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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
2 3
Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmaceutical Sciences, Lonavala, Pune, India Department of Chemistry, Banasthali University, Tonk, India Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Mumbai, India
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 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: [email protected] Fax: þ91-2114-270258
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(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 synthesized 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
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HO
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Results and discussion
F
In silico (docking) studies
N F
OH O
Figure 1. Ezetimibe.
OH
N
O
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.
HO
F
Table 1. Results for molecular docking experiments of standard drug (ezetimibe) and synthesized compounds 4a–4d with NPC1L1 protein. Compound
OH
N
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. 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. Details of the docking methodology are presented in the Experimental section, and the results are summarized in Tables 1 and 2. All the crucial interactions observed between NPC1L1 and ezetimibe were also observed for the representative compounds 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
N
O
F
OH O
II
Docking score 6.381671 8.188506 4.806627 6.107940 5.159371
Ezetimibe 4a 4b 4c 4d
I Table 2. Comparison of the distances of the centroid of ligands backbone and amino acid residues of the active site.
HO
R
HN N R'
X
O
O
Residue
4a (Å)
Ezetimibe (Å)
Ser102 Leu213 His124 Leu103 Leu52 Ala101 Gln206 Thr128 Phe120 Thr106 Leu99 Ile105
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
Figure 3. Target compounds (4a–4h).
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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
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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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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.
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 HN NH2 R'
X
(a)
O
R'
CHO
1
HN N X
O
3
2 (b) HO
R
HN N R'
X
O
O
4a_4h
Compound
R'
R X
4a 4b
N
Cl Ph
4c
Cl
4d
Ph
4e
Cl
4f
N
4g 4h
Cl
Ph Cl Ph
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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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 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 cm1 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
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 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' Compound
X
N
3a
3b
3c
3d
N Cl
Molecular formula
Molecular weight
Melting point (°C)
Yield (%)
C13H11N3O2
241.25
262–264 (264–268) [14]
88.20
C14H12N2O2
240.26
285–287
89.45
C13H11N3O2
241.25
190–192
87.36
C14H11ClN2O2
274.70
280–282 (290–291) [15]
85.67
Table 4. Physical data of synthesized compounds (azetidinones) 4a–4h.
R'
R
Molecular formula
Molecular weight
Melting point (°C)
Yield (%)
Cl Ph
C15H12ClN3O3 C21H17N3O3
317.73 359.38
255–257 (98–110) [14] 198–200
68.66 60.54
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
Cl Ph
C16H12Cl2N2O3 C22H17ClN2O3
351.18 392.83
175–178 148–150
57.32 54.23
Compound 4a 4b
4g 4h a)
N
N Cl
X
Satisfactory elemental analysis (0.4 of the calculated values of %C, %H, and %N obtained). Please refer to the Experimental section.
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Table 5. Antihyperlipaemic activity data for synthesized compounds 4a–4h and correlation with the docking scores. Antihyperlipaemic activitya)
R' Compound
X –
Ezetimibe 4a 4b
R'
4c 4d
R'
4e 4f
R'
4g 4h
R'
X X X X
R
% Reduction in total cholesterol (mg dl1)
% Reduction in triglyceride (mg dl1)
% Change in HDL (mg dl1)
Docking scoreb)
–
52.39 5.18
34.26 5.37
31.50 3.83
6.381671
Cl Ph
51.07 3.09 42.20 1.92
34.40 1.27 31.40 1.27
33.92 0.33 30.26 1.08
8.188506 4.806627
Cl Ph
43.55 2.55 40.16 1.84
24.54 1.33 21.35 1.47
27.44 2.36 23.68 1.29
6.107940 5.159371
Cl Ph
49.42 2.57 47.62 1.97
36.11 1.32 35.66 2.42
24.12 2.38 23.50 3.14
— —
Cl Ph
45.33 1.69 40.08 2.33
30.24 2.07 25.25 1.11
27.62 2.13 18.78 1.52
— —
Results are expressed as mean 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 4a > 4c; (b) reduce serum levels of Tg 4a > ezetimibe > 4c, and (c) enhance serum levels of HDL, 4a > ezetimibe > 4c. a)
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 3.09%, 49.42 2.57%, 47.62 1.97%, and 45.33 1.69%, respectively, which was
comparable to the reduction in % serum cholesterol levels of the animals administered with ezetimibe (52.39 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 1.27%, 36.11 1.32%, and 35.66 2.42%, respectively, which was comparable to the reduction in % serum triglyceride levels of the test animals administered with ezetimibe (34.26 5.37%) (Fig. 7). Further, compound 4a also showed good ability (33.92 0.33% increase) to elevate % serum HDL levels of test animals comparable to the standard ezetimibe (31.5 3.83% increase) (Fig. 8).
Figure 6. % Reduction in total serum cholesterol levels.
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Figure 7. % Reduction in serum triglyceride levels.
Figure 8. % Change in serum HDL levels.
Conclusions 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.
Experimental General All the synthesized compounds were characterized by spectral data (IR, mass, and 1H NMR). The IR spectra of the synthesized ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
compounds were recorded on a Perkin Elmer BX-FTIR spectrophotometer (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. 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). www.archpharm.com
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Computational docking studies 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.
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.
Preparation of protein and ligand structures for docking simulations 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 inhibitorcomplexed 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. 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 distancedependent dielectric, until a gradient of 0.01 kcal mol1 Å1 was achieved.
Receptor grid generation 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 10 Å 10 Å box centered on the centroid of these residues in the crystal complex ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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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.
Chemistry General procedure for the preparation of Schiff’s bases as intermediates 3a–d 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.
N 0 -(4-Hydroxybenzylidene)isonicotinohydrazide (3a) Yield: 88.20%; mp: 262–264°C (lit. 264–268°C) [14]; IR (KBr): 3455, 3083, 1680, 1647, 1280, 850 cm1; 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).
N 0 -(4-Hydroxybenzylidene)benzohydrazide (3b) Yield: 89.45%; mp: 285–287°C; IR (KBr): 3266, 3190, 3058, 1601, 1284, 835 cm1; 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).
N 0 -(4-Hydroxybenzylidene)nicotinohydrazide (3c) Yield: 87.36%; mp: 190–192°C; IR (KBr): 3457, 3287, 3071, 1670, 1647, 1299, 833 cm1; 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 5.44 (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).
N 0 -(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 cm1; 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).
General procedure for the preparation of N-[4-(4hydroxyphenyl)-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 (0.015 mol) was added dropwise over 30 min followed by triethylamine (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 www.archpharm.com
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solution (10% w/v) followed by water and filtered under vacuum, dried, and recrystallized to give the desired compound 4.
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1yl)isonicotinamide (4a) Yield: 68.66%; mp: 255–257°C (lit. 98–110°C) [14]; IR (KBr): 3504, 3351, 3028, 2905, 1732, 1637, 1086, 766 cm1; 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; 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-1yl)isonicotinamide (4b) Yield: 60.54%; mp: 198–200°C; IR (KBr): 3448, 3302, 3011, 2923, 1734, 1663, 1045 cm1; 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).
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1yl)benzamide (4c) Yield: 61.34%; mp: 165–167°C (lit. 160–162°C) [16]; IR (KBr): 3478, 3284, 3032, 2906, 1732, 1660, 1057, 771 cm1; 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-1yl)benzamide (4d) Yield: 56.78%; mp: 136–139°C; IR (KBr): 3466, 3287, 3022, 2941, 1737, 1653, 1045 cm1; 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).
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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-4-chloro-N-(3-chloro-4-(4-hydroxyphenyl)-2oxoazetidin-1-yl)benzamide (4g) Yield: 57.32%; mp: 175–178°C; IR (KBr): 3550, 3318, 3030, 2853, 1734, 1647, 1189, 774 cm1; 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).
Trans-4-chloro-N-(4-(4-hydroxyphenyl)-2-oxo-3phenylazetidin-1-yl)benzamide (4h) Yield: 54.23%; mp: 148–150°C; IR (KBr): 3369, 3276, 3004, 2929, 1736, 1689, 1088, 780 cm1; 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; C22H17ClN2O3 (392.83); requires (found): C, 67.26 (67.35); H, 4.36 (4.27); N, 7.13 (7.28).
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 5°C and humidity of 40–50 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-octylpolyoxyethylene phenol (tyloxapol), was used to induce hyperlipidemia. The animals were divided into eleven groups of six animals of either sex per group. Group I Group II Group III Groups: 4a–4h
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1yl)nicotinamide (4e) Yield: 64.24%; mp: 262–264°C (lit. 195°C) [17]; IR (KBr): 3469, 3299, 3015, 2864, 1740, 1653, 1034, 784 cm1; 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).
Trans-N-(4-(4-hydroxyphenyl)-2-oxo-3-phenylazetidin-1yl)nicotinamide (4f) Yield: 59.11%; mp: 150–153°C; IR (KBr): 3421, 3315, 3038, 2906, 1732, 1645, 1138 cm1; 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
The control group received only vehicle (2% acacia solution as well as normal saline). The cholesterol control group received Triton WR 1339 (200 mg kg1) by i.p. route in normal saline solution. The standard group received Triton WR 1339 (200 mg kg1 i.p.) as well as ezetimibe (1 mg kg1 p.o.) as suspension in 2% w/v acacia (aq.). The test groups 4a–4h received Triton WR 1339 (200 mg kg1 i.p.) as well as test compound (10 mg kg1 p.o.) as suspension in 2% w/v acacia (aq.)
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 www.archpharm.com
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Ezetimibe Analogs
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. The authors have declared no conflict of interest.
References [1] a) K. S. Jain, R. R. Kulkarni, D. P. Jain, Mini-Rev. Med. Chem. 2010, 10, 232–262; b) H. C. McGill, Jr., Geographical Pathology of Atheroslclerosis, Williams and Wilken Co, Baltimore 1985; c) M. L. Overturf, D. S. Loose-Mitchell, Curr. Opin. Lipidol. 1992, 3, 179–185; d) A. Ghatak, O. Asthana, Indian J. Pharmacol. 1995, 27, 14–29; e) P. Schwandt, Klin. Wochenschr 1990, 68, 54–58. [2] a) http://www.americanheart.org/statistics. (American Heart Association: 2001 Heart and Stroke Statistical Update.Dallas Texas: American Heart Association 2000); b) K. S. Jain, M. K. Kathiravan, R. S. Somani, C. J. Shishoo, Bioorg. Med. Chem. 2007, 15, 4674–4699. [3] a) P. D. Mehta, N. P. S. Sengar, A. K. Phathak, Eur. J. Med. Chem. 2010, 45, 5541–5560; b) G. S. Singh, Mini-Rev. Med. Chem. 2004, 4 (69), 93; c) M. Toraskar, V. Kulkarni, V. Kadam, J. Pharm. Res. 2010, 3, 169–173; d) V. Wable, A. Pathak, T. Patil, Med. Chem. 2012, 4, 15. [4] a) J. A. Pfefferkorn, S. D. Larsen, C. V. Huis, R. Sorenson, T. Barton, T. Winters, B. Auerbach, C. Wu, T. J. Wolfram, H. Cai, K. Welch, N. Esmaiel, J. Davis, R. Bousley, K. Olsen, S. B. Mueller, T. Mertz, Bioorg. Med. Chem. Lett. 2008, 18, 546– 553; b) X. Xianxiu, F. Renzhong, C. Jin, C. Shengwu, B. Xu, Bioorg. Med. Chem. Lett. 2007, 17, 101–104; c) Y. Wang, H. W. Huang, H. Zhang, J. Zhou, Lett. Drug Des. Discov. 2011, 8, 500; d) N. Arya, M. K. Munde, J. Dwivedi, A. Y. Jagdale, K. S. Jain, Arch. Pharm. Chem. Life Sci. 2013, 346, 588–595. [5] a) http://www.dailyfinance.com/2013/05/03/fda-approvesmercks-Hpt (FDA approves Merck’s LIPTRUZETTM (ezetimibe and atorvastatin), a new product that can help powerfully lower LDL cholesterol. In a clinical study, LIPTRUZETTM powerfully lowered LDL-C across all doses on average by more than 50%–63% in a dosage range of 10/10 mg/day through 10/80 mg/day.); b) M. K. Kathiravan, M. K. Munde, D. P. Jain, K. S. Jain, Indian Drugs 2009, 46, 91–103; c) www. indstate.edu/theme/mwking/lipid. (This home page belongs to Indiana state university and Carnegie foundation built for the advancement of teaching, Michael, W; King, Ph.D./IU School of Medicine). [6] a) L. Ge, J. Wang, W. Qi, H. H. Miao, J. Cao, Y. X. Qu, B. L. Li, B. L. Song, Cell Metabol. 2008, 7, 508–519; b) A. B. Weinglass, M. Kohler, U. Schulte, J. Liu, E. O. Nketiah, A. Thomas, W. Schmalhofer, B. Williams, W. Bildl, D. R. McMasters, K. Dai, L. Beers, M. E. McCann, G. J. Kaczorowski, M. L. Garcia,
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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
2 3
Department of Pharmaceutical Chemistry, Sinhgad Institute of Pharmaceutical Sciences, Lonavala, Pune, India Department of Chemistry, Banasthali University, Tonk, India Department of Pharmaceutical Chemistry, Bombay College of Pharmacy, Mumbai, India
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 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: [email protected] Fax: þ91-2114-270258
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(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 synthesized 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
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HO
Ezetimibe Analogs
873
Results and discussion
F
In silico (docking) studies
N F
OH O
Figure 1. Ezetimibe.
OH
N
O
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.
HO
F
Table 1. Results for molecular docking experiments of standard drug (ezetimibe) and synthesized compounds 4a–4d with NPC1L1 protein. Compound
OH
N
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. 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. Details of the docking methodology are presented in the Experimental section, and the results are summarized in Tables 1 and 2. All the crucial interactions observed between NPC1L1 and ezetimibe were also observed for the representative compounds 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
N
O
F
OH O
II
Docking score 6.381671 8.188506 4.806627 6.107940 5.159371
Ezetimibe 4a 4b 4c 4d
I Table 2. Comparison of the distances of the centroid of ligands backbone and amino acid residues of the active site.
HO
R
HN N R'
X
O
O
Residue
4a (Å)
Ezetimibe (Å)
Ser102 Leu213 His124 Leu103 Leu52 Ala101 Gln206 Thr128 Phe120 Thr106 Leu99 Ile105
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
Figure 3. Target compounds (4a–4h).
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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
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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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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.
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 HN NH2 R'
X
(a)
O
R'
CHO
1
HN N X
O
3
2 (b) HO
R
HN N R'
X
O
O
4a_4h
Compound
R'
R X
4a 4b
N
Cl Ph
4c
Cl
4d
Ph
4e
Cl
4f
N
4g 4h
Cl
Ph Cl Ph
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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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 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 cm1 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
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 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' Compound
X
N
3a
3b
3c
3d
N Cl
Molecular formula
Molecular weight
Melting point (°C)
Yield (%)
C13H11N3O2
241.25
262–264 (264–268) [14]
88.20
C14H12N2O2
240.26
285–287
89.45
C13H11N3O2
241.25
190–192
87.36
C14H11ClN2O2
274.70
280–282 (290–291) [15]
85.67
Table 4. Physical data of synthesized compounds (azetidinones) 4a–4h.
R'
R
Molecular formula
Molecular weight
Melting point (°C)
Yield (%)
Cl Ph
C15H12ClN3O3 C21H17N3O3
317.73 359.38
255–257 (98–110) [14] 198–200
68.66 60.54
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
Cl Ph
C16H12Cl2N2O3 C22H17ClN2O3
351.18 392.83
175–178 148–150
57.32 54.23
Compound 4a 4b
4g 4h a)
N
N Cl
X
Satisfactory elemental analysis (0.4 of the calculated values of %C, %H, and %N obtained). Please refer to the Experimental section.
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Table 5. Antihyperlipaemic activity data for synthesized compounds 4a–4h and correlation with the docking scores. Antihyperlipaemic activitya)
R' Compound
X –
Ezetimibe 4a 4b
R'
4c 4d
R'
4e 4f
R'
4g 4h
R'
X X X X
R
% Reduction in total cholesterol (mg dl1)
% Reduction in triglyceride (mg dl1)
% Change in HDL (mg dl1)
Docking scoreb)
–
52.39 5.18
34.26 5.37
31.50 3.83
6.381671
Cl Ph
51.07 3.09 42.20 1.92
34.40 1.27 31.40 1.27
33.92 0.33 30.26 1.08
8.188506 4.806627
Cl Ph
43.55 2.55 40.16 1.84
24.54 1.33 21.35 1.47
27.44 2.36 23.68 1.29
6.107940 5.159371
Cl Ph
49.42 2.57 47.62 1.97
36.11 1.32 35.66 2.42
24.12 2.38 23.50 3.14
— —
Cl Ph
45.33 1.69 40.08 2.33
30.24 2.07 25.25 1.11
27.62 2.13 18.78 1.52
— —
Results are expressed as mean 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 4a > 4c; (b) reduce serum levels of Tg 4a > ezetimibe > 4c, and (c) enhance serum levels of HDL, 4a > ezetimibe > 4c. a)
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 3.09%, 49.42 2.57%, 47.62 1.97%, and 45.33 1.69%, respectively, which was
comparable to the reduction in % serum cholesterol levels of the animals administered with ezetimibe (52.39 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 1.27%, 36.11 1.32%, and 35.66 2.42%, respectively, which was comparable to the reduction in % serum triglyceride levels of the test animals administered with ezetimibe (34.26 5.37%) (Fig. 7). Further, compound 4a also showed good ability (33.92 0.33% increase) to elevate % serum HDL levels of test animals comparable to the standard ezetimibe (31.5 3.83% increase) (Fig. 8).
Figure 6. % Reduction in total serum cholesterol levels.
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Figure 7. % Reduction in serum triglyceride levels.
Figure 8. % Change in serum HDL levels.
Conclusions 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.
Experimental General All the synthesized compounds were characterized by spectral data (IR, mass, and 1H NMR). The IR spectra of the synthesized ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
compounds were recorded on a Perkin Elmer BX-FTIR spectrophotometer (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. 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). www.archpharm.com
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Computational docking studies 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.
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.
Preparation of protein and ligand structures for docking simulations 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 inhibitorcomplexed 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. 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 distancedependent dielectric, until a gradient of 0.01 kcal mol1 Å1 was achieved.
Receptor grid generation 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 10 Å 10 Å box centered on the centroid of these residues in the crystal complex ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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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.
Chemistry General procedure for the preparation of Schiff’s bases as intermediates 3a–d 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.
N 0 -(4-Hydroxybenzylidene)isonicotinohydrazide (3a) Yield: 88.20%; mp: 262–264°C (lit. 264–268°C) [14]; IR (KBr): 3455, 3083, 1680, 1647, 1280, 850 cm1; 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).
N 0 -(4-Hydroxybenzylidene)benzohydrazide (3b) Yield: 89.45%; mp: 285–287°C; IR (KBr): 3266, 3190, 3058, 1601, 1284, 835 cm1; 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).
N 0 -(4-Hydroxybenzylidene)nicotinohydrazide (3c) Yield: 87.36%; mp: 190–192°C; IR (KBr): 3457, 3287, 3071, 1670, 1647, 1299, 833 cm1; 1H NMR (400 MHz, CDCl3): d (ppm) ¼ 5.44 (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).
N 0 -(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 cm1; 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).
General procedure for the preparation of N-[4-(4hydroxyphenyl)-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 (0.015 mol) was added dropwise over 30 min followed by triethylamine (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 www.archpharm.com
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solution (10% w/v) followed by water and filtered under vacuum, dried, and recrystallized to give the desired compound 4.
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1yl)isonicotinamide (4a) Yield: 68.66%; mp: 255–257°C (lit. 98–110°C) [14]; IR (KBr): 3504, 3351, 3028, 2905, 1732, 1637, 1086, 766 cm1; 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; 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-1yl)isonicotinamide (4b) Yield: 60.54%; mp: 198–200°C; IR (KBr): 3448, 3302, 3011, 2923, 1734, 1663, 1045 cm1; 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).
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1yl)benzamide (4c) Yield: 61.34%; mp: 165–167°C (lit. 160–162°C) [16]; IR (KBr): 3478, 3284, 3032, 2906, 1732, 1660, 1057, 771 cm1; 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-1yl)benzamide (4d) Yield: 56.78%; mp: 136–139°C; IR (KBr): 3466, 3287, 3022, 2941, 1737, 1653, 1045 cm1; 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).
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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-4-chloro-N-(3-chloro-4-(4-hydroxyphenyl)-2oxoazetidin-1-yl)benzamide (4g) Yield: 57.32%; mp: 175–178°C; IR (KBr): 3550, 3318, 3030, 2853, 1734, 1647, 1189, 774 cm1; 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).
Trans-4-chloro-N-(4-(4-hydroxyphenyl)-2-oxo-3phenylazetidin-1-yl)benzamide (4h) Yield: 54.23%; mp: 148–150°C; IR (KBr): 3369, 3276, 3004, 2929, 1736, 1689, 1088, 780 cm1; 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; C22H17ClN2O3 (392.83); requires (found): C, 67.26 (67.35); H, 4.36 (4.27); N, 7.13 (7.28).
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 5°C and humidity of 40–50 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-octylpolyoxyethylene phenol (tyloxapol), was used to induce hyperlipidemia. The animals were divided into eleven groups of six animals of either sex per group. Group I Group II Group III Groups: 4a–4h
Trans-N-(3-chloro-4-(4-hydroxyphenyl)-2-oxoazetidin-1yl)nicotinamide (4e) Yield: 64.24%; mp: 262–264°C (lit. 195°C) [17]; IR (KBr): 3469, 3299, 3015, 2864, 1740, 1653, 1034, 784 cm1; 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).
Trans-N-(4-(4-hydroxyphenyl)-2-oxo-3-phenylazetidin-1yl)nicotinamide (4f) Yield: 59.11%; mp: 150–153°C; IR (KBr): 3421, 3315, 3038, 2906, 1732, 1645, 1138 cm1; 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
The control group received only vehicle (2% acacia solution as well as normal saline). The cholesterol control group received Triton WR 1339 (200 mg kg1) by i.p. route in normal saline solution. The standard group received Triton WR 1339 (200 mg kg1 i.p.) as well as ezetimibe (1 mg kg1 p.o.) as suspension in 2% w/v acacia (aq.). The test groups 4a–4h received Triton WR 1339 (200 mg kg1 i.p.) as well as test compound (10 mg kg1 p.o.) as suspension in 2% w/v acacia (aq.)
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 www.archpharm.com
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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. The authors have declared no conflict of interest.
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