Design, synthesis, biological evaluation and molecular modelling studies of indole glyoxylamides as a new class of potential pancreatic lipase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.01.012

A series of eighteen indole glyoxylamide analogues were synthesized, characterized and evaluated for their pancreatic lipase inhibitory activity. Porcine pancreatic lipase (Type II) was used with 4-nitrophenyl butyrate (as substrate) for the in vitro assay. Compound 8f exhibited competitive inhibition against pancreatic lipase with IC₅₀ value of 4.92 μM, comparable to that of the standard drug, orlistat (IC₅₀ = 0.99 μM). Compounds 7a-i and 8a-i were subjected to molecular docking into the active site of human PL (PDB ID: 1LPB) wherein compound 8f possessed a potential MolDock score of ?153.037 kcal/mol. Molecular dynamics simulation of 8f complexed with pancreatic lipase, confirmed the role of aromatic substitution in stabilizing the ligand through hydrophobic interactions (maximum observed RMSD = 3.5 ?).

Full text of DOI:10.1016/j.bioorg.2019.01.012

Accepted Manuscript
Design, Synthesis, Biological Evaluation and Molecular Modelling Studies of
Indole Glyoxylamides as a New Class of Potential Pancreatic Lipase Inhibitors
S.N.C. Sridhar, Saksham Palawat, Atish T. Paul
PII:
S0045-2068(18)31293-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.012
YBIOO 2716
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 November 2018
4 January 2019
6 January 2019
Please cite this article as: S.N.C. Sridhar, S. Palawat, A.T. Paul, Design, Synthesis, Biological Evaluation and
Molecular Modelling Studies of Indole Glyoxylamides as a New Class of Potential Pancreatic Lipase Inhibitors,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.012
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Design, Synthesis, Biological Evaluation and Molecular Modelling Studies of Indole
Glyoxylamides as a New Class of Potential Pancreatic Lipase Inhibitors
Sridhar S. N. C., Saksham Palawat, Atish T. Paul1,atish.paul@pilani.bits-pilani.ac.in
Laboratory of Natural Product Chemistry, Department of Pharmacy, Birla Institute of
Technology and Science, Pilani (BITS Pilani), Pilani campus, Pilani - 333 031, Rajasthan, India
1Present Address: Department of Pharmacy, Birla Institute of Technology and Science, Pilani
(Pilani Campus), Pilani - 333 031, Rajasthan, India.
Graphical abstract
Highlights
Eighteen indole glyoxylamides analogues were evaluated for PL inhibitory activity
Compounds 8f exhibited potential competitive PL inhibitory activity (IC50 = 4.92 µM)
Hydrophobic interactions with the lid domain played prominent role in PL inhibition
Abstract
A series of eighteen indole glyoxylamide analogues were synthesized, characterized and
evaluated for their pancreatic lipase inhibitory activity. Porcine pancreatic lipase (Type II) was
used with 4-nitrophenyl butyrate (as substrate) for the in vitro assay. Compound 8f exhibited
competitive inhibition against pancreatic lipase with IC50 value of 4.92 µM, comparable to that
of the standard drug, orlistat (IC50 = 0.99 µM). Compounds 7a-i and 8a-i were subjected to
molecular docking into the active site of human PL (PDB ID: 1LPB) wherein compound 8f
possessed a potential MolDock score of -153.037 kcal/mol. Molecular dynamics simulation of
8f complexed with pancreatic lipase, confirmed the role of aromatic substitution in stabilizing
the ligand through hydrophobic interactions (maximum observed RMSD = 3.5 Å).
Keywords: Indole glyoxylamides, Inhibition Kinetics, Molecular Dynamics, Orlistat,
Pancreatic Lipase.
1. Introduction
1

Obesity is a multifactorial metabolic disorder, defined by an excessive accumulation of
lipids that presents risk to human health [1,2]. Recent decades have observed drastic changes
in the global food system that has transformed obesity into a global epidemic. As per recent
WHO statistics, 650 million adults were found obese, accounting to 13% of the global
population [3]. Further, obesity is associated with various comorbid conditions including
insulin resistance, diabetes mellitus, cardiovascular diseases and certain cancers, that pose
major health problem to obese patients [4]. With an estimated 2.8 million deaths per year,
obesity contributes to fifth leading risk of global deaths [5].
Amongst the several targets that have been explored to treat or prevent obesity [6–13],
pancreatic lipase (PL) is considered as a successful target due to its tolerable side effects.
Human PL (EC 3.1.1.3), encoded by the PNLIP gene, is a primary digestive enzyme secreted
from the pancreatic exocrine [14,15], and is primarily involved in the hydrolysis of
triglycerides [16]. The crystal structure of 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 [17]. This catalytic site is highly restricted by a hydrophobic lid domain that consists
of amino acids Gly 76 - Lys 80, Leu 213 – Met 217 [18]. The hydrolysis of triglyceride esters
by PL is represented by a series of events, initiated through an interfacial activation of the
enzyme by the hydrophobic alkyl chains of triglycerides. This results in the open lid
conformation of PL, and is followed by a nucleophilic attack of Ser 152 on to the carbonyl
carbon of the ester linkage of the triglycerides [19,20].
Orlistat (1), a potent PL inhibitor, is one among the other clinically approved drugs,
widely used for long term treatment of obesity [21]. It is a saturated derivative of lipstatin [22]
and contains a β-lactone (Fig. 1) that inhibits PL covalently, binding to Ser 152 of the active
site [23]. While, orlistat (1) was reported to exhibit tolerable side effects viz., steatorrhea, oily
stools and frequent bowel movements [24], recent reports from FDA has cited severe adverse
effects with long term administration of orlistat, that included hepatotoxicity, acute pancreatitis,
gall stones and renal injuries [25]. These events highlighted necessity for safer and effective
anti-obesity drugs.
Previously, we have synthesized and evaluated twenty-four carbazolyl oxoacetamides
for PL inhibitory activity [26]. These carbazolyl oxoacetamides can be considered a
pharmacophore hybrid of the carbazole nucleus and α-ketoamide functionality, that interact
with lid domain and the active site, respectively. Compounds 2 and 3 from this series exhibited
a potential PL inhibition (IC50 values of 6.31 and 8.72 µM). Further, the possible role of α-
ketoamide to interact covalently with Ser 152 of the active site was also identified (Fig. 1).
2

Nevertheless, an in silico analysis of these molecules highlighted a steric hinderance exhibited
by the carbazole nucleus, that resulted in greater interaction distance between the reactive
carbonyl group of the ketoamide and Ser 152 of the active site.
Based on the above findings, we hypothesized that the replacement of carbazole with
an indole nucleus would reduce this steric hinderance and the interaction distance,
consequently potentiating the PL inhibitory activity. Accordingly, the present study involved
design, synthesis, in vitro evaluation and in silico studies of indole glyoxylamide analogues as
a new class of potential PL inhibitors.
2. Materials and Methods
2.1. Chemistry
The synthesis of all the final compounds 7a-i and 8a-i (Table 1) were carried out as per
the procedure detailed in Scheme 1 [27–29]. Progress of the reactions was followed by Thin
Layer Chromatography (TLC) analysis (Silica gel G60 F254, Merck). Melting points were
determined with electro thermal capillary melting point apparatus (E-Z melting) and are
uncorrected. IR spectra were recorded on Shimadzu IRPrestige 21 FTIR machine using KBr
pellets. 1H and 13C NMR spectra were recorded on a Bruker Avance II 400 spectrometer (400
MHz and 100 MHz, respectively) using DMSO-d6 as solvent. HRMS analysis was performed
on Bruker Compass Data Analysis 4.1 mass instrument.
2.1.1. General procedure for the synthesis of N-benzylindole (5)
To 10 mL DMF taken in a 100 mL round bottomed flask, 25.6 mmol of KOH was added and
stirred vigorously for 15 minutes at room temperature. To this, 8.5 mmol of indole was added
and stirring was continued. After 30 minutes, a solution of benzyl chloride (8.5 mmol) in 5 mL
DMF was added dropwise for over 5 minutes to the reaction mixture, and stirring was
continued overnight. The reaction mixture was then transferred to ice-cold water and stirred
for 5 minutes to obtain a white precipitate, that was filtered and re-precipitated from ethanol to
yield N-benzylindole (5).
2.1.2. General procedure for the synthesis of indolyl oxoacetamides
3

To an ice-cooled solution of indole (4) or its N-benzyl derivative (5) (2.5 mmol) in 5 mL
diethylether, oxalyl chlroide (2.5 mmol) was added dropwise for a period of over 5 min, and
the reaction was continued at 0 °C for 15 minutes. The obtained yellow precipitate of glyoxylyl
chloride derivative (6) was filtered, washed with cold ether, and transferred immediately to a
solution of THF containing appropriate amount of aniline derivative, followed by addition of
triethylamine (7.5 mmol). The reaction was continued at room temperature for 2 hours, and
THF was removed in vacuo to yield crude glyoxylamide. The crude precipitate was then re-
crystallized to obtain compounds 7a-i and 8a-i in 82-93% yields.
2.1.2.1. 2-(1H-indol-3-yl)-2-oxo-N-phenylacetamide (7a)
Yield 89%; Creamy white solid; m.p: 248-250 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.37 (s,
1H), 10.70 (s, 1H), 8.78 (s, 1H), 8.34-8.27 (m, 1H), 7.88 (d, J = 7.4 Hz, 2H), 7.61-7.54 (m,
1H), 7.39 (t, J = 8.0 Hz, 2H), 7.30 (dd, J = 6.1, 3.2 Hz, 2H), 7.15 (t, J = 7.4 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 182.41, 162.85, 139.17, 138.54, 136.96, 129.20, 126.68, 124.69,
124.02, 123.17, 121.69, 120.71, 113.18, 112.43; IR (KBr, v, cm-1): 3742, 3294, 2569, 2361,
1913, 1674, 1612, 1504, 1443, 1319, 1242, 1072, 996, 957, 864, 810, 687, 501, 455; HRMS
(ESI+) calculated for C16H13N2O2 [M+H+], 265.0899; Found 265.0788.
2.1.2.2. 2-(1H-indol-3-yl)-2-oxo-N-(p-tolyl)acetamide (7b)
Yield 81%; Light brown solid; m.p: 225-227 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.34 (s,
1H), 10.58 (s, 1H), 8.78 (s, 1H), 8.32-8.24 (m, 1H), 7.79 (d, J = 7.4 Hz, 2H), 7.60-7.52 (m,
1H), 7.34-7.25 (m, 2H), 6.96 (d, J = 7.4 Hz, 2H), 3.36 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 182.59, 162.31, 156.34, 139.06, 136.82, 131.63, 126.67, 124.00, 123.14, 122.15, 121.69,
114.32, 113.12, 112.47, 55.67; IR (KBr, v, cm-1): 3757, 3402, 2963, 2353, 1883, 1821, 1574,
1435, 1242, 1126, 1011, 818, 640, 471, 417 ; HRMS (ESI+) calculated for C17H15N2O2 [M+H+],
279.1128; Found 279.1114.
2.1.2.3. 2-(1H-indol-3-yl)-2-oxo-N-(3,4-dimethylphenyl)acetamide (7c)
Yield 81%; Greenish yellow solid; m.p: 230-232 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.34
(s, 1H), 10.50 (s, 1H), 8.78 (s, 1H), 8.32-8.25 (m, 1H), 7.68 (s, 1H), 7.56 (d, J = 6.5 Hz, 2H),
7.29 (dd, J = 6.0, 3.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 1H), 2.23 (s, 3H), 2.20 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 182.59, 162.31, 156.34, 139.06, 135.78, 131.63, 126.67, 124.68,
123.34, 122.15, 122.78, 114.28, 113.21, 112.68, 55.67, 53.68; IR (KBr, v, cm-1) 3935, 3341,
4

2978, 2361, 1913, 1682, 1497, 1443, 1327, 1242, 1111, 995, 957, 826, 725, 694, 656, 501;
HRMS (ESI+) calculated for C18H17N2O2 [M+H+], 293.1285; Found 293.1278.
2.1.2.4. 2-(1H-indol-3-yl)-2-oxo-N-(4-methoxyphenyl)acetamide (7d)
Yield 81%; Greenish yellow solid; m.p: 231-233 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.59
(s, 1H), 8.77 (s, 1H), 8.36-8.22 (m, 1H), 7.76 (d, J = 8.4 Hz, 2H), 7.61-7.53 (m, 1H), 7.28 (dd,
J = 6.1, 3.1 Hz, 2H), 7.18 (d, J = 8.2 Hz, 2H), 2.29 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
182.14, 162.78, 139.86, 137.64, 136.07, 133.65, 129.59, 126.96, 123.81, 122.98, 121.63,
120.83, 120.60, 113.44, 112.45, 21.01; IR (KBr, v, cm-1): 3765, 2361, 1875, 1420, 1342, 1250,
1119, 1034, 818, 725, 679, 501, 424; HRMS (ESI+) calculated for C17H15N2O3 [M+H+],
295.1077; Found 295.1064.
2.1.2.5. 2-(1H-indol-3-yl)-2-oxo-N-(4-methoxybenzyl)acetamide (7e)
1
Yield 91%; White solid; m.p: 158-160 °C; H NMR (400 MHz, DMSO-d6) δ 12.31 (s, 1H),
9.24 (t, J = 6.3 Hz, 1H), 8.77 (s, 1H) , 8.34-8.07 (m, 1H), 7.69-7.45 (m, 2H), 7.45-7.13 (m,
4H), 6.90 (d, J = 3.2 H), 4.36 (d, J = 6.1 Hz, 2H), 3.73 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ 182.64, 164.44, 163.97, 158.72, 138.93, 138.90, 137.17, 136.73, 131.45, 129.27, 129.22,
126.64, 125.94, 124.32, 123.91, 123.33, 123.02, 121.74, 121.62, 114.16, 113.21, 113.03,
112.88, 112.66, 55.50, 53.00, 41.96; IR (KBr, v, cm-1): 3757, 3271, 2577, 2369, 1736, 1612,
1497, 1412, 1319, 1242, 1134, 1034, 833, 733, 664, 517, 424; HRMS (ESI+) calculated for
C18H17N2O3 [M+H+], 309.1234; Found 309.1225.
2.1.2.6. 2-(1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide (7f)
Yield 81%; Light brown solid; m.p: 201-203 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.38 (s,
1H), 10.58 (s,1H), 8.84 (s,1H), 8.29 (dd, J = 6.1, 3.1 Hz, 1H), 7.57 (dd, J = 6.1, 3.1 Hz, 1H),
7.35 (s, 2H), 7.30 (dd, J = 6.1, 3.2 Hz, 2H), 3.78 (s, 6H), 3.65 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 182.05, 162.34, 153.16, 139.22, 136.81, 134.60, 134.56, 126.70, 124.06, 123.21,
121.71, 113.14, 112.33, 98.47, 60.59, 56.23; IR (KBr, v, cm-1): 3402, 2832, 2353, 2099, 1867,
1574, 1412, 1342, 1227, 1126, 1103, 818, 671, 463; HRMS (ESI+) calculated for C19H19N2O5
[M+H+], 355.1288; Found 355.1278.
2.1.2.7. 2-(1H-indol-3-yl)-2-oxo-N-(4-bromophenyl)acetamide (7g)
Yield 84%; Yellowish white solid; m.p: 282-285 °C; 1H NMR (400 MHz, DMSO-d6) δ 12.38
(s, 1H), 10.86 (s, 1H), 8.77 (s, 1H), 8.34-8.25 (m, 1H), 7.86 (d, J = 8.9 Hz, 2H), 7.62-7.52 (m,
13
3H), 7.35-7.26 (m, 2H); C NMR (100 MHz, DMSO-d6) δ 182.05, 162.86, 139.16, 137.95,
136.86, 132.05, 126.63, 124.09, 123.24, 122.66, 121.69, 116.54, 113.17, 112.35; IR (KBr, v,
5

cm-1): 3750, 3310, 2978, 2369, 1890, 1659, 1582, 1443, 1242, 1142, 1011, 872, 818, 741, 656,
501; HRMS (ESI+) calculated for C16H12BrN2O2 [M+H+], 343.0004; Found 342.0093.
2.1.2.8. 2-(1H-indol-3-yl)-2-oxo-N-(3,4-chlorophenyl)acetamide (7h)
1
Yield 94%; Pale yellow solid; m.p: 282-284 °C; H NMR (400 MHz, DMSO-d6) δ 12.41 (s,
1H), 11.03 (s, 1H), 8.80 (s, 1H), 8.34-8.23 (m, 2H), 7.85 (dd, J = 8.8, 2.5 Hz, 1H), 7.65 (d, J =
8.9 Hz, 1H), 7.61-7.52 (m, 1H), 7.35-7.26 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 181.50,
162.94, 139.33, 138.70, 136.86, 131.47, 131.15, 126.63, 126.27, 124.14, 123.30, 121.95,
121.68, 120.82, 113.19, 112.27; IR (KBr, v, cm-1): 3927, 3372, 2978, 2353, 1883, 1690, 1574,
1497, 1435, 1227, 1126, 1018, 926, 802, 741, 640; HRMS (ESI+) calculated for C16H11Cl2N2O2
[M+H+], 333.0192; Found 333.0177.
2.1.2.9. 2-(1H-indol-3-yl)-2-oxo-N-(4-fluorophenyl)acetamide (7i)
1
Yield 84%; Pale yellow solid; m.p: 268-270 °C; H NMR (400 MHz, DMSO-d6) δ 12.40 (s,
1H), 10.81 (s, 1H), 8.81 (s, 1H), 8.45-8.18 (m, 1H), 7.93 (dd, J = 8.7, 4.8 Hz, 2H), 7.66-7.50
(m, 1H), 7.42-7.06 (m, 5H); 13C NMR (100 MHz, DMSO-d6) δ 182.26, 162.64, 160.27, 157.87,
139.14, 136.86, 134.97, 134.94, 126.68, 124.05, 123.21, 122.62, 122.54, 121.71, 115.92,
115.70, 113.15, 112.44; IR (KBr, v, cm-1): 3726, 3140, 2554, 2369, 1883, 1751, 1674, 1574,
1497, 1412, 1242, 1142, 1011, 818, 656, 486; HRMS (ESI+) calculated for C16H12FN2O2
[M+H+], 283.0877; Found 283.0862.
2.1.2.10. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-phenylacetamide (8a)
Yield 86%; Creamy white solid; m.p: 142-143 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.70 (s,
1H), 9.01 (s, 1H), 8.44-8.22 (m, 1H), 7.99-7.80 (m, 2H), 7.69-7.57 (m, 1H), 7.47-7.26 (m, 8H),
7.16 (t, J = 7.4 Hz, 1H), 5.63 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 182.20, 162.68, 141.77,
138.49, 137.12, 136.82, 129.23, 129.21, 128.30, 127.88, 127.37, 124.75, 124.21, 123.65,
121.98, 120.72, 112.16, 111.77, 50.34; IR (KBr, v, cm-1): 3935, 3572, 2353, 2122, 2021, 1821,
1589, 1504, 1435, 1381, 1342, 1242, 1150, 1049, 872, 679, 494, 424; HRMS (ESI+) calculated
for C23H19N2O2 [M+H+], 355.1441; Found 355.1428.
2.1.2.11. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(p-tolyl)acetamide (8b)
Yield 88%; Off-white solid;m.p: 160-162 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.64 (s, 1H),
9.02 (s, 1H), 8.37-8.28 (m, 1H), 7.77 (d, J = 8.5 Hz, 2H), 7.67-7.58 (m, 1H), 7.40-7.24 (m,
13
8H), 7.19 (d, J = 8.2 Hz, 2H), 5.62 (s, 2H), 2.29 (s, 3H); C NMR (100 MHz, DMSO-d6) δ
182.27, 162.46, 141.77, 137.13, 136.79, 135.99, 133.78, 129.77, 129.60, 129.23, 128.30,
127.87, 127.39, 124.19, 123.63, 122.00, 120.65, 112.15, 111.79, 50.32, 21.01; IR (KBr, v, cm-1):
6

3942, 3373, 2978, 2361, 2129, 2029, 1813, 1659, 1582, 1435, 1165, 1072, 679, 672, 618, 471;
HRMS (ESI+) calculated for C24H21N2O2 [M+H+], 369.1598; Found 368.1584.
2.1.2.12. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(3,4-dimethylphenyl)acetamide (8c)
Yield 87%; Yellowish white solid; m.p: 180-182 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.55
(s, 1H), 9.04 (s, 1H), 8.37-8.28 (m, 1H), 7.71 (d, J = 2.2 Hz, 1H), 7.67-7.53 (m, 2H), 7.40-7.24
(m, 7H), 7.13 (d, J = 8.2 Hz, 1H), 5.62 (s, 2H), 2.23 (s, 3H), 2.20 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 182.29, 162.40, 141.79, 137.15, 136.83, 136.78, 136.20, 132.59, 130.26, 130.05,
129.22, 128.30, 127.87, 127.40, 124.18, 123.62, 123.28, 122.00, 121.77, 118.20, 116.09,
112.13, 111.99, 111.80, 50.32, 20.16, 19.36; IR (KBr, v, cm-1): 3726, 3140, 2554, 2369, 1883,
1751, 1674, 1574, 1497, 1412, 1242, 1142, 1011, 818, 656, 486, 417; HRMS (ESI+) calculated
for C25H23N2O2 [M+H+], 383.1754; Found 383.1744.
2.1.2.13. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(4-methoxyphenyl)acetamide (8d)
Yield 91%; Creamy yellow solid; m.p: 170-172 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.61
(s, 1H), 9.03 (s, 1H), 8.36-8.27 (m, 1H), 7.84-7.75 (m, 2H), 7.67-7.58 (m, 1H), 7.40-7.24 (m,
7H), 7.00-6.91 (m, 1H), 5.62 (s, 2H), 3.76 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 182.33,
162.15, 156.36, 141.77, 137.14, 136.77, 131.60, 129.23, 128.30, 127.87, 127.40, 124.16,
123.61, 122.14, 121.99, 114.33, 112.13, 111.82, 55.67, 50.31; IR (KBr, v, cm-1): 3927, 3426,
2978, 2369, 2137, 1798, 1558, 1420, 1250, 1157, 1018, 818, 640, 463; HRMS (ESI+) calculated
for C24H21N2O3 [M+H+], 385.1547; Found 385.1536.
2.1.2.14. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(4-methoxybenzyl)acetamide (8e)
1
Yield 90%; White solid; m.p: 168-170 °C; H NMR (400 MHz, DMSO-d6) δ 9.28(t, J = 6.4
Hz, 1H), 9.01 (d, J = 0.8 Hz, 1H), 8.32-8.23 (m, 1H), 7.65-.7.56 (m, 1H), 7.39-7.23 (m, 9H),
6.94-6.84 (m, 2H), 5.60 (s, 2H), 4.36 (d, J = 6.3 Hz, 2H), 3.73 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 182.30, 163.74, 158.74, 141.68, 137.17, 136.70, 131.41, 129.27, 129.22, 128.28,
127.85, 127.38, 124.07, 123.48, 122.04, 114.17, 112.02, 55.52, 50.25, 42.01; IR (KBr, v, cm-
1): 3372, 2963, 2369, 2114, 1883, 1690, 1507, 1427, 1250, 1165, 1003, 802, 687, 447; HRMS
(ESI+) calculated for C25H23N2O3 [M+H+], 399.1703; Found 399.1691.
2.1.2.15. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide (8f)
Yield 83%; Yellow solid; m.p: 180-182 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.59 (s, 1H),
9.11 (s, 1H), 8.37-8.29 (m, 1H), 7.66-7.57 (m, 1H), 7.39-7.24 (m, 9H), 5.64 (s, 2H), 3.79 (s,
6H), 3.66 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 181.73, 162.10, 153.18, 141.93, 137.21,
136.78, 134.69, 134.56, 129.21, 128.27, 127.79, 127.49, 124.22, 123.66, 122.04, 112.15,
111.71, 98.54, 60.61, 56.27, 50.32; IR (KBr, v, cm-1): 3935, 3750, 3433, 2361, 2129, 1875,
7

1558, 1420, 1242, 1096, 1011, 818, 656, 463; HRMS (ESI+) calculated for C26H25N2O5
[M+H+], 445.1758; Found 445.1747.
2.1.2.16. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(4-bromophenyl)acetamide (8g)
Yield 93%; Off-white solid; m.p: 201-203 °C; 1H NMR (400 MHz, DMSO-d6) ), δ 10.89 (s,
1H), 9.02 (s, 1H), 8.45-8.12 (m, 1H), 7.87 (d, J = 3.2 Hz, 2H), 7.67-7.61 (m, 1H), 7.58 (d, J
13
= 2.2 Hz, 2H), 7.43-7.22 (m, 7H) 5.62 (s, 2H); C NMR (100 MHz, DMSO-d6) δ 181.78,
162.71, 141.86, 137.91, 137.07, 136.81, 132.06, 129.23, 128.32, 127.89, 127.36, 124.26,
123.71, 122.91, 122.67, 121.98, 116.60, 112.19, 111.70, 50.36; IR (KBr, v, cm-1): 3950, 3773,
3418, 2932, 2369, 2014, 1690, 1574, 1427, 1204, 1165, 1065, 1011, 818, 733, 656, 463; HRMS
(ESI+) calculated for C23H18BrN2O2 [M+H+], 433.0546; Found 433.0532.
2.1.2.17. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(3,4-dichlorophenyl)acetamide (8h)
1
Yield 92%; White solid; m.p: 189-190 °C; H NMR (400 MHz, DMSO-d6) δ 11.07 (s, 1H),
9.06 (s, 1H), 8.35-8.31 (m, 1H), 8.30 (d, J = 2.4 Hz, 1H), 7.85 (dd, J = 8.8, 2.5 Hz, 1H), 7.71-
13
7.58 (m, 2H), 7.40-7.24 (m, 7H), 5.62 (s, 2H),; C NMR (100 MHz, DMSO-d6) δ 181.20,
162.76, 142.05, 138.67, 137.05, 136.80, 131.49, 131.15, 129.22, 128.32, 128.07, 127.89,
127.39, 126.33, 124.29, 123.75, 122.00, 121.97, 120.84, 112.20, 111.63, 50.39; IR (KBr, v,
cm-1): 3757, 3395, 2940, 2369, 2114, 1906, 1821, 1697, 1582, 1381, 1296, 1196, 1165, 818,
694, 463, 409; HRMS (ESI+) calculated for C23H17Cl2N2O2 [M+H+], 423.0662; Found
423.0651.
2.1.2.18. 2-(1-benzyl-1H-indole-3-yl)-2-oxo-N-(4-fluorophenyl)acetamide (8i)
1
Yield 84%; White solid; m.p: 182-184 °C; H NMR (400 MHz, DMSO-d6) δ 10.83 (s, 1H),
9.03 (s, 1H), 8.38-8.28 (m, 1H), 7.68-7.58 (m, 1H), 7.40-7.18 (m, 11H), 5.62 (s, 2H); 13C NMR
(100 MHz, DMSO-d6) δ 182.01, 162.49, 158.89, 141.83, 137.09, 136.80, 134.94, 134.91,
134.55, 134.52, 129.22, 128.31, 127.88, 127.38, 124.22, 123.67, 122.86, 122.79, 122.61,
122.53, 122.00, 115.98, 115.95, 115.75, 115.73, 112.16, 111.76, 50.35; IR (KBr, v, cm-1): 3750,
3287, 3148, 2932, 2832, 2353, 1890, 1628, 1574, 1512, 1404, 1219, 1157, 818, 679, 501, 471;
HRMS (ESI+) calculated for C23H18FN2O2 [M+H+], 373.1347; Found 373.1336.
2.2. Pancreatic lipase inhibition assay and enzyme kinetics
Orlistat (1), Porcine PL (Type II) and 4-nitrophenyl butyrate were procured from
Sigma-Aldrich (MO, USA). Tris buffer and sodium chloride (molecular biology grade) were
8

procured from Sisco Research Laboratories (MH, India). All other chemicals and solvents
(analytical grade) were used without further purification.
PL inhibition assay and kinetics was performed as per the protocol standardized in our
laboratory [26, 28]. Briefly, a suspension of porcine pancreatic lipase (5 mg/mL) was subjected
to vigorous shaking, followed by centrifugation (4000 rpm, 10 min). and the supernatant was
used afresh as enzyme solution. Stock solutions of the synthesized compounds and orlistat (1)
were prepared in DMSO at linear concentrations ranging from 0.78 - 2000 μg/mL. The final
reaction mixture comprised of 875 μL of buffer, 100 μL of enzyme and 20 μL of compounds
of various stock concentrations, pre-incubated for 5 min at 37 °C, followed by addition of 5 μL
of the substrate (4-nitrophenyl butyrate, 10 mM in acetonitrile). The absorbance of final
mixture was taken on a BioTek EPOCH microplate spectrophotometer (VT, USA) after 5 min
at absorbance maxima of 4-nitrophenol (405 nm). The assay was performed in triplicate and
percentage inhibition was calculated using the formula
% inhibition = [1-( AT ⁄ AE)] x 100
where AE is the absorbance of enzyme control (without inhibitor), and AT is the difference
between the absorbance of test sample, with and without substrate. The IC50 of the compounds
was calculated by plotting linear regression curve.
For the inhibition kinetics, the assay protocol was repeated with varying concentrations
(0, 5 and 10 μM) of 8f and substrate (25, 50, 100 and 200 µM), and a double reciprocal
Lineweaver-Burk plot was plotted to understand the nature of inhibition [31]. Inhibition
constant, Ki, was calculated from Cheng-Prusoff equation [32].
2.3. Molecular modelling studies
Molecular docking studies of the synthesized analogues were performed using Molegro
Virtual Docker 6.0 (CLC bio). The energy minimized structures of these analogues were
obtained using MM2 force field in Chemdraw 3D module of ChemBioOffice v12 (PerkinElmer,
USA), and docked into the crystal structure of human PL (PDB ID: 1LPB) retrieved from
RCSB PDB Data bank [17,33].
Molecular dynamics (MD) simulation of compound 8f in complex with PL was
performed as per the protocol standardized in our laboratory [26,34]. Gromacs 5.0.4 [35],
compiled on a CentOS 7 operating system equipped with Intel(R) Xeon(R) CPU W3565 and
NVIDIA Quadro 4000 Quad-Core Processor was used. CHARMM27 force field [36] was
applied during the MD run, and the topology of the ligand was generated using online tool
9

provided by Swiss Institute of Bioinformatics [37]. Prior to the initiation of MD, the complex
was minimized using Steepest Descent algorithm for 1000 steps, followed by stabilization of
the system to 310 K and 1 atm pressure for 50 ps, using canonical NVT and NPT ensembles.
Parameters like Particle Mesh Ewald method for long-range electrostatics [38], and 14 Å cut-
off for van der Waals and columbic interactions were set during the MD simulation. LINCS
algorithm was applied for calculating bond length [39]. Discovery Studio 4.5 visualizer
(Accelrys, USA) was used to depict graphical representations of the complex.
3. Results and Discussion
3.1. Chemistry
The synthesis of the indole glyoxylamides 7a–i and 8a-i (Table 1) was carried out as
detailed under Scheme 1. Initially, indole (4) or its N-benzylated derivative (5) was allowed to
react with oxalyl chloride under ice-cold conditions to afford respective glyoxylyl chloride
derivative 6 in good yields. Further, the reaction of 6 with various aniline derivatives in the
presence of triethylamine afforded a series of indole glyoxylamides, 7a–i and 8a-i. Structures
of the synthesized analogues were well characterized using IR, NMR (1H and 13C) and HRMS.
In IR spectra of 7a–i and 8a-i, characteristic peaks at ~3700, ~1680 and 1630 cm−1 were
assigned to the amide N-H, α-keto carbonyl and amide carbonyl functionalities, respectively.
13
Further, in C NMR spectra of 7a–i and 8a-i, carbons of carbonyl and amide functionalities
were resonated at ~180 (CO) and ~160 ppm (NHCO).
3.2. Pancreatic lipase inhibition assay and enzyme kinetics
The PL inhibitory activity of the synthesized indole glyoxylamides was evaluated using
porcine PL (Type II) with 4-nitrophenyl butyrate as a substrate. Orlistat (1) was used as
reference standard. As summarized in Table 1, the activity profiles of compounds 7a–i and 8a-i
varied between 4 - 48 µM. Most of the compounds exhibited good (< 15 µM) to moderate (15-
30 µM) activity, while three compounds (7g-i) displayed poor activity (> 30 µM). Analogue 8f
exhibited significantly potent activity against PL, with an IC50 of 4.92 µM in comparison to
10

compound 2 from the previous study. The standard drug, orlistat (1) possessed a very potent
IC50 of 0.99 µM (Fig. 2).
Further, enzyme kinetics was performed for 8f to understand its nature of inhibition,
that in turn would provide adequate information of the ligand’s binding site [40,41]. Three
concentrations (0, 5 and 10 μM) of 8f were taken and the assay protocol was repeated using
four substrate concentrations to plot a double reciprocal Lineweaver-Burk plot (Fig. 3).
As shown in Fig. 3 and Table 2, the plots converged at y-intercept while Km increased
in proportion to inhibitor concentration. These representations indicated that compound 8f
inhibited PL competitively against the substrate similar to orlistat and carbazolyl
oxoacetamides from the previous study [26], highlighting the fact that compound 8f bound to
the active site of PL. Further, compound 8f possessed a Ki value of 3.06 µM.
3.3. Structure Activity Relationship
A preliminary structure-activity relationship of the screened compounds was analysed
based on their PL inhibitory activity. As shown in Fig. 4, the potential of the compounds to
inhibit PL varied with respect to the substitutions on the indole and amide nitrogens viz., R and
Ar respectively. The presence of benzyl substitution on the indole resulted in better PL
inhibition (8a-i) in comparison to their unsubstituted counterparts (7a-i). This might be
attributed to the increased hydrophobicity of the indole, which in turn resulted in stronger
hydrophobic interactions with the lid domain. Similarly, the presence of ring activating groups
on the aryl ring of the amide nitrogen resulted in better potency of the compounds. For instance,
compound 8f that possessed N-benzyl substitution on indole, and trimethoxy substitution on
the aryl ring of the amide, exhibited most potent activity of 4.92 µM. Moreover, the presence
of a methylene bridge between the aryl extension and the amide nitrogen resulted in potential
activity. As represented in Table 1, compound 8d with 4-methoxyphenyl substitution on the
amide nitrogen possessed an IC50 of 10.86 µM, while compound 8e with 4-methoxybenzyl
substitution exhibited an IC50 of 5.86 µM. Nevertheless, a comparatively lower potential of the
sunthesized indole glyoxylamides in comparison to the standard drug, orlistat, might be
attributed to the difference in the chemical reactivity of the respective carbonyl groups (α-
ketoamide and β-lactone) with Ser152 of PL. Also, the presence of long aliphatic chains in
11

orlistat might have provided an additional binding stability to the ligand through enhanced
hydrophobic interactions with the lid domain.
3.4. Molecular modelling
Molecular docking studies were performed to understand the interaction pattern of these
compounds with pocket residues of PL. Compounds 7a–i and 8a-i were docked into the crystal
structure of Human PL (PDB ID: 1LPB) using Molegro Virtual Docker 6.0. Prior to docking,
the grid parameters were validated by redocking the co-crystallised ligand, methoxyundecyl
phosphinic acid (MUP) into the active site of PL. The redocked pose was deviated from co-
crystallised pose by an RMSD of 1.7 Å. A summary of the MolDock scores and interactions
of compounds 7a–i and 8a-i with the active site of PL is represented in Table 3. The docked
poses of these ligands were in accordance to the hypothesis, wherein indole moiety exhibited
hydrophobic interactions with amino acids of the lid domain, while the reactive carbonyl group
of the glyoxylamide existed near to Ser 152 of the active site (Fig. 5).
An analysis of the docking poses of compounds 8a-i highlighted an additional
interaction by the N-benzyl substitution with Phe 77, that was not seen with the unsubstituted
indole counterparts (7a-i). The fact clearly indicated the prominent role of hydrophobic
interactions with lid domain in potentiating PL inhibitory activity. Moreover, no variation was
observed in the hydrophobic interaction pattern exhibited by the indole in comparison to the
carbazole from our previous study. In addition, compounds 8a-i exhibited a prominent π-cation
interaction with Arg 256, that was not seen with 7a-i. Previous reports suggested that the open
lid conformation of Human PL is attained through formation of salt bridge between Arg 256-
Asp 257 and Tyr 267-Lys 268 [16]. Further, the role of π-cation interaction with Arg 256 in
potentiating the activity was established in our previous study [42]. In addition, previous
reports suggested that the presence of donating groups on the ring resulted in stronger π-cation
interaction as compared to the withdrawing groups [43]. This fact is in correlation to the
structure-activity relationship mentioned above, wherein the ring deactivating groups on the
aryl ring of the amide nitrogen resulted in moderate to poor PL inhibitory activity (as seen with
7g-i and 8g-i). The above analysis clearly explained the variation in the MolDock scores of
these compounds, which in turn affected their PL inhibitory activity. The fact was further
confirmed by Pearson correlation analysis wherein a significant correlation (at p < 0.01) was
12

observed between the MolDock scores and IC50 values of these analogues (Pearson’s r =
0.6927). Compound 8f, the most potential analogue among the series exhibited a potential
MolDock score of -153.037 kcal/mol.
In addition, the interaction distance of the reactive carbonyl group from Ser 152 was
found to possess a major role in the PL inhibitory activity. As represented in Fig. 6, this distance
was lesser for compound 8f (3.84 Å) in comparison to compound 2 (4.45 Å), while carbonyl
group of orlistat’s β-lactone existed 3.3 Å from Ser 152. This analysis validated the hypothesis
of the present study wherein the replacement of carbazole (as seen in compound 2) with an
indole nucleus (compound 8f) reduced the interaction distance and potentiated the PL
inhibitory activity (from 6.31 µM to 4.92 µM, respectively).
Molecular docking analysis provided a preliminary outline of the various interactions
required for stable binding conformation of indolyl oxoacetamides. However, it is necessary to
predict the ligand behaviour in motion. Consequently, a 10 ns MD simulation has been
performed for 8f complexed with PL using CHARMM27 force field [36]. The backbone
RMSD remained stable with an overall deviation less than 2.5 Å, validating the MD simulation
(Fig. 7A). Further, 8f remained very stable during the entire MD simulation, with a maximum
deviation of 3.5 Å observed at ~4 ns (Fig. 7B). As summarized in Table 4, 8f 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 potential PL inhibitory activity of the
ligand.
4. Conclusion
In the present study, eighteen indole glyoxylamides were synthesized and screened in vitro to
determine their PL inhibition activity. Compound 8f was found to be the most potent inhibitor
of PL with an IC50 of 4.92 µM. Furthermore, 8f exhibited competitive inhibition similar to
orlistat and carbazolyl oxoacetamides, as evidenced through enzyme kinetic studies. The
results from molecular modelling studies were in alignment to the PL inhibitory activities of
these compounds and highlighted the role of π-π stacking and π-cation interactions with lid
domain and Arg 256 respectively. In conclusion, the present study validated the hypothesis of
replacing the carbazole with an indole that resulted in significant enhancement in the potential
of these indole glyoxylamides to inhibit pancreatic lipase. Further investigations on these
analogues would establish these scaffolds as potential anti-obesity agents.
13

5. Acknowledgements
The authors acknowledge Birla Institute of Technology and Science, Pilani (BITS Pilani),
Pilani Campus, Pilani, Rajasthan, India. Mr. 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)
6. Declarations of Interest: None
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Fig. 1. Rationale for designing indole glyoxylamides as PL inhibitors.
Fig. 2. Linear regression curves for orlistat and 8f obtained from PL inhibition assay.
Fig. 3. Double reciprocal Lineweaver-Burk plot of compound 8f obtained through enzyme kinetic study
(1/V and 1/[S] represent the reciprocals of the reaction velocity and substrate concentration, respectively).
Fig. 4. Structure-activity relationship for indole glyoxylamides.
Fig. 5. (A) 2D interaction diagram of 8f with PL; (B) 3D interaction diagram of 8f highlighting the lid domain
interactions and reactive carbonyl position with reference to Ser 152 (highlighted in green); (C) Representation
of 8f in the binding pocket of PL.
Fig. 6. (A) Superimposition of compound 2 (Brown) and 8f (Green); (B) Superimposition of Orlistat (Brown) and
8f (Green), highlighting the distance of the reactive carbonyl group from Ser 152.
Fig. 7. Backbone RMSD (A) and ligand RMSD (B) retrieved from 10 ns MD simulation of 8f - PL complex.
Scheme 1. Reagents and conditions: (i) C6H5CH2Cl, KOH, DMF, RT, 12 h; (ii) (COCl)2, Et2O, 0 °C, 30 min; (iii)
Ar-NH2, TEA, THF, RT, 2 h.
17

Table 1. In vitro PL inhibitory activity of indole glyoxylamides 7a–i and 8a-i.
O
H
N
Ar
O
N
R
Sr. No Compound
R
Ar
IC50 (µM)
1
2
7a
7b
7c
-H
-H
-H
-H
-H
-H
-H
-H
-H
Phenyl
27.49 ± 1.68
26.13 ± 0.92
26.07 ± 1.14
23.72 ± 0.67
17.39 ± 0.53
17.28 ± 0.49
43.36 ± 1.67
44.27 ± 1.21
47.62 ± 1.48
18.24 ± 0.74
12.9 ± 0.58
10.62 ± 0.66
10.86 ± 0.71
5.83 ± 0.64
4.92 ± 0.29
25.76 ± 0.97
26.72 ± 0.79
24.18 ± 0.86
0.99 ± 0.11
4-methylphenyl
3,4-dimethylphenyl
4-methoxyphenyl
4-methoxybenzyl
3,4,5-trimethoxyphenyl
4-bromophenyl
3,4-dichlorophenyl
4-fluorophenyl
Phenyl
3
4
7d
7e
5
6
7f
7
7g
8
7h
7i
9
10
11
12
13
14
15
16
17
18
8a
-CH2C6H5
-CH2C6H5
-CH2C6H5
-CH2C6H5
-CH2C6H5
8b
8c
4-methylphenyl
3,4-dimethylphenyl
4-methoxyphenyl
4-methoxybenzyl
8d
8e
8f
-CH2C6H5 3,4,5-trimethoxyphenyl
8g
-CH2C6H5
-CH2C6H5
-CH2C6H5
4-bromophenyl
3,4-dichlorophenyl
4-fluorophenyl
8h
8i
Orlistat
Table 2. Vmax and Km values calculated from Lineweaver-Burk plot at different concentrations of 8f [I].
[I] (μM)
Vmax (μM.min-1)
1.789
Km (μM)
82.27
0
5
1.787
123.73
154.20
10
1.789
(Vmax: maximum velocity of the enzyme reaction; Km: Michelis-Menten constant)
Table 3. MolDock score (in kcal/mol) and interaction summary of 7a-i and 8a-i with the active site of the PL.
Compound
MolDock Score
-117.58
H-bond
π-π stacking
π-cation interactions
7a
7b
7c
7d
Phe 77, Ser 152, Leu 153, Phe 215 Phe 215
-
-
-119.927
Phe 77, Ser 152, Leu 153, His 263 Tyr 114, Phe 215
Phe 77, Ser 152, Leu 153, Phe 215 Phe 215, His 263
-131.305
His 263
-
-120.942
Phe 77, Ser 152, Leu 153
Tyr 114, Phe 215
18

7e
7f
-124.531
-140.301
-126.141
-128.118
-110.475
-127.251
-133.192
-148.109
-136.731
-145.138
-153.037
-128.188
-144.217
-129.833
-152.580
Phe 77, His 263
Phe 77, His 263
Asp 79, Arg 256
Phe 77, Ser 152, Leu 153, Phe 215 Tyr 114, Phe 215
Phe 77, Ser 152, Leu 153, Phe 215 Phe 215
-
7g
-
7h
Phe 77, Ser 152, Leu 153, Phe 215 Tyr 114, Phe 215
-
7i
Ser 152
Tyr 114
-
8a
Phe 77, His 151, Ser 152, Leu 153 Phe 77, Tyr 114, Phe 215
Phe 77, His 151, Ser 152, Leu 153 Phe 77, Tyr 114
Asp 79
8b
Arg 256
8c
Phe 77, His 151, Ser 152
Phe 77, Phe 215, His 263
Arg 256
8d
Phe 77, His 151, Ser 152, Leu 153 Phe 77, Tyr 114, Phe 215
Asp 79, Arg 256
Arg 256, His 263
Asp 79, Arg 256, His 263
8e
Phe 77, His 151, Ser 152, Leu 153 Phe 77, Tyr 114, Phe 215, His 263
8f
Gly 76, Phe 77, His 151, Ser 152
Phe 77, Ser 152, Leu 153
Phe 77, Tyr 114, Phe 215, His 263
Phe 77, Tyr 114, His 263
8g
8h
Gly 76, Phe 77, His 151, Ser 152
Phe 77, Tyr 114, Phe 215, His 263
Asp 79, Arg 256, His 263
8i
Phe 77, His 151, Ser 152, Leu 153 Phe 77, Tyr 114
His 151, Ser 152
-
-
Orlistat
-
Table 4. Interaction chart of 8f with PL at different time frames during MD 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
Phe 77, Tyr 114, Phe 215
Phe 77, Tyr 114, Phe 215
Phe 77, Tyr 114, Phe 215
Phe 77, Tyr 114
Arg 256
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Phe 77, Tyr 114, Phe 215
Phe 77, Phe 215
-
-
Phe 215
His 263
Phe 77, Phe 215
-
-
-
Phe 77, Phe 215, His 263
Tyr 114, Phe 215, His 263
Phe 77, Phe 215, His 263
19