Discovery of ASP5878: Synthesis and structure-activity relationships of pyrimidine derivatives as pan-FGFRs inhibitors with improved metabolic stability and suppressed hERG channel inhibitory activity.

Fibroblast growth factor receptor 3 (FGFR3) is an attractive therapeutic target for the treatment of bladder cancer patients harboring genetic alterations in FGFR3. We identified pyrimidine derivative ASP5878 (27) with improved metabolic stability and suppressed human ether-á-go-go related gene (hERG) channel inhibitory activity by the optimization of lead compound 1. Based on prediction of the metabolites of 1, an ether linker was introduced in place of the ethylene linker to improve metabolic stability. Moreover, conversion of the phenyl moiety into the pyrazole ring resulted in the suppression of hERG channel inhibitory activity, possibly due to the weaker π-π stacking interaction with Phe656 in the hERG channel by a reduction in π-electrical density of the aromatic ring. ASP5878 showed potent in vitro FGFR3 enzyme and cell growth inhibitory activity, and in vivo FGFR3 autophosphorylation inhibitory activity. Moreover, ASP5878 did not affect the hERG current up to 10 µM by in vitro patch-clamp assay, and a single oral dose of ASP5878 at 1, 10, and 100 mg/kg did not induce serious adverse effects on the central nervous, cardiovascular, and respiratory systems in dogs. Furthermore, ASP5878 exhibited lower total clearance than hepatic blood flow and high oral bioavailability in rats and dogs, and moderate brain penetration in rats.

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.

ASP5878, a Novel Inhibitor of FGFR1, 2, 3, and 4, Inhibits the Growth of FGF19-Expressing Hepatocellular Carcinoma.

Hepatocellular carcinoma is an aggressive cancer with poor prognosis. Fibroblast growth factor 19, a member of the fibroblast growth factor family, is a ligand for fibroblast growth factor receptor 4. Moreover, it plays a crucial role in the progression of hepatocellular carcinoma. ASP5878 is a novel inhibitor of fibroblast growth factor receptors 1, 2, 3, and 4 that is under development. It inhibits fibroblast growth factor receptor 4 kinase activity with an IC50 of 3.5 nmol/L. ASP5878 potently suppressed the growth of the fibroblast growth factor 19-expressing hepatocellular carcinoma cell lines Hep3B2.1-7, HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878 inhibited the phosphorylation of fibroblast growth factor receptor 4 and its downstream signaling molecules as well as induced apoptosis. Oral administration of ASP5878 at 3 mg/kg induced sustained tumor regression in a subcutaneous xenograft mouse model using Hep3B2.1-7. In HuH-7, an orthotopic xenograft mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival of the mice. These results suggest that ASP5878 is a potentially effective therapeutic agent for hepatocellular carcinoma patients with tumors expressing fibroblast growth factor 19. Mol Cancer Ther; 16(1); 68-75. ©2016 AACR.

ASP5878, a selective FGFR inhibitor, to treat FGFR3-dependent urothelial cancer with or without chemoresistance.

FGF/FGFR gene aberrations such as amplification, mutation and fusion are associated with many types of human cancers including urothelial cancer. FGFR kinase inhibitors are expected to be a targeted therapy for urothelial cancer harboring FGFR3 gene alternations. ASP5878, a selective inhibitor of FGFR1, 2, 3 and 4 under clinical investigation, selectively inhibited cell proliferation of urothelial cancer cell lines harboring FGFR3 point mutation or fusion (UM-UC-14, RT-112, RT4 and SW 780) among 23 urothelial cancer cell lines. Furthermore, ASP5878 inhibited cell proliferation of adriamycin-resistant UM-UC-14 cell line harboring MDR1 overexpression and gemcitabine-resistant RT-112 cell line. The protein expression of c-MYC, an oncoprotein, in gemcitabine-resistant RT-112 cell line was higher than that in RT-112 parental cell line and ASP5878 decreased the c-MYC expression in both RT-112 parental and gemcitabine-resistant RT-112 cell lines. Once-daily oral administration of ASP5878 exerted potent antitumor activities in UM-UC-14, RT-112 and gemcitabine-resistant RT-112 xenograft models without affecting body weight. These findings suggest that ASP5878 has the potential to be an oral targeted therapy against urothelial cancer harboring FGFR3 fusion or FGFR3 point mutation after the acquisition of gemcitabine- or adriamycin-resistance.

Inhibition of antigen-induced airway inflammation and hyperresponsiveness in guinea pigs by a selective antagonist of "chemoattractant receptor homologous molecule expressed on Th2 cells" (CRTH2).

Chemoattractant receptor homologous molecule expressed on T helper type 2 cells (CRTH2) is a PGD2 receptor found on eosinophils, basophils, and Th2 type T cells which exhibits chemotaxis and functions in activation cascades. However, while a number of CRTH2 antagonists, including ramatroban, are known to exert activity in certain animal models, activity in a guinea pig model of EA-induced airway hyperresponsiveness has not been demonstrated. The newly developed CRTH2 antagonist ASP5642 has shown antagonistic activity against human and guinea pig CRTH2 in previous studies and has also been found effective in treating guinea pig models of airway inflammation and airway hyperresponsiveness. While previous studies have used animals such as rats and mice to evaluate CRTH2 antagonist effects, ours is the first attempt to evaluate CRTH2 function in a guinea pig asthma model, which may prove useful in evaluating the compound's effects in humans, given the comparable airway function between the two species taken together, these data from the present study strongly suggest the utility of ASP5642 in investigating the role of CRTH2 in inflammatory responses and as a drug treatment for human asthma.

Discovery of novel and potent CRTH2 antagonists.

High throughput screening of our chemical library for CRTH2 antagonists provided a lead compound 1a. Initial optimization of the lead led to the discovery of a novel, potent and orally bioavailable CRTH2 antagonist 17.

Anti-inflammatory activity of non-nucleoside adenosine deaminase inhibitor FR234938.

Adenosine has anti-inflammatory activity. Adenosine deaminase (EC 3.5.4.4) metabolizes extracellular adenosine, resulting in an exacerbation of inflammation. Consequently, it was hypothesized that adenosine deaminase inhibitors produce anti-inflammatory activity by increasing extracellular adenosine concentration. This group recently developed a non-nucleoside adenosine deaminase inhibitor, FR234938, by using rational structure-based drug design. FR234938 inhibits recombinant human adenosine deaminase enzyme competitively. FR234938 inhibits interleukin (IL)-6-dependent immunoglobulin (Ig) M production by SKW6.4 cells, in the presence of adenosine. Inhibitory effect of FR234938/adenosine combination is blocked by an A2a adenosine receptor antagonist. FR234938 also inhibits anti-type II collagen delayed type hypersensitivity (DTH) in a dose-dependent manner, both in the presence and absence of recombinant human adenosine deaminase. Moreover, FR234938 inhibits tumor necrosis factor (TNF)-alpha and IL-10 production in a lipopolysaccharide (LPS)-induced cytokine production model in mice. These results indicate that FR234938 has potential anti-inflammatory activity. Non-nucleoside adenosine deaminase inhibitor FR234938 has good potential as a new type of anti-rheumatic and anti-inflammatory drug, by modulating host-defense concentrations of adenosine.

Structural basis of compound recognition by adenosine deaminase.

Structural snapshots corresponding to various states enable elucidation of the molecular recognition mechanism of enzymes. Adenosine deaminase has two distinct conformations, an open form and a closed form, although it has so far been unclear what factors influence adaptation of the alternative conformations. Herein, we have determined the first nonligated structure as an initial state, which was the open form, and have thereby rationally deduced the molecular recognition mechanism. Inspection of the active site in the nonligated and ligated states indicated that occupancy at one of the water-binding positions in the nonligated state was highly significant in determining alternate conformations. When this position is empty, subsequent movement of Phe65 toward the space induces the closed form. On the other hand, while occupied, the overall conformation remains in the open form. This structural understanding should greatly assist structure-oriented drug design and enable control of the enzymatic activity.

Rational design of non-nucleoside, potent, and orally bioavailable adenosine deaminase inhibitors: predicting enzyme conformational change and metabolism.

From metabolic considerations and prediction of an inhibitor-induced conformational change, novel adenosine deaminase (ADA) inhibitors with improved activities and oral bioavailability have been developed on the basis of our originally designed non-nucleoside ADA inhibitors. They demonstrated in vivo efficacy in models of inflammation and lymphoma. Furthermore, X-ray crystal structure analysis has revealed a novel induced fit to ADA.

Structure-based design, synthesis, and structure-activity relationship studies of novel non-nucleoside adenosine deaminase inhibitors.

We disclose herein optimization efforts around the novel, highly potent non-nucleoside adenosine deaminase (ADA) inhibitor, 1-[(R)-1-hydroxy-4-(6-(3-(1-methylbenzimidazol-2-yl)propionylamino)indol-1-yl)-2-butyl]imidazole-4-carboxamide 1 (K(i)= 7.7 nM), which we recently reported. Structure-based drug design (SBDD) utilizing the crystal structure of the 1/ADA complex was performed in order to obtain structure-activity relationships (SAR) for this type of compound rationally and effectively. To utilize the newly formed hydrophobic space (F2), replacement of the benzimidazole ring of 1 with a n-propyl chain (4b) or a simple phenyl ring (4c) was tolerated in terms of binding activity, and the length of the methylene-spacer was shown to be optimal at two or three. Replacement of an amide with an ether as a linker was also well tolerated in terms of binding activity and moreover improved the oral absorption (6a and 6b). Finally, transformation of indol-1-yl to indol-3-yl resulted in discovery of a novel highly potent and orally bioavailable ADA inhibitor, 1-[(R)-4-(5-(3-(4-chlorophenyl)propoxy)-1-methylindol-3-yl)-1-hydroxy-2-butyl]imidazole-4-carboxamide 8c.

Structure-based design and synthesis of non-nucleoside, potent, and orally bioavailable adenosine deaminase inhibitors.

We disclose optimization efforts based on the novel non-nucleoside adenosine deaminase (ADA) inhibitor, 4 (K(i) = 680 nM). Structure-based drug design utilizing the crystal structure of the 4/ADA complex led to discovery of 5 (K(i) = 11 nM, BA = 30% in rats). Furthermore, from metabolic considerations, we discovered two inhibitors with improved oral bioavailability [6 (K(i) = 13 nM, BA = 44%) and 7 (K(i) = 9.8 nM, BA = 42%)]. 6 demonstrated in vivo efficacy in models of inflammation and lymphoma.

A highly potent non-nucleoside adenosine deaminase inhibitor: efficient drug discovery by intentional lead hybridization.

We disclose herein the rapid discovery of the first highly potent (Ki = 7.7 nM) non-nucleoside adenosine deaminase (ADA) inhibitor based on the rational hybridization of two structurally distinct leads. Two micromolar inhibitors were discovered by a parallel rational design and random screening program, and individual crystal structures of bovine ADA in complexation with these inhibitors revealed several unknown binding sites and distinct binding modes. Using this information as the starting point, highly effective lead hybridization was achieved in only two structure-based drug design iterations. The conceptual approach illustrated by this example promises to be broadly useful in the search for new chemical entities and can contribute greatly to improve the overall efficiency and speed of drug discovery.

Structure-based de novo design of non-nucleoside adenosine deaminase inhibitors.

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.