Chemicals and instrumentation

Reagents and solvents were bought from Merck (Pty) Ltd and were utilized as provided. Reactions were monitored through thin layer chromatography (TLC), which used Merck 60F254 silica gel plates supported on 0.20 mm thick aluminum sheets. Developed plates were visualized with ultraviolet (UV) light at wavelengths of 254 nm and 366 nm or stained in iodine chamber. A Bruker Biospin 600 MHz spectrometer was used to record the 1H and 13C NMR spectra. Chemical shifts are reported relative to residual solvent peaks (DMSO d6: 2.5 ppm for 1H and 39.5 ppm for 13C). The spectra were processed using MestReNova software. Chemical shifts were quantified in parts per million (ppm), whereas J-coupling constants were recorded in hertz (Hz). Signal multiplicities are expressed as follows: s for a singlet, d for a doublet, dd for the doublet of doublet, ddd for the doublet of the doublet of the doublets, t for a triplet and m for multiplet. High-resolution mass spectra (HRMS) were recorded on Bruker micrOTOF-Q II mass spectrometer using atmospheric pressure chemical ionization (APCI) technique in positive ion mode. The melting points (m. pt.) of the target compounds were ascertained using a Büchi® melting point equipment (Model B-545, AC/DC input 230 V AC). For the Fourier-transform infrared spectroscopy (FTIR), a Bruker ALPHA FTIR spectrometer equipped with a Diamond Crystal ATR (Attenuated Total Internal Reflectance) accessory was employed. High- performance liquid chromatography (HPLC) using an Agilent 1200 series HPLC system equipped with a quaternary pump and an Agilent 1200 series diode array detector was used to ascertain the purity of the compound. A Venusil XBP C18 column (4.60 × 150 mm, 5 µm) was used for separation, and the mobile phase consisted of 10% methanol and 90% MilliQ water at a flow rate of 1 mL/min. The compounds were prepared in HPLC grade acetonitrile (ACN), and 10 µL of the sample was injected. The samples were examined at 280 nm and 400 nm wavelengths with a flow rate adjusted to 1 mL/min.

Synthesis procedure for target compoundsAmide formations General procedure for synthetic route for N-(4-acetylphenyl)-2-(methylamino)benzamide

A round-bottomed flask was charged with p-aminoacetophenone (1 g, 5.64 mmol), 1.5 equiv of N-methylisatoic anhydride (1.14 g, 8.47 mmol) and 10 mL of glacial acetic acid. The mixture was heated at 100 °C overnight. After the reaction was completed, the content was poured into water. The resulting precipitate was then filtered, dried to give intermediate N-(4-acetylphenyl)-2-(methylamino)benzamide, which was used in the next step without further purification.

Aldol reaction

A mixture of N-(4-acetylphenyl)-2-(methylamino)benzamide, 1.5 equiv of benzaldehyde, aqueous solution of sodium hydroxide (NaOH; 20%, 10 mL), and 30 mL of ethanol was stirred at room temperature for 15 h. The reaction progress was monitored by TLC. After the completion of the reaction, the mixture was poured over crushed ice, acidified with diluted HCl and the precipitated chalcone was filtered and dried. The crude compound was purified through recrystallization from 95% ethanol to obtain the desired product of 50.16–80.21% yield.

Compounds characterization(E)-N-(4-(3-(3-fluorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K1)

Brown powder, yield of 50.2% and m. pt. 151.0 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.20 (d, J = 8.2 Hz, 2H), 8.03 (d, J = 15.6 Hz, 1H), 7.95 (d, J = 8.3 Hz, 2H), 7.85 (d, J = 9.9 Hz, 1H), 7.73 (s, 1H), 7.72–7.68 (m, 2H), 7.53–7.47 (m, 1H), 7.38 (t, J = 7.1 Hz, 1H), 7.28 (t, J = 8.1 Hz, 2H), 6.71 (d, J = 8.2 Hz, 1H), 6.66 (t, J = 7.1 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.43, 168.33, 161.67, 150.21, 143.97, 141.77, 137.34, 133.15, 132.15, 130.80, 130.74, 129.68, 129.07, 125.44, 123.48, 119.60, 116.98, 115.07, 114.03, 110.74, 29.37. IR (ATR) Vmax cm−1: 3319 (N-H stretching), 1681 (C=O stretching), 1649 (C=C stretching), 1218 (C-F stretching) and 736 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20FN2O2 375.1503; found 375.1506. Purity (HPLC): 97%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(4-nitrophenyl)acryloyl)phenyl)benzamide (K3)

Mustard powder, yield of 53.47% and m. pt. 189.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.29 (d, J = 7.8 Hz, 2H), 8.21 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 7.8 Hz, 3H), 8.14 (s, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.81 (d, J = 15.6 Hz, 1H), 7.73 (d, J = 7.4 Hz, 1H), 7.38 (t, J = 7.2 Hz, 1H), 6.71 (d, J = 8.1 Hz, 1H), 6.66 (t, J = 7.1 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.31, 168.33, 150.20, 147.97, 144.16, 141.29, 140.41, 133.17, 131.92, 129.79, 129.73, 129.08, 126.09, 123.85, 119.61, 115.04, 114.05, 110.76, 29.38. IR (ATR) Vmax cm−1: 3378 (N-H stretching), 1653 (C=O stretching), 1594 (C=C stretching), 1516 (N-O stretching) and 732 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20N3O4 402.1448; found 402.1440. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(3-nitrophenyl)acryloyl)phenyl)benzamide (K4)

Mustard powder, yield of 80.21% and m. pt. 102.1–102.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.78 (s, 1H), 8.33 (d, J = 7.1 Hz, 1H), 8.26 (d, J = 7.8 Hz, 1H), 8.23 (d, J = 8.1 Hz, 2H), 8.18 (d, J = 15.7 Hz, 1H), 7.96 (d, J = 8.1 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 7.76 (d, J = 7.7 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.38 (t, J = 7.1 Hz, 1H), 7.34 (s, 1H), 6.73–6.64 (m, 2H), 3.31 (s, 3H).13 C NMR (151 MHz, DMSO-d6) δ 187.36, 168.34, 150.23, 148.41, 144.09, 140.73, 136.70, 134.98, 133.16, 132.01, 130.27, 129.80, 129.08, 124.79, 124.47, 122.86, 119.58, 115.04, 114.01, 110.72, 29.36. IR (ATR) Vmax cm−1: 3347 (N-H stretching), 1649 (C=O stretching), 1609 (C=C stretching), 1509 (N-O stretching) and 740 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20N3O4 402.1448; found 402.1443. Purity (HPLC): 97%. Rt: 2.2 min.

(E)-N-(4-(3-(4-fluorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K5)

Brown powder, yield of 86% and m. pt. 171.7–174.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.18–8.08 (m, 2H), 7.97 (d, J = 4.4 Hz, 2H), 7.94 (t, J = 10.7 Hz, 3H), 7.78–7.68 (m, 2H), 7.38 (d, J = 5.6 Hz, 1H), 7.30 (d, J = 4.4 Hz, 3H), 6.73–6.68 (m, 1H), 6.66 (d, J = 5.0 Hz, 1H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.46, 168.32, 164.12, 150.22, 143.83, 142.03, 133.14, 132.31, 131.45, 131.05, 129.57, 129.06, 121.93, 119.60, 115.92, 115.08, 114.02, 110.72, 29.36. IR (ATR) Vmax cm−1: 3356 (N-H stretching), 1648 (C=O stretching), 1608 (C=C stretching), 1334 (C-F stretching) and 740 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20FN2O2 375.1503; found 375.1496. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(3-chlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K6)

Dark brown powder, yield of 50.80% and m. pt. 157.5 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.21 (d, J = 8.0 Hz, 2H), 8.07 (d, J = 11.0 Hz, 2H), 8.04 (s, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.82 (d, J = 5.3 Hz, 1H), 7.71 (t, J = 11.1 Hz, 2H), 7.53–7.46 (m, 2H), 7.38 (t, J = 7.0 Hz, 1H), 6.76–6.63 (m, 2H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.39, 168.33, 150.21, 143.98, 141.54, 137.05, 133.76, 133.15, 132.14, 130.61, 129.94, 129.71, 129.07, 127.81, 127.78, 123.55, 119.59, 115.08, 114.04, 110.74, 29.37. IR (ATR) Vmax cm−1: 3355 (N-H stretching), 1651 (C=O stretching), 1592 (C=C stretching), 737 (C-Cl stretching) and 737 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20ClN2O2 391.1208; found 391.1195. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(o-tolyl)acryloyl)phenyl)benzamide (K7)

Lime green powder, yield of 66.64% and m. pt. 185.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.46 (s, 1H), 8.17 (d, J = 8.2 Hz, 2H), 7.99 (d, J = 6.1 Hz, 2H), 7.98–7.94 (m, 3H), 7.83 (d, J = 15.5 Hz, 1H), 7.76 (d, J = 7.5 Hz, 1H), 7.41 (t, J = 7.4 Hz, 1H), 7.37–7.32 (m, 1H), 7.29 (s, 2H), 6.80 (d, J = 8.1 Hz, 1H), 6.74 (t, J = 7.0 Hz, 1H), 2.84 (s, 3H), 2.45 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 187.62, 168.07, 149.03, 143.77, 140.39, 137.85, 133.39, 133.12, 132.41, 130.73, 130.20, 129.58, 129.16, 126.81, 126.32, 122.94, 119.69, 116.25, 115.30, 111.87, 30.04, 19.31. IR (ATR) Vmax cm−1: 3306 (N-H stretching), 1657 (C=O stretching), 1597 (C=C stretching), 741 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H23N2O2 371.1754; found 371.1736. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-N-(4-(3-(4-bromophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K8)

Light brown powder, yield of 80.13% and m. pt. 190.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.18 (d, J = 8.2 Hz, 2H), 8.01 (s, 1H), 7.98 (s, 1H), 7.95 (d, J = 8.2 Hz, 2H), 7.86 (d, J = 7.9 Hz, 2H), 7.76 (d, J = 7.4 Hz, 1H), 7.71 (s, 1H), 7.69–7.65 (m, 2H), 7.41 (t, J = 7.3 Hz, 1H), 6.81 (d, J = 8.0 Hz, 1H), 6.75 (t, J = 6.9 Hz, 1H), 2.84 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.47, 168.07, 148.99, 143.87, 141.92, 134.10, 133.15, 132.29, 131.83, 130.69, 129.64, 129.18, 123.77, 122.84, 119.68, 116.27, 115.40, 111.96, 30.10. IR (ATR) Vmax cm−1: 3380 (N-H stretching), 1656 (C=O stretching), 1605 (C=C stretching), 673 (C-Br stretching) and 749 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20BrN2O2 435.0703, found 435.0705. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(4-chlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K9)

Mustard powder, yield of 54.92% and m. pt. 167.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.18 (d, J = 8.4 Hz, 2H), 8.00 (s, 1H), 7.96 (d, J = 10.9 Hz, 2H), 7.93 (d, J = 8.2 Hz, 3H), 7.74–7.70 (m, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.38 (t, J = 7.7 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.66 (t, J = 7.4 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.43, 168.33, 150.22, 143.91, 141.78, 134.89, 133.76, 133.15, 132.22, 130.44, 129.62, 129.07, 128.88, 122.78, 119.60, 115.07, 114.02, 110.73, 29.36. IR (ATR) Vmax cm−1: 3321 (N-H stretching), 1652 (C=O stretching), 1590 (C=C stretching), 744 (C-Cl stretching) and 744 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20ClN2O2 391.1208; found 391.1214. Purity (HPLC): 98%. Rt: 2.2 min.

(E)-N-(4-(3-(2-chlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K10)

Lime green powder, yield of 68.65% and m. pt. 175.2 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.50 (s, 1H), 8.22 (d, J = 7.4 Hz, 1H), 8.19 (d, J = 7.9 Hz, 2H), 8.02 (d, J = 6.1 Hz, 2H), 7.97 (t, J = 9.4 Hz, 2H), 7.77 (d, J = 7.8 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.47 (dd, J = 9.4, 4.3 Hz, 2H), 7.42 (t, J = 7.7 Hz, 2H), 6.82 (d, J = 8.3 Hz, 1H), 6.76 (t, J = 7.3 Hz, 1H), 2.84 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 187.38, 168.04, 148.79, 144.01, 137.90, 134.25, 133.15, 132.40, 132.11, 131.83, 129.98, 129.74, 129.20, 128.54, 127.65, 124.81, 119.71, 116.47, 115.62, 112.15, 30.21. IR (ATR) Vmax cm−1: 3314 (N-H stretching), 1658 (C=O stretching), 1602 (C=C stretching) 758 (C-Cl stretching) and 768 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20ClN2O2 391.1208; found 391.1194. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-N-(4-(3-(3,4-dimethoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K11)

Brown powder, yield of 51.54% and m. pt. 188.7 °C 1H NMR (600 MHz, DMSO-d6) δ 10.47 (s, 1H), 8.18 (d, J = 7.4 Hz, 2H), 7.95 (d, J = 7.3 Hz, 2H), 7.86 – 7.82 (m, 1H), 7.77 (d, J = 7.8 Hz, 1H), 7.69 (d, J = 15.4 Hz, 1H), 7.54 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.39 (s, 1H), 7.38 (s, 1H), 7.03 (d, J = 8.2 Hz, 1H), 6.83 (d, J = 8.2 Hz, 1H), 6.77 (t, J = 7.1 Hz, 1H), 3.87 (s, 3H), 3.82 (s, 3H), 2.84 (s, 3H).13C NMR (151 MHz, DMSO-d6) δ 187.48, 168.01, 151.20, 149.04, 148.76, 143.84, 143.56, 133.10, 132.73, 129.46, 129.19, 127.63, 123.79, 119.66, 119.57, 116.59, 115.65, 112.17, 111.61, 110.83, 55.78, 55.61, 30.22. IR (ATR) Vmax cm−1: 3357 (N-H stretching), 1652 (C=O stretching), 1594 (C=C stretching), 1256 (C-C stretching), and 730 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C25H25N2O4 417.1778; found 417.1778. Purity (HPLC): 96%. Rt: 2.2 min.

(E)-N-(4-(3-(3,4-dichlorophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K12)

Mustard powder, yield of 71.92% and m. pt. 195 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.28 (s, 1H), 8.21 (d, J = 8.5 Hz, 2H), 8.07 (d, J = 15.6 Hz, 1H), 7.95 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.0 Hz, 1H), 7.74–7.70 (m, 2H), 7.68 (s, 1H), 7.39 (d, J = 7.2 Hz, 1H), 7.37 (s, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.28, 168.33, 150.23, 144.04, 140.44, 135.69, 133.16, 132.51, 132.06, 131.77, 130.91, 130.02, 129.73, 129.07, 129.03, 124.08, 119.58, 115.05, 114.02, 110.73, 29.36. IR (ATR) Vmax cm−1: 3355 (N-H stretching), 1647 (C=O stretching), 1593 (C=C stretching), 834 (C-Cl stretching) and 741 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H19Cl2N2O2 425.0801; found 425.0801. Purity (HPLC): 95%. Rt: 2.2 min.

E)-2-(methylamino)-N-(4-(3-(4-(trifluoromethyl)phenyl)acryloyl)phenyl)benzamide (K15)

Yellow powder, yield of 63.22% and m. pt. 202.1 °C. 1H NMR (600 MHz, DMSO) δ 10.42 (s, 1H), 8.22 (s, 1H), 8.20 (s, 1H), 8.12 (d, J = 7.8 Hz, 2H), 8.10 (s, 1H), 7.96 (d, J = 8.7 Hz, 2H), 7.84 – 7.79 (m, 2H), 7.77 (s, 1H), 7.73–7.71 (m, 1H), 7.40–7.32 (m, 2H), 6.70 (t, J = 7.2 Hz, 1H), 6.66 (t, J = 7.5 Hz, 1H), 2.81 (d, J = 4.7 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.46, 168.42, 150.28, 144.18, 141.34, 138.86, 133.24, 132.05, 129.82, 129.39, 129.16, 125.71, 125.69, 124.73, 119.67, 119.53, 115.08, 114.07, 110.77, 29.41. IR (ATR) Vmax cm−1: 3364 (N-H stretching), 1653 (C=O stretching), 1592 (C=C stretching), 1319 (C-F stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H20F3N2O2 425.1456; found 425.1456. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(2,4,6-trimethoxyphenyl)acryloyl)phenyl)benzamide (K16)

Maroon powder, yield of 54.08% and m. pt. 132.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.36 (s, 1H), 8.08 (t, J = 12.9 Hz, 1H), 7.99 (d, J = 7.6 Hz, 2H), 7.94–7.88 (m, 3H), 7.71 (d, J = 7.2 Hz, 1H), 7.38 (t, J = 7.0 Hz, 1H), 6.71 (d, J = 8.0 Hz, 1H), 6.66 (t, J = 6.8 Hz, 1H), 6.32 (d, J = 7.5 Hz, 3H), 3.93 (s, 6H), 3.86 (s, 3H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 188.54, 168.25, 163.16, 161.31, 150.05, 143.20, 134.56, 133.25, 133.05, 129.05, 128.91, 120.35, 119.72, 115.33, 114.14, 110.78, 105.21, 91.04, 56.06, 55.52, 29.42. IR (ATR) Vmax cm−1: 3355 (N-H stretching), 1647 (C=O stretching), 1590 (C=C stretching), 1294 (C-O stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C26H27N2O5 447.1908; found 447.1908. Purity (HPLC): 100%. Rt: 2.2 min.

(E)-N-(4-(3-(3-methoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K17)

Grey powder, yield of 51.38% and m. pt. 130.4 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.19 (d, J = 6.3 Hz, 2H), 7.99 (s, 1H), 7.95 (d, J = 7.8 Hz, 2H), 7.72 (s, 1H), 7.70 (s, 1H), 7.49 (s, 1H), 7.44 (s, 1H), 7.41–7.31 (m, 3H), 7.03 (s, 1H), 6.71 (d, J = 7.9 Hz, 1H), 6.66 (s, 1H), 3.84 (s, 3H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.56, 168.32, 159.63, 150.21, 143.84, 143.27, 136.17, 133.13, 132.30, 129.85, 129.59, 129.06, 122.27, 121.54, 119.59, 116.50, 115.09, 114.01, 113.31, 110.71, 55.28, 29.35. IR (ATR) Vmax cm−1: 3371 (N-H stretching), 1652 (C=O stretching), 1602 (C=C stretching), 1289 (C-O stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H23N2O3 387.1695; found 387.1695. Purity (HPLC): 98%. Rt: 2.2 min.

(E)-N-(4-(3-(2-bromophenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K18)

Mustard powder, yield of 61.64% and m. pt. 130.7–134.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.43 (s, 1H), 8.22–8.18 (m, 3H), 7.99 (s, 2H), 7.95 (d, J = 8.7 Hz, 2H), 7.75 (d, J = 7.3 Hz, 1H), 7.73–7.71 (m, 1H), 7.50 (t, J = 7.3 Hz, 1H), 7.41–7.39 (m, 1H), 7.38 (d, J = 7.0 Hz, 1H), 7.34 (d, J = 5.2 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 2.81 (d, J = 4.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.34, 168.42, 150.27, 144.18, 140.66, 134.10, 133.30, 133.24, 132.07, 129.82, 129.16, 128.74, 128.24, 125.33, 124.93, 119.67, 115.08, 114.07, 110.77, 29.42. IR (ATR) Vmax cm-1: 3359 (N-H stretching), 1650 (C=O stretching), 1590 (C=C stretching), 754 (C-Br stretching) and 736 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C23H20BrN2O2 435.0686; found 435.0703. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(2,5-dimethoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K19)

Brown powder, yield of 50.25% and m. pt. 102.2–102.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.18 (s, 2H), 7.98 (d, J = 43.7 Hz, 3H), 7.72 (s, 1H), 7.57 (s, 2H), 7.51–7.28 (m, 2H), 7.05 (s, 2H), 6.70 (s, 1H), 6.67 (s, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 2.81 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.66, 168.31, 153.25, 152.64, 150.20, 143.74, 137.56, 133.11, 132.45, 129.52, 129.06, 123.63, 121.99, 119.59, 117.93, 115.11, 114.01, 113.02, 112.56, 110.70, 56.14, 55.68, 29.35. IR (ATR) Vmax cm−1: 3311 (N-H stretching), 1648 (C=O stretching), 1591 (C=C stretching), 1233 (C-O stretching) and 742 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C25H25N2O4 417.1799; found 417.1799. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(thiophen-3-yl)acryloyl)phenyl)benzamide (K20)

Light mustard powder, yield of 51.82% and m. pt. 114.9 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.15 (d, J = 8.7 Hz, 2H), 8.10 (d, J = 1.7 Hz, 1H), 7.94 (d, J = 8.7 Hz, 2H), 7.79 (d, J = 15.4 Hz, 2H), 7.76 (s, 1H), 7.72 (dd, J = 11.1, 4.2 Hz, 1H), 7.67 (dd, J = 4.7, 2.9 Hz, 1H), 7.38 (t, J = 7.2 Hz, 1H), 7.34 (d, J = 4.5 Hz, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.2 Hz, 1H), 2.81 (d, J = 4.8 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.80, 168.39, 150.25, 143.86, 138.34, 137.20, 133.19, 132.42, 130.34, 129.52, 129.14, 127.66, 126.19, 121.49, 119.64, 115.16, 114.07, 110.75, 29.42. IR (ATR) Vmax cm−1: 3339 (N-H stretching), 1650 (C=O stretching), 1592 (C=C stretching), 2813 (S-H stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C21H19N2O5S 363.1162; found 363.1139. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(thiophen-2-yl)acryloyl)phenyl)benzamide (K21)

Mustard powder, yield of 51.82% and m. pt. 146.3 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.38 (s, 1H), 8.11 (d, J = 8.0 Hz, 2H), 7.95–7.91 (m, 3H), 7.89 (s, 1H), 7.78 (d, J = 4.3 Hz, 1H), 7.72 (d, J = 7.7 Hz, 1H), 7.68 (s, 1H), 7.59 (d, J = 15.3 Hz, 1H), 7.38 (t, J = 7.7 Hz, 1H), 7.20 (s, 1H), 6.71 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 2.82 (s, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.01, 168.30, 150.19, 143.76, 139.79, 136.03, 133.11, 132.48, 132.23, 130.12, 129.39, 129.06, 128.61, 120.32, 119.64, 115.10, 114.02, 110.71, 29.36. IR (ATR) Vmax cm−1: 3322 (N-H stretching), 1645 (C=O stretching), 1593 (C=C stretching), 2360 (S-H stretching) and 735 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C21H19N2O2S 363.1135; found 363.1135. Purity (HPLC): 98%. Rt: 2.2 min.

(E)-N-(4-(3-(benzo[d][1,3]dioxol-5-yl)acryloyl)phenyl)-2-(methylamino)benzamide (K22)

Light yellow powder, yield of 67.01% and m. pt. 135.1 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.17 (d, J = 8.7 Hz, 2H), 7.93 (d, J = 8.7 Hz, 2H), 7.85 (d, J = 15.5 Hz, 1H), 7.74–7.70 (m, 1H), 7.69–7.65 (m, 2H), 7.40–7.31 (m, 3H), 7.00 (d, J = 8.0 Hz, 1H), 6.70 (d, J = 8.3 Hz, 1H), 6.66 (t, J = 7.4 Hz, 1H), 6.11 (s, 2H), 2.81 (d, J = 4.7 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.38, 168.38, 150.25, 149.47, 148.11, 143.83, 143.37, 133.18, 132.53, 129.54, 129.36, 129.14, 125.82, 119.96, 119.63, 115.18, 114.06, 110.75, 108.54, 106.94, 101.65, 29.42. IR (ATR) Vmax cm−1: 3420 (N-H stretching), 1656 (C=O stretching), 1588 (C=C stretching), 1217 (C-O stretching) and 746 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H21N2O4 401.1472; found 401.1472. Purity (HPLC): 99%. Rt: 2.2 min.

(E)-2-(methylamino)-N-(4-(3-(2,4,5-trimethoxyphenyl)acryloyl)phenyl)benzamide (K23)

Yellow powder, yield of 60.09% and m. pt. 121.3–123.0 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.16 (d, J = 8.7 Hz, 1H), 8.06 (d, J = 15.6 Hz, 1H), 7.96–7.93 (m, 1H), 7.92 (s, 1H), 7.88 (d, J = 8.7 Hz, 1H), 7.78 (t, J = 11.9 Hz, 1H), 7.71 (t, J = 7.6 Hz, 1H), 7.53 (s, 1H), 7.37 (dd, J = 16.1, 9.0 Hz, 1H), 7.33 (s, 1H), 6.75 (s, 1H), 6.70 (dd, J = 8.2, 3.8 Hz, 1H), 6.66 (t, J = 7.0 Hz, 1H), 3.92–3.89 (m, 3H), 3.87 (s, 3H), 3.83 (s, 3H), 2.81 (t, J = 4.5 Hz, 3H). 13C NMR (151 MHz, DMSO) δ 186.83, 168.37, 154.19, 152.78, 150.23, 143.11, 137.87, 133.17, 132.85, 129.42, 129.19, 129.13, 119.61, 119.53, 118.64, 115.22, 114.08, 110.97, 110.74, 97.58, 56.44, 55.84, 29.42, 26.46. IR (ATR) Vmax cm−1: 3337 (N-H stretching), 1651 (C=O stretching), 1591 (C=C stretching) 1266 (C-O stretching) and 743 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C26H27N2O5 447.1898; found 447.1898. Purity (HPLC): 95%. Rt: 2.2 min.

(E)-N-(4-(3-(2-methoxyphenyl)acryloyl)phenyl)-2-(methylamino)benzamide (K25)

Light yellow powder, yield of 69.43% and m. pt. 143.8 °C. 1H NMR (600 MHz, DMSO-d6) δ 10.40 (s, 1H), 8.15 (d, J = 8.7 Hz, 2H), 8.05 (d, J = 15.7 Hz, 1H), 7.99 (d, J = 7.1 Hz, 1H), 7.95–7.92 (m, 2H), 7.90 (s, 1H), 7.72 (d, J = 7.1 Hz, 1H), 7.45 (t, J = 7.2 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.34 (d, J = 4.6 Hz, 1H), 7.12 (d, J = 8.3 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.70 (d, J = 8.4 Hz, 1H), 6.66 (t, J = 7.3 Hz, 1H), 3.90 (s, 3H), 2.81 (d, J = 4.9 Hz, 3H). 13C NMR (151 MHz, DMSO-d6) δ 187.73, 168.38, 158.21, 150.25, 143.82, 137.85, 133.20, 132.50, 132.19, 129.54, 129.14, 128.39, 123.07, 121.79, 120.70, 119.68, 115.15, 114.07, 111.80, 110.76, 55.74, 29.42. IR (ATR) Vmax cm−1: 3322 (N-H stretching), 1647 (C=O stretching), 1590 (C=C stretching), 1126 (C-O stretching) and 738 (C-H bending). The HRMS (APCI) m/z [M + H]+ calculated for C24H23N2O3 387.1683; found 387.1683. Purity (HPLC): 98%. Rt: 2.2 min.

In vitro anti-parasitic assay

Anti-parasitic assays were performed as described elsewhere [41, 42]. Briefly, to evaluate anti-Leishmania activity, L. infantum [MHOM/ MA (BE)/67] was used with primary peritoneal mouse macrophages as host cells. 3 × 104 macrophages were infected with 4.5 × 105 parasites per well. Compound dilutions were added after 2 h of infection. After 5 days of incubation, parasite burdens (mean number of amastigotes/macrophage) were assessed microscopically after staining with a 10% Giemsa solution. For T. cruzi, the Tulahuen CL2, β-galactosidase strain (nifurtimox-sensitive) was used and maintained on MRC-5SV2 (human lung fibroblast). 4 × 103 cells were infected with 4 × 104 parasites per well. Parasite burdens were assessed after adding the substrate CPRG (chlorophenol red β-d- galactopyranoside). The change in color was measured spectrophotometrically at 540 nm after 4 h incubation at 37 °C. Drug susceptibility tests for T. brucei were performed using a resazurin assay. Susceptibility assays were performed with T. brucei Squib 42749 or T. b. rhodesiense STIB-90050. T. brucei Squib 427 was seeded at 1.5 × 104 parasites/well and T. b. rhodesiense at 4 × 103 parasites per well, followed by the addition of resazurin after 24 h (T. brucei) or 6 h (T. b. rhodesiense). After the addition of resazurin, plates were incubated for another 24 h followed by fluorescence detection (λex 550 nm, λem 590 nm).

In all assays, parasite growth was compared to untreated-infected controls (100% growth) and noninfected controls (0% growth). Results were expressed as % parasite reduction at the different drug concentrations and used to calculate IC50 values from the dose–response curves.

In vitro cytotoxicity

MRC-5SV2 cell cytotoxicity was evaluated as described elsewhere [41]. Briefly, 1.5 × 105 cells/ mL cells were cultured with compound dilutions at 37 °C and with 5% CO2. Cell growth was compared to untreated-control wells (100% cell growth) and medium-control wells (0% cell growth). After 3 days of incubation, cell viability was assessed fluorimetrically after the addition of 50 μL resazurin per well. After 4 h at 37 °C, fluorescence was measured. The results were expressed as a % reduction in cell growth/ viability compared to control wells and an IC50 value was determined.