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A simple five-step process for the conversion of technical grade (+)-3-carene into 6,6-dimethylbicyclo[3.1.0]hexan-3-one was developed. A robust process was required that delivered the 6,6-dimethylbicyclo[3.1.0]hexan-3-one, minimized chromatography, reduced the excess of silver salts, and avoided toxic chromium oxidants. A simple and scalable process that relies on crystallization and distillation was developed and demonstrated to produce hundreds of grams of 6,6-dimethylbicyclo[3.1.0]hexan-3-one.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

In acute myelogenous leukemia (AML), the FLT3 receptor tyrosine kinase (RTK) is highly expressed with 30% of patients expressing a mutated, constitutively active form of this protein. To inhibit this receptor, VX-322 was developed and found to be very potent against both the FLT3 and c-KIT RTKs with enzyme K(i) values of1 nM and a cellular IC(50) between 1 and 5 nM. It was efficacious in a FLT3-ITD dependent myeloproliferative mouse model, doubling survival compared to other FLT3 inhibitors, with 25% of the mice cured. Upon treatment of primary AML patient blast cells, the dual inhibition of FLT3 and c-KIT was superior to inhibitors targeting a single RTK. Thus, this compound may represent an improved pharmacologic and selectivity profile that could be effective in the treatment of AML.

The MET receptor tyrosine kinase is involved in cell growth, survival, and invasion. Clinical studies with small molecule MET inhibitors have shown the role of biomarkers in identifying patients most likely to benefit from MET-targeted therapy. AMG 337 is an oral, small molecule, ATP-competitive, highly selective inhibitor of the MET receptor. Herein, we describe AMG 337 preclinical activity and mechanism of action in MET-dependent tumor models. These studies suggest MET is the only therapeutic target for AMG 337. In an unbiased tumor cell line proliferation screen (260 cell lines), a closely related analogue of AMG 337, Compound 5, exhibited activity in 2 of 260 cell lines; both were MET-amplified. Additional studies examining the effects of AMG 337 on the proliferation of a limited panel of cell lines with varying MET copy numbers revealed that high-level focal MET amplification (>12 copies) was required to confer MET oncogene addiction and AMG 337 sensitivity. One MET-amplified cell line, H1573 (>12 copies), was AMG 337 insensitive, possibly because of a downstream G12A KRAS mutation. Mechanism-of-action studies in sensitive MET-amplified cell lines demonstrated that AMG 337 inhibited MET and adaptor protein Gab-1 phosphorylation, subsequently blocking the downstream PI3K and MAPK pathways. AMG 337 exhibited potency in pharmacodynamic assays evaluating MET signaling in tumor xenograft models; >90% inhibition of Gab-1 phosphorylation was observed at 0.75 mg/kg. These findings describe the preclinical activity and mechanism of action of AMG 337 in MET-dependent tumor models and indicate its potential as a novel therapeutic for the treatment of MET-dependent tumors. Mol Cancer Ther; 15(7); 1568–79. ©2016 AACR.

Compound 1, (7-methoxy-N-((6-(3-methylisothiazol-5-yl)-[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine) is a potent, selective inhibitor of c-Met (mesenchymal-epithelial transition factor), a receptor tyrosine kinase that is often deregulated in cancer. Compound 1 displayed desirable pharmacokinetic properties in multiple preclinical species. Glutathione trapping studies in liver microsomes resulted in the NADPH-dependent formation of a glutathione conjugate. Compound 1 also exhibited very high in vitro NADPH-dependent covalent binding to microsomal proteins. Species differences in covalent binding were observed, with the highest binding in rats, mice, and monkeys (1100-1300 pmol/mg/h), followed by dogs (400 pmol/mg/h) and humans (144 pmol/mg/h). This covalent binding to protein was abolished by coincubation with glutathione. Together, these in vitro data suggest that covalent binding and glutathione conjugation proceed via bioactivation to a chemically reactive intermediate. The cytochrome (CYP) P450 enzymes responsible for this bioactivation were identified as cytochrome P450 3A4, 1A2, and 2D6 in human and cytochrome P450 2A2, 3A1, and 3A2 in rats. The glutathione metabolite was detected in the bile of rats and mice, thus demonstrating bioactivation occurring in vivo. Efforts to elucidate the structure of the glutathione adduct led to the isolation and characterization of the metabolite by NMR and mass spectrometry. The analytical data confirmed conclusively that the glutathione conjugation was on the 4-C position of the isothiazole ring. Such P450-mediated bioactivation of an isothiazole or thiazole group has not been previously reported. We propose a mechanism of bioactivation via sulfur oxidation followed by glutathione attack at the 4-position with subsequent loss of water resulting in the formation of the glutathione conjugate. Efforts to reduce bioactivation without compromising potency and pharmacokinetics were undertaken in order to minimize the potential risk of toxicity. Because of the exemplary pharmacokinetic/pharmacodynamic (PK/PD) properties of the isothiazole group, initial attempts were focused on introducing alternative metabolic soft spots into the molecule. These efforts resulted in the discovery of 7-(2-methoxyethoxy)-N-((6-(3-methyl-5-isothiazolyl)[1,2,4]triazolo[4,3-b]pyridazin-3-yl)methyl)-1,5-naphthyridin-4-amine (compound 2), with the major metabolic transformation occurring on the naphthyridine ring alkoxy substituent. However, a glutathione conjugate of compound 2 was produced in vitro and in vivo in a manner similar to that observed for compound 1. Furthermore, the covalent binding was high across species (360, 300, 529, 208, and 98 pmol/mg/h in rats, mice, dogs, monkeys, and humans, respectively), but coincubation with glutathione reduced the extent of covalent binding. The second viable alternative in reducing bioactivation involved replacing the isothiazole ring with bioisosteric heterocycles. Replacement of the isothiazole ring with an isoxazole or a pyrazole reduced the bioactivation while retaining the desirable PK/PD characteristics of compounds 1 and 2.

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. We previously showed that O-linked triazolopyridazines can be potent inhibitors of c-Met. Herein, we report the discovery of a related series of N-linked triazolopyridazines which demonstrate nanomolar inhibition of c-Met kinase activity and display improved pharmacodynamic profiles. Specifically, the potent time-dependent inhibition of cytochrome P450 associated with the O-linked triazolopyridazines has been eliminated within this novel series of inhibitors. N-linked triazolopyridazine 24 exhibited favorable pharmacokinetics and displayed potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver PD model. Once-daily oral administration of 24 for 22days showed significant tumor growth inhibition in an NIH-3T3/TPR-Met xenograft mouse efficacy model.

Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.

We have previously shown N-arylnaphthamides can be potent inhibitors of vascular endothelial growth factor receptors (VEGFRs). N-Alkyl and N-unsubstituted naphthamides were prepared and found to yield nanomolar inhibitors of VEGFR-2 (KDR) with an improved selectivity profile against a panel of tyrosine and serine/threonine kinases. The inhibitory activity of this series was retained at the cellular level. Naphthamides 3, 20, and 22 exhibited good pharmacokinetics following oral dosing and showed potent inhibition of VEGF-induced angiogenesis in the rat corneal model. Once-daily oral administration of 22 for 14 days led to 85% inhibition of established HT29 colon cancer and Calu-6 lung cancer xenografts at doses of 10 and 20 mg/kg, respectively.

The overexpression of c-Met and/or hepatocyte growth factor (HGF), the amplification of the MET gene, and mutations in the c-Met kinase domain can activate signaling pathways that contribute to cancer progression by enabling tumor cell proliferation, survival, invasion, and metastasis. Herein, we report the discovery of 8-fluorotriazolopyridines as inhibitors of c-Met activity. Optimization of the 8-fluorotriazolopyridine scaffold through the combination of structure-based drug design, SAR studies, and metabolite identification provided potent (cellular IC50 < 10 nM), selective inhibitors of c-Met with desirable pharmacokinetic properties that demonstrate potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.

Cis -1,3,5-cyclohexanetriol [C 6 H 9 (OH) 3 ] reacts with η 5 -(C 5 R 5 )TiCl 3 [R 5 = H 5 , Me 5 , (Me 3 Si)(H) 4 , 1,3-(Me 3 Si) 2 (H) 3 , (1,3-(t-Bu) 2 (H) 3 ] in the presence of three molar equivalents of triethylamine, affording substituted cyclopentadienyltitanium cis -1,3,5-cyclohexanetrialkoxide complexes with an adamantane-type structure. The complex η 5 -(C 5 Me 5 )Ti(O 3 C 6 H 9 ) is thermally stable to 220°C in the solid state, and a benzene solution of the complex η 5 -(1,3-t-Bu 2 C 5 H 3 )Ti(O 3 C 6 H 9 ) is stable to cleavage of the alkoxide ligand by three equivalents of H 2 O for more than 3 h. Attempts to cleave 1,3-t-Bu 2 C 5 H 4 from η 5 (1,3-t-Bu 2 C 5 H 3 )Ti(O 3 C 6 H 9 ) with electrophiles H + and Br 2 are reported.

A class of 2-acyliminobenzimidazoles has been developed as potent and selective inhibitors of anaplastic lymphoma kinase (ALK). Structure based design facilitated the rapid development of structure–activity relationships (SAR) and the optimization of kinase selectivity. Introduction of an optimally placed polar substituent was key to solving issues of metabolic stability and led to the development of potent, selective, orally bioavailable ALK inhibitors. Compound 49 achieved substantial tumor regression in an NPM-ALK driven murine tumor xenograft model when dosed qd. Compounds 36 and 49 show favorable potency and PK characteristics in preclinical species indicative of suitability for further development.

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

A high-throughput screen of our compound archive revealed a novel class of dual FMS-like tyrosine kinase 3 (FLT3)/c-KIT inhibitors. With the help of molecular modeling, this class was rapidly optimized for both potency against FLT3 and FLT3/c-KIT and excellent potency in cell-based assays, leading to dose-dependent cell death in acute myelogenous leukemia (AML) patient blast samples. Ultimately, the AML patient blast data defined the preferred target profile as we designed and evaluated a set of FLT3 selective and FLT3/c-KIT dual molecules. Further optimization for pharmacokinetic properties resulted in the selection of the dual FLT3/c-KIT inhibitor, N(3)-(4-(trans-4-morpholinocyclohexyl)phenyl)-1-(pyridin-2-yl)-1H-1,2,4-triazole-3,5-diamine, VX-322 (compound 37), to move forward to preclinical evaluation.

1,2,3,4-Tetrahydroisoquinolines and 3,4-dihydroisoquinoline-1(2H)-ones were identified as potent and selective inhibitors of KDR. The discovery, synthesis, and structure-activity relationships of these novel inhibitors are reported. In vitro metabolism and pharmacokinetic profiles of the most interesting compounds are discussed.

A series of naphthyl-based compounds were synthesized as potential inhibitors of vascular endothelial growth factor (VEGF) receptors. Investigations of structure–activity relationships led to the identification of a series of naphthamides that are potent inhibitors of the VEGF receptor tyrosine kinase family. Numerous analogues demonstrated low nanomolar inhibition of VEGF-dependent human umbilical vein endothelial cell (HUVEC) proliferation, and of these several compounds possessed favorable pharmacokinetic (PK) profiles. In particular, compound 48 demonstrated significant antitumor efficacy against established HT29 human colon adenocarcinoma xenografts implanted in athymic mice. A full account of the preparation, structure–activity relationships, pharmacokinetic properties, and pharmacology of analogues within this series is presented.

Signaling through the MET receptor tyrosine kinase and its ligand, hepatocyte growth factor (HGF), promotes cell proliferation, survival, and invasion. Activation of MET signaling is a relatively common hallmark of a diverse range of human cancer types, and as a result, inhibition of MET signaling represents an attractive therapeutic opportunity for the treatment of cancer. In this study, we describe the characterization of AMG 337, a potent and highly selective small molecule ATP-competitive MET kinase inhibitor that demonstrates robust activity in MET-dependent cancer models. In enzymatic assays, AMG 337 inhibited MET kinase activity with an IC50 of < 5nM nM. AMG 337 demonstrated exquisite selectivity for MET when profiled against a diverse panel of over 400 protein and lipid kinases in a competitive binding assay. In cellular assays, AMG 337 inhibited HGF-dependent MET phosphorylation with an IC50 of < 10 nM. To identify predictive genomic markers of response, AMG 337 was profiled in cell viability assays using a diverse panel of over 200 cancer cell lines. Treatment with AMG 337 only affected the viability of two gastric cancer cell lines (SNU-5 and Hs746T), both of which harbor amplification of the MET gene. The AMG 337 IC50 in the two sensitive cell lines was < 50 nM, and > 10 μM in all other tested cell lines. Further studies in an expanded panel of additional cancer cell lines derived from gastric, NSCLC, and esophageal cancer confirmed that the in-vitro anti-proliferative activity of AMG 337 correlated with amplification of MET. In those cell lines, treatment with AMG 337 inhibited downstream PI3K and MAPK signaling pathways, which translated into growth arrest as evidenced by an accumulation of cells in the G1 phase of the cell cycle, a concomitant reduction in DNA synthesis, and the induction of apoptosis. In vivo, oral administration of AMG 337 resulted in robust dose-dependent anti-tumor efficacy in MET amplified gastric cancer xenograft models, with inhibition of tumor growth consistent with the pharmacodynamic modulation of MET signaling. In conclusion, these findings illustrate the potential clinical utility of AMG 337 as a therapeutic agent for the treatment of tumors with evidence of dysregulated MET signaling, including MET amplification. A phase 1 clinical study is currently evaluating the safety, tolerability and pharmacokinectics of AMG 337 in patients with solid tumors. Citation Format: Paul E. Hughes, Yajing Yang, Karen Rex, Yihong Zhang, Paula J. Kaplan-Lefko, Sean Caenepeel, Jodi Moriguchi, Martin Broome, Deborah Choquette, Robert Radinsky, Richard Kendall, Angela Coxon, Isabelle Dussault. AMG 337, a novel, potent and selective MET kinase inhibitor, has robust growth inhibitory activity in MET-dependent cancer models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 728. doi:10.1158/1538-7445.AM2014-728

Anaplastic lymphoma kinase (ALK) is a tyrosine kinase that has been implicated as a driving oncogene in a number of cancers, including non-small cell lung cancer (NSCLC), anaplastic large cell lymphoma (ALCL), neuroblastoma and inflammatory myofibroblastic tumors (IMT). Numerous genetic aberrations at the ALK locus are observed in cancer including point mutations, amplifications, translocations and inversions. Inversions are exemplified by inv(2)(p21;p23), which leads to the constitutively active oncogenic fusion protein EML4-ALK present in ∼5% of NSCLC. Crizotinib, a dual cMet/ALK kinase inhibitor, was recently approved by the FDA for locally advanced or metastatic NSCLC that is ALK-positive, thereby validating ALK as therapeutic target. Here we describe the pharmacological characterization of a novel series of potent, selective and orally bioavailable ALK kinase inhibitors. Members of this series inhibit wild type ALK, NPM-ALK fusion and crizotinib resistant ALK[L1196M] kinase activity at sub-nanomolar concentrations, displaying up to ∼200 fold increased inhibitory activity over crizotinib. Kinase profiling indicate that members of this series display increased selectivity scores relative crizotinib. In Karpas-299 cells, selected compounds inhibited both pY1604 ALK activation (IC50 = 2 nM) and cell proliferation (IC50 = 1 nM). Members of this series were also evaluated in the EML4-ALK expressing NSCLC cell line H3122, and displayed equipotent inhibition of pY1604 ALK activation and inhibition of cell proliferation (both IC50 = 1 nM). Members of this class did not inhibit growth of an ALK negative lymphoma cell line (HT). The in vivo activity of this series was examined in the Karpas-299 ALCL xenograft model. Compound was dosed daily (PO) at 10, 30 and 60 mg/kg. Tumor growth inhibition was observed at all dose levels, and the highest dose level resulted in significant tumor regression (96%, p80% pALK inhibition was observed at the lowest doses tested, and complete inhibition was seen at doses of 30 mg/kg and above. In a direct comparison, members of this series achieved ALK inhibition in these tumors at a ∼15 fold lower plasma concentration than crizotinib. A PK/PD time course study was performed in the Karpas-299 model. A single 60 mg/kg dose of an inhibitor was able to maintain >90% ALK inhibition in tumors up to 24 hours post-dose, indicating significant tumor penetration and sustained ALK kinase inhibition. In conclusion, the described compounds are potent and selective inhibitors of ALK kinase, possess an impressive efficacy profile and drug-like pharmacokinetic properties. These features together indicate the potential for significant advantages over crizotinib. There is a compelling case for their clinical evaluation in patients with ALK-driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1795. doi:1538-7445.AM2012-1795

c-Met is a receptor tyrosine kinase with oncogenic potential for which several small molecule inhibitors are currently being tested in clinical trials. There are suggestions in the literature that c-Met inhibitors could have anti-tumor effects under a variety of contexts, including in tumors in which the MET gene is amplified, contains a gain-of-function mutation or in tumors with receptor overexpression. We have developed ATP-competitive c-Met inhibitors that possess different selectivity profiles and fall into two categories. Class I molecules are highly selective for c-Met while class II molecules are multi-kinase inhibitors. These molecules were profiled in vitro and in vivo to identify biomarkers of c-Met dependence. We show that both classes of molecules inhibited c-Met kinase activity in vitro and in vivo. However, class I and class II molecules differed extensively in their spectrum of anti-tumor activity. The anti-proliferative effects of the different c-Met inhibitors were tested in 359 cancer cell lines in vitro. The activity of class I molecules was exclusively restricted to cell lines that harbor MET amplification. Western blotting in sensitive or resistant cell lines showed that c-Met inhibitors effectively block signaling downstream of c-Met only in MET amplified cell lines. In vivo, class I molecules inhibited only the growth of tumor models that are highly dependent on c-Met activity due to MET amplification or due to a hepatocyte growth factor (HGF)-driven autocrine loop. The same models that were sensitive to class I inhibitors were also sensitive to class II inhibitors. However, class II inhibitors had additional anti-tumor activities in vitro and in vivo in models that did not respond to class I inhibitors. The majority of cell lines that responded only to a class II molecule in vitro required much higher drug concentrations than those required to inhibit the growth of MET amplified cell lines. Finally, the growth inhibitory effects observed in vitro and in vivo were consistent with the selectivity profiles of the different c-Met inhibitors. Together our data demonstrate that MET amplification is a clinically identifiable, potential sensitivity biomarker for selective c-Met kinase inhibitors. c-Met expression alone is not a sensitivity biomarker in preclinical models. The spectrum of c-Met inhibitor sensitive models defined in this work could be used to understand whether other small molecule c-Met inhibitors are exquisitely selective for c-Met or harbor additional activities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3558. doi:10.1158/1538-7445.AM2011-3558