WO2013144532A1 - 3 -cyano- 5 -arylamino-7 -cycloalkylaminopyrrolo [1, 5 -a] pyrimidine derivatives and their use as antitumor agents - Google Patents

Abstract

The invention relates to chemical compounds of Formula (I): or a salt thereof. In some embodiments, the invention relates to inhibitors of CK2. In still further embodiments, the invention relates to pharmaceutical compositions comprising compounds disclosed herein and their use in the prevention and treatment of CK2-related conditions and diseases, e.g., cancer.

Description

3 -CYANO- 5 -ARYLAMINO-7 -CYCLOALKYLAMINOPYRROLO [1 , 5 -A]

PYRIMIDINE DERIVATIVES AND THEIR USE AS ANTITUMOR AGENTS

BACKGROUND

[0001] Protein kinases are a family of structurally and functionally well-characterized mediators of cellular signal transduction networks. In fact, disregulation of kinase activity has been frequently implicated in the origin and manifestation of transformed cellular phenotypes characteristic of cancer.¹ One such protein kinase is CK2 (Casein Kinase 2).

[0002] CK2 exists in a tetrameric form, composed of two catalytic (a and/or α') subunits and two regulatory (β) subunits.²⁵ CK2 is a highly conserved, constitutively active protein Ser/Thr kinase that performs a variety of roles essential to the maintenance of cellular homeostasis.² In particular, CK2 has been shown to play a key role in cell cycle control, cellular differentiation and proliferation³, as well as promoting cell survival via modulation of apoptotic pathways.⁴ In normal cells, the intrinsic level of CK2 activity appears to be tightly regulated. However, elevated levels of CK2 expression or alterations in CK2 function have been implicated in a variety of human diseases, including cancer.⁵' ⁶' ⁷' ²⁶ Furthermore, targeted overexpression of CK2 in transgenic animal models results in neoplastic growth.⁸' ⁹

[0003] A key property of CK2 is that it can undergo dynamic shuttling to different cellular compartments in response to diverse stimuli, ¹⁰' ^u' ²⁷ and has been shown to phosphorylate and regulate the activity and stability of multiple tumor suppressor proteins (including PML, p53, PTEN), as well as oncogenes and transcriptional activators (such as c-Myc, c-Myb, c-Jun, NFKB, β- catenin).² For example, CK2 -mediated phosphorylation of the tumor suppressor PTEN inactivates the phosphatase thereby promoting cell survival signaling via activation of the PI3K pathway.¹² In addition, CK2 acts as a positive regulator of Wnt signaling, mediated in part through phosphorylation of β-catenin, protecting it from proteasomal degradation, thereby supporting Tcf/Lef driven transcription of pro-survival gene products such as Survivin,¹³ c-Myc,¹⁴ and Cyclin Dl .¹⁵

[0004] More recent evidence suggests CK2 can act as suppressor of apoptosis via phosphorylation of pro-apoptotic proteins (including BID, HS1, Max), protecting them from caspase mediated cleavage.³ In addition, CK2 inhibition has been shown to sensitize cells to TRAIL receptor-mediated apoptosis, ionizing radiation and chemotherapeutic agents.²'³

[0005] A variety of molecules have been employed as probes for understanding the potential effects of pharmacological inactivation of CK2 in cells and in vivo.²³ However, despite their utility as probes for validating CK2 as a promising cancer target, the currently available small molecule inhibitors lack the necessary balance of potency, selectivity and drug-like properties for consideration as agents for the treatment of disease in man.

[0006] As such, there is a need for novel compounds that target CK2 with improved properties that would exploit these cancer-linked pathways.

SUMMARY OF THE INVENTION

[0007] The present invention provides novel compounds that target CK2 kinase pathways.

Moreover, methods of use of these compounds in inhibiting CK2, inhibiting cancer, as well as methods of preparation of these compounds are also presented herein. Furthermore, pharmaceutical compositions, combination therapies, and uses thereof are also provided in the present application.

[0008] Accordingly, one aspect of the invention provides a compound of Formula (I):

(I)

wherein:

R¹ is selected from -N(CH₃)(CH₂)₂NH₂, and -N(CH₃)(CH₂)₂N(CH₃)₂; and

R² is C₃_₅cycloalkyl;

and/or a pharmaceutically acceptable salt thereof.

[0009] Compounds of Formulae (I) and/or (II) possess beneficial efficacious, metabolic, pharmacokinetic, and/or pharmacodynamic properties. Compounds of Formulae (I) and/or (II) are useful for their ability to inhibit CK-2 activity and are accordingly also useful in the treatment of diseases or medical conditions mediated alone or in part by CK2 kinase.

[00010] An additional aspect of the invention provides a method of treating cancer. The method comprises administering to a subject, e.g. , a human, diagnosed with, at risk for, or exhibiting symptoms of cancer a pharmaceutical composition comprising a compound of the invention, such that the cancer is treated.

[00011] In particular, compounds of Formulae (I) and/or (II) may be used for the treatment of cancer, including familiary adenomatous polyposis, colon cancer (such as sporadic colon carcinoma), rectum cancer, lung cancer (such as non-small cell lung cancer including the non- squamous and squamous subtypes), hepatocellular cancer, breast cancer, endometrial ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, Barrett's Esophagus, gastric cancer, pancreatic cancer, melanoma, mesothelioma, synovial sarcomas, adrenocortical tumors (such as adrenocortical carcinoma), leukemia, Wilms tumors, hepatoblastoma, and increased bone density.

[00012] Also in accordance with the present invention there are provided methods of using said compounds or pharmaceutically acceptable salts thereof in the treatment of cancer.

[00013] In yet another aspect, the invention provides a method of inhibiting CK2 activity. The method comprises reacting a compound of the invention with CK2 under conditions such that CK2 activity is inhibited.

[00014] Another aspect of the invention provides a pharmaceutical composition which comprises a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, in association with a pharmaceutically-acceptable diluent or carrier.

[00015] In an additional aspect, the present invention provides compounds of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, for use as a medicament.

[00016] In yet another aspect, the invention pertains to the use of compounds of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, for the manufacture of a medicament for the production of a CK2 inhibitory effect in a subject.

[00017] In a further aspect, the invention pertains to the use of compounds of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, for the manufacture of a medicament for the production of an anti-cancer effect in a subject.

[00018] An additional aspect of the invention provides a method of preparation of compounds of Formulae (I) and/or (II), as defined in any one of the examples described herein.

BRIEF DESCRIPTION OF THE DRAWING

[00019] Figure 1 depicts a concentration dependent growth inhibition curve of cells (NCI- H1975) treated with 2-(4-((4-((3-chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6- yl)oxy)piperidin-l-yl)-N-methylacetamide ("Agent 1") and the compound of Example 2 (3 nM), versus Agent 1 alone.

[00020] Figure 2 depicts a concentration dependent growth inhibition curve of cells (NCI- HI 975) treated with Iressa® ("Agent 2") and the compound of Example 2 (3 nM), versus Agent 2 alone.

[00021] Figure 3 depicts a concentration dependent growth inhibition curve of cells (NCI- H1975) treated with Selumetinib ("Agent 3") and the compound of Example 2 (3 nM), versus Agent 3 alone. DETAILED DESCRIPTION OF THE INVENTION

[00022] The present invention provides novel compounds that target CK2 cancer-linked kinase pathways. The present invention, including compounds, methods, and pharmaceutical compositions will be described with reference to the following definitions that, for convenience, are set forth below.

I. DEFINITIONS

[00023] "ALKYL": As used herein, the term "alkyl" means a noncyclic straight chain or branched, unsaturated or saturated hydrocarbon such as those containing from 1 to 10 carbon atoms. In some embodiments, the term "alkyl" contains from 1 to 6 carbon atoms. In some embodiments, the term "alkyl" contains from 7 to 10 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. The alky moiety described herein may be mono- or di-valent (i.e. , a mono- or di-radical).

[00024] In some embodiments, the term "alkyl" means "Ci ALKYL," which refers to a saturated hydrocarbon radical having one carbon atom. In some embodiments, the term "alkyl" means "C2ALKYL," which refers to saturated hydrocarbon radicals having two carbon atoms. In some embodiments, the term "alkyl" means "C3ALKYL," which refers to both straight and branched chain saturated hydrocarbon radicals having one, two, or three carbon atoms. In some embodiments, the term "alkyl" means "C4ALKYL," which refers to both straight and branched chain saturated hydrocarbon radicals having one, two, three or four carbon atoms.

[00025] "C -3 ALKYL": As used herein the term "C^alkyl" refers to both straight and branched chain saturated hydrocarbon radicals having one, two, or three carbon atoms. In some embodiments, "C i _3alkyl" is "Cj alkyl". In some embodiments, "Ci _3alkyl" is "C2alkyl". In some embodiments, "C^alkyl" is "C3alkyl".

[00026] "Ci-2 ALKYL": As used herein the term "Ci_2alkyl" refers to straight chain saturated hydrocarbon radicals having one, or two carbon atoms. In some embodiments, "Ci _2alkyl" is "C ! alkyl". In some embodiments, "C^alkyl" is "C₂alkyl".

[00027] "CANCER": The term "cancer" is art-recognized and used herein to describe, for example, any of various cellular diseases with malignant neoplasms characterized by the proliferation of cells. It is not intended that the diseased cells must actually invade surrounding tissue and metastasize to new body sites. Cancer can involve any tissue of the body and have many different forms in each body area. It encompasses, for example, non-invasive cancers, invasive cancers, and metastatic cancers. Within the context of certain embodiments, whether "cancer is reduced" may be identified by a variety of diagnostic manners known to one skill in the art including, but not limited to, observation the reduction in size or number of tumor masses or if an increase of apoptosis of cancer cells observed (e.g., if more than a 5 % increase in apoptosis of cancer cells is observed for a sample compound compared to a control without the compound). It may also be identified by a change in relevant biomarker or gene expression profile (e.g., measuring changes in prostate-specific antigen (PSA) for prostate cancer, and human epidermal growth factor (her2) for breast cancer, or others.

[00028] "COMBINATION": As used herein, the term "combination," when used in the context of combination therapy, it is to be understood that this refers to simultaneous, separate or sequential administration. In one aspect of the invention "combination" refers to simultaneous administration. In another aspect of the invention "combination" refers to separate administration. In a further aspect of the invention "combination" refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination.

[00029] "C3.5 CYCLOALKYL": As used herein the term "C3.5 cycloalkyl" refers to non- aromatic monocyclic alkyls, which may be saturated, having three, four, or five carbon atoms. In some embodiments, "C3.5 cycloalkyl" is "C3 cycloalkyl." In some embodiments, "C3.5 cycloalkyl" is "C4 cycloalkyl." In some embodiments, "C3.5 cycloalkyl" is "C5 cycloalkyl."

Representative saturated cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.

[00030] "C3 CYCLOALKYL": As used herein the term "C3 cycloalkyl" refers to non- aromatic monocyclic alkyls, which may be saturated, having three carbon atoms. In some embodiments, "C3 cycloalkyl" includes cyclopropyl.

[00031] "C4 CYCLOALKYL": As used herein the term "C4 cycloalkyl" refers to non- aromatic monocyclic alkyls, which may be saturated, having four carbon atoms. In some embodiments, "C4 cycloalkyl" includes cyclobutyl.

[00032] "C5 CYCLOALKYL": As used herein the term "C5 cycloalkyl" refers to non- aromatic monocyclic alkyls, which may be saturated, having five carbon atoms. In some embodiments, "C5 cycloalkyl" includes cyclopentyl. [00033] "OBTAINING": As used herein, the term "obtaining" is used herein to describe the act of acquiring, for example, a material necessary for methods of the present invention. The act of acquiring may include, for example, purchasing the material.

[00034] "PREVENT (ING)" or "PREVENTION": As used herein, the terms "prevent", "preventing" include the prevention of the recurrence, spread or onset. It is not intended that the present invention be limited to complete prevention. In some embodiments, the onset is delayed, or the severity of the disease is reduced.

[00035] "SALT(S)": As used herein, the term "salt(s)" refer to derivatives of the disclosed compounds where the parent compound is modified making acid or base salts thereof. Examples of salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkylamines, or dialkylamines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. In particular embodiment the salts are conventional non-toxic pharmaceutically acceptable salts including the quaternary ammonium salts of the parent compound formed, and non-toxic inorganic or organic acids. Particular salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, sulfanilic, 2- acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, and the like.

[00036] Pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are particular embodiments. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

[00037] "SUBJECT": The term "subject" as used herein is intended to include organisms, e.g. , prokaryotes and eukaryotes, which are capable of suffering from or afflicted with cancer and contain CK2. Examples of subjects include mammals (warm-blooded animals), e.g. , humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g. , a human suffering from, at risk of suffering from, or potentially capable of suffering from cancer. [00038] "TREAT", "TREATING" OR "TREATMENT": The terms "treat", "treating" or "treatment" include administering a therapeutically effective amount of a compound sufficient to reduce or eliminate at least one symptom of the state, disease or disorder, e.g., CK2 related conditions and diseases, e.g., cancer.

II. COMPOUNDS OF THE INVENTION

[00039] One embodiment of the invention provides a compound of Formula (I):

(I)

wherein:

R¹ is selected from -N(CH₃)(CH₂)₂NH₂, and -N(CH₃)(CH₂)₂N(CH₃)₂; and

R² is C₃_₅cycloalkyl;

or a salt, e.g. , pharmaceutical salt, thereof.

[00040] In one embodiment, the invention provides a compound represented by Formula (II):

(Π)

wherein:

R¹ is selected from -N(CH₃)(CH₂)₂NH₂, and -N(CH₃)(CH₂)₂N(CH₃)₂;

or a salt, e.g. , pharmaceutical salt, thereof.

[00041] Additional embodiments of the invention are as follows. These additional embodiments relate to compounds of Formula (I) and/or Formula (II) and/or pharmaceutically acceptable salts thereof. Such specific substituents may be used, where appropriate, with any of the definitions, claims or embodiments defined hereinbefore or hereinafter. R¹ EMBODIMENTS

[00042] In certain embodiments of the invention, R¹ is selected from -N(CH₃)(CH₂)₂NH₂, and -N(CH₃)(CH₂)₂N(CH₃)₂.

[00043] In some embodiments, R¹ is -N(CH₃)(CH₂)₂NH₂. In some embodiments, R is

-N(CH₃)(CH₂)₂N(CH₃)₂.

R² EMBODIMENTS

[00044] In some embodiments, R² is selected from the group consisting of cyclopropyl and cyclobutyl. In some embodiments, R² is cyclopropyl. In some embodiments, R² is cyclobutyl.

R¹ and R² EMBODIMENTS

[00045] In some embodiments, R¹ is -N(CH₃)(CH₂)₂NH₂ and R² is cyclopropyl. In some embodiments, R¹ is -N(CH₃)(CH₂)₂N(CH₃)₂ and R² is cyclopropyl.

[00046] Also provided herein is:

[00047] N-(5-(3-cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5-a]pyrimidin-5-ylamino)-2-((2-

(dimethylamino)ethyl)(methyl)amino)phenyl)acetamide;

[00048] N-(2-((2-aminoethyl)(methyl)amino)-5-(3-cyano-7-(cyclopropylamino) pyrazolo[ 1 ,5- a]pyrimidin-5 -ylamino)phenyl)acetamide; and

[00049] N-(2-((2-aminoethyl)(methyl)amino)-5-(3-cyano-7-(cyclopropylamino) pyrazolo[ 1 ,5- a]pyrimidin-5-ylamino)phenyl)acetamide trifluoroacetic acid salt.

[00050] In some embodiments, the present invention provides any compound listed herein, and, if a free base, a pharmaceutically acceptable salt thereof.

[00051] In one embodiment, provided herein is N-(2-((2-aminoethyl)(methyl)amino)-5-(3- cyano-7-(cyclopropylamino) pyrazolo[ 1 ,5-a]pyrimidin-5-ylamino)phenyl)acetamide.

[00052] In one embodiment, provided herein is N-(2-((2-aminoethyl)(methyl)amino)-5-(3- cyano-7-(cyclopropylamino) pyrazolo[l ,5-a]pyrimidin-5-ylamino)phenyl)acetamide trifluoroacetic acid salt.

[00053] In one embodiment, provided herein is N-(5-(3-cyano-7-

(cyclopropylamino)pyrazolo[l ,5-a]pyrimidin-5-ylamino)-2-((2-(dimethylamino)ethyl)(methyl)- amino)phenyl)acetamide.

[00054] Specific exemplary compounds described herein are set forth in the EXAMPLES below. Those skilled in the art will recognize that compounds described herein, including those set forth in EXAMPLES, can occur in the free, non-salt, form or can occur as salts.

[00055] It is also to be understood that certain compounds of Formulae (I) and/or (II) can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms.

[00056] In addition, the present invention provides compound intermediates of formula (a),

(^a)

[00057] Compounds of formula (a) are useful for preparation of compounds described above, and methods of using these intermediates to make compounds of Formulae (I) and/or (II) and/or pharmaceutically acceptable salts thereof.

[00058] In some embodiments, a precursor compound to a compoud of formula (a) is useful for the preparation of a compound of formula (a).

[00059] It is understood that any embodiment described herein can be combined with any other suitable embodiment described herein to provide additional embodiments. For example, various R1 substituent embodiments can be combined with various R^ substituent embodiments. As would be appreciated by one skilled in the art, any combination of substituents, or group of substituents, is considered to be within the scope of the present invention.

[00060] Certain embodiments of the present invention, and compounds described herein relate to inhibitors of CK2. Moreover, compounds of the present invention, for example, the species noted in the Exemplification section, are potentially useful in the treatment of diseases associated with inhibition of CK2, in particular in the treatment and/or prevention of cancer.

[00061] The following in vitro binding and cellular assays may be used to determine the activity and specificity of compounds of the present invention to bind to CK-2 and inhibit CK-2 function in a cell.

BIOLOGICAL ASSAYS

A. CK2 IN-VITRO MOBILITY SHIFT ASSAY

[00062] Activity of N-terminal 6 X His-tagged recombinant human full length Casein Kinase 2 alpha subunit (CK2) was determined in-vitro using a mobility shift assay on a Caliper LC3000 reader (Caliper, MA), which measures fluorescence of a phosphorylated and unphosphorylated "CK2tide" (BODIPY-FL-RRRDDDSDDD-CONH2, Intonation, Boston, MA) and calculates a ratiometric (i.e., ration of product formed to total product and substrate remaining) value to determine percent turnover. CK2 (48.7kDa, Cat. # 14-445, Upstate/Millipore, Billerica, MA) was expressed in Sf21 insect cells with typical yield >70% purity.

[00063] Phosphorylation of the CK2tide in the presence and absence of compound of interest was determined. 5 μΐ of Enzyme/Substrate/adenosine triphosphate (ATP) mix consisting of 10.5 nM CK2, 3.6uM CK2tide, 180mM NaCl and 62.4uM ATP in 1.2x buffer was pre-incubated with 2 μΐ of compound for 20 minutes at 25 °C. Reactions were initiated with 5 μΐ of Metal mix consisting of 24mM MgCl₂ in 1.2x buffer and incubated at 25 °C for 90 minutes. Reactions were stopped by addition of 5 μΐ of termination buffer consisting of 100 mM HEPES, 121 mM ethylenediamine tetraacetic acid, 0.8% Coatin Reagent 3 (Caliper, MA), and 0.01% Tween. Phosphorylated and unphosphorylated substrate was detected by a Caliper LC3000 reader (Caliper, MA) in the presence of separation buffer consisting of 100 mM HEPES, 16 mM ethylenediamine tetraacetic acid, 0.1% Coatin Reagent 3 (Caliper, MA), 0.015% Brij-35, 5% DMSO, and 5.6 mM MgCl₂. The separation conditions used by the Caliper LC3000 were -1.7 PSI, -500 V upstream voltage, -2000 V downstream voltage, 0.2 second sample duration sampling (sip), 55 second post duration sampling (sip), 10%) laser strength.

[00064] The values for percent inhibition of CK2 enzyme activity were plotted as a function of the compound concentration and the IC50 values were determined, and are presented in TABLE 1 below.

[00065] TABLE 1

* = Data points are geometric means

encompassing multiple test runs

B. HCT- 116 72HR ALAMARBLUE CELL PROLIFERATION ASSAY

[00066] This fluorometric assay determines the effect CK2 inhibitors have on cancer cell growth. The AlamarBlue Assay incorporates a fluorometric growth indicator based on detection of metabolic activity. Specifically, the system incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. A decrease in fluorescence is indicative of cell death. GI50S (Growth Inhibition

Cone. 50%)) are calculated for each compound using a HCT-116 cells only Day 0 fluorescence read as the Min and Day 3 DMSO vehicle control fluorescence as Max. Percent Net Growth for each compound concentration is calculated and the GI50 reported.

[00067] HCT-1 16 cells were seeded at 2500 cells/well in Costar Flat bottomed 96 well plates

(Black wall/clear bottom) in 90 μΐ. of phenol-red free RPMI 1640 supplemented with 10% FBS / 1% L-Glu and incubated overnight in 37 °C, 5% CO2. Compound plates were then treated with lOuL of

10X compound (9pt dose response, triplicates across plates) and incubated for 72hrs at 37 °C, 5% CO2. The AlamarBlue assay was performed by adding 10 uL of reagent to the compound treated cell plates and incubated for 4 hours at 37 °C, 5% CO2. Fluorescence was measured at 535 nm

(excitation) and 590 nm (emission) using a Tecan Ultra plate reader. Percent Net Growth relative to a Day 0 cell only AlamarBlue plate read was calculated for the 72 hr assay plates, the GI50 calculated, and are presented in TABLE 2 below.

[00068] TABLE 2

* = Data points are geometric means

encompassing multiple test runs

C. CELL BASED ASSAY TO DETERMINE ANTI-PROLIFERATIVE EFFECTS ON

CANCER CELLS IN COMBINATION WITH EGFR INHIBITORS

[00069] This fluorometric assay determines the effect CK2 inhibitors have on cancer cell growth in combination with EGFR inhibitors (Agent 1 = 2-(4-((4-((3-chloro-2-fluorophenyl)amino)- 7-methoxyquinazolin-6-yl)oxy)piperidin-l-yl)-N-methylacetamide; Agent 2 = Iressa®; and Agent 3 = Selumetinib). The AlamarBlue Assay (Invitrogen) incorporates a fluorometric growth indicator based on detection of metabolic activity. Specifically, the system incorporates an oxidation- reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. A decrease in fluorescence is indicative of cell death. GI50S (Growth Inhibition Cone. 50%) are calculated for each compound using a cells only Day 0 fluorescence read as the Min and Day 3 or Day 6 DMSO vehicle control fluorescence as Max. Percent Net Growth for each compound concentration is calculated and the GI50 reported.

[00070] NCI-H1975 cells were seeded in Costar Flat bottomed 96 well plates (Black wall/clear bottom) in 90 uL of phenol-red free RPMI 1640 supplemented with 10% FBS / 1% L-Glu and incubated overnight in 37 °C, 5% C0₂. Compound plates were then treated with 10 uL of 10X compound (9pt dose response, triplicates across plates), with and without CK2 (i.e., the compound of Example 2) as a sensitizer at 30nM and 3nM, and then incubated for 3 and 5 days at 37 °C, 5% C0₂. The AlamarBlue assay was performed by adding 10 uL of reagent to the compound treated cell plates and incubated for 2-4 hours at 37 °C, 5% C0₂. Fluorescence was measured at 535 nm (excitation) and 590nm (emission) using a Tecan Ultra plate reader. Percent Net Growth relative to a Day 0 cell only AlamarBlue plate read was calculated for the 3 Day or 6 Day assay plates and the GI₅₀ calculated, and are presented in Figure 1 , Figure 2, and Figure 3 herein.

[00071] Another embodiment of the invention provides a compound selected from the group consisting of any one or more of compounds described in the Exemplification section, or a salt, e.g. , a pharmaceutically acceptable salt, thereof. Moreover, if such compound is represented as a salt, the present invention is intended to include free bases, free acids, or alternative salts of these particular compounds. Additional embodiments comprise compositions and medicaments containing the same (including the aforementioned free bases, free acids, or alternative salts), as well as processes for the preparation and use of such compounds, compositions and medicaments, which are disclosed herein or within the skill or the ordinarily skilled artisan in light of the disclosure herein.

[00072] For example, processes for preparing free bases from salt forms disclosed herein may follow the process described in Example 3, or may be prepared using ordinary skill in the art in light of the present disclosure. Moreover, it should be noted that each of these compounds, and salts thereof, are also intended to be separate embodiments, and in this regard, each species listed in the Exemplification, and salt thereof, should be considered to be an individual embodiment.

[00073] The ordinarily skilled artisan would understand that some compounds of the invention, e.g., Formulae (I) and/or (II), may have chiral centers and/or geometric isomeric centers, and it is to be understood that the invention encompasses all such optical, diastereoisomers and geometric isomers. For example, all optical, diastereoisomers and geometric isomers of compounds of the present invention as described in the Exemplification section are intended to be included within the scope of the present invention.

[00074] It should also be understood that compounds disclosed herein may exist in solid and solution form as tautomers, depending upon the particular compound and the particular composition in which compound(s) is/are contained.

[00075] Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention. By way of example, the R¹ group of Formulae (I) and/or (II) may comprise one or more deuterium atoms. Mixtures of isomeric forms may be separated and/or purified by techniques as would be known to one skilled in this art, including but not limited to column chromatography.

[00076] In certain embodiments, novel compounds that comprise the starting material may be useful in the present invention.

[00077] Additional embodiments comprise compositions and medicaments containing the same, as well as processes for the preparation and use of such compounds, compositions and medicaments.

[00078] Moreover, it should be understood that the present invention is intended to include any novel compound or pharmaceutical composition described herein.

III. GENERAL METHODS OF PREPARATION

[00079] The present invention provides synthetic methodologies for preparing compounds of

Formulae (I) and/or (II). In certain embodiments, compounds of Formulae (I) and/or (II) are further purified.

[00080] An additional embodiment of the invention provides a method of preparation of a compound of Formulae (I) and/or (II), as described herein, e.g. , as defined in any one of the examples described herein. In some embodiments, the method comprises a step of obtaining, e.g. , providing, a compound used in the preparation.

[00081] Compounds of Formulae (I) and/or (II) are generally prepared according to the steps depicted in SCHEME 1A, SCHEME IB, and/or SCHEME 1C set forth below. In SCHEME 1A,

SCHEME IB, and/or SCHEME 1C, R¹ and R² are defined in classes and subclasses as described herein. [00082] SCHEME 1A

-N(CH₃)CH₂CH₂NHBoc

(d) (I)

[00083] As generally depicted in SCHEME 1A, the present invention provides a method for preparing a compound of Formulae (I) and/or (II) comprising providing a 4,6-dichloropyrazolo[l,5- ajpyrimidine compound of formula (a), an amine compound of formula (b), and an aniline compound of formula (c), and further coupling a compound of formula (b) with an aniline compound of formula (c) to provide a tert-butyl carbamate compound of formula (d). Deprotection of a tert- butyl carbamate of formula (d) is performed to give a compound of Formula (I).

[00084] As depicted in SCHEME 1A, a 4,6-dichloropyrazolo[l,5-a]pyrimidine compound of formula (a) is converted to an amine compound of formula (b) upon treatment with a primary amine, either neat or in the presence of a suitable solvent, at a temperature of from about 25° C to about 120° C, with or without the addition of an organic amine base. In some embodiments, a suitable organic amine base is diisopropylethylamine (DIPEA). In some embodiments, an organic solvent is included. Exemplary organic solvents include, but are not limited to ethanol, n-butanol, 1 ,4-dioxane, and N-methylpyrrolidone (NMP). In some embodiments, the temperature of the displacement reaction is 25° C. In some embodiments, the temperature of the displacement reaction is 120° C. [00085] An amine compound of formula (b) is coupled with an aniline compound of formula

(c) under displacement conditions using a suitable inorganic base, in the presence of a suitable organic solvent. In some embodiments, a suitable inorganic base is KF. An exemplary organic solvent includes, but is not limited to N-methylpyrrolidone (NMP). In some embodiments, the temperature of the coupling reaction is 130° C. In some embodiments, the temperature of the coupling reaction is 140° C.

[00086] A tert-butyl carbamate of formula (d) is converted to a compound of Formula (I) upon treatment with a suitable acid. In certain embodiments, a suitable acid is trifluoroacetic acid (TFA). In certain embodiments, a suitable acid is hydrochloric acid.

-N(CH₃)CH₂CH₂OH

Sulfonylation

[00088] As generally depicted in SCHEME IB, the present invention provides a method for preparing a compound of Formula (I) comprising providing a 4,6-dichloropyrazolo[l ,5-a]pyrimidine compound of formula (a), an amine compound of formula (b), an aniline compound of formula (e), and coupling a compound of formula (b) with a compound of formula (e) to form an alcohol compound of formula (f). Conversion of an alcohol compound of formula (f) to a mesylate compound of formula (g) followed by a displacement reaction with an amine (R³-NH-R⁴) gives a compound of Formula (I).

[00089] As depicted in SCHEME IB, an amine compound of formula (b) is coupled with an aniline compound of formula (e) under coupling conditions known to one of ordinary skill in the art in the presence of a suitable palladium catalyst, ligand, a suitable inorganic base and a suitable organic solvent (e.g., Buchwald coupling reaction conditions), to provide an alcohol of formula (f). In some embodiments, a suitable palladium catalyst is Pd₂(dba)₃. In some embodiments a suitable ligand is 9,9-dimethyl-9H-xanthene-4,5-diyl)bis-diphenylphosphine In some embodiments, a suitable inorganic base is Cs₂C0₃. An exemplary organic solvent includes, but is not limited to N,N- dimethylacetamide (DMA). In some embodiments, the temperature of the coupling reaction is about 150° C.

[00090] An alcohol of formula (f) is treated with a sulfonyl halide in the presence of a suitable organic solvent to give a sulfonic acid ester of formula (g), and is further reacted with an amine (R³- NH-R⁴) to give a compound of Formula (I). An exemplary sulfonyl halide includes, but is not limited to, methanesulfonyl chloride. An exemplary organic solvent includes, but is not limited to, pyridine. In some embodiments, the reaction is performed at a temperature of about 0° C.

[00091] A sulfonic acid ester compound of formula (g) is reacted with ammonia, in the presence of a suitable organic solvent to give a compound of Formula (I). In some embodiments, the organic solvent is MeOH. In some embodiments, the reaction is performed at a temperature of about 100° C.

[00092] SCHEME 1C

R¹= -N(CH₃)CH2CH₂NMe2 [00093] As generally depicted in SCHEME 1C, the present invention provides a method for preparing a compound of Formula (I) comprising providing a 4,6-dichloropyrazolo[l ,5-a]pyrimidine compound of formula (a), an amine compound of formula (b), an aniline compound of formula (h), and coupling a compound of formula (b) with a compound of formula (h) to give a compound of Formula (I).

[00094] As depicted in SCHEME 1C, an amine compound of formula (b) is coupled with an aniline compound of formula (h) under coupling conditions known to one of ordinary skill in the art in the presence of a suitable palladium catalyst, ligand, a suitable inorganic base and a suitable organic solvent (e.g., Buchwald coupling reaction conditions), to provide a compound of Formula (I). In some embodiments, a suitable palladium catalyst includes, but is not limited to, Pd₂(dba)₃. In some embodiments a suitable ligand, includes, but is not limited to, 9,9-dimethyl-9H-xanthene-4,5- diyl)bis-diphenylphosphine. In some embodiments, a suitable inorganic base is Cs₂C0₃. An exemplary organic solvent includes, but is not limited to Ν,Ν'-dimethylacetamide (DMA). In some embodiments, the temperature of the coupling reaction is about 150° C.

[00095] Starting materials depicted in SCHEME 1A, SCHEME IB, and/or SCHEME 1C may be prepared as depicted in SCHEME 2 set forth below.

[00097] For preparation of starting materials related to SCHEME 1A, a fluorobenzene compound of formula (i) is coupled to an amine compound of formula j) by displacement to give a nitro compound of formula (k), using a suitable inorganic base in the presence of a suitable organic solvent. In some embodiments, a suitable inorganic base is CS₂CO₃. Exemplary organic solvents include, but are not limited to Ν,Ν'-dimethylformamide (DMF). In some embodiments, the displacement reaction is performed at a temperature of about 60° C. In some embodiments, the displacement reaction is performed at a temperature of about 80° C.

[00098] Reduction of a nitro compound of formula (k) is performed under conditions known to one of ordinary skill in the art using a suitable catalyst, in the presence of a suitable organic solvent (e.g., Pd-catalyzed hydrogenation reaction conditions), to provide an aniline compound of formula (1). Exemplary catalysts include, but are not limited to palladium on carbon (Pd/C). Exemplary organic solvents include, but are not limited to, MeOH. In some embodiments, the hydrogenation reaction is performed at a temperature of about 25° C.

[00099] For preparation of starting materials related to SCHEME IB, a fluorobenzene compound of formula (i) is coupled to an amine compound of formula (m) by displacement to give a nitro compound of formula (n), using a suitable inorganic base in the presence of a suitable organic solvent. Exemplary inorganic bases include, but are not limited to CS₂CO₃. Exemplary organic solvents include, but are not limited to Ν,Ν'-dimethylformamide (DMF). In some embodiments, the displacement reaction is performed at a temperature of about 25° C.

[000100] Reduction of a compound of formula (n) is performed under conditions known to one of ordinary skill in the art using a suitable catalyst, in the presence of a suitable organic solvent (e.g., Pd-catalyzed hydrogenation reaction conditions), to provide an aniline compound of formula (o). Exemplary catalysts include, but are not limited to palladium on carbon (Pd/C). Exemplary organic solvents include, but are not limited to, MeOH. In some embodiments, the hydrogenation reaction is performed at a temperature of about 25° C.

[000101] For preparation of starting materials related to SCHEME 1C, a fluorobenzene compound of formula (i) is coupled to an amine of formula (p) by displacement to give a nitro compound of formula (q), using an inorganic base and organic solvent. In some embodiments, a suitable inorganic base is CS₂CO₃. In some embodiments, the temperature of the displacement reaction is 80° C. Exemplary organic solvents include, but are not limited to Ν,Ν'- dimethylformamide (DMF).

[000102] Reduction of a nitro compound of formula (q) is performed under conditions known to one of ordinary skill in the art using a suitable catalyst, in the presence of a suitable organic solvent (e.g., Pd-catalyzed hydrogenation reaction conditions), to provide an aniline compound of formula (r). Exemplary catalysts include, but are not limited to palladium on carbon (Pd/C). Exemplary organic solvents include, but are not limited to, MeOH. In some embodiments, the hydrogenation reaction is performed at a temperature of about 25° C.

IV. METHODS and USES

[000103] In some embodiments, the present invention provides methods for treating a subject suffering from a CK2 -mediated condition by administering to the subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof.

[000104] In some embodiments, the present invention provides methods for treating or preventing a cancer by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, wherein the cancer is selected from the group consisting of neuroblastoma, intestine carcinoma such as rectum carcinoma, colon carcinoma, familiary adenomatous polyposis carcinoma and hereditary non-polyposis colorectal cancer, esophageal carcinoma, labial carcinoma, larynx carcinoma, hypopharynx carcinoma, tong carcinoma, salivary gland carcinoma, gastric carcinoma, adenocarcinoma, medullary thyroidea carcinoma, papillary thyroidea carcinoma, renal carcinoma, kidney parenchym carcinoma, ovarian carcinoma, cervix carcinoma, uterine corpus carcinoma, endometrium carcinoma, chorion carcinoma, pancreatic carcinoma, prostate carcinoma, testis carcinoma, breast carcinoma, urinary carcinoma, melanoma, brain tumors (such as glioblastoma, astrocytoma, meningioma, medulloblastoma, and peripheral neuroectodermal tumors), Hodgkin lymphoma, non- Hodgkin lymphoma, Burkitt lymphoma, acute lymphatic leukemia ("ALL"), chronic lymphatic leukemia ("CLL"), acute myeloid leukemia ("AML"), chronic myeloid leukemia ("CML"), adult T- cell leukemia lymphoma, hepatocellular carcinoma, gall bladder carcinoma, bronchial carcinoma, small cell lung carcinoma, non-small cell lung carcinoma, multiple myeloma, basalioma, teratoma, retinoblastoma, choroidea melanoma, seminoma, rhabdomyo sarcoma, craniopharyngeoma, osteosarcoma, chondrosarcoma, myosarcoma, liposarcoma, fibrosarcoma, Ewing sarcoma, and plasmocytoma.

[000105] In some embodiments, the present invention provides methods for treating or preventing a condition by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, wherein the condition is selected from the group consisting familiary adenomatous polyposis, colon cancer (such as sporadic colon carcinoma), rectum cancer, lung cancer (such as non-small cell lung cancer including the non-squamous and squamous subtypes), hepatocellular cancer, breast cancer, endometrial ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, Barrett's Esophagus, gastric cancer, pancreatic cancer, melanoma, mesothelioma, synovial sarcomas, adrenocortical tumors (such as adrenocortical carcinoma), leukemia, Wilms tumors, hepatoblastoma, and increased bone density.

[000106] In some embodiments, the present invention provides methods for treating or preventing colon cancer (including colon carcinoma) by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof.

[000107] In some embodiments, the present invention provides methods for treating or preventing lung cancer, particularly non-small cell lung cancer (including the non-squamous and squamous subtypes), by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof.

[000108] In some embodiments, the invention provides methods for treating or preventing a condition associated with abnormal cellular proliferation by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof. Conditions associated with abnormal cellular proliferation include, e.g., benign prostatic hyperplasia, familial adenomatosis polyposis, neurofibromatosis, atherosclerosis, pulmonary fibrosis, arthritis, psoriasis, glomerulonephritis, restenosis following angioplasty or vascular surgery, hypertrophic scar formation, inflammatory bowel disease, transplantation rejection, endotoxic shock, and fungal infections.

[000109] In some embodiments, the present invention provides methods for treating or preventing a condition associated with defective apoptosis by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof. Conditions associated with defective apoptosis include, e.g., viral infections (including but not limited to herpesvirus, poxvirus, Epstein- Barr virus, Sindbis virus and adenovirus), prevention of AIDS development in HIV-infected individuals, autoimmune diseases (including but not limited to systemic lupus erythematosus, rheumatoid arthritis, psoriasis, autoimmune mediated glomerulonephritis, inflammatory bowel disease and autoimmune diabetes mellitus), neurodegenerative disorders (including but not limited to Alzheimer's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, Parkinson's disease, AIDS-related dementia, spinal muscular atrophy and cerebellar degeneration), myelodysplastic syndromes, aplastic anemia, ischemic injury associated with myocardial infarctions, stroke and reperfusion injury, arrhythmia, atherosclerosis, toxin-induced or alcohol related liver diseases, hematological diseases (including but not limited to chronic anemia and aplastic anemia), degenerative diseases of the musculoskeletal system (including but not limited to osteroporosis and arthritis (including osteoarthritis), aspirin- sensitive rhinosinusitis, cystic fibrosis, multiple sclerosis, and kidney diseases (including polycystic kidney disease).

[000110] An additional embodiment of the invention provides a method of treating cancer. The method comprises administering to a subject, e.g. , a human, diagnosed with, at risk for, or exhibiting symptoms of cancer a compound of the invention, e.g., pharmaceutical composition comprising a compound of the invention, such that the cancer is treated. In certain embodiments, the subject is diagnosed with cancer.

[000111] In some embodiments, the present invention provides methods for treating or preventing a cancer by administering to a subject a therapeutically effective amount of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof.

[000112] In yet another embodiment, the invention provides a method of inhibiting CK2, e.g., inhibiting CK2 activity. The method comprises reacting a compound of the invention with CK2 under conditions such that CK2 activity is inhibited.

[000113] In an additional embodiment, the invention provides a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, for use as a medicament.

[000114] In yet another embodiment, the invention pertains to the use of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, for the manufacture of a medicament for the production of a CK2 inhibitory effect in a subject, e.g., human.

[000115] In a further embodiment, the invention pertains to the use of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, for the manufacture of a medicament for the production of an anti-cancer effect in a subject.

[000116] In another embodiment, the invention relates to the use of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for preventing or treating cancer, e.g. , treating.

[000117] In an additional embodiment the present invention provides a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for use in the inhibition of CK2 activity. [000118] In an additional embodiment the present invention provides a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, for use in the manufacture of a medicament for use in the treatment of cancer.

[000119] In some embodiments, the present invention relates to the use of compounds of Formulae (I) and/or (II), or pharmaceutically acceptable salts thereof, for the manufacture of medicaments for treating or preventing a condition selected from the conditions discussed above.

[000120] In some embodiments, the present invention relates to the use of a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating or preventing a condition selected from the group consisting familiary adenomatous polyposis, colon cancer (such as sporadic colon carcinoma), rectum cancer, lung cancer (such as non-small cell lung cancer including the non-squamous and squamous subtypes), hepatocellular cancer, breast cancer, endometrial ovarian cancer, cervical cancer, prostate cancer, bladder cancer, head and neck cancer, Barrett's Esophagus, gastric cancer, pancreatic cancer, melanoma, mesothelioma, synovial sarcomas, adrenocortical tumors (such as adrenocortical carcinoma), leukemia, Wilms tumors, hepatoblastoma, and increased bone density.

V. PHARMACEUTICAL COMPOSITIONS

[000121] In some embodiments, the invention relates to pharmaceutical composition comprising compounds disclosed herein and use in the prevention and treatment of cancer.

[000122] Another embodiment of the invention provides a pharmaceutical composition which comprises a compound of Formulae (I) and/or (II), or a pharmaceutically acceptable salt thereof, as described herein, in association with a pharmaceutically-acceptable diluent or carrier.

[000123] The present pharmaceutical compositions can take the form of solutions, suspensions, emulsion, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. In some embodiments, compounds and/or compositions of the present invention are administered by intravenous (I.V.) administration.

[000124] Administration may be topical, i.e., substance is applied directly where its action is desired, enteral or oral, i.e., substance is given via the digestive tract, parenteral, i.e., substance is given by other routes than the digestive tract such as by injection.

[000125] In a particular embodiment, the active compound and optionally another therapeutic or prophylactic agent are formulated in accordance with routine procedures as pharmaceutical compositions adapted for intravenous administration to human beings. Typically, the active compounds for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, present compositions can also include a solubilizing agent. Compositions for intravenous administration can optionally include a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule. Where the active compound is to be administered by infusion, it can be dispensed, for example, with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the active compound is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[000126] Compositions for oral delivery can be in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups, or elixirs, for example. Orally administered compositions can contain one or more optional agents, for example, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preserving agents, to provide a pharmaceutically palatable preparation. A time delay material such as glycerol monostearate or glycerol stearate can also be used. Oral compositions can include standard vehicles such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Such vehicles are of pharmaceutical grade in particular embodiments.

[000127] Compositions for use in accordance with the present invention can be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, compounds and optionally another therapeutic or prophylactic agent and their physiologically acceptable salts and solvates can be formulated into pharmaceutical compositions for administration by inhalation or insufflation (either through the mouth or the nose) or oral, parenteral or mucosol (such as buccal, vaginal, rectal, sublingual) administration. In one embodiment, local or systemic parenteral administration is used.

[000128] For oral administration, present compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets can be coated by methods well known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); nonaqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.

[000129] In some embodiments, compounds or salts disclosed herein or can be administered as a pharmaceutical composition in which the pharmaceutical composition comprises between 0.1-lmg, 1-10 mg, 10-50 mg, 50-100 mg, 100-500 mg, or 500 mg to 5 g of said compound or salt.

VI. COMBINATION THERAPY

[000130] Compounds of the present invention may be used in methods of the present invention as either a single agent by itself or in combination with other clinically relevant agents or techniques. For example, the anti-cancer treatment defined herein may be applied as a sole therapy or may involve, in addition to compounds of the present invention, conventional surgery or radiotherapy or chemotherapy.

[000131] Such radiotherapy may include one or more of the following categories or radiation:

(i) external radiation therapy using electromagnetic radiation, and intraoperative radiation therapy using electromagnetic radiation;

(ii) internal radiation therapy or brachytherapy; including interstitial radiation therapy or intraluminal radiation therapy; or

(iii) systemic radiation therapy, including but not limited to iodine 131 and strontium 89.

[000132] Such chemotherapy may include one or more of the following categories of antitumor agents:

(i) antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as DNA alkylating agents (for example cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, bendamustine, busulphan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine, capecitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and pemetrexed, tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, liposomal doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin and irinotecan); inhibitors of DNA repair mechanisms such as CHK kinase; DNA- dependent protein kinase inhibitors; inhibitors of poly (ADP-ribose) polymerase (PARP inhibitors); ATM or ATR kinases; and Hsp90 inhibitors such as tanespamycin and retaspimycin;

(ii) compounds that inhibit progression through the cell cycle such as antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine; epothilones such as ixabepilone; taxoids like taxol and taxotere; polo-like kinase inhibitors; and inhibitors of kinesin motor proteins such as Eg5 protein inhibitors); aurora kinase inhibitors (for example AZD1152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459); cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors (for example, flavopiridol/Alvocidib, roscovitine, seliciclib); and inhibitors of centromeric protein function such as CENP-E inhibitors;

(iii) cytostatic agents such as antiestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate); LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin); progestogens (for example megestrol acetate); aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane); inhibitors of 5a-reductase such as finasteride; and inhibitors of CYP17A1, such as abiraterone acetate;

(iv) anti-invasion agents such as c-Src kinase family inhibitors like AZD0530, dasatinib or BMS-354825; bosutinib (SKI-606), metalloproteinase inhibitors like marimastat; inhibitors of urokinase plasminogen activator receptor function; antibodies to heparanase, inhibitors of FAK or focal-adhesion kinase; small molecule inhibitors of MET receptor kinase; and antibodies to MET receptor kinase or the MET ligand hepatocyte growth factor;

(v) inhibitors of tumor, tumor stem cell, and endothelial cell precursor migration, including chemokines and chemokine receptors, such as SDF1, CXCR2 and CXCR4;

(vi) inhibitors of growth factor signaling: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example the anti-erbB2 antibody trastuzumab [Herceptin™], the anti-EGFR antibody panitumumab, the anti-erbBl antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology/haematology, 2005, Vol. 54, pp 11-29); such inhibitors also include tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family and their receptors (for example EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD 1839), N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (CI 1033); combined EGFR and erbB2 tyrosine kinase inhibitors such as lapatinib; and irreversible inhibitors of EGFR and Her2 such as HKI-272 and AZD8931; inhibitors of the hepatocyte growth factor family and their receptors such as MetMab; inhibitors of the insulin growth factor family including small molecule kinase inhibitors and antibodies directed to insulin-like growth factors and insulin-like growth factor receptors; inhibitors of the platelet-derived growth factor family and their receptors such as imatinib and/or nilotinib (AMN107); inhibitors of serine/threonine kinases (for example Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib (BAY 43-9006), tipifarnib (Rl 15777) and lonafarnib (SCH66336)); inhibitors of cell signalling through MEK kinases; AKT kinases, inhibitors of TOR kinases; c-kit inhibitors; abl kinase inhibitors; PI3 kinase inhibitors; Flt3 kinase inhibitors; CSF-1R kinase inhibitors; IGF receptor (insulin-like growth factor) kinase inhibitors; TRK kinase inhibitors; and inhibitors of JAK/STAT signaling such as pirn kinase inhibitors and Jak kinase inhibitors (for example, AZD1480, Ruxolitinib).

(vii) antiangiogenic agents such as those that inhibit the effects of vascular endothelial growth factor [for example the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib (ZD6474), vatalanib (PTK787), sunitinib (SU11248), axitinib (AG-013736), pazopanib (GW 786034) and cediranib (AZD2171); compounds such as those disclosed in International Patent Applications W097/22596, WO 97/30035, WO 97/32856 and WO 98/13354; and compounds that work by other mechanisms (for example linomide, inhibitors of integrin ανβ3 function and angiostatin)], or inhibitors of angiopoietins and their receptors (Tie-1 and Tie-2)

(viii) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(ix) an endothelin receptor antagonist, for example zibotentan (ZD4054) or atrasentan;

(x) antisense therapies, for example those that are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense, oblimerson sodium, an anti-Bcl-2 antisense, antisense to XIAP such as AEG35156, or antisense to survivin;

(xi) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCA1 or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy); approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme; and approaches to increase patient tolerance to chemotherapy or radiotherapy, such as multi-drug resistance gene therapy;

(xii) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumor cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor; approaches to decrease T-cell anergy or regulatory T-cell function; approaches that enhance T-cell responses to tumors, such as blocking antibodies to CTLA4, B7H1 , PD-1 , and agonist antibodies to CD137; PD-1 or B7-H1 , toll-receptor agonists; agonistic antibodies to CD40 such as SGN-40 (Dacetuzumab) or to the Tweak receptor such as PDL-192; agonistic antibodies to FAS; approaches using antibodies to tumor associated antigens, and antibodies that deplete target cell types (e.g. unconjugated anti-CD20 antibodies such as Rituximab, ofatumumab, Obinutuzumab, anti-CD 19 antibodies such as MEDI- 551 , anti-CD52 antibodies such as Alemtuzumab, anti-CD37 antibodies such as TRU-016, anti- CD22 antibodies such as Inotuzumab, radiolabeled anti-CD20 antibodies Bexxar and Zevalin, and anti-CD54 antibody Campath; immunotoxins such as moxetumumab pasudotox), approaches using anti-idiotypic antibodies, approaches that enhance Natural Killer cell function, and approaches that utilize antibody-toxin conjugates (e.g. anti-CD33 antibody Mylotarg). Immune modifiers such as Revlimid (Lenalidomide); approaches using transfected immune cells such as cytokine-transfected dendritic cells; approaches using cytokine-transfected tumor cell lines, approaches using antibodies to tumor associated antigens, and antibodies that deplete target cell types (e.g., unconjugated anti- CD20 antibodies such as Rituximab, radiolabeled anti-CD20 antibodies Bexxar and Zevalin, and anti-CD54 antibody Campath); approaches using anti-idiotypic antibodies; approaches that enhance Natural Killer cell function; and approaches that utilize antibody-toxin conjugates (e.g. anti-CD33 antibody Mylotarg);

(xiii) apoptosis-inducing approaches, including antibodies to death receptor 4 or death receptor 5 or cross reactive antibodies binding to both death receptor 4 and death receptor 5; inhibitors of XIAP and cIAPl and cIAP2; antibodies to FAS; and Bcl-2 family inhibitors;

(xiv) cytokine treatment, including tumor necrosis factor alpha, and recombinant Trail protein, and small molecule or protein mimetics of the Trail protein; or FAS or Tweak ligands or mimetics of these ligands;

(xv) inhibitors of proteasome mediated protein degradation including but not limited to proteasome inhibitors such as Velcade™ , inhibitors of ubiquitin ligases, inhibitors of ubiquitin proteases, inhibitors of protein Neddylation, and inhibitors of protein sumoylation; or (xvi) inhibitors of antigen receptor signaling, including spleen tyrosine kinase (Syk) inhibitors such as fostamatinib (, R788/R406), PRT062607, bruton's tyrosine kinase (Btk) inhibitors such as PCI-32765, AVL-292, Protein kinase C inhibitors such as sortrastaurin, IKK/NFkB inhibitors, PI3-kinase inhibitors such as CAL-101 (GS 1 101), Enzastaurin, AZD8186, BCL6 inhibitors; or

(xvii) regulators of hematopoietic cell trafficking/homing including agents that target CXCR4 such as Plerixafor (rINN and US AN, also known as MOZOBIL, JM 3100 and AMD3100), BKT140, Syk inhibitors such as Fostamatinib, agents that target VLA-4 and agents that target CD44.

[000133] According to this embodiment, there is provided a combination suitable for use in the treatment of CK2 comprising a compound of Formulae (I) and/or (II) as defined herein, and any one of the anti tumor agents listed under (i) - (xvii) above.

[000134] Therefore in some embodiments there is provided a compound of Formulae (I) and/or (II) in combination with an anti-tumor agent selected from one listed under (i) - (xvii) herein above. As illustrated in exemplary Figures 1 , 2, and 3 herein, combination of a compound of the present invention with an anti-tumor agent demonstrates more than an additive effect.

[000135] According to some embodiments, there is provided a pharmaceutical composition which comprises a compound of Formulae (I) and/or (II) in combination with an anti-tumor agent selected from one listed under (i) - (xvii) herein above, in association with a pharmaceutically acceptable diluent or carrier.

[000136] According to some embodiments, there is provided a pharmaceutical composition which comprises a compound of Formulae (I) and/or (II) in combination with an anti-tumor agent selected from one listed under (i) - (xvii) herein above, in association with a pharmaceutically acceptable diluent or carrier for use in the prevention or treatment of cancer.

[000137] According to some embodiments, there is provided a kit comprising a compound of Formulae (I) and/or (II) in combination with an anti-tumor agent selected from one listed under (i) - (xvii) herein above.

[000138] According to some embodiments, there is provided a kit comprising:

a) a compound of Formulae (I) and/or (II) in a first unit dosage form;

b) an anti-tumor agent selected from one listed under (i) - (xvii) herein above; in a second unit dosage form; and

c) container means for containing said first and second dosage forms.

[000139] Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ compounds of this invention, or pharmaceutically acceptable salts thereof, within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

EXEMPLIFICATION

[000140] The following descriptions of experiments, procedures, examples, and intermediates are intended to exemplify embodiments of the invention, and are in no way intended to be limiting.

EXAMPLES

[000141] The invention will now be illustrated by the following non-limiting examples in which, unless stated otherwise:

(i) temperatures are given in degrees Celsius (°C); operations were carried out at room or ambient temperature, that is, at a temperature in the range of 18-25 °C;

(ii) organic solutions were dried over anhydrous sodium sulphate; evaporation of solvent was carried out using a rotary evaporator under reduced pressure (600-4000 Pascals; 4.5-30mmHg) with a bath temperature of up to 60 °C;

(iii) in general, the course of reactions was followed by TLC and reaction times are given for illustration only;

(iv) final products had satisfactory proton nuclear magnetic resonance (NMR) spectra and/or mass spectral data;

(v) yields are given for illustration only and are not necessarily those which can be obtained by diligent process development; preparations were repeated if more material was required;

(vi) when given, NMR data is in the form of delta values for major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard, determined at 400 MHz using perdeuterio dimethyl sulphoxide (DMSO-d₆) as solvent unless otherwise indicated;

(vii) chemical symbols have their usual meanings; SI units and symbols are used;

(viii) solvent ratios are given in volume: volume (v/v) terms; and

(ix) mass spectra were run with an electron energy of 70 electron volts in the chemical ionization (CI) mode using a direct exposure probe; where indicated ionization was effected by electron impact (EI), fast atom bombardment (FAB) or electrospray (ESP); values for m/z are given; generally, only ions which indicate the parent mass are reported; and unless otherwise stated, the mass ion quoted is (MH)⁺; (x) where a synthesis is described as being analogous to that described in a previous example the amounts used are the millimolar ratio equivalents to those used in the previous example;

(xi) the following abbreviations have been used:

DMF N,N-dimethylformamide;

DMA N, N,-dimethylacetamide;

EtOAc ethyl acetate;

EtOH ethanol;

Pd₂(dba)₃ tris(dibenzylideneacetone)dipalladium (0);

DCM dichloromethane;

DMSO dimethylsulfoxide;

TFA trifluoroacetic acid;

MeOH methanol;

Et₃N triethylamine;

NMP 2-methylpyrrolidinone; and

(xii) "ISCO" refers to normal phase flash column chromatography using 12 g and 40 g prepacked silica gel cartridges used according to the manufacturers instruction obtained from ISCO, Inc, 4700 superior street Lincoln, NE, USA.

I. SYNTHETIC EXEMPLIFICATION

A. METHODS OF PREPARATION OF STARTING MATERIAL

5,7-Dihydroxypyrazolo[l ,5-a]pyrimidine-3-carbonitrile

[000143] To a stirring solution of sodium ethoxide in EtOH, generated by portion-wise addition of sodium (5.0 g, 21.7 mmol) to EtOH (50 mL), was added 5-amino-lH-pyrazole-4-carbonitrile (4.3 g, 39.8 mmol) and diethyl malonate and the reaction mixture was stirred at 90 °C for 24 h. The reaction mixture was allowed to cool to 25 °C and the precipitated solids were collected by vacuum filtration. The crude material was then dissolved in ice water and the aqueous mixture was acidified to pH 1-2 with concentrated HC1. Upon standing at room temperature precipitates formed which were collected by vacuum filtration. The precipitates were washed with water and dried under vacuum for 24 h to give 5,7-dihydroxypyrazolo[l ,5-a]pyrimidine-3-carbonitrile (2.5 g, 35 %).

[000144] m/z 111.

5,7-Dichloropyrazolo[l ,5-a]pyrimidine-3-carbonitrile

[000146] Triethylamine (5.51 ml, 39.5 mmol) and N,N-dimethylaniline (20.9 ml, 165.0 mmol) were added to 5,7-dihydroxypyrazolo[l ,5-a]pyrimidine-3-carbonitrile (METHOD 1, 10.0 g, 56.8 mmol) and PC1₅ (14.56 g, 69.9 mmol) in POCI3 (150 ml) and the reaction mixture was heated at 1 15 °C for 15 h. The reaction mixture was concentrated under reduced pressure and poured onto ice. The aqueous mixture was extracted with DCM then washed with brine and the solvents were removed under reduced pressure. The crude residue was purified by column chromatography to give 5,7-dichloropyrazolo[l ,5-a]pyrimidine-3-carbonitrile (7.0 g, 58%).

[000147] m/z 213.

5-Chloro-7-(cyclopropylamino)pyrazolo[l ,5-a]pyrimidine-3-carbonitrile

[000149] 5,7-Dichloropyrazolo[l ,5-a]pyrimidine-3-carbonitrile (METHOD 2, 0.25 g, 1.17 mmol) and cyclopropanamine (0.080 g, 1.41 mmol) in ethanol (5 mL) were heated in a microwave at 120 °C for 20 min. The reaction mixture was allowed to stand at room temperature for 5 h, the product was collected by vacuum filtration and washed with Et20. The product was used in the next step without further purification. [000150] m/z 234.

N-(2-Fluoro-5-nitrophenyl)acetamide

₂

[000152] A mixture of 2-fluoro-5-nitroaniline (9.8 g, 62.7 mmol) and acetic anhydride (60 mL) in a 250-mL flask was stirred at 25 °C overnight. The mixture was concentrated to dryness in vacuo to give a brown solid that was suspended in Et₂0 (30 mL), collected and dried to give N-(2-fluoro-5- nitrophenyl)acetamide as an off- white solid (10.6 g, 85%).

[000153] m/z 199.

METHOD 5

[000154] N-(2-((2-hydroxyethyl)(methyl)amino)-5-nitrophenyl)acetamide

[000155] 2-(methylamino)ethanol (1.9 g, 25.2 mmol) and Cs₂C0₃ were added to a solution of N-(2-fluoro-5-nitrophenyl)acetamide (METHOD 4, 2.5 g, 12.6 mmol) in DMF (50 mL). The mixture was stirred at 25 °C for 14 h. The solvent was removed under reduced pressure to give a brown solid. The solid was suspended in water, collected and re-suspended in MeOH/EtOAc (1 : 1 , 150 mL). The suspension was filtered and the filtrate was concentrated under reduced pressure to give N-(2-((2-hydroxyethyl)(methyl)amino)-5-nitrophenyl)acetamide (1.7 g, 53 %) as a brown solid.

-amino-2-((2-hydroxyethyl)(methyl)amino)phenyl)acetamide

[000157] N-(2-((2-hydroxyethyl)(methyl)amino)-5-nitrophenyl)acetamide (METHOD 5, 0.30 g, 1.2 mmol) was dissolved in MeOH (5 mL). Pd/C (10% by weight, 0.25 g) was added, the mixture was degassed and filled with H₂ (balloon) and stirred under H₂ at 25 °C for 2h. The mixture was filtered through diatomaceous earth (Celite), washed with DCM and MeOH and concentrated under reduced pressure to give N-(5-amino-2-((2-hydroxyethyl)(methyl)amino)phenyl)acetamide (0.26 g, 98%) as an oil.

[000158] m/z 224.

N-(2-((2-(Dimethylamino)ethyl)(methyl)amino)-5-nitrophenyl)acetamide

[000160] A suspension of N-(2-fiuoro-5-nitrophenyl)acetamide (METHOD 4, 0.30 g, 1.51 mmol), Cs₂C0₃ (0.543 g, 1.67 mmol), , ,N2-trimethylethane-l ,2-diamine (0.170 g, 1.67 mmol) and DMF (3.0 mL) in a 50 mL round-bottom flask was stirred at 80 °C for 3 h. The mixture was allowed to cool to 25 °C and concentrated under reduced pressure. The residue was impregnated on silica and purified by chromatography on silica (hexanes/EtOAc then EtOAc/MeOH/Et₃N) to give N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-5-nitrophenyl)acetamide as a viscous brown oil (0.27g, 64% yield).

[000161] m/z 281.

METHOD 8

[000162] N-(5-amino-2-((2-(dimethylamino)ethyl)(methyl)amino)phenyl)acetamide

[000163] N-(2 (2 dimethylamino)ethyl)(methyl)amino)-5-nitrophenyl)acetamide (METHOD 7, 550 mg, 1.96 mmol) was dissolved in MeOH (2 mL). Pd/C (10% by weight, 0.160 g) was added and the mixture was degassed and filled with H₂ (balloon). The reaction mixture was stirred under H₂ at 25 °C for 2h. The mixture was filtered through Celite, washed with MeOH, filtered and concentrated to dryness in vacuo to give N-(5-amino-2-((2- (dimethylamino)ethyl)(methyl)amino)phenyl)acetamide (420 mg, 86% yield) as a purple solid.

[000164] m/z 251.

METHOD 9

[000165] N-(2-((2-acetamido-4-(3-cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5-a]pyrimidin-5- ylamino)phenyl)(methyl)amino)eth l methanesulfonate

[000166] N-(5-(3-cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5-a]pyrimidin-5-ylamino)-2-((2- hydroxyethyl)(methyl)amino)phenyl)acetamide (METHOD 12, 0.330 g, 0.78 mmol) was dissolved in anhydrous pyridine (2.0 mL) and cooled to 0 °C. Methanesulfonyl chloride (0.073 mL, 0.94 mmol) was added and the reaction was stirred at 0 °C for 0.5h. Additional methanesulfonyl chloride (0.073 mL, 0.94 mmol) was added and the mixture was maintained at 0 °C for 0.5 h and allowed to warm to 25 °C. The reaction was diluted with water (20 mL) and extracted with EtOAc (3 x 20 mL). The combined organic phases were dried over Na₂S0₄, filtered and concentrated to dryness in vacuo. The crude product was purified by chromatography on silica using hexanes/EtOAc as eluent to give 2-((2-acetamido-4-(3-cyano-7-(cyclopropylamino)pyrazolo[l ,5-a]pyrimidin-5- ylamino)phenyl)(methyl)amino)ethyl methanesulfonate (0.350 g, 89 %) as a sticky colorless oil%).

[000167] m/z 499. METHOD 10

[000168] tert- utyl 2-((2-acetamido-4-nitro hen l methyl)amino)ethylcarbamate

[000169] A mixture of N-(2-fluoro-5-nitrophenyl)acetamide (METHOD 4, 2.5 g, 12.62 mmol), CS₂CO₃ (9.04 g, 27.76 mmol), and tert-butyl 2-(methylamino)ethylcarbamate hydrochloride (2.92 g, 13.9 mmol) in DMF (10.0 mL) was stirred at 60 °C for 3h. The mixture was allowed to cool to 25 °C and concentrated to dryness under reduced pressure. The residue was dissolved in CH₂C1₂ (50 mL), washed with water, dried over Na₂S0₄, filtered and concentrated under reduced pressure. The residue was purified by chromatography on silica (EtOAc/DCM) to give tert-butyl 2-((2-acetamido- 4-nitrophenyl)(methyl)amino)ethylcarbamate as a yellow solid (4.0 g, 89%).

[0001] m/z 353.

METHOD 1 1

[000170] tert-Butyl 2-((2-acetamido-4-amino hen l (methyl)amino)ethylcarbamate

Boc

[000171] A mixture of tert-butyl 2-((2-acetamido-4-nitrophenyl)(methyl)amino) ethylcarbamate (METHOD 10, 4.0 g, 1 1.35 mmol) and Pd/C (10% by weight, Degussa type, 0.60 g) in MeOH (50 mL) was degassed and flushed with H₂ ( balloon) and stirred at 25 °C overnight. The mixture was filtered through diatomaceous earth (Celite) and washed with MeOH (10 mL). The filtrate was concentrated to dryness in vacuo to give tert-butyl 2-((2-acetamido-4- aminophenyl)(methyl)amino)ethylcarbamate (3.5 g, 95%) as a film.

[000172] m/z 323. METHOD 12

[000173] N-(5-(3-cyano-7-(cyclopropylamino)pyrazolo[l ,5-a]pyrimidin-5-ylamino)-2-((2- hydroxyethyl)(methyl)amino)phenyl acetamide

[000174] A mixture of 5-chloro-7-(cyclopropylamino)pyrazolo[l ,5-a]pyrimidine-3-carbonitrile (METHOD 3, 120 mg, 0.51 mmol), N-(5-amino-2-((2- hydroxyethyl)(methyl)amino)phenyl)acetamide (METHOD 6, 1 15 mg, 0.51 mmol), and cesium carbonate (335 mg, 1.03 mmol), Pd₂(dba)₃, (23.51 mg, 0.03 mmol) and (9,9-dimethyl-9H-xanthene- 4,5-diyl)bis(diphenylphosphine) (29.7 mg, 0.05 mmol) in DMA (3.0 mL) was heated under microwave irradiation at 150 °C for 0.5 h. The reaction was determined to be incomplete by LCMS and was heated at 150 °C for another 2 h. The mixture was diluted with MeOH (2 mL) and filtered. The filtrate was concentrated under reduced pressure and the residue was chromatographed on silica (hexanes then hexanes/EtOAc then EtOAc then 10% MeOH in EtOAc). The resulting oil was triturated with EtOAc (1 mL), the resulting suspension was heated to reflux and filtered while warm.. This collected material was washed with EtOAc (2 mL) and dried at 80 °C under vacuum to afford N-(5-(3-cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5-a]pyrimidin-5-ylamino)-2-((2- hydroxyethyl)(methyl)amino)phenyl)acetamide (1 10 mg, 51 %).

[000175] m/z 421.

METHOD 13

[000176] tert-butyl 2-((2-acetamido-4-(3-cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5- a]pyrimidin-5-ylamino)phenyl)(meth l)amino)ethylcarbamate

[000177] A mixture of potassium fluoride (30.0 mg, 0.52 mmol), 5-chloro-7- (cyclopropylamino)pyrazolo[l ,5-a]pyrimidine-3-carbonitrile (METHOD 3, 121 mg, 0.52 mmol) and tert-butyl 2-((2-acetamido-4-aminophenyl)(methyl)amino)ethylcarbamate (METHOD 1 1 , 200 mg, 0.62 mmol) and NMP was degassed, purged with nitrogen and heated to 130 °C overnight. The mixture was allowed to cool to 25 °C and water and solid K₂CO₃ were added. The solid was collected, dissolved in MeOH/CH₂Cl₂ (20 mL, 1 : 10), dried over Na₂S0₄, filtered and concentrated under reduced pressure. The residue was chromatographed on silica (ISCO) to give tert-butyl 2-((2- acetamido-4-(3-cyano-7-(cyclopropylamino)pyrazolo[l ,5-a]pyrimidin-5- ylamino)phenyl)(methyl)amino) ethylcarbamate (120 mg, 45%).

[000178] m/z 520.

N-(2-methyl-5-nitrophenyl)acetamide

[000180] A solution of 2-methyl-5-nitroaniline (2.5 g, 16.4 mmol) and triethylamine (3.4 mL, 24.6 mmol) in DCM (50 mL) in a 250 mL round-bottomed flask was cooled to 0 °C. Acetic anhydride (2.52 g, 24.6 mmol) was added and the reaction was stirred for 4 h. Additional acetic anhydride (2.52 g, 24.6 mmol) was added at 0 °C and the reaction was allowed to reach 25 °C overnight. The mixture was washed with saturated aqueous NaHC0₃,dried over Na₂S0₄, filtered and concentrated under reduced pressure. The resulting solid was chromatographed on silica using hexanes and EtOAc as eluent to give N-(2-methyl-5-nitrophenyl)acetamide (2.4 g., 75%) as an off- white solid; m/z 195.

METHOD 15

[000181] N-(5-amino-2-methylphenyl)acetamide

[000182] To a solution of N-(2-methyl-5-nitrophenyl)acetamide (Method 14, 0.8 g., 4.1 mmoL) in MeOH (10 mL), was added Pd/C (0.26 g., 10%> by weight). After degassing, the mixture was filled with H₂. The reaction mixture was degassed and stirred under H₂ (1 atm) at 25 °C for 4 h. The mixture was filtered through celite, washed with MeOH and concentrated under reduced pressure to give N-(5 -amino-2-methylphenyl)acetamide

(0.62 g, 91%) as a white solid; m/z 165.

B. METHODS OF PREPARATION OF COMPOUNDS OF FORMULAE (I) and/or (II) EXAMPLE 1

[000183] N-(5-(3-Cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5-a]pyrimidin-5-ylamino)-2-((2- (dimethylamino)ethyl)(methyl)amino)phenyl)acetamide

[000184] A mixture of 5-chloro-7-(cyclopropylamino)pyrazolo[l ,5-a]pyrimidine-3-carbonitrile

(METHOD 3, 80 mg, 0.34 mmol), N-(5-amino-2-((2-

(dimethylamino)ethyl)(methyl)amino)phenyl)acetamide (METHOD 8, 86 mg, 0.34 mmol), CS₂CO₃ (145 mg, 0.45 mmol), Pd₂(dba)₃ (15.6 mg, 0.02 mmol) and 9,9-dimethyl-9H-xanthene-4,5-diyl)bis- diphenylphosphine (19.8 mg, 0.03 mmol) in DMA (0.5 mL) was flushed with N₂ and heated under microwave irradiation at 150 °C for 30 min. The mixture was allowed to cool to 25 °C, concentrated under reduced pressure and purified by chromatography on silica (EtOAc/hexanes then MeOH/EtOAc/Et₃N). The solid that was obtained was washed with EtOAc (1 mL) and Et₂0 (2 mL) to give N-(5-(3-cyano-7-(cyclopropylamino)pyrazolo[ 1 ,5-a]pyrimidin-5-ylamino)-2-((2- (dimethylamino)ethyl)(methyl)amino)phenyl)acetamide (50 mg, 29 %).

[000185] m/z 448.

EXAMPLE 2

[000186] N-(2-((2-aminoethyl)(methyl)amino)-5-(3-cyano-7-(cyclopropylamino) pyrazolo[ 1 ,5- a]pyrimidin-5-ylamino)phenyl)acetamide

[000187] A solution of tert-butyl 2-((2-acetamido-4-(3-cyano-7-(cyclopropylamino) pyrazolo[l,5-a]pyrimidin-5-ylamino)phenyl)(methyl)amino)ethylcarbamate (METHOD 13, 260 mg, 0.5 mmol) in CH₂CI₂ (2 mL) was treated with TFA (1 mL) and the resulting mixture was stirred at 25 °C for 2 h. The mixture was concentrated to dryness under reduced pressure and the residue was purified by reversed-phase semi-preparative HPLC (flow rate = 20 mL/min) using a gradient of MeOH (20-50%) in aqueous formic acid (0.1%, pH = 3) HPLC to give N-(2-((2- aminoethyl)(methyl)amino)-5 -(3 -cyano-7-(cyclopropylamino)pyrazolo [ 1 ,5 -a]pyrimidin-5 - ylamino)phenyl)acetamide (0.075 g) as what was believed to be the its formic acid salt. Further purification by reversed-phase semi-preparative HPLC (flow rate = 20 mL/min) using a gradient of MeOH (60-80%) in aqueous NH₄OH (0.2%, pH =10) gave N-(2-((2-aminoethyl)(methyl)amino)-5- (3 -cyano-7-(cyclopropylamino)pyrazolo [ 1 ,5 -a]pyrimidin-5 -ylamino)phenyl)acetamide (35.0 mg, 16.7 %).

[000188] 1H NMR (300 MHz, CD₃OD) d: 0.72 (m, 2H), 0.88 (m, 2H), 2.21(s, 3H), 2.64 (m, 1H), 2.67 (s, 3H), 2.81 (m, 2H), 3.00 (m, 2H), 5.99 (s, 1H), ), 7.22 (d, 1H), 7.73 (d, 1H), 8.05 (s, 1H), 8.14 (s, 1H).

[000189] m/z 420.

EXAMPLE 3

[000190] N-(2-((2-aminoethyl)(methyl)amino)-5-(3-cyano-7-(cyclopropylamino) pyrazolo[ 1 ,5- a]pyrimidin-5-ylamino)phenyl)acetamide trifluoroacetic acid salt

[000191] A mixture of 2-((2-acetamido-4-(3-cyano-7-(cyclopropylamino)pyrazolo[l ,5- a]pyrimidin-5-ylamino)phenyl)(methyl)amino)ethyl methanesulfonate (METHOD 9, 0.05 g, 0.10 mmol) and ammonia (3.0 mL, 7.0 M in MeOH, 21 mmol) was heated at 100 °C under microwave irradiation for 1 h. The mixture was concentrated under reduced pressure and the residue was dissolved in EtOAc (5 mL). The resulting solution was washed with saturated aqueous NaCl, dried over Na2S04, filtered and concentrated under reduced pressure. The resulting crude product was chromatographed on silica (EtOAc/hexanes then MeOH/EtOAc/Et3N) and further purified by reversed-phase semi-preparative HPLC (gradient of MeCN/H20 with 0.1% TFA) to give N-(2-((2- aminoethyl)(methyl)amino)-5 -(3 -cyano-7-(cyclopropylamino)pyrazolo [ 1 ,5 -a]pyrimidin-5 - ylamino)phenyl)acetamide (0.01 1 g, 20 %) as its trifluoroacetic acid salt.

[000192] m/z 420.

[000193] REFERENCES

1. Cohen, P. Nature Reviews, Drug Discovery 2002, 1 , 309.

2. Duncan, J.S.; Litchfield, D.W. Biochim. Biophys. Acta, 2007, 1784(1), 33-47.

3. Litchfield, D.W. Biochem. J., 2003, 369, 1-15.

4. Ahmad, K.A.; Wang, G.; Unger, G.; Slaton, J.; Ahmed, K. Adv. Enz. Reg. 2008, 48, 179-187.

5. Guerra, B.; Issinger, O.G. Protein kinase CK2 and its role in cellular proliferation, development and pathology. Electrophoresis, 1999, 20, 391-408.

6. G. Stalter, S. Siener, E. Becht, M. Ziegler, K. Remberger, O.-G. Issinger, Asymmetric expression of protein kinase CK2 subunits in human kidney tumors, Biochem. Biophys. Res. Commun. 202 (1994) 141-147.

7. S. Yenice, A.T. Davis, S.A. Goueli, A. Akdas, C. Limas, K.A. Ahmad, Nuclear casein kinase 2 (CK-2) activity in human normal, benign hyperplastic and cancerous prostate, Prostate 24 (1994) 1 1-16.

8. E. Landesman-Bollag, R. Romieu-Mourez, D.H. Song, G.E. Sonenshein, R.D. Cardiff, D.C. Seldin, Protein kinase CK2 in mammary gland tumorigenesis, Oncogene 20 (2001) 3247-3257.

9. D.C. Seldin, P. Leder, Casein kinase II alpha transgene-induce murine lymphoma: relation to theileriosis in cattle, Science 267 (1995) 894-897.

10. M. Faust, M.M., Subcellular localization of protein kinase CK2. A key to its function? Cell Tissue Res. 301 (2000) 329-340.

1 1. Laramas, M.; Pasquier, D.; Filhol, O.; Ringeisen, F.; Descotes, J.L.; Cochet,

C. Eur. J. Cancer., 2007, 43(5), 928-934. 12. S.J. Miller, D.Y. Lou, D.C. Seldin, W.S. Lane, B.G. Neel, Direct identification of PTEN phosphorylation sites, FEBS Lett. 528 (2002) 145-153.

13. J.C. Tapia, D.A. Torres, L. Rodriguez, L. Leyton, F.G. Quest, Casein kinase 2 (CK2) increases survivin expression via enhanced b-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription, Proc. Natl. Acad. Sci. U. S. A. 103 (2006) 15079-15084.

14. T. Sasaki, H. Suzuki, K. Yagi, M. Furuhashi, R. Yao, S. Susa, T. Noda, Y. Arai, K. Miyazono, M. Kato, Lymphoid enhancer factor 1 makes cells resistant to transforming growth factor

-induced repression of c-myc, Cancer Res. 63 (2003) 801-806.

15. D.H. Song, D.J. Sussman, D.C. Seldin, Endogenous protein kinase CK2 participates in Wnt signalling in mammary epithelial cells, J. Biol. Chem. 275 (2000) 23790-23797.

16. Slaton, J.W.; Unger, G.M.; Sloper, D.T.; Davis, A.T.; Ahmed, K. Mol. Cancer Res. 2004, 2, 712.

17. Ahmad, K.A.; Wang, G.; Slaton, J.; Unger, G.; Ahmed, K. Anti-Cancer Drugs 2005, 16, 1037.

18. S. Sarno, M. Ruzzene, P. Frascella, M.A. Pagano, F. Meggio, A. Zambon, M. Mazzorana, G.D. Maira, V. Lucchini, L.A. Pinna, Development and exploitation of CK2 inhibitors, Mol. Cell. Biochem. 274 (2005) 69-76.

19. S. Sarno, S. Mora, F. Meggio, G. Zagotto, D. Dal Ben, P. Ghisellini, R. Battistutta, G. Zanotti, L.A. Pinna, Toward the rational design of protein kinase casein kinase-2 inhibitors, Pharmacol. Ther. 93 (2002) 159-168.

20. M. Farah, K. Parhar, M. Moussavi, S. Eivemark, B. Salh, 5,6- Dichlororibifuranosyl benzimidazole-and apigenin-induced sensitization of colon cancer cells to TNF mediated apoptosis, Am. J. Physiol, Gasterointest. Liver Physiol. 285 (2003) G919-G928.

21. P. Zien, M. Bretner, K. Zastapilo, R. Szyszka, D. Shugar, Selectivity of 4,5,6,7- tetrabromobenzimidazole as an ATP-competitive potent inhibitor of protein kinase CK2 from various sources, Biochem. Biophys. Res. Commun. 306 (2003) 129-133.

22. S. Sarno, E. De Moliner,M. Ruzzene, M.A. Pagano, R. Battistutta, J. Bain, D. Fabbro, M. Schoepfer, M. Elliott, F. Furet, F. Meggio, G. Zanotti, L.A. Pinna, Biochemical and three- dimensional-structural study of the specific inhibition of protein kinase Ck2 by [5-oxo-5,6- dihydroindolo-(l,2-a)quinazolin-7-yl] acetic acid (IQA), Biochem. J. 374 (2003) 639-646.

23. M.A. Pagano, G. Poletto, G. Di Maira, G. Cozza, M. Ruzzene, S. Sarno, J. Bain, M. Elliott, S. Mora, G. Zagotto, F. Meggio, L.A. Pinna, Tetrabromocinnamicacid (TBCA) and related compounds represent a new class of specific protein kinase CK2 inhibitors, ChemBioChem 8 (2006) 129-139.

24. G.; Unger, G.; A. T. Davis; J. W. Slaton; K. Ahmed Curr. Cancer Drug Targets 2004, 4, 77-84.

EQUIVALENTS

[000194] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents were considered to be within the scope of this invention and are covered by the following claims. Moreover, any numerical or alphabetical ranges provided herein are intended to include both the upper and lower value of those ranges. In addition, any listing or grouping is intended, at least in one embodiment, to represent a shorthand or convenient manner of listing independent embodiments; as such, each member of the list should be considered a separate embodiment.

Claims

CLAIMS We claim:

1. A compound of Formula (I)

wherein:

R¹ is selected from -N(CH₃)(CH₂)₂NH₂, and -N(CH₃)(CH₂)₂N(CH₃)₂; and

R² is C₃_₅cycloalkyl;

and/or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein:

R¹ is -N(CH₃)(CH₂)₂NH₂; and

R² is C₃_₅cycloalkyl;

and/or a pharmaceutically acceptable salt thereof.

3. The compound of claim 1 or claim 2, wherein R² is selected from cyclopropyl and cyclobutyl.

4. The compound of any one of claims 1-3, wherein R² is cyclopropyl.

5. A compound of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in claim 1, wherein, R¹ is -N(CH₃)(CH₂)₂NH₂; and R² is cyclopropyl.

6. A compound of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 5, for use as a medicament.

7. A pharmaceutical composition comprising a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-6, and at least one pharmaceutically acceptable carrier, diluent or excipient.

8. Use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-6, in the manufacture of a medicament for use in the production of a CK2 inhibitory effect in a subject.

9. Use of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-6, in the manufacture of a medicament for use in the production of an anti-cancer effect in a subject.

10. A method for treating cancer, said method comprising administering to a subject an effective amount of a compound of Formula (I), as claimed in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.

11. A compound of Formula (I), or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1 to 6, for use in treating cancer.

12. A method of treating cancer comprising administering to a subject diagnosed with, at risk for, or exhibiting symptoms of cancer, a pharmaceutical composition comprising a compound of any one of claims 1-6, such that the cancer is treated.

13. The method of claim 12, wherein said subject is diagnosed with cancer.

14. The method of claim 13, wherein said subject is a human.

15. A method of inhibiting CK2 activity comprising reacting a compound of any one of claims 1- 6 with CK2, under conditions such that CK2 activity is inhibited.

16. A method of preparation a compound of Formula (I), as defined in any one of the examples described herein.

17. A combination of a compound of formula (I) or a pharmaceutically acceptable salt thereof as claimed in any one of claims 1 to 6 and one or more anti-tumor agents selected from antiproliferative/antineoplastic drugs, inhibitors of growth factor signaling, and apoptosis-inducing approaches.