Design, synthesis, biological evaluation and molecular modelling studies of conophylline inspired novel indolyl oxoacetamides as potent pancreatic lipase inhibitors

Article abstract of DOI:10.1039/d0nj02622k

A novel series of 21 indolyl oxoacetamide analogues was designed based on the natural product lead conophylline, and evaluated for their pancreatic lipase inhibitory activity using porcine pancreatic lipase (Type II). Analogues 12c and 12b exhibited comparatively greater potential (IC50 values of 2.95 and 3.26 μM) than conophylline (IC50-3.31 μM), while the standard drug, orlistat, exhibited a potent IC50 value of 0.99 μM. Further, analogues 12b and 12c exhibited reversible competitive inhibition similar to orlistat and conophylline, and possessed Ki values of 1.89 and 1.69 μM, respectively. Molecular docking of these analogues was in agreement with the in vitro results, wherein the MolDock scores exhibited significant correlation with their inhibitory activity. A 10 ns molecular dynamics simulation of 12c complexed with pancreatic lipase confirmed the role of extended alkyl interactions, along with π-π stacking and π-cation interactions, in stabilising the ligand in the active site (maximum observed RMSD ≈ 3.5 ?). ADMET prediction indicated the GI absorption of these analogues to be high; however, they did not possess carcinogenicity and hepatotoxicity in contrast to orlistat and conophylline.

Full text of DOI:10.1039/d0nj02622k

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Title Page
Title:
Design, Synthesis, Biological Evaluation and Molecular Modelling Studies of Conophylline
Inspired Novel Indolyl Oxoacetamides as Potent Pancreatic Lipase Inhibitors
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Authors:
#
#
*
Sridhar S. N. C. , Saksham Palawat , Atish T. Paul .
#
Equal contributing authors
*
Corresponding author
Affiliations:
Laboratory of Natural Product Chemistry, Department of Pharmacy, Birla Institute of
Technology and Science, Pilani (BITS Pilani), Pilani campus, Pilani - 333 031, Rajasthan,
India.
Present Address:
Dr. Atish T. Paul,
Assistant Professor and Head,
Department of Pharmacy,
Birla Institute of Technology and Science, Pilani (Pilani Campus),
Pilani - 333 031, Rajasthan, India.
Email: atish.paul@pilani.bits-pilani.ac.in
1

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DOI: 10.1039/D0NJ02622K
Abstract
A novel series of 21 indolyl oxoacetamide analogues were designed based on the natural
product lead, conophylline, and evaluated for their pancreatic lipase inhibitory activity using
porcine pancreatic lipase (Type II). Analogues 12c and 12b exhibited comparatively greater
potential (IC50 values of 2.95 and 3.26 µM) than conophylline (IC50 - 3.31 µM) while the
standard drug, orlistat exhibited potent IC50 value of 0.99 µM. Further, analogues 12b and 12c
exhibited reversible competitive inhibition similar to orlistat and conophylline, and possessed
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K values of 1.89 and 1.69 µM, respectively. Molecular docking of these analogues was in
i
agreement with the in vitro results, wherein the MolDock scores exhibited significant
correlation with their inhibitory activity. A 10 ns molecular dynamics simulation of 12c
complexed with pancreatic lipase, confirmed the role of extended alkyl interactions along with
π- π stacking and π-cation interactions, in stabilising the ligand in the active site (maximum
observed RMSD ≈ 3.5 Å). ADMET prediction indicated the GI absorption of these analogues
to be high, however, they did not possess carcinogenicity and hepatotoxicity in contrast to
orlistat and conophylline.
Keywords
ADMET, Conophylline, Indolyl Oxoacetamides, Inhibition Kinetics, Molecular Dynamics,
Pancreatic Lipase
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1
. Introduction
Obesity is a chronic, relapsing, progressive, multifactorial metabolic disease
characterized by excessive or abnormal accumulation of fat deposits in the body.1–3 Worldwide,
obesity has tripled since 1975 with recent reports from the World Health Organization (WHO)
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indicating a rapid upsurge in obese population. As per the recent WHO statistics, over 1.9
billion adults were found to be overweight, of which 650 million adults were found obese.4
While obesity is preventable and does not itself impose any risk to the health, a chronic
condition might lead to severe comorbid risks including insulin resistance, diabetes mellitus
(
Type II), hypertension, dyslipidaemia, coronary heart disease, sleep apnea, gall bladder
disease, gout, osteoarthritis, and certain cancers.5
To date, around 15 drugs are approved for the treatment of obesity, however, a majority
of these drugs are directed towards reducing the appetite, and would result in an overall
inhibition of nutrition intake. On the contrary, orlistat (1, Figure 1), a β-lactone containing
lipstatin derivative, acts through reversible covalent inhibition of pancreatic lipase (PL), and is
widely prescribed for long term obesity treatment.6,7 While, orlistat was reported to exhibit
tolerable side effects viz., steatorrhea, oily stools and frequent bowel movements, recent reports
from FDA has cited severe adverse effects with long term administration of orlistat, that
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included hepatotoxicity, acute pancreatitis, gall stones and renal injuries. These events have
highlighted the necessity for safer and effective anti-obesity drugs.
In the recent years, various amide functionality-based pharmacophores, including
tripeptides and rhodanines, were reported to exhibit potential PL inhibition.10–12 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 of PL. Previously, we have identified conophylline
(
2, Figure 1) from the leaves of Tabernaemontana divaricata as a potential natural product lead
for PL inhibition.13,14 Conophylline, a bis-indole alkaloid, exhibited an IC50 of 3.31 µM,
comparable to that of orlistat (IC50 - 0.99 µM). Nevertheless, the lower PL inhibitory potential
of conophylline in comparison to orlistat was attributed to the lack of a highly reactive carbonyl
group in contrary to orlistat, and its structural rigidity yielding in high degree of unfavourable
steric interactions with the active site of Human PL (Figures 1 and 2). Consequent studies that
involved the use of molecular hybridization and scaffold hopping techniques resulted in various
carbazolyl and indolyl oxoacetamides with potential PL inhibitory activity.10,15,16 The α-
ketoamide warhead of these oxoacetamides mimicked the function of the orlistat’s β-lactone,
while the unfavourable steric interactions were significantly reduced (Figures 3 and 4). In
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addition, these studies also established the pivotal role of π-π stackings and π-cation interaction
with the aromatic acids of the lid domain and Arg 256 respectively, for potential PL inhibition.
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Fig. 1 Structures of Orlistat (1) and Conophylline (2) highlighting their respective reactive carbonyl groups
Fig. 2 2D pose of Conophylline (2) highlighting the unfavourable steric interactions (in red)
Nevertheless, the most potent indolyl oxoacetamide (5) exhibited lower, yet comparable
PL inhibitory potential (IC50 - 4.53 µM) in comparison to conophylline (Figure 3). On the
contrary, compound 5 that possessed a 5-methoxy substitution on the indole, did not exhibit
4

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significant difference in its activity compared to its unsubstituted counterpart, 4 (IC50 = 4.92
µM), clearly indicating negligible role of 5-methoxy substitution.10,16 The lower PL inhibitory
potential of these indolyl oxoacetamides in comparison to conophylline was attributed to their
minimal hydrophobic interactions (alkyl/π-alkyl) with the lid domain amino acids (Figure 4).
Considering all the above facts, we hypothesized further developments to the indolyl
oxoacetamides, with particular emphasis on its hydrophobic density and π-cation interactions,
would result in potent PL inhibitors.
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Fig. 3 Schematic representation indicating the series of events in designing the indolyl oxoacetamides
Fig. 4 2D interactions of compound 4 (left) and 5 (right) with Human PL, highlighting no unfavourable bumps
and minimal π-alkyl interactions
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2
. Results and discussion
2
.1. Chemistry
A detailed literature review of various natural products-based PL inhibitors indicated
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the presence of prenyl and geranyl substitutions in potent PL inhibition. Hence, in the present
study, various indolyl oxoacetamides with N-prenyl and N-geranyl substitutions on the indole
moiety were considered, along with N-ethyl, N-benzyl and the unsubstituted analogues.
Further, literature reports suggested that the presence of heteroaryl groups can result in better
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π-cation interaction in comparison to the simple aryl groups. Moreover, the presence of linker
between the aryl extension and the amide, as observed in our previous studies, resulted in
enhanced PL inhibitory activity.10,16 Accordingly, trimethoxybenzyl and 2-(1H-indol-3-
yl)ethyl substitutions were considered for the aryl extension (Ar), apart from various other
substitutions (Scheme 1).
Scheme 1. Reagents and conditions: (i) Ethyl bromide/Benzyl chloride/Prenyl bromide/Geranyl bromide, KOH,
2 2 2
DMF, RT, 12 h; (ii) (COCl) , Et O, 0 °C, 30 min; (iii) Ar-NH , TEA, THF, RT, 2 h.
The syntheses of all the final compounds (Table 1) were carried out as per the procedure
detailed in Scheme 1.10,16,18 Briefly, indole (6) was subjected to N-substitution with various
halides in the presence of KOH as base. The N-benzylindole was obtained as crude precipitate
and purified by recrystallization from ethanol. The N-ethyl, N-prenyl and N-geranyl indoles,
obtained as oily liquids, were extracted using chloroform and purified through column
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chromatography. Various indole analogues (6 and 7) were then allowed to react with oxalyl
chloride under ice-cold conditions to afford the respective glyoxylyl chloride derivative 8 in
good yields. Further, the reaction of 8 with various aniline derivatives in the presence of
triethylamine afforded the final compounds in good yields (68-91%). All these analogues, with
an exception of 9b and 10k, were synthesized for the first time (confirmed via SciFinder).
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Structures of the synthesized analogues were well characterized using IR, NMR ( H
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and C) and HRMS. In the H NMR, the -NH protons of amide functionality resonated at 10-
1 ppm, while the proton at C adjacent to the indole nitrogen resonated at 8-9 ppm. For
analogues 10a-k, the methyl protons of the N-ethyl substitution appeared as a triplet between
1-2 ppm, while the -CH protons appeared as a quartet between 4-5 ppm. For the N-prenyl/N-
geranyl substitutions, the terminal -CH protons resonated at 1.5-2 ppm. For compounds 13a
and 13b, the methylene protons of the N-benzyl substitution appeared as a singlet between 5-
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6 ppm. In C NMR spectra, carbons of carbonyl and amide functionalities were resonated at
180 (CO) and ~160 ppm (NHCO). In addition, for the compounds 11a-c, the terminal methyl
carbons of the N-prenyl substitution were observed around 18 and 25 ppm, while the -CH
carbon attached to the nitrogen of the indole resonated at 45 ppm. Similarly, for the compounds
12a-c, the three methyl carbons as well as the carbons of the -CH -CH - linkers in the N-
carbon attached to the
~
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geranyl substitution resonated in between 16-40 ppm, while the -CH
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nitrogen of the indole resonated at 45 ppm.
2
.2. PL inhibition assay and kinetics
The PL inhibitory activity of the synthesized indolyl oxoacetamides was evaluated as
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per the protocol standardized in our laboratory. Porcine PL (Type II) was used as the enzyme
with 4-nitrophenyl butyrate as a substrate, while orlistat (1) was the reference standard. As
summarized in Table 1, the N-ethyl substituted (10a-k) and the unsubstituted (9a, 9b) indolyl
oxoacetamides exhibited moderate to poor PL inhibitory activity, with their IC50 values varying
between 16 - 40 µM. On the contrary, all the N-benzyl, N-prenyl and N-geranyl substituted
compounds, exhibited potent activity with IC50 ≤ 5 µM. Analogue 12c that possessed N-geranyl
substitution on indole and a 2-(1H-indol-3-yl)ethyl extension on the amide, exhibited the most
potent activity against PL, with an IC50 of 2.95 µM. The standard drug, orlistat (1),
comparatively possessed potent IC50 of 0.99 µM.
In order to further understand the nature of inhibition, analogues 12b and 12c were
subjected to enzyme inhibition kinetics study, that in turn would provide adequate information
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of the ligand’s binding site.20,21 For this, three concentrations each of 12b and 12c (0, 5 and 10
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µM) were taken and the PL inhibition assay was performed against four different substrate
concentrations (25, 50, 100 and 200 µM) to obtain a double reciprocal Lineweaver-Burk plot.
Table 1. In vitro PL inhibitory activity of indolyl oxoacetamides
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IC50 (µM)
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a
3,4,5-trimethoxybenzyl 24.58 ± 1.62 10j
2-(1H-indol-3-yl)ethyl 19.98 ± 1.92 10k
Ethyl
Ethyl
3,4,5-trimethoxybenzyl 18.42 ± 1.36
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b
2-(1H-indol-3-yl)ethyl
3,4,5-trimethoxyphenyl
3,4,5-trimethoxybenzyl
2-(1H-indol-3-yl)ethyl
16.04 ± 0.96
5.48 ± 0.17
4.15 ± 0.48
3.72 ± 0.42
3.99 ± 0.14
3.26 ± 0.38
2.95 ± 0.38
4.26 ± 0.52
3.86 ± 0.49
4.92 ± 0.29
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0a Ethyl
10b Ethyl
0c Ethyl
10d Ethyl
0e Ethyl
0f Ethyl
0g Ethyl
0h Ethyl
phenyl
21.92 ± 2.18 11a Prenyl
19.73 ± 2.01 11b Prenyl
18.85 ± 1.88 11c Prenyl
4-methylphenyl
3,4-dimethylphenyl
4-methoxyphenyl
4-methoxybenzyl
4-bromophenyl
3,4-dichlorophenyl
4-fluorophenyl
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20.63 ± 2.16 12a Geranyl 3,4,5-trimethoxyphenyl
19.74 ± 0.38 12b Geranyl 3,4,5-trimethoxybenzyl
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39.72 ± 2.46 12c Geranyl
28.37 ± 1.72 13a Benzyl
37.68 ± 2.47 13b Benzyl
2-(1H-indol-3-yl)ethyl
3,4,5-trimethoxybenzyl
2-(1H-indol-3-yl)ethyl
3,4,5-trimethoxyphenyl
1
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10i Ethyl 3,4,5-trimethoxyphenyl 17.24 ± 1.82
4
Benzyl
As represented in Figure 5, the convergence of the plots on the y-axis indicated that the
analogues 12b and 12c inhibited PL competitively against the substrate similar to orlistat.
Moreover, the K values increased proportionally with inhibitor concentration, while Vmax
m
remained constant (Table 2) clearly indicating that 12b and 12c exhibited reversible
competitive inhibition. This fact is in line with our previous studies, wherein the indolyl
oxoacetamides 4 and 5 also exhibited a reversible competitive inhibition of PL.10,16 These
results from the enzyme kinetics clearly highlighted the fact that analogues 12b and 12c bound
to the active site of the PL, and exhibited inhibitory constant (K
i
) value of 1.89 and 1.69 μM,
respectively, as calculated using the Cheng-Prusoff equation.22
m i
Table 2. K , Vmax and K values of 12b and 12c retrieved from the enzyme kinetic study
-
1
Code
Km (in μM) at various concentrations of inhibitor
V
max (μM.min )
i
K (μM)
0 μM
69.351
67.14
5 μM
133.250
133.79
10 μM
187.498
184.52
1
2b
1.681
1.549
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2
.3. Structure activity relationship
A preliminary structure-activity relationship of the indolyl oxoacetamides with
reference to their PL inhibitory activity is represented in Figure 6. For analogues 10a-i that
possessed an N-ethyl substitution on the indole, the presence of ring activating groups on the
aryl ring of the amide nitrogen resulted in better potency, and is in line with our previous
studies.10,15,16 Further, the presence of 2-(1H-indol-3-yl)ethyl moiety on the amide (as seen with
1
1c, 12c and 13b) resulted in better PL inhibitory potential, followed by the trimethoxybenzyl
(
11b, 12b and 13a) and trimethoxyphenyl moieties (11a, 12a and 4). Likewise, the presence
of N-prenyl/N-geranyl/N-benzyl substitutions on the indole (11a-c, 12a-c and 13a-b,
respectively) resulted in potent activity with IC50 values less than 5 µM, while the analogues
with N-ethyl substitution (10a-k) and the N-unsubstituted analogues (9a-b) exhibited moderate
to poor PL inhibitory activity. In particular, the presence of N-geranyl substitution (12a-c) has
resulted in comparatively greater potency, and might be attributed to its greater hydrophobicity,
leading to stronger hydrophobic interactions with the lid domain.
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0
1
2
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9
0
1
2
3
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5
6
7
8
9
0
1
2
3
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Fig. 6 Structure-activity relationship for indolyl oxoacetamides
2
.4. Molecular docking and Molecular dynamics
Molecular docking studies of the indolyl oxoacetamides were performed using Molegro
Virtual Docker 6.0, and the energy minimized structures of the ligands were docked in to the
crystal structure of Human PL (PDB ID: 1LPB). A summary of the MolDock scores of the
indolyl oxoacetamides with the active site of PL is represented in Table 3. The MolDock scores
of the analogues exhibited significant correlation to their PL inhibitory activity (Pearson’s r =
0
1
.7658, p < 0.05), with the most potent analogue 12c exhibiting potential MolDock score of -
57.894 kcal/mol. Further, the MolDock scores of the N-benzyl, N-prenyl and the N-geranyl
substituted analogues (11a-c, 12a-c and 13a-b) that exhibited potent IC50 values, possessed
greater MolDock scores compared to a majority of the N-ethyl (10a-i) and the unsubstituted
counterparts (9a).
Various interactions viz., H-bond, π-π stacking, π-cation and alkyl hydrophobic
interactions, exhibited by the indolyl oxoacetamides are summarised in Table 3. A majority of
these analogues exhibited H-bond interactions with Phe 77 and His 263 among others. Further,
the N-ethyl substituted analogues, with few exceptions, exhibited an additional H-bond
interaction with Leu 153. On the contrary, the N-benzyl, N-prenyl and the N-geranyl
substituted analogues exhibited additional H-bond interactions with Gly 76 (as seen with 11c,
1
2a, 13a) and His 151 (as seen with 11c, 12a, 13a, 13b). Distinctively, analogue 12c exhibited
H-bond interaction with Ser 152 of the active site, which was not observed with other analogues
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(
Figure 7). This Ser 152 forms the catalytic triad of the active site along with Asp 176 and His
263, and is involved in the hydrolysis of the triglyceride esters.23
The most common π-π stacking interactions exhibited by the indolyl oxoacetamides
involved Tyr 114 and His 263, followed by Phe 77 and Phe 215. Moreover, all the analogues
with few exceptions (9a and 13a) exhibited intense alkyl interactions with various amino acids.
In particular, the N-geranyl analogues (12b and 12c) exhibited alkyl interactions with various
amino acids of the lid domain viz., Phe 77, Ile 78, Leu 213 and Phe 215. Previous literature
suggests that the catalytic triad of the Human PL is enclosed in the lid domain (inactivated
state), however, is activated by long alkyl chains of triglycerides through hydrophobic
interactions with the lid domain amino acids.24,25 These facts indicated that the N-geranyl
substitution might act similar to these long alkyl chains in stabilization of open lid
conformation, consequently leading to their better inhibitory potential.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Further, a contrasting variation was observed with the π-cation interactions exhibited
by the oxoacetamides in the present study. As summarised in Table 3, the N-ethyl analogues,
with exception for 10j and 10k did not exhibit interaction with Arg 256, which can be, in part,
attributed to their poor PL inhibitory potential. Previous literature suggested that the open lid
conformation of the Human PL is attained through formation of salt bridge between Arg 256-
2
6
Asp 257 and Tyr 267-Lys 268. Further, the role of π-cation interaction with Arg 256 in
potentiating the activity was established in our previous studies.10,15,16 Similar observations can
be drawn with analogues 11c, 12c and 13b, wherein the presence of 2-(1H-indol-3-yl)ethyl
moiety with an ethylene linker might have resulted in better π-cation interaction with Arg 256,
leading to their greater PL inhibitory potential. In comparison, the trimethoxybenzyl
counterparts (11b, 12b and 13a) with a methylene linker exhibited lower inhibition followed
by the trimethoxyphenyl counterparts (11a, 12a and 4) with no linker.
Molecular docking analysis clearly indicated the pivotal role of π-π stacking and alkyl
interactions with the lid domain, as well as the π-cation interaction with Arg 256 in imparting
potential PL inhibitory activity. In an attempt to better understand the role of these interactions,
analogue 12c was subjected to a 10 ns molecular dynamics (MD) simulation in complex with
PL. As represented in Figure 8A, the backbone RMSD remained stable throughout the run,
validating the MD simulation. Further, analogue 12c remained stable in the active site during
the first 3 ns, followed by a steep rise in RMSD from 2 Å to 3 Å (Figure 8B). However, the
ligand remained stable through the rest of the 10 ns run, with a maximum deviation observed
at 7 ns (RMSD ≈ 3.5 Å).
1
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5
6
7
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
Table 3. MolDock scores (in kcal/mol) and the interactions of the indolyl oxoacetamides with the active site of Human PL
#
MolDock H-bond
Score
π-π stacking
π-cation
Alkyl/π-alkyl interactions
interactions
9
a
-128.723
-138.285
-110.872
-118.676
-121.694
-121.499
-117.877
-123.105
-115.959
-119.34
Phe 77, His 263
Phe 77, Phe 215
Phe 215
Arg 256
Ile 78, Ala 178
9b
Phe 77, Phe 215
Arg 256
Ala 178, Pro 180, Arg 256, Ala 259, Ala 260, Leu264
Phe 77, Tyr 114, Ala 178, Pro 180, Ala 260
Ala 178, Pro 180, Phe 215, Arg 256, Ala 260, Leu 264
Ile 78, Ala 178, Pro 180, Phe 215, Arg 256, Ala 260, Leu 264
Ile 78, Ala 178, Pro 180, Phe 215, Leu 264
Tyr 114, Pro 180, Ile 209, Leu 264
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
1
0a
Phe 77, His 151, His 263
Phe 77, His 263
Tyr 114
10b
Tyr 114, His 263
Tyr 114, His 263
Tyr 114, His 263
1
1
1
1
0c
0d
0e
0f
Phe 77, His 263
Phe 77, His 263
Asp 79
Gly 76, Phe 77, Leu 153
Phe 77, Leu 153, His 263
Phe 77, Leu 153, His 263
Phe 77, His 263
Phe 77, Tyr 114, Phe 215 Asp 79
Tyr 114, His 263
Phe 77, Ala 178, Pro 180, Phe 215, Arg 256, Leu 264
Phe 77, Ala 178, Pro 180, Phe 215, Arg 256, Leu 264
Ala 178, Pro 180, Phe 215, Ala 260, Leu 264
Ala 178, Pro 180, Phe 215, Ala 260, Leu 264
Phe 77, Ile 78, Ala 178, Ala 260, Leu264
10g
0h
10i
Tyr 114, His 263
1
Tyr 114, His 263
-119.053
-136.352
-133.292
Phe 77, Leu 153, Arg 256
Phe 77, Arg 256, His 263
Phe 77, His 151, Leu 153,
Arg 256, His 263
Phe 77, Tyr 114, Phe 215
1
1
0j
Phe 77, Phe 215
Phe 77
His 263, Arg 256
0k
Arg 256
Ile 78, Ala 178, Phe 215, Ala 259, Ala 260, Leu 264
1
1
1
1a
1b
1c
-130.409
-154.514
-148.187
Phe 77, Leu 153
Phe 77
-
Phe 77, Ile 78, Ala 178, Pro 180, Phe 215, Leu 264
Ile 78, Ala 178, Phe 215, Ala 260, His 263
Phe 77, His 263
Phe 77, Phe 215
Phe 77
Arg 256
Gly 76, Phe 77, His 151,
Leu 153
Arg 256, His 263
Phe 77, Ile 78, Ala 178, Ala 259, Ala 260, Leu264
1
2a
-156.058
Gly 76, Phe 77, His 151
Phe 77
Asp 79, His 263,
Arg 256
Phe 77, Ile 78, Leu 153, Ala 178, Pro 180, Phe 215, Arg 256
1
1
2b
2c
-153.101
-157.894
Phe 77, Arg 256, His 263
Phe 77, Ser 152
Tyr 114, Phe 215
Tyr 114, His 263
Arg 256
Phe 77, Ile 78, Ala 178, Pro 180, Ile 209, Leu 213, Phe 215
Ile 78, Ala 178, Pro 180, Leu 213, Phe 215, Ala 259, Ala 260,
Leu 264
Asp 79, Arg 256
12

Page 13 of 32
New Journal of Chemistry
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1
1
3a
3b
-135.654
Gly 76, Phe 77, His 151,
Leu 153, Arg 256
Phe 77, Tyr 114, Phe 215 His 263, Arg 256
Ala 178, Ala 260, Leu 264
-152.087
-152.580
Phe 77, His 151, His 263
His 151, Ser 152
Phe 77
-
Arg 256, His 263
-
Ile 78, Ala 178, Ala 259, Ala 260, Leu264
Tyr 114, Phe 215, Trp 252, Arg 256, Ala 259
Orlistat
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Fig. 7 (A) 2D interaction diagram of 12c with PL; (B) 3D interaction diagram highlighting H-bond (green), π-π stacking (pink) and π-cation (brown) interactions of 12c with
the active site of PL; (C) Representation of 12c in the binding pocket of PL, highlighting N-geranyl in close proximity with lid domain (in brown).
13

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3
3
3
3
4
4
4
4
4
4
4
Table 4. Various interactions exhibited by 12c during the 10 ns MD run
Frame H-bond
π-π stacking
π-cation
Alkyl/π-alkyl interactions
0
1
ns
ns
Asp 79
Phe 77
Asp 79
Ile 78
Phe 77, Phe 215
Arg 256
Ile 78, Tyr 114, Ala 178
Phe 77, Tyr 114, Phe 215, Trp 252
Phe 77, Phe 215
-
Arg 256, Ala 259
2 ns
ns
4 ns
-
Ala 178, Ile 209, Val 210, Leu 213, Phe 215, Arg 256
Ile 78, Ala 178, Pro 180, Cys 181, Arg 256
Phe 77, Ile 78, Ala 178, Pro 180, Ile 209, Arg 256, Ala 259, Leu 264
Ile 78, Ala 259
0
1
2
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9
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9
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1
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3
4
5
6
3
Phe 77, 215
Arg 256
Asp 79, His 263 Phe 77, Tyr 114, Phe 215
-
5
6
7
8
ns
ns
ns
ns
Asp 79
Phe 77, Tyr 114
Arg 256
Asp 79
-
Arg 256
Ile 78, Arg 111
Phe 77
His 263
-
-
-
Ile 78, Arg 111, Ala 259, Ala 260, Leu 264
Ile 78, Arg 111, Arg 256, Ala 259, Ala 260, Leu 264
Ile 78, Arg 256, Ala 259, Ala 260, Leu 264
Ile 78, Ala 259, Ala 260
-
-
-
-
-
-
9 ns
0 ns
1
Fig. 8 Backbone RMSD (A) and ligand RMSD (B) retrieved from 10 ns MD simulation of 12c - PL complex.
14

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Various interactions exhibited by 12c during the MD run are summarised in Table 4
and Figure 9. The ligand has maintained stable H-bond interactions with Asp 79 among others
till 7 ns, beyond which all the H-bonds were lost. Similarly, 12c exhibited stable π-π stacking
and π-cation interactions with Phe 77, Tyr 114, Phe 215, and Arg 256, respectively, till 6 ns.
However, 12c exhibited stable alkyl interactions with various amino acids, including the lid
domain, throughout the 10 ns MD run.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Fig. 9 2D representation of various interactions exhibited by 12c with PL during the 10 ns MD simulation.
To summarize, an analysis of the molecular docking and molecular dynamics
simulations in correlation to the PL inhibitory activity of the synthesized analogues, as well as
the conclusions drawn from our previous studies10,15,16 clearly highlighted the following
attributes for a potent PL inhibition; a) highly reactive carbonyl group that can bind covalently
to Ser 152 of the catalytic triad, b) strong π-π stacking interactions with the aromatic amino
acids of the lid domain, viz., Phe 77 and Phe 215, in addition to Tyr 114, c) additional alkyl/π-
alkyl interactions with the lid domain amino acids, viz., Gly 76 - Lys 80 and Leu 213 - Met
2
17, and d) strong π-cation interaction with Arg 256, the amino acid involved in the dynamics
of the lid domain. Analogues 12b and 12c exhibited a majority of these interactions, however,
lacked in π-π stacking with the lid domain. Further enhancement in its aromaticity might result
in greater PL inhibitory activity.
1
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2.5. In silico ADMET prediction
The lipophilicity and ADMET (Absorption, Distribution, Metabolism, Excretion and
Toxicity) profiles of the indolyl oxoacetamides in the present study were predicted using
various tools viz., SwissADME, ProTox-II and ToxTree,27–29 and the results are summarized
in Table 5. Orlistat (1) possessed comparatively higher LogP value and a low gastro-intestinal
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
0
(
GI) absorption which is in accordance with the reported data. On contrary, all the indolyl
oxoacetamides exhibited comparatively lower LogP values and high GI absorption.
Nevertheless, the N-geranyl substituted analogues (12a-c) exhibited greater LogP (4.97-5.53)
compared to conophylline (3.13) and the rest of the analogues (2.40-4.15). Similarly, analogues
1
2a and 12b, exhibited distribution and metabolism profiles similar to that of orlistat. However,
all other analogues including 12c were found to inhibit a majority of the CYP enzymes in
contrast to orlistat.
For the toxicity profile, various parameters viz., oral toxicity, carcinogenicity and
hepatotoxicity were predicted. As summarised in Table 5, orlistat possessed a predicted LD50
of 1300 mg/kg (Class IV), and was also found to be genotoxic. Further, orlistat was predicted
3
1
to be hepatotoxic, which is in line with the FDA reports. On contrary, analogues 4 and 5 from
the previous study possessed very low LD50 values (380 and 78 mg/kg, respectively), while
also exhibiting hepatotoxicity similar to orlistat and conophylline. Nevertheless, the indolyl
oxoacetamides in the present study were predicted to possess comparatively better toxicity
profile, wherein all the analogues existed in Class IV for oral toxicity similar to orlistat and
conophylline, but did not exhibit any carcinogenicity and hepatotoxicity.
1
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Table 5. Lipophilicity and ADMET profile of the indolyl oxoacetamides
Lipophilicity
Absorption
Distribution
Metabolism
Inhibitor for
CYP2 CYP CYP CYP
Toxicity
Oral
LD50
(mg/kg)
1000
1000
1000
1000
2000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1000
1190
380
Carcinogenicity
Liver
#
Consensus
LogPO/W
2.40
GI
absorption
High
High
High
High
High
High
High
High
High
High
High
High
High
High
High
Low
BBB
P-gp
substrate
No
CYP1
A2
Genotoxic
Non-
genotoxic
No
permeant
No
C19
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
2C9
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
2D6
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
3A4
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
9a
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
No
No
Yes
No
No
No
No
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
9
b, 13b
2.85
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
No
No
1
0a-g
0h
0i
10j
0k
11a
1b
2.70 - 3.78
3.02
No
1
No
No
1
2.69
No
No
2.67
No
No
1
3.19
Yes
No
Yes
No
No
3.57
No
1
3.65
No
No
No
No
1
1c
2a
4.09
Yes
No
No
Yes
No
No
1
5.00
No
No
1
2b
4.97
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
1
2c
3a
5.53
No
Yes
No
Yes
Yes
Yes
No
Yes
Yes
Yes
No
No
1
3.59
No
No
13b
4.15
Yes
No
Yes
No
No
Conophylline
3.13
No
4
5
3.62
High
High
Low
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
No
No
3.56
No
No
No
78
No
Orlistat
7.05
No
Yes
No
1300
No
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3
. Conclusion
A series of 21 indolyl oxoacetamides were designed and synthesized as an attempt to
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
optimize the lead 4 from our previous studies. These analogues were evaluated for their PL
inhibitory activity, wherein analogues 12b and 12c possessed greater potential (IC50 values of
3
.26 and 2.95 µM, respectively), compared to the natural product lead, conophylline (IC50 3.31
µM). Further, five more analogues, 11b, 11c, 12a, 13a and 13b, exhibited potent activity (< 5
µM). The MolDock scores of these analogues exhibited significant correlation to their PL
inhibitory activity (Pearson’s r = 0.7658, p < 0.05), with the most potent analogue 12c
exhibiting potential MolDock score of -157.894 kcal/mol. Further, a considerable improvement
in the lipophilicity, in particular, for the N-geranyl substituted analogues (12a, 12b and 12c),
resulted in extended hydrophobic interactions with the lid domain. Similarly, a heteroaryl ring
(
indol-3-yl), extended through an ethylene linker on the amide, also resulted in enhancement
of the PL inhibitory activity. While the ADMET prediction of these analogues indicated a
possible high GI absorption, the toxicity profile has indicated these analogues to be negative
for carcinogenicity and hepatotoxicity, in contrary to orlistat and conophylline. In conclusion,
the present study resulted in conophylline inspired novel indolyl oxoacetamides with potent
pancreatic lipase inhibitory activity comparable to that of orlistat.
4. Materials and Methods
4
.1. Chemistry
The synthesis of all the final analogues were carried out as per the procedure detailed in Scheme
.10,16,18 Progress of the reactions was followed by thin layer chromatography (TLC) analysis
1
(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
1
13
Shimadzu IRPrestige 21 FTIR machine using KBr pellets. H and C NMR spectra were
recorded on a Bruker Avance II 400 spectrometer (400 MHz and 100 MHz, respectively) using
DMSO-d as solvent. HRMS analysis was performed on Bruker Compass Data Analysis 4.1
6
mass instrument. The purity of the synthesized compounds was determined on a Shimadzu
SIL-20AC HT Ultra-Fast Liquid Chromatograph, equipped with SPD-20A UV detector. All
1
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the compounds were through a Hypersil Gold column (Thermofisher, 250 mm x 4.6 mm with
particle size of 5 µm) and the mobile phase used was Acetonitrile : Water (80 : 20% v/v).
.1.1. General procedure for the synthesis of N-substituted indole (7)
4
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Briefly, 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
(6) was added and the stirring was continued. After 30 minutes, a solution of appropriate halide
(
8.5 mmol in 5 mL DMF) was added dropwise for over 5 minutes to the reaction mixture, and
the stirring was continued overnight. The reaction mixture was then transferred to ice-cold
water. The N-benzyl substituted indole analogues were obtained as crude precipitates, and were
purified by recrystallization. The N-ethyl, N-prenyl and N-geranyl substituted indoles were
obtained as oily liquids, which were extracted using chloroform and purified through column
chromatography.
4.1.2. General procedure for the synthesis of indolyl oxoacetamides
Various indole analogues (6 and 7, 2.5 mmol) were stirred at 0°C for 30 minutes in 5
mL diethylether. To this, 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 (8) 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 indolyl oxoacetamide. The crude
precipitate was then re-crystallized from ethanol to obtain the final compounds in 68-91%
yields.
2
-(1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxybenzyl)acetamide (9a)
1
Yield 81%; Brownish white solid; m.p: 239-241°C; H NMR (400 MHz, DMSO-d
6
) δ 12.25
(
s, 1H, -NH of indole), 9.23 (t, J = 6.4 Hz, 1H, -NH of amide), 8.75 (s, 1H, H-2 of indole),
8
.29-8.20 (m, 1H, H-4 of indole), 7.59-7.50 (m, 1H, H-5 of indole), 7.33-7.22 (m, 2H, H-6,7
linked
); C NMR (100 MHz, DMSO-
) δ 182.7, 164.2, 153.2, 138.8, 136.9, 136.7, 135.0, 126.6, 123.9, 123.0, 121.7, 113.0, 112.6,
of indole), 6.68 (s, 2H, H-2,6 of 3,4,5-trimethoxybenzyl), 4.38 (d, J = 6.3 Hz, 2H, -CH
2
13
to amide), 3.76 (s, 6H, 3 and 5-OCH
3
), 3.64 (s, 3H, 4-OCH
3
d
6
-1
1
05.2, 60.4, 56.2, 42.7; IR (KBr, v, cm ) 3736, 2835, 2355, 2318, 2122, 1751, 1595, 1449,
1
9

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1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
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1
362, 1256, 1134, 988, 739, 689, 511; HRMS (ESI ) calculated for C20
H
21
N
2
O [M+H ],
5
DOI: 10.1039/D0NJ02622K
369.1372; Found 369.1398; HPLC Purity: 98.882%, t
R
= 3.594 min.
2
-(1H-indol-3-yl)-2-oxo-N-(2-(1H-indol-3-yl)ethyl)acetamide (9b)
1
Yield 87%; Creamy white solid; m.p: 221-223°C; H NMR (400 MHz, DMSO-d
H, -NH of indole), 10.86 (s, 1H, -NH of 2-(indol-3-yl)ethyl), 8.86 (t, J = 6.0 Hz, 1H, -NH of
amide), 8.79 (s, 1H, H-2 of indole), 8.25 (dt, J = 8.1, 2.8 Hz, 1H), 7.62 (d, J = 7.8 Hz, 1H),
6
) δ 12.22 (s,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
7
.55 (dt, J = 8.0, 2.9 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.33-7.19 (m, 3H), 7.09 (t, J = 7.5 Hz,
H), 7.00 (t, J = 7.4 Hz, 1H), 3.57-3.50 (m, 2H, -CH linked to amide), 2.97 (t, J = 7.6 Hz, 2H,
of 2-(indol-3-yl)ethyl); C NMR (100 MHz, DMSO-d ) δ 182.6, 163.9, 139.0, 136.7,
127.6, 126.7, 123.8, 123.1, 122.9, 121.7, 118.7, 113.0, 112.6, 112.0, 111.8, 39.8, 39.7, 39.5,
1
2
1
3
-CH
2
6
-1
3
8
9.3, 25.3; IR (KBr, v, cm ) 3611, 2922, 2355, 2324, 1792, 1412, 1327, 1256, 1128, 1007,
+
+
26, 810, 673; HRMS (ESI ) calculated for C20
= 3.626 min.
H
18
N
3
O [M+H ], 332.1321; Found 332.1398,
2
HPLC Purity: 95.639%, t
R
2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (10a)
1
Yield 87%; Creamy white solid; m.p: 111-113°C; H NMR (400 MHz, DMSO-d
6
) δ 10.71 (s,
1
H, -NH of amide), 8.85 (s, 1H, H-2 of indole), 8.37-8.28 (m, 1H, H-4 of indole), 7.88 (d, J =
7.4 Hz, 2H, H-3,5 of phenyl), 7.74-7.64 (m, 1H, H-5 of indole), 7.44-7.30 (m, 4H), 7.15 (t, J =
7
.4 Hz, 1H, H-4 of phenyl), 4.38 (q, J = 7.2 Hz, 2H, -CH of ethyl), 1.43 (t, J = 7.2 Hz, 3H, -
2
13
CH
3
of ethyl); C NMR (100 MHz, DMSO-d
6
) δ 181.9, 162.8, 140.9, 138.5, 136.5, 129.2,
-1
1
3
27.3, 124.7, 124.1, 123.5, 121.9, 120.6, 111.7, 111.4, 41.9, 15.6; IR (KBr, v, cm ): 3823,
291, 2689, 2355,2106, 1701, 1555, 1396, 1312, 1219, 1165, 866, 689; HRMS (ESI+)
+
calculated for C18
.756 min.
H
17
N O
2 2
[M+H ], 293.1212; Found 293.1278, HPLC Purity: 98.325%, t
R
=
4
2-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(p-tolyl)acetamide (10b)
1
Yield 84%; Creamy white solid; m.p: 160-162°C; H NMR (400 MHz, DMSO-d
6
) δ 10.53 (s,
1
H, -NH of amide), 8.84 (s, 1H, H-2 of indole), 8.37-8.28 (m, 1H, H-4 of indole), 7.79-7.72
m, 2H, H-5,6 of indole), 7.72-7.63 (m, 1H, H-7 of indole), 7.41-7.29 (m, 2H, H-2,6 of 4-
methylphenyl), 7.19 (d, J = 8.2 Hz, 2H, H-3,5 of 4-methylphenyl), 4.38 (q, J = 7.2 Hz, 2H, -
(
13
CH
2
of ethyl), 2.30 (s, 3H, 4-Me of phenyl), 1.44 (t, J = 7.2 Hz, 3H, -CH
3
of ethyl); C NMR
(
100 MHz, DMSO-d ) δ 182.0, 162.5, 140.9, 136.5, 136.0, 133.7, 129.5, 127.3, 124.0, 123.5,
6
-1
1
22.0, 120.6, 111.6, 111.5, 41.9, 15.5, 15.5; IR (KBr, v, cm ): 3792, 3277, 2978, 2405, 2318,
2
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935, 1838, 1614, 1377, 1312, 1219, 1043, 957, 829; HRMS (ESI ) calculated for C19
H
19
N
2
O
2
+
[M+H ], 307.1368; Found 307.1434, HPLC Purity: 98.236%, t
R
= 5.156 min.
2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(3,4-dimethylphenyl)acetamide (10c)
1
Yield 81%; Creamy yellow solid; m.p: 147-149°C; H NMR (400 MHz, DMSO-d
H), 8.86 (s, 1H), 8.36-8.27 (m, 1H), 7.73-7.64 (m, 2H), 7.56 (dd, J = 8.1, 2.3 Hz, 1H), 7.41-
7.29 (m, 2H), 7.12 (d, J = 8.2 Hz, 1H), 4.38 (q, J = 7.2 Hz, 2H), 2.23 (s, 3H), 2.20 (s, 3H), 1.43
6
) δ 10.53 (s,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
3
(t, J = 7.2 Hz, 3H); C NMR (100 MHz, DMSO-d
6
) δ 182.0, 162.5, 140.9, 136.8, 136.5, 136.2,
1
32.5, 130.0, 127.3, 124.0, 123.5, 121.9, 121.7, 118.1, 111.6 , 111.4, 41.9, 20.1, 19.3, 15.6; IR
-1
(
KBr, v, cm ): 3686, 3312, 2355, 2299, 2106, 1680, 1574, 1504, 1381, 1219, 1184, 991, 789,
+
+
602; HRMS (ESI ) calculated for C20
Purity: 98.127%, t = 5.639 min.
H
21
N
2
O [M+H ], 321.1525; Found 321.1599, HPLC
2
R
2
-(1-ethyl-1H-indol-3-yl)- 2-oxo-N-(4-methoxyphenyl)acetamide (10d)
1
Yield 73%; Yellowish green solid; m.p: 178-180°C; H NMR (400 MHz, DMSO-d
6
) δ 10.51
(
s, 1H, -NH of amide), 8.89 (s, 1H, H-2 of indole), 8.34-8.26 (m, 1H, H-4 of indole), 7.73 (d,
J = 8.4 Hz, 2H, H-2,6 of 4-methoxyphenyl), 7.72-7.63 (m, 1H, H-5 of indole), 7.41-7.29 (m,
2
H, H-6,7 of indole), 7.19 (d, J = 8.2 Hz, 2H, H-3,5 of 4-methoxyphenyl), 4.37 (q, J = 7.1 Hz,
1
3
2H, -CH
2
of ethyl), 3.80 (s, 3H, -OCH
3
), 1.44 (t, J = 7.1 Hz, 3H, -CH of ethyl); C NMR (100
3
MHz, DMSO-d
6
) δ 182.0, 162.2, 156.3, 140.9, 136.4, 131.6, 127.3, 124.0, 123.5, 122.1, 122.0,
-1
1
1
14.3, 111.6, 111.5, 55.6, 41.9, 15.5; IR (KBr, v, cm ): 3645, 3291, 2830, 2355, 2318, 1711,
+
595, 1381, 1222, 1165, 1097, 1022, 851, 799, 673, 440; HRMS (ESI ) calculated for
+
C
19
H
19
N
2
O
3
[M+H ], 323.1317; Found 323.1386, HPLC Purity: 98.302%, t
R
= 4.533 min.
2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(4-methoxybenzyl)acetamide (10e)
1
Yield 68%; Creamy white solid; m.p: 90-93°C; H NMR (400 MHz, DMSO-d
6
) δ 10.56 (t, J =
6.2 Hz, 1H, -NH of amide), 8.87 (s, 1H, H-2 of indole), 8.37-8.24 (m, 1H, H-4 of indole), 7.68
(
d, J = 8.4 Hz, 2H, H-2,6 of 4-methoxybenzyl), 7.72-7.63 (m, 1H, H-5 of indole), 7.41-7.29
m, 2H, H-6,7 of indole), 7.17 (d, J = 8.2 Hz, 2H, H-3,5 of 4-methoxyphenyl), 4.48 (d, J = 6.1
(
Hz, 2H, -CH
2
- linked to amide), 4.37 (q, J = 7.1 Hz, 2H, -CH
2
of ethyl), 3.80 (s, 3H, 4-OCH
3
),
13
1.44 (t, J = 7.1 Hz, 3H, -CH
3
of ethyl); C NMR (100 MHz, DMSO-d ) δ 182.0, 163.8, 158.7,
6
1
1
8
3
40.8, 136.4, 131.4, 129.2, 127.3, 123.9, 123.3, 122.0, 114.1, 111.6, 111.5, 55.5, 41.9, 41.8,
-1
5.5; IR (KBr, v, cm ): 3736, 3343, 2355, 2334, 1842, 1736, 1504, 1468, 1362, 1184, 1028,
+
+
26, 673, 530, 440; HRMS (ESI ) calculated for C20
37.1535, HPLC Purity: 87.899%, t = 4.282 min.
H
21
N
2
O [M+H ], 337.1474; Found
3
R
2
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1
1
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
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-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(4-bromophenyl)acetamide (10f)
1
Yield 79%; Light yellow solid; m.p: 178-180°C; H NMR (400 MHz, DMSO-d
6
) δ 10.87 (s,
1
H, -NH of amide), 8.84 (s, 1H, H-2 of indole), 8.36-8.27 (m, 1H, H-4 of indole), 7.86 (d, J =
8
.9 Hz, 2H, H-2,6 of 4-bromophenyl), 7.74-7.64 (m, 1H, H-5 of indole), 7.58 (d, J = 8.9 Hz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
H, H-3,5 of 4-bromophenyl), 7.41-7.30 (m, 2H, H-6,7 of indole), 4.38 (q, J = 7.2 Hz, 2H, -
13
CH
2
of ethyl), 1.43 (t, J = 7.2 Hz, 3H, -CH
3
of ethyl); C NMR (100 MHz, DMSO-d ) δ 181.5,
6
1
62.8, 141.0, 137.9, 132.0, 127.3, 124.1, 123.6, 119.2, 116.5, 111.7, 100.8, 41.9, 15.5; IR
-1
(
KBr, v, cm ): 3611, 3312, 2922, 2355, 2106, 1857, 1686, 1524, 1418, 1381, 1240, 1150, 1007,
+
+
7
73, 667; HRMS (ESI ) calculated for C18
= 5.605 min.
H16BrN
2
O [M+H ], 371.0317; Found 371.0377,
2
HPLC Purity: 86.783%, t
R
2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(3,4-dichlorophenyl)acetamide (10g)
1
Yield 91%; Light yellow solid; m.p: 162-163°C; H NMR (400 MHz, DMSO-d
6
) δ 11.05 (s,
1H, -NH of amide), 8.88 (s, 1H, H-2 of indole), 8.35-8.26 (m, 2H, H-4,5 of indole), 7.84 (dd,
J = 8.9, 2.4 Hz, 1H, H-6 of indole), 7.75-7.61 (m, 2H, H-7 of indole and H-2 of 3,4-
dichlorophenyl), 7.42-7.30 (m, 2H, H-5,6 of 3,4-dichlorophenyl), 4.39 (q, J = 7.2 Hz, 2H, -
13
CH
2
of ethyl), 1.43 (t, J = 7.2 Hz, 3H, -CH
3
of ethyl); C NMR (100 MHz, DMSO-d ) δ 181.0,
6
162.9, 141.2, 138.6, 136.5, 131.4, 131.1, 127.3, 126.2, 124.2, 123.6, 121.9, 121.9, 120.8, 111.7,
-1
1
1
3
11.2, 41.9, 15.6; IR (KBr, v, cm ): 3844, 3567, 3327, 2972, 2355, 2112, 1695, 1574, 1402,
+
[M+H+],
219, 1134, 1007, 935, 814, 667; HRMS (ESI ) calculated for C18
H
15Cl
2
N
2
O
2
61.0432; Found 361.0493, HPLC Purity: 90.746%, t = 6.451 min.
R
2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(4-fluorophenyl)acetamide (10h)
1
Yield 91%; Brown solid; m.p: 150-152°C; H NMR (400 MHz, DMSO-d
6
) δ 10.81 (s, 1H, -
NH of amide), 8.85 (s, 1H, H-2 of indole), 8.36-8.27 (m, 1H, H-4 of indole), 7.96 – 7.85 (m,
2H, H-5,6 of indole), 7.74-7.65 (m, 1H, H-7 of indole), 7.41-7.30 (m, 2H, H-2,6 of 4-
fluorophenyl), 7.29-7.18 (m, 2H, H-3,5 of 4-fluorophenyl), 4.38 (q, J = 7.2 Hz, 2H, -CH of
2
13
ethyl), 1.43 (t, J = 7.2 Hz, 3H, -CH
3
of ethyl); C NMR (100 MHz, DMSO-d
6
) δ 181.8, 162.6,
1
60.2, 157.8, 141.0, 136.5, 134.9, 127.3, 124.1, 123.5, 122.5, 121.9, 115.9, 111.7, 41.9, 15.6;
-1
IR (KBr, v, cm ) 3879, 3611, 3331, 2922, 2355, 2318, 2087, 1857, 1807, 1611, 1412, 1260,
+
+
1
3
113, 1057, 814, 652; HRMS (ESI ) calculated for C18
11.1176, HPLC Purity: 91.492%, t = 4.718 min.
H16FN
2
O [M+H ], 311.1118; Found
2
R
2
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2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide (10i)
1
Yield 84%; Creamy yellow solid; m.p: 153-155°C; H NMR (400 MHz, DMSO-d
6
) δ 10.57 (s,
1
H, -NH of amide), 8.93 (s, 1H, H-2 of indole), 8.38-8.29 (m, 1H, H-4 of indole), 7.73-7.65
(m, 1H, H-5 of indole), 7.40-7.32 (m, 4H), 4.40 (q, J = 7.2 Hz, 2H, -CH
2
of ethyl), 3.79 (s, 6H,
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
13
3
and 5-OCH
3
), 3.34 (s, 3H, 4-OCH
3
), 1.43 (t, J = 7.2 Hz, 3H, -CH
3
of ethyl); C NMR (100
MHz, DMSO-d ) δ 181.4, 162.2, 153.1, 141.1, 136.4, 135.6, 134.6, 127.4, 124.1, 123.5, 122.0,
6
-1
1
1
11.6, 111.3, 98.4, 60.6, 56.2, 41.9, 15.6; IR (KBr, v, cm ) 3611, 3312, 2340, 2318, 2106,
+
736, 1645,1574, 1412, 1234, 1128, 1007, 829, 652, 633; HRMS (ESI ) calculated for
+
C
21
H
23
N
2
O
5
[M+H ], 383.1529; Found 383.1600, HPLC Purity: 90.447%, t
R
= 4.404 min.
2-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxybenzyl)acetamide (10j)
1
Yield 77%; Brownish white solid; m.p: 145-147°C; H NMR (400 MHz, DMSO-d
6
) δ 9.24 (t,
J = 6.3 Hz, 1H, -NH of amide), 8.84 (s, 1H, H-2 of indole), 8.27 (dd, J = 6.5, 1.9 Hz, 1H, H-5
of indole), 7.66 (dd, J = 6.6, 1.5 Hz, 1H, H-6 of indole), 7.39-7.26 (m, 2H, H-4,7 of indole),
6
.68 (s, 2H, H-2,6 of 3,4,5-trimthoxybenzyl), 4.41-4.28 (m, 4H, -CH
CH of ethyl), 3.76 (s, 6H, 3 and 5-OCH ), 3.64 (s, 3H, 4-OCH ), 1.41 (t, J = 7.2 Hz, 3H, -CH
of ethyl).; C NMR (100 MHz, DMSO-d ) δ 182.1, 164.0, 153.2, 140.7, 136.9, 136.4, 135.0,
127.3, 123.9, 123.4, 122.0, 111.7, 111.5, 105.2, 60.4, 56.2, 42.7, 41.8, 15.5; IR (KBr, v, cm )
723, 3601, 2901, 2355, 2318, 2106, 1717, 1589, 1396, 1240, 1184, 1128, 1007, 810, 689;
2
linked to amide and -
2
3
3
3
1
3
6
-1
3
+
+
HRMS (ESI ) calculated for C22
H
25
N
2
O [M+H ], 397.1685; Found 397.1755, HPLC Purity:
5
9
4.475%, t = 4.053 min.
R
2
-(1-ethyl-1H-indol-3-yl)-2-oxo-N-(2-(1H-indol-3-yl)ethyl)acetamide (10k)
1
Yield 80%; Creamy white solid; m.p: 168-170°C; H NMR (400 MHz, DMSO-d
6
) δ 10.86 (s,
1
H, -NH of 2-(indol-3-yl)ethyl), 8.86 (t, J = 6.0 Hz, 1H, -NH of amide), 8.82 (s, 1H, H-2 of
indole), 8.32-8.21 (m, 1H), 7.70-7.58 (m, 2H), 7.39-7.26 (m, 3H), 7.21 (d, J = 2.3 Hz, 1H),
7
2
.08 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.00 (ddd, J = 8.0, 7.0, 1.1 Hz, 1H), 4.35 (q, J = 7.3 Hz,
H, -CH of N-ethyl), 3.58-3.47 (m, 2H, -CH linked to amide), 2.96 (t, J = 7.5 Hz, 2H, -CH
2
2
2
1
3
of 2-(indol-3-yl)ethyl), 1.41 (t, J = 7.2 Hz, 3H, -CH
3
of N-ethyl); C NMR (100 MHz, DMSO-
d
1
2
6
) δ 182.2, 163.9, 140.8, 136.7, 136.4, 127.6, 127.3, 123.9, 123.3, 123.1, 122.0, 121.4, 118.7,
-1
18.7, 112.0, 111.8, 111.6, 111.5, 41.8, 39.8, 25.3, 15.6; IR (KBr, v, cm ) 3611, 2922, 2355,
+
324, 1792, 1412, 1327, 1256, 1128, 1007, 826, 810, 673; HRMS (ESI ) calculated for
+
C
22
H
22
N
3
O
2
[M+H ], 360.1634; Found 360.1710, HPLC Purity: 92.347%, t
R
= 4.180 min.
2
3

New Journal of Chemistry
Page 24 of 32
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7
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
View Article Online
DOI: 10.1039/D0NJ02622K
2
-(1-(3-methylbut-2-en-1-yl)-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide
(11a)
1
Yield 74%; Creamy yellow solid; m.p: 172-174°C; H NMR (400 MHz, DMSO-d
H, -NH of amide), 8.89 (s, 1H, H-2 of indole), 8.36-8.28 (m, 1H, H-4 of indole), 7.64-7.55
m, 1H, H-5 of indole), 7.39-7.30 (m, 4H), 5.43-5.35 (m, 1H), 4.98 (d, J = 7.0 Hz, 2H, -N-
CH ), 3.78 (s, 6H, 3- and 5-OCH ), 3.66 (s, 3H, 4-OCH ), 1.86 (s, 3H, -CH of N-prenyl), 1.74
of N-prenyl); C NMR (100 MHz, DMSO-d ) δ 181.4, 162.1, 153.1, 141.2, 137.4,
36.6, 134.6, 134.5, 127.5, 124.1, 123.6, 122.0, 119.6, 111.9, 111.3, 98.4, 60.6, 56.2, 45.1,
6
) δ 10.57 (s,
1
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
2
3
3
3
1
3
(s, 3H, -CH
3
6
1
2
-1
5.8, 18.4; IR (KBr, v, cm ) 3752, 2922, 2355, 2318, 2016, 1751, 1686, 1518, 1377, 1256,
+
+
1128, 1057, 829, 789, 683; HRMS (ESI ) calculated for C24
H
27
N
2
O [M+H ], 423.1842; Found
5
4
23.1910, HPLC Purity: 84.224%, t
R
= 5.326 min.
2
-(1-(3-methylbut-2-en-1-yl)-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxybenzyl)acetamide
(11b)
Yield 79%; White solid; m.p: 179-181°C; H NMR (400 MHz, DMSO-d
Hz, 1H, -NH of amide), 8.80 (s, 1H, H-2 of indole), 8.32-8.24 (m, 1H, H-4 of indole), 7.62-
.53 (m, 1H, H-5 of indole), 7.32 (ddd, J = 6.5, 4.0, 1.7 Hz, 2H, H-6,7 of indole), 6.67 (s, 2H,
H-2,6 of 3,4,5-trimethoxybenzyl), 5.38 (t, J = 7.0 Hz, 1H, -C=CH- of N-prenyl), 4.93 (d, J =
.0 Hz, 2H, -CH linked to amide), 4.36 (d, J = 6.3 Hz, 2H, -N-CH - of prenyl), 3.76 (s, 6H, 3
and 5-OCH ), 3.64 (s, 3H, 4-OCH ), 1.84 (s, 3H, -CH of N-prenyl), 1.74 (s, 3H, -CH of N-
prenyl); C NMR (100 MHz, DMSO-d ) δ 182.0, 164.0, 153.2, 140.8, 137.7, 136.8, 136.6,
35.0, 127.3, 123.9, 123.4, 122.0, 119.4, 111.7, 111.6, 105.1, 60.4, 56.2, 44.9, 42.7, 25.8, 18.4;
1
6
) δ 9.25 (t, J = 6.3
7
7
2
2
3
3
3
3
1
3
6
1
-1
IR (KBr, v, cm ) 3773, 3474, 2922, 2355, 2318, 2127, 1680, 1412, 1256, 1165, 1134, 1007,
+
+
8
10, 689; HRMS (ESI ) calculated for C25
= 4.704 min.
H N
29 2
O [M+H ], 437.1998; Found 437.2074,
5
HPLC Purity: 89.895%, t
R
2
-(1-(3-methylbut-2-en-1-yl)-1H-indol-3-yl)-2-oxo-N-(2-(1H-indol-3-yl)ethyl)acetamide
(11c)
1
Yield 83%; Light brown solid; m.p: 187-189°C; H NMR (400 MHz, DMSO-d
6
) δ 10.73 (s,
1H, -NH of 2-(indol-3-yl)ethyl), 8.89 (t, J = 6.1 Hz, 1H, -NH of amide), 8.76 (s, 1H, H-2 of
indole), 8.32-8.21 (m, 1H), 7.72 (dd, J = 7.4, 1.5 Hz, 1H), 7.53 (dd, J = 7.4, 1.5 Hz, 1H), 7.31-
7
7
.22 (m, 3H), 7.21 (d, J = 2.3 Hz, 1H), 7.08 (ddd, J = 8.1, 6.9, 1.2 Hz, 1H), 7.00 (ddd, J = 8.0,
.0, 1.1 Hz, 1H), 5.37-5.29 (m, 1H, -N-C-CH= of prenyl), 4.93 (d, J = 7.0 Hz, 2H, -N-CH of
linked to amide), 2.96 (t, J = 7.5 Hz, 2H, -CH of 2-(indol-3-
2
prenyl), 3.58-3.47 (m, 2H, -CH
2
2
2
4

Page 25 of 32
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13
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yl)ethyl), 1.84 (s, 3H, -CH
DMSO-d ) δ 182.0, 163.7, 160.4, 140.9, 137.6, 136.7, 136.6, 127.6, 127.6, 127.4, 123.9, 123.3,
23.1, 123.0, 122.0, 121.4, 119.5, 118.7, 112.0, 44.9, 39.7, 25.8, 25.3, 25.2, 18.4; IR (KBr, v,
3
of N-prenyl), 1.74 (s, 3H, -CH
3
of N-prenyl); C NMR (100 MHz,
DOI: 10.1039/D0NJ02622K
6
1
-
1
cm ) 3595, 3299, 2355, 2318, 1611, 1499, 1381, 1256, 1128, 972, 814, 773, 689, 530; HRMS
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
+
+
(
ESI ) calculated for C25
H
26
N
3
O [M+H ], 400.1947; Found 400.2017, HPLC Purity:
2
94.230%, t
R
= 8.520 min.
(
E)-2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphen
yl)acetamide (12a)
Yield 80%; Cream yellow solid; m.p: 86-88°C; H NMR (400 MHz, DMSO-d
-NH of amide), 8.90 (s, 1H, H-2 of indole), 8.37-8.28 (m, 1H, H-4 of indole), 7.62-7.53 (m,
1
6
) δ 10.57 (s, 1H,
1
H, H-5 of indole), 7.39-7.30 (m, 4H, H-6,7 of indole and H-2,6 of 3,4,5-trimethoxyphenyl),
.37 (t, J = 6.8 Hz, 1H, -N-C-CH=), 5.06-4.95 (m, 3H), 3.78 (s, 6H, 3 and 5-OCH ), 3.65 (s,
), 2.11-1.99 (m, 4H), 1.85 (s, 3H, -CH of N-geranyl), 1.55 (s, 3H, terminal -CH
of N-geranyl), 1.51 (s, 3H, terminal -CH of N-geranyl); C NMR (100 MHz, DMSO-d
5
3
3H, 4-OCH
3
3
3
1
3
3
6
) δ
1
1
81.4, 162.1, 153.1, 141.2, 140.8, 136.7, 134.5, 131.5, 127.5, 124.0, 123.6, 121.9, 119.5, 111.9,
-1
11.3, 99.9, 98.6, 98.4, 60.5, 56.2, 56.1, 45.0, 26.1, 25.8, 17.9, 16.6; IR (KBr, v, cm ) 3574,
3240, 2371, 2318, 1807, 1651, 1412, 1346, 1256, 1128, 1022, 845, 758, 637; HRMS (ESI+)
+
calculated for C29
.928 min.
H
35
2
N O
5
[M+H ], 491.2468; Found 491.2525, HPLC Purity: 79.412%, t
R
=
8
(
E)-2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxybenzyl)
acetamide (12b)
Yield 72%; Yellowish white solid; m.p: 72-74°C; H NMR (400 MHz, DMSO-d
1
6
) δ 9.26 (t, J
=
6.3 Hz, 1H, -NH of amide), 8.81 (s, 1H, H-2 of indole), 8.32-8.24 (m, 1H, H-4 of indole),
7.62- 7.53 (m, 1H, H-5 of indole), 7.36-7.31 (m, 2H, H-6,7 of indole), 6.66 (s, 2H, H-2,6 of
,4,5-trimethoxybenzyl), 5.06-4.95 (m, 3H, -N-CH -CH= of geranyl), 4.36 (d, J = 6.3 Hz, 2H,
CH linked to amide), 3.78 (s, 6H, 3 and 5-OCH ), 3.65 (s, 3H, 4-OCH ), 2.11-1.99 (m, 2H),
.06-2.01 (m, 3H), 1.87 (s, 3H, -CH of N-geranyl), 1.56 (s, 3H, terminal -CH of N-geranyl),
of N-geranyl); C NMR (100 MHz, DMSO-d ) δ 182.0, 163.9,
53.2, 140.9, 140.9, 136.8, 136.6, 135.0, 131.5, 127.4, 124.0, 123.9, 123.4, 121.9, 119.4, 111.8,
3
2
-
2
3
3
2
3
3
1
3
1.52 (s, 3H, terminal -CH
3
6
1
1
2
-1
11.6, 105.1, 60.4, 56.2, 44.9, 42.7, 26.1, 25.8, 17.9, 16.6; IR (KBr, v, cm ) 3736, 3331, 2851,
+
318, 1751, 1611, 1504, 1472, 1377, 1234, 1134, 922, 814, 633; HRMS (ESI ) calculated for
+
C
30
H
37
N
2
O
5
[M+H ], 505.2624; Found 505.2695, HPLC Purity: 94.219%, t
R
= 7.311 min.
2
5

New Journal of Chemistry
Page 26 of 32
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1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
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5
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View Article Online
DOI: 10.1039/D0NJ02622K
(
E)-2-(1-(3,7-dimethylocta-2,6-dien-1-yl)-1H-indol-3-yl)-2-oxo-N-(2-(1H-indol-3-yl)ethyl)
acetamide (12c)
Yield 69%; Light brown solid; m.p: 93-95°C; H NMR (400 MHz, DMSO-d
1
6
) δ 10.87 (s, 1H,
-
NH of 2-(indol-3-yl)ethyl), 8.86 (t, J = 6.0 Hz, 1H, -NH of amide), 8.82 (s, 1H, H-2 of indole),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
.31-8.22 (m, 1H), 7.61 (d, J = 7.9 Hz, 1H), 7.58-7.53 (m, 1H), 7.39-7.25 (m, 3H), 7.20 (d, J
= 2.3 Hz, 1H), 7.12-7.02 (m, 1H), 6.99 (td, J = 7.5, 7.0, 1.1 Hz, 1H), 5.36 (t, J = 6.6 Hz, 1H, -
N-C-CH= of geranyl), 5.07-4.98 (m, 1H), 4.94 (d, J = 7.0 Hz, 2H, -N-CH of geranyl), 3.51
dt, J = 8.1, 6.4 Hz, 2H, -CH linked to amide), 2.95 (t, J = 7.6 Hz, 2H, -CH
yl)ethyl), 2.11-2.01 (m, 4H), 1.84 (s, 3H, -CH of N-geranyl), 1.56 (s, 3H, -CH
of N-geranyl); C NMR (100 MHz, DMSO-d
36.7, 136.6, 131.5, 127.6, 127.4, 125.3, 124.0, 123.8, 123.3, 123.1, 122.0, 121.4, 119.4, 118.7,
2
(
2
2
of 2-(indol-3-
of N-geranyl),
3
3
1
3
1.52 (s, 3H, -CH
3
6
) δ 182.0, 163.7, 141.0, 140.8,
1
1
-1
12.0, 111.8, 111.7, 111.6, 44.9, 34.8, 30.8, 26.7, 25.8, 25.3, 18.0, 16.6; IR (KBr, v, cm ) 3630,
3296, 2866, 2355, 2303, 1802, 1696, 1362, 1256, 1128, 1007, 951, 814, 779, 673; HRMS
+
+
(ESI ) calculated for C30
H
34
N
3
O [M+H ], 468.2573; Found 468.2642, HPLC Purity:
2
9
5.948%, t = 7.847 min.
R
2
-(1-benzyl-1H-indol-3-yl)-2-oxo-N-(3,4,5-trimethoxybenzyl)acetamide (13a)
1
Yield 73%; Yellowish white solid; m.p: 167-169°C; H NMR (400 MHz, DMSO-d
6
) δ 9.27 (t,
J = 6.3 Hz, 1H, -NH of amide), 8.99 (s, 1H, H-2 of indole), 8.33-8.24 (m, 1H, H-4 of indole),
.65-7.56 (m, 1H, H-5 of indole), 7.39-7.24 (m, 7H, H-6,7 of indole and aromatic protons of
N-benzyl), 6.68 (s, 2H, H-2,6 of 3,4,5-trimethoxybenzyl), 5.60 (s, 2H, -CH of N-benzyl), 4.38
d, J = 6.3 Hz, 2H, -CH linked to amide), 3.76 (s, 6H, 3 and 5-OCH ), 3.64 (s, 3H, 4-OCH );
13C NMR (100 MHz, DMSO-d
) δ 182.3, 163.9, 153.2, 141.6, 137.1, 136.9, 136.7, 135.7,
35.0, 129.2, 128.2, 127.8, 127.3, 124.1, 123.4, 122.0, 112.0, 105.2, 60.4, 56.2, 50.2, 42.7; IR
7
2
(
2
3
3
6
1
-1
(KBr, v, cm ) 3736, 3418, 2953, 2374, 2318, 1857,1792, 1464, 1306, 1128, 1043, 814, 795,
+
+
4
74; HRMS (ESI ) calculated for C27
H
27
N
2
O [M+H ], 459.1842; Found 459.1907, HPLC
5
Purity: 98.649%, t = 4.402 min.
R
2
-(1-benzyl-1H-indol-3-yl)-2-oxo-N-(2-(1H-indol-3-yl)ethyl)acetamide (13b)
1
Yield 76%; Brown solid; m.p: 187-189°C; H NMR (400 MHz, DMSO-d
6
) δ 10.73 (s, 1H, -
NH of 2-(indol-3-yl)ethyl), 9.07 (s, 1H, -NH of amide), 8.89 (s, 1H, H-2 of indole), 8.69 (dd,
J = 7.4, 1.4 Hz, 1H), 7.62 (dd, J = 7.5, 1.5 Hz, 1H), 7.53 (dd, J = 7.3, 1.5 Hz, 1H), 7.38-7.23
(m, 7H), 7.16 (s, 1H), 7.06 (m, 2H), 7.02-6.94 (m, 1H), 5.56 (s, 2H, -CH
2
of N-benzyl), 3.49
(
t, J = 5.4 Hz, 2H, -CH linked to amide), 2.81 (t, J = 5.4 Hz, 2H, -CH of 2-(indol-3-yl)ethyl);
2
2
2
6

Page 27 of 32
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5
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9
13C NMR (100 MHz, DMSO-d
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6
) δ 183.0, 165.7, 137.2, 136.5, 135.0, 134.6, 129.5, 128.0,
DOI: 10.1039/D0NJ02622K
127.6, 126.7, 123.9, 122.6, 122.2, 122.1, 121.2, 119.4, 118.7, 115.9, 112.5, 111.4, 110.8, 51.5,
-
1
4
8
0.4, 26.0; IR (KBr, v, cm ) 3611, 2922, 2355, 2324, 1792, 1412, 1327, 1256, 1128, 1007,
+
+
26, 810, 673; HRMS (ESI ) calculated for C27
= 4.566 min.
H
24
N
3
O [M+H ], 422.1790; Found 422.1862,
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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4
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5
6
0
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0
HPLC Purity: 79.639%, t
R
4.2. 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
procured from Sisco Research Laboratories (MH, India). All other chemicals and solvents
(analytical grade) were used without further purification.
The procedure for PL inhibition assay and kinetics was performed as per the protocol
19
standardized in our laboratory. 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 the 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 the 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 the 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 the percentage inhibition was calculated using the
formula
%
inhibition = [1-( A
T
⁄ A
E
)] x 100
where A
E
is the absorbance of enzyme control (without inhibitor), and A is the difference
T
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
of 12b and 12c (0, 5 and 10 μM) and substrate (25, 50, 100 and 200 µM), and a double
32
reciprocal Lineweaver-Burk plot was plotted to understand the nature of inhibition. The
, was calculated from Cheng-Prusoff equation.22
inhibition constant, K
i
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DOI: 10.1039/D0NJ02622K
4
.3. Molecular modelling studies
Molecular docking of the indolyl oxoacetamides was performed using Molegro Virtual
3
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Docker 6.0. Prior to docking, all the ligands were subjected to energy minimization using
Molecular Mechanics 2 (MM2) force field in Chem3D module of ChemBioOffice v12
0
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(
PerkinElmer, USA). The crystal structure of Human PL (PDB ID: 1LPB) was retrieved from
2
3
the RCSB PDB Data bank, and the energy minimized ligands were docked in to the active
site using previously validated grid parameters.16
Molecular dynamics (MD) simulation of compound 12c in complex with PL was
performed as per the protocol standardized in our laboratory.10,16 Gromacs 5.0.4, compiled on
a CentOS 7 operating system equipped with Intel(R) Xeon(R) CPU W3565 and NVIDIA
Quadro 4000 Quad-Core Processor was used for the purpose of MD simulation.34
35
CHARMM27 force field was applied during the MD run, and the topology of the ligand 12c
3
6
was generated using online tool provided by Swiss Institute of Bioinformatics. Prior to the
initiation of the 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 the
canonical NVT and NPT ensembles. Parameters like Particle Mesh Ewald method for long-
3
7
range electrostatics, and 14 Å cut-off for van der Waals and columbic interactions were set
during the MD simulation. LINCS algorithm was applied for the calculation of bond length.38
Discovery Studio 4.5 visualizer (Accelrys, USA) was used to depict the graphical
representations of the complex.
4
.4. ADMET prediction
The in silico ADMET prediction of the synthesized analogues alongside orlistat (1),
conophylline (2) and the indolyl oxoacetamides (4 and 5) was generated using various online
tools and freeware viz., SwissADME, ProTox-II and ToxTree (v 3.1.0). Parameters such as
consensus LogPO/W, GI absorption, blood-brain barrier (BBB) permeability and effect on CYP
27
enzymes were predicted using SwissADME. Toxicity parameters including oral toxicity
predicted as LD50) and Hepatotoxicity were predicted using ProTox-II,28 while
(
carcinogenicity parameters viz., Genotoxicity and Non-genotoxicity were predicted using
ToxTree.29
5
. Acknowledgements
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DOI: 10.1039/D0NJ02622K
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 HR-MS 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 fellowship (SRF - File No: 09/719 (0088)/2018-EMR-I).
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6. Conflicts of Interest: There are no conflicts to declare.
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