Design, synthesis, and biological study of 4-[(2-nitroimidazole-1H-alkyloxyl)aniline]-quinazolines as EGFR inhibitors exerting cytotoxicities both under normoxia and hypoxia

Introduction

EGFR is a trans-membrane protein that belongs to the HER family of receptor tyrosine kinases,¹ its overexpression has been confirmed in many solid carcinomas, which include non-small-cell lung carcinoma,² colon carcinoma,³ ovarian carcinoma,⁴ head and neck carcinoma,⁵ and so on. On the other hand, hypoxia is unavoidable in solid carcinomas, it makes carcinomas resistant to radiotherapy and chemotherapy.⁶ In particular, EGFR is massively overexpressed when carcinomas are under hypoxia.^7,8 The dual functions of EGFR overexpression and tumor hypoxia exhibit a great challenge to carcinoma treatment. In other words, if a drug can inhibit EGFR, and also be activated under carcinoma hypoxia, it may exert a stronger effect. Recently, many small molecule EGFR inhibitors have been successfully developed and introduced onto the market, these drugs include gefitinib (1),⁹ erlotinib (2),¹⁰ lapatinib (3),¹¹ osimertinib (4),¹² etc (Figure 1). In addition, reagents targeting carcinoma-hypoxia have been massively explored, including the 2-nitroimidazole derivative pimonidazole (5),¹³ nimorazole (6),¹⁴ and so on (Figure 1). Moreover, compounds that have been combined with hypoxia-activated groups and other functional moieties have been widely reported.^15,16,17 For example, the hypoxia-activated prodrug TH-302 (7, Figure 1) is 270-fold more effective in carcinoma hypoxia than in normoxia;¹⁸ the hypoxia-activated EGFR inhibitor tarloxotinib (8, Figure 1) has been advanced to a phase II clinical study due to its excellent potency.¹⁹

Figure 1 Structures/depicted binding models of EGFR inhibitors (1–4, the depicted binding models of inhibitors with EGFR are shown in different colors), structures of 2-nitroimidazole derivatives (5 and 6) and clinically studied hypoxia-activated compounds (7 and 8).

EGFR inhibitors compete with ATP to bind to kinase, blocking signal transduction and playing an inhibitory role.²⁰ Three main interaction regions, the solvent region, the hinge region, and the backpocket region, are involved in the inhibitor-EGFR kinase bindings (Figure 1).²¹ In carcinoma hypoxic environment, 2-nitroimidazole can be reduced to hydroxylamine intermediates through four-electron reduction. The intermediates are further reduced to free radicals under hypoxia, and covalently bind to nucleophilic proteins, amino acids, and other components to target tumors. In normoxia, the presence of oxygen prevents the 2-nitroimidazole group from being reduced and activated, and it exists in the prototype state, thus ensuring its safety in normal tissue (Figure 2).²²

Figure 2 Reductive activation mechanism of 2-nitroimidazole in carcinoma hypoxic environment.

In order to get novel EGFR inhibitors that exert more potency in tumor hypoxia than in normoxia, we designed a series of compounds by combining 2-nitroimidazole group and 4-anilinoquinazolines (Figure 3). It has been confirmed that the aniline group of 4-anilinoquinazoline was positioned in the backpocket region of EGFR kinase, and this space was enough to accommodate bulky moieties,²³ so we incorporated 2-nitroimidazole group into the aniline position herein (Figure 3). Meanwhile, different kinds of side chains at C-7 and C-8 of the quinazolines were chosen, referencing the structures of gefitinib and erlotinib. Through investigation of the aniline substituents and the styles of the side chain, the structure-activity relationships (SARs) of these compounds were studied.

Figure 3 Our design strategy for EGFR inhibitors.

Methods and materials

Chemistry

Melting points were determined by a B-540 Büchi melting-point apparatus, nuclear magnetic resonance (NMR) spectra were recorded on BRUKER AVIII 500 MHz or BRUKER AVII 400 MHz spectrometer (500 or 400 MHz for ¹H NMR, 100 MHz for ¹³C NMR). Mass spectra were obtained on the Finnigan LCQ DecaXP ion trap mass spectrometer.

Procedure for the synthesis of substituted nitrobenzene (10a-d)

Dihalide alkane (30 mmol) was added to a stirred solution of substituted phenol (9a-c) (10 mmol) and K₂CO₃ (5 mmol) in DMF (10 mL). The reaction mixture was heated to 60°C and stirred overnight. After the reaction was completed, the mixture was cooled to room temperature (rt) and filtered, the filtrate was removed in vacuo. Finally, silica gel column chromatography was used to purify the residue, and pure 10a-d were obtained.

1-(2-Bromoethoxy)-2-chloro-4-nitrobenzene (10a): white solid. Yield: 64%, mp: 56°C–58°C. ¹H NMR (500 MHz, CDCl₃) δ 8.33–8.30 (m, 1H), 8.16 (dd, J =9.0, 2.5 Hz, 1H), 6.99 (d, J =9.0 Hz, 1H), 4.45 (t, J =6.5 Hz, 2H), 3.73 (t, J =6.5 Hz, 2H). ESI-MS m/z: 280 [M+1]⁺.

1-(3-Bromopropoxy)-2-chloro-4-nitrobenzene (10b): white solid. Yield: 71%, mp: 52°C–54°C. ¹H NMR (500 MHz, CDCl₃) δ 8.30 (d, J =2.5 Hz, 1H), 8.17 (dd, J =9.0, 2.5 Hz, 1H), 7.02 (d, J =9.0 Hz, 1H), 4.30 (t, J =5.5 Hz, 2H), 3.67 (t, J =6.0 Hz, 2H), 2.46–2.39 (m, 2H). ESI-MS m/z: 296 [M+1]⁺.

1-(2-Chloroethoxy)-3-nitrobenzene (10c): white solid. Yield: 59%, mp: 57°C–59°C (lit. 59°C–60°C).²⁴

1-(2-Bromoethoxy)-4-nitrobenzene (10d): white solid. Yield: 51%, mp: 59°C–61°C (lit. 62°C–64°C).²⁵

Procedure for the synthesis of substituted aniline (11a-d)

To a solution of 10a-d (10 mmol) in methanol (20 mL), and 10% Pd/C was added. Then 10a-d were reduced by hydrogen at atmospheric pressure for about 2 hours and the catalyst was removed by filtration. The filtrate was evaporated to afford 11a-d in the yields >93%.

4-(2-Bromoethoxy)-3-chloroaniline (11a): brown solid. Yield: 95%, mp: 68°C–70°C. ¹H NMR (500 MHz, CDCl₃) δ 6.83 (d, J =8.5 Hz, 1H), 6.73 (d, J =3.0 Hz, 1H), 6.52 (dd, J =8.5, 3.0 Hz, 1H), 4.24 (t, J =6.5 Hz, 2H), 3.62 (t, J =6.5 Hz, 2H), 3.53 (br, 2H). ESI-MS m/z: 252 [M+1]⁺.

4-(3-Bromopropoxy)-3-chloroaniline (11b): brown solid. Yield: 93%, mp: 62°C–64°C. ¹H NMR (500 MHz, CDCl₃) δ 6.80 (d, J =8.5 Hz, 1H), 6.74 (d, J =3.0 Hz, 1H), 6.54 (dd, J =8.5, 3.0 Hz, 1H), 4.07 (t, J =5.5 Hz, 2H), 3.65 (t, J =6.5 Hz, 2H), 3.25 (br, 2H), 2.34–2.28 (m, 2H). ESI-MS m/z: 266 [M+1]⁺.

3-(2-Chloroethoxy)-aniline (11c): colorless oil. Yield: 96%. ¹H NMR (500 MHz, CDCl₃) δ 7.07 (t, J =8.0 Hz, 1H), 6.38–6.32 (m, 2H), 6.30 (t, J =2.5 Hz, 1H), 4.19 (t, J =6.0 Hz, 2H), 3.79 (t, J =6.0 Hz, 2H), 3.58 (br, 2H). ESI-MS m/z: 172 [M+1]⁺.

4-(2-Bromoethoxy)-aniline (11d): white solid. Yield: 100%, mp: 89°C–91°C. ¹H NMR (500 MHz, CDCl₃) δ 6.79–6.75 (m, 2H), 6.67–6.61 (m, 2H), 4.21 (t, J =6.5 Hz, 2H), 3.59 (t, J =6.5 Hz, 2H), 3.44 (br, 2H). ESI-MS m/z: 216 [M+1]⁺.

Procedure for the synthesis of 2-nitroimidazole derivative (12a-d)

To a stirred solution of substituted aniline (11a-d) (10 mmol) and powdered K₂CO₃ (5 mmol) in DMF (10 mL), 2-nitroimidazole (1.36g, 12 mmol) was added. The reaction mixture was heated to 70°C and stirred for about 10 hours. After the reaction was completed, the mixture was cooled to rt and filtered, the filtrate was removed in vacuo. Then methylene chloride was used to dissolve the obtained residue, which was further washed with water. Finally, the organic solvent was dried over sodium sulfate and concentrated to obtain crude 12a-d, which were further purified by column chromatography to get pure materials.

3-Chloro-4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)aniline (12a): yellow solid. Yield: 55%, mp: 127°C–129°C. ¹H NMR (500 MHz, CDCl₃) δ 7.38 (d, J =1.0 Hz, 1H), 7.17 (d, J =1.0 Hz, 1H), 6.70 (d, J =3.0 Hz, 1H), 6.67 (d, J =8.5 Hz, 1H), 6.50 (dd, J =8.5, 3.0 Hz, 1H), 4.85–4.80 (m, 2H), 4.31–4.26 (m, 2H), 2.75 (br, 2H). ESI-MS m/z: 283 [M+1]⁺.

3-Chloro-4-(3-(2-nitro-1H-imidazol-1-yl)propoxy)aniline (12b): yellow solid. Yield: 53%, mp: 136°C–138°C. ¹H NMR (500 MHz, CDCl₃) δ 7.17 (s, 1H), 7.11 (s, 1H), 6.76 (d, J =2.5 Hz, 1H), 6.72 (d, J =8.5 Hz, 1H), 6.53 (dd, J =8.5, 3.0 Hz, 1H), 4.71 (t, J =6.5 Hz, 2H), 3.89 (t, J =5.5 Hz, 2H), 3.53 (br, 2H), 2.36–2.30 (m, 2H). ESI-MS m/z: 297 [M+1]⁺.

3-(2-(2-Nitro-1H-imidazol-1-yl)ethoxy)aniline (12c): brown solid. Yield: 58%, mp: 51°C–53°C. ¹H NMR (500 MHz, CDCl₃) δ 7.23 (d, J =0.5 Hz, 1H), 7.13 (d, J =1.0 Hz, 1H), 7.02 (t, J =8.0 Hz, 1H), 6.30 (dd, J =8.0, 2.0 Hz, 1H), 6.22 (dd, J =8.0, 2.5 Hz, 1H), 6.15 (t, J =2.0 Hz, 1H), 4.77 (dd, J =12.5, 8.0 Hz, 2H), 4.27 (dd, J =10.5, 5.5 Hz, 2H), 4.04–3.10 (br, 2H). ESI-MS m/z: 249 [M+1]⁺.

4-(2-(2-Nitro-1H-imidazol-1-yl)ethoxy)aniline (12d): brown solid. Yield: 48%, mp: 48°C–50°C. ¹H NMR (500 MHz, CDCl₃) δ 7.24 (d, J =1.0 Hz, 1H), 7.14 (t, J =3.0 Hz, 1H), 6.69–6.62 (m, 2H), 6.62–6.57 (m, 2H), 4.77 (dd, J =14.5, 10.0 Hz, 2H), 4.25 (dd, J =13.5, 8.5 Hz, 2H), 3.47 (br, 2H). ESI-MS m/z: 249 [M+1]⁺.

Procedure for the synthesis of 4-(3-((4-chloro-6-methoxyquinazolin-7-yl)oxy)propyl)morpholine (13)

Compound 13 was prepared following the published synthesis route.²⁶ Briefly, commercially available 6-methoxy-7-(3-morpholinopropoxy)quinazolin-4(3H)-one (2.0 g, 6.3 mmol) was mixed with SOCl₂ (15 mL) and DMF (0.2 mL), then the mixture was heated at reflux temperature for 5 hours. The volatiles were removed under reduced pressure. The residue was dissolved in CH₂Cl₂ (50 mL) and the organic layer was washed with aqueous NaHCO₃ solution and brine, and dried over Na₂SO₄, filtered and evaporated to give the crude product 13, which was purified by silica gel column chromatography to get pure intermediate 13 as a white solid. Yield: 48%, mp: 111°C–113°C. ¹H NMR (CDCl₃, 500 MHz) δ 8.86 (s, 1H), 7.39 (s, 1H), 7.35 (s, 1H), 4.30 (t, 2H, J =6.4 Hz), 4.05 (s, 3H), 3.85 (m, 4H), 2.33–2.94 (m, 6H), 2.20 (m, 2H). ESI-MS m/z: 338 [M+1]⁺.

Procedure for the synthesis of 4-chloro-6,7-bis(2-methoxyethoxy)quinazoline (14)²⁷

Compound 14 was prepared in the same way as compound 13, but the was changed from 6-methoxy-7-(3-morpholinopropoxy)quinazolin-4(3H)-one to 6,7-bis(2-methoxyethoxy)quinazolin-4(3H)-one. White solid. Yield: 52%, mp: 105°C–107°C. ¹H NMR (400 MHz, CDCl₃) δ 8.78 (s, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 4.30–4.23 (m, 4H), 3.91–3.74 (m, 4H), 3.43 (s, 3H), 3.42 (s, 3H). ESI-MS m/z: 313 [M+1]⁺.

Procedure for the synthesis of 4-(3-((4-chloro-7-methoxyquinazolin-6-yl)oxy)propyl)morpholine (15)²⁸

Compound 15 was prepared in the same way as compound 13, but the start material was changed from 6-methoxy-7-(3-morpholinopropoxy)quinazolin-4(3H)-one to 7-methoxy-6-(3-morpholinopropoxy)quinazolin-4(3H)-one. White solid. Yield: 63%, mp: 119°C–121°C. ¹H NMR (400 MHz, CDCl₃) δ 8.78 (s, 1H), 7.31 (s, 1H), 7.25 (s, 1H), 4.30–4.14 (m, 2H), 3.98 (s, 3H), 3.75–3.61 (m, 4H), 2.62–2.48 (m, 2H), 3.47–2.30 (m, 4H), 2.12–2.03 (m, 2H). ESI-MS m/z: 338 [M+1]⁺.

Procedure for the synthesis of the target compounds (16a-l)

To a stirred solution of 2-nitroimidazole derivative (12a-d) (1.0 mmol) in isopropyl alcohol (10 mL), compound 13, 14 or 15 (1.0 mmol) was added. The mixture was reacted in reflux temperature and stirred for about 2 hours. After completion, the mixture was washed with saturated NaHCO₃ solution and CH₂Cl₂, then the CH₂Cl₂ solvent was dried by anhydrous Na₂SO₄, which was concentrated to obtain crude products 16a-l. They were further purified by column chromatography to get pure materials.

N-(3-Chloro-4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-amine (16a): yellow solid. Yield: 83%, mp: 203°C–205°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.41 (s, 1H), 8.42 (s, 1H), 7.91 (d, J =2.5 Hz, 1H), 7.77 (s, 1H), 7.70 (s, 1H), 7.67 (dd, J =9.0, 2.5 Hz, 1H), 7.20 (s, 1H), 7.16 (d, J =10.0 Hz, 2H), 4.87 (t, J =5.0 Hz, 2H), 4.43 (t, J =5.0 Hz, 2H), 4.16 (t, J =6.5 Hz, 2H), 3.93 (s, 3H), 3.61–3.54 (m, 4H), 2.47–2.30 (m, 6H), 1.98–1.91 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.6, 154.1, 153.3, 149.6, 149.5, 147.3, 145.4, 134.2, 128.8, 128.1, 124.2, 122.5, 121.2, 114.4, 109.1, 108.3, 102.4, 68.0, 67.2, 66.7 (2), 56.7, 55.2, 53.8 (2), 49.0, 26.1. ESI-MS m/z: 584 [M+1]⁺.

N-(3-Chloro-4-(3-(2-nitro-1H-imidazol-1-yl)propoxy)phenyl)-6-methoxy-7-(3-morpholinopropoxy)quinazolin-4-amine (16b): yellow solid. Yield: 81%, mp: 92°C–94°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.42 (s, 1H), 8.43 (s, 1H), 7.92 (d, J =2.5 Hz, 1H), 7.78 (s, 1H), 7.67 (dd, J =9.0, 2.5 Hz, 1H), 7.65 (d, J =1.0 Hz, 1H), 7.19–7.14 (m, 3H), 4.60 (t, J =7.0 Hz, 2H), 4.16 (t, J =6.5 Hz, 2H), 4.09 (t, J =6.0 Hz, 2H), 3.94 (s, 3H), 3.57 (t, J =4.5 Hz, 4H), 2.44 (t, J =7.0 Hz, 2H), 2.40–2.34 (m, 4H), 2.33–2.26 (m, 2H), 2.00–1.89 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.7, 154.1, 153.3, 150.1, 149.5, 147.4, 145.2, 134.0, 128.4, 128.2, 124.3, 122.6, 121.4, 114.5, 109.1, 108.4, 102.5, 67.2, 66.7 (2), 66.6, 56.7, 55.2, 53.8 (2), 47.3, 29.8, 26.1. ESI-MS m/z: 598 [M+1]⁺.

6-Methoxy-7-(3-morpholinopropoxy)-N-(3-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)quinazolin-4-amine (16c): yellow solid. Yield: 84%, mp: 149°C–151°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.38 (s, 1H), 8.44 (s, 1H), 7.80 (s, 1H), 7.71 (d, J =1.0 Hz, 1H), 7.43 (dd, J =8.0, 1.5 Hz, 1H), 7.39 (t, J =2.0 Hz, 1H), 7.26 (t, J =8.0 Hz, 1H), 7.18 (d, J =1.0 Hz, 1H), 7.16 (s, 1H), 6.65 (dd, J =8.0, 2.0 Hz, 1H), 4.82 (t, J =5.0 Hz, 2H), 4.36 (t, J =5.0 Hz, 2H), 4.17 (t, J =6.5 Hz, 2H), 3.94 (s, 3H), 3.57 (t, J =4.5 Hz, 4H), 2.44 (t, J =7.0 Hz, 2H), 2.41–2.32 (m, 4H), 1.99–1.90 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.4, 156.7, 154.1, 153.2, 149.5, 147.5, 145.4, 141.3, 129.7, 128.6, 128.1, 115.5, 109.4, 109.3, 109.2, 108.3, 102.5, 67.2, 66.7, 66.7 (2), 56.8, 55.2, 53.8 (2), 49.1, 26.1. ESI-MS m/z: 550 [M+1]⁺.

6-Methoxy-7-(3-morpholinopropoxy)-N-(4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)quinazolin-4-amine (16d): yellow solid. Yield: 79%, mp: 152°C–154°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.36 (s, 1H), 8.35 (s, 1H), 7.79 (s, 1H), 7.71 (d, J =1.0 Hz, 1H), 7.64–7.57 (m, 2H), 7.19 (d, J =1.0 Hz, 1H), 7.13 (s, 1H), 6.94–6.87 (m, 2H), 4.81 (t, J =5.0 Hz, 2H), 4.35 (t, J =5.0 Hz, 2H), 4.15 (t, J =6.5 Hz, 2H), 3.92 (s, 3H), 3.57 (t, J =4.0 Hz, 4H), 2.46–2.41 (m, 2H), 2.41–2.33 (m, 4H), 2.01–1.85 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 157.0, 154.6, 153.9, 153.5, 149.3, 147.2, 145.5, 133.4, 128.6, 128.1, 124.7 (2), 114.8 (2), 109.1, 108.3, 102.6, 67.2, 67.0, 66.7 (2), 56.7, 55.2, 53.8 (2), 49.1, 26.1. ESI-MS m/z: 550 [M+1]⁺.

N-(3-Chloro-4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (16e): yellow solid. Yield: 84%, mp: 92°C–94°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.39 (s, 1H), 8.42 (s, 1H), 7.90 (s, 1H), 7.80 (s, 1H), 7.70 (s, 1H), 7.66 (dd, J =9.0, 2.0 Hz, 1H), 7.20–7.16 (m, 3H), 4.87 (t, J =4.5 Hz, 2H), 4.43 (t, J =4.5 Hz, 2H), 4.28–4.23 (m, 4H), 3.79–3.69 (m, 4H), 3.35(s, 3H), 3.33(s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.7, 154.1, 153.3, 149.7, 148.6, 147.3, 145.4, 134.2, 128.8, 128.1, 124.2, 122.5, 121.3, 114.5, 109.2, 108.8, 103.8, 70.6, 70.6, 68.9, 68.5, 68.0, 58.9, 58.8, 49.0. ESI-MS m/z: 559 [M+1]⁺.

N-(3-Chloro-4-(3-(2-nitro-1H-imidazol-1-yl)propoxy)phenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (16f): yellow solid. Yield: 89%, mp: 90°C–92°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.41 (br, 1H), 8.43 (s, 1H), 7.91 (d, J =2.5 Hz, 1H), 7.82 (s, 1H), 7.69–7.63 (m, 2H), 7.19 (s, 1H), 7.18 (d, J =1.0 Hz, 1H), 7.16 (d, J =9.0 Hz, 1H), 4.60 (t, J =7.0 Hz, 2H), 4.26 (t, J =9.0 Hz, 4H), 4.09 (t, J =6.0 Hz, 2H), 3.76 (t, J =9.0 Hz, 2H), 3.73 (t, J =9.0 Hz, 2H), 3.35 (s, 3H), 3.33 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.7, 154.1, 153.4, 150.2, 148.6, 147.3, 145.1, 133.9, 128.4, 128.2, 124.3, 122.6, 121.4, 114.5, 109.3, 108.8, 103.9, 70.6, 70.5, 68.9, 68.5, 66.6, 58.9, 58.8, 47.3, 29.8. ESI-MS m/z: 573 [M+1]⁺.

6,7-Bis(2-methoxyethoxy)-N-(3-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)quinazolin-4-amine (16g): yellow solid. Yield: 81%, mp: 183°C–185°C. ¹H NMR (400 MHz, DMSO-d₆) δ 9.39 (s, 1H), 8.46 (s, 1H), 7.86 (s, 1H), 7.73 (d, J =1.2 Hz, 1H), 7.44 (dd, J =8.0, 1.2 Hz, 1H), 7.41 (t, J =2.0 Hz, 1H), 7.27 (t, J =8.0 Hz, 1H), 7.22 (s, 1H), 7.20 (d, J =1.2 Hz, 1H), 6.66 (dd, J =8.0, 2.0 Hz, 1H), 4.84 (t, J =5.2 Hz, 2H), 4.38 (t, J =5.2 Hz, 2H), 4.32–4.24 (m, 4H), 3.81–3.72 (m, 4H), 3.37 (s, 3H), 3.35 (s, 3H). ¹³C NMR (100 MHz, CDCl₃) δ 158.2, 156.3, 154.7, 153.3, 149.0, 147.1, 140.1, 129.9 (2), 128.3, 127.2, 114.8, 110.0, 109.1, 108.5, 108.1, 102.7, 71.0, 70.5, 69.3, 68.4, 66.2, 59.3, 59.3, 49.4. ESI-MS m/z: 525 [M+1]⁺.

6,7-Bis(2-methoxyethoxy)-N-(4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)quinazolin-4-amine (16h): white solid. Yield: 83%, mp: 196°C–198°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.34 (s, 1H), 8.35 (s, 1H), 7.82 (s, 1H), 7.71 (d, J =1.0 Hz, 1H), 7.62–7.58 (m, 2H), 7.19 (d, J =1.0 Hz, 1H), 7.16 (s, 1H), 6.94–6.88 (m, 2H), 4.81 (t, J =5.0 Hz, 2H), 4.35 (t, J =5.0 Hz, 2H), 4.29–4.21 (m, 4H), 3.78–3.74 (m, 2H), 3.74–3.69 (m, 2H), 3.35 (s, 3H), 3.33 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆) δ 157.0, 154.6, 154.0, 153.5, 148.4, 147.2, 145.5, 133.3, 128.6, 128.1, 124.7 (2), 114.8 (2), 109.2, 108.7, 104.0, 70.6, 70.6, 68.9, 68.5, 67.0, 58.9, 58.8, 49.1. ESI-MS m/z: 525 [M+1]⁺.

N-(3-Chloro-4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (16i): yellow solid. Yield: 65%, mp: 132°C–134°C. ¹H NMR (500 MHz, DMSO-d₆) δ 9.56 (s, 1H), 8.46 (s, 1H), 7.96 (d, J =2.5 Hz, 1H), 7.91 (s, 1H), 7.77–7.69 (m, 2H), 7.27–7.13 (m, 3H), 4.90 (t, J =5.0 Hz, 2H), 4.45 (t, J =5.0 Hz, 2H), 4.23 (t, J =5.5 Hz, 2H), 3.94 (s, 3H), 3.88–3.60 (m, 4H), 3.44–3.22 (m, 6H), 2.24–2.04 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.8, 154.9, 153.2, 149.7, 148.4, 147.0, 145.4, 134.2, 128.8, 128.1, 124.3, 122.6, 121.2, 114.4, 109.2, 107.6, 104.0, 68.0, 67.4, 64.7, 56.4 (2), 54.6, 52.3, 49.0 (2), 24.4. ESI-MS m/z: 584 [M+1]⁺.

N-(3-Chloro-4-(3-(2-nitro-1H-imidazol-1-yl)propoxy)phenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (16j): yellow solid. Yield: 59%, mp: 107°C–109°C. ¹H NMR (500 MHz, CDCl₃) δ 8.63 (s, 1H), 7.90 (d, J =2.5 Hz, 1H), 7.70 (br, 1H), 7.57 (dd, J =9.0, 2.5 Hz, 1H), 7.35 (s, 1H), 7.25 (s, 1H), 7.18 (s, 1H), 7.13 (d, J =0.5 Hz, 1H), 6.91 (d, J =9.0 Hz, 1H), 4.74 (t, J =6.5 Hz, 2H), 4.26 (t, J =6.5 Hz, 2H), 4.04–3.96 (m, 5H), 3.83 (t, J =4.5 Hz, 4H), 2.74 (t, J =7.0 Hz, 2H), 2.70–2.61 (m, 4H), 2.44–2.36 (m, 2H), 2.24–2.16 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.7, 154.9, 153.3, 150.2, 148.7, 147.3, 145.4, 133.9, 128.4, 128.2, 124.4, 122.7, 121.4, 114.4, 109.2, 107.8, 103.2, 67.6, 66.6 (2), 66.5, 56.3, 55.4, 53.9 (2), 47.3, 29.8, 26.3. ESI-MS m/z: 598 [M+1]⁺.

7-Methoxy-6-(3-morpholinopropoxy)-N-(3-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)quinazolin-4-amine (16k): yellow solid. Yield: 58%, mp: 60°C–62°C. ¹H NMR (400 MHz, CDCl₃,): 8.61 (s, 1H, CH), 7.82 (br, 1H), 7.49 (t, J =2.0 Hz, 1H), 7.35–7.27 (m, 4H), 7.25 (s, 1H), 7.15 (d, J =1.2 Hz, 1H), 4.84 (t, J =9.6 Hz, 2H), 4.39 (t, J =9.6 Hz, 2H), 4.24 (t, J =6.8 Hz, 2H), 3.99 (s, 3H), 3.79 (t, J =9.2 Hz, 4H), 2.69 (t, J =7.2 Hz, 2H), 2.62 (t, J =9.2 Hz, 4H), 2.17–2.15 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 158.4, 156.7, 154.9, 153.2, 148.7, 147.5, 145.5, 141.2, 129.7, 128.6, 128.1, 115.6, 109.4 (2), 109.3, 107.8, 103.3, 67.7, 66.7, 66.6 (2), 56.3, 55.4, 53.9 (2), 49.1, 26.3. ESI-MS m/z: 550 [M+1]⁺.

7-Methoxy-6-(3-morpholinopropoxy)-N-(4-(2-(2-nitro-1H-imidazol-1-yl)ethoxy)phenyl)quinazolin-4-amine (16l): yellow solid. Yield: 55%, mp: 212°C–214°C. ¹H NMR (400 MHz, CDCl₃) δ 8.58 (s, 1H), 7.61–7.56 (m, 2H), 7.40 (s, 1H), 7.27 (d, J =1.2 Hz, 2H), 7.24 (s, 1H), 7.22 (s, 1H), 7.17 (d, J =1.2 Hz, 1H), 6.93–6.84 (m, 2H), 4.87–4.82 (m, 2H), 4.44–4.31 (m, 2H), 4.23 (t, J =6.8 Hz, 2H), 4.00 (s, 3H), 3.79–3.75 (m, 4H), 2.65 (t, J =5.6 Hz, 2H), 2.60–2.54 (m, 4H), 2.19–2.10 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆) δ 156.6, 154.3, 154.2, 153.0, 148.1, 146.8, 145.0, 132.9, 128.2, 127.7, 124.4 (2), 114.37 (2), 108.8, 107.3, 102.8, 67.2, 66.5, 66.2 (2), 55.8, 55.0, 53.5 (2), 48.7, 25.9. ESI-MS m/z: 550 [M+1]⁺.

In vitro enzymatic activity assay

Activity of kinases was determined using Z’-Lyte Kinase Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions.²⁹ The compounds were first tested the inhibition percentage at the concentration of 1.0×10⁻⁶ mol/L. If the inhibition percentage was higher than 50%, these compounds were further chosen to test their IC₅₀ values. For the IC₅₀ values, ten concentration gradients from 5.1×10⁻¹¹ to 1.0×10⁻⁶ mol/L were set for the tested compounds. The experiments were performed according to the instructions of the manufacturer.

Cytotoxicity assay

The cytotoxicity assay was performed as described previously.^16,17 Two human cancer cell lines (A549 and HT-29) were purchased from Cell Bank of China Science Academy (Shanghai, People's Republic of China). The cells were cultured in RPMI-1640 (Thermo Fisher Scientific) medium with heat-inactivated 10% FBS, penicillin (100 units/mL), and streptomycin (100 mg/mL) and incubated at 37°C. The cytotoxic activity in vitro was measured using sulforhodamine B (SRB) assay. All the compounds were dissolved in DMSO at the concentration 10.0 mg/mL and were then diluted to the appropriate concentrations. Cells were plated in 96-well plates (5×10³ per well) for 24 hours and subsequently treated with different concentrations of all tested compounds under normoxia or hypoxia for 72 hours, respectively. Cells were then washed with PBS and fixed with 10% (w/v) trichloroacetic acid at 4°C for 1 hour. After washing, the cells were stained for 30 minutes with 0.4% SRB dissolved in 1% acetic acid. Then the cells were washed with 1% acetic acid five times, and protein-bound dye was extracted with 10 mmol unbuffered Tris base. The absorbance was measured at 515 nm using a multiscan spectrum (Thermo Electron Co., Vantaa, Finland). The inhibition rate on cell proliferation of each well was calculated as (A515 control cells - A515 treated cells)/A515 control cells ×100%, and the IC₅₀ values were determined by Logit method.

Molecular modeling

According to the docking mode of compound 16i with EGFR, the co-crystal structure of EGFR/lapatinib was chosen as the template (PDB ID: 1XKK). The docking procedure was carried out by using C-DOCKER protocol within Discovery Studio 2.1. The detailed process has been described previously, and the pictures were prepared using Pymol.^16,17

Results and discussion

Synthesis

Synthesis of the target compounds 16a-l followed the general pathway shown in Scheme 1. The start materials 9a-c reacted with α, ω-dihalide alkane in the presence of potassium carbonate to afford 10a-d, then they were reduced with hydrogen to generate 11a-d. Coupling 11a-d with 2-nitroimidazole in the presence of potassium carbonate to obtain intermediates 12a-d. In addition, compounds 13, 14, and 15 were prepared following the published synthesis route.^26,27,28 Finally, condensing 12a-d with 13 in isopropanol to furnish the target compounds 16a-l. Compounds 16e-h and 16i-l were obtained in a similar way by condensing 12a-d with compound 14 and 15, respectively.

Scheme 1 Reagents and conditions: i) α, ω-dihalide alkane, K2CO3, DMF, 60°C, overnight; ii) H2, Pd/C, MeOH, room temperature, 2 hours; iii) 2-nitroimidazole, K2CO3, DMF, 70°C, 10 hours; iv) 12a-d, isopropanol, reflux, 2 hours.

Enzymatic activity

The inhibitory activity of compound 16a-l against EGFR was summarized in Table 1. Half of the tested compounds (16a, 16b, 16e, 16f, 16i, and 16j) manifested potent activities, with the IC₅₀ values ranging from 0.12–0.40 μM. As expected, the meta-chloride substituent on aniline was crucial to the EGFR inhibitory activity. For example, compounds 16d, 16h, and 16l, bearing no chloride group on the aniline, only exhibited the inhibited rate lower than 50% at 1.0 μM. While meta-chloride-bearing compounds 16a, 16e, and 16i displayed the IC₅₀ values of 0.19, 0.23, and 0.12 μM, respectively. The bindings of lapatinib/EGFR indicate that the chloride atom of lapatinib takes up a small cavity comprised by Leu788, Thr790, and Ala743,²³ it seems that the chloride atom of this series exerts similar effects. The position of linker impacts activities as well, compounds with 2-nitroimidazole-1H-alkoxy moieties at the meta- position exhibited decreased enzymatic inhibitory activities compared to those at para- positions (16c vs 16d, 16g vs 16h, and 16k vs 16l, inhibition% at 1.0 μM: 24% vs 42%, 28% vs 44%, and 30% vs 45%, respectively). The length of the alkyloxyl linker between aniline and 2-nitroimidazole also had obvious impact on the enzymatic inhibitory activities. The compromised EGFR inhibitory activities of compounds 16b, 16f, and 16j (IC₅₀: 0.33, 0.40, and 0.18 μM, respectively) compared with 16a, 16e, and 16i (IC₅₀: 0.19, 0.23, and 0.12 μM, respectively) indicated that shorter linkers (n=1) were more favorable than longer ones (n=2), probably because longer linkers restricted the molecules from adapting the kinase. Moreover, the types and positions of groups adjusting physicochemical properties affected activities as well. Compounds with 6-morpholinopropoxyl-7-methoxy side chains (16i-l) exhibited more potent EGFR inhibitory activities than those with 6, 7-dimethoxylethoxy (16e-h) or 6-methoxyl-7-morpholinopropoxy (16a-d) ones.

Table 1 EGFR inhibitory activity and cytotoxicity of compounds 16a-l

In vitro cytotoxicity

The in vitro cytotoxicities of compounds 16a-l were evaluated on human non-small-cell lung cancer A549 cells and human colorectal adenocarcinoma HT-29 cells under normoxic and hypoxic conditions (Table 1). Seven of all the 12 new compounds (16a, 16b, 16e, 16f, 16i, 16j, and 16l) exhibited good anti-proliferation activities. The SARs analysis indicated that both the introduction of 2-nitroimidazole-1H-alkoxy moieties at the aniline- para-position and the 3ʹ-chloro substitution were favorable in terms of cytotoxicity (16a >16c, 16e >16g, and 16i >16k), and the results were consisted with the enzymatic inhibitory activities. Almost all of these compounds manifested comparable to more potent cytotoxicities against A549 and HT-29 cells under normoxia compared to under hypoxia. Although the difference in cytotoxicity was not obvious between hypoxia and normoxia, considering EGFR expression was significantly up-regulated under hypoxia, which could partly offset the cytotoxicity generated in that circumstance,¹⁷ it was speculated that the 2-nitroimidazole moieties underwent a bio-reductive activated process and exerted cytotoxicity especially under tumor hypoxia. Particularly, the most potent compound, 16i, exhibited corresponding IC₅₀ values of 1.59 and 1.09 μM under normoxia and hypoxia against A549 cells, 2.46 and 1.35 μM under normoxia and hypoxia against HT-29 cells, which were superior to the positive control lapatinib.

Molecular docking study

Figure 4 (A) Proposed binding models of compound 16i in complex with EGFR. (B) Co-crystal structures of lapatinib in complex with EGFR (PDB code: 1XKK). (C) Comparison of the bindings of compound 16i (purplish red) and lapatinib (cyan).

The possible binding modes of these compounds in complex with EGFR were explored through a molecular modeling study between 16i and the reported EGFR crystal structure (PDB ID: 1XKK). As illustrated in Figure 4, compound 16i bound in the ATP-binding pocket of EGFR, which is similar to that of lapatinib. The 2-nitroimidazole group of 16i was surrounded by the residues of Thr854, Phe856, Met766, and Leu777. Two hydrogen bonds were formed between the two oxygen atoms of 2-nitroimidazole and the residues of Arg776 and Leu777 of EGFR. In addition, the 3ʹ-chloro group of the aniline was inserted in a pocket formed by the residues of Ala743, Thr790, and Leu788 (Figure 4A). Although compounds with two carbon linkers between aniline and 2-nitroimidazole moiety exhibited good EGFR inhibitory activity, this linker still appeared crowed and repulsed the 4-anilinoquinazoline group to the more “out” positione than lapatinib (Figure 4C). This caused the quinazoline group to not form hydrogen bonds with Thr790 or Thr854, which was observed in lapatinib-EGFR complex (Figure 4B). We speculate that the loss of these important interactions led compound 16i to display weaker EGFR inhibitory activity than lapatinib (IC₅₀: 0.12 μM vs 0.011 μM, Table 1). These results provide guidance for the further optimization of this series of compounds.

Conclusion

For the purpose of getting novel EGFR inhibitors and taking advantage of the carcinoma hypoxia circumstance, a series of small molecule inhibitors was designed and synthesized by incorporating 2-nitroimidazole group into the aniline of the 4-anilinoquinazolines. The SARs studies revealed that the introduction of chloride at the meta- position and the 2-nitroimidazole-1H-alkoxy moiety at para- position of the aniline were crucial to the EGFR inhibitory activity. Meanwhile, the optimal alkyl linker between aniline and 2-nitroimidazole moiety was two carbons in these compounds. The anti-proliferative activities were consistent with the EGFR inhibitory activities, and almost all the new compounds exhibited more potent cytotoxicities under hypoxia than normoxia. In particular, the most promising compound, 16i, exhibited good EGFR inhibitory activity (IC₅₀ value of 0.12 μM) and more potent cytotoxicities (A549 cells: IC₅₀ values of 1.59 and 1.09 μM under normoxia and hypoxia, respectively; HT-29 cells: IC₅₀ values of 2.46 and 1.35 μM under normoxia and hypoxia, respectively) than lapatinib. Moreover, the docking study confirmed the binding models between 16i and EGFR. These results provide new insights for development of EGFR inhibitors exerting activities both under normoxia and hypoxia.