EP1042509A1 - Techniques permettant d'utiliser des banques chimiques pour rechercher de nouveaux inhibiteurs des kinases - Google Patents

Techniques permettant d'utiliser des banques chimiques pour rechercher de nouveaux inhibiteurs des kinases

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Publication number
EP1042509A1
EP1042509A1 EP98964881A EP98964881A EP1042509A1 EP 1042509 A1 EP1042509 A1 EP 1042509A1 EP 98964881 A EP98964881 A EP 98964881A EP 98964881 A EP98964881 A EP 98964881A EP 1042509 A1 EP1042509 A1 EP 1042509A1
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Prior art keywords
mrna transcripts
compounds
chain
cell
oligonucleotides
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EP98964881A
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German (de)
English (en)
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Nathanael S. Gray
Peter Schultz
Lisa Wodicka
Laurent Meijer
David J. Lockhart
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Centre National de la Recherche Scientifique CNRS
University of California
Affymetrix Inc
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Centre National de la Recherche Scientifique CNRS
University of California
Affymetrix Inc
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Publication of EP1042509A1 publication Critical patent/EP1042509A1/fr
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/16—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 two nitrogen atoms
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    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Phosphorylation of serine, threonine and tyrosine residues by protein kinases represents one of the most common post-translational regulatory modifications of proteins. More than 200 protein kinases have been described, following either purification to homogeneity or molecular cloning (see, Hunter, T. (1991), Methods Enzymol, 200:3-37; Hanks, S.K., et al. (1991), Methods Enzymol, 200:38-81 ; Hanks, S.K. 1991), Curr. Opin. Struct. Biol, 1 :369-383; and Hubbard, M.J., et al. (1993) Trends Biochem. Sci., 18:172-177).
  • CDK Cyclin-dependent kinases
  • CDKl also known as CDC2
  • CDK3 for which the regulatory cyclin has not yet been identified, all these CDKs proteins are regulated by the transient association with one member of the cyclin family, i.e., cyclin A (CDC2, CDK2), B1-B3 (CDC2), D1-D3 (CDK2, CDK4, CDK5, CDK6), E (CDK2).
  • cyclin A CDC2, CDK2
  • B1-B3 CDC2
  • D1-D3 CDK2, CDK4, CDK5, CDK6
  • E CDK2
  • CDK complexes CDK complexes: d/S transition (CDK2/cyclin E, CDK3/unknown cyclin, CDK4/cyclin Dl- D3, CDK6/cyclin D3), S phase (CDK2/cyclin A), G 2 (CDC2/cyclin A), G 2 /M transition (CDC2/cyclins B).
  • CDKs are able to phosphorylate many proteins involved in cell cycle events, including histones, lamins and tumor suppressor proteins, such as the retinoblastoma gene product pRb (see, Norbury, C, et al., supra, Matsushime, H., et al. (1992), Cell, 71:323-334, Nigg, E.E. (1993), Curr. Opin. Cell. Biol, 5:187-193).
  • enzyme activity is tightly controlled by multiple mechanisms.
  • Thr-161 in CDC2 requires complex formation with regulatory cyclin proteins as described above, followed by an activating phosphorylation on Thr-161 in CDC2 or the corresponding Thr in the other CDKs (see, e.g., Gould, K.L., et al. (1991), EMBO J, 10:3297-3309; Desai, D., et al. (1992), Mol. Biol. Cell, 3:571-582; Solomon, M.J., et al. (1992), Mol. Biol. Cell, 3:13-27).
  • enzyme activity is negatively regulated by phosphorylations at Tyr-15 and/or Thr- 14 (see, e.g., Solomon, M.J., et al, supra; Gu, Y., et al. (1992), EMBO J., 11:3995-4005; Krek, W., et al. (1991), EMBOJ., 10:3331-3341; Norbury, C., et ⁇ /. (1991), EMSO J, 10:3321-3329; Parker, L.L., et al. (1992), Proc. Nat 7. Acad. Sci. U.S.A., 89:2917-2921; McGowan, C.H., et al.
  • CDKs are a promising target for developing inhibitors with antineoplastic effects and for the treatment of cell-pro liferative diseases.
  • the purine ring system is a key structural element of the substrates and ligands of many biosynthetic, regulatory and signal transduction proteins including cellular kinases, G proteins and polymerases. As such, the purine ring system has been a good starting point in the search for inhibitors of many biomedically significant processes.
  • a relatively selective inhibitor, olomoucine (Figure 1), was identified that competitively inhibited CDK2/cyclin A with an IC 50 of 7 ⁇ M (see, Vesely, J., et al, (1994) Eur. J. Biochem., 224:771-786).
  • the present invention provides for methods of identifying compounds which modulate cell proliferation.
  • the methods comprise the steps of (i) treating at least one cell with at least one compound, (ii) isolating a plurality of mRNA transcripts from said cell, and (iii) comparing a plurality of mRNA transcripts from a cell not treated with the compound to the mRNA transcripts from the treated cell, whereby a decrease in the number of mRNA transcripts indicates an inhibition of cell proliferation.
  • the compounds are inhibitors of cyclin-dependent kinases.
  • the mRNA transcripts are converted to cRNA.
  • the mRNA transcripts encode proteins associated with cell proliferation.
  • the mRNA is isolated by hybridization under stringent conditions to oligonucleotide probes of about 15 to about 50 nucleotides complementary to nucleic acids which encode proteins associated with cell proliferation.
  • the oligonucleotides are linked to a solid support in a high density array.
  • a method of determining the identity of proteins that modulate cell proliferation during or after exposure to chemical or genetic challenges comprises the steps of (i) isolating mRNA transcripts generated from cells after exposure to compounds known to modulate cellular proliferation, (ii) isolating mRNA transcripts generated from cells not exposed to said compounds, (iii) comparing the total number of mRNA transcripts from both treated and untreated cells, and (iv) determining which proteins are encoded by mRNA transcripts present in differing amounts in treated or untreated cells.
  • the compounds are cyclin-dependent kinase inhibitors.
  • the mRNA transcripts are converted to cRNA.
  • the mRNA is isolated by hybridization under stringent conditions to oligonucleotides of about 15 to about 50 nucleotides in length which are complementary to nucleic acids that encode proteins associated with cell proliferation.
  • the oligonucleotides are linked to a solid supporte in a high density array.
  • a method of determining proteins associated with increased drug resistance comprises the steps of (i) isolating mRNA transcripts generated from drug-resistant cells after exposure to drugs known to inhibit cellular proliferation, (ii) isolating mRNA transcripts generated from non-drug resistant cells exposed to said drugs, (iii) comparing the total number of mRNA transcripts from both drug-resistant and non-resistant cells, and (iv) determining which proteins are encoded by mRNA transcripts present in increased amounts in the drug- resistant cells.
  • the compounds are cyclin-dependent kinase inhibitors.
  • the mRNA transcripts are converted to cRNA.
  • the mRNA is isolated by hybridization under stringent conditions to oligonucleotides of about 15 to about 50 nucleotides in length which are complementary to nucleic acids that encode proteins associated with cell proliferation.
  • the oligonucleotides are linked to a solid supporte in a high density array.
  • Figure 1 sets forth the structure of olomoucine and the numbering scheme for the purine nucleus.
  • FIGS. 2 and 3 illustrate the IC 50 for representative compounds from Table 1.
  • Figure 4 A provides a scheme for the combinatorial synthesis of 2,6,9- trisubstituted purines from a 2, 6, or 9 linked purine scaffold using amination and alkylation chemistries.
  • Figure 5 shows schematic drawing of CDK2 -purvalanol B interactions.
  • Protein side chain contacts are indicated by lines connecting the respective residue box while interactions to main chain atoms are shown as lines to the specific main chain atoms.
  • Van der Waals contacts are indicated by thin dotted lines, and hydrogen bonds by dashed lines. For hydrogen bonds the distances between the non-hydrogen atoms are indicated in angstroms.
  • Figure 6 shows representative transcripts observed to change more than two fold for triplicate hybridizations for each of two independent experiments: (A) names of the genes whose mRNA levels change in common to compound 52 and flavopiridol and (B) transcript changes that may result from Pho85p kinase inhibition observed in either the compound 52 or flavopiridol profiles; and (C) transcripts that change for cdc28- 4, cdc28-4 and compound 52, cdc28-4 and flavopiridol, and compound 52.
  • the present invention provides a combinatorial approach to modifying the purine scaffold to better aid in the search for potent and specific inhibitors of various purine-utilizing enzymes.
  • CDKs cyclin-dependent kinases
  • CDK/cyclin complexes are negatively regulated in response to a variety of antiproliferative signals including myogenic (Parker, Science 59:66 (1994)), myeloid (Liu, et al, Genes Dev. 10, 142-153 (1996)), contact inhibition, and DNA damage checkpoints ( El-Deiry, Cell 75, 817-825 (1993)).
  • ATP binding site was targeted by screening combinatorial libraries of 2, 6, 9-trisubstituted purines.
  • R groups e.g., R 1 , R 2 , R 4 and R 3
  • R 1 , R 2 and R 3 can be identical or different (e.g., R 1 , R 2 and R 3 may all be substituted alkyls or R 1 and R 2 may be a substituted alkyl and R 3 may be an aryl, etc.).
  • R group will generally have the structure which is recognized in the art as corresponding to R groups having that name.
  • representative R groups as enumerated above are defined herein. These definitions are intended to supplement and illustrate, not preclude, the definitions known to those of skill in the art.
  • alkyl is used herein to refer to a branched or unbranched, saturated or unsaturated, monovalent hydrocarbon radical having from 1-12 carbons and preferably, from 1-6 carbons. When the alkyl group has from 1-6 carbon atoms, it is referred to as a "lower alkyl.”
  • Suitable alkyl radicals include, for example, methyl, ethyl, w-propyl, z ' -propyl, 2-propenyl (or allyl), n-butyl, t-butyl, /-butyl (or 2-methylpropyl), etc.
  • Substituted alkyl refers to alkyl as just described including one or more functional groups such as lower alkyl, aryl, acyl, halogen (i.e., alkylhalos, e.g., CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, aryloxy, aryloxyalkyl, mercapto, both saturated and unsaturated cyclic hydrocarbons, heterocycles and the like. These groups may be attached to any carbon of the alkyl moiety.
  • aryl is used herein to refer to an aromatic substituent which may be a single aromatic ring or multiple aromatic rings which are fused together, linked covalently, or linked to a common group such as a methylene or ethylene moiety.
  • the common linking group may also be a carbonyl as in benzophenone.
  • the aromatic ring(s) may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone among others.
  • arylalkyl is used herein to refer to a subset of “aryl” in which the aryl group is attached through an alkyl group as defined herein.
  • Substituted aryl refers to an aryl as just described and including one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto and both saturated and unsaturated cyclic hydrocarbons fused to the aromatic ring(s), linked covalently or linked to a common group such as a methylene or ethylene moiety.
  • the linking group may also be a carbonyl such as in cyclohexyl phenyl ketone.
  • substituted aryl encompasses "substituted arylalkyl.”
  • Substituted arylalkyl defines a subset of "substituted aryl” wherein the substituted aryl group is attached through an alkyl group as defined herein.
  • halogen is used herein to refer to fluorine, bromine, chlorine and iodine atoms.
  • hydroxy is used herein to refer to the group COH.
  • amino is used herein to refer to the group CNRRN, where R and RN may independently be hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl or acyl.
  • alkoxy is used herein to refer to the COR group, where R is a lower alkyl, substituted lower alkyl, aryl, substituted aryl, arylalkyl or substituted arylalkyl wherein the alkyl, aryl, substituted aryl, arylalkyl and substituted arylalkyl groups are as described herein.
  • Suitable alkoxy radicals include, for example, methoxy, ethoxy, phenoxy, substituted phenoxy, benzyloxy, phenethyloxy, t-butoxy, etc.
  • alkylamino denotes secondary and tertiary amines wherein the alkyl groups may be either the same or different and may consist of straight or branched, saturated or unsaturated hydrocarbons.
  • heterocyclic is used herein to describe a monovalent group having a single ring or multiple condensed rings from 1-12 carbon atoms and from 1-4 heteroatoms selected from nitrogen, sulfur or oxygen within the ring.
  • heterocycles are, for example, tetrahydrofuran, morpholine, piperidine, pyrrolidine, thiophene, pyridine, isoxazole, phthalimide, pyrazole, indole, furan, benzo-fused analogs of these rings, etc.
  • substituted heterocyclic as used herein describes a subset of “heterocyclic” wherein the heterocycle nucleus is substituted with one or more functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
  • functional groups such as lower alkyl, acyl, halogen, alkylhalos (e.g., CF 3 ), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy, mercapto, etc.
  • pharmaceutically acceptable salt refers to those salts of compounds which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, ⁇ -toluenesulfonic acid, salicylic acid and the like.
  • Pharmaceutically acceptable salts include, for example, alkali metal salts, such as sodium and potassium, alkaline earth salts and ammonium salts.
  • purine compounds of present invention can be “administered” by any conventional method such as, for example, parenteral, oral, topical and inhalation routes as described herein.
  • An amount sufficient or “an effective amount” is that amount of a given purine analog which exhibits the binding/inhibitory activity of interest or, which provides either a subjective relief of a symptom(s) or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • “Complementary” refers to the topological compatibility or matching together of interacting surfaces of a ligand molecule and its receptor.
  • the receptor and its ligand can be described as complementary, and furthermore, the contact surface characteristics are complementary to each other.
  • a "ligand” is a molecule that is recognized by a particular receptor.
  • ligands that can be investigated by this invention include, but are not restricted to, cRNA, mRNA and other oligonucleotides, agonists and antagonists for cell membrane receptors, toxins and venoms, viral epitopes, hormones (e.g., steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates, cofactors, (e.g., opiates, etc.), lectins, sugars, oligosaccharides, proteins, and monoclonal antibodies.
  • Nucleic acids include DNA and RNA, as well as individual nucleotides and oligonucleotides.
  • RNA is mRNA.
  • cRNA The complement of mRNA. Its preparation is well known to those of skill and is described in Gray, et al, Science 281:533 (1998) which is hereby incorporated in its entirety for all purposes.
  • cRNA is used synonymously with mRNA.
  • stringent hybridization conditions or “stringency” refers to conditions in a range from about 5°C to about 20°C or 25°C below the melting temperature (Tm) of the target sequence and a probe with exact or nearly exact complementarity to the target.
  • Tm melting temperature
  • the melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half-dissociated into single strands.
  • Tm 81.5 + 0.41 (% G + C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter
  • stringent hybridization conditions are salt concentrations less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion at pH 7.0 to 8.3, and temperatures at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 65°C for long probes (e.g., greater than 50 nucleotides).
  • stringent conditions may also be achieved with the addition of destabilizing agents such as formamide, in which case lower temperatures may be employed.
  • genetic challenge refers to an aberration in the DNA of the cell.
  • An example of a genetic challenge is a mutation, either a single nucleotide exchange, an addition of one or more nucleotides, or a deletion of one or more nucleotides. Mutations are induced by techniques well known in the art, e.g., UV irradiation, and exposure to compounds known to cause knicks and cuts in either one or both strands of DNA.
  • Chemical challenges are the addition of compounds which, in addition to causing mutations in DNA also cause aberrations in cell proliferation, metabolism and catabolism.
  • Such compounds include, but are not limited to, the purine analogs of this invention.
  • a "receptor” is a molecule that has an affinity for a given ligand.
  • Receptors may be naturally-occurring or manmade molecules. Also, they can be employed in their unaltered state or as aggregates with other species.
  • Receptors may be attached, covalently or noncovalently, to a binding member, either directly or via a specific binding substance.
  • Examples of receptors which can be employed by this invention include, but are not restricted to, oligonucleotides, cell membrane receptors, monoclonal antibodies and antisera reactive with specific antigenic determinants (such as on viruses, cells or other materials), drugs, sugars, polysaccharides, cells, cellular membranes, and organelles.
  • Receptors are sometimes referred to in the art as anti-ligands. As the term receptors is used herein, no difference in meaning is intended.
  • a "ligand receptor pair" is formed when two macromolecules have combined through molecular recognition to form a complex.
  • “Monomer” is a member of the set of small molecules which can be joined together to form a polymer.
  • the set of monomers includes but is not restricted to, for example, the set of common nucleotides, the set of synthetic nucleotides, the set of nucleotide analogs and the set of pentoses and hexoses.
  • monomers refers to any member of a basis set for synthesis of a polymer. For example, dimers of nucleotides form a basis set of 400 monomers for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer.
  • Random energy is energy which may be selectively applied including energy having a wavelength of between 10 " and 10 4 meters including, for example, electron beam radiation, gamma radiation, x-ray radiation, ultraviolet radiation, visible light, infrared radiation, microwave radiation, and radio waves.
  • Irradiation refers to the application of radiation to a surface.
  • substrate refers to a material having a rigid or semi-rigid surface.
  • at least one surface of the substrate will be substantially flat, although in some embodiments it may be desirable to physically separate synthesis regions for different polymers with, for example, wells, raised regions, etched trenches, or the like. According to other embodiments, small beads may be provided on the surface which may be released upon completion of the synthesis.
  • protective group refers to a material which is bound to a monomer unit and which may be spatially removed upon selective exposure to an activator such as electromagnetic radiation.
  • an activator such as electromagnetic radiation.
  • protective groups with utility herein include nitroveratryloxy carbonyl, nitrobenzyloxy carbonyl, dimethyl dimethoxybenzyloxy carbonyl, 5-bromo-7-nitroindolinyl, o-hydroxy- alpha -methyl cinnamoyl, and 2-oxymethylene anthraquinone.
  • Other examples of activators include ion beams, electric fields, magnetic fields, electron beams, x-ray, and the like.
  • predefined region refers to a predefined region is a localized area on a surface which is, was, or is intended to be activated for formation of a polymer.
  • the predefined region may have any convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc.
  • predefined regions are sometimes referred to simply as “regions.”
  • substantially pure refers to a polymer that is considered to be “substantially pure” within a predefined region of a substrate when it exhibits characteristics that distinguish it from other predefined regions. Typically, purity will be measured in terms of biological activity or function as a result of uniform sequence. Such characteristics will typically be measured by way of binding with a selected ligand or receptor.
  • the purine ring is a key structural element of the substrates and ligands of many biosynthetic, regulatory and signal transduction proteins including cellular protein kinases, G proteins and polymerases.
  • the present invention provides purine analogs which can be used to inhibit such proteins and, thus, many biomedically important processes. More particularly, the present invention provides purine analogs that inhibit, inter alia, protein kinases and other cellular processes. As such, the purine analogs of the present invention can be used to block cell-cycle progression, cellular proliferation, and apoptosis as well as other cellular processes.
  • the purine analogs of the present invention are active in the subnanomolar and submicromolar ranges.
  • the present invention provides for methods of screening purine analogs having the generally formula:
  • R 1 , R 2 , R 4 and R 5 are independently selected and are functional groups including, but not limited to, H, C ⁇ -C 8 straight-chain, branched-chain, saturated and unsaturated alkyl, CpC 8 straight-chain, branched-chain, saturated and unsaturated substituted alkyl, aryl and substituted aryl.
  • R 1 and R 2 are independently selected and are functional groups including, but not limited to, H, aryl, substituted aryl, C ⁇ -C 8 straight-chain, saturated alkyl substituted with aryl and C ⁇ -C 8 straight-chain, saturated alkyl substituted with substituted aryl;
  • R 3 is a functional group including, but not limited to, -Cs branched-chain saturated alkyl and C ⁇ -C 8 branched-chain unsaturated alkyl;
  • R 4 and R 5 are independently selected and are functional groups including, but not limited to, H, C ⁇ -C 8 straight-chain, branched-chain, saturated and unsaturated alkyl, C ⁇ -C 8 straight- chain, branched-chain, saturated and unsaturated substituted alkyl, aryl and substituted aryl.
  • R 1 and R 2 are independently selected and are functional groups including, but not limited to, H, unsubstituted aryl and substituted aryl; R 3 is isopropyl; and R 4 and R 3 are independently selected and are functional groups including, but not limited to, H, C ⁇ -C 8 saturated and unsaturated branched-chain alkyl and C ⁇ -C 8 saturated and unsaturated branched-chain substituted alkyl.
  • R 5 are independently selected and are functional groups including, but not limited to, H, and
  • X is a member selected from the group consisting of H, OH, CH 2 OH, C(O)NH2, SH, COOH or a pharmaceutically acceptable salt thereof, and COOR 7 , wherein R 7 is lower alkyl; and R 6 is a member selected from the group consisting of H, C ⁇ -C 8 straight - chain alkyl, C ⁇ -C 8 branched-chain alkyl, CpC 8 straight-chain substituted alkyl, CpC branched-chain substituted alkyl.
  • R and R are independently selected and are functional groups including, but not limited to, H and aryl substituted in at least one of positions 3, 4, or 5 with a member independently selected from the group consisting of halogen, alkoxy, trihalomethyl, amino, hydroxyl, thiol, sulfonic acid, sulfonic acid, amide, ester and carboxylic acid.
  • Table 1 sets forth purine compounds in accordance with the present invention which are particularly preferred. The compounds in this table and throughout this specification are refened to by code numbers, which are used for convenience only, and are strictly arbitrary for purposes of this invention.
  • IC 50 S can be compared with other known small molecule inhibitors of CDK2 (see, Figures 2 and 3). It will be readily appreciated by those of skill in the art that depending on the substituents, the purine analogs of the present invention can be a racemic mixture or either of a pair of diastereomers or enantiomers.
  • the purine analogs of the present invention can be synthesized in a variety of ways, using conventional synthetic chemistry techniques.
  • the compounds of the present invention are prepared according to Scheme I, wherein R 1 , R 2 , R 3 R 4 , and R 3 are as defined above.
  • the use of appropriate organic solvents, temperature and time conditions for running the reactions are within the level of skill in the art. Reactions of this type are generally described by Norman, et al, J. Am. Chem. Soc. 118:7430-7431 (1996); and Gray, et al, Tetrahedron Letters 38:1161-1164 (1997), the teachings of which are incorporated herein by reference.
  • suitable synthesis reactions are illustrated herein by the representative examples. Necessary starting materials can be obtained by standard procedures of organic chemistry.
  • a purine derivative with a halogen at the 2-position is alkylated at the 9-position with an alcohol using the Mitsonubo alkylation. Following the alkylation, the purine derivative is aminated at the 6-position with an amine.
  • the purine analogs can be purified (e.g., by TLC), characterized (e.g., by Reverse Phase HPLC) and analyzed (e.g., by high resolution spectroscopy using, for example, 1H NMR or FAB-MS).
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by combining a number of chemical "building blocks" such as reagents.
  • the "building blocks” can be combined either through chemical or biological synthesis.
  • a linear combinatorial chemical library such as an oligonucleotide library is formed by combining a set of chemical building blocks called nucleotides in every possible way for a given compound length (i.e., the number of nucleotides in a nucleic acid compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, (1991) Int. J. Pept. Prot. Res. 37: 487-493, Houghton, et al. (1991) Nature 354: 84-88).
  • Peptide synthesis is by no means the only approach envisioned.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to; peptoids (PCT Publication No WO 91/19735, 26 Dec.
  • nucleic acid libraries see, e.g., Strategene, Corp.
  • peptide nucleic acid libraries see, e.g,, U.S. Patent 5,539,083 antibody libraries (see, e.g., Vaughn, et al. (1996) Nature Biotechnology 14(3): 309-314), and PCT/US96/10287)
  • carbohydrate libraries see, e.g., Liang, et al. (1996) Science 274:1520-1522, and U.S. Patent 5,593,853
  • small organic molecule libraries see, e.g., benzodiazepines: Baum (1993) C&EN, Jan 18, page 33; isoprenoids: U.S.
  • Patent 5,569,588; thiazolidinones and metathiazanones U.S. Patent 5,549,974; pyrrolidines: U.S. Patents 5,525,735 and 5,519,134; morpholino compounds: U.S. Patent 5,506,337; benzodiazepines: 5,288,514; and the like).
  • a number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate 11 , Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif) which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
  • a typical starting point is a 2-fluoro-6- chloropurine framework ( Figure 4A).
  • Substitution chemistry is then used to install amines or other functional groups at the 2- and 6-positions and, in a preferred embodiment, a Mitsunobu reaction is employed to alkylate the N-9 position of the purine core. See, Mitsonobu, Synthesis 1-28 (1981); and Toyota, et al, Heterocycles 36:1625- 1630 (1993).
  • This type of substitution chemistry allows introduction of a wide range of primary and secondary functional groups, while the Mitsunobu reaction tolerates primary and secondary alcohols lacking additional acidic hydrogens.
  • Newly appended groups are then modified combinatorially in subsequent steps using a variety of chemistries including acylation, reductive animation, and Suzuki coupling reactions (Backes, et al, J. Am. Chem. Soc. 116:11171-11172 (1994)).
  • a variety of chemistries including acylation, reductive animation, and Suzuki coupling reactions (Backes, et al, J. Am. Chem. Soc. 116:11171-11172 (1994)).
  • one position of the purine ring is held invariant to allow attachment to a solid support.
  • Libraries are then synthesized in a spatially-separated format using either a pin apparatus (Geysen, et al, Immunol. Methods 102:(1987) or a polystyrene resin, and then screened for activity.
  • the compounds are screened for kinase inhibitory activity.
  • the most basic type of screen for inhibition of activity is to assay for binding to the target compound, in the instant invention, protein kinases. From the ability to bind to the target, one can predict whether the compound being assayed will inhibit the kinase by competing for the enzyme's natural substrate. However, this type of assay is not fool-proof and some measure of functional activity is desired.
  • Purine analogs suitable for use in the methods of the present invention can readily be identified using in vitro and in vivo activity screening assays. Such assays may screen for the ability of a particular compound to inhibit malignant tumor cell growth or to abolish tumorigenicity of malignant cells in vitro or in vivo.
  • tumor cell lines can be exposed to varying concentrations of a purine analog of interest, and the viability of the cells can be measured at set time points using the Alamar BlueTM assay (commercially available from BioSource, International of Camarillo, California).
  • Alamar BlueTM dye When Alamar BlueTM dye is added to the culture medium, the dye is reduced by cellular mitochondrial enzymes and yields a soluble product with substantially enhanced fluorescence. This fluorescence is then measured with a fiuorimeter, whereby the signal is directly proportional to the cell number. Using this information, IC 5 o values 1 for the compounds of interest can be readily be calculated.
  • MDA MB 231 (breast), MCF-7 (breast), MDA MB 468 (breast), Siha (squamous cell carcinoma), A549 (non-small cell lung), HL-60 (leukemia) Ovcar-3
  • IC 50 is the concentration of compound lethal to 50% of a cell culture as compared to a control culture. (ovarian), etc.
  • the purine analogs of the present invention can be screened on the National Cancer Institute panel of 60 human tumor cell lines (see, Appendix I).
  • other in vitro and/or in vivo assays to screen for anti-tumor and/or anti-cancer activity known to and used by the skilled artisan can also be employed to identify effective purine analogs useful in the methods of the present invention.
  • the effect on mRNA transcription in the presence of the compounds of this invention is measured.
  • the compounds are added to cells in culture. After an incubation for a suitable time, the cells are solubilized in a chaotropic agent, such as guanidine hydrochloride (see, Sambrook, et al. MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989) (“Sambrook”) or CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, F. Ausubel, et al, ed. Greene Publishing and Wiley-Interscience, New York (1987) ("Ausubel”).
  • a chaotropic agent such as guanidine hydrochloride
  • mRNA is then isolated by techniques well known in the art (see, Sambrook, supra) and quantified. Quantification of mRNA can be done by agarose gel electrophoresis, UN. absorption, northern blotting, and other techniques that are standard in the field of molecular biology.
  • oligonucleotides present in the mR ⁇ A of a cell are screened for hybridization with oligonucleotides provided in a solid phase array. This technique provides the artisan with known oligonucleotides which represent known mR ⁇ A.
  • the present invention provides methods and apparatus for the preparation and use of a substrate having a plurality of polymer sequences in predefined regions. These polymer sequences are then used as a screen for purine analog activity.
  • the invention is described herein primarily with regard to the preparation of molecules containing sequences of nucleotides, but could readily be applied in the preparation of other polymers.
  • Such polymers include, for example, both linear and cyclic polymers of nucleic acids, polysaccharides, phospholipids, and peptides, heteropolymers in which a known drug is covalently bound to any of the above, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or other polymers which will be apparent upon review of this disclosure.
  • the invention herein is used in the screening of Saccharomyces cerevesiae cRNA binding.
  • the invention preferably provides for the use of a substrate "S” with a surface.
  • Linker molecules "L” are optionally provided on a surface of the substrate.
  • the purpose of the linker molecules is to facilitate receptor recognition of the synthesized polymers.
  • the linker molecules may be chemically protected for storage purposes.
  • a chemical storage protective group such as t-BOC (t-butoxycarbonyl) may be used in some embodiments, particularly when assembling peptides on the substrate.
  • Such chemical protective groups would be chemically removed upon exposure to, for example, acidic solution and would serve to protect the surface during storage and be removed prior to polymer preparation.
  • a functional group with a protective group P 0 is provided on the substrate or a distal end of the linker molecules.
  • the protective group P 0 may be removed upon exposure to radiation, electric fields, electric cunents, or other activators to expose the functional group.
  • the radiation is ultraviolet (UV), infrared (IR), or visible light.
  • the protective group may alternatively be an electrochemically-sensitive group which may be removed in the presence of an electric field.
  • ion beams, electron beams, or the like may be used for deprotection.
  • the exposed regions and, therefore, the area upon which each distinct polymer sequence is synthesized are smaller than about 1 cm or less than 1 mm .
  • the exposed area is less than about 10,000 ⁇ m" or, more preferably, less than 100 ⁇ m 2 and may, in some embodiments, encompass the binding site for as few as a single molecule.
  • each polymer is preferably synthesized in a substantially pure form.
  • the surface is contacted with a first monomer unit Mi which reacts with the functional group which has been exposed by the deprotection step.
  • the first monomer includes a protective group Pi. Pi may or may not be the same as P 0 .
  • known first regions of the surface may comprise the sequence:
  • second regions of the surface (which may include the first region) are exposed to light and contacted with a second monomer M 2 (which may or may not be the same as Mi) having a protective group P 2 .
  • P 2 may or may not be the same as Po and Pi.
  • different regions of the substrate may comprise one or more of the following sequences:
  • the above process is repeated until the substrate includes desired polymers of desired lengths.
  • the location of each sequence will be known.
  • the protective groups are removed from some or all of the substrate and the sequences are, optionally, capped with a capping unit C.
  • the process results in a substrate having a surface with a plurality of polymers of the following general formula:
  • a plurality of locations on the substrate polymers contain a common monomer subsequence. For example, it may be desired to synthesize a sequence S-M 1 -M2-M 3 at first locations and a sequence S-M -M 2 -M 3 at second locations. The process would commence with irradiation of the first locations followed by contacting with Mi-P, resulting in the sequence S-Mj-P at the first location. The second locations would then be irradiated and contacted with M 4 -P, resulting in the sequence S- M 4 -P at the second locations.
  • both the first and second locations would be irradiated and contacted with the dimer M2-M 3 , resulting in the sequence S-M 1 -M 2 -M 3 at the first locations and S-M -M 2 -M 3 at the second locations.
  • common subsequences of any length could be utilized including those in a range of 2 or more monomers, 2 to 100 monomers, 2 to 20 monomers, and a most preferred range of 2 to 3 monomers.
  • a set of masks is used for the first monomer layer and, thereafter, varied light wavelengths are used for selective deprotection.
  • first regions are first exposed through a mask and reacted with a first monomer having a first protective group Pi, which is removable upon exposure to a first wavelength of light (e.g., IR).
  • Second regions are masked and reacted with a second monomer having a second protective group P 2 , which is removable upon exposure to a second wavelength of light (e.g., UN).
  • a second wavelength of light e.g., UN
  • the polymers prepared on a substrate according to the above methods will have a variety of uses including, for example, screening for biological activity.
  • the substrate containing the sequences is exposed to an unlabeled or labeled drug, oligonucleotide, including mRNA or cRNA, receptor such as an antibody, receptor on a cell, phospholipid vesicle, and/or any one of a variety of other receptors.
  • the hybridization under stringent conditions of nucleic acid, such as mRNA or cRNA to oligonucleotides on the surface of the anay is desired.
  • Hybridization under stringent conditions is defined as maintaining hybridization in 0.2X SSC at 65°C for 15 minutes.
  • the positions of the hybridized nucleic acids is determined. This can be done by a variety of techniques well known to one of skill, but in a preferred embodiment is through biotin labeling, of the nucleic acid. From the location of the bound nucleic acid, the identity of the oligonucleotide is discovered and thus the identity of the nucleic acid hybridized to the oligonucleotide.
  • the receptor molecules may bind with one or more polymers on the substrate.
  • the presence of the labeled receptor and, therefore, the presence of a sequence which binds with the receptor is detected in a preferred embodiment through the use of autoradiography, detection of fluorescence with a charge-coupled device, fluorescence microscopy, or the like.
  • the sequence of the polymer at the locations where the receptor binding is detected may be used to determine all or part of a sequence which is complementary to the receptor.
  • the compounds of the present invention are useful for treating a wide variety of cancers.
  • cancers include, by way of example and not limitation, carcinomas such as pharynx, colon, rectal, pancreatic, stomach, liver, lung, breast, skin, prostate, ovary, cervical, uterine and bladder cancers; leukemias; lymphomas; gliomas; retinoblastomas; and sarcomas.
  • mammalian subjects include, but are not limited to, humans, laboratory animals, domestic pets and farm animals.
  • the purine analogs of the present invention are used to treat a neurodegenerative disease, the method comprising administering to a mammal having such a disease, a therapeutically effective amount of a compound having the general formula: or a pharmaceutically acceptable salt thereof.
  • Neurodegenerative diseases which can be treated using the purine analog compounds of the present invention include, but are not limited to, neurodegenerative pathologies involving multiple neuronal systems and or brainstem including Alzheimer's disease, AIDS-related dementia, Leigh's disease, diffuse Lewy body disease, epilepsy, multiple system atrophy, Guillain-Barre syndrome, lysosomal storage disorders such as lipofuscmosis, late-degenerative stages of Down's syndrome, Alper's disease, vertigo as result of CNS degeneration, etc.
  • Other neurodegenerative diseases which can be treated using the purine analogs of the present invention will be readily apparent to those of skill in the art.
  • the purine analogs of the present invention can be used to inhibit undesirable proliferation, including, as described above, cancer, psoriasis, growth of fungi, parasites, viruses, plants, etc.
  • the purine analogs of the present invention have apoptosis- inducing effects in actively dividing cells and, thus, can be advantageously used to treat various disease states associated with undesirable proliferation.
  • Such uses are described, for example, in Meijer, L., Trends in Cell Biology (1986) 6:393-397, the teachings of which are incorporated herein by reference for all purposes.
  • the purine analogs of the present invention can be used in vitro as molecular tools and probes.
  • CDK inhibitors arrest cells both in Gi and late G 2 /early prophase, they can be used to synchronize cells when used preferably in combination with another synchronizing agent/method (e.g., when used in combination with aphidicolin).
  • immobilized CDK inhibitors can be used for affinity purification depletion of CDKs from cellular extracts.
  • Such purine analogs will be particularly useful for massive purification of expressed CDKs (for crystallography or screening purposes).
  • such purine analogs are useful for comparative analysis of CDKs extracted from cells at difference developmental or cell- cycle stages (variation of concentration, kinase activity, post-translational modifications, etc.).
  • the compounds, i.e., purine analogs, of the present invention can be administered to a mammal, e.g., a human patient, alone, in the form of a pharmaceutically acceptable salt, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount, e.g., at doses effective to inhibit a protein kinase or a cellular process or achieve amelioration of symptoms of a disease associated with a protein kinase.
  • the compounds of this invention can be incorporated into a variety of formulations for therapeutic administration. More particularly, the compounds of the present invention can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, pills, powders, granules, dragees, gels, slurries, ointments, solutions, suppositories, injections, inhalants and aerosols. As such, administration of the compounds can be achieved in various ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, intracheal, etc., administration.
  • the compound can be administered in a local rather than systemic manner, for example via injection of the compound directly into a solid tumor, often in a depot or sustained release formulation.
  • the compounds can be administered in a targeted drug delivery system, for example, in a liposome coated with tumor-specific antibody. Such liposomes will be targeted to and taken up selectively by the tumor.
  • the purine analogs of the present invention can be administered alone, in combination with each other, or they can be used in combination with other known compounds (e.g., other drugs, such as anti-cancer drugs, anti-mitotics, anti- inflammatories, antibiotics, corticosteroids, vitamins, etc.). More particularly, the compound of the present invention can be used in conjunctive therapy with other known chemotherapeutic or antineoplastic agents (e.g., vinca alkaloids, antibiotics, antimetabolites, platinum coordination complexes, etc.).
  • other drugs such as anti-cancer drugs, anti-mitotics, anti- inflammatories, antibiotics, corticosteroids, vitamins, etc.
  • chemotherapeutic or antineoplastic agents e.g., vinca alkaloids, antibiotics, antimetabolites, platinum coordination complexes, etc.
  • the compounds of the present invention can be used in conjunctive therapy with a vinca alkaloid compound, such as vinblastine, vincristine, taxol, etc.; an antibiotic, such as adriamycin (doxorubicin), dactinomycin (actinomycin D), daunorubicin (daunomycin, rubidomycin), bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C), etc.; an antimetabolite, such as methotrexate, cytarabine (AraC), azauridine, azaribine, fluorodeoxyuridine, deoxycoformycin, mercaptopurine, etc.
  • a vinca alkaloid compound such as vinblastine, vincristine, taxol, etc.
  • an antibiotic such as adriamycin (doxorubicin), dactinomycin (actinomycin D), daunorubicin (daunomycin
  • the compounds of the present invention can be used in conjunctive therapy with other known chemotherapeutic or antineoplastic compounds.
  • the compounds may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • compositions described herein can be manufactured in a manner that is known to those of skill in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the following methods and excipients are merely exemplary and are in no way limiting.
  • the compounds can be formulated into preparations by dissolving, suspending or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other similar oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • the compounds of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the compounds can be formulated readily by combining with pharmaceutically acceptable carriers that are well known in the art.
  • Such carriers enable the compounds to be formulated as tablets, pills, dragees, capsules, emulsions, lipophilic and hydrophilic suspensions, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
  • Pharmaceutical preparations for oral use can be obtained by mixing the compounds with a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pynolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro ethane, carbon dioxide or other suitable gas, or from propellant-free, dry-powder inhalers.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro ethane, carbon dioxide or other suitable gas, or from propellant-free, dry-powder inhalers.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro ethane, carbon dioxide or other suitable gas
  • propellant-free, dry-powder inhalers e
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols or other glycerides, all of which melt at body temperature, yet are solid at room temperature.
  • rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter, carbowaxes, polyethylene glycols or other glycerides, all of which melt at body temperature, yet are solid at room temperature.
  • the compounds may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • hydrophobic pharmaceutical compounds may be employed.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophobic drugs.
  • Certain organic solvents such as dimethylsulfoxide also may be employed, although usually at the cost of greater toxicity.
  • the compounds may be delivered using a sustained-release system, such as semipermeable matrices of solid hydrophobic polymers containing the therapeutic agent.
  • sustained-release materials have been established and are well known by those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks up to over 100 days.
  • compositions also may comprise suitable solid or gel phase carriers or excipients.
  • suitable solid or gel phase carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in a therapeutically effective amount.
  • the amount of composition administered will, of course, be dependent on the subject being treated, on the subject's weight, the severity of the affliction, the manner of administration and the judgment of the prescribing physician. Determination of an effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.
  • a therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 as determined in cell culture, or the IC 100 as determined in cell culture (i.e., the concentration of compound that is lethal to 100% of a cell culture). Such information can be used to more accurately determine useful doses in humans.
  • Initial dosages can also be estimated from in vitro or in vivo data.
  • Initial dosages can also be formulated by comparing the effectiveness of the compounds described herein in cell culture assays with the effectiveness of known drugs. For instance, when used as anticancer agents, initial dosages can be formulated by comparing the effectiveness of the compounds described herein in cell culture assays with the effectiveness of known anti-cancer drugs such as vincristine. In this method, an initial dosage can be obtained by multiplying the ratio of effective concentrations obtained in cell culture assay for the a compound of the present invention and a known anti-cancer drug by the effective dosage of the known anti-cancer drug.
  • an initial effective dosage of the compound of the present invention would be one -half the known dosage for vincristine.
  • toxicity and therapeutic efficacy of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD 50 , (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effect is the therapeutic index and can be expressed as the ratio between LD 50 and ED 50 .
  • Compounds which exhibit high therapeutic indices are preferred.
  • the data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See, e.g., Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1, p. 1). Dosage amount and interval may be adjusted individually to provide plasma levels of the active compound which are sufficient to maintain therapeutic effect. Usual patient dosages for oral administration range from about 50-2000 mg/kg/day, commonly from about 100-1000 mg/kg/day, preferably from about 150-700 mg/kg/day and most preferably from about 250-500 mg/kg/day. Preferably, therapeutically effective serum levels will be achieved by administering multiple doses each day. In cases of local administration or selective uptake, the effective local concentration of the drug may not be related to plasma concentration. One having skill in the art will be able to optimize therapeutically effective local dosages without undue experimentation.
  • Example 1 illustrates a general synthetic scheme for producing the purine derivatives of the invention on a solid support.
  • the solid-phase synthesis strategy exemplified by Scheme 2 involves attaching the growing compound to the solid-support
  • Example 2 illustrates a generalized synthetic route to purine derivatives on a solid support.
  • the solid-phase synthesis strategy exemplified by Scheme 3 involves attaching the growing compound to the solid-support via the side-chain at position 9 of the purine ring structure.
  • Example 3 illustrates a general route to purine derivatives synthesized on a solid support.
  • the route exemplified by Scheme 4 involves attaching the growing compound to the solid-support via the substituent at the 6-position of the purine ring.
  • Example 4 details the alkylation of position 9 of a purine nucleus.
  • the synthetic route is summarized in Scheme 5.
  • the reaction was quenched by adding water (500 ⁇ L) to the reaction mixture.
  • the solvent was removed in vacuo to yield a viscous yellow oil.
  • the oil was azeotroped with CH2CI 2 (2 x 10 mL) to remove trace THF.
  • Purification was effected by column chromatography on silica gel eluted with CH 2 CI 2 .
  • the CH 2 CI 2 was removed from the desired fraction.
  • the desired product was isolated in 57% yield as a white powder.
  • Example 5 illustrates the synthetic route to animation of the 6-position of the purine ring system. The synthetic route is illustrated in Scheme 6.
  • Example 6 details the amination of the 2-position of the purine ring system.
  • the synthetic route is illustrated in Scheme 7.
  • This example illustrates a CDK2/cyclinA microtiter protein kinase assay which can be used to screen the purine analogs of the present invention for inhibitory activity.
  • Buffer A 80 mM Tris (pH-7.2) mM MgCl 2
  • Stepwise Description of Assay a. Prepare solutions of inhibitors at three times the desired final assay concentration in ddH 2 O with 15% DMSO by volume. b. Dispense 20 ⁇ L of inhibitors to the well of a microtiter-formatted assay tray. c. Thaw Histone HI solution (1 mL aliquot), ATP solution (1 mL aliquot) and CDK2 solution (192 ⁇ L aliquot). d. Dilute 192 ⁇ L of CDK2 solution into 2.1 mL of buffer A. Swirl to mix. Dispense 20 ⁇ L of this solution to each well using a multichannel pipetman.
  • ii) Load the nitrocellulose paper onto the dot blot apparatus. Load 100 ⁇ L of water into each well of the dot blot to rehydrate the membrane. Apply a weak vacuum to remove the excess water, but do not dry out the membrane. iii) Add 35 ⁇ L of 10% TC A to each well of the dot blot. g. Using the multichannel pipetman, transfer 35 ⁇ L of the reaction mixtures to each well of the dot blot in the same fashion as the ATP was dispensed (to insure equal reaction times). h. Add an additional 35 ⁇ L of 10% TCA and apply a weak vacuum until the wells are free of liquid.
  • Example 8 demonstrates the identification of a CDK2 inhibitor from a purine library.
  • Substitution chemistry was used to install amines at the 2- and 6- positions and a Mitsunobu (Mitsonobu, Synthesis 1-28 (1981) and Toyota, et al, Heterocycles 36, 1625-1630 (1993)) reaction was employed to alkylate the N-9 position of the purine core.
  • the substitution chemistry allowed introduction of primary and secondary amines bearing a wide range of functional groups, while the Mitsunobu reaction tolerated primary and secondary alcohols lacking additional acidic hydrogens.
  • Newly appended groups were modified combinatorially in subsequent steps using a variety of chemistries including acylation, reductive amination, and Suzuki coupling reactions (Backes, et al, J. Am. Chem. Soc.
  • Inhibitor (20 ⁇ L, 15% DMSO in H 2 O) was introduced to a solution containing CAK activated CDK2/cyclinA (20 ⁇ L, 0.3 mg/ml, 80 mM Tris, pH 7.2, 40 mM MgCl 2 ) in a 96-well microtiter array.
  • the kinase reaction was initiated by the addition of substrate histone HI, ATP mixture (20 ⁇ L, 0.22 mg/mL histone HI, 10 mM HEPES, pH 7.2, 45 ⁇ M ATP, 150 ⁇ g/mL BSA, 1.5 mM DTT, 0.1 vol % ⁇ - 32 P-ATP, 10 ⁇ Ci/mL).
  • reaction mixtures were transferced to 96-well dot-blot apparatus and quenched by the addition 35 ⁇ L of 10% TCA.
  • the phosphorylated histone HI was immobilized onto a nitrocellulose membrane, washed with 10% TCA followed by H 2 O and quantitated by densitometry on a phosphoimager.
  • Table 2 IC50 values for Purvalanol A and B for a variety of purified kinases.
  • IC 0 data for these series of compounds indicated that the inhibitory effects of these substituents are approximately additive.
  • the most potent inhibitor found was 2- (lR-isopropyl-2-hydroxyethyl)-6-(3-chloroanilino)-9-isopropylpurine (purvalanol A, Fig. 4D) or its water soluble 6-(3-chloro-4-carboxyanilino) analog (purvalanol B, Fig. 4D).
  • These inhibitors have IC 0 's against CDK2/cyclinA of 70 and 6 nM, respectively. This conesponds to a 1000-fold improvement over olomoucine and a 30-fold improvement over flavopiridol (Fig.
  • purvalanol A was tested on the NCI's panel of 60 human tumor cell lines (leukemia, non-small cell lung cancer, colon cancer, renal cancer, prostate cancer, and breast cancer). See Appendix I.
  • the average GI 50 (50% growth inhibition) of 2 ⁇ M is substantially higher than that observed for flavopiridol, which uniformly inhibited cell lines with an average GI 50 of 72 nM. This result may reflect poorer bioavailability of purvalanol A or the possibility that flavopiridol's mode of action involves inhibition of additional targets.
  • Example 9 details the structural analysis of the CDK2 -purvalanol complex.
  • the crystal structure of the human CDK2 -purvalanol B complex was determined to 2 05 A resolution and compared to CDK2-hgand complexes containing bound olomoucine (Schulze-Gahmen, et al Proteins Structure, Function, and Genetics 22 378-391 (1995)), roscovitme (De Azevedo, et al , Eur J Biochem 243 518-526 (1997)), flavopindol (De Azevedo, et al , Proc Nat ' I Acad Sci USA 93 2735-2740 (1996)) and ATP (Schultze-Gahmen, et al , J Med Chem 39 4540-4546 (1996)) (Fig 5)
  • CDK2-purvalanol B complex Refinement of the CDK2-purvalanol B complex was started from the coordinates of the highly refined CDK2-ATP model All refinement steps were earned out using the program X-PLOR (A T Brunger, Yale Univ Press, Version 3 0, 1991) Molecular replacement followed by ngid body refinement was necessary to successivefully reonent and reposition the CDK2 molecule m the unit cell of the frozen crystal
  • the CDK2 model was further refined using several rounds of conjugated- gradient energy minimization At this stage the electron density corresponding to purvalanol B was clearly visible from 2Fo-Fc and Fo-Fc Founer maps and the inhibitor could be added to the model Several rounds of both X-ray restrained energy minimization and molecular dynamics in the resolution range 7-2.05 A, alternated with model building using the program O, where necessary to improve the model.
  • the overall geometry of purvalanol B bound to CDK2 resembled that of the related adenine-substituted inhibitors in the CDK2-olomoucine and CDK2 -roscovitine complexes, with the purine ring and its C2, N6 and N9 substituents occupying similar binding pockets.
  • the purine ring made mostly hydrophobic and van der Waals contacts with CDK2 residues and a pair of conserved hydrogen bonds from the N7 imidazole nitrogen to the backbone NH of Leu83 and between the N6 amino group and the backbone carbonyl of Leu83.
  • the C2-side chain of purvalanol B bound in the ATP ribose binding pocket, with the R-isopropyl group closely packed against backbone atoms of the glycine- rich loop and the hydroxyl group making a hydrogen bond with the backbone carbonyl of Glnl31.
  • the R-isopropyl side chain of purvalanol B led to a significant repositioning of the C2 substituent relative to the R-ethyl substituent of roscovitine. This repositioning left open a pocket in the active site lined by the polar side chains of Lys33, Asnl32 and Aspl45. Some electron density was visible in this region, most likely due to the binding of an ethyleneglycol molecule.
  • the 3- chloroanilino group at N6 of purvalanol B pointed towards the outside of the ATP- binding pocket and occupied a region not occupied by any parts of the ATP in the CDK2- ATP complex. Interactions in this region were largely responsible for the increased affinity and selectivity of the inhibitors compared to ATP, as was further demonstrated by the binding of flavopiridol, whose phenyl ring is also bound here.
  • the 3-chloroanilino group of the inhibitor is bound at a slighly different position compared to the benzylamino groups in CDK2-olomoucine and CDK2- roscovitine, allowing for an optimized packing of the phenyl ring against the side chains of He 10 and Phe82. Further stabilization of the binding of the 3-chloroanilino group came from a hydrogen bond with the side chain of Asp86, which existed in about two thirds of the molecules in the CDK2 -purvalanol B crystals. In the other conformation, the phenyl ring of the 3-chloroanilino group was flipped approximately 160 degrees with the chlorine atom located at the opposite site, away from the carboxylate group of Asp86.
  • N9 substituents of the three adenine-substitued inhibitors bound in a small hydrophobic pocket formed by the side chains of Vail 8, Ala31, Phe80, Leul34 and Alal44. Binding was most favorable for the isopropyl group of purvalanol B and roscovitine, whereas the methyl group of olomoucine was found to be too small to occupy the pocket completely.
  • Example 10 shows cellular effects of inhibition by purines and flavopiridol.
  • the cellular effects of the compounds were determined by measuring changes in mRNA levels in yeast following treatment with compounds. mRNA transcript profiles were obtained in Saccharomyces cerevisiae because of the availability of high density oligonucleotide expression anays (Lockhart, et al, Nat. Biotech. 14:1675-1680 (1996); and Wodicka, et al, Nat. Biotech. 15:1359-1367 (1997)), and because the yeast cyclin dependent kinase (CDC28) is highly homologous to human CDK2.
  • a strain was employed with three drug sensitizing deletions (ergo, pdr5, snq2). This strain showed 50% growth inhibition (GI 50 ) for compound 52 and flavopiridol at concentrations of 20 ⁇ M and 7 ⁇ M, respectively.
  • Three cultures (1 lOmL, YPD) were inoculated with single colonies of YRP1 (MATa, erg6::LEU2, pdr5::TRPl, snq2::HIS6) and grown at 30°C with constant agitation in a water bath incubator.
  • Yeast cultures were grown to late log phase and treated with 25 ⁇ M concentrations of the inhibitors for two hours after which cellular poly (A)+ mRNA was isolated and converted to biotin-labeled cRNA.
  • the labeled cRNA was then hybridized to a set of four anays containing more than 260,000 25-mer oligonucleotides.
  • the identities of open reading frames (ORFs) were obtained from the following public databases: Yeast Protein Database (quest7.proteome.edu) and Saccharomyces Genome Database (genome- www, stanford.edu). Transcripts that showed a significant and reproducible change in concentration (two to three-fold) in cells treated with the two compounds between three independent hybridizations were examined further.
  • CDK activity has been implicated in transcriptional regulation of histone genes such as HTA2 and HTB2 (Van Wijnen, et al, Proc. Nat 7 Acad. Sci. USA 91:12882-12886 (1994)) and EGT2, a gene involved in the timing of cell- separation after cytokinesis.
  • HTA2 and HTB2 Van Wijnen, et al, Proc. Nat 7 Acad. Sci. USA 91:12882-12886 (1994)
  • EGT2 a gene involved in the timing of cell- separation after cytokinesis.
  • Other genes involved in cell cycle progression such as YDR247 (a putative negative regulator of meiosis), RAD16 (involved in G 2 repair of inactive genes), YBR214 (similar to the mocl protein of S.
  • pombe which is involved in meiosis and mitosis and RLM1 (a target of Mpklp which is regulated by Cdc28p kinase activity) were induced.
  • the changes in expression levels of these genes are consistent with predominant Gj/S inhibition, in accord with FACS determined DNA content measurements previously reported for analogous purine derivatives (Brooks, et al, J. Biol. Chem. 272:29207-29211 (1997)).
  • Compound 52 and flavopiridol also had similar effects on the transcript levels of many genes involved in cellular metabolism.
  • genes that are involved in glycolysis PDC5, PFK26, YAL061 W, an alcohol dehydrogenase), the citric acid cycle (ALD4, ALD5), glycogen metabolism (PGM2, YPR184W, a putative debranching enzyme), gluconeogenesis (PCK1) and a probable sugar transporter (HXT5) were induced.
  • cdc28p was the intended target of both compound 52 and flavopiridol, more than half of the changes in transcript levels that resulted from exposure to the two compounds were distinct. For example, of the approximately fifty genes whose transcript levels were decreased at least three-fold in response to compound 52, fourteen were ribosomal proteins (including RPL4A, RPL26B, RPS24A). This was found to be consistent with the observed up regulation of protein kinase A, which has an established role in modulating ribosomal protein synthesis (Griffioen, et al., FEMS Microbiol Lett. 123: 137-44 (1994)). In contrast, no ribosomal protein transcript levels decreased more than three-fold for flavopiridol.
  • Compound 52 also uniquely affected YMR116C (a determinant of cell size), a cytosine/purine permease and CLB2 (G 2 /M- phase specific cyclin).
  • YMR276W which encodes a ubiquitin like protein involved in duplication of the spindle pole body
  • CLN2 which encodes a Gj/S specific cyclin.
  • the differential effects of the two compounds resulted from different cellular bioavailability or their effects on other cellular targets not specifically examined in vitro such as the additional yeast CDKs KIN28 (involved in mRNA transcription) and PH085 (phosphate regulation).
  • transcripts induced in cdc28-4 were ones involved in stress signaling (Ruis & Schuller, BioEssays 17:959-965 (1995)): heat shock elements (HSEs), stress response elements (STREs), and members of the major facilitator superfamily (MFSs).
  • HSEs heat shock elements
  • STREs stress response elements
  • MFSs major facilitator superfamily
  • the cdc28-13 strain contains an arginine to asparagine mutation at residue 283 near the C-terminus which does not significantly affect kinase activity at the permissive temperature but does cause cell cycle anest when switched to the nonpermissive temperature (LoRincz & Reed, Mol. Cell Biol. 6:4099-4103 (1986)).
  • the cdc28-13 strain showed very few changes in mRNA transcripts when compared to wild type at the permissive temperature. The levels of only 11 mRNAs changed by more than two-fold, consistent with the observation that this mutant possesses essentially wildtype kinase activity at 25°C.
  • the nearly identical gene expression patterns obtained for the cdc28-13 and isogenic wildtype CDC28 strain demonstrate the reproducibility of these experiments.
  • CDC28 Since CDC28 is an essential gene, the transcript profile of two cdc28 temperature sensitive strains (cdc28-4 and cdc28-13) and their isogenic wild-type (wt) strains were measured under permissive growth conditions (25°C). Under these conditions cdc28-4 grew at essentially wild type rates which approximated the small degree of growth inhibition observed for the two hour compound treatments used to prepare the inhibitor profiles.
  • the cdc28-4 strain contains a single histidine to tyrosine mutation at position 128 which when mapped onto the human CDK2 crystal structure is located adjacent to the ATP binding site.
  • Cdc28p specific kinase activity is greatly reduced as measured by an immunoprecipitation phosphorylation assay (Reed, et al, Proc. Nat 'I Acad. Sci. USA 82:4055-4059 (1985)).
  • the cdc28-4 mutant When switched to the nonpermissive temperature, the cdc28-4 mutant anests early in the cell cycle as large unbudded cells. Since Cdc28p activity is high during S phase and mitosis, the mutation in cdc28-4 might be expected to simulate the effects of chemically inhibiting the kinase during these two key points in the cell cycle.
  • the specific mechanism of Cdc28p inactivation may differ significantly from that resulting from a competitive active site inhibitor.
  • Example 11 demonstrates screening purine libraries against other cellular targets.
  • the recombinant JNK-his6 fusion was produced in E. coli and purified by Ni-agarose chromatography.
  • a 30 ⁇ L kinase reaction contained 20 mM MgCl , 20 mM Tris/HCl pH 7.6, 20 ⁇ M ATP (cold), 66 nM JNK, 0.5 ⁇ L ⁇ - 32 P-ATP, 1 ⁇ g GSTc-Jun(l- 79), and the indicated concentrations of inhibitors.
  • the reaction was carried out at 30°C for 30 min.
  • the phosphorylated GSTc-Jun was separated by SDS-PAG ⁇ , and phosphorylated bands were quantified by phosphoimager analysis.
  • V5C D- 706230 -Y / I I E2c ⁇ je ⁇ ment ID: 9809NS65 ⁇ 5 i Test Type: 08 [ Units; Molar

Abstract

La synthèse d'inhibiteurs sélectifs pour des protéines kinases spécifiques fournirait de nouveaux outils permettant d'analyser les voies de transduction des signaux, ainsi qu'éventuellement de nouveaux agents thérapeutiques. Nous avons inventé une approche du développement d'inhibiteurs sélectifs des protéines kinases en nous basant sur le mode de fixation inattendu des purines 2,6,9-trisubstituées sur le site de liaison de l'ATP de la CDK2 humaine. L'inhibiteur le plus puissant, le purvalanol B (IC50 = 6 nM), se fixe avec une affinité 30 fois supérieure à celle de l'inhibiteur CDK2 connu, le flavopiridol. Nous avons étudié les effets cellulaires de cette classe de composés et les avons comparé à ceux du flavopiridol en observant les modifications du taux d'expression de l'ARNm, pour tous les gènes contenus dans les cellules traitées de Saccharomyces cerevisiae, au moyen d'un réseau haute densité de sondes oligonucléotidiques.
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DE60205376T2 (de) 2001-06-27 2006-04-06 Cyclacel Ltd. 2,6,9-substituierte purinderivate und ihre verwendung bei der behandlung proliferativer krankheiten
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US7005445B2 (en) 2001-10-22 2006-02-28 The Research Foundation Of State University Of New York Protein kinase and phosphatase inhibitors and methods for designing them
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WO2006105386A1 (fr) * 2005-03-30 2006-10-05 Genentech, Inc. Inhibiteurs de cdk2
US20090269772A1 (en) * 2008-04-29 2009-10-29 Andrea Califano Systems and methods for identifying combinations of compounds of therapeutic interest
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