Design, synthesis and biological evaluation of N-substituted indole-thiazolidinedione analogues as potential pancreatic lipase inhibitors

Article abstract of DOI:10.1111/cbdd.13846

Pancreatic Lipase (PL) is a key enzyme responsible for the digestion of 50%–70% of dietary triglycerides, hence its inhibition is considered as a viable approach for the management of obesity. A series of indole-TZD hybrid analogues were synthesized, characterized and evaluated for their PL inhibitory activity. Knoevenagel condensation of various substituted indole-3-carboxaldehyde with substituted thiazolidinediones resulted in the formation of titled analogues. Analogues 6d and 6e exerted potent PL inhibitory activity (IC₅₀-6.19 and 8.96?μM, respectively). Further, these analogues exerted a competitive mode of PL inhibition. Moreover, molecular modelling studies were in agreement with the in vitro results (Pearson's r?=.8682, p?<.05). The fluorescence spectroscopic analysis further supported the strong binding affinity of these analogues with PL. A molecular dynamics study (20?ns) indicated that these analogues were stable in a dynamic environment. Thus, the present study highlighted the potential role of indole-thiazolidinedione hybrid analogues as potential PL inhibitors and further optimization might result in the development of new PL inhibitory lead candidates.

Full text of DOI:10.1111/cbdd.13846

Received: 20 January 2021
Revised: 20 March 2021
Accepted: 5 April 2021
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DOI: 10.1111/cbdd.13846
R E S E A R C H A R T I C L E
Design, synthesis and biological evaluation of N-substituted
indole-thiazolidinedione hybrid analogues as potential pancreatic
lipase inhibitors
Ginson George
Prashant S. Auti
Atish T. Paul
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Laboratory of Natural Product Chemistry,
Department of Pharmacy, Birla Institute
of Technology and Science, Pilani (BITS
Pilani), Pilani Campus, India
Abstract
Pancreatic Lipase (PL) is a key enzyme responsible for the digestion of 50%–70% of
dietary triglycerides, hence its inhibition is considered as a viable approach for the
management of obesity. A series of indole-TZD hybrid analogues were synthesized,
characterized and evaluated for their PL inhibitory activity. Knoevenagel
condensation of various substituted indole-3-carboxaldehyde with substituted
thiazolidinediones resulted in the formation of titled analogues. Analogues 6d and
6e exerted potent PL inhibitory activity (IC50-6.19 and 8.96 µM, respectively).
Further, these analogues exerted a competitive mode of PL inhibition. Moreover,
molecular modelling studies were in agreement with the in vitro results (Pearson's
r = .8682, p < .05). The fluorescence spectroscopic analysis further supported the
strong binding affinity of these analogues with PL. A molecular dynamics study
(20 ns) indicated that these analogues were stable in a dynamic environment. Thus,
the present study highlighted the potential role of indole-thiazolidinedione hybrid
analogues as potential PL inhibitors and further optimization might result in the
development of new PL inhibitory lead candidates.
Correspondence
Atish T. Paul, Laboratory of Natural
Product Chemistry, Department of
Pharmacy, Birla Institute of Technology and
Science, Pilani (BITS Pilani), Pilani, India.
Email: atish.paul@pilani.bits-pilani.ac.in
K E Y W O R D S
enzyme inhibitors, fluorescence spectroscopy, molecular modelling, pancreatic lipase
1
INTRODUCTION
Since obesity is caused mainly due to the chronic imbal-
ance between energy intake and expenditure associated with
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According to the position statement of the World Obesity
Federation, obesity is defined as a chronic relapsing pro-
gressive disease process (Bray et al., 2017). In 2016, 13%
of the global adult population were reported as obese pop-
ulation, while 39% of adults were reported as overweight
(WHO, 2020). The prevalence of children's and adolescent's
obesity is also increasing and hence UNICEF in its new global
assessment of child malnutrition warned about the high rates
of childhood obesity (Ivanovitch et al., 2020). Though obe-
sity is a preventable condition, more than 4.7 billons adults
are prone to premature death due to obesity and its related
co-morbidities (WHO, 2020).
the sedentary lifestyle, current anti-obesity strategies are
mainly focussed on the reduction in energy intake and expen-
diture (Srivastava & Apovian, 2018). Pancreatic Lipase (PL)
is a vital enzyme in triglyceride digestion, where it accounts
for 50%–70% fat degradation. Dietary triglycerides are the
main source of the energy, thus the inhibition of digestion
of dietary triglycerides and their subsequent fat absorption
viz. modulating the PL is considered as a prominent strategy
for obesity management (Birari et al., 2009; Lowe, 2002).
Orlistat is the only specific PL inhibitor used clinically for
managing obesity. However, it is reported to possessed numer-
ous adverse effects such as hepatotoxicity and nephrotoxicity
Chem Biol Drug Des. 2021;00:1–11.
wileyonlinelibrary.com/journal/cbdd
© 2021 John Wiley & Sons A/S.
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GEORGE Et al.
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(Elizebeth et al., 2013; Filippatos et al., 2008). Hence, there
is an urgent need to develop newer PL inhibitory based anti-
obesity drugs.
Currently, numerous natural/herbal products have also
been explored for the management of obesity that resulted
in the identification of potential structural scaffolds for PL
inhibition (Liu et al., 2020; Sridhar et al., 2019). Amongst
these, indole-containing scaffolds contribute a significant
share in PL inhibition (Figure 1; Birari et al., 2009; George
et al., 2019; Sridhar et al., 2020).
the five-membered heterocycles possessed these active war-
heads for the PL inhibition. For instance, thiazolidinedione
and rhodanine analogues with a nitrogen centred diamide
linkage exerted a potential PL inhibitory activity (Chauhan
et al., 2019; George et al., 2020; Sridhar, Bhurta, et al., 2017).
The structural diversity at two positions of thiazolidinedione
and rhodanine offers an additional advantage for the fur-
ther incorporation of essential pharmacophoric features co-
verts these as attractive structural scaffolds in the area of PL
inhibitors.
Further, numerous chemical warheads that mimic the nat-
ural esters of triglycerides have also been explored as poten-
tial pharmacophoric features for PL inhibition. This includes
fluorinated ketone, α-keto esters, α-keto amides (Sridhar,
Bhurta, et al., 2017) and chalcones (Huo et al., 2021). These
reactive ester or ester mimicking carbonyl groups acts as
electrophiles for Ser 152 in the active site, thereby it is help-
ful for the inactivation of PL.
Past research has demonstrated the importance of five-
membered nitrogen heterocycles on PL inhibitory profiles.
For instance, oxadiazole (Point et al., 2012), 1,2,4-triazole
(Bekircan et al., 2014; Kahveci et al., 2017), triaza-
phospholinoalkanes (Hamzaoui et al., 2015), rhodanine
(Chauhan et al., 2019), thiazolidinedione (Sridhar, Bhurta,
et al., 2017) etc. have explored for PL inhibition. Some of
Numerous chemical entities consisting of these individ-
ual scaffolds have been reported previously. For instance,
indole glyoxamide analogues, tryptophan-containing amino
acids derivatives (Sridhar et al., 2020; Stefanucci, Dimmito,
et al., 2019; Stefanucci, Luisi, et al., 2019), diaryl substituted
pyrazolyl thiazolidinediones and rhodanine acetic acid con-
taining analogues (Chauhan et al., 2019; Sridhar, Bhurta,
et al., 2017) have been explored for their PL inhibition
(Figure 2).
By considering the promising activity of indole and thi-
azolidinedione moieties in the PL inhibition, hybrid ana-
logues of these scaffolds might be results in the formation of
potential bioactive PL inhibitory analogues.
Thus, based on the above reports and numerous
pharmacophoric requirements, the authors designed
FIGURE 1 Structures of some indole and five-membered nitrogen heterocycles containing PL inhibitors

GEORGE Et al.
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FIGURE 2 Structures of some synthetic PL inhibitors containing indole or thiazolidinediones
PL inhibition activities were further supported by the spec-
troscopical and molecular modelling studies.
2
EXPERIMENTAL SECTION
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The experimental procedures for synthesis and biological
evaluation for analogues (6a to 6ab) have been discussed
elsewhere [(George et al., 2020; Sridhar, Ginson, et al., 2017)
Supplementary Data].
3
RESULTS AND DISCUSSION
Chemistry
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3.1
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FIGURE 3 The rationale involved in the design of indole-
The synthesis of all the titled analogues 6a to 6ab was car-
ried out as per the procedure illustrated in Scheme 1. The
key starting materials, TZD (3) was synthesized by the
condensation reaction of thiourea (1) with chloroacetic
acid (2) under ice-cold condition followed by acidifica-
tion and refluxing. N-arylated analogues of TZD (4) and
thiazolidinedione hybrid analogues as PL inhibitors
indole-thiazolidinedione hybrid analogues (Figure 3) via a
molecular hybridization approach followed by the evaluation
of their PL inhibitory potential. Furthermore, the obtained

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GEORGE Et al.
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SCHEME 1 Synthesis of the title
analogues. Reagents and conditions (i) 0–
5°C, 30 min, RT; (ii) HCl, H2O, 110–120°C,
10–12 h; (iii) NaH, DMF, overnight; (iv)
Piperidine, Glacial acetic acid, EtOH, 6-8h,
Reflux
indole-3-carboxaldehyde (5a–5f) were obtained by the treat-
ment of the corresponding starting materials (3 or 5) with
NaH in anhydrous THF, followed by the addition of vari-
ous alkyl/aryl halides (George et al., 2020). The final hybrid
analogues (6a to 6ab) were prepared by Knoevenagel con-
densation, wherein 4 and 5a–f derivatives in ethanol were
refluxed in the presence of piperidine and glacial acetic
acid. Apart from the piperidine/glacial acetic acid, L-proline
catalysed Knoevenagel condensation was also reported for
the synthesis of potential indole-thiazolidinedione hybrids
(Riyaz et al., 2011).
confirmed the condensation of TZD and indole scaffolds.
All the analogues showed a prominent signal at δ 15–51
due to the presence of various alkyl groups. Finally, the as-
signed structures of various analogues were confirmed by
their mass spectra where characteristic [M + H]+ peak was
observed.
3.2
In vitro PL inhibitory potential
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evaluation, enzyme kinetics and structural
activity relationship
The products (6a to 6ab) were obtained in good yield
with a buff yellowish colour. Further, the synthesized an-
The in vitro PL inhibitory activity of the synthesized
analogues were evaluated by a standardized assay protocol
(George et al., 2020; Sridhar, Bhurta, et al., 2017). As
summarized in Table 1, synthesized analogues displayed
potential to moderate PL inhibition (IC50-6.19 to 45.00 µM).
Most of the analogues exhibited good (<15 µM) to moderate
(15–30 µM) activity, while few analogues displayed poor
activity (>30 µM). Analogue 6d exhibited the most potent
activity against PL, with an IC50 of 6.19 µM, followed by 6e
(IC50-8.96 µM). Orlistat (positive control) exhibited a potent
PL inhibitory activity (IC50-0.86 µM).
The inhibition mode of the synthesized analogues was eval-
uated by performing an enzyme inhibition kinetic experiment
of the topmost active analogues (6d and 6e). The obtained
Lineweaver–Burk (LB) plots (Lineweaver & Burk, 1934)
were converged at its first quadrant and intersected at one
point, which indicated that these analogues exerted a compet-
itive inhibition (Figure 4). A proportionate increase in the ap-
parent Km values without affecting the Vmax further indicated
the scope of a reversible competitive inhibition (Table 2).
Inhibition constant (Ki) for the values were deduced as 3.931
and 5.691 µM (retrieved from the Cheng–Prusoff equation;
Burlingham & Widlanski, 2003), proving that these molecules
have a strong affinity towards PL. From the enzyme kinetics
study, it can be concluded that the synthesized analogues were
bound to the active site of the PL during the inhibition.
1
alogues were characterized by ATR spectroscopy, H, 13C
NMR spectroscopy, and mass spectrometry (HRMS - ESI
mode). The obtained data were in good agreement with their
structural identity. The IR spectrum of the title analogues
(6a to 6ab) showed a strong absorption band at 1,697–1,746
and 1,653–1,699 cm−1 due to two carbonyl groups. The
disappearance of methylene proton (δ value-3.95 ppm) of
TZD and the appearance of arylidene (=CH-) peak further
confirmed the formation of the hybrid analogues. Arylidene
proton in the target analogues can exist as ‘E’ and ‘Z’ config-
uration. Yu Momose et al. revealed that the arylidene proton
in ‘Z’ configuration resonates at 7.4 to 8.2 ppm, while the
‘E’ configuration resonates at 6.2 to 6.8 ppm. Anisotropic
effects of TZD carbonyl group resulted in the deshielding
of arylidene proton. Further, the intramolecular hydrogen
bonds between the TZD carbonyl group and arylidene pro-
ton favoured the formation of a thermodynamically stable
‘Z’ configuration (Momose et al., 1991; Sridhar, Bhurta,
et al., 2017). In the present work, arylidene groups proton
appeared as singlet or multiplet at >8.26 ppm, indicating
that the products predominantly formed were in ‘Z’ con-
figuration. -CH2 proton attached to the indole resonated in
the range of 5.23 to 5.54 ppm (δ). All other protons were
observed in expected regions. The appearance of three
C=O peaks in 13C NMR (δ value-165 to 173 ppm) further

GEORGE Et al.
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GEORGE Et al.
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FIGURE 4 Double reciprocal Lineweaver–Burk plots of analogues 6d, 6e and orlistat
TABLE 2 Km, Vmax and Ki values of
Km (apparent) at different
6d and 6e retrieved from the PL enzyme
concentration (µM)
kinetics
Vmax (µM/
Ki
µM
Nature of
inhibition
Code
0 µM
0.625 µM
5 µM
min)
6d
6e
Orlistat
84.545
88.567
79.757
109.672
111.585
87.301a
148.084
132.383
119.756b
1.413
1.670
1.956
3.931
5.691
0.528
Competitive
Competitive
Competitive
a0.0625 µM.
b4 µM orlistat concentration.
FIGURE 5 Generalized SAR of the
synthesized indole-TZD hybrid analogues
(6a–6ab)
Based on the in vitro PL inhibition assay, a preliminary
structure-activity relationship of the screened analogues has
been deduced. As represented in Figure 5, the presence of
aromatic functionalities on the indole significantly increased
the PL inhibition activity of analogues in comparison with
the alkyl/unsubstituted counterparts. For instance, 6d with
a benzyl substitution exhibited higher PL inhibitory activ-
ity (IC50-6.19 µM) than the unsubstituted/alkyl substitu-
tions (6a–6c; IC50 values 23.42 and 10.52 µM, respectively).
Further, an additional substitution on the aryl ring resulted in
the variation in the PL inhibitory potential. Ring deactivat-
ing groups resulted in the weakening of the potency, wherein
-NO2 substitution resulted in the poorest activity (IC50 value
ranges 18.29 and 36.73 µM, respectively). A similar kind
of trend has been observed with the TZD ring substitutions
wherein electron-withdrawing substituents exerts a reduc-
tion of the PL inhibitory potential. Analogue 6k possessing
a 4-chlorobenzyl substituent on amidic nitrogen exhibited a
weaker PL inhibition (IC50-9.14 µM) than the 6d that lacked
electron-withdrawing groups in the aromatic functionality
(IC50-6.19 µM). Nitro substituted analogues (6y and 6ab)
exhibited comparatively lesser activity (IC50-18.76 and
36.73 µM, respectively) than the -Cl substituted analogues
(6k and 6n; IC50-9.14 and 31.63 µM, respectively).

GEORGE Et al.
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3.3
with PL
Fluorescence quenching measurements
Fluorescence quenching is described by the Stern–Volmer
equation as follows:
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F0
The intrinsic fluorescence exerted by various aromatic amino
acids provides the necessary information about the structural
and dynamic properties of a protein. Thus, the association re-
action between a protein and a bioactive molecule can be in-
vestigated with the help of fluorescence spectroscopy. Seven
tryptophan residues (Trp 17, Trp 30, Trp 85, Trp 106, Trp 252,
Trp 338 and Trp 402) cause the major intrinsic fluorescence
properties to the PL (Zhu et al., 2018). The effects of the syn-
thesized analogues (6d and 6e) in the fluorescence quenching
of PL were evaluated and the results are depicted in Figure 6,
Supplementary Data and Table 3. The screened analogues
exerted a concentration dependant quenching effect on the
PL fluorescence. Further, with the increased concentrations
of screened analogues, the fluorescence intensity of PL was
considerably decreased that suggested the microenvironment
changes results due to the PL-analogues interactions.
= 1 + Kqꢀo [Q] = 1 + KSV [Q] .
F
where F and F0 are the fluorescence intensities of PL with or
without quencher, respectively. [Q] represents the concentration
of the quencher. Ksv denotes the Stern–Volmer quenching con-
stant, while Kq is the bimolecular quenching constant, τ0 is the
average lifetime of the fluorophore molecule without quencher
(10−8 s). The values of Ksv, Kq are determined from the linear
regression plot of F0/F versus [Q].
Fluorescence quenching is generally classified into
two types, namely static quenching and dynamic quench-
ing (Ladokhin, 2000; Lakowicz, 2006). Formation of
non-fluorescent ground state complex (fluorophore
and quencher) is termed as static quenching, while the
quencher molecules deactivated fluorophore in the solu-
tion is denoted as dynamic quenching. Moreover, a lin-
ear Stern–Volmer plot usually indicates a single way of
quenching as either dynamic or static quenching wherein
the fluorophores are all equally accessible to the quencher.
An upward curvature in the Stern–Volmer plot suggests
the probability of occurrence of both mechanisms, static
and dynamic quenching effects. Further, static quenching
exhibit a diminished quenching constant with an increase
in temperature, while dynamic quenching results in incre-
ment in the quenching constant with the increment of tem-
perature (Ladokhin, 2000; Lakowicz, 2006). In the present
work, screened analogues were in good agreement with
the linear Stern–Volmer plot indicating that the predomi-
nance of a single fluorescence quenching mechanism, that
is static or dynamic quenching mechanisms.
a
75000
60000
45000
30000
h
15000
0
The Kq value reflects the accessibility of fluorophore to
the quencher or quenching efficiency of analogues towards a
protein. For a typical dynamic quenching mechanism, the Kq
value is expected to ≈1 × 1010 M−1 S−1, while a higher value
indicates the static mechanism.
350
400
450
500
Wavelength (nm)
FIGURE 6 Representative fluorescence spectra of PL in the
presence of 6d at various concentrations (pH 7.4)
TABLE 3 Fluorescence quenching parameters for the interactions of analogues with PL
#
T (K)
KSV (×104 M−1
)
Kq (×1010 M−1 s−1
)
R2
Kb (×104 M−1 s−1
)
n
R2
6d
298
304
310
298
304
310
1.98
1.53
1.23
1.80
1.65
1.42
124.53
96.23
77.36
113.21
103.77
89.31
.9893
.999
.9978
.9971
.9970
.9951
3.32
2.19
1.74
3.60
2.05
1.37
0.836
0.899
0.892
0.808
0.940
1.048
.9882
.9948
.9938
.9970
.9870
.9918
6e
Note: Stern–Volmer quenching constant (Ksv), Bimolecular quenching constant (Kq), binding constant (Kb) and the number of binding sites (n) at different
temperatures.

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GEORGE Et al.
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FIGURE 7 (a) 2D interaction diagram of 6d in the active site of PL (1LPB); (b) RMSD of 6d-PL complex retrieved from 20 ns MD
simulations
As represented in Table 3, an inverse correlation between
the Ksv and temperature has been obtained, suggesting that
the quenching effects on the PL may be mainly attributed to
static quenching. Furthermore, Kq values were in the range
of 77 to 125 (1010 M−1 s−1) supporting the static mechanism-
based quenching wherein both analogues formed a complex
with PL.
3.4
Molecular docking and dynamics
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To find out the putative mode of structural interactions respon-
sible for the PL inhibition, molecular docking simulation of
the analogues were performed with the crystal structure of the
PL-Colipase complex (PDB ID: 1LPB, 2.46 Å resolution n) via
Molegro Virtual Docker 6.0 (Thomsen & Christensen, 2006)
with validated grid parameters (George et al., 2020; Sridhar,
Bhurta, et al., 2017). The obtained MolDock scores and the
various interactions exhibited by the hybrid analogues are rep-
resented in Figure 7 and Supplementary Data.
PL is a glycoprotein of 50 KDa, comprised of 449 amino
acids. The larger N-terminal contains 1–336 amino acids
with a catalytic triad, while the C-terminal comprises the
residues from 337 to 449. Co-lipase is a crucial enzyme for
the enzymatic activity of PL that bound to the C-terminus.
Structurally, the active site of PL comprised of a catalytic
triad of Ser 152-Asp 176- His 263 that is enclosed in a hy-
drophobic lid domain (Egloff et al., 1995). Previous reports
also suggested that the modification of Ser 152 residue leads
to its inactivation and hence is considered as a prominent
amino acid for PL inhibition (Hadvary et al., 1991; Lüthi-
Peng et al., 1992). During the lipolysis process, at a hostile
lipid-water interface, PL forms a complex with co-lipase that
results in the opening of the lid domain and successive inter-
actions with the active site amino acids. To achieve an active
conformation, the open lid structure of the PL requires the
interactions of Arg 256 and Asp 257 with the core residues
(Birari et al., 2011; Lowe, 2002).
For a static quenching mechanism, the binding constant
and number of binding sites can be identified by the modified
Stern–Volmer equation as follows.
F0
log = logKb + n log [Q] .
F
where Kb is the binding constant and ‘n’ is the number of bind-
ing sites. Values of these parameters were determined from the
double log graph of log F0/F versus log [Q] (Ladokhin, 2000;
Lakowicz, 2006).
Based on the modified Stern–Volmer equation, the num-
ber of binding sites and binding constants were calculated
that further supported the inhibitory potential of these ana-
logues. The number of the binding sites (n) for the ligand-PL
complex was approximately equal to 1 (0.808 to 1.048),
suggesting the presence of the single binding site, that was
in agreement with the competitive inhibition, deduced from
the enzyme inhibition kinetics. Moreover, a higher binding
force of these analogues with PL has been identified from the
strong binding constant (Kb) that were in the range 1.37 to
3.60(×104 M−1 s−1).

GEORGE Et al.
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From the docking pose of ligand (Figure 7; Supplementary
During the MD run of the orlistat, H bonding was pre-
dominantly formed with Gly-76, Phe-77, His-151, Ser-152
while π-alkyl interactions were observed with Ile-78, Pro-
180, Leu-213, Trp-252, Arg-256, Ala-259 etc.
In case of 6d, initially, H bond interactions were formed
with the Ser 152 whereas prominent π–π interactions with lid
domain amino acids (Phe 77, Tyr 114 and Phe 215) were ob-
served throughout the simulation. Apart from that, π-sulphur
interactions were also exhibited with lid domain and catalytic
triad amino acids.
In case of 6e, H-bond has formed with the Ser 152 during
the later stages of the simulations, whereas π–π interactions
were prominent during the entire tenure. Numerous π-alkyl
interactions were observed, wherein various amino acids
such as Ile 78, Pro 180, Arg 256, Ala 259, Ala 260, and Leu
264 were involved. Additionally, 6e exerted π-sulphur inter-
action with Phe 77, Tyr 114, His-151, Phe 215 and His-263.
Data), it is clearly observed that these analogues formed in-
teractions such as hydrogen bonding, π–π stacking, π-sulphur
and π-cation with the active site amino acids. Interestingly,
the PL inhibitory variation trends across different substituents
and their MolDock scores exhibited a significant correlation
(Pearson's r = .8682, p < .05). For instance, 6d, the most ac-
tive analogue from the series (IC50-6.19 µM) possessed a top
docking score of −115.619 kcal/mol while, 6e, the second
most active (IC50-8.96 µM) in the series exhibited a MolDock
score of −108.706 kcal/mol. The majority of the molecules
exhibited a consistent H-bond interaction with Gly76, Phe77,
His 151 and Ser152. Further, prominent π–π interactions
were observed with the lid domain amino acid (Phe 77, Phe
215). An π-sulphur interaction has been observed with the
catalytic triad amino acid, His 263. Moreover, π-cation inter-
action with Arg 256 has also been observed that suggested
the probable role of these analogues for the opening of lid
domain amino acid.
Orlistat possessing a reactive lactone ring, will break
during the interaction with PL and results in the formation
of reversible covalent bond inhibition (Egloff et al., 1995).
Orlistat interacted with 1LPB via numerous hydrogens
bonding (with Phe-77, Ser-152 and His-263) as well as π-
alkyl interactions (Supplementary Data). Since orlistat
contains long alkyl chains, numerous π-alkyl interactions
(Tyr-114, Trp-252, Arg-256, Phe-258, Ala-259) were ob-
served in the active site of 1LPB. However, synthesized ana-
logues exerted a lesser extent of π-alkyl interaction. Overall,
the Mol Dock score of orlistat (−127.750 kcal/mol) was sig-
nificantly higher than that of synthesized analogues (−82.373
to −115.619 kcal/mol). This might be due to the variations in
the π-alkyl interaction as well as the presence of the reactive
constrained lactone ring in orlistat.
4
CONCLUSION
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By considering the PL inhibitory potential of indole and
TZD analogues for PL inhibition, the present study aimed
to develop indole-TZD hybrid analogues as a potential PL
inhibitor. A series of indole-TZD hybrid analogues (6a to
6ab) have been synthesized and evaluated for their in vitro
PL inhibition activity. Amongst the screened analogues,
6d exhibited a potential PL inhibition (IC50-6.19 µM), fol-
lowed by 6e (IC50-8.16 µM). Enzyme kinetics established
a reversible competitive type of PL inhibition by these
synthesized analogues. Fluorescence study further sup-
ported these facts, wherein, the presence of one binding
site has been confirmed. Apart from this, a greater binding
efficiency of these analogues with PL also suggested by
the fluorescence spectroscopic studies. Molecular dock-
ing results further supported the PL inhibitory potential
of titled analogues, wherein significant correlations be-
tween the PL inhibition and MolDock score (Pearson
correlation r = .8682, p < .05) were obtained. Moreover,
the MD simulation revealed a stable confirmation of both
analogues throughout the run. Preliminary investigation
revealed that the electron-withdrawing groups on indole-
thiazolidinedione hybrids exerted decremental PL inhibi-
tory potentials. Thus, substitutions with electron-donating
groups might be helpful for the further PL inhibitory po-
tentials. Apart from that, the overall hydrophobicity of the
hybrid analogues can also be potentiated by incorporating
numerous long-chain carbon atoms similar to the reported
scaffolds. For instance, numerous natural products con-
taining isoprenoids (prenyl/geranyl/farnesyl) have been
explored due to their potent PL inhibitory activities. In
conclusion, the present study shed lights into the poten-
tial of indole-TZD hybrid analogues and further detailed
Various interactions of the analogues with PL (1LPB)
were identified by using molecular modelling studies.
Further, a 20 ns MD simulation has been performed for orli-
stat, 6d and 6e, that provides information about the stability
of the binding mode and interactions in a dynamic environ-
ment. Orlistat, 6d and 6e exhibited a stable binding confir-
mation throughout the MD simulation run (Supplementary
Data, Figure 7). Orlistat exerted a maximum deviation of
≈ 0.4 nm (throughout the MD run) while analogues 6d and
6e exhibited a maximum deviation of ≈ 0.8 nm (at ≈ 12.5
and 7.5 ns), respectively. Furthermore, a stable radius of
gyration indicated the compactness of the 1LPB during the
MD run with studied molecules. The obtained interactions
during the course MD simulation of these analogues 1LPB
complex during 20 ns of MD simulation are summarized in
Supplementary Data. The ligand analogues exhibited numer-
ous interactions such as H bonding, π–π interaction, π-alkyl
and π-sulphur interactions, while orlistat exerted H bonding
and π-alkyl interactions.

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GEORGE Et al.
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ACKNOWLEDGEMENTS
The authors acknowledge Birla Institute of Technology
and Science, Pilani (BITS Pilani), Pilani Campus, Pilani,
Rajasthan, India, for providing NMR, high-performance
computing facilities 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
programme. 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).
CONFLICT OF INTEREST
The authors declare there are no conflicts of interests.
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SUPPORTING INFORMATION
Additional supporting information may be found online in
the Supporting Information section.
How to cite this article: George G, Auti PS, Paul AT.
Design, synthesis and biological evaluation of
N-substituted indole-thiazolidinedione hybrid
analogues as potential pancreatic lipase inhibitors.
Chem Biol Drug Des. 2021;00:1–11. https://doi.
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