Preclinical Characterization of Linrodostat Mesylate, a Novel, Potent, and Selective Oral Indoleamine 2,3-Dioxygenase 1 Inhibitor.

Tumors can exploit the indoleamine 2,3-dioxygenase 1 (IDO1) pathway to create an immunosuppressive microenvironment. Activated IDO1 metabolizes tryptophan into immunosuppressive kynurenine, leading to suppressed effector T-cell (Teff) proliferation, allowing for tumor escape from host immune surveillance. IDO1 inhibition counteracts this immunosuppressive tumor microenvironment and may improve cancer outcomes, particularly when combined with other immunotherapies. Linrodostat mesylate (linrodostat) is a potent, selective oral IDO1 inhibitor that occupies the heme cofactor-binding site to prevent further IDO1 activation and is currently in multiple clinical trials for treatment of patients with advanced cancers. Here, we assess the in vitro potency, in vivo pharmacodynamic (PD) activity, and preclinical pharmacokinetics (PKs) of linrodostat. Linrodostat exhibited potent cellular activity, suppressing kynurenine production in HEK293 cells overexpressing human IDO1 and HeLa cells stimulated with IFNγ, with no activity against tryptophan 2,3-dioxygenase or murine indoleamine 2,3-dioxygenase 2 detected. Linrodostat restored T-cell proliferation in a mixed-lymphocyte reaction of T cells and allogeneic IDO1-expressing dendritic cells. In vivo, linrodostat reduced kynurenine levels in human tumor xenograft models, exhibiting significant PD activity. Linrodostat demonstrated a PK/PD relationship in the xenograft model, preclinical species, and samples from patients with advanced cancers, with high oral bioavailability in preclinical species and low to moderate systemic clearance. Our data demonstrate that linrodostat potently and specifically inhibits IDO1 to block an immunosuppressive mechanism that could be responsible for tumor escape from host immune surveillance with favorable PK/PD characteristics that support clinical development.

Discovery of Imidazopyridines as Potent Inhibitors of Indoleamine 2,3-Dioxygenase 1 for Cancer Immunotherapy.

Indoleamine 2,3-dioxygenase 1 (IDO1) has been identified as a target for small-molecule immunotherapy for the treatment of a variety of cancers including renal cell carcinoma and metastatic melanoma. This work focuses on the identification of IDO1 inhibitors containing replacements or isosteres for the amide found in BMS-986205, an amide-containing, IDO1-selective inhibitor currently in phase III clinical trials. Detailed subsequently are efforts to identify a structurally differentiated IDO1 inhibitor via the pursuit of a variety of heterocyclic isosteres, leading to the discovery of highly potent, imidazopyridine-containing IDO1 inhibitors.

Enhanced antitumor immunity by a novel small molecule HPK1 inhibitor.

BACKGROUND: Hematopoietic progenitor kinase 1 (HPK1 or MAP4K1) has been demonstrated as a negative intracellular immune checkpoint in mediating antitumor immunity in studies with HPK1 knockout and kinase dead mice. Pharmacological inhibition of HPK1 is desirable to investigate the role of HPK1 in human immune cells with therapeutic implications. However, a significant challenge remains to identify a small molecule inhibitor of HPK1 with sufficient potency, selectivity, and other drug-like properties suitable for proof-of-concept studies. In this report, we identified a novel, potent, and selective HPK1 small molecule kinase inhibitor, compound K (CompK). A series of studies were conducted to investigate the mechanism of action of CompK, aiming to understand its potential application in cancer immunotherapy. METHODS: Human primary T cells and dendritic cells (DCs) were investigated with CompK treatment under conditions relevant to tumor microenvironment (TME). Syngeneic tumor models were used to assess the in vivo pharmacology of CompK followed by human tumor interrogation ex vivo. RESULTS: CompK treatment demonstrated markedly enhanced human T-cell immune responses under immunosuppressive conditions relevant to the TME and an increased avidity of the T-cell receptor (TCR) to recognize viral and tumor-associated antigens (TAAs) in significant synergy with anti-PD1. Animal model studies, including 1956 sarcoma and MC38 syngeneic models, revealed improved immune responses and superb antitumor efficacy in combination of CompK with anti-PD-1. An elevated immune response induced by CompK was observed with fresh tumor samples from multiple patients with colorectal carcinoma, suggesting a mechanistic translation from mouse model to human disease. CONCLUSION: CompK treatment significantly improved human T-cell functions, with enhanced TCR avidity to recognize TAAs and tumor cytolytic activity by CD8+ T cells. Additional benefits include DC maturation and priming facilitation in tumor draining lymph node. CompK represents a novel pharmacological agent to address cancer treatment resistance.

Discovery and Preclinical Evaluation of BMS-986242, a Potent, Selective Inhibitor of Indoleamine-2,3-dioxygenase 1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-containing dioxygenase enzyme implicated in cancer immune response. This account details the discovery of BMS-986242, a novel IDO1 inhibitor designed for the treatment of a variety of cancers including metastatic melanoma and renal cell carcinoma. Given the substantial interest around this target for cancer immunotherapy, we sought to identify a structurally differentiated clinical candidate that performs comparably to linrodostat (BMS-986205) in terms of both in vitro potency and in vivo pharmacodynamic effect in a mouse xenograft model. On the basis of its preclinical profile, BMS-986242 was selected as a candidate for clinical development.

Conformational-Analysis-Guided Discovery of 2,3-Disubstituted Pyridine IDO1 Inhibitors.

IDO1 inhibitors have shown promise as immunotherapies for the treatment of a variety of cancers, including metastatic melanoma and renal cell carcinoma. We recently reported the identification of several novel heme-displacing IDO1 inhibitors, including the clinical molecules linrodostat (BMS-986205) and BMS-986242. Both molecules contain quinolines that, while being present in successful medicines, are known to be potentially susceptible to oxidative metabolism. Efforts to swap this quinoline with an alternative aromatic system led to the discovery of 2,3-disubstituted pyridines as suitable replacements. Further optimization, which included lowering ClogP in combination with strategic fluorine incorporation, led to the discovery of compound 29, a potent, selective IDO1 inhibitor with robust pharmacodynamic activity in a mouse xenograft model.

Design, synthesis and structure-activity relationships of novel biarylamine-based Met kinase inhibitors.

Biarylamine-based inhibitors of Met kinase have been identified. Lead compounds demonstrate nanomolar potency in Met kinase biochemical assays and significant activity in the Met-driven GTL-16 human gastric carcinoma cell line. X-ray crystallography revealed that these compounds adopt a bioactive conformation, in the kinase domain, consistent with that previously seen with 2-pyridone-based Met kinase inhibitors. Compound 9b demonstrated potent in vivo antitumor activity in the GTL-16 human tumor xenograft model.

Identification of pyrrolo[2,1-f][1,2,4]triazine-based inhibitors of Met kinase.

An amide library derived from the pyrrolo[2,1-f][1,2,4]triazine scaffold led to the identification of modest inhibitors of Met kinase activity. Introduction of polar side chains at C-6 of the pyrrolotriazine core provided significant improvements in in vitro potency. The amide moiety could be replaced with acylurea and malonamide substituents to give compounds with improved potency in the Met-driven GTL-16 human gastric carcinoma cell line. Acylurea pyrrolotriazines with substitution at C-5 demonstrated single digit nanomolar kinase activity. X-ray crystallography revealed that the C-5 substituted pyrrolotriazines bind to the Met kinase domain in an ATP-competitive manner.

Apoptotic and cytostatic farnesyltransferase inhibitors have distinct pharmacology and efficacy profiles in tumor models.

BMS-214662 and BMS-225975 are tetrahydrobenzodiazepine-based farnesyltransferase inhibitors (FTIs) that have nearly identical structures and very similar pharmacological profiles associated with farnesyltransferase (FT) inhibition. Despite their similar activity against FT in vitro and in cells, these compounds differ dramatically in their apoptotic potency and tumor-regressing activity in vivo. BMS-214662 is the most potent apoptotic FTI known and exhibits curative responses in mice bearing a variety of staged human tumor xenografts such as HCT-116 human colon tumor. By contrast, BMS-225975 does not cause tumor regression and at best causes partial tumor growth inhibition in staged HCT-116 human colon tumor xenografts. Lack of tumor regression activity in BMS-225975 was attributable to its relatively weak apoptotic potency, not to poor cell permeability or pharmacokinetics. Both compounds were equally effective in inhibiting Ras processing and causing accumulation of a variety of nonfarnesylated substrates of FT in HCT-116 cells. Because BMS-225975 has poor apoptotic activity compared with BMS-214662 but inhibits FT to the same extent as BMS-214662, it is very unlikely that FT inhibition alone can account for the apoptotic potency of BMS-214662. Clearly distinct patterns of sensitivities in a cell line panel were obtained for the apoptotic FTI BMS-214662 and the cytostatic FTI BMS-225975. Activation of the c-Jun-NH(2)-terminal kinase pathway was readily observed with BMS-214662 but not with BMS-225975. We developed a highly sensitive San-1 murine xenograft tumor model that is particularly useful for evaluating the in vivo activity of cytostatic FTIs such as BMS-225975.

Discovery of the pyrrolo[2,1-f][1,2,4]triazine nucleus as a new kinase inhibitor template.

The pyrrolo[2,1-f][1,2,4]triazine nucleus was identified as a novel kinase inhibitor template which effectively mimics the well-known quinazoline kinase inhibitor scaffold. Attachment of a 4-((3-chloro-4-fluorophenyl)amino) substituent to the template provided potent biochemical inhibitors of the tyrosine kinase activity of EGFR, as well as inhibition of cellular proliferation of the human colon tumor cell line DiFi. Attachment of a 4-((3-hydroxy-4-methylphenyl)amino) substituent provided potent inhibitors of VEGFR-2 which also showed effects on the VEGF-dependent proliferation of human umbilical vein endothelial cells. Biological activity was maintained with substitution at positions 5 or 6, but not 7, suggesting that the former positions are promising sites for introducing side chains which modulate physicochemical properties. Preliminary inhibition studies with varying ATP concentrations suggest that, like the quinazoline-based kinase inhibitors, the pyrrolotriazine-based inhibitors bind in the ATP pocket.

Discovery of N-(4-(2-amino-3-chloropyridin-4-yloxy)-3-fluorophenyl)-4-ethoxy-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamide (BMS-777607), a selective and orally efficacious inhibitor of the Met kinase superfamily.

Substituted N-(4-(2-aminopyridin-4-yloxy)-3-fluoro-phenyl)-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carboxamides were identified as potent and selective Met kinase inhibitors. Substitution of the pyridine 3-position gave improved enzyme potency, while substitution of the pyridone 4-position led to improved aqueous solubility and kinase selectivity. Analogue 10 demonstrated complete tumor stasis in a Met-dependent GTL-16 human gastric carcinoma xenograft model following oral administration. Because of its excellent in vivo efficacy and favorable pharmacokinetic and preclinical safety profiles, 10 has been advanced into phase I clinical trials.

Discovery of pyrrolopyridine-pyridone based inhibitors of Met kinase: synthesis, X-ray crystallographic analysis, and biological activities.

Conformationally constrained 2-pyridone analogue 2 is a potent Met kinase inhibitor with an IC50 value of 1.8 nM. Further SAR of the 2-pyridone based inhibitors of Met kinase led to potent 4-pyridone and pyridine N-oxide inhibitors such as 3 and 4. The X-ray crystallographic data of the inhibitor 2 bound to the ATP binding site of Met kinase protein provided insight into the binding modes of these inhibitors, and the SAR of this series of analogues was rationalized. Many of these analogues showed potent antiproliferative activities against the Met dependent GTL-16 gastric carcinoma cell line. Compound 2 also inhibited Flt-3 and VEGFR-2 kinases with IC50 values of 4 and 27 nM, respectively. It possesses a favorable pharmacokinetic profile in mice and demonstrates significant in vivo antitumor activity in the GTL-16 human gastric carcinoma xenograft model.

Pyrrolopyridazine MEK inhibitors.

The synthesis and SAR of a series of pyrrolopyridazine MEK inhibitors are reported. Optimal activity was achieved by incorporation of a 4-phenoxyaniline substituent at C4 and an acylated amine at C6.

Design, synthesis, and structure-activity relationships of tetrahydroquinoline-based farnesyltransferase inhibitors.

Tetrahydroquinoline-based small molecule inhibitors of farnesyltransferase (FT) have been identified. Lead compounds were shown to have nanomolar to sub-nanomolar activity in biochemical assays with excellent potency in a Ras-mutated cellular reversion assay. BMS-316810 (9e), a 0.7 nM FT inhibitor, was orally-active in a nude mouse tumor allograft efficacy study.