4.1. Chemistry

All commercially available solvents and reagents were used without further purification. NMR spectra were recorded on an Inova 500 spectrometer (Varian, Palo Alto, CA, USA). The chemical shifts (δ) are reported in parts per million downfield from tetramethylsilane (TMS), which was used as internal standard, and the spectra were recorded in hexadeuteriodimethylsulphoxide (DMSO-d₆). Infrared spectra were recorded on a Vector 22 spectrometer (Bruker, Bremen, Germany) in Nujol mulls. The main bands are given in cm⁻¹. Positive-ion electrospray ionization (ESI) mass spectra were recorded on a double-focusing MAT 95 instrument (Finnigan, Waltham, MA, USA) with BE geometry. Melting points (mp) were determined on a SMP1 Melting Point apparatus (Stuart Scientific, Stone, UK) and were uncorrected. All reported products showed NMR spectra in agreement with the assigned structures. The purity of the tested compounds was determined by combustion elemental analyses conducted by the Microanalytical Laboratory of the Department of Chemical and Pharmaceutical Sciences of the University of Ferrara with a MT-5 CHN recorder elemental analyzer (Yanagimoto, Kyoto, Japan), and the values found were within 0.4% of theoretical values. Compounds 6, 9 [47] 11–15, 19, 21, 23, 27, 29–37 and 49–51 were prepared as previously described [48].

A mixture of acid 10 (0.33 g, 1 mmol), EDCI (1.92 g, 1.1 mmol), and HOBt (0.13 g, 1 mmol) in dry MeCN (10 mL) was stirred at room temperature for 30 min and then treated with the appropriate phenol (1 mmol). The mixture was stirred at room temperature for an additional 24 h. Then the solution was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed with brine (2 × 5 mL), 5% aqueous sodium hydroxide (2 × 5 mL), and water (2 × 5 mL). The organic layer was dried over anhydrous sodium sulfate. Concentration of the dried extract yielded a residue which was triturated with di-isopropyl ether. The formed precipitate was filtered off and purified by crystallization from the adequate solvent to give the ester derivatives 11–24.

2-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (16). Yield 86%. Mp 179–180 °C (MeCN). ¹H NMR (DMSO-d₆): δ 2.38 (s, 3H, CH₃), 6.98–7.41 (m, 5H, aryl), 7.52 d, 1H, J = 8.1 Hz, pyridyl), 7.46 (d, 1H, J = 8.4 Hz, aryl), 8.26 (d, 1H, J = 8.4 Hz, aryl), 8.88 (d, 1H, J = 8.1 Hz, pyridyl), 9.98 (s, 1H, NH), 10.06 (s, 1H, OH). IR (Nujol) 3330, 1712, 1615, 1590 cm⁻¹. m/z 423 (M + H)⁺. Anal. Calcd for C₂₀H₁₄ClF₃N₂O₃ (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.87, %H 3.32, %N 6.61.

3-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (17). Yield 87%. Mp 188-190 °C (MeCN). ¹H NMR (DMSO-d₆): δ 2.27 (s, 3H, CH₃), 7.30-7.66 (m, 7H, aryl), 8.11 (d, 1H, J = 8.5 Hz, aryl), 8.79 (d, 1H, J = 8.0 Hz, pyridyl), 9.88 (s, 1H, NH), 10.05 (s, 1H, OH). IR (Nujol) 3294, 1712, 1620, 1590 cm⁻¹. m/z 423 (M + H)⁺. Anal. Calcd for C₂₀H₁₄ClF₃N₂O₃ (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.78, %H 3.33, %N 6.60.

4-Hydroxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (18). Yield 63%. Mp 184-186 °C (EtOH). ¹H NMR (DMSO-d₆): δ 2.27 (s, 3H, CH₃), 6.83 (d, 2H, J = 9.0 Hz, aryl), 7.13 (d, 2H, J = 7.0 Hz, aryl), 7.37 (m, 3H, aryl and pyridyl), 8.14 (d, 1H, J = 9.0 Hz, aryl), 8.73 (d, 1H, J = 7.5 Hz, pyridyl), 9.55 (s, 1H, NH), 9.86 (s, 1H, OH). IR (Nujol) 3474, 3345, 1687, 1617, 1589 cm⁻¹. m/z 423 (M + H)⁺. Anal. Calcd for C₂₀H₁₄ClF₃N₂O₃ (422.78) %C 56.82, %H 3.34, %N 6.63. Found %C 56.79, %H 3.36, %N 6.66.

3,4-Dimethoxyphenyl-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (20). Yield 52%. Mp 127-129 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.30 (s, 3H, CH₃), 3.79 (s, 3H, CH₃), 3.86 (s, 3H, CH₃), 6.85–7.98 (m, 6H, aryl and pyridyl), 8.24 (d, 1H, J = 8.5 Hz, aryl), 8.76 (d, 1H, J = 8.0 Hz, pyridyl), 10.51 (s, 1H, NH). IR (Nujol) 3345, 3278, 1748, 1699, 1622, 1608, 1591 cm⁻¹. m/z 467 (M + H)⁺. Anal. Calcd for C₂₂H₁₈ClF₃N₂O₄ (466.84) %C 56.60, %H 3.89, %N 6.00. Found %C 56.65, %H 3.90, %N 6.02.

3,4,5-Trimethoxyphenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (22). Yield 98%. Mp 138-140 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.26 (s, 3H, CH₃), 3.67 (s, 3H, CH₃), 3.76 (s, 6H, CH₃), 6.73 (s, 2H, aryl), 7.28 (d, 1H, J = 8.5 Hz, aryl), 7.35–7.40 (m, 2H, aryl and pyridyl), 8.08 (d, 1H, J = 8.5 Hz, aryl), 8.72 (d, 1H, J = 7.5 Hz, pyridyl), 9.89 (s, 1H, NH). IR (Nujol) 3331, 3293, 1697, 1618, 1590 cm⁻¹. m/z 497 (M + H)⁺. Anal. Calcd for C₂₃H₂₀ClF₃N₂O₄ (496.86) %C 55.60, %H 4.06, %N 5.64. Found %C 55.54, %H 4.07, %N 5.66.

4-(Methylthio)phenyl 2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinate (24). Yield 63%. Mp 125–127 °C (2-propanol). ¹H NMR (DMSO-d₆): δ (s, 3H, CH₃), 7.41–7.52 (m, 10H, aryl and pyridyl), 8.24 (d, 1H, J = 8.8 Hz, aryl), 8.87 (d, 1H, J = 8.1 Hz, pyridyl), 10.03 (s, 1H, NH). IR (Nujol) 3294, 1712, 1620, 1590 cm⁻¹. m/z 453 (M + H)⁺. Anal. Calcd for C₂₁H₁₆ClF₃N₂O₂S (452.88) %C 55.69, %H 3.56, %N 6.19. Found %C 55.74, %H 3.58, %N 6.17.

A mixture of acid 10 (0.33 g, 1 mmol), EDCI (1.92 g, 1.1 mmol), and HOBt (0.13 g, 1 mmol) in dry MeCN (10 mL) was stirred at room temperature for 30 min and then treated with the appropriate amine (1 mmol). The mixture was stirred at room temperature for an additional 24 h. Then the solution was evaporated to dryness in vacuo. The residue was dissolved in ethyl acetate (20 mL) and washed sequentially with brine (2 × 5 mL), 10% aqueous sodium carbonate (2 × 5 mL), 10% aqueous citric acid (2 × 5 mL), and water (2 × 5 mL). The organic layer was dried over anhydrous magnesium sulfate. Concentration of the dried extracts yielded a solid residue which was washed with ethyl ether, filtered off, and dried to give the amide derivatives in analytically pure form without additional purification by crystallization if not elsewhere indicated.

2-((5-Chloro-2-methylphenyl)amino)-N-hydroxy-6-(trifluoromethyl)nicotinamide (25). Yield 61%. Mp 154–155 °C. ¹H NMR (DMSO-d₆): δ 2.37 (s, 3H, CH₃), 7.36–7.68 (m, 3H, aryl and pyridyl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.86 (d, 1H, J = 7.7 Hz, pyridyl), 9.87 (s, 1H, NH), 10.59 (s, 1H, NH) 12.01 (s, 1H, OH). IR (Nujol) 3350, 1686, 1618, 1589 cm⁻¹. m/z 346 (M + H)⁺. Anal. Calcd for C₁₄H₁₁ClF₃N₃O₂ (345.70) %C 48.64, %H 3.21, %N 12.15. Found %C 48.69, %H 3.20, %N 12.18.

Ethyl 2-(2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamido)acetate (26). Yield 96%. Mp 128-130 °C. ¹H NMR (DMSO-d₆): δ 1.22 (t, J = 7.1 Hz, 3H, CH₃), 2.39 (s, 3H, CH₃), 4.27 (q, J = 7.1 Hz, 2H, CH₂), 4.15 (d, J = 4.8 Hz, 2H, CH₂), 7.25–7.39 (m, 3H, aryl and pyridyl), 8.20 (d, 1H, J = 8.0 Hz, aryl), 8.56 (d, 1H, J = 7.3 Hz, pyridyl), 9.59 (s, 1H, NH), 10.69 (s, 1H, NH). IR (Nujol) 3429, 1737, 1722, 1655 cm⁻¹. m/z 416 (M + H)⁺. Anal. Calcd for C₁₈H₁₇ClF₃N₃O₃ (415.79) %C 52.00, %H 4.12, %N 10.11. Found %C 52.05, %H 4.10, %N 10.08.

2-((5-Chloro-2-methylphenyl)amino)-N-(4-(methylthio)phenyl)-6-(trifluoromethyl)nicotinamide (28). Yield 64%. Mp 152-154 °C. ¹H NMR (DMSO-d₆): δ 2.27 (s, 3H, CH₃), 2.48 (s, 3H, CH₃), 7.23 (d, 1H, J = 8.5 Hz, aryl), 7.28 (m, 3H, aryl), 7.36 (d, 1H, J = 7.5 Hz, pyridyl), 7.65 (d, 2H, J = 7.5 Hz, aryl), 8.15 (d, 1H, J = 8.5 Hz, aryl), 8.46 (d, 1H, J = 7.5 Hz, pyridyl), 10.35 (s, 1H, NH), 10.64 (s, 1H, NH). IR (Nujol) 3380, 3300, 1639, 1588 cm⁻¹. m/z 452 (M + H)⁺. Anal. Calcd for C₂₁H₁₇ClF₃N₃OS (451,89) %C 55.82, %H 3.79, %N 9.30.

N-(2-chlorobenzyl)-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamide (38). Yield 93%. Mp 169-170 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.39 (s, 3H, CH₃), 4.72 (d, J = 5.2 Hz, 2H, CH₂), 7.36–7.61 (m, 6H, aryl and pyridyl), 8.35 (d, 1H, J = 8.8 Hz, aryl), 8.56 (d, 1H, J = 8.0 Hz, pyridyl), 9.68 (s, 1H, NH), 10.90 (s, 1H, NH). IR (Nujol) 3300, 1636, 1613, 1597 cm⁻¹. m/z 450 (M + H)⁺. Anal. Calcd for C₂₁H₁₆ClF₃N₃O (449.85) %C 58.74, %H 4.26, %N 9.34. Found %C 58.69, %H 4.27, %N 9.30.

2-((5-Chloro-2-methylphenyl)amino)-N-(2-methoxybenzyl)-6-(trifluoromethyl)nicotinamide (39). Yield 53%. Mp 148-150 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H, CH₃), 3.96 (s, 3H, CH₃), 4.63 (d, J = 5.1 Hz, 2H, CH2), 7.02–7.48 (m, 6H, aryl and pyridyl), 8.37 (d, 1H, J = 8.6 Hz, aryl), 8.55 (d, 1H, J = 7.7 Hz, pyridyl), 9.52 (s, 1H, NH), 11.00 (s, 1H, NH). IR (Nujol) 3315, 1639, 1617, 1599 cm⁻¹. m/z 450 (M + H)⁺. Anal. Calcd for C₂₁H₁₆ClF₃N₃O (449.85) %C 58.74, %H 4.26, %N 9.34. Found %C 58.79, %H 4.24, %N 9.37.

N-(4-chlorobenzyl)-2-((5-chloro-2-methylphenyl)amino)-6-(trifluoromethyl)nicotinamide (40). Yield 46%. Mp 130–131 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.28 (s, 3H, CH₃), 4.52 (d, J = 5.5 Hz, 2H, CH₂), 7.24–7.40 (m, 7H, aryl and pyridyl), 8.23 (d, 1H, J = 9.0 Hz, aryl), 8.40 (d, 1H, J = 7.5 Hz, pyridyl), 9.60 (t, J = 5.5 Hz, 1H, NH), 10.96 (s, 1H, NH). IR (Nujol) 3283, 1634, 1612, 1599 cm⁻¹. m/z 452 (M + H)⁺. Anal. Calcd for C₂₁H₁₆ClF₃N₃O (451.89) %C 55.52, %H 3.55, % N 9.25. Found %C 55.58, %H 3.54, % N 9.22.

2-((5-Chloro-2-methylphenyl)amino)-N-(4-fluorobenzyl)-6-(trifluoromethyl)nicotinamide (41). Yield 43%. Mp 184–185 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H, CH₃), 4.63 (d, J = 5.5 Hz, 2H, CH₂), 7.25–7.54 (m, 7H, aryl and pyridyl), 8.35 (d, 1H, J = 8.7 Hz, aryl), 8.51 (d, 1H, J = 7.5 Hz, pyridyl), 9.69 (s, 1H, NH), 10.97 (s, 1H, NH). IR (Nujol) 3276, 1635, 1613 cm⁻¹. m/z 438 (M + H)⁺. Anal. Calcd for C₂₁H₁₆ClF₄N₃O (437.82) %C 55.52, %H 3.55, %N 9.25. Found %C 55.57, %H 3.54, %N 9.23.

2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)-N-(4-(trifluoromethyl)benzyl)nicotinamide (42). Yield 62%. Mp 170–171 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.38 (s, 3H, CH₃), 4.74 (d, J = 5.5 Hz, 2H, CH₂), 7.37 (d, 1H, J = 8.8 Hz, aryl), 7.43 (s, 1H aryl), 7.84 (d, 1H, J = 7.8 Hz, pyridyl), 7.70 (d, 2H, J = 8.3 Hz, aryl), 7.82 (d, 2H, J = 8.3 Hz, aryl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.54 (d, 1H, J = 7.8 Hz, pyridyl), 9.78 (s, 1H, NH), 10.92 (s, 1H, NH). IR (Nujol) 3325, 1635, 1613, 1597 cm⁻¹. m/z 488 (M + H)⁺. Anal. Calcd for C₂₁H₁₆ClF₄N₃O (487.83) %C 54.17, %H 3.31, %N 8.61.

2-((5-Chloro-2-methylphenyl)amino)-N-(4-methoxybenzyl)-6-(trifluoromethyl)nicotinamide (43). Yield 39%. Mp 126-128 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H, CH₃), 3.84 (2, 3H, CH₃), 4.58 (d, J = 5.3 Hz, 2H, CH₂), 7.02 (d, 1H, J = 7.1 Hz, aryl), 7.43 (s, 1H aryl), 7.34–7.45 (m, 6H, aryl and pyridyl), 8.35 (d, 1H, J = 8.8 Hz, aryl), 8.48 (d, 1H, J = 7.8 Hz, pyridyl), 9.63 (s, 1H, NH), 11.00 (s, 1H, NH). IR (Nujol) 3292, 1634, 1612, 1598 cm⁻¹. m/z 450 (M + H)⁺. Anal. Calcd for C₂₂H₁₉ClF₃N₃O₂ (449.85) %C 58.74, %H 4.86, %N 9.34. Found %C 58.68, %H 4.88, %N 9.37.

2-((5-Chloro-2-methylphenyl)amino)-N-(4-methylbenzyl)-6-(trifluoromethyl)nicotinamide (44). Yield 95%. Mp 189–190 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.39 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 4.60 (d, J = 5.3 Hz, 2H, CH2), 7.25–7.47 (m, 6H, aryl and pyridyl), 9.66 (s, 1H, NH), 10.99 (s, 1H, NH). IR (Nujol) 3277, 1634, 1612, 1598 cm⁻¹. m/z 434 (M + H)⁺. Anal. Calcd for C₂₂H₁₉ClF₃N₃O (433.85) %C 60.90, %H 4.41, %N 9.69. Found %C 60.97, %H 4.43, %N 9.66.

2-((5-Chloro-2-methylphenyl)amino)-N-(2,4-dichlorobenzyl)-6-(trifluoromethyl)nicotinamide (45). Yield 65%. Mp 153–155 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.38 (s, 3H, CH₃), 4.69 (d, J = 5.5 Hz, 2H, CH₂), 7.36–7.57 (m, 5H, aryl and pyridyl), 7.76, (s, 1H, aryl), 8.34 (d, 1H, J = 8.7 Hz, aryl), 8.54 (d, 1H, J = 7.8 Hz, pyridyl), 9.69 (s, 1H, NH), 10.88 (s, 1H, NH). IR (Nujol) 3304, 1635, 1614, 1598 cm⁻¹. m/z 489 (M + H)⁺. Anal. Calcd for C₂₁H₁₅ClF₃N₃O (488.72) %C 51.91, %H 3.09, %N 8.60. Found %C 51.96, %H 3.09, %N 8.64.

2-((5-Chloro-2-methylphenyl)amino)-N-(3,4-dichlorobenzyl)-6-(trifluoromethyl)nicotinamide (46). Yield 79%. Mp 179–180 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.39 (s, 3H, CH₃), 4.65 (d, J = 5.5 Hz, 2H, CH₂), 7.27–7.82 (m, 6H, aryl and pyridyl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.53 (d, 1H, J = 7.3 Hz, pyridyl), 9.80 (s, 1H, NH), 10.92 (s, 1H, NH). IR (Nujol) 3283, 1644, 1615, 1597 cm⁻¹. m/z 489 (M + H)⁺. Anal. Calcd for C₂₁H₁₅ClF₃N₃O (488.72) %C 51.91, %H 3.09, %N 8.60. Found %C 51.85, %H 3.10, %N 8.63.

2-((5-Chloro-2-methylphenyl)amino)-N-(4-hydroxy-3-methoxybenzyl)-6-(trifluoromethyl)nicotinamide (47). Yield 95%. Mp 142–143 °C (2-propanol). ¹H NMR (DMSO-d₆): δ 2.33 (s, 3H, CH₃), 3.85 (s, 3H, CH₃), 4.53 (d, J = 5.4 Hz, 2H, CH₂), 6.83–7.50 (m, 6H, aryl and pyridyl), 8.34 (d, 1H, J = 8.8 Hz, aryl), 8.54 (d, 1H, J = 7.8 Hz, pyridyl), 9.04 (s, 1H, OH), 9.60 (s, 1H, NH), 11.01 (s, 1H, NH). IR (Nujol) 3305, 1645, 1616 cm⁻¹. m/z 466 (M + H)⁺. Anal. Calcd for C₂₂H₁₉Cl₃F3N₃O (465.85) %C 56.72, %H 4.11, %N 9.02. Found %C 56.67, %H 4.12, %N 9.06.

(2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)pyridin-3-yl)(4-phenylpiperazin-1-yl)methanone (48). Yield 81%. Mp 118–120 °C (n-hexane). ¹H NMR (DMSO-d₆): δ 2.25 (s, 3H, CH₃), 3.17–3.21 (m, 4H, CH₂), 3.36–3.41 (m, 4H, CH₂), 6.80–7.32 (m, 8H, aryl and pyridyl), 7.55 (d, 1H, J = 8.5 Hz, aryl), 7.84 (d, 1H, J = 7.0 Hz, pyridyl), 8.55 (s, 1H, NH). IR (Nujol) 3365, 1630, 1614, 1599 cm⁻¹. m/z 475 (M + H)⁺. Anal. Calcd for C₂₄H₂₂ClF₃N₄O (474.91) %C 60.70, %H 4.67, %N 11.80. Found %C 60.64, %H 4.69, %N 11.84.

(2-((5-Chloro-2-methylphenyl)amino)-6-(trifluoromethyl)pyridin-3-yl)(4-(3,4-dichlorophenyl)piperazin-1-yl)methanone (52). Yield 87%. Mp 165–167°C (MeCN). ¹H NMR (DMSO-d₆): δ 2.27 (s, 3H, CH₃), 3.41–3.45 (m, 4H, CH₂), 3.73–3.75 (m, 4H, CH₂), 7.05–7.55 (m, 6H, aryl and pyridyl), 7.65 (d, 1H, J = 8.4 Hz, aryl), 7.96 (d, 1H, J = 7.7 Hz, pyridyl), 8.65 (s, 1H, NH). IR (Nujol) 3323, 1638, 1609 cm⁻¹. m/z 544 (M + H)⁺. Anal. Calcd for C₂₄H₂₀Cl₃F₃N₄O (543.80) %C 53.01, %H 3.71, %N 10.30. Found %C 53.07, %H 3.70, %N 10.27.