Frank Stieber

EMD 1214063 and EMD 1204831 Constitute a New Class of Potent and Highly Selective c-Met Inhibitors

Purpose: The mesenchymal–epithelial transition factor (c-Met) receptor, also known as hepatocyte growth factor receptor (HGFR), controls morphogenesis, a process that is physiologically required for embryonic development and tissue repair. Aberrant c-Met activation is associated with a variety of human malignancies including cancers of the lung, kidney, stomach, liver, and brain. In this study, we investigated the properties of two novel compounds developed to selectively inhibit the c-Met receptor in antitumor therapeutic interventions. Experimental Design: The pharmacologic properties, c-Met inhibitory activity, and antitumor effects of EMD 1214063 and EMD 1204831 were investigated in vitro and in vivo, using human cancer cell lines and mouse xenograft models. Results: EMD 1214063 and EMD 1204831 selectively suppressed the c-Met receptor tyrosine kinase activity. Their inhibitory activity was potent [inhibitory 50% concentration (IC50), 3 nmol/L and 9 nmol/L, respectively] and highly selective, when compared with their effect on a panel of 242 human kinases. Both EMD 1214063 and EMD 1204831 inhibited c-Met phosphorylation and downstream signaling in a dose-dependent fashion, but differed in the duration of their inhibitory activity. In murine xenograft models, both compounds induced regression of human tumors, regardless of whether c-Met activation was HGF dependent or independent. Both drugs were well tolerated and induced no substantial weight loss after more than 3 weeks of treatment. Conclusions: Our results indicate selective c-Met inhibition by EMD 1214063 and EMD 1204831 and strongly support clinical testing of these compounds in the context of molecularly targeted anticancer strategies. Clin Cancer Res; 19(11); 2941–51. ©2013 AACR.

Identification and optimization of pyridazinones as potent and selective c-Met kinase inhibitors.

In a high-throughput screening campaign for c-Met kinase inhibitors, a thiadiazinone derivative with a carbamate group was identified as a potent in vitro inhibitor. Subsequent optimization guided by c-Met-inhibitor X-ray structures furnished new compound classes with excellent in vitro and in vivo profiles. The thiadiazinone ring of the HTS hit was first replaced by a pyridazinone followed by an exchange of the carbamate hinge binder with a 1,5-disubstituted pyrimidine. Finally an optimized compound, 22 (MSC2156119), with excellent in vitro potency, high kinase selectivity, long half-life after oral administration and in vivo anti-tumor efficacy at low doses, was selected as a candidate for clinical development.

Design and synthesis of isoquinolines and benzimidazoles as RAF kinase inhibitors

RAF kinase plays a critical role in the RAF-MEK-ERK signaling pathway and inhibitors of RAF could be of use for the treatment of various cancer types. We have designed potent RAF-1 inhibitors bearing novel bicyclic heterocycles as key structural elements for the interaction with the hinge region. In both series exploration of the SAR was focussed on the substitution of the phenyl ring, which binds to the induced fit pocket. Overall, it was confirmed that incorporation of lipophilic substituents was needed for potent Raf inhibition and a number of potent analogues were obtained.

Abstract 1787: The c-Met inhibitors EMD 1214063 and EMD 1204831 are effective in combination with EGFR and VEGF inhibitors in NSCLC models

c-Met is a receptor tyrosine kinase that has hepatocyte growth factor as its ligand. Evidence from biochemical and human genetic studies indicate that c-Met is one of the most frequently activated tyrosine kinases in human cancer. Dysregulated c-Met signaling can result in the development of tumors highly dependent on c-Met. Furthermore, enhanced c-Met signaling is likely involved in conferring resistance to epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF) inhibitors. The orally available EMD 1214063 and EMD 1204831 inhibit c-Met activity in a potent and highly selective fashion, and are currently being evaluated in cancer patients. Here we report the activity of these compounds in combination with inhibitors of EGFR and VEGF in various tumor xenograft models. EMD 1214063 was tested alone and in combination with EGFR inhibitors (erlotinib and cetuximab) in xenografts of erlotinib-resistant tumor cells, namely the lung cancer H1975 and the NCI-H441 non-small-cell lung cancer (NSCLC) cell lines. The H1975 cell line expresses moderate amounts of c-Met, and EMD 1214063 was inactive as a single agent (treatment group/control group [T/C] 78%; tumor growth delay [TGD] 2 days). Cetuximab was active with a T/C of 13% inducing a TGD of 21 days. The combination of EMD 1214063 and cetuximab was active with a T/C of α1% and a TGD of 42 days. The NCI-H441 cell line is characterized by constitutive c-Met overexpression and is sensitive to c-Met inhibitors in vivo. EMD 1214063 (100 mg/kg) was active inducing a T/C of 20%, while erlotinib alone (30 mg/kg) was not efficacious. However, the combination of EMD 1214063 with erlotinib enhanced tumor growth inhibition, induced partial tumor regression in 2/9 mice, and delayed tumor re-growth. These data are compatible with the hypothesis that cMet pathway activation confers resistance to EGFR inhibitors. We additionally investigated the combination of either EMD 1214063 or EMD 1204831 with VEGF inhibitors in NSCLC models. In NCI-H441 xenografts, EMD 1204831 produced a modest T/C (66%) at the suboptimal dose of 25 mg/kg/bid, similarly to aflibercept at 40 mg/kg (T/C 55%). In contrast, the combination of both compounds yielded a T/C of 1% with partial responses in 2/10 mice. In the EBC-1 model, treatment with EMD 1214063 (10 mg/kg) or with the antiVEGF antibody B20-4.1 (20 mg/kg) as single agents resulted in a T/C of β21% (with partial regression in 1/10 mice) and 19%, respectively. In contrast, the combination of EMD 1214063 and B20-4.1 exhibited enhanced anti-tumor activity with a T/C of −72% and partial regressions in 10/10 mice. These data support the hypothesis that EMD 1214063 or EMD 1204831 may be effective in NSCLC patients in combination with VEGF-targeting agents. In conclusion, our findings provide a rationale to evaluate the efficacy of the combination of EMD 1214063 or EMD 1204831 with EGFR and VEGF inhibitors in NSCLC patients. 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 1787. doi:1538-7445.AM2012-1787

Abstract 2786: Identification and preclinical characterization of EMD 1204831 – A selective c-Met kinase inhibitor in clinical phase 1

The involvement of the mesenchymal endothelial transition factor (c-Met) in the primary event of oncogenic transformation and the secondary ability to mediate metastatic spread has been convincingly demonstrated in preclinical and early clinical settings. The clinical benefits of c-Met kinase inhibitors with various modes of actions and selectivity profiles are currently under investigation hoping that inhibitors of c-Met might emerge as valuable cancer therapeutics in the future. During an HTS run 3-(diethylamino)propyl N-[3-[[5-(3,4-dimethoxyphenyl)-2-oxo-6H-1,3,4-thiadiazin-3-yl]methyl]phenyl]carbamate was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): >100 µg/ml, IC 50 (cMet enzyme): 30 nM, IC 50 (cMet A549): 800 nM) providing a valid starting point. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting with the main chain nitrogen atom of Met1160 within the hinge region and with the main chain nitrogen of Asp1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1204831 was identified as development compound. EMD1204831 is currently being investigated in a phase 1 clinical trial. The pyridazinone EMD1204831 inhibits enzymatic and cellular c-Met kinase activity with IC 50 values of 12 nM and 15 nM, respectively. EMD1204831 displayed an exquisite selectivity when tested in vitro against a panel of more than 400 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes. EMD 1204831 demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models, including U87-MG glioblastoma cells (autocrine HGF expression), TPR-Met-transformed mouse fibroblasts (oncogenic Met fusion protein) or Hs746T gastric cancer cells (c-Met gene amplification and HGF-independent activation). Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. PK/PD analysis revealed efficient, dose- and time-dependent inhibition of c-Met phosphorylation, reduction of IL-8 and cyclin D1 expression as well as an induction of the cell cycle inhibitor p27. The overall profile of EMD1204831 including first time structural disclosure, some structure activity relationships, in vitro potency, selectivity profile and in-vivo data will be presented. 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 2786. doi:10.1158/1538-7445.AM2011-2786

Abstract 5777: EMD 1214063, an exquisitely selective c-Met kinase inhibitor in clinical phase 1

The role of the receptor tyrosin kinase c-Met in tumor progression, metastasis and aggressiveness has been convincingly demonstrated in preclinical and early clinical settings. Several compounds with different selectivity profiles inhibiting c-Met are currently under preclinical/clinical investigation and might emerge as valuable cancer therapeutics in the future. During an HTS run N-(3-(3,6-Dihydro-5-(3,4-dimethoxyphenyl)-2-oxo-2H-1,3,4-thiadiazin-3-ylmethyl)-phenyl)-carbaminic acid-(3-(N,N-diethylamino)-propylester) (1) was identified as an attractive lead structure with an interesting overall profile (clogD (7.4): 2.5, S (pH 7.4): >100 µg/ml, IC 50 (c-Met in vitro): 30 nM, IC 50 (c-Met cellular): 800 nM) providing a valid starting point for lead optimization. The co-crystal structure of 1 revealed the binding mode and the essential structural features. The initial HTS hit was bound in a DFG-in conformation interacting namely with the main chain nitrogen atom of methionine 1160 within the hinge region and with the main chain nitrogen of aspartic acid 1222. After subsequent optimization of potency, efficacy, PK properties and the safety profile of thiadiazinone 1, EMD1214063 was identified as candidate for further development and is currently investigated in a phase 1 clinical trial. The pyridazinone EMD1214063 inhibits enzymatic and cellular c-Met kinase activity with IC 50 values in the low nanomolar range. This compound displayed an exquisite selectivity when tested in vitro against a panel of more than 250 potential off-targets, including kinases, GPCRs, ion channels, transporters and various enzymes and demonstrated excellent anti-tumor activity in vivo in a variety of xenograft models. Depending on the particular model, complete regressions were observed with doses as low as 6 mg/kg/d administered per os. The overall profile of EMD1214063 including first time disclosure of the precise structure, synthesis, structure activity relationships, in vitro potency, selectivity profile, pharmacokinetic and in vivo data will be discussed. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5777.

Abstract 3622: Preclinical characterization of EMD1214063, a potent and highly selective inhibitor of the c-Met kinase in Phase I clinical trials

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DCnnc-Met, the receptor for hepatocyte growth factor (HGF), is a well characterized receptor tyrosine kinase, which is crucial for cell functions, such as survival, proliferation, motility and migration. Activating point mutations of c-Met have been identified in human malignancies, including renal carcinoma and lung cancer and constitutive activation of the HGF/c-Met pathway leads to tumor development and progression in cancer animal models. In the light of these findings, c-Met has become a key target for oncology therapeutics.nnIn order to develop c-Met specific inhibitors, we have conducted a biochemical HTS run with the recombinant kinase domain of c-Met. This approach has enabled us to identify a new class of c-Met inhibitors. Our report will focus on pharmacological and pre-clinical data, characterising EMD1214063, a promising therapeutic candidate currently under clinical testing.nnInhibition of the c-Met kinase by EMD1214063 was potent and highly selective. In in vitro kinase reactions and binding assays, EMD1214063 at a dose of 1μM, inhibited only 5 additional kinases in a panel of more than 250 different potential off-targets more than 50%. In vitro, EMD1214063 interfered with survival, anchorage-independent growth and HGF-induced migration of tumor cells, by blocking HGF-dependent as well as constitutive phosphorylation of c-Met.nnIn vivo, oral administration of EMD1214063 resulted in a strong inhibition - ranging from delayed growth to complete regression - of HGF-dependent and -independent c-Met driven tumor xenografts. Such anti-tumor activity was observed at a broad dose range, suggesting that EMD1214063 has a wide therapeutic index. The inhibitor was well tolerated, as indicated by the lack of significant weight loss in treated mice. The mechanism of action of EMD1214063 was investigated in vivo in a series of PK/PD studies. Administration of a single dose of EMD1214063 induced a complete and long-lasting inhibition of c-Met phosphorylation in the tumor xenografts. Furthermore, it strongly reduced the plasma levels of tumor-derived IL-8. c-Met phosphorylation and plasma IL-8 levels decreased as a function of the drug concentration in the tumor and plasma.nnTaken together, the high c-Met selectivity, potency and in vivo efficacy of EMD1214063 render this compound a very promising candidate for the treatment of patients bearing c-Met-driven tumors.nnCitation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3622.

Abstract B252: Identification of a new class of highly potent and selective c‐Met kinase inhibitors

The role of the receptor tyrosin kinase c‐Met in tumor progression and metastasis has been shown in preclinical studies and in early clinical settings. Several c‐Met inhibitors with different selectivity profiles are currently in preclinical/clinical investigation and might provide effective cancer therapeutics in the future. A biochemical HTS run using the recombinant kinase domain of c‐Met enabled us to identify N‐(3‐(3,6‐Dihydro‐5‐(3,4‐dimethoxyphenyl)‐2‐oxo‐2H‐1,3,4‐thiadiazin‐3‐ylmethyl)‐phenyl)‐carbaminic acid‐(3‐(N,N‐diethylamino)‐propylester) representing as new chemical class of kinase inhibitors. This HTS hit inhibited c‐Met in vitro kinase activity and HGF‐induced c‐Met phosphorylation in A459 lung cancer cells with IC50 values well below 1 µM. The analysis of co‐crystal structures defined the molecular requirements for potency and selectivity and guided the subsequent chemical optimization. As a result of the lead optimization process, two candidate compounds with distinct pharmacological properties were synthesized, EMD 1214063 and EMD 1204831. Both compounds are highly potent and efficient inhibitors of c‐Met in vitro kinase activity and c‐Met auto‐phosphorylation induced in vitro by HGF stimulation or by c‐Met overexpression. Both compounds displayed an exquisite selectivity profile when tested in vitro against a panel of more than 250 potential off‐targets, including kinases, GPCRs, ion channels, transporters and various enzymes. EMD 1214063 and EMD 1204831 exhibited excellent anti‐tumor activity in vivo in a variety of xenograft models, including U87‐MG glioblastoma cells (autocrine HGF production), TPR‐Met‐transformed mouse fibroblasts (oncogenic Met fusion protein) or Hs746T gastric and EBC‐1 lung cancer cells (c‐Met gene amplification and HGF‐independent activation). In c‐Met‐driven models complete regressions were observed with doses as low as 6 mg/kg/d administered per os. PK/PD analysis revealed efficient, dose‐ and time‐dependent inhibition of c‐Met phosphorylation, reduction of IL‐8 and cyclin D1 expression as well as an induction of the cell cycle inhibitor p27. The pharmacodynamic effects of EMD 1214063 were longlasting and allowed various schedule variations without compromising its anti‐tumor activity, while c‐Met inhibition by EMD 1204831 was rather transient, allowing optimal anti‐tumor activity when the compound was applied once or twice daily. The profile of EMD 1214063 and EMD 1204831 including synthesis, structure activity relationships, X‐ray structures and a comprehensive pharmacological in vitro and in vivo characterization will be presented. Furthermore, potential pharmacodynamic and predictive biomarkers will be discussed. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B252.