Design, synthesis and evaluation of rhodanine derivatives as aldose reductase inhibitors

Article abstract of DOI:10.1111/cbdd.12369

Aldose reductase (ALR) enzyme plays a significant role in conversion of excess amount of glucose into sorbitol in diabetic condition, inhibitors of which decrease the secondary complication of diabetes mellitus. To understand the structural interaction of inhibitors with ALR enzyme and develop more effective ALR inhibitors, a series of substituted 5-phenylbenzoate containing N-substituted rhodanine derivatives were synthesized and evaluated for their in vitro ALR inhibitory activity. Docking studies of these compounds were carried out, which revealed that the 5-phenylbenzoate moiety deeply influenced the key π-π stacking while 4-oxo-2-thioxothiazolidines contributed in hydrogen bond interactions. The phenyl ring of benzylidene system occupied in specific pocket constituted from Phe115, Phe122, Leu300 and Cys303 while the rhodanine ring forms a tight net of hydrogen bond with Val47 at anionic binding site of the enzyme. The structural insights obtained from the docking study gave better understanding of rhodanine and macromolecular interaction and will help us in further designing and improving of ALR inhibitory activity of rhodanine analogs. Series of substituted 5-phenylbenzoate rhodanine analogs were synthesized and evaluated for their ALR inhibitory activity. The docking study of synthesized compounds was carried out, which revealed that the 5-phenylbenzoate moiety deeply influenced the key π-π stacking while 4-oxo-2-thioxothiazolidines contributed in hydrogen bond interactions.

Full text of DOI:10.1111/cbdd.12369

Received Date : 15-Jan-2014
Revised Date : 14-Apr-2014
Accepted Date : 17-May-2014
Article type : Research Article
Design, Synthesis of Rhodanine Derivatives and In-vitro Evaluation as
Aldose Reductase Inhibitors
Yogesh P. Agrawal1∗, Mona Y. Agrawal1 and Arun K Gupta2
1Drug Design and Development Laboratory, Govt. College of Pharmacy, Ratnagiri, 415612, India
2Department of Pharmaceutical Chemistry, RKDF Institute of Pharmaceutical Sci., Indore 452010, India
Abstract: - Aldose reductase enzyme plays a significant role in conversion of excess amount of
glucose into sorbitol in diabetic condition, inhibitors of which decreases the secondary
complication of Diabetes Mellitus. To understand the structural interaction of inhibitors with
aldose reductase (ALR) enzyme and develop more effective aldose reductase inhibitors, a
series of substituted 5-phenylbenzoate containing N-substituted rhodanine derivatives were
synthesized and evaluated for their in vitro ALR inhibitory activity. Docking studies of these
compounds were carried out which revealed that the 5-phenylbenzoate moiety deeply
influenced the key π-π stacking while 4-oxo-2-thioxothiazolidines contributed in hydrogen
bond interactions. The phenyl ring of benzylidene system occupied in specific pocket
constituted from Phe115, Phe122, Leu300 and Cys303 while the rhodanine ring forms a tight
net of hydrogen bond with Val47 at anionic binding site of the enzyme. The structural
insights obtained from the docking study gave better understanding of rhodanine and
∗ Corresponding to:
Government College of Pharmacy, Ratnagiri, 415612, India
Ph. No.:- +919423720983; E mail: yogeshpagrawal@yahoo.co.in
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
an 'Accepted Article', doi: 10.1111/cbdd.12369
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macromolecular interaction and will help us in further designing and improving of aldose
reductase inhibitory activity of rhodanine analogs.
Keywords: Rhodanine, Aldose Reductase Inhibitors, Docking, Diabetes Mellitus.
Introduction
Diabetes mellitus (DM) is a common chronic disease in constant growth that is taking on
epidemic proportions especially in developing countries. More than 220 million people
worldwide suffer from DM and this figure is expected to increase to 400 million cases by
20301. Diabetic complications, such as neuropathy, retinopathy, nephropathy or cataract, are
serious and disabling pathologies associated with DM2. Hyperglycemia is a typical condition
of DM and plays a crucial role in the development and advancement of these complications
which arise from acute and reversible changes in cellular metabolism as well as from
irreversible long-term damage in biological macromolecules.
Prospective studies have highlighted that good control of blood glucose levels delays
the onset or slows the progression of diabetic complications3. Despite the wide availability of
effective oral antidiabetic drugs, it is difficult to keep glycemia under tight control and thus
the onset of long-term damage is unavoidable. Considerable efforts have been made to
identify effective agents that are able to counteract the biological mechanisms responsible for
the development of diabetic complications but so far very few drugs have been marketed4.
Numerous mechanisms like non-enzymatic glycation of proteins, glucose auto-oxidation, the
activation of protein kinase C (PKC) isoforms and polyol pathway triggered by the chronic
exposure to high levels of glucose5. Moreover these biochemical pathways sustain in various
tissue types and increases in hyperglycemic induced oxidative stress which is observed in
both clinical and experimental DM and leads to the deleterious effects on cellular functions
and signaling underlying the appearance of both macro and microangiopathy5-8. In
hyperglycemic conditions, the auto-oxidation reactions of glucose and other α-
hydroxyaldehydes produce reactive oxygen species (ROS) which can damage biomolecules,
cellular membranes and tissues by promoting intermolecular cross-linking and fragmentation
reactions as well as lipid oxidation5, 6. Moreover the highly reactive dicarbonyl compounds
produced in the course of glucose auto-oxidation reactions lead to the glycation of
macromolecules which culminates in the synthesis of advanced glycation end-products
(AGEs)7, 8. The significant increase of glucose flux through the polyol pathway increases
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both cellular oxidative and osmotic stress, thereby significantly contributing to tissue injury
in which the glucose up-take is insulin independent linked to DM4,5,7
.
Aldose reductase (E.C.1.1.1.21), a ubiquitous enzyme which has been identified in
brain, kidney, liver, lens and skeletal muscle tissue, is an aldo-keto reductase that catalyzes
the NADPH-dependent reduction of glucose into sorbitol in the first step of the polyol
pathway which further dehydrogenase through an NAD+ dependent reaction into fructose.
Although not determinant, the osmotic stress generated by sorbitol accumulation has been
proposed as contributing to the development of tissue damage above all in the lens8,9.
Moreover, the increase in the NADPH/NADP+ ratio promotes the activation of PKC isoforms
which are found to be crucial mediators of biochemical and functional alterations triggered
by hyperglycemia. Thus, the oxidative stress triggered by the glucose-oxidation process and
upheld by increased aldose reductase activity is rightly considered to be a major and unifying
mechanism responsible for the onset of diabetic complications5. Over the last three decades,
numerous ALR inhibitors (ARIs) have been identified; most of them belong to either
carboxylic acid (such as epalrestat; Figure 1) or hydantoin (such as sorbinil, fidarestat;
Figure 1) classes of compounds10-14. However, many of the clinically tested ARIs proved to
be inadequate as drug candidates because of adverse pharmacokinetics, toxic side effects or
low efficacy. At present, epalrestat is the only ARI available on the market10,11,15
.
Literature reveals that in last few years, numerous 5-arylidene-2, 4-thiazolidinediones
(Figure 1) derivatives produced appreciable ALR inhibition similar to that identified16,17 but
their effectiveness generally decreases in-vivo, probably due to their poor penetrability to key
target tissues, in particular peripheral nerves18-20. Thus, to develop new ARIs, with pKa values
higher than those of carboxylic acids, the 2-oxo group of 2,4-thiazolidinediones was
bioisosterically replaced to 4-oxo-2-thioxothiazolidines which may alter the physicochemical
properties and enhanced the potential with better bioavailability for the successful
development of new clinical agents21-22
.
In the present study new active analogues of 5-arylidene-4-oxo-2-thioxothiazolidines
were identified, designed and synthesized as potent in-vitro ALR inhibitors. In particular, 5-
benzylidene moieties bearing hydroxy, methoxy, nitro, chloro and/or dichloro substituted
benzoates were prepared which is potentially able to enhance stability of enzyme inhibitor
complex.
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Method and Material
Docking
The detailed interaction study of synthesized rhodanine analogs and aldose reductase enzyme
was carried out through Glide version 5.5 module of Schrodinger suite23. Grid based ligand
docking with energetics (Glide) was used for ligand docking in XP (extra precision) mode.
This docking method is fast and accurate and consists of mainly two steps.
A) Ligand preparation: The initial structure of ligands were drawn on ChemDraw ultra 11.0
and converted to 3D by using Chem3D ultra 11.0 and then minimized by using MM2 force
field and truncated newton minimizer corresponding to RMS gradient 0.100 and saved as
mole file. Subsequently these mole files were imported in to the Maestro workspace and
combined to one molecule file as sdf format. This file was used for ligands preparation with
the help of LigPrep module of the Schrodinger suite keeping state generation pH condition
7 2 value and output file used for docking study.
B) Protein preparation: The X-ray crystal structure of the aldose reductase enzyme (2PDG)
was retrieved from protein data bank24. All the water molecules were removed from the
enzyme and subsequently protein was prepared in Maestro 9.0 using protein preparation
wizard. Co-factor (NAP) and ligand (47D) of enzyme were prepared using “Generate Het
States” module of protein preparation wizard and default Het States of NAP & 47D were
used for docking. Hydrogen atoms were added to the protein. The H-bonds were optimized
using sample water orientations. Finally, the protein structure (hydrogen only) was
minimized to the default Root Mean Square Deviation (RMSD) value of 0.30Å. Receptor
grid was generated using centroid of work space ligand. The XYZ coordinates of centroid are
16.93, -6.27, & 14.46 respectively. The active site was defined as an enclosing box and
bounding box around the assigned center (16.93, -6.27, & 14.46) of lengths 10 Å & 30 Å
respectively. The grid was used for the next step of docking.
Docking protocol was validated by comparing the RMSD of crystal structure
conformation of ligand with docked conformation of the ligand obtain through docking
methodology. The RMSD value of these two conformers was found to be 1.91Å which is
under acceptable limit. The minimized structures of ligand’s and protein were subsequently
used in docking simulation. The conformation flexibility of ligands were considered by
exhaustive conformational search within glide augmented by heuristic screen that removed
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conformations which were not suitable for aldose reductase binding or had long range
hydrogen bonds, whereas aldose reductase conformation remained fixed. An exhaustive
systemic search of the conformational space and a series of hierarchical filters to locate the
possible position of ligands in the docking simulation were done. The shape and properties
were represented on grid by different fields, which provided more accurate position and
orientation of ligands (termed pose) in the Aldose reductase. Ten poses were calculated per
ligand molecule, which were docked to Aldose reductase. The resulting dock conformations
were analyzed using Glide pose viewer tool. The conformation/poses that made the
maximum number of interaction were considered to further analysis. G score method was
considered for selection of the compounds.
Biological Evaluation
In- vitro aldose reductase inhibitory activity
A purified goat lens extract was prepared in accordance with the method of Hayman and
Kinoshita with slight modifications25. Lenses were quickly removed from goat eye following
euthanasia and homogenized (Glass-Potter) in 5 volume of cold deionized water. The
homogenate was centrifuged at 10,400 rpm at 0-4°C for 30min. Saturated ammonium sulfate
solution was added to the supernatant fraction to form a 40% solution, which was stirred for
30min at 0-4ºC and subsequently centrifuged at 10,400 rpm for 20 min. Recovered
supernatant was subsequently treated with saturated ammonium sulfate solution to make a
50% solution and follow the above mentioned stirring and centrifugation condition. The
obtained supernatant was adjusted to 75% salt saturation by saturated ammonium sulfate
solution and repeated the stirring and centrifugation procedure. The precipitate recovered
from the 75% saturated fraction, possessing ALR enzyme. The precipitate was dissolved in
0.05M NaCl and dialyzed overnight in 0.05M NaCl. The dialyzed material was used for the
enzymatic assay.
Chemistry
The purification of intermediates and final compounds was carried out through
recrystallization and column chromatography technique. Melting points were recorded by
open capillary method on melting points apparatus and are uncorrected. TLC controls were
1
carried out on precoated silica gel plates. H NMR spectra were recorded on Bruker Avance
II 400 magnetic resonance spectrometer from Central Drug Research Institute, Lucknow.
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Chemical shifts are given in δ units (ppm) relative to internal standard TMS and refer to
DMSO-D6 solutions. The IR spectra of the intermediates and synthesized compounds were
recorded on FTLA 2000 ABB spectrophotometer at SCOPE, Indore. MS spectral data were
recorded from SAIF Punjab University, Chandigarh using THERMO Finnigan LCQ
Advantage max ion trap MS for the entire synthesized compound. Unless stated otherwise, all
materials were obtained from commercial suppliers and used without further purification.
Results
Docking Analysis
The docking results for all the compounds are summarized in Table 1. Visual inspection of
the top-ranked poses for every ligand–protein combination showed that, in contrast to the
cross-docking of the crystal structure ligands, the compounds often had similar poses in
2PDG protein. Glide score was calculated based on number of parameters such as hydrogen
bonds (H bond), hydrophobic (Lipo), van der Waals (vdW), columbic (coul), polar
interaction in the binding-site (site), metal-binding term (metal) and penalty for buried polar
group (Bury P), and freezing rotable bonds (Rot B). Among these compounds, compound C-2
having lowest Glide score when docked into 2PDG, suggesting that the π-π stacking and
hydrogen bonds interaction with the amino acid. 2D representation of docking interaction of
proto type design compound & 3D docked poses view using PyMOL26 interface are shown in
Figure 2 & 3. The phenyl ring of benzylidene system takes the position in specific pocket
constituted from Phe115, Phe122, Leu300 and Cys303 and interacts with Trp111 through π-π
stacking. At the same time rhodanine occupied position in the anionic binding site and
nitrogen of rhodanine forms a hydrogen bond with O of Val47.
Aldose Reductase Inhibition
All the synthesized rhodanine derivatives were evaluated for their ability to inhibit the in
vitro reduction of D,L-glyceraldehydes by partially purified ALR from goat lenses; sorbinil
was used as a reference drug (Table 1). Among the synthesized compounds, (4-((4-oxo-2-
thioxothiazolidin-5-ylidene) methyl) phenyl)-2-chlorobenzoate (C-2) showed IC50 value; 1.82
µM (Table 1) which was very similar to that of sorbinil. In contrast, analogues C-3 and C-9,
bearing an 4-Chloro and 2,4-dichloro benzoate substituent in the 5-arylidene moiety produced
no or only moderate ALR inhibition. In particular, 4-((4-oxo-2-thioxothiazolidin-5-
ylidene)methyl) phenyl 4-methoxybenzoate (C-6) ( 2.57 µM) proving to be more or same
active than the corresponding 4-((4-oxo-2-thioxothiazolidin-5-ylidene)methyl) phenyl 3,4-
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dimethoxybenzoate (C-4) (2.68 µM) whereas its 2-methoxy derivative (C-6) was shown to be
5 times less effective at the same dose.
The displacement of methyl group of C-5 from para to ortho position in C-7 of the 5-
benzylidene ring markedly increased inhibitory effectiveness, while replacement of phenyl
substitution with benzyl moiety which are totally inefficacious as an ALR inhibitors.
Conclusion
In conclusion, rhodanine derivatives were synthesized and evaluated as aldose reductase
inhibitors. Preliminary structure activity relationship revealed that rhodanine derivatives are
having improved aldose reductase inhibitory activity as compare to N-acetic acid rhodanine
derivatives. Electron withdrawing group are favourable at 2nd position of terminal phenyl ring
whereas electron withdrawing group at 4th position of terminal phenyl ring decreases activity.
Similarly di-substituted electronegative derivatives are having less activity while
electropositive and bulky group at 4th position showed comparable activity. Methylene bridge
between benzylidene and terminal phenyl group decreases activity. N-acetic acid rhodanine
derivatives showed that electron withdrawing group at terminal phenyl ring increases activity,
while methoxy substitution showed comparable activity. Docking study reveal that
synthesized compounds are having the same orientation like ligand protein complex (2PDG).
Phenyl ring of benzylidene system takes the position in specific pocket constituted from
Phe115, Phe122, Leu300 and Cys303 and interacts with Trp111 through π-π stacking which
is similar to that of 1,3-benzothiazolyl moiety of ligand co-crystal structure. Among the
synthesized rhodanine derivatives, C-2 and C-6 exhibited ALR inhibiting affinity at micro
molar concentration which will be used as starting points for a future drug discovery
program.
General Method of Synthesis
Synthesis of substituted benzoyl chloride (A1-12):
Different substituted benzoic acid derivatives (0.01mol) were refluxed with thionyl chloride
for 3-4 hr and monitored the reaction through TLC. After completion of reaction, evaporate
excess of thionyl chloride under reduced pressure, the crude solid was used as such in next
step. (Scheme 1)
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Synthesis of substituted 4-formylphenyl benzoate (B1-12):
A cooled solution of benzoyl chloride analogs was mixed slowly with triethylamine
(0.03mol) with constant stirring, followed by adding p-hydroxy benzaldehyde (0.01mol).
Stirred the reaction mixture at 0°C for 2 hr. and continued stirring at RT for overnight. After
evaporation of the solvent under reduced pressure, the crude solid was washed with saturated
solution of sodium bicarbonate, brine solution and water. Organic phase was separated and
pass through anhydrous Na2SO4 and re-crystallized using ethanol. (Scheme 1)
Synthesis of substituted 4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl
benzoate (C1–12):
All the rhodanine derivatives (C1–C12) were synthesized by Knoevenagel condensation27,28
by reacting rhodanine with prepared 4–formyl phenyl benzoate derivatives. A mixture of
derivatives of 4–formyl phenyl benzoate (0.0025mol) and rhodanine (0.0025mol) in glacial
acetic acid containing catalytic amount of sodium acetate (0.080 gm.) was stirred and heat at
100-105°C for 10-12 hr. After completion of reaction, cooled to RT and filtered the
crystalline product, washed with cold acetic acid and purified by column chromatography.
(Scheme 1)
Synthesis of substituted 2-(5-(4-(benzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-
yl) acetic acid derivatives (C13-C24):
Equimolar concentration of derivative of 4–formyl phenyl benzoate (0.025mol) and
rhodanine-N-acetic acid (0.025mol) were taken in round-bottomed flask containing glacial
acetic acid. To this a catalytic amount of sodium acetate (0.080 gm.) was added. The reaction
mixture was stirred and heat at 100-105°C for 10-12 h. Progress of the reaction mixture was
checked through TLC. After completion of reaction, mixture was kept aside for overnight at
RT. Crystalline product was filtered, washed with cold acetic acid and purified by column
chromatography. (Scheme 1)
All the compounds were analyzed and assigned structures were confirmed by spectroscopic
data as follows.
1. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl) benzoate (C-1): Yield 64%;
MP 218–222 °C; IR (ATR) 3132 (C-H), 1686 (C=C), 1165 (C-O), 1731 (C=O), 3132 (N-
1
H); H NMR (DMSO) δ 9.7 (s, 1H, N-H), 8.16- 8.18 (t, 2H, arom. CH), 7.55– 7.80 (m,
5H, arom. CH), 7.35-7.50 (m, 2H, arom. CH), 7.25 (s, 1H, ethylene CH); MS (ESI+): 342
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[M+H]+
2. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-2-chlorobenzoate (C-2):
Yield 52%; MP 210–214 °C; IR (ATR) 3033 (C-H), 1685 (C=C), 1159 (C-O), 1748
1
(C=O), 3648 (N-H), 736 (C-Cl); H NMR (DMSO) δ 9.65 (s, 1H, N-H), 7.9-8.1 (d, 1H,
arom. CH), 7.55-7.75 (m, 5H, arom. CH), 7.45-7.55 (m, 2H, arom. CH), 7.38 (s, 1H,
ethylene CH); MS (ESI+): 375 [M]+
3. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-4-chlorobenzoate (C-3):
Yield 69%; MP 215–220 °C; IR (ATR) 2984 (C-H), 1676 (C=C), 1267 (C-O), 1732
1
(C=O), 1590 (N-H), 759 (C-Cl). H NMR (DMSO) δ 9.85 (s, 1H, N-H), 7.95-8.25 (m,
2H, arom. CH), 7.6-7.75 (m, 4H, arom. CH), 7.3-7.5 (m, 2H, arom. CH), 7.15 (s, 1H,
ethylene CH); MS (ESI+): 374.1 [M]+
4. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-3,4-dimethoxybenzoate
(C-4): Yield 45%; MP 248–250 °C; IR (ATR) 3061 (C-H), 1697 (C=C), 1138 (C-O),
1734 (C=O), 3181 (N-H), 1212 (-OCH3). 1H NMR (DMSO) δ 9.95 (s, 1H, N-H), 7.8-8.0
(m, 2H, arom. CH), 7.61-7.75 (m, 2H, arom. CH), 7.35-7.50 (m, 2H, arom. CH), 7.1 (s,
1H, ethylene CH), 6.9-7.2 (m, 1H, arom. CH), 3.9-4.25 (d, 6H, methyl CH). MS (ESI+):
402[M+H]+
5. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-4-methylbenzoate (C-5):
Yield 36%; MP 198–200 °C; IR (ATR) 3055 (C-H), 1683 (C=C), 1220 (C-O), 1732
1
(C=O), 3647 (N-H). H NMR (DMSO) δ 9.75 (s, 1H, N-H), 8.0 (t, 2H, arom. CH), 7.5-
7.8 (m, 2H, arom. CH), 7.3-7.5 (m, 4H, arom. CH), 7.3 (s, 1H, ethylene CH), 2.5 (s, 3H,
methyl CH). MS (ESI+): 356.6[M+H]+
6. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-4-methoxybenzoate (C-6):
Yield 39%; MP 202–208 °C; IR (ATR) 3077 (C-H), 1698 (C=C), 1165 (C-O), 1748
(C=O), 3647 (N-H), 1204 (-OCH3). 1H NMR (DMSO) δ 10.05 (s, 1H, N-H), 7.7-8.1 (m,
2H, arom. CH), 7.5-7.7 (m, 2H, arom. CH), 7.3-7.5 (m, 2H, arom. CH), 7.03 (s, 1H,
ethylene CH), 6.9-7.2 (m, 2H, arom. CH), 3.9 (s, 3H, methyl CH). MS (ESI+): 372
[M+H]+
7. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-2-methylbenzoate (C-7):
Yield 47%; MP 212–215 °C; IR (ATR) 2998(C-H), 1683 (C=C), 1166 (C-O), 1733
1
(C=O), 3647 (N-H). H NMR (DMSO) δ 9.90 (s, 1H, N-H), 8.1 (d, 1H, arom. CH), 7.6-
7.8 (m, 3H, arom. CH), 7.4-7.55 (m, 4H, arom. CH), 7.35 (s, 1H, ethylene CH), 2.5-2.6
(m, 3H, methyl CH). MS (ESI+): 356.2 [M+H]+
8. (4-((4-oxo-2-thioxothiazolidin-5-ylidene)methyl) phenyl)-2-methoxybenzoate (C-8):
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Yield 71%; MP 228–230 °C; IR (ATR) 3037 (C-H), 1658 (C=C), 1125 (C-O), 1708
(C=O), 3587 (N-H), 1244 (-OCH3).1H NMR (DMSO) δ 9.80 (s, 1H, N-H), 7.85-8.15 (d,
1H, arom. CH), 7.5-7.70 (m, 3H, arom. CH), 7.3-7.45 (m, 2H, arom. CH), 7.2 (s, 1H,
ethylene CH), 6.8-7.1 (m, 2H, arom. CH), 3.8 (s, 3H, methyl CH). MS (ESI+): 372
[M+H]+
9. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-2,4-dichlorobenzoate (C-
9): Yield 57%; MP 231–235 °C; IR (ATR) 3084 (C-H), 1546 (C=C), 1252 (C-O), 1712
(C=O), 1610 (N-H), 785 (C-Cl).1H NMR (DMSO) δ 10.0 (s, 1H, N-H), 7.8-8.1 (d, 1H,
arom. CH), 7.5-7.7 (m, 4H, arom. CH), 7.35-7.45 (m, 2H, arom. CH), 7.3 (s, 1H,
ethylene CH). MS (ESI+): 410 [M+H]+
10. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-4-nitrobenzoate (C-10):
Yield 38%; MP 238–241 °C; IR (ATR) 3025 (C-H), 1653 (C=C), 1260 (C-O), 1710
(C=O), 3627 (N-H). 1H NMR (DMSO) δ 10.15 (s, 1H, N-H), 8.0-8.3 (m, 4H, arom. CH),
7.65-7.85 (m, 2H, arom. CH), 7.25-7.55 (m, 2H, arom. CH), 7.1 (s, 1H, ethylene CH).
MS (ESI+): 386.8[M]+
11. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-3,5-dinitrobenzoate (C-
11): Yield 31%; MP 210–213 °C; IR (ATR) 2945 (C-H), 1663 (C=C), 1242 (C-O), 1718
1
(C=O), 3627 (N-H). H NMR (DMSO) δ 9.90 (s, 1H, N-H), 8.9-9.3 (d, 2H, arom. CH),
8.6 (s, 1H, arom. CH), 7.69-7.85 (m, 2H, arom. CH), 7.5-7.65 (m, 2H, arom. CH), 7.2 (s,
1H, ethylene CH). MS (ESI+): 431 [M]+
12. (4-((4-oxo-2-thioxothiazolidin-5-ylidene) methyl) phenyl)-2-phenylacetate (C-12):
Yield 30%; MP 224–230 °C; IR (ATR) 3032 (C-H), 1661 (C=C), 1175 (C-O), 1721
1
(C=O), 3172 (N-H). H NMR (DMSO) δ 10.05 (s, 1H, N-H), 7.7- 7.95 (t, 2H, arom.
CH), 7.3– 7.6 (m, 7H, arom. CH), 7.25 (s, 1H, ethylene CH), 3.7 (s, 2H, methylene CH).
MS (ESI+): 356.8 [M+H]+
13. 2-(5-(4-(benzoyloxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid (C-13):
Yield 71%; MP 109-111°C; IR (cm-1) 3142 (C-H), 1696 (C=C), 1175 (C-O), 1721
1
(C=O), 3142 (N-H); H NMR (DMSO) (δ) 11.2 (s, 1H, O-H), 8.16- 8.18 (t, 2H, arom.
CH), 7.55– 7.8 (m, 5H, arom. CH), 7.38-7.50 (m, 2H, arom. CH), 7.33-7.35 (d, 1H,
ethylene CH), 3.68 (s, 2H, methylene); MS (ESI+): 401.7 [M+H]+
14. 2-(5-(4-(2-chlorobenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-14): Yield 56%; MP 98-101°C; IR (cm-1) 3043 (C-H), 1695 (C=C), 1169 (C-O),
1758 (C=O), 3658 (N-H), 746 (C-Cl); 1H NMR (DMSO) (δ) 11.05 (s, 1H, O-H), 7.8-8.1
(d, 1H, arom. CH), 7.65-7.75 (m, 5H, arom. CH), 7.45-7.55 (m, 2H, arom. CH), 7.38-
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7.45 (d, 1H, ethylene CH), 3.34 (s, 2H, methylene); MS (ESI+): 433.1 [M]+
15. 2-(5-(4-(4-chlorobenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-15): Yield 65%; MP 102-104°C; IR (cm-1) 2994 (C-H), 1686 (C=C), 1277 (C-
1
O), 1722 (C=O), 1600 (N-H), 769 (C-Cl); H NMR (DMSO) (δ) 10.87 (s, 1H, O-H),
7.95-8.25 (m, 2H, arom. CH), 7.6-7.75 (m, 4H, arom. CH), 7.3-7.55 (m, 2H, arom. CH),
7.15-7.25(d, 1H, ethylene CH), 3.58 (s, 2H, methylene); MS (ESI+): 433.2 [M+H]+
16. 2-(5-(4-(3,4-dimethoxybenzoyloxy)
benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)
acetic acid (C-16): Yield 70%; MP 100-102°C; IR (cm-1) 3071 (C-H), 1687 (C=C),
1
1148 (C-O), 1724 (C=O), 3191 (N-H), 1222 (-OCH3); H NMR (DMSO) (δ) 11.04 (s,
1H, O-H), 7.8-8.0 (m, 2H, arom. CH), 7.61-7.75 (m, 2H, arom. CH), 7.35-7.50 (m, 2H,
arom. CH), 7.1-7.2 (d, 1H, ethylene CH), 6.9-7.2 (m, 1H, arom. CH), 3.9-4.25 (d, 6H,
methyl CH), 3.67 (s, 2H, methylene); MS (ESI+): 460 [M+H]+
17. 2-(5-(4-(4-methylbenzoyloxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)
acetic
acid (C-17): Yield 50%; MP 99-105°C; IR (cm-1) 3065 (C-H), 1693 (C=C), 1230 (C-
1
O), 1726 (C=O), 3657 (N-H); H NMR (CDCl3) (δ) 11.09 (s, 1H, O-H), 8.0-8.2 (t, 2H,
arom. CH), 7.58-7.8 (m, 2H, arom. CH), 7.3-7.5 (m, 4H, arom. CH), 7.2-7.3 (d, 1H,
ethylene CH), 2.5 (s, 3H, methyl CH), 3.36 (s, 2H, methylene); MS (ESI+): 411.5 [M-H]+
18. 2-(5-(4-(4-methoxybenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-18): Yield 63%; MP 113-115°C; IR (cm-1) 3047 (C-H), 1668 (C=C), 1135 (C-
O), 1718 (C=O), 3597 (N-H), 1254 (-OCH3); 1H NMR (DMSO) (δ) 11.07 (s, 1H, O-H),
8.1-8.34 (m, 2H, arom. CH), 7.6-8.0 (m, 2H, arom. CH), 7.3-7.5 (m, 2H, arom. CH),
7.16-7.3 (d, 1H, ethylene CH), 6.71-7.0 (m, 2H, arom. CH), 3.8 (s, 3H, methyl CH), 3.54
(s, 2H, methylene); MS (ESI+): 430.2 [M+H]+
19. 2-(5-(4-(2-methylbenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-19): Yield 64%; MP 94-97°C; IR (cm-1) 2988(C-H), 1673 (C=C), 1156 (C-O),
1
1723 (C=O), 3637 (N-H); H NMR (CDCl3) (δ) 10.99 (s, 1H, O-H), 8.1 (d, 1H, arom.
CH), 7.6-7.8 (m, 3H, arom. CH), 7.2-7.3 (m, 4H, arom. CH), 6.8-7.1 (d, 1H, ethylene
CH), 2.5-2.6 (m, 3H, methyl CH), 3.64 (s, 2H, methylene); MS (ESI+): 412.8 [M]+
20. 2-(5-(4-(2-methoxybenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-20): Yield 69 %; MP 92-94°C; IR (cm-1) 3067 (C-H), 1688 (C=C), 1155 (C-O),
1728 (C=O), 3657 (N-H), 1214 (-OCH3); 1H NMR (CDCl3) (δ) 10.91 (s, 1H, O-H), 7.7-
8.15 (d, 1H, arom. CH), 7.5-7.60 (m, 3H, arom. CH), 7.3-7.5 (m, 2H, arom. CH), 7.01-
7.03 (d, 1H, ethylene CH), 6.78-6.91 (m, 2H, arom. CH), 3.9 (s, 3H, methyl CH), 3.40 (s,
2H, methylene); MS (ESI+): 430 [M+H]+
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21. 2-(5-(4-(2,4-dichlorobenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-21): Yield 55%; MP 88-91°C; IR (cm-1) 3094 (C-H), 1556 (C=C), 1262 (C-O),
1718 (C=O), 1620 (N-H), 795 (C-Cl); 1H NMR (DMSO) (δ) 10.87 (s, 1H, O-H), 8.3-8.4
(d, 1H, arom. CH), 7.5-8.1 (m, 4H, arom. CH), 7.48-7.55 (m, 2H, arom. CH), 7.4-7.5 (d,
1H, ethylene CH), 3.62 (s, 2H, methylene); MS (ESI+): 465.6 [M-H]+
22. 2-(5-(4-(4-nitrobenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic acid
(C-22): Yield 58%; MP 111-114°C; IR (cm-1) 3035 (C-H), 1663 (C=C), 1270 (C-O),
1
1720 (C=O), 3637 (N-H); H NMR (CDCl3) (δ) 11.14 (s, 1H, O-H), 7.7-8.1 (m, 4H,
arom. CH), 7.41-7.65 (m, 2H, arom. CH), 7.25-7.4 (m, 2H, arom. CH), 7.1-7.2 (d, 1H,
ethylene CH), 3.55 (s, 2H, methylene); MS (ESI+): 445.3 [M+H]+
23. 2-(5-(4-(3,5-dinitrobenzoyloxy) benzylidene)-4-oxo-2-thioxothiazolidin-3-yl) acetic
acid (C-23): Yield 70%; MP 95-97°C;IR (cm-1) 2955 (C-H), 16673 (C=C), 1252 (C-O),
1
1721 (C=O), 3637 (N-H); H NMR (DMSO) (δ) 10.92 (s, 1H, O-H), 7.9-8.3 (d, 2H,
arom. CH), 8.5 (s, 1H, arom. CH), 7.69-7.85 (m, 2H, arom. CH), 7.5-7.65 (m, 2H, arom.
CH), 7.2-7.35 (d, 1H, ethylene CH), 3.67 (s, 2H, methylene); MS (ESI+): 489.5[M]+
24. 2-(5-(4-(2-phenylacetoxy)benzylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic acid (C-
24): Yield 72 %; MP 107-110°C; IR (cm-1) 3042 (C-H), 1671 (C=C), 1185 (C-O), 1711
1
(C=O), 3182 (N-H); H NMR (CDCl3) (δ) 10.95 (s, 1H, O-H), 8.22- 8.38 (t, 2H, arom.
CH), 7.8– 8.19 (m, 7H, arom. CH), 7.5-7.8 (d, 1H, ethylene CH), 3.4 (s, 2H, methylene
CH), 3.8 (s, 2H, methylene); MS (ESI+): 412.2 [M-H]+
In-vitro Enzymatic Assay
ALR activity was assayed at 30ºC in a reaction mixture containing 0.75mL of 10mM D,L-
glyceraldehyde, 0.5mL of 0.104mM NADPH, 0.75mL of 0.1M sodium phosphate buffer (pH
= 6.2), 0.3mL of enzyme extract and 0.7mL of deionized water in a total volume of 3mL. All
the above reagents, except D,L-glyceraldehyde, were incubated at 30ºC for 10 min; the
substrate was then added to start the reaction, which was monitored for 5min. Enzyme
activity was calibrated by diluting the enzymatic solution in order to obtain an average
reaction rate of 0.011 0.0010 absorbance units/min for the sample.
The sensitivity of aldose reductase to different compounds was tested in the above assay
conditions in the presence of inhibitors dissolved at proper concentration in DMSO (1 % v/v).
IC50 values were determined by non linear regression analysis. Each log dose-inhibition curve
was generated using at least seven concentrations of inhibitor causing an inhibition between
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10 and 90. The 95% confidence limits (95% CL) were calculated using GraphPad Prism
software. The inhibitory activity of the compounds is shown in Table 1.
Conflict of interest
The authors report no conflict of interest.
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Table 1: Glide Score and Aldose Reductase Inhibitory activity of Synthesized Compounds
S
O
C
S
N
O
Ar
O
R1
aAldose Reductase
Inhibitory activity
(IC50 in µM)
Compound
No.
Glide
Score
R1
Ar
C-1
C-2
C-3
C-4
C-5
H
H
H
H
H
C6H5-
-10.34
-11.87
-10.64
-9.19
2.47 (2.28-2.91)
1.82 (1.13-1.49)
5.89 (5.63-6.29)
2.68 (2.43-2.87)
5.82 (5.32-6.09)
2-Cl-C6H4-
4-Cl-C6H4-
3,4-(OCH3)2-C6H3-
4-CH3-C6H4-
-11.09
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C-6
C-7
H
4-OCH3-C6H4-
2-CH3-C6H4-
2-OCH3-C6H4-
2-(Cl)2-C6H3-
4-NO2-C6H4-
3,5-(NO2)2-C6H3-
C6H5-CH2-
-10.34
-11.84
-09.93
-9.14
2.57 (2.37-2.71)
3.65 (3.32-3.94)
4.50 (4.36-4.89)
12.5 (11.03-13.09)
15.54 (14.43-16.07)
9.73 (9.14-10.26)
b14% (12.5 µM)
16.9 (15.75-17.62)
3.17 (2.88-3.41)
7.02 (6.13-7.49)
b51 % (12.5 µM)
10.18 (9.43-10.87)
15.02 (14 -15.09)
4 .57 (4.37-4.71)
b34% (12.5 µM)
12.01 (11.36-12.49)
11.5 (11.03-12.09)
5.40 (4.97-6.03)
6.13 (6.1-6.26)
H
C-8
H
C-9
H
C-10
C-11
C-12
C-13
C-14
C-15
C-16
C-17
C-18
C-19
C-20
C-21
C-22
C-23
C-24
Standard
H
-10.58
-11.08
-9.73
H
H
-CH2COOH
-CH2COOH
-CH2COOH
C6H5-
-11.34
-11.73
-12.15
-11.11
-10.85
-12.13
-8.61
2-Cl-C6H4-
4-Cl-C6H4-
-CH2COOH 3,4-(OCH3)2-C6H3-
-CH2COOH
-CH2COOH
-CH2COOH
-CH2COOH
-CH2COOH
-CH2COOH
-CH2COOH
-CH2COOH
4-CH3-C6H4-
4-OCH3-C6H4-
2-CH3-C6H4-
2-OCH3-C6H4-
2,4-(Cl)2-C6H3-
4-NO2-C6H4-
3,5-(NO2)-C6H3-
C6H5-CH2-
-12.34
-7.94
-5.15
-5.03
-3.82
Sorbinil
1.32 (1.12-1.65)
a
b
Fifty percent inhibitory concentration in µM with 95% confidence limit; Percentage inhibition at the
concentration given in the parenthesis
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OH
O
NH
O
HN
O
O
F
N
S
S
O
Epalrestat
Sorbinil
O
OH
NH
F
O
HN
O
F
F
N
S
N
F
O
CONH2
Lidorestat
Fidarestat
O
R1
1 R= H; R1= H
1a R= H; R1= OH
S
N
R
2 R= CH2COOCH3; R1= H
3 R= CH2COOH; R1= H
O
5-arylidene-2,4-thiazolidinediones
Figure 1: Structure of aldose reductase inhibitors
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This article is protected by copyright. All rights reserved.

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HO
R
R
a
OH
Cl
+
O
C
O
C
O
C
H
A1-12
b
S
R
O
S
S
O
N
S
C
O
R
R1
C
N
O
O
O
c
OHC
R1
B1-12
C1-12; R1=H
C13-24;R1=CH2COOH
Reagents and conditions: (a) SOCl2, Reflux; (b) (C2H5)N, CH2Cl2, RT;(c) CH3COONa, CH3COOH, Reflux.
Scheme 1: General synthesis scheme of rhodanine derivatives
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