Arch Pharm Res Vol 32, No 4, 481-487, 2009 DOI 10.1007/s12272-009-1401-0
Synthesis and Docking Studies of New 1,4-Dihydropyridines Containing 4-(5)-Chloro-2-ethyl-5-(4)-Imidazolyl Substituent as Novel Calcium Channel Agonist Asghar Davood1, Ali reza Nematollahi1, Maryam Iman2, and Abbas Shafiee3 1
Department of Medicinal Chemistry, Pharmaceutical Sciences Branch, Islamic Azad University, Tehran, Iran, 2Department of Pharmaceutical Sciences, Faculty of Pharmacy, Mashad University of Medical Sciences, Mashad, Iran, and 3 Department of Chemistry, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, 14174, Iran (Received August 29, 2008/Revised March 4, 2009/Accepted March 6, 2009)
1,4-Dihydropyridines have been recognized as calcium channel agonist. Three new analogues of Bay K8644 in which the ortho trifluromethyl phenyl group at position 4 is replaced by the 4-(5)-Chloro-2-ethyl-5-(4)-imidazolyl substituent, were designed and synthesized as calcium channel agonist. For this propose, the structures of designed compounds were drawn by HYPERCHEM program. Conformations of the compounds were optimized through semiempirical method followed by PM3 calculation. Then the crystalin stucture of L-type calcium channel was obtained from the Protein Data Bank (PDB) server. Docking calculations were carried out using Auto-Dock.4 program. The good interaction of our 1,4-DHP derivatives showed that they can be as possible calcium channel agonist agents. Finally compounds were synthesized according to a modified Hantzsch condensation procedure. Key words: Calcium channel agonist, Dihydropyridines, Chloroimidazole, Nifedipine analogues, (S)Bay K 8644
Selected by Editors INTRODUCTION Calcium channels are important in excitation secretion coupling, because increase in the cytoplasmic concentration of free Ca2+ plays a key role in the regulation of Ca-dependent enzyme activity and hormone release (Rasmussen and Barret, 1984). Calcium channel is a widely in various neuronal, muscle, endocrine, thyroid and other cell types (Doyle et al., 1998). The dihydropyridine (S)-Bay K8644 (Fig. 1) which acted in the opposite manner to calcium channel antagonists, such as nifedipine and verapamil, in Correspondence to: Asghar Davood, Assistant professor of Medicinal Chemistry, Pharmaceutical Sciences Branch, Islamic Azad university, Tehran, Iran Tel: (0098-21)22609043 E-mail:
[email protected]
Fig. 1. Structure of Ca2+ channel activator (S)-Bay K8644
that it was positively inotropic and caused vasoconstriction (Perozo et al., 1999). In our previous studies we confirmed that 4-(5)-Chloro-2-ethyl-5-(4)-imidazolyl substituent is bioisoster of nitrophenyl in nifedipine (Davood et al., 2001). Based on the above mentioned subject we designed novel dihyropyridines as a calcium channel activator with using of 4-(5)-Chloro -2-ethyl-5-(4)-imidazolyl substituent in the C4 of dihydropyridine ring. By considering the obtained results from our com-
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putational studies, Molecular modeling and DOCKING, we synthesized new DHPs as a calcium channel agonist.
MATERIALS AND METHODS Molecular modeling The structure of DHP’s were built with standard bond length and angle using Hyperchem (version 7, Hypercube Inc.) and then the Semi-empirical molecular orbital calculations (PM3) of the structure were performed using the Hyperchem program and the among all energy minima conformers (two favored tautomers, Fig. 2), the global minimum of compounds were consider in docking calculations.
Fig. 2. Two favored tautomers of compound 6a (A, B)
Docking study Docking studies were carried out by using the program AUTODOCK 4. This program starts with a ligand molecule in an arbitrary conformation, orientation, and position and finds favorable dockings in a protein-binding site using both simulating annealing and genetic algorithms. The program AutoDockTools (ADT), which has been released as an extension suite to the Python Molecular Viewer, was used to prepare the protein and the ligand. For the macromolecule (L-type calcium channel, that was generated by resorting to multi body molecular
dynamics simulations, was downloading from the PDB bank server [PDB entry 1T0J]), polar hydrogens were added, and then Kollman United Atom charges and atomic solvation parameters were assigned. The grid maps of docking studies were computed using the AutoGrid4 included in the Autodock4 distribution. Grid center was centered on the active site was obtained by trial and error and previous study by Cosconati et al and 60x60x60 points with grid spacing of 0.375 were calculated. The GA-LS method was adopted to perform the molecular docking. The parameters for GA were defined as follows: a maximum number of 250,000 energy evaluations; a maximum number of generations of 27,000; mutation and crossover rates of 0.02 and 0.8, respectively. Pseudo-Solis & Wets parameters were used for local search and 300 iterations of Solis & Wets local search were imposed. The number of docking runs was set to 50. Both Autogrid and Autodock computations were performed on Cygwin. After docking, all structures generated were assigned to clusters based on a tolerance of 1 A ° all-atom RMSD from the lowest-energy structure. Hydrogen bonding and hydrophobic interactions between docked potent agents and macromolecule were analyzed using ADT (Version 1.50).
Chemistry Unsymmetrical DHPs 6a-c (Table I), analogues of Bay K8644, were synthesized according to Scheme 1. These compounds were prepared by a modified Hantzch condensation procedure reported by (Meyer et al., 1981), in which 4-(5)-Chloro-2-ethyl-5-(4)imidazolyl substituent 3 was reacted with alkyl 3aminocrotonates 5a-c (Li et al., 1998) and nitroacetone. The compound 3 could be prepared in three steps from propionaldehyde, dihydroxyacetone and ammonia (Weidenhagen and Herrmann,1935; Duncan et al., 1994). Alkyl 3-aminocrotonates 5a,b and the 3-oxobuthanoic acid esters were prepared according to the literature. Compound 5c was obtained from Merck.
Table I. Physical properties of unsymmetrical DHPs Comp
R
MP (oC)
Reflux (h)
Solvents of crystallization
Yield %
Molecular formula(1)
6a 6b 6c
Methyl Ethyl Isopropyl
232-237 179-189 115-122
36 28 36
Ether Ether Ether
5 22 9
C14H17ClN3O4 C15H19ClN3O4 C16H21ClN3O4
(1) Micro analytical analyses were within ±0.4% of theoretical values.
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Scheme 1. Synthesis of new 1,4-dihydropyridines containing 4-(5)-chloro-2-ethyl-5-(4)-imidazolyl substituent
RESULTS AND DISCUSSION Molecular modeling Molecular geometry of our designed compounds has been calculated by the semi-empirical method using PM3. An important observation in the 1,4dihydropyridine series was that very small mole-
cular changes produced potent calcium channel activators, molecules with vasoconstrictor, cardio acceleratory, and secretagogue activity. A broad structure-activity relationship for 1,4-dihydropyridine action at the L-type channel has been derived from pharmacological and radioligand binding studies in smooth and cardiac muscle and is summarized in
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Fig. 3, 4. In comparison of our outcomes with the refrence calcium channel activator, (S)-Bay k8644, if nitrogen atom of NO2 group be place flat in order to dihydropyridine ring, we can expect the agonist activity.
Compund
R1 Port
R2 Starboard
Nifedipine (R)-Bay K8644 (S)-Bay K8644
COOMe COOMe NO2
COOMe NO2 COOMe
R3
teractions between these groups and makes the DHP ring more flexible than in un-substituted molecules. Now, Based on above subjects we superimpose our compounds on (S)-Bay K 8644, the results showed that good overlay be exist that Fig. 5 be as a sample. By consideration obtained results we can expect our compounds could be as a novel calcium channel activator.
Effect
NO2 Antagonist CF3 Antagonist CF3 Agonist
Fig. 3. Structure and activity of dihydropyridine ligands. Note designations of the different sides of the boat-like structure
Fig. 5. Superimposition of 6b( cyan ball stick) and (S)-Bay K 8644 (violet ball stick)
Fig. 4. Structure-activity relationship for 1,4-dihydropyridines active at the L-type Ca2+ channel
The results indicate that all of the molecules have the semi-boat conformation of the DHP ring.The dihydrocycle in ortho-aryl derivatives is more flexible. The portside and starboard substituents in the DHP ring (Fig. 3) are known to determine the agonist and antagonist properties of DHPs. As you observe when small groups like Cl and CF3 in Oposition situate in same side with NO2 group, the agonist enantiomer was obtained. These substituents are situated near from mean plane of 1,4-dihydropyridine ring in an equilibrium conformation (Fig. 4) and are drawn together. The dihydrocycle bending in the direction of planar conformation leads to a decrease of unfavorable non-bonded in-
Docking Flexible docking of all data sets used for the computational study was carried out on the active site of L-type calcium channel. The orientation of the most potent calcium channel activator, compound 6b, in the active site of L-type calcium channel (LCC) was examined by a docking experiment (Huey and Morris, 2007) (Fig. 6). This docking studies shows the oxygen atom of nitro group forms a hydrogen bonding interaction with the NH of HIS 363 (distance=1.993) and the binding energy of 6b was -6.89 Kcal/mol. Other obtained results from docking are listed in Table II that reveal all of compounds posses good and acceptable binding energy. Just as predicted NO2 has main role in interaction with receptor and aryl ring play stereoselectivity role. These observations provide a good estimation for the potent activator activity of these compounds.
EXPERIMENTAL General Reagents and solvents were obtained from Merck
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5-(4)-Chloro-2-ethyl 4(5)hydroxymethylimidazole (2) White solid (21 g, 56%), m.p. 130-131oC. IR (KBr): ν cm-1 3100 (OH). 1H-NMR (DMSO-d6): δ = 1.20 (t, 3H, CH3), 2.60 (q, 2H, CH2), 4.35 (d, 2H, CH2OH), 5.10 (t, 1H, OH), 11.05 (s, 1H, NH). Anal. Calcd. for C6H9Cl N2O: C, 44.87; H,5.65; N, 17.44, found C, 44.68; H, 5.80; N, 17.63.
Fig. 6. Docked structure of 6b in Model of LCC. DHPs are displayed as sticks, and Hydrogen bonds are represented with dashed green lines (Docking study by using ADT program and LCC model obtained from PDB server) Table II. Docking results obtained from AutoDock4 software Comp. 6a 6b 6c Bay k 8644
Binding energy (Kcal/mol)
(1)
-5.72 -6.89 -6.11 -6.43
1) The predicted binding energy (Kcal/mol)
(Darmstadt,Germany). Melting points were determined using a Thomas Hoover capillary apparatus (Philadelphia, PA, USA) and were uncorrected. 1HNMR and 13C-NMR spectra were recorded on a Bruker FT-500MHz spectrometer (Bruker) and TMS was used as an internal standard. Infrared spectra were acquired on a Nicolet 550-FT spectrometer (Nicolet, Madison, WI, USA). Mass spectra were measured with a Finnigan TSQ-70 spectrometer (Thermo Electron Company) at 70 eV. Elemental analysis was carried out with a Perkin-Elmer model 240-C apparatus (Perkin-Elmer). The results of elemental analysis (C, H, N) were within ±0.4% of the calculated amounts.(Davood et al., 2006) 2-Ethyl-4-(5)-hydroxymethylimidazole (1) White crystals: m. p. 86-87oC. IR (KBr): ν cm-1 3160 (OH). 1H-NMR (DMSO-d6): δ = 1.18 (t, 3H, J= 7.4 Hz, CH3), 2.61 (q, 2H, J = 7.4 Hz, CH2), 4.34 (s, 2H, CH2OH), 4.54 (s, 1H, OH), 6.75 (s, 1H, imidazole). Anal. Calcd. for C6H10N2O: C, 57.12; H, 7.99; N, 22.21, found C, 57.33; H, 7.75; N, 22.40.
5-(4)-Chloro-2-ethylimidazole-4(5)-carboxaldehyde (3) White crystals: m.p. 107-110oC. IR (KBr): ν cm-1 1670 (CO). 1H-NMR (CDCl3): δ = 1.41 (t, 3H, CH3), 2.90 (q, 2H, CH2), 9.65 (s, 1H, CHO), 11.45 (s, 1H, NH). Anal. Calcd. for C6H7ClN2O: C, 45.44; H, 4.45; N, 17.66, found C, 45.29;H, 4.27; N, 17.49. 1-Nitropropan-2-one (4) Light green crystals. m.p. = 45-47°C IR(KBr) : ν 1728 (CO), 1588, 1357 (NO2) cm-1, 1H-NMR (CDCl3) : δ = 5.34 (s, 2H, CH2NO3), 2.42 (s, 3H, CH3CO). Note: The nitro acetone was unstable compound that decompose rapidly at 250oC, therefore in all reaction must be control the temperature. The ether solution was stable in cool temperature.
Methyl-3-aminobut-2-enoate (5a) A solution of methyl acetoacetate (21.6 g, 0.186 mol) and amoniumacetate (71.68 g, 0.930 mol) in absolute ethanol (310 mL) was reflux for 24 hours. The solvent was removed under reduced pressure and the residue was dissolved in water (200 mL) and extracted with CHCl3 (3×100 mL). The organic layer was dried with (Na2SO4), and evaporated under reduced pressure. The residue was crystallized from n-hexane to give the title compound (18.3 g, 98%) as colorless layer crystals: mp = 80-82°C IR (KBr): ν 3370 (NH2), 1660 (CO) cm-1; 1HNMR (CDCl3) : δ 4.55 (s, 1H, 2-H), 3.61 (s, 3H, COOCH3), 1.87 (s, 3H, CH3(NH2)C=C). Ethyl -3-aminobut-2-enoate (5b) A solution of ethyl acetoacetate (25 g, 0.192 mol) and amoniumacetate (73.99 g, 0.950 mol) in absolute ethanol (320 mL) was reflux for 30 hours. The solvent was removed under reduced pressure and the residue was dissolved in water (200 mL) and extracted with CHCl3 (3×100 mL). The organic layer was dried with (Na2SO4), and evaporated under reduced pressure. The residue was purified with fractional distillation and obtained title compound as transparent liquid (20 g, 81%). m.p. 32-35°C 1HNMR (CDCl3) : δ 4.52 (s, 1H, 2-H), 4.05 (q, 2H,
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COOCH2CH3), 1.91 (s, 3H, CH3(NH2)C=C), 1.22 (t, 3H, COOCH2CH3).
General procedure for preparation of 1,4Dihydro-2,6-dimethyl-5-nitro-4-[4-(5)-Chloro-2ethyl-5-(4)-imidazolyl]-3-pyridinecarboxylic acid, alkyl ester (6a-c) A solution of compound 3 (0.3 g, 1.893 mmol), nitroacetone (0.244 g, 1.893 mmol), and alkyl 3aminocrotonate (1.893 mmol) in methanol (4 mL) was reflux for several hours. The reaction mixture was purified with preparative TLC (silica gel, chloroform: methanol 20: 1). The crude product was crystallized from ether to give the desired compound. 1,4-Dihydro-2,6-dimethyl-5-nitro-4-[4-(5)-Chloro2-ethyl-5-(4)-imidazolyl]-3-pyridinecarboxylic acid, methyl ester (6a) Using the general procedure and methyl 3-aminocrotonate provide the title compound after 36 h reflux: yellow crystals, yield 5%; mp = 232-237oC IR (KBr): ν 3304 (NH), 1686 (CO), 1646, 1314 (NO2) cm-1; 1HNMR (CDCl3) : δ 1.26(t, 3H, J = 7.1 Hz, CH3CH2-imidazole), 2.32 and 2.53 (2s, 6H, C2, C6CH3), 2.65 (q, 3H, J = 7.1 Hz, CH3CH2-imidazole), 3.71 (s, 3H, COOCH3), 5.38 (s, 1H, 4-H-DHP ), 9.04 (br, 1H, NH-DHP); Mass: m/z (%) 340 (M+, 3), 194 (40), 164 (100), 83 (10). Molecular Formula = C14H17ClN4O4 Calculated = C (49.35%) H (5.03%) N (16.44%) Found = C (49.37%) H (5.04%) N (16.46%). 1,4-Dihydro-2,6-dimethyl-5-nitro-4-[4-(5)-Chloro2-ethyl-5-(4)-imidazolyl]-3-pyridinecarboxylic acid, ethyl ester (6b) Using the general procedure and ethyl 3-aminocrotonate provide the title compound after 28 h reflux: yellow crystals, yield 22%; mp = 179-182oC IR (KBr): ν 3324 (NH), 1706 (CO), 1653, 1315 (NO2) cm-1; 1HNMR (CDCl3+DMSO-d6) : δ 0.71 (t, 6H, J = 7.1 Hz, CH3CH2-imidazole and COOCH2CH3), 1.81 and 2.01 (2s, 6H, C2, C6-CH3), 2.67 (br, 2H, CH3CH2imidazole), 3.62 (q, 2H, J = 7.1 Hz, COOCH2CH3), 4.82 (s, 1H, 4-H-DHP ), 8.78 (br, 1H, NH-DHP), 10.99 (br, 1H, NH-imidazole); Mass: m/z (%) 354 (M+, 30), 309 (6), 262 (5), 225 (7), 210 (95), 178 (100), 151 (25), 128 (12), 83 (17), 77 (7). Molecular Formula = C15H19ClN4O4 Calculated = C (50.78%) H (5.40%) N (15.79%) Found = C (50.75%) H (5.43%) N (15.82%). 1,4-Dihydro-2,6-dimethyl-5-nitro-4-[4-(5)-Chloro2-ethyl-5-(4)-imidazolyl]-3-pyridinecarboxylic acid, iso-propyl ester (6c) Using the general procedure and iso-propyl 3-
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aminocrotonate provide the title compound after 36 h reflux: yellow crystals, yield 9%; mp = 115-122oC IR (KBr): ν 3308 (NH), 1705 (CO), 1654, 1311 (NO2) cm-1; 1HNMR (CDCl3) : δ 0.90-1.39 (m, 9H, CH3CH2imidazole and COOCH(CH3)2), 2.30 and 2.49 (2s, 6H, C2, C6-CH3), 4.71-5.11 (m, 1H, CH(CH3)2), 5.25 (s, 1H, 4-H-DHP), 9.70 (br, 1H, NH-DHP), 11.88 (br, 1H, NH-imidazole); Mass: m/z (%) 368 (M+, 100), 308 (12), 280 (10), 222 (60), 192 (19), 128 (9), 67 (13), 56 (43), 42 (98). Molecular Formula = C16H21ClN4O4 Calculated = C (52.10%) H (5.74%) N (15.19%) Found = C (52.15%) H (5.77%) N (15.17%).
CONCLUSIONS Thus, 1,4-dihydropyrimidine ring in the biological active DHP derivatives possesses a degree of conformational flexibility and can change its conformation easily to create the most favorable conditions for effective interactions with biological receptor. These results of molecular modeling and docking study allow the assumption that these compounds can be possible Ca channel agonist. Further study might be required for biological evaluation. This study might provide a foundation for design and synthesize novel Ca channel agonist agents. Based on our docking studies, compounds 6a-c will be evaluated later as calcium channel agonist in the ginea pig.
ACKNOWLEDGEMENTS This research was supported by grants from the research council of Isfahan and Tehran Universities of Medical Sciences and the Iran National Science Foundation (INSF). We thank Professor Arthur J. Olson for his kindness in offering us the AutoDock 4 program. The technical assistance of Medicinal Chemistry Department of AZAD university in performing the computational studies is gratefully acknowledged.
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imidazolyl Substituent. Arch Pharm Chem Life Sci, 339, 299-304 (2006). Doyle, D. A., Cabral, J. M., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R., Science, 280, 69-77 (1998). Duncan, B. Judd, Michael D. Dowle, David Middlemiss, David I. C. Scopes, Barry C. Ross, Torquil I. Jack, Martin Pass, Elvira Tranquillini, Julie E. Hobson, Bromobenzofuran-Based Non-peptide Antagonists of Angiotensin II: GR138950, a Potent Antihypertensive Agent with High Oral Bioavailability. J. Med. Chem., 37(19), 3108-3120 (1994). Huey, R. and Morris, G. M., Using AutoDock 4 with AutoDockTools. (2007).
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Li, A. H., Moro, S., Melman, N., Ji, X. D., and Jacobson, K. A., Structure-activity relationships and molecular modeling of 3, 5-diacyl-2,4-dialkylpyridine derivatives as selective A3 adenosine receptor antagonists. J. Med. Chem., 41, 3186-3201 (1998). Perozo, E., Cortes, D. M., and Cuello, L. G., Structural rearrangements underlying K+-channel activation gating. Science, 285,73-78, (1999). Rasmussen, H. and Barret, P. Q., Calcium messenger system: an integrated view. Physiol. Rev., 64, 938-984 (1984). Weidenhagen, R. and Herrmann, R., Eine neue synthese von imidazol-derivaten. Ber. Dtsch. Chem. Ges, 68, 953961 (1935).
