Following the discovery of NVP-BEZ235, our first dual pan-PI3K/mTOR clinical compound, we sought to identify additional phosphoinositide 3-kinase (PI3K) inhibitors from different chemical classes with a different selectivity profile. The key to achieve these objectives was to couple a structure-based design approach with intensive pharmacologic evaluation of selected compounds during the medicinal chemistry optimization process. Here, we report on the biologic characterization of the 2-morpholino pyrimidine derivative pan-PI3K inhibitor NVP-BKM120. This compound inhibits all four class I PI3K isoforms in biochemical assays with at least 50-fold selectivity against other protein kinases. The compound is also active against the most common somatic PI3Ka mutations but does not significantly inhibit the related class III (Vps34) and class IV (mTOR, DNA-PK) PI3K kinases. Consistent with its mechanism of action, NVP-BKM120 decreases the cellular levels of p-Akt in mechanistic models and relevant tumor cell lines, as well as downstream effectors in a concentrationdependent and pathway-specific manner. Tested in a panel of 353 cell lines, NVP-BKM120 exhibited preferential inhibition of tumor cells bearing PIK3CA mutations, in contrast to either KRAS or PTEN mutant models. NVP-BKM120 shows dose-dependent in vivo pharmacodynamic activity as measured by significant inhibition of p-Akt and tumor growth inhibition in mechanistic xenograft models. NVP-BKM120 behaves synergistically when combined with either targeted agents such as MEK or HER2 inhibitors or with cytotoxic agents such as docetaxel or temozolomide. The pharmacological, biologic, and preclinical safety profile of NVP-BKM120 supports its clinical development and the compound is undergoing phase II clinical trials in patients with cancer. Mol Cancer Ther; 11(2); 317-28. Ó2011 AACR.

T he phosphoinositide-3-kinase (PI3K) family of lipid kinases is involved in a diverse set of cellular functions, including cell growth, proliferation, motility, differentiation, glucose transport, survival, intracellular trafficking, and membrane ruffling. 1 PI3K's can be categorized into class I, II, or III, depending on their subunit structure, regulation, and substrate selectivity. 2 Class IA PI3K's are activated by receptor tyrosine kinases and consist of a regulatory subunit (p85) and a catalytic subunit (p110). There are three catalytic isoforms: p110R, β, and δ. A single class IB PI3K, activated by GPCRs, consists of only one member: a p110γ catalytic subunit and a p101 regulatory subunit. The primary in vivo substrate of the class I PI3K's is phosphatidylinositol (4,5) diphosphate (PtdIns(4,5)P2), which upon phosphorylation at the 3-position of the inositol ring to form phosphatidylinositol triphosphate (3,4,5)P3 (PIP3) serves as a second messenger by activating a series of downstream effectors that mediate the cellular functions mentioned above. The PI3K isoforms have different distributions and share similar cellular functions, which are context dependent. In particular, p110R pathway deregulation has been demonstrated in ovarian, breast, colon, and brain cancers. 3,4 Inhibitors of PI3KR represent an intriguing therapeutic modality for these indications, and as such, there is much interest in generating suitable molecules to test this hypothesis in the clinic. 5À10 We have previously reported on a series of 6-hydroxyphenyl-2-morpholino pyrimidines, 11 as potent pan class I PI3K inhibitors that exhibit high selectivity toward protein kinases (serine/threonine and tyrosine kinases). We have further reported on non-phenol containing heterocyclic, morpholino pyrimidines 12 such as compound 1 which demonstrate in vivo PI3K pathway modulation and modest tumor growth inhibition. Described herein are our efforts to identify potent morpholino pyrimidinyl inhibitors of class I PI3Ks that exhibit potency and pharmacokinetic properties which allow for maximal pathway modulation in vivo and have druglike properties suitable for clinical development. These efforts culminated in the identification of 15, NVP-BKM120.Aminopyrimidine 1 and analogues such as 3 (Figure 1) exhibit low or sub-nanomolar biochemical potency and sub-micromolar cellular potency against PI3KR. Even with high rodent CL values, such analogues can demonstrate PI3K pathway modulation in mouse xenograft models. 12 During our exploration of the C 6 position, it was noted that C 6 aminopyridine analogue 4, while being less potent than 3 against PI3KR (>10Â potency loss), exhibited a markedly reduced (>9Â) rat CL value, increased %F, and increased oral AUC. Thus, superior pharmacokinetic properties were achievable within this scaffold and the challenge remaining was to retain this kind of pharmacokinetic profile while optimizing all the other attributes (potency, solubility, permeability, safety) necessary for advancement. To address this challenge, ...

Purpose: PIM kinases have been shown to act as oncogenes in mice, with each family member being able to drive progression of hematologic cancers. Consistent with this, we found that PIMs are highly expressed in human hematologic cancers and show that each isoform has a distinct expression pattern among disease subtypes. This suggests that inhibitors of all three PIMs would be effective in treating multiple hematologic malignancies.Experimental Design: Pan-PIM inhibitors have proven difficult to develop because PIM2 has a low K m for ATP and, thus, requires a very potent inhibitor to effectively block the kinase activity at the ATP levels in cells. We developed a potent and specific pan-PIM inhibitor, LGB321, which is active on PIM2 in the cellular context.Results:LGB321 is active on PIM2-dependent multiple myeloma cell lines, where it inhibits proliferation, mTOR-C1 signaling and phosphorylation of BAD. Broad cancer cell line profiling of LGB321 demonstrates limited activity in cell lines derived from solid tumors. In contrast, significant activity in cell lines derived from diverse hematological lineages was observed, including acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), multiple myeloma and non-Hodgkin lymphoma (NHL). Furthermore, we demonstrate LGB321 activity in the KG-1 AML xenograft model, in which modulation of pharmacodynamics markers is predictive of efficacy. Finally, we demonstrate that LGB321 synergizes with cytarabine in this model. Conclusions:We have developed a potent and selective pan-PIM inhibitor with single-agent antiproliferative activity and show that it synergizes with cytarabine in an AML xenograft model. Our results strongly support the development of Pan-PIM inhibitors to treat hematologic malignancies.

Proviral insertion of Moloney virus (PIM) 1, 2, and 3 kinases are serine/threonine kinases that normally function in survival and proliferation of hematopoietic cells. As high expression of PIM1, 2, and 3 is frequently observed in many human malignancies, including multiple myeloma, nonHodgkins lymphoma, and myeloid leukemias, there is interest in determining whether selective PIM inhibition can improve outcomes of these human cancers. Herein, we describe our efforts toward this goal. The structure guided optimization of a singleton high throughput screening hit in which the potency against all three PIM isoforms was increased >10,000-fold to yield compounds with pan PIM K i s < 10 pM, nanomolar cellular potency, and in vivo activity in an acute myeloid leukemia Pimdependent tumor model is described. KEYWORDS: Proviral insetion site in Moloney murine leukemia virus kinases inhibitors, Pim1 kinase inhibitor, Pim2 kinase inhibitor, Pim3 kinase inhibitor, pan-Pim kinase inhibitors P roviral insertion site of Moloney murine leukemia virus kinases, or PIM 1, 2, and 3 kinases are constitutively active serine/threonine kinases that normally function in the survival, proliferation, and differentiation of hematopoietic cells in response to growth factors and cytokines. 1,2 PIM's play redundant roles in oncogenesis and, therefore, suggest that a pan-PIM kinase inhibitor may be clinically useful. 3 In human disease, high expression and/or dysfunction of the three PIMs has been implicated in the progression of hematopoetic and solid tumor cancers. 1,2 In addition to cancer, PIM kinases have been reported to play a role in several autoimmune diseases. 4 Not surprisingly, PIM kinases have emerged as attractive therapeutic targets and have elicited several groups to investigate and report novel inhibitors of PIM 5−10 including the clinical compounds SGI-1776 6 and AZD1208, 7 Figure 1. Pim kinases share a high level of sequence homology within the family (>61%) and all share the unique feature of being the only kinases with a proline in the hinge, 11 which results in only one hydrogen bond interaction with ATP. As the ATP K m for PIM2 is 10−100× lower than that for PIM1 and PIM3, cell active pan PIM inhibitors have been more challenging to identify than PIM 1/3 inhibitors. Herein, we describe potent and selective cell active inhibitors of all three PIM kinases. A representative of this compound series, 5c, has suitable PK properties and was recently used to establish a PK/PD efficacy relationship in a PIM2 driven multiple myeloma xenograft model. 12 Here we also demonstrate efficacy in the AML EOL-1 xenograft model. Letter pubs.acs.org/acsmedchemlett

Pan proviral insertion site of Moloney murine leukemia (PIM) 1, 2, and 3 kinase inhibitors have recently begun to be tested in humans to assess whether pan PIM kinase inhibition may provide benefit to cancer patients. Herein, the synthesis, in vitro activity, in vivo activity in an acute myeloid leukemia xenograft model, and preclinical profile of the potent and selective pan PIM kinase inhibitor compound 8 (PIM447) are described. Starting from the reported aminopiperidyl pan PIM kinase inhibitor compound 3, a strategy to improve the microsomal stability was pursued resulting in the identification of potent aminocyclohexyl pan PIM inhibitors with high metabolic stability. From this aminocyclohexyl series, compound 8 entered the clinic in 2012 in multiple myeloma patients and is currently in several phase 1 trials of cancer patients with hematological malignancies.

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Phospoinositide-3-kinases (PI3K) are important oncology targets due to the deregulation of this signaling pathway in a wide variety of human cancers. A series of 2-morpholino, 4-substituted, 6-(3-hydroxyphenyl) pyrimidines have been reported as potent inhibitors of PI3Ks. Herein, we describe the structure-guided optimization of these pyrimidines with a focus on replacing the phenol moiety, while maintaining potent target inhibition and improving in vivo properties. A series of 2-morpholino, 4-substituted, 6-heterocyclic pyrimidines, which potently inhibit PI3K, were discovered. Within this series a compound, 17, was identified with suitable pharmacokinetic (PK) properties, which allowed for the establishment of a PI3K PK/pharmacodynamic-efficacy relationship as determined by in vivo inhibition of AKT Ser473 phosphorylation and tumor growth inhibition in a mouse A2780 tumor xenograft model.KEYWORDS phosphoinositide 3-kinase alpha, PI3K/AKT pathway T he phospoinositide-3-kinase (PI3K) family of lipid kinases is involved in a diverse set of cellular functions, including cell growth, proliferation, motility, differentiation, glucose transport, survival intracellular trafficking, and membrane ruffling. 1 PI3Ks can be categorized in class I, II, or III, depending on their subunit structure, regulation, and substrate selectivity. 2 Class IA PI3Ks are activated by receptor tyrosine kinases and consist of a regulatory subunit (p85) and a catalytic subunit (p110). There are three catalytic isoforms: p110 R, β, and δ. A single class IB PI3K, activated by G protein-coupled receptor, consists of only one member: a p110 γ catalytic subunit and a p101 regulatory subunit. The primary in vivo substrate of the class I PI3Ks is phosphatidylinositol (4,5) diphosphate, which, upon phosphorylation at the 3-position of the inositol ring to form phosphatidylinositol triphosphate (3,4,5)P3, serves as a second messenger by activating a series of downstream effectors that mediate the cellular functions mentioned above. The PI3K isoforms have different distributions and share similar cellular functions, which are context dependent. In particular, p110R pathway deregulation has been demonstrated in ovarian, breast, colon, and brain cancers. 3,4 Inhibitors of PI3KR represent an intriguing therapeutic modality for these indications, and as such, there is much interest in generating suitable molecules to test this hypothesis in the clinic. [5][6][7][8][9] We have reported phenolic mopholino pyrimidines, 10 such as compound 1 (Figure 1), as potent pan class I PI3K inhibitors that exhibit high selectivity toward other serine/threonine as well as tyrosine kinases. While exhibiting potent in vitro properties, the in vivo potential of such compounds may be limited due to the presence of the phenol moiety. Described herein are our efforts to identify potent morpholino pyrimidinyl inhibitors of PI3K that do not require a phenol group and exhibit PK properties suitable for achieving in vivo target modulation and efficacy.The importance of the...

Equimolar Ge x P x Se100–2x ternary glasses have been synthesized over a wide composition range, 4% < x < 25%, and examined in Raman scattering, modulated DSC, and volumetric experiments. Modulated DSC experiments show the enthalpy of relaxation at T g to display a square-well-like reversibility window with an onset (end) of 9.0% (18.0%) respectively, thus, fixing the onset of the rigidity transition, near x r = 9.0% and the stress transition, near x s = 18.0%. These findings show that the Intermediate-Phase (IP) resides in the 9% < x < 18% range. Melt fragility index, m(x) display a Gaussian-like minimum with m(x) < 20 for IP compositions and with m > 20 for non-IP ones. Fragility index results show a global minimum of m = 14 near the center of the window, x = 14, underscoring IP melts to be superstrong. Molar volumes, V m (x), of glasses reveal a Gaussian-like minimum, for IP compositions in relation to the non-IP ones. Melt fragility indices are closely correlated to the glass enthalpy of relaxation, and they show that superstrong melts yield IP glasses, while fragile melts yield flexible phase (FP) or stressed-rigid phase (SRP) glasses upon cooling. Special synthesis of glasses permitted the variance of Ge or P content, ⟨Δx⟩, across 1.5-g-sized batch compositions could be reduced to less than 0.1%. The homogeneous nature of glasses led to abrupt rigidity- and stress-elastic phase transitions in harmony with the percolative nature of these transitions as predicted by theory. Availability of the topological phases (TPs) in Ge x Se100‑x glasses, and P x Se100‑x glasses from earlier work, when combined with the presently measured TPs in the equimolar Ge x P x Se100–2x glasses, has permitted constructing a global TP diagram for the Ge–P–Se composition triangle. The global plot will assist in the choice of appropriate glass compositions for select applications of these materials. In spite of homogenization of these melts/glasses, compositional trends in T g(x) and Raman vibrational modes show that the P–P bearing local structural units of P4Se3 monomers and ethylene-like P2(Se1/2)4 units are decoupled from the network backbone in the IP and in the SRP.