Design, synthesis,: In silico molecular modelling studies and biological evaluation of novel indole-thiazolidinedione hybrid analogues as potential pancreatic lipase inhibitors

Article abstract of DOI:10.1039/d0nj05649a

Pancreatic lipase (PL) is a key enzyme responsible for the digestion of dietary triglycerides; hence its inhibition is considered as a promising target for the management and/or treatment of obesity. A new series of indole-thiazolidinedione (TZD) hybrid analogues were synthesized using a molecular hybridisation approach and evaluated for their anti-obesity effects via PL inhibition. The targeted analogues were synthesized via the condensation reaction between various substituted isatin with TZD in the presence of aqueous KOH in methanol. Amongst the synthesized analogues, 7k and 7m exhibited a potential PL inhibitory activity (IC50-7.30 and 9.51 μM, respectively). Kinetic study of these potent analogues revealed their competitive mode of enzyme inhibition. This fact was confirmed via fluorescence spectroscopy which further suggested the presence of one binding site for the synthesized analogues. Molecular docking of the synthesized analogues was performed using human PL (PDB ID: 1LPB). The obtained MolDock scores were aligned with the in vitro PL inhibitory activity (Pearson's r = 0.9108, p < 0.05). Moreover, a stable conformation of the 1LPB-ligands suggested the stability of these complexes in the dynamic environment. These studies provided a basis for the potential role of the indole-TZD hybrids in PL inhibition and further optimization might result in the development of new lead candidates for obesity treatment.

Full text of DOI:10.1039/d0nj05649a

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Design, synthesis, in silico molecular modelling
studies and biological evaluation of novel indole-
thiazolidinedione hybrid analogues as potential
pancreatic lipase inhibitors†
Cite this: New J. Chem., 2021,
45, 1381
Ginson George, Prashant S. Auti and Atish T. Paul
*
Pancreatic lipase (PL) is a key enzyme responsible for the digestion of dietary triglycerides; hence its
inhibition is considered as a promising target for the management and/or treatment of obesity. A new
series of indole-thiazolidinedione (TZD) hybrid analogues were synthesized using a molecular hybridisation
approach and evaluated for their anti-obesity effects via PL inhibition. The targeted analogues were
synthesized via the condensation reaction between various substituted isatin with TZD in the presence of
aqueous KOH in methanol. Amongst the synthesized analogues, 7k and 7m exhibited a potential PL
inhibitory activity (IC50 – 7.30 and 9.51 mM, respectively). Kinetic study of these potent analogues revealed
their competitive mode of enzyme inhibition. This fact was confirmed via fluorescence spectroscopy which
further suggested the presence of one binding site for the synthesized analogues. Molecular docking of
the synthesized analogues was performed using human PL (PDB ID: 1LPB). The obtained MolDock scores
were aligned with the in vitro PL inhibitory activity (Pearson’s r = 0.9108, p o 0.05). Moreover, a stable
conformation of the 1LPB-ligands suggested the stability of these complexes in the dynamic environment.
These studies provided a basis for the potential role of the indole-TZD hybrids in PL inhibition and further
optimization might result in the development of new lead candidates for obesity treatment.
Received 19th November 2020,
Accepted 12th December 2020
DOI: 10.1039/d0nj05649a
rsc.li/njc
Amongst these, inhibition of Pancreatic Lipase (PL) has been
validated as a promising target, owing to its crucial role in
1. Introduction
Obesity is a chronic multifactorial metabolic disease that dietary fat digestion. After the ingestion of fatty meals, the
significantly contributes towards morbidity, mortality and pancreas produces PL (glycoprotein of 50 KDa) that is released
socio-economic burden across the globe. Notably, the prevalence into the duodenum, where it is responsible for the breakdown
of obesity is increasing alarmingly not only across the borders of (50–70%) of dietary triglycerides into monoglycerides and free
countries but also within different age groups. World Health fatty acids.4,5 The lipolytic activity of the PL is maintained by
Organization (WHO) reported a rapid upsurge (tripled since the catalytic triad (Ser152, Asp176, and His263) in the active
1975) in the obesity population. In 2016, 650 million adults pocket.6,7 Currently, orlistat, a saturated lipstatin derivative, is
and 38.3 million children under the age of five were identified as the only PL inhibitory drug approved by the US Food and Drug
obese. Though obesity is a preventable condition, over 4 million Administration (FDA) for the long-term treatment of obesity.8,9
people die each year due to overweight and obese conditions.1–3 However, it possesses severe side effects including gastrointestinal
This scenario clearly indicates that overweightness and obesity disturbances, oily stools, hepatotoxicity, acute pancreatitis, gall
are growing in epidemic proportions and significant develop- stones, renal injuries etc.10 These events have aroused great
ments in the medical fraternity are required for the prevention/ interest in the medicinal chemistry domain for the discovery of
treatment of the same.
more efficacious PL inhibitors as drug leads or candidates.
In recent years, various amide functionality-based analogues
Along with diet regulation and exercise, numerous strategies
have been explored for the prevention/management of obesity. have been evaluated for PL inhibitory potentials. These include
tripeptides, thiazolidinediones (TZDs), rhodanines etc.7,11,12
TZDs represent a renowned class of anti-diabetic medications,
used in the treatment of type II diabetes mellitus (Fig. 1).
Laboratory of Natural Product Chemistry, Department of Pharmacy, Birla Institute
of Technology and Science, Pilani (BITS Pilani), Pilani Campus, Pilani - 333 031,
Furthermore, they have also been explored for their anti-obesity
Rajasthan, India. E-mail: atish.paul@pilani.bits-pilani.ac.in
potential via PTP1B inhibition.13,14 It is noteworthy that TZDs
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0nj05649a
contains a nitrogen centred diamide linkage, that can mimic the
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Fig. 1 Structures of some TZD and indole containing analogues reported for metabolic disease conditions with their mechanisms. The inset represents
the pharmacophoric design of the target analogues.
chemical environment similar to the natural esters in trigly- modifications were performed by considering the essential
cerides; hence it can act as an electrophile for Ser 152 of the pharmacophoric features for PL inhibition. Accordingly,
active site of PL.11 Therefore, a potential PL inhibitor can be the present study involves the synthesis, in silico molecular
developed by molecular hybridisation of TZD with other hetero- modelling studies and biological evaluation of novel indole-
cyclic pharmacophores.
thiazolidinedione hybrids as potential PL inhibitors. Further-
Indole based heterocycles are one of the integral basic struc- more, the authors hypothesize that the hybrid designing strategy
tural units constituted in the essential amino acid, tryptophan. will be helpful for the identification of potential PL inhibitors.
The indole functionality is unique due to its structural and
chemical properties. The electron rich property of indole nucleus
provides intra-aromatic ring-stacking effects and p–cationic inter-
actions with numerous proteins, which has resulted in the
potential activities with numerous enzymes. Indole containing
2. Results and discussion
2.1 Chemistry
scaffolds exhibit a broad range of pharmacological activities15,16 The route for the synthesis of the titled analogues is depicted in
such as anti-inflammatory, anti-diabetic, anti-malarial, and anti- Scheme 1. TZD (3) was prepared according to the literature.
tubercular. The potential of the indole scaffold in PL inhibition Condensation of thiourea (1) with chloroacetic acid (2) at 0–5 1C
has been explored previously and has resulted in the identification resulted in the formation of 2-iminothiazolidine-4-dione (white
of various alkaloids and peptides as potential leads. For instance, ppt) that upon further refluxing with conc. HCl for 10–12 h
indole alkaloids from Murraya koenigii leaves17 and numerous resulted in the formation of 2,4-TZD (3) as white fine crystals.
indoles (tryptophan) containing tripeptide analogues7,18 showed N (3)-alkylation of TZDs was carried out by treating TZD with
potential PL inhibition. Apart from these, the present authors also NaH followed by the addition of alkyl halides (methyl iodide,
have reported the PL inhibitory potential of numerous indole benzyl bromide). After the specified time, the reaction mixture
containing alkaloids such as echitamine19 and conophylline.20
was poured into ice-cold water resulting in the formation of
Hence, based on the wide array of activities of these phar- N-substituted 2,4-TZD (4).11,21 Treatment of isatin (5) with
macophores and the molecular hybridization technique, TZD- various halides in the presence of NaH in DMF resulted
indole hybrid analogues were designed, and further structural in the formation of N-substituted isatin (6). Treatment of
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Scheme 1 Synthesis of the title analogues. Reagents and conditions (i) rt, 15 min; (ii) HCl, H2O, 100–110 1C, 10 h; (iii) NaH, DMF, 12 h; (iv) KOH, MeOH,
2–3 h, RT.
N-substituted 2,4-TZD (4) with N-substituted/isatin (5, 6) in with the carbonyl functionality of TZD. Apart from that,
methanol and few drops of 40% KOH resulted in the formation the configuration can be ascribed due to the favourable
of the corresponding indole-TZD hybrid analogues (7).22,23
electrostatic interactions between the C2 carbonyl oxygen atom
The products were obtained in good yield with a vermilion red of isatin and partially positively charged ‘‘S’’ of TZD
colour. Furthermore, the synthesized analogues were charac- heterocycles.21,23–25
terised by ATIR, 1H, 13C NMR spectroscopy and mass spectro-
2.2 PL inhibition assay, kinetics and structural activity
relationship
metry. The obtained data were in good agreement with their
structural identity. The IR spectrum of the title analogues showed
strong absorption bands at 1731–1746 and 1663–1680 cmꢀ1 that The in vitro PL inhibitory activity of the synthesized title analo-
were assigned to the carbonyl groups. The disappearance of the gues was evaluated by a standardised assay protocol11,19 and the
methylene proton (d value – 3.95 ppm) of TZD further confirmed results are summarised in Table 1. The assay protocol consisted
its reaction with isatin. –CH2 proton attached to the TZD reso- of porcine PL Type II (enzyme), 4-nitrophenyl butyrate (substrate)
nated in the range of 4.95 to 5.00 ppm (d), while the –CH3 proton and orlistat (standard). The amount of p-nitrophenol released by
resonated at a d range of 3.14 to 3.34 ppm. –CH2 proton attached PL was measured at its maximum wavelength (405 nm). As
to the indoline-2-one resonated in the range of 4.97 to 5.12 ppm summarised in Table 1, synthesized analogues displayed
(d). All other protons were observed in the expected regions. potential to moderate PL inhibition (IC50 – 7.30 to 28.37 mM).
The appearance of three CQO peaks in 13C NMR (d value – Orlistat, a commercially available PL inhibitor, exhibited a potent
165 to 170 ppm) further confirmed the reaction progress of TZD PL inhibitory activity (IC50ꢀ-0.86 mM)
and isatin scaffolds. All the analogues showed a prominent
To study the inhibition mode against the PL, enzyme
signal at d 27–49 due to the presence of –CH3, –CH2 groups. inhibition kinetics analysis of the topmost active analogues
A ‘‘cis’’ isomeric configuration was obtained between arylidene (7k and 7m) was performed. The inhibition types, number of
double bonds amongst the isatin and TZD. This fact can be binding sites etc. can be deduced from the Lineweaver–Burk
supported by the downfield shift of H4 of isatin nucleus (d value (LB) plots.26 Hence, a LB plot was constructed by performing an
E 9 ppm), which might be due to the hydrogen bond formation in vitro PL inhibition assay, wherein various concentrations of
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Table 1 In vitro PL inhibitory activity of the synthesized analogues (7a–7n)
a
a
IC50 (mM)
Comp.
Structure
IC50 (mM)
Comp.
Structure
7a
28.37 ꢁ 1.42
7b
18.43 ꢁ 0.39
7c
7e
21.79 ꢁ 1.98
14.19 ꢁ 0.53
7d
12.22 ꢁ 1.21
12.10 ꢁ 1.25
7f
7g
17.06 ꢁ 1.06
7h
26.61 ꢁ 1.34
7i
14.65 ꢁ 1.53
7j
17.46 ꢁ 1.22
7k
7.30 ꢁ 0.80
7l
11.70 ꢁ 1.66
7m
9.51 ꢁ 1.19
7n
12.27 ꢁ 1.38
Orlistat
0.86 ꢁ 0.065
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Fig. 2 Double reciprocal Lineweaver–Burk plots of 7k and 7m obtained through the enzyme kinetic study.
the analogues were evaluated against different concentrations analogues exerted their PL inhibitory activity in a competitive
of the substrate (25, 50, 100 and 200 mM).
manner.
In the case of a structural activity relationship, the substitution
As represented in Fig. 2, the plots were converged at its first
quadrant and intersected at one point, which indicated that of TZD and isatin scaffolds with various alkyl/aryl groups exhibited
analogues 7k and 7m exerted their PL inhibitory activity in a a variable PL inhibitory potential. It is noteworthy that the
competitive manner. Furthermore, a proportionate increase in extension of–CH3 of TZD with a denser aromatic heterocycle
the apparent Km values without affecting the Vmax further indi- resulted in a higher PL inhibitory potential than the simple alkyl
cated the scope of reversible competitive inhibition (Table 2). chains. For instance, analogue 7k exhibited a significant potential
Inhibition constant (Ki) values were deduced as 4.637 and 6.041 (IC50 – 7.30 mM) compared to analogue 7d which lacks benzyl
mM (retrieved from the Cheng–Prusoff equation27), proving that extension on the TZD scaffold (IC50 – 12.22 mM). Furthermore, the
these molecules have a strong affinity towards PL. From the substitution of the –NH of isatin with various alkyl/aryl groups
enzyme kinetics study, it can be concluded that the synthesized resulted in potential activity. Unsubstituted isatin derivatives
Table 2 Km, Vmax and Ki values of 7k and 7m retrieved from the PL enzyme kinetics
Km (apparent) at different concentrations (mM)
Code
0 mM
0.625 mM
5 mM
Vmax (mM minꢀ1
)
Ki (mM)
Nature of inhibition
7k
7m
87.053
87.053
121.438
103. 378
170.487
127.486
1.439
1.316
4.637
6.041
Competitive
Competitive
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inhibitory activity. Amongst the various benzyl substitutions, the
p-NO2 counterpart displayed a comparatively lesser activity (ana-
logue 7g and 7n IC50 = 17.06 and 12.07 mM, respectively) than the
unsubstituted benzyl part (analogue 7d and 7k IC50= 12.22 and
7.30 mM, respectively). Furthermore, –Br substituted analogues
exhibited comparatively higher activity (analogue 7f and 7m with
–Br substitutions; IC50= 12.10 and 9.51 mM, respectively) than the
–Cl substituted analogues (analogue 7e and 7l with –Cl substitu-
tions; IC50= 14.19 and 11.10 mM, respectively). Based on these
observations, a preliminary structural activity relationship was
deduced and is represented in Fig. 3.
Thus, it can be concluded that the substitutions at TZD and
isatin scaffolds are essential for exerting a potential PL inhibi-
tory activity.
Fig. 3 Structure–activity relationship of synthesized Indole-TZD Hybrids.
2.3 Fluorescence quenching measurements with PL
(7a and 7h) exhibited a moderate inhibitory potential towards the The fluorescence property of PL originates from various aro-
PL (IC50 – 28.37 and 26.61 mM, respectively). Introduction of matic amino acids such as tryptophan, phenylalanine and
various substituents to the –NH of isatin displayed significant tyrosine while intrinsic fluorescence is mainly contributed by
activity (IC50 o 20 mM). Alkyl substitutions (analogues 7b and 7c) the tryptophan residues. Furthermore, changes in emission
exhibited a lesser activity (IC50 – 18.43 to 21.79) than the denser spectra of tryptophan are observed with the various structural
substitutions (analogues 7i and 7J; IC50 – 7.30 to 17.36 mM, and dynamic properties of a macromolecule. Thus, the associa-
respectively). Moreover, the presence of the electron-withdrawing tion reaction between bioactive molecules with a protein can
groups in the benzyl substitutions resulted in a decremental PL be investigated via intrinsic fluorescence spectroscopy.28,29
Fig. 4 (A and B) The fluorescence spectra of PL in the presence of 7k and 7m at various concentrations (pH 7.4, T = 298 K); (C and D) Stern–Volmer plots
for the quenching of 7k and 7m on the PL at 298, 304, and 310 K.
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Table 3 Binding constant and the number of binding sites (n) at different temperatures
Code
T (K)
KSV (ꢂ 104 Mꢀ1
)
Kq (ꢂ 1010 Mꢀ1 sꢀ1
)
R2
Kb (ꢂ 104 Mꢀ1 sꢀ1
)
n
R2
7k
298
304
310
298
304
310
1.6
1.4
1.1
1.6
1.2
0.8
103.14
88.68
71.07
101.13
75.47
52.20
0.9920
0.9941
0.9992
0.9936
0.9921
0.9908
3.22
2.82
2.00
3.94
3.03
1.79
0.841
0.822
0.822
0.717
0.747
0.787
0.9898
0.9933
0.9937
0.9868
0.9992
0.9945
7m
As shown in Fig. 4, the analogues exerted a quenching effect to indicating the formation of PL-quencher complexes. Fluores-
PL fluorescence. Moreover, the fluorescence intensity was cence quenching is classified into two types, namely static
decreased with the increase in the quencher concentration quenching and dynamic quenching.30,31
Fig. 5 (A and B) Binding pose and 2D interaction diagram of 7k and 7m in the active site of PL; (C) 2D interaction diagram of orlistat in the active
site of PL.
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The excited state fluorophore is deactivated by the quencher
molecule in solution and is denoted as dynamic quenching
while the formation of a non-fluorescent ground state complex
of the fluorophore-quencher is described as static quenching.
A negative correlation between quenching constant and tem-
perature highlights dynamic quenching while a diminished
quenching constant with an increase in temperature repre-
sented static quenching.30,31
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F0
log ¼ log Kb þ n log½Qꢃ
F
where Kb is the binding constant and n is the number of
binding sites. Values of these parameters were determined
from the double log graph of log F0/F vs. log [Q].32 The
obtained values of these parameters are represented in
Table 3. The values of ‘‘n’’ at the corresponding temperature
were in the range of 0.71 to 0.84, which indicated the presence
of only one binding site in PL. This result further supported
the competitive inhibition on the enzyme kinetics of PL.
Additionally, the Kb values were in the range of 1.79 to 3.22 ꢂ
Fluorescence quenching is described by the Stern–Volmer
equation as follows:
F0
¼ 1 þ Kqto½Qꢃ ¼ 1 þ KSV½Qꢃ
104 Mꢀ1 ꢀ1, which indicates the strong binding forces of these
s
F
analogues with PL.
where F and F0 are the fluorescence intensities of PL with or
without the quencher, respectively. [Q] represents the concen-
tration of the quencher. KSV denotes the Stern–Volmer quenching
2.4 Molecular docking and dynamics
constant, while Kq is the bimolecular quenching constant, and t0 To decipher the degree of energetics and structural interactions
is the average lifetime of the fluorophore molecule without the responsible for PL inhibition, the binding mode of the analo-
quencher (10ꢀ8 s). The values of KSV and Kq are determined from gues has been evaluated by using a molecular docking simula-
the linear regression plot of F0/F vs. [Q].
tion. Synthesized analogues were docked with the crystal
As represented in Table 3, an inverse correlation between the structure of the PL-colipase complex (PDB ID: 1LPB, 2.46 Å
KSV and temperature has been obtained, which suggests that the resolution) via Molegro Virtual Docker 6.033 with validated grid
quenching effects on the PL may be mainly attributed to static parameters11,34 and the obtained results are summarised in
quenching rather than dynamic quenching. A similar kind of Fig. 5 and Table 4. The MolDock scores of the analogues
result was previously reported by Yan-Qin Li et al., wherein exhibited significant correlation to their PL inhibitory activity
quercetin, isoquercetin, and rutin exhibited an inverse correla- (Pearson’s r = 0.9108, p o 0.05) and these activities were exerted
tion with the temperature and fluorescence intensity of the PL. The via various interactions such as hydrogen bonding, p–p stacking,
term Kq reflects the quenching efficiency or accessibility of the p–sulfur, p–cations and p–sigma etc. Structurally, the active sites
fluorophore by the quencher; thus, it provides information about of 1LPB consist of a highly restricted catalytic triad of Ser 152-Asp
the formation of the complex. The Kq value near to 1 ꢂ 1010
M
ꢀ1 sꢀ1 176 – His 263 that is enclosed within a hydrophobic lid domain
is typical for dynamic quenching. In the present study, Kq values comprised of Gly 76-Lys 80 and Leu 213-Met 217. During
were 22 to 50-fold higher (1010
M
ꢀ1 sꢀ1) than dynamic quenching, the triglyceride ester hydrolysis process, the hydrophobic lid
thus suggesting that both analogues formed a complex with PL, domain confirmation is opened by the interfacial activation
thereby quenching the fluorescence intensity via
a
static phenomenon.7,35 All the analogues interacted with the hydro-
phobic lid domain (Phe 77, Phe 215) as well as the catalytic
mechanism.28,32
For static quenching, the binding constant and number of triad amino acids (Ser 152, His 263). It is also noteworthy
binding sites were calculated by the modified Stern–Volmer that the benzyl substituted analogues (having potential PL
equation as follows.
inhibition) exerted an additional alkyl/p–alkyl interaction over
Table 4 Mol Dock scores (in kcal molꢀ1) and the interactions of the synthesized analogues (7a–n) with the human PL active site
No.
Mol Dock score
H-Bond
p–p
p–Cation
Alkyl/p-alkyl
p-Sulfur
7a
7b
7c
7d
7e
7f
7g
7h
7i
ꢀ62.79
ꢀ69.36
ꢀ70.77
ꢀ90.278
ꢀ95.74
ꢀ89.95
ꢀ81.79
ꢀ66.84
ꢀ73.82
ꢀ72.62
ꢀ99.74
ꢀ95.43
Phe-77, His-151
Phe-77, His-151
Phe-77, His-151
Phe-77, His-151
Phe-77, His-151
Phe-77, His-151
Phe-77, His-151
Phe-77, His-151
Phe-77
Phe-77, Phe-215
Phe-77, Phe-215
Phe-77, Phe-215
Phe-77, Tyr-114
Phe-77, Tyr-114
Phe-77, Tyr-114
Phe-77, Tyr-114
—
Phe-77, Phe-215
Phe-77, Phe-215
Phe-77, Tyr-114
Phe-77, Tyr-114
His-151, His-263
His-151, His-263
His-151, His-263
His-151, His-263
His-151, His-263
His-151, His-263
His-151, His-263
His-263
Ser-152, His-263
Ser-152, His-263
Ser-152, His-263
His-263
His-151, Ala-178, Leu-264
His-151, Ala-178, Phe-215, His-263, Leu-264
Phe-77, His-151, Ala-178, Leu-264
His-151, Leu-153, Ala-178, Leu-264
Tyr-114, His-151, Leu-153, Ala-178, Leu-264
Tyr-114, His-151, Leu-153, Ala-178, Leu-264
Gly-76, His-151, Leu-153, Ala-178, Leu-264
Arg-256, Ala-260
Phe-77, Ala-178, Phe-215, Arg-256, Ala-259
Phe-77, Ala-178, Arg-256, Ala-259
His-263
His-263
His-263
His-263
His-263
His-263
His-263
His-263
His-263
His-263
His-263
Ser -152,
His-263
Ser -152,
His-263
Ser -152,
His-263
7j
7k
7l
Phe-77
Phe-77
Phe-77
Leu-153, Ala-178, Pro-180, Arg-256, Ala-259
Leu-153, Ala-178, Arg-256, Ala-259, Ala-260
7m
7n
ꢀ94.85
ꢀ91.86
Phe-77
Phe-77
Phe-77, Phe-215
His-263
His-263
Phe-77, Ala-178, Arg-256, Ala-259
Phe-77, Tyr-114,
Phe-215
Leu-153, Ala-178, Arg-256, Ala-259, Ala-260
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Fig. 6 RMSD of ligands and radius of gyrations retrieved from 20 ns MD simulations of the PL-ligand complex (A)- 7k; (B)-7m.
the methyl-substituted counterpart. The previous literature of the binding mode and interactions in a dynamic environ-
suggests that the catalytic triad of human PL is enclosed in ment has been evaluated by an optimised molecular
the lid domain (inactivated state); potential role of Arg 256 in dynamic (MD) simulation.11,34 In the MD run (GROMACS39),
the opening of the hydrophobic lid domain has been explored RMSD values reflect the stability of the complex structure
by various groups.36–38 Thus, the drastic potentiation in the PL while the RMSF value refers to the flexibility of a certain
inhibitory activity between the methyl and benzyl substituted region of the macromolecule. Both the analogues (7k and
analogue might be due to its interaction ability with the 7m) exhibited stable binding confirmation throughout
hydrophobic lid domain (open confirmation) via alkyl/p–alkyl the simulation with a maximum deviation of E0.8 nm at
interactions with Arg 256. In the case of orlistat, two types of E10 ns (Fig. 6). The interactions of these molecules
interactions were primarily involved. A hydrogen bond inter- with 1LPB through the 20 ns MD run are summarised in
action with Phe 77, Ser 152 and hydrophobic interaction with Table 5.
Tyr 114, Phe 215 and Arg 256 etc. were formed during the
The ligand analogues exhibited numerous interactions such
docking simulations. The above results from the molecular as H bonding, p–p stalking and p–alkyl interactions etc. Initi-
docking further support the hypothesis, wherein both the ally, H bond interactions were formed with the lid domain
aromatic substitutions on isatin and TZD were helpful for amino acids (Phe 77, Ile 78 and Asp 79), later these turned to a
the lid domain interactions while the diamide linkage in TZD stable interaction with Arg 256. p–p stackings were observed
interacted with Ser 152.
with the aromatic amino acids (Phe 77, Phe 215) and p–alkyl
Molecular modelling study suggested the probable role interactions with Ile 78, Arg 256, Ala 259, Ala 260, Leu 264, etc.
of the analogues for imparting various interactions with Furthermore, a stable radius of gyration indicated the compact-
active site amino acids of 1LPB. Furthermore, the stability ness of 1LPB during the MD run with test analogues.
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Table 5 Various interactions exhibited by 7k and 7m during the 20 ns MD run
Time (ns)
H bond
p–p Stacking
p–Alkyl
7k
0
2
4
6
Phe77, Asp 79
—
Arg 256
—
Arg 256
—
Arg 256
Arg 256
Arg 256
—
Phe 77, Phe 215
Phe 215
—
Phe 215, Phe 258
Phe 215, His 263
Phe 215
—
Phe 77
Phe 77, Phe 215
Phe 215
Phe 215, Phe 258
Ile 78, Arg 256, Ala 259
Ile 78, Ile 209, Arg 256, Ala 259, Ala 260, Leu 264
Ile 78, Ala 259, Ala 260
Ile 78, Arg 256, Ala 259, Ala 260
Ile 78, Leu 213, Ala 259, Ala 260, Leu 264
Ile 78, Ala 259, Ala 260, Leu 264
Ile 78, Ala 259, Ala 260, Leu 264
Ile 78, Ala 259, His 263, Leu 264
Ile 78, Ala 259, Ala 260, Leu 264
Ile 78, Arg 256, Ala 259, Ala 260, Leu 264
Ile 78, Arg 256, Ala 259, Ala 260, Leu 264
8
10
12
14
16
18
20
7m
0
—
Phe77
Ile 78
Phe 77, Tyr 114, Phe 215
Phe 77, Phe 215
Ala 178, Ala 259
Arg 256, Ala 259
2
4
6
8
10
12
14
16
18
20
Ile 78, Arg 256
Ile 78
Ile 78
Phe 77, Phe 215
Phe 215, Trp 252
Phe 77, Phe 215, Trp 252, Thr 255
Phe 77, Phe 215
Phe 77
Phe 77, Phe 215, Thr 255
Phe 77, Trp 252
Phe 77, Phe 215, Trp 252
Phe 77, Phe 215, Trp 252
Arg 256, Ala 259, Ala 260
Ile 209, Leu 213, Arg 256, Ala 259, Ala 260
Leu 213, Arg 256, Ala 260
Arg 256, Ala 259, Ala 260
Ala 260
Arg 256, Ala 259, Ala 260
Ile 78, Ala 259, Ala 260
Arg 256, Ala 260
Ile 78
Ile 78, Arg 256
Arg 256
Arg 256
Arg 256
Arg 256
Ala 259, Ala 260
3. Conclusion
4. Materials and methods
4.1 Chemistry
Inspired by the bioactive molecules consisting of indole and TZD
pharmacophoric units, a series of indole-TZD hybrids were The synthesis of all the final analogues was carried out as per the
synthesized and evaluated for their PL inhibitory potential. The procedure detailed in Scheme 1. The progress of the reactions
condensation reaction between substituted isatin with substi- was monitored by thin-layer chromatography (TLC) analysis by
tuted TZDs in methanol in the presence of a catalytic amount of using commercially available pre-coated aluminium plates (silica
KOH resulted in the formation of the targeted analogues. gel G60 F254, Merck). All the evaporation procedures were carried
Amongst the series, analogues 7k and 7m exhibited a potential out under reduced pressure on the Heidolph-rotary evaporator.
PL inhibition (IC50 – 7.30 and 9.51 mM, respectively). Enzyme Melting points were determined with OptiMelt (Stanford
kinetics established a reversible competitive type of PL inhibition Research Systems) in one end sealed capillaries and were uncor-
by these synthesized analogues. Fluorescence study further rected. ATIR spectra were recorded on a Bruker Alpha AT-IR
supported these facts, wherein, the number of binding sites spectrophotometer. 1H NMR and 13C NMR spectra were recorded
was identified as E1. Apart from this, it also suggested the on a Bruker Avance II 400 spectrophotometer CDCl3 or DMSO-d6
potential interaction ability of these analogues with the PL. and chemical shift (d) values are given in ppm relative to TMS
Molecular docking results also corroborated the PL inhibitory (tetramethylsilane) as an internal standard. Splitting patterns are
potential of these analogues, wherein significant correlations designated as follows; s, singlet; d, doublet; dd, doublet of
were obtained between the PL inhibition and MolDock score doublets; t, triplet; m, multiplet. High-resolution mass data were
(Pearson correlation r = 0.9108, p o 0.05). For instance, analogue obtained on an Agilent 6545 Q-TOF LC/MS (ESI).
7k, that exhibited the most potential activity (IC50 = 7.30 mM),
also demonstrated a maximum MolDock of ꢀ99.74 kcal molꢀ1
.
4.1.1 General procedure for the synthesis of thiazolidine-
2,4-dione (3)
Furthermore, a variation in the PL inhibition activity was
observed with various TZD substitutions. Replacement of methyl
groups with the bulkier benzyl functionalities exhibited a sig- A 6 mL aqueous solution of 5.6 g of chloroacetic acid (2) was
nificant improvement in the PL inhibitory activity. The denser added to 6 mL aqueous solution of 4.56 g of thiourea (1) and
TZD substitution resulted in the formation of p–alkyl interaction stirred for 15 min at room temperature, resulting in a white
that helped in the opening of the lid domain amino acids, which precipitate accompanied by considerable cooling. The resulting
are essential for the PL inhibition. Furthermore, the MD simula- precipitate was treated with conc. HCl (5 mL) and the reaction
tion revealed a stable confirmation of both analogues through- mixture was refluxed for 10 h at 100–110 1C by stirring. The
out the run. In conclusion, the study suggested the PL inhibitory mixture was then cooled, and the resulting white needles were
potential of a new class of indole-TZD analogues and further filtered, washed with water to remove traces of HCl and
optimisation might result in a clinically useful PL inhibitor.
recrystallised from ethanol.11
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4.1.2 General procedure for the synthesis of N-substituted Hz, 1H, –H7 of Indolin-2-one), 3.33 (s, 3H, –CH3), 3.32 (s, 3H,
thiazolidine-2,4-dione (4a,b)
–CH3); 13C NMR (100 MHz, CDCl3) d 170.20, 167.47, 165.82,
145.00, 132.56, 130.70, 128.67, 127.39, 123.20, 119.86, 108.39,
27.46, 26.45; IR (ATR) u 2353, 1731, 1664, 1600, 1477, 1427,
1363, 1274, 1140, 1075, 1007, 870, 750, 667 cmꢀ1; HRMS (ESI+)
calculated for C13H10N2O3S [M + H], 275.0412; found 275.0475.
(Z)-5-(1-Ethyl-2-oxoindolin-3-ylidene)-3-methylthiazolidine-2,4-
dione (7c). Yield: 75%; vermilion red solid; mp = 142–144 1C;
1H NMR (400 MHz, CDCl3) d 8.96 (dd, J = 7.9, 1.3 Hz, 1H, –H4 of
Indolin-2-one), 7.43 (td, J = 7.7, 1.3 Hz, 1H, –H5 of Indolin-2-one),
7.14 (td, J = 7.7, 1.1 Hz, 1H, –H6 of Indolin-2-one), 6.91 (d, J = 7.8
Hz, 1H, –H7 of Indolin-2-one), 3.86 (q, J = 7.2 Hz, 2H, –CH2
attached to Indolin-2-one), 3.29 (s, 3H, –CH3 attached to TZD),
1.33 (t, J = 7.2 Hz, 3H, –CH3 attached Indolin-2-one); 13C NMR
(100 MHz, CDCl3) d 170.24, 167.05, 165.79, 144.12, 132.49, 130.51,
128.84, 127.46, 122.94, 120.02, 108.49, 35.14, 27.43, 12.76; IR
(ATR) u 2355, 1735, 1664, 1600, 1462, 1423, 1357, 1276, 1228,
1142, 1071, 1003, 942, 749, 655 cmꢀ1; HRMS (ESI+) calculated for
C14H12N2O3S [M + H], 289.0569; found 289.0631.
The synthesis of N-substituted thiazolidine-2,4-dione (4a,b) was
performed by the condensation of thiazolidine-2,4-dione (3)
with various alkyl (methyl iodide, a)/aryl halides (benzyl bromide, b)
in the presence of NaH in anhydrous DMF. A mixture of
thiazolidine-2,4-dione (3) (1 eq.) and NaH (1.5 eq.) was stirred
vigorously for 30 min at room temperature under nitrogen
conditions. To this, various alkyl/aryl halides (a, b; 1.5 eq.) were
added, and the stirring was continued overnight. Completion of
the reaction was monitored via thin-layer chromatography and
the solution was poured into ice-cold water. The obtained
precipitate was filtered and recrystallised from the appropriate
solvents.34
4.1.3 General procedure for the synthesis of N-substituted indole
analogues (6a–f)
Briefly, to 10 mL DMF taken in a 100 mL round-bottomed flask,
NaH (1.2 eq.) was added and stirred vigorously for 30 min. at
room temperature. To this, 1.0 eq. isatin (5) was added and the
stirring was continued. After 30 min., a solution of appropriate
halide (1.5 eq.) was added dropwise for over 5 min. to the
reaction mixture, and the stirring was continued overnight.
The reaction mixture was then transferred to ice-cold water.
The N-substituted isatin (6a–f) analogues were obtained as
crude precipitates that were purified by recrystallization from
ethanol or by column chromatography.34
(Z)-5-(1-Benzyl-2-oxoindolin-3-ylidene)-3-methylthiazolidine-
2,4-dione (7d). Yield: 70%; vermilion red solid; mp = 164–
1
165 1C; H NMR (400 MHz, CDCl3) d 8.99 (dd, J = 7.9, 1.2 Hz,
1H, –H4 of Indolin-2-one), 7.38–7.29 (m, 6H), 7.13 (td, J = 7.8,
1.1 Hz, 1H, –H6 of Indolin-2-one), 6.83–6.79 (m, 1H), 5.01 (s,
2H, –CH2 attached to Indolin-2-one), 3.31 (s, 3H, –CH3 attached
to TZD); 13C NMR (100 MHz, CDCl3) d 170.09, 167.59, 165.77,
144.17, 135.09, 132.50, 131.05, 128.93, 128.75, 127.95, 127.33,
127.22, 123.21, 120.02, 109.39, 44.15, 27.49 IR (ATR) u 2915,
2850, 2355, 1733, 1669, 1603, 1464, 1424, 1359, 1278, 1189,
1081, 1011, 748, 677 cmꢀ1; HRMS (ESI+) calculated for
4.1.4 General procedure for the synthesis of thiazolidinedione-
indole analogues (7a–n)
C
19H14N2O3S [M + H], 351.0725; found 351.0770.
To a mixture of 1.5 eq. of 1,3-thiazolidine-2,4-dione (4a,b) and
(Z)-5-(1-(4-Chlorobenzyl)-2-oxoindolin-3-ylidene)-3-methyl-
1.0 eq. of isatin analogues (5, 6a–f) in methanol (20 mL), few thiazolidine-2,4-dione (7e). Yield: 65%; vermilion red solid;
drops of 40% KOH were added and the reaction mixture was mp = 218–220 1C; 1H NMR (400 MHz, CDCl3) d 9.00 (dd, J = 7.9,
stirred at room temperature. A solid was separated out of the 1.2 Hz, 1H, –H4 of Indolin-2-one), 7.40–7.30 (m, 3H), 7.28–7.24
reaction mixture within 15–20 min and stirring was continued (m, 2H), 7.15 (td, J = 7.8, 1.1 Hz, 1H, –H6 of Indolin-2-one), 6.82–
for 2–3 h to complete the reaction. The crude precipitate was 6.74 (m, 1H), 4.98 (s, 2H, –CH2 attached to Indolin-2-one), 3.32
then re-crystallized from the chloroform–methanol mixture or (s, 3H, –CH3 attached to TZD); 13C NMR (100 MHz, CDCl3) d
underwent column chromatography resulting in final analogues 169.94, 167.60, 165.74, 143.82, 133.89, 133.60, 132.50, 131.34,
(7a–n).22,40
129.13, 128.87, 128.72, 127.02, 123.40, 120.05, 109.18, 43.52,
(Z)-3-Methyl-5-(2-oxoindolin-3-ylidene)thiazolidine-2,4-dione 27.52; IR (ATR) u 2926, 2855, 2356, 1738, 1674, 1607, 1428, 1359,
(7a). Yield: 55%; vermilion red solid; mp = 278–280 1C; 1H NMR 1285, 1184, 1088, 1014, 872.5, 795, 752 cmꢀ1; HRMS (ESI+)
(400 MHz, DMSO-d6) d 11.26 (s, 1H, –NH), 8.83 (d, J = 7.9 Hz, calculated for C19H13ClN2O3S [M + H], 385.0335; found 385.0382.
1H, -H4 of Indolin-2-one), 7.42 (t, J = 7.7 Hz, 1H, –H5 of Indolin-
(Z)-5-(1-(4-Bromobenzyl)-2-oxoindolin-3-ylidene)-3-methyl-
2-one), 7.11 (t, J = 7.8 Hz, 1H, –H6 of Indolin-2-one), 6.97 (d, J = thiazolidine-2,4-dione (7f). Yield: 60%; vermilion red solid;
1
7.8 Hz, 1H, –H7 of Indolin-2-one), 3.14 (s, 3H, –CH3); 13C NMR mp = 188–200 1C; H NMR (400 MHz, CDCl3) d 9.01 (dd, J =
(100 MHz, DMSO-d6) d 170.52, 168.83, 166.18, 144.33, 133.17, 7.9, 1.2 Hz, 1H, –H4 of Indolin-2-one), 7.51–7.45 (m, 2H), 7.36
130.63, 128.33, 127.22, 122.57, 120.33, 111.01, 27.78; IR (ATR) u (td, J = 7.8, 1.3 Hz, 1H, –H6 of Indolin-2-one), 7.22–7.12
3139, 3071, 2884, 2353, 1680, 1616, 1427, 1351, 1293, 1233, (m, 3H), 6.78 (dt, J = 7.8, 0.8 Hz, 1H, –H7 of Indolin-2-one),
1081, 1006, 753, 673 cmꢀ1; HRMS (ESI+) calculated for 4.97 (s, 2H, –CH2 attached to Indolin-2-one), 3.33 (s, 3H, –CH3
C12H8N2O3S [M + H], 261.0256; found 261.0251.
attached to TZD); 13C NMR (100 MHz, CDCl3) d 170.11, 167.51,
(Z)-3-Methyl-5-(1-methyl-2-oxoindolin-3-ylidene)thiazolidine- 166.01, 144.01, 138.37, 135.79, 133.00, 132.12, 131.96, 130.13,
2,4-dione (7b). Yield: 82%; vermilion red solid; mp = 254– 130.02, 128.28, 125.97, 123.38, 121.24, 119.86, 110.29, 43.02,
1
258 1C; H NMR (400 MHz, CDCl3) d 8.98 (d, J = 7.7 Hz, 1H, 27.89; IR (ATR) u 2356, 1733, 1674, 1608, 1467, 1427, 1357,
–H4 of Indolin-2-one), 7.46 (t, J = 7.8 Hz, 1H, –H5 of Indolin-2- 1286, 1182, 1082, 1010, 943, 752, 672 cmꢀ1; HRMS (ESI+)
one), 7.17 (t, J = 7.7 Hz, 1H, –H6 of Indolin-2-one), 6.91 (d, J = 7.8 calculated for C19H13BrN2O3S [M + H], 428.9830; found 428.9862.
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(Z)-3-Methyl-5-(1-(4-nitrobenzyl)-2-oxoindolin-3-ylidene)- CDCl3) d 170.00, 167.60, 165.46, 144.16, 135.07, 134.96,
thiazolidine-2,4-dione (7g). Yield: 62%; vermilion red solid; mp = 132.52, 131.03, 128.91, 128.89, 128.87, 128.82, 128.33, 127.93,
242–245 1C; 1H NMR (400 MHz, CDCl3) d 9.05 (dd, J = 7.9, 1.1 Hz, 127.40, 127.28, 123.20, 120.03, 109.36, 44.91, 44.12; IR (ATR) u
1H, –H4 of Indolin-2-one), 8.29–8.18 (m, 2H), 7.53–7.46 (m, 2H), 3886, 2918, 2852, 1668, 1601, 1456, 1371, 1331, 1180, 1072,
7.38 (td, J = 7.8, 1.3 Hz, 1H, –H6 of Indolin-2-one), 7.19 (td, J = 7.8, 1020, 963, 746, 697 cmꢀ1
; HRMS (ESI+) calculated for
1.1 Hz, 1H), 6.75 (d, J = 7.8 Hz, 1H, –H7 of Indolin-2-one), 5.12
C
25H18N2O3S [M + H], 427.1038; found 427.1109.
(s, 2H, –CH2 attached to Indolin-2-one), 3.34 (s, 3H, –CH3 attached
(Z)-3-benzyl-5-(1-(4-chlorobenzyl)-2-oxoindolin-3-ylidene)-
to TZD); 13C NMR (100 MHz, CDCl3) d 169.70, 167.67, 165.67, thiazolidine-2,4-dione (7l). Yield: 62%; vermilion red solid;
147.74, 143.36, 142.41, 132.57, 131.92, 129.08, 128.05, 126.64, mp = 158–160 1C; 1H NMR (400 MHz, CDCl3) d 9.01 (dd, J = 7.9,
124.24, 123.74, 120.11, 108.88, 43.52, 27.59; IR (ATR) u 2916, 1.1 Hz, 1H, –H4 of Indolin-2-one), 7.50–7.46 (m, 2H), 7.39–7.29
2849, 2356, 1732, 1670, 1603, 1518, 1426, 1346, 1280, 1184, (m, 6H), 7.26–7.22 (m, 2H), 7.14 (td, J = 7.8, 1.1 Hz, 1H, –H6 of
1087, 1014, 868, 747, 674 cmꢀ1; HRMS (ESI+) calculated for Indolin-2-one), 6.76 (dd, J = 7.9, 1.0 Hz, 1H, –H7 of Indolin-2-
C19H13N3O5S [M + H], 396.0576; found 396.0627.
one), 4.98 (d, J = 5.6 Hz, 4H, –CH2); 13C NMR (100 MHz, CDCl3)
(Z)-3-Benzyl-5-(2-oxoindolin-3-ylidene)thiazolidine-2,4-dione d 169.85, 167.57, 165.40, 143.80, 134.92, 133.87, 133.59, 132.52,
(7h). Yield: 52%; vermilion red solid; mp = 298–300 1C; 1H NMR 131.32, 129.12, 129.00, 128.91, 128.88, 128.82, 128.68, 128.36,
(400 MHz, DMSO-d6) d 11.27 (s, 1H, –NH), 8.81 (d, J = 7.9 Hz, 127.15, 123.37, 120.04, 109.15, 44.94, 43.49; IR (ATR) u 3878,
1H,-H4 of Indolin-2-one), 7.45–7.29 (m, 6H), 7.10 (t, J = 7.7 Hz, 3729, 3572, 2908, 2843, 2356, 1734, 1667, 1603, 1469, 1428, 1338,
1H, –H6 of Indolin-2-one), 6.97 (d, J = 7.8 Hz, 1H, –H7 of 1144, 1078, 871, 795, 749, 694 cmꢀ1; HRMS (ESI+) calculated for
Indolin-2-one), 4.89 (s, 2H); 13C NMR (100 MHz, DMSO-d6) d C25H18N2O3S [M + H], 461.0648; found 461.0721.
175.30, 173.52, 170.67, 149.25, 143.68, 140.59, 138.81, 138.12,
(Z)-3-Benzyl-5-(1-(4-bromobenzyl)-2-oxoindolin-3-ylidene)-
133.87, 133.21, 132.99, 132.94, 127.35, 125.06, 115.81, 49.49; IR thiazolidine-2,4-dione (7m). Yield: 68%; vermilion red solid;
(ATR) u 3852, 3746, 3150, 2918, 2852, 1744, 1665, 1540, 1453, mp = 170–173 1C; 1H NMR (400 MHz, CDCl3) d 9.01 (dd, J = 7.9,
1379, 1331, 1228, 1146, 1071, 1028, 955, 889, 741, 684 cmꢀ1
;
1.2 Hz, 1H, –H4 of Indolin-2-one), 7.52–7.44 (m, 4H), 7.40–7.31
HRMS (ESI+) calculated for C18H12N2O3S [M + H], 337.0569; (m, 4H), 7.23–7.09 (m, 3H), 6.79–6.72 (m, 1H), 4.97 (d, J = 13.0 Hz,
found 337.0637.
4H, –CH2); 13C NMR (100 MHz, CDCl3) d 169.84, 167.57, 165.39,
(Z)-3-Benzyl-5-(1-methyl-2-oxoindolin-3-ylidene)thiazolidine- 143.78, 134.93, 134.12, 132.52, 132.08, 131.33, 129.00, 128.89,
2,4-dione (7i). Yield: 83%; vermilion red solid; mp = 186–188 1C; 128.83, 128.37, 127.13, 123.39, 121.94, 120.04, 109.15, 44.95,
1H NMR (400 MHz, CDCl3) d 8.94 (d, J = 7.9 Hz, 1H, –H4 of 43.54; IR (ATR) u 3781, 3705, 2356, 1666, 1602, 1476, 1344,
Indolin-2-one), 7.51–7.33 (m, 6H), 7.12 (t, J = 7.8 Hz, 1H, –H6 of 1184, 1143, 1073, 1011, 960, 872, 749, 693 cmꢀ1; HRMS (ESI+)
Indolin-2-one), 6.85 (d, J = 7.8 Hz, 1H, –H7 of Indolin-2-one), calculated for C25H17BrN2O3S [M + H], 505.0143; found 505.0217.
4.96 (s, 2H, –CH2), 3.29 (s, 3H, –CH3); 13C NMR (100 MHz,
(Z)-3-Benzyl-5-(1-(4-nitrobenzyl)-2-oxoindolin-3-ylidene)-
CDCl3) d 170.09, 167.38, 165.41, 144.95, 134.99, 132.53, 130.61, thiazolidine-2,4-dione (7n). Yield: 65%; vermilion red
128.98, 128.93, 128.80, 128.74, 128.72, 128.33, 127.43, 123.14, solid; mp = 220–222 1C; 1H NMR (400 MHz, CDCl3) d 9.04
119.80, 108.34, 44.88, 26.42; IR (ATR) u 3852, 3741, 3614, 3031, (d, J = 7.9 Hz, 1H, –H4 of Indolin-2-one), 8.20 (d, J = 8.3 Hz,
1746, 1663, 1522, 1465, 1376, 1332, 1148, 1064, 871, 749, 692 cmꢀ1
;
2H), 7.52–7.43 (m, 4H), 7.35 (t, J = 7.9 Hz, 4H), 7.17 (t, J = 7.8 Hz,
HRMS (ESI+) calculated for C19H14N2O3S [M + H], 351.0725; found 1H), 6.72 (d, J = 7.8 Hz, 1H), 5.10 (s, 2H, –CH2), 4.98 (s, 2H, –CH2);
351.0796.
13C NMR (100 MHz, CDCl3) d 169.60, 167.62, 165.31, 147.72,
(Z)-3-Benzyl-5-(1-ethyl-2-oxoindolin-3-ylidene)thiazolidine-2,4- 143.34, 142.42, 134.86, 132.57, 131.88, 129.19, 128.98, 128.91,
dione (7j). Yield: 78%; vermilion red solid; mp = 178–180 1C; 128.84, 128.41, 128.02, 126.74, 124.22, 123.70, 120.09, 108.85,
1H NMR (400 MHz, CDCl3) d 8.98 (ddd, J = 7.9, 1.3, 0.5 Hz, 1H, 45.01, 43.48; IR (ATR) u 3779, 1742, 1672, 1605, 1518, 1467, 1340,
–H4 of Indolin-2-one), 7.50–7.28 (m, 6H), 7.13 (td, J = 7.8, 1.1 Hz, 1190, 1137, 1082, 1031, 873, 746, 690 cmꢀ1; HRMS (ESI+)
1H, –H6 of Indolin-2-one), 6.89 (ddd, J = 7.9, 1.1, 0.6 Hz, 1H, –H7 calculated for C25H17N3O5S [M + H], 472.0889; found 472.0967.
of Indolin-2-one), 4.97 (s, 2H, -CH2), 3.85 (q, J = 7.2 Hz, 2H,
–CH2), 1.31 (t, J = 7.2 Hz, 3H, –CH3); 13C NMR (100 MHz, CDCl3) d
4.2 PL Inhibitory assay
170.16, 167.06, 165.47, 144.11, 135.01, 132.51, 130.49, 129.04, Porcine PL (Type II), p-nitrophenyl butyrate and orlistat were
128.98, 128.90, 128.87, 128.80, 128.63, 128.61, 128.49, 128.31, procured from Sigma-Aldrich (St. Louis, MO, USA). Sodium chloride
127.64, 122.95, 120.05, 108.45, 44.85, 35.11, 12.75; IR (ATR) u (molecular biology grade) and Tris buffer were procured from Sisco
3740, 1733, 1667, 1600, 1461, 1428, 1375, 1335, 1232, 1149, 1074, Research Laboratories (MH, India). All other chemicals and solvents
1019, 748, 700 cmꢀ1; HRMS (ESI+) calculated for C20H16N2O3S were in analytical grade. The PL inhibition assay was performed as
[M + H], 365.0882; 365.0948.
(Z)-3-Benzyl-5-(1-benzyl-2-oxoindolin-3-ylidene)thiazolidine- prepared immediately before use.
per the standardized protocol.11,34 The enzyme solutions were
2,4-dione (7k). Yield: 72%; vermilion red solid; mp = 185–
Briefly, crude porcine PL (5 mg mLꢀ1) was suspended in
187 1C; 1H NMR (400 MHz, CDCl3) d 9.01 (d, J = 7.9 Hz, 1H, –H4 Tris-HCl buffer (pH 7.4). The mixture was subjected to vigorous
of Indolin-2-one), 7.49 (d, J = 6.6 Hz, 2H), 7.33 (ddt, J = 15.5, shaking, followed by centrifugation (3000g, 10 min) and the
11.4, 6.1 Hz, 9H), 7.13 (t, J = 7.8 Hz, 1H), 6.80 (d, J = 7.9 Hz, 1H), supernatant was collected. Stock solutions of the orlistat
5.01 (s, 2H, –CH2), 4.99 (s, 2H,-CH2); 13C NMR (100 MHz, and synthesized analogues were prepared in DMSO. A linear
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concentration (0.78–2000 mg mLꢀ1) was used as stock solutions. docking, the structures of the analogues were constructed in
The 875 mL of Tris–HCl buffer and 100 mL of PL solution were ChemDraw Professional 15.0 (PerkinElmer, USA) and were sub-
preincubated (5 min) with different 20 mL concentrations of test jected to energy minimization using the Molecular Mechanics
solutions at 37 1C. Furthermore, 5 mL of the substrate 2 (MM2) force field in the Chem3D module (PerkinElmer, USA).
(4-nitrophenyl butyrate, 10 mM in acetonitrile) was added and The energy minimized ligands were docked into the active site of
the amount of released p-nitrophenol was measured at 405 nm 1LPB using the previously validated grid parameters.11,34 The 2D
[BioTek EPOCH microplate spectrophotometer (VT, USA)]. The interpretation of the ligand interactions was constructed by the
percentage inhibition was calculated using the formula
BIOVIA Discovery Studio 4.5 visualizer (Accelrys, USA).
Molecular dynamics (MD) simulations were conducted in a
% inhibition = [1 ꢀ (AT/AE)] ꢂ 100
high-performance computing facility using
a standardized
where AE is the absorbance of enzyme control (without inhibitor), protocol.11,34 Molecular dynamic simulations were performed on
and AT is the difference between the absorbance of the test sample, a docked ligand–PL complex using GROMACS-5.1.4 packages39
with and without the substrate. IC50 values were calculated from with a CHARMM27 force field.42 The topology of ligands was
the slope of the linear regression curve.
obtained from an online tool provided by the Swiss Institute of
Bioinformatics.43 Bound structures were solvated using a TIP3P
water model in a cubical box. Five sodium ions were added to
4.3 PL inhibition kinetics
Enzyme inhibition kinetics was performed for the most active neutralize the system. A steepest descent algorithm was used for
analogues (7k and 7m) to determine their nature of inhibition. the energy minimisation of the system while the system was
The procedure for the kinetics study was performed as per the stabilised by using canonical NVT and NPT ensembles. A 20 ns
standardized protocol.11,34 Briefly, the PL inhibition assay standard MD simulation was performed keeping similar para-
protocol was repeated at four different substrate concentrations meters as per our previous MD simulation studies and trajectories
(25, 50, 100 and 200 mM) using increasing concentrations of the were stored. The analyses were performed using GROMACS
inhibitor. The absorbance of the final reaction mixture was inbuilt analysis tools.
taken in a kinetic mode (16 readings for 30 min.) and the
respective concentration of 4-nitrophenol was calculated from
its calibration curve. The velocity of the reaction was then
Conflicts of interest
calculated as the slope of the graph for concentration vs. time.
A double reciprocal Lineweaver–Burk plot with reciprocals of
The authors declare that there are no conflicts of interest.
velocity and substrate concentration (on y-axis and x-axis, respec-
tively) was plotted to understand the nature of inhibition.26 The Acknowledgements
inhibition constant, Ki, was calculated using the Cheng–Prusoff
The authors acknowledge Birla Institute of Technology and
equation.27
Science, Pilani (BITS Pilani), Pilani Campus, Pilani, Rajasthan,
4.4 Fluorescence quenching studies
India, for providing NMR, high-performance computing facili-
ties and other necessary laboratory facilities. The authors also
acknowledge the Department of Science and Technology (DST),
New Delhi, India, for sponsoring the HRMS facility at BITS
Pilani under the FIST program. Mr Ginson George is thankful
to the Council for Scientific and Industrial Research (CSIR),
New Delhi, India, for providing a fellowship (SRF – File No: 09/
719 (0100)/2019-EMR-I).
Effects of the synthesized analogues on the fluorescence
quenching of the PL were evaluated by the previously reported
methods with necessary modifications.28,41 Standard stock
solutions (2 mg mLꢀ1) of the most active analogues were
prepared by dissolution in DMSO. A standard solution of PL
(5 mg mLꢀ1) was prepared in pH 7.4 Tris–HCl buffer. Different
concentrations of inhibitors (0.15625–100 mg mLꢀ1) were mixed
along with 10 mM of PL and their fluorescence intensity was
recorded. For the titration studies, an excitation wavelength of
290 nm was selected, and the emission wavelength range was
from 305 to 500 nm using a cuvette of 1.0 cm pathlength. The
slit widths of excitation and emission were set as 1.5 nm. The
fluorescence spectra were recorded at three temperatures 298,
304 and 310 K by a spectrofluorometer (Fluorolog-3 fluores-
cence spectrophotometer, Horiba Jobin Yvon Inc., France) and
a linear Stern–Volmer plot was used for the identification of the
quenching mechanism of the inhibitors.
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