Synthesis, evaluation and molecular modelling studies of 2-(carbazol-3-yl)-2-oxoacetamide analogues as a new class of potential pancreatic lipase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.11.031

A series of twenty four 2-(carbazol-3-yl)-2-oxoacetamide analogues were synthesized, characterized and evaluated for their pancreatic lipase (PL) inhibitory activity. Porcine PL was used against 4-nitrophenyl butyrate (method A) and tributyrin (methods B and C) as substrates during the PL inhibition assay. Compounds 7e, 7f and 7p exhibited potential PL inhibitory activity (IC₅₀values of 6.31, 8.72 and 9.58 μM, respectively in method A; and X_i50of 21.85, 21.94 and 26.2, respectively in method B). Further, inhibition kinetics of 7e, 7f and 7p against PL, using method A, revealed their competitive nature of inhibition. A comparison of the inhibition profiles of the top three compounds in methods B and C, provided a preliminary idea of covalent bonding of the compounds with Ser 152 of PL. Molecular docking studies of the compounds 7a–x into the active site of human PL (PDB ID: 1LPB) was in agreement with the in vitro results, and highlighted probable covalent bond formation with Ser 152 apart from hydrophobic interactions with the lid domain. Molecular dynamics simulation of 7e complexed with PL, further confirmed the role of aromatic groups in stabilising the ligand (RMSD ? 4 ?). The present study led to the identification of 2-(carbazol-3-yl)-2-oxoacetamide analogues 7a–x as a new class of potential PL inhibitors.

Full text of DOI:10.1016/j.bmc.2016.11.031

Accepted Manuscript
Synthesis, Evaluation and Molecular Modelling Studies of 2-(Carbazol-3-yl)-2-
oxoacetamide Analogues as a New Class of Potential Pancreatic Lipase Inhib-
itors
S.N.C. Sridhar, Ginson George, P.O. Venkataramana Reddy, Mukund P.
Tantak, Dalip Kumar, Atish T. Paul
PII:
S0968-0896(16)31220-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.031
BMC 13398
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
9 September 2016
16 November 2016
17 November 2016
Please cite this article as: Sridhar, S.N.C., George, G., Venkataramana Reddy, P.O., Tantak, M.P., Kumar, D., Paul,
A.T., Synthesis, Evaluation and Molecular Modelling Studies of 2-(Carbazol-3-yl)-2-oxoacetamide Analogues as
a New Class of Potential Pancreatic Lipase Inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://
dx.doi.org/10.1016/j.bmc.2016.11.031
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title Page
Title:
Synthesis, Evaluation and Molecular Modelling Studies of 2-(Carbazol-3-yl)-2-oxoacetamide
Analogues as a New Class of Potential Pancreatic Lipase Inhibitors
Authors:
a
a
b
b
Sridhar S. N. C. , Ginson George , Venkataramana Reddy P. O. , Mukund P. Tantak , Dalip
b
a
Kumar , Atish T. Paul .
Affiliations:
a
Department of Pharmacy, Birla Institute of Technology and Science Pilani (Pilani campus),
Pilani - 333 031, Rajasthan, India.
b
Department of Chemistry, Birla Institute of Technology and Science Pilani (Pilani campus),
Pilani - 333 031, Rajasthan, India.
Corresponding authors:
Dr. Dalip Kumar
dalipk@pilani.bits-pilani.ac.in
Dr. Atish T. Paul
atish.paul@pilani.bits-pilani.ac.in
Present Address:
Dr. Atish T. Paul,
Assistant Professor,
3
121-C, Department of Pharmacy,
Birla Institute of Technology and Science Pilani (Pilani campus),
Pilani - 333 031, Rajasthan, India.
1

Abstract
A series of twenty-four 2-(carbazol-3-yl)-2-oxoacetamide analogues were synthesized,
characterized and evaluated for their PL inhibitory activities. Porcine PL was used against 4-
nitrophenyl butyrate and tributyrin as substrates during the PL inhibition assay. Compounds
7
e, 7f and 7p exhibited potential PL inhibitory activity (IC50 values of 6.31, 8.72 and 9.58
µM, respectively in method A; and Xi50 of 21.85, 21.94 and 26.2, respectively in method B).
Further, inhibition kinetics of 7e, 7f and 7p against PL revealed their competitive nature of
inhibition. A comparison of the inhibition profiles of the top three compounds in method B
and C, provided a preliminary idea of covalent bonding of the compounds with Ser 152 of
PL. Molecular docking studies of the compounds 7a-x into the active site of human PL (PDB
ID: 1LPB) was in agreement with the in vitro results, and highlighted probable covalent bond
formation with Ser 152 apart from hydrophobic interactions with the lid domain. Molecular
dynamics simulation of 7e complexed with PL, further confirmed the role of aromatic groups
in stabilising the ligand (RMSD ≤ 4 Å). The present study led to the identification of 2-
(
carbazol-3-yl)-2-oxoacetamide analogues 7a-x as a new class of potential PL inhibitors.
Highlights



2-(carbazol-3-yl)-2-oxoacetamide analogues were evaluated for PL inhibitory activity
Compounds 7e, 7f and 7p exhibited potential PL inhibitory activity
Probable covalent bond formation of the compounds with Ser 152 of PL was
determined

Hydrophobic interactions with the lid domain played an important role in PL
inhibition
Keywords
Carbazolyl Oxoacetamides, Inhibition Kinetics, Molecular Dynamics, Orlistat, Pancreatic
Lipase.
2

1
. Introduction
Obesity is a multifactorial metabolic disorder, defined by an abnormal or excessive
accumulation of lipids that presents risk to human health [1,2]. Recent statistical reports of
World Health Organisation (WHO) on obesity has projected a rapid growth in the obese
population with 600 million obese adults worldwide, accounting to 13% of global population
[
3]. Further, obesity is associated with various comorbid conditions including insulin
resistance, diabetes mellitus, cardiovascular diseases and certain cancers, that poses major
health problem to the obese patients [4]. With an estimated 2.8 million deaths per year,
obesity contributes to the 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 to be a successful and valid target considerably due to its
tolerable side effects. The human PL (EC 3.1.1.3) is a primary digestive enzyme secreted
from the exocrine glands of pancreas, and is primarily involved in the hydrolysis of ester
bonds of the triglycerides [14]. The crystal structure of the human PL (PDB ID: 1LPB) is
composed of 449 amino acids, with the catalytic triad Ser 152 - Asp 176 - His 263 defining
the active site of the enzyme [15]. This catalytic site is highly restricted and is surrounded by
the lid domain which consists of the amino acids Gly 76 - Lys 80, Leu 213 – Met 217 and the
disulfide bridge between Cys 238 and Cys 262 [16]. The hydrolysis of the triglyceride esters
by PL is represented by a series of events, initiated through an interfacial activation by the
hydrophobic alkyl chains of the triglycerides, resulting in the open lid conformation followed
by the nucleophilic attack of Ser 152 on to the carbonyl carbon of the ester linkage of the
triglycerides [17,18].
Orlistat (1), a PL inhibitor, remains to be the only drug approved for long term
treatment of obesity [19]. It is a saturated derivative of lipstatin [20] and contains a β-lactone
(
Fig. 1), that inhibits PL covalently, binding to Ser 152 of the active site [21]. Orlistat (1) was
reported to exhibit tolerable side effects [22], however, its long term administration causes
severe adverse effects including hepatotoxicity, gall stones, kidney stones and acute
pancreatitis [23]. In 2010, the United States Food and Drug Administration (USFDA)
approved a revised label for orlistat (1) with reference to cases of severe liver injury reported
with the use of this medication [24]. These events highlighted the requirement of safe and
effective drugs for the treatment of obesity.
Carbazoles represent an important class of indole alkaloids that have gained immense
significance in recent years due to their wide range of pharmacological activities. They have
3

been reported to exhibit PL inhibitory activity, for example; mahanimbine (2), koenimbin and
koenigicine, with IC50 values of 17.9, 168.6 and 428.6 µM, respectively [25]. However, these
molecules did not contain an ester or ester mimicking group which is essential for potential
PL inhibition. Scaffolds including fluorinated ketones, α-keto esters, α-ketoamides and 1,2-
diketones have been found to possess reactive carbonyl groups acting as an electrophile for
Ser 152 [26,27]. The role of α-ketoamide as an ester mimicking group was previously
described, where the replacement of ester with ketoamide in the triacylglycerol analogues (3)
led to potential PL inhibition [28–30].
Furthermore, literature review suggests the requirement of hydrophobic aromatic
moieties attached to the ester/ester mimicking group, essential to attain the open lid
conformation in PL. For example, substitution of phenoxybenzyl group on 1,3,4-oxadiazol-2-
one was reported to exhibit potential PL inhibition as compared to its unsubstituted derivative
without any PL inhibition activity [31].
Based on the above findings, we hypothesized the carbazolyl oxoacetamides to
possess potential PL inhibitory activity. These carbazolyl oxoacetamides can be considered a
pharmacophore hybrid of the carbazole nucleus and α-ketoamide functionality, present in a
single molecule (Fig. 1). Accordingly, the present study reports synthesis, in vitro evaluation
and in silico studies of 2-(carbazol-3-yl)-2-oxoacetamide analogues 7a-x as a new class of
potential PL inhibitors.
4

Figure 1. Rationale for selecting 2-(carbazol-3-yl)-2-oxoacetamides as PL inhibitors.
2
2
. Materials and Methods
.1. Chemistry
The syntheses of all the final compounds 7a-x (Table 1) were carried out as per the
procedure developed previously in our laboratory [32]. Briefly, the reactions were carried out
in a CEM focused microwave. 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 ABB Bomen MB 3000 FTIR machine using KBr
5

1
13
pellets. H and C NMR spectra were recorded on a Bruker Avance II 400 spectrometer (400
MHz and 100 MHz, respectively) using DMSO-d and CDCl as solvents. HRMS analysis
6
3
was performed on Bruker Compass Data Analysis 4.1 mass instrument. Purity of all the final
compounds was > 97% as determined by WATERS 515 HPLC system with a Sunfire C-18
column (5 µm, 4.6 × 250 mm) and PDA detector, using a flow rate of 1 mL/min. and a
gradient of acetonitrile.
2
.1.1. General procedure for the synthesis of carbazolyl oxoacetamides (7a-x)
To a solution of carbazolyl-2-oxoacetic acid 6 (0.275 mmol) in DMF (2 mL), HATU (0.12 g,
.317 mmol), N,N-diisopropylethylamine (0.09 g, 0.687 mmol) and an appropriate
0
aryl/heteroarylamine (0.303 mmol) was taken in a 10 mL microwave tube. The reaction
mixture was irradiated in microwave oven for 45 min at 70 °C. Upon completion of the
reaction, as confirmed by TLC, reaction contents were poured into ice-cold water (30 mL)
and stirred for 20 min at 25 °C. Suspension so obtained was filtered, dried and purified
through column chromatography on silica gel using ethylacetate:hexane (3:7) as eluent to
afford pure yellow coloured solids (7a-x) in 70-91% yields (Scheme 1).
Scheme 1. Reagents and conditions: (a) KOH, CH
AlCl , DCM, 0 °C to RT, 3 h; (c) LiOH, THF:H O (1:1), RT, 2 h; (d) ArNH
min, MW.
3
I/C
2
H
5
I/4-ClC
6
H
4
CH
2
Cl, DMF, RT, 13 h; (b) Cl(CO)
2
OEt,
3
2
2
, HATU, DIPEA, DMF, 70 °C, 45
6

Table 1
Chemical Structures and physicochemical properties of 2-(carbazol-3-yl)-2-oxoacetamides 7a-x.
Compound
R
Ar
M.P (°C)
191-193
172-173
106-107
168-170
136-137
183-184
202-203
116-118
211-213
129-131
178-179
221-222
171-173
199-201
202-203
207-208
> 300
% Yield
70
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
4-Chlorobenzyl
H
Phenyl
4-Methylphenyl
3-Methoxyphenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
4-Fluorophenyl
4-Pyridyl
72
73
d
e
72
70
f
71
g
h
i
75
91
N,N-dimethylaminophenyl
6-Quinolyl
71
j
70
k
l
2-(5-Methyl)thiazolyl
Phenyl
74
70
m
n
o
p
q
r
H
3-Methoxyphenyl
4-Methoxyphenyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
4-Pyridyl
72
H
70
H
71
H
72
H
71
Methyl
Phenyl
165-166
187-188
159-160
110-111
175-176
173-174
191-192
78
s
Methyl
3,4,5-Trimethoxyphenyl
Phenyl
80
t
Ethyl
82
u
v
w
x
Ethyl
3,4-Dimethoxyphenyl
3,4,5-Trimethoxyphenyl
N,N-dimethylaminophenyl
6-Quinolyl
85
Ethyl
86
Ethyl
73
Ethyl
78
2
.1.1.1. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-phenylacetamide (7a).
-1
Yield 70%; M.p: 191-193 °C; IR (KBr, v, cm ): 3335, 3090, 3052, 2916, 1682, 1653, 1589,
1
1
520, 1435, 1307, 1250, 1134, 1011, 825, 795; H NMR (400 MHz, CDCl
3
) δ 9.47 (d, J = 1.3
Hz, 1H), 9.15 (s, 1H), 8.58 (dd, J = 8.8, 1.7 Hz, 1H), 8.25 (dd, J = 7.6, 1.1 Hz, 1H), 7.78 (dd,
7

J = 8.6, 1.0 Hz, 2H), 7.54–7.50 (m, 1H), 7.45 (dd, J = 8.3, 7.5 Hz, 3H), 7.41–7.36 (m, 3H),
1
3
7
.26 (t, J = 2.6 Hz, 2H), 7.08 (d, J = 8.6 Hz, 2H), 5.55 (s, 2H); C NMR (100 MHz, CDCl )
3
δ 185.9, 160.0, 144.2, 141.2, 136.9, 134.6, 133.7, 129.8, 129.3, 129.2, 127.7, 127.0, 126.2,
+
1
25.2, 125.0, 123.6, 123.1, 121.1, 121.1, 120.0, 109.5, 108.8, 46.24; HRMS (ESI )
+
+
Calculated for C27
H20ClN
2
O
2
[M + H] , 439.1213; Found 439.1208 and 461.1025 [M + Na] ;
Anal. RP-HPLC t
R
= 4.641 min, purity 98.55%.
2
.1.1.2. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-(p-tolyl)acetamide (7b).
-1
Yield 72%; M.p: 172-173 ºC; IR (KBr, v, cm ): 3325, 3094, 3055, 2916, 1682, 1651, 1620,
1
1
9
582, 1520, 1443, 1327, 1265, 1149; H NMR (400 MHz, CDCl
3
) δ 9.48 (d, J = 1.6 Hz, 1H),
.09 (s, 1H), 8.58 (dd, J = 8.8, 1.7 Hz, 1H), 8.27–8.23 (m, 1H), 7.67–7.64 (m, 2H), 7.53-7.49
(
m, 1H), 7.44–7.35 (m, 3H), 7.26 (t, J = 2.2 Hz, 3H), 7.24 (s, 1H), 7.09 (d, J = 8.6 Hz, 2H),
1
3
5
1
1
4
9
.55 (s, 2H), 2.39 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 186.0, 159.9, 144.2, 141.1, 134.9,
34.6, 134.3, 133.7, 129.9, 129.7, 129.2, 127.7, 126.9, 126.1, 125.1, 123.6, 123.1, 121.1,
+
+
21.0, 119.9, 109.5, 108.7, 46.2, 21.0; HRMS (ESI ) Calculated for C28
H21ClN
2
O
2
[M + H] ,
+
53.1369; Found 453.1365 and 475.1183 [M + Na] ; Anal. RP-HPLC t
8.10%.
R
= 5.317 min, purity
2
.1.1.3. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(3-methoxyphenyl)-2-oxoacetamide
(
7c).
-1
Yield 73%; M.p: 106-107 ºC; IR (KBr, v, cm ): 3333, 3055, 2932, 2862, 1693, 1659, 1589,
1
1
528, 1381, 1335, 1265, 1203, 1142, 1088, 1041,849, 795, 725, 687, 679; H NMR (400
MHz, CDCl
3
) δ 9.43 (s, 1H), 9.11 (s, 1H), 8.57–8.54 (m, 1H), 8.23 (d, J = 7.7 Hz, 1H), 7.52–
7
2
1
1
.46 (m, 2H), 7.40–7.33 (m, 3H), 7.31–7.28 (m, 2H), 7.24–7.19 (m, 2H), 7.06 (d, J = 8.3 Hz,
1
3
H), 6.77 (dd, J = 7.8, 1.6 Hz, 1H), 5.52 (s, 2H), 3.87 (s, 3H); C NMR (100 MHz, CDCl ) δ
3
85.8, 160.3, 159.9, 144.2, 141.1, 138.1, 134.6, 133.7, 129.9, 129.7, 129.2, 127.7, 126.9,
26.1, 124.9, 123.5, 123.1, 121.1, 121.0, 112.2, 111.1, 109.5, 108.7, 105.5, 55.4, 46.2;
+
+
HRMS (ESI ) Calculated for C28
H22ClN
2
O
3
[M + H] , 469.1319; Found 469.1313 and
+
4
91.1133 [M + Na] ; Anal. RP-HPLC t
R
= 4.550 min, purity 98.36%.
2
.1.1.4. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(4-methoxyphenyl)-2-oxoacetamide
(
7d).
8

-1
Yield 72%; M.p: 168-170 ºC; IR (KBr, v, cm ): 3348, 3055, 2924, 1682, 1643, 1620, 1582,
1
1
528, 1443, 1327, 1250, 1149; H NMR (400 MHz, CDCl
3
) δ 9.45 (d, J = 1.5 Hz, 1H), 9.04
(
s, 1H), 8.56 (dd, J = 8.8, 1.7 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.69–7.65 (m, 2H), 7.51-7.47
m, 1H), 7.42–7.32 (m, 4H), 7.25–7.23 (m, 1H), 7.06 (d, J = 8.5 Hz, 2H), 6.97–6.94 (m, 2H),
(
1
3
5
1
1
4
.52 (s, 2H), 3.84 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 186.1, 159.8, 157.0, 144.2, 141.1,
34.6, 133.7, 130.1, 129.7, 129.2, 127.7, 126.9, 126.1, 125.1, 123.6, 123.1, 121.6, 121.1,
+
+
21.0, 114.4, 109.5, 108.7, 55.5, 46.2; HRMS (ESI ) Calculated for C28
H22ClN
2
O
3
[M + H] ,
+
+
69.1319; Found 469.1310, 471.1271 [M + H + 2] and 491.1129 [M + Na] ; Anal. RP-
HPLC t = 4.368 min, purity 98.67%.
R
2
.1.1.5. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(3,4-dimethoxyphenyl)-2-
oxoacetamide (7e).
-1
Yield 70%; M.p: 136-137 ºC; IR (KBr, v, cm ): 3317, 3055, 2924, 2847, 1690, 1651, 1589,
1
1
520, 1458, 1412, 1381, 1335, 1242, 1203, 1134, 1026, 849, 795, 748, 663, 602, 540; H
NMR (400 MHz, CDCl
3
) δ 9.43 (s, 1H), 9.08 (s, 1H), 8.56 (d, J = 8.7 Hz, 1H), 8.23 (d, J =
7
2
2
1
1
C
.7 Hz, 1H), 7.55 (d, J = 2.0 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.40–7.33 (m, 3H), 7.24 (s,
H), 7.16 (dd, J = 8.5, 2.1 Hz, 1H), 7.06 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 8.6 Hz, 1H), 5.51 (s,
1
3
H), 3.96 (s, 3H), 3.90 (s, 3H); C NMR (100 MHz, CDCl ) δ 186.0, 159.7, 149.2, 146.5,
3
44.2, 141.1, 134.6, 133.7, 130.6, 129.7, 129.2, 127.7, 126.9, 126.1, 125.1, 123.6, 123.1,
+
21.1, 121.0, 112.1, 111.4, 109.5, 108.7, 104.5, 56.1, 56.0, 46.2; HRMS (ESI ) Calculated for
+
+
29
H
24ClN
2
O
4
[M + H] , 499.1424; Found 499.1424, 501.1390 [M + H + 2] and 521.1238
= 4.031 min, purity 98.17%.
+
[
M + Na] ;Anal. RP-HPLC t
R
2
.1.1.6. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)
acetamide (7f).
-1
Yield 71%; M.p: 183-184 ºC; IR (KBr, v, cm ): 3356, 2932, 1690, 1651, 1597, 1528, 1504,
1
1
1
7
6
1
1
450, 1412, 1373, 1335, 1296, 1250, 1126, 1011, 802; H NMR (400 MHz, CDCl
3
) δ 9.43 (s,
H), 9.15 (s, 1H), 8.57 (dd, J = 8.8, 1.6 Hz, 1H), 8.25 (d, J = 7.6 Hz, 1H), 7.55–7.49 (m, 1H),
.40 (t, J = 8.6 Hz, 3H), 7.29-7.25 (m, 3H), 7.08 (d, J = 8.6 Hz, 3H), 5.53 (s, 2H), 3.94 (s,
1
3
H), 3.89 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 185.8, 159.9, 153.5, 144.2, 141.1, 135.3,
34.6, 133.7, 133.0, 129.7, 129.1, 127.7, 127.0, 126.0, 124.9, 123.5, 123.1, 121.1, 121.0,
+
+
09.5, 108.8, 97.5, 61.0, 56.2, 46.2; HRMS (ESI ) Calculated for C30
H26ClN
2
O
5
[M + H] ,
9

+
5
9
29.1530; Found 529.1531 and 551.1344 [M + Na] ; Anal. RP-HPLC t
R
= 4.181 min, purity
8.17%.
2
.1.1.7. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(4-fluorophenyl)-2-oxoacetamide
(
7g).
-1
Yield 75%; M.p: 202-203 ºC; IR (KBr, v, cm ): 3310, 3055, 2935, 1690, 1651, 1589, 1535,
1
1
497, 1381, 1335, 1265, 1211, 1142, 833, 705; H NMR (400 MHz, CDCl
3
) δ 9.43 (d, 1.5
Hz, 1H), 9.13 (s, 1H), 8.55 (dd, J = 8.8, 1.6 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.74–7.71 (m,
H), 7.53–7.46 (m, 1H), 7.40–7.33 (m, 3H), 7.24 (s, 2H), 7.14–7.03 (m, 4H), 5.51 (s, 2H);
2
1
3
C NMR (100 MHz, CDCl ) δ 185.7, 159.9, 144.2, 141.2, 134.6, 133.7, 133.0, 133.0, 129.7,
3
1
1
29.2, 127.7, 127.0, 126.2, 124.9, 123.6, 123.1, 121.8, 121.7, 121.1, 116.1, 115.9, 113.4,
09.5, 108.8, 46.2.
2
.1.1.8. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (7h).
-1
Yield 91%; M.p: 116-118 ºC; IR (KBr, v, cm ): 3340, 2924, 1696, 1659, 1489, 1327, 1265,
1
1
142, 1021, 803; H NMR (400 MHz, CDCl
3
) δ 9.40 (s, 1H), 9.32 (s, 1H), 8.61 (d, J = 5.0
Hz, 2H), 8.53 (dd, J = 8.8, 1.4 Hz, 1H), 8.22 (d, J = 7.5 Hz, 1H), 7.69 (d, J = 6.1 Hz, 2H),
7
5
1
1
4
.50 (t, J = 7.4 Hz, 1H), 7.31–7.35 (m, 3H), 7.24 (t, J = 4.8 Hz, 2H), 7.06 (d, J = 8.3 Hz, 2H),
1
3
.52 (s, 2H); C NMR (100 MHz, CDCl
3
) δ 184.6, 160.5, 151.0, 144.4, 143.8, 141.2, 134.5,
33.8, 129.8, 129.2, 127.7, 127.2, 126.2, 124.5, 123.5, 123.3, 121.2, 121.1, 113.8, 109.6,
+
+
08.9, 46.3; HRMS (ESI ) Calculated for C26
H19ClN
3
O
2
[M + H] , 440.1166; Found
+
40.1171 and 442.1141 [M + H + 2] ; Anal. RP-HPLC t
R
= 4.306 min, purity 98.02%.
2
.1.1.9. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(4-(dimethylamino)phenyl)-2-
oxoacetamide (7i).
-1
Yield 71%; M.p: 211-213 ºC; IR (KBr, v, cm ): 3333, 3047, 2924, 2854, 2800, 1674, 1643,
1
1
582, 1520, 1443, 1350, 1265, 1136, 1011, 949, 895, 825, 795, 748, 694, 617; H NMR (400
MHz, CDCl
3
) δ 9.46 (d, J = 1.3 Hz, 1H), 8.98 (s, 1H), 8.56 (dd, J = 8.8, 1.7 Hz, 1H), 8.24–
8
.21 (m, 1H), 7.63–7.60 (m, 2H), 7.50-7.46 (m, 1H), 7.42–7.30 (m, 4H), 7.24 (s, 1H), 7.06 (d,
10

+
J = 8.6 Hz, 2H), 6.79–6.76 (m, 2H), 5.52 (s, 2H), 2.97 (s, 6H); HRMS (ESI ) Calculated for
+
+
C
29
H25ClN
3
O [M + H] , 482.1635; Found 482.1621 and 484.1609 [M + H + 2] .
2
2
.1.1.10. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-2-oxo-N-(quinolin-6-yl)acetamide (7j).
-1
Yield 70%; M.p: 129-131 ºC; IR (KBr, v, cm ): 3356, 2932, 1690, 1651, 1597, 1528, 1504,
1
1
1
450, 1412, 1373, 1335, 1296, 1250, 1126, 1011, 802; H NMR (400 MHz, CDCl
3
) δ 9.44 (d,
.3 Hz, 2H), 8.89 (dd, J = 4.3, 1.6 Hz, 1H), 8.61–8.55 (m, 2H), 8.25–8.18 (m, 2H), 8.16 (d, J
9.0 Hz, 1H), 7.79 (dd, J = 9.0, 2.4 Hz, 1H), 7.52–7.41 (m, 2H), 7.41–7.30 (m, 3H), 7.23 (t,
=
1
3
J = 2.1 Hz, 2H), 7.05 (d, J = 8.5 Hz, 2H), 5.49 (s, 2H); C NMR (100 MHz, CDCl
3
) δ 185.4,
60.3, 149.5, 145.4, 144.3, 141.1, 136.4, 134.9, 134.5, 133.7, 130.2, 130.1, 129.8, 129.2,
28.8, 127.7, 127.1, 126.2, 124.8, 123.5, 123.4, 123.2, 121.9, 121.1, 116.5, 109.5, 108.8,
1
1
4
4
+
+
6.2; HRMS (ESI ) Calculated for C30
H
21ClN
3
O
2
[M + H] , 490.1322; Found 490.1327 and
+
92.1304 [M + H + 2] ; Anal. RP-HPLC t
R
= 4.911 min, purity 97.89%.
2
.1.1.11. 2-(9-(4-Chlorobenzyl)-9H-carbazol-3-yl)-N-(5-methylthiazol-2-yl)-2-
oxoacetamide (7k).
-1
Yield 74%; M.p: 178-179 ºC; IR (KBr, v, cm ): 3317, 3055, 2932, 1690, 1651, 1589, 1528,
1
1
489, 1257, 1142, 1011, 802; H NMR (400 MHz, CDCl
3
) δ 10.63 (s, 1H), 9.48 (d, J = 1.3
Hz, 1H), 8.60 (dd, J = 8.8, 1.5 Hz, 1H), 8.25 (d, J = 8.2 Hz, 1H), 7.52 (t, J = 7.3 Hz, 1H),
7
3
1
4
4
.46–7.32 (m, 4H), 7.26 (s, 1H), 7.08 (d, J = 8.3 Hz, 2H), 6.70 (s, 1H), 5.53 (s, 2H), 2.44 (s,
1
3
H); C NMR (100 MHz, CDCl
3
) δ 182.9, 159.3, 156.1, 148.4, 144.4, 141.2, 134.5, 133.8,
29.7, 129.2, 128.7, 127.7, 127.1, 126.2, 124.6, 123.5, 123.3, 121.2, 109.6, 109.2, 108.9,
+
+
6.3, 17.1; HRMS (ESI ) Calculated for C25
H19ClN
3
O S [M + H] , 460.0886; Found
2
60.0888; Anal. RP-HPLC t = 4.562 min, purity 99.82%;
R
2
.1.1.12. 2-(9H-Carbazol-3-yl)-2-oxo-N-phenylacetamide (7l).
-1
Yield 70%; M.p: 221-222 ºC; IR (KBr, v, cm ): 3348, 3055, 2932, 1674, 1651, 1589, 1528,
1
1
443, 1335, 1296, 1265, 1257, 1165, 1134, 1011, 787, 748, 687, 602, 548; H NMR (400
MHz, CDCl
3
) δ 9.41 (s, 1H), 9.12 (s, 1H), 8.55 (dd, J = 8.7, 1.5 Hz, 1H), 8.43 (s, 1H), 8.18
(
d, J = 7.7 Hz, 1H), 7.76 (d, J = 7.7 Hz, 2H), 7.53–7.46 (m, 3H), 7.43 (t, J = 7.9 Hz, 2H),
11

1
3
7
1
1
3
.36–7.30 (m, 1H), 7.21 (t, J = 7.4 Hz, 1H); C NMR (100 MHz, CDCl
3
) δ 187.4, 162.4,
43.9, 140.6, 137.5, 128.8, 128.3, 126.4, 125.1, 124.6, 124.0, 123.1, 122.8, 120.4, 120.2,
+
+
20.0, 111.4, 110.9; HRMS (ESI ) Calculated for C20
H
15
N
2
O
2
[M + H] , 315.1133; Found
+
15.1129 and 337.0949 [M + Na] ; Anal. RP-HPLC t
R
= 3.335 min, purity 98.25%.
2
.1.1.13. 2-(9H-Carbazol-3-yl)-N-(3-methoxyphenyl)-2-oxoacetamide (7m).
-1
Yield 72%; M.p: 171-173 ºC; IR (KBr, v, cm ): 3379, 3317, 3070, 2962, 2831, 1690, 1651,
1
1
597, 1535, 1497, 1450, 1335, 1242, 1203, 1149, 1034, 795, 687; H NMR (400 MHz,
CDCl
3
) δ 9.40 (s, 1H), 9.10 (s, 1H), 8.55 (dd, J = 8.7, 1.4 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J =
7
.8 Hz, 1H), 7.49 (dd, J = 8.5, 3.8 Hz, 3H), 7.35–7.29 (m, 2H), 7.22 (d, J = 8.0 Hz, 1H), 6.77
+
(
dd, J = 7.9, 1.8 Hz, 1H), 6.38–6.21 (m, 1H), 3.87 (s, 3H); HRMS (ESI ) Calculated for
+
+
C
21
H
17
N
2
O [M + H] , 345.1239; Found 345.1237 and 367.1058 [M + Na] ; Anal. RP-HPLC
3
t
R
= 3.251 min, purity 98.37%.
2
.1.1.14. 2-(9H-Carbazol-3-yl)-N-(4-methoxyphenyl)-2-oxoacetamide (7n).
-1
Yield 70%; M.p: 199-201 ºC; IR (KBr, , cm ): 3294, 3256, 3094, 2962, 2831, 1692, 1659,
1
1
597, 1504, 1450, 1412, 1335, 1281, 1234, 1134, 1103, 1011, 802, 795, 679; H NMR (400
MHz, CDCl
3
) δ 9.41 (s, 1H), 9.04 (s, 1H), 8.54 (dd, J = 8.7, 1.7 Hz, 1H), 8.43 (s, 1H), 8.17
(
d, J = 7.8 Hz, 1H), 7.71–7.67 (m, 1H), 7.67–7.65 (m, 1H), 7.48 (dd, J = 5.8, 2.1 Hz, 3H),
+
7
.34–7.30 (m, 1H), 6.98–6.96 (m, 1H), 6.96–6.93 (m, 1H), 3.84 (s, 3H); HRMS (ESI )
+
+
Calculated for C21
H
17
N
2
O
3
[M + H] , 345.1239; Found 345.1238 and 367.1059 [M + Na] ;
Anal. RP-HPLC t
R
= 3.138 min, purity 99.62%.
2
.1.1.15. 2-(9H-Carbazol-3-yl)-N-(3,4-dimethoxyphenyl)-2-oxoacetamide (7o).
-1
Yield 71%; M.p: 202-203 ºC; IR (KBr, v, cm ): 3472, 3286, 3086, 2924, 2839, 1692, 1659,
1
1
9
1
589, 1450, 1335, 1219, 1134, 1018, 849, 795, 733, 671, 617; H NMR (400 MHz, CDCl
3
) δ
.41 (s, 1H), 9.07 (s, 1H), 8.55 (dd, J = 8.7, 1.7 Hz, 1H), 8.43 (s, 1H), 8.18 (d, J = 7.8 Hz,
H), 7.56 (d, J = 2.4 Hz, 1H), 7.51–7.45 (m, 3H), 7.35-7.31 (m, 1H), 7.16 (dd, J = 8.6, 2.4
1
3
Hz, 1H), 6.90 (d, J = 8.6 Hz, 1H), 3.96 (s, 3H), 3.91 (s, 3H); C NMR (100 MHz, CDCl
3
) δ
1
1
86.0, 159.7, 149.2, 146.5, 143.4, 139.9, 130.6, 129.6, 126.9, 126.1, 125.1, 123.6, 123.3,
+
20.9, 120.9, 112.1, 111.4, 111.1, 110.5, 104.4, 56.1, 56.0; HRMS (ESI ) Calculated for
12

+
+
C
22
H
19
N
2
O [M + H] , 375.1344; Found 375.1348 and 397.1164 [M + Na] ; Anal. RP-HPLC
4
t
R
= 2.996 min, purity 98.66%.
2
.1.1.16. 2-(9H-Carbazol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide (7p).
-1
Yield 72%; M.p: 207-208 ºC; IR (KBr, v, cm ): 3340, 3301, 3055, 2932, 2839, 1690, 1651,
1
1
597, 1528, 1458, 1412, 1373, 1335, 1234, 1203, 1126, 995, 802, 733, 617; H NMR (400
MHz, CDCl
3
) δ 9.39 (s, 1H), 9.08 (s, 1H), 8.55 (dd, J = 8.7, 1.7 Hz, 1H), 8.47 (s, 1H), 8.19
(
d, J = 7.8 Hz, 1H), 7.51–7.45 (m, 3H), 7.35–7.31 (m, 1H), 7.07 (s, 2H), 3.93 (s, 6H), 3.86 (s,
1
3
3
H); C NMR (100 MHz, CDCl
3
) δ 185.9, 160.0, 153.5, 143.5, 140.0, 135.3, 133.1, 129.5,
+
1
26.9, 126.1, 124.9, 123.6, 123.3, 120.9, 120.8, 111.1, 110.6, 97.6, 61.0, 56.2; HRMS (ESI )
+
+
Calculated for C23
H
21
N
2
O
5
[M + H] , 405.1450; Found 405.1451 and 427.1273 [M + Na] ;
Anal. RP-HPLC t
R
= 3.113 min, purity 98.72%.
2
.1.1.17. 2-(9H-Carbazol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (7q).
-1
Yield 71%; M.p ˃300 ºC; IR (KBr, v, cm ): 3286, 3217, 3094, 2962, 2800, 1705, 1659,
1
1
605, 1566, 1489, 1420, 1342, 1311, 1250, 1196, 1157, 1126, 1011, 802, 787, 748, 679; H
NMR (400 MHz, DMSO-d
6
) δ 11.97 (s, 1H), 11.34 (s, 1H), 8.91 (d, J = 1.4 Hz, 1H), 8.56 (d,
J = 6.0 Hz, 2H), 8.31 (d, J = 7.8 Hz, 1H), 8.10 (dd, J = 8.6, 1.7 Hz, 1H), 7.78 (d, J = 6.3 Hz,
2
7
1
H), 7.66 (d, J = 8.6 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.49 (t, J = 8.2 Hz, 1H), 7.27 (t, J =
1
3
.9 Hz, 1H); C NMR (100 MHz, DMSO-d
6
) δ 188.6, 165.8, 151.1, 145.2, 144.2, 141.0,
+
28.1, 127.3, 124.7, 123.6, 123.0, 123.0, 121.5, 120.6, 114.6, 112.2, 112.0; HRMS (ESI )
+
Calculated for C19
H
14
N
3
O
2
[M + H] , 316.1086; Found 316.1085; Anal. RP-HPLC t
R
= 3.133
min, purity 98.12%.
2
.1.1.18. 2-(9-Methyl-9H-carbazol-3-yl)-2-oxo-N-phenylacetamide (7r).
-1
Yield 78%; M.p: 165-166 ºC; IR (KBr, v, cm ): 3333, 2924, 2854, 1682, 1643, 1610, 1528,
1
1
1
2
1
1
443, 1366, 1304, 1257, 1149, 1026, 895; H NMR (400 MHz, CDCl
3
) δ 9.40 (d, J = 1.4 Hz,
H), 9.18 (s, 1H), 8.60 (dd, J = 8.8, 1.7 Hz, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 7.6 Hz,
H), 7.56 (t, J = 8.2 Hz, 1H), 7.47–7.41 (m, 4H), 7.35 (t, J = 7.9 Hz, 1H), 7.23 (t, J = 7.4 Hz,
1
3
H), 3.87 (s, 3H); C NMR (100 MHz, CDCl
3
) δ 185.8, 160.2, 144.6, 141.7, 136.9, 129.5,
29.2, 126.7, 126.0, 125.1, 124.4, 123.3, 122.7, 120.8, 120.6, 120.0, 109.1, 108.4, 29.4; MS
+
+
(
ESI ) Calculated for C21
H
17
N
2
O
2
[M + H] , 329.12; Found 329.12; Anal. RP-HPLC t =
R
3
.461 min, purity 98.76%;
13

2
.1.1.19. 2-(9-Methyl-9H-carbazol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide
(
7s).
-1
Yield 80%; M.p: 187-188 ºC; IR (KBr, v, cm ): 3310, 2924, 2854, 1683, 1659, 1607, 1589,
1
1
1
1
504,1450, 1373, 1327, 1227, 1126, 895; H NMR (400 MHz, CDCl
3
) δ 9.35 (d, J = 1.6 Hz,
H), 9.17 (s, 1H), 8.57 (dd, J = 8.8, 1.6 Hz, 1H), 8.18 (d, J = 7.7 Hz, 1H), 7.54 (t, J = 8.2 Hz,
H), 7.43–7.38(m, 2H), 7.34 (t, J = 7.2 Hz, 1H), 7.08 (s, 2H), 3.92 (s, 6H), 3.88 (s, 3H), 3.84
1
3
(
s, 3H); C NMR (100 MHz, CDCl
3
) δ 185.7, 160.1, 153.4, 144.5, 141.6, 135.2, 133.1,
1
29.4, 126.7, 125.9, 124.3, 123.2, 122.7, 120.8, 120.5, 109.1, 108.4, 97.5, 61.0, 56.2, 29.3;
+
+
MS (ESI ) Calculated for C24
H
23
N
2
O
5
[M + H] , 419.16; Found 419.18; Anal. RP-HPLC t =
R
2
.837 min, purity 99.34%.
2
.1.1.20. 2-(9-Ethyl-9H-carbazol-3-yl)-2-oxo-N-phenylacetamide (7t).
-1
Yield 82%; M.p 159-160 ºC; IR (KBr, v, cm ): 3310, 2924, 2854, 1690, 1643, 1589,
1
1
1
7
1
1
1
3
535,1497, 1443, 1381, 1335, 1257, 1149, 756; H NMR (400 MHz, CDCl
3
) δ 9.44 (d, J =
.3 Hz, 1H), 9.18 (s, 1H), 8.62 (dd, J = 8.8, 1.7 Hz, 1H), 8.22 (d, J = 7.7 Hz, 1H), 7.79 (d, J =
.6 Hz, 2H), 7.59–7.53 (m, 1H), 7.49–7.43 (m, 4H), 7.35 (t, J = 7.4 Hz, 1H), 7.26–7.21 (m,
1
3
H), 4.42 (q, J = 7.2 Hz, 2H), 1.50 (t, J = 7.3 Hz, 3H); C NMR (100 MHz, CDCl
3
) δ 185.8,
60.2, 143.7, 140.7, 136.9, 129.5, 129.2, 126.6, 126.2, 125.1, 124.3, 123.5, 122.9, 121.0,
+
+
20.5, 119.9, 109.1, 108.4, 37.9, 13.8; MS (ESI ) Calculated for C22
43.14; Found 343.19; Anal. RP-HPLC t = 2.870 min, purity 98.74%.
H
19
N
2
O [M + H] ,
2
R
2
.1.1.21. N-(3,4-Dimethoxyphenyl)-2-(9-ethyl-9H-carbazol-3-yl)-2-oxoacetamide (7u).
-1
Yield 85%; M.p: 110-111 ºC; IR (KBr, v, cm ): 3338, 2924, 2854, 1724, 1659, 1589, 1443,
1
1
9
7
2
1
1
1
381, 1327, 1250, 1142, 1026, 768; H NMR (400 MHz, CDCl
3
) δ 9.41 (d, J = 1.4 Hz, 1H),
.15 (s, 1H), 8.60 (dd, J = 8.8, 1.6 Hz, 1H), 8.20 (d, J = 7.7 Hz, 1H), 7.58 (d, J = 2.4 Hz, 1H),
.54 (t, J = 7.2 Hz, 1H), 7.44 (t, J = 8.6 Hz, 2H), 7.34 (t, J = 7.4 Hz, 1H), 7.18 (dd, J = 8.6,
.4 Hz, 1H), 6.90 (d, J = 8.7 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 3.98 (s, 3H), 3.92 (s, 3H),
1
3
.47 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl
3
) δ 185.9, 160.0, 149.2, 146.4, 143.6,
40.6, 130.6, 129.4, 126.6, 126.1, 124.4, 123.5, 122.9, 121.0, 120.4, 112.0, 111.3, 109.1,
+
+
08.3, 104.4, 56.0, 37.9, 13.8; MS (ESI ) Calculated for C24
= 2.849 min, purity 99.51%.
H
23
N
2
O [M + H] , 403.16;
4
Found 403.21; Anal. RP-HPLC t
R
2
.1.1.22. 2-(9-Ethyl-9H-carbazol-3-yl)-2-oxo-N-(3,4,5-trimethoxyphenyl)acetamide (7v).
14

-1
Yield 86%; M.p: 175-176 ºC; IR (KBr, v, cm ): 3279, 2932, 2854, 1736, 1659, 1607, 1589,
1
1
1
7
2
1
1
504,1450, 1389, 1342, 1234, 1126,1049, 995; H NMR (400 MHz, CDCl
3
) δ 9.39 (d, J =
.4 Hz, 1H), 9.18 (s, 1H), 8.59 (dd, J = 8.8, 1.6 Hz, 1H), 8.20 (d, J = 7.7 Hz, 1H), 7.54 (t, J =
.7 Hz, 1H), 7.47–7.41 (m, 2H), 7.34 (t, J = 7.7 Hz, 1H), 7.10 (s, 2H), 4.38 (q, J = 7.2 Hz,
1
3
H), 3.93 (s, 6H), 3.88 (s, 3H), 1.47 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl ) δ
3
85.7, 160.1, 153.5, 143.6, 140.6, 135.2, 133.1, 129.4, 126.6, 126.0, 124.2, 123.4, 122.9,
+
21.0, 120.5, 109.1, 108.3, 97.6, 61.0, 56.2, 37.9, 13.8; MS (ESI ) Calculated for C25
H
25
N
2
O
5
+
[
M + H] , 433.17; Found 433.18; Anal. RP-HPLC t
R
= 2.866 min, purity 98.57%.
2
.1.1.23. N-(4-(dimethylamino)phenyl)-2-(9-ethyl-9H-carbazol-3-yl)-2-oxoacetamide
(
7w).
-1
Yield 73%; M.p: 173-174 ºC; IR (KBr, v, cm ): 3290, 2924, 2854, 1723, 1666, 1582,1448,
1
1
350, 1234, 1126, 1065, 895; H NMR (400 MHz, CDCl
3
) δ 9.45 (d, J = 1.3 Hz, 1H), 9.04
(
(
(
s, 1H), 8.63 (dd, J = 8.8, 1.6 Hz, 1H), 8.21 (d, J = 7.7 Hz, 1H), 7.65 (d, J = 9.0 Hz, 2H), 7.55
t, J = 7.6 Hz, 1H), 7.49–7.43 (m, 2H), 7.34 (t, J = 7.0 Hz, 1H), 6.80 (d, J = 9.0 Hz, 2H), 4.41
1
3
q, J = 7.2 Hz, 2H), 2.99 (s, 6H), 1.49 (t, J = 7.2 Hz, 3H); C NMR (100 MHz, CDCl
3
) δ
1
1
86.3, 159.8, 148.3, 143.6, 140.6, 129.5, 126.7, 126.6, 126.1, 124.7, 123.6, 122.8, 121.4,
+
21.0, 120.4, 112.9, 109.1, 108.3, 40.8, 37.9, 13.8; MS (ESI ) Calculated for C24
H
24
N
3
2
O [M
+
+
H] , 386.18; Found 386.24; Anal. RP-HPLC t
R
= 2.862 min, purity 99.71%.
2
.1.1.24. 2-(9-Ethyl-9H-carbazol-3-yl)-2-oxo-N-(quinolin-6-yl)acetamide (7x).
-1
Yield 78%; M.p: 191-192 ºC; IR (KBr, v, cm ): 3348, 2924, 2854, 1690, 1651, 1582,1466,
1
1
327, 1234, 1126, 1065, 887; H NMR (400 MHz, CDCl
3
) δ 9.49 (s, 1H), 9.42 (d, J = 1.3
Hz, 1H), 8.89 (dd, J = 4.2, 1.7 Hz, 1H), 8.64–8.56 (m, 2H), 8.19 (t, J = 7.1 Hz, 2H), 8.14 (d, J
8.0 Hz, 1H), 7.80 (dd, J = 9.0, 2.4 Hz, 1H), 7.57–7.52 (m, 1H), 7.47–7.40 (m, 3H), 7.34 (t,
=
1
3
J = 7.5 Hz, 1H), 4.37 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.3 Hz, 3H); C NMR (100 MHz,
CDCl ) δ 185.3, 160.5, 149.8, 145.8, 143.7, 140.6, 136.0, 134.9, 130.5, 129.5, 128.8, 126.7,
26.2, 124.2, 123.4, 123.2, 122.9, 121.8, 121.0, 120.6, 116.5, 109.2, 108.4, 37.9, 13.8; MS
3
1
+
+
(
ESI ) Calculated for C25
H
20
N
3
O
2
[M + H] , 394.15; Found 394.16; Anal. RP-HPLC t =
R
2
.852 min, purity 98.11%.
2
.2. Pancreatic Lipase Inhibition Assay and Enzyme Kinetics
15

Orlistat (1), Porcine PL (Type II) and 4-nitrophenyl butyrate were procured from
Sigma-Aldrich, USA. Tris buffer, MOPS, Calcium chloride, Sodium chloride (Molecular
Biology grade) and Sodium taurodeoxycholate (NaTDC) were procured from Sisco Research
Laboratories, India. Tributyrin was procured from TCI Chemicals, India. All other chemicals
and solvents (analytical grade) were used without further purification. The procedure for PL
inhibition assay involved three methods, A, B and C. Method A was performed to obtain
preliminary inhibitory activities of the compounds 7a-x, while methods B and C were
performed to validate the potential of the top three compounds (7e, 7f and 7p), as well as to
understand their possible covalent interaction with Ser 152 of PL.
2
.2.1. Method A and Enzyme Kinetics
The procedure for method A was performed as per the previously reported literature
with minor modifications [33]. Briefly, 50 mg of porcine pancreatic lipase was suspended in
0 mL of Tris-HCl buffer (containing 2.5 mmol of Tris and 2.5 mmol of NaCl, adjusted to
1
pH 7.4 with HCl). The solution was subjected to vigorous shaking for 15 min, followed by
centrifugation (4000 rpm, 291 K for 10 min). The supernatant was collected and used afresh
as the enzyme solution.
Stock solutions of the synthesized compounds and orlistat (1) were prepared in
DMSO at linear concentrations ranging from 1.56 - 2000 μg/mL and 0.78 – 1000 μg/mL,
respectively. 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 310
K, followed by addition of 10 μL of the substrate (4-nitrophenyl butyrate, 10 mM in
acetonitrile). The amount of DMSO in the final concentration did not exceed 2%. The
absorbance of the final mixture was taken in microplate spectrophotometer (EPOCH,
BioTek) 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 = [(A -A
E T
E
) ⁄ A ] 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, and was compared to that of
orlistat (reference standard).
For the enzyme inhibition kinetics, the assay protocol (Method A) was repeated for
orlistat, 7e, 7f and 7p, each at four different concentrations (0, 5, 10 and 20 μM) using four
different substrate concentrations (25, 50, 100 and 200 µM), and a double reciprocal
16

Lineweaver-Burk plot was plotted to understand the nature of inhibition [34]. The inhibition
constants, K , were calculated from Dixon plot [35].
i
2
.2.2. Method B
The procedure for method B was performed as per previously reported protocols
31,36]. Briefly, 0.5 ml of tributyrin was mechanically stirred in 14.5 ml of buffer (containing
.3 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM CaCl , and 4 mM NaTDC) to obtain an
[
0
2
emulsion. For the enzyme solution, 5 mg/mL of porcine pancreatic lipase was prepared in 10
mM MOPS (pH 7) containing 150 mM NaCl. The enzyme solution was incubated with
various inhibitors viz. orlistat, 7e, 7f and 7p at various molar excess (X
C. The final concentration of PPL in the incubation medium was equivalent to 25 µg/mL.
Enzymatic activity was recorded at 37 °C by measuring the amount of free fatty acid
i
) for 30 minutes at 25
°
released from the tributyrin emulsion, using 0.1 N NaOH with a pH-Stat (AT-38C, Spectralab
Instruments, India) adjusted to a fixed end point value. Samples from the incubation medium
containing different inhibitor molar excess (X ) were injected into the pH-Stat reaction vessel,
i
and the lipase activity was measured at 1 minute intervals for a total period of 5 minutes. All
experiments were conducted in triplicate, and the activities were expressed as international
units; 1 U = 1 µmol of FFA released per minute. The concentration of the inhibitor molar
excess which reduced the lipase activity by 50% (Xi50) was then calculated with reference to
the change in % residual activity vs. inhibitor molar excess (X
.2.3. Method C
For method C, the assay protocol was similar to method B, but NaTDC (4 mM) was
i
).
2
added to the incubation medium instead of the reaction mixture [37]. This method was
performed to understand the possible mode of interaction of the top three compounds, 7e, 7f
and 7p, with pancreatic lipase.
2
.3. Molecular Modelling studies
A total of 24 2-carbazol-3-yl) oxoacetamide analogues were subjected to molecular
docking studies on human PL (PDB ID: 1LPB). The structures of the compounds were drawn
using Chemdraw 2D module of ChemBioOffice v12 (PerkinElmer) and subjected to energy
minimization using OPLS 2005 force field in LigPrep module of Schrodinger Suite. Glide 5.9
17

module of Schrodinger Suite was used for docking the energy minimized structures into the
crystal structure of human PL.
In order to understand the behaviour of the ligand in a dynamic environment,
molecular dynamics (MD) simulation was conducted for 7e in complex with PL. For this
purpose, Gromacs 5.0.4 [38], 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 [39] was applied during the MD run, and the topology of the ligand
was generated using online tool provided by Swiss Institute of Bioinformatics [40]. Prior to
the initiation of the MD, the complex was minimized using Steepest Descent algorithm for
1
000 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-range electrostatics [41], 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 [42]. Visual Molecular Dynamics 1.9.2 [43] and Discovery Studio
4
.5 visualizer (Accelrys) were used to analyse and visualise the MD trajectory file, and to
depict the graphical representations of the complex.
3
3
. Results and Discussion
.1. Chemistry
The syntheses of the 2-(carbazol-3-yl)-2-oxoacetamide analogues 7a–x (Table 1) was
carried out as per Scheme 1 previously optimized in our laboratory [32]. Initially, carbazole 4
was allowed to react with ethyl chlorooxoacetate in the presence of anhydrous AlCl3,
followed by ester hydrolysis using LiOH to afford the respective carbazolyl oxoacetic acid
derivative 6 in good yield (Scheme 1). Further, the reaction of 6 and aryl/heteroarylamines in
the
presence
of
O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HATU) at 70 °C under microwave irradiation produced 7 in 70-91%
yields. Scope and generality of this protocol was further demonstrated by coupling 6 with
various arylamines, and prepared a series of carbazolyl oxoacetamides 7a-x. Structures of the
synthesized 2-(carbazol-3-yl)-2-oxoacetamides were well characterized using spectroscopic
1
13
techniques, including IR, NMR ( H and C) and HRMS. HPLC analysis of carbazolyl
oxoacetamides 7a-x indicated the purity of all the compounds was greater than 97%. In IR
−
1
spectra of 7a-x, characteristic peaks at ~1690 and 1655 cm were assigned to the carbonyl
18

1
3
and amide functionalities, respectively. Further, in C NMR spectra of 7a-x, carbons of
carbonyl and amide functionalities were resonated at ~180 (CO) and ~160 ppm (NHCO).
3
3
.2. Inhibition studies and Enzyme Kinetics
.2.1. Method A
The preliminary PL inhibitory activity of the synthesized 2-(carbazol-3-yl)-2-
oxoacetamides was evaluated against porcine PL (Type II) using 4-nitrophenyl butyrate as a
substrate. Orlistat (1) was used as the reference standard. Table 2 represents the activity
profiles of the compounds screened for PL inhibition. From the Table 2, it is clear that most
of the compounds exhibited good (< 15 µM) to moderate (15-30 µM) activity, while few
compounds displayed poor activity (> 30 µM). Analogue 7e exhibited the most potential
activity against PL, with an IC50 of 6.31 µM, followed by 7f and 7p (8.72 and 9.58 µM,
respectively). Orlistat (1), however, possessed a very potent IC50 of 0.99 µM.
Table 2
In vitro PL inhibitory activities of 2-(carbazol-3-yl)-2-oxoacetamides 7a-x.
Sr. No Compound
IC50 (µM)
13.91 ± 2.10
13.53 ± 1.67
26.61 ± 0.97
14.83 ± 2.17
6.31 ± 0.56
8.72 ± 0.47
27.98 ± 2.25
19.87 ± 1.08
14.78 ± 1.50
23.05 ± 2.49
32.76 ± 6.05
29.73 ± 5.62
0.99 ± 0.11
Sr. No
13
Compound
IC50 (µM)
33.02 ± 2.30
19.31 ± 0.80
44.12 ± 5.18
9.58 ± 1.24
29.35 ± 2.17
36.18 ± 1.88
23.12 ± 2.06
24.22 ± 0.30
16.37 ± 2.09
11.37 ± 1.12
56.18 ± 4.34
15.48 ± 1.87
1
2
3
4
5
6
7
8
9
7a
7b
7m
7n
7o
7p
7q
7r
7s
14
7c
15
7d
16
7e
17
7f
18
7g
19
7h
20
7t
7i
21
7u
7v
7w
7x
1
0
1
2
7j
22
1
1
7k
23
7l
24
Orlistat
19

Further, in an attempt to understand the nature of inhibition, kinetic studies were
performed for 7e, 7f, 7p and orlistat (1). While IC50 represents the inhibition potential of the
ligand against enzyme, the nature of inhibition provides adequate information of the ligand’s
binding site [44,45]. The nature of inhibition was determined through the use of Lineweaver-
Burk plot.
1
Km
1
1
ꢁ
ꢅ
(Equation 1)
ꢀ
ꢀmaꢂ ꢃSꢄ
ꢀmaꢂ
Equation 1 represents the Lineweaver-Burk equation, where V represents velocity of
the reaction, Vmax is maximum velocity, [S] is substrate concentration and K is the
Michaelis Menten constant [34]. As shown in Supplementary data, (Fig. S1.) and Table 3, the
plots converged at y-intercept while the K value increased in proportion to the inhibitor
m
m
concentration. These representations indicated that the standard drug, orlistat (1) as well as
the ligands 7e, 7f and 7p inhibited PL competitively against the substrate, highlighting the
fact that the ligands bound to the active site of the PL.
Table 3
V
max, K
m
and K
concentrations [I] of orlistat, 7e, 7f and 7p.
(in µM) at different inhibitor concentrations [I]
i
(inhibition constant) values calculated from Lineweaver-Burk plots at different inhibitory
K
m
Code
0 µM
92.36
92.36
92.36
92.36
5 µM
196.52
129.15
125.98
122.77
10 µM
228.49
156.15
152.38
150.93
20 µM
252.51
178.52
164.97
163.38
V
max (µM/min)
i
K (µM) Nature of inhibition
Orlistat
15.25
9.8
Competitive
Competitive
Competitive
Competitive
7
e
15.25
17.5
19.6
21.6
7
f
15.50
7
p
15.00
3
.2.2. Method B
In an attempt to validate the potential of the compounds 7e, 7f and 7p, the PL
inhibition assay was repeated using tributyrin, owing to its better suitability as a substrate for
pancreatic lipase [46]. The residual activities of the enzyme at different inhibitor molar
excess (X ) were determined using pH-Stat reaction vessel. The specific activity of PPL in the
i
absence of inhibitor was found to be 9440 ± 123.16 U per mg of Protein. Orlistat exhibited a
potent Xi50 of 3.14, while 7e, 7f and 7p exhibited Xi50 of 21.85, 21.94 and 26.2, respectively
(
Fig. 2).
20

1
1
10
00
A
B
7e
1
1
20
00
7
f
7p
9
8
0
0
8
6
4
2
0
0
0
0
0
70
6
5
4
3
0
0
0
0
20
1
0
0
0
10
20
30
40
50
60
70
0
50
100
150
200
250
300
350
Inhibitor molar excess (Xi)
Inhibitor molar excess (Xi)
i
Figure 2. Effects of increasing inhibitor molar excess (X ) of orlistat (A); and compounds 7e, 7f and 7p (B) on
the residual activity (%) of PL.
A comparison of the inhibitory activities of the top three compounds viz., 7e, 7f and
p, with that of orlistat (1), with reference to method A and method B, represented similar
7
line of results, validating the potency of the top three compounds.
.2.3. Method C
The results from the inhibition kinetics highlighted the fact that orlistat (1), as well as
3
the top three compounds 7e, 7f and 7p, bound to the active site of PL. Recently, Tiss et. al.
reported the role of bile salts to affect the strength of covalent bond formed between the PL
and orlistat (1). The presence of NaTDC in the incubation medium containing the enzyme
and orlistat (1) increased the covalent bond strength, which was evident through a reduction
in the activity of the PL, while the presence of the NaTDC in the reaction mixture exerted the
opposite effect [37]. Hence, the residual activity of PL was determined in the presence of 7e,
7
f and 7p, pre-incubated with the enzyme in the presence of 4mM of NaTDC for 30 min at
2
5 °C. The results obtained were compared with that of method B, wherein the NaTDC was
added to the reaction mixture. Figure 3 represents the variation in the residual activity of the
enzyme with reference to method B (NaTDC in the reaction mixture) and method C (NaTDC
in the incubation medium). The residual activities of the PL were reduced with 7e, 7f and 7p,
incubated with 4 mM of NaTDC. These results indicated that the compounds 7e, 7f and 7p
probably exhibited a covalent interaction with Ser 152 of PL, similar to orlistat (1).
21

7
f
1
1
20
00
7
e
1
1
20
00
Method B
Method C
Method B
Method C
8
6
4
0
0
0
8
6
4
2
0
0
0
0
0
20
0
0
50
100
150
200
250
300
350
0
50
100
150
200
250
300
350
Inhibitor molar excess (Xi)
Inhibitor molar excess (Xi)
22

7
p
1
1
20
00
Method B
Method C
8
6
4
2
0
0
0
0
0
0
50
100
Inhibitor molar excess (Xi)
Figure 3. Comparative graphical plots of 7e, 7f and 7p representing better inhibitory activities in method C.
150
200
250
300
350
23

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 Figure 4, the potential of the compounds to
inhibit PL varied with respect to the substitutions on the carbazolyl and amide nitrogen viz., R
and Ar respectively. The presence of an electron withdrawing group on the carbazolyl
nitrogen increased the PL inhibition activity of compounds. This might be attributed to the
decrease in the polarity of carbazole in the presence of withdrawing group, which in turn
resulted in stronger hydrophobic interactions with the lid domain. Similarly, the presence of
an electron-donating group on the amide nitrogen potentiated the PL inhibitory activity of the
compounds. 3,4-Dimethoxy and 3,4,5-trimethoxy analogues exhibited comparatively stronger
inhibition
activity
when
compared
to
other
derivatives.
24

Figure 4. Structure-activity relationship for 2-(carbazol-3-yl)-2-oxoacetamide.
3
.4. Molecular modelling
Molecular docking studies provide insight into the interactions of the compounds with
the pocket residues of PL. In the present study, all the synthesized compounds (7a-x) were
docked into the crystal structure of the PL (PDB ID: 1LPB) using Glide 5.9 module of
Schrodinger suite. Prior to the docking, the grid parameters were validated by redocking the
co-crystallised ligand, methoxyundecyl phosphinic acid (MUP). The redocked pose was
deviated from the co-crystallised pose by an RMSD of 1.7 Å (Fig. 5).
25

Figure 5. Re-docked pose of MUP (Brown) over co-crystallised MUP (Grey).
Table 4 summarizes the XP GScore and interactions of 2-(carbazol-3-yl)-2-
oxoacetamides with the pocket residues of human PL. Compound 7e possessed a top docking
score of -10.196 kcal/mol and exhibited interactions with Phe 77, Tyr 114, His 263 and Arg
2
56 (Fig. 7). Moreover, the XP GScore was much in alignment with the IC50 values of the in
2
vitro assay (R = 0.8659), validating the docking parameters.
Table 4
XP GScore and interaction summary of 7a-x with PL.
Compound
XP GScore
-9.578
-9.44
H-bond
Phe 77
Phe 77
ND
Pi-Pi stacking interactions
Phe 77, His 263
Pi-cation
Arg 256
Arg 256
Arg 256
Arg 256
Arg 256
Arg 256
Arg 256
-
7
7
a
b
Phe 77, His 263
7
c
-8.774
-9.766
-10.196
-9.514
-8.917
-8.928
-9.518
Phe 77
7
d
Phe 77
Phe 77
Phe 77
His 263
ND
Phe 77, Tyr 114, His 263
Phe 77, Tyr 114, His 263
Phe 77, His 263
7
e
7
f
7
7
g
Phe 77, His 263
h
Phe 77, Tyr 114, Phe 215
Phe 77, His 263
7
i
Phe 77
-
26

7
j
-9.217
-8.516
-8.389
-8.451
-9.219
-7.803
-9.908
-8.607
-8.531
-9.292
-8.804
-9.092
-9.261
-9.174
-9.23
ND
Phe 77, Tyr 114, Phe 215
Phe 77, His 263
Tyr 114, Arg 256
His 263
-
7
k
Phe 77
His 263
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
Phe 77
His 263
Arg 256
7
l
-
7
m
Arg 256
7
n
o
p
q
His 263
Arg 256
7
His 151, Phe 215, His 263
Phe 77
-
7
7
-
His 263, Arg 256
Phe 77, His 263
Phe 77
-
7
r
-
7
s
Arg 256
Arg 256
Arg 256
Arg 256
Arg 256
Arg 256
-
7
t
His 263
7
u
v
His 263
7
Phe 77, His 263
Phe 77, His 263
His 263
7
w
7
x
Orlistat
-10.680
-
As shown in Table 4 and Figure 6, the ligands exhibited pi-pi stacking interactions
with amino acids of the lid domain in addition to H-bond interaction with Phe 77. Further, the
presence of aromatic group (4-chlorobenzyl substitution on carbazole nitrogen) facilitated an
additional pi-pi interaction with Tyr 114 as evident from binding pose analysis. Moreover,
most of the ligands exhibited an additional pi-cation interaction with Arg 256. Literature
study revealed that Arg 256 plays a key role in the transformation of PL from closed to an
open lid conformation. Lowe et al. reported that Arg 256 and Asp 257 makes a salt bridge
with Tyr 267 and Lys 268 in the open lid conformation [47]. Birari et al. also suggested the
role of Arg 256 interaction with isoliquiritigenin (4) for its potential PL inhibitory activity
(
IC50 = 7.3 µM) [48]. The above findings from the molecular docking studies are in
accordance to our hypothesis, wherein the aromatic wings viz., carbazole nucleus and the aryl
extension at N-arylglyoxamide interacted with the amino acids of the lid domain, probably
resulting in the open lid conformation.
27

Figure 6. Representation of 7e in the binding pocket of PL (A); 2D interaction diagram of 7e with PL (B); 3D
interaction diagram of 7e highlighting the lid domain interactions (C).
Further, the binding mode of 7e was superimposed on that of orlistat (1), to
understand the probability of a covalent bond formation of compounds 7e, 7f and 7p. The 3D
structure of orlistat adopted the shape of a fork (Fig. 7A), which was in accordance to the
docking pose described by Point et. al [31]. Figure 7B represents the 3D superimposition
diagram of 7e, on to orlistat (1) within the active site of PL. As shown in Figure 7B, the
reactive carbonyl groups of both the molecules were overlapping each other with a minor
deviation (< 1 Å), confirming a probable covalent interaction of the compound 7e, similar to
that of orlistat (1).
28

Figure 7. Representation of fork shaped orlistat in the binding pocket of PL (A); Superimposition of orlistat
Brown) and 7e (Grey) highlighting the overlap of the reactive carbonyl groups (Green) (B).
(
Docking studies provided a preliminary idea of 2-(carbazol-3-yl)-2-oxoacetamide
interactions with the protein. However, it is necessary to predict the ligand behaviour in
motion. A 10 ns molecular dynamics simulation has been performed for 7e in complex with
PL using CHARMM force field [39]. The RMSD of the protein backbone remained stable
during the entire run with a maximum deviation of 2.5 Å recorded around 8 ns (Fig. S2,
Supplementary Data). Furthermore, the ligand remained very stable during the first 4 ns
(
RMSD < 2 Å), after that it slightly deviated by 0.5 Å during the next 3 ns, and achieved a
final RMSD of 4 Å in the last 3 ns (Fig. S2, Supplementary Data).
29