Discovery of novel fibroblast growth factor receptor 1 kinase inhibitors by structure-based virtual screening

Abstract

Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC(50) values of 23 and 50 microM. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 microM. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

Figure 1

Examples of inhibitors of FGFR1 kinase with available crystal structures; indolinones 1 and 2, the pyridopyrimidine 3, and 4.

Figure 2

The indolinone 1 bound in the ATP binding site of FGFR1 kinase. Hydrogen bonds formed with Glu562 and Ala564, which are in the hinge region, are highlighted.

Figure 3

Crystal structures of FGFR1 kinase with 4 illustrating the two conformations for the nucleotide binding loop: extended or up (blue) and down (brown).

Figure 4

Overlays of the crystal structure (grey) with the XP GLIDE pose (green) for 1, 2, 3, and 4. Hydrogen bonds with Glu562 and Ala564 are highlighted.

Figure 5

XP Glide score frequency for 40,000 compounds from the ZINC collection in black and known active compounds in red (scaled by 300) for the conformation with (A) the nucleotide binding loop up and (B) with the nucleotide binding loop down.

Figure 6

Structures of the purchased compounds obtained by docking into the conformation of FGFR1 kinase with the nucleotide binding loop up. Though 7 was ordered, it turned out to be the isomer 10. Samples of both 7 and 10 were synthesized.

Figure 7

Structures of the purchased compounds obtained by docking into the conformation of FGFR1 kinase with the nucleotide binding loop down. Compounds with stereocenters are racemic mixtures.

Figure 8

Computed structures for the complexes of FGFR1 kinase with 10 (A) and 40 (B). Selected backbone and side-chain atoms of the kinase are shown; carbon atoms of the inhibitors are colored green. Hydrogen bonds are highlighted with black lines.

Scheme 1^a

^aReagents and conditions: (a) 1. HCHO, HCl, 50 °C, 3h; 2. MeOH, ref lux, 3h, 57% two steps (b) 1,3-dibromopropane, K₂CO₃, DMF, 80 °C, 1h (43–60%) (c) K₂CO₃, DMF, 80 °C, 1h (44–91%) (d) thiohydantoin, pipiridine, MeOH, ref lux (54–74%) (e) pseudothiohydantoin, AcONa, AcOH, 170 °C, 15 min, MW, 53%

Scheme 2^a

^aReagents and conditions: (a) S, piperidine, EtOH, 50 °C, 4h (23–60%) (b) p-Chloranil, 1,4-Dioxane, 90 °C, 5h (c) 3,4-dimethoxybenzaldehyde or 3-methoxybenzaldehyde, conc HCl, n-BuOH, 80 °C, 8H (48–67%) (d) ethyl 2-(4-formyl-2-methoxyphenoxy)acetate (52), conc HCl, n-BuOH, ref lux, 2h (30–62%) (e) 5N NaOH, EtOH, reflux, 2h (30%-Quant.)