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

In this study we screened twelve newly synthesised N-(substituted phenyl)-2-chloroacetamides for antimicrobial potential relying on quantitative structure-activity relationship (QSAR) analysis based on the available cheminformatics prediction models (Molinspiration, SwissADME, PreADMET, and PkcSM) and verified it through standard antimicrobial testing against Escherichia coli, Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), and Candida albicans. Our compounds met all the screening criteria of Lipinski's rule of five (Ro5) as well as Veber's and Egan's methods for predicting biological activity. In antimicrobial activity tests, all chloroacetamides were effective against Gram-positive S. aureus and MRSA, less effective against the Gram-negative E. coli, and moderately effective against the yeast C. albicans. Our study confirmed that the biological activity of chloroacetamides varied with the position of substituents bound to the phenyl ring, which explains why some molecules were more effective against Gram-negative than Gram-positive bacteria or C. albicans. Bearing the halogenated p-substituted phenyl ring, N-(4-chlorophenyl), N-(4-fluorophenyl), and N-(3-bromophenyl) chloroacetamides were among the most active thanks to high lipophilicity, which allows them to pass rapidly through the phospholipid bilayer of the cell membrane. They are the most promising compounds for further investigation, particularly against Gram-positive bacteria and pathogenic yeasts.

The growing spread and resistance of various pathogens call for developing new promising antimicrobial agents. One such group of agents that have received attention due to a wide variety of biological activities (such as analgesic, antipyretic, antimicrobial, bactericidal, fungicidal, hypoglycaemic, and antitumor) and applications in agriculture are chloroacetamides (1, 2, 3, 4, 5, 6, 7, 8, 9, 10). Their biological activity is driven primarily by their chemical structure, i.e. the type of functional groups that bind to active sites on receptors of bacterial and fungal strains and promote desired intermolecular interactions. Knowing the relation between specific structures and their activity allows us to predict the biological activity of newly synthesised structures. As there is ample evidence (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) that N-substituted chloroacetamides are highly effective and selective as microbial reagents, we were encouraged to develop new N-(substituted phenyl)2-chloroacetamide analogues (19) (Figure 1) with an aim to improve their selectivity, lipophilicity, and antimicrobial activity. This study is therefore an extension on a series of newly synthesised N-(substituted phenyl)-2-chloroacetamides with the aim to determine how the chemical structure of their substituted functional residues contributes to their antimicrobial activity.

Figure 1

Structural formula of the investigated N-(substituted phenyl)-2-chloroacetamides

To do that, we applied quantitative structure-activity relationship (QSAR) analysis as well as Lipinski’s rule of five (Ro5) (20) and its extensions such as Veber’s (21) and Egan’s (22) methods. The assessment of biological activity took into account molecular descriptors, biophysiochemical properties, and biophysical-kinetic parameters. In addition, the synthesised derivatives were evaluated in vitro for antimicrobial activity against some of the most common pathogens.

Materials and methods

Synthesis of N-(substituted phenyl)-2-chloroacetamides

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).

Characterisation methods and spectral analysis

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 (δH=0 ppm) in the 1H NMR spectra and to residual solvent signal (δC=39.5 ppm) in the 13C NMR spectra. Full spectral characterisation of all 12 chloroacetamides is given in Tables 1–3.

Table 1

Melting point and yield of N-(substituted phenyl) chloroacetamides

Compound	Substituent	Melting point (ºC)	Yield (%)
SP1	H	136–137	86
SP2	4-CH3	160–162	89
SP3	4-OCH3	117–119	84
SP4	4-Cl	166–168	65
SP5	4-Br	178–180	88
SP6	4-F	128–130	83
SP7	4-I	192–195	72
SP8	4-CH3CO	144–145	64
SP9	4-OH	144–146	76
SP10	4-CN	180–183	56
SP11	3-CN	165–170	61
SP12	3-Br	110–113	83

SP1 – N-phenyl chloroacetamide; SP2 – N-(4-methylphenyl) chloroacetamide; SP3 – N-(4-metoxylphenyl) chloroacetamide; SP4 – N-(4-chlorophenyl) chloroacetamide; SP5 – N-(4-bromophenyl) chloroacetamide; SP6 – N-(4-fluorophenyl) chloroacetamide; SP7– N-(4-iodophenyl) chloroacetamide; SP8 – N-(4-acetylphenyl) chloroacetamide; SP9 – N-(4-hydroxyphenyl) chloroacetamide; SP10 – N-(4-cyanophenyl) chloroacetamide; SP11 – N-(3-cyanophenyl) chloroacetamide; SP12 – N-(3-bromophenyl) chloroacetamide

Table 3

1H and 13C NMR spectral data

N-phenyl chloroacetamide (SP1)	1H NMR (CDCl3): δ 4.272 (2H, s, Cl-CH2), 7.057–7.130 (1H, t, JHH = 7.4 Hz, Ar-4H), 7.302–7.380 (2H, t, JHH = 7.8 Hz, Ar-H), 7.597–7.636 (2H, d, JHH = 7.8 Hz, Ar-H), 10.321 (1H, s, NH). 13C NMR (CDCl3): δ 43.833 (Cl-CH2), 119.651 (C2,C5), 124.130 (C4) 129.119 (C3,C5), 138.751 (C1), 164.934 (C=O).
N-(4-methylphenyl) chloroacetamide (SP2)	1H NMR (CDCl3): δ 2.255 (2H, s, CH3), 4.421 (1H, s, Cl-CH2), 7.111–7.153 (2H, d, JHH = 8.2 Hz, Ar-H), 7.473–7.515 (2H, d, JHH = 8.2 Hz, Ar-H), 10.222 (1H, s, NH). 13C NMR (CDCl3): δ 20.655 (CH3), 43.797 (Cl-CH2), 119.614 (C2,C6), 129.483 (C3,C5), 133.088 (C1), 136.238 (C4), 164.643 (C=O).
N-(4-metoxylphenyl) chloroacetamide (SP3)	1H NMR (CDCl3): δ 3.729 (2H, s, OCH3), 4.229 (1H, s, Cl-CH2), 6.886–6.948 (2H, d, JHH = 9.0 Hz, Ar-H), 7.481–7.560 (2H, d, JHH = 9.0 Hz, Ar-H), 10.177 (1H, s, NH).). 13C NMR (CDCl3): δ 43.742 (Cl-CH2), 55.359 (OCH3), 114.189 (C3,C5), 121.217 (C2,C6), 131.814 (C1), 155.885(C4), 164.424 (C=O).
N-(4-chlorophenyl) chloroacetamide (SP4)	1H NMR (CDCl3): δ 4.280 (1H, s, Cl-CH2), 7.358–7.431 (2H, d, JHH = 9.0 Hz, Ar-H), 7.613–7.686 (2H, d, JHH = 9.0 Hz, Ar-H), 10.445 (1H, s, NH), 13C NMR (CDCl3): δ 43.741 (Cl-CH2), 121.162 (C2,C6), 129.010 (C3,C5), 137.677 (C1), 165.061 (C=O).
N-(4-bromophenyl) chloroacetamide (SP5)	1H NMR (CDCl3): δ 4.274 (1H, s, Cl-CH2), 7.495–7.616 (4H, m, Ar-H), 10.447 (1H, s, N-H). 13C NMR (CDCl3): δ 43.742 (Cl-CH2), 115.736 (C4), 121.526 (C2,C6), 131.923 (C3,C5), 138.095 (C1), 165.061 (C=O).
N-(4-fluorophenyl) chloroacetamide (SP6)	1H NMR (CDCl3): δ 4.369 (1H, s, Cl-CH2), 7.122–7.226 (2H, t, JHH = 9.0 Hz, Ar-H)), 7,588–7,675 (2H, m, Ar-H), 10,337 (1H, s, NH). 13C NMR (CDCl3): δ 43.688 (Cl-CH2), 115.463–115.900 (C3,C5), 121.381 (C2,C6), 135.073 (C1), 160.983 (C4), 164.861 (C=O).
N-(4-iodophenyl) chloroacetamide (SP7)	1H NMR (CDCl3): δ 4.263 (1H, s, Cl-CH2), 7.425–7.4709 (2H, d, JHH = 9.0 Hz, Ar-H), 7,658–7,701 (2H, d, JHH = 9.0 Hz, Ar-H), 10.416 (1H, s, NH). 13C NMR (CDCl3): δ 43.760 (Cl-CH2), 87.732 (C4), 121.745 (C2,C6), 137.750–138.551 (C3,C5), 165.043 (C=O).
N-(4-acetylphenyl) chloroacetamide (SP8)	1H NMR (CDCl3): δ 2.544 (3H, s, CH3), 4.328 (1H, s, Cl-CH2), 7,723–7,768 (2H, d, JHH = 9.0 Hz, Ar-H), 7.945–7.990 (2H, d, JHH = 9.0 Hz, Ar-H), 10.646 (1H, s, NH). 13C NMR (CDCl3): δ 26.645 (CH3), 43.833 (Cl-CH2), 118.868 (C2,C6), 129.793 (C3,C5), 132.451 (C4), 143.030 (C1), 165.462 (C=O), 196.798 (COCH3).
N-(4-hydroxyphenyl) chloroacetamide (SP9)	1H NMR (CDCl3): δ 4.280 (2H, s, Cl-CH2), 4.684 (1H, s, OH), 7.139–7.184 (2H, d, JHH = 9.0 Hz, Ar-H), 7.625–7.686 (2H, d, JHH = 8.8 Hz, Ar-H). 13C NMR (CDCl3): δ 43.706 (Cl-CH2), 120.671(C3,C5), 122.091 (C2,C6), 136.7489 (C1), 146.180 (C4), 164.989–166.791 (C=O).
N-(4-cyanophenyl) chloroacetamide (SP10)	1H NMR (CDCl3): δ 4.319 (2H, s, Cl-CH2), 7.552–7.619 (2H, d, JHH = 9.0 Hz, Ar-H), 7.782–7.877 (2H, d, JHH = 9.0 Hz, Ar-H), 10.745 (1H, s, 2-H). 13C NMR (CDCl3): δ 43.669 (Cl-CH2), 111.985 (C3), 118.668 (CN), 122.309 (C2), 124.203 (C6), 127.662 (C4), 130.321–130.594 (C5), 139.516 (C1), 165.589 (C=O).
N-(3-cyanophenyl) chloroacetamide (SP11)	1H NMR (CDCl3): δ 4.339 (1H, s, Cl-CH2), 7.552–7.619 (2H, d, JHH = 5.6 Hz, Ar-H), 7.782–7.813 (1H, m, Ar-H), 8.094 (1H, s, Ar-H), 10.745 (1H, s, NH). 13C NMR (CDCl3): δ 43.669 (Cl-CH2), 111.985 (C3), 118.668 (CN), 122.309 (C2), 124.203 (C6), 127.662 (C4), 130.321–130.594 (C5), 139.516 (C1), 165.589 (C=O).
N-(3-bromophenyl) chloroacetamide (SP12)	1H NMR (CDCl3): δ 4.286 (3H, s, Cl-CH2), 7.285–7.358 (2H, m, Ar-H), 7.470– 7.571 (1H, m, Ar-H), 7.962 (1H, s, Ar-H), 10.489 (1H, s, N-H). 13C NMR (CDCl3): δ 43.688 (Cl-CH2), 118.376 (C2), 121.836 (C5), 121.927 (C6), 126.697 (C4), 131.067 (C3), 140.262 (C1), 165.243 (C=O).

QSAR analysis

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.

Microbial strains and growth conditions

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.

MIC assay

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.

Statistical analysis

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).

Results

Biological profile of N-(substituted phenyl)-2-chloroacetamides

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 Å2, 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).

Table 4

Physicochemical properties of the studied chloroacetamides

Compound	Molecular weight (g/ mol)	Number of atoms	Number of rotatable bonds	Number of hydrogen bond donors	Number of hydrogen bond acceptors	Molar refractivity	Topological polar surface area (Å2)
SP1	169.61	11	3	1	2	45.55	29.10
SP2	183.63	12	3	1	2	50.52	29.10
SP3	199.63	13	4	1	3	52.04	38.33
SP4	204.05	12	3	1	2	50.56	29.10
SP5	248.50	12	3	1	2	53.25	29.10
SP6	187.60	12	3	1	2	45.51	29.10
SP7	295.50	12	3	1	2	58.27	29.10
SP8	211.64	14	4	1	3	55.75	46.17
SP9	185.61	12	3	2	3	47.57	49.33
SP10	194.62	13	3	1	3	50.27	52.89
SP11	194.62	13	3	1	3	50.27	52.89
SP12	248.50	12	3	1	3	53.25	29.10
Levetiracetam	156.23	11	3	1	2	48.17	46.33
Piracetam	142.16	10	2	1	2	38.76	63.40

SP1 – N-phenyl chloroacetamide; SP2 – N-(4-methylphenyl) chloroacetamide; SP3 – N-(4-metoxylphenyl) chloroacetamide; SP4 – N-(4-chlorophenyl) chloroacetamide; SP5 – N-(4-bromophenyl) chloroacetamide; SP6 – N-(4-fluorophenyl) chloroacetamide; SP7 – N-(4-iodophenyl) chloroacetamide; SP8 – N-(4-acetylphenyl) chloroacetamide; SP9 – N-(4-hydroxyphenyl) chloroacetamide; SP10 – N-(4-cyanophenyl) chloroacetamide; SP11 – N-(3-cyanophenyl) chloroacetamide; SP12 – N-(3-bromophenyl) chloroacetamide

Table 5

Partition coefficients of the studied chloroacetamides

Compound	logP (22)	logPo/w(XLOGP3) (23)	logPo/w(WLOGP) (23)	logPo/w(MLOGP) (23)
SP1	1.72	1.63	1.67	1.84
SP2	2.17	1.99	1.98	2.15
SP3	1.78	1.65	1.68	1.54
SP4	2.40	2.26	2.33	2.42
SP5	2.53	2.32	2.44	2.56
SP6	1.89	1.73	2.23	2.27
SP7	2.81	2.28	2.28	2.71
SP8	1.62	1.86	1.88	1.47
SP9	1.24	1.27	1.38	1.23
SP10	1.45	1.82	1.54	1.18
SP11	1.48	1.35	1.54	1.18
SP12	2.51	2.93	2.44	2.56
Levetiracetam	0.69	0.62	-0.03	0.28
Piracetam	-1.32	-1.54	-1.29	-0.96

SP1 – N-phenyl chloroacetamide; SP2 – N-(4-methylphenyl) chloroacetamide; SP3 – N-(4-metoxylphenyl) chloroacetamide; SP4 – N-(4-chlorophenyl) chloroacetamide; SP5 – N-(4-bromophenyl) chloroacetamide; SP6 – N-(4-fluorophenyl) chloroacetamide; SP7 – N-(4-iodophenyl) chloroacetamide; SP8 – N-(4-acetylphenyl) chloroacetamide; SP9 – N-(4-hydroxyphenyl) chloroacetamide; SP10 – N-(4-cyanophenyl) chloroacetamide; SP11 – N-(3-cyanophenyl) chloroacetamide; SP12 – N-(3-bromophenyl) chloroacetamide

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).

Table 6

QSAR pharmacokinetic profiles of the selected compounds related to absorption properties

Compound	SwissADME	pkCSM	SwissADME	PreADMET	SwissADME	PreADMET
	Gastrointestinal absorption	Intestinal absorption (%)	the compound penetrates the blood-brain barrier	The compound penetrating the blood-brain barrier (cbrain/cblood)	the compound is a P-gp inhibitor	the compound is a P-gp inhibitor
	SP1	High	91.156	Yes	0.902206	No	No
SP2	High	91.692	Yes	2.16896	No	No
SP3	High	93.810	Yes	0.612824	No	No
SP4	High	91.969	Yes	1.65555	No	No
SP5	High	91.902	Yes	1.79202	No	No
SP6	High	91.217	Yes	1.07913	No	No
SP7	High	90.802	Yes	1.52595	No	No
SP8	High	92.635	Yes	0.546121	No	No
SP9	High	90.745	Yes	0.975597	No	No
SP10	High	92.986	Yes	0.975597	No	No
SP11	High	92.817	Yes	0.975597	No	No
SP12	High	92.405	Yes	1.79204	No	No
Levetiracetam	High	86.852	No	0.440234	No	No
Piracetam	High	86.061	No	0.165163	No	No

SP1 – N-phenyl chloroacetamide; SP2 – N-(4-methylphenyl) chloroacetamide; SP3 – N-(4-metoxylphenyl) chloroacetamide; SP4 – N-(4-chlorophenyl) chloroacetamide; SP5 – N-(4-bromophenyl) chloroacetamide; SP6 – N-(4-fluorophenyl) chloroacetamide; SP7 – N-(4-iodophenyl) chloroacetamide; SP8 – N-(4-acetylphenyl) chloroacetamide; SP9 – N-(4-hydroxyphenyl) chloroacetamide; SP10 – N-(4-cyanophenyl) chloroacetamide; SP11 – N-(3-cyanophenyl) chloroacetamide; SP12 – N-(3-bromophenyl) chloroacetamide

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 7

QSAR biophysical-kinetic profiles of the compounds related to metabolism properties

Prediction tool	SwissADME	pkCSM	Swiss ADME	pkCSM	SwissADME	pkCSM	SwissADME	pkCSM	SwissADME	pkCSM
Compound	Inhibits CYP1A2	Inhibits CYP1A2	Inhibits CYP2C19	Inhibits CYP2C19	Inhibits CYP2C9	Inhibits CYP2C9	Inhibits CYP2D6	Inhibits CYP2D6	Inhibits CYP3A4	Inhibits CYP3A4
SP1	Yes	No	No	No	No	No	No	No	No	No
SP2	Yes	Yes	No	No	No	No	No	No	No	No
SP3	Yes	Yes	No	No	No	No	No	No	No	No
SP4	Yes	Yes	No	No	No	No	No	No	No	No
SP5	Yes	Yes	No	No	No	No	No	No	No	No
SP6	Yes	Yes	No	No	No	No	No	No	No	No
SP7	Yes	Yes	No	No	No	No	No	No	No	No
SP8	Yes	Yes	No	No	No	No	No	No	No	No
SP9	No	No	No	No	No	No	No	No	No	No
SP10	Yes	Yes	No	No	No	No	No	No	No	No
SP11	Yes	Yes	No	No	No	No	No	No	No	No
SP12	Yes	Yes	No	No	No	No	No	No	No	No
Levetiracetam	No	No	No	No	No	No	No	No	No	No
Piracetam	No	No	No	No	No	No	No	No	No	No

Sri –A-phenyl chloroacetannde; SP2 –A-(4-methylphenyl) chloroacetannde; SP3 – N-(4-metoxylphenyl) chloroacetaminde; SP4 – N-(4-chlorophenyl) chloroacetamide; SP5 – N-(4-bromophenyl) chloroacetamide; SP6 – N-(4-fluorophenyl) chloroacetamide; SP7 – N-(4-iodophenyl) chloroacetamide; SP8 – N-(4-acetylphenyl) chloroacetamide; SP9 – N-(4-hydroxyphenyl) chloroacetamide; SP10 –A-(4-cyanophenyl) chloroacetamide; SP11 – N-(3-cyanophenyl) chloroacetamide; SP12 – N-(3-bromophenyl) chloroacetamide

Antimicrobial activity

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.

Table 8

Minimum inhibitory, bactericidal, and fungicidal concentrations of N-(substituted phenyl)-2-chloroacetamides (means ± standard errors)

Tested substances	R	C. albicans	E. coli	S. aureus	MRSA
		MIC (μg/mL)
SP1	4-H	190±40c	920±80c	90±20c	50±0cd
SP2	4-CH3	330±110c	3330±330ab	60±0c	60±0cd
SP3	4-OCH3	190±40c	540±110c	110±10c	190±40bc
SP4	4-Cl	60±0c	3670±330ab	60±0c	90±20bcd
SP5	4-Br	330±80c	4000±0a	60±0c	60±0cd
SP6	4-F	110±10c	500±140c	150±50bc	110±10bcd
SP7	4-I	830±170c	2670±330b	40±10c	40±10d
SP8	4-COCH3	330±80c	330±80c	190±40bc	90±20bcd
SP9	4-OH	2660±670a	270±20c	130±0c	40±10d
SP10	3-CN	290±40c	190±40c	40±10c	90±20bcd
SP11	4-CN	230±20c	1000±290c	750±140a	220±20ab
SP12	3-Br	100±20c	500±140c	80±20c	90±20bcd
Ant/Myc	–	2000±0ab	90±10c	40±10c	70±20cd
MBC/MFC (μg/mL)
SP1	4-H	500±0c	2000±0b	250±0bcd	120±0c
SP2	4-CH3	670±170c	4000±0a	170±40cd	310±110c
SP3	4-OCH3	330±80c	1000±0c	250±0bcd	330±80c
SP4	4-Cl	2000±0b	Nd	130±0d	750±140bc
SP5	4-Br	4000±0a	Nd	420±80bcd	750±140bc
SP6	4-F	330±80c	1000±0c	750±140bc	250±0c
SP7	4-I	3000±580ab	4000±0a	130±0d	290±110c
SP8	4-COCH3	670±170c	670±170d	750±140bc	330±80c
SP9	4-OH	4000±0a	500±0d	330±80bcd	170±40c
SP10	3-CN	500±0c	420±80d	130±0d	250±0c
SP11	4-CN	500±0c	2000±0b	2330±330a	1330±330ab
SP12	3-Br	670±170c	1000±0c	210±40cd	330±80c
Ant/Myc	–	Nd	130±30e	100±0d	100±0c

*Values followed by the same letter in each column and isolate were not significantly different (P<0.05, Tukey’s HSD test). Ant/Myc – rifampicin or nystatin. Nd – not determined (above the highest concentration applied of 4000 μg/mL)

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).

Discussion

Predicting biological activity of newly synthesised small molecules to be screened for medicinal use takes into account simple molecular properties such as molecular weight and the number of hydrogen/rotatable bonds, which determine the size, polarity, and flexibility of a compound (28, 29). The most common screening criterion is Lipinski’s rule of five (Ro5). Its name stems from the following cheminformatics filters: MW≤500 g/mol, number of HBD≤5, number of HBA≤10, and logP≤5), whose aim is to filter out compounds that do not satisfy the most common oral absorption parameters. Although it does not predict whether a compound will be biologically active, Ro5 does not allow more than one deviation from the set parameters (20). This rule was later extended by other empirical thresholds for potential bioactivity, such as those proposed by Veber (21) (Nrot≤10, TPSA≤140 Å2, number of HBD/ HBA≤12) and Egan (22) (WlogP≤5.88 and TPSA≤131.6 Å2).

Chloroacetamides synthesised and tested in our study met all of these criteria (Tables 4 and 5). The introduction of a p-substituted phenyl ring and the chlorine atom into the acetamide fragment increased the molecular weight of the synthesised compounds compared to commercial levetiracetam and piracetam, and their topological polar surface area in the range of 29.10–52.89 Å2 and the number of rotatable bonds not exceeding 4 promise good biological activities (30). Veber (31) demonstrated that molecules with TPSA≤140 Å2 display efficient permeability. Higher partition coefficient logP will allow biologically active chloroacetamides higher efficiency by passive diffusion as well as effective binding to the active receptor sites (32). Besides, almost no divergence in logP (-Br, -CN) was observed for compounds with the same substituent in different positions (3). Likewise, all analysed chloroacetamide molecules showed higher absorption probability (Table 3) thanks to small size and low polarity (29).

In antimicrobial activity tests, all N-(substituted phenyl)-2-chloroacetamides were effective against Gram-positive bacteria, less effective against the Gram-negative E. coli, and moderately effective against the C. albicans yeast (Table 8). This was expected, given the differences in the cell wall structure and composition of these species. Ertan et al. (5) reported good bactericidal and fungicidal effects of organic acetamide derivatives N-(2-hydroxy-4 (or 5 )-nit r o / a m in o phenyl ) be n z a mid e s a n d phenylacetamides, whose structure differs from our compounds. Their MICs (around 250 μg/mL) were higher than ours against C. albicans and E. coli and similar against S. aureus. Similar findings were reported by a study of biologically active 2-chloro-N-alkyl/aryl acetamide derivatives (12). However, as neither study tested their respective compounds against MRSA, ours seems to be the first in this respect. More recently, Sharma et al. (33) identified 2-((4-bromophenyl)amino)-N-(4-(4-bromophenyl)thiazol-2-yl)acetamide, N-(4-(4-bromophenyl) thiazol-2-yl)-2-((4-chloro-3-nitrophenyl) amino)acetamide, and N-(4-(4-bromophenyl)thiazol-2-yl)-2-((2-chloro-4-nitrophenyl)amino)acetamide as effective against S. aureus, E. coli, and C. albicans, with the lowest MICs ranging from 13 to 27 μmol/L. They also reported that the improved antimicrobial activity was owed to the presence of electron withdrawing groups (–Br, –Cl, –NO2) at the ortho, meta, and para-position of the ring B and to N-acylation with the synthesised compounds. Another study (34) indicated high activity of 2-(2-methylquinoxalin-3-ylthio)-N-(benzo[d]thiazol-2-yl)acetamide and 2-(2-methylquinoxalin-3-ylthio)–N-cyclohexylacetamide against E. coli, S. aureus, and C. albicans.

Compounds SP1–SP6, SP9, SP10, and SP12 showed the highest antimicrobial activity in our study, much thanks to their structure (lipophilicity/hydrophobicity) and no more than one hydrogen or nitrogen atom (35). Higher logP values may have facilitated their penetration through the bacterial/fungal cell membrane and microbial death. Good biological activity of SP4–SP7 and SP12 can also be attributed to the presence of halogenated substituents (3, 36).

Jablonkai et al. (37) demonstrated that the biological activity of chloroacetamides varies with the position of substituents bound to the phenyl ring. This may explain different activity of the compounds SP5 and SP12 or SP10 and SP11 against the tested strains. Furthermore, different susceptibility of the tested pathogens to compound SP10 may be the consequence of their morphological characteristics determining compound penetration into the microbial/fungal cell.

Conclusion

Judging from the cheminformatics prediction models such as Molinspiration, SwissADME, PreADMET, and PkcSM, our twelve newly synthesised N-(substituted phenyl)-2-chloroacetamides met all the empirical criteria for good biological activity. Standard antimicrobial activity tests showed they were highly effective against Gram-positive bacteria, less effective against the Gram-negative E. coli, and moderately effective against the C. albicans yeast. Bearing the halogenated p-substituted phenyl ring, N-(4-chlorophenyl) chloroacetamide, N-(4-fluorophenyl) chloroacetamide, and N-(3-bromophenyl) chloroacetamide were among the most active thanks to high lipophilicity, which allows them to pass rapidly through the phospholipid bilayer of the cell membrane. They are the most promising compounds for further investigation, particularly against Gram-positive bacteria and yeasts. Our findings have set the path for the preparation of new, improved N-(p-substituted phenyl)-2-chloroacetamides and for better understanding of the structure-activity relationship, which should extend research to more different bacterial and fungal strains in the future.

Table 2

Characterisation of investigated N-(substituted phenyl)-2-chloroacetamides

Comp	R	IR (KBr) νmax (cm-1)
SP1	H	3267 (N-H); 3207, 3145, 3098 (C-H aromatic ring); 2947 (C-H); 1671 (C=O); 1618 (C=C); 1557 (N-H deformation); 1498, (C-H bending); 1443 (C-H bending); 1344 (C-H); 1251 (C-N); 749 (N-H).
SP2	4-CH3	3273 (N-H); 3204, 3135, 3090 (C-H aromatic ring); 2954 (C-H); 1673 (C=O); 1616 (C=C); 1554 (N-H); 1402 (C-H); 1343 (C-H); 1251 (C-N); 818 (N-H).
SP3	4-OCH3	3295 (N-H); 3139, 3073 (C-H aromatic ring); 2957 (C-H); 2909 2835 (C-H); 1663 (C=O); 1612 (C=C); 1547 (N-H); 1510 (N-H); 1465 (C-H); 1413 (C-H); 1247 (C-N); 830 (N-H).
SP4	4-Cl	3264(N-H); 3199, 3131, 3082 (C-H aromatic ring); 3005, 2952(C-H); 1669 (C=O); 1614 (C=C); 1551 (N-H); 1490 (C-H); 1400 (C-H); 1248 (C-N); 825 (N-H).
SP5	4-Br	3263 (N-H); 3194 (C-H); 3125, 3077 (C-H aromatic ring); 3000 2953 (C-H); 1669 (C=O); 1549 (N-H); 1488 (C-H); 1395 (C-H); 1248 (C-N); 822 (N-H).
SP6	4-F	3275, 3221 (N-H); 3165 (C-H aromatic ring); 2947 (C-H); 1668 (C=O); 1508 (N-H); 1406 (C-H); 1292; 1212 (C-N); 832 (N-H).
SP7	4-I	3309, 3270 (N-H); 3194, 3077 (C-H aromatic ring); 2936 (C-H); 2953 (C-H); 1672 (C=O); 1610 (N-H); 1543 (C-H); 1392–1089 (CH); 1245 (C-N); 817 (N-H).
SP8	4-COCH3	3325, 3286 (N-H); 3196, 3109 (C-H aromatic ring); 2922, 2857 (C-H); 1707 (C=O); 1655 (C=C); 1599 (N-H); 1539 (C-H); 1283 (C-O); 1252 (C-N); 834 (N-H).
SP9	4-OH	3296 (O-H); 3144 (N-H); 3098 (C-H); 1677 (C=O); 1508 (N-H); 1313 (C-H); 1211 (C-N); 820 (N-H).
SP10	4-CN	3265 (N-H); 3192, 3119 (C-H); 2946 (C-H); 2226 (C?N); 1681 (C=O); 1603 (C=C); 1539 (N-H); 1408, 1345 (C-H); 1256 (C-N); 839 (N-H).
SP11	3-CN	3265 (N-H); 3096 (C-H); 2964 C-H); 2232 (C?N); 1678 (C=O); 1610 (C=C); 1561 (N-H); 1485 (C-H); 1293 (C-N); 1089 (C-H); 799 (N-H).
SP12	3-Br	3268 (N-H); 3193, 3127 (C-H); 2945 (C-H); 1679 (C=O); 1594 (N-H); 1424 (C-H); 1249 (C-N); 779 (N-H).

SP1 – N-phenyl chloroacetamide; SP2 – N-(4-methylphenyl) chloroacetamide; SP3 – N-(4-metoxylphenyl) chloroacetamide; SP4 – N-(4-chlorophenyl) chloroacetamide; SP5 – N-(4-bromophenyl) chloroacetamide; SP6 – N-(4-fluorophenyl) chloroacetamide; SP7 – N-(4-iodophenyl) chloroacetamide; SP8 – N-(4-acetylphenyl) chloroacetamide; SP9 – N-(4-hydroxyphenyl) chloroacetamide; SP10 – N-(4-cyanophenyl) chloroacetamide; SP11 – N-(3-cyanophenyl) chloroacetamide; SP12 – N-(3-bromophenyl) chloroacetamide