Hydrazonoyl Chlorides as Precursors for Synthesis of Novel Bis-Pyrrole Derivatives

A convenient synthesis of some novel bis-pyrrole derivatives via hydrazonoyl halides is described. Antimicrobial evaluation of some selected examples of the synthesized products was carried out. The bis-pyrrole derivative having chloro substituents showed good activity against all of the used microbes. The molecular docking of the bis-pyrrole derivatives was performed by the Molecular Operating Environment (MOE) program.


Chemistry
Treatment of compound 1 with a α-ketohydrazonoyl halide (2a) [28,29] in (1:2 molar ratios), in dry benzene at refluxing temperature in the presence of triethylamine (2 molar ratio) afforded the bis-pyrrole derivative 5a (Scheme 1). The structure of compound 5a was elucidated from its spectroscopic, as well as elemental analytical data. Its 1 H-NMR spectrum revealed a triplet signal at δ 1.13 (J = 7.1 Hz) due to CH 3 protons, two singlet signals at δ 2.17 and 2.34 due to two CH 3 protons and a quartet signal at δ 4.14 (J = 7.1 Hz) due to CH 2 protons, a singlet signal δ 4.31 due to NCH 2 protons, in addition to an aromatic multiplet in the region 7.40-7.68 ppm. This spectroscopic analysis allows ruling out structures 6a and 7a. To account for the formation of the product 5a, it is assumed that the reaction initially proceeds via an initial nucleophilic substitution to give the intermediate 3a, which underwent intramolecular cyclization followed by the elimination of two water molecules to afford the final product 5a.
Molecules 2016, 21, 326 2 of 9 underwent intramolecular cyclization followed by the elimination of two water molecules to afford the final product 5a.
Prompted by the aforementioned results, and to generalize this reaction, the behavior of the hydrazonoyl halides 2b-e [30] towards compound 1 was studied, under the same experimental conditions, which led to the respective bis-pyrrole derivatives 5b-e (Scheme 2).
Prompted by the aforementioned results, and to generalize this reaction, the behavior of the hydrazonoyl halides 2b-e [30] towards compound 1 was studied, under the same experimental conditions, which led to the respective bis-pyrrole derivatives 5b-e (Scheme 2). underwent intramolecular cyclization followed by the elimination of two water molecules to afford the final product 5a. NH Prompted by the aforementioned results, and to generalize this reaction, the behavior of the hydrazonoyl halides 2b-e [30] towards compound 1 was studied, under the same experimental conditions, which led to the respective bis-pyrrole derivatives 5b-e (Scheme 2).

Antimicrobial Evaluation
Gram-positive and Gram-negative standard bacterial strains were used in this study to screen synthesized compounds for their potential antibacterial activities. The Gram-positive bacteria were Staphylococcus aureus and Bacillus subtilis. The Gram-negative bacteria were Pseudomonas aeruginosa and Escherichia coli. Four species of fungi known to cause different types of mycoses were also used Scheme 2. Synthesis of bis-pyrrole derivatives 5b-e.

Antimicrobial Evaluation
Gram-positive and Gram-negative standard bacterial strains were used in this study to screen synthesized compounds for their potential antibacterial activities. The Gram-positive bacteria were Staphylococcus aureus and Bacillus subtilis. The Gram-negative bacteria were Pseudomonas aeruginosa and Escherichia coli. Four species of fungi known to cause different types of mycoses were also used to test the antifungal activities of synthesized compounds in this study. These fungal species were Aspergillus fumigatus, Geotrichum candidum, Syncephalastrum racemosum, and Candida albicans. Inhibition zone diameter (IZD) in mm was used a criterion for the antimicrobial activity using the agar diffusion well method. The fungicide Itraconazole and the bactericides Penicillin G and Streptomycin were used as references to evaluate the potency of the tested compounds under the same conditions. The results are depicted in Table 1. The results revealed that most of the tested compounds revealed better activity against the Gram-positive bacteria rather than the Gram-negative bacteria. Also, all compounds exhibited almost no activity against Candida albicans. Compound 5d was found to be the most potent relative to the standard drug, Itraconazole, against Aspergillus fumigates.
Additionally, compound 5d has a high degree of antibacterial activity against Gram-positive bacteria Staphylococcus aureus (MSSA) and Bacillus subtilis. All the tested compounds except 5d exhibited no activity against Pseudomonas aeruginosa. The structure antimicrobial activity relationship of the synthesized compounds revealed that the maximum activity was attained with compound 5d, having chloro substituent in the para position of the phenyl ring.

Docking and Molecular Modeling
Molecular docking is used to predict the binding mode of ligands within the binding site of target proteins [31]. To validate and specify the target protein for the anti-bacterial activity of newly synthesized bis-pyrrole derivatives E. coli Enoyl reductase protein was selected and downloaded from the Protein Data Bank (PDB ID: 1LXC) [32].
Docking studies of compound 5a into the active site of E. Coli Enoyl reductase Enzyme showed van der Waals bonding between Phe203 and the phenyl ring (Figure 1), while docking studies of compound 5b showed no interactions with E. coli Enoyl reductase Enzyme (Figure 2).  Similarly, docking conformation of compound 5c in the active site of E. coli Enoyl reductase Enzyme showed good interactions with the active site residues of this protein. Compound 5c formed hydrogen bond interaction between carbonyl group moiety, as it acts as a hydrogen bond acceptor with the side chain of Phe203 residue (2.47 Å) with a strength of 45%. Furthermore, it showed van der Waals interaction with Lys163 ( Figure 3).   Similarly, docking conformation of compound 5c in the active site of E. coli Enoyl reductase Enzyme showed good interactions with the active site residues of this protein. Compound 5c formed hydrogen bond interaction between carbonyl group moiety, as it acts as a hydrogen bond acceptor with the side chain of Phe203 residue (2.47 Å) with a strength of 45%. Furthermore, it showed van der Waals interaction with Lys163 ( Figure 3). Similarly, docking conformation of compound 5c in the active site of E. coli Enoyl reductase Enzyme showed good interactions with the active site residues of this protein. Compound 5c formed hydrogen bond interaction between carbonyl group moiety, as it acts as a hydrogen bond acceptor with the side chain of Phe203 residue (2.47 Å) with a strength of 45%. Furthermore, it showed van der Waals interaction with Lys163 ( Figure 3). Docking studies of compound 5d showing hydrogen bond interaction between nitrogen atom of azo group moiety, as it acts as a hydrogen bond acceptor with the side chain of Asn155 (2.49 Å) with a strength of 11% (Figure 4). while docking studies of compound 5e showing hydrogen bond interaction between carbonyl group moiety, as it acts as a hydrogen bond acceptor with the side chain of Phe203 (2.96 Å) with a strength of 15% ( Figure 5).  Docking studies of compound 5d showing hydrogen bond interaction between nitrogen atom of azo group moiety, as it acts as a hydrogen bond acceptor with the side chain of Asn155 (2.49 Å) with a strength of 11% (Figure 4). while docking studies of compound 5e showing hydrogen bond interaction between carbonyl group moiety, as it acts as a hydrogen bond acceptor with the side chain of Phe203 (2.96 Å) with a strength of 15% ( Figure 5). Docking studies of compound 5d showing hydrogen bond interaction between nitrogen atom of azo group moiety, as it acts as a hydrogen bond acceptor with the side chain of Asn155 (2.49 Å) with a strength of 11% (Figure 4). while docking studies of compound 5e showing hydrogen bond interaction between carbonyl group moiety, as it acts as a hydrogen bond acceptor with the side chain of Phe203 (2.96 Å) with a strength of 15% ( Figure 5).

General
Melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. IR spectra were recorded in potassium bromide discs on Shimadzu FTIR 8101 PC infrared spectrophotometer (Shimadzu, Tokyo, Japan). The NMR spectra were recorded on a BRUKER VX-500 NMR spectrometer (Varian, Palo Alto, CA, USA). 1 H spectra were run at 500 MHz and 13 C spectra were run at 125 MHz in deuterated dimethylsulphoxide (DMSO-d6) using TMS as an internal standard. Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer (Shimadzu, Tokyo, Japan) at 70 e.V. Elemental analyzes were measured by using a German made Elementar vario LIII CHNS analyzer (GmbH & Co. KG, Hanau, Germany). The biological evaluation of the products was carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt. Diethyl (Z,Z)-3,3′-(ethane-1,2-diyldiimino)-dibut-2-enoate (1) [26,27], hydrazonoyl chlorides (2a) [28,29], and 2b-e [30] were prepared according to the procedures reported literature.

Synthesis of Bis-Pyrrole Derivatives 5a-e
General procedure: To a solution of compound 1 (0.284 g, 1 mmol) and the appropriate hydrazonoyl halide 2a-e (2 mmol) in dry benzene (20 mL), was added triethylamine (0.2 mL, 2 mmol) and the reaction mixture was heated under reflux for 6 h. After cooling, the mixture was filtered off to remove the precipitated triethylamine hydrochloride and the solvent was distilled under reduced pressure. The oil residue was triturated with MeOH and the solid product was collected by filtration, washed with methanol, and recrystallized for purification to afford the corresponding bis-pyrrole derivatives 5a-e.

General
Melting points were measured on a Gallenkamp melting point apparatus and are uncorrected. IR spectra were recorded in potassium bromide discs on Shimadzu FTIR 8101 PC infrared spectrophotometer (Shimadzu, Tokyo, Japan). The NMR spectra were recorded on a BRUKER VX-500 NMR spectrometer (Varian, Palo Alto, CA, USA). 1 H spectra were run at 500 MHz and 13 C spectra were run at 125 MHz in deuterated dimethylsulphoxide (DMSO-d 6 ) using TMS as an internal standard. Chemical shifts were related to that of the solvent. Mass spectra were recorded on a Shimadzu GCMS-QP 1000 EX mass spectrometer (Shimadzu, Tokyo, Japan) at 70 e.V. Elemental analyzes were measured by using a German made Elementar vario LIII CHNS analyzer (GmbH & Co. KG, Hanau, Germany). The biological evaluation of the products was carried out in the Medical Mycology Laboratory of the Regional Center for Mycology and Biotechnology of Al-Azhar University, Cairo, Egypt. Diethyl (Z,Z)-3,3 1 -(ethane-1,2-diyldiimino)-dibut-2-enoate (1) [26,27], hydrazonoyl chlorides (2a) [28,29], and 2b-e [30] were prepared according to the procedures reported literature.

Synthesis of Bis-Pyrrole Derivatives 5a-e
General procedure: To a solution of compound 1 (0.284 g, 1 mmol) and the appropriate hydrazonoyl halide 2a-e (2 mmol) in dry benzene (20 mL), was added triethylamine (0.2 mL, 2 mmol) and the reaction mixture was heated under reflux for 6 h. After cooling, the mixture was filtered off to remove the precipitated triethylamine hydrochloride and the solvent was distilled under reduced pressure. The oil residue was triturated with MeOH and the solid product was collected by filtration, washed with methanol, and recrystallized for purification to afford the corresponding bis-pyrrole derivatives 5a-e. N-methyl-N-((1-methyl-1H-indol-2-yl)methyl) acrylamide (PDB ID: AYM)) (NAD), that was obtained from Protein Data Bank.

Conclusions
A new simple approach to bis-pyrrole derivatives from hydrazonoyl halides has been achieved. The maximum antimicrobial activity was attained with compound 5d, having chlorine in the para position. The molecular docking of bis-pyrrole derivatives 5a-e was performed using the MOE program.