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Introduction: During our quest to find novel kinase inhibitor motifs for our protein kinase research projects we discovered a novel series of macrocyclic structures which possess unique but tunable kinase inhibitory profiles. Here we present the chemical structure of SB1317 and the SAR studies leading to the discovery of this novel CDK/FLT3/JAK2 inhibitor, currently in clinical trials for hematological malignancies. Methods/Results: The unusual structure of SB1317 is characterized by a biaryl connection to the hinge-binding 2-anilinopyrimidine and an allylic tertiary amine – homo allylic ether linker forming an 18-membered macrocyclic ring. Combination of the biaryl motif and unsaturation in the linker confers limited conformational freedom and may explain the specific biological activity observed with this series. Following extensive SAR studies a single preferred compound, SB1317, emerged with potent CDK (IC50 against CDKs 1, 2 and 9 = 9, 5 and 3 nM, respectively), FLT3 (IC50 = 19nM) and JAK2 (IC50 = 19nM) potency. SB1317 has low molecular weight and good physicochemical properties conferring good oral exposure and prompting its further evaluation in vitro and in in vivo cancer models. Following promising efficacy data in these studies, SB1317 was nominated as the preferred candidate for progression to the clinic. SB1317 was synthesized via a ring-closing metathesis (RCM) strategy where the macrocycle was formed from coupling the two halves of the molecule to provide a diene which was then closed by RCM using either Grubbs or Zhan catalysts. Crucially the RCM reaction requires acidic conditions to neutralize the basic centre to allow the metathesis to proceed. Conclusions: SB1317 is a low molecular weight compact macrocyclic structure with a unique spectrum of kinase activity suggesting it may have particular utility in the treatment of hematological malignancies and potential for solid tumor therapy as well. SB1317 is currently being developed in the clinic by Tragara Pharmaceuticals (San Diego, CA) under the name TG02. 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 3591. doi:10.1158/1538-7445.AM2011-3591

Introduction: Janus kinase 2 (JAK2) and fms-related tyrosine kinase 3 (FLT3) are important tyrosine kinases in hematopoietic progenitor cell differentiation and proliferation. Aberrant signaling of JAK2 or FLT3 is implicated in various myeloid malignancies. SB1518, a new chemical entity, is an inhibitor of both JAK2 (IC50 = 22 nM) and its JAK2V617F mutant (IC50 = 19 nM) as well as FLT3 (IC50 = 22 nM) and its mutant FLT3D835Y (IC50 = 6 nM). It is currently being evaluated in Phase I and II clinical trials for advanced hematologic malignancies and is showing promising therapeutic effects. Here we present a pharmacological evaluation of SB1518 in in vitro and in vivo models of myeloid disease that provide a rationale for its selection for clinical development. Methods: The inhibitory effect of SB1518 on JAK2, FLT3 and STAT signaling was evaluated by Western-blot analysis in cell lines with different JAK2/FLT3 mutation status. Cell proliferation assays using the CellTiterGlo® assay were performed on a panel of different cancer cell lines and primary cells from patients. Selected cells were treated with SB1518 to determine effects on apoptosis (Caspase 3/7 activation) and cell cycle (propidium iodide stain). SET-2 cells (a megakaryoblastic cell line derived from an ET-patient bearing JAK2V617F) and MOLM-13 cells (an AML cell line bearing FLT3-ITD) were used to establish subcutaneous tumors in nude mice to test the anti-tumor efficacy of SB1518 after oral daily dosing. Results: SB1518 inhibits phosphorylation of JAK2 at Y221, FLT3 and of STAT proteins and shows anti-proliferative activity in cell lines driven by mutant or wt JAK2 or FLT3-ITD (IC50 range of 47-348 nM), leading to cell cycle arrest (in G0/1) and induction of apoptosis. Cells bearing JAK2 or FLT-3 mutations or genetic alterations leading to high expression of JAK2, are the most sensitive to the anti-proliferative effects of SB1518, as are primary cells isolated from PV or AML patients. In these cells SB1518 induces cell cycle arrest and apoptosis. SB1518 has favorable physicochemical properties and pharmacokinetics allowing oral dosing. It significantly blocks tumor growth in a subcutaneous model of megakaryoblastic AML (SET-2 cells, TGI = 61% at 150 mg/kg b.i.d.). In a model of late stage, aggressive AML (MOLM-13), SB1518 not only blocked tumor growth (TGI = 81%, at 150 mg/kg b.i.d.), but also significantly reduced lung metastasis. In both models SB1518 was very well tolerated and did not induce cytopenia. Conclusions: SB1518, a novel, orally bio-available small molecule inhibitor of JAK2/FLT3, shows promising activities in various hematological cancer cell lines as well as primary cells from patients. It is efficacious in mouse xenograft models of FLT-3 dependent MOLM-13 cells and JAK2-dependent SET-2 cells. SB1518 is currently undergoing evaluation in Phase 2 clinical trials in myelofibrosis (post-PV, post-ET and primary) and has shown encouraging clinical activity. 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 3570. doi:10.1158/1538-7445.AM2011-3570

Introduction: Here we present the unique chemical structure of SB1518 and SAR studies leading to the discovery of this exciting novel JAK2/FLT3 inhibitor, currently in clinical trials for myelofibrosis and lymphoma. Methods/Results: As part of our innovative chemical strategy towards creating novel kinase scaffolds we discovered a series of unusual small molecular weight macrocycles which were optimized for selective JAK2 inhibitory potency culminating in the discovery of SB1518, which is selective for JAK2 (IC50 = 22nM) and its V617F mutant (IC50 = 19nM) over JAK1 (IC50 > 1µM) and JAK3 (IC50 > 0.5µM). FLT3 was identified as an additional kinase target (IC50 = 22nM and 6nM for its D835Y mutant) giving a broader potential in other hematological malignancies. The exciting and novel chemical structures of these compounds led us into a full lead optimization program to take full advantage of the unique nature of these molecules. SAR studies modifying the macrocyclic linker allowed unwanted protein kinase activity to be tuned out, whilst retaining a narrow spectrum of desired JAK2 kinase activity and selectivity over JAK1 and JAK3, a distinguishing feature. SB1518 and analogues were synthesized via a ring-closing metathesis (RCM) strategy: coupling of the two halves of the molecule provided a diene which was closed by RCM using either Grubbs or Zhan catalysts. SAR investigations, led by structure-based design, influenced the choice and position of a critical solubilising side-chain. Preferred compounds had improved aqueous solubility and high permeability. SB1518 is very stable both chemically and metabolically with a long half life in human microsomal preparations. With the desired balance of potency and oral pharmacokinetics, SB1518 was selected for further profiling. SB1518 demonstrated potent activity in cell lines driven by mutant JAK2 or FLT3 and was efficacious at very well tolerated oral doses in tumor models generated with these cell lines. Based on its attractive preclinical profile, SB1518 was selected for clinical development. Conclusions: Discovery of a new series of small molecule macrocycles of a unique and highly novel chemical structure are presented as inhibitors of JAK2 and FLT3 kinases. SAR studies directed us towards an optimal linker and solubilising side chain fine-tuning the molecular structure. SB1518 was selected as a development candidate with an attractive and distinguishing JAK2 selective inhibitory profile and additional activity against FLT3. SB1518 has shown encouraging clinical activity demonstrating that its attractive preclinical profile translates to the clinic. 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 3564. doi:10.1158/1538-7445.AM2011-3564

Kinase inhibitors have found applications in multiple oncology settings due to their ability to target key signaling pathways in many different cancers. In general, the broad-spectrum kinase inhibitors have yielded better clinical outcomes than more selective ones because they block more than one pathway critical for tumor growth. We describe herein the pharmacological profile of TG02, a multi-kinase inhibitor being developed by Tragara Pharmaceuticals, which combines cell cycle regulatory and transcriptional CDK inhibition with activity against kinase targets important in the etiology of various leukemias. In vitro potency against the kinases was evaluated using recombinant enzymes with synthetic substrates. Potency on intracellular signaling pathways was assessed by Western blot analyses of the phosphorylated substrates in cell lysates. Functional potency was assessed using proliferation assays performed on human tumor cell lines as well as hematopoietic progenitors expanded from patients' blood. Evaluation of pharmacokinetics, pharmacodynamic biomarkers and anti-tumor efficacy was performed in nude mouse xenograft models of leukemia. TG02 was shown to be a potent inhibitor of several key CDKs (IC50 = 19, 11, 37 & 10nM vs. CDK1, 2, 7 & 9) as well as two other kinases implicated in hematological malignancies, FLT3 and JAK2, with similar potency. The CDK spectrum includes both cell cycle regulatory and transcriptional CDKs. TG02 potently inhibits proliferation across a broad panel of human liquid tumor cell lines (IC50 from 68 to 230 nM). Comparison with reference inhibitors that block only one of the kinase targets of interest demonstrated the significant added benefit of combined CDK and JAK2 targeting. Inhibition of signaling by the CDK and FLT3-JAK2 pathways in leukemia cell lines was detected at the same range (0.10 to 0.35 μM). TG02 was generally cleared from the blood within 8 hours of oral administration but was retained in tumor masses at supratherapeutic levels for 24-48 hours, depending on dose. Accordingly, pathway-related biomarkers were markedly suppressed for 24-72 hours after dosing. In a murine model of FLT3-mutated leukemia (MV4-11), TG02 induced tumor regression after oral daily dosing (40 mg/kg for 21 days). Furthermore, in an orthotopic model of AML with WT FLT3 and JAK2 (HL60), TG02 significantly prolonged median survival time (59 days vs. 40 days) after treatment at 100 mg/kg p.o. 2d on/5d off. Proliferation assays using hematopoietic progenitors expanded from patients with JAK2 mutations or FLT3 wild-type or mutant acute myeloid leukemia revealed higher efficacy of TG02 compared to reference inhibitors targeting only one of the kinases of interest, again demonstrating the benefits of co-targeting CDKs and additional kinases. TG02 is a multi-kinase inhibitor with a previously unreported spectrum of targets that shows promising preclinical activity for the treatment of hematological malignancies. 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 2542.

Background: Dysregulation of the PI3K signaling pathway, either through activating mutations, amplification of PI3K or deletion of PTEN has been linked to a wide range of hyperproliferative diseases. The development of small molecule modulators of key proteins in this signaling cascade has focused mainly on mTOR or PI3K. Recent investigations have shown a significant overlap in the activity of small molecule inhibitors against these two proteins. Inhibitors that are currently progressing through preclinical and early clinical development have been shown to affect both mTOR and PI3K to some extent. However, most reported inhibitors do not inhibit both of these kinases in a close dose range. Our drug discovery program has focused on equipotent inhibition of both targets. SB2312 is a novel compound with very favorable drug-like properties, giving rise to equivalent targeting of both mTOR and PI3K in cells and tumor tissue and resulting in a more efficient inhibition of this pathway compared to reference compounds currently under evaluation in clinical trials. Methods: In vitro-IC50 for mTOR and PI3K were determined using recombinant enzymes. Inhibitory activity of SB2312 on the mTOR/PI3K signaling pathway was determined by Western-blot analysis, Surefire assays, and proliferation assays in various cancer cell lines. Efficacious doses of SB2312 or reference compounds were selected after pharmacokinetic and biomarker analysis of PC3 tumor-bearing mice, treated with single oral doses. Anti-tumor efficacy of SB2312 and reference compounds was evaluated in mouse xenograft models after continuous daily oral dosing. Results: SB2312 is a potent and selective dual mTOR and PI3K inhibitor (IC50: 55 nM and 18 nM, respectively) and has no significant activity on a large panel of protein kinases or enzymes and receptors. Efficient and equivalent inhibition of mTOR and PI3K in human cell lines was demonstrated by blockade of the phosphorylation of S6KT389 (IC50= 70 nM) and AKTS473 (IC50= 21 nM). SB2312 inhibits the proliferation of a broad range of human tumor cell lines independently of their PI3K pathway mutation status (IC50= 112–882 nM) and is also effective against rapamycin-resistant cell lines. SB2312 has very favorable physicochemical and pharmacokinetic properties, with high oral bioavailability and tumor exposure. Efficient and sustained inhibition of PI3K and mTOR was demonstrated in tumor tissues from a PC3 tumor xenograft model, along with dose-dependent anti-tumor activity at very well tolerated doses. Tumor growth inhibition was 53%, 105%, and 117% at 11, 33, and 80 mg/kg SB2312, respectively, without any body weight loss at any of these doses. Summary: Herein, we report SB2312, a novel, potent and selective dual mTOR/PI3K inhibitor with excellent drug-like properties. Our studies show superior and sustained blockade of the mTOR/PI3K pathway and high antitumor efficacy at well tolerated doses. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B138.