Design, synthesis and biological evaluation of some 2-azetidinone derivatives as potential antihyperlipidemic agents

Article abstract of DOI:10.1002/ardp.201300262

In an effort to develop new molecules with improved antihyperlipidemic activity, eight new 2-azetidinone analogs (4a-4h) of ezetimibe were designed through in silico docking experiments with the crystal structure of the Niemann-Pick C1-like 1 protein (NPC

Full text of DOI:10.1002/ardp.201300262

872
Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
Full Paper
Design, Synthesis and Biological Evaluation of Some
2-Azetidinone Derivatives as Potential Antihyperlipidemic Agents
Nikhilesh Arya1,2, Jaya Dwivedi2, Vijay M. Khedkar3, Evans C. Coutinho3, and Kishor S. Jain1
1
Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmaceutical Sciences, Lonavala, Pune,
India
2
Department of Chemistry, Banasthali University, Tonk, India
Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Mumbai, India
3
In an effort to develop new molecules with improved antihyperlipidemic activity, eight new 2-azetidinone
analogs (4a–4h) of ezetimibe were designed through in silico docking experiments with the crystal
structure of the Niemann-Pick C1-like 1 protein (NPC1L1). Synthesis and further antihyperlipdemic
evaluation of this series in the Triton WR 1339 induced hyperlipidemic rat model showed some of the
molecules to exhibit significant lipid-lowering effects comparable to ezetimibe. Correlation between the
observed biological activity and the in silico molecular docking scores of the compounds was observed.
Keywords: Antihyperlipidemic / 2-Azetidinone / Ezetimibe / Molecular docking / NPC1L1
Received: July 17, 2013; Revised: August 17, 2013; Accepted: August 29, 2013
DOI 10.1002/ardp.201300262
Introduction
(NPC1L1) protein, present in the brush border membrane of
small intestine, which is actively involved in the absorption of
dietary cholesterol, is blocked by ezetimibe [6]. Thus, NPC1L1
protein has become an important target for the design of
novel ligands as potential drugs with antihyperlipidemic
activity, and many analogs of ezetimibe have been synthe-
sized and evaluated for antihyperlipidemic activity.
Nicotinic acid (niacin) II (Fig. 2), which occurs naturally in
food as vitamin B3, is also useful as a drug for reducing the
amount of “bad cholesterol [LDL, VLDL]” and triglycerides in
the blood. It also has favorable good cholesterol or high
density lipoprotein (HDL) elevating effects [7].
There are numerous examples of hybrid molecules wherein
dual bioactivities have been incorporated into a single
chemical entity [8]. It was therefore thought worthwhile to
combine the features of I and II to evolve out novel analogs of
ezetimibe (Fig. 3).
In the present study, it was planned to design a series of
such hybrid structures to be taken up for synthesis and
antihyperlipaemic evaluation. Initially in silico docking
experiments with a few representative structures at the
active site of the NPC1L1 (PDB: 3QNT), to assess the binding
and fit (docking scores and energies) of such structures with
respect to that of ezetimibe, were undertaken. Based on these
results the series was expanded for the synthesis and
evaluation of antihyperlipaemic activity. Also, whether a
It is well established that elevated lipid levels in blood are a
potential risk factor for coronary heart disease (CHD) and
stroke, as they have a direct link with atherosclerosis. Various
antihyperlipeamic drugs primarily act by controlling blood
lipid levels, through various mechanisms, at different stages
of lipid formation, transportation, absorption, metabolism,
and reuptake [1, 2].
2-Azetidinones (b-lactams) as a class of compounds are
subject of various reviews, due to wide-ranging biological
activities, including antihyperlipidemic activity, exhibited
by them [3]. Several derivatives based on this scaffold have
been regularly designed, synthesized and evaluated for
antihyperlipaemic activity [4].
Ezetimibe (Eze) I (Fig. 1), representing 2-azetidinones, has
emerged as a selective cholesterol absorption inhibitor drug
capable of reducing blood LDL-C levels. It is used as
monotherapy or in conjunction with the statins [5]. Ezetimibe
reduces LDL-C levels in both cases. Niemann-Pick C1-like 1
Correspondence: Prof. Kishor S. Jain, Department of Pharmaceutical
Chemistry, Sinhgad Institute of Pharmaceutical Sciences, S.No.309/310,
Kusgaon (Bk.), Off. Mumbai-Pune Expressway, Lonavala, Pune – 410 401,
Maharashtra, India.
E-mail: drkishorsjain@gmail.com
Fax: þ91-2114-270258
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
Ezetimibe Analogs
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Results and discussion
HO
F
In silico (docking) studies
Docking studies were performed for four different compound
structures 4a–4d, as well as ezetimibe, in order to postulate a
hypothetical binding model for their interaction with NPC1L1
using the X-ray crystal structure (PDB ID: 3QNT) of the latter
with its co-crystallized ligand N-acetyl-D-glucosamine in its
active site [9]. This ligand was removed and replaced one by
one with ezetimibe and 4a–4d to assess their individual
docking interactions.
N
OH
F
O
Figure 1. Ezetimibe.
The amino acids in the active site of NPC1L1, which are in
proximity and involved in interactions (van der Waals,
hydrophobic, H-bonding) with these ligands, are Leu52,
Ser102, Ile105, Leu213, Leu99, Ala101, Leu103, Thr106,
Phe120, Gln206, etc.
OH
N
O
Details of the docking methodology are presented in the
Experimental section, and the results are summarized in
Tables 1 and 2.
Figure 2. Nicotinic acid.
correlation existed between the docking scores of a few
representative compounds and their actually observed
biological activity was verified. The present work describes
the design, synthesis, and investigation of antihyperlipidemic
properties of new derivatives of 2-azetidinone (4a–4h), as
ezetimibe analogs.
All the crucial interactions observed between NPC1L1 and
ezetimibe were also observed for the representative com-
pounds 4a–4d, indicating a fairly consistent binding mode for
all the synthesized analogs. Table 2 shows a comparison of the
distances between centroid of the ligand 4a and ezetimibe and
the various amino acid residues of the active site. The docking
Table 1. Results for molecular docking experiments of standard
drug (ezetimibe) and synthesized compounds 4a–4d with
NPC1L1 protein.
HO
F
Compound
Docking score
OH
N
Ezetimibe
ꢀ6.381671
ꢀ8.188506
ꢀ4.806627
ꢀ6.107940
ꢀ5.159371
4a
4b
4c
4d
N
O
OH
F
O
II
I
Table 2. Comparison of the distances of the centroid of ligands
backbone and amino acid residues of the active site.
Residue
4a (Å)
Ezetimibe (Å)
Ser102
Leu213
His124
Leu103
Leu52
Ala101
Gln206
Thr128
Phe120
Thr106
Leu99
07.94
06.73
14.75
09.94
06.81
10.38
12.20
16.63
12.96
11.97
12.73
09.86
08.75
08.16
17.37
11.00
06.74
09.75
13.56
19.54
18.75
13.65
14.15
10.96
HO
R
N
HN
R'
O
O
X
Ile105
Figure 3. Target compounds (4a–4h).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
score obtained for 4a was the most favorable, due to the
stronger interactions and higher proximity with the active
site residues. A three-dimensional representation of the
optimized docked model of 4a and that of ezetimibe in the
active site of NPC1L1 is depicted in Fig. 4. The corresponding
two-dimensional interaction diagram of the docked model
(Fig. 5) shows amino acid residues within active site of 4a and
that of ezetimibe. It is observed from Fig. 4 that the docked
ligand exhibited a hydrogen bonding interaction with Leu 52
at a distance of 3.343 Å. The docked ezetimibe is also involved
in similar hydrogen bonding interaction with Leu 52, but at a
slightly higher distance of 4.997 Å, suggesting a more favored
affinity for compound 4a. Also, compound 4a showed a strong
coulombic interaction with Leu 52, Leu 99, and Ala 101, as
well as significant van der Waals interaction with Leu 52, Ile
105, and Leu 213. Although several factors beyond the
Figure 4. Representation of the 3D docking poses of ezetimibe and 4a with NPC1L1 (PDB: 3QNT).
Figure 5. Two-dimensional representation of the optimized docking models of ezetimibe and 4a at the active site of NPC1L1. Most of
the amino acid residues are closer to the centroid of 4a than ezetimibe (Table 2).
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
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predicted docking interactions and energies with a particular
target govern the biological activity of a compound in vivo, the
above docking results support the structure-based design of
the new derivatives with the rank order for docking scores as:
4a > ezetimibe > 4c > 4d > 4b.
On this basis it was decided to undertake the synthesis and
evaluation of these four compounds, 4a–4d, as well as other
analogs, 4e–4h, based on these compounds.
Chemistry
The synthetic methodology adopted for the preparation of
target compounds, 4a–4h, is summarized in Scheme 1. The
first step involves the formation of Schiff’s bases (3a–3d)
through the nucleophilic attack by the hydrazide 1 at the
carbonyl carbon of 4-hydroxybenzaldehyde 2, which is
affected by refluxing equimolar amounts of the reactants
in ethanol under acid catalysis. These intermediates, 3a–3d,
HO
HO
NH2
HN
(a)
N
HN
R'
X
O
R'
CHO
X
O
1
3
2
(b)
HO
R
N
HN
R'
O
O
X
_
4a 4h
Compound
R
R'
N
X
4a
4b
4c
4d
4e
4f
Cl
Ph
Cl
Ph
Cl
Ph
Cl
Ph
N
4g
4h
Cl
Scheme 1. Reagents and conditions: (a) EtOH/reflux, 4–5 h; (b) ClCH2COCl/PhCH2COCl, Et3N, dioxane, 0–5°C, 24–48 h.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
are formed in good yields and purity (Scheme 1, Table 3). The
subsequent step involved reaction of 3a–3d with acyl
chlorides (chloroacetyl chloride/phenylacetyl chloride) in
dioxane under basic conditions (triethyl amine) at 0–5°C
over 24–48 h to afford the target compounds, 4a–4h
(Scheme 1, Table 4).
the compounds 4a–4h was all trans (J ¼ ꢁ2 Hz is characteristic
of trans-coupling between methine protons, H-C(3) and H-C(4),
whereras for the cis-configuration it is reported to be as high
as J ¼ ꢁ5–6 Hz) [12].
Pharmacological activity
The structural assignments of the intermediates 3a–3d
and target compounds 4a–4h are based on their correct
spectral data and elemental analysis. The IR spectra of the
2-azetidinones (4a–4h) exhibit a strong absorption around
1735 cmꢀ1 characteristic of the b-lactam ring [10]. The C-(3),
C-(4)-cis/trans arrangement for the newly synthesized
2-azetidinones was deduced on the basis of their 1H NMR
data [11]. The 1H NMR spectra coupling constants between
H-C(3) and H-C(4) of these compounds were at about 2 Hz,
indicating that the relative configuration at C-(3) and C-(4) of
Increased levels of lipids (triglyceride, cholesterol) and
lipoproteins (LDL and VLDL) in the blood are characteristic
of the condition of hyperlipidemia. Triton WR 1339
(Tyloxapol1) induced hyperlipidemia in Wistar albino
rat [13] was the model used for the biological evaluation of
title compounds. The lipid profile (cholesterol, triglycerides,
and HDL) for the hyperlipidemic and control Wistar albino
rats was studied by administering the test compounds (4a–4h)
and the standard drug, ezetimibe, p.o. (Table 5). In all, eleven
animal groups of six animals each were formed for the study
Table 3. Physical data for intermediates (Schiff’s bases) 3a–3d.
R'
Molecular
formula
Molecular
weight
Melting
point (°C)
Yield
(%)
X
Compound
3a
N
C13H11N3O2
241.25
240.26
262–264 (264–268) [14]
285–287
88.20
89.45
3b
C14H12N2O2
3c
C13H11N3O2
241.25
274.70
190–192
87.36
85.67
N
Cl
3d
C14H11ClN2O2
280–282 (290–291) [15]
Table 4. Physical data of synthesized compounds (azetidinones) 4a–4h.
R'
Molecular
formula
Molecular
weight
Melting
point (°C)
Yield
(%)
X
Compound
R
4a
4b
Cl
Ph
C15H12ClN3O3
C21H17N3O3
317.73
359.38
255–257 (98–110) [14]
198–200
68.66
60.54
N
4c
4d
Cl
Ph
C16H13ClN2O3
C22H18N2O3
316.74
358.39
165–167 (160–162) [16]
136–139
61.34
56.78
4e
4f
Cl
Ph
C15H12ClN3O3
C21H17N3O3
317.73
359.38
262–264 (195) [17]
150–153
64.24
59.11
N
4g
4h
Cl
Ph
C16H12Cl2N2O3
C22H17ClN2O3
351.18
392.83
175–178
148–150
57.32
54.23
Cl
a)Satisfactory elemental analysis (ꢂ0.4 of the calculated values of %C, %H, and %N obtained). Please refer to the Experimental section.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
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Table 5. Antihyperlipaemic activity data for synthesized compounds 4a–4h and correlation with the docking scores.
Antihyperlipaemic activitya)
% Reduction in total
cholesterol
% Reduction in
triglyceride
% Change
in HDL
R'
Docking
scoreb)
X
Compound
R
(mg dlꢀ1
)
(mg dlꢀ1
)
(mg dlꢀ1
)
Ezetimibe
–
–
52.39 ꢂ 5.18
34.26 ꢂ 5.37
31.50 ꢂ 3.83
ꢀ6.381671
4a
4b
Cl
Ph
51.07 ꢂ 3.09
34.40 ꢂ 1.27
33.92 ꢂ 0.33
ꢀ8.188506
R'
R'
R'
R'
42.20 ꢂ 1.92
31.40 ꢂ 1.27
30.26 ꢂ 1.08
ꢀ4.806627
X
X
X
X
4c
4d
Cl
Ph
43.55 ꢂ 2.55
40.16 ꢂ 1.84
24.54 ꢂ 1.33
21.35 ꢂ 1.47
27.44 ꢂ 2.36
23.68 ꢂ 1.29
ꢀ6.107940
ꢀ5.159371
4e
4f
Cl
Ph
49.42 ꢂ 2.57
47.62 ꢂ 1.97
36.11 ꢂ 1.32
35.66 ꢂ 2.42
24.12 ꢂ 2.38
23.50 ꢂ 3.14
—
—
4g
4h
Cl
Ph
45.33 ꢂ 1.69
40.08 ꢂ 2.33
30.24 ꢂ 2.07
25.25 ꢂ 1.11
27.62 ꢂ 2.13
18.78 ꢂ 1.52
—
—
a)
Results are expressed as mean ꢂ standard error, statistically significant (p < 0.05, t-test, n ¼ 6). The results of the eight compounds
selected for screening were correlated statistically. Triton WR 1339 model with regression r ¼ 0.9, i.e., showed good correlation.
b)
The order of docking scores ¼ 4a > ezetimibe > 4c compares with the ability to (a) reduce serum levels of total cholestrol
ezetimibe ꢃ 4a > 4c; (b) reduce serum levels of Tg 4a > ezetimibe > 4c, and (c) enhance serum levels of HDL, 4a > ezetimibe > 4c.
to assess and compare the effects of test compounds and the
standard drug on the test animals. The first three groups
were the control group (Group-I), cholesterol control group
(Group-II), and standard group (Group-III). The remaining
eight groups were named as Group 4a–4h, respectively, and
administered with respective test compounds p.o. Triton was
administered i.p. to the animals of the test as well as standard
groups.
Of the eight compounds, 4a–4h, tested, compounds 4a, 4e,
4f, and 4g effected reduction in % serum cholesterol levels
of the test animals, up to 51.07 ꢂ 3.09%, 49.42 ꢂ 2.57%,
47.62 ꢂ 1.97%, and 45.33 ꢂ 1.69%, respectively, which was
comparable to the reduction in % serum cholesterol levels of
the animals administered with ezetimibe (52.39 ꢂ 5.18%) (Fig. 6).
Compounds 4a, 4e, and 4f have also been shown to
cause good % reduction in serum triglyceride levels of
the test animals, up to 34.40 ꢂ 1.27%, 36.11 ꢂ 1.32%, and
35.66 ꢂ 2.42%, respectively, which was comparable to the
reduction in % serum triglyceride levels of the test animals
administered with ezetimibe (34.26 ꢂ 5.37%) (Fig. 7).
Further, compound 4a also showed good ability
(33.92 ꢂ 0.33% increase) to elevate % serum HDL levels of test
animals comparable to the standard ezetimibe (31.5 ꢂ 3.83%
increase) (Fig. 8).
Figure 6. % Reduction in total serum cholesterol levels.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
Figure 7. % Reduction in serum triglyceride levels.
Figure 8. % Change in serum HDL levels.
Conclusions
compounds were recorded on a Perkin Elmer BX-FTIR spectro-
photometer (Massachusetts, USA) in potassium bromide discs.
The 1H NMR were taken on a NMR Bruker Avance II 400 MHz
spectrometer (California, USA) using CDCl3 as solvent and TMS as
an internal standard (chemical shift in d ppm). Mass spectra were
obtained on a Shimadzu GCMS-QP2010 spectrometer (Kyoto,
Japan). The purity of the compounds was monitored by elemental
analysis and thin-layer chromatography. Elemental analyses for
compounds were obtained using Flash EA 1112 Thermofinnigan
instrument (San Diego, USA). Thin layer chromatography (TLC)
silica gel 60 F254 plates (200 mm; Merck, Darmstadt, Germany)
were used to assess preliminary purity of compounds. All melting
points were taken in open capillaries using a Veego electronic
melting point apparatus model MP-D (Mumbai, India) containing
silicon oil bath with stirrer and are uncorrected.
A
novel series of 2-azetidinone derivatives has been
designed and prepared on the basis of a docking study at
NPC1L1. The synthesized compounds were characterized and
evaluated in Triton WR 1339 induced hyperlipidemic rats,
for antihyperlipidemic activity. The present investigation
showed significant antihyperlipidemic activity in some of
these compounds, which is comparable to the standard
drug, ezetimibe. A certain degree of correlation between
the docking scores and antihyperlipaemic activity of the
compounds vis-a-vis the standard drug is also observed.
Triton WR 1339 (Sigma, Bangalore, India) was procured
commercially. The total lipid profile was determined by using
Infinite liquid cholesterol solution ready to use diagnostic kits
(Accurex India Biomedicals, Mumbai, India). All the biological
activity carried out in this study was approved by the Institutional
Animal Ethics Committee (SIPS/IAEC/2012-13/08).
Experimental
General
All the synthesized compounds were characterized by spectral
data (IR, mass, and 1H NMR). The IR spectra of the synthesized
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Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
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Computational docking studies
in order to explore a large region of the protein. Default values
were used for the van der Waals scaling, and partial charges were
assigned from the input structure, rather than from the force
field, by selecting the use input partial charges option.
All the molecular modeling studies were carried out on Intel Xeon
based system with the Linux Enterprise OS. The computations
were executed with the Schrödinger molecular modeling package
(Schrödinger, Inc., USA). Maestro, Version 9.1, Schrödinger, LLC,
New York, NY, 2010.
Chemistry
General procedure for the preparation of Schiff’s bases as
intermediates 3a–d
Docking
The Glide (Grid-Based Ligand Docking with Energetics) module
incorporated in the Schrödinger molecular modeling package
was used to perform molecular docking studies. Flexibility of the
ligand as well as protein was considered during docking, allowing
a flip for 5- and 6-membered rings. A maximum of 10 poses were
generated for each molecule. Default settings were used for all the
remaining parameters. The minimized poses generated by
docking were scored using the Glide Extra-Precision (XP) scoring
function equipped with a variety of force field-based parameters
accounting for solvation and repulsive interactions, lipophilic,
hydrogen bonding interactions, metal–ligand interactions, as
well as contributions from coulombic and van der Waals
interaction energies, all incorporated in the empirical energy
functions.
A mixture of equimolar quantities of p-hydroxybenzaldehyde 2
and aromatic acid hydrazide 1 was refluxed for 3 h in ethanol
(50 mL) with few drops of glacial acetic acid. On completion of
reaction (TLC), the reaction mixture was cooled and poured onto
ice–water mixture (50 mL). The solid separated was washed with
sodium thiosulfate solution followed by water and filtered under
vacuum, dried, and recrystallized to give the desired compound 3.
N0-(4-Hydroxybenzylidene)isonicotinohydrazide (3a)
Yield: 88.20%; mp: 262–264°C (lit. 264–268°C) [14]; IR (KBr): 3455,
3083, 1680, 1647, 1280, 850 cmꢀ1 1H NMR (400 MHz, CDCl3):
;
d (ppm) ¼ 5.51 (s, 1H, OH), 6.46–6.60 (m, 4H, Ar–H), 6.83–7.01
–
(m, 4H, Ar–H), 8.51 (s, 1H, –CH N–), 9.59 (s, 1H, –NH–CO); MS (TOF):
–
m/z ¼ 241; C13H11N3O2 (241.25); requires (found): C, 64.72 (64.88);
H, 4.60 (4.66); N, 17.42 (17.51).
Preparation of protein and ligand structures for
docking simulations
N0-(4-Hydroxybenzylidene)benzohydrazide (3b)
The X-ray structure of Niemann-Pick C1-Like 1 (NPC1L1) protein
(PDB code: 3QNT) was used as the target structure [9] to endeavor
the molecular docking studies. Coordinates of the inhibitor-
complexed protein were prepared for Glide calculations by
running the protein preparation wizard applying the OPLS-2005
force field. The crystallographic waters were removed and
hydrogens were added to the structure corresponding to pH
7.0 considering the appropriate ionization states for both the
acidic and basic amino acid residues. The prepared structure was
then subjected to energy minimization until the average root
mean square deviation (r.m.s.d.) reached 0.3 Å.
Yield: 89.45%; mp: 285–287°C; IR (KBr): 3266, 3190, 3058, 1601,
1284, 835 cmꢀ1; 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 5.67 (s, 1H,
OH), 6.61–6.75 (m, 5H, Ar–H), 6.86–6.97 (m, 4H, Ar–H), 8.59 (s, 1H,
–
–CH N–), 9.14 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 240; C14H12N2O2
–
(240.26); requires (found): C, 69.99 (70.15); H, 5.03 (5.14); N, 11.66
(11.57).
N0-(4-Hydroxybenzylidene)nicotinohydrazide (3c)
Yield: 87.36%; mp: 190–192°C; IR (KBr): 3457, 3287, 3071, 1670,
1
1647, 1299, 833 cmꢀ1; H NMR (400 MHz, CDCl3): d (ppm) ¼ 5.44
The initial 3D structures of the new derivatives of 2-azetidinone
(4a–d), as ezetimibe analogs were built in Maestro Suite and then
optimized by the LigPrep module in the Schrodinger Suite. This
tool applies corrections to the structures (adjusts the bond
lengths and bond angles), generates variations on the structures,
eliminates unwanted structures, and optimizes the structures.
The ligand partial charges were ascribed using the OPLS-2005
force-field and possible ionization states were assigned at a
target pH of 7.0. The ligand geometries were further optimized by
energy minimization using the LBFGS method and a distance-
dependent dielectric, until a gradient of 0.01 kcal molꢀ1 Åꢀ1 was
achieved.
(s, 1H, OH), 6.87–6.99 (m, 4H, Ar–H), 7.10–7.24 (m, 4H, Ar–H), 8.44
–
(s, 1H, –CH N–), 9.77 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 241;
–
C13H11N3O2 (241.25); requires (found): C, 64.72 (64.60); H, 4.60
(4.54); N, 17.42 (17.48).
N0-(4-Hydroxybenzylidene)-4-chlorobenzohydrazide (3d)
Yield: 85.67%; mp: 280–282°C (lit. 290–291°C) [15]; IR (KBr): 3384,
3289, 3045, 1622, 1272, 896, 752 cmꢀ1; 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 5.75 (s, 1H, OH), 6.70–6.83 (m, 4H, Ar–H), 6.90–7.06
–
(m, 4H, Ar–H), 8.38 (s, 1H, –CH N–), 9.37 (s, 1H, –NH–CO); MS (TOF):
–
m/z ¼ 274; C14H11ClN2O2 (274.7); requires (found): C, 61.21 (61.30);
H, 4.04 (4.16); N, 10.20 (10.29).
Receptor grid generation
General procedure for the preparation of N-[4-(4-
hydroxyphenyl)-2-oxo-3-substituted-azetidin-1-yl]-
arylamides 4a–h
To a vigorously stirred mixture of 3 (0.01 mol) in anhydrous DMF
(50 mL) at 0–5°C chloroacetyl chloride/phenylacetyl chloride
(0.015mol) was added dropwise over 30 min followed by triethyl-
amine (0.03 mol). The reaction mixture was further stirred for 3 h
at 0–5°C and then left at RT. On completion of the reaction (TLC),
the reaction mixture was poured onto ice–water (100 mL), the
solid separated washed with aqueous sodium bicarbonate
After ensuring that the protein and ligands were in the correct
form, the receptor grid was generated to define the active pocket
for docking using the Receptor Grid Generation tool in Glide. It uses
two cubical boxes that share a common center to organize the
calculations: a larger enclosing and a smaller binding box. Grid
file was generated by defining the centroid of the residues Leu52,
Thr106, Leu99, Ala101, Ser102, Ile105, Phe120, His124, Thr128,
Gln206, Leu103, and Leu213 forming the active site of NPC1L1 [9].
The binding region was defined by a 10 Å ꢄ 10 Å ꢄ 10 Å box
centered on the centroid of these residues in the crystal complex
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

880
N. Arya et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
solution (10% w/v) followed by water and filtered under vacuum,
dried, and recrystallized to give the desired compound 4.
6.78–6.99 (m, 4H, Ar–H), 7.05–7.14 (m, 4H, Ar–H), 7.18–7.27 (m, 5H,
Ar–H); 7.83 (s, 1H, –NH–CO–); MS (TOF): m/z ¼ 359; C21H17N3O3
(359.38); requires (found): C, 70.18 (70.25); H, 4.77 (4.83); N, 11.69
(11.82).
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1-
yl)isonicotinamide (4a)
Trans-4-chloro-N-(3-chloro-4-(4-hydroxyphenyl)-2-
Yield: 68.66%; mp: 255–257°C (lit. 98–110°C) [14]; IR (KBr): 3504,
3351, 3028, 2905, 1732, 1637, 1086, 766 cmꢀ1; 1H NMR (400 MHz,
CDCl3): d (ppm) ¼ 4.70 (d, 1H, –CH–Cl), 4.85 (d, J ¼ 2.4 Hz, 1H,
–CH–N–), 5.32 (s, 1H, OH), 6.77–6.89 (m, 4H, Ar–H), 6.97–7.08
(m, 4H, Ar–H), 7.62 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 317;
oxoazetidin-1-yl)benzamide (4g)
Yield: 57.32%; mp: 175–178°C; IR (KBr): 3550, 3318, 3030, 2853,
;
1734, 1647, 1189, 774 cmꢀ1 1H NMR (400 MHz, CDCl3):
d (ppm) ¼ 4.72 (d, 1H, –CH–Cl), 4.96 (d, J ¼ 2.4 Hz, 1H, –CH–N–),
5.61 (s, 1H, OH), 6.77–6.89 (m, 4H, Ar–H), 6.97–7.08 (m, 4H, Ar–H),
7.62 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 351; C16H12Cl2N2O3 (351.18);
requires (found): C, 54.72 (54.79); H, 3.44 (3.57); N, 7.98 (7.86).
C15H12ClN3O3 (317.73); requires (found): C, 56.70 (56.64);
H, 3.81 (3.92); N, 13.23 (13.29).
Trans-N-(4-(4-hydroxyphenyl)-2-oxo-3-phenylazetidin-1-
yl)isonicotinamide (4b)
Trans-4-chloro-N-(4-(4-hydroxyphenyl)-2-oxo-3-
phenylazetidin-1-yl)benzamide (4h)
Yield: 60.54%; mp: 198–200°C; IR (KBr): 3448, 3302, 3011, 2923,
1734, 1663, 1045 cmꢀ1; 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 4.64
(d, 1H, –CH–Ph), 4.89 (d, J ¼ 2.4 Hz, 1H, –CH–N–), 5.36 (s, 1H, OH),
6.81–6.92 (m, 4H, Ar–H), 6.97–7.10 (m, 4H, Ar–H), 7.13–7.25 (m, 5H,
Ar–H); 7.75 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 359; C21H17N3O3
(359.38); requires (found): C, 70.18 (70.14); H, 4.77 (4.89); N, 11.69
(11.66).
Yield: 54.23%; mp: 148–150°C; IR (KBr): 3369, 3276, 3004, 2929,
1736, 1689, 1088, 780 cmꢀ1 1H NMR (400 MHz, CDCl3): d
;
(ppm) ¼ 4.84 (d, 1H, –CH–Ph), 5.24 (d, J ¼ 2.5 Hz, 1H, –CH–N–),
5.70 (s, 1H, OH), 6.65–6.81 (m, 5H, Ar–H), 6.91–7.15 (m, 4H, Ar–H),
7.22–7.40 (m, 4H, Ar–H); 7.60 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 392;
C
22H17ClN2O3 (392.83); requires (found): C, 67.26 (67.35); H, 4.36
(4.27); N, 7.13 (7.28).
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1-
yl)benzamide (4c)
Pharmacological activity
Wistar albino rats (170–200 g) of either sex were selected at
random for the experiments. The animals were housed at a
temperature of 30 ꢂ 5°C and humidity of 40–50 ꢂ 5% with 12-h
light and 12-h dark cycles and were given food and water ad
libitum, unless specified otherwise.
Triton WR 1339, a surfactant, chemically iso-octylpolyoxy-
ethylene phenol (tyloxapol), was used to induce hyperlipidemia.
The animals were divided into eleven groups of six animals of
either sex per group.
Yield: 61.34%; mp: 165–167°C (lit. 160–162°C) [16]; IR (KBr): 3478,
3284, 3032, 2906, 1732, 1660, 1057, 771 cmꢀ1; 1H NMR (400 MHz,
CDCl3): d (ppm) ¼ 4.59 (d, 1H, –CH–Cl), 4.89 (d, J ¼ 2.2 Hz, 1H,
–CH–N–), 5.64 (s, 1H, OH), 6.88–7.02 (m, 5H, Ar–H), 7.10–7.21
(m, 4H, Ar–H), 7.58 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 316;
C16H13ClN2O3 (316.74); requires (found): C, 60.67 (60.60);
H, 4.14 (4.20); N, 8.84 (8.75).
Trans-N-(4-(4-hydroxyphenyl)-2-oxo-3-phenylazetidin-1-
yl)benzamide (4d)
Group I
Group II
The control group received only vehicle (2%
acacia solution as well as normal saline).
The cholesterol control group received Triton WR
1339 (200 mg kgꢀ1) by i.p. route in normal
saline solution.
The standard group received Triton WR 1339
(200 mg kgꢀ1 i.p.) as well as ezetimibe (1 mg
kgꢀ1 p.o.) as suspension in 2% w/v acacia (aq.).
Yield: 56.78%; mp: 136–139°C; IR (KBr): 3466, 3287, 3022, 2941,
1737, 1653, 1045 cmꢀ1; 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 4.68
(d, 1H, –CH–Ph), 5.11 (d, J ¼ 2.4 Hz, 1H, –CH–N–), 5.59 (s, 1H, OH),
6.78–6.95 (m, 5H, Ar–H), 6.99–7.10 (m, 4H, Ar–H), 7.15–7.45 (m, 5H,
Ar–H); 7.90 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 358; C22H18N2O3
(358.39); requires (found): C, 73.73 (73.65); H, 5.06 (5.17); N, 7.82
(7.91).
Group III
Groups: 4a–4h The test groups 4a–4h received Triton WR 1339
(200 mg kgꢀ1 i.p.) as well as test compound
(10 mg kgꢀ1 p.o.) as suspension in 2% w/v acacia
(aq.)
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1-
yl)nicotinamide (4e)
Yield: 64.24%; mp: 262–264°C (lit. 195°C) [17]; IR (KBr): 3469, 3299,
3015, 2864, 1740, 1653, 1034, 784 cmꢀ1 1H NMR (400 MHz,
;
CDCl3): d (ppm) ¼ 4.38 (d, 1H, –CH–Cl), 4.78 (d, J ¼ 2.1 Hz, 1H,
–CH–N–), 5.54 (s, 1H, OH), 6.65–6.86 (m, 4H, Ar–H), 6.92–7.11
(m, 4H, Ar–H), 7.79 (s, 1H, –NH–CO); MS (TOF): m/z ¼ 317;
C15H12ClN3O3 (317.73); requires (found): C, 56.70 (56.88);
H, 3.81 (3.66); N, 13.23 (13.18).
Blood samples were collected from the retro-orbital plexus of
the eyes of the animals, initially and after 24 h. The samples were
analyzed for the serum levels of cholesterol (total), triglyceride,
and HDL.
The authors acknowledge the contributions of Prof. M. N. Navale,
President and Dr. (Mrs.) S. M. Navale, Secretary, Sinhgad Technical
Education Society, Pune, India for providing encouragement and
facilities to carry out the synthetic work and basic spectroscopic
analysis and Principal, Bombay College of Pharmacy, Mumbai, India
Trans-N-(4-(4-hydroxyphenyl)-2-oxo-3-phenylazetidin-1-
yl)nicotinamide (4f)
Yield: 59.11%; mp: 150–153°C; IR (KBr): 3421, 3315, 3038, 2906,
1732, 1645, 1138 cmꢀ1; 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 4.64
(d, 1H, –CH–Ph), 4.88 (d, J ¼ 2.2 Hz, 1H, –CH–N–), 5.60 (s, 1H, OH),
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 872–881
Ezetimibe Analogs
881
for providing facilities to carry out in the silico experiments. The
spectral analyses were done at SAIF, Panjab University, Chandigarh,
India and University of Pune, Maharashtra, India, respectively.
PNAS 2008, 105, 11140–11145; c) M. G. Calvo, J. M. Lisnock,
H. G. Bull, B. E. Hawes, D. A. Burnett, M. P. Braun, J. H. Crona,
H. R. Davis, Jr., D. C. Dean, P. A. Detmers, M. P. Graziano,
M. Hughes, D. E. MacIntyre, A. Ogawa, K. A. O’Neill, S. P.
N. Iyer, D. E. Shevell, M. M. Smith, Y. S. Tang,
A. M. Makarewicz, F. Ujjainwalla, S. W. Altmann,
K. T. Chapman, N. A. Thornberry, PNAS 2005, 102, 8132–8137.
The authors have declared no conflict of interest.
[7] E. T. Bodor, S. Offermanns, Br. J. Pharmacol. 2008, 153, S68–
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