Raf inhibitors and uses thereof

This application claims the benefit of U.S. Provisional Application No. 60/792,314, filed Apr. 17, 2006, the contents of which are incorporated herein by reference in their entirety.

There are three RAF isoforms in humans: A-RAF, B-RAF and C-RAF (Marais and Marshall. Cancer Surv. 27:101-125 (1996)). These serine/threonine protein kinases are components of a conserved signaling pathway downstream of the membrane-bound small G protein RAS, which is activated by growth factors, hormones, and cytokines (Robinson and Cobb, Curr. Opin. Cell Biol. 9:180-186 (1997)). RAS stimulates RAF activation, which then leads to activation of the MEK kinase and subsequently the ERK kinase. Depending on the cellular context, this pathway mediates diverse biological functions such as cell growth, survival and differentiation predominantly through the regulation of transcription, metabolism and cytoskeletal rearrangements.

The RAS-RAF signaling pathway has long been associated with human cancers because oncogenic mutations in the ras gene occur in at least 15% of all human cancers (Davies, H. et al., Nature 417:949-954 (2002)), and the downstream kinase ERK is hyperactivated in 30% of cancers (Allen, et al., Semin. Oncol. 30:105-116 (2003)). However, for more than a decade, the RAF proteins had been considered to be important in cancer only because of their position downstream of RAS. This view was changed radically when activating mutations of B-RAF were found at a high frequency in human cancer, implicating B-RAF as a critical initiator and promoter of malignancy (Davies, H. et al., Nature 417:949-954 (2002)).

Activating mutations in the B-RAF protooncogene underlie 70% of melanomas, 50% of papillary thyroid cancers and 10% of colon cancers (Tuveson, et al., Cancer Cell 4:95-98 (2003); and Xing, Endocrine-Related Cancer: 12:245-262 (2005). Approximately 90% of these mutations occur as a single-nucleotide substitution that converts a valine to glutamate at amino acid 600 (V600E) in the kinase domain of B-RAF. This mutation increases the basal kinase activity of B-RAF, resulting in the activation of the MEK and ERK proteins that ultimately leads to uncontrolled tumor cell growth. Significantly, B-RAF and RAS mutations are usually mutually exclusive in the same tumor types, suggesting that these genes are on the same oncogenic signaling pathway and that RAS acts to activate B-RAF in these tumors.

Recent studies have found that knockdown of mutant B-RAF by small interference RNA in human melanoma cells inhibits both MEK and ERK kinases, causing growth arrest and ultimately promoting apoptosis (Sharma, et al., Cancer Res. 65:2412-2421 (2005); and Wellbrock et al., Cancer Res. 64:2338-2342 (2004)). In addition, data obtained from a short-hairpin RNA xenograft models targeting mutant B-RAF have shown that tumor regression resulting from B-RAF suppression is inducible, reversible, and tightly regulated (Hoeflich et al., Cancer Res. 66:999-1006 (2006). Taken together, gain-of-function B-RAF signaling is strongly associated with in vivo tumorigenicity, confirming B-RAF as an important target for cancer therapeutics.

The references cited herein are not admitted to be prior art to the claimed invention.

The present invention provides a compound of formula I or pharmaceutically acceptable salts thereof

wherein

X is O, S(O)_p;

p is an integer from 0 to 2;

R₁ is halogen, —CN, —NO₂, —OH, —O—(C₁-C₈ substituted or unsubstituted alkyl), —O—(C₁-C₈ fluoroalkyl), —(CH₂)_0-3—CO₂H, —(CH₂)_0-3—C(O)O-alkyl, -(C₁-C₈ substituted or unsubstituted alkyl), —(C₁-C₈ fluoroalkyl), —(C₃-C₈ cycloalkyl), —(C₃-C₈ fluorocycloalkyl), O-aryl(substituted or unsubstituted), —O-heteroaryl (substituted or unsubstituted), —NR₆R₇, —N₈—C(O)R₉, —NR₈—C(O)-fluoroalkyl, —NR₈—C(O)— aryl(substituted or unsubstituted), —(CH₂)_0-3—C(O)NR₆R₇, —NR₈SO₂R₉, —NR₉C(O)NR₆R₇, —NR₈C(S)NR₆R₇, —OSO₂NR₆R₇, —C(N—OH)NH₂, —C(N—OH)R₈, —CH₂OR₉, —OC(O)NR₆R₇, —SR₉, or —C(O)NR₈SO₂R₉;

R₂ is hydrogen, halogen, —CN, —NO₂, —OH, —O—(C₁-C₈ substituted or unsubstituted alkyl), —O—(C₁-C₈ fluoroalkyl), —(CH₂)_0-3—CO₂H, —(CH₂)_0-3—C(O)O-alkyl, —(C₁-C₈ substituted or unsubstituted alkyl), —(C₁-C₈ fluoroalkyl), —(C₃-C₈ cycloalkyl), —(C₃-C₈ fluorocycloalkyl), O-aryl(substituted or unsubstituted), —O-heteroaryl (substituted or unsubstituted), —NR₆R₇, —NR₉—C(O)R₉, —NR₉—C(O)-fluoroalkyl, —NR₈—C(O)-aryl (substituted or unsubstituted), —(CH₂)_0-3—C(O)NR₆R₇, —NR₈SO₂R₉, —NR₈C(O)NR₆R₇, —NR₈C(S)NR₆R₇, —OSO₂NR₆R₇, —C(N—OH)NH₂, —C(N—OH)R₈, —CH₂OR₈, —OC(O)NR₆R₇, —SR₉, or —C(O)NR₈SO₂R₉, and R₁, R₂, taken together, may form a ring;

R₃ and R₄ are independently hydrogen, substituted or unsubstituted lower alkyl, —COOH, —COO₈, or —C(O)NR₁₀R₁₁;

R₅ is selected from the group consisting of C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, heterocyclyl, and aryl; wherein said C₁-C₁₀ alkyl, C₃-C₁₀ cycloalkyl, heterocyclyl, and aryl groups may be substituted with one or more substituents independently selected from the group consisting of: hydroxyl group, —COOH, oxo, fluorine, thiol, cyano, nitro, —NR₆R₇, C₁-C₆ alkylthio, arylthio, C₁-C₆ alkyl, C₁-C₈ fluoro-substituted alkyl, C₁-C₆ alkoxy, C₁-C₈ fluoro-substituted alkoxy, —O-aryl, —OC(═O)-alkyl, —OC(═O)-aryl, C₃-C₈ cycloalkyl, C₃-C₈ fluoro-substituted cycloalkyl, C₃-C₈ cycloalkyloxy, C₃-C₈ fluoro-substituted cycloalkyloxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, aryl, —C(═O)—NR₈R₉, C₁-C₆ alkylcarbonyl, C₃-C₈ cycloalkylcarbonyl, heterocyclylcarbonyl, —C(═O)-aryl, —C(═O)—O-aryl, C₁-C₆ alkoxycarbonyl, C₁-C₆ fluoro-substituted alkoxycarbonyl, C₃-C₈ cycloalkyloxycarbonyl, heterocyclyloxycarbonyl, C₁-C₆ alkylsulfonyl, arylsulfonyl, and heterocyclyl;