Solid supported microwave induced synthesis of imidazole–pyrimidine hybrids: Antimicrobial evaluation and docking study as 14DM-CPY51 inhibitors

2.2.1 Conventional method

Substituted ethyl 1-formyl-1,2,3,6-tetrahydro-4-methyl-6-phenyl-2-oxo/thioxopyrimidine-5-carboxylates 1a–g (25 mmol), Benzil 2 (25 mmol, 5.25 g) and ammonium acetate (10 g) were dissolved in glacial acetic acid and refluxed for 10–12 h. After refluxing, the reaction mixture was left overnight and filtered to remove any precipitate. Water (300 mL) was then added to the filtrate and the first crop of crude material was obtained. The filtrate was neutralized with ammonium hydroxide and a second crop of the solid was collected. The two crops of the precipitate were combined, dried and recrystallized from ethanol.

2.2.2 Microwave irradiation method

Substituted ethyl 1-formyl-1,2,3,6-tetrahydro-4-methyl-6-phenyl-2-oxo/thioxopyrimidine-5-carboxylates 1a–g (25 mmol), Benzil 2 (25 mmol, 5.25 g), ammonium acetate (10 g) and acidic alumina (1.0 g) were mixed thoroughly in an agate mortar. The mixture was placed in a beaker, 2-drops of glacial acetic acid was added. The beaker was kept in a microwave oven at maximum power level for 8 min with intermittent interval. The progress of reaction was monitored by TLC using benzene:ethyl acetate (8:2). The dried mass was diluted in cold water to dissolve acidic alumina and crude product was recovered, washed with ammonium hydroxide. The obtained mass was dried and recrystallized from ethanol (Scheme 1).

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Scheme 1

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2.3.1 Methodology

All molecular techniques used in this manuscript were performed on Argus Lab ver. 4.0-work system. The starting 3D structure (Boscott and Grant, 1994) of the cytochrome P450sterol 14DM CPY51 of C. albicans was downloaded from the Protein Data Bank (http://www.rcsb.org) as PDB files (PDB entry: 1ea1).

2.3.2 1ea1

The file containing the crystal structure of cytochrome P450α-sterol demethylase (14DM) with its selective inhibitor i.e. Fluconazole (470TPF) in the active site (PDB entry 1ea1, six ligand) was downloaded. It is monomer structure with only chain A consisting of 449 residues. This chain A has 470YPF, water and 1 heme (HEM) groups. The chain A with residues, water and the hetero groups (HEM) within a radius of 5 Å was refined and cleaned by checking the hybridization, valence of the ligand and introducing H-atoms to the protein residues. 1ea1 carries net charge 2 and 3590 atoms. The active sites of residues of 1ea1 are shown in (Fig. 2).

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Figure 2

Active site residues of cytochrome P450 (14DM) CPY51 with Fluconazole (470TPF) hydrogens are suppressed for clarity.

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2.3.3 Docking and binding evaluation

In the automated module of Argus Lab ver. 4.0 work systems, the ligand was docked into the active site of cytochrome P450 14α-sterol demethylase (14DM) from C. albicans using Argus dock with a fast, simplified potential of mean force (PMF). The docking is carried with flexible ligand into a rigid protein active site. The general procedure for docking process starts with the addition of energy minimized target ligand on the enzyme. The active site and the ligand were specified in the program. The different starting parameters were optimized by using 15 × 15 × 15 box located at the centre of the target active site using a united atom (explicit hydrogen are not considered) potential of mean force (PMF) with a docking algorithm that has a population of 50 chromosomes and runs for 6000 generations. The process of docking is repeated until a constant value of docking score is reached. This takes about 12,000–18,000 generation. The final results are parameterized in terms of docking score in kcal/mol. The docked ligand–P450 (14DM) CPY51 complex is interpreted by looking at the H-bonding or hydrophobic interaction of the ligand with the amino acid residues in the active site. The same procedure was followed for docking of different substituted 4,5-imidazolyl pyrimidine into the active site of cytochrome P450 14α-sterol demethylase CPY51 enzymes.

2.3.4 Validation of PMF method

To ensure the validation of the programme, before docking the test compounds, the docking of Fluconazole (470TPF) into the active site of P450 14DM was performed. This selective inhibitor of cytochrome P450 binds in the active site with a binding score of −9.1128 cal/mol. The docked structure of Fluconazole in the active site of cytochrome P450 is shown in (Fig. 2).

2.3.5 Docking of known selective and non-selective cytochrome P450 demethylase inhibitors into the active site of 1ea1

The non-selective behavior of Clotrimazole, Miconazole, Griesofulvin and selective behavior of Fluconazole towards cytochrome P450 (14DM) CY51 is in reasonable agreement with their binding energy (docking score) with 1ea1 as calculated from the docking of these compounds in 1ea1 active site. During binding of this known antifungal drug in the binding pocket of cytochrome P450, the conformational placement of amino acid residues in the active site is observed.

2.3.6 Docking of imidazolylpyrimidine derivatives into active site of 1ea1

The docking of synthesized substituted 4,5-diphenyl imidazolyl pyrimidine derivatives (Scheme 1) 3a–g in 14DM active site has been studied.

2.4.1 In vitro antibacterial activity

As the sensitivity was not observed at conc. <100 μg disc⁻¹, the antibacterial activity of compounds 3a–g has been assayed at concentration of 100 μg disc⁻¹ against strains of Gram +ve and Gram −ve pathogenic bacteria (Salmonella typhi, Pseudomonas aurogenosa, Klebsiella pneumonae and Staphylococcus aureus). Initially, susceptibility testing was carried out by measuring the inhibitory zone diameter on Muller–Hinton agar, with conventional paper disc diffusion method (Fioraventi et al., 1997a,b), and the inhibitory zone diameters were read and rounded off to the nearest whole numbers (mm) for analysis. The inhibitory effects of compounds 3a–g against these organisms were compared with standard drug i.e. Norfloxacin.

2.4.2 In vitro antifungal activity

As the sensitivity was not observed at conc. <100 μg disc⁻¹, the antifungal activities of compounds 3a–g have been assayed in vitro at a concentration 100 μg disc⁻¹ against C. albicans. Griesofulvin was used as standard fungicide for the antifungal test. Muller–Hinton agar was used as basal medium for test fungi (Fioraventi et al., 1997a,b). Glass Petri dishes were sterilized and 10 ml of sterilized melted MH agar medium (45 °C) was poured into each Petri dish. After solidification of the medium, small portion of mycelium of C. albicans was spread carefully over the centre of each MH agar plate with the help of spreader. Thus, fungus was transferred to each plate. The plates were then incubated at (27 °C) and after half an hour of incubation they were ready for use. The prepared discs of test sample were placed gently with sterile forceps on the solidified agar plate, freshly seeded with the test organisms. The plates were then incubated at 37.5 °C for 24 h. Dimethyl formamide (DMF) was used as a solvent to prepare desired solutions of the compounds initially.

3.1.1 A-single crystal XRD 3a

Yellow coloured needle shape single crystals suitable for X-ray diffraction were grown from methanol using the slow evaporation technique. Diffraction intensity data were collected with a FR590 MACH3 single crystal diffractometer using Mo Kα monochromated radiation (λ = 0.70930) at room temperature (299 K). Crystal data, data collection and structure refinement for the compound are listed in (Table 2). The final atomic coordinates for all atoms and a complete listing of bond distance and angles are tabulated.

An integration type of absorption correction was applied to data sets. The structure was resolved by direct methods using the solution program SHELXS97 in the WinGX package (Sheldrick, 1997) and refined by a full-matrix least-squares procedure on F² using SHELXS97. All non-hydrogen atoms were refined, first with isotropic and then anisotropic parameters. Hydrogen atoms bonded to carbon were included using a riding model, starting from calculated positions. An ORTEP-3 (Farrugia, 1997) drawing of the molecule with the atomic numbering scheme is shown in (Fig. 3).

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Figure 3

An ORTEP drawing of the molecule 3a showing the atomic numbering system.

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3.3.1 In vitro antibacterial and antifungal activities

The screening results revealed that in addition to 3a–c, the compound 3d was found to be the most active against S. aureus amongst all the tested compounds. S. typhi is highly sensitive to the compound 3b and 3e–g while moderately sensitive to Compounds 3a and 3c. The results depicted in Table 5 suggested that P. aurogenosa and K. pneumonae are highly resistant to the synthesized compounds. In vitro antifungal studies revealed that the compounds 3b and 3c displayed significant activity while rest of the compounds showed moderate to low activity (Table 5).

Similarly preliminary studies in order to verify how the synthesized azole compounds interact at the target enzyme cytochrome P450-dependent lanosterol 14α-demethylase (P450 14DM, CPY51) in the ergosterol biosynthesis pathway have been carried out by computational approach and structure–activity relationship.

3.3.2 Structure–activity relationship

A comparative study involving the interaction of known antimicrobial drug viz. Clotrimazole, Miconazole, Fluconazole, and Griesofulvin in the active site pocket of cytochrome P450 (1ea1) was made for better understanding of their antibacterial and antifungal action. Structure–activity relationship, a powerful stencil used for tailoring effective lead molecules, was studied. We tested the entire synthesized compounds against bacteria and fungus with moderate to high antimicrobial activity. All compounds can be generalized into a single frame work and expected to show their potency as synthetic inhibitor, depending upon the structural features essential for binding in the cavity of cytochrome P450 (1ea1). All newly synthesized molecules (3a–g) could be divided into three segments viz. imidazole central core structure (ring A), characteristics substituted pyrimidine nucleus (ring B) and two phenyl rings (rings C and D). Our hybrid molecule considered as a template scaffold in which one can insert substituted aromatic ring as a substituent of ring B to enhance the specificity towards microorganism as antifungal activities. These hybrid molecules possesses ring A as imidazole subunit, ring B constitute substituted pyrimidine ring at C-2 position of imidazole ring while the attached two phenyl ring at C-4 and C-5 position of main imidazole moiety raised antifungal activity (Fig. 8).

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Figure 8

Structure–activity relationship.

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It is interesting to note that the compounds 3b, 3c and 3f exhibit significant antifungal activity revealing that the presence of electron withdrawing groups like –Cl, NO₂ as substituent at different position on aromatic ring attached to ring B improved the efficacy of the compounds. The two phenyl rings are essential for modulation of the hydrophobic interaction which enhanced its antifungal activity. Other compounds, exhibited moderate activity against fungi and bacteria due to the presence of –OCH₃ and –OH group on substituted aromatic ring.