WO2010059593A1 - Procédés et compositions pour le traitement d'affections ophtalmiques - Google Patents

Procédés et compositions pour le traitement d'affections ophtalmiques Download PDF

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WO2010059593A1
WO2010059593A1 PCT/US2009/064717 US2009064717W WO2010059593A1 WO 2010059593 A1 WO2010059593 A1 WO 2010059593A1 US 2009064717 W US2009064717 W US 2009064717W WO 2010059593 A1 WO2010059593 A1 WO 2010059593A1
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substituted
unsubstituted
alkyl
aryl
cycloalkyl
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PCT/US2009/064717
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English (en)
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Troy Edward Wilson
Christian Rommel
Yi Liu
Pingda Ren
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Intellikine, Inc.
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Priority to US13/127,973 priority Critical patent/US20110269779A1/en
Publication of WO2010059593A1 publication Critical patent/WO2010059593A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents

Definitions

  • Aberrant neovascularization or vascular permeability in the eye is a major cause of many ocular disorders including age-related macular degeneration (AMD), retinopathy of prematurity (ROP), ischemic retinal vein occlusion, diabetic retinopathy (DR), and neovascular glaucoma (NVG).
  • AMD and DR are amongst the most common causes of severe, irreversible vision loss in humans.
  • central vision loss is secondary to angiogenesis, a process by which new blood vessels from preexisting vasculature are developed and/or vascular permeability properties are altered.
  • Inflammation is another cause of many eye diseases.
  • Neovascular stimuli include tissue hypoxia, inflammatory cell infiltration and penetration barrier breakdown which can increase cytokine production, expression of cell adhesion molecules such as integrins, and upregulation of proteinase activity. These stimuli in turn result in angiogenesis, which can disrupt the organizational structure of the neural retina, damage the integrity of the inner limiting membranes surrounding the vitreous, and disrupt endothelial tight junctions leading to increased vascular leakage and retinal edema.
  • T cells which activates the complement cascade and produces reactive oxygen species, which facilitate lipid peroxidation, which in turn, is toxic to neuronal cells such as photoreceptor and retinal pigment epithelial cells.
  • PDT photodynamic therapy
  • the effects of photocoagulation on AMD are achieved through the thermal destruction of retinal cells.
  • PDT usually requires slow infusion of a specific wavelength absorbing dye, followed by application of non-thermal laser light that is absorbed by the dye which leads to localized destruction or perturbation of cells that have absorbed a significant fraction of the dye.
  • PDT treatment is often repeated several times a year and is associated with side effects including headaches, blurring, decreased visual acuity, and gaps in vision in approximately 4% of patients. Subjects receiving PDT must also avoid direct sunlight for up to 5 days to avoid sunburn.
  • Newer treatments include VEGF and VEGFR antagonists such as monoclonal antibodies (ranibizumab) or antibody fragments (bevacizumab) and nucleic acid aptamers (pegaptanib) that bind to and inhibit the interaction between VEGF and its receptor.
  • VEGF and VEGFR antagonists such as monoclonal antibodies (ranibizumab) or antibody fragments (bevacizumab) and nucleic acid aptamers (pegaptanib) that bind to and inhibit the interaction between VEGF and its receptor.
  • pegaptanib nucleic acid aptamers
  • Xalatan a prostaglandin FP2 agonist, which facilitates drainage of the intraocular fluid through the uveoscleral route.
  • uveoscleral outflow thought to represent only 20% of the total outflow, with the remaining 80% draining through the trabecular meshwork.
  • vascular endothelial growth factor vascular endothelial growth factor
  • PDGF vascular endothelial growth factor
  • FGF vascular endothelial growth factor
  • TNF vascular endothelial growth factor
  • IGF vascular endothelial growth factor receptor
  • the present invention provides methods and compounds for modulating the vascular endothelial growth factor receptor (VEGF-R) signaling pathway in an eye, or cells derived from an eye.
  • VEGF-R vascular endothelial growth factor receptor
  • an agent that can reduce T cell activation, thereby curbing the local inflammatory response is provided for treatment of dry-type AMD.
  • the methods includes administering to a subject one or more antagonists of a PB K or pharmaceutically acceptable salt thereof.
  • the one or more antagonists of PI3K inhibit one or more of PBK ⁇ , PDK ⁇ , PI3K ⁇ , or PI3K ⁇ with an IC50 less than about l ⁇ M, 75OnM, 50OnM, 25OnM, 15OnM, 10OnM, 5OnM, 25nM, 1OnM or about InM.
  • the one or more antagonists of PI3K inhibit two or three or more of PI3K ⁇ , PI3K ⁇ , PI3K ⁇ , or PI3K ⁇ with an IC50 less than about l ⁇ M, 75OnM, 50OnM, 25OnM, 15OnM, 10OnM, 5OnM, 25nM, 1OnM or about InM.
  • the present invention provides a method for ameliorating, treating, or mitigating an ophthalmic disease by administering one or more antagonists of mTOR and a PI3K antagonists or pharmaceutically acceptable salt thereof to the eye.
  • mTOR antagonist inhibits both mTORCl and mTORC2.
  • the mTOR antagonist and the PI3K antagonist inhibit their target kinases with an IC50 of less than about l ⁇ M, 75OnM, 50OnM, 25OnM, 15OnM, 10OnM, 5OnM, 25nM, 1OnM or about InM.
  • the present invention provides methods and compositions for inhibiting white blood cells by administering one or more pyrazolopyrimidine compounds or pharmaceutically acceptable salts thereof to the eye.
  • the pyrazolopyrimidine compound inhibits white blood cell proliferation (e.g. B-cells, T-cells, macrophages, neutrophils, or microlia) with an IC50 less than about l ⁇ M, 75OnM, 50OnM, 25OnM, 15OnM, 10OnM, 5OnM, 25nM, 1OnM or about InM.
  • the present invention provides methods and compositions for facilitating drainage of the intraocular fluid through the trabecular meshwork, thereby lowering the intraocular pressure and alleviating the symptoms of glaucoma.
  • methods are provided for treating ophthalmic disease by administering one or more of a pyrazolopyrimidine PI3K or mTOR antagonists to the eye of a subject.
  • methods are further provided for administering the compounds of the present invention via eye drop, intraocular injection, intravitreal injection, topically, or through the use of a drug eluting device, microcapsule, implant, or microfluidic device.
  • the compounds of the present invention are administered with a carrier or excipient that increases the intraocular penetrance of the compound such as an oil and water emulsion with colloid particles having an oily core surrounded by an interfacial film.
  • the colloid particles include at least one cationic agent and at least one non-ionic sufactant such as a poloxamer, tyloxapol, a polysorbate, a polyoxyethylene castor oil derivative, a sorbitan ester, or a polyoxyl stearate.
  • the cationic agent is an alkylamine, a tertiary alkyl amine, a quarternary ammonium compound, a cationic lipid, an amino alcohol, a biguanidine salt, a cationic compound or a mixture thereof.
  • the cationic agent is a biguanidine salt such as chlorhexidine, polyaminopropyl biguanidine, phenformin, alkylbiguanidine, or a mixture thereof.
  • the quaternary ammonium compound is a benzalkonium halide, lauralkonium halide, cetrimide, hexadecyltrimethylammonium halide, tetradecyltrimethylammonium halide, dodecyltrimethylammonium halide, cetrimonium halide, benzethonium halide, behenalkonium halide, cetalkonium halide, cetethyldimonium halide, cetylpyridinium halide, benzododecinium halide, chlorallyl methenamine halide, rnyristylalkonium halide, stearalkonium halide or a mixture of two or more thereof.
  • cationic agent is a benzalkonium chloride, lauralkonium chloride, benzododecinium bromide, benzethenium chloride, hexadecyltrimethylammonium bromide, tetradecyltrimethylammonium bromide, dodecyltrimethylammonium bromide or a mixture of two or more thereof.
  • the oil phase is mineral oil and light mineral oil, medium chain triglycerides (MCT), coconut oil; hydrogenated oils comprising hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate castor oil or hydrogenated soybean oil; polyoxyethylene hydrogenated castor oil derivatives comprising poluoxyl-40 hydrogenated castor oil, polyoxyl-60 hydrogenated castor oil or polyoxyl-100 hydrogenated castor oil.
  • MCT medium chain triglycerides
  • coconut oil hydrogenated oils comprising hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate castor oil or hydrogenated soybean oil
  • polyoxyethylene hydrogenated castor oil derivatives comprising poluoxyl-40 hydrogenated castor oil, polyoxyl-60 hydrogenated castor oil or polyoxyl-100 hydrogenated castor oil.
  • the present invention provides a drug eluting device for treatment of ophthalmic disease.
  • the drug eluting device includes one or more compounds of the present invention for slow release.
  • the drug eluting device further includes a non-biodegradable polymer from which the one or more compounds are slowly released non-biodegradable compounds include silicone, acrylates, polyethylenes, polyurethane, polyester, polypropylene, polytetrafluoroethylene, poly ether ketone, nylon, collagen, polyethylene terepthalate, polycarbonate, or polyimide.
  • the drug eluting device includes a biodegradable polymer from which one or more compounds are slowly released biodegradable polymers include polyglycolide, polylactide, poly ⁇ -caprolactone, polyglyconate, polyhydroxybutyrate, polyhydroxyvalerate, and polydioxanone.
  • the drug eluting device is composed of biodegradable and non-biodegradable components.
  • the drug eluting device is composed of a non-ferrous metal suitable for implant into the eye of a subject.
  • the one or more antagonists are of Formula I:
  • R 1 is hydrogen, R 3 -substituted or unsubstituted alkyl, R 3 -substituted or unsubstituted heteroalkyl, R 3 -substituted or unsubstituted cycloalkyl, R 3 -substituted or unsubstituted heterocycloalkyl, R 3 -substituted or unsubstituted aryl, or R 3 -substituted or unsubstituted heteroaryl;
  • R 2 is halogen, R 4 -substituted aryl, or substituted or unsubstituted heteroaryl;
  • said one or more antagonists are of Formula XV
  • R 1 and R 2 are independently halogen, -CN, - OR 5 , -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroarylene; R 1 and R 2 are independently halogen, -CN, - OR 5 , -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstit
  • Figure 1 illustrates the PI3K/mTOR pathway.
  • Figure 2 illustrates a mechanism for angiogenesis involving mTOR activation.
  • the term "effective amount” or “therapeutically effective amount” refers to that amount of a compound described herein that is sufficient to effect the intended application including but not limited to disease treatment, as defined below.
  • the therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art.
  • the term also applies to a dose that will induce a particular response in target cells, e.g. reduction of platelet adhesion and/or cell migration.
  • the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.
  • treatment or “treating,” or “palliating” or “ameliorating” is used interchangeably herein. These terms refer to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit and/or a prophylactic benefit.
  • therapeutic benefit is meant eradication or amelioration of the underlying disorder being treated.
  • a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the patient, notwithstanding that the patient may still be afflicted with the underlying disorder.
  • compositions may be administered to a patient at risk of developing a particular disease, or to a patient reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of a physical examination, functional (self) evaluation, and/or any form of vision evaluation.
  • the certain methods presented herein successfully treat vision loss due to aberrant neovascularization or vascular permeability in the eye by decreasing the incidence of angiogenesis and or preventing the incidence of angiogenesis.
  • the "therapeutic effecf ' as used herein, encompasses a therapeutic benefit and/or a prophylactic benefit as described above.
  • a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
  • the terms "co-administration,” “administered in combination with,” and their grammatical equivalents, as used herein, encompass administration of two or more agents to an animal so that both agents and/or their metabolites are present in the animal at the same time. Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which both agents are present.
  • pharmaceutically acceptable salt refers to salts derived from a variety of organic and inorganic counter ions well known in the art and include, by way of example only, sodium, potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and when the molecule contains a basic functionality, salts of organic or inorganic acids, such as hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and the like.
  • antagonists are used interchangeably, and they refer to a compound having the ability to inhibit a biological function of a target protein, whether by inhibiting the activity or expression of the target protein. Accordingly, the terms “antagonist” and “inhibitors” are defined in the context of the biological role of the target protein. While preferred antagonists herein specifically interact with (e.g. bind to) the target, compounds that inhibit a biological activity of the target protein by interacting with other members of the signal transduction pathway of which the target protein is a member are also specifically included within this definition. A preferred biological activity inhibited by an antagonist is associated with the development, growth, or spread of a tumor.
  • agonist refers to a compound having the ability to initiate or enhance a biological function of a target protein, whether by inhibiting the activity or expression of the target protein. Accordingly, the term “agonist” is defined in the context of the biological role of the target polypeptide. While preferred agonists herein specifically interact with (e.g. bind to) the target, compounds that initiate or enhance a biological activity of the target polypeptide by interacting with other members of the signal transduction pathway of which the target polypeptide is a member are also specifically included within this definition.
  • agent or “biologically active agent” refers to a biological, pharmaceutical, or chemical compound or other moiety.
  • Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a vitamin derivative, a carbohydrate, a toxin, or a chemotherapeutic compound.
  • Various compounds can be synthesized, for example, small molecules and oligomers (e.g., oligopeptides and oligonucleotides), and synthetic organic compounds based on various core structures.
  • various natural sources can provide compounds for screening, such as plant or animal extracts, and the like. A skilled artisan can readily recognize that there is no limit as to the structural nature of the agents of the present invention.
  • Signal transduction is a process during which stimulatory or inhibitory signals are transmitted into and within a cell to elicit an intracellular response.
  • a modulator of a signal transduction pathway refers to a compound which modulates the activity of one or more cellular proteins mapped to the same specific signal transduction pathway.
  • a modulator may augment (agonist) or suppress (antagonist) the activity of a signaling molecule.
  • an "anti-angiogenesis agent”, “anti-angiogenetic agent”, “antiangiogenetic agent”, “anti- angiogenetic drug”, or “anti-angiogenesis drug” refers to any agent useful in the treatment of a condition characterized by excessive or abnormal angiogenesis.
  • Antiangiogenetic therapy means the administration of one or more antiangiogenetic drugs and/or other agents to a patient suffering from a disorder characterized by excessive or abnormal angiogenesis by various methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, transdermal, buccal, instillation, continuous release, sustained release, ocular inserts, inhalation or in the form of a suppository.
  • cell proliferation refers to a phenomenon by which the cell number has changed as a result of division. This term also encompasses cell growth by which the cell morphology has changed (e.g., increased in size) consistent with a proliferative signal.
  • selective inhibition or “selectively inhibit” as applied to a biologically active agent refers to the agent's ability to preferentially reduce the target signaling activity as compared to off-target signaling activity, via direct or interact interaction with the target.
  • mTORCl/mTORC2 inhibitor refers to such compounds that inhibit both mTORCl and mTORC2. These compounds are distinguished from mTOR inhibitors such as rapamycin and rapamcyin analogues (i.e. rapalogues) due to their ability to inhibit mTORCl and mTORC2. In some embodiments, the inhibition of both mTORCl and mTORC2 can be measured by assaying the effects of the compounds of the present invention on the phosphorylation of Akt.
  • Subject refers to an animal, such as a mammal, for example a human.
  • the methods described herein can be useful in both human therapeutics and veterinary applications.
  • the patient is a mammal, and in some embodiments, the patient is human.
  • salts are meant to include salts of active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein.
  • base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent.
  • pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt.
  • acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent.
  • Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like.
  • inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and
  • Prodrug is meant to indicate a compound that may be converted under physiological conditions or by solvolysis to a biologically active compound described herein.
  • prodrug refers to a precursor of a biologically active compound that is pharmaceutically acceptable.
  • a prodrug may be inactive when administered to a subject, but is converted in vivo to an active compound, for example, by hydrolysis.
  • the prodrug compound often offers advantages of solubility, tissue compatibility or delayed release in a mammalian organism (see, e.g., Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).
  • Bundgard, H. Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam).
  • a discussion of prodrugs is provided in Higuchi, T., et al., "Pro-drugs as Novel Delivery Systems," A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated in full by reference herein.
  • prodrug is also meant to include any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of an active compound, as described herein may be prepared by modifying functional groups present in the active compound in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent active compound.
  • Prodrugs include compounds wherein a hydroxy, amino or mercapto group is bonded to any group that, when the prodrug of the active compound is administered to a mammalian subject, cleaves to form a free hydroxy, free amino or free mercapto group, respectively.
  • prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of an alcohol or acetamide, formamide and benzamide derivatives of an amine functional group in the active compound and the like.
  • in vivo refers to an event that takes place in a subject's body.
  • in vitro refers to an event that takes places outside of a subject's body.
  • an in vitro assay encompasses any assay run outside of a subject assay.
  • in vitro assays encompass cell-based assays in which cells alive or dead are employed.
  • In vitro assays also encompass a cell-free assay in which no intact cells are employed.
  • structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by 13 C- or 14 C-enriched carbon are within the scope of this invention.
  • the compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds.
  • the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( 3 H), iodine-125 ( 125 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.
  • R of an acyloxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -
  • R of an acyloxy group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR 3 , -
  • alkyl refers to an (aryl)alkyl — radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for aryl and alkyl respectively.
  • Alkoxy refers to a (alkyl)O-radical, where alkyl is as described herein and contains 1 to 10 carbons (e.g., Ci-Cio alkyl). Whenever it appears herein, a numerical range such as “1 to 10" refers to each integer in the given range; e.g., "1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In some embodiments, it is a Ci-C 4 alkoxy group. A alkoxy moiety may be substituted by one or more of the substituents described as suitable substituents for an alkyl radical.
  • Alkyl refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, containing no unsaturation, having from one to ten carbon atoms (e.g., C 1 -C 10 alkyl).
  • a numerical range such as “1 to 10” refers to each integer in the given range; e.g., "1 to 10 carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated.
  • Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, sec-butyl isobutyl, tertiary butyl, pentyl, isopentyl, neopentyl, hexyl, septyl, octyl, nonyl, decyl, and the like.
  • the alkyl is attached to the rest of the molecule by a single bond, for example, methyl (Me), ethyl (Et), «-propyl, 1 -methylethyl (zso-propyl), «-butyl, «-pentyl, 1 , 1 -dimethylethyl ( ⁇ -butyl), 3-methylhexyl, 2-methylhexyl, and the like.
  • an alkyl group is optionally substituted by one or more of substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -
  • an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention.
  • a "lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.
  • Alkylaryl refers to an -(alkyl)aryl radical where aryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for aryl and alkyl respectively.
  • Alkylhetaryl refers to an -(alkyl)hetaryl radical where hetaryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for aryl and alkyl respectively.
  • Alkylheterocyclyl refers to an -(alkyl) heterocycyl radical where alkyl and heterocyclyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heterocyclyl and alkyl respectively.
  • alkene refers to a group consisting of at least two carbon atoms and at least one carbon- carbon double bond
  • an "alkyne” moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond.
  • the alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic.
  • alkenyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one double bond, and having from two to ten carbon atoms (ie. C 2 -Ci O alkenyl). Whenever it appears herein, a numerical range such as “2 to 10" refers to each integer in the given range; e.g., "2 to 10 carbon atoms” means that the alkenyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In certain embodiments, an alkenyl comprises two to eight carbon atoms.
  • an alkenyl comprises two to five carbon atoms (e.g., C 2 -C 5 alkenyl).
  • the alkenyl is attached to the rest of the molecule by a single bond, for example, ethenyl (i.e., vinyl), prop-1-enyl (i.e., allyl), but-1-enyl, pent-1-enyl, penta-l,4-dienyl, and the like.
  • an alkenyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR 3 , - SR 3 , -OC(O)-R 3 , -N(R 3 ) 2 , -C(O)R 3 , -C(O)OR 3 , -OC(O)N(R 3 ) 2 , -C(O)N(R 3 ) 2 , -N(R 3 )C(O)OR 3 , -N(R 3 )C(O)R 3 , - N(R 3 )C(O)N(R 3 )N(R 3 )OR
  • alkenyl-cycloalkyl refers to an -(alkenyl)cycloalkyl radical where alkenyl and cyclo alkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for alkenyl and cycloalkyl respectively.
  • Alkynyl refers to a straight or branched hydrocarbon chain radical group consisting solely of carbon and hydrogen atoms, containing at least one triple bond, having from two to ten carbon atoms (ie. C 2 -C 10 alkynyl). Whenever it appears herein, a numerical range such as “2 to 10" refers to each integer in the given range; e.g., "2 to 10 carbon atoms” means that the alkynyl group may consist of 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms. In certain embodiments, an alkynyl comprises two to eight carbon atoms.
  • an alkynyl has two to five carbon atoms (e.g., C 2 -Cs alkynyl).
  • the alkynyl is attached to the rest of the molecule by a single bond, for example, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like.
  • an alkynyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -
  • Alkynyl-cycloalkyl refers to refers to an -(alkynyl)cycloalkyl radical where alkynyl and cyclo alkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for alkynyl and cycloalkyl respectively.
  • Amino refers to a -N(R 3 ) 2 radical group, where each R 3 is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification.
  • R 3 is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl, unless stated otherwise specifically in the specification.
  • a -N(R 3 ) 2 group has two Ra other than hydrogen they can be combined with the nitrogen atom to form a A-, 5-, 6-, or 7-membered ring.
  • -N(R 3 ) 2 is meant to include, but not be limited to, 1 -pyrrolidinyl and 4-morpholinyl.
  • an amino group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR 3 , -
  • Amide refers to a chemical moiety with formula -C(O)N(R) 2 or -NHC(O)R, where R is selected from the group consisting of hydrogen, alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). In some embodiments it is a C 1 -C 4 amido or amide radical, which includes the amide carbonyl in the total number of carbons in the radical.
  • the R 2 of - N(R) 2 of the amide may optionally be taken together with the nitrogen to which it is attached to form a 4-, 5-, 6-, or 7- membered ring.
  • an amido group is optionally substituted independently by one or more of the substituents as described herein for alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclyl.
  • An amide may be an amino acid or a peptide molecule attached to a compound of
  • Aromatic or "aryl” refers to an aromatic radical with six to ten ring atoms (e.g., C 6 -C 10 aromatic or
  • C 6 -Ci O aryl which has at least one ring having a conjugated pi electron system which is carbocyclic (e.g., phenyl, fluorenyl, and naphthyl).
  • carbocyclic e.g., phenyl, fluorenyl, and naphthyl.
  • a numerical range such as “6 to 10" refers to each integer in the given range; e.g., "6 to 10 ring atoms” means that the aryl group may consist of 6 ring atoms, 7 ring atoms, etc., up to and including 10 ring atoms.
  • the term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of ring atoms) groups.
  • an aryl moiety is optionally substituted by one or more substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -
  • R 3 is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.
  • Cyano refers to a -CN radical.
  • Cycloalkyl refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, or partially unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms
  • cycloalkyl group may consist of 3 carbon atoms, etc., up to and including 10 carbon atoms. In some embodiments, it is a C 3 -Cg cycloalkyl radical. In some embodiments, it is a C 3 -C 5 cycloalkyl radical.
  • cycloalkyl groups include, but are not limited to the following moieties: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloseptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl, and the like.
  • a cycloalkyl group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR 3 , -
  • each R 3 is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.
  • Cycloalkyl-alkenyl refers to a -(cycloalkyl) alkenyl radical where cycloalkyl and heterocyclyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heterocyclyl and cycloalkyl respectively.
  • Cycloalkyl-heterocyclyl refers to a -(cycloalkyl) heterocycyl radical where cycloalkyl and heterocyclyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heterocyclyl and cycloalkyl respectively.
  • Cycloalkyl-heteroaryl refers to a -(cycloalkyl) heteroaryl radical where cycloalkyl and heterocyclyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heterocyclyl and cycloalkyl respectively
  • Ester refers to a chemical radical of formula -COOR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). Any amine, hydroxy, or carboxyl side chain on the compounds described herein can be esterified. The procedures and specific groups to make such esters are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis,
  • an ester group is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, trifluoromethyl, trifluoromethoxy, nitro, trimethylsilanyl, -OR a , -
  • R 3 is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.
  • Fluoroalkyl refers to an alkyl radical, as defined above, that is substituted by one or more fluoro radicals, as defined above, for example, trifluoromethyl, difluoromethyl, 2,2,2-trifluoroethyl, l-fluoromethyl-2-fluoroethyl, and the like.
  • the alkyl part of the fluoroalkyl radical may be optionally substituted as defined above for an alkyl group.
  • Halo means fluoro, chloro, bromo or iodo.
  • haloalkyl include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof.
  • haloalkyl include alkyl, alkenyl, alkynyl and alkoxy structures that are substituted with one or more halo groups or with combinations thereof.
  • fluoroalkyl and fluoroalkoxy include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine.
  • Heteroalkyl “heteroalkenyl” and “heteroalkynyl” include optionally substituted alkyl, alkenyl and alkynyl radicals and which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof.
  • a numerical range may be given, e.g. C 1 -C 4 heteroalkyl which refers to the chain length in total, which in this example is 4 atoms long. For example, a -
  • CH 2 OCH 2 CH 3 radical is referred to as a "C 4 " heteroalkyl, which includes the heteroatom center in the atom chain length description. Connection to the rest of the molecule may be through either a heteroatom or a carbon in the heteroalkyl chain.
  • a heteroalkyl group may be substituted with one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR 3 , - SR a , -OC(O)-R 3 , -N(R a ) 2 , -C(O)R 3 , -C(O)OR 3 , -C(O)N(R 3 ) 2 , -N(R 3 )C(O)OR 3 , -N(R 3 )C(O)R 3 , -N(R 3 )S(O) t R 3 (where t is 1 or 2), -S(O) 4 OR 3 (where t is 1 or
  • Heteroalkylaryl refers "to an -(heteroalkyl)aryl radical where heteroalkyl and aryl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heteroalkyl and aryl respectively.
  • Heteroalkylheteroaryl refers "to an -(heteroalkyl)heteroaryl radical where heteroalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heteroalkyl and heteroaryl respectively
  • Heteroalkylheterocyclyl refers "to an -(heteroalkyl)heterocyclyl radical where heteroalkyl and heteroaryl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heteroalkyl and heterocyclyl respectively
  • Heteroalkylcycloalkyl refers "to an -(heteroalkyl) cycloalkyl radical where heteroalkyl and cycloalkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for heteroalkyl and cycloalkyl respectively
  • Heteroaryl or, alternatively, “heteroaromatic” refers to a 5- to 18-membered aromatic radical (e.g., C 5 -C 13 heteroaryl) that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur, and which may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system.
  • a numerical range such as “5 to 18” refers to each integer in the given range; e.g., "5 to 18 ring atoms” means that the heteroaryl group may consist of 5 ring atoms, 6 ring atoms, etc., up to and including 18 ring atoms.
  • heteroaryl refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom.
  • the polycyclic heteroaryl group may be fused or non- fused.
  • the heteroatom(s) in the heteroaryl radical is optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • the heteroaryl is attached to the rest of the molecule through any atom of the ring(s).
  • heteroaryls include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzindolyl, 1,3-benzodioxolyl, benzofuranyl, benzooxazolyl, benzo[d]thiazolyl, benzothiadiazolyl, benzo[ ⁇ ][l,4]dioxepinyl, benzo[b][l,4]oxazinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzoxazolyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzofurazanyl, benzothiazolyl, benzothienyl (benzothiophenyl), benzothieno[3,2-d]pyrimidinyl
  • a heteraryl moiety is optionally substituted by one or more substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR a , -
  • Heteroarylalkyl or “hetarylalkyl” refers to an (heteroaryl)alkyl — radical where heteroaryl and alkyl are as disclosed herein and which are optionally substituted by one or more of the subsituents described as suitable substituents for aryl and alkyl respectively.
  • Heterocycloalkyl or “heterocyclylalkyl” refers to a radical of the formula -R c -heterocyclyl where R c is an alkyl chain as defined above. If the heterocyclyl is a nitrogen- containing heterocyclyl, the heterocyclyl is optionally attached to the alkyl radical at the nitrogen atom.
  • the alkylene chain of the heterocyclylalkyl radical is optionally substituted as defined above for an alkylene chain.
  • the heterocyclyl part of the heterocyclylalkyl radical is optionally substituted as defined above for a heterocyclyl group.
  • Heterocyclyl refers to a stable 3- to 18-membered non-aromatic ring (e.g., C 3 -C 18 heterocyclyl) radical that comprises two to twelve carbon atoms and from one to six heteroatoms selected from nitrogen, oxygen and sulfur.
  • a numerical range such as “3 to 18” refers to each integer in the given range; e.g., "3 to 18 ring atoms” means that the heteroaryl group may consist of 3 ring atoms, 4 ring atoms, etc., up to and including 18 ring atoms. In some embodiments, it is a C 5 -C 10 heterocyclyl.
  • the heterocyclyl radical is a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems.
  • the heteroatoms in the heterocyclyl radical may be optionally oxidized.
  • One or more nitrogen atoms, if present, are optionally quaternized.
  • the heterocyclyl radical is partially or fully saturated.
  • the heterocyclyl may be attached to the rest of the molecule through any atom of the ring(s).
  • heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1 -oxoxo
  • a heterocylyl moiety is optionally substituted by one or more substituents which independently are: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR 3 , -
  • Heteroalicyclic refers to a cycloalkyl radical that includes at least one heteroatom selected from nitrogen, oxygen and sulfur.
  • the radicals may be fused with an aryl or heteroaryl.
  • heteroalicyclic also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the oligosaccharides.
  • a heteroalicyclic group is optionally substituted by one or more of substituents which are independently: alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, hydroxy, halo, cyano, nitro, oxo, thioxo, trimethylsilanyl, -OR a , -
  • each R 3 is independently hydrogen, alkyl, fluoroalkyl, carbocyclyl, carbocyclylalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl, heteroaryl or heteroarylalkyl.
  • Mercaptyl refers to a (alkyl)S- or (H)S- radical.
  • Moiety refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule.
  • Niro refers to the -NO 2 radical.
  • Oxa refers to the -O- radical.
  • it is a C 1 -C 10 sulfonamido, wherein each R in sulfonamido contains 1 carbon, 2 carbons,
  • a sulfonamido group is optionally substituted by one or more of the subsituents described for alkyl, cycloalkyl, aryl, heteroaryl respectively
  • a sulfonate group is optionally substituted on R by one or more of the substituents described for alkyl, cycloalkyl, aryl, heteroaryl respectively.
  • Thiocyanato refers to a -CNS radical.
  • “Substituted” means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from acyl, alkyl, alkylaryl, cycloalkyl, aralkyl, aryl, carbohydrate, heteroaryl, heterocyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, ester, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, phosphate, silyl, sulfinyl, sulfonyl, sulfonamidyl, sulfoxyl, sulfonate, and amino, including mono- and di- substituted amino groups, and the protected derivatives thereof.
  • subsituents themselves may be substituted, for example, a cycloakyl substituent may have a halide substituted at one or more ring carbons, and the like.
  • the protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, above.
  • a “size-limited substituent” or “ size-limited substituent group,” as used herein means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 2O alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 4 -C 8 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
  • a "lower substituent” or " lower substituent group,” as used herein means a group selected from all of the substituents described above for a “substituent group,” wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 5 -C 7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
  • the compounds presented herein may possess one or more chiral centers and each center may exist in the R or S configuration.
  • the compounds presented herein include all diastereomeric, enantiomeric, and epimeric forms as well as the appropriate mixtures thereof. Stereoisomers may be obtained, if desired, by methods known in the art as, for example, the separation of stereoisomers by chiral chromatographic columns.
  • the methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of compounds having the structure of Formula (I), as well as active metabolites of these compounds having the same type of activity.
  • the compounds described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the compounds presented herein are also considered to be disclosed herein.
  • Compounds described can contain one or more asymmetric centers and may thus give rise to diastereomers and optical isomers.
  • the present invention includes all such possible diastereomers as well as their racemic mixtures, their substantially pure resolved enantiomers, all possible geometric isomers, and pharmaceutically acceptable salts thereof.
  • the above Formula I is shown without a definitive stereochemistry at certain positions.
  • the present invention includes all stereoisomers of Formula I and pharmaceutically acceptable salts thereof. Further, mixtures of stereoisomers as well as isolated specific stereoisomers are also included. During the course of the synthetic procedures used to prepare such compounds, or in using racemization or epimerization procedures known to those skilled in the art, the products of such procedures can be a mixture of stereoisomers.
  • the present invention includes all manner of rotamers and conformationally restricted states of a compound of the invention.
  • This invention pertains to the use of a distinct class of biologically active agents that exhibit selective inhibition of certain protein kinases, and the uses of these agents for treatment of diseases mediated by such protein kinases.
  • the present invention provides methods of treating ocular disorders by inhibiting angiogenesis.
  • the invention provides a method for inhibiting angiogenesis by administering an inhibitor of one or more type I phosphatidylinositol 3 -kinases (PI3 -kinase), wherein the one or more type I PI3 -kinase is selected from the group consisting of PI3 -kinase ⁇ , PI3 -kinase ⁇ , PI3 -kinase ⁇ , and PI3-kinase ⁇ .
  • PI3 -kinase type I phosphatidylinositol 3 -kinases
  • the method for inhibiting angiogenesis includes administration of a selective inhibitor of one or more type I PI3 -kinase selected from the group consisting of PI3 -kinase ⁇ , PI3- kinase ⁇ , PI3 -kinase ⁇ , and PI3 -kinase ⁇ .
  • the present invention provides a method for inhibiting angiogenesis comprising contacting a cell with a biologically active agent that selectively inhibits mTorCl and/or mTorC2 activity relative to one or more type I phosphatidylinositol 3-kinases (PI3-kinase), wherein the one or more type I PI3-kinase is selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • PI3-kinase type I phosphatidylinositol 3-kinases
  • the present invention provides a method for inhibiting angiogenesis comprising contacting a cell with a biologically active agent that inhibits mTorCl and/or mTorC2 activity as well as one or more type I phosphatidylinositol 3-kinases (PI3 -kinase), wherein the one or more type I PI3-kinase is selected from the group consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , PI3-kinase ⁇ , and PI3-kinase ⁇ .
  • a kinase antagonist which is a compound of Formula I, or a pharmaceutically acceptable salt thereof:
  • R 1 is hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 1 when R 1 is substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, or substituted heteroaryl, it is substituted by R 3 .
  • R 2 is halogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R when R is substituted aryl or substituted heteroaryl, it is R 4 -substituted aryl, or R 4 -substituted or unsubstituted heteroaryl.
  • R 2 is substituted aryl, it is R 4 -substituted aryl.
  • R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , and R 33 are independently hydrogen, R 35 -substituted or unsubstituted alkyl, R 35 -substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, R 35 -substituted or unsubstituted heterocycloalkyl, R 35 - substituted or unsubstituted aryl, or R 35 -substituted or unsubstituted heteroaryl.
  • R 19 , R 34 and R 35 are independently hydrogen, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 36 is halogen, -NR 37 R 38 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 37 and R 38 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 1 is hydrogen. In some embodiments, R 1 is halogen. In some embodiments, R 1 is unsubstituted alkyl. In some embodiments, R 1 is substituted alkyl. In some embodiments, R 1 is unsubstituted heteroalkyl. In some embodiments, R 1 is substituted heteroalkyl. In some embodiments, R 1 is unsubstituted cycloalkyl. In some embodiments, R 1 is substituted cycloalkyl. In some embodiments, R 1 is unsubstituted heterocycloalkyl. In some embodiments, R 1 is substituted heterocycloalkyl. In some embodiments, R 1 is unsubstituted aryl.
  • R 1 is substituted aryl. In some embodiments, R 1 is unsubstituted heteroaryl. In some embodiments, R 1 is substituted heteroaryl. [00122] In some embodiments, when R 1 is substituted alkyl, it is substituted by halogen. In some embodiments, when R 1 is substituted alkyl, it is substituted by -CN. In some embodiments, when R 1 is substituted alkyl, it is substituted by -OR 5 . In some embodiments, when R 1 is substituted alkyl, it is substituted by -S(O) n R 6 . In some embodiments, when R 1 is substituted alkyl, it is substituted by -NR 7 R 8 .
  • R 1 when R 1 is substituted alkyl, it is substituted by -NR 16 S(O) 2 R 17 . In some embodiments, when R 1 is substituted alkyl, it is substituted by -S(O) 2 NR 18 . In some embodiments, when R 1 is substituted alkyl, it is substituted by R 19 - substituted alkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by unsubstituted alkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by R 19 -substituted heteroalkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by unsubstituted heteroalkyl.
  • R 1 when R 1 is substituted alkyl, it is substituted by R 19 -substituted cycloalkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by R 19 -substituted heterocycloalkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by unsubstituted heterocycloalkyl. In some embodiments, when R 1 is substituted alkyl, it is substituted by R 19 -substituted aryl.
  • R 1 when R 1 is substituted alkyl, it is substituted by unsubstituted aryl. In some embodiments, when R 1 is substituted alkyl, it is substituted by R 19 -substituted heteroaryl. In some embodiments, when R 1 is substituted alkyl, it is substituted by unsubstituted heteroaryl. [00123] In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by halogen. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by -CN. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by -OR 5 .
  • R 1 when R 1 is substituted heteroalkyl, it is substituted by -NR 1 "-C(O)R 1 ⁇ In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by -NR 12 -C(O)-OR 13 . In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by - C(O)NR 14 R 15 . In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by -NR 16 S(O) 2 R 17 . In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by -S(O) 2 NR 18 .
  • R 1 when R 1 is substituted heteroalkyl, it is substituted by R 19 -substituted alkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by unsubstituted alkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by R 19 -substituted heteroalkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by unsubstituted heteroalkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by R 19 -substituted cycloalkyl.
  • R 1 when R 1 is substituted heteroalkyl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by R 19 -substituted heterocycloalkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by unsubstituted heterocycloalkyl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by R 19 -substituted aryl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by unsubstituted aryl.
  • R 1 when R 1 is substituted heteroalkyl, it is substituted by R 19 -substituted heteroaryl. In some embodiments, when R 1 is substituted heteroalkyl, it is substituted by unsubstituted heteroaryl.
  • R 1 when R 1 is substituted cycloalkyl, it is substituted by halogen. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by -CN. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by -OR 5 . In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by -S(O) n R 6 . In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by -NR 7 R 8 . In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by -C(O)R 9 .
  • R 1 when R 1 is substituted cycloalkyl, it is substituted by -NR 16 S(O) 2 R 17 . In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by -S(O) 2 NR 18 . In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by R 19 -substituted alkyl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by unsubstituted alkyl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by R 19 -substituted heteroalkyl.
  • R 1 when R 1 is substituted cycloalkyl, it is substituted by unsubstituted heteroalkyl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by R 19 -substituted cycloalkyl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by R 19 -substituted heterocycloalkyl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by unsubstituted heterocycloalkyl.
  • R 1 when R 1 is substituted cycloalkyl, it is substituted by R 19 -substituted aryl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by unsubstituted aryl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by R 19 -substituted heteroaryl. In some embodiments, when R 1 is substituted cycloalkyl, it is substituted by unsubstituted heteroaryl.
  • R 1 when R 1 is substituted heterocycloalkyl, it is substituted by halogen. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by -CN. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by -OR 5 . In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by -S(O) n R 6 . In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by -NR 7 R 8 . In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by -C(O)R 9 .
  • R 1 when R 1 is substituted heterocycloalkyl, it is substituted by -NR 16 S(O) 2 R 17 . In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by -S(O) 2 NR 18 . In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by R 19 -substituted alkyl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by unsubstituted alkyl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by R 19 -substituted heteroalkyl.
  • R 1 when R 1 is substituted heterocycloalkyl, it is substituted by unsubstituted heteroalkyl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by R 19 -substituted cycloalkyl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by R 19 -substituted heterocycloalkyl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by unsubstituted heterocycloalkyl.
  • R 1 when R 1 is substituted heterocycloalkyl, it is substituted by R 19 -substituted aryl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by unsubstituted aryl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by R 19 -substituted heteroaryl. In some embodiments, when R 1 is substituted heterocycloalkyl, it is substituted by unsubstituted heteroaryl.
  • R 1 when R 1 is substituted aryl, it is substituted by halogen. In some embodiments, when R 1 is substituted aryl, it is substituted by -CN. In some embodiments, when R 1 is substituted aryl, it is substituted by -OR 5 . In some embodiments, when R 1 is substituted aryl, it is substituted by -S(O) n R 6 . In some embodiments, when R 1 is substituted aryl, it is substituted by -NR 7 R 8 . In some embodiments, when R 1 is substituted aryl, it is substituted by -C(O)R 9 .
  • R 1 when R 1 is substituted aryl, it is substituted by - S(O) 2 NR 18 . In some embodiments, when R 1 is substituted aryl, it is substituted by R 19 -substituted alkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by unsubstituted alkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by R 19 -substituted heteroalkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by unsubstituted heteroalkyl.
  • R 1 when R 1 is substituted aryl, it is substituted by R 19 -substituted cycloalkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by R 19 -substituted heterocycloalkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by unsubstituted heterocycloalkyl. In some embodiments, when R 1 is substituted aryl, it is substituted by R 19 - substituted aryl.
  • R 1 when R 1 is substituted aryl, it is substituted by unsubstituted aryl. In some embodiments, when R 1 is substituted aryl, it is substituted by R 19 -substituted heteroaryl. In some embodiments, when R 1 is substituted aryl, it is substituted by unsubstituted heteroaryl. [00127] In some embodiments, when R 1 is substituted heteroaryl, it is substituted by halogen. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by -CN. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by -OR 5 .
  • R 1 when R 1 is substituted heteroaryl, it is substituted by -NR 1 "-C(O)R 1 ⁇ In some embodiments, when R 1 is substituted heteroaryl, it is substituted by -NR 12 -C(O)-OR 13 . In some embodiments, when R 1 is substituted heteroaryl, it is substituted by - C(O)NR 14 R 15 . In some embodiments, when R 1 is substituted heteroaryl, it is substituted by -NR 16 S(O) 2 R 17 . In some embodiments, when R 1 is substituted heteroaryl, it is substituted by -S(O) 2 NR 18 .
  • R 1 when R 1 is substituted heteroaryl, it is substituted by R 19 -substituted alkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by unsubstituted alkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by R 19 -substituted heteroalkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by unsubstituted heteroalkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by R 19 -substituted cycloalkyl.
  • R 1 when R 1 is substituted heteroaryl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by R 19 -substituted heterocycloalkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by unsubstituted heterocycloalkyl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by R 19 -substituted aryl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by unsubstituted aryl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by R 19 -substituted heteroaryl. In some embodiments, when R 1 is substituted heteroaryl, it is substituted by unsubstituted heteroaryl.
  • R is halogen. In some embodiments, R is unsubstituted alkyl. In some embodiments, R 2 is substituted alkyl. In some embodiments, R 2 is unsubstituted heteroalkyl. In some embodiments, R 2 is substituted heteroalkyl. In some embodiments, R 2 is unsubstituted cycloalkyl. In some embodiments, R 2 is substituted cycloalkyl. In some embodiments, R 2 is unsubstituted heterocycloalkyl. In some embodiments, R 2 is substituted heterocycloalkyl. In some embodiments, R 2 is unsubstituted aryl. In some embodiments, R 2 is substituted aryl. In some embodiments, R 2 is unsubstituted heteroaryl. In some embodiments, R 2 is substituted heteroaryl.
  • R 2 when R 2 is substituted aryl, it is substituted by halogen, In some embodiments, when R is substituted aryl, it is substituted by -CN. In some embodiments, when R is substituted aryl, it is substituted by -OR 20 . In some embodiments, when R 2 is substituted aryl, it is substituted by -S(O) q R 21 , where the symbol q represents an integer from 0 to 2 In some embodiments, when R 2 is substituted aryl, it is substituted by -NR 22 R 23 . In some embodiments, when R 2 is substituted aryl, it is substituted by -C(O)R 24 .
  • R 2 when R 2 is substituted aryl, it is substituted by -S(O) 2 NR 33 . In some embodiments, when R 2 is substituted aryl, it is substituted by R 34 -substituted alkyl. In some embodiments, when R is substituted aryl, it is substituted by unsubstituted alkyl. In some embodiments, when R is substituted aryl, it is substituted by R 34 -substituted or unsubstituted heteroalkyl. In some embodiments, when R 2 is substituted aryl, it is substituted by R 34 -substituted heteroalkyl.
  • R 2 when R 2 is substituted aryl, it is substituted by unsubstituted heteroalkyl. In some embodiments, when R 2 is substituted aryl, it is substituted by R 34 -substituted cycloalkyl. In some embodiments, when R 2 is substituted aryl, it is substituted by unsubstituted cycloalkyl. In some embodiments, when R 2 is substituted aryl, it is substituted by R 34 -substituted heterocycloalkyl. In some embodiments, when R 2 is substituted aryl, it is substituted by unsubstituted heterocycloalkyl.
  • R 2 when R 2 is substituted aryl, it is substituted by R 34 -substituted aryl. In some embodiments, when R 2 is substituted aryl, it is substituted by unsubstituted aryl. In some embodiments, when R 2 is substituted aryl, it is substituted by R 34 -substituted heteroaryl. In some embodiments, when R 2 is substituted aryl, it is substituted by unsubstituted heteroaryl.
  • R 36 is halogen. In some embodiments, R 36 is -NR 37 R 38 . In some embodiments, R 36 is unsubstituted alkyl. In some embodiments, R 36 is substituted alkyl. In some embodiments, R 36 is unsubstituted heteroalkyl. In some embodiments, R 36 is substituted heteroalkyl. In some embodiments, R 36 is unsubstituted cycloalkyl. In some embodiments, R 36 is substituted cycloalkyl. In some embodiments, R 36 is unsubstituted heterocycloalkyl. In some embodiments, R 36 is substituted heterocycloalkyl. In some embodiments, R 36 is unsubstituted aryl.
  • R 36 is substituted aryl. In some embodiments, R 36 is unsubstituted heteroaryl. In some embodiments, R 36 is substituted heteroaryl. [00131] In some embodiments, R 37 and R 38 are independently hydrogen, or unsubstituted alkyl. [00132] In some embodiments, R 36 is -NH 2 , and the kinase antagonist is of the formula:
  • R 1 , R 2 , and X are as defined above in Formula I.
  • R 1 is hydrogen, R 3 -substituted or unsubstituted alkyl, R 3 - substituted or unsubstituted heteroalkyl, R 3 -substituted or unsubstituted cycloalkyl, R 3 -substituted or unsubstituted heterocycloalkyl, R 3 -substituted or unsubstituted aryl, or R 3 -substituted or unsubstituted heteroaryl.
  • R 2 is halogen, R 4 -substituted aryl, or substituted or unsubstituted heteroaryl.
  • NR 27 -C(O)-OR 28 , -C(O)NR 29 R 30 , -NR 31 S(O) 2 R 32 , -S(O) 2 NR 33 , R 34 -substituted or unsubstituted alkyl, R 34 - substituted or unsubstituted heteroalkyl, R 34 -substituted or unsubstituted cycloalkyl, R 34 -substituted or unsubstituted heterocycloalkyl, R 34 -substituted or unsubstituted aryl, or R 34 -substituted or unsubstituted heteroaryl.
  • the symbol q represents an integer from 0 to 2.
  • R 29 , R 30 , R 31 , R 32 , and R 33 are independently hydrogen, R 35 -substituted or unsubstituted alkyl, R 35 -substituted or unsubstituted heteroalkyl, unsubstituted cycloalkyl, R 35 -substituted or unsubstituted heterocycloalkyl, R 35 - substituted or unsubstituted aryl, or R 35 -substituted or unsubstituted heteroaryl.
  • R 19 , R 34 and R 35 are independently hydrogen, halogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , and R 33 are independently hydrogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.
  • R 20 , R 21 , R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , R 28 , R 29 , R 30 , R 31 , R 32 , and R 33 are independently hydrogen, unsubstituted alkyl, or unsubstituted heteroalkyl.
  • R 1 is R 3 -substituted or unsubstituted alkyl, R 3 -substituted or unsubstituted cycloalkyl, or R 3 -substituted or unsubstituted aryl. In other embodiments, R 1 is R 3 -substituted or unsubstituted alkyl, or R 3 -substituted or unsubstituted cycloalkyl. In some embodiments, R 1 is R 3 -substituted or unsubstituted
  • R 1 is R 3 -substituted or unsubstituted C 1 -C 4 alkyl, or R 3 -substituted or unsubstituted cyclopentyl.
  • R 1 is methyl or unsubstituted C 3 -C 6 branched alkyl (e.g. isopropyl, isobutyl, etc.).
  • R 3 is R 19 -substituted or unsubstituted alkyl, R 19 -substituted or unsubstituted cycloalkyl, or R 19 -substituted or unsubstituted aryl. In other embodiments, R 3 is R 19 -substituted or unsubstituted alkyl, R 19 -substituted or unsubstituted cycloalkyl, or R 19 -substituted or unsubstituted aryl. In some embodiments, R 3 is R 19 -substituted or unsubstituted alkyl, or R 19 -substituted or unsubstituted cycloalkyl.
  • R 19 is unsubstituted alkyl or unsubstituted cycloalkyl. In some embodiments, R 19 is unsubstituted Ci-C 4 alkyl or unsubstituted cyclopentyl. [00143] In some embodiments, R 2 is R 4 -substituted aryl, or R 4 -substituted or unsubstituted heteroaryl.
  • R 2 may be R 4 -substituted phenyl, R 4 -substituted or unsubstituted naphthyl, R 4 -substituted or unsubstituted pyridinyl, R 4 -substituted or unsubstituted pyrimidinyl, R 4 -substituted or unsubstituted thiophenyl, R 4 -substituted or unsubstituted furanyl, R 4 -substituted or unsubstituted indolyl, R 4 -substituted or unsubstituted benzoxadiazolyl, R 4 -substituted or unsubstituted benzodioxolyl, R 4 -substituted or unsubstituted benzodioxanyl, R 4 -substituted or unsubstituted thianaphthanyl, R 4
  • R 2 is R 4 -substituted phenyl, R 4 -substituted or unsubstituted pyrrolepyridinyl, R 4 -substituted or unsubstituted quinolinyl, R 4 -substituted or unsubstituted indazolyl, R 4 -substituted or unsubstituted quinolinyl, R 4 -substituted or unsubstituted indolyl, or R 4 -substituted or unsubstituted naphthyl.
  • R 4 is halogen, -CN, -OR 20 , or -NR 22 R 23 . In other embodiments, R 4 is halogen, or -OR 20 . In some embodiments, R 20 is hydrogen, alkyl or aryl. In various embodiments, R 20 is hydrogen. In some embodiments, R 4 is-OH.
  • R 2 is a moiety of one of the following structures:
  • R 2 is 5-hydroxy indol-2-yl. In some embodiments, R 2 is indol-2-yl.
  • R is a moiety of Formula IV:
  • ring A is aromatic; W 1 , W 2 , and W 3 are independently CR 4 or N; W 4 is CH, N, or O, W 5 is N, O, NH or NR a ; W 6 is CR 4 or N; no two adjacent ring atoms are oxygen and/or sulfur and no more than two adjacent ring atoms are nitrogen.
  • ring A comprises an oxygen ring atom and a nitrogen ring atom.
  • W 4 is CH, W 5 is NH and W 6 is N or CR 4 ; W 4 is CH, W 5 is NR a and W 6 is N or CR 4 ; W 4 is CH or N, W 5 is O and W 6 is N or CR 4 ; W 4 is N, W 5 is NH and W 6 is CR 4 ; W 4 is N, W 5 is O and W 6 is N or CR 4 ; or W 4 is O, W 5 is N and W 6 is N or CR 4 . In some embodiments, W 4 is N, W 5 is O and W 6 is CR 4 .
  • R 2 is a moiety of Formula V:
  • W 1 , W 2 , and W 3 are independently CH, CR 4 or N; W 4 and W 6 are independently CH, CR 4 , or N; and W 7 is CH or N; and wherein no more than two adjacent ring atoms are N.
  • R 1 is R 3 - substituted or unsubstituted alkyl, or R 3 - substituted or unsubstituted heterocycloalkyl
  • R 2 is a moiety of Formula VI:
  • ring A is a five membered aromatic ring; W 1 , W 2 , and W 3 are independently CR 4 or N; W 5 is N, O, NH or NR 4 ; and wherein R 4 is -C(O)Oalkyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • ring A further comprises no more than three heteroatoms.
  • R 1 is alkyl.
  • R 1 is isopropyl.
  • R 1 is substituted heterocycloalkyl.
  • R 2 is a moiety of the following structure:
  • Each R 4 is as defined above in the description of Formulae I and II.
  • Ring A is a substituted or unsubstituted heteroaryl or substituted or unsubstituted heterocycloalkyl. In some embodiments, ring A is a 6 to 7 membered heterocycloalkyl or 6 to 7 membered heteroaryl. Thus, in some embodiments, ring A is partially or fully unsaturated 6- or 7- membered ring.
  • R may be hydrogen or unsubstituted Ci-Cio alkyl. In some embodiments, R is hydrogen or unsubstituted Ci-C 4 alkyl. R 20 may also simply be hydrogen or methyl.
  • R 2 is of one of the following formulae:
  • R 4 is absent, halogen, unsubstituted C 1 -C 4 alkyl, or -OR 20 .
  • the halogen may be F, Cl, or Br. In some embodiments, the halogen is F or Cl. In other embodiments, the halogen is F. In some embodiments, R 20 is hydrogen or unsubstituted Ci-C 4 alkyl.
  • R is 6-hydroxynaphthyl, unsubstituted 7-azaindole, unsubstituted indolyl, unsubstituted indazolyl, or unsubstituted quinolinyl.
  • R 2 is one of the following formulae:
  • R 20 is as defined above. It is noted that, in accordance with the description of R 20 above, each R 20 is optionally different.
  • the symbol z is an integer from 1 to 5 (e.g. 1 or 2).
  • R 20 is hydrogen or unsubstituted Ci-Ci 0 alkyl (e.g. Ci-C 5 alkyl such as methyl or ethyl).
  • R 2 is a moiety of one of the following formulae:
  • R 20 is as defined above, for example, in the description of Formulae I, II, X, and XI above.
  • each substituted group described above for the compounds of the present invention is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, aryl(Ci-C 6 )alkyl, and heteroaryl(Ci-C 6 )alkyl described above is substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. Alternatively, at least one or all of these groups are substituted with at least one lower substituent group.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 2O alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 4 -C 8 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 5 -C 7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl.
  • the present invention provides compound of Formula Ia or Ib for use in the methods of the invention:
  • Z is NR 3 R 4 , halo, H, ,OH, alkyl, alkyloxy, or haloalkyl;
  • R 1 is Q-C 6 alkyl or C 4 -C 7 cycloalkyl, wherein said alkyl or said cycloalkyl is optionally substituted with mono- or di-alkoxy, mono- or di-halophenyl, mono- or di-(C 1 . 4 )alkoxy phenyl, mono- or di-(C 1 . 4 )alkyl phenyl, phenyl, carboxyl, tert-b ⁇ xty ⁇ carboxyl, phosphoryl, (Ci_ 6 )alkyl, (C 4 .
  • R 2a is indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, or phenyl, wherein said phenyl is substituted with at least one of OH, -NR 3 R 4 , -C(O)NR 6 R 7 , -CN, NO 2 -C(O)OH, -C(0)0-alkyl, (Ci_C 4 )alkyl, halo, haloalkyl or haloaryl; and wherein said indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl, or pyrrolyl is optionally substituted with OH, -NR 3 R 4 , -C(O)NR 6 R 7 , -CN, NO 2 , -C(O)O-R 3 , (Ci_C 4 )alkyl, halo, haloalkyl or halo
  • R 2b is indolyl, thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, or phenyl wherein said indoyl, thiazolyl, benzyl, biphenylyl, thiophenyl, pyrrolyl, phenyl is optionally substituted with -OH, -NR 3 R 4 , -C(O)NR 6 R 7 , -CN, NO 2 , -C(O)O-R 3 , (C r C 4 )alkyl, halo, haloalkyl, or haloaryl; [00174] R 3 and R 4 are independently H, Ci-C 6 alkyl, t-Boc, morpholino(Ci-C 4 )alkyl, carboxy(C r C 3 )alkyl, (Ci-C 4 )alkoxycarbonyl(Ci-C 3 )alkyl
  • R 5 are independently H, -OH, halo, optionally monosubstituted (Ci-C 6 )alkyl, optionally monosubstituted (Ci-C 4 )alkoxycarbonyl, optionally monosubstituted (Ci-C 4 )alkanoyl, carbamoyl, optionally monosubstituted (Ci-C 4 )alkyl carbamoyl, phenyl, halophenyl, optionally monosubstituted (Ci-C 4 )alkylphenyl, optionally monosubstituted (Ci-C 4 )alkoxyphenyl, or optionally monosubstituted perhalo(Ci-C 4 )alkylphenyl, wherein said optional substitution is (Ci-C 4 )alkyl, OH, or halogen; [00176] R ⁇ and R 7 are independently H, alkyl, aryl, heteroaryl, alkylaryl,
  • Y is N.
  • R 2a or R 2b is phenyl substituted with mono, di or tri-OH.
  • the phenyl is further substituted with a halo.
  • the halo is F.
  • R 2a or R 2b is indolyl.
  • R 2a or R 2b is 2-indolyl.
  • R 1 is 2-methyl-propane.
  • R 3 and R 4 are H.
  • R 5 is H.
  • R 6 is H and R 7 is methyl.
  • R 2a is, independently, phenyl substituted at a meta position with -CH 3 , tert- butyl, -CF 3 or halo.
  • R 2a is, independently, phenyl substituted at a meta position with halo, alkyl, haloalkyl, haloaryl, aryl, O-alkyl, CN, NO 2 , CO-O-R 3 , CO-N(R 3 ) 2 .
  • Z is F, Br Cl, or I.
  • the compounds of Formula Ia or Formula Ib include: 3-(4-amino-7-isopropyl-
  • the invention provides a PI3-kinase antagonists which is a compound of Formula
  • L 1 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene.
  • R 1 and R 2 are independently halogen, -CN, -OR 5 , -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2.
  • R 3 and R 4 are independently hydrogen, halogen, -CN, -OR 5 , -S(O) n R 6 , -NR 7 R 8 , -C(O)R 9 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl, where n is an integer from 0 to 2.
  • R 5 is independently hydrogen, -C(O)R 10 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 6 is independently hydrogen, -NR 11 R 12 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. Where n is 1 or 2, R 6 is other than hydrogen.
  • R 7 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 8 is independently hydrogen, -S(O) n R 13 , - C(O)R 14 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 9 is independently -NR 15 R 16 , hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 10 is independently hydrogen, -NR 17 R 18 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 14 is independently hydrogen, -NR 19 R 20 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and
  • R 11 , R 12 , R 13 , R 15 , R 16 , R 17 , R 18 , R 19 , and R 20 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.
  • R 1 is halogen, substituted or unsubstituted halo(Ci-C 6 )alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted aryl(Ci-C 6 )alkyl, or substituted or unsubstituted heteroary ⁇ Q-C ⁇ alkyl.
  • R 1 is halogen, substituted or unsubstituted phenyl, substituted or unsubstituted furanyl, substituted or unsubstituted pyrrolyl, substituted or unsubstituted thiophenyl, or substituted or unsubstituted benzothiophenyl, substituted or unsubstituted indolyl, substituted or unsubstituted quinolinyl, substituted or unsubstituted pyridinyl, substituted or unsubstituted IH- pyrrolo[2,3-c]pyridinyl, substituted or unsubstituted lH-pyrrolo[2,3- ⁇ ]pyridinyl, substituted or unsubstituted thiazolyl, substituted or unsubstituted imidazolyl, substituted or unsubstituted oxazolyl, substituted or unsubstituted isoxazolyl, substituted or
  • R 1 is phenyl, furanyl, pyrrolyl, thiophenyl, or benzothiophenyl, each of which are optionally substituted with one or more R 21 substituent(s).
  • R 21 is independently (1) or (2) as defined in this paragraph.
  • R 21 is halogen, -CN, -OR 22 , -C(O)R 23 , -NR 24 R 25 , -S(O) W NR 26 R 27 , or - S(O) W R 28 .
  • the symbol w is an integer from 0 to 2.
  • R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , and R 28 are independently hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyl-alkyl, heterocycloalkyl- alkyl, arylalkyl, or heteroarylalkyl, optionally substituted with unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl, unsubstituted heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted heteroarylalkyl.
  • R 21 is (Ci-Cio)alkyl, 2 to 10 membered heteroalkyl, C 3 -C 8 cycloalkyl, 3 to 8 membered heterocycloalkyl, aryl or heteroaryl optionally substituted with halogen, -OH, -CN, -NH 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl, unsubstituted heterocycloalkyl- alkyl, unsubstituted arylalkyl, or unsubstituted heteroarylalkyl.
  • R 1 is phenyl substituted at the meta and para positions, or substituted at the meta and meta positions. That is, R 1 is a 4,5-substituted phenyl or a 3,5-substituted phenyl. In some related embodiments, the 4,5-substituted phenyl or 3,5-substituted phenyl is substituted, independently, with R 21 (as defined in the previous paragraph). In some embodiments, R 21 is halogen or -OR 22 . In other embodiments, R 21 is fluorine and in various embodiments, R 22 is hydrogen or unsubstituted C 1 -C 4 alkyl (e.g. methyl). In other embodiments, R 1 is phenyl substituted para position (i.e. a 4-substituted phenyl).
  • L 1 is substituted or unsubstituted alkylene (e.g. a substituted or unsubstituted alkynylene.
  • L 1 is substituted or unsubstituted methylene, substituted or unsubstituted ethylene, substituted or unsubstituted propylene, substituted or unsubstituted butylenes, substituted or unsubstituted ethynylene, or substituted or unsubstituted prop-2-ynylene.
  • R 1 is - CN, -OR 5 , NR 7 R 8 , R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted aryl, R 21 - substituted or unsubstituted heteroaryl, R 21 -substituted or unsubstituted C 1 -C 4 alkyl.
  • R 21 may be halogen, - OR 22 , -NR 24 R 25 , or unsubstituted C 1 -C 4 alkyl.
  • R 5 , R 7 , R 8 , R 22 , R 24 and R 25 are independently hydrogen or unsubstituted C 1 -C 4 alkyl (e.g. methyl).
  • R 2 is halogen, -OH, -CN, -NH 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl, unsubstituted heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted heteroarylalkyl.
  • R 2 is halogen or unsubstituted alkyl.
  • R 2 is fluorine or unsubstituted C 1 -C 4 alkyl (e.g. methyl).
  • R 3 is halogen, -OH, -CN, -NH 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl, unsubstituted heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted heteroarylalkyl.
  • R 3 is unsubstituted C 1 -C 4 alkyl (e.g. methyl).
  • R 4 is halogen, -OH, -CN, -NH 2 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, unsubstituted cycloalkyl-alkyl, unsubstituted heterocycloalkyl-alkyl, unsubstituted arylalkyl, or unsubstituted heteroarylalkyl.
  • R 2 and R 3 are independently unsubstituted C 1 -C 4 alkyl, R 4 is NH 2 , q is 1, and z is l.
  • each substituted group described above in the compound of Formula XV is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted cycloalkyl-alkyl, substituted heterocycloalkyl-alkyl, substituted arylalkyl, and/or substituted heteroarylalkyl, described above in the compounds of Formula XV is substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size- limited substituent group. Alternatively, at least one or all of these groups are substituted with at least one lower substituent group.
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted C 1 -C 2O alkyl, including those alkyl groups forming part of a cycloalkyl-alkyl (i.e. a cycloalkyl-(C 1 -C 20 )alkyl), heterocycloalkyl-alkyl (i.e. a heterocycloalkyl-(C 1 -C 20 )alkyl), arylalkyl (i.e. an aryl- (Ci-C 2 o)alkyl), or substituted heteroarylalkyl (i.e.
  • Each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl.
  • Each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 4 -C 8 cycloalkyl, including those cycloalkyl groups forming part of a cycloalkyl-alkyl (i.e. a GrCg cycloalkyl-alkyl, or a C 4 -C 8 cycloalkyl-(Ci-C 2 o)alkyl).
  • Each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl, including those heterocycloalkyl groups forming part of a heterocycloalkyl-alkyl (i.e. a 4 to 8 membered heterocycloalkyl- alkyl, or a 4 to 8 membered heterocycloalkyl-(Ci-C 2 o)alkyl).
  • each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C 8 alkyl
  • each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl
  • each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C 5 -C 7 cycloalkyl
  • each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl, including cycloalkyl-alkyl groups, heterocycloalkyl-alkyl groups, heteroarylalkyl groups, and arylalkyl groups, as described in the preceding paragraph.
  • R 1 is unsubstituted or substituted aryl. In some embodiments of Formula XV, R 1 is aryl which is substituted at the meta and para positions, or substituted at the meta and meta positions.
  • the substituents independently are (1) halogen, -CN, -OR 22 , -C(O)R 23 , - NR 24 R 25 , -S(O) W NR 26 R 27 , or -S(O) W R 28 , wherein w is an integer from 0 to 2, and R 22 , R 23 , R 24 , R 25 , R 26 , R 27 , and R 28 are independently hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyl- alkyl, heterocycloalkyl-alkyl, arylalkyl, or heteroarylalkyl, optionally substituted with unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl,
  • L 1 is a covalent bond.
  • L 1 is substituted alkylene, and is substituted by unsubstituted or substituted cycloalkyl, unsubstituted or substituted aryl, -OR', -NR'-C(O)NR"R'", -NR'R", -CN, wherein R', R", R'" and R"" each independently are hydrogen or substituted or unsubstituted alkyl.
  • R is unsubstituted or substituted alkyl.
  • R 3 is unsubstituted or substituted alkyl.
  • R 4 is NR 7 R 8 .
  • R 4 is NH 2 .
  • a +++ indicates an IC50 of less than 1 ⁇ M; a ++ indicates an IC50 of from 1 ⁇ M to 50 ⁇ M; and a + indicates and IC50 of more than 50 ⁇ M.
  • the symbol +++ represents an IC50 of less than 1 ⁇ M; the symbol ++ represents an IC50 value froml ⁇ M to 100 ⁇ M; and + represents an IC50 value of more than 100 ⁇ M.
  • Scheme 1 depicts the synthesis of 2-( 4-amino-lH-pyrazolo[3,4-d]pyrimidin-3-yl) iodide.
  • Cyano substituted aminopyrazole 1-1 is heated with formamide at 160 0 C for 5 hours to yield 2-( 4-amino-lH- pyrazolo[3,4-d]pyrimidine (compound 1-2) in 90% yield.
  • This intermediate is reacted with N-iodosuccinimide in dimethylformamide at 80 0 C for 16 hours, to produce 2-( 4-amino-lH-pyrazolo[3,4-d]pyrimidin-3-yl) iodide
  • Compound 2-4 is synthesized as shown in Scheme 2.
  • Compound 1-3 is reacted with isopropyl bromide in dimethylformamide with potassium carbonate at 80 0 C, to provide the 1 -isopropyl pyrazolopyrimidine intermediate, compound 2-1.
  • This intermediate is reacted with the protected indolyl boronic acid species 2-2, using tetrakistriphenylphosphine palladium catalysis in DME-water solvent at 80 0 C for 4-5 hours, to produce the Suzuki coupling product, compound 2-3. Removal of the protecting groups with acid in dioxane yields the product (Cpd. 2-4).
  • Scheme 4 illustrates synthetic routes to certain compounds listed in Table 5. Using the information provided in Scheme 4, and the detailed synthesis information of certain compounds provided below, one skilled in the art would immediately recognize the synthetic routes to the compounds of the present invention. Detailed Synthesis of Certain Compounds
  • compositions for ocular administration In some embodiments, the invention provides a pharmaceutical composition for ocular administration containing a compound of the present invention, and a pharmaceutical excipient suitable for ocular administration.
  • the invention provides a liquid pharmaceutical composition for ocular administration containing: (i) an effective amount of a compound of the present invention; (ii) an effective amount of a second agent; and (iii) a pharmaceutical excipient suitable for ocular administration.
  • the composition further contains: (iv) an effective amount of a third agent.
  • Pharmaceutical compositions of the invention suitable for ocular administration can be presented as discrete dosage forms, such as drops or sprays each containing a predetermined amount of an active ingredient a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion.
  • Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient into association with the carrier, which constitutes one or more necessary ingredients.
  • the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then if necessary, dissolving or diluting in a liquid diluent.
  • This invention further encompasses anhydrous pharmaceutical compositions and dosage forms comprising an active ingredient, since water can facilitate the degradation of some compounds.
  • water may be added (e.g., 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf- life or the stability of formulations over time.
  • Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained.
  • anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.
  • An active ingredient can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
  • the carrier can take a wide variety of forms depending on the form of preparation desired for administration.
  • any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, preservatives, coloring agents, and the like in the case of liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used.
  • Carriers suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystalline cellulose, and mixtures thereof.
  • natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrroli
  • Lubricants which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e.g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof.
  • Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, or mixtures thereof.
  • a lubricant can optionally be added, in an amount of less than about 1 weight percent of the pharmaceutical composition.
  • the essential active ingredient therein may be combined with various coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.
  • Surfactant which can be used to form pharmaceutical compositions and dosage forms of the invention include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed. [00259] A suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10.
  • HLB hydrophilic-lipophilic balance
  • Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions.
  • Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable.
  • lipophilic (i.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10.
  • Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and derivatives thereof; lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-g
  • preferred ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophospholipids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
  • Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidylcholine, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG-phosphatidylethanolamine, PVP-phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono/diacetylated tartaric acid esters of mono/diglycerides, citric acid esters of mono/diglycerides, cholylsarcosine, caproate, caprylate
  • Hydrophilic non-ionic surfactants may include, but not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; polyoxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; polyglycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene steas; poly
  • the polyol may be glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, or a saccharide.
  • Other hydrophilic-non-ionic surfactants include, without limitation, PEG-10 laurate, PEG- 12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG-15 stearate, PEG-32 distearate, PEG-40 stearate, PEG-100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glycerol
  • Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and mixtures thereof.
  • preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.
  • the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present invention and to minimize precipitation of the compound of the present invention. This can be especially important for compositions for injection.
  • a solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.
  • solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methylcellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (glycofurol) or methoxy PEG ; amides and other nitrogen-containing compounds such as 2-pyrrolidone, 2-piperidone,
  • solubilizers may also be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N-methylpyrrolidone, N- hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.
  • the amount of solubilizer that can be included is not particularly limited.
  • the amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art.
  • the solubilizer can be in a weight ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug, and other excipients.
  • very small amounts of solubilizer may also be used, such as 5%, 2%, 1% or even less.
  • the solubilizer may be present in an amount of about 1% to about 100%, more typically about 5% to about 25% by weight.
  • the composition can further include one or more pharmaceutically acceptable additives and excipients.
  • additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.
  • an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability, or for other reasons.
  • Examples of pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like.
  • bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thiogly colic acid, toluene sulfonic acid, uric acid, and the like.
  • a pharmaceutically acceptable acid such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino
  • Salts of polyprotic acids such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used.
  • the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals, alkaline earth metals, and the like.
  • Example may include, but not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.
  • Suitable acids are pharmaceutically acceptable organic or inorganic acids.
  • suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like.
  • suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p- toluene sulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thioglycolic acid, toluenesulfonic acid, uric acid and the like.
  • the composition includes an ophthalmic emulsion to increase the bioavailability or intraocular penetrance of the compounds by electrostatic attraction between the emulsions's positive charge and the negative charges carried at the eye surface.
  • emulsions must be stabilized to provide a useful shelf- life.
  • One known approach to stabilize an emulsion is to confer an electrostatic charge to the droplets surface which will result in droplet repulsion and less droplet coalescence. Colloidal particles dispersed in a solution are electrically charged due to their ionic characteristics and dipole attributes.
  • U.S. Pat. No. 6,007,826 discloses a cationic oil-in-water emulsion which comprises colloid particles with a positively charged interfacial film.
  • the interfacial film is formed by cationic lipids (0.05-3% by weight) such as C 10-Cl 4 primary alkylamines (disclosed are stearylamine or oleylamine), C10-C24 primary alkanolamine or a cholesterol betainate; phospholipids (0.5-3%) and non-ionic surfactants from the group consisting of poloxamers, tyloxapol, polysorbate, and polyoxyethylene fatty acid esters (0.05-3%).
  • the concentration of the oily core is maintained within the 3-20% range.
  • U.S. Patent Application No. 12/089609 discloses a cationic oil-in-water emulsion which provides enhanced stability of the drug compounds emulsified therein.
  • the cation content within the formulation should not exceed 0.1%, preferably not exceed 0.05% and even more preferably should not exceed 0.03%.
  • Quaternary amines such as benzalkonium chloride, benzododecinium bromide and benzethonium chloride are allowed by health authorities for ophthalmic administration up to concentration of approximately 0.03% (Furrer et al., Eur. J. Pharm. Biopharm. 2002, 53:263-280).
  • cationic-oil-in-water emulsions comprise one or more PI3K antagonists or mTORCl/mTORC2 inhibitors, at least one cationic agent and at least one non ionic surfactant selected from selected from the group consisting of poloxamers, tyloxapol, polysorbates, polyoxy ethylene castor oil derivatives, sorbitan esters, polyoxyl stearates and a mixture of two or more thereof.
  • the emulsion may further comprise an antiinflammatory compound, preferably a non steroidal anti-inflammatory compound or a omega-3 fatty acid.
  • Antiinflammatory agents may be chosen in the group comprising COX-2 inhibitors, salicylates, 2-arylpropionic acids, N-arylanthranilic acids, oxicams, sulphonanilides, pyrazolidines derivatives, arylalkanoic acids, 3- benzolphenylacetic acids and derivatives; steroids such as cortisone, hydrocortisone, prednisone, prednisolone, methylprednisone, fluoromethalone, medrysone, betamethasone, loteprednol, flumethasone, mometasone, testosterone, methyltestosterone, danazol, beclomethasone, dexamethasone, dexamethasone palmitate, tramcinolone, triamcinolone acetonide, fluocinolone, fluocinolone acetonide, difluprednate, rimexolone.
  • steroids such as cortisone, hydrocortisone,
  • the oil phase comprises one or more components selected from the group comprising or consisting of mineral oil such as for example petrolatum and liquid paraffin, and light mineral oil, medium chain triglycerides (MCT) which is generally defined as a triglyceride oil in which the carbohydrate chain has about 8-12 carbon atoms, coconut oil; hydrogenated oils comprising hydrogenated cottonseed oil, hydrogenated palm oil, hydrogenate castor oil or hydrogenated soybean oil; polyoxyethylene hydrogenated castor oil derivatives comprising poluoxyl-40 hydrogenated castor oil, polyoxyl- 60 hydrogenated castor oil or polyoxyl- 100 hydrogenated castor oil.
  • mineral oil such as for example petrolatum and liquid paraffin
  • MCT medium chain triglycerides
  • the present invention provides methods and compositions for treating diseases and conditions related to angiogenesis, especially angiogenesis mediated by ocular cells or taking place inside a subject's eye.
  • Angiogenesis can be regulated by signal transduction pathways which include a variety of signaling molecules such as kinases.
  • Kinases can generally be classified into protein kinases and lipid kinases, and certain kinases exhibit dual specificities. Protein kinases are enzymes that phosphorylate other proteins and/or themselves (i.e., autophosphorylation).
  • Protein kinases can be generally classified into three major groups based upon their substrate utilization: tyrosine kinases which predominantly phosphorylate substrates on tyrosine residues (e.g., erb2, PDGF receptor, EGF receptor, VEGF receptor, src, abl), serine/threonine kinases which predominantly phosphorylate substrates on serine and/or threonine residues (e.g., mTorCl, mTorC2, ATM, ATR, DNA-PK, Akt), and dual-specificity kinases which phosphorylate substrates on tyrosine, serine and/or threonine residues.
  • tyrosine kinases which predominantly phosphorylate substrates on tyrosine residues (e.g., erb2, PDGF receptor, EGF receptor, VEGF receptor, src, abl), serine/threonine kinases which predominantly phosphorylate substrates
  • Lipid kinases are enzymes that catalyze the phosphorylation of lipids within cells. These enzymes, and the resulting phosphorylated lipids and lipid derived biologically active organic molecules, play a role in many different physiological processes, including cell proliferation, migration, adhesion, and differentiation.
  • a particular group of lipid kinases comprises membrane lipid kinases, i.e., kinases that catalyze the phosphorylation of lipids contained in or associated with cell membranes.
  • phosphinositide(s) kinases such as PI3-kinases, PI4-Kinases
  • diacylglycerol kinases phosphoinositide 3-kinases
  • sphingosine kinases phosphoinositide 3-kinases
  • the PI3K family comprises 15 kinases with distinct substrate specificities, expression patterns, and modes of regulation (Katso et al., 2001).
  • the class I PI3Ks (pi 10a, pi lO ⁇ , pi lO ⁇ , and pi lO ⁇ ) are typically activated by tyrosine kinases such as VEGF- R or G-protein coupled receptors to generate PIP3, which engages downstream effectors such as the Akt/PDKl pathway, mTOR, the Tec family kinases, and the Rho family GTPases.
  • the class II and III PB-Ks play a key role in intracellular trafficking through the synthesis of PI(3)P and PI(3,4)P2.
  • the PIKKs are protein kinases that control cell growth (mTORCl) or monitor genomic integrity (ATM, ATR, DNA-PK, and hSmg-1).
  • Downstream mediators of the PBK signal transduction pathway include Akt and mammalian target of rapamycin (mTOR).
  • Akt posseses a pleckstrin homology (PH) domain that binds PIP3, leading to Akt kinase activation.
  • Akt phosphorylates many substrates and is a central downstream effector of PBK for diverse cellular responses. Full activation of Akt typically requires phosphorylation of T308 in the activation loop and S473 in a hydrophobic motif.
  • Akt Akt-like protein kinase
  • mTOR serine -threonine kinase related to the lipid kinases of the PBK family. mTOR has been implicated in a wide range of biological processes including cell growth, cell proliferation, cell motility and survival. Disregulation of the mTOR pathway has been reported in various types of cancer. mTOR is a multifunctional kinase that integrates growth factor and nutrient signals to regulate protein translation, nutrient uptake, autophagy, and mitrochondrial function.
  • Dysregulation of signaling pathways mediated by many kinases is a key factor in the development of human diseases.
  • Aberrant or excessive protein kinase activity or expression has been observed in many disease states including ocular diseases and conditions, immune disorders, benign and malignant proliferative diseases, disorders such as allergic contact dermatitis, rheumatoid arthritis, osteoarthritis, inflammatory bowel diseases, chronic obstructive pulmonary disorder, psoriasis, multiple sclerosis, asthma, disorders related to diabetic complications, and inflammatory complications of the cardiovascular system such as acute coronary syndrome.
  • methods are provided for treating diseases and conditions related to aberrant growth and/or permeability of the vasculature of the eye.
  • Diseases and conditions related to aberrant growth and/or permeability of the vasculature of the eye include but are not limited to age-related macular degeneration, retinopathy of prematurity, ischemic retinal vein occlusion, diabetic retinopathy, and neovascular glaucoma.
  • methods for treating aberrant growth and/or permeability of the vasculature of the eye include the use of inhibitors of the vascular endothelial growth factor (VEGF) signal transduction pathway and/or the hypoxia inducible factor (HIF) signal transduction pathway.
  • said methods include the use of, or administration of, antagonists (e.g. inhibitors) of PBK, which is an important downstream mediator of VEGF and HIF- ⁇ signaling.
  • PBK antagonists of the present invention include compounds that are specific for a single class of PBK such as PBK ⁇ , PBK ⁇ , PBK ⁇ , or PBK ⁇ .
  • Compounds of the present invention that specifically inhibit a single class of PBK may in some cases provide enhanced treatment modalities by leading to a lower number, severity, and/or frequency of side-effects.
  • antagonists are administered that are specific for a single class of PBK, for example, they inhibit one class of PBK with an IC50 that is at least 2-fold lower than for another class of PBK.
  • specific PBK antagonists of the present invention may be administered that inhibit a first class of PBK such as, for example, PBK ⁇ with a IC50 of less than about 1, 5, 10, 50, 100, 150, 200, 300, 400, 500, 750, or less than about 100OnM, while at that antagonist concentration exhibiting less than about 1%, 2%, 3%, 4%, 5%, 10%, 20%, 25%, 30%, 35%, or less than about 40% inhibition of another class of PI3K.
  • PI3K ⁇ antagonists are administered to the eye of a subject to inhibit inflammatory responses that lead to activation of the VEGF signaling pathway.
  • compounds of the present invention are administered and they are specific for more than one class of PI3K.
  • PI3K antagonists of the present invention may substantially and specifically inhibit classes I, II, or III of PI3K.
  • pan-specific PI3K inhibitors refers to PI3K antagonist compounds of the present invention that inhibit two or more classes or isoforms of PDKs.
  • the administration of antagonists of different classes of PI3Ks provides the ability to inhibit angiogenesis at multiple points in the signal transduction pathway. Such multi-point inhibition may in some cases provide additive or even synergistic effects as compared to the administration of compounds that are directed to a single isoform.
  • the administered compounds may selectively inhibit one or more members of type I or class I phosphatidylinositol 3-kinases (PI3-kinase) with an IC50 value of about 100 nM, 50 nM, 10 nM, 5 nM, 100 pM, 10 pM or even 1 pM, or less as ascertained in an in vitro kinase assay.
  • PI3-kinase phosphatidylinositol 3-kinases
  • the administered compounds may selectively inhibit one or two members of type I or class I phosphatidylinositol 3-kinases (PI3-kinase) consisting of PI3-kinase ⁇ , PI3-kinase ⁇ , PI3- kinase ⁇ , and PI3-kinase ⁇ .
  • PI3-kinase phosphatidylinositol 3-kinases
  • some of the subject compounds selectively inhibit PI3-kinase ⁇ as compared to all other type I PI3 -kinases.
  • the administered compounds selectively inhibit PI3- kinase ⁇ as compared to all other type I PI3 -kinases.
  • the administered compounds selectively inhibit PI3 -kinase ⁇ as compared to all other type I PI3 -kinases. In other aspects, some of the subject compounds selectively inhibit PI3 -kinase ⁇ and PI3 -kinase ⁇ as compared to the rest of the type I PI3 -kinases. In yet other aspects, some of the subject compounds selectively inhibit PI3 -kinase ⁇ and PI3 -kinase ⁇ as compared to the rest of the type I PI3-kinases.
  • an inhibitor selectively inhibits one or more members of type I PI3 -kinase inhibitor, or an inhibitor that selectively inhibits one or more members of the type I PI3 -kinases mediated signaling, alternatively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC 50 ) with respect to a given type I PI3-kinase, that is at least at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least 1000-fold, at least 10, 100-fold, or lower, than the inhibitor's IC 50 with respect to the rest of the other type I PI3 -kinases.
  • IC 50 50% inhibitory concentration
  • compounds that antagonize PI3K ⁇ and PI3K ⁇ can inhibit inflammatory responses that lead to or exacerbate aberrant neovascularization and/or vascular permeability.
  • Agonist against PI3K ⁇ can have a direct inhibitory effect on angiogenesis.
  • antagonists that inhibit multiple isoforms of class I PI3K provides methods for inhibiting signal transduction pathways that lead to neovascularization and/or vascular permeability at multiple points along the pathway.
  • the compounds of the present invention that are administered inhibit kinases other than PI3Ks including but not limited to mTOR, Akt, Src, MAPK, MEK, Jun Kinases, receptor tyrosine kinases such as VEGF-R, DNA-PK, AbI, Hck, EGFR, EphB4, and PDGFR.
  • methods for administering a combination of compounds of the present invention are provided that inhibit VEGF signaling at several points such as but not limited to PI3K signaling, VEGF-R signaling, GPCR signaling, and inflammatory-mediated vascular permeability and neovascularization.
  • VEGF VEGF
  • Cells of ocular origin include but are not limited to extrafoveal cells, subfoveal cells, juxtafoveal cells, scleral cells, conjunctival cells, retinal cells, retinal pigmented epithelial cells, lens cells, muscle cells, neurons, rod cells, cone cells, cells of the cornea, cells of the pupil, cells of the iris, cells of the optic nerve, cells of the choroidal layer, and vascular epithelial cells within and surrounding the eye.
  • cells of ocular origin may be assayed in vitro to determine the effect of the methods and compounds of the present invention on VEGF signaling, in other cases, cells of ocular origin are assayed in vivo to determine the effect of the methods and compounds of the present invention on VEGF signaling.
  • Methods for assaying the effect of PI3K or mTOR inhibitors on VEGF signaling are well known in the art and include, but are not limited to, the use of angiogenesis assays such as described in Auerbach, R. et al. Clinical Chemistry. 49:1, 32-40; the use of VEG-F signaling assays such as the HTScan® VEGF Receptor 3 Kinase Assay Kit from cell signaling technologies; cell permeability assays; the assays described in US Patent Application Publication No. 2003/020,208; the cornea micropocket angiogenesis assay.
  • modulation of VEGF signaling or reducing angiogenesis is assayed by measuring kinase activity in vitro or in vivo by the use of anti-phospho protein specific antibodies such as, but not limited to, anti-phospho VEGF-R, anti-phsopho Akt, anti-phospho S6, anti-phospho PRAS40, and anti-phospho 4EBP; anti-phospho RET, anti- phospho FOXO3A, anti-phospho FOXOl, and anti-phospho GSK3b.
  • inhibition of VEGF and/or HIF signaling is indicated by the successful inhibition of aberrant angiogenesis and/or vascular permeability in a subject suffering from an eye disease or condition.
  • methods are provided for modulating, mitigating, or treating an ophthalmic disease by administering one or more compounds that inhibit both mTORCl and mTORC2.
  • the compounds are administered to the eye; and in other cases, the compounds are administered systemically.
  • some the compounds of the present invention to be administered inhibit both mTORCl and mTORC2.
  • the compounds inhibit mTORCl and mTORC2 with an IC50 of between less than about InM to less than about l ⁇ M, including about InM, 2nM, 4nM, 8nM, 1OnM, 15nM, 2OnM, 4OnM, 8OnM, 10OnM, 15OnM, 20OnM, 25OnM, 50OnM, 75OnM, and about 100OnM.
  • Methods for determining the level of kinase inhibition are provided herein and include for example in vitro kinase assays, in vivo kinase assays, and cell proliferation assays.
  • inhibition of mTORCl and mTORC2 is determined by measuring Akt phosphorylation.
  • the administration of mTORCl/mTORC2 inhibitors antagonizes or inhibits the VEGF and/or HIF signaling pathway.
  • the antagonists of mTOR reduce angiogenesis in the eye or vascular permeability in cells or tissues of ocular origin.
  • methods are provided for inhibiting angiogenesis in the eye by administering a combination of an mTORCl/mTORC2 inhibitor and one or more PI3K antagonists.
  • the combination of mTORCl /mTORC2 inhibitor and one or more PI3K antagonists inhibits mTORCl/mTORC2 and one or more of PI3K ⁇ , PI3K ⁇ , PI3K ⁇ , or PI3K ⁇ with an IC50 of less than about l ⁇ M.
  • the combination inhibits mTORCl/mTORC2 and one or more of PDK ⁇ , PDK ⁇ , PI3K ⁇ , or PI3K ⁇ with an IC50 of less than about 75OnM, 50OnM, 35OnM, 25OnM, 20OnM, 15OnM, 10OnM, 5OnM, 25nM, 1OnM, 5nM, or less than about InM.
  • IC50 of less than about 75OnM, 50OnM, 35OnM, 25OnM, 20OnM, 15OnM, 10OnM, 5OnM, 25nM, 1OnM, 5nM, or less than about InM.
  • white blood cells such as but not limited to macrophages, neutrophils, and microglia.
  • White blood cell induced inflammatory responses have been implicated in such ocular diseases and conditions as, for example, sarcoidosis, sympathetic ophthalmia, Vogt-Koyanagi-Harada disease, age-related macular degeneration, uveitis, and other ocular diseases mediated by inflammation, vascular permeability, and/or angiogenesis.
  • white blood cell induced inflammatory responses lead to aberrant neovascularization and/or vascular permeability due to activation of the VEGF signaling pathway.
  • the inflammatory response mediated by white blood cells may in some cases be inhibited by reducing white blood cell activation (e.g. proliferation and/or cytokine release), or by inhibiting the VEGF signaling pathway.
  • methods are provided for administration of the compounds of the present invention to the eye such that B-cell activation, leukocyte activation, macrophage activation, neutrophil activation, or microglial cell activation is inhibited.
  • the compounds administered inhibit the activation or proliferation of one or more types of white blood cell with an IC50 of less than about 25OnM, including less than about InM, 5nM, 1OnM, 25nM, 5OnM, 10OnM, 15OnM, 20OnM, and less than about 25OnM.
  • Methods for assaying B-cell proliferation include the method described by Hashimoto et al. J Immunol Methods. 1986 Jun 10;90(l):97-103. Hashimoto et al. describe a method for rapid colorimetric determination of alkaline phosphatase (APase) activity that can be used in conjunction with a multiwell scanning photometer to quantitatively measure lymphokine-dependent B cell proliferation.
  • Other methods for B-cell proliferation assays include Storek et al. J Immunol Methods. 1992 JuI 6;151(l-2):261-7. Storek et al.
  • lymphocyte proliferation include tritiated thymidine incorporation, BrDu incorporation, or EdU incorporation.
  • Methods for assaying neutrophil activation include those described in Lieberman et al. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, ⁇ Nov. 1996, p. 654-662. Lieberman et al. describe two methods for assaying neutrophil activation. In the first method, neutrophils activation is measured by assaying the cellular debris fraction of neutrophil lysate in a microtiter plate for NADPH oxidase activity using luminol as a substrate. In the second method, the uptake and oxidation of the non-fluorescent dye dihydrorhodamine 123 to the fluorescent rhodamine 123 by activated neutrophils is monitored by flow cytometry.
  • white blood cell activation may be measured using any one of a number of general techniques known in the art for assaying cell proliferation. Such techniques include the MTT assay, the XTT assay, and measuring uptake of BrdU or an analogue thereof such as EdU, and tritiated thymidine incorporation assays.
  • white blood cell activation in vivo may be measured by administration of the compounds of the present invention, such as for example daily for about two weeks and harvesting white blood cells and/or lymphoid tissues for analysis.
  • a reagent e.g. BrDu or EdU
  • Methods of Administration e.g. BrDu or EdU
  • compositions for use in the methods of the invention may be administered via any viable delivery method or route, however in some cases, local administration is preferred. It is contemplated that all local routes to the eye may be used including topical, subconjunctival, periocular, retrobulbar, subtenon, intracameral, intravitreal, intraocular, subretinal, juxtascleral and suprachoroidal administration. Systemic or parenteral administration may be feasible including but not limited to intravenous, subcutaneous, and oral delivery.
  • the most preferred method of administration will be intravitreal or subtenon injection of solutions or suspensions, or intravitreal or subtenon placement of bioerodible or non-bioerodible devices, or by topical ocular administration of solutions or suspensions, or posterior juxtascleral administration of a gel or cream formulation.
  • topical administration all the formulations for topical ocular administration used in the field of ophthalmology (e.g., eye drops, inserts, eye packs, impregnated contact lenses, pump delivery systems, dimethylsulfoxide (DMSO)-based solutions suspensions, liposomes, and eye ointment) and all the formulations for external use in the fields of dermatology and otolaryngology (e.g., ointment, cream, gel, powder, salve, lotion, crystalline forms, foam, and spray) may be utilized as is known in the art.
  • DMSO dimethylsulfoxide
  • Eye drops may be prepared by dissolving the active ingredient in a sterile aqueous solution such as physiological saline, buffering solution, etc., or by combining powder compositions to be dissolved before use.
  • Other vehicles may be chosen, as is known in the art, including but not limited to: balance salt solution, saline solution, water soluble polyethers such as polyethyene glycol, polyvinyls, such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methylcellulose, petroleum derivatives such as mineral oil and white petrolatum, animal fats such as lanolin, polymers of acrylic acid such as carboxypolymethylene gel, vegetable fats such as peanut oil and polysaccharides such as dextrans, and glycosaminoglycans such as sodium hyaluronate.
  • additives ordinarily used in the eye drops can be added.
  • Such additives include isotonizing agents (e.g., sodium chloride, etc.), buffer agent (e.g., boric acid, sodium monohydrogen phosphate, sodium dihydrogen phosphate, etc.), preservatives (e.g., benzalkonium chloride, benzethonium chloride, chlorobutanol, etc.), thickeners (e.g., saccharide such as lactose, mannitol, maltose, etc.; e.g., hyaluronic acid or its salt such as sodium hyaluronate, potassium hyaluronate, etc.; e.g., mucopolysaccharide such as chondroitin sulfate, etc.; e.g., sodium polyacrylate, carboxyvinyl polymer, crosslinked polyacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, methyl cellulose
  • compositions of the present invention may be administered to the ocular surface via a pump-catheter system, or released from within a continuous or selective release device such as, e.g., membranes such as, but not limited to, those employed in the OcusertTM System (Alza Corp, Palo Alto, Calif.).
  • the pharmaceutical compositions can be incorporated within, carried by or attached to contact lenses which are then worn by the subject.
  • the pharmaceutical compositions can be sprayed onto ocular surface.
  • the compounds of the invention may be attached releasably to biocompatible polymers for use in sustained release formulations on, in or attached to inserts for topical or systemic administration.
  • the controlled release from a biocompatible polymer may be utilized with a water soluble polymer to form a instillable formulation, as well.
  • Ocular conditions, diseases and disorders may in some cases be treated by introducing slow release drug-containing microcapsules or implants directly into the anterior and/or posterior chambers of the eye.
  • the microcapsules may be formulated to include one or more drugs which may be released over an extended period of time at a therapeutically effective dosage into the vitreous humor.
  • the primary element of the capsule may be the polymeric encapsulating agent or lipid encapsulating agent.
  • the compositions may further be biocompatible, and in some cases biodegradable.
  • the polymeric compositions may be organic esters or ethers, which when degraded result in physiologically acceptable degradation products, including the monomers.
  • anhydrides, amides, orthoesters or the like, by themselves or in combination with other monomers may also find use.
  • the polymers may be addition or condensation polymers, particularly condensation polymers.
  • the polymers may be cross-linked or non-cross-linked, usually not more than lightly cross-linked, generally less than 5%, usually less than 1%.
  • the polymers will include oxygen and nitrogen, particularly oxygen.
  • the oxygen may be present as oxy, e.g. hydroxy or ether, carbonyl, e.g.
  • the drug eluting devices may be made of biodegradable polymers such as polyglycolide, polylactide, poly ⁇ -caprolactone, polyglyconate, polyhydroxybutyrate, polyhydroxyvalerate, and polydioxanone polymers. These biodegradable polymers as well as others described herein provide for slow release of compounds as the device degrades in vivo.
  • drug eluting devices may be made using polymers of hydroxyaliphatic carboxylic acids, either homo- or copolymers, and polysaccharides, Included among the polyesters of interest are polymers of D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid, polycaprolactone, and combinations thereof.
  • L-lactate a slowly eroding polymer may be achieved, while erosion may be substantially enhanced with the lactate racemate.
  • the drug eluting device may include a polysaccharides such as for example calcium alginate, or functionalized celluloses, particularly carboxymethylecellulose esters characterized by being water insoluble, with a molecular weight of about 5 kD to 500 kD, etc.
  • a polysaccharides such as for example calcium alginate, or functionalized celluloses, particularly carboxymethylecellulose esters characterized by being water insoluble, with a molecular weight of about 5 kD to 500 kD, etc.
  • Other polymers of interest include polyvinyl alcohol, esters and ethers, which are biocompatible and may be biodegradable.
  • characteristics of the polymers include biocompatibility, compatibility with the drug, ease of encapsulation, a halflife in the physiological environment of at least 6 hrs, preferably greater than one day, polymers that do not significantly enhance the viscosity of the vitreous, water insoluble, and the like.
  • the drug eluting device may include a protein polymer such as for example collagen, matrigel, gelatin or other large molecular weight protein.
  • Protein polymers have the advantage of being in some cases particularly biocompatible because they are derived from natural sources.
  • the biodegradable polymers which form the microencapsulated particles may in some cases be subject to enzymatic or hydrolytic instability.
  • Water soluble polymers may be cross-linked with hydrolytic or biodegradable unstable cross-links to provide useful water insoluble polymers.
  • the degree of stability can be varied widely, depending upon the choice of monomer, whether a homopolymer or copolymer is employed, employing mixtures of polymers, where the polymers may be employed as varying layers or mixed.
  • the rate of release of the drug will be primarily diffusion controlled, depending upon the surrounding membrane or monolithic polymer structure, rather than breakdown of the particle.
  • the selected particles will have lifetimes at least equal to the desired period of administration, preferably at least twice the desired period of administration, and may have lifetimes of 5 to 10 times the desired period of administration.
  • the period of administration may be at least 3 days, more usually at least 7 days, generally at least about 15 days and may be 20 days or more.
  • the drug eluting device may be made up of non-biodegradable polymers or constituents including but not limited to silicone, acrylates, polyethylenes, polyurethane, polyester, polypropylene, polytetrafluoroethylene, poly ether ketone, nylon, collagen, polyethylene terepthalate, polycarbonate, and polyimide polymers, or non-ferrous metals including biocompatible allows of steel and titanium.
  • Non-biodegradable devices may be constructed to contain a drug containing reservoir and one or more pores for release of therapeutic agents. In other cases, non-biodegradable devices are swelled with a solution of therapeutic agent which is then released upon implantation.
  • non-biodegradable devices may be coated with a biodegradable or other coating containing therapeutic agent.
  • the coating may further possess suitable characteristics for slow release of the therapeutic compound contained therein.
  • the coating may be biodegradable or bioerodable, or the coating may contain a plurality of micropores, or the coating may have an reversible affinity for the therapeutic agent such that the therapeutic agent is slowly released at equillibrium.
  • the particles may be substantially homogeneous as to composition and physical characterstics or heterogeneous.
  • particles can be prepared where the center may be of one material and the surface have one or more layers of the same or different composition, where the layers may be cross-linked, of different molecular weight, different density or porosity, or the like.
  • the center could be a polylactate coated with a polylactate -poly glycolate copolymer, so as to enhance the rate of initial degradation. Most ratios of lactate to glycolate employed will be in the range of about 1:0-1.
  • the center could be polyvinyl alcohol coated with polylactate, so that on degradation of the polylactate the center would dissolve and be rapidly washed out of the eye.
  • any pharmacologically active agent for which sustained release is desirable may be employed.
  • the drug will be sufficiently soluble in the vitreous to be presented at a pharmacologically effective dose.
  • Pharmacologic agents which may find use include the compounds of the present invention as well as those found in U.S. Pat. Nos. 4,474,451, columns 4-6 and 4,327,725, columns 7-8, which disclosures are incorporated herein by reference in their entirety.
  • Drugs of particular interest include the compounds of the present invention as well as hydrocortisone (5-20mcg/l as plasma level), gentamycin (6-lOmcg/ml in serum), 5-fluorouracil (.about.30mg/kg body weight in serum), sorbinil, IL-2, TNF, Phakan-a (a component of glutathione), thioloa-thiopronin, Bendazac, acetylsalicylic acid, trifluorothymidine, interferon ( ⁇ , ⁇ and ⁇ ), immune modulators, e.g. lymphokines, monokines, and growth factors, etc.
  • hydrocortisone 20mcg/l as plasma level
  • gentamycin 6-lOmcg/ml in serum
  • 5-fluorouracil .about.30mg/kg body weight in serum
  • sorbinil IL-2
  • TNF a component of glutathione
  • Phakan-a a component of glutathione
  • anti-glaucoma drugs such as the beta-blockers: timolol maleate, betaxolol and metipranolol; mitotics: pilocarpine, acetylcholine chloride, isofluorophate, demacarium bromide, echothiophate iodide, phospholine iodide, carbachol, and physostigimine; epinephrine and salts, such as dipivefrin hydrochloride; and dichlorphenamide, acetazolamide and methazolamide; anti-cataract and anti- diabetic retinopathy drugs, such as aldose reductase inhibitors: tolrestat, lisinopril, enalapril, and statil; thiol cross-linking drugs other than those considered previously; anti-cancer drugs, such as retinoic acid, methotrexate, adriamycin, bleo
  • cycloplegic and mydriatic agents such as atropine, cyclogel, scopolamine, homatropine and mydriacyl.
  • Other agents include anticholinergics, anticoagulants, antifibrinolytic agents, antihistamines, antimalarials, antitoxins, chelating agents, hormones, immunosuppressives, thrombolytic agents, vitamines, salts, desensitizing agents, prostaglandins, amino acids, metabolites and antiallergenics.
  • the amount of drug employed in the device may vary widely depending on the effective dosage required and rate of release. Usually the drug may be from about 1 to 80, more usually 20 to 40 weight percent of the microcapsule.
  • agents may be employed in the formulation for a variety of purposes.
  • buffering agents and preservatives may be employed.
  • Water soluble preservatives include sodium bisulfite, sodium thiosulfate, ascorbate, benzalkonium chloride, chlorobutanol, thimerosal, phenylmercuric borate, parabens, benzyl alcohol and phenylethanol. These agents may be present in amounts of from 0.001 to 5% by weight and preferably 0.01 to 2%.
  • Suitable water soluble buffering agents are alkali or alkaline earth, carbonates, phosphates, bicarbonates, citrates, borates, acetates, succinates and the like, such as sodium phosphate, citrate, borate, acetate, bicarbonate and carbonate. These agents may be present in amounts sufficient to maintain a pH of the system of between 2 to 9 and preferably 4 to 8. As such the buffering agent may be as much as 5% on a weight to weight basis of the total composition.
  • the particles may be of a narrow or broad range in size, normally not exceeding 300 ⁇ m, so as to be capable of being administered with an 18 gauge needle. Usually, the particle range will not differ by greater than about 200% of the average particle size, more usually not greater than about 100%.
  • the average particle size will usually be in the range of 5 ⁇ m to 2 mm, more usually in the range of 10 ⁇ m to 1 mm. In some instances the particles will be selected to have an average diameter in the range of 1-2 mm to provide large depots, while in other instances the particles will have average diameter in the range of about 25-500 ⁇ m, to provide smaller depots.
  • the size of the particle can be used to control the rate of release, period of treatment and drug concentration in the eye. In some situations mixtures of particles may be employed employing the same or different pharmacological agent. In this way in a single administration a course of drug treatment may be achieved, where the pattern of release may be greatly varied.
  • Various techniques may be employed to produce the encapsulated drugs.
  • Useful techniques include solvent- evaporation methods, phase separation methods, interfacial methods and the like.
  • solvent- evaporation methods may be employed.
  • the preformed rate controlling polymer may be dissolved in a volatile substantially water-immiscible solvent, such as chloroform, methylene chloride, or benzene.
  • the water immiscible sovlent may be modified with a small amount of a water-miscible organic cosolvent, particularly an oxygenated solvent, such as acetone, methanol, ethanol, etc.
  • the water-miscible organic cosolvent will be less than about 40 vol %, usually less than about 25 vol %.
  • the drug may then be added to the polymer- solvent solution. Depending upon the nature of the drug, one may have the drug dispersed in the viscous polymer- solvent mixture or a solid dispersion of drug particles, where the drug may in some cases have been pulverized to obtain a fine powder, sometimes a micro fine powder particularly of a size of less than about 1 mm, usually less than about 0.5 mm, and may be about 0.5 ⁇ m or smaller.
  • the amount of polymer employed in the medium may vary with the size of the particle desired, whether additional coatings will be added, the viscosity of the solution, the solubility of the polymer and the like. Usually, the concentration of polymer may be in the range of 10 to 80 weight percent. The ratio of drug to polymer may vary with the desired rate of release, the amount of drug generally varying in the range of 1 to 80 weight percent of the polymer.
  • the dispersion or solution obtained above may be added to a rapidly stirred aqueous solution comprising water and a dispersing agent, which may be a protective colloid.
  • a dispersing agent which may be a protective colloid.
  • macromolecular dispersing agents are agents such as poly(vinyl alcohol) (1-5%) or non-ionic detergents, such as Span detergents.
  • the volume of the organic phase may be smaller than the aqueous phase, generally being in a volume ratio of from about 1 : 1 to 10 3 of organic to aqueous phase, and an oil-in- water emulsion is produced.
  • the rate of stirring may selected to produce the appropriate droplet size and stirring may be continued throughout the next step.
  • the microencapsulation vessel may be closed and a mild vacuum is applied to the system to evaporate the volatile organic solvent.
  • the solvent may be evaporated slowly, since too rapid evaporation results in bubbles and blow holes formed in the microcapsule walls.
  • the rate of evaporation may be determined empirically, using the experience reported in the literature.
  • the vacuum may be in the range of about 3 to 10 mm Hg.
  • the resulting microcapsules can be centrifuged, washed completely with water, collected, e.g., filtration, and drained.
  • the microcapsules may then be subdivided with sieves to isolate particles of a size range of the desired diameter.
  • the process may be carried out conveniently at room temperature, but cooling or heating may be employed in specific situations to optimize the process.
  • the ratio of drug to polymer may adjusted to produce optimized compositions, since the final product will normally result in the initial ratio.
  • the initial bulk viscosity of the drug-polymer-solvent mixture and of the aqueous dispersing medium, along with the stir rate production of microcapsules with the desired size may be optimized.
  • the composition of dissolved organic solvent and the rate of solvent evaporation can be tested to produce microcapsules with larger or smaller crystals of drug in the microcapsules.
  • the microcapsules should not be exposed to the aqueous dispersing medium for excessively long periods during the solvent- evaporation step.
  • each batch of microcapsules may be relatively narrow. However, when desired, the size-fractions may be further refined by a physical separation process such as dry or wet sieving.
  • a weighed sample of microcapsules may be added to a measured volume of a solution containing four parts by weight of ethanol and six parts by weight of deionized water. The mixture may be maintained at 37° C. and stirred slowly to maintain the microcapsules suspended. The appearance of the dissolved drug as a function of time may be followed spectrophotometrically until the absorbance becomes constant or until greater than 90% of the drug has been released.
  • the drug concentration after 1 h in the medium is indicative of the amount of free unencapsulated drug in the dose, while the time required for 90% drug to be released is related to the expected duration of action of the dose in vivo.
  • one day of drug release in vitro is approximately equal to 35 days of release in vivo. While release may not be uniform, preferably the release will be free of larger fluctuations from some average value which allows for a relatively uniform release.
  • microcapsules may be administered into the eye in a variety of ways, including injection, infusion, trocar, etc.
  • Various techniques for introducing materials into the anterior and/or posterior chambers are well known, see, for example, Liu et al., 1987, supra, and references cited therein.
  • the doses of the active agents in the compositions used for the above described purposes will vary, but will be in effective amounts to inhibit or cause regression of neovascularization or angiogenesis and to provide neuroprotection to the retinal tissues.
  • the doses of the one or more kinase inhibitor in the compositions of the invention will be in an effective amount to treat or prevent the progression of AMD, DR, sequela associated with retinal ischemia, and macular and/or retinal edema.
  • the term "pharmaceutically effective amount” refers to an amount of one or more kinase inhibitor which will effectively treat AMD, DR, and/or retinal edema, or inhibit or cause regression of neovascularization or angiogenesis, in a human patient.
  • the doses used for any of the above -described purposes will generally be from about 0.01 to about 100 milligrams per kilogram of body weight (mg/kg), administered one to four times per day.
  • the compositions When the compositions are dosed topically, they will generally be in a concentration range of from 0.001 to about 5% w/v, with 1-2 drops administered 1-4 times per day.
  • the compounds will generally be in a concentration range of from 0.001% to about 10% w/v. Examples
  • Example 1 Expression and Inhibition Assays of pll ⁇ /p85 ⁇ , pll ⁇ /p85 ⁇ , pll ⁇ /p85 ⁇ , and pllO ⁇ : [00332]
  • Class I PB-Ks can be either purchased (pi 10 ⁇ /p85 ⁇ , pi 10 ⁇ /p85 ⁇ , pi 10 ⁇ /p85 ⁇ from Upstate, and pi lO ⁇ from Sigma) or expressed as previously described (Knight et al., 2004). IC50 values are measured using either a standard TLC assay for lipid kinase activity (described below) or a high-throughput membrane capture assay.
  • Kinase reactions are performed by preparing a reaction mixture containing kinase, inhibitor (2% DMSO final concentration), buffer (25 mM HEPES, pH 7.4, 10 mM MgC12), and freshly sonicated phosphatidylinositol (100 ⁇ g/ml). Reactions are initiated by the addition of ATP containing 10 ⁇ Ci of ⁇ -32P-ATP to a final concentration 10 or 100 ⁇ M and allowed to proceed for 5 minutes at room temperature. For TLC analysis, reactions are then terminated by the addition of 105 ⁇ l IN HCl followed by 160 ⁇ l CHC13:MeOH (1: 1).
  • the biphasic mixture is vortexed, briefly centrifuged, and the organic phase is transferred to a new tube using a gel loading pipette tip precoated with CHCI 3 .
  • This extract is spotted on TLC plates and developed for 3 - 4 hours in a 65:35 solution of n-propanol:lM acetic acid.
  • the TLC plates are then dried, exposed to a phosphorimager screen (Storm, Amersham), and quantitated.
  • kinase activity is measured at 10 - 12 inhibitor concentrations representing two-fold dilutions from the highest concentration tested (typically, 200 ⁇ M).
  • IC50 determinations are repeated two to four times, and the reported value is the average of these independent measurements.
  • kits or systems for assaying PI3-K activities are avaiable.
  • the commercially available kits or systems can be used to screen for inhibitors and/or agonists of PB-Ks including but not limited to PI 3-Kinase ⁇ , ⁇ , ⁇ , and ⁇ .
  • Anr exemplary system is PI 3-Kinase (human) HTRFTM Assay from Upstate.
  • the assay can be carried out according to the procedures suggested by the manufacturer. Briefly, the assay is a time resolved FRET assay that indirectly measures PIP3 product formed by the activity of a PI3-K.
  • the kinase reaction is performed in a microtitre plate (e.g., a 384 well microtitre plate).
  • the total reaction volume is approximately 20ul per well.
  • each well receives 2ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration.
  • approximately 14.5ul of a kinase/PIP2 mixture (diluted in IX reaction buffer) is added per well for a final concentration of 0.25-0.3ug/ml kinase and lOuM PIP2.
  • the plate is sealed and incubated for 15 minutes at room temperature.
  • 3.5ul of ATP (diluted in IX reaction buffer) is added per well for a final concentration of lOuM ATP.
  • the plate is sealed and incubated for 1 hour at room temperature.
  • the reaction is stopped by adding 5ul of Stop Solution per well and then 5ul of Detection Mix is added per well.
  • the plate is sealed, incubated for 1 hour at room temperature, and then read on an appropriate plate reader. Data is analyzed and IC50s are generated using GraphPad Prism 5.
  • the compounds described herein can be assayed in triplicate against recombinant full-length AbI or AbI (T3151) (Upstate) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 200 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 0.5 mg/mL BSA.
  • the optimized AbI peptide substrate EAIYAAPF AKKK is used as phosphoacceptor (200 ⁇ M). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 3 Expression and Inhibition Assays of Hck
  • the compounds described herein can be assayed in triplicate against recombinant full-length Hck in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 200 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 0.5 mg/mL BSA.
  • the optimized Src family kinase peptide substrate EIYGEFKKK is used as phosphoacceptor (200 ⁇ M). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • the compounds described herein can be assayed in triplicate against recombinant full-length Src or Src (T338I) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 200 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 0.5 mg/mL BSA.
  • the optimized Src family kinase peptide substrate EIYGEFKKK is used as phosphoacceptor (200 ⁇ M). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets were dried and the transferred radioactivity quantitated by phosphorimaging.
  • DNA-PK can be purchased from Promega and assayed using the DNA-PK Assay System (Promega) according to the manufacturer's instructions.
  • the compounds described herein can be tested against recombinant mTOR (Invitrogen) in an assay containing 50 mM HEPES, pH 7.5, ImM EGTA, 10 mM MgC12, 2.5 mM, 0.01% Tween, 10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • Rat recombinant PHAS-1/4EBP1 (Calbiochem; 2 mg/mL) is used as a substrate. Reactions are terminated by spotting onto nitrocellulose, which is washed with IM NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • kits or systems for assaying mTOR activity are commercially available.
  • Invitrogen' s LanthaScreenTM Kinase assay to test the inhibitors of mTOR disclosed herein.
  • This assay is a time resolved FRET platform that measures the phosphorylation of GFP labeled 4EBP 1 by mTOR kinase.
  • the kinase reaction is performed in a white 384 well microtitre plate.
  • the total reaction volume is 20ul per well and the reaction buffer composition is 5OmM HEPES pH7.5, 0.01% Polysorbate 20, ImM EGTA, 1OmM MnC12, and 2mM DTT.
  • each well receives 2ul of test compound in 20% dimethylsulphoxide resulting in a 2% DMSO final concentration.
  • 8ul of mTOR diluted in reaction buffer is added per well for a
  • lOul of an ATP/GFP-4EBP 1 mixture (diluted in reaction buffer) is added per well for a final concentration of lOuM ATP and 0.5uM GFP-4EBP1.
  • the plate is sealed and incubated for 1 hour at room temperature.
  • the reaction is stopped by adding lOul per well of a Tb-anti- pT46 4EBP 1 antibody/EDTA mixture (diluted in TR-FRET buffer) for a final concentration of 1.3nM antibody and 6.7mM EDTA.
  • the plate is sealed, incubated for 1 hour at room temperature, and then read on a plate reader set up for LanthaScreenTM TR-FRET. Data is analyzed and IC50s are generated using GraphPad Prism 5.
  • Example 7 In Vivo Effect of mTor Inhibitors on Kinase Substrate Phosphorylation
  • mice are treated with an mTORCl/mTORC2 inhibitor and rapamycin, and the acute effect of these drugs on insulin signaling in fat, skeletal muscle and liver tissue are examined.
  • mice 30 minutes for the rapamycin-treated mouse or about 10 min for the mTORCl/mTORC2 and vehicle treated mice, approximately 25OmU of insulin in about 100 ⁇ l of saline is injected IP. Typically 15 minutes after the insulin injection, the mice are sacrificed by CO2 asphyxiation followed by cervical dislocation. Tissues are harvested and frozen on liquid nitrogen in about 200 ⁇ l of cap lysis buffer. The frozen tissue is thawed on ice, manually disrupted with a mortar and pestle, and then further processed with a micro tissue-homogenizer
  • Rapamycin often stimulates the phosphorylation of Akt, probably by relieving feedback inhibition from S6K to the insulin receptor substrate 1 (IRSl), a key signaling molecule that links activation of the insulin receptor to PI3K activation.
  • IRSl insulin receptor substrate 1
  • Rapamycin and mTORCl/mTORC2 inhibitors differentially affect the mTorCl substrates S6K and 4EBP1 in vivo. S6 phosphorylation is equally inhibited by rapamycin and mTORCl/mTORC2 inhibitor in all tissues examined. mTORCl/mTORC2 inhibitor is effective at blocking the phosphorylation of 4EBP 1 on both T37/46 and S65 in all tissues examined. While rapamycin is more effective at inhibiting the phosphorylation of 4EBP 1 in vivo than in cell culture experiments, rapamycin never blocks 4EBP 1 phosphorylation as completely as mTORCl/mTORC2 inhibitor.
  • Rapamycin has been a powerful pharmacological tool allowing the discovery of mTor's central role in the control of protein synthesis. Since the discovery of a rapamycin-insensitive mTor complex there has been a significant effort to develop pharmacological tools for studying this complex.
  • mTORCl/mTORC2 inhibitors used to chemically dissect the effects of mTor kinase inhibition toward mTorCl and mTorC2 activity.
  • results of the present invention are expected to shown through the use of these inhibitors that the inhibition of mTor kinase activity is sufficient to prevent the phosphorylation of Akt at S473, under the conditions tested, providing further evidence that mTorC2 may be the kinase responsible for Akt hydrophobic motif phosphorylation.
  • results of the present invention further are expected to provide that phosphorylation at T308 is probably linked to phosphorylation at S473, as had been observed in experiments where mTorC2 was disabled by RNAi, but not homologous recombination.
  • results of the present invention are expected to provide that phosphorylation of the mTorCl substrate 4EBP 1 is partially resistant to rapamycin treatment at concentrations that fully inhibit p70S6K while mTORCl/mTORC2 inhibitor completely inhibits both p70S6K and 4EBP 1. Consequently, the enhanced block of cell proliferation by the mTORCl/mTORC2 inhibitor compared with rapamycin may reflect in part its ability to more efficiently inhibit eIF4E-dependent translation control.
  • Example 8 Expression and Inhibition Assays of Vascular endothelial growth receptor [00346]
  • the compounds described herein can be tested against recombinant KDR receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 0.1% BME, 10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/niL
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with IM NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 9 Expression and Inhibition Assays of Ephrin receptor B4 (EphB4)
  • the compounds described herein can be tested against recombinant Ephrin receptor B4 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 0.1% BME, 10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • Poly E-Y (Sigma; 2 mg/niL) is used as a substrate. Reactions are terminated by spotting onto nitrocellulose, which is washed with IM NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 10 Expression and Inhibition Assays of Epidermal growth factor receptor (EGFR) [00348] The compounds described herein can be tested against recombinant EGF receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 0.1% BME, 10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA. Poly E-Y (Sigma; 2 mg/niL) is used as a substrate. Reactions are terminated by spotting onto nitrocellulose, which is washed with IM NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 11 Expression and Inhibition Assays of KIT
  • the compounds described herein can be tested against recombinant KIT kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, ImM DTT, 1OmM MnC12, 10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • Poly E-Y Sigma; 2 mg/niL
  • Reactions are terminated by spotting onto nitrocellulose, which is washed with IM NaCl/1% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 12 Expression and Inhibition Assays of RET
  • the compounds described herein can be tested against recombinant RET kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 2.5mM DTT,10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • the optimized AbI peptide substrate EAIY AAPF AKKK is used as phosphoacceptor (200 ⁇ M). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 13 Expression and Inhibition Assays of Platelet derived growth factor receptor (PDGFR) [00351]
  • PDGFR Platelet derived growth factor receptor
  • the compounds described herein can be tested against recombinant PDG receptor kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 2.5mM DTT,10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • the optimized AbI peptide substrate EAIYAAPF AKKK is used as phosphoacceptor (200 ⁇ M). Reactions are terminated by spotting onto phosphocellulose sheets, which are washed with 0.5% phosphoric acid (approximately 6 times, 5-10 minutes each). Sheets are dried and the transferred radioactivity quantitated by phosphorimaging.
  • Example 14 Expression and Inhibition Assays of FMS-related tyrosine kinase 3 (FLT-3)
  • FMS-related tyrosine kinase 3 FMS-related tyrosine kinase 3
  • the compounds described herein can be tested against recombinant FLT-3 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 2.5mM DTT,10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P- ATP), and 3 ⁇ g/mL BSA.
  • the optimized AbI peptide substrate EAIY AAPF AKKK is used as phosphoacceptor (200 ⁇ M).
  • TIE2 TEK receptor tyrosine kinase
  • the compounds described herein can be tested against recombinant TIE2 kinase domain (Invitrogen) in an assay containing 25 mM HEPES, pH 7.4, 10 mM MgC12, 2mM DTT, 1OmM MnC12, 10 ⁇ M ATP (2.5 ⁇ Ci of ⁇ -32P-ATP), and 3 ⁇ g/mL BSA.
  • Cells comprising components of the Akt/mTOR pathway including but not limited to L6 myoblasts, B- ALL cells, B-cells, T-cells, leukemia cells, bone marrow cells, pi 90 transduced cells, philladelphia chromosome positive cells (Ph+), and mouse embryonic fibroblasts, are typically grown in cell growth media such as DMEM supplemented with fetal bovine serum and/or antibiotics, and grown to confluency.
  • cell growth media such as DMEM supplemented with fetal bovine serum and/or antibiotics
  • said cells are serum starved overnight and incubated with one or more compounds disclosed herein or about 0.1% DMSO for approximately 1 minute to about 1 hour prior to stimulation with insulin (e.g. 100 nM) for about 1 minutes to about 1 hour.
  • insulin e.g. 100 nM
  • Cells are lysed by scraping into ice cold lysis buffer containing detergents such as sodium dodecyl sulfate and protease inhibitors (e.g., PMSF).
  • the solution After contacting cells with lysis buffer, the solution is briefly sonicated, cleared by centrifugation, resolved by SDS-PAGE, transferred to nitrocellulose or PVDF and immunoblotted using antibodies to phospho- Akt S473, phospho- Akt T308, Akt, and ⁇ -actin (Cell Signaling Technologies).
  • one or more compounds of the present disclosure inhibit insulin stimulated phosphorylation of Akt at S473.
  • some compounds disclosed herein additionally inhibit insulin stimulated phosphorylation of Akt at T308.
  • Such class of compounds can inhibit Akt more effectively than rapamycin and may be indicative of mTORC2 inhibitors or inhibitors of upstream kinases such as PI3K or Akt.
  • Example 17 B Cell Activation and Proliferation Assay
  • the ability of one or more subject compounds to inhibit B cell activon and proliferation is determined according to standard procedures known in the art. For example, an in vitro cellular proliferation assay is established that measures the metabolic activity of live cells. The assay is performed in a 96 well microtiter plate using Alamar Blue reduction. Balb/c splenic B cells are purified over a Ficoll-PaqueTM PLUS gradient followed by magnetic cell separation using a MACS B cell Isolation Kit (Miletenyi). Cells are plated in 90ul at 50,000 cells/well in B Cell Media (RPMI + 10%FBS + Penn/Strep + 5OuM bME + 5mM HEPES).
  • a compound disclosed herein is diluted in B Cell Media and added in a lOul volume. Plates are incubated for 30min at 37C and 5% CO 2 (0.2% DMSO final concentration). A 50ul B cell stimulation cocktail is then added containing either lOug/ml LPS or 5ug/ml F(ab')2 Donkey anti-mouse IgM plus 2ng/ml recombinant mouse IL4 in B Cell Media. Plates are incubated for 72 hours at 37 0 C and 5% CO 2 . A volume of 15uL of Alamar Blue reagent is added to each well and plates are incubated for 5 hours at 37C and 5% CO 2 . Alamar Blue fluoresce is read at 560Ex/590Em, and IC50 values are calculated using GraphPad Prism 5.
  • Example 18 Cell Culture of Epithelial Cells of Ocular Origin
  • Ocular epithelial cells are obtained within 5 days postmortem post-mortem from corneas preserved under cold storage conditions in Optisol (Bausch and Lomb, Irvine, CA) or from corneal biopsy from living donors. The tissue is washed with phosphate -buffered saline and incubated in Dispase II (Roche Diagnostics, Basel, Switzerland) at 37°C for 30 minutes, and the epithelial surface is gently scraped to separate the epithelium from the underlying stroma. The separated epithelium is then incubated and pipetted in trypsin- ethylenediaminetetraacetic acid to obtain a single cell suspension. The trypsin is then neutralized with corneal epithelium culture medium.
  • Corneal epithelium culture medium is composed of Dulbecco modified Eagle medium:F12 basal media in a 2:1 ratio containing 10% irradiated fetal bovine serum, hydrocortisone 0.4 ⁇ g/mL, cholera toxin 0.1 nmol, recombinant human insulin 5 ⁇ g/mL, and epidermal growth factor 10 ng/niL, and the antimicrobials penicillin (100 IU/mL), streptomycin (100 ⁇ g/mL), and amphotericin B (0.25 ⁇ g/mL). Cells are maintined by sub-culturing at a 1 :4 ratio after reaching 80% confluency.
  • Ocular epithelial cells are screened for inhibition of proliferation or toxicity by contacting a test compound with the cells and assaying for viability using the commercially available MTT assay (Promega).
  • Example 19 Cell Culture of Endothelial Cells of Ocular Origin
  • All tissues are maintained at 4°C in storage medium (Optisol; Chiron Vision, Irvine, CA) for less than 10 days before study.
  • the tissue is rinsed three times with DMEM containing 50 mg/mL gentamicin and 1.25 mg/mL amphotericin B.
  • the central cornea is removed by a trephine of 8-mm diameter.
  • the Descemet's membrane and corneal endothelial cells are stripped from the posterior surface of the peripheral corneoscleral tissue under a dissecting microscope and digested at 37°C for 1.5 to 16 hours with 2 mg/mL collagenase A in supplemented hormonal epithelial medium (SHEM), which is made of an equal volume of HEPES -buffered DMEM and Ham's F12 supplemented with 5% FBS, 0.5% dimethyl sulfoxide, 2 ng/mL mouse EGF, 5 ⁇ g/mL insulin, 5 ⁇ g/mL transferrin, 5 ng/mL selenium, 0.5 ⁇ g/mL hydrocortisone, 1 nM cholera toxin, 50 ⁇ g/mL gentamicin, and 1.25 ⁇ g/mL amphotericin B.
  • SHEM hormonal epithelial medium
  • HCECs formed aggregates, which are collected by centrifugation at 2000 rpm for 3 minutes to remove the digestion solution.
  • Descemet's membrane strips are also digested in 10 mg/mL Dispase II in SHEM and trypsin/EDTA for up to 3 hours.
  • HCEC aggregates are preserved in KSFM with complete supplement (storage medium 1), DMEM/F12 with KSFM supplements (storage medium T), or DMEM/F12 with SHEM supplements without FBS (storage medium 3). All these media are serum free, one of the major differences among them is the calcium concentration, which is 0.09 mM in storage medium 1, but is 1.05 mM in storage media 2 and 3.
  • HCEC aggregates are stored in a tissue culture incubator at 37°C for up to 3 weeks. Cell viability is determined (Live and Dead assay; Invitrogen) and also evaluated by subculturing them in SHEM. Expansion of Isolated HCEC Aggregates
  • the resultant HCEC aggregates are then cultured in SHEM with or without additional growth factors such as 40 ng/mL bFGF, 0.1 mg/mL BPE, and 20 ng/mL NGF on a plastic dish under 37°C and 5% CO2.
  • the media are changed every 2 to 3 days.
  • Some HCEC aggregates are pretreated with trypsin/EDTA at 37°C for 10 minutes to dissociate endothelial cells before the aforementioned cultivation.
  • HCEC aggregates are embedded in OCT and subjected to frozen sectioning. Cryosections of 4 ⁇ m are air-dried at room temperature (RT) for 30 minutes, and fixed in cold acetone for 10 minutes at -20 0 C. Sections used for immunostaining are rehydrated in PBS, and incubated in 0.2% Triton X-100 for 10 minutes. After three rinses with PBS for 5 minutes each and preincubation with 2% BSA to block nonspecific staining, the sections are incubated with anti-laminin 5, type IV collagen, perlecan, ZO-I, and connexin 43 (all at 1: 100) antibodies for 1 hour.
  • RT room temperature
  • HCECs cultured in 24-well plates or chamber slides are fixed in 4% paraformaldehyde for 15 minutes at RT and stained with anti-ZO-1 and connexin 43 antibodies as just described.
  • HCEC aggregates are incubated with cell-viability assay reagents for 15 minutes at RT. Live cells are distinguished by green fluorescence staining of the cell cytoplasm, and dead cells are stained with red fluorescence in the nuclei.
  • the TUNEL assay is performed according to the manufacturer's instructions. Briefly, cross-sections of HCEC aggregates are fixed in 4% paraformaldehyde for 20 minutes at RT and permeabilized with 1% Triton X-100.
  • Samples are then incubated for 60 minutes at 37°C with exogenous TdT and fluorescein-conjugated dUTP, for repair of nicked 3'-hydroxyl DNA ends.
  • Cells are treated with DNase I as the positive control, whereas negative control cells are incubated with a buffer lacking the rTdT enzyme.
  • the apoptotic nuclei are labeled with green fluorescence.
  • Eyes are cut in half along their equator and the neural retina is dissected from the anterior part of the eye in buffered saline solution, according to standard methods known in the art. Briefly, the retina, ciliary body, and vitreous are dissected away from the anterior half of the eye in one piece, and the retina is gently detached from the clear vitreous. Each retina is dissociated with papain (Worthington Biochemical Corporation, Lakewood, NJ.), followed by inactivation with fetal bovine serum (FBS) and addition of 134 Kunitz units/ml of DNasel.
  • papain Worthington Biochemical Corporation, Lakewood, NJ.
  • FBS fetal bovine serum
  • the enzymatically dissociated cells are triturated and collected by centrifugation, resuspended in Dulbecco's modified Eagle's medium (DMEM)/F12 medium (Gibco BRL, Invitrogen Life Technologies, Carlsbad, Calif.) containing 25 ⁇ g/ml of insulin, 100 ⁇ g/ml of transferrin, 60 ⁇ M putrescine, 30 nM selenium, 20 nM progesterone, 100 U/ml of penicillin, 100 ⁇ g/ml of streptomycin, 0.05 M Hepes, and 10% FBS.
  • DMEM Dulbecco's modified Eagle's medium
  • F12 medium Gibco BRL, Invitrogen Life Technologies, Carlsbad, Calif.
  • Dissociated primary retina 1 cells are plated onto Poly-D-lysine- and Matrigel- (BD, Franklin Lakes, NJ.) coated glass coverslips that are placed in 24-well tissue culture plates (Falcon Tissue Culture Plates, Fisher Scientific, Pittsburgh, Pa.). Cells are maintained in culture for 5 days to one month in 0.5 ml of media (as above, except with only 1% FBS) at 37° C. and 5% CO2. Immunocytochemistry Analysis
  • the retina 1 neuronal cells are cultured for 1, 3, 6, and 8 weeks in the presence and absence of test compounds of the present invention, and the cells are analyzed by immunohistochemistry at each time point. Immunocytochemistry analysis is performed according to standard techniques known in the art. Rod photoreceptors are identified by labeling with a rhodopsin-specilic antibody (mouse monoclonal, diluted 1:500; Chemicon, Temecula, Calif.).
  • An antibody to mid-weight neurofilament (NFM rabbit polyclonal, diluted 1: 10,000, Chemicon) is used to identify ganglion cells; an antibody to ⁇ 3 -tubulin (G7121 mouse monoclonal, diluted 1 : 1000, Promega, Madison, Wis.) is used to generally identify interneurons and ganglion cells, and antibodies to calbindin (AB1778 rabbit polyclonal, diluted 1:250, Chemicon) and calretinin (AB5054 rabbit polyclonal, diluted 1:5000, Chemicon) are used to identify subpopulations of calbindin- and calretinin- expressing interneurons in the inner nuclear layer.
  • the retina 1 cell cultures are fixed with 4% paraformaldehyde (Polysciences, Inc, Warrington, Pa.) and/or ethanol, rinsed in Dulbecco's phosphate buffered saline (DPBS), and incubated with primary antibody for 1 hour at 37° C.
  • DPBS Dulbecco's phosphate buffered saline
  • the cells are then rinsed with DPBS, incubated with a secondary antibody (Alexa 488- or Alexa 568-conjugated secondary antibodies (Molecular
  • DAPI 4',6-diamidino-2-phenylindole
  • Example 21 Matrigel Plug Angiogenesis Assay.
  • Matrigel containing test compounds are injected subcutaneously or intraocularly, where it solidifies to form a plug.
  • the plug is recovered after 7-21 days in the animal and examined histologically to determine the extent to which blood vessels have entered it.
  • Angiogenesis is measured by quantification of the vessels in histologic sections.
  • fluorescence measurement of plasma volume is performed using fluorescein isothiocyanate (FITC)-labeled dextran 150.
  • FITC fluorescein isothiocyanate
  • a pocket is made in the cornea, and a plug containing an angiogenesis inducing formulation (e.g.
  • VEGF vascular endothelial growth factor
  • FGF vascular endothelial growth factor
  • tumor cells when introduced into this pocket, elicits the ingrowth of new vessels from the peripheral limbal vasculature.
  • Slow-release materials such as ELVAX (ethylene vinyl copolymer) or Hydron are used to introduce angiogenesis inducing substances into the corneal pocket.
  • a sponge material is used.
  • the effect of putative inhibitors on the locally induced (e.g., sponge implant) angiogenic reaction in the cornea e.g., by FGF, VEGF, or tumor cells.
  • the test compound is administered orally, systemically, or directly to the eye.
  • Systemic administration is by bolus injection or, more effectively, by use of a sustained- release method such as implantation of osmotic pumps loaded with the test inhibitor.
  • Administration to the eye is by any of the methods described herein including but not limited to eye drops, topical administration of a cream, emulsion, or gel, intravitreal injection.
  • the vascular response is monitored by direct observation throughout the course of the experiment using a stereomicroscope in mice.
  • Definitive visualization of the corneal vasculature is achieved by administration of fluorochrome-labeled high-molecular weight dextran. Quantification is performed by measuring the area of vessel penetration, the progress of vessels toward the angiogenic stimulus over time, or in the case of fluorescence, histogram analysis or pixel counts above a specific (background) threshold.
  • results are expected to indicate one or more compounds disclosed herein that inhibit angiogenesis and are thus expected to be useful in treating ocular disorders related to aberrant angiogenesis and/or vascular permeability.
  • the assay plate is prepared by placing a collagen plug in the bottom of each well with 5-10 cell spheroids per collagen plug each spheroid containing 400-500 cells. Each collagen plug is covered with 1 lOO ⁇ l of storage medium per well and stored for future use (1-3 days at 37°C, 5% CO 2 ). The plate is sealed with sealing. Test compounds are dissolved in 200 ⁇ l assay medium with at least one well including a VEGF positive control and at least one well without VEGF or test compound as a negative control. The assay plate is removed from the incubator and storage medium is carefully pipeted away. Assay medium containing the test compounds are pipeted onto the collagen plug.
  • the plug is placed in a humidified incubator for (37°C, 5% CO 2 ) 24-48 hours.
  • Angiogenesis is quantified by counting the number of sprouts, measuring average sprout length, or determining cumulative sprout length.
  • the assay can be preserved for later analysis by removing the assay medium, adding ImI of 10% paraformaldehyde in Hanks BSS per well, and storing at 4°C. The results are expected to identify compounds that inhibit angiogenesis in various cell types tested, including cells of ocular origin.

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Abstract

La présente invention concerne des entités ou des composés chimiques, ainsi que des compositions pharmaceutiques correspondantes, capables de moduler la transduction du signal par certaines protéines kinases telles que la mTor kinase ou la tyrosine kinase et/ou par des lipides kinases telles que la PB kinase, dans un tissu oculaire. L'invention porte également sur des procédés d'utilisation desdites compositions pour moduler les activités d'une ou plusieurs desdites kinases, et particulièrement dans le cadre d'applications thérapeutiques.
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