Synthesis and biological evaluation of 4-(2′,4′-difluorobiphenyl-4-yl)-6-arylpyrimidin- 2-amine derivatives

Chemistry

The desired compounds 5a–g were synthesized as shown in Scheme 1 using the synthetic methodology described previously for similar derivatives [15], [16]. The key intermediates 3a–g were prepared by the Claisen-Schmidt condensation method as reported previously [17]. The structures of the synthesized compounds were characterized by elemental analysis, IR, ¹H NMR, ¹³C NMR spectroscopy, and mass spectrometry. The spectral analysis is fully consistent with the given structures. In several cases, the tentative assignments made by analysis of ¹H NMR and ¹³C NMR spectra were verified by analysis of the ¹H-¹³C COSY spectra. The electrospray ionization mass spectra of all products show the expected peak at [M+1]⁺.

Scheme 1

Antimicrobial evaluation

Compounds 5a–g were tested against Gram-positive bacteria (Bacillus subtilis and Staphylococcus aureus), Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae), and fungi (Candida albicans) by the disc diffusion method. Additional evaluation was carried out on compounds 5a–g to determine their minimum inhibitory concentration (MIC) values using the two-fold serial dilution method. The results are illustrated in Figures 1 and 2 (Tables S1 and S2 in the Supplementary Material). The mean zone of inhibition produced by these compounds against tested bacterial and fungal strains ranged from 7 mm to 16 mm. Under similar conditions, gentamycin, used as a positive control, produced a mean zone of inhibition ranging between 17 mm and 20 mm. It can be seen that the fluorine-substituted derivative 5b possesses 75%–80% of the activity of gentamycin against all bacterial strains. Compound 5g with methoxy substitution at the ortho position exhibits superior activity towards the fungal strain C. albicans. The MIC results support the conclusion above that compound 5b has a pronounced antibacterial activity. All other derivatives, 5a and 5c–g, exert only moderate activities against the tested organisms.

Figure 1

Antimicrobial activity of compounds 5a–g obtained by disc diffusion method.

Figure 2

Antimicrobial activity of compounds 5a–g obtained by two-fold serial dilution method.

Cytotoxicity

Compounds 5a–g were assayed for their in vitro cytotoxic activity against the Hep-2 laryngeal carcinoma cell line by the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay with maintaining a control (with solvent only). The results are shown in Figure 3. In general, the pyrimidine derivatives exhibit good cytotoxic activity against various cell lines like melanoma, colon cancer, ovarian cancer, and breast cancer [18]. The results also indicate that the compounds are strongly inhibitory against the Hep-2 cell line. Based on the substitution on the phenyl ring, the cytotoxic activity decreases in the order of 4-F>H>2-OCH₃>4-Br>4-Cl>4-CH₃>3-NO₂. It is evident that compound 5b with lipophilic fluorine substitution exhibits the most potent inhibitory activity with a cytotoxicity of 70.5%. The results are in agreement with previous findings that incorporation of fluorine enhances the pharmacological activity of the modified molecule [19].

Figure 3

Cell viability of compounds 5a–g.

Molecular docking studies

Molecular docking is a promising tool to gain insight into the binding of the ligand and protein. Docking studies were made with protein carotenoid dehydrosqualene synthase from S. aureus complexed with BPH-673 (3ACX) and human folate receptor alpha in complex with folic acid (4LRH). Docking results of the ligands were expressed as a G score (Table 1), which is an empirical function that includes many factors like hydrophobicity, hydrogen bonding, and rotation penalty, among others. The docked ligands were ranked based on various types of interactions between the ligand and protein. The results reveal that all ligands can accommodate the active pocket of the receptor and show an excellent G score ranging from –10.9 to –9.9. The 3D- and 2D-binding modes of the best ligand 5g are shown in Figure 4.

Figure 4

3D and 2D images of the complex of compound 5g with 4LRH protein residues.

Antimicrobial assays

The clinical isolates of bacterial strains Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, and a fungal strain Candida albicans were obtained from the Department of Microbiology, Rajah Muthiah Medical College and Hospital, Annamalai University, Annamalainagar, Tamilnadu, India. The strains were inoculated on a sterile medium and sub-cultured on Mueller Hinton agar plates and maintained on agar at 4°C. Gentamycin was used as a positive control for bacteria and fungi.

Disc diffusion assay

Antimicrobial activity was assayed using the standard disc diffusion method. Inhibition zones were measured and compared with the standard positive controls. All the tests were carried out in triplicate.

Minimum inhibitory concentration (MIC)

The dilution susceptibility testing method was used for MIC determination with reference to the literature [20]. The test compounds were dissolved in 1 mL of chloroform. The growth was indicated by the measure of a white pellet on a well bottom.

Cell viability assay

The cytotoxicity of the synthesized compounds was tested against Hep-2 cell lines using the MTT assay [21]. The MTT assays were performed four times independently, and each independent experiment was done in triplicate. The percentage of survival was calculated using the formula:

% Survival =  [live cell number (test)/live cell number (control)] × 100.

Cytotoxicity (%) = 100 − % survival

Molecular docking

Molecular docking was performed with the aid of Maestro v. 9.3.5 of the Schrödinger software suite, 2011. The 3D crystallographic structures of proteins (PDB ID: 4LRH, 3ACX) were retrieved from Protein Data Bank (www.rcsb.org/pdb). The protein structures were pre-processed and refined by Protein Preparation Wizard. Further, they were minimized by Optimized Potential for Liquid Simulations (OPLS)-2005 force field until the root mean square deviation (RMSD) reached the value of 0.3 Å. The ligands were optimized by a LigPrep program using the OPLS-2005 force field to generate the lowest energy state of ligands. The molecular docking studies of the ligand and protein were carried out by GLIDE. The best fit ligands with the target protein were ranked based on G score.

Synthesis of 4-(2′,4′-difluorobiphenyl-4-yl)-6-arylpyrimidin-2-amines 5a–g

A solution of (2E)-1-(2′,4’-difluorobiphenyl-4-yl)-3-arylprop-2-en-1-one (3a–g, 0.01 mol) and guanidine nitrate (4, 0.01 mol) in ethanol (30 mL) was treated with aqueous sodium hydroxide (10%, 2 mL) and the mixture was heated under reflux for 2 h. An additional amount of the sodium hydroxide solution was added and the mixture was heated for an additional 6 h. Progress of the reaction was monitored by thin layer chromatography (TLC). After cooling, the mixture was poured into crushed ice and the resultant solid was filtered out, washed with water, dried, and crystallized from ethanol.