ChemistryGeneral

Reaction progress was monitored by TLC on silica gel plates (Merck 60, F254, 0.2 mm). Organic solutions were dried over Na2SO4. Evaporation refers to the removal of solvent on a rotary evaporator under reduced pressure. All melting points were determined on a Büchi 530 capillary melting point apparatus and are uncorrected. IR spectra were recorded with a Perkin Elmer Spectrum RXI FT-IR System spectrophotometer, with compound as a solid in a KBr disc or nujol. 1H NMR (300 MHz) and APT (75 MHz) spectra were recorded with a Bruker AC-E spectrometer at room temperature; chemical shifts (δ) are expressed as ppm values. Microanalyses data (C, H, N) were obtained by an Elemental Vario EL III apparatus and were within ±0.4% of the theoretical values. Yields refer to purified products and are not optimized. The names of the compounds were obtained using Chem Draw Ultra 12.0 software (CambridgeSoft).

General procedure for preparation of benzoyl chlorides 18, 23a, e, 24b–d and 33a, d [21]

4-Nitrobenzoyl chloride 18 was commercially available. Substituted benzoyl chlorides 23a, c, 24b–d and 33a, d were obtained by refluxing for 5 h the appropriate acid derivatives 21a, c, 22b–d and 32a, d (0.01 mole) with thionyl chloride (7.25 mL). After evaporation under reduced pressure, the crude liquid residue was used for subsequent reactions without purification.

General procedure for preparation of compounds 20a, e [22, 23]

A solution of ethyl 1-R-5-amino-1H-pyrazole-4-carboxylate 19a, e (0.01 mole) in acetonitrile (50 mL) was heated under reflux with the 4-nitrobenzoyl chloride 18 (0.01 mole) for 7 h. The solid which separated was collected then recrystallized from ethanol to give compounds 20a, e that were identical in all respect (mp, mixed mp, Rf, IR, 1H-NMR) with an authentic specimen of ethyl 1-methyl-5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylate 20a [22] and ethyl 5-(4-nitrobenzamido)-1-phenyl-1H-pyrazole-4-carboxylate 20e [23].

General procedure for preparation of ethyl compounds 25a, e [23, 24] and 31

To a solution of ethyl 1-R-5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylate 20a, e (0.013 moles) or 1-methyl-5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylic acid 31 (0.017 moles) in warm ethanol (200 mL) 300 mg of 10% Pd-C as catalyst was added. The mixture was left under hydrogenation in a Parr apparatus at 50 psi for 24 h. The suspension was filtered, and the filtrate was concentrated to a small volume affording a compound which was identical in all respect (mp, mixed mp, Rf, IR, 1H-NMR) with an authentic specimen of ethyl 5-(4-aminobenzamido)-1-methyl-1H-pyrazole-4-carboxylate 25a [24] and ethyl 5-(4-aminobenzamido)-1-phenyl-1H-pyrazole-4-carboxylate 25e [23]. Compound 31 was isolated as white crystalline product.

5-(4-aminobenzamido)-1-methyl-1H-pyrazole-4-carboxylic acid (31): yields: 68%; mp 248–250 °C. I.R. (cm−1): 3470–2593 (NH2, NH, OH); 1680 (CO); 1672 (CO). 1H NMR (DMSO-d6) (δ): 3.63 (3H, s, CH3); 5.89 (2H, s, exchangeable, NH2); 6.60–7.81 (5H, set of signals, C6H4 and pyrazole H-3); 9.85 (1H, s, exchangeable, NH); 12.27 (1H, broad, exchangeable, OH); 13C NMR (DMSO-d6) (δ): 36.54, 108.26, 113.01, 119.50, 130.30, 140.09, 140.20, 153.28, 163.91, 166.10. Anal. Calc. for C12H12N4O3: C, 55.38%; H, 4.65%; N, 21.53%. Found: C, 55.40%; H, 4.67%; N, 21.40.

General procedure for preparation of 1-Methyl-5-(4-(3-phenylpropanamido)benzamido)-1H-pyrazole-4-carboxylate 26a, e, f and ethyl 5-(4-(2-(2-R2-4-R1-phenoxy)acetamido)benzamido)-1-methyl-1H-pyrazole-4-carboxylate 27b, e, d

A solution of 5-(4-aminobenzamido)-1-R-1H-pyrazole-4-carboxylate 25a, e (4 mmol) and the appropriate cinnamoyl chlorides 23a, c (4 mmol) in acetonitrile (20 mL) was refluxed for 8 h. The solvent was partially evaporated under reduced pressure until a product precipitates. The residue was collected and recrystallized from ethanol to give pure 26a, e, f. Compounds 27b–d were obtained with the same procedure using 5-(4-aminobenzamido)-1-R-1H-pyrazole-4-carboxylate 25a, e (1.74 mmol) and the appropriate 2-phenoxyacetyl chlorides 24b–d (4 mmol) in acetonitrile (20 mL).

Ethyl 5-(4-cinnamamidobenzamido)-1-methyl-1H-pyrazole-4-carboxylate 26a: yields 82%, mp 195–200 °C; I.R (Nujol) cm−1 3389–3273 (NH), 1694 (CO) 1660 (CO), 1H NMR (CHCl3) δ: 1.32 (t, 3H, CH3); 3.88 (s, 3H, CH3); 4.276 (q, 2H, CH2); 6.597 (d, 1H, J = 15.9 Hz, olefinic CH); 7.26–7.99 (m, 15H, ArH and olefinic CH); 8.23 (s, 1H, pyrazole H3); 9.29 (s, 1H, exchangeable, NH). 13 C NMR(δ) (CDCl3) 14.35, 38.56 60,46, 104.40, 119.56, 120.35, 127.71, 128.08, 128.97, 129.11, 130.32, 134.32, 139.49, 140.66, 142.46, 143.31, 164.08, 164.37, 165.07. Anal. Calc. for C23H22N4O4: C, 66.02%; H, 5.30%; N, 13.39%. Found: C, 65.68%; H, 5.01%; N, 13.05.

Ethyl 5-(4-(3-(4-chlorophenyl)acrylamido)benzamido)-1-methyl-1H-pyrazole-4-carboxylate 26c: yields 103,5%, mp 205–207 °C; I.R (Nujol) cm−1 3558–3254 (NH), 1708-1891-1655 (CO), 1H NMR (DMSO) δ: 1.14 (s, 3H, CH3); 4.13 (q, 2H, CH2); 6.87 (d, 1H, J = 15.9 Hz, olefinic CH); 7.51–7.90 (m, 15H, ArH and olefinic CH); 8.01 (s, 1H, pyrazole H3); 10.33 (s, 1H, exchangeable, NH); 10.56 (s, 1H, exchangeable, NH). 13 C NMR(δ) (CDCl3) 14.59, 36.46, 39.17, 39.45, 39.73., 40.01, 40.29, 40.56, 40.84, 108.09, 119.04, 123.18, 127.86, 129.53, 129.58, 130.00, 134.02, 134.89, 139.42, 140.03, 143.27, 162.19, 164.23, 165.85. Anal. Calc. for C23H21ClN4O4: C, 61.00%; H, 4.67%; N, 12.37%. Found: C, 60.69%; H, 4.70%; N, 12.32.

Ethyl 5-(4-(3-(4-chlorophenyl)acrylamido)benzamido)-1-phenyl-1H-pyrazole-4-carboxylate 26f: yields 60%, mp 250–255 °C; I.R (Nujol) cm−1 3583-3356-3212 (NH), 1894 (CO) 1698 (CO), 1H NMR (DMSO) δ: 1.14 (s, 3H, CH3); 4.18 (q, 2H, CH2); 6.88 (d, 1H, J = 15.9 Hz, olefinic CH); 7.40–7.94 (m, 15H, ArH and olefinic CH); 8.19 (s, 1H, pyrazole H3); 10.41 (s, 1H, exchangeable, NH); 10.56 (s, 1H, exchangeable, NH). 13 C NMR(δ) (DMSO) 14.56, 39.16, 39.44, 39.71, 39.99., 40.27, 40.55, 40.83, 110.63, 119.09, 123.16, 124.21, 127.88, 128.88, 129.37, 129.57, 129.73, 129.99, 134.01, 134.90, 138.41, 139.19, 140.03, 141.77, 143.27,162.04, 164.22, 166.44. Anal. Calc. for C28H23ClN4O4: C, 65.31%; H, 4.50%; N, 10.88%. Found: C, 65.55%; H, 4.84%; N, 11.22.

Ethyl 1-methyl-5-(4-(2-(o-tolyloxy)acetamido)benzamido)-1H-pyrazole-4-carboxylate 27b: yields 40%; mp. 183–85 °C. I.R. (cm−1): 3330 (NH); 3210 (NH); 1715(CO); 1664 (CO). 1 H NMR (DMSO-d6) (δ): 1.20 (3H, t, CH3); 2.18 (3H, s, CH3); 3.67 (3H, s, CH3); 4.20 (2H, q, CH2); 4.70 (2H, s, CH2); 6.85–8.01 (m, 9H, 2 x C6H4 and pyrazole H3); 10.30 (1H, s, exchangeable, NH); 10.38 (1H, s, exchangeable, NH). Anal. Calc. for C23H24N4O5: C, 63.29%; H, 5.54%; N, 12.84%. Found: C, 63.09%; H, 5.15%; N, 12.57.

Ethyl 5-(4-(2-(4-chlorophenoxy)acetamido)benzamido)-1-methyl-1H-pyrazole-4-carboxylate 27c: yields 44%; mp 168–70 °C. I.R. (cm−1): 3341 (NH); 3228 (NH); 1716 (CO); 1666 (CO). 1H NMR (DMSO-d6) (δ): 1.13 (3H, t, CH3); 3.69 (3H, s, CH3); 4.12 (2H, q, CH2); 4.77 (2H, s, CH2); 7.03–8.02 (9 H, m, 2 x C6H4 and pyrazole H3); 10.33 (1H, s, exchangeable, NH); 10.43 (1H, s, exchangeable, NH). Anal. Calc. for C22H21ClN4O5: C, 57.84%; H, 4.36%; N, 12.26%. Found: C, 57.66%; H, 4.98%; N, 12.52.

Ethyl 5-(4-(2-(2,4-dichlorophenoxy)acetamido)benzamido)-1-methyl-1H-pyrazole-4-carboxylate 27d: yields 37%; mp 188–90 °C. I.R. (cm−1): 3386 (NH); 1712 (CO); 1681 (CO). 1 H NMR (DMSO-d6) (δ): 1.12 (3H, t, CH3); 3.68 (3H, s, CH3); 4.12 (2H, q, CH2); 4.92 (2H, s, CH2); 7.12–8.01 (8 H, m, C6H3, C6H4 and pyrazole H3); 10.33 (1H, s, exchangeable, NH); 10.51 (1H, s, exchangeable, NH). 13 C NMR(δ) (DMSO) 14.59, 36.46, 59.92, 68.21, 108.10, 115.83, 119.17, 122.96, 125.56, 128.17, 128.52, 129.52, 129.89, 139.39, 140.05, 142.45, 153.11, 162.19, 165.81, 166.76. Anal. Calc. for C22H20Cl2N4O5: C, 53.78%; H, 4.10%; N, 11.40%. Found: C, 54.09%; H, 3.81%; N, 11.02.

General procedure for preparation of 1-R-5-(4-(3-phenylpropanamido)benzamido)-1H-pyrazole-4-carboxylic acid 28a, e, f

To a solution of ethyl 1-R-1H-pyrazole-4-carboxylates 26a, e, f (3.2 mmoles) in ethanol (18.75 ml), a solution aqueous 4% of NaOH (22.5 ml) was added. The reaction mixture is heated under reflux for 15 ‘, then left at room temperature for 12 h.

After this time, the ethanol was removed under reduced pressure and the remaining aqueous solution was acidified with 1 M HCl until complete precipitation of the acids. Finally, the precipitate was filtered and crystallized with ethanol to give compounds 28a, e, f.

5-(4-cinnamamidobenzamido)-1-methyl-1H-pyrazole-4-carboxylic acid (28a): yields 80% mp 228–232 °C; I.R (Nujol) cm−1 3254 (NH) 1698 (CO); 1H NMR (DMSO) δ: 3.68 (d, 1H, J = 15.9 Hz, olefinic CH); 7.05–7.94 (m, 15H, ArH and olefinic CH); 8.05 (s, 1H, pyrazole H3); 10.35 (s, 1H, exchangeable, NH); 10.38 (s, 1H, exchangeable, NH); 12.35 (s, 1H, broad, exchangeable, OH). 13C NMR(δ) (DMSO) 36.50, 108.77, 118.91, 122.72, 127.64, 128.28, 129.50, 130.39, 135.14, 139.37, 140.32, 141.01,143.57, 163.71, 164.56, 165.84. Anal. Calc. for C21H18N4O4: C, 64.61%; H, 4.65%; N, 14.35%. Found: C, 64.74%; H, 4.45%; N, 14.43.

5-(4-cinnamamidobenzamido)-1-phenyl-1H-pyrazole-4-carboxylic acid (28e): yields 80% mp 230–232 °C; I.R (Nujol) cm−1 3308 (NH) 1764, 1681, 1655 (CO); 1H NMR (DMSO) δ: 3.68 (d, 1H, J = 15.9 Hz, olefinic CH); 7.41–7.91 (m, 15H, ArH and olefinic CH); 8.15(s, 1H, pyrazole H3); 10.37 (s, 1H, NH); 10.54 (s, 1H, NH); 12.56 (s, 1H, broad, exchangeable, OH). 13C NMR (δ) (CDCl3) 39.14, 39.42, 39.70, 39.98, 40.26, 40.53, 40.81, 111.36, 119.08, 122.34, 124.15, 127.87, 128.31, 128.74, 129.37, 129.53, 129.67, 130.47, 135.04, 138.60, 139.08, 141.46, 142.09, 143.31, 163.57, 164.42, 166.33. Anal. Calc. for C26H20N4O4: C, 69.02%; H, 4.46%; N, 12.38%. Found: C, C, 68.79%; H, 4.27%; N, 12.60%.

5-(4-(3-(4-chlorophenyl)acrylamido)benzamido)-1-phenyl-1H-pyrazole-4-carboxylic acid 28f: yields 70% mp 155–160 °C; I.R (Nujol) cm−1 3579, 3185 (NH) 1693, 1625 (CO); 1H NMR (DMSO) δ: 3.90 (d, 1H, J = 15.9 Hz, olefinic CH); 7.40–7.92 (m, 15H, ArH e olefinic CH); 8.14 (s, 1H, pyrazole H3); 10.35 (s, 1H, NH); 10.59 (s, 1H, NH); 12.51 (s, 1H, NH), 12.46 (s, 1H, broad, exchangeable, OH). 13C NMR(δ) (DMSO) 119.05, 122.41, 124.14, 125.76, 127.86, 128.30, 128.71, 129.34, 129.52, 129.66, 130.44, 130.92, 135.06, 138.60, 139.09, 141.37, 142.06, 142.32, 163.55, 164.42, 166.32. Anal. Calc. for C26H19ClN4O4: C, 64.14%; H, 3.93%; N, 11.51%. Found: C, 64.25%; H, 3.60%; N, 11.73.

General procedure for preparation of 4-cinnamamido-N-(1-methyl-1H-pyrazol-5-yl)benzamide 29e

The acid 5-(4-cinnamamido)-1phenyl-1H-pyrazole-4-carboxylic 28e has been decarboxylated by melting to obtain the corresponding compound 29e that has been purified by crystallization.

4-cinnamamido-N-(1-phenyl-1H-pyrazol-5-yl)benzamide 29e: yields 70% mp 195–197 °C; I.R (Nujol) cm−1 3585, 3254 (NH), 1741 (CO), 1679 (CO); 1H NMR (DMSO) δ: 6.48 (s, 1H, pyrazole H3); 6.86 (d, 1H, J = 15.9 Hz, olefinic CH); 7.35–7.89 (m, 15H, ArH e olefinic CH, pyrazole H4); 10.29 (s, 1H, NH); 10.53 (s, 1H, exchangeable, NH). 13C NMR(δ) (DMSO) 104.72, 119.01, 122.28, 132.72, 127.78, 128.05, 128.32, 129.28, 129.55, 129.58, 130.49, 135.01, 136.53, 139.39, 140.21, 141.44, 143.14, 164.39, 165.92. Anal. Calc. for C25H20N4O4: C, 73.51%; H, 4.94%; N, 13.72%. Found: C, 73.73%; H, 5.03%; N, 13.35.

General procedure for preparation of 1-methyl-5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylic acid 30

Compound 30 is known [25] but was prepared in a different way. To a solution of ethyl 1-methyl-5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylate 20a (12.3 mmoles) in ethanol (24 ml), a solution aqueous 8% of NaOH (24 ml) was added. The reaction mixture was left at room temperature for 12 h.

After this time, the ethanol was removed under reduced pressure and the remaining aqueous solution was acidified with 1 M HCl until complete precipitation of the acids. Finally, the precipitate was filtered and crystallized with ethanol to give a compound which was identical in all respect (mp, mixed mp, Rf, IR, 1H-NMR) with an authentic specimen of 1-methyl-5-(4-nitrobenzamido)-1H-pyrazole-4-carboxylic acid 30 [25].

General procedure for preparation of 5-(4-(3-(2-R2-4-R1-phenyl)acrylamido)benzamido)-1-methyl-1H-pyrazole-4-carboxylic acids 34a–d

A suspension of 5-(4-aminobenzamido)-1-methyl-1H-pyrazole-4-carboxylic acid 31 (1.73 mmol) and the appropriate 2-phenoxyacetyl chloride 33a–d (1.73 mmol) in acetonitrile (60 mL) was refluxed for 8 h. The reaction mixture was filtered, then the solvent was partially evaporated under reduced pressure until a product precipitates. The residue was collected and recrystallized from ethanol to give pure 34a, c, d. Compound 34b, which separated directly from the reaction mixture, was directly crystallized from ethanol.

5--1-methyl-1H-pyrazole-4-carboxylic acid (34a): yield 14%; mp 230–34 °C. I.R. (cm−1): 3259–2605 multiple bands (NH, OH); 1685 (CO); 1655 (CO). 1H-NMR (DMSO) (δ): 3.67(3H, s, CH3); 4.78 (2H, s, CH2); 7.03–8.02 (9 H, 2 x C6H4 e pyrazole H-3); 10.36 (1H, s, exchangeable NH); 10.44 (1H, s, exchangeable NH); 12.40 (1H, broad, exchangeable OH). 13C-NMR (DMSO-d6) (δ): 36.39, 67.54, 108.68, 116.93, 119.49, 125.50, 128.15, 129.45, 129.77, 139.12, 140.37, 142.21, 157.00, 163.65, 166.01, 167.38. Anal. Calc. for C20H17ClN4O5: C, 56.02%; H, 4.00%; N, 13.07%. Found: C, 55.75%; H, 4.34%; N, 12.76.

5--1-methyl-1H-pyrazole-4-carboxylic acid (34b): yields 34%; mp 245–47 °C. I.R. (cm−1): 3388–2671 (multiple bands, NH, OH); 1701 (broad, CO);. 1H NMR (DMSO-d6) (δ): 3.66 (3H, s, CH3); 4.92 (2H, s, CH2); 7.12–8.02(8 H, m, C6H3, C6H4 e pyrazole H3); 10.30 (1H, s, exchangeable, NH); 10.51 (1H, s, exchangeable NH); 12.25 (s, 1H, broad, exchangeable, OH). Anal. Calc. for C20H16Cl2N4O5: C, 51.85%; H, 3.48%; N, 12.09%. Found: C, 52.03%; H, 3.86%; N, 12.00.

1-methyl-5--1H-pyrazole-4-carboxylic acid (34c): yields 16%; mp 235–37 °C. I.R. (cm−1): 3220–2507 (multiple bands, NH, OH); 1677 (CO); 1659 (CO). 1H NMR (DMSO-d6) (δ): 2.23 (3H, s, CH3); 3.66 (3H, s, CH3); 4.70 (2H, s, CH2); 6.89–8.02 (9 H, m, 2 x C6H4 and pyrazole H3); 10.30 (1H, s, exchangeable, NH); 10.38 (1H, s, exchangeable, NH); 12.31 (s, 1H, broad, exchangeable, OH). Anal. Calc. for C21H20N4O5: C, 61.76%; H, 4.94%; N, 13.72%. Found: C, 61.82%; H, 4.91%; N, 13.41.

1-methyl-5--1H-pyrazole-4-carboxylic acid (34d): yields 13%; mp 233–35 °C. I.R. (cm−1): 3223–2507 (multiple bands NH, OH); 1712 (CO); 1697 (CO). 1H NMR (DMSO-d6) (δ): 2.24 (3H, s, CH3); 3.70 (3H, s, CH3); 4.75 (2H, s, CH2); 6.87–8.04 (9 H, m, 2xC6H4 and pyrazole H3); 10.31 (1H, s, exchangeable NH); 10.39 (1H, s, exchangeable NH); 12.29 (s, 1H, broad, exchangeable, OH). Anal. Calc. for C21H20N4O5: C, 61.76%; H, 4.94%; N, 13.72%. Found: C, 61.68%; H, 4.56%; N, 14.04.

BiologyCell lines and culture conditions

Triple negative breast cancer MDA-MB231 cells, obtained from Istituto Scientifico Tumori (Genoa, Italy), were grown as monolayers in DMEM medium. supplemented with 10% (v/v) fetal bovine serum (FCS), 2 mM glutamine and 1% non-essential amino acids. The cells were grown at 37 °C in a humidified atmosphere containing 5% CO2 as previously reported [26]. For the experiments, cells were plated on 96-well plates, then were allowed to adhere overnight in culture medium before the treatment with chemicals or vehicle only. Stock solutions of the synthesized benzamido derivatives were prepared in DMSO and stored at −20 °C. In each experiment, the compounds were diluted to their final concentrations in the culture medium. The final concentration of DMSO never exceeded 0.04%, a concentration which had no discernible effects MDA-MB231 cells in comparison with the control.

Cell viability assay

For the evaluation of the effects of benzamides derivatives on cell viability, MDA-MB231 breast cancer cells were plated in 96-well plate (8 × 103/well) in the presence of different concentrations of the compounds (5–25 μM). After 48 h cell viability was determined by a colorimetric assay incubating the cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), MTT reagent (11 mg/mL in PBS, 20 µL) was added to each well and incubated for another 2 h at 37 °C. Then, the colored crystal of produced formazan was dissolved in 100 µL of lysis buffer (20% sodium dodecyl sulphate in 50% N,N-dimethylformamide, pH 4.0). The absorbance was measured by a microplate reader (OPSYS MR, Dynex Technologies, Chantilly, VA, USA) at 540 nm with a reference wavelength of 630 nm. Cell viability was measured as the percentage of the optical density (OD) values of treated cells compared with untreated cells as control. Each experiment was performed in triplicate. We reported in Table 2 the absorbances of three experiments and the mean value used for the Fig. 6.

Table 2 Values of absorbances (ABS) at 540 nm and mean values measured by a microplate reader (OPSYS MR, Dynex Technologies, Chantilly, VA, USA) with a reference wavelength of 630 nm

For these experiments, MDA-MB231 cells were plated in 96-well plate (8 × 103/well) in the presence of different concentrations of the compounds. After 48 h cell viability was determined by a colorimetric assay incubating the cells with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as reported [10, 11]. MTT is yellow tetrazolium salt that can be reduced to purple formazan by mitochondrial enzymes of living cells. The absorbance of the formazan was measured by a microplate reader (OPSYS MR, Dynex Technologies, Chantilly, VA, USA) at 540 nm with a reference wavelength of 630 nm and cell viability was quantified as the percentage of the optical density (OD) values of treated cells compared with that of untreated control cells. Each experiment was performed in triplicate.

Microbial strains

The reference strains Staphylococcus aureus ATCC 25923, Enterococcus faecalis ATCC 29212, Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 25922 and Candida albicans ATCC 10231 were used in the determination of Minimum Inhibitory Concentrations (MICs), and Inhibition of Biofilm Formation (IBF) tests. The bacterial strains were cultured aerobically in Muller-Hinton broth (MHB) or tryptic soy agar (TSA) [27]. Fungal C. albicans strain was cultured aerobically on Sabouraud (BS) broth or agar medium [28].

Determination of Minimum Inhibitory Concentrations (MICs)

MICs were determined by a microdilution method. Briefly, a series of solutions were prepared with a range of concentrations from 100 to 1.5 µg/mL (obtained by two-fold serial dilution). The serial dilutions were made in Mueller–Hinton broth (MH) (Sigma Aldrich) in a 96-wells plate, starting from a stock solution of 100 µg/mL in MH [29]. To each well, 10 µL of a bacterial suspension from a culture grown at 37 °C for 24 h on Tryptic Soy Agar (TSA), containing ~106 cfu/mL was added. A growth control and negative control, consisting respectively of bacterial strains in the medium without tested substances, and the medium without both substance and inoculum were also included in the 96-wells plate [30]. A substance control, consisting only of the substance solution in the medium without bacterial inoculum were added to evaluate the absorbance of substance at the tested concentrations. The plate was incubated at 37 °C for 24 h, the MICs were determined by a microplate reader (Glomax Multidetection System TM297 Promega, Milano Italy) as the lowest concentration of compound whose OD, read at 570 nm, was comparable with the negative control wells (broth only, without inoculum) [27]. Antifungal activity against C. albicans ATCC 10231 was evaluated by using a micro-method described above, using Sabouraud broth (BS) (Sigma-Aldrich) as growth medium.

Inhibition of biofilm formation (crystal violet method)

Compounds 26a, c, f, 27b–d, 28a, e, f, 29a and 34a–d were tested for their ability to interfere with biofilm formation of C. albicans ATCC 10231 and above mentioned bacterial strains. The yeast was grown in Sabouraud broth (BS) containing 2% (w/v) glucose overnight at 37 °C. After the incubation time, 2.5 µL of fungal suspension (containing ~106 cfu/mL) was placed into each well of a sterile flat-bottom 96-well loaded with 200 µL of BS with 2% glucose, supplemented with a screening concentration of 100 µg/mL of each substance [31]. The plates were incubated at 37 °C for 24 h; after this incubation time, the medium was removed, the plates were washed twice with sterile NaCl 0.9%, air-dried and then each well was filled with 100 µL of crystal violet solution (0.1%) for 15 min. The plate was then washed three times with water, and the crystal violet was dissolved in 200 µl of ethanol by pipetting up and down. Each assay was performed in triplicate and repeated at least twice. The plate was read at 570 nm using a microplate reader (Glomax Multidetection System TM297 Promega, Milano, Italy). Inhibition percentages at screening concentration (or at lower concentrations in the case of activity higher than 50% of each sample) were obtained by comparing the OD of control wells with that of the sample wells, by using the following formula:

$$}( \% )=(}\,}\,}-}\,})/}\,}\,})\times 100.$$

BIC50 (the concentration at which the percentage of inhibition of biofilm formation is equal to 50%) was calculated using AAT Bioquest, Inc. Quest Graph™ IC50 Calculator (v.1), retrieved from https://www.aatbio.com/tools/ic50-calculator-v1.

Inhibition of bacterial biofilms was determined by using the method described above, using Tryptose broth (TS) (Sigma-Aldrich) enriched with 2% w/v of glucose as growth and test medium [32].