Structure-activity relationship study of central pyridine-derived TYK2 JH2 inhibitors: Optimization of the PK profile through C4' and C6 variations.

Efforts directed at improving potency and preparing structurally different TYK2 JH2 inhibitors from the first generation of compounds such as 1a led to the SAR study of new central pyridyl based analogs 2-4. The current SAR study resulted in the identification of 4h as a potent and selective TYK2 JH2 inhibitor with distinct structural differences from 1a. In this manuscript, the in vitro and in vivo profiles of 4h are described. The hWB IC50 of 4h was shown as 41 nM with 94% bioavailability in the mouse PK study.

Long-Acting Tumor-Activated Prodrug of a TGFßR Inhibitor.

Inhibition of TGFß signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFß receptor (TGFßR) inhibitors in cancer therapy. To restrict the activity of TGFßR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFßR1 inhibitor 1 were prepared by appending 1 to a serine protease substrate and a half-life extension fatty acid carbon chain. The prodrugs were shown to be selectively metabolized in tumor tissues relative to the heart and blood and demonstrated a prolonged favorable increase in the tumor-to-heart ratio of the active drug in tissue distribution studies. Once-weekly administration of the most tissue-selective compound 10 provided anti-tumor efficacy comparable to the parent compound and reduced systemic exposure of the active drug.

Discovery of BMS-986202: A Clinical Tyk2 Inhibitor that Binds to Tyk2 JH2.

A search for structurally diversified Tyk2 JH2 ligands from 6 (BMS-986165), a pyridazine carboxamide-derived Tyk2 JH2 ligand as a clinical Tyk2 inhibitor currently in late development for the treatment of psoriasis, began with a survey of six-membered heteroaryl groups in place of the N-methyl triazolyl moiety in 6. The X-ray co-crystal structure of an early lead (12) revealed a potential new binding pocket. Exploration of the new pocket resulted in two frontrunners for a clinical candidate. The potential hydrogen bonding interaction with Thr599 in the pocket was achieved with a tertiary amide moiety, confirmed by the X-ray co-crystal structure of 29. When the diversity search was extended to nicotinamides, a single fluorine atom addition was found to significantly enhance the permeability, which directly led to the discovery of 7 (BMS-986202) as a clinical Tyk2 inhibitor that binds to Tyk2 JH2. The preclinical studies of 7, including efficacy studies in mouse models of IL-23-driven acanthosis, anti-CD40-induced colitis, and spontaneous lupus, will also be presented.

Identification of Imidazo[1,2-b]pyridazine Derivatives as Potent, Selective, and Orally Active Tyk2 JH2 Inhibitors.

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

Identification of potent tricyclic prodrug S1P1 receptor modulators.

Recently, our research group reported the identification of prodrug amino-alcohol 2 as a potent and efficacious S1P1 receptor modulator. This molecule is differentiated preclinically over the marketed drug fingolimod (Gilenya 1), whose active phosphate metabolite is an S1P1 full agonist, in terms of pulmonary and cardiovascular safety. S1P1 partial agonist 2, however, has a long half-life in rodents and was projected to have a long half-life in humans. The purpose of this communication is to disclose highly potent partial agonists of S1P1 with shorter half-lives relative to the clinical compound 2. PK/PD relationships as well as their preclinical pulmonary and cardiovascular safety assessment are discussed.

Structure-Based Design of Selective Janus Kinase 2 Imidazo[4,5-d]pyrrolo[2,3-b]pyridine Inhibitors.

Early hit to lead work on a pyrrolopyridine chemotype provided access to compounds with biochemical and cellular potency against Janus kinase 2 (JAK2). Structure-based drug design along the extended hinge region of JAK2 led to the identification of an important H-bond interaction with the side chain of Tyr 931, which improved JAK family selectivity. The 4,5-dimethyl thiazole analogue 18 demonstrated high levels of JAK family selectivity and was identified as a promising lead for the program.

Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms.

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors.

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

9H-Carbazole-1-carboxamides as potent and selective JAK2 inhibitors.

The discovery, synthesis, and characterization of 9H-carbazole-1-carboxamides as potent and selective ATP-competitive inhibitors of Janus kinase 2 (JAK2) are discussed. Optimization for JAK family selectivity led to compounds 14 and 21, with greater than 45-fold selectivity for JAK2 over all other members of the JAK kinase family.

Optimization of benzodiazepinones as selective inhibitors of the X-linked inhibitor of apoptosis protein (XIAP) second baculovirus IAP repeat (BIR2) domain.

The IAPs are key regulators of the apoptotic pathways and are commonly overexpressed in many cancer cells. IAPs contain one to three BIR domains that are crucial for their inhibitory function. The pro-survival properties of XIAP come from binding of the BIR domains to the pro-apoptotic caspases. The BIR3 domain of XIAP binds and inhibits caspase 9, while the BIR2 domain binds and inhibits the terminal caspases 3 and 7. While XIAP BIR3 inhibitors have previously been reported, they also inhibit cIAP1/2 and promote the release of TNFα, potentially limiting their therapeutic utility. This paper will focus on the optimization of selective XIAP BIR2 inhibitors leading to the discovery of highly potent benzodiazepinone 36 (IC50 = 45 nM), which has high levels of selectivity over XIAP BIR3 and cIAP1 BIR2/3 and shows efficacy in a xenograft pharmacodynamic model monitoring caspase activity while not promoting the release of TNFα in vitro.

Benzazepinones and benzoxazepinones as antagonists of inhibitor of apoptosis proteins (IAPs) selective for the second baculovirus IAP repeat (BIR2) domain.

XIAP is a key regulator of apoptosis, and its overexpression in cancer cells may contribute to their survival. The antiapoptotic function of XIAP derives from its BIR domains, which bind to and inhibit pro-apoptotic caspases. Most known IAP inhibitors are selective for the BIR3 domain and bind to cIAP1 and cIAP2 as well as XIAP. Pathways activated upon cIAP binding contribute to the function of these compounds. Inhibitors selective for XIAP should exert pro-apoptotic effects through competition with the terminal caspases. This paper details our synthetic explorations of a novel XIAP BIR2-selective benzazepinone screening hit with a focus on increasing BIR2 potency and overcoming high in vivo clearance. These efforts led to the discovery of benzoxazepinone 40, a potent BIR2-selective inhibitor with good in vivo pharmacokinetic properties which potentiates apoptotic signaling in a manner mechanistically distinct from that of known pan-IAP inhibitors.

Identification of a phenylacylsulfonamide series of dual Bcl-2/Bcl-xL antagonists.

A series of phenylacylsulfonamides has been prepared as antagonists of Bcl-2/Bcl-xL. In addition to potent binding affinities for both Bcl-2 and Bcl-xL, these compounds were shown to induce classical markers of apoptosis in isolated mitochondria. Overall weak cellular potency was improved by the incorporation of polar functionality resulting in compounds with moderate antiproliferative activity.

Pyrazole and pyrimidine phenylacylsulfonamides as dual Bcl-2/Bcl-xL antagonists.

5-Butyl-1,4-diphenyl pyrazole and 2-amino-5-chloro pyrimidine acylsulfonamides were developed as potent dual antagonists of Bcl-2 and Bcl-xL. Compounds were optimized for binding to the I88, L92, I95, and F99 pockets normally occupied by pro-apoptotic protein Bim. An X-ray crystal structure confirmed the proposed binding mode. Observation of cytochrome c release from isolated mitochondria in MV-411 cells provides further evidence of target inhibition. Compounds demonstrated submicromolar antiproliferative activity in Bcl-2/Bcl-xL dependent cell lines.

Pyrrolo[1,2-f]triazines as JAK2 inhibitors: achieving potency and selectivity for JAK2 over JAK3.

SAR studies of pyrrolo[1,2-f]triazines as JAK2 inhibitors is presented. Achieving JAK2 inhibition selectively over JAK3 is discussed.

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.

The antiangiogenic activity in xenograft models of brivanib, a dual inhibitor of vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 kinases.

Tumor angiogenesis is a complex and tightly regulated network mediated by various proangiogenic factors. The fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) family of growth factors, and associated tyrosine kinase receptors have a major influence in tumor growth and dissemination and may work synergistically to promote angiogenesis. Brivanib alaninate is the orally active prodrug of brivanib, a selective dual inhibitor of FGF and VEGF signaling. Here, we show that brivanib demonstrates antitumor activity in a broad range of xenograft models over multiple dose levels and that brivanib alaninate shows dose-dependent efficacy equivalent to brivanib in L2987 human tumor xenografts. Brivanib alaninate (107 mg/kg) reduced tumor cell proliferation as determined by a 76% reduction in Ki-67 staining and reduced tumor vascular density as determined by a 76% reduction in anti-CD34 endothelial cell staining. Furthermore, Matrigel plug assays in athymic mice showed that brivanib alaninate inhibited angiogenesis driven by VEGF or basic FGF alone, or combined. Dynamic contrast-enhanced magnetic resonance imaging, used to assess the effects of brivanib alaninate on tumor microcirculation, showed a marked decrease in gadopentetate dimeglumine contrast agent uptake at 107 mg/kg dose, with a reduction in area under the plasma concentration-time curve from time 0 to 60 minutes at 24 and 48 hours of 54% and 64%, respectively. These results show that brivanib alaninate is an effective antitumor agent in preclinical models across a range of doses, and that efficacy is accompanied by changes in cellular and vascular activities.

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.

Metabolism of 5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737): identification of an unusual N-acetylglucosamine conjugate in the cynomolgus monkey.

5-Isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737) is a potent and selective vascular endothelial growth factor receptor-2 antagonist. In this study, liquid chromatography/tandem mass spectrometry and NMR were used to investigate the biotransformation of BMS-645737 in vitro and in the cynomolgus monkey, dog, mouse, and rat. Metabolic pathways for BMS-645737 included multistep processes involving both oxidation and conjugation reactions. For example, the 2-methyl-1H-pyrrolo moiety underwent cytochrome P450-catalyzed hydroxylation followed by oxidation to a carboxylic acid and then conjugation with taurine. Alternatively, the 5-methyl-1,3,4-oxadiazol-2-yl moiety was metabolized by hydroxylation and then conjugation with sulfate. The pyridin-5-yl group underwent direct glucuronidation in hepatocytes (dog, monkey, human) and conjugation with N-acetylglucosamine in the monkey. Conjugation with glutathione and processing along the mercapturic acid pathway was a minor metabolic pathway in vivo, although BMS-645737 did not form conjugates in the presence of glutathione-supplemented liver microsomes. Other minor biotransformation pathways included oxidative dehydrogenation, dihydroxylation, and hydrolytic opening of the oxadiazole ring followed by either deacetylation or hydrolysis of the resulting diacyl hydrazide. Whereas previous studies have shown the formation of N-acetylglucosamine conjugates of alcohols, arylamines, and other small molecules, this report describes the biotransformation of a heterocyclic aromatic amine via direct conjugation with N-acetylglucosamine.

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.

Discovery of orally active pyrrolopyridine- and aminopyridine-based Met kinase inhibitors.

A series of acylurea analogs derived from pyrrolopyridine and aminopyridine scaffolds were identified as potent inhibitors of Met kinase activity. The SAR at various positions of the two kinase scaffolds was investigated. These studies led to the discovery of compounds 3b and 20b, which demonstrated favorable pharmacokinetic properties in mice and significant antitumor activity in a human gastric carcinoma xenograft model.