Discovery of ASP6918, a KRAS G12C inhibitor: Synthesis and structure-activity relationships of 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivatives as covalent inhibitors with good potency and oral activity for the treatment of solid tumors.

Although KRAS protein had been classified as an undruggable target, inhibitors of KRAS G12C mutant protein were recently reported to show clinical efficacy in solid tumors. In our previous report, we identified 1-{2,7-diazaspiro[3.5]non-2-yl}prop-2-en-1-one derivative (1) as a KRAS G12C inhibitor that covalently binds to Cys12 of KRAS G12C protein. Compound 1 exhibited potent cellular pERK inhibition and cell growth inhibition against a KRAS G12C mutation-positive cell line and showed an antitumor effect on subcutaneous administration in an NCI-H1373 (KRAS G12C mutation-positive cell line) xenograft mouse model in a dose-dependent manner. In this report, we further optimized the substituents on the quinazoline scaffold based on the structure-based drug design from the co-crystal structure analysis of compound 1 and KRAS G12C to enhance in vitro activity. As a result, ASP6918 was found to exhibit extremely potent in vitro activity and induce dose-dependent tumor regression in an NCI-H1373 xenograft mouse model after oral administration.

Discovery and biological evaluation of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as covalent inhibitors of KRAS G12C with favorable metabolic stability and anti-tumor activity.

RAS protein plays a key role in cellular proliferation and differentiation. RAS gene mutation is a known driver of oncogenic alternation in human cancer. RAS inhibition is an effective therapeutic treatment for solid tumors, but RAS protein has been classified as an undruggable target. Recent reports have demonstrated that a covalent binder to KRAS protein at a mutated cysteine residue (G12C) is effective for the treatment of solid tumors. Here, we report a series of 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one derivatives as potent covalent inhibitors against KRAS G12C identified throughout structural optimization of an acryloyl amine moiety to improve in vitro inhibitory activity. From an X-ray complex structural analysis, the 1-{2,7-diazaspiro[3.5]nonan-2-yl}prop-2-en-1-one moiety binds in the switch-II pocket of KRAS G12C. Further optimization of the lead compound (5c) led to the successful identification of 1-[7-[6-chloro-8-fluoro-7-(5-methyl-1H-indazol-4-yl)-2-[(1-methylpiperidin-4-yl)amino]quinazolin-4-yl]-2,7-diazaspiro[3.5]nonan-2-yl]prop-2-en-1-one (7b), a potent compound with high metabolic stabilities in human and mouse liver microsomes. Compound 7b showed a dose-dependent antitumor effect on subcutaneous administration in an NCI-H1373 xenograft mouse model.

Synthesis and structure-activity relationships of pyrimidine derivatives as potent and orally active FGFR3 inhibitors with both increased systemic exposure and enhanced in vitro potency.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of patients with bladder cancer harboring genetic alterations in FGFR3. We identified pyrimidine derivative 20b, which induced tumor regression following oral administration to a bladder cancer xenograft mouse model. Compound 20b was discovered by optimizing lead compound 1, which we reported previously. Specifically, reducing the molecular size of the substituent at the 4-position and replacing the linker of the 5-position in the pyrimidine scaffold resulted in an increase in systemic exposure. Furthermore, introduction of two fluorine atoms into the 3,5-dimethoxyphenyl ring enhanced FGFR3 inhibitory activity. Molecular dynamics (MD) simulation of 20b suggested that the fluorine atom interacts with the main chain NH moiety of Asp635 via a hydrogen bond.

Structure-based drug design of 1,3,5-triazine and pyrimidine derivatives as novel FGFR3 inhibitors with high selectivity over VEGFR2.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of bladder cancer. We identified 1,3,5-triazine derivative 18b and pyrimidine derivative 40a as novel structures with potent and highly selective FGFR3 inhibitory activity over vascular endothelial growth factor receptor 2 (VEGFR2) using a structure-based drug design (SBDD) approach. X-ray crystal structure analysis suggests that interactions between 18b and amino acid residues located in the solvent region (Lys476 and Met488), and between 40a and Met529 located in the back pocket of FGFR3 may underlie the potent FGFR3 inhibitory activity and high kinase selectivity over VEGFR2.

Effect of Fms-like tyrosine kinase 3 (FLT3) ligand (FL) on antitumor activity of gilteritinib, a FLT3 inhibitor, in mice xenografted with FL-overexpressing cells.

Therapeutic effects of FLT3 inhibitors have been reported in acute myeloid leukemia (AML) with constitutively activating FLT3 mutations, including internal tandem duplication (ITD) and point mutation, which are found in approximately one-third of AML patients. One of the critical issues of treatment with FLT3 inhibitors in FLT3-mutated AML is drug resistance. FLT3 ligand (FL) represents a mechanism of resistance to FLT3 inhibitors, including quizartinib, midostaurin, and sorafenib, in AML cells harboring both wild-type and mutant FLT3 (FLT3 wt/FLT3 mut). Here, we investigated the effect of FL on the efficacy of gilteritinib, a FLT3 inhibitor, in AML-derived cells in vitro and in mice. In contrast to other FLT3 inhibitors, FL stimulation had little effect on growth inhibition or apoptosis induction by gilteritinib. The antitumor activity of gilteritinib was also comparable between xenograft mouse models injected with FL-expressing and mock MOLM-13 cells. In the FLT3 signaling analyses, gilteritinib inhibited FLT3wt and FLT3-ITD to a similar degree in HEK293 and Ba/F3 cells, and similarly suppressed FLT3 downstream signaling molecules (including ERK1/2 and STAT5) in both the presence and absence of FL in MOLM-13 cells. Co-crystal structure analysis showed that gilteritinib bound to the ATP-binding pocket of FLT3. These results suggest that gilteritinib has therapeutic potential in FLT3-mutated AML patients with FL overexpression.

Structural insights into the novel inhibition mechanism of Trypanosoma cruzi spermidine synthase.

Trypanosoma cruzi causes Chagas disease, a severe disease affecting 8-10 million people in Latin America. While nifurtimox and benznidazole are used to treat this disease, their efficacy is limited and adverse effects are observed. New therapeutic targets and novel drugs are therefore urgently required. Enzymes in the polyamine-trypanothione pathway are promising targets for the treatment of Chagas disease. Spermidine synthase is a key enzyme in this pathway that catalyzes the transfer of an aminopropyl group from decarboxylated S-adenosylmethionine (dcSAM) to putrescine. Fragment-based drug discovery was therefore conducted to identify novel, potent inhibitors of spermidine synthase from T. cruzi (TcSpdSyn). Here, crystal structures of TcSpdSyn in complex with dcSAM, trans-4-methylcyclohexylamine and hit compounds from fragment screening are reported. The structure of dcSAM complexed with TcSpdSyn indicates that dcSAM stabilizes the conformation of the `gatekeeping' loop to form the putrescine-binding pocket. The structures of fragments bound to TcSpdSyn revealed two fragment-binding sites: the putrescine-binding pocket and the dimer interface. The putrescine-binding pocket was extended by an induced-fit mechanism. The crystal structures indicate that the conformation of the dimer interface is required to stabilize the gatekeeping loop and that fragments binding to this interface inhibit TcSpdSyn by disrupting its conformation. These results suggest that utilizing the dynamic structural changes in TcSpdSyn that occur upon inhibitor binding will facilitate the development of more selective and potent inhibitors.

Addressing phototoxicity observed in a novel series of biaryl derivatives: discovery of potent, selective and orally active phosphodiesterase 10A inhibitor ASP9436.

We synthesized several biaryl derivatives as PDE10A inhibitors to prevent phototoxicity of 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (1) and found that the energy difference between the energy-minimized conformation and the coplanar conformation of the biaryl moiety helped facilitate prediction of the phototoxic potential of biaryl compounds. Replacement of the quinoline ring of 1 with N-methyl benzimidazole increased this energy difference and prevented phototoxicity in the 3T3 NRU test. Further optimization identified 1-methyl-5-(1-methyl-3-{[4-(1-methyl-1H-benzimidazol-4-yl)phenoxy]methyl}-1H-pyrazol-4-yl)pyridin-2(1H)-one (38b). Compound 38b exhibited good selectivity against other PDEs, and oral administration of 38b improved visual-recognition memory deficit in mice at doses of 0.001 and 0.003mg/kg in the novel object recognition test. ASP9436 (sesquiphosphate of 38b) may therefore be used for the treatment of schizophrenia with a low risk of phototoxicity.

Structures of complexes of type 5 17ß-hydroxysteroid dehydrogenase with structurally diverse inhibitors: insights into the conformational changes upon inhibitor binding.

Type 5 17ß-hydroxysteroid dehydrogenase (17ß-HSD5) is an aldo-keto reductase expressed in the human prostate which catalyzes the conversion of androstenedione to testosterone. Testosterone is converted to 5α-dihydrotestosterone, which is present at high concentrations in patients with castration-resistant prostate cancer (CRPC). Inhibition of 17ß-HSD5 is therefore considered to be a promising therapy for treating CRPC. In the present study, crystal structures of complexes of 17ß-HSD5 with structurally diverse inhibitors derived from high-throughput screening were determined. In the structures of the complexes, various functional groups, including amide, nitro, pyrazole and hydroxyl groups, form hydrogen bonds to the catalytic residues His117 and Tyr55. In addition, major conformational changes of 17ß-HSD5 were observed following the binding of the structurally diverse inhibitors. These results demonstrate interactions between 17ß-HSD5 and inhibitors at the atomic level and enable structure-based drug design for anti-CRPC therapy.