Synthesis of Aniline Precursors

Aniline precursors were synthesized as described in figure 37.

Figure 37: Synthesis of Aniline Precursors.

7.2.8.1.1 3-Methyl-4-Nitrobenzoic acid chloride (1)

Oxalyl Chloride (22.6 mL of a 1 M solution in CH2Cl2) was added dropwise to a stirred solution of 3-methyl-4-nitrobenzoic acid (10.0 g, 55.2 mmol) in THF (330 mL) at RT. The reaction mixture was stirred at RT for 2 h. The reaction mixture was concentrated under reduced pressure. The resulting residue was distilled under high vacuum to give 1 as a yellow solid, which was used without further analysis. Yield 10.0 g, 91 %.

7.2.8.1.2 N-(2-Hydroxy-6-methylphenyl)-3-methyl-4-nitrobenzamine (2a)

111

2-Hydroxy-6-methyl aniline (R¹= CH3) (8.64 g in 50 mL THF, 23.7 mmol) was added dropwise to a stirred solution of 1 (7.0 g, 10.8 mmol) in THF (250 mL) at RT over 10 min. The reaction mixture was stirred overnight for 18 h and afterwards concentrated under reduced pressure.

The remaining residue was dissolved in ethyl acetate (250 mL) and was washed with 1 N aq.

HCl (3 x 75 mL), sat. aq. NaHCO3 (3 x 75 mL), sat. aq. NaCl (1 x 50 mL), dried (MgSO4) and concentrated under reduced pressure to give 2a as a red solid.

Yield: 9.51 g, 95 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 9.74 (s, 1H), 9.70 (s, 1H), 8.09 (d, J = 8.4, 2.0 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H), 7.98 (dd, J = 8.4, 2.0 Hz, 1H), 7.63 (dd, J = 8.1, 1.5 Hz 1H), 7.09-7.05 (m, 1H), 6.94 (dd, J = 8.1, 1.5 Hz, 1H), 6.84 (td, J = 7.6, 1.4 Hz), 2.58 (s, 3H).

13C NMR (75 MHz, d6-DMSO, 298 K) δ 163.7 (s), 153.3 (s), 150.4 (s), 138.4 (s), 136.8 (s), 132.7 (s), 132.0 (d), 127.4 (d), 126.5 (d), 124.4 (d), 123.4 (s), 120.4 (d), 113.6 (d), 19.4 (q), 17.9 (q).

HRMS (ESI+): Calcd for C15H14N2O2Na: 309.0846 [M+ Na]⁺ Found 309.0839.

7.2.8.1.3 N-(2-Hydroxyphenyl)-3-methyl-4-nitrobenzamine (2b)

2-Aminophenol (R¹= H) (0.995 g in 5 mL THF, 23.7 mmol) was added dropwise to a stirred solution of 1 (2.0 g, 10.8 mmol) in THF (25 mL) at RT over 10 min. The reaction mixture was stirred overnight for 18 h. The reaction mixture was concentrated under reduced pressure and the remaining residue was dissolved in ethyl acetate (250 mL) and was washed with 1N aq. HCl (3 x 75 mL), sat. aq. NaHCO3 (3 x 75 mL), sat. aq. NaCl (1 x 50 mL), dried (MgSO4) and reduced under reduced pressure to give 2b as a red solid.

Yield: 2.65 g, 90 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 9.74 (s, 1H), 9.70 (s, 1H), 8.09 (d, J = 8.4 Hz, 1H), 8.07 (d, J = 2.0 Hz, 1H), 7.98 (dd, J = 8.3, 2.0 Hz, 1H), 7.63 (dd, J = 8.0, 1.6 Hz, 1H), 7.09-7.05 (m, 1H), 6.94 (dd, J = 8.0, 1.4 Hz, 1H), 6.84 (td, J = 7.6, 1.4 Hz, 1H), 2.58 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 163.7 (s), 150.5 (s), 149.9 (s), 138.4 (s), 132.7 (s), 131.9 (d), 126.4 (d), 126.2 (d), 125.2 (s) 124.9 (d), 124.4 (d), 118.9 (d), 116.0 (d), 19.3 (q).

112

7.2.8.1.4 4-Methyl-2-(3-methyl-4-nitrophenyl benzoxazole (3a)

p-toluenesulfonic acid monohydrate (12.4 g, 65.3 mmol) was added in one portion to a stirred suspension of 2a (8.5 g, 29.7 mmol) in toluene (300 mL) at RT. The reaction mixture was heated at 110 °C for 3 h. The reaction mixture was cooled and washed with sat. aq.

NaHCO3 (3 x 50 mL), water (1 x 50 ml), sat. aq. NaCl (1 x 50 mL), dried (MgSO4) and concentrated under reduced pressure to give 3a as a red solid.

Yield: 7.85 g, 99 %

1H NMR (300 MHz, CDCl3, 298 K) δ 8.29 (d, J = 1.9 Hz 1H), 8.23 (dd, J = 8.5 Hz, 1.9 Hz, 1H), 8.12 (d, J = 8.5 Hz 1H), 7.44 (d, J = 8.5 Hz 1H), 7.31 (t, J = 7.8 Hz, 1H), 7.20 (dt, J = 7.4 Hz, 1H), 2.73 (s, 3H), 2.70 (s, 3H).

13C NMR (75 MHz, CDCl3, 298 K) δ 159.9 (s), 150.7 (s), 150.3 (s), 141.2 (s), 134.3 (s), 131.6 (d), 131.3 (s), 131.2 (s), 125.8 (d) 125.7 (d), 125.5 (d), 125.3 (d), 108.0 (s), 20.4 (q), 16.5 (q).

HRMS (ESI+): Calcd for C15H14N2O2Na: 309.0846 [M+ Na]⁺ Found 309.0839.

7.2.8.1.5 4-Methyl-2-(4-nitrophenyl benzoxazole (3b)

p-toluenesulfonic acid monohydrate (6.92 g, 36.4 mmol) was added in one portion to a stirred suspension of 2b (4.5 g, 16.5 mmol) in toluene (200 mL) at RT. The reaction mixture was heated at 110°C for 3 h. The reaction mixture was cooled and washed with sat. aq.

NaHCO3 (3 x 50 mL), water (1 x 50 ml), sat. aq. NaCl (1 x 50 mL), dried (MgSO4) and concentrated under reduced pressure to give 3b as a red solid.

Yield: 3.92 g, 93 %

1H NMR (300 MHz, CDCl3, 298 K) δ 8.28-8.26 (m, 1H), 8.21 (dd, J = 8.4 Hz, 2.1, 1H), 8.11 (d, J

= 8.4 Hz, 1H), 7.85-7.78 (m, 1H), 7.66-7.60 (m, 1H), 7.47-7.40 (m, 2H), 2.71 (s, 3H).

13C NMR (75 MHz, CDCl3, 298 K) δ 160.7 (s), 150.9 (s), 150.6 (s), 141.7 (s), 134.4 (s), 131.7 (d), 131.0 (s), 126.2 (d), 125.8 (d) 125.4 (d), 125.1 (d), 120.5 (d), 110.9 (d), 20.4 (q).

HRMS (ESI+): Calcd for C13H11N2O3: 255.0764 [M+ H]⁺ Found 255.0753.

113 7.2.8.2 Synthesis of CG3 05 A02 Analogues

The CG3 05 A02 Analogues were synthesized as described in figure 38.

Figure 38: Synthesis of CG3 05 A02 Analogues.

7.2.8.2.1 2-Methyl-4-(4-methylbenzoxazol-2-yl)benzamine (4a)

3a (6.05 g, 22.5 mmol) was suspended in ethanol (300 mL) and water (50 mL). Ammonium chloride (6.03 g, 113 mmol), followed by iron powder (6.30 g, 113 mmol) were added both on one portion at RT. The reaction mixture was heated to 80 °C for 2 h. The reaction mixture was cooled to RT and concentrated under reduced pressure. The remaining residue was dissolved in ethyl acetate (500 mL) and washed with sat. aq. NaHCO3 (2 x 50 mL), sat. aq.

NaCl (1 x 50 mL), dried (MgSO4) and again concentrated under reduced pressure. The residue was dry loaded onto silica gel and purified using column chromatography (SiO2) using a gradient elution 10% EtOAc in Cyclohexane  50 % EtOAc to give 4a as a beige solid.

Yield: 4.84 g, 90 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 7.79 (dd, J = 2.0 Hz 0.9 Hz, 1H), 7.76 (dd, J = 8.3 Hz, 2.1, 1H), 7.51-7.42 (m, 1H), 7.18 (t, J = 7.7 Hz 1H), 7.12 (dt, J = 2.0 Hz 0.9 Hz, 1H), 6.74 (d, J = 8.3 Hz, 1H), 5.70 (s, 2H), 2.54 (s, 3H), 2.16 (s, 3H).

114

13C NMR (75 MHz, d6-DMSO, 298 K) δ 162.9 (s), 150.5 (s), 149.5 (s), 141.2 (s), 129.3 (d), 128.6 (d), 126.5 (s), 124.7 (s), 123.6 (d), 120.9 (d),113.5 (d), 113.2 (s), 107.5 (s), 17.3 (q), 16.3 (q).

HRMS (ESI+): Calcd for C15H14N2ONa: 261.0998 [M+ Na]⁺ Found 261.0999.

7.2.8.2.2 4-(Benzoxazol-2-yl)-2-methylaniline (4b)

3b (3.42 g, 13.5 mmol) was suspended in ethanol (150 mL) and ammonium chloride (3.60 g, 67.3 mmol), followed by iron powder (3.76 g, 67.3 mmol) were added both on one portion at RT. The reaction mixture was heated to 80 °C for 2 h. The reaction mixture was cooled to RT and concentrated under reduced pressure. The remaining residue was dissolved in ethyl acetate (200 mL) and washed with sat. aq. NaHCO3 (2 x 20 mL), sat. aq. NaCl (1 x 20 mL), dried (MgSO4) and concentrated under reduced pressure. The residue was dry loaded onto silica gel and purified using column chromatography (SiO2) using a gradient elution 10 % EtOAc in Cyclohexane  50 % EtOAc to give 4b as a colorless solid.

Yield: 2.38 g, 79 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 7.80 (dd, J = 2.1, 0.9 Hz, 1H), 7.76 (dd, J = 8.3, 2.1 Hz, 1H), 7.68-7.63 (m, 2H), 7.33-7.27 (m, 2H), 6.75 (d, J = 8.3 Hz, 1H), 5.72 (s, 2H), 2.16 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 163.6 (s), 150.6 (s), 149.9 (s), 142.1 (s), 129.3 (d), 126.6 (d), 124.2 (d), 123.9 (d), 120.9 (s), 118.6 (d), 113.5 (d), 113.0 (s), 110.2 (d), 17.3 (q).

HRMS (ESI+): Calcd for C14H13N2O: 225.1022 [M+ H]⁺ Found 225.1023.

7.2.8.2.3 4-(4-Methylbenzoxazol-2-yl)benzamine (4c)

Polyphosphoric Acid (50 mL) was added to a round-bottomed flask and heated to 120 °C. 2-hydroxy-6-methylaniline (5.02 g, 40.8 mmol) was added, followed by 4-Aminobenzoic acid (5.59 g, 40.8 mmol) with mechanical stirring. After dissolution of the solids, the reaction mixture was heated to 220 °C for 4 h. The reaction mixture was cooled and neutralized carefully with cooled sodium hydroxide (2 M) and the resulting precipitate was filtered and dried overnight under high vacuum (P2O5). The residue was dry loaded onto silica gel and purified using column chromatography (SiO2) using a gradient elution 10 % EtOAc in Cyclohexane  50 % EtOAc to give 4c as a colorless solid.

115 Yield: 5.48 g, 60 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 7.89-7.85 (m, 2H), 7.45 (dt, J = 8.0 0.9 Hz, 1H),7.18 (t, J = 7.7 Hz 1H), 7.12 (dt, J = 7.4, 0.9 Hz, 1H), 6.72-6.69 (m, 2H), 5.94 (s, 2H), 2.53 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 162.8 (s), 152.3 (s), 149.5 (s), 142.2 (s), 128.8 (d), 128.6 (s), 124.7 (d) 123.6 (d), 113.5 (d), 113.0 (s), 107.5 (d), 16.2 (q).

HRMS (ESI+): Calcd for C14H13N2O: 225.1022 [M+ H]⁺ Found 225.1027.

7.2.8.2.4 4-(Benzoxazol-2-yl)benzamine (4d)

Polyphosphoric Acid (160 mL) was added to a round-bottomed flask and heated to 140°C.

Aminophenol (11.55 g, 106 mmol) was added, followed by 4-Aminobenzoic acid (14.52 g, 106 mmol) with mechanical stirring. After dissolution of the solids, the reaction mixture was heated to 220°C for 4 hours. The reaction mixture was cooled and neutralized carefully with cooled sodium hydroxide (2M) and the resulting precipitate was filtered and dried overnight under high vacuum (P2O5). The residue was dry loaded onto silica gel and purified using column chromatography (SiO2) using a gradient elution 10% EtOAc in Cyclohexane  50%

EtOAc to give 4d as a colorless solid.

Yield: 11.8 g, 53 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 7.90-7.85 (m, 2H), 7.66-7.64 (m, 2H),7.34-7.27 (m, 2H), 6.74-6.68 (m, 2H), 5.98 (s, 2H).

13C NMR (75 MHz, d6-DMSO, 298 K) δ 163.6 (s), 152.5 (s), 149.8 (s), 142.1 (s), 128.9 (d), 124.3 (d) 124.0 (d), 118.7 (d), 113.5 (d), 112.7 (s), 110.2 (d).

HRMS (ESI+): Calcd for C13H11N2O: 211.0866 [M+ H]⁺ Found 211.0883.

7.2.8.2.5 2-Methoxy-N-(2-methyl-4-(4-methylbenzol-2-yl)phenylbenzamine (5a)

4a (0.50 g, 2.10 mmol) was dissolved in acetonitrile (25 mL), DIPEA (914 µL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (423 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5a as a colorless solid.

116 Yield: 0.578 g, 74 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 10.12 (s, 1H), 8.40 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.06 (dd, J = 8.5, 2.0 Hz, 1H), 8.00 (d, J = 7.8, 1.8 Hz, 1H), 7.63-7.53 (m, 2H), 7.33-7.25 (m, 2H), 4.05 (s, 3H), 2.59 (s, 3H), 2.47 (s, 3H).

13C NMR (75 MHz, d6-DMSO, 298 K) δ 163.1 (s), 161.5 (s), 157.0 (s), 149.8 (s), 140.8 (s), 140.0 (s), 133.4 (d), 131.1 (d), 129.6 (s), 129.1 (d), 128.9 (s), 125.7 (d), 125.1 (d), 124.9 (d), 121.9 (s), 121.7 (d), 121.0 (d), 112.4 (d), 108.0 (d), 56.4 (q), 17.6 (q), 16.3 (q).

LCMS (ESI) 373.34 [M+H]⁺ Rt = 15.6 mins (99 %)

HRMS (ESI+): Calcd for C23H20N2O3Na: 395.1366 [M+ Na]⁺. Found 395.1366.

7.2.8.2.6 N-(2-Methyl-4-(4-methylbenzoxazol-2-yl)phenylbenzamine (5b)

4b (0.688 g, 2.50 mmol) was dissolved in acetonitrile (50 mL), DIPEA (1.39 mL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (423 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.736 g, 86 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 10.02 (s, 1H), 8.23-8.16 (m, 2H), 8.10-8.04 (m, 2H), 8.02-7.96 (m, 2H), 7.80-7.73 (m, 2H), 7.65-7.52 (m, 3H), 7.43-7.36 (m, 2H).

13C NMR (75 MHz, d6-DMSO, 298 K) δ 165.4 (s), 161.4 (s), 149.9 (s), 140.8 (s), 139.8 (s), 134.3 (s), 133.8 (s), 131.7 (d), 129.7 (s), 129.1 (d), 128.4 (d), 127.7 (d), 126.4 (d), 125.13 (d), 125.09 (d), 125.0 (d), 123.5 (s), 108.1 (d), 17.9 (q), 16.2 (q).

HRMS (ESI+): Calcd for C22H19N2O2: 343.1441 [M+H]⁺. Found 343.1438.

7.2.8.2.7 N-(4-(Benzoxazol-2-yl)-2-methylphenyl)-2-methoxybenzamine (5c)

117

4b (0.688 g, 2.50 mmol) was dissolved in acetonitrile (20 mL), DIPEA (676 µL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (313 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.510 g, 92 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 10.12 (s, 1H), 8.40 (d, J = 8.5 Hz, 1H), 8.12 (dd, J = 2.0, 0.9 Hz, 1H), 8.07 (dd, J = 8.5, 2.0 Hz, 1H), 8.01 (dd, J = 7.5, 2.0 Hz, 1H), 7.80-7.74 (m, 2H), 7.59 (ddd, J = 8.5, 7.5, 2.0 Hz, 1H), 7.44-7.37 (m, 2H), 7.28 (d, J = 8.5 Hz, 1H), 7.15 (td, J = 7.5 Hz, 1H), 4.05 (s, 3H), 2.47 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 163.1 (s), 162.2 (s), 157.0 (s), 150.1 (s), 141.6 (s), 140.1 (s), 133.4 (d), 131.0 (d), 129.1 (d), 128.9 (s), 125.7 (d), 125.1 (d), 124.7 (d), 121.9 (s), 121.7 (d), 121.5 (s), 121.0 (d), 119.5 (d), 112.4 (d), 110.7 (d), 56.4 (q), 17.5 (q).

LCMS (ESI) 359.2 [M+H]⁺ Rt = 14.4 min

HRMS (ESI+): Calcd for C23H19N2O: 359.1390 [M+H]⁺. Found 359.1394.

7.2.8.2.8 N-(4-(Benzoxazol-2-yl)-2-methylphenyl)benzamine (5d)

4b (0.50 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (388 µL, 5.13 mmol) was added in one portion and then benzoyl chloride (423 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.636 g, 87 %

1H NMR (500 MHz, d6-DMSO, 298 K) δ 10.03 (s, 1H), 8.15-8.13 (m, 1H), 8.07 (dd, J = 8.3, 2.1 Hz, 1H), 8.03-8.00 (m, 2H), 7.82-7.77 (m, 2H), 7.72 (d, J = 8.3 Hz, 1H), 7.64-7.60 (m, 1H), 7.56 (dd, J = 8.3, 6.7 Hz, 2H), 7.45-7.39 (m, 2H), 2.40 (s, 3H).

13C NMR (125 MHz, d6-DMSO, 298 K) δ 165.4 (s), 162.1 (s), 150.2 (s), 141.6 (s), 140.0 (s), 134.3 (s), 133.9 (s), 131.8 (d), 129.2 (d), 128.4 (d), 127.7 (d), 126.4 (d), 125.3 (d), 125.2 (d), 124.8 (d), 123.4 (s), 119.7 (d), 110.8 (d), 17.9 (q).

LCMS (ESI) 329.23 [M+H]⁺ Rt = 13.5 mins (90 %)

HRMS (ESI+): Calcd for C21H16N2O2Na: 351.1104 [M+ Na]⁺. Found 351.1115.

118

7.2.8.2.9 2-Methoxy-N-(4-(4-methylbenzoxazol-2-yl)phenylbenzamine (5e)

4b (0.5 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (964 µL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (450 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.631 g, 79 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 10.45 (s, 1H), 8.18 (d, J = 8.6 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.64 (dd, J = 7.5, 1.8 Hz, 1H), 7.58-7.55 (m, 1H), 7.53 (ddd, J = 8.6, 7.4, 1.8 Hz 1H), 7.30 (t, J = 7.4 Hz 1H), 7.22-7.19 (m, 2H), 7.09 (td, J = 7.4, 0.9 Hz, 1H), 3.91 (s, 3H), 2.59 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 165.0 (s), 161.5 (s), 156.5 (s), 149.8 (s), 142.1 (s), 140.8 (s), 132.2 (d), 129.6 (d), 128.0 (d), 125.1 (d), 124.84 (d), 124.80 (s), 121.2 (s), 120.5 (d), 119.7 (d), 112.0 (d), 55.9 (q), 16.2 (q).

LCMS (ESI) 359.18 [M+H]⁺ Rt = 14.7 min

HRMS (ESI+): Calcd for C22H19N2O2: 359.1390 [M+ H]⁺. Found 359.1383.

7.2.8.2.10 N-(4-(4-Methylbenzoxazol-2-yl)phenylbenzamine (5f)

4b (0.5 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (964 µL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (388 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.658g, 90 %

1H NMR (500 MHz, d6-DMSO, 298 K) δ 10.58 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 8.06 (d, J = 8.4 Hz, 2H), 8.02-7.96 (m, 2H), 7.65-7.59 (m, 1H), 7.58-7.53 (m, 3H), 7.28 (t, J = 7.8 Hz 1H), 7.19 (d, J = 7.4 Hz, 1H), 2.58 (s, 3H).

119

13C NMR (125 MHz, d6-DMSO, 298 K) δ 165.9 (s), 161.5 (s), 149.8 (s), 142.4 (s), 140.8 (s), 134.6 (s), 131.8 (d), 129.6 (s), 128.4 (d), 127.9 (d), 127.8 (d), 125.1 (d), 124.8 (d), 121.4 (s), 120.3 (d), 108.0 (d), 16.2 (q).

LCMS (ESI) 329.18 [M+H]⁺ Rt = 13.7 min (99 %)

HRMS (ESI+): Calcd for C21H16N2O2Na: 351.1104 [M+ Na]⁺. Found 351.1105.

7.2.8.2.11 N-(4-(benzoxazol-2-yl)phenyl)-2-methoxybenzamine (5g)

4b (0.65 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (1.34 mL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (623 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 90 min and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.883 g, 83 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 10.48 (s, 1H), 8.19 (d, J = 8.8 Hz, 2H), 7.99 (d, J = 8.8 Hz 2H), 7.81-7.73 (m, 2H), 7.65 (dd, J = 7.6, 1.8 Hz, 1H), 7.53 (ddd, J = 9.0, 7.4, 1.8 Hz, 1H), 7.43-7.37 (m, 2H), 7.20 (d, J = 8.4 Hz, 1H), 7.09 (td, J = 7.4, 1.0 Hz, 1H), 3.91 (s, 3H).

13C NMR (75 MHz, d6-DMSO, 298 K) δ 165.0 (s), 162.2 (s), 156.5 (s), 150.1 (s), 142.3 (s), 141.6 (s), 132.2 (d), 129.6 (d), 128.2 (d), 125.2 (d), 124.8 (d), 124.7 (d), 121.1 (s), 120.5 (d), 119.7 (d), 119.5 (d), 112.0 (d), 110.7 (d), 55.9 (q).

HRMS (ESI+): Calcd for C21H16N2O3Na: 367.1053 [M+ Na]⁺. Found 367.1057.

7.2.8.2.12 N-(4-(Benzoxazol-2-yl)phenyl)benzamine (5h)

4b (0.7 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (1.45 mL, 5.13 mmol) was added in one portion and then benzoyl chloride (580 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

120 Yield: 0.836 g, 80 %

1H NMR (300 MHz, d6-DMSO, 298 K) δ 10.59 (s, 1H), 8.23-8.16 (m, 2H), 8.10-8.04 (m, 2H), 8.02-7.96 (m, 2H), 7.80-7.73 (m, 2H), 7.65-7.52 (m, 3H), 7.43-7.36 (m, 2H).

13C NMR (75 MHz, d6-DMSO, 298 K) δ 165.9 (s), 162.2 (s), 150.1 (s), 142.5 (s), 141.6 (s), 134.6 (s), 131.8 (d), 128.4 (d), 128.0 (d), 127.8 (d), 125.1 (d), 124.7 (d), 121.2 (s), 120.3 (d), 119.5 (d), 110.7 (d).

LCMS (ESI) 315.10 [M+H]⁺ Rt = 13.6 min (99 %).

HRMS (ESI+): Calcd for C20H14N2O2Na: 337.0947 [M+ Na]⁺. Found 337.0959 7.2.8.3 Synthesis of the Control Compounds

The control compounds were synthesized as described in figure 39.

Figure 39: Synthesis of the control compounds.

7.2.8.3.1 4-Methoxy-N-(2-methyl-4-(4-methylbenzol-2-yl)phenylbenzamine (5i)

4b (0.50 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (914 µL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (423 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

121 Yield: 0.620 g, 79 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 9.84 (s, 1H), 8.11 (dd, J = 2.1, 0.9 Hz, 1H), 8.05 (dd, J = 8.3, 2.1 Hz 1H), 8.00 (d, J = 8.8 Hz, 2H), 7.70 (m, J = 8.3 Hz, 1H), 7.58-7.56 (m, 1H), 7.30 (t, J = 7.8 Hz, 1H), 7.22-7.20 (m, 1H), 7.08 (d, J = 8.8 Hz, 2H), 3.85 (s, 3H), 2.60 (s, 3H), 2.39 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 164.8 (s), 162.0 (s), 161.4 (s), 149.9 (s), 140.8 (s), 140.0 (s), 133.7 (s), 129.68 (s), 129.64 (d), 129.1 (d), 126.35 (s), 126.30 (d), 125.11 (d), 125.04 (d), 124.96 (d), 123.3 (s), 113.6 (d), 108.0 (d), 55.4 (q), 17.9 (q), 16.2 (q).

LCMS (ESI) 373.22 [M+H]⁺ Rt = 13.8 min (98 %)

HRMS (ESI+): Calcd for C23H21N2O3: 373.1547 [M+H]⁺. Found 373.1545.

7.2.8.3.2 3-Methoxy-N-(2-methyl-4-(4-methylbenzol-2-yl)phenylbenzamine (5j)

4b (0.688 g, 2.50 mmol) was dissolved in acetonitrile (25 mL), DIPEA (914 µL, 5.13 mmol) was added in one portion and then 2-methoxybenzoyl chloride (423 µL in 5 mL acetonitrile, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 1 h and the resulting precipitate was filtered and washed with cold acetonitrile to give 5b as a colorless solid.

Yield: 0.566 g, 72 %

1H NMR (600 MHz, d6-DMSO, 298 K) δ 9.98 (s, 1H), 8.12 (d, J = 2.0 Hz, 1H), 8.05 (dd, J = 8.3, 2.0 Hz 1H), 7.68 (d, J = 8.3 Hz, 1H), 7.59 (dt, J = 7.8, 1.0 Hz, 1H), 7.56 (d, J = 8.3 Hz, 1H), 7.55-7.53 (m, 1H), 7.46 (t, J = 7.8 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 7.22-7.19 (m, 1H), 7.18 (ddd, J = 8.3, 2.7, 1.0 Hz, 1H), 3.84 (s, 3H), 2.59 (s, 3H), 2.39 (s, 3H).

13C NMR (200 MHz, d6-DMSO, 298 K) δ 165.1 (s), 161.4 (s), 159.2 (s), 149.9 (s), 140.8 (s), 139.7 (s), 135.7 (s), 134.0 (s), 129.7 (s), 129.7 (d), 129.6 (d), 129.1 (d), 126.5 (d), 125.12 (d), 125.07 (d), 124.99 (d), 123.6 (s), 119.9 (d), 117.5 (d), 112.9 (d), 108.0 (d), 55.3 (q), 17.9 (q), 16.2 (q).

LCMS (ESI) 373.22 [M+H]⁺ Rt = 13.9 min (95 %)

HRMS (ESI+): Calcd for C23H21N2O3: 373.1547 [M+H]⁺. Found 373.1551.

122

7.2.8.3.3 N-(2-Methyl-4-(4-methylbenzoxazol-2-yl)phenyl)acetamide (6)

4b (0.25 g, 2.50 mmol) was dissolved in acetonitrile (15 mL), DIPEA (817 µL, 5.13 mmol) was added in one portion and then acetyl chloride (224 µL, 3.15 mmol) was added dropwise over 10 min with stirring at 0 °C. The reaction mixture was stirred 3 h and reaction mixture. The residue purified using column chromatography (SiO2) using a gradient elution 10 % EtOAc in Cyclohexane  75 % EtOAc to give 6 as a colorless solid.

Yield: 83 mg, 31 %

1H NMR (400 MHz, d6-DMSO, 298 K) δ 9.41 (s, 1H), 8.03 (d, J = 2.1 Hz 1H), 7.98 (dd, J = 8.5, 2.1 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.54 (d, J = 8.1 Hz, 1H), 7.28 (t, J = 7.8 Hz, 1H), 7.19 (dt, J = 7.5, 1.0 Hz, 1H), 2.57 (s, 3H), 2.35 (s, 3H), 2.13 (s, 3H).

13C NMR (100 MHz, d6-DMSO, 298 K) δ 166.6 (s), 161.4 (s), 149.8 (s), 140.8 (s), 139.9 (s), 130.9 (s), 129.6 (s), 129.1 (d), 125.09 (d), 125.08 (d), 124.9 (d), 124.1 (d), 108.0 (d), 23.6 (q), 17.8 (q), 16.2 (q).

HRMS (ESI+): Calcd for C17H16N2O2Na: 303.1104 [M+ Na]⁺. Found 303.1106.

7.2.8.3.4 2-Methoxy-N-methyl-N-(2-methyl-4-(4-methylbenzol-2-yl)phenylbenzamine (7)

5a (0.438 mg, 0.934 mmol) was dissolved in dry THF (15 mL) under argon at room temperature. Subsequently, the reaction mixture was cooled to 0 °C and potassium t-butoxide (934 µL of a 1M solution in ^tBuOH, 0.934 mmol) was added dropwise over 10 min.

The reaction mixture was maintained at 0 °C for further 30 min, after which time methyl iodide (64 µL, 1.03 mmol) was added in one portion. The reaction mixture was allowed to warm to RT and stirred overnight for 16 h. The reaction mixture was quenched with water and concentrated under reduced pressure. The resulting residue was dissolved in ethyl acetate (100 mL) and washed with water (2 x 20 mL) and sat. aq. NaCl (1 x 10 mL), dried (MgSO4) and concentrated under reduced pressure. The residue was purified using column chromatography (SiO2) using a gradient elution 20 % EtOAc in cyclohexane  50 % EtOAc to give 7 as a colorless solid.

123 Yield: 280 mg, 78 %

1H NMR (2.5:1 rotamer ratio, asterisks denote minor rotamer peaks, 600 MHz, d6-DMSO, 298 K) δ 8.17* (d, J = 2.1 Hz, 1H), 8.11* (d, J = 8.1, 2.1 Hz, 1H), 7.95 (d, J = 2.1 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.60-7.57* (m, 1H), 7.51 (dt, J = 8.1, 0.9 Hz, 2 x 1H), 7.47* (ddd, J = 8.3, 7.4, 1.7 Hz, 1H), 7.37* (d, J = 7.3 Hz, 1H), 7.32* (t, J = 7.8 Hz, 1H), 7.30-7.26 (m, 2 x 1H), 7.28 (t, J = 7.8 Hz, 2 x 1H), 7.24-7.21 (m, 1H), 7.19 (dt, J = 7.5, 1.0 Hz, 1H), 7.17-7.15* (m, 1H), 7.15* (dd, J = 1.7, 0.9 Hz, 1H), 7.14* (d, J = 1.7 Hz, 1H), 7.08* (td, J = 7.4, 0.9 Hz, 1H), 6.80 (td, J = 7.4, 0.9 Hz, 1H), 6.78-6.75 (m, 1H), 3.91* (s, 3H), 3.64 (s, 3H), 3.27 (s, 3H), 3.06* (s, 3H), 2.60* (s, 3H), 2.54 (s, 3H), 2.39* (s, 3H), 2.35 (s, 3H).

13C NMR (asterisks denote minor rotamer peaks, 200 MHz, d6-DMSO, 298 K) δ 167.7 (s), 161.1* (s), 160.8 (s), 155.0 (s), 154.6* (s), 150.0* (s), 149.9 (s), 145.2* (s), 144.9 (s), 140.7*

(s), 140.6 (s), 136.8* (s), 136.3 (s), 130.7* (d), 130.3 (d), 129.9* (s), 129.8 (s), 129.5 (d), 129.4* (d), 129.2 (d), 128.4 (d), 125.4 (s), 125.3 (s), 125.21 (d), 125.19 (d), 125.16 (d), 124.7 (d), 120.8* (d), 119.7 (d), 111.5* (d), 110.8 (d), 108.1* (d), 108.0 (d), 55.7* (q), 54.9 (q), 38.4*

(q), 35.6 (q), 17.3 (q), 17.2* (q), 16.2 (q), 16.1* (q).

LCMS (ESI) 387.27 [M+H]⁺ Rt = 13.7 min (99 %) 7.2.9 Handling of compounds

The small molecules used during the screening as well as the re-synthesized hits and derivatives were dissolved in anhydrous DMSO. The ComGenex library compounds were stored as 10 mM stock solutions as well as ready-to-use 2 mM solutions in 96-well plates at – 20 °C. Solid compound stock powders of in-house synthesized compounds were stored at 4

°C in the dark. 10 mM stock solutions of the in-house synthesized compounds were stored at room temperature in the dark. The compounds were synthesized by Dr. Jeffrey Hannam from the Famulok lab.

Im Dokument The role of guanine nucleotide exchange factors (GEFs) in EGF-receptor signalling (Seite 110-123)