WO2009097014A2 - Therapeutic kinase modulators - Google Patents

Therapeutic kinase modulators Download PDF

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Publication number
WO2009097014A2
WO2009097014A2 PCT/US2008/074820 US2008074820W WO2009097014A2 WO 2009097014 A2 WO2009097014 A2 WO 2009097014A2 US 2008074820 W US2008074820 W US 2008074820W WO 2009097014 A2 WO2009097014 A2 WO 2009097014A2
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Prior art keywords
compound
protein
protein kinase
formula
ring
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PCT/US2008/074820
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French (fr)
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WO2009097014A3 (en
Inventor
Fabrice Pierre
Mustapha Haddach
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Cylene Pharmaceuticals, Inc.
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Priority to CA2698463A priority Critical patent/CA2698463A1/en
Publication of WO2009097014A2 publication Critical patent/WO2009097014A2/en
Publication of WO2009097014A3 publication Critical patent/WO2009097014A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
    • G01N2333/91215—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases with a definite EC number (2.7.1.-)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

  • the invention relates in part to molecules having certain biological activities that include, but are not limited to, inhibiting cell proliferation, and modulating serine-threonine protein kinase activity
  • Molecules of the invention can modulate casein kinase (CK) activity (e g , CK2 activity)
  • CK casein kinase
  • the invention also relates in part to methods for using such molecules
  • the present invention in part provides chemical compounds having certain biological activities that include, but are not limited to, inhibiting cell proliferation, inhibiting angiogenesis, modulating protein kinase activity and modulating polymerase activity
  • Certain molecules can modulate casein kinase 2 (CK2) activ ity and can affect biological functions that include but are not limited to, inhibiting gamma phosphate transfer from ATP to a protein or peptide substrate, inhibiting angiogenesis, inhibiting cell proliferation and inducing cell apoptosis, for example
  • the present invention also in part prov ides methods for preparing novel chemical compounds, and analogs thereof, and methods of using the foregoing
  • group labeled ⁇ represents a 5-6 membered aromatic or heteroaromatic ring fused onto the ring containing Q 1 , wherein ⁇ is a 6-membered aryl ⁇ ng optionally containing one or more nitrogen atoms as ⁇ ng members, or a five membered aryl ⁇ ng selected from thiophene and thiazole,
  • the invention also includes the pharmaceutically acceptable salts of compounds of formula (A).
  • Formula (A) represents a fused tricyclic ring system which is linked through Q 1 to a group R 10 , which is further described below.
  • the invention provides compounds of Formula (I):
  • each Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 ;
  • each R 3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R 3 is independently halo, CF 3 , OR, NR 2
  • Z 5 is CR 6A or N; each of R 6A , R 6B and R 6C is independently H or an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R 6A , R 6B and R 6C can be halo, CF 3 , OR, NR 2 , NROR, NRNR 2 , SR, SOR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRCOOR, NRCOR, CN, COOR, CONR 2 , 0OCR, COR, or
  • R 10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
  • Z 5 is N or CR 6A , such that the ring containing Z 5 is independently an optionally substituted pyridine or pyrimidine ring.
  • one or more ring nitrogen atoms within the ring containing Z 5 may be arranged as follows:
  • R 6A , R 6B , and R 6C is independently selected from the R 6 substituents defined above with respect to compounds of Formula (I).
  • each of Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , where each R 3 is independently selected from the R 3 substituents defined above with respect to compounds of Formula (I).
  • At least one of Z 1 , Z 2 , Z 3 , and Z 4 is a nitrogen atom.
  • One or more ring nitrogen atoms can be positioned in the ring containing Z 1 , Z 2 , Z 3 , and Z 4 such that each ring is independently an optionally substituted pyridine, pyrimidine or pyridazine ring.
  • each of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , where each R 3 is independently selected from the R 3 substituents defined above with respect to compounds of Formula (I).
  • one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , and the others are CH.
  • one of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , and the others are CH.
  • Z 2 is CR 3 and Z 1 , Z 3 , and Z 4 are CH.
  • At least one R 3 substituent is a polar substituent, such as a carboxylic acid or a salt, an ester or a bioisostere thereof.
  • at least one R 3 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof, for example.
  • at least one R 3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or other carboxy bioisostere.
  • at least one R 3 is a carboxylic acid-containing substituent or a salt thereof. This polar substituent is sometimes referred to herein as R 3P .
  • polar substituent refers to any substituent having an electric dipole, and optionally a dipole moment (e.g., an asymmetrical polar substituent has a dipole moment and a symmetrical polar substituent does not have a dipole moment).
  • Polar substituents include substituents that accept or donate a hydrogen bond, and groups that would carry at least a partial positive or negative charge in aqueous solution at physiological pH levels.
  • a polar substituent is one that can accept or donate electrons in a non- covalent hydrogen bond with another chemical moiety.
  • a polar substituent is selected from a carboxy, a carboxy bioisostere or other acid-derived moiety that exists predominately as an anion at a pH of about 7 to 8.
  • Other polar substituents include, but are not limited to, groups containing an OH or NH, an ether oxygen, an amine nitrogen, an oxidized sulfur or nitrogen, a carbonyl, a nitrile, and a nitrogen-containing or oxygen-containing heterocyclic ring whether aromatic or non-aromatic.
  • the polar substituent represented by R 3 is a carboxylate or a carboxylate bioisostere.
  • Carboxylate bioisostere or “carboxy bioisostere” as used herein refers to a moiety that is expected to be negatively charged to a substantial degree at physiological pH.
  • the carboxylate bioisostere is a moiety selected from the group consisting of:
  • each R 7 is independently H or an optionally substituted member selected from the group consisting of Cj. io alkyl, C M0 alkenyl, C 2 .io heteroalkyl, C 3 . 8 carbocyclic ring, and C 3 . 8 heterocyclic ring optionally fused to an additional optionally substituted carbocyclic or heterocyclic ring; or R 7 is a C MO alkyl, C 2 - I o alkenyl, or C 2 . io heteroalkyl substituted with an optionally substituted C 3 . 8 carbocyclic ring or C 3 .g heterocyclic ring.
  • the polar substituent is selected from the group consisting of carboxylic acid, carboxylic ester, carboxamide, tetrazole, triazole, carboxymethanesulfonamide, oxadiazole, oxathiadiazole, thiazole, aminothiazole and hydroxythiazole.
  • At least one R 3 present is a carboxylic acid or a salt, or ester or a bioisostere thereof.
  • at least one R 3 present is a carboxylic acid- containing substituent or a salt, ester or bioisostere thereof.
  • the R 3 substituent may be a Cl-ClO alkyl or C2-C10 alkenyl linked to a carboxylic acid (or salt, ester or bioisostere thereof), for example, and in some embodiments, the R 3 substituent is not NHCOOCH 2 CH 3 .
  • a polar substituent may be at any position on the ring containing Z'-Z 4 in Formula (I), and the ring may include one, two, three or four polar substituents.
  • at least one of Z'-Z 4 may be CR 3 and one of the R 3 substituents may be a polar substituent (e.g., a carboxylate or carboxylic acid ester, or a tetrazole) arranged at any one of the positions in the ring containing Z'-Z 4 :
  • one of Z'-Z 4 is CR 3 and the others of Z 1 -Z 4 are CH, where R 3 is a polar substituent (i.e., R 3P ), for example, a carboxylate or carboxylic acid, ester, or amide, or a tetrazole, arranged at any one of the positions in the ring containing Z 1 -Z 4 :
  • R 3 is a polar substituent (i.e., R 3P ), for example, a carboxylate or carboxylic acid, ester, or amide, or a tetrazole, arranged at any one of the positions in the ring containing Z 1 -Z 4 :
  • the polar substituent represented by R 3 in some embodiments is a carboxy (e.g., COOH), carboxyalkyl (e.g., carboxymethyl), tetrazole or amide (e.g., -CONH 2, .CONHc-Pr) substituent.
  • R 3 represents a carboxylate bioisostere.
  • R 3 at position Z 2 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof.
  • Z 2 is CR 3 , where R 3 is COOH or COOMe.
  • it is CONR 2 , where each R is independently H or C M alkyl; sometimes, it is CONH 2 or CONHc-Pr.
  • R 10 is an optionally substituted five-membered or six- membered carbocyclic or heterocyclic aromatic ring.
  • R 10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring.
  • the optionally substituted six-membered carbocyclic or heterocyclic aromatic ring is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, and optionally substituted variants of the foregoing.
  • R 10 is an optionally substituted phenyl or pyridyl ring ring.
  • R 10 is phenyl substituted with one or more halo, CN, C M alkyl, C M alkoxy, NH 2 , or NHAc substitutents.
  • R 10 is an optionally substituted five-membered heterocyclic aromatic ring.
  • the optionally substituted five-membered heterocyclic aromatic ring is selected from pyrrole, imidazole, pyrazole, triazole, tetrazole, thiophene, thiazole, thiadiazole, isothiazole, furan, oxazole, isoxazole, oxadiazole and optionally substituted variants thereof.
  • R 10 is an optionally substituted pyrazole, isoxazole or thiophene; in specific embodiments, it is an N'-methyl-pyrazol-4-yl substituent or an optionally substituted 2- or 3-thiophene substituent.
  • optional substituents when present on a five-membered heterocyclic aromatic ring include one or more halo, CN, or C M alkyl groups.
  • R 6B , R 6C and R 6A are independently selected from the list defined above with respect to compounds of Formula (I).
  • at least one of R 6B and R 6C is H.
  • both R 6B and R 6C are H.
  • R 6B is thiomethoxy (-SMe) and R 6C is H.
  • Z 5 is N, and R 6B is SR, SO 2 R, or NR 2 , where each R is independently H or C 14 alkyl; in some such embodiments, R 6B is SMe, SO 2 Me or NHc-Pr.
  • Z 5 is R 6A , and each of R 6A , R 6B and R 6C is H.
  • the invention provides compounds of Formula (II):
  • each Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 ;
  • each R 3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R 3 is independently halo, CF 3 , OR
  • Z 5 is CR 6A or N; each of R 6A and R 6B is independently H or an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R 6A and R 6B can be halo, CF 3 , OR, NR 2 , NROR, NRNR 2 , SR, SOR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRCOOR, NRCOR, CN, COOR, CONR 2 , OOCR, COR, or NO 2 ; wherein each R is independently H
  • R 10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
  • Embodiments described with respect to compounds of Formula (I) for substituents Z 1 , Z 2 , Z 3 , Z 4 , Z 5 , R 6A , R 6B , R 3 and R 10 may also be applied to compounds of Formula (II).
  • Z 5 is N or CR 6A , such that the ring containing Z 5 is independently an optionally substituted thiophene or thiazole ring.
  • one or more heteroatoms within the ring containing Z 5 may be arranged as follows:
  • each of R 6A and R 6B are independently selected from the R 6 substituents defined above with respect to compounds of Formula (II).
  • each of Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , where each R 3 is independently selected from the R 3 substituents defined above with respect to compounds of Formula (I) or (II).
  • At least one of Z'-Z 4 is a nitrogen atom.
  • One or more ring nitrogen atoms can be positioned in the ring containing Z'-Z 4 such that each ring is independently an optionally substituted pyridine, pyrimidine or pyridazine ring.
  • each of Z 1 , Z 2 , Z 3 and Z 4 is CR 3 , where each R 3 is independently selected from the R 3 substituents defined above with respect to compounds of Formula (II).
  • at least one of Z 1 , Z 2 , Z 3 and Z 4 is CR 3 , and the others are CH.
  • one of Z 1 , Z 2 , Z 3 and Z 4 is CR 3 , and the others are CH.
  • Z 2 is CR 3
  • Z 1 , Z 3 and Z 4 are CH.
  • At least one R 3 substituent is a polar substituent, such as a carboxylic acid or a salt, an ester or a bioisostere thereof.
  • a polar substituent may be at any position on the ring containing Z'-Z 4 , and the ring may include one, two, three or four polar substituents.
  • At least one of Z'-Z 4 may be CR 3 and one of the R 3 substituents may be a polar substituent (e.g., a carboxylate or carboxylic acid ester, or a tetrazole) arranged at any one of the positions in the ring, as described for compounds of Formula (I).
  • at least one R 3 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof, for example.
  • at least one R 3 is a carboxylic acid-containing substituent or a salt thereof.
  • R 10 is an optionally substituted five-membered or six- membered carbocyclic or heterocyclic aromatic ring.
  • R 10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring.
  • the optionally substituted six-membered carbocyclic or heterocyclic aromatic ring is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, and optionally substituted variants of the foregoing.
  • R 10 is an optionally substituted phenyl ring.
  • R 10 is an optionally substituted five-membered heterocyclic aromatic ring.
  • the optionally substituted f ⁇ ve- membered heterocyclic aromatic ring is selected from pyrrole, imidazole, pyrazole, triazole, tetrazole, thiophene, thiazole, thiadiazole, isothiazole, furan, oxazole, isoxazole, oxadiazole and optionally substituted variants thereof.
  • R 6B and R 6A are independently selected from the list defined above with respect to compounds of Formula (II).
  • at least one of R 6A and R 6B is H.
  • both R 6A and R 6B are H.
  • R 6B is halo or NR 2 .
  • Z 5 is CR 6A , where R 6A is H, and R 6B is H or halo.
  • Z 5 is N, and R 6B is H or NR 2 .
  • each of Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , where each R 3 is independently selected from the R 3 substituents defined above with respect to compounds of Formula (I).
  • At least one of Z 1 , Z 2 , Z 3 , and Z 4 is a nitrogen atom.
  • one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , and the others are CH.
  • one of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 , and the others are CH.
  • Z 2 is CR 3 and Z 1 , Z 3 , and Z 4 are CH.
  • At least one R 3 substituent is a polar substituent, such as a carboxylic acid or a salt, an ester or a bioisostere thereof.
  • at least one R 3 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof, for example.
  • at least one R 3 is a carboxylic acid-containing substituent or a salt thereof.
  • R 3 at position Z 2 is COOH, COOR or CONR 2 , where each R is independently H or C M alkyl.
  • R 3 at position Z 2 is COOH, COOMe or CONHc-Pr.
  • Z 2 is CR 3 where R 3 is F.
  • R 10 is an optionally substituted five-membered heterocyclic aromatic ring or six-membered carbocyclic or heterocyclic aromatic ring.
  • R 10 is an optionally substituted phenyl or pyridine ring. In some embodiments, it is an unsubstituted phenyl ring.
  • R 10 is phenyl substituted with one or more halo, CN, C M alkyl, C) ⁇ alkoxy, NH 2 , or NHAc substitutents.
  • R 10 is an optionally substituted five-membered heterocyclic aromatic ring.
  • R 10 is an optionally substituted pyrazole, isoxazole or thiophene ring.
  • R 6A , R 6B , and R 6C where present in compounds of Formula (III), (IV), (V) and (VI) is independently selected from the list defined above with respect to compounds of Formula (I).
  • at least one of R 6B and R 6C is H.
  • both R 6B and R 60 are H.
  • R 6B is SR, SO 2 R, or NR 2 , where each R is independently H or C M alkyl; in some such embodiments, R 6B is SMe, SO 2 Me or NHc-Pr.
  • R 6A , R 6B and R 6C are H.
  • compositions comprising a compound of .
  • Pharmaceutical compositions can be utilized in treatments descnbed herein
  • identifying a candidate molecule that interacts with a CK2 protein which comprise contacting a composition containing a CK2 protein and a compound desc ⁇ bed herein with a candidate molecule under conditions in which the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein
  • the protein is a CK2 protein, such as a CK2 protein comp ⁇ sing the amino acid sequence of SEQ ID NO 1 , 2 or 3 or a substantially identical variant thereof, for example SEO ID NQ 1 (NP 001886, casein kinase II alpha 1 subunit isoform a [Homo sapiensl)
  • the protein is in a cell or in a cell-free system.
  • the protein, the compound or the molecule in some embodiments is in association with a solid phase.
  • the interaction between the compound and the protein is detected via a detectable label, where in some embodiments the protein comprises a detectable label and in certain embodiments the compound comprises a detectable label.
  • the interaction between the compound and the protein sometimes is detected without a detectable label.
  • Also provided are methods for modulating the activity of a CK2 protein which comprise contacting a system comprising the protein with a compound described herein in an amount effective for modulating the activity of the protein.
  • methods for modulating the activity of a Pirn protein or a Fit protein which comprise contacting a system comprising the protein with a compound described herein in an amount effective for modulating the activity of the protein.
  • the activity of the protein is inhibited, and sometimes the protein is a CK2 protein, such as a CK2 protein comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3 or a substantially identical variant thereof, for example.
  • the system is a cell, and in other embodiments the system is a cell-free system.
  • the protein or the compound may be in association with a solid phase in certain embodiments.
  • the cells sometimes are in a cell line, such as a cancer cell line (e.g., breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, ovary cancer cell line), for example.
  • the cancer cell line is a breast cancer, prostate cancer or pancreatic cancer cell line.
  • the cells sometimes are in a tissue, can be in a subject, at times are in a tumor, and sometimes are in a tumor in a subject.
  • the method further comprises inducing cell apoptosis. Cells sometimes are from a subject having macular degeneration.
  • the cell proliferative condition is a tumor-associated cancer.
  • the cancer sometimes is of the breast, prostate, pancreas, lung, colorectum, skin, or ovary.
  • the cell proliferative condition is a non-tumor cancer, such as a hematopoietic cancer, for example.
  • the cell proliferative condition is macular degeneration in some embodiments.
  • kits for treating cancer or an inflammatory disorder in a subject in need of such treatment comprising: administering to the subject a therapeutically effective amount of a therapeutic agent as described herein; and administering to the subject a molecule that inhibits CK2, Pirn or Rt in an amount that is effective to enhance a desired effect of the therapeutic agent.
  • the therapeutic agent sometimes is a compound of formula TA 1 - 1 , TA2, TA3-1, TA4-1 , TA5- 1 or TA6-l as described herein, or a pharmaceutically acceptable salt of one of these compounds.
  • the molecule that inhibits CK2, Pirn or FH is a compound of Formula (I), (II), (III), (IV), (V), or (VI) as described herein, or a pharmaceutically acceptable salt thereof.
  • the molecule that inhibits CK2, Pirn or Fit is a known compound, or a compound provided herein, or a pharmaceutically acceptable salt of one of these compounds.
  • the desired effect of the therapeutic agent that is enhanced by the molecule that inhibits CK2, Pirn or Fit is a reduction in cell proliferation.
  • the desired effect of the therapeutic agent that is enhanced by the molecule that inhibits CK2, Pirn or Fit is an increase in apoptosis in at least one type of cell.
  • the therapeutic agent in certain embodiments is:
  • the therapeutic agent and the molecule that inhibits CK2, Pirn or Fit are administered at substantially the same time.
  • the therapeutic agent and molecule that inhibits CK2 sometimes are used concurrently by the subject.
  • the therapeutic agent and the molecule that inhibits CK2, Pirn or Fit are combined into one pharmaceutical composition in certain embodiments.
  • Some embodiments are directed to a pharmaceutical composition comprising a therapeutic agent of any of formulas TA 1 - 1 , TA2, TA3- 1 , TA 4- 1 , TA5- 1 or TA6 admixed with a molecule that inhibits CK2, Pirn or Fit, or a pharmaceutically acceptable salt thereof.
  • the molecule that inhibits CK2, Pirn or Fit is a compound of Formula (I), (II), (III), (IV), (V), or (VI) as described herein, or a pharmaceutically acceptable salt thereof.
  • the therapeutic agent is a compound of formula TA2 or a pharmaceutically acceptable salt thereof.
  • a therapeutic composition in certain embodiments comprises a therapeutically effective amount of a therapeutic agent of the formula TA2:
  • a CK modulator or a pharmaceutically acceptable salt thereof or a specific isomer or mixture of isomers thereof, admixed with an amount of a CK modulator or a pharmaceutically acceptable salt of a CK modulator; and where the amount of the CK modulator or the pharmaceutically acceptable salt of a CK modulator is an amount that is effective to enhance a desired effect of the therapeutic agent.
  • compositions comprising a compound described herein and an isolated protein.
  • the protein sometimes is a CK2 protein, such as a CK2 protein comprising the amino acid sequence of SEQ ED NO: 1, 2 or 3 or a substantially identical variant thereof, for example.
  • the protein is a Pirn protein.
  • the protein is a Fit protein.
  • Certain compositions comprise a compound described herein in combination with a cell.
  • the cell may be from a cell line, such as a cancer cell line. In the latter embodiments, the cancer cell line is sometimes a breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, ovary cancer cell line.
  • Compounds of Formula (I), (II), (III), (IV), (V), and (VI) can exert biological activities that include, but are not limited to, inhibiting cell proliferation, modulating protein kinase activity and modulating polymerase activity.
  • Compounds of Formula (I), (II), (III), (IV), (V), and (VI) can modulate CK2 activity, for example. Such compounds therefore can be utilized in multiple applications by a person of ordinary skill in the art.
  • compounds described herein may find uses that include, but are not limited to, (i) modulation of protein kinase activity (e.g., CK2 activity), (ii) modulation of cell proliferation, (iii) modulation of apoptosis, and (iv) treatments of cell proliferation related disorders (e.g., administration alone or co-administration with another molecule).
  • protein kinase activity e.g., CK2 activity
  • modulation of cell proliferation e.g., CK2 activity
  • modulation of cell proliferation e.g., CK2 activity
  • modulation of cell proliferation e.g., apoptosis
  • treatments of cell proliferation related disorders e.g., administration alone or co-administration with another molecule.
  • the group takes up two available valences, so the total number of substituents that may be included is reduced according to the number of available valences.
  • the compounds of the invention often have ionizable groups so as to be capable of preparation as salts.
  • a pharmaceutically acceptable salt may also be used.
  • These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases.
  • the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases.
  • Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.
  • the compounds may contain both an acidic and a basic functional group, in which case they may have two ionized groups and yet have no net charge.
  • the compounds of the invention contain one or more chiral centers.
  • the invention includes each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers and tautomers that can be formed.
  • the compounds of the invention may also exist in more than one tautomeric form; the depiction herein of one tautomer is for convenience only, and is also understood to encompass other tautomers of the form shown.
  • alkyl straight-chain, branched-chain and cyclic monovalent hydrocarbyl radicals, and combinations of these, which contain only C and H when they are unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like.
  • the total number of carbon atoms in each such group is sometimes described herein, e.g., when the group can contain up to ten carbon atoms it can be represented as 1- 1OC or as Cl-ClO or Cl-10.
  • heteroatoms N, O and S typically
  • the numbers describing the group though still written as e.g. C1-C6, represent the sum of the number of carbon atoms in the group plus the number of such heteroatoms that are included as replacements for carbon atoms in the backbone of the ring or chain being described.
  • the alkyl, alkenyl and alkynyl substituents of the invention contain 1-lOC (alkyl) or 2- 1OC (alkenyl or alkynyl). Preferably they contain 1-8C (alkyl) or 2-8C (alkenyl or alkynyl). Sometimes they contain 1-4C (alkyl) or 2-4C (alkenyl or alkynyl).
  • a single group can include more than one type of multiple bond, or more than one multiple bond; such groups are included within the definition of the term "alkenyl” when they contain at least one carbon- carbon double bond, and are included within the term "alkynyl" when they contain at least one carbon-carbon triple bond.
  • Alkyl, alkenyl and alkynyl groups are often optionally substituted to the extent that such substitution makes sense chemically.
  • Alkyl, alkenyl and alkynyl groups can also be substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which can be substituted by the substituents that are appropriate for the particular group.
  • Heteroalkyl “heteroalkenyl”, and “heteroalkynyl” and the like are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the 'hetero' terms refer to groups that contain 1-3 O, S or N heteroatoms or combinations thereof within the backbone residue; thus at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group.
  • heteroforms of alkyl, alkenyl and alkynyl groups are generally the same as for the corresponding hydrocarbyl groups, and the substituents that may be present on the heteroforms are the same as those described above for the hydrocarbyl groups.
  • substituents that may be present on the heteroforms are the same as those described above for the hydrocarbyl groups.
  • such groups do not include more than two contiguous heteroatoms except where an oxo group is present on N or S as in a nitro or sulfonyl group.
  • alkyl as used herein includes cycloalkyl and cycloalkylalkyl groups
  • cycloalkyl may be used herein to describe a carbocyclic non-aromatic group that is connected via a ring carbon atom
  • cycloalkylalkyl niay be used to describe a carbocyclic non-aromatic group that is connected to the molecule through an alkyl linker.
  • heterocyclyl may be used to describe a non-aromatic cyclic group that contains at least one heteroatom as a ring member and that is connected to the molecule via a ring atom, which may be C or N; and “heterocyclylalkyl” may be used to describe such a group that is connected to another molecule through a linker.
  • the sizes and substituents that are suitable for the cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. As used herein, these terms also include rings that contain a double bond or two, as long as the ring is not aromatic.
  • acyl encompasses groups comprising an alkyl, alkenyl, alkynyl, aryl or arylalkyl radical attached at one of the two available valence positions of a carbonyl carbon atom
  • heteroacyl refers to the corresponding groups wherein at least one carbon other than the carbonyl carbon has been replaced by a heteroatom chosen from N, O and S.
  • Acyl and heteroacyl groups are bonded to any group or molecule to which they are attached through the open valence of the carbonyl carbon atom. Typically, they are C1 -C8 acyl groups, which include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, which include methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl.
  • the hydrocarbyl groups, aryl groups, and heteroforms of such groups that comprise an acyl or heteroacyl group can be substituted with the substituents described herein as generally suitable substituents for each of the corresponding component of the acyl or heteroacyl group.
  • Aromatic moiety or "aryl” moiety refers to a monocyclic or fused bicyclic moiety having the well-known characteristics of aromaticity; examples include phenyl and naphthyl.
  • heteroaryl refers to such monocyclic or fused bicyclic ring systems which contain as ring members one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits aromaticity in 5-membered rings as well as 6-membered rings. Any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition.
  • the ring systems contain 5-12 ring member atoms.
  • the monocyclic heteroaryls contain 5-6 ring members, and the bicyclic heteroaryls contain 8-10 ring members.
  • Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as, for example, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, triazole, tetrazole, thiophene, thiazole, isothiazole, thiadiazole, furan, oxazole, isoxazole, oxadiazole, oxathiadiazole, and the fused bicyclic moieties formed by fusing one of these monocyclic groups with a phenyl ring or with any of the heteroaromatic monocyclic groups to form a C8-C10 bicyclic group such as indole, benzimidazole, indazole, benzotriazole, isoquinoline, quinoline, benzothiazole, benzofur
  • Aryl and heteroaryl moieties may be substituted with a variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of these, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halo, OR, NR 2 , SR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRCOOR, NRCOR, CN, C ⁇ CR, COOR, CONR 2 , OOCR, COR, and NO 2 , wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, C
  • an arylalkyl substituent may be substituted on the aryl portion with substituents described herein as typical for aryl groups, and it may be further substituted on the alkyl portion with substituents described herein as typical or suitable for alkyl groups.
  • arylalkyl and “heteroarylalkyl” refer to aromatic and heteroaromatic ring systems which are bonded to their attachment point through a linking group such as an alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers.
  • the linker is C1-C8 alkyl or a hetero form thereof.
  • These linkers may also include a carbonyl group, thus making them able to provide substituents as an acyl or heteroacyl moiety.
  • An aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group may be substituted with the same substituents described above for aryl groups.
  • an arylalkyl group includes a phenyl ring optionally substituted with the groups defined above for aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane.
  • a heteroarylalkyl group preferably includes a C5-C6 monocyclic heteroaryl group that is optionally substituted with the groups described above as substituents typical on aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, or it includes an optionally substituted phenyl ring or C5-C6 monocyclic heteroaryl and a C1-C4 heteroalkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane.
  • substituents may be on either the alkyl or heteroalkyl portion or on the aryl or heteroaryl portion of the group.
  • the substituents optionally present on the alkyl or heteroalkyl portion are the same as those described above for alkyl groups generally; the substituents optionally present on the aryl or heteroaryl portion are the same as those described above for aryl groups generally.
  • Arylalkyl groups as used herein are hydrocarbyl groups if they are unsubstituted, and are described by the total number of carbon atoms in the ring and alkylene or similar linker.
  • a benzyl group is a C7-arylalkyl group
  • phenylethyl is a C8-arylalkyl.
  • Heteroarylalkyl refers to a moiety comprising an aryl group that is attached through a linking group, and differs from “arylalkyl” in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S.
  • the heteroarylalkyl groups are described herein according to the total number of atoms in the ring and linker combined, and they include aryl groups linked through a heteroalkyl linker; heteroaryl groups linked through a hydrocarbyl linker such as an alkylene; and heteroaryl groups linked through a heteroalkyl linker.
  • C7-heteroarylalkyl would include pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.
  • Alkylene refers to a divalent hydrocarbyl group; because it is divalent, it can link two other groups together. Typically it refers to -(CHa) n - where n is 1-8 and preferably n is 1-4, though where specified, an alkylene can also be substituted by other groups, and can be of other lengths, and the open valences need not be at opposite ends of a chain. Thus -CH(Me)- and -C(Me )2- may also be referred to as alkylenes, as can a cyclic group such as cyclopropan- l,l-diyl.
  • the substituents include those typically present on alkyl groups as described herein.
  • any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group or any heteroform of one of these groups that is contained in a substituent may itself optionally be substituted by additional substituents.
  • the nature of these substituents is similar to those recited with regard to the primary substituents themselves if the substituents are not otherwise described.
  • R 7 is alkyl
  • this alkyl may optionally be substituted by the remaining substituents listed as embodiments for R 7 where this makes chemical sense, and where this does not undermine the size limit provided for the alkyl per se; e.g., alkyl substituted by alkyl or by alkenyl would simply extend the upper limit of carbon atoms for these embodiments, and is not included.
  • each such alkyl, alkenyl, alkynyl, acyl, or aryl group may be substituted with a number of substituents according to its available valences; in particular, any of these groups may be substituted with fluorine atoms at any or all of its available valences, for example.
  • Heteroform refers to a derivative of a group such as an alkyl, aryl, or acyl, wherein at least one carbon atom of the designated carbocyclic group has been replaced by a heteroatom selected from N, O and S.
  • the heteroforms of alkyl, alkenyl, alkynyl, acyl, aryl, and arylalkyl are heteroalkyl, heteroalkenyl, heteroalkynyl, heteroacyl, heteroaryl, and heteroarylalkyl, respectively. It is understood that no more than two N, O or S atoms are ordinarily connected sequentially, except where an oxo group is attached to N or S to form a nitro or sulfonyl group.
  • Halo as used herein includes fluoro, chloro, bromo and iodo. Fluoro and chloro are often preferred.
  • Amino refers to NH 2 , but where an amino is described as “substituted” or “optionally substituted”, the term includes NR' R" wherein each R' and R" is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group or a heteroform of one of these groups, and each of the alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl groups or heteroforms of one of these groups is optionally substituted with the substituents described herein as suitable for the corresponding group.
  • R' and R" are linked together to form a 3-8 membered ring which may be saturated, unsaturated or aromatic and which contains 1-3 heteroatoms independently selected from N, O and S as ring members, and which is optionally substituted with the substituents described as suitable for alkyl groups or, if NR'R" is an aromatic group, it is optionally substituted with the substituents described as typical for heteroaryl groups.
  • carbocyclic and heterocyclic structures encompass compounds having monocyclic, bicyclic or multiple ring systems, and may also comprise aryl or heteroaryl rings.
  • heteroatom refers to any atom that is not carbon or hydrogen, such as nitrogen, oxygen or sulfur.
  • heterocycles include but are not limited to pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, triazole, tetrazole, thiophene, thiazole, isothiazole, thiadiazole, furan, oxazole, isoxazole, oxadiazole, oxathiadiazole, benzimidazole, benzofuran, isobenzofuran, indole, benzothiazole, benzotriazole, isoquinoline, quinoline, pyrazolopyridine, quinazoline, quinoxaline, cinnoline, tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran, 1
  • inorganic substituent refers to substituents that do not contain carbon or contain carbon bound to elements other than hydrogen (e.g., elemental carbon, carbon monoxide, carbon dioxide, and carbonate).
  • inorganic substituents include but are not limited to nitro, halogen, azido, cyano, sulfonyls, sulfinyls, sulfonates, phosphates, etc.
  • a candidate molecule or compound described herein may be in a therapeutically effective amount in a formulation or medicament, which is an amount that can lead to a biological effect, such as apoptosis of certain cells (e.g., cancer cells), reduction of proliferation of certain cells, or lead to ameliorating, alleviating, lessening, or removing symptoms of a disease or condition, for example.
  • the terms also can refer to reducing or stopping a cell proliferation rate (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor).
  • microorganism examples include but are not limited to virus, bacterium and fungus.
  • apoptosis refers to an intrinsic cell self-destruction or suicide program.
  • cells undergo a cascade of events including cell shrinkage, blebbing of cell membranes and chromatic condensation and fragmentation. These events culminate in cell conversion to clusters of membrane-bound particles (apoptotic bodies), which are thereafter engulfed by macrophages.
  • the invention in part provides pharmaceutical compositions comprising at least one compound within the scope of the invention as described herein, and methods of using compounds described herein.
  • the invention in part provides methods for identifying a candidate molecule that interacts with a CK2 protein, which comprises contacting a composition containing a CK2 protein and a molecule described herein with a candidate molecule and determining whether the amount of the molecule described herein that interacts with the protein is modulated, whereby a candidate molecule that modulates the amount of the molecule described herein that interacts with the protein is identified as a candidate molecule that interacts with the protein.
  • the invention provides methods for identifying a candidate molecule that interacts with a Pirn or Fit protein, which comprises contacting a composition containing a Pirn or Fit protein and a molecule described herein with a candidate molecule and determining whether the amount of the molecule described herein that interacts with the protein is modulated, whereby a candidate molecule that modulates the amount of the molecule described herein that interacts with the protein is identified as a candidate molecule that interacts with the protein.
  • Also provided are methods for modulating the activity of a CK2 protein which comprises contacting a system comprising the protein with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein.
  • the system in such embodiments can be a cell-free system or a system comprising cells.
  • methods for reducing cell proliferation, and optionally inducing apoptosis which comprises contacting cells with a compound described herein in an amount effective to reduce proliferation of the cells.
  • the cells in such embodiments can be in a cell line, in a tissue or in a subject (e.g., a research animal or human).
  • compositions comprising a compound described herein in combination with a protein or cell, such as an isolated protein (e.g., isolated CK2 or other serine-threonine protein kinase protein) or a cell in a cell line (e.g., HCT-1 16 cell line).
  • a protein or cell such as an isolated protein (e.g., isolated CK2 or other serine-threonine protein kinase protein) or a cell in a cell line (e.g., HCT-1 16 cell line).
  • Serine-threonine protein kinases catalyze the transfer of a gamma phosphate from adenosine triphosphate to a serine or threonine amino acid in a peptide or protein substrate.
  • methods which comprise contacting a system comprising a serine-threonine protein kinase protein with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein.
  • the activity of the serine-threonine protein kinase is the catalytic activity of the protein (e.g., catalyzing the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate).
  • methods for identifying a candidate molecule that interacts with a serine-threonine protein kinase comprise: contacting a composition containing a serine- threonine protein kinase and a compound described herein with a candidate molecule under conditions in which the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein.
  • Systems in such embodiments can be a cell-free system or a system comprising cells (e.g., in vitro).
  • the protein, the compound or the molecule in some embodiments is in association with a solid phase.
  • the interaction between the compound and the protein is detected via a detectable label, where in some embodiments the protein comprises a detectable label and in certain embodiments the compound comprises a detectable label.
  • the interaction between the compound and the protein sometimes is detected without a detectable label.
  • Protein kinases catalyze the transfer of a gamma phosphate from adenosine triphosphate to a serine or threonine amino acid (serine/threonine protein kinase), tyrosine amino acid (tyrosine protein kinase), tyrosine, serine or threonine (dual specificity protein kinase) or histidine amino acid (histidine protein kinase) in a peptide or protein substrate.
  • methods which comprise contacting a system comprising a protein kinase protein with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein kinase.
  • the activity of the protein kinase is the catalytic activity of the protein (e.g., catalyzing the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate).
  • provided are methods for identifying a candidate molecule that interacts with a protein kinase which comprise: contacting a composition containing a protein kinase and a compound described herein with a candidate molecule under conditions in which the compound and the protein kinase interact, and determining whether the amount of the compound that interacts with the protein kinase is modulated relative to a control interaction between the compound and the protein kinase without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein kinase relative to the control interaction is identified as a candidate molecule that interacts with the protein kinase.
  • Systems in such embodiments can be a cell-free system or a system comprising cells (e.g., in vitro).
  • the protein kinase, the compound or the molecule in some embodiments is in association with a solid phase.
  • the interaction between the compound and the protein kinase is detected via a detectable label, where in some embodiments the protein kinase comprises a detectable label and in certain embodiments the compound comprises a detectable label.
  • the interaction between the compound and the protein kinase sometimes is detected without a detectable label.
  • compositions of matter comprising a protein kinase and a compound described herein.
  • the protein kinase in the composition is a serine-threonine protein kinase or a tyrosine protein kinase.
  • the protein kinase is a protein kinase fragment having compound-binding activity.
  • the protein kinase in the composition is, or contains a subunit (e.g., catalytic subunit, SH2 domain, SH3 domain) of, CK2, Pirn subfamily protein kinase (e.g., PDvIl, PIM2, PDvO) or Fit subfamily protein kinase (e.g, FLTl , FLT3, FLT4).
  • the composition is cell free and sometimes the protein kinase is a recombinant protein.
  • the protein kinase can be from any source, such as cells from a mammal, ape or human, for example.
  • the protein kinase is a human protein kinase.
  • the serine-threonine protein kinase can be from any source, such as a mammal, ape or human, for example.
  • serine-threonine protein kinases that may be inhibited by compounds disclosed herein include without limitation human versions of CK2, CK2 ⁇ 2, Pirn subfamily kinases (e.g., PIMl, PIM2, PIM3), CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2, ZIPK and ZIPK.
  • a serine-threonine protein kinase sometimes is a member of a subfamily containing one or more of the following amino acids at positions corresponding to those listed in human CK2: leucine at position 45, methionine at position 163 and isoleucine at position 174.
  • Examples of such protein kinases include without limitation human versions of CK2, STKlO, HIPK2, HIPK3, DAPK3, DYK2 and PIM- 1.
  • Examples of tyrosine protein kinases that can be inhibited, or may potentially be inhibited, by compounds disclosed herein include without limitation human versions of Fit subfamily members (e.g., FLTl , FLT2, FLT3, FLT3 (D835Y), FLT4).
  • DYRK2 An example of a dual specificity protein kinase that can be inhibited, or may potentially be inhibited, by compounds disclosed herein includes without limitation DYRK2. Nucleotide and amino acid sequences for serine-threonine protein kinases and reagents are publicly available (e.g., World Wide Web URLs ncbi.nlm.nih.gov/sites/entrez/ and Invitrogen.com).
  • nucleotide sequences can be accessed using the following accession numbers: NM_002648.2 and NP_002639.1 for PIMl ; NM_006875.2 and NP_006866.2 for PIM2; XM_938171.2 and XP_943264.2 for PIM3; NM_0041 19.2 and NP_0041 10.2 for FLT3; NM_002020.3 and NP_00201 1.2 for FLT4; and NM_002019.3 and NP_002010.2 for FLTl.
  • the invention also in part provides methods for treating a condition related to aberrant cell proliferation.
  • methods of treating a cell proliferative condition in a subject which comprises administering a compound described herein to a subject in need thereof in an amount effective to treat the cell proliferative condition.
  • the subject may be a research animal (e.g., rodent, dog, cat, monkey), optionally containing a tumor such as a xenograft tumor (e.g., human tumor), for example, or may be a human.
  • a cell proliferative condition sometimes is a tumor or non-tumor cancer, including but not limited to, cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart (e.g., leukemia, lymphoma, carcinoma).
  • cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart e.g., leukemia, lymphoma, carcinoma.
  • methods of treating pain in a subject which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the pain.
  • methods of treating inflammation in a subject which comprises administering a compound described herein to a subject in need thereof in an amount effective to treat the inflammation.
  • the subject may be a research animal (e.g., rodent, dog, cat, monkey), for example, or may be a human.
  • Conditions associated with inflammation and pain include without limitation acid reflux, heartburn, acne, allergies and sensitivities, Alzheimer's disease, asthma, atherosclerosis, bronchitis, carditis, celiac disease, chronic pain, Crohn's disease, cirrhosis, colitis, dementia, dermatitis, diabetes, dry eyes, edema, emphysema, eczema, fibromyalgia, gastroenteritis, gingivitis, heart disease, hepatitis, high blood pressure, insulin resistance, interstitial cystitis, joint pain/arthritis/rheumatoid arthritis, metabolic syndrome (syndrome X), myositis, nephritis, obesity, osteopenia, osteoporosis, Parkinson's disease, periodontal disease, polyarteritis, polychondritis, psoriasis, scleroderma, sinusitis, Sjogren's syndrome, spastic colon, systemic candidia
  • a compound herein reduces inflammation or pain which comprise contacting a system with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of a pain signal or inflammation signal.
  • methods for identifying a compound that reduces inflammation or pain which comprise: contacting a system with a compound of Formula (I), (II), (III), (FV), (V) or (VI); and detecting a pain signal or inflammation signal, whereby a compound that modulates the pain signal relative to a control molecule is identified as a compound that reduces inflammation of pain.
  • pain signals are formalin-stimulated pain behaviors and examples of inflammation signals include without limitation a level of a pro-inflammatory molecule.
  • the invention also in part pertains to methods for modulating angiogenesis in a subject, and methods for treating a condition associated with aberrant angiogenesis in a subject.
  • methods for determining whether a compound herein modulates angiogenesis which comprise contacting a system with a compound described herein in an amount effective for modulating (e.g., inhibiting) angiogenesis or a signal associated with angiogenesis.
  • Signals associated with angiogenesis are levels of a pro-angiogenesis growth factor such as VEGF.
  • Methods for assessing modulation of angiogenesis also are known, such as analyzing human endothelial tube formation (BD BioCoatTM Angiogenesis System from BD Biosciences).
  • identifying a compound that modulates angiogenesis which comprise contacting a system with a compound of Formula (I), (II), (III), (IV), (V) or (VI) or a pharmaceutically acceptable salt thereof; and detecting angiogenesis in the system or an angiogenesis signal, whereby a compound that modulates the angiogenesis or angiogenesis signal relative to a control molecule is identified as a compound that modulates angiogenesis.
  • methods for treating an angiogenesis condition which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the angiogenesis condition.
  • Angiogenesis conditions include without limitation solid tumor cancers, varicose disease and the like.
  • the invention also in part pertains to methods for modulating an immune response in a subject, and methods for treating a condition associated with an aberrant immune response in a subject.
  • methods for determining whether a compound herein modulates an immune response which comprise contacting a system with a compound described herein in an amount effective for modulating (e.g., inhibiting) an immune response or a signal associated with an immune response.
  • Signals associated with immunomodulatory activity include, e.g., stimulation of T-cell proliferation, suppression or induction of cytokines, including, e.g., interleukins, interferon- ⁇ and TNF.
  • identifying a compound that modulates an immune response comprise contacting a system with a compound of Formula (I), (II), (III), (IV), (V) or (VI) or a pharmaceutically acceptable salt thereof; and detecting immunomodulatory activity in a system, or a signal associated with immunomodulatory activity, whereby a compound that modulates the immune response relative to a control molecule is identified as an immune response modulatory compound.
  • methods for treating a condition associated with an aberrant immune response in a subject which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the condition.
  • an immune response may be modulated by administering a compound herein in combination with a molecule that modulates (e.g., inhibits) the biological activity of an mTOR pathway member or member of a related pathway (e.g., mTOR, PI3 kinase, AKT).
  • a molecule that modulates e.g., inhibits
  • the biological activity of an mTOR pathway member or member of a related pathway e.g., mTOR, PI3 kinase, AKT.
  • the molecule that modulates the biological activity of an mTOR pathway member or member of a related pathway is rapamycin.
  • a composition comprising a compound described herein in combination with a molecule that modulates the biological activity of an mTOR pathway member or member of a related pathway, such as rapamycin, for example.
  • the compound is a compound of Formula (I), (II), (III), (IV), (V) or (VI) in one of the Tables provided herein, or a pharmaceutically acceptable salt of one of these compounds.
  • Any suitable formulation of a compound described above can be prepared for administration.
  • Any suitable route of administration may be used, including, but not limited to, oral, parenteral, intravenous, intramuscular, transdermal, topical and subcutaneous routes.
  • the mode of administration, and the type of treatment desired e.g., prevention, prophylaxis, therapy; the compounds are formulated in ways consonant with these parameters.
  • Preparation of suitable formulations for each route of administration are known in the art. A summary of such formulation methods and techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, which is incorporated herein by reference.
  • each substance or of the combination of two substances will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like.
  • the substances to be administered can be administered also in liposomal compositions or as microemulsions.
  • formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions.
  • Suitable excipients include, for example, water, saline, dextrose, glycerol and the like.
  • Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.
  • Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration.
  • Oral administration is also suitable for compounds of the invention. Suitable forms include syrups, capsules, tablets, as is understood in the art.
  • the appropriate dosage of a compound described above often is 0.01-150 mg/kg, and sometimes 0.1-100 mg/kg. Dosage levels are dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration; however, optimization of such parameters is within the ordinary level of skill in the art.
  • Compounds of the invention may be used alone or in combination with another therapeutic agent.
  • the invention provides methods to treat conditions such as cancer and inflammation by administering to a subject in need of such treatment a therapeutically effective amount of a therapeutic agent that binds to certain DNA segments and administering to the same subject a CK2 modulator in an amount that is effective to enhance the activity of the therapeutic agent.
  • a CK2 modulator is an agent that inhibits or enhances a biological activity of a CK2 protein, and is generically referred to hereafter as a "modulator.”
  • a Pirn or Fit modulator is an agent that inhibits or enhances a biological activity of a Pirn protein or a FH protein.
  • the therapeutic agent and the modulator may be administered together, either as separate pharmaceutical compositions or admixed in a single pharmaceutical composition.
  • the therapeutic agent and the modulator may also be administered separately, including at different times and with different frequencies, as long as the modulator is administered at a time that increases the potency of the therapeutic agent.
  • the modulator may be administered by any known route, such as orally, intravenously, intramuscularly, nasally, and the like; and the therapeutic agent may also be administered by any conventional route. In many embodiments, at least one and optionally both of the modulator and the therapeutic agent may be administered orally.
  • the modulator and the therapeutic agent are administered at the same time, whether in separate dosages or admixed in a single dosage.
  • the modulator and therapeutic agent are preferably combined into a single pharmaceutical composition, so the treated patient may receive a single oral dosage or a single injection, for example.
  • the amount of each of these materials to be administered will vary with the route of administration, the condition of the subject, other treatments being administered to the subject, and other parameters.
  • the therapeutic agents of the invention may, of course, cause multiple desired effects; and the amount of modulator to be used in combination with the therapeutic agent should be an amount that increases one or more of these desired effects.
  • the modulator is to be administered in an amount that is effective to enhance a desired effect of the therapeutic agent.
  • An amount is "effective to enhance a desired effect of the therapeutic agent", as used herein, if it increases by at least about 25% at least one of the desired effects of the therapeutic agent alone.
  • it is an amount that increases a desired effect of the therapeutic agent by at least 50% or by at least 100% (i.e., it doubles the effective activity of the therapeutic agent.) In some embodiments, it is an amount that increases a desired effect of the therapeutic agent by at least 200%.
  • the amount of a modulator that increases a desired effect of a therapeutic agent may be determined using in vitro methods, such as cell proliferation assays.
  • the therapeutic agents of the invention are useful to counter hyperproliferative disorders such as cancer, thus they reduce cell proliferation.
  • a suitable amount of a modulator could be the amount needed to enhance an antiproliferative effect of a therapeutic agent by at least 25% as determined in a cell proliferation assay.
  • the modulator used in the present invention enhances at least one desired effect produced by the therapeutic agent it is used with, thus the combinations of the inveniton provide a synergistic effect, not merely an additive effect.
  • the modulators themselves are at times useful for treating the same types of conditons, and thus may also have some direct effect in such assays. In that event, the "amount effective to increase a desired effect" must be a synergistic enhancement of the activity of the therapeutic agent that is attributable to enhancement by the modulator of an effect of the therapeutic agent, rather than a simple additive effect that would be expected with separate administration of the two materials.
  • the modulator can be used in an amount (concentration) that would not be expected to have any apparent effect on the treated subject or the in vitro assay, so the increased effect achieved with the combination is directly attributable to a synergistic effect.
  • the present invention includes methods and compositions for treating a patient having a cell proliferation disorder or an inflammatory disorder with a therapeutic agent as described herein, and a "modulator" described above, where the timing of administration of the modulator permits it to enhance a desired effect of the therapeutic agent .
  • a "modulator" as described above may be used in combination with a therapeutic agent that can act by binding to regions of DNA that can form certain quadruplex structures.
  • the therapeutic agents have anticancer activity on their own, but their activity is enhanced when they are used in combination with a modulator. This synergistic effect allows the therapeutic agent to be administered in a lower dosage while achieving equivalent or higher levels of at least one desired effect.
  • the appropriate dosage of a modulator such as a compound of Formula (I), (II), (III), (IV), (V), or (VI) as described herein, is typically between about 0.01-15 mg/kg, and about 0.1-10 mg/kg. Dosage levels are dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration; however, optimization of such parameters is within the ordinary level of skill in the art.
  • a modulator may be separately active for treating a cancer.
  • the dosage of a modulator will frequently be two-fold to ten-fold lower than the dosage required when the modulator is used alone to treat the same condition or subject. Determination of a suitable amount of the modulator for use in combination with a therapeutic agent is readily determined by methods known in the art.
  • the therapeutic agents of the invention are compounds that bind to certain motifs in nucleic acids.
  • the therapeutic agent to be used can be selected from several different classes of compounds, such as those that bind to quadruplex-forming regions of DNA.
  • the therapeutic agents are useful for the treatment of cancer and other indications such as inflammatory disorders, and methods for making and using them are known in the art. Several preferred classes of these therapeutic agents are described below. Each class of therapeutic agents can be used in combination with any active CK modulator, including but not limited to those disclosed herein.
  • the therapeutic agent can be a compound of formula (TAl-I):
  • V is H, halo, or NR 1 R 2 ;
  • A is H, fluoro, or NR 1 ⁇ ;
  • Z is O, S, NR 1 or CH 2 ;
  • X is OR 2 , NR 1 R 2 , halo, azido, or SR 2 ; n is 1-3; wherein in NR 1 R 2 , R 1 and R 2 may form a double bond or a ring, each of which is optionally substituted;
  • R 1 is H or a Ci. 6 alkyl
  • W is an optionally substituted aryl or heteroaryl, which may be monocyclic or fused with a single or multiple ring and optionally containing a heteroatom; or a compound having formula (TA 1-2):
  • V, A, X, Z and U are as defined in formula TAl-I, and W is selected from the group consisting of
  • the therapeutic agent is a compound having formula (TAl- IA): or a pharmaceutically acceptable salt, esters or prodrug thereof, or a specific isomer or mixture of isomers thereof.
  • the therapeutic agent of the combinations of the invention is a compound of this formula:
  • V is H, halo, or NR 1 R 2 ; AisH, fluoro, orNR' 2 ; Z is O, S, NR 1 OrCH 2 ; UiSOR 2 OrNR 1 R 2 ; X is OR 2 , NR 1 R 2 , halo, azido, or SR 2 ; n is 1-3; wherein in NR 1 R 2 , R 1 and R 2 may form a double bond or a ring, each of which is optionally substituted;
  • R 1 isHoraC,. 6 alkyl
  • R is H or a C
  • R 2 is an optionally substituted heterocyclic ring, aryl or heteroaryl
  • W is an optionally substituted aryl or heteroaryl, which may be monocyclic or fused with a single or multiple ring and optionally containing a heteroatom; or a compound having formula (TA3-2)
  • V, A, X, Z and U are as defined in formula 1 , and W is selected from the group consisting of
  • the therapeutic agent of the combinations of the invention is a compound of this formula:
  • B, X, A, or V is absent if Z 2 , Z 3 , or Z 4 , respectively, is N , and independently H, halo, azido, R 2 , CH 2 R 2 , SR 2 , OR 2 or NR 1 R 2 if Z 2 , Z 3 , or Z 4 , respectively, is C; or
  • a and V, A and X, or X and B may form a carbocyclic ring, heterocyclic ring, aryl or heteroaryl, each of which may be optionally substituted and/or fused with a cyclic ring;
  • Z 1 , Z 2 , Z 3 and Z 4 are C or N, provided any two N are non-adjacent;
  • W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused to an optionally substituted saturated or unsaturated ring;
  • said saturated or unsaturated ring may contain a heteroatom and is monocyclic or fused with a single or multiple carbocyclic or heterocyclic rings;
  • R 4 is H, a C] - 1O alkyl or C 2 .io alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R 3 and R 4 together with N may form an optionally substituted ring; • each R 5 is a substituent at any position on ring W; and is H, OR 2 , amino, alkoxy, amido, halogen, cyano or an inorganic substituent; or R 5 is C ⁇ _6 alkyl, C 2 .
  • B may be absent when Z 1 is N, or is H or a halogen when Z 1 is C.
  • W together with N and Z form a group having the formula selected from the group consisting of
  • R 6 is H, or a substituent known in the art, including but not limited to hydroxyl, alkyl, alkoxy, halo, amino, or amido; and ring S and ring T may be saturated or unsaturated.
  • W together with N and Z forms a 5- or 6-membered ring that is fused to a phenyl. In other embodiments, W together with N and Z forms a 5- or 6-membered ring that is optionally fused to another ring, when U is NR 1 R 2 , provided U is not NH 2 . In certain embodiments, W together with N and Z forms a 5- or 6-membered ring that is not fused to another ring, when U is NR 1 R 2 (e.g., NH 2 ).
  • the compounds of the present invention have the general formula (TA4-2A) or (TA4-2B):
  • A, B, V, X, U, Z, Z 1 , Z 2 , Z 3 , Z 4 and n are as described for TA4-1 ;
  • R 6 is H, Ci- 6 alkyl, hydroxyl, alkoxy, halo, amino or amido;
  • Z and Z 5 may optionally form a double bond.
  • U may be NR l 1 rR> 2', wherein R 1 is H, and R 2 is a C M0 alkyl optionally substituted with a heteroatom, a C3.6 cycloalkyl, aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S.
  • R 2 may be a Ci -I0 alkyl substituted with an optionally substituted morpholine, thiomorpholine, imidazole, aminodithiadazole, pyrrolidine, piperazine, pyridine or piperidine.
  • R 1 and R 2 together with N form an optionally substituted piperidine, pyrrolidine, piperazine, morpholine, thiomorpholine, imidazole, or aminodithiazole.
  • the therapeutic agent to be combined with a CK modulator can be selected from compounds having this formula:
  • V, X, and Y are absent if attached to a heteroatom other than Nitrogen, and independently H, halo, azido, R 2 , CH 2 R 2 , SR 2 , OR 2 or NR 1 R 2 when attached to C or N; or wherein V and X, or X and Y may form a carbocyclic ring, heterocyclic ring, aryl or hdteroaryl, each of which may be optionally substituted and/or fused with a cyclic ring;
  • Z 1 , Z 2 and Z 3 are C, N, O or S;
  • W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused to an optionally substituted aryl or heteroaryl, wherein said aryl or heteroaryl may be monocyclic or fused with a single or multiple ring, and wherein said ring optionally contains a heteroatom;
  • R 1 and R 3 are independently H or C
  • R 4 is H, a Ct-io alkyl or C 2 . ⁇ o alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R 3 and R 4 together with N may form an optionally substituted ring; each R 5 is a substituent at any position on W; and is H, OR 2 , amino, alkoxy, amido, halogen, cyano or an inorganic substituent; or R 5 is C ⁇ _ ⁇ alkyl, C 2 - 6 alkenyl, -CONHR 1 , each optionally substituted by halo, carbonyl or one or more non-adjacent heteroatoms; or two adjacent R 5 are linked to obtain a 5-6 membered optionally substituted carbocyclic or heterocyclic ring, optionally fused to an additional optionally substituted carbocyclic or heterocyclic ring; and n is 1-6.
  • ring T may form an optionally substituted 5- membered ring selected from the group consisting of:
  • W together with N and Z may form an optionally substituted 5- or 6-membered aryl or heteroaryl ring that is fused to an optionally substituted aryl or heteroaryl selected from the group consisting of:
  • W together with N and Z may form a group having the formula selected from the group consisting of
  • R 6 is H, or a substituent known in the art, including but not limited to hydroxyl, alky], alkoxy, halo, amino, or amido;
  • Ring S and M may be saturated or unsaturated.
  • W together with N and Z may form a 5- or 6-membered ring that is fused to a phenyl.
  • the compounds of the present invention have the general formula (TA5-2A) or (TA5-2B): wherein U, V, W, X, Y, Z, Z ⁇ Z 2 , Z 3 , R 5 and n are as described above for TA5-1 ;
  • Z and Z 4 may optionally form a double bond.
  • U may be SO 2 NR 1 R 2 , wherein R 1 is H, and R 2 is a C
  • R 2 may be a C]. 10 alkyl substituted with an optionally substituted morpholine, thiomo ⁇ holine, imidazole, aminodithiadazole, pyrrolidine, piperazine, pyridine or piperidine.
  • R 1 and R 2 together with N form an optionally substituted piperidine, pyrrolidine, piperazine, morpholine, thiomo ⁇ holine, imidazole, or aminodithiazole.
  • U is SO 2 NR'-(CR'2)n-NR 3 R 4 ; n is 1-4; each R 1 is H or alkyl; and R 3 and R 4 in NR 3 R 4 together form an optionally substituted piperidine, pyrrolidine, piperazine, mo ⁇ holine, thiomo ⁇ holine, imidazole, or aminodithiazole.
  • U is SO 2 NH-(CH 2 ) n -NR 3 R 4 wherein R 3 and R 4 together with N form an optionally substituted pyrrolidine, which may be linked to (CH 2 ) n at any position in the pyrrolidine ring. In one embodiment, R 3 and R 4 together with N form an N-methyl substituted pyrrolidine.
  • the present invention provides compounds having formula (TA5-1), (TA5-2A) or (TA5-2B), wherein: each of V and Y if present is independently H or halogen (e.g., chloro or fluoro);
  • X is -(ROR 1 R', wherein R 3 is C or N and wherein in each -(ROR 1 1 DR2" , R 1 and R' together may form an optionally substituted aryl or heteroaryl ring;
  • Z is NH or N-alkyl (e.g., N-CH 3 ); W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused with an optionally substituted aryl or heteroaryl ring; and
  • U is -SO 2 R 5 R 6 -(CH 2 ) n -CHR 2 -NR 3 R 4 , wherein R 5 is CR 1 or N; R 1 is H or alkyl; R 6 is H or C
  • the present invention provides compounds having formula (TA5- 1), (TA5-2A) or (TA5-2B), wherein:
  • V and Y if present is H or halogen (e.g., chloro or fluoro);
  • X if present is -(R 5 )R : R 2 , wherein R 5 is C or N and wherein in each -(R 5 )R ! R 2 , R 1 and R 2 together may form an optionally substituted aryl or heteroaryl ring;
  • Z is NH or N-alkyl (e.g., N-CH 3 );
  • U is -SO 2 R 5 R 6 -(CH 2 ) n -CHR 2 -NR 3 R 4 ,
  • R 5 is CR 1 or N
  • R 6 is H or alkyl and wherein in the -CHR 2 -NR 3 R 4 moiety each R 3 or R 4 together with the C may form an optionally substituted heterocyclic or heteroaryl ring, or wherein in the -CHR 2 - NR 3 R 4 moiety each R 3 or R 4 together with the N may form an optionally substituted carbocyclic, heterocyclic, aryl or heteroaryl ring.
  • the compounds of the present invention have the general formula (TA5-3):
  • the compounds of the present invention have the general formula (TA5-4A) or (TA5-4B):
  • the therapeutic agent for the combinations of the invention can be a compound of the formula:
  • X is H, OR", NR 1 R', halogen, azido, SR' or CH 2 R;
  • A is H, halogen, NR 1 R', SR', OR", CH 2 R", azido or NR 1 - (CR 2 ) n - NR J R 4
  • Z is O, S, NR 1 or CH 2 ;
  • U is R", OR", NR > 1 1 ⁇ R> 2' or NR 1 - (CR' 2 ) n - NR 3 J ⁇ R> 4 4 . provided U is not H;
  • W is an optionally substituted aryl or heteroaryl, which may be monocyclic or fused with a single or multiple ring optionally containing a heteroatom; wherein R 1 and R 2 together with N in NR 1 R 2 , and R 3 and R 4 together with N in NR 3 R 4 may independently form an optionally substituted 5-6 membered ring containing N, and optionally O or S;
  • R 1 and R 3 are independently H or a C
  • R 2 and R 4 are independently H, or a C MO alkyl or C 2 - 1 0 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with a substituted or unsubstituted aryl, heteroaryl, carbocyclic, or heterocyclic ring; or R 2 is an optionally cycloalkyl, substituted heterocyclic ring, aryl or heteroaryl;
  • W may be selected from the group consisting of
  • Q, Q 1 , Q 2 , and Q 3 are independently CH or N;
  • R 5 is as defined in formula 1.
  • each W in the above formula TA6- 1 or TA6-1A may be an optionally substituted phenyl, pyridine, biphenyl, naphthalene, phenanthrene, quinoline, isoquinoline, quinazoline, cinnoline, phthalazine, quinoxaline, indole, benzimidazole, benzoxazole, benzthiazole, benzofuran, anthrone, xanthone, acridone, fluorenone, carbazolyl, pyrimido[4,3-b]furan, pyrido[4,3-b]indole, pyrido[2,3-6]indole, dibenzofuran, acridine or acridizine.
  • W is an optionally substituted phenyl.
  • the present invention utilizes the above therapeutic agents in combination with at least one modulator.
  • Modulators that can be utilized in combination with a therapeutic agent described above include compounds having structures of Formula (I), (II), (III), (IV), (V) and (VI) as described herein.
  • the present invention utilizes the above therapeutic agents in combination with at least one modulator.
  • compounds of the invention may be used in combination with PARP inhibitors.
  • PARP inhibitors are known in the art, and are disclosed, for example, in CR. Calebrese, et al., Clin. Cancer Res. vol. 9, 271 1-18 (2003); S.J. Veuger, et al., Cancer Res. vol. 63, 6008-15 (2003); CR. Calabrese et al., J. Nat 'I. Cancer Inst. 96( 1 ), 56-67 (2004); "Potent Novel PARP Inhibitors," Expert Reviews in Molecular Medicine, vol. 7(4) (March 2005); and P.
  • PARP inhibitors disclosed in these documents are suitable for use in the methods and compositions of the present invention.
  • Additional PARP inhibitors that can be used include, for example, 10-(4- methyl-piperazin-l-ylmethyl)-2H-7-oxa-l ,2-diaza-benzo[de]anthracen-3-one (GPI 15427) and 2-(4-methyl-piperazin-l-yl)-5H-benzo[c][ l,5]naphthyridin-6-one (GPI 16539). See Di Paola, et al., Eur. J. Pharmacology, 527(1-3), 163-71 (2005).
  • Representative, but non-limiting, examples of PARP inhibitors that are suitable for use in the invention include the known compounds shown hereafter, including the pharmaceutically acceptable salts thereof, and individual isomers or mixtures of isomers thereof.
  • Modulators that can be utilized in combination with a therapeutic agent described above also include compounds having structures of Formula (I), (II), (III), (IV), (V) and (VI) as described herein.
  • the compound TAl-IA is a preferred therapeutic agent for use in the methods and compositions of the invention. More detail on suitable methods for its formulation and administration are provided in U.S. Provisional Application Serial No. 60/803,864 to Lim, et al., which was filed on June 3, 2006.
  • compositions comprising at least one therapeutic agent within the scope of the invention as described herein in combination with at least one modulator.
  • the composition may comprise a diluent or other pharmaceutically acceptable excipients.
  • the appropriate dosage of the therapeutic agent is typically 0.01-15 mg/kg, preferably 0.1- 10 mg/kg. Dosage levels are dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration; however, optimization of such parameters is within the ordinary level of skill in the art.
  • the dosage of a modulator is typically between about 0.01-150 mg/kg, and about 0.1-100 mg/kg.
  • a modulator may be separately active for treating a cancer.
  • the dosage of a modulator will frequently be two-fold to ten-fold lower than the dosage required when the modulator is used alone to treat the same condition or subject. Determination of a suitable amount of the modulator for use in combination with a therapeutic agent is readily determined by methods known in the art.
  • Also provided are methods for modulating the activity of a CK protein which comprises contacting a system comprising the CK protein with a composition described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein.
  • the system in such embodiments can be a cell-free system or a system comprising cells.
  • methods for reducing cell proliferation, and optionally inducing apoptosis which comprises contacting cells with a composition or a combination therapy as described herein, wherein a therapeutic agent is administered in an amount effective to reduce proliferation of the cells, and a CK inhibitor is administered in an amount sufficient to enhance the efficacy of the therapeutic agent.
  • the cells in such embodiments can be in a cell line, in a tissue or in a subject (e.g., a research animal or human).
  • the invention also in part provides methods for treating a condition related to aberrant cell proliferation.
  • methods of treating a cell proliferative condition in a subject which comprises administering a therapeutic agent described herein and a CK inhibitor described herein to a subject in need of treatment for a cell proliferative disorder; the therapeutic agent and the CK inhibitor are administered in amounts effective to treat the cell proliferative condition.
  • the subject may be a research animal (e.g., rodent, dog, cat, monkey), optionally containing a tumor such as a xenograft tumor (e.g., human tumor), for example, or may be a human.
  • a cell proliferative condition sometimes is a tumor or non-tumor cancer, including but not limited to, cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart (e.g., leukemia, lymphoma, carcinoma).
  • cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart e.g., leukemia, lymphoma, carcinoma
  • any suitable formulation of the therapeutic agent and the CK inhibitor can be prepared for administration, either together or separately. Any suitable route of administration may be used for each component, including but not limited to oral, parenteral, intravenous, intramuscular, transdermal, topical and subcutaneous routes.
  • the two substances used together may be administered separately or together. When administered together, they may be in separate dosage forms, or they may be combined into a single combination drug.
  • pharmaceutical compositions comprising a therapeutic agent as described herein and at least one CK inhibitor, and a pharmaceutically acceptable excipient.
  • Ethyl 3-bromo-4-pyridine carboxylate 1.15 g, 5.0 mmol
  • 2-amino-4- methoxycarbonyl-phenylboronic acid (1.04 g, 4.5 mmol)
  • sodium acetate (1.64 g, 20 mmol)
  • l ,r-bis(diphenylphosphino)ferrocene palladium (II) chloride complexed with dichloromethane
  • dimethylformamide 7.5 mL
  • Methyl 2-acetamido-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylate was prepared according to the procedure used in process 2, starting from methyl 2-acetamido-5- bromothiazole-4-carboxylate. Methyl 2-acetamido-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline- 7-carboxylate was isolated as a black solid (106 mg, 37% yield). LCMS (ES): 95% pure, m/z 318 [M+1] + . Process 28
  • Compounds of Formula (II) are prepared by routes analogous to those described herein for preparation of compounds of Formula (I).
  • the halogenated products prepared in processes 20, 21 , 22, and 30, may be converted to compounds of Formula (II) under conditions substantially as described in Example 4, or under conditions known to those of skill in the art.
  • Test compounds in aqueous solution are added at a volume of 10 microliters, to a reaction mixture comprising 10 microliters Assay Dilution Buffer (ADB; 2OmM MOPS, pH 7.2, 25 mM beta-glycerolphosphate, 5 mM EGTA, 1 mM sodium orthovanadate and 1 mM dithiothreitol), 10 microliters of substrate peptide (RRRDDDSDDD, dissolved in ADB at a concentration of 1 mM), 10 microliters of recombinant human CK2 (25 ng dissolved in ADB; Upstate).
  • ADB Assay Dilution Buffer
  • RRRDDDSDDD substrate peptide
  • Reactions are initiated by the addition of 10 microliters of ATP Solution (90% 75 mM MgCl 2 , 75 micromolar ATP dissolved in ADB; 10% [ ⁇ - 33 P]ATP (stock 1 mCi/100 ⁇ l; 3000 Ci/mmol (Perkin Elmer) and maintained for 10 minutes at 30 degrees C.
  • the reactions are quenched with 100 microliters of 0.75% phosphoric acid, then transferred to and filtered through a phosphocellulose filter plate (Millipore). After washing each well 5 times with 0.75% phosphoric acid, the plate is dried under vacuum for 5 min and, following the addition of 15 ul of scintillation fluid to each well, the residual radioactivity is measured using a Wallac luminescence counter.
  • a representative cell-proliferation assay protocol using Alamar Blue dye (stored at 4°C, use 20ul per well) is described hereafter.
  • 96-well plate setup and compound treatment a. Split and trypsinize cells.
  • H EMPTY d Add 100 ⁇ l of 2X drug dilution to each well in a concentration shown in the plate layout above. At the same time, add 100 ⁇ l of media into the control wells (wells B lO to B 12). Total volume is 200 ⁇ l /well.
  • e Incubate four (4) days at 37°C, 5% CO2 in a humidified incubator.
  • f. Add 20 ⁇ l Alamar Blue reagent to each well.
  • g. Incubate for four (4) hours at 37°C, 5% CO2 in a humidified incubator.
  • h Record fluorescence at an excitation wavelength of 544nm and emission wavelength of 590nm using a microplate reader.
  • cells are cultured with a test compound for approximately four days, the dye is then added to the cells and fluorescence of non-reduced dye is detected after approximately four hours.
  • Different types of cells can be utilized in the assays (e.g., HCT-1 16 human colorectal carcinoma cells, PC-3 human prostatic cancer cells and MiaPaca human pancreatic carcinoma cells).
  • the human leukemia Jurkat T-cell line is maintained in RPMI 1640 (Cambrex) supplemented with 10% fetal calf serum and 50 ng/ml Geutamycin. Before treatment cells are washed, resuspended at a density of about 10 6 cells/milliliter in medium containing 1 % fetal calf serum and incubated in the presence of indicated mounts of drug for two hours.
  • Cells are recovered by centrifugation, lysed using a hypotonic buffer (20 mM Tris/HCl pH 7.4; 2 mM EDTA; 5 mM EGTA; 10 mM mercaptoethanol; 1OmM NaF; 1 uM Okadaic acid; 10% v/v glycerol; 0.05% NP-40; 1% Protease Inhibitor Cocktail) and protein from the cleared lysate is diluted to 1 microgram per microliter in Assay Dilution Buffer (ADB; 2OmM MOPS, pH 7.2, 25mM ⁇ -glycerolphosphate, 5mM EGTA, ImM sodium orthovanadate and ImM dithiothreitol).
  • ADB Assay Dilution Buffer
  • the pharmacokinetics properties of drugs are investigated in ICR mice following an intravenous (FV) bolus and oral (PO) doses of drug at 5 mg/kg and 25 mg/kg respectively. Blood samples are collected at predetermined times and the plasma separated. Plasma is separated from the blood samples collected at 5, 15 and 30 minutes and 1 , 2, 4, 8 and 24 hours post-dose.
  • FV intravenous
  • PO oral
  • Drug levels are quantified by the LC/MS/MS method described below.
  • Noncompartmental pharmacokinetic analysis is applied for intravenous administration.
  • a linear trapezoidal rule is used to compute AUC(O- 24).
  • /2 and Co are calculated using the last three and the first three data points, respectively
  • Bioanalysis is performed using a Quattro Micro LC/MS/MS instrument in the MRM detection mode, with an internal standard (IS). Briefly, 15 ⁇ L plasma samples are prepared for analysis using protein precipitation with 120 ⁇ L of acetonitrile. The supernatants are transferred into a 96 well plate and subjected to LC-MS/MS analysis using a Phenomenex Polar-RP HPLC column. The mobile phases are 10 mM NH 4 HCO 3 in water (Solution-A) and 10 mM NH 4 HCO 3 in methanol (Solution-B). The column is initially equilibrated with 25 % Solution-B and followed with 100% Solution B over 5 minutes. The method has a dynamic range from 1 to 10,000 ng/mL. Quantitation of the analytes is performed in the batch mode with two bracketing calibration curves according to the bioanalytical sample list.
  • Compounds described herein are profiled for in vitro modulatory activity against protein kinases other than CK2.
  • the in vitro analysis is conducted using known protocols (e.g., assay protocols described at world-wide web address upstate.com/img/pdf/KP_Assay Protocol_BookIet_v3.pdf).
  • Compounds described herein are screened in the assays and prioritized based upon modulatory activity against protein kinases other than CK2 and specificity for CK2.
  • test compounds in aqueous solution are added at a volume of 5 ⁇ l, to a reaction mixture comprising 5 ⁇ l of 5x Reaction buffer (4OmM MOPS, pH 7.0, ImM EDTA), 2.5 ⁇ l of recombinant human PDvI-I solution (10 ng), 2.5 ⁇ l of substrate peptide (KKRNRTLTK) and 10 ⁇ l of ATP solution - 98% (75 mM MgC12 37.5 uM ATP) 2% ([ ⁇ - 33P]ATP: 3000Ci/mmol - Perkin Elmer).
  • 5x Reaction buffer 4OmM MOPS, pH 7.0, ImM EDTA
  • 2.5 ⁇ l of recombinant human PDvI-I solution 10 ng
  • substrate peptide KKRNRTLTK
  • ATP solution - 98% 75 mM MgC12 37.5 uM ATP
  • the reactions are incubated for 10 min at 30 0 C, quenched with 100 ⁇ l of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore). After washing each well 5 times with 0.75% Phosphoric acid, Scintillation fluid (15 ⁇ l) is added to each well. The residual radioactivity is measured using a luminescence counter.
  • Test compounds are tested further for activity against other protein kinases.
  • Selected kinase inhibition ICsodata are determined using standardized radiometric kinase assays for each individual kinase, which entail filter binding of 33 P labeled substrate proteins by the kinase of interest. Each IC 50 value is determined over a range of 10 drug concentrations. Representative reaction conditions are available from the World Wide Web URL upstate.corn/discovery/services/ic50_profiler.q.
  • each Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 ; each R 3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2-
  • each R 3 is independently halo, CF 3 , CFN, OR, NR 2 , NROR, NRNR 2 , SR, SOR, SO 2 R,
  • C2-C8 heteroalkyl C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl,
  • each of R 6A , R 6B and R 6C can be halo, CF 3 , OR, NR 2 , NROR, NRNR 2 , SR, SOR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRCOOR, NRCOR, CN, COOR, CONR 2 , 0OCR, COR, or NO 2 ; wherein each R is independently H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6- ClO
  • R 10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
  • each of each Z 1 , Z 3 and Z 4 iiss CCHH,, aanndd ZZ 22 iiss CCRR 33 ,, wwhhcere R 3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
  • A6 The compound of any one of embodiments A1-A4, wherein at least one of Z'-Z 4 is N.
  • A7 The compound of any one of embodiments A1-A6, wherein R 10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring.
  • A12 The compound of any one of embodiments Al-Al 1, wherein at least one of R 6B or R 6C is H.
  • A13 The compound of any one of embodiments A1-A12, wherein R 6B and R 6C are H.
  • a 14 The compound of any one of embodiments Al -A 12, wherein R 6B is H, SR, SO 2 R or NR 2 , where each R is independently H or C M alkyl.
  • a 15 The compound of of any one of embodiments A3-A14, wherein R 6 ⁇ is H.
  • R 3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
  • B5. The compound of any one of embodiments B1-B4, wherein R 1 10 i,s an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring.
  • B6. The compound of embodiment B5, wherein R 10 is an optionally substituted phenyl or pyridyl ring.
  • R 6C is H.
  • B 12 The compound of any one of embodiments B l-B lO, wherein R 6B is H, SR, SO 2 R or NR?, where each R is independently H or C M alkyl.
  • each Z 1 , Z 2 , Z 3 , and Z 4 is N, CH, or CR 3 , provided that none, one or two of Z 1 , Z 2 , Z 3 , and Z 4 are N; and further provided that one or more of Z 1 , Z 2 , Z 3 , and Z 4 is CR 3 ;
  • each R 3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C 1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R 3 is independently halo, CF 3 , OR, NR 2 , NfR
  • Z 5 is CR 6A or N; each of R 6A and R 6B is independently H or an optionally substituted C1 -C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R 6A and R 6B can be halo, CF 3 , OR, NR 2 , NROR, NRNR 2 , SR, SOR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRCOOR, NRCOR, CN, COOR, CONR 2 , OOCR, COR, or NO 2 ; wherein each R is independently
  • R 10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
  • each of each Z 1 , Z 3 and Z 4 is CH, and Z 2 is CR 3 , where R 3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
  • a pharmaceutical composition comprising a compound of any one of embodiments Al -A 15 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a compound of any one of embodiments Bl -B 13 and a pharmaceutically acceptable excipient.
  • a pharmaceutical composition comprising a compound of any one of embodiments Cl -C 13 and a pharmaceutically acceptable excipient.
  • a method for identifying a candidate molecule that interacts with a casein kinase 2 (CK2) protein comprises: contacting a composition containing a CK2 protein and a compound having a structure of Formula (I), (II), (III), (FV), (V), or (VI) with a candidate molecule under conditions in which the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein.
  • CK2 casein kinase 2
  • a method for modulating the activity of a CK2 protein comprises contacting a system comprising the protein with a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) in an amount effective for modulating the activity of the protein.
  • F6 The method of any one of embodiments F1-F5, wherein the protein, the compound or the molecule is in association with a solid phase.
  • a method for inhibiting cell proliferation comprises contacting cells with a compound having a structure of Formula (I), (H), (III), (IV), (V), or (VI) in an amount effective to inhibit proliferation of the cells.
  • G2 The method of embodiment Gl, wherein the cells are in a cancer cell line.
  • cancer cell line is a breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, or ovary cancer cell line.
  • G5 The method of any one of embodiments G1-G4, wherein contacting cells with a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) induces cell apoptosis.
  • a method for treating a condition related to aberrant cell proliferation comprises administering a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to treat the cell proliferative condition.
  • a method for identifying a candidate molecule that interacts with a protein kinase which comprises: contacting a composition containing a protein kinase and a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) under conditions in which the compound and the protein interact with a candidate molecule, and determining whether the amount of the compound that interacts with the protein kinase is modulated relative to a control interaction between the compound and the protein kinase without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein kinase relative to the control interaction is identified as a candidate molecule that interacts with the protein kinase.
  • J4 The method of embodiment Jl , J2, or J3, wherein the protein kinase is a human serine-threonine protein kinase selected from the group consisting of CK2, CK2 ⁇ 2, PIM 1 , PIM2, PIM3, CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK.
  • the protein kinase is a human serine-threonine protein kinase selected from the group consisting of CK2, CK2 ⁇ 2, PIM 1 , PIM2, PIM3, CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK.
  • J5. The method of embodiment J2, wherein the protein kinase is a serine-threonine protein kinase which contains one or more of the following amino acids at positions corresponding to those listed in human CK2: leucine at position 45, methionine at position 163 and isoleucine at position 174.
  • J6. The method of embodiment J5, wherein the serine threonine protein kinase is selected from the group consisting of CK2, STKlO, HIPK2, HIPK3, DAPK3, DYK2 and PIM-I .
  • J7 The method of embodiment J 1 , J2, or J3, wherein the protein kinase is a human tyrosine protein kinase selected from the group consisting of FLTl , FLT2, FLT3, FLT3 (D835Y), and FLT4.
  • the protein kinase is a human tyrosine protein kinase selected from the group consisting of FLTl , FLT2, FLT3, FLT3 (D835Y), and FLT4.
  • JlO The method of any one of embodiments J1-J9, wherein the protein kinase activity is the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate.
  • a method for modulating a protein kinase activity which comprises contacting a system comprising a protein kinase protein with a compound of Formula (I), (II), (III), (IV), (V), or (VI) in an amount effective for modulating the activity of the protein kinase.
  • the protein kinase is a human serine- threonine protein kinase selected from the group consisting of CK2, CK2 ⁇ 2, PIMl, PIM2, PIM3, CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK.
  • K5. The method of embodiment K2, wherein the protein kinase is a human tyrosine protein kinase selected from the group consisting of FLTl , FLT2, FLT3, FLT3 (D835Y), and FLT4.
  • a method for treating pain or inflammation in a subject which comprises administering a compound of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to treat the pain or the inflammation.
  • a method for inhibiting angiogenesis in a subject which comprises administering a compound of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to inhibit the angiogenesis.
  • composition of matter comprising a protein kinase and a compound of Formula (I), (II), (UI), (IV) 1 (V), or (VI).
  • composition of embodiment Nl wherein the protein kinase is a human serine-threonine kinase or a human tyrosine kinase.
  • a compound of Formula (I), (II), (HI), (IV), (V), or (VI) which is a compound in one of the Tables provided herein or a pharmaceutically acceptable salt thereof.

Abstract

The invention relates in part to molecules having certain biological activities that include, but are not limited to, inhibiting cell proliferation, and modulating protein kinase activity. Molecules of the invention can modulate casein kinase (CK) activity. The invention also relates in part to methods for using such molecules.

Description

THERAPEUTIC KINASE MODULATORS
Field of the Invention
[0001] The invention relates in part to molecules having certain biological activities that include, but are not limited to, inhibiting cell proliferation, and modulating serine-threonine protein kinase activity Molecules of the invention can modulate casein kinase (CK) activity (e g , CK2 activity) The invention also relates in part to methods for using such molecules
Disclosure of the Invention
[0002] The present invention in part provides chemical compounds having certain biological activities that include, but are not limited to, inhibiting cell proliferation, inhibiting angiogenesis, modulating protein kinase activity and modulating polymerase activity Certain molecules can modulate casein kinase 2 (CK2) activ ity and can affect biological functions that include but are not limited to, inhibiting gamma phosphate transfer from ATP to a protein or peptide substrate, inhibiting angiogenesis, inhibiting cell proliferation and inducing cell apoptosis, for example The present invention also in part prov ides methods for preparing novel chemical compounds, and analogs thereof, and methods of using the foregoing Also provided are compositions compπsing the above-descπbed molecules in combination with other agents, and methods for using such molecules in combination with other agents
[0003] The compounds of the invention have the general formula (A)
Figure imgf000002_0001
wherein the group labeled α represents a 5-6 membered aromatic or heteroaromatic ring fused onto the ring containing Q1, wherein α is a 6-membered aryl πng optionally containing one or more nitrogen atoms as πng members, or a five membered aryl πng selected from thiophene and thiazole,
Q' IS C-R10, Q2 IS N, and the bond between Q1 and Q2 is a double bond, and wherein Z'-Z4 and R10 are as defined below for Formula (I).
[0004] The invention also includes the pharmaceutically acceptable salts of compounds of formula (A). Thus in each compound of the invention, Formula (A) represents a fused tricyclic ring system which is linked through Q1 to a group R10, which is further described below.
[0005] Compounds related to Formula (A), wherein Q1 is carbon substituted with various groups connected through a heteroatom have been previously disclosed by the present applicants in U.S. Application Serial No. 11/849,230, to Chua et al., which was filed August 31, 2007, and is entitled SERINE-THREONINE PROTEIN KINASE AND PARP MODULATORS. In the present invention, it has surprisingly been found that compounds of Formula (A) wherein the group attached to Q1 is not connected through a heteroatom provide unexpectedly beneficial results.
[0006] In one aspect, the invention provides compounds of Formula (I):
Figure imgf000003_0001
and pharmaceutically acceptable salts, esters, prodrugs and tautomers thereof; wherein: each Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3; each R3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R3 is independently halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, COOH, polar substituent, carboxy bioisostere, CONR2, OOCR, COR, or NO2;
Z5 is CR6A or N; each of R6A, R6B and R6C is independently H or an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R6A, R6B and R6C can be halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, 0OCR, COR, or NO2; wherein each R is independently H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6- ClO aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and wherein two R on the same atom or on adjacent atoms can be linked to form a 3-8 membered ring, optionally containing one or more N, O or S; and each R group, and each ring formed by linking two R groups together, is optionally substituted with one or more substituents selected from halo, =0, =N-CN, =N-OR', =NR\ OR', NR'2, SR', SO2R', SO2NRS, NR1SO2R', NR'CONRS, NR'COOR', NR'COR', CN, COOR', C0NR'2, 0OCR', COR', and NO2, wherein each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2- C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1 -C4 alkyl, Cl- C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =O; and wherein two R' can be linked to form a 3-7 membered ring optionally containing up to three heteroatoms selected from N, O and S; and
R10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
[0007] In compounds of Formula (I), Z5 is N or CR6A, such that the ring containing Z5 is independently an optionally substituted pyridine or pyrimidine ring. For example, one or more ring nitrogen atoms within the ring containing Z5 may be arranged as follows:
Figure imgf000004_0001
where each of R6A, R6B, and R6C is independently selected from the R6 substituents defined above with respect to compounds of Formula (I).
[0008] In compounds of Formula (I), each of Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3, where each R3 is independently selected from the R3 substituents defined above with respect to compounds of Formula (I).
[0009] In certain embodiments, at least one of Z1, Z2, Z3, and Z4 is a nitrogen atom. One or more ring nitrogen atoms can be positioned in the ring containing Z1, Z2, Z3, and Z4 such that each ring is independently an optionally substituted pyridine, pyrimidine or pyridazine ring. In other embodiments, each of Z1, Z2, Z3, and Z4 is CR3, where each R3 is independently selected from the R3 substituents defined above with respect to compounds of Formula (I). In further embodiments, one or more of Z1, Z2, Z3, and Z4 is CR3, and the others are CH. In specific embodiments, one of Z1, Z2, Z3, and Z4 is CR3, and the others are CH. In certain preferred embodiments, Z2 is CR3 and Z1, Z3, and Z4 are CH.
[0010] In certain embodiments, at least one R3 substituent is a polar substituent, such as a carboxylic acid or a salt, an ester or a bioisostere thereof. In some embodiments, at least one R3 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof, for example. In certain embodiments, at least one R3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or other carboxy bioisostere. In specific embodiments, at least one R3 is a carboxylic acid-containing substituent or a salt thereof. This polar substituent is sometimes referred to herein as R3P.
[0011] The term "polar substituent" as used herein refers to any substituent having an electric dipole, and optionally a dipole moment (e.g., an asymmetrical polar substituent has a dipole moment and a symmetrical polar substituent does not have a dipole moment). Polar substituents include substituents that accept or donate a hydrogen bond, and groups that would carry at least a partial positive or negative charge in aqueous solution at physiological pH levels. In certain embodiments, a polar substituent is one that can accept or donate electrons in a non- covalent hydrogen bond with another chemical moiety. In certain embodiments, a polar substituent is selected from a carboxy, a carboxy bioisostere or other acid-derived moiety that exists predominately as an anion at a pH of about 7 to 8. Other polar substituents include, but are not limited to, groups containing an OH or NH, an ether oxygen, an amine nitrogen, an oxidized sulfur or nitrogen, a carbonyl, a nitrile, and a nitrogen-containing or oxygen-containing heterocyclic ring whether aromatic or non-aromatic. In some embodiments, the polar substituent represented by R3 is a carboxylate or a carboxylate bioisostere.
[0012] "Carboxylate bioisostere" or "carboxy bioisostere" as used herein refers to a moiety that is expected to be negatively charged to a substantial degree at physiological pH. In certain embodiments, the carboxylate bioisostere is a moiety selected from the group consisting of:
Figure imgf000006_0001
and salts and prodrugs of the foregoing, wherein each R7 is independently H or an optionally substituted member selected from the group consisting of Cj. io alkyl, CM0 alkenyl, C2.io heteroalkyl, C3.8 carbocyclic ring, and C3.8 heterocyclic ring optionally fused to an additional optionally substituted carbocyclic or heterocyclic ring; or R7 is a CMO alkyl, C2-Io alkenyl, or C2. io heteroalkyl substituted with an optionally substituted C3.8 carbocyclic ring or C3.g heterocyclic ring.
[0013] In certain embodiments, the polar substituent is selected from the group consisting of carboxylic acid, carboxylic ester, carboxamide, tetrazole, triazole, carboxymethanesulfonamide, oxadiazole, oxathiadiazole, thiazole, aminothiazole and hydroxythiazole.
[0014] In some embodiments, at least one R3 present is a carboxylic acid or a salt, or ester or a bioisostere thereof. In certain embodiments, at least one R3 present is a carboxylic acid- containing substituent or a salt, ester or bioisostere thereof. In the latter embodiments, the R3 substituent may be a Cl-ClO alkyl or C2-C10 alkenyl linked to a carboxylic acid (or salt, ester or bioisostere thereof), for example, and in some embodiments, the R3 substituent is not NHCOOCH2CH3.
[0015] A polar substituent may be at any position on the ring containing Z'-Z4 in Formula (I), and the ring may include one, two, three or four polar substituents. In certain embodiments, at least one of Z'-Z4 may be CR3 and one of the R3 substituents may be a polar substituent (e.g., a carboxylate or carboxylic acid ester, or a tetrazole) arranged at any one of the positions in the ring containing Z'-Z4:
Figure imgf000007_0001
where R >3JPP i •s a polar substituent and each R , 3JAA, R3B, R3C and R3D is H or is independently selected from the R3 substituents, as defined above with respect to compounds of Formula (I).
[0016] In specific embodiments, one of Z'-Z4 is CR3 and the others of Z1 -Z4 are CH, where R3 is a polar substituent (i.e., R3P), for example, a carboxylate or carboxylic acid, ester, or amide, or a tetrazole, arranged at any one of the positions in the ring containing Z1 -Z4:
Figure imgf000008_0001
[0017] The polar substituent represented by R3 in some embodiments is a carboxy (e.g., COOH), carboxyalkyl (e.g., carboxymethyl), tetrazole or amide (e.g., -CONH2, .CONHc-Pr) substituent. In other embodiments, R3 represents a carboxylate bioisostere. In preferred embodiments, R3 at position Z2 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof. In specific embodiments, Z2 is CR3 , where R3 is COOH or COOMe. In other embodiments, it is CONR2, where each R is independently H or CM alkyl; sometimes, it is CONH2 or CONHc-Pr.
[0018] In compounds of Formula (I), R10 is an optionally substituted five-membered or six- membered carbocyclic or heterocyclic aromatic ring. In certain embodiments, R10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring. In some such embodiments, the optionally substituted six-membered carbocyclic or heterocyclic aromatic ring is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, and optionally substituted variants of the foregoing. In particular embodiments, R10 is an optionally substituted phenyl or pyridyl ring ring. In some embodiments, it is an unsubstituted phenyl ring. In other embodiments, R10 is phenyl substituted with one or more halo, CN, CM alkyl, C M alkoxy, NH2, or NHAc substitutents.
[0019] In other embodiments, R10 is an optionally substituted five-membered heterocyclic aromatic ring. In some such embodiments, the optionally substituted five-membered heterocyclic aromatic ring is selected from pyrrole, imidazole, pyrazole, triazole, tetrazole, thiophene, thiazole, thiadiazole, isothiazole, furan, oxazole, isoxazole, oxadiazole and optionally substituted variants thereof. In particular embodiments, R10 is an optionally substituted pyrazole, isoxazole or thiophene; in specific embodiments, it is an N'-methyl-pyrazol-4-yl substituent or an optionally substituted 2- or 3-thiophene substituent. In certain embodiments, optional substituents when present on a five-membered heterocyclic aromatic ring include one or more halo, CN, or CM alkyl groups.
[0020] Each of R6B, R6C and R6A, where present, is independently selected from the list defined above with respect to compounds of Formula (I). In certain embodiments, at least one of R6B and R6C is H. In other embodiments, both R6B and R6C are H. In some embodiments, R6B is thiomethoxy (-SMe) and R6C is H. In certain embodiments, Z5 is N, and R6B is SR, SO2R, or NR2, where each R is independently H or C14 alkyl; in some such embodiments, R6B is SMe, SO2Me or NHc-Pr. In other embodiments, Z5 is R6A, and each of R6A, R6B and R6C is H.
[0021] In another aspect, the invention provides compounds of Formula (II):
Figure imgf000009_0001
Formula (II) and pharmaceutically acceptable salts, esters, prodrugs and tautomers thereof; wherein: each Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3; each R3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R3 is independently halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, COOH, polar substituent, carboxy bioisostere, CONR2, OOCR, COR, or NO2;
Z5 is CR6A or N; each of R6A and R6B is independently H or an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R6A and R6B can be halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, or NO2; wherein each R is independently H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6- ClO aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and wherein two R on the same atom or on adjacent atoms can be linked to form a 3-8 membered ring, optionally containing one or more N, O or S; and each R group, and each ring formed by linking two R groups together, is optionally substituted with one or more substituents selected from halo, =0, =N-CN, =N-0R', =NR\ OR', NR'2; SR', SO2R', SO2NR\, NR1SO2R', NR'CONR'2, NR'COOR', NR'COR', CN, COOR', C0NR'2, OOCR', COR', and NO2, wherein each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C l -C6 acyl, C2- C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, Cl- C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =O; and wherein two R' can be linked to form a 3-7 membered ring optionally containing up to three heteroatoms selected from N, O and S; and
R10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
[0022] Embodiments described with respect to compounds of Formula (I) for substituents Z1, Z2, Z3, Z4, Z5, R6A, R6B, R3 and R10 may also be applied to compounds of Formula (II).
[0023] In compounds of Formula (II), Z5 is N or CR6A, such that the ring containing Z5 is independently an optionally substituted thiophene or thiazole ring. For example, one or more heteroatoms within the ring containing Z5 may be arranged as follows:
Figure imgf000010_0001
where each of R6A and R6B are independently selected from the R6 substituents defined above with respect to compounds of Formula (II). [0024] In compounds of Formula (II), each of Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3, where each R3 is independently selected from the R3 substituents defined above with respect to compounds of Formula (I) or (II).
[0025] In certain embodiments, at least one of Z'-Z4 is a nitrogen atom. One or more ring nitrogen atoms can be positioned in the ring containing Z'-Z4 such that each ring is independently an optionally substituted pyridine, pyrimidine or pyridazine ring. In other embodiments, each of Z1, Z2, Z3 and Z4 is CR3, where each R3 is independently selected from the R3 substituents defined above with respect to compounds of Formula (II). In further embodiments, at least one of Z1, Z2, Z3 and Z4 is CR3, and the others are CH. In other embodiments, one of Z1, Z2, Z3 and Z4 is CR3, and the others are CH. In some such embodiments, Z2 is CR3, and Z1, Z3 and Z4 are CH.
[0026] In certain embodiments of Formula (II), at least one R3 substituent is a polar substituent, such as a carboxylic acid or a salt, an ester or a bioisostere thereof. In compounds of Formula (II), a polar substituent may be at any position on the ring containing Z'-Z4, and the ring may include one, two, three or four polar substituents. In certain embodiments, at least one of Z'-Z4 may be CR3 and one of the R3 substituents may be a polar substituent (e.g., a carboxylate or carboxylic acid ester, or a tetrazole) arranged at any one of the positions in the ring, as described for compounds of Formula (I). In certain embodiments of Formula (II), at least one R3 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof, for example. In some embodiments, at least one R3 is a carboxylic acid-containing substituent or a salt thereof.
[0027] In compounds of Formula (II), R10 is an optionally substituted five-membered or six- membered carbocyclic or heterocyclic aromatic ring. In certain embodiments of Formula (II), R10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring. In some such embodiments, the optionally substituted six-membered carbocyclic or heterocyclic aromatic ring is selected from phenyl, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, and optionally substituted variants of the foregoing. In certain embodiments, R10 is an optionally substituted phenyl ring.
[0028] In other embodiments of Formula (II), R10 is an optionally substituted five-membered heterocyclic aromatic ring. In some such embodiments, the optionally substituted fϊve- membered heterocyclic aromatic ring is selected from pyrrole, imidazole, pyrazole, triazole, tetrazole, thiophene, thiazole, thiadiazole, isothiazole, furan, oxazole, isoxazole, oxadiazole and optionally substituted variants thereof.
[0029] Each of R6B and R6A, where present, is independently selected from the list defined above with respect to compounds of Formula (II). In certain embodiments, at least one of R6A and R6B is H. In other embodiments, both R6A and R6B are H. In other embodiments, R6B is halo or NR2. In certain embodiments, Z5 is CR6A, where R6A is H, and R6B is H or halo. In other embodiments, Z5 is N, and R6B is H or NR2.
[0030] Also provided herein are compounds for formula (I) having the structures of formulae (in), (IV), (V) and (VI):
Figure imgf000012_0001
and pharmaceutically acceptable salts, esters, and tautomers thereof; wherein R6A, R6B, R6C, R10, Z1 , Z2, Z3, Z4 and R3 are defined as for Formula (I). [0031] In compounds of Formula (III) and (IV), each of Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3, where each R3 is independently selected from the R3 substituents defined above with respect to compounds of Formula (I). [0032] In certain embodiments of Formula (III) or (IV), at least one of Z1, Z2, Z3, and Z4 is a nitrogen atom. In further embodiments, one or more of Z1, Z2, Z3, and Z4 is CR3, and the others are CH. In specific embodiments, one of Z1, Z2, Z3, and Z4 is CR3, and the others are CH. In certain preferred embodiments, Z2 is CR3 and Z1, Z3, and Z4 are CH.
[0033] In certain embodiments of Formula (III), (IV), (V) and (VI), at least one R3 substituent is a polar substituent, such as a carboxylic acid or a salt, an ester or a bioisostere thereof. In some embodiments, at least one R3 is a carboxylic acid-containing substituent or a carboxylate bioisostere, or a salt or ester thereof, for example. In certain embodiments, at least one R3 is a carboxylic acid-containing substituent or a salt thereof. In some embodiments, R3 at position Z2 is COOH, COOR or CONR2, where each R is independently H or CM alkyl. In specific embodiments, R3 at position Z2 is COOH, COOMe or CONHc-Pr. In other embodiments, Z2 is CR3 where R3 is F.
[0034] In compounds of Formula (III), (IV), (V) and (VI), R10 is an optionally substituted five-membered heterocyclic aromatic ring or six-membered carbocyclic or heterocyclic aromatic ring. In certain embodiments, R10 is an optionally substituted phenyl or pyridine ring. In some embodiments, it is an unsubstituted phenyl ring. In other embodiments, R10 is phenyl substituted with one or more halo, CN, CM alkyl, C)^ alkoxy, NH2, or NHAc substitutents. In certain embodiments, R10 is an optionally substituted five-membered heterocyclic aromatic ring. In some such embodiments, R10 is an optionally substituted pyrazole, isoxazole or thiophene ring.
[0035] Each of R6A, R6B, and R6C, where present in compounds of Formula (III), (IV), (V) and (VI) is independently selected from the list defined above with respect to compounds of Formula (I). In certain embodiments, at least one of R6B and R6C is H. In other embodiments, both R6B and R60 are H. In certain embodiments of Formula (IV) and (VI), R6B is SR, SO2R, or NR2, where each R is independently H or CM alkyl; in some such embodiments, R6B is SMe, SO2Me or NHc-Pr. In other embodiments of Formula (III) and (V), R6A, R6B and R6C are H.
[0036] Other embodiments described herein with respect to compounds of Formula (I) also may be applied to compounds of Formula (III), Formula (IV), Formula (V) and Formula (VI).
[0037] Also provided herein is a pharmaceutical composition comprising a compound of . Formula (A), Formula (I), Formula (H), Formula (III), Formula (IV), Formula (V), or Formula (VI), as descπbed herein and a pharmaceutically acceptable carrier or excipient Pharmaceutical compositions can be utilized in treatments descnbed herein
[0038] Provided also are methods for identifying a candidate molecule that interacts with a CK2 protein, which comprise contacting a composition containing a CK2 protein and a compound descπbed herein with a candidate molecule under conditions in which the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein
[0039] In certain embodiments, the protein is a CK2 protein, such as a CK2 protein compπsing the amino acid sequence of SEQ ID NO 1 , 2 or 3 or a substantially identical variant thereof, for example SEO ID NQ 1 (NP 001886, casein kinase II alpha 1 subunit isoform a [Homo sapiensl)
1 msgpvpsrar vytdvnthrp reywdyeshv vewgnqddyq lvrklgrgky sevfeainit
61 nnekvvvkil kpvkkkkikr eikilenlrg gpniitladi vkdpvsrtpa lvfehvnntd
121 fkqlyqtltd ydirfymyei lkaldychsm gimhrdvkph nvmidhehrk lrlidwglae
181 fyhpgqeynv rvasryfkgp ellvdyqmyd ysldmwslgc mlasmifrke pffhghdnyd
241 qlvriakvlg tedlydyidk ynieldprfn dilgrhsrkr werfvhsenq hlvspealdf
301 ldkllrydhq srltareame hpyfytvvkd qarmgsssmp ggstpvssan mmsgissvpt
361 psplgplags pviaaanplg mpvpaaagaq q
SEQ ID NO 2 (NP 808227. casein kinase II alpha 1 subunit isoform a [Homo sapiensl)
1 msgpvpsrar vytdvnthrp reywdyeshv vewgnqddyq lvrklgrgky sevfeainit
61 nnekvvvkil kpvkkkkikr eikilenlrg gpniitladi vkdpvsrtpa lvfehvnntd
121 fkqlyqtltd ydirfymyei lkaldychsm gimhrdvkph nvmidhehrk lrlidwglae
181 fyhpgqeynv rvasryfkgp ellvdyqmyd ysldmwslgc mlasmifrke pffhghdnyd
241 qlvriakvlg tedlydyidk ynieldprfn dilgrhsrkr werfvhsenq hlvspealdf
301 ldkllrydhq srltareame hpyfytvvkd qarmgsssmp ggstpvssan mmsgissvpt
361 psplgplags pviaaanplg mpvpaaagaq q
SEQ ID NO 3 (NP 808228, casein kinase II alpha 1 subunit isoform b [Homo sapiensl)
1 myeilkaldy chsmgimhrd vkphnvmidh ehrklrlidw glaefyhpgq eynvrvasry 61 fkgpellvdy qmydysldmw slgcmlasmi frkepffhgh dnydqlvria kvlgtedlyd 121 yidkynield prfndilgrh srkrwerfvh senqhlvspe aldfldkllr ydhqsrltar 181 eamehpyfyt vvkdqarmgs ssmpggstpv ssanmmsgis svptpsplgp lagspviaaa 241 nplgmpvpaa agaqq [0040] In certain embodiments the protein is in a cell or in a cell-free system. The protein, the compound or the molecule in some embodiments is in association with a solid phase. In certain embodiments, the interaction between the compound and the protein is detected via a detectable label, where in some embodiments the protein comprises a detectable label and in certain embodiments the compound comprises a detectable label. The interaction between the compound and the protein sometimes is detected without a detectable label.
[0041] Also provided are methods for modulating the activity of a CK2 protein, which comprise contacting a system comprising the protein with a compound described herein in an amount effective for modulating the activity of the protein. Also provided are methods for modulating the activity of a Pirn protein or a Fit protein which comprise contacting a system comprising the protein with a compound described herein in an amount effective for modulating the activity of the protein. In certain embodiments the activity of the protein is inhibited, and sometimes the protein is a CK2 protein, such as a CK2 protein comprising the amino acid sequence of SEQ ID NO: 1, 2 or 3 or a substantially identical variant thereof, for example. In certain embodiments, the system is a cell, and in other embodiments the system is a cell-free system. The protein or the compound may be in association with a solid phase in certain embodiments.
[0042] Provided also are methods for inhibiting cell proliferation, which comprise contacting cells with a compound described herein in an amount effective to inhibit proliferation of the cells. The cells sometimes are in a cell line, such as a cancer cell line (e.g., breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, ovary cancer cell line), for example. In some embodiments, the cancer cell line is a breast cancer, prostate cancer or pancreatic cancer cell line. The cells sometimes are in a tissue, can be in a subject, at times are in a tumor, and sometimes are in a tumor in a subject. In certain embodiments, the method further comprises inducing cell apoptosis. Cells sometimes are from a subject having macular degeneration.
[0043] Also provided are methods for treating a condition related to aberrant cell proliferation, which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the cell proliferative condition. In certain embodiments the cell proliferative condition is a tumor-associated cancer. The cancer sometimes is of the breast, prostate, pancreas, lung, colorectum, skin, or ovary. In some embodiments, the cell proliferative condition is a non-tumor cancer, such as a hematopoietic cancer, for example. The cell proliferative condition is macular degeneration in some embodiments.
[0044] Provided also are methods for treating cancer or an inflammatory disorder in a subject in need of such treatment, comprising: administering to the subject a therapeutically effective amount of a therapeutic agent as described herein; and administering to the subject a molecule that inhibits CK2, Pirn or Rt in an amount that is effective to enhance a desired effect of the therapeutic agent. The therapeutic agent sometimes is a compound of formula TA 1 - 1 , TA2, TA3-1, TA4-1 , TA5- 1 or TA6-l as described herein, or a pharmaceutically acceptable salt of one of these compounds. In certain embodiments, the molecule that inhibits CK2, Pirn or FH is a compound of Formula (I), (II), (III), (IV), (V), or (VI) as described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the molecule that inhibits CK2, Pirn or Fit is a known compound, or a compound provided herein, or a pharmaceutically acceptable salt of one of these compounds. In some embodiments, the desired effect of the therapeutic agent that is enhanced by the molecule that inhibits CK2, Pirn or Fit is a reduction in cell proliferation. In certain embodiments, the desired effect of the therapeutic agent that is enhanced by the molecule that inhibits CK2, Pirn or Fit is an increase in apoptosis in at least one type of cell. The therapeutic agent in certain embodiments is:
Figure imgf000016_0001
or a pharmaceutically acceptable salt thereof, or a specific isomer or mixture of isomers thereof. [0045] In some embodiments, the therapeutic agent and the molecule that inhibits CK2, Pirn or Fit are administered at substantially the same time. The therapeutic agent and molecule that inhibits CK2 sometimes are used concurrently by the subject. The therapeutic agent and the molecule that inhibits CK2, Pirn or Fit are combined into one pharmaceutical composition in certain embodiments. Some embodiments are directed to a pharmaceutical composition comprising a therapeutic agent of any of formulas TA 1 - 1 , TA2, TA3- 1 , TA 4- 1 , TA5- 1 or TA6 admixed with a molecule that inhibits CK2, Pirn or Fit, or a pharmaceutically acceptable salt thereof. In some embodiments, the molecule that inhibits CK2, Pirn or Fit is a compound of Formula (I), (II), (III), (IV), (V), or (VI) as described herein, or a pharmaceutically acceptable salt thereof.
[0046] In some embodiments the therapeutic agent is a compound of formula TA2 or a pharmaceutically acceptable salt thereof. A therapeutic composition in certain embodiments comprises a therapeutically effective amount of a therapeutic agent of the formula TA2:
Figure imgf000017_0001
or a pharmaceutically acceptable salt thereof, or a specific isomer or mixture of isomers thereof, admixed with an amount of a CK modulator or a pharmaceutically acceptable salt of a CK modulator; and where the amount of the CK modulator or the pharmaceutically acceptable salt of a CK modulator is an amount that is effective to enhance a desired effect of the therapeutic agent.
[0047] Also provided are compositions comprising a compound described herein and an isolated protein. The protein sometimes is a CK2 protein, such as a CK2 protein comprising the amino acid sequence of SEQ ED NO: 1, 2 or 3 or a substantially identical variant thereof, for example. In some embodiments, the protein is a Pirn protein. In other embodiments, the protein is a Fit protein. Certain compositions comprise a compound described herein in combination with a cell. The cell may be from a cell line, such as a cancer cell line. In the latter embodiments, the cancer cell line is sometimes a breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, ovary cancer cell line.
[0048] These and other embodiments of the invention are described in the description that follows.
Modes of Carrying Out the Invention
[0049] Compounds of Formula (I), (II), (III), (IV), (V), and (VI) can exert biological activities that include, but are not limited to, inhibiting cell proliferation, modulating protein kinase activity and modulating polymerase activity. Compounds of Formula (I), (II), (III), (IV), (V), and (VI) can modulate CK2 activity, for example. Such compounds therefore can be utilized in multiple applications by a person of ordinary skill in the art. For example, compounds described herein may find uses that include, but are not limited to, (i) modulation of protein kinase activity (e.g., CK2 activity), (ii) modulation of cell proliferation, (iii) modulation of apoptosis, and (iv) treatments of cell proliferation related disorders (e.g., administration alone or co-administration with another molecule).
[0050] "Optionally substituted" as used herein indicates that the particular group or groups being described may have no non-hydrogen substituents, or the group or groups may have one or more non-hydrogen substituents. If not otherwise specified, the total number of such substituents that may be present is equal to the number of H atoms present on the unsubstituted form of the group being described. Where an optional substituent is attached via a double bond, such as a carbonyl oxygen (=O), the group takes up two available valences, so the total number of substituents that may be included is reduced according to the number of available valences.
[0051] The compounds of the invention often have ionizable groups so as to be capable of preparation as salts. In that case, wherever reference is made to the compound, it is understood in the art that a pharmaceutically acceptable salt may also be used. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art. In some cases, the compounds may contain both an acidic and a basic functional group, in which case they may have two ionized groups and yet have no net charge.
[0052] In some cases, the compounds of the invention contain one or more chiral centers. The invention includes each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers and tautomers that can be formed. The compounds of the invention may also exist in more than one tautomeric form; the depiction herein of one tautomer is for convenience only, and is also understood to encompass other tautomers of the form shown.
[0053] As used herein, the terms "alkyl," "alkenyl" and "alkynyl" include straight-chain, branched-chain and cyclic monovalent hydrocarbyl radicals, and combinations of these, which contain only C and H when they are unsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl, cyclopentylethyl, 2-propenyl, 3-butynyl, and the like. The total number of carbon atoms in each such group is sometimes described herein, e.g., when the group can contain up to ten carbon atoms it can be represented as 1- 1OC or as Cl-ClO or Cl-10. When heteroatoms (N, O and S typically) are allowed to replace carbon atoms as in heteroalkyl groups, for example, the numbers describing the group, though still written as e.g. C1-C6, represent the sum of the number of carbon atoms in the group plus the number of such heteroatoms that are included as replacements for carbon atoms in the backbone of the ring or chain being described.
[0054] Typically, the alkyl, alkenyl and alkynyl substituents of the invention contain 1-lOC (alkyl) or 2- 1OC (alkenyl or alkynyl). Preferably they contain 1-8C (alkyl) or 2-8C (alkenyl or alkynyl). Sometimes they contain 1-4C (alkyl) or 2-4C (alkenyl or alkynyl). A single group can include more than one type of multiple bond, or more than one multiple bond; such groups are included within the definition of the term "alkenyl" when they contain at least one carbon- carbon double bond, and are included within the term "alkynyl" when they contain at least one carbon-carbon triple bond.
[0055] Alkyl, alkenyl and alkynyl groups are often optionally substituted to the extent that such substitution makes sense chemically. Typical substituents include, but are not limited to, halo, =0, =N-CN, =N-OR, =NR, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, C≡CR, COOR, CONR2, OOCR, COR, and NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, or C5-C10 heteroaryl, and each R is optionally substituted with halo, =0, =N-CN, =N-OR', =NR', OR', NR* 2, SR', SO2R1, SO2NR'2, NR'S02R', NR'CONRS, NR'COOR', NR'COR', CN, C≡CR', COOR', CONRS, OOCR', COR', and NO2, wherein each R' is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl. Alkyl, alkenyl and alkynyl groups can also be substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which can be substituted by the substituents that are appropriate for the particular group.
[0056] "Acetylene" substituents are 2- 1OC alkynyl groups that are optionally substituted, and are of the formula -C≡C-Ra, wherein Ra is H or C1-C8 alky], C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each Ra group is optionally substituted with one or more substituents selected from halo, =0, =N-CN, =N-OR\ =NR\ OR', NR'2( SR', SO2R', SO2NR'2, NR1SO2R', NR'CONRS, NR'COOR', NR'COR', CN, COOR', C0NR'2, OOCR', COR', and NO2, wherein each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2-C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, C1-C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =O; and wherein two R' can be linked to form a 3- 7 membered ring optionally containing up to three heteroatoms selected from N, O and S. In some embodiments, Ra of -C≡C-Ra is H or Me.
[0057] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl" and the like are defined similarly to the corresponding hydrocarbyl (alkyl, alkenyl and alkynyl) groups, but the 'hetero' terms refer to groups that contain 1-3 O, S or N heteroatoms or combinations thereof within the backbone residue; thus at least one carbon atom of a corresponding alkyl, alkenyl, or alkynyl group is replaced by one of the specified heteroatoms to form a heteroalkyl, heteroalkenyl, or heteroalkynyl group. The typical and preferred sizes for heteroforms of alkyl, alkenyl and alkynyl groups are generally the same as for the corresponding hydrocarbyl groups, and the substituents that may be present on the heteroforms are the same as those described above for the hydrocarbyl groups. For reasons of chemical stability, it is also understood that, unless otherwise specified, such groups do not include more than two contiguous heteroatoms except where an oxo group is present on N or S as in a nitro or sulfonyl group.
[0058] While "alkyl" as used herein includes cycloalkyl and cycloalkylalkyl groups, the term "cycloalkyl" may be used herein to describe a carbocyclic non-aromatic group that is connected via a ring carbon atom, and "cycloalkylalkyl" niay be used to describe a carbocyclic non-aromatic group that is connected to the molecule through an alkyl linker. Similarly, "heterocyclyl" may be used to describe a non-aromatic cyclic group that contains at least one heteroatom as a ring member and that is connected to the molecule via a ring atom, which may be C or N; and "heterocyclylalkyl" may be used to describe such a group that is connected to another molecule through a linker. The sizes and substituents that are suitable for the cycloalkyl, cycloalkylalkyl, heterocyclyl, and heterocyclylalkyl groups are the same as those described above for alkyl groups. As used herein, these terms also include rings that contain a double bond or two, as long as the ring is not aromatic.
[0059] As used herein, "acyl" encompasses groups comprising an alkyl, alkenyl, alkynyl, aryl or arylalkyl radical attached at one of the two available valence positions of a carbonyl carbon atom, and heteroacyl refers to the corresponding groups wherein at least one carbon other than the carbonyl carbon has been replaced by a heteroatom chosen from N, O and S. Thus heteroacyl includes, for example, -C(=O)OR and -C(=O)NR2 as well as -C(=O)-heteroaryl.
[0060] Acyl and heteroacyl groups are bonded to any group or molecule to which they are attached through the open valence of the carbonyl carbon atom. Typically, they are C1 -C8 acyl groups, which include formyl, acetyl, pivaloyl, and benzoyl, and C2-C8 heteroacyl groups, which include methoxyacetyl, ethoxycarbonyl, and 4-pyridinoyl. The hydrocarbyl groups, aryl groups, and heteroforms of such groups that comprise an acyl or heteroacyl group can be substituted with the substituents described herein as generally suitable substituents for each of the corresponding component of the acyl or heteroacyl group.
[0061] "Aromatic" moiety or "aryl" moiety refers to a monocyclic or fused bicyclic moiety having the well-known characteristics of aromaticity; examples include phenyl and naphthyl. Similarly, "heteroaromatic" and "heteroaryl" refer to such monocyclic or fused bicyclic ring systems which contain as ring members one or more heteroatoms selected from O, S and N. The inclusion of a heteroatom permits aromaticity in 5-membered rings as well as 6-membered rings. Any monocyclic or fused ring bicyclic system which has the characteristics of aromaticity in terms of electron distribution throughout the ring system is included in this definition. It also includes bicyclic groups where at least the ring which is directly attached to the remainder of the molecule has the characteristics of aromaticity. Typically, the ring systems contain 5-12 ring member atoms. Preferably the monocyclic heteroaryls contain 5-6 ring members, and the bicyclic heteroaryls contain 8-10 ring members.
[0062] Typical heteroaromatic systems include monocyclic C5-C6 aromatic groups such as, for example, pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, triazole, tetrazole, thiophene, thiazole, isothiazole, thiadiazole, furan, oxazole, isoxazole, oxadiazole, oxathiadiazole, and the fused bicyclic moieties formed by fusing one of these monocyclic groups with a phenyl ring or with any of the heteroaromatic monocyclic groups to form a C8-C10 bicyclic group such as indole, benzimidazole, indazole, benzotriazole, isoquinoline, quinoline, benzothiazole, benzofuran, pyrazolopyridine, quinazoline, quinoxaline, cinnoline, and the like.
[0063] Aryl and heteroaryl moieties may be substituted with a variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of these, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halo, OR, NR2, SR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, C≡CR, COOR, CONR2, OOCR, COR, and NO2, wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and each R is optionally substituted as described above for alkyl groups. The substituent groups on an aryl or heteroaryl group may of course be further substituted with the groups described herein as suitable for each type of such substituents or for each component of the substituent. Thus, for example, an arylalkyl substituent may be substituted on the aryl portion with substituents described herein as typical for aryl groups, and it may be further substituted on the alkyl portion with substituents described herein as typical or suitable for alkyl groups.
[0064] Similarly, "arylalkyl" and "heteroarylalkyl" refer to aromatic and heteroaromatic ring systems which are bonded to their attachment point through a linking group such as an alkylene, including substituted or unsubstituted, saturated or unsaturated, cyclic or acyclic linkers. Typically the linker is C1-C8 alkyl or a hetero form thereof. These linkers may also include a carbonyl group, thus making them able to provide substituents as an acyl or heteroacyl moiety. An aryl or heteroaryl ring in an arylalkyl or heteroarylalkyl group may be substituted with the same substituents described above for aryl groups. Preferably, an arylalkyl group includes a phenyl ring optionally substituted with the groups defined above for aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane. Similarly, a heteroarylalkyl group preferably includes a C5-C6 monocyclic heteroaryl group that is optionally substituted with the groups described above as substituents typical on aryl groups and a C1-C4 alkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl groups or heteroalkyl groups, or it includes an optionally substituted phenyl ring or C5-C6 monocyclic heteroaryl and a C1-C4 heteroalkylene that is unsubstituted or is substituted with one or two C1-C4 alkyl or heteroalkyl groups, where the alkyl or heteroalkyl groups can optionally cyclize to form a ring such as cyclopropane, dioxolane, or oxacyclopentane.
[0065] Where an arylalkyl or heteroarylalkyl group is described as optionally substituted, the substituents may be on either the alkyl or heteroalkyl portion or on the aryl or heteroaryl portion of the group. The substituents optionally present on the alkyl or heteroalkyl portion are the same as those described above for alkyl groups generally; the substituents optionally present on the aryl or heteroaryl portion are the same as those described above for aryl groups generally. •
[0066] "Arylalkyl" groups as used herein are hydrocarbyl groups if they are unsubstituted, and are described by the total number of carbon atoms in the ring and alkylene or similar linker. Thus a benzyl group is a C7-arylalkyl group, and phenylethyl is a C8-arylalkyl.
[0067] "Heteroarylalkyl" as described above refers to a moiety comprising an aryl group that is attached through a linking group, and differs from "arylalkyl" in that at least one ring atom of the aryl moiety or one atom in the linking group is a heteroatom selected from N, O and S. The heteroarylalkyl groups are described herein according to the total number of atoms in the ring and linker combined, and they include aryl groups linked through a heteroalkyl linker; heteroaryl groups linked through a hydrocarbyl linker such as an alkylene; and heteroaryl groups linked through a heteroalkyl linker. Thus, for example, C7-heteroarylalkyl would include pyridylmethyl, phenoxy, and N-pyrrolylmethoxy.
[0068] "Alkylene" as used herein refers to a divalent hydrocarbyl group; because it is divalent, it can link two other groups together. Typically it refers to -(CHa)n- where n is 1-8 and preferably n is 1-4, though where specified, an alkylene can also be substituted by other groups, and can be of other lengths, and the open valences need not be at opposite ends of a chain. Thus -CH(Me)- and -C(Me )2- may also be referred to as alkylenes, as can a cyclic group such as cyclopropan- l,l-diyl. Where an alkylene group is substituted, the substituents include those typically present on alkyl groups as described herein. [0069] In general, any alkyl, alkenyl, alkynyl, acyl, or aryl or arylalkyl group or any heteroform of one of these groups that is contained in a substituent may itself optionally be substituted by additional substituents. The nature of these substituents is similar to those recited with regard to the primary substituents themselves if the substituents are not otherwise described. Thus, where an embodiment of, for example, R7 is alkyl, this alkyl may optionally be substituted by the remaining substituents listed as embodiments for R7 where this makes chemical sense, and where this does not undermine the size limit provided for the alkyl per se; e.g., alkyl substituted by alkyl or by alkenyl would simply extend the upper limit of carbon atoms for these embodiments, and is not included. However, alkyl substituted by aryl, amino, alkoxy, =0, and the like would be included within the scope of the invention, and the atoms of these substituent groups are not counted in the number used to describe the alkyl, alkenyl, etc. group that is being described. Where no number of substituents is specified, each such alkyl, alkenyl, alkynyl, acyl, or aryl group may be substituted with a number of substituents according to its available valences; in particular, any of these groups may be substituted with fluorine atoms at any or all of its available valences, for example.
[0070] "Heteroform" as used herein refers to a derivative of a group such as an alkyl, aryl, or acyl, wherein at least one carbon atom of the designated carbocyclic group has been replaced by a heteroatom selected from N, O and S. Thus the heteroforms of alkyl, alkenyl, alkynyl, acyl, aryl, and arylalkyl are heteroalkyl, heteroalkenyl, heteroalkynyl, heteroacyl, heteroaryl, and heteroarylalkyl, respectively. It is understood that no more than two N, O or S atoms are ordinarily connected sequentially, except where an oxo group is attached to N or S to form a nitro or sulfonyl group.
[0071] "Halo", as used herein includes fluoro, chloro, bromo and iodo. Fluoro and chloro are often preferred.
[0072] "Amino" as used herein refers to NH2, but where an amino is described as "substituted" or "optionally substituted", the term includes NR' R" wherein each R' and R" is independently H, or is an alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl group or a heteroform of one of these groups, and each of the alkyl, alkenyl, alkynyl, acyl, aryl, or arylalkyl groups or heteroforms of one of these groups is optionally substituted with the substituents described herein as suitable for the corresponding group. The term also includes forms wherein R' and R" are linked together to form a 3-8 membered ring which may be saturated, unsaturated or aromatic and which contains 1-3 heteroatoms independently selected from N, O and S as ring members, and which is optionally substituted with the substituents described as suitable for alkyl groups or, if NR'R" is an aromatic group, it is optionally substituted with the substituents described as typical for heteroaryl groups.
[0073] As used herein, the term "carbocycle" refers to a cyclic compound containing only carbon atoms in the ring, whereas a "heterocycle" refers to a cyclic compound comprising a heteroatom. The carbocyclic and heterocyclic structures encompass compounds having monocyclic, bicyclic or multiple ring systems, and may also comprise aryl or heteroaryl rings.
[0074] As used herein, the term "heteroatom" refers to any atom that is not carbon or hydrogen, such as nitrogen, oxygen or sulfur.
[0075] Illustrative examples of heterocycles include but are not limited to pyridine, pyridazine, pyrimidine, pyrazine, triazine, pyran, pyrrole, pyrroline, imidazole, imidazoline, pyrazole, pyrazoline, triazole, tetrazole, thiophene, thiazole, isothiazole, thiadiazole, furan, oxazole, isoxazole, oxadiazole, oxathiadiazole, benzimidazole, benzofuran, isobenzofuran, indole, benzothiazole, benzotriazole, isoquinoline, quinoline, pyrazolopyridine, quinazoline, quinoxaline, cinnoline, tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran, 1 ,3- dihydro-isobenzofuran, 4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one, octahydro-pyrrolo[3,4 b]pyridine, piperazine, morpholine, thiomorpholine, imidazolidine-2,4- dione, l,3-dihydrobenzimidazol-2-one, tetrahydro-thiophene- 1 ,1 -dioxide, diazepine, diazabicyclo[2.2. l]heptane, 2,5 diazabicyclo[2.2.1]heptane, 2,3,4,4a,9,9a hexahydro-l H-β- carboline, oxirane, oxetane, dioxane, aziridine, azetidine, lactones, and lactams.
[0076] As used herein, the term "inorganic substituent" refers to substituents that do not contain carbon or contain carbon bound to elements other than hydrogen (e.g., elemental carbon, carbon monoxide, carbon dioxide, and carbonate). Examples of inorganic substituents include but are not limited to nitro, halogen, azido, cyano, sulfonyls, sulfinyls, sulfonates, phosphates, etc.
[0077] The terms "treat" and "treating" as used herein refer to ameliorating, alleviating, lessening, and removing symptoms of a disease or condition. A candidate molecule or compound described herein may be in a therapeutically effective amount in a formulation or medicament, which is an amount that can lead to a biological effect, such as apoptosis of certain cells (e.g., cancer cells), reduction of proliferation of certain cells, or lead to ameliorating, alleviating, lessening, or removing symptoms of a disease or condition, for example. The terms also can refer to reducing or stopping a cell proliferation rate (e.g., slowing or halting tumor growth) or reducing the number of proliferating cancer cells (e.g., removing part or all of a tumor). These terms also are applicable to reducing a titre of a microorganism in a system (i.e., cell, tissue, or subject) infected with a microorganism, reducing the rate of microbial propagation, reducing the number of symptoms or an effect of a symptom associated with the microbial infection, and/or removing detectable amounts of the microbe from the system. Examples of microorganism include but are not limited to virus, bacterium and fungus.
[0078] As used herein, the term "apoptosis" refers to an intrinsic cell self-destruction or suicide program. In response to a triggering stimulus, cells undergo a cascade of events including cell shrinkage, blebbing of cell membranes and chromatic condensation and fragmentation. These events culminate in cell conversion to clusters of membrane-bound particles (apoptotic bodies), which are thereafter engulfed by macrophages.
[0079] The invention in part provides pharmaceutical compositions comprising at least one compound within the scope of the invention as described herein, and methods of using compounds described herein. For example, the invention in part provides methods for identifying a candidate molecule that interacts with a CK2 protein, which comprises contacting a composition containing a CK2 protein and a molecule described herein with a candidate molecule and determining whether the amount of the molecule described herein that interacts with the protein is modulated, whereby a candidate molecule that modulates the amount of the molecule described herein that interacts with the protein is identified as a candidate molecule that interacts with the protein. In other embodiments, the invention provides methods for identifying a candidate molecule that interacts with a Pirn or Fit protein, which comprises contacting a composition containing a Pirn or Fit protein and a molecule described herein with a candidate molecule and determining whether the amount of the molecule described herein that interacts with the protein is modulated, whereby a candidate molecule that modulates the amount of the molecule described herein that interacts with the protein is identified as a candidate molecule that interacts with the protein.
[0080] Also provided are methods for modulating the activity of a CK2 protein, which comprises contacting a system comprising the protein with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein. The system in such embodiments can be a cell-free system or a system comprising cells. Also provided are methods for reducing cell proliferation, and optionally inducing apoptosis, which comprises contacting cells with a compound described herein in an amount effective to reduce proliferation of the cells. The cells in such embodiments can be in a cell line, in a tissue or in a subject (e.g., a research animal or human). In related embodiments, provided are compositions comprising a compound described herein in combination with a protein or cell, such as an isolated protein (e.g., isolated CK2 or other serine-threonine protein kinase protein) or a cell in a cell line (e.g., HCT-1 16 cell line).
[0081] Provided also are methods for modulating a serine-threonine protein kinase activity. Serine-threonine protein kinases catalyze the transfer of a gamma phosphate from adenosine triphosphate to a serine or threonine amino acid in a peptide or protein substrate. Thus, included herein are methods which comprise contacting a system comprising a serine-threonine protein kinase protein with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein. In some embodiments, the activity of the serine-threonine protein kinase is the catalytic activity of the protein (e.g., catalyzing the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate). In certain embodiments, provided are methods for identifying a candidate molecule that interacts with a serine-threonine protein kinase, which comprise: contacting a composition containing a serine- threonine protein kinase and a compound described herein with a candidate molecule under conditions in which the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein. Systems in such embodiments can be a cell-free system or a system comprising cells (e.g., in vitro). The protein, the compound or the molecule in some embodiments is in association with a solid phase. In certain embodiments, the interaction between the compound and the protein is detected via a detectable label, where in some embodiments the protein comprises a detectable label and in certain embodiments the compound comprises a detectable label. The interaction between the compound and the protein sometimes is detected without a detectable label. [0082] Provided also are methods for modulating a protein kinase activity. Protein kinases catalyze the transfer of a gamma phosphate from adenosine triphosphate to a serine or threonine amino acid (serine/threonine protein kinase), tyrosine amino acid (tyrosine protein kinase), tyrosine, serine or threonine (dual specificity protein kinase) or histidine amino acid (histidine protein kinase) in a peptide or protein substrate. Thus, included herein are methods which comprise contacting a system comprising a protein kinase protein with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein kinase. In some embodiments, the activity of the protein kinase is the catalytic activity of the protein (e.g., catalyzing the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate). In certain embodiments, provided are methods for identifying a candidate molecule that interacts with a protein kinase, which comprise: contacting a composition containing a protein kinase and a compound described herein with a candidate molecule under conditions in which the compound and the protein kinase interact, and determining whether the amount of the compound that interacts with the protein kinase is modulated relative to a control interaction between the compound and the protein kinase without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein kinase relative to the control interaction is identified as a candidate molecule that interacts with the protein kinase. Systems in such embodiments can be a cell-free system or a system comprising cells (e.g., in vitro). The protein kinase, the compound or the molecule in some embodiments is in association with a solid phase. In certain embodiments, the interaction between the compound and the protein kinase is detected via a detectable label, where in some embodiments the protein kinase comprises a detectable label and in certain embodiments the compound comprises a detectable label. The interaction between the compound and the protein kinase sometimes is detected without a detectable label.
[0083] Provided also are compositions of matter comprising a protein kinase and a compound described herein. In some embodiments, the protein kinase in the composition is a serine-threonine protein kinase or a tyrosine protein kinase. In certain embodiments, the protein kinase is a protein kinase fragment having compound-binding activity. In some embodiments, the protein kinase in the composition is, or contains a subunit (e.g., catalytic subunit, SH2 domain, SH3 domain) of, CK2, Pirn subfamily protein kinase (e.g., PDvIl, PIM2, PDvO) or Fit subfamily protein kinase (e.g, FLTl , FLT3, FLT4). In certain embodiments the composition is cell free and sometimes the protein kinase is a recombinant protein. The protein kinase can be from any source, such as cells from a mammal, ape or human, for example. In certain embodiments, the protein kinase is a human protein kinase.
[0084] The serine-threonine protein kinase can be from any source, such as a mammal, ape or human, for example. Examples of serine-threonine protein kinases that may be inhibited by compounds disclosed herein include without limitation human versions of CK2, CK2α2, Pirn subfamily kinases (e.g., PIMl, PIM2, PIM3), CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2, ZIPK and ZIPK. A serine-threonine protein kinase sometimes is a member of a subfamily containing one or more of the following amino acids at positions corresponding to those listed in human CK2: leucine at position 45, methionine at position 163 and isoleucine at position 174. Examples of such protein kinases include without limitation human versions of CK2, STKlO, HIPK2, HIPK3, DAPK3, DYK2 and PIM- 1. Examples of tyrosine protein kinases that can be inhibited, or may potentially be inhibited, by compounds disclosed herein include without limitation human versions of Fit subfamily members (e.g., FLTl , FLT2, FLT3, FLT3 (D835Y), FLT4). An example of a dual specificity protein kinase that can be inhibited, or may potentially be inhibited, by compounds disclosed herein includes without limitation DYRK2. Nucleotide and amino acid sequences for serine-threonine protein kinases and reagents are publicly available (e.g., World Wide Web URLs ncbi.nlm.nih.gov/sites/entrez/ and Invitrogen.com). For example, various nucleotide sequences can be accessed using the following accession numbers: NM_002648.2 and NP_002639.1 for PIMl ; NM_006875.2 and NP_006866.2 for PIM2; XM_938171.2 and XP_943264.2 for PIM3; NM_0041 19.2 and NP_0041 10.2 for FLT3; NM_002020.3 and NP_00201 1.2 for FLT4; and NM_002019.3 and NP_002010.2 for FLTl.
[0085] The invention also in part provides methods for treating a condition related to aberrant cell proliferation. For example, provided are methods of treating a cell proliferative condition in a subject, which comprises administering a compound described herein to a subject in need thereof in an amount effective to treat the cell proliferative condition. The subject may be a research animal (e.g., rodent, dog, cat, monkey), optionally containing a tumor such as a xenograft tumor (e.g., human tumor), for example, or may be a human. A cell proliferative condition sometimes is a tumor or non-tumor cancer, including but not limited to, cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart (e.g., leukemia, lymphoma, carcinoma).
[0086] Also provided are methods for treating a condition related to inflammation or pain. For example, provided are methods of treating pain in a subject, which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the pain. Provided also are methods of treating inflammation in a subject, which comprises administering a compound described herein to a subject in need thereof in an amount effective to treat the inflammation. The subject may be a research animal (e.g., rodent, dog, cat, monkey), for example, or may be a human. Conditions associated with inflammation and pain include without limitation acid reflux, heartburn, acne, allergies and sensitivities, Alzheimer's disease, asthma, atherosclerosis, bronchitis, carditis, celiac disease, chronic pain, Crohn's disease, cirrhosis, colitis, dementia, dermatitis, diabetes, dry eyes, edema, emphysema, eczema, fibromyalgia, gastroenteritis, gingivitis, heart disease, hepatitis, high blood pressure, insulin resistance, interstitial cystitis, joint pain/arthritis/rheumatoid arthritis, metabolic syndrome (syndrome X), myositis, nephritis, obesity, osteopenia, osteoporosis, Parkinson's disease, periodontal disease, polyarteritis, polychondritis, psoriasis, scleroderma, sinusitis, Sjogren's syndrome, spastic colon, systemic candidiasis, tendonitis, urinary track infections, vaginitis, inflammatory cancer (e.g., inflammatory breast cancer) and the like. Methods for determining effects of compounds herein on pain or inflammation are known. For example, formalin- stimulated pain behaviors in research animals can be monitored after administration of a compound described herein to assess treatment of pain (e.g., Li et ai, Pain 115(1-2): 182-90 (2005)). Also, modulation of pro-inflammatory molecules (e.g., IL-8, GRO-alpha, MCP-I , TNFalpha and iNOS) can be monitored after administration of a compound described herein to assess treatment of inflammation (e.g., Parhar et ai, Int J Colorectal Dis. 22(6): 601-9 (2006)), for example. Thus, also provided are methods for determining whether a compound herein reduces inflammation or pain, which comprise contacting a system with a compound described herein in an amount effective for modulating (e.g., inhibiting) the activity of a pain signal or inflammation signal. Provided also are methods for identifying a compound that reduces inflammation or pain, which comprise: contacting a system with a compound of Formula (I), (II), (III), (FV), (V) or (VI); and detecting a pain signal or inflammation signal, whereby a compound that modulates the pain signal relative to a control molecule is identified as a compound that reduces inflammation of pain. Non-limiting examples of pain signals are formalin-stimulated pain behaviors and examples of inflammation signals include without limitation a level of a pro-inflammatory molecule.
[0087] The invention also in part pertains to methods for modulating angiogenesis in a subject, and methods for treating a condition associated with aberrant angiogenesis in a subject. Thus, provided are methods for determining whether a compound herein modulates angiogenesis, which comprise contacting a system with a compound described herein in an amount effective for modulating (e.g., inhibiting) angiogenesis or a signal associated with angiogenesis. Signals associated with angiogenesis are levels of a pro-angiogenesis growth factor such as VEGF. Methods for assessing modulation of angiogenesis also are known, such as analyzing human endothelial tube formation (BD BioCoat™ Angiogenesis System from BD Biosciences). Provided also are methods for identifying a compound that modulates angiogenesis, which comprise contacting a system with a compound of Formula (I), (II), (III), (IV), (V) or (VI) or a pharmaceutically acceptable salt thereof; and detecting angiogenesis in the system or an angiogenesis signal, whereby a compound that modulates the angiogenesis or angiogenesis signal relative to a control molecule is identified as a compound that modulates angiogenesis. Also provided are methods for treating an angiogenesis condition, which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the angiogenesis condition. Angiogenesis conditions include without limitation solid tumor cancers, varicose disease and the like.
[0088] The invention also in part pertains to methods for modulating an immune response in a subject, and methods for treating a condition associated with an aberrant immune response in a subject. Thus, provided are methods for determining whether a compound herein modulates an immune response, which comprise contacting a system with a compound described herein in an amount effective for modulating (e.g., inhibiting) an immune response or a signal associated with an immune response. Signals associated with immunomodulatory activity include, e.g., stimulation of T-cell proliferation, suppression or induction of cytokines, including, e.g., interleukins, interferon-γand TNF. Methods of assessing immunomodulatory activity are known in the art. Provided also are methods for identifying a compound that modulates an immune response, which comprise contacting a system with a compound of Formula (I), (II), (III), (IV), (V) or (VI) or a pharmaceutically acceptable salt thereof; and detecting immunomodulatory activity in a system, or a signal associated with immunomodulatory activity, whereby a compound that modulates the immune response relative to a control molecule is identified as an immune response modulatory compound. Also provided are methods for treating a condition associated with an aberrant immune response in a subject, which comprise administering a compound described herein to a subject in need thereof in an amount effective to treat the condition. Conditions characterized by an aberrant immune response include without limitation, organ transplant rejection, asthma, autoimmune disorders, including rheumatoid arthritis, multiple sclerosis, myasthenia gravis, systemic lupus erythematosus, Crohn's disease, and ulcerative colitis. In certain embodiments, an immune response may be modulated by administering a compound herein in combination with a molecule that modulates (e.g., inhibits) the biological activity of an mTOR pathway member or member of a related pathway (e.g., mTOR, PI3 kinase, AKT). In certain embodiments the molecule that modulates the biological activity of an mTOR pathway member or member of a related pathway is rapamycin. In certain embodiments, provided herein is a composition comprising a compound described herein in combination with a molecule that modulates the biological activity of an mTOR pathway member or member of a related pathway, such as rapamycin, for example.
[0089] In preferred embodiments of the present invention, the compound is a compound of Formula (I), (II), (III), (IV), (V) or (VI) in one of the Tables provided herein, or a pharmaceutically acceptable salt of one of these compounds.
[0090] Any suitable formulation of a compound described above can be prepared for administration. Any suitable route of administration may be used, including, but not limited to, oral, parenteral, intravenous, intramuscular, transdermal, topical and subcutaneous routes. Depending on the subject to be treated, the mode of administration, and the type of treatment desired — e.g., prevention, prophylaxis, therapy; the compounds are formulated in ways consonant with these parameters. Preparation of suitable formulations for each route of administration are known in the art. A summary of such formulation methods and techniques is found in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co., Easton, PA, which is incorporated herein by reference. The formulation of each substance or of the combination of two substances will generally include a diluent as well as, in some cases, adjuvants, buffers, preservatives and the like. The substances to be administered can be administered also in liposomal compositions or as microemulsions. [0091] For injection, formulations can be prepared in conventional forms as liquid solutions or suspensions or as solid forms suitable for solution or suspension in liquid prior to injection or as emulsions. Suitable excipients include, for example, water, saline, dextrose, glycerol and the like. Such compositions may also contain amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, such as, for example, sodium acetate, sorbitan monolaurate, and so forth.
[0092] Various sustained release systems for drugs have also been devised, and can be applied to compounds of the invention. See, for example, U.S. patent No. 5,624,677, the methods of which are incorporated herein by reference.
[0093] Systemic administration may also include relatively noninvasive methods such as the use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral administration is also suitable for compounds of the invention. Suitable forms include syrups, capsules, tablets, as is understood in the art.
[0094] For administration to animal or human subjects, the appropriate dosage of a compound described above often is 0.01-150 mg/kg, and sometimes 0.1-100 mg/kg. Dosage levels are dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration; however, optimization of such parameters is within the ordinary level of skill in the art.
Therapeutic Combinations
[0095] Compounds of the invention may be used alone or in combination with another therapeutic agent. The invention provides methods to treat conditions such as cancer and inflammation by administering to a subject in need of such treatment a therapeutically effective amount of a therapeutic agent that binds to certain DNA segments and administering to the same subject a CK2 modulator in an amount that is effective to enhance the activity of the therapeutic agent. A CK2 modulator is an agent that inhibits or enhances a biological activity of a CK2 protein, and is generically referred to hereafter as a "modulator." A Pirn or Fit modulator is an agent that inhibits or enhances a biological activity of a Pirn protein or a FH protein. The therapeutic agent and the modulator may be administered together, either as separate pharmaceutical compositions or admixed in a single pharmaceutical composition. The therapeutic agent and the modulator may also be administered separately, including at different times and with different frequencies, as long as the modulator is administered at a time that increases the potency of the therapeutic agent. The modulator may be administered by any known route, such as orally, intravenously, intramuscularly, nasally, and the like; and the therapeutic agent may also be administered by any conventional route. In many embodiments, at least one and optionally both of the modulator and the therapeutic agent may be administered orally.
[0096] In some embodiments, the modulator and the therapeutic agent are administered at the same time, whether in separate dosages or admixed in a single dosage. Where the frequency of administration of the two materials can be adjusted to match, the modulator and therapeutic agent are preferably combined into a single pharmaceutical composition, so the treated patient may receive a single oral dosage or a single injection, for example.
[0097] The amount of each of these materials to be administered will vary with the route of administration, the condition of the subject, other treatments being administered to the subject, and other parameters. The therapeutic agents of the invention may, of course, cause multiple desired effects; and the amount of modulator to be used in combination with the therapeutic agent should be an amount that increases one or more of these desired effects. The modulator is to be administered in an amount that is effective to enhance a desired effect of the therapeutic agent. An amount is "effective to enhance a desired effect of the therapeutic agent", as used herein, if it increases by at least about 25% at least one of the desired effects of the therapeutic agent alone. Preferably, it is an amount that increases a desired effect of the therapeutic agent by at least 50% or by at least 100% (i.e., it doubles the effective activity of the therapeutic agent.) In some embodiments, it is an amount that increases a desired effect of the therapeutic agent by at least 200%.
[0098] The amount of a modulator that increases a desired effect of a therapeutic agent may be determined using in vitro methods, such as cell proliferation assays. The therapeutic agents of the invention are useful to counter hyperproliferative disorders such as cancer, thus they reduce cell proliferation. Thus, for example, a suitable amount of a modulator could be the amount needed to enhance an antiproliferative effect of a therapeutic agent by at least 25% as determined in a cell proliferation assay.
[0100] The modulator used in the present invention enhances at least one desired effect produced by the therapeutic agent it is used with, thus the combinations of the inveniton provide a synergistic effect, not merely an additive effect. The modulators themselves are at times useful for treating the same types of conditons, and thus may also have some direct effect in such assays. In that event, the "amount effective to increase a desired effect" must be a synergistic enhancement of the activity of the therapeutic agent that is attributable to enhancement by the modulator of an effect of the therapeutic agent, rather than a simple additive effect that would be expected with separate administration of the two materials. In many cases, the modulator can be used in an amount (concentration) that would not be expected to have any apparent effect on the treated subject or the in vitro assay, so the increased effect achieved with the combination is directly attributable to a synergistic effect.
[0101] The present invention includes methods and compositions for treating a patient having a cell proliferation disorder or an inflammatory disorder with a therapeutic agent as described herein, and a "modulator" described above, where the timing of administration of the modulator permits it to enhance a desired effect of the therapeutic agent .
[0102] In certain embodiments, a "modulator" as described above may be used in combination with a therapeutic agent that can act by binding to regions of DNA that can form certain quadruplex structures. In such embodiments, the therapeutic agents have anticancer activity on their own, but their activity is enhanced when they are used in combination with a modulator. This synergistic effect allows the therapeutic agent to be administered in a lower dosage while achieving equivalent or higher levels of at least one desired effect.
[0103] For administration to animal or human subjects, the appropriate dosage of a modulator, such as a compound of Formula (I), (II), (III), (IV), (V), or (VI) as described herein, is typically between about 0.01-15 mg/kg, and about 0.1-10 mg/kg. Dosage levels are dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration; however, optimization of such parameters is within the ordinary level of skill in the art.
[0104] A modulator may be separately active for treating a cancer. For combination therapies described above, when used in combination with a therapeutic agent, the dosage of a modulator will frequently be two-fold to ten-fold lower than the dosage required when the modulator is used alone to treat the same condition or subject. Determination of a suitable amount of the modulator for use in combination with a therapeutic agent is readily determined by methods known in the art. [0105] The therapeutic agents of the invention are compounds that bind to certain motifs in nucleic acids. The therapeutic agent to be used can be selected from several different classes of compounds, such as those that bind to quadruplex-forming regions of DNA. The therapeutic agents are useful for the treatment of cancer and other indications such as inflammatory disorders, and methods for making and using them are known in the art. Several preferred classes of these therapeutic agents are described below. Each class of therapeutic agents can be used in combination with any active CK modulator, including but not limited to those disclosed herein.
[0106] In one aspect, the therapeutic agent can be a compound of formula (TAl-I):
Figure imgf000036_0001
and pharmaceutically acceptable salts, esters and prodrugs thereof; wherein V is H, halo, or NR1R2;
A is H, fluoro, or NR1^;
Z is O, S, NR1 or CH2;
U iS OR2 Or NR1R2;
X is OR2, NR1R2, halo, azido, or SR2; n is 1-3; wherein in NR1R2, R1 and R2 may form a double bond or a ring, each of which is optionally substituted;
R1 is H or a Ci.6 alkyl;
R2 is H or a CM0 alkyl or C2.10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R2 is an optionally substituted heterocyclic ring, aryl or heteroaryl; R5 is a substituent at any position on W; and is H, OR2, C).6 alkyl, C2-6 alkenyl, each optionally substituted by halo, =0 or one or more heteroatoms; or R5 is an inorganic substituent; and
W is an optionally substituted aryl or heteroaryl, which may be monocyclic or fused with a single or multiple ring and optionally containing a heteroatom; or a compound having formula (TA 1-2):
Figure imgf000037_0001
wherein V, A, X, Z and U are as defined in formula TAl-I, and W is selected from the group consisting of
Figure imgf000037_0002
Figure imgf000038_0001
wherein Q, Q1, Q2, and Q3 are independently CH or N; Y is independently O, CH, =0 or NfR1 ; and R5 is as defined in formula 1.
[0107] Compounds of this structure, and methods for making and using them, are described in U.S. Patent Application Serial No. 1 1/106,909, to Whitten, et al., which is entitled SUBSTITUTED QUINOBENZOXAZINE ANALOGS AND METHODS OF USING THEREOF, and was filed on April 15, 2005.
[0108] In a specific embodiment of the therapeutic agents of formula (TA 1 - 1 ), the therapeutic agent is a compound having formula (TAl- IA):
Figure imgf000039_0001
or a pharmaceutically acceptable salt, esters or prodrug thereof, or a specific isomer or mixture of isomers thereof.
[0109] In another aspect, the therapeutic agent of the combinations of the invention is a compound of this formula:
Figure imgf000039_0002
and pharmaceutically acceptable salts, esters and prodrugs thereof; wherein V is H, halo, or NR1R2; AisH, fluoro, orNR'2; Z is O, S, NR1OrCH2; UiSOR2OrNR1R2; X is OR2, NR1R2, halo, azido, or SR2; n is 1-3; wherein in NR1R2, R1 and R2 may form a double bond or a ring, each of which is optionally substituted;
R1 isHoraC,.6alkyl; R is H or a C|.ιo alkyl or C2-io alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R2 is an optionally substituted heterocyclic ring, aryl or heteroaryl;
R5 is a substituent at any position on W; and is H, OR2,
Figure imgf000040_0001
alkyl, C2.6 alkenyl, each optionally substituted by halo, =O or one or more heteroatoms; or R5 is an inorganic substituent; and
W is an optionally substituted aryl or heteroaryl, which may be monocyclic or fused with a single or multiple ring and optionally containing a heteroatom; or a compound having formula (TA3-2)
Figure imgf000040_0002
wherein V, A, X, Z and U are as defined in formula 1 , and W is selected from the group consisting of
Figure imgf000040_0003
Figure imgf000041_0001
wherein Q, Q1, Q2, and Q3 are independently CH or N; Y is independently O, CH, =0 or NR1 ; and R5 is as defined in formula 1.
[0110] The preparation and activity of these compounds of formula (TA3-1) are described in U.S. Patent Application Serial No. 60/81 1,992, filed June 8, 2006, to Nagasawa, et al., entitled QUINOLONE ANALOGS DERIV ATIZED WITH SULFONIC ACID, SULFONATE OR SULFONAMIDE.
[0111] In another aspect, the therapeutic agent of the combinations of the invention is a compound of this formula:
Figure imgf000042_0001
and pharmaceutically acceptable salts, esters and prodrugs thereof; wherein B, X, A, or V is absent if Z2, Z3, or Z4, respectively, is N , and independently H, halo, azido, R2, CH2R2, SR2, OR2 or NR1R2 if Z2, Z3, or Z4, respectively, is C; or
A and V, A and X, or X and B may form a carbocyclic ring, heterocyclic ring, aryl or heteroaryl, each of which may be optionally substituted and/or fused with a cyclic ring;
Z is O, S, NR1, CH2, or C=O;
Z1, Z2, Z3 and Z4 are C or N, provided any two N are non-adjacent;
W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused to an optionally substituted saturated or unsaturated ring; said saturated or unsaturated ring may contain a heteroatom and is monocyclic or fused with a single or multiple carbocyclic or heterocyclic rings;
U is R2, OR2, NR1R2, NR1 - (CR'2)n - NR3R4, or N=CR1R2, wherein in N=CR1R2 R1 and R2 together with C may form a ring, provided U is not H, and when U is OH, OR2 or NH2, then at least one of Z'-Z4 is N; in each NR1R2, R1 and R2 together with N may form an optionally substituted ring; in NR3R4, R3 and R4 together with N may form an optionally substituted ring; R1 and R3 are independently H or Cι_6 alkyl; each R2 is H, or a Ci-I0 alkyl or C2.io alkenyl each optionally substituted with a halogen, one or more non-adjacent heteroatoms, a carbocyclic ring, a heterocyclic ring, an aryl or heteroaryl, wherein each ring is optionally substituted; or R" is an optionally substituted carbocyclic ring, heterocyclic ring, aryl or heteroaryl;
R4 is H, a C]- 1O alkyl or C2.io alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R3 and R4 together with N may form an optionally substituted ring; • each R5 is a substituent at any position on ring W; and is H, OR2, amino, alkoxy, amido, halogen, cyano or an inorganic substituent; or R5 is Cι_6 alkyl, C2.6 alkenyl, C2-6 alkynyl, -CONHR1, each optionally substituted by halo, carbonyl or one or more non-adjacent heteroatoms; or two adjacent R5 are linked to obtain a 5-6 membered optionally substituted carbocyclic or heterocyclic ring that may be fused to an additional optionally substituted carbocyclic or heterocyclic ring; and n is 1-6.
[0112] In the above formula (TA4-1), B may be absent when Z1 is N, or is H or a halogen when Z1 is C.
[0113] In the above formula (TA4-1), W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused to an optionally substituted aryl or heteroaryl selected from the group consisting of:
Figure imgf000043_0001
Figure imgf000044_0001
wherein each Q, Q1, Q2, and Q3 is independently CH or N; Y is independently O, CH, C=O or NR1 ; n and R5 is as defined above.
[0114] In other embodiments, W together with N and Z form a group having the formula selected from the group consisting of
Figure imgf000044_0002
wherein Z is O, S, CR1, NR1, or C=O; each Z5 is CR6, NR1, or C=O, provided Z and Z5 if adjacent are not both NR1; each R1 is H, Ci.6 alkyl, COR2 or S(O)PR2 wherein p is 1-2;
R6 is H, or a substituent known in the art, including but not limited to hydroxyl, alkyl, alkoxy, halo, amino, or amido; and ring S and ring T may be saturated or unsaturated.
[0115] In some embodiments, W together with N and Z forms a 5- or 6-membered ring that is fused to a phenyl. In other embodiments, W together with N and Z forms a 5- or 6-membered ring that is optionally fused to another ring, when U is NR1R2, provided U is not NH2. In certain embodiments, W together with N and Z forms a 5- or 6-membered ring that is not fused to another ring, when U is NR1R2 (e.g., NH2).
[0116] In yet another embodiment, the compounds of the present invention have the general formula (TA4-2A) or (TA4-2B):
Figure imgf000045_0001
wherein A, B, V, X, U, Z, Z1, Z2, Z3, Z4 and n are as described for TA4-1 ;
Z5 is O, NR1, CR6, or C=O;
R6 is H, Ci-6 alkyl, hydroxyl, alkoxy, halo, amino or amido; and
Z and Z5 may optionally form a double bond.
[0117] In the above formula (TA4-1), (TA4-2A) and (TA4-2B), U may be NR l1rR> 2', wherein R1 is H, and R2 is a CM0 alkyl optionally substituted with a heteroatom, a C3.6 cycloalkyl, aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S. For example, R2 may be a Ci-I0 alkyl substituted with an optionally substituted morpholine, thiomorpholine, imidazole, aminodithiadazole, pyrrolidine, piperazine, pyridine or piperidine. In other examples, R1 and R2 together with N form an optionally substituted piperidine, pyrrolidine, piperazine, morpholine, thiomorpholine, imidazole, or aminodithiazole.
[0118] The compounds of formula (TA4-1), and methods of making and using them, are described in U.S. Patent Application No. 1 1/228,636, to Whitten, et al., entitled QUINOLONE ANALOGS, and filed on September 16, 2005.
[0119] In yet another aspect, the therapeutic agent to be combined with a CK modulator can be selected from compounds having this formula:
Figure imgf000046_0001
and pharmaceutically acceptable salts, esters and prodrugs thereof; wherein V, X, and Y are absent if attached to a heteroatom other than Nitrogen, and independently H, halo, azido, R2, CH2R2, SR2, OR2 or NR1R2 when attached to C or N; or wherein V and X, or X and Y may form a carbocyclic ring, heterocyclic ring, aryl or hdteroaryl, each of which may be optionally substituted and/or fused with a cyclic ring;
Z1, Z2 and Z3 are C, N, O or S;
Z is O, S, NR2, CH2 or C=O;
W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused to an optionally substituted aryl or heteroaryl, wherein said aryl or heteroaryl may be monocyclic or fused with a single or multiple ring, and wherein said ring optionally contains a heteroatom;
U is -C(=O)R2, -COOR2, -CONR1R2, -CONR1 - (CR'2)n - NR3R4, SO3R2, SO2NR1R2, SO2NR1NR1R2, SO2NR1OR2, SO2NRl-(CR1 2)n-NR3R4 or SO2NR1NR1^CR1 S)n-NR3R4 or SO2NR'-O-(CR1 2)n-NR3R; wherein in each NR1R2, R1 and R2 together with N may form an optionally substituted ring; in NR3R4, R3 and R4 together with N may form an optionally substituted ring;
R1 and R3 are independently H or C|.6 alkyl; each R2 is H, or a CMO alkyl or C2-10 alkenyl each optionally substituted with a halogen, one or more non-adjacent heteroatoms selected from N, O and S, a carbocyclic ring, a heterocyclic ring, an aryl or heteroaryl, wherein each ring is optionally substituted; or R" is an optionally substituted carbocyclic ring, heterocyclic ring, aryl or heteroaryl; or R2 is COR1 or S(O)xR1 wherein x is 1-2;
R4 is H, a Ct-io alkyl or C2.ιo alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O and S, and optionally substituted with a carbocyclic or heterocyclic ring; or R3 and R4 together with N may form an optionally substituted ring; each R5 is a substituent at any position on W; and is H, OR2, amino, alkoxy, amido, halogen, cyano or an inorganic substituent; or R5 is Cι_δ alkyl, C2-6 alkenyl, -CONHR1, each optionally substituted by halo, carbonyl or one or more non-adjacent heteroatoms; or two adjacent R5 are linked to obtain a 5-6 membered optionally substituted carbocyclic or heterocyclic ring, optionally fused to an additional optionally substituted carbocyclic or heterocyclic ring; and n is 1-6.
[0120] In the above formula (TA5- 1), ring T may form an optionally substituted 5- membered ring selected from the group consisting of:
Figure imgf000048_0002
Figure imgf000048_0001
Figure imgf000048_0003
Figure imgf000048_0004
[0121] In the above formula (TA5-1), W together with N and Z may form an optionally substituted 5- or 6-membered aryl or heteroaryl ring that is fused to an optionally substituted aryl or heteroaryl selected from the group consisting of:
Figure imgf000049_0001
Figure imgf000050_0001
wherein each Q, Q1, Q2, and Q3 is independently CH or N; P is independently O, CH, C=O or NR1 ; n and R5 is as defined above.
[0122] In other embodiments of these compounds, W together with N and Z may form a group having the formula selected from the group consisting of
Figure imgf000050_0002
wherein Z is O1 S, NR2, CH2 or C=O; each Z4 is CR6, NR2, or C=O;
R6 is H, or a substituent known in the art, including but not limited to hydroxyl, alky], alkoxy, halo, amino, or amido; and
Ring S and M may be saturated or unsaturated.
[0123] In some embodiments, W together with N and Z may form a 5- or 6-membered ring that is fused to a phenyl.
[0124] In yet another embodiment, the compounds of the present invention have the general formula (TA5-2A) or (TA5-2B):
Figure imgf000051_0001
wherein U, V, W, X, Y, Z, Z\ Z2, Z3, R5 and n are as described above for TA5-1 ;
Z4 is CR6, NR2, or C=O; and
Z and Z4 may optionally form a double bond.
[0125] In the above formula (TA5-1), (TA5-2A) and (TA5-2B), U may be SO2NR1R2, wherein R1 is H, and R2 is a C|. io alkyl optionally substituted with a heteroatom, a C3.6 cycloalkyl, aryl or a 5-14 membered heterocyclic ring containing one or more N, O or S. For example, R2 may be a C].10 alkyl substituted with an optionally substituted morpholine, thiomoφholine, imidazole, aminodithiadazole, pyrrolidine, piperazine, pyridine or piperidine. In other examples, R1 and R2 together with N form an optionally substituted piperidine, pyrrolidine, piperazine, morpholine, thiomoφholine, imidazole, or aminodithiazole.
[0126] In other embodiments of these compounds, U is SO2NR'-(CR'2)n-NR3R4; n is 1-4; each R1 is H or alkyl; and R3 and R4 in NR3R4 together form an optionally substituted piperidine, pyrrolidine, piperazine, moφholine, thiomoφholine, imidazole, or aminodithiazole. In some examples, U is SO2NH-(CH2)n-NR3R4 wherein R3 and R4 together with N form an optionally substituted pyrrolidine, which may be linked to (CH2)n at any position in the pyrrolidine ring. In one embodiment, R3 and R4 together with N form an N-methyl substituted pyrrolidine.
[0127] In one embodiment, the present invention provides compounds having formula (TA5-1), (TA5-2A) or (TA5-2B), wherein: each of V and Y if present is independently H or halogen (e.g., chloro or fluoro);
X is -(ROR1R', wherein R3 is C or N and wherein in each -(ROR 11DR2" , R1 and R' together may form an optionally substituted aryl or heteroaryl ring; Z is NH or N-alkyl (e.g., N-CH3); W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused with an optionally substituted aryl or heteroaryl ring; and
U is -SO2R5R6-(CH2)n-CHR2-NR3R4, wherein R5 is CR1 or N; R1 is H or alkyl; R6 is H or C|.ιo alkyl and wherein in the -CHR2-NR3R4 moiety each R3 or R4 together with the C may form an optionally substituted heterocyclic or heteroaryl ring, or wherein in the -CHR2-NR3R4 moiety each R3 or R4 together with the N may form an optionally substituted carbocyclic, heterocyclic, aryl or heteroaryl ring.
[0128] In another embodiment, the present invention provides compounds having formula (TA5- 1), (TA5-2A) or (TA5-2B), wherein:
V and Y if present is H or halogen (e.g., chloro or fluoro);
X if present is -(R5)R:R2, wherein R5 is C or N and wherein in each -(R5)R!R2 , R1 and R2 together may form an optionally substituted aryl or heteroaryl ring;
Z is NH or N-alkyl (e.g., N-CH3);
W together with N and Z forms an optionally substituted 5- or 6-membered ring that is fused with an optionally substituted aryl or heteroaryl ring; and
U is -SO2R5R6-(CH2)n-CHR2-NR3R4,
R5 is CR1 or N;
R6 is H or alkyl and wherein in the -CHR2-NR3R4 moiety each R3 or R4 together with the C may form an optionally substituted heterocyclic or heteroaryl ring, or wherein in the -CHR2- NR3R4 moiety each R3 or R4 together with the N may form an optionally substituted carbocyclic, heterocyclic, aryl or heteroaryl ring.
[0129] In yet another embodiment, the compounds of the present invention have the general formula (TA5-3):
Figure imgf000053_0001
wherein U, V, X, Y, Z, Z , 11,
Figure imgf000053_0002
Z\ R3 and n are as described above. [0130] In yet another embodiment, the compounds of the present invention have the general formula (TA5-4A) or (TA5-4B):
Figure imgf000053_0003
wherein U, V, X, Z, R and n are as described above for TA5-1.
[0131] Compounds of Formula (TA5-1 ), and methods for making and using them, are described in U.S. Patent Application Serial No. 60/81 1 ,990, to Pierre, et al., entitled PYREDINONE ANALOGS, which was filed June 8, 2006, and in U.S. Provisional Patent Application to Nagasawa, et al., filed on March 1 , 2007, having attorney docket no. 53223- 3003001.
[0132] In still another aspect, the therapeutic agent for the combinations of the invention can be a compound of the formula:
Figure imgf000054_0001
and pharmaceutically acceptable salts, esters and prodrugs thereof, wherein X is H, OR", NR1R', halogen, azido, SR' or CH2R;
A is H, halogen, NR1R', SR', OR", CH2R", azido or NR1 - (CR 2)n - NRJR4
Z is O, S, NR1 or CH2;
U is R", OR", NR > 11πR> 2' or NR1 - (CR'2)n - NR 3JπR> 44 . provided U is not H;
W is an optionally substituted aryl or heteroaryl, which may be monocyclic or fused with a single or multiple ring optionally containing a heteroatom; wherein R1 and R2 together with N in NR1R2, and R3 and R4 together with N in NR3R4 may independently form an optionally substituted 5-6 membered ring containing N, and optionally O or S;
R1 and R3 are independently H or a C|.6 alkyl; and
R2 and R4 are independently H, or a CMO alkyl or C2-10 alkenyl optionally containing one or more non-adjacent heteroatoms selected from N, O, and S, and optionally substituted with a substituted or unsubstituted aryl, heteroaryl, carbocyclic, or heterocyclic ring; or R2 is an optionally cycloalkyl, substituted heterocyclic ring, aryl or heteroaryl;
R5 is a substituent at any position of W and is H, halo, cyano, azido, -CONHR1, OR2, or C].6 alkyl or C2_6 alkenyl, each optionally substituted by halo, =O or one or more heteroatoms; provided X and A both are not H, and further provided that R5 is cyano or -CONHR1 when A is H, halogen or NR1R2; or a compound having formula (TA6-1A)
Figure imgf000055_0001
and pharmaceutically acceptable salts, esters and prodrugs thereof; A is H, halogen, azido, SR2, OR2, CH2R2, NR1R2, or NR1 - (CR'2)n - NR3R4; Z, U, W, R1, R2, R3 and R4 are as defined in formula TA6- 1 ; and R5 is a substituent at any position of W and is H, halo, cyano, azido, -CONHR1, OR2, or
C i-6 alkyl or C2-6 alkenyl, each optionally substituted by halo, =O or one or more heteroatoms; wherein each optionally substituted moiety in formula TA6-1 and - IA is substituted with one or more halo, cyano, azido, acetyl, amido, OR2, NR1R2, carbamate, Ci.10 alkyl, C2 10 alkenyl, each optionally substituted by halo, =O, aryl or one or more heteroatoms selected from N, O and
S; or is substituted with an aryl, a carbocyclic or a heterocyclic ring.
[0133] In the above formula TA6- 1 or TA6- 1 A, W may be selected from the group consisting of
Figure imgf000055_0002
Figure imgf000056_0001
wherein Q, Q1, Q2, and Q3 are independently CH or N;
Y is independently O, CH, =O or NR1; and
R5 is as defined in formula 1.
[0134] In some embodiments of these compounds, each W in the above formula TA6- 1 or TA6-1A may be an optionally substituted phenyl, pyridine, biphenyl, naphthalene, phenanthrene, quinoline, isoquinoline, quinazoline, cinnoline, phthalazine, quinoxaline, indole, benzimidazole, benzoxazole, benzthiazole, benzofuran, anthrone, xanthone, acridone, fluorenone, carbazolyl, pyrimido[4,3-b]furan, pyrido[4,3-b]indole, pyrido[2,3-6]indole, dibenzofuran, acridine or acridizine. In one embodiment, W is an optionally substituted phenyl.
[0135] The compounds of formula (TA6-1), and methods for making and using them, are described in US Patent Application Serial No. 1 1/404,947, to Whitten, et al., which was filed on April 14, 2006, and is entitled QUINOBENZOXAZINE ANALOGS AND METHODS OF USING THEREOF.
[0136] The present invention utilizes the above therapeutic agents in combination with at least one modulator. Modulators that can be utilized in combination with a therapeutic agent described above include compounds having structures of Formula (I), (II), (III), (IV), (V) and (VI) as described herein.
[0137] The present invention utilizes the above therapeutic agents in combination with at least one modulator. For example, compounds of the invention may be used in combination with PARP inhibitors. Examples of PARP inhibitors are known in the art, and are disclosed, for example, in CR. Calebrese, et al., Clin. Cancer Res. vol. 9, 271 1-18 (2003); S.J. Veuger, et al., Cancer Res. vol. 63, 6008-15 (2003); CR. Calabrese et al., J. Nat 'I. Cancer Inst. 96( 1 ), 56-67 (2004); "Potent Novel PARP Inhibitors," Expert Reviews in Molecular Medicine, vol. 7(4) (March 2005); and P. Jagtap, Nature Rev.: Drug Discovery, vol. 4, 421-40 (20045). The PARP inhibitors disclosed in these documents are suitable for use in the methods and compositions of the present invention. Additional PARP inhibitors that can be used include, for example, 10-(4- methyl-piperazin-l-ylmethyl)-2H-7-oxa-l ,2-diaza-benzo[de]anthracen-3-one (GPI 15427) and 2-(4-methyl-piperazin-l-yl)-5H-benzo[c][ l,5]naphthyridin-6-one (GPI 16539). See Di Paola, et al., Eur. J. Pharmacology, 527(1-3), 163-71 (2005). Representative, but non-limiting, examples of PARP inhibitors that are suitable for use in the invention include the known compounds shown hereafter, including the pharmaceutically acceptable salts thereof, and individual isomers or mixtures of isomers thereof.
Figure imgf000058_0001
R = 3-NH2 AG14361 R = 2-OH
Figure imgf000058_0002
CEP-6800 BGP-15
Figure imgf000059_0001
Figure imgf000060_0001
Figure imgf000061_0001
Figure imgf000061_0002
[0138] Modulators that can be utilized in combination with a therapeutic agent described above also include compounds having structures of Formula (I), (II), (III), (IV), (V) and (VI) as described herein.
[0139] The compound TAl-IA is a preferred therapeutic agent for use in the methods and compositions of the invention. More detail on suitable methods for its formulation and administration are provided in U.S. Provisional Application Serial No. 60/803,864 to Lim, et al., which was filed on June 3, 2006.
[0140] The invention also in part provides pharmaceutical compositions comprising at least one therapeutic agent within the scope of the invention as described herein in combination with at least one modulator. Optionally, the composition may comprise a diluent or other pharmaceutically acceptable excipients.
[0141] For administration to animal or human subjects, the appropriate dosage of the therapeutic agent is typically 0.01-15 mg/kg, preferably 0.1- 10 mg/kg. Dosage levels are dependent on the nature of the condition, drug efficacy, the condition of the patient, the judgment of the practitioner, and the frequency and mode of administration; however, optimization of such parameters is within the ordinary level of skill in the art.
[0142] Similarly, the dosage of a modulator, such as a compound of Formula (I), (II), (III), (IV), (V) or (VI) as described herein, is typically between about 0.01-150 mg/kg, and about 0.1-100 mg/kg. A modulator may be separately active for treating a cancer. For combination therapies described above, when used in combination with a therapeutic agent, the dosage of a modulator will frequently be two-fold to ten-fold lower than the dosage required when the modulator is used alone to treat the same condition or subject. Determination of a suitable amount of the modulator for use in combination with a therapeutic agent is readily determined by methods known in the art.
[0143] Also provided are methods for modulating the activity of a CK protein, which comprises contacting a system comprising the CK protein with a composition described herein in an amount effective for modulating (e.g., inhibiting) the activity of the protein. The system in such embodiments can be a cell-free system or a system comprising cells. Also provided are methods for reducing cell proliferation, and optionally inducing apoptosis, which comprises contacting cells with a composition or a combination therapy as described herein, wherein a therapeutic agent is administered in an amount effective to reduce proliferation of the cells, and a CK inhibitor is administered in an amount sufficient to enhance the efficacy of the therapeutic agent. The cells in such embodiments can be in a cell line, in a tissue or in a subject (e.g., a research animal or human).
[0144] The invention also in part provides methods for treating a condition related to aberrant cell proliferation. For example, provided are methods of treating a cell proliferative condition in a subject, which comprises administering a therapeutic agent described herein and a CK inhibitor described herein to a subject in need of treatment for a cell proliferative disorder; the therapeutic agent and the CK inhibitor are administered in amounts effective to treat the cell proliferative condition. The subject may be a research animal (e.g., rodent, dog, cat, monkey), optionally containing a tumor such as a xenograft tumor (e.g., human tumor), for example, or may be a human.
[0145] A cell proliferative condition sometimes is a tumor or non-tumor cancer, including but not limited to, cancers of the colorectum, breast, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart (e.g., leukemia, lymphoma, carcinoma).
[0146] Any suitable formulation of the therapeutic agent and the CK inhibitor can be prepared for administration, either together or separately. Any suitable route of administration may be used for each component, including but not limited to oral, parenteral, intravenous, intramuscular, transdermal, topical and subcutaneous routes. The two substances used together (CK inhibitor and therapeutic agent) may be administered separately or together. When administered together, they may be in separate dosage forms, or they may be combined into a single combination drug. Thus, provided herein are pharmaceutical compositions comprising a therapeutic agent as described herein and at least one CK inhibitor, and a pharmaceutically acceptable excipient.
[0147] All references cited herein, including patents, patent applications, and publications, are hereby incorporated by reference in their entireties, whether previously specifically incoφorated or not. As used herein, the terms "a", "an", and "any" are each intended to include both the singular and plural forms.
[0148] The following examples illustrate and do not limit the invention.
Example 1 Preparation of Methyl S-chlorobenzorc^.όlnaphthyridine-δ-carboxylate
Process 1
Figure imgf000063_0001
[0149] 3-Bromo-4-pyridine carboxylic acid (3.0 g, 14.9 mmol) in ethanol (100 mL) was treated with concentrated sulfuric acid (5 mL). The mixture was brought to reflux at which time everything went into solution. After 12 hours at reflux, LCMS indicated that the reaction was complete. The reaction mixture was cooled to room temperature and concentrated on a rotary evaporator to a third of its original volume. The mixture was then diluted with 250 mL of ethyl acetate and washed twice with saturated aqueous sodium bicarbonate. Concentration on a rotary evaporator yielded 3.25 g of the ethyl ester as a yellowish oil which was sufficiently pure enough for subsequent chemical transformations. LCMS (ESI) 216.2 (M+l)+. Process 2
Figure imgf000064_0001
[0150] Ethyl 3-bromo-4-pyridine carboxylate 1.15 g, 5.0 mmol), 2-amino-4- methoxycarbonyl-phenylboronic acid (1.04 g, 4.5 mmol), sodium acetate (1.64 g, 20 mmol), l ,r-bis(diphenylphosphino)ferrocene palladium (II) chloride (complexed with dichloromethane) ( 182 mg, 0.25 mmol) and dimethylformamide (7.5 mL) were combined in a flask. The flask was evacuated and filled with nitrogen twice and heated to 1250C with stirring for 12 hours or until LCMS indicated the absence of any starting material. The mixture was cooled to room temperature and water ( 100 mL) was added to form a brown precipitate. The precipitate was filtered to yield 637 mg of methyl 5-oxo-5,6-dihydrobenzo[c][2,6]naphthyridine- 8-carboxylate. LCMS (ESI) 255.4 (M+ 1)+.
Figure imgf000064_0002
[0151] Methyl 5-oxo-5,6-dihydrobenzo[c][2,6]naphthyridine-8-carboxylate (200 mg, 0.787 mmol) was combined with phosphorus oxychloride ( 1 mL) and heated to reflux. After 2 hours, LCMS indicated the absence of any starting material. The volatiles were removed under reduced pressure. The residue was taken up in dichloromethane (50 mL) and washed twice with saturated aqueous sodium bicarbonate. The organic phase was dried over sodium sulfate and concentrated on a rotary evaporator to give methyl 5-chlorobenzo[c][2,6]naphthyridine-8- carboxylate (140 mg) as a grayish solid. LCMS (ESI) 273.3 (M+l)+.
Example 2 Preparation of Methyl S-chloropyrimidoK.S-clαuinoline-δ-carboxylate
Process 4
Figure imgf000065_0001
[0152] S-Bromopyrimidine^-carboxylic acid (prepared according to the procedure described in U.S patent 4,1 10,450) (1.0 eq, 6.14 g, 30.2 mmol) was suspended in CH2Cl2 ( 100 ml). Oxalylchloride (1.1 eq, 2.9 ml, 33.0 mmol) was added followed by 2 drops of DMF. The mixture was stirred at room temperature overnight and the volatiles were removed in vacuo. The residue was taken in MeOH (50 ml) and heated. After evaporation of MeOH in vacuo the compound was dissolved in CH2Cl2 and poured on a prepacked silica gel column. The material was eluted using 20% Ethyl acetate in hexanes. Evaporation of the solvent provided methyl-5- bromopyrimidine-4-carboxylate as a light orange crystalline solid (2.54 g, 39% yield). LCMS (ES): 95% pure, m/z 217 [M]+; 219 [M+2]+; 1H NMR (CDCl3, 400 MHz) δ 4.04 (s, 3H), 9.02 (s, IH), 9.21 (s, IH) ppm.
Process 5
Figure imgf000065_0002
[0153] Sodium acetate (4.0 eq, 1.92 g, 23.41 mmol) and 1 ,1 '- bis(diphenylphosphino)ferrocene palladium (II) chloride (complexed with dichloromethane) (0.05 eq, 214 mg, 0.29 mmol) were added to a mixture of methyl-5-bromopyrimidine-4- carboxylate (1.0 eq, 1.27 g, 5.85 mmol), and 2-amino-4-(methoxycarbonyl)phenylboronic acid hydrochloride (1.0 eq, 1.35 g, 5.85 mmol) in anydrous DMF (10 ml). The mixture was stirred under nitrogen atmosphere at 1200C for 18 hours. Water and brine were added and the resulting solid impurities filtered off. The material was extracted with CH2Cl2 (4x) and the combined extracts dried over Na2SO,). After evaporation Of CH2Cl2, the remaining DMF was evaporated by heating the residue in vacuo. The resulting solid was triturated in CH2Cl2, filtered and dried to provide methyl 5-oxo-5,6-dihydropyrimido[4,5-c]quinoline-8-carboxylate as a beige solid (127 mg, 8.5% yield). LCMS (ES): >80% pure, m/z 256 [M+ 1]+; 1H NMR (DMSO-^s, 400 MHz) δ 3.79 (s, 3H), 7.81 (d, J = 8.0, IH), 8.68 (d, J = 8.8, IH), 9.49 (s, IH), 10.19 (s, I H), 12.37 (s, IH) ppm.
Figure imgf000066_0001
[0154] In a vial, methyl 5-oxo-5,6-dihydropyrimido[4,5-c]quinoline-8-carboxylate ( 1 .0 eq,
151 mg, 0.59 mmol) was mixed in toluene (1 ml) with DIEA (1.5 eq, 155 ul, 0.89 mmol) and POCI3 (5 eq, 270 ul, 3.0 mmol). The mixture was stirred at 12O0C for 1 hour and cooled down to room temperature. After adding ice and water the compound was extracted with CH2CI2 (4x). The solution was filtered over Na2SO4 and filtered through a pad of celite. After evaporation of the volatiles, the material was triturated in a mixture of ethyl acetate and hexanes, filtered and dried to afford methyl 5-chloropyrimido[4,5-c]quinoline-8-carboxylate as a light brown fluffy solid (1 15 mg, 71% yield). LCMS (ES): 95% pure, m/z 274 [M+ 1 ]+. 1H NMR (DMSO-^6, 400 MHz) δ 3.96 (s, 3H), 8.37 (dd, 7 = 1.6, 7 = 8.4, I H), 8.60 (d, 7 = 1.6, IH), 9.15 (d, J = 8.8, I H), 9.74 (s, IH), 10.61 (s, IH) ppm.
Example 3 Preparation of methyl 5-chloro-3-(methylthio)pyrimido[4,5-clquinoline-8-carboxylate
Process 7
Figure imgf000066_0002
[0155] Methyl-5-bromo-2-(methylthio)pyrimidine-4-carboxylate was prepared according to the procedure used in Process 4 for the preparation of methyl-5-bromopyrimidine-4-carboxylate. LCMS (ES): >90% pure, m/z 263 [M]+, 265 [M+2]+; 1H NMR (CDCl3, 400 MHz) δ 2.59 (s, 3H), 4.00 (s, 3H), 8.71 (s, IH) ppm. Process 8
Figure imgf000067_0001
[0156] In a microwave vessel, methyl 5-bromo-2-(methylthio)pyrimidine-4-carboxylate (1.0 eq, 274 mg, 1.18 mmol), 2-amino-4-(methoxycarbonyl)phenylboronic acid hydrochloride ( 1.2 eq, 329 mg, 1.42 mmol), and sodium acetate (3.0 eq, 291 mg, 3.55 mmol) were mixed in anhydrous DMF (2 ml). The mixture was degassed by bubbling nitrogen gas in the solution for 10 min and the reaction heated under microwaves at 12O0C for 30 min. After cooling down the expected material crashed out of NMP. The solid was filtered, suspended in water filtered and dried. The material was triturated in AcOEt and filtered give a yellow solid. The same procedure was repeated 9 times using the same amounts of materials to provide methyl 3-(methylthio)-5- oxo-5,6-dihydropyrimido[4,5-c]quinoline-8-carboxylate (283 mg, 10% yield). LCMS (ES): >95% pure, m/z 302 [M+l ]+, 1H NMR (DMSO-^6, 400 MHz) 52.71 (s, 3H), 3.89 (s, 3H), 7.80 (dd, 7 = 1.6, 7 = 8.4, IH), 7.97 (d, J = 1.6, IH), 8.59 (d, J = 8.8, I H), 9.98 (s, IH), 12.34 (s, IH) ppm.
Process 9
Figure imgf000067_0002
[0157] Methyl 3-(methylthio)-5-oxo-5,6-dihydropyrimido[4,5-c]quinoline-8-carboxylate
(1.0 eq, 279 mg, 0.926 mmol) was suspended in toluene (2 ml). POCl3 (2 ml) and DIEA (0.5 ml) were added and the mixture stirred at 12O0C for 5 hours. The volatiles were removed in vacuo and CH2CI2 was added. The organic phase was washed with saturated aqueous sodium bicarbonate, washed with water and dried over Na2SO4. The solution was filtered through a pad of celite and the solvents removed in vacuo. The material was triturated in hexanes and AcOEt, filtered and dried to provide methyl 5-chloro-3-(methylthio)pyrimido[4,5-c]quinoline-8- carboxylate as a beige solid (184 mg, 63% yield). LCMS (ES): >95% pure, m/z 320 [M+l]+, 322 [M+3]+.
Example 4 Preparation of methyl 3-(methylthio)-5-phenylpyrimidor4,5-clquinoline-8-carboxylate
Figure imgf000068_0001
• [0158] Methyl 5-chloro-3-(methylthio)pyrimido[4,5-c]quinoline-8-carboxylate (1.0 eq, 56 mg, 0.175 mmol) was mixed with benzeneboronic acid (2.0 eq, 43 mg, 0.35 mmol), PdCl2(dppf) (0.1 eq, 13 mg, 0.0178 mmol) and cesium carbonate (2.0 eq, 1 14 mg, 0.35 mmol) in dioxane (0.5 ml). The mixture was stirred for 10 minutes at 12O0C under microwave heating. Water was added and the resulting precipitate was filtered and dried. After trituration in ethylacetate and hexanes, methyl 3-(methylthio)-5-phenylpyrimido[4,5-c]quinoline-8-carboxylate was isolated by filtration (53 mg, 84% yield). LCMS (ES): >90% pure, m/z 362 [M+l]+.
Example 5 Preparation of methyl 3-(methylsulfonyl)-5-phenylpyrimidor4.5-clquinoline-8-carboxylate
Figure imgf000068_0002
[0159] methyl 3-(methylthio)-5-phenylpyrimido[4,5-c]quinoline-8-carboxylate ( 1.0 eq, 44 mg, 0.1219 mmol) was dissolved in CH2Cl2 ( 1 ml), mcpba (3.0 eq, 82 mg, 0.366 mmol) was added and the mixture stirred at room temperature for 2.5 hours. CH2Cl2 was added and the organic phase washed with saturated NaHCO3, then water. After drying over Na2SO4 and evaporation of the solvent, methyl 3-(methylsulfonyl)-5-phenylpyrimido[4,5-c]quinoline-8- carboxylate was isolated as a solid. LCMS (ES): >95% pure, m/z 394 [M+ 1]+.
Example 6 Preparation of methyl S-fcvclopropylaminoVS-phenylpyrimidoKS-ciquinol i ne-8-carboxylate
Figure imgf000069_0001
[0160] methyl 3-(methylsulfonyl)-5-phenylpyrimido[4,5-c]quinoline-8-carboxylate ( 1.0 eq, 1 17 mg, 0.298 mmol) was mixed in a vial with cylopropylamine (3.0 eq, 62 ul, 0.89 mmol) in NMP (2 ml). The mixture was stirred at 6O0C for 50 minutes. Water was added and the resulting solid filtered. The material was suspended in AcOEt/hexanes and filtered to afford methyl 3- (cyclopropylamJno)-5-phenylpyrimido[4,5-c]quinoline-8-carboxylate as a yellow solid (86 mg, 78% yield). LCMS (ES): >95% pure, m/z 371 [M+ 1]+.
Example 7 Preparation of 3-(cvclopropylamino)-5-phenylpyrimidof4.5-clquinoline-8-carboxylic acid
Figure imgf000069_0002
[0161] methyl 3-(cyclopropylamino)-5-phenylpyrimido[4,5-c]quinoline-8-carboxylate (1.0 eq, 81 mg, 0.219 mmol) was stirred in EtOH (3 ml) and 6N NaOH (1 ml) at 6O0C for 15 hours. Water was added and the mixture acidified using 6N HCl. The solid was filtered, washed with water and dried. After trituration in MeOH and filtration, 3-(cyclopropylamino)-5- phenylpyritnido[4,5-c]quinoline-8-carboxylic acid was isolated as yellow solid (57 mg, 73% yield). ). LCMS (ES): >95% pure, m/z 357 [M+ 1]+.
Example 8
Preparation of N-cvclopropyl-3-(cvclopropylamino)-5-phenylpyrimidol4■5-clquinoline-8- carboxamide
Figure imgf000070_0001
[0162] 3-(cyclopropylamino)-5-phenylpyrimido[4,5-c]quinoline-8-carboxylic (1.0 eq, 25 mg) was reacted in NMP (0.5 ml), HOBLH2O (2.0 eq, 19 mg), DIEA (4.0 eq, 50 ul), EDCI (2.0 eq, 27 mg) and 4.0 equivalent of cyclopropylamine at 7O0C for 1.5 hours. Water was added and the solid isolated by filtration. After trituration in AcOEt/hexanes, filtration and drying, N- cyclopropyl-3-(cyclopropylamino)-5-phenylpyrimido[4,5-c]quinoline-8-carboxamide was isolated as a solid (21 mg). LCMS (ES): >95% pure, m/z 396 [M+ 1]+.
Example 9 Preparation of Methyl 5-phenylbenzoMf2,61naphthyridine-8-carboxylate
Figure imgf000070_0002
[0163] Methyl 5-phenylbenzo[c][2,6]naphthyridine-8-carboxylate was prepared from methyl 5-chlorobenzo[c][2,6]naphthyridine-8-carboxylate using the same protocol as described in Example 4 (91% yield). LCMS (ES): >95% pure, m/z 315 [M+ 1 ]+. Example 10 Preparation of 5-phenylbenzofcir2.61naphthyridine-8-carboxylic acid
Figure imgf000071_0001
[0164] methyl 5-phenylbenzo[c][2,6]naphthyridine-8-carboxylate (147 mg, 0.468 mmol) was reacted with EtOH (3 ml) and 6N NaOH (1 ml) at 6O0C for 2 hours. Water was added and the mixture acidified using 6N HCl. The solid was filtered and washed with water. The material was triturated in AcOEt/hexanes, filtered and dried to afford 5- phenylbenzo[c][2,6]naphthyridine-8-carboxylic acid as an off-white solid (121 mg, 86% yield). LCMS (ES): >95% pure, m/z 301 [M+ 1]+.
Example 1 1
[0165] The molecules depicted in the following table were prepared using similar chemistries in the presence of appropriate boronic acids or esters, as well as appropriate amines. Table 1. Representative compounds and data
Figure imgf000071_0002
Figure imgf000072_0001
Figure imgf000073_0001
Figure imgf000074_0001
Figure imgf000075_0001
Figure imgf000076_0001
Figure imgf000077_0001
Figure imgf000078_0001
Figure imgf000079_0001
Figure imgf000080_0001
Figure imgf000081_0001
Figure imgf000082_0001
Figure imgf000083_0001
Figure imgf000084_0001
Figure imgf000085_0001
Figure imgf000086_0001
Figure imgf000087_0002
Example 12
Figure imgf000087_0001
[0166] Methyl 5-(l -methyl- lH-pyrazol-4-yl)-3-(methylthio)pyrimido[4,5-c)quinoline-8- carboxylate was prepared using the same protocol as described in Example 4. LCMS (ES): m/z 366 [M+l]+.
Example 13
Figure imgf000088_0001
[0167] Methyl 5-(l-methyl-lH-pyrazol-4-yl)benzo[c][2,6]naphthyridine-8-carboxylate was prepared was prepared using the same protocol as described in Example 4. LCMS (ES): m/z 319 [M+l ]+.
Example 14 Processes for Synthesizing Compounds of Formula (H)
Process 10
Figure imgf000088_0002
[0168] 2-bromo-3-thiophene carboxylic acid ( 1.0 eq, 12.56 g, 60.66 mmol) was suspended in CH2Cl2 (200 ml). Oxalyl chloride (1.1 eq, 5.9 ml, 67.16 mmol) and 5 drops of DMF were added, inducing formation of gas. The mixture was stirred overnight at room temperature and the volatiles were removed in vacuo. The resulting solid was suspended in dry methanol ( 150 ml) and the mixture heated to ebullition. Evaporation of the solvents afforded methyl 2-bromo- 3-thiophene carboxylate (13.16 g, 98% yield) as a crude brown oil. LCMS (ES): 99% pure, m/z not detected; 1H NMR (CDCl3, 400 MHz) δ 3.88 (s, 3H), 7.23 (d, J = 5.6, 1 H), 7.56 (d, J = 5.6, IH) ppm.
Process 1 1
Figure imgf000089_0001
[0169] In a microwave vessel, methyl 2-bromo-3-thiophene carboxylate ( 1.0 eq, 260 mg, 1.18 mmol), 2-amino-4-(methoxycarbonyl)phenylboronic acid hydrochloride (1.1 eq, 300 mg, 1.30 mmol), sodium acetate (3.0 eq, 292 mg, 3.56 mmol) and PdCl2(dppf) (0.05 eq, 31 mg, 0.059 mmol) were mixed together in anhydrous DMF (2 ml). The mixture was heated in a microwave oven at 1200C for 10 mn. Water was added and the solid filtered and dried. The material was suspended in CH2Ch , filtered and dried to afford methyl 4-oxo-4,5- dihydrothieno[3,2-c]quinoline-7-carboxylate as a yellow solid (152 mg, 50% yield). LCMS (ES): 95% pure, m/z 260 [M+ 1]+ ; 1H NMR (CDCl3, 400 MHz) δ 3. 99 (s, 3H), 7.54 (d, J = 5.2, IH), 7.79 (d, 7 = 4.8, IH), 7.86 (d, J = 8.4, IH), 7.91 (dd, 7 = 8.4, 7 = 1.6, I H), 8.03 (d, J = 1.2, IH) ppm.
Figure imgf000089_0002
[0170] Methyl 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate ( 1.0 eq, 618 mg, 2.38 mmol) was suspended in 10 ml of a mixture of MeOH, THF, and water (l : l : l , v:v:v). LiOH (2.0 eq, 1 14 mg, 4.76 mmol) was added and the mixture was stirred at room temperature for 2 hours. An additional amount of LiOH (1 14 mg) was added and the mixture was stirred for an hour. LiOH (50 mg) was added and the mixture stirred for an additional 2 hours. Water was added and the solution filtered through a pad of celite. The pad of celite was thoroughly washed with aqueous 1 N NaOH. The solution was acidified with 6 N aqueous HCl to induce precipitation of the expected material. Filtration and drying afforded 4-oxo-4,5-dihydrothieno[3,27c]quinoline-7- carboxylic acid as a yellow solid (562 mg, 96% yield). LCMS (ES): 95% pure, m/z 246 [M+ 1 ]+ I 1H NMR (DMSO-^, 400 MHz) 57.61 (d, 7 = 5.2, IH), 7.73 (dd, J = 1.6, 7 = 8.0, I H), 7.88 (d, 7 = 5.6, IH), 7.92 (d, 7 = 8.4, IH), 8.02 (d, 7 = 1.6, I H), 1 1.92 (s, I H), 13.21 (br. s, IH) ppm.
Figure imgf000090_0001
[0171] 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylic acid ( 1.0 eq, 38 mg, 0.155 mmol) was suspended in dioxane (1 ml). LiAlH4 (7.0 eq, 40 mg, 1.05 mmol) was added and the mixture stirred at 1000C for 45 mn. Water was added, then MeOH and CH2Cl2. The solid salts were filtered off and washed with MeOH and CH2Cl2. After evaporation of the volatiles in vacuo, the material was dissolved in a mixture of NMP, MeOH and water and was purified by preparative HPLC. Genevac evaporation afforded 7-(hydroxymethyl)thieno[3,2-c]quinolin- 4(5H)-one as an off-white solid (12 mg, 34%). LCMS (ES): 95% pure, m/z 232 [M+l ]+ ; 1H NMR (DMSO-J6, 400 MHz) δ 4.56 (s, 2H), 7.15 (d, J = 7.6, IH), 7.39 (br s, IH), 7.55 (d, 7 = 5.2, IH), 7.73 (d, 7 = 5.2, IH), 7.76 (d, 7 = 8.0, IH), 1 1.73 (s, IH) ppm.
Figure imgf000090_0002
[0172] Methyl 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate ( 1.0 eq, 17 mg, 0.066 mmol) was suspended in a mixture of chloroform (0.3 ml) and acetic acid (0.1 ml). NBS was added (9.5 eq, 112 mg, 0.63 mmol) and the mixture stirred at 7O0C for 16 hours. Water and aqueous ammonia was added and the material was extracted with CH2Cl2 (2x). The combined extracts were dried over Na2SC>4 and the solvent removed in vacuo to provide methyl 2-bromo- 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate (17 mg, 76%). LCMS (ES): >85% pure, m/z 338 [M]+, 340 [M+2]+; 1H NMR (CDCI3/CD3OD, 9: 1, 400 MHz) δ 3.99 (s, 3H), 7.30 (m, 1 H), 7.69 (d, J = 8.4, 1 H), 7.45 (m, 1 H), 7.88 (br d, J = 8, 1 H), 8.05 (br s, 1 H) ppm.
Process 15
Figure imgf000091_0001
[0173] Methyl 2-bromo-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate ( 1.0 eq, 17 mg, 0.050 mmol) was suspended in a 1 : 1 : 1 mixture of MeOH/THF/water (0.6 ml). LiOH (39 mg) was added and the mixture stirred at room temperature for one hour. Water and 6N HCl was added and the resulting precipitate was filtered. The material was purified by preparative HPLC. Genevac evaporation provided 2-bromo-4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylic acid as a solid (2.1 mg, 13% yield). LCMS (ES): >95% pure, m/z 324 [M]+, 326[M+2]+; 1H NMR (DMSO-J6, 400 MHz) δ 7.75 (s, IH), 7.75 (dd, J = 1.6, J = 8.0, IH), 7.90 (d, J = 8.4, 1 H), 8.03 (d, J = 1.6, 1 H), 12.06 (s, 1 H) ppm.
Figure imgf000091_0002
[0174] In a closed vessel, Methyl 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate (44 mg, 0.170 mmol) was suspended in concentrated aqueous ammonia ( I mI). The mixture was stirred at 1000C overnight. Aqueous IN NaOH was added and the mixture stirred at room temperature for 2 hours. The solid was filtered and dried to provide 4-oxo-4,5- dihydrothieno[3,2-c]quinoline-7-carboxamide as a brown solid (13 mg, 32% yield). LCMS (ES): 95% pure, m/z 245 [M+ 1]+.
Process 17
Figure imgf000092_0001
[0175] In a microwave vessel, methyl 2-bromo-3-thiophene carboxylate (1.0 eq, 64 mg, 0.29 mmol), 2-amino phenyl boronic acid (1.2eq, 48 mg, 0.35 mmol), sodium acetate (3.0 eq, 71 mg, 0.86 mmol) and PdCl2(dppf) (0.1 eq, 15 mg, 0.028 mmol) were mixed together in anhydrous DMF (0.2 ml). The mixture was heated in a microwave oven at 12O0C for 5 mn. The material was purified by preparative HPLC. Acetonitrile was evaporated, and the compound was extracted with CH2Cl2 (3x). The combined extracts were washed with water, dried over Na2SO4, and the solvents removed in vacuo. Recrystallization in EtOH provided thieno[3,2-c]quinolin- 4(5H)-one as a tan crystalline solid (7 mg, 12% yield). LCMS (ES): 95% pure, m/z 202 [M+ 1 ]+ ; 1H NMR (CDCI3/CD3OD, 9: 1, 400 MHz) δ 7.28 (m, IH), 7.33 (m, IH), 7.43-7.50 (m, 2H), 7.74 (d, J = 4.4, IH), 7.82 (d, J = 7.6, IH) ppm
Process 18
Figure imgf000092_0002
[0176] In a microwave vessel, methyl 2-bromo-3-thiophene carboxylate (1.0 eq, 250 mg, 1.13 mmol), 2-amino-3-cyanophenyl boronic acid HCl (1.1 eq, 250 mg, 1.24 mmol), sodium acetate (3.0 eq, 278 mg, 3.39 mmol) and PdCl2(dppf) (0.007 eq, 4.3 mg, 0.0082 mmol) were mixed together in anhydrous DMF (2.5 ml). The mixture was heated in a microwave oven at 12O0C for 10 mn. Water was added and the material extracted with CH2Cl2. The organic extracts were washed with brine, dried over Na2SO4 and the solvents removed in vacuo. The resulting solid was sonicated in AcOEt, filtered and dried to afford 4-oxo-4,5-dihydrothieno[3,2- c]quinoline-7-carbonitrile as a beige solid ( 121 mg, 48% yield). LCMS (ES): 95% pure, m/z 227 [M+l]+.
Figure imgf000093_0001
[0177] 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carbonitrile (1.0 eq, 20 mg, 0.088 mmol) was dissolved in anhydrous DMF (0.15 ml). Sodium azide (4.0 eq, 23 mg, 0.354 mmol) and ammonium chloride (4.0 eq, 19 mg, 0.354 mmol) were added and the mixture stirred at 12O0C overnight. The reaction mixture was cooled down and water was added. Addition of aqueous 6N HCl induced formation of a precipitate. After filtration and drying in vacuo, 7-(lH-tetrazol-5- yl)thieno[3,2-c]quinolin-4(5H)-one was isolated as a greenish solid (18 mg, 76% yield). ). LCMS (ES): 95% pure, m/z 270 [M+l]+, 242 [M+l-N2]+; 1H NMR (DMSCW6, 400 MHz) δ 7.64 (d, y = 5.2, IH), 7.86 (dd, 7 = 1.6, 7 = 8.4, IH), 7.89 (d, J = 5.2, IH), 8.09 (d, J = 8.0, IH), 8.16 (d, J = 1.6, IH), 12.03 (s, IH) ppm.
Figure imgf000093_0002
[0178] Methyl 4-oxo-4,5-dihydrothieno[3,2-c]quinoline-7-carboxylate ( 1.0 eq, 1.50 g, 5.79 mmol) was suspended in dry toluene (15 ml). POCl3 (1.2 eq, 0.64 mmol, 6.99 mmol) and DIEA (0.8 eq, 0.81 mmol, 4.65 mmol) were added and the mixture vigorously stirred at 1200C for 3 hours under nitrogen atmosphere. The mixture was hydrolyzed by addition of ice and water. The compound was extracted with CH2Cb (4x). The combined extracts were dried over Na2SO4 and the black solution filtered through a pad of celite. After evaporation of the volatiles in vacuo, the resulting solid was triturated in a mixture of AcOEt and hexanes. Filtration and drying provided methyl 4-chlorothieno[3,2-c]quinoline-7-carboxylate as a yellow fluffy solid ( 1.14 g, 71 % yield). LCMS (ES): 95% pure, m/z 278 [M+l ]+, 1H NMR (CDCl3, 400 MHz) δ 4.01 (s, 3H), 7.72 (d, 7 = 4.8, I H), 7.74 (d, J = 5.2, I H), 8.14 (d, J = 8.4, I H), 8.25 (d, 7 = 8.4, I H), 8.85 (d, J = 1.6, IH) ppm.
Figure imgf000094_0001
[0179] 4-chlorothieno[3,2-c]quinoline was prepared according to the procedure used in process 16, starting from thieno[3,2-c]quinolin-4(5H)-one. 4-chlorothieno[3,2-c]quinoline was isolated as a solid (71 mg, 93% yield). LCMS (ES): 95% pure, m/z 220 [M+ 1 ]+, 223 [M+3]+.
Figure imgf000094_0002
[0180] 4-chlorothieno[3,2-c]quinoline-7-carbonitrile was prepared according to the procedure used in process 16. 4-chlorothieno[3,2-c]quinoline-7-carbonitrile was isolated as a yellow fluffy solid (833 mg, 77% yield). LCMS (ES): 95% pure, m/z 245 [M+ 1 ]+, 247 [M+3]+
Process 23
Figure imgf000094_0003
[0181] Methyl 2-amino-4-bromothiazole-4-carboxylate ( 1.0 eq, 100 mg, 0.42 mmol) was dissolved in anhydrous DMF (0.8 ml). The mixture was heated to 8O0C under nitrogen atmosphere. To the hot mixture, a solution of tert-Buty\ nitrite (1.2 eq, 60 ul, 0.50 mmol) in DMF (0.8 ml) was added dropwise. After a few minutes, absence of gas evolution indicated completion of the reaction. The mixture was cooled down and poured onto a prepacked silica gel column. Flash chromatography using hexanes, then AcOEt/hexanes (2:8), provided methyl 5- bromothiazole-4-carboxylate as a yellow solid (49 mg, 53% yield). LCMS (ES): 95% pure, m/z 222 [M]+, 224 [M+2]+.
Process 24
Figure imgf000095_0001
[0182] In a microwave vessel, methyl 5-bromothiazole-4-carboxylate ( 1.0 eq, 97 mg, 0.44 mmol), 2-amino-3-methoxycarbonyl phenyl boronic acid HCl (1.1 eq, 1 1 1 mg, 0.48 mmol), sodium acetate (3.0 eq, 107 mg, 1.31 mmol) and PdCl2(dppf) (0.05 eq, 1 1 mg, 0.022 mmol) were mixed together in anhydrous DMF ( 1 ml). The mixture was heated in a microwave oven at 1200C for 10 mn. Water was added and the material extracted with CH2Ck The combined extracts wre washed with brine, dried over Na2SC^ and the solvents removed by evaporation. The material was dissolved in a mixture Of CH2Cl2 and MeOH and the solution filtered through a pad of celite. Evaporation of the volatiles afforded crude methyl 4-oxo-4,5- dihydrothiazolo[4,5-c]quinoline-7-carboxylate as a black solid (44 mg, 39% yield). A small part of the compound was subjected to preparative HPLC for analytical purpose. LCMS (ES): 95% pure, m/z 261 [M+l ]+.
Process 25
Figure imgf000095_0002
[0183] Methyl 4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylate (35 mg, 0.12 mmol) and LiOH (60 mg, 0.83 mmol) were stirred in a (1 : 1 : 1 , v:v:v) mixture of THF, MeOH and water (0.6 ml) for 2 hours. 6N aqueous NaOH was added and the solution filtered through celite. The solution was acidified and the resulting solid filtered. Preparative HPLC purification and genevac evaporation provided 4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylic acid as a white solid (0.8 mg). LCMS (ES): 95% pure, m/z 247 [M+ 1 ]+.
Process 26
Figure imgf000096_0001
[0184] Methyl 2-amino-4-bromothiazole-4-carboxylate (1.0 eq, 2.0 g, 8.44 mmol) was dissolved in CH2Cb (4 ml). Acetic anhydride (1.5 eq, 1.2 ml, 12.66 mmol) and triethylamine (1.1 eq, 1.3 ml, 9.28 mmol) were added and the mixture stirred at 1000C for one hour. The resulting solid was filtered, triturated in AcOEt and then filtered again. After drying, methyl 2- acetamido-5-bromothiazole-4-carboxylate was isolated as a beige solid (1.81 g, 77% yield). LCMS (ES): 95% pure, m/z 280 [M+l]+. 1H NMR (CDCl3, 400 MHz) δ 2.25 (s, 3H), 3.95 (s, 3H) ppm.
Process 27
Figure imgf000096_0002
[0185] Methyl 2-acetamido-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylate was prepared according to the procedure used in process 2, starting from methyl 2-acetamido-5- bromothiazole-4-carboxylate. Methyl 2-acetamido-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline- 7-carboxylate was isolated as a black solid (106 mg, 37% yield). LCMS (ES): 95% pure, m/z 318 [M+1]+. Process 28
Figure imgf000097_0001
[0186] 2-acetamido-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylic acid was prepared according to the procedure in process 3, starting from . Methyl 2-acetamido-4-oxo- 4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylate. -acetamido-4-oxo-4,5-dihydrothiazolo[4,5- c]quinoline-7-carboxylic acid was isolated as a black solid ( 14 mg, 44% yield). LCMS (ES): 95% pure, m/z 304 [M+ 1]+, 1H NMR (DMSO-^6, 400 MHz) δ 2.22 (s, 3H), 7.74 (dd, J = 1.2, J = 8.0, IH), 7.89 (d, 7 = 8.4, I H), 8.03 (d, J = 1.6, I H), 12.07 (s, IH), 12.80 (s, IH) ppm.
Process 29
Figure imgf000097_0002
[0187] 2-acetamido-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylic acid ( 102 mg, 0.34 mmol) was stirred at 12O0C in aqueous 6N HCl overnight. Water was added and the compound was filtered and dried to provide 2-amino-4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline- 7-carboxylic acid as a black solid (76 mg, 86% yield). LCMS (ES): 95% pure, m/z 262 [M+ 1 ]+, 1H NMR (DMSO-^5, 400 MHz) δ 7.60 (d, 7 = 8.4, 1 H), 7.70 (dd, J = 1.2, J = 8.0, 1 H), 7.99 (d, 7 = 1.2, IH), 1 1.94 (s, I H) ppm.
Process 30
Figure imgf000098_0001
[0188] Methyl 4-oxo-4,5-dihydrothiazolo[4,5-c]quinoline-7-carboxylate ( 1.0 eq, 0.62 g, 2.38 mmol) was suspended in toluene. DIEA ( 1.5 eq, 122 ul, 3.57 mmol) and POCl3 (2.3 eq, 507 ul, 5.47 mmol) were added and the mixture vigorously stirred at 12O0C for 1 hour. Water, ice and CH2CI2 were added and the resulting emulsion filtered through celite. The organic phase was decanted and the aqueous phase further extracted with CH2Cb. The combined organic extracts were dried over Na2SC>4 and the solvent removed in vacuo to afford methyl 4- chlorothiazolo[4,5-c]quinoline-7-carboxylate (0.31 g, 47% yield). LCMS (ES): >90% pure, m/z 279[M+1]+.
Conversion of intermediates to compounds of Formula (H)
[0189] Compounds of Formula (II) are prepared by routes analogous to those described herein for preparation of compounds of Formula (I). For example, the halogenated products prepared in processes 20, 21 , 22, and 30, may be converted to compounds of Formula (II) under conditions substantially as described in Example 4, or under conditions known to those of skill in the art.
Example 15 Modulation of CK2 activity in cell-free in vitro assays
[0190] Modulatory activity of compounds described herein is assessed in vitro in cell-free CK2 assays. These assays are described hereafter.
CK2 Assay
[0191] Test compounds in aqueous solution are added at a volume of 10 microliters, to a reaction mixture comprising 10 microliters Assay Dilution Buffer (ADB; 2OmM MOPS, pH 7.2, 25 mM beta-glycerolphosphate, 5 mM EGTA, 1 mM sodium orthovanadate and 1 mM dithiothreitol), 10 microliters of substrate peptide (RRRDDDSDDD, dissolved in ADB at a concentration of 1 mM), 10 microliters of recombinant human CK2 (25 ng dissolved in ADB; Upstate). Reactions are initiated by the addition of 10 microliters of ATP Solution (90% 75 mM MgCl2, 75 micromolar ATP dissolved in ADB; 10% [γ-33P]ATP (stock 1 mCi/100 μl; 3000 Ci/mmol (Perkin Elmer) and maintained for 10 minutes at 30 degrees C. The reactions are quenched with 100 microliters of 0.75% phosphoric acid, then transferred to and filtered through a phosphocellulose filter plate (Millipore). After washing each well 5 times with 0.75% phosphoric acid, the plate is dried under vacuum for 5 min and, following the addition of 15 ul of scintillation fluid to each well, the residual radioactivity is measured using a Wallac luminescence counter.
Example 16 Cell Proliferation Modulatory Activity
[0192] A representative cell-proliferation assay protocol using Alamar Blue dye (stored at 4°C, use 20ul per well) is described hereafter. 96-well plate setup and compound treatment a. Split and trypsinize cells. b. Count cells using hemocytometer. c. Plate 4,000-5,000 cells per well in 100 μl of medium and seed into a 96-well plate according to the following plate layout. Add cell culture medium only to wells B lO to B 12. Wells Bl to B9 have cells but no compound added.
1 2 4 5 7 8 10 11
3 6 9 12
A EMPTY
NO COMPOUND Medium
D
ADDED Only
C 1OnM 10OnM 1 uM 1OuM Control
D 1OnM 10OnM 1 uM 1OuM Compi
E 1OnM 10OnM 1uM 1OuM Comp2
F 1OnM 10OnM 1uM 1OuM Comp3
G 1OnM 10OnM 1uM 1OuM Comp4
H EMPTY d. Add 100 μl of 2X drug dilution to each well in a concentration shown in the plate layout above. At the same time, add 100 μl of media into the control wells (wells B lO to B 12). Total volume is 200 μl /well. e. Incubate four (4) days at 37°C, 5% CO2 in a humidified incubator. f. Add 20μl Alamar Blue reagent to each well. g. Incubate for four (4) hours at 37°C, 5% CO2 in a humidified incubator. h. Record fluorescence at an excitation wavelength of 544nm and emission wavelength of 590nm using a microplate reader.
In the assays, cells are cultured with a test compound for approximately four days, the dye is then added to the cells and fluorescence of non-reduced dye is detected after approximately four hours. Different types of cells can be utilized in the assays (e.g., HCT-1 16 human colorectal carcinoma cells, PC-3 human prostatic cancer cells and MiaPaca human pancreatic carcinoma cells).
Example 17 Modulation of Endogenous CK2 Activity
[0193] The human leukemia Jurkat T-cell line is maintained in RPMI 1640 (Cambrex) supplemented with 10% fetal calf serum and 50 ng/ml Geutamycin. Before treatment cells are washed, resuspended at a density of about 106 cells/milliliter in medium containing 1 % fetal calf serum and incubated in the presence of indicated mounts of drug for two hours. Cells are recovered by centrifugation, lysed using a hypotonic buffer (20 mM Tris/HCl pH 7.4; 2 mM EDTA; 5 mM EGTA; 10 mM mercaptoethanol; 1OmM NaF; 1 uM Okadaic acid; 10% v/v glycerol; 0.05% NP-40; 1% Protease Inhibitor Cocktail) and protein from the cleared lysate is diluted to 1 microgram per microliter in Assay Dilution Buffer (ADB; 2OmM MOPS, pH 7.2, 25mM β-glycerolphosphate, 5mM EGTA, ImM sodium orthovanadate and ImM dithiothreitol). To 20 microliters of diluted protein is added 10 microliters of substrate peptide (RRRDDDSDDD, dissolved in ADB at a concentration of 1 mM) and 10 microliters of PKA Inhibitor cocktail (Upstate). Reactions are initiated by the addition of 10 microliters of ATP Solution (90% 75 mM MgCl2, 100 uM ATP dissolved in ADB; 10% [gamma-33P]ATP (stock 1 mCi/100 microliters; 3000Ci/mmol (Perkin Elmer)) and maintained for 15 min at 32 degrees C. The reactions are quenched with 100 microliters of 0.75% phosphoric acid, then transferred to and filtered through a phosphocellulose filter plate (Millipore). After washing each well 5 times with 0.75% phosphoric acid, the residual radioactivity is measured using a Wallac luminescence counter.
Example 18 Evaluation of Pharmacokinetic Properties
[0194] The pharmacokinetics properties of drugs are investigated in ICR mice following an intravenous (FV) bolus and oral (PO) doses of drug at 5 mg/kg and 25 mg/kg respectively. Blood samples are collected at predetermined times and the plasma separated. Plasma is separated from the blood samples collected at 5, 15 and 30 minutes and 1 , 2, 4, 8 and 24 hours post-dose.
[0195] Drug levels are quantified by the LC/MS/MS method described below. Noncompartmental pharmacokinetic analysis is applied for intravenous administration. A linear trapezoidal rule is used to compute AUC(O- 24). The terminal t|/2 and Co are calculated using the last three and the first three data points, respectively
[0196] Bioanalysis is performed using a Quattro Micro LC/MS/MS instrument in the MRM detection mode, with an internal standard (IS). Briefly, 15 μL plasma samples are prepared for analysis using protein precipitation with 120 μL of acetonitrile. The supernatants are transferred into a 96 well plate and subjected to LC-MS/MS analysis using a Phenomenex Polar-RP HPLC column. The mobile phases are 10 mM NH4HCO3 in water (Solution-A) and 10 mM NH4HCO3 in methanol (Solution-B). The column is initially equilibrated with 25 % Solution-B and followed with 100% Solution B over 5 minutes. The method has a dynamic range from 1 to 10,000 ng/mL. Quantitation of the analytes is performed in the batch mode with two bracketing calibration curves according to the bioanalytical sample list.
Example 19 Modulation of non-CK2 Protein Kinase Activity
[0197] Compounds described herein are profiled for in vitro modulatory activity against protein kinases other than CK2. The in vitro analysis is conducted using known protocols (e.g., assay protocols described at world-wide web address upstate.com/img/pdf/KP_Assay Protocol_BookIet_v3.pdf). Compounds described herein are screened in the assays and prioritized based upon modulatory activity against protein kinases other than CK2 and specificity for CK2.
Example 20 Modulation of protein kinase activity in cell-free in vitro assay
[0198] In a PIM-I assay, test compounds in aqueous solution are added at a volume of 5 μl, to a reaction mixture comprising 5 μl of 5x Reaction buffer (4OmM MOPS, pH 7.0, ImM EDTA), 2.5 μl of recombinant human PDvI-I solution (10 ng), 2.5 μl of substrate peptide (KKRNRTLTK) and 10 μl of ATP solution - 98% (75 mM MgC12 37.5 uM ATP) 2% ([γ- 33P]ATP: 3000Ci/mmol - Perkin Elmer). The reactions are incubated for 10 min at 30 0C, quenched with 100 μl of 0.75% Phosphoric acid, then transferred to and filtered through a Phosphocellulose filter plate (Millipore). After washing each well 5 times with 0.75% Phosphoric acid, Scintillation fluid (15 μl) is added to each well. The residual radioactivity is measured using a luminescence counter.
[0199] Test compounds are tested further for activity against other protein kinases. Selected kinase inhibition ICsodata are determined using standardized radiometric kinase assays for each individual kinase, which entail filter binding of 33P labeled substrate proteins by the kinase of interest. Each IC50 value is determined over a range of 10 drug concentrations. Representative reaction conditions are available from the World Wide Web URL upstate.corn/discovery/services/ic50_profiler.q.
Example 21 BIOLOGICAL ACTIVITY
[0200] Biological activity data for representative compounds of the invention against CK2 and in a variety of cell lines is provided in Table 2 below. Table 2. Representative Compounds and Data
O K)
Figure imgf000103_0001
Figure imgf000103_0002
Table 2. Representative Compounds and Data
O
Figure imgf000104_0001
Table 2. Representative Compounds and Data
Figure imgf000105_0001
Table 2. Representative Compounds and Data
O Ul
Figure imgf000106_0001
Figure imgf000106_0002
Table 2. Representative Compounds and Data
O
Figure imgf000107_0002
Figure imgf000107_0001
Table 2. Representative Compounds and Data
O
Figure imgf000108_0001
Table 2. Representative Compounds and Data
O
Figure imgf000109_0001
Figure imgf000109_0002
Table 2. Representative Compounds and Data
O
Figure imgf000110_0001
Table 2. Representative Compounds and Data
Figure imgf000111_0001
Table 2. Representative Compounds and Data
Figure imgf000112_0001
Table 2. Representative Compounds and Data
Figure imgf000113_0001
Table 2. Representative Compounds and Data
M
Figure imgf000114_0001
Table 2. Representative Compounds and Data
Figure imgf000115_0001
Table 2. Representative Compounds and Data
Figure imgf000116_0001
Table 2. Representative Compounds and Data
Figure imgf000117_0001
Table 2. Representative Compounds and Data
Figure imgf000118_0001
Table 2. Representative Compounds and Data
Figure imgf000119_0001
Table 2. Representative Compounds and Data
VO
Figure imgf000120_0001
Figure imgf000120_0002
Table 2. Representative Compounds and Data
K)
O
Figure imgf000121_0001
Figure imgf000121_0002
Table 2. Representative Compounds and Data
Figure imgf000122_0001
Table 2. Representative Compounds and Data
K) κ>
Figure imgf000123_0001
Figure imgf000123_0002
Table 2. Representative Compounds and Data
K)
Figure imgf000124_0001
Table 2. Representative Compounds and Data
Figure imgf000125_0001
Table 2. Representative Compounds and Data
b!
Figure imgf000126_0002
Figure imgf000126_0001
Table 2. Representative Compounds and Data
K)
Figure imgf000127_0001
Table 2. Representative Compounds and Data
K)
Figure imgf000128_0001
Table 2. Representative Compounds and Data
K)
00
Figure imgf000129_0001
Table 2. Representative Compounds and Data
K)
Figure imgf000130_0001
Table 2. Representative Compounds and Data
O
Figure imgf000131_0001
Table 2. Representative Compounds and Data
Figure imgf000132_0001
Table 2. Representative Compounds and Data
K)
Figure imgf000133_0001
Figure imgf000133_0002
Table 2. Representative Compounds and Data
M M
Figure imgf000134_0001
Table 2. Representative Compounds and Data
Figure imgf000135_0001
Example 22 Exemplary Embodiments of the Invention
[0201] Some representative embodiments of the invention are set forth hereafter, but are not to be taken as limiting the scope of the invention as described herein.
Embodiments
Al . A compound of Formula (I):
Figure imgf000136_0001
and pharmaceutically acceptable salts, esters, and tautomers thereof; wherein: each Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3; each R3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2-
C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R3 is independently halo, CF3, CFN, OR, NR2, NROR, NRNR2, SR, SOR, SO2R,
SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, COOH, polar substituent, carboxy bioisostere, CONR2, 0OCR, COR, or NO2; Z5 is CR6A or N; each of R6A, R6B and R6C is independently H or an optionally substituted C l -C8 alkyl,
C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl,
C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C 12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R6A, R6B and R6C can be halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, 0OCR, COR, or NO2; wherein each R is independently H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6- ClO aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and wherein two R on the same atom or on adjacent atoms can be linked to form a 3-8 membered ring, optionally containing one or more N, O or S; and each R group, and each ring formed by linking two R groups together, is optionally substituted with one or more substituents selected from halo, =0, =N-CN, =N-OR', =NR', OR', NR'2, SR', SO2R', SO2NRS, NR1SO2R', NR'C0NR'2, NR'COOR', NR'COR', CN, COOR', CONR'2, OOCR', COR', and NO2, wherein each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C1-C6 acyl, C2- C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7- 12 arylalkyl, or C6-12 heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, Cl - C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =O; and wherein two R' can be linked to form a 3-7 membered ring optionally containing up to three heteroatoms selected from N, O and S; and
R10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
A2. The compound of embodiment Al, wherein Z5 is N.
A3. The compound of embodiment Al, wherein Z5 is CR6Λ.
A4. The compound of embodiment Al, A2, or A3, wherein at least one R3 is a polar substituent, wherein said polar substituent is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
A5. The compound of any one of embodiments A1-A4, wherein each of each Z1, Z3 and Z4 iiss CCHH,, aanndd ZZ22 iiss CCRR33,, wwhhcere R3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere. A6. The compound of any one of embodiments A1-A4, wherein at least one of Z'-Z4 is N.
A7. The compound of any one of embodiments A1-A6, wherein R10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring.
A8. The compound of embodiment A7, wherein R10 is an optionally substituted phenyl or pyridyl ring.
A9. The compound of embodiment A8, wherein R10 is phenyl optionally substituted with one or more halo, CN, CM alkyl, C M alkoxy, NH2, or NHAc substituents.
AlO. The compound of any one of embodiments A1-A6, wherein R10 is an optionally substituted five-membered heterocyclic aromatic ring.
Al 1. The compound of embodiment AlO, wherein R10 is an optionally substituted pyrazole, isoxazole or thiophene ring.
A12. The compound of any one of embodiments Al-Al 1, wherein at least one of R6B or R6Cis H.
A13. The compound of any one of embodiments A1-A12, wherein R6B and R6C are H.
A 14. The compound of any one of embodiments Al -A 12, wherein R6B is H, SR, SO2R or NR2, where each R is independently H or CM alkyl.
A 15. The compound of of any one of embodiments A3-A14, wherein R6Λ is H.
Bl . The compound of embodiment Al having the structure of Formula (III), (IV), (V), or (VI):
Figure imgf000139_0001
and pharmaceutically acceptable salts, esters, and tautomers thereof.
B2. The compound of embodiment B 1 , wherein at least one R3 is a polar substituent, wherein said polar substituent is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
B3. The compound of embodiment B l or B2, wherein: the compound has the Formula (III) or (IV) and each of each Z1, Z3 and Z4 is CH, and Z2 is CR3; or the compound has the Formula (V) or (VI); and
R3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
B4. The compound of of embodiment B 1 or B2, wherein at least one of Z -Z is N.
B5. The compound of any one of embodiments B1-B4, wherein R 1 10 i,s an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring. B6. The compound of embodiment B5, wherein R10 is an optionally substituted phenyl or pyridyl ring.
B7. The compound of embodiment B6, wherein R10 is phenyl optionally substituted with one or more halo, CN, CM alkyl, C M alkoxy, NH2, or NHAc substituents.
B8. The compound of embodiment B5, wherein R10 is an optionally substituted five- membered heterocyclic aromatic ring.
B9. The compound of embodiment B8, wherein R10 is an optionally substituted pyrazole, isoxazole or thiophene ring.
B 10. The compound of any one of embodiments B 1 -B9, wherein at least one of R 6B or
R6C is H.
BI l. The compound of any one of embodiments Bl-B lO, wherein R6B and R6C are H.
B 12. The compound of any one of embodiments B l-B lO, wherein R6B is H, SR, SO2R or NR?, where each R is independently H or CM alkyl.
B 13. The compound of any one of embodiments B 1 -B 12 having Formula (III) or (V), wherein R6A is H.
Cl. A compound of Formula (H):
Figure imgf000140_0001
and pharmaceutically acceptable salts, esters, and tautomers thereof; wherein: each Z1, Z2, Z3, and Z4 is N, CH, or CR3, provided that none, one or two of Z1, Z2, Z3, and Z4 are N; and further provided that one or more of Z1, Z2, Z3, and Z4 is CR3; each R3 is independently an optionally substituted C1-C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C 1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R3 is independently halo, CF3, OR, NR2, NfROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, COOH, polar substituent, carboxy bioisostere, CONR2, OOCR, COR, or NO2;
Z5 is CR6A or N; each of R6A and R6B is independently H or an optionally substituted C1 -C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C12 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl group, or each of R6A and R6B can be halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, OOCR, COR, or NO2; wherein each R is independently H or C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6- ClO aryl, C5-C10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl, and wherein two R on the same atom or on adjacent atoms can be linked to form a 3-8 membered ring, optionally containing one or more N, O or S; and each R group, and each ring formed by linking two R groups together, is optionally substituted with one or more substituents selected from halo, =O, =N-CN, =N-0R', =NR\ OR', NRS, SR', SO2R', SO2NR'2, NR'S02R', NR'CONRS, NR'COOR', NR'COR', CN, COOR', C0NR'2, OOCR', COR', and NO2, wherein each R' is independently H, C1-C6 alkyl, C2-C6 heteroalkyl, C l -C6 acyl, C2- C6 heteroacyl, C6-C10 aryl, C5-C10 heteroaryl, C7-12 arylalkyl, or C6-12 heteroarylalkyl, each of which is optionally substituted with one or more groups selected from halo, C1-C4 alkyl, Cl- C4 heteroalkyl, C1-C6 acyl, C1-C6 heteroacyl, hydroxy, amino, and =0; and wherein two R' can be linked to form a 3-7 membered ring optionally containing up to three heteroatoms selected from N, O and S; and
R10 is an optionally substituted five-membered or six-membered carbocyclic or heterocyclic aromatic ring.
C2. The compound of embodiment Cl , wherein Z5 is N.
C3. The compound of embodiment Cl, wherein Z5 is CR6A
C4. The compound of embodiment Cl , C2, or C3, wherein at least one R3 is a polar substituent, wherein said polar substituent is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
C5. The compound of any one of embodiments C1-C4, wherein each of each Z1, Z3 and Z4 is CH, and Z2 is CR3, where R3 is a carboxylic acid, carboxylate salt, ester, carboxamide, tetrazole or carboxy bioisostere.
C6. The compound of any one of embodiments C 1 -C4, wherein at least one of Z1 -Z4 is N.
C7. The compound of any one of embodiments C1-C6, wherein R10 is an optionally substituted six-membered carbocyclic or heterocyclic aromatic ring.
C8. The compound of embodiment C7, wherein R10 is an optionally substituted phenyl or pyridyl ring.
C9. The compound of embodiment C7, wherein R10 is an optionally substituted five- membered heterocyclic aromatic ring.
ClO. The compound of embodiment C9, wherein R10 is an optionally substituted pyrazole, isoxazole or thiophene ring. CI l . The compound of any one of embodiments Cl-ClO, wherein Z5 is R6A and R6A is H.
C12. The compound of any one of embodiments Cl-ClO, wherein Z5 is N.
C13. The compound of any one of embodiments Cl-ClO, wherein R6B is H, halo, or NR2, where each R is independently H or CM alkyl.
Dl. A pharmaceutical composition comprising a compound of any one of embodiments Al -A 15 and a pharmaceutically acceptable excipient.
D2. A pharmaceutical composition comprising a compound of any one of embodiments Bl -B 13 and a pharmaceutically acceptable excipient.
D3. A pharmaceutical composition comprising a compound of any one of embodiments Cl -C 13 and a pharmaceutically acceptable excipient.
El. A method for identifying a candidate molecule that interacts with a casein kinase 2 (CK2) protein, which method comprises: contacting a composition containing a CK2 protein and a compound having a structure of Formula (I), (II), (III), (FV), (V), or (VI) with a candidate molecule under conditions in which the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein.
E2. The method of embodiment El, wherein the CK2 protein comprises the amino acid sequence of SEQ ID NO: 1 , SEQ ID NO:2 or SEQ ID NO:3, or a substantially identical variant thereof. E3. The method of embodiment El or E2, wherein the protein is in a cell.
E4. The method of embodiment El or E2, wherein the protein is in a cell-free system.
E5. The method of any one of embodiments E1-E4, wherein the protein, the compound or the molecule is in association with a solid phase.
E6. The method of any one of embodiments E1-E5, wherein the interaction between the compound and the protein is detected via a detectable label.
E7. The method of any one of embodiments E1-E5, wherein the interaction between the compound and the protein is detected without a detectable label.
Fl. A method for modulating the activity of a CK2 protein, which method comprises contacting a system comprising the protein with a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) in an amount effective for modulating the activity of the protein.
F2. The method of embodiment Fl, wherein the activity of the protein is inhibited.
F3. The method of embodiment Fl or F2, wherein the CK2 protein comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO:2 or SEQ ID NO:3, or a substantially identical variant thereof.
F4. The method of embodiment Fl, F2 or F3, wherein the system is a cell.
F5. The method of embodiment Fl , F2 or F3, wherein the system is a cell-free system.
F6. The method of any one of embodiments F1-F5, wherein the protein, the compound or the molecule is in association with a solid phase.
Gl . A method for inhibiting cell proliferation, which method comprises contacting cells with a compound having a structure of Formula (I), (H), (III), (IV), (V), or (VI) in an amount effective to inhibit proliferation of the cells. G2. The method of embodiment Gl, wherein the cells are in a cancer cell line.
G3. The method of embodiment G2, wherein the cancer cell line is a breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, or ovary cancer cell line.
G4. The method of embodiment Gl, G2 or G3, wherein the cells are in a tumor in a subject.
G5. The method of any one of embodiments G1-G4, wherein contacting cells with a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) induces cell apoptosis.
G6. The method of embodiment G 1 , wherein the cells are from an eye of a subject having macular degeneration.
G7. The method of embodiment Gl, wherein the cells are in a subject having macular degeneration.
Hl. A method for treating a condition related to aberrant cell proliferation, which method comprises administering a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to treat the cell proliferative condition.
H2. The method of embodiment Hl , wherein the cell proliferative condition is a tumor- associated cancer.
H3. The method of embodiment H2, wherein the cancer is of the colorectum, breast, ovary, lung, liver, pancreas, lymph node, colon, prostate, brain, head and neck, skin, liver, kidney, blood and heart.
H4. The method of embodiment Hl, wherein the cell proliferative condition is a non- tumor cancer. H5. The method of embodiment H4, wherein the non-tumor cancer is a hematopoietic cancer.
H6. The method of embodiment Hl, wherein the cell proliferative condition is macular degeneration.
Jl . A method for identifying a candidate molecule that interacts with a protein kinase, which comprises: contacting a composition containing a protein kinase and a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) under conditions in which the compound and the protein interact with a candidate molecule, and determining whether the amount of the compound that interacts with the protein kinase is modulated relative to a control interaction between the compound and the protein kinase without the candidate molecule, whereby a candidate molecule that modulates the amount of the compound interacting with the protein kinase relative to the control interaction is identified as a candidate molecule that interacts with the protein kinase.
J2. The method of embodiment Jl , wherein the protein kinase is serine-threonine protein kinase or a tyrosine protein kinase.
J3. The method of embodiment Jl or J2, wherein the protein kinase is a human serine-threonine protein kinase or a human tyrosine protein kinase.
J4. The method of embodiment Jl , J2, or J3, wherein the protein kinase is a human serine-threonine protein kinase selected from the group consisting of CK2, CK2α2, PIM 1 , PIM2, PIM3, CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK.
J5. The method of embodiment J2, wherein the protein kinase is a serine-threonine protein kinase which contains one or more of the following amino acids at positions corresponding to those listed in human CK2: leucine at position 45, methionine at position 163 and isoleucine at position 174. J6. The method of embodiment J5, wherein the serine threonine protein kinase is selected from the group consisting of CK2, STKlO, HIPK2, HIPK3, DAPK3, DYK2 and PIM-I .
J7. The method of embodiment J 1 , J2, or J3, wherein the protein kinase is a human tyrosine protein kinase selected from the group consisting of FLTl , FLT2, FLT3, FLT3 (D835Y), and FLT4.
J8. The method of any one of embodiments J1-J7, wherein the protein, the compound or the molecule is in association with a solid phase.
J9. The method of any one of embodiments J1-J8, wherein the interaction between the compound and the protein is detected via a detectable label.
JlO. The method of any one of embodiments J1-J9, wherein the protein kinase activity is the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate.
Kl . A method for modulating a protein kinase activity, which comprises contacting a system comprising a protein kinase protein with a compound of Formula (I), (II), (III), (IV), (V), or (VI) in an amount effective for modulating the activity of the protein kinase.
K2. The method of embodiment Kl, wherein the protein kinase is serine-threonine protein kinase or a tyrosine protein kinase.
K3. The method of embodiment K2, wherein the protein kinase is a human serine- threonine protein kinase or a human tyrosine protein kinase.
K4. The method of embodiment K3, wherein the protein kinase is a human serine- threonine protein kinase selected from the group consisting of CK2, CK2α2, PIMl, PIM2, PIM3, CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK. K5. The method of embodiment K2, wherein the protein kinase is a human tyrosine protein kinase selected from the group consisting of FLTl , FLT2, FLT3, FLT3 (D835Y), and FLT4.
Ll . A method for treating pain or inflammation in a subject, which comprises administering a compound of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to treat the pain or the inflammation.
Ml . A method for inhibiting angiogenesis in a subject, which comprises administering a compound of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to inhibit the angiogenesis.
Nl. A composition of matter comprising a protein kinase and a compound of Formula (I), (II), (UI), (IV)1 (V), or (VI).
N2. The composition of embodiment Nl , wherein the protein kinase is a human serine-threonine kinase or a human tyrosine kinase.
N3. The composition of embodiment N2, wherein the protein kinase is selected from the group consisting of CK2, CK2α2, PIMl, PIM2, POvD, CDKl/cyclinB, c-RAF, Mer, MELK, HIPK3, HIPK2, ZIPK, FLTl, FLT2, FLT3, FLT3 (D835Y), and FLT4.
Pl . A compound of Formula (I), (II), (HI), (IV), (V), or (VI) which is a compound in one of the Tables provided herein or a pharmaceutically acceptable salt thereof.
Ql . A compound of Formula (I), selected from the group consisting of:
Figure imgf000149_0001
or a pharmaceutically acceptable salt thereof.

Claims

Claims
1. A compound of Formula (I):
Figure imgf000150_0001
and pharmaceutically acceptable salts, esters, and tautomers thereof; wherein: each Z1, Z2, Z\ and Z4 is N, CH, or CR\ provided that none, one or two of Z1, Z2, Z\ and Z4 are N; and further provided that one or more of Z1, Z", Z\ and Z4 is CR1; each R1 is independently an optionally substituted C l -C8 alkyl, C2-C8 heteroalkyl, C2- C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C1-C8 acyl, C2-C8 heteroacyl, C6-C 10 aryl, C5-C12 heteroaryl, C7-C 12 arylalkyl, or C6-C12 heteroarylalkyl group, or each R3 is independently halo, CF3, CFN, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, COOH, polar substituent, carboxy bioisostere, CONR2, 0OCR, COR, or NO2;
Z5 is CR( A or N; each of R(lA, R(lB and R(l< is independently H or an optionally substituted C 1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C 1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl, C5-C 12 heteroaryl, C7-C12 arylalkyl, or C6-C 12 heteroarylalkyl group, or each of R(lA, R(lB and R< ( can be halo, CF3, OR, NR2, NROR, NRNR2, SR, SOR, SO2R, SO2NR2, NRSO2R, NRCONR2, NRCOOR, NRCOR, CN, COOR, CONR2, 0OCR, COR, 01 NO2; wherein each R is independently H or C 1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C 1-C8 acyl, C2-C8 heteroacyl, C6- C lO aryl, C5-C 10 heteroaryl, C7-C12 arylalkyl, or C6-C12 heteroarylalkyl. and wherein two R on the same atom or on adjacent atoms can be linked to form a 3-8 membered ring, optionall) containing one or more N, O or S, and each R group, and each ring formed b\ linking two R groups together, is optionalh substituted with one or more substituents selected from halo, =0, =N-CN, =N-OR', =NR', OR', NR;, SR', SO.R', SO.NR;, NR'SO.R', NRCONR;, NRCOOR', NRCOR', CN, COOR', CONR';, 0OCR', COR', and NO;, wherein each R' is independent!) H, C1-C6 alk\l, C2-C6 heteroalk\l, C1-C6 ac\l, C2- Cb heteroac\l, C6-C10 ar\l, C5-C10 heteroanl, C7-12 an, IaIk)I. orC6-12 heteroai) IaIk)I, each of which is optionall) substituted with one or more groups selected from halo, C1-C4 alk\l, Cl- C4 heteroalk)l, C1-C6 ac\l, C1-C6 heteroac\l, h\drow, amino, and =0, and wherein two R' can be linked to form a 3-7 membered ring optionall) containing up to three heteroatoms selected from N, O and S, and
R1" is an optionall) substituted fi\e-membered or sivmembered carboc\chc or heteroc)clic aromatic ring.
2. The compound of claim 1, wherein Z^ is N.
3. The compound of claim 1, wherein Z^ is CR( Λ
4. The compound of claim 1, wherein at least one R' is a polar substituent, wherein said polar substituent is a carbowlic acid, carbowlate salt, ester, carbo\amide, tetrazole or carbow bioisostere.
5. The compound of claim 1, wherein each of each Z1, Z' and Z1 is CH, and Z" is CR\ where R' is a carbowlic acid, carbow late salt, ester, carbo\amide, tetrazole or carbow bioisostere.
6. The compound of claim 1, wherein at least one of Z -Z is N.
7. The compound of claim 1, wherein R1" is an optionall) substituted sivmembered carbocNclic or heteroc\chc aromatic πns.
8. The compound of claim 7, wherein R1" is an optionalh substituted phen\l or p\πd\l ring.
9. The compound of claim 8, wherein R1" is phen\l optionalh substituted with one or more halo, CN, Ci j alk\ 1, Ci j alkow , NH;, or NHAc substituents.
10. The compound of claim 1, wherein R > i(ι is an optionalh substituted fi\e- membered heteroc\chc aromatic ring.
11. The compound of claim 10, wherein R1" is an optionalh substituted puazole, iso\azole or thiophene ring.
12. The compound of claim 1 , w herein at least one of R( B or R( ( is H.
< B
13. The compound of claim 1, wherein R and R are H.
14. Tlie compound of claim 2, wherein R( is H, SR, SO;R or NR;, where each R is independent!) H or Ci j alk\l.
15. Tlie compound of claim 3, wherein R is H.
16. Tlie compound of claim 1 having the structure of Formula (III), (IV), (V), or
(VI)
Figure imgf000152_0001
Figure imgf000153_0001
and pharmaceutical!) acceptable salts, esters, and tautomers thereof.
17, A compound of Formula (II)
Figure imgf000153_0002
and pharmaceutically acceptable salts, esters, and tautomers thereof, wherein each Z1, Z", Z\ and Z"1 is N, CH, or CR\ provided that none, one or two of Z1, Z", Z \ and ZJ are N, and further pro\ ided that one or more of Z1, Z", Z\ and ZJ is CR\ each R' is independenth an optionalh substituted C1-C8 alk\l, C2-C8 heteroalk\l, C2- C8 alkeml, C2-C8 heteroalkeml, C2-C8 alk\n\l, C2-C8 heteroalk\n\l, C1-C8 ac\l, C2-C8 heteroac\l, C6-C10 an,l, C5-C12 heteroanl, C7-C12 an, IaIb1I, orC6-C12 heteroaiΛ lalk\ 1 group, or each R' IS independentl} halo, CF,, OR, NR2, NROR, NRNR2, SR, SOR, SO;R, SO;NR;, NRSO2R, NRCONR;, NRCOOR, NRCOR, CN, COOR, COOH, polar substituent, bioisostere, CONR;, 0OCR, COR, or NO;.
Z\sCR<ΛorN. each of R( Λ and R( B is independentl) H or an optionalh substituted C1-C8 alk\l, C2-C8 heteroalk\l, C2-C8 alkeml, C2-C8 heteroalkeml, C2-C8 alk\ml, C2-C8 heteroalk\n\l, C1-C8 ac\l,C2-C8heteiOac\l,C6-C10ai-}l,C5-C12heteiOai-}l,C7-C12ar\lalk\l,orC6-C12 heteroai) IaIk) 1 group. or each of R( Λ and R( B can be halo, CF,, OR, NR2, NROR, NRNR2, SR, SOR, SO.R, SO.NR;, NRSO.R, NRCONR;, NRCOOR, NRCOR, CN, COOR, CONR;, 0OCR, COR, or
NO;. wherein each R is independent!) H 01CI-C8 alk\l, C2-C8 heteroalk\l, C2-C8 alkeml, C2-C8 heteroalkeml, C2-C8 alk\n\l, C2-C8 heteroalk\n\l, C1-C8 ac\l, C2-C8 heteroac\l, C6- ClO ar\l. C5-C10 heteroarλl, C7-C12 an, IaIk)I. orC6-C12 heteroan, IaIk)I, and wherein two R on the same atom or on adjacent atoms can be linked to form a 3-8 membered ring, optionall) containing one or more N, O or S, and each R group, and each ring formed b\ linking two R groups together, is optionall) substituted with one or more substituents selected from halo, =0, =N-CN, =N-0R', =NR', OR', NR;, SR', SO.R', SO.NR;, NR SO.R', NRCONR;, NRCOOR', NRCOR', CN, COOR', CONR';, 0OCR', COR', and NO;, wherein each R' is independent!) H, C1-C6 alk)l, C2-C6 heteroalk)l, C1-C6 ac\l, C2- Cb heteroac)l, C6-C10 ar\l, C5-C10 heteroanl, C7-12 an, IaIk)I. orC6-12 heteroan, IaIk)I, each of which is optionall) substituted with one or more groups selected from halo, C1-C4 alk\l, Cl- C4 heteroalk)l, C1-C6 ac_\l, C1-C6 heteroac\l, h\dro\\, amino, and =0, and wherein two R' can be linked to form a 3-7 membered ring optionall) containing up to three heteroatoms selected from N, O and S, and
R1" is an optionall) substituted fi\e-membered or sivmembered carboc\chc or heteroc)clic aromatic ring.
18. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutical!) acceptable e\cipient.
19. A pharmaceutical composition comprising a compound of claim 17 and a pharmaceutical!) acceptable e\cipient.
20. A method for identifying a candidate molecule that interacts with a casein kinase 2 (CK2) protein, which method comprises contacting a composition containing a CK2 protein and a compound having a structure of Formula < I ), < II ), < III ), < IV ), (V). or ( VI ) w ith a candidate molecule under conditions in w Inch the compound and the protein interact, and determining whether the amount of the compound that interacts with the protein is modulated relative to a control interaction between the compound and the protein without the candidate molecule, wherein a candidate molecule that modulates the amount of the compound interacting with the protein relative to the control interaction is identified as a candidate molecule that interacts with the protein.
21. The method of claim 20, wherein the CK2 protein comprises the amino acid sequence of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3, or a substantiallv identical variant thereof.
22. The method of claim 20, wherein the protein is in a cell.
23. The method of claim 20, wherein the protein is in a cell-free svstem.
24. The method of claim 20, wherein the protein, the compound or the molecule is in association with a solid phase.
25. The method of claim 20, wherein the interaction between the compound and the protein is detected via a detectable label.
26. The method of claim 20, wherein the interaction between the compound and the protein is detected without a detectable label.
27. A method for modulating the activitv of a CK2 protein, which method comprises contacting a svstem comprising the protein with a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) in an amount effective for modulating the activitv of the protein.
28. The method of claim 27, wherein the activitv of the protein is inhibited.
29. The method of claim 27, wherein the CK2 protein comprises the amino acid sequence of SEQ ID NO 1, SEQ ID NO 2 or SEQ ID NO 3, or a substantiallv identical variant thereof.
30. The method of claim 27, wherein the svstem is a cell.
31. The method of claim 27, wherein the svstem is a cell-free svstem.
32. The method of claim 27, wherein the protein, the compound or the molecule is in association with a solid phase.
33. A method for inhibiting cell proliferation, which method comprises contacting cells w ith a compound ha\ ing a structure of Formula ( I ), ( II ), ( III ), ( I V ), ( V ), or ( VI ) in an amount effective to inhibit proliferation of the cells.
34. The method of claim 33, wherein the cells are in a cancer cell line.
35. The method of claim 34, wherein the cancer cell line is a breast cancer, prostate cancer, pancreatic cancer, lung cancer, hemopoietic cancer, colorectal cancer, skin cancer, or ovarv cancer cell line.
36. The method of claim 33, wherein the cells are in a tumor in a subject.
37. The method of claim 33, wherein contacting cells with a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) induces cell apoptosis.
38. The method of claim 33, wherein the cells are from an eve of a subject having macular degeneration.
39. The method of claim 33, wherein the cells are in a subject having macular degeneration.
40. A method for treating a condition related to aberrant cell proliferation, which method comprises administering a compound having a structure of Formula (I), (II), (III), (IV), (V), or (VI) to a subject in need thereof in an amount effective to treat the cell proliferative condition.
41. The method of claim 40, w herein the cell proliferativ e condition is a tumor- associated cancer.
42. The method of claim 41 , w herein the cancer is of the colorectum, breast, o\ arv , lung, liv er, pancreas, lv mph node, colon, prostate, brain, head and neck, skin, liv er, kidnev , blood and heart.
43. The method of claim 40, w herein the cell proliferativ e condition is a non-tumor cancer.
44. The method of claim 43, w herein the non-tumor cancer is a hematopoietic cancer.
45. The method of claim 40, w herein the cell proliferativ e condition is macular degeneration.
46. A method for identifv ing a candidate molecule that interacts w ith a protein kinase, w hich comprises contacting a composition containing a protein kinase and a compound hav ing a structure of Formula ( I ), ( II ), ( III ), ( IV ), ( V ), or ( VI ) under conditions in w Inch the compound and the protein interact w ith a candidate molecule, and determining w hether the amount of the compound that interacts w ith the protein kinase is modulated relativ e to a control interaction betw een the compound and the protein kinase w ithout the candidate molecule, w herebv a candidate molecule that modulates the amount of the compound interacting w ith the protein kinase relativ e to the control interaction is identified as a candidate molecule that interacts w ith the protein kinase.
47. The method of claim 46, w herein the protein kinase is serine-threonine protein kinase or a tv rosine protein kinase.
48. The method of claim 46, w herein the protein kinase is a human serine-threonine protein kinase or a human tv rosine protein kinase.
49. The method of claim 48, w herein the protein kinase is a human serine-threonine protein kinase selected from the group consisting of CK2, CK2α2, PIM l , PIM2, PIM3, CDK l/c\ clinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK.
50. The method of claim 47, w herein the protein kinase is a serine-threonine protein kinase w hich contains one or more of the follow ing amino acids at positions corresponding to those listed in human CK2 leucine at position 45, methionine at position 163 and isoleucine at position 174.
51. The method of claim 50, w herein the serine threonine protein kinase is selected from the group consisting of CK2, STK lO, HIPK2, HIPK3, DAPK3, DYK2 and PIM- I .
52. The method of claim 48, w herein the protein kinase is a human tuosine protein kinase selected from the group consisting of FLTl , FLT2, FLT3, FLT3 ( D835Y ), and FLT4.
53. The method of claim 46, w herein the protein, the compound or the molecule is in association w ith a solid phase.
54. The method of claim 46, w herein the interaction betw een the compound and the protein is detected v ia a detectable label.
55. The method of claim 46, w herein the protein kinase actiwt\ is the transfer of a gamma phosphate from adenosine triphosphate to a peptide or protein substrate.
56. A method for modulating a protein kinase actiwt\ , w hich comprises contacting a s) stem comprising a protein kinase protein w ith a compound of Formula < I ), < II ), < III ), < I V ), < V ), or ( VI ) in an amount effective for modulating the actiwt\ of the protein kinase.
57. The method of claim 56, w herein the protein kinase is serine-threonine protein kinase or a ty rosine protein kinase.
58. The method of claim 56, wherein the protein kinase is a human serine-threonine protein kinase or a human tvrosine protein kinase.
59. The method of claim 58, wherein the protein kinase is a human serine-threonine protein kinase selected from the group consisting of CK2, CK2α2, PIMl, PIM2, PIM3, CDKl/cvclinB, c-RAF, Mer, MELK, HIPK3, HIPK2 and ZIPK.
60. The method of claim 58, wherein the protein kinase is a human tvrosine protein kinase selected from the group consisting of FLTl, FLT2, FLT3, FLT3 (D835Y), and FLT4.
61. A method for treating pain or inflammation in a subject, which comprises administering a compound of Formula ( I ), ( II ), ( III ), ( IV ), ( V ), or ( VI Mo a subject in need thereof in an amount effective to treat the pain or the inflammation.
62. A method for inhibiting angiogenesis in a subject, which comprises administering a compound of Formula ( I ), ( II ), ( III ), ( I V ), ( V ), or ( VI ) to a subject in need thereof in an amount effective to inhibit the angiogenesis.
63. A composition of matter comprising a protein kinase and a compound of Formula (I). (II). (III). (IV). (V). or (VI).
64. The composition of claim 63, wherein the protein kinase is a human serine- threonine kinase or a human tvrosine kinase.
65. The composition of claim 64, wherein the protein kinase is selected from the group consisting of CK2, CK2α2, PIMl, PIM2, PIM3, CDKl/cvclinB, c-RAF, Mer, MELK, HIPK3, HIPK2, ZIPK, FLTl, FLT2, FLT3, FLT3 (D835Y), and FLT4.
66. A compound of Formula ( I ), ( II ), ( III ), ( I V ), ( V ), or ( VI ) w Inch is a compound in one of the Tables provided herein or a pharmaceutical!) acceptable salt thereof.
67. A compound of Formula ( I ), selected from the group consisting of:
Figure imgf000160_0001
or a phaπnaceuticall) acceptable salt thereof.
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