Synthesis and Antimicrobial Activity of Amino Acids Conjugated Diphenylmethylpiperazine Derivatives

A series of amino acid conjugated diphenylmethylpiperazine derivatives were synthesized by coupling diphenylmethylpiperazine with different Boc-amino acids using EDCI/HOBt as coupling agent and NMM as base. The synthesized compounds were characterized by H-NMR and elemental analysis. The Boc-deblocked derivatives were tested for their antimicrobial activity. We are here reporting that Phe and Trp conjugated diphenylmethylpiperazine showed equally good antibacterial activities as that of conventional antimicrobial drugs.

Currently there is a tendency to use amino acid/peptidyl residues during the prodrug design process.The literature reports that bioactive compounds show enhanced activity when linked to amino acids [18][19][20][21][22][23] .The presence of an unusual amino acid and heterocyclic building blocks has stimulated interest in new synthetic methodologies and strategies to obtain a target structure.In this connection, we have synthesized novel diphenylmethylpiperazine derivatives by the coupling of diphenylmethylpiperazine with various N-protected amino acids.The present study was undertaken with a view to find the efficacy of these diphenylmethylpiperazine derivatives as antimicrobial agents.

Materials and methods
All the amino acids used were of L-configuration unless mentioned.All Boc-amino acids and HOBt were purchased from Advanced Chem.Tech.(Louisville, Kentuky, USA).EDCI and NMM were purchased from Sigma Chemicals Co. (St.Louis, MO).All solvents and reagents used for the synthesis and analysis were of analytical grade.TLC was carried out on precoated silica gel plates (Merck) using chloroform/methanol/acetic acid (95:5:3) as an eluting system. 1 H NMR spectra were obtained on a 300 MHz Bruker FT-NMR Spectrometer instrument by using CDCl 3 as solvent and TMS as an internal standard.
The diphenylmethylpiperazine was synthesized as previously reported using standard procedure 24 .The compound 4 was coupled with different Boc-amino acids using EDCI/HOBt as coupling agent and NMM as base (Scheme 1).

General procedure for the coupling of N-Boc amino acids with diphenylmethylpiperazine
To the stirred solution of Boc-amino acid (2 mmol) and HOBt (0.31 g, 2 mmol) in DMF (10 mL) cooled to 0 ˚C, added NMM (0.22 mL, 2 mmol), EDCI (0.42 g, 2 mmol) and diphenylmethylpiperazine (0.5 g, 2 mmol).After 20 minutes, the pH of solution was adjusted to 8 by the addition of NMM and the reaction mixture was stirred overnight while slowly warming to room temperature.The reaction mixture was quenched with water (2 mL) and the solvent was condensed.The residue was dissolved in chloroform (25 mL), washed with 5% NaHCO 3 (3 x 20 mL), H 2 O (1 x 20 mL) followed by 0.1N cold HCl (3 x 20 mL) and brine solution (3 x 20 mL), dried over anhydrous Na 2 SO 4 .The chloroform was removed under reduced pressure to obtain the desired products.The analytical data of these compounds are presented in (Table 1).
Where, R= Side chains of amiono acids

Deprotection of Boc group
The Boc group was deblocked by treating the protected compounds (1 mmol) with 4 N HCl in dioxane (10 mL / g of the compound) for 1.5 hours.Excess HCl and dioxane were removed under reduced pressure, triturated with ether, filtered, washed with ether and dried (yield, 100%).

Antimicrobial assay Antibacterial activity
In vitro antibacterial assays were performed against Staphylococcus aureus, E coli, Klebesiella pnemoniae and Pseudomonas auregenosa by using agar well diffusion method 25 .All the synthesized compounds were tested in triplicate, streptomycin was used as positive control and water as negative control.The zone of inhibition area was measured in mm (Table 2).

Antifungal activity
In vitro antifungal assays were performed against Aspergillus niger, Aspergillus flavus and Fusarium monoliforme by using agar well diffusion method. 26The fungicidal activity of the synthesized compounds was assessed by comparing the zone of fungal growth in treated plates with that of control plates in mm (Table 2).

Results and Discussion
We have synthesized new class of diphenylmethylpiperazine derivatives 5(a-l) by coupling of diphenylmethylpiperazine with N-protected Boc-amino acids using EDCI as a coupling agent, HOBt was used to avoid the racemization at the C-terminal amino acids, NMM was used to maintain the pH to 8. The product obtained was gummy and characterized by TLC, elemental analysis and 1 H NMR.
All the synthesized compounds 5(a-l) showed antibacterial activity against strains of both gram +ve and gram -ve bacteria such as Staphylococcus aureus, Klebesiella pnemoniae, Pseudomonas auregenosa and E coli and showed antifungal activity was against Aspergillus niger, Aspergillus flavus and Fusarium monoliforme.Among the synthesized compounds 5e and 5j showed equally good antibacterial activity as that of conventional antimicrobial drugs.Their enhancement of antibacterial activity may be due to the presence of aromatic and heterocyclic moiety of the amino acid residues.Even though amino acids and diphenylmethylpiperazine which taken in isolation were inactive or weakly active towards these bacterial strains, synthesized compounds showed activity.

Conclusion
In an effort to discover new diphenylmethylpiperazine analogues as antimicrobial molecule, we found that compounds 5e and 5j showed good antibacterial activity and all other compounds showed a moderate activity.But conversely some of the compounds 5i, 5e, 5f and 5j showed moderate antifungal activity and the remaining showed a mild antifungal activity.The results of the present study indicate that compounds 5i, 5j, 5e and 5f might be of interest for the identification of new antimicrobial molecules.Further work on conjugation of aromatic and heterocyclic moiety to the diphenylmethylpiperazine is in progress.

Table 1 .
Analytical data of the synthesized compounds.

Table 2 .
Antibacterial and antifungal activity of the synthesized compounds against various bacterial and fungal strains.