Design, synthesis, biological evaluation, and molecular modeling studies of rhodanine derivatives as pancreatic lipase inhibitors

Article abstract of DOI:10.1002/ardp.201900029

A series of rhodanine-3-acetic acid derivatives were synthesized via Knoevenagel condensation of rhodanine-3-acetic acid with various substituted aromatic aldehydes. The synthesized derivatives were screened in vitro for understanding the inhibitory potential towards pancreatic lipase (PL), a key enzyme responsible for the digestion of dietary fats. Derivative 8f exhibited a potential inhibitory activity towards PL (IC₅₀=5.16 μM), comparable to that of the standard drug, orlistat (0.99 μM). An increase in the density of the aromatic ring resulted in potential PL inhibition. The enzyme kinetics of 8f exhibited a reversible competitive-type inhibition, similar to that of orlistat. Derivative 8f exhibited a MolDock score of -125.19 kcal/mol in docking studies, and the results were in accordance with their PL inhibitory potential. Furthermore, the reactive carbonyl group of 8f existed at a distance adjacent to Ser152 (≈3 ?) similar to that of orlistat. Molecular dynamics simulation (10 ns) of the 8f-PL complex revealed a stable binding conformation of 8f in the active site of PL (maximum root mean square displacement of ≈2.25 ?). The present study identified novel rhodanine-3-acetic acid derivatives with promising PL inhibitory potential, and further lead optimization might result in potent PL inhibitors.

Full text of DOI:10.1002/ardp.201900029

Received: 25 January 2019
Revised: 27 June 2019
Accepted: 2 July 2019
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DOI: 10.1002/ardp.201900029
F U L L P A P E R
Design, synthesis, biological evaluation, and molecular
modelingstudiesofrhodaninederivativesaspancreaticlipase
inhibitors
Divya Chauhan1
Vikramdeep Monga1
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Ginson George2
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S. N. C. Sridhar2
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Rohit Bhatia1
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Atish T. Paul2
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1Department of Pharmaceutical Chemistry,
ISF College of Pharmacy, Ghal Kalan, Moga,
Punjab, India
Abstract
A series of rhodanine‐3‐acetic acid derivatives were synthesized via Knoevenagel
condensation of rhodanine‐3‐acetic acid with various substituted aromatic aldehydes.
The synthesized derivatives were screened in vitro for understanding the inhibitory
potential towards pancreatic lipase (PL), a key enzyme responsible for the digestion
of dietary fats. Derivative 8f exhibited a potential inhibitory activity towards PL
(IC50 =5.16 µM), comparable to that of the standard drug, orlistat (0.99 µM). An
increase in the density of the aromatic ring resulted in potential PL inhibition. The
enzyme kinetics of 8f exhibited a reversible competitive‐type inhibition, similar to
that of orlistat. Derivative 8f exhibited a MolDock score of ‒125.19 kcal/mol in
docking studies, and the results were in accordance with their PL inhibitory potential.
Furthermore, the reactive carbonyl group of 8f existed at a distance adjacent to
Ser152 (≈3 Å) similar to that of orlistat. Molecular dynamics simulation (10 ns) of the
8f‒PL complex revealed a stable binding conformation of 8f in the active site of PL
(maximum root mean square displacement of ≈2.25 Å). The present study identified
novel rhodanine‐3‐acetic acid derivatives with promising PL inhibitory potential, and
further lead optimization might result in potent PL inhibitors.
2Laboratory of Natural Product Chemistry,
Department of Pharmacy, Birla Institute of
Technology and Science (Pilani campus), Pilani,
Rajasthan, India
Correspondence
Atish T. Paul, Laboratory of Natural Product
Chemistry, Department of Pharmacy, Birla
Institute of Technology and Science (Pilani
campus), Pilani, 333 031 Rajasthan, India.
Email: atish.paul@pilani.bits-pilani.ac.in
Vikramdeep Monga, Department of
Pharmaceutical Chemistry, ISF College of
Pharmacy, Ghal Kalan, Moga, 142001 Punjab,
India.
Email: vikramdeepmonga@gmail.com and
vikramdeepmonga@yahoo.com
K E Y W O R D S
enzyme kinetics, molecular modeling, obesity, pancreatic lipase, rhodanine
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1
INTRODUCTION
and we are living in a world where obesity kills more people than being
underweight.[4] With over 2.8 million deaths per year, obesity ranks fifth
Obesity is a complex and multifactorial disease that arises mainly due to
the modern lifestyle and increased consumption of high‐fat diets.[1]
Obesity is characterized by excessive deposition of lipids in the body,
which results from long term imbalance between the calories consumed
and calories expended.[2] Moreover, obesity is associated with various
comorbid conditions including diabetes mellitus, musculoskeletal
disorders, and cardiovascular diseases, and so on, posing a major health
risk to obese patients.[3] As per the World Health Organization (WHO),
the prevalence of obesity has nearly tripled between 1975 and 2016
among global deaths.[5] Hence, obesity has become a global threat to
developed countries as well as developing countries.
Numerous therapeutic targets and medications are available for the
treatment of obesity. Among them, the reduction in fat digestion that
would reduce the absorption of fat in the GI tract is considered to be a
promising target for the prevention of obesity. Pancreatic lipase (PL) or
triacylglycerol lipase (EC 3.1.1.3), a key enzyme responsible for the
digestion of dietary lipids from the duodenum, has been identified as the
crucial target for regulation of lipid absorption.[6] Orlistat, a saturated
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Arch Pharm Chem Life Sci. 2019;e1900029.
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derivative of lipstatin, remains the only PL inhibitor approved for long‐
term treatment of obesity. However, recent reports from the Food and
Drug Administration indicated severe adverse effects with long term
administration of orlistat, including hepatotoxicity, acute pancreatitis,
and so on.[7] Thus, the discovery of safer and effective PL inhibitors is
highly desirable.
have designed structural derivatives of thiazolidine‐2,4‐dione, that is,
the rhodanine class of scaffolds possessing a 2‐thioxo group instead
of the 2‐oxo group as present in TZD, and explored them as possible
PL inhibitors. Accordingly, the present study involved the design,
synthesis, characterization, in vitro evaluation, and molecular
modeling studies of novel thioxo‐thiazolidinone acetic acid (rhoda-
nine acetic acid [RAA]) derivatives as potent PL inhibitors.
In recent years, five‐membered nitrogen heterocycles have gained
prominent significance in PL inhibition. Examples include oxadia-
zoles[8,9] and 1,3‐pyrazoles[10], and so on, which exhibited potential PL
inhibition. By considering this fact and as a part of our drug discovery
program, we have identified diaryl substituted pyrazole clubbed
thiazolidinediones (TZD) as potential PL inhibitors in our previous
study, wherein the nitrogen‐centered diamide interacted with Ser152
and diaryl pyrazole wing interacted with Phe77 and Phe215 of the lid
domain (Gly76–Lys80 and Leu213–Met217).[11]
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
The synthetic strategy for various arylidene derivatives of rhodanine‐
3‐acetic acid (RAA, 8a‒n) is represented in Scheme 1. Rhodanine‐3‐
acetic acid intermediate (RAA, 3) required for the preparation of
target derivatives was synthesized by cyclizing the glycine with carbon
disulfide and sodium chloroacetate under acidic conditions, using a
previously reported procedure.[17] Treatment of benzyl bromide with
different commercially available hydroxyl‐substituted aromatic alde-
hydes (5‐chloro‐2‐hydroxybenzaldehyde, 5‐chloro‐2‐hydroxy‐3‐meth-
oxybenzaldehyde, 2‐hydroxy‐5‐nitrobenzaldehyde, and 2‐hydroxy‐1‐
naphthaldehyde) in the presence of anhydrous K2CO3 in dry acetone
afforded the intermediates 6a‒d. The target compounds 8a‒n (Table 1)
were synthesized by Knoevenagel condensation of rhodanine‐3‐acetic
acid (3) with various substituted aromatic aldehydes (6a‒d and 7). To
simplify the ease of preparation and isolation of various target
derivatives, we explored different reaction conditions involving
various solvents and catalytic bases for this reaction, including acetic
acid/sodium acetate, toluene/piperidine, and ethanol/piperidine.[18,19]
It was found that the ethanol/piperidine system was the most practical
one, with short reaction times (2‒4 hr) and straightforward product
isolation. Under these conditions, various rhodanine derivatives were
successfully synthesized and in all cases, the product was precipitated
from the ethanolic solution upon cooling at room temperature, and in
turn was collected by simple filtration. Thus, all final derivatives were
obtained as solids in satisfactory yields (53–87%). Remarkably, this
procedure was also applicable to benzaldehydes containing a free
phenolic hydroxyl group (e.g., 4). The Knoevenagel condensation
reaction of RAA with aromatic aldehydes provided only the
Rhodanines and related substances like 2,4‐thiazolidinediones are
often considered as bioisosteric derivatives.[12] The molecules based on
the 2‐thioxo‐thiazolidine‐4‐one (rhodanine) core, as an imperious
privileged scaffold, are known for their broad range of pharmacological
profiles.[12–14] The structural diversity among 2‐thioxo‐1,3‐thiazolidine‐
4‐one derivatives is exclusively located in two positions, that is, the first
is the N‐3 position and the second is the nature of arylidene moiety at
the C‐5 position. Introduction of the lipophilic or hydrophilic or aromatic
group at one of the two key positions (or at both) offers a panel of
possibilities for drug design and also for the physicochemical property
studies. Epalrestat (Figure 1) is a rhodanine‐3‐acetic acid derivative and
acts as a potent aldose reductase inhibitor, which has been approved for
the treatment of diabetic complications, such as neuropathy, nephro-
pathy, and cataract. Furthermore, rhodanine derivatives have been
reported as potential PTP1B inhibitors for the treatment of diabetes
and obesity.[15] Recently, the therapeutic potential of rhodanine
derivatives as potential antidyslipidaemic agents has been demon-
strated by lowering the low‐density lipoprotein cholesterol, very low‐
density lipoprotein cholesterol and triglycerides, while increasing the
level of high‐density lipoprotein cholesterol, thereby decreasing the
atherogenic index.[16]
In the present study, the derivatives were designed based on the
concept of molecular hybridization. Considering the above facts and
pharmacophoric requirements for the design of PL inhibitors, we
FIGURE 1 Representation for
deciphering the pharmacophoric design of
pancreatic lipase (PL) inhibitors

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SCHEME 1 Synthesis of the target
compounds 8a‒n. Reagents and conditions:
(i) NH4OH, CS2, KOH, room temperature,
1 hr; ClCOONa, room temperature, 3 hr.
(ii) 1 N HCl, 110°C, reflux, 1 hr. (iii)
Acetone, K2CO3, reflux, 2 hr. (iv) EtOH,
AcOH, 80‒90°C, reflux, 3‒4 hr
TABLE 1 Physicochemical data of various rhodanine‐3‐acetic acid derivatives (8a–n)
S.No
1.
Compound number
8a
Structures
Chemical formula
Color
Yield (%)
Melting point (°C)
Rfa
C12H8FNO3S2
Yellow
67
231–234
0.79
2.
8b
C14H13NO5S2
Orange
72
240–243
0.76
3.
4.
8c
8d
C15H15NO6S2
Yellow
Yellow
83
59
193–196
250–252
0.68
0.70
C16H11NO3S2
5.
6.
8e
8f
C16H11NO3S2
Orange
Brown
63
87
235–237
0.70
0.62
(Lit. 238–239)3
C16H11NO4S2
248–251
7.
8g
C12H8ClNO4S2
Yellow
63
245–248
(Lit. 243–247)4
0.81
(Continues)

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TABLE 1 (Continued)
S.No
8.
Compound number
Structures
Chemical formula
Color
Yield (%)
Melting point (°C)
Rfa
8h
C13H10ClNO5S2
Orange
74
248–250
0.78
9.
8i
C12H8N2O6S2
Dark brown
82
227–230
0.80
10.
11.
8j
C13H10N2O7S2
Green
Yellow
55
75
245–248
229–231
0.76
0.80
8k
C19H14ClNO4S2
12.
8l
C20H16ClNO5S2
Yellow
78
244–246
0.74
13.
8m
C19H14N2O6S2
Yellow‐brown
79
212–215
0.82
14.
8n
C23H17NO4S2
Yellow
47
233–235
0.76
aToluene/ethyl acetate/formic acid = 4:0.8:0.2.
thermodynamically more stable Z isomer, as determined by the
chemical shift of the methine proton.[20,21] The physicochemical
properties of various synthesized derivatives are given in Table 1.
The synthesized derivatives were characterized by FTIR, 1H, and
13C NMR, mass spectroscopy and elemental analysis data, which fully
supported their structural identity. The IR spectrum of title
derivatives (8a‒n) showed strong absorption bands in the range of
1,712–1,722 and 1,692–1,706 cm−1 due to two C═O groups
stretching. Derivatives 8a‒n showed typical absorption at
3,410–3,480 cm−1 due to OH group stretching, whereas a band due
to C═S group stretching vibrations appeared at 1,317–1,353 cm−1
.
Furthermore, the absorption band appeared in the range of

CHAUHAN ET AL.
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1,573–1,603 cm−1 due to aromatic C═C stretching. In the 1H NMR
spectrum, the disappearance of a peak due to the methylene group
and appearance of an arylidene (═CH–) peak confirms the
Knoevenagel condensation between various aromatic aldehydes
and rhodanine‐3‐acetic acid. The methine proton of the arylidene
group appeared either as a singlet or multiplet in the range of δ
7.55–7.93. In the 1H NMR spectrum ═CH– proton was deshielded
more (δ = 7.5–7.9 ppm) as expected in the Z‐form, relative to the
slightly shielded protons of the E‐form (δ = 6.2–6.3 ppm), thus
indicating that all the derivatives were obtained exclusively in Z‐
form. This downfield movement of the methine proton in (Z) C5‐
arylidene analogs were reported to be due to the deshielding effect
of the adjacent carbonyl group.[20] Moreover, the Z‐isomer is
thermodynamically more stable because of the intramolecular
hydrogen bond that can be formed between the hydrogen atom of
═CH and the oxygen atom of the carbonyl group in RAA.[21] A broad
singlet due to the proton of the carboxylic group appeared in the
range of δ 12.98–13.47, whereas the proton of the phenolic group in
derivatives 8f–j appeared as a singlet in the range of δ 10.83–11.33.
Protons of the methylene group (–N–CH2) in various title derivatives
8a–n resonated as a singlet at δ 4.60–4.86 ppm. In derivatives 8k–n,
another singlet due to the methylene protons of the benzylic group
(–CH2C6H5) resonated at δ = 5.30–5.44 ppm. All other protons were
observed in the expected regions. In 13C‐NMR, signals due to two
carbonyl carbons of RAA appeared at δ = 167.51–168.10 and
166.23–166.94 ppm, respectively. The signal due to thiocarbonyl
group resonated at δ = 189.01–193.97. All the derivatives showed
prominent signals at δ = 128.97–136.51 due to arylidene carbon
(–CH═). Furthermore, the derivatives 8a–n also showed character-
istic signals in the range of δ = 45.54–46.70 due to the carbon of the
CH2 group attached to the N‐3 position of the RAA ring. In
derivatives 8k–n, the signal due to benzylic carbon (–CH2C6H5)
appeared in the range of δ = 70.90–75.93 ppm. Finally, the assigned
structures of various derivatives were confirmed by their mass
spectra, where a characteristic [M+H]+ peak was observed. All
derivatives gave satisfactory elemental analysis.
TABLE 2 PL inhibitory activity of the synthesized rhodanine
derivatives 8a–n
Compound
Ar
IC50 (µM)a
8a
8b
8c
8d
8e
8f
3‐Flurophenyl
47.29 0.91
22.95 0.74
9.34 0.35
16.14 0.45
47.72 1.19
5.16 0.31
11.38 1.52
10.64 0.79
53.19 2.06
13.56 1.17
26.77 1.70
13.52 0.93
2,5‐Dimethoxy phenyl
2,3,4‐Trimethoxy phenyl
2‐Naphthyl
1‐Naphthyl
2‐Hydroxy naphthyl
5‐Chloro‐2‐hydroxyphenyl
5‐Chloro‐2‐hydroxy‐3‐methoxyphenyl
2‐Hydroxy‐5‐nitrophenyl
2‐Hydroxy‐3‐methoxy‐5‐nitrophenyl
2‐(Benzyloxy)‐5‐chlorophenyl
8g
8h
8i
8j
8k
8l
2‐(Benzyloxy)‐5‐chloro‐3‐
methoxyphenyl
8m
2‐(Benzyloxy)‐5‐nitrophenyl
17.51 1.45
25.12 2.54
0.99 0.11
8n
2‐(Benzyloxy)naphthyl
Orlistat
–
Abbreviations: PL, pancreatic lipase; SEM, standard error of mean.
aAll experiments were performed in triplicate and the results are
expressed as mean SEM.
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2.2.2
Enzyme kinetics study
To understand the nature of inhibition, an enzyme kinetics study
for the most active derivative (8f) was performed by using a
protocol standardized in our laboratory.[22] The protocol was
performed at three concentration levels of the inhibitor and a
double reciprocal Lineweaver–Burk plot was acquired. As
represented in Figure 2 and Table 3, the Km values increased in
proportion to inhibitor concentration, indicating a reversible
competitive inhibition similar to that of orlistat from the previous
study. Furthermore, derivative 8f exhibited an inhibition constant
(Ki) value of 2.472 µM as calculated from the Cheng–Prusoff
equation.[23]
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2.2
Biological activity
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2.2.1
In vitro PL inhibitory activity
The synthesized derivatives were subjected to in vitro PL
inhibition assay as per the protocol standardized in our
laboratory.[22] PL inhibitory activities of various synthesized
derivatives (8a‒n) are represented in Table 2. Derivative 8f
exhibited the most potent PL inhibitory activity of 5.16 µM,
comparable to that of orlistat (0.99 µM), followed by derivative
8c (9.34 µM). Derivatives 8h, 8g, 8l, 8j, 8d, and 8m also exhibited
a potential PL inhibitory activity of 10.64, 11.38, 13.52, 13.56,
16.14, and 17.51 µM, respectively. Derivatives 8b, 8k, and 8n
exhibited moderate activity (>20 µM) and all other derivatives
(such as 8a, 8e, and 8i) exhibited weak PL inhibitory activity
(>40 µM).
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2.2.3
Structure–activity relationship
Based on the PL inhibitory potential of the synthesized derivatives, a
preliminary structural activity relationship has been derived. As shown
in Figure 3, the potential of the derivatives to inhibit PL varied with
respect to the ring density and the substitutions on the aromatic ring.
Previously, it was reported that the hydrophobic aromatic moieties
attached with the reactive carbonyl groups help in opening the
conformation of the lid domain of PL. For instance, derivative 8f

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2
1.5
1
FIGURE 2 Lineweaver‒Burk plot of 8f
representing reversible competitive
inhibition
High Inhibitor (5 µM)
Medium Inhibitor (2.5 µM)
Low Inhibitor (1.25 µM)
No Inhibitor
0.5
0
-0.03
-0.02
-0.01
0
0.01
0.02
0.03
1/S
0.04
0.05
-0.5
TABLE 3 Vmax and Km values calculated from the Lineweaver–
Burk plot for different concentrations of 8f
position compared to derivative 8h exhibited a similar kind of reduction
in the inhibitory activity (IC50 =10.64 [8h] to 13.56 µM). In contrast, the
introduction of an additional electron‐donating methoxy group at the
third position of the phenyl ring resulted in compounds 8h and 8j
(IC50 = 10.64 and 13.56 µM) with higher PL inhibitory activities
compared to the nonmethoxy counterparts 8g and 8i (IC50 =11.38
and 53.19 µM), respectively. Masking of the free hydroxyl group
(in 8g, 8h, and 8f) with benzyl substitution provided derivatives
8k, 8l, and 8n, which display a reduced PL inhibitory potential as
compared to their non benzylated counterpart with the exception of
derivative 8m, in which the inhibitory potency was significantly
improved (IC50 = 17.51 µM) compared to its parent counterpart (8i).
I (µM)
0
Vmax (µM/min)
0.07401
Km (µM)
64.120
64.629
65.285
66.289
1.25
2.5
0.07423
0.07535
5.0
0.07652
possessing a dense aromatic ring (bulky 2‐hydroxy naphthyl substitu-
tion) exhibited the maximum PL inhibitory potential (IC50 =5.16 µM).
Interestingly, a drastic variation in the activity was observed with the
removal of the 2‐hydroxy group and with respect to the change in the
attachment position of the dense aromatic ring (naphthyl ring).
Derivative 8d, with a 2‐naphthyl ring substitution, exhibited a higher
PL inhibitory potential (IC50 = 16.14 µM) as compared to 8e possessing
1‐naphthyl ring substitution (IC50 =47.72 µM). Substitution with 2,3,4‐
trimethoxyphenyl ring imparted potential inhibitory activity to the
derivative 8c (IC50 =9.16 µM) as compared to that of dimethoxy
substitution (8b, IC50 =22.95 µM). Surprisingly, the presence of the Cl
group (8g and 8h) at the fifth position of the phenyl ring resulted in a
significant inhibitory effect with IC50 values of 11.38 and 10.64 µM,
respectively. Furthermore, results demonstrated that presence of
electron donating groups (such as –OCH3, –CH3, and –OH) on the aryl
rings resulted in potent to good PL inhibitory activity as compared to
that of electron withdrawing groups (such as –NO2, –F). Replacement of
–Cl (8g) with a more polarized electron withdrawing –NO2 substituent
furnished derivative 8i with weak inhibitory activity (IC50 = 53.19 µM).
Apart from this, derivative 8j possessing an additional nitro group at
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2.3
Docking and molecular dynamics simulations
Docking studies were performed using a Molegro Virtual Docker
(v 6.0) with validated grid parameters (see experimental data) for
understanding the interactions of the derivatives with the active
site amino acids of human PL (PDB ID: 1LPB). Structurally, the
active site of human PL consists of the catalytic triad, Ser152/
Asp176/His263, which is enclosed within
a hydrophobic lid
domain comprised of Gly76/Lys80 and Leu213/Met217.[11] The
whole series of derivatives docked into the active site of PL, and
MolDock scores along with key interactions exhibited by various
derivatives are summarized in Table 4. The docked poses of these
ligands were according to the hypothesis, wherein substituted
aromatic moieties exhibited a hydrophobic interaction with the
amino acid of the lid domain (Tyr114) while the reactive carbonyl
group of TZD ring existed near the Ser152. The docking scores
were in alignment to the PL inhibitory activity wherein derivative
8f exhibited a potential docking score of −125.19 kcal/mol. The
binding mode of the most active derivative 8f in the active site of
PL is shown in Figure 4a. The major H‐bond interactions included
Gly76, Phe77, His151, and Ser152. Apart from, pi–pi interactions
played a prominent role in the potential PL inhibitory activity
wherein a significant pi–pi interaction of the aromatic ring of 8f
with His263 was observed. Moreover, a variation in the position
and substitution on the aryl ring resulted in a diverse interaction
with the synthesized derivatives. For instance, derivative 8d
exhibited a pi–pi interaction with Phe215 of the lid domain,
FIGURE 3 Preliminary SAR of the synthesized derivatives
depicting the PL inhibitory activity (IC50) versus substitution. PL,
pancreatic lipase; SAR, structure‐activity relationship

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TABLE 4 Summary of MolDock scores and interactions exhibited by various synthesized compounds 8a–n
Compounds
MolDock Score (kcal/mol)
−113.29
H bond
Pi–pi
Pi–cation
His263
–
8a
Asp79, His151, Ser152
Gly76, Phe77, His151, Ser152
Phe77, His 151, Ser152, Phe215
Gly76, Phe77, His151, Ser152
Gly76, Phe77, His151
Phe215
Tyr114
Tyr114
Phe215
Tyr114
Phe215
Tyr 114
Tyr114
Tyr114
Tyr114
Phe215
–
8b
8c
−117.21
−121.33
His263
His263
–
8d
8e
−119.73
−110.25
8f
−125.19
Gly76, Phe77, His151, Ser152
Gly76, Phe77, Asp79, Ser152
Gly76, Phe77, His151, Ser152
Gly76, Asp79, Ser152, His263
Gly76, Asp79, Ser152, His263
Gly76, Phe77, His151, His263
Ser152
His263
8g
−119.57
His151, His263
8h
8i
−119.27
–
111.26
–
8j
−120.23
–
8k
8l
−117.67
–
−118.77
Arg256
8m
8n
Orlistat
−122.19
Gly76, Asp79, Ser152, His263
Phe77, Ser152, Leu153
His151, Ser152
Tyr114
Tyr114, Phe215
–
–
−116.92
His263
−152.58
–
[8,22,24]
which was not observed with 8e. The fact can be further justified
wherein derivative 8d exhibited a potential activity in compar-
ison to 8e. These results are in accordance to previous reports
that highlight the importance of aromatic moieties in hydro-
phobic interactions and the crucial impact of the reactive
carbonyl group in interaction with Ser152.
To understand
the probable role of the reactive carbonyl group in interaction
with Ser152 of the active site of PL, the binding pose of 8f was
superimposed with that of orlistat (Figure 4b) and
a two
dimensional representation of interaction mode of derivative 8f
FIGURE 4 (a) Binding pose of 8f in the
active site of PL; (b) overlay diagram of 8f
(yellow) over orlistat (white) representing
the distance of reactive carbonyl carbon
from Ser152; (c) two‐dimensional
interaction diagram of 8f. PL, pancreatic
lipase

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The initial MD run indicated a higher degree of pi–sulfur
interactions with the lid domain of the active site, which vanished
during the course of run time. However, derivative 8f predominantly
made stable pi–pi interactions with the amino acids of the lid domain
(Tyr114 and Phe215). Furthermore, derivative 8f made a variable H‐
bond interaction with Ser152 and Tyr114 throughout the MD.
|
2.3.1
In silico drug‐likeness predictions
Drug likeness is the complex balance between various molecular
properties and structural features that are helpful to predict
how similar is the designed derivative to the known drugs. The
drug‐likeness behavior of the designed compounds was estimated
via predicting their physico‐chemical parameters and this tool is
now widely used by various researchers to correlate the results
of biological findings with the physicochemical properties.[25,26]
In silico drug likeness prediction of various designed and
synthesized derivatives was carried out by using a Swiss ADME
predictor tool as depicted in Table 6. Percentage absorption (%
ABS) was calculated by using the formula %ABS = 109 ‒ (0.345 ×
TPSA). The synthesized derivatives displayed moderate absorp-
tion in the range of 50‒80%. The results showed (Table 6) that
the ILogP of all the derivatives was below 5.0, LogS values were
found to be around −5.0 with the molecular weight <500. Hence,
the various title derivatives showed no violation of the Lipinski
rule of five. These predictions revealed that the synthesized
analogs could be considered as drug candidates.
FIGURE 5 Ligand root mean square displacement (RMSD) of
derivative 8f acquired through the 10 ns MD run. MD, molecular
dynamics
is given in Figure 4c. Reactive carbonyl groups of the derivatives
existed approximately in equidistance from Ser152 (3.3 and 3.6 Å
for orlistat and derivative 8f, respectively) and also derivative 8f
fitted best within the cavity, which accounts for its highest
activity. Molecular docking results were in accordance with the
PL inhibitory activity exhibited by the derivatives and the binding
pose analysis validated the rationale for designing the rhodanine
based PL inhibitors.
A 10 ns molecular dynamics (MD) simulation of the 8f‒PL complex
was performed for understanding the binding mode and interaction in
a dynamic environment by using the CHARMM27 force field (see
section 4).[11,24] Derivative 8f exhibited a stable binding conformation
throughout the MD run, as confirmed through the ligand root mean
square displacement (RMSD) and exhibited a maximum of 2.25 Å at
2 ns (Figure 5). The interactions of the ligand with the active site of PL
in the dynamic environment are summarized in Table 5.
|
2.3.2
In silico toxicity prediction
Toxicity prediction is an essential tool to predict the safety of a
derivative. The safety profile of various synthesized derivatives was
predicted using PreADME and ProTox web‐based tools. The
predicted data (Table 7) revealed that the designed derivatives are
noncarcinogenic as shown by their negative values on carcinomouse
and carcinorat models. Medium risk for hERG (human ether‐a‐go‐go‐
related gene) inhibition indicated that the designed derivatives have
a minimum risk on the cardiac action potential. As predicted by
protox, the minimum lethal dose (LD50) for various derivatives was in
the range of 350–1,500 mg/kg that lies under Class 4. Derivatives
belonging to Class 4 are not harmful in this LD50 range even if
swallowed. From the toxicity predictions, it is evident that the
designed derivatives were safer for future studies.
TABLE 5 Summary of interactions exhibited by 8f through the
10 ns molecular dynamics run
Time
Pi‐pi
frame (ns) H bond interaction
Pi‐cation
Pi‐sulfur
0
1
Asp79
Phe215
–
Phe77, His151,
His263
Arg256 Tyr114, His263, Ala 178
Leu153
His151, His263
2
3
4
His263
Ser152
Ser152
–
–
–
Tyr114
Phe77
–
Ile 209
Tyr114
Ile209,
Pro180
|
3
CONCLUSION
5
6
7
8
9
Ser152
–
–
–
–
–
Pro180
Ile209
Pro180
–
Phe77
–
In the current study,
a series of novel substituted 2‐thioxo‐
Tyr114
Tyr114
Ser152
–
thiazolidin‐4‐one acetic acid derivatives were designed, synthesized,
and evaluated for their PL inhibitory activity. Of the derivatives, 8f
exhibited the best in vitro PL inhibitory activity (5.16 0.31 µM)
comparable to that of orlistat (0.99 0.11 µM). Enzyme inhibition
experiments showed that the derivatives acted as reversible
–
Phe77, Tyr114,
Phe215
–
His263
10
–
Phe77
–
–

CHAUHAN ET AL.
9 of 14
|
TABLE 6 Prediction of in silico drug likeness properties of compounds 8a–n
Compounds
TPSA
MW
RoB
<10
3
HBD
<5
1
HBA
<10
4
ILogP (MlogP)a
<5
LogSb
>−4
% ABS
–
Rule
8a
8b
8c
8d
8e
8f
≤140
<500
115.00
133.46
142.69
82.91
297.33
339.39
369.28
299.34
329.39
345.39
329.78
359.86
340.33
370.36
434.94
464.96
430.45
435.52
1.61
−3.40
−3.40
−3.49
−3.89
−4.43
−4.29
−3.70
−3.79
−3.18
−3.26
−4.83
−4.74
−4.79
−5.87
69.32
62.95
59.77
80.39
69.32
62.34
62.34
59.16
46.53
43.35
66.14
62.95
50.33
62.95
5
1
5
2.35
6
1
6
2.55
3
1
3
1.83
115.00
135.23
135.23
144.46
181.05
190.28
124.23
133.46
170.05
124.23
3
1
3
2.10
3
2
4
2.04
8g
8h
8i
3
2
4
1.78
4
2
5
2.09
4
2
6
1.05
8j
5
2
7
1.64
8k
8l
6
1
4
2.42
7
1
5
2.32
8m
8n
7
1
6
2.34
6
1
4
2.86
Abbreviations: ABS, percentage absorption; HBD, hydrogen hydrogen bind donors; MW, molecular weight; RoB, number of rotatable bonds; TPSA,
topological polar surface area.
aLogarithm of compound partition coefficient between n‐octanol and water.
bLogarithm of water solubility.
competitive inhibitors towards PL. Molecular docking studies of the
synthesized derivatives revealed that the carbonyl group of the
rhodanine moiety interacted with Ser152 whereas various aromatic
substitutions were involved in the interaction with the amino acids of
the lid domain. The obtained molecular docking studies results were
in accordance with the in vitro results. A 10 ns MD simulation of
derivative 8f in the active site of PL revealed a stable binding
confirmation with RMSD ≤ 2.25 Å. Furthermore, in silico drug‐like-
ness of the derivatives followed the Lipinski rule of five and the
toxicity data predicted the derivatives to be safe. To conclude, the
present study identified RAA derivatives as a novel class of potential
PL inhibitors, and further studies with generated hits might be
helpful for the design and development of potent clinical PL
inhibitors.
TABLE 7 In silico toxicity prediction of compounds 8a–n
Compound
Carcinomouse
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Negativea
Carcinorat
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
Negativeb
hERG inhibition
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
Medium riskc
ProTox predicted LD50 (mg/kg)
ProTox predicted class
Class 4
8a
8b
8c
8d
8e
8f
350
350
350
350
1,400
450
350
350
350
350
350
350
350
350
Class 4
Class 4
Class 4
Class 4
Class 4
8g
8h
8i
Class 4
Class 4
Class 4
8j
Class 4
8k
8l
Class 4
Class 4
8m
8n
Class 4
Class 4
aCompound computationally predicted to be safe in carcinomouse.
bCompound computationally predicted to be safe carcinorat.
cNo significant effect on the electrical activity of the heart.
Class 4: Not harmful on swallowing at LD50 range of 350–1,500 mg/kg.

10 of 14
CHAUHAN ET AL.
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|
4
EXPERIMENTAL
COOH), 167.26 (C═O), 44.76 (N–CH2), 35.94 (CH2); MS (ESI) m/z (%):
192.0 [M+H]+; Anal. calcd. for C5H5NO3S2: C, 31.40; H, 2.64; N, 7.32.
Found: C, 31.52; H, 2.51; N, 7.24.
|
4.1
Chemistry
|
4.1.1
General
Solvents and organic reagents were purchased from Sigma‐Aldrich,
Merck, and HiMedia and were used without further purification. All
the evaporations were carried out under reduced pressure on a
Buchi‐rotary evaporator. Reactions were monitored by thin‐layer
chromatography (TLC) using commercially available precoated
aluminum plates (Merck Kieselgel 60 F254 silica, 0.2 mm) with UV
light (254 nm), iodine, and vanillin–sulfuric acid as derivatization
agents. Melting points were recorded with a COMPLAB melting point
apparatus and uncorrected. IR spectra (KBr disks) were recorded on
a NICOLET‐380 FT‐IR spectrophotometer. 1H NMR and 13C NMR
spectra were recorded on a Bruker AM 300 (300 MHz) or a Bruker
Avance III 500 (500 MHz; 13C NMR at 75 and 125 MHz) spectro-
meter (Bruker Biosciences) using CDCl3 or DMSO‐d6 as the solvent
at room temperature within the range. Chemical shifts (δ) are
expressed as parts per million (ppm), using tetramethylsilane as the
internal standard. Splitting patterns are designated as follows; s,
singlet; d, doublet; dd, doublet of doublets; t, triplet; m, multiplet.
Chemical shift values (δ) are given in ppm. Mass spectra (MS; please
see the Supporting Information) were taken in ESI mode on an
Agilent 1100 LC–MS (Agilent, Palo Alto, CA). The results of elemental
analyses (% C, H, N) for all the synthesized derivatives were within
0.4% range of the theoretical value.
|
4.1.3
General procedure for the synthesis of
various O‐benzylated aromatic aldehydes
To a solution of aromatic aldehyde (1.52 g) in 8 ml of anhydrous
acetone contained in a round‐bottom flask was added potassium
carbonate (2.76 g) followed by addition of different alkyl/aryl halides
(1.1 eq). The reaction mixture was refluxed at 70°C with continuous
stirring until the completion of the reaction as evidenced by TLC.
Upon completion, the reaction mixture was poured into water and
extracted twice with chloroform (2 × 30 ml). The organic layers were
combined and washed with water (25 ml). The solvent was removed
in vacuum and the solid product so obtained was purified by
recrystallization from absolute ethanol to afford various O‐benzy-
lated aromatic aldehydes in satisfactory yield.
2‐(Benzyloxy)‐5‐chlorobenzaldehyde (6a)
White solid; yield 74%; m.p.: 86–88°C; IR (KBr) νmax (cm−1): 3,060
(aromatic C–H stretching), 2,921 (aliphatic C–H stretching), 1,710
(C═O), 1,609 (C═C), 1,332 (C═S), 1,262 (C–O stretching), 810 (C–Cl);
1H‐NMR (300 MHz, DMSO‐d6): 10.31 (s, 1H, CHO), 7.69–7.65 (m,
2H, Ar–H), 7.62–7.29 (m, 6H, Ar–H), 5.28 (s, 2H, –OCH2–).
The InChI codes of the investigated derivatives together with
biological activity data are provided as Supporting Information.
2‐(Benzyloxy)‐5‐chloro‐3‐methoxybenzaldehyde (6b)
Off white solid; yield 81%; m.p.: 64–67°C; IR (KBr) νmax (cm−1): 3,037
(aromatic C–H stretching), 2,943 (aliphatic C–H stretching), 1,703
(C═O), 1,612 (C═C), 1,356 (C═S), 1,278 (C–O stretching), 799 (C–Cl);
1H‐NMR (300 MHz, DMSO‐d6): 10.01 (s, 1H, CHO), 7.47–7.32 (m,
6H, Ar‐H), 7.05–7.02 (d, J = 8.4 Hz, 1H, Ar–H), 5.16 (s, 2H, –OCH2–).
|
4.1.2
General procedure for the
synthesis of rhodanine‐3‐acetic acid
(= 2‐[4‐oxo‐2‐thioxothiazolidin‐3‐yl]acetic acid)
In a round‐bottom flask equipped with a magnetic stirrer, glycine
(24.0 g; 320 mmol) was dissolved with 25% ammonium hydroxide
solution (70 ml) in water (10 ml). Then, carbon disulfide (24.3 g;
320 mmol) was added and the reaction mixture was stirred
vigorously for 1 hr. An aqueous solution of sodium chloroacetate
(37.1 g; 320 mmol) was then added dropwise to the solution over
15 min and stirring was continued at 25°C for 3 hr. Furthermore, the
reaction mixture was acidified with dilute HCl until pH 1.0 and
refluxed for 1 hr and neutralized with saturated NaHCO3 solution.
The resultant solution was acidified again with dilute HCl. The
cyclized product was extracted in ethyl acetate, dried over
anhydrous sodium sulfate, evaporated under reduced pressure, and
the residue was purified by recrystallization from ethyl acetate.
Molecular formula C5H5NO3S2; yellow solid, yield: 62%; m.p.
144–147°C (Lit. 145–148°C)[17]; IR (KBr) νmax (cm−1): 3,513 (–OH
stretching); 2,993 (aliphatic C–H stretching); 1,744 and 1,698 (C═O);
1,345 (C═S); 1,214 (C–N); 1H NMR (300 MHz, DMSO‐d6): δ (ppm)
2‐(Benzyloxy)‐5‐nitrobenzaldehyde (6c)
Creamish solid; yield 55%; m.p.: 135–137°C; IR (KBr) νmax (cm−1):
3,012 (aromatic C–H stretching), 2,932 (aliphatic C–H stretching),
1,697 (C═O), 1,591 (C═C), 1,523 and 1,380 (–NO2), 1,317 (C═S),
1,282 (C–O stretching); 1H‐NMR (300 MHz, DMSO‐d6): 10.64 (s, 1H,
CHO), 8.40–8.37 (d, J = 8.7 Hz, 1H, Ar–H), 8.31 (s, 1H, Ar–H),
7.49–7.37 (m, 6H, Ar–H), 5.42 (s, 2H, OCH2).
2‐(Benzyloxy)‐1‐naphthaldehyde (6d)
White solid; yield 68%; m.p.:112–114°C; IR (KBr) νmax (cm−1): 3,048
(aromatic C–H stretching), 2,926 (aliphatic C–H stretching), 1,708
(C═O), 1,601 (C═C), 1,332 (C═S), 1,262 (C–O stretching); 1H‐NMR
(300 MHz, DMSO‐d6): 10.84 (s, 1H, CHO), 9.12–9.09 (d, J = 8.7 Hz,
1H, Ar–H), 8.27–8.24 (d, J = 9.3 Hz, 1H, Ar–H), 7.94–7.91 (d,
J = 8.1 Hz, 1H, Ar–H), 7.83–7.78 (m, 2H, Ar–H), 7.69–7.33 (m, 6H,
Ar–H), 5.46 (s, 2H, –OCH2–).
10.02 (bs, 1H, –COOH); 4.71 (s, 2H, N–CH2–); 4.12 (s, 2H, –CH2); 13
NMR (DMSO‐d6, 75 MHz): δ (ppm) 202.68 (C═S), 173.73 (C═O,
C

CHAUHAN ET AL.
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|
4.1.4
General procedure for the synthesis of
119.56, 109.32, 62.34 (OCH3), 61.03 (OCH3), 56.70 (OCH3), 45.45
(N–CH2); MS (ESI) m/z (%): 370.0 [M+H]+; Anal. calcd. for
C15H15NO6S2: C, 48.77; H, 4.09; N, 3.79. Found: C, 48.64; H, 3.98;
N, 3.86.
arylidene derivatives of rhodanine‐3‐acetic acid
(8a‒n)
A mixture of rhodanine‐3‐acetic acid (20 mmol, 10.9 g) and sub-
stituted aromatic aldehyde (20 mmol) was suspended in absolute
alcohol and to this a catalytic amount of piperidine (16 mmol, 1.4 g)
and glacial acetic acid was added. The mixture was then allowed to
reflux with stirring at 80°C. The completion of the reaction was
monitored with the help of TLC. On completion, the reaction mixture
was concentrated, and poured in ice‐cold water and acidified with
glacial acetic acid to pH 3–4 to afford a solid product, which was
filtered and dried. The crude product was recrystallized from ethanol
to get pure product in satisfactory yield.[19]
(Z)‐2‐(5‐(Naphthalen‐1‐ylmethylene)‐4‐oxo‐2‐thioxothiazolidin‐3‐
yl)acetic acid (8d)
IR (KBr) νmax (cm−1): 3,419 (O–H), 3,005 (aromatic C–H stretching),
2,905 (aliphatic C–H stretching), 1,722 (C═O), 1,706 (C═O), 1,594
(C═C), 1,332 (C═S), 1,195 (C–O); 1H‐NMR (500 MHz, DMSO‐d6): δ
13.47 (bs; 1H OH), 8.55 (s, 1H, Ar–H), 8.22–8.20 (d, J = 7.8 Hz, 1 H,
Ar–H), 8.15‐8.13 (d, J = 8.2 Hz, 1H; Ar–H), 8.07–8.05 (d; J = 8.2 Hz;
1H, Ar–H), 7.77–7.66 (m, 4H, 3Ar–H, and –CH═), 4.79 (s, 2H,
N–CH2); 13C‐NMR (100 MHz, DMSO‐d6): δ 194.28 (C=S), 167.76
(COOH), 166.26 (CO), 133.80, 132.13 (–CH═), 131.52, 131.17,
130.44, 129.43, 128.23, 127.76, 127.48, 126.27, 125.70, 123.93,
45.52 (–N–CH2); MS (ESI) m/z (%): 330.0 [M+H]+; Anal. calcd. for
C16H11NO3S2: C, 58.34; H, 3.37; N, 4.25. Found: C, 58.57; H, 3.31; N,
4.18.
(Z)‐2‐(5‐(3‐Fluorobenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐3‐yl)acetic
acid (8a)
IR (KBr) νmax (cm−1): 3,446 (O–H), 2,985 (aromatic C–H stretching),
2,919 (aliphatic C–H stretching), 1,714 and 1,703 (C═O), 1,578
(C═C), 1,323 (C═S), 1,273 (C–O), 1195 (C–N); 1H‐NMR (300 MHz,
DMSO‐d6): δ 13.53 (s, 1H, OH), 7.90 (s, 1H, –CH═), 7.62–7.47 (m, 3H,
Ar–H), 7.42–7.36 (m, 1H, Ar–H), 4.75 (s, 2H, N–CH2); 13C‐NMR
(75 MHz, DMSO‐d6): δ 193.40 (C═S), 167.67 (COOH), 166.69 (C═O),
(Z)‐2‐(5‐(Naphthalen‐2‐ylmethylene)‐4‐oxo‐2‐thioxothiazolidin‐3‐
yl)acetic acid (8e)
IR (KBr) νmax (cm−1): 3,423 (O–H stretching), 3,093 (aromatic C–H
stretching), 2922 (aliphatic C–H stretching), 1,720 and 1,704 (C═O),
1,593 (C═C), 1,353 (C═S), 1,199 (C–N); 1H‐NMR (300 MHz, DMSO‐
d6): δ 13.76 (bs, 1H, OH), 8.21 (s, 1H, Ar–H), 8.10–7.97 (m, 4H, Ar–H
and –CH═), 7.74–7.59 (m, 3H, Ar–H), 4.74 (s, 2H, N–CH2); 13C‐NMR
(75 MHz, DMSO‐d6): δ 193.73 (C=S), 167.73 (COOH), 166.86 (CO),
134.34, 134.04 (–CH═), 133.23, 132.54, 130.88, 129.67, 129.46,
128.94, 128.22, 127.76, 126.77, 122.54, 45.71 (–N–CH2); MS (ESI)
m/z (%): 329.9 [M+H]+; Anal. calcd. for C16H11NO3S2: C, 58.34; H,
3.37; N, 4.25. Found: C, 58.45; H, 3.48; N, 4.27.[25]
164.40
(C–F),
161.15,
135.58–135.48
(–CH═),
132.87,
132.08–131.97 (d, 1C), 126.66, 123.96, 118.53–117.73 (m, 1C),
46.22 (N–CH2). MS (ESI) m/z (%): 298.0 [M+H]+; Anal. calcd. for
C12H8FNO3S2: C, 48.47; H, 2.71; N, 4.71. Found: C, 48.31; H, 2.63; N,
4.77.
(Z)‐2‐(5‐(2,5‐Dimethoxybenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐3‐
yl(acetic acid (8b)
IR (KBr) νmax (cm−1): 3,480 (O–H), 3,032 (aromatic C–H stretching),
2,914 (aliphatic C–H stretching), 1,713 and 1,695 (C═O), 1,584
(C═C), 1,329 (C═S), 1,188 (C–N); 1H‐NMR (300 MHz, DMSO‐d6): δ
7.89 (s, 1H, –CH═), 7.10 (s, 2H, Ar–H), 6.93 (s, 1H, Ar–H), 4.56 (s, 2H,
N–CH2), 3.86 (s, 3H, OCH3), 3.77 (s, 3H, OCH3); 13C‐NMR (75 MHz,
DMSO‐d6): δ 194.01 (C═S), 167.87 (COOH), 167.09 (C═O), 153.66
(C–OCH3), 153.00 (C–OCH3), 128.83 (–CH═), 123.10, 122.18,
119.35, 114.68, 113.69, 56.53 (OCH3), 56.04 (OCH3), 46.91
(N–CH2); MS (ESI) m/z (%): 340.0 [M+H]+; Anal. calcd. for
C14H13NO5S2: C, 49.55; H, 3.86; N, 4.13. Found: C, 49.71; H, 3.72;
N, 4.07.
(Z)‐2‐(5‐((2‐Hydroxynaphthalen‐1‐yl)methylene)‐4‐oxo‐2‐
thioxothiazolidin‐3‐yl)acetic acid (8f)
IR (KBr) νmax (cm−1): 3,410 (O–H), 3,057 (aromatic C–H stretching),
2,962 (aliphatic C–H stretching), 1,716 and 1,696 (C═O), 1,594
(C═C), 1,338 (C═S), 1,276 (C–O stretching); 1H‐NMR (500 MHz,
DMSO‐d6): δ 13.47 (bs, 1H, COOH), 8.59 (s, 1H, Ar–H), 8.26−8.24 (d,
J = 8.1 Hz, 1H, Ar–H), 8.19–8.17 (d, J = 8.7 Hz, 1H, Ar–H), 8.12–8.10
(d, J = 8.4 Hz, 1H, Ar–H), 7.81–7.69 (m, 3H, Ar–H and –CH═), 5.80 (s,
1H, OH), 4.84 (s, 2H, N–CH2); 13C‐NMR NMR (125 MHz, DMSO‐d6):
δ 193.86 (C═S), 167.72 (COOH), 166.89 (CO), 154.63 (C–OH),
133.72, 133.13 (–CH═), 131.76, 130.48, 129.86, 129.16, 128.64,
127.92, 126.16, 123.47, 118.35, 45.71 (–N–CH2); MS (ESI) m/z (%):
346.0 [M+H]+; Anal. calcd. for C16H11NO4S2: C, 55.64; H, 3.21; N,
4.06. Found: C, 55.52; H, 3.42; N, 4.12.
(Z)‐2‐(4‐Oxo‐2‐thioxo‐5‐(2,3,4‐trimethoxybenzylidene)‐
thiazolidin‐3‐yl)acetic acid (8c)
IR (KBr) νmax (cm−1): 3,440 (O–H), 3,019 (aromatic C–H stretching),
2,929 (aliphatic C–H stretching), 1,703 & 1,697 (C═O), 1,603 (C═C),
1,347 (C═S), 1,209 (C–O); 1H‐NMR (300 MHz, CDCl3): δ 13.46 (bs,
1H, OH), 7.87 (s, 1H, –CH═), 7.27–7.24 (d, J = 9.0 Hz, 1H, Ar–H),
7.03–7.00 (d, J = 9.0 Hz, 1H, Ar–H), 4.72 (2H, N–CH2), 3.89 (s, 6H,
2OCH3), 3.78 (s, 3H, OCH3); 13C‐NMR (75 MHz, CDCl3): δ 194.10
(C═S), 167.81 (COOH), 166.97 (C═O), 157.28 (COCH3), 153.65
(C–OCH3), 142.18 (C–OCH3), 129.51 (–CH═), 126.29, 120.14,
(Z)‐2‐(5‐(5‐Chloro‐2‐hydroxybenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐
3‐yl)acetic acid (8g)
IR (KBr) νmax (cm−1): 3,472 (O–H), 2,985 (aromatic C–H stretching),
2,929 (aliphatic C–H stretching), 1,714 and 1,703 (C═O), 1,573
(C═C), 1,329 (C═S), 785 (C–Cl); 1H‐NMR (300 MHz, DMSO‐d6):

12 of 14
CHAUHAN ET AL.
|
δ 13.54 (bs, 1H, COOH), 7.90 (s, 1H, –CH═), 7.47–7.37 (m, 2H, Ar–H),
7.01–6.98 (d, J = 8.6 Hz, 1H, Ar–H), 4.83 (s, 2H, N–CH2); 13C‐NMR
(75 MHz, DMSO‐d6): δ 193.97 (C═S), 167.72 (COOH), 166.92 (C═O),
156.69 (C–OH), 133.05 (–CH═), 129.51, 128.99, 123.77 (C–Cl),
122.73, 121.87, 118.54, 45.67 (N–CH2); MS (ESI) m/z (%): 330.7
[M+H]+; Anal. calcd. for C12H8ClNO4S2 (329.78): C, 43.70; H, 2.45; N,
4.25. Found: C, 43.76; H, 2.32; N, 4.34.[20]
(Z)‐2‐(5‐(2‐(Benzyloxy)‐5‐chlorobenzylidene)‐4‐oxo‐2‐thioxothiazoli-
din‐3‐yl)acetic acid (8k)
IR (KBr) νmax (cm−1): 3,423 (O–H), 3,068 (aromatic C–H stretching),
2,953 (aliphatic C–H stretching), 1,718 and 1,698 (C=O), 1,583
(C═C), 1,337 (C═S), 1,284 (C–O); 1H‐NMR (300 MHz, DMSO‐d6):
δ 7.91 (s, 1H, –CH═), 7.57–7.53 (dd, J = 2.4 and 8.7 Hz, 2H, Ar–H),
7.48–7.26 (m, 6H, Ar–H), 5.30 (s, 2H, OCH2), 4.63 (s, 2H, N–CH2);
13C‐NMR NMR (75 MHz, DMSO‐d6):
δ 193.56 (C═S), 167.70
(COOH), 166.82 (C═O), 156.44 (C–OCH2–), 136.44, 132.76
(–CH═), 129.29, 129.06 (2CH), 128.65, 128.16 (2CH), 127.35,
125.65, 124.44, 123.97 (C–Cl), 115.87, 70.90 (O–CH2), 46.27
(N–CH2); MS (ESI) m/z (%): 420.0 [M+H]+; Anal. calcd. for
C19H14ClNO4S2: C, 54.35; H, 3.36; N, 3.34. Found: C, 54.47; H,
3.48; N, 3.26.
(Z)‐2‐(5‐(5‐Chloro‐2‐hydroxy‐3‐methoxybenzylidene)‐4‐oxo‐2‐thiox-
othiazolidin‐3‐yl)acetic acid (8h)
IR (KBr) νmax (cm−1): 3,473 (O–H), 3,017 (aromatic C–H stretch-
ing), 2,967 (aliphatic C–H stretching), 1,716 and 1,701 (C═O),
1583 (C═C), 1,323 (C═S), 1,273 (C–O stretching); 1H‐NMR
(300 MHz, DMSO‐d6): 7.92 (s, 1H, –CH═), 7.19 (s, 1H, Ar–H),
6.96 (s, 1H, Ar–H), 4.70 (s, 2H, N–CH2), 3.88 (s, 3H, OCH3);
13C‐NMR (75 MHz, DMSO‐d6): δ 193.92 (C═S), 167.73 (COOH),
166.90 (C═O), 149.53 (C–OMe), 146.51 (C–OH), 128.87 (–CH═),
123.80 (C–Cl), 122.80, 121.29, 120.18, 115.07, 56.99 (OCH3),
45.69 (N–CH2); MS (ESI) m/z (%): 359.9 [M+H]+; Anal. calcd. for
(Z)‐2‐(5‐(2‐(Benzyloxy)‐5‐chloro‐3‐methoxybenzylidene)‐4‐oxo‐
2‐thioxothiazolidin‐3‐yl)acetic acid (8l)
IR (KBr) νmax (cm−1): 3,467 (O–H), 3,033 (aromatic C–H stretching),
2,937 (aliphatic C–H stretching), 1,718 and 1,692 (C═O), 1,589
(C═C), 1,345 (C═S), 1,282 (C–O), 796 (C–Cl); 1H‐NMR (300 MHz,
CDCl3): δ 12.98 (bs, 1H, OH), 7.68 (s, 1H, –CH═), 7.54–7.37 (m, 5H,
Ar–H), 7.01–6.98 (m, 2H, Ar–H), 5.40 (s, 2H, OCH2), 4.67 (s, 2H,
N–CH2), 3.89 (s, 3H, OCH3); 13C‐NMR (75 MHz, DMSO‐d6): δ 189.01
(C═S), 167.68 (COOH), 166.38 (CO), 154.61 (C–OCH2–), 149.58
(C–OCH3–), 145.85, 136.50 (–CH═), 130.75, 129.43–128.49 (5C),
124.84, 118.94, 117.54, 116.29, 75.93 (OCH2), 57.05 (OCH3), 45.54
(N–CH2); MS (ESI) m/z (%): 450.0 [M+H]+; Anal. calcd. for
C20H16ClNO5S2: C, 53.39; H, 3.58; N, 3.11. Found: C, 53.57; H,
3.64; N, 3.17.
C
13H10ClNO5S2: C, 43.40; H, 2.80; N, 3.89. Found: C, 43.32; H,
2.56; N, 3.84.
(Z)‐2‐(5‐(2‐Hydroxy‐5‐nitrobenzylidene)‐4‐oxo‐2‐thioxothiazolidin‐
3‐yl)acetic acid (8i)
IR (KBr) νmax (cm−1): 3,462 (O–H), 2,995 (aromatic C–H stretching),
2,942 (aliphatic C–H stretching), 1,712 and 1,701 (C═O), 1592 (C=C),
1,510 and 1,371 (–NO2), 1,322 (C═S), 1262 (C–O stretching); 1H‐
NMR (300 MHz, DMSO‐d6): δ 13.10 (1H, bs, COOH), 8.30 (s, 1H,
Ar–H), 8.23–8.21 (d, J = 8.6 Hz, 1H, Ar–H), 7.96 (s, 1H, –CH=),
7.03–7.00 (d, J = 8.6 Hz, 1H, Ar–H), 4.79 (s, 2H, N–CH2); 13C‐NMR
(75 MHz, DMSO‐d6): δ 193.06 (C═S), 167.24 (COOH), 166.27 (C═O),
162.77 (C–OH), 139.72 (C–NO2), 128.19 (–CH═), 127.50, 125.89,
123.43, 120.10, 116.70, 44.98 (N–CH2); MS (ESI) m/z(%): 339.2 [M‒
H]+, 341.0 [M+H]+; Anal. calcd. for C12H8N2O6S2: C, 42.35; H, 2.37;
N, 8.23. Found: C, 42.52; H, 2.48; N, 8.33.
(Z)‐2‐(5‐(2‐(Benzyloxy)‐5‐nitrobenzylidene)‐4‐oxo‐2‐thioxothiazoli-
din‐3‐yl)acetic acid (8m)
IR (KBr) νmax (cm−1): 3,457 (O–H), 3,017 (aromatic C–H stretching),
2,954 (aliphatic C–H stretching), 1,715 and 1,702 (C═O), 1,582
(C═C), 1,547 and 1,342 (–NO2), 1,317 (C═S), 1,276 (C–O); 1H‐NMR
(300 MHz, DMSO‐d6): δ 8.38–8.35 (dd, J = 2.7 and 9.0 Hz, 1H, Ar–H),
8.28–8.27 (m, 1H, Ar–H), 7.92 (s. 1H, –CH=), 7.51–7.34 (m, 6H,
Ar–H), 5.45 (s, 2H, OCH2), 4.60 (s, 2H, N–CH2); 13C‐NMR (NMR
(75 MHz, DMSO‐d6): δ 193.17 (C═S), 167.55 (COOH), 166.78 (C═O),
162.10 (C–OCH2–), 141.44 (C–NO2), 135.82 (–CH═), 129.16 (2C),
128.91 (2C), 128.35 (2C), 126.07, 125.84, 125.32, 122.80, 114.40,
71.62 (O–CH2), 46.70 (N–CH2); MS (ESI) m/z (%): 431.0 [M+H]+; Anal.
calcd. for C19H14N2O6S2: C, 53.01; H, 3.28; N, 6.51. Found: C, 53.12;
H, 3.37; N, 6.57.
(Z)‐2‐(5‐(2‐Hydroxy‐3‐methoxy‐5‐nitrobenzylidene)‐4‐oxo‐2‐
thioxothiazolidin‐3‐yl)acetic acid (8j)
IR (KBr) νmax (cm−1): 3,432 (O–H), 3,043 (aromatic C–H stretch-
ing), 2,923 (aliphatic C–H stretching), 1,719 and 1,706 (C═O),
1,586 (C═C), 1,532 and 1,366 (–NO2), 1,328 (C═S), 1,269 (C–O
stretching); 1H‐NMR (300 MHz, DMSO‐d6): δ 13.37 (bs, 1H,
COOH), 7.96–7.95 (m, 1H, Ar–H), 7.88 (s, 1H, –CH═), 7.59 (s, 1H,
Ar–H), 4.72 (s, 2H, N–CH2), 3.86 (s, 3H, OCH3); 13C‐NMR
(75 MHz, DMSO‐d6): δ 193.91 (C═S), 167.71 (COOH), 166.92
(C═O), 157.53 (C–OH), 151.22 (C–OMe), 142.97 (C–NO2),
133.31 (–CH═), 126.37, 123.74, 122.02, 121.17, 116.52, 45.62
(N–CH2); MS (ESI) m/z (%): 371.0 [M+H]+, 393.0 [M+Na+]; Anal.
calcd. for C13H10N2O7S2: C, 42.16; H, 2.72; N, 7.56. Found: C,
42.28; H, 2.85; N, 7.63.
(Z)‐2‐(5‐((2‐(Benzyloxy)naphthalen‐1‐yl)methylene)‐4‐oxo‐
2‐thioxothiazolidin‐3‐yl)acetic acid (8n)
IR (KBr) νmax (cm−1): 3,449 (O–H), 3,033 (aromatic C–H stretching),
2,937 (aliphatic C–H stretching), 1,718 and 1,692 (C═O), 1,589
(C═C), 1,345 (C═S), 1,282 (C–O); 1H‐NMR (300 MHz, CDCl3):
δ 9.29–9.26 (d, J = 8.4 Hz, 1H, Ar–H), 8.02–7.99 (d, J = 9.0 Hz, 1H,
Ar–H), 7.76–7.73 (d, J = 7.8 Hz, 1H, Ar–H), 7.63–7.24 (m, 9H, 8Ar–H
and –CH═), 5.31 (s, 2H, OCH2), 4.72 (s, 2H, N–CH2); 13C‐NMR NMR

CHAUHAN ET AL.
13 of 14
|
(75 MHz, CDCl3): δ 192.07 (C═S), 164.07 (COOH), 163.19 (C═O),
151.19 (C–OCH2–), 137.48, 135.96 (–CH═), 131.58, 129.90, 128.82
(3C), 128.72, 128.43, 128.24 (3C), 127.39, 125.03, 124.94, 117.30,
113.99, 71.53 (O–CH2), 45.54 (N–CH2); MS (ESI) m/z (%): 436.0
[M+H]+; Anal. calcd. for C23H17NO4S2: C, 63.43; H, 3.93; N, 3.22.
Found: C, 63.47; H, 4.08; N, 3.34.
ChemBioOffice v16 (Perkin–Elmer). Before docking, the grid para-
meters were validated by redocking the cocrystallized ligand
(subjected to energy minimized), MUP into the active site of PL.
The redocked pose was deviated from the cocrystallized pose by an
RMSD of 1.7 Å. The obtained docked poses were analyzed for their
MolDock scores, while the various interactions exhibited by the
ligands with the active site were visualized in a Discovery Studio
Visualizer (Dassault Systemes Biovia).
|
4.2
Biological assays
|
4.2.1
PL inhibitory assay
|
4.3.2
Molecular dynamics simulations
The assay procedure for method A was performed as per the
protocol optimized in our laboratory.[22,24] Briefly, 50 mg of porcine
PL was suspended in 10 ml of Tris‐HCl buffer (containing 2.5 mmol of
Tris and 2.5 mmol of NaCl) and adjusted to pH 7.4 with HCl. The
solution was shaken vigorously for 15 min, followed by centrifugation
(4,000 rpm, 291 K for 10 min). The supernatant was collected and
used afresh as the enzyme solution. Stock solutions of orlistat and
derivatives 8a–n were prepared in dimethylsulfoxide (DMSO) at
linear concentrations ranging from 0.78 to 1,000 μg/ml. The final 1 ml
reaction mixture comprised 875 μl of the buffer, 100 μl enzyme and
20 μl of the inhibitors of various stock concentrations. The mixture
was incubated for 5 min at 37°C, followed by addition of 5 μl of the
substrate (4‐nitrophenyl butyrate, 10 mM in acetonitrile). The final
concentration of DMSO in the mixture did not exceed 2%.
Absorbance was detected using an EPOCH microplate spectro-
photometer (BioTek, VT) after 5 min at the absorbance maxima of 4‐
nitrophenol (405 nm). The assay was performed in triplicate and the
percentage inhibition was calculated using the formula
Molecular dynamics represents a useful technique for understanding
the ligand interactions and its binding stability in dynamic
a
environment. Hence, the MD simulations for the most potent ligands
were performed using a GROningen Machine for Chemical Simula-
tions (GROMACS 5.0.4), compiled on a CentOS 7 operating system
equipped with Intel(R) Xeon(R) CPU W3565 and an NVIDIA Quadro
4000 Quad‐Core Processor was used. A CHARMM27 force field was
applied during the MD run and the topology of the ligands was
generated using the online tool provided by the Swiss Institute of
Bioinformatics.[28,29] Before the initiation of the MD, the complex
was minimized using the Steepest Descent algorithm for 1,000 steps,
followed by stabilization of the system to 310 K and 1 atm pressure
for 50 ps, using the canonical constant Number of atoms, Volume,
and Temperature (NVT) and constant number of atoms, pressure, and
temperature (NPT) ensembles. Parameters like the Particle Mesh
Ewald method for long‐range electrostatics and 14 Å cutoff for van
der Waals and columbic interactions were set during the MD
simulation.[30] LINCS algorithm was applied for the calculation of
[31]
% Inhibition = ([AE − AT]/AE) × 100
bond length.
Discovery Studio visualizer was used to depict the
graphical representations of the complex.
where AE is the absorbance of enzyme control (without inhibitor) and
AT is the difference between the absorbance of test samples, with
and without substrate. The IC50 values of the inhibitors were
calculated by plotting the linear regression curve (% inhibition vs.
concentration).
|
4.3.3
In silico drug likeness predictions
The drug likeness predicts the oral bioavailability of the derivatives on
the basis of various structural and physicochemical properties. In the
present study, the drug likeness of the synthesized derivatives was
predicted using Lipinski’s rule of five, which describes that for a
molecule to act as a therapeutic candidate, it must possess five
characteristics which are (a) number of hydrogen bond donors should
not be more than 5, (b) number of hydrogen bond acceptors should not
be more than 10, (c) molecular mass should be less than 500 daltons, (d)
LogP (octanol–water partition coefficient) should not be greater than 5.
Drug likeness was predicted by online Swiss ADME tools.
For the enzyme inhibition kinetics, the assay protocol was
repeated using 8f (1.25, 2.5, and 5 μM) at four different substrate
concentrations (25, 50, 100, and 200 µM) and a double reciprocal
Lineweaver–Burk plot was plotted to understand the nature of
inhibition. The inhibition constant, Ki, was calculated from the
Cheng–Prusoff equation.[23]
|
4.3
Molecular modeling studies
Molecular docking
|
4.3.1
Molecular docking was performed with the aid of Molegro Virtual
Docker 6.0 (CLC Bio, Denmark) and the crystal structure of the open
lid conformation of the human PL was retrieved from the RCSB
Protein Data Bank (PDB Code: 1LPB).[26,27] Before the docking, the
structures of the ligand were drawn using the ChemDraw 2D module
and the energy minimized structures were obtained using the
molecular mechanics 2 force field in the ChemDraw 3D module of
|
4.3.4
In silico toxicity prediction
The toxicity analysis of the synthesized derivatives was done by using
preADME and protox online software. PreADME predicts the toxicity
profile on carcinorats, carcinomice, and human ether related gene
factor (hERG) models, whereas ProTox predicts the LD50 dose as well
as the toxicity class.

14 of 14
CHAUHAN ET AL.
|
ACKNOWLEDGMENTS
[15] Z. Liu, Q. Chai, Y. Li, Q. Shen, L. Ma, L. Zhang, X. Wang, L. Sheng, J. Li,
J. Li, J. Shen, Acta Pharmacol. Sin. 2010, 31, 1005.
[16] A. K. Singh, A. C. Tripathi, A. Tewari, V. Chawla, S. K. Saraf, Med.
Chem. Res. 2017, 26, 1535.
The authors (D. C., R. B., and V. M.) acknowledge the I. S. F. College of
Pharmacy, Moga, Punjab (India) for providing the necessary
laboratory facilities. The authors (G. G., S. N. C., and A. P.)
acknowledge the Birla Institute of Technology and Science, Pilani
(BITS Pilani), Pilani Campus, Pilani, Rajasthan (India) for providing the
necessary laboratory facilities and fellowship to G. G.
[17] S. Wang, Y. Zhao, W. Zhu, Y. Liu, K. Guo, P. Gong, Arch. Pharm. 2012,
345, 73.
[18] Y. Ohishi, T. Mukai, M. Nagahara, M. Yajima, N. Kajikawa, K.
Miyahara, T. Takano, Chem. Pharm. Bull. 1990, 38, 1911.
[19] D. Hardej, C. R. Ashby Jr, N. S. Khadtare, S. S. Kulkarni, S. Singh, T. T.
Talele, Eur. J. Med. Chem. 2010, 45, 5827.
[20] M. Krátký, J. Vinšová, J. Stolaříková, Bioorg. Med. Chem. 2017, 25,
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CONFLICT OF INTEREST
[21] D. K. Aneja, P. Lohan, S. Arora, C. Sharma, K. R. Aneja, O. Prakash,
Org. Med. Chem. Lett. 2011, 1, 15.
The authors declare that there are no conflict of interest.
[22] S. N. C. Sridhar, G. Ginson, P. O. Venkataramana Reddy, M. P. Tantak,
D. Kumar, A. T. Paul, Bioorg. Med. Chem. 2017, 25, 609.
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[24] S. N. C. Sridhar, S. Mutya, A. T. Paul, Med. Chem. Res. 2017, 26,
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ORCID
Vikramdeep Monga
http://orcid.org/0000-0003-4004-6241
[25] M. J. Naim, M. J. Alam, F. Nawaz, V. G. M. Naidu, S. Aaghaz, M. Sahu,
N. Siddiqui, O. Alam, Bioorg. Chem. 2017, 73, 24.
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Hadda, H. M. Sirat, Y. T. Zheng, M. Sankaranarayanan, Bioorg. Chem.
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