Patent for “Inhibitors human phosphatidylinositol 3kinase delta”

Chemical Products – Chanchal Sadhu, Ken Dick, Jennifer Treiberg, C. Gregory Sowell, Edward A. Kesicki, Amy Oliver, Icos Corp

Search Patent for “Inhibitors human phosphatidylinositol 3kinase delta”

Abstract for “Inhibitors human phosphatidylinositol 3kinase delta”

“Methods to inhibit phosphatidylinositol-3kinase delta (PI3K)? Methods of treating diseases such as inflammation and disorders of immunity in which PI3K is involved, including methods for inhibiting phosphatidylinositol 3-kinase delta isoform (PI3K) These methods are designed to treat diseases such as inflammation and disorders of immunity that involve PI3K? The methods use active agents that inhibit PI3K? but not in significant ways. Are there compounds that inhibit PI3K’? Activity, as well as compounds that inhibit PI3K selectively. activity. How can PI3K be used? Inhibitory compounds are also provided to stop the growth and proliferation of cancer cells. The invention also provides methods for using PI3K. Inhibitors to inhibit PI3K-mediated processes in vitro or in vivo.”

Background for “Inhibitors human phosphatidylinositol 3kinase delta”

“Cell signaling via three-phosphorylatedphosphoinositides has been implicated with a variety cellular processes (e.g. malignant transformation, growth factor signals, inflammation and immunity) (see Rameh, J. Biol Chem 274:8347-8350 (1999), for a review). The enzyme that generates these phosphorylated signals products, phosphatidylinositol 3kinase (PI3K), was initially identified as an activity that is associated with viral oncoproteins (GFRTK) and growth factor receptor tyrosine kinases. It phosphorylates phosphatidylinositol (3?-hydroxyl) of the inositol ring. (Panayotou et.

“The levels of phosphatidylinositol-3,4,5-triphosphate (PIP3), the primary product of PI 3-kinase activation, increase upon treatment of cells with a variety of agonists. The activation of PI 3-kinase is thought to play a role in cell responses such as cell growth, differentiation and apoptosis. (Parker et.al., Current Biology 5:577-99 (1995); Yao, Science 267:2003-05 (1995).) Although the downstream targets for phosphorylated lipids that are generated by PI 3-kinase activation are not well understood, evidence suggests that FYVE-finger and pleckstrin-homology-containing proteins can be activated when they bind to various phosphatidylinositol lipids. (Sternmark et.al., J Cell Sci. 112:4175?83 (1999); Lemmon et.al., Trends Cell Biol. 7:237-42 (42). In vitro, PIP3 activates some forms of protein kinase C, while PKB (the PKC-related protein kinase) is activated by PI 3kinase (Burgering et.al., Nature, 376,599-602 (1995 )).”).

According to their substrate specificities, the PI-3-kinase enzyme group has been divided into three classes. Class I PI3Ks 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 PI3Ks phosphorylate PI and phosphatidylinositol-4-phosphate, whereas Class III PI3Ks can only phosphorylate PI.”

“The initial purification of PI 3-kinase and molecular cloning revealed it to be a heterodimer composed of p85 subunits and p110 (Otsu et.al., Cell 65:91-104 (1991); Hiles et.al., Cell 70:419-19-29 (1992).) Four distinct Class I PI3Ks were identified since then. They are referred to as PI3K?,?,?,?,and?. Each of them consists of a 110 kDa catalytic and regulatory subunit. Three of the catalytic units, p110, p110, and p110, are more specific. Each interacts with the same regulatory unit, p85, while p110 and p110 are distinct. interacts with a distinct regulatory unit, p101. The patterns of expression for each of these PI3Ks are different in human cells and tissues, as described below. Although there is a lot of information available about the cellular functions and roles of PI3Ks in the human body, it is still not clear what the specific roles of each of these PI3Ks play.

“Cloning of bovines p110?” has been described. This protein was found to be related to Saccharomyces cerevisiae’s protein Vps34p. It is involved in vacuolar protein production. Recombinant p110 The recombinant p110? was also demonstrated to associate with p85 to produce a PI3K activation in transfected COS-1 cell lines. See Hiles et al., Cell, 70, 419-29 (1992).”

Hu et. al., Mol Biol, 13 :7677-88 (1993) describes the cloning and characterization of a second human isoform of p110, called p110? This isoform, also known as p110, is thought to associate with cells’ p85. The mRNA was found in many tissues, including human and mouse umbilical vein endothelial cell, Jurkat human leukemic cells, 293 human embryonic renal cells, 293 mouse 3T3 cells, HeLa cells and NBT2 rats bladder carcinoma cells. This isoform appears to be important in signaling pathways, as evidenced by its wide expression.

“Identification of p110?” Chantry et. al., J Biol Chem 272:19236-41 (1997). The human p110 was found to be a phospholipin-like substance. The expression of this isoform is tissue-restricted. It is found in high amounts in lymphocytes and lymphoid tissue, suggesting that it might be involved in PI 3-kinase-mediated signaling within the immune system. More information about the P110? U.S. Pat. also contains details about the P110? Nos. Nos. 5,858,753, 5,822,910, and 5,985,589. Also see Vanhaesebroeck and al., Proc Natl Acad Sci USA 94:4330-5 (1997) and international publication WO97/46688.

“In each of PI3K??, &? subtypes, the p85 subunit acts to localize PI 3-kinase to the plasma membrane by interaction of its SH2 domain with phosphorylated tyrosine residues (present in an appropriate sequence context) (Rameh et al., Cell, 83:821-30 (1995). “In each of the PI3K?,?, and? subtypes, p85 acts to localize PI 3-kinase into the plasma membrane through the interaction between its SH2 domain and phosphorylated tyrosine (present in an appropriate context context in target proteins) (Rameh et.al., Cell. 83:821-30 (1995). There are two forms of p85: p85? which is widely expressed and p85? which is more common in lymphoid and brain tissues (Volinia and al., Oncogene 7:789-93 (1992).) The p85 subunit is associated with the PI 3-kinase p110??,?, or??? These enzymes are catalytically active and stable only if they have catalytic subunits. The binding of Ras proteins increases PI 3-kinase activities.

“The Cloning of P110” The PI3K enzyme family was complexer (Stoyanov et. al. Science, 269.690-93 (1995). The p110 isoform? The p110 isoform is closely related with p110? p110 and p110 are closely related. (45-48% identity within the catalytic domain, but as noted does NOT make use of p85 to target subunits. Instead, p110? It also contains a domain called a?pleckstrin domain homology domain? Near its amino terminus. This domain allows interaction with p110? This domain allows interaction of p110? with the??

“The p101 regulatory unit for PI3Kgamma, was initially cloned from swine and the human ortholog was identified later (Krugmann et.al., J Biol Chem 274:17152-8 (1999). Interaction of the N-terminal regions of p101 and p110 PI3K activation appears to be crucial activation via G? Previously mentioned.”

International publication WO 96/25488 describes a constitutively active PI3K oligopeptide. The publication describes the preparation of a Chimeric Fusion Protein. This protein is composed of a 102-residue p85 fragment known as the interSH2 (iSH2) area. It is linked to the N-terminus by a linker region. The p85 iSH2domain is apparently capable of activating PI3K activity in a way comparable to intact p85 (Klippel et.al., Mol Cell Biol. 14:2675-85 (1994 )).”).

“The amino acid identity and activity of PI 3-kinases are two ways to define them. Other members of this growing gene group include Vps34, TOR1, andTOR2 of Saccharomyces Cerevisiae (and mammalian homologs FRAP/mTOR), ataxia Telangiectasia Gene Product (ATR) as well as the catalytic subunit DNA-dependent proteinkinase (DNAPK). “See generally Hunter, Cell, 83.1-4 (1995).

“PI 3-kinase appears to also be involved in leukocyte activation in several aspects. It has been demonstrated that a p85-associated PI-3kinase activity can physically associate with CD28’s cytoplasmic domain, which is an important costimulatory molecule in the activation T-cells to respond to antigen (Pages et. al., Nature 369:327-29 (1994); Rudd and Immunity, 4:527-34) (1996). The threshold for activation by antigen is lower and the proliferative response is more intense. These effects are associated with increases in transcription of a variety of genes, including interleukin-2, which is an important T-cell growth factor (Fraser et. al. Science, 251(3)13-16 (1991). Mutations in CD28 that make it incompatible with PI-3kinase cause a failure of IL2 production. This suggests a crucial role for PI-3kinase during T cell activation.

“Specific inhibitors against specific enzymes are invaluable tools to decipher the functions of each enzyme. Two compounds, LY294002 (wortmannin), have been extensively used as PI-3-kinase inhibitors. These compounds are not specific PI3K inhibitors because they don’t distinguish between the four Class I PI3K members. The IC50 values for wortmannin against each Class I PI3-kinase are between 1-10 nM. The IC50 values of LY294002 against each one of these PI-kinases are approximately 1?M (Fruman et. al. Ann Rev Biochem 67:481-507 (1998)). These compounds are therefore not useful in studying the roles of Class I PI3-kinases.

Based on experiments using wortmannin, it is clear that PI-3 kinase function is also required for certain aspects of leukocyte signaling via G-protein coupled receptors. (Thelen et al. Proc Natl Acad Sci USA 91:4960-64 (1994). It has also been demonstrated that wortmannin, LY294002, and other compounds block neutrophil migration as well as superoxide production. These compounds don’t distinguish between the various PI3K forms, so it is not clear which PI3K type or isoforms are responsible for these phenomena.

“”

“Considering the above, it is evident that there is not much knowledge about the structural and functional characteristics of the PI-3 kinase enzymes. This includes subcellular localization, activation state, substrate affinities and so on. It remains to be determined how these enzymes function in normal and diseased tissues. Particularly, what is the role of PI3K? Its function in leukocytes is not known and its role in human physiology is unknown. It has been difficult to separate the activities of different PI3K isoforms from coexpression in these tissues. It is possible to separate the activities of different PI3K enzymes if there are inhibitors with selective inhibition properties. Applicants do not know of any such specific or selective inhibitors of PI3K.

“There is a need for structural characterization of the PI3K in the art.” polypeptide. Functional characterization of PI3K is also needed. Our understanding of PI3K is also needed. Further research is needed to understand the structural interactions between PI3K and its regulatory subunit. To better understand the functions of each isozyme, it is also necessary to develop selective or specific inhibitors for PI3K. Is it possible to develop selective or specific inhibitors for PI3K? These are important for examining the role of this enzyme and the development of pharmaceuticals that modulate its activity.”

“One aspect the invention is to provide compounds which can inhibit the biological activities of human PI3K?” The invention also provides compounds that inhibit PI3K. While inhibiting PI3K? selectively, they have a relatively low inhibitory power against other PI3K forms. The invention also provides methods for characterizing human PI3K? function. The invention also provides methods for selectively modulating human PI3K. activity and thereby promoting medical treatments for diseases mediated via PI3K? dysfunction. The artisan with ordinary skill in the art will also be able to see other aspects and benefits of the invention.

“It was discovered that the present invention can achieve these and other aspects. In one aspect, it is a method to disrupt leukocyte function. This involves contacting leukocytes using a compound that selectively inhibits Phosphatidylinositol 3-kinase Delta (PI3K). activity in the leukocytes. The method allows for the inclusion of cells from any group, including neutrophils and B lymphocytes as well as T lymphocytes and basophils.

“For example, if the neutrophils are present in the leukocytes, the method may include disrupting at most one neutrophil function from the group of stimulated superoxide, stimulated exocytosis and chemotactic movement. The preferred method does not significantly disrupt bacterial phagocytosis and bacterial killing by neutrophils. If the leukocytes are composed of B lymphocytes, then the method may include disrupting the proliferation of B lymphocytes and antibody production by B lymphocytes. If the leukocytes are composed of T lymphocytes, then the method may include disrupting the proliferation of these lymphocytes. If the leukocytes are composed of basophils, then the method may include disrupting histamine production by the basophils.

“In the methods described in the invention, a selective inhibitor of PI3K is used. It is preferable that the compound used to inhibit PI3K be at least 10 times more selective. Comparatively to other Type I PI3K forms in a cell-based test. It is preferable that the compound be at least 20-fold selective to inhibit PI3K. Comparable to other Type I PI3K forms in a cell-based assay. Even better, the compound should be at least 50-fold selective to inhibit PI3K. Comparative to other Type I PI3K forms in a biochemical assay.”

“Preferred Select Compounds Useful According to the Methods” includes compounds with the following structure (I).

“”

“wherein A can be an optionally substituted monocyclic, or bicyclic system of ring systems containing at minimum two nitrogen atoms and at least one aromatic ring;”

“X” is chosen from the group consisting C(Rb), CH2CHRb and CH?C (Rb);

“Y” is selected from the following groups: null, SO, SO2, NH and O, C(??O), OC?O, C(??O)O and NHC (?O) CH2S.

“R1 and R2, independently, are selected from the group consisting of hydrogen, C1-6alkyl, aryl, heteroaryl, halo, NHC(?O)C1-3alkyleneN(Ra)2, NO2, ORa, CF3, OCF3, N(Ra)2, CN, OC(?O)Ra, C(?O)Ra, C(?O)ORa, arylORb, Het, NRaC(?O)C1-3alkyleneC(?O)ORa, arylOC1-3-alkyleneN(Ra)2, arylOC(?O)Ra, C1-4alkyleneC(?O)ORa, OC1-4alkyleneC(?O)ORa, C1-4alkyleneOC1-4alkyleneC(?O)ORa, C(?O)NRaSO2Ra, C1-4alkyleneN(Ra)2, C2-6alkenyleneN(Ra)2, C(?O)NRaC1-4alkyleneORa, C(?O) NRaC1-4alkyleneHet, OC2-4-alkyleneN(Ra)2, OC1-4alkyleneCH(ORb)CH2N(Ra)2, OC1-4alkyleneHet, OC2-4alkyleneORa, OC2-4alkyleneNRaC(?O)ORa, NRaC1-4alkyleneN(Ra)2, NRaC(?O)Ra, NRaC(?O)N(Ra)2, N(SO2C1-4alkyl)2, NRa(SO2C1-4alkyl), SO2N(Ra)2, OSO2CF3, C1-3alkylenearyl, C1-4alkyleneHet, C1-6alkyleneORb, C1-3alkyleneN(Ra)2, C(?O)N(Ra)2, NHC(?O)C1-C3alkylenearyl, C3-8cycloalkyl, C3-8heterocycloalkyl, arylOC1-3-alkyleneN(Ra)2, arylOC(?O)Rb, NHC(?O)C1-3alkyleneC3-8-heterocycloalkyl, NHC(?O)C1-3alkyleneHet, OC1-4alkyleneOC1-4alkyleneC(?O)ORb, C(?O)C1-4alkyleneHet, and NHC(?O)haloC1-6alkyl;”

“Or R1 and R2 can be combined to form a 3-, 4-membered alkylene/alkenylene chain component of an 5- or 6-membered rings, optionally containing at most one heteroatom.

“R3 is selected from the group consisting of optionally substituted hydrogen, C1-6alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C1-4alkylenecycloalkyl, C2-6alkenyl, C1-3alkylenearyl, arylC1-3alkyl, C(?O)Ra, aryl, heteroaryl, C(?O)OR, C(?O)N(Ra)2, C(?S)N(Ra)2, SO2Ra, SO2N(Ra)2, S(?O)Ra, S(?O)N(Ra)2, C(?O)NRaC1-4-alkyleneORa, C(?O)NRaC1-4alkyleneHet, C(?O)C1-4alkylenearyl, C(?O)C1-4alkyleneheteroaryl, C1-4alkylenearyl optionally substituted with one or more of halo, SO2N(Ra)2, N(Ra)2, C(?O)ORa, NRaSO2CF3, CN, NO2, C(?O)Ra, ORa, C1-4alkyleneN(Ra)2, and OC1-4alkyleneN(Ra)2, C1-4-alkyleneheteroaryl, C1-4alkyleneHet, C1-4alkyleneC(?O)?C1-4alkylenearyl, C1-4alkyleneC(?O)C1-4alkyleneheteroaryl, C1-4alkyleneC(?O)Het, C1-4alkyleneC(?O)N(Ra)2, C1-4alkyleneORa, C1-4alkyleneNRaC(?O)Ra, C1-4alkyleneO?C1-4alkyleneORa, C1-4alkyleneN(Ra)2, C1-4alkyleneC(?O)?ORa, and C1-4alkyleneOC1-4alkyleneC(?O)ORa;”

“Ra is selected from the group consisting of hydrogen, C1-6alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C1-3alkyleneN(Rc)2, aryl, arylC1-3alkyl, C1-3-alkylenearyl, heteroaryl, heteroarylC1-3alkyl, and C1-3alkyleneheteroaryl;”

“Or two Ra groups can be combined to form a 5- or 6-membered ring. Optionally, it may contain at least one heteroatom.

“Rb is selected from the group consisting of hydrogen, C1-6alkyl, heteroC1-3alkyl, C1-3alkyleneheteroC1-3alkyl, arylheteroC1-3alkyl, aryl, heteroaryl, arylC1-3alkyl, heteroarylC1-3alkyl, C1-3alkylenearyl, and C1-3alkyleneheteroaryl;”

“Rc” is chosen from the following: hydrogen, C1-6alkyl and C3-8cycloalkyl. aryl and heteroaryl.

“Het is a heterocyclic ring with 5 or 6 members, which can be saturated, partially, or fully unsaturated. It contains at least one heteroatom from the group consisting oxygen, nitrogen, or sulfur and can optionally be substituted with C1-4alkyl, or C(??O)ORa.

“and pharmaceutically acceptable salts or solvates (e.g. hydrates) thereof.”

“wherein the compound has at most about a 10 fold selective inhibition of PI3K?” relative to other Type-I PI3K forms in a cell-based test.”

“Another embodiment of the invention consists in administering an effective amount to an animal that is suffering from a medical condition caused by neutrophils. This compound selectively inhibits phosphatidylinositol 3-kinase Delta (PI3K). activity in neutrophils. The method can be used to treat a variety of medical conditions, including those that are characterized by an unfavorable neutrophil function. These conditions include stimulated superoxide, stimulated exocytosis and chemotactic movement. The preferred method is that phagocytic activity and bacterial killing by neutrophils are substantially uninhibited.

“In another embodiment, the invention consists of contacting osteoclasts using a compound that selectively inhibits Phosphatidylinositol 3-kinase Delta (PI3K). osteoclasts. The method allows the compound to be made up of a moiety that preferentially binds with bone.

“In another embodiment, the invention consists of administering an effective amount to an animal of a compound that inhibits the phosphatidylinositol 3kinase delta. (PI3K) The compound inhibits phosphatidylinositol 3-kinase delta (PI3K) activity in osteoclasts. Osteoporosis is a preferred bone-resorption disorder that can be treated according to this method.

“In another embodiment, the invention consists of contacting cancer cells with a compound that inhibits phosphatidylinositol 3-kinase Delta (PI3K)?” activity in cancer cells. This can be used to inhibit the growth and proliferation of certain cancers, such as multiple myelomas, lymphomas, or leukemias.

“Another embodiment of the invention is a method for inhibiting kinase activation of a phosphatidylinositol-3-kinase Delta (PI3K)? Contacting the PI3K is one way to make a polypeptide. polypeptide containing a compound with the generic structure (I).

“Preferred compounds that are useful according to the method include compounds chosen from the following group:

“”

“wherein Y is chosen from the group consisting null, S and NH;

“R4” is chosen from the group consisting H, halo and NO2, OH. OCH3, CH3, CH3, and CF3

“R5” is chosen from the group that includes OCH3, H, and halo.

“Or R4 and R5 along with C-6, C-7, and C-7 of a quinazoline-ring system define a 5- to 6-membered aromatic rings optionally containing one, more, or all O, N or S atoms.

“R6” is chosen from the following: phenyls, alkoxyphenyls, alkylphenyls, alkylphenyls, biphenyls, benzyls, pyridinyls, morpholinyls, and C1-C6alkyl.

“Rd, independently, is selected from the group consisting of NH2, halo, C1-3alkyl, S(C1-3alkyl), OH, NH(C1-3alkyl), N(C1-3alkyl)2, NH(C1-3alkylenephenyl), and”

“and”

“q is 1 or 2.”

“Subject to the condition that at least one R4 or R5 is not H, when R6 contains phenyl or 2-chlorophenyl.”

“More preferable, the compound is chosen from the group consisting:

“”

“wherein Y is chosen from the group consisting null, S and NH;

“R7” is chosen from the group that includes H, halo (OH), OCH3, CH3, or CF3;

“R8” is chosen from the group that includes OCH3, H, and halo.

“Or R7, R8 along with C-6, C-7, and C-7 of a quinazoline-ring system define a 5- to 6-membered aromatic rings optionally containing one, more, or all O, N or S atoms.

“R9” is chosen from the following group: C1-C6alkyl phenyl and halophenyl.

“Rd, independently, is selected from the group consisting of NH2, halo, C1-3alkyl, S(C1-3alkyl), OH, NH(C1-3alkyl), N(C1-3alkyl)2, NH(C1-3alkylenephenyl); and”

“q is 1 or 2.”

“Subject to the condition that at least one R7 or R8 is distinct from 6-halo and 6,7-dimethoxy groups and that R9 differs from 4-chlorophenyl.”

“Another embodiment of the invention involves contacting leukocytes using a compound with a general structure (I) to disrupt their leukocyte function.

“In another embodiment, the invention consists of a class or compounds that have been observed inhibiting PI3K?” Activity in biochemical assays and cell-based assays. They are expected to have therapeutic benefits in medical conditions where PI3K is involved. Activity is too high or incontinent. The invention provides a class compositions having the structure (II).

“Preferably, compounds have a general structural (IV).”

“”

“wherein Y is chosen from the group consisting null, S and NH;

“R10” is chosen from the group consisting H, halo OH, OCH3, CH3, CF3, and CF3

“R11” is chosen from the group that includes OCH3, H, and halo.

“Or R10, R11, C-6, and C-7 of a quinazoline-ring system define a 5-or 6-membered aromatic rings optionally containing one, more O, N or S atoms;

“R12” is chosen from the following group: C1-C6alkyl phenyl and halophenyl.

“Rd, independently, is selected from the group consisting of NH2, halo, C1-3alkyl, S(C1-3alkyl), OH, NH(C1-3alkyl), N(C1-3alkyl)2, NH(C1-3alkylenephenyl), and”

“q is 1 or 2.”

“provided that:”

“(a) At least one R10 or R11 is distinct from the 6-halo and 6,7-dimethoxy group;

“(b) R12 differs from 4-chlorophenyl;

“(c) At least one of R10 or R11 is different than H when R12 contains phenyl, 2-chlorophenyl, and X is S.”

These and other features and benefits of the invention will be apparent from the detailed description and examples given herein. Although the examples and detailed description are intended to aid in understanding the invention, they are not meant to limit its scope.

“The invention contains compounds that inhibit selectively the activity of PI3K?” Further, the invention provides methods for inhibiting PI3K? The invention also provides methods for inhibiting PI3K activity. isozyme in cells, particularly leukocytes and osteoclasts as well as cancer cells. These methods can be used in vitro, ex vivo and in vivo.

Methods of selectively modulating PI3K are particularly beneficial. activity in the clinic to treat disease or disorders mediated via PI3K activity. Treatment of disorders or diseases characterized by excessive or inadvertent PI3K can be done. PI3K selective modulators can be used to treat PI3K activity According to the invention

“Other methods include the ability to further characterize the physiological role for the isozyme. The invention also provides pharmaceutical compositions that contain selective PI3K? inhibitors. Articles of manufacture containing a selective PI3K are also provided. Instructions for using the compound and an inhibitor compound (or pharmaceutical composition comprising it) are also provided. These and other useful embodiments are described in detail.

The use of compounds that inhibit, or preferably specifically inhibit, PI3K activity is a benefit in the methods described. Cells, both in vitro and in vivo. Endogenous PI3K is a cell that has been used in these methods. a recombinant introduction of one or more polynucleotides encoding a PI3K into cells is not possible. A biologically active fragment of a polypeptide is also possible. Exogenous PI3K is also possible. In this case, one or more polynucleotides encode PI3K. Recombinant techniques have allowed the introduction of a biologically active fraction or all of it into the cell.

“The cells can be grown in vivo (i.e. in living subjects such as animals or humans) where there is a PI3K. An inhibitor can be used to treat PI3K? The subject’s activity. The cells can also be isolated from a single cell or from a whole tissue for ex vivo and in vitro methods. The invention also includes the step of contacting a PI3K. The invention can be used to inhibit an enzyme or a biologically active fraction thereof. What is the PI3K? The PI3K can be a purified enzyme or an isolated enzyme. This enzyme is obtained from a natural source, such as cells or tissues that naturally express a PI3K. polypeptide without modification by recombinant tech) or from cells that have been modified to express exogenous enzyme.

“The term’selective Pi3K’? inhibitor? referred to as a compound that inhibits PI3K This isozyme works better than any other members of the PI3K isozyme family. A?selective? PI3K? inhibitor? It is believed that the compound is more selective for PI3K. compared to compounds that are generically and conventionally designated PI3K inhibiters, e.g. wortmannin and LY294002. LY294002 and wortmannin are also considered ‘nonselective PI3K inhibitors. Any compound that can selectively regulate PI3K negatively? As selective PI3K inhibitors, compounds of any type can regulate PI3K’s expression and activity. Inhibitors are used in the invention. Any compound that can selectively inhibit PI3K may also be used. As selective PI3K inhibitors, compounds that have acceptable pharmacological properties and expression can be used. Inhibitors in the therapeutic methods according to the invention

The relative effectiveness of compounds acting as inhibitors can be determined by first determining the concentration at which each compound inhibits an enzyme activity or other biological activity and then comparing the results. Typically, the concentration that inhibits 50% or more of an enzyme activity is preferred. This is known as the 50% inhibitory concentration, or?IC50. Using the most common techniques in the art, IC50 can be determined. An IC50 is generally determined by measuring enzyme activity in the presence of a variety of inhibitors. The inhibitor concentrations are then plotted against the experimentally determined enzyme activity values. The IC50 value is the concentration of an inhibitor that exhibits 50% enzyme activity. This is a comparison to the activity without any inhibitor. Analogously, other inhibitory levels can be determined by appropriate activity determinations. In some cases, it may be possible to set a 90% inhibitory concentration (IC90, etc.).

“Accordingly, a “selective” PI3K? inhibitor? Alternatively, a compound with a 50% inhibition rate (IC50) in relation to PI3K could be described. preferably less than 10 times, but more preferably between 20 and 30 folds, the IC50 value for any of the Class I PI3K members. The term “specific PI3K”? inhibitor? can be understood as a selective inhibitor of PI3K? An inhibitor compound with an IC50 in relation to PI3K preferably 50-fold or more, preferably 100-fold or more, more preferably 200-fold or more, but still lower than the IC50 for any other PI3K Class I members.

“The invention includes methods for inhibiting leukocyte function. The invention provides methods for inhibiting or suppressing the functions of neutrophils, T and B lymphocytes, and neutrophils. Unexpectedly, inhibition of PI3K has been discovered to be a factor in neutrophils. Activity inhibits neutrophil functions. The compounds of the invention can be used to inhibit classical neutrophil functions like stimulated superoxide, stimulated exocytosis and chemotactic movement. The invention allows for suppression of certain functions in neutrophils while not significantly affecting the functions of other cells. The selective PI3K does not significantly inhibit the phagocytosis by neutrophils. The invention contains inhibitor compounds.

“The invention also includes methods to inhibit neutrophil functions without significantly inhibiting phagocytosis. Any function that is mediated via PI3K can be considered as a neutrophil function. activation or expression. These functions include stimulated superoxide, stimulated exocytosis, or degranulation, chemotactic movement, adhesion (e.g., roll/tether neutrophils, activation of neutrophil activity and/or latching neutrophils to the endothelium), transmural emigration or migration through the endothelium into peripheral tissues. These functions are collectively known as?inflammatory functions? These functions are often related to the neutrophil response to inflammation. These inflammatory functions can be distinguished from those of the neutrophils, such as phagocytosis or killing of bacteria. The invention also includes methods for treating diseases in which some or all of the inflammatory functions of neutrophils is abnormal or unfavorable.

It has been further established by the invention of PI3K. plays a role for the stimulation of lymphocyte proliferation, including T cells and B cells. Moreover, PI3K? It appears that PI3K plays a role in stimulating the secretion antibodies by B cells. Is there a selective inhibitor of PI3K? These inhibitor compounds were used to prove that inhibition of PI3K can stop these phenomena. The invention also includes methods for inhibiting lymphocyte proliferation and inhibiting B lymphocyte antibody production. The invention also allows for treatment of diseases in which any one or more of these functions is abnormal or unfavorable.

“It is now clear that PI3K?” Activity can be selectively inhibited to aid in the treatment of a PI3K-mediated disease. This will also reduce or eliminate complications typically associated with concomitant inhibitions of other Class I PI 3-kinases. This embodiment can be illustrated by using members of a group of compounds that have been shown to selectively inhibit PI3K. Comparatively to other PI3K forms

“The methods described in this embodiment can be used with compounds of the general structure (III). The preferred methods use compounds that have been empirically shown to inhibit PI3K by at least 10 fold. Comparable to other PI3K forms. The following compounds can be used to practice the methods:

“It was further determined that the methods described in the invention can be beneficially applied using members of a group of compounds that exhibit the PI3K enzyme. Inhibitory activity is facilitated by PI3K inhibition. activity in diseases that are mediated by it. This embodiment can, for example, be used to practice the methods of the invention using compounds having the general structural (I).

“”

“wherein A can be an optionally substituted monocyclic, or bicyclic system of ring systems containing at minimum two nitrogen atoms and at least one aromatic ring;”

“X” is chosen from the group consisting C(Rb), CH2CHRb and CH?C (Rb);

“Y” is selected from the following groups: null, SO, SO2, NH and O, C(??O), OC?O, C(??O)O and NHC (?O) CH2S.

“R1 and R2, independently, are selected from the group consisting of hydrogen, C1-6alkyl, aryl, heteroaryl, halo, NHC(?O)C1-3alkyleneN(Ra)2, NO2, ORa, CF3, OCF3, N(Ra)2, CN, OC(?O)Ra, C(?O)Ra, C(?O)ORa, arylORb, Het, NRaC(?O)C1-3alkyleneC(?O)ORa, arylOC1-3-alkyleneN(Ra)2, arylOC(?O)Ra, C1-4alkyleneC(?O)ORa, OC1-4alkyleneC(?O)ORa, C1-4alkyleneOC1-4alkyleneC(?O)ORa, C(?O)NRaSO2Ra, C1-4alkyleneN(Ra)2, C2-6alkenyleneN(Ra)2, C(?O)NRaC1-4alkyleneORa, C(?O)NRaC1-4alkyleneHet, OC2-4-alkyleneN(Ra)2, OC1-4alkyleneCH(ORa)CH2N(Ra)2, OC1-4-alkyleneHet, OC2-4alkyleneORa, OC2-4alkyleneNRaC(?O)ORa, NRaC1-4alkyleneN(Ra)2, NRaC(?O)Ra, NRaC(?O)N(Ra)2, N(SO2C1-4alkyl)2, NRa(SO2C1-4alkyl), SO2N(Ra)2, OSO2CF3, C1-3alkylenearyl, C1-4alkyleneHet, C1-6alkyleneORb, C1-3alkyleneN(Ra)2, C(?O)N(Ra)2, NHC(?O)C1-C3alkylenearyl, C3-8cycloalkyl, C3-8heterocycloalkyl, arylOC1-3-alkyleneN(Ra)2, arylOC(?O)Rb, NHC(?O)C1-3alkyleneC3-8-heterocycloalkyl, NHC(?O)C1-3alkyleneHet, OC1-4alkyleneOC1-4alkyleneC(?O)ORb, C(?O)C1-4alkyleneHet, and NHC(?O)haloC1-6alkyl;”

“Or R1 and R2 can be combined to form a 3-, 4-membered alkylene/alkenylene chain component of an 5- or 6-membered rings, optionally containing at most one heteroatom.

“R3 is selected from the group consisting of optionally substituted hydrogen, C1-6alkyl, C3-8cycloalkyl, C3-8heterocycloalkyl, C1-4alkylenecycloalkyl, C2-6alkenyl, C1-3alkylenearyl, arylC1-3alkyl, C(?O)Ra, aryl, heteroaryl, C(?O)ORa, C(?O)N(Ra)2, C(?S)N(Ra)2, SO2Ra, SO2N(Ra)2, S(?O)Ra, S(?O)N(Ra)2, C(?O)NRaC1-4-alkyleneORa, C(?O)NRaC1-4alkyleneHet, C(?O)C1-4alkylenearyl, C(?O)C1-4alkyleneheteroaryl, C1-4alkylenearyl optionally substituted with one or more of halo SO2N(Ra)2, N(Ra)2, C(?O)ORa, NRaSO2CF3, CN, NO2, C(?O)Ra, ORa, C1-4alkyleneN(Ra)2, and OC1-4alkyleneN(Ra)2, C1-4alkyleneheteroaryl, C1-4alkyleneHet, C1-4alkylene-C(?O)C1-4alkylenearyl, C1-4alkyleneC(?O)C1-4alkyleneheteroaryl, C1-4alkyleneC(?O)Het, C1-4alkyleneC(?O)?N(Ra)2, C1-4alkyleneORa, C1-4alkyleneNRaC(?O)Ra, C1-4alkyleneOC1-4alkyleneORa, C1-4alkyleneN(Ra)2, C1-4alkyleneC(?O)ORa, and C1-4alkyleneOC1-4alkylene-C(?O)ORa;”

Summary for “Inhibitors human phosphatidylinositol 3kinase delta”