WO2019067396A1 - Compositions d'inhibiteur de kinase zap-70, procédés et utilisations de celles-ci - Google Patents

Compositions d'inhibiteur de kinase zap-70, procédés et utilisations de celles-ci Download PDF

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WO2019067396A1
WO2019067396A1 PCT/US2018/052557 US2018052557W WO2019067396A1 WO 2019067396 A1 WO2019067396 A1 WO 2019067396A1 US 2018052557 W US2018052557 W US 2018052557W WO 2019067396 A1 WO2019067396 A1 WO 2019067396A1
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substituted
unsubstituted
zap
compound
group
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Mark J. Burk
Brandon CHEN
Jingyi Li
Shawn BACHAN
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Snap Bio, Inc.
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    • 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
    • C07D498/22—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 four or more hetero rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains three hetero rings
    • C07D487/14—Ortho-condensed systems

Definitions

  • the field of invention covers potent and selective inhibitors of the protein kinase ZAP-70, ZAP-70 inhibitor compositions of general Formulas (I)-(VI), pharmaceutical formulations, methods for their preparation, and uses thereof, including uses aimed at transplantation therapy and treating autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and lupus, as well as treating cancer malignancies such as T cell-associated leukemias and lymphomas.
  • autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, and lupus
  • cancer malignancies such as T cell-associated leukemias and lymphomas.
  • Protein kinases constitute a large family of structurally related enzymes that are responsible for catalyzing the phosphorylation of specific amino acid residues in proteins and thereby controlling a variety of signal transduction processes within the cell in response to external stimuli (Ubersax, J. A., Ferrell, J.E., Nai. Rev. Mol. Cell Biol., 2007, 8, 530-541; Shchemelinin, I. et al. Folia Biol., 2006, 52, 81-101). Protein kinases are thought to have evolved from a common ancestral gene due to the conservation of their structure and catalytic function (Manning, G., et al., Science, 2002, 298, 1912-1934).
  • kinases there are at least 518 kinases in the human body and aimost all kimtses contain a similarly structured ATP-binding pocket and catalytic domain containing 250-300 amino acids.
  • Kinases are categorized into families according to the substrates they phosphorylate (e.g., the hydroxy! group of protein-tyrosine residues, protein-serine/threonine residues, or lipids).
  • protein kinases mediate intracellular signaling by effecting a phosphoryl transfer from a nucleoside triphosphate to a protein acceptor that is involved in a signaling pathway. These phosphorylation events act as molecular on/off switches that can modulate or regulate the target protein biological function. These phosphorylation events are ultimately triggered in response to a variety of exlracelliilar and other stimuli.
  • Examples of such stimuli include environmental and chemical stress signals (e.g., osmotic shock, heat shock, ultraviolet radiation, bacterial endotoxin, and H202), cytokines (e.g., interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha)), and growth factors (e.g., granulocyte macrophage-colony-srimulating factor (GM-CSF), and fibroblast growth, factor (FGF)).
  • IL-1 interleukin-1
  • TNF-alpha tumor necrosis factor alpha
  • growth factors e.g., granulocyte macrophage-colony-srimulating factor (GM-CSF), and fibroblast growth, factor (FGF)
  • An exlracelliilar stimulus may affect one or more cellular responses related to cell growth, migration, differentiation, secretion of hormones, activation of transcription factors, muscle contraction, glucose metabolism, control of protein synthesis, and regulation of the cell cycle.
  • Autoimmune diseases develop when the immune sy stem attacks healthy cells as foreign, leading to symptoms such as inflammation, swelling, pain, stiffness, rashes, blisters, fatigue, and fever (Wang, L., et al., J. Intern. Med., 2015, 278(4), 369-95).
  • Autoimmune diseases can affect almost any part of the body, including the heart, brain, nerves, muscles, skin, eyes, joints, lungs, kidneys, glands, the digestive tract, and blood vessels.
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • psoriasis inflammatory bowel disease
  • SLE systemic lupus erythematosus
  • Autoimmune diseases include: Acute disseminated Encephalomyelitis, Acute motor axonal neuropathy, Addison's disease. Adiposis dolorosa. Adult-onset Still's disease, Alopecia areata, Angioedema, Ankylosing Spondylitis, Anti-Glomerular Basement Membrane nephritis, Anti-neutrophil cytoplasmic antibody-associated vasculitis, Anti-N-Methyl-D-Aspartate Receptor Encephalitis, Antiphospholipid syndrome, Autisynthetase syndrome, Aplastic anemia, Autoimmune Angioedema, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune, neutropenia.
  • Autoimmune oophoritis Autoimmune orchitis, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune polyendocrine syndrome type 2, Autoimmune polyendocrine syndrome type 3, Autoimmune progesterone dermatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura, Autoimmune thyroiditis, Autoimmune urticarial, Autoimmune uveitis, Balo concentric sclerosis, Behcet's disease, Bickerstaffs encephalitis, Bullous pemphigoid, Celiac disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan sy ndrome, Cold agglutinin disease, Complex regional pain syndrome, CREST syndrome, Crohn's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellit
  • Eosinophilic fasciitis Epidermolysis bullosa aequisita, Erythema nodosum, Essential mixed cryoglobulinemia, Evans syndrome, Felty syndrome, Fibromyalgia, Gastritis, Gestational pemphigoid.
  • Giant cell arteritis Goodpasture syndrome, Graves' disease, Graves ophthalmopathy, Guillain-Barre syndrome, Hashimoto's encephalopathy, Henoch-Schonlein purpura, Hidradenitis suppurativa, Idiopathic infiammatory demyelinating diseases, IgG4-related systemic disease, Inclusion body myositis, Inflammatory Bowel Disease (IBD), Intermediate uveitis, Interstitial cystitis, Juvenile Arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease, Lupus nephritis, Lupus vasculitis, Lyme disease (Chronic), Meniere's disease, Microscopic colitis, Microscopic polyangiitis, Mixed connective tissue disease, Mooren's
  • ADs present before or during a woman's reproductive years, they can have effects on fetal and maternal outcomes, such as pregnancy loss in women with systemic lupus erythematosus, vasculitis and type 1 diabetes, and infertility in women with rheumatoid arthritis.
  • fetal and maternal outcomes such as pregnancy loss in women with systemic lupus erythematosus, vasculitis and type 1 diabetes, and infertility in women with rheumatoid arthritis.
  • ADs are of considerable personal and public health burdens and the reasons for their high prevalence, gender and ethnic disparities and rising incidence and prevalence remain unclear (Lerner, A.L., et al, Int. 1 Celiac Disease, 2015, 3, 151- 155).
  • T cells are critical members of the human adaptive immune system which normally is activated when foreign organisms or toxic substances are detected in the body by antigen-presenting cells such as macrophages, dendritic cells, and B cells (Owen, J. A. et al., Kuby Immunology, 7th Ed., W.H. Freeman & Co.: New York, 2013).
  • antigen-presenting cells such as macrophages, dendritic cells, and B cells
  • T ceils are reactive toward self-antigens, but these self-reactive T cells are usually either killed prior to becoming fully activated within the immune system, or removed from their role within the immune system by regulatory cells.
  • regulatory cells When these mechanisms fail, it is possible to have a reservoir of self-reactive T cells that become functional within the immune system.
  • T cell-targeted therapeutic interventions that inhibit the ability of T cells to mediate the patholog of autoimmune diseases offer a promising approach to address these chronie debilitating ailments.
  • T cell activity is associated with most autoimmune diseases and specific types of cancer.
  • MS multiple sclerosis
  • multiple sclerosis is caused by demyelination of nerve cells in the brain and spinal cord and there is no known cure (See: Compston, A., et al, Lancet, 2008; 372: 1502- 1517). While the causes have not been definitively established, there is ample evidence that indicates T cells are responsible for the demyelination.
  • T cells are not able to pass through the intact blood-brain-barrier (BBB), but viral or bacterial infections can cause temporary openings that allow T cells to enter the central nervous system. Once T cells cross, they do not recognize myelin as self and engage in a response leading to degradation. T cells are alway s found in abundance in MS lesions. A T cell-specific drug that crosses the BBB could be an effective treatment for MS.
  • Rheumatoid arthritis is another autoimmune disease that afflicts millions worldwide. Over- reactive T cells are believed to play a role in the pathology of RA (See: Cope, A.P., et al., Clin. Exp. Rheumatol., 2007; 25 (Suppl. 46), S4-S 11). Direct evidence that implicates CD4+ T cell involvement in A is the observed association of specific variants of MHC-DRB 1 T cell presenting complexes with RA pathogenesis See: Ponchei, F., et al., Int. J. Clin. Rheumatol., 2012, 7(1), 37-53). A selective drug thai inhibits T cell receptor signaling could be an effective treatment for RA.
  • autoimmune diseases focuses on relieving symptoms and preventing complications because there is no curative therapy.
  • Some drugs can suppress immune system activity. These drugs can help control the disease process and preserve organ function. For instance, these drugs are used to control inflammation in affected kidneys in people with lupus to keep the kidneys working.
  • Medicines used to suppress inflammation include chemotherapy agents, such as methotrexate, given at lower doses than for cancer treatment, as well as drugs used in patients who have had an organ transplant to protect against rejection (e.g., cyclosporine or FK-506).
  • TNF alpha anti-tumor necrosis factor alpha
  • TNF alpha inhibitors are difficult to manufacture and store (poor long-term stability and short shelf life), are ineffective in up to 40% of AD patients, are not suitable for treating MS due to inability to cross blood-brain- barrier, they decline over time in effectiveness due to anti-drug antibodies formed against these large proteinaceous molecules, and they must be administered by injection (Lis et al, Arch. Med. Sci., 2014; 10, 1 175-- ! 185).
  • JAK kinases are being introduced (e.g., tofacitinib) to suppress the immune system by blocking JAK-STAT signal transduction pathway within immune cells, as well as other cell types.
  • JAK kinases are a family of intracellular, non-receptor tyrosine comprising JAK 1, JAK2, JAK3 and ⁇ 2 (tyrosine kinase 2), and were first described more than 20 years ago, but complexities associated with their activation, regulation and pleiotropic signaling functions are still being explored. See, e.g., Babon, J.J., et al., Bioehem J., 2014; 462(1), 1-13.
  • Disrupted or dysregulated JAK-STAT functionality may result in a variety of disorders, including immune deficiency syndromes and cancers.
  • Aaronson et al. Science 2002, 296, 1653- 1655.
  • a primary challenge with developing kinase inhibitors aimed at chronic, non-life threatening, non- oncological indications is to define the degree of inhibitor selectivity required across the kinome that will limit the off-target side effects and associated dose limitations of a potential new drug.
  • Designing compounds that target the disease-associated kinase of interest and also exhibit a high degree of selectivity across the kinome is generally difficult due to the highly conserved nature of the ATP-binding pocket of kinase active sites.
  • JAK inhibitors are administered orally, but target many cell types and can display undesirable dose-limiting side effects such as severe allergic reactions, susceptibility to serious infections, and possible malignancies. Accordingly, there is an urgent need for new, more effective drugs that treat autoimmune diseases, but which target only kinases related to the disease and do not inhibit other kinases leading to toxicity and undesirable side effects.
  • T cell-specific therapies such as kinase inhibitors that target kinases expressed only in T cells, could offer a precise therapeutic mechanism for targeting the main mediator of autoimmune diseases such as RA, MS, and others.
  • T cell-targeted therapeutics could enable new treatments for cancers that are associated with T cell dysfunction and malignancy, such as T cell lymphomas and leukemias.
  • T cell lymphomas and leukemias For example, four types of T cell lymphoma affect T cells and account for 10-15% of non-Hodgkin l mphoma cases (See: Vose, J.M., Hematol./Oncol. Clin.
  • inhibition of ZAP-70 will suppress T cell function and will serve as an effective means of managing organ transplant rejection by administering to an individual a pharmaceutical composition comprising a therapeutically effective amount of an inhibitor of the T cell-specific kinase ZAP-70, as described herein.
  • Compounds of the invention can inhibit ZAP-70 through a reactive functional group attached to the inhibitor, W, which forms a eovalent bond with the sulfur group of cysteine (Cys) 346 of ZAP-70.
  • Compounds of the inventions can be used to selectively inhibit ZAP-70 and specifically target T cell-associated diseases.
  • substituted or unsubstituted alkyl substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxy, substituted or unsubstituted heieroalkyl, substituted or unsubstituted cycloaikyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or an optionai substiruent selected, for example, haloaikyl, alkenyl, arylalkyl, alkoxyalkyl, hydroxyalkyl, nionoalkylaminoalkyl, dialkylaminoalkyl, acylaminoalkyl, acyloxyalkyl, cyanoalkyl, amidinoalkyl, carboxyaikyl, alko y carbony!alky!, aminocarbon
  • Eac E', F', G', and M' is independently C or ;
  • R' is H or linear or branched substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloaikyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;
  • R 2 is linear or branched substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted cycloaikyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted ar l or substituted or unsubstituted heteroaryl;
  • glycose optionally may be substituted with O-acyl, O-methyl, amino, mono- and di-alkylamino, or acylamino substituents; aminoglycose sugars contain an amino group (- N(R ) 2 ) attached directly to the sugar carbons.
  • Non-natural glycose sugars can contain substituents not typically found in nature, such as, for example, fluoro, cyano, or azido substituted sugars.
  • Glycose sugars also may be additionally substituted on their carbon backbone with groups such as A defined above;
  • Linker L is a linker consisting of a linear, branched or cyclic chain of atoms that connect a glycosyl group to W.
  • Linkers L can be 0-20 atoms in length, typically consisting of substituted or unsubstituted atoms such as C, N, O, P, and S;
  • W is a reactive functional group that reversibly or irreversibly interacts with amino acid residues of a kinase in a manner that causes an attractive engagement, such as the formation of a covending bond.
  • Examples include acrylamide or aery late groups, or a halomethyacyl group which reacts with and form covending bonds to a sulfur atom of specific cysteine residues in or near the catalytic pocket of a kinase.
  • Another example is a halosulfonyi or halosulfonate group that reacts with and forms covIER bonds to an oxygen atom of specific tyrosine, serine or threonine residues in or near the catalytic pocket of a kinase.
  • Compounds of Formula (1) are themselves useful as protein kinase inhibitors. As noted above, kinase inhibitors are useful for treating a variety of conditions including cancer, central nervous system disorders, Alzheimer's, cardiovascular disease, dermatological diseases, inflammation, autoimmune diseases such as rheumatoid arthritis, and diabetic complications. Compounds of Formula (I) are specifically useful for inhibiting ZAP-70 kinase.
  • compounds having the structure of Formula (II) or a pharmaceutically acceptable salt, solvate, hydrate, N-oxide, prodrug, stereoisomer, enantiomer, mixture of enautiomers, mixture of diastereomers, iso topic variants, and metabolites thereof which are examples representing kinase inhibitors, or intermediates for the preparation of compounds of Formula (I), wherein:
  • Each A, B, C, D, A', B', C, D ⁇ E, F, G, M, E', F', G ⁇ M', R l , R 2 , R 3 , Glycose, Y, and Z is as defined above;
  • kinase inhibitors are themselves useful as protein kinase inhibitors or represent intermediates useful for the preparation of compounds of Formula (I) exhibiting kinase inhibitory activity.
  • kinase inhibitors are useful for treating a variety of conditions including cancer, central nervous system disorders, Alzheimer's, cardiovascular disease, dermatological diseases, inflammation, autoimmune diseases such as rheumatoid artliritis, and diabetic complications,
  • Each A, B, C, D, A', B', C ⁇ D', E, F, G, M, E', F', G ⁇ M', R , R ⁇ R ! , Y, and Z is as defined above;
  • Q and R of Formula (III) are both H.
  • kinase inhibitors are themselves useful as protein kinase inhibitors or represent intermediates useful for the preparation of compounds of Formulas (I) and (II) exliibiting kinase inhibitory activity.
  • kinase inhibitors are useful for treating a variety of conditions including cancer, central nervous system disorders, Alzheimer's, cardiovascular disease, dermatological diseases, inflammation, autoimmune diseases such as rheumatoid artliritis, and diabetic complications.
  • A, B, C, and D each are independently H, F, CI, Br, I, -OH, -CN, -N 3 , -OR 3 , -N0 2 , -NH 2 , - CH 2 NH 2 , ⁇ CH 2 N(R 3 ) 2 , -CH.-SR .
  • E', F', G', or M' are independently nitrogen.
  • E and F are nitrogen.
  • E * and F' are nitrogen.
  • E and F are nitrogen.
  • E and G are nitrogen.
  • E' and G' are nitrogen.
  • E and M are nitrogen. In another embodiment, E' and M' are nitrogen. In another embodiment, F and M are nitrogen. In another embodiment, F' and M' are nitrogen,
  • kits for producing bisindole alkaloids and analogs of Formulas (I)-(III) through a coupled transcription/translation (TX-TL) cell-free biosynthesis (CFB) system wherein reactions are conducted by adding bisindole alkaloid pathway genes to cell-free extracts containing metabolic enzymes, sails, co-factors, amino acids, sugars, nucleotides, and precursor molecules such as tryptophan and/or tryptophan derivatives, and wherein optionally the mixture is capable of in vitro transcription, translation and-'or coupled transcription translation to produce molecules of Formula (III) where Q and R independently are either hydrogen, a giycose group attached to one indole nitrogen atom, or together form a giycose that bridges both indole nitrogen atoms.
  • Compounds of Formula (I) and (II) subsequently are produced from compounds of Formula (III) thus obtained through chemical transformittions that introduce the linkers (L)
  • cell-free extracts are created by growing and breaking open cells, removing cell membrane and cell wall materials, and digesting native DNA and/or RNA, wherein the cells derive from different kingdoms, phyla, classes, orders, families, genera or species and the cells are a prokaryotic or a eukaryotic cell; or, a bacterial cell, a fungal cell, an algae cell, an Archaeal cell, a yeast cell, an insect cell, a plant cell, a mammalian cell or a human cell.
  • provided herein are methods to produce bisindole alkaloids and analogs of Formula (III) through cell-free reactions involving the use of isolated enzymes corresponding to the natural or unnatural pathway enzymes for bisindole alkaloid synthesis, wherein tryptophan and/or tryptophan derivatives are combined with such enzymes to afford molecules of Formula (III), which subsequently are converted to compounds of Formula (1) and (II) by chemical introduction of linkers (L) and reactive functional groups (W) attached to the gly cosyl group.
  • compositions comprising a compound disclosed herein, e.g., a compound of Formula I, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and one or more pharmaceutically acceptable excipients.
  • a compound disclosed herein e.g., a compound of Formula I, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof; and one or more pharmaceutically acceptable excipients.
  • a method of treating, preventing, or ameliorating one or more symptoms of a T cell-associated or ZAP-70 kinase-mediated disorder, disease, or condition in a subject comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formula I, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.
  • a compound disclosed herein e.g., a compound of Formula I, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.
  • a method of modulating ZAP-70 kinase activity comprising contacting a ZAP-70 kinase in vitro or in vivo with a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formula T, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereoniers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof.
  • a compound disclosed herein e.g., a compound of Formula T, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereoniers, or isotopic variant thereof.
  • Also provided herein is a method of treating, preventing, or ameliorating one or more symptoms of a ZAP-70 kinase-mediated disorder, disease, or condition in a subject, comprising administering to the subject a therapeutically effective amount of a compound disclosed herein, e.g., a compound of Formula I, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diaste isomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof, wherein the compound partially or completely inhibits ZAP-70 activity and displays increased potency against ZAP-70 kinase and/or increased selectivity for ZAP-70 kinase relative to Syk kinase and other kinases.
  • a compound disclosed herein e.g., a compound of Formula I, including a stereoisomer, enantiomer, mixture of enantiomers, mixture of diaste isomers, or isotopic
  • FIGs. la-IE schematically represent an embodiment of Formula (I) or (IV) wherein W is a reactive functional group that is attached to L directly through a C, N, or O and which is selected representative and exemplar)' list of structures corresponding to W, wherein each wavy line indicates the point of attachment to L.
  • FIGs. 2A-2E schematically represent an embodiment of Formulas (V) and (VI), a represe tative and exemplary list of structures corresponding to L wherein L is connected to W, as defined in the application, and wherein the alkyl chains of L are represented as linear, but can be cy disrupt or branched, and each carbon can be substituted or unsubstituted, and wherein each alkyl chain of L can contain one or snore heteroatoms, and/or one or more carbon-carbon double bonds (OC), and/or one or more carbon-oxygen double bonds (OO) and'or one or more carbon-carbon triple bonds (OC) inserted at any position and with any isomeric form possible and known to those skilled in the art, and in all cases the aromatic ring of L can be substituted or unsubstituted aryl or heteroaryl.
  • FIGS. 3 A-3K represent embodiments of the various compounds.
  • FIG. 4 is a graphical representation of a Kd plot of compound 2.
  • FIG. 5 is a western blot analysis showing reduction of SLP76 phosphorylation in Jurkat cells by Compounds 2 and 3.
  • FIG. 6 is a graphical representation of Compound 2 IC 50 data measured by inhibition of SLP-76 phosphorylation.
  • the individual lias been diagnosed with or is suspected of suffering from a cancer or autoimmune disease that is mediated by ZAP-70 kinase
  • methods for treating autoimmune conditions characterized by abnormal, inflammation, swelling, pain, joint stiffness, rashes, blisters, fatigue, and fever.
  • Autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), psoriasis, vasculitis, ankylosing spondylitis, inflammatory bowel disease (IBD), and systemic lupus erythematosus (SUE) can affect almost any part of the body, including the heart, brain, nerves, muscles, skin, eyes, joints, lungs, kidneys, glands, the digestive tract, and blood vessels and they collectively impact up to an estimated 8% of the global population (Cooper, G.S., et al, J. Autoimmun. 2009, 33, 197-207; Ramos, P.S., et al, J.
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • psoriasis vasculitis
  • ankylosing spondylitis inflammatory bowel disease
  • IBD inflammatory bowel disease
  • SUE systemic lupus erythematos
  • ADs Human Genetics, 2015, 60, 657-664. Patients often endure lifelong debilitating symptoms, loss of organ function, reduced productivity at work and high medical expenses. Collectively, ADs are of considerable personal and public health burdens and the reasons for their high prevalence, gender and ethnic disparities and rising incidence and prevalence remain unclear (Lierier, A.L., et al., Int. J. Celiac Disease, 2015, 3, 151 -155).
  • T cell lymphoma such as extranodal T cell lymphoma, cutaneous T cell lymphomas (Sezary sy ndrome and My cosis fungoides), anaplastic large cell lymphoma, and angioimmunoblastic T cell lymphoma
  • T cell leukemia such as large granular lymphocytic leukemia, adult T-cell leukemia/lymphoma, and T ⁇ cell prolymphocytic leukemia.
  • T cells also called T lymphocytes
  • T cells act as central players in the adaptive immune system by transmitting extracellular signals that activate the T cell receptor to the nucleus, thus leading to an appropriate cellular response to the stimulus (e.g., cytokine or protein production).
  • cytokine or protein production e.g., cytokine or protein production.
  • T cells There are five major types of T cells, i.e. helper T-cell (Th), cytotoxic T-cells, memory T-cells, regulatory T-cells (Tregs) and natural killer (N ) T- cells.
  • Helper T-cells aid other white blood cells (WBC) in different immunological processes such as cytotoxic T-celi and macrophage activation and maturation of B-cells to memory cells and plasma cells etc.
  • WBC white blood cells
  • Cytotoxic T- cells have a specific function, which is to destroy virally infected cells or tumor cells and also are involved in organ transplant rejection.
  • Memory T-cells are responsible for developing immunological memory against specific kinds of antigens.
  • Regulatory T-cells maintain immunological tolerance and homeostasis by regulating and balancing the T ceil types and suppressing the overproduction of autoreactive T cells.
  • T eeiis A13 subtypes of T eeiis collectively function to protect the host from different pathogenic attacks. Balanced immune response of T-cells is crucial, as their augmented response may lead to autoimmune disease conditions whereas greatly diminished response a lead to serious infection and death. Thus, it is imperative that under normal circumstances T-cells act in a precise manner to meet the protective demands of a given situation. When T cell types are not kept in balance, for example, when over-reactive or self-reactive T cells are not suppressed properly, or are allowed to proliferate, diseases such as autoimmune diseases and cancer occur.
  • T lymphocytes are activated when a foreign species (e.g., bacteria, virus, or fungi) or substance (e.g., protein toxins) has entered the body from the external environment, for example by inhalation, ingestion or injection (Smith-Garvin, I.E., et al., Annu. Rev. Immunol, 2009, 27, 591-619).
  • Antigens are absorbed by endocytosis or phagocytosis into most cells of our body, although certain cells called antigen-presenting cells (APCs), such as macrophages, B cells, or dendritic cells, specialize in this function. When antigens are internalized, they are processed or degraded into fragments (e.g., peptides).
  • APCs then present these fragments to T helper cells by the use of class II major histocompatibility (MHC) proteins on their surface, which binds the antigen fragment (peptide) and presents it to the T cell receptor.
  • MHC major histocompatibility
  • Normal somatic cells similarly can present antigens to T cells via analogous MHC class 1 proteins to signal that they have been invaded by antigens.
  • T cells are specific for the peptide :MHC complex and thus become activated, which initiates the signal cascade to the T cell nucleus leading to the secretion of cytokines, substances such as interleukins (ILs), that activate cytotoxic T lymphocytes (CTLs), antibody-secreting B cells, macrophages and other immune cells (See: Cantrell, D.A., Immunology-, 2002, 105, 369-374; Malissen, B., et al., Nat. Immunol., 2014, 15, 790- 797)
  • ILs interleukins
  • CTLs cytotoxic T lymphocytes
  • B cells macrophages and other immune cells
  • Each type of T cell displays specific imunoglobulin-like glycoproteins on their surface that facilitates recognition and differential engagement with other cell types.
  • the two main T cell-specific gly coproteins are called cluster of differentiation (CD) 4 and CDS.
  • CD4 is a co-receptor that assists the T cell receptor (TCR) of T helper cells in communicating with an antigen-presenting cell by interacting with its MHC class II domains (See: Zhu, J., et al., Blood J., 2008, 1 12, 1557- 1569).
  • TCR T cell receptor
  • MHC class II domains See: Zhu, J., et al., Blood J., 2008, 1 12, 1557- 1569.
  • CD4 amplifies the signal generated by the TCR by recruiting the tyrosine kinase Lck, which is essential for activating many molecular components of the signaling cascade of an activated T cell.
  • both the TCR complex and CD4 are recruited to bind to different regions of the ⁇ ⁇ ⁇ molecxde ( ⁇ / ⁇ and ⁇ 2, respectively ). Close proximity between the TCR complex and CD4 in this situation means the Lck kinase, bound to the cytoplasmic tail of CD4, is able to ryrosine-phosphor late the immune receptor tyrosine activation motifs (ITAMs) present on the cytoplasmic domains of TCR.
  • ITAMs immune receptor tyrosine activation motifs
  • Phosphorylated IT AM motifs recruit and activate SH2 domain-containing protein tyrosine kinases (PTK) such as ZAP-70 to further mediate downstream signal transduction via tyrosine phosphorylation, leading to transcription factor activation, including NF-kB, and consequent T cell activation.
  • PTK SH2 domain-containing protein tyrosine kinases
  • Regulatory T cells express either CD4 or CDS, but not both.
  • CDS is the co-receptor expressed on the surface of cytotoxic T cells, which interact with MHC class I complexes on infected somatic cells to initiate an immune response through TCR signaling in a similar fashion (See: Zhang, N. and Bevan, M., Immunity, 2011, 35, 161- 168; Wang, W.M., et al., Immunology and Cell Biology, 2009, 87, 192-193).
  • the natural function of CD8+ T cells is related to protection against viral infections and tumors.
  • CD8+ T ceils perform this function by inflicting cytotoxic damage to target cells that express MHC class I molecules and the relevant antigenic peptide.
  • CD8+ cells have a great potential to cause tissue damage.
  • activated CD8 ⁇ T cells can produce very high levels of tumor necrosis factor (TNF) and interferon IFN-y, which may contribute directly and/or indirectly to target cell destruction in autoimmune diseases.
  • TNF tumor necrosis factor
  • IFN-y interferon IFN-y
  • Two recent independent observations indicate a role for CD8+ T cells in disease progression of RA and associated inflammation.
  • synovial CD8+ T cells contain significant frequencies of IFN- ⁇ producing effector cells that might contribute to sustained inflammation by secreting proinflammatory cytokines.
  • a subgroup of CD8 ⁇ T cells, which co-express CD57 accumulates with duration of disease in the peripheral blood and the synovial fluid.
  • CD8+ T cells may regulate the structural integrity and functional activity of germinal center-like structures in ectopic lymphoid follicles within the synovial membrane.
  • activated CD8+ T cells are involved in aggravating pathologic responses in rheumatoid synovitis (Skapenko, A, et al., Arthritis Res T er., 2005, 7(Suppl 2): S4-S 14).
  • Autoimmune diseases develop when the immune system attacks healthy cells as foreign, leading to symptoms such as inflammation, swelling, pain, stiffness, rashes, blisters, fatigue, and fever (Wang, L., et al., J. Intern. Med., 2015, 278(4), 369-95).
  • Autoimmune diseases can affect almost any part of the body, including the heart, brain, nerves, muscles, skin, eyes, joints, lungs, kidney s, glands, the digestive tract, and blood vessels.
  • the mechanisms resulting in the destruction of tissue and the loss of organ function during the course of an autoimmune disease are essentially the same as in protective immunity against invasive microorganisms.
  • CD4+ T cells In addition to CD8+ T cells, activated CD4+ T cells are of fundamental importance in initiating, controlling, and driving these specific immune responses (See: Cope, A.P., et al., Clin. Exp. Rheumatol., 2007; 25 (Suppl. 46), S4-S 11). Once activated, CD4+ T cells differentiate into specialized effector cells and become the central regulators of specific immune responses. In RA a number of observations are consistent with the hy pothesis that CD4+ T cells play a dominant role in the immuno-pathogenesis of the disease.
  • activated CD4+ T cells can be found in the inflammator infiltrates of the rheumatoid synovium (See; Van Boxel J.A., Paget S.A., New Engl. J. Med., 1975, 293, 517-520).
  • CD4+ T cells play an important role in a variety of animal models of inflammatory arthritis, and tissue-damaging autoimmunity can be induced by transfer of CD4 ⁇ T cells from sick animals into healthy syngeneic recipients.
  • T-cell directed therapies have clearly conferred clinical benefit in RA, although the positive effect appears to diminish over time.
  • MS Multiple sclerosis
  • the damage caused by demyelination disrupts the ability of parts of the nervous system to communicate, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems.
  • T cells gain entry into the brain via disruptions in the blood- brain barrier (BBB), which is a part of the capillary system that typically prevents the entry of T cells into the central nervous system. It is believed that the BBB becomes permeable to T cells and other immune system cells during viral or bacterial infections. After the BBB repairs itself, typically once the infection has cleared, T cells remain trapped inside the brain. The T cells recognize myelin as foreign and attack it, and the attack of myelin also initiates the inflammatory processes, which triggers other immune cells and the release of soluble factors like cytokines and antibodies.
  • BBB blood- brain barrier
  • CD4+ T cells including Thl and Th 17 cells are considered the most significant mediators in the pathogenesis of MS and its key animal model, experimental allergic encephalomyelitis (EAE). While subsets of CD4+ T cells are common targets in therapeutic efforts directed at MS, recent pathology reports also indicate an important role for CD8+ T cells.
  • CD8+ T cells are the most numerous lymphocyte subpopulations found in MS patient lesions and in normal appearing MS tissue, regardless of MS subtype or stage of lesion formation. CD8+ T-cells also have been observed in close proximity to damaged neurons in axons both in MS patients. Currently there is no cure for multiple sclerosis. Thus, a T cell-targeted therapeutic agent could represent an effective means to treat MS.
  • Psoriasis is a common, chronic T cell-mediated inflammatory autoimmune skin disease, affecting approximately 2% of the worldwide population (See: Cai, Y. et al., Cell. Mol. Immunol., 2012, 9, 302-309).
  • Psoriasis vulgaris is the most common type of psoriasis, manifested as dry, red raised plaques with adherent silvety scales. Histologically, psoriasis is characterized by hyperproliferation and aberrani differentiation of keratinocytes, dilated, hyperplastic blood vessels as well as an inflammatory infiltration of leukocytes, predominantly into the dermis.
  • T cell-targeted drugs such as anti-CD4 monoclonal antibody and cytotoxic T lymphocyte-associated antigen 4-immunoglobulin were also observed to have a significant therapeutic efficacy in psoriasis treatment.
  • CD4+ T ceils from psoriatic lesions have been shown to enhance keratinocyte proliferation via secretion of interferon- ⁇ (1FN ⁇ ) and the establishment of psoriasis xenograft animal model in severe combined immunodeficient mouse further cosrfirms the importance of T ceils in psoriasis development.
  • psoriatic plaques and peripheral blood of psoriatic patients there were large numbers of CD4+ Thl and CD8+ cytotoxic T cells type 1 (Tel) cells as well as elevated cytokine levels of IFN- ⁇ , tumor necrosis factor (TNF)-alpha and IL-12, which well-defined psoriasis as a Thl cell-mediated disease.
  • TNF tumor necrosis factor
  • Thl7 lL-17-producing CD4+ Th cells
  • Thl7 cells and their related cytokines such as 1L- 17A, TL- 17F, 1L-22, 1L-21 and IL-26 play essential roles in a variety of chronic inflammatory diseases, including psoriasis.
  • Thl7 cells and their downstream effector molecules which include IL-17A, 1L-17F, IL-22, IL-21 and TNF-alpha, are found at increased levels in psoriatic skin and circulation.
  • Intradermal injection of IL-23 or IL-21 in mice can stimulate keratinocytes proliferation and cause epidermal hyperplasia (acanthosis), which is one of the most significant features in human psoriasis (See: Chatnian, F., ei al., Cutr. Opin, Rheumatol., 2004, 16, 331-337).
  • acanthosis epidermal hyperplasia
  • Psoriasis is considered to be an organ-specific T cell-driven inflammatory disease and T cells, and importantly T helper (Th) cells, play a dominant pathogenic role in the initiation and maintenance of psoriasis.
  • Th T helper
  • a T cell-targeted therapeutic agent could represent an effective means to treat psoriasis.
  • Chronic intestinal inflammation due to noninfectious causes represents a growing health issue all over the world.
  • Celiac disease as well as inflammatory bowel diseases (IBD) like Crohn's disease (CD) and ulcerative colitis (UC) and microscopic colitis involve uncontrolled T-cell activation and T-cell-mediated damage as comnion denominators (See: Hisamatsu, H., et al., Inflamm. Intest. Dis., 2016, I, 52-62).
  • Chrome intestinal inflammation clinically manifests itself as frequent recurrent vomiting, periods of (non-bloody) diarrhea and/or obstipation, abdominal pain, rectal bleeding, internal cramps and spasm, nausea, fever, weight loss and overall developmental delay in children.
  • CD Crohn's disease and ulcerative colitis are the two main forms of IBD (See: Xavier, R.J., Podolsky, D.K., Nature, 2007, 448, 427-434).
  • IBD the vast amount of potential antigens and the corresponding antigen-specific T cells makes it unlikely to find universal triggers, like in celiac disease.
  • CD is a chronic inflammatory disease that could involve the entire digestive organ, especially the small bowel and the colon, leading to the progressive destruction of the alimentary tract. Inflammation in CD observed in the mucosal, submucosal and muscular layers cause intestinal complications such as fisrulae, perforation and stricture.
  • Chronic inflammation in ulcerative colitis mainly affects the colon and rectum. Endoscopic and radiological studies have shown continuous and diffuse mucosal inflammation from the rectum to the proximal colon. In UC, fistula formation is relatively rare compared to CD, since the mucosal layer is a main target of inflammation.
  • Another form of IBD microscopic colitis, is mainly an inflammation of the large intestine, though the terminal ileum can be involved. The term 'microscopic ' refers to the fact that diagnosis demands microscopic examination. The patients suffer from chronic non-bloody diarrhea. In the local immunity of the intestinal mucosa, imbalances of T-cell subsets [e.g. T hi, Th2, Thl7, Treg, natural killer T (NKT) cells] in the intestinal mucosa are hallmarks of IBD.
  • T-cell subsets e.g. T hi, Th2, Thl7, Treg, natural killer T (NKT) cells
  • JAK inhibitors Janus Kinases
  • Tofacitinib (CP-690,550) is an oral inhibitor of JAK 1, 2 and 3, which interferes with Th2 and Thl7 differentiation and blocks the secretion of 1L-17 and IL-22 (See: Danese, S., et al., Am. J. Physiol. Gastrointest. Liver Physiol., 2016, 310(3), G155-G162; Yoshida, H., et al, Biochem. Biophys. Res. Commun., 2012, 418(2), 234-240).
  • T cell-targeted therapeutic agent could represent an effective means to treat TBD.
  • Celiac disease is a long term autoimmune disorder primarily affecting the small intestine that occurs in people who are genetically predisposed to an immune reaction to gluten. Celiac disease has been shown to lead to an increased risk of both adenocarcinoma and lymphoma of the small bowel, enteropathy -associated T- cell lymphoma (EATL) or other non-Hodgkin's lymphomas (See: Barker J.M., Liu E., Adv. Pediatr., 2008, 55, 349-365). In celiac disease, the driving role of T cells in the lamina limbalium mainly specific for two defined antigens is well established.
  • celiac disease represents a prototypic CD4+ T cell-dependent disease with chronic intestinal inflammation.
  • Celiac disease is diagnosed by severe crypt hyperplasia and villous atrophy in the small intestine, with specific antibodies, primarily autoantibodies, directed at the tissue transglutaminase and at giiadin.
  • HLA human leukocyte antigen
  • T cells recognize and are activated by gluten-derived peptides that are deaminated by the tissue transglutaminase and effectively presented by HLA-DQ variants.
  • Antigen-specific activated effector CD4+ T cells release pro-inflammatory cytokines, predominantly interferon- ⁇ (IFNy) and interleukin-21 (IL- 21).
  • IFNy interferon- ⁇
  • IL- 21 interleukin-21
  • T-cell subsets are involved in sustaining the heterogeneous cytokine milieu maintaining or counteracting the local inflammation.
  • lEL intraepithelial lymphocytes
  • TCR ⁇ T-cell receptor
  • CD4 - CDS - y5TCR+ T cells See: Bodd M., et ;iL Mucosal Immunol, 2010, 3, 594-601; Nil sen E.M., et ;iL Gut, 1995, 37, 766-776.
  • TCR ⁇ T-cell receptor
  • a T cell-targeted therapeutic agent could represent an effective means to treat celiac disease.
  • Chronic lymphocytic leukemia is one of the most common human leukemias. CLL affects B cell lymphocytes, which originate in the bone marrow, develop in the lymph nodes, and normally fight infection by producing antibodies. In CLL, B cells grow in an uncontrolled maimer and accumulate in the bone marrow and blood, where they crowd out healthy blood cells. CLL is a disease that occurs largely in older adults, although, in rare cases, it can occur in teenagers and occasionally in children who may have an inherited predisposition for the disease. Most people are diagnosed without symptoms as the result of a routine blood test that shows a high white blood cell count.
  • CLL results in swollen lymph nodes, spleen, and liver, and eventually anemia and serious infections.
  • Modem DNA analysis lias distinguished two major types of CLL, with different survival times. People with CLL that is positive for the marker ZAP-70 have an average survival of 8 years, while those negative for ZAP-70 have an average survival of more than 25 years. ZAP-70 is found to be overexpressed in approximately 50% of cases of CLL and is associated with a ver poor prognosis (See: Herishanu, Y., et al., Leukemia (2005) 19, 1289-1291).
  • a T cell-targeted therapeutic agent especially one that targets ZAP-70, could represent an effective means to treat CLL (See: Dielschneider, R. I- ' . , et al, Cell Death and Disease, 2014, 5, e l439; doi: 10.1038/cddis.2014.391).
  • Lymphoma is the most common blood cancer.
  • the two main forms of lymphoma are Hodgkin lymphoma and non-Hodgkin lymphoma (NHL).
  • Lymphoma occurs when cells of the immune system called lymphocytes, a type of white blood cell, grow and multiply uncontrollably. Cancerous lymphocytes can travel to many parts of the body, including the lymph nodes, spleen, bone marrow, blood, or other organs, and form a mass called a tumor.
  • the body has two main types of lymphocytes that can develop into lymphomas: B- lymphocytes (B cells) and T-lymphocytes (T cells).
  • Peripheral T cell lymphoma consists of a group of rare and usually aggressive (fast-growing) HLs that develop from mature T cells (See: Vose, J.M., HematoL/Oncol. Clin, North. Amer., 2008, 22, 997-1005). Most T-cell lymphomas are PTCLs, which collectively account for about 10 percent to 15 percent of all NHL cases in the United States. The three most common subtypes of PTCL, peripheral T-cell lymphoma not otherwise specified (PTCL-NOS), anaplastic large-cell lymphoma (ALCL), and angioimmunoblastic T-cell lymphoma (AITL), account for approximately 70 percent of all PTCLs in the United States.
  • PTCL-NOS peripheral T-cell lymphoma not otherwise specified
  • ALCL anaplastic large-cell lymphoma
  • AITL angioimmunoblastic T-cell lymphoma
  • ALCL is an aggressive T cell lymphoma, accounting for about three percent of all lymphomas in adults (about 15 percent to 20 percent of all PTCLs) and between 10 percent and 30 percent of all lymphomas in children. ALCL can appear in the skin or in other organs throughout the body (systemic ALCL).
  • AITL is an aggressive T-cell lymphoma that accounts for about two percent of all NHL cases (about 10 percent to 15 percent of all PTCLs) in the United States. This type of lymphoma often responds to milder therapies, such as steroids, although it often progresses and requires chemotherapy and other medications.
  • the frontline treatment regimen for PTCL is typically a combination chemotherapy, such as CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone), EPOCH (etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone), or other multi-drug regimens that exhibit serious toxic side-effects.
  • CHOP cyclophosphamide, doxorubicin, vincristine, prednisone
  • EPOCH etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone
  • other multi-drug regimens that exhibit serious toxic side-effects. Because most patients with PTCL will relapse, high-dose chemotherapy followed by an autologous (in which patients receive their own stem cells) stem cell transplant is often recommended, although there is no definitive and positive clinical data to support a transplant as beneficial, in 2009, the U.S.
  • praiatrexate Food and Drug Administration (FDA) approved praiatrexate (Folotyn) for the treatment of patients with relapsed (disease returns after treatment) or refractoiy (disease does not respond to treatment) PTCL.
  • Praiatrexate was the first drug approved specifically for patients with PTCL.
  • Clinical trials are in development to see if praiatrexate is effective when combined with other drags commonly used in the treatment of both T-cell and B-celi lymphomas, including gemcitabine (Gemzar) and bexarotene (Targretiti).
  • the FDA also approved romidepsin (Istodax) for the treatment of relapsed or refractory PTCL.
  • romidepsin in combination with chemotherapies commonly used in PTCL, including CHOP and ICE (ifosfamide, carboplatin, etoposide).
  • Gemcitabine appears effective against some forms of relapsed PTCL and is often given in combination with other chemotherapies, including vinorelbine (Navelbine) and doxorubicin (Doxil) in a regimen called GND,
  • Other chemotherapy regimens used for relapsed or refractory PTCLs include DHAP (dexainethasone, cytarabine, cisplatin) and ESHAP (etoposide, methylprednisolone, cytarabme, and cisplatin).
  • DHAP diexainethasone, cytarabine, cisplatin
  • ESHAP etoposide, methylprednisolone, cytarabme, and cisplatin.
  • T cell leukemia describes several rare and aggressive types of lymphoid leukemia which affect T cells.
  • the main types of include large granular lymphocytic leukemia, adult T cell leukemia/lymphoma (See: Graham, R.L., et al, Proc. Bayl. Univ. Med. Cent., 2014, 27(3),235-238), and T cell prolymphocytic leukemia (See: Graham, R.L., et al., T cell prolymphocytic leukemia, Proc. Bayl. Univ. Med. Cent., 2013, 26(1), 19-21).
  • LGL leukemia Large granular lymphocytic (LGL) leukemia is a chronic lymphoproliferative disorder that exhibits an unexplained, chronic (> 6 months) elevation in large granular lymphocytes (LGLs) in the peripheral blood. It is divided in two main categories: T cell LGL leukemia (T-LGLL) and natural-killer (NK)-cell LGL leukemia (NK-LGL)L. T cell large granular lymphocyte leukemia is characterized by involvement of c totoxic-T cells. It is also known by the name T cell chronic lymphocytic leukemia.
  • ATLL adult T cell leukemia/lymphoma
  • HTLV- 1 Human T cell leukemia/lympho tropic virus type 1
  • ATLL The cumulative risk of ATLL development among HTLV-1 carriers is estimated to be 2.5% to 5% over the course of a 70-year life span. Affected individuals are usually exposed to the virus early in life. Viral transmission predominantly occurs through breast milk, sexual intercourse, and exposure to peripheral blood and blood products. The disease has a long latency, and thus ATLL occurs only in adults with onset ages ranging from 20 to 80, with an average age of 58 years. In HTLV- 1 -infected lymphocytes, the p40 tax viral protein has been shown to lead to transcriptional activation of many genes inducing proliferation and inhibiting apoptosis in vivo.
  • HTLV- 1 basic leucine zipper factor (HBZ)
  • HBZ HTLV- 1 basic leucine zipper factor
  • T cell prolymphocytic leukemia is a mature T cell leukemia with aggressive behavior and predilection for blood, bone marrow, lymph nodes, liver, spleen, and skin involvement.
  • T-PLL is a very rare leukemia, primarily affecting adults over the age of 30. It represents 2% of all small lymphocy tic leukemias in adults.
  • T-PLL is an extremely rare aggressive disease, and patients are not expected to live normal lifespans.
  • the median survival time was 7.5 months after diagnosis. More recently, some patients have survived five years and more, although the median survival is still low.
  • T cell leukemias are relatively rare but aggressive cancers that are difficult to treat since they do not respond to most available chemotherapeutic drugs.
  • a T cell-targeted therapeutic agent could represent an effective means to treat T cell leukemias.
  • Zeta-Chain Associated Protein Kinase 70 kDa (Human ZAP-70, EC:2.7.10.2, GenBank Accession Number AAH39039.1) is a 70 kDa tyrosine kinase that belongs to the Syk kinase family of protein kinases.
  • ZAP-70 is a 619 amino acid protein expressed predominantly in T cells and natural killer (NK) cells and initiates signaling by binding to the zeta subunits of an activated T cell receptor (TCR), thus playing a critical role in normal TCR-derived signaling, and the activation and development of T-cells (See: Au-Yeung, B.B., et al., Immunological Reviews, 2009, 228, 41 -57; Bene, M.C., et al, Cytometry Part B, 2006, 70B, 204-208). Selective inhibition of ZAP-70 represents a mechanism to specifically target T cell hyperactivity associated with autoimmune diseases and numerous malignant cancers.
  • ZAP-70 comprises of three domains that include two Src homology (SH2) domains present at the N- terminal and a kinase domain at the C-terminus of the protein.
  • SH2 domains present at the N- terminal
  • a kinase Lck phosphorylates the cytosolic CD 3 and ⁇ chains of the TCR.
  • the SH2 domains of ZAP-70 help recruit the kinase and bind to the diphosphorylated immunoreceptor tyrosine-based activating motifs (ITAMs) located on the CD3 domain and ⁇ chain dimers of T cell receptors.
  • ITAMs diphosphorylated immunoreceptor tyrosine-based activating motifs
  • ZAP-70 Such interaction of ZAP-70 with ITAMs is required for the phosphorylation and activation of ZAP-70, also by Lck, which transfers a phosphate group from ATP to tyrosine residues 292 (Y292), 315 (Y3 I5) and 319 (Y319) of ZAP- 70.
  • the activated ZAP-70 in turn phosphorylates a transmembrane protein linker of activated T-cells (LAT) that acts as a linker between initial TCR signal and the downstream events of the T-cell signaling cascade.
  • LAT is phosphorylated by ZAP-70 at tyrosine residues Y!
  • LAT binds to the SH2 domain containing leukocyte protein of 76 kDa (SLP-76) via GRB2 -related adapter protein-2 (GADS) constituting a trimeric complex SLP-76/GADS/LAT which (complex) is further phosphorylated and activated by ZAP-70, which phosphorylates SLP-76 at tyrosine residues Y113, Y128 and Y145.
  • SLP-76 leukocyte protein of 76 kDa
  • GADS GRB2 -related adapter protein-2
  • ZAP-70 can also act as a scaffold protein for recruiting further signaling molecules to activated TCR complex (See: Wang, H., et al., Cold Spring Harb. Perspect. Biol, 2010, 2, 1-10, doi: 10.110 l/cshperspect.a002279).
  • the activated trimeric complex SLP-76/GADS/LAT alleviates the migration of auto -inhibited kinase ITK from the cytoplasm to the membrane where it binds tlirough its PH domain.
  • ITK interleukin-1 kinase
  • SLP-76/GADS/ZAP-70 complex completely activates ITK by the trans-phosphorylation of tyrosine (Y51 1 ; present in the activation loop of ITK) by Lck enzyme and auto-phosphorylation of tyrosine 180 residue in SH3 domain by ITK itself.
  • the activated ITK then interacts with the SH2 domain of PLCyl lipase and activates the lipase by phosphorylating tyrosines 775 and 783 amino acid residues.
  • IP3 inositol triphosphate
  • DAG diacyl glycerol
  • IP3 is a small polar molecule that is released into the cytosol, where it directs the release of Ca(ll) from intracellular stores.
  • ⁇ 3 accumulates rapidly and transiently, and subsequently binds to its intracellular receptor, IP3R, located in the endoplasmic reticulum mobilizing Ca(II) from internal stores.
  • the cytoplasmic calcium activates calmodulin.
  • the activated Ca(II)/calmodulin complex further binds to and stimulates the calcium dependent serine threonine phosphatase calcineurin.
  • Activated calcineurin activates cytoplasmic nuclear factor of activated T cells, (NFAT transcription factor) via dephosphorylation.
  • the activated cNFAT then translocates into the nucleus and governs the expression of pro-inflammatory proteins such as Cox-2 and cytokines such as interleukin 2 (IL-2) that uitiinately promotes the proliferation and differentiation of T-cells.
  • pro-inflammatory proteins such as Cox-2 and cytokines such as interleukin 2 (IL-2) that uitiinately promotes the proliferation and differentiation of T-cells.
  • IL-2 interleukin 2
  • DAG In the D AG-mediated pathway, DAG, on the other hand, triggers protein kinase C- ⁇ (PKC- ⁇ ), to stimulate the inhibitor of kappa B kinase (IKK)- induced release of activated NF ⁇ KB that translocates to the nucleus to initiate transcription leading to growth and differentiation of T ⁇ cells. DAG also initiates niitogen-activated protein kinases 1 and 2 (MEKi and MEK2)/Mitogen-activated protein kinases 3 and 1 (ERK1 and ERK2) cascade that, in turn, activates several transcription factors including AP-1 that plays a vital role in transcription, ultimately leading to immune response (T-cell proliferation and differentiation).
  • PKC- ⁇ protein kinase C- ⁇
  • IKK kappa B kinase
  • Both SH2 domains have a central antiparaltel B sheet flanked by two alpha-helices.
  • the linker-SH2 region commences as a ⁇ strand followed by a coiled coil of two antiparallel alpha-helices that forms the stem of overall Y-shape.
  • ⁇ - Chain establishes contact with both N terminal and C-terminal SH2 domains with head to tail binding orientation i.e. the amino terminus of the peptide is in contact with C-terminal SI-I2 domain. Binding of phosphorylated peptides to SH2 domain has been explained as socket and ping conformation, where phosphotyrosine (pY) and the pY ⁇ 3 residues are prongs of the plug. The C-tenninal phosphotyrosine binds to the pocket formed by both SH2 domains.
  • the methylaniino NH of glycosidic ring forms a hydrogen bond with the carbonyl oxygen of Arg465 of the catalytic loop similar to that formed by the ribose moiety of ATP.
  • One indole ring resides in the gatekeeper pocket whereas She other indole ring was found to fill a small lipophilic pocket.
  • indolocarbazole moiety was found to engage in important van der Waals interactions with Leu344, Gly345, Val352, Ala367, Lys369, Val399, Met414, Glu415 and Met4 !6 from N- terminal lobe and Ala417, Gly420, Pro421, Arg465, Asn466, Leu468, Ser478 and Asp479 from C-terminal lobe.
  • ZAP-70 kinase contains a cysteine residue at position 346 (Cys346) that resides within the phosphate- binding loop (P-loop) region, which is proximal to the ATP-tainding pocket and could serve as a basis for targeting with an inhibitor containing a group that forms a covalent interaction with Cys346, For example, a properly placed aery late or acr lamide functional, group could form a covalent bond with Cys346, thus resulting in selective irreversible inhibition of ZAP-70.
  • Cys346 cysteine residue at position 346
  • P-loop phosphate- binding loop
  • T cell aciivation, proliferation, effector function, and memory responses by CD4 ⁇ and CD8+ T cells have been shown to be highly dependent on ZAP-70 catalytic activity (See: Levin, S.E., et al., J Biol Chem, 2008, 283, 15419-15430; ).
  • TCR signal transduction is absent in ZAP-70-deficient Jurkat ceil lines and similar impairments are seen in peripheral CD4 T cells of ZAP-70 deficient SCID patients.
  • a series of in vitro and in vivo studies by Weiss and co-workers have definitively demonstrated the biological impact of selectively inhibiting the catalytic activity of the kinase domain of ZAP-70.
  • Treg cells even in the presence of 3- MB-PP1, apparently have TCR-induced phosphorylation of ZAP-70 on Tyr315, followed by the association of this phosphorylated residue with CrkII-C3G, thus faciiitating the activation of Rapl, resulting in increased LFA- 1 adhesion to TCAM- 1 expressed on antigen-presenting cells (APCs), which enhances their close proximity to APCs and target conventional T cells, where they can utilize multiple mechanisms of suppression. Consistently, Treg cells do not require ZAP-70 catalytic function to respond to IL-2 and ZAP-70 scaffold- mediated activation of integrin adhesion is sufficient to enable Treg cell suppression.
  • APCs antigen-presenting cells
  • inhibitor of ZAP- 70 kinase activity might be utilized to dampen the response of pathogenic conventional T cells that are involved in autoimmune disease or allograft rejection, without compromising the T ceil suppressive regulatory function of Treg cells.
  • a selective ZAP-70 inhibitor may be used to specifically target T cell-associated diseases such as autoimmune diseases and T cell lymphomas and leukemias.
  • upstream signaling effectors of ZAP-70 include, but are not limited to, T cell receptors, T cell receptor CDS chains, T cell receptor alpha and beta chains, T cell receptor ⁇ -chains, CD4 co- receptor protein, antigen-presenting ceils, MHC class II antigen molecules, MHC class I antigen molecules, B7 protein, CD28 receptor protein, CD40 receptor protein, CD40L protein, and Lck kinase.
  • downstream signaling effectors of ZAP-70 include, but are not limited to, substrates of ZAP-70 kinase, such as LAT, SLP-76, GADS, ITK kinase, PLCy!
  • lipase PIP2, inositol triphosphate ( ⁇ 3), diacyl glycerol (DAG), calcium(II), calmodulin, NFAT, interleukin 2 (IL-2), inhibitor of kappa B kinase (IKK), NF- ⁇ , Ras-GRP, Ras, mitogen-activated protein kinases 1 and 2 (MEKl and MEK2) Mitogen-activated protein kinases 3 and 1 (ERK1 and ERK2), INK, p38, and AP-1.
  • DAG diacyl glycerol
  • IKK inhibitor of kappa B kinase
  • Inhibitors of the kinase ZAP-70 are potentially useful for reducing or abolishing the T cell receptor- based signaling pathways required for differentiation, activation, and proliferation of T cells.
  • ZAP-70 inhibitor no potent and selective inhibitors of ZAP-70 have been developed and reported to date (Kaur, M., et al., Cellular Signalling, 2014, 26, 2481-2492).
  • a major challenge has been developing molecules that are potent inhibitors of ZAP-70, but do not inhibit other kinases, especially the homologous spleen tyrosine kinase (Syk), which similarly regulates TCR signaling in B cells of the immune system, inhibiting both T cell and B cell signaling would significantly compromise the immune system and render it subject to serious infectious and other diseases, and in many cases would be lethal.
  • Syk homologous spleen tyrosine kinase
  • ZAP-70 lias been characterized by X-ray crystallography and was shown to have a narrow ATP- binding pocket that challenges inhibitor design. Perhaps the most important barrier to inhibitor design is that the analogous kinase Syk, which functions in B cells, exhibits 71% overall homology with ZAP-70 and 77% homology in the kinase domain. Therefore, developing a ZAP-70 selective inhibitor has encountered significant obstacles. However, the in vitro and in vivo biological effects of selective ZAP-70 inhibition have been demonstrated by mutating the gatekeeper methionine (Met) 413 residue of wild type murine ZAP-70 (corresponding to Met4 !
  • a selective ZAP-70 inhibitor may be used to specifically target T cell-associated diseases.
  • Novartis Ag claimed a range of 2,4-di(hetero)aryl-armnorjyrimidine derivatives as ZAP-70 inhibitors (Baenteli, R, et al, WO2005026158A1; Garcia-Echeverria, C, et al., WO2005016894A1; Baenteli, R., et al., US Patent 8,283,356B2, Garcia-Echeverria, C, et al., US Patent 7,893,074; Baenteli, R., et al., US Patent 7,671,063B2; Baenteli, R., et al., US Patent 8,431,589B2).
  • ZAP-70 e.g., stau
  • ZAP-70 inhibitors that treat one or more symptoms associated with autoimmune diseases or T cell-associated cancers.
  • pharmaceutical compositions comprising a ZAP-70 inhibitor (e.g., a ZAP-70 inhibitor compound described herein) for reversing or reducing one or more of the negative symptoms and/or positive symptoms associated with autoimmune diseases or T cell-associated cancers.
  • pharmaceutical compositions comprising a ZAP-70 inhibitor (e.g., a ZAP- 70 inhibitor compound described herein) for halting or delaying the progression of negative symptoms and/or positive sy mptoms associated with autoimmune diseases or T cell-associated cancers.
  • Described herein is the use of a ZAP-70 inhibitor for manufacture of a medicament for treatment of one or more symptoms of autoimmune diseases or T cell-associated cancers.
  • a ZAP-70 inhibitor described herein reduces or inhibits the activity of one or more of ZAP-70 while not affecting the activity of Syk. In some embodiments, a ZAP-70 inhibitor described herein substantially reduces or inhibits the activity of ZAP-70. In some embodiments, a ZAP-70 inhibitor described herein is a substantially complete inhibitor of ZAP-70. As used herein, “substantially complete inhibition” means, for example, >95% inhibition of ZAP-70. In other embodiments, “substantially complete inhibition” means, for example, >90% inhibition of ZAP-70. in some other embodiments, “substantially complete inhibition” means, for example, >80% inhibition of ZAP-70.
  • a ZAP-70 inhibitor described herein is a partial inhibitor of ZAP-70.
  • partial inhibition means, for example, between about 40% to about 60% inhibition of ZAP-70. In other embodiments, “partial inhibition” means, for example, between about 50% to about 70% inhibition of ZAP-70.
  • a ZAP-70 inhibitor substantially inliibits or partially inhibits the activity of ZAP-70 while not affecting the activity of Syk, it means, for example, less than about 10% inhibition of Syk when Syk is contacted with the same concentration of the ZAP-70 inhibitor.
  • a ZAP-70 inhibitor siibstantialiy inhibits or partially inhibits the activity of ZAP-70 while not affecting the activity of Syk
  • a ZAP-70 inhibitor substantially inhibits or partially inhibits the activity of ZAP-70 while not affecting the activity of Syk it means, for example, less than about 1% inhibition of Syk when Syk is contacted with the same concentration of the ZAP-70 inhibitor as used for ZAP-70.
  • -CH 2 SR 1 substituted or unsubstituted aSkyl, substituted or unsubstituted aikenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, or an optional substituent selected, for example, haloalkyl, aikenyl, arylaikyl, alkoxyalkyl, hydroxyalkyl, monoalkyiaminoaikyl, dialkylaminoalkyl, acylaminoalkyl, acy
  • Each E, F, G, and M is independently C or N;
  • Each E', F", G', and M' is independently C or N;
  • R' is H or linear or branched substituted or unsubstituted alkyl, substituted or unsubstituted alkenyi, substituted or unsubstituted cycloalkyi, substituted or unsubstituted cycloalkenyL substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;
  • R 2 is linear or branched substituted or unsubstituted alkyl, substituted or unsubstituted alkenyi, substituted or unsubstituted cycloalkyi, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl;
  • Each R 3 is independently H, linear or branched substituted or unsubstituted alkyl. substituted or unsubstituted alkenyi, substituted or unsubstituted cycloalkyi, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted acyl (-C(0)R 1 ), or two R* together with the atoms to which they are attached form a substituted or unsubstituted heterocycle;
  • Glycose is a natural or non-natural cyclic furanosyl or pyranosyl sugar group containing a five or six carbon ring, respectively, or alternatively ring contracted (4 carbon) or ring expanded (7 carbon) derivatives, such as those derived from natural sugar groups including, but not limited to, allosyl, altrosyl, glucosyl, mannosyl, gulosyl, idosyl, galactosyl, talosyl, digitoxosyl, olivosyl, arabinosyl, xylosyl, lyxosyl, rhamnosly, ribosyl, deoxyfurananosyl, deoxypyranosyl, ristosaminyl, and deoxyribosyl, wherein the glycose group bridges both indole nitrogen atoms of Formula (II).
  • glycose optionally may be substituted with O-acyl, O-methyl, amino, mono- and di-alkylainino, or N-acylamino substituents; aminoglycose sugars contain an amino group (- N(R 3 ) 2 ) attached directly to the sugar carbons.
  • Non-natural glycose sugars can contain substituents not typically found in nature, such as, for example, fluoro, cyano, or azido substituted sugars.
  • Glycose sugars also may be additionally substituted on their carbon backbone with groups such as A defined above;
  • L is a linker consisting of a linear, branched or cyclic chain of atoms that connect a glycosyl group to W. Linkers L can be 0-20 atoms in length, typically consisting of substituted or unsubstituted atoms such as C, N, O, P, and S;
  • W is a reactive fu ctional group that reversibly or irreversibly interacts with amino acid residues of a kinase in a manner that causes an attractive engagement, such as the formation of a covending bond.
  • Examples include aerylamide or aery late groups, or a haloniethyacyi group which reacts with and form covending bonds to a sulfur atom of specific cysteine residues in or near the catalytic pocket of a kinase.
  • Another example is a halosuifonyl or halo sulfonate group that reacts with and forms covending bonds to an oxygen atom of specific ty rosine, serine or threonine residues in or near the catalytic pocket of a kinase.
  • Compounds of Formula (I) are themselves useful as protein kinase inliibitors. As noted above, kinase inhibitors are useful for treating a variety of conditions including cancer, central nervous system disorders, Alzheimer's, cardiovascular disease, dermatological diseases, inflammation, autoimmune diseases such as rheumatoid arthritis, and diabetic complications. Compounds of Formula (I) are specifically useful for inhibiting ZAP-70 kinase.
  • Formula (II) compounds having the structure of Formula (II) or a pharmaceutically acceptable salt, solvate, hydrate, N-oxide, prodrug, stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, isotopic variants, and metabolites thereof, which are examples representing kinase inhibitors, or intermediates for the preparation of compounds of Formula (I), wherein: Formula (II)
  • Each A, B, C, D, A ⁇ B', C ⁇ D', E, F, G, M, E ⁇ F ⁇ G', M ⁇ R 1 , R 2 , R 3 , Glycose, Y, and Z is as defined above;
  • kinase inliibitors are useful for treating a variety of conditions including cancer, central nervous system disorders, Alzheimer's, cardiovascular disease, dermatological diseases, inflammation, autoimmune diseases such as rheumatoid arthritis, and diabetic complications.
  • Each A, B, C, D, A', B ⁇ C ⁇ I)', E, F, G, M, E ⁇ F', G', M', R 1 , R 2 , R 3 , Y, and Z is as defined above;
  • Q and R of Formula (III) are both H.
  • kinase inhibitors are themselves useful as protein kinase inhibitors or represent intermediates useful for the preparation of compounds of Formulas (I) and (IT) exhibiting kinase inhibitory activity.
  • kinase inhibitors are useful for treating a variety of conditions including cancer, central nervous system disorders, Alzheimer's, cardiovascular disease, dermatological diseases, inflammation, autoimmune diseases such as rheumatoid arthritis, and diabetic complications.
  • a structure of Formula (TV) or a pharmaceutically acceptable salt, solvate, hydrate, N-oxide, prodrug, stereoisomer, enantiomer, mixture of enantiomers, mixture of diasteieomers, isotopic variants, and metabolites thereof which in some embodiments can be inhibitors of the kinase ZAP-70, wherein:
  • Each A, B, C, D, A', B ⁇ C ⁇ D ⁇ E, F, G, M, E ⁇ F ⁇ G', ⁇ ', R 1 , R 2 , R 3 , L, W, Y, and Z is as defined above;
  • Each R* is H, substituted or unsubstituted alkyl, substituted or unsubstituted cycioaik i, substituted or unsubstituted heterocycloalkyi substituted or unsubstituted aryl or substituted or unsubstituted heteroatyl;
  • Each X is H, -OH, -OR 3 , N(R') 2 , haiogen, -N 3 , nitro, substituted or unsubstituted aikyi, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted alkoxy, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or uusubstituted
  • heterocycloalkyl substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl
  • A, B, C, and D each are independently H, F, CI, Br, I, -OH, -CN, -N 3 , -OR 3 , -N0 2 , -N3 ⁇ 4, - CH 2 NH 2 , -CH 2 N(R 3 ),, -C3 ⁇ 4SR !
  • E', F', G', or M' are independently nitrogen.
  • E and F are nitrogen.
  • E * and F' are nitrogen.
  • E and F are nitrogen.
  • E and G are nitrogen.
  • £' and G" are nitrogen.
  • E and M are nitrogen.
  • E' and M' are nitrogen.
  • F and M are nitrogen.
  • F' and M' are nitrogen.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain containing from 1-6 carbons, a linear or branched and substituted or unsubstituted alkenyl chain containing from 1-6 carbons, or a linear or branched and substituted or unsubstituted alkynyl chain containing from 1-6 carbons.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain, alkenyl chain, or alkynyl chain containing from 1-6 carbons optionally substituted with O or N terminal groups.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alky l chain, alkenyl chain, or alkynyl chain containing from 1-6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted aryl or heteroaryl group.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain, alkenyl chain, or alky ny l chain containing from 1 -6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted heterocy loa!ky 1 gro up.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alky l chain, alkenyl chain, or alkynyl chain containing from 1-6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted aiy 1 or heteroar l group.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alky! cliain, alkenyl chain, or alkynyl chain containing from 1 -6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted piperazine, substituted or unsubstituted piperidine, substituted or unsubstituted pyrrolidine or substituted or unsubstituted morpholine.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain, alkenyl chain, or alky ny l chain containing from 1 -6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted aryl or heteroaryl group which has W attached to carbon position 2 of the aryl or heteroaryl group relative to the attachment point of L to the aryl or heteroaryl group.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain, alkenyl chain, or alky nyl chain containing from 1 -6 carbons optionally substituted with O or N terminal, groups and containing a substituted or unsubstituted aryl or heteroaryl group which lias W attached to carbon position 3 of the aryl or heteroaryl group relative to the attachment point of L to the aryl or heteroaryl group.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain, alkenyl chain, or alk nyl chain containing from 1-6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted ary l or heteroaryl group which has W attached to carbon position 4 of the aryl or heteroaryl group relative to the attachment point of L to the aryl or heteroaryl group.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alky l chain, alkenyl cliain, or alkynyl chain containing from 1-6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted aryl, wherein the aryl group is phenyl.
  • L is a linker chain containing a linear or branched and substituted or unsubstituted alkyl chain, alkenyl chain, or alky ny l chain containing from I -6 carbons optionally substituted with O or N terminal groups and containing a substituted or unsubstituted heteroaryl group, wherein the heteroaryl group is 1 -H-pyrrole, pyrazole, imidazole, 1,2,4-triazole, 1,2,3- triazole, tetrazole, furan, thiophene, oxazole, isoxazole, isothiazole, thiazole, 1,2,5-oxadiazole, 1,2,3- oxadiazole, 1 ,3,4-thiadiazole, 1,2,5-thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, or 1,2,
  • [0098] in one embodiment is a compound of Formulas (I) or (IV), wherein W is a reactive functional group that is covIERly linked to L and is capable of forming a covalent bond with the sulfur atom of a cysteine amino acid residue in or near the ATP -binding site of a kinase.
  • In one embodiment is a compound of Formulas (I) or (IV), wherein W is a reactive functional group that is covIERly linked to L and is capable of forming a covalent bond with the sulfur atom of a cysteine amino acid residue 346 (Cys 346) of ZAP-70 kinase.
  • [0100] in one embodiment is a compound of Formulas (I) or (IV), wherein W is a reactive functional group that is covIERly linked to L through C, N, O, or S atoms and which has at least one double bond capable forming a covIER bond with the sulfur atom of a cysteine amino acid residue 346 (Cys 346) of ZAP-70 kinase.
  • [0101] is a compound of Formulas (I) or (IV), wherein W is an acrylo l group.
  • NHC(0)CH CHCH 2 N(CH 3 )-R 1 , -X! liCO . i CiMCH -R . -NHC(0)C ⁇ CH, - ⁇ i!Ci(»C C-R . - NHC(0)OC-C3 ⁇ 4-R l , -NHC(0)OC-C3 ⁇ 4N(CH 3 )-R ! , -C(0)C ⁇ CH, -C(0)OC-R S , -C3 ⁇ 4OC(0)OCH, - ( II. ⁇ )( (()3 ⁇ 4( ⁇ OR . -( (OiCi! ( 11-. -( i H-!l Cii-R .
  • N(CH 3 )C(0)CH-CHCH 2 N(CH 3 )-R 1 , N(CH 3 )C(0)CH-CHCH 2 N(CH 3 ) 2 , -N(CH 3 )(CO)C( CH 2 )CH 2 -R l , - N(CH 3 )C(0)C ⁇ C-R ! , -N(CH 3 )C(0)C ⁇ C-CH 2 -R 1 , -XfOOCiOiC I- ⁇ ;Cih)-R .
  • -OC(0)CH CH 2 , -
  • [0104] is a compound of Formulas (I) or (IV), wherein W is a reactive functional group thai is attached to L directly through a C, N, or O and which, is selected from the following representative and exemplary list of structures corresponding to W is shown in Figure 1, wherein each wavy line indicates the point of attachment to L;
  • [0105] in one embodiment is a compound of Formulas (I) or (IV), wherein W is reactive functional group tliat is capable of forming a covending bond with the oxygen atom of a tyrosine amino acid residue in or near the ATP -binding site of ZAP-70 kinase.
  • W is reactive functional group that is capable of forming a covending bond with the oxygen atom of a tyrosine amino acid residue in or near the ATP- binding site of ZAP-70 kinase, wherein W is -S0 2 F, -OS0 2 F, -NHS0 2 F, -N(CH3)S0 2 F, -N(CH 2 CH 3 )S0 2 F, -N(Aryl)S0 2 F, or -N(Heteroaryi)S0 2 F.
  • compounds having the structure of Formulas (V) and (VI), or a pharmaceutically accepiable salt, solvate, hydrate, N-oxide, prodrug, stereoisomer, enanliorner, mixture of enantiomers, mixture of diastereomers, isotopic variants, and metabolites thereof are most preferred, which in some embodiments can be inhibitors of the kinase ZAP-70, and wherein each A, B, C, D, A', B', C, D', E, F,
  • L is -C3 ⁇ 4-, -CH(R 4 )-, -C(R 4 ) 2 -, -O, -NH-, -N(R J ) ⁇ , or -S- and L is connected directly to W, as defined above, wherein R 4 is H or linear or branched substituted or unsubstituted alky!, substituted or unsubstituted alkenyi, substituted or unsubstituted c cioalk l, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, or where two R 1 taken together with the atoms to which they are attached form a substituted or unsubstituted cycioalkyl or heterocycle, and where R J is independently
  • substituted or unsubstituted alkenyi, substituted or unsubstituted cycioalky l, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted heterocycloalkyl substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted acyl (-C( 0)R l );
  • a further aspect of this invention are exemplary' compounds having the structures shown in Figure 3, wherein the structures provided are representative and are not meant to be comprehensive, and wherein indole aromatic substitution can occur in either a symmetric manner on both indole pheny l rings, as depicted, or in an asymmetric manner wherein each phenyl ring is differently substituted, and wherein the aromatic ring of L can be substituted or unsubstituted aryl or heteroaryl:
  • a ZAP-70 inhibitor is a small molecule.
  • a "small molecule” is an organic molecule that is less than about 5 kilodaltons (kDa) in size. In some embodiments, the small molecule is less than about 4 kDa, 3 kDa, about 2 kDa, or about 1 kDa.
  • the small molecule is less than about 800 daitons (Da), about 600 Da, about 500 Da, about 400 Da, about 300 Da, about 200 Da, or about 100 Da, In some embodiments, a small molecule is less than about 4000 g/mol, less than about 3000 g/mol, 2000 g/mol, less than about 1500 g mol, less than about 1000 g/mol, less than aboul 800 g/mol, or less than about 500 g mol. In some embodiments, small molecules are non-polymeric.
  • small molecules are not proteins, polypeptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, or proteoglycans, but include peptides of up to about 40 amino acids.
  • a derivative of a small moleciiie refers So a molecule thai shares the same structural core as the original small molecule, but which is prepared by a series of chemical reactions that vary and form a derivative of the original snsali molecule.
  • a pro-drag of a small molecule is a derivative of that small molecule.
  • An analog of a small molecule refers to a molecule that shares the same or similar structural core as the original ssnall molecule, and which is synthesized by a similar or related route, or art-recognized variation, as the original small molecule.
  • compounds described herein have one or more chiral centers. As such, all stereoisomers are envisioned herein.
  • compounds described herein are present in optically active or racemic forms. It is to be understood that the compounds described herein encompass racemic, optically-active, regioisoineric and stereoisomeric forms, or combinations thereof that possess the therapeutically useful properties described herein.
  • Preparation of optically active forms is achieve in any suitable manner, including by way of non-limiting example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase, in some embodiments, mixtures of one or more isomer are utilized as the therapeutic compound described herein.
  • compounds described herein contain one or more chiral centers. These compounds are prepared by any means, including enantioselective synthesis and/or separation of a mixture of enantiomers and/or diastereomers. Resolution of compounds and isomers thereof is achieved by any means including, by way of non-limiting example, chemical processes, enzymatic processes, fractional crystallization, distillation, chromatography, and the like.
  • pharmaceutically acceptable salts described herein include, by way of non-limiting example, a nitrate, chloride, bromide, phosphate, sulfate, acetate, hexafiuorophosphate, citrate, gluconate, benzoate, propionate, butyrate, sulfosalic late, maleate, laurate, malate, fumarate, succinate, tartrate, amsonate, pamoate, p toluenenesulfonate, mesylate and the like.
  • pharmaceutically acceptable salts include, by way of non-limiting example, alkaline earth metal salts (e.g., calcium or magnesium), alkali metal salts (e.g., sodium-dependent or potassium), ammonium salts and the like.
  • Compounds described herein also include isotopically-labeled compounds wherein one or more atoms is replaced by an atom having the same atomic number, but an atomic mass or mass number different from the atomic mass or mass number usually found in nature.
  • isotopes suit- able for inclusion in the compounds described herein include and are not limited to 2H, 3H, 11C, 13C, 14C, 36C1, 18F, 1231, 12,51, 13N, 15N, 150, 170, 180, 32P, 35S or the like.
  • isotopically-labeled compounds are useful in drug and/or substrate tissue distribution studies.
  • substitution with heavier isotopes such as deuterium affords certain therapeutic advantages resulting from greater metabolic stability (for example, increased in vivo half-life or reduced dosage requirements).
  • substitution with positron emitting isotopes, such as 11C, 18F, 150, and 13N is useful in Positron Emission Topography (PET) studies for examining substrate receptor occupancy .
  • PET Positron Emission Topography
  • halo and "halogen” as used herein to identify substituent moieties, represent fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.
  • alkyl represents a cyclic, linear or branched chain saturated hydrocarbon group, which in the case of straight and branched chains, preferably has from one to four carbon atoms (C1-C4 alkyl) such as methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec -butyl, tert- butyl, n-pentyl, n-hexyl, and the like and in the case of a cyclic hydrocarbon preferably hits from three to seven carbon atoms, such as cyclopropyl and cyclohexyl.
  • C1-C4 alkyl such as methyl, ethyl, n-propyl, isopropyl, cyclopropyl, n-butyl, isobutyl, sec -butyl, tert- butyl, n-pentyl, n-he
  • substituted alkyl is intended to include an alky l group substituted with a substituent group that is not H,
  • An "alkyl” group refers to an aliphatic hydrocarbon group. Reference to an alkyl group includes “saturated alkyl” and/or “unsaturated alkyl”. The alkyl group, whether saturated or unsaturated, includes branched, straight chain, or cyclic groups.
  • alkyl includes methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, t-butyl, pentyl, iso-pentyl, neo- pentv'l, and hexyl.
  • alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like.
  • a "lower alkyl” is a C1 -C6 alkyl.
  • cycloalkyl refers to a cyclic hydrocarbon chain, wherein the cycloalkyl is optionally substituted with one or more substituenis as described herein.
  • monocyclic or polycyciic cycloalkyl groups may be saturated or unsaturated, but non-aromittic, and'or spiro and'or non-spiro, and ' or bridged, and/or non-bridged, and/or fused bicyclic groups, wherein each of the atoms forming the ring (i.e. skeletal atoms) is a carbon atom.
  • eycloalkyls are saturated, or partially unsaturated.
  • eycloalkyls are fused with an aromatic ring.
  • Cycloalkyl groups include groups having from 3 to 10 ring atoms:
  • Monocyclic eycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl, cyclopentenyl, cyclohexenyi, cyclohexadienyl, cycloheptenyl.
  • Bicyclic eycloalkyls include, but are not limited to tetrahydronaphthyl, indanyl, tetrahydropentalene, bicycto[2.1, 1 [hexyl, bicyclo[2.2.1 jheptyl, decalinyl, or the like.
  • Polycyciic eycloalkyls include adamantane, norbornane or the like.
  • cycloalkyl includes "unsaturated nonaromatic carbocyclyl” or “nonaromatic unsaturated carbocyclyl” groups both of which refer to a nonaromatic carbocycie, as defined herein that contains at least one carbon-carbon double bond or one carbon-carbon triple bond.
  • the eycloalkyl lias from 3 to 20 (C3-C20), from 3 to 15 (C3-C15), from 3 to 10 (C3-C 10), or from 3 to 6 (C3-C6) carbon atoms.
  • lialoalkyl is one such substituted alkyl, substituted with one or more halo atoms, and preferably is a CI to CI O alkyl substituted with one to five halo atoms.
  • lialoalkyl groups include, but are not limted to: difluoromethyl, dichloromefhyl, trifluoroinethyl, 2,2,2-trifluoroethyl, and pentafluroethyl.
  • alkoxy used alone or in combination, is an alkyl, preferably a CI to C4 alkyl, covalently bonded to the parent molecuie through an -O- linkage alone or in combination.
  • alkoxy groups are mefhoxy, ethoxy, propoxy, isopropoxy, butoxy and t-butoxy.
  • alkoxycarbonyl is, for example, t- butoxycarbonyl or BOC.
  • An "alkoxy” group refers to a (alky 1)0— group, where alky! is as defined herein.
  • aryl refers to an aromatic ring wherein each of the atoms forming the ring is a carbon atom.
  • Aryl rings described herein include rings having five, six, seven, eight, nine, or more than nine carbon atoms. .Aryl groups are optionally substituted. Examples of ary l groups include, but are not limited to phenyl, and naphthalenyl.
  • aryl when used alone or in combination represents a substituted or unsubstituted phenyl, biphenyl, or naphthyl.
  • Aryl may optionally be substituted with one or more substituents that are independently selected from hydroxy, carboxy, alkoxy, preferably a CI to CIO alkoxy, an alkyl, preferably a Cl-ClO alkyl, a haloalkyi, nitro, -NR2R3, -NHCO(C !
  • aryloxy is one such aryl covalently bonded through an -O- linkage.
  • aryialkyi can be considered a substituted alkyl and represents -(CH2)maryl with m being an integer of generally 1 to 3, and preferably is benzyl.
  • alkylaryl can be considered a substituted aryl and may, for example, represent a moiety such as aryl(CH2 ' ) consult- CH3 where m is an integer of generally 0 to 6.
  • alkenyl refers to a two to ten carbon, linear or branched hydrocarbon containing one or more carbon-carbon double bonds, preferably one or two double bonds, wherein the alkenyl group is optionally substituted with one or more substituents as described herein.
  • alkenyl include ethylenyl, propylenyl, 1,3-butadienyl, and 1,3,5-hexatrienyl.
  • alkynyl refers to a linear or branched hydrocarbon, which contains one or more carbon- carbon triple bond(s), wherein the alkynyl is optionally substituted with one or more substituents as described herein.
  • C2-C6 alkynyl refers to a linear unsaturated monovalent hydrocarbon radical of 2 to 6 carbon atoms or a branched unsaturated monovalent hydrocarbon radical of 3 to 6 carbon atoms.
  • the alky nyl is a linear monovalent hydrocarbon of 2 to 20 (C2-C20), 2 to 15 (C2-C15), 2 to 10 (C2-C10), or 2 to 6 (C2-C6) carbon atoms, or a branched monovalent hydrocarbon of 3 to 20 (C3-C20), 3 to 15 (C3-C15), 3 to 10 (C3-C10), or 3 to 6 (C3-C6) carbon atoms.
  • alkynyl groups include, but are not limited to, ethynyl (-CCH), propynyl (including all isomeric forms, e.g., 1-propynyi (C-CCH3) and propargyl (CH2CCH)), and butynyl (including all isomeric forms, e.g., 1-butyn-l-yl and 2-butyn-l-yl).
  • the acyl moiety of an acylamino or acylanunoalkyl group is derived from an aikanoic acid containing a maximum of 10, preferably a maximum of 6, carbon atoms (e.g., acetyl, propionyl or buryryl) or from an aromatic carboxylic acid (e.g. benzoyl).
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quatemized form of a basic nitrogen.
  • a "heteroalkyl” group substitutes any one of the carbons of the alkyl group with a heteroatom having the appropriate number of hydrogen atoms attached (e.g., a CH, group to an NH group or an O group).
  • An "amide” is a chemical moiety with formula -C(0)NHR or -NHC(0)R. where R is selected from alkyl, cycloalkyl, aryl, heteroaiyi (bonded through a ring carbon) and heteroaJicyclic (bonded through a ring carbon).
  • esters refers to a chemical moiety with formula— C(0)OR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroary l and heteroalicyclic.
  • heterocycle or heterocyclic group can be a stable, saturated, partially unsaturated, or aromatic 5- or 6-membered heterocyclic group.
  • the heterocyclic ring consists of carbon atoms and from one to three heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur.
  • a heteroary l group is heterocycle that is aromatic, such as pyridine.
  • the heterocyclic group can be optionally substituted with one to four substituents independently selected from halogen, alkyl, aryl, hydroxy, alkoxy, haloalkyl, nitro, amino, acylamino, monoalkylamino, dialkylamino, alkylthio, alkylsulfinyl and aikylsulfonyl or, when the heterocyclyl group is an aromatic nitrogen-containing heterocyclic group, the nitrogen atom can carry an oxide group.
  • heterocyclic groups examples include imidazolyL imidazolinyl, thiazolinyl, pyridyl, indolyl, fury , pyrirrddinyl, morpholinyl, pyridazinyl, pyrazinyl, triazinyl and triazolyl.
  • Two or more heterocycles may be fused to form polyheterocycles such as, for example, azaindole or purine.
  • heteroaryl or, alternatively, “heteroaromatic” refers to an ary l group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur.
  • An N-containing “heteroaromatic” or “heteroary l” moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom.
  • heteroaryl groups are monocyclic or polycyclic.
  • Examples of monocyclic heteroaryl groups include and are not limited to pyrrole, furan, thiophene, pyrazole, imidazole, isoxazole and are referred to as pyrrolyl, furarryl, thiophenyl, pyrazolyl, irnidazolyl, and isoxazoly groups.
  • heteroaiyi refers to a cyclic aromatic compound containing 5 or 6 atoms with at least one heteroatom wherein the heteroaiyi group derives from, for example, without limitation 1-H-pyrrole, pyrazole, imidazole, 1 ,2,4-triazole, 1,2,3 -triazole, tetrazole, furan, thiophene, oxazole, isoxazole, isothiazole, thiazole, 1,2,5- oxadiazole, 1,2,3-oxadiazole, 1,3,4-thiadiazole, 1,2,5-thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, or 1,2,4-triazine.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, irnidazolyl, pyrazolyl, triazolyl, tetrazolyi, oxazol l, isoxazoiyl, oxadiazolyl, thiazolyl, isothiazolyi, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, and pieridinyl.
  • heteroaryl rings may be fused with another aryl or heteroaryl ring to form polyheteroaryls such as, for example, indole, benzofuran, benzoxazole, quinolone, isoquinoline, quinoxaline, quinazoline, cinnoline, or 1,8-naphtholine.
  • heteroaryl as used herein aiso includes groups in which a heteroaroniatic ring is fused to one or more aryl, cyeloaliphatic, or helerocyclyl rings, where the radical or point of attachment is on the heieroaromalic ring.
  • Nonlsmiting examples of heteroaryl groups that contain two or more fused rings include indol l, isoindolyl, benzothierryl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benztMazolyl, quinolyl, isoquinolyl, einnolinyi, phthalazinyl, quinazolinyl, quinoxalinyl, 4H-quinolizinyl, carbazolyl, aeridinyl, phenazinyl, phenotMazinyS, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, arid pyridof2,3-bl-l,4-oxazin-3(4H)-one.
  • heteroaryl group may be mono-or bicyclic.
  • heteroary l may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaroniatic,” any of which terms include rings that are optionally substituted.
  • heteroaroniatic any of which terms include rings that are optionally substituted.
  • heteroarylkyl refers to an aikyl group substituted by a heteroaryl, wherein the alkyl and heteroaryl portions independently are optionally substituted.
  • a heterocycloalkyl group may be fused with another heterocycloalkyl or heteroaryl group to form a polyheterocycle, such as, for example, indoiine or 2,3-dihydrobenzolfuran.
  • heterocycloalkyl refers to heteroaroniatic and heteroaiicyclic groups containing one to four ring heteroatoins each selected from O, S and N.
  • each heterocyclic group lias from 4 to 10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent O or S atoms.
  • Non-aromatic heterocyclic groups include groups having 3 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system.
  • the heterocyclic groups include benzo-fused ring systems.
  • An example of a 3- membered heterocyclic group is aziridinyl (derived from aziridine).
  • An example of a 4-membered heterocyclic group is azetidinyi (derived from azetidine).
  • An example of a 5-membered heterocyclic group is thiazolyl.
  • An example of a 6-membered heterocyclic group is pyridyl, and an example of a 10-memhered heterocyclic group is quinolinyl.
  • non-aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyS, terrahydrothiopyranyl, piperidino, morpholino, tliiomorpholino, thioxanyl, piperazinyl, aziridinyl, azetidinyi, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, tiiiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6-tetrahydropyridinyl, 2- pyrrolinyl, 3-pyrroIinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3 -dioxo
  • aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyi, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyi, indolyl, benzimidazolyl, benzofuranyS, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl
  • giycose and "glycosyl” refer to a natural or non-natural four, five, six, or seven carbon sugar group, including those derived from natural sugar groups such as allosyi, altrosyl, glucosyl, mannosyl, gulos l, idosyi, galactosyl, talos l, digitoxosyl, olivosyi, arabinosyl, xyiosyl, lyxosyl, rhamnosly, ribosyl, deoxyfiirananosyi, deoxypyranosyl, and deoxyribosyl.
  • Sugars may be additionally substituted or unsubstituted on their carbon backbone with groups such as group A defined above in Formula (1).
  • a!kylglycose represents a giycose moiety linked indirectly to the indolyl N of Formula (T) through a C2 to C4 alkyl chain linker attached to the glycosyl C I carbon.
  • Non-natural sugars can contain substituents not typically found in nature, such as fluoro, cyano, or azido substituted sugars.
  • the giycose may be substituted with O-acyl, O- methyl, amino, mono- and di-alkylamino, or acylamino substituents; aminoglycose refers to sugars containing an amino group (-N(R3)2) attached directly to the sugar carbons.
  • Glycosy l groups can be linked to one or both indolyl N as in Formulas (i), (II), and (IV- VI) or alternatively a single glycosyl group can form a bridge and be linked at different carbons to the two different indolyl N atoms of same molecule, represented by Q and R of Formula (III) which taken together as a single group thus forms bridge the two indoly l N atoms, as in Formulas (I) and (IT).
  • tryptophan derivative or “tryptophan analog” refers to the amino acid tryptophan that is substituted with one or more substituents other than hydrogen on one or more of its aromatic rings, and such that the substituents correspond to the definitions provided for A, B, C, D, A', B', C, and D', and/or a tryptophan that is substituted at the ring positions with C or N as defined for E, F, G, M, E', F', G', and M' above.
  • substituted means a substituent or function group or groups, such as those described for A, are attached to carbon of the main hy drocarbon scaffold in place of hydrogen.
  • LG leaving group
  • a leaving group is any group or atom that enhances the electrop licity of the atom to which it is attached for easy displacement by a nucleophilic group or atom.
  • Examples of preferred leaving groups are triflate (-OS02CF3), mesylate, tosy late, imidate, chloride, bromide, and iodide.
  • — NH protective groups and “protecting group” when used in a similar context, and as used in the specification and claims, refers to sub-class of amino protecting groups that are commonly employed to block or protect the— NH functionality while reacting other functional groups on the compound.
  • the species of protecting group employed in carrying out the method of the present invention is not critical so long as the derivatized— NH group is stable to the condition(s) of subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule.
  • T. W. Greene and P. Wuts, Protective Groups in Organic Synthesis, Chapter 7, pages 385-394 and 397-403, provide a list of commonly employed protecting groups for indoles and maleimides.
  • Preferred indole protecting groups are trimethyisilylethoxymethy!, benzyl, tosyl, carbamate, amide, alkyl or aryl sulfonamide, while maleimide protecting groups include alkoxy, benzyl, dialkoxybenzyl, benzyloxyaikyl or allyl.
  • protected — H defines a group substituted with an— NH protecting group as defined.
  • hydroxy protecting group refers to one of the ether or ester derivatives of the hydroxy group commonly employed to block or protect the hydroxy group while reactions are carried out on other functional groups on a compound.
  • the species of hydroxy protecting group employed is not critical so long as the derivatized hy droxy group is stable to the condition of subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule.
  • Preferred hydroxy protecting groups are tertbutyldiphenylsilyloxy (TBDPS), tert-butyldimethylsilyloxy (TBDMS), tripherrylmefhyl (trityl), mono- or dimethoxytrityl, or an alky! or aryl ester.
  • amino protecting group refers to substituents of an amino group commonly employed to block or protect the amino functionality while reacting other functional groups on the compound.
  • the species of amino-protecting group employed in carrying out the method of the present invention is not critical so long as the derivatized amino group is stable to the condition(s) of subsequent reaction(s) and can be removed at the appropriate point without disrupting the remainder of the molecule.
  • Preferred amino-protecting groups are t- butoxyearbonyL phthalimide, a cyclic alky I, and benzyloxycarbonyl.
  • activated maleimide refers to a 3,4-disubstituted maleimide (pyrroly 1 -2,5-dione) or 2,3,4-trisubstituted maleimide, substituted with at least one leaving group that facilitates reaction with a reagent and especially with an optionally N-substituted organometallic-3-indole.
  • indolylmaleimide embraces a genus of compounds having as their root structure a 3- (indol-3-yl)- pyrroly 1 -2,5-dione and includes the subgenus of "bisindolyimaleimides” having as their root structure a 3,4-(indo l -3-yl)-pyrrolyl-2,5-dione, wherein the indol-3-yl moiety or moieties is/are optionally N- substituted, may optionally be substituted on the fused 6-membered aromatic ring of the indolyl moiety and may optionally be substituted at position 2 of the indol-3-yl moiety or moieties.
  • indolocarbazole refers to an alkaloid compound containing two indole rings derived from tryptophan and a fused maleimide or lactam functionality, and derivatives thereof.
  • the most frequently isolated natural indolocarbazoles are indolo(2,3-a)carbazoles and the most common subgroup are the indoio(2,3-a)pyrrole(3,4-c)carbazoles.
  • compounds of the invention may contain "optionally substituted” moieties.
  • substituted whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an “optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substitiLent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • the term "optionally substituted” or “substituted” means that the referenced group substituted with one or more additional group(s).
  • the one or more additional group(s) are individually and independently selected from amide, ester, alkyl, cycloalkyl, heteroa!kyl, aryl, heteroaryl, heteroalicyclic, hydroxy, aikoxy, aryioxy, alkylfhio, asylthio, alkylsulfoxide, aryisiilfoxide, ester, alkylsulfone, aiylsulfone, cyano, halogen, alkoyl, alkoyloxo, isoeyanato, fhioeyanato, isotMocyanato, nitro, haloalkyl, haloalkoxy, fluoroalkyl, amino, alkyl-amino, dialkyl-amino, amido.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in certain embodiments, their recovery, purification, and use for o ne or snore of the purposes disclosed herein,
  • solubilizing group refers to a chemical moiety that promotes the solubility of a compound to which it is attached.
  • Suitable solubilizing groups include, for example, saturated heterocyclic rings, such as morpholino, piperazinyl, and piperadinyl, and amino groups, such as dimethyl amino and methoxypropylamino.
  • structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
  • an “isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, hydrogen (1H), deuterium (2H), tritium (3H), carbon- 11 (11C), carbon-12 (12C) carbon- 13 ( 13C), carbon- 14 (14C), nitrogen-13 (13N), nitrogen-14 (14N), nitrogen-15 (15N), oxygen-14 (140), oxygen- 15 (150), oxygen-16 ( 160), oxygen- 17 (170), oxygen-18 (180) fluorine- 17 (17F), fluorine- 18 (18F), phosphorus-31 (3 IP), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-32 (32S), sulfur-33 (33S), sulfur-34 (34S), sulfur-35 (35S), sulfur-36 (36S), cMorine-35 (
  • an "isotopic variant" of a compound contains unnatural, proportions of one or more isotopes, including, but not 25 limited to, hydrogen ( 1H), deuterium (2H), carbon-12 (12C), carbon- 13 (13C), nitrogen-14 (14N), nitrogen- 15 (15N), oxygen-16 ( 160) oxygen- 17 (170), oxygen- 18 (180) fluorine-17 ( 17F), phosphorus-31 (3 IP), sulfur-32 (32S), suifur-33 (33S), sulfur-34 (34S), sulfur-36 (36S), chlorine-35 (35C1), chlorine-37 (37C1), bromine-79 (79Br), bromine-81 (81Br), and iodine- 127 (1271).
  • an "isotopic variant" of a compound is in an unstable form, that is, radioactive.
  • an "isotopic variant” of a compound contains unnatural proportions of one or more isotopes, including, but not limited to, tritium (3H), carbon- l i (11C), carbon- 14 (14C), nitrogen-13 ( 13N), oxygen-14 ( 140), oxygen- 15 (150), fiuorine-18 (18F), phosphorus-32 (32P), phosphorus-33 (33P), sulfur-35 (35S), cMorine-36 (36C1), iodine-123 (1231) iodine-125 (1251), iodine-129 (1291) and iodine-131 (1311).
  • any hydrogen can be 2H, as example, or any carbon can be 13C, as example, or any nitrogen can be 15N, as example, and any oxygen can be 180, as example, where feasible according to the judgment of one of skill in the art.
  • an "isotopic variant" of a compound contains an unnatural proportion of deuterium.
  • structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopicallv enriched atoms.
  • compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a 13C- or 14C-enriched carbon are within the scope of this invention.
  • Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.
  • solvate refers to a complex or aggregate formed by one or more molecules of a solute, e.g., a compound provided herein, and one or more molecules of a solvent, which present in a stoichiometric or non-stoichiometric amount.
  • Suitable solvents include, but are not limited to, water, MeOH, ethanol, n-propanol, isopropanol, and acetic acid.
  • the solvent is pharmaceutically acceptable.
  • the complex or aggregate is in a crystalline form.
  • the complex or aggregate is in a noncrystalline form.
  • the solvent is water
  • the solvate is a hydrate. Examples of hydrates include, but are not limited to, a hemihydrate, monohydrate, dihydraie, trihydrate, tetrahydrate, and pentahydrate.
  • naturally occurring or “natural” or “native” when used in connection with naturally occurring biological materials such as nucleic acid molecules, amino acids, polypeptides, small molecule natural products, host cells, and the like, refers to materials that are found in or isolated directly from Nature and are not changed or manipulated by humans.
  • non-naturally occurring or “non-natural” or “unnatural” or “non-native” refers to a material that is not known to exist or not found in Nature or that has been structurally modified or synthesized by humans.
  • si-synthesis refers to modifying a natural material synthetically to create a new variant, derivative, or analog of the original natural material.
  • derivative or “analog” refer to a structural variant of compound that derives from a natural or nan-natural material.
  • optically active and “enantiomerically active” refer to a collection of molecules, which has an enantiomeric excess of no less than about 50%, no less than about 70%, no less than about 80%, no less than about 90%, no less than about 91%, no less than about 92%, no less than about 93%, no less than about 94%, no less than about 95%, no less than about 96%, no less than about 97%, no less than about 98%, no less than about 99%, no less than about 99.5%, or no less than about 99.8%.
  • the compound comprises about 95% or more of one enantiomer and about 5% or less of the other enantiomer based on the total weight of the racemate in question.
  • the prefixes R and S are used to denote the absolute configuration of the molecule about its chiral centers).
  • the symbols (+) and (-) are used to denote the optical rotation of the compound, that is, the direction in which a plane of polarized light is rotated by the optically active compound.
  • the (-) prefix indicates that the compound is levorotatory, that is, the compound rotates the plane of polarized light to the left or counterclockwise.
  • the (+) prefix indicates that the compound is dextrorotatory, that is, the compound rotates the plane of polarized light to the right or clockwise.
  • the sign of optical rotation, (+) and (-) is not related to the absolute configuration of the molecule, R and S.
  • a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant thereof; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug thereof has the same meaning as the phrase "a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant of the compound referenced therein; a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of the compound referenced therein; or a pharmaceutically acceptable salt, solvate, hydrate, or prodrug of a stereoisomer, enantiomer, mixture of enantiomers, mixture of diastereomers, or isotopic variant of the compound referenced therein.
  • the term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means within 50%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range.
  • active ingredient and “active substance” refer to a compound, which is administered, alone or in combination with one or more pharmaceutically acceptable excipients, to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder, disease, or condition.
  • active ingredient and active substance may be an optically active isomer or an isotopic variant of a compound described herein.
  • drug refers to a compound, or a pharmaceutical composition thereof, which is administered to a subject for treating, preventing, or ameliorating one or more symptoms of a disorder, disease, or condition.
  • subject refers to an animal, including, but not limited to, a primate (e.g., human), cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse.
  • primate e.g., human
  • cow, pig, sheep, goat horse
  • dog cat
  • rabbit rat
  • patient are used interchangeably herein in reference, for example, to a mammalian subject, such as a human subject, in one embodiment, a human.
  • patient means an animal, preferably a mammal, and most preferably a human.
  • treat means to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.
  • prevent are meant to include a method of delaying and/or precluding the onset of a disorder, disease, or condition, and/or its attendant symptoms; barring a subject from acquiring a disorder, disease, or condition; or reducing a subject's risk of acquiring a disorder, disease, or condition.
  • terapéuticaally effective amount are meant to include the amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the sy mptoms of the disorder, disease, or condition being treated.
  • therapeutically effective amount also refers to the amount of a compound that is sufficient to elicit the biological or medical response of a biological molecule (e.g., a protein, enzyme, RNA, or DNA), cell, tissue, system, animal, or human, which, is being sought by a researcher, veterinarian, medical doctor, or clinician.
  • a biological molecule e.g., a protein, enzyme, RNA, or DNA
  • IC 3 ⁇ 4 > or "EC 50” refers an amount, concentration, or dosage of a compound that is required for 50% inhibition of a maximal response in an assay that measures such response.
  • CC50 refers an amount, concentration, or dosage of a compound that results in 50% reduction of the viability of a host.
  • the CC50 of a compound is the amount, concentration, or dosage of the compound that is required to reduce the viability of cells treated with the compound by 50%, in comparison with cells untreated with the compound.
  • Kd refers to the equilibrium dissociation constant for a ligand and a protein, which is measured to assess the binding strength that a small molecule ligand (such as a small molecule drug) has for a protein, such as a kinase.
  • the dissociation constant, Kd is commonly used to describe the affinity between a ligand and a protein; i.e., how tightly a ligand binds to a particular protein, and is the inverse of the association constant.
  • Ligand-protein affinities are influenced by non-covalent intermolecular interactions between the two molecules such as hydrogen bonding, electrostatic interactions, hydrophobic and van der Waals forces.
  • K i is the inhibitor constant or inhibition constant, which is the equilibrium dissociation constant for an enzyme inhibitor, and provides an indication of the potency of an inhibitor.
  • biologically active refers to a characteristic of any substance that lias activity in a biological system and/or organism For instance, a substance that, when administered to an organism, has a bioiogicai effect on that organism is considered to be biologically active.
  • a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a "biologically active" portion.
  • an effective amount is an amount, which when administered systemicaily, is sufficient to effect beneficial or desired results, such as beneficial or desired clinical results, or other desired effects that lead to an improvement of the disease condition.
  • An effective amount is also an amount that produces a prophylactic effect, e.g., an amount that delays, reduces, or eliminates the appearance of a pathological or undesired condition associated with an autoimmune disease or cancer.
  • An effective amount is optionally administered in one or more administrations.
  • an "effective amount" of a composition described herein is an amount that is sufficient to palliate, alleviate, ameliorate, stabilize, reverse or slow the progression of an autoimmune disease or cancer,
  • an "effective amount” includes any ZAP-70 inhibitor used alone or in conjunction with one or more agents used to treat a disease or disorder.
  • An "effective amount" of a therapeutic agent as described herein will be determined by a patient's attending phy sician or other medical care provider. Factors which influence what a therapeutically effective amount will be include, the absorption profile (e.g., its rate of uptake into the brain or other tissues) of the ZAP-70 inhibitor, time elapsed since the initiation of disease, and the age, physical condition, existence of other disease states, and nutritional status of an individual being treated. Additionally, other medication the patient is receiving, used in combination with a ZAP-70 inhibi tor, will typically affect the determination of the therapeutically effective amount of the therapeutic agent to be administered.
  • the term "inhibitor” refers to a molecule which is capable of inhibiting (including partially inhibiting or ailosteric inhibition) one or more of the bioiogicai activities of a target molecule, e.g., a ZAP-70 kinase, inhibitors, for example, act by reducing or suppressing the activity of a target molecule and/or reducing or suppressing signal transduction.
  • a ZAP-70 inhibitor described herein causes substantially complete inhibition of ZAP-70.
  • the phrase "partial inhibitor” refers to a molecule which can induce a partial response for example, by partially reducing or suppressing the activity of a target molecule and/or partially reducing or suppressing signal transduction.
  • a partial inhibitor mimics the spatial arrangement, electronic properties, or some other physrcochermcal and/or biological property of the inhibitor.
  • a partial inhibitor competes with the inhibitor for occupancy of the target molecule and provides a reduction in efficacy, relative to the inhibitor alone.
  • a ZAP-70 inhibitor described herein is a partial inhibitor of ZAP-70. In some embodiments, a ZAP-70 inhibitor described herein is an allosteric modulator of ZAP-70. In some embodiments, a ZAP-70 inhibitor binds to the kinase domain of ZAP-70. In some embodiments, the ZAP-70 inhibitor described herein blocks the ATP binding site of ZAP-70. In some embodiments, a ZAP-70 inhibitor is a "Type II" kinase inhibitor. In some embodiments a ZAP-70 inhibitor stabilizes ZAP-70 in its inactive conformation. In some embodimenis, a ZAP-70 inhibitor stabilizes the "DFG-out" conformation of ZAP-70.
  • ZAP-70 iniiibitors reduce, abolish, and/or remove the binding between ZAP-70 and at least one of its natural binding partners (e.g. T cell receptor-CD3 complex zeta chains, lymphocyte- specific protein tyrosine kinase (Lck)).
  • binding between ZAP-70 and at least one of its natural partners is stronger in the absence of a ZAP-70 inhibitor (by e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30°A or 20%)) than in the presence of a ZAP-70 inhibitor.
  • ZAP-70 inhibitors inhibit the phosphotransferase activity of ZAP-70, e.g., by binding directly to the catalytic site or by altering the conformation of ZAP-70 such that the catalytic site becomes inaccessible to substrates.
  • ZAP-70 iniiibitors inhibit the ability of ZAP-70 to phosphorylate at least one of its target substrates, e.g., Linker of activated T cells (LAT), SH2 domain containing leukocyte phosphoprotein of 76 kDa (SLP-76), or itself, ZAP-70 iniiibitors include inorganic and/or organic compounds.
  • ZAP-70 inhibitors described herein decrease signal transduction from the T cell receptor (TCR) to the nucleus of a T cell. In some embodimenis, ZAP-70 inhibitors described herein decrease the calcium concentration inside T cells. In some embodiments, ZAP-70 inhibitors described herein decrease phosphorylation and activation of kinase ITK. In some embodiments, ZAP-70 inhibitors described herein decrease phosphorylation of phospholipase C gamma 1 (PLCyl) in T cells. In some embodiments, ZAP-70 inhibitors described herein decrease production of interleukin 2 (IL-2) in T cells. In some embodiments, ZAP- 70 inhibitors described herein decrease CD8+ T cell proliferation.
  • TCR T cell receptor
  • ZAP-70 inhibitors described herein decrease the calcium concentration inside T cells. In some embodiments, ZAP-70 inhibitors described herein decrease phosphorylation and activation of kinase ITK. In some embodiments, ZAP-70 inhibitors described herein decrease
  • ZAP-70 inhibitors described herein decrease production of inositol triphosphate in T ceils, in some embodiments, ZAP-70 inhibitors described herein decrease production of diacyiglycerol in T cells after TCR activation. In some embodiments, ZAP-70 inhibitors described herein decrease TCR-dependent MAP kinase signaling. In some embodiments, ZAP-70 iniiibitors described herein decrease TCR-induced calcium signaling. In some embodiments, ZAP-70 inhibitors described herein decrease CD4+ T cell proliferation. In some embodimenis, ZAP-70 iniiibitors described herein decrease CD8 ⁇ T cell proliferation.
  • ZAP-70 inhibitors described herein decrease T helper cell 1 (Thl) proliferation. In some embodiments, ZAP-70 inhibitors described herein decrease T helper cell 2 (Tli2) proliferation. In some embodiments, ZAP-70 inhibitors described herein decrease T helper cell 17 (Thl7) proliferation, in some embodiments, ZAP-70 iniiibitors described herein reduce the activity of cytotoxic T cells. In some embodiments, ZAP-70 inhibitors described herein decrease the production of tumor necrosis factor (TNF). In some embodiments, ZAP-70 inhibitors described herein decrease production of interferon gamma. In some embodiments, ZAP-70 inhibitors described herein do not impact the activity of regulator ⁇ ' T cells (Tregs).
  • TNF tumor necrosis factor
  • ZAP-70 inhibitors described herein decrease production of interferon gamma. In some embodiments, ZAP-70 inhibitors described herein do not impact the activity of regulator ⁇ ' T cells (Tregs).
  • ZAP inhibitors described herein decrease the activation and proliferation of T cells but not B cells. In some embodiments, ZAP inhibitors described herein decrease the activation and proliferation of T cells and B cells. In some embodiments, ZAP-70 inhibitors described herein do not decrease Treg proliferation. In some embodiments, ZAP-70 inhibitors described herein decrease nuclear translocation of the transcription factors NFAT, NF-KB, c-Jun, c-Fos, and AP- 1.
  • a ZAP-70 inhibitor suitable for the methods described herein is a direct ZAP- 70 inhibitor. In some embodiments, a ZAP-70 inhibitor suitable for the methods described herein is an indirect ZAP-70 inhibitor. In some embodiments, a ZAP-70 inhibitor suitable for the methods herein decreases ZAP-70 activity relative to a basal level of ZAP-70 activity by about 1.1 fold to about 1000 fold, e.g., to about 1.2 fold, 1.5 fold, 1.6 fold, 1.7 fold, 2.0 fold, 3.0 fold, 5.0 fold, 6.0 fold, 7.0 fold, 8.5 fold, 9.7 fold, 10 fold, 12 fold, 14 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold.
  • the ZAP-70 inhibitor is a reversible ZAP-70 inhibitor. In other embodiments, the ZAP-70 inhibitor is an irreversible ZAP- 70 inhibitor.
  • Direct ZAP-70 inhibitors are optionally used for the manufacture of a medicament for treating an autoimmune disease. Direct ZAP-70 inhibitors are optionally used for the manufacture of a medicament for treating T cell-associated lymphomas and leukemias.
  • a ZAP-70 inhibitor used for the methods described herein has an in vitro IC 50 , defined as inhibitory concentration where 50% of ZAP-70 activity is remaining after contacting a ZAP-70 inhibitor with ZAP-70 kinase, or dissociation constant (Kd), or inhibitory constant (Ki) of less than 100 ⁇ (e.g., less than 10 ⁇ , less than 5 ⁇ , less than 4 ⁇ , less than 3 ⁇ , less than 1 uM, less than 0.8 ⁇ , less than 0.6 ⁇ , less than 0.5 ⁇ , less than 0.4 ⁇ , less than 0.3 ⁇ , less than less than 0.2 ⁇ .
  • Kd dissociation constant
  • Ki inhibitory constant
  • expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription of DNA into messenger RNA); (2) processing of an RNA transcript (e.g., by splicing, editing. 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; (4) post-translational modification of a polypeptide or protein.
  • ZAP-70 polypeptide or "ZAP-70 protein” or “ZAP-70” or “ZAP-70 kinase” refers to a protein that belongs in the Syk family of human kinases.
  • a representative example of ZAP-70 amino acid sequences includes, but is not limited to, human ZAP-701 (GcnBank Accession Number AAH39039.1 ), Human ZAP-70 also has numerous truncated isoforms that have been identified and ZAP- 70. homologues exist throughout the animal kingdom.
  • a ZAP-70 polypeptide comprises an amino acid sequence that is at least 60% to 100% identical, e.g., at least 75%, 80%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 70% to about 100% identical to sequences of GenBank Accession Number ⁇ 39039.1,
  • a human ZAP-70 gene comprises a nucleotide sequence that is at least 70% to 100% identical, e.g., at least 75%, 80%, 85%. 86%, 87%, 88%, 90%, 91%, 92%, 94%, 95%, 96%, 97%, 98%, or any other percent from about 70% to about 100% identical to sequences of GenBank Accession Number NG007727.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • Gapped BLAST is utilized as described in Altschul, S.F., et al. Nucleic Acids Res., 1997, 25, 3389-3402.
  • the default parameters of the respective programs e.g., BLAST and NBLAST
  • Proteins suitable for use in the methods described herein also includes proteins having between 1 to 15 amino acid changes, e.g., 1 , 2, 3. 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions, deletions, or additions, compared to the amino acid sequence of any protein ZAP-70 inhibitor described herein.
  • the altered amino acid sequence is at least 75% identical, e.g., 77%, 80%, 82%, 85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any protein ZAP-70 inhibitor described herein.
  • sequence-variant proteins are suitable for the methods described herein as long as the altered amino acid sequence retains sufficient biological activity to be functional in the compositions and methods described herein.
  • the substitutions should be conservative amino acid substitutions.
  • a "conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff, S., et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919). Accordingly, the BLOSUM62 substitution frequencies are used to define conservative amino acid substitutions that may be introduced into the amino acid sequences described or described herein. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater thanl .
  • an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, I, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • ZAP-70 activity includes, but is not limited So, at least one of ZAP-70 protein-protein interactions, ZAP-70 phosphotransferase activity (intermolecular or intermolecular), translocation, etc of one or more ZAP-70 isofonns.
  • a "ZAP-70 inhibitor” refers to any molecule, compound, or composition that directly or indirectly decreases the ZAP-70 activity.
  • ZAP-70 inhibitors inhibit, decrease, and/or abolish the level of a ZAP-70 mRNA and/or protein or the half -life of ZAP-70 mRNA and/or protein, such inhibitors are referred to as "clearance agents".
  • a ZAP-70 inhibitor is a ZAP-70 antagonist that inhibits, decreases, and/or abolishes an activity of ZAP-70.
  • a ZAP-70 inhibitor also disrupts, inhibits, or abolishes the interaction between ZAP-70 and its natural binding partners (e.g., a substrate for ZAP-70 kinase, for CD3 protein, or for Lck kinase) or a protein that is a binding partner of ZAP-70 in a pathological condition, as measured using standard methods.
  • ZAP-70 inhibitors reduce, abolish, and/or remove the binding between ZAP-70 and at least one of its natural binding partners (e.g., CD3 zeta chain, LCK). In some instances, binding between ZAP-70 and at least one of its natural binding partners is stronger in the absence of a ZAP-70 inhibitor (by e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20%) than in the presence of a ZAP-70 inhibitor. In some embodiments, ZAP-70 inhibitors prevent, reduce, or abolish binding between ZAP-70 and a protein that abnormally accumulates or aggregates in cells or tissue in a disease state.
  • ZAP-70 inhibitors prevent, reduce, or abolish binding between ZAP-70 and a protein that abnormally accumulates or aggregates in cells or tissue in a disease state.
  • binding between ZAP-70 and at least one of the proteins that aggregates or accumulates in a cell or tissue is stronger in the absence of a ZAP-70 inhibitor (by e.g., 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20%) than in the presence of an inhibitor.
  • An "individual” or an “individual,” as used herein, is a mammal. In some embodiments, an individual is an animal, for example, a rat, a mouse, a dog or a monkey. In some embodiments, an individual is a human patient. In some embodiments an "individual" or an “individual” is a human.
  • an individual suffers from an autoimmune disease or T cell-associated cancer or is suspected to be suffering from an autoimmune disease or T cell-associated cancer or is pre-disposed to an autoimmune disease or T cell- associated cancer.
  • a pharmacological composition comprising a ZAP-70 inhibitor is "administered peripherally" or "peripherally administered.”
  • these terms refer to any form of administration of an agent, e.g., a therapeutic agent, to an individual that is not direct administration to the central nervous system, i.e., that brings the agent in contact with the non-brain side of the blood-brain barrier.
  • Peripheral administration includes intravenous, intra-arterial, subcutaneous, intramuscular, intraperitoneal, transdermal, by inhalation, transbuccal, intranasal, rectal, oral, parenteral, sublingual, or transnasal.
  • a ZAP-70 inhibitor is administered by an intracerebral route.
  • polypeptide and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a protein, and vice versa.
  • the terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturaliy occuning amino acid, e.g., an amino acid analog.
  • the terms encompass amino acid chains of any length, including full length proteins (i.e., antigens), wherein She amino acid residues are linked by covalent peptide bonds.
  • amino acid refers to naturally occurring and non-naturaliy occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occuning amino acids.
  • Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine.
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, hotnoserine, norieucine, methionine sulfoxide, methionine methyl suUbniiim. Such analogs have modified R groups (such as, norieucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
  • nucleic acid refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a maimer similar to naturally occuning nucleotides.
  • oligonucleotide analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like).
  • PNA peptidonucleic acid
  • analogs of DNA used in antisense technology phosphorothioates, phosphoroamidates, and the like.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base andlor deoxyinosine residues (Batzer, M.A., et ai., Nucleic Acid Res., 1991, 19, 5081-1585; Ohtsuka, E. et a!., J. Biol. Chem,, 1985, 260, 2605-2608; and Rossolini, G.M., et ai., Mol. Cell. Probes, 1994, 8, 91-98).
  • isolated and purified refer to a material that is substantially or essentially removed from or concentrated in its natural environment.
  • an isolated nucleic acid is one that is separated from the nucleic acids that normally flank it or other nucleic acids or components (proteins, lipids, etc.) in a sample.
  • a polypeptide is purified if it is substantially removed from or concentrated in its natural environment. Methods for purification and isolation of nucleic acids and proteins are documented methodologies.
  • antibody describes an immunoglobulin whether natural or partly or wholly synthetically produced.
  • the term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antigen binding domain.
  • CDR grafted antibodies are also contemplated by this term.
  • the term antibody as used herein will also be understood to mean one or more fragments of an antibody that retain the ability to specifically bind to an antigen, (See generally: Holliger, P. et al., Nature Biotech. 2005, 23 (9), 1 126-1129).
  • Non-limiting examples of such antibodies include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ah')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward, E.S., et aL, Nature, 1989, 341, 544-546), which consists of a VH domain: and (vi) an isolated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • a F(ah')2 fragment a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they are optionally joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which, the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g.. Bird, R.E., et aL, Science 1988, 242, 423-426; Huston, J.S., et aL, Proc. Natl. Acad. Sci. USA, 1988, 85, 5879-5883; and Osboum, J.K., et aL, Nat. Biotechnol.
  • scFv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term antibody.
  • Any VH and VL sequences of specific scFv is optionally linked to human immunoglobulin constant region cDNA or genomic sequences, in order to generate expression vectors encoding complete IgG molecules or other isotypes.
  • VH and VL are also optionally used in the generation of Fab, Fv or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology.
  • Other forms of single chain antibodies, such as diabodies are also encompassed.
  • F(aW),” and “Fab”' moieties are optionally produced by treating immunoglobulin (monoclonal antibody) with a protease such as pepsin and papain, and includes an antibody fragment generated by digesting immuno-globulin near the disulfide bonds existing between the lunge regions in each of the two H chains.
  • immunoglobulin monoclonal antibody
  • protease such as pepsin and papain
  • papain cleaves IgG upstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate two homologous antibody fragments in which an L chain composed of VL (L chain variable region) and CL (L chain constant region), and an H chain fragment composed of VH (H chain variable region) and CHyl (yl region in the constant region of H chain) are connected at their C terminal regions through a disulfide bond.
  • Each of these two homologous antibody fragments is called Fab'.
  • Pepsin also cleaves IgG downstream of the disulfide bonds existing between the hinge regions in each of the two H chains to generate an antibody fragment slightly larger than the fragment in which the two above-mentioned Fab' are connected at the hinge region. This antibody fragment is called F(ab')2.
  • the Fab fragment also contains the constant domain of the light chain and the first constant domain (CHI) of the heavy chain.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH I domain including one or more cysteine(s) from the antibody hinge region.
  • Fab'-SH is the designation herein for Fab' in which, the cysteine residue(s) of the constant domains bear a free thiol group.
  • F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are documented. "Fv” is the minimum antibody fragment which contains a 25 complete antigen-recognition and antigen binding site.
  • This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions coirfer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • Single-chain Fv or “sFv” antibody fragments comprise a VH, a VL, or both a VH and VL domain of an antibody, wherein both domains are present in a single polypeptide chain.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the sFv to form the desired structure for antigen binding.
  • a "chimeric" antibody includes an antibody derived from a combination of different mammals.
  • the mammal is, for example, a rabbit, a mouse, a rat, a goat, or a human.
  • the combination of different mammals includes combinations of fragments from human and mouse sources.
  • an antibody described or described herein is a monoclonal antibody (MAb), typically a chimeric human-mouse antibody derived by humanization of a mouse monoclonal antibody.
  • MAb monoclonal antibody
  • Such antibodies are obtained from, e.g., transgenic mice that have been "engineered” to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice synthesize human antibodies specific for human antigens, and the mice are used to produce human antibody-secreting hybridomas.
  • enzymes can be assayed based on their ability to act upon a detectable substrate.
  • a compound can be assayed based on its ability to bind to a particular target molecule or molecules.
  • the term “modulating” or “modulate” refers to an effect of altering a biological activity (i.e. increasing or decreasing the activity), especially a biological activity associated with a particular biomolecule such as a protein kinase.
  • a biological activity i.e. increasing or decreasing the activity
  • an inhibitor of a particular biomolecule modulates the activity of that biomolecule, e.g., an enzyme, by decreasing the activity of the biomolecule, such as an enzyme.
  • Such activity is typically indicated in terms of an inhibitory concentration ⁇ l(V.) of the compound for an inhibitor with respect to, for example, an enzy me.
  • the term "contacting" means that the compound(s) are caused to be in sufficient proximity to a particular molecule, complex, cell, tissue, in an organism, or other specified material that potential binding interactions and/or chemical reaction between the compound and other specified material can occur.
  • a number of different assays for kinase activity can be utilized for assaying for active modulators and/or determining specificity of a modulator for a particular kinase or group of kinases.
  • assays mentioned in the Examples below, one of ordinary skill in the art will know of other assays that can be utilized and can modify an assay for a particular application. For example, numerous papers concerning kinases describe assays that can be used. Additional alternative assays can employ binding determinations.
  • this sort of assay can be formatted either in a fluorescence resonance energy transfer (FRET) format, or using an AlphaScreen (amplified luminescent proximity homogeneous assay) format by varying the donor and acceptor reagents that are attached to srreptavidin or the phospho -specific antibody.
  • FRET fluorescence resonance energy transfer
  • AlphaScreen amplified luminescent proximity homogeneous assay
  • biopharmaceutical properties refers to the pharmacokinetic action of a compound or complex of the present invention, including the dissolution, absorption and distribution of the compound on administration to a subject.
  • certain solid forms of compounds of the invention such as amorphous complexes of compounds of the invention, are intended to provide improved dissolution and absorption of the active compound, which is typically reflected in improved Cmax, the maximum achieved concentration in the plasma after administration of the drug) and improved AUC (i.e. area under the curve of drug plasma concentration vs. time after administration of the drug).
  • the term "therapeutically effective” or “effective amount” indicates that the materials or amount of material is effective to prevent, alleviate, or ameliorate one or more symptoms of a disease or medical condition, and/or to prolong the survival of the subject being treated.
  • the invention also includes prodrugs (generally pharmaceutically acceptable prodrugs), active metabolic derivatives (active metabolites), and their pharmaceutically acceptable salts.
  • Prodrugs are compounds or pharmaceutically acceptable salts thereof which, when metabolized under physiological, conditions or when converted by solvol sis, yield the desired active compound.
  • Prodrugs include, without limitation, esters, amides, carbamates, carbonates, ureides, solvates, or hydrates of the active compound.
  • the prodrug is inactive, or less active than the active compound, but may provide one or more advantageous handling, administration, and/or metabolic properties.
  • some prodrugs are esters of the active compound: during metabolysis, the ester group is cleaved to yield the active drug.
  • Esters include, for example, esters of a carboxylic acid group, or S-acyl or 0-acyl derivatives of thiol, alcohol, or phenol groups.
  • a common example is an alkyl ester of a carboxylic acid.
  • Prodrugs may also include variants wherein an NH group of the co pound has undergone acylation, such as the 7 -position of the pyrrolo[2,3-dlpyrimidine ring, the 1 -position of the lH-pyrrolo[2,3-b]pyridine ring, or the nitrogen of the sulfonamide group of compounds as described herein, where cleavage of the acyl group provides the free ⁇ group of the active drug.
  • prodrugs are activated enzymaticaily to yield the active compound, or a compound may undergo further chemical reaction to yield the active compound.
  • Prodrugs may proceed from prodrug form to active form in a single step or may have one or more intermediate forms which may themselves have activity or may be inactive.
  • prodrugs can be conceptually divided into two non-exclusive categories, bioprecursor prodrugs and carrier pro-drugs.
  • bioprecursor prodrugs are compounds that are inactive or have low activity compared to the corresponding active drug compound that contain one or more protective groups and are converted to an active form by metabolism or solvolysis. Both the active drug form and any released metabolic products should have acceptably low toxicity.
  • Oxidative reactions are exemplified without limitation by reactions such as oxidation of alcohol, carbonyl, and acid functionalities, hydrox lation of aliphatic carbons, hvdroxylation of alicyclic carbon atoms, oxidation of aromatic carbon atoms, oxidation of carbon-carbo double bonds, oxidation of nitrogen-containing functional groups, oxidation of silicon, phosphorus, arsenic, and sulfur, oxidative N-dealkylation, oxidative O- and S-dealkylation, oxidative deamination, as well as other oxidative reactions.
  • Reductive reactions are exemplified without limitation by reactions such, as reduction of carbonyl functionalities, reduction of alcohol functionalities and carbon-carbon double bonds, reduction of nitrogen-containing functional groups, and other reduction reactions.
  • Reactions without change in the oxidation state are exemplified without limitation by reactions such as hydrolysis of esters and ethers, hydrolytic cleavage of carbon-nitrogen single bonds, hydrolytic cleavage of non-aromatic heterocycles, hydration and dehydration at multiple bonds, new atomic linkages resulting from dehydration reactions, hydrolytic dehalogenation, removal of hydrogen halide molecule, and other such reactions.
  • Carrier prodrugs are drug compounds that contain a transport moiety, e.g., that improves uptake and/or localized delivery to a site(s) of action.
  • a transport moiety e.g., that improves uptake and/or localized delivery to a site(s) of action.
  • the linkage between the drag moiety and the transport moiety is a covalent bond
  • the prodrug is inactive or less active than the drug compound
  • the prodrug, and any release transport moiety are acceptably non-toxic.
  • the transport moiety is intended to enhance uptake, typically the release of the transport moiety should be rapid.
  • it is desirable to utilize a moiety that provides slow release e.g., certain polymers or other moieties, such as cyclodextrins.
  • carrier prodrugs are often advantageous for orally administered drugs.
  • the transport moiety provides targeted delivery of the drag, for example the drag may be conjugated to an antibody or antibody fragment.
  • Carrier prodrugs can, for example, be used to improve one or more of the following properties: increased lipophilicity, increased duration of pharmacological effects, increased site-specificity, decreased toxicity and adverse reactions, and/or improvement in drug formulation (e.g., stability , water solubility, suppression of an undesirable organoleptic or physiochemical property).
  • lipophilicity can be increased by esterification of hydroxy! groups with lipophilic carboxylic acids, or of carboxylic acid groups with alcohols, e.g., aliphatic alcohols. Wermuth, su ra.
  • Metabolites e.g., active metabolites
  • pro-drugs as described above, e.g., bioprecursor prodrugs.
  • metabolites are pharmacologically active compounds or compounds that further metabolize to pharmacologically active compounds that are derivatives resulting from metabolic processes in the body of a subject.
  • active metabolites are such pharmacologically active derivative compounds.
  • prodrugs the prodrug compound is generally inactive or of lower activity than the metabolic product.
  • the parent compound may be either an active compound or may be an inactive prodrug.
  • one or more alkoxy groups can be metabolized to hydroxy!
  • glucuronidation groups while retaining pharmacologic activity and or carboxyl groups can be esterified, e.g., glucuronidation.
  • a derivative compound resulting from metabolic glucuronidation may be inactive or of low activity, and can be further metabolized to provide an active metabolite.
  • Metabolites of a compound may be identified using routine techniques known in the art, and their activities determined using tests such as those described herein. See, e.g., Bertolini et al, 1997, J. Med. Chem., 40:2011-2016; Shan et al., 1997, J Pharm Sci 86(7):756-757; Bagshawe, 1995, Drag Dev. Res., 34:220-230; Wermuth, supra.
  • stereoisomers include geometric isomers, such as cis- or trans- orientation of substituents on adjacent toluenesulfonate carbons of a double bond. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the present invention. Unless specified to the contrary, all such stereoisomeric forms are included within the formulae provided herein.
  • a chiral compound of the present invention is in a form that contains at least 80% of a single isomer (60% enantiomeric excess ("e.e.") or diastereomeric excess (“d.e.”)), or at least 85% (70% e.e. or d.e.), 90% (80% e.e. or d.e.), 95% (90% e.e. or d.e.), 97.5% (95% e.e. or d.e.), or 99% (98% e.e. or d.e.).
  • an optically pure compound having one chiral center is one that consists essentially of one of the two possible enantiomers (i.e., is enantiomerically pure), and an optically pure compound having more than one chiral center is one that is both diastereomerically pure and enantiomerically pure.
  • the compound is present in optically pure form, such optically pure form being prepared and/or isolated by methods known in the art (e.g. by recrystallization techniques, chiral synthetic techniques (including synthesis from optically pure starting materials), and chromatographic separation using a chiral. column.
  • compositions described herein can be in the form of pharmaceutically acceptable salts, or can be formulated as pharmaceutically acceptable salts.
  • Contemplated pharmaceutically acceptable salt forms include, without limitation, mono, bis, iris, tetrakis, and so on.
  • Pharmaceutically acceptable salts are non-toxic in the amounts and concentrations at which they are administered. The preparation of such salts can facilitate the pharmacological use by altering the physical characteristics of a compound without preventing it from exerting its physiological effect. Useful alterations in so physical properties include lowering the melting point to facilitate transmucosal administration and increasing the solubility to facilitate administering higher concentrations of the drug.
  • a compound of the invention may possess a sufficiently acidic, a sufficiently basic, or botii functional groups, acid/base and accordingly can react with any of a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.
  • the term "pharmaceutically acceptable salt” refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit risk ratio.
  • Pharmaceutically acceptable salts are well known in the ait. For example, pharmaceutically acceptable salts are described in S. M. Berge et al., J. Pharmaceutical Sciences, 1977, 66, 1-19, which is incorporated herein by reference.
  • Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases.
  • suitable inorganic and organic acids and bases include those derived from suitable inorganic and organic acids and bases.
  • pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange.
  • salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2- mtphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pect
  • Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and quaternary ammonium, N(C1-C4 alky)4, salts.
  • Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternar ammonium, and amine cations formed using counterfoils such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alk l sulfonate and ar l sulfonate.
  • the compounds and salts may exist in different crystal or polymorphic forms, or may be formulated as co-crystals, or may be in an amorphous form, or may be any combination thereof (e.g. partially crystalline, partially amorphous, or mixtures of polymorphs) all of which are intended to be within the scope of the present invention and specified formulae.
  • salts are formed by addition, i.e.
  • co-crystals are a new chemical species that is formed between neutral compounds, resulting in the compound and an additional molecular species in the same cry stal structure.
  • compounds of the invention are complexed with an acid or a base, including base addition salts such as ammonium, diethylamide, ethanolamine, ethylenediamine, diethanolamine, butylamine, piperazine, meglu-besylaie, camsylate, citrate, formate, fumarate, glutarate, hydrochlorate, maleate, mesylate, nitrate, oxalate, phosphate, succinate, sulfate, tartrate, thiocyanate and tosylate; and amino acids such as alanine, arginine, asparagine, aspariic acid, cysteine, glutamine, glutamic acid, glycine, Mstidine, stearowet, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine or valine.
  • base addition salts such as ammoni
  • an amorphous complex is preferably formed rather than a crystalline material such as a typical salt or co-crystal, in some instances, the amorphous form of the complex is facilitated by additional processing, such as by spray-drying, mechano-chemical methods such as roller compaction, or microwave irradiation of the parent compound mixed with the acid or base.
  • additional processing such as by spray-drying, mechano-chemical methods such as roller compaction, or microwave irradiation of the parent compound mixed with the acid or base.
  • Such methods may also include addition of ionic and/or succinate), non-ionic polymer systems, including, but not limited to, hydroxypropyl methyl cellulose acetate succinate (HPMCAS) and methacrylic acid copolymer (e.g. Eudragit® L10055), that further stabilize the amorphous nature of the complex.
  • HPMCAS hydroxypropyl methyl cellulose acetate succinate
  • methacrylic acid copolymer e.g. Eu
  • amorphous complexes provide several advantages. For example, lowering of the melting temperature relative to the free base faciiitiates additional processing, such as hot melt extrusion, to further improve the biopharrnaceutical properties of the compound. Also, the amorphous complex is readily friable, which provides improved compression for loading of the sohd into capsule or tablet form. Additionally, the formulae are intended to cover hydrated or solvated as well as unhydrated or unsolvated forms of the identified structures. For example, the indicated compounds include both hydrated and non-hydrated forms. Other examples of solvates include the structures in combination with a suitable solvent, such as isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, or ethanolamine.
  • a suitable solvent such as isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, or ethanolamine.
  • pharmaceutically acceptable carrier refers to a pharmaceutically - acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, solvent, or encapsulating material.
  • each component is “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit risk ratio.
  • compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcelluiose, poiyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.
  • ion exchangers alumina, aluminum stearate, lecithin
  • serum proteins such as human serum albumin
  • buffer substances such as phosphates, glycine, sorbic acid
  • the invention provides a composition comprising a compound of this invention or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • the amount of compound in compositions of this invention is such that is effective to measurably inliibit a target protein kinase, pariicularly ZAP-70, or a mutant thereof, in a biological sample or in a patient.
  • the amount of compound in compositions of this invention is such that is effective to measurably inliibit ZAP-70, or a mutant thereof, in a biological sample or in a patient.
  • a composition of this invention is formulated for administration to a patient in need of such composition.
  • a composition of this invention is formulated for oral administration to a patient.
  • a "pharmaceutically acceptable derivative” means any non-toxic salt, ester, salt of an ester or other derivative of a compound of this invention that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention or an inhibitorily active metabolite or residue thereof.
  • inhibitors as used herein, the term "inhibitorily active metabolite or residue thereof" means that a metabolite or residue thereof is also an inhibitor of ZAP70 or a mutant thereof.
  • compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccaily, vaginally or via an implanted reservoir.
  • parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.
  • compositions are administered orally, intraperitoneally or intravenously.
  • Sterile injectable forms of the compositions of this invention may be aqueous or oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3- butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed or synthetic mono- or di- glycerides.
  • Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically -acceptable oils, such as olive oil or castor oil, especially in their poly-oxyethylated versions.
  • These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxy methyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions.
  • Other commonly used surfactants such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation.
  • compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets aqueous suspensions or solutions.
  • carriers commonly used include lactose and cornstarch.
  • Lubricating agents such as magnesium stearate, are also typically added.
  • useful diluents include lactose and dried cornstarch.
  • aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.
  • compositions of this invention may be administered in the form of suppositories for rectal administration.
  • suppositories for rectal administration.
  • suitable non- irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug.
  • suitable non- irritating excipient include cocoa butter, beeswax and polyethylene glycols.
  • compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. Topical application for the lower intestinal tract can be in a rectal suppository formulation (see above) or in suitable enema formulation. Topically -transdermal patches may also be used. For topical applications, provided pharmaceutically acceptable compositions may be foniiulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers.
  • Carriers for topical administration of compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water.
  • provided pharmaceutically acceptable compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers.
  • Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • compositions may be formulated as inicronized suspensions in isotonic, pH adjusted sterile saline, or, preferably, as solutions in isotonic, pH adjusted sterile saline, either with or without a preservative such as benzylalkonium chloride.
  • the pharmaceutically acceptable compositions may be formulated in an ointment such as petrolatum.
  • Pharmaceutically acceptable compositions of this invention may also be administered by nasal aerosol or inhalation.
  • compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorptio promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.
  • compositions of this invention are formulated for oral administration.
  • the amount of compounds of the present invention that may be combined with the carrier materials to produce a composition in a single dosage form will vary depending upon the host treated, the particular mode of administration.
  • provided compositions should be formulated so that a dosage of between 0.01-100 mg/kg body weight/day of the inhibitor can be administered to a patient receiving these compositions.
  • a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated.
  • an inhibitor is defined as a compound that binds to and-'or inhibits a target protein kinase with measurable affinity .
  • an inhibitor has an IC3 ⁇ 4> and/or binding constant of less than about 50 ⁇ , less than about 1 uM, less than about 500 ⁇ , less than about 100 uM, less than about 10 ⁇ , or less than about ⁇ ⁇ .
  • a compound of the present invention may be tethered to a detectable moiety.
  • a detectable moiety may be attached to a provided compound via a suitable substituent.
  • suitable substituent refers to a moiety that is capable of eovaient attachment to a detectable moiety.
  • moieties are well known to one of ordinary skill in the art and include groups containing, e.g., a carboxylate moiety, an amino moiety, a thiol moiety, or a hydroxy! moiety, to name but a few.
  • moieties be directly attached to a provided compound or via a tethering group, such as a bivalent saturated or unsaturated hydrocarbon chain.
  • such moieties may be attached via click chemistry.
  • such moieties may be attached via a 1,3- cycloaddition of an azide with an alkyne, optionally in the presence of a copper catalyst.
  • Methods of using click chemistry are known in the art and include those described by Rostovtsev, V.V, et al., Angew. Chem. Int. Ed. Engl. 2002, 41 , 2596-2599 and Sun, X,-L span et al., Bioconjugate Chem., 2006, 17, 52-57.
  • detectable moiety is used interchangeably with the term “label” and relates to a moiety capable of being detected, e.g., primary labels and secondary labels.
  • Primary labels such as radioisotopes (e.g., tritium, 32P, 33P, 35S, or 14C), mass-tags, and fluorescent labels are signal generating reporter groups which can be detected without further modifications.
  • Detectable moieties also include luminescent and phosphorescent groups.
  • secondary label refers to moieties such as biotin and various protein antigens that require the presence of a second intermediate for production of a detectable signal.
  • the secondary intermediate may include streptavidin-enzyme conjugates.
  • antigen labels secondary intermediates may include antibody-enzyme conjugates.
  • Some fluorescent groups act as secondary labels because they transfer energy to another group in the process of non-radiative fluorescent resonance energy- transfer (FRET), and the second group produces the detected signal.
  • FRET non-radiative fluorescent resonance energy- transfer
  • fluorescent label fluorescent dye
  • fluoroplrore refer to moieties that absorb light energy at a defined excitation wavelength and emission of light energy at a different wavelength.
  • fluorescent labels include, but are not limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, ASexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S, BODIPY dyes (BOD1PY FL, BOD1PY R6G, BODIPY TMR, BODIPY TR, BOD1PY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591 , BODIPY 630/650, BODIPY 650/665), Carboxy-rhodamine 6G, carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumadin 343, Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dap
  • mass-tag refers to any moiet that is capable of being uniquely detected by virtue of its mass using mass spectrometry (MS) detection techniques.
  • mass-tags include elecirophore release tags such as N4344'-
  • mass-tags include, but are not limited to, nucleotides, dideoxynucieo tides, oligonucleotides of varying length and base composition, oligopeptides, oligosaccharides, and other synthetic polymers of varying length and monomer composition.
  • a large variety of organic molecules, both neutral and charged (biomolecules or sy nthetic compounds) of an appropriate mass range ( 100-2000 Daltons) may also be used as mass-tags.
  • measurable affinity and “measurably inhibit,” as used herein, means a measurable change in a protein kinase activity between a sample comprising a compound of the present invention, or composition thereof, and protein, and an equivalent sample comprising the protein kinase, in the absence of said compound, or composition thereof.
  • the term "irreversibie” or “irreversible inhibitor” or “covalent inhibitor” or covalent irreversible inhibitor” refers to an inhibitor (i.e. a compound) that is able to be covalently bonded to a cysteine residue or tyrosine residue in a target protein kinase in a substantially non-reversible manner. That is, whereas a reversible inhibitor is able to bind to (but is generally unable to form a covalent bond with) a target protein kinase, and therefore can become dissociated from target protein kinase, an irreversibie inhibitor ill remain substantially bound to the target protein kinase once covalent bond formation has occurred.
  • Irreversible inhibitors usually display time dependency, whereby the degree of inhibition increases with the time with which the inhibitor is in contact with the enzyme.
  • an irreversible inhibitor will remain substantially bound to target protein kinase once covalent bond formation has occurred and will remain bound for a time period that is longer than the life of the protein.
  • Methods for identifying if a compound is acting as an irreversibie inliibitor are known to one of ordinal ⁇ ' skill in the art. Such methods include, but are not limited to, enzyme kinetic analysis of t e inhibition profile of the compound with target protein kinase, the use of mass spectrometry of the protein drug target modified in the presence of the inliibitor compound, discontinuous exposure, also known as "washout” studies, and the use of labeling, such as radiolabeled inhibitor, to show covalent modification of the enzyme, as well as other methods known to one of skill in the art.
  • reactive functional groups can act as "reactive functional groups.”
  • the term “reactive functional group” or “reactive functional group group” refers to a functional group present on a compound of the present invention wherein that functional group is capable of covalently binding to an amino acid residue (such as cysteine,lysine, histidine, tyrosine, or other residues capable of being covalently modified) present in the binding pocket of the target protein, thereby irreversibly inWbiting the protein.
  • an amino acid residue such as cysteine,lysine, histidine, tyrosine, or other residues capable of being covalently modified
  • a conjugate comprising a protein kinase that contains a cysteine residue in the ATP binding site and an inhibitor that binds to the ATP binding site.
  • the inhibitor is covendingiy and irreversibly bonded to the cysteine residue in the ATP -binding site of the protein kinase such that the activity of the protein kinase is irreversibly inhibited.
  • the conjugates described herein have a variety of uses.
  • the amount of conjugated target polypeptide relative to unconjugated target polypeptide in a biological sample obtained from a patient that has been treated with an irreversible inhibitor can be used as a biomarker to monitor dosing and efficacy in inhibiting polypeptide activity.
  • the conjugates can be used to tailor dosing of irreversible inhibitors (e.g., quantity administered and/or time interval between administrations) to obtain the desired therapeutic effect.
  • certain protein kinases contain certain non-conserved cysteines that are present in or near the ATP binding site.
  • the common non-conserved cysteines are targets for covalent modification in the conjugates of the invention.
  • ZAP-70 contains a non-conserved cysteine at position 346 of the protein, and Cys346 represents a possible target for covalent irreversible modification using an appropriately designed irreversible ZAP-70 inhibitor.
  • the conjugates contain a protein kinase or portion thereof.
  • the protein kinase or portion thereof is a human protein kinase or portion thereof.
  • the invention encompasses conjugates that contain a protein kinase or portion thereof from any desired species, such as a rodent (mouse, rat) or primate (macaque, chimpanzee). It is well-known in the art that there may be two or more sequences for a particular kinase that differ in amino acid sequence and the sequence variation may be due to natural sequence variation, such as allelic variants or naturally arising mutations.
  • the conjugates of the invention encompass all forms of protein kinases, including allelic variants and mutant proteins.
  • Irreversible inliibitors that form a covalent bond with a target Cys residues can selectively form conjugates with protein kinases that contain the target cysteine residue.
  • Irreversible inhibitors that are suitable for forming a conjugate of the invention comprise a binding moiety that binds in or near the ATP binding site of a protein kinase, a linker L, and a reactive functional group moiety W.
  • the reactive functional group moiety can react with a target cysteine of the protein kinase, and is provided by the group -L-W.
  • the conjugate can comprise any suitable chemical moiety that binds in or near the ATP-binding site of a protein kinase.
  • Many suitable chemical moieties that bind the ATP- binding site of kinases are well-known in the art.
  • the binding modes of many such chemical moieties are known and can be used to design additional moieties that bind the ATP binding site using conventional methods of structure based design. For example, tofacitinib, ceritinib, midostaurin, nilotinib, imatinib, sorafenib (U.S. Pat. No. 7,235,576), VX-680 (U.S. Pat. No.
  • the conjugate can comprise any suitable chemical moiety that binds in or near the ATP binding site of the protein kinase.
  • the invention relates to conjugates comprising ZAP-70 kinase that contains a cysteine residue in the ATP binding site at position 346 and an inhibitor as disclosed herein, wherein the inhibitor is covendingiy and irreversibly bonded to the cysteine residue in the ATP binding site of the protein kinase such that the activity of the protein kinase is irreversibly inhibited.
  • the conjugate comprises an inhibitors disclosed herein and ZAP-70 kinase, wherein the inhibitor is covalently and irreversibly bonded to cysteine residue 346 in the ATP binding site of ZAP-70.
  • Covalent irreversible kinase inhibitors typically are developed by structure-guided incorporation of an elecfrophilic moiety into an inhibitor designed to react with an nucieophilic amino acid residue, such as cysteine, lysine, or tyrosine in or near the kinase ATP-binding or active site (See: Man, R., et al., Bioorg. Med. Chem. Lett., 2014, 24, 33-39).
  • covalent irreversible inhibitors already possess sub-micromola binding affinity to the kinase target of interest and irreversible reaction with a uniquely placed nucieophilic residue can afford significant improvement in affinity and selectivity for the kinase of interest.
  • Covalent irreversible inhibition targeting the highly nucieophilic cysteine thiolate involves the use of electrophilic "reactive functional groups" that can react with nucieophilic amino acid residues such as cysteine, lysine or tyrosine.
  • the Michael addition reaction is the most widely utilized reaction to achieve irreversible binding.
  • Functional groups typically introduced to undergo this addition reaction include acrylamid.es, acrylates, vinyl sulfonates, quinones, alkynyi amides and propargylic acid derivatives.
  • a second frequently employed chemistry uses nucieophilic displacement or addition to ⁇ -halo ketones, thiocyanates, alkynes, nitriles, epoxides, and sulfonyl fluorides (See: Chen, W., et al., J. Am, Chem. Soc. 2016, 138, 7353-7364). Examples of covalent irreversible kinase inhibitors have been described (See, for example: Bridges, A.J., US Patent No.
  • Covalent inhibitors initially bind non-covalently and then if the trajectory of the reactive moiety is appropriate, covalent bond formation takes place to permanently disable enzymatic activity. Kinase function, thus inhibited, is only restored following expression of new protein, the kinetics of which vat)' dramatically for different kinases.
  • Covalent irreversible kinase inhibitors provide advantages for inhibiting the activity of kinases such as ZAP-70. For example, irreversible inhibitors produce sustained inhibition of a target protein. Once protein activity is inhibited sufficient it is only regained through the production of new target protein.
  • an irreversible kinase inhibitor can provide significant improvements in potency and selectivity relative to reversible kinase inhibitors.
  • Covalent kinase inhibitors is that high, selectivity for a given target kinase can be obtained using a combination of both noncovalent and covalent binding.
  • a properly designed covalent irreversible kinase inhibitor that has one dominant mode of binding in, for example, the ATP binding site of a kinase typically only forms a covalent bond with a kinase that possesses a cy steine at a particular position in or near the ATP-binding site. Therefore, non-covalent recognition only needs to enable discrimination beiween kinases that possess an equivalently placed cysteine residue, and thus covalent irreversible inhibition can render high selectivity in addition to high potency.
  • covalent reversible inhibitors which contain an additional electrophilic group on the reactive reactive functional group, such as for example a nitrile substituent attached to the alpha carbon of an acrylamide group (See: Serafimova, I.M., et al., Nat. Chem. Bio., 2012, 8, 471-476).
  • Acrylamide- based kinase inhibitors react with cysteine residues in or near the ATP-binding site of kinases, but also may react irreversibly with glutathione and may therefore occasionally react with proteins other than the desired target, especially proteins with hyper- reactive cysteines (See, for example, Wissner, A. et al., J. Med.
  • This invention relates to compounds that irreversibly inhibit ZAP-70 kinase and to pharmaceutically acceptable salts and compositions thereof. It is believed that the reactive functional group groups in the compounds described herein are particularly suitable for covalently binding to cysteine residue 346 in the binding site of ZAP-70.
  • the compounds disclosed herein are inhibitors of ZAP-70 kinase.
  • the covalent irreversible kinase inhibitor is formulated for topical administration.
  • the irreversible inhibitor can be formulated for delivery topical delivery to the lung (e.g., as an aerosol, such as a dry powder or liquid formulation), as a cream, ointment, lotion or the like for topical application to the skin to treat psoriasis, or as an ocular formulation for topical application to the eye to treat an ocular disease.
  • Such a formulation can contain an irreversible inhibitor and a pharmaceutically acceptable carrier. Additional components, such as preservatives, and agents to increase viscosity of the formulation such as natural or sy nthetic polymers may also be present.
  • the ocular formulation can be in any suitable form, such as a liquid, an ointment, a hydrogel or a powder.
  • An effective amount of the irreversible inlubitor is administered topically, for example to the eye, lung or skin.
  • An effective amount for topical delivery is an amount to have the desired effect, such as an amount sufficient to substantially inhibit the activity of the disease target, or an amount sufficient to slow or prevent disease progression.
  • the compounds of the invention comprise a linker L and a reactive functional group group W that together is provided by -L-W in the formulas described herein.
  • W is a reactive functional group that reversibly or irreversibly interacts with amino acid residues of a kinase in a manner that causes an attractive engagement, such as the formation of a eova!ent bond. Examples include acrylamide or acryiate groups, or a halomethyacyi group which react with and form covalent bonds to a sulfur atom of specific cysteine residues in or near the catalytic pocket of a kinase.
  • halosulfonyl or halosulfonate group thai reacts with and forms covalent bonds to an oxygen atom of specific tyrosine, serine or threonine residues in or near the catalytic pocket of a kinase
  • ZAP-70-mediated disorders or conditions as used herein means any disease or other deleterious condition in which. ZAP-70, or a mutant thereof, are known to play a role.
  • another embodiment of the present invention relates to treating or lessening the severity of one or more diseases in which one or more of ZAP-70 or a mutant thereof, are known to play a role.
  • the present invention relates to a method of treating or lessening the severity of a disease or condition selected from a proliferative disorder, wherein said method comprises administering to a patient in need thereof a compound or composition according to the present invention.
  • the cancer is associated with a solid tumor.
  • the cancer is breast cancer, glioblastoma, lung cancer, cancer of the head and neck, colorectal cancer, bladder cancer, or non-small cell lung cancer.
  • Some embodiments provide a method for treating or lessening the severity of one or more disorders selected from squamous ceil carcinoma, salivary gland carcinoma, ovarian carcinoma, or pancreatic cancer.
  • the cancer is associated with a soluble tumor, such as a leukemia, lymphoma or myeloma.
  • the present invention provides a method for treating or lessening the severity of one or more immunological or hypersensitivity disorders, such as asthma, allergy, transplant rejection, graft versus host disease, and autoimmune diseases such as rheumatoid arthritis, amyotrophic lateral sclerosis, and multiple sclerosis, as well as in solid and hematologic malignancies such as leukemias, lymphomas, and myelomas, wherein said method comprises administering to a patient in need thereof a composition according to the present invention.
  • additional therapeutic agents which are normally administered to treat that condition, may also be present in the compositions of this invention.
  • additional therapeutic agents that are normally administered to treat a particular disease, or condition, are known as "appropriate for the disease, or condition, being treated.”
  • compounds of the present invention, or a pharmaceutically acceptable composition thereof are administered in combination with chemo therapeutic agents to treat proliferative diseases and cancer.
  • chemotherapeutic agents include, but are not limited to, Adriamycin, dexamefhasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxoi, interferons, platinum derivatives, taxane (e.g., paclitaxel), vinca alkaloids (e.g., vinblastine), anthraeyclines (e.g., doxorubicin), epipodoliphySIotoxins (e.g., etoposide), cisplatin, an mTOR inhibitor (e.g., a rapamycin), methotrexate, aclinomycin D, dolastalin 10, colchicine, emetine, trimetrexate, metoprine, cyclosporin, daunorubicin, teniposide, amphotericin, alkylating agents (e.g., chlorambucil), 5 -fluorouracil, campthothecin
  • a compound of the present invention is administered in combination with a biologic agent, such as Avastin or ⁇ ⁇ ( ' F! B! X.
  • a biologic agent such as Avastin or ⁇ ⁇ ( ' F! B! X.
  • compounds of the present invention, or a pharmaceutically acceptable composition thereof are administered in combination with an antiproliferative or chemotherapeutic agent selected from any one or more of Abarelix, aldesleukin, Aldesleukin, Alemtuzumab, Alitretinoin, Aliopurinol, Altretamine, Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine, BCG Live, Bevacuzimab, Fluorouracil, Bexarotene, Bleomycin, Bortezoniib, Busulfan, Calusterone, Capecitabine, Camptothecin, Carboplaiin, Canntistine, Celecoxib, Cetuxuna
  • MS Multiple S
  • compounds of the present invention are administered in combination with a monoclonal antibody or with an siRNA therapeutic.
  • Those additional agents may be administered separately is from an inventive compound containing composition, as part of a multiple dosage regimen. Alternatively, those agents may be part of a single dosage form, mixed together with a compound of this invention in a single composition. If administered as pari; of a multiple dosage regime, the two active agents may be submitted simultaneously, sequentially or within a period of time from one another normally within five hours from one another. [0243] As used herein, the term “combination,” “combined,” and related terms refers to the simultaneous or sequential administration of therapeutic agents in accordance with this invention.
  • a compound of the present invention may be administered with another therapeutic agent simultaneously or sequentially in separate unit dosage forms or together in a single unit dosage form
  • the present invention provides a single unit dosage form comprising a compound of Formula (I)-(VI), an additional therapeutic agent, and a pharmaceutically acceptable carrier, adjuvant, or vehicle.
  • the amount of both, an inventive compound and additional therapeutic agent in those compositions which comprise an additional therapeutic agent as described above
  • compositions of this invention should be formulated so that a dosage of between 0,01 - 100 mg/kg body weight/day of an inventive compound can be administered.
  • compositions which comprise an additional therapeutic agent that additional therapeutic agent and the compound of this invention may act synergisticaliy. Therefore, the amount of additional therapeutic agent in such compositions will be less than that required in a monotherapy utilizing only that therapeutic agent. In such compositions a dosage of between 0.01-1,000 mg/kg body weight/day of the additional therapeutic agent can be administered.
  • the amount of additional therapeutic agent present in the compositions of this invention will be no more than the amount that would normally be administered in a composition comprising that therapeutic agent as the only agent.
  • the amount of additional therapeutic agent in the presently disclosed compositions will range from about 50% to 100% of the amount normally present in a composition comprising that agent as the only therapeutically active agent.
  • compositions for coating an implantable medical device such as prostheses, artificial valves, vascular grafts, stents and catheters.
  • Drug resistance is emerging as a significant challenge for targeted therapies.
  • drug resistance has been reported for Gleevec® and Iressa®, as well as several other kinase inhibitors in development.
  • Drug resistance for example, has been reported for inhibitors cKit and EGFR kinases used for cancer treatment, it has been reported that irreversible inhibitors may be effective against drug resistant forms of protein kinases (See: Kwak, E.L., et al, Proc. Nat. Acad. Sci. USA, 2005, 102, 7665-7670).
  • Compounds of the present invention may be effective inhibitors of drug resistant forms of protein kinases.
  • the term “clinical drug resistance” refers to the loss of susceptibility of a drug target to drug treatment as a consequence of mutations in the drug target.
  • the term “resistance” refers to changes in the wild-type nucleic acid sequence coding a target protein, and/or the protein sequence of the target, which changes decrease or abolish the inhibitory effect of the inhibitor on the target protein. Examples of kinases that are inhibited by the compounds and compositions described herein and against which the methods described herein are useful against ZAP -70, or a mutant thereof.
  • the activity of a compound utilized in this invention as an inhibitor of a target kinase, in particular ZAP-70, or a mutant thereof, may be assayed in vitro, in vivo or in a cell line.
  • In vitro assay s include more assays that determine inhibition of either the phosphorylation activity and/or the subsequent functional consequences, or ATPase activity of activated target kinase, or a mutant thereof.
  • Alternate in vitro assays quantitate the ability of the inhibitor to bind to a target kinase, e.g., ZAP-70.
  • Inhibitor binding may be measured by radioiabelling the inMbitor prior to binding, isolating the inhibitor/target kinase complex and determining the amount of radiolabel bound.
  • inhibitor binding may be determined by running a competition experiment where new inhibitors are incubated with target kinase bound to known radioligands.
  • Detailed conditions for assaying a compound utilized in this invention as an inhibitor of certain kinases, or a mutant thereof, are set forth in the Examples below.
  • Protein kinases are a class of enzymes that catalyze the transfer of a phosphate group from ATP or GTP to an acceptor amino acid residue (e.g., tyrosine, serine, and threonine) residue located on a protein substrate.
  • Receptor kinases act to transmit signals from the outside of a cell to the inside by activating secondary messaging effectors via a phosphorylation event.
  • a variety of cellular processes are promoted by these signals, including proliferation, carbohydrate utilization, protein synthesis, angiogenesis, cell growth, and cell survival .
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein.
  • treatment may be administered after one or more symptoms have developed.
  • treatment may be administered in the absence of symptoms.
  • treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.
  • the present invention provides a method for treating ZAP-70-mediated disorders comprising the step of administering to a patient in need thereof a compound of the present invention, or pharmaceutically acceptable composition thereof.
  • ZAP-70 inhibitors are designed to cross the blood brain barrier by introducing lipophilic substituents (e.g., an alkyl or -CF3 group) into the chemical structure of the inhibitor (See, for example: Heffron, T.P., J. Med. Chem, 2016, 59, 10030-10066).
  • a ZAP-70 inhibitor is optionally administered in combination with a blood brain barrier facilitator.
  • an agent that facilitates the transport of a ZAP-70 inhibitor is covalently attached to the ZAP-70 inhibitor.
  • ZAP-70 inhibitors described herein are modified by covalent attachment to a lipophilic carrier or co -formulation with a lipophilic carrier.
  • a ZAP-70 inhibitor is covalently attached to a lipophilic carrier, such as e.g., DHA, or a laity acid.
  • a ZAP-70 inhibitor is covalently attached to artificial low density lipoprotein particles.
  • carrier systems facilitate the passage of ZAP-70 inhibitors described herein across the blood-brain barrier and include but are not limited to, the use of a dihydropyridine pyridinium salt carrier redox system for delivery of drug species across the blood brain barrier.
  • a ZAP-70 inhibitor described herein is coupled to a lipophilic phosphonate derivative.
  • ZAP-70 inhibitors described herein are conjugated to PEG-oligomers/ polymers or aprotinin derivatives and analogs.
  • an increase in influx of a ZAP-70 inhibitor described herein across the blood brain barrier is achieved by modifying A ZAP-70 inhibitor described herein (e.g., by reducing or increasing the number of charged groups on the compound) and enhancing affinity for a blood brain barrier transporter.
  • a ZAP-70 inMbitor is co administered with an agent that reduces or inhibits efflux across the blood brain barrier, e.g. an inhibitor of P-glycoprotein pump (PGP) mediated efflux (e.g., cyclosporin, SCH66336 (lonafarnib, Schering)).
  • PGP P-glycoprotein pump
  • compounds of this invention are optionally administered in combination with a ZAP-70 clearance agent.
  • compounds of this invention are optionally administered in combination with a compound that directly or indirectly decreases the activation or activity of the upstream effectors of ZAP-70.
  • a co mpound that inhibits the activity of LCK or ' ICR is used in combination, thereby reducing the activation of ZAP-70 kinase.
  • LCK inhibitor 7-cyclopenty1-5-(4-phenoxyphenyi)-7H-pyrrolo[2,3 ⁇ d]pyrimidin-4-amine could reduce ITAM and ZAP-70 phosphorylation at tyrosine residues 292 (Y292), 3 15 (Y315) and 319 (Y3 19) of ZAP-70, and thus decrease the activity or activation of ZAP-70 (Arnold, L.D., et al, Bioorg. Med. Chem. Lett., 2000, 10, 2167- 2170; Calderwood, D.J., et al., Bioorg. Med. Chem.
  • ZAP-70 activation is also decreased by small molecules that bind directly to ITAMs.
  • ZAP-70 inhibitors are used in combination with agents that bind directly to LAT or SLP-76 and prevent ZAP-70 from phosphor)' rating tyrosine residues in these downstream effectors (LAT tyrosine residues Y110, 127, 132, 171, 191, and 226; SLP-76 tyrosine residues Yl 13, 128 and 145).
  • compounds of the invention are optionally administered in combination with a compound that decreases the level of ZAP-70 including a peptide, polypeptide, or small molecule that inhibits dephosphorylation of a downstream target of ZAP-70, such thai phosphorylation of the downstream target remains at a level that leads to downregulation of ZAP-70 levels.
  • ZAP-70 activity is reduced or inhibited via activation and/or inhibition of an upstream regulator and/or downstream target of ZAP-70.
  • the protein expression of a ZAP-70 is downregulated.
  • the amount of ZAP-70 in a cell is decreased.
  • a compound that decreases ZAP-70 protein levels in cells also decreases the activity of ZAP-70 in the cells. In some embodiments a compound that decreases ZAP-70 protein levels does not decrease ZAP-70 activity in cells. In some embodiments a compound that increases ZAP-70 activity in cells decreases ZAP-70 protein levels in the cells.
  • any combination of a ZAP-70 inhibitor and second therapeutic agent is compatible with any method described herein.
  • the ZAP-70 inhibitor compositions described herein are also optionally used in combination with other therapeutic reagents that are selected for their therapeutic value for the condition to be treated.
  • the compositions described herein and, in embodiments where combinational therapy is employed, other agents do not have to be administered in the same pharmaceutical composition, and, because of different physical and chemical characteristics, are optionally administered by different routes.
  • the initial administration is generally made according to established protocols, and then, based upon the observed effects, the dosage, modes of administration and times of administration subsequently modified.
  • a ZAP-70 inhibitor composition described herein in combination with another therapeutic agent.
  • another therapeutic agent such as nausea, then it is appropriate to administer an anti-nausea agent in combination with the initial therapeutic agent.
  • the therapeutic effectiveness of a ZAP-70 inhibitor is enhanced by administration of an adjuvant (i.e., by itself the adjuvant has minimal therapeutic benefit, but in combination with another therapeutic agent, the overall therapeutic benefit to the patient is enhanced).
  • the benefit experienced by a patient is increased by administering a ZAP -70 inhibitor with another therapeutic agent (which, also includes a therapeutic regimen) that also has therapeutic benefit.
  • another therapeutic agent which, also includes a therapeutic regimen
  • the overall benefit experienced by the patient is either simply additive of the two therapeutic agents or the patient experiences a sy nergistic benefit.
  • Therapetitically-eifective dosages vary when the drags are used in treatment combinations. Suitable methods for experimentally determining therapeutically-effective dosages of drugs and other agents include, e.g., the use of metronomic dosing, i.e., providing more frequent, lower doses in order to minimize toxic side effects. Combination treatment further includes periodic treatments that start and stop at various times to assist with the clinical management of the patient.
  • the multiple therapeutic agents (one of which is a ZAP-70 inhibitor described herein) is administered in any order, or even simultaneously. If simultaneously, the multiple therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). In some embodiments, one of the therapeutic agents is given in multiple doses, or both are given as multiple doses. If not simultaneous, the timing between the multiple doses optionally varies from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations is also envisioned.
  • the pharmaceutical agents which make up the combination therapy disclosed herein are optionally a combined dosage form or in separate dosage forms intended for substantially simultaneous administration.
  • the pharmaceutical agents that make up the combination therapy are optionally also be administered sequentially, with either therapeutic compound being administered by a regimen calling for two step administration.
  • the two-step administration regimen optionally calls for sequential administration of the active agents or spaced- apart administration of the separate active agents.
  • the time period between the multiple administration steps ranges from, a few minutes to several hours, depending upon the properties of each pharmaceutical agent, such as potency, solubility, bioavailability, plasma half-life and pharmacokinetic profile of the pharmaceutical agent.
  • Orcadian variation of the target molecule concentration is optionally used to determine the optimal dose interval.
  • a ZAP-70 inhibitor is optionally used in combination with procedures that provide additional or synergistic benefit to the patient.
  • patients are expected to find therapeutic and/or prophy lactic benefit in the methods described herein, wherein pharmaceutical composition of a ZAP-70 inhibitor and/or combinations with other therapeutics are combined with genetic testing to determine whether that individual is a carrier of a mutant gene that is correlated with certain diseases or conditions.
  • a ZAP-70 inhibitor and additional therapies are optionally administered before, during or after the occurrence of a disease or condition, and the timing of administering the composition containing a ZAP-70 inhibitor varies in some embodiments.
  • the ZAP-70 inhibitor is used as a prophylactic and administered continuously to individual with a propensity to develop conditions or diseases in order to prevent the occurrence of a disease or condition.
  • ZAP-70 inhibitors and compositions are optionally administered to an individual during or as soon as possible after the onset of the symptoms.
  • the administration of the compounds are optionally initiated within the first 48 hours of the onset of the symptoms, preferably within the first 48 hours of the onset of the symptoms, more preferably within the first 6 hours of the onset of the symptoms, and most preferably within 3 hours of the onset of the symptoms.
  • the initial administration is optionally via any route practical, such as, for example, an intravenous injection, a bolus injection, infusion over 5 minutes to about 5 hours, a pill, a capsule, transdermal patch, buccal delivery, and the like, or combination thereof a ZAP-70 inhibitor is optionally administered as soon as is practicable after the onset of a disease or condition is detected or suspected, and for a length of time necessary for the treatment of the disease, such as, for example, from about 1 month to about 3 months. For ciironic non-life-threatening diseases, treatment duration may be extended for years. The length of treatment optionally varies for each disease and each individual, and the length is then determined using the known criteria.
  • the ZAP-70 inhibitor or a formulation containing the ZAP-70 inhibitor can be administered for at least 2 weeks, preferably about 1 month to about 10 years, and more preferably from about 1 month to about 5 y ears for treatment of cancer.
  • the ZAP-70 inhibitor may be administered for the remaining lifespan of the individual.
  • the particular choice of compounds depends upon the diagnosis of the attending physicians and their judgment of the condition of an individual and the appropriate treatment protocol.
  • the compounds are optionaily administered concurrently (e.g., simultaneously, essentially simultaneously or within the same treatment protocol) or sequentially, depending upon the nature of the disease, disorder, or condition, the condition of an individual, and the actual choice of compounds used.
  • the determination of the order of administration, and the number of repetitions of administration of each therapeutic agent during a treatment protocol is based on an evaluation of the disease being treated and the condition of an individual.
  • therapeutically-effective dosages vary when the drags are used in treatment combinations. Methods for experimentally determining therapeutically-effective dosages of dmgs and other agents for use in combination treatment regimens are described in the literature.
  • dosages of the co -administered compounds vary depending on the type of co-drug employed, on the specific drag employed, on the disease or condition being treated and so forth.
  • the compound provided herein is optionally administered either simultaneously with the biologically active agent(s), or sequentially. In certain instances, if administered sequentially, the attending physician will decide on the appropriate sequence of therapeutic compound described herein in combination with the additional therapeutic agent.
  • the multiple therapeutic agents are optionally administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents are optionally provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills), in certain instances, one of the therapeutic agents is optionally given in multiple doses. In other instances, both are optionaily given as multiple doses. If not simultaneous, the timing between the multiple doses is any suitable timing, e.g., from more than zero weeks to less than four weeks.
  • the additional therapeutic agent is utilized to achieve reversal or amelioration of symptoms of a disease or disorder, whereupon the therapeutic agent described herein (e.g., a compound of Formula (I)-(VT)) is subsequently administered.
  • the combination methods, compositions and formulations are not to be limited to the use of only two agents; the use of multiple therapeutic combinations is also envisioned.
  • a dosage regimen to treat, prevent, or ameliorate the condition(s) for which relief is sought is modified in accordance with a variety of factors. These factors include the disorder from which an individual suffers, as well as the age, weight, sex, diet, and medical condition of an individual. Thus, in various embodiments, the dosage regimen actually employed varies and deviates from the dosage regimens set forth lie rein.
  • compositions comprising a therapeutically effective amount of any compound described herein (e.g., a compound of Formulas (I)-(VI)).
  • Pharmaceutical compositions are formulated using one or more physiologically acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which are used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • a summary of pharmaceutical compositions is found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999).
  • compositions that include ZAP-70 inhibitors and a pharmaceutically acceptable diluentis), excipientis), or carrier(s).
  • the ZAP-70 inhibitor is optionally administered as pharmaceutical compositions in which it is mixed with other active ingredients, as in combination therapy.
  • the pharmaceutical compositions includes other medicinal or pharmaceutical agents, carriers, adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure, and/or buffers.
  • the pharmaceutical compositions also contain other therapeutically valuable substances.
  • a pharmaceutical composition refers to a mixture of a ZAP-70 inhibitor with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and/or excipients.
  • the pharmaceutical composition facilitates administration of the ZAP-70 inhibitor to an organism.
  • therapeutically effective amounts of a ZAP-70 inhibitor are administered in a pharmaceutical composition to a mammal having a condition, disease, or disorder to be treated.
  • the mammal is a human.
  • a therapeutically effective amount varies depending on the severity and stage of the condition, the age and relative health of an individual, the potency of the ZAP-70 inhibitor used and other factors.
  • the ZAP-70 inhibitor is optionally used singly or in combination with one or more therapeutic agents as components of mixtures.
  • compositions described herein are optionally administered to art individual by multiple administration routes, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular), intranasal, buccal, topical, rectal, or transdermal administration route.
  • parenteral e.g., intravenous, subcutaneous, intramuscular
  • intranasal e.g., buccal
  • topical e.g., topical, rectal, or transdermal administration route.
  • Example 38 describes a parenteral formulation
  • Example 39 describes an oral formulation of compounds of the invention
  • the pharmaceutical formulations described herein include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast smelt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multi-particiilate formulations, and mixed immediate and controlled release formulations.
  • the pharmaceutical compositions will include at least one ZAP -70 inhibitor, as an active ingredient in free-acid or free-base form, or in a pharmaceutically acceptable salt form.
  • the methods and pharmaceutical compositions described herein include the use of -oxides, crystalline forms (also known as polymorphs), as well as active metabolites of these ZAP -70 inhibitors having the same type of activity.
  • ZAP-70 inhibitors exist as tautomers. All tautoniers are included within the scope of the compounds presented herein.
  • the ZAP-70 inhibitor exists in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, etlianol, and the like. The solvated forms of the ZAP-70 inhibitors presented herein are also considered to be disclosed herein.
  • Carrier materials include any commonly used excipients in pharmaceutics and should be selected on the basis of compatibility with compounds disclosed herein, such as, a ZAP-70 inhibitor, and the release profile properties of the desired dosage form.
  • Exemplar)' carrier materials include, e.g., binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers. Stabilizers, lubricants, wetting agents, diluents, and the like.
  • compositions described herein which include a ZAP-70 inhibitor, are formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by a patient to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multi-particulate formulations, and mixed immediate release and controlled release formulations.
  • aqueous oral dispersions liquids, gels, syrups, elixirs, slurries, suspensions and the like
  • solid oral dosage forms including but not limited to, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills,
  • a formulation comprising a ZAP-70 inhibitor is a solid drug dispersion.
  • a solid dispersion is a dispersion of one or more active ingredients in an inert earner or matrix at solid state prepared by the melting (or fusion), solvent, or melting-solvent methods. (See: Chiou, W.L., Riegelman, S., J. Pharm. Sci, 1971 , 60, 1281-1302), The dispersion of one or more active agents in a solid diluent is achieved without mechanical mixing.
  • Solid dispersions are also called solid-state dispersions, in some embodiments, any compound described herein (e.g., a compound of Formula (I)-(V1) is formulated as a spray dried dispersion (SDD).
  • An SDD is a single pliase amorphous molecular dispersion of a drug in a polymer matrix. It is a solid solution prepared by dissolving the drug and a polymer in a solvent (e.g., acetone, methanol or the like) and spray drying the solution. The solvent rapidly evaporates from droplets which rapidly solidifies the polymer and drug mixture trapping the drug in amorphous form as an amorphous molecular dispersion.
  • amorphous dispersions are filled in capsules and/or constituted into oral powders for reconsrirution.
  • Solubility of an SDD comprising a drug is higher than the solubility of a crystalline form of a drug or a non-SDD amorphous form of a drug.
  • ZAP-70 inhibitors are administered as SDDs constituted into appropriate dosage forms as described herein.
  • Pharmaceutical preparations for oral use are optionally obtained by mixing one or more solid excipient with a ZAP-70 inhibitor, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • Suitable excipients include, for example, fillers such as sugars, including lactose, sucrose, rriannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanfh, methyleellulose, microcrystalline cellulose, hydroxypropylmefhylcellulose, sodium carboxymethylcellulose; or others such as: polyvinylpyrrolidone (PVP or povidone) or calcium phosphate.
  • PVP polyvinylpyrrolidone
  • povidone povidone
  • disintegrating agents are added, such as the cross-linked croscarmellose sodium, polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions are generally used, which optionally contain gum arable, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments are optionally added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • the solid dosage forms disclosed herein are in the form of a tablet, (including a suspension tablet, a fast-melt tablet, a bite-disintegration tablet, a rapid- disintegration tablet, an effervescent tablet, or a caplet), a pill, a powder (including a sterile packaged powder, a dispensable powder, or an effervescent powder) a capsule (including both soft or hard capsules, e.g., capsules made from animal-derived gelatin or plant-derived I-IPMC, or "spri nkle capsules"), solid dispersion, solid solution, bioerodible dosage form, controlled release formulations, pulsatile release dosage forms, multiparticulate dosage forms, pellets, granules, or an aerosol.
  • Example 40 describes an oral solid dosage formulation that is a tablet.
  • the pharmaceutical formulation is in the form of a powder. In still other embodiments, the pharmaceutical formulation is in the form of a tablet, including but not limited to, a fast-melt tablet. Additionally, pharmaceutical formulations of a ZAP-70 inhibitor are optionally administered as a single capsule or in multiple capsule dosage form. In some embodiments. The pharmaceutical formulation is administered in two, or three, or four, capsules or tablets. In another aspect, dosage forms include microencapsulated formulations. In some embodiments, one or more other compatible materials are present in the microencapsulation material.
  • Exempiasy materials include, but are not limited to, pH modifiers, erosion facilitators, anti-foaming agents, anti-oxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegration agents, filling agents, surfactants, solubilizers, stabilizers, lubricants, wetting agents, and diluents.
  • Exemplary microencapsulation materials useful for delaying the release of the formulations including a ZAP-70 inhibitor include, but are not limited to, hydroxypropyl cellulose ethers (HPC) such as Klucel ⁇ or Nisso HPC, low-substituted hydroxypropyl cellulose ethers (L-HPC), hydroxypropyl methyl cellulose ethers (HPMC) such as Seppifilm-LC, Phannacoat®, Metolose SR, Methocel®-E, Opadry YS, PrimaFlo, Benecel MP824, and Benecel MP843, methyleellulose polymers such as Methocel@-A.
  • HPC hydroxypropyl cellulose ethers
  • L-HPC low-substituted hydroxypropyl cellulose ethers
  • HPMC hydroxypropyl methyl cellulose ethers
  • Seppifilm-LC Phannacoat®
  • Metolose SR Methocel®-E
  • hydroxypropylmethylcellulose acetate stearate Aqoat HF-LS, HF-LG, HF-MS
  • Metolose® EthylcelMoses (EC) and mixtures thereof such as E461, Ethocel®, Aqualon®-EC, Surelease®, Polyvinylalcohol (PVA) such as Opadry AMB, hydroxyelhylceliuloses such as Natrosol®, carboxymethycelluloses, and sodium salts of carboxymethylcelluloses (CMC) such as Aqualon®-CMC
  • CMC carboxymethylcelluloses
  • polyvinyl alcohol and polyethylene glycol co-polymers such as Kollicoat IR®, monoglycerides (Myverol), triglycerides (KLX), polyethylene glycols, modified food starch, acrylic polymers and mixtures of acrylic polymers with cellulose ethers such as Eudragit ⁇ EPO, Eudragit® L30D-55, Eudragit® FS 30
  • Controlled release refers to the release of the ZAP-70 inhibitor from a dosage form in which it is incorporated according to a desired profile over an extended period of time.
  • Controlled release profiles include, for example, sustained release, prolonged release, pulsatile release, and delayed release profiles.
  • immediate release compositions controlled release compositions allow delivery of an agent to an individual over an extended period of time according to a predetermined profile.
  • Such release rates provide therapeutically effective levels of agent for an extended period of time and thereby provide a longer period of pharmacologic response while minimizing side effects as compared to conventional rapid release dosage forms.
  • Such longer periods of response provide for many inherent benefits that are not achieved with the corresponding short acting, immediate release preparations.
  • the formulations described herein, which include a ZAP-70 inhibitor are delivered using a pulsatile dosage form.
  • a pulsatile dosage form is capable of providing one or more immediate release pulses at predetermined time points after a controlled lag time or at specific sites.
  • Pulsatile dosage forms including the formulations described herein, which include a ZAP-70 inhibitor are optionally administered using a variety of pulsatile formulations that include, but are not limited to, those described in U.S. Pat. Nos. 5,01 1 ,692, 5,017,381, 5,229,135, and 5,840,329.
  • Other pulsatile release dosage forms suitable for use with the present formulations include, but are not limited to, for example, U.S. Pat. Nos. 4,871,549, 5,260,068, 5,260.069, 5,508,040, 5,567,441 and 5,837,284.
  • Liquid formulation dosage forms for oral administration are optionally aqueous suspensions selected from the group including, but not limited to, pharmaceutically acceptable aqueous oral dispersions, emulsions, solutions, elixirs, gels, and syrups. See, e.g., Singh et al., Encyclopedia of Pharmaceutical Technology, 2nd Ed., pp. 754-757 (2002).
  • the liquid dosage forms optionally include additives, such as: (a) disintegrating agents; (b) dispersing agents; (c) wetting agents; (d) at least one preservative, (e) viscosity enhancing agents, (f) at least one sweetening agent, and (g) at least one flavoring agent.
  • the aqueous dispersions further includes a crystal-forming inhibitor.
  • the pharmaceutical formulations described herein are self-emulsifying drug delivery systems (SEDDS).
  • SEDDS self-emulsifying drug delivery systems
  • Emulsions are dispersions of one immiscible phase in another, usually in the form of droplets.
  • emulsions are created by vigorous mechanical dispersion.
  • SEDDS as opposed to emulsions or rnicroemulsions, spontaneously form emulsions when added to an excess of water without any external mechanical dispersion or agitation.
  • An advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally , water or the aqueous phase is optionally added just prior to administration, which ensures stability of an unstable or hydrophobic active ingredient.
  • the SEDDS provides an efiective delivery system for oral and parenteral delivery of hydrophobic active ingredients.
  • SEDDS provides improvements in the bioavailability of hydrophobic active ingredients.
  • Methods of producing self-emulsifying dosage forms include, but are not limited to, for example, U.S. Pal. Nos. 5,858,401, 6,667,048, and 6,960,563.
  • Suitable intranasal formulations include those described in, for example, U.S. Pat. Nos. 4,476,116, 5,116,817 and amounts of water in addition to the active ingredient. Minor amounts of other ingredients such as pH adjusters, emulsifiers or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing and solubilizing agents are optionally present.
  • the ZAP-70 inhibitor is optionally in a form as an aerosol, a mist or a powder.
  • Pharmitceutical compositions described herein are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., difluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., difluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit is determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges o such as, by way of example only, gelatin for use in an inhaler or insufflator are formulated containing a powder mix of the ZAP-70 inhibitor and a suitable powder base such as lactose or starch.
  • a powder mix of the ZAP-70 inhibitor and a suitable powder base such as lactose or starch.
  • a suitable powder base such as lactose or starch.
  • Example 20e describes an inhalation formulation.
  • buccal formulations that include a ZAP-70 inhibitor include, but are not limited to, U.S. Pat. Nos. 4,229,447, 4,596,795, 4,755,386, and 5,739, 136.
  • the buccal dosage forms described herein optionally further include a bioerodible (hydrolysable) polymeric earner that also serves to adhere the dosage form to the buccal mucosa.
  • the buccal dosage form is fabricated so as to erode gradually over a predetermined time period, wherein the delivery of the ZAP-70 inhibitor, is provided essentially throughout.
  • the biodegradatable (hydrol sable) polymeric carrier generally comprises hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the buccal mucosa.
  • hydrophilic (water-soluble and water-swellable) polymers that adhere to the wet surface of the buccal mucosa.
  • polymeric carriers useful herein include acrylic acid polymers and co, e.g., those known as "carbomers” (Carbopol®, which may be obtained from B.F. Goodrich, is one such polymer).
  • compositions optionally take the form of tablets, lozenges, or gels formulated in a conventional manner
  • Examples 41 and 42 describe sublingual formulations.
  • Transdermal formulations of a ZAP-70 inhibitor are administered through the skin.
  • the transdermal formulaiions described herein include at least three components: (1) a formulation of a ZAP-70 inhibitor, (2) a penetration enhancer; and (3) an aqueous adjuvant.
  • transdermal formulations include components such as, but not limited to, gelling agents, creams and ointment bases, and the like.
  • the transdermal formulation further includes a woven or non-woven backing to material to enhance absorption and prevent the removal of the transdermal formulation from the skin, in other embodiments, the transdermal formulations described herein maintain a saturated or supersaturated state to promote diffusion into the skin.
  • formulations suitable for transdermal administration of a ZAP-70 inhibitor employ transdermal delivery devices and transdermal delivery patches and are lipophilic emulsions or buffered, aqueous solutions, dissolved and/or dispersed in a polymer or an adhesive. Such patches are optionally constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents. Still further, transdermal delivery of the ZAP-70 inhibitor is optionally accomplished by means of iontophoretie patches and the like. Additionally, transdermal patches provide controlled delivery of the ZAP-70 inhibitor. The rate of absorption is optionally slowed by using rate-controlling membranes or by trapping the ZAP-70 inhibitor within a polymer matrix or gel.
  • absorption enhancers are used to increase absorption.
  • An absorption enhancer or earner includes absorbable pharmaceutically acceptable solvents to assist passage through the skin.
  • transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the ZAP-70 inhibitor optionally with carriers, optionally a rate controlling barrier to deliver the ZAP-70 inhibitor to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin.
  • Formulations that include a ZAP-70 inhibitor suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions.
  • suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles including water, ethanol, polyols (propyleneglycol, polyethylenegiyeol, glycerol, cremophor and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.
  • Formulations suitable for subcutaneous injection also contain optional additives such as preserving, wetting, emulsify ing, and dispensing agents,
  • a ZAP-70 inhibitor is optionally formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution. Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution. Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • appropriate formulations include aqueous or non-aqueous solutions, preferably with physiologically compatible buffers or excipients.
  • Parenteral injections optionally involve bolus injection or continuous infusion.
  • Formulations for injection are optionally presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative.
  • the pharmaceutical composition described herein are in a form suitable for parenteral injection as a sterile suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and or dispersing agents.
  • Pharmaceutical formulations for parenteral administration include aqueous solutions of the ZAP-70 inhibitor in water soluble form. Additionally, suspensions of the ZAP-70 inhibitor are optionally prepared as appropriate oily injection suspensions.
  • the ZAP-70 inhibitor is administered topically and formulated into a variety of topically administrable compositions, such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments.
  • topically administrable compositions such as solutions, suspensions, lotions, gels, pastes, medicated sticks, balms, creams or ointments.
  • Such pharmaceutical compositions optionally contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.
  • the ZAP-70 inhibitor is also optionally formulated in rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas, containing conventional suppository bases such as cocoa butter or other glycosides, as well as synthetic polymers such as polyvinylpyrrolidone, PEG, and the like.
  • rectal compositions such as enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, or retention enemas
  • conventional suppository bases such as cocoa butter or other glycosides
  • synthetic polymers such as polyvinylpyrrolidone, PEG, and the like.
  • a low-melting wax such as, but not limited to, a mixture of fatty acid glycerides, optionally in combination with cocoa butter is first melted. Examples of Methods of Dosing and Treatment Regimens
  • the ZAP-70 inhibitor is optionally used in the preparation of medicaments for the prophylactic and/or therapeutic treatment of a disease or disorder that would benefit, at least in part, from amelioration of symptoms.
  • a method for treating any of the diseases or conditions described herein in an individual in need of such treatment involves administration of pharmaceutical compositions containing at least one ZAP- 70 inhibitor described herein, or a pharmaceutically acceptable salt, pharmaceutically acceptable N-oxide, pharmaceutically active metabolite, pharmaceutically acceptable prodrug, or pharmaceutically acceptable solvate thereof in therapeutically effective amounts to said individual.
  • the administration of the ZAP-70 inhibitor is optionally administered chronically, that is, for an extended period of time, including throughout the duration of the patient's life in order to ameliorate or otherwise control or limit the symptoms of the patient's disease or condition.
  • the administration of the ZAP-70 inhibitor is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday ").
  • the length of the drug holiday optionally varies between 2 da s and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days. 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday includes from 10%- 100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • the pharmaceutical compositions described herein are in unit dosage forms suitable for single administration of precise dosages.
  • the formulation is divided into unit doses containing appropriate quantities of one or more ZAP-70 inhibitor.
  • the unit dosage is in the form of a package containing discrete quantities of the formulation.
  • Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules.
  • aqueous suspension compositions are packaged in single-dose non-reclosable containers. Alternatively, multiple-dose re-closable containers are used, in which case it is typical to include a preservative in the composition.
  • formulations for parenteral injection are presented in unit dosage form, which include, but are not limited to ampoules, or in multi dose containers, with an added preservative.
  • the daily dosages appropriate for the ZAP-70 inhibitor are from about 0.01 to about 2.5 mg/kg per body weight.
  • An indicated daily dosage in the larger mammal, including, but not limited to, humans, is in the range from about 0.5 mg to about 1000 mg, conveniently administered in divided doses, including, but not limited to, up to four times a day or in extended release form.
  • Suitable unit dosage forms for oral administration include from about 1 to about 500 mg active ingredient, from about 1 to about 250 mg of active ingredient, or from about 1 to about 100 mg active ingredient.
  • Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index which is expressed as the ratio between LD50 and ED50.
  • ZAP-70 inhibitors exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human.
  • the dosage of such ZAP-70 inhibitors lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity.
  • the dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.
  • Small molecule ZAP-70 inhibitors are optionally identified in high-throughput in vitro or cellular assays as described, for example, in US Patent Nos. 8,283,356 B2, 7,671,063 B2, and 8,431,589 B2.
  • ZAP-70 inhibitors suitable for the methods described herein are available from a variety of sources including both natural (e.g., bacterial culture, soil or plant extracts) and synthetic.
  • candidate ZAP-70 inhibitors are isolated from a combinatorial library, i.e., a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks.”
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks called amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks, as desired.
  • each member of a library may be singular and/or may be part of a mixture (e.g. a "compressed library”).
  • the library may comprise purified compounds and/or may be "dirty" (i.e., containing a quantity of impurities).
  • Preparation and screening of combinatorial chemical libraries are documented methodologies (See: Cabilly, S.
  • Combinatorial chemical libraries include, but are not limited to: diversomers such as hydantoins, benzodiazepines, and dipeptides, as described in, e.g., DeWitt, S.H., et al., Proc. Natl. Acad. Sci. USA., 1993, 90, 6909-69 13; analogous organic syntheses of small compound libraries, as described in Chen, C, et al., J. Am.
  • Any of the above devices are optionally used to generate combinatorial libraries for identification and characterization of ZAP-70 inhibitors which mimic the manual s nthetic operations performed by small molecule ZAP-70 inhibitors suitable for the methods described herein. Any of the above device are optionally used to identify and characterize small molecule ZAP-70 inhibitors suitable for the methods disclosed herein.
  • the identification of potential ZAP-70 inhibitors is determined by, for example, assaying the in vitro kinase activity of ZAP-70 in the presence of candidate inhibitors.
  • ZAP-70 and/or a characteristic ZAP-70 fragment produced by recombinant means is contacted with a substrate in the presence of a phosphate donor (e.g., ATP) containing radiolabeled phosphate, and ZAP-70-dependent incorporation is measured.
  • a phosphate donor e.g., ATP
  • Substrate includes any substance containing a suitable hydroxy! moiety that can accept the y- phosphate group from a donor molecule such as ATP in a reaction catalyzed by ZAP-70.
  • the substrate may be an endogenous substrate of ZAP-70, i.e. a naturally occurring substance that is phosphoryiated in unmodified cells by naturally-occurring ZAP-70 (e.g., LAT or SLP-76) or any other substance that is not normally phosphoryiated by ZAP-70 in physiological conditions, but may be phosphor iated in the employed conditions.
  • the substrate may be a protein or a peptide, and the phosphrylation reaction may occur on a serine and/or tlireonine residue of the substrate.
  • specific substrates, which are commonly employed in such assays include, but are not limited to, histone proteins and myelin basic protein.
  • ZAP-70 inhibitors are identified using 1MAP ⁇ technology or LanthaScreen technology.
  • Detection of ZAP-70 dependent phosphorylation of a substrate can be quantified by a number of means other than measurement of radiolabeled phosphate incorporation.
  • incorporation of phosphate groups may affect physiochemical properties of the substrate such as electrophoretic mobility, chromatographic properties, light absorbance, fluorescence, and phosphorescence.
  • monoclonal or polyclonal antibodies can be generated which selectively recognize phosphoryiated forms of the substrate from non-phosphorylated forms whereby allowing antibodies to function as an indicator of ZAP-70 kinase activity.
  • High-throughput ZAP-70 kinase assays can be performed in, for example, microtiter plates with each well containing ZAP-70 kinase or an active fragment thereof, substrate covalently linked to each well, P32 radiolabled ATP and a potential ZAP-70 inhibitor candidate.
  • Microliter plates can contain 96 wells or 1536 wells for large scale screening of combinatorial library compounds. After the phosphorylation reaction has completed, the plates are washed leaving the bound substrate. The plates are then detected for phosphate group incorporation via autoradiography or antibody detection.
  • Candidate ZAP-70 inhibitors are identified by their ability to decrease the amount of ZAP-70 phosphotransferase ability upon a substrate in comparison with ZAP- 70 phosphotransferase ability alone.
  • the identification of potential ZAP-70 inhibitors may also be determined, for example, via in vitro competitive binding assays on the catalytic sites of ZAP-70 such as the ATP binding site and/or the substrate binding site.
  • a known protein kinase inhibitor with high affinity to the ATP binding site is used such as staurosporine.
  • Staurosporine is immobilized and may be fluorescently labeled, radiolabeled or in any manner that allows detection.
  • the labeled staurosporine is introduced to recombinantly expressed ZAP-70 protein or a fragment thereof along with potential ZAP-70 inhibitor candidates.
  • the candidate is tested for its ability to compete, in a concentration-dependant manner, with the immobilized staurosporine for binding to the ZAP-70 protein.
  • the amount of staurosporine bound ZAP-70 is inversely proportional to the affinity of the candidate inhibitor for ZAP-70. Potential inhibitors would decrease the quantifiable binding of staurosporine to ZAP-70. See e.g., Fabian et al (2005) Nat. Biotech., 23 :329. Candidates identified from this competitive binding assay for the ATP binding site for ZAP-70 would then be further screened for selectivity against other kinases for ZAP-70 specificity.
  • the identification of potential ZAP-70 inhibitors may also be determined, for example, by in cyto assays of ZAP-70 activity in the presence of the inhibitor candidate.
  • cyto assays of ZAP-70 activity Various cell lines and tissues may be used, including cells specifically engineered for this purpose.
  • cyto screening of inhibitor candidates may assay ZAP-70 activity by monitoring the downstream effects of ZAP-70 activity as well as other cellular responses such as growth, growth arrest, differentiation, or apoptosis.
  • ZAP-70-mediated phosphorylation of a downstream target of ZAP-70 can be observed in cell based assays by first treating various cell lines or tissues with ZAP-70 inhibitor candidates followed by lysis of the cells and detection of ZAP-70 mediated events.
  • Cell lines used in this experiment e.g., Jurkat T cell lines
  • ZAP-70 mediated events include, but are not limited to, ZAP-70 mediated phosphorylation of downstream ZAP-70 mediators.
  • phosphorylation of downstream ZAP-70 mediators can be detected using antibodies that specifically recognize the phosphorylated ZAP-70 mediator but not the unphosphorylated form. These antibodies have been described in the literature and have been extensively used in kinase screening campaigns.
  • CROs ZAP-70 kinase assay sen/ices, including DiscoverX, Inc, (San Diego, California), Reaction Biology Corporation (Malvern, Pennsy lvania), and Carna Biosciences (Tokyo, Japan).
  • the identification of potential ZAP-70 inhibitors may also be determined, for example, by in vivo assays involving the use of animal models, including transgenic animals that have been engineered to have specific defects or carry markers that can be used to measure the ability of a candidate substance to reach and/or affect different cells within the organism. For example, mice have been engineered with Syk-deficient B cells that overexpress ZAP-70, leading to complementation and survival.
  • the compounds of the invention are therefore potentially useful in the prevention or treatment of disorders or diseases where ZAP-70 inhibition plays a role, e.g. diseases or disorders mediated by T lymphocytes, or acute or chronic rejection of organ or tissue alio- or xenografts.
  • the compounds of the invention are potentially useful in the treatment and/or prevention of acute or chronic inflammatory diseases or disorders or autoimmune diseases e.g.
  • sarcoidosis fibroid lung, idiopathic interstitial pneumonia, obstructive airways disease, including conditions such as asthma, intrinsic asthma, extrinsic asthma, dust asthma, particularly chronic or inveterate asthma (for example, late asthma and airway hyper-responsiveness), bronchitis, including bronchial asthma, infantile asthma, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, nephrotic syndrome lupus, Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, type T diabetes meiiitus and complications associated therewith, type II adult onset diabetes mellitus, uveitis, nephrotic syndrome, steroid dependent and steroid-resistant nephrosis, palmoplantar pustulosis, allergic encephalomyelitis, glomerulonephritis, psoriasis, psoriatic arthritis, atopic e
  • necrotizing enterocohtis renal diseases including interstitial nephriiis, Goodpasture's syndrome hemolytic uremic syndrome and diabetic nephropathy, nervous diseases selected from multiple myositis, Guillain-Barre syndrome, Meniere's disease and radiculopathy, collagen disease including scleroderma, Wegener's granuloma and Sjogren' syndrome, chronic autoimmune liver diseases including autoimmune hepatitis, primary biliary cirrhosis and sclerosing cholangitis), partial liver resection, acute liver necrosis (e.g.
  • the compounds of the invention are potentially useful for treating T cell cancers and tumors of the blood and lymphatic system (e.g. Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS- related lymphomas, malignant immunoproliferative diseases, multiple myeloma and malignant plasma cell neoplasms, lymphoid leukemia, acute or chronic myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specified cell type, leukemia of unspecified cell type, other and unspecified malignant neoplasms of lymphoid, haematopoietic and related tissues, for example diffuse large cell lymphoma, T cell lymphoma or cutaneous T cell lymphoma).
  • Hodgkin's disease e.g. Hodgkin's disease, Non-Hodgkin's lymphoma, Burkitt's lymphom
  • Myeloid cancer includes e.g. acute or chronic myeloid leukaemia. Where a tumor, a tumor disease, a carcinoma or a cancer are mentioned, also metastasis in the original organ or tissue and/or in any other location are implied alternatively or in addition, whatever the location of the tumor and/or metastasis.
  • the required dosage will of course vary depending on the mode of administration, the particular condition to be treated and the effect desired. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.02 to 25 mg kg per body weight.
  • An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.2 mg to about 2 g, conveniently administered, for example, in divided doses up to four times a day or in retard form.
  • Suitable unit dosage forms for oral administration comprise from ca.0.1 to 500 mg active ingredient.
  • the compounds of the invention may be administered by any conventional route, in particular parenterally, for example in the form of injectable solutions or suspensions, enterally, e.g. orally, for example in the form of tablets or capsules, topically, e.g. in the form of lotions, gels, ointments or creams, or in a nasal or a suppository form.
  • Topical administration is e.g. to the skin.
  • a further form of topical administration is to the eye.
  • Pharmaceuticai compositions comprising a compound of the invention in association with at least one pharmaceutical acceptable carrier or diluent may be manufactured in conventional maimer by mixing with a pharmaceutically acceptable carrier or diluent.
  • the compounds of formula I may be administered in free form or in pharmaceutically acceptable salt form, e.g. as indicated above.
  • Such salts may be prepared in conventional manner and exhibit the same order of activity as the free compounds.
  • the present invention also provides:
  • a pharmaceutical composition e.g. for use in any of the indications herein before set forth, comprising a compound of Formulas (I)-(VI) or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable diluents or carriers therefor.
  • a method for the treatment of any of particular indication hereinbefore set forth, in a subject in need thereof which comprises administering to the subject an effective amount of a compound of Formula (l)-(VI) or a pharmaceutically acceptable salt thereof;
  • a compound of Formula (I)-(VI) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a disease or condition in which. ZAP-70 activation plays a role or is implicated; e.g. as discussed above.
  • the compounds of Formula (!-( VI) may be administered as the sole active ingredient or in conjunction with, e.g. as an adjuvant to, other drugs e.g. in immunosuppressive or immunomoduiating regimens or other anti -inflammatory agents, e.g. for the treatment or prevention of alio- or xenograft acute or chronic rejection or inflammatory or autoimmune disorders, a chemotherapeutic agent or an anti-infective agent, e.g.
  • an anti-viral agent such as e.g. an anti-retroviral agent or an antibiotic.
  • the compounds of formula I may be used in combination with a calcineurin inhibitor, e.g. cyclosporin A, ISA 247 or FK 506; an roTOR inhibitor, e.g. rapamycin, CC 1779, ABT578, bioliiniis-7, bioIimus-9, TAFA-93, AP23573, AP23464, or AP2384I; an ascomycin having immunosuppressive properties, e.g.
  • ABT-281, ASM981, etc. corticosteroids; cathepsin S inhibitors; cyclophosphamide; azathioprine; methotrexate; leflunomide; mizoribine; mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an immunosuppressive homologue, analogue or derivative thereof; a sphiiigosine-l-phosphate receptor agonist, e.g. FTY720 or an analog thereof, e.g Y-36018; monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD7, CDS, CD l la CD18, CD25, CD27, CD28, CD40.
  • a compound of Formula (I)-(V1) may also be used in combination with other antiproliferative agents.
  • antiproliferative agents include, but are not limited to:
  • aromatase inhibitors e.g. steroids, especially exemestane arid formestane and, in particular, nonsteroids, especially anunoglutethimide, vorozole, fadrozole, anastrozole and, very especially, letrozole;
  • antiestrogens e.g. tamoxifen, fulvestrant, raloxifene and raloxifene hydrochloride;
  • topoisomerase T inhibitors e.g. topotecan, irinotecan, 9-mtrocamptothecin and the macro molecular camptothecin conjugate PNU-166148 (compound Al in W099/17804);
  • topoisomerase I inhibitors e.g. the antracyclines doxorubicin (including liposomal formulation, e.g. CAELYXTm), epirabicin, idarubicin and nemorubicin, the anthraquinones mitoxantrone and Iosoxantrone, and the podophillotoxines etoposide and teniposide;
  • microtubule active agents e.g. the taxanes paclitaxel and docetaxel, the vinca alkaloids, e.g., vinblastine, especially vinblastine sulfate, vincristine especially vincristine sulfate, and vinorelbine, discodermolide and epothiiones, such as epothilone B and D;
  • vinca alkaloids e.g., vinblastine, especially vinblastine sulfate, vincristine especially vincristine sulfate, and vinorelbine
  • discodermolide and epothiiones such as epothilone B and D
  • alkylating agents e.g. cyclophosphamide, ifosfamide and melphalan;
  • COX-2 inhibitors e.g. celecoxib (Celebrex ⁇ ), rofecoxib (Vioxx®) and lumiracoxib (COX189);
  • antineoplastic anii metabolites e.g. 5-fiuorouracil, tegafur, capecitabine, cladribine, cytarabine, fludarabine phospliate, fluorouridine, gemcitabine, 6-mercaptopurine, hydroxyurea, methotrexate, edatrexate and salts of such compounds, and furthermore ZD 1694 (RALTITREXEDTM), LY23 15 14 (ALIMTATM), LY264618 (LOMOTREXOLTM) and OGT719;
  • platin compounds e.g. carboplatin, cis-platin and oxaliplatin;
  • VEGF Vascular Endothelial Growth Factor
  • EGF Vascular Endothelial Growth Factor
  • c-Src Epidermal Growth Factor
  • PDGF Platelet-derived Growth Factor
  • IGF-IR Insulin-like Growth Factor I Receptor
  • CDKs Cyciin-dependent kinases
  • gonadorelin agonists e.g. abareiix, goserelin and goserelin acetate
  • anti -androgens e.g. bicalutamide (CASODEXTM);
  • bisphosphonates e.g. etridonic acid, clodronic acid, tiludronic acid, pamidronic acid, alendronic acid, ibandronic acid, risedronic acid and zoledronic acid;
  • antiproliferative antibodies e.g. trastuzumab (HerceptinTM), Trastuzumab-DMl, erlotinib (TarcevaTm), bevacizumab (AvastinTm), rituximab (Rituxan®), PR064553 (anti-CD40) and 2C4 Antibody;
  • temozolornide (TEMODAL®).
  • TEMODAL® temozolornide
  • a method as defined above comprising co-administration, e.g., concomitantly or in sequence, of a therapeutically effective amount of (a) a compound of Formula (T)-(VI) or acceptable salt thereof, and b) a second drag substance, said second drug substance being, for example, for use in any of the particular indications hereinbefore set forth.
  • a combination comprising a therapeutically effective amount of a ZAP-70 kinase inhibitor, e.g. a compound of Formula (I ' )-(VI) or a pharmaceutically acceptable salt thereof, and a second dmg substance, said second drug substance being for example as disclosed above.
  • a ZAP-70 kinase inhibitor e.g. a compound of Formula (I)-(VT)
  • other immunosuppressive i munomodulatoiy, anti-inflammatory or antineoplastic agent
  • dosages of the coadministered drag or agent will of course vary depending on the type of co-drug or agent employed, or the specific drag or agent used, or the condition being treated and so forth.
  • methods and systems for synthesis of compounds and compositions of the present invention are in vitro cell-free biosynthesis (CFB) systems that serve as a platform to produce proteins and small molecule metabolites using the the cells enzymes and metabolic machinery without the living cell (See: Hodginan, C.E., Jewett, M. C, Metab. Eng., 2012, 14(3), 261-269).
  • CFB cell-free biosynthesis
  • Cell-free biosynthesis systems provided herein have numerous applications for drug discovery by allowing rapid expression of natural biosynthetic genes and pathways and by allowing activity screening without the need for plasmid based cloning and in vivo propagation, thus enabling rapid process/product pipelines (creation of small molecule libraries).
  • a key feature of the CFB methods and systems used herein is that biosynthesis pathway flux to a target compound can be optimized by directing resources to user defined objectives and consequently allows for the exploration of a large sequence space. Central metabolism, oxidative phosphorylation, and protein synthesis can be co-activated by the user. The lack of a cell wall also provides for the ability to easily screen toxic metabolites, proteins, and small molecules. Cell-free biosynthesis methods involving in vitro transcription translation (TX-TL) have been used to produce ( 1) proteins (See, for example: Carlson, E.D., et al wider Biotechnol. Adv., 2012, 30(5),1185- 1194; Swartz, I, et al., US Patent No.
  • the CFB methods and sy stems can be used to rapidly prototype novel complex biocircuits as well as metabolic pathways. Protein expression from multiple DNA pieces, including linear and plasmid based DNA, can be performed.
  • the CFB methods and systems enable modulating concentrations of DNA encoding individual pathway enzymes and testing the related effect on metabolite production.
  • the ability to express multi-enzyme pathways using linear DNA in the CFB methods and systems bypasses the need for in vivo selection and propagation of plasmids.
  • Linear DNA fragments can be assembled in 1 to 3 hours (hrs) via isothermal or Golden Gate assembly techniques and be immediately used for a CFB reaction. The CFB reaction can take place in several hours, e.g.
  • linear DN A provides a valuable platform for rapid prototyping libraries of DNA genes.
  • mechanisms of regulation and transcription exogenous to E.coii such as the tet repressor and T7 NA polymerase, or other host cell extracts, can be supplemented as defined by the user to generate and maximize endogenous properties, diversity or production.
  • the CFB methods and systems further enhance diversity and production of target compounds by modifying endogenous properties including mR A and DNA degradation rates.
  • ATP regeneration sy stems that allow for the recycling of inorganic phosphate, a strong inhibitor of protein synthesis, are manipulated in the CFB methods and systems.
  • Redox potential including e.g., NAD/NADH, NADP/NADPH
  • CFB Redox potential
  • methods for modifying redox and availability of specific cofactors which in turn enables the user to selectively modulate any reaction in the CFB system.
  • CFB methods and systems enable in vitro cell-free transcription/translation systems (TX-TL) and function as rapid prototyping platforms for the synthesis, modification and identification of products, e.g., natural products (NPs) or natural product analogs (NPAs), from biosynthetic pathway genes.
  • NPs natural products
  • NPAs natural product analogs
  • CFB systems are used for the combinatorial biosynthesis of natural products and natural product analogs, such as those provided in the present invention
  • CFB systems are used for the rapid prototyping of complex biosynthetic pathways as a way to rapidly assess combinatorial designs for the synthesis of compounds of Formulas (l)-(VI).
  • these CFB systems are multiplexed for high-throughput automation for rapid prototyping of natural product pathway genes, the natural products they encode and synthesize, and natural product analogs, such as the compounds of Formulas (1)-(V1) provided in the present invention.
  • the CFB methods and systems are described in Culler, S. et al, PCT Application WO2017/031399 A l , and is incorporated herein by reference.
  • the CFB compositions, methods, and systems can be used to rapidly produce analogs of known compounds, for example natural product analogs and secondary metabolic structural analogs, such as compounds of Formulas (I)-(VI).
  • Representative biosynthetic enzymes described in this application include, for example, amino acid sequence identifiers (SEQ ID NOs) for VioA (SEQ ID NO: 1), VioB (SEQ ID NO: 2), RebC (SEQ ID NO: 3 ), RebP (SEQ ID NO: 4), StaP (SEQ ID NO: 5), Sta C (SEQ ID NO: 6), RebO (SEQ ID NO: 7), RebD (SEQ ID NO: 8), RebG (SEQ ID NO: 9), StaO (SEQ ID NO: 10), StaD (SEQ ID NO: 11), StaG (SEQ ID NO: 12), and StaN (SEQ ID NO: 13).
  • SEQ ID NOs amino acid sequence identifiers for VioA (SEQ ID NO: 1), VioB (SEQ
  • methods provided herein include a cell-free (in vitro) biosynthesis (CFB) method for making, synthesizing or altering the structure of compounds of Formulas (l)-(Vl).
  • CFB methods can produce in the TX-TL- extract or extract mixture at least two or more of the altered compounds to create a library of altered compounds; preferably the library is a natural product analog library, prepared, synthesized or modified by the CFB method.
  • practicing the invention comprises use of any conventional technique commonly used in molecular biology, microbiology, and recombinant DNA, which are within the skill of the art.
  • Such techniques are known to those of skill in the art and are described in numerous texts and reference works (See e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual,” Second Edition, Cold Spring Harbor, 1989; and Ausubel et al., "Current Protocols in Molecular Biology," 1987).
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.
  • compounds of Formula (III) may be prepared by reacting indoie-3-acetamide derivatives with methyl indole-3-glyoxylates in the presence of potassium tert-butoxide in THF solvent, as shown in Scheme 2 and as reported in the literature (See, for example: Paul, et al., Tetrahedron Lett., 1999, 40, 1109-1112; Paul et al., J. Org. Chem. 1998, 63, 6053-6058; Faul et al., J. Org. Chem. 1999, 64, 2465-2470).
  • Indole-3-acetamide and indole-3-glyoxylate derivatives are readily prepared from a wide range of available substituted indoles. Ring closure of the initially formed bisiiidoiomaieimide derivatives affords the indolo[2,3- a]carbazoles represented by Formula (I), which can be accomplished with a variety oxidants [O], including Pd(OAc)2, PdC12, hv/02 or 12, DDQ, CuC12, or Pd(OTf)2 (See, for example: Faul et al., J. Org. Chem. 1999, 64, 2465-2470).
  • the inside functionality can be reduced to the lactam functionality using standard reagents such as sodium boro ydride or zinc amalgam.
  • Q and/or R groups can be added by standard methods through reactions with the indole N-I-I functionality (e.g., through alkylation or acylation reactions).
  • compounds of Formula (Hi) may be prepared directly by reacting tryptophan derivatives in a process involving cell-free biosynthesis (CFB) as shown in Scheme 4.
  • CFB cell-free biosynthesis
  • enzymes are used to condense two tryptophan molecules or tryptophan derivatives to directly produce compounds of Formula (I) where Q and R are hydrogen.
  • the enzymes required for these transformations have been elucidated and enzymes from various pathways can be used to generate indolocarbazoie derivatives.
  • Certain enzymes are known to catalyze transformations and facilitate pathways to produce natural indolocarbazoie.
  • VioA amino oxidase
  • VioB chromopyrrolic acid synthase
  • StaO amino oxidase
  • StaD chromopyrrolic acid synthase
  • StaP cytochrome P450 nionooxygenase
  • StaC flavin hydroxylase
  • RebO amino oxidase
  • RebD chromopyrrolic acid synthase
  • RebP cytochrome P450 nionooxygenase
  • RebC flavin hydroxylase
  • the enzymes VioA and Vio B of the vioiacein pathway, StaO, StaD, StaP, and StaC of the staurosporine pathway, and/or RebO, RebD, RebP, and RebC of the rebeccamycin pathway are used in a ceil-free biosy nthesis process to produce compounds of Formula (III) by combining and transforming two of the same or different substituted tryptophan derivatives, as outlined in Scheme 4.
  • Staurosporine pathway enzymes (StaO, StaD, StaP, and StaC), for example, can be used to directly produce compounds of Formula (III) wherein Q, R, Y and Z are hy drogen.
  • compounds of Formula (III), wherein Q and R are hydrogen may be transformed in a cell-free biosynthesis process, or a cell-based biosynthesis process, to introduce a glycose group onto one indole nitrogen atom (Q or R of Formula (III) is a glycose unit) or alternatively a glycose group that bridges both, indole nitrogen atoms (Q and R of Formuia (III) together form the glycose, as in Formula (II)), as shown in Scheme 5.
  • Enzymes used to introduce glycose groups can be, for example, StaG (N-glycosyltransferase) and StaN (cytochrome P450 nionooxygenase) of the staurosporine pathway, or RebG (N-glycosyltransferase) of the rebeccamycin pathway, which can introduce a variety of sugar-based glycose groups that are activated as thymidine diphosphate (TOP) or uridine diphosphate (See, for example, Sanchez et al., Chem. Comn.ua, 2009, 4118— 4120).
  • TOP thymidine diphosphate
  • uridine diphosphate See, for example, Sanchez et al., Chem. Comn.ua, 2009, 4118— 4120.
  • StaG or RebG are used alone to introduce a gly cose group onto one indole nitrogen atom of Formula (III), or they are used in combination with StaN to introduce a glycose group that bridges the two indole nitrogen atoms of Formula (III), as in Formula (II).
  • Certaiir glycose groups also can be introduced chemically into compounds of Formula (III), wherein Q and R are hydrogen (See, for example, Wood et al., J. Am. Chem. Soc. 1997, 119, 9641-9651; Wood, J.L., et al., J. Am. Chem. Soc, 1997, 1 19, 9652-9661).
  • Glycose groups often have functional groups such as -OH, -NH2, and -C02R, which can be used to introduce a linker (L) and reactive functional group (W) group that is connected to either a leaving group (LG) or a nucleophilic group (Nu).
  • L linker
  • W reactive functional group
  • LG leaving group
  • Nu nucleophilic group
  • a linker and a reactive functional group is introduced by standard chemical methods through a nucleophilic group such as an amino group, as depicted in Scheme 6, which, produces an amide compound of Formula (V).
  • a linker and a reactive functional group is introduced by standard chemical methods through a nucleophilic group such as a hydroxy! group, as depicted in Scheme 7, which produces an ester compound of Formula (V).
  • Analytical I-IPLC was perfomed using an Agilent 1 100 instnsment with a variable wavelength detector.
  • LC/MS was performed on an Applied Biosy stems 3200 APCI triple quadrupole mass spectrometer with alternating positive and negative ion scans.
  • High resolution mass spectrometry was performed using a Thermo Fisher Q Exactive MS instrument.
  • GC-MS was performed using an Agilent 6890N instrument equipped with a 5973N inert mass selective detector.
  • Ion chromatography was performed using a Metrohni 940 Professional IC Vario instrument. 1H NMR was performed on a Jeol JNM- ECS-400 at 400 MHz or a Broker DRX-600 at 600 MHz.
  • Microwave reactions were performed in a Biotage Initiator using the instrument software to control heating time and pressure. Hydrogenation reactions were performed on an H-Cube using the commercially available catalyst cartridges. Silica gel chromatography was performed either manually using standard columns or using pre-packed Sep-Pak silica cartridges from Waters. Preparative HPLC was performed on a Waters 1525/2487 with UV detection at 220 run and manual collection.
  • Solvent abbreviations include dichloromethane (DCM), ethyl acetate (EtOAc), acetonitrile (ACN), diisopropylethylamine (DIEA), trifiuoroacetie acid (TFA), dimethylformamide (DMF), tetrahydrofuran (THF), [0365] Analytical LC/MS Method A:
  • Step 1 Synthesis of l-N-Boc-3-N'-acryloyl-l,3-propanecUamine
  • Step 2 Synthesis of N-acryloyl-l,3-propanediamine-hydrochloride salt
  • Step 3 Synthesis of (9a, 10b,12a)-2,3,9,10,l l , 12-hexahydro-10-hydroxy
  • Product Rf - 0.5 (10% MeOH/ CH2C12). Each fraction was checked by UV and HPLC (100% acetonitrile, CIS column). The fractions containing product in high purity were collected and evaporated to dryness to obtain the product as a waxy solid (5 mg, 22%).
  • N-acryloyl intermediate N-(3-hydroxypropyl)prop-2-enamide was purchased from Enamine Ltd. (Monmouth Junction, NJ). Othenvise, the procedure described in Step 3 of Example 1 was employed.
  • Step 1 Synthesis of N-[2 -(hydroxy methyl)phenyl]prop-2-enamide
  • Step 2 Synthesis of N-[2-(bromomethyl)phenyl]prop-2-enamide
  • Step 3 Synthesis of (9a,10b,12a) ⁇ 2,3,9, !0,l l,12-hexahydro ⁇ !0 ⁇ hyd ⁇
  • Step 1 Synthesis of N-[2-(2-hydroxyethyl)phenyl]prop-2-enamide
  • Step 2 Synthesis of (9a, 10b,12a)-2,3,9,10,l 1, 12-hexahydro-10-hydroxy-10-(2-N-acryioyl-2-amino-l- ethyloxyphenylcarbonyl)-9-methyl-9, 12-ep
  • the reaction was diluted with DCM (50 mL), transferred to a separatory funnel, and washed successively with 5% HCI (5 mL), IM NaHC03 (5 mL), water (2x 5 mL), then dried (Na2S04), filtered, and concentrated on a rotovap.
  • the liquid residue was purified two on a silica column eluted with DCM/MeOH (0 to 10% MeOH gradient). The fractions were monitored by HPLC ( 100% ACN, 290 nm). The samples with product were combined and evaporated to afford a colorless viscous oil (0.005 g, 20%).
  • Step 1 Synthesis of N-[2-(3-hydroxypropyl)phenyl]prop-2-enamide
  • Step 2 Synthesis of 3-[2-(prop-2-enamido)phenyi]propyl methanesulfonate
  • Step 3 Synthesis of (9a,10b,12a)-2,3,9,10,n,12-hexahydro-10-hydrox ⁇ --10-(2-N-acryloyl-2-aniino-l- pro ylox5 ⁇ he y ⁇ carbonyl)-9-met yl-9,12-epox - lH-diindolo[l ,2,3-fg:3 2 ⁇ -ki] y ⁇ "oio 3,4- i][ l ,6]benzodiazocin-l-one (52)
  • Codon-optimized DNA encoding the sequences for the proteins VioA and VioB from Chromobacterium violaceum, RebC and RebP from Lechevalieria aerocolonigenes, and StaC and StaP from Streptomyces Longisporoflavus DSM 10189 were synthesized (Thermo Fisher, Carlsbad, CA) and individually cloned into a pZE expression vector behind a T7 promoter (Expressys). The resulting plasmids encoding genes for VioA, VioB, RebC, RebP, SlaC and StaP proteins were used with or without a C- teiminal strep tag.
  • Biosynthetic enzymes were selected from a list of VioA, VioB, RebC, and RebP, with SEQ ID NO:s 1 -4, or homologous enzymes, representative examples of which are provided in Table 2. Enzymes were produced and isolated by synthesizing individual codon-optimized genes bearing sequences for C-terminal strep-tags and cloning into the pZE expression vector (Expressys). Following transformation of the plasmids into E. coli NEB5alpha (New England BioLabs, Ipswich, MA), the strains were cultivated in 5 L aerated fermenters and the cell mass was isolated by centrifugation and lyzed using a cell homogenizer. Individual enzymes were purified using Strep-Tactin resins (Strep-Tactin ⁇ Superflow® high capacity cartridge, IBA Lifesciences) following the manufacturer's instructions.
  • the biosynthesis reaction was performed as follows: To 4 mL Tris buffer (0.1 mM, pH 8.0) was added purified enzymes VioA (3 ⁇ ), VioB (3 ⁇ ), RebP (3 ⁇ ), and RebC (1.5 ⁇ ). The reaction was initiated by adding 5-fluorotryptophan (lmM), along with 5 mM NADH, 1 ⁇ Ferredoxin-NADP ⁇ Reductase from Spinacia oieracea (Sigma), and 20 ⁇ . ⁇ Spinach ferrodoxin, and the mixture was incubated at 25 OC for 24 hours. Upon completion, the reaction was treated with MeOH (4 mL) and centrifuged to remove precipitated protein.
  • Two different assays for measuring kinase inhibitor activity were employed for assessing the effectiveness of compounds of the present invention for inhibiting ZAP-70 and Syk kinases.
  • One assay was a competition-based assay, commercialized by Eurofins DiscoverX and known as KINOMEscanTM, wherein binding affinity (Kd) was measured by displacement of an immobilized inhibitor of known affinity (e.g., staurosporine) and quantify ing the amount of kinase remaining attached to the support following treatment with the inhibitor of interest.
  • the second assay was a standard biochemical assay employing radiolabeled ATP, in which inhibitory concentration of 50% of kinase activity (iC 50 ) was measured by quantifying the amount of radiolabel incorporation into a standard peptide substrate.
  • Inhibitor binding constants were measured by using active site-dependent competition binding assays essentially as described in Karaman, M.W., et al., Nat. BiotechnoL 2008, 26, 127-132).
  • ZAP-70 and Syk kinases were labeled with a chimeric double-stranded DNA tag containing the NF-kB binding site (50 - GGGAATTCCC-30 ) fused to an ampiicon for qPCR readout, which then were cloned in a modified version of the commercially available T7 Select 10 vector and strain (Novagen).
  • DNA-tagged kinase-T7 phage clones were grown in parallel in 24- or 96-well blocks in an E.
  • E. coli was grown to log phase and infected with T7 phage from a frozen stock (multiplicity of infection ca. 0.1) and incubated with shaking at 32 oC until lysis (ca. 90 min). The lysates were centrifuged (6,000g) and filtered (0.2 mm) to remove cell debris. Extracts were used directly in binding assays without any enzyme purification steps at a >10,000-fold overall stock dilution (final DNA-tagged enzyme concentration ⁇ 0.1 nM).
  • Streptavidin-coated magnetic beads were treated with biotinylated staurosporine for 30 min at 25 oC to generate affinity resins for kinase assays.
  • the remaining streptavidin sites of the liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% T ween 20, 1 mM DTT) to remove unbound ligand and to reduce nonspecific phage binding.
  • Test compounds (compounds of the invention and positive controls) were prepared as 1 ,000 x stock solutions in DMSO and rapidly diluted into the aqueous environment (0.1 % DMSO final). DMSO (0, 1%) was added to control assays lacking a test compound. Ail reactions were earned out in polyst ene 96-weil plates that had been pretreated with blocking buffer in a final volume of 0.1 ml.
  • Binding reactions were assembled by combining tagged-kinase extracts, liganded affinity beads and test compounds prepared as 100 x stocks in DMSO. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in 384-weli piates, whereas Kd determinations were performed in 96-well piates. Assay plates were incubated in 1 x binding buffer (20% SeaBlock, 0. 17 x PBS, 0.05% Tween 20, 6 mM DTT) at 25 °C with shaking for 1 h, which was sufficient to establish equilibrium. The affinity beads were washed four times with wash buffer (1 x PBS, 0.05% Tween 20, 1 mM DTT) to remove unbound phage/protein.
  • 1 x binding buffer (20% SeaBlock, 0. 17 x PBS, 0.05% Tween 20, 6 mM DTT
  • the beads were resuspended in elution buffer (1 x PBS, 0.05% Tween 20, 2 mM nonbiotin lated affinity ligand) and incubated at 25 oC with shaking for 30 min in order to elute bound kinase.
  • the phage titer and kinase concentration in the eluaies was measured by standard plaque assays and quantitative PGR, respectively.
  • Kds were determined using 11 serial threefold dilutions of test compound and a DMSO control. For each assay the affinity probe concentrations were optimized to ensure that true thermodynamic inhibitor Kd values were measured. Binding constants were calculated based on phage concentration in the eluates as described in Fabian, M.A., et al., Nat. BiotechnoL, 2005, 23, 329-336.
  • KTNOMEscanTM is based on a competition binding assay that quantitatively measures the ability of a compound to compete with an immobilized, active-site directed ligand.
  • the assay is performed by combining three components: DNA-tagged kinase; immobilized ligand; and a test compound. The ability of the test compound to compete with the immobilized ligand is measured via quantitative PGR of the DNA tag.
  • kinase-tagged T7 phage strains were prepared in an E. coli host derived from the BL2 I strain. E. coli were grown to log-phase and infected with T7 phage and incubated with shaking at 32°C until lysis. The ly sates were centrifuged and filtered to remove ceil debris. StreptavidLin-coated magnetic beads were treated with biotinylated small molecule ligands for 30 minutes at room temperature to generate affinity resins for kinase assays.
  • the iiganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinases, iiganded affinity beads, and test compounds in Ix binding buffer (20% SeaBlock, 0.17x PBS, 0.05%) Tween 20, 6 mM DTT). Test compounds were prepared as 11 IX stocks in 100% DMSO. Kds were determined using an ! .1 -point 3-fold compound dilution series with three DMSO control points. All compounds for Kd measurements are distributed by acoustic transfer (non-contact dispensing.
  • Binding constants were calculated with a standard dose-response curve using the Hill equation:
  • compounds of the invention exhibit Kd values in the range of 0.1 nM to 2 ⁇ I . preferably in the range 0.1 nM to 10 nM.
  • Compound 2 of Example 1 exhibits a Kd for ZAP-70 of 3.9 nM.
  • Positive control inhibitor staurosporine has a Kd of 44 nM vs ZAP-70 using this assay.
  • compounds of this invention exhibit Kd in the range of 10 nM to 10 uM, preferably in the range 100 nM to 10 uM.
  • Compound 2 of Example 1 exhibits a Kd for Syk of 13 nM.
  • Compound of E9 of Example 9 exhibits a Kd for Syk of >10 uM.
  • Positive control inhibitor staurosporine has a Kd of 14 nM vs Syk using this assay.
  • each of the Compounds 2, 3, 10, 50, 51 and 52 was dissolved in 100% dimethyl sulfoxide (DMSO) to y ield 10 niM stock.
  • DMSO dimethyl sulfoxide
  • the binding affinityof each compound against ZAP-70 tyrosine kinase was measured with She KINOMEsean TM tecSmology, a competition binding kinase assay developed by Eurofins DiscoverX (Fremont, CA). Each assay was run in triplicate.
  • Figure 4 shows a plot of the amount of kinase measured by qPCR (Signal; y-axis) against the corresponding compound concentration in nM in log 10 scale (x-axis), which was used to calculate the Kd of 3,9 nM for Compound 2.
  • Table 3 lists the Kd values for Compounds 2, 3, 10, 50, 51, and 52.
  • the basic biochemical assay employs radiolabeled ATP to measure the kinase-catalyzed transfer of radioactive phosphorus to a tyrosine-containing peptide substrate, according to the general equation:
  • the reaction was then terminated by the addition of one-third volume of stop reagent (0,25 mM EDTA and 33 mM ATP in dH20). A 15 mL aliquot was removed, spotted onto a P-81 filtermat ion exchange paper and washed sequentially with 10% (w/v) chloroacetic acid and dH20 to remove ATP.
  • the bound 33P-polyGlu-Tyr was quantified by scintillation counting and the disintegrations per minute (dpm) obtained, being directly proportional to the amount of 33P- polyGIu-Tyr produced by ZAP 70, were used to determine the IC» fo each compound.
  • compounds of this invention exhibit ICjo in the range of 10 nM to 10 uM, preferably in the range 100 nmM to 10 ⁇ .
  • Compound 3 exhibits an 1C 50 for Syk of 16 nM.
  • Compound E10 exhibits an IC 30 of >10 ⁇ .
  • Positive control inhibitor staurosporine has an IC 50 of 0.2 nM vs Syk using this assay .
  • compounds of the invention exhibit IC JO values in the range of 0.1 nM to 2 ⁇ , preferably in the range 0.1 nM to 10 nM.
  • Compound 3 of Example 2 exhibits an IC st> for ZAP-70 of 130 nM.
  • Positive control inhibitors staurosporine had an IC 50 of 14 nM vs ZAP-70 using this assay, while K252a exhibited an IC 50 of 208 nM.
  • the Jurkat cells were washed once with PBS, lysed with 50 ⁇ of IX cell lysis buffer (20 mM Tris-HCl (pH7.5), 150 mM NaCl, 1 n Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ⁇ -glycerophosphate, 1 mM Na3 V04, 1 ug/ml leupeptin, and 1 mM PMSF).
  • the lysate samples were centrifuged at 12000 rpm for 10 minutes. The supernatants were transferred to a new set of eppendorf tubes. 4X LDS sample buffer was added to the cell r sates.
  • the cell lysate samples were subjected to SDS-PAGE with 12% Bis-Tris gel and transferred onto nitrocellulose membrane by iBiot dry blotting system.
  • the membrane was blocked with 3% BSA for 1 hour and probed with anti-phospho-SLP-76(Ser376), and anti-SLP-76 antibody, respectively.
  • Anti-rabbit TgG !RDye 680RD and anti-rabbit IgG IRDye 800CW secondary antibodies were used to detect ihe primary antibodies.
  • the membranes were scanned with Ll-COR Odyssey Fc Imaging System. The specific bands of interest were quantified by Ll-COR Image Studio Lite software.
  • the ICJO curves were plotted and IC 50 values were calculated using the GraphPad Prism 4 program based on a sigmoidal dose-response equation.
  • SLP-76 also known as lymphocyte eytosolic protein 2 (I.CP2), is an immediate downstream substrate of the ZAP-70 protein tyrosine kinase following T ceil receptor (TCR) ligation in the leukemic T cell line Jurkat.
  • TCR T ceil receptor
  • BALB/c mice are purchased from Taconic or Hilltop Lab Animals.
  • DBA/lJbomTac mice are obtained from Taconic. Animals are maintained in accordance with the Guide for the Care and Use of Laboratory Animals. Ail study protocols are approved by the Institutional Animal Care and Use Committee.
  • ZAP-70 inhibitors of the invention are prepared for in vivo oral administration in vehicle (phosphate-buffered saline: ThermoFisher Scientific Inc., Waltham, MA) containing 0.5% methylcellulose/0.025% Tween 20 (Sigma-Aldrich, St. Louis, MO),
  • Bovine type II collagen (Chondrex) is dissolved in 0,0 IN acetic acid and emulsified in an equal volume of complete Freud's adjuvant containing 1 mg mL of heat-kilted Mycobacterium tuberculosis (Sigma). Arthritis is induced in IQ-week-okl male DBA/lJBomTac mice by the initial immunization with 200 mg/100 mL emulsion by an intradermal injection in the base of the tail. A boost 21 d later with an aqueous solution of 200 mg/ 100 mL CIT is administered intraperitoneally .
  • mice are scored weekly, beginning 3 wk after primary Cll immunization, for signs of developing arthritis. The severity of the arthritis is assessed using a visual scoring system. Each paw is scored on a graded scale from 0 to 3 : 0, normal paw; 1, swelling and/or redness of one toe or finger joint; 2, swelling of two or more toes or joints, or increased swelling; 3, severe swelling and/or ankylosis throughout the entire paw. Each paw was graded and the four scores were added such that the maximal score per mouse is 12.
  • mice Treatment is initiated when >10% of mice demonstrated clinical signs of disease.
  • blood is collected for anti-collagen II EL1SA testing and paws are collected for histopathoiogy.
  • Anti-Type II Collagen Antibodv ELISA Anti-Type II Collagen Antibodv ELISA.
  • IgG antibody levels against the immunogen are measured by standard ELISA methodology using peroxidase-conjugated secondary antibody and substrate ABTS. Serum dilutions, 1/1,000, are chosen after preliminary assays. The optical density is measured at 405 run using a Spectramax Plus 384 plate reader (Molecular Devices Corporation), The anti-type II collagen concentrations are determined by reference to standard curves of murine IgG, IgGl, IgG2a, or IgG2b (Southern Biotechnology Associates, Inc.).
  • Histological Techniques For histological processing, paws are fixed in phosphate buffer containing 10% formaldehyde and decalcified in sodium citrate. Paws are processed by routine methods to paraffin blocks. Specimens are sectioned at 6 mm and stained with hematoxylin and eosin according to the manufacturer's protocol ( Sigma- Aldrich). The sections are evaluated for the degree of synovial hyperplasia, inflammatory cell infiltrate, cartilage damage, pannus formation, bone erosion, fibrillation, and ankylosis. The severity of the disease in the joint sections is graded using a scoring system from 0 to 5: 0, within normal limits; 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe.
  • Histopathologic scores are assigned as follows: as follows: as follows: grade 0 - no abnormal findings; grade 1 - synoviocyte hypertrophy, slight synovial membrane fibrosis, slight-to-mild inflammatory cell infiltrates into the synovial membrane/articular capsule and/or joint space; grade 2 - grade 1 plus mild-to-moderate inflammatory cell infiltrates, pannus formation (if present) minimal with superficial cartilage erosion; grade 3 - grade 2 plus marked inflammatory cell infiltrates and fibrosis, mild-to-severe erosion of the cartilage extending into subchondral bone: and grade 4 - loss of joint integrity through erosion or destruction with bone remodeling, massive inflammatory cell infiltrates, fibrosis, and ankylosis.
  • the arthritis severity score for each paw is weighted based on the number of joints per paw receiving a specific score. Each pa is graded and the score for four paws are added such that the maximal score per mouse was 20.
  • mice Male BALB/c and BALB/cBy mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All mice are housed in a specific pathogen-free environment and maintained in accordance with the Guide for the Care and Use of Laboratory. Animals are used between 6 and 8 weeks of age. AH protocols are approved by the Institutional Animal Care and Use Committee. Reagents
  • Recombinant mouse IL-23 is purchased from eBiosciences, Anti-mouse TL-12/23 p40 (clone C I 7.8), and isotype control antibody (rat IgG2a), are purchased from BioLegend (San Diego, C A).
  • mice are each injected intra- deraially every other day with 150 ng of mouse recombinant IL-23 (eBiosciences) or PBS in a total volume of 25 ⁇ . Ear swelling is measured in triplicate using a micrometer (Mitutoyo) right before each IL-23 challenge.
  • mice are euthanized and ears are collected for H&E staining, pSTAT3 iroinunohistocheroical staining, and evaluated for cytokine gene expression.
  • This dosing regimen was optimized to achieve robust skin inflammation in mice. Control mice are treated similarly with a control vehicle cream (Vaseline Lanette cream; Fagron). At the days indicated, the ear thickness is measured in triplicate using a micrometer (Mitutoyo).
  • mice are administered 16 mg kg of either anti ⁇ p40 Ab twice per week i.p. or 3-30 mg/kg of ZAP-70 inhibitor/vehicle twice daily (BID) by oral gavage for the duration of the study. Mice are monitored for external signs of skin lesions twice per week. At termination of the study, mouse ear, back skin, lymph nodes, and spleen are collected for further ex-vivo studies. Macroscopic evaluation: Mouse ear thickness is monitored daily in the IL-23 intra-dermal injection and imiquimod models using a micrometer (Mitutoyo).
  • 0 no skin or ear abnormalities
  • 0.5 slight erythema on either the ears or eyelids
  • 1 mild to moderate erythema on the ears or ey elids, with mild thickening of the ear ( ⁇ 2% of the body surface)
  • 2 moderate to severe erythema on 2-10% of the body surface, mild scaling
  • 3 severe erythema and scaling on 10-20% of the body surface
  • 4 very severe and extensive erythema, and scaling on 20-40% of the body surface.
  • 5 very severe and extensive erythema, and scaling on 40-60% of the body surface.
  • 6 very severe and extensive erythema, and scaling on greater than 60% of the body surface.
  • Tissues are processed into paraffn tissue blocks using routine methods, sectioned, or serially sectioned to obtain consecutive levels.
  • the sections are stained with hematoxylin and eosin (H&E).
  • H&E hematoxylin and eosin
  • paraffn t ssue sections are stained with antibodies specific for pSTAT3 or isotype control.
  • Skin cytokines are extracted from pulverized frozen mouse ears with T-Per tissue extraction buffer supplemented with protease inhibitors (Thermo Scientific, Waltham, MA). Specific immunoassays are used to determine tissue levels of IL-6 (MSD, Roekvpository, MD), IL-17.A (Miilipore, Billerica, MA), IL-22 and IL-23 (R&D Systems, Minneapolis, N). Data are expressed as picograms of cytokine per milligram of total protem in the extract.
  • RNA is isolated from the mouse ears using the Qiagen Rneasy kit (Qiagen, Valencia, CA). Quantitative RT-PCR for various transcripts is performed using pre-qualified primers and probes to IL- l a, TL- 1B, 1L-7, IL-22, IL-17A, 1L-17F, IL-6, IL-23pl9, IFNy, CXCL-10, S 100A8, IL-21R, IL-22R, IL-12Ra, IL- 12RB (Applied Biosystems, Foster City, CA ). The ACt method is used to normalize transcripts to GAPDH.
  • Example 36 Treatment of Multiple Sclerosis by Administration of a ZAP -70 Inhibitor Compound Disclosed Herein in an Animal Model
  • mice Female C57BL/6ByJ and SJLB J mice are purchased from The Jackson Laboratory (Bar Harbor, ME). All mice are housed in a specific pathogen-free environment and maintained in accordance with the Guide for the Care and Use of Laboratory. Animals are used between 8 and 12 weeks of age. Ail protocols are approved by the Institutional Animal Care and Use Committee.
  • ZAP-70 inhibitors of the invention are prepared for in vivo oral administratio in vehicle (phosphate-buffered saline: Thermo Fisher Scientific inc., Waltham, MA) containing 0.5% methyicellulose/0.025% Tween 20 (Sigma-Aldrich, St. Louis, MO).
  • EAE is induced by immunizing C57BL/6ByJ and SJLByJ mice s.c. with 200 of myelin oligodendrocyte glycoprotein (MOG) peptide 35-55 or MOG1-121 peptide emulsified in complete Freund's adjuvant (Chondrex,Redmond, WA) containing 4 mg/ml My cobacterium tuberculosis.
  • MOG myelin oligodendrocyte glycoprotein
  • mice are anesthetized and perfused intracardially with Ringer's solution and 4% paraformaldehyde (PFA).
  • PFA paraformaldehyde
  • the lumbar part of spinal cords are dissected and fixed in 4% PFA overnight at 4°C, followed by incubation in 30% sucrose for 24 h.
  • Cryosections (14-um thick) are stained with hematoxylin & eosin (H&E) or Luxol fast blue (LFB). Quantification is performed as described below.
  • CNS lymphocyte populations from the spleen and peripheral blood is performed by flow cytometry analysis of single cell suspensions. Isolation of resident CNS (brain and spinal cord) lymphocytes is performed using perfused animal CNS tisue that is digested with collagenase and DNAse followed by mononuclear cell separation by Percoli density gradient centrifugation Sigma- Aldrich; Si. Louis, MO). Mononuclear cells are collected and used for flow cytometry analysis.
  • Staimng of immune cells is performed by incubating 1 ⁇ 106 cells with fluorescently -labeled antibodies against mouse cell surface markers including CD4 (RM4-5), CDS 53-6.7) CD44 (1M7), CD62L (Mel- 14), CD25 (PC61.5), FR4 (12A5), B220 (RA3-6B2), N 1.1 (PK 136), and, GR-1 (RB6.8C5) purchased from Ebioscience (San Diego, CA) or BD Bioscience (San Jose, CA). Peripheral blood is evaluated by staining 50 ⁇ of whole blood with the indicated antibodies followed by removal of contaminating red blood cells with FACs lysis buffer (BD Bioscience) as described by the manufacturer. Fluorescence intensities are determined using an LSR-IT flow cytometer (BD Bioscience) and data analysis is performed using Fiowjo software (Treestar Inc., Ashland, OR).
  • Percent control for ail tissues is calculated by dividing the number of total cells for each population by the mean ioial number of the same cellular population from the vehicle-treated control group [total cells in tesi animal/mean of total cells in control group 3 ⁇ 4 100],
  • Intracellular cytokine staining is performed by culturing splenocytes in vitro with 1 ⁇ ' ⁇ of MOG35-55 peptide for 5 b in the presence of GolgiStop (BD Bioscience; San Jose, CA) as described by the manufacturer. Ceils are co-stained with anti-CD4, anti-IFNy and anti-IL-17 antibodies (Ebioscience, San Diego, CA). Levels of cytokine release are evaluated by culturing splenocy tes in vitro for 48 h with 1 ⁇ g of MOG35-55 peptide or irrelevant peptide Ova323-339).
  • Supematants are then collected and analyzed for IL- 1 ⁇ , JL-2, IL-4, IL-5, IL-6, IL-10, TL- 17, IFNy and TNFa using an Aushon multiplex array (Aushon Biosysiems, Bilierica, MA) following the manufacture 's instructions.
  • Aushon multiplex array Aushon Biosysiems, Bilierica, MA
  • MBP myelin basic protein
  • Peroxidazed-1 Biocare Medical PX968M, Concord, CA
  • Tissues are blocked with serum-free Protein Block (Dako X0909, Carpinteria, CA) and primary rat anti-mouse MBP antibody (Abeam Ab7349, Cambridge, MA) or control rat IgG (Vector Laboratories 1-4000, Burlingame, CA) is added.
  • Axonal damage is evaluated by staining for non-phosphorylated neurofilament heavy chain (SMI-32). Tissues are deparaffinized and rehydrated followed by heat-induced antigen retrieval in Decloaking ChamberTM NxGen (Biocare Medical DC2012, Concord, CA). After heat treatment, endogenous peroxidase is blocked using Peroxidazed-1 (Biocare Medical PX968M, Concord, CA).
  • SMI-32 non-phosphorylated neurofilament heavy chain
  • Tissues are blocked with Rodent Block M (Biocare Medical RBM961, Concord, CA) and incubated with primary antibody consisting of mouse anti-non-phosphoryiated neurofilaments (BioLegend SMI-32P, Dedliam, MA) or control mouse IgG I (BD Pliarmingen 557273, San Jose, CA) at 1 ⁇ ' ⁇ .
  • Secondary antibody containing anti-mouse IgG HRP R.T.U. Biocare Medical MM510
  • SMI-32 stained slides are used for pathological scoring of axonal damage.
  • mice Male BALB/c mice are purchased from Charles River Laboratories (Wilmington, MA). All mice are housed in a specific pathogen-free environment and maintained in accordance with the Guide for the Care and Use of Laboratory. Animals are used between 8 and 12 weeks of age. All protocols are approved by the Institutional Animal Care and Use Committee.
  • ZAP-70 inhibitors of the invention are prepared for in vivo oral administration in vehicle (phosphate-buffered saline: Thermo Fisher Scientific Inc., Waltham, MA) containing 0.5% methy!celluiose/0.025% Tween 20 ( Sigma- Aldrich, St. Louis, MO).
  • Unconjugated and biotinylated monoclonal rat anti-mouse IL-2 (clones JES6-1A12/JES6-5H4), IL-4 (B VD4-1D11/BVD6-24G2), IL-10 (JES5-2A5/SXC-1), and IFN-'y (R4-6A2/XMG1.2) antibodies and mouse rlL-2 (specific activity - 2.5 x 106 BtLMP U/mg), IL-4 (107 U/rag by CTLL- 2.4 assay), IL-10 (5 x 105 U/mg), and IFN-g ( 107 U/mg) are purchased from Thermo Fisher (Waltham, MA) and/or Sigma Aldrich (St.
  • hamster anti-mouse CD3 ⁇ (clone 145-2C11) and hamster anti-mouse CD28 (clone 37.51) antibodies are obtained from BD Biosciences (San Diego, CA). 2,4,6,-Trinitrobenzene sulfonic acid (TNBS) is purchased from Sigma Aldrich (St. Louis, MO).
  • TNBS 2,4,6,-Trinitrobenzene sulfonic acid
  • TNBS 2,4,6,-trinitrobenzene sulfonic acid
  • the procedure for the induction of colitis involves carefully inserting a flexible rubber catheter (3.5 French) into the colon of unanesthetized BALB/c mice such that the tip is 4 cm proximal to the anal verge.
  • 0.5 mg of the hapten reagent TNBS in 50% elhanol to break the intestinal epithelial barrier
  • mice receives 50% ethanol alone using the same technique described above.
  • the total injection volume is 100 mL in both groups allowing TNBS or ethanol to reach the entire colon, including the caecum and appendix. Animals are then kept in a vertical position for 45 s for drug delivery and returned to their cages.
  • mice In order to examine the effect of ZAP-70 inhibitors of the invention on the course and outcome of TNBS -induced colitis in mice, BALB/c mice are treated with ZAP-70 (25 nig kg) orally by gavage daily starting on day -1 and continuing until the mice are sacrificed. A group of TNBS -challenged mice are treated with vehicle alone.
  • mice To monitor the weight changes, the weight of the mice is recorded daily. On days 7 and 14 after administration of TNBS (or vehicle), groups of mice are sacrificed by C02 inhalation, and colonic inflammation was assessed. Immediately after sacrifice, the distal colon is excised and examined for macroscopic changes. Tissues were removed and embedded in paraffin. Paraffin sections were made and stained with hematoxylin and eosin. The diameter of the colon of each mouse is measured with the help of a caliper. After measurements of the diameter, the distal 8 cm of colon is excised, blotted dry, and weighed.
  • TNBS or vehicle
  • LP lymphocytes are isolated from freshly obtained colonic specimens. After removal of the Peyer's patches, the colon is washed in Ca/Mg-free HBSS, cut into 0.5-cm pieces and incubated twice in HBSS containing EDTA (0.37 mg/rnl) and DTT (0. 145 mg ml) at 37oC for 15 min.
  • the tissue is digested further in RPMI containing collagenase D (400 U/ml) arid DNase I (0.1 mg/ml) (Boehringer Mannheim Biochemicals, Indianapolis, IN) in a shaking incubator at 37oC LP cells are then layered on a 40-100% Percoll gradient (Pharmacia, Uppsala, Sweden), and lymphocyte-enriched populations are isolated from the cells at the 40- 100% interface.
  • Enriched CD4+ T cell populations are obtained by negative selection using mouse CD4+ T cell isolation columns (isocell; Pierce Chemical Co., Rockford, 1L).
  • the resultant cells which are analyzed by flow cytometry (FACSeanj Becton Dickinson & Co., Mountain View, CA) contained >85%) CD4+ cells.
  • Cell cultures of LP CD4+ T cells are performed in complete medium consisting of RPMI 1640 (Whittaker Bioproducts, Walkersville, MD) supplemented with 3 mM L-glutamine, 10 rriM Hepes buffer, 10 mg/ml gentamycin (Whittaker), 100 U/mL each of penicillin and streptomycin (Whittaker), 0.05 mM 2-ME (Sigma Aldrich Chemical Co.), and 10% FCS.
  • cytokine production To measure cytokine production, 24-well plates (Costar Corp., Cambridge, MA) are coated with 10 mg/mL murine anti-CD3e antibody in carbonate buffer (pH 9.6) overnight at 4oC, 105 LP CD4+ T cells are then cultured in 1 ml of complete medium in precoated or uncoated wells, and 1 mg/ml soluble anti-CD28 antibody is added to the anti-CD3e- coated wells. Culture supernatants are removed after 48 h and assayed for cytokine concentration.
  • Cytokine concentrations are determined by specific ELISA per the manufacturer's recommendation (BD Biosciences) using Immulon 4 96-well microtiter plates (Dynatech Laboratories Inc., Chantilly, VA). ODs are measured on a Dy natech MR 5000 ELISA reader at a wavelength of 490 nm.
  • a parenteral pharmaceutical composition of the compounds of the invention that are suitable for administration by injection, the compounds can be formulated as a mixture and incorporated into a dosage unit form.
  • a typical 5 mg/mL parenteral formulation of a compound of the invention proportionally contains, in addition to the compound itself (0.5%), propylene glycol (40%), ethyl alcohol (10%), sodium benzoate/benzoic acid (5%), benzyl alcohol ( 1.5%), and water (43%).
  • a typical 25 mg oral (capsule) formulation of a compound of the invention contains, in addition to the compound itself, polyoxyl 40 hydrogenated castor oil, gelatin, polyethylene glycol 400, glycerin 85%, dehydrated alcohol, corn oil mono-di-triglycerides, titanium dioxide, vitamin E, ferric oxide yellow, ferric oxide red, carmine, hypromellose 2910, propylene glycol, and purified water.
  • a pharmaceutical composition of the compounds of the invention are suitable for oral solid dosage (tablet) administration
  • the compounds can be formulated as a mixture and incorporated into a dosage unit form.
  • a typical 50 mg oral solid dosage formulation of a compound of the invention can be prepared by granulating and compacting into a solid mixture that contains, in addition to the compound itself, excipients, binders and fillers that include modified starch, polyethylene glycol 400, stearyl citrate, polyvinylpyrrolidone, lecithin, mannitol, sorbitol, sage extract, calcium phosphate and gelatin.
  • a pharmaceutical composition for buccal delivery such as a hard lozenge
  • a pharmaceutical composition for buccal delivery such as a hard lozenge

Abstract

La présente invention concerne des composés représentés par les formules (I) et (V) et des compositions associées utilisables en tant qu'inhibiteurs de la kinase ZAP-70. L'invention concerne également des procédés de synthèse et des procédés d'utilisation d'inhibiteurs de ZAP-70 dans le traitement d'individus souffrant d'un rejet de greffe d'organe, de maladies auto-immunes et d'un cancer.
PCT/US2018/052557 2017-09-26 2018-09-25 Compositions d'inhibiteur de kinase zap-70, procédés et utilisations de celles-ci WO2019067396A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4037694A4 (fr) * 2019-10-04 2023-10-25 Dana-Farber Cancer Institute, Inc. Médicaments à base d'immunomodulateur de type imide en tant qu'agonistes de la protéine kinase 70 associée à la chaîne zêta (zap70) et leurs utilisations
US11893301B2 (en) 2020-09-10 2024-02-06 Snap Inc. Colocated shared augmented reality without shared backend

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63295588A (ja) * 1987-01-22 1988-12-01 Kyowa Hakko Kogyo Co Ltd 生理活性物質ｋ−２５２の誘導体
WO2008122038A1 (fr) * 2007-04-02 2008-10-09 President And Fellows Of Harvard College Autophagie régulatrice
WO2009112490A1 (fr) * 2008-03-11 2009-09-17 Cellzome Limited Sulfonamides en tant qu'inhibiteurs de zap-70
WO2012092339A2 (fr) * 2010-12-28 2012-07-05 The Children's Hospital Of Philadelphia Conception de promédicaments libérables par hydrolyse pour des formulations de nanoparticules à libération prolongée
CN106083830A (zh) * 2016-06-01 2016-11-09 中国海洋大学 双吲哚马来酰亚胺衍生物及其制备方法和用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63295588A (ja) * 1987-01-22 1988-12-01 Kyowa Hakko Kogyo Co Ltd 生理活性物質ｋ−２５２の誘導体
WO2008122038A1 (fr) * 2007-04-02 2008-10-09 President And Fellows Of Harvard College Autophagie régulatrice
WO2009112490A1 (fr) * 2008-03-11 2009-09-17 Cellzome Limited Sulfonamides en tant qu'inhibiteurs de zap-70
WO2012092339A2 (fr) * 2010-12-28 2012-07-05 The Children's Hospital Of Philadelphia Conception de promédicaments libérables par hydrolyse pour des formulations de nanoparticules à libération prolongée
CN106083830A (zh) * 2016-06-01 2016-11-09 中国海洋大学 双吲哚马来酰亚胺衍生物及其制备方法和用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SLATER M J ET AL: "INDOLOCARBAZOLES: POTENT, SELECTIVE INHIBITORS OF HUMAN CYTOMEGALOVIRUS REPLICATION", BIOORGANIC & MEDICINAL CHEMISTRY, PERGAMON, GB, vol. 7, no. 6, 1 January 1999 (1999-01-01), pages 1067 - 1074, XP001074630, ISSN: 0968-0896, DOI: 10.1016/S0968-0896(99)00032-2 *
XIAMIN CHENG ET AL: "A tuned affinity-based staurosporine probe for in situ profiling of protein kinases", CHEMICAL COMMUNICATIONS, vol. 50, no. 22, 1 January 2014 (2014-01-01), pages 2851, XP055529047, ISSN: 1359-7345, DOI: 10.1039/c4cc00184b *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4037694A4 (fr) * 2019-10-04 2023-10-25 Dana-Farber Cancer Institute, Inc. Médicaments à base d'immunomodulateur de type imide en tant qu'agonistes de la protéine kinase 70 associée à la chaîne zêta (zap70) et leurs utilisations
US11893301B2 (en) 2020-09-10 2024-02-06 Snap Inc. Colocated shared augmented reality without shared backend

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