WO2000016626A1

WO2000016626A1 - A method of treating cancer

Info

Publication number: WO2000016626A1
Application number: PCT/US1999/022224
Authority: WO
Inventors: Samuel L. Graham; Kenneth S. Koblan; David C. Heimbrook; Allen I. Oliff; Steven M. Stirdivant
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 1998-09-24
Filing date: 1999-09-23
Publication date: 2000-03-30
Anticipated expiration: 2001-03-24
Also published as: AU6162499A

Abstract

The instant invention provides for a method of treating cancer which comprises administering to a mammal a composition which comprises a first compound which is an HMG-CoA reductase inhibitor and a second compound which is a prenyl-protein transferase inhibitor, and which is efficacious in vivo as an inhibitor of the growth of cancer cells characterized by a mutated K4B-Ras protein.

Description

TITLE OF THE INVENTION

A METHOD OF TREATING CANCER

BACKGROUND OF THE INVENTION The present invention relates to methods of treating cancer which comprise administering to a patient in need thereof a combination of an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and an inhibitor of prenyl-protein transferase. Chemotherapy, the systematic administration of antineoplastic agents that travel throughout the body via the blood circulatory system, along with and often in conjunction with surgery and radiation treatment, has for years been widely utilized in the treatment of a wide variety of cancers. Unfortunately, the available chemotherapeutic drugs often fail patients because they kill many healthy cells and thus bring on serious side effects that limit the doses physicians can administer.

Prenylation of proteins by prenyl-protein transferases represents a class of post-translational modification (Glomset, J. A., Gelb, M. H., and Farnsworth, C. C. (1990), Trends Biochem. Sci. 15, 139-142; Maltese, W. A. (1990), FASEB J. 4, 3319-3328). This modification typically is required for the membrane localization and function of these proteins. Prenylated proteins share characteristic C-terminal sequences including CAAX (C, Cys; A, an aliphatic amino acid; X, another amino acid), XXCC, or XCXC. Three post- translational processing steps have been described for proteins having a C-terminal CAAX sequence: addition of either a 15 carbon (farnesyl) or 20 carbon (geranylgeranyl) isoprenoid to the Cys residue, proteolytic cleavage of the last 3 amino acids, and methylation of the new C-terminal carboxylate (Cox, A. D. and Der, C. J. (1992a), Critical Rev. Oncogenesis 3:365-400; Newman, C. M. H. and Magee, A. I. (1993), Biochim. Biophys. Ada 1155:79-96). Some proteins may also have a fourth modification: palmitoylation of one or two Cys residues N-terminal to the farnesylated Cys. While some mammalian cell proteins terminating in XCXC are carboxymethylated, it is not clear whether carboxy methylation follows prenylation of proteins terminating with a XXCC motif (Clarke, S. (1992), Annu. Rev. Biochem. 61, 355- 386). For all of the prenylated proteins, addition of the isoprenoid is the first step and is required for the subsequent steps (Cox, A. D. and Der, C. J. (1992a), Critical Rev. Oncogenesis 3:365-400; Cox, A. D. and Der, C. J. (1992b) Current Opinion Cell Biol. 4:1008-1016).

Three enzymes have been described that catalyze protein prenylation: farnesyl-protein transferase (FPTase), geranylgeranyl- protein transferase type I (GGPTase-I), and geranylgeranyl-protein transferase type-II (GGPTase-II, also called Rab GGPTase). These enzymes are found in both yeast and mammalian cells (Clarke, 1992; Schafer, W. R. and Rine, J. (1992) Annu. Rev. Genet. 30:209-237). Each of these enzymes selectively uses farnesyl diphosphate or geranyl- geranyl diphosphate as the isoprenoid donor and selectively recognizes the protein substrate. FPTase farnesylates CAAX-containing proteins that end with Ser, Met, Cys, Gin or Ala. For FPTase, CAAX tetra- peptides comprise the minimum region required for interaction of the protein substrate with the enzyme. The enzymological characterization of these three enzymes has demonstrated that it is possible to selectively inhibit one with little inhibitory effect on the others (Moores, S. L., Schaber, M. D., Mosser, S. D., Rands, E., O'Hara, M. B., Garsky, V. M., Marshall, M. S., Pompliano, D. L., and Gibbs, J. B., J. Biol. Chem., 266:17438 (1991), U.S. Pat. No. 5,470,832).

The prenylation reactions have been shown genetically to be essential for the function of a variety of proteins (Clarke, 1992; Cox and Der, 1992a; Gibbs, J. B. (1991). Cell 65: 1-4; Newman and Magee, 1993; Schafer and Rine, 1992). This requirement often is demonstrated by mutating the CAAX Cys acceptors so that the proteins can no longer be prenylated. The resulting proteins are devoid of their central biological activity. These studies provide a genetic "proof of principle" indicating that inhibitors of prenylation can alter the physiological responses regulated by prenylated proteins. The Ras protein is part of a signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation. Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein. In the inactive state, Ras is bound to GDP. Upon growth factor receptor activation, Ras is induced to exchange GDP for GTP and undergoes a conformational change. The GTP-bound form of Ras propagates the growth stimulatory signal until the signal is terminated by the intrinsic GTPase activity of Ras, which returns the protein to its inactive GDP bound form (D.R. Lowy and D.M. Willurnsen, Ann. Rev. Biochem. £2:851-891 (1993)). Activation of Ras leads to activation of multiple intracellular signal transduction pathways, including the MAP Kinase pathway and the Rho/Rac pathway (Joneson et al, Science 272:810-812).

Mutated ras genes are found in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias. The protein products of these genes are defective in their GTPase activity and constitutively transmit a growth stimulatory signal.

The Ras protein is one of several proteins that are known to undergo post-translational modification. Farnesyl-protein transferase utilizes farnesyl pyrophosphate to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group (Reiss et al, Cell, f52:81-88 (1990); Schaber et al, J. Biol. Chem., 265:14701-14704 (1990); Schafer et al, Science, 249:1133-1139 (1990); Manne et al, Proc. Natl. Acad. Sci USA, 87:7541-7545 (1990)).

Ras must be localized to the plasma membrane for both normal and oncogenic functions. At least 3 post-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or "Cys-Aaa-Aaa-Xaa" box (Cys is cysteine, Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willurnsen et al, Nature 310:583-586 (1984)). Depending on the specific sequence, this motif serves as a signal sequence for the enzymes farnesyl-protein transferase or geranylgeranyl-protein transferase, which catalyze the alkylation of the cysteine residue of the CAAX motif with a Cl5 or C20 isoprenoid, respectively. (S. Clarke.,

Ann. Rev. Biochem. 61:355-386 (1992); W.R. Schafer and J. Rine, Ann. Rev. Genetics 30:209-237 (1992)). Other farnesylated proteins include the Ras-related GTP- binding proteins such as RhoB, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin. James, et al., J. Biol. Chem. 269, 14182 (1994) have identified a peroxisome associated protein Pxf which is also farnesylated. James, et al., have also suggested that there are farnesylated proteins of unknown structure and function in addition to those listed above.

Inhibitors of farnesyl-protein transferase (FPTase) have been described in two general classes. The first class includes analogs of farnesyl diphosphate (FPP), while the second is related to protein substrates (e.g., Ras) for the enzyme. The peptide derived inhibitors that have been described are generally cysteine containing molecules that are related to the CAAX motif that is the signal for protein prenylation. (Schaber et al., ibid; Reiss et. al., ibid; Reiss et al., PNAS, 88:132-736 (1991)). Such inhibitors may inhibit protein prenylation while serving as alternate substrates for the farnesyl-protein transferase enzyme, or may be purely competitive inhibitors (U.S. Patent 5,141,851, University of Texas; N.E. Kohl et al, Science, 260:1934-1937 (1993); Graham, et al., J. Med. Chem., 37, 725 (1994)).

Mammalian cells express four types of Ras proteins (H-, N-, K4A-, and K4B-Ras) among which K4B-Ras is the most frequently mutated form of Ras in human cancers. The genes that encode these proteins are abbreviated H-røs, N-ras , K4A-rαs and K4B- ras respectively. H-ras is an abbreviation for Harvey-ras. K4A-rαs and K4B-røs are abbreviations for the Kirsten splice variants of ras that contain the 4A and 4B exons, respectively. Inhibition of farnesyl-protein transferase has been shown to block the growth of H-ras-transformed cells in soft agar and to modify other aspects of their transformed phenotype. It has also been demonstrated that certain inhibitors of farnesyl-protein transferase selectively block the processing of the H-Ras oncoprotein intracellularly (N.E. Kohl et al. , Science, 260:1934-1937 (1993) and G.L. James et al., Science, 260:1937-1942 (1993). Recently, it has been shown that an inhibitor of farnesyl-protein transferase blocks the growth of H-røs -dependent tumors in nude mice (N.E. Kohl et al., Proc. Natl. Acad. Sci U.S.A., 92:9141-9145 (1994) and induces regression of mammary and salivary carcinomas in H-røs transgenic mice (N.E. Kohl et al, Nature Medicine, 1:792-797 (1995).

Indirect inhibition of farnesyl-protein transferase in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, NJ) and compactin (Hancock et al., ibid; Casey et al, ibid; Schafer et al, Science 245:319 (1989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids including farnesyl pyrophosphate. Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in cultured cells. However, inhibition of cell growth in vitro by lovastatin is not specific to cells transformed by mutated Ras proteins (DeClue, J.E. et al, Cancer Research, 51:712-717 (1991)). It has also been observed that concentrations of lovastatin which inhibit 50% of sterol biosynthesis in vitro show no inhibitory activity against protein prenylation (Sinensky, M. et al. J. Biol. Chem.265: 19937 (1990)). It has been disclosed that the lysine-rich region and terminal CVIM sequence of the C-terminus of K4B-Ras confer resistance to inhibition of the cellular processing of that protein by certain selective FPTase inhibitors. (James, et al., J. Biol. Chem. 270, 6221 (1995)) Those FPTase inhibitors were effective in inhibiting the processing of H-Ras proteins. James et al., suggested that prenylation of the K4B-Ras protein by GGTase contributed to the resistance to the selective FPTase inhibitors. (Zhang et al, J. Biol. Chem. 272 : 10232-239 (1997); Rowell et al, J. Biol. Chem. 272 :14093-14097 (1997); Whyte et al, J. Biol. Chem. 272 :14459-14464 (1997)). Several groups of scientists have recently disclosed compounds that inhibit both FPTase and GGTase (Lerner, et al., J. Biol. Chem. 270, 26770 (1995); and Graham, et al., J. Med. Chem. 37, 725 (1994)).

It has been disclosed that d-limonene and its metabolites may be used in combination with an HMG-CoA reductase inhibitor in the treatment of cancer (Japanese Pat. Publ. 07-316076). d-Limonene is described as an inhibitor of protein-farnesyl transferase in JP 07-316076, but the same group of scientists contemporaneously states that it has not been directly demonstrated that the compound is an inhibitor of farnesyl - protein transferase (British J. Cancer, 69:1015-1020 (1994)). There have been numerous scientific publications which describe d-limonene as a "weak inhibitor" of farnesyl-protein transferase (eg., M. H. Gelb et al. Cancer Letters 91:169-175 (1991); K. R. Stayrook et al. Anticancer Researchl8:823-828 (1998)). Pharmaceutical compositions that comprise compounds which are dual inhibitors of squalene synthetase and protein farensyl- transferase and compounds which are HMG-CoA reductase inhibitor have been generally described (PCT Publs. WO 96/33159 and WO 96/34850). It is the object of the present invention to provide a therapeutic composition useful in the treatment of cancer that comprises a first compound which is an HMG-CoA reductase inhibitor and a second compound which is a prenyl-protein transferase inhibitor, and which is efficacious in vivo as an inhibitor of the growth of several types of cancer cells, especially of those characterized by a mutated K4B-Ras protein.

It is also the object of the instant invention to provide a method of treating cancer which utilizes such a composition.

SUMMARY OF THE INVENTION

A method of treating cancer is disclosed which is comprised of administering to a mammalian patient in need of such treatment an effective amount of a therapeutic composition that comprises a first compound which is an HMG-CoA reductase inhibitor and a second compound which is a prenyl-protein transferase inhibitor.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1: Western Analysis SDS-PAGE Electrophoresis of PSN-1 cell lysates: The figure shows an X-ray film that was exposed to a PVDF membrane following transfer from a SDS-PAGE electro- phoresis gel. The Western blot was developed with Kirsten-ras specific monoclonal antibody, c-K-ras Ab-1 (Calbiochem). The proteins were isolated from the lysates of PSN-1 cells that had been exposed to vehicle (lane 1), 3 μM Compound 1 (Example 1) (lane 2) or a combination of 3 μM Compound 1 and 1 μM of simvastatin (lane 3). Details of the assay procedure can be found in Example 15.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method of treating cancer which is comprised of administering to a mammalian patient in need of such treatment an effective amount of a therapeutic composition that comprises a first compound which is an HMG-CoA reductase inhibitor and a second compound which is a prenyl-protein transferase inhibitor. The present method of treating cancer by simultaneously inhibiting protein prenylation and production of mevalonic acid offers advantages over previously disclosed methods which utilize a prenyl-protein transferase inhibitor alone, in that the dosage of the inhibitor of prenyl- protein transferase can be reduced. Any compounds which act as an HMG-CoA reductase inhibitor and any compounds which inhibit prenyl- protein transferase can be used in the instant method. Preferably the compounds utilized in the instant combination are an HMG-CoA reductase inhibitor and an inhibitor of prenyl-protein transferase which is efficacious in vivo as an inhibitor of the growth of cancer cells, including those characterized by a mutated K4B-Ras protein. More preferably the compounds utilized in the instant combination are an HMG-CoA reductase inhibitor and a dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I.

When practicing the present method the HMG-CoA reductase inhibitor and the inhibitor of prenyl-protein transferase may be administered either sequentially in any order or simultaneously. However, it has been found that administration of the HMG-CoA reductase inhibitor from one to several days prior to administration of the inhibitor of prenyl-protein transferase may be advantageous. It is anticipated that the therapeutic effect of the instant compositions may be achieved with smaller amounts of the prenyl- protein transferase inhibitor than would be required if such a prenyl- protein transferase inhibitors were administered alone, thereby avoiding adverse toxicity effects which might result from administration of an amount of the prenyl-protein transferase inhibitor sufficient to achieve the same therapeutic effect. It is further anticipated that the therapeutic effect of the instant compositions may be achieved with amounts of an inhibitor of HMG-CoA reductase that are known to be tolerated in man (see Thibault, A., Proc. Am. Assoc. Cancer Res., Vol. 35, Abstract 1351 (1994)). It is also anticipated that the instant compositions will achieve a synergistic therapeutic effect or will exhibit unexpected therapeutic advantage over the effect of any of the component compounds if administered alone. The terms prenyl-protein transferase inhibitor and inhibitor of prenyl-protein transferase refer to compounds which antagonize, inhibit or counteract the expression of the gene coding a prenyl-protein transferase or the activity of the protein product thereof. Prenyl-protein transferases include farnesyl-protein transferase and geranylgeranyl-protein transferase.

The terms farnesyl-protein transferase inhibitor and inhibitor of farnesyl-protein transferase likewise refer to compounds which antagonize, inhibit or counteract the expression of the gene coding farnesyl-protein transferase or the activity of the protein product thereof.

The term selective as used herein refers to the inhibitory activity of the particular compound against a prenyl-protein transferase activity. The extent of selectivity of the two inhibitors that comprise the method of the instant invention may effect the advantages that the method of treatment claimed herein offers over previously disclosed methods of using a combination of an HMG-CoA reductase inhibitor and compounds which are described as inhibitor of farnesyl-protein transferase. In particular, use of two independent pharmaceutically active components that have complementary, essentially non- overlapping activities allows the person utilizing the instant method of treatment to independently and accurately vary the inhibitory activity of the combination without having to synthesize a single drug having a particular pharmaceutical activity profile. Preferably, for example, a selective inhibitor of prenyl-protein transferase exhibits at least 20 times greater activity against prenyl-protein transferase when comparing its activity against another receptor or enzymatic activity (such as squalene synthetase), respectively. More preferably the selectivity is at least 100 times or more.

It is preferred that the inhibitors of prenyl-protein transferase which are efficacious in vivo as an inhibitor of the growth of cancer cells characterized by a mutated K4B-Ras protein utilized in the instant invention are efficacious in vivo as inhibitors of both farnesyl- protein transferase and geranylgeranyl-protein transferase type I (GGTase-I). Preferably, such a dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I, which may be termed a Class II prenyl-protein transferase inhibitor, is characterized by: a) an ICQQ (a measurement of in vitro inhibitory activity) of less than about 1 μM for inhibiting the transfer of a geranylgeranyl

G residue to a protein or peptide substrate comprising a CAAX motif by geranylgeranyl-protein transferase type I in the presence of a modulating anion; and b) an IC 0 (a measurement of in vitro inhibitory activity) of less than about 500 nM against transfer of a farnesyl residue to a protein or

F peptide substrate comprising a CAAX motif by farnesyl-protein transferase. Preferably, such a Class II prenyl-protein transferase inhibitor is also characterized by: c) inhibition of the cellular prenylation of greater than (>) about 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I at a concentration of less than (<)10 μM. More preferably, such a Class II prenyl-protein transferase inhibitor is also characterized by: c) inhibition of the cellular prenylation of greater than (>) about 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I at a concentration of less than (<)5 μM. r-^χ

The term "CAAX " will refer to such motifs that may be geranylgeranylated by GGTase-I. In particular, such "CAAX " motifs include (the corresponding human protein is in parentheses): CVIM (K4B-Ras) (SEQ.ID.: 1), CVLL (mutated H-Ras) (SEQ.ID.: 2), CWM (N-Ras) (SEQ.ID.: 3), CIIM (K4A-Ras) (SEQ.ID.: 4), CLLL (Rap-IA) (SEQ.ID.: 5), CQLL (Rap-IB) (SEQ.ID.: 6), CSIM (SEQ.ID.: 7), CALM (SEQ.ID.: 8), CKVL (RhoB) (SEQ.ID.: 9), CLIM (PFX) (SEQ.ID.: 10) and CVIL (Rap2B) (SEQ.ID.: 12). Preferably, the CAAX motif is rx

CVIM (SEQ.ID.: 1). It is understood that some of the "CAAX " containing protein or peptide substrates may also be farnesylated by farnesyl-protein transferase.

The modulating anion may be selected from any type of molecule containing an anion moiety. Preferably the modulating anion is selected from a phosphate or sulfate containing anion. Particular examples of modulating anions useful in the instant GGTase-I inhibition assay include adenosine 5'-triphosphate (ATP), 2'-deoxyadenosine 5 '-triphosphate (dATP), 2'-deoxycytosine 5 '-triphosphate (dCTP), β-glycerol phosphate, pyrophosphate, guanosine 5 '-triphosphate (GTP), 2'-deoxyguanosine 5 '-triphosphate (dGTP), uridine 5'-triphosphate, dithiophosphate, 3'-deoxythymidine 5 '-triphosphate, tripolyphosphate, D-myo-inositol 1,4, 5 -triphosphate, chloride, guanosine 5'-monophosphate, 2 '-deoxy uanosine 5'-monophosphate, orthophosphate, formycin A, inosine diphosphate, trimetaphosphate, sulfate and the like. Preferably, the modulating anion is selected from adenosine 5 '-triphosphate, 2'-deoxyadenosine 5 '-triphosphate, 2'-deoxycytosine 5'-triphosphate, β-glycerol phosphate, pyrophosphate, guanosine 5'-triphosphate, 2'-deoxyguanosine 5'-triphosphate, uridine 5 '-triphosphate, dithiophosphate, 3'-deoxythymidine 5'-triphosphate, tripolyphosphate, D-myo-inositol 1,4,5-triphosphate and sulfate. Most preferably, the modulating anion is selected from adenosine 5'-triphosphate, β-glycerol phosphate, pyrophosphate, dithiophosphate and sulfate.

A method for measuring the activity of the inhibitors of prenyl-protein transferase utilized in the instant methods against transfer of a geranylgeranyl residue to protein or peptide substrate comprising a CAAX motif by geranylgeranyl-protein transferase type I in the presence of a modulating anion is described in Example 11.

F As used herein, the term "CAAX " is used to designate a protein or peptide substrate that incorporates four amino acid C- terminus motif that is farnesylated by farnesyl-protein transferase. In

F particular, such "CAAX " motifs include (the corresponding human protein is in parentheses): CVLS (H-ras) (SEQ.ID.: 11), CVIM (K4B-Ras)

(SEQ.ID.: 1), CWM (N-Ras) (SEQ.ID.: 3), CKVL (RhoB) (SEQ.ID.: 9),

CLIM (PFX) (SEQ.ID.: 10) and CNIQ (Rap2A) (SEQ.ID.: 13). It is

F understood that certain of the "CAAX " containing protein or peptide substrates may also be geranylgeranylated by GGTase-I. A method for measuring the activity of the inhibitors of prenyl-protein transferase, as well as the instant combination compositions, utilized in the instant methods against transfer of a

F farnesyl residue to a protein or peptide substrate comprising a CAAX motif by farnesyl-protein transferase is described in Example 10. Examples of assay cells that may be utilized to determine inhibition of cellular processing of newly synthesized protein that is a substrate of an enzyme that can modify the K4B-Ras protein C- terminus include 3T3, C33a, PSN-1 (a human pancreatic carcinoma cell line) and K-røs-transformed Rat-1 cells. Preferred assay cell line has been found to be PSN-1. The preferred newly synthesized protein, whose percentage of processing is assessed in this assay, is selected from K4B-Ras and Rapl.

A method for measuring the activity of the inhibitors of prenyl-protein transferase, as well as the instant combination compositions, utilized in the instant methods against the cellular processing of newly synthesized protein that is a substrate of an enzyme that can modify the K4B-Ras protein C-terminus after incubation of assay cells with the compound of the invention transferase is described in Example 14 and 15.

A Class II prenyl-protein transferase inhibitor may also be characterized by: a) an ICQQ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells of less than 5 μM.

A Class II prenyl-protein transferase inhibitor may also be characterized by: a) an ICgg (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells between 0.1 and 100 times the IC5_Q for inhibiting the farnesylation of the protein hDJ in cells; and b) an ICg_Q (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

A Class II prenyl-protein transferase inhibitor may also be characterized by: a) an ICQQ (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than 2 fold lower but less than 20,000 fold lower than the inhibitory activity (IC50) against H-rαs-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and b) an IC5_Q (a measurement of in vitro inhibitory activity) against

H-rαs-CVLL dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

A Class II prenyl-protein transferase inhibitor may also be characterized by: a) an ICQQ (a measurement of in vitro inhibitory activity) against

H-Ras dependent activation of MAP kinases in cells greater than 10-fold lower but less than 2,500 fold lower than the inhibitory activity (IC50) against H-ms-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and b) an IC5_Q (a measurement of in vitro inhibitory activity) against

H-rαs-CVLL dependent activation of MAP kinases in cells greater than 5 fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

A method for measuring the activity of the inhibitors of prenyl-protein transferase, as well as the instant combination compositions, utilized in the instant methods against Ras dependent activation of MAP kinases in cells is described in Example 13. It is preferred that the therapeutic compositions which are efficacious in vivo as an inhibitor of the growth of cancer cells characterized by a mutated K4B-Ras protein utilized in the instant invention are efficacious in vivo in the inhibition of both farnesylation and geranylgeranylation of the K4B-Ras protein. Preferably, such a composition, which may be termed a Class II prenyl-protein transferase inhibiting therapeutic composition, is characterized by the following in vitro activity in the assays described in the Examples (Criteria A): a) inhibition of the cellular prenylation of greater than (>) about 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the composition of the invention.

Examples of assay cells that may be utilized to determine inhibition of cellular processing of newly synthesized protein that is a substrate of an enzyme that can modify the K4B-Ras protein C-terminus include 3T3, C33a, PSN-1 (a human pancreatic carcinoma cell line) and K-røs-transformed Rat-1 cells. Preferred assay cell lines have been found to be PSN-1. The preferred newly synthesized protein, whose percentage of processing is assessed in this assay, is selected from K4B-Ras and Rapl.

It is preferred that the concentration of the instant composition that is tested when evaluating whether the instant therapeutic composition is characterized by Criteria A is a concentration that includes a concentration of less than (<) 5 μM of the prenyl-protein transferase inhibitor.

Methods for measuring the activity of the therapeutic composition utilized in the instant methods against the cellular processing of newly synthesized protein that is a substrate of an enzyme that can modify the K4B-Ras protein C-terminus after incubation of assay cells with the composition of the instant invention is described in Examples 14 and 15.

A Class II prenyl-protein transferase inhibiting therapeutic composition may also be characterized by (Criteria B): b) inhibition of greater than (>) about 50% of the K4B-Ras dependent activation of MAP kinases in cells.

It is preferred that the concentration of the instant composition that is tested when evaluating whether the instant therapeutic composition is characterized by Criteria B is a concentration that includes a concentration of < 5 μM of the prenyl-protein transferase inhibitor and at a concentration of < 1 μM of the HMG-CoA reductase inhibitor. More preferably, the concentration of the instant composition that is tested for evaluating Criteria B is a concentration that includes a concentration of < 5 μM of the prenyl-protein transferase inhibitor and at a concentration of < 100 nM of the HMG-CoA reductase inhibitor. A Class II prenyl-protein transferase inhibiting therapeutic composition may also be characterized by (Criteria C): c) that produces an IC5_Q (a measurement of in vitro inhibitory activity) for inhibition of H-Ras dependent activation of MAP kinases in cells at least 2 fold lower but less than 20,000 fold lower than the inhibitory activity (IC50) against H-rαs-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells. It is preferred that the concentration of the instant composition that is tested when evaluating whether the instant therapeutic composition is characterized by Criteria C is a concentration that includes a concentration of < 5 μM of the prenyl-protein transferase inhibitor and at a concentration of < 1 μM of the HMG-CoA reductase inhibitor. More preferably, the concentration of the instant composition that is tested for evaluating Criteria C is a concentration that includes a concentration of < 5 μM of the prenyl-protein transferase inhibitor and at a concentration of < 100 nM of the HMG-CoA reductase inhibitor.

Examples of assay cells that may be utilized to determine inhibition of Ras dependent activation of MAP kinases in cells include 3T3, C33a, PSN-1 (a human pancreatic carcinoma cell line) and K-røs- transformed Rat-1 cells. Preferred assay cell line have been found to be C33a.

A method for measuring the activity of the therapeutic composition utilized in the instant methods against Ras dependent activation of MAP kinases in cells is described in Example 13. It has been surprisingly found that combining a first compound that is an HMG-CoA reductase inhibitor and a second compound which is a dual inhibitor of farnesyl protein transferase and geranylgeranyl-protein transferase Type I, will require a smaller amount of the dual inhibitor of farnesyl protein transferase and geranylgeranyl-protein transferase Type I to exhibit any of the Criteria A, B and C than is required to exhibit any of the Criteria A, B and C hereinabove when the dual inhibitor is tested in the absence of an HMG- CoA reductase inhibitor. Such an enhanced therapeutic effect is also observed when the HMG-CoA reductase inhibitor is administered prior to the administration of the farnesyl-protein transferase inhibitor.

The term "synergistic" as used herein means that the effect achieved with the methods and compositions of this invention is greater than the sum of the effects that result from methods and compositions comprising the prenyl-protein transferase inhibitor and HMG-CoA reductase inhibitor of this invention separately and in the amounts employed in the methods and compositions hereof. Such synergy between the two active ingredients enabling smaller doses to be given and preventing or delaying the build up of multi-drug resistance. The preferred therapeutic effect provided by the instant composition is the treatment of cancer and specifically the inhibition of cancerous tumor growth and/or the regression of cancerous tumors. Cancers which are treatable in accordance with the invention described herein include cancers of the brain, breast, colon, genitourinary tract, prostate, skin, lymphatic system, pancreas, rectum, stomach, larynx, liver and lung. More particularly, such cancers include histiocytic lymphoma, lung adenocarcinoma, pancreatic carcinoma, colo-rectal carcinoma, small cell lung cancers, bladder cancers, head and neck cancers, acute and chronic leukemias, melanomas, and neurological tumors.

The composition of this invention is also useful for inhibiting other proliferative diseases, both benign and malignant, wherein Ras proteins are aberrantly activated as a result of oncogenic mutation in other genes (i.e., the ras gene itself is not activated by mutation to an oncogenic form) with said inhibition being accomplished by the administration of an effective amount of the instant composition to a mammal in need of such treatment. For example, the composition is useful in the treatment of neurofibromatosis, which is a benign proliferative disorder. The composition of the instant invention is also useful in the prevention of restenosis after percutaneous transluminal coronary angioplasty by inhibiting neointimal formation (C. Indolfϊ et al. Nature medicine, 1:541-545(1995).

The instant composition may also be useful in the treatment and prevention of polycystic kidney disease (D.L. Schaffner et al.

American Journal of Pathology, 142:1051-1060 (1993) and B. Cowley, Jr. et al. FASEB Journal, 2:A3160 (1988)).

The instant composition may also inhibit tumor angio- genesis, thereby affecting the growth of tumors (J. Rak et al. Cancer Research, 55:4575-4580 (1995)). Such anti-angiogenesis properties of the instant composition may also be useful in the treatment of certain forms of vision deficit related to retinal vascularization.

The instant composition may also be useful in the treatment of certain viral infections, in particular in the treatment of hepatitis delta and related viruses (J.S. Glenn et al. Science, 256:1331-1333 (1992). The instant composition may also be useful in the inhibition of proliferation of vascular smooth muscle cells and therefore useful in the prevention and therapy of arteriosclerosis and diabetic vascular pathologies. The instant composition may comprise a combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor, either alone or, preferably, in combination with pharmaceutically acceptable carriers, excipients or diluents, according to standard pharmaceutical practice. The composition may be administered to mammals, preferably humans. The instant composition can be administered orally or parenterally, including the intravenous, intramuscular, intraperitoneal, subcutaneous, rectal and topical routes of administration.

The pharmaceutical compositions containing the active ingredients may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, microcrystalline cellulose, sodium crosscarmellose, corn starch, or alginic acid; binding agents, for example starch, gelatin, polyvinyl- pyrrolidone or acacia, and lubricating agents, for example, magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to mask the unpleasant taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a water soluble taste masking material such as hydroxypropylmethyl- cellulose or hydroxypropylcellulose, or a time delay material such as ethyl cellulose, cellulose acetate buryrate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water soluble carrier such as polyethyleneglycol or an oil medium, for example peanut oil, liquid paraffin, or olive oil. Aqueous suspensions contain the active material in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, ethylcellulose, hydroxypropylmethyl- cellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethylene-oxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose, saccharin or aspartame.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as butylated hydroxyanisol or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid. The pharmaceutical compositions of the invention may also be in the form of an oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring agents, preservatives and antioxidants. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, flavoring and coloring agents and antioxidant.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solutions. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.

The sterile injectable preparation may also be a sterile injectable oil-in-water microemulsion where the active ingredient is dissolved in the oily phase. For example, the active ingredient may be first dissolved in a mixture of soybean oil and lecithin. The oil solution then introduced into a water and glycerol mixture and processed to form a microemulation.

The injectable solutions or microemulsions may be introduced into a patient's blood-stream by local bolus injection. Alternatively, it may be advantageous to administer the solution or microemulsion in such a way as to maintain a constant circulating concentration of the instant compound. In order to maintain such a constant concentration, a continuous intravenous delivery device may be utilized. An example of such a device is the Deltec CADD-PLUS™ model 5400 intravenous pump.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension for intramuscular and subcutaneous administration. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The instant compositions may also be administered in the form of a suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non- irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.

For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the combination of a dual inhibitors of farnesyl-protein transferase and geranylgeranyl-protein transferase and an HMG-CoA reductase inhibitor are employed. (For purposes of this application, topical application shall include mouth washes and gargles.)

The compositions of the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles and delivery devices, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in the art. To be administered in the form of a transdermal delivery system, the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen. As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specific amounts, as well as any product which results, directly or indirectly, from combination of the specific ingredients in the specified amounts. The combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor of the instant method may also be co-administered with other well known therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, the instant composition may be useful in further combination with known anti- cancer and cytotoxic agents. Similarly, the instant composition may be useful in further combination with agents that are effective in the treatment and prevention of neurofibromatosis, restinosis, poly cystic kidney disease, infections of hepatitis delta and related viruses and fungal infections. The instant combination an inhibitor of prenyl- protein transferase and an HMG-CoA reductase inhibitor may also be useful in combination with other inhibitors of parts of the signaling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation.

The instant combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor may be utilized in combination with farnesyl pyrophosphate competitive inhibitors of the activity of farnesyl-protein transferase or in combination with a compound which has Raf antagonist activity. The instant combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor may also be co-administered with compounds that are selective inhibitors of geranylgeranyl protein transferase or selective inhibitors of farnesyl-protein transferase. The composition of the instant invention may also be co-administered with other well known cancer therapeutic agents that are selected for their particular usefulness against the condition that is being treated. Included in such combinations of therapeutic agents are combinations of the instant combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor and an antineoplastic agent. It is also understood that the instant combination of a combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor may be used in conjunction with other methods of treating cancer and/or tumors, including radiation therapy and surgery.

If formulated as a fixed dose, such combination products employ the combinations of this invention within the dosage range described below and the other pharmaceutically active agent(s) within its approved dosage range. Combinations of the instant invention may alternatively be used sequentially with known pharmaceutically acceptable agent(s) when a multiple combination formulation is inappropriate.

Radiation therapy, including x-rays or gamma rays which are delivered from either an externally applied beam or by implantation of tiny radioactive sources, may also be used in combination with a combination of an inhibitor of prenyl-protein transferase and an HMG-CoA reductase inhibitor.

Additionally, compositions of the instant invention may also be useful as radiation sensitizers, as described in WO 97/38697, published on October 23, 1997, and herein incorporated by reference. In particular, the composition of the instant invention may be administered to a patient in need prior to the application of radiation therapy. In another embodiment of the instant method of treatment, an HMG-CoA reductase inhibitor is administered prior to the administration of an inhibitor of prenyl-protein transferase, and administration of radiation therapy is either at the same time as administration of the inhibitor of prenyl-protein transferase or after the administration of the inhibitor of prenyl-protein transferase.

The instant composition may also be useful in combination with an integrin antagonist for the treatment of cancer, as described in U.S. Ser. No. 09/055,487, filed April 6, 1998, which is incorporated herein by reference.

As used herein the term an integrin antagonist refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to an integrin(s) that is involved in the regulation of angiogenisis, or in the growth and invasiveness of tumor cells. In particular, the term refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβ3 integrin, which selectively antagonize, inhibit or counteract binding of a physiological ligand to the αvβδ integrin, which antagonize, inhibit or counteract binding of a physiological ligand to both the αvβ3 integrin and the αvβδ integrin, or which antagonize, inhibit or counteract the activity of the particular integrin(s) expressed on capillary endothelial cells. The term also refers to antagonists of the αvβ6, αvβ8, αlβl, α2βl, αδβl, α6βl and α6β4 integrins. The term also refers to antagonists of any combination of αvβ3, αvβδ, αvβ6, αvβ8, αlβl, α2βl, αδβl, α6βl and α6β4 integrins. The instant compounds may also be useful with other agents that inhibit angiogenisis and thereby inhibit the growth and invasiveness of tumor cells, including, but not limited to angiostatin and endostatin. When a composition according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms. In one exemplary application, a suitable amount of an inhibitor of prenyl-protein transferase and a suitable amount of an HMG-CoA reductase inhibitor are administered to a mammal undergoing treatment for cancer. Administration occurs in an amount of each type of inhibitor of between about 0.1 mg/kg of body weight to about 60 mg/kg of body weight per day, preferably of between O.δ mg/kg of body weight to about 40 mg/kg of body weight per day. A particular daily therapeutic dosage that comprises the instant composition includes from about 10 mg to about 3000mg of an inhibitor of prenyl-protein transferase and about O.lmg to about 3000 mg of an HMG-CoA reductase δ inhibitor. Preferably, the daily dosage comprises from about 10 mg to about lOOOmg of an inhibitor of prenyl-protein transferase and about 0.3mg to about 160 mg of an HMG-CoA reductase inhibitor.

Examples of an antineoplastic agent include, in general, microtubule-stabilising agents ( such as paclitaxel (also

10 known as Taxol®), docetaxel (also known as Taxotere®), or their derivatives); alkylating agents, anti-metabolites; epidophyllotoxin; an antineoplastic enzyme; a topoisomerase inhibitor; procarbazine; mitoxantrone; platinum coordination complexes; biological response modifiers and growth inhibitors; hormonal/anti-hormonal lδ therapeutic agents and haematopoietic growth factors.

Example classes of antineoplastic agents include, for example, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, the taxanes, the epothilones, discodermolide, the pteridine family of drugs,

20 diynenes and the podophyllotoxins. Particularly useful members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro- methotrexate, mitomycin C, porfiromycin, δ-fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside,

2δ podophyllotoxin or podo-phyllotoxin derivatives such as etoposide, etoposide phosphate or teniposide, melphalan, vinblastine, vincristine, leurosidine, vindesine, leurosine, paclitaxel and the like. Other useful antineoplastic agents include estramustine, cisplatin, carboplatin, cyclophosphamide, bleomycin, gemcitibine, ifosamide,

30 melphalan, hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons and interleukins.

A compound which inhibits HMG-CoA reductase is used to practice the instant invention. Compounds which have inhibitory activity for HMG-CoA reductase can be readily identified by using assays well-known in the art. For example, see the assays described or cited in U.S. Patent 4,231,938 at col. 6, and WO 84/02131 at pp. 30-33. The terms δ "HMG-CoA reductase inhibitor" and "inhibitor of HMG-CoA reductase" have the same meaning when used herein.

Examples of HMG-CoA reductase inhibitors that may be used include but are not limited to lovastatin (MEVACOR®; see US Patent No. 4,231,938; 4,294,926; 4,319,039), simvastatin (ZOCOR®; see US

10 Patent No. 4,444,784; 4,820,8δ0; 4,916,239), pravastatin (PRAVACHOL®; see US Patent Nos. 4,346,227; 4,δ37,8δ9; 4,410,629; δ,030,447 and 5,180,589), fluvastatin (LESCOL®; see US Patent Nos. 5,3δ4,772; 4,911,165; 4,929,437; 5,189,164; 5,118,853; 5,290,946; 5,3δ6,896), atorvastatin (LIPITOR®; see US Patent Nos. 5,273,995; 4,681,893; 5,489,691; 5,342,9δ2) lδ and cerivastatin (also known as rivastatin and BAYCHOL®; see US Patent No. δ, 177,080). The structural formulas of these and additional HMG-CoA reductase inhibitors that may be used in the instant methods are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 8δ-89 (δ February 1996) and US Patent Nos.

20 4,782,084 and 4,88δ,314. The term HMG-CoA reductase inhibitor as used herein includes all pharmaceutically acceptable lactone and open- acid forms (i.e., where the lactone ring is opened to form the free acid) as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such salts, esters,

2δ open-acid and lactone forms is included within the scope of this invention. An illustration of the lactone portion and its corresponding open-acid form is shown below as structures I and II.

actone O fpen-Acid I II

In HMG-CoA reductase inhibitor's where an open-acid form can exist, salt and ester forms may preferably be formed from the open-acid, and δ all such forms are included within the meaning of the term "HMG-CoA reductase inhibitor" as used herein. Preferably, the HMG-CoA reductase inhibitor is selected from lovastatin and simvastatin, and most preferably simvastatin. Herein, the term "pharmaceutically acceptable salts" with respect to the HMG-CoA reductase inhibitor shall

10 mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base, particularly those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium, as well as those salts formed from amines lδ such as ammonia, ethylenediamine, N-methylglucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, l-p-chlorobenzyl-2-pyrrolidine-l'-yl-methylbenzimidazole, diethylamine, piperazine, and tris(hydroxymethyl)aminomethane. Further examples

20 of salt forms of HMG-CoA reductase inhibitors may include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate,

2δ glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate.

Ester derivatives of the described HMG-CoA reductase δ inhibitor compounds may act as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, may cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy.

Prenyl-protein transferase inhibitor compounds that are 10 useful in the methods of the instant invention and are identified by the properties described hereinabove include:

(a) a compound represented by formula I:

lδ wherein:

Rla is selected from: hydrogen or Ci-Cβ alkyl;

Rl° is independently selected from: 20 a) hydrogen, b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(R¹⁰)2 or C2-C6 alkenyl, c) Ci-Cβ alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl^O-, or -N(RlO)2;

2δ

R³ and R⁴ selected from H and CH3;

R2 is selected fromH; unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, \ . NR⁶R⁷

T 0 ; or Cl-5 alkyl, unbranched or branched, unsubstituted or substituted with one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR^6a, Sθ2R^6a, or

5) •\ NR⁶R⁷

T 0 ^; and R2 and R³ are optionally attached to the same carbon atom;

10

R° and R^ are independently selected from:

H; Ci-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle, unsubstituted or substituted with: a) Cχ-4 alkoxy, lδ b) halogen, c) perfluoro-Ci-4 alkyl, or d) aryl or heterocycle;

R^6a is selected from: 20 Ci-4 alkyl or C3-6 cycloalkyl, unsubstituted or substituted with: a) Ci-4 alkoxy, b) halogen, or c) aryl or heterocycle; 2δ

R° is independently selected from: a) hydrogen, b) C 1-C6 alkyl, C2-C6 alkenyl, C2-C6 al ynyl, C 1-C6 perfluoroalkyl, F, Cl, R¹⁰O-, Rl0c(O)NRl°-, CN, NO2,

30 (RlO)2N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or R11OC(O)NR10- and c) C1 -C6 alkyl substituted by Ci-Cβ perfluoroalkyl, R¹⁰O-, R¹⁰C(O)NR¹0-, (RlO)2N-C(NR¹⁰)-, R!0C(O)-, -N(R¹⁰)2, or Rl¹OC(O)NR¹0-;

δ R^^a is hydrogen or methyl;

RIO is independently selected from hydrogen, Ci-Cρ alkyl, Ci-Cg perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

10 RU is independently selected from Cχ-C6 alkyl and aryl;

Al and A² are independently selected from: a bond, -CH=CH-, -C=C-, -C(O)-, -C(O)NRl0-, O, -N(RlO)-, or S(0)_m;

lδ V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl,

20 c) aryl, d) C1-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, and provided that V is not hydrogen if A^ is S(0)_m and V is not hydrogen if 2δ A¹ is a bond, n is 0 and A² is S(0)_m;

X is -CH2- or -C(=0)-;

Z is selected from: 30 1) a unsubstituted or substituted group selected from aryl, heteroaryl, arylmethyl, heteroarylmethyl, arylsulfonyl, heteroarylsulfonyl, wherein the substituted group is substituted with one or more of the following: a) Cχ-4 alkyl, unsubstituted or substituted with: Cχ-4 alkoxy, NR^R⁷, C3-6 cycloalkyl, unsubstituted or substituted aryl, heterocycle, HO, -S(0)mR , or -C(0)NR6R⁷, b) aryl or heterocycle, δ c) halogen, d) OR6> e) NR6R7, f) CN, g) N0₂, 10 h) CF3; i) -S(0)_mR6a j) -C(0)NR6R⁷, or k) C3-C6 cycloalkyl; or 2) unsubstituted Ci-Cβ alkyl, substituted Ci-Cρ alkyl, lδ unsubstituted C3-C6 cycloalkyl or substituted C3-C6 cycloalkyl, wherein the substituted Cχ-C6 alkyl and substituted C3-C6 cycloalkyl is substituted with one or two of the following: a) Cl-4 alkoxy, b) NR6R7,

20 c) C3-6 cycloalkyl, d) -NR6C(0)R⁷, e) HO, f) -S(0)_mR6a, g) halogen, or

2δ h) perfluoroalkyl;

m is 0, 1 or 2; n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; and

30 r is 0 to δ, provided that r is 0 when V is hydrogen;

provided that the substituent (R⁸)_r- V - A¹(CR^la2)_nA²(C ¹a2)n - is not H; b) the inhibitors of farnesyl-protein transferase are illustrated by the formula II:

wherein:

Rla is selected from: hydrogen or Cχ-C6 alkyl;

Rl° is independently selected from: a) hydrogen, 10 b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(R¹⁰)2 or C2-C6 alkenyl, c) Cχ-C6 alkyl unsubstituted or substituted by unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl, Rl^O-, or -N(R¹⁰)2; lδ

Rlc is selected from: a) hydrogen, b) unsubstituted or substituted Cχ-Cβ alkyl wherein the substituent on the substituted Cl-Cβ alkyl is selected from

20 unsubstituted or substituted aryl, heterocyclic, C3-C10 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, Rl°0-, R¹¹S(0)_m-, Rl0C(O)NR!0-, (RlO)₂N-C(0)-, CN, (RlO)2N-C(NR¹⁰)-, R!0C(O)-, R!0θC(O)-, N3, -N(RlO)2, and R¹¹OC(O)-NR¹0-, and

2δ c) unsubstituted or substituted aryl;

R3 and R⁴ independently selected from H and CH3;

R² is selected fromH; OR¹*⁾; \ ^NR⁶R⁷

O or Ci-5 alkyl, unbranched or branched, unsubstituted or substituted with one or more of:

1) aryl,

2) heterocycle,

3) OR⁶,

4) SR⁶a, Sθ2R^6a or

⁵) \ _^NR⁶R⁷

O and R², R³ and R⁴ are optionally attached to the same carbon atom;

10

R⁶ and R⁷ are independently selected from: H; Cχ.4 alkyl, C3-6 cycloalkyl, aryl, heterocycle, unsubstituted or substituted with: a) Cχ-4 alkoxy, b) halogen, or lδ c) aryl or heterocycle;

R^6a is selected from:

Ci-4 alkyl or C3-6 cycloalkyl, unsubstituted or substituted with: 20 a) C1-4 alkoxy, b) halogen, or c) aryl or heterocycle;

R8 is independently selected from: 2δ a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C(O)NR¹⁰-, CN, NO2, (RlO)2N-C(NR¹⁰)-, R¹⁰C(O)-, -N(R¹⁰)2, or RHOC(0)NR¹⁰-, and 30 c ) C 1-C6 alkyl substituted by C χ-C6 perfluoroalkyl, R¹⁰O-,

R¹⁰C(O)NR¹⁰-, (R¹⁰)2N-C(NR¹⁽ )-, Rl0c(O)-, -N(RlO)2, or RHθC(O)NRl0-;

R^a is hydrogen or methyl;

RIO is independently selected from hydrogen, Cχ-Cβ alkyl, Cl-Cβ δ perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

RU is independently selected from Cχ-C6 alkyl and aryl;

R!² is selected from: H; unsubstituted or substituted Cχ-8 alkyl, unsubstituted or substituted aryl or unsubstituted or substituted 10 heterocycle, wherein the substituted alkyl, substituted aryl or substituted heterocycle is substituted with one or more of:

1) aryl or heterocycle, unsubstituted or substituted with: a) Ci-4 alkyl, lδ b) (CH2)_pOR⁶, c) (CH2)pNR⁶R⁷, d) halogen, e) CN, f) aryl or heteroaryl,

20 g) perfluoro-Ci-4 alkyl, h) SR⁶a S(0)R^6a, Sθ2R^6a,

2) C3-6 cycloalkyl,

3) OR⁶, 4) SR^6a, S(0)R⁶a or Sθ2R^6a,

5) — NR⁶R⁷

— 0^ . NR⁶R⁷

8) T 0

9) — 0. .0R⁶

T 0

10) \ ^ NR⁶R⁷ 0

11) — S0₂-NR⁶R⁷

13)

^ ^R6

0

14)

0

15) N₃, 16) F,

17) perfluoro-C_1-4-alkyl, or 18) C_1-6-alkyl;

δ A and A² are independently selected from: a bond, -CH=CH-, -C=C-, -C(O)-, -C(O)NR!0-, -NR¹⁰C(O)-, O, - RICK or S(0)_m;

V is selected from: a) hydrogen, 0 b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) C1-C2O alkyl wherein from 0 to 4 carbon atoms are replaced with a heteroatom selected from O, S, and N, and e) C2-C2O alkenyl, and provided that V is not hydrogen if A^ is S(0)m and V is not hydrogen if δ Al is a bond, n is 0 and A² is S(0) ;

X is -CH2- or -C(=0)-;

X¹ is a bond, -C(=0)-, -NR⁶C(=0)-, -NR⁶-, -O- or -S(=0)_m-;

10 Y is selected from: a) hydrogen, b) Rl O-, RllS(0)_m-, R¹⁰C(O)NR¹⁰-, (R¹⁰)2N-C(O)-, CN, NO2, (R¹⁰)2N-C(NR¹⁰)-, R¹²C(0)-, R¹⁰OC(O)-, N3, F, -N(R¹⁰)2, or

RllOC(0)NR¹⁰-, lδ c) unsubstituted or substituted Cχ-Cβ alkyl wherein the substituent on the substituted Cχ-C6 alkyl is selected from unsubstituted or substituted aryl, R¹⁰O-, R¹⁰C(O)NR¹⁰-, (R!0)2N-C(O)-, Rl°C(0)- and R¹⁰OC(O)-;

20 Z is an unsubstituted or substituted aryl, wherein the substituted aryl is substituted with one or more of the following:

1) Cχ-4 alkyl, unsubstituted or substituted with: a) Cχ-4 alkoxy,

2δ b) NR⁶R⁷, c) C3-6 cycloalkyl, d) aryl, substituted aryl or heterocycle, e) HO, f) -S(0)_mR⁶a or

30 g) -C(0)NR⁶R⁷,

2) aryl or heterocycle,

3) halogen,

4) OR⁶> δ) NR⁶R⁷> 6) CN,

7) N0₂,

8) CF₃;

9) -S(0)_mR^6a, δ 10) -C(0)NR⁶R⁷, or

11) C3-C6 cycloalkyl;

m is 0, 1 or 2; n is 0, 1, 2, 3 or 4; 0 p is 0, 1, 2, 3 or 4; and r is 0 to δ, provided that r is 0 when V is hydrogen; and v is 0, 1 or 2;

(c) a compound represented by formula III:

lδ wherein:

Rl is independently selected from: hydrogen or C1-C6 alkyl;

R² is independently selected from: a) hydrogen, b) substituted or unsubstituted aryl, substituted or

20 unsubstituted heterocycle, C3-C10 cycloalkyl, Rl"0- or

C2-C6 alkenyl, c) Cχ-C6 alkyl unsubstituted or substituted by aryl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, Rl°0-,

2δ

R3 is selected from: a) hydrogen, b) Cχ-C6 alkyl unsubstituted or substituted by C2-C6 alkenyl, R1<>0-, R¹¹S(0)_m-, RlOC^NRiO-, CN, N3, (R!0)2N-C(NR¹0)-, R¹⁰C(O)-, -N(RlO)2, or R¹¹OC(0)NR¹⁰-, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C3-C10 cycloalkyl, δ C2-C6 alkenyl, fluoro, chloro, R¹²0-, R^{1 :L}S(0)_m-,

R¹0C(O)NR¹⁰-, CN, NO2, (RlO)2N-C(NRlO)-, RlOC(O)-, N3, -N(RlO)2, or

RHOC(O)NR¹0-, and d) Cχ-C6 alkyl substituted with an unsubstituted or

10 substituted group selected from substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic and C3-C10 cycloalkyl;

R4 and Rδ are independently selected from: lδ a) hydrogen, b) Cχ-C6 alkyl unsubstituted or substituted by

C2-C6 alkenyl, RlOO-, R¹¹S(0)_m-, R¹⁰C(O)NRl0-, CN, N3, (RiO)2N-C(NR¹0)-, R¹⁰C(O)-, -N(Rl )₂, or RU0C(0)NR¹⁰-, c) substituted or unsubstituted aryl, substituted or 20 unsubstituted heterocycle, C3-C10 cycloalkyl,

C2-C6 alkenyl, fluoro, chloro, R¹⁰O-, R¹¹S(0)_m-, R¹0c(O)NR¹0-, CN, NO2, (R¹⁰)2N-C(NRlO)-, R!0C(O)-, N3, -N(Rl°)2, or RllOC(0)NR¹⁰-, and d) Cχ-C6 alkyl substituted with an unsubstituted or 2δ substituted group selected from substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic and C3-C10 cycloalkyl;

R⁶ is independently selected from: 30 a) hydrogen, b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, Cχ-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, allyloxy, Rl0C(O)NRl0-, CN, NO2, (R!0)2N-C(NRlO)-, RlOc(O)-, -N(R!0)2, (R¹²)2NC(0)- or R OC(0)NR¹⁰-, and c) Ci-Cβ alkyl substituted by CI-CQ perfluoroalkyl, R¹⁰O-, R¹⁰C(O)NRl0-, (RlO)2N-C(NR¹⁰)-, R¹⁰C(O)-, -N(R¹⁰)2, or Rl¹OC(O)NR¹0-; δ

R⁷ is independently selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle,

10 d) unsubstituted or substituted cycloalkyl, and e) Cχ-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl; wherein heterocycle is selected from pyrrolidinyl, lδ imidazolyl, pyridinyl, thiazolyl, pyridonyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;

R8 is selected from: a) hydrogen, 20 b) Cι-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, R¹⁰C(O)NR¹⁰-, CN, NO2, (R¹⁰)2N-C(NRl°)-, Rl°C(0)-, R¹⁰OC(O)-, -N(Rl°)2, or

R OC(0)NRl°-, and c) Cχ-C6 alkyl substituted by C1-C6 perfluoroalkyl, R¹⁰O-, 25 Rl0C(O)NRl0-, (RlO)2N-C(NR¹⁰)-, R¹⁰C(O)-, R1°OC(0)-,

-N(R!0)2, or RⁿOC(O)NRl0-;

R9 is selected from: a) hydrogen, 30 b) C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl,

F, Cl, R¹⁰O-, R¹¹S(0)_m-, R¹⁰C(0)NR!0-, CN, NO2, (R¹⁰)2N-C(NRl°)-, Rl0_C(O)-, RlOθC(O)-, -N(R¹⁰)2, or R¹¹OC(0)NR¹⁰-, and c) Cχ-C6 alkyl unsubstituted or substituted by Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, Rl¹S(0)_m-, R¹⁰C(O)NRl0-, CN, (R¹0)2N-C(NRlO)-, RlOc(O)-, RlOOC(O)-, -N(R¹⁰)2, or R OC(O)NRl0-;

5

RlO is independently selected from hydrogen, Cχ-C6 alkyl, Cχ-C6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

RU is independently selected from Cχ-Cβ alkyl and aryl;

10

R^² is independently selected from hydrogen, Cχ-Cβ alkyl, Cχ-C6 alkyl substituted with CO2R¹⁰, Cχ-Cβ alkyl substituted with aryl, Cχ-Cβ alkyl substituted with substituted aryl, Cχ-Cβ alkyl substituted with heterocycle, C -C6 alkyl substituted with lδ substituted heterocycle, aryl and substituted aryl;

Al and A² are independently selected from: a bond, -CH=CH-, -C≡C-, -C(O)-, -C(0)NR⁷-, -NR⁷C(0)-, -S(0)2NR⁷-, -NR⁷S(0)2-, O, -N(R⁷)-, or S(0)_m;

20

A3 is selected from: a bond, -C(0)NR⁷-, -NR⁷C(0)-, -S(0)2NR⁷-, -NR⁷S(0)2- or -N(R⁷)-;

A⁴ is selected from: a bond, O, -N(R⁷)- or S; 2δ

V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl,

30 quinolinyl, isoquinolinyl, and thienyl, c) aryl, d) Cχ-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a heteroatom selected from O, S, and N, and e) C2-C20 alkenyl, and provided that V is not hydrogen if A^ is S(0)_m and V is not hydrogen if Al is a bond, n is 0 and A² is S(0)_m;

Z is independently (Rl)2 or O; δ m is 0, 1 or 2; n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; q is 0 or 1; and

10 r is 0 to δ, provided that r is 0 when V is hydrogen;

d) a compound represented by formula A:

wherein: lδ

Rla is selected from: hydrogen or Cl-C6 alkyl;

Rib is independently selected from: a) hydrogen, 20 b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(R¹⁰)2 or C2-C6 alkenyl, c) Cχ-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl^O-, or -N(RlO)2;

2δ R a, R2b and R3 are independently selected from: a) hydrogen, b) Cχ-C6 alkyl unsubstituted or substituted by C2-C6 alkenyl, Rl^O-, RllS(0)_m-, R¹⁰C(O)NRl0-, CN, N3, (RlO)2N-C(NRlO)-, RlOC(O)-, RlOOC(O)-, -N(RlO)2, or

30 RllOC(O)NR¹0-, c) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or substituted cycloalkyl, alkenyl, R¹⁰O-, R¹¹S(0)_nl-, R!0C(O)NR¹⁰-, CN, N02, (R¹⁰)2N-C(NR¹0)-, R¹⁰C(O)-, R¹0θC(O)-, N3, -N(R¹⁰)2, halogen or

RllOC(0)NR¹⁰-, and d) C -C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocyclic and C3-C10 cycloalkyl;

10

R4 is

R^ is hydrogen;

lδ R8 is selected from: a) hydrogen, b) Cχ-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, Rl0C(O)NR¹⁰-, CN, Nθ2, (RlO)2N-C(NR¹⁰)-, R¹⁰C(O)-, R!0OC(O)-, -N(R¹⁰)2, or

20 RllOC(0)NR¹⁰-, and c) Cχ-Cβ alkyl substituted by C -C6 perfluoroalkyl, R¹⁰O-, R¹⁽ C(0)NR¹⁰-, (R¹0)2N-C(NR¹⁰)-, RlOC(O)-, R¹⁰OC(O)-, -N(Rl°)2, or RllOC(0)NR¹⁰-;

2δ R^a is independently selected from Cχ-Cβ alkyl and aryl;

RlO is independently selected from hydrogen, Cχ-Cβ alkyl, C -C6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

30 RU is independently selected from C -C6 alkyl, benzyl and aryl; Al and A² are independently selected from: a bond, -CH=CH-, -C≡C-, -C(O)-, -C(0)NR8-, -NRδC(O)-, O, -N(R⁸)-, -S(0)2N(R8)-, -N(R8)S(0)2-, or S(0)_m;

δ V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl,

10 c) aryl, d) Cχ-C20 alkyl wherein from 0 to 4 carbon atoms are replaced with a heteroatom selected from O, S, and N, and e) C2-C2O alkenyl, provided that V is not hydrogen if A^ is S(0)m and V is not hydrogen if lδ Al is a bond, n is 0 and A² is S(0)_m;

Z is H2 or O; m is 0, 1 or 2; n is 0, 1, 2, 3 or 4;

20 p is independently 0, 1, 2, 3 or 4; and r is 0 to δ, provided that r is 0 when V is hydrogen;

or the pharmaceutically acceptable salts thereof.

In a further embodiment of the formula I compounds 2δ of this invention, the inhibitors of farnesyl-protein transferase are illustrated by the formula I-a:

Z

wherein: Rl" is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(R¹⁰)2 or C2-C6 alkenyl, c) Cχ-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl^O-, or -N(RlO)2;

R² is selected from H; unsubstituted or substituted aryl or Cχ-5 alkyl,

10 unbranched or branched, unsubstituted or substituted with one or more of:

1) aryl,

2) heteroaryl,

3) OR⁶, or lδ 4) SR^6a;

R⁶ and R⁷ are independently selected from: Cχ_4 alkyl, aryl, and heteroaryl, unsubstituted or substituted with: a) Cχ-4 alkoxy,

20 b) halogen, c) perfluoro-Cχ-4 alkyl, or d) aryl or heteroaryl;

R ^a is selected from: 2δ C -4 alkyl, unsubstituted or substituted with: a) Cχ-4 alkoxy, or b) aryl or heteroaryl;

R° is independently selected from: 30 a) hydrogen, b) C χ-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C χ-Cβ perfluoroalkyl, F, Cl, R¹⁰O-, R¹0C(O)NR¹0-, CN, NO2, (RlO)2N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or RUOC(0)NR¹⁰-, and c) Cχ-Cβ alkyl substituted by Cχ-C6 perfluoroalkyl, RlOO-, R!0C(O)NR1°-, (R^1{ )2N-C(NR¹⁰)-, R¹⁰C(O)-, -N(Rl°)2, or RllOC(0)NR¹⁰-;

δ RlO is independently selected from hydrogen, Cχ-Cβ alkyl, Cχ-Cβ perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

RU is independently selected from Cl-Cβ alkyl and aryl;

10 X is -CH2- or -C(=0)-;

Z is an unsubstituted or substituted group selected from aryl, arylmethyl and arylsulfonyl, wherein the substituted group is substituted with one or more of the following: lδ a) Cχ_4 alkyl, unsubstituted or substituted with:

Cχ-4 alkoxy, NR⁶R⁷, C3-6 cycloalkyl, unsubstituted or substituted aryl, heterocycle, HO, -S(0)_mR^6a, or

-C(0)NR⁶R⁷, b) aryl or heterocycle,

20 c) halogen, d) OR⁶, e) NR⁶R⁷> f) CN, g) NO2, 25 h) CF3; i) -S(0)_mR⁶a, j) -C(0)NR⁶R⁷, or k) C3-C6 cycloalkyl;

30 m is 0, 1 or 2; and p is 0, 1, 2, 3 or 4; and r is 0 to 3;

or the pharmaceutically acceptable salts thereof; In another embodiment of this invention, the inhibitors of farnesyl-protein transferase are illustrated by the formula Il-a:

wherein:

5

Rio is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(Rl°)2 or C2-C6 alkenyl, 10 c) Cχ-C6 alkyl unsubstituted or substituted by unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl, Rl^O-, or -N(R¹⁰)2;

Rlc is selected from: lδ a) hydrogen, b) unsubstituted or substituted Cχ-Cβ alkyl wherein the substituent on the substituted Cχ-Cβ alkyl is selected from unsubstituted or substituted aryl, heterocyclic, C3-C 0 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, RlOO-, RllS(0)_m-,

20 R¹0C(O)NRl°-, (Rl°)2N-C(0)-, CN, (R¹⁰)2N-C(NR¹⁰)-,

R¹⁰C(O)-, R¹⁰OC(O)-, N3, -N(R¹⁰)2, and R¹¹OC(0)-NR¹⁰-, and c) unsubstituted or substituted aryl;

2δ R⁶, R⁷ and R^7a are independently selected from:

H; Cχ-4 alkyl, C3-6 cycloalkyl, aryl, heterocycle, unsubstituted or substituted with: a) Cχ_4 alkoxy, b) halogen, or c) aryl or heterocycle;

R^6a is selected from:

Cχ-4 alkyl or C3-6 cycloalkyl, unsubstituted or substituted with: a) Cχ_4 alkoxy, b) halogen, or c) aryl or heterocycle;

10

R8 is independently selected from: a) hydrogen, b) Cχ-C₆ alkyl, C₂-C6 alkenyl, C₂-C₆ alkynyl, Cχ-Cβ perfluoroalkyl, F, Cl, R¹⁰O-, Rl0c(O)NR¹⁰-, CN, NO2, lδ (RlO)₂N-C(NR¹⁰)-, R¹⁰C(O)-, -N(R!0)2, or RHOC(0)NR¹⁰-, and c) Cχ-C6 alkyl substituted by Cχ-C6 perfluoroalkyl, R^O-, R¹⁰C(O)NR¹⁰-, (RlO)₂N-C(NRl°)-, R¹⁰C(O)-, -N(RlO)2, or RllOC(0)NR¹⁰-;

20

RlO is independently selected from hydrogen, Cχ-Cβ alkyl, Cχ-Cβ perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

RU is independently selected from C1-C6 alkyl and substituted or 2δ unsubstituted aryl;

Rl² is selected from: H; unsubstituted or substituted Cχ-8 alkyl, unsubstituted or substituted aryl or unsubstituted or substituted heterocycle, 30 wherein the substituted alkyl, substituted aryl or substituted heterocycle is substituted with one or more of:

1) aryl or heterocycle, unsubstituted or substituted with: a) Cχ-4 alkyl, b) halogen,

3δ c) CN, d) perfluoro-Cχ-4 alkyl,

2) C3-6 cycloalkyl,

3) OR⁶, 4) SR^6a, S(0)R^6a, or Sθ2R^6a,

6)

^ ^0Rβ

0

7) N₃-

8) F,

9) perfluoro-Ci_-4-alkyl, or

10) C_1-6-alkyl;

X¹ is a bond, -C(=0)- or -S(=0)_m-;

Y is selected from: 10 a) hydrogen, b) Rl O-, RllS(0)_m-, Rl0C(O)NR¹0-, (RlO)₂N-C(0)-, CN, NO2, (R!0)2N-C(NR¹⁰)-, R¹²C(0)-, RlOθC(O)-, N3, F, -N(Rl°)2, or

RllOC(0)NR¹⁰-, c) unsubstituted or substituted C -C6 alkyl wherein the lδ substituent on the substituted Cχ-Cβ alkyl is selected from unsubstituted or substituted aryl, R¹⁰O-, R¹⁰C(O)NR¹⁰-, (R!0)2N-C(O)-, Rl°C(0)- and RiOOrø)-;

Z is an unsubstituted or substituted aryl, wherein the

20 substituted aryl is substituted with one or more of the following: 1) C -4 alkyl, unsubstituted or substituted with: a) Cχ-4 alkoxy, b) NR⁶R⁷ c) C3-6 cycloalkyl, d) aryl, substituted aryl or heterocycle, e) HO, f) -S(0)_mR^6a, or δ g) -C(0)NR⁶R⁷,

2) aryl or heterocycle,

3) halogen,

4) OR⁶. δ) NR⁶R⁷>

10 6) CN,

7) N0₂,

8) CF3;

9) -S(0)_mR^6a,

10) -C(0)NR⁶R⁷, or lδ 11) C3-C6 cycloalkyl;

m is 0, 1 or 2; p is 1 or 2 ; r is 0 to 3; and

20 v is 0, 1 or 2;

or a pharmaceutically acceptable salt thereof.

In a further embodiment of this invention, the inhibitors of farnesyl-protein transferase are illustrated by the formula Ill-a:

wherein:

R² is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(R¹⁰)2 or C2-C6 alkenyl, c) Cχ-Cβ alkyl unsubstituted or substituted by aryl, δ heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(RlO)2;

R3 is selected from: a) hydrogen, b) Cχ-Cβ alkyl unsubstituted or substituted by C2-C6 10 alkenyl, R^O-, RllS(0)_m-, R¹⁰C(O)NR¹0-, CN, N3,

(R¹⁰)2N-C(NRlO)-, R10_C(O)-, -N(R¹⁰)2, or R¹¹OC(0)NR¹⁰-, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, fluoro, chloro, R¹²0-, R S(0)_m-, lδ R¹0C(O)NR¹0-, CN, NO2, (R¹⁰)2N-C(NR¹⁰)-,

R^!0C(O)-, N3, -N(R¹⁰)2, or R^llo ONR¹⁰-, and d) C -C6 alkyl substituted with an unsubstituted or substituted group selected from substituted or unsubstituted aryl, substituted or unsubstituted

20 heterocyclic and C3-C 0 cycloalkyl;

R4 and Rδ are independently selected from: a) hydrogen, b) Cχ-Cβ alkyl unsubstituted or substituted by RlOO- or

2δ -N(R¹⁰)2, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, fluoro, chloro, Rl°0-, R¹lS(0)_m-, Rl0C(O)NR¹0-, CN, NO2, (R¹⁰)2N-C(NRlO).,

30 RiOC(O)-, N3, -N(RlO)₂, or RUOC(0)NR¹⁰-, and d) Cχ-C6 alkyl substituted with an unsubstituted or substituted group selected from substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic and C3-C10 cycloalkyl;

3δ R6 is independently selected from: a) hydrogen, b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, Cχ-C6 alkyl, C2-C6 alkenyl, δ C2-C6 alkynyl, Cχ-Cβ perfluoroalkyl, F, Cl, R¹⁰O-, allyloxy,

Rl°C(O)NRl0-, CN, NO2, (R¹⁰)2N-C(NR¹⁰)-, RlOc(O)-, -N(R¹⁰)2, (R¹²)2NC(0)- or R¹¹OC(0)NR¹⁰-, and c) Cχ-C6 alkyl substituted by C -C6 perfluoroalkyl, R¹⁰O-, Rl°C(O)NR¹0-, (RlO)₂N-C(NR¹⁰)-, R!0C(O)-,

10 -N(R¹⁰)2, or R¹¹OC(0)NR¹⁰-;

R⁷ is independently selected from a) hydrogen, b) unsubstituted or substituted aryl, lδ c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) Cχ-C6 alkyl substituted with hydrogen or an unsubstituted or substituted group selected from aryl, heterocycle and cycloalkyl; 20 wherein heterocycle is selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, indolyl, quinolinyl, isoquinolinyl, and thienyl;

R8 is independently selected from: 2δ a) hydrogen, b) Cχ-Cβ alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, R¹0C(O)NR¹⁰-, CN, NO2, (R¹⁰)2N-C(NR¹⁰)-, R¹⁰C(O)-, -N(RlO)₂, or RHOC(0)NR¹⁰-, and 30 c) Cχ-Cβ alkyl substituted by Cχ-Cβ perfluoroalkyl, Rl°0-,

Rl c(O)NRl0-, (RlO)2N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or Rl¹OC(0)NR¹⁰-; Rl is independently selected from hydrogen, C -C6 alkyl, Cχ-C6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;

RU- is independently selected from Cχ-Cβ alkyl and aryl;

R^ is independently selected from hydrogen, Cχ-Cρ alkyl, Cχ-C6 alkyl substituted with Cθ2R* , Cχ-C6 alkyl substituted with aryl, C -C6 alkyl substituted with substituted aryl, Cχ-Cβ alkyl substituted with heterocycle, Cχ-Cβ alkyl substituted with

10 substituted heterocycle, aryl and substituted aryl;

lδ Z is independently H2 or O; m is 0, 1 or 2; and n is 0, 1, 2, 3 or 4; p is 0, 1, 2, 3 or 4; q is 0 or 1; and

20 r is 0 to 3;

or the pharmaceutically acceptable salts thereof.

In a further embodiment of the formula A compounds of this invention, the inhibitors of farnesyl-protein transferase are 2δ illustrated by the formula A-i:

wherein:

Rl° is independently selected from: 30 a) hydrogen, b) aryl, heterocycle, cycloalkyl, R¹⁰O-, -N(R¹⁰)2 or C2-C6 alkenyl, c) Cχ-C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl O-, or -N(RlO)2; δ

R ^a and R^2D are independently selected from: a) hydrogen, b) C -C6 alkyl unsubstituted or substituted by

C2-C6 alkenyl, R¹⁰O-, R¹¹S(0)_m-, R¹0C(O)NR¹0-, CN, N3, 10 (R¹0)₂N-C(NR¹0)-, RlOC(O)-, RlOθC(O)-, -N(R10)_2J or

R¹¹OC(O)NR¹0-, c) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or substituted cycloalkyl, alkenyl, RlOO-, R¹¹S(0)_m-, lδ Rl0c(O)NR¹⁰-, CN, NO2, (R¹⁰)2N-C(NR¹⁰)-,

R¹⁰C(O)-, R¹⁰OC(O)-, N3, -N(RlO)₂, halogen or

R¹¹OC(0)NR¹⁰-, and d) Cχ-C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocyclic and 20 C3-CX0 cycloalkyl;

R4 is

R° is hydrogen;

2δ

R8 is independently selected from: a) hydrogen, b) Cχ-Cβ alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Cχ-C6 perfluoroalkyl, F, Cl, R¹⁰O-, R10C(0)NR10-, CN, Nθ2, (R¹⁰)2N-C(NRlO)-, R10_C(O)-, -N(RlO)2, or RHOC(0)NR¹⁰-, and c) Cχ-C6 alkyl substituted by Cχ-C6 perfluoroalkyl, R¹⁰O-, R¹⁰C(O)NR¹⁰-, (R¹0)₂N-C(NR¹⁰)-, R¹⁰C(O)-, -N(R¹⁰)2, or R1¹OC(O)NR10-_;

RI is independently selected from hydrogen, C -C6 alkyl, substituted or unsubstituted Cχ-Cβ aralkyl and substituted or unsubstituted aryl;

10

RU is independently selected from Cχ-Cβ alkyl, benzyl and aryl;

Z is H2 or O; m is 0, 1 or 2; lδ n is 0, 1, 2, 3 or 4; p is independently 0, 1 or 2; and r is 0 to δ;

or the pharmaceutically acceptable salts thereof.

20

Specific compounds which are inhibitors of prenyl-protein transferases and are therefore useful in the present invention include:

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-[(3-pyridyl)methoxyethyl)]-4-(l- 2δ naphthoyl)piperazine

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-(benzyloxymethyl)-4-(l- naphthoyl)piperazine

30 l-[2(R)-Amino-3-mercaptopropyl]-2(S)-(benzyloxymethyl)-4-[7-(2,3- dihydrobenzofuroyl)] piperazine

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-(benzylcarbamoyl)-4-(l- naphthoyl)piperazine

3δ 1- [2(R)-Amino-3-mercaptopropyl] -2(S)- [4-(δ-dimethylamino-l- naphthalenesulfonamido)-l-butyl]-4-(l-naphthoyl)piperazine

N-[2(S)-(l-(4-Nitrophenylmethyl)-lH-imidazol-δ-ylacetyl)amino-3(S)- δ methylpentyl] -N-1-naphthylmethyl-glycyl-methionine

N-[2(S)-(l-(4-Nitrophenylmethyl)-lH-imidazol-δ-ylacetyl)amino-3(S)- methylpentyl]-N-l-naphthylmethyl-glycyl-methionine methyl ester

10 N-[2(S)-([l-(4-cyanobenzyl)-lH-imidazol-δ-yl]acetylamino)-3(S)- methylpentyl] -N-( l-naphthylmethyl)glycyl-methionine

N-[2(S)-([l-(4-cyanobenzyl)-lH-imidazol-δ-yl]acetylamino)-3(S)- methylpentyl] -N-( l-naphthylmethyl)glycyl-methionine methyl ester lδ

2(S)-rι-Butyl-4-(l-naρhthoyl)-l-[l-(2-naphthylmethyl)imidazol-δ- ylm ethyl] -piperazine

2(S)-τz-Butyl-l-[l-(4-cyanobenzyl)imidazol-δ-ylmethyl]-4-(l- 20 naphthoyl)piperazine

l-{[l-(4-cyanobenzyl)-lH-imidazol-δ-yl]acetyl}-2(S)-n- butyl-4-( l-naphthoyl)piperazine

2δ l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2-piperazinone

l-phenyl-4-[l-(4-cyanobenzyl)-lH-imidazol-δ-ylethyl]-piperazin-2-one

l-(3-trifluoromethylphenyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-δ- 30 ylmethyl]-piperazin-2-one

l-(3-bromophenyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-δ-ylmethyl]- piperazin-2-one δ(S)-(2-[2,2,2-trifluoroethoxy]ethyl)-l-(3-trifluoromethylphenyl)- 4-[l-(4- cyanobenzyl)-4-imidazolylmethyl]-piperazin-2-one

l-(δ,6,7,8-tetrahydronaphthyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-δ- δ ylmethyl]-piperazin-2-one

l-(2-methyl-3-chlorophenyl)-4-[l-(4-cyanobenzyl)-4-imidazolylmethyl)]- piperazin-2-one

10 2(RS)-{[l-(Naphth-2-ylmethyl)-lH-imidazol-δ-yl)] acetyl}amino-3-(t- butoxycarbonyl)amino- N-(2-methylbenzyl) propionamide

N-{l-(4-Cyanobenzyl)-lH-imidazol-δ-ylmethyl}-4(R)-benzyloxy-2(S)-{N'- acetyl-N'-3-chlorobenzyl}aminomethylpyrrolidine lδ

N-{l-(4-Cyanobenzyl)-lH-imidazol-δ-ylethyl}-4(R)-benzyloxy-2(S)-{N'- acetyl-N'-3-chlorobenzyl}aminomethyl pyrrolidine

l-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl] pyrrolidin-2(S)-ylmethyl]-(N- 20 2-methylbenzyl)-glycine N'-(3-chlorophenylmethyl) amide

l-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl] pyrrolidin-2(S)-ylmethyl]-(N- 2-methylbenzyl)-glycine N'- methyl-N'-(3-chlorophenylmethyl) amide

2δ (S)-2-[(l-(4-Cyanobenzyl)-δ-imidazolylmethyl)amino]-N-

(benzyloxycarbonyl)-N-(3-chlorobenzyl)-4-(methanesulfonyl)butanamine

l-(3,δ-Dichlorobenzenesulfonyl)-3(S)-[Ν-(l-(4-cyanobenzyl)-lH-imidazol-δ- ylethyl)carbamoyl] piperidine

30

N-{[l-(4-Cyanobenzyl)-lH-imidazol-δ-yl]methyl}-4-(3-methylphenyl)-4- hydroxy piperidine,

N-{[l-(4-Cyanobenzyl)-lH-imidazol-δ-yl]methyl}-4-(3-chlorophenyl)-4 3δ hydroxy piperidine, 4-[l-(4-cyanobenzyl)-δ-imidazolylmethyl]-l-(2,3-dimethylphenyl)- piperazine-2,3-dione

l-(2-(3-Trifluoromethoxyphenyl)-pyrid-δ-ylmethyl)-δ-(4- δ cyanobenzyl)imidazole

4-{δ-[l-(3-Chloro-phenyl)-2-oxo-l,2-dihydro-pyridin-4-ylmethyl]-imidazol- l-ylmethyl}-2-methoxy-benzonitrile

10

3(R)-3-[l-(4-Cyanobenzyl)imidazol-δ-yl-ethylamino]-δ-phenyl-l-(2,2,2- trifluoroethyl)-H-benzo[e] [1,4] diazepine

3(S)-3-[l-(4-Cyanobenzyl) imidazol-δ-yl]-ethylamino]-δ-phenyl-l-(2,2,2- lδ trifluoroethyl)-H-benzo[e] [l,4] diazepine

N-[l-(4-Cyanobenzyl)-lH-imidazol-δ-ylacetyl)pyrrolidin-2(S)-ylmethyl]- N- (l-naphthylmethyl)glycyl-methionine

20 N-[l-(4-Cyanobenzyl)-lH-imidazol-δ-ylacetyl)pyrrolidin-2(S)-ylmethyl]- N- (l-naphthylmethyl)glycyl-methionine methyl ester

2(S)-(4-Acetamido-l-butyl)-l-[2(R)-amino-3-mercaptoρropyl]-4-(l- naphthoyDpiperazine

2δ

2(RS)-{[l-(Naphth-2-ylmethyl)-lH-imidazol-δ-yl)] acetyl}amino-3-(t- butoxycarbonyl)amino- N-cyclohexyl-propionamide

l-{2(R,S)-[l-(4-cyanobenzyl)-lH-imidazol-δ-yl]propanoyl}-2(S)- - 30 butyl-4-(l-naphthoyl)piperazine

l-[l-(4-cyanobenzyl)imidazol-δ-ylmethyl]-4-(diphenylmethyl)piperazine

l-(Diphenylmethyl)-3(S)-[N-(l-(4-cyanobenzyl)-2-methyl-lH-imidazol-δ- 3δ ylethyl)-N-(acetyl)aminomethyl] piperidine N-[l-(lH-Imidazol-4-ylpropionyl)pyrrolidin-2(S)-ylmethyl]- N-(2- chlorobenzyl)glycyl-methionine

N-[l-(lH-Imidazol-4-ylpropionyl)pyrrolidin-2(S)-ylmethyl]- N-(2- δ chlorobenzyDglycyl-methionine methyl ester

3(R)-3-[l-(4-Cyanobenzyl)imidazol-δ-yl-methylamino]-δ-phenyl-l-(2,2,2- trifluoroethyl)-H-benzo[e] [1,4] diazepine

10 l-(3-trifluoromethoxyphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone

l-(2,δ-dimethylphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone lδ l-(3-methylphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2-piperazinone

l-(3-iodophenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2-piperazinone

20 l-(3-chlorophenyl)-4-[l-(4-cyano-3-methoxybenzyl)imidazolylmethyl]-2- piperazinone

l-(3-trifluoromethoxyphenyl)-4-[l-(4-cyano-3-methoxybenzyl)imidazolyl methyl] -2-piperazinone

25

4-[((l-(4-cyanobenzyl)-5-imidazolyl)methyl)amino]benzophenone

l-(l-{[3-(4-cyano-benzyl)-3H-imidazol-4-yl]-acetyl}-pyrrolidin-2(S)- ylmethyl)-3(S)-ethyl-pyrrolidine-2(S)-carboxylic acid 3-chloro- 30 benzylamide

or the pharmaceutically acceptable salt thereof.

Compounds within the scope of this invention which have been identified as inhibitors of prenyl-protein transferases and are 3δ therefore useful in the present invention, and methods of synthesis thereof, can be found in the following patents, pending applications and publications, which are herein incorporated by reference:

U.S. Pat. No. δ,736,539 (April 7, 1998); WO 95/00497 (January 5, 1995) U.S. Pat. No. δ,652,2δ7 (July 29, 1997); WO 96/10034 (April 4, 1996)

WO 96/30343 (October 3, 1996); USSN 08/412,829 filed on March 29, 199δ; and USSN 08/470,690 filed on June 6, 199δ; and USSN 08/600,728 filed on February 28, 1996; U.S. Pat. No. δ,661,161 (August 26, 1997); U.S. Pat. No. δ,756,528 (May 6, 1998); WO 96/39137 (December 12, 1996); WO 96/37204 (November 28, 1996); USSN 08/449,038 filed on May 24, 199δ;

USSN 08/648,330 filed on May lδ, 1996; WO 97/18813 (May 29, 1997); USSN 08/749,2δ4 filed on November lδ, 1996; WO 97/3866δ (October 23, 1997); USSN 08/831,308 filed on April 1, 1997; WO 97/36889 (October 9, 1997); USSN 08/823,923 filed on March 25, 1997; WO 97/36901 (October 9, 1997); USSN 08/827,483 filed on March 27, 1997; WO 97/36879 (October 9, 1997); USSN 08/823,920 filed on March 25, 1997; WO 97/36605 (October 9, 1997); USSN 08/823,934 filed on March 25, 1997; WO 98/28980, (July 9, 1998); USSN 08/997,171 filed on December 22, 1997; and

USSN 60/014,791 filed on April 3, 1996; USSN 08/831,308, filed on April 4, 1997.

All patents, publications and pending patent applications identified are hereby incorporated by reference.

The following compounds, which are described as inhibitors of farnesyl-protein transferase, are particularly useful when combined with an HMG-CoA reductase inhibitor in a composition that is useful in the treatment of cancer:

(±)-6-[amino(4-chlorophenyl)(l-methyl-lH-imidazol-δ-yl)methyl]-4-(3- chlorophenyl)-l-methyl-2(lH)-quinolinone (Compound J)

(-)-6-[amino(4-chlorophenyl)(l-methyl-lH-imidazol-δ-yl)methyl]-4-(3- chlorophenyl)-l-methyl-2(lH)-quinolinone (Compound J-A; designated "comp. 74" in WO 97/21701)

(+)-6-[amino(4-chlorophenyl)(l-m ethyl- lH-imidazol-δ-yl)methyl] -4-(3- chlorophenyl)-l-methyl-2(lH)-quinolinone (Compound J-B; designated "comp. 7δ" in WO 97/21701)

or a pharmaceutically acceptable salt thereof. The syntheses of these compounds are specifically described in PCT Publication WO 97/21701, in particular on pages 19-28. The preferred compound among these compounds to use in combination with an HMG-CoA reductase inhibitor is Compound J-B. Compounds which are described as inhibitors of farnesyl- protein transferase and may therefore useful in the present invention, and methods of synthesis thereof, can be found in the following patents, pending applications and publications, which are herein incorporated by reference: WO 9δ/32987 published on 7 December 199δ; U. S. Pat. No. δ,420,245; U. S. Pat. No. δ,δ23,430 U. S. Pat. No. δ,δ32,3δ9 U. S. Pat. No. 5,510,510 U. S. Pat. No. 5,δ89,48δ U. S. Pat. No. δ,602,098 European Pat. Publ. 0 618 221; European Pat. Publ. 0 67δ 112;

European Pat. Publ. 0 604 181;

European Pat. Publ. 0 696 δ93; δ WO 94/193δ7;

WO 9δ/08δ42 ;

WO 96/11917;

WO 9δ/12612;

WO 9δ/12δ72; 0 WO 9δ/10δl4 and U.S. Pat. No. δ,661,lδ2;

WO 9δ/10δlδ;

WO 9δ/10δl6;

WO 9δ/24612;

WO 9δ/34δ35; 5 WO 95/2δ086;

WO 96/0δδ29;

WO 96/06138;

WO 96/06193;

WO 96/16443; 0 WO 96/21701;

WO 96/214δ6;

WO 96/22278;

WO 96/24611;

WO 96/24612; δ WO 96/06168;

WO 96/0δl69;

WO 96/00736 and U.S. Pat. No. 5,571,792 granted on November 5, 1996;

WO 96/17861;

WO 96/331δ9 0 WO 96/348δ0

WO 96/348δl

WO 96/30017

WO 96/30018

WO 96/30362 3δ WO 96/30363 O 96/31111 O 96/31477 O 96/31478 O 96/31501 O 97/00252 WO 97/03047 WO 97/03060 WO 97/04785 WO 97/02920 WO 97/17070 WO 97/23478 WO 97/26246 WO 97/30053 WO 97/44360 WO 98/02436 and U. S. Pat. No 6,532,359 granted on July 2, 1996.

With respect to the compounds of formulas I through A-i the following definitions apply: The term "alkyl" refers to a monovalent alkane

(hydrocarbon) derived radical containing from 1 to 15 carbon atoms unless otherwise defined. It may be straight, branched or cyclic. Preferred straight or branched alkyl groups include methyl, ethyl, propyl, isopropyl, butyl and t-butyl. Preferred cycloalkyl groups include cyclopentyl and cyclohexyl.

When substituted alkyl is present, this refers to a straight, branched or cyclic alkyl group as defined above, substituted with 1-3 groups as defined with respect to each variable.

Heteroalkyl refers to an alkyl group having from 2-15 carbon atoms, and interrupted by from 1-4 heteroatoms selected from O, S and N.

The term "alkenyl" refers to a hydrocarbon radical straight, branched or cyclic containing from 2 to 15 carbon atoms and at least one carbon to carbon double bond. Preferably one carbon to carbon double bond is present, and up to four non-aromatic (non- resonating) carbon-carbon double bonds may be present. Examples of alkenyl groups include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, isoprenyl, farnesyl, geranyl, geranylgeranyl and the like. Preferred alkenyl groups include ethenyl, propenyl, butenyl and cyclohexenyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkenyl group may contain double bonds and may be substituted when a substituted alkenyl group is provided.

The term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic, containing from 2 to 15 carbon atoms and at least one carbon to carbon triple bond. Up to three carbon- carbon triple bonds may be present. Preferred alkynyl groups include ethynyl, propynyl and butynyl. As described above with respect to alkyl, the straight, branched or cyclic portion of the alkynyl group may contain triple bonds and may be substituted when a substituted alkynyl group is provided. Aryl refers to aromatic rings e.g., phenyl, substituted phenyl and like groups as well as rings which are fused, e.g., naphthyl and the like. Aryl thus contains at least one ring having at least 6 atoms, with up to two such rings being present, containing up to 10 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms. The preferred aryl groups are phenyl and naphthyl. Aryl groups may likewise be substituted as defined below. Preferred substituted aryls include phenyl and naphthyl substituted with one or two groups. With regard to the prenyl-protein transferase inhibitors, "aryl" is intended to include any stable monocyclic, bicyclic or tricyclic carbon ring(s) of up to 7 members in each ring, wherein at least one ring is aromatic. Examples of aryl groups include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl and the like. The term "heteroaryl" refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S or N, in which a carbon or nitrogen atom is the point of attachment, and in which one additional carbon atom is optionally replaced by a heteroatom selected from O or S, and in which from 1 to 3 additional carbon atoms are optionally replaced by nitrogen heteroatoms. The heteroaryl group is optionally substituted with up to three groups. Heteroaryl thus includes aromatic and partially aromatic groups which contain one or more heteroatoms. Examples of this type are thiophene, purine, imidazopyridine, pyridine, oxazole, thiazole, oxazine, pyrazole, tetrazole, imidazole, pyridine, pyrimidine, pyrazine and triazine. Examples of partially aromatic groups are tetrahydro- imidazo [4, δ-c] pyridine, phthalidyl and saccharinyl, as defined below.

With regard to the prenyl-protein transferase inhibitors, the term heterocycle or heterocyclic, as used herein, represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic or stable 11-lδ membered tricyclic heterocycle ring which is either saturated or unsaturated, and which consists of carbon atoms and from one to four heteroatoms selected from the group consisting of N, O, and S, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring. The heterocyclic ring may be attached at any heteroatom or carbon atom which results in the creation of a stable structure. Examples of such heterocyclic elements include, but are not limited to, azepinyl, benzimidazolyl, benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl, benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl, cinnolinyl, dihydrobenzofuryl, dihydro-benzothienyl, dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl, imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl, isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl, isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyridyl N-oxide, pyridonyl, pyrazinyl, pyrazolidinyl, pyrazolyl, pyrimidinyl, pyrrolidinyl, pyrrolyl, quinazolinyl, quinolinyl, quinolinyl N-oxide, quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl, tetrahydro-quinolinyl, thiamorpholinyl, thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl, thienothienyl, and thienyl. 5 Preferably, heterocycle is selected from imidazolyl, 2-oxopyrrolidinyl, piperidyl, pyridyl and pyrrolidinyl.

With regard to the prenyl-protein transferase inhibitors, the terms "substituted aryl", "substituted heterocycle" and "substituted cycloalkyl" are intended to include the cyclic group which is substituted 10 with 1 or 2 substitutents selected from the group which includes but is not limited to F, Cl, Br, CF3, NH2, N(Cχ-Cβ alkyl)2, NO2, CN, (Cχ-C6 alkyl)0-, -OH, (Cχ-C6 alkyl)S(0)_m-, (Cχ-Cβ alkyl)C(0)NH-, H2N-C(NH)-, (C1-C6 alkyl)C(O)-, (Cχ-C6 alkyl)OC(O)-, N3/C1-C6 alkyl)OC(0)NH- and Cχ-C20 alkyl. lδ The compounds used in the present method may have asymmetric centers and occur as racemates, racemic mixtures, and as individual diastereomers, with all possible isomers, including optical isomers, being included in the present invention. Unless otherwise specified, named amino acids are understood to have the natural "L"

20 stereoconfiguration.

When various "R" substituents are combined to form - (CH2)u -, cyclic moieties are formed. Examples of such cyclic moieties include, but are not limited to:

2δ In addition, such cyclic moieties may optionally include a heteroatom(s). Examples of such heteroatom-containing cyclic moieties include, but are not limited to:

The pharmaceutically acceptable salts of the compounds of this invention include the conventional non-toxic salts of the compounds of this invention as formed, e.g., from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like: and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenyl-acetic, glutamic, benzoic, salicylic, sulfanilic, 2-acetoxy-benzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.

It is intended that the definition of any substituent or variable (e.g., Rl , Z, n, etc.) at a particular location in a molecule be independent of its definitions elsewhere in that molecule. Thus, -N(RlO)2 represents -NHH, -NHCH3, -NHC2H5, etc. It is understood that substituents and substitution patterns on the compounds of the instant invention can be selected by one of ordinary skill in the art to provide compounds that are chemically stable and that can be readily synthesized by techniques known in the art as well as those methods set forth below. The pharmaceutically acceptable salts of the compounds of this invention can be synthesized from the compounds of this invention which contain a basic moiety by conventional chemical methods. Generally, the salts are prepared by reacting the free base with stoichiometric amounts or with an excess of the desired salt- forming inorganic or organic acid in a suitable solvent or various combinations of solvents.

Peptidyl compounds useful in the instant methods can be synthesized from their constituent amino acids by conventional peptide synthesis techniques, and the additional methods described below. Standard methods of peptide synthesis are disclosed, for example, in the following works: Schroeder et al, "The Peptides", Vol. I, Academic Press 1965, or Bodanszky et al, "Peptide Synthesis", Interscience Publishers, 1966, or McOmie (ed.) "Protective Groups in Organic Chemistry", Plenum Press, 1973, or Barany et al, "The

Peptides: Analysis, Synthesis, Biology" 2, Chapter 1, Academic Press, 1980, or Stewart et al, "Solid Phase Peptide Synthesis", Second Edition, Pierce Chemical Company, 1984. Also useful in exemplifying syntheses of specific unnatural amino acid residues are European Pat. Appl. No. 0 350 163 A2 (particularly page 51-52) and J. E. Baldwin et al. Tetrahedron, 50:5049-5066 (1994). With regards to the synthesis of such peptidyl compounds containing a (β-acetylamino)alanine residue at the C-terminus, use of the commercially available Nα-Z-L-

2,3-diaminopropionic acid (Fluka) as a starting material is preferred. Abbreviations used in the description of the chemistry and in the Examples that follow are:

Ac2θ Acetic anhydride;

Boc t-Butoxycarbonyl; DBU l,8-diazabicyclo[5.4.0]undec-7-ene;

DMAP 4-Dimethylaminopyridine;

DME 1 ,2-Dimethoxy ethane ;

DMF Dimethylformamide; EDC 1 - (3-dimethylaminopropyl)- 3 -ethy 1-carbodiimide- hydrochloride; HOBT 1-Hydroxybenzotriazole hydrate;

Et3N Triethylamine;

EtOAc Ethyl acetate;

FAB Fast atom bombardment;

HOOBT 3-Hydroxy- 1 ,2,2-benzotriazin-4(3#)-one;

HPLC High-performance liquid chromatography;

MCPBA m-Chloroperoxybenzoic acid;

MsCl Methanesulfonyl chloride;

NaHMDS Sodium bis(trimethylsilyl)amide;

Py Pyridine;

TFA Trifluoroacetic acid;

THF Tetrahydrofuran.

The compounds are useful in various pharmaceutically acceptable salt forms. The term "pharmaceutically acceptable salt" refers to those salt forms which would be apparent to the pharmaceutical chemist, i.e., those which are substantially non-toxic and which provide the desired pharmacokinetic properties, palatability, absorption, distribution, metabolism or excretion. Other factors, more practical in nature, which are also important in the selection, are cost of the raw materials, ease of crystallization, yield, stability, hygroscopicity and flowability of the resulting bulk drug. Conveniently, pharmaceutical compositions may be prepared from the active ingredients in combination with pharmaceutically acceptable carriers.

Pharmaceutically acceptable salts include conventional non-toxic salts or quarternary ammonium salts formed, e.g., from non-toxic inorganic or organic acids. Non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, trifluoroacetic and the like.

The pharmaceutically acceptable salts of the compounds useful in the instant invention can be synthesized by conventional chemical methods. Generally, the salts are prepared by reacting the free base or acid with stoichiometric amounts or with an excess of the desired salt-forming inorganic or organic acid or base, in a suitable solvent or solvent combination.

The dual inhibitors of farnesyl-protein transferase and geranylgeranyl-protein transferase type I of formula (I) can be synthesized in accordance with Schemes 1-11, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures. Substituents R, R^a and R^, as shown in the Schemes, represent the substituents R^, R3, R4_{? an}d R5; however their point of attachment to the ring is illustrative only and is not meant to be limiting.

These reactions may be employed in a linear sequence to provide the compounds of the invention or they may be used to synthesize fragments which are subsequently joined by the alkylation reactions described in the Schemes.

Synopsis of Schemes 1-11:

The requisite intermediates are in some cases commercially available, or can be prepared according to literature procedures, for the most part.

Piperazin-5-ones can be prepared as shown in Scheme 1. Thus, the protected suitably substituted amino acid IV can be converted to the corresponding aldehyde V by first forming the amide and then reducing it with LAH. Reductive amination of Boc-protected amino aldehydes V gives rise to compound VI. The intermediate VI can be converted to a piperazinone by acylation with chloroacetyl chloride to give VII, followed by base-induced cyclization to VIII. Deprotection, followed by reductive alkylation with a protected imidazole carboxalde- hyde leads to IX, which can be alkylated with an arylmethylhalide to give the imidazolium salt X. Final removal of protecting groups by either solvolysis with a lower alkyl alcohol, such as methanol, or treatment with triethylsilane in methylene chloride in the presence of trifluoroacetic acid gives the final product XI.

The intermediate VIII can be reductively alkylated with a variety of aldehydes, such as XII. The aldehydes can be prepared by standard procedures, such as that described by O. P. Goel, U. Krolls, M. Stier and S. Kesten in Organic Syntheses. 1988, 67, 69-75, from the appropriate amino acid (Scheme 2). The reductive alkylation can be accomplished at pH 5-7 with a variety of reducing agents, such as sodium triacetoxyborohydride or sodium cyanoborohydride in a solvent such as dichloroethane, methanol or dimethylformamide. The product XIII can be deprotected to give the final compounds XIV with trifluoro- acetic acid in methylene chloride. The final product XIV is isolated in the salt form, for example, as a trifluoroacetate, hydrochloride or acetate salt, among others. The product diamine XIV can further be selectively protected to obtain XV, which can subsequently be reductively alkylated with a second aldehyde to obtain XVI. Removal of the protecting group, and conversion to cyclized products such as the dihydroimidazole XVII can be accomplished by literature procedures.

Alternatively, the imidazole acetic acid XVIII can be converted to the acetate XIX by standard procedures, and XIX can be first reacted with an alkyl halide, then treated with refluxing methanol to provide the regiospecifically alkylated imidazole acetic acid ester XX (Scheme 3). Hydrolysis and reaction with piperazinone VIII in the presence of condensing reagents such as l-(3-dimethylaminopropyl)- 3-ethylcarbodiimide (EDC) leads to acylated products such as XXI. If the piperazinone VIII is reductively alkylated with an aldehyde which also has a protected hydroxyl group, such as XXII in Scheme 4, the protecting groups can be subsequently removed to unmask the hydroxyl group (Schemes 4, 5). The alcohol can be oxidized under standard conditions to e.g. an aldehyde, which can then be reacted with a variety of organometallic reagents such as Grignard reagents, to obtain secondary alcohols such as XXIV. In addition, the fully deprotected amino alcohol XXV can be reductively alkylated (under conditions described previously) with a variety of aldehydes to obtain secondary amines, such as XXVI (Scheme 5), or tertiary amines. The Boc protected amino alcohol XXIII can also be utilized to synthesize 2-aziridinylmethylpiperazinones such as XXVII (Scheme 6). Treating XXIII with l,l'-sulfonyldiimidazole and sodium hydride in a solvent such as dimethylformamide led to the formation of aziridine XXVII. The aziridine reacted in the presence of a nucleophile, such as a thiol, in the presence of base to yield the ring-opened product XXVIII. In addition, the piperazinone VIII can be reacted with aldehydes derived from amino acids such as O-alkylated tyrosines, according to standard procedures, to obtain compounds such as XXX (Scheme 7). When R' is an aryl group, XXX can first be hydrogenated to unmask the phenol, and the amine group deprotected with acid to produce XXXI. Alternatively, the amine protecting group in XXX can be removed, and O-alkylated phenolic amines such as XXXII produced.

Scheme 8 illustrates the use of an optionally substituted homoserine lactone XXXIII to prepare a Boc-protected piperazinone XXXVII. Intermediate XXXVII may be deprotected and reductively alkylated or acylated as illustrated in the previous Schemes. Alternatively, the hydroxyl moiety of intermediate XXXVII may be mesylated and displaced by a suitable nucleophile, such as the sodium salt of ethane thiol, to provide an intermediate XXXVIII. Intermediate XXXVII may also be oxidized to provide the carboxylic acid on intermediate IXL, which can be utilized form an ester or amide moiety.

N-Aralkyl-piperazin-5-ones can be prepared as shown in Scheme 9. Reductive amination of Boc-protected amino aldehydes V (prepared from III as described previously) gives rise to compound XL. This is then reacted with bromoacetyl bromide under Schotten-Baumann conditions; ring closure is effected with a base such as sodium hydride in a polar aprotic solvent such as dimethylformamide to give XLI. The carbamate protecting group is removed under acidic conditions such as trif uoroacetic acid in methylene chloride, or hydrogen chloride gas in methanol or ethyl acetate, and the resulting piperazine can then be carried on to final products as described in Schemes 1-7.

The isomeric piperazin-3-ones can be prepared as described in Scheme 10. The imine formed from arylcarboxamides XLII and 2-aminoglycinal diethyl acetal (XLIII) can be reduced under a variety of conditions, including sodium triacetoxyborohydride in dichloroethane, to give the amine XLIV. Amino acids I can be coupled to amines XLIV under standard conditions, and the resulting amide XLV when treated with aqueous acid in tetrahydrofuran can cyclize to the unsaturated XLVI. Catalytic hydrogenation under standard conditions gives the requisite intermediate XLVII, which is elaborated to final products as described in Schemes 1-7.

Amino acids of the general formula IL which have a sidechain not found in natural amino acids may be prepared by the reactions illustrated in Scheme 11 starting with the readily prepared imine XLVIII.

SCHEME 1

IV DMF, Et₃N, pH 7

LAH, Et₂0 BocN

SCHEME 1 (continued

NaHC0₃ VII VIII

X

XI SCHEME 2

HCIH

VIM

NHBoc

XIII

XIV

XV SCHEME 2 (continued)

SCHEME 3

XIX 1) ArCH₂X CH₃CN reflux_ 2) CH₃0H, reflux

XIX

SCHEME 3 (continued

SCHEME 4

HCI H

XXII

XXIII

SCHEME 5

R

CF₃C0₂H

XXIII

R

R'CHO

XXV

R

i N N-Ar

NH O

R'CH;

XXVI

SCHEME 6

XXIII

SCHEME 7

^H°X⁾

ocNH C0₂CH₃

SCHEME 7 (continued)

XXX HCI ETOAc

1)20%Pd(OH)₂ CH₃OH, CH₃C0₂H

2) HCI, EtOAc

XXXII

XXXI SCHEME 8

CICH₂CH₂CI XXXIV

Ar

XXXV

XXXVI SCHEME 8 (continued)

XXXVIII IXL

SCHEME 9

pH6

V

XL

XLI

r

SCHEME 10

NaBH(OAc)₃

ArCHO + NH₂CH₂CH(OC_; >H_{5 2} -

XLIV

EDC . HCI, HOBT

DMF, Et₃N, pH 7

XLV

XLVI

SCHEME 11

1. KOtBu, THF _R

/-C0₂Et ^R2χ >-C0₂Et

=N ~ H₂N

Ph^' 2. 5% aqueous HCI _HC) XLVI 11

BocHN

2. LiAIH₄, Et₂0

IL

Reactions used to generate the compounds of the formula (II) are prepared by employing reactions as shown in the Schemes 16- 37, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures. Substituents R^a and R^D, as shown in the Schemes, represent the substituents R2, R3, R4_{? an}d R5; substituent "sub" represents a suitable substituent on the substituent Z. The point of attachment of such substituents to a ring is illustrative only and is not meant to be limiting.

Synopsis of Schemes 16-37:

The requisite intermediates utilized as starting material in the Schemes hereinbelow are in some cases commercially available, or can be prepared according to literature procedures. In Scheme 16, for example, a suitably substituted Boc protected isonipecotate LI may be deprotonated and then treated with a suitably substituted alkylating group, such as a suitably substituted benzyl bromide, to provide the gem disubstituted intermediate LIII. Deprotection and reduction provides the hydroxymethyl piperidine LIV which can be utilized is synthesis of compounds of the invention or which may be nitrogen-protected and methylated to give the intermediate LV.

As shown in Scheme 17, the protected piperidine intermediate LIII can be deprotected and reductively alkylated with aldehydes such as l-trityl-4-imidazolyl-carboxaldehyde or l-trityl-4-imidazolylacetaldehyde, to give products such as LVI. The trityl protecting group can be removed from LVI to give LVII, or alternatively, LVI can first be treated with an alkyl halide then subsequently deprotected to give the alkylated imidazole LVIII.

The deprotected intermediate LIII can also be reductively alkylated with a variety of other aldehydes and acids as shown above in Schemes 4-7.

An alternative synthesis of the hydroxymethyl intermediate LIV and utilization of that intermediate in the synthesis of the instant compounds which incorporate the preferred imidazolyl moiety is illustrated in Scheme 18. Scheme 19 illustrates the reductive alkylation of intermediate LIV to provide a 4-cyanobenzylimidazolyl substituted piperidine. The cyano moiety may be selectively hydrolyzed with sodium borate to provide the corresponding amido compound of the instant invention.

Scheme 20 alternative preparation of the methyl ether intermediate LV and the alkylation of LV with a suitably substituted imidazolylmethyl chloride to provide the instant compound. Preparation of the homologous l-(imidazolylethyl)piperidine is illustrated in Scheme 21.

Specific substitution on the piperidine of the compounds of the instant invention may be accomplished as illustrated in Scheme 22. Thus, metal-halogen exchange coupling of a butynyl moiety to an isonicotinate, followed by hydrogenation, provides the 2-butylpiperidine intermediate that can then undergo the reactions previously described to provide the compound of the instant invention. Incorporation of a 4-amido moiety for LV is illustrated in Scheme 23.

Scheme 24 illustrates the synthesis of the instant compounds wherein the moiety Z is attached directly to the piperidine ring. Thus the piperidone LIX is treated with a suitably substituted phenyl Grignard reagent to provide the gem disubstituted piperidine LX. Deprotection provides the key intermediate LXI. Intermediate LXI may be acetylated as described above to provide the instant compound LXII (Scheme 25). As illustrated in Scheme 26, the protected piperidine

LX may be dehydrated and then hydroborated to provide the 3- hydroxypiperidine LXIII. This compound may be deprotected and further derivatized to provide compounds of the instant invention (as shown in Scheme 27) or the hydroxyl group may be alkylated, as shown in Scheme 26, prior to deprotection and further manipulation. The dehydration product may also be catalytically reduced to provide the des-hydroxy intermediate LXV, as shown in Scheme 28, which can be processed via the reactions illustrated in the previous Schemes. Schemes 29 and 30 illustrate further chemical manipulations of the 4-carboxylic acid functionality to provide instant compounds wherein the substituent Y is an acetylamine or sulfonamide moiety.

Scheme 31 illustrates incorporation of a nitrile moiety in the 4-position of the piperidine of the compounds of formula II. Thus, the hydroxyl moiety of a suitably substituted 4-hydroxypiperidine is substituted with nitrile to provide intermediate LXVI, which can undergo reactions previously described in Schemes 17-21.

Scheme 32 illustrates the preparation of several pyridyl intermediates that may be utilized with the piperidine intermediates such as compound LI in Scheme 16 to provide the instant compounds. Scheme 33 shows a generalized reaction sequence which utilizes such pyridyl intermediates.

Compounds of the instant invention wherein χl is a carbonyl moiety may be prepared as shown in Scheme 34. Inter- mediate LXVII may undergo subsequent reactions as illustrated in Schemes 17-21 to provide the instant compounds. Preparation of the instant compounds wherein χl is sulfur in its various oxidation states is shown in Scheme 35. Intermediates LXVIII-LXXI may undergo the previously described reactions to provide the instant compounds. Scheme 36 illustrated preparation of compounds of the formula A wherein Y is hydrogen. Thus, suitably substituted isonipecotic acid may be treated with N,0-dimethylhydroxylamine and the intermediate LXXII reacted with a suitably substituted phenyl Grignard reagent to provide intermediate LXXIII. That intermediate may undergo the reactions previously described in Schemes 17-21 and may be further modified by reduction of the phenyl ketone to provide the alcohol LXXIV.

Compounds of the instant invention wherein χl is an amine moiety may be prepared as shown in Scheme 37. Thus the

N-protected 4-piperidinone may be reacted with a suitably substituted aniline in the presence of trimethylsilylcyanide to provide the 4-cyano- 4-aminopiperidine LXXV. Intermediate LXXV may then be converted in sequence to the corresponding amide LXXVI, ester LXXVII and alcohol LXXVIII. Intermediates LXXVI-LXXVIII can be deprotected and can then undergo the reactions previously described in Schemes 17-21 to provide the compounds of the instant invention.

SCHEME 16

SCHEME 17

SCHEME 18

NaHMDS

SCHEME 19

SCHEME 20

HCI, EtOAc

SCHEME 21

HCI, EtOAc

SCHEME 22

SCHEME 23

SCHEME 24

SCHEME 25

SCHEME 26

dine

LX

LXI 11

LXIV

SCHEME 27

LXI II

Tr

SCHEME 28

ridine

LXV

EDC ^• HCI

HOBt DMF

SCHEME 29

SCHEME 29 (continued)

SCHEME 30

SCHEME 31

HCI, EtOAc

Sub

SCHEME 32

SCHEME 32 (continued)

SCHEME 33

LiAIH,

SCHEME 34

R — CH , CH₃CH₂

SCHEME 35

LIAIH,

Oxone

SCHEME 36

HCI, EtOAc

iPr₂NEt

SCHEME 37

LXXVI

LiAIH,

- Ill Compounds of this invention of formula (III) are prepared by employing the reactions shown in the following Reaction Schemes 38-51, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures. Some key bond-forming and peptide modifying reactions are:

Reaction A Amide bond formation and protecting group cleavage using standard solution or solid phase methodologies.

Reaction B Preparation of a reduced peptide subunit by reductive alkylation of an amine by an aldehyde using sodium cyanoborohydride or other reducing agents.

Reaction C Alkylation of a reduced peptide subunit with an alkyl or aralkyl halide or, alternatively, reductive alkylation of a reduced peptide subunit with an aldehyde using sodium cyanoborohydride or other reducing agents.

Reaction D Peptide bond formation and protecting group cleavage using standard solution or solid phase methodologies.

Reaction E Preparation of a reduced subunit by borane reduction of the amide moiety.

These reactions may be employed in a linear sequence to provide the compounds of the invention or they may be used to synthesize fragments which are subsequently joined by the alkylation reactions described in the Reaction Schemes and in Reaction Schemes 43-51 hereinbelow. REACTION SCHEME 38

Reaction A. Coupling of residues to form an amide bond

REACTION SCHEME 39

Reaction B. Preparation of reduced peptide subunits by reductive alkylation

NaCNBH,

REACTION SCHEME 40

Reaction C. Alkylation/reductive alkylation of reduced peptide subunits

R^yCH, NaCNBH₃

R^E

REACTION SCHEME 41

Reaction D. Coupling of residues to form an amide bond

NR C^UnRD

REACTION SCHEME 42

Reaction E. Preparation of reduced dipeptides from peptides

where RA and RB are R2, R3 or R5 as previously defined; RC and RD are R^ or Rl2; XL is a leaving group, e.g., Br, I- or MsO-; and Ry is defined such that R7 is generated by the reductive alkylation process. In addition to the reactions described in Reaction Schemes 26-30, other reactions used to generate the compounds of formula (III) of this invention are shown in the Reaction Schemes 43-51. All of the substituents shown in the Reaction Schemes, represent the same substituents as defined hereinabove. The substituent "Ar" in the Reaction Schemes represents a carbocyclic or heterocyclic, substituted or unsubstituted aromatic ring.

These reactions may be employed in a linear sequence to provide the compounds of the invention or they may be used to synthesize fragments which are subsequently joined by the alkylation reactions described in the Reaction Schemes. The sequential order whereby substituents are incorporated into the compounds is often not critical and thus the order of reactions described in the Reaction Schemes are illustrative only and are not limiting.

Synopsis of Reaction Schemes 43-51: The requisite intermediates are in some cases commercially available, or can be readily prepared according to known literature procedures, including those described in Reaction Schemes 38-42 hereinabove.

Reaction Scheme 43 illustrates incorporation of the cyclic amine moiety, such as a reduced prolyl moiety, into the compounds of the formula III of the instant invention. Reduction of the azide LXXXI provides the amine LXXXII, which may be mono- or di-substituted using techniques described above. As an example, incorporation of a naphthylmethyl group and an acetyl group is illustrated.

As shown in Reaction Scheme 44, direct attachment of a aromatic ring to a substituted amine such as LXXXIII is accomplished by coupling with a triarylbismuth reagent, such as tris(3-chlorophenyl) bismuth.

Reaction Scheme 45 illustrates the use of protecting groups to prepare compounds of the instant invention wherein the cyclic amine contains an alkoxy moiety. The hydroxy moiety of key intermediate LXXXIVa may be further converted to a fluoro or phenoxy moiety, as shown in Reaction Scheme 46. Intermediates LXXXV and LXXXVI may then be further elaborated to provide the instant compounds. Reaction Scheme 474 illustrates syntheses of instant compounds wherein the variable

is a suitably substituted α-hydroxybenzyl moiety. Thus the protected intermediate aldehyde is treated with a suitably substituted phenyl Grignard reagent to provide the enantiomeric mixture LXXXVII. Treatment of the mixture with 2-picolinyl chloride allows chromatographic resolution of compounds LXXXVIII and IXC. Removal of the picolinoyl group followed by deprotection provides the optically pure intermediate XC which can be further processed as described hereinabove to yield the instant compounds.

Syntheses of imidazole-containing intermediates useful in synthesis of instant compounds wherein the variable p is 0 or 1 and Z is H2 are shown in Reaction Scheme 48 and 49. Thus the mesylate XCI can be utilized to alkylate a suitably substituted amine or cyclic amine, while aldehyde XCII can be used to similarly reductively alkylate such an amine. Reaction Scheme 50 illustrates the syntheses of imidazole-containing intermediates wherein the attachment point of the -(CR22)p-C(Z)- moiety to W (imidazolyl) is through an imidazole ring nitrogen. Reaction Scheme 51 illustrates the synthesis of an intermediate wherein an R2 substituent is a methyl.

REACTION SCHEME 43

REACTION SCHEME 43 (continued)

REACTION SCHEME 44

R

REACTION SCHEME 45

REACTION SCHEME 45 (continued)

n

REACTION SCHEME 46

REACTION SCHEME 47

LXXXVI

OTBS l Chloride

LXXXVII

OTBS OTBS

REACTION SCHEME 47 (continued)

OTBS

XC

OH

REACTION SCHEME 48

REACTION SCHEME 49

Ac₂0 pyridine *

XCII REACTION SCHEME 50

Ni(PPh₃)₂CI₂

+ BrZnCH₂Ar tf

Tr

TfOCH₂C0₂CH₃

XCVII

(MeS0₂)₂0 B₃N

CVIII REACTION SCHEME 51

XCVI The prenyl transferase inhibitors of formula (A) can be synthesized in accordance with Reaction Scheme below, in addition to other standard manipulations such as ester hydrolysis, cleavage of protecting groups, etc., as may be known in the literature or exemplified in the experimental procedures. Some key reactions utilized to form the aminodiphenyl moiety of the instant compounds are shown.

The reactions may be employed in a linear sequence to provide the compounds of the invention or they may be used to synthesize fragments which are subsequently joined by the alkylation reactions described in the Reaction Scheme.

A method of forming the benzophenone intermediates, illustrated in Reaction Scheme 52, is a Stille reaction with an aryl stannane. Such amine intermediates may then be reacted as illustrated hereinabove with a variety of aldehydes and esters/acids. REACTION SCHEME 52

n-Bu)₃

Tr EXAMPLES

Examples provided are intended to assist in a further understanding of the invention. Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof.

The standard workup referred to in the examples refers to solvent extraction and washing the organic solution with 10% citric acid, 10% sodium bicarbonate and brine as appropriate. Solutions were dried over sodium sulfate and evaporated in vacuo on a rotary evaporator.

EXAMPLE 1

1 -(3-Chlorophenyl)-4- [ 1 -(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone dihydrochloride (Compound 1)

Step A: Preparation of l-triphenylmethyl-4-(hydroxymethyl)- imidazole To a solution of 4-(hydroxymethyl)imidazole hydrochloride (35.0 g, 260 mmol) in 250 mL of dry DMF at room temperature was added triethylamine (90.6 mL, 650 mmol). A white solid precipitated from the solution. Chlorotriphenylmethane (76.1 g, 273 mmol) in 500 mL of DMF was added dropwise. The reaction mixture was stirred for 20 hours, poured over ice, filtered, and washed with ice water. The resulting product was slurried with cold dioxane, filtered, and dried in vacuo to provide the titled product as a white solid which was sufficiently pure for use in the next step.

Step B: Preparation of l-triphenylmethyl-4-(acetoxymethyl)- imidazole

Alcohol from Step A (260 mmol, prepared above) was suspended in 500 mL of pyridine. Acetic anhydride (74 mL, 780 mmol) was added dropwise, and the reaction was stirred for 48 hours during which it became homogeneous. The solution was poured into 2 L of EtOAc, washed with water (3 x 1 L), 5% aq. HCI soln. (2 x 1 L), sat. aq. NaHCθ3, and brine, then dried (Na2Sθ4), filtered, and concentrated in vacuo to provide the crude product. The acetate was isolated as a white powder which was sufficiently pure for use in the next reaction.

Step C: Preparation of l-(4-cyanobenzyl)-5-(acetoxymethyl)- imidazole hydrobromide A solution of the product from Step B (85.8 g, 225 mmol) and α-bromo-/?-tolunitrile (50.1 g, 232 mmol) in 500 mL of EtOAc was stirred at 60°C for 20 hours, during which a pale yellow precipitate formed. The reaction was cooled to room temperature and filtered to provide the solid imidazolium bromide salt. The filtrate was concentrated in vacuo to a volume 200 mL, reheated at 60°C for two hours, cooled to room temperature, and filtered again. The filtrate was concentrated in vacuo to a volume 100 mL, reheated at 60°C for another two hours, cooled to room temperature, and concentrated in vacuo to provide a pale yellow solid. All of the solid material was combined, dissolved in 500 mL of methanol, and warmed to 60°C. After two hours, the solution was reconcentrated in vacuo to provide a white solid which was triturated with hexane to remove soluble materials. Removal of residual solvents in vacuo provided the titled product hydrobromide as a white solid which was used in the next step without further purification.

Step D: Preparation of l-(4-cyanobenzyl)-5-(hydroxymethyl)- imidazole

To a solution of the acetate from Step C (50.4 g, 150 mmol) in 1.5 L of 3: 1 THF/water at 0°C was added lithium hydroxide monohydrate (18.9 g, 450 mmol). After one hour, the reaction was concentrated in vacuo, diluted with EtOAc (3 L), and washed with water, sat. aq. NaHC03 and brine. The solution was then dried (Na2Sθ4), filtered, and concentrated in vacuo to provide the crude product as a pale yellow fluffy solid which was sufficiently pure for use in the next step without further purification.

Step E: Preparation of l-(4-cyanobenzyl)-5- imidazolecarboxaldehyde

To a solution of the alcohol from Step D (21.5 g, 101 mmol) in 500 mL of DMSO at room temperature was added triethylamine (56 mL, 402 mmol), then Sθ3-pyridine complex (40.5 g, 254 mmol). After 45 minutes, the reaction was poured into 2.5 L of EtOAc, washed with water (4 x 1 L) and brine, dried (Na2Sθ4), filtered, and concentrated in vacuo to provide the aldehyde as a white powder which was sufficiently pure for use in the next step without further purification.

Step F: Preparation of N-(3-chlorophenyl)ethylenediamine hydrochloride

To a solution of 3-chloroaniline (30.0 mL, 284 mmol) in 500 mL of dichloromethane at 0°C was added dropwise a solution of 4 N HCI in 1,4-dioxane (80 mL, 320 mmol HCI). The solution was warmed to room temperature, then concentrated to dryness in vacuo to provide a white powder. A mixture of this powder with 2-oxazolidinone (24.6 g, 282 mmol) was heated under nitrogen atmosphere at 160°C for 10 hours, during which the solids melted, and gas evolution was observed. The reaction was allowed to cool, forming the crude diamine hydrochloride salt as a pale brown solid.

Step G: Preparation of N-(fer -butoxycarbonyl)-N'-(3- chlorophenyDethylenediamine

The amine hydrochloride from Step F (ca. 282 mmol, crude material prepared above) was taken up in 500 mL of THF and 500 mL of sat. aq. ΝaHCθ3 soln., cooled to 0°C, and άi-tert- butylpyrocarbonate (61.6 g, 282 mmol) was added. After 30 h, the reaction was poured into EtOAc, washed with water and brine, dried (Na2S04), filtered, and concentrated in vacuo to provide the titled carbamate as a brown oil which was used in the next step without further purification.

Step H: Preparation of N-[2-(ter?-butoxycarbamoyl)ethyl]-N-(3- chlorophenyl)-2-chloroacetamide

A solution of the product from Step G (77 g, ca. 282 mmol) and triethylamine (67 mL, 480 mmol) in 500 mL of CH2CI2 was cooled to 0°C. Chloroacetyl chloride (25.5 mL, 320 mmol) was added dropwise, and the reaction was maintained at 0°C with stirring. After 3 h, another portion of chloroacetyl chloride (3.0 mL) was added dropwise. After 30 min, the reaction was poured into EtOAc (2 L) and washed with water, sat. aq. NH4CI soln, sat. aq. NaHCθ3 soln., and brine. The solution was dried (Na2Sθ4), filtered, and concentrated in vacuo to provide the chloroacetamide as a brown oil which was used in the next step without further purification.

Step I: Preparation of 4-(tert-butoxycarbonyl)-l-(3- chlorophenyl)-2-piperazinone To a solution of the chloroacetamide from Step H (ca.

282 mmol) in 700 mL of dry DMF was added K2CO3 (88 g, 0.64 mol). The solution was heated in an oil bath at 70-75°C for 20 hrs., cooled to room temperature, and concentrated in vacuo to remove ca. 500 mL of DMF. The remaining material was poured into 33% EtOAc/hexane, washed with water and brine, dried (Na2S04), filtered, and concentrated in vacuo to provide the product as a brown oil. This material was purified by silica gel chromatography (25- 50% EtOAc/hexane) to yield pure product, along with a sample of product (ca. 65% pure by HPLC) containing a less polar impurity.

Step J: Preparation of l-(3-chlorophenyl)-2-piperazinone

Through a solution of Boc-protected piperazinone from Step I (17.19 g, 55.4 mmol) in 500 mL of EtOAc at -78°C was bubbled anhydrous HCI gas. The saturated solution was warmed to 0°C, and stirred for 12 hours. Nitrogen gas was bubbled through the reaction to remove excess HCI, and the mixture was warmed to room temperature. The solution was concentrated in vacuo to provide the hydrochloride as a white powder. This material was taken up in 300 mL of CH2CI2 and treated with dilute aqueous NaHCθ3 solution. The aqueous phase was extracted with CH2CI2 (8 x 300 mL) until tic analysis indicated complete extraction. The combined organic mixture was dried (Na2S04), filtered, and concentrated in vacuo to provide the titled free amine as a pale brown oil.

Step K: Preparation of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl) imidazolylmethyll-2-piperazinone dihydrochloride

To a solution of the amine from Step J (55.4 mmol, prepared above) in 200 mL of 1,2-dichloroethane at 0°C was added 4 A powdered molecular sieves (10 g), followed by sodium triacetoxyborohydride (17.7 g, 83.3 mmol). The imidazole carboxaldehyde from Step E of Example 1 (11.9 g, 56.4 mmol) was added, and the reaction was stirred at 0°C. After 26 hours, the reaction was poured into EtOAc, washed with dilute aq. NaHCθ3, and the aqueous layer was back-extracted with EtOAc. The combined organics were washed with brine, dried (Na2S04), filtered, and concentrated in vacuo. The resulting product was taken up in 500 mL of 5: 1 benzene:CH2θ2, and propylamine (20 mL) was added. The mixture was stirred for 12 hours, then concentrated in vacuo to afford a pale yellow foam. This material was purified by silica gel chromatography (2-7% MeOH/CH2θ2), and the resultant white foam was taken up in CH2CI2 and treated with 2.1 equivalents of 1 M HCl/ether solution. After concentrated in vacuo, the product dihydrochloride was isolated as a white powder.

Examples 2-5 (Table 1) were prepared using the above protocol, which describes the synthesis of the structurally related compound 1 -(3-chlorophenyl)-4- [ 1 -(4-cyanobenzyl)-imidazolylmethyl]-2- piperazinone dihydrochloride. In Step F, the appropriately substituted aniline was used in place of 3-chloroaniline.

Table 1: 1 - Aryl-4- [ 1 - (4-cy anobenzyl)imidazolylmethy 1] -2- piperazinones

FAB mass spectrum CHN

Example X (M+l) Analysis

3-OCF₃ 456 C23H20 F₃N5θ2^«2.0HCl^«0.60H2θ calcd; C, 51.24; H, 4.34; N, 12.99. found; C, 51.31; H, 4.33; N, 12.94.

2,5-(CH₃)2 400 C24H25N5O^«2.00HCl^«0.65H₂O calcd; C, 59.54; H, 5.89; N, 14.47 found; C, 59.54; H, 5.95; N, 14.12.

3-CH3 386 C23H23N5O*2.0HCl-0.80H₂O calcd; C, 58.43; H, 5.67; N, 14.81. found; C, 58.67; H, 6.00; N, 14.23.

3-1 498 C22H20N5θI^«2.25HCl-0.90H2θ calcd; C, 44.36; H, 4.07; N, 11.76. found; C, 44.37; H, 4.06; N, 11.42. EXAMPLE 6

l-(3-chlorophenyl)-4-[l-(4-cyano-3-methoxybenzyl)imidazolylmethyl]-

2-piperazinone dihydrochloride

Step A: Preparation of Methyl 4-Amino-3-hydroxybenzoate

Through a solution of 4-amino-3-hydroxybenzoic acid (75 g, 0.49 mol) in 2.0 L of dry methanol at room temperature was bubbled anhydrous HCI gas until the solution was saturated. The solution was stirred for 48 hours, then concentrated in vacuo. The product was partitioned between EtOAc and saturated aq. NaHC0₃ solution, and the organic layer was washed with brine, dried (Na₂S0₄), and concentrated in vacuo to provide the titled compound (79 g, 96% yield).

Step B: Preparation of Methyl 3-Hydroxy-4-iodobenzoate

A cloudy, dark solution of the product from Step A (79 g, 0.47 mol), 3N HCI (750 mL), and THF (250 mL) was cooled to 0°C. A solution of NaN0₂ (35.9 g, 0.52 mol) in 115 mL of water was added over ca. 5 minutes, and the solution was stirred for another 25 minutes. A solution of potassium iodide (312 g, 1.88 mol) in 235 mL of water was added all at once, and the reaction was stirred for an additional 15 minutes. The mixture was poured into EtOAc, shaken, and the layers were separated. The organic phase was washed with water and brine, dried (Na₂S0₄), and concentrated in vacuo to provide the crude product (148 g). Purification by column chromatography through silica gel (0%-50% EtOAc/hexane) provided the titled product (96 g, 73% yield).

Step C: Preparation of Methyl 4-Cyano-3-hydroxybenzoate A mixture of the iodide product from Step B (101 g, 0.36 mol) and zinc(II)cyanide (30 g, 0.25 mol) in 400 mL of dry DMF was degassed by bubbling argon through the solution for 20 minutes. Tetrakis(triphenylphosphine)palladium (8.5 g, 7.2 mmol) was added, and the solution was heated to 80°C for 4 hours. The solution was cooled to room temperature, then stirred for an additional 36 hours. The reaction was poured into EtO Ac/water, and the organic layer was washed with brine (4x), dried (Na₂S0₄), and concentrated in vacuo to provide the crude product. Purification by column chromatography through silica gel (30%-50%

EtOAc/hexane) provided the titled product (48.8 g, 76% yield).

Step D: Preparation of Methyl 4-Cyano-3-methoxybenzoate

Sodium hydride (9 g, 0.24 mol as 60% wt. disp. mineral oil) was aded to a solution of the phenol from Step C (36.1 g, 204 mmol) in 400 mL of dry DMF at room temperature. Iodomethane was added (14 mL. 0.22 mol) was added, and the reaction was stirred for 2 hours. The mixture was poured into EtO Ac/water, and the organic layer was washed with water and brine (4x), dried (Na₂S0₄), and concentrated in vacuo to provide the titled product (37.6 g, 96% yield).

Step E: Preparation of 4-Cvano-3-methoxybenzyl Alcohol

To a solution of the ester from Step D (48.8 g, 255 mmol) in 400 mL of dry THF under argon at room temperature was added lithium borohydride (255 mL, 510 mmol, 2M THF) over 5 minutes. After 1.5 hours, the reaction was warmed to reflux for 0.5 hours, then cooled to room temperature. The solution was poured into EtOAc/lN HCI soln. [CAUTION], and the layers were separated. The organic layer was washed with water, sat Na₂C0₃ soln. and brine (4x), dried (Na₂S0₄), and concentrated in vacuo to provide the titled product (36.3 g, 87% yield).

Step F: Preparation of 4-Cyano-3-methoxybenzyl Bromide A solution of the alcohol from Step E (35.5 g,

218 mmol) in 500 mL of dry THF was cooled to 0°C. Triphenylphosphine was added (85.7 g, 327 mmol), followed by carbontetrabromide (108.5 g, 327 mmol). The reaction was stirred at 0°C for 30 minutes, then at room temperature for 21 hours. Silica gel was added (ca. 300 g), and the suspension was concentrated in vacuo. The resulting solid was loaded onto a silica gel chromatography column. Purification by flash chromatography (30%-50% EtOAc/hexane) provided the titled product (42 g, 85% yield).

Step G: Preparation of l-(4-cyano-3-methoxybenzyl)-5-

(acetoxymethyl)-imidazole hydrobromide

The titled product was prepared by reacting the bromide from Step F (21.7 g, 96 mmol) with the imidazole product from Step B of Example 1 (34.9 g, 91 mmol) using the procedure outlined in Step C of Example 1. The crude product was triturated with hexane to provide the titled product hydrobromide (19.43 g, 88% yield).

Step H: Preparation of l-(4-cyano-3-methoxybenzyl)-5- (hydroxymethyl)-imidazole

The titled product was prepared by hydrolysis of the acetate from Step G (19.43 g, 68.1 mmol) using the procedure outlined in Step D of Example 1. The crude titled product was isolated in modest yield (11 g, 66% yield). Concentration of the aqueous extracts provided solid material (ca. 100 g) which contained a significant quantity of the titled product , as judged by H NMR spectroscopy.

Step I: Preparation of l-(4-cyano-3-methoxybenzyl)-5- imidazolecarboxaldehyde

The titled product was prepared by oxidizing the alcohol from Step H (11 g, 45 mmol) using the procedure outlined in Step E of Example 1. The titled aldehyde was isolated as a white powder (7.4 g, 68% yield) which was sufficiently pure for use in the next step without further purification.

Step J: Preparation of l-(3-chlorophenyl)-4-[l-(4-cyano-3- methoxybenzyl)imidazolylmethyl]-2-piperazinone dihydrochloride The titled product was prepared by reductive alkylation of the aldehyde from Step I (859 mg, 3.56 mmol) and the amine (hydrochloride) from Step K of Example 1 (800 mg, 3.24 mmol) using the procedure outlined in Step H of Example 1. Purification by flash column chromatography through silica gel (50%-75% acetone CH₂C1₂) and conversion of the resulting white foam to its dihydrochloride salt provided the titled product as a white powder

(743 mg, 45% yield). FAB ms (m+1) 437.

Anal. Calc. for C23H23ClN5θ2^«2.0HCM⁾.35CH2Cl2: C, 51.97; H, 4.80; N, 12.98.

Found: C, 52.11; H, 4.80; N, 12.21.

EXAMPLE 7

1 - (3-trifluoromethoxyphenyl)-4- [ 1 - (4-cy ano-3 - methoxybenzyDimidazolyl methyl! -2-piperazinone dihydrochloride 1 -(3-trifluoromethoxy-phenyl)-2-piperazinone hydrochloride was prepared from 3-trifluoromethoxyaniline using Steps F-J of Example 1. This amine (1.75 g, 5.93 mmol) was coupled to the aldehyde from Step I of Example 6 (1.57 g, 6.52 mmol) using the procedure outlined in Step H of Example 1. Purification by flash column chromatography through silica gel (60%- 100% acetone CH₂C1₂) and conversion of the resulting white foam to its dihydrochloride salt provided the titled product as a white powder (1.947 g, 59% yield). FAB ms (m+1) 486.

Anal. Calc. for C24H23F3N5θ3^»2.0HCl-0.60H2θ:

C, 50.64; H, 4.46; N, 12.30. Found: C, 50.69; H, 4.52; N, 12.13.

EXAMPLE 8

4-[((l-(4-cyanobenzyl)-5-imidazolyl)methyl)amino]benzophenone hydrochloride The titled product was prepared by reductive alkylation of the aldehyde from Step E of Example 1 (124 mg, 0.588 mmol) and 4-aminobenzophenone (116 mg, 0.588 mmol) using the procedure outlined in Step K of Example 1. Purification by flash column chromatography through silica gel (2-6% MeOH/CH₂Cl₂) and conversion to the hydrochloride salt provided the titled product as a white solid (126 mg, 50% yield). FAB ms (m+1) 393.11.

Anal. Calc. for C25H2θN5θ-1.40HCl-0.40H2θ: C, 66.62; H, 4.96; N, 12.43. Found: C, 66.73; H, 4.94; N, 12.46.

EXAMPLE 9

N-{ l-(4-Cyanobenzyl)-lH-imidazol-5-ylethyl}-4(R)-benzyloxy-2(S)- { N'-acetyl-N'-3-chlorobenzyl }aminomethylpyrolidine

Step A: 4(R)-Hydroxyproline methyl ester

A suspension of 4(R)-hydroxyproline (35.12g, 267.8 mmol) in methanol (500ml) was saturated with gaseous hydrochloric acid. The resulting solution was allowed to stand for 16hrs and the solvent evaporated in vacuo to afford the title compound as a white solid.

!H NMR CD3OD δ 4.60 (2H, m), 3.86(3H, s), 3.48(1H, dd, J=3.6 and 12.0Hz), 3.23(1H, d, J=12.0Hz), 2.43(1H, m) and 2.21(1H, m) ppm.

Step B: N-t-Butoxycarbonyl-4(R)-hydroxyproline methyl ester

To a solution of 4(R)-hydroxyproline methyl ester (53.5g, 268mmol), and triethylamine (75ml, 540mmol), in CH2CI2 (500ml), at 0°C, was added a solution of di-t-butyl dicarbonate (58.48, 268mmol), in CH2CI2 (75ml). The resulting mixture was stirred for 48hrs at room temperature. The solution was washed with 10% aqueous citric acid solution, saturated NaHCθ3 solution, dried (Na2S04) and the solvent evaporated in vacuo. The title compound was obtained as a yellow oil and used in the next step without furthur purification. iH NMR CD3OD δ 4.40-4.30 (2H, m), 3.75(3H, m), 3.60-3.40(2H, m), 2.30(1H, m), 2.05(1H, m) and 1.55-1.40(9H, m) ppm.

Step C: N-t-Butoxycarbonyl-4(R)-t-butyldimethylsilyloxy proline methyl ester

To a solution of N-t-butoxycarbonyl-4(R)-hydroxy proline methyl ester (65.87g, 268mmol), and triethylamine (41ml, 294mmol), in CH2CI2 (536ml), at 0°C, was added a solution of t-butyldimethyl silylchloide (42.49g, 282mmol), in CH2CI2 (86ml).

The resulting mixture was stirred for 16hrs at room temperature.

The solution was washed with 10% aqueous citric acid solution, saturated NaHCθ3 solution, dried (Na2S04) and the solvent evaporated in vacuo. The title compound was obtained as a yellow oil and used in the next step without furthur purification. iH NMR CD3OD δ 4.60-4.40 (2H, m), 3.75(3H, m), 3.60-3.20(2H, m), 2.30-1.90(2H, m), 1.45-1.40(9H, m), 0.90-0.85(9H, m), 0.10-

0.00(6H, m) ppm.

Step D: N-t-Butoxycarbonyl-4(R)-t-butyldimethylsilyloxy-2(S)- hydroxymethylpyrrolidine

A solution of N-t-butoxycarbonyl-4-(R)-t-butyldimethyl- silyloxy proline methyl ester (86.65g, 241mmol), in THF (150ml), was added over 90 minutes to a solution of lithium aluminum hydride (247ml of a 1M solution in THF, 247mmol), under argon, so that the temperature did not exceed 12°C. Stirring was continued for 50 mins and then EtOAc (500ml) was added cautiously, followed by sodium sulphate decahydrate (34g), and the resulting mixture stirred for 16 hrs at room temperature. Anhydrous Na₂S0₄ (34g) was added and the mixture stirred an additional 30 min and then filtered. The solids were washed with EtOAc (800ml), the filtrates combined and the solvent evaporated in vacuo. The title compound was obtained as a colourless oil and used in the next step without further purification. Step E: N-t-Butoxycarbonyl-4(R)-t-butyldimethylsilyloxy-2(S)- methanesulfonyloxymethylpyrrolidine

To a solution of N-t-butoxycarbonyl-4(R)-t-butyldimethyl- silyloxy-2(S)-hydroxymethylpyrrolidine (50.0g, 150.8 mmol) and triethylamine (42.0 ml, 300 mmol) in CH₂C1₂(1 1) was added methane sulfonyl chloride (12.4ml, 160 mmol) over a period of 5 minutes and stirring was continued for 1 hour. The solvent was evaporated in vacuo diluted with EtOAc (800 mL) and washed sequentially with aqueous citric acid and NaHC0₃. The organic extracts were dried (Na₂S0₄), evaporated in vacuo and the residue purified by chromatography (Si0₂, 15% EtOAc in hexanes). The title compound was obtained as a pale yellow solid. FAB Mass spectrum, m/z = 410(M+1). !H NMR CDCI3 δ 4.60-4.00 (4H, m), 3.60-3.30(2H, m), 2.98(3H, s), 2.05-2.00(2H, m), 1.48-1.42(9H, m),0.90-0.80(9H, m), 0.10-0.00(6H, m) ppm.

Step F: Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)-azidomethylpyrrolidine

In a flask protected by a safety screen, a solution of N-t-butoxycarbonyl-4(S)-t-butyldimethylsilyloxy-2(S)-methane- sulfonyloxy methyl pyrrolidine(10.40g, 25.39mmol) and tetrabutyl- ammonium azide (8.18g, 28.7mmol) in toluene (250ml) was stirred at 80°C for 5 hr. The reaction was cooled to room temperature and diluted with EtOAc (250ml), washed with water and brine and dried (Na2Sθ4).

The solvent was evaporated in vacuo to afford the title compound as a yellow oil which was used in the next step without furthur purification. !H NMR CDCI3 δ 4.60-3.20 (6H, m), 2.05-1.90(2H, m), 1.47(9H, s), 0.87(9H, s) and 0.10-0.00(6H, m) ppm.

Step G: Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)-aminomethylpyrrolidine

A solution of N-t-butoxycarbonyl-4(R)-t-butyldimethyl- silyloxy-2(S)-azidomethylpyrrolidine (9.06g, 25.39mmol) in EtOAc (120ml) was purged with argon and 10% palladium on carbon (1.05g) added. The flask was evacuated and stirred under an atmosphere of hydrogen (49 psi) for 16hrs. The hydrogen was replaced by argon, the catalyst removed by filtration and the solvent evaporated in vacuo. The residue was chromatographed (Siθ2, 2.5 to 5% saturated NH4OH in acetonitrile, gradient elution), to afford the title compound as an oil. iH NMR(CDCl3, 400 MHz) δ 4.40-2.60 (6H, s), 2.05-1.80(2H, m), 1.46(9H, s), 1.36(2H, s), 0.87(9H, s), 0.10-0.00(6H, m)ppm.

Step H: Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)- { N'-3- chlorobenzy 1 } aminomethy lpyrrolidine To a slurry of 3-chlorobenzaldehyde (1.2 ml, 10.6 mmol), crushed 3 A molecular sieves (9.5g) and the amine from step G (3.50g, 10.6mmol) in methanol (150 ml) was added sodium cyanoborohydride (11.0ml of a 1M solution in THF, l l.Ommol) at room temperature. The pH was adjusted to 7 by the addition of glacial acetic acid (0.68ml, 12mmol) and the reaction was stirred for 16 hrs. The reaction was filtered and the filtrate evaporated in vacuo. The residue was partitioned between EtOAc and saturated NaHC03 solution and the organic extract washed with brine, dried (Na2S04), and the solvent evaporated in vacuo. The residue was purified by chromatography (Siθ2, 2.5% MeOH in CH2CI2) to provide the title compound as an oil. lHNMR(CDCl3, 400 MHz) δ 7.40-7.10(4H, m), 4.36(1H, s), 4.15- 3.90(2H, m), 3.90-3.30(2H, m), 2.85-2.60(2H, m), 2.05-1.90(2H, m), 1.44(9H, s), 0.87(9H, s) and 0.06(6H, m) ppm.

Step I: Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)- { N'-3-chlorobenzyl-

N'-acetyl ) -aminomethylpyrrolidine

To a solution of N-t-butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)- { N'-3-chlorobenzyl } -aminomethyl pyrrolidine (3.80g, 8.35 mmol) in CH2CI2 (85ml) and triethylamine (2.40ml, 17.0 mmol) at 0°C was added acetyl chloride (0.60ml, 8.44 mmol). The reaction was stirred at room temperature for lhr, diluted with water and extracted with CH2CI2. The extracts were washed with brine, dried (Na2Sθ4) and the solvent evaporated in vacuo. The residue was purified by chromatography (Siθ2, 10 to 25% EtOAc in CH2CI2, gradient elution). iHNMR (CDCI3, 400 MHz) δ 7.40-7.00(4H, m), 5.10-3,00(8H, m), 2.20-1.70(5H, m), 1.50-1.30(9H, m), 0.87(9H, s) and 0.06(6H, m) ppm.

Step J: Preparation of N-t-Butoxycarbonyl-4(R)-hydroxy-2(S}-

{N' -3-chlorobenzyl-N'-acetyl }-aminomethylpyrrolidine To a solution of N-t-butoxycarbonyl-4(R)-t-butyl- dimethylsilyloxy -2(S)-{N'-3-chlorobenzyl-N'-acetyl}-aminomethyl- pyrrolidine (4.02g, 8.09 mmol) in THF (80ml) at 0°C was added tetrabutylammonium fluoride (9.00ml of a 1M solution in THF,

9.00mmol). The reaction was stirred at 0°C for lhr and then at room temperature for 30min. The reaction was quenched by the addition of a saturated NH4CI solution (50ml), dilution with EtOAc. The organic extracts were washed with brine, dried (Na2S04) and the solvent evaporated in vacuo. The residue purified by chromatography (Siθ2, 3 to 5% MeOH in CH2CI2, gradient elution) to afford the title compound as a foam. iHNMR (CDCI3, 400 MHz) δ 7.40-7.00(4H, m), 5.00-4,00(4H, m), 4.00-3.10(4H, m), 2.30-1.60(5H, m) and 1.50-1.30(9H, m) ppm.

Step K: N-t-Butoxycarbonyl-4(R)-benzyloxyoxy-2(S)- { N'- acetyl-N'-3-chlorobenzyl}aminomethylpyrrolidine

To a solution of N-t-Butoxycarbonyl-4(S)-hydroxy-2(S)- {N'-acetyl-N' 3-chlorobenzyl}aminomethylpyrrolidine (701mg, 1.83 mmol) in DMF (9ml) at 0°C was added sodium hydride (1 lOmg of a 60% dispersion in mineral oil, 2.75mmol). After 15 min benzyl bromide (0.435ml, 3.66mmol), was added and the reaction stirred at room temperature for 16 hrs. The reaction was quenched with saturated NaHCθ3 solution (2ml) and extracted with ethyl acetate. The organic extract was washed with brine and dried (Na2Sθ4), and the solvent evaporated in vacuo. The residue was purified by chromatography (Siθ2, 25 to 50% EtOAc in CH2CI2, gradient elution) to afford the title compound as a foam.

Step L: 4(S)-Benzyloxy-2(S)- { N^*-acetyl-N'-3-chlorobenzyl }- aminomethylpyrrolidine hydrochloride A solution of the product from step K (0.834g, 1.76 mmol) in EtOAc (25 ml) at 0°C was saturated with gaseous hydrogen chloride. The resulting solution was allowed to stand at room temperature for 30min. The solvent was evaporated in vacuo to afford the title compound as a white solid.

Step M: Preparation of lH-Imidazole-4- acetic acid methyl ester hydrochloride.

A solution of lH-imidazole-4-acetic acid hydrochloride

(4.00g, 24.6 mmol) in methanol (100 ml) was saturated with gaseous hydrogen chloride. The resulting solution was allowed to stand at room temperature (RT) for 18hr. The solvent was evaporated in vacuo to afford the title compound as a white solid. iH NMR(CDCl3, 400 MHz) δ 8.85(1H, s),7.45(lH, s), 3.89(2H, s) and

3.75(3H, s) ppm.

Step N: Preparation of 1- (Triphenylmethyl)- lH-imidazol-4-ylacetic acid methyl ester

To a solution of the product from Step M (24.85g, 0.141 mol) in dimethyl formamide (DMF) (115ml) was added triethylamine (57.2 ml, 0.412mol) and triphenylmethyl bromide (55.3g, 0.171mol) and the suspension was stirred for 24hr. After this time, the reaction mixture was diluted with ethyl acetate (EtOAc) (1 1) and water (350 ml). The organic phase was washed with sat. aq. NaHC03 (350 ml), dried (Na2Sθ4) and evaporated in vacuo. The residue was purified by flash chromatography (Siθ2, 0-100% ethyl acetate in hexanes; gradient elution) to provide the title compound as a white solid. iH NMR (CDCI3, 400 MHz) δ 7.35(1H, s), 7.31(9H, m), 7.22(6H, m), 6.76(1H, s), 3.68(3H, s) and 3.60(2H, s) ppm.

Step O: Preparation of [l-(4-cyanobenzyl)-lH-imidazol-5-yl]acetic acid methyl ester

To a solution of the product from Step N (8.00g, 20.9 mmol) in acetonitrile (70 ml) was added bromo-p-tolunitrile (4.10g, 20.92 mmol) and heated at 55°C for 3 hr. After this time, the reaction was cooled to room temperature and the resulting imidazolium salt (white precipitate) was collected by filtration. The filtrate was heated at 55°C for 18hr. The reaction mixture was cooled to room temperature and evaporated in vacuo. To the residue was added EtOAc (70 ml) and the resulting white precipitate collected by filtration. The precipitated imidazolium salts were combined, suspended in methanol (100 ml) and heated to reflux for 30 min. After this time, the solvent was removed in vacuo, the resulting residue was suspended in EtOAc (75ml) and the solid isolated by filtration and washed (EtOAc). The solid was treated with sat aq NaHCθ3 (300ml) and CH2CI2 (300ml) and stirred at room temperature for 2 hr. The organic layer was separated, dried (MgS04) and evaporated in vacuo to afford the title compound as a white solid: lHNMR(CDCl3, 400 MHz) δ 7.65(1H, d, J=8Hz), 7.53(1H, s), 7.15(1H, d, J=8Hz), 7.04QH, s), 5.24(2H, s), 3.62(3H, s) and 3.45(2H, s) ppm.

Step P: Preparation of (l-(4-Cyanobenzyl)-lH-imidazol-5-yl)- ethanol

To a stirred solution of the ester from step O, (1.50g, 5.88 mmol), in methanol (20ml) at 0°C, was added sodium borohydride (l.Og, 26.3mmol) portionwise over 5 minutes. The reaction was stirred at 0°C for 1 hr and then at room temperature for an additional 1 hr. The reaction was quenched by the addition of sat.NH4Cl solution and the methanol was evaporated in vacuo.. The residue was partitioned between EtOAc and sat NaHC03 solution and the organic extracts dried (MgS04), and evaporated in vacuo.. The residue was purified by chromatography (Siθ2, 4 to 10% methanol in methylene chloride, gradient elution) to afford the title compound as a solid. iH NMR CDC13 δ 7.64(2H, d, J=8.2Hz), 7.57QH, s), 7.11(2H, d, J=8.2Hz), 6.97(1H, s), 5.23(2H, s), 3.79(2H, t, J=6.2Hz) and 2.66(2H, t, J=6.2Hz) ppm.

Step 0: l-(4-Cyanobenzyl)-imidazol-5-yl-ethylmethanesulfonate A solution of (l-(4-Cyanobenzyl)-lH-imidazol-5-yl)- ethanol (0.500 g, 2.20 mmol) in methylene chloride (6.0 ml) at

0°C was treated with Hunig's base (0.460ml, 2.64mmol) and methane sulfonyl chloride (0.204ml, 2.64mmol). After 2 hrs, the reaction was quenched by addition of saturated NaHC03 solution (50ml) and the mixture was extracted with methylene chloride (50ml), dried (MgS04) and the solvent evaporated in vacuo. The title compound was used without furthur purification. iH NMR CDC13 δ 7.69 (IH, s) 7.66(2H, d, J=8.2Hz), 7.15 (2H, d, J=8.2Hz), 7.04(1H, s), 5.24(2H, s), 4.31(2H, t, J=6.7Hz), 2.96(3H, s), and 2.88(2H, t, J=6.6Hz)ppm.

Step R: N{ l-(4-Cyanobenzyl)- lH~imidazol-5-ylethyl }-4(R)- benzyloxyoxy-2(S)- { N'-acetyl-N'-3- chlorobenzyl } aminomethylpyrrolidine

A mixture of 4(R)-benzyloxy-2(S)-{N'-acetyl-N'-3- chlorobenzyl-aminomethyljpyrrolidine (199mg, 0.486mmol), the mesylate from step Q (140mg, 0.458 mmol), potassium carbonate (165mg, 1.19mmol), and sodium iodide (289mg, 1.93mmol) in DMF (1.5ml), were heated at 55°C for 16 hrs. The cooled mixture was diluted with EtOAc, washed with NaHCθ3 solution and brine, dried (Na2S04) and the solvent evaporated in vacuo. The residue was purified by preparative HPLC (C-18, 95:5 to 5:95 water in acetonitrile containing 0.1% TFA, gradient elution). The title compound was obtained as a white solid after lyophillisation. Anal, calc'd for C34H36N5O2CI 3.00 TFA, 0.85 H2O:

C, 51.14; H, 4.37, N, 7.45. Found: C, 51.15; H, 4.42; N, 6.86.

FAB HRMS exact mass calc'd for C34H37N5O2CI 582.263579(MH+),

Found: 582.263900.

EXAMPLE 10

In vitro inhibition of ras farnesyl transferase

Transferase Assays. Isoprenyl-protein transferase activity assays are carried out at 30 °C unless noted otherwise. A typical reaction contains (in a final volume of 50 μL): [³H]farnesyl diphosphate, Ras protein , 50 mM HEPES, pH 7.5, 5 mM MgCl₂, 5 mM dithiothreitol, 10 μM ZnCl₂, 0.1% polyethyleneglycol (PEG) (15,000- 20,000 mw) and isoprenyl-protein transferase. The FPTase employed in the assay is prepared by recombinant expression as described in Omer, C.A., Krai, A.M., Diehl, R.E., Prendergast, G.C., Powers, S., Allen, CM., Gibbs, J.B. and Kohl, N.E. (1993) Biochemistry 32:5167-5176. After thermally pre-equilibrating the assay mixture in the absence of enzyme, reactions are initiated by the addition of isoprenyl-protein transferase and stopped at timed intervals (typically 15 min) by the addition of 1 M HCI in ethanol (1 mL). The quenched reactions are allowed to stand for 15 m (to complete the precipitation process). After adding 2 mL of 100% ethanol, the reactions are vacuum-filtered through Whatman GF/C filters. Filters are washed four times with 2 mL aliquots of 100% ethanol, mixed with scintillation fluid (10 mL) and then counted in a Beckman LS3801 scintillation counter. For inhibition studies, assays are run as described above, except inhibitors are prepared as concentrated solutions in 100% dimethyl sulfoxide and then diluted 20-fold into the enzyme assay mixture. Substrate concentrations for composition or inhibitor IC50 determinations are as follows: FTase, 650 nM Ras-CVLS (SEQ.ID.NO.: 1), 100 nM farnesyl diphosphate.

The compounds useful in the instant invention described in the above Examples 1-9 were tested for inhibitory activity against human FPTase by the assay described above and were found to have IC50 of < l μM.

EXAMPLE 11

Modified/?! vitro GGTase inhibition assay

The modified geranylgeranyl-protein transferase inhibition assay is carried out at room temperature. A typical reaction contains (in a final volume of 50 μL): [³H]geranylgeranyl diphosphate, biotinylated Ras peptide, 50 mM HEPES, pH 7.5, a modulating anion (for example 10 mM glycerophosphate or 5mM ATP), 5 mM MgCl₂, 10 μM ZnCl₂, 0.1% PEG (15,000-20,000 mw), 2 mM dithiothreitol, and geranylgeranyl-protein transferase type I(GGTase). The GGTase- type I enzyme employed in the assay is prepared as described in U.S. Pat. No. 5,470,832, incorporated by reference. The Ras peptide is derived from the K4B-Ras protein and has the following sequence: biotinyl-GKKKKKKSKTKCVIM (single amino acid code) (SEQ. ID.NO.: 2). Reactions are initiated by the addition of GGTase and stopped at timed intervals (typically 15 min) by the addition of 200 μL of a 3 mg/mL suspension of streptavidin SPA beads (Scintillation Proximity Assay beads, Amersham) in 0.2 M sodium phosphate, pH 4, containing 50 mM EDTA, and 0.5% BSA. The quenched reactions are allowed to stand for 2 hours before analysis on a Packard TopCount scintillation counter.

For inhibition studies, assays are run as described above, except compositions or inhibitors are prepared as concentrated solutions in 100% dimethyl sulf oxide and then diluted 25-fold into the enzyme assay mixture. IC₅0 values are determined with Ras peptide near KM concentrations. Enzyme and substrate concentrations for inhibitor IC₅₀ determinations are as follows: 75 pM GGTase-I, 1.6 μM Ras peptide, 100 nM geranylgeranyl diphosphate. EXAMPLE 11

Cell-based vitro ras farnesylation assay The cell line used in this assay is a v-ras line derived from either Ratl or NIH3T3 cells, which expressed viral Ha-ras p21. The assay is performed essentially as described in DeClue, J.E. et al. , Cancer Research 51:712-717. (1991). Cells in 10 cm dishes at 50-75% confluency are treated with the test compound or composition (final concentration of solvent, methanol or dimethyl sulfoxide, is 0.1%). After 4 hours at 37°C, the cells are labeled in 3 ml methionine-free DMEM supple-mented with 10% regular DMEM, 2% fetal bovine serum and 400 mCi[35S]methionine (1000 Ci/mmol). After an additional 20 hours, the cells are lysed in 1 ml lysis buffer (1% NP40/20 mM HEPES, pH 7.5/5 mM MgCl2/lmM DTT/10 mg/ml aprotinen 2 mg/ml leupeptin/2 mg/ml antipain/0.5 mM PMSF) and the lysates cleared by centrifugation at 100,000 x g for 45 min. Aliquots of lysates containing equal numbers of acid-precipitable counts are bought to 1 ml with IP buffer (lysis buffer lacking DTT) and immuno- precipitated with the ras-specific monoclonal antibody Y 13-259 (Furth, M.E. et al., J. Virol. 43:294-304, (1982)). Following a 2 hour antibody incubation at 4°C, 200 ml of a 25% suspension of protein A-Sepharose coated with rabbit anti rat IgG is added for 45 min. The immuno- precipitates are washed four times with IP buffer (20 nM HEPES, pH 7.5/1 mM EDTA 1% Triton X-100.0.5% deoxycholate/0.1 %/SDS/ 0.1 M NaCl) boiled in SDS-PAGE sample buffer and loaded on 13% acrylamide gels. When the dye front reached the bottom, the gel is fixed, soaked in Enlightening, dried and autoradiographed. The intensities of the bands corresponding to farnesylated and nonfarnesylated ras proteins are compared to determine the percent inhibition of farnesyl transfer to protein. EXAMPLE 12

Cell-based vitro growth inhibition assay

To determine the biological consequences of FPTase inhibition, the effect of the compounds or compositions of the instant invention on the anchorage-independent growth of Ratl cells transformed with either a v-ras, v-raf, or v-mos oncogene is tested. Cells transformed by v-Raf and v-Mos maybe included in the analysis to evaluate the specificity of compounds or instant compositions for Ras-induced cell transformation.

Rat 1 cells transformed with either v-ras, v-raf, or v-mos are seeded at a density of 1 x 10⁴ cells per plate (35 mm in diameter) in a 0.3% top agarose layer in medium A (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum) over a bottom agarose layer (0.6%). Both layers contain 0.1% methanol or an appropriate concentration of the compound or instant composition (dissolved in methanol at 1000 times the final concentration used in the assay). The cells are fed twice weekly with 0.5 ml of medium A containing 0.1 % methanol or the concentration of the instant compound. Photomicrographs are taken 16 days after the cultures are seeded and comparisons are made.

EXAMPLE 13

Construction of SEAP reporter plasmid pDSElOO

The SEAP reporter plasmid, pDSElOO was constructed by ligating a restriction fragment containing the SEAP coding sequence into the plasmid pCMV-RE-AKI. The SEAP gene is derived from the plasmid pSEAP2-Basic (Clontech, Palo Alto, CA). The plasmid pCMV-RE-AKI was constructed by Deborah Jones (Merck) and contains 5 sequential copies of the 'dyad symmetry response element' cloned upstream of a 'CAT-TATA' sequence derived from the cytomegalovirus immediate early promoter. The plasmid also contains a bovine growth hormone poly-A sequence. The plasmid, pDSElOO was constructed as follows. A restriction fragment encoding the SEAP coding sequence was cut out of the plasmid pSEAP2-Basic using the restriction enzymes EcoRl and Hpal. The ends of the linear DNA fragments were filled in with the Klenow fragment of E. coli DNA Polymerase I. The 'blunt ended' DNA containing the SEAP gene was isolated by electrophoresing the digest in an agarose gel and cutting out the 1694 base pair fragment. The vector plasmid pCMV-RE-AKI was linearized with the restriction enzyme Bgl- II and the ends filled in with Klenow DNA Polymerase I. The SEAP DNA fragment was blunt end ligated into the pCMV-RE-AKI vector and the ligation products were transformed into DH5-alpha E. coli cells (Gibco-BRL). Transformants were screened for the proper insert and then mapped for restriction fragment orientation. Properly oriented recombinant constructs were sequenced across the cloning junctions to verify the correct sequence. The resulting plasmid contains the SEAP coding sequence downstream of the DSE and CAT-TATA promoter elements and upstream of the BGH poly- A sequence.

Alternative Construction of SEAP reporter plasmid. pDSElOl The SEAP repotrer plasmid, pDSElOl is also constructed by ligating a restriction fragment containing the SEAP coding sequence into the plasmid pCMV-RE-AKI. The SEAP gene is derived from plasmid pGEM7zf(-)/SEAP.

The plasmid pDSElOl was constructed as follows: A restriction fragment containing part of the SEAP gene coding sequence was cut out of the plasmid pGEM7zf(-)/SEAP using the restriction enzymes Apa I and Kpnl. The ends of the linear DNA fragments were chewed back with the Klenow fragment of E. coli DNA Polymerase I. The "blunt ended" DNA containing the truncated SEAP gene was isolated by electrophoresing the digest in an agarose gel and cutting out the 1910 base pair fragment. This 1910 base pair fragment was ligated into the plasmid pCMV-RE-AKI which had been cut with Bgl-II and filled in with E. coli Klenow fragment DNA polymerase. Recombinant plasmids were screened for insert orientation and sequenced through the ligated junctions. The plasmid pCMV-RE-AKI is derived from plasmid pCMVIE-AKI-DHFR (Whang , Y., Silberklang, M., Morgan, A., Munshi, S., Lenny, A.B., Ellis, R.W., and Kieff, E. (1987) J. Virol., 61, 1796-1807) by removing an EcoRI fragment containing the DHFR and Neomycin markers. Five copies of the fos promoter serum response element were inserted as described previously (Jones, R.E., Defeo-Jones, D., McAvoy, E.M., Vuocolo, G.A., Wegrzyn, R.J., Haskell, K.M. and Oliff, A. (1991) Oncogene, 6, 745-751) to create plasmid pCMV-RE-AKI.

The plasmid pGEM7zf(-)/SEAP was constructed as follows. The SEAP gene was PCRed, in two segments from a human placenta cDNA library (Clontech) using the following oligos.

Sense strand N-terminal SEAP : 5'

GAGAGGGAATTCGGGCCCTTCCTGCAT GCTGCTGCTGCTGCTGCTGCTGGGC 3' (SEQ.ID.NO.:3)

Antisense strand N-terminal SEAP: 5' GAGAGAGCTCGAGGTTAACCCGGGT

GCGCGGCGTCGGTGGT 3' (SEQ.ID.NO.:4)

Sense strand C-terminal SEAP: 5' GAGAGAGTCTAGAGTTAACCCGTGGTCC CCGCGTTGCTTCCT 3' (SEQ.ID.NO.:5)

Antisense strand C-terminal SEAP: 5' GAAGAGGAAGCTTGGTACCGCCACTG GGCTGTAGGTGGTGGCT 3' (SEQ.ID.NO.:6)

The N-terminal oligos (SEQ.ID.NO.: 4 and SEQ.ID.NO.: 5) were used to generate a 1560 bp N-terminal PCR product that contained EcoRI and Hpal restriction sites at the ends. The Antisense N-terminal oligo (SEQ.ID.NO.: 4) introduces an internal translation STOP codon within the SEAP gene along with the Hpal site. The C-terminal oligos (SEQ.ID.NO.: 5 and SEQ.ID.NO.: 6) were used to amplify a 412 bp C-terminal PCR product containing Hpal and Hindlll restriction sites. The sense strand C-terminal oligo (SEQ.ID.NO.: 5) introduces the internal STOP codon as well as the Hpal site. Next, the N-terminal amplicon was digested with EcoRI and Hpal while the C-terminal amplicon was digested with Hpal and Hindlll. The two fragments comprising each end of the SEAP gene were isolated by electrophoresing the digest in an agarose gel and isolating the 1560 and 412 base pair fragments. These two fragments were then co-ligated into the vector pGEM7zf(-) (Promega) which had been restriction digested with EcoRI and Hindlll and isolated on an agarose gel. The resulting clone, pGEM7zf(-)/SEAP contains the coding sequence for the SEAP gene from amino acids.

Construction of a constitutively expressing SEAP plasmid pCMV-SEAP An expression plasmid constitutively expressing the SEAP protein was created by placing the sequence encoding a truncated SEAP gene downstream of the cytomegalovirus (CMV) IE- 1 promoter. The expression plasmid also includes the CMV intron A region 5' to the SEAP gene as well as the 3' untranslated region of the bovine growth hormone gene 3' to the SEAP gene.

The plasmid pCMVIE-AKI-DHFR (Whang et al, 1987) containing the CMV immediate early promoter was cut with EcoRI generating two fragments. The vector fragment was isolated by agarose electrophoresis and religated. The resulting plasmid is named pCMV- AKI. Next, the cytomegalovirus intron A nucleotide sequence was inserted downstream of the CMV IE1 promter in pCMV-AKI. The intron A sequence was isolated from a genomic clone bank and subcloned into pBR322 to generate plasmid ρl6T-286. The intron A sequence was mutated at nucleotide 1856 (nucleotide numbering as in Chapman, B.S., Thayer, R.M., Vincent, K.A. and Haigwood, N.L., Nuc. Acids Res. 19, 3979-3986) to remove a Sacl restriction site using site directed mutagenesis. The mutated intron A sequence was PCRed from the plasmid pl6T-287 using the following oligos.

Sense strand: 5' GGCAGAGCTCGTTTAGTGAACCGTCAG 3' (SEQ.ID.NO.: 7)

Antisense strand: 5' GAGAGATCTCAAGGACGGTGACTGCAG 3' (SEQ.ID.NO.: 8)

These two oligos generate a 991 base pair fragment with a Sad site incorporated by the sense oligo and a Bgl-II fragment incorporated by the antisense oligo. The PCR fragment is trimmed with Sad and Bgl-II and isolated on an agarose gel. The vector pCMV-AKI is cut with Sad and Bgl-II and the larger vector fragment isolated by agarose gel electrophoresis. The two gel isolated fragments are ligated at their respective Sad and Bgl-II sites to create plasmid pCMV-AKI- InA.

The DNA sequence encoding the truncated SEAP gene is inserted into the pCMV-AKI-InA plasmid at the Bgl-II site of the vector. The SEAP gene is cut out of plasmid pGEM7zf(-)/SEAP

(described above) using EcoRI and Hindlll. The fragment is filled in with Klenow DNA polymerase and the 1970 base pair fragment isolated from the vector fragment by agarose gel electrophoresis. The pCMV- AKI-InA vector is prepared by digesting with Bgl-II and filling in the ends with Klenow DNA polymerase. The final construct is generated by blunt end ligating the SEAP fragment into the pCMV-AKI-InA vector. Transformants were screened for the proper insert and then mapped for restriction fragment orientation. Properly oriented recombinant constructs were sequenced across the cloning junctions to verify the correct sequence. The resulting plasmid, named pCMV- SEAP, contains a modified SEAP sequence downstream of the cytomegalovirus immediately early promoter IE-1 and intron A sequence and upstream of the bovine growth hormone poly-A sequence. The plasmid expresses SEAP in a constitutive manner when transfected into mammalian cells.

Cloning of a Myristylated viral-H-ras expression plasmid A DNA fragment containing viral-H-ras can be PCRed from plasmid "H-l" (Ellis R. et al. J. Virol. 36, 408, 1980) or "HB-11 (deposited in the ATCC under Budapest Treaty on August 27, 1997, and designated ATCC 209,218) using the following oligos.

Sense strand:

5'TCTCCTCGAGGCCACCATGGGGAGTAGCAAGAGCAAGCCTAA GGACCCCAGCCAGCGCCGGATGACAGAATACAAGCTTGTGGTG G 3'. (SEQ.ID.NO.: 9)

Antisense:

5'CACATCTAGATCAGGACAGCACAGACTTGCAGC 3'. (SEQ.ID.NO.: 10)

A sequence encoding the first 15 aminoacids of the v-src gene, containing a myristylation site, is incorporated into the sense strand oligo. The sense strand oligo also optimizes the 'Kozak' translation initiation sequence immediately 5' to the ATG start site. To prevent prenylation at the viral-ras C-terminus, cysteine 186 would be mutated to a serine by substituting a G residue for a C residue in the C-terminal antisense oligo. The PCR primer oligos introduce an Xhol site at the 5' end and a Xbal site at the 3 'end. The Xhol-Xbal fragment can be ligated into the mammalian expression plasmid pCI (Promega) cut with Xhol and Xbal. This results in a plasmid in which the recombinant myr-viral-H-ras gene is constitutively transcribed from the CMV promoter of the pCI vector.

Cloning of a viral-H-ras-CVLL expression plasmid

A viral-H-ras clone with a C-terminal sequence encoding the amino acids CVLL can be cloned from the plasmid "H-l" (Ellis R. et al. J. Virol. 36, 408, 1980) or "HB-11 (deposited in the ATCC under Budapest Treaty on August 27, 1997, and designated ATCC 209,218) by PCR using the following oligos.

Sense strand:

5'TCTCCTCGAGGCCACCATGACAGAATACAAGCTTGTGGTGG- 3' (SEQ.ID.NO.: 11)

Antisense strand: 5'CACTCTAGACTGGTGTCAGAGCAGCACACACTTGCAGC-3' (SEQ.ID.NO.: 12)

The sense strand oligo optimizes the 'Kozak' sequence and adds an Xhol site. The antisense strand mutates serine 189 to leucine and adds an Xbal site. The PCR fragment can be trimmed with Xhol and Xbal and ligated into the Xhol-Xbal cut vector pCI (Promega). This results in a plasmid in which the mutated viral-H-ras-CVLL gene is constitutively transcribed from the CMV promoter of the pCI vector.

Cloning of c-H-ras-Leu61 expression plasmid

The human c-H-ras gene can be PCRed from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers.

Sense strand:

5'-GAGAGAATTCGCCACCATGACGGAATATAAGCTGGTGG-3' (SEQ.ID.NO.: 13)

Antisense strand: 5'-GAGAGTCGACGCGTCAGGAGAGCACACACTTGC-3' (SEQ.ID.NO.: 14)

The primers will amplify a c-H-ras encoding DNA fragment with the primers contributing an optimized 'Kozak' translation start sequence, an EcoRI site at the N-terminus and a Sal I stite at the C-terminal end. After trimming the ends of the PCR product with EcoRI and Sal I, the c-H-ras fragment can be ligated ligated into an EcoRI -Sal I cut mutagenesis vector pAlter-1 (Promega). Mutation of glutamine-61 to a leucine can be accomplished using the manufacturer's protocols and the following oligonucleotide:

5'-CCGCCGGCCTGGAGGAGTACAG-3' (SEQ.ID.NO.: 15)

After selection and sequencing for the correct nucleotide substitution, the mutated c-H-ra -Leu61 can be excised from the pAlter- 1 vector, using EcoRI and Sal I, and be directly ligated into the vector pCI (Promega) which has been digested with EcoRI and Sal I. The new recombinant plasmid will constitutively transcribe c-H-ra -Leu61 from the CMV promoter of the pCI vector.

Cloning of a c-N-ras- Val- 12 expression plasmid

The human c-N-ras gene can be PCRed from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers.

Sense strand:

5'-GAGAGAATTCGCCACCATGACTGAGTACAAACTGGTGG-3' (SEQ.ID.NO.: 16)

Antisense strand:

5'-GAGAGTCGACTTGTTACATCACCACACATGGC-3' (SEQ.ID.NO.: 17)

The primers will amplify a c-N-ras encoding DNA fragment with the primers contributing an optimized 'Kozak' translation start sequence, an EcoRI site at the N-terminus and a Sal I stite at the C-terminal end. After trimming the ends of the PCR product with EcoRI and Sal I, the c-N-ras fragment can be ligated into an EcoRI -Sal I cut mutagenesis vector p Alter- 1 (Promega). Mutation of glycine-12 to a valine can be accomplished using the manufacturer's protocols and the following oligonucleotide:

5'-GTTGGAGCAGTTGGTGTTGGG-3' (SEQ.ID.NO.: 18) After selection and sequencing for the correct nucleotide substitution, the mutated c-N-ras-Val-12 can be excised from the pAlter- 1 vector, using EcoRI and Sal I, and be directly ligated into the vector pCI (Promega) which has been digested with EcoRI and Sal I. The new recombinant plasmid will constitutively transcribe c-N-ras-Val-12 from the CMV promoter of the pCI vector.

Cloning of a c-K-ms-Val-12 expression plasmid

The human c-K-ras gene can be PCRed from a human cerebral cortex cDNA library (Clontech) using the following oligonucleotide primers.

Sense strand:

5'-GAGAGGTACCGCCACCATGACTGAATATAAACTTGTGG-3' (SEQ.ID.NO.: 19)

Antisense strand:

5'-CTCTGTCGACGTATTTACATAATTACACACTTTGTC-3' (SEQ.ID.NO.: 20)

The primers will amplify a c-K-ras encoding DNA fragment with the primers contributing an optimized 'Kozak' translation start sequence, a Kpnl site at the N-terminus and a Sal I site at the C-terminal end. After trimming the ends of the PCR product with Kpn I and Sal I, the c-K-ras fragment can be ligated into a Kpnl -Sal I cut mutagenesis vector p Alter- 1 (Promega). Mutation of cysteine- 12 to a valine can be accomplished using the manufacturer's protocols and the following oligonucleotide:

5'-GTAGTTGGAGCTGTTGGCGTAGGC-3' (SEQ.ID.NO.: 21)

After selection and sequencing for the correct nucleotide substitution, the mutated c-K-ras-Val-12 can be excised from the pAlter- 1 vector, using Kpnl and Sal I, and be directly ligated into the vector pCI (Promega) which has been digested with Kpnl and Sal I. The new recombinant plasmid will constitutively transcribe c-K-ras-Val-12 from the CMV promoter of the pCI vector. SEAP assay

Human C33A cells (human epitheial carcenoma - ATTC collection) are seeded in 10cm tissue culture plates in DMEM + 10% fetal calf serum + IX Pen/Strep + IX glutamine + IX NEAA. Cells are grown at 37°C in a 5% Cθ2 atmosphere until they reach 50 -80% of confluency.

The transient transfection is performed by the CaP04 method (Sambrook et al., 1989). Thus, expression plasmids for H-ras, N-ras, K-ras, Myr-ras or H-ras-CVLL are co-precipitated with the DSE-SEAP reporter construct. For 10cm plates 600μl of CaCl₂-DNA solution is added dropwise while vortexing to 600μl of 2X HBS buffer to give 1.2ml of precipitate solution (see recipes below). This is allowed to sit at room temperature for 20 to 30 minutes. While the precipitate is forming, the media on the C33A cells is replaced with DMEM (minus phenol red; Gibco cat. # 31053-028)+ 0.5% charcoal stripped calf serum + IX (Pen/Strep, Glutamine and nonessential aminoacids). The CaP04-DNA precipitate is added dropwise to the cells and the plate rocked gently to distribute. DNA uptake is allowed to proceed for 5-6 hrs at 37°C under a 5% C02 atmosphere.

Following the DNA incubation period, the cells are washed with PBS and trypsinized with 1ml of 0.05% trypsin. The 1 ml of trypsinized cells is diluted into 10ml of phenol red free DMEM + 0.2% charcoal stripped calf serum + IX (Pen/Strep, Glutamine and NEAA ). Transfected cells are plated in a 96 well microtiter plate (lOOμl/well) to which drug, diluted in media, has already been added in a volume of lOOμl. The final volume per well is 200μl with each drug concentration repeated in triplicate over a range of half-log steps.

Incubation of cells and test compound or composition is for 36 hrs at 37° under C02- At the end of the incubation period, cells are examined microscopically for evidence of cell distress. Next, lOOμl of media containing the secreted alkaline phosphatase is removed from each well and transferred to a microtube array for heat treatment at 65°C for 1 hr to inactivate endogenous alkaline phosphatases (but not the heat stable secreted phosphatase).

The heat treated media is assayed for alkaline phosphatase by a luminescence assay using the luminescence reagent CSPD® (Tropix, Bedford, Mass.). A volume of 50 μl media is combined with 200 μl of CSPD cocktail and incubated for 60 minutes at room temperature. Luminesence is monitored using an ML2200 microplate luminometer (Dynatech). Luminescence reflects the level of activation of the fos reporter construct stimulated by the transiently expressed protein.

DNA-CaPO_A precipitate for 10cm. plate of cells Ras expression plasmid (lμg/μl) lOμl

DSE-SEAP Plasmid ( 1 μg/μl) 2μl Sheared Calf Thymus DNA ( 1 μg/μl) 8μl

2M CaCl₂ 74μl dH₂0 506μl

2X HBS Buffer 280mM NaCl lOmM KC1

1.5mM Na₂HP0₄ 2H₂0

12mM dextrose

50mM HEPES Final pH = 7.05

Luminesence Buffer (26ml)

Assay Buffer 20ml

Emerald Reagent™ (Tropix) 2.5ml lOOmM homoarginine 2.5ml

CSPD Reagent® (Tropix) 1.0ml

Assay Buffer

Add 0.05M Na₂C0₃ to 0.05M NaHCQ₃ to obtain pH 9.5. Make ImM in MgCl₂

EXAMPLE 14

The processing assays employed in this example and in

Example 15 are modifications of that described by DeClue et al [Cancer Research 51, 712-717, 1991].

K4B-Ras processing inhibition assay PSN-1 (human pancreatic carcinoma) cells are used for analysis of protein processing. Subconfluent cells in 100 mm dishes are fed with 3.5 ml of media (methionine-free RPMI supplemented with 2% fetal bovine serum or cysteine-free/methionine-free DMEM supplemented with 0.035 ml of 200 mM glutamine (Gibco), 2% fetal bovine serum, respectively) containing the desired concentration of test compound, lovastatin or solvent alone. Cells treated with lovastatin (5- 10 μM), a compound that blocks Ras processing in cells by inhibiting a rate-limiting step in the isoprenoid biosynthetic pathway, serve as a positive control. Test compounds or compositions are prepared as lOOOx concentrated solutions in DMSO to yield a final solvent concentration of 0.1%. Following incubation at 37°C for two hours 204 μCi/ml [35s]Pro-Mix (Amersham, cell labeling grade) is added. After introducing the label amino acid mixture, the cells are incubated at 37°C for an additional period of time (typically 6 to 24 hours). The media is then removed and the cells are washed once with cold PBS. The cells are scraped into 1 ml of cold PBS, collected by centrifugation (10,000 x g for 10 sec at room temperature), and lysed by vortexing in 1 ml of lysis buffer (1% Nonidet P-40, 20 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.5% deoxycholate, 0.1% SDS, 1 mM DTT, 10 μg/ml AEBSF, 10 μg/ml aprotinin, 2 μg/ml leupeptin and 2 μg/ml antipain). The lysate is then centrifuged at 15,000 x g for 10 min at 4°C and the supernatant saved.

For immunoprecipitation of Ki4B-Ras, samples of lysate supernatant containing equal amounts of protein are utilized. Protein concentration is determined by the bradford method utilizing bovine serum albumin as a standard. The appropriate volume of lysate is brought to 1 ml with lysis buffer lacking DTT and 8 μg of the pan Ras monoclonal antibody, Yl 3-259, added. The protein/antibody mixture is incubated on ice at 4°C for 24 hours. The immune complex is collected on pansorbin (Calbiochem) coated with rabbit antiserum to rat IgG (Cappel) by tumbling at 4°C for 45 minutes. The pellet is washed 3 times with 1 ml of lysis buffer lacking DTT and protease inhibitors and resuspended in 100 μl elution buffer (10 mM Tris pH 7.4, 1% SDS). The Ras is eluted from the beads by heating at 95 °C for 5 minutes, after which the beads are pelleted by brief centrifugation (15,000 x g for 30 sec. at room temperature).

The supernatant is added to 1 ml of Dilution Buffer 0.1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 10 mM Tris pH 7.4) with 2 μg Kirsten-ras specific monoclonal antibody, c-K-ras Ab-1 (Calbiochem). The second protein/antibody mixture is incubated on ice at 4°C for 1-2 hours. The immune complex is collected on pansorbin (Calbiochem) coated with rabbit antiserum to rat IgG (Cappel) by tumbling at 4°C for 45 minutes. The pellet is washed 3 times with 1 ml of lysis buffer lacking DTT and protease inhibitors and resuspended in Laemmli sample buffer. The Ras is eluted from the beads by heating at 95°C for 5 minutes, after which the beads are pelleted by brief centrifugation. The supernatant is subjected to SDS-PAGE on a 12% acrylamide gel (bis-acrylamide:acrylamide, 1:100), and the Ras visualized by fluorography.

hDJ processing inhibition assay

PSN-1 cells are seeded in 24- well assay plates. For each compound to be tested, the cells are treated with a minimum of seven concentrations in half-log steps. The final solvent (DMSO) concentration is 0.1%. A vehicle-only control is included on each assay plate. The cells are treated for 24 hours at 37 °C / 5% C0₂.

The growth media is then aspirated and the samples are washed with PBS. The cells are lysed with SDS-PAGE sample buffer containing 5% 2-mercaptoethanol and heated to 95°C for 5 minutes. After cooling on ice for 10 minutes, a mixture of nucleases is added to reduce viscosity of the samples.

The plates are incubated on ice for another 10 minutes. The samples are loaded onto pre-cast 8% acrylamide gels and electrophoresed at 15 mA/gel for 3-4 hours. The samples are then transferred from the gels to PVDF membranes by Western blotting.

The membranes are blocked for at least 1 hour in buffer containing 2% nonfat dry milk. The membranes are then treated with a monoclonal antibody to HDJ-2 (Neomarkers Cat. # MS-225), washed, and treated with an alkaline phosphatase-conjugated secondary antibody. The membranes are then treated with a fluorescent detection reagent and scanned on a phosphorimager.

For each sample, the percent of total signal corresponding to the unprenylated species of HDJ (the slower-migrating species) is calculated by densitometry. Dose-response curves and IC50 values are generated using 4-parameter curve fits in SigmaPlot software.

EXAMPLE 15

K4B-Ras processing inhibition assay

PSN- 1 (human pancreatic carcinoma) cells are used for analysis of protein processing. Subconfluent cells in 150 mm dishes are fed with 20 ml of media (RPMI supplemented with 15% fetal bovine serum) containing the desired concentration of test composition, prenyl- protein transferase inhibitor, HMG-CoA reductase inhibitor or solvent alone. Cells treated with lovastatin (5-10 μM), a compound that blocks Ras processing in cells by inhibiting a rate-limiting step in the isoprenoid biosynthetic pathway, serve as a positive control. Test compounds or compositions are prepared as lOOOx concentrated solutions in DMSO to yield a final solvent concentration of 0.1%.

The cells are incubated at 37°C for 24 hours, the media is then removed and the cells are washed twice with cold PBS. The cells are scraped into 2 ml of cold PBS, collected by centrifugation (10,000 x g for 5 min at 4°C) and frozen at -70 °C. Cells are lysed by thawing and addition of lysis buffer (50 mM HEPES, pH 7.2, 50 mM NaCl, 1% CHAPS, 0.7 μg/ml aprotinin, 0.7 μg/ml leupeptin 300 μg/ml pefabloc, and 0.3 mM EDTA). The lysate is then centrifuged at 100,000 x g for 60 min at 4°C and the supernatant saved. The supernatant may be subjected to SDS-PAGE, HPLC analysis, and/or chemical cleavage techniques.

The lysate is applied to a HiTrap-SP (Pharmacia Biotech) column in buffer A (50 mM HEPES pH 7.2) and resolved by gradient in buffer A plus 1 M NaCl. Peak fractions containing Ki4B-Ras are pooled, diluted with an equal volume of water and immunoprecipitated with the pan Ras monoclonal antibody, Y 13-259 linked to agarose. The protein/antibody mixture is incubated at 4°C for 12 hours. The immune complex is washed 3 times with PBS , followed by 3 times with water. The Ras is eluted from the beads by either high pH conditions (pH>10) or by heating at 95°C for 5 minutes, after which the beads are pelleted by brief centrifugation. The supernatant may be subjected to SDS- PAGE, HPLC analysis, and/or chemical cleavage techniques.

EXAMPLE 16

Rapl processing inhibition assay

Protocol A:

Cells are labeled, incubated and lysed as described in Example 14. For immunoprecipitation of Rapl, samples of lysate supernatant containing equal amounts of protein are utilized. Protein concentration is determined by the bradford method utilizing bovine serum albumin as a standard. The appropriate volume of lysate is brought to 1 ml with lysis buffer lacking DTT and 2 μg of the Rapl antibody, Rapl/Krevl (121) (Santa Cruz Biotech), is added. The protein antibody mixture is incubated on ice at 4°C for 1 hour. The immune complex is collected on pansorbin (Calbiochem) by tumbling at 4°C for 45 minutes. The pellet is washed 3 times with 1 ml of lysis buffer lacking DTT and protease inhibitors and resuspended in 100 μl elution buffer (10 mM Tris pH 7.4, 1% SDS). The Rapl is eluted from the beads by heating at 95 °C for 5 minutes, after which the beads are pelleted by brief centrifugation (15,000 x g for 30 sec. at room temperature).

The supernatant is added to 1 ml of Dilution Buffer (0.1% Triton X-100, 5 mM EDTA, 50 mM NaCl, 10 mM Tris pH 7.4) with 2 μg Rapl antibody, Rapl/Krevl (121) (Santa Cruz Biotech). The second protein/antibody mixture is incubated on ice at 4°C for 1-2 hours. The immune complex is collected on pansorbin (Calbiochem) by tumbling at 4°C for 45 minutes. The pellet is washed 3 times with 1 ml of lysis buffer lacking DTT and protease inhibitors and resuspended in Laemmli sample buffer. The Rapl is eluted from the beads by heating at 95°C for 5 minutes, after which the beads are pelleted by brief centrifugation. The supernatant is subjected to SDS-PAGE on a 12% acrylamide gel (bis-acrylamide: acrylamide, 1:100), and the Rapl visualized by fluorography.

Protocol B:

PSN-1 cells are passaged every 3-4 days in 10cm plates, splitting near-confluent plates 1:20 and 1:40. The day before the assay is set up, 5x 10⁶ cells are plated on 15cm plates to ensure the same stage of confluency in each assay. The media for these cells is RPM1 1640

(Gibco), with 15% fetal bovine serum and lx Pen/Strep antibiotic mix. The day of the assay, cells are collected from the 15cm plates by trypsinization and diluted to 400,000 cells/ml in media. 0.5ml of these diluted cells are added to each well of 24-well plates, for a final cell number of 200,000 per well. The cells are then grown at 37°C overnight.

The compounds or compositions to be assayed are diluted in DMSO in 1/2-log dilutions. The range of final concentrations to be assayed is generally 0.1-100μM. Four concentrations per compound is typical. The compounds are diluted so that each concentration is lOOOx of the final concentration (i.e., for a lOμM data point, a lOmM stock of the compound is needed). 2μL of each lOOOx compound stock is diluted into 1ml media to produce a 2X stock of compound. A vehicle control solution (2μL DMSO to 1ml media), is utilized. 0.5 ml of the 2X stocks of compound are added to the cells. After 24 hours, the media is aspirated from the assay plates.

Each well is rinsed with 1ml PBS, and the PBS is aspirated. 180μL SDS-PAGE sample buffer (Novex) containing 5% 2-mercaptoethanol is added to each well. The plates are heated to 100°C for 5 minutes using a heat block containing an adapter for assay plates. The plates are placed on ice. After 10 minutes, 20μL of an RNAse/DNase mix is added per well. This mix is 1 mg/ml DNasel (Worthington Enzymes), 0.25mg/ml Rnase A (Worthington Enzymes), 0.5M Tris-HCl ρH8.0 and 50mM MgCl₂. The plate is left on ice for 10 minutes. Samples are then either loaded on the gel, or stored at -70°C until use. Each assay plate (usually 3 compounds, each in 4-point titrations, plus controls) requires one 15-well 14% Novex gel. 25μl of each sample is loaded onto the gel. The gel is run at 15mA for about 3.5 hours. It is important to run the gel far enough so that there will be adequate separation between 21kd (Rapl) and 29kd (Rab6). The gels are then transferred to Novex pre-cut PVDF membranes for 1.5 hours at 30V (constant voltage). Immediately after transferring, the membranes are blocked overnight in 20ml Western blocking buffer (2% nonfat dry milk in Western wash buffer (PBS + 0.1% Tween-20). If blocked over the weekend, 0.02% sodium azide is added. The membranes are blocked at 4°C with slow rocking.

The blocking solution is discarded and 20ml fresh blocking solution containing the anti Rap la antibody (Santa Cruz Biochemical SC1482) at 1:1000 (diluted in Western blocking buffer) and the anti Rab6 antibody (Santa Cruz Biochemical SC310) at 1:5000 (diluted in Western blocking buffer) are added. The membranes are incubated at room temperature for 1 hour with mild rocking. The blocking solution is then discarded and the membrane is washed 3 times with Western wash buffer for 15 minutes per wash. 20ml blocking solution containing 1: 1000 (diluted in Western blocking buffer) each of two alkaline phosphatase conjugated antibodies (Alkaline phosphatase conjugated Anti-goat IgG and Alkaline phosphatase conjugated anti-rabbit IgG [Santa Cruz Biochemical]) is then added. The membrane is incubated for one hour and washed 3x as above. About 2ml per gel of the Amersham ECF detection reagent is placed on an overhead transparency (ECF) and the PVDF membranes are placed face-down onto the detection reagent. This is incubated for one minute, then the membrane is placed onto a fresh transparency sheet. The developed transparency sheet is scanned on a phosphorimager and the Rap la Minimum Inhibitory Concentration is determined from the lowest concentration of compound or composition that produces a detectable Rap la Western signal. The Rap la antibody used recognizes only unprenylated/unprocessed Rap la, so that the precence of a detectable Rap la Western signal is indicative of inhibition of Rap la prenylation.

EXAMPLE 17

In vivo tumor growth inhibition assay (nude mouse)

In vivo efficacy as an inhibitor of the growth of cancer cells may be confirmed by several protocols well known in the art. Examples of such in vivo efficacy studies are described by N. E. Kohl et al. (Nature Medicine, 1:792-797 (1995)) and N. E. Kohl et al. (Proc. Nat. Acad. Sci. U.S.A., 91:9141-9145 (1994)).

Rodent fibroblasts transformed with oncogenically mutated human Ha-ras or Ki-ras (10⁶ cells/animal in 1 ml of DMEM salts) are injected subcutaneously into the left flank of 8-12 week old female nude mice (Harlan) on day 0. The mice in each oncogene group are randomly assigned to a vehicle, compound or combination treatment group. Animals are dosed subcutaneously starting on day 1 and daily for the duration of the experiment. Alternatively, the test combination composition or prenyl-protein transferase inhibitor may be administered by a continuous infusion pump. Compound, compound combination or vehicle is delivered in a total volume of 0.1 ml. Tumors are excised and weighed when all of the vehicle-treated animals exhibited lesions of 0.5 - 1.0 cm in diameter, typically 11-15 days after the cells were injected. The average weight of the tumors in each treatment group for each cell line is calculated.

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for achieving a therapeutic effect in a mammal in need thereof which comprises an amount of a first compound which is an inhibitor of HMG-CoA reductase and an amount of a second compound which is an inhibitor of prenyl-protein transferases.

2. The composition according to Claim 1 wherein the inhibitor of HMG-CoA reductase is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and cerivastatin, and the pharmaceutically acceptable lactone, open acid, salt and ester forms thereof.

3. The composition according to Claim 1 wherein the inhibitor of prenyl-protein transferase is a dual inhibitor of farnesyl- protein transferase and geranylgeranyl-protein transferase type I.

4. The composition according to Claim 3 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl- protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) of less than about 1 μM for inhibiting the transfer of a geranylgeranyl residue to a protein or peptide substrate comprising a CAAX motif by geranylgeranyl-protein transferase type I in the presence of a modulating anion; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) of less than about 500 nM against transfer of a farnesyl residue to a protein or

F peptide substrate comprising a CAAX motif by farnesyl-protein transferase.

5. The composition according to Claim 4 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl- protein transferase type I is further characterized by: c) inhibition of the cellular prenylation of greater than (>) 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I at a concentration of less than (<) 10 μM.

6. The composition according to Claim 3 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl- protein transferase type I is characterized by: d) an IC₅₀ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells of less than 5 μM.

7. The composition according to Claim 3 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl- protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells between 0.1 and 100 times the IC₅₀ for inhibiting the farnesylation of the protein hDJ in cells; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

8. The composition according to Claim 3 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl- protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than 2 fold lower but less than 20,000 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and b) an IC₅₀ (^a measurement of in vitro inhibitory activity) against

H-ras-CVLL dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

9. The composition according to Claim 3 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl- protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than

10- fold lower but less than 2,500 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) against H-ras-CVLL dependent activation of MAP kinases in cells greater than 5 fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

10. The composition according to Claim 1 which is characterized by: a) inhibition of the cellular prenylation of greater than (>) about 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the compounds of the invention.

11. The composition according to Claim 1 which is characterized by: b) inhibition of greater than about 50% the K4B-Ras dependent activation of MAP kinases in cells.

12. The composition according to Claim 1 which is characterized by: c) inhibition of H-Ras dependent activation of MAP kinases in cells at least about 2 fold lower but less than about 20,000 fold lower than the inhibitory activity (IC50) against H-ras-CVLL

(SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells.

13. The composition according to Claim 1 wherein the inhibitor of prenyl-protein transferase is selected from:

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-[(3-pyridyl)methoxyethyl)]-4- (l-naphthoyl)piperazine

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-(benzyloxymethyl)-4-[7-(2,3- dihydrobenzofuroyl)]piperazine

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-[4-(5-dimethylamino-l- naphthalenesulfonamido)- 1 -butyl]-4-( 1 -naphthoyl)piperazine

N-[2(S)-(l-(4-Nitrophenylmethyl)-lH-imidazol-5-ylacetyl)amino-3(S)- methylpentyl] -N- 1 -naphthylmethyl-glycyl-methionine

N-[2(S)-(l-(4-Nitrophenylmethyl)-lH-imidazol-5-ylacetyl)amino-3(S)- methylpentyl] -N- 1 -naphthylmethyl-glycyl-methionine methyl ester N-[2(S)-([l-(4-cyanobenzyl)-lH-imidazol-5-yl]acetylamino)-3(S)- methylpentyl]-N-(l-naphthylmethyl)glycyl-methionine

N-[2(S)-([l-(4-cyanobenzyl)-lH-imidazol-5-yl]acetylamino)-3(S)- methylpentyl]-N-(l-naphthylmethyl)glycyl-methionine methyl ester

2(S)-π-Butyl-4-( 1 -naphthoyl)- 1 - [ 1 -(2-naphthylmethyl)imidazol-5- y lmethy 1] -piperazine

2(S)-«-Butyl- 1 - [ 1 -(4-cyanobenzyl)imidazol-5-ylmethyl] -4-( 1 - naphthoyl)piperazine

1 - { [ 1 -(4-cyanobenzyl)- 1 H-imidazol-5-yl]acetyl } -2(S)-n- butyl-4-( 1 -naphthoyl)piperazine

1 -(3-chlorophenyl)-4- [ 1 -(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone

l-phenyl-4-[l -(4-cyanobenzyl)- lH-imidazol-5-ylethyl]-piperazin-2-one

l-(3-trifluoromethylphenyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-5- ylmethyl]-piperazin-2-one

l-(3-bromophenyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-5-ylmethyl]- piperazin-2-one

5(S)-(2-[2,2,2-trifluoroethoxy]ethyl)-l-(3-trifluoromethylρhenyl)- 4-[l- (4-cyanobenzyl)-4-imidazolylmethyl]-piperazin-2-one

l-(5,6,7,8-tetrahydronaphthyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-5- ylmethyl]-piperazin-2-one

l-(2-methyl-3-chlorophenyl)-4-[l-(4-cyanobenzyl)-4- imidazolylmethyl)]-piperazin-2-one 2(RS)- { [ 1 -(Naphth-2-ylmethyl)- 1 H-imidazol-5-yl)] acetyl } amino-3-(t- butoxycarbonyl)amino- N-(2-methylbenzyl) propionamide

N- { 1 -(4-Cyanobenzyl)- 1 H-imidazol-5-ylmethyl } -4(R)-benzyloxy-2(S)- { N'-acetyl-N'-3-chlorobenzyl } aminomethylpyrrolidine

N-{ l-(4-Cyanobenzyl)-lH-imidazol-5-ylethyl}-4(R)-benzyloxy-2(S)- {N'-acetyl-N'-3-chlorobenzyl}aminomethyl pyrrolidine

l-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl] pyrrolidin-2(S)- ylmethyl]-(N-2-methylbenzyl)-glycine N'-(3-chlorophenylmethyl) amide

l-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl] pyrrolidin-2(S)- ylmethyl]-(N-2-methylbenzyl)-glycine N'-methyl-N'-(3- chlorophenylmethyl) amide

(S)-2-[(l-(4-Cyanobenzyl)-5-imidazolylmethyl)amino]-N- (benzyloxycarbonyl)-N-(3-chlorobenzyl)-4- (methanesulfonyl)butanamine

1 -(3 ,5-Dichlorobenzenesulfonyl)-3 (S)- [N-( 1 -(4-cyanobenzyl)- 1 H- imidazol-5-ylethyl)carbamoyl] piperidine

N- { [ 1 -(4-Cy anobenzyl)- 1 H-imidazol-5 -y 1] methyl } -4- (3 -methylphenyl)- 4-hydroxy piperidine,

N- { [ 1 - (4-Cy anobenzyl)- 1 H-imidazol- 5-yl] methyl } -4- (3 -chlorophenyl)-4 hydroxy piperidine,

4-[l-(4-cyanobenzyl)-5-imidazolylmethyl]-l-(2,3-dimethylphenyl)- piperazine-2,3-dione

l-(2-(3-Trifluoromethoxyphenyl)-pyrid-5-ylmethyl)-5-(4- cyanobenzyl)imidazole 4- { 5-[ 1 -(3-Chloro-phenyl)-2-oxo- 1 ,2-dihydro-pyridin-4-ylmethyl]- imidazol- 1 -y lmethyl } -2-methoxy-benzonitrile

3 (R)-3- [ 1 -(4-Cy anobenzyl)imidazol-5-yl-ethylamino] -5-phenyl- 1 -(2,2,2- trifluoroethyl)-H-benzo [e] [ 1 ,4] diazepine

3(S)-3-[l-(4-Cyanobenzyl) imidazol-5-yl]-ethylamino]-5-phenyl-l- (2,2,2-trifluoroethyl)-H-benzo[e] [ 1 ,4] diazepine

N-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl)pyrrolidin-2(S)- ylmethyl]- N-(l-naphthylmethyl)glycyl-methionine

N-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl)pyrrolidin-2(S)- ylmethyl]- N-(l-naphthylmethyl)glycyl-methionine methyl ester

2(S)-(4-Acetamido- 1 -butyl)- 1 -[2(R)-amino-3-mercaptopropyl]-4-( 1 - naphthoyl)piperazine

2(RS)-{ [l-(Naphth-2-ylmethyl)-lH-imidazol-5-yl)] acetyl}amino-3-(t- butoxycarbonyl)amino- N-cyclohexyl-propionamide

1 - { 2(R,S)- [ 1 -(4-cyanobenzyl)- 1 H-imidazol-5-yl]propanoyl } -2(S)-n- butyl-4-( 1 -naphthoyl)piperazine

1 - [ 1 - (4-cy anobenzyl)imidazol-5-ylmethyl] -4- (diphenylmethyl)piperazine

1 -(Diphenylmethyl)-3 (S)- [N- ( 1 -(4-cy anobenzyl)-2-methyl- 1 H-imidazol- 5-ylethyl)-N-(acetyl)aminomethyl] piperidine

N-[l-(lH-Imidazol-4-ylpropionyl)pyrrolidin-2(S)-ylmethyl]- N-(2- chlorobenzyl)glycyl-methionine

N-[l-(lH-Imidazol-4-ylpropionyl)pyrrolidin-2(S)-ylmethyl]- N-(2- chlorobenzyl)glycyl-methionine methyl ester 3(R)-3-[l-(4-Cyanobenzyl)imidazol-5-yl-methylamino]-5-phenyl-l- (2,2,2-trifluoroethyl)-H-benzo[e] [ 1 ,4] diazepine

1 - (3 -trifluoromethoxyphenyl)-4- [ 1 -(4-cy anobenzyl)imidazolylmethyl] - 2-piperazinone

l-(2,5-dimethylphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone

l-(3-methylphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone

l-(3-iodophenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2-piperazinone

l-(3-chlorophenyl)-4-[l-(4-cyano-3-methoxybenzyl)imidazolylmethyl]- 2-piperazinone

1 - (3 -trifluoromethoxyphenyl)-4- [ 1 - (4-cy ano-3- methoxybenzyl)imidazolyl methyl] -2-piperazinone

4-[((l-(4-cyanobenzyl)-5-imidazolyl)methyl)amino]benzophenone

l-(l-{ [3-(4-cyano-benzyl)-3H-imidazol-4-yl]-acetyl}-pyrrolidin-2(S)- ylmethyl)-3(S)-ethyl-pyrrolidine-2(S)-carboxylic acid 3-chloro- benzylamide

or the pharmaceutically acceptable salt thereof.

14. A pharmaceutical composition for achieving a therapeutic effect in a mammal in need thereof which comprises an amount of a first compound which is an inhibitor of HMG-CoA reductase and an amount of a second compound which is:

(+)-6-[amino(4-chlorophenyl)(l-methyl-lH-imidazol-5-yl)methyl]-4-(3- chlorophenyl)-l-methyl-2(lH)-quinolinone or the pharmaceutically acceptable salt thereof.

15. The composition according to Claim 1 wherein the amount of the inhibitor of HMG-CoA reductase is between about 0.1 mg and about 3000 mg.

16. The composition according to Claim 15 wherein the amount of the inhibitor of HMG-CoA reductase is between about 0.3 and about 160 mg.

17. The composition according to Claim 3 wherein the amount of the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is between about 10 mg and about 3000 mg.

18. The composition according to Claim 17 wherein the amount of the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is between about 10 mg and about 1000 mg.

19. A method of inhibiting the growth of cancer cells which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition which comprises an amount of a first compound which is an inhibitor of HMG-CoA reductase and an amount of a second compound which is an inhibitor of prenyl-protein transferase.

20. The method according to Claim 19 wherein the inhibitor of HMG-CoA reductase is selected from: lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and cerivastatin, and the pharmaceutically acceptable lactone, open acid, salt and ester forms thereof. 21. The method according to Claim 19 wherein the inhibitor of prenyl-protein transferase is a dual inhibitor of farnesyl- protein transferase and geranylgeranyl-protein transferase type I.

22. The method according to Claim 21 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) of less than about 1 μM against transfer of a geranylgeranyl residue to f-^χ a protein or peptide substrate comprising a CAAX motif by geranylgeranyl-protein transferase type I in the presence of a modulating anion; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) of less than about 500 nM against transfer of a farnesyl residue to a protein or

F peptide substrate comprising a CAAX motif by farnesyl-protein transferase.

23. The method according to Claim 21 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is characterized by: c) inhibition of the cellular prenylation of greater than (>) 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I at a concentration of less than (<)10 μM.

24. The method according to Claim 21 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is characterized by: d) an IC₅₀ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells of less than 5 μM. 25. The method according to Claim 21 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells between 0.1 and 100 times the IC₅₀ for inhibiting the farnesylation of the protein hDJ in cells; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) for inhibiting K4B-Ras dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

26. The method according to Claim 21 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than 2 fold lower but less than 20,000 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) against H-ras-CVLL dependent activation of MAP kinases in cells greater than 5-fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

27. The method according to Claim 21 wherein the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is characterized by: a) an IC₅₀ (a measurement of in vitro inhibitory activity) against H-Ras dependent activation of MAP kinases in cells greater than 10- fold lower but less than 2,500 fold lower than the inhibitory activity (IC50) against H-ras-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells; and b) an IC₅₀ (a measurement of in vitro inhibitory activity) against

H-ras-CVLL dependent activation of MAP kinases in cells greater than 5 fold lower than the inhibitory activity (IC50) against expression of the SEAP protein in cells transfected with the pCMV-SEAP plasmid that constitutively expresses the SEAP protein.

28. The method according to Claim 19 wherein the composition is characterized by: a) inhibition of the cellular prenylation of greater than (>) about 50% of the newly synthesized K4B-Ras protein after incubation of assay cells with the compounds of the invention.

29. The method according to Claim 19 wherein the composition is characterized by: b) inhibition of greater than about 50% the K4B-Ras dependent activation of MAP kinases in cells.

30. The method according to Claim 19 wherein the composition is characterized by: c) inhibition of H-Ras dependent activation of MAP kinases in cells at least about 2 fold lower but less than about 20,000 fold lower than the inhibitory activity (IC50) against H-ras-CVLL

(SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells.

31. The method according to Claim 19 wherein the inhibitor of prenyl-protein transferase is selected from:

l-[2(R)-Amino-3-mercaptopropyl]-2(S)-[(3-pyridyl)methoxyethyl)]-4- ( 1 -naphthoyl)piperazine l-[2(R)-Amino-3-mercaptopropyl]-2(S)-(benzyloxymethyl)-4-(l- naphthoyl)piperazine

1 - [2(R)- Amino-3 -mercaptopropyl] -2(S )- [4-(5 -dimethylamino- 1 - naphthalenesulfonamido)- 1 -butyl] -4- ( 1 -naphthoyl)piperazine

N-[2(S)-(l-(4-Nitrophenylmethyl)-lH-imidazol-5-ylacetyl)amino-3(S)- methylpentyl] -N- 1 -naphthylmethyl-glycyl-methionine methyl ester

N-[2(S)-([l-(4-cyanobenzyl)-lH-imidazol-5-yl]acetylamino)-3(S)- methylpentyl]-N-(l-naphthylmethyl)glycyl-methionine

N-[2(S)-([l-(4-cyanobenzyl)-lH-imidazol-5-yl]acetylamino)-3(S)- methylpentyl]-N-( 1 -naphthylmethyl)glycyl-methionine methyl ester

2(S)-n-Butyl-4-(l-naphthoyl)-l-[l-(2-naphthylmethyl)imidazol-5- y lmethyl] -piperazine

2(S)-«-B utyl- 1 -[ 1 -(4-cyanobenzyl)imidazol-5-ylmethyl] -4-( 1 - naphthoyl)piperazine

l-{ [l-(4-cyanobenzyl)-lH-imidazol-5-yl]acetyl}-2(S)-n- butyl-4-( 1 -naphthoyl)piperazine

l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone l-phenyl-4-[l -(4-cyanobenzyl)- lH-imidazol-5-ylethyl]-piperazin-2-one

l-(3-bromophenyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-5-ylmethyl]- piperazin-2-one

5(S)-(2-[2,2,2-trifluoroethoxy]ethyl)-l-(3-trifluoromethylphenyl)- 4-[l- (4-cyanobenzyl)-4-imidazolylmethyl]-piperazin-2-one

l-(5,6,7,8-tetrahydronaphthyl)-4-[l-(4-cyanobenzyl)-lH-imidazol-5- ylmethyl] -piperazin-2-one

l-(2-methyl-3-chlorophenyl)-4-[l-(4-cyanobenzyl)-4- imidazolylmethyl)]-piperazin-2-one

2(RS)- { [ 1 -(Naρhth-2-ylmethyl)- lH-imidazol-5-yl)] acetyl } amino-3-(t- butoxycarbonyl)amino- N-(2-methylbenzyl) propionamide

N-{ l-(4-Cyanobenzyl)-lH-imidazol-5-ylmethyl}-4(R)-benzyloxy-2(S)- { N'-acetyl-N'-3-chlorobenzyl } aminomethylpyrrolidine

N-{ l-(4-Cyanobenzyl)-lH-imidazol-5-ylethyl}-4(R)-benzyloxy-2(S)- { N'-acetyl-N'-3-chlorobenzyl } aminomethyl pyrrolidine

1 -[ 1 -(4-Cyanobenzyl)- lH-imidazol-5-ylacetyl] pyrrolidin-2(S)- ylmethyl]-(N-2-methylbenzyl)-glycine N'-(3-chlorophenylmethyl) amide

l-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl] pyrrolidin-2(S)- ylmethyl]-(N-2-methylbenzyl)-glycine N'-methyl-N'-(3- chlorophenylmethyl) amide (S)-2-[(l-(4-Cyanobenzyl)-5-imidazolylmethyl)amino]-N-

(benzyloxycarbonyl)-N-(3-chlorobenzyl)-4-

(methanesulfonyl)butanamine

N-{[l-(4-Cyanobenzyl)-lH-imidazol-5-yl]methyl}-4-(3-methylphenyl)- 4-hydroxy piperidine,

N- { [ 1 -(4-Cyanobenzyl)- lH-imidazol-5-yl]methyl }-4-(3-chlorophenyl)-4 hydroxy piperidine,

4- [ 1 -(4-cyanobenzyl)-5-imidazolylmethyl]- 1 -(2,3-dimethylphenyl)- piperazine-2,3-dione

l-(2-(3-Trifluoromethoxyphenyl)-pyrid-5-ylmethyl)-5-(4- cyanobenzyl)imidazole

4- { 5-[ 1 -(3-Chloro-phenyl)-2-oxo- 1 ,2-dihydro-pyridin-4-ylmethyl]- imidazol- 1 -ylmethyl } -2-methoxy-benzonitrile

3(R)-3-[l-(4-Cyanobenzyl)imidazol-5-yl-ethylamino]-5-ρhenyl-l-(2,2,2- trifluoroethyl)-H-benzo[e][l,4] diazepine

3(S)-3-[l-(4-Cyanobenzyl) imidazol-5-yl]-ethylamino]-5-ρhenyl-l- (2,2,2-trifluoroethyl)-H-benzo[e] [ 1 ,4] diazepine

N-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl)pyrrolidin-2(S)- ylmethyl]- N-( 1 -naphthylmethyl)glycyl-methionine

N-[l-(4-Cyanobenzyl)-lH-imidazol-5-ylacetyl)pyrrolidin-2(S)- ylmethyl]- N-(l-naphthylmethyl)glycyl-methionine methyl ester 2(S)-(4-Acetamido- 1 -butyl)- l-[2(R)-amino-3-mercaptopropyl]-4-(l - naphthoyl)piperazine

2(RS)- { [ 1 -(Naρhth-2-ylmethyl)- 1 H-imidazol-5-yl)] acetyl } amino-3-(t- butoxycarbonyl)amino- N-cyclohexyl-propionamide

l-{2(R,S)-[l-(4-cyanobenzyl)-lH-imidazol-5-yl]propanoyl}-2(S)-n- butyl-4-( 1 -naphthoyl)piperazine

l-[l-(4-cyanobenzyl)imidazol-5-ylmethyl]-4-(diphenylmethyl)piperazine

l-(Diphenylmethyl)-3(S)-[N-(l-(4-cyanobenzyl)-2-methyl-lH-imidazol- 5-ylethyl)-N-(acetyl)aminomethyl] piperidine

N-[l-(lH-Imidazol-4-ylpropionyl)pyrrolidin-2(S)-ylmethyl]- N-(2- chlorobenzyl)glycyl-methionine methyl ester

3(R)-3-[l-(4-Cyanobenzyl)imidazol-5-yl-methylamino]-5-phenyl-l- (2,2,2-trifluoroethyl)-H-benzo[e] [ 1 ,4] diazepine

1 -(3-trifluoromethoxyphenyl)-4- [ 1 -(4-cyanobenzyl)imidazolylmethyl] - 2-piperazinone

l-(2,5-dimethylphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone

l-(3-methylphenyl)-4-[l-(4-cyanobenzyl)imidazolylmethyl]-2- piperazinone

1 -(3-iodophenyl)-4- [ 1 -(4-cyanobenzyl)imidazolylmethyl]-2-piperazinone l-(3-chlorophenyl)-4-[l-(4-cyano-3-methoxybenzyl)imidazolylmethyl]- 2-piperazinone

1 -(3-trifluoromethoxyphenyl)-4- [ 1 -(4-cyano-3- methoxybenzyDimidazolyl methyl] -2-piperazinone

4-[((l-(4-cyanobenzyl)-5-imidazolyl)methyl)amino]benzophenone

l-(l-{[3-(4-cyano-benzyl)-3H-imidazol-4-yl]-acetyl}-pyrrolidin-2(S)- ylmethyl)-3(S)-ethyl-pyrrolidine-2(S)-carboxylic acid 3-chloro- benzylamide

or the pharmaceutically acceptable salt thereof.

32. A method of inhibiting the growth of cancer cells which comprises administering to a mammal in need thereof a therapeutically effective amount of a composition which comprises an amount of a first compound which is an inhibitor of HMG-CoA reductase and an amount of a second compound which is:

(+)-6-[amino(4-chlorophenyl)(l-methyl-lH-imidazol-5-yl)methyl]-4-(3- chlorophenyl)- 1 -methyl-2( l/7)-quinolinone

or the pharmaceutically acceptable salt thereof.

33. The method according to Claim 19 wherein the amount of the inhibitor of HMG-CoA reductase is between about 0.1 mg/day and about 3000 mg/day.

34. The method according to Claim 33 wherein the amount of the inhibitor of HMG-CoA reductase is between about 0.3 mg/day and about 160 mg/day.

35. The method according to Claim 21 wherein the amount of the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is between about 10 mg/day and about 3000 mg/day.

36. The method according to Claim 35 wherein the amount of the dual inhibitor of farnesyl-protein transferase and geranylgeranyl-protein transferase type I is between about 10 mg/day and about 1000 mg/day.

37. A method of inhibiting the growth of cancer cells which comprises administering to a mammal in need thereof a therapeutically effective amount of an amount of a first compound which is an inhibitor of HMG-CoA reductase and an amount of a second compound which is an inhibitor of prenyl-protein transferase.

38. The method according to Claim 37 wherein the inhibitor of HMG-CoA reductase is administered prior to the administration of the inhibitor of prenyl-protein transferase.

39. A method of inhibiting the growth of cancer cells in a mammal which comprises administering to said mammal an amount of a first compound which is an inhibitor of HMG-CoA reductase and an amount of a second compound which is an inhibitor of prenyl-protein transferase and applying to the mammal radiation therapy.

40. The method according to Claim 41 wherein the amount of an inhibitor of prenyl-protein transferase and the radiation therapy are administered simultaneously.

41. The method according to Claim 41 wherein the amount of the inhibitor of HMG-CoA reductase and the amount of an inhibitor of prenyl-protein transferase are administered first and the radiation therapy is administered after the the amount of an inhibitor of prenyl- protein transferase has been administered.