Drugs in Clinical Pipeline: CX-6258

CX-6258 [(E)-5-chloro-3-((5-(3-(4-methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)indolin-2-one] is an oral, small molecule inhibitor from a novel class of oxindole-based derivatives that selectively inhibit all three Pim (Provirus Integration site for Moloney murine leukemia virus) kinases and show robust in vivo anticancer activity in Pim kinase related xenograft models [1]. The pan-PIM inhibitory activity of CX-6258 (IC₅₀ PIM-1, -2, -3 = 5, 25, 16 nM, respectively) is further strengthen with a good inhibition values for Flt3 kinase (IC₅₀ = 0.134 uM). Since Flt-3 regulates the Pim kinases in leukemia, its level of inhibition is important information for the interpretation of cellular activity performed with Pim kinases inhibitors. CX-6258 was also shown to be a reversible inhibitor of Pim-1 (K_i=0.005 uM).

Cylene Pharma disclosed CX-6258 in 2011, when it was one the few pan-PIM kinase inhibitors. It was the first one with nanomolar inhibition against Pim-3 kinase. With Pim-3 isoform reported to be overexpressed in pancreatic, gastric, and colon cancer, CX-6258 could be beneficial as an anticancer agent against these malignancies.

The activity of CX-6258 is as follows:

IC₅₀ (Pim-1 enzyme assay) = 5 nM; K_i = 5 nM

IC₅₀ (Pim-2 enzyme assay) = 25 nM

IC₅₀ (Pim-3 enzyme assay) = 16 nM

Common Name: CX-6258

Synonyms:  CX-6258; CX 6258; CX6258

IUPAC Name: (E)-5-chloro-3-((5-(3-(4-methyl-1,4-diazepane-1-carbonyl)phenyl)furan-2-yl)methylene)indolin-2-one

CAS Number: 1202916-90-2; 1353858-99-7 (hydrochloride hydrate)

SMILES: CN1CCCN(C(C2=CC(C3=CC=C(/C=C4C(C=C(Cl)C=C5)=C5NC\4=O)O3)=CC=C2)=O)CC1

Mechanism of Action: Kinase Inhibitor; pan-PIM Kinase Inhibitor

Indication: Various Cancers

Development Stage: Pre-Clinical

Company: Cylene Pharma

Pim kinases (Provirus Integration site for Moloney murine leukemia virus) are a family of serine/threonine kinases that regulate cell survival. This family of kinases is composed of three different isoforms (Pim-1, Pim-2, and Pim-3) that share 60-70% sequence identity in their kinase domains. The Pim kinases are tightly regulated at the level of transcription and translation,5 and their expression is mediated by the JAK/STAT signaling pathway, which is activated by various cytokines and hormones. In addition, several oncogenes, including Flt3-ITD and Bcr/Abl, were shown to upregulate the expression of Pims. The Pim kinase family members are considered oncogenes and have been implicated in tumorigenesis either alone or operating synergistically with c-Myc. Pim kinases are known to suppress apoptosis by the direct phosphorylation and inhibition of pro-apoptotic Bcl-2 antagonist of cell death (BAD). Overexpression of Pim kinases has been reported in leukemia and lymphoma tumors. They were also found to be overexpressed in solid tumors, including pancreatic cancer and prostate cancer.

To assess its selectivity profile, CX-6258 was screened against 107 kinases where using a concentration of 0.5 µM, only Pim-1, Pim-2, Pim-3, and Flt-3 were inhibited by more than 80%. The antiproliferative activity of CX-6258 was examined against a panel of cell lines derived from human solid tumors and hematological malignancies. CX-6258 demonstrated robust antiproliferative potencies against all cell lines tested. Cell lines derived from acute leukemias were the most sensitive to CX-6258.

In mechanistic cellular assays with MV-4-11 human AML cells, CX-6258 caused dose dependent inhibition of the phosphorylation of two pro-survival proteins, Bad and 4E-BP1, at the Pim kinase specific sites S112 and S65 and T37/46, respectively. Using ELISA against phospho-Flt3 (Y591), researchers demonstrated that CX-6258 does not inhibit Flt3 activity in MV-4-11 cells at relevant concentrations (IC50 greater than 10 µM) indicating that the effect of CX-6258 on phosphorylation of Bad and 4E-BP1 results from direct inhibition of Pim kinases rather than being a secondary consequence of Flt3 inhibition.

Combinations of CX-6258 with doxorubicin (10:1 molar ratio) and CX-6258 with paclitaxel (100:1 molar ratio) produced synergistic PC3 cell killing with combination index (CI₅₀) values equal to 0.4 and 0.56, respectively. When used as single agent, the IC₅₀ values for PC3 inhibiton were 13, doxorubicin, and paclitaxel 452 nM, 114 nM, and 2.5 nM, respectively, supporting the role of Pim kinases in modulating the chemosensitivity of cancer cells.

References:
1. Haddach, M.; et. al. Discovery of CX-6258. A Potent, Selective, and Orally Efficacious pan-Pim Kinases Inhibitor. ACS Med Chem Lett 2011, 3(2), 135-139.

PIM447 | pan-PIM Kinase Inhibitor | LGH447

PIM447 [N-(4-((1R,3S,5S)-3-Amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide] is a novel, specific pan-Pim kinase inhibitor in development for the treatment of patients with multiple myeloma (MM) and other hematologic malignancies. PIM447 demonstrated significant tumor growth inhibition in xenograft mouse models of MM as compared with control animals, supporting the clinical development of PIM447 in MM patients. PIM447 is currently in several phase 1 trials of cancer patients with hematological malignancies.

PIM447: 2D and 3D Structure

The biochemical potency for PIM447 was assessed in a high ATP AlphaScreen assay using [ATP]’s of 2800, 500, and 2800 µM for PIMs 1, 2, and 3, respectively. Under these conditions, the K_i ’s of PIM447 were determined to be 6, 18, and 9 pM for PIMs 1, 2, and 3, respectively [1]. The kinase selectivity of PIM447 was determined in biochemical assays for a panel of 68 diverse protein kinases includeding PIM2 as well as 9 lipid kinases. In this panel, only PIM2 was significantly inhibited by PIM447 with an IC₅₀ of less than 0.00035 µM. PIM447 also inhibited GSK3ß (IC₅₀ = 1.3 uM), PKN1 (IC₅₀ = 4.9 uM), and PKCt (IC₅₀ = 4.1 uM), but at a significantly lower potency with IC₅₀.

Additional kinase profiling of PIM447 at 1 µM against 442 kinases using the KINOMEscan binding displacement assay, indicated high kinase selectivity for PIM1,2 and 3.

Novartis  is developing PIM447 (earlier named as LGH447), in relapsed, refractory MM, and further suggest that it may be effective in the treatment of AML and other hematological malignancies, either as a single agent or in combination with other select therapeutic agents.

PIM447 Synthesis

US9079889B2: The patent reports many possible routes for the synthesis. The route appears to be an industrial one. Also see Ref. 1.

PIM Kinase

The PIM (Provirus Integration site for Moloney leukemia) kinase gene family encodes 3 serine/threonine protein kinases (PIM-1,2,3) that have roles in cell cycle progression and survival. In human disease, elevated levels of PIM1 and PIM2 are associated with hematologic malignancies, with MM showing the highest level of PIM2 expression. In preclinical studies, a majority of MM cell lines proved sensitive in vitro to LGH447-mediated PIM inhibition, exhibiting a dose-dependent decrease in cell proliferation.

PIM1, PIM2, and PIM3 are a closely related family of constitutively active serine/threonine kinases. PIM kinases were first identified as common integration sites in MMLV-induced murine T-cell lymphomas, with these viral insertions resulting in transcriptional activation and expression of the kinase. Similar unbiased insertional mutagenesis screens were used to demonstrate that the three kinases could substitute for each other in this oncogenic process, indicating that inhibition of all three PIM kinases may be necessary to achieve a therapeutic benefit. In human cancers, increased expression and activity of PIM kinases has been detected in many hematopoietic malignancies and solid tumors (prostate, lung, liver) and it is thought that this contributes to cancer cell survival and proliferation in these malignancies.

Activity of PIM447

PIM447 (also LGH447), a potent and specific pan-PIM inhibitor is shown to be active in PIM2-dependent Multiple Myeloma (MM) cell lines by inhibiting proliferation, mTOR-C1 signaling and phosphorylation of BAD. In addition, LGH447 inhibited proliferation of several AML cell lines. Furthermore, LGH447 inhibits tumor growth in several mouse subcutaneous xenograft models of MM and AML, where modulation of the pharmacodynamic marker phospho-S6 Ribosomal Protein was used to predict efficacy. LGH447 significantly reduced the bone tumor burden in a disseminated orthotopic human xenograft model of MM. Finally, researchers have demonstrated increased activity of LGH447 in combination with the PI3K inhibitor BYL719 in a MM model and with Cytarabine in an AML model.

Identifications:

1H NMR (Estimated) for PIM447

References:

1. Burger, M. T.; et. al. Identification of N-(4-((1R,3S,5S)-3-Amino-5-methylcyclohexyl)pyridin-3-yl)-6-(2,6-difluorophenyl)-5-fluoropicolinamide (PIM447), a Potent and Selective Proviral Insertion Site of Moloney Murine Leukemia (PIM) 1, 2, and 3 Kinase Inhibitor in Clinical Trials for Hematological Malignancies. J Med Chem 2015, 58(21), 8373-8386.
2. Burger, M. T.; et. al. Kinase inhibitors and methods of their use. US9079889B2