Development of Potent Inhibitors of Receptor Tyrosine Kinases by Ligand-Based Drug Design and Target-Biased Phenotypic Screening

Abstract

Pyrazolopyrimidines with potent antiproliferative properties were developed by an adaptive strategy that applies ligand-based design and phenotypic screening iteratively and is informed by biochemical assays. To drive development toward specific oncopathways, compounds were tested against cancer cells that overexpress, or not, AXL kinase. Identified phenotypic hits were found to inhibit oncotargets AXL, RET, and FLT3. Subsequent optimization generated antiproliferative lead compounds with unique selectivity profiles, including selective AXL inhibitors and a highly potent inhibitor of FLT3.

Figure 1

Medicinal chemistry campaign based on the cocrystal structure of 52 and MER (PDB 2G15(15)) performed by Wang and co-workers for the discovery of MER inhibitor UNC569 and the 6-methylaminopyrazolo[3,4-d]pyrimidine scaffold explored in this work.

Scheme 1. Four-Step Synthetic Route for the Preparation of Compounds 7a–j from Commercially Available 6-Chloro-1H-pyrazolo[3,4-d]pyrimidine, 3

Figure 2

EC₅₀ values of 7a–j and the positive control foretinib against AXL^– (red) and AXL⁺ (blue) cells. Cell viability assay: PrestoBlue reagent at day 5. Error bars: ±SD from n = 2.

Scheme 2. Synthetic Route for the Preparation of Triazole-Containing Compounds 10a–g from Intermediate 5

Figure 3

EC₅₀ values of 10a–g and reference hit 7f against AXL^– (red) and AXL⁺ (blue) cells. Cell viability assay: PrestoBlue reagent at day 5. Error bars: ±SD from n = 2.

Figure 4

Structure, molecular weight (MW), and cLogP of derivatives 12a–f.

Figure 5

EC₅₀ values of 12a–f and reference hit 10a against AXL^– (red) and AXL⁺ (blue) cells. Cell viability assay: PrestoBlue reagent at day 5. Error bars: ±SD from n = 2.

Figure 6

Structure, MW, and cLogP of derivatives 13a–d.

Figure 7

EC₅₀ values of 13a–d and BGB324 against MV4-11 and MOLM-13 cells. Cell viability assay: PrestoBlue reagent at day 5. Error bars: ±SD from n = 2.

Figure 8

Map of the human kinome screened for 13a at 1 μM. Green circles denote <65% inhibition. Red circles denote >65% inhibition (>98% for the largest one).