WO2018081719A1 - Inhibiteurs à petites molécules de nek2 et utilisations associées - Google Patents

Inhibiteurs à petites molécules de nek2 et utilisations associées Download PDF

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WO2018081719A1
WO2018081719A1 PCT/US2017/059061 US2017059061W WO2018081719A1 WO 2018081719 A1 WO2018081719 A1 WO 2018081719A1 US 2017059061 W US2017059061 W US 2017059061W WO 2018081719 A1 WO2018081719 A1 WO 2018081719A1
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cancer
compound
nek2
compounds
cell
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PCT/US2017/059061
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English (en)
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Hong-Yu Li
Brendan FRETT
Ichiro Nakano
Haiyong Han
Wenhao Hu
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Arizona Board Of Regents On Behalf Of The University Of Arizona
East China Normal University
The Uab Research Foundation
The Translational Genomics Research Institute
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Publication of WO2018081719A1 publication Critical patent/WO2018081719A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems

Definitions

  • This invention is in the field of medicinal chemistry.
  • the invention relates to a new class of small-molecules having an imidazole pyrimidine structure which function as inhibitors of NEK2 protein, and their use as therapeutics for the treatment of cancer and other diseases.
  • Pancreatic cancer is now the third leading cause of cancer related death in the United States.
  • Pancreatic ductal adenocarcinoma (PDAC) which accounts for the majority (>90%) of pancreatic cancer cases, has the worst mortality rate of all malignancies.
  • PDAC Pancreatic ductal adenocarcinoma
  • survival of patients with advanced PDAC has not improved, with median survival still peaking at 9 months (see, Vincent, A., et al, (2011) Lancet 378, 607-620. The majority of patients' tumors relapse after 6 months of treatment.
  • Nek2 a kinase involved in the progression of aneuploidy, is highly overexpressed in several cancer types including breast, liver, prostate, cervical, testicular, and pancreatic cancers (see, e.g., Rahib, L., et al, (2014) Cancer Res 74, 2913-2921; Conroy, T., et al., (2011) N Engl J Med 364, 1817-1825; Von Hoff, D. D., et al., (2013) N Engl J Med 369, 1691-1703; Zhou, W., et al, (2013) Cancer Cell 23, 48- 62).
  • recent publications indicate that Nek2 overexpression indirectly activates Akt and efflux drug pumps (see, e.g., Zhou, W., et al, (2013) Cancer Cell 23, 48-62).
  • Nek2 is highly expressed in PDAC cell lines and tumor tissues (see, Example I) and PDAC cell growth rate is associated with Nek2 expression and activity (see, Example II).
  • Nek2 Increased expression of Nek2 has been identified in breast, liver, prostate, testicular, and hematological carcinomas, which positions Nek2 as a widespread oncogene with no current targeted treatment (see, e.g., de Vos, S., et al, (2003) Lab Invest 83, 271-285; Kokuryo, T., et al, (2007) Cancer Res 67, 9637-9642; Tsunoda, N., et al., Cancer Sci 100, 11 1-1 16; Hayward, D. G., et al., (2004) Cancer Res 64, 7370-7376).
  • Nek2 a genetically diverse, heterogeneous population of cancer cells. These cells will all carry various traits that give the cancer a selective advantage during chemotherapy, metastasis, and nutrient accumulation. Since overexpression of Nek2 has been shown to propagate chromosomal abnormalities and more aggressive tumors harbor higher expression levels of Nek2, the kinase is likely significant in the tumorigenesis of many life threatening cancers. The development of a clinical candidate for the Nek2 inhibition could further provide the target validation and treatment of other Nek2 driven tumors.
  • the present invention addresses this need and provides a new class of small-molecules having an imidazole pyrimidine structure that function as inhibitors of NEK2 protein.
  • compounds 3a, 3b, 4, and 4a were identified as Nek2 inhibitors (see, Example III):
  • the present invention contemplates that exposure of animals (e.g., humans) suffering from cancer (e.g., and/or cancer related disorders) to therapeutically effective amounts of drug(s) having an imidazole pyrimidine structure (e.g., small molecules having an imidazole pyrimidine structure) that inhibit the activity of NEK2 will inhibit the growth of cancer cells or supporting cells outright and/or render such cells as a population more susceptible to the cell death-inducing activity of cancer therapeutic drugs or radiation therapies.
  • drug(s) having an imidazole pyrimidine structure e.g., small molecules having an imidazole pyrimidine structure
  • the present invention contemplates that inhibitors of NEK2 activity satisfy an unmet need for the treatment of multiple cancer types, either when administered as monotherapy to induce cell growth inhibition, apoptosis and/or cell cycle arrest in cancer cells, or when administered in a temporal relationship with additional agent(s), such as other cell death- inducing or cell cycle disrupting cancer therapeutic drugs or radiation therapies (combination therapies), so as to render a greater proportion of the cancer cells or supportive cells susceptible to executing the apoptosis program compared to the corresponding proportion of cells in an animal treated only with the cancer therapeutic drug or radiation therapy alone.
  • additional agent(s) such as other cell death- inducing or cell cycle disrupting cancer therapeutic drugs or radiation therapies (combination therapies)
  • combination treatment of animals with a therapeutically effective amount of a compound of the present invention and a course of an anticancer agent produces a greater tumor response and clinical benefit in such animals compared to those treated with the compound or anticancer drugs/radiation alone. Since the doses for all approved anticancer drugs and radiation treatments are known, the present invention contemplates the various combinations of them with the present compounds.
  • the present invention relates to imidazole pyrimidine compounds useful for inhibiting NEK2 activity (e.g., thereby facilitating cell apoptosis), and increasing the sensitivity of cells to inducers of apoptosis and/or cell cycle arrest.
  • Certain imidazole pyrimidine compounds of the present invention may exist as stereoisomers including optical isomers.
  • the invention includes all stereoisomers, both as pure individual stereoisomer preparations and enriched preparations of each, and both the racemic mixtures of such stereoisomers as well as the individual diastereomers and enantiomers that may be separated according to methods that are well known to those of skill in the art.
  • imidazole pyrimidine compounds encompassed within
  • Formula I is not limited to a particular chemical moiety for Rl, R2, and R3.
  • the particular chemical moiety for Rl, R2, and R3 independently include any chemical moiety that permits the resulting compound to inhibit NEK2 activity.
  • the particular chemical moiety for Rl, R2, and R3 independently include any chemical moiety that permits the resulting compound to inhibit NEK2 related Akt activity.
  • the particular chemical moiety for Rl, R2, and R3 independently include any chemical moiety that permits the resulting compound to inhibit NEK2 related efflux drug pump related activity.
  • Rl is selected from
  • R4 is selected from H, CH 3 , CH 2 CH 3 , or CH(CH 3 ) 2 .
  • R2 and R3 are independently selected from hydrogen or (Ci- C3)alkyl, (Ci-C3)alkoxy, (Ci-C3)alkamino, or R2 and R3 are formed in a ring structure between each other.
  • the invention further provides processes for preparing any of the compounds of the present invention through following at least a portion of the techniques recited the Examples.
  • the invention also provides the use of compounds to induce cell cycle arrest and/or apoptosis in cells containing functional NEK2 proteins.
  • the invention also relates to the use of compounds for sensitizing cells to additional agent(s), such as inducers of apoptosis and/or cell cycle arrest, and chemoprotection of normal cells through the induction of cell cycle arrest prior to treatment with chemotherapeutic agents.
  • additional agent(s) such as inducers of apoptosis and/or cell cycle arrest
  • chemoprotection of normal cells through the induction of cell cycle arrest prior to treatment with chemotherapeutic agents.
  • the compounds of the invention are useful for the treatment, amelioration, or prevention of disorders, such as those responsive to induction of apoptotic cell death, e.g., disorders characterized by dysregulation of apoptosis, including hyperproliferative diseases such as cancer.
  • the compounds can be used to treat, ameliorate, or prevent cancer that is characterized by resistance to cancer therapies (e.g., those cancer cells which are chemoresistant, radiation resistant, hormone resistant, and the like).
  • cancer is pancreatic cancer (e.g., pancreatic ductal adenocarcinoma (PDAC)), breast cancer, liver cancer, prostate cancer, testicular cancer, and/or hematological carcinoma.
  • PDAC pancreatic ductal adenocarcinoma
  • the compounds can be used to treat hyperproliferative diseases characterized by expression of functional NEK2 and/or NEK2 related proteins.
  • the invention also provides pharmaceutical compositions comprising the compounds of the invention in a pharmaceutically acceptable carrier.
  • kits comprising a compound of the invention and instructions for administering the compound to an animal.
  • the kits may optionally contain other therapeutic agents, e.g., anticancer agents or apoptosis-modulating agents.
  • FIG. 1 Immunostaining of Nek2 in a PDAC case (top) and normal pancreas (bottom).
  • FIG. 2 Nek2 siRNA inhibits pancreatic cancer cell growth.
  • A Knockdown of Nek2 protein (two isoforms) by two siRNA sequences, Nek2_5 and Nek2_6.
  • Nek siRNA treatment reduced the growth rate of PL45 (B) and BxPC-3 (C).
  • FIG. 3 Drug dose response curves of Compound 3a in four pancreatic cancer ceils lines.
  • FIG. 4 Drug dose response curves of Compound 3a ( IM-038A) in MIA PaCa-2 pancreatic cancer cell line and its derived nab-paclitaxel resistant (MIA PaCa-2 Nab-p-R) and gemcitabine resistant (MIA PaCa-2 Gem-R) cell lines.
  • FIG. 5 A schematic of the formulation of compounds 1, 2, 3, and 4.
  • FIG. 6 (A) Tumor volume as a function of days post inoculation of Compounds 4 and 4a. (B) Relative change of body weight percentage as a function of days post inoculation of Compounds 4 and 4a.
  • FIG. 7 shows evaluation of NIM-038A in a normal pancreatic ductual epithelial cell line.
  • NIM-038A was much less sensitive to normal pancreatic ductual epithelial cell line than to cancer cells.
  • anticancer agent refers to any therapeutic agents (e.g., chemotherapeutic compounds and/or molecular therapeutic compounds), antisense therapies, radiation therapies, or surgical interventions, used in the treatment of hyperproliferative diseases such as cancer (e.g. , in mammals, e.g.., in humans).
  • therapeutic agents e.g., chemotherapeutic compounds and/or molecular therapeutic compounds
  • antisense therapies e.g., radiation therapies, or surgical interventions, used in the treatment of hyperproliferative diseases such as cancer (e.g. , in mammals, e.g.., in humans).
  • prodrug refers to a pharmacologically inactive derivative of a parent "drug” molecule that requires biotransformation (e.g. , either spontaneous or enzymatic) within the target physiological system to release, or to convert (e.g. , enzymatically,
  • Prodrugs are designed to overcome problems associated with stability, water solubility, toxicity, lack of specificity, or limited bioavailability.
  • Exemplary prodrugs comprise an active drug molecule itself and a chemical masking group (e.g. , a group that reversibly suppresses the activity of the drug).
  • Some prodrugs are variations or derivatives of compounds that have groups cleavable under metabolic conditions. Prodrugs can be readily prepared from the parent compounds using methods known in the art, such as those described in A Textbook of Drug Design and
  • prodrugs become pharmaceutically active in vivo or in vitro when they undergo solvolysis under physiological conditions or undergo enzymatic degradation or other biochemical transformation (e.g. , phosphorylation, hydrogenation, dehydrogenation, glycosylation).
  • Prodrugs often offer advantages of water solubility, tissue compatibility, or delayed release in the mammalian organism. (See e.g. , Bundgard, Design of Prodrugs, /?/?. 7-9, 21-24, Elsevier, Amsterdam (1985); and Silverman, The Organic Chemistry of Drug Design and Drug Action, pp. 352-401, Academic Press, San Diego, CA (1992)).
  • Common prodrugs include acid derivatives such as esters prepared by reaction of parent acids with a suitable alcohol (e.g.
  • a suitable carboxylic acid e.g., an amino acid
  • amides prepared by reaction of the parent acid compound with an amine amides prepared by reaction of the parent acid compound with an amine, basic groups reacted to form an acylated base derivative (e.g. , a lower alkylamide), or phosphorus-containing derivatives, e.g.
  • salts of the compounds of the present invention refers to any salt (e.g. , obtained by reaction with an acid or a base) of a compound of the present invention that is physiologically tolerated in the target animal (e.g. , a mammal). Salts of the compounds of the present invention may be derived from inorganic or organic acids and bases.
  • acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, gly colic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, sulfonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like.
  • Other acids such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the invention and their pharmaceutically acceptable acid addition salts.
  • bases include, but are not limited to, alkali metal (e.g. , sodium) hydroxides, alkaline earth metal (e.g. , magnesium) hydroxides, ammonia, and compounds of formula NW ⁇ 4 + , wherein W is C 1-4 alkyl, and the like.
  • alkali metal e.g. , sodium
  • alkaline earth metal e.g. , magnesium
  • W is C 1-4 alkyl
  • salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate,
  • flucoheptanoate glycerophosphate, hemisulfate, heptanoate, hexanoate, chloride, bromide, iodide, 2 -hydroxy ethanesulfonate, lactate, maleate, mesylate, methanesulfonate,
  • salts include anions of the compounds of the present invention compounded with a suitable cation such as Na + , NH 4 + , and NW (wherein W is a C 1-4 alkyl group), and the like.
  • a suitable cation such as Na + , NH 4 + , and NW (wherein W is a C 1-4 alkyl group), and the like.
  • salts of the compounds of the present invention are contemplated as being pharmaceutically acceptable.
  • salts of acids and bases that are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.
  • solvate refers to the physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. This physical association often includes hydrogen bonding. In certain instances, the solvate is capable of isolation, for example, when one or more solvate molecules are incorporated in the crystal lattice of the crystalline solid. "Solvate” encompasses both solution-phase and isolable solvates.
  • Exemplary solvates include hydrates, ethanolates, and methanolates.
  • a therapeutically effective amount refers to that amount of the therapeutic agent sufficient to result in amelioration of one or more symptoms of a disorder, or prevent advancement of a disorder, or cause regression of the disorder.
  • a therapeutically effective amount will refer to the amount of a therapeutic agent that decreases the rate of tumor growth, decreases tumor mass, decreases the number of metastases, increases time to tumor progression, or increases survival time by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.
  • sensitize and “sensitizing,” as used herein, refer to making, through the administration of a first agent (e.g. , an imidazole pydrimidine compound of the invention), an animal or a cell within an animal more susceptible, or more responsive, to the biological effects (e.g. , promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis) of a second agent.
  • a first agent e.g. , an imidazole pydrimidine compound of the invention
  • biological effects e.g. , promotion or retardation of an aspect of cellular function including, but not limited to, cell division, cell growth, proliferation, invasion, angiogenesis, necrosis, or apoptosis
  • the sensitizing effect of a first agent on a target cell can be measured as the difference in the intended biological effect (e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis) observed upon the administration of a second agent with and without administration of the first agent.
  • the intended biological effect e.g., promotion or retardation of an aspect of cellular function including, but not limited to, cell growth, proliferation, invasion, angiogenesis, or apoptosis
  • the response of the sensitized cell can be increased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least 300%, at least about 350%, at least about 400%, at least about 450%, or at least about 500% over the response in the absence of the first agent.
  • dysregulation of apoptosis refers to any aberration in the ability of (e.g. , predisposition) a cell to undergo cell death via apoptosis.
  • Dysregulation of apoptosis is associated with or induced by a variety of conditions, non-limiting examples of which include, autoimmune disorders (e.g.
  • systemic lupus erythematosus erythematosus
  • rheumatoid arthritis graft-versus-host disease
  • myasthenia gravis myasthenia gravis
  • Sjogren's syndrome chronic inflammatory conditions
  • hyperproliferative disorders e.g., tumors, B cell lymphomas, or T cell lymphomas
  • viral infections e.g. , herpes, papilloma, or HIV
  • other conditions such as osteoarthritis and atherosclerosis.
  • NEK2 refers to wild-type NEK2 expressed at normal, high, or low levels and mutant NEK2 that retains at least about 5% of the activity of wild-type NEK2, e.g. , at least about 10%, about 20%, about 30%, about 40%, about 50%, or more of wild-type activity.
  • NEK2-related protein refers to proteins that have partial sequence homology (e.g., at least 5%, 10%, 25%, 50%, 75%, 85%, 95%, 99%, 99.999%) with NEK2, have tumor suppressor activity, and are inhibited by interaction with a compound of the present invention (e.g., an imidazole pydrimidine compound of the present invention).
  • a compound of the present invention e.g., an imidazole pydrimidine compound of the present invention.
  • hyperproliferative disease refers to any condition in which a localized population of proliferating cells in an animal is not governed by the usual limitations of normal growth.
  • hyperproliferative disorders include tumors, neoplasms, lymphomas and the like.
  • a neoplasm is said to be benign if it does not undergo invasion or metastasis and malignant if it does either of these.
  • a "metastatic" cell means that the cell can invade and destroy neighboring body structures.
  • Hyperplasia is a form of cell proliferation involving an increase in cell number in a tissue or organ without significant alteration in structure or function.
  • Metaplasia is a form of controlled cell growth in which one type of fully differentiated cell substitutes for another type of differentiated cell.
  • autoimmune disorder refers to any condition in which an organism produces antibodies or immune cells which recognize the organism's own molecules, cells or tissues.
  • Non-limiting examples of autoimmune disorders include autoimmune hemolytic anemia, autoimmune hepatitis, Berger's disease or IgA nephropathy, celiac sprue, chronic fatigue syndrome, Crohn's disease, dermatomyositis, fibromyalgia, graft versus host disease, Grave's disease, Hashimoto's thyroiditis, idiopathic thrombocytopenia purpura, lichen planus, multiple sclerosis, myasthenia gravis, psoriasis, rheumatic fever, rheumatic arthritis, scleroderma, Sjogren's syndrome, systemic lupus erythematosus, type 1 diabetes, ulcerative colitis, vitiligo, and the like.
  • neoplastic disease refers to any abnormal growth of cells being either benign (non-cancerous) or malignant (cancerous).
  • normal cell refers to a cell that is not undergoing abnormal growth or division. Normal cells are non-cancerous and are not part of any hyperproliferative disease or disorder.
  • anti-neoplastic agent refers to any compound that retards the proliferation, growth, or spread of a targeted (e.g. , malignant) neoplasm.
  • prevent refers to a decrease in the occurrence of pathological cells (e.g. , hyperproliferative or neoplastic cells) in an animal.
  • the prevention may be complete, e.g. , the total absence of pathological cells in a subject.
  • the prevention may also be partial, such that the occurrence of pathological cells in a subject is less than that which would have occurred without the present invention.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable vehicle” encompasses any of the standard pharmaceutical carriers, solvents, surfactants, or vehicles. Suitable pharmaceutically acceptable vehicles include aqueous vehicles and nonaqueous vehicles. Standard pharmaceutical carriers and their formulations are described in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, 19th ed. 1995.
  • PKs Protein kinases
  • PKs are enzymes that catalyze the phosphorylation of specific serine, threonine or tyrosine residues in cellular proteins. These post-translational modifications of substrate proteins act as a molecular switch, playing a central role in diverse biological processes, such as control of cell growth, metabolism, tumor microenviroment, differentiation, and apoptosis. Aberrant, excessive or more generally inappropriate PK activity has been observed in several disease states including malignant proliferative disorders. For examples, Zydelig® (Idelaisib) has been used as a medicine for hematological malignancies as a PI3K inhibitor.
  • Nek2 NIMA related kinase 2
  • mitotic kinase a mitotic kinase
  • elevated Nek2 activity has been implemented in cellular processes that directly lead to increased levels of drug-efflux, ATP -binding cassette (ABC) containing proteins, resulting in chemo-refractory malignancies in patients.
  • ABSC ATP -binding cassette
  • numerous cancers such as pancreatic, liver, prostate, cervical testicular, brain, and breast cancers, aberrant Nek2 activity contributes to detrimental mitotic errors, which promotes malignant processes and more aggressive phenotypes.
  • Nek2 Knockout of Nek2 effectively reduces the growth of Nek2 positive cancers in vitro and in vivo. Furthermore, knock-down of Nek2 expression re-sensitizes cancer cells that have developed resistance to paclitaxel, doxorubicin, or bortezomib. Further enhancing its clinical relevance, Nek2 inhibition can lower Akt activity by re-enabling the PPl/Akt regulatory system. Therefore, a NEK2 inhibitor can be generally effective for all cancers, but especially for the brain cancer and pancreatic cancers. Experiments conducted during the course of developing embodiments for the present invention further examined the role of Nek2. It was shown that Nek2 is highly expressed in PDAC cell lines and tumor tissues (see, Example I) and PDAC cell growth rate is associated with Nek2 expression and activity (see, Example II).
  • Nek2 Increased expression of Nek2 has been identified in breast, liver, prostate, testicular, and hematological carcinomas, which positions Nek2 as a widespread oncogene with no current targeted treatment (see, e.g., de Vos, S., et al, (2003) Lab Invest 83, 271 -285; Kokuryo, T., et al, (2007) Cancer Res 67, 9637-9642; Tsunoda, N., et al, Cancer Sci 100, 1 1 1-116; Hayward, D. G., et al., (2004) Cancer Res 64, 7370-7376).
  • Nek2 a genetically diverse, heterogeneous population of cancer cells. These cells will all carry various traits that give the cancer a selective advantage during chemotherapy, metastasis, and nutrient accumulation. Since overexpression of Nek2 has been shown to propagate chromosomal abnormalities and more aggressive tumors harbor higher expression levels of Nek2, the kinase is likely significant in the tumorigenesis of many life threatening cancers. The development of a clinical candidate for the Nek2 inhibition could further provide the target validation and treatment of other Nek2 driven tumors.
  • the present invention addresses this need and provides a new class of small-molecules having an imidazole pyrimidine structure which function as inhibitors of NEK2 protein.
  • Nek2 inhibitors see, Example III:
  • the present invention contemplates that exposure of animals (e.g., humans) suffering from cancer (e.g., and/or cancer related disorders) to therapeutically effective amounts of drug(s) having an imidazole pyrimidine structure (e.g., small molecules having an imidazole pyrimidine structure) that inhibit the activity of NEK2 will inhibit the growth of cancer cells or supporting cells outright and/or render such cells as a population more susceptible to the cell death-inducing activity of cancer therapeutic drugs or radiation therapies.
  • drug(s) having an imidazole pyrimidine structure e.g., small molecules having an imidazole pyrimidine structure
  • the invention further relates to methods of treating, ameliorating, or preventing disorders in a patient, such as those that are responsive to induction of apoptosis, comprising administering to the patient a compound of the invention and additional agent(s), e.g. , an inducer of apoptosis.
  • disorders include those characterized by a dysregulation of apoptosis and those characterized by the proliferation of cells expressing functional NEK2 proteins (e.g., pancreatic cancer).
  • imidazole pyrimidine compounds encompassed within
  • Formula I is not limited to a particular chemical moiety for Rl, R2, and R3.
  • the particular chemical moiety for Rl, R2, and R3 independently include any chemical moiety that permits the resulting compound to inhibit NEK2 activity.
  • the particular chemical moiety for Rl, R2, and R3 independently include any chemical moiety that permits the resulting compound to inhibit NEK2 related Akt activity.
  • the particular chemical moiety for Rl , R2, and R3 independently include any chemical moiety that permits the resulting compound to inhibit NEK2 related efflux drug pump related activity.
  • Rl is selected from
  • R4 is selected from H, CH 3 , CH 2 CH 3 , or CH(CH 3 ) 2 .
  • R2 and R3 are independently selected from hydrogen or (Ci- C3)alkyl, (Ci-C3)alkoxy, (Ci-C3)alkamino, or R2 and R3 are formed in a ring structure between each other.
  • An important aspect of the present invention is that compounds of the invention induce cell cycle arrest and/or apoptosis and also potentiate the induction of cell cycle arrest and/or apoptosis either alone or in response to additional apoptosis induction signals. Therefore, it is contemplated that these compounds sensitize cells to induction of cell cycle arrest and/or apoptosis, including cells that are resistant to such inducing stimuli.
  • the NEK2 inhibitors of the present invention e.g., imidazole pydrimidine compounds
  • the inhibitors can be used to induce apoptosis in any disorder that can be treated, ameliorated, or prevented by the induction of apoptosis.
  • the inhibitors can be used to induce apoptosis in cells comprising functional NEK2 and/or NEK2-related proteins.
  • compositions and methods of the present invention are used to treat diseased cells, tissues, organs, or pathological conditions and/or disease states in an animal (e.g. , a mammalian patient including, but not limited to, humans and veterinary animals).
  • an animal e.g. , a mammalian patient including, but not limited to, humans and veterinary animals.
  • various diseases and pathologies are amenable to treatment or prophylaxis using the present methods and compositions.
  • a non-limiting exemplary list of these diseases and conditions includes, but is not limited to, pancreatic cancer, breast cancer, prostate cancer, lymphoma, skin cancer, colon cancer, melanoma, malignant melanoma, ovarian cancer, brain cancer, primary brain carcinoma, head and neck cancer, glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lung cancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma, bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal cortex carcinoma, malignant pancreatic insulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant hypercalcemia, cervical hyperplasia, leuk
  • the cancer cells being treated are metastatic. In other embodiments, the cancer cells being treated are resistant to anticancer agents. In other embodiments, the disorder is any disorder having cells having NEK2 protein and/or NEK2 -related protein expression.
  • Some embodiments of the present invention provide methods for administering an effective amount of a compound of the invention and at least one additional therapeutic agent (including, but not limited to, chemotherapeutic antineoplastics, apoptosis-modulating agents, antimicrobials, antivirals, antifungals, and anti-inflammatory agents) and/or therapeutic technique (e.g. , surgical intervention, and/or radiotherapies).
  • the additional therapeutic agent(s) is an anticancer agent.
  • suitable anticancer agents are contemplated for use in the methods of the present invention. Indeed, the present invention contemplates, but is not limited to,
  • anticancer agents such as: agents that induce apoptosis;
  • polynucleotides e.g. , anti-sense, ribozymes, siRNA
  • polypeptides e.g. , enzymes and antibodies
  • biological mimetics alkaloids; alkylating agents; antitumor antibiotics;
  • antimetabolites include hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g. , antibodies conjugated with anticancer drugs, toxins, defensins); toxins; radionuclides; biological response modifiers (e.g. , interferons (e.g., IFN-a) and interleukins (e.g. , IL-2)); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce tumor cell
  • chemotherapeutic compounds and anticancer therapies suitable for coadministration with the disclosed compounds are known to those skilled in the art.
  • anticancer agents comprise agents that induce or stimulate apoptosis.
  • Agents that induce apoptosis include, but are not limited to, radiation (e.g. , X-rays, gamma rays, UV); tumor necrosis factor (TNF)-related factors (e.g. , TNF family receptor proteins, TNF family ligands, TRAIL, antibodies to TRAIL-Rl or TRAIL-R2); kinase inhibitors (e.g.
  • epidermal growth factor receptor (EGFR) kinase inhibitor vascular growth factor receptor (VGFR) kinase inhibitor, fibroblast growth factor receptor (FGFR) kinase inhibitor, platelet- derived growth factor receptor (PDGFR) kinase inhibitor, and Bcr-Abl kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g. , HERCEPTIN, RITUXAN, ZEVALIN, and AVASTIN); anti -estrogens (e.g., raloxifene and tamoxifen); anti-androgens (e.g. , flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids);
  • EGFR epidermal growth factor receptor
  • VGFR vascular growth factor receptor
  • FGFR fibroblast growth factor receptor
  • PDGFR platelet- derived growth factor receptor
  • COX-2 cyclooxygenase 2
  • COX-2 inhibitors e.g. , celecoxib, meloxicam, NS-398, and non-steroidal anti-inflammatory drugs (NSAIDs)
  • anti-inflammatory drugs e.g., butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL,
  • PEDIAPRED phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL
  • cancer chemotherapeutic drugs e.g. , irinotecan (CAMPTOSAR), CPT-11, fludarabine (FLUDARA), dacarbazine (DTIC), dexamethasone, mitoxantrone, MYLOTARG, VP- 16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL); cellular signaling molecules; ceramides and cytokines; staurosporine, and the like.
  • irinotecan CAMPTOSAR
  • CPT-11 CPT-11
  • fludarabine FLUDARA
  • compositions and methods of the present invention provide a compound of the invention and at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g. , herbs and other plant and/or animal derived compounds).
  • at least one anti-hyperproliferative or antineoplastic agent selected from alkylating agents, antimetabolites, and natural products (e.g. , herbs and other plant and/or animal derived compounds).
  • Alkylating agents suitable for use in the present compositions and methods include, but are not limited to: 1) nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethylenimines and methylmelamines (e.g. , hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g. , busulfan); 4) nitrosoureas (e.g. , carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozocin
  • nitrogen mustards e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil
  • 2) ethylenimines and methylmelamines e.
  • streptozotocin streptozotocin
  • DTIC dacarbazine
  • antimetabolites suitable for use in the present compositions and methods include, but are not limited to: 1) folic acid analogs (e.g. , methotrexate (amethopterin)); 2) pyrimidine analogs (e.g. , fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode- oxyuridine; FudR), and cytarabine (cytosine arabinoside)); and 3) purine analogs (e.g. , mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2 ' -deoxy coformy cin)).
  • folic acid analogs e.g. , methotrexate (amethopterin)
  • pyrimidine analogs e.g. , fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorode- oxyuridine
  • chemotherapeutic agents suitable for use in the
  • compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g. , vinblastine (VLB), vincristine); 2) epipodophyllotoxins (e.g. , etoposide and teniposide); 3) antibiotics (e.g. , dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g. , interferon-alfa); 6) platinum coordinating complexes (e.g., cisplatin (cis-DDP) and carboplatin); 7)
  • vinca alkaloids e.g. , vinblastine (VLB), vincristine
  • epipodophyllotoxins
  • anthracenediones e.g. , mitoxantrone
  • substituted ureas e.g., hydroxyurea
  • methylhydrazine derivatives e.g. , procarbazine (N-methylhydrazine; MIH)); 10) adrenocortical suppressants (e.g. , mitotane ( ⁇ , ⁇ '-DDD) and aminoglutethimide); 11) adrenocorticosteroids (e.g. , prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g. , diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g. , tamoxifen); 15) androgens (e.g., testosterone propionate and
  • fluoxymesterone 16) antiandrogens (e.g., flutamide): and 17) gonadotropin-releasing hormone analogs (e.g. , leuprolide).
  • antiandrogens e.g., flutamide
  • gonadotropin-releasing hormone analogs e.g. , leuprolide
  • any oncolytic agent that is routinely used in a cancer therapy context finds use in the compositions and methods of the present invention.
  • the U.S. Food and Drug Administration maintains a formulary of oncolytic agents approved for use in the United States. International counterpart agencies to the U.S.F.D.A. maintain similar formularies.
  • Table 2 provides a list of exemplary antineoplastic agents approved for use in the U.S. Those skilled in the art will appreciate that the "product labels" required on all U.S. approved chemotherapeutics describe approved indications, dosing information, toxicity data, and the like, for the exemplary agents.
  • Cisplatin Platinol Bristol-Myers Squibb PtCl 2 H 6 N 2
  • Daunorubicin HC1 Daunomycin Cerubidine Wyeth Ayerst, Madison,
  • Mitomycin C Mutamycin Bristol-Myers Squibb mitomycin C Mitozytrex SuperGen, Inc., Dublin,
  • Anticancer agents further include compounds which have been identified to have anticancer activity. Examples include, but are not limited to, 3-AP, 12-O-tetradecanoylphorbol- 13-acetate, 17AAG, 852A, ABI-007, ABR-217620, ABT-751, ADI-PEG 20, AE-941, AG- 013736, AGRO100, alanosine, AMG 706, antibody G250, antineoplastons, AP23573, apaziquone, APC8015, atiprimod, ATN-161, atrasenten, azacitidine, BB-10901, BCX-1777, bevacizumab, BG00001, bicalutamide, BMS 247550, bortezomib, biyostatin-1, buserelin, calcitriol, CCI-779, CDB-2914, cefixime, cetuximab, CG0070, cilengitide, clofarabine, combreta
  • anticancer agents and other therapeutic agents those skilled in the art are referred to any number of instructive manuals including, but not limited to, the Physician's Desk Reference and to Goodman and Gilman's "Pharmaceutical Basis of Therapeutics" tenth edition, Eds. Hardman et al, 2002.
  • the present invention provides methods for administering a compound of the invention with radiation therapy.
  • the invention is not limited by the types, amounts, or delivery and administration systems used to deliver the therapeutic dose of radiation to an animal.
  • the animal may receive photon radiotherapy, particle beam radiation therapy, other types of radiotherapies, and combinations thereof.
  • the radiation is delivered to the animal using a linear accelerator.
  • the radiation is delivered using a gamma knife.
  • the source of radiation can be external or internal to the animal.
  • External radiation therapy is most common and involves directing a beam of high-energy radiation to a tumor site through the skin using, for instance, a linear accelerator. While the beam of radiation is localized to the tumor site, it is nearly impossible to avoid exposure of normal, healthy tissue. However, external radiation is usually well tolerated by animals.
  • Internal radiation therapy involves implanting a radiation-emitting source, such as beads, wires, pellets, capsules, particles, and the like, inside the body at or near the tumor site including the use of delivery systems that specifically target cancer cells (e.g. , using particles attached to cancer cell binding ligands). Such implants can be removed following treatment, or left in the body inactive.
  • Types of internal radiation therapy include, but are not limited to, brachy therapy, interstitial irradiation, intracavity irradiation, radioimmunotherapy, and the like.
  • the animal may optionally receive radiosensitizers (e.g., metronidazole, misonidazole, intra-arterial Budr, intravenous iododeoxy uridine (IudR), nitroimidazole, 5-substituted-4- nitroimidazoles, 2H-isoindolediones, [[(2-bromoethyl)-andno]methyl]-nitro-lH-imidazole-l- ethanol, nitroaniline derivatives, DNA-affinic hypoxia selective cytotoxins, halogenated DNA ligand, 1,2,4 benzotriazine oxides, 2-nitroimidazole derivatives, fluorine-containing nitroazole derivatives, benzamide, nicotinamide, acridine-intercalator, 5-thiotretrazole derivative, 3-nitro- 1,2,4-triazole, 4,5-dinitroimidazole derivative, hydroxylated texaphrins
  • Any type of radiation can be administered to an animal, so long as the dose of radiation is tolerated by the animal without unacceptable negative side-effects. Suitable types of
  • radiotherapy include, for example, ionizing (electromagnetic) radiotherapy (e.g. , X-rays or gamma rays) or particle beam radiation therapy (e.g. , high linear energy radiation).
  • Ionizing radiation is defined as radiation comprising particles or photons that have sufficient energy to produce ionization, i.e. , gain or loss of electrons (as described in, for example, U.S. 5,770,581 incorporated herein by reference in its entirety).
  • the effects of radiation can be at least partially controlled by the clinician.
  • the dose of radiation is fractionated for maximal target cell exposure and reduced toxicity.
  • the total dose of radiation administered to an animal is about .01 Gray (Gy) to about 100 Gy.
  • about 10 Gy to about 65 Gy e.g., about 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy, 50 Gy, 55 Gy, or 60 Gy
  • a complete dose of radiation can be administered over the course of one day
  • the total dose is ideally fractionated and administered over several days.
  • radiotherapy is administered over the course of at least about 3 days, e.g. , at least 5, 7, 10, 14, 17, 21, 25, 28, 32, 35, 38, 42, 46, 52, or 56 days (about 1-8 weeks).
  • a daily dose of radiation will comprise approximately 1-5 Gy (e.g. , about 1 Gy, 1.5 Gy, 1.8 Gy, 2 Gy, 2.5 Gy, 2.8 Gy, 3 Gy, 3.2 Gy, 3.5 Gy, 3.8 Gy, 4 Gy, 4.2 Gy, or 4.5 Gy), or 1-2 Gy (e.g. , 1.5-2 Gy).
  • the daily dose of radiation should be sufficient to induce destruction of the targeted cells.
  • radiation is not administered every day, thereby allowing the animal to rest and the effects of the therapy to be realized.
  • radiation desirably is administered on 5 consecutive days, and not administered on 2 days, for each week of treatment, thereby allowing 2 days of rest per week.
  • radiation can be administered 1 day/week, 2 days/week, 3 days/week, 4 days/week, 5 days/week, 6 days/week, or all 7 days/week, depending on the animal's responsiveness and any potential side effects.
  • Radiation therapy can be initiated at any time in the therapeutic period. In one embodiment, radiation is initiated in week 1 or week 2, and is administered for the remaining duration of the therapeutic period. For example, radiation is administered in weeks 1- 6 or in weeks 2-6 of a therapeutic period comprising 6 weeks for treating, for instance, a solid tumor. Alternatively, radiation is administered in weeks 1-5 or weeks 2-5 of a therapeutic period comprising 5 weeks.
  • These exemplary radiotherapy administration schedules are not intended, however, to limit the present invention.
  • Antimicrobial therapeutic agents may also be used as therapeutic agents in the present invention. Any agent that can kill, inhibit, or otherwise attenuate the function of microbial organisms may be used, as well as any agent contemplated to have such activities. Antimicrobial agents include, but are not limited to, natural and synthetic antibiotics, antibodies, inhibitory proteins (e.g. , defensins), antisense nucleic acids, membrane disruptive agents and the like, used alone or in combination. Indeed, any type of antibiotic may be used including, but not limited to, antibacterial agents, antiviral agents, antifungal agents, and the like.
  • a compound of the invention and one or more therapeutic agents or anticancer agents are administered to an animal under one or more of the following conditions: at different periodicities, at different durations, at different concentrations, by different administration routes, etc.
  • the compound is administered prior to the therapeutic or anticancer agent, e.g. , 0.5, 1, 2, 3, 4, 5, 10, 12, or 18 hours, 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, or 4 weeks prior to the administration of the therapeutic or anticancer agent.
  • the compound is administered after the therapeutic or anticancer agent, e.g.
  • the compound and the therapeutic or anticancer agent are administered concurrently but on different schedules, e.g. , the compound is administered daily while the therapeutic or anticancer agent is administered once a week, once every two weeks, once every three weeks, or once every four weeks. In other embodiments, the compound is administered once a week while the therapeutic or anticancer agent is administered daily, once a week, once every two weeks, once every three weeks, or once every four weeks.
  • compositions within the scope of this invention include all compositions wherein the compounds of the present invention are contained in an amount which is effective to achieve its intended purpose. While individual needs vary, determination of optimal ranges of effective amounts of each component is within the skill of the art.
  • the compounds may be administered to mammals, e.g. humans, orally at a dose of 0.0025 to 50 mg/kg, or an equivalent amount of the pharmaceutically acceptable salt thereof, per day of the body weight of the mammal being treated for disorders responsive to induction of apoptosis. In one embodiment, about 0.01 to about 25 mg/kg is orally administered to treat, ameliorate, or prevent such disorders.
  • the dose is generally about one-half of the oral dose.
  • a suitable intramuscular dose would be about 0.0025 to about 25 mg/kg, or from about 0.01 to about 5 mg/kg.
  • the unit oral dose may comprise from about 0.01 to about 1000 mg, for example, about
  • the unit dose may be administered one or more times daily as one or more tablets or capsules each containing from about 0.1 to about 10 mg, conveniently about 0.25 to 50 mg of the compound or its solvates.
  • the compound may be present at a concentration of about 0.01 to 100 mg per gram of carrier. In a one embodiment, the compound is present at a concentration of about 0.07-1.0 mg/ml, for example, about 0.1-0.5 mg/ml, and in one embodiment, about 0.4 mg/ml.
  • the compounds of the invention may be administered as part of a pharmaceutical preparation containing suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.
  • suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the compounds into preparations which can be used pharmaceutically.
  • the preparations particularly those preparations which can be administered orally or topically and which can be used for one type of administration, such as tablets, dragees, slow release lozenges and capsules, mouth rinses and mouth washes, gels, liquid suspensions, hair rinses, hair gels, shampoos and also preparations which can be administered rectally, such as suppositories, as well as suitable solutions for administration by intravenous infusion, injection, topically or orally, contain from about 0.01 to 99 percent, in one embodiment from about 0.25 to 75 percent of active compound(s), together with the excipient.
  • compositions of the invention may be administered to any patient who may experience the beneficial effects of the compounds of the invention.
  • mammals e.g., humans, although the invention is not intended to be so limited.
  • Other patients include veterinary animals (cows, sheep, pigs, horses, dogs, cats, and the like).
  • the compounds and pharmaceutical compositions thereof may be administered by any means that achieve their intended purpose.
  • administration may be by parenteral, subcutaneous, intravenous, intramuscular, intraperitoneal, transdermal, buccal, intrathecal, intracranial, intranasal, or topical routes.
  • administration may be by the oral route.
  • the dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.
  • compositions of the present invention are manufactured in a manner which is itself known, for example, by means of conventional mixing, granulating, dragee- making, dissolving, or lyophilizing processes.
  • pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipients, optionally grinding the resulting mixture and processing the mixture of granules, after adding suitable auxiliaries, if desired or necessary, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or polyvinyl pyrrolidone.
  • fillers such as saccharides, for example lactose or sucrose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, for example tricalcium phosphate or calcium hydrogen phosphate, as well as binders such as starch paste, using, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methyl cellulose,
  • disintegrating agents may be added such as the above- mentioned starches and also carboxymethyl-starch, cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Auxiliaries are, above all, flow-regulating agents and lubricants, for example, silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
  • Dragee cores are provided with suitable coatings which, if desired, are resistant to gastric juices.
  • concentrated saccharide solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • suitable cellulose preparations such as acetylcellulose phthalate or hydroxypropylmethyl- cellulose phthalate, are used.
  • Dye stuffs or pigments may be added to the tablets or dragee coatings, for example, for identification or in order to characterize combinations of active compound doses.
  • Other pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer such as glycerol or sorbitol.
  • the push-fit capsules can contain the active compounds in the form of granules which may be mixed with fillers such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds are in one embodiment dissolved or suspended in suitable liquids, such as fatty oils, or liquid paraffin.
  • stabilizers may be added.
  • Possible pharmaceutical preparations which can be used rectally include, for example, suppositories, which consist of a combination of one or more of the active compounds with a suppository base.
  • Suitable suppository bases are, for example, natural or synthetic triglycerides, or paraffin hydrocarbons.
  • gelatin rectal capsules which consist of a combination of the active compounds with a base.
  • Possible base materials include, for example, liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form, for example, water-soluble salts and alkaline solutions.
  • suspensions of the active compounds as appropriate oily injection suspensions may be administered.
  • Suitable lipophilic solvents or vehicles include fatty oils, for example, sesame oil, or synthetic fatty acid esters, for example, ethyl oleate or triglycerides or polyethylene glycol-400.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension; these substances include, for example, sodium carboxymethyl cellulose, sorbitol, and/or dextran.
  • the suspension may also contain stabilizers.
  • the topical compositions of this invention are formulated in one embodiment as oils, creams, lotions, ointments and the like by choice of appropriate carriers.
  • Suitable carriers include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohol (greater than C 12 ).
  • the carriers may be those in which the active ingredient is soluble.
  • Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired.
  • transdermal penetration enhancers can be employed in these topical formulations. Examples of such enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762; each herein
  • Ointments may be formulated by mixing a solution of the active ingredient in a vegetable oil such as almond oil with warm soft paraffin and allowing the mixture to cool.
  • a vegetable oil such as almond oil
  • a typical example of such an ointment is one which includes about 30% almond oil and about 70% white soft paraffin by weight.
  • Lotions may be conveniently prepared by dissolving the active ingredient, in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.
  • compositions, and methods of the present invention are compositions, and methods of the present invention.
  • Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered in clinical therapy and which are obvious to those skilled in the art are within the spirit and scope of the invention.
  • Nek2 gene copy number and expression were conducted to further understand the clinical relevance of Nek2 in PDAC.
  • Table 1 DNA microarray based gene expression profiling of PDAC cell lines and tumor tissues revealed that Nek2 is highly expressed in PDAC at the transcription level (average of 28 and 41 fold increases in cell lines and tumor tissues, respectively).
  • Array CGH based gene copy number profiling shows that in some pancreatic cancer cell lines and tumor tissues Nek2 has low copy number amplification.
  • Tissue microarray based immunohistochemistry analysis also indicates that the Nek2 protein is highly expressed in pancreatic tumor tissues.
  • Fig. 1 shows examples of a normal pancreatic duct with negative staining compare to a strongly positive PDAC case.
  • Nek2 siRNA For purposes of understatinding Nek2 as a potenital drug target, further experiments examined the effect of Nek2 siRNA on the growth of two PDAC cell lines, PL45 and BxPC-3. As shown in Fig. 2A, the two siRNA sequences used in the experiments effectively knocked down the expression of Nek2 in both cell lines (>90% reduction in protein level compared to Allstar not-targeting siRNA control). Fig. 2B and 2C show the realtime growth curves of cells unpon siRNA treatment. For both cell lines, the Nek2 siRNA treated samples grow much slower than the non-targeting siRNA control. Such results demonstrate that PL45, which expresses higher levels of Nek2, is more sensitive to Nek2 knockdown. These results indicate that inhibition of Nek2 function will have antitumor effects in pancreatic cancer. Example III.
  • Compound 3a was first tested in a panel of 4 pancreatic cancer cells lines for growth inhibition using the sulforhodamine B (SRB) assay. Cells were treated with a serial dilution of the compound for 72 hours and the cell density was measured by the SRB assay. Compound 3a was active against gemcitabine and nab-paclitaxel resistant MIA PaCa-2 cell-lines with IC5 0 S of 0.3 ⁇ . It also potently inhibited proliferation of several other PDAC cell lines (AsPC-1, IC5 0 : 0.064 ⁇ ; PL45, IC 50 : 0.031 ⁇ ; MIA PaCa-2, IC 50 : 0.161 ⁇ ).
  • SRB sulforhodamine B
  • the enantiomer 3b is 10-fold less potent on MIA PaCa-2, gemcitabine resistant MIA PaCa-2, and nab-paclitaxel resistant MIA PaCa-2 cell lines with IC5 0 S of 3.5 ⁇ .
  • CFPAC-1 ceils are the most sensitive to NIM-038A with an IC5 0 value of 1.5 nM followed by PL45 and AsPC-1 with IC 50 values of 31.4 nM and 64.0 nM, respectively.
  • Capan-2 is the less sensitive with an IC5 0 of 1.6 ⁇ .
  • nab- paclitaxel Abraxane
  • gemcitabine nab- paclitaxel
  • Patients respond to the combination initially, but all eventually develop resistance.
  • a pair of cell lines were established that are resistant to either nab- paclitaxel or gemcitabine (Fig. 4A and 4B).
  • the cell lines were developed from the MIA PaCa-2 pancreatic cancer cell line (MIA PACa-2 parental in Fig. 4) which is very sensitive to both nab- paclitaxel and gemcitabine (IC50 2.5 nM and 36.4 nM, respectively).
  • the nab-paclitaxel resistant line (MIA PaCa-2 Nab-p-R) is 42 fold more resistant to nab-paclitaxel with an IC5 0 of 523.8 nM (Fig. 4A).
  • the gemcitabine resistant line (MIA PaCa-2 Gem-R) is 950 fold more resistant to gemcitabine with an IC5 0 of 34.6 ⁇ (Fig. 4B).
  • Compound 3a showed very similar potency in all three cell lines with IC50 values of 161.6 nM, 231.4 nM, and 312.3 nM in the parental, nab-paclitaxel resistant, and gemcitabine resistant cell lines, respectively (Fig. 4C).
  • tumor volume was significantly reduced (/? ⁇ 0.05) with a TGI of 49.5% and 36.0%, respectively.
  • compounds 4 and 4a were well tolerated and did not cause any significant body weight loss ( ⁇ 10.0%) (Fig. 6B).
  • a gold standard in this tumor model at 10 mg/kg, compounds 4 and 4a at 20 mg/kg were slightly less active but exhibited good antitumor efficacy.
  • the compounds of the present invention can be prepared by a variety of procedures, some of which are illustrated in the Schemes below. It will be recognized by one of skill in the art that the individual Steps in the following schemes may be varied to provide the compounds of Formula (I). The particular order of steps required to produce the compounds of Formula (I) and (II) is dependent upon the particular compound being synthesized, the starting compound, and the relative lability of the substituted moieties.
  • step a depicts the standard boron ester formation with bis(pinacolato)diboron in situ, preferably catalyzed by a palladium catalyst (Pd2(dba) 3 ) and a ligand such as P(Cy) 3 to give formula (lb).
  • Pd2(dba) 3 palladium catalyst
  • P(Cy) 3 ligand
  • the bromine atom of 3-bromo-7-chloroimidazo[l ,2-a]pyridine is used as a more active leaving group in the combination of boronic ester or tin analogues in the presence of a suitable catalyst, preferably Pd 2 (dba)3, and a suitable base such as sodium carbonate, to further compounds of Formula (Ic) (Suzuki reaction see: Miyaura, N. et al Synth. Commun., 1981, 513-518).
  • Step b and step c repeat the boronic ester/or acids formation and the Suzuki reaction, respectively to give formula (Ie).
  • Methyl 2-mercaptoacetate (4.31 mL, 47.1 mmol) was added to MeOH (94 mL) and cooled in an ice bath. Following, sodium hydride (3.39 g, 85 mmol) was slowly added and the reaction was stirred for 1 hour at room temperature. The reaction was cooled to -10 C and ethyl propiolate (5.01 mL, 48.0 mmol) was slowly added. The reaction was stirred at 0 °C for 1 hour, room temperature for 1 hour, and heated to 45 °C for three hours.
  • Methyl 3-hydroxythiophene-2-carboxylate (5 g, 26.8 mmol) and l-(2- (trifluoromethyl)phenyl)ethan-l-ol (5.61 g, 29.5 mmol) were dissolved in DCM (80 mL) and cooled in an ice bath. Following, triphenylphosphine (10.18 g, 38.9 mmol) was added and DEAD (6.97 g, 38.9 mmol) was added dropwise. After the addition, the reaction was removed from the ice bath and stirred at room temperature for 12 hours. The crude reaction was condensed and adsorbed onto silica and purified via flash chromatography using Hexanes/EtOAC. The title compound was isolated as white solid/crystals methyl 3- (l-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (6.12 g, 63.7%).
  • Methyl 3-(l-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (0.150 mg, 0.454 mmol) was dissolved in THF (5 mL) and cooled to -20 °C. Following, trimethyl borate (0.103 mL, 0.908 mmol) was added. LDA (0.908 mL, 1.362 mmol) was added to the reaction dropwise over the course of 10 minutes. After about 30 minutes, all starting material was consumed, and the boronic acid was generated. In a separate reaction vessel, lb (0.454 mmol) was dissolved in 7:3 THFAVater and degassed with argon.
  • Methyl 5 7 l-(2-(dimethylamino)ethyl)-lH-pyrazol-4-yl)imidazo[l,2-a]pyridin- -yl)-3-(l-(2-(trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate
  • reaction mixture was stirred at 120 °C for 30 min under microwave irradiation.
  • the reaction mixture was filtered over a pad of celite and concentrated under vacuum.
  • the residue was purified with column chromatography (2-4% MeOH /DCM) to afford methyl 5-(7-(l-(2-(dimethylandno)ethyl)-lH-pyrazol-4-yl)imidazo[l,2-a]pyridin-3-yl)-3-(l-(2- (trifluoromethyl)phenyl)ethoxy)thiophene-2-carboxylate (233 mg, 40%) as a yellow solid.
  • Kinase activity was measured in a microfluidics assay that monitors the separation of a phosphorylated product from substrate.
  • the assay was run using a 12-sipper chip on a Caliper EZ Reader II (PerkinElmer®, Walthman, USA) with separation buffer (100 mM HEPES, 10 mM EDTA, 0.015% Brij-35, 0.1% CR-3 [PerkinElmer®, Walthman, USA]).
  • the NEK2 enzyme (InvitrogenTM, Grand Island, USA) was diluted in kinase buffer to a concentration of 2 nM and 5 of the enzyme mixture was transferred to the assay plate. The inhibitors/NEK2 enzyme were incubated for 60 minutes with minor shaking. A substrate mix was prepared containing ATP (Ambresco®, Solon, USA) and 5FAM tagged NEK2 peptide (PerkinElmer®, Walthman, USA) dissolved in kinase buffer, and 5 of the substrate mix was added to the assay plate. Running concentrations were as follows: ATP (190 ⁇ ), peptide (1.5 ⁇ ),
  • Prisim 6 and fit to an exponential one-phase decay line and IC5 0 values were obtained from the half-life value of the curve.
  • IC5 0 values were generated in duplicate and error was calculated from the standard deviation between values. All examples showed activity with less than IC 50 of l OO nM.
  • Dose-response growth curves and IC5 0 doses were graphed using the curve-fitting PRISM (GraphPad software). To compare tumour growth we used an unpaired Student's- ⁇ test (InStat program, GraphPad software). P values were statistically significant at p ⁇ 05.
  • Fig. 7 shows evaluation of NIM-038A in a normal pancreatic ductual epithelial cell line. As shown, NIM-038A was much less sensitive to normal pancreatic ductual epithelial cell line than to cancer cells.

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Abstract

La présente invention concerne le domaine de la chimie médicale. En particulier, l'invention concerne une nouvelle classe de petites molécules qui présentent la structure d'imidazole pyrimidine et qui agissent comme inhibiteurs de la protéine NEK2, ainsi que leur utilisation comme agents thérapeutiques pour le traitement du cancer et d'autres maladies.
PCT/US2017/059061 2016-10-31 2017-10-30 Inhibiteurs à petites molécules de nek2 et utilisations associées WO2018081719A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110003202A (zh) * 2019-03-25 2019-07-12 华东师范大学 咪唑并[1,2-a]吡啶类衍生物的制备方法和应用
WO2022006292A1 (fr) * 2020-06-30 2022-01-06 Bioventures, Llc Chimères ciblant la protéolyse de nek2 destinées à être utilisées dans une maladie maligne

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040019003A1 (en) * 2002-01-24 2004-01-29 Chiron Corporation Nek2 inhibitors
US7915305B2 (en) * 2006-01-23 2011-03-29 Vertex Pharmaceuticals Incorporated Thiophene-carboxamides useful as inhibitors of protein kinases
US8030327B2 (en) * 2004-11-08 2011-10-04 Mds K.K. Fused imidazole derivative
CN105017245A (zh) * 2014-04-30 2015-11-04 华东师范大学 一种咪唑并吡啶化合物及其制备方法和应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040019003A1 (en) * 2002-01-24 2004-01-29 Chiron Corporation Nek2 inhibitors
US8030327B2 (en) * 2004-11-08 2011-10-04 Mds K.K. Fused imidazole derivative
US7915305B2 (en) * 2006-01-23 2011-03-29 Vertex Pharmaceuticals Incorporated Thiophene-carboxamides useful as inhibitors of protein kinases
CN105017245A (zh) * 2014-04-30 2015-11-04 华东师范大学 一种咪唑并吡啶化合物及其制备方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110003202A (zh) * 2019-03-25 2019-07-12 华东师范大学 咪唑并[1,2-a]吡啶类衍生物的制备方法和应用
WO2022006292A1 (fr) * 2020-06-30 2022-01-06 Bioventures, Llc Chimères ciblant la protéolyse de nek2 destinées à être utilisées dans une maladie maligne

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