MXPA05013150A - 3-substituted indoles and derivatives thereof as therapeutic agents - Google Patents

Abstract

The present invention provides indoles of Formula I:wherein R1 and R2 have any of the values defined therefor in the specification, and pharmaceutically acceptable salts thereof, that are useful as agents in the treatment of diseases and conditions, including inflammatory diseases, cardiovascular diseases, and cancers. Also provided are pharmaceutical compositions comprising one or more compounds of Formula I.

Description

INDICATIONS 3-SUSTAINED AND DERIVED FROM THEM AS THERAPEUTIC AGENTS BACKGROUND OF THE INVENTION Phosphoinositide-3-kinases (P13Ks) are a family of lipid kinases that phosphorylate phosphoinositols in 3'-OH to generate PI-3-P (phosphatidylinositol 3-phosphate), PI-3.4-P2 and PI-3,4,5-P3. A class of PI3Ks that are stimulated by growth factors include Pl3Ka, PI3Kβ and P13K5. A separate class of P13Ks were activated by coupled G protein receptors and include PI3K ?. The PI3Ks stimulated by the growth factor (ie PI3Ka), have been implicated in cell proliferation and cancer. The PI3K? has shown that it is involved in signaling cascades. For example, the PI3K? it is activated in response to ligands such as C5a, fMLP, ADP and IL-8. Also, the PI3K? it has been implicated in immune diseases (Hirsh et al., Science 2000; 1049-1053). The null macrophages Pl3K? they show a reduced chemotactic response and a reduced ability to combat inflammation (Hirsch et al., 2000, supra). Also, the PI3K? it has also been implicated in thrombolytic diseases (ie, thromboembolism, ischemic diseases, heart attacks and stroke) (Hirsh et al., FASEB J. 2000; 15 (11): 2019-2021 and Hirsh et al., FASEB J. July 9 2001; 10.1096 / fj.00-0810fje (cited in this document as Hirsh et al., 2001).

Inhibitors of PI3Ks members have been developed for the treatment of human disease (see, for example, WO 01/81346, WO 01/53266 and WO 01/83456. There is a need for additional compounds which can inhibit PI3Ks to be used as pharmaceutical agents.

SUMMARY OF THE INVENTION In one aspect, the present invention provides indoles of the formula I: or a pharmaceutically acceptable salt thereof; wherein R2 is U or a C1-C3 alkyl and wherein R1 is an unsubstituted phenyl or a phenyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C4 alkyl, methyl, 0-CC alkyl, methoxy, halo, Cl, Br and I. Examples of the compounds of the formula II include but are not limited to: 5,6-dimethoxy-3-o-tolyl-sulphani-1 H-indole-2-carboxylic acid (2H-) tetrazol-5-yl) -amide; 5,6-Dimethoxy-1-methyl-3-phenylisuphanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 5,6-Dimethoxy-3-phenylsufinanyl-1 H -indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 5,6-Dimethoxy-3- (3-methoxy-phenylsulfanyl) -1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 1-Ethyl-5,6-dimethoxy-3-phenyl-1H-indole-2-carboxylic acid (1H-tetrazol-5-yl) -amide and 5,6-dimethoxy-3-phenyl-1-propyl-1 acid H-indoI-2-carboxylic acid (1 H-tetrazol-5-yl) -amide.

In certain embodiments of Formula I, R is an unsubstituted phenyl - a compound of the Formula II: Examples of Formula II include, but are not limited to: ,6-Dimethoxy-1-methyl-3-phenylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 5,6-Dimethoxy-3-phenylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 1-Ethyl-5,6-dimethoxy-3-phenyl-1H-indole-2-carboxylic acid (1H-tetrazol-5-yl) -amide and 5,6-Dimethoxy-3-phenyl-1-propyl-1 acid H-Indole-2-carboxylic acid (1 H-tetrazol-5-yl) -amide.

In certain embodiments of Formula I, R 1 is a phenyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C 4 alkyl, methyl, 0 C 4 alkyl, methoxy, Cl, Br and I - a compound of Formula III: Examples of the compounds of Formula III include but are not limited to: 5,6-Dimethoxy-3-o-tolylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazoI-5-yl) -amide; 3- (3,4-Dichloro-phenylsulfanyl) -5,6-dimethoxy-1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide and 1-Acid 5,6-Dimethoxy-3- ( 3-methoxy-phenylsulfanyl) -1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide.

In certain embodiments of Formula I, R is a C -C3 alkyl - a compound of the Formula IV: p.

Examples of the compounds of Formula IV include but are not limited to: 5,6-Dimethoxy-1-methyl-3-phenylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 1-Ethyl-5,6-dimethoxy-3-phenyl-1 H-indole-2-carboxylic acid (1 H-tetrazol-5-yl) -amide and 5,6-Dimetoxy-3-phenyl-1-propyl acid -1H-indole-2-carboxylic acid (1 H-tetrazol-5-yl) -amide.

In another aspect, the invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound of Formulas I-IV and a pharmaceutically acceptable carrier. In certain embodiments, these compositions are useful in the treatment of a mediated PI3K disease or condition. The compounds of the invention can also be combined in a pharmaceutical composition which also comprises compounds that are useful for the treatment of cancer, a thrombolytic disease, heart disease, stroke, an inflammatory disease such as rheumatoid arthritis or another disease mediated by PI3K.

In another aspect, the present invention provides methods for treating a subject suffering from a PI3K-mediated disease or condition comprising: administering to a subject suffering from a disease or PI3K-mediated condition, a pharmaceutical composition comprising a therapeutically effective amount of a compound of Formulas I-IV and a pharmaceutically acceptable carrier. In certain embodiments, the mediated PI3K disease or condition is selected from the group consisting of: rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases and autoimmune diseases. In other embodiments, the PI3k condition or disease mediated is selected from the group consisting of: cardiovascular diseases, arterioscles, hypertension, deep vein thrombosis, stroke, myocardial infarction, unstable angina, thromboembolism, pulmonary embolism, thrombolytic diseases, acute arterial ischemia, peripheral thrombotic occlusions and coronary artery disease. In some embodiments, the PI3K mediated disease or condition is selected from the group consisting of: cancer, colon cancer, glioblastoma, endometrial carcinoma, hepatocellular cancer, lung cancer, melanoma, renal cell carcinoma, thyroid carcinoma, cell lymphoma, diseases lymphoproliferatives, small cell lung cancer, squamous cell lung carcinoma, glioma, breast cancer, pate cancer, ovarian cancer, cervical cancer and leukemia. In another embodiment, the mediated PI3K disease or condition is selected from the group consisting of: type II diabetes. In other embodiments, the PI3K-mediated condition or disease is selected from the group consisting of: respiratory diseases, bronchitis, asthma, and chronic obstructive pulmonary disease. In certain modalities, the subject is a human.

DEFINITIONS As used in this document, the following terms have the meaning ascribed to them unless otherwise specified.

A "PI3K medal condition or disease" is characterized by the participation of one or more PI3Ks or a PI3P phosphatase (ie, PTEN, etc.) in the beginning, manifestation of one or more symptoms or markers of disease, severity or progress of a disease or condition. Conditions or diseases mediated by PI3K include but are not limited to: rheumatoid arthritis, osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases, pulmonary fibs, autoimmune diseases, cardiovascular diseases, arterioscles, hypertension, deep vein thrombosis, stroke, myocardial infarction, angina unstable, thromboembolism, pulmonary embolism, thrombolytic diseases, acute arterial ischemia, peripheral thrombotic occlusions, coronary artery disease, cancer, breast cancer, glioblastoma, endometrial carcinoma, hepatocellular carcinoma, colon cancer, lung cancer, melanoma, renal cell carcinoma, thyroid carcinoma, small cell lung cancer, squamous cell lung carcinoma, glioma, pate cancer, ovarian cancer, cervical cancer, leukemia, cell lymphoma, lymphoproliferative diseases, type II diabetes, respiratory diseases, bronchitis, asthma and pu disease chronic obstructive lmonar.

A PI3K is an enzyme that is capable of phosphorylating the 3'-0H of a phosphoinositol to generate PI3P. PI3Ks include but are not limited to PI3Ka, PI3Kß, PI3K? and PI3Kd. A PI3K typically comprises at least one catalytic subunit (ie p110?) And may further comprise a regulatory subunit (ie p101, etc.).

The term "alkyl group" or "alkyl" includes branched or linear carbon chain radicals. The term "alkylene" refers to a biradical of a substituted or unsubstituted alkane. For example, a "C-M alkyl" is an alkyl group having 1 to 4 carbon atoms. Examples of the straight chain alkyl groups include but are not limited to methyl, ethyl, n-propyl, n-butyl, etc. Examples of branched chain alkyl groups include but are not limited to isopropyl, tert-butyl, isobutyl, etc. Examples of alkylene groups include but are not limited to -CH2-, -CH2-CH2-, -CH2-CH (CH3) -CH2- and - (CH2)? -4. The alkylene groups can be substituted with groups as set forth below for the alkyl.

In addition, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl portions having substituents that replace a hydrogen at one or more carbons (i.e. replacing a hydrogen at 1, 2, 3 or 4 carbons) of the hydrocarbon column. Such substituents may include but are not limited to C2-C6 alkenyl, C2-C6 alkynyl, halo, I, Br, Cl, F, -OH, -COOH, sulfhydryl, (CrCr-S- alkyl, C6-alkylsulfinyl, nitro, cyano, trifluoromethyl, NH 2, = 0, = S, = N-CN, = N-OH, C 1 -C 6 alkylsulfinyl, nitro, cyano, trifluoromethyl, -NH 2, = 0, = N-CN, = N-OH, -OCH 2 F , -OCHF2, -OCF3, -SCF3, -S02-NH2, C-Cβ alkoxy, -C (0) 0- (alkylated dC-β), -C (0) -NH2-, C (0) -N ( H) -alkyl d-Cß, -C (0) -N (C 6 alkyl) 2, -OC (0) -NH 2, -C (0) -H, -C (O) -, (d-C6 alkyl) , -C (S) - (C6 alkyl), -NR70R72, wherein R70 and R72 are each independently selected from H, d-Cß alkyl, C2-C6 alkenyl, C2-C6 alkynyl and C (0) alkyl- C C6.

Typical substituted alkyl groups are furthermore aminomethyl, 2-nitroethyl, 4-cyanobutyl, 2,3-dichloropentyl and 3-hydroxy-5-carboxyhexyl, 2-aminoethyl, pentachloroethyl, trifluoromethyl, 2-diethylaminoethyl, 2-dimethylaminopropyl, ethoxycarbonylmethyl, methanylsulfanylmethyl , methoxymethyl, 3-hydroxypentyl, 2-carboxybutyl, 4-chlorobutyl and pentafluoroethyl. "alkoxy" refers to the aforementioned alkyl groups linked through oxygen, examples of which include methoxy, ethoxy, isopropoxy, fe-butoxy, and the like. In addition, alkoxy refers to polyethers such as 0- (CH2) 2-0-CH3 and the like. The term "alkoxy" is intended to include both substituted and unsubstituted alkoxy groups. The alkoxy groups can be substituted on the carbon atoms with groups such as those established for the alkyl. Typical substituted alkoxy groups include aminomethoxy, trifluoromethoxy, 2-diethylaminoethoxy, 2-ethoxycarbonylethoxy, 3-hydroxypropoxy and the like.

"Alkanoyl" groups are alkyl bonded through a carbonyl, i.e. C alquilo-C6-C (0) - alkyl. Such groups include formyl, acetyl, propionyl, butyryl and isobutyryl. The term "alkanoyl" is intended to include both substituted and unsubstituted alkanoyl groups. The alkanoyl groups can be substituted with groups such as those established for alkyl.

"Halo" includes fluorine, chlorine, bromine and iodine.

"Alkenyl" means linear or branched hydrocarbon radicals having 2 or more carbon atoms and comprising at least one carbon-carbon double bond and includes ethenyl, 3-buten-1-yl, 2-ethenyl-butyl, 3-hexen-1- il and the like. The term "alkenyl" is intended to include amnes substituted and unsubstituted alkenyl groups. A "C2-C6 alkenyl" is an alkenyl group having from 2 to 6 carbon atoms. The alkenyl groups may be substituted with groups such as those set forth above for the alkyl. The term "alkenylene" refers to a biradical of a substituted or unsubstituted alkene. Examples of the alkenylene groups include but are not limited to -CH = CH-, -CH = CH-CH2- and - (CH2) 1.6-CH = CH-CH2-.

"Alkynyl" means straight and branched hydrocarbon radicals having 2 or more carbon atoms and comprising at least one carbon-carbon triple bond and including ethinyl, 3-butin-1-yl, propinyl, 2-butin-1-yl, 3-pentyl-1-yl and the like. The term "alkynyl" is intended to include both substituted and unsubstituted alkynyl groups. The alkynyl groups can be substituted with groups such as those set forth above for the alkyl. In certain embodiments, a branched chain or straight chain alkynyl group has 6 or fewer carbon atoms in its column (ie C2-C6 for the straight chain, C3-C6 for the branched chain). The term C2-C6 includes alkynyl groups containing 2 to 6 carbon atoms. The term "alkynylene" refers to a biradical of a substituted or unsubstituted alkyne. Examples of the alkynylene groups include but are not limited to -CH = CH-, -C = C-CH2 and- (CH2) 1-6-C = C-CH2-.Some of the compounds of the present invention can exist as stereoisomers including enantiomers, diastereomers and geometric isomers. Geometric isomers include compounds of the present invention having alkenyl groups, which may exist as entgegen or zusammen conformations, in which case all geometric forms thereof, both entgegen and zusammen, cis and trans and mixtures thereof, are within the scope of the present invention. Some compounds of the present invention have cycloalkyl groups that can be substituted on more than one carbon atom, in which case all geometric forms thereof, both cis and trans and mixtures thereof, are within the scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to Índoles of the Formulas I-IV, wherein R1 and R2 have any of the values defined in this specification and the pharmaceutical salts thereof which are useful as agents in the treatment of diseases and conditions, including inflammatory diseases, cardiovascular diseases and cancers. Also provided are pharmaceutical compositions comprising one or more compounds of Formulas I-IV.

PREPARATION OF COMPOUNDS The compounds of the present invention (ie, the compounds of Formulas I-1V) can be prepared by applying the synthetic methodology known in the art and the synthetic methodology outlined in the subsequent syntheses.

Synthesis In Synthesis I, the compounds of formula I can be synthesized using the synthetic synthesis depicted. The ethyl ester of 1 H-indole-2-carboxylic acid 2, the ethyl ester of (5,6-dimethoxy-1H-indole-2-carboxylic acid (Lancaster Synthesis Inc., Windham, NH) is reacted in dichloromethane with N -chlorosuccinimide (NCS) treated with R1-SH (ie, 2-methyl benzenethiol, 3,4-dichlorobenzenethiol, 3,4-diciorobenzenethiol, benzenethiol, 3-methoxybenzenethiol, etc.) to produce 4 (ie ethyl ether of the acid 5,6-Dimethoxy-3-o-tolylsulfanyl-1H-indole-2-carboxylic acid) The ester 4 is subsequently saponified with an inorganic base such as LiOH or NaOH in MeOH and THF solution, dioxane and water or methanol and water. to produce carboxylic acid 6 (ie 5,6-dimethoxy-3-o-tolylsulfanyl-1 H-indole-2-carboxylic acid).

The carboxylic acid 6 is subsequently coupled to 5-aminotetrazole by reaction with 4-methylmorpholine (NMM), 1-hydroxybenzotriazole (HOBT) and 1- [3-dimethylamino) propyl] -3-ethylcarbodiimide hydrochloride (EDCI) in a solvent such as tetrahydrofuran to give 8 (ie, 5,6-dimethoxy-3-o-tolylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) amide).

Synthesis 2 As shown in Synthesis 2, the compounds of formula 4 can be alkylated in the indole nitrogen by reaction with a base of sodium hydride (NaH) and a C3 alkyl halide such as iodomethane in a solvent such as THF and N, N-dimethylformamide to produce the N-alkylated indole 20 (ie 5,6-dimethoxy-1-methyl-3-phenylsulfanyl-1H-indole-2-carboxylic acid ethyl ester). The acid is then saponified 22 as described in Synthesis 1. The aminotetrazole was subsequently coupled to 22 as described in Synthesis 1 to produce 24.

ASSESSMENT OF COMPOUNDS The compounds of the present invention (ie, compounds of Formulas I-IV and pharmaceutically acceptable salts thereof) can be tested for their ability to inhibit a PI3K. Examples of these tests are set forth below and include in vitro and in vivo tests of PI3K activity.

In certain embodiments of the present invention are compounds that selectively inhibit one or more PI3Ks as compared to one or more enzymes including but not limited to a cyclic nucleotide-dependent protein kinase, PDGF, a tyrosine kinase, a MAP kinase, a MAP kinase kinase, a MEKK, a cylin dependent protein kinase. In other embodiments of the invention are compounds that selectively inhibit a PI3K as compared to another PI3K. For example, in certain embodiments, the compounds of the present invention display the ability to selectively inhibit PI3K? as compared to PI3Ka p Pl3Kß. A compound selectively inhibits a first enzyme as compared to a second enzyme, when the IC50 of the compound towards the first enzyme is less than the IC50 of the compound towards the second compound. The IC50 can be measured, for example in a PI3K in vitro test.

In the presently preferred embodiments, the compounds of the present invention can be tested for their ability to inhibit PI3K activity in an in vitro or in vivo test (see below).

The PI3K tests were carried out in the presence or absence of a PI3K inhibitor compound and the amount of enzyme activity is compared by a determination of the inhibitory activity of PI3K inhibitor compound.

Samples that do not contain a PI3K inhibitor compound are assigned a relative PI3K activity value of 100. Inhibition of PI3K activity is achieved when the PI3K activity in the presence of a PI3K inhibitor compound is lower than the control sample (ie say non-inhibitory compound). The IC50 of a compound is the concentration of compound that exhibits 50% of the activity of the control sample. In certain embodiments, the compounds of the present invention have an IC50 of less than about 100 μM. In other embodiments, the compounds of the present invention have an IC50 of about 200 nM or less.

The PI3K tests? have been described in the art (see for example Leopoldt et al., J. Biol .. Chem., 1998; 273: 7024-7029). Typically, a sample that contains a complex of p101 and the protein p110? are combined with Gß and G proteins? (ie protein G subunits ß? /? 2). Radiolabelled ATP (ie? 32P-ATP) is subsequently added to this mixture.The lipid substrates are formed by the creation of PIP2 which contains lipid micelles.The reactions are subsequently initiated by adding the lipid and the enzyme mixtures and they stop with the addition of H3PO4. the products lipids are subsequently transferred to a filter plate fiberglass and washed with H3P04 several times. the presence of radioactive lipid product (PIP3) can be measured using radiometric methods that are well known in The technique.

The activity of PI3Ks regulated by the growth factor can also be measured using a lipid kinase test. For example, PI3Ka can be measured using samples containing a catalytic and regulatory subunit. An activation peptide (ie pY peptide, SynPep Corp.) was added to the sample with radiolabeled ATP. The PIP2 containing the lipid micelles are subsequently added to the sample to initiate the reaction. Reactions were worked on and analyzed as described by APRA PI3K test? just described. The tests were also carried out using cell extracts (Susa et al., J. Biol .. Che., 1992; 267: 22951-22956).

SALTS AND SOLVATOS PHARMACEUTICALLY ACCEPTABLE The compounds to be used in the present invention can exist in unsolvated forms as well as in solvated forms including the hydrated forms. In general, solvated forms, including hydrated forms, are equivalent to unsolvated forms and are intended to be within the scope of the present invention.

The compounds of the present invention (ie, the compounds of Formulas I-IV) are capable of further forming both pharmaceutically acceptable salts, including but not limited to basic salts and / or addition acids. The pharmaceutically acceptable salts of the compounds of the formula (I) include the basic salts and addition acids (including bisalts) thereof. Examples of the appropriate salts can be found for example in Stahl and Wermuth, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Wiley-VCH, Weinheim, Germany (2002) and Berge et al., "Pharmaceutical Salts", J. of Pharmaceutical Science, 1977; 66: 1-19.

Acidic salts of pharmaceutically acceptable addition compounds of Formulas l-IV include nontoxic salts derived from inorganic acids such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydriodic, phosphorous and the like, as well as the salts derived from organic acids, such as mono and bi-carboxylic acids, phenyl substituted alkanoic acids, hydroxy alkanoic acids, alkanedithic acids, aromatic acids, aromatic sulfonic acids and aliphatic acids, etc. Said salts further include acetate, aspartate, benzoate, besylate (benzenesulfonate), bicarbonate / carbonate, bisulfate, caprylate, camsylate (camphor sulfate), chlorobenzoate, citrate, edisilate (1,2-ethane bisulfonate), dihydrogenphosphate, dinitrobenzoate, esylate ( sulfonate ethane), fumarate, gluceptate, gluconate, glucuronate, hybienate, hydrochloride / chloride, hydrobromide / bromide, hydrothide / iodide, isobuthyrate, monohydrogen phosphate, isethionate, D-lactate, L-lactate, malate, maleate, malonate, mandelate, mesylate methanesulfonate), metaphosphate, methylbenzoate, methylisulfate, 2-napsolate (2-naphthalene sulfonate), nicotinate, nitrate, orotate, oxalate, palmoate, phenylacetate, phosphate, phthalate, propionate, pyrophosphate, pyrosulfate, saccharate, sebacate, stearate, suberate, sulfate succinate, sulfite, D-tartrate, L-tartrate, tosylate (toluene sulfonate) and xinafoate salts and the like of the compounds of Formulas I-IV. Also contemplated are the salts of the amino acids such as arginate, gluconate, galacturonate and the like.

The acid addition salts of the basic compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt in a conventional manner. The free base form can be regenerated by contacting the salt form with a base and isolating the free base in a conventional manner. The free base forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free bases for purposes of the present invention.

The pharmaceutically acceptable basic addition salts are formed with metals or amines, such as alkaline earth metal or alkali hydroxides or organic amines. Examples of metals used as cations are aluminum, calcium, magnesium, potassium, sodium and the like. Examples of the appropriate amines include arginine, choline, chloroprocaine, N.N'-dibenzylethylenediamine, diethylamine, diethanolamine, diolamine, ethylenediamine (ethane-1,2-diamine), glycine, lysine, meglumine, N-methylglucamine, olamine, procaine (benzathine) and tromethamine.

The basic addition salts of the acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form can be regenerated by contacting the salt form with an acid and isolating the free acid in a conventional manner. The free-form forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention.

PHARMACEUTICAL COMPOSITIONS AND METHODS OF ADMINISTRATION This invention provides compositions comprising a therapeutically effective amount of a compound of Formulas I-IV or a pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier, diluent or excipient therefor. The phrase "pharmaceutical composition" refers to a composition suitable for administration in medical or veterinary use. The phrase "therapeutically effective amount" means an amount of a compound or pharmaceutically acceptable salt thereof sufficient to inhibit, arrest or allow an improvement in the disease or condition being treated when administered alone or in conjunction with another agent or treatment. pharmaceutical in a particular subject or subject population. For example, in a human or in another mammal, a therapeutically effective amount may be determined experimentally in a laboratory or clinical setting or may be the amount required by the statutes of the Food and Drug Administration of the United States or an equivalent foreign agency, for the particular disease and the subject that is being treated.

It should be appreciated that the determination of the appropriate dosage forms, the dosage amounts and the routes of administration are within the levels of the medical and pharmaceutical art and are described below.

A compound of the present invention can be formulated as a pharmaceutical composition in the form of a syrup, an elixir, a suspension, a powder, a granule, a tablet, a capsule, a troche, an aqueous solution, a cream, an ointment, a lotion, a gel, an emulsion, etc. Preferably, a compound of the present invention will cause a decrease in symptoms or an indication of disease associated with a PI3K mediated disease as measured quantitatively or qualitatively.

To prepare pharmaceutical compositions from the compounds of the present invention, pharmaceutically acceptable carriers can be solid or liquid. Solid form preparations include powders, tablets, pills, capsules, caches, suppositories and dispersible granules. A solid carrier may be one or more substances which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents or an encapsulating material.

In powders, the carrier is a finely divided solid that is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier which has the necessary binding properties in appropriate proportions and is compacted in the desired shape and size.

The powders and tablets contain from 1% to 95% e (w / w) of the active compound. In certain embodiments, the active compound ranges from 5% to 70% (w / w). Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter and the like. The term "preparation" is intended to include the formulation of the active compound with the encapsulating material as a carrier that provides a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is also in association with it. . Similarly, capsules and troches are included. Tablets, powders, capsules, pills, cachets and troches can be used as solid dosage forms suitable for oral administration.

For the preparation of suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first mixed and the active component dispersed homogeneously as by agitation. The homogeneous mixture subsequently fused is poured into molds of suitable size, allowing them to cool and therefore solidify.

Liquid form preparations include solutions, suspensions and emulsions, for example propylene glycol / water or water solutions. For parenteral injection, liquid preparations can be formulated in aqueous polyethylene glycol solution.

Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding colorants, flavors, stabilizers and thickening agents as desired. Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as synthetic or natural gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents.

Also included are solid form preparations which are intended to be rapidly converted prior to use to liquid form preparations for oral administration. Said liquid forms include solutions, suspensions and emulsions. These preparations may contain, in addition to the active component, colorants, flavors, stabilizers, binders, natural and artificial sweeteners, dispersants, thickeners, solubilizing agents and the like.

The pharmaceutical preparation is preferably in a unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packed tablets, capsules and powders in ampoules or injections. Also, the unit dosage form may be a capsule, tablet, cachet or tablet by itself may be the appropriate number of any of these packaged forms.

The amount of the active component in a unit dose preparation can be varied or adjusted from 0.1 mg to 1000 mg, preferably 1.0 mg to 100 mg or from 1% to 95% (w / w) of a unit dose, in accordance with particular application and the potency of the active compound. The composition can, if desired, also contain other compatible therapeutic agents.

The pharmaceutically acceptable carriers are determined in part by the particular composition being administered as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition, Gennaro et al., Eds. Lippincott Williams and Wilkins, 2000).

A compound of the present invention, alone or in combination with other appropriate components, can be made in aerosol formulations (ie, they can be "nebulized") to be administered via inhalation. The aerosol formulations can be placed in acceptable pressurized propellants, such as dichlorodifluoromethane, propane nitrogen and the like.

Formulations suitable for parenteral administration, such as for example by intravenous, intramuscular, intradermal and subcutaneous routes, include isotonic, non-aqueous or aqueous sterile injection solutions which may contain antioxidants, binders, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient and the nonaqueous and aqueous sterile suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. In the practice of this invention, the compositions can be administered for example by intravenous, oral, topical, intraperitoneal, intravesical or intrathecally infusion. The formulations of the compounds can be presented in sealed multi-dose or single dose containers, such as ampules and injections. Injectable solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

The dose administered to a subject in the context of the present invention should be sufficient to affect a beneficial therapeutic response in the subject in time. The term "subject" refers to a member of the mammalian class. Examples of mammals include no human limit, primates, chimpanzees, rodents, mice, rats, rabbits, horses, cattle, dogs, cats, sheep and cows.

The dose will be determined by the efficacy of the particular compound employed and the condition of the subject, as well as the body weight or surface area of the subject to be treated. The size of the dose will also be determined by the existence, nature of any adverse side effects that accompany the administration of a particular compound in a particular subject. In determining the effective amount of the compound to be administered in the treatment or prophylaxis of the disease being treated, the physician may evaluate factors such as the circulating plasma levels of the compound, the toxicities of the compound and / or the progress of the disease. , etc. In general, the equivalent dose of a compound is from about 1 μg / kg to 100 mg / kg for a typical subject. Many of the different administration methods are known to those skilled in the art.

For administration, the compounds of the present invention may be administered in a proportion determined by factors that may be included but not limited to the LD50 of the compound, the pharmacokinetic profile of the compound, the contraindicated drugs as applied to the dough and to health in general of the subject.

Examples of a typical tablet, parenteral and patch formulation include the following: EXAMPLE 1 FORMULATION OF THE TABLET Formulation of the Tablet Ingredient Quantity Compound of the Formulas I-IV 50 mg Lactose 80 mg Corn starch (for mixing) 10 mg Corn starch (for pasta) 8 mg Magnesium stearate (1%) 2 mg 150 mg The compounds of the present invention (ie a compound of Formulas I-IV or a pharmaceutically acceptable salt thereof) can be mixed with lactose and corn starch (for mixing) and mixed uniformly for a powder. The corn starch (for pasta) is suspended in 6 ml of water and heated with agitation to form a paste. The paste is added to the mixed powder and the mixture is granulated. The wet granules are passed through a hard screen No. 8 and dried at 50 ° C. The mixture is lubricated with 1% magnesium stearate and compressed into a tablet. The tablets are administered to a patient in the ratio of 1 to 4 each day for the treatment of a PI3K-mediated condition or disorder.

EXAMPLE 1 FORMULATION OF PARENTERAL SOLUTION In a solution of 700 ml of propylene glycol and 200 ml of water for Injection, 20.0 g of a compound of the present invention can be added. The mixture is stirred and the pH is adjusted to 5.5 with hydrochloric acid. The volume was adjusted to 1000 ml with water for injection. The solution was sterilized, filled in 5.0 ml injections, each containing 2.0 ml (40 mg of the compound of the invention) and sealed under nitrogen. The solution was administered by injection to a subject suffering from a condition or PI3K disorder mediated and in need of treatment.

EXAMPLE 1 PATCH FORMULATION Ten milligrams of a compound of the present invention can be mixed with 1 ml of propylene glycol and 2 mg of adhesive with acrylic-based polymer containing a resinous crosslinking agent. The mixture was applied to an impermeable support (30 cm2) and applied to the upper back of a patient for the prolonged-release treatment of a PI3k-mediated disease or condition.

METHODS FOR THE TREATMENT OF MEDIATED PI3K CONDITIONS AND DISEASES The compounds of the present invention and pharmaceutical compositions comprising a compound of the present invention can be administered to a subject suffering from a PI3K-mediated condition or disorder. Disorders and P13K mediated diseases can be treated prophylactically, acutely and chronically using the compounds of the present invention, depending on the nature of the disease or condition. Typically, the host or subject in each of these methods is human although other mammals may also benefit from the administration of a compound of the present invention.

In therapeutic applications, the compounds of the present invention can be prepared and administered in a wide variety of parenteral and oral dosage forms. The term "administer" refers to the method of contacting a compound with a subject. In addition, the compounds of the present invention can be administered by injection, that is intravenous, intramuscular, intracutaneous, subcutaneous, intraduodenal, parenteral or intraperitoneally. Also, the compounds described herein can be administered by inhalation, for example, intranasally. Additionally, the compounds of the present invention can be administered transdermally, topically, via implantation, transdermally, topically and via implantation. In certain embodiments, the compounds of the present invention are administered orally. The compounds can also be administered rectally, buccally, intravaginally, ocularly, andially or by insufflation.

The compounds used in the pharmaceutical method of the invention can be administered in initial doses of about 0.001 mg / kg to about 100 mg / kg daily. In certain embodiments, the daily dose range is from about 0.1 mg / kg to about 10 mg / kg. The doses, however, can be varied depending on the requirements of the subject, the severity of the condition being treated and the compound being used. The determination of the appropriate dose for a particular situation is known to the person skilled in the art. Generally, treatment starts with smaller doses, which are less than the optimal dose of the compound. Then, the dose was increased by small increments until the optimum effect was reached under the circumstances. For convenience, the oral daily dose can be divided and administered in portions during the day if desired. The term "treatment" includes the prophylactic, chronic or acute alleviation or reduction of at least one symptom or characteristic associated with or caused by the disorder being treated. For example, the treatment may include the reduction of various symptoms of a disease, inhibition of the pathological progress of a disease or the complete eradication of a disease. The compounds of the present invention can be co-administered to a subject. The term "co-administration" means the administration of two or more different pharmaceutical agents or treatments (i.e., radiation treatment) that are administered to a subject by the combination in the same pharmaceutical composition or separate pharmaceutical compositions. In addition, co-administration involves the administration at the same time of a simple pharmaceutical composition comprising two or more pharmaceutical agents or the administration of two or more different compositions to the same subject in the same or at different times. For example, a subject who was administered a first dose comprising a compound of the present invention at 8 a.m. and then CELEBREX® is administered at 1-12 hours later, that is to say at 6 p.m. on the same day he has been co-administered a compound of the present invention and CELEBREX®. Alternatively, for example, a subject could be administered a single dose comprising a compound of the present invention and CELEBREX®.

In addition, the compounds of the invention can also be co-administered with compounds that are useful for the treatment of cancer (ie, cytotoxic drugs such as TAXOL®, taxotere, GLEEVEC® (Imatinib mesylate), adriamycin, daunomycin, cisplatin, etoposide , a vinca alkaloid, vinblastine, vincristine, methotrexate or adriamycin, daunomycin, cis-platinum, etoposide and alkaloids such as vincristine, farnesyl transferase inhibitors, endostatin and angiostatin, VEGF inhibitors, and antimetabolites such as methotrexate.The compounds of the present invention can also be used in combination with a taxane derivative, a platinum coordination complex, a nucleoside analogue, an anthracycline, a topoisomerase inhibitor or an aromatase inhibitor). Radiation treatments can also be co-adhered with a compound of the present invention for the treatment of cancers.

The compounds of the invention can be co-administered with compounds that are useful for the treatment of a thrombolytic disease, heart disease, stroke, etc. (ie aspirin, streptokinase, pyosminogen activator in tissue, urokinase, anticoagulants, antiplatelet drugs say PLAVIX®, clopidogrei bisulfate), a statin (ie, LIPITOR® (calcium from atorvastatin), ZOCOR® (Simvastatin), CRESTOR® (Rosuvastatin), etc.), a beta-blocker (ie, Atenolol), NORVASC ® (amlodipine besylate) and an ACE inhibitor (ie Accupril® (Quinapril hydrochloride), Lisinopril, etc.).

The compounds of the present invention can also be co-administered for the treatment of hypertension with compounds such as ACE inhibitors, lipid reducing agents such as statins, LIPITOR® (calcium of atorvastatin), calcium channel blockers such as NORVASC® ( amlodipine besilate). The compounds of the present invention can also be used in combination with fibrates, beta-blockers, NEPI inhibitors, Angiotensin-2 receptor antagonists and platelet aggregation inhibitors.

For the treatment of inflammatory diseases, including rheumatoid arthritis, the compounds of the invention can be co-administered with agents such as TNF-a inhibitors such as monoclonal anti-TNFa antibodies (such as REMICADE®, CDP-870 and HUMIRA ™ (adalimumab ) and the immunoglobulin-TNF receptor fusion molecules (such as ENBREL®, IL-1 inhibitors, receptor antagonists or soluble IL-1Ra (ie KINERET ™ or ICE inhibitors), non-spheroidal anti-inflammatory agents (NSAIDS), piroxicam, diclofenac, naproxen, flurbiprofen, fenoprofen, ketoprofen ibuprofen, fenamates, mefenamic acid, indomethacin, sulindac, apazone, pyrazolones, phenylbutazone, aspirin, COX-2 inhibitors (such as CELEBREX®- (celecoxib), VIOXX® (rofecoxib), BEXTRA® (valdecoxib) and etoricoxib, metalloprotease inhibitors (preferably selective MMP-13 inhibitors), NEUROTIN®, pregabalin, low dose methotrexate, leflunomide, hydroxychloroquine, d-penicillamine, au ranofin or parenteral or oral gold.

The compounds of the invention can be co-administered with the existing therapeutic agents for the treatment of osteoarthritis. Suitable agents to be used in combination include standard non-spheroidal anti-inflammatory agents (herein NSAIDs) such as piroxicam, diclofenac, propionic acids such as naproxen, flurbiprofen, fenprofen, ketoprofen and ibuprofen, fenamates such as phenylbutazone, salicylates such as aspirin. , COX-2 inhibitors such celecoxib, valdecoxib, rofecoxib and etoricoxib, analgesics and intra-articular therapies such as corticosteroids and hyaluronic acids such as hyalgan and sinvisc.

The compounds of the invention can also be co-administered with antiviral agents such as Viracept, AZT, acyclovir and famciclovir and antisepsis compounds such as Valant.

The compounds of the present invention can also be co-administered with CNS agents such as antidepressants (such as sertralline), anti-Parkinsonism drugs (such as deprenil, L-Dopa, Requip, Mirapex, MAOB inhibitors such as selegine and rasagiline, comP inhibitors such as Tasmar, A-2 inhibitors, dopamine re-uptake inhibitors, NMDA antagonists, agonists of Nicotine, Dopamine agonists and inhibitors of neuronal nitric oxide synthase) and anti-Alzheimer's drugs such as donepezil, tacrlna, NEUROTIN®, pregabalin, COX-2 inhibitors, propentofylline or metrifonate.

The compounds of the present invention can be additionally co-administered with osteoporosis agents such as EVISTA® (raloxifene hydrochloride) droloxifen, lasofoxifen or fosomax and immunosuppressive agents such as FK-506 and rapamycin.

EXAMPLES EXAMPLES 1-7 Intermediate 1: 5,6-Dimethoxy-3-o-tolylsulfanyl-1H-indole-2-carboxylic acid ethyl ester To a solution of -78 ° C dichloromethane (30 ml) and N-chlorosuccinimide was added 2-methyl benzenethiol (0.709, 6.01 mmol) per drop. The reaction was allowed to warm to 0 ° C and subsequently stirred for 30 minutes. 5,6-Dimethoxy-1H-indole-2-carboxylic acid ethyl ester (Lancaster Synthesis Inc., Windham, NH) (1.5 g, 6.01 mmol) in dichloromethane (15 mL) was added dropwise. The reaction was warmed to room temperature and stirred overnight. The reaction was concentrated under reduced pressure. Methanol (5 ml) was added to the residue and a solid formed. The solid was collected, washed with diethyl ether (20 ml) and air dried to yield a beige solid (1.46 g, 66%). MS: M + -1 = 370.2 Da.

Intermediate 2: 5,6-Dimethoxy-3-o-tolylsulfanyl-1H-indole-2-carboxylic acid To a solution at room temperature of Intermediate 1 (0.500 g, 1.35 mmol) in methanol (15 ml) and tetrahydrofuran was added 1 N of lithium hydroxide (2.96 ml, 2.96 mmol). The reaction was stirred overnight and then 1 N of lithium hydroxide (2.96 mL, 2.96 mmol) was added. The reaction was again heated and stirred overnight. The reaction was diluted with ethyl acetate (200 ml) and subsequently washed twice with brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure to yield a crude solid. The solid was collected and washed with 1: 1 hexanes / diethyl ether and subsequently air dried to provide the pure product (0.260 g, 56%). MS: M + -1 = 342.1 Da.

Example 1. 5,6-Dimethoxy-3-o-tolylsulfanyl-1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide.

To a solution at room temperature of Intermediate 2 (0.220 g, 0.641 mmol) in tetrahydrofuran (10 mL) was added 4-methylmorpholine (0.14 mL, 1.28 mmol), 1-hydroxybenzotriazole (0.129 g, 0.96 mmol) and 1- hydrochloride. [3-dimethylamino) propyl] -3-ethylcarbodiimide (0.183 g, 0.96 mmol) and 5-aminotetrazole (0.054, 0.64 mmol). The reaction was allowed to stir overnight. The reaction was subsequently diluted with ethyl acetate (200 ml). The organic layer was washed twice with 5% citric acid and then once with brine. The organic layer was dried over magnesium sulfate, filtered and then concentrated under reduced pressure to yield a crude solid. The solid was collected and washed with diethyl ether to yield the title product (0.128 g, 49%).

Example 2. 3- (3,4-Dichloro-phenylsulfanyl) -5,6-dimethoxy-1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide. The title compound was synthesized in a manner similar to that described for Example 1 using 3,4-dichlorobenzenethiol in place of 2-methylene benzenethiol. The product was obtained as a yellow powder (0.132 g, 23%).

Intermediate 3: 5,6-Dimethoxy-1-methyl-3-phenylsulfanyl-1H-indole-2-carboxylic acid ethyl ester. To a solution of tetrahydrofuran at 0 ° C of 5,6-dimethoxy-3-phenylsulfanyl-1H-indole-2-carboxylic acid ethyl ester (0.500 g, 1.40 mmol) was added sodium hydride (95%, 0.037 g, 1.54 mmol) followed by iodomethane (0.096 mL, 1.54 mmol). The ethyl ester of 5,6-dimethoxy-3-phenylisulfanyl-1 H-indole-2-carboxylic acid was synthesized in a manner analogous to Intermediate 1 using benzene thiol instead of 2-methyl benzenethiol. N, N-Dimethylformamide was added to homogenize the reaction. The reaction was allowed to warm to room temperature and stirred overnight. The reaction was diluted with ethyl acetate (100 mL). The organic layers were washed with 1 N hydrochloric acid (25 mL) and brine (25 mL), dried over magnesium sulfate, filtered and concentrated under reduced pressure to yield the title compound, which was used without further purification.

Example 3. 5,6-Dimethoxy-1-methyl-3-phenylsulfanyl-1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide. The title compound was synthesized in a manner similar to that described for Example 1 using Intermediary 3 instead of Intermediary 1. The product was obtained as a white powder (0.025 g, 11%).

Example 4. 5,6-Dimethoxy-3-phenylsuIFanyl-1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide. The title compound was synthesized in a manner similar to that described for Example 1 using benzenethiol in place of 2-methyl-benzenethiol. The product was obtained as a yellow powder (0.079 g, 33%).

Example 5. 5,6-Dimethoxy-3- (3-methoxy-phenylsulfanyl) -1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide. The title compound was synthesized in a manner similar to that described for Example 1 using 3-methoxybenzenethiol in place of 2-methyl-benzenethiol. The product was obtained as a yellow powder (0.024 g, 16%).

Example 6. 1-Ethyl-5,6-dimethoxy-3-phenyl-1H-indole-2-carboxylic acid (1H-tetrazol-5-yl) -amide The title compound was synthesized in a manner similar to that described for Example 3 using iodo-ethane instead of iodo-methane as in the synthesis of Intermediate 3.

Example 7. 5,6-Dimethoxy-3-phenyl-1-propyl-1H-indole-2-carboxylic acid (1H-tetrazol-5-yl) -amide.

The title compound was synthesized in a manner similar to that described for Example 3 using 1-iodo-propane instead of iodo-methane as in the synthesis of Intermediate 3.

EXAMPLE 1 BIOLOGICAL Expression of the PI3Ky Protein and Purification Protocol The spodtera frugiperda cells, cultured in the ESF921 medium were co-infected with baculovirus expressing the glu-labeled p101 and the baculovirus expressing an HA-labeled p110? in a 3: 1 ratio of baculovirus p101 to baculovirus p110 ?. Sf9 cells were cultured for 1 x 10 7 total cells / ml in 10 L of the blockers and cultured 48-72 hours post-infection. Samples of the infected cells were subsequently tested for the expression of p101 / p110? PI3 by immunoprecipitation and Western Hybridization analysis methods.

To purify PI3K ?, 4 volumes at room temperature of hypotonic lysis stabilizer (1 mM MgCl2, 1 mm DTT, 5 mM EGTA, 1 mM Pefabloc, 0.5 μM aprotinin, 5 μM leupeptin, 2 μM pepstatin, 5 μM E64, pH 8) per gram of cell paste, was poured into frozen cell granules with stirring, subsequently used in a 400 psi nitrogen "pump" (599 HC T316, Parr Instrument Co Moline, IL). The NaCl was added at 150 mM and sodium cholate was added at 1% and mixed for another 45 minutes. The lysates were clarified by centrifugation for 25 minutes at 14,000 rpm. The lysates were then loaded onto Sepharose beds of the anti-glu-linked Protein G (Covance Research Products, Richmond, CA) using 200 ml resin / 50 g cell paste. The column was washed with 15 volumes of wash binder (1 mM DTT, 0.2 mM EGTA, 150 mM NaCl, 1% sodium cholate, pH 8). The PI3K? it was eluted with 6 column volumes of the wash binder containing 100 μg / ml of a peptide competent to bind the glu tag. Column fractions with the eluted protein (determined by taking the OD280 readings) were collected and dialysed in 0.2 mM EGTA, 1 mM DTT, 1 mM Pefabloc, 5 μM leupetin, 0.5% sodium cholate, 150 mM NaCl and 50% glycerol, pH 8. Fractions were stored at -80 ° C until further use.

EXAMPLE 2 BIOLOGICAL Expression of Protein G Subunits Spodtera frugiperda cells were co-infected with the baculovirus expressing a G-labeled gamma protein and the baculovirus expressing a β2 G protein at a 1: 1 ratio of the baculovirus ßi of glu-labeled G protein for the baculovirus ß2 of protein G. Sf9 cells were cultured in 10 L bioreactors and harvested 48-72 hours post-infection. Samples of the infected cells were tested for ß ^ ß2 expression of the G protein by Western Blot analysis as described below. The cell lysates were homogenized and loaded onto a column of glu-labeled beads as in Biological Example 1 and compete outside the column with a glu peptide and processed as described in Biological Example 1.

BIOLOGICAL EXAMPLE 3 Western Hybridization Assay Protein samples were run on an 8% Tris-Glycine gel and transferred to a 45 μM nitrocellulose membrane. Hybridizations were subsequently blocked with 5% albumin in bovine serum (BSA) and 5% ovalbumin in TBST (50 mM Tris, 200 mM NaCl, 0.1% Tween 20, pH 7.4) for 1 hour at room temperature and were incubated overnight at 4 ° C with primary antibody diluted 1: 1000 in TBST with 0.5% BSA. The primary antibodies for p110 ?, p110a, p110β, p85a, ßi protein G and subunits? 2 of the G protein were purchased from Santa Cruz Biotechnology Inc. Santa Cruz CA. Antibodies of the p101 subunit were developed in Research Genetics Inc. Huntsville, AL based on a p101 peptide antigen.

After incubation with the primary antibody, the hybridizations were washed with TBST and incubated for 2 hours at room temperature with goat-anticlock conjugate HRP (Bio-Rad Laboratories, Inc., Hercules, CA, product number 170-6515 ), diluted 1: 10,000 in TBST with 0.5% BSA. The antibodies were detected with ECL ™ detection reagents (Amersham Biosciences Corp. Piscataway, New Jersey) and quantified on a Kodak ISO400F analyzer.

BIOLOGICAL EXAMPLE 4 Immunoprecipitation 100 μL of the cell passage of Biological Example 1 or 2 was thawed and used on ice with 400 μl of hypotonic lysis binder (25 mM tris, 1 mM DTT, 1 mM EDTA, 1 mM Pefabloc, 5 μM leupeptin, 5 μM E-64 (Roche), 1% Nonidet P40, pH 7.5-8). The lysate was incubated for 2 hours at room temperature with glu-labeled beds (Covance Research Products, Cambridge, England, product number AFC-115P). The beads were washed 3 times in water binder (20 mM Tris, pH 7.8-8, 150 mM NaCl2, 0.5% NP40) and the protein eluted off the beds by heating 2 times the sample binder (Invitrogen, Corporation, Carlsbad, CA product number LC1676).

BIOLOGICAL EXAMPLE 5 Test Kinease of Pl3K? In Vitro The inhibitory properties of the compounds of Table 1 were tested in an in vitro PI3K test. In a 96-well polypropylene plate, each well was labeled with 2 μl of 50 times the desired final concentration of the compound in DMSO. The protein p101 / p110? purified recombinant (0.03 μg - 2.7 nM) and subunits ^ lz of protein G (0.09 μg -57.7 nM) for each reaction were combined in the test binder (30 mM HEPES, 100 mM NaCl, 1 mM EGTA and 1 mM DTT). ATP and [? -32P-ATP] (0.09 μCi) were added to this mixture so that the final concentration of ATP in the reaction was 20 μM. The lipid micelles were formed by sonicate phosphatidylinositol-4,5-diphosphate (PIP2), phosphatidylethanolamine (PE) and Na-cholate in the test binder for 10 minutes, adding MgCl2 and incubating on ice for 20 minutes, for the final concentrations of 25 μM of PIP2, 300 μM of PE, 0.02% of Na-cholate and 10 mM of MgCl2 in the reaction. The reactions were initiated by adding volumes of lipids and enzyme mixture in a total volume of 50 μL, allowing to run for 20 minutes at room temperature and stopped with 100 μL of 75 mM H3P0. The lipid product was transferred to a glass fiber filter plate and washed with 75 mM H3P04 several times. The presence of the radioactive lipid product (PIP3) was measured by adding Wallac Optiphase mixture to each well and counting on a Wallac 1450 Trilux plate reader (PerkinElmer Life Sciences Inc. Boston MA 02118). The IC50 for each compound tested was reported in μM in Table 1: Table 1 Example No. IC50 (μM)? 0.585 2 1.845 3 0.550 4 0.210 5 0.725 6 6.6 7 4.145 It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested by those skilled in the art and are included within the spirit and purity of this application and the scope of the appended claims. All publications, patents and patent applications cited in this document are incorporated in full as a reference for any purpose.

Claims (13)

1. A compound of the Formula or a pharmaceutically acceptable salt thereof: wherein R2 is H or a C3 alkyl and wherein R1 is an unsubstituted phenyl or a phenyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of: C- alkyl? -C, methyl, alkyl-0 C C4, methoxy, halo, Cl, Br and I.

2. The compound according to claim 1, wherein R 1 is an unsubstituted phenyl.

3. The compound according to claim 1, wherein R 1 is a phenyl substituted with 1, 2 or 3 substituents independently selected from the group consisting of: Cr C 4 alkyl, methyl, 0 CrC 4 alkyl, methoxy, halo, Cl, Br e l.

4. The compound according to claim 1, wherein said compound is selected from the group consisting of: 5,6-Dimethoxy-3-o-tolylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) )-amide; 3- (3,4-Dichloro-phenylsulfanyl) -5,6-dimethoxy-1H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 5,6-Dimethoxy-1-methyl-3-phenylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 5,6-Dimethoxy-3-phenylsulfanyl-1 H-indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 5,6-Dimethoxy-3- (3-methoxy-phenylsulfanyl) -1 H -indole-2-carboxylic acid (2H-tetrazol-5-yl) -amide; 1-Ethyl-5,6-dimethoxy-3-phenyl-1 H-indole-2-carboxylic acid (1 H-tetrazol-5-yl) -amide and 5,6-dimethoxy-3-phenyl-1-propyl acid -1H-indole-2-carboxylic acid (1 H-tetrazol-5-yl) -amide.

5. A method for treating a subject suffering from a PI3K-mediated condition or disease comprising: administering to said subject suffering from the PI3K-mediated condition or disorder, a pharmaceutical composition comprising a pharmaceutically effective amount comprising a compound in accordance with claim 1 and a pharmaceutically acceptable carrier.

6. The method according to claim 5, wherein said PI3K-mediated condition or disease is selected from the group consisting of: rheumatoid arthritis, psoriasis, psoriatic arthritis, osteoarthritis, inflammatory diseases and autoimmune diseases.

7. The method according to claim 5, wherein said condition or PI3K mediated disorder is selected from the group consisting of: cardiovascular diseases, arteriosclerosis, hypertension, deep vein thrombosis, stroke, myocardial infarction, unstable angina, thromboembolism, pulmonary embolism, thrombotic diseases, acute arterial ischemia, peripheral thrombotic occlusions and coronary artery disease.

8. The method according to claim 5, wherein said disease or PI3K medal condition is selected from the group consisting of: cancer, breast cancer, glioblastoma, endometrial carcinoma, heptocellular carcinoma, colon cancer, lung cancer, melanoma, carcinoma renal cell carcinoma, thyroid carcinoma, small cell lung cancer, squamous cell lung carcinoma, glioma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, leukemia, cell lymphoma and lymphoproliferative diseases.

9. The method according to claim 5, wherein said PI3K-mediated condition or disorder is selected from the group consisting of: type II diabetes.

10. The method according to claim 5, wherein said PI3K-mediated condition or disorder is selected from the group consisting of: respiratory diseases, bronchitis, asthma and chronic obstructive pulmonary disease.

11. The method according to claim 5, wherein said compound is a compound of any of claims 1-4.

12. A pharmaceutical composition comprising: a therapeutically effective amount of a compound according to claim 1 and a pharmaceutically acceptable carrier.

13. A pharmaceutical composition comprising: a therapeutically effective amount of a compound according to any of claims 1-4 and a pharmaceutically acceptable carrier.