Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D498/04—Ortho-condensed systems
• • 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
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • 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
  • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
  • A61K31/5375—1,4-Oxazines, e.g. morpholine
  • A61K31/538—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with carbocyclic ring systems
• • 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
  • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
  • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
  • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
  • C07D403/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
  • C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
  • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
  • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D413/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains two hetero rings
  • C07D498/06—Peri-condensed systems

Definitions

• the present invention relates generally to 2,4-pyrimidinediamine compounds, pharmaceutical compositions comprising the compounds, intermediates and synthetic methods of making the compounds and methods of using the compounds and compositions in a variety of contexts, such as in the treatment or prevention of autoimmune diseases and/or the symptoms associated therewith.
• Crosslinking of Fc receptors such as the high affinity receptor for IgE (FceRI) and/or the high affinity receptor for IgG (Fc ⁇ RI) activates a signaling cascade in mast, basophil and other immune cells that results in the release of chemical mediators responsible for numerous adverse events.
• Fc receptors such as the high affinity receptor for IgE (FceRI) and/or the high affinity receptor for IgG (Fc ⁇ RI) activates a signaling cascade in mast, basophil and other immune cells that results in the release of chemical mediators responsible for numerous adverse events.
• Fc receptors such as the high affinity receptor for IgE (FceRI) and/or the high affinity receptor for IgG (Fc ⁇ RI) activates a signaling cascade in mast, basophil and other immune cells that results in the release of chemical mediators responsible for numerous adverse events.
• Fc receptors such as the high affinity receptor for IgE (FceRI) and/or the high affinity receptor for IgG (Fc ⁇ RI) activates a
• Fc ⁇ RI comprises an array of cellular proteins. Among the most important intracellular signal propagators are the tyrosine kinases. And, an important tyrosine kinase involved in the signal transduction pathways associated with crosslinking the FceRI and/or Fc ⁇ RI receptors, as well as other signal transduction cascades, is Syk kinase (see Valent et al, 2002, Intl. J. Hematol 75(4):257-362 for review). As the mediators released as a result of FceRI and Fc ⁇ RI receptor cross-linking are responsible for, or play important roles in, the manifestation of numerous adverse events, the availability of compounds capable of inhibiting the signaling cascade(s) responsible for their release would be highly desireable. Moreover, owing to the critical role that Syk kinase plays these and other receptor signaling cascade(s), the availability of compounds capable of inhibiting Syk kinase would also be highly desirable.
• the present invention provides novel 2,4-pyrimidinediamine compounds that, as will be discussed in more detail below, have myriad biological activities.
• the compounds generally comprise a 2,4-pyrimidinediamine "core" having the following structure and numbering convention:
• the compounds of the invention are substituted at the C2 nitrogen (N2) to form a secondary amine and are optionally further substituted at one or more of the following positions: the C4 nitrogen (N4), the C5 position and/or the C6 position.
• the substituent forms a secondary amine.
• the substituent at N2, as well as the optional substituents at the other positions, may range broadly in character and physico- chemical properties.
• the substituent(s) may be a branched, straight-chained or cyclic alkyl, a branched, straight-chained or cyclic heteroalkyl, a mono- or polycyclic aryl a mono- or polycyclic heteroaryl or combinations of these groups.
• N2 and/or N4 substituents may be attached directly to their respective nitrogen atoms, or they may be spaced away from their respective nitrogen atoms via linkers, which may be the same or different.
• the nature of the linkers can vary widely, and can include virtually any combination of atoms or groups useful for spacing one molecular moiety from another.
• the linker may be an acyclic hydrocarbon bridge (e.g, a saturated or unsaturated alkyleno such as methano, ethano, etheno, propano, prop[l]eno, butano, but[l]eno, but[2]eno, buta[l,3]dieno, and the like), a monocyclic or polycyclic hydrocarbon bridge (e.g., [l,2]benzeno, [2,3]naphthaleno, and the like), a simple acyclic heteroatomic or heteroalkyldiyl bridge (e.g., -O-, -S-, -S-O-, -NH-, -PH-, -C(O)-, -C(O)NH-, -S(O)-, -S(O) 2 -, -S(O)NH-, -S(O) 2 NH-, -O-CH 2 -, -CH 2
• substituents at the N2, N4, C5 and/or C6 positions, as well as the optional linkers, may be further substituted with one or more of the same or different substituent groups.
• the nature of these substituent groups may vary broadly.
• suitable substituent groups include branched, straight-chain or cyclic alkyls, mono- or polycyclic aryls, branched, straight-chain or cyclic heteroalkyls, mono- or polycyclic heteroaryls, halos, branched, straight-chain or cyclic haloalkyls, hydroxyls, oxos, thioxos, branched, straight-chain or cyclic alkoxys, branched, straight-chain or cyclic haloalkoxys, trifluoromethoxys, mono- or polycyclic aryloxys, mono- or polycyclic heteroaryloxys, ethers, alcohols, sulfides, thioethers, sulfanyls,
• L 1 and L 2 are each, independently of one another, selected from the group consisting of a direct bond and a linker; R 2 and R 4 are described infra; R 5 is selected from the group consisting of R 6 , (C1-C6) alkyl optionally substituted with one or more of the same or different R groups, (C1-C4) alkanyl optionally substituted with one or more of the same or different R 8 groups, (C2-C4) alkenyl optionally substituted with one or more of the same or different R groups and (C2-C4) alkynyl optionally substituted with one or more of the same or different R groups; each R 6 independently is selected from the group consisting of hydrogen, an electronegative group, -OR d , -SR d , (C1-C3) haloalkyloxy, (C1-C3) perhaloalkyloxy, -NR C R C , halogen, (
• each R a is independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C3-C8) cycloalkyl, cyclohexyl, (C4-C11) cycloalkylal
• R 5 is F and R 6 is hydrogen.
• the present invention provides prodrugs of the 2,4- pyrimidinediamine compounds.
• Such prodrugs may be active in their prodrug form, or may be inactive until converted under physiological or other conditions of use to an active drug form.
• one or more functional groups of the 2,4- pyrimidinediamine compounds are included in promoieties that cleave from the molecule under the conditions of use, typically by way of hydrolysis, enzymatic cleavage or some other cleavage mechanism, to yield the functional groups.
• promoieties that cleave from the molecule under the conditions of use, typically by way of hydrolysis, enzymatic cleavage or some other cleavage mechanism, to yield the functional groups.
• primary or secondary amino groups may be included in an amide promoiety that cleaves under conditions of use to generate the primary or secondary amino group.
• the prodrugs of the invention include special types of protecting groups, termed "progroups,” masking one or more functional groups of the 2,4-pyrimidinediamine compounds that cleave under the conditions of use to yield an active 2,4-pyrimidinediamine drug compound.
• Functional groups within the 2,4-pyrimidinediamine compounds that may be masked with progroups for inclusion in a promoiety include, but are not limited to, amines (primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, carbonyls, phenols, catechols, diols, alkynes, phosphates, etc.
• promoieties suitable for masking such functional groups to yield promoieties that are cleavable under the desired conditions of use are known in the art. All of these progroups, alone or in combinations, may be included in the prodrugs of the invention. Specific examples of promoieties that yield primary or secondary amine groups that can be included in the prodrugs of the invention include, but are not limited to amides, carbamates, imines, ureas, phosphenyls, phosphoryls and sulfenyls.
• promoieties that yield sulfanyl groups include, but are not limited to, thioethers, for example S-methyl derivatives (monothio, dithio, oxythio, aminothio acetals), silyl thioethers, thioesters, thiocarbonates, thiocarbamates, asymmetrical disulfides, etc.
• promoieties that cleave to yield hydroxyl groups that can be included in the prodrugs of the invention include, but are not limited to, sulfonates, esters and carbonates.
• promoieties that yield carboxyl groups that can be included in the prodrugs of the invention included, but are not limited to, esters (including silyl esters, oxamic acid esters and thioesters), amides and hydrazides.
• the prodrugs of the invention are compounds according to structural formula (I) in which the protecting group of R c and R d is a progroup.
• the prodrugs of the invention are compounds according to structural formula (II):
• the present invention provides compositions comprising one or more compounds and/or prodrugs of the invention and an appropriate carrier, excipient or diluent.
• the exact nature of the carrier, excipient or diluent will depend upon the desired use for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use.
• the present invention provides intermediates useful for synthesizing the 2,4-pyrimidinediamine compounds and prodrugs of the invention.
• the intermediates are 4-pyrimidineamines according to structural formula (III):
• R 4 , R 5 , R 6 and L 2 are as previously defined for structural formula (I); LG is a leaving group such as, for example, -S(O) 2 Me, -SMe or halo (e.g., F, CI, Br, I); and R 4c is hydrogen, a progroup, an alkyl group or as described herein.
• the intermediates are 2-pyrimidineamines according to structural formula (IN):
• the intermediates are 4-amino- or 4-hydroxy-2- pyrimidineamines according to structural formula (V):
• the intermediates are N4-substituted cytosines according to structural formula (VI):
• the present invention provides methods of synthesizing the 2,4-pyrimidinediamine compounds and prodrugs of the invention.
• the method involves reacting a 4-pyrimidineamine according to structural formula (III) with an amine of the formula HR 2c N-L 1 -R 2 , where L 1 , R 2 and R 2c are as previously defined for structural formula (IN) to yield a 2,4-pyrimidinediamine according to structural formula (I) or a prodrug according to structural formula (II).
• the method involves reacting a 2-pyrimidineamine according to structural formula (IN) with an amine of the formula R 4 -L 2 - ⁇ HR 4c where L 4 , R 4 and R 4c are as previously defined for structural formula (III) to yield a 2,4- pyrimidinediamine according to structural formula (I) or a prodrug according to structural formula (II).
• the method involves reacting a 4-amino-2- pyrimidineamine according to structural formula (V) (in which R 7 is an amino group) with an amine of the formula R 4 -L 2 -NHR 4c , where L 2 , R 4 and R 4c are as defined for structural formula (III), to yield a 2,4-pyrimidinediamine according to structural formula (I) or a prodrug according to structural formula (II).
• the 4-amino-2-pyrimidineamine may be reacted with a compound of the formula R 4 -L 2 -LG, where R 4 and L 2 are as previously defined for structural formula (I) and LG is a leaving group.
• the method involves halogenating a 4-hydroxy-2- pyrimidineamine according to structural formula (V) (R 7 is a hydroxyl group) to yield a 2- pyrimidineamine according to structural formula (IV) and reacting this pyrimidineamine with an appropriate amine, as described above.
• the method involves halogenating an N4-substituted cytosine according to structural formula (VI) to yield a 4-pyrimidineamine according to structural formula (III) and reacting this pyrimidineamine with an appropriate amine, as described above.
• the 2,4-pyrimidinediamine compounds of the invention are potent inhibitors of degranulation of immune cells, such as mast, basophil, neutrophil and/or eosinophil cells.
• the present invention provides methods of regulating, and in particular inhibiting, degranulation of such cells.
• the method generally involves contacting a cell that degranulates with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to regulate or inhibit degranulation of the cell.
• the method may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with cellular degranulation.
• biochemical data confirm that the 2,4-pyrimidinediamine compounds exert their degranulation inhibitory effect, at least in part, by blocking or inhibiting the signal transduction cascade(s) initiated by crosslinking of the high affinity Fc receptors for IgE ("FceRI”) and/or IgG (“Fc ⁇ RI”).
• FceRI high affinity Fc receptors for IgE
• Fc ⁇ RI IgG
• the 2,4-pyrimidinediamine compounds are potent inhibitors of both FceRI-mediated and Fc ⁇ RI-mediated degranulation.
• the 2,4-pyrimidine compounds may be used to inhibit these Fc receptor signalling cascades in any cell type expressing such FceRI and/or Fc ⁇ RI receptors including but not limited to macrophages, mast, basophil, neutrophil and/or eosinophil cells.
• the methods also permit the regulation of, and in particular the inhibition of, downstream processes that result as a consequence of activating such Fc receptor signaling cascade(s).
• downstream processes include, but are not limited to, FceRI-mediated and/or Fc ⁇ RI-mediated degranulation, cytokine production and/or the production and/or release of lipid mediators such as leukotrienes and prostaglandins.
• the method generally involves contacting a cell expressing an Fc receptor, such as one of the cell types discussed above, with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvent, N-oxide and/or composition thereof, effective to regulate or inhibit the Fc receptor signaling cascade and/or a downstream process effected by the activation of this signaling cascade.
• an Fc receptor such as one of the cell types discussed above
• the method may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with the Fc receptor signaling cascade, such as diseases effected by the release of granule specific chemical mediators upon degranulation, the release and/or synthesis of cytokines and/or the release and/or synthesis of lipid mediators such as leukotrienes and prostaglandins.
• the present invention provides methods of treating and/or preventing diseases characterized by, caused by or associated with the release of chemical mediators as a consequence of activating Fc receptor signaling cascades, such as FceRI and/or Fc ⁇ RI- signaling cascades.
• the methods may be practiced in animals in veterinary contexts or in humans.
• the methods generally involve administering to an animal subject or human an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to treat or prevent the disease.
• a 2,4-pyrimidinediamine compound or prodrug of the invention or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to treat or prevent the disease.
• activation of the FceRI or Fc ⁇ RI receptor signaling cascade in certain immune cells leads to the release and/or synthesis of a variety of chemical substances that are pharmacological mediators of a wide variety of diseases. Any of these diseases may be treated or prevented according to the methods of the invention.
• activation of the FceRI or Fc ⁇ RI signaling cascade leads to the immediate (i.e., within 1-3 min. of receptor activation) release of preformed mediators of atopic and or Type I hypersensitivity reactions (e.g., histamine, proteases such as tryptase, etc.) via the degranulation process.
• preformed mediators of atopic and or Type I hypersensitivity reactions e.g., histamine, proteases such as tryptase, etc.
• Such atopic or Type I hypersensitivity reactions include, but are not limited to, anaphylactic reactions to environmental and other allergens (e.g., pollens, insect and/or animal venoms, foods, drugs, contrast dyes, etc.), anaphylactoid reactions, hay fever, allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, eczema, urticaria, mucosal disorders, tissue disorders and certain gastrointestinal disorders.
• allergens e.g., pollens, insect and/or animal venoms, foods, drugs, contrast dyes, etc.
• anaphylactoid reactions hay fever, allergic conjunctivitis, allergic rhinitis, allergic asthma, atopic dermatitis, eczema, urticaria, mucosal disorders, tissue disorders and certain gastrointestinal disorders.
• the immediate release of the preformed mediators via degranulation is followed by the release and/or synthesis of a variety of other chemical mediators, including, among other things, platelet activating factor (PAF), prostaglandins and leukotrienes (e.g., LTC4) and the de novo synthesis and release of cytokines such as TNF ⁇ , IL-4, IL-5, IL-6, IL-13, etc.
• PAF platelet activating factor
• prostaglandins and leukotrienes e.g., LTC4
• cytokines such as TNF ⁇ , IL-4, IL-5, IL-6, IL-13, etc.
• the first of these two processes occurs approximately 3-30 min. following receptor activation; the latter approximately 30 min. - 7 hrs. following receptor activation.
• These "late stage” mediators are thought to be in part responsible for the chronic symptoms of the above-listed atopic and Type I hypersensitivity reactions, and in addition are chemical mediators of inflammation and inflammatory diseases (e.g., osteoarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, idiopathic inflammatory bowel disease, irritable bowel syndrome, spastic colon, etc.), low grade scarring (e.g., scleroderma, increased fibrosis, keloids, post-surgical scars, pulmonary fibrosis, vascular spasms, migraine, reperfusion injury and post myocardial infarction), and sicca complex or syndrome. All of these diseases may be treated or prevented according to the methods of the invention.
• inflammatory diseases e.g., osteoarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, idiopathic inflammatory bowel disease, irritable bowel syndrome, spastic colon, etc.
• low grade scarring e.
• Additional diseases which can be treated or prevented according to the methods of the invention include diseases associated with basophil cell and/or mast cell pathology.
• diseases associated with basophil cell and/or mast cell pathology include, but are not limited to, diseases of the skin such as scleroderma, cardiac diseases such as post myocardial infarction, pulmonary diseases such as pulmonary muscle changes or remodeling and chronic obstructive pulmonary disease (COPD) and diseases of the gut such as inflammatory bowel syndrome (spastic colon).
• the 2,4-pyrimidinediamine compounds of the invention are also potent inhibitors of the tyrosine kinase Syk kinase.
• the present invention provides methods of regulating, and in particular inhibiting, Syk kinase activity.
• the method generally involves contacting a Syk kinase or a cell comprising a Syk kinase with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to regulate or inhibit Syk kinase activity.
• the Syk kinase is an isolated or recombinant Syk kinase.
• the Syk kinase is an endogenous or recombinant Syk kinase expressed by a cell, for example a mast cell or a basophil cell.
• the method may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with Syk kinase activity. While not intending to be bound by any particular theory of operation, it is believed that the 2,4-pyrimdinediamine compounds of the invention inhibit cellular degranulation and/or the release of other chemical mediators primarily by inhibiting Syk kinase that gets activated through the gamma chain homodimer of FceRI (see, e.g., FIG. 2). This gamma chain homodimer is shared by other Fc receptors, including Fc ⁇ RI, Fc ⁇ RIII and FcoRI.
• the 2,4-pyrimidinediamine compounds described herein may be used to regulate, and in particular inhibit, the signaling cascades of Fc receptors having this gamma chain homodimer, such as FceRI, Fc ⁇ RI, Fc ⁇ RIII and Fco-RI, as well as the cellular responses elicited through these receptors.
• Syk kinase is known to play a critical role in other signaling cascades.
• Syk kinase is an effector of B-cell receptor (BCR) signaling (Turner et al, 2000, Immunology Today 21:148-154) and is an essential component of integrin beta(l), beta(2) and beta(3) signaling in neutrophils (Mocsai et al, 2002, Immunity 16:547-558).
• BCR B-cell receptor
• integrin beta(l), beta(2) and beta(3) signaling in neutrophils Mocsai et al, 2002, Immunity 16:547-558.
• the 2,4-pyrimidinediamine compounds described herein are potent inhibitors of Syk kinase, they can be used to regulate, and in particular inhibit, any signaling cascade where Syk plays a role, such as, fore example, the Fc receptor, BCR and integrin signaling cascades, as well as the cellular responses elicited through these signaling cascades.
• cellular responses regulated or inhibited will depend, in part, on the specific cell type and receptor signaling cascade, as is well known in the art.
• Non- limiting examples of cellular responses that may be regulated or inhibited with the 2,4-pyrimidinediamine compounds include a respiratory burst, cellular adhesion, cellular degranulation, cell spreading, cell migration, phagocytosis (e.g., in macrophages), calcium ion flux (e.g., in mast, basophil, neutrophil, eosinophil and B-cells), platelet aggregation, and cell maturation (e.g., in B- cells).
• the present invention provides methods of regulating, and in particular inhibiting, signal transduction cascades in which Syk plays a role.
• the method generally involves contacting a Syk-dependent receptor or a cell expressing a Syk- dependent receptor with an amount of a 2,4-pyrimidinediamine compound or prodrug of the invention, or an acceptable salt, hydrate, solvate, N-oxide and/or composition thereof, effective to regulate or inhibit the signal transduction cascade.
• the methods may also be used to regulate, and in particular inhibit, downstream processes or cellular responses elicited by activation of the particular Syk-dependent signal transduction cascade.
• the methods may be practiced to regulate any signal transduction cascade where Syk is not known or later discovered to play a role.
• the methods may be practiced in in vitro contexts or in in vivo contexts as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by or associated with activation of the Syk-dependent signal transduction cascade.
• diseases characterized by, caused by or associated with activation of the Syk-dependent signal transduction cascade.
• Non-limited examples of such diseases include those previously discussed.
• Cellular and animal data also confirm that the 2,4-pyrimidinediamine compounds of the invention may also be used to treat or prevent autoimmune diseases and/or symptoms of such diseases.
• the methods generally involve administering to a subject suffering from an autoimmune disease or at risk of developing an autoimmune disease an amount of a 2,4- pyrimidinediamine method or prodrug of the invention, or an acceptable salt, N-oxide, hydrate, solvate or composition thereof, effective to treat or prevent the autoimmune disease and/or its associated symptoms.
• Autoimmune diseases that can be treated or prevented with the 2,4-pyrimidinediamine compounds include those diseases that are commonly associated with nonanaphylactic hypersensitivity reactions (Type II, Type III and/or Type IV hypersensitivity reactions) and/or those diseases that are mediated, at least in part, by activation of the Fc ⁇ R signaling cascade in monocyte cells.
• Such autoimmune disease include, but are not limited to, those autoimmune diseases that are frequently designated as single organ or single cell-type autoimmune disorders and those autoimmune disease that are frequently designated as involving systemic autoimmune disorder.
• diseases frequently designated as single organ or single cell-type autoimmune disorders include: Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis and membranous glomerulopathy.
• Non-limiting examples of diseases often designated as involving systemic autoimmune disorder include: systemic lupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis- dermatomyositis, systemic sclerosis, polyarteritis nodosa, multiple sclerosis and bullous pemphigoid.
• FIG. 1 provides a cartoon illustrating allergen-induced production of IgE and consequent release of preformed and other chemical mediators from mast cells
• FIG. 2 provides a cartoon illustrating the Fc ⁇ Rl signal transduction cascade leading to degranulation of mast and/or basophil cells
• FIG. 3 provides a cartoon illustrating the putative points of action of compounds that selectively inhibit upstream FceRI-mediated degranulation and compounds that inhibit both FceRI-mediated and ionomycin-induced degranulation.
• Alkyl by itself or as part of another substituent refers to a saturated or unsaturated branched, straight-chain or cyclic monovalent hydrocarbon radical having the stated number of carbon atoms (i.e., C1-C6 means one to six carbon atoms) that is derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene or alkyne.
• Typical alkyl groups include, but are not limited to, methyl; ethyls such as ethanyl, ethenyl, ethynyl; propyls such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-l-yl, prop-l-en-2-yl, prop-2-en-l-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl, prop-1-yn-l-yl , prop-2-yn-l-yl, etc.; butyls such as butan-1-yl, butan-2-yl, 2-methyl-propan-l-yl, 2-methyl-propan-2-yl, cyclobutan-1-yl, but-1-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-yl, but-2-en-l-yl , but-2
• alkanyl alkenyl
• alkynyl alkynyl
• the alkyl groups are (C1-C6) alkyl.
• Alkanyl by itself or as part of another substituent refers to a saturated branched, straight-chain or cyclic alkyl derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane.
• Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyls such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl, etc.; butanyls such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-l-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl, etc.; and the like.
• the alkanyl groups are (C1-C6) alkanyl.
• Alkenyl by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl having at least one carbon-carbon double bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkene.
• the group may be in either the cis or trans conformation about the double bond(s).
• Typical alkenyl groups include, but are not limited to, ethenyl; propenyls such as prop-1-en-l-yl , prop-l-en-2-yl, prop-2-en-l-yl, prop-2-en-2-yl, cycloprop-1-en-l-yl; cycloprop-2-en-l-yl ; butenyls such as but-1-en-l-yl, but-l-en-2-yl, 2-methyl-prop-l-en-l-yl, but-2-en-l-yl, but-2-en-2-yl, buta-l,3-dien-l-yl, buta-l,3-dien-2-yl, cyclobut-1-en-l-yl, cyclobut-l-en-3-yl, cyclobuta-l,3-dien-l-yl, etc.; and the like.
• the alkenyl group is (C2-C6) alkenyl.
• "Alkynyl" by itself or as part of another substituent refers to an unsaturated branched, straight-chain or cyclic alkyl having at least one carbon-carbon triple bond derived by the removal of one hydrogen atom from a single carbon atom of a parent alkyne.
• Typical alkynyl groups include, but are not limited to, ethynyl; propynyls such as prop-1-yn-l-yl , prop-2-yn-l-yl, etc.; butynyls such as but-1-yn-l-yl, but-l-yn-3-yl, but-3-yn-l-yl , etc.; and the like.
• the alkynyl group is (C2-C6) alkynyl.
• Alkyldiyl by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic divalent hydrocarbon group having the stated number of carbon atoms (i.e., C1-C6 means from one to six carbon atoms) derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene or alkyne, or by the removal of two hydrogen atoms from a single carbon atom of a parent alkane, alkene or alkyne.
• the two monovalent radical centers or each valency of the divalent radical center can form bonds with the same or different atoms.
• Typical alkyldiyl groups include, but are not limited to, methandiyl; ethyldiyls such as ethan-l,l-diyl, ethan-l,2-diyl, ethen-l,l-diyl, ethen-l,2-diyl; propyldiyls such as propan-l,l-diyl, propan-l,2-diyl, propan-2,2-diyl, propan-l,3-diyl, cyclopropan-l,l-diyl, cyclopropan-l,2-diyl, prop-l-en-l,l-diyl, prop-l-en-l,2-diyl, prop-2-en-l,2-diyl, prop-l-en-l,3-diyl, cycloprop-l-en-l,2-diyl, prop-2-en-l,2-
• alkanyldiyl alkenyldiyl and/or alkynyldiyl
• alkylidene alkylidene
• saturated acyclic alkanyldiyl groups in which the radical centers are at the terminal carbons, e.g., methandiyl (methano); ethan-l,2-diyl (ethano); propan-l,3-diyl (propano); butan-l,4-diyl (butano); and the like (also referred to as alkylenos, defined infra).
• Alkyleno by itself or as part of another substituent refers to a straight-chain saturated or unsaturated alkyldiyl group having two terminal monovalent radical centers derived by the removal of one hydrogen atom from each of the two terminal carbon atoms of straight-chain parent alkane, alkene or alkyne.
• the locant of a double bond or triple bond, if present, in a particular alkyleno is indicated in square brackets.
• Typical alkyleno groups include, but are not limited to, methano; ethylenos such as ethano, etheno, ethyno; propylenos such as propano, prop[l]eno, propa[l,2]dieno, prop[l]yno, etc.; butylenos such as butano, but[l]eno, but[2]eno, buta[l,3]dieno, but[l]yno, but[2]yno, buta[l,3]diyno, etc.; and the like. Where specific levels of saturation are intended, the nomenclature alkano, alkeno and/or alkyno is used.
• the alkyleno group is (C1-C6) or (C1-C3) alkyleno. Also preferred are straight-chain saturated alkano groups, e.g., methano, ethano, propano, butano, and the like.
• Heteroalkyl Heteroalkanyl
• Heteroalkenyl Heteroalkynyl
• Heteroalkyldiyl and ⁇ eteroalkyleno by themselves or as part of another substituent refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkyleno groups, respectively, in which one or more of the carbon atoms are each independently replaced with the same or different heteratoms or heteroatomic groups.
• Typical heteroatoms and/or heteroatomic groups which can replace the carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR'-, -PH-, -S(O)-, -S(O) 2 -, -S(O) NR'-, -S(O) 2 NR'-, and the like, including combinations thereof, where each R' is independently hydrogen or (C1-C6) alkyl.
• “Cycloalkyl” and "Heterocycloalkyl” by themselves or as part of another substituent refer to cyclic versions of "alkyl” and "heteroalkyl” groups, respectively.
• a heteroatom can occupy the position that is attached to the remainder of the molecule.
• Typical cycloalkyl groups include, but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like.
• Typical heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl, piperidin- 2-yl, etc.), mo holinyl (e.g., morpholin-3-yl, morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl, piperazin-2-yl, etc.), and the like.
• tetrahydrofuranyl e.g., tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, etc.
• piperidinyl e.g., piperidin-1-yl, piperidin- 2-yl, etc.
• mo holinyl e.g., morpholin-3-yl,
• Acyclic Heteroatomic Bridge refers to a divalent bridge in which the backbone atoms are exclusively heteroatoms and/or heteroatomic groups.
• Typical acyclic heteroatomic bridges include, but are not limited to, -O-, -S-, -S-O-, -NR'-, -PH-, -S(O)-, -S(O) -, -S(O) NR'-, -S(O) NR'-, and the like, including combinations thereof, where each R' is independently hydrogen or (C1-C6) alkyl.
• Parent Aromatic Ring System refers to an unsaturated cyclic or polycyclic ring system having a conjugated ⁇ electron system.
• parent aromatic ring system fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, fluorene, indane, indene, phenalene, tetrahydronaphthalene, etc.
• Typical parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, indacene, -.-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and the like, as well
• Aryl by itself or as part of another substituent refers to a monovalent aromatic hydrocarbon group having the stated number of carbon atoms (i.e., C5-C15 means from 5 to 15 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system.
• Typical aryl groups include, but are not limited to, groups derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexalene, ⁇ -f-indacene, -.-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like, as well as the various
• the aryl group is (C5-C15) aryl, with (C5-C10) being even more preferred.
• Particularly preferred aryls are cyclopentadienyl, phenyl and naphthyl.
• “Arylaryl” by itself or as part of another substituent refers to a monovalent hydrocarbon group derived by the removal of one hydrogen atom from a single carbon atom of a ring system in which two or more identical or non-identical parent aromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent aromatic ring systems involved.
• Typical arylaryl groups include, but are not limited to, biphenyl, triphenyl, phenyl-naphthyl, binaphthyl, biphenyl-naphthyl, and the like. Where the number of carbon atoms in an arylaryl group are specified, the numbers refer to the carbon atoms comprising each parent aromatic ring.
• (C5-C15) arylaryl is an arylaryl group in which each aromatic ring comprises from 5 to 15 carbons, e.g., biphenyl, triphenyl, binaphthyl, phenylnaphthyl, etc.
• each parent aromatic ring system of an arylaryl group is independently a (C5-C15) aromatic, more preferably a (C5-C10) aromatic.
• arylaryl groups in which all of the parent aromatic ring systems are identical, e.g., biphenyl, triphenyl, binaphthyl, trinaphthyl, etc.
• Biaryl by itself or as part of another substituent refers to an arylaryl group having two identical parent aromatic systems joined directly together by a single bond.
• Typical biaryl groups include, but are not limited to, biphenyl, binaphthyl, bianthracyl, and the like.
• the aromatic ring systems are (C5-C15) aromatic rings, more preferably (C5-C10) aromatic rings.
• a particularly preferred biaryl group is biphenyl.
• “Arylalkyl” by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp carbon atom, is replaced with an aryl group.
• Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan- 1 -yl, 2-phenylethen- 1 -yl, naphthylmethyl,
• the arylalkyl group is (C6-C21) arylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C6) and the aryl moiety is (C5-C15).
• the arylalkyl group is (C6-C13), e.g., the alkanyl, alkenyl or alkynyl moiety of the arylalkyl group is (C1-C3) and the aryl moiety is (C5-C10).
• "Parent Heteroaromatic Ring System” refers to a parent aromatic ring system in which one or more carbon atoms are each independently replaced with the same or different heteroatoms or heteroatomic groups. Typical heteroatoms or heteroatomic groups to replace the carbon atoms include, but are not limited to, N, NH, P, O, S, S(O), S(O) 2 , Si, etc.
• parent heteroaromatic ring systems fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as, for example, benzodioxan, benzofuran, chromane, chromene, indole, indoline, xanthene, etc.
• parent heteroaromatic ring system those recognized rings that include common substituents, such as, for example, benzopyrone and 1 -methyl- 1, 2,3, 4-tetrazole.
• parent heteroaromatic ring system benzene rings fused to cyclic polyalkylene glycols such as cyclic polyethylene glycols.
• Typical parent heteroaromatic ring systems include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxan, benzodioxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxaxine, benzoxazole, benzoxazoline, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthy
• Heteroaryl by itself or as part of another substituent refers to a monovalent heteroaromatic group having the stated number of ring atoms (e.g., “5-14 membered” means from 5 to 14 ring atoms) derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system.
• Typical heteroaryl groups include, but are not limited to, groups derived from acridine, benzimidazole, benzisoxazole, benzodioxan, benzodiaxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole, ⁇ -carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenazine, phthalazine, pter
• the heteroaryl group is a 5-14 membered heteroaryl, with 5-10 membered heteroaryl being particularly preferred.
• Heteroaryl-Heteroaryl by itself or as part of another substituent refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a ring system in which two or more identical or non-identical parent heteroaromatic ring systems are joined directly together by a single bond, where the number of such direct ring junctions is one less than the number of parent heteroaromatic ring systems involved.
• Typical heteroaryl-heteroaryl groups include, but are not limited to, bipyridyl, tripyridyl, pyridylpurinyl, bipurinyl, etc. Where the number of atoms are specified, the numbers refer to the number of atoms comprising each parent heteroaromatic ring systems.
• 5-15 membered heteroaryl-heteroaryl is a heteroaryl-heteroaryl group in which each parent heteroaromatic ring system comprises from 5 to 15 atoms, e.g., bipyridyl, tripuridyl, etc.
• each parent heteroaromatic ring system is independently a 5-15 membered heteroaromatic, more preferably a 5-10 membered heteroaromatic.
• heteroaryl-heteroaryl groups in which all of the parent heteroaromatic ring systems are identical.
• "Biheteroaryl" by itself or as part of another substituent refers to a heteroaryl-heteroaryl group having two identical parent heteroaromatic ring systems joined directly together by a single bond.
• Typical biheteroaryl groups include, but are not limited to, bipyridyl, bipurinyl, biquinolinyl, and the like.
• heteroaromatic ring systems are 5-15 membered heteroaromatic rings, more preferably 5-10 membered heteroaromatic rings.
• Heteroarylalkyl by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal or sp 3 carbon atom, is replaced with a heteroaryl group. Where specific alkyl moieties are intended, the nomenclature heteroarylalkanyl, heteroarylakenyl and/or heteroarylalkynyl is used.
• the heteroarylalkyl group is a 6-21 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety of the heteroarylalkyl is (C1-C6) alkyl and the heteroaryl moiety is a 5-15-membered heteroaryl.
• the heteroarylalkyl is a 6-13 membered heteroarylalkyl, e.g., the alkanyl, alkenyl or alkynyl moiety is (C1-C3) alkyl and the heteroaryl moiety is a 5-10 membered heteroaryl.
• Halogen or “Halo” by themselves or as part of another substituent, unless otherwise stated, refer to fluoro, chloro, bromo and iodo.
• Haloalkyl by itself or as part of another substituent refers to an alkyl group in which one or more of the hydrogen atoms is replaced with a halogen.
• haloalkyl is meant to include monohaloalkyls, dihaloalkyls, trihaloalkyls, etc. up to perhaloalkyls.
• (C1-C2) haloalkyl includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.
• the above-defined groups may include prefixes and/or suffixes that are commonly used in the art to create additional well-recognized substituent groups.
• alkyloxy or “alkoxy” refers to a group of the formula -OR
• alkylamine refers to a group of the formula -NHR
• dialkylamine refers to a group of the formula -NR"R
• each R is independently an alkyl
• haloalkoxy or “haloalkyloxy” refers to a group of the formula -OR”', where R"' is a haloalkyl.
• Protecting group refers to a group of atoms that, when attached to a reactive functional group in a molecule, mask, reduce or prevent the reactivity of the functional group.
• a protecting group may be selectively removed as desired during the course of a synthesis.
• protecting groups can be found in Greene and Wuts, Protective Groups in Organic Chemistry, 3 rd Ed., 1999, John Wiley & Sons, NY and Harrison et al, Compendium of Synthetic Organic Methods, Vols. 1-8, 1971-1996, John
• Representative amino protecting groups include, but are not limited to, formyl, acetyl, trifluoroacetyl, benzyl, benzyloxycarbonyl ("CBZ”), tert-butoxycarbonyl (“Boc”), trimethylsilyl ("TMS”), 2-trimethylsilyl-ethanesulfonyl (“TES”), trityl and substituted trityl groups, allyloxycarbonyl, 9-fluorenylmethyloxycarbonyl (“FMOC”), nitro- veratryloxycarbonyl (“NVOC”) and the like.
• hydroxyl protecting groups include, but are not limited to, those where the hydroxyl group is either acylated or alkylated such as benzyl and trityl ethers, as well as alkyl ethers, tetrahydropyranyl ethers, trialkylsilyl ethers (e.g., TMS or TIPPS groups) and allyl ethers.
• Prodrug refers to a derivative of an active 2,4-pyrimidinediamine compound (drug) that requires a transformation under the conditions of use, such as within the body, to release the active 2,4-pyrimidinediamine drug. Prodrugs are frequently, but not necessarily, pharmacologically inactive until converted into the active drug.
• Prodrugs are typically obtained by masking a functional group in the 2,4-pyrimidinediamine drug believed to be in part required for activity with a progroup (defined below) to form a promoiety which undergoes a transformation, such as cleavage, under the specified conditions of use to release the functional group, and hence the active 2,4-pyrimidinediamine drug.
• the cleavage of the promoiety may proceed spontaneously, such as by way of a hydrolysis reaction, or it may be catalyzed or induced by another agent, such as by an enzyme, by light, by acid or base, or by a change of or exposure to a physical or environmental parameter, such as a change of temperature.
• the agent may be endogenous to the conditions of use, such as an enzyme present in the cells to which the prodrug is administered or the acidic conditions of the stomach, or it may be supplied exogenously.
• a wide variety of progroups, as well as the resultant promoieties, suitable for masking functional groups in the active 2,4-pyrimidinediamines compounds to yield prodrugs are well-known in the art.
• a hydroxyl functional group may be masked as a sulfonate, ester or carbonate promoiety, which may be hydrolyzed in vivo to provide the hydroxyl group.
• An amino functional group may be masked as an amide, carbamate, imine, urea, phosphenyl, phosphoryl or sulfenyl promoiety, which may be hydrolyzed in vivo to provide the amino group.
• a carboxyl group may be masked as an ester (including silyl esters and thioesters), amide or hydrazide promoiety, which may be hydrolyzed in vivo to provide the carboxyl group.
• Nitrogen protecting groups and nitrogen pro-drugs of the invention may include lower alkyl groups as well as amides, carbamates, etc. Other specific examples of suitable progroups and their respective promoieties will be apparent to those of skill in the art.
• Progroup refers to a type of protecting group that, when used to mask a functional group within an active 2,4-pyrimidinediamine drug to form a promoiety, converts the drug into a prodrug.
• Progroups are typically attached to the functional group of the drug via bonds that are cleavable under specified conditions of use.
• a progroup is that portion of a promoiety that cleaves to release the functional group under the specified conditions of use.
• an amide promoiety of the formula -NH-C(O)CH 3 comprises the progroup -C(O)CH 3 .
• Fc Receptor refers to a member of the family of cell surface molecules that binds the Fc portion (containing the specific constant region) of an immunoglobulin.
• Each Fc receptor binds immunoglobulins of a specific type.
• the Fee. receptor (“Fc ⁇ R”) binds IgA
• the FceR binds IgE
• the Fc ⁇ R binds IgG.
• the Fc ⁇ R family includes the polymeric Ig receptor involved in epithelial transport of IgA IgM, the myeloid specific receptor RCGRI (also called CD89), the ⁇ cai ⁇ R and at least two alternative IgA receptors (for a recent review see Monteiro & van de Winkel, 2003, Annu. Rev. Immunol, advanced e-publication.
• the FcoRI is expressed on neutrophils, eosinophils, moncytes/macrophages, dendritic cells and kupfer cells.
• the Fc ⁇ RI indues one alpha chain and the FcR gamma homodimer that bears an activation motif (IT AM) in the cytoplasmic domain and phosphorylates Syk kinase.
• the FceR family includes two types, designated FceRI and FceRII (also known as CD23).
• FceRI is a high affinity receptor (binds IgE with an affinity of about lO ⁇ M "1 ) found on mast, basophil and eosinophil cells that anchors monomeric IgE to the cell surface.
• the FceRI possesses one alpha chain, one beta chain and the gamma chain homodimer discussed above.
• the FceRII is a low affinity receptor expressed on mononuclear phagocytes, B lymphocytes, eosinophils and platelets.
• the FceRII comprises a single polypeptide chain and does not include the gamma chain homodimer.
• the Fc ⁇ R family includes three types, designated Fc ⁇ RI (also known as CD64),
• Fc ⁇ RII also known as CD32
• Fc ⁇ RIII also known as CD16
• Fc ⁇ RI is a high affinity receptor (binds IgGl with an affinity of 10 M " ) found on mast, basophil, mononuclear, neutrophil, eosinophil, deudritic and phagocyte cells that anchors nomomeric IgG to the cell surface.
• the Fc ⁇ RI includes one alpha chain and the gamma chain dimer shared by FcoRI and FceRI.
• the Fc ⁇ RII is a low affinity receptor expressed on neutrophils, monocytes, eosinophils, platelets and B lymphocytes.
• the Fc ⁇ RII includes one alpha chain, and does not include the gamma chain homodimer discussed above.
• the Fc ⁇ RIII is a low affinity (bindes IgGl with an affinity of 5xl0 5 M _1 ) expressed on NK, eosinophil, macrophage, neutrophil and mast cells. It comprises one alpha chain and the gamma homodimer shared by FcoRI, FceRI and Fc ⁇ RI. Skilled artisans will recognize that the subunit structure and binding properties of these various Fc receptors, cell types expressing them, are not completely characterized.
• IgE-Induced Degranulation or “FceRI-Mediated Degranulation” refers to degranulation that proceeds via the IgE receptor signal transduction cascade initiated by crosslinking of Fc ⁇ Rl -bound IgE.
• the crosslinking may be induced by an IgE-specific allergen or other multivalent binding agent, such as an anti-IgE antibody.
• the FceRI signaling cascade leading to degranulation may be broken into two stages: upstream and downstream.
• the upstream stage includes all of the processes that occur prior to calcium ion mobilization (illustrated as "Ca 2+ " in FIG. 2; see also FIG. 3).
• the downstream stage includes calcium ion mobilization and all processes downstream thereof.
• Compounds that inhibit FceRI-mediated degranulation may act at any point along the FceRI-mediated signal transduction cascade.
• Compounds that selectively inhibit upstream FceRI-mediated degranulation act to inhibit that portion of the FceRI signaling cascade upstream of the point at which calcium ion mobilization is induced.
• compounds that selectively inhibit upstream FceRI-mediated degranulation inhibit degranulation of cells such as mast or basophil cells that are activated or stimulated with an IgE-specific allergen or binding agent (such as an anti-IgE antibody) but do not appreciably inhibit degranulation of cells that are activated or stimulated with degranulating agents that bypass the FceRI signaling pathway, such as, for example the calcium ionophores ionomycin and A23187.
• IgG- Induced Degranulation or "Fc ⁇ RI-Mediated Degranulation” refers to degranulation that proceeds via the Fc ⁇ RI signal transduction cascade initiated by crosslinking of Fc ⁇ RI-bound IgG.
• the crosslinking may be induced by an IgG-specific allergen or another multivalent binding agent, such as an anti-IgG or fragment antibody.
• an IgG-specific allergen or another multivalent binding agent such as an anti-IgG or fragment antibody.
• the Fc ⁇ RI signaling cascade also leads to degranulation which may be broken into the same two stages: upstream and downstream. Similar to FceRI-mediated degranulation, compounds that selectively inhibit upstream Fc ⁇ RI-mediated degranulation act upstream of the point at which calcium ion mobilization is induced.
• compounds that selectively inhibit upstream Fc ⁇ RI-mediated degranulation inhibit degranulation of cells such as mast or basophil cells that are activated or stimulated with an IgG-specific allergen or binding agent (such as an anti-IgG antibody or fragment) but do not appreciably inhibit degranulation of cells that are activated or stimulated with degranulating agents that bypass the Fc ⁇ RI signaling pathway, such as, for example the calcium ionophores ionomycin and A23187.
• Ionophore-Induced Degranulation or “Ionophore-Mediated Degranulation” refers to degranulation of a cell, such as a mast or basophil cell, that occurs upon exposure to a calcium ionophore such as, for example, ionomycin or A23187.
• Spyk Kinsase refers to the well-known 72kDa non-receptor (cytoplasmic) spleen protein tyrosine kinase expressed in B-cells and other hematopoetic cells.
• Syk kinase includes two consensus Src-homology 2 (SH2) domains in tandem that bind to phosphorylated immunoreceptor tyrosine-based activation motifs ("ITAMs"), a "linker” domain and a catalytic domain (for a review of the structure and function of Syk kinase see Sada et al, 2001, J. Biochem. (Tokyo) 130:177-186); see also Turner et al, 2000, Immunology Today 21 : 148-154). Syk kinase has been extensively studied as an effector of B-cell receptor (BCR) signaling (Turner et al, 2000, supra). Syk kinase is also critical for tyrosine phosphorylation of multiple proteins which regulate important pathways leading from immunoreceptors, such as Ca mobilization and mitogen-activated protein kinase
• Syk kinase also plays a critical role in integrin signaling in neutrophils (see, e.g., Mocsai et al. 2002, Immunity 16:547-558).
• Syk kinase includes kinases from any species of animal, including but not limited to, homosapiens, simian, bovine, porcine, rodent, etc., recognized as belonging to the Syk family. Specifically included are isoforms, splice variants, allelic variants, mutants, both naturally occuring and man-made.
• Syk kinases are well known and available from GENBANK. Specific examples of mRNAs encoding different isoforms of human Syk kinase can be found at GENBANK accession no. gi
• Syk mimics As a consequence of this structural similarity, such kinases, referred to herein as “Syk mimics,” are expected to catalyze phosphorylation of substrates phosphorylated by Syk.
• Syk mimics signal transduction cascades in which such Syk mimics play a role and biological responses effected by such Syk mimics and Syk mimic-dependent signaling cascades may be regulated, and in particular inhibited, with the 2,4-pyrimidinediamine compounds described herein.
• Syk-Dependent Signaling Cascade refers to a signal transduction cascade in which Syk kinase plays a role.
• Non- limiting examples of such Syk-dependent signaling cascades include the FcoRI, FceRI, Fc ⁇ RI, Fc ⁇ RIII, BCR and integrin signaling cascades.
• Autoimmune Disease refers to those diseases which are commonly associated with the nonanaphylactic hypersensitivity reactions (Type II, Type III and/or Type IN hypersensitivity reactions) that generally result as a consequence of the subject's own humoral and/or cell-mediated immune response to one or more immunogenic substances of endogenous and/or exogenous origin.
• Such autoimmune diseases are distinguished from diseases associated with the anaphylactic (Type I or IgE-mediated ) hypersensitivity reactions.
• the 2,4-Pyri idinediamine Compounds are generally 2,4-pyrimidinediamine compounds according to structural formula (I):
• L and L are each, independently of one another, selected from the group consisting of a direct bond and a linker; R 2 and R 4 are as described in the following embodiments and examples; R 5 is selected from the group consisting of R 6 , (C1-C6) alkyl optionally substituted o with one or more of the same or different R groups, (C1-C4) alkanyl optionally substituted with one or more of the same or different R groups, (C2-C4) alkenyl optionally substituted with one or more of the same or different R groups and (C2-C4) alkynyl optionally substituted with one or more of the same or different R groups; each R 6 independently is selected from the group consisting of hydrogen, an electronegative group, -OR d , -SR d , (C1-C3) haloalkyloxy, (C1-C3) perhaloalkyloxy, -NR C R C , hal
• L 1 and L 2 represent, independently of one another, a direct bond or a linker.
• the substituents R 2 and/or R 4 may be bonded either directly to their respective nitrogen atoms or, alternatively, spaced away from their respective nitrogen atoms by way of a linker.
• the identity of the linker is not critical and typical suitable linkers include, but are not limited to, (C1-C6) alkyldiyls, (C1-C6) alkanos and (C1-C6) heteroalkyldiyls, each of which may be • ft ft optionally substituted with one or more of the same or different R groups, where R is as previously defined for structural formula (I).
• L 1 and L 2 are each, independently of one another, selected from the group consisting of a direct bond, (C1-C3) alkyldiyl optionally substituted with one or more of the same or different R a , suitable R b or R 9 groups and 1-3 membered heteroalkyldiyl optionally substituted with one or more of the same or different R a , suitable R b or R 9 groups, wherein R 9 is selected from the group consisting of (C1-C3) alkyl, -OR a , -C(O)OR a , (C5-C10) aryl optionally substituted with one or more of the same or different halogens, phenyl optionally substituted with one or more of the same or different halogens, 5-10 membered heteroaryl optionally substituted with one or more of the same or different halogens and 6 membered heteroaryl optionally substituted with one or more of the same or different halogens; and R a
• R groups that may be used to substitute L and L include -OR a , -C(O)OR a , phenyl, halophenyl and 4-halophenyl, wherein R a is as previously defined for structural formula (I).
• L 1 and L 2 are each, independently of one another, selected from the group consisting of methano, ethano and propano, each of which may be optionally monosubstituted with an R 9 group, where R 9 is as previously defined above.
• specific R a groups that may be included in R 9 groups are selected from the group consisting of hydrogen, (C1-C6) alkyl, phenyl and benzyl.
• L 1 and L 2 are each a direct bond such that the 2,4-pyrimidinediamine compounds of the invention are compounds according to structural formula (la):
• R , R , R , R , R c , R , m and n are as previously defined, R is , wherein each
• R independently of the others, is methyl or (C1-C6) alkyl and R is
• X is selected from the group consisting of N and CH
• Y is selected from the group consisting of O, S, SO, SO 2 , SONR 36 , NH, NR 35 and NR 37 ,Z is selected from the group consisting of O, S, SO, SO 2 , SONR 36 , NH, NR 35 and NR 37 .
• Each R 36 is independently selected from the group consisting of hydrogen and (C1-C6) alkyl. Each R is independently selected from the group consisting of hydrogen and a progroup.
• R 38 is selected from the group consisting of (C1-C6) alkyl and (C5-C14) aryl.
• Y is O
• Z is NH
• X is N.
• R 5 can be halogen and R 6 is a hydrogen.
• R 5 , R 6 , R 8 , R a , R b , R c , R d , m, n, R 35 , R 36 , R 37 , R 38 , X, Y and Z are as previously defined, R 2 wherein each R , independently of the others, is methyl or (C1-C6) alkyl
• Y is O
• Z is NH
• X is N
• R 5 can be halogen and R 6 is a hydrogen.
• Y is O
• Z is NH
• X is N
• each R 31 is methyl. 1 -y
• L , L , R 5 , R 6 , R 8 , R a , R b , R c , R d , m, n, R 31 , R 35 , R 36 , R 37 , R 38 , X, Y and Z are as previously defined,
• R is or and yy is 1-6.
• Y is O
• Z is NH
• X is N.
• R can be halogen and R is a hydrogen.
• R ⁇ R , R 8 , R a , R B , R c , R°, m. n, R ,3"5, R E> 3 i 6 ⁇ , D R3"7, D R38 , X, Y and Z are as previously defined, R z
• R 2 phenyl ring can be at the 2, 3, 4, 5 or 6 positions.
• Y is O
• Z is NH
• X is N.
• R 5 can be halogen and R 6 is a hydrogen.
• Substitution about the R 2 phenyl ring can be at the 2,3, 2,4, 2, 5, 2,6, 3,4, 3,5, 3,6, 4,5, 4,6 or 5,6 positions, with the proviso that the following compounds are not included: N4-(2,2-Dimethyl-3-oxo-4H-5-pyrid[l,4]oxazin-6-yl)-N2-(3-chloro-4- methoxyphenyl)-5-fluoro-2,4-pyrimidinediamine; N4-(2,2-Dimethyl-3-oxo-4H-5-pyrid[l,4]oxazin-6-yl)-N2-(3,5-dimethoxyphenyl)-5- fluoro-2,4-pyrimidinediamine; N2-(3,4-Dichlorophenyl)-N4-(2,2-dimethyl-3-oxo-4H-5-pyrid[l,4]oxazin-6-yl)-5- fluoro-2,4-pyrimidinediamine;
• each R b independently is selected from (C1-C6) alkoxy, (Cl-16) alkyl, (C1-C6) perhaloalkyls, halogens, carboxylic acid, carboxylic ester, carboxamides, sulfonamides and imidazoles.
• L , L , R 5 , R 6 , R 8 , R a , R b , R c , R d , m, n, R 35 , R 36 , R 37 , R 38 , X, Y and Z are as previously defined, R 2
• R phenyl group trisubstituted with three R b groups and rfi.
• 'M Substitution about the R phenyl ring can be at the 2,3,4, 2,3,5, 2,3,6, 2,4,5, 2,4,6, 2,5,6,
• each R b independently is selected from (C1-C6) alkoxy, (Cl-16) alkyl, (C1-C6) perhaloalkyls, halogens, carboxylic acid, carboxylic esters, carboxamides, sulfonamides.
• the compounds disclosed in U.S. Patent Application Serial are selected from (C1-C6) alkoxy, (Cl-16) alkyl, (C1-C6) perhaloalkyls, halogens, carboxylic acid, carboxylic esters, carboxamides, sulfonamides.
• R 5 of the pyrimidine ring is a halogen atom, such as fluorine
• R 6 of the pyrimidine ring is a hydrogen atom.
• L and L are covalent bonds for the above-identified embodiments. Also specifically described are combinations of the above first through eight embodiments.
• the 2,4-pyrimidinediamine compounds described herein may include functional groups that can be masked with progroups to create prodrugs.
• prodrugs are usually, but need not be, pharmacologically inactive until converted into their active drug form.
• many of the active 2,4-pyrimidinediamine compounds described in TABLE 1 include promoieties that are hydro lyzable or otherwise cleavable under conditions of use.
• ester groups commonly undergo acid- catalyzed hydrolysis to yield the parent carboxylic acid when exposed to the acidic conditions of the stomach, or base-catalyzed hydrolysis when exposed to the basic conditions of the intestine or blood.
• 2,4- pyrimidinediamines that include ester moieties may be considered prodrugs of their corresponding carboxylic acid, regardless of whether the ester form is pharmacologically active.
• ester-containing 2,4-pyrimidinediamines of the invention are active in their ester, "prodrug" form.
• any available functional moiety may be masked with a progroup to yield a prodrug.
• Functional groups within the 2,4-pyrimidinediamine compounds that may be masked with progroups for inclusion in a promoiety include, but are not limited to, amines (primary and secondary), hydroxyls, sulfanyls (thiols), carboxyls, etc.
• Myriad progroups suitable for masking such functional groups to yield promoieties that are cleavable under the desired conditions of use are known in the art.
• the prodrugs of the invention are compounds according to structural formula (I) in which R c and R d may be, in addition to their previously-defined alternatives, a progroup.
• R c and R d may be, in addition to their previously-defined alternatives, a progroup.
• the compounds and prodrugs of the invention may include one or more chiral centers and/or double bonds and as a consequence may exist as stereoisomers, such as double-bond isomers (i.e., geometric isomers), enantiomers and diasteromers and mixtures thereof, such as racemic mixtures.
• the compounds and prodrugs of the invention may exist in several tautomeric forms, including the enol form, the keto form and mixtures thereof.
• the 2,4-pyrimidinediamine compounds and prodrugs of the invention may be in the form of salts.
• Such salts include salts suitable for pharmaceutical uses ("pharmaceutically-acceptable salts"), salts suitable for veterinary uses, etc.
• Such salts may be derived from acids or bases, as is well-known in the art.
• the salt is a pharmaceutically acceptable salt.
• pharmaceutically acceptable salts are those salts that retain substantially one or more of the desired pharmacological activities of the parent compound and which are suitable for administration to humans.
• Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids or organic acids.
• Inorganic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, hydrohalide acids (e.g., hydrochloric acid, hydrobromic acid, hydriodic, etc.), sulfuric acid, nitric acid, phosphoric acid, and the like.
• hydrohalide acids e.g., hydrochloric acid, hydrobromic acid, hydriodic, etc.
• sulfuric acid nitric acid
• phosphoric acid phosphoric acid
• Organic acids suitable for forming pharmaceutically acceptable acid addition salts include, by way of example and not limitation, acetic acid, trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, oxalic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, palmitic acid, benzoic acid, 3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, alkylsulfonic acids (e.g., methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, etc.), arylsulfonic acids (e.g., benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenes
• Pharmaceutically acceptable salts also include salts formed when an acidic proton present in the parent compound is either replaced by a metal ion (e.g., an alkali metal ion, an alkaline earth metal ion or an aluminum ion), an ammonium ion or coordinates with an organic base (e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, mo ⁇ holine, piperidine, dimethylamine, diethylamine, etc.).
• a metal ion e.g., an alkali metal ion, an alkaline earth metal ion or an aluminum ion
• an ammonium ion or coordinates with an organic base e.g., ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, mo ⁇ holine, piperidine, dimethylamine, diethylamine, etc.
• the 2,4-pyrimidinediamine compounds and of the invention, as well as the salts thereof may also be in the form
• the compounds and prodrugs of the invention may be synthesized via a variety of different synthetic routes using commercially available starting materials and/or starting materials prepared by conventional synthetic methods. Suitable exemplary methods that may be routinely adapted to synthesize the 2,4-pyrimidinediamine compounds and prodrugs of the invention are found in U.S. Patent No. 5,958,935, U.S. Patent Application 10/355,543, filed January 31, 2003 (US Publication US20040029902-A1), WO 03/063794, published August 1, 2003, U.S. Patent Application 10/631,029, filed July 29, 2003 and WO 2004/014382, published February 19, 2004, the disclosures of which are inco ⁇ orated herein by reference.
• R 2 , R 4 , R 5 , R 6 , L 1 and L 2 are as previously defined for structural formula (I),
• X is a halogen (e.g., F, CI, Br or I) and Y and Y' are each, independently of one another, selected from the group consisting of O and S.
• uracil or thiouracil 2 is dihalogenated at the 2- and 4-positions using standard halogenating agent POX 3 (or other standard halogenating agent) under standard conditions to yield 2,4-bishalo pyrimidine 4.
• the halide at the C4 position is more reactive towards nucleophiles than the halide at the C2 position.
• This differential reactivity can be exploited to synthesize 2,4-pyrimidinediamines according structural formula (I) by first reacting 2,4-bishalopyrimidine 4 with one equivalent of amine 10, yielding 4N-substituted-2-halo-4-pyrimidineamine 8, followed by amine 6 to yield a 2,4-pyrimidinediamine according structural formula (I).
• 2N,4N-bis(substituted)-2,4- pyrimidinediamines 12 and 14 can be obtained by reacting 2,4-bishalopyrimidine 4 with excess 6 or 10, respectively.
• the C4 halide is more reactive towards nucleophiles, as illustrated in the Scheme.
• the identity of the R 5 substituent may alter this reactivity. For example, when R 5 is trifluoromethyl, a 50:50 mixture of 4N-substituted-4-pyrimidineamine 8 and the corresponding 2N-substituted-2- pyrimidineamine is obtained.
• the regioselectivity of the reaction can be controlled by adjusting the solvent and other synthetic conditions (such as temperature), as is well-known in the art.
• the reactions depicted in Scheme (I) may proceed more quickly when the reaction mixtures are heated via microwave.
• the following conditions may be used: heat to 175°C in ethanol for 5-20 min. in a Smith Reactor (Personal Chemistry) in a sealed tube (at 20 bar pressure).
• the uracil or thiouracil 2 starting materials may be purchased from commercial sources or prepared using standard techniques of organic chemistry.
• uracils and thiouracils that can be used as starting materials in Scheme (I) include, by way of example and not limitation, uracil (Aldrich #13,078-8; CAS Registry 66-22-8); 2- thio-uracil (Aldrich #11,558-4; CAS Registry 141-90-2); 2,4-dithiouracil (Aldrich #15,846- 1; CAS Registry 2001-93-6); 5-acetouracil (Chem.
• Additional 5-, 6- and 5,6-substituted uracils and/or thiouracils are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA (www.generalintermediates.com) and/or Interchim, France (www.interchim.com). or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.
• Amines 6 and 10 may be purchased from commercial sources or, alternatively, may be synthesized utilizing standard techniques. For example, suitable amines may be synthesized from nitro precursors using standard chemistry. Specific exemplary reactions are provided in the Examples section. See also Nogel, 1989, Practical Organic Chemistry, Addison Wesley Longman, Ltd. and John Wiley & Sons, Inc.
• amines 6 and 10 and/or substituents R 5 and/or R 6 on uracil or thiouracil 2 may include functional groups that require protection during synthesis.
• the exact identity of any protecting group(s) used will depend upon the identity of the functional group being protected, and will be apparent to these of skill in the art.
• Guidance for selecting appropriate protecting groups, as well as synthetic strategies for their attachment and removal, may be found, for example, in Greene & Wuts, Protective Groups in Organic Synthesis, 3d Edition, John Wiley & Sons, Inc., New York (1999) and the references cited therein (hereinafter "Greene & Wuts").
• Scheme (I) utilizing 5-fluorouracil (Aldrich #32,937-1) as a starting material is illustrated in Scheme (la), below:
• R 2 , R 4 , L 1 and L 2 are as previously defined for Scheme (I).
• 5-fluorouracil 3 is halogenated with POCl 3 to yield 2,4-dichloro- 5-fluoropyrimidine 5, which is then reacted with excess amine 6 or 10 to yield N2,N4-bis substituted 5-fluoro-2,4-pyrimidinediamine 11 or 13, respectively.
• non-bis 2N,4N-disubstituted-5-fluoro-2,4-pyrimidinediamine 9 may be obtained by reacting 2,4- dichloro-5-fluoropyrimidine 5 with one equivalent of amine 10 (to yield 2-chloro-N4- substituted-5-fluoro-4-pyrimidineamine 7) followed by one or more equivalents of amine 6.
• the 2,4-pyrimidinediamine compounds of the invention may be synthesized from substituted or unsubstituted cytosines as illustrated in Schemes (Ila) and (lib), below: Scheme (Ila) acylation, silylation, etc POX 3 (other halogenating agents) 20 22 24
• Protected cytosine 22 is halogenated at the C2 position using a standard halogenation reagent under standard conditions to yield 2-chloro-4N-protected-4-pyrimidineamine 24. Reaction with amine 6 followed by deprotection of the C4 exocyclic amine and reaction with amine 10 yields a 2,4-pyrimidinediamine according to structural formula (I). Alternatively, referring to Scheme (lib), cytosine 20 may be reacted with amine 10 or protected amine 21 to yield N4-substituted cytosine 23 or 27, respectively.
• cytosines may then be halogenated as previously described, deprotected (in the case of N4-substituted cytosine 27) and reacted with amine 6 to yield a 2,4-pyrimidinediamine according to structural formula (I).
• cytosines that may be used as starting materials in Schemes (Ha) and (lib) include, but are not limited to, cytosine (Aldrich #14,201-8; CAS Registry 71-30-7); N 4 -acetylcytosine (Aldrich #37,791-0; CAS Registry 14631-20-0); 5-fluorocytosine (Aldrich #27,159-4; CAS Registry 2022-85-7); and 5-(trifluoromethyl)- cytosine.
• cytosines useful as starting materials in Schemes (Ila) are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA (www.generalintermediates.com) and/or Interchim, France (www.interchim.com), or may be prepared using standard techniques. Myriad textbook references teaching suitable synthetic methods are provided infra.
• the 2,4-pyrimidinediamine compounds of the invention may be synthesized from substituted or unsubstituted 2-amino-4-pyrimidinols as illustrated in Scheme (III), below: Scheme (III)
• 2-amino-4-pyrimidinols 30 that can be used as starting materials in Scheme (III) include, but are not limited to, 2-amino-6-chloro-4-pyrimidinol hydrate (Aldrich #A4702-8; CAS Registry 00000-00-0) and 2-amino-6-hydroxy-4-pyrimidinol (Aldrich #A5040-1; CAS Registry 56-09-7).
• 2-amino-4-pyrimidinols 30 useful as starting materials in Scheme (III) are available from General Intermediates of Canada, Inc., Edmonton, Alberta, CA
• 2,4-pyrimidinediamine compounds of the invention may be prepared from substituted or unsubstituted 4-amino-2-pyrimidinols as illustrated in Scheme (IV), below:
• R 2 , R 4 , R 5 , R 6 , L 1 and L 2 are as previously defined for Scheme (I) and Z represents a leaving group.
• Z represents a leaving group.
• the C2-hydroxyl of 4-amino-2-pyrimidinol 40 is more reactive towards nucleophiles than the C4-amino such that reaction with amine 6 yields N2-substituted-2,4-pyrimidinediamine 42.
• Subsequent reaction with compound 44, which includes a good leaving group Z, or amine 10 yields a 2,4-pyrimidinediamine according to structural formula (I).
• Compound 44 may include virtually any leaving group that can be displaced by the C4-amino of N2-substituted-2,4-pyrimidinediamine 42.
• Suitable leaving groups Z include, but are not limited to, halogens, methanesulfonyloxy (mesyloxy; "OMs"), trifluoromethanesulfonyloxy ("OTf ') and ?-toluenesulfonyloxy (tosyloxy; "OTs”), benzene sulfonyloxy ("besylate”) and metanitro benzene sulfonyloxy ("nosylate”).
• halogens methanesulfonyloxy (mesyloxy; "OMs”), trifluoromethanesulfonyloxy ("OTf ') and ?-toluenesulfonyloxy (tosyloxy; "OTs”), benzene sulfonyl
• 2,4-pyrimidinediamine compounds of the invention can be prepared from 2-chloro-4-aminopyrimidines or 2-amino-4-chloropyrimidines as illustrated in Scheme (V), below: Scheme (V)
• pyrimidines 50 and 54 suitable for use as starting materials in Scheme (V) are commercially available, including by way of example and not limitation, 2-amino-4,6-dichloropyrimidine (Aldrich #A4860-1; CAS Registry 56-05-3); 2-amino-4-chloro-6-methoxy-pyrimidine (Aldrich #51,864-6; CAS Registry 5734-64-5); 2-amino-4-chloro-6-methylpyrimidine (Aldrich #12,288-2; CAS Registry 5600-21-5); and 2-amino-4-chloro-6-methylthiopyrimidine (Aldrich #A4600-5; CAS Registry 1005-38-5).
• R 5 and R 6 are as previously defined for structural formula (I).
• dicarbonyl 53 is reacted with guanidine to yield 2-pyrimidineamine 51.
• Reaction with peracids like m-chloroperbenzoic acid, trifluoroperacetic acid or urea hydrogen peroxide complex yields N-oxide 55, which is then halogenated to give 4-chloro- 2-pyrimidineamine 50.
• the corresponding 4-halo-2-pyrimidineamines may be obtained by using suitable halogenation reagents.
• the 2,4-pyrimidinediamine compounds of the invention can be prepared from substituted or unsubstituted uridines as illustrated in Scheme (VI), below:
• R 2 , R 4 , R 5 , R 6 , L 1 , L 2 and X are as previously defined for Scheme (I) and the superscript PG represents a protecting group, as discussed in connection with Scheme (lib).
• uridine 60 has a C4 reactive center such that reaction with amine 10 or protected amine 21 yields N4-substituted cytidine 62 or 64, respectively.
• N4-substituted 62 or 64 Acid-catalyzed deprotection of N4-substituted 62 or 64 (when "PG” represents an acid-labile protecting group) yields N4-substituted cytosine 28, which may be subsequently halogenated at the C2-position and reacted with amine 6 to yield a 2,4-pyrimidinediamine according to structural formula (I).
• Cytidines may also be used as starting materials in an analogous manner, as illustrated in Scheme (VII), below: Scheme (VII)
• N2,N4-bis-substituted-2,4-pyrimidinediamines can be prepared in a manner analogous to Scheme (VIII) by employing 2,4-dichloro- 5 -methylpyrimidine or 2,4-dichloro- pyrimidine as starting materials. In this instance, the mono-substituted pyrimidineamine corresponding to compound 81 is not obtained. Instead, the reaction proceeds to yield the N2,N4-bis-substituted-2,4-pyrimidinediamine directly. Referring to Scheme (IX), 2,4,5,6-tetrachloropyrimidine 90 (Aldrich #24,671-9;
• N2,N4-bis-substituted-5,6,-dichloro-2,4-pyrimidinediamine 92 may be further reacted at the C6 halide with, for example a nucleophilic agent 94 to yield compound 95.
• compound 92 can be converted into N2,N4-bis-subsituted-5- chloro-6-aryl-2,4-pyrimidinediamine 97 via a Suzuki reaction.
• 2,4-Pyrimidinediamine 95 may be converted to 2,4-pyrimidinediamine 99 by reaction with Bn SnH.
• 2,4-pyrimidinediamines according to the invention synthesized via the exemplary methods described above or by other well-known means, may also be utilized as starting materials and/or intermediates to synthesize additional 2,4-pyrimidinediamine compounds of the invention.
• Scheme (X) illustrates A specific example is illustrated in Scheme (X), below: Scheme (X)
• R 4 , R 5 , R 6 , L 2 and R a are as previously defined for structural formula (I). Each R a is independently an R a , and may be the same or different from the illustrated R a .
• carboxylic acid or ester 100 may be converted to amide 104 by reaction with amine 102.
• R a may be the same or different than R a of acid or ester 100.
• carbonate ester 106 maybe converted to carbamate 108.
• R 4 , R 5 , R 6 , L 2 and R c are as previously defined for structural formula (I).
• amide 110 or 116 may be converted to amine 114 or 118, respectively, by borane reduction with borane methylsulfide complex 112.
• Other suitable reactions for synthesizing 2,4-pyrimidinediamine compounds from 2,4-pyrimidinediamine starting materials will be apparent to those of skill in the art.
• substituents R 2 , R 4 , R 5 , R 6 , L 1 and/or L 2 may include functional groups requiring protection.
• Prodrugs according to structural formula (II) may be prepared by routine modification of the above-described methods. Alternatively, such prodrugs may be prepared by reacting a suitably protected 2,4-pyrimidinediamine of structural formula (I) with a suitable progroup. Conditions for carrying out such reactions and for deprotecting the product to yield a prodrug of formula (II) are well-known.
• Active 2,4-pyrimidinediamine compounds of the invention inhibit Fc receptor signalling cascades that lead to, among other things, degranulation of cells.
• the compounds inhibit the FceRI and/or Fc RI signal cascades that lead to degranulation of immune cells such as neutrophil, eosinophil, mast and/or basophil cells.
• allergen-induced disorders including, for example, allergic rhinitis and asthma.
• allergens which may be, among other things, pollen or parasites
• allergen-specific IgE antibodies are synthesized by B-cells activated by IL-4 (or IL-13) and other messengers to switch to IgE class specific antibody synthesis.
• IL-4 or IL-13
• the Fc ⁇ RI -bound IgEs Upon binding of antigen, the Fc ⁇ RI -bound IgEs are cross-linked and the IgE receptor signal transduction pathway is activated, which leads to degranulation of the cells and consequent release and/or synthesis of a host of chemical mediators, including histamine, proteases (e.g., tryptase and chymase), lipid mediators such as leukotrienes (e.g., LTC4), platelet-activating factor (PAF) and prostaglandins (e.g., PGD2) and a series of cytokines, including TNF- ⁇ , IL-4, IL-13, IL-5, IL-6, IL-8, GMCSF, VEGF and TGF- ⁇ .
• chemical mediators including histamine, proteases (e.g., tryptase and chymase), lipid mediators such as leukotrienes (e.g., LTC4), platelet-activ
• the Fc ⁇ RI is a heterotetrameric receptor composed of an IgE-binding alpha-subunit, a beta subunit, and two gamma subunits (gamma homodimer).
• Cross-linking of FceRI-bound IgE by multivalent binding agents induces the rapid association and activation of the Src-related kinase Lyn.
• Lyn phosphorylates immunoreceptor tyrosine-based activation motifs (ITAMS) on the intracellular beta and gamma subunits, which leads to the recruitment of additional Lyn to the beta subunit and Syk kinase to the gamma homodimer.
• ITAMS immunoreceptor tyrosine-based activation motifs
• receptor-associated kinases which are activated by intra- and intermolecular phosphorylation, phosphorylate other components of the pathway, such as the Btk kinase, LAT, and phospholipase C-gamma PLC-gamma).
• Activated PLC-gamma initiates pathways that lead to protein kinase C activation and Ca 2+ mobilization, both of which are required for degranulation.
• Fc ⁇ RI cross-linking also activates the three major classes of mitogen activated protein (MAP) kinases, i.e. ERK1/2, JNK1/2, and p38.
• MAP mitogen activated protein
• the FcyRI signaling cascade is believed to share some common elements with the FceRI signaling cascade.
• the FcyRI includes a gamma homodimer that is phosphorylated and recruits Syk, and like FceRI, activation of the FcyRI signaling cascade leads to, among other things, degranulation.
• Fc receptors that share the gamma homodimer, and which can be regulated by the active 2,4-pyrimidinediamine compounds include, but are not limited to, Fco-RI and FcyRIII.
• the ability of the 2,4-pyrimidinediamine compounds of the invention to inhibit Fc receptor signaling cascades may be simply determined or confirmed in in vitro assays.
• Suitable assays for confirming inhition of FceRI-mediated degranulation are provided in the Examples section.
• cells capable of undergoing FceRI-mediated degranulation such as mast or basophil cells, are first grown in the presence of IL-4, Stem Cell Factor (SCF), IL-6 and IgE to increase expression of the FceRI, exposed to a 2,4- pyrimidinediamine test compound of the invention and stimulated with anti-IgE antibodies (or, alternatively, an IgE-specific allergen).
• SCF Stem Cell Factor
• the amount of a chemical mediator or other chemical agent released and/or synthesized as a consequence of activating the FceRI signaling cascade may be quantified using standard techniques and compared to the amount of the mediator or agent released from control cells (i.e., cells that are stimulated but that are not exposed to test compound).
• the concentration of test compound that yields a 50% reduction in the quantity of the mediator or agent measured as compared to control cells is the IC 50 of the test compound.
• the origin of the mast or basophil cells used in the assay will depend, in part, on the desired use for the compounds and will be apparent to those of skill in the art.
• mast or basophil cells are human or other animal which constitutes an accepted or known clinical model for the particular disease.
• the mast or basophil cells may be derived from a wide variety of animal sources, ranging from, for example, lower mammals such as mice and rats, to dogs, sheep and other mammals commonly employed in clinical testing, to higher mammals such as monkeys, chimpanzees and apes, to humans.
• cells suitable for carrying out the in vitro assays include, but are not limited to, rodent or human basophil cells, rat basophil leukemia cell lines, primary mouse mast cells (such as bone marrow-derived mouse mast cells “BMMC”) and primary human mast cells isolated from cord blood (“CHMC”) or other tissues such as lung.
• BMMC bone marrow-derived mouse mast cells
• CHMC primary human mast cells isolated from cord blood
• Methods for isolating and culturing these cell types are well-known or are provided in the Examples section (see, e.g., Demo et al, 1999, Cytometry 36(4):340-348 and copending application Serial No. 10/053,355, filed November 8, 2001, the disclosures of which are incorporated herein by reference).
• the mediator or agent quantified is not critical. The only requirement is that it be a mediator or agent released and/or synthesized as a consequence of initiating or activating the Fc receptor signaling cascade. For example, referring to FIG. 1, activation of the FceRI signaling cascade in mast and/or basophil cells leads to numerous downstream events. For example, activation of the FceRI signal cascade leads to the immediate release (i.e., within 1-3 min. following receptor activation) of a variety of preformed chemical mediators and agents via degranulation.
• the mediator or agent quantified may be specific to granules (i.e., present in granules but not in the cell cytoplasm generally).
• granule-specific mediators or agents that can be quantified to determine and/or confirm the activity of a 2,4- pyrimidinediamine compound of the invention include, but are not limited to, granule- specific enzymes such as hexosaminidase and tryptase and granule-specific components such as histamine and serotonin. Assays for quantifying such factors are well-known, and in many instances are commercially available.
• tryptase and/or hexosaminidase release may be quantified by incubating the cells with cleavable substrates that fluoresce upon cleavage and quantifying the amount of fluorescence produced using conventional techniques.
• cleavable fluorogenic substrates are commercially available.
• the 2,4- pyrimidinediamine compounds of the invention may also be assessed for activity by quantifying the amount of one or more of these mediators released and/or synthesized by activated cells. Unlike the granule-specific components discussed above, these "late stage" mediators are not released immediately following activation of the FceRI signaling cascade.
• the activated cell culture is incubated for a time sufficient to result in the synthesis (if necessary) and release of the mediator being quantified.
• PAF and lipid mediators such as leukotriene C4 are released 3-30 min. following FceRI activation.
• the cytokines and other late stage mediators are released approx. 4-8 hrs. following FceRI activation. Incubation times suitable for a specific mediator will be apparent to those of skill in the art. Specific guidance and assays are provided in the Examples section.
• the amount of a particular late stage mediator released may be quantified using any standard technique. In one embodiment, the amount(s) may be quantified using ELISA assays.
• ELISA assay kits suitable for quantifying the amount of TNF ⁇ , IL-4, IL-5, IL-6 and/or IL-13 released are available from, for example, Biosource International, Inc., Camarillo, CA 93012 (see, e.g., Catalog Nos. KHC3011, KHC0042, KHC0052, KHC0061 and KHC0132).
• ELISA assay kits suitable for quantifying the amount of leukotriene C4 (LTC4) released from cells are available from Cayman Chemical Co., Ann Arbor, MI 48108 (see, e.g., Catalog No. 520211).
• active 2,4-pyrimidinediamine compounds of the invention will exhibit IC 50 s with respect to FceRI-mediated degranulation and/or mediator release or synthesis of about 20 ⁇ M or lower, as measured in an in vitro assay, such as one of the in vitro assays described above or in the Examples section.
• an in vitro assay such as one of the in vitro assays described above or in the Examples section.
• compounds which exhibit lower IC 50 s for example on the order of 10 ⁇ M, 1 ⁇ M, 100 nM, 10 nM, 1 nM, or even lower, are particularly useful.
• Skilled artisans will also appreciate that the various mediators discussed above may induce different adverse effects or exhibit different potencies with respect to the same adverse effect.
• the lipid mediator LTC4 is a potent vasoconstrictor - it is approximately 1000-fold more potent at inducing vasoconstriction than histamine.
• cytokines can also cause tissue remodeling and cell proliferation.
• compounds which inhibit the release of all three types of mediators — granule- specific, lipid and cytokine — are useful for treating or preventing immediate Type I hypersensitivity reactions as well as the chronic symptoms associated therewith.
• Compounds of the invention capable of inhibiting the release of more than one type of mediator may be identified by determining the IC 50 with respect to a mediator representative of each class using the various in vitro assays described above (or other equivalent in vitro assays).
• Compounds of the invention which are capable of inhibiting the release of more than one mediator type will typically exhibit an IC 50 for each mediator type tested of less than about 20 ⁇ M.
• a compound which exhibits an IC 50 of 1 ⁇ M with respect to histamine release (IC 50 hlstam ⁇ ne ) and an IC 50 of 1 nM with respect to leukotriene LTC4 synthesis and or release (IC 50 LTC4 ) inhibits both immediate (granule-specific) and late stage mediator release.
• 1 ⁇ M inhibits immediate (granule-specific), lipid and cytokine mediator release.
• IC 50 s of one representative mediator of each class skilled artisans will appreciate that the IC 50 s of a plurality, or even all, mediators comprising one or more of the classes may be obtained.
• the quantity(ies) and identity(ies) of mediators for which IC 50 data should be ascertained for a particular compound and application will be apparent to those of skill in the art.
• Similar assays may be utilized to confirm inhibition of signal transduction cascades initiated by other Fc receptors, such as FcoRI, Fc ⁇ RI and/or Fc ⁇ RIII signaling, with routine modification.
• the ability of the compounds to inhibit Fc ⁇ RI signal transduction may be confirmed in assays similar to those described above, with the exception that the Fc ⁇ RI signaling cascade is activated, for example by incubating the cells with IgG and an IgG-specific allergen or antibody, instead of IgE and an IgE-specific allergen or antibody.
• Suitable cell types, activating agents and agents to quantify to confirm inhibition of other Fc receptors, such as Fc receptors that comprise a gamma homodimer will be apparent to those of skill in the art.
• One particularly useful class of compounds includes those 2,4-pyrimidinediamine compounds that inhibit the release of immediate granule-specific mediators and late stage mediators with approximately equivalent IC 50 s.
• IC 50 s for each mediator type are within about a 10-fold range of one another.
• Another particularly useful class of compounds includes those 2,4-pyrimidinediamine compounds that inhibit the release of immediate granule-specific mediators, lipid mediators and cytokine mediators with approximately equivalent IC 50 s. In a specific embodiment, such compounds inhibit the release of the following mediators with approximately equivalent
• IC 50 s histamine, tryptase, hexosaminidase, IL-4, IL-5, IL-6, IL-13, TNF ⁇ and LTC4.
• Such compounds are particularly useful for, among other things, ameliorating or avoiding altogether both the early and late stage responses associated with atopic or immediate Type I hypersensitivity reactions.
• the ability to inhibit the release of all desired types of mediators will reside in a single compound.
• mixtures of compounds can also be identified that achieve the same result.
• a first compound which inhibits the release of granule specific mediators may be used in combination with a second compound which inhibits the release and/or synthesis of cytokine mediators.
• degranulation of mast and/or basophil cells can be induced by other agents.
• ionomycin a calcium ionophore that bypasses the early FceRI or Fc ⁇ RI signal transduction machinery of the cell, directly induces a calcium flux that triggers degranulation.
• activated PLC ⁇ initiates pathways that lead to, among other things, calcium ion mobilization and subsequent degranulation. As illustrated, this Ca 2+ mobilization is triggered late in the FceRI signal transduction pathway.
• ionomycin directly induces Ca 2+ mobilization and a Ca 2+ flux that leads to degranulation.
• ionophores that induce degranulation in this manner include A23187.
• the ability of granulation-inducing ionophores such as ionomycin to bypass the early stages of the FceRI and/or Fc ⁇ RI signaling cascades may be used as a counter screen to identify active compounds of the invention that specifically exert their degranulati on- inhibitory activity by blocking or inhibiting the early FceRI or Fc ⁇ RI signaling cascades, as discussed above.
• Compounds which specifically inhibit such early FceRI or Fc ⁇ RI- mediated degranulation inhibit not only degranulation and subsequent rapid release of histamine, tryptase and other granule contents, but also inhibit the pro-inflammatory activation pathways causing the release of TNF ⁇ , IL-4, IL-13 and the lipid mediators such as LTC4.
• compounds which specifically inhibit such early FceRI and/or Fc ⁇ RI- mediated degranulation block or inhibit not only acute atopic or Type I hypersensitivity reactions, but also late responses involving multiple inflammatory mediators.
• Compounds of the invention that specifically inhibit early FceRI and/or Fc ⁇ RI- mediated degranulation are those compounds that inhibit FceRI and/or Fc ⁇ RI-mediated degranulation (for example, have an IC 50 of less than about 20 ⁇ M with respect to the release of a granule-specific mediator or component as measured in an in vitro assay with cells stimulated with an IgE or IgG binding agent) but that do not appreciably inhibit ionophore-induced degranulation.
• compounds are considered to not appreciably inhibit ionophore-induced degranulation if they exhibit an IC 50 of ionophore- induced degranulation of greater than about 20 ⁇ M, as measured in an in vitro assay.
• active compounds that exhibit even higher IC 50 s of ionophore-induced degranulation, or that do not inhibit ionophore-induced degranulation at all, are particularly useful.
• compounds are considered to not appreciably inhibit ionophore-induced degranulation if they exhibit a greater than 10-fold difference in their IC 50 s of FceRI and/or Fc ⁇ RI-mediated degranulation and ionophore-induced degranulation, as measured in an in vitro assay.
• Assays suitable for determining the IC 50 of ionophore- induced degranulation include any of the previously-described degranulation assays, with the modification that the cells are stimulated or activated with a degranulation-inducing calcium ionophore such as ionomycin or A23187 (A.G. Scientific, San Diego, CA) instead of anti-IgE antibodies or an IgE-specific allergen.
• a degranulation-inducing calcium ionophore such as ionomycin or A23187 (A.G. Scientific, San Diego, CA) instead of anti-IgE antibodies or an IgE-specific allergen.
• Specific assays for assessing the ability of a particular 2,4-pyrimidinediamine compound of the invention to inhibit ionophore- induced degranulation are provided in the Examples section.
• compounds which exhibit a high degree of selectivity of FceRI-mediated degranulation find particular use, as such compounds selectively target the FceRI cascade and do not interfere with other degranulation mechanisms.
• compounds which exhibit a high degree of selectivity of Fc ⁇ RI- mediated degranulation find particular use, as such compounds selectively target the Fc ⁇ RI cascade and do not interfere with other degranulation mechanisms.
• Compounds which exhibit a high degree of selectivity are generally 10-fold or more selective for FceRI- or Fc ⁇ RI -mediated degranulation over ionophore-induced degranulation, such as ionomycin- induced degranulation.
• the activity of the 2,4-pyrimidinediamine compounds of the invention may also be confirmed in biochemical or cellular assays of Syk kinase activity.
• Syk kinase phosphorylates LAT and PLC-gammal, which leads to, among other things, degranulation. Any of these activities may be used to confirm the activity of the 2,4-pyrimidinediamine compounds of the invention.
• the activity is confirmed by contacting an isolated Syk kinase, or an active fragment thereof with a 2,4-pyrimidinediamine compound in the presence of a Syk kinase substrate (e.g., a synthetic peptide or a protein that is known to be phophorylated by Syk in a signaling cascade) and assessing whether the Syk kinase phosphorylated the substrate.
• a Syk kinase substrate e.g., a synthetic peptide or a protein that is known to be phophorylated by Syk in a signaling cascade
• the assay may be carried out with cells that express a Syk kinase.
• the cells may express the Syk kinase endogenously or they may be engineered to express a recombinant Syk kinase.
• the cells may optionally also express the Syk kinase substrate.
• the active compounds of the invention inhibit Fc receptor signaling cascades, especially those Fc receptors including a gamma homodimer, such as the FceRI and/or Fc ⁇ RI signaling cascades, that lead to, among other things, the release and/or synthesis of chemical mediators from cells, either via degranulation or other processes.
• the active compounds are also potent inhibitors of Syk kinase.
• the active compounds of the invention may be used in a variety of in vitro, in vivo and ex vivo contexts to regulate or inhibit Syk kinase, signaling cascades in which Syk kinase plays a role, Fc receptor signaling cascades, and the biological responses effected by such signaling cascades.
• the compounds may be used to inhibit Syk kinase, either in vitro or in vivo, in virtually any cell type expressing Syk kinase. They may also be used to regulate signal transduction cascades in which Syk kinase plays a role.
• Such Syk-dependent signal transduction cascades include, but are not limited to, the FceRI, Fc ⁇ RI, Fc ⁇ RIII, BCR and integrin signal transduction cascades.
• the compounds may also be used in vitro or in vivo to regulate, and in particular inhibit, cellular or biological responses effected by such Syk-dependent signal transduction cascades.
• Such cellular or biological responses include, but are not limited to, respiratory burst, cellular adhesion, cellular degranulation, cell spreading, cell migration, cell aggregation, phagcytosis, cytokine synthesis and release, cell maturation and Ca flux.
• the compounds may be used to inhibit Syk kinase in vivo as a therapeutic approach towards the treatment or prevention of diseases mediated, either wholly or in part, by a Syk kinase activity.
• Syk kinase mediated diseases that may be treated or prevented with the compounds are those discussed in more detail, below.
• the active compounds may be used to regulate or inhibit the Fc receptor signaling cascades and/or FceRI- and/or Fc ⁇ RI-mediated degranulation as a therapeutic approach towards the treatment or prevention of diseases characterized by, caused by and/or associated with the release or synthesis of chemical mediators of such Fc receptor signaling cascades or degranulation.
• Such treatments may be administered to animals in veterinary contexts or to humans.
• Diseases that are characterized by, caused by or associated with such mediator release, synthesis or degranulation, and that can therefore be treated or prevented with the active compounds include, by way of example and not limitation, atopy or anaphylactic hypersensitivity or allergic reactions, allergies (e.g., allergic conjunctivitis, allergic rhinitis, atopic asthma, atopic dermatitis and food allergies), low grade scarring (e.g., of scleroderma, increased fibrosis, keloids, post-surgical scars, pulmonary fibrosis, vascular spasms, migraine, reperfusion injury and post myocardial infarction), diseases associated with tissue destruction (e.g., of COPD, cardiobronchitis and post myocardial infarction), diseases associated with tissue inflammation (e.g.
• autoimmune diseases irritable bowel syndrome, spastic colon and inflammatory bowel disease
• inflammation and scarring cellular and animal empirical data confirm that the 2,4-pyrimidinediamine compounds described herein are also useful for the treatment or prevention of autoimmune diseases, as well as the various symptoms associated with such diseases.
• the types of autoimmune diseases that may be treated or prevented ⁇ tf-th the 1 2,4-pynm ⁇ tnfed ⁇ amine compounds generally include those disorders involving tissue injury that occurs as a result of a humoral and or cell-mediated response to immunogens or antigens of endogenous and/or exogenous origin.
• Type II hypersensitivity reactions generally result from the release of pharmacologically active substances, such as histamine, from mast and/or basophil cells following contact with a specific exogenous antigen.
• Type I reactions play a role in numerous diseases, including allergic asthma, allergic rhinitis, etc.
• Type II hypersensitivity reactions also referred to as cytotoxic, cytolytic complement-dependent or cell-stimulating hypersensitivity reactions
• Type II hypersensitivity reactions Diseases that are commonly associated with Type II hypersensitivity reactions include, but are not limited, to autoimmune hemolytic anemia, erythroblastosis fetalis and Goodpasture's disease.
• Type III hypersensitivity reactions (also referred to as toxic complex, soluble complex, or immune complex hypersensitivity reactions) result from the deposition of soluble circulating antigen-immunoglobulin complexes in vessels or in tissues, with accompanying acute inflammatory reactions at the site of immune complex deposition.
• Non-limiting examples of prototypical Type III reaction diseases include the Arthus reaction, rheumatoid arthritis, serum sickness, systemic lupus erythematosis, certain types of glomerulonephritis, multiple sclerosis and bullous pemphingoid.
• Type IV hypersensitivity reactions (frequently called cellular, cell-mediated, delayed, or tuberculin-type hypersensitivity reactions) are caused by sensitized T- lymphocytes which result from contact with a specific antigen.
• diseases cited as involving Type IV reactions are contact dermatitis and allograft rejection including, but not limited to, heart transplant.
• Autoimmune diseases associated with any of the above nonanaphylactic hypersensitivity reactions may be treated or prevented with the 2,4-pyrimidinediamine compounds of the invention.
• the methods may be used to treat or prevent those autoimmune diseases frequently characterized as single organ or single cell-type autoimmune disorders including, but not limited to: Hashimoto's thyroiditis, autoimmune hemolytic anemia, autoimmune atrophic gastritis of pernicious anemia, autoimmune encephalomyelitis, autoimmune orchitis, Goodpasture's disease, autoimmune thrombocytopenia, sympathetic ophthalmia, myasthenia gravis, Graves' disease, primary biliary cirrhosis, chronic aggressive hepatitis, ulcerative colitis and membranous glomerulopathy, as well as those autoimmune diseases frequently characterized as involving systemic autoimmune disorder, which include but are not limited to: systemic lupus erythematosis, rheumatoid arthritis, Sjogren's syndrome, Reiter's syndrome, polymyositis- dermatomyositis, systemic sclerosis, polyarteritis nodosa
• autoimmune diseases are associated with severe symptoms, the amelioration of which provides significant therapeutic benefit even in instances where the underlying autoimmune disease may not be ameliorated. Many of these symptoms, as well as their underlying disease states, result as a consequence of activating the Fc ⁇ R signaling cascade in monocyte cells.
• the 2,4-pyrimidinediamine compounds described herein are potent inhibitors of such Fc ⁇ R signaling in monocytes and other cells, the methods find use in the treatment and/or prevention of myriad adverse symptoms associated with the above-listed autoimmune diseases.
• RA rheumatoid arthritis
• RA is characterized by chronically inflamed synovium that is densely crowded with lymphocytes.
• the synovial membrane which is typically one cell layer thick, becomes intensely cellular and assumes a form similar to lymphoid tissue, including dentritic cells, T-, B- and NK cells, macrophages and clusters of plasma cells.
• the methods may be used to treat or ameliorate any one, several or all of these symptoms of RA.
• the methods are considered to provide therapeutic benefit (discussed more generally, infra) when a reduction or amelioration of any of the symptoms commonly associated with RA is achieved, regardless of whether the treatment results in a concomitant treatment of the underlying RA and/or a reduction in the amount of circulating rheumatoid factor ("RF").
• RF circulating rheumatoid factor
• SLE systemic lupus erythematosis
• SLE systemic lupus erythematosis
• symptoms such as fever, joint pain (arthralgias), arthritis, and serositis (pleurisy or pericarditis).
• the methods are considered to provide therapeutic benefit when a reduction or amelioration of any of the symptoms commonly associated with SLE are achieved, regardless of whether the treatment results in a concomitant treatment of the underlying SLE.
• multiple sclerosis (MS) cripples the patient by disturbing visual acuity; stimulating double vision; disturbing motor functions affecting walking and use of the hands; producing bowel and bladder incontinence; spasticity; and sensory deficits (touch, pain and temperature sensitivity).
• MS multiple sclerosis
• the methods are considered to provide therapeutic benefit when an improvement or a reduction in the progression of any one or more of the crippling effects commonly associated with MS is achieved, regardless of whether the treatment results in a concomitant treatment of the underlying MS.
• the active compounds When used to treat or prevent such diseases, the active compounds may be administered singly, as mixtures of one or more active compounds or in mixture or combination with other agents useful for treating such diseases and/or the symptoms associated with such diseases.
• the active compounds may also be administered in mixture or in combination with agents useful to treat other disorders or maladies, such as steroids, membrane stablizers, 5LO inhibitors, leukotriene synthesis and receptor inhibitors, inhibitors of IgE isotype switching or IgE synthesis, IgG isotype switching or IgG synthesis, ⁇ -agonists, tryptase inhibitors, aspirin, COX inhibitors, methotrexate, anti-TNF drugs, retuxin, PD4 inhibitors, p38 inhibitors, PDE4 inhibitors, and antihistamines, to name a few.
• the active compounds may be administered er se in the form of prodrugs or as pharmaceutical compositions, comprising an active compound or prodrug.
• Pharmaceutical compositions comprising the active compounds of the invention (or prodrugs thereof) may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making levigating, emulsifying, encapsulating, entrapping or lyophilization processes.
• the compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically.
• the active compound or prodrug may be formulated in the pharmaceutical compositions per se, or in the form of a hydrate, solvate, N-oxide or pharmaceutically acceptable salt, as previously described.
• such salts are more soluble in aqueous solutions than the corresponding free acids and bases, but salts having lower solubility than the corresponding free acids and bases may also be formed.
• Pharmaceutical compositions of the invention may take a form suitable for virtually any mode of administration, including, for example, topical, ocular, oral, buccal, systemic, nasal, injection, transdermal, rectal, vaginal, etc., or a form suitable for administration by inhalation or insufflation.
• the active compound(s) or prodrug(s) may be formulated as solutions, gels, ointments, creams, suspensions, etc. as are well-known in the art.
• Systemic formulations include those designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal oral or pulmonary administration.
• Useful injectable preparations include sterile suspensions, solutions or emulsions of the active compound(s) in aqueous or oily vehicles.
• the compositions may also contain formulating agents, such as suspending, stabilizing and/or dispersing agent.
• the formulations for injection may be presented in unit dosage form, e.g., in ampules or in multidose containers, and may contain added preservatives.
• the injectable formulation may be provided in powder form for reconstitution with a suitable vehicle, including but not limited to sterile pyrogen free water, buffer, dextrose solution, etc., before use.
• the active compound(s) may be dried by any art-known technique, such as lyophilization, and reconstituted prior to use.
• penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are known in the art.
• the pharmaceutical compositions may take the form of, for example, lozenges, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalhne cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate).
• binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
• fillers e.g., lactose, microcrystalhne cellulose or calcium hydrogen phosphate
• lubricants e.g., magnesium stearate, talc or silica
• Liquid preparations for oral administration may take the form of, for example, elixirs, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
• Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, cremophoreTM or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
• suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
• emulsifying agents e.g., lecithin or acacia
• non-aqueous vehicles e.g
• the preparations may also contain buffer salts, preservatives, flavoring, coloring and sweetening agents as appropriate.
• Preparations for oral administration may be suitably formulated to give controlled release of the active compound or prodrug, as is well known.
• the compositions may take the form of tablets or lozenges formulated in conventional manner.
• the active compound(s) may be formulated as solutions (for retention enemas) suppositories or ointments containing conventional suppository bases such as cocoa butter or other glycerides.
• the active compound(s) or prodrug(s) can be conveniently delivered in the form of an aerosol spray from pressurized packs or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas.
• a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, fluorocarbons, carbon dioxide or other suitable gas.
• the dosage unit may be determined by providing a valve to deliver a metered amount.
• Capsules and cartridges for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
• a suitable powder base such as lactose or starch.
• a specific example of an aqueous suspension formulation suitable for nasal administration using commercially-available nasal spray devices includes the following ingredients: active compound or prodrug (0.5-20 mg/ml); benzalkonium chloride (0.1-0.2 mg/mL); polysorbate 80 (TWEEN® 80; 0.5-5 mg/ml); carboxymethylcellulose sodium or microcrystalhne cellulose (1-15 mg/ml); phenylethanol (1-4 mg/ml); and dextrose (20-50 mg/ml).
• the pH of the final suspension can be adjusted to range from about pH5 to pH7, with a pH of about pH 5.5 being typical.
• Another specific example of an aqueous suspension suitable for admimstration of the compounds via inhalation, and in particular for such administration of a compound of the invention contains 1-20 mg/mL of the compound or prodrug, 0.1-1% (v/v) Polysorbate 80 (TWEEN®80), 50 mM citrate and or 0.9% sodium chloride.
• the active compound(s) or prodrug(s) may be formulated as a solution, emulsion, suspension, etc. suitable for administration to the eye.
• a variety of vehicles suitable for administering compounds to the eye are known in the art.
• the active compound(s) or prodrug(s) can be formulated as a depot preparation for administration by implantation or intramuscular injection.
• the active ingredient may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g., as a sparingly soluble salt.
• transdermal delivery systems manufactured as an adhesive disc or patch which slowly releases the active compound(s) for percutaneous abso ⁇ tion may be used.
• permeation enhancers may be used to facilitate transdermal penetration of the active compound(s).
• Suitable transdermal patches are described in for example, U.S. Patent No. 5,407,713.; U.S. Patent No. 5,352,456; U.S. Patent No. 5,332,213; U.S. Patent No. 5,336,168; U.S. Patent No. 5,290,561; U.S. Patent No. 5,254,346; U.S. Patent No. 5,164,189; U.S. Patent No. 5,163,899; U.S. Patent No. 5,088,977; U.S. Patent No. 5,087,240; U.S. Patent No. 5,008,110; and U.S. Patent No. 4,921,475.
• Liposomes and emulsions are well-known examples of delivery vehicles that may be used to deliver active compound(s) or prodrug(s).
• Certain organic solvents such as dimethylsulfoxide (DMSO) may also be employed, although usually at the cost of greater toxicity.
• DMSO dimethylsulfoxide
• the pharmaceutical compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active compound(s).
• the pack may, for example, comprise metal or plastic foil, such as a blister pack.
• the pack or dispenser device may be accompanied by instructions for administration.
• the active compound(s) or prodrug(s) of the invention, or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat or prevent the particular disease being treated.
• the compound(s) may be administered therapeutically to achieve therapeutic benefit or prophylactically to achieve prophylactic benefit.
• therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated and/or eradication or amelioration of one or more of the symptoms associated with the underlying disorder such that the patient reports an improvement in feeling or condition, notwithstanding that the patient may still be afflicted with the underlying disorder.
• admimstration of a compound to a patient suffering from an allergy provides therapeutic benefit not only when the underlying allergic response is eradicated or ameliorated, but also when the patient reports a decrease in the severity or duration of the symptoms associated with the allergy following exposure to the allergen.
• therapeutic benefit in the context of asthma includes an improvement in respiration following the onset of an asthmatic attack, or a reduction in the frequency or severity of asthmatic episodes.
• Therapeutic benefit also includes halting or slowing the progression of the disease, regardless of whether improvement is realized.
• the compound may be administered to a patient at risk of developing one of the previously described diseases.
• the compound may be administered prior to administration of the drug to avoid or ameliorate an allergic response to the drug.
• prophylactic administration may be applied to avoid the onset of symptoms in a patient diagnosed with the underlying disorder.
• a compound may be administered to an allergy sufferer prior to expected exposure to the allergen.
• Compounds may also be administered prophylactically to healthy individuals who are repeatedly exposed to agents known to one of the above-described maladies to prevent the onset of the disorder.
• a compound may be administered to a healthy individual who is repeatedly exposed to an allergen known to induce allergies, such as latex, in an effort to prevent the individual from developing an allergy.
• a compound may be administered to a patient suffering from asthma prior to partaking in activities which trigger asthma attacks to lessen the severity of, or avoid altogether, an asthmatic episode.
• the amount of compound administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular active compound, etc. Determination of an effective dosage is well within the capabilities of those skilled in the art. Effective dosages may be estimated initially from in vitro assays.
• an initial dosage for use in animals may be formulated to achieve a circulating blood or serum concentration of active compound that is at or above an IC 50 of the particular compound as measured in as in vitro assay, such as the in vitro CHMC or BMMC and other in vitro assays described in the Examples section.
• IC 50 of the particular compound as measured in as in vitro assay
• Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound is well within the capabilities of skilled artisans.
• the reader is referred to Fingl & Woodbury, "General Principles," In: Goodman and Gilman 's The Pharmaceutical Basis of Therapeutics, Chapter 1, pp. 1-46, latest edition, Pagamonon Press, and the references cited therein.
• Initial dosages can also be estimated from in vivo data, such as animal models.
• Suitable animal models of systemic mastocytosis are described in O'Keefe et al, 1987, J. Vet. Intern. Med. l(2):75-80 and Bean-Knudsen et al, 1989, Vet. Pathol. 26(l):90-92.
• Suitable animal models of hyper IgE syndrome are described in Claman et al, 1990, Clin. Immunol. Immunopathol. 56(l):46-53.
• Suitable animal models of B-cell lymphoma are described in Hough et al, 1998, Proc. Natl. Acad. Sci. USA 95:13853-13858 and Hakim et al, 1996, J. Immunol. 157(12):5503-5511.
• Suitable animal models of atopic disorders such as atopic dermatitis, atopic eczema and atopic asthma are described in Chan et al, 2001, J. Invest. Dermatol. 117(4):977-983 and Suto et al, 1999, Int. Arch. Allergy Immunol. 120(Su ⁇ pl l):70-75. Ordinarily skilled artisans can routinely adapt such information to determine dosages suitable for human administration. Additional suitable animal models are described in the Examples section.
• Dosage amounts will typically be in the range of from about 0.0001 or 0.001 or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the compound, its bioavailability, the mode of administration and various factors discussed above. Dosage amount and interval may be adjusted individually to provide plasma levels of the compound(s) which are sufficient to maintain therapeutic or prophylactic effect.
• the compounds may be administered once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician.
• the effective local concentration of active compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective local dosages without undue experimentation.
• the compound(s) will provide therapeutic or prophylactic benefit without causing substantial toxicity. Toxicity of the compound(s) may be determined using standard pharmaceutical procedures. The dose ratio between toxic and therapeutic (or prophylactic) effect is the therapeutic index. Compounds(s) that exhibit high therapeutic indices are preferred.
• the 2,4-Pyrimidinediamine Compounds of the Invention Inhibit FceRI Receptor-Mediated Degranulation
• CHMC cultured human mast cells
• BMMC mouse bone marrow derived cells
• Inhibition of release and/or synthesis of lipid mediators was assessed by measuring the release of leukotriene LTC4 and inhibition of release and/or synthesis of cytokines was monitored by quantifying TNF- ⁇ , IL-6 and IL-13. Tryptase and hexosaminidase were quantified using fluorogenic substrates as described in their respective examples. Histamine, TNF ⁇ , IL-6, IL-13 and
• LTC4 were quantified using the following commercial ELISA kits: histamine (Immunotech #2015, Beckman Coulter), TNF ⁇ (Biosource #KHC3011), IL-6 (Biosource #KMC0061), IL-13 (Biosource #KHC0132) and LTC4 (Cayman Chemical #520211). The protocols of the various assays are provided below. 7.2.1 Culturing of Human Mast and Basophil Cells Human mast and basophil cells were cultured from CD34-negative progenitor cells as described below (see also the methods described in copending U.S. application Serial No. 10/053,355, filed November 8, 2001, the disclosure of which is inco ⁇ orated herein by reference).
• STEMPRO-34 complete medium 250 mL STEMPRO-34TM serum free medium (“SFM”; GibcoBRL, Catalog No. 10640) was added to a filter flask. To this was added 13 mL STEMPRO-34 Nutrient Supplement (“NS”; GibcoBRL, Catalog No. 10641) (prepared as described in more detail, below). The NS container was rinsed with approximately 10 mL SFM and the rinse added to the filter flask. Following addition of 5 mL L-glutamine (200 mM; Mediatech, Catalog No. MT 25-005-CI and 5 mL 100X penicillin streptomycin ("pen-strep"; HyClone, Catalog No.
• the most variable aspect of preparing the CM is the method by which the NS is thawed and mixed prior to addition to the SFM.
• the NS should be thawed in a 37° C water bath and swirled, not vortexed or shaken, until it is completely in solution. While swirling, take note whether there are any lipids that are not yet in solution. If lipids are present and the NS is not uniform in appearance, return it to the water bath and repeat the swirling process until it is uniform in appearance. Sometimes this component goes into solution immediately, sometimes after a couple of swirling cycles, and sometimes not at all. If, after a couple of hours, the NS is still not in solution, discard it and thaw a fresh unit. NS that appears non-uniform after thaw should not be used.
• CD34+ cells A starting population of CD34-positive (CD34+) cells of relatively small number (1-5 x 10° cells) was expanded to a relatively large number of CD34-negative progenitor cells (about 2-4 x 10 cells) using the culture media and methods described below.
• the CD34+ cells (from a single donor) were obtained from Allcells (Berkeley, CA). Because there is a degree of variation in the quality and number of CD34+ cells that Allcells typically provides, the newly delivered cells were transferred to a 15 mL conical tube and brought up to 10 mL in CM prior to use. On day 0, a cell count was performed on the viable (phase-bright) cells and the cells were spun at 1200 ⁇ m to pellet.
• the cells were resuspended to a density of 275,000 cells/mL with CM containing 200 ng/mL recombinant human Stem Cell Factor ("SCF"; Peprotech, Catalog No. 300-07) and 20 ng/mL human flt-3 ligand (Peprotech, Catalog No. 300-19) ("CM SCF/flt-3 medium").
• SCF Stem Cell Factor
• CM SCF/flt-3 medium 20 ng/mL human flt-3 ligand
• the density of the culture was checked by performing a cell count and the culture was diluted to a density of 275,000 cells/mL with fresh CM/SCF/flt-3 medium.
• the culture was transferred to a sterile tube and a cell count was performed.
• the cells were spun at 1200 ⁇ m and resuspended to a density of 275,000 cells/mL with fresh CM/SCF/flt-3 medium. This cycle was repeated, starting from day 0, a total of 3-5 times over the expansion period.
• the contents of all of the flasks are combined into a single container prior to performing a cell count. This ensures that an accurate cell count is achieved and provides for a degree of uniformity of treatment for the entire population.
• Each flask is checked separately for contamination under the microscope prior to combining to prevent contamination of the entire population.
• the culture can begin to go into decline (i.e., approximately 5- 10% of the total number of cells die) and fail to expand as rapidly as before.
• the cells are then monitored on a daily basis during this time, as complete failure of the culture can take place in as little as 24 hours.
• the cells are counted, spun down at 850 ⁇ m for 15 minutes, and resuspended at a density of 350,000 cells/mL in CM/SCF/flt-3 medium to induce one or two more divisions out of the culture.
• the cells are monitored daily to avoid failure of the culture. When greater than 15% cell death is evident in the progenitor cell culture and some debris is present in the culture, the CD34-negative progenitor cells are ready to be differentiated.
• CD34-Negative Progenitor Cells into Mucosal Mast Cells A second phase is performed to convert the expanded CD34-negative progenitor cells into differentiated mucosal mast cells.
• These mucosal cultured human mast cells (“CHMC") are derived from CD34+ cells isolated from umbilical cord blood and treated to form a proliferated population of CD34-negative progenitor cells, as described above.
• CHMC mucosal cultured human mast cells
• the resuspension cycle for the culture was the same as that described above, except that the culture was seeded at a density of 425,000 cells/mL and 15% additional media was added on about day four or five without performing a cell count.
• the cytokine composition of the medium was modified such that it contained SCF (200 ng/mL) and recombinant human IL-6 (200 ng/mL; Peprotech,
• CM/SCF/IL-6 medium Phases I and II together span approximately 5 weeks. Some death and debris in the culture is evident during weeks 1-3 and there is a period during weeks 2-5 during which a small percentage of the culture is no longer in suspension, but is instead attached to the surface of the culture vessel.
• Phase I when the culture is to be resuspended on day seven of each cycle, the contents of all flasks are combined into a single container prior to performing a cell count to ensure uniformity of the entire population. Each flask is checked separately for contamination under the microscope prior to combining to prevent contamination of the entire population. When the flasks are combined, approximately 75% of the volume is transferred to the communal container, leaving behind about 10 mL or so in the flask.
• the flask containing the remaining volume was rapped sha ⁇ ly and laterally to dislodge the attached cells.
• the rapping was repeated at a right angle to the first rap to completely dislodge the cells.
• the flask was leaned at a 45 degree angle for a couple of minutes before the remaining volume was transferred to the counting vessel.
• the cells were spun at 950 ⁇ m for 15 min prior to seeding at 35-50 mL per flask (at a density of 425,000 cells/mL).
• CD34-Negative Progenitor Cells into Connective Tissue-Type Mast Cells A proliferated population of CD34-negative progenitor cells is prepared as above and treated to form a tryptase/chymase positive (connective tissue) phenotype. The methods are performed as described above for mucosal mast cells, but with the substitution of IL-4 for IL-6 in the culture medium. The cells obtained are typical of connective tissue mast cells.
• CD34-Negative Progenitor Cells A proliferated population of CD34-negative progenitor cells is prepared as described in Section 7.2.1.3, above, and used to form a proliferated population of basophil cells.
• the CD34-negative cells are treated as described for mucosal mast cells, but with the substitution of IL-3 (at 20-50 ng/mL) for IL-6 in the culture medium.
• Leukotriene C4 is also quantified using an ELISA kit on appropriately diluted supernatant samples (determined empirically for each donor cell population so that the sample measurement falls within the standard curve) following the supplier's instructions.
• CHMC High Cell Density IgE Activation Degranulation (Tryptase, Histamine), Leukotriene (LTC4), and Cytokine (TNFalpha, IL-13) Assays Cultured human mast cells (CHMC) are sensitized for 5 days with IL-4 (20 ng/ml), SCF (200 ng/ml), IL-6 (200 ng/ml), and Human IgE (CP 1035K from Cortx Biochem, 100-500ng/ml depending on generation) in CM medium. After sensitizing, cells are counted, pelleted (1000 ⁇ m, 5-10 minutes), and resuspended at 1-2 xlO 6 cells/ml in MT buffer.
• EDTA (10 mM final)/anti-phosphotyrosine antibody (IX final)/fluorescent phosphopeptide tracer (0.5X final) was added in FP Dilution Buffer to stop the reaction for a total volume of 40 uL according to manufacturer's instructions (PanVera Co ⁇ oration)
• the plate was incubated for 30 minutes in the dark at room temperature. Plates were read on a Polarion fluorescence polarization plate reader (Tecan). Data were converted to amount of phosphopeptide present using a calibration curve generated by competition with the phosphopeptide competitor provided in the Tyrosine Kinase Assay Kit, Green (PanVera Co ⁇ oration).
• IC deposition at specific anatomic sites central nervous system (CNS) for experimental autoimmune encephalomyelitis (EAE) and synovium for collagen-induced arthritis (CIA) leads to activation of cells expressing surface Fc ⁇ R and FceR, notably mast cells, macrophages, and neutrophils, which results in cytokine release, and neutrophil , chemotaxis.
• Activation of the inflammatory response is responsible for downstream effector responses, including edema, hemorrhage, neutrophil infiltration, and release of pro- inflammatory mediators.
• the consequences of these IC-triggered events are difficult to identify in autoimmune disorders; nonetheless, many investigators have demonstrated that inhibition of the Fc ⁇ R signaling pathway in these animal models has resulted in a significant reduction in disease onset and severity.
• IC-mediated acute inflammatory tissue injury is implicated in a variety of human autoimmune diseases, including vasculitis syndrome, sick serum syndrome, systemic lupus erythematosus (SLE), rheumatoid arthritis, Goodpasture's syndrome, and glomerulonephritis.
• the classical experimental model for IC-mediated tissue injury is the reverse passive Arthus reaction.
• the RPA reaction model is a convenient in vivo method to study localized inflammation, induced by ICs, without systemic effects.
• Aspects of the mouse RPA reaction model resemble the inflammatory response of patients with rheumatoid arthritis, SLE and glomerulonephritis.
• test compounds are administered at several timepoints prior to administration of Abs and Ags.
• a solution of rabbit anti-OVA IgG (50 ⁇ g in 25 ⁇ l/mouse) is injected intradermally, and immediately following is an intravenous injection of chicken egg albumin ( 20 mg/kg of body weight) in a solution containing 1% Evans blue dye.
• the degree of edema and hemorrhage is measured in the dorsal skin of C57BL/6 mice using the Evan's Blue dye as an indicator of local tissue damage.
• Purified polyclonal rabbit IgG is used as a control.
• Pretreatment time in which the test compounds are administered prior to Ab/Ag challenge, depends on the pharmacokinetic (PK) properties of each individual compound. Four hours after induction of Arthus reaction, mice are euthanized, and tissues are harvested for assessment of edema. This model system allows us to rapidly screen the in vivo activity of many inhibitors.
• PK pharmacokinetic
• Table 2 7.5.2 The Compounds are effective in Mouse Collagen Antibody Induced Arthritis Model
• the in vivo efficacy of compoundstowards autoimmune diseases can be demonstrated in a mouse model of collagen antibody-induced arthritis (CAIA).
• CAIA collagen antibody-induced arthritis
• CIA Model Collagen-induced arthritis
• Administration of type II collagen into mice or rats results in an immune reaction that characteristically involves inflammatory destruction of cartilage and bone of the distal joints with concomitant swelling of surrounding tissues.
• CIA is commonly used to evaluate compounds that might be of potential use as drugs for treatment of rheumatoid arthritis and other chronic inflammatory conditions.
• Arthrogen-CIA® Arthritis-inducing Monoclonal Antibody Cocktail (Chemicon International Inc.) is administered intravenously to Balb/c mice (2mg/mouse) on Day 0. Forty- eight hours later, 100 ⁇ l of LPS (25 ⁇ g) is injected intraperitoneally. On Day 4, toes may appear swollen. By Day 5, one or two paws (particular the hind legs) begin to appear red and swollen. On Day 6, and thereafter, red and swollen paws will remain for at least 1-2 weeks.
• the clinical signs of inflammation are scored to evaluate the intensity of edema in the paws.
• the severity of arthritis is recorded as the sum score of both hind paws for each animal (possible maximum score of 8).
• the degree of inflammation with involved paws is evaluated by measurement of diameter of the paws. Body weight changes are monitored. Animals can be treated at the time of induction of arthritis, beginning on Day 0. Test compounds and control compounds can be administered once a day (q.d.) or twice a day (b.i.d.), via per os (PO), depending on previously established PK profiles.
• mice are euthanized and the paws are transected at the distal tibia using a guillotine and weighed.
• the mean ⁇ standard error of the mean (SEM) for each group is determined each day from individual animal clinical scores, and hind paw weights for each experimental group are calculated and recorded at study termination. Histopathological evaluation of paws are obtained.
• CIA collagen-induced arthritis
• RA rheumatoid arthritis
• IgG-containing IC are abundant in the synovial tissue of patients with RA. While it is still debated what role these complexes play in the etiology and pathology of the disease, IC communicate with the hematopoetic cells via the Fc ⁇ R.
• CIA is a widely accepted animal model of RA that results in chronic inflammatory synovitis characterized by pannus formation and joint degradation. In this model, intradermal immunization with native type II collagen, emulsified with incomplete Freund's adjuvant, results in an inflammatory polyarthritis within 10 or 11 days and subsequent joint destruction in 3 to 4 weeks.
• Hind limbs would be scored daily for clinical arthritis severity using a standardized method based on the degree of joint inflammation. High resolution digital radiographs of hind limbs can be obtained at the conclusion of the study (Day 28). These limbs can also be analyzed for histopathologic changes. IgG antibodies to native CII can be measured in quadruplicate by ELISA.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• General Health & Medical Sciences (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Epidemiology (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Immunology (AREA)
• Rheumatology (AREA)
• Engineering & Computer Science (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Physical Education & Sports Medicine (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Plural Heterocyclic Compounds (AREA)
• Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
• Heterocyclic Carbon Compounds Containing A Hetero Ring Having Nitrogen And Oxygen As The Only Ring Hetero Atoms (AREA)

Applications Claiming Priority (6)

Publications (2)

Family

ID=34119815

Family Applications (2)

Family Applications After (1)

Country Status (21)

Cited By (39)

Families Citing this family (142)

Citations (3)

Family Cites Families (280)