Design, synthesis, evaluation, and molecular modeling studies of indolyl oxoacetamides as potential pancreatic lipase inhibitors

Article abstract of DOI:10.1002/ardp.202000048

A series of indolyl oxoacetamide analogs was synthesized, characterized, and evaluated for their pancreatic lipase inhibitory activity using porcine pancreatic lipase (type II) and 4-nitrophenyl butyrate. Compound 8d exhibited a potent inhibition, with an IC₅₀ value of 4.53 μM, followed by 8c (IC₅₀ = 5.12 μM), compared with the standard drug, orlistat (IC₅₀ = 0.99 μM). Furthermore, analogs 8c and 8d exhibited a reversible competitive inhibition, similar to orlistat. Molecular docking studies of the compounds 7a–f and 8a–f were in agreement with the in vitro results, wherein 8d exhibited a potential MolDock score of ?163.052 kcal/mol. A 10-ns molecular dynamics simulation of 8d complexed with pancreatic lipase confirmed the role of π–π stacking and π–cation interactions with the lid domain and Arg 256, respectively, in stabilizing the ligand at the active site (maximum observed root mean square deviation ≈ 2 ?). The present study led to the identification of novel indolyl oxoacetamides (8a–d) as potential pancreatic lipase inhibitory leads that might further result in enhanced potency through lead optimization.

Full text of DOI:10.1002/ardp.202000048

Received: 19 February 2020
Revised: 12 April 2020
Accepted: 11 May 2020
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DOI: 10.1002/ardp.202000048
F U L L P A P E R
Design, synthesis, evaluation, and molecular modeling studies
of indolyl oxoacetamides as potential pancreatic lipase
inhibitors
S. N. C. Sridhar | Saksham Palawat | Atish T. Paul
Laboratory of Natural Product Chemistry,
Department of Pharmacy, Birla Institute of
Abstract
Technology and Science, Pilani (BITS Pilani),
A series of indolyl oxoacetamide analogs was synthesized, characterized, and eval-
uated for their pancreatic lipase inhibitory activity using porcine pancreatic lipase
(type II) and 4‐nitrophenyl butyrate. Compound 8d exhibited a potent inhibition,
with an IC50 value of 4.53 µM, followed by 8c (IC50 = 5.12 µM), compared with the
standard drug, orlistat (IC50 = 0.99 µM). Furthermore, analogs 8c and 8d exhibited a
reversible competitive inhibition, similar to orlistat. Molecular docking studies of the
compounds 7a–f and 8a–f were in agreement with the in vitro results, wherein 8d
exhibited a potential MolDock score of −163.052 kcal/mol. A 10‐ns molecular
dynamics simulation of 8d complexed with pancreatic lipase confirmed the role
of π–π stacking and π–cation interactions with the lid domain and Arg 256, re-
spectively, in stabilizing the ligand at the active site (maximum observed root mean
square deviation ≈ 2 Å). The present study led to the identification of novel indolyl
oxoacetamides (8a–d) as potential pancreatic lipase inhibitory leads that might
further result in enhanced potency through lead optimization.
Pilani Campus, Pilani, Rajasthan, India
Correspondence
Atish T. Paul, Laboratory of Natural Product
Chemistry, Department of Pharmacy, Birla
Institute of Technology and Science, Pilani
(BITS Pilani), Pilani Campus, Pilani, Rajasthan
333031, India.
Email: atish.paul@pilani.bits-pilani.ac.in
K E Y W O R D S
ADME, indolyl oxoacetamides, inhibition kinetics, molecular dynamics, orlistat, pancreatic
lipase
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1
INTRODUCTION
encoded by the PNLIP gene, is a primary digestive enzyme secreted from
the exocrine glands of the pancreas,[11,12] and it is involved in the hy-
Recent decades have observed drastic changes in the global food
system that has transformed obesity into a global epidemic. Obesity,
a multifactorial metabolic disorder characterized by an excessive
deposition of fats in the body, is observed among 13% of the global
population, as indicated by the recent WHO statistics.[1] Further-
more, obesity is associated with various comorbid conditions in-
cluding diabetes mellitus and cardiovascular diseases, which pose
major health problems to the obese patients.[2]
drolysis of the triglycerides.[13] The crystal structure of the human PL
(PDB ID: 1LPB) is composed of 449 amino acids, with the catalytic triad
Ser 152–Asp 176–His 263 defining the active site of the enzyme.[14] This
catalytic site is highly restricted by a hydrophobic lid domain that consists
of amino acids Gly 76–Lys 80 and Leu 213–Met 217.[15]
Orlistat (1), a potent PL inhibitor, is one of the clinically approved
drugs widely used for long‐term treatment of obesity.[16] It contains a
β‐lactone (Figure 1), which binds covalently to Ser 152 of the active
site.[17] Although orlistat (1) was reported to exhibit tolerable side
effects,[18] recent reports from FDA have cited severe adverse ef-
fects with long‐term administration of orlistat, which included
Pancreatic lipase (PL) is one of the several targets that have been
explored to treat or prevent obesity,[3–10] and it is considered as a suc-
cessful target due to its tolerable side effects. The human PL (EC 3.1.1.3),
These authors contributed equally to this study.
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Arch Pharm. 2020;e2000048.
wileyonlinelibrary.com/journal/ardp © 2020 Deutsche Pharmazeutische Gesellschaft
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FIGURE 1 Rationale for designing indolyl oxoacetamides as pancreatic lipase inhibitors (carbazole and indole moieties are highlighted in
blue, and β‐lactone and α‐ketoamide warheads are highlighted in red)
hepatotoxicity and acute pancreatitis.[19] These events highlighted
the necessity for safer and effective anti‐obesity drugs.
Initially, 5‐methoxyindole (4) or its N‐benzyl derivative (5) was made
to react with oxalyl chloride to obtain a yellowish‐orange precipitate
of glyoxylyl chloride derivative (6). Furthermore, the reaction of
compound 6 with various aniline derivatives in the presence of
triethylamine afforded a series of indolyl oxoacetamides, 7a–f and
In the recent years, various amide functionality‐based pharmaco-
phores, including tripeptides and rhodanines, were reported as potential
PL inhibitors.[20–22] The carbonyl groups of these amides mimic that of
the natural esters in the triglycerides, thus acting as electrophiles for Ser
152 of the active site. In particular, scaffolds with 1,3‐diketoamide and
α‐ketoamide functionality possessed a greater PL inhibitory potential,
owing to the greater reactivity of their carbonyl groups.[23,24]
TABLE 1 Pancreatic lipase inhibitory activity of indolyl
oxoacetamides 7a–f and 8a–f
Previously, we have synthesized and evaluated 24 carbazolyl
oxoacetamides for PL inhibitory activity.[25] As represented in
Figure 1, these carbazolyl oxoacetamides consisted of a carbazole
moiety and an α‐ketoamide warhead, which exhibited a dual inter-
action with the lid domain and the active site of PL, respectively.
Compounds 2 and 3 from this series possessed a potential PL in-
hibition (IC50 values of 6.31 and 8.72 µM, respectively). Nevertheless,
an in silico analysis of these molecules highlighted a steric hindrance
exhibited by the carbazole moiety, which resulted in a greater
interaction distance between the reactive carbonyl group of the
ketoamide and Ser 152 of the active site.
Compound
R
Ar
IC50 (µM)
7a
–H
3,4‐dimethylphenyl
4‐methoxyphenyl
4‐methoxybenzyl
3,4,5‐trimethoxyphenyl
3,4‐dichlorophenyl
4‐fluorophenyl
18.26 0.58
18.12 0.43
17.93 0.39
16.38 0.26
34.62 0.94
37.83 0.98
9.14 0.69
6.28 0.24
5.12 0.38
4.53 0.47
23.21 0.93
19.48 0.76
0.99 0.11
7b
7c
–H
On the basis of the above findings, we hypothesized that the
replacement of carbazole with an indole moiety would reduce this
steric hindrance and the interaction distance, consequently enhan-
cing the PL inhibitory activity. Accordingly, the present study in-
volved synthesis, in vitro evaluation, and in silico studies of indolyl
oxoacetamide analogs as a new class of potential PL inhibitors.
–H
7d
7e
–H
–H
7f
–H
8a
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
–CH2C6H5
3,4‐dimethylphenyl
4‐methoxyphenyl
4‐methoxybenzyl
3,4,5‐trimethoxyphenyl
3,4‐dichlorophenyl
4‐fluorophenyl
8b
8c
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2
RESULTS AND DISCUSSION
8d
8e
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2.1
Chemistry
8f
The syntheses of all the final compounds, 7a–f and 8a–f (Table 1),
were carried out as per the procedure detailed in Scheme 1.[26–29]
Orlistat

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SCHEME 1 The synthesis of the target
compounds 7a–f and 8a–f. Reagents and
conditions: (i) C6H5CH2Cl, KOH, DMF, RT,
12 hr; (ii) (COCl)2, Et2O, 0°C, 30 min; (iii)
Ar–NH2, TEA, THF, RT, 2 hr
8a–f in 82–93% yields. Structures of the synthesized analogs were
well characterized using infrared (IR), nuclear magnetic resonance
(NMR; 1H and 13C), and high‐resolution mass spectrometry (HRMS;
see Supporting Information Data). In IR spectra, characteristic peaks
at ~3,700, ~1,680, and 1,630 cm−1 were assigned to the amide N–H,
α‐ketocarbonyl, and amide carbonyl functionalities, respectively.
Furthermore, in 13C‐NMR spectra of 7a–f and 8a–f, carbons of car-
bonyl and amide functionalities resonated at ~180 (CO) and
~160 ppm (NHCO), respectively.
TABLE 2 Km, Vmax, and Ki values of 8c and 8d retrieved from the
kinetic study
Km (in µM) at different
inhibitor concentrations (I)
Vmax
(µM/
min)
Nature of
Code 10 µM
5 µM
0 µM
88.22
74.02
Ki (µM) inhibition
8c
8d
150.92
168.08
122.77
134.63
1.880 3.26
1.650 2.70
Competitive
Competitive
Abbreviations: Ki, inhibition constant; Km, Michaelis–Menten constant;
max, velocity maximum.
V
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2.2
PL inhibition assay and kinetics
better activity against PL amongst other compounds of the series,
with an IC50 value of 4.53 µM, followed by 8c (IC50 = 5.12 µM). The
standard drug, orlistat (1), comparatively possessed a potent IC50
value of 0.99 µM (see Supporting Information Data).
Compounds 7a–f and 8a–f were subjected to PL inhibition assay as
per the protocol standardized in our laboratory.[30] Porcine pan-
creatic lipase (type II) was used with 4‐nitrophenyl butyrate as the
substrate, and the absorbance was recorded on a microplate reader
for subsequent determination of % inhibition and IC50 values. As
summarized in Table 1, the activity profiles of the compounds varied
between 4 and 38 µM. Most of the compounds exhibited a good
(<15 µM) to moderate (15–30 µM) activity, whereas compounds 7e
and 7f displayed a poor activity (>30 µM). Analog 8d exhibited a
Furthermore, enzyme kinetics was performed for 8c and 8d
to understand the nature of inhibition, which, in turn, would
provide adequate information of the ligand's binding site.[31,32] As
represented in Figure 2 and Table 2, the plots converged at the
y‐intercept, whereas the Km value increased in proportion to
the inhibitor concentration. These representations indicated that the
FIGURE 2 Double‐reciprocal
Lineweaver–Burk plots for 8c and 8d obtained
through the enzyme kinetic study

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compounds inhibited PL competitively against the substrate, similar
to orlistat (1) and carbazolyl oxoacetamides from the previous
study.[25] These results from the enzyme kinetics clearly highlighted
the fact that the ligands are bound to the active site of the PL.
the ligands exhibited a dual interaction with the amino acids of the
lid domain and the active site, similar to that of the carbazolyl
oxoacetamides (Figure 4).
An analysis of the docking poses of 8a–f highlighted an additional
π–π stacking interaction with Phe 77, which was not observed with 7a–f.
This fact clearly highlighted the prominent role of hydrophobic interac-
tions with the lid domain in potentiating PL inhibitory activity. Moreover,
compounds 8a–f exhibited a prominent π–cation interaction with Arg
256, which was not observed with 7a–f. Previous reports suggested that
the open‐lid conformation of human PL is attained through the formation
of the salt bridge between Arg 256–Asp 257 and Tyr 267–Lys 268.[13]
Furthermore, the role of π–cation interaction with Arg 256 in po-
tentiating the activity was established in our previous study.[24] In addi-
tion, previous reports suggested that the presence of donating groups on
the ring resulted in a stronger π–cation interaction as compared with the
withdrawing groups.[34] This fact is in correlation to the structure–activity
relationship, wherein the activating groups on the aryl ring of the amide
nitrogen resulted in a potential PL inhibitory activity (as observed with
7a–d and 8a–d).
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2.3
Structure–activity relationship
A preliminary structure–activity relationship of these compounds was
analyzed on the basis of their PL inhibitory activity. As shown in Figure 3,
the presence of N‐benzyl substitution resulted in a significant variation in
the PL inhibitory activities, with 8a–f exhibiting a potential inhibition,
compared to their unsubstituted counterparts (7a–f). This might be at-
tributed to the increased hydrophobicity of the indole, which, in turn,
resulted in stronger hydrophobic interactions with the lid domain. Simi-
larly, the presence of ring‐activating groups on the aryl ring of the amide
nitrogen resulted in better potency, with analog 8c (possessing tri-
methoxy substitution) exhibiting greater potency in the series. Moreover,
the presence of a methylene bridge between the aryl extension and the
amide nitrogen resulted in a potential activity. As represented in Table 1,
analog 8b with 4‐methoxyphenyl substitution on the amide nitrogen
possessed an IC50 value of 6.28 µM, whereas analog 8c with
4‐methoxybenzyl substitution exhibited an IC50 value of 5.12 µM.
Furthermore, the docked poses of compound 2 and compound
8d were superimposed on each other, and the interaction distance of
the reactive carbonyl group from Ser 152 was calculated. As re-
presented in Figure 5, the interaction distance was lesser for com-
pound 8d (3.41 Å), compared with compound 2 (4.45 Å), validating
the hypothesis of the present study, wherein the replacement of
carbazole (as observed in compound 2) with an indole nucleus (as in
8d) reduced the interaction distance and potentiated the PL
inhibitory activity (from 6.31 to 4.53 µM).
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2.4
Molecular modeling
Molecular docking of compounds 7a–f and 8a–f was performed as per
the validated grid parameters[24] using Molegro Virtual Docker 6.0 (CLC
Bio), and the crystal structure of human PL (PDB ID: 1LPB) was re-
trieved from the RCSB PDB data bank.[14,33] As summarized in Table 3,
the MolDock scores of compounds 7a–f and 8a–f were in agreement
with their PL inhibitory activity, with the most potent compound 8d
exhibiting a potential docking score of −163.052 kcal/mol. Moreover,
The molecular docking analysis provided a preliminary outline of
the various interactions required for a stable binding conformation of
the ligands. However, it is necessary to predict ligand behavior in
motion. Consequently, a 10‐ns molecular dynamics (MD) simulation
has been performed for 8c and 8d complexed with PL. The MD
simulation was performed on Gromacs 5.0.4 using CHARMM27 force
FIGURE 3 Structure–activity relationship
for indolyl oxoacetamides

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TABLE 3 The MolDock score (in kcal/mol) and interaction summary of indolyl oxoacetamides
#
MolDock score
−141.281
−141.105
−131.126
−140.953
−144.163
−123.261
−155.561
−160.657
−160.577
−163.052
−159.287
−136.758
−152.580
H‐bond
π–π stacking interactions
Phe 215
π–cation
7a
Phe 77, Ser 152, Leu 153, Phe 215
Phe 77, Ser 152, Leu 153, Phe 215
Phe 77, Ser 152, Phe 215
–
7b
7c
Tyr 114, Phe 215
–
Tyr 114, Phe 215
Asp 79
7d
7e
7f
Phe 77, Ser 152, Leu 153
Tyr 114, Phe 215
–
–
–
Phe 77, Ser 152, Leu 153, Phe 215
Phe 77, Ser 152, Leu 153, Phe 215
Gly 76, Phe 77, His 151, Ser 152
Gly 76, Phe 77, His 151, Ser 152
Gly 76, Phe 77, His 151, Ser 152, Leu 153
Gly 76, Phe 77, His 151, Ser 152
Gly 76, Phe 77, His 151, Ser 152, Phe 215
Gly 76, Phe 77, His 151, Ser 152
His 151, Ser 152
Tyr 114, Phe 215, His 263
Phe 215, His 263
8a
Phe 77, Phe 215, His 263
Phe 77, Phe 215, His 263
Phe 77, Tyr 114, Phe 215, His 263
Phe 77, Phe 215, His 263
Phe 77, His 263
Asp 79, Arg 256, His 263
Asp 79, Arg 256, His 263
Asp 79, Arg 256
Asp 79, Arg 256, His 263
Asp 79, His 263
8b
8c
8d
8e
8f
Phe 77, Phe 215, His 263
–
Asp 79, His 263
Orlistat
–
field as per the protocol standardized in our laboratory.[20,28]
As represented in Figure 6, 8c and 8d exhibited stable binding
conformations during the entire MD simulation, with a maximum
deviation of 4 and 2 Å, respectively, observed at ~7 ns.
Asp 79 and Arg 256 throughout the MD run. These interactions,
however, were not observed with compound 8c.
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In silico absorption, distribution, metabolism,
and excretion (ADME) prediction
Furthermore, both the ligands exhibited prominent hydrophobic
interactions with Phe 77 and Phe 215 of the lid domain, in addition to
Tyr 114, substantiating the role of aromatic wings for a potential PL
inhibitory activity (Tables 4 and 5). Nevertheless, the greater stability of
8d over 8c (as observed with their root mean square deviation) can be
attributed to the stable π–cation and H‐bond interactions exhibited by
8d. As summarized in Tables 4 and 5, 8d exhibited prominent H‐bond
interactions with Phe 77 and Ser 152, and π–cation interactions with
2.5
The ADME profiles of the synthesized analogs, as well as orlistat (1),
were predicted using SwissADME,[35] and the results are summarized
in Table 6.
Orlistat (1) possessed a low gastrointestinal (GI) absorption, which
is in accordance with the reported data.[36] In contrast, all the indolyl
FIGURE 4 (a) The two‐dimensional interaction diagram of 8d with pancreatic lipase (PL). (b) The three‐dimensional interaction diagram of 8d,
highlighting the lid domain interactions (pink), H‐bonds (green), and π–cation interactions (brown). (c) Representation of 8d in the binding pocket of PL

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FIGURE 5 The superimposition of compounds
2 (brown) and 8d (green), highlighting the
distance of the reactive carbonyl group from
Ser 152
oxoacetamide analogs in the present study possessed a high GI ab-
sorption. Moreover, these analogs were found to possess a broad CYP
inhibitory profile, compared with orlistat. Nevertheless, a further lead
optimization to enhance the lipophilicity of these indolyl oxoacetamides
might result in a better ADME profile, similar to that of orlistat.
256, respectively. The ADME prediction of these analogs indicated a
possible high GI absorption. In conclusion, the present study resulted
in novel indolyl oxoacetamide‐based leads (8a–d) with potent pan-
creatic
lipase inhibition.
A further lead optimization to enhance their
lipophilicity might result in an improved efficacy of these analogs.
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3
CONCLUSION
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4
EXPERIMENTAL
The present study reported the PL inhibitory activities of indolyl
oxoacetamides, wherein compounds 8a–d possessed a potential
activity (< 10 µM). The most potent compound of the series, 8d,
exhibited a competitive inhibition with an IC50 value of 4.53 µM. The
results from molecular modeling studies were in alignment with the
PL inhibitory activities of the compounds and highlighted the role of
π–π stacking and π–cation interactions with the lid domain and Arg
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4.1
Chemistry
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4.1.1
General
The syntheses of all the final compounds, 7a–f and 8a–f were carried
out as per the procedure detailed in Scheme 1.[26–29] The progress of
FIGURE 6 Ligand root mean square deviation of 8c and 8d retrieved from a 10‐ns molecular dynamics simulation

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TABLE 4 The interaction chart of 8c with pancreatic lipase at different time frames during molecular dynamics simulation
Time frame (ns)
H‐bond
π–π stacking
π–cation interactions
0
1
2
3
4
5
6
7
8
9
10
Phe 77, Ser 152
Phe 77, Tyr 114, Phe 215
Phe 77, Phe 215
Phe 77, Phe 215
Phe 77, Tyr 114
Phe 77, Tyr 114, Phe 215
Phe 77, Phe 215
Phe 215
Arg 256
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
Phe 77, Phe 215
Phe 77, His 263
Tyr 114, Phe 215
Phe 215
the reactions was monitored by thin‐layer chromatography analysis
(Silica gel G60 F254; Merck). Melting points were determined with
electrothermal capillary melting point apparatus (E–Z melting) and
vigorously for 15 min at room temperature. To this, 8.5 mmol of
5‐methoxyindole (4) was added and the stirring was continued. After
30 min, a solution of benzyl chloride (8.5 mmol) in 5 ml of DMF was
added dropwise to the reaction mixture for over 5 mim, and the
stirring was continued overnight. The reaction mixture was then
transferred to ice‐cold water and stirred for 5 min to obtain a white
precipitate, which was filtered and reprecipitated from ethanol to
yield N‐benzyl‐5‐methoxyindole (5).
were uncorrected. IR spectra were recorded on
a Shimadzu
IRPrestige 21 FTIR machine using KBr pellets. 1H‐ and 13C‐NMR
spectra were recorded on a Bruker Avance II 400 spectrometer (400
and 100 MHz, respectively) using dimethyl sulfoxide (DMSO)‐d6 as
solvent. The HRMS analysis was performed on a Bruker Compass
Data Analysis 4.1 mass instrument.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting Information.
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4.1.3
General procedure for the synthesis of
indolyl oxoacetamides
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4.1.2
General procedure for the synthesis of
To an ice‐cooled solution of 5‐methoxyindole (4) or its N‐benzyl
derivative (5) (2.5 mmol) in 5 ml diethyl ether, oxalyl chloride
(2.5 mmol) was added dropwise for over 5 min, and the reaction was
continued at 0°C for 15 min. The obtained yellow precipitate of
glyoxylyl chloride derivative (6) was filtered, washed with cold
N‐benzyl‐5‐methoxyindole (5)
To 10 ml of dimethylformamide (DMF) taken in a 100‐ml round‐
bottomed flask, 25.6 mmol of KOH was added, and it was stirred
TABLE 5 The interaction chart of 8d with pancreatic lipase at different time frames during molecular dynamics simulation
Time frame (ns)
H‐bond
π–π stacking
π–cation interactions
Asp 79, Arg 256
Asp 79, Arg 256
Asp 79, Arg 256
Asp 79, Arg 256
Asp 79, Arg 256
Asp 79, Arg 256
Asp 79
0
1
2
3
4
5
6
7
8
9
10
Phe 77, Ser 152
His 263
Phe 77, Ser 152
His 263, Phe 215
Tyr 114, Phe 215, His 263
Tyr 114, Phe 215, His 263
Tyr 114, Phe 215, His 263
Tyr 114, Phe 215, His 263
Tyr 114, His 263
Phe 77, Phe 215
Phe 77
Phe 77, Ser 152
Phe 77
Phe 77
Phe 77
Phe 77, Ser 152
–
–
–
–
Arg 256
–
Phe 77
Arg 256
Phe 77
–

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TABLE 6 ADME profiles of analogs 7a–f, 8a–f, and orlistat (1) predicted using SwissADME
CYP inhibitory profile
#
GI absorption
High
BBB permeant
Pgp substrate
CYP1A2
Yes
Yes
Yes
Yes
Yes
Yes
No
CYP2C19
Yes
Yes
Yes
No
CYP2C9
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
CYP2D6
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
CYP3A4
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
7a
Yes
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
7b
7c
High
High
No
7d
7e
7f
High
No
High
Yes
Yes
Yes
Yes
Yes
No
Yes
No
High
8a
High
Yes
Yes
Yes
Yes
Yes
Yes
No
8b
8c
High
No
High
No
8d
8e
8f
High
No
High
Yes
Yes
No
No
High
No
Orlistat
Low
No
Abbreviations: ADME, absorption, distribution, metabolism, and excretion; BBB, blood–brain barrier; GI, gastrointestinal.
ether, and transferred immediately to a solution of tetrahydrofuran
(THF) containing an appropriate amount of aniline derivative, fol-
lowed by the addition of triethylamine (7.5 mmol). The reaction was
continued at room temperature for 2 hr, and THF was removed in
vacuo to yield crude oxoacetamide. The crude precipitate was then
recrystallized in ethanol to obtain compounds 7a–f and 8a–f in
82–93% yields.
1,142, 1,018, 1,011, 818, 656, 463, and 424; HRMS (ESI+) calculated
for C18H17N2O4 [M+H+], 325.1183; found 325.1169.
2‐(5‐Methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(4‐methoxybenzyl)acetamide (7c)
Yield 94%; light brown solid; m.p: 201–203°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 12.15 (s, 1H), 9.20 (t, J = 6.3 Hz, 1H), 8.69 (s, 1H), 7.75
(d, J = 2.57 Hz, 1H), 7.44 (d, J = 8.8 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H),
6.94–6.86 (m, 3H), 4.35 (d, J = 6.3 Hz, 2H), 3.80 (s, 3H), and 3.73
(s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ: 182.44, 164.06, 158.72,
156.43, 138.87, 131.48, 131.46, 129.16, 127.60, 114.17, 113.78,
113.32, 112.53, 103.82, 55.72, 55.51, and 41.93; IR (KBr, v, cm−1):
3,767, 3,242, 2,926, 2,717, 2,362, 2,138, 1,992, 1,876, 1,814, 1,582,
1,443, 1,250, 1,135, 1,027, 818, and 486; HRMS (ESI+) calculated for
C19H19N2O4 [M+H+], 339.1339; found 339.1324.
2‐(5‐Methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3,4‐dimethylphenyl)‐
acetamide (7a)
Yield 93%; brownish yellow solid; m.p: 210–212°C; 1H‐NMR
(400 MHz, DMSO‐d6) δ: 12.24 (s, 1H), 10.49 (s, 1H), 8.70 (s, 1H),
7.81 (d, J = 2.5 Hz, 1H), 7.67 (d, J = 2.1 Hz, 1H), 7.55 (d, J = 2.2 Hz, 1H),
7.46 (d, J = 8.8 Hz, 1H), 7.12 (d, J = 8.2 Hz, 1H), 6.93 (dd, J = 8.8,
2.6 Hz, 1H), 3.82 (s, 3H), 2.23 (s, 3H), and 2.20 (s, 3H); 13C‐NMR
(100 MHz, DMSO‐d6) δ: 182.36, 162.61, 156.52, 138.94, 136.83,
136.23, 132.51, 131.56, 130.05, 127.63, 121.72, 118.14, 113.88,
113.42, 112.32, 103.78, 55.71, 20.15, and 19.35; IR (KBr, v, cm−1):
3,935, 3,719, 3,672, 2,970, 2,353, 2,129, 2,006, 1,813, 1,582, 1,435,
1,342, 1,111, 1,057, 995, 679, 501, and 455; HRMS (ESI+) calculated
for C19H19N2O3 [M+H+], 323.1390; found 323.1375.
2‐(5‐Methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3,4,5‐trimethoxyphenyl)‐
acetamide (7d)
Yield 86%; brownish yellow solid; m.p: 207–209°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 12.27 (d, J = 2.9 Hz, 1H), 10.56 (s, 1H), 8.76 (d, J = 3.0 Hz,
1H), 7.81 (s, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.34 (s, 2H), 6.93 (d, J = 8.8 Hz,
1H), 3.82 (s, 3H), 3.78 (s, 6H), and 3.36 (s, 3H); 13C‐NMR (100 MHz,
DMSO‐d6) δ: 181.86, 162.42, 156.56, 153.17, 139.11, 134.62, 134.55,
131.54, 127.70, 113.90, 113.41, 112.21, 103.85, 98.42, 60.59, 56.23,
and 55.71; IR (KBr, v, cm−1): 3,935, 3,696, 2,955, 2,361, 2,021, 1,579,
1,443, 1,126, 1,049, 995, 872, 679, 656, and 455; HRMS (ESI+) calcu-
lated for C20H21N2O6 [M+H+], 385.1394; found 385.1385.
2‐(5‐Methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(4‐methoxyphenyl)‐
acetamide (7b)
Yield 82%; greenish yellow solid; m.p: 242–244°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 12.24 (s, 1H), 10.56 (s, 1H), 8.73 (d, J = 2.8 Hz, 1H),
7.92–7.71 (m, 3H), 7.47 (d, J = 8.8 Hz, 1H), 7.02–6.88 (m, 3H), 3.82
(s, 3H), and 3.75 (s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ: 182.37,
162.35, 156.52, 156.33, 138.96, 131.65, 131.55, 127.67, 122.09,
114.33, 113.87, 113.39, 112.36, 103.81, 55.70, and 55.65; IR (KBr, v,
cm−1): 3,750, 3,418, 2,978, 2,369, 1,805, 1,651, 1,558, 1,420, 1,296,
2‐(5‐Methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3,4‐dichlorophenyl)‐
acetamide (7e)
Yield 87%; yellow solid; m.p: 272–274°C; 1H‐NMR (400 MHz, DMSO‐d6)
δ: 12.32 (s, 1H), 11.01 (s, 1H), 8.73 (s, 1H), 8.26 (s, 1H), 7.88–7.78 (m, 2H),

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7.63 (d, J = 8.8 Hz, 1H), 7.47 (d, J = 8.8 Hz, 1H), 6.93 (dd, J = 8.8, 2.6 Hz,
1H), and 3.82 (s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ: 181.25, 162.98,
156.62, 139.19, 138.70, 131.59, 131.48, 131.13, 127.65, 126.25, 121.87,
120.73, 113.94, 113.45, 112.15, 103.85, and 55.70; IR (KBr, v, cm−1):
3,742, 3,341, 2,832, 2,361, 1,697, 1,566, 1,504, 1,404, 1,265, 1,250,
1,188, 1,119, 1,034, 818, and 679; HRMS (ESI+) calculated for
C17H13Cl2N2O3 [M+H+], 363.0298; found 363.0287.
(100 MHz, DMSO‐d6) δ: 182.06, 163.83, 158.74, 156.80, 141.58,
137.17, 131.54, 131.42, 129.21, 128.42, 128.27, 127.81, 114.17,
113.32, 112.88, 111.73, 104.22, 55.78, 55.50, 50.42, and 41.99; IR
(KBr, v, cm−1): 3,750, 3,132, 2,824, 2,361, 1,690, 1,574, 1,504, 1,396,
1,242, 1,165, 1,042, 856, 802, 648, and 517; HRMS (ESI+) calculated
for C26H25N2O4 [M+H+], 429.1809; found 429.1799.
2‐(1‐Benzyl‐5‐methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3,4,5‐trimethoxyphen-
yl)acetamide (8d)
2‐(5‐Methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3‐fluorophenyl)‐
acetamide (7f)
Yield 83%; light yellow solid; m.p: 160–162°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 10.59 (s, 1H), 9.04 (s, 1H), 7.84 (d, J = 2.6 Hz, 1H), 7.51
(d, J = 8.9 Hz, 1H), 7.39–7.25 (m, 7H), 6.93 (dd, J = 8.9, 2.6 Hz, 1H), 5.60
(s, 2H), 3.81 (s, 3H), 3.78 (s, 6H), and 3.66 (s, 3H); 13C‐NMR (100 MHz,
DMSO‐d6) δ: 181.48, 162.16, 156.92, 153.17, 141.80, 137.22, 134.60,
134.58, 131.57, 129.20, 128.53, 128.26, 127.75, 113.40, 113.04,
111.41, 104.26, 98.37, 60.60, 56.22, 55.78, and 50.46; IR (KBr, v,
cm−1): 3,873, 2,986, 2,832, 2,369, 2,106, 1,929, 1,798, 1,643, 1,543,
1,504, 1,342, 1,111, 1,057, 679, 586, 501, and 463; HRMS (ESI+)
calculated for C27H27N2O6 [M+H+], 475.1864; found 475.1858.
Yield 82%; greenish yellow solid; m.p: 242–245°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 10.76 (s, 1H), 8.73 (s, 1H), 7.96–7.86 (m, 2H), 7.83
(d, J = 2.5 Hz, 1H), 7.49 (d, J = 8.8 Hz, 1H), 7.23 (m, 2H), 6.93 (dd, J = 8.8,
2.5 Hz, 1H), and 3.82 (s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ: 181.88,
162.77, 160.23, 157.83, 156.52, 139.41, 135.01, 131.95, 127.81,
122.51, 122.43, 115.93, 115.71, 114.07, 113.30, 112.29, 103.84, and
55.68; IR (KBr, v, cm−1): 3,719, 3,163, 2,832, 2,353, 1,666, 1,574, 1,412,
1,265, 1,227, 1,142, 1,026, 926, 810, 664, 486, and 424; HRMS (ESI+)
calculated for C17H14FN2O3 [M+H+], 313.0983; found 313.0970.
2‐(1‐Benzyl‐5‐methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3,4‐
2‐(1‐Benzyl‐5‐methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(3,4‐dichlorophen-
yl)acetamide (8e)
dimethylphenyl)acetamide (8a)
Yield 83%; off‐white solid; m.p: 168–170°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 10.52 (s, 1H), 8.96 (s, 1H), 7.83 (d, J = 2.5 Hz, 1H), 7.70
(s, 1H), 7.60–7.46 (m, 2H), 7.41–7.24 (m, 5H), 7.12 (d, J = 8.2 Hz, 1H),
6.93 (dd, J = 9.0, 2.6 Hz, 1H), 5.58 (s, 2H), 3.81 (s, 3H), 2.23 (s, 3H),
and 2.20 (s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ: 182.05, 162.44,
156.88, 141.64, 137.16, 136.84, 136.21, 132.56, 131.60, 130.06,
129.21, 128.43, 128.29, 127.83, 121.70, 118.14, 113.41, 113.01,
111.52, 104.16, 55.78, 50.49, 20.15, and 19.36; IR (KBr, v, cm−1):
3,927, 3,750, 2,932, 2,777, 2,361, 2,006, 1,913, 1,852, 1,713, 1,582,
1,474, 1,435, 1,342, 1,265, 1,165, 1,111, and 1,057; HRMS (ESI+)
calculated for C26H25N2O3 [M+H+], 413.1862; found 413.1849.
Yield 87%, creamy yellow solid; m.p: 191–193°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 11.05 (s, 1H), 8.98 (s, 1H), 8.28 (d, J = 2.4 Hz, 1H), 7.84
(dd, J = 8.7, 2.5 Hz, 2H), 7.65 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 9.0 Hz,
1H), 7.40–7.24 (m, 5H), 6.94 (dd, J = 8.9, 2.6 Hz, 2H), 5.58 (s, 2H), and
3.81 (s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ: 180.98, 162.86,
156.98, 141.86, 138.68, 137.07, 131.61, 131.48, 131.18, 129.21,
128.43, 128.32, 127.86, 126.29, 121.89, 120.78, 113.46, 113.10,
111.33, 104.22, 55.78, and 50.55; IR (KBr, v, cm−1): 3,757, 2,978,
2,770, 2,361, 1,875, 1,713, 1,582, 1,497, 1,342, 1,265, 1,165, 1,057,
864, 679, and 463; HRMS (ESI+) calculated for C24H19Cl2N2O3
[M+H+], 453.0767; found 453.0755.
2‐(1‐Benzyl‐5‐methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(4‐methoxyphenyl)‐
acetamide (8b)
2‐(1‐Benzyl‐5‐methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(4‐fluorophenyl)‐
acetamide (8f)
Yield 89%; yellowish‐white solid; m.p: 152–154°C; 1H‐NMR
(400 MHz, DMSO‐d6) δ: 10.58 (s, 1H), 8.95 (s, 1H), 7.86–7.75
(m, 3H), 7.52 (d, J = 8.9 Hz, 1H), 7.40–7.24 (m, 5H), 7.02–6.89
(m, 3H), 5.58 (s, 1H), 3.81 (s, 3H), and 3.76 (s, 3H); 13C‐NMR
(100 MHz, DMSO‐d6) δ: 182.09, 162.20, 156.88, 156.36, 141.61,
137.15, 131.62, 131.60, 129.21, 128.45, 128.29, 127.83, 122.08,
114.34, 113.39, 113.00, 111.56, 104.20, 55.78, and 50.49; IR (KBr, v,
cm−1): 3,922, 3,775, 3,443, 2,972, 2,500, 2,362, 2,130, 1,822, 1,582,
1,428, 1,119, 1,057, 996, 834, 641, and 509; HRMS (ESI+) calculated
for C25H23N2O4 [M+H+], 415.1652; found 415.1641.
Yield 86%; white solid; m.p: 197–199°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 10.82 (s, 1H), 8.96 (s, 1H), 7.97–7.82 (m, 3H), 7.52
(d, J = 8.9 Hz, 1H), 7.40–7.18 (m, 7H), 6.94 (dd, J = 8.9, 2.6 Hz, 1H),
5.57 (s, 2H), and 3.81 (s, 3H); 13C‐NMR (100 MHz, DMSO‐d6) δ:
181.77, 162.55, 160.26, 157.87, 156.92, 141.66, 137.09, 134.95,
131.61, 129.21, 128.44, 128.31, 127.84, 122.54, 122.46, 115.96,
115.74, 113.43, 113.03, 111.49, 104.19, 55.76, and 50.52; IR (KBr, v,
cm−1): 3,742, 3,140, 2,361, 1,875, 1,674, 1,636, 1,504, 1,389, 1,265,
1,211, 1,057, 833, 664, 501, and 424; HRMS (ESI+) calculated for
C24H20FN2O3 [M+H+], 403.1452; found 403.1439.
2‐(1‐Benzyl‐5‐methoxy‐1H‐indol‐3‐yl)‐2‐oxo‐N‐(4‐methoxybenzyl)‐
acetamide (8c)
|
4.2
Pancreatic lipase inhibition assay and enzyme
Yield 91%; light brown solid; m.p:160–162°C; 1H‐NMR (400 MHz,
DMSO‐d6) δ: 9.26 (t, J = 6.4 Hz, 1H), 8.95 (s, 1H), 7.79 (s, 1H), 7.49 (d,
J = 8.9 Hz, 1H), 7.39–7.24 (m, 7H), 6.97–6.86 (m, 3H), 5.55 (s, 2H),
4.36 (d, J = 6.3 Hz, 2H), 3.79 (s, 3H), and 3.73 (s, 3H); 13C‐NMR
kinetics
Orlistat (1), porcine PL (type II), and 4‐nitrophenyl butyrate were
procured from Sigma‐Aldrich (MO). Tris buffer and sodium chloride

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(molecular biology grade) were procured from Sisco Research
Laboratories (MH, India). All other chemicals and solvents (analytical
grade) were used without further purification.
algorithm was applied for the calculation of the bond length.[44]
Discovery Studio 4.5 visualizer (Accelrys) was used to depict the gra-
phical representations of the complex.
The procedure for the PL inhibition assay and kinetics was
performed as per the protocol standardized in our laboratory.[25,30]
Briefly, a suspension of porcine PL (5 mg/ml) was subjected to vig-
orous shaking, followed by centrifugation (4,000 rpm, 10 min), and
the supernatant was used afresh as the enzyme solution. Stock so-
lutions of the synthesized compounds and orlistat were prepared in
DMSO at linear concentrations ranging from 0.78 to 2,000 μg/ml.
The final reaction mixture consisted of 875 μl of buffer, 100 μl of
enzyme, and 20 μl of the compounds of various stock concentrations;
it was preincubated for 5 min at 37°C, followed by the addition of 5 μl
of the substrate (4‐nitrophenyl butyrate, 10 mM in acetonitrile). The
absorbance of the final mixture was taken on a BioTek EPOCH mi-
croplate spectrophotometer (VT) after 5 min at absorbance maxima
of 4‐nitrophenol (405 nm). The assay was performed in triplicate and
the percentage inhibition was calculated using the following formula:
|
4.4
The in silico ADME prediction
The in silico ADME prediction of the synthesized analogs, 7a–f and
8a–f, alongside orlistat (1) was assessed using the online tool
SwissADME.[35] Various parameters, including GI absorption,
blood–brain barrier permeability, and effect on CYP enzymes, were
considered for the ADME prediction.
ACKNOWLEDGMENTS
The authors acknowledge Birla Institute of Technology and
Science, Pilani (BITS Pilani), Pilani Campus, Pilani, Rajasthan, India,
for providing NMR and other necessary laboratory facilities. The
authors also acknowledge Department of Science and Technology
(DST), New Delhi, India, for sponsoring HRMS facility at BITS Pilani
under FIST program. Dr. S N C Sridhar acknowledges the Council
for Scientific and Industrial Research (CSIR), New Delhi, India, for
providing Senior Research Fellowship (File No: 09/719(0088)/
2018‐EMR‐I).
% Inhibition = [1 − (AT/AE)] × 100,
where AE is the absorbance of the enzyme control (without inhibitor)
and AT is the difference between the absorbance of the test sample,
with and without substrate. The IC50 value of the compounds was
calculated by plotting a linear regression curve.
CONFLICT OF INTERESTS
For the inhibition kinetics, the assay protocol was repeated with
varying concentrations (0, 5, and 10 μM) of 8c and 8d, and substrate
(25, 50, 100, and 200 µM), and a double‐reciprocal Lineweaver–Burk
plot was plotted to understand the nature of inhibition.[37] The inhibi-
tion constant, Ki, was calculated using the Cheng–Prusoff equation.[38]
The authors declare that there are no conflicts of interests.
ORCID
Atish T. Paul
http://orcid.org/0000-0002-8143-5721
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4.3
Molecular modeling studies
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SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
How to cite this article: Sridhar SNC, Palawat S, Paul AT.
Design, synthesis, evaluation, and molecular modeling studies
of indolyl oxoacetamides as potential pancreatic lipase
inhibitors. Arch Pharm. 2020;e2000048.
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J. Med. Chem. 2017, 136, 184.
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