Synthesis of 3-[ 4-( 2-furoyl )-1-piperazinyl ]-N-( substituted ) propanamides as promising antibacterial agents with mild cytotoxicity

Purpose: To evaluate the antibacterial activity and cytotoxicity of a series of molecules with amalgamation of furoyl, piperazine and amide moieties. Methods: New derivatives, namely 3-[4-(2-furoyl)-1-piperazinyl]-N-(substituted) propanamides, were synthesized and evaluated for their antibacterial activity and toxicity to mammalian cells. The synthesis was initiated by treating different aryl/aralkyl amines (1a-u) with 3-bromopropionyl chloride (2) to obtain the solid electrophiles 3a-u, which were collected by filtration. Thereafter, the different N-aryl/aralkyl-3bromopropionamides (3a-u) and 2-furoyl-1-piperazine (4) at equimolar ratios were allowed to react in acetonitrile and in the presence of a base, K2CO3, to form the target compounds, 5a-u. Structural elucidation was carried out using EI-MS (electron impact mass spectrometry), IR (infrared) and 1H-NMR (proton nuclear magnetic resonance). The antibacterial activity of the synthesized compounds was evaluated against various bacterial strains. Furthermore, hemolysis was determined to assess cytotoxicity using bovine red blood cells. Results: Molecules 5g, 5a, 5p, 5g and 5i were found to be potent agents against S. aureus, S. typhi, P. aeruginosa, E. coli and B. subtilis with respective minimum inhibitory concentration (MIC) values of 8.34 ± 0.55, 8.37 ± 0.12, 8.65 ± 0.57, 8.97 ± 0.12 and 9.24 ± 0.50 μM, compared to 7.80 ± 0.19, 7.45 ± 0.58, 7.14 ± 0.58, 7.16 ± 0.58 and 7.29 ± 0.90 μM for the reference standard, ciprofloxacin. The most active compounds, 5a, 5g, 5i and 5p, showed a hemolysis of 15.48, 8.03, 5.52 and 4.35 %, respectively. Conclusion: The synthesized compounds exhibit good antibacterial activity. The hemolysis data indicate that these compounds have a low toxicity level. However, in vivo studies are required to ascertain their potentials as new drug candidates.

These valuable biological activities of furan and amide moieties prompted us to introduce some new molecules with the amalgamation of these moieties and to evaluate their potential as antibacterial agents.

EXPERIMENTAL General
Chemicals and solvents were from Sigma Aldrich and Alfa Aesar and purchased through a local supplier.All solvents were further used without purification.A Griffin and George melting point apparatus was utilized to read the melting points of synthesized compounds, and melting points were uncorrected.Thin layer chromatography (TLC) run on silica-coated aluminum plates helped to confirm product formation.The mobile phase was a mixture of ethyl acetate and nhexane and spots were visualized under a UV lamp at 254 nm.A Jasco-320-A spectrometer was used to record IR peaks by the KBr pellet method.Bruker spectrometers were used to record 1 H-NMR signals at 500 MHz in CHCl 3 -d 1 , and a JMS-HX-110 spectrometer recorded EIMS signals.

Antibacterial activity assay
The broth microdilution method was employed to evaluate the antibacterial activity of the synthesized compounds.The synthesized compounds at different concentrations were mixed with selected bacterial strains, and the change in absorbance before and after incubation was noted [12,13].

Evaluation of hemolytic activity
Hemolytic activity was studied using a previously reported method [14,15].A blood sample was collected from a cow, and red blood cells (RBCs) were isolated for use.Triton X-100 and phosphate buffer saline (PBS) were employed as the positive and negative controls, respectively.

Statistical analysis
Microsoft Excel 2010 was utilized for statistical analysis of results which were expressed as mean ± SEM (n = 3).

Antibacterial and hemolytic activities
All synthesized molecules were screened against Gram-positive and Gram-negative bacteria and were found to be good to excellent inhibitors.The results are given as % inhibition and MIC values in Tables 1 and 2. The % hemolysis of the synthesized compounds is also given in Table 2.The results for compounds were compared with that for Triton X-100 and phosphate-buffered saline (PBS).propanamides showed notable activity against both Grampositive and Gram-negative bacterial strains compared to ciprofloxacin.A comparison of the two compounds 5a bearing an unsubstituted phenyl group and 5l bearing an aliphatic cyclohexyl group showed that 5a was more active against S. typhi, E. coli and S. aureus, but 5l, more against P. aeruginosa and B. subtilis.The presence of an aromatic system at the nitrogen of the propanamoyl group had a positive effect on the bioactivity of the synthesized molecule.The hemolysis results for compound 5a showed that it is more toxic than 5l.The increase in the distance of the unsubstituted phenyl ring from the nitrogen of the amidic group resulted in an increase in bioactivity potential but this effect was found to be the reverse for a too long aliphatic chain.Therefore, compound 5e bearing a benzyl group was found to be more active than 5a bearing a phenyl group, but 5f bearing a phenylethyl group was the least active.Furthermore, 5e was the least toxic compound among these three.No considerable difference in bioactivity potential was found between the three compounds 5b-d, bearing a methyl-substituted phenyl group at the ortho, meta and para positions.When a 2-methylphenyl group (compound 5b) was replaced by a 2-ethylphenyl group (compound 5g), the bioactivity potential was found to be increased and also with low toxicity.The presence of an ethoxy group at the ortho position of the phenyl ring (5i) did not favor higher bioactivity.
A similar order of bioactivity was found for 4methylphenyl (compound 5d) and 4-ethylphenyl (compound 5h), but 5h was more toxic.4-Ethoxyphenyl (compound 5j) had a little less antibacterial activity compared to 5h but it possessed much less toxicity.This shows that the substitution of ethyl at the ortho position of the phenyl ring (compound 5g) resulted in more activity with low toxicity as compared to that at the para position (compound 5h).A similar pattern of bioactivity was observed for the ethoxy group at the ortho and para positions.Among those with dimethylphenyl groups, compounds 5n-q, bearing one methyl at the ortho position and a second at other positions turned out to be less effective compared to compounds 5r and 5s, bearing one methyl at the meta position and a second at other positions.Among compounds 5n-q, 5n was the most active, but it showed high toxicity.The other three showed comparable potential, with no considerable effect of variation in position of substitution.Both compounds 5r and 5s also showed a similar order of activity.
Overall, all synthesized molecules showed impressive antibacterial activity against S. typhi.The aromatic rings were responsible for more interaction, and the alkyl groups in better positions accounted for higher antibacterial activity.The compounds exhibiting low toxicity could be further subjected to in vivo study to assess their application in a drug design program.

CONCLUSION
The findings of this study show that almost all the derivatives are active against all the strains of Gram-positive and Gram-negative bacteria tested.Only a few of the synthesized molecules possess moderate cytotoxicity.Thus, these molecules have some potential as therapeutic agents, but further studies are required in this regard.

Table 2 :
Antibacterial activity (MIC) and hemolytic activity of the synthesized compounds Minimum inhibitory concentration (MIC) was calculated using different concentrations (ranging 5 -30 μg/well) and EZ-Fit software (Perrella Scientific Inc. Amherst, NH, USA)