Synthesis, Spectral and Anthelmintic Activity Studies on Some Novel Imidazole Derivatives

Present study describes the synthesis of a novel series of 3,5-diiodo-4(5-nitro-1H-2-imidazolyl)benzoyl amino acids and di/tri/tetrapeptides using diisopropylcarbodiimide/dicyclohexylcarbodiimide (DIPC/DCC) as coupling agents and N-methylmorpholine/triethylamine (NMM/TEA) as bases. Structure elucidation of all the newly synthesized compounds was done by elemental analysis and IR, H NMR, C NMR and mass spectral data. Synthesized imidazolopeptides were screened for their anthelmintic activity and found to possess moderate to good bioactivity against earthworms Megascoplex konkanensis, Pontoscotex corethruses and Eudrilus eugeniea when compared to reference drugs – albendazole and mebendazole at dose level of 2 mg mL.


Experimental
Melting points were determined by open capillary method and are uncorrected.Amino acids, di-tert-butylpyrocarbonate (Boc 2 O), DIPC, DCC, TFA, NMM and TEA were procured from SpectroChem Ltd, Mumbai, India.IR spectra were recorded on Shimadzu 8700 fourier transform infrared spectrophotometer using a thin film supported on KBr pellets for solids and CDCl 3 as solvent for semisolids. 1 H NMR and 13 C NMR spectra were recorded on Bruker AC NMR spectrometer (300 MHz) using CDCl 3 as solvent and TMS as internal standard.Mass spectra were recorded on Jeol JMS DX 303 Mass spectrometer operating at 70 eV.Elemental analysis of all compounds was performed on Elementar vario EL III.Purity of all the compounds was checked by TLC on precoated silica gel G plates.Optical rotation of synthesized peptide derivatives was measured on automatic polarimeter (Optics Tech, Ghaziabad, India) in a 2 dm tube at 25°C using sodium D-light and methanol as solvent.

Preparation of peptides
Amino acid methyl ester hydrochloride/dipeptide methyl ester (0.01 mol) was dissolved in CH 2 Cl 2 (20 mL).To this, TEA (0.021 mol) was added at 0°C and the reaction mixture was stirred for 15 min.Boc-amino acid/dipeptide (0.01 mol) in CH 2 Cl 2 (20 mL) and DCC (0.01 mol) were added with stirring.After 24h, the reaction mixture was filtered and the residue was washed with CHCl 3 (30 mL) and added to the filtrate.The filtrate was washed with 5% NaHCO 3 and saturated NaCl solutions.The organic layer was dried over anhydrous Na 2 SO 4 , filtered and evaporated in vacuum.The crude product was recrystallized from a mixture of chloroform and n-hexane followed by cooling at 0°C.Resulting Boc-di/tri/tetrapeptide methyl ester (0.01 mol) was dissolved in CHCl 3 (15 mL) and treated with trifluoroacetic acid (0.020 mol).The mixture was stirred at room temperature for 1h and washed with saturated NaHCO 3 solution.The resulting organic layer was dried over anhydrous Na 2 SO 4 and concentrated under reduced pressure.Finally, crude product was purified by recrystallization with CHCl 3 and petroleum ether (B.p. 40-60°C) to get the deprotected di/tri/tetrapeptide methyl esters.

Results and Discussion
All peptide derivatives (4-15) were synthesized in good yields using DIPC/DCC as coupling agents and NMM/TEA as bases.Presence of bands at 3302-2497, 1698, 1545, 1348, 590 cm -1 in IR spectra of compound (3) clearly indicated presence of functional groups like -COOH, -NO 2 and -I but no free -NH 2 group which was present in the starting material (1).Furthermore, IR spectra of peptide derivatives (4-15) showed amide I and amide II bands at 1663-1638 cm -1 and 1537-1529 cm -1 indicating formation of peptide bonds and accomplishment of coupling reaction.This fact was further confirmed by appearance of broad singlets at 9.98-6.32ppm (for imino proton of CO-NH moiety) in 1 H NMR spectra and singlets at 179.4-165.1 ppm (for carbonyl carbon of CO-NH moiety) in 13 C NMR spectra of compounds (4-15).Moreover, presence of nitro and iodo groups in peptide analogs of compound (3) was indicated by appearance of medium bands at 1549-1540 cm -1 and 1349-1341 cm -1 (asymmetric and symmetric NO 2 stretching), and at 589-585 cm -1 (C−I str) in IR spectra.Mass spectra of peptide ester derivatives showed molecular ion peaks along with isotopic peaks at m/z values, consistent with their respective molecular formulas.All peptide ester derivatives showed easily distinguishable R-C≡O + ion peaks at M -31 along with characteristic fragmentation pattern after and before the carbonyl moiety in their respective structures.Furthermore, [CH 3 O + ] and [CH 3 OCO + ] fragment ion peaks appeared at m/z values 31 and 59 in mass spectra of synthesized peptide ester derivatives.Structures of hydrolyzed derivatives (12-15)  were confirmed by appearance of strong bands at 1711-1705 cm -1 (C=O str, COOH) in IR spectra and broad singlets at 9.95-9.25 ppm (for hydroxyl proton of COOH) in 1 H NMR spectra.This fact was further supported by disappearance of medium to strong bands at 1751-1749 cm -1 (C=O str, ester) and 1273-1270 cm -1 (C−O str, ester) in IR spectra and singlets at 54.4-51.4ppm (for carbonyl carbon of OCH 3 ) in 13 C NMR spectra of compounds (12-15).All hydrolyzed peptide derivatives showed peaks at M -17 and M -45 in their mass spectra by loss of OH and COOH respectively.Moreover, [COOH + ] fragment ion peak appeared at m/z value 45 in mass spectra of compounds (14, 15) along with characteristic fragmentation pattern after and before the carbonyl moiety.The newly synthesized compounds were also analyzed for C, H and N content and the results revealed the variation by a factor of ± 0.03 from calculated values.

Biological activity
All the synthesized compounds were subjected to anthelmintic activity studies 30 against three different species of earthworms M. konkanensis, P. corethruses and E. eugeniea at 2 mg mL −1 concentration.Tween 80 (0.5%) in distilled water was used as control whereas albendazole and mebendazole were used as a reference compounds.The paralyzing and death times were noted and their mean was calculated for triplicate sets.The death time was ascertained by placing the earthworms in warm water (50 °C) which stimulated the movement, if the worm was alive.The results of anthelmintic studies are tabulated in Table 1.Almost, all the newly synthesized compounds were found to exhibit moderate to good anthelmintic activity against all the three earthworm species.Comparison of anthelmintic activity data revealed that hydrolyzed peptide derivatives (12-15) are slightly more active than their corresponding ester derivatives (5, 9-11).Compound (9) and its hydrolyzed derivative (13) were found to exhibit higher bioactivity against all three earthworm species, in comparison to standard drugs-albendazole and mebendazole whereas compound (4) displayed least anthelmintic activity.Moreover, compounds (6, 9, 11, 13 and 15) bearing tryptophan and histidine residues in their amino acid chains were found to possess good activity against all earthworms species whereas compounds (7, 8, 10 and 14) showed only moderate level of activity.

Conclusions
Present investigation describes successful synthesis of title compounds via coupling reaction in good yields.For peptide coupling, method employing DIPC/NMM proved to be better than DCC/TEA method utilizing DMF as solvent, providing 6-10% extra yields.Diisopropyl carbodiimide proved to be a good coupling agent both economically and yieldwise.Greater anthelmintic activity was found in derivatives with tryptophan and histidine constituents in their amino acid chain.Hydrolyzed derivatives exhibited more anthelmintic activity in comparison to their corresponding methyl ester analogs.Among tested compounds (9, 13 and 15) possessed better anthelmintic activity as compared to standard drugs.On passing toxicity tests, these compounds may prove good candidates for clinical studies and may be potential anthelmintic agents of future.