Characterisation of twelve newly synthesised N-(substituted phenyl)-2-chloroacetamides with QSAR analysis and antimicrobial activity tests

N-(substituted phenyl)-2-chloroacetamides – namely N-phenyl chloroacetamide (SP1), N-(4-methylphenyl) chloroacetamide (SP2), N-(4-metoxylphenyl) chloroacetamide (SP3), N-(4-chlorophenyl) chloroacetamide (SP4), N-(4-bromophenyl) chloroacetamide (SP5), N-(4-fluorophenyl) chloroacetamide (SP6), N-(4-iodophenyl) chloroacetamide (SP7), N-(4-acetylphenyl) chloroacetamide (SP8), N-(4-hydroxyphenyl) chloroacetamide (SP9), N-(4-cyanophenyl) chloroacetamide (SP10), N-(3-cyanophenyl) chloroacetamide (SP11), and N-(3-bromophenyl) chloroacetamide (SP12) – were synthesised following the method described in our earlier article (19).

The chemical structure and purity of the synthesised compounds were verified by melting point, Fourier-transform infrared (FTIR), and ¹H and ¹³C nuclear magnetic resonance (NMR) spectroscopy. FTIR spectra were recorded in transmission mode using a Bomem MB 100 (ABB Bomem Inc., Quebec, Canada) spectrometer. ¹H and ¹³C NMR spectra were determined in deuterated dimethylsulphoxide (DMSO-d₆), used as the solvent, and recorded on a Bruker AC-250 spectrometer (Bruker Corporation, Billerica, MA, USA) at 200 MHz using tetramethylsilane (TMS) as internal standard. Chemical shifts were determined with respect to distorsionless enhancement by polarisation transfer (DEPT), two-dimensional ¹H to ¹³C heteronuclear correlation (HETCOR), and selective insensitive nuclei enhancement by polarisation transfer (INEPT) long-range experiments and are expressed in ppm with respect to TMS (δ_H=0 ppm) in the ¹H NMR spectra and to residual solvent signal (δ_C=39.5 ppm) in the ¹³C NMR spectra. Full spectral characterisation of all 12 chloroacetamides is given in Tables 1–3.

Molecular descriptors, i.e. molecular weight (MW), hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), molecular hydrophobicity/partition coefficient (logP), number of rotatable bonds (Nrot), and topological polar surface area (TPSA) of the twelve synthesised chloroacetamides were obtained using the available computational web tools, namely Molinspiration (23) and SwissADME (24), while their biophysical-kinetic parameters related to absorption and metabolism were obtained using SwissADME (24), PreADMET (25), and PkcSM (26) designed to predict absorption, distribution, metabolism, excretion (ADME) and bioactivity of tested molecules.

Antibacterial activity was tested against Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and methicillin resistant S. aureus (MRSA) ATCC 33591 and antifungal activity against Candida albicans ATCC 10231. The bacterial strains were cultured in the Luria Bertani (LB) medium (HiMedia, Mumbai, India) and C. albicans in tryptic soy broth (TSB) (Biomedics, Madrid, Spain). The bacterial strains and the yeast were cultured overnight at 37 °C. Suspensions were adjusted to 0.5 McFarland standard turbidity (BioMérieux, Marcy-l’Étoile, France), which corresponds to 10⁸ CFU/mL.

Minimum inhibitory (MIC), minimum bactericidal (MBC), and minimum fungicidal concentrations (MFC) for the 12 N-(substituted phenyl)-2-chloroacetamides (SP1–12) were determined using the broth microdilution method. The final concentration of each sample in the first well was 4000 μg/mL, while the concentration of the solvent dimethyl sulphoxide (DMSO) was 5 %. Twofold serial dilutions of the chloroacetamide samples were made with LB and TSB in 96-well microtitre plates in the concentration range from 32 to 4000 μg/mL. Besides negative control (untreated bacteria and fungi) we also used sterility control (containing only the culture medium) and positive control, treated with rifampicin and nystatin. The final concentration of rifampicin and nystatin in the first well was 400 and 2000 μg/mL, respectively. Each well, except for the sterility control, was inoculated with 20 μL of bacterial and yeast culture (1×10⁸ CFU/mL), reaching a final volume of 200 μL. At the end, 22 μL of resazurin (oxidation-reduction indicator of cell growth) was added to each well. The plates were incubated at 37 °C for 24 h. All tests were performed in a lighted environment, but the plates were incubated in the dark. Resazurin is a blue non-fluorescent and non-toxic dye that becomes pink and fluorescent when reduced to resorufin by oxidoreductases from viable cells (27). MIC was determined as no change in colour. MBC and MFC, which were obtained by sub-culturing test dilutions from each well without colour change on agar plates and incubating them for 24 h, corresponded to the lowest concentration that showed no bacterial or yeast growth. The results were expressed in μg/mL.

For the analysis of variance (ANOVA) we used the Kolmogorov-Smirnov test for the normality of residuals and Levene’s test for homogeneity of variance. For mean separation for MIC, MBC, and MFC we used Tukey’s honest significant difference (HSD) test. Significance was set at P<0.05. All dilutions were tested in duplicate with three repetitions. Statistical analyses were run on STATISTICA v.7 (StatSoft, Inc., Tulsa, OK, USA) and IBM SPSS Statistics v.20 (SPSS, Inc., Chicago, IL, USA).

Table 4 shows that compounds containing the 4-COCH₃ (SP8), 4-OH (SP9), 3-CN (SP10), and 4-CN (SP11) groups within the phenyl core had their TPSA in the optimal interval from 46.17 to 52.89 Ų, which is the most favourable for high permeability. N-(4-bromophenyl)-2-chloroacetamoide (SP5) showed the highest lipophilicity, and the compound carrying the p-OH-substituent (SP9) the lowest (Table 5).

The best predisposition for optimal intestinal absorption was seen in the derivatives containing electron-donor substituent (compound SP3) and strong electron-acceptor/ halogen substituents (compounds SP8 and SP10–12), and these properties were significantly higher than those observed for commercial drugs levetiracetam and piracetam (Table 6).

The above mentioned web tools Molinspiration (23), SwissADME (24), PreADMET (25), and PkcSM (26) predicted that the investigated chloroacetamides would not significantly inhibit the activity of P-glycoprotein (P-gp or ABCB1) (Table 7). Regarding CYP450 inhibition, CYP1A2 showed the highest probability to be inhibited by all tested chloroacetamides.

Table 8 shows the results of antibacterial and antifungal activity of the tested chloroacetamides. DMSO, which was used as negative/solvent control, did not show any inhibitory effect on the tested strains. The most sensitive strains, with MIC mainly lower than 100 μg/mL, were S. aureus and MRSA. The MIC of most compounds ranged between 40 and 130 μg/mL for these pathogens and did not significantly differ from rifampicin. N-(4-iodophenyl) chloroacetamide (SP7) showed the strongest activity against both Gram-positive strains (MIC 40 μg/mL). E. coli was the most resistant strain, as the MIC of half of the tested compounds (SP1, SP2, SP4, SP5, SP7, and SP11) ranged from 920 to 4000 μg/mL. All compounds save for SP1 and SP11 were significantly less effective than rifampicin.

The yeast strain C. albicans showed moderate susceptibility to chloroacetamides compared to Gram-positive bacterial strains, with MICs mostly below 500 μg/ mL, but much higher than to positive control nystatin, with the MIC of 2000 μg/mL. Only N-(4-hydroxyphenyl) chloroacetamide (SP9) had a higher MIC of 2660 μg/mL. The best and statistically significant inhibitory activity vs nystatin against C. albicans was observed for SP4, SP6, and SP12, with MICs ranging from 60 to 100 μg/mL and similar to the one against Gram-positive bacterial strains. In general, N-(4-cyanophenyl) chloroacetamide (SP10) showed significant inhibitory activity (lowest MICs) against all tested bacterial strains and yeast taken together. SP4 and SP12 showed the strongest inhibitory activity against C. albicans and Gram-positive strains. It is interesting to note that SP7 and SP9, which strongly inhibited Gram-positive strains, were completely ineffective against C. albicans.

MICs for rifampicin control were in the range evidenced for most chloroacetamides against Gram-positive strains, and quite lower against E. coli. Chloroacetamide MBCs and MFCs were at least twice as high as their MICs, varying from 120 to over 4000 μg/mL in the case of MBCs for SP4 and SP5 against E. coli, which was the highest concentration we applied in testing (Table 8).