Farnesyl pyrophosphate analogs

Details Farnesyl pyrophosphate analogs Farnesyl pyrophosphate analogs

1999-12-15

2001-09-04

Vollano, Jean F. (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Phosphorus esters

C560S020000

Reexamination Certificate

active

06284910

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to farnesyl pyrophosphate analogs and their use in modulating cellular chemistry. More specifically, the invention relates to unsubstituted and substituted anilinogeranyl pyrophosphates (AGPP), unsubstituted and substituted benzyloxyalkylisoprenyl pyrophosphates, the processes of making the same, intermediates for use in the process of making the same, and the methods of using the FPP analogs in substitution for farnesyl pyrophosphate (FPP) or as farnesyl transferase inhibitors (FTIs).
BACKGROUND OF THE INVENTION
Farnesyl pyrophosphate 1 (FPP) and its C
20
homolog, geranylgeranyl pyrophosphate (GGPP) are isoprenoids that are involved in a number of cellular processes including cholesterol biosynthesis, glycoprotein biosynthesis, vitamin and cofactor synthesis and protein prenylation. Recently, the enzyme farnesyltransferase (FTase) which can utilize FPP or GGPP as a substrate depending on the nature of the &bgr; subunit, has attracted considerable interest as a possible target for the design of chemotherapeutic agents.
An area of interest is the continued search for proteins that are post-translationally modified in cells as part of normal metabolism. Farnesylation has been shown to be required for the normal function of various proteins including Ras.
Mutated forms of the cellular Ras genes are among the most common genetic abnormalities in human cancer, occurring in 30% of all neoplasms. This frequency indicates an important role for aberrant Ras function in carcinogenesis (1). Ras proteins are synthesized as cytosolic precursors which localize to the inner leaflet of the plasma membrane only after undergoing a series of well defined posttranslational modifications (2). The first and obligatory step in this processing is the transfer by protein farnesyltransferase (FTase) of the 15-carbon isoprene farnesyl from farnesylpyrophosphate (FPP) to a cysteine residue located at the Ras C-terminus via a thioether linkage (2,3). This Cys residue forms part of the prenylation recognition sequence, CAAX (where A is an aliphatic residue and X is most often Met, Ser, or Gln), present in all Ras proteins (4). Prenylation is followed by proteolytic removal of the COOH-terminal tripeptide and carboxymethylation of the now terminal, prenylated cysteine (2). These modifications render the C-terminus hydrophobic, which is thought to promote the association of these proteins with the plasma membrane. Studies employing site-directed mutants within the Ras CAAX motif (5,6) or inhibitors of 3-hydroxyl-3-methylglutaryl CoA reductase (7), the rate limiting enzyme in isoprenoid biosynthesis, have demonstrated that isoprenylation is required for Ras proteins to become membrane associated and to induce cellular transformation.
Three distinct enzymes responsible for protein isoprenylation have been isolated and biochemically characterized (2). In addition to FTase, two geranylgeranyl-transferases (GGTases) have been isolated and have been shown to modify specific protein substrates. While the CAAX GGTase (also known as GGTase-1) geranylgeranylates proteins which end in a CAAL sequence, where C is cysteine, A is usually an aliphatic amino acid, and L is leucine (8,9), the Rab GGTase (also known as GGTase-2) catalyzes the attachment of two geranylgeranyl groups to paired carboxyl-terminal cysteines in members of the Rab family of GTP-binding proteins (10). These proteins terminate in a CC or CXC motif.
A number of basic issues pertaining to the biological function of protein isoprenylation, and the contribution of lipid modification in general, remain to be answered. While farnesylation of Ras is absolutely required for its biological effects and membrane localization, it is unknown whether the prenyl group functions as a hydrophobic membrane association signal (11-16) or by targeted protein-protein recognition (17,18). The functional significance of protein farnesylation versus geranylgeranylation to the biological activity of prenylated proteins is also unknown. Studies employing inhibitors of FTase have highlighted the importance of isoprenylation to the membrane association and activity of the Ras family of signal transduction proteins. However, inhibition of protein prenylation precludes relating the effects of many of the downstream events such as further modification of the target protein by palmitoylation, proteolysis and carboxymethylation to the biological function of Ras.
Extensive research studies have established an important contribution of aberrant Ras function to human carcinogenesis (88, 89) Consequently, there is considerable interest in the development of inhibitors of Ras function for use in cancer treatment. Since Ras function is absolutely dependent on lipid modification and tight association with the plasma membrane, the development of drugs that block Ras membrane association has attracted the greatest interest (89-91). FTase has proven to be a biochemical target for the development of inhibitors of post-translational processing of Ras that act as potent anti-Ras drugs.
Several general approaches have been employed in the identification and development of specific FTase inhibitors (reviewed in 88). First, high throughput random screens have been used to identify natural or library compounds that inhibit the ability of FTase to catalyze the addition of farnesyl to recombinant Ras in vitro. Second, Ras CaaX tetrapeptide sequences alone are sufficient to signal modification by farnesylation and can serve as potent inhibitors of FTase activity in vitro. Therefore, a variety of CaaX-based peptidomimetic compounds have been synthesized. Consequently, the potent and selective FTIs identified to date include a diverse collection of structurally unrelated compounds. However, the structural requirements of the prenyl group have not been uncovered.
The initial characterization of FTIs was done in fibroblast model systems transformed by oncogenic H-Ras (92, 93). These studies clearly showed that FTIs can potently and specifically inhibit H-Ras farnesylation, without affecting the modification of geranylgeranylated proteins. FTI inhibition of H-Ras processing correlated directly with inhibition of H-Ras-induced signaling, morphologic and growth transformation (94). Similar dramatic results were seen in in vivo tumor models using Ras-transformed rodent cells grown as xenografted tumors in nude mice (95), and in viral H-Ras transgenic mice in which salivary and mammary tumors occur stochastically in a high percentage of mice carrying the transgene (96, 97). An unexpected, but desired, aspect of these studies is the apparent lack of normal cell toxicity. This was unexpected because farnesylation is required for normal Ras function, which is critical for normal cell viability (97). The reason(s) for the relative resistance of normal cells to FTIs is presently not known.
Because of previous finding that FTIs were capable of inhibiting Ras transformation in animal model systems, and because of the widespread tendency to view FTIs as Ras inhibitors rather than FTase inhibitors, many investigators assumed that human tumors cells that contained mutated Ras proteins would be more sensitive to FTI action than those that did not. However, studies have shown that ras mutation status is not predictive of FTI sensitivity (98). Indeed, it is clear that these drugs interfere with the processing of other farnesylated proteins in addition to Ras. There is now evidence that FTIs may block Ras transformation, in part, by inhibiting the function of other farnesylated proteins, one or more of which may represent the critical target for FTI action (87, 99, 100).
It is clear that the cellular mechanism by which FTIs exert their biological activity is incompletely understood (88). Two central issues appear that must be addressed before the biochemical mechanism by which FTIs function can be understood. First, it is necessary to understand the biological significance of the isoprenoid lipid in the context of the function of a fully processed prenyl protein. For FTIs,

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