Synthesis and antimicrobial activities of novel peptide deformylase inhibitors

A new series of N-formylhydroxylamine compounds were designed, optimized with the AutoDock 4.0.1 to investigate the interactions between the target compounds and the amino acid residues of the Escherichia coli PDF•Ni enzyme, and then synthesized through multi-step sequence starting from diethyl malonate. The structures of the compounds were characterized on the basis of spectral (FT-IR, 1H NMR and mass) analysis. All the synthesized compounds have been screened for their antimicrobial activities. It was found that the compounds 11c , 11d , 11f and 11g exhibited potent inhibitory activity against S. aureus in vitro .


Introduction
2] There is, therefore, an urgent need to identify new antibiotics to combat infectious diseases.One of the new targets receiving widespread interest from both academic and industrial researcher groups is Peptide deformylase (PDF), [3][4][5] which is an iron containing metalloenzyme responsible for the removal of the N-formyl group from the terminal methionine residue following protein synthesis in bacteria, 6 and this enzyme is a high priority target for antibiotic design. 79] The N-formyl hydroxylamine BB-3497 is an effective inhibitor (IC50 = 7nM) of the Escherichia coli PDF•Ni enzyme, exhibiting potent antibacterial activity both in vitro and in vivo. 10n order to reduce the peptide structural features to find more active compounds, we referred to the structure of the BB-3497, which was reported in the literature, 11 and designed a new series of N-formylhydroxylamine derivatives.Our intention is to synthesize new active compounds and detect their antibacterial activities by introducing one benzimidazole ring [12][13] to serve as a hydrogen bond donor or accepter.At the meantime, in order to investigate the interactions between our designed compounds and the amino acid residues of the Escherichia coli PDF•Ni enzyme, a molecular docking study was also performed.After the optimization of the designed compounds, they were synthesized with our own designed multi-step reaction route and their antibiotic activities were tested in vitro also.

Molecular docking study
The molecular docking study was performed using the AutoDock 4.0.1 software. 14The crystallographic structure of Escherichia coli PDF•Ni enzyme which is retrieved from the RSCB Protein Data Bank (PDB code 1G2A) serves as docking receptor, 15 and all the synthesized compounds are selected as ligand molecules.The 50 docking runs for each ligand were clustered on the basis of root mean square deviation (RMSD = 1.0 Å) between the Cartesian coordinates of the ligand atoms and were ranked according to the binding free energy.The structure with the lowest binding free energy and the most cluster members was chosen for the optimum docking conformation.
Docking results show that all the designed molecules have similar orientations in the binding pocket of PDF enzyme, except that the terminal substituents on the benzimidazole ring have relatively large conformational differences because of their diversity on atomic composition and chemical property.The functional group HCO-NOH in all molecules are chelated to Ni 2+ ion and can effectively form hydrogen bonds with Gln50, Leu91 and Glu133, which is very similar to inhibitor BB-3497. 16The binding modes of BB-3497 and the synthesized compound 11c bound to active site of PDF•Ni enzyme are shown in Figure 1.It should be noted that on the benzimidazole portion of 11c, the N 3 atom is hydrogen bonded to the amine hydrogen of Gly89 and the bonding length is 1.96 Å, and another hydrogen bond which located at the O atom of methoxyl and residue Arg97 also forms, with a bond length of 1.84 Å.These strong hydrogen-bonding interactions are concomitant with the introduction of the benzimidazole ring, which means that this portion can increase the binding affinity between the target molecule and the Escherichia coli PDF•Ni enzyme.

Figure 1.
The binding conformation of inhibitor BB-3497 16 and 11c with E. coli PDF, shown in part A and B respectively.

Chemistry
The target compounds were prepared using the reaction sequence as shown in Scheme 1, 2. The chemical structures of the synthesized compounds were confirmed by means of their IR, 1 H NMR and mass spectral analysis.

Antibacterial activity
All the synthesized compounds were evaluated for their antimicrobial activities.The results are summarized in Table 1.All the target compounds exhibit weak inhibitory activity against K. pneumonia.Compounds 11a, 11b and 11e show weak to moderate antibacterial effect against S. aureus, while compounds 11c, 11d, 11f and 11g show high activity comparable to that of the positive drug.The presence of electron-withdrawing groups on the benzene ring in generally increased the antimicrobial activity against S. aureus compared to compounds with electron-donating groups.Based upon the results, it is necessary to optimize the target molecule by substituting a series of electron-withdrawing groups on the benzene ring.Taken together, these results confirmed the mode of action of the target compounds and directed our design in the future.

Experimental Section
General.All solvents used were of analytical grade.Melting points were determined in open capillary tubes and were uncorrected.The 1 H NMR spectra were recorded on a Bruker Avance DPX300 spectrometers with CDCl3 as the solvent and TMS as the internal standard.The IR spectra were measured on a Bruker Vector FTIR spectrophotometer with KBr pellets or film.The mass spectra were obtained with an Agilent 6510 Q-TOF spectrometer.

Synthesis of N-(benzyloxy)formamide (3).
Sodium hydroxide (5.00 g, 125.0 mmol) and ethyl formate (23.19 g, 313.5 mmol) were added to a stirred mixture of O-benzylhydroxylamine (10.00 g, 62.7 mmol) in anhydrous methanol (120 ml), and the resulting mixture was stirred at room temperature for 16 h.The reaction mixture was concentrated to remove methanol and then was extracted with dichloromethane (3 × 40 ml), and the combined extracts were washed with water, 2 mol/L HCl and brine successively and then dried over anhydrous MgSO4 followed by evaporation.The crude product was distilled under the reduced pressure to result in compound 3 in the form of a colorless oil.Yield: 70%.

Synthesis of tert-butyl (1H-benzo[d]imidazol-2-yl)methylcarbamate (7a). Typical procedure
Benzene-1, 2-diamine (1.05 g, 9.72 mmol) and N-(tert-butoxycarbonyl)glycine (1.71 g, 9.72 mmol) were dissolved in 30 ml of THF and cooled to 0 °C.Into the above solution was added N, N'-dicyclohexylcarbodiimide (2.41 g, 11.7 mmol ) in batches and the mixture was stirred at 0 °C for half an hour and then at room temperature overnight.The reaction mixture was filtrated and evaporated to afford a brown oil, which was purified by a silica-gel column chromatography (dichloromethane / methanol, 25:1 by volume) to get a yellow solid (2.24 g).
The solid was dissolved in 20 ml of acetic acid and the solution was stirred at 72 °C for 8 h.The acetic acid was removed under reduced pressure and the crude compound was purified by a silica-gel column chromatography (dichloromethane / methanol, 25:

Table 1 .
The antibacterial activity of the target compounds (MIC) Compound 4 (5.01 g, 16.3 mmol) and LiOH•H2O (0.79 g, 17.9 mmol) were stirred in 75 ml of 2:1 (volume ratio) methanol/water at room temperature for 24 h.The reaction mixture was concentrated to remove methanol, and the residue was diluted with water (10 ml).The mixture was then extracted with ethyl acetate (3 × 20 ml), and the aqueous phase was acidified to pH < 3 with concentrated HCl.