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Definitions

• the present invention relates generally to phosphatidylinositol 3-kinase (PI3K) enzymes, and more particularly to selective inhibitors of PI3K ⁇ activity and methods of using such inhibitors.
• PI3K phosphatidylinositol 3-kinase
• PI3K originally was identified as an activity associated with viral oncoproteins and growth factor receptor tyrosine kinases that phosphorylates phosphatidylinositol (PI) and its phosphorylated derivatives at the 3'-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol 2:358-60 (1992)).
• PIP3 phosphatidylinositol-3,4,5-triphosphate
• PI 3-kinase activation is believed to be involved in a range of cellular responses including cell growth, differentiation, and apoptosis (Parker et al., Current Biology, 5:577-99 (1995); Yao et al., Science, 267:2003-05 (1995)).
• PKC protein kinase C
• 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
• Class III PI3Ks can only phosphorylate PI.
• PI 3-kinase 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 PDKs have been identified, designated PI3K ⁇ , ⁇ , ⁇ , and ⁇ , each consisting of a distinct 110 kDa catalytic subunit and a regulatory subunit.
• pllO ⁇ three of the catalytic subunits, i.e., pllO ⁇ , pllO ⁇ , and pl lO ⁇ , each interact with the same regulatory subunit, p85; whereas pllO ⁇ interacts with a distinct regulatory subunit, plOl.
• pllO ⁇ interacts with a distinct regulatory subunit, plOl.
• bovine pi 10a Cloning of bovine pi 10a has been described. This protein was identified as related to the Saccharomyces cerevisiae protein: Vps34p, a protein involved in vacuolar protein processing. The recombinant pi 10a product was also shown to associate with p85 ⁇ , to yield a PI3K activity in transfected COS-I cells. See Hiles et al., Cell, 70, 419-29 (1992).
• pi lO ⁇ The cloning of a second human pi 10 isoform, designated pi lO ⁇ , is described in Hu et al., MoI Cell Biol, 13:7677-88 (1993).
• This isoform is said to associate with p85 in cells, and to be ubiquitously expressed, as pllO ⁇ mRNA has been found in numerous human and mouse tissues, as well as in human umbilical vein endothelial cells, Jurkat human leukemic T cells, 293 human embryonic kidney cells, mouse 3T3 fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells.
• pllO ⁇ isoform is broadly important in signaling pathways.
• 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 (Rameh et al., Cell, 53:821-30 (1995)).
• target proteins phosphorylated tyrosine residues (present in an appropriate sequence context) in target proteins.
• p85 ⁇ Two isoforms of p85 have been identified, p85 ⁇ , which is ubiquitously expressed, and p85 ⁇ , which is primarily found in the brain and lymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)).
• association of the p85 subunit to the PI 3-kinase pllO ⁇ , ⁇ , or ⁇ catalytic subunits appears to be required for the catalytic activity and stability of these enzymes.
• the binding of Ras proteins also upregulates PI 3-kinase activity.
• pllO ⁇ contains an additional domain termed a "pleckstrin homology domain" near its amino terminus. This domain allows interaction of pllO ⁇ with the ⁇ subunits of heterotrimeric G proteins and this interaction appears to regulate its activity.
• plOl regulatory subunit for PI3Kgamma was originally cloned in swine, and the human ortholog identified subsequently (Krugmann et al., J Biol Chem, 274:17152-8 (1999)). Interaction between the N-terminal region of plOl with the N-terminal region of pllO ⁇ appears to be critical for the PI3K ⁇ activation through G ⁇ mentioned above.
• a constitutively active PI3K polypeptide is described in International Publication No. WO 96/25488.
• This publication discloses preparation of a chimeric fusion protein in which a 102-residue fragment of p85 known as the inter-SH2 (iSH2) region is fused through a linker region to the N-terminus of murine pi 10.
• the p85 iSH2 domain apparently is able to activate PI3K activity in a manner comparable to intact p85 (Klippel et al., MoI Cell Biol, 74:2675-85 (1994)).
• PI 3-kinases can be defined by their amino acid identity or by their activity. Additional members of this growing gene family include more distantly related lipid and protein kinases including Vps34 TORI, TOR2 of Saccharomyces cerevisiae (and their mammalian homologs such as FRAP and mTOR), the ataxia telangiectasia gene product (ATR), and the catalytic subunit of DNA-dependent protein kinase (DNA-PK). See generally, Hunter, Cell, 83:1-4 (1995).
• PI 3-kinase also appears to be involved in a number of aspects of leukocyte activation.
• a p85-associated PI 3-kinase activity has been shown to physically associate with the cytoplasmic domain of CD28, which is an important costimulatory molecule for the activation of T- cells in response to antigen (Pages et al., Nature, 369:327-29 (1994); Rudd, Immunity, 4:527-34 (1996)).
• Activation of T cells through CD28 lowers the threshold for activation by antigen and increases the magnitude and duration of the proliferative response.
• interleukin-2 IL2
• T cell growth factor an important T cell growth factor
• PI 3-kinase inhibitors Two compounds, LY294002 and wortmannin, have been widely used as PI 3-kinase inhibitors. These compounds, however, are nonspecific PI3K inhibitors, as they do not distinguish among the four members of Class I PI 3- kinases.
• the IC 50 values of wortmannin against each of the various Class I PI 3-kinases are in the range of 1-10 nM.
• the IC 50 values for LY294002 against each of these PI 3- kinases is about 1 ⁇ M (Fruman et al., Ann Rev Biochem, 67:481-507 (1998)). Hence, the utility of these compounds in studying the roles of individual Class I PI 3-kinases is limited.
• LY294002 wortmannin [0016] Based on studies using wortmannin, evidence exists that PI 3-kinase function also is required for some aspects of leukocyte signaling through G-protein coupled receptors (Thelen et al., Proc Natl Acad Sci USA, 91 :4960-64 (1994)). Moreover, it has been shown that wortmannin and LY294002 block neutrophil migration and superoxide release. However, because these compounds do not distinguish among the various isoforms of PDK, it remains unclear which particular PI3K isoform or isoforms are involved in these phenomena.
• WO 2005/112935 discloses selective inhibitors of PI3K ⁇ , and indicates that they are useful to treat solid tumors such as carcinomas and sarcomas, as well as cancers involving vascular or lymphoreticular systems, lymphomas and hematological cancers such as myeloma and leukemia. It demonstrates that a selective inhibitor of PI3K ⁇ reduced tumor growth rates and vascularization significantly, and that when combined with a radiation treatment, the PI3K ⁇ inhibitor had a pronounced synergistic effect for reducing tumor vasculature development. Thus the compounds of the invention are useful to treat tumors by inhibiting angiogenesis, and they can be combined with other tumor treatments to provide a synergistic effect.
• PI3K ⁇ inhibitors While some selective PI3K ⁇ inhibitors are thus known, a need remains for additional therapeutic agents useful to treat proliferative disorders, such as cancer, and excessive or destructive immune reactions, such as asthma, rheumatoid arthritis, multiple sclerosis, and lupus.
• the present invention provides novel compounds that are potent inhibitors of PI3K ⁇ , and are highly selective for the delta isoform and much less active against other isoforms of PI3K. These compounds are useful for the treatment of disorders associated with excessive activity, accumulation or production of hematopoietic cells, especially lymphocytes and leukocytes, including lymphomas, leukemias, and excessive immune response disorders.
• One aspect of the present invention is to provide compounds that can inhibit the biological activity of human PI3K ⁇ . Another aspect of the present invention is to provide compounds that inhibit PI3K ⁇ selectively compared to the other PI3K isoforms and that have a good bioavailability. Still another aspect of the invention is to provide a method of selectively modulating human PI3K ⁇ activity, and thereby promote medical treatment of diseases mediated by PI3K ⁇ function or dysfunction. Yet another aspect of the invention is to provide a method of characterizing the function of human PI3K ⁇ .
• Another aspect of the present invention is to provide a method of disrupting leukocyte function comprising contacting leukocytes with a compound that selectively inhibits phosphatidylinositol 3-kinase delta (PI3K ⁇ ) activity in the leukocytes.
• the leukocytes can comprise cells selected from the group consisting of neutrophils, B lymphocytes, T lymphocytes, and basophils.
• the method comprises disrupting at least one neutrophil function selected from the group consisting of stimulated superoxide release, stimulated exocytosis, and chemotactic migration.
• the method does not substantially disrupt bacterial phagocytosis or bacterial killing by the neutrophils.
• the leukocytes comprise B lymphocytes
• the method comprises disrupting proliferation of the B lymphocytes or antibody production by the B lymphocytes.
• the leukocytes comprise T lymphocytes
• the method comprises disrupting proliferation of the T lymphocytes.
• the leukocytes comprise basophils
• the method comprises disrupting histamine release by the basophils.
• the PI3K ⁇ inhibitor is selective. It is preferred that the PDK ⁇ inhibitor is at least about 10-fold selective for inhibition of PDK ⁇ relative to other Type I PI3K isoforms in a biochemical assay. Preferably, the compound is at least about 20-fold selective, and more preferably, 30-fold selective, for inhibition of PI3K ⁇ relative to other Type I PI3K isoforms in a biochemical assay. In several embodiments, the compound is at least about 50-fold selective for inhibition of PI3K ⁇ relative to PI3K ⁇ in a biochemical assay.
• Compounds of the present invention are capable of inhibiting PDK ⁇ activity and have a structural formula (I):
• U, V, W, and Z independently, are selected from the group consisting of CR a , N, NR b , and O, or wherein at least one of U, V, W and Z is N, and the others of U, V, W and Z are selected from the group consisting of CR a , NR b , S, and O, and wherein at least one, but not all, of U, V, W, and Z is different from CR a ;
• A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms as ring memebers, and at least one ring of the system is aromatic;
• R 1 is selected from the group consisting of H, substituted or unsubstituted Ci-ioalkyl, substituted or unsubstituted C 2 _ioalkenyl, substituted or unsubstituted C 2 _ioalkynyl, substituted or unsubstituted Ci_ 6 perfluoroalkyl, substituted or unsubstituted C 3 _ 8 cycloalkyl, substituted or unsubstituted Cs-gheterocycloalkyl, substituted or unsubstituted substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylCi ⁇ alkyleneOR 6 , substituted or unsubstituted heteroarylCi ⁇ alkyleneN(R d ) 2 , substituted or unsubstituted heteroarylCi- 4 alkyleneOR e , substituted or unsubstituted Ci_ 3
• R d is selected from the group consisting of H, substituted or unsubstituted Ci-ioalkyl, substituted or unsubstituted C 2 _ioalkenyl, substituted or unsubstituted C 2 _ioalkynyl, substituted or unsubstituted C 3 _ 8 cycloalkyl, substituted or unsubstituted C 3 _ 8 heterocycloalkyl, substituted or unsubstituted Ci_3alkyleneN(R e ) 2 , aryl, substituted or unsubstituted arylCi_3alkyl, substituted or unsubstituted Ci_ 3 alkylenearyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylCi ⁇ alkyl, and substituted or unsubstituted Ci_ 3 alkyleneheteroaryl; or two R d groups are taken together with the nitrogen to which they are attached to form
• R e is selected from the group consisting of H, substituted or unsubstituted Ci_ 6 alkyl, substituted or unsubstituted C 3 _ 8 cycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, or two R e groups are taken together with the nitrogen to which they are attached to form a 5- or 6-membered ring, optionally containing a second heteroatom that is N, O or S; said A, R 1 , R a , R b , R c , and R d , independently, are optionally substituted with one to three substituents selected from the group consisting of Ci_ioalkyl, C 2 _ioalkenyl, C 2 _ioalkynyl, C3_8cycloalkyl, Ci_6alkyleneOR e , Ci_ 4 alkyleneN(R e ) 2 , aryl, Ci_ 3 alkylenearyl,
• Another aspect of the present invention is to provide a method of treating a medical condition mediated by neutrophils comprising administering to a mammal in need thereof a therapeutically effective amount of a compound that selectively inhibits phosphatidylinositol 3-kinase delta (PI3K ⁇ ) activity in the neutrophils.
• exemplary medical conditions that can be treated according to the method include those conditions characterized by an undesirable neutrophil function selected from the group consisting of stimulated superoxide release, stimulated exocytosis, and chemotactic migration.
• phagocytic activity or bacterial killing by the neutrophils is substantially uninhibited.
• Still another aspect of the present invention is to provide a method of disrupting a function of osteoclasts comprising contacting osteoclasts with a compound that selectively inhibits PI3K ⁇ activity in the osteoclasts.
• the compound comprises a moiety that preferentially binds to bone.
• Another aspect of the present invention is to provide a method of ameliorating a bone-resorption disorder in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of a compound that inhibits PI3K ⁇ activity in osteoclasts of the mammal.
• a preferred bone-resorption disorder amenable to treatment according to the method is osteoporosis.
• Another aspect of the present invention is to provide a method of ameliorating an excessive or undesired immune response in a mammal in need thereof comprising administering to the mammal a therapeutically effective amount of a compound that inhibits PI3K ⁇ activity in osteoclasts of the mammal.
• a therapeutically effective amount of a compound that inhibits PI3K ⁇ activity in osteoclasts of the mammal include but are not limited to asthma, rheumatoid arthritis, lupus erythematosus, and multiple sclerosis.
• disorders include disorders such as autoimmune thyroiditis, multiple sclerosis, some forms of diabetes, and Reynaud's syndrome; transplant rejection disorders such as GVHD and allograft 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.
• CIBD chronic inflammatory bowel disease
• CIBD chronic inflammatory bowel disease
• Crohn's disease 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 include fever and myalgias due to infection.
• Yet another aspect of the present invention is to provide a method of inhibiting the growth or proliferation of cancer cells of hematopoietic origin comprising contacting the cancer cells with a compound that selectively inhibits PI3K ⁇ activity in the cancer cells.
• the method can be advantageous in inhibiting the growth or proliferation of cancers selected from the group consisting of lymphomas, multiple myelomas, and leukemias.
• Another aspect of the present invention is to provide a method of inhibiting kinase activity of a PI3K ⁇ polypeptide comprising contacting the PI3K ⁇ polypeptide with a compound having a structural formula (I).
• Still another aspect of the present invention is to provide a method of disrupting leukocyte function comprising contacting leukocytes with a compound having a structural formula (I).
• Another aspect of the present invention is to provide compounds having a structural formula (I) that inhibit PI3K ⁇ activity in biochemical and cell-based assays, and exhibit a therapeutic benefit in treating medical conditions wherein PI3K ⁇ activity is excessive or undesirable.
• the present invention provides compounds that selectively inhibit the activity of PI3K ⁇ .
• the present invention further provides methods of inhibiting PI3K ⁇ activity, including methods of selectively modulating the activity of the PI3K ⁇ isozyme in cells, especially leukocytes, osteoclasts, and cancer cells.
• the methods include in vitro, in vivo, and ex vivo applications.
• the invention provides pharmaceutical compositions comprising a selective PI3K ⁇ inhibitor.
• articles of manufacture comprising a selective PI3K ⁇ inhibitor compound (or a pharmaceutical composition comprising the compound) and instructions for using the compound.
• Other methods of the invention include enabling the further characterization of the physiological role of the isozyme.
• the methods described herein benefit from the use of compounds that selectively inhibit, and preferably specifically inhibit, the activity of PI3K ⁇ in cells, including cells in vitro, in vivo, or ex vivo.
• Cells treated by methods of the present invention include those that express endogenous PI3K ⁇ , wherein endogenous indicates that the cells express PI3K ⁇ absent recombinant introduction into the cells of one or more polynucleotides encoding a PI3K ⁇ polypeptide or a biologically active fragment thereof.
• the present methods also encompass use of cells that express exogenous PI3K ⁇ , wherein one or more polynucleotides encoding PI3K ⁇ or a biologically active fragment thereof have been introduced into the cell using recombinant procedures.
• the cells can be in vivo, i.e., in a living subject, e.g., a mammal, including humans, wherein a PI3K ⁇ inhibitor can be used therapeutically to inhibit PI3K ⁇ activity in the subject.
• the cells can be isolated as discrete cells or in a tissue, for ex vivo or in vitro methods.
• In vitro methods encompassed by the invention can comprise the step of contacting a PI3K ⁇ enzyme or a biologically active fragment thereof with an inhibitor compound of the invention.
• the PI3K ⁇ 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 PI3K ⁇ polypeptide absent modification by recombinant technology) or isolated from cells modified by recombinant techniques to express exogenous enzyme.
• a natural source e.g., cells or tissues that normally express a PI3K ⁇ polypeptide absent modification by recombinant technology
• isolated from cells modified by recombinant techniques to express exogenous enzyme e.g., cells or tissues that normally express a PI3K ⁇ polypeptide absent modification by recombinant technology
• selective PDK ⁇ inhibitor refers to a compound that inhibits the PI3K ⁇ isozyme more effectively than other isozymes of the PI3K family.
• a "selective PI3K ⁇ inhibitor” compound is understood to be more selective for PI3K ⁇ than compounds conventionally and generically designated PI3K inhibitors, e.g., wortmannin or LY294002. Concomitantly, wortmannin and LY294002 are deemed “nonselective PI3K inhibitors.”
• compounds of the present invention selectively negatively regulate PI3K ⁇ expression or activity and possess acceptable pharmacological properties for use in the therapeutic methods of the invention.
• the relative efficacies of compounds as inhibitors of an enzyme activity can be established by determining the concentrations at which each compound inhibits the activity to a predefined extent, then comparing the results.
• the preferred determination is the concentration that inhibits 50% of the activity in a biochemical assay, i.e., the 50% inhibitory concentration or "IC 50 .”
• IC 50 determinations can be accomplished using conventional techniques known in the art. In general, an IC 50 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 is taken as the IC 50 value.
• 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., IC 90 .
• Compounds of the present invention exhibit an IC 50 value vs. PI3K ⁇ of about 10 ⁇ M or less. In several embodiments, the compounds have an IC 50 vs. PI3K ⁇ of less than 5 ⁇ M. In other embodiments, the compounds have an IC 50 value vs. PI3K ⁇ of less than l ⁇ m, for example, down to 1 nm.
• a "selective PI3K ⁇ inhibitor” alternatively can be understood to refer to a compound that exhibits a 50% inhibitory concentration (IC 50 ) with respect to PI3K ⁇ that is at least 10-fold, preferably at least 20-fold, and more preferably at least 30-fold, lower than the IC 50 value with respect to any or all of the other Class I PI3K family members.
• PI3K ⁇ inhibitor can be understood to refer to a selective PI3K ⁇ inhibitor compound that exhibits an IC 50 with respect to PI3K ⁇ that is at least 50-fold, preferably at least 100-fold, more preferably at least 200- fold, and still more preferably at least 500-fold, lower than the IC 50 with respect to any or all of the other PI3K Class I family members.
• the most preferred compounds of the present invention therefore, have a low IC 50 value vs. PI3K ⁇ (i.e., is a potent inhibitor), and are selective with respect to inhibiting PI3K ⁇ compared to the other PI3K isoforms.
• the present invention provides a method of inhibiting leukocyte function. More particularly, the present invention provides methods of inhibiting or suppressing functions of neutrophils and T and B lymphocytes.
• inhibition of PI3K ⁇ activity inhibits functions of neutrophils.
• the compounds of the present invention elicit inhibition of classical neutrophil functions, such as stimulated superoxide release, stimulated exocytosis, and chemotactic migration.
• a method of the present invention permits suppression of certain functions of neutrophils, while not substantially affecting other functions of these cells. For example, it has been observed that phagocytosis of bacteria by neutrophils is not substantially inhibited by the selective PI3K ⁇ inhibitor compounds of the present invention.
• the present invention includes methods of inhibiting neutrophil functions, without substantially inhibiting phagocytosis of bacteria.
• Neutrophil functions suitable for inhibition according to the present method include any function mediated by PI3K ⁇ activity or expression. Such functions include, without limitation, stimulated superoxide release, stimulated exocytosis or degranulation, chemotactic migration, adhesion to vascular endothelium (e.g., tethering/rolling of neutrophils, triggering of neutrophil activity, and/or latching of neutrophils to endothelium), transmural diapedesis, or emigration through the endothelium to peripheral tissues.
• these functions can be collectively termed "inflammatory functions," as they are typically related to neutrophil response to inflammation.
• the inflammatory functions of neutrophils can be distinguished from the bacterial killing functions exhibited by these cells, e.g., phagocytosis and killing of bacteria. Accordingly, the present invention further includes methods of treating disease states in which one or more of the inflammatory functions of neutrophils are abnormal or undesirable.
• PI3K ⁇ plays a role in the stimulated proliferation of lymphocytes, including B cells and T cells. Moreover, PI3K ⁇ appears to play a role in stimulated secretion of antibodies by B cells. Selective PI3K ⁇ inhibitor compounds of the present invention have been employed to establish that these phenomena can be abrogated by inhibition of PI3K ⁇ .
• the present invention includes methods of inhibiting lymphocyte proliferation, and methods of inhibiting antibody production by B lymphocytes. Other methods enabled by the present invention include methods of treating disease states in which one or more of these lymphocyte functions are abnormal or undesirable.
• PDK ⁇ activity can be inhibited selectively or specifically to facilitate treatment of a PI3K ⁇ -mediated disease, while reducing or eliminating complications that typically are associated with concomitant inhibition of the activity of other Class I PI 3-kinases.
• methods of the invention can be practiced using members of a class of compounds that have been found to exhibit selective inhibition of PDK ⁇ relative to other PI3K isoforms.
• the methods of this invention can be practiced using compounds having a general structural formula (I).
• Preferred methods employ compounds that have been determined to exhibit at least a 10-fold selective inhibition of PI3K ⁇ relative to other PI3K isoforms.
• the methods can be practiced using a racemic mixture of the compounds or a specific enantiomer.
• the S enantiomer is sometimes preferred when X represents a chiral center, such as CHR C where R° is not H.
• the methods of the invention can be practiced using members of a class of compounds that exhibit PI3K ⁇ inhibitory activity, thereby facilitating inhibition of PDK ⁇ activity in diseases mediated thereby.
• the methods of the invention can be practiced using compounds having the general structural formula (I):
• U, V, W, and Z independently, are selected from the group consisting of CR a , N, NR b , and O, or wherein at least one of U, V, W and Z is N, and the others of U, V, W and Z are selected from the group consisting of CR a , NR b , S, and O, and wherein at least one, but not all, of U, V, W, and Z is different from CR a ;
• A is an optionally substituted monocyclic or bicyclic ring system containing at least two nitrogen atoms as ring memebers, and at least one ring of the system is aromatic;
• R 1 is selected from the group consisting of H, substituted or unsubstituted Ci-ioalkyl, substituted or unsubstituted C 2 _ioalkenyl, substituted or unsubstituted C 2 _ioalkynyl, substituted or unsubstituted Ci_ 6 perfluoroalkyl, substituted or unsubstituted C 3 _ 8 cycloalkyl, substituted or unsubstituted Cs-gheterocycloalkyl, substituted or unsubstituted substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted arylCi ⁇ alkyleneOR 6 , substituted or unsubstituted heteroarylCi ⁇ alkyleneN(R d ) 2 , substituted or unsubstituted heteroarylCi- 4 alkyleneOR e , substituted or unsubstituted Ci_ 3
• R d is selected from the group consisting of H, substituted or unsubstituted Ci.ioalkyl, substituted or unsubstituted C 2 _ioalkenyl, substituted or unsubstituted C 2 _ioalkynyl, substituted or unsubstituted C 3 _ 8 cycloalkyl, substituted or unsubstituted C 3 _ 8 heterocycloalkyl, substituted or unsubstituted Ci_3alkyleneN(R e ) 2 , aryl, substituted or unsubstituted arylCi_ 3 alkyl, substituted or unsubstituted Ci_ 3 alkylenearyl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heteroarylCi ⁇ alkyl, and substituted or unsubstituted Ci_ 3 alkyleneheteroaryl; or two R d groups are taken together with the nitrogen to which they are attached to form
• R e is selected from the group consisting of H, substituted or unsubstituted Ci_ 6 alkyl, substituted or unsubstituted C 3 _ 8 cycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl, or two R e groups are taken together with the nitrogen to which they are attached to form a 5- or 6-membered ring, optionally containing a second heteroatom that is N, O or S; said A, R 1 , R a , R b , R c , and R d , independently, are optionally substituted with one to three substituents selected from the group consisting of Ci_ioalkyl, C 2 _ioalkenyl, C 2 _ioalkynyl, C 3 _ 8 cycloalkyl, C 3 _ 8 heterocycloalkyl, C ⁇ alkyleneOR 6 , C ⁇ alkyleneNCR 6 ) ⁇ aryl, C ⁇
• the compounds of the present invention are selective inhibitors of PDK ⁇ activity.
• the compounds exhibit inhibition of PDK ⁇ in biochemical assays, and selectively disrupt function of PI3K ⁇ -expressing cells in cell-based assays.
• the present compounds have demonstrated an ability to inhibit certain functions in neutrophils and other leukocytes, as well as functions of osteoclasts.
• alkyl is defined as straight chained or branched hydrocarbon groups or cyclic hydrocarbon groups containing the indicated number of carbon atoms, typically methyl, ethyl, and straight chain and branched propyl and butyl groups, and cyclopropyl, cyclopentyl and cyclohexyl groups, as well as combination of straight chain, branched chain and cyclic groups, e.g., cyclopropylmethyl and norbornyl.
• the hydrocarbon group can contain up to 16 carbon atoms, preferably one to eight carbon atoms.
• alkyl includes cyclic, bicyclic, and "bridged alkyl,” i.e., a C 6 -C 16 bicyclic or polycyclic hydrocarbon group, for example, norbornyl, adamantyl, bicyclo[2.2.2]octyl, bicyclo[2.2.1]heptyl, bicyclo[3.2.1]octyl, or decahydronaphthyl.
• cycloalkyl is defined as a cyclic C 3 -C 8 hydrocarbon group, e.g., cyclopropyl, cyclobutyl, cyclohexyl, and cyclopentyl.
• alkenyl is defined identically as “alkyl,” except the hydrocarbon groups contain at least one carbon-carbon double bond.
• alkynyl defined identically as “alkyl,” except the hydrocarbon groups contain at least one carbon-carbon triple bond.
• Cycloalkenyl is defined similarly to cycloalkyl, except at least one carbon-carbon double bond is present in the ring.
• perfluoroalkyl is defined as an alkyl group wherein each hydrogen atom is replaced by fluorine.
• alkylene is defined as an alkyl group having a substituent, for example, the term “Ci_ 3 alkylenearyl” refers to an alkyl group containing one to three carbon atoms, and substituted with an aryl group.
• alkylene when used without description of another group can refer to a divalent alkyl group, which can link two other structural features together, for example CH 2 and (CH 2 ) 3 are 1-carbon and 3-carbon alkylene groups.
• halo or halogen is defined herein to include fluorine, bromine, chlorine, and iodine. Often , fluoro or chloro is preferred.
• haloalkyl is defined herein as an alkyl group substituted with one or more halo substituents, either fluoro, chloro, bromo, iodo, or combinations thereof.
• halocycloalkyl is defined as a cycloalkyl group having one or more halo substituents.
• aryl alone or in combination, is defined herein as a monocyclic or polycyclic aromatic group, preferably a monocyclic or bicyclic aromatic group, e.g., phenyl or naphthyl.
• an "aryl” group can be unsubstituted or substituted, for example, with one or more, and in particular one to three, halo, alkyl, phenyl, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl.
• aryl groups include phenyl, naphthyl, biphenyl, tetrahydronaphthyl, chlorophenyl, fluorophenyl, aminophenyl, methylphenyl, methoxyphenyl, trifluoromethylphenyl, nitrophenyl, carboxyphenyl, and the like.
• arylCi_ 6 alkyl and “heteroarylCi_ 6 alkyl” are defined as an aryl or heteroaryl group having a C ⁇ alkyl substituent.
• heteroaryl is defined herein as a monocyclic or bicyclic ring system containing one or two aromatic rings and containing at least one nitrogen, oxygen, or sulfur atom in an aromatic ring, and which can be unsubstituted or substituted, for example, with one or more, and in particular one to three, substituents, like halo, alkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, haloalkyl, nitro, amino, alkylamino, acylamino, alkylthio, alkylsulfinyl, and alkylsulfonyl.
• heteroaryl groups include thienyl, furyl, pyridyl, oxazolyl, quinolyl, isoquinolyl, indolyl, triazolyl, isothiazolyl, isoxazolyl, imidazolyl, benzothiazolyl, pyrazinyl, pyrimidinyl, thiazolyl, and thiadiazolyl.
• Cs-sheterocycloalkyl is defined as monocyclic ring system containing one or more heteroatoms selected from the group consisting of oxygen, nitrogen, and sulfur.
• Nonlimiting examples of "C 3 _ 8 heterocycloalkyl” groups include 1,3-dioxolane, 2-pyrazoline, pyrazolidine, pyrrolidine, piperazine, a pyrroline, 2H-pyran, 4H-pyran, morpholine, thiopholine, piperidine, 1,4- dithiane, and 1,4-dioxane.
• hydroxy is defined as -OH.
• alkoxy is defined as -OR, wherein R is C1-C8 alkyl, C2-C8 alkenyl or C2-C8 alkynyl; each alkyl, alkenyl and alkynyl group is optionally substituted.
• alkoxyalkyl is defined as an alkyl group wherein a hydrogen has been replaced by an alkoxy group.
• (alkylthio) alkyl is defined similarly as alkoxyalkyl, except a sulfur atom, rather than an oxygen atom, is present.
• hydroxyalkyl is defined as a hydroxy group appended to an alkyl group.
• amino is defined as -NH 2
• alkylamino is defined as -NR 2 , wherein at least one R is alkyl and the second R is alkyl or hydrogen.
• alkylthio is defined as -SR, wherein R is alkyl.
• alkylsulfinyl is defined as R-SO, wherein R is alkyl.
• alkylsulfonyl is defined as R-SO 2 , wherein R is alkyl.
• amino is defined as -NH 2
• alkylamino is defined as -NR 2 , wherein at least one R is alkyl, alkenyl or alkynyl, and the second R is alkyl, alkenyl, alkynyl or hydrogen.
• nitro is defined as -NO 2 .
• trifluoromethyl is defined as -CF 3 .
• trifluoromethoxy is defined as -OCF 3 .
• cyano is defined as -CN.
• Alkyl, alkenyl and alkynyl groups are often substituted to the extent that such substitution makes sense chemically.
• Alkyl, alkenyl and alkynyl groups can also be substituted by C1-C8 acyl, C2-C8 heteroacyl, C6-C10 aryl or C5-C10 heteroaryl, each of which can be substituted by the substituents that are appropriate for the particular group.
• Aryl and heteroaryl moieties may be substituted with a variety of substituents including C1-C8 alkyl, C2-C8 alkenyl, C2-C8 alkynyl, C5-C12 aryl, C1-C8 acyl, and heteroforms of these, each of which can itself be further substituted; other substituents for aryl and heteroaryl moieties include halo,OR, NR 2 , SR, SO 2 R, SO 2 NR 2 , NRSO 2 R, NRCONR 2 , NRCOOR, NRCOR, CN, COOR, CONR 2 , 0OCR, COR, and NO 2 , wherein each R is independently H, C1-C8 alkyl, C2-C8 heteroalkyl, C2-C8 alkenyl, C2-C8 heteroalkenyl, C2-C8 alkynyl, C2-C8 heteroalkynyl, C6-C10 aryl, C5-C10 ary
• an arylalkyl substituent may be substituted on the aryl portion with substituents described herein as typical for aryl groups, and it may be further substituted on the alkyl portion with substituents described herein as typical or suitable for alkyl groups.
• Heteroforms' refers to a modified alkyl, alkenyl, aryl, etc. wherein at least one heteroatom selected from N, O and S replaces at least one carbon atom in the hydrocarbon group being described.
• Y is selected from the group consisting of null (i.e., Y is absent, so it represents a bond between X and A), S, and NH.
• X contains a chiral carbon, such as when X is CH(CH 3 ) or CH(CH 2 CH 3 ), it is often preferable to use the S-enantiomer of X. In other embodiments, the R-enantiomer may be used.
• X is CHR C or CHR C CHR C
• Y is NR C
• two of the R c groups cyclize to form a ring.
• the S enantiomer is often preferred in certain embodiments, and in some embodiments the R enantiomer is preferred.
• -X-Y- taken together can represent a ring such as this:
• the A ring can be monocyclic or bicyclic.
• Monocyclic A ring systems are aromatic.
• Bicyclic A ring systems contain at least one aromatic ring, but both rings can be aromatic.
• Examples of A ring systems include, but are not limited to, imidazolyl, pyrazolyl, 1,2,3-triazolyl, pyridizinyl, pyrimidinyl, pyrazinyl, 1,3,5-triazinyl, purinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 1,8-naphthyridinyl, pteridinyl, lH-indazolyl, and benzimidazolyl.
• A comprises at least one pyrimidine ring, e.g., they include purine and pteridine ring systems as well as imidazolpyrimidines, pyrazolopyrimidines and pyrrolopyrimidines.
• A is represented by an optionally substituted ring system selected from the group consisting of
• the A ring system optionally can be substituted with one to three, and preferably one or two, substituents selected from the group consisting of N(R e ) 2 , halo, Ci_ 3 haloalkyl, Ci_ 3 alkyl, S(Ci -3 alkyl), and OR e .
• substituents include, but are not limited to, NH 2 , NH(CH 3 ), N(CH 3 ) 2 , NHCH 2 C 6 H 5 , NH(C 2 H 5 ), Cl, F, CH 3 , CF 3 , SCH 3 , and OH.
• Especially preferred A rings include
• n is 0.
• preferred ring systems include, but are not limited to,
• the ring systems are unsubstituted (i.e., R a and R are hydro) or they may be substituted with substituents suitable for aryl or heteroaryl groups, preferably with one or more of Ci- 6 alkyl, halo, C ⁇ alkoxy, CF 3 , C 3 _ 8 cycloalkyl, aryl, or heteroaryl.
• substituents suitable for aryl or heteroaryl groups preferably with one or more of Ci- 6 alkyl, halo, C ⁇ alkoxy, CF 3 , C 3 _ 8 cycloalkyl, aryl, or heteroaryl.
• a substituent is on the atom represented by V, and in some embodiments it is on the atom represented by W.
• R 1 in formula (I) is selected from the group consisting of optionally substituted C h alky!, aryl, heteroaryl, C 3 _ 8 cycloalkyl, C 3 _ 8 heterocycloalkyl, C ! _ 4 alkyleneC 3 _ sheterocycloalkyl, Ci ⁇ alkylenecycloalkyl, and Ci ⁇ alkylenearyl.
• Specific R 1 groups include, but are not limited to, optionally substituted forms of:
• R 1 is preferably a phenyl group, heteroaryl group, or C 3 _ 8 cycloalkyl or C 3 _ 8 heterocycloalkyl, each of which is unsubstituted or is substituted with up to three substituents.
• the pyrimidin-4-one ring structure can be a 5,6-fused bicyclic or a 6,6-fused bicyclic system, and the following numbering of the ring structure is used for convenience:
• the purine ring structure is sometimes present as group A in formula (I) and for convenience the numbering of its ring structure, is
• A represents purine
• it is sometimes attached to Y at position 9 of the purine, and it is sometimes attached at position 6 of the purine.
• Y often represents S, NH or NR C
• the purine group is often further substituted at position 9 by, for example, an amine.
• the present invention includes all possible stereoisomers and geometric isomers of compounds of structural formula (I) having an asymmetric center, and includes not only racemic compounds, but also the optically active isomers as well.
• a compound of structural formula (I) When a compound of structural formula (I) is desired as a single enantiomer, it can be obtained either by resolution of the final product or by stereospecific synthesis from either isomerically pure starting material or use of a chiral auxiliary reagent. For example, see Z. Ma et al., Tetrahedron: Asymmetry, 8(6), pages 883-888 (1997). Resolution of the final product, an intermediate, or a starting material can be achieved by any suitable method known in the art. Specific stereoisomers exhibit an excellent ability to inhibit kinase activity of PI3K ⁇ .
• prodrug refers to compounds that are rapidly transformed in vivo to a compound having structural formula (I) or (II), for example, by hydrolysis. Prodrug design is discussed generally in Hardma et al. (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics, 9th ed., pp. 11-16 (1996). A thorough discussion of prodrugs is provided in Higuchi et al., Prodrugs as Novel Delivery Systems, Vol. 14, ASCD Symposium Series, and in Roche (ed.), "Bioreversible Carriers in Drug Design," American Pharmaceutical Association and Pergamon Press (1987).
• prodrugs can be converted into a pharmacologically active form through hydrolysis of, for example, an ester or amide linkage, thereby introducing or exposing a functional group on the resultant product.
• Prodrugs can be designed to react with an endogenous compound to form a water-soluble conjugate that further enhances the pharmacological properties of the compound, for example, increased circulatory half-life.
• prodrugs can be designed to undergo covalent modification on a functional group with, for example, glucuronic acid, sulfate, glutathione, amino acids, or acetate.
• the resulting conjugate can be inactivated and excreted in the urine, or rendered more potent than the parent compound.
• High molecular weight conjugates also can be excreted into the bile, subjected to enzymatic cleavage, and released back into the circulation, thereby effectively increasing the biological half-life of the originally administered compound.
• the PI3K ⁇ protein, as well as fragments thereof possessing biological activity, can be used for screening putative negative regulator compounds in any of a variety of drug screening techniques.
• a negative regulator of PI3K ⁇ is a compound that diminishes or abolishes the ability of PI3K ⁇ to carry out any of its biological functions.
• An example of such compounds is an agent that decreases the ability of a PI3K ⁇ polypeptide to phosphorylate phosphatidylinositol or to target appropriate structures within a cell.
• the selectivity of a compound that negatively regulates PI3K ⁇ activity can be evaluated by comparing its activity on the PI3K ⁇ to its activity on other proteins.
• Selective negative regulators include, for example, antibodies and other proteins or peptides that specifically bind to a PI3K ⁇ polypeptide, oligonucleotides that specifically bind to PI3K ⁇ polypeptides, and other nonpeptide compounds (e.g., isolated or synthetic organic molecules) that specifically interact with PI3K ⁇ polypeptides. Negative regulators also include compounds as described above, but which interact with a specific binding partner of PI3K ⁇ polypeptides.
• cytoplasmic regions of PDK ⁇ polypeptides that contact other proteins and/or localize PI3K ⁇ within a cell (1) cytoplasmic regions of PDK ⁇ polypeptides that contact other proteins and/or localize PI3K ⁇ within a cell;
• one target for development of modulators is the identified regulatory interaction of p85 with pi lO ⁇ , which can be involved in activation and/or subcellular localization of the pllO ⁇ moiety.
• Still other selective modulators include those that recognize specific regulatory or PDK ⁇ -encoding nucleotide sequences. Modulators of PDK ⁇ activity can be therapeutically useful in treatment of a wide range of diseases and physiological conditions in which aberrant PDK ⁇ activity is involved.
• the invention provides methods of characterizing the potency of a test compound as an inhibitor of PDK ⁇ polypeptide, said method comprising the steps of (a) measuring activity of a PDK ⁇ polypeptide in the presence of a test compound; (b) comparing the activity of the PDK ⁇ polypeptide in the presence of the test compound to the activity of the PDK ⁇ polypeptide in the presence of an equivalent amount of a reference compound (e.g., a PDK ⁇ inhibitor compound of the present invention, wherein a lower activity of the PDK ⁇ polypeptide in the presence of the test compound than in the presence of the reference indicates that the test compound is a more potent inhibitor than the reference compound, and a higher activity of the PDK ⁇ polypeptide in the presence of the test compound than in the presence of the reference indicates that the test compound is a less potent inhibitor than the reference compound.
• a reference compound e.g., a PDK ⁇ inhibitor compound of the present invention
• the invention further provides methods of characterizing the potency of a test compound as an inhibitor of PDK ⁇ polypeptide, comprising the steps of (a) determining an amount of a control compound (e.g., a PDK ⁇ inhibitor compound of the present invention) that inhibits an activity of a PI3K ⁇ 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 PI3K ⁇ 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.
• a control compound
• the method uses a reference inhibitory amount which is the amount of the compound than inhibits the activity of the PI3K ⁇ polypeptide by 50%, 60%, 70%, 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 PI3K ⁇ 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 further provides methods of identifying a negative regulator of PI3K ⁇ activity, comprising the steps of (i) measuring activity of a PI3K ⁇ polypeptide in the presence and absence of a test compound, and (ii) identifying as a negative regulator a test compound that decreases PI3K ⁇ activity and that competes with a compound of the invention for binding to PI3K ⁇ .
• the invention provides methods for identifying compounds that inhibit PI3K ⁇ activity, comprising the steps of (i) contacting a PI3K ⁇ polypeptide with a compound of the present invention in the presence and absence of a test compound, and (ii) identifying a test compound as a negative regulator of PI3K ⁇ activity wherein the compound competes with a compound of the invention for binding to PI3K ⁇ .
• the invention therefore provides a method of screening for candidate negative regulators of PDK ⁇ activity and/or to confirm the mode of action of candidate such negative regulators. Such methods can be employed against other PI3K isoforms in parallel to establish comparative activity of the test compound across the isoforms and/or relative to a compound of the invention.
• the PI3K ⁇ polypeptide can be a fragment of pi lO ⁇ that exhibits kinase activity, i.e., a fragment comprising the catalytic site of pi lO ⁇ .
• the PI3K ⁇ polypeptide can be a fragment from the pi lO ⁇ -binding domain of p85 and provides a method to identify allosteric modulators of PI3K ⁇ .
• the methods can be employed in cells expressing cells expressing PDK ⁇ 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 PI3K ⁇ polypeptide and the agent being tested then can be measured.
• Human PI3K 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.
• HTS high throughput screening
• 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.
• Chemical libraries can contain known compounds, proprietary structural analogs of known compounds, or compounds that are identified from natural product screening.
• Natural product libraries are collections of materials isolated from naturals sources, typically, microorganisms, animals, plants, or marine organisms. Natural products are isolated from their sources by fermentation of microorganisms followed by isolation and extraction of the fermentation broths or by direct extraction from the microorganisms or tissues (plants or animal) themselves. Natural product libraries include polyketides, nonribosomal peptides, and variants (including nonnaturally occurring variants) thereof. For a review, see Cane et al., Science, 282:63-68 (1998).
• Combinatorial libraries are composed of large numbers of related compounds, such as peptides, oligonucleotides, or other organic compounds as a mixture. Such compounds are relatively straightforward to design and prepare by traditional automated synthesis protocols, PCR, cloning, or proprietary synthetic methods. Of particular interest are peptide and oligonucleotide combinatorial libraries.
• Still other libraries of interest include peptide, protein, peptidomimetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
• combinatorial chemistry and libraries created thereby see Myers, Curr Opin Biotechnol, 5:701-07 (1997).
• Therapeutic Uses of Inhibitors of PDK ⁇ Activity see Myers, Curr Opin Biotechnol, 5:701-07 (1997).
• the invention provides a method for selectively or specifically inhibiting PI3K ⁇ activity therapeutically or prophylactically.
• the method comprises administering a selective or specific inhibitor of PDK ⁇ activity in an amount effective therefor.
• the method can be employed to treat humans or animals who are or can be subject to any condition whose symptoms or pathology is mediated by PI3K ⁇ expression or activity.
• Treating 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.
• 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.
• Inflammatory disorder 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 associated with an 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.
• 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 (GVHD), are further examples of inflammatory reactions of the specific defense system.
• GVHD graft versus host disease
• nonspecific defense system refers to inflammatory disorders that are mediated by leukocytes that are incapable of immunological memory (e.g., granulocytes, and macrophages).
• 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.
• ARDS adult (acute) respiratory distress syndrome
• 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
• Autoimmune disease 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 refers to any symptoms, tissue damage, or loss of tissue function resulting from allergy.
• Arthritic disease refers to any disease that is characterized by inflammatory lesions of the joints attributable to a variety of etiologies.
• Dermatis 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 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 neutrophils, basophils, and eosinophils), mast cells, dendritic cells, Langerhans cells, and endothelial cells).
• a stimulus including, but not limited to
• TNF ⁇ tumor necrosis factor alpha
• LPS lipopolysaccharide
• TNF ⁇ is an extraordinarily potent and promiscuous activator of inflammatory processes, being involved in activation of neutrophils and various other cell types, induction of leukocyte/endothelial cell adhesion, pyrexia, enhanced MHC class I production, and stimulation of angiogenesis.
• superoxide release can be stimulated by formyl-Met-Leu-Phe (fMLP) or other peptides blocked at the N-terminus by formylated methionine.
• fMLP formyl-Met-Leu-Phe
• Such peptides normally are not found in eukaryotes, but are fundamentally characteristic of bacteria, and signal the presence of bacteria to the immune system.
• Leukocytes expressing the fMLP receptor are stimulated to migrate up gradients of these peptides (i.e., chemotaxis) toward loci of infection.
• compounds of the present invention inhibit stimulated superoxide release by neutrophils in response to either TNF ⁇ or fMLP.
• Other functions of neutrophils including stimulated exocytosis and directed chemotactic migration, also have been shown to be inhibited by the PI3K ⁇ inhibitors of the invention. Accordingly, compounds of the present invention can be expected to be useful in treating disorders, such as inflammatory disorders, that are mediated by any or all of these neutrophil functions.
• the present invention enables methods of treating such diseases as arthritic diseases, such as rheumatoid arthritis, monoarticular arthritis, osteoarthritis, gouty arthritis, spondylitis; Behcet 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, vernal 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, e
• 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.
• 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 neutrophils in turn results in generation of reactive oxygen metabolites, which damage components of the involved tissue or organ.
• 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.
• vascular stroke including global and focal ischemia
• hemorrhagic shock myocardial ischemia or infarction
• organ transplantation organ transplantation
• cerebral vasospasm cerebral vasospasm.
• 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. It is expected that inhibition of PI3K ⁇ activity will result in reduced amounts of reperfusion injury in such situations.
• 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.
• 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 in 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.
• selective PDK ⁇ inhibitors of the present invention can be employed in methods of treating diseases of bone, especially diseases in which osteoclast function is abnormal or undesirable.
• compounds of the present invention inhibit osteoclast function in vitro. Accordingly, the use of such compounds and other PDK ⁇ selective inhibitors can be of value in treating osteoporosis, Paget's disease, and related bone resorption disorders.
• the present invention includes methods of using PI3K ⁇ 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.
• 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; leukemias, such as lymphocytic leukemias, chronic myeloid (myelogenous) leukemias, and the like.
• the present PI3K ⁇ inhibitory compounds can be used to inhibit or control the growth or proliferation of chronic myeloid (myelogenous) leukemia cells.
• the invention includes a method of suppressing a function of basophils and/or mast cells, thereby enabling treatment of diseases or disorders characterized by excessive or undesirable basophil and/or mast cell activity.
• a present compound can be used to selectively inhibit the expression or activity of PI3K ⁇ in the basophils and/or mast cells.
• the method employs a PI3K ⁇ inhibitor in an amount sufficient to inhibit stimulated histamine release by the basophils and/or mast cells.
• a present selective PI3K ⁇ inhibitors 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.
• allergic disorders including disorders such as chronic obstructive pulmonary disease (COPD), asthma, ARDS, emphysema, and related disorders.
• COPD chronic obstructive pulmonary disease
• asthma asthma
• ARDS emphysema
• emphysema emphysema
• a compound of the present invention can be administered as the neat chemical, but it is typical, and preferable, to administer the compound in the form of a pharmaceutical composition or formulation.
• the present invention also provides pharmaceutical compositions that comprise a chemical or biological compound (“agent") that is active as a modulator of PI3K ⁇ 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.
• 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.
• An optimal pharmaceutical formulation can 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.
• these pharmaceutical compositions can be formulated and administered systemically or locally.
• compositions are formulated to contain suitable pharmaceutically acceptable carriers, and optionally can 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.
• 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.
• penetrants appropriate to the particular barrier to be permeated are used in the formulation.
• penetrants are generally known in the art.
• the formulation can include stabilizing materials, such as polyols (e.g., sucrose) and/or surfactants (e.g., nonionic surfactants), and the like.
• 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.
• 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 ® 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.
• 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).
• compositions comprising the agent in dosages suitable for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art.
• 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.
• 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 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 arabic 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,
• 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.
• 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.
• 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 are 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.
• suitable acids for example, Berge et al., / Pharm Sci, 66:1 (1977), describe pharmaceutically acceptable salts in detail.
• 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, fumarate, hydrochloride, 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
• Basic addition salts can be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a carboxylic acid-containing moiety with a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or organic primary, secondary, or tertiary amine.
• a suitable base such as the hydroxide, carbonate, or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or organic primary, secondary, or tertiary amine.
• Pharmaceutically acceptable basic addition salts include, but are not limited to, cations based on alkali metals or alkaline earth metals such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like, and nontoxic quaternary ammonium and amine cations including ammonium, tetramethylammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, ethylammonium, diethylammonium, triethylammonium, and the like.
• Other representative organic amines useful for the formation of base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine, and the like.
• Basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain alkyl halides such as decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; arylalkyl halides such as benzyl and phenethyl bromides; and others. Products having modified solubility or dispersibility are thereby obtained.
• lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
• dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates
• 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, solvates, quaternary derivatives, and prodrugs, thereof.
• compositions comprising a compound of the invention formulated in a pharmaceutically acceptable carrier can be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
• 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 also are contemplated.
• a 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.
• conditions indicated on the label can include treatment of inflammatory disorders, cancer, and the like.
• compositions comprising an inhibitor of PDK ⁇ activity can be administered to the subject by any conventional method, including 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 administration.
• 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, e.g., POWDERJECT .
• 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.
• administration can be accomplished by inhalation or deep lung administration of sprays, aerosols, powders, and the like.
• parenteral administration is typically preferred.
• Formulations of the compounds to optimize them for biodistribution following parenteral administration would be desirable.
• the PI3K ⁇ inhibitor compounds can be administered before, during, or after administration of chemotherapy, radiotherapy, and/or surgery.
• the therapeutic index of the PI3K ⁇ 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.
• 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, 129:51 (1992); Trail et al., Science, 261:212 (1993); and Rowlinson- Busza et al., Curr Opin Oncol, 4:1142 (1992)).
• PI3K ⁇ inhibitor compounds and radioisotopes or chemotherapeutic agents can be conjugated to the same anti-tumor antibody.
• the PI3K ⁇ inhibitors can be delivered by any suitable method. Focal administration can be desirable, such as by intraarticular injection. In some cases, it can be desirable to couple the compounds to a moiety that can target the compounds to bone.
• a PI3K ⁇ 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)).
• the agents used in the methods of the invention should readily penetrate the blood brain barrier when peripherally administered. Compounds that cannot penetrate the blood brain barrier, however, can still be effectively administered by an intravenous route.
• 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.
• a therapeutically effective dose can be estimated initially from biochemical and/or cell-based assays. Then, dosage can be formulated in animal models to achieve a desirable circulating concentration range that modulates PI3K ⁇ expression 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 in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (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 LD 50 /ED 50 .
• 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 dosage for human use.
• the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
• 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.
• effective amount refers to an amount sufficient to modulate PI3K ⁇ 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 1000 mg/kg.
• dosage units of the active agent comprise from about 0.01 mg to about 1000 mg, preferably from about 0.1 mg to about 100 mg, depending upon the indication, route of administration, and severity of the condition, for example.
• a suitable dose can be calculated according to body weight, body surface area, or organ size.
• the final dosage regimen is determined by the attending physician in view of good medical practice, considering various factors that modify the action of drugs, e.g., the specific activity of the compound, 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 depends 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, continuous infusion, sustained release depots, or combinations thereof, as required to maintain desired minimum level of the agent.
• Short- acting pharmaceutical compositions i.e., short half- life
• Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks.
• PI3K heterodimeric complexes consisting of a pi 10 catalytic subunit and a p85 regulatory subunit were overexpressed using the BAC-TO-BAC ® HT baculovirus expression system (GIBCO/BRL), and then purified for use in biochemical assays.
• pi 105 A FLAG ® -tagged version of human pi 105 (SEQ ID NO: 1) (see Chantry et al., / Biol Chem, 272: 19236-41 (1997)) was subcloned using standard recombinant DNA techniques into the BamHl-Xbal site of the insect cell expression vector pFastbac HTb (Life Technologies, Gaithersburg, MD), such that the clone was in frame with the His tag of the vector.
• the FLAG ® system is described in U.S. Patent Nos.
• pi 10a Similar to the method used for pi 105, described above, a FLAG ® -tagged version of pllO ⁇ (see Volinia et al., Genomics, 24(3 ):427-477 (1994)) was subcloned in BamHl-Hindlll sites of pFastbac HTb (Life Technologies) such that the clone was in frame with the His tag of the vector.
• pl lO ⁇ A pllO ⁇ (see Hu et al., MoI Cell Biol, 73:7677-88 (1993)) clone was amplified from the human MARATHON ® Ready spleen cDNA library (Clontech, Palo Alto CA) according to the manufacturer's protocol using the following primers: 5' Primer
• the 5' primer was built to contain a FLAG ® tag in frame with the pi lO ⁇ sequence.
• the FLAG ® -pll0 ⁇ sequence was subcloned using standard recombinant techniques into the EcoRl-Notl sites of pFastbac HTa (Life Technologies), such that the clone was in frame with the His tag of the vector.
• pl lO ⁇ The pllo ⁇ cDNA (see Stoyanov et al., Science, 269:690-93 (1995)) was amplified from a human Marathon Ready spleen cDNA library (Clontech) according to the manufacturer's protocol using the following primers:
• a FLAG ® tag was subsequently attached to the 5' end of the pi lO ⁇ sequence and was cloned in the S ⁇ mHl-Spel sites of pFastbac HTb (Life Technologies) using standard recombinant DNA techniques, with the FLAG ® -1 lO ⁇ sequence in-frame with the His tag of the vector.
• p85 ⁇ A BamHl-EcoRl fragment of FLAG ® -tagged p85 cDNA (see Skolnik et al., Cell, 65:83-89 (1991)) was subcloned into the Bam ⁇ l-EcoRl sites of the vector pFastbac dual (Life Technologies).
• Recombinant baculoviruses containing the above clones were generated using manufacturer's recommended protocol (Life Technologies). Baculoviruses expressing His-tagged pllO ⁇ , pllO ⁇ , or pl lO ⁇ catalytic subunit and p85 subunit were coinfected into Sf21 insect cells. To enrich the heterodimeric enzyme complex, an excess amount of baculovirus expressing p85 subunit was infected, and the His-tagged pi 10 catalytic subunit complexed with p85 was purified on nickel affinity column. Since pi lO ⁇ does not associate with p85, Sf21 cells were infected with recombinant baculoviruses expressing His-tagged pllO ⁇ only. In an alternate approach, plOl can be cloned into baculovirus, to permit coexpression with its preferred binding partner pllO ⁇ .
• the 72-hour post- infected Sf21 cells (3 liters) were harvested and homogenized in a hypotonic buffer (20 mM HEPES-KOH, pH 7.8, 5 mM KCl, complete protease inhibitor cocktail (Roche Biochemicals, Indianapolis, IN), using a Dounce homogenizer.
• the homogenates were centrifuged at 1,000 x g for 15 min.
• the supernatants were further centrifuged at 10,000 x g for 20 min, followed by ultracentrifugation at 100,000 x g for 60 min.
• the soluble fraction was immediately loaded onto 10 mL of HITRAP ® nickel affinity column (Pharmacia, Piscataway, NJ) equilibrated with 50 mL of Buffer A (50 mM HEPES-KOH, pH 7.8, 0.5 M NaCl, 10 mM imidazole).
• Buffer A 50 mM HEPES-KOH, pH 7.8, 0.5 M NaCl, 10 mM imidazole.
• the column was washed extensively with Buffer A, and eluted with a linear gradient of 10-500 mM imidazole. Free p85 subunit was removed from the column during the washing step and only the heterodimeric enzyme complex eluted at 250 mM imidazole.
• a high throughput screen of a proprietary chemical library was performed to identify candidate inhibitors of PI3K ⁇ activity.
• PI3K ⁇ catalyzes a phosphotransfer from ⁇ -[ 32 P]ATP to PIP 2 /PS liposomes at the D3' position of the PIP 2 lipid inositol ring.
• This reaction is MgCl 2 dependent and is quenched in high molarity potassium phosphate buffer pH 8.0 containing 30 mM EDTA.
• This reaction is performed in the presence or absence of library compounds.
• the reaction products (and all unlabelled products) are transferred to a 96-well, prewetted PVDF filter plate, filtered, and washed in high molarity potassium phosphate. Scintillant is added to the dried wells and the incorporated radioactivity is quantitated.
• the 3X assay stocks of substrate and enzyme were made and stored in a trough (for robotics assays) or a 96-well, V-bottom, polypropylene plate (for manual assays). Reagents were stable for at least 3 hours at room temperature.
• the 3X substrate for the HTS contained 0.6 mM Na 2 ATP, 0.10 mCi/mL ⁇ -[ 32 P] ATP (NEN, Pittsburgh, PA), 6 ⁇ M PIP 2 /PS liposomes (Avanti Polar Lipids, Inc., Atlanta, GA), in 20 mM HEPES, pH 7.4.
• the 3X enzyme stock for the HTS contained 1.8 nM PI3K ⁇ , 150 ⁇ g/mL horse IgG (used only as a stabilizer), 15 mM MgCl 2 , 3 mM DTT in 20 mM HEPES, pH 7.4.
• HTS chemical high throughput screen
• DMSO dimethyl sulfoxide
• the reaction was initiated with 20 ⁇ L of 3X enzyme, incubated at room temperature for 10 minutes. This dilution established a final concentration of 200 ⁇ M ATP in the reaction volume.
• the reaction was stopped with 150 ⁇ L quench buffer (1.0 M potassium phosphate pH 8.0, 30 mM EDTA). A portion of the quenched solution (180 ⁇ L) then was transferred to a PVDF filter plate (Millipore #MAIP NOB prewetted with sequential 200 ⁇ L washes of 100% methanol, water, and finally 1.0 M potassium phosphate pH 8.0 wash buffer).
• the PVDF filter plate was aspirated under moderate vacuum (2-5 mm Hg), washed with 5 x 200 ⁇ L of wash buffer, and then dried by aspiration. The filter was subsequently blotted, allowed to air dry completely, and inserted into a Wallac counting cassette with 50 ⁇ L of Ecoscint scintillation cocktail added per well. The incorporated radioactivity was quantitated, and data were analyzed, after normalizing to the enzyme positive control (set at 100%), to identify the curve intersection at the 50% inhibition value to estimate IC 50 values for the inhibitors. The inhibitors also were subjected to selectivity assays against PI3K ⁇ and PI3K ⁇ (see assay protocol in Example 9).
• PI3K ⁇ is expressed at significant levels in leukocytes, it is important to study the effects of the PI3K ⁇ -selective inhibitor on leukocyte functions. Accordingly, the effects of PI3K ⁇ inhibition in several types of leukocytes were examined. Neutrophils were examined to determine the effects that selective inhibition of PI3K ⁇ might elicit (Example 3, below). It surprisingly was found that selective inhibition of PI3K ⁇ activity appears to be significantly associated with inhibition of some but not all functions characteristic of activated neutrophils. In addition, the effects of PI3K ⁇ inhibition on B cell and T cell function also were tested (Examples 4-5, below).
• Superoxide generation is one of the hallmarks of neutrophil activation.
• a variety of activators potentiate superoxide generation by neutrophils.
• the effect of a present PI3K ⁇ inhibitor on superoxide generation by three different agonists: TNFl ⁇ , IgG, and fMLP, each representing separate classes of activator, is measured.
• Superoxide generated by the neutrophils is measured by monitoring a change in absorbance upon reduction of cytochrome C by modification of the method described by Green et al., (pp. 14.5.1-14.5.11 in Supp. 12, Curr Protocols Immunol (Eds., Colligan et al.) (1994)), as follows.
• activated neutrophils In addition to superoxide generation, activated neutrophils also respond by releasing several proteases that are responsible for the destruction of tissues and cartilage during inflammation. As an indication of protease release, the effect of present compound on elastase exocytosis is measured. Elastase exocytosis is quantitated by modification of the procedure described by Ossanna et al. (/ Clin Invest, 77: 1939-1951 (1986)), as follows.
• Purified human neutrophils (0.2 x 10 6 ) (treated with either DMSO or a serial dilution of a present compound in DMSO) are stimulated with fMLP in PBS containing 0.01 mg/mL cytochalasin B, 1.0 ⁇ M sodium azide (NaNs), 5 ⁇ g/mL L-methionine and 1 ⁇ M fMLP for 90 min at 37 0 C in a 96- well plate.
• Absorbance at 410 nm is monitored for 2 hr in a 96- well plate reader. To measure the relative amounts of elastase excytosed, all absorbance values are normalized to the values without any inhibitor.
• PI3K ⁇ inhibitor compounds of the present invention inhibit fMLP-induced elastase exocytosis significantly, and do so in a dose- dependent fashion. D. Measurement of fMLP-induced human neutrophil migration
• Neutrophils have the intrinsic capacity to migrate through tissues, and are one of the first cell types to arrive at the sites of inflammation or tissue injury. The effect of the present compounds on neutrophil migration towards a concentration gradient of fMLP is measured. The day before the migration assays are performed, 6-well plates are coated with recombinant ICAM- 1/Fc fusion protein (Van der Vieren et al., Immunity, 3:683-690 (1995)) (25 ⁇ g/mL in bicarbonate buffer, pH 9.3) and left overnight at 4 0 C.
• 1% agarose solution in RPMI-1640 with 0.5% bovine serum albumin (BSA), is added to wells with or without an inhibitor, and plates are placed into a refrigerator before punching holes in the gelled agarose to create plaques (1 central hole surrounded by 6 peripheral ones per well).
• BSA bovine serum albumin
• Human neutrophils are obtained as described above, and resuspended in RPMI medium supplemented with 0.5% BSA at 5 x 10 cells/mL. After combining equal volumes of neutrophil suspension and medium (either with DMSO or a serial dilution of the test compound in DMSO), neutrophils are aliquoted into the peripheral holes, while the central hole received fMLP (5 ⁇ M). Plates are incubated at 37 0 C in the presence of 5% CO 2 for 4 hr, followed by termination of migration by the addition of 1% glutaraldehyde solution in D-PBS. After removing the agarose layer, wells are washed with distilled water and dried.
• PI3K ⁇ inhibitor compounds of the present invention have an effect on neutrophil migration, inhibiting this activity in a dose-dependent manner.
• PI3K ⁇ inhibitor compounds of the present invention affect certain neutrophil functions, whether the compounds affect neutrophil-mediated bacterial killing is of interest.
• the effect of the compounds on neutrophil-mediated Staphylococcus aureus killing is studied according to the method described by Clark and Nauseef (pp. 7.23.4-7.23.6 in Vol. 2, Supp. 6, Curr Protocols Immunol (Eds., Colligan et al.) (1994)).
• Purified human neutrophils (5 x 10 6 cells/mL) (treated with either DMSO or a serial dilution of present compound in DMSO) are mixed with autologous serum. Overnight-grown S.
• aureus cells are washed, resuspended in HBSS, and added to the serum-opsonized neutrophils at a 10: 1 ratio.
• Neutrophils are allowed to internalize the bacteria by phagocytosis by incubation at 37 0 C for 20 min.
• the noninternalized bacteria are killed by 10 units/mL lysostaphin at 37 0 C for 5 min and the total mixture is rotated at 37 0 C.
• Samples are withdrawn at various times for up to 90 min and the neutrophils are lysed by dilution in water. Viable bacteria are counted by plating appropriate dilutions on trypticase-soy-agar plate and counting the S. aureus colonies after overnight growth.
• Heparinized blood 200 mL from healthy volunteers is mixed with an equal volume of D-PBS, layered on 10 x 10 mL FICOLL-PAQUE ® (Pharmacia), and centrifuged at 1600 rpm for 30 min at room temperature.
• Peripheral blood mononuclear cells PBMC
• FBS fetal bovine serum
• FBS fetal bovine serum
• cells are incubated with DYNAL ® Antibody Mix (B cell kit) (Dynal Corp., Lake Success, NY) for 20 min at 4-8 0 C.
• B cells are aliquoted at 50-75 x 10 3 cells/well into 96- well plate with or without inhibitor, to which IL-2 (100 U/mL) and PANSORB IN ® (Calbiochem) Staphylococcus aureus cells (1:90,000) were added. Part of the media is removed after 24-36 hr, and fresh media (with or without inhibitor) and IL-2 is added. Cultures are incubated at 37 0 C, in the presence of a CO 2 incubator for additional 7 days. Samples from each condition (in triplicate) are removed, and analyzed for IgG and IgM, as measured by ELISA.
• IL-2 100 U/mL
• PANSORB IN ® Calbiochem Staphylococcus aureus cells
• IMMULON ® 4 96-well plates are coated (50 ⁇ L/well) with either 150 ng/mL donkey antihuman IgG (H+L) (Jackson ImmunoResearch, West Grove PA), or 2 ⁇ g/mL donkey antihuman IgG+IgM (H+L) (Jackson ImmunoResearch) in bicarbonate buffer, and left overnight at 4 0 C.
• B cells are stimulated using PANSORB IN ® .
• the effect compounds of the present invention on B cell proliferation response when they are stimulated through their cell surface IgM using anti-IgM antibody also was measured.
• Murine splenocytes (Balb/c) are plated into 96-well microtiter plates at 2 x 10 5 cells per well in 10% FBS/RPMI. Appropriate dilutions of test inhibitor in complete medium are added to the cells and the plates are incubated for 30-60 minutes prior to the addition of stimulus.
• an F(ab') 2 preparation of goat antibody specific for the ⁇ -chain of mouse IgM is added to the wells at a final concentration of 25 ⁇ g/mL.
• the plates are incubated at 37 0 C for 3 days and 1 ⁇ Ci of [ 3 H]- thymidine is added to each well for the final four hours of culture.
• the plates are harvested onto fiber filters, washed, and the incorporation of radiolabel is determined using a beta counter (Matrix 96, Packard Instrument Co., Downers Grove, IL) and expressed as counts per minute (CPM).
• Compounds of the present invention inhibit anti-IgM-stimulated B cell proliferation in a dose-dependent manner. Because compounds of the present invention inhibit B cell proliferation, it is envisioned that these compounds and other PI3K ⁇ inhibitors could be used to suppress undesirable proliferation of B cells in clinical settings. For example, in B cell malignancy, B cells of various stages of differentiation show unregulated proliferation. Based on the results shown above, one can infer that PDK ⁇ selective inhibitors could be used to control, limit, or inhibit growth of such cells.
• T cell proliferation in response to costimulation of CD3+CD28 is measured.
• T cells are purified from healthy human blood by negative selection using antibody coated magnetic beads according to the manufacturer's protocol (Dynal) and resuspended in RPMI. The cells are treated with either DMSO or a serial dilution of a present compound in DMSO and plated at 1 x 10 cells/well on a 96-well plate precoated with goat antimouse IgG. Mouse monoclonal anti-CD3 and anti-CD28 antibodies then are added to each well at 0.2 ng/mL and 0.2 ⁇ g/mL, respectively.
• the plate is incubated at 37 0 C for 24 hr and [ 3 H] -thymidine (1 ⁇ Ci/well) is added. After another 18-hr incubation, the cells are harvested with an automatic cell harvester, washed, and the incorporated radioactivity was quantified.
• the present PDK ⁇ inhibitor compounds inhibited anti-CD3- and anti- CD28-induced proliferation of T cells, an effect is not as strong as an effect on B cells or on some of the functions of neutrophils.
• mouse bone marrow cells are isolated and differentiated to osteoclasts by treating the cells with Macrophage Colony Stimulating Factor "1 (mCSF 1 ) and Osteoprotegerin Ligand (OPGL) in serum- containing medium ( ⁇ MEM with 10% heat-inactivated FBS; Sigma) for 3 days. On day four, when the osteoclasts had developed, the medium is removed and cells are harvested.
• mCSF 1 Macrophage Colony Stimulating Factor "1
• OPGL Osteoprotegerin Ligand
• the osteoclasts are plated on dentine slices at 10 cells/well in growth medium, i.e., ⁇ MEM containing 1% serum and 2% BSA with 55 ⁇ g/mL OPGL and 10 ng/mL mCSF "1 .
• the medium is changed to 1% serum and 1% BSA, with or without osteopontin (25 ⁇ g/mL) and the PI3K inhibitors (100 nM).
• the medium is changed every 24 hours with fresh osteopontin and the inhibitors.
• the medium is removed, and the dentine surfaces are washed with water to remove cell debris and stained with acid hematoxylin. Excess stain is washed and the pit depths are quantitated using confocal microscopy.
• the present PI3-kinase inhibitors had an inhibitory effect on osteoclast function. Both the nonspecific inhibitors LY294002 and wortmannin inhibited osteoclast activity. However, the present PDK ⁇ inhibitor compounds had a greater effect, and in some cases almost completely inhibited osteoclast activity.
• the compounds of the present invention can be prepared by the following schemes and examples. Additional methods and examples that can readily be adapted by those skilled in the art for making the compounds of the invention are disclosed in WO 2005/113554 and in a U.S. Provisional Application entitled THIENOPYRIMIDINONES FOR TREATMENT OF INFLAMMATORY DISORDERS AND CANCERS, by White, et al., filed on November 13, 2006 under attorney docket no. 61608-3000100; and in U.S. Patent Nos. 6,518,277; 6,667,300; 6,949,535; and 6,800,620, and in published U.S. Patent Application US 2006/0106038.
• A3 l-Methyl-5-nitro-lH-pyrazole-4-carboxylic acid phenylamide.
• a solution of commercially available l-methyl-5-nitro-lH-pyrazole-4-carboxylic acid (2.5 g, 14.6 mmol) in toluene (10 mL) was treated with DMF (3 drops), followed by addition of thionyl chloride (5.3 mL, 73.0 mmol). The resulting mixture was heated at reflux for 16 h, then cooled to room temperature. The cooled solution then was concentrated by rotary evaporation to provide a residue.
• A4 S-Amino-l-methyl-lH-pyrazole ⁇ -carboxylic acid phenylamide.
• a solution of l-methyl-5-nitro-lH-pyrazole-4-carboxylic acid phenylamide (1.5 g, 6.09 mmol) in acetic acid (25 mL) was treated with zinc dust (2.39 g, 36.5 mmol) at room temperature.
• the resulting mixture was allowed to stir at room temperature for 20 min., then filtered through filter paper.
• the filtrate was concentrated by rotary evaporation to provide a residue that was dissolved in methylene chloride (20 mL).
• A5 N-(2-Methyl-4-phenylcarbomoyl-2H-pyrazolo-3-yl)-propionimidic acid methyl ester.
• A6 6-Ethyl-l-methyl-5-phenyl-l,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one.
• a solution of N-(2-methyl-4-phenylcarbamoyl-2H-pyrazolo-3-yl)-propionimidic acid methyl ester (760 mgs, 2.65 mmol) in trimethyl orthopropionate (5 mL) was treated with acetic acid (0.30 mL) at room temperature. The resulting solution was heated at reflux for 4 d. Reaction was allowed to cool to room temperature, and a white precipitate formed. The reaction mixture was filtered through filter paper, and the product was recovered as a tan solid.
• LC/MS API-ESI, AcOH 0.05%) m/z 255 (MH+).
• A7 3-Bromo-6-(l-bromo-ethyl)-l-methyl-5-phenyl-l,5-dihydro-pyrazolo[3,4- d]pyrimidin-4-one.
• acetic acid 11 mL
• potassium acetate 1.35 g, 13.7 mmol
• bromine 0.704 mL, 13.7 mL
• C2 5-Amino-3-methyl-isoxazole-4-carboxylic acid phenylamide.
• a solution of commercially available 5-amino-3-methyl-isoxazole-4-carboxylic acid (200 mg, 1.41 mmol) and aniline (321 ⁇ L, 3.52 mmol) in pyridine (2.5 mL) was cooled to -2O 0 C, then treated with phosphorous oxychloride (164 ⁇ L, 1.76 mmol). After 25 minutes at -2O 0 C, the reaction mixture was allowed to warm to room temperature for 16 h.
• C3 5-(2-Chloro-acetylamino)-3-methyl-isoxazole-4-carboxylic acid phenylamide.
• a solution of 5-amino-3-methyl-isoxazole-4-carboxylic acid phenylamide (103 mg, 0.474 mmol) in methylene chloride (2 mL) was treated with chloroacetyl chloride (37 ⁇ L, 0.474 mmol), and stirred at room temperature for 16 h. The solution then was treated with DIEA (83 mL, 0.474 mmol).
• C4 6-Chloromethyl-3-methyl-5-phenyl-5H-isoxazolo[5,4-d]pyrimidin-4-one.
• a solution of 5-(2-chloro-acetylamino)-3-methyl-isoxazole-4-carboxylic acid phenylamide (45 mg, 0.153 mmol) in phosphorous oxychloride (7 mL) was heated in a sealed tube at 13O 0 C for 1 h 40 min. The reaction mixture then was concentrated by rotary evaporation to provide a tan residue. The residue was dissolved in ethyl acetate (1OmL), and treated with saturated aqueous sodium bicarbonate solution (10 mL). The organic layer was separated, dried over magnesium sulfate, filtered, and concentrated by rotary evaporation to provide pure product without purification.
• LC/MS (AP-ESI, AcOH 0.05%) m/z 276 (MH+).
• F2 3-Amino-pyridine-2-carboxylic acid phenylamide.
• DIEA 3-Amino-pyridine-2-carboxylic acid phenylamide.
• Aniline 3.3 mL. 36.2 mmol was added, and the reaction mixture was cooled to -2O 0 C. After 10 minutes, POCI 3 and the reaction was allowed to stir for 4 h. Water (50 mL) was added, and the reaction mixture was allowed to stir for 18 h.
• F4 2-(l-Chloro-ethyl)-3-phenyl-3H-pyrido[3,2-d]pyrimidin-4-one.
• a solution of 3- (2-chloro-propionylamino)-pyridine-2-carboxylic acid phenylamide (625 mg, 2.06 mmol) in phosphorous oxychloride (5 mL) was heated in a sealed tube at 125 0 C for 48 h, then cooled to room temperature and concentrated to provide a residue.
• F5 2-(l-Amino-ethyl)-3-phenyl-3H-pyrido[3,2-d]pyrimidin-4-one.
• a solution of 2- (l-chloro-ethyl)-3-phenyl-3H-pyrido[3,2-d]pyrimidin-4-one (304 mg, 1.06 mmol) in 7N NH 3 /Me0H was heated in a sealed tube at 85 0 C for 26.5 h, then cooled to room temperature and concentrated to provide the product.
• LC/MS (AP-ESI, AcOH 0.05%) m/z 267 (MH+).
• Compound 1 6-[l-(6-Amino-purin-9-yl)-ethyl]-3-bromo-l-methyl-5-phenyl-l,5- dihydro-pyrazolo[3,4-d]pyrimidin-4-one.
• General procedure A stirring solution of 3-bromo-6-(l- bromo-ethyl)-l-methyl-5-phenyl-l,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one (45 mg, 0.109 mmol) in DMF (2 mL) was treated with adenine (15 mg, 0.111 mmol), followed by the addition of potassium carbonate (15 mg, 0.109 mmol).
• Compound 2 3-Bromo-l-methyl-5-phenyl-6-[l-(9H-purin-6-ylsulfanyl)-ethyl]-l,5- dihydro-pyrazolo[3,4-d]pyrimidin-4-one.
• the ratios of the IC 50 values of the compounds for PI3K ⁇ , PI3K ⁇ , and PI3K ⁇ relative to PDK ⁇ are given, respectively, as “Alpha/Delta Ratio,” “Beta/Delta Ratio,” and “Gamma/Delta Ratio.”
• test compounds were weighed out and dissolved into 10-50 mM stocks in 100% DMSO (depending on their respective solubilities) and stored at -2O 0 C. Compounds were thawed (to room temperature or 37 0 C), diluted to 300 ⁇ M in water from which a 3- fold dilution series into water was done. From these dilutions, 20 ⁇ L was added into the assay wells alongside water blanks used for the enzyme (positive) control and the no enzyme (background) control. The rest of the assay was essentially done according to the selectivity assay protocol in Example 2.

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