Late-stage modification of complex drug: Base-controlled Pd-catalyzed regioselective synthesis and bioactivity of arylated osimertinibs

Abstract

Achieving regioselective synthesis in complex molecules with multiple reactive sites remains a tremendous challenge in synthetic chemistry. Regiodivergent palladium-catalyzed C─H arylation of complex antitumor drug osimertinib with various aryl bromides via the late-stage functionalization strategy was demonstrated here. This reaction displayed a switch in regioselectivity under complete base control. Potassium carbonate (K₂CO₃) promoted the arylation of acrylamide terminal C(sp²)-H, affording 34 derivatives. Conversely, sodium tert-butoxide (t-BuONa) mediated the aryl C(sp²)-H arylation of the indole C2 position, providing 27 derivatives. The derivative 3r containing a 3-fluorophenyl group at the indole C2 position demonstrated similar inhibition of EGFR^T790M/L858R and superior antiproliferative activity in H1975 cells compared to osimertinib, as well as similar antiproliferative activity in A549 cells and antitumor efficacy in xenograft mouse model bearing H1975 cells. This approach provides a "one substrate-multi reactions-multiple products" strategy for the structural modification of complex drug molecules, creating more opportunities for the fast screening of pharmaceutical molecules.

Fig. 1.. Overview of osimertinib.

(A) Structure of osimertinib and its active sites. (B) Two-dimensional binding mode of osimertinib to EGFR found in the x-ray structure. HR, hydrophobic region; PB, phosphate binding site.

Fig. 2.. Single-crystal x-ray structures of compounds 3a and 2v.

(A) 3a and (B) 2v.

Fig. 3.. Compounds 3e and 3r inhibited cell proliferation and induced apoptosis of H1975 cells.

(A to C) Colony formation assay of H1975 cells treated with 3e, 3r, and osimertinib. (D) Hoechst 33258 staining assay in H1975 cells treated with 3e, 3r, and osimertinib. Scale bar, 40 μm. (E and F) H1975 cells were treated with 3e, 3r, and osimertinib for 24 hours, and apoptosis ratios were determined by flow cytometry analysis of Annexin V/PI double staining. (G to J) Western blot analysis of Bax, Bcl-2, and cleaved caspase-3 in H1975 cells treated with 3e, 3r, and osimertinib for 24 hours. β-Actin was used as a loading control. ns, not significant.

Fig. 4.. Compounds 3e and 3r inhibit migration in H1975 cells.

(A and B) H1975 cells were treated with 3e, 3r, and osimertinib. The scratch assay was used to measure migration capabilities of the cells. The wound closure ratio represents the level of cell migration ability. Scale bar, 100 μm. (C to E) Western blot analysis of E-cadherin and MMP-2 in H1975 cells treated with 3e, 3r, and osimertinib for 24 hours. β-Actin was used as a loading control. (F and G) Immunofluorescence analysis of MMP-2 and E-cadherin levels in H1975 cells treated with 3e, 3r, and osimertinib for 24 hours. Scale bar, 10 μm.

Fig. 5.. In vivo antitumor effects of osimertinib and 3r on H1975 (n = 8 per group).

(A) Tumor tissues, (B) tumor volumes, and (C) tumor weight following treatment by oral administration with osimertinib and 3r. (D) Western blot analysis of xenograft tumor tissues from vehicle-, osimertinib-, and 3r-treated nude mice for expression of Ki67, p-EGFR, and EGFR. β-Actin was used as a loading control. (E) Quantification of Ki67 by Western blot analysis. (F) Body weights following treatment by oral administration with osimertinib and 3r. (G) Representative hematoxylin and eosin staining in heart, liver, spleen, lung, and kidney sections. Scale bar, 40 μm. Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control groups.