MXPA05013475A - Compounds having inhibitive activity of phosphatidylinositol 3-kinase and methods of use thereof - Google Patents

Abstract

Compounds inhibiting phosphatidylinositol 3-kinase (PI 3-K) activities and methods of preparing and using thereof in treating diseases are disclosed. Compounds inhibiting PI 3-K activity and methods of using PI 3-K inhibitory compounds to inhibit cancer cell grwoth or to treat disorders of immunity and inflammation, in which PI 3-K plays a role in leukocyte function are also provided.

Description

COMPOUNDS THAT HAVE INHIBITOR ACTIVITY OF PHOSPHATRODYLINOSITOL 3-KINASE AND METHODS OF USE OF THEM BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates, in general, to enzymes phosphatidylinositol 3-kinase (Pl 3-K), and more particularly to inhibitors of the activity of Pl 3-K and methods of use of those materials.

RELATED TECHNIQUE The behavior of all cellular communications is governed by signaling systems which translate external signals such as hormones, neurotransmitters and growth factors into intracellular secondary messengers. Phosphoinositide polyphosphates (PIPn) are key lipid secondary messengers in cell signaling (Martin, Ann. Rev. Cell Dev. Biol., 14: 231-2614 (1998)). Because their activity is determined by their phosphorylation status, the enzymes that modify these lipids are central to the correct execution of signaling events (Leslie, et al., Chem Rev, 101: 2365-80. (2001)) . Disturbances in these processes are common to many disease states, including cancer, diabetes, inflammation and cardiovascular disease. The production of phosphoinositide polyphosphate PI (3,4,5) P3 or PTP by phosphatidylinositol 3-kinase (Pl 3-K) is important in the pathways that govern proliferation, differentiation, apoptosis and cell migration. Alterations that affect the correct regulation of PIP3 levels and the levels of their lipid products are associated with a variety of cancer types (Phillips et al., Cancer 83: 41-47. (1998), Shayesteh, et al. , Nat Genet, 21: 99-102. (1999), Ma, et al., Oncogene, 19: 2739-44. (2000)). Mutations affecting the regulation of Pl 3-K signaling contribute to abnormal proliferation and tumorigenesis (Li, et al., Science, 275: 1943-7. (1997), Teng, et al., Cancer Res, 57: 5221-5 (1997)) (Shayesteh, et al., Nat Genet, 21: 99-102. (1999), Ma, et al., Oncogene, 19: 2739-44. (2000)). When activated by tyrosine kinase receptors in response to stimulation with growth factor, Pl 3-K catalyzes the formation of PIP3. By increasing the cellular levels of PIP3, PI3-K induces the formation of defined molecular complexes that act on signal transduction pathways. Most notably, the activity of Pl 3-K suppresses apoptosis and promotes cell survival through activation of its downstream target, PKB / Akt (Franke, et al., Cell, 81: 727-36. (1995), Datta, et al., J Biol Chem, 271: 30835-9. (nineteen ninety six)). Lipid phosphatases PTEN and SHIP are two enzymes that both act to decrease cellular levels of PIP3 by conversion to PI (4,5) P2 or PI (3,4) P2. Currently, the family of Pl 3-kinases enzymes has been divided into three classes based on their substrate specificities. Pl 3-K of class I can phosphorylate phosphatidylinositol (Pl), phosphatidyl inositol-4-phosphate, and phosphatidylinositol-4,5-bisphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3, 4 -biphosphate, and phosphatidylinositol-3,4,4,5-triphosphate, respectively. Pl 3-K of Class II phosphorylate Pl and phosphatidylinositol-4-phosphate, while Pl 3-K of Class III can only phosphorylate Pl. Eight separate isoforms of Pl 3-K have been characterized in humans. The initial molecular purification and cloning of Pl 3-kinase revealed that this was a heterodimer consisting of p85 and pOl subunits (Otsu et al., Cell, 65: 91-104 (1991); Hiles et al., Cell, 70: 419-29 (1992)). Since then, four distinct classes of Pl 3-K of Class I have been identified, designated Pl 3-K alpha, beta, delta, and gamma, each consisting of a different catalytic subunit of 110 kDa, more specifically , three of the catalytic subunits, that is, alpha pllO, pllO beta and pllO delta, each interact with the same regulatory subunit, p85; while the gamma pllO interacts with a different regulatory subunit, the plOl. In each of the subtypes of Pl 3-kinase alpha, beta, and delta, the p85 subunit acts to locate Pl 3-kinase in the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in a context of appropriate sequence) in white proteins. Two isoforms of p85 have been identified, p85 alpha, which is ubiquitously expressed, and p85 beta, which is found mainly in brain and lymphoid tissues. The association of the p85 subunit with the catalytic subunits pllO alpha, beta, or delta of Pl 3-kinase seems to be a requirement for the catalytic activity and stability of these enzymes. In addition, the binding of Ras proteins also upregulates the activity of Pl 3-kinase. Although all the information that has accumulated in the recent past on the cellular functions of Pl 3-kinase in general, in particular for Pl 3-K alpha and Pl 3-K gamma, the roles played by individual isoforms do not They have been clearly defined. Details related to pllO isoform may also be found in U.S. Patent Nos. 5,858,753; 5,822,910; and 5,985,589.

Specific inhibitors against individual members of a family of enzymes provide invaluable tools for deciphering the functions of each enzyme. The experimental use of PI 3-K inhibitors has contributed to the current understanding of the role of PI 3-K activity in normal function and disease. The main pharmacological tools used with this capability are ortmanin (Powis, et al., Cancer Res, 54: 2419-23. (199), and bioflavenoid compounds, including quercetin (Matter et al., Biochem. Biophys. Res. Commun. 186: 624-631. (1992)) and LY294002 (Vlahos, et al., J Biol Chem, 269: 5241-8. (1994).) The concentrations of wortmanin required to inhibit Pl 3-K fluctuate from 1-100 nM, and inhibition occurs via the covalent modification of the catalytic site (Wymann et al., Mol.Cell. Biol. 16: 1722-1733. (1996).) The bioflavenoid quercetin effectively inhibits Pl 3-K with an IC50 of 3.8 μM, but has poor selectivity, since it also shows inhibitory activity towards Pl 4-kinase, and several protein kinases.LY294002 is a synthetic compound produced using quercetin as a model, inhibits Pl 3-K with an IC 50 of 100 M (Vlahos, et al., J Biol Chem, 269: 5241-8. (1994)) Both quercetin and LY294002 are competitive inhibitors. However, only LY294002 shows specificity for the inhibition of Pl 3-K and does not affect other types of kinases. Both wortmanin and LY294002 have been used extensively to characterize the biological roles of Pl 3-K, however, none shows selectivity. by individual isoforms of Pl 3-K. Consequently, the utility of these compounds in the study of the roles of the individual Class I Pl 3-kinases is limited. It is expected that Pl 3-K inhibitors are a new type of drugs useful for cell proliferation disorders, in particular as antitumor agents. As inhibitors of Pl 3-K, the wortmannin [H. Yano et al., J. Biol. Chem., 263, 16178 (1993)] and LY294002 [J. Vlahos et al., J. Biol. Chem., 269, 5241 (1994)] which is represented by the following formula, are known. However, the creation of Pl 3-K inhibitors having a more potent cancer cell growth inhibiting activity is desirable. Because many oncogenic signaling pathways are mediated by Pl 3-K, inhibitors of the white activity of Pl 3-K may have application for the treatment of cancer. Studies using comparative genomic hybridization revealed several regions of a recurrent abnormal DNA sequence number copy that can code for genes involved in the genesis or progress of ovarian cancer. A region that was found to be increased in the number of copies in approximately 40% of an ovary and other cancers contains the PIK3CA gene, which codes for the pllO catalytic subunit of Pl 3-K alpha. This association between PIK3CA copy number and PI 3-kinase activity makes PIK3CA a candidate oncogene because a wide range of cancer-related functions have been associated with Pl 3-kinase-mediated signaling. PIK3CA is frequently found to increase in the number of copies in ovarian cancers, and an increased number of copies is associated with increased transcription of PIK3CA, expression of the pllO-alpha protein, and activity of PI 3- kinase (Shayesteh, et al., Nature Genet, 21: 99-102, (1999)). In addition, the treatment of ovarian cancer cell lines that exhibit an increase in Pl 3rK activity and activation of Akt with a Pl 3-kinase inhibitor decreased proliferation and increased apoptosis (Shayesteh, et al., Nature Genet 21: 99-102, (1999), Yuan et al., Oncogene 19: 2324-2330. (2000)). Thus, Pl 3-K alpha has an important role in ovarian cancer. In cervical cancer cell lines harboring amplified PIK3CA, expression of the gene product increased and was associated with a high activity of Pl 3-kinase (Ma et al., Oncogene 19: 2739-2744, (2000)). Thus, increased expression of Pl 3-kinase alpha in cervical cancer can promote cell proliferation and reduce apoptosis. In addition, the lipid phosphatase mutation and PTEN tumor suppressor, a 3 'phosphatase that degrades PIP3, is one of the most common cancer-associated mutations, and is particularly associated with glioblastoma, prostate, endometrial and breast cancers (Li et al., Science 275: 1943-1947 (1997), Teng et al., Cancer Res. 57: 5221-5225. (1997), Ali et al., J. National Cancer Institute, 91: 1922-1932. (1999), Simpson and Parsons, Exp. Cell Res. 264: 29-41 (2002)). The activity of PI3-K suppresses apoptosis and promotes cell survival to a large extent through activation of its downstream target, PKB / Akt (Franke et al., Cell 81: 727-736. (1995), Dattaet al. .,. J Biol Chem 271: 30835-30839 (1996)). Activation and amplification of Akt is present in many cancers (Testa and Bellicosa, Proc. Nati, Acad. Sci. USA 98: 10983-10985. (2002)). Treatment with PI 3-K inhibitors has been shown to block the proliferation of several cancer cell lines, and to be an effective treatment for tumor xenograft models in addition to ovarian carcinoma. Akt is activated in a majority of non-small cell lung cancer cell lines, and treatment with Pl 3-K inhibitors produces a proliferative disruption in those cells (Brognard et al., Cancer Res. 60: 6353-6358 (2000), Lee et al., J. Biol. Chem. Electronic publication, (2003)). The Pl 3-K / Akt pathway is also constitutively activated in a majority of human pancreatic cancer cell lines, and treatment with Pl 3-K inhibitors induced apoptosis in those cell lines. Decrease in tumor growth and metastasis was also observed after treatment with Pl 3-K inhibitors in a pancreatic cancer xenograft model (Perugini et al., J. Surg. Res. 90: 39-44 (2000), Bondar et al., Mol. Cancer Ther. 1: 989-997 (2002)).

Treatment with LY204002 induced an arrest of growth and apoptosis in human malignant glioma cells defnt in PTEN (Shingu et al., J. Neurosurg, 98: 154-161 (2003)). LY294002 produces a growth arrest in human colon cancer cell lines and suppression of tumor growth in colon carcinoma xenografts in mice (Semba et al., Clin Cancer Res. 8: 1957-1963. (2002)). PI3-K inhibitors inhibit the independent growth of the anchor in vi tro and the in vivo metastasis of liver cancer cells (Nakanishi et al., Cancer Res. 62: 2971-2975. (2002)). Treatment of Burkitt's lymphoma cells with LY294002 induces apoptosis (Brennan et al., Oncogene 21: 1263-1271 (2002)). LY294002 has also been shown to induce apoptosis in cells resistant to multiple drugs (Nicholson et al., Cancer Lett., 190: 31-36 (2003)). Thus, Pl 3-K inhibitors can be suitable therapeutic agents for many tumors exhibiting activated or increased levels of Pl 3-K or PKB / Akt as well as for tumors that are defnt in PTEN. Several studies have shown that agents targeting the Pl 3-K pathway can improve the standard chemotherapeutic effects in a variety of cancer types. Thus, Pl 3-K inhibitors may have value as novel adjuvant therapies for certain cancers. Pl 3-K inhibitors induce apoptosis in pancreatic carcinoma cells exhibiting constitutive phosphorylation and activation of AKT, and suboptimal doses produce additive inhibition of tumor growth when combined with a suboptimal dose of gemcitabine (Ng, et al., Cancer Res. , 60: 5451-5 (2000, Bondar, et al., Mol Cancer, Ther, 1: 989-97. (2002)). Inhibition of Pl 3-K also increases the response of pancreatic carcinoma cells to the agent Non-steroidal inflammatory (NSAID) sulindac (Yip-Schneider, et al., J Gastrointest Surg, 7: 354-63. (2003)). In a pancreatic cancer mouse xenograft model, a combination of wortmanin with gemcitabine also showed an increase in the efficacy in the induction of tumor apoptosis in relation to the treatment with each agent alone (Ng, et al., Clin Cancer Res, 7: 3269-75. (2001)). In a nude mouse xenograft model of ovarian cancer, the combined treatment with LY294002 and pa clitaxal resulted in an increase in the efficacy of paclitaxal-induced apoptosis of tumor cells, and allows the use of lower levels of LY294002, resulting in lower dermatological toxicity (Hu, et al., Cancer Res, 62: 1087-92. (2002)). The HL60 human leukemia cells show sensitization to cytotoxic drug treatment and Fas-induced apoptosis when treated with PI 3-K inhibitors, suggesting a role for the inhibition of Pl 3-K in the treatment of myeloid leukemia. drug-resistant acute (O 'Gorman, et al., Leukemia, 14: 602-11. (2000, O' Gorman, et al., Leuk Res, 25: 801-11. (2001)). Pl 3-K increases the apoptotic effects of sodium butyrate, gemcitabine, and 5-fluorouracil in aggressive colon cancer cell lines (Wang, 'et al., Clin Cancer Res, 8: 1940-7. (2002)) LY294002 potentiates the apoptosis induced by doxorubicin, trastumazab, paclitaxal, tamoxifen, and etoposide in breast cancer cell lines that exhibit PTEN mutations or erbB2 overexpression (Clark, et al., Mol Cancer Ther, 1: 707- 17. (2002).) Inhibition of Pl 3-K potentiates the effect of etoposide to induce apoptosis in cancer cells. small-cell lung disease (Krystal, et al., Mol Cancer, Ther, 1: 913-22. (2002)). In addition to improving the effects of chemotherapeutic agents for cancer treatment, PI 3-K inhibitors can also improve the tumor response to radiation treatment. Pl 3-K inhibitors reverse radioresistance in breast cancer cells transfected with constitutively active H-ras (Liang, et al., Mol Cancer, Ther, 2: 353-60. (2003)), and PI 3-K inhibitors improve radiation-induced apoptosis and cytotoxicity in vascular endothelial tumor cells (Edwards, et al. , Cancer Res, 62: 4671-7. (2002)). In this way, PI 3-K inhibitors could be used to improve the response to radiotherapy, both in tumor cells and in the tumor vasculature. U.S. Patent No. 6,403,588 describes imidazopyridine derivatives having excellent inhibitory activity of Pl 3-K and inhibitory activity of cancer cell growth. U.S. Patent No. 5,518,277 describes compounds that inhibit the activity of Pl 3-K delta, including compounds that selectively inhibit the activity of Pl 3-K delta. However, all these compounds have a structure different from those of the present invention.

THE INVENTION It has been recognized that it would be advantageous to develop inhibitors of Pl 3 -K polypeptides. In particular, Pl 3-K inhibitors are desirable to explore the roles of Pl 3 -K isozymes and to develop drugs to modulate the activity of the isozymes. One embodiment of the present invention is to provide a compound that is useful as an inhibitor of phosphatidylinositol 3-kinase (PI3-K) having a general structure represented by Formula I, Formula II, or Formula III; Formula Formula II Formula wherein n may be an integer selected from 0 to 2. In one aspect, R and R2 may each independently be a portion or entity selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, hetaryl, aralkyl, hetaralkyl, substituted alkyl with at least one substituent, aryl substituted with at least one substituent, hetaryl substituted with at least one substituent, aralkyl substituted with at least one substituent, and substituted hetaralkyl with at least one substituent. In another aspect, R3 may be a portion selected from the group consisting of hydrogen, alkyl, alkenyl, aralkyl, alkyl substituted with at least one substituent, aralkyl substituted with at least one substituent, CO-R5, S02-R5; C0-0-R5, CO-N-R4, and R5. In a further aspect, R and R5 can each independently be a portion selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, aralkyl, aryl, alkyl substituted with at least one substituent, cycloalkyl substituted with a substituent, aryl substituted with at least one substituent, and aralkyl substituted with at least one substituent. One embodiment of the present invention is a compound which is useful as an inhibitor of phosphatidylinositol 3-kinase (Pl 3-K) having a general structure represented by Formula I, II, or III wherein alkyl, cycloalkyl or aralkyl is a C1-15 alkyl group, C3_8 cycloalkyl, C2-I8 alkenyl, or aralkyl substituted by 1 to 5 substituents selected from the group consisting of the nitro, hydroxy, cyano, carbamoyl, mono- or di-alkyl C groups. Carbamoyl, carboxy, C? -4-carbonyl alkoxy, sulfo, halogen, C? _4 alkoxy, phenoxy, halofenoxy, C? _ alkylthio, mercapto, phenylthio, pyridylthio, C-4-4 alkylsulfinyl, alkylsulfonyl, C? _4, amino, C 1-3 alkanoylamino, mono- or dialkylamino of C? _4, cyclic amino of 4 to 6 members, C 1-3 alkanoyl, benzoyl and heterocyclic groups of 5 to 10 members. Another embodiment of the present invention is a compound which is useful as an inhibitor of phosphatidylinositol 3-kinase (Pl 3K) having a general structure represented by Formula I, II or III, wherein the alkyl is a straight-chain hydrocarbon or branched having from 1 to 15 carbon atoms, the aryl is an aromatic cyclic hydrocarbon group having from 6 to 14 carbon atoms, the hetaryl is a 5- or 6-membered monocyclic heterocyclic group containing from 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen or a bicyclic fused heterocyclic group containing from 1 to β selected heteroatoms of oxygen, sulfur and nitrogen, the substituted aryl is an aryl group of Ce-? which is substituted by 1 to 4 substituents selected from the group consisting of halogen groups, C__4 alkyl, C ?4 haloalkyl, C? ___ haloalkoxy, C ?4 alkoxy, C-4-4 alqu alkylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-alkylamino of C__4, formyl, mercapto, C ?4-carbonyl alkyl, C-carbonyl alkoxy, sulfo, C? _4 alkylsulfonyl, carbamoyl, mono- or di-alkyl C? -4-carbamoyl, oxo and thioxo; and the substituted hetaryl is a hetaryl which is substituted by 1 to 4 substituents selected from the group consisting of halogen, C ___ alkyl- C 1-4 haloalkyl, C 1-4 haloalkoxy, C 1-4 alkoxy, C 1 alkylthio 4, hydroxy, carboxy, cyano, nitro, amino, mono- or di-alkylamino of C _ _. 4, formyl, mercapto, C? -4-carbonyl alkyl, C 1-4 alkoxy-carbonyl, sulfo, alkylsulfonyl of C1-4, carbamoyl, mono- or di-C 1 -4 -carbamoyl alkyl, oxo and thioxo. Another embodiment of the present invention is a compound which is useful as an inhibitor of phosphatidyl inositol 3-kinase (Pl 3-K) having a general structure represented by Formula I, II or III, where Ri and R2 are each independently a member selected from the group consisting of C___ alkyl, phenyl, naphthyl, hetaryl substituted with C1-6 alkyl and phenyl substituted with C6_6 alkyl; R3 is a member selected from the group consisting of H, C? _6 alkyl, aralkyl substituted by C1-6 alkyl, aralkyl groups, CO-R5, or S02-Rs; CO-O-R5, CO-N-R4, and R5; and R4 and R5 may be a member selected from the group consisting of H, C1-6 alkyl, substituted Ci-g alkyl, cycloalkyl and aralkyl groups. Another embodiment of the present invention is a compound which is useful as an inhibitor of phosphatidyl inositol 3-kinase (Pl 3-K) having a general structure represented by Formula I, II or III where n is 1; Ri is a member selected from the group consisting of straight chain Ci-g alkyl groups, branched chain C?-6 alkyl and phenyl; R 2 is a member selected from the group consisting of phenyl, C 1 -C 6 alkylphenyl, Ci-s dialkylphenyl, C 6 -alkoxyphenyl, halophenyl, dihalophenyl and nitrophenyl groups; R3 is a member selected from hydrogen groups, straight chain C6-6 alkyl and branched chain C6-6 alkyl; R4 is a phenyl substituted with at least one substituent selected from the group consisting of aryloxy, alkylaryloxy, halo aryloxy, straight chain C6-6 alkyl, branched chain C6-6 alkyl, C6-6 alkoxy, haloaryl of C? _6, and C? -4 haloalkylaryl; and R5 is a straight or branched chain C6-6 alkyl group. The preferred embodiment of the present invention is a compound which is useful as an inhibitor of phosphatidylinositol 3-kinase (Pl 3-K) having a general structure represented by Formula I, II or III, where Ri is a phenyl group or terbutyl; R2 is a member selected from the group consisting of the methylphenyl, dimethylphenyl, tertbutyl, methoxyphenyl, chloro phenyl, dichlorophenyl, fluorophenyl, and nitrophenyl groups; R3 is hydrogen; R is a phenyl substituted with at least one substituent selected from the group consisting of phenoxy, benzyloxy, halofenoxy, straight chain C6_6 alkyl, branched chain C1_6 alkyl, C___ alkoxy, C6_6 haloophenyl groups , and halo-alkylphenyl of C? _4; and R5 is a straight or branched chain C1-6 alkyl group. The most preferred embodiment of the present invention is a compound which is useful as an inhibitor of phosphatidylinositol 3-kinase (Pl 3-K) having a general structure represented by Formula I, II or III, where Ri is phenyl or terbutyl; R2 is a member selected from the group consisting of methylphenyl, dimethyl phenyl, tertbutyl, methoxyphenyl, chlorophenyl, dichlorophenyl fluorophenyl and nitrophenyl; R3 is hydrogen; R 4 is a phenyl substituted with at least one substituent selected from the group consisting of phenoxy, benzyloxy, halophenoxy, straight-chain C-6 alkyl, branched-chain C 1-6 alkyl, C-6 alkoxy, Ci halophenyl groups -e, and halo-alkylphenyl of C? _4; and R5 is a methyl group. The present invention also relates to novel pharmaceutical compositions, particularly Pl 3-K inhibitors and antitumor agents, which comprises a compound of the present invention and a pharmaceutically acceptable excipient or carrier. A further aspect of the present invention relates to methods of treating disorders (especially cancers) influenced by Pl 3-K, wherein an effective amount of a compound of the present invention is administered to humans or animals. The additional features and advantages of the invention will be apparent from the following detailed description, taken in conjunction with the accompanying drawings, which together illustrate, by way of example, the features of the invention.

DETAILED DESCRIPTION Reference will now be made to the exemplary embodiments illustrated in the drawings, and specific language will be used herein to describe them. However, it should be understood that it is not intended to limit the scope of the present invention therefore. Further alterations and modifications of the features of the invention illustrated herein, and additional applications of the principles of the invention as illustrated herein, which will occur to one of skill in the relevant art and having this description in mind, are considered within of the scope of the invention. One embodiment of the present invention relates to novel compounds which are useful as inhibitors of Pl 3-K and antitumor agents. The compounds of the present invention are represented by one of the following general formulas: Formula I Formula II Formula III wherein n can be an integer selected from 0 to 2. In one aspect, Ri and R2 can each independently be a member selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, hetaryl, aralkyl, hetaralkyl, alkyl substituted with less a substituent, aryl substituted with at least one substituent, hetaryl substituted with at least one substituent, aralkyl substituted with at least one substituent, and hetaralkyl substituted with at least one substituent. In another aspect, R3 may be a member selected from the group consisting of hydrogen, alkyl, alkenyl, aralkyl, alkyl substituted with at least one substituent, aralkyl substituted with at least one substituent, CO-R5, S02-R5; CO-O-R5, CO-N-R4, and R5. In a further aspect, R4 and R5 can each independently be a member selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, aralkyl, aryl, alkyl substituted with at least one substituent, cycloalkyl substituted with one substituent, aryl substituted with at least one substituent, and aralkyl substituted with at least one substituent. According to the invention, the compound according to Formula I, Formula II and / or Formula III can be substituted with several portions or entities, where any of those are used. Accordingly, the alkyl may be C1-15 alkyl of chain. linear or branched. In one aspect, the cycloalkyl may be C3_8 cycloalkyl. In another aspect, the alkenyl can be a straight or branched chain C2 is alkenyl. In yet another aspect, the aralkyl may be a carbomiocyclic or aromatic carbobicyclic aromatic substituted with a C1-15 alkyl of straight or branched chain. In yet another aspect, any of the substituents may be selected from the group consisting of nitro, hydroxy, cyano, carbamoyl, mono- or di-alkyl of C? _4-carbamoyl, carboxy, C-4-alkoxy, sulfo, halogen C4-4 alkoxy, phenoxy, halofenoxy, C1-4 alkylthio, mercapto, phenylthio, pyridylthio, C1-4 alkylsulfinyl, C1-4 alkylsulfonyl, amino, C1-3 alkanoylamino, mono- or di- C? _ alkylamino, 4- to 6-membered cyclic amino, C? _3 alkanoyl, benzoyl and 5- to 10-membered heterocyclic groups.

In another embodiment, R1-5 of Formula I, Formula II and / or Formula III may each be selected individually from a variety of portions or entities where any of those are used, where entities may be optionally substituted with the minus one substituent. Accordingly, the aryl can be an aromatic carbombocyclic or aromatic carbobicyclic. In one aspect, the hetaryl can be a heteromonocyclic or aromatic heterobicyclic aromatic compound containing from 1 to 4 heteroatoms or from 1 to 6 heteroatoms selected from oxygen, sulfur and nitrogen. In another aspect, the aralkyl may be a carbomiocyclic or aromatic carbobicyclic aromatic compound substituted with a straight or branched chain C 1 -5 alkyl group. In a further aspect, the substituent may be selected from the group consisting of halogen, C ?4 alkyl, Ci-4 haloalkyl, C ?4 haloalkoxy, C__4 alkoxy, C? -4 alkylthio, hydroxy, carboxy, cyano, nitro, amino, mono or dialkylamino of C ?4, formyl, mercapto, C 1-4 alkylcarbonyl, C? -4 -carbonyl alkoxy, sulfo, C? _4 alkylsulfonyl, carbamoyl, mono- or di-C? _4 alkyl, -carbamoyl, oxo and thioxo. In one aspect, R and R2 may each be a member independently selected from the group consisting of hydrogen, straight or branched chain C6-6 alkyl, phenyl, naphthyl, hetaryl, C6_6alkyl substituted with at least one alkylphenyl of straight or branched chain C__6, phenyl substituted with at least one substituent, and benzyl. In one aspect, R3 may be a member selected from the group consisting of hydrogen, C? _6 alkyl, aralkyl, C? _6 alkyl substituted with at least one substituent, CO-R5 or SO2-R5; CO-O-R5, CO-N-R4 and R5. In another aspect, R4 and R5 may each independently be a member selected from the group consisting of hydrogen, C__6 alkyl, C? ~6 alkyl substituted with at least one substituent, cycloalkyl, phenyl, phenyl substituted with at least one substituent, benzyl, and aralkyl groups. In a further embodiment, the conjugated portions thereof may not be substituted or substituted with at least one substituent. In one aspect, the alkyl may be C1-15 straight or branched chain. In another aspect, the alkenyl can be a straight or branched chain C2-i8 alkenyl. In a further aspect, the aryl may be a carbomonocyclic or aromatic carbobicyclic aromatic group. In yet another aspect, the cycloalkyl may be a C3-8 alkyl ring. In yet another aspect, the hetaryl may be a heteromonocyclic or aromatic heterobicyclic aromatic compound containing from 1 to 6 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen. In yet another aspect, the aralkyl may be a carbomonocyclic or aromatic carbobicyclic aromatic group and may be substituted with straight or branched chain C? -15 alkyl. In yet another aspect, the hetaralkyl may be a heteromonocyclic or heterobicyclic aromatic compound containing from 1 to 4 heteroatoms or from 1 to 6 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen substituted with straight or branched chain C? -i5. In addition, any of the substituents can independently be a member selected from the group consisting of halogen, C__4 alkyl, C ?4 haloalkyl, C ?4 haloalkoxy, C alco- alkoxy, C? _4 alkylthio, phenoxy, halophenoxy phenylthio, pyridylthio, hydroxy, carboxy, cyano, nitro, amino, C__3 alkanoylamino, mono- or di-alkylamino of C? -_, cyclic amino of 4 to 6 members, formyl, mercapto, C? -4- alkyl carbonyl, C 4 -carbonyl alkoxy, sulfo, C___ alkylsulfinyl, C? -4 -4 alkylsulfonyl, C?-3 alkanoyl, benzoyl, C mono -4-carbamoyl mono- or di-alkyl, oxo, thioxo, a Heterocyclic compound of 5 to 10 members, and combinations thereof. In a more specific embodiment, the portions may be substituted or not, with at least one substituent. Accordingly, Ri and R2 can each independently be a member selected from the group consisting of straight or branched chain C6-6 alkyl, phenyl, naphthyl, straight or branched chain C-C6 alkyl substituted with at least one substituent and phenyl substituted with at least one substituent. In another aspect, R 3 may be a member selected from hydrogen, C 1 g straight or branched chain alkyl, C 6 aralkyl and C 1 6 alkyl substituted with at least one substituent. In another aspect, R 4 and R 5 can each independently be a member selected from the group consisting of hydrogen, straight or branched chain C 1-6 alkyl, straight or branched chain C 1-6 alkyl substituted with at least one substituent, cycloalkyl, phenyl, phenyl substituted with at least one substituent, aralkyl of C6-6, and aralkyl of C1-6 substituted with at least one substituent. In still another aspect, any of the substituents may be a member selected from the group consisting of methyl, halogen, halophenyloxy, methoxy, ethyloxy phenoxy, benzyloxy, trifluoromethyl, t-butyl and nitro. In one aspect, Ri can be selected from the group consisting of a straight or branched chain C? _ Alquilo alkyl and phenyl. In another aspect, R2 can be selected from the group consisting of phenyl, C__6 alkylphenyl, C__6 dialkylphenyl, C__6 alkoxyphenyl, halophenyl, dihalophenyl and nitrophenyl. In a further aspect, R3 may be selected from hydrogen and straight or branched chain C__6 alkyl. In yet another aspect, R 4 may be a phenyl substituted with at least one substituent selected from the group consisting of phenoxy, benzyloxy, halofenoxy, straight or branched chain C 1-6 alkyl, C 1-6 alkoxy, halophenyl, and halo-C 1 alkyl. In a further aspect, R5 may be straight or branched chain C6-6 alkyl. In another aspect, Ri can be phenyl or t-butyl; R2 may be a member selected from the group consisting of methylphenyl, dimethylphenyl, t-butyl, methoxyphenyl, chlorophenyl, dichlorophenyl, fluorophenyl, and nitrophenyl; R3 can be hydrogen; R may be a phenyl substituted with at least one substituent selected from the group consisting of chloro, fluoro, phenoxy, benzyloxy, chlorophenoxy, methoxy, ethoxy, and trifluoromethyl; and R5 can be a methyl. The terms "substituted alkyl, cycloalkyl, alkenyl, or aralkyl" mean: C 1 -C 8 alkylcycloalkyl, C 2__ 8 alkenyl or aralkyl groups which may be substituted by 1 to 5 substituents selected from the group consisting of (i) nitro , (ii) hydroxy, (iii) cyano, (iv) carbamoyl, (v) mono- or dialkyl of C_4-carbamoyl, (vi) carboxy, (vii) C4_4-carbonyl alkoxy, (viii) sulfo, (ix) halogen, (x) C 4 4 alkoxy, (xi) phenoxy, (xii) halofenoxy, (xiii) C 1-4 alkylthio, (xiv) mercapto, (xv) phenylthio, (xvi) pyridylthio (xvii) C__4 alkylsulfinyl, (xviii) C? _ alkylsulfonyl, (xix) amino, (xx) C? _3 alkanoylamino, (xxi) C 1-4 mono- or dialkylamino, (xxii) cyclic amino 4 - to 6 members (xxiii) alkanoyl of C? _3, (xxiv) benzoyl and (xxv) heterocyclic groups of 5- to 10 members. It should be noted that, as used in this specification and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In the description and claim of the present invention, the following terminology will be used according to the definitions set forth below. The term "alkyl", unless otherwise stated, means straight or branched hydrocarbon chain having 1 to 15, preferably 1 to 6 carbon atoms, and a methyl or ethyl group is more preferable. The term "aryl", unless otherwise stated, is used throughout the specification to refer to a group of aromatic cyclic hydrocarbon. An aryl having 6 to 14 carbon atoms is preferable. This may be partially saturated. Preferred examples of those aryls are the phenyl and naphthyl groups. The term "hetaryl", unless otherwise stated, is used throughout the specification to refer to a 5- or 6-membered monocyclic or heterocyclic group containing from 1 to 4 heteroatoms selected from oxygen, sulfur and nitrogen, or a fused bicyclic heterocyclic group containing from 1 to 6 heteroatoms selected from oxygen, sulfur and nitrogen, each of which may be substituted by 1 to 4 substituents selected from the group consisting of (i) halogen groups, (ii) C 1-4 alkyl, (iii) C 1-4 haloalkyl, (iv) C? _4 haloalkoxy, (v) C? _4 alkoxy, (vi) C? _4 alkylthio, (vii) hydroxy, (viii) carboxy, (ix) cyano, (x) nitro, (xi) amino, (xii) mono- or di-alkylamino of C? _4, (xiii) formyl, (xiv) mercapto, (xv) C? _4 -carbonyl alkyl, (xvi) C? -4-carbonyl alkoxy, (xvii) sulfo, (xviii) C__4 alkylsulfonyl, (xix) carbamoyl, (xx) mono- or di ^-C ?4-carbamoyl alkyl, (xxi) oxo and (xxii) thioxo. The term "substituted aryl" is used throughout the specification to refer to an aryl group of C6-? 4, which may be substituted by 1 to 4 substituents selected from the group consisting of the (i) halogen groups, (ii) ) alkyl of C? _4, (iii) haloalkyl of C? _4, (iv) haloalkoxy of C _4, (v) alkoxy of C? _4, (vi) alkylthio of C? -4, (vii) hydroxy, (viii) carboxy, (ix) cyano, (x) nitro, (xi) amino, (xii) mono- or di-alkylamino of C? _, (xiii) formyl, (xiv) mercapto, (xv) C? _4- alkyl- carbonyl, (xvi) C? -4-carbonyl alkoxy, (xvii) sulfo, (xviii) C? __ alkylsulfonyl, (xix) carbamoyl, (xx) mono- or di-alkyl of C_-carbamoyl, (xxi) oxo and (xxii) thioxo. The aryl may be substituted in any position thereon. Accordingly, when the aryl is a phenyl, the phenyl ring may be substituted in the para, meta, or ortho position, and any combination thereof. The term "substituted hetaryl" is used throughout the specification to refer to hetaryl as described above which may be substituted by 1 to 4 substituents selected from the group consisting of the (i) halogen groups, (ii) C? _4 alkyl, (iii) C___ haloalkyl, (iv) C? _4 haloalkoxy, (v) C? _4 alkoxy, (vi) C 1 -4 alkylthio, (vii) hydroxy, ( viii) carboxy, (ix) cyano, (x) nitro, (xi) amino, (xii) mono- or dialkylamino of C1-.4, (xiii) formyl, (xiv) mercapto, (xv) alkyl of C? 4-carbonyl, (xvi) C ?4-carbonyl alkoxy, (xvii) sulfo, (xviii) C 1-4 alkylsulfonyl, (xix) carbamoyl, (xx) mono- or di-C 4 alkylcarbamoyl, (xxi) oxo and (xxii) thioxo. The term "halo" or "halogen" is used to describe a substituent which is a chlorine and fluorine. Additionally, halogen can be a bromine when functionally possible. The compounds of the present invention may be geometric isomers or tautomers depending on the type of substituents. The present invention also covers these isomers in separate forms and mixtures thereof. In addition, some of the compounds may contain an asymmetric carbon in the molecule; in that case the isomers could be present. The present invention also encompasses mixtures of these optical isomers and the isolated forms of the isomers. Some of the compounds of the invention can form salts. There is no particular limitation as long as the salt forms are pharmacologically acceptable. Specific examples of acid addition salts are the salts of inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid, etc., organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid, glutamic acid, etc. Specific examples of basic salts include salts with inorganic bases containing metals such as sodium, potassium, magnesium, calcium, aluminum, etc., or salts with 1 Organic bases such as methylamine, ethylamine, ethanolamine, lysine, ornithine, etc. The present invention also encompasses various hydrates and solvates of the compounds or salts thereof of the invention as well as polymorphisms thereof. Hereinafter, the representative processes for producing the compounds of the present invention are described. In these processes, the functional groups present in the initial or intermediate materials can be adequately protected with protective groups, depending on the type of functional group. In view of the preparation techniques, it may be advantageous to protect the functional groups with groups that can be easily reverted to the original functional group. When required, the protective groups are removed to give the desired products. Examples of these functional groups are amino, hydroxy, carboxy groups, etc. Examples of groups that can be used to protect those functional groups are shown in, for example Greene and Wuts, "Protective Groups in Organic Synthesis", second edition. The general procedures for synthesizing the compounds pyrazolo [3, 4-b] quinolin-5-one and pyrazolo [3, -b] pyridin-6-one are illustrated as follows: The reaction vessel was charged with aminopyrazole (1.0 mmol) dissolved in ethyl alcohol (10 mL). The appropriate aldehyde (1.0 mmol) and the dimedone (1.0 mmol) were added to the above solution while stirring at room temperature. The reaction mixture was heated to 80 ° C and refluxed for 6-8 h. The reaction vessel was then cooled to room temperature, and the solvent was removed under reduced pressure in a rotary evaporator. The residue was triturated with n-hexane to induce crystallization. The solid product was filtered, washed abundantly with n-hexane and dried under ambient conditions. Performance: 30-75% Purity: 90-95%.

The reaction vessel was charged with aminopyrazole (1.0 mmol) dissolved in ethyl alcohol (10 mL). The appropriate aldehyde (1.0 mmol) and Meldrum's acid (1.0 mmol) were added to the above solution while being stirred at room temperature. The reaction mixture was heated to 80 ° C and refluxed for 6-8 h. The reaction vessel was then cooled to room temperature, and the solvent was removed under reduced pressure in a rotary evaporator. The residue was purified by flash column chromatography. Performance: 50-75% Purity: 90-95%. The desired compound of the present invention can also be prepared by functional group transformation methods well known to those skilled in the art, which may depend on the type of substituent. The order of the reactions, or the like, can be changed appropriately according to the objective compound and the type of reaction to be employed. The other compounds of the present invention and the starting compounds can be easily produced from suitable materials in the same manner as the above processes or by methods well known to those skilled in the art. Each of the reaction products obtained by the production methods mentioned above are isolated and purified as the free base or salt thereof. The salt can be produced by usual salt formation methods. The isolation and purification steps can be carried out using conventional chemical techniques such as extraction, concentration, evaporation, crystallization, filtration, recrystallization, various types of chromatographies and the like. Various forms of isomers can be isolated by conventional methods making use of the physicochemical differences between the isomers. For example, racemic compounds can be separated by conventional optical resolution methods (e.g., by forming diastereomeric salts with a conventional optically active acid such as tartaric acid, etc. and then optically resolving the salts) to give optically pure isomers. A mixture of diastereomers can be separated by conventional means, for example, fractional crystallization or chromatography. In addition, an optical isomer of an appropriate optically active starting compound can also be synthesized. Table 1 lists the structure of representative compounds of the present invention.

One embodiment of the present invention relates to compounds that inhibit the activity of Pl 3-K alpha. The invention provides methods for inhibiting the activity of Pl 3-K alpha, including methods for modulating the activity of Pl 3 -K alpha in cells, especially cancer cells. Of particular benefit are the methods for modulating the activity of Pl 3-K alpha in the clinical setting to alleviate diseases or disorders mediated by the activity of Pl 3-K alpha. Thus, the treatment of diseases or disorders characterized by excessive or inappropriate Pl 3-K alpha activity can be effected through the use of Pl 3-K alpha modulators according to the present invention. The compounds of the present invention may also show inhibitory activity against other isms of Pl 3-K, including Pl 3-K beta, gamma, and delta. Therefore, the present invention also provides methods that allow further characterization of the physiological role of each Pl 3 -K isozyme. In addition, the invention provides pharmaceutical compositions comprising Pl 3-K inhibitors and methods of making and using those Pl 3-K inhibitor compounds. The methods described herein benefit from the use of compounds that inhibit, and that specifically inhibit specifically, the activity of an ism of Pl 3-K in cells. Cells useful in the methods include those expressing endogenous Pl 3-K, where endogenous indicates that the cells express the missing recombinant introduction of Pl 3-K in cells of one or more polynucleotides that encode an ism of Pl 3-K or a biologically active fragment thereof. The methods also encompass the use of cells expressing exogenous Pl 3-K isms where one or more polynucleotides encoding isms of Pl 3-K or a biologically active fragment thereof, have been introduced into the cell using recombinant methods. Of particular advantage, the cells can be in vivo, i.e., in a living subject, eg, an animal or human, where the Pl 3-K inhibitor can be used therapeutically to inhibit the activity of Pl 3. -K on the subject. Alternatively, the cells can be isolated as discrete cells or in a tissue, by ex vivo or in vi tro methods. The in vitro methods also encompassed by the invention may comprise the step of contacting a Pl 3-K enzyme, or a biologically active fragment thereof, with an inhibitor compound of the invention. The Pl 3-K enzyme can include a purified and isolated enzyme, where the enzyme is isolated from a natural source (for example, cells or tissues that normally express an absent modification of the Pl 3-K polypeptide by recombinant technology) or isolated of cells modified by recombinant techniques to express the exogenous enzyme. The relative efficiencies of the compounds as inhibitors of the enzymatic activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined degree and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% activity in a biochemical assay, i.e. 50% inhibitory concentration or "IC50". The IC50 determinations can be made using conventional techniques known in the art. In general, the IC50 can be determined by measuring the activity of a given enzyme in the presence of a range of concentrations of the inhibitor under study. The experimentally obtained values of the enzymatic activity are then plotted against the inhibitor concentrations used. The concentration of the inhibitor that allows 50% of the enzymatic activity (in comparison with the activity in the absence of any inhibitor) is taken as the IC 50 value. Analogously, other inhibitory concentrations can be defined through appropriate activity determinations. For example, in some scenarios it may be desirable to establish an inhibiting concentration of 90%, ie, IC90, etc. The compounds of the present invention exhibit kinase inhibitory activity, especially Pl 3-K inhibitory activity and therefore, can be used to inhibit abnormal cell growth in which Pl 3-K plays a role. In this way, the compounds are effective in the treatment of disorders with which the actions of Pl 3-K on abnormal cell growth are associated, such as restenosis, atherosclerosis, bone disorders, arthritis, diabetic retinopathy, psoriasis, prosthetic hypertrophy. benign, atherosclerosis, inflammation, angiogenesis, immunological disorders, pancreatitis, kidney disease, cancer, etc. In particular, the compounds of the present invention possess excellent cancer cell growth inhibitory effects and are effective to treat cancers, preferably all types of solid cancers and malignant lymphocytes, and especially leukemia, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, gastric cancer and brain tumors, etc. Accordingly, the invention provides methods for characterizing the potency of a test compound as an inhibitor of the Pl 3-K polypeptide, the method comprising the steps of (a) measuring the activity of a Pl 3 -K polypeptide in the presence of a test compound; (b) comparing the activity of the PI3 polypeptide in the presence of the test compound with the activity of the Pl 3 -K polypeptide in the presence of an equivalent amount of a reference compound (e.g., an inhibitor compound of Pl 3-K a of the invention as described herein), where the lower activity of the Pl 3-K polypeptide in the presence of the test compound in the presence of the reference compound indicates that the test compound is a more potent inhibitor than the reference compound, and a higher activity of the Pl 3-K polypeptide in the presence of the test compound which in the presence of the reference compound indicates that the test compound is a less potent inhibitor than the reference compound. The invention further provides methods for characterizing the potency of a test compound as an inhibitor of the P? 3-K comprising the steps of (a) determining the amount of a control compound (e.g., an inhibitor compound of Pl 3-K alpha of the invention as described herein) that inhibits an activity of a Pl 3 polypeptide -K in an inhibition reference percentage, thereby defining a reference inhibitory amount for the control compound; (b) determining the amount of a test compound that inhibits the activity of a Pl 3-K polypeptide at a percentage of reference inhibition, thereby defining a reference inhibitory amount for the test compound; (c) comparing the reference inhibitory amount for the test compound with the reference inhibitory amount for the control compound, where a lower reference inhibitory amount for the test compound than for the control compound indicates that the compound test is a more potent inhibitor than the control compound, and a higher reference inhibitory amount for the test compound than for the control compound indicates that the test compound is a less potent inhibitor than the control compound. In one aspect, the method uses a reference inhibitory amount, which is the amount of compound that inhibits the activity of the Pl 3-K alpha polypeptide by 50%, 60%, 70% or 80%. In another aspect the method employs a reference inhibitory amount which is the amount of the compound that inhibits the activity of the Pl 3 -K alpha polypeptide in 90%, 95% or 99%. Those methods comprise determining the reference inhibitory amount of the compounds in an in vitro biochemical assay, in an in vitro cell-based assay, or in an in vivo assay. The invention further provides methods for identifying a negative regulator of Pl 3-K alpha activity comprising the steps of (i) measuring the activity of a PI3 alpha polypeptide in the presence and absence of a test compound, and (ii) ) identifying as a negative regulator a test compound which decreases the activity of Pl 3-K alpha and which competes with a compound of the invention by binding to Pl 3 -K-alpha. In addition, the invention provides methods for identifying compounds that inhibit Pl 3 -K alpha activity, comprising the steps of (i) contacting a Pl 3-K alpha polypeptide with a compound of the invention in the presence and absence of a test compound, and (ii) identifying a test compound as a negative regulator of Pl 3 -K alpha activity, where the compound competes with a compound of the invention for binding to Pl 3-K alpha. The invention further provides a method for separating candidate negative regulators from the activity of Pl 3-K alpha and / or to confirm the mode of action of candidates as negative regulators. These methods can be used against other isoforms of Pl 3-K in parallel to establish the comparative activity of the test compounds through the isoforms and / or in relation to a compound of the invention. In those methods, the Pl 3 -K 'polypeptide can be a fragment of the peptide that exhibits kinase activity or a fragment of the binding domain that provides a method for identifying allosteric modulators of the peptide. The methods can be used in cells that express the peptide Pl 3-K or its subunits, either endogenously or exogenously. Accordingly, the polypeptide employed in those methods can be solution-free, fixed on a solid support, modified to be presented according to the cell surface, or located intra-cellularly. Modulation of the activity or formation of binding complexes between the Pl 3-K polypeptide and the agent being tested can then be measured. The human Pl 3-K polypeptides are suitable for biochemical or cell-based high-throughput (HTS) separation assays according to methods known and practiced in the art, including melanophore assay systems to investigate receptor-ligand interactions , yeast-based assay systems, and mammalian cell expression systems. For a review, see Jayawickreme and Kost, Curr Opin Biotechnol, 8: 629-34 (1997). The automated and miniaturized HTS assays are comprised as described, for example, in Houston and Banks, Curr Opin Biotechnol, 8: 734-40 (1997). These HTS assays are used to separate libraries of compounds to identify particular compounds that exhibit a desired property. Any library of compounds, including chemical libraries, libraries of natural products and combined libraries comprising oligopeptides, oligonucleotides or other random or designed organic compounds, may be used. The present invention also provides a method for inhibiting the activity of Pl 3-K therapeutically or prophylactically. The method comprises administering an inhibitor of the activity of Pl 3-K in an effective amount thereof to treat humans or animals that can be subjected to any condition whose symptoms or pathological is mediated by the expression or activity of Pl 3-K. "Treat" as used herein refers to preventing the occurrence of a disorder in an animal that may be predisposed to the disorder but has not yet been diagnosed as having this disorder.; inhibit the disorder; that is, to counteract their development; reverse the disorder; that is, to produce its regression; or relieve the disorder, that is, reduce the severity of the symptoms associated with the disorder. "Disorder" is intended to encompass disorders, diseases, conditions, medical syndromes and the like, without limitation. The methods of the invention encompass several ways to treat an animal subject, preferably a mammal, more preferably a primate, and even more preferably a human. Among the animals that can be treated are, for example, companion animals (pets), including dogs and cats; farm animals, including cattle, horses, sheep, pigs and goats; laboratory animals, including rats, mice, rabbits, guinea pigs and primates, non-humans and zoo specimens. Non-mammalian animals include, for example, birds, fish, reptiles, and amphibians. In one aspect, the method of the invention can be employed to treat subjects therapeutically or prophylactically having or who may be subject to an inflammatory disorder. One aspect of the present invention is derived from the involvement of Pl 3-K in the mediating aspects of inflammatory processes. Without pretending to be limited by any theory, we have the theory that, because inflammation involves processes that are typically mediated by chemotactic activation and transmigration of leukocytes (for example, neutrophils, lymphocytes, etc.) and because Pl 3-K can mediate this phenomenon, Pl 3-K antagonists can be used to suppress the damage associated with inflammation. "Inflammation" as used herein refers to a protective, localized response, caused by damage or destruction of tissues, which serves to destroy, dilute, or wall (sequester) the harmful agent and the damaged tissue. The inflammation is associated, in a remarkable way, with the influx of leukocytes and / or chemotaxis of neutrophils. Inflammation can result from infection with pathogenic organisms and viruses and noninfectious media such as trauma or reperfusion after myocardial infarction or stroke, immune response to external antigens and autoimmune responses. Accordingly, inflammatory disorders suitable for the invention encompass disorders associated with reactions of the specific defense system as well as with reactions of the non-specific defense system. Therapeutic methods of the present invention include methods for the treatment of disorders associated with inflammatory cell activation. "Inflammatory cell activation" refers to the induction by a stimulus (including but not limited to, cytokines, antigens or autoantibodies) of a proliferative cell response, the production of soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive amines), or the expression on the cell surface of new or an increased number of mediators (including, but not limited to, major histocompatibility antigens or cell adhesion) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes (ie, polymorphonuclear leukocytes such as neutrophils, basophils, and eosinophils), mast cells, dendritic cells, Langerhans cells, and endothelial cells ). It will be appreciated by those skilled in the art that the activation of one or a combination of those phenotypes in those cells may contribute to the onset, perpetuation, or exacerbation of an inflammatory disorder. In a further aspect, the invention includes methods of using the Pl 3-K inhibitory compounds to inhibit the growth or proliferation of cancer cells of hematopoietic origin, preferably cancer cells of lymphoid origin, and more preferably cancer cells related to or derivatives of B lymphocytes or B lymphocyte progenitors. Cancers suitable to be treated using the methods of the present invention include, without limitation, lymphomas, for example, malignant neoplasms of lymphoid and reticuloendothelial tissues, such as Burkitt's lymphoma, Hodgkin's lymphoma, lymphomas non-Hodgkins, lymphocytic lymphomas and the like; multiple myelomas; as well as leukemias such as lymphocytic leukemias, chronic myeloid (myelogenous) leukemias, and the like. A compound of the present invention can be administered as a pure chemical compound, but typically refers to administering a compound in the form of a pharmaceutical composition or formulation. Accordingly, the present invention also provides pharmaceutical compositions comprising a chemical or biological compound ("agent") that is active as a modulator of the activity of Pl 3-K and a biocompatible excipient, adjuvant or pharmaceutical carrier. The composition may include the agent as the sole active entity or in combination with other agents, such as oligo- or polynucleotides, oligo- or polypeptides, drugs or hormones mixed with excipients or other pharmaceutically acceptable carriers. The excipients or vehicles and other ingredients can be considered pharmaceutically acceptable as long as they are compatible with other ingredients of the formulation and not harmful to the recipient thereof. Techniques for the formulation and administration of pharmaceutical compositions can be found in Reminton's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co, Easton, Pa. , 1990. The pharmaceutical compositions of the present invention can be made using any conventional method, for example, mixing, dissolving, granulating, tabletting, levigating, emulsifying, encapsulating, capturing, melt centrifugation, spray drying or lyophilization. However, optimal pharmaceutical formulations will be determined by one skilled in the art depending on the route of administration and the desired dose. These formulations can influence the physical state, stability, speed, release in vivo, and rate of in vivo elimination of the agent administered. Depending on the condition being treated, those pharmaceutical compositions can be formulated and administered systemically or locally. The pharmaceutical compositions are formulated to contain suitable pharmaceutically acceptable excipients and may optionally comprise excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The mode of administration will generally determine the nature of the excipient or vehicle. For example, formulations for parenteral administration may comprise aqueous solutions of the active compounds in water-soluble form. Suitable excipients or vehicles for parenteral administration can be selected from saline, buffered saline solution, dextrose, water and other physiologically compatible solutions. Preferred excipients or vehicles for parenteral administration are physiologically compatible buffers such as Hank's solution, Ringer's solution or physiologically buffered saline solution. For tissue or cellular administration, appropriate penetrants are used for the particular barrier to be infiltrated into the formulation. These penetrants are generally known in the art. For preparations comprising proteins, the formulation may include stabilizing materials, such as polyols (e.g., sucrose) and / or surfactants (e.g., nonionic surfactants), and the like. Alternatively, formulations for parenteral use may comprise dispersions or suspensions of the active compounds prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, and synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain stabilizers or suitable agents that increase the solubility of the compounds to allow the preparation of highly concentrated solutions. Aqueous polymers that provide pH-sensitive solubilization and / or sustained release of the active agent can also be used as coating or matrix structures, for example, methacrylic polymers, such as the EUDRAGIT.RTM series. available from Rohm America Inc. (Piscataway, N.J.). Emulsions may also be used, for example, oil-in-water or water-in-oil dispersions, optionally stabilized by an emulsifying or dispersing agent (surface active materials).; surfactants). The suspensions may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth gum and mixtures thereof. Liposomes containing the active agent can also be used for parenteral administration. Liposomes are generally derived from phospholipids or other lipid substances. The compositions in liposome form may also contain other ingredients, such as stabilizers, preservatives, excipients and the like. Preferred lipids include phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods of liposome formation are known in the art. See, for example, Prescott (Ed.), Methods in Cell Biology, Vol. XIV, p. 33, Academic Press, New York (1976). Pharmaceutical compositions comprising the agent in a dose suitable for oral administration can be formulated using pharmaceutically acceptable excipients or vehicles well known in the art. Preparations formulated for oral administration may be in the form of tablets, pills, capsules, sachets, dragees, lozenges, lipids, gels, syrups, suspensions, elixirs, suspensions or powders. As an illustration, pharmaceutical preparations for oral use can be obtained by combining the active compounds with a solid excipient, optionally grinding the reaction mixture, and processing the mixture of granules, after the addition of suitable auxiliaries if desired, to obtain tablets or dragee cores. Oral formulations may employ excipients or liquid carriers similar in type to those described for parenteral use, for example, buffered aqueous solutions, suspensions, and the like. Preferred oral formulations include tablets, dragees, gelatin capsules. Those preparations may contain one or more excipients, which include, without limitation: a) diluents, such as sugars, including lactose, dextrose, sucrose, mannitol or sorbitol; b) binders, such as magnesium aluminum silicate, corn starch, wheat, rice, potato, etc .; c) cellulose materials, such as methylcellulose, hydroxypropylcellulose, and sodium carboxymethylcellulose, polyvinylpyrrolidone, gums, such as gum arabic and tragacanth gum, and proteins, such as gelatin and collagen; d) disintegrating or solubilizing agents such as the cross-linked polyvinyl pyrrolidone, starches, agar, alginic acid or a salt thereof, such as sodium alginate, or effervescent compositions; e) lubricants, such as silica, talc, stearic acid or its magnesium or calcium salt, and polyethylene glycol; f) flavors and sweeteners; g) dyes or pigments, for example, to identify the product or to characterize the amount (dose) of active compound and h) other ingredients, such as preservatives, stabilizers, leavening agents, emulsifying agents, solution promoters, salts to regulate the pressure osmotic, and shock absorbers. Gelatin capsules include pressure-adjustable capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Pressure-adjusting capsules can contain the active ingredients mixed with fillers, binders, lubricants and / or stabilizers, etc. In soft capsules, the active compounds can be dissolved or suspended in suitable fluids, such as fatty oils, liquid paraffin or liquid polyethylene glycol or with or without stabilizers.

Dragee cores can be provided with suitable coatings as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, lacquer solutions and suitable organic solvents or mixtures of solvents. The pharmaceutical composition can be provided as a salt of the active agent. The salts tend to be more soluble in aqueous solvents or other protonic solvents than in the corresponding free acid or base forms. Pharmaceutically acceptable salts are well known in the art. Compounds containing acidic portions can form pharmaceutically acceptable salts with suitable cations. The pharmaceutically acceptable cations include, for example, alkali (for example, sodium or potassium) and alkaline earth (for example, calcium or magnesium) cations. The compounds of structural formulas I-III of the present invention can form pharmaceutically acceptable acid addition salts with suitable acids. For example, Berge et al describe pharmaceutically acceptable salts in detail in J Pharm Sci, 66: 1 (1977). The salts can be prepared in itself during the isolation and final purification of the compounds of the invention or separately by reacting a free base function with a suitable acid. In light of the above, any reference to the compounds of the present invention appearing herein is intended to include compounds of the structural formula described above as well as pharmaceutically acceptable salts and solvates, as well as prodrugs thereof. Compositions comprising a compound of the present invention formulated in a pharmaceutically acceptable excipient may be prepared, placed in an appropriate container, and labeled for the treatment of an indicated condition. Accordingly, an article of manufacture, such as a container comprising a dosage form of a compound of the invention and a label containing instructions for the use of the compound, was also contemplated. Devices were also contemplated under the invention. For example, the kit may comprise a dosage form of a pharmaceutical composition and a package insert containing instructions for the use of the composition in the treatment of a medical condition. In any case, the conditions indicated on the label may include the treatment of inflammatory disorders, cancer, etc. Pharmaceutical compositions comprising an inhibitor of Pl 3 -K activity can be administered to the subject by any conventional method, including by parenteral and enteral techniques. Patterns of parenteral administration include those in which the composition is administered by a different route through the gastrointestinal tract, for example, by intravenous, intraarterial, intraperitoneal, intramedullary, intramuscular, intraarticular, intrathecal, and intraventricular injections. Methods of enteral administration include, for example, oral (including buccal and sublingual) and rectal administration. Transepithelial administration modalities include, for example, transmucosal administration and transdermal administration. Transmucosal administration includes, for example, enteral administration as well as nasal, inhalation, and deep pulmonary administration; vaginal administration; and rectal administration. Transdermal administration includes passive or active transdermal or transcutaneous modalities, including, for example, patches and iontophoresis devices, as well as topical applications of pastes, balms or ointments. Parenteral administration can also be effected using high pressure techniques. Surgical techniques include the implantation of reservoir compositions (reservoirs), osmotic pumps and the like. A preferred route of administration for the treatment of inflammation may be local or topical delivery for localized disorders such as arthritis., or systemic release for distributed disorders, for example, intravenous release for reperfusion injury or for systemic conditions such as septicemia. For other diseases, including those involving the respiratory tract, for example, chronic obstructive pulmonary disease, asthma and emphysema, administration can be effected by inhalation or deep pulmonary administration of sprays, aerosols, powders and the like. For the treatment of neoplastic diseases, especially leukemias and other distributed cancers, parenteral administration is typically preferred. Formulations of the compounds to optimize them for biodistribution after parenteral administration would be desirable. The Pl 3-K inhibitor compounds can be administered before, during or after the administration of chemotherapy, radiotherapy and / or surgery. As noted above, the characteristics of the agents themselves and the formulation of the agent can have an influence on the physical state, in vivo release stability, and in vivo clearance rate of the agent administered. This pharmacokinetic and pharmacodynamic information can be collected through preclinical studies in vi tro and in vivo, later confirmed in humans during the course of clinical trials. Thus, for any compound used in the method of the invention, a therapeutically effective dose can be estimated initially through biochemical and / or cell-based assays. Then, the dose can be formulated in animal models to achieve a desirable concentration range in circulation that modulates the expression or activity of Pl 3-K. When human studies are conducted, additional information will emerge regarding the appropriate dose levels and the duration of treatment for various diseases and conditions. The toxicity and therapeutic efficacy of these compounds can be determined by standard pharmaceutical procedures using cell cultures or animal experiments, for example, to determine the LD50 (the lethal dose at 50% of the population) and the ED50 (the therapeutically effective dose at 50% of the population). The dose ratio between toxic and therapeutic effects is the "therapeutic index", which is typically expressed as the LD50 / DE5o ratio. Compounds that exhibit large therapeutic indices, i.e., that the toxic dose is substantially greater than the effective dose, are more preferred. The data obtained from these cell culture assays and additional animal studies can be used to formulate a range of doses for human use. The dose of these compounds preferably falls within a range of calculation concentrations that include ED50 with little or no toxicity. For the methods of the present invention, any effective administration regimen regulating timing and sequence of doses may be used. Doses of the agent preferably include pharmaceutical dosage units comprising an effective amount of the agent. As used herein, "effective amount" refers to an amount sufficient to modulate the expression or activity of Pl 3-K and / or derive a measurable change in a physiological parameter of the subject through the administration of one or more units of pharmaceutical dose. Exemplary dose levels for a "human subject" are in the range of about 0.001 milligram of active agent per kilogram of body weight (mg / kg) to about 100 mg / kg Typically, the dosage units of the active agent comprise about 0.01. mg to about 10,000 mg, preferably from about 0.1 mg to about 1,000 mg, depending on the indication, route of administration, etc.

Depending on the route of administration, an adequate dose can be calculated according to body weight, body surface area, or organ size. The final dosage regimen will be determined by the doctor who pays attention in view of good medical practice, considering the different factors that modify the action of the drugs, for example, the specific activity of the agent, the identity and severity of the state of disease, the patient's response, the age, condition, body weight, sex and diet of the patient, and the severity of any infection. Additional factors that may be taken into account include time and frequency of administration, drug combinations, reaction sensitivities and tolerance / response to therapy. Further refining of the appropriate dose for the treatment involving any of the formulations mentioned herein is routinely performed by one skilled in the art without undue experimentation, especially in light of the dose information and assays described, as well as the pharmacokinetic data observed in clinical trials in humans. Appropriate doses can be determined through the use of established tests to determine the concentration of the agent in a body fluid or other sample along with the dose response data. The frequency of dosing will depend on the pharmacokinetic parameters of the agent and the route of administration. The dose and administration are adjusted to provide sufficient levels of the active portion to maintain the desired effect. Accordingly, the pharmaceutical compositions can be administered in a single dose, multiple discrete doses, by continuous infusion, as sustained release reservoirs, or combinations thereof, as required to maintain the desired minimum level of the agent. The short-acting pharmaceutical compositions (ie, short half-life) can be administered once a day or more than once a day (eg, two, three or four times a day). The long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks. Pumps, such as subcutaneous, intraperitoneal or subdural pumps, may be preferred for continuous infusion. The following Examples are provided to further assist in understanding the invention, and presuppose an understanding of conventional methods well known to those skilled in the art to which the examples pertain. These methods are described in detail in numerous publications including, for example, Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), Ausubel et al. (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); and Ausubel et al. (Eds.), Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons, Inc. (1999). The particular materials and conditions described hereinafter are intended to exemplify particular aspects of the invention and should not be construed as limiting the reasonable scope thereof.

EXAMPLE 1 Synthesis and Characterization of 964076 3-t-butyl-4- (2-chlorophenyl) -7,7-dimethyl-1- (4-methoxyphenyl) -4,7,8,9-tetrahydro-1-pyrazolo [3 , 4-b] quinolin-5 (ßH) -one The reaction vessel was charged with l- (4-methoxyphenyl) -3-t-butyl-5-aminopyrazole (500 mg, 2.03 mmol) dissolved in ethyl alcohol (20 mL). Next, (2-chloro-benzaldehyde (218 mL, 2.43 mmol) and dimedone (285 mg, 1.0 mmol) were added to the above solution while stirring at room temperature.The reaction mixture was heated to 80 ° C and maintained at reflux for 6 h The reaction vessel was then cooled to room temperature, and the solvent was removed under reduced pressure in a rotary evaporator, the residue was triturated with n-hexane to induce crystallization, the solid product was redissolved and purified. additionally by column chromatography producing a pure product (110 mg) which was then characterized by NMR: lE NMR (CDC13): 0.8, 1.02, 1.1, 1.23, 2.03, 2.14, 3.85, 5.67, 6.32, 7.02, 7.14, 7.23 , 7.44.

Example 2 Synthesis and Characterization of 964028 3-t-butyl-4- (4-methylphenyl) -7,7-dimethyl-1- (4-methylphenyl) -4,7,8,9-tetrahydro-l / i-pyrazolo [3, 4-b] quinolin-5 (SE) -one The reaction vessel was charged with l- (4-methylphenyl) -3-t-butyl-5-aminopyrazole (180 mg, 0.78 mmol) dissolved in ethyl alcohol (10 mL). Then p-tolualdehyde (110 mg, 0.94 mmol) and dimedone (110 mg, 0.78 mmol) were added to the above solution while stirring at room temperature. The reaction mixture was heated to 80 ° C and maintained at reflux for 6 h. The reaction vessel was then cooled to room temperature, and the solvent was removed under reduced pressure in a rotary evaporator. The residue was triturated with n-hexane to induce crystallization. The solid product (178 mg) was filtered, washed and dried under ambient conditions, which was then characterized by NMR: XH NMR (CDC13): 0.82, 1.03, 1.14, 1.23, 2.25, 2.41, 5.40, 6.22, 7.00, 7.18 , 7.31, 7.43.

Example 3 Synthesis and Characterization of 1,3-Di-t-butyl-4-p-trifluoromethylphenyl-1,4,5,7-tetrahydro-pyrazolo [3,4-b] pyridin-6-one The reaction vessel was charged with 1,3-di-t-butyl-5-aminopyrazole (50 mg, 0.26 mmol) dissolved in ethyl alcohol (5 mL). Then p-trifluoromethylbenzaldehyde (44.6 mg, 0.26 mmol) and Meldrum's acid (36 mg, 0.26 mmol) were added to the above solution while stirring at room temperature. The reaction mixture was heated to 80 ° C and maintained at reflux for 6 h. The reaction vessel was then cooled to room temperature, and the solvent was removed under reduced pressure in a rotary evaporator. The residue was purified by chromatography on silica, eluting with a mixture of hexane and ethyl acetate. The solid product (70 mg) was isolated and then characterized by NMR: 1 H NMR (CDC 13): 1.12, 1.59, 2.67, 3.12, 4.40, 7.14, 7.51, 8.23.

Example 4 Synthesis and Characterization of l, 3-Di-t-butyl-4- (3,4-dimethoxy-phenyl) -4,6,7,8-tet.rahidro-li? -l, 2,8-triaza -s-indacen-5-one The reaction vessel was charged with?, J-a? -t-butyl-5-aminopyrazole (40 mg, 0.20 mmol) dissolved in ethyl alcohol (5 mL). Then 3,4-dimethoxybenzaldehyde (34 mg, 0.20 mmol) and 1,3-cyclopentadione (36 mg, 0.26 mmol) were added to the above solution while stirring at room temperature. The reaction mixture was heated to 80 ° C and maintained at reflux for 6 h. The reaction vessel was then cooled to room temperature, the solvent was removed under reduced pressure in a rotary evaporator. The residue was purified by chromatography on silica, eluting with a mixture of hexanes and ethyl acetate. The solid product (60 mg) was isolated and then characterized by NMR: XH NMR (CDC13): 0.95, 1.56, 2.27, 2.49, 3.59, 3.69, 3.71, 5.00, 6.58, 3.61, 6. 78.

Example 5 Synthesis and Characterization of 4- (3,4-Bis-benzyloxy-phenyl) -1,3-di-t-butyl-4,6,7,8,9,10-hexahydro-l # -l, 2 , 10-triaza-hepta [f] inden-5-one cycle t-butyl-5-aminopyrazole (40 mg, 0.20 mmol) dissolved in ethyl alcohol (5 mL). 3,4-Dibenzyloxybenzaldehyde (65 mg, 0.20 mmol) and 1,3 cycle heptadione (36 mg, 0.26 mmol) were then added to the above solution while stirring at room temperature. The reaction mixture was heated to 80 ° C and maintained at reflux for 6 h. The reaction vessel was then cooled to room temperature, and the solvent was removed under reduced pressure in a rotary evaporator. The residue was purified by chromatography on silica, eluting with a mixture of hexanes and ethyl acetate. The solid product (18 mg) was isolated and then characterized by NMR: aH NMR (CDC13): 1.03, 1.64, 2.42, 5.07, 5.28, 6.72, 7.31, 7.37.

Example 6 Synthesis and Characterization of 1- (1,3-Di-tert-butyl-4-p-tolyl-4,7-dihydro-lH-pyrazolo [3,4-pyridin-5-yl] -eta? ona The reaction vessel is charged with 1,3-di-t-butyl-5-aminopyrazole (40 mg, 0.20 mmol) dissolved in ethyl alcohol (5 mL). P-Tolualdehyde (23 mg) is added, 0.20 mmol) and 1,3 pentadione (36 mg, 0.26 mmol) to the above solution while stirring at room temperature. The reaction mixture is heated to 80 ° C and maintained at reflux for 6 hours. The reaction vessel is then cooled to room temperature, and the solvent removed under reduced pressure in a rotary evaporator. The residue is purified by chromatography on silica, eluting with a mixture of hexanes and ethyl acetate.

Example 7 Isolation and Purification of Recombinant Pl 3 -K Polypeptide Recombinant heterodimeric Pl 3-K alpha, consisting of a pllO catalytic subunit and a p85-labeled subunit regulator, was expressed in Sf9 cells using the baculovirus expression system. The recombinant plasmids or expression constructs were obtained from the laboratory of Dr. Alex Toker, Harvard University. The method is well known to those skilled in the art and is also described in Stoyanov et al., Science 269, 690-693 (1995) and Stoyanova et al., Biochem. J. 324: 489-495. (1997).

The harvested cell mass was resuspended in 3 ml of Buffer A (20 mM Tris pH 7.0, 150 mM NaCl, 10 mM EDTA, 20 mM Sodium Fluoride, 5 mM Sodium Pyrophosphate, 10% Glycerol, 0.1% Igapal) containing inhibitors of protease (PMSF lmM, NaV03 ImM, Leupeptin 1 μg / ml, Pepstatin 1 μg / ml.). The suspension was incubated for 1 hour at 4 ° C without rotation to break the cells, and then vortexed gently to ensure cell lysis. The solution was centrifuged at 14,000 g for 15 minutes, and the supernatant was diluted by the addition of 10 ml of Buffer A. The diluted supernatant was added to 3 ml of Glutathione-agarose resin (Pharmacia) pre-equilibrated in Buffer A, and incubated for 1 hour at 4 ° C with rotation. The resin was poured into a column and washed with 35 ml of Buffer A, and the protein was eluted using 10 mM Glutathione in Buffer A. Twenty 0.5 ml fractions were collected and the presence of protein was evaluated on SDS PAGE Tris Glycine gel to 12% (Invitrogen). Fractions containing the target protein were pooled and concentrated using a Microsep 30K concentrator (Pall-Gelman). The concentrated protein was diluted with 3 ml of Final Buffer (20 mM Tris pH 7.4, 100 mM NaCl, 1 mM EDTA) and concentrated twice more to remove any detergent. The protein was diluted in 50% glycerol and stored at -20 ° C.

Example 8 Assay and Separation of Pl 3-K Activity for Pl 3-K Inhibitors Vectors for the expression of GST-GRP1-PH were obtained from Mark Lemmon, University of Pennsylvania. (Kavran, et al., J Biol Chem, 273: 30497-30508 (1998)). The expression and purification of E. coli protein was carried out as follows: A LB / amp plate was detached from an E coli pattern with frozen glycerol containing the expression vector and grown overnight at 37 °. C. A single colony was removed and inoculated in 20 ml of medium containing LB 100 μg / ml ampicillin, and grown overnight. The overnight culture was added to one liter of LB medium containing 100 μg / ml of ampicillin and grown until the D.O. 600 was between 0.8-1.0. Protein expression was induced by the addition of 0.1 mm IPTG, the cultures continued to grow overnight at 37 ° C. The cells were harvested by centrifugation at 4,000 g for 20 minutes. The sediments were stored frozen at -80 ° C until the purification of protein was carried out. The purification of GST-labeled protein was carried out as follows. The pellets were suspended in 25 ml of Buffer A (50 mM Tris pH 7.5, 1 mM BME, lmM EDTA, lmM EGTA, 1 mM NaV03, 50mM Sodium Fluoride, 5mM Sodium Pyrophosphate, 0.27M Sucrose) with protease inhibitors ( PMSF lmM, 0.5 μg / ml of Leupeptin, 0.7 μg / ml of Pepstatin). The cells were used by sonication for 3 minutes, and Triton x-100 was added to a final concentration of 0.01%. The mixture was clarified by centrifugation at 10,000 rpm for 15 minutes. The supernatant was mixed with 5 ml of Glutathione-agarose resin (Amersham), pre-equilibrated in Buffer A. The protein was allowed to bind to the resin for 1 hour at 4 ° C with rotation. The resin was transferred to a column and washed with 30 ml of Buffer A. The protein was eluted using mM glutathione (Sigma) in Buffer A. Twenty 1 ml fractions were collected and protein levels were assessed by SDS-PAGE on 12% Tris-Glycine gels (Invitrogen). Fractions containing pure protein were pooled and stored at -20 ° C. The reactions of Pl 3-kinase were carried out in a reaction buffer - containing 5 mM HEPES, pH 7, 2.5 mM MgCl 2, and 25 μM ATP, containing 50 ng of recombinant Pl 3-K with 10 picomoles of diCβ Pl (4,5) P2 (Echelon Biosciences) as the substrate. The reactions were allowed to proceed at room temperature for 1-3 hours, then stopped by the addition of EDTA to a final concentration of 10 mM. The final reaction volumes were 10 μl. The compounds to be tested for inhibition were added to a final concentration of 1 μM of DMSO standards. The final concentration of DMSO was 1%. The conversion of the substrate to Pl (3,4,5) P3 was determined using a competition assay using the Amplified Luminescent Proximity Homogeneous Assay (ALPHA ©) technology developed by Perkin Elmer. 0.25 picomoles of recombinant GST-Grpl-PH domain protein and 0.25 picomoles of biotinylated diCβ Pl were added. (3,4,5) P3 (Echelon Biosciences) to each reaction mixture.

The donor and acceptor pearls of the Detection Team (PerkinElmer) from GST (Glutathione-S-Transferase) AlphaScreen® were added to a final concentration of 20 μg / ml. The final volume was 25 μl. The reactions were incubated at 37 ° C for two hours, and the luminescent signal was read on a Fusion a microplate reader. Percent inhibition of enzyme activity was determined by comparison with controls without enzyme (100% inhibition) and DMSO alone (0% inhibition). An alternative method used for the detection of substrate conversion to PI (3,4,5) P3 was a competitive Fluorescence Polarization assay. 125 picomoles of recombinant GST-Grpl-PH domain and 0.25 picomoles of TAMRA-I (1, 3, 4, 5) P4 (Echelon Biosciences) were added to each reaction mixture. The final volume was 25 μl. Polarization values were measured on a microplate reader using 550 nm excitation emission / 580 nm polarization filters. The BODIPY-TMR-I (1,3,4,5) P4 or the BODIPY-TMR-PI (3, 4, 5) P3 could be a substitute for the fluorescence tracers in this assay. Percent inhibition of enzyme activity was determined by comparison of controls without enzyme (100% inhibition) and DMSO alone (0% inhibition).

Example 9 Determination of IC50 for Pl 3-K inhibitors A library of potential Pl 3-K inhibitors was tested for its activity against Pl 3-K alpha in the following manner. Of the identified active compounds, twelve were selected as representative of the different chemical groups present in the library and subjected to further analysis. The IC50 values for the selected compounds of the present invention were determined. Enzyme activity assays were performed as described above, in the presence of a range of compound concentrations to allow the determination of IC 50 values. The activity and percent inhibition of the enzyme were determined using the AlphaScreen® luminescent assay or a Fluorescence Polarization assay as described above. These inhibitors also show activities against other isoforms of Pl 3-K, including Pl 3-K beta, gamma and delta.

EXAMPLE 10 Characterization of the Effects of Pl 3-K Inhibitors on Cancer Cells The selected compounds were tested for their selectivity against paired ovarian cancer cell lines and breast cancer. The cell line of ovarian cancer SK0V3 does not present alterations in the signaling of Pl 3-K and should be more sensitive to the antiproliferative effects produced by the treatment with inhibitors of Pl 3-K, while the cellular line 0VCAR3, which presents alterations in the signaling of Pl 3-K, via the amplification of the activity of Pl 3-K, it would be sensitive. SK0V3 cells were seeded in 96-well cell culture plates (Greiner) at a density of 20,000 cells per well in McCoys 5A media (GibcoBRL) with 10% fetal sheep serum and 20 mM L-glutamine. 0VCAR3 cells were seeded at a density of 15,000 per well in RPMI 1640 media (GibcoBRL) containing 20 mM l-glutamine, 0.01 mg / ml bovine insulin, 10 mM Hepes pH 7.4, 1 mM sodium pyruvate, 2.5 g / L of glucose, and 20% of fetal sheep serum. After 24 hours, the compounds were added to the cell media at a final concentration of 1 μm, and the cells were grown in the presence of the compounds for 48 hours, in media containing 0.5% fetal sheep serum. Viability was determined using a series of MTT cell proliferation (R and D systems) and by comparison with controls with DMSO alone (100% viability). Compounds that resulted in reduced viability may act as inhibitors of cell proliferation or inducers of apoptosis (programmed cell death). Compounds representative of structural group 096 within the library showed selective effects on cell proliferation and viability. The compounds present in the library that have been identified as inhibitors of Pl 3-K using in vi tro separation, and which were also structurally related to the compounds of the present invention that showed specific cellular effects on viability, were tested for their activity against paired ovarian cancer cell lines. Many of these also show selective cellular effects on cell growth. Table 2 summarizes the results of two separate cell proliferation experiments for selected compounds of the present invention. The selected compounds were evaluated against ovarian cancer cell lines paired at a range of concentrations to determine the effective concentrations for growth inhibition. Table 2. Summary of two different experiments in which compounds of the present invention were tested for the selective effects on paired ovarian cancer cell lines.

The PI 3-K inhibitors that show this activity profile were effective against a number of tumor cell lines and tumor types in which the Pl 3-K signaling is altered, either by amplification of the P3 activity K, or by mutations that affect the regulation of Pl 3-K activity, including mutations in the PTEN tumor suppressor gene. These include cancers of the breast, prostate, colon and ovary. Pl 3-K inhibitors were also evaluated for their selectivity against breast cancer cell lines. The MDA-MB-468 cell line is mutant of PTEN, a negative regulator of Pl-K signaling, Pl 3-K signaling is abnormally activated in those cells, while the cell line MDA-MB-231 shows a normal expression and activity of PTEN and the signaling of Pl 3-K is regulated in a normal manner.

MDA-MB-468 and MDA-MB-231 cells were seeded in 96-well cell culture plates (Greiner) at a density of 20,000 cells per well in RPMI media (GibcoBRL) with 10% fetal sheep serum and L -glutamine at 20 mM. After 24 hours, the compounds were added to cell media at final concentrations ranging from 10 nM to 100 μM, and the cells were grown in the presence of the compounds for 48 hours in RMPI media containing 0.5% fetal sheep serum and 20 mM L-glutamine. The vial was determined using a cell proliferation assay MTT (Systems R and D) and by comparison with controls with DMSO alone (100% viability) Compounds that result in induced viability can act by inhibiting cell proliferation or inducing apoptosis ( programmed cell death.) Compounds representative of 096 structural groups within the library showed selective effects on cell proliferation and viability.The selected compounds were evaluated against paired breast cancer cell lines at concentration ranges to determine effective concentrations for the inhibition of growth.

EXAMPLE 11 Effects on Pl 3-K mediated signaling through PKB / Akt by Pl 3-K inhibitors Because the phosphorylation and activation of PKB / Akt is dependent on the activity of Pl 3-K, the inhibitors of Pl 3-K decrease cellular levels of phospho-Akt. MDA-MD-468 cells showed constitutively high levels of phospho-Akt as a result of abnormal activation of Pl 3 -K signaling. The effect of treatment with PI inhibitors 3-K on phospho-Akt levels in those cells was determined as follows. Cells were grown in 6-well cell culture plates at a density of 5 x 10 5 cells per well in RPMI media containing 10% fetal sheep serum and 2 mM L-glutamine. Twenty-four hours later, the media was removed and replaced with serum-free RPMI containing 2 mM L-glutamine. The cells were deprived of serum overnight. The compounds were diluted in serum-free media to a final concentration of 50 μM and added to the cells. The cells were incubated in the presence of Pl 3-K inhibitors for 4 hours. Phospho-Akt levels were determined using one of the following methods. To determine phospho-Akt levels using electro-immunoblotting, cells were washed twice with PBS and used in ice-cooled lysis buffer (1% Triton X-100, 50mM Hepes pH 7.4, 150mM NaCl, 1.5mM MgC12, EGTA ImM, 100mM NaF, 10 mM sodium pyrophosphate, Na (subscript: 3) VO (subscript: 4) lmM, 10% glycerol, phenylmethylsulfonyl fluoride lmM, and 10 μg / ml aprotinin). The total protein concentration was determined using a BCA assay. 30μg of total cell lysate protein was diluted in Laemmli sample buffer and loaded on a 10% acrylamide gel, subjected to SDS-PAGE, and transferred to a PVDF membrane. The membrane was blocked with 5% bovine serum albumin and then incubated at 4 ° C overnight with antibody. The membrane was washed with TBS-T (Tris-HCl lOmM pH 7.4, 150mM NaCl, and 0.1% Tween-20) and incubated with antibody conjugated with HRP (diluted in 5% milk in TBS-T) at room temperature for 1 h. The membrane was thoroughly washed and the proteins visualized by chemiluminescent detection. The effects of the compounds on the phospho-Akt levels were observed as relative differences in the amount of phosphor-Akt detected by electro-immunoblotting. The effects on phospho-Akt cell levels after treatment with Pl 3-K inhibitors were quantified using the phospho-Akt PathScan ELISA (Cell Signaling Technologies), a sandwich ELISA for the detection of phospho-Akt. The equipment was used according to the manufacturer's protocol. Absorbance at 450 nm was determined for each sample and used directly as equivalents of phospho-Akt levels. The percent decrease in phospho-Akt levels was determined by normalizing in relation to white samples (0%) and control samples treated with DMSO alone (100%). Treatment with Pl 3-K inhibitors resulted in a 20-60% decrease in phospho-Akt levels as determined by this assay. These data show that these compounds are capable of affecting cell signaling mediated by Pl 3-K. Table 3 summarizes the data of several compounds of this structural group, including the IC50 for the inhibition of the in vitro enzymatic activity, the cellular ICsom and the antiproliferative activity against altered tumor cells in the signaling mediated by Pl 3-K, and the effects-on the cellular levels of phospho-Akt.

Table 3. Summary of the data for compounds of this invention Example 12 Effects on the growth of tumor cells in 3-D culture systems by Pl 3-K inhibitors. Pl 3-K inhibitors are tested for their effects on the growth of tumor cells in a three-dimensional matrix that more closely resembles the environment of a tumor than other cell culture models. The MDA-MB-468 cells are mixed in a matrix solution, such as Matrigel (BD Biosciences) at 2 x 106 cells / ml and 100 μl of this mixture added to each well of a 24-well cell culture plate. Each well is 6.5 mm in diameter and 2xl05 cells are added per well. Once the matrix is solidified, the RMPI media containing 10% fetal sheep serum and 2 mM L-glutamine are added to each well. After approximately 14 days of culture, the compounds are added to the cell media at final concentrations ranging from lOnM to 100 μM, and the cells are allowed to grow in the presence of the compounds for 7 days in RMPI media containing 0.5% serum of fetal sheep and 20 mM L-glutamine. After this treatment, cell growth in the three-dimensional matrix can be measured using a cell viability assay such as the Cell Proliferation Assay in CellTiter 96 solution (Promega, G3582). 1.2 ml of test solution are added per well, the cells are incubated for 3 hours. The absorbance is determined at 550 nm for each well and is used directly as equivalent to the number of cells. In addition, living and dead cells can be distinguished and observed using fluorescence microscopy after staining with fluorescein diacetate (Sigma), which labels living cells, and propidium iodide (Sigma), which marks dead cells. . The Pl 3-K inhibitors of the present invention show antiproliferative effects in this tumor cell growth model, as shown by the representative data in Table 6, which compares the antiproliferative effects of an inhibitor compared to the effects of the laboratory PI 3-K inhibitor LY294002. The Pl 3-K inhibitors of the present invention also show improved anti-proliferative activity when combined with other cancer drugs, for example paclitaxel or doxorubicin.

Table 4. Effect of Pl 3-K inhibitors in a three-dimensional model of tumor cell growth.

Example 13 Inhibition of Tumor Growth The in vivo efficacy of a cancer cell growth inhibitor can be confirmed by several protocols well known in the art. Human tumor cells that are deregulated via the Pl 3-K pathway, for example, LnCaP, PC3, C33a, OVCAR-3, MDA-MB-468 are injected subcutaneously on the flank of hairless or hairless mice on day 0 The mice are assigned to a group with vehicles, with compound or combined treatment. The administration of the compound can begin on day 1-7.

Subcutaneous administration can be carried out daily or a day if and a day not during the duration of the experiment, or the compound can be provided by a continuous infusion pump. The size of the subcutaneous tumors can be verified through the course of the experiment. The tumors are excised and weighed at the conclusion of the experiment and the average weight of the tumors in each treatment group is calculated. Alternatively, cell lines such as OVCAR-3 can be injected intraperitoneally into the abdominal cavity of male nude mice. Subcutaneous, intravenous or intraperitoneal administration can be carried out daily or a day if and a day not during the duration of the experiment, or the compound can be provided by a continuous infusion pump. The tumors are excised and weighed at the conclusion of the experiment and the average weight of the tumors for each treatment group is calculated. Pl 3-K inhibitors show better activity against tumor growth when combined with other cancer drugs such as paclitaxel or doxorubicin. It should be understood that the arrangements referred to above are only illustrative of the application of the principles of the present invention. Numerous modifications and alternative arrangements may be contemplated without departing from the spirit and scope of the present invention. Although the present invention has been shown in the drawings and fully described above with particularity and detail in relation to what is currently considered to be the most practical and preferred embodiment of the invention, it will be apparent to those skilled in the art that numerous modifications can be made without departing from the principles and concepts of the invention as set forth in the claims.

Claims (19)

1. CLAIMS 1. Compound having a general structure represented by Formula I, Formula II, or Formula III;
2. Formula Formula II Formula where n is an integer selected from 0 to 2; Ri and P-2 are each independently a member selected from the group consisting of hydrogen, alkyl, alkenyl, aryl, hetaryl, aralkyl, hetaralkyl, alkyl substituted with at least one substituent, aryl substituted with at least one substituent, hetaryl substituted with at least one substituent, aralkyl substituted with at least one substituent, and hetaralkyl substituted with at least one substituent; R3 is a member selected from the group consisting of hydrogen, alkyl, alkenyl, aralkyl, alkyl substituted with at least one substituent, aralkyl substituted with at least one substituent, CO-R5, S02-R5; CO-O-R5, CO-N-R4, and R5; and R4 and R5 are each independently a member selected from the group consisting of hydrogen, alkyl, alkenyl, cycloalkyl, aralkyl, aryl, alkyl substituted with at least one substituent, cycloalkyl substituted with at least one substituent, aryl substituted with at least one substituent, and aralkyl substituted with at least one substituent. 2. Compound according to claim 1, with reference to R? _5, when the following are used; alkyl is a straight or branched chain C? _? alkyl; cycloalkyl is a C3_8 cycloalkyl, alkenyl is a straight or branched chain C2-? 8 alkenyl, aralkyl is a carbomiocyclic or aromatic carbobicyclic aromatic compound substituted with straight or branched chain C __? 5 alkyl; and the substituent is selected from the group consisting of nitro, hydroxy, cyano, carbamoyl, mono- or di-alkyl of C? _4-carbamoyl, carboxy, C? _4-carbonyl alkoxy, sulfo, halogen, C-alkoxy? -4, phenoxy, halo phenoxy, Cilt _4 alkylthio, mercapto, phenylthio, pyridylthio, C? ~ 4 alkylsulfinyl, C? _4 alkylsulfonyl, amino, C 1-3 alkanoylamino, mono- or di-alkylamino of C? _4, cyclic amino of 4 to 6 members, C ?_3 alkanoyl, benzoyl and 5- to 10-membered heterocyclic groups.
3. 3. Compound according to claim 1, with reference to R__5, when the following are used; aryl is an aromatic or aromatic carbobicyclic carbomonocyclic; hetaryl is an aromatic heteromonocyclic or heterobicyclic aromatic group containing from 1 to 6 heteroatoms selected from oxygen, sulfur and nitrogen; the aralkyl is aromatic carbobicyclic or aromatic carbobicyclic substituted with a linear or branched C? _? 5 alkyl chain; and the substituent is a member selected from the group consisting of halogen, C? _4 alkyl, C? _4 haloalkyl, C? _4 haloalkoxy, C1_4 alkoxy, C? _4 alkylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-alkylamino of C? _4, formyl, mercapto, C4-carbsnyl alkyl, C? _4-carbonyl alkoxy, sulfo, C1-4 alkylsulfonyl, carbamoyl, mono- or di-alkyl C? _ 4-carbamoyl, oxo and thioxo.
4. 4. The compound according to claim 1, wherein n is 1; Ri and R2 are each independently a member selected from the group consisting of hydrogen, straight or branched chain C? _6 alkyl, phenyl, naphthyl, hetaryl substituted with C? _g alkyl with at least one substituent, an alkylphenyl chain linear or branched C1-6, phenyl substituted with at least one substituent, benzyl, and benzyl substituted with at least one substituent; R3 is a member selected from the group consisting of hydrogen, C? _s alkyl, aralkyl, C? _6 alkyl substituted with at least one substituent, CO-R5, or S02-R5; CO-O-R5, CO-N-R4, and R5; R and R5 are each independently a member selected from the group consisting of hydrogen, C? _6 alkyl, C? -6 alkyl substituted with at least one substituent, cycloalkyl, phenyl and phenyl substituted with at least one substituent, aralkyl, benzyl and benzyl substituted with at least one substituent; and the substituent is a member selected from the group consisting of halogen, C alquilo ~4alkyl, C? _4 haloalkyl, C halo _haloalkoxy, C? _4alkoxy, C? ~ 4alkylthio, phenoxy, halofenoxy, phenylthio, pyridylthio, hydroxy, carboxy, cyano, nitro, amino , C alca _ alkanoylamino, C mono _ mono- or di-alkylamino, cyclic amino of 4 to 6 'members, formyl, mercapto, C? _4carbonyl, C? _4-carbonyl alkoxy, sulfo, C1-4 alkyl sulfinyl, C1-4 alkylsulfonyl, C3_3 alkanoyl, benzoyl, C7-4-carbamoyl mono- or di-alkyl, oxo, thioxo, a 5- to 10-membered heterocyclic compound.
5. 5. The compound according to claim 1, wherein n is 1, and with reference to R1-5, when the following are used; alkyl is C1-15 straight or branched chain, alkenyl is a straight or branched chain C2_? 8 alkenyl; aryl is a carbomiocyclic or aromatic carbobicyclic aromatic group; cycloalkyl can be an alkyl ring of C3_8, hetaryl is an aromatic heteromonocyclic or aromatic heterobicyclic compound containing from 1 to 6 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen; Aralkyl is carbomiocyclic or aromatic carbobicyclic aromatic alkyl and is substituted with straight or branched chain C 1 - 5 alkyl; hetaralkyl is a heteromonocyclic or heterobicyclic aromatic compound containing from 1 to 6 heteroatoms selected from the group consisting of oxygen, sulfur and nitrogen substituted with straight or branched chain C1-15 alkyl; and the substituent is a member selected from the group consisting of halogen, C? -4 alquiloalkyl, C halo-4 halo haloalkyl, C--. halo haloalkoxy, C-4-4 alco alkoxy, C-4-4 alqu alquiltiltiltiltiltiltiltiltiltiltilt,,,,,, fen fen fen fen fen fen fen fen fen, phenoxy, halofenoxy, phenylthio, pyridylthio, hydroxy, carboxy, cyano, nitro, amino, C? _3 alkanoylamino, C_-4 mono- or di-alkylamino, 4 to 6 membered cyclic amino, formyl, mercapto, C ?4-carbonyl alkyl , C ?4-carbonyl alkoxy, sulfo, C? _4 alkylsulfinyl, C? _4 alkylsulfonyl, C 1-3 alkanoyl, benzoyl, C 1 -4-carbamoyl mono- or di-alkyl, oxo, thioxo, a Heterocyclic compound of 5 to 10 members.
6. 6. The compound according to claim 1, wherein n is 1; Ri and R2 are each independently a member selected from the group consisting of straight or branched chain C? _6 alkyl, phenyl, benzyl, naphthyl, straight or branched chain C?-Alkyl substituted with at least one substituent; phenyl substituted with at least one substituent, and benzyl substituted with at least one substituent; R3 is a member selected from hydrogen, straight or branched chain C6 alkyl, C6-6 aralkyl and C6-6 alkyl substituted with at least one substituent; R4 and R5 are each independently a member selected from the group consisting of hydrogen, straight or branched chain C1-6alkyl, straight or branched chain C6-6alkyl substituted with at least one substituent, cycloalkyl, phenyl, phenyl substituted with at least one substituent, benzyl, and benzyl substituted with at least one substituent; and the substituent is a member selected from the group consisting of methyl, halogen, halophenyloxy, methoxy, ethyloxy phenoxy, benzyloxy, trifluoromethyl, t-butyl and nitro.
7. 7. The compound according to claim 1, wherein n is 1; Ri is a member selected from the group consisting of a straight or branched chain C 1-6 alkyl and phenyl; R 2 is a member selected from the group consisting of phenyl, C 1-6 alkylphenyl, C 1-6 dialkylphenyl, C 1-6 alkoxyphenyl, halophenyl, dihalophenyl and nitrophenyl; R3 is a member selected from hydrogen and straight or branched chain C__6 alkyl; R 4 is a phenyl substituted with at least one substituent selected from the group consisting of halogen, phenoxy, benzyloxy, halofenoxy, straight or branched chain C 1-6 alkyl, C 1 e alkoxy, halo C 1-4 alkyl; and R5 is straight or branched chain C6-6 alkyl.
8. 8. Compound according to claim 1, wherein n is 1; Ri is phenyl or t-butyl; R2 is a member selected from the group consisting of methylphenyl, dimethylphenyl, t-butyl, methoxyphenyl, chlorophenyl, dichlorophenyl, fluoro phenyl, and nitrophenyl; R3 is hydrogen; R 4 is a phenyl substituted with at least one substituent selected from the group consisting of chloro, fluoro, phenoxy, benzyloxy, chlorophenoxy, methoxy, ethoxy, and trifluoromethyl; and R5 is a methyl.
9. 9. A compound according to any of claims 1 to 8, wherein the compound has an IC50 of less than 10 μM in in vitro inhibition of Pl .3-K activity or an IC50 of less than 20 μM in the cellular inhibition of the activity of Pl 3-K.
10. 10. A pharmaceutical composition comprising the compound or a salt thereof according to any of claims 1 to 8 and a pharmaceutically acceptable excipient or carrier.
11. 11. Method for separating and characterizing the potency of a test compound as an inhibitor of the phosphatidylinositol 3-kinase (Pl 3-K) polypeptide, the method comprising the steps of (a) measuring the activity of a Pl 3-K polypeptide in presence of a test compound according to one of claims 1 to 8; (b) comparing the activity of the Pl 3-K polypeptide in the presence of the test compound with the activity of the Pl 3 -K polypeptide in the presence of an equivalent amount of a Pl 3-K inhibitor known as the reference compound, where the lower activity of the Pl 3-K polypeptide in the presence of the test compound which in the presence of the reference compound indicates that the test compound is a more potent inhibitor than the reference compound, and a higher activity of the Pl-3 polypeptide K in the presence of the test compound which in the presence of the reference compound indicates that the test compound is a less potent inhibitor than the reference compound.
12. 12. Use of a compound or a salt thereof according to claims 1 to 8, for preparing a pharmaceutical composition for treating a disorder in which Pl 3-K plays a role.
13. 13. Use according to claim 12, wherein the disorder is a cancer or an immunity disease "and inflammation
14. 14. Use according to claim 12, wherein the disorder is the disturbance of the function of Pl 3-K in leukocytes.
15. 15. Method for inhibiting the growth of cancer cells, comprising contacting the cancer cells with an effective amount of the compound or the salt thereof according to any of the claims 1 to 8. The method according to claim 15, wherein the cancer cells are altered in Pl 3 -K-mediated signaling via the PTEN mutation, amplification of the PIK3CA gene or mutations in the Pl 3-kinase. The method of claim 15, wherein the cancers include breast, prostate, colon, lung, ovarian and other cancers that have altered 3-K Pl activities. 18. Method for effecting Pl 3 -K mediated signaling in cells, comprising contacting the cells with an effective amount of the compound or a salt thereof according to any of claims 1 to 8. 19. Method according to claim 18 , wherein the compound affects the phosphorylation mediated by Pl 3 -K Akt.