DESIGN, SYNTHESIS AND VIRTUAL SCREENING OF CERTAIN 2-PYRAZOLIN-5-ONE AND PYRAZOLIDINE-3, 5-DIONE DERIVATIVES AS POTENTIAL PPAR AGONISTS
HTML Full TextDESIGN, SYNTHESIS AND VIRTUAL SCREENING OF CERTAIN 2-PYRAZOLIN-5-ONE AND PYRAZOLIDINE-3, 5-DIONE DERIVATIVES AS POTENTIAL PPARg AGONISTS
Mohamed A. H. Ismaila,*, Dalal A. Abou El Ella, Khaled A. M. Abouzid, and Maiy Jaballah
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, El Khalifa ElMaamoon St., 11566, Abbasseya, Cairo, Egypt
ABSTRACT
In the quest for novel PPARg agonists as putative drugs for the treatment of type 2 diabetes, a new series of 2-pyrazolin-5-one and pyrazolidine-3, 5-dione derivatives, were designed and synthesized, as analogues to the anti-diabetic thiazolidinedione agents (TZDs). Extensive molecular modeling studies for the designed molecules were performed; including their compare-fit studies on the generated and validated PPARg agonist's hypothesis and their molecular docking on the binding sites of the 3D structure of the PPARg receptors.
Keywords:
PPARg, Pyrazolin-5-one, Pyrazolidine-3, 5-dione, |
Design
INTRODUCTION: Diabetes is the fourth leading killer disease in the developed world. There are more than 200 million diabetics worldwide, accounting for a huge economic and social burden 1, 2. Thus, the development of new effective therapeutic agents is the major thrust for research and is germane to both the national and international scenario. Type 2 diabetes mellitus (T2DM) is the most important type of diabetes, as more than 80% of diabetics are of this class.
The peroxisome proliferator-activated receptors (PPARs) are legitimate molecular targets for the development of the antidiabetic agents. The reported synthetic ligands such as rosiglitazone (I) and pioglitazone (II) 3, 4 had high affinity for PPAR-γ receptor, belong to the thiazolidinediones (TZDs) class of antidiabetic agents (Fig. 1) and have significantly improved the clinical situation of Type 2 diabetics.
Unfortunately, theses agents have serious side effects of hepatotoxicity, weight gain, and edema. This situation emphasized the need to identify strategies to develop new antihyperglycemic agents that could retain the insulin sensitizing properties of TZDs through PPARγ agonistic activities with minimum or no adverse side effects. This necessitated a search for highly effective and safe antihyperglycemic agents, particularly those that normalize both insulin and glucose levels.
FIGURE 1: ROSIGLITAZONE (I) AND; PIOGLITAZONE (II)
A survey of various classes of PPARg agonists, 3D-QSAR studies and crystal structure information reveals that the pharmacophoric features of these agents essentially consists of three parts:
- an acidic head group,
- central aromatic region and
- terminal lipophillic side chain and a linker ( 2) 5a. These features are clearly represented in the highly potent lead molecule (cpd 154329) 5a.
The TZD ring of rosiglitazone is reported to make three important H-bonds with His323, Tyr473 and His449 that are important for the activation of the receptor 5b. This observation was instrumental in the design of a variety of non-TZD PPAR agonists based on free carboxyl group, oxazolidinedione, tetrazoles, etc 6. However, such reports are limited and most of the reported PPARg agonists still belong to either ‘glitazone’ class (possessing TZD ring) or ‘glitazar’ class (possessing free carboxylic acid). Thus, we set our objective to explore newer head groups for designing novel series of PPARg ligands.
FIGURE 2 5: PROPOSED LEAD FEATURES OF PPARg AGONIST AGENTS REPRESENTED ON CPD (154329)
The design of new molecules (4a-c, 5-8, 9a-c, 10a, b, 11a, b and 12a, b) in this investigation was based on, firstly, the ligand receptor binding knowledge and bioisoteric replacement of TZD head group by 2-pyrazolin-5-one and pyrazolidinedione ring systems, or the potentially active malonate precursors, as it was reported earlier that malonate derivatives provide potent PPAR g agonists, as well as, isosteric replacements in both the central aromatic (flat hydrophobic region) and the terminal hydrophobic side chain (Fig. 2).
Secondly, the perfect selection of the final PPARg agonist active hits was peformed by virtual screening through molecular modeling simulation studies. Such studies involved two important techniques:
- Generation and validation of novel PPARg agonist hiphop hypothesis, derived from frozen bioactive conformations of reported PPARg agonists, using cerius2 and catalyst software modules, followed by utilizing the compare-Fit studies between the hypothesis and the proposed test set targeted molecules. The molecules having high fit values with low conformation energies are considered active hits.
- The promising test set compounds which showed high fit values to the generated hypothesis were subjected to further molecular modeling studies employing the docking program Molsoft (ICM 3.4-8c) in order to investigate the binding mode of the designed compounds and evaluate their binding affinities through the calculation of their docking scores.
The above two virtual screening studies indicated that the molecules (4a-c, 5-8, 9a-c, 10a,b, 11a, b and 12a, b) have high potential PPARg agonist activities. Thus, these molecules are synthesized adopting Schemes 1 and 2; in order to be available for further in-vivo testing for their effect on T2DMs.
RESULTS:
- Synthesis of 2-pyrazolin-5-one, pyrazolidine-3,5-diones: In scheme 1, the synthesis of the designed 2-pyrazoline-5-one derivatives (4a, b-8) was carried out through uncatalysed Knoevenagel condensation of the substituted aldehydes (2a-e) with 3-methylpyrazolin-5(4H)-one and 3-methyl-1-phenyl-2-pyrazolin-5(4H)-one molecules to produce 4a, b-8 following the procedure described by Mohit 6.
In scheme 2, condensation of aldehydes (3a-c) with 3-methylpyrazolin-5(4H)-one and 3-methyl-1-phenyl-2-pyrazolin-5(5H)-one molecules yielded the pyrazoline derivative (9a-c). Pyrazolidine-3,5-diones, (12a, b), were synthesized starting from the aldehyde derivatives (2a, b) through knoevenagel condensation with diethyl malonate to produce the arylidene derivatives 7 (10a, b) followed by catalytic reduction of the benzylidene double bond to produce the aryl methyl derivatives (11a, b). The latter intermediates were reacted with hydrazine hydrate in presence of sodium ethoxide to produce (12a, b).
SCHEME 1: Compounds: 2a, R= 4(3H)-Quinazolonyl; 2b, R= 2-Methyl-4(3H)-quinazolonyl; 2c, R= 4-(N-acetamido)phenyl; 2d, Benzo[d]thiazole-2-thiolyl; 2e, R=4-(3-Chlorophenyl)piperazin-1-yl. Reagents: A= 3-substituted-propyl chloride, acetone or DMF, K2CO3; B= 3-methylpyrazolin-5(4H)-one or 3-methyl-1-phenyl-2-pyrazolin-5(4H)-one, H2O/ Ethanol, pipredine
SCHEME 2: Compounds: 3a, R= 4-Cl; 3b, R= 4-CH3; 3c, R= 3,5-CH3; 9a, R= 4-Cl, X= H; 9b R= 4-Cl, X= Ph; 9c, R= 4-CH3, X=Ph; 9d, R= 3,5(CH3)2, X= Ph; 10a, R= 4-CH3; 10b, R= 3,5(CH3)2. Reagents: C= p-hydroxybenzaldehyde, acetone, K2CO3; D= 3-methylpyrazolin-5(4H)-one or 3-methyl-1-phenyl-2-pyrazolin-5(4H)-one, H2O/ Ethanol, pipredine; E= Diethyl malonate, benzene, acetic acid, pipredine; F= abs. Ethanol, H2/pd; G= abs. ethanol, hydrazine hydrate, sodium ethoxide
- Generation of PPARg Hiphop Hypothesis: The docked leads, Rosiglitazone (I), 3 AZ242 (III), 8 YPA101(IV), 9 570200(V), 10 54441(VI),11 DRF 101(VII) 12 (Figure 3), at the PARg binding site , were selected from their binding sites and used without any conformational changes to generate the common feature hypotheses of the PPARg Using the protein tool deck in Cerius 2 software, such frozen bioactive conformers of the mentioned lead compounds, were used to generate the common feature hypotheses (by default), where 10 hypotheses were generated.
The assessment of the ideal hypothesis among the generated ones indicated that hypothesis no. 7 was the ideal one, as it encompasses the crucial constraint features for selective PPARg agonists; where the orientation of the two ring aromatic features were perpendicular to each other, which coincide with the gauche conformation adopted by the ligands in the PPARg binding site as demonstrated in Fig. 4a, 4b, 5. In addition, hypothsis no. 7 had more consistent simulation fitting values to the respective ligands and the test set compounds than other hypotheses.
Such an ideal hypothesis, that is reported here as the first time by our research team, encompassed four features namely; two hydrogen bond acceptor (HBA1 and HBA2), two ring aromatic features (RA1 and RA2) (figures 4a, b). Herein, we report the range of constraint angles and distances between all features; (Table 1) and Figures (4a, 4b, 5).
TABLE 1: CONSTRAINT DISTANCES AND ANGLES BETWEEN FEATURES OF THE GENERATED PPARg AGONIST HYPOTHESIS
Dimensions | Our Catalyst ACEI Hypothesis (values are recorded in ranges) |
Constraint distances (Å) between different features | RA2-HBA1,10.159-12.159; RA2-HBA2,10.625-12.625;
RA1-RA2, 6.144-8.144; RA1-HBA1, 5.808-7.808; RA1-HBA2, 4.951-6.951; HBA1-HBA2, 3.433-5.433 |
Constraint angles (o) between different features | HBA1-RA2-RA1,41.2988-51.2998; RA2-RA1-HBA2, 119.897-129.898;
RA2-RA1-HBA1, 101.199-111.2; RA1-HBA2-HBA1, 36.8986-86.8993. |
FIGURE 3: STRUCTURES OF PPARg AGONIST LEAD COMPOUNDS USED FOR THE GENERATION OF PPARg AGONIST HYPOTHESIS.
FIGURE 4A: CONSTRAINT DISTANCES OF PPARg AGONIST HYPOTHESIS
FIGURE 4B: CONSTRAINT ANGLES OF PPARg AGONIST HYPOTHESIS
FIGURE 5: MAPPING OF PPARg AND TetVIII
Further validation of the PPARg agonist hypothesis was assessed through the following:
- The database search study for examining the mapping of such PPARg agonist hypothesis with the molecular structures of the provided databases with Catalyst 4.8, namely NCI 2000, Maybridge 2001, and MiniMaybridge, which contain 296092 compounds, revealed that only 467 molecules (~002% of the total), have been retrieved from the databases (table 2), including several anti-T2DMs agents. Such a low percentage of the recognized database molecules by our hypothesis, may indicate a reasonable selectivity for this hypothesis.
TABLE 2: MAPPING OF CATALYST DATABASE MOLECULES WITH PPARg AGONIST HYPOTHESIS
Database | Total numbers of molecules | Number of retrieved (mapped) hits. | % of retrieved (mapped) hits |
NCI2000 | 238819 | 250 | 0.001 |
Maybridge2001 | 55273 | 193 | 0.003 |
MiniMaybridge | 2000 | 24 | 0.012 |
Total | 296092 | 467 | 0.002 |
- Further validation and evaluation of the predictive power of the hypothesis was performed through mapping of compounds having potent PPARg agonistic activity; obtained from literature, viz; Tet (VIII) 13, DRH (IX) 14, CO1(X) 15, 3EA(XI) 16, PLB(XII) 17 (Figure 6). The mapping results indicated high fit values with these compounds (table 3).
FIGURE 6: KNOWN PPARg , EVALUATION SET COMPOUNDS, WHICH WERE MAPPED BY OUR PPARg AGONIST HYPOTHESIS
TABLE 3: COMPARE/FIT AND CONFORMATIONAL ENERGY VALUES OF THE BEST FITTED CONFORMERS OF THE EVALUATION SET COMPOUNDS (VIII-XII) AND PPARg AGONIST HYPOTHESIS
Cpd. | No. of conformers | Conf. energy | Fit Value/4 |
Tet VIII13 | 151 | 10.10 | 3.45 |
DRH IX14 | 205 | 14.64 | 2.93 |
COI X15 | 81 | 12.99 | 2.5 |
3EA XI16 | 229 | 4.75 | 2. 79 |
PLB XII17 | 72 | 8.6 | 2.95 |
Results of The Compare/Fit studies of compounds (4a-c, 5-8, 9a-c, 10a,b, 11a,b, and 12a,b): The compare/fit score values mentioned in table 4, revealed that most of the designed molecules showed high fitting affinities to the generated ideal new PPARg receptor agonist hypothesis, indicated that all of these molecules have promising activity as PPARg agonists and compounds 7, 8, 11a & 12a are the most promising hits. Accordingly, further molecular docking studies of 7, 8, 11a & 12a with the 3D PPARg receptor binding sites would be performed.
TABLE 4: COMPARE/FIT AND CONFORMATIONAL ENERGY VALUES OF THE BEST FITTED CONFORMERS OF 2-PYRAZOLIN-5-ONE AND 3,5-PYRAZOLIDINEDIONES AND THE PPARg AGONIST HYPOTHESIS
Compounds | Number of conformers | Conf. energy at the agonist hyothesis
(Kcal mol-1) |
Fitting values with PPARg agonist hypothesis |
Rosiglitazone (I) | 1 | - | 3.99 |
9b | 78 | 9.02 | 2.49 |
9c | 81 | 16.13 | 2.36 |
9d | 118 | 5.95 | 2.88 |
9a | 77 | 16 | 2.87 |
4a | 116 | 2.3 | 2.86 |
4b | 130 | 9.47 | 2.84 |
5 | 122 | 12.92 | 2.94 |
6 | 124 | 12.14 | 2.43 |
7 | 160 | 5.5 | 2.89 |
8 | 178 | 6.355 | 2.98 |
11a | 204 | 7.21 | 3.54 |
11b | 211 | 6.165 | 3.45 |
12a | 137 | 12.7 | 3.22 |
12b | 191 | 15.46 | 3.28 |
- Docking results: Molecular modeling docking studies using Molsoft software between the 3D PPARg receptor binding sites and small molecules were performed. The docking scores (displayed in (DG) energy terms) of binding of a ligand to a receptor, is based upon the virtual calculation of various interaction of various small molecular ligand with protein. The present study adopted rigid receptor/flexible ligand approach between the 3D PPARg receptor binding sites and small molecules of a lead drug and the designed molecules. Five potential energy maps combining hydrophobicity, electrostatics, hydrogen bond formation, and two van der Waal parameters were selected by default, to get the docking score 18. The Molsoft could be used to deduce the binding affinity (DG values) of the lead molecule; Rosiglitazone (1) and the binding affinities of the promising test set molecules (7, 8, 11a & 12a) with the targeted PPARg The study indicated that these test set molecules have potential agonist activity similar or higher than Rosiglitazone. Table 5 shows the recorded docking score of Rosiglitazone in comparizone to the active hit molecules (7, 8, 11a & 12a).
TABLE 5: FITTING VALUES WITH PPARg AGONIST HYPOTHESIS AND DOCKING SCORE ENERGIES AT THE PPARg BINDING SITE OF COMPOUNDS (I, 7, 8, 11a, 12a)
Compounds | Fitting values with PPARg agonist hypothesis | Docking score/ G(Kcal/mol) | Amino acid residues and corresponding hydrogen bond s length in Å |
Rosiglitazone (I) | 3.99 | -96.35 | S289 : 1.90 A
H323: 1.75 A H449: 1.87 A Y473 : 2.27 A |
7 | 2.89 | -95.22 | R288 : 2.68 A
S342 : 1.70 A R288: 1.71 A S289: 1.93 A |
8 | 2.98 | -89.41 | H323: 1.68 A
H449: 2.45 A Y473: 2.24 A |
11a | 3.54 | -114.46 | R288: 1.48 A
R288: 1.86 A S342: 2.35 A S342 :2.66 A |
12a | 3.22 | -95.9 | S289: 2.53 A
H323: 2.24 A Q286 : 2.62 A Y473: 2.41 A |
All selected compounds (7, 8, 11a, 12a) showed similar or relatively high docking scores in the PPARg binding pocket compared to rosiglitazone (I). Compound 12a, form hydrogen bonding interaction to the same aminoacid residues as those that interact with Rosiglitazone (I) in the PPARg binding pocket; 18 (Fig. 6a).
FIGURE 6A: SHOWS SIGNIFICANT ALIGNMENT INDICATED BY SUPERIMPOSITION OF THE MIDDLE PHENOXY RING AND SIMILARITIES IN THE HEAD GROUP PYRAZOLIDINE-3, 5-DIONE IN 12A (PINK) AND THIAZOLIDINEDIONE RING OF ROSIGLITAZONE (WHITE). The carbonyl oxygen of rosiglitazone (I) is considered as the most essential pharmacophore for the binding with PPARg. Projection of pyrazolidine-3, 5-dione ring in 12a, toward thiazolidinedione region of I as illustrated, prompted to visualize its importance to act as a head group.
However, compound 11a that showed the highest docking score, makes hydrogen bonding interactions with amino acid residue no. S342 and R288 indicating a different binding mode than that of Rosiglitazone (Fig. 6b).
FIGURE 6B: COMPOUND 11A DOCKED AT PPARg BINDING POCKET WITH HYDROGEN BOND FORMATION TO RESIDUES S342, R288
Conclusion of Molecular Modeling Studies: Comparing the Fit values of the virtual screening of the generated and validated PPARg agonist hypothesis obtained by using Catalyst HipHop modules, together with the docking score of ICM-Pro (as illustrated in table (5) indicated that the fit values for the selected compounds (7, 8, 11a, 12a) with the ideal valid hypothesis were coincide with their docking scores. The binding of the compound (11a) exhibit a different hydrogen bond interaction when compared to Rosiglitazone, through forming hydrogen bond interaction with amino acid residues; R288 and S342. Such binding showed high affinity to the receptor than that of Rosiglitazone. This may indicate that such new binding mode may increase the PPARg agonistic activity.
Accordingly, these active hit molecules were synthesized to be available for future in vitro and in vivo evaluation as anti-type 2 diabetes mellitus (anti-T2DM).
Experimental: Melting points were determined with a Stuart Scientific apparatus and are uncorrected. FT-IR spectra were recorded on a Perkin-Elmer spectrophotometer and measured by m0 cm_1 scale using KBr cell. 1H NMR spectra were measured in d scale on a JEOL 270 MHz spectrometer. Unless otherwise stated, the spectra were obtained on solutions in DMSO and referred to TMS. The electron impact (EI) mass spectra were recorded on Finnigan Mat SSQ 7000 (70 ev) mass spectrometer. The peak intensities, in parentheses, are expressed as percentage abundance.
Analytical thin layer chromatography (TLC) was performed on Merk Kieselgel 60PF254 silica on Aluminum backed sheets. All Rf values were recorded from the center of the spots. All TLC solvent proportions were measured volume by volume. Column chromatography was performed using Merk Silica gel (mesh size = 0.032–0.064 mm). All reagents and solvents were purified and dried by standard techniques. Solvents were removed under reduced pressure in a rotary evaporator. Elemental microanalysis was performed at the Microanalytical Center, Ain-Shams University. Catalytic hydrogenation was performed under atomospheric pressure at the National Research Centre, Dr. Nabil Aboul-enin laboratory. The preparation of 3-Methyl-1-phenyl-1H-pyrazolin-5(4H)-one, 19 3-Methylpyrazol-5-one, 20 N-[4-(3-chloropropoxy) phenyl] acetamide, 21 Benzo[d] thiazole-2-thiol, 22 3-(3-Chloropropyl)-2- substituted quinazoline-4(3H)-on 23 was performed according to the reported procedures.
- Preparationof4-[3-(2-substituted-3,4-dihydro-4-oxoquinazolin-3-yl)-propoxy]benzald-ehyde (2a, b):
General Procedure: A mixture of the 3-(3-Chloropropyl)-2- substituted quinazoline-4(3H)-one (15 mmol of each), few specks of KI, 4-hydroxybenzaldehyde (1.83 g, 15 mmol) and anhydrous K2CO3 (2.75 g, 20 mmol) in dry acetone (30 ml) was refluxed for 8 hrs, cooled and filtered. The filtrate was distilled under vacuum till dryness, the residue was washed with 10% NaOH solution, extracted with ethylacetate. The organic layer was dried over anhydrous Na2SO4, then concentrated in vacuo. The resulting solid was separated, crystallized from acetone.
- For compound (2a): It was separated as white crystals (66% yield), m.p. 110° (Found: C, 70.35; H, 5.12; N, 8.98, C18H16N2O3 requires C, 70.12; H, 5.23; N, 9.09) MS (EI): m/z 308 (M+, 40%); IR(FT): 1684 (CO aldehydic). 1H NMR; 2.18 (m, 2H, J=7 Hz, -N-CH2-CH2-CH2-O-), 3.56 (t, 2H, J= 7Hz,-N-CH2-CH2-CH2-O-), 4.15 (t, 2H, J=7 Hz , -N-CH2-CH2-CH2-O-), 6.78-6.90 (m, 3H, Ar-H), 7.51-7.82 (m, 3H, Ar-H ), 8.13-8.15 (d, 2H, Ar-H ), 8.35 (S, 1H, quinazolyl-2H-), 9.94 (S, 1H, -CHO).
- For compound (2b): It was separated as faint yellowish white crystals. (53% yield), m.p. 107° (Found: C, 71.1; H, 5.43; N, 9.47, C19H18N2O3 requires C, 70.79; H: 5.63; N, 8.69) MS (EI): m/z 322 (M+, 4.5%); IR (FT): 1695(CO aldehydic). 1H NMR; 2.17(m, 2H, J=7 Hz , -N-CH2-CH2-CH2-O-, 3H, CH3), 3.58 (t, 2H, J= 7Hz,-N-CH2-CH2-CH2-O-), 4.20 (t, 2H, J=7 Hz , -N-CH2-CH2-CH2-O-), 6.92-7.07 (d, 2H, Ar-H ), 7.43-7.64 (m, 3H, Ar-H ), 7.74-7.84 (m, 3H, Ar-H ), 9.94 (S, 1H, -CHO).
- Preparation of N-[4-[3-(4-Formylphenoxy) propoxy] phenyl]acetamide (2c): To a mixture of N-[4-(3-chloropropoxy)phenyl]acetamide (3.41 g, 15 mmol), anhydrous K2CO3 (20 mmol) in DMF (20 mL) and few specks of KI, 4-hydroxybenzaldehyde (15 mmol) was added. The reaction mixture was stirred in a water bath at 70°C for 36 h, cooled then evaporated till near dryness under vaccum, ice cold water and brine were added where white solid was precipitated. The precipitate was filtered, washed with water and 10% NaOH solution, dried, then crystallized from acetone-ether mixture to produce the title compound (9).
- For compound (2c): It was separated as faint yellowish white crystals. (90% yield), m.p. 120° (Found: C, 65.12; H, 5.67; N, 11.21, C18H19NO4 requires C, 65.38; H: 5.76; N, 11.44) MS (EI): m/z 313 (M+, 4.5%); IR (FT): 3249 (NH Stretching). 1H NMR; 1.97 (s, 3H, NH-CO-CH3), 2.15 (m, 2H, J=6Hz, O-CH2-CH2-CH2-O), 4.06 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 4.22 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 6.85-7.12 ( m, 4H, Ar-H), 7.43-7.82 (m, 4H, Ar-H), 9.74 (s, 1H, NH)(D2O exchangeable), 10.01 (s, 1H, CHO)
- Preparation of 4-[3- (Benzo[d]thiazol-2-ylthio) propoxy]benzaldehyde (2d): The title compound was prepared from 4-(3-chloropropoxy) benzaldehyde (15 mmol) and 2-mercapto-benzothiazole (15 mmol) following the same general procedure of 3a, b, the reaction was stirred at 70°C for 72 h. The resulting yellow solid was crystallized from dry acetone. It was separated as faint yellow crystals. (40% yield), m.p. 112° (Found: C, 62.02; H, 4.49; N, 5.12, C17H15NO2S3 requires C, 61.98; H: 4.58; N, 4.25) MS (EI): m/z 329 (M+, 45%); IR (FT): 1695 (CO aldehydic). 1H NMR; 2.28 (m, 2H, J=9Hz, S-CH2-CH2-CH2-O), 3.22 (t, 2H, J=9Hz, S-CH2-CH2-CH2-O) 4.22 (t, 2H, J=9Hz, S-CH2-CH2-CH2-O), 7.03-7.24 (m, 3H, Ar-H), 7.37-7.47 (m, 2H, Ar-H), 7.69-7.86 (m, 3H, Ar-H), 9.89 (s, 1H, CHO).
- Preparation of 4-[3-[4-(3-Chlorophenyl)piperazin-1-yl]propoxy]benzaldehyde(2e): The title compound was prepared from 1-(3-Chlorophenyl)-4-(3-chloropropyl)piperazine.HCl (4.63 g, 15 mmol) and 4-hydroxybenzaldehyde (15 mmol) using the same general procedure described for the preparation of 5a, b The resulting solid was crystallized from dry acetone and separated as white solid. (80% yield), m.p. 116° (Found: C, 67.01; H, 6.51; N, 7.92, C20H23ClN2O2 requires C, 66.94; H: 6.46; N, 7.81) MS (EI): m/z 358 (M+, 45%); IR (FT): 1695 (CO aldehydic). 1H NMR; 1.94 (m, 2H, J=6Hz, N-CH2-CH2-CH2-O),3.13-3.18 (m, 8H, 4CH2 piperazine), 3.45 ((t, 2H, J=6Hz, N-CH2-CH2-CH2-O), 4.13 (t, 2H, J=6Hz, N-CH2-CH2-CH2-O), 6.74-6.85 (m, 3H, Ar-H), 7.09-7.21 (m, 3H, Ar-H), 7.83 (d, 2H, Ar-H), 9.85 (s, 1H, CHO).
- Preparation of 4-[4-[4-{2-(Arylamino)oxoethoxy} phenyl]methylene]-3-methyl-1-phenyl-1H-pyrazol -5(4H)-one (9b-d): General procedure: To a stirring solution of 2a-c (0.35 g, 2 mmol) in ethanol 95% (8 mL), the corresponding (2 mmol each) were added. After complete addition, the mixture was stirred for 24 hours at room temp. The reaction mixture was filtered, dried, washed with ether, then crystallized from acetone-ether mixture.
- For compound (9b): It was separated as white crystals. (70% yield), m.p. 165° (Found: C, 68.95; H, 4.96; N, 9.97, C25H22ClN3O2 requires C, 69.52; H: 5.13; N, 9.97) MS (EI): m/z 445 (M+, 75%); IR (FT): 1665 (CO amidic). 1H NMR; 2.11 (s, 3H, CH3), 4.79 (s, 2H, CO-CH2-O), 7.12-7.35 (m, 6H, Ar-H), 7.45 (s, 1H, CH=C), 7.64-7.71 (m, 5H, Ar-H), 7.72-7.85 (m, 3H, Ar-H), 9.5 (s, 1H, NH) D2O exchangeable
- For compound (9c): It was separated as faint yellowish white crystals. (85% yield), m.p. 143° (Found: C, 75.7; H, 6.23; N, 10.32, C26H25N3O2 requires C, 75.89; H: 6.12; N, 10.21) MS (EI): m/z 425 (M+, 75%); IR (FT): 1665 (CO amidic). 1H NMR; 2.11 (s, 3H, CH3), 2.28 (s, 3H, CH3), 4.70 (s, 2H, CO-CH2-O), 7.11-7.21 (m, 6H, Ar-H), 7.38-7.51 (m, 4H, Ar-H, CH=C), 7.71-7.90 (m, 4H, Ar-H), 9.5 (s, 1H, NH) D2O exchangeable.
- For compound (9d): It was separated as faint white crystals. (65% yield), m.p. 137° (Found: C, 76.36; H, 6. 3; N, 10.01, C27H27N3O2 requires C, 76.21; H: 6.4; N, 9.87) MS (EI): m/z 439 (M+, 75%); IR (FT): 1660 (CO amidic). 1H NMR; 2.11 (s, 3H, CH3), 2.23 (s, 3H, CH3), 2.30 (s, 3H, CH3), 4.65 (s, 2H, CO-CH2-O), 6.94 (d, 2H, Ar-H), 7.05-7.17 (m, 3H Ar-H), 7.38-7.68 (m, 5H, Ar-H, CH=C), 7.72-7.82 (m, 3H, Ar-H), 9.5 (s, 1H, NH) D2O exchangeable.
- Preparation of 2-Substituted-3-[3-[4-{(3-methyl-4,5-dihydro-5-oxo-1-phenyl-1H-pyrazol-4-ylidene)methylene}phenoxy]propyl]quinazolin-4(3H)-one(4a,b),4-[3-4-{(3-Methyl-5-oxo-1-phenyl -1H-pyrazolin-4(5H) ylidene) methyl} phenoxy] propoxy] acetanilide (7), 4-[[4-{3-(Benzo[d] thiazol-2-ylthio)propoxy} phenyl] methylene]-3-methyl-1-phenyl-1H-pyrazol-5(4H)-one (5): The title compounds were prepared from 3-methyl-1-phenyl(5H)pyrazolin-5-one (2 mmol) and the corresponding aldehydes (2a-d) (2 mmol) using the same general procedure described for the preparation of 9b-d to give crude products which were further recrystallized from acetone-ether mixture.
- For compound (4a): (66% yield), m.p. 190° (Found: C, 72.37; H, 4.96; N, 11.87, C28H24N4O3 requires C, 72.4; H: 5.21; N, 12.06) MS (EI): m/z 464 (M+, 20%); IR (FT): 1665 (CO amidic). 1H NMR; 2.11 (s, 3H, CH3), 2.21 (m, 2H, J=7 Hz, N-CH2-CH2-CH2-O-), 4.18 (t, 2H, J=7 Hz, -N-CH2-CH2-CH2-O-), 7.18-7.23 (m, 4H , Ar-H), 7.41 (m, 3H, Ar-H, (CH=C-), 7.52-7.63 (m,3H, Ar-H), 7.65-7.71 (d, 2H, Ar-H), 7.81 (d, 2H, Ar-H), 8.36 (S, 1H, quinazolyl-2H-).
- For compound (4b): (53% yield), m.p. 196° (Found: C, 72.54; H, 5.39; N, 11.82, C29H26N4O3 requires C, 72.79; H: 5.48; N, 11.71) MS (EI): m/z 478 (M+, 5%); IR (FT): 1665 (CO amidic). 1H NMR; 2.08 (s, 3H, CH3), 2.11 (s, 3H, CH3), 2.21 (m, 2H, J=7 Hz, N-CH2-CH2-CH2-O-), 4.18 (t, 2H, J=7 Hz, -N-CH2-CH2-CH2-O-), 7.18-7.23 (m, 4H , Ar-H), 7.41 (m, 3H, Ar-H, CH=C-)), 7.52-7.63 (m,3H, Ar-H), 7.65-7.71 (d, 2H, Ar-H), 7.81 (d, 2H, Ar-H).
- For compound (7): ).(0.71g, 76%yield, m.p. 170°C(Found: C, 71.45; H, 5.6; N, 9.04, C28H27N3O4 requires C, 71.62; H: 5.8; N, 8.94) MS (EI): m/z 469 (M+, 75%); IR (FT): 1665 (CO amidic). 1H NMR; 1.98 (s, 3H, NH-CO-CH3), 2.11 (s, 3H, CH3), 2.23 (m, 2H, J=6Hz, O-CH2-CH2-CH2-O), 4.05 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 4.22 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 6.84-7.16 (m, 6H, Ar-H), 7.39-7.67 (m, 3H, Ar-H, CH=C), 7.71-7.88 (m, 5H, Ar-H), 9.74 (s, 1H, NH) D2O exchangeable, 10.01 (s, 1H, CHO).
- For compound (5): (0.68g, 60% yield), m.p. 185°C (Found: C, 66.92; H, 4.65; N, 8.57, C27H23N3O2S2 requires C, 66.87; H: 4.77; N, 8.65) MS (EI): m/z 485 (M+, 25%); IR (FT): 1660 (CO amidic). 1H NMR; 2.01 (s, 3H, CH3), 2.25 (m, 2H, J=9Hz, S-CH2-CH2-CH2-O), 3.45 (t, 2H, J=9Hz, S-CH2-CH2-CH2-O), 4.19 (t, 2H, J=9Hz, S-CH2-CH2-CH2-O), 7.01-7.32 (m, 4H, Ar-H), 7.34-7.73 (m, 5H, Ar-H, CH=C), 7.82-7.99 (m, 5H, Ar-H).
- Preparation of 4-[4-[2-N-(4-Chlorophenyl) acetamido)phenoxy]methylene]-3-methyl -1H-pyrazo-5(4H)-one (9a), [4-[2-N-(4-Chlorophenyl) acetamido)phenoxy]methylene]-3-methyl -1H-pyrazo-5(4H)-one(4a),4-[3-[4-{(3-Methyl-4,5-dihydro-5-oxo-1H-pyrazol-4-ylidene)methyl} phenoxy]propoxy]acetanilide(8), 4-[4-[3-{4-(3-Chlorophenyl)piperazin-1-yl}propoxy]benzyli dene]-3-methyl-1H-Pyrazol-5(4H)-one (6): General procedure: A stirred mixture of 3-methylpyrazolin-5-(4H)-one (10 mmol) and 3a, 2c, 2e (10 mmol) in absolute ethanol (20 mL) containing 5-6 drops of piperidine was refluxed for 7 hrs. After cooling, the solution was evaporated in vacuo, the residue was recrystallized from CHCl3/hexane.
- For compound (9a): It was separated as white solid, (54% yield), p. 189°C(Found: C, 61.84; H, 4.43; N, 11.28, C19H16ClN3O3 requires C, 61.71; H: 4.36; N, 11.36) MS (EI): m/z 369 (M+, 25%); IR (FT): 1680 (CO amidic). 1H NMR; 1.47 (s, 3H, CH3), 4.55 (s, 2H, CH2), 6.88 (d, 2H, Ar-H), 7.03 (d, 2H, Ar-H), 7.24-7.33 (m, 4H, Ar-H), 7.42 (s, 1H, CH=C), 8.73 (s, 1H, pyrazol NH) D2O exchangeable.
- For compound (8): It was separated as white solid, (46% yield), p. 170°C(Found: C, 66.98; H, 5.7; N, 10.54, C19H16ClN3O3 requires C, 67.16; H: 5.89; N, 10.68) MS (EI): m/z 393 (M+, 40%); IR (FT): 1675 (CO amidic). 1H NMR; 1.47(s, 3H, CH3), 1.99 (s, 3H, NH-CO-CH3), 2.18 (m, 2H, J=6Hz, O-CH2-CH2-CH2-O), 3.95 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 4.07 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 6.75-6.98 (m, 4H, Ar-H), 7.45-7.65 (m, 5H, Ar-H, CH=C), 8.75 (s, 1H, pyrazol NH) D2O exchangeable, 9.73(s, 1H, NHCOCH3) D2O exchangeable. 4.07 (t, 2H, J=6Hz, O-CH2-CH2-CH2-O), 6.75-6.98 (m, 4H, Ar-H), 7.45-7.65 (m, 5H, Ar-H, CH=C), 8.75 (s, 1H, pyrazol NH) D2O exchangeable, 9.73(s, 1H, NHCOCH3) D2O exchangeable.
- For compound (6): It was separated as white solid, (65% yield), m.p.150°C (Found: C, 65.96; H: 6.15; N, 12.58, C24H27ClN4O2 requires C, 65.67; H: 6.2; N, 12.76) MS (EI): m/z 439 (M+, 40%); IR (FT): 1680 (CO amidic). 1H NMR; 1.49(s, 3H, CH3), 1.94 (p, 2H, J=6Hz, O-CH2-CH2-CH2-O), 3.15 (m, 4H, 2CH2 piperazine, 2H, N-CH2-CH2-CH2-O), 4.16 (t, 2H, J=6Hz, N-CH2-CH2-CH2-O), 6.75-6.98 (m, 4H, Ar-H), 7.45-7.65 (m, 3H, Ar-H, CH=C), 8.67 (s, 1H, pyrazol NH) D2O exchangeable
- Preparation of Diethyl 2-[4-(2-arylamino-2-oxoethoxy)benzylidene]malonate (10a,b): General procedure: A mixture of (2b,c) (32.75 mmol) alternatively and diethyl malonate (5.35 mL, 39.3 mmol) in benzene (50 mL) containing piperidinium acetate (1.41 g, 9.82 mmol) was refluxed in Dean-Stark trap for 5 h. The solution was concentrated in vacuo, cooled in a refrigerator and filtered. The residue was crystallized from ether.
- For compound (10a): It was separated as faint yellowish white crystals. (75% yield), p. 85C (Found: C, 66.59; H, 5.84; N, 4.51, C23H25NO6 requires C, 67.14; H: 6.12; N, 3.4) MS (EI): m/z 411 (M+, 30%); IR (FT): 1770 (CO of ester). 1H NMR; 1.18-1.24 (m,6H,, 2(COOCH2CH3)), 2.22 (s, 3H, CH3), 4.16-4.30 (m, 4H, 2(COOCH2CH3)), 4.73(s, 2H, CO-CH2-O-), 7.03-7.11 (m, 4H, Ar-H), 7.45-7.49 (m, 4H, Ar-H), 7.65 (s, 1H, CH=C-), 9.33 (s,1H, NH) D2O exchangeable.
- For compound (10b): It was separated as faint yellowish white crystals. (86% yield), m.p. 115°C (Found: C, 67.84; H, 6.3; N, 3.1, C23H25NO6 requires C, 67.75; H: 6.40; N, 3.29) MS (EI): m/z 425 (M+, 60%); IR (FT): 1770 (CO of ester). 1H NMR; 1.15-1.26 (m, 6H, 2(COOCH2CH3)), 2.10 (s, 3H, CH3), 2.23 (s, 3H, CH3), 4.09-4.30 (m, 4H, 2(COOCH2CH3)), 4.78 (s, 2H, CO-CH2-O-), 6.98 (d, 2H, J=6Hz, Ar-H), 7.09 (d, 2H, Ar-H), 7.21 (s, 1H, Ar-H) 7.49 (d, 2H, J=6Hz, Ar-H), 7.65 (s, 1H, CH=C-), 9.33 (s,1H, NH) D2O exchangeable.
- Preparation of diethyl2-[4-(2-arylamino-2-oxoethoxy)benzyl]malonate (11a,b): Suspension of compounds (10a,b) (4.23 mol each ) in absolute ethanol was stirred in the presence of 10% palladium on charcoal (0.6g) under atmosphere of hydrogen at room temperature for 24 hrs. The solution was filtered through Celite, and the filtrate was evaporated under vacuum to produce a light yellow solid. The product was crystallized with ether.
- For compound (11a): It was separated as white crystals. (80% yield), p. 70°C (Found: C, 66.93; H, 6.43; N, 3.27, C23H27NO6 requires C, 66.81; H: 6.58; N, 3.39) MS (EI): m/z 413 (M+, 70%); IR (FT): 1770 (CO of ester). 1H NMR; 1.09(t, 6H, J=9Hz, 2(COOCH2CH3)) 2.24 (s, 3H, CH3), 3.02 (d, 2H, J=6Hz, CH2-CH-(COO)2-), 3.77 (t, 1H, J=6Hz, CH2-CH-(COO)2-), 4.08 (q, 4H, J=9Hz, 2(COOCH2CH3)), 4.66 (s, 2H, CO-CH2-O-), 6.90 (d, 2H, Ar-H), 7.14 (d, 2H, Ar-H), 7.17-7.22 (m, 4H, Ar-H), 9.33 (s,1H, NH)D2O exchangeable.
- Compound (11b): It was separated as white crystals. (86% yield), m.p. 105°C (Found: C, 68.13; H, 5.96; N, 3.78, C24H29NO6 requires C, 67.43; H: 5.96; N, 3.28) MS (EI): m/z 427 (M+, 30%); IR (FT): 1770 (CO of ester). 1H NMR; 1.11 (t, 6H, J=9Hz, 2(COOCH2CH3)) 2.09 (s, 3H, CH3), 2.24 (s, 3H, CH3), 3.02 (d, 2H, J=6Hz, CH2-CH-(COO)2-), 3.85 (t, 1H, J=6Hz, CH2-CH-(COO)2-), 4.10 (q, 4H, J=9Hz, 2(COOCH2CH3)), 4.66 (s, 2H, CO-CH2-O-) 6.90-7.14 (m, 4H, Ar-H), 7.17 (d, 2H, Ar-H), 7.22 (s, 1H, Ar-H), 9.33 (s,1H, NH) D2O exchangeable.
- Preparation of N-aryl-2-[4-[(3,5-dioxopyrazolidin-4-yl)methyl]phenoxy]acetanilide (12a, b):
General procedure: To stirring solution of (11a,b) (20 mmol each), add hydrazine hydrate 99% (2.5g, 50 mmol), then reflux for 30 minutes, a precipitate is formed. After cooling, a solution of sodium ethoxide (0.02 atom/ g) in a absolute ethanol (20 ml) was added, then the reaction mixture was refluxed once more for 1 h. After cooling, the solution was evaporated under vacuum, the residue was dissolved in 20% HCl solution. A solid was precipitated. The precipitate was filtered, dried and crystallized from THF.
- For compound (12a): It was separated as white crystals. (64% yield), m.p. 219°C (Found: C, 64.67; H, 5.35; N, 11.72, C19H19N3O4 requires C, 64.58; H: 5.42; N, 11.89) MS (EI): m/z 353 (M+, 50%); IR (FT): 1670 (CO amidic). 1H NMR; l2.23, (s, 3H, CH3), 2.98 (d, 2H, J=6Hz, CH2-CH-CO), 3.25 (t, 1H, J=6Hz, CH2-CH-CO), 4.64 (s, 2H, CO-CH2-O), 6.78-7.07( m, 4H, Ar-H), 7.17-7.27 (m, 4H, Ar-H).
- For compound (12b): It was separated as white crystals. (54% yield), m.p. 220°C (Found: C, 65.27; H, 5.82; N, 11.37, C20H21N3O4 requires C, 65.38; H: 5.76; N, 11.44) MS (EI): m/z 367(M+, 30%); IR (FT): 1675 (CO amidic). 1H NMR; 1.11 (t, 6H, J=9Hz, 2(COOCH2CH3)), 2.09 (s, 3H, CH3), 2.24 (s, 3H, CH3), 3.02 (d, 2H, J=6Hz, CH2-CH-(COO)2-), 3.85 (t, 1H, J=6Hz, CH2-CH-(COO)2-), 4.10 (q, 4H, J=9Hz, 2(COOCH2CH3)), 4.66 (s, 2H, CO-CH2-O-), 6.90-7.14 (m, 4H, Ar-H), 7.17 (d, 2H, Ar-H), 7.22 (s, 1H, Ar-H), 9.33 (s,1H, NH) D2O exchangeable.
Hypothesis Generation Experiments: Molecular modeling work was performed on Silicon Graphic (SGI), Fuel workstation (500 MHz, R 14000 ATM processor, 512 MB memory) using the Catalyst package of Molecular Simulation (version 4.8), under an IRIX 6.8 operating system, at Faculty of Pharmacy, Ain Shams University. A generalized visualizer, confirm, info, HipHop, Compare/fit, force field was used throughout.
The frozen bioactive conformation of the training set molecules were isolated from their respective PDB files using the protein tools deck in Cerius2 module. Using Cerius2 3D-Sketcher, the 3D structures of corrected bioactive conformations of the training set were saved and transformed to Tripos. Mol2 format.
The training molecules with their associated conformational models were submitted to catalysis by using default common features hypothesis generation by using HipHop commands. The chemical function groups (features) used in this generation step included H-bond acceptor, ring aromatic groups, and hydrogen bond donor groups.
The training set molecules: Molecules were built within the catalyst and conformational models for each compound were generated automatically using the poling algorithm. This emphasizes representative coverage over a 20 kcal mol-1 energy range above the estimated global energy minimum and the best searching procedure was chosen.
Molsoft Molecular Modeling Experiments: The docking algorithm implemented in ICM optimizes the entire ligand in the receptor field, applying a multistart Monte Carlo minimization procedure in internal coordinate space. The number of Monte Carlo steps and iterations of the local energy minimization was determined automatically by an adaptive algorithm depending on the size and number of flexible torsions in the ligand 18.
REFRENCES:
- King, H.; Aubert, W.; Herman, W. Diabetes Care, 1998, 21, 1413-1431.
- Zimmet, P.; Alberti, K.G.; Shaw, J. Nature, 2001, 414, 782.
- J. Ram, Prog. Drug Res. 60 (2003), pp. 93–132.
- Lehmann, J. M.; Moore, L. B.; Smith-Oliver, T. A.; Wilkison, W. O.; Willson T. M. Biol. Chem., 1995, 270, 12953.
- (a) Kulkarni, S. S.; Gediya, L. K.; Kulkarni, V. M. Bioorg. Med. Chem. 1999, 7,1475; (b) Nolte, R. T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert, M. H.; Kurokawa, R.; Rosenfeld, M. G.; Willson, T. M.; Glass, C. K.; Milburn, M. V. Nature 1998, 395, 137.
- (a) Momose, Y.; Maekawa, T.; Yamano, T.; Kawada, M.; Odaka, H.; Ikeda, H.; Sohda, T. Med. Chem. 2002, 45, 1518; (b) Henke, B. R.; Blanchard, S. G.; Brackeen, M. F.; Brown, K. K.; Cobb, J. E.; Collins, J. L.; Harrington, W. W., Jr.; Hashim, M. A.; Hull-Ryde, E. A.; Kaldor, I.; Kliewer, S. A.; Lake, D. H.; Leesnitzer, L. M.; Lehmann, J. M.; Lenhard, J. M.; Orband-Miller, L. A.; Miller, J. F.; Mook, R. A., Jr.; Noble, S. A.; Oliver, W., Jr.; Parks, D. J.; Plunket, K. D.; Szewczyk, J. R.; Willson, T. M. J. Med. Chem. 1998, 41, 5020; (c) Momose, Y.; Maekawa, T.; Odaka, H.; Ikeda, H.; Yamano, T.; Sohda, T. Chem. Pharm. Bull. 2002, 50, 100.
- Wang, L.F.; Zhang, H.Y. Med. Chem. Lett., 2003., 13, 3789.
- Fan, X.; Zhang, X.; Zhou, L. Med. Chem. Lett., 2006, 16, 3224.
- Ahn, J. H.; Iung, S. H.; Yang, S. D. Bioorg. Med. Chem. Lett, 2004., 14(17), 4461.
- Matsueda, H.; Ohsumi, K. Med. Chem. Lett, 2003., 13(14), 2269.
- Cronet, P.; Peterson, J. F.; Folmer, R.; Structrure (Camb.) , 2001, 9, 699.
- Sauerberg, P.; Pettersson, I.; Med. Chem. , 2001, 45, 789.
- Burgermeister, E.; Schnoebelen, A. Endocrinol. , 2006, 20, 809.
- Shio, Q.; Dropinski, J. F.; Mekeever, B. M.; 48, 4457, (2005).
- Edrup, S.; Pettersson, I.; Nygaard, L.; Med. Chem. , 2003, 46, 1306.
- Hulin, B.; Newton, L. S.; Lewis, D. M.; Genereux, P. E.; Gibbs, E. M.; Clark, D.A.; J. Med. Chem., 1996,39, 3897.
- Pochetti, G.; Godio, C.; Mitro, N.; Biol. Chem. , 2007, 6.
- Totrov, M.; Abagyan, R. Proteins: Struct., Funct., Genet., S1, 1997, 215.
- Chen, W.-C.; Chou, Y. C.; Med. Chem. , 2005, 48, 8194.
- Sydow-Backman, M.; Gustavsson, A. L; David, A. T.;. Wright, A. S. Biol. Chem. , 2004, 279, 41124.
- Furnis BS, Hannaford AJ and Smith PW: Text book of practical organic chemistry, John Wiley Inc. 3rd edition, 1974; 807.
- Vogel A: Text book of practical organic chemistry, 3rd edition 1974: 238.
- Lazer ES, Wong HC and Graham AG: Synthesis and .alpha.2-adrenoceptor antagonist activity of some disulfonamido-benzoquinolizines. Journal of Medicinal Chemistry 1989; 32: 179-182.
How to cite this article:
Ismaila AH Md, El Ella DAA, Abouzid KAM and Jaballah M: Design, Synthesis and Virtual Screening of Certain 2-Pyrazolin-5-one and Pyrazolidine-3, 5-dione Derivatives as potential PPARg Agonists. Int J Pharm Sci Res. 3(10); 3746-3757
Article Information
28
3746-3757
829KB
1093
English
Ijpsr
Mohamed A. H. Ismaila,*, Dalal A. Abou El Ella, Khaled A. M. Abouzid, and Maiy Jaballah
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, El Khalifa ElMaamoon St., 11566, Abbasseya, Cairo, Egypt
mhismaeel@yahoo.com
27 May, 2012
22 August, 2012
26 September, 2012
http://dx.doi.org/10.13040/IJPSR.0975-8232.3(10).3746-57
01 October, 2012