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
Structural formula of the investigated
To do that, we applied quantitative structure-activity relationship (QSAR) analysis as well as Lipinski’s
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-
Melting point and yield of
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 –
Characterisation of investigated
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 –
1H and 13C NMR spectral data
1H NMR (CDCl3): δ 4.272 (2H, s, Cl-CH2), 7.057–7.130 (1H, t, |
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1H NMR (CDCl3): δ 2.255 (2H, s, CH3), 4.421 (1H, s, Cl-CH2), 7.111–7.153 (2H, d, |
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1H NMR (CDCl3): δ 3.729 (2H, s, OCH3), 4.229 (1H, s, Cl-CH2), 6.886–6.948 (2H, d, |
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1H NMR (CDCl3): δ 4.280 (1H, s, Cl-CH2), 7.358–7.431 (2H, d, |
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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). | |
1H NMR (CDCl3): δ 4.369 (1H, s, Cl-CH2), 7.122–7.226 (2H, t, |
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1H NMR (CDCl3): δ 4.263 (1H, s, Cl-CH2), 7.425–7.4709 (2H, d, |
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1H NMR (CDCl3): δ 2.544 (3H, s, CH3), 4.328 (1H, s, Cl-CH2), 7,723–7,768 (2H, d, |
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1H NMR (CDCl3): δ 4.280 (2H, s, Cl-CH2), 4.684 (1H, s, OH), 7.139–7.184 (2H, d, |
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1H NMR (CDCl3): δ 4.319 (2H, s, Cl-CH2), 7.552–7.619 (2H, d, |
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1H NMR (CDCl3): δ 4.339 (1H, s, Cl-CH2), 7.552–7.619 (2H, d, |
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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). |
Molecular descriptors, i.e. molecular weight (MW), hydrogen bond donor (HBD), hydrogen bond acceptor (HBA), molecular hydrophobicity/partition coefficient (log
Antibacterial activity was tested against
Minimum inhibitory (MIC), minimum bactericidal (MBC), and minimum fungicidal concentrations (MFC) for the 12
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
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.
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 –
Partition coefficients of the studied chloroacetamides
Compound | log |
log |
log |
log |
---|---|---|---|---|
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 –
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).
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 –
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.
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 –
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
Minimum inhibitory, bactericidal, and fungicidal concentrations of
Tested substances | 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 |
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
MICs for rifampicin control were in the range evidenced for most chloroacetamides against Gram-positive strains, and quite lower against
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
Chloroacetamides synthesised and tested in our study met all of these criteria (Tables 4 and 5). The introduction of a
In antimicrobial activity tests, all
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 log
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.
Judging from the cheminformatics prediction models such as Molinspiration, SwissADME, PreADMET, and PkcSM, our twelve newly synthesised