It has long been established that proteins are reversibly modified in response to many extracellular and intracellular stimuli. One such mechanism is the phosphorylation of proteins by ATP, wherein a phosphate group is added to the hydroxyl group-bearing side chain of serine, threonine, or tyrosine residues. This reaction is catalyzed by enzymes known as protein kinases, which transfer the γ-phosphate group from ATP to the side-chain hydroxyl groups of substrate proteins. The reaction is reversible in a hydrolysis reaction catalyzed in situ by phosphatase enzymes. These phosphorylation and hydrolysis reactions have been established as critical to intracellular signaling processes, regulation of cellular functions, and activation or deactivation of cellular processes.
In mammals, protein kinases tend to fall within three groups: the serine-threonine kinases (S/TKs); the tyrosine kinases (TKs); and the relatively dual function kinases that act as both S/TKs and TKs. TKs have been identified as associated with cell proliferation, activation, or differentiation, and excessive TK activity has been observed in many disease states including benign and malignant proliferative disorders and immune system disorders. Certain TKs have also been identified as mediators of angiogenesis and therefore involved in the progression of cancer and other diseases involving inappropriate vascularization. For example, it has been found that chronic niyelogenous leukemia (CML) is a result of a chromosomal abnormality resulting in production of an a typical TK in the form of a BCR-ABL fusion protein. Inhibitors targeting that fusion protein have been made, and one such inhibitor, imatinib mesylate (the mesylate salt of 4-[(4-methyl-1-piperazinyl)methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrinmidinyl]amino]-phenyl]benzamide), is now commercially available under the tradename Gleevec. See, e.g., Carroll et al. (1997) Blood 90:4947, Zimmerman et al. (1997) Bioorg. Med. Chem. Lett. 7:187, Bridges (2001) Chem. Rev. 101:2541, and U.S. Pat. No. 5,521,184 to Zimmerman.
A representative and important family of S/TK kinases are known as “protein kinase C” (PKC), which was identified in 1977 (Takai et al. (1977) J. Biol. Chem. 252:7603). PKC has 12 isoforms that fall into three groups, the conventional or c-PKCs, activated by diacylglycerol and calcium, the novel or n-PKCs, which do not require calcium for activation, and the a typical or a-PKCs, which require neither calcium nor diacylglycerol for activation. See Bridges (2001), supra, Nishizuka (1992) Science 258:607, and Dekker et al. (1994) Trends Biochem. Sci. 19:73. PKC and its various isoforms have been associated with a variety of disorders and diseases, including cancer, CNS disorders, Alzheimer's disease, cardiovascular disease, dermatological disorders, inflammation, autoimmune diseases such as rheumatoid arthritis, and diabetic complications.
Staurosporin, an indolocarbazole natural product, was identified as the first potent inhibitor of PKC, exhibiting an IC50 value of 2.7 nM. Tamaoki et al. (1986) Biochem. Biophys. Res. Commun. 135:397. Staurosporin is known to induce programmed cell death and has been used in conjunction with other anti-cancer drugs. Jacobson et al. (1996) J. Cell Biol. 133:1041.

While exhibiting inhibition in the low-nanomolar range, however, staurosporin inhibits five of the PKC isoforms with an IC50 below 10 nM, and inhibits many other kinases as well. The compound is not, therefore, useful as a selective PKC inhibitor.
Several additional inhibitors of PKC have been investigated for their inhibitory activity on the proliferation of several tumor cell lines. For example, the phorbol ester and bryostatins are known to bind and regulate PKC competitively with diacylglycerol. See, e.g., Wender et al. (1988) Proc. Natl. Acad. Sci. USA 85:7197, Wender et al. (1986) Proc. Natl. Acad. Sci. USA 83:4214, Wender et al. (1998) Pure Appl. Chem. 70:539, and Wender et al. (1998) J. Am. Chem. Soc. 120:4534. Bryostatin 1, for example, has been established as a potent activator of the c-PKCs.

Although the bryostatins have been known for some time, their low natural abundance, difficulty in isolation, and severely limited availability through total synthesis have impeded efforts to advance their clinical development. Chemically synthesized simplified analogues of the bryostatins have been disclosed, however, and have exhibited PKC inhibitory activity; see U.S. Patent Application Publication No. 2002/0137789 A1 to Wender et al.
Other classes of compounds known to bind to and regulate PKC are indolo[2,3-α]carbazoles and bisindolylmaleimides.

One recently developed bisindolylmaleimide, LY 333531, has been established as particularly selective for the β isoforms of PKC:

See Engel et al. (2000) Intl. J. Pharmaceutics 198(2):239, Jirousec et al. (1996) J. Med. Chem. 39(14):2664, U.S. Pat. No. 5,859,261 to Faul et al., and U.S. Pat. No. 6,117,861 to Engel et al.
While a number of kinase inhibitors have, accordingly, been studied and developed, there is an ongoing need for potent inhibitors that can be readily modified so as to achieve selectivity with respect to a particular kinase, and that can be readily synthesized from relatively simple starting materials. Optimal kinase inhibitors would also be quite potent while exhibiting very low toxicity.
The present invention is the result of extensive, systematic research in the design of novel kinase inhibitors in the form of isoindolone analogs, particularly analogs that derive from the use of staurosporin as a pharmacophoric template. To the best of applicants' knowledge, the compounds, compositions, and methods of the invention are completely unknown and completely unsuggested by the art.
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