2.5.3. General procedure for the synthesis of N-Boc- protected 3-amino-
coumarins (7a-d)
To a solution of 3-N-acetyl aminocoumarin (6a-d) (10mmoles, 2.61
g, 1 eq) in tetrahydrofuran (THF) (50 ml) was added Boc-anhydride
((Boc)2O) (20mmoles, 4.5 ml, 2 eq), dimethyl amino pyridine (DMAP,
2mmoles, 0.2 gm, 0.2 eq) and refluxed at temperature 60 ◦C for 4 h
under nitrogen atmosphere. The reaction mixture was cooled to room
temperature and volume was doubled with the addition of methanol
followed by the addition of hydrazine hydrate (40mmoles, 1.28 ml, 4 eq)
to carryout in situ deacetylation of intermediate 3-(N-acetyl-N-Boc)
amino coumarin to N-Boc-3-amino coumarin. At the end, excess of
methanol and hydrazine hydrate were removed under reduced pressure
and the reaction mixture was column chromatographed using the 10%
ethylacetate : 90% petroleum ether to give N-Boc-protected 3-aminocou-
marin (7a-d).
2.5.4.1. 3-aminocoumarins (8a). Dark brown solid, 89% yield, melting
ꢀ 1
point 137–138 ◦C, I.R (KBr) (cm ): 3427 , 3319 (N H str, NH2), 1705
–
(C O str, OC = O, lactone), 1H NMR (CDCl3) (400 MHz): δ ppm:
–
–
7.35–7.25 (m, H-5, H-7, H-8 , 3H), 7.22–7.17 (m, H-6, 1H), 6.61 δ (s, H-
4, 1H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 159.0 (C-2), 148.3 (C-
8a), 133.7 (C-3), 125.7 (C-7), 125.2 (C-5), 124.9 (C-6), 122.2 (C-4a),
115.8 (C-8), 108.1 (C-4), DIP Mass: Molecular ion peak at m/z 162 [M +
H]+.
2.5.3.1. N-Boc-3-aminocoumarin (7a).ꢀ C1ream colour, 80% yield,
2.5.4.2. 6-Bromo-3-aminocoumarin (8c). Off-white colour, 83% yield,
ꢀ 1
melting point 102–103 ◦C, I.R (KBr) (cm ): 3091 (N H str, NH), 1703
melting point 195–196 ◦C, I.R (KBr) (cm ): 3435, 3350 (N H str,
–
–
(C O str, C O, lactone), 1633(C O str, NH-C O), 1H NMR (CDCl3)
NH ), 1712 (C O str, C O, lactone), 1643 (N H bending, NH2), 1H
–
–
–
–
–
–
–
–
–
–
–
–
–
2
(400 MHz): δ ppm: 8.25 (s, broad peak, –NH, 1H), 7.46 (dd, H-7, 1H, 1H,
NMR (CDCl3) (400 MHz): δ ppm: 7.42 (d, H-5, 1H), 7.38 (distorted
doublet , H-7, 1H), 7.16 (distorted doublet , H-8, 1H), 6.60 (s, H-4 , 1H),
4.38 (broad peak, –NH2 , 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm:
158.6 (C-2), 147.2 (C-8a), 134.6 (C-3), 127.8 (C-7), 127.1 (C-5), 124.6
(C-4a), 117.9 (C-8), 116.8 (C-6), 106.2 (C-4), DIP Mass: Molecular ion
peak atm/z 240 [M + H]+, M + 2 peak at m/z 242 [M + H + 2]+.
J = 8.4 Hz, 1.6 Hz), 7.41 (m, H-5, H-8, 2H), 7.32 (s, H-4, 1H), 7.28 (dd,
H-6, 1H, J = 8 Hz, 1.2 Hz), 1.55 (s, 3xCH3 groups of tbutyl , 9H), 13
C
NMR (DMSO‑d6) (100.6 MHz): δ ppm: 157.9 (C-2), 152.9 (C-8a),149.9
(–NH-CO-), 129.7 (C-3), 128.0 (C-7), 125.3 (C-5), 125.0 (C-6), 122.6 (C-
4a), 120.0 (C-8), 116.2 (C-4), 80.9 (-O-CO-), 28.3 (3xCH3 groups), DIP
Mass (-ve mode): Molecular ion peak at m/z 260 [Mꢀ H]-.
2.5.4.3. 6-Chloro-3-aminocoumarin (8b). Off-white colour, 81% yield,
I.R (KBr) (cmꢀ 1): 3429, 3408 (N H str, NH ), 1720 (C O str, OC = O,
–
–
2.5.3.2. 6-Bromo-N-Boc-3-aminocoumarin(7b). Cream colour, 85%
–
2
ꢀ 1
yield, melting point 111–112 ◦C, I.R (KBr) (cm ): 3414 (N H str),
lactone), 1H NMR (CDCl3) (400 MHz): δ ppm: 7.12 (t, H-5, 3H), 6.85 (m,
H-7, H-8 1H), 6.67 (s, H-4, 1H), 4.25 (broad peak, –NH2, 2H), 13C NMR
(DMSO‑d6) (100.6 MHz): δ ppm: 158.6 (C-2), 147.2 (C-8a), 134.6 (C-3),
127.8 (C-7), 127.1 (C-5), 124.6 (C-4a), 117.9 (C-8), 116.8 (C-6), 106.2C-
4), DIP Mass: Molecular ion peak atm/z 196 [M + H]+, M + 2 peak at m/
z 198 [M + H + 2]+.
–
1
–
–
1722 (C O str, OC = O, lactone), H NMR (CDCl ) (400 MHz): δ ppm:
8.18 (s, –NH, 1H), 7.58 (s, H-4, 1H), 7.45 (distort3ed doublet, H-7, 1H),
7.40 (s, H-5, 1H),7.18 (distorted doublet, H-8, 1H), 1.48 (s, CH3 of Boc,
3H), 1.46 (s, 2xCH3 of Boc, 6H), 13C NMR (DMSO‑d6) (100.6 MHz): δ
ppm: 157.4 (C-2), 152.8 (C-8a), 148.8 (–NH-CO-), 131.9 (C-3), 130.0 (C-
7), 126.0 (C-5), 122.2 (C-4a), 120.8 (C-8), 118.4 (C-6), 117.1 (C-4), 81.1
(3◦-carbon of Boc), 28.3 (3xCH3 groups), DIP Mass: Molecular ion peak
atm/z 362 [M + Na]+, M + 2 peak at m/z 364 [M + Na + 2]+.
2.5.4.4. 8-Methoxy-3-aminocoumarin (8d). Off-white colour, 91%
yield, I.R (KBr) (cmꢀ 1): 3452 , 3361 (N H str, NH ), 1705 (C O str, OC
–
–
–
= O, lactone), 1H NMR (CDCl3) (400 MHz): δ ppm2: 7.96 (d, H-5, 1H, J =
10 Hz), 7.76 (d, H-7, 1H, J = 11.2 Hz), 7.48 (d, H-6, 1H, J = 9.6 Hz, 10
2.5.3.3. 6-Chloro-N-Boc-3-aminocoumarin (7c). Cream colour, 87%
10

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
Hz), 6.59 (s, H-4, 1H), 4.72 (s, -OMe group , 3H), 4.38 (broad peak,
–NH2, 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 157.0 (C-2), 146.8
(C-8), 141.0 (C-8a), 133.9 (C-3), 125.6 (C-6), 122.3 (C-4a), 120.1 (C-5),
119.5 (C-7), 114.3 (C-4), 56.5 (–OCH3), ESI Mass: quasimolecular ion
peak at m/z 192 [M + H]+.
was added KF in excess, refluxed at 80 ◦C for 14 h under nitrogen at-
mosphere. In the end, the reaction mixture was cooled to room tem-
perature, water was added (to remove KF) and the aqueous solution was
extracted with ethyl acetate, concentrated to give the crude reaction
mass which upon column chromatography using 17% ethyl acetate :
83% petroleum ether gave pure 5-cycloalkylidene-1,3-thiazol-2,4-dione
(3a-d). To the ethanolic solution of 3a-d, ethanolic KOH was added at
room temperature and stirred for half-an-hour to get the pure potassium
salt of 5-cycloalkylidene-1,3-thiazolidine-2,4-diones (4a-d).
2.5.5. General procedure for the synthesis of 3-N-
bromoacetylaminocoumarins (9 a-d)
To a solution of 3-aminocoumarin (8a-d) (10mmoles, 1.61 g, 1 eq) in
dry.dichloromethane maintained at 0 ◦C under nitrogen atmosphere for
half-an-hour was added bromoacetyl bromide (taken a little excess i.e,
upto 1.5 eq, 15mmoles, 13 ml) dissolved in dry.dichloromethane drop
wise followed by the addition of a base triethylamine (0.2 eq) and stirred
it for 24 h at room temperature. In the end, distillation of the solvent
under vaccum gave crude product followed by column chromatography
with 40% ethylacetate : 60% petroleum ether gave pure 3-N-bromoa-
cetyl aminocoumarins (9a-d).
2.5.6.1. 5-cyclopentylidene-1,3-thiazolidine-2,4-diones (4a). Off-white
colour, 60% yield, melting point 145–146 ◦C, I.R (KBr) (cmꢀ 1): 2954
3
1
–
–
(Sp C H str), 1683 (4C = O str, 2C = O str), 1639 (C C str), H NMR
–
(CDCl3) (400 MHz): δ ppm: 2.74–2.70 (m, H1-2′, H1-5′, 2H, J = 6.0 Hz),
2.15–2.12 (m, H2-2′, H2-5′, 2H, J = 7.6 Hz, 5.6 Hz), 1.67–1.59 (m, H1-3′
& H2-3′, H1-4′& H2-4′, 4H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm:
182.4 (C-4), 175.7 (C-2), 143.5 (C-1′), 128.5 (C-5), 36.8 (C-2′), 33.3 (C-
5′), 27.5 (C-3′), 25.6 (C-4′), DIP Mass: Molecular ion peak at m/z 182 [M
+ H]+.
2.5.5.1. 3-N-bromoacetyl aminocoumarin (9a). Brown colour, 84%
ꢀ 1
yield, melting point 199–200 ◦C, I.R (KBr) (cm ): 3313 (N H str, N-
–
1
–
–
–
–
HCO), 1702 (C O str, 2 OC = O), 1678 (C O str, NHC = O), H NMR
2.5.6.2. 5-cyclohexylidene-1,3-thiazolidine-2,4-diones
(4b). Pinkish
white colour, 78% yield, melting point 138–139 ◦C, I.R (KBr) (cmꢀ 1):
(CDCl3) (400 MHz): δ ppm: 9.62 (broad peak, S, –NH, 1H), 8.66 δ (S, H-
4, 1H), 7.54–7.45 (m, H-5, H-7, 2H), 7.34–7.29 (m, H-6, H-8, 2H), 4.15 δ
(S, –CH2 of bromoacetamide, 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ
ppm: 172.2 (–NH-CO-), 157.9 (C-2), 148.6 (C-8a), 129.6 (C-3), 129.3 (C-
7), 127.3 (C-5), 124.8 (C-6), 121.6 (C-4a), 121.1 (C-8), 118.2 (C-4), 61.9
(–CH2-Br), DIP Mass (-ve mode): Molecular ion peak at m/z 280 [Mꢀ H]-,
M + 2 peak at m/z 282 [Mꢀ H + 2]-.
1
–
–
3489 (N H str, imide), 1740–1700 (C O str, 4C = O, 2C = O), H NMR
–
(DMSO‑d6) (400 MHz): δ ppm: 2.84–2.79 (m, He-2′, He-6′, 2H, J = 5.6
Hz), 2.24–2.19 (m, Ha-2′, Ha-6′, 2H, J = 5.6 Hz), 1.54 (m, He-3′ &Ha-3′,
He-4′& Ha-4′, He-5′ &Ha-5′, 6H), 13C NMR (DMSO‑d6) (100.6 MHz): δ
ppm: 165.4 (C-4), 154.3 (C-2), 132.2 (C-1′), 122.8 (C-5), 29.9(C-2′),
27.9 (C-6′), 27.4 (C-3′), 27.0 (C-5′), 26.0 (C-4′), DIP Mass: Molecular ion
peak at m/z 197 [M]+.
2.5.5.2. 6-Bromo-3-N-(2-bromoacetyl) amino coumarin (9c). Brown
colour, 90% yield, melting point 201–202 ◦C, I.R (KBr) (cmꢀ 1): 3315
2.5.6.3. 5-(4-Methylcyclohexylidene)-1,3-thiazolidine-2,4-dione
(4c).
–
–
–
Off-white colour, 80% yield, melting point 133–134 ◦C, I.R (KBr)
–
(N H str, NH-CO), 1710 (C O str, OC = O, lactone), 1672 (C O str,
–
NHC = O, amide), 1H NMR (CDCl3) (400 MHz): δ ppm: 9.38 (s, –NH,
1H), 8.61 (s, H-4, 1H), 8.05 (m, H-7, 1H), 7.66 (m, H-5, 1H), 7.390 (m,
H-8, 1H), 4.079 (s, –CO-CH2, 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ
ppm: 167.0 (–NH-CO-), 157.3 (C-2), 149.3 (C-8a), 132.6 (C-3), 130.4 (C-
7), 125.5 (C-5), 123.3 (C-4a), 121.9 (C-8), 118.5 (C-6), 117.1 (C-4), 30.2
(–CH2-Br), DIP Mass: Molecular ion peak atm/z 385 [M + H + Na]+.
–
(cmꢀ 1): 3149 (N H str, imide), 1730 (C O str, 4C = O), 1672 (C
O
–
–
–
–
1
–
–
str, 2C = O), 1610 (C C str), H NMR (CDCl ) (400 MHz): δ ppm: 8.36
3
(broad peak, –NH, 1H), 4.168–4.115 (double triplet, He-2′, 1H, 2J =
17.2 Hz, 3J1 = 14.4 Hz, 3J2 = 14.0 Hz), 2.270 (q, Ha-2′, He-6′, 2H, 2J =
8.8 Hz, 3J = 4.4 Hz), 2.15 δ-2.07 (triplet doublet, Ha-6′, 1H, 2J = 14.0
Hz, 3J = 13.6 Hz, 13.2 Hz, 4.8 Hz, 4.4 Hz), 1.97–1.87 δ (m, Ha-3′ & He-3′,
2H), 1.71–1.65 (m, H-4′, 1H), 1.20–1.13 (m, Ha-5′& He-5′, 2H), 0.95 (d,
–CH3, 3H, J = 6.8 Hz), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 167.2
(C-2), 166.7 (C-4), 156.5 (C-1′), 117.6 (C-1), 36.4(C-3′), 35.5 (C-5′),
35.4 (C-2′), 31.4 (C-6′), 29.3 (C-4′), 21.6 (CH3 on C-4′), DIP Mass: Mo-
lecular ion peak at m/z 212 [M + H]+.
2.5.5.3. 6-Chloro-3-N-(2-bromoacetyl) amino coumarin (9b). Brown
colour, 82% yield, melting point 218–219 ◦C, I.R (KBr) (cmꢀ 1): 3315
–
–
–
–
(N H str, NH-CO), 1712 (C O str, OC = O, lactone), 1672 (C O str,
–
NHC = O), 1H NMR (CDCl3) (400 MHz): δ ppm: 9.31 (s, –NH, 1H), 8.62
(s, H-4, 1H), 7.50 (s, H-5, 1H), 7.41 (dd, H-7, 1H, J = 8.8 Hz), 7.28 (d, H-
8, 1H, J = 8.8 Hz), 4.08 (s, H-3′, 2H), 13C NMR (DMSO‑d6) (100.6 MHz):
δ ppm: 172.2 (–NH-CO), 157.9 (C-2), 148.6 (C-8a), 129.6 (C-3), 129.3
(C-6), 127.4 (C-7), 124.8 (C-5), 121.6 (C-4a), 121.1 (C-8), 118.3 (C-4),
61.9 (–CH2-Br), DIP Mass: Molecular ion peak at m/z 317 [M]+.
2.5.6.4. 5-(Tetrahydrothiopyran-4-ylidene) thiazolidine-2,4-dione (4d).
Brown colour, 80% yield, melting point 163–164 ◦C, I.R (KBr) (cmꢀ 1):
–
–
–
–
3415 (N H str, NH,imide), 1666 (C O str, 4C = O), 1614 (C C str, 2C
–
= O), 1H NMR (CDCl3) (400 MHz): δ ppm: 3.48 (t, H1-5′& H2-5′, 2H),
2.84 (t, H1-3′& H2-3′, 2H), 2.82 (t, H1-6′& H2-6′, 2H), 2.60 (t, H1-2′& H2-
2′, 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 182.1 (C-2), 174.5 (C-
4), 138.0 (C-4′), 132.4(C-5), 30.9 (C-5′), 30.1 (C-3′), 29.3(C-2′ & C-6′),
DIP Mass: Molecular ion peak at m/z 214 [M + H]+.
2.5.5.4. 8-Methoxy-3-N-(2-bromoacetyl) amino coumarin (9d). Brown
colour, 87% yield, melting point 173–174 ◦C, I.R (KBr) (cmꢀ 1): 3327
–
–
–
–
(N H str, N-HCO), 1708 (C O str, OC = O, lactone), 1681 (C O str, 3
–
NHC = O, amide), 1H NMR (CDCl3) (400 MHz): δ ppm: 9.28 (s, broad
peak, –NH, 1H), 8.69 (s, H-4, 1H), 7.22 (distorted triplet, H-6, 1H), 7.12
(distorted doublet, H-5, 1H), 7.08 (distorted doublet, H-7, 1H) , 4.23 (s,
–CO-CH2 –Br, 2H), 3.99 (s, -OMe, 3H), 13C NMR (DMSO‑d6) (100.6
MHz): δ ppm: 168.9 (–NH-CO-), 167.0 (C-2), 157.5 (C-8), 146.7 (C-8a),
133.0 (C-3), 125.4 (C-6), 120.4 (C-4a), 119.8 (C-5), 116.2 (C-4), 112.9
(C-7), 56.5 (–OCH3), 28.4 (–CH2-Br), DIP Mass: Molecular ion peak atm/
z 336 [M + H + Na]+, M + 2 peak at m/z 338 [M + H + Na + 2]+.
2.5.7. General procedure for the synthesis of 2-[5- cycloalkylidene ꢀ 2,4-
dioxothiazolidin-3-yl)-N-(3- coumarinyl) acetamides(10 a-n)
To
a
solution of 3-N-bromoacetyl aminocoumarins(9a-d)
(10mmoles, 2.80 g, 1 eq) and potassium salt of 5-cycloalkylidene-1,3-
thiazolidine-2,4-dione (4a-d) (12mmoles, 2.19 g, 1.2 eq) in dry.
dichloromethane, triethyl amine base (3–4 drops) was added under ni-
trogen, refluxed the mixture at 80 ◦C for 6–12 h with TLC monitoring. At
the end reaction mixture was concentrated under vaccum and the crude
was column chromatographed using 14–22% ethylacetate : 86–78%
petroleum ether to give 10 (a-n).
2.5.6. General procedure for the synthesis of potassium salt of 5-cycloal-
kylidene-1,3-thiazolidine-2,4-diones (4a-d)
To a solution of 1,3-thiazolidine-2,4-dione (10mmoles, 1.17 g, 1 eq),
cycloalkanone (15mmoles, 1.5 eq) in dry.dimethylformamide (30 ml)
11

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
2.5.7.1. 2-[5-cyclopentylidene-2,4-dioxothiazolidene-3-yl)-N-(3-coumar-
H1-3′′&H2-3′′, H1-5′′&H2-5′′, 4H), 0.85 (m, H1-4′′&H2-4′′, 2H), 13C NMR
(DMSO‑d6) (100.6 MHz): δ ppm: 172.0 (C-2′), 167.0 (–NH-CO-), 157.7
(C-2 & C-4′), 150.3 (C-1′′& C-8a), 130.4 (C-3), 128.5 (C-7), 125.4 (C-5),
124.9 (C-6), 124.6 (C-4a), 119.7 (C-8a), 116.3 (C-4 &C-5′), 30.2 (C-2′′),
29.55 (C-6′′), 29.50 (C-3′′), 29.1 (C-5′′), 27.0 (C-4′′), DIP Mass: Molecular
ion peak at m/z 399 [M + H]+.
inyl)]acetamide(10a). Creamcolour, 75% yield, melting point
ꢀ 1
216–217 ◦C, I.R (KBr) (cm ): 3327 (N H str, amide), 1730–1670
–
1
–
–
(C O str, lactone, amide & imide), H NMR (CDCl ) (400 MHz): δ ppm:
3
8.57 (s, H-4, 1H), 8.41 (broad singlet, –NH, 1H), 7.48–7.41 (m, H-5, H-7,
2H), 7.36–7.26 (m, H-6, H-8, 2H), 4.59 (s, –CO-CH2, 2H), 3.00 (m, H1-
2′′& H2-2′′, 2H, J = 6.8 Hz), 2.46–2.43 (m, H1-5′′& H2-5′′, 2H), 1.89–1.81
(m, H1-3′′& H2-3′′, H1-4′′&H2-4′′, 4H), 13C NMR (DMSO‑d6) (100.6
MHz): δ ppm: 167. 4 (C-2′), 166.2 (–NH-CO-), 163.8 (C-4′), 162.5 (C-2),
157.7 (C-1′′), 150.2 (C-8a), 130.3 (C-3), 128.4 (C-7), 125.4 (C-5), 124.8
(C-6), 124.5 (C-4a), 119.8 (C-8), 116.3 (C-4), 113.7 (C-5′), 44.1 (–CO-
CH2-N(-3◦-)), 36.5 (C-2′′), 34.9 (C-5′′), 27.1 (C-3′′), 25.4 (C-4′′), DIP
Mass: Molecular ion peak at m/z 385[M + H]+.
2.5.7.6. N-[(6-Chloro-3-coumarinyl)-2-(5-cyclohexylidene-2,4-dioxothia-
zolidin-3-yl)] Acetamide (10f). Creamcolour, 65% yield, melting point
ꢀ 1
193–194 ◦C, I.R (KBr) (cm ): 3315 (N H str, NHCO), 1712 (C O str,
–
3
–
–
1
–
OC = O, lactone), 1674 (C O str, amide, imide), H NMR (CDCl ) (400
–
MHz): δ ppm: 9.62 (broad peak, –NH, 1H), 8.64 (s, H-4, 1H), 7.50 (s, H-
5, 1H), 7.39 (m, H-7, 1H), 7.27 (m, H-8, 1H), 4.12 (s, –CO-CH2, 2H), 2.59
(m, He-2′′, 1H), 2.31 (m, He-5′′, 1H), 2.02 (m, Ha-2′′, 1H), 1.60 (m, Ha-5′′,
1H), 1.29 (m, H1-3′′& H2-3′′, H1-5′′&H2-5′′, 4H), 0.88 (m, H1-4′′&H2-4′′,
2.5.7.2. N-[(6-Cholro-3-coumarinyl)-2-(5-cyclopentylidene-2,4-dioxothia-
13
zolidin-3-yl)]acetamide (10b). Cream colour, 80% yield, melting point
2H), C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 172.2 (C-2′), 167.0
ꢀ 1
218–219 ◦C, I.R (KBr) (cm ): 3327 (N H str, NHCO), 1730–1670
(–NH-CO-CH2), 157.9 (C-4′), 157.4 (C-2), 148.9 (C-1′′), 148.6 (C-8a),
129.8 (C-3), 127.5 (C-7), 129.2 (C-6),125.6 (C-5), 124.8 (C-4a), 123.4
(C-8), 118.3 (C-4), 118.2 (C-5′), 62.0 (–CO-CH2), 31.7 (C-2′′), 30.2 (C-
6′′), 29.4 (C-3′′), 29.1 (C-5′′), 23.7 (C-4′′), DIP Mass: Molecular ion peak
at m/z 433 [M + H]+.
–
1
–
–
(C O str, lactone, amide, imide), H NMR (CDCl ) (400 MHz): δ ppm:
9.52 (s, –NH, 1H), 8.62 (s, H-4, 1H), 7.40 (s, H-53, 1H), 7.38 (distorted
doublet, H-7, 1H), 7.25 (distorted doublet, H-8, 1H), 4.65 (s, –CO-CH2
group, 2H), 3.04–2.98 (m, H1-2′′&H2-2′′, 2H), 2.52–2.42 (m, H1-5′′&H2-
5′′, 2H), 1.92–1.80 (m, H1-3′′&H2-3′′, H1-4′′&H2-4′′, 4H), 13C NMR
(DMSO‑d6) (100.6 MHz): δ ppm: 172.2 (C-2′), 167.0 (–NH-CO-), 157.9
(C-4′), 157.4 (C-2), 148.9 (C-1′′), 148.6 (C-8a), 129.8 (C-3), 129.2 (C-6),
127.5 (C-7), 125.6 (C-5), 124.8 (C-4a), 123.4 (C-8), 118.3 (C-4), 118.2
(C-5′), 62.0 (–CH2-N (3◦)), 36.5 (C-2′′), 34.9 (C-5′′), 27.1 (C-3′′), 25.4 (C-
4′′), DIP Mass (-ve mode): Molecular ion peak in the negative mode at m/
z 417 [Mꢀ H]-and M + 2 peak at m/z 419 [Mꢀ H + 2]-.
2.5.7.7. 2-[5-(4-Methylcyclohexylidene-2,4-dioxothiazolidin-3-yl)-N-(3-
coumarinyl)] acetamide (10 g). Light cream colour, 76% yield, melting
ꢀ 1
1
point 219–220 ◦C, I.R (KBr) (cm ): 1730 (C O str, 2 OC = O, lactone),
–
3
–
–
–
1681 (C O str, amide, imide), H NMR (CDCl ) (400 MHz): δ ppm: 8.65
(s, H-4, 1H), 8.33 (s, broad peak, –NH, 1H), 7.45 (d, H-5, H-8, 2H, J =
7.2 Hz), 7.34 (d, H-7, 1H, J = 8.4 Hz), 7.31 (dd, H-6, 1H, J = 8.0 Hz, 1.2
Hz), 4.55 (S, –CO-CH2, 2H), 4.21–4.16 (double triplet, He-2′′, 1H),
2.34–2.30 (dd, Ha-2′′, He-6′′, 2H, 2J = 10.8 Hz, 10.6 Hz, 3J = 4.4 Hz, 4.0
Hz), 2.16 (double of triplet, Ha-6′′, 1H, 2J = 14.4 Hz, 14.0 Hz, 3J = 4.8
Hz, 4.8 Hz, 4.4 Hz), 1.97–1.89 (m, Ha-3′′&He-3′′, 2H), 1.76–1.65 (m, H-
4′′, 1H), 1.26–1.16 (m, Ha-5′′&He-5′’, 2H), 0.95 (d, CH3-4′′, 3H, J = 6.4
2.5.7.3. N-[(6-Bromo-3-coumarinyl)-2-(5-cyclopentylidene-2,4-dioxothia-
zolidin-3-yl)] Acetamide (10c). Creamcolour, 79% yield, melting point
ꢀ 1
191–192 ◦C, I.R (KBr) (cm ): 1724–1681 (C O str, lactone, amide,
–
–
1
–
imide), 1369 (C O str, 2 OC-O, lactone), H NMR (CDCl3) (400 MHz): δ
13
ppm: 8.55 (s, H-4, 1H), 8.34 (s, broad peak, –NH, 1H), 7.57 (d, H-5, 1H),
7.55 (dd, H-7, 1H, J = 8.8 Hz, 2.4 Hz), 7.22 (d, H-8, 1H, J = 8.4 Hz), 4.55
(s, –CO-CH2, 2H), 3.00 (m, H1-2′′&H2-2′′, 2H), 2.45 (m, H1-5′′&H2-5′′,
2H), 1.86 (m, H1-3′′&H2-3′′, H1-4′′& H2-4′′, 4H), 13C NMR (DMSO‑d6)
(100.6 MHz): δ ppm: 167.4 (C-2′), 166.3 (-N-CO-C), 163.8 (C-4′), 162.6
(C-2), 157.3 (C-1′′), 149.2 (C-8a), 132.5 (C-3), 130.3 (C-7),125.5 (C-
5),123.2 (C-4a), 122.0 (C-8), 118.5 (C-6), 117.1 (C-4), 113.6 (C-5′), 44.1
(–CO-CH2-N), 36.5 (C-2′′), 35.0 (C-5′′), 27.1 (C-3′′), 25.4 (C-4′′), DIP
Mass: Molecular ion peak at m/z 463 [M + H]+ and isotopic peak at m/z
465 [M + H + 2]+.
Hz), C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 166.7 (C-2′), 166.4
(–NH-CO-),164.3 (C-1′’), 158.7 (C-4′), 157.3 (C-2), 148.8 (C-8a), 129.7
(C-3), 129.2 (C-6), 127.3 (C-7), 125.5 (C-5), 123.3 (C-4a), 121.5 (C-8),
118.2 (C-4), 115.4 (C-5′), 41.1 (3◦-N-CH2-), 36.4 (C-3′′), 35.6 (C-5′′),
35.4 (C-4′′), 31.4 (C-2′′), 29.8 (C-6′′), 21.6 (CH3-4′′), DIP Mass: Molecular
ion peak at m/z 413 [M + H]+.
2.5.7.8. N-(6-Chloro-3-coumarinyl)-2-[5-(4-methyl-cyclohexylidene)-2,4-
dioxothiazolidin-3-yl] Acetamide (10 h). Off white colour, 60% yield,
ꢀ 1
melting point 201–202 ◦C, I.R (KBr) (cm1 ): 1730 (C O str, 2 OC = O,
–
–
–
–
lactone), 1681 (C O str, amide, imide), H NMR (CDCl ) (400 MHz): δ
2.5.7.4. 2-[5-Cyclopentylidene-2,4-dioxothiazolidin-3-yl)-N-(8-methoxy-
ppm:8.57 (s, H-4, 1H), 8.40 (s, broad peak, –NH, 1H),7.431–7.39 (m, H-5,
H-7, 2H),7.26 (d, H-8, 1H), 4.55 (s, –CO-CH2, 2H) , 4.19–4.15(double
triplet, He-2′′, 1H), 2.34–2.30 (dd, Ha-2′′, He-6′′, 2H), 2.21–2.13 (double
of triplet, Ha-6′′, 1H, 2J = 14.0 Hz, 13.6 Hz, 13.2 Hz, 3J = 4.8 Hz, 4.4 Hz),
1.97–1.89 (m, Ha-3′′&He-3′′, 2H), 1.75–1.67 (m, H-4′′, 1H), 1.27–1.23
(m, Ha-5′′&He-5′′, 2H), 0.95 (d, CH3-4′′, 3H, J = 6.4 Hz), 13C NMR
(DMSO‑d6) (100.6 MHz): δ ppm: 166.7 (C-2′), 166.4 (–NH-CO-), 164.3
(C-1′′), 158.7 (C-4′), 157.3 (C-2), 148.8 (C-8a), 129.7 (C-3), 129.2 (C-6),
127.3 (C-7), 125.5 (C-5), 123.3 (C-4a), 121.5 (C-8), 118.2 (C-4), 115.4
(C-5′), 44.1 (3◦-N-CH2), 36.4 (C-3′′), 35.6 (C-5′′), 35.4 (C-4′′), 31.4 (C-
2′′), 29.9 (C-6′′), 21.6 (CH3-4′′), DIP Mass: Molecular ion peak at m/z 447
[M + H]+, M + 2 peak atm/z 449 [M + H + 2]+.
3-coumarinyl)] acetamide (10d). Off white colour, 79% yield, melting
ꢀ 1
point 209–210 ◦C, I.R (KBr) (cm ): 3302 (N H str, NHCO), 1733–1681
–
1
–
–
(C O str, lactone, amides, imide), H NMR (CDCl ) (400 MHz): δ ppm:
8.62 (s, H-4, 1H), 8.41 (s, –NH, 1H), 7.23 (t, H-6, 13H), 7.03 (d, H-5, H-7,
2H), 4.55 (s, –CO-CH2, 2H), 3.96 (s, -OMe group , 3H), 3.00 (m, H1-
2′′&H2-2′′, 2H), 2.45 (m, H1-5′′&H2-5′′, 2H), 1.87 (m, H1-3′′& H2-3′′, H1-
4′′& H2-4′′, 4H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 167.4 (C-2′),
166.3 (-N-CO-C), 163.9 (C-4′), 162.6 (C-2), 157.4 (C-1′′, C-8), 146.7 (C-
8a), 139.4 (C-3), 125.4 (C-6), 124.7 (C-4a), 119.7 (C-5), 117.0 (C-4),
113.7 (C-7), 112.8 (C-5′), 56.5 (–OCH3), 44.1(–CO-CH2-N), 36.5 (C-2′′),
34.9(C-5′′), 27.1 (C-3′′), 25.4 (C-4′′), DIP Mass: Molecular ion peak at m/
z 415 [M + H]+.
2.5.7.9. N-(6-Bromo-3-coumarinyl)-2-[5-(4-methyl-cyclohexylidene)-2,4-
2.5.7.5. 2-[5-Cyclohexylidene-2,4-dioxothiazolidin-3-yl)-N-(3-coumar-
dioxothiazolidin-3-yl] Acetamide (10i). Light cream colour, 77% yield,
ꢀ 1
melting point 197–198 ◦C, I.R (KBr) (cm ): 3294 (N H str, NHCO),
–
inyl)] acetamide (10e). Yellowcolour, 75% yield, melting point
ꢀ 1
182–183 ◦C, I.R (KBr) (cm ): 3315 (N H str, NHCO), 1712 (C O str, 2
1722–1660 (C O str, lactone, amide, imide), 1H NMR (CDCl3) (400
–
–
–
–
–
1
–
OC = O, lactone), 1674 (C O str, amide, imide), H NMR (CDCl ) (400
MHz): δ ppm: 8.57 (s, H-4, 1H), 8.40 (s, broad peak, –NH, 1H),
7.59–7.58 (m, H-5, 1H), 7.55 (distorted doublet, H-7, 1H), 7.21 (dis-
torted doublet, H-8, 1H), 4.55 (s, –CO-CH2, 2H), 4.19–4.15 (double
triplet, He-2′′, 1H), 2.34–2.30 (dd, Ha-2′′, He-2′′, 2H), 2.21–2.13 (doublet
–
3
MHz): δ ppm: 8.98 (s, broad peak, –NH, 1H), 8.65 (s, H-4, 1H), 7.53 (d,
H-5, 1H), 7.45 (d, H-8, 1H), 7.35 (t, H-6, H-7, 2H), 4.02 (s, –CO-CH2-,
2H), 2.38 (m, H1-2′′& H2-2′′, 2H), 2.03 (m, H1-5′′& H2-5′′, 2H), 1.60 (m,
12

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
of triplet, Ha-6′′, 1H, 2J = 14.0 Hz, 13.6 Hz , 13.2 Hz , 3J = 4.8 Hz, 4.4
Hz), 1.97–1.89 (m, Ha-3′′& He-3′′, 2H), 1.75–1.67 (m, H-4′′, 1H),
1.27–1.23 (m, Ha-5′′& He-5′′, 2H), 0.95 (d, CH3-4′′, 3H, J = 6.4 Hz), 13C
NMR (DMSO‑d6) (100.6 MHz): δ ppm: 166.5 (C-2′), 166.2 (–NH-CO-),
164.3 (C-1′′), 157.3 (C-4′), 155.3 (C-2), 149.2 (C-8a), 132.5 (C-3), 130.3
(C-6), 125.5 (C-7), 123.2 (C-5), 122.0 (C-4a), 118.5 (C-8), 117.7 (C-4),
117.1 (C-5′), 79.6 (N-CH2-), 44.1 (C-3′′), 38.4 (C-5′′), 32.5 (C-4′′), 29.8
(C-2′′), 29.3 (C-6′′&CH3-4′′), DIP Mass: Molecular ion peak at m/z 491
[M + H]+, M + 2 peak at m/z 493 [M + H + 2]+.
2′′&H2-2′′, 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 171.1 (C-2′),
170.8 (–NH-CO-), 168.9 (C-4′), 162.2 (C-2), 159.0 (C-4′’), 153.6 (C-8a),
137.0 (C-3), 134.4 (C-7), 129.8 (C-5), 127.8 (C-4a), 126.2 (C-8), 123.0
(C-6),122.6 (C-4), 122.3 (C-5′), 48.6(3o– N-CH2), 43.1 (C-5′′), 37.4 (C-
3′′), 35.0 (C-6′′), 34.4 (C-2′′), DIP Mass: Molecular ion peak at m/z 494
[M]+ , M + 2 peak at m/z 496 [M + 2]+.
2.5.7.14. 2-[5-(Tetrahydrothiopyran-4-ylidene)-2,4-dioxothiazolidin-3-
yl)-N-(8-methoxy-3-coumarinyl)] acetamide (10n). Off white colour,
ꢀ 1
83% yield, melting point 221–222 ◦C, I.R (KBr) (cm ): 3294 (N H str,
–
1
–
–
2.5.7.10. N-(8-Methoxy-3-coumarinyl)-2-[5-(4-methyl-cyclohexylidene)-
NHCO), 1730–1678 (C O str, lactone, amide, imide), H NMR (CDCl )
3
2,4-dioxo-thiazolidin-3-yl] acetamide (10j). Creamcolour, 71% yield,
(400 MHz): δ ppm: 8.61 (s, H-4, 1H), 8.32 (s, broad peak, –NH, 1H), 7.23
(t, H-6, 1H, J = 8 Hz) , 7.02 distorted doublet, H-5, H-7, 2H, J = 8 Hz),
4.56 (s, –CO-CH2, 2H), 3.98 (s, -OMe group , 3H), 3.53 (m, H1-5′′&H2-5′′,
2H), 2.88 (m, H1-3′′&H2-3′′, 2H), 2.83 (m, H1-6′′&H2-6′′, 2H), 2.66 (m,
H1-2′′&H2-2′′, 2H), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 166.5 (C-
2′), 166.2 (–NH-CO-), 164.3 (C-4′), 157.4 (C-2), 155.2 (C-8), 146.7 (C-
4′′), 139.4 (C-8a), 125.4 (C-6), 134.0 (C-3), 124.8 (C-4a), 19.7 (C-
5),117.7 (C-4), 117.0 (C-5′), 112.8 (C-7), 56.5 (–OCH3), 44.1 (-N-CH2),
38.4 (C-5′′), 32.5 (C-3′′), 29.8 (C-6′′), 29.3 (C-2′′), DIP Mass: Molecular
ion peak at m/z 447 [M + H]+.
ꢀ 1
melting point 258–259 ◦C., I.R (KBr) (cm ): 3304 (N H str, NHCO),
–
1735–1683 (C O str, lactone, amide, imide), 1H NMR (CDCl3) (400
–
–
MHz): δ ppm: 8.62 (s, H-4, 1H), 8.35 (s, broad, –NH, 1H),7.22 (distorted
triplet, H-6, 1H, J = 8.0 Hz, 7.6 Hz),7.03 (doublet of doublet, H-5, 1H, 3J
= 8.4 Hz, 7.6 Hz, 4J = 1.6 Hz, 0.8 Hz), 7.01 (dd, H-7, 1H, 3J = 8.0 Hz, 4J
= 1.2 Hz), 4.54 (s, –CO-CH2, 2H), 4.19–4.16 (doublet of triplet, He-2′′,
1H), 3.96 (s, -OMe, 3H), 2.33–2.31 (dd, Ha-2′′, He-6′′, 2H), 2.20–2.12
(doublet of triplet, Ha-6′′, 1H), 1.97–1.89(m, Ha-3′′&He-3′′, 2H),
1.72–1.67 (m, H-4′′, 1H), 1.20–1.09 (m, Ha-5′′&He-5′′, 2H), 0.95 (d, CH3-
4′′, 3H, 3J = 6.8 Hz), 13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 166.7 (C-
2′), 166.3 (NH-CO–),164.3 (C-1′′), 158.7 (C-4′), 157.4 (C-2), 146.7 (C-8),
139.4 (C-8a), 125.4 (C-3), 125.0 (C-6), 124.7 (C-4a), 120.4 (C-5), 119.7
(C-4), 115.4 (C-7), 112.8 (C-5′), 56.5 (8-OCH3), 44.1 (N-CH2), 36.4 (C-
3′′), 35.6 (C-5′′), 35.4 (C-4′′), 31.4 (C-2′′), 29.9 (C-6′′), 21.6 (4′′-CH3), ESI
Mass: Molecular ion peak at m/z 443 [M + H]+.
2.6. Biological assay methodology
2.6.1. Materials
DL-Glyceraldehyde, Lithium sulfate, β-mercaptoethanol, Butylated
hydroxytoluene, NADPH, Sorbinil, Fidarestat were procured from Sigma
Chemicals (MO, USA). All other chemicals and solvents were of
analytical grade and obtained from SRL (Mumbai, India).
2.5.7.11. 2-[5-(Tetrahydrothiopyran-4-ylidene)-2,4-dioxothiazolidin-3-
yl)-N-(3-coumarinyl)] acetamide (10 k). Creamcolour, 78% yield,
ꢀ 1
melting point 240–241 ◦C, I.R (KBr) (cm ): 3315 (N H str, NHCO),
–
1730–1680 (C O str, lactone, amide, imide), 1H NMR (CDCl3) (400
–
–
2.6.2. Purification of aldose reductase (ALR2)
Partial purification of rat lens AR was performed essentially as
described earlier [51]. Eyeballs were removed from 9 weeks old SD male
rats obtained from the National Center for Laboratory Animal Sciences,
Hyderabad. Lenses were then separated from the eye ball and homog-
enized with 10 volumes of 100 mM potassium phosphate buffer pH 6.2.
The homogenate was centrifuged at 12,000g at 41˚C for 30 min. The
supernatant was precipitated with ammonium sulfate at 40%, 50% and
75% salt saturations. After which pellet from the previous step, pos-
sessing AR activity, was dispersed in 75% ammonium sulfate and stored
at ꢀ 80˚C in small aliquots.
MHz): δ ppm: 8.65 (s, H-4, 1H), 8.34 (s, broad peak, –NH, 1H), 7.46 (dd,
H-5, H-8, 2H, 3J = 8.4 Hz, 6.8 Hz), 7.35 (dt, H-6, H-7, 2H, J = 10 Hz, 8.4
Hz), 4.56 (s, –CO-CH2, 2H), 3.52 (m, H1-5′′&H2-5′′, 2H), 2.88 (m, H1-
3′′&H2-3′′, 2H), 2.82 (m, H1-6′′&H2-6′′, 2H), 2.66 (m, H1-2′′&H2-2′′, 2H),
13C NMR (DMSO‑d6) (100.6 MHz): δ ppm: 166.5 (C-2′), 166.1 (–NH-CO-
), 164.3 (C-4′), 157.7 (C-2), 155.2 (C-4′′), 150.2 (C-8a), 130.3 (C-3),
128.4 (C-7), 125.4 (C-5), 124.9 (C-6), 124.4 (C-4a), 119.8 (C-8), 117.7
(C-4), 116.3 (C-5′), 44.1 (30-N-CH2), 38.4 (C-5′′), 32.5 (C-3′′), 29.8 (C-
6′′), 29.3 (C-2′′),DIP Mass: Molecular ion peak at m/z 417 [M + H]+.
2.5.7.12. N-(6-Chloro-3-coumarinyl)-2-[5-(4-tetrahydrothiopyran-4-yli-
2.6.3. Purification of aldehyde reductase (ALR1)
dene)-2,4-dioxo thiazolidin-3-yl] acetamide (10 l). Creamcolour, 82%
ꢀ 1
yield, melting point 220–221 ◦C., I.R (KBr) (cm ): 3437 (N H str, 3
ALR1 was partially purified from bovine kidney following the pre-
viously described methods [52]. Briefly, freshly collected (Chengicherla
Abattoir, Hyderabad) bovine kidney was homogenized in 5 vol of 10 mM
sodium phosphate buffer, pH 7.2 containing 0.25 M sucrose, 2.0 mM
EDTA, 2.5 mM 2-mercaptoethanol. The homogenate was centrifuged at
16 000 × g for 20 min and the supernatant was subjected to ammonium
sulfate precipitation. Precipitate obtained between 45% and 75% satu-
ration was dissolved in the above buffer and dialyzed extensively against
the same buffer. The dialyzed material was removed by centrifugation
and the supernatant used as the source of ALR1.
–
–
–
–
–
NHCO), 1724 (C O str, 2 OC = O, lactone), 1681 (C O str, amide,
imide), 1H NMR (CDCl3) (400 MHz): δ ppm: 8.56 (s, H-4, 1H), 8.40 (s,
broad peak, –NH, 1H), 7.42 (m, H-7, H-5, 2H), 7.28 (d, H-8, 1H), 4.56 (s,
–CO-CH2, 2H), 3.52 (m, H1-5′′&H2-5′′, 2H), 2.88 (m, H1-3′′&H2-3′′, 2H),
2.82 (m, H1-6′′&H2-6′′, 2H), 2.68 (m, H1-2′′&H2-2′′, 2H), 13C NMR
(DMSO‑d6) (100.6 MHz): δ ppm: 166.5 (C-2′), 166.3 (–NH-CO-), 164.3
(C-4′), 157.3 (C-2), 155.3 (C-4′′), 148.8 (C-8a), 129.8 (C-3), 129.2 (C-6),
127.4 (C-7), 125.5 (C-5), 123.4 (C-4a), 121.6 (C-8), 118.2 (C-4), 117.7
(C-5′), 44.1 (-3◦-N-CH2), 38.4 (C-5′′), 32.5 (C-3′′), 29.8 (C-6′′), 29.3 (C-
2′′), DIP Mass: Molecular ion peak at m/z 451 [M + H]+ , M + 2 peak at
m/z 453 [M + H + 2]+
.
2.6.4. Inhibition of ALR1 &ALR2
Synthesised compounds were dissolved in 1% dimethyl sulfoxide
(DMSO) and diluted with deionized H2O and inhibitory activity were
tested against ALR1 & ALR2. To correct for the nonenzymatic oxidation
of NADPH, the rate of NADPH oxidation in the presence of all the re-
action mixture components except the substrate was subtracted from
each experimental rate.The assay mixture of 1 ml consisted of 50 mM
phosphate buffer (pH 6.2), 0.4 M lithium sulfate, 5 mM of β-mercap-
toethanol, 10 mM DL-glyceraldehyde, 0.1 mM NADPH and rat lens AR
preparation. The reaction mixture was incubated at 37 ◦C for 5 min and
the reaction was initiated by adding NADPH. Change in the absorbance
2.5.7.13. N-(6-Bromo-3-coumarinyl)-2-[5-(tetrahydrothiopyran-4-yli-
dene)-2,4-dioxo thiazolidin-3-yl] acetamide (10 m). Creamcolour, 84%
ꢀ 1
yield, melting point 177–178 ◦C, I.R (KBr) (cm ):3304 (N H str,
–
1
–
–
NHCO), 1716–1695 (C O str, lactone, amide, imide), H NMR (CDCl )
3
(400 MHz): δ ppm: 8.55 (s, H-4, 1H), 8.32 (s, broad peak, –NH, 1H), 7.58
(t, H-7, 1H), 7.55 (d, H-5, 1H , 3J = 2 Hz), 7.21 (distorted doublet, H-8,
3
1H, J = 8.4 Hz), 4.55 (s, –CO-CH2, 2H), 3.53 (m, H1-5′′&H2-5′′, 2H),
2.88 (m, H1-3′′&H2-3′′, 2H), 2.82 (m, H1-6′′&H2-6′′, 2H), 2.68 (m, H1-
13

V. Kumar Pasala et al.
Bioorganic Chemistry 114 (2021) 104970
at 340 nm due to NADPH oxidation was measured by double beam
spectrophotometer (Hitachi U-2910). For inhibition studies, stocks of
compounds 10a-n were prepared in 1% DMSO and various concentra-
tions (1 to 100 µM) and added to the reaction mixture. The percent in-
hibition was calculated considering ALR1 & ALR2 activities as 100% in
the absence of inhibitor. The concentration of compounds resulting in
50% inhibition (IC50) was determined by nonlinear regression analysis
of log concentration of compounds versus percentage inhibition. Most
dose–response curves were generated using at least three concentrations
of compound with inhibitory activity between 20% and 80%, with three
replicates at each concentration. The 95% confidence limits (95% CL)
were calculated from t values for n = 5, where n is the total number of
determinations.
protein carbonyls was calculated by using molar extinction coefficient
(
ε
365 nm = 21 mMꢀ 1 cmꢀ 1).
2.7. Molecular docking studies
The 3D crystal structure of ALR1 (PDB: 3FX4) and ALR2 (PDB: 1US0)
were collected from protein data bank (https://www.rcsb.org/struc
ture/3FX4 & https://www.rcsb.org/structure/1US0). Both the pro-
teins were preprocessed, optimized and their energy was minimized by
protein preparation wizard. The receptor grid was generated around the
co-crystal ligand without removing the co-factors (NAP and NDP) by
employing the Glide module. The ligand docking studies were carried
¨
out by Glide module of Schrodinger, LLC, New York, NY, 2017.
2.6.5. Animal experiments and induction of diabetes
2.7.1. Ligand preparation
Three month old male Sprague-Dawley rats with an average body
weight of 218 ± 6 g were used in this study. All the rats were fed ad
libitum with AIN-93 diet. The control group rats (Group I, n = 8) received
sham and the experimental rats received a single i.p injection of STZ (32
mg/kg) in 0.1 M citrate buffer pH 4.5. After 72 h, fasting blood glucose
levels were monitored and animals having blood glucose levels greater
than 150 mg/dl were included in the experiment. Diabetic rats have
been divided into four groups; Group II was fed on a regular diet and
considered as diabetic control group. Whereas the other two groups (III
& IV) of rats which were fed on AIN-93 diet have received compound
10c 10 µM/kg body weight (Group-III, n = 6), 100 µM/kg body weight
(Group-IV, n = 6) and Sorbinil100µM/kg body weight (Group-V, n = 6)
through the diet, respectively. Blood glucose levels of all the rats were
recorded week-wise after an overnight fasting. Cataract progression in
experimental rats was monitored on a weekly basis using slit-lamp mi-
croscope (VISIL 02 Excel) and scoring of cataract formation was per-
formed as described previously. At the end of 8 weeks, body weight was
taken and the rats were sacrificed by CO2 asphyxiation. Rat lenses were
dissected by posterior approach and used for the biochemical analysis of
GSH (reduced glutathione), TBARS (Thiobarbituric acid reactive spe-
cies), Protein carbonyl levels and SOD (Superoxide dismutase), Catalase
and GPx (Glutathione peroxidase) enzyme activities were estimated at
end of the experiment. Animal care and experimental protocols were
approved by Purpose of Control and Supervision of Experiments on
Animals under guideline (CPCSEA) to Osmania University Animal
Ethical Committee, Dept. of Zoology (Ethical Committee Ref. No: 383/
01).
The considered molecules were built in 3D coordinates and their
most stable (lower energy) conformations were calculated, by geomet-
rical optimization of their structure. The ligands were sketched in 2D
environment of maestro, then converted to 3D format and prepared by
¨
the Ligprep module. (LigPrep, Schrodinger, LLC, New York, NY, 2017).
2.7.2. Preparation of protein structures
The crystal structure of ALR1 and ALR2 was considered to get in-
sights into the binding modes of ligands. The protein X-ray structure was
prepared using the Protein Preparation Wizard implemented in Maestro
11.4 (Schrodinger, LLC, New York, NY, 2017) with the following steps
(i) the missing side chains were added to the crystal structure by
Schrodinger’s Prime 3.0. (ii) Hydrogen bond atoms were added and
water molecules beyond 0 Å of the co-crystallized ligand were removed.
(iii) Protonation states of entire systems were adjusted to the pH range of
7.0+/-4.0 using Epik. (iv) Hydrogen bond networks and flip orienta-
tions/tautomeric states of Gln, Asn, and His residues were optimized. (v)
The geometry optimization was performed to a maximum root mean
square deviation (RMSD) of 0.3 Å with the OPLS2005 force field.
Declaration of Competing Interest
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.
Acknowledgements
2.6.6. Biochemical analysis
We thank Prof. G. L. David Krupadanam (Osmania University) for
guidance, inspiration and constructive discussions. We also thank Prof.
Ch. Prasad Rao and Prof. M. Vittal, Osmania University for their
encouragement. We thank immensely UGC, DST-PURSE, UPE-FAR and
CFRD-OU for the financial assistance and facilities, infrastructure
respectively.
2.6.6.1. Estimation of sorbitol in the lens. Lenses were dissected from
different groups of the rats and homogenized in 9 volumes of 0.8 M
perchloric acid. The homogenate was centrifuged at 5,000 g at 4 ◦C for
10 min and the pH of the supernatant was adjusted to 3.5 with 0.5 M
potassium carbonate. Estimation of sorbitol in the supernatant was
performed as described earlier using JASCO FP-750 spectrofluorimeter.
Appendix A. Supplementary data
2.6.6.2. Estimation of reduced glutathione. The reduced glutathione was
estimated by the method of Ellman (1959). Total reaction was measured
at 412 nm against blank and values were compared with a standard
curve of GSH.
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104970.
References
2.6.6.3. Estimation of TBARS levels. The level of thiobarbituric reactive
substance (TBARS) in the lens homogenate was analyzed according to
the method (Ohkawa et al. 1979 and Matsunami et al. 2010). The ab-
sorption of supernatant was measured spectrophotometrically at 535
nm.
[1] P.A. Kumar, G.B. Reddy, Exp. Eye Res. (2006) 1–2.
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