REFERENCE TO THE SEQUENCE LISTING

The entire contents of the ASCII text file entitled “SNA0003WO_Sequence_Listing.txt,” created on Oct. 31, 2018 and having a size of 33 kilobytes is incorporated herein by reference in its entirety.

BACKGROUND

The search for new therapeutic agents has been greatly aided in recent years by a better understanding of the structure of enzymes and other biomolecules associated with diseases. One important class of enzymes that has been the subject of extensive study over the past twenty years is protein kinases.

Protein kinases constitute a large family of structurally related enzymes that are responsible for catalyzing the phosphorylation of specific amino acid residues in proteins and thereby controlling a variety of signal transduction processes within the cell in response to external stimuli (Ubersax, J. A., Ferrell, J. E., Nat. Rev. Mol. Cell Biol., 2007, 8, 530-541; Shchemelinin, I. et al., Folia Biol., 2006, 52, 81-101). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function (Manning, G., et al., Science, 2002, 298, 1912-1934). There are at least 518 kinases in the human body and almost all kinases contain a similarly structured ATP-binding pocket and catalytic domain containing 250-400 amino acids. Kinases are categorized into families according to the substrates they phosphorylate (e.g., the hydroxyl group of protein-tyrosine residues, protein-serine/threonine residues, or lipids).

In general, protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of extracellular and other stimuli. Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H2O2), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α)), and growth factors (e.g., granulocyte macrophage-colony-stimulating factor (GM-CSF), and fibroblast growth factor (FGF)). An extracellular stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggered by protein kinase-mediated events as described above (Roskoski, R. Jr., Pharmacol. Res., 2015, 100, 1-23; Fleuren, E. D. G., et al., Nat. Rev. Cancer, 2016, 16, 83-98). These diseases include, but are not limited to, cancer, autoimmune diseases, inflammatory diseases, bone diseases, metabolic diseases, neurological and neurodegenerative diseases, cacardiovascular diseases, allergies and asthma, Alzheimer's disease, and hormone-related diseases. Accordingly, there remains a need to find new safe and efficacious protein kinase inhibitors that are useful as therapeutic agents.

Cancer is a disease that derives from cells and tissues that overcome inherent cell growth checkpoints and regulators, thus leading to transformation into a malignant state of uncontrolled proliferation. As indicated above, kinases play a central role in regulating and transducing signals from the cell surface to the nucleus in order to elicit a response to extracellular stimuli. Aberrant kinase-mediated signaling, such as that propogated by overactive or abnormally abundant kinases, is often associated with cell growth, proliferation, migration, and survival, which are known to be key drivers of cancer pathology. Many types of cancer are known, and include the following:

Endodermal cancers are serious malignancies of tissues that derive from inner embryonic endoderm layer, which includes the stomach, liver, colon, gallbladder, prostate, and pancreas. Several of these cancers typically have a poor prognosis when advanced beyond Stage II upon diagnosis. For example, it is estimated that 41,000 adults in the United States will be diagnosed with primary liver cancer and 29,000 deaths will occur from this disease in 2017. The incidence of liver cancer has tripled since 1980 and it is now the 5th most common cause of cancer death among men and the general 5-year survival rate is only 18%. Similarly, 28,000 adults in the United States will be diagnosed with stomach cancer and 10,960 deaths (6,720 men and 4,240 women) will occur from this disease will occur in 2017. The general 5-year survival rate for people with stomach cancer is 30%. Also, an estimated 53,000 adults in the United States will be diagnosed with pancreatic cancer and 41,780 deaths will occur from this disease in 2017. Pancreatic ductal adenocarcinoma makes up the vast majority (90%) of all pancreatic malignancies and remains a disease with very poor prognosis and high morbidity. Pancreatic cancer is the fourth leading cause of cancer death in the US and prognosis is poor because pancreatic cancer is difficult to diagnose, with an estimated 1-year survival rate of 29%, and a 5-year survival rate of just 7%. Survival rates decrease further if the cancer is not diagnosed early and there is an important need for new therapeutics that enable more effective treatment of endodermal cancers at all stages (For endodermal cancer statistics, see the websites: www.cancer.net and www.cancer.org).

The PIM (proviral integration site for Moloney murine leukemia virus) kinases are a family of three constitutively active proto-oncogenic serine/threonine kinases, PIM1, PIM2, and PIM3, which have been shown to regulate signaling associated with several important normal biological processes, including cell survival, proliferation, differentiation, and apoptosis. However, when these processes become disrupted or hyperactivated, they manifest the hallmarks of cancer. The PIM kinases promote cell survival and downregulate cell apoptosis and, accordingly, have been shown to be involved directly in signaling mechanisms associated with tumorigenesis. Importantly, PIM1 and PIM3 kinases have been found to be widely and aberrantly expressed, particularly in endodermal cancers of the human colon, stomach, liver, prostate, and pancreas. Inhibition of PIM 1 and/or PIM3, for example with targeted short hairpin shRNA, was shown to significantly increase levels of apoptosis and reduce proliferation of endodermal cancer cell lines and of endodermal tumors in mice (See: Li Y Y., et al., Cancer Res., 2006, 66(13), 6741-6747; Zheng H C, et al., J. Cancer Res. Clin. Oncol., 2008, 134(4), 481-488; Fujii C, et al., Int. J. Cancer, 2005, 114(2), 209-218; Popivanova B. K., et al., Cancer Sci., 2007, 98(3), 321-328; Qu, Y., et al., Med. Sci. Monit., 2016, 22, 4254-4260; Xu, J., et al., J. Exp. Clin. Can. Res., 2016, 35 (133), 1-12, DOI 10.1186/s13046-016-0406-z.). In addition, PIM1 and/or PIM3 overexpression has been shown to be involved in other malignancies, such as cancer of the ovaries (Zhuang, H., et al., Asian Pac. J. Cancer Prev., 2015, 16(8), 3325-3331) and in glioblastomas (Quan, J., et al., Cell. Mol. Biol., 2015, 61(1), 42-50).

Due to the aforementioned poor prognosis and limited therapeutic options associated with endodermal and other cancers that overexpress PIM1 and/or PIM3, development of selective inhibitors of PIM1 and/or PIM3 kinase represents a novel strategy for specifically treating cancers, especially those of the stomach, liver, colon, prostate, esophagus, pancreas, and other endodermal organs, with drugs, either alone or in combination with other therapeutic agents, that exhibit higher efficacy, lower toxicity, and lower susceptibility to resistance relative to existing treatment modalities.

SUMMARY

This disclosure provides potent and selective inhibitors of the family of three protein kinases referred to as PIM (Proviral Integration site of Moloney murine leukemia virus), PIM inhibitor compositions of general Formulas (I)-(II) set out below, pharmaceutical formulations, methods for their preparation, and uses thereof, including uses aimed at specifically targeting endodermal cancers through selective inhibition of PIM3.

Described herein are compounds, compositions, and methods for treating an individual suffering with cancers of the endodermal organs, including the cecum, intestine, stomach, thymus, liver, pancreas, lungs, esophagus, gallbladder, thyroid, lungs, and prostate, and which are manifested in a variety of forms, such as by way of example, esophageal adenocarcinoma, squamous cell carcinoma, nasopharyngeal carcinoma, gastric adenocarcinoma, pancreatic ductal adenocarcinoma, hepatocellular carcinoma, gallbladder adenocarcinoma, prostatic adenocarcinoma, colorectal adenocarcinoma, gastrointestinal stromal tumors (GIST), and gastrointestinal carcinoid tumors or the like, by administering to an individual a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of the PIM kinases, as described herein. Compounds of the invention can inhibit PIM reversibly by competitively binding potently and selectively to the ATP-binding pocket of the kinase. Certain compounds of the inventions can be used to selectively inhibit PIM3, and to a lesser degree PIM1 and PIM2, and specifically target cancers that overexpress the PIM3 kinase as a means of survival, growth, and proliferation leading to cancer pathology and disease progression. Other compounds of the invention inhibit PIM1 and PIM3, and to a lesser degree PIM2, and therefore are useful for diseases that overexpress these kinases. Yet other compounds of the invention target all three PIM kinases (PIM1-3) with approximately the same degree of potency and are useful for treating diseases where all three PIM kinases are involved in disease.

During the course of our research, we unexpectedly discovered during a broad kinase screening initiative that the natural products acryriaflavin A and K252a (See: Nakano, H. and Omura, S., J. Antibiot., 2009, 62, 17-26) have high affinity for the kinase PIM3. For example, when the affinity of arcyriaflavin A was analyzed against 450 kinases using the kinase screening platform KINOMEscan® (DiscoverX, Inc., San Diego, Calif.), we discovered that this molecule bound tightly to PIM3 with a Kdof 2 nM and had an order of magnitude lower affinity for both PIM1 (Kd=21 nM) and PIM2 (Kd=19 nM). Relative to arcyriaflavin A, K252a was shown to have a slightly reduced binding affinity for PIM3 (Kdca. 10 nM), and even lower for PIM1 and PIM2. Biochemical assays performed in the presence of ATP (Reaction Biology Corporation, Malvern, Pa.) confirmed potent inhibition of PIM3 by arcyriaflavin A with IC50=0.13 nM.



Similarly, a broad screen of kinases against a panel of known kinase inhibitors unexpectedly revealed that ruboxistaurin (See: Jirousek, M. R., et al., J. Med. Chem., 1996, 39, 2664-2671), a drug that was clinically developed as a potent PKC inhibitor for diabetic retinopathy and neuropathy, was also a potent and selective inhibitor of PIM3 (See: Karaman, M. W., et al., Nat. Biotechnol., 2008, 26, 127-132; Davis, M. I., et al., Nat. Biotech., 2011, 11, 1046-1051). Ruboxistaurin (LY333531) is a dialkylated macrocyclic analog of the known natural product arcyriarubin A. These results suggested that further optimization of these lead molecules would enable the development of potent and selective inhibitors of PIM3 as novel treatments for patients afflicted with serious endodermal cancers.

In one aspect of this invention is a compound having the structure of Formula (I) or a pharmaceutically acceptable salt, solvate, hydrate, N-oxide, prodrug, stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, isotopic variants, and metabolites thereof; wherein Formula (I) is as defined in the Detailed Description below.

In another embodiment of the invention are compounds having the structure of Formula (II) or a pharmaceutically acceptable salt, solvate, hydrate, N-oxide, prodrug, stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, isotopic variants, and metabolites thereof, which are examples representing kinase inhibitors, wherein Formula (II) is as defined in the Detailed Description below.

Also provided herein are pharmaceutical compositions comprising a compound disclosed herein, e.g., a compound of Formulas (I) and (II), including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and one or more pharmaceutically acceptable excipients.

Further provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a disorder, disease, or condition involving PIM3 expression in a subject, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formulas (I) or (II), including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

Also provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a disorder, disease, or condition in a subject, including cancers of endodermal organs such as the stomach, liver, colon, pancreas, prostate, and gallbladder, as well as other cancers involving PIM3 expression, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formulas (I) or (II), including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

Additionally provided herein is a method of modulating PIM3 kinase activity, comprising contacting a PIM3 kinase in vitro or in vivo with a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formulas (I) or (II), including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.

Also provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a PIM3 kinase-mediated disorder, disease, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formulas (I) or (II), including a stereoisomer enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, wherein the compound partially or completely inhibits PIM3 activity and displays increased potency against PIM3 kinase and/or increased selectivity for inhibiting PIM3 kinase relative to PIM1 kinase, PIM2 kinases and other kinases known to be present in the human body.

