Synthesis, In-Vitro Antibacterial, Antifungal, and Molecular Modeling of Potent Anti-Microbial Agents with a Combined Pyrazole and Thiophene Pharmacophore

Ethyl 5-acetyl-4-methyl-2-(phenylamino)thiophene-3-carboxylate (2) and there derivatives 3a–c, 4, 6a–c and 9a–f were synthesized. The structure of compound 2 was deduced by 1H-NMR, 13C-NMR, FT-IR, MS, microanalysis, and single-crystal X-ray crystallography. The compound crystallized in the monoclinic system, with space group P21/c and cell coordinates a = 8.5752(16) Å, b = 21.046(4) Å, c = 8.2941(12) Å, β = 101.131(6)°, V = 1468.7(4) Å3, and Z = 4. Compounds 2, 3a–c, 4, 5a–c and 9a–f were subjected into in vitro antimicrobial activity tests. Compounds 3a and 3c were more potent than standard drug amphotericin B, showing MIC values of 23.8 ± 0.42 and 24.3 ± 0.68, respectively, against Aspergillus fumigatus while the standard drug MIC was 23.7 ± 0.1. Compound 3c was also more potent (MIC 24.8 ± 0.64) than the standard drug amphotericin B (MIC 19.7 ± 0.2) against Syncephalastrum racemosum. Compounds 4 and 9f also showed promising anti-microbial activity. Molecular modeling was performed for the most active compounds.


Introduction
Heterocyclic compounds possessing the thiophene core have attracted tremendous interest in the field of medicinal chemistry due to their diverse and wide range of biological properties, including analgesic [1], antidepressant [2], anti-inflammatory [3], antimicrobial [4] and anticonvulsant activities [5][6][7][8]. The thiophene moiety is an integral part of the structure of different antiepileptic drugs (AEDs), for example etizolam, brotizolam, and tiagabine. It is worthy to mention that it has been established that the higher activity of sodium phethenylate has been attributed to the fact the structure contains a thiophene ring. Consequently, the synthesis of novel thiophene analogues has gained much importance in medicinal chemistry due to their potential as labile pro-drugs [7][8][9].
Enaminones are versatile precursors that have a lot of synthetic applications in organic chemistry. Enaminones are key synthons for the synthesis of a wide variety of naturally occurring alkaloids [10,11], and nitrogen-containing heterocycles [12][13][14][15]. They have also been employed as important intermediates for the synthesis of pharmaceutical drugs with antiviral, larvicidal [16] and anticonvulsant properties [17][18][19]. Due to their rich applications, many efficient approaches to these compounds have been developed. In continuation of our research program [20][21][22][23][24] studying the synthesis of novel heterocyclic compound which may be biologically active, herein, we report the synthesis of some novel heterocyclic compounds incorporating a combination of thiophene and pyrazole pharmacophores. The structure of the key intermediate 5-acetyl-4-methyl-2-(phenylamino)thiophene-3-carboxylate (2) was unambiguously deduced by the single-crystal X-ray diffraction technique. New series (3a-c, 4, 6a-c and 9a-f) from the key intermediate 2 were synthesized. The antimicrobial activities of the synthesized compounds were also examined and the molecular modeling of the most active products is discussed.

Synthesis of Compounds 2, 3a-c, 4, 6a-c and 9a-f
Ethyl 5-acetyl-4-methyl-2-(phenylamino)thiophene-3-carboxylate (2) was synthesized in 92% yield as shown in Scheme 1. Reaction of ethyl acetoacetate with phenyl isothiocyanate in the presence of K2CO3 under reflux in DMF, followed by addition of chloroacetone furnished the product 2. Compound 2 later reacted with aromatic amines using a catalytic amount of ZnCl2 in refluxing EtOH for 2 h to afford regioselectively the Schiff's bases 3a-c in excellent yields of up to 99%. The structure of 2 was deduced by combined use of IR, 1 H-NMR, 13 C-NMR, and mass spectral data. In addition, the assigned structure of 2 was unambiguously established via a single-crystal X-ray diffraction study. Scheme 1. Synthesis of ethyl 5-acetyl-4-methyl-2-(phenylamino)thiophene-3-carboxylate (2) and there derivatives 3a-c.
Next, Condensation of 2 with dimethylformamide dimethyl acetal (DMF-DMA) furnished enaminone 4 in excellent yield 93% (Scheme 2). Reaction of enaminone 4 with an N-nucleophile such as hydrazine hydarate, phenyl hydrazine and p-chlorophenyl hydrazine in EtOH under reflux for 4-6 h in the presence of a catalytic amount of ZnCl2 as a Lewis acid afforded 6a-c, respectively (Scheme 3). The formation of compounds 6a-c would involve an initial addition of the amino group in the hydrazine to the activated double bond in the enaminone derivative 4, followed by deamination to an intermediate which then undergoes cyclization and aromatization via loss of water affording the final isolable pyrazole derivatives 6a-c. The utility of enaminone 4 in the synthesis of annulated heterocycles was further explored via its reaction with (Z)-ethyl 2-chloro-2-(2-phenylhydrazono)acetate derivatives to afford 9a-f in very good yield (Scheme 3). Spectral data (IR, NMR, MS) and elemental analysis were consistent with the proposed structures of isolated products 9a-f. It is assumed that these products were formed via a [2+3] cycloaddition reaction and initial formation of a nonisolable pyrazole derivative 8, followed by elimination of NHMe2 to give the desired products 9a-f.

X-ray Crystal Structure of Compound 2
Slow evaporation of an ethanol solution of pure compound 2 afforded colorless crystals. A single crystal of approximate dimensions 0.33 × 0.17 × 0.14 mm was selected for the X-ray diffraction technique Data were collected on a Bruker Kappa APEXII Duo diffractometer equipped with a CCD detector and graphite monochromatic Mo Kα radiation (λ = 0.71073 Å) at 100 K. Cell refinement and data reduction were performed by Bruker SAINT. SHELXS-97 [25,26] was used to solve the structure (Tables 1-3).

Antimicrobial Activity
To investigate the biological activity of the newly prepared compounds, the cup-plate agar diffusion method was adopted by using sterile filter paper discs (6 mm in diameter). Tested compounds were dissolved in DMSO and loaded on the discs at concentrations of 5 mg/mL. The discs were then placed in Petri dishes and were charged with different Gram-positive and Gram negative bacterial strains Pseudomoas aeruginosa and Escherichia coli for Gram-negative bacteria and Staphylococcus pneumonia and Bacillis subtilis for Gram-positive, and Aspergillus fumigates, Syncephalastrum racemosum, Geotricum candidum and Candida albicans for fungi. Results of the biological activity are displayed in Table 4; results are expressed as mm inhibition. Table 4. Antibacterial and antifungal activity of synthesized compound (zone of inhibition in diameter in mm). 12  The results shown in Table 1 reveal that compounds 2, 3a-c, 4, 6a-c and 9a-f exhibit moderate to high activity against both fungi and Gram-positive bacteria. On the other hand, compounds 3a, 3c and 9f were the most active against the tested fungi. Results also show that compounds 3a and 3c with MIC values of 23.8 ± 0.42 and 24.3 ± 0.68, respectively, were more potent than the standard drug amphotericin B (MIC 23.7 ± 0.1) against Aspergillus fumigatus. On the other hand, 3c (MIC 24.8 ± 0.64) was also more potent than the standard drug (amphotericin B, MIC

Molecular Modeling
To understand the mechanism of the antimicrobial and antifungal activities of the compounds synthesized, molecular modelling and docking studies were performed on the X-ray crystal structure of the E.  (Table 5).  (Figure 3). Figure 4 shows that compound 9f was superimposed with co-crystalline clorobiocin in the active site of the E. coli 24 kDa domain (Figure 4).  Regarding cytochrome P450 14α-sterol demethylase, the fourteen tested compounds revealed MolDock scores between −139.61 to −221.17 (Table 5). Compound 9b was found to have best MolDock score of −221.17 and form four hydrogen bonding interactions with Arg96 (2.73, 2.79, 2.92 Å) and Gln72 (3.53 Å) ( Figure 5).

General
All melting points were recorded on a Gallenkamp melting point apparatus in open glass capillaries and are uncorrected. All the chemicals were purchased from Fluka Chemie GmbH (Buchs, Switzerland) and Sigma-Aldrich (Gillingham, Dorset, UK) and were used without further purification, unless otherwise stated. IR spectra were recorded as KBr pellets on a 6700 FT-IR Nicolet spectrophotometer (Thermo Fisher Scientific, Madison, WI, USA). Elemental analysis was carried out on a 2400 Elemental Analyzer, CHN mode (Perkin Elmer, Waltham, MA, USA). The NMR spectra were recorded on an Avance AV-600 NMR spectrometer (Bruker, International Equipment Trading Ltd 960 Woodlands Parkway, Vernon Hills, IL, USA). 1 H-NMR (600 MHz) and 13 C-NMR (150 MHz) were run in deuterated dimethyl sulphoxide (DMSO-d6) or deuterated dimethylformamide (DMF-d7). MS were recorded using a JMS-600 H instrument (Peabody, MA, USA).

Preparation of Ethyl 5-Acetyl-4-methyl-2-(phenylamino)thiophene-3-carboxylate (2)
A mixture of ethyl acetoacetate (1, 13 g, 0.1 mol) and anhydrous potassium carbonate (25 g) in DMF (30 mL) was stirred vigorously at RT for 5 min then phenyl isothiocyanate (13.5 mL, 0.1 mol) was added with continued stirring for 30 min. The resulting reaction mixture was cooled in an ice bath, and chloroacetone (13.9 mL, 0.1 mol) was added over 15 min with continued stirring. The cooling bath was subsequently removed and the mixture was stirred for 2 h. The solid product 2 was precipitated by addition of H2O, collected by filtration, washed with water, and dried.

General Preparation of Compounds 3a-c (GP1)
Compound 2 (0.303 g, 1 mmol) was fused with the appropriate aniline derivative (1 mmol, 1 equiv.) for 10 min, then EtOH (10 mL) was added to the reaction mixture, followed by ZnCl2 (0.2 gm) and the reaction mixture was refluxed for 6 h. The solid product was collected by filtration and recrystallized from EtOH to afforded 3a-c.

General Preparation of compounds 6a-c (GP2)
A mixture of 4 (1 mmol) and the appropriate hydrazine derivative (1 mmol, 1 equiv.) was refluxed for 6 h in EtOH (10 mL). The solid product was collected by filtration and recrystallized from EtOH to afforded 6a-c.

Antibacterial Activity of Compounds 2, 3a-c, 4, 6a-c and 9a-f
Samples of 2, 3a-c, 4, 6a-c and 9a-f were screened in vitro for antibacterial activity against various bacterial strains namely, Escherichia coli and Pseudomoas aeruginosa (Gram-negative bacteria) and Bacillis subtilis and Staphylococcus pneumonia (Gram-positive bacteria). The antibacterial activity was measured by the agar well diffusion method according to a reported procedure [27].

Molecular Modeling
For the docking of ligands into the proteins' active sites and for estimating the binding affinities of docked compounds, the X-ray crystal structure of the E. coli 24 kDa domain in complex with clorobiocin (PDB code: 1KZN) and the crystal structure of cytochrome P450 14α-sterol demethylase (Cyp51) from Mycobacterium tuberculosis in complex with fluconazole (PDB 1EA1) were obtained from the Brookhaven Protein Data Bank [28] and loaded to the Molegro Virtual Docker (MVD2013.6.0.0 [win32]) program (fully functional free trial version with time limiting license [29]). The non-bonded oxygen atoms of water, present in the crystal structure, were removed. ChemBio3D Ultra 10 [30] was used to draw the 3D structures of the different ligands. Ligands were further pre-optimized using the free version of MarvinSketch 4.1.13 from Chemaxon Ltd. [31] with the MM force field and saved in Tripos mol2 file format. MolDock score functions were used with a 0.3 Å grid resolution. The binding sites were defined to any residues with 10 Å distant from the cocrystallized clorobiocin and fluconazole in the complex crystal structure of the enzymes [32,33].

Conclusions
The synthesis in excellent yield and characterization of the new compound ethyl 5-acetyl-4-methyl-2-(phenylamino)thiophene-3-carboxylate (2) was reported. The structure of 2 was deduced by single-crystal X-ray diffraction. New enaminone derivatives 4 and a series of novel pyrazole derivatives 6a-c and 9a-f were reported. All synthesized products have been examined for anti-microbial activity and shown promising results. Also the molecular docking of the synthesized compounds was discussed.