In vitro studies of potent aldose reductase inhibitors: Synthesis, characterization, biological evaluation and docking analysis of rhodanine-3-hippuric acid derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.103640

Inhibitors of aldose reductase are rate-limiting enzymes and could play a key role to prevent the complications of diabetes. In our attempt to develop novel inhibitors of aldose reductase, the derivatives of rhodanine-3-hippuric acid-pyrazole hybrid were synthesized and characterised by spectral data. The biological studies reveal that all the compounds show an excellent activity against ALR2 with IC₅₀ values ranging from 0.04 to 1.36 μM. Among these the synthesised compounds 6a-m, 6g and 6e showed specific inhibitory activity with IC₅₀ values of 0.04 and 0.06 μM respectively against ALR2 and found to be more potent than epalrestat (IC₅₀ = 0.87 μM), the only aldose reductase inhibitor currently used in the therapy. Molecular docking analysis using the AR-NADP⁺ complex as a receptor was performed with all the synthesized compounds. All the compounds exhibit a well-defined binding mode within the AR active site, similarly to previous described AR inhibitors, with the anion head group bound to the catalytic center, blocking thus its activity. By forming hydrogen bonds with Tyr48 and His110 of the protein from ALR2 (PDB ID: 2FZD), the compounds 6g and 6e interrupt the proton donation mechanism, which is necessary for the catalytic activity of ALR2.

Full text of DOI:10.1016/j.bioorg.2020.103640

Journal Pre-proofs
In vitro studies of potent aldose reductase inhibitors: synthesis, characteriza-
tion, biological evaluation and docking analysis of rhodanine-3-hippuric acid
derivatives
Stephen Kumar Celestina, Kaveri Sundaram, Subban Ravi
PII:
S0045-2068(19)31319-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103640
YBIOO 103640
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Bioorganic Chemistry
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Accepted Date:
10 August 2019
28 November 2019
28 January 2020
Please cite this article as: S. Kumar Celestina, K. Sundaram, S. Ravi, In vitro studies of potent aldose reductase
inhibitors: synthesis, characterization, biological evaluation and docking analysis of rhodanine-3-hippuric acid
derivatives, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103640
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In vitro studies of potent aldose reductase inhibitors: synthesis,
characterization, biological evaluation and docking analysis of rhodanine-3-
hippuric acid derivatives
Stephen Kumar Celestinaa, Kaveri Sundarama,* and Subban Ravia
aDepartment of Chemistry, Karpagam Academy of Higher Education, Coimbatore-641021,
Tamil Nadu, India

Graphical abstract

Highlights
 Novel rhodanine-3-hippuric acid derivatives were synthesized.
 In vitro aldose reductase inhibitory activity was carried out for the synthesized
compounds.
 Compounds 6g and 6e are found to be more active and the IC50 values are 0.04 µM and
0.06 µM.
 All the compounds are well occupied in the receptor cavity.

In vitro studies of potent aldose reductase inhibitors: synthesis,
characterization, biological evaluation and docking analysis of rhodanine-3-
hippuric acid derivatives
Stephen Kumar Celestina, Kaveri Sundaram* and Subban Ravi
*Department of Chemistry, Karpagam Academy of Higher Education, Coimbatore-641021,
Tamil Nadu, India
Abstract
Inhibitors of aldose reductase are rate-limiting enzymes and could play a key role to prevent
the complications of diabetes. In our attempt to develop novel inhibitors of aldose reductase,
the derivatives of rhodanine-3-hippuric acid-pyrazole hybrid were synthesized and
characterised by spectral data. The biological studies reveal that all the compounds show an
excellent activity against ALR2 with IC50 values ranging from 0.04 -1.36 µM. Among these
the synthesised compounds 6a-m, 6g and 6e showed specific inhibitory activity with IC50
values of 0.04 and 0.06 µM respectively against ALR2 and found to be more potent than
epalrestat (IC50 = 0.87 μM), the only aldose reductase inhibitor currently used in the therapy.
Molecular docking analysis using the AR-NADP+ complex as a receptor was performed with
all the synthesized compounds. All the compounds exhibit a well-defined binding mode within
the AR active site, similarly to previous described AR inhibitors, with the anion head group
bound to the catalytic center, blocking thus its activity. By forming hydrogen bonds with Tyr48
and His110 of the protein from ALR2 (PDB ID: 2FZD), the compounds 6g and 6e interrupt
the proton donation mechanism, which is necessary for the catalytic activity of ALR2.
Keywords: Aldose reductase, rhodanine-3-hippuric acid, pyrazole pharmacophore, molecular
docking
1. Introduction
Diabetes mellitus is a major health threat in most of the developed and developing countries
and a chronic disease that causes mainly microvascular and macrovascular complications [1].
The international diabetes federations predict that approximately 600 million adults will have
diabetes by the year 2035 [2]. The main problem of diabetes is to maintain the normal level of
glucose among the patients [3]. The high level of glucose present in the blood of diabetic
patients affects the functions of many tissues including the retina in the eye, kidney and
peripheral nerves and results in long term complications [4]. It is believed that the diabetic

complications are due to the accumulation of a significant amount of sugar caused by polyol
pathway [5,6].
In the polyol pathway, the first step is the rate-determining step in which the aldose reductase
enzyme ALR2 catalyses the formation of sorbitol by the reduction of glucose and converts
NADPH into NADP+. Therefore, aldose reductase inhibition in the rate-determining step of the
polyol pathway, is a useful method of planning for the prevention and treatment of
complications arising due to chronic diabetes [7-10]. Tireless efforts in this approach have led
to the identification of a large number of AR inhibitors to treat the complications of diabetes
[10-13]. Broadly, two classes of ARIs have been developed and evaluated: (a) acetic acid
derivatives like alrestatin, tolrestat, zopolrestat and epalrestat and (b) Cyclic imides like
sorbinil [14]. In spite of the intense efforts made in this direction, only a few thiazolidine-2,4-
diones such as glitazones, ciglitazone, rosiglitazone and pioglitazone have entered into clinical
trials [15-19]. Apart from this all other developed ARIs are not successful in clinical trials
either due to side effects or poor pharmacokinetic properties or both [20]. Thus, it is the need
of the hour to develop novel ARIs with less toxic profile and improved efficacy.
We are fascinated with the recent reports on hybrid systems like pyrazole-rhodanine scaffolds
as potent aldose reductase inhibitors. They are rhodanine and pyrazole hybrid compounds and
non-carboxylic acid ARIs. The N-atom of the rhodanine ring is substituted with substituted
phenyl ring. The 5th position of the rhodanine ring is substituted with 1,3-diarylpyrazole
system. Also, only a limited number of substituents are used in the 1,3- diarylpyrazole system
and that too in the para position. Further, in their docking study there is no interaction with the
three key residues Trp111, Tyr48 and His110 available for binding at the substrate active site
of ALR2 [21]. According to Costantino et al. the minimum requirement to inhibit ALR2 is to
form hydrogen bonds with Trp111, Tyr48 and His110 [22]. In our earlier study we developed
a series of carboxylic acid ARIs with rhodanine scaffold and had substitution in the 5th position
with differently substituted arylidene ring. Prompted by this, in order to increase the efficacy
of the rhodanine-pyrazole hybrid compounds in the present work, we worked on carboxylic
acid ARIs with the pyrazole-rhodanine hybrid system with structural variations in both meta
and para positions of the aromatic ring of the 1,3- diarylpyrazole moiety. To get an insight into
the interaction between the potent inhibitors inside the active site of ALR2 molecular docking
was carried out.

2. Result and discussions
2.1. Chemistry
The target compounds 6a-m were prepared by a general procedure as outlined in scheme 1.
The pharmacophore rhodanine-3-hippuric acid 5 was achieved by the reaction of 4-
aminohippuric acid with cabon disulphide and chloroacetic acid. A series of pyrazole
derivatives 4a-m were synthesized according to the Vilsmeier-Haack procedure from
phenylhydrazine hydrochloride 1 and substituted acetophenones 2a-m. Rhodanine-3-hippuric
acid 5 was subjected to Knoevenagel condensation reaction with 4a-m to afford the target
compounds 6a-m. The structure of the desired compounds was elucidated by readily available
1
analytical techniques including IR, H and 13C NMR and elemental analysis. Among the
synthesized compounds, 6b was taken as a representative compound for spectroscopic
discussion. In the IR spectrum of rhodanine-3-hippuric acid derivatives, a strong absorption
band at 3400 cm-1 is due to –OH group. The NH stretching vibration band appeared at 2900
cm-1. The characteristic band of the carbonyl group appeared at 1710 cm-1 and the peaks at
1536 cm-1 and 1346 cm-1 belong to C=N and C=S respectively. In the 1H NMR spectrum of
newly synthesized compound 6b a singlet was observed at δ 3.88 (s, 2H) corresponding to the
N-CH2 protons of hippuric acid moiety. The exocyclic double bond of olefinic =C-H was
resonated at δ 7.77 ppm showing a Z configuration [23] and the strong distinctive group of =C-
H in the pyrazole ring was observed at δ 8.84 ppm. Moreover, the peak at 9.01 ppm was
assigned to NH proton of hippuric acid moiety, whereas the rest of the signals appeared
multiplet due to aromatic protons. The 13C NMR spectra exhibited signals for the newly
synthesized compounds at the expected region. The characteristic signal at 193.11 ppm due to
thiocarbonyl of rhodanine moiety and signals at 171.12 ppm and 165.22 ppm due to two
carbonyls of hippuric acid moiety (–COOH and –CO-NH) were also observed. In addition, 13C
NMR spectra showed a distinct peak at 166.36 ppm assigned to keto carbonyl group of
rhodanine moiety. Moreover, the resonance at 152.69 ppm belongs to C=N group of pyrazole
ring, whereas the signal at 138.53 ppm is assigned to exocyclic olefinic carbon. Further, the
peak at 41.28 ppm points to N-CH2 functionality and the rest of the signals from 115.61 ppm
to 137.65 ppm due to aromatic and pyrazole ring carbons. All other derivatives exhibit identical
results.
2.2. Aldose Reductase Inhibitors
The newly synthesized rhodanine-3-hippuric acid derivatives 6a-m were screened for their
effective inhibitory activity against aldose reductase enzyme, extracted from rat kidney tissue

and purified. The acetic acid group [24] and phenyl group [21] on N3 position of the rhodanine
ring, according to previously published works, lead to good activity against aldose reductase.
In the present work, we introduced a hippuric acid moiety consisting of benzimido and acetic
acid group on N-3 position of rhodanine nucleus, which promisingly exhibit a potential against
aldose reductase enzyme. The in vitro inhibitory evaluation indicates that a great number of
compounds are generally proved to have potential against ALR2 with sub micromolar level
concentrations. Epalrestat has been employed as positive standard.
The IC50 values of the derivatives 6a-m were generally shown to be from 0.04 to 1.36 μM
(Table 1). Among all the tested compounds, compound 6g that carried fluro atom on meta
position is found to have excellent activity against ALR2 with the IC50 value of 0.04μM, which
is 21.5 times more than standard drug epalrestat (IC50 = 0.87 μM). On the other hand, the para
substituent of fluoro derivative 6h has less activity (IC50 = 0.36 μM) than meta substituent, but
it has 2-fold more activity than epalrestat. Further, compounds 6e and 6i incorporated with
methyl and nitro substituents in meta position showed an IC50 value of 0.06 and 0.08 μM
respectively, which is about 14.5 and 11.8 times more efficient respectively than standard drug.
However, its para derivatives 6f and 6j show slightly more activity than standard drug with the
0.64 and 0.76μM respectively. Compound 6a bearing bromo atom on the meta position exhibits
inhibition with IC50 value of 0.90 μM against aldose reductase and its IC50 value is slightly
lower than that of standard reference. The same observation is also made in the compound 6b,
which contains bromo atom on the para position showing the activity with the IC50 value of
1.12 μM. The ALR2 inhibitory activity of chloro substituent 6c displays a remarkable activity
with the IC50 values of 0.87 μM, compared to the standard inhibitor epalrestat. However, its
para substituent 6d shows more decreased inhibition against ALR2 with the IC50 value of 1.36
μM. Compound 6k with methyl group on meta position shows a moderate inhibitory activity
when compared to the standard epalrestat for ALR2 which shows an IC50 value of 0.98 μM.
On the other hand, the para substituent of compound 6l shows a weak inhibitory activity (IC50:
1.10 μM). Among the series, the compounds 6e, 6f, 6g, 6h, 6i and 6j are found to be more
potent inhibitors, particularly 6g and 6e are found to have excellent inhibition activity for
ALR2. Irrespective of the electron withdrawing and electron donating nature of the groups, the
compounds showed very good activity, but the activity depends upon the position in which the
substituents are present. Compounds having phenyl ring with meta substituents are more active
than the compounds having the phenyl ring in the para position.
2.3. Molecular Docking

Aldose reductase belongs to the enzyme of aldo-keto reductase superfamily. Aldose reductase,
in crystallized complex with ligand and site directed mutagenesis, allows the structure of
enzyme to be identified. The enzyme is composed of 315 amino acid residues [25]. The docking
studies were performed using X-ray crystal structures of the human ALR2. Based on these
studies, the ALR2 binding site can be separated into 3 regions: Anion binding pocket,
Specificity pocket, Hydrophobic pocket [26].
Docking studies were performed to investigate the binding mode in the series of compounds
using Autodock vina software [27,28]. All the compounds were docked into the binding pocket
of the Human ALR2 (PDB ID: 2FZD). From all the docking result (Table 1), the compounds
6g and 6e docked well in the active site of ALR2 with a better docking score -11.4 and -
11.3kcal/mol respectively, compared to other compounds, and they have high binding affinity
(Fig.2). All the synthesized compounds are compared with the reference compound epalrestat.
From our observation of docking results, the compound 6g and 6e are found to be more active
ALR2 inhibitors.
All the compounds are well occupied in the receptor cavity and form hydrophobic and
hydrogen bonding interactions. In hydrogen bonding interactions, the electron deficient
hydrogen in binding site is linked by a covalent bond to an electronegative atom in a drug or
vice versa. In synthesized compounds, the electron deficient hydrogen atom in the active site
of Tyr48, Gln183 and Lys77 is binding with the electronegative oxygen atom in acid group.
Electronegative oxygen in amide group is binding with hydrogen in His110. The lone pairs are
in sp2 hybridized orbitals. The nitrogen in hippuric acid ring cannot act as a hydrogen bond
acceptor because the lone pair interacts with the carbonyl group. Trp20 is binding with
thiocarbonyl group. The active site of Trp219, Phe122 and Trp20 is binding the target with
hydrophobic interactions. This type of interactions mainly happens in aromatic rings because
the binding region of the aromatic ring is a narrow slot rather than a planar surface. By forming
hydrogen bonds with Tyr48 and His110 the compounds interrupt the proton donation
mechanism, which is necessary for the catalytic activity of ALR2. Tyr48, His110 and Trp111,
the three key residues for binding along with the positively charged nicotinamide ring of the
oxidized cofactor form a positively charged anionic binding site that recognizes and binds
negatively charged inhibitors like carboxylates. This is the advantage we had in the present
series of compounds synthesised. In addition to this all the compounds establishes a huge
number of hydrophobic contacts with the enzyme due to the presence of an aromatic ring

neighbouring to the carboxylate system. Similar interactions were reported for the
commercially available ALR2 inhibitors like Zopolrestat and Sorbinil.
With respective to the compounds reported as in the reference [21] in their docking study there
is no interaction with the three key residues Trp111, Tyr48 and His110 available for binding at
the substrate active site of ALR2. According to Costantino et al. the minimum requirement to
inhibit ALR2 is to form hydrogen bonds with Trp111, Tyr48 and His110 [22]. For the
compounds 6a-m, these interactions were possible only because of the hippuric acid moiety.
The synthesised compounds 6a-m in the present study inhibited ALR2 in an uncompetitive
manner and the IC50 values are shown in the table. Results from molecular docking studies
were consistent with the pattern of inhibition of ALR2 and their specificity. When molecular
docking studies of 6a-m was carried out with proteins of ALR1 (PDB id: 3H4G), the binding
results are not satisfactory. This suggests that 6a-m are poor inhibitor of closely related
members of the aldo–keto reductase (AKR) superfamily, particularly aldehyde reductase
(ALR1). These results suggest that compounds 6g and 6e are promising agents to prevent or
treat diabetic complications.
Drug-like properties
Further drug-like properties of these compounds were predicted and analysed (Table 2). All
the synthesised compounds obey Lipinski rule with respect to the physic-chemical parameters
like cLog P (5<), number of H-bond donor (2 to 3) and number of H-bond acceptor (5 to 7)
and deviate only with respect to the molecular weight. The molecules violating more than one
of these rules may have problems with bioavailability. This showed that all the synthesised
compounds under investigation have good permeability and absorption.
3. Conclusion
In the present work, we synthesized a new series of pyrazole-rhodanine-3-hippuric acid
derivatives and developed aldose reductase inhibitors. All the tested compounds are confirmed
to be active, showing IC50 value in the sub micromolar range 0.04 – 1.36 μM. Particularly, the
compounds 6g and 6e are found to be more active than epalrestat. Docking studies were
performed for all the synthesized compounds on aldose reductase receptor. Among all the
compounds, 6g and 6e showed good interaction and docking score -11.4 and -11.3 kcal/mol
respectively. The enzymatic inhibition results proved that the pyrazole and rhodanine-3-
hippuric acid core is more favourable with the enzyme. The replacement of electron
withdrawing group and donating groups play better role with aldose reductase enzyme. Results

obtained through our analysis show that derivatives of pyrazole-rhodanine-3-hippuric acid
hybrid scaffolds may act as lead compounds to inhibit diabetic complications.
4. Experimental method
4.1. Chemistry
The compounds synthesized in the present study were characterized by spectral and elemental
analyses. Melting point was observed in a BUNA instrument and uncorrected. NMR spectra
were measured on a Bruker-400 spectrometer using TMS as standard and DMSO as solvent.
IR spectra were recorded on a Perkin Elmer spectrum RX I. Elemental analyses were observed
using a Perkin Elmer 240°C Elemental Analyzer.
4.2. Method for the synthesis of compound 3
An equimolar ratio of Phenyl hydrazine hydrochloride 1 and Sodium Acetate each of 0.01mol
were dissolved in ethanol. Then substituted aromatic methyl ketone 2 (0.01mol) was added
reflexed for 1hr. The crystallized product was used for further process without purification.
4.3. Method for the synthesis of compound 4
The compound 3 (0.001mol) was added to the cold solution of dimethyl formamide. Then
phosphorous oxychloride (0.004mol) was added dropwise with stirring and set for reflux for
about 5hrs. Afterwards, it was poured into cold water and neutralized with sodium bicarbonate.
Then the formed precipitate was filtered and dried.
4.4. Method for the synthesis of compound 5
p-amino hippuric acid (0.01mol) is dissolved in water and kept it to the stirrer. While stirring,
solution of potassium hydroxide (0.02 mol) was added. The solution was cooled to 0°C, the
solution of CS2 (0.01mol) was added. The mixture was kept for stirring for about 3hrs and
placed in a refrigerator for next 24hrs. Then the water solution of chloroacetic acid (0.01mol)
was added and the mixture was stirred for 3hrs. subsequently, the solution of hydrochloric acid
was added and reflux it for 2hrs. While cooling, a precipitate was appeared and then it was
filtered and dried.
4.5. Method for the synthesis of compound 6a-m
A mixture of compound 4 (0.001 mol) and 5 (0.001 mol) was refluxed in glacial acetic acid
and sodium acetate (0.001 mol) for 4-6hrs. After completion of the reaction, the reaction
mixture was poured into ice water and formed precipitate was filtered and dried.

4.5.1.
2-(4-((E)-5-((3-(3-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)benzamido)acetic acid (6a)
Yellow solid; Yield: 67%; mp: 245°C; IR (KBr) cm-1: 3412 (OH), 1714 (C=O), 1532 (C=N), 1341
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.864 (s, 1H, NHhippuric ring), 8.078-7.782 (m, 4H, ArH), 7.624-
7.538 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.158-7.042 (m, 4H, ArH), 3.981 (s, 2H, HN-CH2); 13C
NMR (100 MHz, DMSO-d6): δ 193.46 (C=S), 171.45 (COOHhippuric ring), 166.94 (NH-C=Ohippuric
ring), 165.86 (C=Orhodanine ring), 154.12 (C=Npyrazole ring), 138.96-114.68 (ArH and =C-H), 41.16
(CH2 hippuric ring); Anal. Calc. for C28H19BrN4O4S2: C, 54.28; H, 3.09; Br, 12.90; N, 9.04; O, 10.33; S,
10.35; Found: C, 54.22; H, 3.13; Br, 12.94; N, 9.06; O, 10.36; S, 10.39.
4.5.2.
2-(4-((E)-5-((3-(4-bromophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl) benzamido) acetic acid (6b)
Yellow solid; Yield: 69%; mp: 256°C; IR (KBr) cm-1: 3415 (OH), 1705 (C=O), 1528 (C=N), 1349
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.849 (s, 1H, NHhippuric ring), 8.065-7.752 (m, 5H, ArH), 7.618-
7.420 (m, 10H, ArH, =C-Hpyrazole ring and =C-H), 3.980 (s, 2H, HN-CH2); 13C NMR (100 MHz,
DMSO-d6): δ 193.11 (C=S), 171.12 (COOHhippuric ring), 166.36 (NH-C=Ohippuric ring), 165.92
(C=Orhodanine ring), 152.69 (C=Npyrazole ring), 138.53-115.61 (ArH and =C-H), 41.28 (CH2 hippuric
ring); Anal. Calc. for C28H19BrN4O4S2: C, 54.28; H, 3.09; Br, 12.90; N, 9.04; O, 10.33, S, 10.35; Found:
C, 54.24; H, 3.12; Br, 12.96; N, 9.09; O, 10.32, S, 10.31.
4.5.3.
2-(4-((E)-5-((3-(3-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)benzamido)acetic acid (6c)
Yellow solid; Yield: 63%; mp: 240°C; IR (KBr) cm-1: 3414(OH), 1718 (C=O), 1536 (C=N), 1345
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.868 (s, 1H, NHhippuric ring), 8.074-8.000 (m, 4H, ArH), 7.614-
7.522 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.156-7.048 (m, 4H, ArH), 3.984 (s, 2H, HN-CH2); 13C
NMR (100 MHz, DMSO-d6): δ 193.49 (C=S), 171.25 (COOHhippuric ring), 166.29 (NH-C=Ohippuric
ring), 165.72 (C=Orhodanine ring), 154.32 (C=Npyrazole ring), 138.46-114.72 (ArH and =C-H), 41.38
(CH2 hippuric ring); Anal.Calc. for C28H19ClN4O4S2: C, 58.48, H, 3.33, Cl, 6.19, N, 9.74, O, 11.13, S,
11.15; Found: C, 58.42, H, 3.37, Cl, 6.21, N, 9.76, O, 14.13, S, 11.18.
4.5.4.
2-(4-((E)-5-((3-(4-chlorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)benzamido)acetic acid (6d)
Yellow solid; Yield: 66%; mp: 255°C; IR (KBr) cm-1: 3428 (OH), 1712 (C=O), 1528 (C=N), 1354
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.862 (s, 1H, NHhippuric ring), 8.079-7.782 (m, 4H, ArH), 7.612-
7.511 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.162-7.052 (m, 4H, ArH), 3.962 (s, 2H, HN-CH2); 13C

NMR (100 MHz, DMSO-d6): δ 193.44 (C=S), 171.05 (COOHhippuric ring), 166.59 (NH-C=Ohippuric
ring), 165.92 (C=Orhodanine ring), 154.18 (C=Npyrazole ring), 138.76-114.72 (ArH and =C-H), 41.26
(CH2 hippuric ring); Anal. Calc. for C28H19ClN4O4S2: C, 58.48; H, 3.33; Cl, 6.17; N, 9.74; O, 11.13;
S,11.15; Found: C, 58.52; H, 3.35; Cl, 6.19; N, 9.78; O, 11.16; S,11.12.
4.5.5.
2-(4-((E)-4-oxo-5-((1-phenyl-3-m-tolyl-1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-3-
yl)benzamido)acetic acid (6e)
Yellow solid; Yield: 61%; mp: 260°C; IR (KBr) cm-1: 3412 (OH), 1713 (C=O), 1522 (C=N), 1350
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.861 (s, 1H, NHhippuric ring), 8.072-8.001 (m, 4H, ArH), 7.618-
7.534 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.453-7.346 (m, 4H, ArH), 3. 978 (s, 2H, HN-CH2),
2.382 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6): δ 193.62 (C=S), 171.14 (COOHhippuric ring),
166.48 (NH-C=Ohippuric ring), 165.96 (C=Orhodanine ring), 154.38 (C=Npyrazole ring), 138.82-114.72
(ArH and =C-H), 41.26 (CH2 hippuric ring), 20.96 (CH3); Anal. Calc. for C29H22N4O4S2: C, 62.80; H,
4.00; N, 10.10; O, 11.54; S, 11.56; Found: C, 62.78; H, 4.05; N, 10.12; O, 11.58; S, 11.53.
4.5.6.
2-(4-((E)-4-oxo-5-((1-phenyl-3-p-tolyl-1H-pyrazol-4-yl)methylene)-2-
thioxothiazolidin-3-yl)benzamido)acetic acid (6f)
Yellow solid; Yield: 64%; mp: 260 °C; IR (KBr) cm-1: 3412 (OH), 1714(C=O), 1524 (C=N), 1345
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.864 (s, 1H, NHhippuric ring), 8.092-8.007 (m, 4H, ArH), 7.604-
7.538 ( m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.458-7.376 (m, 4H, ArH), 3.972 (s, 2H, HN-CH2),
2.397 (s, 3H, -CH3); 13C NMR (100 MHz, DMSO-d6): δ 193.19 (C=S), 171.09 (COOHhippuric ring),
166.45 (NH-C=Ohippuric ring), 165.89 (C=Orhodanine ring), 154.07 (C=Npyrazole ring), 138.82-115.57
(ArH and =C-H), 41.25 (CH2 hippuric ring), 20.86 (CH3); Anal. Calc. for C29H22N4O4S2: C, 62.80; H,
4.00; N, 10.10; O, 11.54; S, 11.56; Found: C, 62.84; H, 4.03; N, 10.08; O, 11.58; S, 11.52.
4.5.7. 2-(4-((E)-5-((3-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)4-oxo-2-thioxothiazolidin-3-
yl)benzamido)acetic acid (6g)
Yellow solid; Yield: 69%; mp: 255°C; IR (KBr) cm-1: 3421 (OH), 1714 (C=O), 1534 (C=N), 1348
1
(C=S); H NMR (400 MHz, DMSO-d6): δ 8.864 (s, 1H, NHhippuric ring), 8.078-8.002 (m, 4H, ArH),
7.622-7.54 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.159-7.042 (m, 4H, ArH), 3.988 (s, 2H, HN-CH2);
13C NMR (100 MHz, DMSO-d6): δ 193.56 (C=S), 171.54 (COOHhippuric ring), 166.86 (NH-
C=Ohippuric ring), 165.92 (C=Orhodanine ring), 154.42 (C=Npyrazole ring), 138.86-115.76 (ArH and =C-
H), 41.36 (CH2 hippuric ring); Anal. Calc. for C28H19FN4O4S2: C, 60.20; H, 3.43; F, 3.40; N, 10.03; O,
11.46; S, 11.48; Found: C, 60.18; H, 3.45; F, 3.43; N, 10.07; O, 11.48; S, 11.42.

4.5.8.
2-(4-((E)-5-((3-(4-fluorophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)benzamido)acetic acid (6h)
Yellow solid; Yield: 70%; mp: 257°C; IR (KBr) cm-1: 3424 (OH), 1716 (C=O), 1524 (C=N), 1356
1
(C=S); H NMR (400 MHz, DMSO-d6): δ 8.869 (s, 1H, NHhippuric ring), 8. 072-7.782 (m, 4H, ArH),
7.624- 7.532 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.153-7.042 (m, 4H, ArH), 3.964 (s, 2H, HN-
CH2); 13C NMR (100 MHz, DMSO-d6): δ 193.54 (C=S), 171.38 (COOHhippuric ring), 166.86 (NH-
C=Ohippuric ring), 165.74 (C=Orhodanine ring), 154.28 (C=Npyrazole ring), 138.86-115.56 (ArH and =C-
H), 41.34 (CH2 hippuric ring); Anal. Calc. for C28H19FN4O4S2: C,60.20; H, 3.43; F, 3.40; N, 10.03; O,
11.46; S, 11.48; Found: C,60.25; H, 3.41; F, 3.43; N, 10.08; O, 11.42; S, 11.50.
4.5.9. 2-(4-((E)-5-((3-(3-nitrophenyl)-1-phenyl-1H-pyrazol-4-methylene)-4-oxo-2-thioxothiazolidin-3-
yl)benzamido)acetic acid (6i)
Yellow solid; Yield: 65%; mp 240°C; IR (KBr) cm-1: 3412 (OH), 1714 (C=O), 1536 (C=N), 1345
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.862 (s, 1H, NHhippuric ring), 8.074-8.000 (m, 4H, ArH), 7.624-
7.531 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.159-7.048 (m, 4H, ArH), 3.984 (s, 2H, HN-CH2); 13C
NMR (100 MHz, DMSO-d6): δ 193.64 (C=S), 171.58 (COOHhippuric ring), 166.69 ( NH-C=Ohippuric
ring), 165.68 (C=Orhodanine ring), 154.26 (C=Npyrazole ring), 138.86-115.72 (ArH and =C-H), 41.54
(CH2 hippuric ring); Anal. Calc. for C28H19N5O6S2: C, 57.43; H, 3.27; N, 11.96; O, 16.39; S, 10.95; Found:
C, 57.42; H, 3.29; N, 11.92; O, 16.41; S, 10.97.
4.5.10.
2-(4-((E)-5-((3-(4-nitrophenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl)benzamido)acetic acid (6j)
Yellow solid; Yield: 63%; mp: 250°C; IR (KBr) cm-1: 3422 (OH), 1724 (C=O), 1570 (C=N), 1358
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.868 (s, 1H, NHhippuric ring), 8.065-8.000 (m, 4H, ArH), 7.604-
7.538 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.164-7.042 (m, 4H, ArH), 3.964 (s, 2H, HN-CH2). 13C
NMR (100 MHz, DMSO-d6): δ 193.42 (C=S), 171.36 (COOHhippuric ring), 166.86 (NH-C=Ohippuric
ring), 165.74 (C=Orhodanine ring), 154.22 (C=Npyrazole ring), 138.86-115.56 (ArH and =C-H), 41.62
(CH2 hippuric ring); Anal.Calc. for C28H19N5O6S2: C, 57.43; H, 3.27; N, 11.96; O, 16.39; S, 10.95; Found:
C, 57.41; H, 3.26; N, 11.92; O, 16.41; S, 10.93.
4.5.11.
2-(4-((E)-5-((3-(3-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl) benzamido)acetic acid (6k)
Yellow solid; Yield: 68%; mp: 265°C; IR (KBr) cm-1: 3416 (OH), 1718 (C=O), 1528 (C=N), 1348
1
(C=S); H NMR (400 MHz, DMSO-d6): δ 8.866 ( s, 1H, NHhippuric ring ), 8.063-8.004 (m, 4H, ArH),
7.608-7.518 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.156-7.048 (m, 4H, ArH), 3.972 (s, 2H, HN-

CH2), 3.829 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6): δ 193.52 (C=S), 171.36
(COOHhippuric ring), 166.76 (NH-C=Ohippuric ring), 165.82 (C=Orhodanine ring), 154.48 (C=Npyrazole ring),
138.69-115.86 (ArH and =C-H), 55.86 (OCH3), 41.38 (CH2
ring); Anal. Calc. for
hippuric
C29H22N4O5S2: C, 61.04, H, 3.89, N, 9.82, O, 14.02, S, 11.24; Found: C, 61.01, H, 3.92, N, 9.84, O,
14.05, S, 11.26.
4.5.12.
2-(4-((E)-5-((3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene)-4-oxo-2-
thioxothiazolidin-3-yl) benzamido)acetic acid (6l)
Yellow solid; Yield: 65%; mp: 263°C ;IR (KBr) cm-1: 3422 (OH), 1714 (C=O), 1522 (C=N),
1351(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.852 (s, 1H, NHhippuric ring ), 8.084-7.896 (m, 4H, ArH),
7.614-7.410 (m, 7H, ArH, =C-Hpyrazole ring and =C-H), 7.151- 7.059 (m, 4H, ArH), 3.961( s, 2H,HN-
CH2), 3.828 (s, 3H,-OCH3); 13C NMR (100 MHz, DMSO-d6): δ 193.83 (C=S), 171.08
(COOHhippuric ring), 166.46 (NH-C=Ohippuric ring), 164.12 (C=Orhodanine ring), 154.26 (C=Npyrazole ring),
138.77-115.54 (ArH and =C-H), 55.28 (OCH3), 41.26 (CH2
ring); Anal. Calc. for
hippuric
C29H22N4O5S2: C, 61.04, H, 3.89, N, 9.82; O, 14.02; S, 11.24; Found: C, 61.06, H, 3.92, N, 9.83; O,
14.06; S, 11.26.
4.5.13.
2-(4-((E)-4-oxo-5-((1,3-diphenyl-1H-pyrazol-4-yl)methylene)-2-thioxothiazolidin-3-
yl)benzamido)acetic acid 6m)
Yellow Solid; Yield: 63%; mp: 240°C; IR (KBr) cm-1: 3418 (OH), 1716 (C=O), 1536 (C=N), 1346
(C=S); 1H NMR (400 MHz, DMSO-d6): δ 8.861 (s, 1H, NHhippuric ring), 8.062-7.781 (m, 4H, ArH), 7.628-
7.541 (m, 8H, ArH, C-Hpyrazole ring and =C-H), 7.149-7.052 (m, 4H, ArH), 3.979 (s, 2H, HN-CH2); 13C
NMR (100 MHz, DMSO-d6): δ 193.16 (C=S), 171.32 (COOHhippuric ring), 166.89 ( NH-C=O hippuric
ring), 165.74 (C=Orhodanine ring), 154.03 (C=Npyrazole ring), 138.93-114.56 (ArH and =C-H), 41.32
(CH2 hippuric ring); Anal. Calc. for C28H20N4O4S2: C, 62.21; H, 3.73; N, 10.36; O, 11.84; S, 11.86; Found:
C, 62.18; H, 3.78; N, 10.31; O, 11.86; S, 11.84.
4.6. Inhibitory method of aldose reductase
The enzyme ALR2 was isolated from the rat kidney removed from the freshly slaughtered
animals in the local slaughter house. Tissues (100-200g) were homogenised in a volumes of
10mM sodium phosphate buffer (pH 7.2), 2.0 mM EDTA dipotassium salt and 2.5 mM β-
mercaptothiol for 20 min. Homogenate was centrifuged at 10,000 rpm for 15 minutes at 4 °C
to remove insoluble material. Precipitated material containing lipids was discarded. Pure ALR2
was precipitated by addition of powdered ammonium sulphate to 80% saturation. After
centrifugation, supernatant was discarded and the precipitated enzyme was redissolved in

50mM NaCl and dialysed over night against 4 litres 50 nM NaCl. After dialysis, the volume of
the sample was recorded, treated with liquid nitrogen and samples stored at -80 °C for further
analysis.
The in vitro method of enzyme inhibition of aldose reductase was carried out by Hayman and
Kinoshita method [29]. For this assay the mixture contained 0.1 M Pottasium Phosphate buffer,
0.104 mM NADPH, distilled water, 0.2ml of enzyme source and 10 mM DL-Glyceraldehyde.
The sample compound was incubated along with the enzyme at 37°C for 20 mins. The reaction
mixture was begun by NADPH addition. Due to the oxidation of NADPH there was a change
in the absorbance at 340 nm, and it was measured by ultra violet-visible spectrometer. The
sample compound for inhibitory activity was predicted based on the decrease in the NADPH
absorbance at 340 nm. To correct for the non-enzymatic oxidation of NADPH, the rate of
NADPH oxidation in the presence of all the reaction mixture components except the substrate
was subtracted from each experimental rate. Most of the dose response curves were generated
using atleast five concentration of the compound with inhibitory activity between 20 % and
80%, with three replicates at each concentration, and the IC50 values were calculated by least
square analysis of the linear portion of the log (dose) versus response curves (r2 >0.95). The
experiments were performed in triplicate.
Percentage of inhibition = [T/C]-100 X 100,
C ¼ absorbance of control and T ¼ absorbance of test reaction
The 50% inhibitory concentration (IC50) was calculated.
4.7. Molecular docking method
The molecular interaction behind the inhibition mechanism of compound 6a-m and molecular
docking studies were carried out using Auto Dock Vina software. To perform with 2FZD
protein was downloaded from PDB in three-dimensional formation, and to remove the co-
crystallized ligand from the protein, the structures of the 6a-m ligand were drawn using
Chem3D ultra. In addition, 3D structures were established to apply partial charges and energy
minimization. It is found that the prediction of binding affinity by molecular docking is
consistent with the experimental study. Chimera software has been used to observe the
interactions.
Acknowledgement

We thank Karpagam Academy of Higher Education for providing instrumentation
specialities to do the present research work. The authors are thankful to SAIF-STIC, Cochin
University of Science and Technology for their instrumentation support.
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R
NH2 .HCl
+
O
HN
CH3COONa
EtOH, H2O
N
DMF
NHN C
3a-m
N
POCl3
CHO
R
4a-m
R
1
2a-m
R
O
O
O
O
CH3COONa
CH3COOH
N
N
N
S
S
4a-m
N
+
NH
NH
HO
HO
S
S
O
O
5
R: a = 3-Br
R: b = 4-Br
R: c = 3-Cl
R: d = 4-Cl
R: e = 3-CH3
R: f = 4-CH3
R: g = 3-F
R: h = 4-F
6a-m
R: i = 3-NO2
R: j = 4-NO2
R: k = 3-OCH3
R: l = 4-OCH3
R: m = H
Scheme 1. Synthesis of substituted 5- benzylidene pyrazole rhodanine-3-hippuric acid 6a-m.

Fig. 1. Polyol Pathway of diabetic complications

Fig. 2. 3D and 2D structure of compound 6e and 6g with 2FZD. Non-polar hydrogen bond has
been removed for clarity in 3D structure. In 2D the green circle indicates the H-Bond
interaction, pink colour indicates Pi-Pi stacked interaction, yellow circle indicates Pi-S
interaction

Table 1
In vitro aldose reductase inhibitory activity and binding affinity of the
compounds 6a-m.
Compounds
R
IC50 [µM]
Binding Energy
[kcal/mol]
-11.2
6a
3-Br
4-Br
0.90
1.12
0.87
1.36
0.06
0.64
0.04
0.36
0.08
0.76
0.98
1.10
1.16
0.87
6b
-10.1
-11.2
-10.7
-11.3
-10.7
-11.4
-11.0
-11.3
-9.9
6c
3-Cl
6d
4-Cl
6e
3-CH3
4-CH3
3-F
6f
6g
6h
4-F
6i
3-NO2
4-NO2
3-OCH3
4-OCH3
H
6j
6k
-10.8
-9.0
6l
6m
-11.0
-9.7
Epalrestat
-

Table 2
Drug-like properties of the compounds 6a-m.
Compounds cLogP Solubility TPSA No. of H-
bond donor
No. of H-
bond
Molar
Refractivity
acceptor
5
6a
6b
6c
6d
6e
6f
4.59
4.60
4.52
4.55
4.31
4.34
4.29
4.34
1.87
2.12
4.00
4.01
3.96
-7.20
-7.20
-6.89
-6.89
-6.59
-6.59
-6.45
-6.45
-6.07
-6.07
-6.37
-6.37
-6.29
161.92
161.92
161.92
161.92
161.92
161.92
161.92
161.92
211.58
211.58
171.15
171.15
161.92
2
2
2
2
2
2
2
2
3
3
2
2
2
161.30
161.30
158.61
158.61
158.57
158.57
153.56
153.56
160.85
160.85
160.10
160.10
153.60
5
5
5
5
5
6
6
7
7
6
6
5
6g
6h
6i
6j
6k
6l
6m

Conflict of Interest
The authors declare that they have no conflicts of interest.