WO2005002514A2

WO2005002514A2 - Compounds having inhibitibe activity of phosphatidylinositol 3-kinase and methods of use thereof

Info

Publication number: WO2005002514A2
Application number: PCT/US2004/019131
Authority: WO
Inventors: Beth E. Drees; Leena Chakravarty; Glenn D. Prestwich; Gyorgy Dorman; Mariann Kavecz; Andras Lukacs; Laszlo Urge; Ferenc Darvas
Original assignee: Echelon Biosciences Incorporated; Comgenex, Rt
Priority date: 2003-06-13
Filing date: 2004-06-14
Publication date: 2005-01-13
Also published as: WO2005002514A3

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 growth or to treat disorders of immunity and inflammation, in which PI 3-K plays a role in leukocyte function are also provided.

Description

COMPOUNDS HAVING INHIBITIVE ACTIVITY OF PHOSPHATIDYLINOSITOL 3-KINASE AND METHODS OF USE THEREOF

BACKGROUND OF THE INVENTION Field of the Invention The present invention relates generally to phosphatidylinositol 3-kinase (PI 3-K) enzymes, and more particularly to inhibitors of PI 3-K activity and to methods of using such materials. Related Art The behavior of all cellular communications is governed by signaling systems which translate external signals such as hormones, neurotransmitters, and growth factors into intracellular second messengers. Phosphoinositide polyphosphates (PIPn) are key lipid second messengers in cellular signaling (Martin, Ann. Rev. Cell Dev. Biol., 14:231- 2614 (1998)). Because their activity is determined by their phosphorylation state, the enzymes that modify these lipids are central to the correct execution of signaling events (Leslie, et al., Chem Rev, 101:2365-80. (2001)). Disruptions in these processes are common to many disease states, including cancer, diabetes, inflammation, and cardiovascular disease. The production of the phosphoinositide polyphosphate PI(3,4,5)P₃ or PIP₃ by phosphatidylinositol 3-kinase (PI 3-K) is important in pathways governing cell proliferation, differentiation, apoptosis, and migration. Alterations which affect 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 which affect the regulation of PI 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 growth factor stimulation, PI 3-K catalyzes the formation of PIP₃. By increasing cellular levels of PIP₃, PI 3-K induces the formation of defined molecular complexes that act in signal transduction pathways. Most notably, PI 3-K activity 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. (1996)). The lipid phosphatases PTEN and SHIP are two enzymes that both act to decrease the cellular levels of PIP₃ by conversion either to PI(4,5)P₂ or PI(3,4)P₂.

Presently, the PI 3-kinase enzyme family has been divided into three classes based on their substrate specificities. Class I PI 3-Ks can phosphorylate phosphatidylinositol (PI), phosphatidylinositol-4-phosphate, and phosphatidylinositol- 4,5-biphosphate (PIP2) to produce phosphatidylinositol-3-phosphate (PIP), phosphatidylinositol-3,4-biphosphate, and phosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI 3-Ks phosphorylate PI and phosphatidylinositol-4-phosphate, whereas Class III PI 3-Ks can only phosphorylate PL Eight separate isoforms of PI 3-K have been characterized in humans.

The initial purification and molecular cloning of PI 3-kinase revealed that it was a heterodimer consisting of p85 and pi 10 subunits (Otsu et al., Cell, 65:91-104 (1991); Hiles et al., Cell, 70:419-29 (1992)). Since then, four distinct Class I PI 3-Ks have been identified, designated PI 3-K alpha, beta, delta, and gamma, each consisting of a distinct 110 kDa catalytic subunit and a regulatory subunit. More specifically, three of the catalytic subunits, i.e., pi 10 alpha, pi 10 beta and pi 10 delta, each interact with the same regulatory subunit, ρ85; whereas pi 10 gamma interacts with a distinct regulatory subunit, plOl. In each of the PI 3-Kinase alpha, beta, and delta subtypes, the p85 subunit acts to localize PI 3-kinase to the plasma membrane by the interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins Two isoforms of p85 have been identified, p85 alpha, which is ubiquitously expressed, and p85 beta, which is primarily found in the brain and lymphoid tissues. Association of the p85 subunit to the PI 3-kinase pi 10 alpha, beta, or delta catalytic subunits appears to be required for the catalytic activity and stability of these enzymes. In addition, the binding of Ras proteins also upregulates PI 3-kinase activity. Though a wealth of information has been accumulated in recent past on the cellular functions of PI 3 -kinases in general, in particular for PI 3-K alpha and PI 3-K gamma, the roles played by the individual isoforms are have yet to be clearly defined. Details concerning the pi 10 isoform also can 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. Experimental usage of PI

3-K inhibitors has contributed to the current understanding of the role of PI 3-K activity in normal function and in disease. The major pharmacological tools used in this capacity are wortmannin (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 (Nlahos, et al., J Biol Chem, 269:5241-8. (1994)).

The concentrations of wortmannin needed to inhibit PI 3-Ks range from 1-100 nM, and inhibition occurs via covalent modification of the catalytic site (Wymann et al., Mol. Cell. Biol. 16:1722-1733. (1996)). The bioflavenoid quercetin effectively inhibits PI 3-K with an IC₅o of 3.8 μM, but has poor selectivity, as it also shows inhibitory activity toward PI 4-kinase, and several protein kinases. LY294002 is a synthetic compound made using quercetin as a model, inhibits PI 3-K with an IC₅o of 100 μM (Nlahos, et al., J Biol Chem, 269:5241-8. (1994)). Both quercetin and LY294002 are competitive inhibitors of the ATP binding site of PI 3-K, however, only LY294002 shows specificity for inhibition of PI 3-K and does not affect other types of kinases. Both wortmannin and LY294002 have been used extensively to characterize the biological roles of PI 3-K, however, neither shows selectivity for individual PI 3-K isoforms. Hence, the utility of these compounds in studying the roles of individual Class I PI 3-kinases is limited. The PI 3-K inhibitors are expected to be a new type of medication useful for cell proliferation disorders, in particular as antitumor agents. As PI 3-K inhibitors, wortmannin [H. Yano et al., J. Biol. Chem., 263, 16178 (1993)] and LY294002 [J. Nlahos et al., J. Biol. Chem., 269, 5241(1994)] which is represented by the formula below, are known. However, creation of PI 3-K inhibitors having more potent cancer cell growth inhibiting activity is desired. Because many oncogenic signaling pathways are mediated by PI 3-K, inhibitors that target PI 3-K activity may have application for the treatment of cancer. Studies using comparative genomic hybridization revealed several regions of recurrent abnormal DΝA sequence copy number that may encode genes involved in the genesis or progression of ovarian cancer. One region found to be increased in copy number in approximately 40% of ovarian and other cancers contains the PIK3CA gene, which encodes the pi 10 alpha catalytic subunit of PI 3-K alpha. This association between the PIK3CA copy number and PI 3-kinase activity makes PIK3CA a candidate oncogene because a broad range of cancer-related functions have been associated with PI 3-kinase- mediated signaling. PIK3CA is frequently increased in copy number in ovarian cancers, and increased copy number is associated with increased PIK3CA transcription, pi 10- alpha protein expression, and PI 3-kinase activity (Shayesteh, et al., Nature Genet. 21 : 99-102, (1999)). Furthermore, treatment of ovarian cancer cell lines exhibiting increased

PI 3-K activity and Akt activation with a PI 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, PI 3-K alpha has an important role in ovarian cancer. In cervical cancer cell lines harboring amplified PIK3CA, the expression of the gene product was increased and was associated with high PI 3-kinase activity (Ma et al., Oncogene 19: 2739-2744, (2000)). Thus, increased expression of PI 3-kinase alpha in cervical cancer may promote cell proliferation and reduce apoptosis. In addition, mutation of the lipid phosphatase and tumor suppressor PTEN, a 3' phosphatase that breaks down 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)). PI 3-K activity suppresses apoptosis and promotes cell survival largely 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)). Akt activation and amplification is present in many cancers (Testa and Bellicosa, Proc. Natl. Acad. Sci. USA 98:10983-10985. (2002)). Treatment with PI 3-K inhibitors has been shown to block 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 PI 3-K inhibitors causes proliferative arrest in these cells (Brognard et al., Cancer Res. 60:6353-6358. (2000), Lee et al, J. Biol. Chem. electronic publication, (2003)). The PI 3-K/ Akt pathway is also constitutively activated in a majority of human pancreatic cancer cell lines, and treatment with PI 3-K inhibitors induced apoptosis in these cell lines. Decreased tumor growth and metastasis was also observed upon treatment with PI 3-K inhibitors in a xenograft model of pancreatic cancer (Perugini et al., J. Surg. Res. 90:39-44 (2000), Bondar et al., Mol. Cancer Ther. 1:989- 997 (2002)). Treatment with LY204002 induced growth arrest and apoptosis in PTEN- deficient human malignant glio a cells (Shingu et al., J. Neurosurg. 98:154-161.

(2003)). LY294002 produces 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)). Inhibitors of PI 3-K inhibit in vitro anchorage- independent growth and 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 also has been shown to induce apoptosis in multi-drug resistant cells (Nicholson et al., Cancer

Lett. 190:31-36. (2003)). Thus, PI 3-K inhibitors maybe suitable therapeutics agents for many tumors exhibiting activated or increased levels of PI 3-K or PKB/Akt as well as for tumors which are PTEN-deficient. Several studies have demonstrated that agents which target the PI 3-K pathway can enhance the effects of standard chemotherapeutic agents in a variety of cancer types. Thus, PI 3-K inhibitors may have value as novel adjuvant therapies for certain cancers. PI 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 PI 3-K also increases the responsiveness of pancreatic carcinoma cells to the non-steroidal anti-inflammatory agent (NSAID) sulindac (Yip-Schneider, et al, J

Gastrointest Surg, 7:354-63. (2003)). hi a mouse xenograft model of pancreatic cancer, a combination of wortmannin with gemcitabine also showed increased efficacy in induction of tumor apoptosis relative to treatment with each agent alone (Ng, et al., Clin Cancer Res, 7:3269-75. (2001)). In an athymic mouse xenograft model of ovarian cancer, combined treatment with LY294002 and paclitaxal results in increased efficacy of paclitaxal-induced apoptosis of tumor cells, and allows the use of decreased levels of LY294002, resulting in less dermatological toxicity (Hu, et al., Cancer Res, 62:1087-92. (2002)). 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 PI 3-K inhibition in treating drug resistant acute myeloid leukemia (O'Gorman, et al., Leukemia, 14:602-11. (2000, O'Gorman, et al, Leuk Res, 25:801-11. (2001)). Inhibition of PI 3-K enhances the apoptotic effects of sodium butyrate, gemcitabine, and 5-fluoruracil in aggressive colon cancer cell lines (Wang, et al., Clin Cancer Res, 8:1940-7. (2002)). LY294002 potentiates apoptosis induced by doxorubicin, trastumazab, paclitaxal, tamoxifen, and etoposide in breast cancer cell lines exhibiting PTEN mutations or erbB2 overexpression (Clark, et al., Mol Cancer Ther, 1:707-17. (2002)). Inhibition of PI 3-K potentiates the effect of etoposide to induce apoptosis in small cell lung cancer cells (Krystal, et al., Mol Cancer Ther, 1:913-22. (2002)). In addition to enhancing the effects of chemotherapeutic agents for cancer treatment, PI 3-K inhibitors also may enhance tumor response to radiation treatment. Inhibitors of PI 3-K revert 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 enhance radiation-induced apoptosis and cytotoxicity in tumor vascular endothetial cells (Edwards, et al., Cancer Res, 62:4671-7. (2002)). Thus, PI 3-K inhibitors could be used to enhance response to radiotherapy, both in tumor cells and in tumor vasculature. US Patent No. 6,403,588 discloses imidazopyridine derivatives having excellent PI 3-K inhibiting activity and cancer cell growth inhibiting activity. US Patent No.

5,518,277 discloses compounds that inhibit PI 3-K delta activity, including compounds that selectively inhibit PI 3-Kdelta activity. However, all of these compounds have a structure different from those of the present invention.

SUMMARY OF THE INVENTION It has been recognized that it would be advantageous to develop inhibitors of PI 3-K polypeptides. In particular, inhibitors of PI 3-K are desirable for exploring the roles of PI 3-K isozymes and for development of pharmaceuticals to modulate the activity of the isozymes. One aspect of the present invention is to provide compounds that can inhibit the biological activity of human PI 3-K alpha. Another aspect of the invention is to provide compounds that inhibit other PI 3-K isoforms, including PI 3-K beta, gamma and delta. Another aspect of the invention is to provide methods of synthesizing and using these PI 3-K inhibitors. One embodiement of the present invention provides a compound which is useful as a phosphatidylinositol 3-kinase (PI 3-K) inhibitor having a general structural of formulas I and II:

Formula I Formula II

wherein R\ can be selected from the group consisting of CH=CH-R₅, CO-OR₅, aryl, hetaryl, substituted aryl and substituted hetaryl groups; R₂ and R₃ can be each independently selected from the group consisting of alkyl, substituted alkyl, aryl, hetaryl, aralkyl, substituted aryl, and substituted hetaryl groups; can be selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O-R₅, CO-N-R₆, R₅, alkyl, aralkyl, and cycloalkyl; R₅ can be selected from the group consisting of H, alkyl, aryl, hetaryl, substituted aryl and substituted hetaryl groups; and R₆ can be selected from the group consisting of H, aryl, hetaryl, substituted aryl and substituted hetaryl groups. hi another embodiment of the present invention, wherein

can be a C_1-15 alkylester or C _2-18 alkenyl. With reference to R_1-6 independent, when ever used said alkyl can be a straight or branched chain C_1-15 alkyl or C _2-18 alkenyl; said substituted alkyl can be a straight or branched chain C_1-15 alkyl or C _2-18 alkenyl substituted by 1 to 5 substituents selected from the group consisting of nitro, hydroxy, cyano, carbamoyl, mono- or di-C_1- alkyl-carbamoyl, carboxy, C_1- alkoxy-carbonyl, sulfo, halogen, C _1-4 alkoxy, phenoxy, halophenoxy, C_1-4 alkylthio, mercapto, phenylthio, pyridylthio, C_1-4 alkylsulfinyl, Cμ alkylsulfonyl, amino, C_1-3 alkanoylamino, mono- or di-C_1-4 alkylamino, 4- to 6-membered cyclic amino, C_1-3 alkanoyl, benzoyl, and 5- to 10- membered heterocyclic groups; said aryl can be a carbomonocyclic aromatic or carbobicyclic aromatic group; said hetaryl can be a heteromonocyclic aromatic or heterobicyclic aromatic moiety containing 1 to 4 hetero-atoms selected from oxygen, sulfur and nitrogen; said aralkyl can be a carbomonocyclic aromatic or carbobicyclic aromatic substituted with a straight or branched chain C_1-15 alkyl or C _2-18 alkenyl group; said substituted aryl can be an carbomonocyclic aromatic or carbobicyclic aromatic substituted with a substituted by 1 to 4 substituents selected from the group consisting of halogen, C_1-4 alkyl, Cι_-4 haloalkyl, C_1-4 haloalkoxy, C_1-4 alkoxy, C_1-4 alkylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C_1-4 alkylamino, formyl, mercapto, C_1-4 alkyl- carbonyl, C_1-4 alkoxy-carbonyl, sulfo, C_1-4 alkylsulfonyl, carbamoyl, mono- or di-C_1-4 alkyl-carbamoyl, oxo and thioxo; and said substituted hetaryl can be a heteromonocyclic aromatic or heterobicyclic aromatic containing 1 to 6 hetero-atoms selected from oxygen, sulfur and nitrogen, each of which can be substituted with 1 to 4 substituents selected from the group consisting of halogen, C_1-4 alkyl, C_1-4 haloalkyl, Cμ₄ haloalkoxy, C_1-4 alkoxy, C_1- alkylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-Cι_-4 alkylamino, formyl, mercapto, C_1-4 alkyl-carbonyl, C_1-4 alkoxy-carbonyl, sulfo, C_1-4 alkylsulfonyl, carbamoyl, mono- or di-C_1-4 alkyl-carbamoyl, oxo and thioxo groups. In another embodiment, the R_\ can be an C_1-6 alkylester or C _2-6 alkenyl; R₂ and R₃ can be each independently selected from the group consisting of straight or branched chain C_1-4 alkyl, a 5- or 6-membered heteromonocylic aromatic having at least one sulphur hetero-atom, a 5- or 6-membered heteromonocyclic aromatic having at least one sulphur hetero atom and substituted with at least one substituent, a 5- or 6-membered heteromonocylic aromatic having at least one oxygen hetero-atom, 5- or 6-membered heteromonocyclic aromatic having at least one oxygen hetero atom and substituted with at least one substituent, a 5- or 6-membered heteromonocylic aromatic having at least one nitrogen hetero-atom, a 6- membered carbomonocyclic aromatic fused with a 5- membered carbomonocyclic ring, a 6-membered carbomonocyclic aromatic fused with a substituted 5-membered carbomonocyclic ring and substituted with at least one substituent, bicycloheptenyl, a 5- to 7-membered carbomonocyclic aromatic, a 5- to 7- membered carbomonocyclic aromatic substituted with at least one substituent, a 6- membered heteromonocyclic aromatic having at least one nitrogen hetero-atom fused with a 5-membered heteromonocylic aromatic having at least one nitrogen hetero-atom, a 6-membered heteromonocyclic aromatic having at least one nitrogen fused with a 5- membered heteromonocyclic aromatic and substituted with at least one substituent, and C_1- alkylester substituted 5- to 7-membered carbomonocyclic aryl C_1-4 alkyl; I can be selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O-R₅, CO-N-R₆; R₅ can be selected from the group consisting straight or branched chain C^o alkyl, C_1-8 alkenyl substituted with at least one substituent, a 5- or 6-membered heteromonocylic aromatic having at least one sulphur hetero-atom, a 5- or 6-membered heteromonocylic aromatic having at least one oxygen hetero-atom, a 5- or 6-membered heteromonocyclic aromatic having at least one oxygen hetero atom and substituted with at least one substituent, a 5- or 6-membered heteromonocylic aromatic having at least one nitrogen hetero-atom fused with a 5- or 6- membered carbomonocyclic aromatic, a 6- membered carbomonocyclic aromatic fused with a 6-membered carbomonocyclic aromatic, a camphoryl, a camphoryl substituted with at least one substitutent, a 5- to 7-membered carbomonocyclic aromatic, a 5- to 7-membered carbomonocyclic aromatic substituted with at least one substituent, a 5- or 6- membered heteromonocyclic aromatic having at least one nitrogen hetero-atom, C_1-4 alkylester substituted 5- to 7-membered carbocyclic aryl C_1- alkyl, phenylcyclo-C_3-6 alkyl, C_1- alkyl-carbamoylphenyl; alkyl-alkylate, and phenylalkenyl substituted with at least one substituent; and R₆ can be a 5- to 7-membered carbomonocyclic aromatic or a substituted 5- to 7-membered carbomonocyclic aromatic. Any of the moieties described herein can be substituted with a substituent that can be selected from the group consisting of halogen, C_1-4 alkyl, C_1-4 haloalkyl, C_1-4 haloalkoxy, C_1-4 alkoxy, C_1-4 alkylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C_1-4 alkylamino, formyl, mercapto, C_1-4 alkyl-carbonyl, C_1-4 alkoxy-carbonyl, sulfo, Cι_-4 alkylsulfonyl, carbamoyl, mono- or di-C_1-4 alkyl-carbamoyl, oxo and thioxo groups. In an other embodiment, R_\ can be a C_1-6 alkylester or C _2-6 alkenyl; R₂ and R₃ can be each independently selected from the group consisting of straight or branched chain C_1-6 alkyl, thiophenyl, substituted thiophenyl, indenyl, substituted indenyl, bicyclo 2.2.1. heptenyl, phenyl, substituted phenyl^* pyridinyl, furanyl, substituted furanyl, pyrazolopyramidine, substituted pyrazolopyramidine, and C_1-6 alkylester substituted benzyl; can be selected from the group consisting of CO-R5, or SO₂-R₅; CO-O-R5, CO-N-R₆; R₅ can be selected from the group consisting of phenyl, substituted phenyl, benzyl, substituted benzyl, furanyl, substituted furanyl, diphenyl-C_1-6 alkyl, camphor, substituted camphor, substituted phenylalkenyl, thiophenyl, substituted thiophenyl, ρhenoxy-C_1-6 alkyl, phenylthio-C_1-6 alkyl, C_1-6 alkyl-

alkylate, phenylcyclo-C_3-6 alkyl, quinolinyl, naphthaline, and acetylamide phenyl; and R₆ can be phenyl or substituted phenyl. In another embodiment, R\ can be a C_1-3 alkylester or C _2- alkenyl. With respect to R₂ and R₃, the substituted thiophenyl, substituted indenyl, substituted phenyl, substituted furanyl, and substituted pyrazolopyramidine can be each independently substituted with at least one substituent selected from the group consisting of halogen, straight or branched chain CM alkyl. With respect to R₅, the substituted phenyl, substituted benzyl, substituted furanyl, substituted camphor, substituted phenyl- Cι_-4 alkenyl, and substituted thiophenyl, can be each independently substituted with at least one substituent selected from the group consisting of halogen, straight or branched chain C _O alkyl, C_1-4 haloalkyl, C_1-4 alkoxy, nitro, phenoxy-C_1-4 alkyl. Additionally, R₆ can be phenyl substituted with at least one C_1-4 alkoxy. In yet another embodiment, R_\ can be an ethyl ester or an ethylenyl; R₂ and R₃ can be each independently selected from the group consisting of methyl, ethyl, thiophenyl, chlorothiophenyl, bromothiophenyl, dimethyl-tertbutyl indenyl, bicyclo 2.2.1. heptenyl, phenyl, chlorophenyl, flurophenyl, pyridinyl, furanyl, methylfuranyl, dimethylpyrazolopyramidine, methylpropylesterbenzyl; R can be an CO-R₅ or SO₂-R₅; R₅ can be selected from the group consisting of an methoxyphenyl, dimethoxyphenyl, pentanylphenyl, chlorothiophenyl, furanyl, methylphenyl, diphenylmethyl, chlorophenyl, dichlorophenyl, flurophenyl, bromophenyl, butylesterphenyl, dimethylcamphor, phenylethylenyl, octanyl, tertbutylphenyl, trifluromethylphenyl, di-trifluromethylphenyl, nitrochlorophenyl, butylphenyl, naphthyl, nitrophenylethylenyl, acetylamide-phenyl, nitromethylphenyl, phenylthiomethyl, phenylcyclopentyl, quinolinyl, thiophenyl, dinitrophenyl, ethylbutyrate, and chloropropyl. In one aspect, only one of R₂ and R₃ is an aromatic. The present invention further relates to novel pharmaceutical compositions, particularly to PI 3-K inhibitors and antitumor agents, comprising a compound of the present invention and a pharmaceutically acceptable carrier. A further aspect of the present invention relates to treatment methods of disorders (especially cancers) influenced by PI 3-K, wherein an effective amount of a compound of the present invention is administered to humans or animals. Additional features and advantages of the invention will be apparent from the detailed description which follows, taken in conjunction with the accompanying drawings, which together illustrate, by way of example, 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 the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended. Alterations and further modifications of the inventive features illustrated herein, and additional applications of the principles of the inventions as illustrated herein, which would occur to one skilled in the relevant art and having possession of this disclosure, are to be considered within the scope of the invention. An aspect of the present invention relates to novel compounds which are useful as PI 3-K inhibitors and antitumor agents. The compounds of the present invention are represented by the general formulas I and II:

Formula I Formula II

In accordance therewith, Ri can be a member selected from the group consisting of CH=CH-R₅, CO-OR₅, aryl, hetaryl, substituted aryl and substituted hetaryl groups; additionally, Ri can be hydrogen, alkyl, substituted alkyl, sulfonyl, substituted sulfonyl, carbonyl, ketyl, aralkyl, substituted aralkyl, CO-NHR₅, -O-CO-R₅, -OH, OR₅, etc. R₂ and R₃ can each be independently selected from the group consisting of alkyl, aryl, hetaryl, and aralkyl groups. -R₄ can be selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O-R₅, CO-N-R₆, R₅, -NH-CO-R₅, NH-R₅, -NR₅R_δ, -O-R₅, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkyl. R₅ can be a member selected from the group consisting of H, alkyl, aryl, and hetaryl groups. R₆ can be a member selected from the group consisting of H, aryl, and hetaryl groups. Optionally, each of the aforementioned Ri-Re groups is independently substituted with at least one substituent. In an additional optional aspect, only one of the R₂ and R₃ groups can be aromatic. As such, with reference to R_1-6 independently can be various moieties. In one aspect, any of the alkyl moieties can be a straight or branched chain CM_S alkyl. In another aspect, any of the alkenyl moieties can be a straight or branched chain C _2-18 alkenyl. In still another aspect, any of the aryl moieties can be a carbomonocyclic aromatic or carbobicyclic aromatic group. In yet another aspect, any of the hetaryl moieties can be a heteromonocyclic aromatic or heterobicyclic aromatic moiety containing 1 to 4 hetero-atoms selected from oxygen, sulfur and nitrogen. As such, only one of the aromatic rings in the bicylic ring system has to include a hetero atom, but optionally, both rings can include hetero-atoms. In an additional aspect, any of the aralkyl moieites can be a carbomonocyclic aromatic or carbobicyclic aromatic substituted with a straight or branched chain C_1-15 alkyl or C _-18 alkenyl group. In a further aspect, any of the moieties can be substituted with at least one substituent that can be a member selected from the group consisting of halogen, C_1-4 alkyl, C_1-4 haloalkyl, C_\. ₄ haloalkoxy, C_1-4 alkoxy, C_1-4 alkylthio, phenylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C_1-4 alkylamino, formyl, mercapto, C_1-4 alkyl-carbonyl, C_1-4 alkoxy- carbonyl, sulfo, C_1- alkylsulfonyl, carbamoyl, mono- or di-C_1-4 alkyl-carbamoyl, oxo and thioxo groups. In another embodiment, R_\ can be a member selected from the group consisting of CO-O-R₅, C ₂-₆ alkenyl, and carbomonocyclic aromatic. Also, the R₂ and R₃ can be each independently selected from the group consisting of straight or branched chain Cι_-4 alkyl, sulphur containing heteromonocyclic aromatic, sulphur containing heteromonocyclic aromatic substituted with at least one substituent, oxygen containing heteromonocyclic aromatic, oxygen containing heteromonocyclic aromatic substituted with at least one substituent, nitrogen containing heteromonocyclic aromatic, 6-membered carbomonocyclic aromatic fused with a 5-membered carbomonocyclic ring, 6-membered carbomonocyclic aromatic fused with a 5-membered carbomonocyclic ring and substituted with at least one substituent, bicycloheptenyl, carbomonocyclic aromatic, carbomonocyclic aromatic substituted with at least one substituent, 6-membered nitrogen containing heteromonocyclic aromatic fused with a 5-membered nitrogen containing heteromonocylic aromatic, 6-membered nitrogen containing heteromonocyclic aromatic fused with a 5-membered nitrogen containing heteromonocyclic aromatic and substituted with at least one substituent, and straight or branched chain C_1-4 alkoxybenzyl.

Additionally, j can be a member selected from the group consisting of CO-R₅, or SO - R₅; CO-O-R₅, CO-N-R₆,. Further, R₅ can be a member selected from the group consisting of straight or branched chain C_1-9 alkyl, straight or branched chain C_1-9 alkyl substituted with at least one substituent, C_2- alkenyl substituted with at least one substituent, sulphur containing heteromonocylic aromatic, oxygen containing heteromonocylic aromatic, oxygen containing heteromonocyclic aromatic substituted with at least one substituent, 5- or 6-membered nitrogen containing heteromonocylic aromatic fused with a carbomonocyclic aromatic, carbomonocyclic aromatic fused with a carbomonocyclic aromatic, camphoryl, camphoryl substituted with at least one substitutent, carbomonocyclic aromatic, carbomonocyclic aromatic substituted with at least one substituent, 5- or 6 membered nitrogen containing heteromonocyclic aromatic,

C_1-4 alkoxybenzyl, phenylcyclo-C₃.₆ alkyl, C_M alkyl-carbamoylphenyl; CM alkyl- -₆ alkylate, and phenylalkenyl substituted with at least one substituent. Furthermore, R₆ can be a carbomonocyclic aromatic or a carbomonocyclic aromatic substituted with at least one substituent. In accordance with the moieties described herein, any of the moieties can be substituted with a substituent selected from the group consisting of halogen, C_M alkyl, C_M haloalkyl, C_1-4 haloalkoxy, C_1-4 alkoxy, Cι_-4 alkylthio, phenylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C_1-4 alkylamino, formyl, mercapto, C_1-4 alkyl-carbonyl, C_1-4 alkoxy-carbonyl, sulfo, C_1-4 alkylsulfonyl, carbamoyl, mono- or di-C_1-4 alkyl-carbamoyl, oxo and thioxo groups. In another embodiment of the present invention, R_t can be selected from the group consisting of CO-O-R_5; C _2-4 alkenyl and phenyl. In one aspect, R₂ and R₃ can be each independently selected from the group consisting of straight or branched chain Cι- alkyl, thiophenyl, thiophenyl substituted with at least one substituent, indenyl, indenyl substituted with at least one substituent, bicyclo 2.2. L heptenyl, phenyl, phenyl substituted with at least one substituent, pyridinyl, furanyl, furanyl substituted with at least one substitutent, pyrazolopyramidine, pyrazolopyramidine substituted with at least one substituent, and C_1-6 alkyloxybenzyl. In another aspect, Rj can be selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O-R₅, CO-N-R₆. In yet another aspect, R₅ can be selected from the group consisting of straight or branched chain C_1-9 alkyl, straight or branched chain C_1-9 alkyl substituted with at least one substituent, phenyl, phenyl substituted with at least one substituent, benzyl, benzyl substituted with at least one substituent, furanyl, furanyl substituted with at least one substitutent, diphenyl-C_1-6 alkyl, camphor, camphor substituted with at least one substituent, phenylalkenyl substituted with at least one substitutent, thiophenyl, thiophenyl substituted with at least one substituent, phenylthio-CH₂-, C alkyl- CM alkylate, phenylcyclopropyl, C_1-4 alkoxy, quinolinyl, naphthalenyl, and acetylamidylphenyl. In still another aspect, R₆ can be phenyl or phenyl substituted with at least one substituent. In accordance with any of the aspects described herein, the moieties can each be individually substituted with any of a variety of substituents. As such, the R₂ and R₃ substituents can be each independently selected from the group consisting of halogen and straight or branched chain C alkyl. Also, the R₅ substituents can be selected from the group consisting of halogen, straight or branched chain CM_O alkyl, C haloalkyl, Cι-₄ alkoxy, trifluro C alkyl, C alkylamidyl, nitro, phenyl, phenoxy-C_M alkyl.

Additionally, the R₆ substituent can be C_M alkoxy. In a detailed embodiment, each of the R_1-6 moieties can be varied accordingly to obtain the desired physiological functionalities described herein. In accordance therewith, R_\ can be an ethyl ester, phenyl, or an ethylenyl. In one aspect, R₂ and R₃ can be each independently selected from the group consisting of methyl, ethyl, thiophenyl, chlorothiophenyl, bromothiophenyl, dimethyl t-butyl indenyl, bicyclo 2.2. L heptenyl, phenyl, chlorophenyl, flurophenyl, pyridinyl, furanyl, methylfuranyl, dimethylpyrazolopyramidme, and methylpropylesterbenzyl. In another aspect, can be an CO-R₅ or SO₂-R₅. In yet another aspect, R₅ can be selected from the group consisting of methyl, ethyl, methoxyphenyl, dimethoxyphenyl, pentanylphenyl, chlorothiophenyl, furanyl, methylphenyl, diphenylmethyl, chlorophenyl, dichlorophenyl, flurophenyl, bromophenyl, butyloxyphenyl, dimethylcamphor, phenylethylenyl, octanyl, t- butylphenyl, trifluromethylphenyl, di-trifluromethylphenyl, nitrochlorophenyl, butylphenyl, naphthalinyl, nitrophenylethylenyl, acetylamidylphenyl, nitromethylphenyl, phenylthiomethyl, phenoxymethyl, phenylcyclopentanyl, quinolinyl, thiophenyl, dinitrophenyl, isobutyloxybenxyl, ethylpropylate, benzyloxymethyl, and chloropropyl. In a further aspect, R₆ can be a phenyl or dimethoxyphenyl. In an alternative embodiment, Ri and R , when combined, can form a ring structure which is a member selected from the group consisting of saturated or partially saturated alicyclic or heterocyclic ring systems R₂ and R₃ are each independently a member selected from the group consisting of alkenyl, alkynyl, NH-CO-, NH-R₅, - NR₅Re, -O-R₅, alkyl, substituted alkyl, aryl, aralkyl, substituted aryl, and substituted hetaryl groups; R₂ and R₃, when combined, can form a ring structure which is a member selected from the group consisting of saturated hydrocarbons (-(CH₂)_n-) or partially or fully unsaturated hydrocarbones wherein (-(C_nH_2n-_2x)_n, x is an integer between 5-8, (where x is the number of double bonds) and the hydrocarbons can be connected via - CON(R₅)-, -CO- or ~N(R₅)-X, oxygen, sulfur, ~N(R₅)CO~, ~CO(R₅)~, -CO- or - N(R₅)~; C₄H₇-_mRe X- wherein m is an integer of 0 to 4, X is O,S or N and can be positioned at any location along the carbon chain; and R₂ and R₃ can also form a spiro heterocyclic ring system which can be saturated, partially or fully unsaturated; It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. The term "alkyl" means a straight or branched hydrocarbon chain having 1 to 10, preferably 1 to 6 carbon atoms, and is more preferably a methyl or ethyl group. The term "substituted alky, cycloalkyl, alkenyl, or aralkyl" means: C_1-15 alkyl, C ₃_₈ cycloalkyl, C _2-18 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 di-C_1-4 alkyl-carbamoyl, (vi) carboxy, (vii) C_M alkoxy- carbonyl, (viii) sulfo, (ix) halogen, (x) C _1-4 alkoxy, (xi) phenoxy, (xii) halophenoxy, (xiii) C alkylthio, (xiv) mercapto, (xv) phenylthio, (xvi) pyridylthio, (xvii) C_1-4 alkylsulfinyl, (xviii) C_1-4 alkylsulfonyl, (xix) amino, (xx) C_1-3 alkanoylamino, (xxi) mono- or di-Cι- alkylamino, (xxii) 4- to 6-membered cyclic amino, (xxiii) C_1-3 alkanoyl, (xxiv) benzoyl and (xxv) 5- to 10-membered heterocyclic group. The term "aryl" is used throughout the specification to mean an aromatic cyclic hydrocarbon group. An aryl having 6 to 14 carbon atoms is preferable. It may be partially saturated. Preferred examples of such aryls are phenyl and naphthyl groups. The term "hetaryl" is used throughout the specification to mean a 5- or 6- membered monocyclic heterocyclic group containing 1 to 4 hetero-atoms selected from oxygen, sulfur and nitrogen or a fused bicyclic heterocyclic group containing 1 to 6 hetero-atoms 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, (ii) C alkyl, (iii) C_M haloalkyl, (iv) C_M haloalkoxy, (v) C_1-4 alkoxy, (vi) CM alkylthio, (vii) hydroxy, (viii) carboxy, (ix) cyano, (x) nitro, (xi) amino, (xii) mono- or di-C_1-4 alkylamino, (xiii) formyl, (xiv) mercapto, (xv) C_1-4 alkyl-carbonyl, (xvi) C_1- alkoxy- carbonyl, (xvii) sulfo, (xviii) C_1-4 alkylsulfonyl, (xix) carbamoyl, (xx) mono- or di-C_1- alkyl-carbamoyl, (xxi) oxo and (xxii) thioxo. The term "substituted aryl" is used throughout the specification to mean a C_6-1 aryl group which may be substituted by 1 to 4 substituents selected from the group consisting of (i) halogen, (ii) C_1-4 alkyl, (iii) C_M haloalkyl, (iv) C_M haloalkoxy, (v) C alkoxy, (vi) CM alkylthio, (vii) hydroxy, (viii) carboxy, (ix) cyano, (x) nitro, (xi) amino,

(xii) mono- or di-Cι.4 alkylamino, (xiii) formyl, (xiv) mercapto, (xv) C_M alkyl-carbonyl,

(xvi) C alkoxy-carbonyl, (xvii) sulfo, (xviii) C_M alkylsulfonyl, (xix) carbamoyl, (xx) mono- or di-C_M alkyl-carbamoyl, (xxi) oxo and (xxii) thioxo. The term "substituted hetaryl" is used throughout the specification to mean a hetaryl as described above which may be substituted by 1 to 4 substituents selected from the group consisting of (i) halogen, (ii) C alkyl, (iii) C₁-₄ haloalkyl, (iv) CM haloalkoxy, (v) CM alkoxy, (vi) C alkylthio, (vii) hydroxy, (viii) carboxy, (ix) cyano,

(x) nitro, (xi) amino, (xii) mono- or di-Ci-₄ alkylamino, (xiii) formyl, (xiv) mercapto, (xv) C alkyl-carbonyl, (xvi) CM alkoxy-carbonyl, (xvii) sulfo, (xviii) C.sub.1-4 alkylsulfonyl, (xix) carbamoyl, (xx) mono- or di-C₁-₄ alkyl-carbamoyl, (xxi) oxo and (xxii) thioxo. The term, "fused" as used herein with respect to two or more ring chemical structures relates to rings that share a common bond. Accordingly, two rings that are fused share two atoms. As such, quinoline and naphthaline represent chemical moieties that have fused rings. The compounds of the present invention may be geometric isomers or tautomers depending upon the type of substituents. The present invention also covers these isomers in separated forms and the mixtures thereof. Furthermore, some of the compounds may contain an asymmetric carbon in the molecule; in such case stereoisomers could be present. The present invention also embraces mixtures of these optical isomers and the isolated forms of the isomers. Some of the compounds of the invention may form salts. There is no particular limitation so long as the formed salts are pharmacologically acceptable. Specific examples of acid addition salts are 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 organic bases such as methylamine, ethylamine, ethanolamine, lysine, ornithine, etc. The present invention further embraces various hydrates and solvates to the compounds (I) or salts thereof of the invention as well as polymorphisms thereof. Hereinafter, representative processes for producing the compounds of the present invention are described. In these processes, functional groups present in the starting materials or intermediates may be suitably protected with protective groups, depending upon the kind of functional group. In view of the preparation techniques, it may be advantageous to protect the functional groups with groups that can readily be reverted to the original functional group. When required, the protective groups are removed to give the desired products. Examples of such functional groups are amino, hydroxy, carboxy groups, etc. Examples of the groups which may be used to protect these functional groups are shown in, e.g., Greene and Wuts, "Protective Groups in Organic Synthesis", second edition. The General procedure for synthesizing 2-thioxo-oxazolidine compounds is illustrated as follows:

SCIVL T*„

Step 1. Ring closure KOtBu (2J mmol) was dissolved in absolute tetrahydrofuran (THF; 3 mL) under a dry and inert atmosphere and the mixture was cooled to -70°C in a dry ice-acetone bath. A THF solution of the corresponding isocyanate and the oxo-component (pre- dissolved in 2 mL of THF) was carefully added while continuously stirring. The reaction mixture was stirred for an additional 30 min at the same temperature. The mixture was then allowed to warm gradually to room temperature. After the reaction was completed by TLC, acetic acid (2.5 mmol) was added to the reaction vessel and the mixture was evaporated. The residue was dissolved in a mixture of CHC1₃ and water. The organic layer was separated, dried and concentrated under reduced pressure resulting in a crude oil, which was purified by flash chromatography. If the K-salt of the product incidentally precipitated from the mixture, it was isolated by filtration. The pure product was then obtained from the collected solid portions by dissolving them in methanol followed by acidification with acetic acid.

Yield: 40-50 %; Purity: 85-90 %

Step 2: N-acylation and carbamoylation The experiments were performed in lJ-dichloroethane, either at room temperature or with slight heating to ca. 50°C, with the addition of 1.0 eq. of acyl chloride in the presence of 1J molar equivalent of triethylamine. The reaction was completed within 3-5 hours. The final products were purified by liquid-liquid extraction and isolated after evaporation of the organic solvents. Yield: 70-90 %, Purity: 85-90 % The desired compound of the present invention may also be prepared by functional group transformation methods well known to those skilled in the art, which may depend on the kind of substituent. The order of the reactions, or the like, may be appropriately changed in accordance with the aimed compound and the type of reaction to be employed. The other compounds of the present invention and starting compounds can be easily produced from suitable materials in the same manner as in the above processes or by methods well known to those skilled in the art. Each of the reaction products obtained by the aforementioned production methods are isolated and purified as the free base or salt thereof. The salt can be produced by usual salt forming methods. The isolation and purification steps are carried out by employing conventional chemical techniques such as extraction, concentration, evaporation, crystallization, filtration, recrystallization, various types of chromatography and the like. Narious forms of isomers can be isolated by conventional procedures making use of physicochemical differences among isomers. For instance, racemic compounds can be separated by means of 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, e.g., fractional crystallization or chromatography. In addition, an optical isomer can also be synthesized from an appropriate optically active starting compound. Table 1 list the structure of representative compounds of the present invention.

One embodiment of the present invention relates to compounds that inhibit the activity of PI 3-K alpha. The invention further provides methods of inhibiting PI 3-K alpha activity, including methods of modulating the activity of the PI 3-K alpha isozyme in cells, especially leukocytes, cardiomyocytes, osteoclasts, and cancer cells. Of particular benefit are methods of modulating PI 3-K alpha activity in the clinical setting in order to ameliorate disease or disorders mediated by PI 3-K alpha activity. Thus, treatment of diseases or disorders characterized by excessive or inappropriate PI 3-K alpha activity can be treated tlirough use of modulators of PI 3-K alpha according to the present invention. The compounds of the present invention may also show inhibitive activity against other PI 3-K isoforms, including PI 3-K beta, gamma, and delta. Therefore, the present invention also provides methods enabling the further characterization of the physiological role of each PI 3-K isozyme. Moreover, the invention provides pharmaceutical compositions comprising PI 3-K inhibitors and method of manufacturing and using such PI 3-Kinhibitor compounds (or a pharmaceutical composition comprising the compound). The methods described herein benefit from the use of compounds that inhibit, and preferably specifically inhibit, the activity of a PI 3-K isoform in cells, including cells in vitro, in vivo, or ex vivo. Cells useful in the methods include those that express endogenous PI 3-K, wherein endogenous indicates that the cells express PI 3-K absent recombinant introduction into the cells of one or more polynucleotides encoding a PI 3-K isoform polypeptide or a biologically active fragment thereof. Methods also encompass use of cells that express exogenous PI 3-K isoforms wherein one or more polynucleotides encoding a PI 3-K isoforms or a biologically active fragment thereof, have been introduced into the cell using recombinant procedures. Of particular advantage, the cells can be in vivo, i.e., in a living subject, e.g., an animal or human, wherein a PI 3-K inhibitor can be used therapeutically to inhibit PI 3-K activity in the subject. Alternatively, the cells can be isolated as discrete cells or in a tissue, for ex vivo or in vitro methods. In vitro methods also encompassed by the invention can comprise the step of contacting a PI 3-K enzyme, or a biologically active fragment thereof, with an inhibitor compound of the invention. The PI 3-K enzyme can include a purified and isolated enzyme, wherein the enzyme is isolated from a natural source (e.g., cells or tissues that normally express a PI 3-K polypeptide absent modification by recombinant technology) or isolated from cells modified by recombinant techniques to express exogenous enzyme. The relative efficacies of compounds as inhibitors of an enzyme activity (or other biological activity) can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent and then comparing the results. Typically, the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC₅o." IC₅o determinations can be accomplished using conventional techniques known in the art. In general, an IC₅o 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 enzyme activity then are plotted against the inhibitor concentrations used. The concentration of the inhibitor that shows 50% enzyme activity (as compared to the activity in the absence of any inhibitor) is taken as the IC₅o value. Analogously, other inhibitory concentrations can be defined through appropriate determinations of activity. For example, in some settings it can be desirable to establish a 90% inhibitory concentration, i.e., IC₉o, etc. The compounds of the present invention exhibit kinase inhibitory activity, especially PI 3-K inhibitory activity and therefore, can be utilized to inhibit abnormal cell growth in which PI 3-K plays a role. Thus, the compounds are effective in the treatment of disorders with which abnormal cell growth actions of PI 3-K are associated, such as restenosis, atherosclerosis, bone disorders, arthritis, diabetic retinopathy, psoriasis, benign prostatic hypertrophy, atherosclerosis, inflammation, angiogenesis, immunological disorders, pancreatitis, kidney disease, cancer, etc. In particular, the compounds of the present invention possess excellent cancer cell growth inhibiting effects and are effective in treating cancers, preferably all types of solid cancers and malignant lymphomas, and especially, leukemia, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, renal cancer, gastric cancer, brain tumors, etc. Accordingly, the invention provides methods of characterizing the potency of a test compound as an inhibitor of the PI 3-K polypeptide, said method comprising the steps of (a) measuring activity of a PI 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 to the activity of the PI 3-K polypeptide in the presence of an equivalent amount of a reference compound (e.g., a PI 3-Kα. inhibitor compound of the invention as described herein), wherem lower activity of the PI 3-K polypeptide in the presence of the test compound than in the presence of the reference compound indicates that the test compound is a more potent inhibitor than the reference compound, and higher activity of the PI 3-K polypeptide in the presence of the test compound than 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 of characterizing the potency of a test compound as an inhibitor of the PI 3-K polypeptide, comprising the steps of (a) determining an amount of a control compound (e.g., a PI 3-Kα inhibitor compound of the invention as described herein) that inhibits an activity of a PI 3-K polypeptide by a reference percentage of inhibition, thereby defining a reference inhibitory amount for the control compound; (b) determining an amount of a test compound that inhibits an activity of a PI 3-K polypeptide by a reference percentage of inhibition, thereby defining a reference inhibitory amount for the test compound; (c) comparing the reference inhibitory amount for the test compound to the reference inhibitory amount for the control compound, wherein a lower reference inhibitory amount for the test compound than for the control compound indicates that the test compound 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 the compound than inhibits the activity of the PI 3-Kα polypeptide by 50%, 60%, 10%, or 80%. In another aspect the method employs a reference inhibitory amount that is the amount of the compound that inhibits the activity of the PI 3-K α polypeptide by 90%, 95%, or 99%. These 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 fiirther provides methods of identifying a negative regulator of PI

3-K α activity, comprising the steps of (i) measuring activity of a PI3 α polypeptide in the presence and absence of a test compound, and (ii) identifying as a negative regulator a test compound that decreases PI 3-K α activity and that competes with a compound of the invention for binding to PI 3-Kα Furthermore, the invention provides methods for identifying compounds that inhibit PI 3-K α activity, comprising the steps of (i) contacting a PI 3-Kα 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 PI 3-K α activity wherein the compound competes with a compound of the invention for binding to PI 3-K α. The invention therefore provides a method for screening for candidate negative regulators of PI 3-K α activity and/or to confirm the mode of action of candidates such negative regulators. Such methods can be employed against other PI 3- K isoforms in parallel to establish comparative activity of the test compound across the isoforms and/or relative to a compound of the invention. In these methods, the PI 3-K polypeptide can be a fragment of the peptide that exhibits kinase activity or a fragment from the binding domain that provides a method to identify allosteric modulators of the peptide. The methods can be employed in cells expressing PI 3-K peptide or its subunits, either endogenously or exogenously.

Accordingly, the polypeptide employed in such methods can be free in solution, affixed to a solid support, modified to be displayed on a cell surface, or located intracellularly. The modulation of activity or the formation of binding complexes between the PI 3-K polypeptide and the agent being tested then can be measured. Human PI 3-K polypeptides are amenable to biochemical or cell-based high throughput screening (HTS) 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). Automated and miniaturized HTS assays also are comprehended as described, for example, in Houston and Banks, Curr Opin Biotechnol, 8:734-40 (1997). Such HTS assays are used to screen libraries of compounds to identify particular compounds that exhibit a desired property. Any library of compounds can be used, including chemical libraries, natural product libraries, and combinatorial libraries comprising random or designed oligopeptides, oligonucleotides, or other organic compounds. The present invention also provides a method for inhibiting PI 3-K activity therapeutically or prophylactically. The method comprises administering an inhibitor of PI 3-K activity in an amount effective therefor in treating humans or animals who are or can be subject to any condition whose symptoms or pathology is mediated by PI 3-

Kexpression or activity. "Treating" as used herein refers to preventing a disorder from occurring in an animal that can be predisposed to the disorder, but has not yet been diagnosed as having it; inhibiting the disorder, i.e., arresting its development; relieving the disorder, i.e., causing its regression;, or ameliorating the disorder, i.e., reducing the severity of symptoms associated with the disorder. "Disorder" is intended to encompass medical disorders, diseases, conditions, syndromes, and the like, without limitation. The methods of the invention embrace various modes of treating an animal subject, preferably a mammal, more preferably a primate, and still more preferably a human. Among the mammalian 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 nonhuman primates, and zoo specimens. Nonmammalian 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 who have or can be subject to an inflammatory disorder. One aspect of the present invention derives from the involvement of PI 3-K in mediating aspects of the inflammatory process. Without intending to be bound by any theory, it is theorized that, because inflammation involves processes are typically mediated by leukocyte (e.g., neutrophils, lymphocyte, etc.) activation and chemotactic transmigration, and because PI 3-K can mediate such phenomena, antagonists of PI 3-K can be used to suppress injury associated with inflammation. "Inflammatory disorder" as used herein can refer to any disease, disorder, or syndrome in which an excessive or unregulated inflammatory response leads to excessive inflammatory symptoms, host tissue damage, or loss of tissue function. "Inflammatory disorder" also refers to a pathological state mediated by influx of leukocytes and/or neutrophil chemotaxis. "Inflammation" as used herein refers to a localized, protective response elicited by injury or destruction of tissues, which serves to destroy, dilute, or wall off (sequester) both the injurious agent and the injured tissue.

Inflammation is notably associated with influx of leukocytes and/or neutrophil chemotaxis. Inflammation can result from infection with pathogenic organisms and viruses and from noninfectious means such as trauma or reperfusion following myocardial infarction or stroke, immune response to foreign antigen, and autoimmune responses. Accordingly, inflammatory disorders amenable to the invention encompass disorders associated with reactions of the specific defense system as well as with reactions of the nonspecific defense system. As used herein, the term "specific defense system" refers to the component of the immune system that reacts to the presence of specific antigens. Examples of inflammation resulting from a response of the specific defense system include the classical response to foreign antigens, autoimmune diseases, and delayed type hypersensitivity response mediated by T-cells. Chronic inflammatory diseases, the rejection of solid transplanted tissue and organs, e.g., kidney and bone marrow transplants, and graft versus host disease (GNHD), are further examples of inflammatory reactions of the specific defense system. The term "nonspecific defense system" as used herein refers to inflammatory disorders that are mediated by leukocytes that are incapable of immunological memory (e.g., granulocytes, and macrophages). Examples of inflammation that result, at least in part, from a reaction of the nonspecific defense system include inflammation associated with conditions such as adult (acute) respiratory distress syndrome (ARDS) or multiple organ injury syndromes; reperfusion injury; acute glomerulonephritis; reactive arthritis; dermatoses with acute inflammatory components; acute purulent meningitis or other central nervous system inflammatory disorders such as stroke; thermal injury; inflammatory bowel disease; granulocyte transfusion associated syndromes; and cytokine-induced toxicity. "Autoimmune disease" as used herein refers to any group of disorders in which tissue injury is associated with humoral or cell-mediated responses to the body's own constituents. "Allergic disease" as used herein refers to any symptoms, tissue damage, or loss of tissue function resulting from allergy. "Arthritic disease" as used herein refers to any disease that is characterized by inflammatory lesions of the joints attributable to a variety of etiologies. "Dermatitis" as used herein refers to any of a large family of diseases of the skin that are characterized by inflammation of the skin attributable to a variety of etiologies. "Transplant rejection" as used herein refers to any immune reaction directed against grafted tissue, such as organs or cells (e.g., bone marrow), characterized by a loss of function of the grafted and surrounding tissues, pain, swelling, leukocytosis, and thrombocytopenia. The 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 auto-antibodies) of a proliferative cellular response, the production of soluble mediators (including but not limited to cytokines, oxygen radicals, enzymes, prostanoids, or vasoactive amines), or cell surface expression of new or increased numbers of mediators (including, but not limited to, major histocompatability antigens or cell adhesion molecules) in inflammatory cells (including but not limited to monocytes, macrophages, T lymphocytes, B lymphocytes, granulocytes (i.e., polymorphonuclear leukocytes such as neufrophils, basophils, and eosinophils), mast cells, dendritic cells, Langerhans cells, and endothelial cells). It will be appreciated by persons skilled in the art that the activation of one or a combination of these phenotypes in these cells can contribute to the initiation, perpetuation, or exacerbation of an inflammatory disorder. The present invention enables methods of treating arthritic diseases, such as rheumatoid arthritis, monoarticular arthritis, osteoarthritis, gouty arthritis, spondylitis; Behcets disease; sepsis, septic shock, endotoxic shock, gram negative sepsis, gram positive sepsis, and toxic shock syndrome; multiple organ injury syndrome secondary to septicemia, trauma, or hemorrhage; ophthalmic disorders such as allergic conjunctivitis, venereal conjunctivitis, uveitis, and thyroid-associated ophthalmopathy; eosinophilic granuloma; pulmonary or respiratory disorders such as asthma, chronic bronchitis, allergic rhinitis, ARDS, chronic pulmonary inflammatory disease (e.g., chronic obstructive pulmonary disease), silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis, emphysema, pneumonia, bronchiectasis, and pulmonary oxygen toxicity; reperfusion injury of the myocardium, brain, or extremities; fibrosis such as cystic fibrosis; keloid formation or scar tissue formation; atherosclerosis; autoimmune diseases, such as systemic lupus erythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud's syndrome; and transplant rejection disorders such as GNHD and allo graft rejection; chronic glomerulonephritis; inflammatory bowel diseases such as chronic inflammatory bowel disease (CIBD), Crohn's disease, ulcerative colitis, and necrotizing enterocolitis; inflammatory dermatoses such as contact dermatitis, atopic dermatitis, psoriasis, or urticaria; fever and myalgias due to infection; central or peripheral nervous system inflammatory disorders such as meningitis, encephalitis, and brain or spinal cord injury due to minor trauma; Sjogren's syndrome; diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterial pneumonia; antigen-antibody complex mediated diseases; hypovolemic shock; Type I diabetes mellitus; acute and delayed hypersensitivity; disease states due to leukocyte dyscrasia and metastasis; thermal injury; granulocyte transfusion-associated syndromes; and cytokine-induced toxicity. The method can have utility in treating subjects who are or can be subject to reperfusion injury, i.e., injury resulting from situations in which a tissue or organ experiences a period of ischemia followed by reperfusion. The term "ischemia" refers to localized tissue anemia due to obstruction of the inflow of arterial blood. Transient ischemia followed by reperfusion characteristically results in neutrophil activation and transmigration through the endothelium of the blood vessels in the affected area. Accumulation of activated neufrophils in turn results in generation of reactive oxygen metabolites, which damage components of the involved tissue or organ. This phenomenon of "reperfusion injury" is commonly associated with conditions such as vascular stroke (including global and focal ischemia), hemorrhagic shock, myocardial ischemia or infarction, organ transplantation, and cerebral vasospasm. To illustrate, reperfusion injury occurs at the termination of cardiac bypass procedures or during cardiac arrest when the heart, once prevented from receiving blood, begins to reperfuse. With respect to the nervous system, global ischemia occurs when blood flow to the entire brain ceases for a period. Global ischemia can result from cardiac arrest.

Focal ischemia occurs when a portion of the brain is deprived of its normal blood supply. Focal ischemia can result from thromboembolytic occlusion of a cerebral vessel, traumatic head injury, edema, or brain tumor. Even if transient, both global and focal ischemia can cause widespread neuronal damage. Although nerve tissue damage occurs over hours or even days following the onset of ischemia, some permanent nerve tissue damage can develop in the initial minutes following the cessation of blood flow to the brain. Ischemia also can occur in the heart from myocardial infarction and other cardiovascular disorders in which the coronary arteries have been obstructed as a result of atherosclerosis, thrombus, or spasm. Accordingly, the invention is believed to be useful for treating cardiac tissue damage, particularly damage resulting from cardiac ischemia or caused by reperfusion injury, in mammals. In another aspect, inhibitors of PI 3-K activity, such as the compounds of the present invention, can be employed in methods of treating diseases of bone, especially diseases in which osteoclast function is abnormal or undesirable. Accordingly, the use of the compounds of the present invention can be of value in treating osteoporosis, Paget's disease, and related bone resorption disorders. hi a further aspect, the invention includes methods of using PI 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 derived from B lymphocytes or B lymphocyte progenitors. Cancers amenable to treatment using the methods of the present invention include, without limitation, lymphomas, e.g., malignant neoplasms of lymphoid and reticuloendothelial tissues, such as Burkitt's lymphoma, Hodgkins'lymphoma, non-Hodgkins lymphomas, lymphocytic lymphomas and the like; multiple myelomas; as well as leukemias such as lymphocytic leukemias, chronic myeloid (myelogenous) leukemias, and the like. In another aspect, the invention includes a method for suppressing the function of basophils and/or mast cells, and thereby enabling treatment of diseases or disorders characterized by excessive or undesirable basophil and/or mast cell activity. According to the method, a compound of the invention can be used that inhibits the expression or activity of phosphatidylinositol 3-kinase in the basophils and/or mast cells. Preferably, the method employs a PI 3-K inhibitor in an amount sufficient to inhibit stimulated histamine release by the basophils and/or mast cells. Accordingly, the use of such compounds can be of value in treating diseases characterized by histamine release, i.e., allergic disorders, including disorders such as chronic obstructive pulmonary disease (COPD), asthma, ARDS, emphysema, and related disorders. A compound of the present invention can be administered as the neat chemical, but it is typically preferable to administer the compound in the form of a pharmaceutical composition or formulation. Accordingly, the present invention also provides pharmaceutical compositions that comprise a chemical or biological compound ("agent") that is active as a modulator of PI 3-K activity and a biocompatible pharmaceutical carrier, adjuvant, or vehicle. The composition can include the agent as the only active moiety or in combination with other agents, such as oligo- or polynucleotides, oligo- or polypeptides, drugs, or hormones mixed with excipient(s) or other pharmaceutically acceptable carriers. Carriers and other ingredients can be deemed pharmaceutically acceptable insofar as they are compatible with other ingredients of the formulation and not deleterious to the recipient thereof. Techniques for formulation and administration of pharmaceutical compositions can be found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co,

Easton, Pa., 1990. The pharmaceutical compositions of the present invention can be manufactured using any conventional method, e.g., mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, melt-spinning, spray- drying, or lyophilizing processes. However, the optimal pharmaceutical formulation will be determined by one of skill in the art depending on the route of administration and the desired dosage. Such formulations can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agent. Depending on the condition being treated, these pharmaceutical compositions can be formulated and administered systemically or locally. The pharmaceutical compositions are formulated to contain suitable pharmaceutically acceptable carriers, and can optionally comprise excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. The administration modality will generally determine the nature of the carrier. For example, formulations for parenteral administration can comprise aqueous solutions of the active compounds in water-soluble form. Carriers suitable for parenteral administration can be selected from among saline, buffered saline, dextrose, water, and other physiologically compatible solutions. Preferred carriers for parenteral administration are physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiologically buffered saline. For tissue or cellular administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. For preparations comprising proteins, the formulation can include stabilizing materials, such as polyols (e.g., sucrose) and/or surfactants (e.g., nonionic surfactants), and the like. Alternatively, formulations for parenteral use can 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 can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Optionally, the suspension also can contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions. Aqueous polymers that provide pH-sensitive solubilization and/or sustained release of the active agent also can be used as coatings or matrix structures, e.g., methacrylic polymers, such as the EUDRAGIT.RTM. series available from Rohm America Inc. (Piscataway, NJ.).

Emulsions, e.g., oil-in-water and water-in-oil dispersions, also can be used, optionally stabilized by an emulsifying agent or dispersant (surface active materials; surfactants). Suspensions can contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethlyene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, gum tragacanth, and mixtures thereof. Liposomes containing the active agent also can be employed for parenteral administration. Liposomes generally are derived from phospholipids or other lipid substances. The compositions in liposome form also can contain other ingredients, such as stabilizers, preservatives, excipients, and the like. Preferred lipids include phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods of forming liposomes are known in the art. See, e.g., Prescott (Ed.), Methods in Cell Biology, Vol. XIV, p. 33, Academic Press, New York (1976). The pharmaceutical compositions comprising the agent in dosages suitable for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art. The preparations formulated for oral administration can be in the form of tablets, pills, capsules, cachets, dragees, lozenges, liquids, gels, syrups, slurries, elixirs, suspensions, or powders. To illustrate, pharmaceutical preparations for oral use can be obtained by combining the active compounds with a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Oral formulations can employ liquid carriers similar in type to those described for parenteral use, e.g., buffered aqueous solutions, suspensions, and the like. Preferred oral formulations include tablets, dragees, and gelatin capsules. These preparations can 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, starch from corn, wheat, rice, potato, etc.; c) cellulose materials, such as methylcellulose, hydroxypropylmethyl cellulose, and sodium carboxymethylcellulose, polyvinylpyrrolidone, gums, such as gum arable and gum tragacanth, and proteins, such as gelatin and collagen; d) disintegrating or solubilizing agents such as 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) flavorants and sweeteners; g) colorants or pigments, e.g., to identify the product or to characterize the quantity (dosage) of active compound; and h) other ingredients, such as preservatives, stabilizers, swelling agents, emulsifying agents, solution promoters, salts for regulating osmotic pressure, and buffers. Gelatin capsules include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating such as glycerol or sorbitol. Push-fit capsules can contain the active ingredient(s) 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 with or without stabilizers. Dragee cores can be provided with suitable coatings such as concentrated sugar solutions, which also can contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. The pharmaceutical composition can be provided as a salt of the active agent. Salts tend to be more soluble in aqueous or other protonic solvents than the corresponding free acid or base forms. Pharmaceutically acceptable salts are well known in the art. Compounds that contain acidic moieties can form pharmaceutically acceptable salts with suitable cations. Suitable pharmaceutically acceptable cations include, for example, alkali metal (e.g., sodium or potassium) and alkaline earth (e.g., calcium or magnesium) cations. Compounds of structural formula (I) that contain basic moieties 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 situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable acid. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate, digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fiimarate, hydrochlori.de, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isothionate), lactate, maleate, methanesulfonate or sulfate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, phosphate or hydrogen phosphate, glutamate, bicarbonate, p-toluenesulfonate, and undecanoate. Examples of acids that can be employed to form pharmaceutically acceptable acid addition salts include, without limitation, such inorganic acids as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and such organic acids as oxalic acid, maleic acid, succinic acid, and citric acid. In light of the foregoing, any reference to compounds of the present invention appearing herein is intended to include compounds of structural formula I, 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 carrier can be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. Accordingly, there also is contemplated an article of manufacture, such as a container comprising a dosage form of a compound of the invention and a label containing instructions for use of the compound. Kits are also contemplated under the invention. For example, the kit can comprise a dosage form of a pharmaceutical composition and a package insert containing instructions for use of the composition in treatment of a medical condition. In either case, conditions indicated on the label can include treatment of inflammatory disorders, cancer, etc. Pharmaceutical compositions comprising an inhibitor of PI 3-K activity can be administered to the subject by any conventional method, including by parenteral and enteral techniques. Parenteral administration modalities include those in which the composition is administered by a route other than through the gastrointestinal tract, for example, intravenous, intraarterial, intraperitoneal, intramedullary, intramuscular, intraarticular, intrathecal, and intraventricular injections. Enteral administration modalities 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 lung administration; vaginal administration; and rectal admimstration. Transdermal administration includes passive or active transdermal or transcutaneous modalities, including, for example, patches and iontophoresis devices, as well as topical application of pastes, salves, or ointments. Parenteral administration also can be accomplished using a high-pressure technique. Surgical techniques include implantation of depot (reservoir) compositions, osmotic pumps, and the like. A preferred route of administration for treatment of inflammation can be local or topical delivery for localized disorders such as arthritis, or systemic delivery for distributed disorders, e.g., intravenous delivery for reperfusion injury or for systemic conditions such as septicemia. For other diseases, including those involving the respiratory tract, e.g., chronic obstructive pulmonary disease, asthma, and emphysema, administration can be accomplished by inhalation or deep lung 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 following parenteral administration would be desirable. The PI 3-K inhibitor compounds can be administered before, during, or after administration of chemotherapy, radiotherapy, and/or surgery. Moreover, the therapeutic index of the PI 3-K.delta. inhibitor compounds can be enhanced by modifying or derivatizing the compounds for targeted delivery to cancer cells expressing a marker that identifies the cells as such. For example, the compounds can be linked to an antibody that recognizes a marker that is selective or specific for cancer cells, so that the compounds are brought into the vicinity of the cells to exert their effects locally, as previously described (see for example, Pietersz et al., Immunol Rev, 129:57 (1992); Trail et al., Science, 261:212 (1993); and Rowlinson-Busza et al., Curr Opin Oncol, 4:1142 (1992)). Tumor-directed delivery of these compounds enhances the therapeutic benefit by minimizing potential nonspecific toxicities that can result from radiation treatment or chemotherapy. In another aspect, PI 3-K inhibitor compounds and radioisotopes or chemotherapeutic agents can be conjugated to the same anti-tumor antibody. For the treatment of bone resorption disorders or osteoclast-mediated disorders, the PI 3-K inhibitors can be delivered by any suitable method. Focal administration may be desirable, such as by intraarticular injection. In some cases, it may be desirable to couple the compounds to a moiety that can target the compounds to bone. For example, a PI 3-K inhibitor can be coupled to compounds with high affinity for hydroxyapatite, which is a major constituent of bone. This can be accomplished, for example, by adapting a tetracycline-coupling method developed for targeted delivery of estrogen to bone (Orme et al., Bioorg Med Chem Lett, 4(11): 1375-80 (1994)). To be effective therapeutically in modulating central nervous system targets, the agents used in the methods of the invention should readily penetrate the blood brain barrier when administered peripherally. Compounds that cannot penetrate the blood brain barrier, however, can still be effectively administered by an intravenous route. As noted above, the characteristics of the agent itself and the formulation of the agent can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agent. Such pharmacokinetic and pharmacodynamic information can be collected through preclinical in vitro and in vivo studies, 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 from biochemical and/or cell-based assays. Then, the dosage can be formulated in animal models to achieve a desirable circulating concentration range that modulates PI 3- Kexpression or activity. As human studies are conducted, further information will emerge regarding the appropriate dosage levels and duration of treatment for various diseases and conditions. Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures using cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅o (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the "therapeutic index," which typically is expressed as the ratio LD50/ED50 Compounds that exhibit large therapeutic indices, i.e., the toxic dose is substantially higher than the effective dose, are preferred. The data obtained from such cell culture assays and additional animal studies can be used in formulating a range of dosages for human use. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. For the methods of the present invention, any effective administration regimen regulating the timing and sequence of doses can 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 PI 3-K expression or activity and/or derive a measurable change in a physiological parameter of the subject through administration of one or more of the pharmaceutical dosage units. Exemplary dosage levels for a human subject are of the order of from about 0.001 milligram of active agent per kilogram body weight (mg/kg) to about 100 mg/kg. Typically, dosage units of the active agent comprise from about 0.01 mg to about 10,000 mg, preferably from about 0J mg to about 1,000 mg, depending upon the indication, route of administration, etc. Depending on the route of administration, a suitable dose can be calculated according to body weight, body surface area, or organ size. The final dosage regimen will be determined by the attending physician in view of good medical practice, considering various factors that modify the action of drugs, e.g., the agent's specific activity, the identity and severity of the disease state, the responsiveness of the patient, the age, condition, body weight, sex, and diet of the patient, and the severity of any infection. Additional factors that can be taken into account include time and frequency of administration, drug combinations, reaction sensitivities, and tolerance/response to therapy. Further refinement of the dosage appropriate for treatment involving any of the formulations mentioned herein is done routinely by the skilled practitioner without undue experimentation, especially in light of the dosage information and assays disclosed, as well as the pharmacokinetic data observed in human clinical trials. Appropriate dosages can be ascertained through use of established assays for determining concentration of the agent in a body fluid or other sample together with dose response data. The frequency of dosing will depend on the pharmacokinetic parameters of the agent and the route of administration. Dosage and administration are adjusted to provide sufficient levels of the active moiety or 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 depots, or combinations thereof, as required to maintain the desired minimum level of the agent. Short-acting pharmaceutical compositions (i.e., short half-life) can be administered once a day or more than once a day (e.g., two, three, or four times a day). Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks. Pumps, such as subcutaneous, intraperitoneal, or subdural pumps, can be preferred for continuous infusion. The following Examples are provided to further aid in understanding the invention, and pre-suppose an understanding of conventional methods well-known to those persons having ordinary skill in the art to which the examples pertain. Such 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 ed., John Wiley & Sons, Inc. (1999). The particular materials and conditions described hereunder are intended to exemplify particular aspects of the invention and should not be construed to limit the reasonable scope thereof.

Example 1 Synthesis and Characterization of 935665 2-thioxo-3 - [(3 -trifluoromethyl)-benzenesulfonyl)] - 5 -methyl-5 - [2,7-dimethyl- pyrazolo[l,5a]pyrimidine 6-yl]-4-oxazolidine-carboxylic acid ethyl ester

5 Step 1: KOtBu ( 1J5 g, 21 mmol) was dissolved in abs. t etrahydrofuran (THF; 3 mL) under dry and inert atmosphere and the mixture was cooled to -70°C in a dry ice-acetone bath. A THF solution (30 mL) of ethyl isothiocyanatoacetate (2.9 g, 20 mmol) and 2,7- dimethyl-6-acetyl-pyrazolo[l,5a]pyrimidine (3,78 g, 20 mmol) was then carefully added 10 to the basic media while stirring rapidly. The reaction mixture was stirred for an additional 30 min at the s a e t emperature t hen the m ixture w as allowed to w arm u p gradually to room temperature. After 24 h stirring at r.t, acetic acid (24 mmol) was added to the reaction vessel and the mixture was evaporated. The residue was taken up in a mixture of CHC13 and 15 water. The organic layer was separated, dried and concentrated under reduced pressure resulting in a crude oil, which was purified by flash chromatography used in the next step.

Step 2: The compound obtained in Step 1 (334 mg, 1 mmol) was dissolved in 1,2- 20 dichloroethane and 153 microL (1J mmol) triethyl amine was added, followed by the addition of 3-trifluoromethyl-benzenesulfonyl chloride (245 mg, 1 mmol). The reaction was completed within 3 hours at r.t. The final products were purified by liquid-liquid extraction and isolated after evaporation of the organic solvents yielding 232 mg product, which was then characterized by NMR: 1H NMR (CDC13): 1.18, 1.32, 1.36, 1.40, 1.80,

2.49, 2.95, 4.38, 4.40, 4.42, 4.44, 5.33, 6.50, 7.20, 7.57, 7.82, 8.16, 8.41.

Example 2 Synthesis and Characterization of 935653 2-thioxo-3-(4-chloro-benzenesulfonyl)-5-methyl-5-[2, 7-dimethyl- pyrazolo[l,5a]pyrimidine-6-yl]-4-oxazolidine-carboxylic acid ethyl ester

Step 1

As shown under Example 1/Step 1.

Step 2: The compound obtained in Example 1./ Step 1 (81.4 mg, 0.243 mmol) was dissolved in 1,2-dichloroethane (2.4 mL) and 37 microL (0.268 mmol) triethyl amine was added, followed by the addition of 4-chloro-benzenesulfonyl chloride (53 mg, 0J43 mmol). After stirring overnight at r.t and additional portion of sulfonylating reagent (8 mg) was added together with 5 microL triethyl amine and the reaction mixture was stirred for another 5 h at 80 C. After allowing to cool down, the crude products were purified by liquid-liquid extraction and isolated after evaporation of the organic solvents yielding 18 mg product, which was then characterized by NMR: 1H NMR (CDC13): 0.70, 2.05, 2.55, 2.85, 3.77, 5.29, 6.48, 7.25, 7.58, 8.07, 8.48. Example 3 Isolation and purification of Recombinant PI 3-K polypeptide Recombinant heterodimeric PI 3-K alpha, consisting of a pi 10 catalytic subunit and a GST-tagged p85 regulatory subunit, was expressed in Sf9 cells using a baculovirus expression system. Expression constructs were obtained from the lab 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 pellet was re-suspended in 3 ml of Buffer A (20mM Tris pH 7.0, 150mM NaCI, 1 OmM EDTA, 20mM Sodium Fluoride, 5mM Sodium

Pyrophosphate, 10% Glycerol, 0.1% Igapal) containing protease inhibitors (ImM PMSF,

ImM NaNO₃, Leupeptin lug/ml, Pepstatin lug/ml.) The suspension was incubated for lhour at 4°C with rotation to break the cells, and then vortexed gently to ensure cell lysis.

The solution was centrifuged at 14,000g for 15 minutes, and the supernatant was diluted by the addition of 10ml of Buffer A. The diluted supernatant was added to 3ml 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 35ml of

Buffer A, and the protein was eluted using lOmM Glutathione in Buffer A. Twenty,

0.5ml fractions were collected and the presence of protein was assessed on 12% SDS- PAGE Tris Glycine gel (Invitrogen). Fractions containing target protein were pooled and concentrated using a Microsep 30K concentrator (Pall-Gelman). The concentrated protein was diluted with 3 ml of Final Buffer (20mM Tris pH 7.4, lOOmM NaCI, ImM

EDTA) and concentrated twice more to remove any detergent. The protein was diluted in 50% glycerol and stored at -20°C.

Example 4 PI 3-K Activity Assay and Screen for PI 3-K Inhibitors Vectors for expression of GST-GRP1-PH were obtained from Mark Lemmon,

University of Pennsylvania. (Kavran, et al, J Biol Chem, 273:30497-30508 (1998)). Protein expression and purification from E. coli was carried out as follows: A LB/amp plate was streaked from a frozen glycerol stock of E coli containing the expression vector and grown overnight at 37°C. A single colony was picked and inoculated into 20 ml of

LB media containing lOOug/ml of ampicillin, and grown overnight. The overnight culture was added to 1 Liter of LB media containing lOOug/ml of ampicillin and grown until the O.D. 600 was between 0.8-1.0. Protein expression was induced by the addition of 0J mM IPTG, and cultures continued to grow overnight at 37°C. Cells were harvested by centrifugation at 4,000g for 20 minutes. Pellets were stored frozen at -80°C until protein purification was carried out. The purification of GST-tagged protein was performed as follows: the pellets were resupended in 25 ml of Buffer A (50mM Tris pH

7.5, ImM BME, ImM EDTA, ImM EGTA, ImM NaNO3, 50mM Sodium Fluoride,

5mM Sodium Pyrophosphate, 0J7M Sucrose) with protease inhibitors (ImM PMSF,

0.5ug/ml Leupeptin, OJug/ml Pepstatin). The cells were lysed 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 10,000rpm for 15 minutes. The supernatant was mixed with 5 ml 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 into a column and washed with 30 ml of Buffer A. The protein was eluted using lOmM Glutathione (Sigma) in Buffer A. Twenty, 1ml fractions were collected and protein levels assessed by SDS-PAGE on 12% Tris-Glycine gels (Invitrogen). The fractions containing purified protein were pooled and stored at -20 C°. PI 3-kinase reactions were performed in a reaction buffer containing 5 mM HEPES, pH 7, 2.5 mM MgCl₂, and 25 μM ATP, containing 50 ng of recombinant PI 3-K with 10 picomoles of diC₈ PI(4,5)P₂ (Echelon Biosciences) as the substrate. The reactions were allowed to proceed at room temperature for 1-3 hours, then quenched 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 concenfration of 1 μM from stocks in DMSO. The final concentration of DMSO was 1%. Conversion of the substrate to PI(3,4,5)P₃ was determined using a competition assay using Amplified Luminescent Proximity Homogeneous Assay (ALPHA®) technology developed by Perkin Elmer. 0J5 picomoles of recombinant GST-Grpl-PH domain protein and 0.25 picomoles of biotinylated diC₆ PI(3,4,5)P₃ (Echelon Biosciences) were added to each reaction mixture. Donor and Acceptor beads from the AlphaScreen® GST (Glutathione-S-Transferase) Detection Kit (PerkinElmer) 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 α microplate reader. Percent inhibition of enzyme activity was determined by comparison to no enzyme (100 % inhibition) and DMSO alone (0% inhibition) controls. An alternate method used for detecting substrate conversion to PI(3,4,5)P₃ was a competitive Fluorescence Polarization assay. 125 picomoles of recombinant GST-Grpl- PH domain protein and .25 picomoles of TAMRA-I(1,3,4,5)P (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/580 nm polarizing emission filters. BODIPY-TMR-I(l,3,4,5)P4 or BODIPY-TMR-PI(3,4,5)P3 could substitute as the fluorescent tracers in this assay. Percent inhibition of enzyme activity was determined by comparison to no enzyme (100 % inhibition) and DMSO alone (0% inhibition) controls.

Example 5 Determination of IC₅o for PI 3-K Inhibitors A library of potential PI 3-K inhibitors was tested for activity against PI 3-K alpha in the following mamier. IC₅o values were determined for the selected represented compounds of the present invention. Enzyme activity assays were performed as previously described, in the presence of a range of compound concentrations to allow determination of IC₅₀ values. Enzyme activity and percent inhibition was determined using the AlphaScreen® luminescent assay or a Fluorescence Polarization assay as previously described. These inhibitors may also show activity against other PI 3-K isoforms, including PI 3-K beta, gamma, and delta.

Example 6 Characterization of Effects of PI 3-K Inhibitors on Cancer Cells Selected compounds were tested for selective activity against paired ovarian cancer and breast cancer cell lines. The ovarian cancer cell line SKON3 is not altered in PI 3-K signaling and should be less sensitive to the anti-proliferative effects produced by treatment with PI 3-K inhibitors, while the ONCAR3 cell line, which is altered in PI 3-K signaling, via amplification of PI 3-K activity, should be sensitive. SKON3 cells were seeded in 96- well cell culture plates (Greiner) at a density of 20,000 cells per well in McCoys 5 A media (GibcoBRL) with 10% fetal calf serum and 20 mM L-glutamine. ONCAR3 cells were seeded at a density of 15,000 cells per well in RPMI 1640 media (GibcoBRL) containing 20 mM 1-glutamine, 0.01 mg/ml bovine insulin, 10 mM Hepes pH 7.4, 1 mM sodium pyruvate, 2.5 g/L glucose, and 20 % fetal calf serum. After 24 hours, compounds were added to cell media to 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 calf serum. Viability was determined using a MTT cell proliferation assay (R and D Systems) and comparison to DMSO alone controls (100% viability). Compounds which result in reduced viability may act either by inhibiting cell proliferation or by inducing apoptosis (programmed cell death). Compounds representative of the 093 structural groups within the library showed selective effects on cell proliferation and viability. Compounds present in the library which had been identified as PI 3-K inhibitors using the in vitro screen, and which were also structurally related to the compounds of the present invention that showed cell-specific effects on viability, were tested for activity against the paired ovarian cancer cell lines. Many of these also show similar selective effects on cell growth. Table 2 summarizes the results of two separate cell proliferation experiments for selected compounds of the present invention.

Table 2. Summary of two different experiments in selected compounds tested for selective effects on paired ovarian cancer cell lines.

PI 3-K inhibitors which show this activity profile may be effective against a number of tumor cell lines and tumor types in which PI 3-K signaling is altered, either by amplification of PI 3-K activity, or by mutations which effect regulation of PI 3-K activity, including mutations in the tumor suppressor PTEN gene. These include breast, prostate, colon, and ovarian cancers. PI 3-K inhibitors were also evaluated for selective activity against breast cancer cell lines. The cell line MDA-MB-468 is mutant of PTEN, a negative regulator of PI 3- K signaling, and PI 3-K signaling is abnormally activated in these cells, while the cell line MDA-MB-231 shows normal expression and activity of PTEN and PI 3-K signaling is normally regulated. 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 RMPI media (GibcoBRL) with 10% fetal calf serum and 20 mM L-glutamine. After 24 hours, compounds were added to cell media to a 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 calf serum and 20 mM L-glutamine. Viability was determined using a MTT cell proliferation assay (R and D Systems) and comparison to DMSO alone controls (100%) viability). Compounds which result in reduced viability may act either by inhibiting cell proliferation or by inducing apoptosis (programmed cell death). Compounds of the present invention within the library showed selective effects on cell proliferation and viability. Selected compounds were evaluated against the paired breast cancer cell lines at a range of concentrations to determine effective concentrations for growth inhibition.

Example 7 Effects on PI 3-K mediated signaling through PKB/Akt by PI 3-K inhibitors Because phosphorylation and activation of PKB/Akt is dependent on PI 3-K activity, PI 3-K inhibitors decrease the cellular levels of phospho-Akt. MDA-MB-468 cells show constitutively high levels of phospho-Akt as a result of abnormal activation of PI 3-K signaling. The effect of freatment with PI 3-K inhibitors on phospho-Akt levels in these cells was determined as follows. Cells were plated into 6-well cell culture dishes at a density of 5 x 10⁵ cells per well in RMPI media containing 10% fetal calf serum and 2 mM L-glutamine. Twenty-four hours later, media was removed and replaced with serum-free RMPI containing 2 mM L-glutamine. The cells were serum-starved overnight. Compounds were diluted into serum-free media to a final concentration of 50 μM and added to the cells. The cells were incubated in the presence of PI 3-K inhibitors for

4 hours. Phospho-AJkt levels were determined using one of the following methods. To determine phospho-Akt levels using immunoblotting, cells were washed twice with PBS and lysed in ice-cold lysis buffer (1 % Triton X- 100, 50mM Hepes pH 7.4, 150 mM NaCI, 1.5mM MgC12, ImM EGTA, lOOmM NaF, lOmM Sodium Pyrophosphate,

ImM Na(subscript: 3)VO(subscript: 4), 10% glycerol, ImM phenylmethylsulfonyl fluoride, and 10 ug/ml aprotinin). Total protein concentration was determined using a

BCA assay. 30ug of total cell lysate protein was diluted into Laemmli sample buffer and loaded onto 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 in TBS-T (lOmM Tris-HCl pH 7.4, 150mM NaCI, and 0.1% Tween-20) and incubated with HRP-conjugated antibody (diluted in 5% milk in TBS-T) at room temperature for lh.

The membrane was washed extensively and the proteins were visualized by chemiluminscent detection. The compounds effects on phospho- Akt levels were observed as relative differences in the amount of phospho-Akt detected by immunoblotting. Effects on cellular levels of phospho-Akt following treatment with PI 3-K inhibitors were quantified using the PathScan phospo-Akt ELISA (Cell Signalling Technologies)., a sandwich ELISA for detection of phospho-Akt. The kit was used according to the manufacturer protocol. Absorbance at 450 nm was determined for each sample and used directly as equivalent of phosphor- Akt levels. Percent decreases in phosphor- Akt levels were determined by normalizing relative to blank samples (0%) and confrol samples treated with DMSO alone (100%). Treatment with PI 3-K inhibitors resulted in a 20-60% decrease in phospho-Akt levels as determined by this assay. This data shows that these compounds are capable of affecting cellular PI 3-K mediated signaling. Table 3 summarizes the data for several compounds of this structural group, including IC₅₀ for inhibition of enzyme activity in vitro, cellular mIC₅o and anti- proliferative activity against tumor cells altered in PI 3-K mediated signaling, and effects on cellular levels of phospho-Akt. Table 3. Characterization of compounds of the present invention in vitro and biological activities assays

Example 8. Effects on tumor cells grown in 3-D culture systems by PI 3-K inhibitors

PI 3-K inhibitors are assayed for effects on tumor cells grown in three-dimensional matrix that more closely mimics the environment of a tumor than other cell culture models. MDA-MB-468 cells are mixed in a matrix solution, such as Matrigel (BD

Biosciences) at 2 x 10 ⁶ 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 2 xlO⁵ cells are added per well. Once the matrix is solidified, RMPI media containing 10% fetal calf serum and 2 mM L-glutamine is added to each well. After approximately 14 days of culture, the compounds are added to cell media at final concentrations ranging from 10 nM to 100 μM, and the cells are grown in the presence of the compounds for 7 days in RMPI media containing 0.5% fetal calf serum and 20 mM L-glutamine. Following this treatment, cell growth in the three dimensional matrix can be measured using a cell viability assay such as the CellTiter 96 One Solution Cell

Proliferation Assay (Promega, G3582). 1.2 ml of assay solution is added per well, the cells are incubated for 3 hours. Absorbance at 550 nm is detennined for each well and used directly as being equivalent of cell number, hi addition, live and dead cells can be distinguished and observed using fluorescence microscopy after staining with Fluorescein diacetate (Sigma), which labels live cells, and propidium iodide (Sigma), which labels dead cells. The PI 3-K inhibitors of the present invention show anti-proliferative effects in this model of tumor cell growth, which compares the anti-proliferative effects of one inhibitor compared to the effects of the benchmark PI 3-K inhibitor LY294002. The PI 3- K inhibitors of the present invention also show enhanced anti-proliferative activity when combined with other cancer drugs, for example paclitaxel or doxorubicin.

Example 9 Inhibition of Tumor Growth The in vivo efficacy of an inhibitor of the growth of cancer cells may be confirmed by several protocols well known in the art. Human tumor cells which are deregulated in the PI 3-K pathway, for example, LnCaP, PC3, C33a, OVCAR-3, MDA- MB-468 are injected subcutaneously into the flank of nude mice on day 0. Mice are assigned to a vehicle, compound, or combination freatment group. Compound administration may begin on day 1-7. Subcutaneous administration may be done every day or every other day for the duration of the experiment, or the compound may be delivered by a continuous infusion pump. The size of subcutaneous tumors can be monitored throughout the course of the experiment. The tumors are excised and weighed at the conclusion of the experiment and the average weight of tumors for each treatment group is calculated. Alternatively, cell lines such as OVCAR-3 may be injected intraperitoneally into the abdominal cavity of female nude mice. Subcutaneous, intravenous, or intraperitoneal administration may be done every day or every other day for the duration of the experiment, or the compound may be delivered 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. The PI 3-K inhibitors show enhanced activity against tumor growth when combined with other cancer drugs, for example paclitaxel or doxorubicin. It is to be understood that the above-referenced arrangements are only illustrative of the application of the principles of the present invention. Numerous modifications and alternative arrangements can be devised without departing from the spirit and scope of the present invention. While the present invention has been shown in the drawings and is fully described above with particularity and detail in connection with what is presently deemed to be the most practical and preferred embodiment(s) of the invention, it will be apparent to those of ordinary skill 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

We claim: 1. A compound having a general structure represented by Formula I or Formula ii;

Formula I Formula II wherein R₁ is independently a member selected from the group consisting of CH=CH-R₅, CO-OR₅, CO- NHR5, -O-CO-R₅, -OH, OR₅, hydrogen, alkyl, alkenyl, sulfonyl, carbonyl, ketyl, aralkyl, aryl, and hetaryl; R₂ and R₃ are each independently a member selected from the group consisting of alkyl, aryl, hetaryl, and aralkyl; R₄ is a member selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O- R₅, CO-N-R₆, R₅, -NH-CO-R₅, NH-R₅, -NR₅Re, -O-R₅, alkyl, alkenyl, alkynyl, aralkyl, and cycloalkyl; R₅ is a member selected from the group consisting of hydrogen, alkyl, aryl, and hetaryl; R₆ is a member selected from the group consisting of hydrogen, aryl, and hetaryl; and optionally, each of the aforementioned R_t-R₆ groups can independently be substituted with at least one substituent.

2. A compound according to claim 1, with reference to

independently, whenever the following are used; alkyl is a straight or branched chain C_1-15 alkyl; alkenyl is a straight or branched chain C _2-18 alkenyl; aryl is a carbomonocyclic aromatic or carbobicyclic aromatic; hetaryl is a heteromonocyclic aromatic or heterobicyclic aromatic moiety containing 1 to 4 hetero-atoms selected from oxygen, sulfur and nitrogen; aralkyl is a carbomonocyclic aromatic or carbobicyclic aromatic substituted with a straight or branched chain C_1-15 alkyl; and substituent is a member selected from the group consisting of halogen, C_1-4 alkyl, C_1- haloalkyl, C_1-4 haloalkoxy, C_1-4 alkoxy, C_1-4 alkylthio, phenylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C_1-4 alkylamino, formyl, mercapto, C_1-4 alkyl-carbonyl, C_1-4 alkoxy-carbonyl, sulfo, C_1- alkylsulfonyl, carbamoyl, mono- or di-C_1- alkyl- carbamoyl, oxo and thioxo.

3. A compound according to claim 1, wherein R_\ is independently a member selected from the group consisting of CO-OR₅, C _2-6 alkenyl, and carbomonocyclic aromatic; R₂ and R₃ are each independently a member selected from the group consisting of straight or branched chain C_1-4 alkyl, sulphur containing heteromonocyclic aromatic, sulphur containing heteromonocyclic aromatic substituted with at least one substituent, oxygen containing heteromonocyclic aromatic, oxygen containing heteromonocyclic aromatic substituted with at least one substituent, nitrogen containing heteromonocyclic aromatic, 6-membered carbomonocyclic aromatic fused with a 5-membered carbomonocyclic ring, 6-membered carbomonocyclic aromatic fused with a 5-membered carbomonocyclic ring and substituted with at least one substituent, bicycloheptenyl, carbomonocyclic aromatic, carbomonocyclic aromatic substituted with at least one substituent, 6-membered nitrogen containing heteromonocyclic aromatic fused with a 5- membered nitrogen containing heteromonocylic aromatic, 6-membered nitrogen containing heteromonocyclic aromatic fused with a 5-membered nitrogen containing heteromonocyclic aromatic and substituted with at least one substituent, and straight or branched chain C_1-4 alkyloxybenzyl; R is a member selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O- R₅, and CO-N-R₆; R₅ is a member selected from the group consisting of straight or branched chain C_1-9 alkyl, straight or branched chain C_1-9 alkyl substituted with at least one substituent, C_2-4 alkenyl substituted with at least one substituent, sulphur containing heteromonocylic aromatic, oxygen containing heteromonocylic aromatic, oxygen containing heteromonocyclic aromatic substituted with at least one substituent, nitrogen containing heteromonocylic aromatic fused with a carbomonocyclic aromatic, carbomonocyclic aromatic fused with a carbomonocyclic aromatic, camphoryl, camphoryl substituted with at least one substitutent, carbomonocyclic aromatic, carbomonocyclic aromatic substituted with at least one substituent, nitrogen containing heteromonocyclic aromatic,

C_1-4 alkoxybenzyl, phenylcyclo-C_3-6 alkyl, C_1-4 alkyl-carbamoylphenyl; C_1-6 alkyl- Cι_-6 alkylate, phenylalkenyl, and phenylalkenyl substituted with at least one substituent; and R₆ is a carbomonocyclic aromatic or carbomonocyclic aromatic substituted with at least one substituent.

4. A compound according to claim 3, wherein the substituent is a member selected from the group consisting of halogen, CM alkyl, C_1- haloalkyl, C_1- haloalkoxy, C_M alkoxy, Cμ alkylthio, phenylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C_1-4 alkylamino, formyl, mercapto, C alkyl-carbonyl, C alkoxy-carbonyl, sulfo, Cι-₄ alkylsulfonyl, carbamoyl, mono- or di-C₁.₄ alkyl-carbamoyl, oxo and thioxo.

5. A compound according to claim 1, wherein R} is independently a member selected from the group consisting of CO-OR₅, C ₂.₄ alkenyl and phenyl; R₂ and R₃ are each independently a member selected from the group consisting of straight or branched chain Cμ alkyl, thiophenyl, thiophenyl substituted with at least one substituent, indenyl, indenyl substituted with at least one substituent, bicyclo 2.2. L heptenyl, phenyl, phenyl substituted with at least one substituent, pyridinyl, furanyl, furanyl substituted with at least one substitutent, pyrazolopyramidine, pyrazolopyramidine substituted with at least one substituent, and CM alkoxybenzyl; _t is a member selected from the group consisting of CO-R₅, or SO₂-R₅; CO-O- R₅, and CO-N-R₆, R₅ is a member selected from the group consisting of straight or branched chain

C]-₉ alkyl, straight or branched chain Q-₉ alkyl substituted with at least one substituent, C_2-4 alkenyl substituted with at least one substituent, phenyl, phenyl substituted with at least one substituent, benzyl, benzyl substituted with at least one substituent, furanyl, furanyl substituted with at least one substitutent, phenylbenzyl, camphor, camphor substituted with at least one substituent, phenylethylenyl, phenylethylenyl substituted with at least one substitutent, thiophenyl, thiophenyl substituted with at least one substituent, benzyloxy, C alkoxyphenyl, CM alkyphenyl ether, phenylthio-CH₂-, CM alkyl-d-₄ alkylate, phenylcyclopropyl, quinolinyl, naphthalenyl, and acetylamidylphenyl; R₆ is phenyl or phenyl substituted with at least one substituent; and substituent is a member selected from the group consisting of halogen, Cμ₄ alkyl, C_M haloalkyl, C haloalkoxy, C₁-₄ alkoxy, CM alkylthio, phenylthio, hydroxy, carboxy, cyano, nitro, amino, mono- or di-C₁-₄ alkylamino, formyl, mercapto, C₁-₄ alkyl-carbonyl, C alkoxy-carbonyl, sulfo, Cι-₄ alkylsulfonyl, carbamoyl, mono- or di-C_M alkyl- carbamoyl, oxo and thioxo.

6. A compound according to claim 5, wherein Ri is selected from the group consisting of CO-OR₅, C ₂.₄ alkenyl and phenyl; with respect to R and R₃, the substituent is a member selected from the group consisting of halogen and straight or branched chain C_M alkyl; with respect to R₅, the substituent is a member selected from the group consisting of halogen, straight or branched chain C _O alkyl, C haloalkyl, C alkoxy, trifluro C₁.₄ alkyl, C_M alkylamidyl, nitro, phenyl, and phenoxy-C_1- alkyl; and with respect to R₆, the substituent is C_1-4 alkoxy.

7. A compound according to claim 1, wherein R_\ is CO-OR₅ or ethylenyl; R₂ and R₃ are each independently a member selected from the group consisting of methyl, ethyl, thiophenyl, chlorothiophenyl, bromothiophenyl, dimethyl t-butyl indenyl, bicyclo 2.2. L heptenyl, phenyl, chlorophenyl, flurophenyl, pyridinyl, furanyl, methylfuranyl, dimethylpyrazolopyramidme, and methylpropylesterbenzyl;

R₅ is a member selected from the group consisting of methyl, ethyl, methoxyphenyl, dimethoxyphenyl, pentanylphenyl, chlorothiophenyl, furanyl, methylphenyl, diphenylmethyl, chlorophenyl, dichlorophenyl, flurophenyl, bromophenyl, butyloxyphenyl, dimethylcamphor, phenylethylenyl, octanyl, t- butylphenyl, trifluromethylphenyl, di-trifluromethylphenyl, nitrochlorophenyl, butylphenyl, naphthalinyl, nifrophenylethylenyl, acetylamidylphenyl, nitromethylphenyl, phenylthiomethyl, phenoxymethyl, phenylcyclopentanyl, quinolinyl, thiophenyl, dinitrophenyl, isobutyloxybenxyl, ethylpropylate, benzyloxymethyl, and chloropropyl; and R₆ is phenyl or dimethoxyphenyl.

8. A compound according to one of the claims 1 to 7, wherein only one of R₂ and R₃ is aromatic.

9. A compound according to one of the claims 1 to 8, wherein said compound has an IC5₀ less than 10 μM in an in vitro inhibition of P 13-K activity or an IC₅o less than 20 μM in cellular inhibition of P 1 3-K activity.

10. A pharmaceutical composition comprising the compound or a salt thereof according to one of the claims 1 to 8 and a pharmaceutically acceptable carrier.

11. A method of screening and characterizing the potency of a test compound as an inhibitor of phosphatidylinositol 3-kinase (PI 3-K) polypeptide, said method comprising the steps of (a) measuring activity of a PI 3-K polypeptide in the presence of a test compound according to one of the claims 1 to 8; (b) comparing the activity of the PI 3-K polypeptide in the presence of the test compound to the activity of the PI 3-K polypeptide in the presence of an equivalent amount of a known PI 3-K inhibitor as a reference compound, wherein lower activity of the PI 3-K polypeptide in the presence of the test compound than in the presence of the reference compound indicates that the test compound is a more potent inhibitor than the reference compound, and higher activity of the PI 3-K polypeptide in the presence of the test compound than in the presence of the reference compound indicates that the test compound is a less potent inhibitor than the reference compound.

12. A method to treat a disorder in which P 1 3-K plays a role, comprising administering to a patient with said disorder an effective amount of the compound or a salt thereof according to one of the claims 1 to 8.

13. A method of claim 11, wherein the disorder is a cancer or a disease of immunity and inflammation.

14. A method of claim 11, wherein the disorder is disruption of PI 3-K function in leukocytes.

15. A method for inhibiting growth of cancer cells, comprising contacting said cancer cells with an effective amount of the compound or a salt thereof according to one of the claims 1 to 8.

16. A method according to claim 14, wherein said cancer cells are altered in PI 3-K mediated signaling via mutation in PTEN, amplification of the PIK3CA gene or mutations in PI 3 -Kinase.

17. A method according to claim 14, wherein said cancers include breast, prostate, colon, lung, ovarian, and other cancers having altered PI 3-K activities.

18. A method for affecting PI 3-K mediated signaling in cells comprising contacting said cells with an effective amount of the compound or a salt thereof according to one of the claims 1 to 8.

19. A method according to claim 17, wherein said compounds affect PI 3-K mediated phosphorylation of Akt.