+

WO2000016626A1 - Methode de traitement du cancer - Google Patents

Methode de traitement du cancer Download PDF

Info

Publication number
WO2000016626A1
WO2000016626A1 PCT/US1999/022224 US9922224W WO0016626A1 WO 2000016626 A1 WO2000016626 A1 WO 2000016626A1 US 9922224 W US9922224 W US 9922224W WO 0016626 A1 WO0016626 A1 WO 0016626A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
ras
protein
inhibitor
protein transferase
Prior art date
Application number
PCT/US1999/022224
Other languages
English (en)
Inventor
Samuel L. Graham
Kenneth S. Koblan
David C. Heimbrook
Allen I. Oliff
Steven M. Stirdivant
Original Assignee
Merck & Co., Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB9824575.6A external-priority patent/GB9824575D0/en
Application filed by Merck & Co., Inc. filed Critical Merck & Co., Inc.
Priority to AU61624/99A priority Critical patent/AU6162499A/en
Publication of WO2000016626A1 publication Critical patent/WO2000016626A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • 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.
  • HMG-CoA reductase 3-hydroxy-3-methylglutaryl-CoA reductase
  • prenyl-protein transferase an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase
  • 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.
  • 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.
  • 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.
  • FPTase farnesyl-protein transferase
  • GGPTase-I geranylgeranyl- protein transferase type I
  • Rab GGPTase geranylgeranyl-protein transferase type-II
  • 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.
  • 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.
  • Ras functions like a G-regulatory protein.
  • Ras In the inactive state, Ras is bound to GDP.
  • Ras 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)).
  • 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.
  • 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.
  • FPTase farnesyl-protein transferase
  • the first class includes analogs of farnesyl diphosphate (FPP), while the second is related to protein substrates (e.g., Ras) for the enzyme.
  • FPP farnesyl diphosphate
  • 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)).
  • H-r ⁇ s H-r ⁇ s
  • N-ras N-ras
  • K4A-r ⁇ s K4A-r ⁇ s
  • K4B-ras H-ras
  • lovastatin 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.
  • 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)).
  • d-limonene as a "weak inhibitor" of farnesyl-protein transferase (eg., M. H. Gelb et al.
  • 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).
  • 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.
  • 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 ⁇ M Compound 1 (Example 1) (lane 2) or a combination of 3 ⁇ M Compound 1 and 1 ⁇ M of simvastatin (lane 3). Details of the assay procedure can be found in Example 15.
  • 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.
  • 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.
  • 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.
  • the HMG-CoA reductase inhibitor and the inhibitor of prenyl-protein transferase may be administered either sequentially in any order or simultaneously.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 ⁇ M for inhibiting the transfer of a geranylgeranyl
  • F peptide substrate comprising a CAAX motif by farnesyl-protein transferase.
  • 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 ⁇ M.
  • 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 ⁇ M. r- ⁇
  • CAAX will refer to such motifs that may be geranylgeranylated by GGTase-I.
  • 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).
  • CVIM K4B-Ras
  • 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.
  • the modulating anion is selected from a phosphate or sulfate containing anion.
  • 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), ⁇ -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,
  • the modulating anion is selected from adenosine 5 '-triphosphate, 2'-deoxyadenosine 5 '-triphosphate, 2'-deoxycytosine 5'-triphosphate, ⁇ -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.
  • the modulating anion is selected from adenosine 5'-triphosphate, ⁇ -glycerol phosphate, pyrophosphate, dithiophosphate and sulfate.
  • 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.
  • CAAX motifs include (the corresponding human protein is in parentheses): CVLS (H-ras) (SEQ.ID.: 11), CVIM (K4B-Ras)
  • CAAX containing protein or peptide substrates may also be geranylgeranylated by GGTase-I.
  • F farnesyl residue to a protein or peptide substrate comprising a CAAX motif by farnesyl-protein transferase is described in Example 10.
  • 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-r ⁇ s-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 ⁇ M.
  • ICQQ a measurement of in vitro inhibitory activity
  • 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.
  • ICgg a measurement of in vitro inhibitory activity
  • ICg Q a measurement of in vitro inhibitory activity
  • 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-r ⁇ s-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
  • IC50 inhibitory activity
  • a Class II prenyl-protein transferase inhibitor may also be characterized by: a) an ICQQ (a measurement of in vitro inhibitory activity) against
  • IC5 Q a measurement of in vitro inhibitory activity
  • Example 13 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.
  • 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.
  • assay cells examples include 3T3, C33a, PSN-1 (a human pancreatic carcinoma cell line) and K-r ⁇ s-transformed Rat-1 cells.
  • PSN-1 a human pancreatic carcinoma cell line
  • K-r ⁇ s-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.
  • 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 ⁇ M of the prenyl-protein transferase inhibitor.
  • 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.
  • 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 ⁇ M of the prenyl-protein transferase inhibitor and at a concentration of ⁇ 1 ⁇ M 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 ⁇ M 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-r ⁇ s-CVLL (SEQ.ID.NO.: 1) dependent activation of MAP kinases in cells.
  • 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 ⁇ M of the prenyl-protein transferase inhibitor and at a concentration of ⁇ 1 ⁇ M 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 ⁇ M of the prenyl-protein transferase inhibitor and at a concentration of ⁇ 100 nM of the HMG-CoA reductase inhibitor.
  • assay cells examples include 3T3, C33a, PSN-1 (a human pancreatic carcinoma cell line) and K-r ⁇ s- transformed Rat-1 cells.
  • Preferred assay cell line have been found to be C33a.
  • Example 13 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.
  • 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.
  • 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. Indolf ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • dispersing or wetting agents may be a naturally-occurring phosphatide, for example le
  • 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.
  • preservatives for example ethyl, or n-propyl p-hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p-hydroxybenzoate
  • flavoring agents such as sucrose, saccharin or aspartame.
  • 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.
  • compositions may be in the form of a sterile injectable aqueous solutions.
  • 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.
  • 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.
  • a continuous intravenous delivery device may be utilized.
  • An example of such a device is the Deltec CADD-PLUSTM 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.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • 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.
  • suitable non- irritating excipient include cocoa butter, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol.
  • 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.
  • topical application shall include mouth washes and gargles.
  • 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.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • 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.
  • 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.
  • the instant composition may be useful in further combination with known anti- cancer and cytotoxic agents.
  • 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.
  • 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.
  • combinations of therapeutic agents include 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.
  • 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.
  • 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.
  • the composition of the instant invention may be administered to a patient in need prior to the application of radiation therapy.
  • 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.
  • 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.
  • 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.
  • the term refers to compounds which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ v ⁇ 3 integrin, which selectively antagonize, inhibit or counteract binding of a physiological ligand to the ⁇ v ⁇ integrin, which antagonize, inhibit or counteract binding of a physiological ligand to both the ⁇ v ⁇ 3 integrin and the ⁇ v ⁇ 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 ⁇ v ⁇ 6, ⁇ v ⁇ 8, ⁇ l ⁇ l, ⁇ 2 ⁇ l, ⁇ l, ⁇ 6 ⁇ l and ⁇ 6 ⁇ 4 integrins.
  • the term also refers to antagonists of any combination of ⁇ v ⁇ 3, ⁇ v ⁇ , ⁇ v ⁇ 6, ⁇ v ⁇ 8, ⁇ l ⁇ l, ⁇ 2 ⁇ l, ⁇ l, ⁇ 6 ⁇ l and ⁇ 6 ⁇ 4 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.
  • 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. ⁇ 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 ⁇ inhibitor.
  • 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.
  • antineoplastic agent examples include, in general, microtubule-stabilising agents (such as paclitaxel (also
  • Taxol® docetaxel
  • docetaxel also known as Taxotere®
  • alkylating agents 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 l ⁇ 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,
  • diynenes and the podophyllotoxins are particularly useful members of those classes include, for example, doxorubicin, carminomycin, daunorubicin, aminopterin, methotrexate, methopterin, dichloro- methotrexate, mitomycin C, porfiromycin, ⁇ -fluorouracil, 6-mercaptopurine, gemcitabine, cytosine arabinoside,
  • 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,
  • melphalan hexamethyl melamine, thiotepa, cytarabin, idatrexate, trimetrexate, dacarbazine, L-asparaginase, camptothecin, CPT-11, topotecan, ara-C, bicalutamide, flutamide, leuprolide, pyridobenzoindole derivatives, interferons and interleukins.
  • HMG-CoA reductase 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 ⁇ "HMG-CoA reductase inhibitor” and "inhibitor of HMG-CoA reductase” have the same meaning when used herein.
  • HMG-CoA reductase inhibitors examples 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
  • 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,
  • HMG-CoA reductase inhibitor 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 ⁇ all such forms are included within the meaning of the term "HMG-CoA reductase inhibitor” as used herein.
  • the HMG-CoA reductase inhibitor is selected from lovastatin and simvastatin, and most preferably simvastatin.
  • pharmaceutically acceptable salts with respect to the HMG-CoA reductase inhibitor shall
  • 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 l ⁇ 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.
  • a suitable organic or inorganic base particularly those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc and tetramethylammonium
  • 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,
  • Ester derivatives of the described HMG-CoA reductase ⁇ 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:
  • Rla is selected from: hydrogen or Ci-C ⁇ alkyl
  • Rl° is independently selected from: 20 a) hydrogen, b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(R 10 )2 or C2-C6 alkenyl, c) Ci-C ⁇ alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
  • R 3 and R 4 selected from H and CH3;
  • R2 is selected fromH; unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, ⁇ .
  • NR 6 R 7 is selected fromH; unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, ⁇ .
  • NR 6 R 7 is selected fromH; unsubstituted or substituted aryl, unsubstituted or substituted heteroaryl, ⁇ .
  • R2 and R 3 are optionally attached to the same carbon atom;
  • R° and R ⁇ are independently selected from:
  • 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; 2 ⁇
  • 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, R 10 O-, Rl0c(O)NRl°-, CN, NO2,
  • ⁇ R ⁇ a is hydrogen or methyl
  • RIO is independently selected from hydrogen, Ci-C ⁇ alkyl, Ci-Cg perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • 10 RU is independently selected from C ⁇ -C6 alkyl and aryl;
  • V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl,
  • 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) C ⁇ -4 alkyl, unsubstituted or substituted with: C ⁇ -4 alkoxy, NR ⁇ R 7 , C3-6 cycloalkyl, unsubstituted or substituted aryl, heterocycle, HO, -S(0)mR , or -C(0)NR6R 7 , b) aryl or heterocycle, ⁇ c) halogen, d) OR6> e) NR6R7, f) CN, g) N0 2 , 10 h) CF3; i) -S(0) m R6a j) -C(0)NR6R 7 , or k) C3-C6 cycloalkyl
  • n 0, 1, 2, 3 or 4
  • p 0, 1, 2, 3 or 4
  • r is 0 to ⁇ , provided that r is 0 when V is hydrogen;
  • Rla is selected from: hydrogen or C ⁇ -C6 alkyl
  • Rl° is independently selected from: a) hydrogen, 10 b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(R 10 )2 or C2-C6 alkenyl, c) C ⁇ -C6 alkyl unsubstituted or substituted by unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(R 10 )2; l ⁇
  • Rlc is selected from: a) hydrogen, b) unsubstituted or substituted C ⁇ -C ⁇ alkyl wherein the substituent on the substituted Cl-C ⁇ alkyl is selected from
  • R3 and R 4 independently selected from H and CH3;
  • R 2 is selected fromH; OR 1 * ) ; ⁇ ⁇ NR 6 R 7
  • Ci-5 alkyl unbranched or branched, unsubstituted or substituted with one or more of:
  • R 2 , R 3 and R 4 are optionally attached to the same carbon atom;
  • R 6 and R 7 are independently selected from: H; C ⁇ .4 alkyl, C3-6 cycloalkyl, aryl, heterocycle, unsubstituted or substituted with: a) C ⁇ -4 alkoxy, b) halogen, or l ⁇ c) aryl or heterocycle;
  • R 6a is selected from:
  • R8 is independently selected from: 2 ⁇ a) hydrogen, b) C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl, F, Cl, R 10 O-, R 10 C(O)NR 10 -, CN, NO2, (RlO)2N-C(NR 10 )-, R 10 C(O)-, -N(R 10 )2, or RHOC(0)NR 10 -, and 30 c ) C 1-C6 alkyl substituted by C ⁇ -C6 perfluoroalkyl, R 10 O-,
  • R 10 C(O)NR 10 -, (R 10 )2N-C(NR 1( )-, Rl0c(O)-, -N(RlO)2, or RH ⁇ C(O)NRl0-;
  • R ⁇ a is hydrogen or methyl
  • RIO is independently selected from hydrogen, C ⁇ -C ⁇ alkyl, Cl-C ⁇ ⁇ perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • RU is independently selected from C ⁇ -C6 alkyl and aryl
  • R! 2 is selected from: H; unsubstituted or substituted C ⁇ -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:
  • aryl or heterocycle unsubstituted or substituted with: a) Ci-4 alkyl, l ⁇ b) (CH2) p OR 6 , c) (CH2)pNR 6 R 7 , d) halogen, e) CN, f) aryl or heteroaryl,
  • 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 ⁇ Al is a bond, n is 0 and A 2 is S(0) ;
  • 10 Y is selected from: a) hydrogen, b) Rl O-, RllS(0) m -, R 10 C(O)NR 10 -, (R 10 )2N-C(O)-, CN, NO2, (R 10 )2N-C(NR 10 )-, R 12 C(0)-, R 10 OC(O)-, N3, F, -N(R 10 )2, or
  • 20 Z is an unsubstituted or substituted aryl, wherein the substituted aryl is substituted with one or more of the following:
  • n 0, 1, 2, 3 or 4
  • 0 p 0, 1, 2, 3 or 4
  • r is 0 to ⁇ , provided that r is 0 when V is hydrogen; and v is 0, 1 or 2;
  • Rl is independently selected from: hydrogen or C1-C6 alkyl
  • R 2 is independently selected from: a) hydrogen, b) substituted or unsubstituted aryl, substituted or
  • C2-C6 alkenyl c) C ⁇ -C6 alkyl unsubstituted or substituted by aryl, heterocycle, C3-C10 cycloalkyl, C2-C6 alkenyl, Rl°0-,
  • R3 is selected from: a) hydrogen, b) C ⁇ -C6 alkyl unsubstituted or substituted by C2-C6 alkenyl, R1 ⁇ >0-, R 11 S(0) m -, RlOC ⁇ NRiO-, CN, N3, (R!0)2N-C(NR 1 0)-, R 10 C(O)-, -N(RlO)2, or R 11 OC(0)NR 10 -, c) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C3-C10 cycloalkyl, ⁇ C2-C6 alkenyl, fluoro, chloro, R 12 0-, R 1 :L S(0) m -,
  • R 1 0C(O)NR 10 -, CN, NO2, (RlO)2N-C(NRlO)-, RlOC(O)-, N3, -N(RlO)2, or
  • R4 and R ⁇ are independently selected from: l ⁇ a) hydrogen, b) C ⁇ -C6 alkyl unsubstituted or substituted by
  • R 6 is independently selected from: 30 a) hydrogen, b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C ⁇ -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl, F, Cl, R 10 O-, allyloxy, Rl0C(O)NRl0-, CN, NO2, (R!0)2N-C(NRlO)-, RlOc(O)-, -N(R!0)2, (R 12 )2NC(0)- or R OC(0)NR 10 -, and c) Ci-C ⁇ alkyl substituted by CI-CQ perfluoroalkyl, R 10 O-, R 10 C(O)NRl0-, (RlO)2N-C(NR 10 )-, R 10 C(O)-, -N(R 10 )2, or Rl 1 OC(O)NR 1 0-;
  • R 7 is independently selected from a) hydrogen, b) unsubstituted or substituted aryl, c) unsubstituted or substituted heterocycle,
  • R8 is selected from: a) hydrogen, 20 b) C ⁇ -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl, F, Cl, R 10 O-, R 10 C(O)NR 10 -, CN, NO2, (R 10 )2N-C(NRl°)-, Rl°C(0)-, R 10 OC(O)-, -N(Rl°)2, or
  • R9 is selected from: a) hydrogen, 30 b) C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl,
  • RlO is independently selected from hydrogen, C ⁇ -C6 alkyl, C ⁇ -C6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • RU is independently selected from C ⁇ -C ⁇ alkyl and aryl
  • R ⁇ 2 is independently selected from hydrogen, C ⁇ -C ⁇ alkyl, C ⁇ -C6 alkyl substituted with CO2R 10 , C ⁇ -C ⁇ alkyl substituted with aryl, C ⁇ -C ⁇ alkyl substituted with substituted aryl, C ⁇ -C ⁇ alkyl substituted with heterocycle, C -C6 alkyl substituted with l ⁇ substituted heterocycle, aryl and substituted aryl;
  • A3 is selected from: a bond, -C(0)NR 7 -, -NR 7 C(0)-, -S(0)2NR 7 -, -NR 7 S(0)2- or -N(R 7 )-;
  • a 4 is selected from: a bond, O, -N(R 7 )- or S; 2 ⁇
  • V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl,
  • Z is independently (Rl)2 or O; ⁇ 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
  • Rla is selected from: hydrogen or Cl-C6 alkyl
  • Rib is independently selected from: a) hydrogen, 20 b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(R 10 )2 or C2-C6 alkenyl, c) C ⁇ -C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
  • R a, R2b and R3 are independently selected from: a) hydrogen, b) C ⁇ -C6 alkyl unsubstituted or substituted by C2-C6 alkenyl, Rl ⁇ O-, RllS(0) m -, R 10 C(O)NRl0-, CN, N3, (RlO)2N-C(NRlO)-, RlOC(O)-, RlOOC(O)-, -N(RlO)2, or
  • RllOC(O)NR 1 0- c) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or substituted cycloalkyl, alkenyl, R 10 O-, R 11 S(0) nl -, R!0C(O)NR 10 -, CN, N02, (R 10 )2N-C(NR 1 0)-, R 10 C(O)-, R 1 0 ⁇ C(O)-, N3, -N(R 10 )2, halogen or
  • R ⁇ is hydrogen
  • R8 is selected from: a) hydrogen, b) C ⁇ -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl, F, Cl, R 10 O-, Rl0C(O)NR 10 -, CN, N ⁇ 2, (RlO)2N-C(NR 10 )-, R 10 C(O)-, R!0OC(O)-, -N(R 10 )2, or
  • RllOC(0)NR 10 - and c) C ⁇ -C ⁇ alkyl substituted by C -C6 perfluoroalkyl, R 10 O-, R 1( C(0)NR 10 -, (R 1 0)2N-C(NR 10 )-, RlOC(O)-, R 10 OC(O)-, -N(Rl°)2, or RllOC(0)NR 10 -;
  • R ⁇ a is independently selected from C ⁇ -C ⁇ alkyl and aryl
  • RlO is independently selected from hydrogen, C ⁇ -C ⁇ alkyl, C -C6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • 30 RU is independently selected from C -C6 alkyl, benzyl and aryl;
  • ⁇ V is selected from: a) hydrogen, b) heterocycle selected from pyrrolidinyl, imidazolyl, pyridinyl, thiazolyl, pyridonyl, 2-oxopiperidinyl, indolyl, quinolinyl, isoquinolinyl, and thienyl,
  • 20 p is independently 0, 1, 2, 3 or 4; and r is 0 to ⁇ , provided that r is 0 when V is hydrogen;
  • Rl is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(R 10 )2 or C2-C6 alkenyl, c) C ⁇ -C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(RlO)2;
  • R 2 is selected from H; unsubstituted or substituted aryl or C ⁇ -5 alkyl,
  • R 6 and R 7 are independently selected from: C ⁇ _4 alkyl, aryl, and heteroaryl, unsubstituted or substituted with: a) C ⁇ -4 alkoxy,
  • R a is selected from: 2 ⁇ C -4 alkyl, unsubstituted or substituted with: a) C ⁇ -4 alkoxy, or b) aryl or heteroaryl;
  • R° is independently selected from: 30 a) hydrogen, b) C ⁇ -C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C ⁇ perfluoroalkyl, F, Cl, R 10 O-, R 1 0C(O)NR 1 0-, CN, NO2, (RlO)2N-C(NRlO)-, RlOC(O)-, -N(RlO)2, or RUOC(0)NR 10 -, and c) C ⁇ -C ⁇ alkyl substituted by C ⁇ -C6 perfluoroalkyl, RlOO-, R!0C(O)NR1°-, (R 1 ⁇ )2N-C(NR 10 )-, R 10 C(O)-, -N(Rl°)2, or RllOC(0)NR 10 -;
  • ⁇ RlO is independently selected from hydrogen, C ⁇ -C ⁇ alkyl, C ⁇ -C ⁇ perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • RU is independently selected from Cl-C ⁇ alkyl and aryl
  • 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: l ⁇ a) C ⁇ _4 alkyl, unsubstituted or substituted with:
  • inhibitors of farnesyl-protein transferase are illustrated by the formula Il-a:
  • Rio is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(Rl°)2 or C2-C6 alkenyl, 10 c) C ⁇ -C6 alkyl unsubstituted or substituted by unsubstituted or substituted aryl, heterocycle, cycloalkyl, alkenyl, Rl ⁇ O-, or -N(R 10 )2;
  • Rlc is selected from: l ⁇ a) hydrogen, b) unsubstituted or substituted C ⁇ -C ⁇ alkyl wherein the substituent on the substituted C ⁇ -C ⁇ alkyl is selected from unsubstituted or substituted aryl, heterocyclic, C3-C 0 cycloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, RlOO-, RllS(0) m -,
  • R 6 , R 7 and R 7a are independently selected from:
  • R 6a is selected from:
  • R8 is independently selected from: a) hydrogen, b) C ⁇ -C 6 alkyl, C 2 -C6 alkenyl, C 2 -C 6 alkynyl, C ⁇ -C ⁇ perfluoroalkyl, F, Cl, R 10 O-, Rl0c(O)NR 10 -, CN, NO2, l ⁇ (RlO) 2 N-C(NR 10 )-, R 10 C(O)-, -N(R!0)2, or RHOC(0)NR 10 -, and c) C ⁇ -C6 alkyl substituted by C ⁇ -C6 perfluoroalkyl, R ⁇ O-, R 10 C(O)NR 10 -, (RlO) 2 N-C(NRl°)-, R 10 C(O)-, -N(RlO)2, or RllOC(0)NR 10 -;
  • RlO is independently selected from hydrogen, C ⁇ -C ⁇ alkyl, C ⁇ -C ⁇ perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • RU is independently selected from C1-C6 alkyl and substituted or 2 ⁇ unsubstituted aryl;
  • Rl 2 is selected from: H; unsubstituted or substituted C ⁇ -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:
  • Y is selected from: 10 a) hydrogen, b) Rl O-, RllS(0) m -, Rl0C(O)NR 1 0-, (RlO) 2 N-C(0)-, CN, NO2, (R!0)2N-C(NR 10 )-, R 12 C(0)-, RlO ⁇ C(O)-, N3, F, -N(Rl°)2, or
  • Z is an unsubstituted or substituted aryl, wherein the
  • substituted aryl is substituted with one or more of the following: 1) C -4 alkyl, unsubstituted or substituted with: a) C ⁇ -4 alkoxy, b) NR 6 R 7 c) C3-6 cycloalkyl, d) aryl, substituted aryl or heterocycle, e) HO, f) -S(0) m R 6a , or ⁇ g) -C(0)NR 6 R 7 ,
  • 20 v is 0, 1 or 2;
  • inhibitors of farnesyl-protein transferase are illustrated by the formula Ill-a:
  • R 2 is independently selected from: a) hydrogen, b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(R 10 )2 or C2-C6 alkenyl, c) C ⁇ -C ⁇ alkyl unsubstituted or substituted by aryl, ⁇ heterocycle, cycloalkyl, alkenyl, RlOO-, or -N(RlO)2;
  • R3 is selected from: a) hydrogen, b) C ⁇ -C ⁇ alkyl unsubstituted or substituted by C2-C6 10 alkenyl, R ⁇ O-, RllS(0) m -, R 10 C(O)NR 1 0-, CN, N3,
  • R4 and R ⁇ are independently selected from: a) hydrogen, b) C ⁇ -C ⁇ alkyl unsubstituted or substituted by RlOO- or
  • R6 is independently selected from: a) hydrogen, b) substituted or unsubstituted aryl, substituted or unsubstituted heterocycle, C ⁇ -C6 alkyl, C2-C6 alkenyl, ⁇ C2-C6 alkynyl, C ⁇ -C ⁇ perfluoroalkyl, F, Cl, R 10 O-, allyloxy,
  • Rl°C(O)NRl0- CN, NO2, (R 10 )2N-C(NR 10 )-, RlOc(O)-, -N(R 10 )2, (R 12 )2NC(0)- or R 11 OC(0)NR 10 -, and c) C ⁇ -C6 alkyl substituted by C -C6 perfluoroalkyl, R 10 O-, Rl°C(O)NR 1 0-, (RlO) 2 N-C(NR 10 )-, R!0C(O)-,
  • R 7 is independently selected from a) hydrogen, b) unsubstituted or substituted aryl, l ⁇ c) unsubstituted or substituted heterocycle, d) unsubstituted or substituted cycloalkyl, and e) C ⁇ -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: 2 ⁇ a) hydrogen, b) C ⁇ -C ⁇ alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl, F, Cl, R 10 O-, R 1 0C(O)NR 10 -, CN, NO2, (R 10 )2N-C(NR 10 )-, R 10 C(O)-, -N(RlO) 2 , or RHOC(0)NR 10 -, and 30 c) C ⁇ -C ⁇ alkyl substituted by C ⁇ -C ⁇ perfluoroalkyl, Rl°0-,
  • Rl is independently selected from hydrogen, C -C6 alkyl, C ⁇ -C6 perfluoroalkyl, 2,2,2-trifluoroethyl, benzyl and aryl;
  • RU- is independently selected from C ⁇ -C ⁇ alkyl and aryl;
  • R ⁇ is independently selected from hydrogen, C ⁇ -C ⁇ alkyl, C ⁇ -C6 alkyl substituted with C ⁇ 2R* , C ⁇ -C6 alkyl substituted with aryl, C -C6 alkyl substituted with substituted aryl, C ⁇ -C ⁇ alkyl substituted with heterocycle, C ⁇ -C ⁇ alkyl substituted with
  • A3 is selected from: a bond, -C(0)NR 7 -, -NR 7 C(0)-, -S(0)2NR 7 -, -NR 7 S(0)2- or -N(R 7 )-;
  • l ⁇ 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
  • the inhibitors of farnesyl-protein transferase are 2 ⁇ illustrated by the formula A-i:
  • Rl° is independently selected from: 30 a) hydrogen, b) aryl, heterocycle, cycloalkyl, R 10 O-, -N(R 10 )2 or C2-C6 alkenyl, c) C ⁇ -C6 alkyl unsubstituted or substituted by aryl, heterocycle, cycloalkyl, alkenyl, Rl O-, or -N(RlO)2; ⁇
  • R a and R 2D are independently selected from: a) hydrogen, b) C -C6 alkyl unsubstituted or substituted by
  • R 11 OC(O)NR 1 0-, c) unsubstituted or substituted aryl, unsubstituted or substituted heterocycle, unsubstituted or substituted cycloalkyl, alkenyl, RlOO-, R 11 S(0) m -, l ⁇ Rl0c(O)NR 10 -, CN, NO2, (R 10 )2N-C(NR 10 )-,
  • R 11 OC(0)NR 10 -, and d) C ⁇ -C6 alkyl substituted with an unsubstituted or substituted group selected from aryl, heterocyclic and 20 C3-CX0 cycloalkyl;
  • R° is hydrogen
  • R8 is independently selected from: a) hydrogen, b) C ⁇ -C ⁇ alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C ⁇ -C6 perfluoroalkyl, F, Cl, R 10 O-, R10C(0)NR10-, CN, N ⁇ 2, (R 10 )2N-C(NRlO)-, R10 C (O)-, -N(RlO)2, or RHOC(0)NR 10 -, and c) C ⁇ -C6 alkyl substituted by C ⁇ -C6 perfluoroalkyl, R 10 O-, R 10 C(O)NR 10 -, (R 1 0) 2 N-C(NR 10 )-, R 10 C(O)-, -N(R 10 )2, or R1 1 OC(O)NR10- ;
  • RI is independently selected from hydrogen, C -C6 alkyl, substituted or unsubstituted C ⁇ -C ⁇ aralkyl and substituted or unsubstituted aryl;
  • RU is independently selected from C ⁇ -C ⁇ alkyl, benzyl and aryl;
  • alkyl refers to a monovalent alkane
  • hydrocarbon (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.
  • substituted alkyl 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.
  • 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.
  • alkenyl groups examples 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.
  • 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.
  • "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.
  • aryl groups include phenyl, naphthyl, anthracenyl, biphenyl, tetrahydronaphthyl, indanyl, phenanthrenyl and the like.
  • 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.
  • 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.
  • partially aromatic groups are tetrahydro- imidazo [4, ⁇ -c] pyridine, phthalidyl and saccharinyl, as defined below.
  • heterocycle or heterocyclic represents a stable 5- to 7-membered monocyclic or stable 8- to 11-membered bicyclic or stable 11-l ⁇ 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.
  • 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, oxadiazoly
  • substituted aryl substituted heterocycle
  • 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(C ⁇ -C ⁇ alkyl)2, NO2, CN, (C ⁇ -C6 alkyl)0-, -OH, (C ⁇ -C6 alkyl)S(0) m -, (C ⁇ -C ⁇ alkyl)C(0)NH-, H2N-C(NH)-, (C1-C6 alkyl)C(O)-, (C ⁇ -C6 alkyl)OC(O)-, N3/C1-C6 alkyl)OC(0)NH- and C ⁇ -C20 alkyl.
  • 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.
  • named amino acids are understood to have the natural "L"
  • cyclic moieties 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:
  • cyclic moieties may optionally include a heteroatom(s).
  • 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.
  • 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.
  • any substituent or variable e.g., Rl , Z, n, etc.
  • -N(RlO)2 represents -NHH, -NHCH3, -NHC2H5, etc.
  • 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
  • compositions are useful in various pharmaceutically acceptable salt forms.
  • 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.
  • pharmaceutical compositions may be prepared from the active ingredients in combination with pharmaceutically acceptable carriers.
  • Non-toxic 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.
  • Piperazin-5-ones can be prepared as shown in Scheme 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • acidic conditions such as trif uoroacetic acid in methylene chloride, or hydrogen chloride gas in methanol or ethyl acetate
  • 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.
  • 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.
  • 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.
  • aldehydes such as l-trityl-4-imidazolyl-carboxaldehyde or l-trityl-4-imidazolylacetaldehyde
  • 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.
  • Scheme 18 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.
  • Scheme 24 illustrates the synthesis of the instant compounds wherein the moiety Z is attached directly to the piperidine ring.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 E Preparation of a reduced subunit by borane reduction of the amide moiety.
  • Reaction B Preparation of reduced peptide subunits by reductive alkylation
  • 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.
  • 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.
  • 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.
  • incorporation of a naphthylmethyl group and an acetyl group is illustrated.
  • 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 ⁇ -hydroxybenzyl moiety.
  • the protected intermediate aldehyde is treated with a suitably substituted phenyl Grignard reagent to provide the enantiomeric mixture LXXXVII.
  • Reaction Scheme 48 and 49 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.
  • 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.
  • 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 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.
  • 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.
  • 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
  • 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 ⁇ -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
  • Step E Preparation of l-(4-cyanobenzyl)-5- imidazolecarboxaldehyde
  • 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. ⁇ aHC ⁇ 3 soln., cooled to 0°C, and ⁇ i-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
  • Step I Preparation of 4-(tert-butoxycarbonyl)-l-(3- chlorophenyl)-2-piperazinone To a solution of the chloroacetamide from Step H (ca.
  • Step K Preparation of l-(3-chlorophenyl)-4-[l-(4-cyanobenzyl) imidazolylmethyll-2-piperazinone dihydrochloride
  • Examples 2-5 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.
  • Step F the appropriately substituted aniline was used in place of 3-chloroaniline.
  • 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.
  • Step F Preparation of 4-Cyano-3-methoxybenzyl Bromide A solution of the alcohol from Step E (35.5 g,
  • Step G Preparation of l-(4-cyano-3-methoxybenzyl)-5-
  • 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 CH 2 C1 2 ) and conversion of the resulting white foam to its dihydrochloride salt provided the titled product as a white powder
  • Step B N-t-Butoxycarbonyl-4(R)-hydroxyproline methyl ester
  • Step C N-t-Butoxycarbonyl-4(R)-t-butyldimethylsilyloxy proline methyl ester
  • the resulting mixture was stirred for 16hrs at room temperature.
  • Step D N-t-Butoxycarbonyl-4(R)-t-butyldimethylsilyloxy-2(S)- hydroxymethylpyrrolidine
  • Step E N-t-Butoxycarbonyl-4(R)-t-butyldimethylsilyloxy-2(S)- methanesulfonyloxymethylpyrrolidine
  • Step F Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)-azidomethylpyrrolidine
  • Step H Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)- ⁇ N'-3- chlorobenzy 1 ⁇ aminomethy lpyrrolidine
  • 3-chlorobenzaldehyde 1.2 ml, 10.6 mmol
  • crushed 3 A molecular sieves 9g
  • the amine from step G 3.50g, 10.6mmol
  • methanol 150 ml
  • sodium cyanoborohydride (11.0ml of a 1M solution in THF, l l.Ommol
  • Step I Preparation of N-t-Butoxycarbonyl-4(R)-t- butyldimethylsilyloxy-2(S)- ⁇ N'-3-chlorobenzyl-
  • Step K N-t-Butoxycarbonyl-4(R)-benzyloxyoxy-2(S)- ⁇ N'- acetyl-N'-3-chlorobenzyl ⁇ aminomethylpyrrolidine
  • Step L 4(S)-Benzyloxy-2(S)- ⁇ N * -acetyl-N'-3-chlorobenzyl ⁇ - aminomethylpyrrolidine hydrochloride
  • EtOAc 25 ml
  • EtOAc 25 ml
  • 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.
  • Step P Preparation of (l-(4-Cyanobenzyl)-lH-imidazol-5-yl)- ethanol
  • 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
  • Step R N ⁇ l-(4-Cyanobenzyl)- lH ⁇ imidazol-5-ylethyl ⁇ -4(R)- benzyloxyoxy-2(S)- ⁇ N'-acetyl-N'-3- chlorobenzyl ⁇ aminomethylpyrrolidine
  • Isoprenyl-protein transferase activity assays are carried out at 30 °C unless noted otherwise.
  • a typical reaction contains (in a final volume of 50 ⁇ L): [ 3 H]farnesyl diphosphate, Ras protein , 50 mM HEPES, pH 7.5, 5 mM MgCl 2 , 5 mM dithiothreitol, 10 ⁇ M ZnCl 2 , 0.1% polyethyleneglycol (PEG) (15,000- 20,000 mw) and isoprenyl-protein transferase.
  • PEG polyethyleneglycol
  • 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).
  • the modified geranylgeranyl-protein transferase inhibition assay is carried out at room temperature.
  • a typical reaction contains (in a final volume of 50 ⁇ L): [ 3 H]geranylgeranyl diphosphate, biotinylated Ras peptide, 50 mM HEPES, pH 7.5, a modulating anion (for example 10 mM glycerophosphate or 5mM ATP), 5 mM MgCl 2 , 10 ⁇ M ZnCl 2 , 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 ⁇ L 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.
  • streptavidin SPA beads Scintillation Proximity Assay beads
  • compositions or inhibitors are prepared as concentrated solutions in 100% dimethyl sulf oxide and then diluted 25-fold into the enzyme assay mixture.
  • IC 5 0 values are determined with Ras peptide near KM concentrations.
  • Enzyme and substrate concentrations for inhibitor IC 50 determinations are as follows: 75 pM GGTase-I, 1.6 ⁇ M Ras peptide, 100 nM geranylgeranyl diphosphate.
  • 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%).
  • 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.
  • 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
  • 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.
  • 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
  • Rat 1 cells transformed with either v-ras, v-raf, or v-mos are seeded at a density of 1 x 10 4 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Antisense strand N-terminal SEAP 5' GAGAGAGCTCGAGGTTAACCCGGGT
  • 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 introduces the internal STOP codon as well as the Hpal site.
  • 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.
  • 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.
  • 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 ⁇ l6T-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)
  • 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
  • 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 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente invention concerne une méthode de traitement du cancer par administration d'une certaine composition à un mammifère. Cette composition renferme un premier composé qui est un inhibiteur de la HMG-CoA réductase et un second composé qui est un inhibiteur de la prényl-transférase, et qui est efficace in vivo en tant qu'inhibiteur de croissance des cellules cancéreuses caractérisées par une protéine K4B-Ras mutée.
PCT/US1999/022224 1998-09-24 1999-09-23 Methode de traitement du cancer WO2000016626A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU61624/99A AU6162499A (en) 1998-09-24 1999-09-23 A method of treating cancer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US10162398P 1998-09-24 1998-09-24
US60/101,623 1998-09-24
GB9824575.6 1998-11-09
GBGB9824575.6A GB9824575D0 (en) 1998-11-09 1998-11-09 A method of treating cancer

Publications (1)

Publication Number Publication Date
WO2000016626A1 true WO2000016626A1 (fr) 2000-03-30

Family

ID=26314647

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/022224 WO2000016626A1 (fr) 1998-09-24 1999-09-23 Methode de traitement du cancer

Country Status (2)

Country Link
AU (1) AU6162499A (fr)
WO (1) WO2000016626A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1487489A2 (fr) * 2002-03-04 2004-12-22 Medimmune, Inc. PROCEDES PERMETTANT DE PREVENIR OU DE TRAITER DES TROUBLES PAR ADMINISTRATION D'UN ANTAGONISTE DE L'INTEGRINE avb3 ASSOCIE A UN INHIBITEUR DE LA REDUCTASE HMG-COA OU UN BISPHOSPHONATE

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BR. JR. CANCER, vol. 69, no. 6, 1994, pages 1015 - 1020 *
CHEMICAL ABSTRACTS, vol. 121, 1994, Columbus, Ohio, US; abstract no. 195205T, KAWATA ET AL.: "Modulation of the Mevalonate Pathway and Cell Growth by Pravastatin and D-Limonene in a Human Hepatoma Cell Line (HepG2)" XP002923669 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1487489A2 (fr) * 2002-03-04 2004-12-22 Medimmune, Inc. PROCEDES PERMETTANT DE PREVENIR OU DE TRAITER DES TROUBLES PAR ADMINISTRATION D'UN ANTAGONISTE DE L'INTEGRINE avb3 ASSOCIE A UN INHIBITEUR DE LA REDUCTASE HMG-COA OU UN BISPHOSPHONATE
EP1487489A4 (fr) * 2002-03-04 2008-10-01 Medimmune Inc PROCEDES PERMETTANT DE PREVENIR OU DE TRAITER DES TROUBLES PAR ADMINISTRATION D'UN ANTAGONISTE DE L'INTEGRINE avb3 ASSOCIE A UN INHIBITEUR DE LA REDUCTASE HMG-COA OU UN BISPHOSPHONATE

Also Published As

Publication number Publication date
AU6162499A (en) 2000-04-10

Similar Documents

Publication Publication Date Title
US6387903B1 (en) Inhibitors of prenyl-protein transferase
US6358956B1 (en) Inhibitors of prenyl-protein transferase
US20020052363A1 (en) Inhibitors of prenyl-protein transferase
WO2000051614A1 (fr) Inhibiteurs de prenyl-proteine transferases
EP1014984A1 (fr) Inhibiteurs de prenyl-proteine-transferases
WO2001060368A1 (fr) Inhibiteurs de la prenyle proteine transferase
US6376496B1 (en) Inhibitors of prenyl-protein transferase
US6103487A (en) Method of treating cancer
WO2001060458A2 (fr) Inhibiteurs de la prenyl-proteine transferase
US6335343B1 (en) Inhibitors of prenyl-protein transferase
US6355643B1 (en) Inhibitors of prenyl-protein transferase
WO2000016626A1 (fr) Methode de traitement du cancer
WO2000016778A1 (fr) Procede de traitement du cancer
WO1999010525A1 (fr) Procede pour le traitement du cancer
WO1999010329A1 (fr) Inhibiteurs de prenyl-proteine-transferase
EP1158984A1 (fr) Inhibiteurs de prenyl-proteine transferase
WO1999010523A1 (fr) Procede de traitement du cancer
WO1999010524A1 (fr) Procede pour le traitement du cancer
WO2001077116A1 (fr) Inhibiteurs de prenyl-proteine transferase

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载