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WO2003047569A1 - Compositions et methodes permettant d'inhiber des prenyltransferases - Google Patents

Compositions et methodes permettant d'inhiber des prenyltransferases Download PDF

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WO2003047569A1
WO2003047569A1 PCT/US2002/038511 US0238511W WO03047569A1 WO 2003047569 A1 WO2003047569 A1 WO 2003047569A1 US 0238511 W US0238511 W US 0238511W WO 03047569 A1 WO03047569 A1 WO 03047569A1
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substituted
unsubstituted
heteroaryl
compound
aryl
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Jon H. Come
Krishna K. Murthi
Zhongguo Wang
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Agennix USA Inc
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GPC Biotech Inc
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
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    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/36—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/81—Amides; Imides
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    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/54—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members
    • C07D233/64—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having two double bonds between ring members or between ring members and non-ring members with substituted hydrocarbon radicals attached to ring carbon atoms, e.g. histidine
    • C—CHEMISTRY; METALLURGY
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    • C07D241/00—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings
    • C07D241/02—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings
    • C07D241/04—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
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    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
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    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
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    • C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings

Definitions

  • Ras proteins including Ha-Ras, Ki4a-Ras, Ki4b-Ras and N-Ras, are part of a signalling pathway that links cell surface growth factor receptors to nuclear signals initiating cellular proliferation.
  • Biological and biochemical studies of action indicate that Ras functions like a regulatory G-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 stimulation 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 et al. (1993) Ann. Rev. Biochem. 62:851-891; Barbacid (1987) Ann. Rev. Biochem. 56:779).
  • Ras must be localized to the plasma membrane to achieve its normal function.
  • Covalent modification by lipids contributes to membrane interactions and biological activities of a rapidly expanding group of proteins, including Ras (see, for example, Maltese (1990) E4SER J4:3319; and Glomset et al. (1990) Trends Biochem Sci 15:139).
  • Ras see, for example, Maltese (1990) E4SER J4:3319; and Glomset et al. (1990) Trends Biochem Sci 15:139.
  • farnesyl (15-carbon) or geranylgeranyl (20-carbon) isoprenoids can be attached to specific proteins, with geranylgeranyl being the predominant isoprenoid found on proteins (Farnsworth et al. (1990) Science 247:320).
  • FPTase and GGPTase-I are ⁇ / ⁇ heterodimeric enzymes that share a common subunit; the ⁇ subunits are distinct but share approximately 30% amino acid similarity (Brown et al (1993) Nature 366:14-15; Zhang et al. (1994) J. Biol. Chem. 269:3175-3180; Zhang et al. (1994) J. Biol. Chem. 269:23465-23470; Yokoyama et al. (1995) Biochem. 34:1344-1354).
  • GGPTase II has different ⁇ and ⁇ subunits and complexes with a third component (R ⁇ P, Rab Escort Protein) that presents the protein substrate to the ⁇ / ⁇ catalytic subunits.
  • R ⁇ P Rab Escort Protein
  • Each of these enzymes selectively uses farnesyl diphosphate or geranylgeranyl 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 tetrapeptides comprise the minimum region required for interaction of the protein substrate with the enzyme.
  • GGPTase-II modifies XXCC and XCXC proteins, while the interaction between GGPTase-II and its protein substrates is more complex, requiring protein sequences in addition to the C-terminal amino acids for recognition.
  • 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 et al. (1991) J. Biol. Chem. 266:17438).
  • Some prenyltransferases have been recombinantly prepared (Omer et al.
  • FPTase covalently modifies the Cys thiol group of the Ras CAAX box (Reiss et al. (1990) Cell 62:81-88; Schaber et al. (1990) J. Biol. Chem. 265:14701-14704; Schafer et al, (1990) Science 249:1133-1139; Manne et al. (1990) Proc. Natl. Acad. Sci USA 87:7541-7545).
  • Other farnesylated proteins include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin. James et al. (1994) J.
  • GGPTase-I transfers the prenyl group from geranylgeranyl diphosphate to the sulphur atom in the Cys residue within the CAAX sequence.
  • Proteins of the Ras superfamily, including Rhol, Rho2, Rsrl/Budl, and Cdc42 appear to be GGPTase substrates (Madaule et al. (1987) PNAS 84:779-783; Bender et al.
  • Fungal infections of humans range from superficial conditions, usually caused by dermatophytes or Candida species, that affect the skin (such as dermatophytoses) to deeply invasive and often lethal infections (such as candidiasis and cryptococcosis).
  • Pathogenic fungi occur worldwide, although particular species may predominate in certain geographic areas.
  • the incidence of fungal infections has increased dramatically, as have the numbers of potentially invasive species. Indeed, fungal infections, once dismissed as a nuisance, have begun to spread so widely that they are becoming a major concern in hospitals and health departments.
  • Fungal infections occur more frequently in people whose immune system is compromised or suppressed (e.g., because of organ transplantation, cancer chemotherapy, or the human immunodeficiency virus), who have been treated with broad-spectrum antibacterial agents, or who have been subject to invasive procedures (catheters and prosthetic devices, for example). Fungal infections are now important causes of morbidity and mortality of hospitalized patients: the frequency of invasive candidiasis has increased tenfold to become the fourth most common blood culture isolate (Pannuti et al. (1992) Cancer 69:2653).
  • Invasive pulmonary aspergillosis is a leading cause of mortality in bone-marrow transplant recipients (Pannuti et al , supra), while Pneumocystis carinii pneumonia is the cause of death in many patients with acquired immunodeficiency syndrome (AIDS) in North America and Europe (Hughes (1991) Pediatr Infect. Dis J. 10:391). Many opportunistic fungal infections cannot be diagnosed by usual blood culture and must be treated empirically in severely immuno-compromised patients (Walsh et al. (1991) Rev. Infect. Dis. 13 :496). The fungi responsible for life-threatening infections include Candida species
  • the fungal cell wall is a structure that is both essential for the fungus and absent from mammalian cells, and consequently may be an ideal target for antifungal agents.
  • Polyoxins and the structurally related nikkomycins (both consist of a pyrimidine nucleoside linked to a peptide moiety) inhibit chitin synthase competitively, presumably acting as analogs of the substrate uridine diphosphate (UDP)-N-acetylglucosamine (chitin is an N-acetylglucosamine homopolymer), causing inhibition of septation and osmotic lysis.
  • UDP uridine diphosphate
  • chitin is an N-acetylglucosamine homopolymer
  • the target of polyoxins and nikkomycins is in the inner leaflet of the plasma membrane; they are taken up by a di
  • glucans are the major components that strengthen the cell wall.
  • the glucosyl units within these glucans are arranged as long coiling chains of ⁇ - (l,3)-linked residues, with occasional sidechains that involve ⁇ -(l,6) linkages.
  • Three ⁇ -(l,3) chains running in parallel can associate to form a triple helix, and the aggregation of helices produces a network of water-insoluble fibrils.
  • ⁇ -(l,3)-glucan is required to maintain the integrity and form of the cell wall (Kurtz et al. (1994) Antimicrob Agents Chemother 38:1408-1489), and, in P.
  • ⁇ -(l,3)-glucan is produced by a synthase composed of at least two subunits (Tkacz, J.S. (1992), in Emerging Targets in Antibacterial and Antifungal Chemotherapy 495-523 (Sutcliffe and Georgopapadakou, Eds., Chapman & Hall); and Kang et al. (1986) PNAS 83:5808- 5812).
  • One subunit is localized to the plasma membrane and is thought to be the catalytic subunit, while the second subunit binds GTP and associates with and activates the catalytic subunit (Mol et al. (1994) J Biol Chem 269:31267-31274).
  • the cell wall of many fungi is required to maintain cell shape and integrity.
  • the main structural component responsible for the rigidity of the yeast cell wall is 1,3- ⁇ -linked glucan polymers with some branches through 1,6- ⁇ - linkages.
  • the biochemistry of the yeast enzyme catalyzing the synthesis of 1,3- ⁇ - glucan chains has been studied extensively.
  • a pair of closely related proteins (Gscl/Fksl and Gsc2/Fks2) had previously been described as subunits of the 1,3 - ⁇ - glucan synthase (GS) (Inoue et al. (1995) supra; Douglas et al. (1994) PNAS 91 :12907; Drgonova et al. (1996) Science 272:277).
  • GTP-binding protein is a regulatory subunit that stimulates this enzyme (Mol et al. (1994) J. Biol. Chem. 269:31267; Qadota et al. (1996) Science 272:279).
  • Rho-like GTPase activities are critically involved in cell wall integrity, hyphael formation, and other cellular functions critical to pathogenesis.
  • Fungal Rhol GTPase is required for glucan synthase activity, copurifies with 1,3- ⁇ -glucan synthase, and is found to associate with the Gscl/Fksl subunit of this complex in vivo.
  • Rhol is a regulatory subunit of 1,3- ⁇ -glucan synthase, and accordingly Rhol, and the resulting enzyme complex, are potential therapeutic targets for development of antifungal agents.
  • Rhol is required for protein kinase C (PKCl) mediated MAPK activation, and confers upon PKCl the ability to be stimulated by phosphatidylserine (PS), indicating that Rhol controls signal transmission through PKCl.
  • PKCl protein kinase C
  • PS phosphatidylserine
  • Loss of PKCl activity results in cell lysis.
  • Prenylation of Rhol by GGPTase is a critical step to maintenance of cell wall integrity in yeast, and loss of Rhol prenylation results in cell lysis.
  • Ras genes are found in many human cancers. Transforming Ras genes are the oncogenes most frequently identified in human cancers. Clinical investigations have identified activated Ras genes in a wide variety of human neoplasms, including carcinomas, sarcomas, leukemias, and lymphomas. It is estimated that 40% of all human colon cancers and 95% of human pancreatic cancers contain activated Ras oncogenes (Kuzumaki (1991) Anticancer Res. 11 :313- 320). Mammalian cells express at least four types of Ras proteins, Ha-Ras, Ki4a-
  • Ras, Ki4b-Ras and N-Ras are defective in their GTPase activity and constitutively transmit a growth stimulation signal. Mutations to the Ras proto-oncogene translate into amino acid substitutions in the GTP binding domain, activating the Ras protein and biasing this molecular switch in the "on" position.
  • the Ras transformed cell behaves like a cell with a faulty switch, signaling extracellular hormone binding when none is present. Cells transformed in this way grow and differentiate in an abnormal manner.
  • Ras must be localized to the plasma membrane by prenylation to produce any oncogenic effect.
  • Prenyltransferases including FPTase, GGPTase I, and GGPTase II, are therefore potential targets for anticancer, antitumor, and other like agents. It was therefore one object of this invention to identify compounds that antagonize prenylation of low molecular weight G-proteins such as Ras.
  • One aspect of the present invention relates to methods for treating or preventing fungal infections and infections involving other eukaryotic parasites of plants or animals, using compounds that inhibit the biological activity of a prenyltransferase.
  • the prenyltransferase that is inhibited is GGPTase.
  • the present invention also relates to the novel compositions of matter used in such methods.
  • the subject inhibitors can be used for the treatment of mycotic infections in animals; as additives in feed for livestock to promote weight gain; as disinfectant formulations; and as in agricultural applications to prevent or treat fungal infection of plants.
  • the practice of the subject method utilizes inhibitors which are selective inhibitors of the fungal or parasitic prenyltransferase relative to any human prenyltransferases.
  • the method can be used for treating a nosocomial fungal and skin/wound infection involving fungal organisms, including, among others, the species Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Epidermophyton, Hendersonula, Histoplasma, Microsporum, Paecilomyces, Paracoccidioides, Pneumocystis, Trichophyton, and Trichosporium.
  • the method can be used for treating an animal or plant parasites, such as infections involving liver flukes, nematodes or the like.
  • the inhibitors of prenyltransferases may be used to treat cancer, neoplasms, or other forms or types of aberrant hyperproliferation or unwanted proliferation.
  • the practice of the subject method utilizes prenyltransferase inhibitors which are selective inhibitors of human GGPTase I, GGPTase II, or FPTase.
  • the present invention also relates to the novel compositions of matter used in such methods.
  • the practice of the subject method utilizes prenyltransferase inhibitors which are selective inhibitors of a specific prenyltransferase, such as GGPTase I, GGPTase II, or FPTase.
  • treatment using the inhibitors of the present invention comprises the administration of a pharmaceutical composition of the invention in a therapeutically effective amount to an individual in need of such treatment.
  • the compositions may be administered parenterally by intramuscular, intravenous, intraocular, intraperitoneal, or subcutaneous routes; inhalation; orally, topically and intranasally.
  • Figures 1-31 present various illustrative reaction schemes for preparing prenyltransferase inhibitors useful in the methods and compositions of the present invention.
  • Figure 32 is a demonstration of the effect of a prenyltransferase inhibitor on prenylation state of CaRHOl. Detailed Description of the Invention
  • the present invention relates to methods for treating and/or preventing fungal infections using compounds that specifically inhibit the biological activity of fungal enzymes involved in cell wall integrity, hyphael formation, and other cellular functions critical to pathogenesis.
  • prenylation of Rhol -like phosphatases by a geranylgeranylproteintransferase (GGPTase) activity can be critical to maintenance of cell wall integrity in yeast.
  • GGPTase geranylgeranylproteintransferase
  • prenylation of, inter alia, Rhol -like GTPase(s) is required for sufficient glucan synthase activity.
  • the present invention relates to methods of treating and preventing cancer, neoplasms, and other aberrant hyperproliferation by using compounds that specifically inhibit the biological activity of prenyltransferases, including FTPase, GGPTase I, and GGPTase II.
  • proteins encoded by mutant Ras genes are able to transform cells to a malginant phenotype.
  • Inhibition by compounds of the present invention may suppress the oncogenic potential of any protein encoded by a mutant Ras gene.
  • the present invention also relates to the novel compositions of matter used in such methods.
  • prenyltransferases Different substrate specificity among prenyltransferases allows for preparation of inhibitors of the present invention having improved therapeutic indexes. That is, certain inhibitors may inhibit some prenyltransferases and not others. As a result, inhibitors only for prenyltransferases encoded by oncogenes, and not wildtype enzymes, may be employed. Some of the reasons why different prenyltransferases may exhibit different specificity include the following. The ⁇ subunits for FTPase and GGPTase I are distinct, and such subunits contribute significantly to the activity of the enzyme.
  • prenyltransferase selectively, which should allow for improved therapeutic indexes for any inhibitor when administered specifically for any cancer, neoplasm, or aberrant hyperproliferative disorder resulting from mutation of a particular gene encoding a prenyltransferase.
  • the present invention provides methods and compositions for inhibiting prenyltransferases using small molecule (e.g., less than about 1000 amu) inhibitors.
  • the preferred inhibitors inhibit a targeted prenyltransferase with a K; of 10 ⁇ M or less, more preferably 1 ⁇ M or less, and even more preferably with a Kj less than 100 nM, 10 nM, or even 1 nM.
  • the subject method preferably employs prenyltransferase inhibitors, such as inhibitors of FPTase, GGPTase I, or GGPTase II to treat cancer, neoplasms and other aberrant hyperproliferative disorders.
  • prenyltransferase inhibitors such as inhibitors of FPTase, GGPTase I, or GGPTase II to treat cancer, neoplasms and other aberrant hyperproliferative disorders.
  • the chemotherapeutic properties of the compounds of the present invention may be determined from cell-based assays, as well as by other methods, including, inter alia, growth inhibition assays, flow cytometry analyses, and other standard assays known to those skilled in the art.
  • Preferred anticancer agent pharmaceutical preparation would provide a dose less than the ED 50 for modulation of prenyltansferase activity of nonmutated genes as compared to oncogenic ones, more preferably at least 1 order of magnitude less, more preferably at least 2, 3 or 4 orders of magnitude less.
  • Another parameter useful in identifying and measuring the effectiveness of the prenyltransferase inhibitor compounds of the invention as anticancer agents is the determination of the kinetics of the activity of such compounds. Such a determination can be made by determining the effect of an inhibitor, e.g., anticancer or antifungal activity, as a function of time.
  • the compounds display kinetics which result in efficient lysis of a fungal cell.
  • the compounds are fungicidal.
  • the compounds display kinetics which result in at least slowing of cell proliferation, or more preferably, cell death for any oncogenic cell.
  • the subject method preferably employs prenyltransferase inhibitors which are selective for a fungal enzyme relative to the host animals' prenyltransferase, e.g., the Kj for inhibition of the fungal enzyme is at least one order of magnitude less than the Kj for inhibition any prenyltransferase from human (or other animal), and even more preferably at least two, three, or even four orders of magnitude less.
  • the practice of the subject method in vivo in animals utilizes inhibitors with therapeutic indexes of at least 10, and more preferably at least 100 or 1000.
  • inhibitors for use as antifungal agents inhibit fungal GGPTase.
  • the antifungal properties of the compounds of the present invention may be determined from a fungal lysis assay, as well as by other methods, including, mter alia, growth inhibition assays, fluorescence-based fungal viability assays, flow cytometry analyses, and other standard assays known to those skilled in the art.
  • the assays for growth inhibition of a microbial target can be used to derive an ED 5Q value for the compound, that is, the concentration of compound required to kill 50% of the fungal sample being tested.
  • Preferred antifungal agent pharmaceutical preparation whether for topical, injection or oral delivery (or other route of administration), would provide a dose less than the ED 5 ⁇ for modulation of FPTase and/or GGPTase activity in the host (mammal), more preferably at least 1 order of magnitude less, more preferably at least 2, 3 or 4 orders of magnitude less.
  • growth inhibition by an antifungal compound of the invention may also be characterized in terms of the minimum inhibitory concentration (MIC), which is the concentration of compound required to achieve inhibition of fungal cell growth.
  • MIC minimum inhibitory concentration
  • Such values are well known to those in the art as representative of the effectiveness of a particular antifungal agent against a particular organism or group of organisms.
  • cytolysis of a fungal population by an antifungal compound can also be characterized, as described above by the minimum inhibitory concentration, which is the concentration required to reduce the viable fungal population by 99.9%.
  • the value of MIC5 0 defined as the concentration of a compound required to reduce the viable fungal population by 50%, can also be used.
  • the compounds of the present invention are selected for use based, wter alia, on having MIC values of less than 25 ⁇ g/mL, more preferably less than 7 ⁇ g/mL, and even more preferably less than 1 ⁇ g/mL against a desired fungal target, e.g., Candida albicans.
  • the preferred anticancer compounds of the invention display selective toxicity to target cells having mutated genes encoding for prenyltransferases over the wildtype form. Determination of the toxic dose (or "LD 5 o") can be carried out using protocols well known in the field of pharmacology.
  • tissue culture assays e.g., the present compounds can be evaluated according to standard methods known to those skilled in that art (see for example Gootz, T. D. (1990) Clin. Microbiol. Rev. 3:13-31).
  • assay methods include, inter alia, trypan blue exclusion and MTT assays (Moore et al. (1994) Compound Res. 7:265-269).
  • a specific cell type may release a specific metabolite upon changes in membrane permeability
  • that specific metabolite may be assayed, e.g., the release of hemoglobin upon the lysis of red blood cells (Srinivas et al. (1992) J Biol. Chem. 267:7121-7127).
  • the compounds of the invention are preferably tested against primary cells, e.g., using human skin fibroblasts (HSF) or fetal equine kidney (FEK) cell cultures, or other primary cell cultures routinely used by those skilled in the art. Permanent cell lines may also be used, e.g., Jurkat cells.
  • the subject compounds are selected for use in animals, or animal cell/tissue culture based at least in part on having LD 5 o's at least one order of magnitude greater than the MIC or ED 5Q as the case may be, and even more preferably at least two, three and even four orders of magnitude greater. That is, in preferred embodiments where the subject compounds are to be administered to an animal, a suitable therapeutic index is preferably greater than 10, and more preferably greater than 100, 1000 or even 10,000.
  • the preferred antifungal compounds of the invention display selective toxicity to target microorganisms and minimal toxicity to mammalian cells. Determination of the toxic dose (or "LD 5 o") can be carried out using protocols well known in the field of pharmacology. Ascertaining the effect of a compound of the invention on mammalian cells is preferably performed using tissue culture assays, e.g., the present compounds can be evaluated according to standard methods known to those skilled in that art (see for example Gootz, T. D. (1990) Clin. Microbiol. Rev. 3:13-31). For mammalian cells, such assay methods include, ter alia, trypan blue exclusion and MTT assays (Moore et al.
  • a specific cell type may release a specific metabolite upon changes in membrane permeability
  • that specific metabolite may be assayed, e.g., the release of hemoglobin upon the lysis of red blood cells (Srinivas et al. (1992) J. Biol. Chem. 267:7121-7127).
  • the compounds of the invention are preferably tested against primary cells, e.g., using human skin fibroblasts (HSF) or fetal equine kidney (FEK) cell cultures, or other primary cell cultures routinely used by those skilled in the art. Permanent cell lines may also be used, e.g., Jurkat cells.
  • the subject compounds are selected for use in animals, or animal cell/tissue culture based at least in part on having LD 5 o's at least one order of magnitude greater than the MIC or ED 50 as the case may be, and even more preferably at least two, three, and even four orders of magnitude greater. That is, in preferred embodiments where the subject compounds are to be administered to an animal, a suitable therapeutic index is preferably greater than 10, and more preferably greater than 100, 1000 or even 10,000.
  • the invention is also directed to methods for treating a microbial infection in a host using the compositions of the invention.
  • the compounds provided in the subject methods exhibit broad antifungal activity against various fungi and can be used as agents for treatment and prophylaxis of fungal infectious diseases.
  • the subject method can be used to treat or prevent nosocomial fungal and skin/wound infection involving fungal organisms, including, among others, the species Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Epidermophyton, Hendersonula, Histoplasma, Microsporum, Paecilomyces, Paracoccidioides, Pneumocystis, Trichophyton, and Trichosporium.
  • treatment of such fungal infections comprises the administration of a pharmaceutical composition of the invention in a therapeutically effective amount to an individual in need of such treatment.
  • the compositions may be administered parenterally by intramuscular, intravenous, intraocular, intraperitoneal, or subcutaneous routes; inhalation; orally, topically and intranasally.
  • the subject inhibitors of the present invention may also be used to inhibit neoplastic growth or proliferative disorders in tissue culture.
  • the subject inhibitors, and corresponding antifungal methods are also particularly useful in inhibiting unwanted fungal growth in tissue culture, especially those used for production of recombinant proteins or vectors for use in gene therapy.
  • the invention is also directed to pharmaceutical compositions containing one or more of the inhibitory compounds of the invention as the active ingredient which may be administered to a patient.
  • the invention is also directed to pharmaceutical compositions containing one or more of the antimicrobial compounds of the invention as the active ingredient which may be administered to a host animal.
  • inhibitors of prenyltransferases There have been a number of reports on methods for detecting inhibitors of prenyltransferases and uses of such inhibitors. For example, inhibition of farnesyl- protein transferase has been shown to block the growth of Ras-transformed cells in soft agar and to modify other aspects of their transformed phenotype. It has also been demonstrated that certain inhibitors of famesyl-protein transferase selectively block the processing of the Ras oncoprotein intracellularly (N. E.
  • prenyltransferase inhibitors exhibit varying degrees of inhibition of different prenyltransferases (Lerner et al. (1997) Oncogene 15:1283-1288). Some reports describe specific inhibitors of FPTase that do not inhibit GGPTase (Garcia et al. (1993) J. Biol. Chem. 268:18415-18418; Ratemi et al. (1996) J. Org. Chem. 61 :6296-6301). Conversely, inhibitors of GGPTase and not FPTase have also been reported (Macchia et al. (1996) J Med. Chem. 39:1352-1356; Lerner et al. (1995) J. Biol. Chem. 270:26770-26773).
  • prenyltransferase inhibitors have been reported. For example, it has recently been reported that famesyl-protein transferase inhibitors are inhibitors of proliferation of vascular smooth muscle cells and are therefore useful in the prevention and therapy of arteriosclerosis and diabetic disturbance of blood vessels (JP H7-112930). In addition, inhibition of protein geranylgeranylation causes a superinduction of nitric-oxide synthase-2 by interleukin- 1 -beta in vacular smooth muscle cells (Finder et al. (1997) J. Biol. Chem. 272:13484-13488).
  • abnormal proliferation and “unwanted proliferation” are interchangeable and refer to proliferation of cells which is undesired, e.g., such as may arise it due to transformation and/or immortalization of the cells, e.g., neoplastic or hyperplastic.
  • fungi and "yeast” are used interchangeably herein and refer to the art recognized group of eukaryotic protists known as fungi. That is, unless clear from the context, "yeast” as used herein can encompass the two basic morphologic forms of yeast and mold and dimorphisms thereof.
  • antimicrobial refers to the ability of the inhibitors of the invention to prevent, inhibit or destroy the growth of microbes such as bacteria, fungi, protozoa and viruses.
  • prodrug is intended to encompass compounds which, under physiological conditions, are converted into the inhibitor agents of the present invention.
  • a common method for making a prodrug is to select moieties which are hydrolyzed under physiological conditions to provide the desired biologically active drug.
  • the prodrug is converted by an enzymatic activity of the patient or alternatively of a target fungi.
  • ED 50 means the dose of a drug which produces 50% of its maximum response or effect. Alternatively, it may refer to the dose which produces a pre-determined response in 50% of test subjects or preparations.
  • LD 5 o means the dose of a drug which is lethal in 50% of test subjects.
  • therapeutic index refers to the therapeutic index of a drug defined as LD 50 /ED 50 .
  • structure-activity relationship refers to the way in which altering the molecular structure of drugs alters their interaction with a receptor, enzyme, etc.
  • acylamino is art-recognized and refers to a moiety that can be represented by the general formula:
  • R 9 is as defined above, and R'j ⁇ represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -R.8 > where m and Rg are as defined above.
  • aliphatic group refers to a straight-chain, branched-chain, or cyclic aliphatic hydrocarbon group and includes saturated and unsaturated aliphatic groups, such as an alkyl group, an alkenyl group, and an alkynyl group.
  • alkenyl and alkynyl refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double or triple bond respectively.
  • alkoxyl or "alkoxy” as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto.
  • Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like.
  • An "ether” is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O-alkenyl, -O-alkynyl, -O-(CH2) m -Rg, where m and Rg are described above.
  • alkyl refers to the radical of saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl-substituted cycloalkyl groups, and cycloalkyl-substiruted alkyl groups.
  • a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chains, C3-C30 for branched chains), and more preferably 20 or fewer.
  • preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.
  • alkyl (or “lower alkyl) as used throughout the specification, examples, and claims is intended to include both “unsubstituted alkyls” and “substituted alkyls”, the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone.
  • Such substituents can include, for example, a- halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety.
  • the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate.
  • the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF3, -CN and the like.
  • Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl- substituted alkyls, -CF3, -CN, and the like.
  • lower alkyl as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, “lower alkenyl” and “lower alkynyl” have similar chain lengths. Throughout the application, preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.
  • alkylthio refers to an alkyl group, as defined above, having a sulfur radical attached thereto.
  • the "alkylthio" moiety is represented by one of -S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2) m -Rg, wherein m and Rg are defined above.
  • Representative alkylthio groups include methylthio, ethylthio, and the like.
  • amine and “amino” are art-recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula: / 10 1 +
  • R9, R10 and R' 10 each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2) m -R8 > or R9 and RI Q taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure;
  • Rg represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and
  • m is zero or an integer in the range of 1 to 8.
  • only one of R9 or Ri 0 can be a carbonyl, e.g., R9, Ri Q and the nitrogen together do not form an imide.
  • R9 and RJ Q each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2) m -Rg.
  • alkylamine as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R9 and RI Q is an alkyl group.
  • amino is art-recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:
  • R9, RJ Q are as defined above.
  • Preferred embodiments of the amide will not include imides which may be unstable.
  • aralkyl refers to an alkyl group substituted with an aryl group (e.g., an aromatic or heteroaromatic group).
  • aryl as used herein includes 5-, 6-, and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like.
  • aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or “heteroaromatics.”
  • the aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN, or the like.
  • aryl also includes polycyclic ring systems having two or more cyclic rings in which two or more carbons are common to two adjoining rings (the rings are "fused rings") wherein at least one of the rings is aromatic, e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.
  • carrier refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon.
  • carbonyl is art-recognized and includes such moieties as can be represented by the general formula:
  • R ⁇ represents a hydrogen, an alkyl, an alkenyl, -(CH2) m -Rg or a pharmaceutically acceptable salt
  • R' ⁇ 1 represents a hydrogen, an alkyl, an alkenyl or -(CH2) m -Rg, where m and Rg are as defined above.
  • X is an oxygen and R ⁇ ⁇ or R ⁇ is not hydrogen
  • the formula represents an "ester”.
  • X is an oxygen, and R ⁇ ⁇ is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when R ⁇ ⁇ is a hydrogen, the formula represents a "carboxylic acid".
  • heteroatom as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are boron, nitrogen, oxygen, phosphorus, sulfur and selenium.
  • heterocyclyl or “heterocyclic group” refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles.
  • Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, o
  • the heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate,
  • nitro means -NO2; the term “halogen” designates - F, -Cl, -Br or -I; the term “sulfhydryl” means -SH; the term “hydroxyl” means -OH; and the term “sulfonyl” means -SO2-.
  • a phenethylazaryl portion is a subunit having a structure according to the general formula:
  • A represents a substituted or unsubstituted aryl or heteroaryl ring
  • U represents a carbon or nitrogen atom, preferably an sp -hybridized carbon atom, to which the linkage is attached
  • K represents a nitrogen-containing heteroaryl ring.
  • A represents a phenyl ring, preferably bearing from 1-3 substituents, even more preferably a disubstituted phenyl ring such as a 2,4- disubstituted phenyl ring.
  • A is a phenyl ring substituted with at least one halogen atom.
  • the phenyl ring is substituted with a halogen atom at an ortho and a para position.
  • K is a substituted or unsubstituted pyridine, imidazole, pyrrole, or triazole ring, preferably an imidazole or triazole ring.
  • K represents an unsubstituted imidazole or triazole ring linked through a nitrogen atom of the ring.
  • the phenethylazaryl portion has the formula: wherein Y represents CH or N;
  • U represents a nitrogen or carbon atom, such as an sp 3 -hybridized carbon atom, to which the linkage is attached; and R 7 represents from 0 to 5 substituents on the ring to which it is attached, preferably from 1 to 3 substitutents, independently selected from fluoro, chloro, bromo, iodo, nitro, and cyano.
  • R includes at least two halogen substituents, e.g., Cl and/or F, preferably located at an ortho and a para position on the phenyl ring.
  • R 7 consists of two halogen substituents, e.g., Cl and/or F, located at an ortho and a para position on the phenyl ring.
  • a “phosphonamidite” can be represented in the general formula:
  • Q ⁇ represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N.
  • Qi represents an S
  • the phosphoryl moiety is a "phosphorothioate”.
  • polycyclyl or “polycyclic group” refer to two or more rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining rings, e.g., the rings are "fused rings". Rings that are joined through non-adjacent atoms are termed "bridged" rings.
  • Each of the rings of the polycycle can be substituted with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.
  • substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphate, phosphonate, phosphinate, carbonyl
  • protecting group means temporary substituents which protect a potentially reactive functional group from undesired chemical transformations.
  • protecting groups include esters of carboxylic acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively.
  • the field of protecting group chemistry has been reviewed (Greene, T.W.; Wuts, P.G.M. Protective Groups in Organic Synthesis, 2 nd ed.; Wiley: New York, 1991).
  • a “selenoalkyl” refers to an alkyl group having a substituted seleno group attached thereto.
  • Exemplary “selenoethers” which may be substituted on the alkyl are selected from one of -Se-alkyl, -Se-alkenyl, -Se-alkynyl, and -Se-(CH2) m -Rg, m and Rg being defined above.
  • the term "substituted" is contemplated to include all permissible substituents of organic compounds.
  • the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds.
  • Illustrative substituents include, for example, those described herein above.
  • the permissible substituents can be one or more and the same or different for appropriate organic compounds.
  • the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.
  • substitution or “substituted with” includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
  • sulfonate is art-recognized and includes a moiety that can be represented by the general formula: 0
  • R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.
  • sulfoxido or "sulfinyl”, as used herein, refers to a moiety that can be represented by the general formula:
  • R44 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.
  • Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.
  • each expression e.g., alkyl, m, n, etc., when it occurs more than once in any structure, is intended to be independent of its definition elsewhere in the same structure.
  • triflyl, tosyl, mesyl, and nonaflyl are art-recognized and refer to trifluoromethanesulfonyl, »-toluenesulfonyl, methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively.
  • triflate, tosylate, mesylate, and nonaflate are art-recognized and refer to trifluoromethanesulfonate ester, p- toluenesulfonate ester, methanesulfonate ester, and nonafluorobutanesulfonate ester functional groups and molecules that contain said groups, respectively.
  • Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl, -toluenesulfonyl and methanesulfonyl, respectively.
  • a more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry; this list is typically presented in a table entitled Standard List of Abbreviations. The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference.
  • Certain compounds of the present invention may exist in particular geometric or stereoisomeric forms.
  • the present invention contemplates all such compounds, including cis- and trans-isomers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof, as falling within the scope of the invention.
  • Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, as well as mixtures thereof, are intended to be included in this invention.
  • a particular enantiomer of a compound of the present invention may be prepared by asymmetric synthesis, or by derivation with a chiral auxiliary, where the resulting diastereomeric mixture is separated and the auxiliary group cleaved to provide the pure desired enantiomers.
  • diastereomeric salts may be formed with an appropriate optically active acid or base, followed by resolution of the diastereomers thus formed by fractional crystallization or chromatographic means well known in the art, and subsequent recovery of the pure enantiomers.
  • Contemplated equivalents of the compounds described above include compounds which otherwise correspond thereto, and which have the same general properties thereof (e.g., the ability to inhibit hedgehog signaling), wherein one or more simple variations of substituents are made which do not adversely affect the efficacy of the compound.
  • the compounds of the present invention may be prepared by the methods illustrated in the general reaction schemes as, for example, described below, or by modifications thereof, using readily available starting materials, reagents and conventional synthesis procedures. In these reactions, it is also possible to make use of variants which are in themselves known, but are not mentioned here.
  • hydrocarbon is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom.
  • permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted.
  • the present invention makes available a novel method for inhibiting cell growth by selectively inhibiting the activity of prenyltransferases.
  • the compounds presented below, useful in the subject methods, have been divided into three sections. Variables, such as W, X, and Ri, may have different definitions and preferred substituents in different sections.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula I:
  • Y is O or S, preferably O;
  • Ri represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R 2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH2) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH2) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or two R
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • S(O)-, or -S(O)2-, or two M taken together represent substituted or unsubstituted ethene or ethyne; q represents an integer from 0 to 3; and n, individually for each occurence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor Rn includes a linkage to a phenethylazaryl moiety.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula II:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Ar represents an aryl or heteroaryl ring, e.g., a substituted or unsubstituted phenyl ring;
  • Y is O or S, preferably O;
  • R independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH2) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH2) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or two R3
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • q represents an integer from 0 to 3; and
  • n individually for each occurence, represents an integer from 0 to 10,
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-methylimidazolyl, and may, in certain embodiments wherein Q is imidazolyl, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q represents pyridyl
  • Q may be attached at the meta- position or the para-position, for example.
  • At least one occurrence of R 3 is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R 3 are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • R represents a substituent other than H, and in certain embodiments, the two substituents of the nitrogen to which R is attached are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • R 4 is absent.
  • Rj represents one substituted or unsubstituted alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group, e.g., located adjacent to the nitrogen bound to M.
  • the composition is substantially pure or enriched in the diastereomer wherein the stereocenter where i is attached has the S designation.
  • R ⁇ and R 2 are absent for all occurrences.
  • a subject compound is substantially pure or enriched in one or more diastereomers of the above-described compounds.
  • the compound is substantially pure or enriched in a diastereomer wherein the stereocenter where M is attached to the nitrogen- and sulfur-bearing substituent is R.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula III:
  • Y is O or S, preferably O;
  • Ri represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH2) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted or unsubsti
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • a substituted or unsubstituted methylene group such as -CH 2 -, -CHF-, -CHOH-, -CH
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula IV :
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Ar represents an aryl or heteroaryl ring, e.g., a substituted or unsubstituted phenyl ring;
  • Y is O or S, preferably O;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH2) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or
  • S(O)-, or -S(O) 2 -, or two M taken together represent substituted or unsubstituted ethene or ethyne;
  • q represents an integer from 0 to 3, preferably from 1-2;
  • n individually for each occurence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor R 4 includes a linkage to a phenethylazaryl moiety.
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-methylimidazolyl, and may, in certain embodiments, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q may be attached at the meta-position or the para-position, for example.
  • At least one occurrence of R is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R 3 are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R 3 are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • R 3 represents a substituent other than H, and in certain embodiments, the two substituents of the nitrogen to which R 3 is attached are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • Ar represents a substituted or unsubstited phenyl ring, e.g., phenyl, m-chlorophenyl, etc.
  • j is absent.
  • R-j represents one substituted or unsubstituted alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group, e.g., located adjacent to the nitrogen bound to M.
  • the composition is substantially pure or enriched in the diastereomer wherein the stereocenter where R 4 is attached has the S designation.
  • R ⁇ and R 2 are absent for all occurrences.
  • a subject compound is substantially pure or enriched in one or more diastereomers of the above-described compounds.
  • the compound is substantially pure or enriched in a diastereomer wherein the stereocenter where M is attached to the nitrogen- and sulfur-bearing substituent is R. 21
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula V:
  • X represents O, S, or NR , preferably NR 3 ;
  • Y is O or S, preferably O;
  • Z is H or OH;
  • R ⁇ represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R 2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH2) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • S(O)-, or -S(O) -, or two M taken together represent substituted or unsubstituted ethene or ethyne;
  • q represents an integer from 0 to 3;
  • x and y represent, independently, 0, 1, or 2;
  • n individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor t includes a linkage to a phenethylazaryl moiety.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula VI:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Ar represents an aryl or heteroaryl ring, e.g., a substituted or unsubstituted phenyl ring;
  • Y is O or S, preferably O;
  • Z is H or OH;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH2) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha
  • R 8 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • S(O)-, or -S(O) 2 -, or two M taken together represent substituted or unsubstituted ethene or ethyne;
  • q represents an integer from 0 to 3;
  • x and y represent, independently, 0, 1, or 2;
  • n individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor R 4 includes a linkage to a phenethylazaryl moiety.
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-mefhylimidazolyl, and may, in certain embodiments wherein Q is imidazolyl, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q may be attached at the meta- position or the para-position, for example.
  • at least one occurrence of R 3 is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R 3 are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R 3 are identical, e.g., both benzyl, m- chlorobenzyl, etc.
  • R represents a substituent other than H, and in certain embodiments, the two substituents of the nitrogen to which R 3 is attached are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • R-t is absent.
  • R 4 represents one substituted or unsubstituted alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group, e.g., located adjacent to the carbon bound to M.
  • the sum of x and y is two, e.g., x is 2 and y is 0, or each of x and y is one.
  • Z is OH
  • the occurrence of M bound to the carbon bearing Z preferably represents a substituted or unsubstituted methylene group.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula VII:
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • Y is O or S, preferably O;
  • Z represents H or OH
  • Ri represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R 2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • S(O)-, or -S(O) 2 -, or two M taken together represent substituted or unsubstituted ethene or ethyne;
  • q represents an integer from 0 to 3, preferably from 1-2;
  • x and y represent, independently, 0, 1, or 2;
  • n individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor R 4 includes a linkage to a phenethylazaryl moiety.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula IX:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Ar represents an aryl or heteroaryl ring, e.g., a substituted or unsubstituted phenyl ring;
  • Y is O or S, preferably O;
  • Z represents H or OH
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH2) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH2) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or two R
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • S(O)-, or -S(O) 2 -, or two M taken together represent substituted or unsubstituted ethene or ethyne;
  • q represents an integer from 0 to 3, preferably from 1-2;
  • x and y represent, independently, 0, 1, or 2;
  • n individually for each occurrence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor R 4 includes a linkage to a phenethylazaryl moiety.
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-methylimidazolyl, and may, in certain embodiments, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q may be attached at the meta-position or the para-position, for example.
  • at least one occurrence of R 3 is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R 3 are identical, e.g., both benzyl, m- chlorobenzyl, etc.
  • R 3 represents a substituent other than H, and in certain embodiments, the two substituents of the nitrogen to which R 3 is attached are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • R 4 is absent.
  • i is a substituted or unsubstituted alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group.
  • Ri and R 2 are absent for all occurrences.
  • the sum of x and y is two, e.g., x is 2 and y is 0, or each of x and y is one.
  • Z is OH
  • the occurrence of M bound to the carbon bearing Z preferably represents a substituted or unsubstituted methylene group.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula X:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Ar independently for each occurrence, represents an aryl or heteroaryl ring, e.g., a substituted or unsubstituted phenyl ring;
  • V is H or OH;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • Y is O or S, preferably O;
  • Z represents H or OH
  • Ri represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R 2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH2) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH 2 ) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • q represents an integer from 0 to 3, preferably from 1-2;
  • x and y represent, independently, 0, 1, or
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula XI:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Ar represents an aryl or heteroaryl ring, e.g., a substituted or unsubstituted phenyl ring;
  • V is H or OH
  • Y is O or S, preferably O;
  • Z represents H or OH
  • R independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH2) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural amino acid residue (e.g., an alpha-amino acid residue), or two
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • a substituted or unsubstituted methylene group such as -CH 2 -
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to the carbon bearing M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-methylimidazolyl, and may, in certain embodiments, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q represents pyridyl
  • Q may be attached at the meta- position or the para-position, for example.
  • Ar bound to the carbon bearing Q represents a substituted or unsubstituted aryl ring, such as a benzene ring.
  • Ar bound to the carbon bearing Q includes at least two aryl rings, e.g., fused (such as naphthyl), linked (such as biphenyl), or tethered (such as a diphenyl ether or diphenyl amine, etc.).
  • At least one occurrence of R 3 is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R 3 are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R 3 are identical, e.g., both benzyl, m- chlorobenzyl, etc.
  • R 3 represents a substituent other than H, and in certain embodiments, the two substituents of the nitrogen to which R is attached are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • Ar represents a substituted or unsubstituted phenyl ring, e.g., phenyl, m-chlorophenyl, etc.
  • the sum of x and y is two, e.g., x is 2 and y is 0, or each of x and y is one.
  • the occurrence of M bound to the carbon bearing Z preferably represents a substituted or unsubstituted methylene group.
  • V is OH
  • the occurrence of M bound to the carbon bearing V preferably represents a substituted or unsubstituted methylene group.
  • R 4 is absent.
  • i represents one substituted or unsubstituted alkyl, aryl, heteroaryl, aralkyl, or heteroaralkyl group, e.g., located adjacent to the carbon bound to M.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula XII:
  • Y is O or S, preferably O;
  • Ri represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH2) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g.,
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • a substituted or unsubstituted methylene group such as -CH 2 -, -CHF-, -CHOH-, -CH(Me)-
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula XIII:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Y is O or S, preferably O;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH2) n heterocyclyl (
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • S(O)-, or -S(O) 2 -, or two M taken together represent substituted or unsubstituted ethene or ethyne; q represents an integer from 0 to 3; and n, individually for each occurence, represents an integer from 0 to 10, preferably from 0 to 5, wherein preferably neither R 3 nor i includes a linkage to a phenethylazaryl moiety.
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-mefhylimidazolyl, and may, in certain embodiments wherein Q is imidazolyl, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q represents pyridyl
  • Q may be attached at the meta- position or the para-position, for example.
  • At least one occurrence of R 3 is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R 3 are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R 3 are identical, e.g., both benzyl, m-chlorobenzyl, etc.
  • an occurrence of M directly bound to the pyrrolidine ring represents NR 8 , e.g., NMe, NBn, or NH.
  • R 8 represents, for example, H or substituted or unsubstituted alkyl or aralkyl, e.g., H,
  • R 4 is absent. In certain embodiments, Rj and R 2 are absent for all occurrences.
  • a subject compound is substantially pure or enriched in one or more diastereomers of the above-described compounds.
  • the compound is substantially pure or enriched in a diastereomer wherein the stereocenter where M is attached to the ring has the designation R.
  • the compound is substantially pure or enriched in a diastereomer wherein the stereocenter where M is attached to the nitrogen- and sulfur-bearing substituent is R. In certain embodiments, both of these stereocenters are of the R designation.
  • the compound is substantially pure or enriched in an enantiomer or diastereomer wherein the stereocenter where M is attached to the ring has the designation S.
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula XIV: wherein
  • Y is O or S, preferably O;
  • Ri represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • R2 independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • X represents O, S, or NR 3 , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH2) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or un
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • a substituted or unsubstituted methylene group such as -CH 2 -, -CHF-, -CHOH-, -CH
  • the subject method can be practiced using an inhibitor of a prenyltransferase represented by the general formula XV:
  • Q represents a substituted or unsubstituted heteroaryl moiety containing at least one nitrogen atom in the ring structure, such as a pyridyl or imidazolyl ring;
  • Y is O or S, preferably O;
  • X represents O, S, or NR , preferably NR 3 ;
  • R 3 independently for each occurrence, represents H, substituted or unsubstituted lower alkyl, lower alkenyl, lower alkynyl, cycloalkyl, cycloalkylalkyl, e.g., -(CH 2 ) n cycloalkyl (e.g., substituted or unsubstituted), heterocyclyl, heterocyclylalkyl, e.g., -(CH 2 ) n heterocyclyl (e.g., substituted or unsubstituted), aryl, aralkyl, e.g., -(CH2) n aryl (e.g., substituted or unsubstituted), heteroaryl, heteroaralkyl, e.g., -(CH 2 ) n heteroaryl (e.g., substituted or unsubstituted), or a natural or unnatural
  • Rg independently for each occurrence, represents H or substituted or unsubstituted alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, heterocyclylalkyl, aralkyl, aryl, heteroaryl, heteroaralkyl;
  • a substituted or unsubstituted methylene group such as -CH 2 -, -CHF-, -CHOH-, -CH
  • Q represents substituted or unsubstituted imidazolyl, oxazolyl, pyrrolyl, pyridyl, or thiazolyl.
  • Q may be attached to M at nitrogen, or may include an alkyl or aralkyl substituent on nitrogen, e.g., methyl, benzyl, etc.
  • Q represents pyridyl, imidazolyl, or N-methylimidazolyl, and may, in certain embodiments, be attached to the subject inhibitor at the 5-position of the imidazole ring.
  • Q may be attached at the meta-position or the para-position, for example.
  • at least one occurrence of R 3 is an aralkyl group, e.g., a substituted or unsubstituted benzyl group.
  • both occurrences of R 3 are aralkyl, e.g., substituted or unsubstituted benzyl, groups.
  • R 3 may represent a benzyl group substituted with one or more halogens.
  • both occurrences of R 3 are identical, e.g., both benzyl, both m-chlorobenzyl, etc.
  • M q -NR 8 includes an amide, urea, carbamate, or amine linkage, e.g., -CH 2 NR 8 - or -
  • R 8 represents, for example, H or substituted or unsubstituted alkyl or aralkyl, e.g., H, Me, Bn, etc.
  • a subject compound is substantially pure or enriched in one or more diastereomers of the above-described compounds.
  • the compound is substantially pure or enriched in a diastereomer wherein the amine-bearing stereocenter of the pyrrolidine ring has the designation R.
  • the compound is substantially pure or enriched in a diastereomer wherein the stereocenter where M is attached to the nitrogen- and sulfur-bearing substituent is R. In certain embodiments, both of these stereocenters are of the R designation.
  • the compound is substantially pure or enriched in an enantiomer or diastereomer wherein the amine-bearing stereocenter of the pyrrolidine ring has the designation S.
  • substituents that include an aryl or heteroaryl moiety do not include a second aryl or heteroaryl moiety, e.g., such substituents are unsubstituted or substituted with one or more of a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkyl, an alkenyl, an alkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfon
  • the ability of fungal cells to transport ectopically added compounds, paricularly inhibitors of the present invention can be enhanced by conjugation of the compound with an amino acid residue or oligopeptide (preferably a dipeptide or tripeptide) which is itself taken up by the a cell in a permease-mediated transport mechanism.
  • an amino acid residue or oligopeptide preferably a dipeptide or tripeptide
  • another aspect of the invention features prenyltransferase inhibitors which include a "permease tag", e.g., which comprises an amino acid residue, dipeptide or tripeptide which facilitates permease- mediated transport of the inhibitor into the fungal pathogen.
  • Such compounds can have desirable pharmacokinetic properties due to, for example, increased bioavailability and/or increased selectivity.
  • the permease tag does not increase the cellular uptake of the inhibitor by mammalian cells to any greater degree than it does for cellular uptake by the fungal pathogen, though in the most preferred embodiments, the permease tag increases the uptake by fungal cells to a greater degree than for uptake by mammalian cells.
  • the permease tag is removed from the inhibitor as a result of its permease-mediated transport into the fungal pathogen.
  • the amino acid or oligopeptide of the permease tag includes a free N-terminal amine, or a group hydrolyzable thereto under the conditions that the pathogen is contacted with the inhibitor.
  • the permease tag facilitates permease-mediated transport by an alanine transporter of the fungal pathogen.
  • the inhibitor is derivatized at a free amine with L- alanine, or a dipeptide or tripeptide including L-alanine.
  • the L-alanine moiety is attached to the prenyltransferase inhibitor through an amide linkage through either an amine or carboxyl group of the inhibitor, and provides the complementary functionality in the permease tag.
  • the L-alanine containing permease tag is provided by derivatization of a free amine on the inhibitor with a carboxyl group on an L-alanine containing oligopeptide, with the oligopeptide providing a free amine (or a group which is hydrolyzable thereto)
  • Candida permeases are known in the art, and appropriate permease tags can be generated for facilitating uptake of the subject inhibitors by other permease-mediated mechanisms.
  • the permease tag can be selected to increase uptake of the inhibitor by any one of the following Candida permeases:
  • permease tags can be selected to increase permease-mediated uptake by a mechanism relying on a Candida homolog of any one of the following S. cerevisae permeases:
  • GNP1 high-affinity glutamine permease
  • the present invention provides pharmaceutically acceptable compositions which comprise a therapeutically effective amount of one or more compounds of the subject invention, such as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and/or diluents for use in the treatment of fungal infections.
  • the pharmaceutical compositions of the present invention may be specially formulated for administration in solid or liquid form, including those adapted for the following: (1) oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam.
  • the pharmaceutical preparations may be non-pyrogenic, i.e., do not elevate the body temperature of a patient.
  • therapeutically effective amount means that amount of a compound, material, or composition comprising an inhibitor of the subject invention which is effective for producing some desired therapeutic effect. Such therapeutic effect may result from, for example, inhibition of aberrant hyperproliferation of a cell resulting from transformation of a Ras-related gene, or alternatively, by inhibiting fungal cell wall biosynthesis.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compounds from one organ, or portion of the body, to another organ, or portion of the body.
  • a pharmaceutically acceptable material, composition or vehicle such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject compounds from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide;
  • certain embodiments of the present subject compounds may contain a basic functional group, such as amino or alkylamino, and are, thus, capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids.
  • pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic acid addition salts of such inhibitors of prenyltransferases. These salts can be prepared in situ during the final isolation and purification of the compounds of the present invention, or by separately reacting a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and isolating the salt thus formed.
  • Representative salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, napthylate, mesylate, glucoheptonate, lactobionate, and laurylsulphonate salts and the like.
  • sulfate bisulfate
  • phosphate nitrate
  • acetate valerate
  • oleate palmitate
  • stearate laurate
  • benzoate lactate
  • phosphate tosylate
  • citrate maleate
  • fumarate succinate
  • tartrate napthylate
  • mesylate glucoheptonate
  • lactobionate lactobionate
  • laurylsulphonate salts and the like See, for example,
  • the compounds of the present invention may contain one or more acidic functional groups and, thus, are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases.
  • pharmaceutically acceptable salts refers to the relatively non-toxic, inorganic and organic base addition salts of an inhibitor of prenyltransferases. These salts can likewise be prepared in situ during the final isolation and purification of the compounds of the present invention, or by separately reacting the purified compound in its free acid form with a suitable base, such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable organic primary, secondary or tertiary amine.
  • Representative alkali or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like.
  • Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like. (See, for example, Berge et al., supra)
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • water soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like
  • oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), le
  • Formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration.
  • the amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of inhibitor which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 1 percent to about ninety-nine percent of active ingredient, preferably from about 5 percent to about 70 percent, most preferably from about 10 percent to about 30 percent.
  • Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing into association an inhibitor of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • Formulations of the invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a compound of the present invention as an active ingredient.
  • An inhibitor of the present invention may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cety
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered inhibitor moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulations so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
  • compositions may be sterilized by, for example, filtration through a bacteria- retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions which can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active inhibitor(s) of the present invention, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active inhibitor.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active inhibitor.
  • Formulations of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
  • Dosage forms for the topical or transdermal administration of a compound of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
  • the ointments, pastes, creams and gels may contain, in addition to an active prenyltransferase inhibitor, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound of the present invention to the body.
  • dosage forms can be made by dissolving or dispersing th einhibitor of the present invention in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the drug across the skin. The rate of such flux can be controlled by either providing a rate controlling membrane or dispersing the compound of the present invention in a polymer matrix or gel.
  • Opthalmic formulations, eye ointments, powders, solutions and the like, are also contemplated as being within the scope ofthis invention.
  • compositions of this invention suitable for parenteral administration comprise one or more inhibitors of the invention in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and other antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • the absorption of the inhibitor in order to prolong the therapeutic effect of an inhibitor, it is desirable to slow the absorption of the inhibitor from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of abso ⁇ tion of the inhibitor then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered inhibitor form is accomplished by dissolving or suspending the inhibitor in an oil vehicle.
  • Injectable depot forms are made by forming microencapsuled matrices of the subject inhibitors in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably,
  • the preparations of the present invention may be given orally, parenterally, topically, or rectally. They are of course given by forms suitable for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administration is preferred.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal injection and infusion.
  • systemic administration means the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.
  • the prenyltransferase inhibitors useful in the subject method may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the present invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response, e.g., antifungal or anticancer activity, for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • desired therapeutic response e.g., antifungal or anticancer activity
  • the selected dosage level will depend upon a variety of factors including the activity of the particular prenyltransferase inhibitor employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular inhibitor employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a potent prenyltransferase inhibitor e.g., having an EC 5Q in the range of 1 mM to sub-nanomolar
  • a suitable daily dose of a potent prenyltransferase inhibitor will be that amount of the compound which is the lowest dose effective to produce a therapeutic effect.
  • Such an effective dose will generally depend upon the factors described above.
  • intravenous, mtracerebroventricular and subcutaneous doses of the compounds of this invention for a patient when used for the indicated antifungal effects, will range from about 0.0001 to about lOOOmg per kilogram of body weight per day, though preferably 0.5 to 300mg per kilogram.
  • the effective daily dose of the active inhibitor may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • the inhibitor agent is formulated for oral administration, as for example in the form of a solid tablet, pill, capsule, caplet or the like (collectively hereinafter “tablet") or an aqueous solution or suspension.
  • the inhibitor agent of the present invention may be, for example, an anticancer agent or an antifungal agent.
  • the tablets are preferably formulated such that the amount of inhibitor agent (or inhibitor agents) provided in 20 tablets, if taken together, would provide a dose of at least the median effective dose (ED50), e.g., the dose at which at least 50% of individuals exhibited a therapeutic affect.
  • ED50 median effective dose
  • the therapeutic effect would be a quantal effect of inhibition of fungal cell growth or protection (e.g., a statistically significant reduction in infection).
  • the tablets are formulated such that the total amount of inhibitor agent (or inhibitor agents) provided in 10, 5, 2 or 1 tablets would provide at least an ED50 dose to a patient (human or non-human mammal).
  • the amount of inhibitor agent (or inhibitor agents) provided in 20, 10, 5 or 2 tablets taken in a 24 hour time period would provide a dosage regimen providing, on average, a mean plasma level of the inhibitor agent(s) of at least the ED50 concentration (the concentration for 50% of maximal effect of, e.g., inhibiting fungal cell growth), though preferably less than 100 times the ED50, and even more preferably less than 10 or 5 times the ED50.
  • a single dose of tablets (1-20 tablets) provides about .25 mg to 1250 mg of an inhibitor agent(s).
  • the inhibitor agents can be formulated for parenteral administration, as for example, for subcutaneous, intramuscular or intravenous injection, e.g., the inhibitor agent can be provided in a sterile solution or suspension (collectively hereinafter "injectable solution").
  • injectable solution is preferably formulated such that the amount of antifungal agent (or antifungal agents) provided in a 200 cc bolus injection would provide a dose of at least the median effective dose, though preferably less than 100 times the ED50, and even more preferably less than 10 or 5 times the ED50.
  • the injectable solution is formulated such that the total amount of antifungal agent (or antifungal agents) provided in 100, 50, 25, 10, 5, 2.5, or 1 cc injections would provide an ED50 dose to a patient, and preferably less than 100 times the ED50, and even more preferably less than 10 or 5 times the ED50.
  • the amount of inhibitor agent (or inhibitor agents) provided in a total volume of lOOcc, 50, 25, 5 or 2cc to be injected at least twice in a 24 hour time period would provide a dosage regimen providing, on average, a mean plasma level of the inhibitor agent(s) of at least the ED50 concentration, though preferably less than 100 times the ED50, and even more preferably less than 10 or 5 times the ED50.
  • a single dose injection provides about .25 mg to 1250 mg of inhibitor agent.
  • the inhibitor agent may be provided in a sterile dilute solution or suspension (collectively hereinafter "i.v. injectable solution”).
  • i.v. injectable solution is preferably formulated such that the amount of inhibitor agent (or inhibitor agents) provided in a IL solution would provide a dose, if administered over 15 minutes or less, of at least the median effective dose, though preferably less than 100 times the ED50, and even more preferably less than 10 or 5 times the ED50. More preferably, the i.v.
  • injectable solution is formulated such that the total amount of inhibitor agent (or inhibitor agents) provided in IL solution administered over 60, 90, 120 or 240 minutes would provide an ED50 dose to a patient, though preferably less than 100 times the ED50, and even more preferably less than 10 or 5 times the ED50.
  • a single i.v. "bag” provides about .25 mg to 5000 mg of inhibitor agent per liter i.v. solution, more preferably .25 mg to 2500 mg, and even more preferably .25 mg to 1250 mg.
  • an inhibitor agent may be, for example, an antifungal agent or an anticancer agent.
  • the preferred antifungal agent pharmaceutical preparation would provide a dose less than the ED50 for modulation of FPTase, GGPTase, and/or other prenyltransferase activity in the host, more preferably at least 1 order of magnitude less, and more preferably at least 2, 3 or 4 orders magnitude less.
  • the preferred anticancer agent pharmaceutical preparation would provide a dose less than the ED50 for modulation of any patient's prenyltransferase other than the prenyltransferase corresponding to any oncogene that is responsible for any aberrant hyperproliferation, cancer or the like, more preferably at least 1 order of magnitude less, more preferably at least 2, 3 or 4 orders magnitude less.
  • An ED50 dose, for a human, is based on a body weight of from 10 lbs to 250 lbs, though more preferably for an adult in the range of 100 to 250 lbs.
  • Potential inhibitors may be assessed for ED50 values for any inhibition, induing for example anticancer or antifungal activity, using any of a number of well known techniques in the art.
  • the inhibitors selected for use in the subject method will be orders of magnitude better inhibitors of a prenyltransferase that prenylates the protein product of an oncogene, e.g., a particular Ras protein, as compared to other prenyltransferases that prenylate protein products expressed from nonmutated genes of the patient.
  • an oncogene e.g., a particular Ras protein
  • the inhibitors that may be selected for use in the subject method may be better inhibitors, on the order of magnitudes, of a fungal GGPTase or other prenyltransferase than a mammalian GGPTase or other prenyltransferase, and/or have greater membrane permeance through a fungal cell wall than a mammalian cell membrane.
  • compositions of matter of the present invention that are candidate inhibitors of prenyltransferase will be screened for activity in appropriate assays. Compounds that display desired characteristics in a given assay may serve as lead compounds for the discovery of more potent inhibitors.
  • GGPTase I compounds active against fungal prenyltransferases, e.g., GGPTase I, will be screened independently against mammalian prenyltransferases. Additionally, compounds against any particular mammalian prenyltransferase will be screened against other mammalian prenyltransferases. The present invention is not limited in terms of the methods relied upon for pinpointing potent inhibitors. Compounds selected based on their activity in vitro will be screened subsequently in vivo.
  • a candidate inhibitor can be tested in an assay comprising a prenylation reaction system that includes a prenyltransferase, such as FPTase, GGPTase I, and GGPTase II; a suitable protein for prenylation by the particular prenyltransferase of the assay, or a portion thereof, which serves as a prenylation target substrate; and an activated moiety to serve as the isoprenoid donor which can be covalently attached to the prenylation substrate by the prenyltransferase.
  • a prenyltransferase such as FPTase, GGPTase I, and GGPTase II
  • a suitable protein for prenylation by the particular prenyltransferase of the assay, or a portion thereof, which serves as a prenylation target substrate which serves as a prenylation target substrate
  • the level of prenylation of the target substrate brought about by the system is measured in the presence and absence of a candidate agent, and a statistically significant decrease in the level prenylation is indicative of a potential activity for the candidate agent of interest.
  • the prenyltransferase is a fungal GGPTase
  • the suitable protein for prenylation is a fungal GTPase protein or portion thereof
  • the activated moiety is a geranylgeranyl moiety.
  • the prenylation system is designed for use with mammalian GGPTase, mammalian FTPase, or fungal FTPase.
  • the level of prenylation of the target protein can be measured by determining the actual concentration of substrate:isoprenoid conjugates formed; or inferred by detecting some other quality of the target substrate affected by prenylation, including membrane localization of the target.
  • the present assay comprises an in vivo prenylation system, such as a cell able to conduct the target substrate through at least a portion of a isoprenoid conjugation pathway.
  • the present assay comprises an in vitro prenylation system in which at least the ability to transfer isoprenoids to the target protein is constituted. Still other embodiments provide assay formats which detect protein- protein interaction between the prenyltransferase and a target protein, rather than enzymatic activity er se. Cell-free Assay Formats
  • a reaction mixture is generated to include a polypeptide for prenylation, such as Ras or other protein having GTPase-activity, candidate inhibitor(s) of interest, and a polypeptide having prenylation activity, such as FPTase, GGPTase I, or GGPTase II or a protion thereof retaining enzymatic activity.
  • a polypeptide for prenylation such as Ras or other protein having GTPase-activity
  • candidate inhibitor(s) of interest such as FPTase, GGPTase I, or GGPTase II or a protion thereof retaining enzymatic activity.
  • Detection and quantification of the enzymatic conversion of the polypeptide for prenylation or the formation of complexes containing the polypeptide for prenylation and the polypeptide having prenylation activity provide a means for determining a compound's efficacy at inhibiting (or potentiating) the complex bioactivity of any prenyltransferase.
  • the efficacy of the compound can be assessed by generating dose response curves from data obtained using various concentrations of the test compound.
  • a control assay may also be performed to provide a baseline for comparison.
  • the subject drug screening assay comprises a prenylation system, e.g., a reaction mixture which enzymatically conjugates isoprenoids to a target protein, which is arranged to detect inhibitors of the prenylation of a Rho-like GTPase.
  • a prenylation system e.g., a reaction mixture which enzymatically conjugates isoprenoids to a target protein, which is arranged to detect inhibitors of the prenylation of a Rho-like GTPase.
  • a prenylation system e.g., a reaction mixture which enzymatically conjugates isoprenoids to a target protein, which is arranged to detect inhibitors of the prenylation of a Rho-like GTPase.
  • a cell-free prenylation system one or more cell lysates including a prenyltransferase, a Rho-like GTPase (or substrate analog thereof), and an activate
  • Lysates can be derived from cells expressing one or more of the relevant proteins, and mixed appropriately (or split) where no single lysate contains all the components necessary for generating the prenylation system.
  • one or more of the components, especially the substrate target are recombinantly produced in a cell used to generate a lysate, or added by spiking a lysate mixture with a purified or semi-purified preparation of the substrate.
  • the prenyltransferase is either fungal or mammalian FTPase, GGPTase I, or GGPTase II. In other preferred embodiments, the prenyltransferase is fungal GGPTase.
  • the prenylation systems can be derived from any number of cell types, ranging from bacterial cells to yeast cells to cells from metazoan organisms including insects and mammalian cells.
  • a fungal prenylation system can be reconstituted by mixing cell lysates derived from insect cells expressing prenyltransferase subunits cloned into baculoviral expression vectors.
  • the exemplary GGPTase-I expression vectors described below can be recloned into baculoviral vectors (e.g., pVL vectors), and recombinant GGPTase-I produced in transfected Spodoptera fungiperda cells.
  • the level of activity can be assessed by enzymatic activity, or by quantitating the level of expression by detecting, e.g., an exogenous tag added to the recombinant protein.
  • Substrate and activated geranylgeranyl diphosphate can be added to the lysate mixtures.
  • the transfected cells can be cells which lack an endogenous GGPTase activity, or the substrate can be chosen to be particularly sensitive to prenylation by the exogenous fungal GGPTase relative to any endogenous activity of the cells. In other embodiments, other prenyltransferases are employed.
  • the prenylation system comprises a reconstituted protein mixture of at least semi-purified proteins.
  • semi- purified it is meant that the proteins utilized in the reconstituted mixture have been previously separated from other cellular proteins.
  • the proteins involved in conjugation of geranylgeranyl moieties to a target protein, together with the target protein are present in the mixture to at least 50% purity relative to all other proteins in the mixture, and more preferably are present at 90-95% purity.
  • the reconstituted protein mixture is derived by mixing highly purified proteins such that the reconstituted mixture substantially lacks other proteins which might interfere with or otherwise alter the ability to measure specific prenylation rates of the target GTPase substrate.
  • prenylation systems derived from purified proteins may have certain advantages over cell lysate based assays. Unlike the reconstituted protein system, the prenylation activity of a cell-lysate may not be readily controlled. Measuring kinetic parameters is made tedious by the fact that cell lysates may be inconsistent from batch to batch, with potentially significant variation between preparations. In vitro evidence indicates that prenyltransferases have the ability to cross-prenylate CAAX-related sequences, so that prenyltransferase not of interest present in a lysate may provide an unwanted kinetic parameter.
  • GDI guanine nucleotide dissociation inhibitor
  • the purified protein mixture includes a purified preparation of the substrate polypeptide and a isoprenoid moiety (or analog thereof) under conditions which drive the conjugation of the two molecules.
  • the mixture can include a fungal GGPTase I complex including RAM2 and CDC43 subunits, a geranylgeranyl diphosphate, a divalent cation, and a substrate polypeptide, such as may be derived from Rhol.
  • Prenylation of the target regulatory protein via an in vitro prenylation system in the presence and absence of a candidate inhibitor, can be accomplished in any vessel suitable for containing the reactants. Examples include microtitre plates, test tubes, and micro-centrifuge tubes. In such embodiments, a wide range of detection means can be practiced to score for the presence of the prenylated protein.
  • the products of a prenylation system are separated by gel electrophoresis, and the level of prenylated substrate polypeptide assessed, using standard electrophoresis protocols, by measuring an increase in molecular weight of the target substrate that corresponds to the addition of one or more isoprenoid moieties.
  • one or both of the target substrate and isoprenoid group can be labeled with a radioisotope such as 35 S, , C, or 3 H, and the isotopically labeled protein bands quantified by autoradiographic techniques.
  • Standardization of the assay samples can be accomplished, for instance, by adding known quantities of labeled proteins which are not themselves subject to prenylation or degradation under the conditions which the assay is performed.
  • other means of detecting electrophoretically separated proteins can be employed to quantify the level of prenylation of the target substrate, including immunoblot analysis using antibodies specific for either the target substrate or isoprenoid epitopes.
  • one embodiment of the present assay comprises the use of a biotinylated target substrate in the conjugating system.
  • a biotinylated target substrate in the conjugating system.
  • biotinylated GGPTase substrates have been described in the art (c.f. Yokoyama et al. (1995) Biochemistry 34:1344-1354).
  • the biotin label is detected in a gel during a subsequent detection step by contacting the electrophoretic products (or a blot thereof) with a streptavidin-conjugated label, such as a streptavidin linked fluorochrome or enzyme, which can be readily detected by conventional techniques.
  • a reconstituted protein mixture is used (rather than a lysate) as the conjugating system
  • prenylated and unprenylated substrate can be separated by other chromatographic techniques, and the relative quantities of each determined.
  • HPLC can be used to quantitate prenylated and unprenylated substrate (Pickett et al. (1995) Analytical Biochem 225:60-63), and the effect of a test compound on that ratio determined.
  • an immunoassay or similar binding assay is used to detect and quantify the level of prenylated target substrate produced in the prenylation system.
  • Many different immunoassay techniques are amenable for such use and can be employed to detect and quantitate the conjugates.
  • the wells of a microtitre plate (or other suitable solid phase) can be coated with an antibody which specifically binds one of either the target substrate or isoprenoid groups. After incubation of the prenylation system with and without the candidate agent, the products are contacted with the matrix bound antibody, unbound material removed by washing, and prenylated conjugates of the target substrate specifically detected.
  • a detectable anti-isoprenoid antibody can be used to score for the presence of prenylated target substrate on the matrix.
  • the prenylation substrate can be immobilized throughout the reaction, such as by cross-linking to activated polymer, or sequestered to the well walls after the development of the prenylation reaction.
  • a Rho-like GTPase e.g., a fungal Rhol , Rho2, Cdc42 or Rsrl/Budl
  • a Rho-like GTPase is cross-linked to the polymeric support of the well, the prenylation system set up in that well, and after completion, the well washed and the amount of geranylgeranyl sidechains attached to the immobilized GTPase detected.
  • wells of a microtitre plate are coated with streptavidin and contacted with a developed prenylation system under conditions wherein a biotinylated substrate binds to and is sequestered in the wells. Unbound material is washed from the wells, and the level of prenylated target substrate is detected in each well.
  • a variety of techniques for detecting the level of prenylation of the immobilized substrate For example, by the use of dansylated (described infra) or radiolabelled isoprenoid moieties in the reaction mixture, addition of appropriate scintillant to the wells will permit detection of the label directly in the microtitre wells.
  • the substrate can be released and detected, for example, by any of those means described above, e.g., by radiolabel, gel electrophoresis, etc.
  • Reversibly bound substrate such as the biotin- conjugated substrate set out above, is particularly amenable to the latter approach.
  • only the isoprenoid moiety is released for detection.
  • the thioether linkage of the isoprenoid with the substrate peptide sequence can be cleaved by treatment with methyl iodide.
  • the released isoprenoid products can be detected, e.g., by radioactivity, HPLC, or other convenient format.
  • Other isoprenoid derivatives include detectable labels which do not interfere greatly with the conjugation of that group to the target substrate.
  • the assay format provides fluorescence assay which relies on a change in fluorescent activity of a group associated with a prenyltransferase substrate to assess test compounds against a prenyltransferase.
  • prenylation activity of any prenyltransferase may be measured by a modified version of the continuous fluorescence assay described for farnesyl transferases (Cassidy et al, (1985) Methods Enzymol. 250: 30-43; Pickett et al. (1995) Analytical Biochem 225:60-63; and Stirtan et al. (1995) Arch Biochem Biophys 321 : 182-190).
  • dansyl-Gly-Cys-Ile-Ile-Leu d-GCIIL
  • geranylgeranyl diphosphate are added to assay buffer, along with the test agent or control.
  • This mixture is preincubated at 30 °C for a few minutes before the reaction is initiated with the addition of GGPTase enzyme.
  • the sample is vigorously mixed, and an aliquot of the reaction mixture immediately transferred to a prewarmed cuvette, and the fluorescence intensity measured for 5 minutes.
  • Useful excitation and emission wavelengths are 340 and 486 nm, respectively, with a bandpass of 5.1 nm for both excitation and emission monochromators.
  • fluorescence data are collected with a selected time increment, and the inhibitory activity of the test agent is determined by detecting a decrease in the initial velocity of the reaction relative to samples which lack a test agent.
  • the prenyltransferase activity against a particular substrate can be detected in the subject assay by using a phosphocellulose paper absorption system (Roskoski et al. (1994) Analytical Biochem 222:275-280), or the like.
  • a phosphocellulose paper absorption system Rosham et al. (1994) Analytical Biochem 222:275-280
  • several basic residues can be added, preferably to the amino-terminal side of the target sequence of the peptide, to produce a peptide with a minimal minimum charge of +2 or +3 at pH less than 2. This follows the strategy used for the phosphocellulose absorption assay for protein kinases.
  • the transfer of a [H 3 ] isoprenoid group from [H 3 ]-isoprenoid pyrophosphate to acceptor peptides can be measured under conditions similar to the farnesyl transferase reactions described by Reiss et al (Reiss et al, (1990) Cell 62: 81-88).
  • the transfer of the [H 3 ] geranylgeranyl group from [H 3 ] -geranylgeranyl pyrophosphate to KLKCAIL can be measured.
  • Reaction mixtures can be generated to contain 50 mM Tris-HCL (pH 7.5), 50 ⁇ M ZnCl 2 , 20 mM KC1, 1 mM dithiothreitol, 250 ⁇ M KLKCAIL, 0.4 ⁇ M [H 3 ] geranylgeranyl pyrophosphate, and 10-1000 ⁇ g/ml of purified fungal GGPTase protein. After incubation, e.g., for 30 minutes at 37 °C, samples are applied to Whatman P81 phosphocellulose paper strips.
  • the strips are washed in ethanol/phosphoric acid (prepared by mixing equal volumes of 95% ethanol and 75 mM phosphoric acid) to remove unbound isoprenoids.
  • ethanol/phosphoric acid prepared by mixing equal volumes of 95% ethanol and 75 mM phosphoric acid
  • the samples are air dried, and radioactivity can be measured by liquid scintillation spectrometry. Background values are obtained by using reaction mixture with buffer in place of enzyme. An added feature of this strategy is that it produces hydrophilic peptides that are more readily dissolved in water.
  • compounds for use in the subject method can be detected using a screening assay derived to include a whole cell expressing a GTPase protein, e.g., Ras, along with a prenyltransferase, e.g., FTPase, GGPTase I, and GGPTase II.
  • the reagent cell is a mammalian cell that has been engineered to express one or more of these proteins from mammalian recombinant genes.
  • the reagent cell is a fungal cell that has been engineered to express one or more of these proteins from fungal recombinant genes.
  • the reagent cell may be manipulated so that the recombinant gene(s) complement a loss-of-function mutation to the homologous gene in the reagent cell.
  • the reagent cell is a non-pathogenic cell which has been engineered to express one or more of these proteins from recombinant genes cloned from a pathogenic fungus.
  • non-pathogenic fungal cells such as S. cerevisae
  • a non-pathogenic yeast cell is engineered to express a Rho-like GTPase, e.g., Rhol, and at least one of the subunits of a GGPTase, e.g., RAM2 and/or Cdc43, derived from a fungal protein.
  • reagent cells One salient feature to such reagent cells is the ability of the practitioner to work with a non-pathogenic strain rather than the pathogen itself. Another advantage derives from the level of knowledge, and available strains, when working with such reagent cells as S. cerevisae.
  • compounds for use in the subject method can be detected using a screening assay derived to include a whole cell expressing a substrate for a prenyltransferase, e.g., a GTPase protein, along with a prenyltransferase.
  • the reagent cell is a mammalian cell which has been engineered to express one or more of these proteins from recombinant mammalian genes.
  • the reagent cell is a non-fungal cell which has been engineered to express one or more of these proteins from recombinant mammalian genes.
  • the reagent cell is a non-pathogenic cell which has been engineered to express one or more of these proteins from recombinant genes cloned from a pathogenic fungus.
  • non-pathogenic fungal cells such as S. cerevisae
  • a non-pathogenic yeast cell is engineered to express a Rho- like GTPase, e.g., Rhol, and at least one of the subunits of a GGPTase, e.g., RAM2 and/or Cdc43, derived from a fungal protein.
  • the reagent cell may be manipulated such that the recombinant gene(s) complement a loss-of-function mutation to the homologous gene in the reagent cell.
  • test agent to alter the activity of a prenyltransferase may be detected by analysis of the cell or products produced by the cell.
  • agonists and antagonists of the GTPase biological activity can be detected by scoring for alterations in growth or viability of the cell.
  • Other embodiments will permit inference of the level of GTPase activity based on, for example, detecting expression of a reporter, the induction of which is directly or indirectly dependent on the activity of a Rho-like GTPase.
  • General techniques for detecting each are well known, and will vary with respect to the source of the particular reagent cell utilized in any given assay.
  • quantification of proliferation of cells in the presence and absence of a candidate agent can be measured with a number of techniques well known in the art, including simple measurement of population growth curves.
  • turbidimetric techniques i.e., absorption/transmission of light of a given wavelength through the sample
  • measurement of abso ⁇ tion of light at a wavelength between 540 and 600 nm can provide a conveniently fast measure of cell growth.
  • ability to form colonies in solid medium e.g., agar
  • solid medium e.g., agar
  • a GTPase substrate protein such as a histone
  • a fusion protein which permits the substrate to be isolated from cell lysates and the degree of acetylation detected.
  • GTPase has been affected by the added agent.
  • the ability of an agent to create a lytic phenotype which is mediated in some way by a recombinant GTPase protein can be assessed by visual microscopy.
  • test agent on reagent cell can be assessed by measuring levels of expression of specific genes, e.g., by reverse transcription-PCR.
  • Another method of scoring for effect on protein activity of interest, e.g., GTPase is by detecting cell-type specific marker expression through immunofluorescent staining. Many such markers are known in the art, and antibodies are readily available.
  • the target cell in order to enhance detection of cell lysis for fungal inhibitors, can be provided with a cytoplasmic reporter which is readily detectable, either because it has "leaked” outside the cell, or substrate has “leaked” into the cell, by perturbations in the cell wall.
  • Preferred reporters are proteins which can be recombinantly expressed by the target cell, do not interfere with cell wall integrity, and which have an enzymatic activity for which chromogenic or fluorogenic substrates are available.
  • a fungal cell can be constructed to recombinantly express the ⁇ -galactosidase gene from a construct (optionally) including an inducible promoter.
  • ⁇ -galactosidase activity can be scored using such colorimetric substrates as 5-bromo-4-chloro-3-indolyl- ⁇ -D- galactopyranoside or fluorescent substrates such as methylumbelliferyl- ⁇ -D- galactopyranoside.
  • the membrane localization resulting from prenylation of a GTPase can be exploited to generate the cell-based assay.
  • the subject assay can be derived with a reagent cell having: (i) a reporter gene construct including a transcriptional regulatory element which can induce expression of the reporter upon interaction of the transcriptional regulatory protein portion of the above fusion protein.
  • a gal 4 protein can be fused with a Rhol polypeptide sequence which includes the CAAX prenylation target.
  • prenylation of the fusion protein will result in partitioning of the fusion protein at the cell surface membrane. This provides a basal level of expression of the reporter gene construct.
  • partitioning is lost and, with the concomitant increase in nuclear concentration of the protein, expression from the reporter construct is increased.
  • the cell is engineered such that inhibition by fungal inhibitors of the GGPTase activity does not result in cell lysis.
  • fungal inhibitors of the GGPTase activity does not result in cell lysis.
  • mutation of the C- terminus of Rhol and cdc42 can provide proteins which are targets of farsenyl transferase rather than geranylgeranyl transferase.
  • such mutants can be used to render the GGPTase I activity dispensable.
  • GGPTase substrate/transcription factor fusion protein in such cells as YOT35953 cells (Ohya et al., vide supra) generates a cell whose viability vis- ⁇ -vis the GGPTase activity is determined by the reporter construct, if at all, rather than by prenylation of an endogenous Rho-like GTPase by the GGPTase.
  • the reporter gene product can be derived to have no effect on cell viability, providing for example another type of detectable marker (described, infra).
  • Such cells can be engineered to express an exogenous GGPTase activity in place of an endogenous activity, or can rely on the endogenous activity.
  • the Call mutant YOT35953 cell can be further manipulated to express a Call homolog from, e.g., a fungal pathogen or a mammalian cell.
  • the leakage assay can be utilized to detect expression of the reporter protein.
  • the reporter gene may encode ⁇ -galactosidase, and inhibition of the GGPTases activity scored for by the presence of cells which take up substrate due to loss of cell wall integrity, and convert substrate due to the expression of the reporter gene.
  • the reporter gene is a gene whose expression causes a phenotypic change which is screenable or selectable. If the change is selectable, the phenotypic change creates a difference in the growth or survival rate between cells which express the reporter gene and those which do not.
  • the phenotype change creates a difference in some detectable characteristic of the cells, by which the cells which express the marker may be distinguished from those which do not.
  • the marker gene is coupled to GTPase-dependent activity, be it membrane association, or a downstream signaling pathway induced by a GTPase complex, so that expression of the marker gene is dependent on the activity of the GTPase. This coupling may be achieved by operably linking the marker gene to a promoter responsive to the therapeutically targeted event.
  • GTPase-responsive promoter indicates a promoter which is regulated by some product or activity of the fungal GTPase.
  • transcriptional regulatory sequences responsive to signals generated by PKC/GTPase, GS/GTPase and/or other GTPase complexes, or to signals by other proteins in such complexes which are interrupted by GTPase binding can be used to detect function of Rho-like GTPases such as Rhol and cdc42.
  • suitable positively selectable (beneficial) genes include the following:
  • suitable positively selectable (beneficial) genes include the following: URA3, LYS2, HIS3, LEU2, TRP1; ADE1, 2, 3, 4, 5, 7,
  • IGP dehydratase imidazoleglycerol phosphate dehydratase gene
  • HIS3 imidazoleglycerol phosphate dehydratase gene
  • HIS3 is preferred because it is both quite sensitive and can be selected over a broad range of expression levels.
  • the cell is auxotrophic for histidine (requires histidine for growth) in the absence of activation. Activation of the gene leads to synthesis of the enzyme and the cell becomes prototrophic for histidine (does not require histidine). Thus the selection is for growth in the absence of histidine. Since only a few molecules per cell of IGP dehydratase are required for histidine prototrophy, the assay is very sensitive.
  • the marker gene may also be a screenable gene.
  • the screened characteristic may be a change in cell mo ⁇ hology, metabolism or other screenable features.
  • Suitable markers include beta-galactosidase (Xgal, C 1 FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)), alkaline phosphatase, horseradish peroxidase, exo-glucanase (product of yeast exbl gene; nonessential, secreted); luciferase; bacterial green fluorescent protein; (human placental) secreted alkaline phosphatase (SEAP); and chloramphenicol transferase (CAT).
  • a preferred screenable marker gene is ⁇ -galactosidase; for in yeast cells, for example, expression of the enzyme converts the colorless substrate Xgal into a blue pigment.
  • Call-1 cells or the like e.g., impaired for certain prenyltransferase activities, are suitable for use in assays to detect GS inhibitors, as such cells are more sensitive to the effects of GS inhibitors.
  • the benefits to enhanced sensitivity include speedier development of assay readouts, and the further prejudicing of the assay towards GS inhibitors rather than other targets which may not provide cytotoxicity.
  • the latter can provide the ability to identify potential hits which may not themselves be potent GS inhibitors, but which can be manipulated, e.g., by combinatorial chemistry approaches, to provide potent and specific GS inhibitors.
  • yet another embodiment of the subject assay utilizes a side-by-side comparison of the effect of a test agent on (i) a cell which prenylates a Rho-like GTPase by adding geranylgeranyl moieties, and (ii) a cell which prenylates an equivalent Rho-like GTPase by adding farnesyl moieties.
  • the assay makes use of the ability to suppress GGPTase I defects in yeast by altering the C-terminal tail of Rhol and cdc42 to become substrate targets of farnesyl transferase (see Ohya et al., supra).
  • the assay is arranged by providing a yeast cell in which the target Rho-like GTPases is prenylated by a GGPTase activity of the cell. Both the GGPTase and GTPase can be endogenous to the "test" cell, or one or both can be recombinantly expressed in the cell. The level of prenylation of the GTPase is detected, e.g., cell lysis or other means described above.
  • test compound to inhibit the addition of geranylgeranyl groups to the GTPase in the first cell is compared against the ability of test compound to inhibit the farnesylation of the GTPase in a control cell.
  • the "control" cell is preferably identical to the test cell, with the exception that the targeted GTPase(s) are mutated at their CAAX sequence to become substrates for FPTases rather than GGPTases.
  • Agents which inhibit prenylation in the test cell but not the control cell are selected as potential antifungal agents. Such differential screens can beakily sensitive to inhibitors of GGPTase I prenylation of Rho-like GTPases.
  • the test cell is derived from the S.
  • cerivisae cell YOT35953 (Ohya et al., supra) or the like which is defective in GGPTase subunit cdc43.
  • the cell is then engineered with a cdc43 subunit from a fungal pathogen such as Candida albicans to generate the test cell, and additionally with the mutated Rho-like GTPases to generate the control cell.
  • assays can be used to identify compounds that have favorable therapeutic indexes.
  • anticancer agents can be identified by the present assays which inhibit particular prenyltransferases, and thereby may treating conditions resulting from mutations in the specific gene encoding that prenyltransferase.
  • antifungal agents can be identified by the present assays which inhibit proliferation of yeast cells or other lower eukaryotes, but which have a substantially reduced effect on mammalian cells, thereby improving therapeutic index of the drug as an anti-mycotic agent.
  • differential screening assays can be used to exploit the difference in protein interactions and/or catalytic mechanism of different prenyltransferases in order to identify agents which display a statistically significant increase in specificity for inhibiting certain prenylation reactions relative to others.
  • lead compounds which act specifically on the certain prenylation reactions can be developed.
  • differential screening assays can be used to exploit the difference in protein interactions and/or catalytic mechanism of mammalian and fungal GGPTases in order to identify agents which display a statistically significant increase in specificity for inhibiting the fungal prenylation reaction relative to the mammalian prenylation reaction.
  • lead compounds which act specifically on the prenylation reaction in pathogens, such as fungus involved in mycotic infections can be developed.
  • the present assays can be used to screen for agents which may ultimately be useful for inhibiting the growth of at least one fungus implicated in such mycosis as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinosporidosis, nocaidiosis, para-actinomycosis, penicilliosis, monoliasis, or sporotrichosis.
  • mycosis as candidiasis, aspergillosis, mucormycosis, blastomycosis, geotrichosis, cryptococcosis, chromoblastomycosis, coccidioidomycosis, conidiosporosis, histoplasmosis, maduromycosis, rhinospor
  • the present assay can comprise comparing the relative effectiveness of a test compound on inhibiting the prenylation of a mammalian GTPase protein with its effectiveness towards inhibiting the prenylation of a GTPase from a yeast selected from the group consisting of Candida albicans, Candida stellatoidea, Candida glabrata, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida pseudotropicalis, Candida guilliermondii, or Candida rugosa.
  • the present assay can be used to identify antifungal agents which may have therapeutic value in the treatment of aspergillosis by selectively targeting, relative to human cells, GTPase homologs from yeast such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus.
  • yeast such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus.
  • the GTPase system to be screened can be derived from yeast such as Rhizopus arrhizus, Rhizopus oryzae, Absidia corymbifera, Absidia ramosa, or Mucor pusillus.
  • Sources of other assay reagents for includes the pathogen Pneumocystis carinii.
  • Cyanogen bromide e.g., DCM, DMF, THF, toluene.
  • Base neutral solvents (e.g., DCM, toluene, THF).
  • NH2NHR NH2NHR
  • neutral solvents e.g., DCM, DMF, THF, toluene
  • H2NOH neutral solvents (e.g., DCM, DMF, THF, toluene).
  • neutral solvents e.g., DCM, DMF, THF, toluene
  • Carbonyldiimidazole neutral solvents (e.g., DCM, DMF, THF, toluene).
  • simple turbidimetric assays e.g., measuring the Agoo of a culture
  • spotting compounds on fungal lawns can be used to screen a library of the subject compounds for those having inhibitory activity toward a particular fungal strain.
  • a combinatorial library for the pu ⁇ oses of the present invention is a mixture of chemically related compounds which may be screened together for a desired property.
  • the preparation of many related compounds in a single reaction greatly reduces and simplifies the number of screening processes which need to be carried out. Screening for the appropriate physical properties can be done by conventional methods.
  • the substrate aryl groups used in the combinatorial reactions can be diverse in terms of the core aryl moiety, e.g., a variegation in terms of the ring structure, and/or can be varied with respect to the other substituents.
  • a library of candidate antifungal diversomers can be synthesized utilizing a scheme adapted to the techniques described in the Still et al. PCT publication WO 94/08051, e.g., being linked to a polymer bead by a hydrolyzable or photolyzable group e.g., located at one of the positions of the candidate antifungals or a substituent of a synthetic intermediate.
  • the library is synthesized on a set of beads, each bead including a set of tags identifying the particular diversomer on that bead.
  • the bead library can then be "plated" on a lawn of fungi for which an inhibitor is sought.
  • the diversomers can be released from the bead, e.g., by hydrolysis. Beads surrounded by areas of no, or diminished, fungal growth, e.g., a "halo", can be selected, and their tags can be "read” to establish the identity of the particular diversomer.
  • MS mass spectrometry
  • the libraries of the subject method can take the multipin library format.
  • Geysen and co-workers (Geysen et al. (1984) PNAS 81 :3998-4002) introduced a method for generating compound libraries by a parallel synthesis on polyacrylic acid-grated polyethylene pins arrayed in the microtitre plate format.
  • the Geysen technique can be used to synthesize and screen thousands of compounds per week using the multipin method, and the tethered compounds may be reused in many assays.
  • Appropriate linker moieties can also been appended to the pins so that the compounds may be cleaved from the supports after synthesis for assessment of purity and further evaluation (c.fi, Bray et al.
  • a variegated library of compounds can be provided on a set of beads utilizing the strategy of divide-couple-recombine (see, for example, Houghten (1985) PNAS 82:5131-5135; and U.S. Patents 4,631,211; 5,440,016; 5,480,971).
  • the beads are divided into separate groups equal to the number of different substituents to be added at a particular position in the library, the different substituents coupled in separate reactions, and the beads recombined into one pool for the next iteration.
  • the divide-couple-recombine strategy can be carried out using an analogous approach to the so-called "tea bag” method first developed by Houghten, where compound synthesis occurs on resin sealed inside porous polypropylene bags (Houghten et al. (1986) PNAS 82:5131-5135). Substituents are coupled to the compound-bearing resins by placing the bags in appropriate reaction solutions, while all common steps such as resin washing and deprotection are performed simultaneously in one reaction vessel. At the end of the synthesis, each bag contains a single compound.
  • a scheme of combinatorial synthesis in which the identity of a compound is given by its locations on a synthesis substrate is termed a spatially addressable synthesis.
  • the combinatorial process is carried out by controlling the addition of a chemical reagent to specific locations on a solid support (Dower et al. (1991) Annu Rep Med Chem 26:271-280; Fodor, S.P.A. (1991) Science 251 :767; Pirrung et al. (1992) U.S. Patent No. 5,143,854; Jacobs et al (1994) Trends Biotechnol 12:19-26).
  • the spatial resolution of photolithography affords miniaturization. This technique can be carried out through the use protection/deprotection reactions with photolabile protecting groups.
  • a synthesis substrate is prepared for coupling through the covalent attachment of photolabile nitroveratryloxycarbonyl (NVOC) protected amino linkers or other photolabile linkers.
  • Light is used to selectively activate a specified region of the synthesis support for coupling. Removal of the photolabile protecting groups by light (deprotection) results in activation of selected areas. After activation, the first of a set of amino acid analogs, each bearing a photolabile protecting group on the amino terminus, is exposed to the entire surface. Coupling only occurs in regions that were addressed by light in the preceding step. The reaction is stopped, the plates washed, and the substrate is again illuminated through a second mask, activating a different region for reaction with a second protected building block.
  • NVOC photolabile nitroveratryloxycarbonyl
  • the pattern of masks and the sequence of reactants define the products and their locations. Since this process utilizes photolithography techniques, the number of compounds that can be synthesized is limited only by the number of synthesis sites that can be addressed with appropriate resolution. The position of each compound is precisely known; hence, its interactions with other molecules can be directly assessed. In a light-directed chemical synthesis, the products depend on the pattern of illumination and on the order of addition of reactants. By varying the lithographic patterns, many different sets of test compounds can be synthesized simultaneously; this characteristic leads to the generation of many different masking strategies.
  • the subject method utilizes a compound library provided with an encoded tagging system.
  • a recent improvement in the identification of active compounds from combinatorial libraries employs chemical indexing systems using tags that uniquely encode the reaction steps a given bead has undergone and, by inference, the structure it carries.
  • this approach mimics phage display libraries, where activity derives from expressed peptides, but the structures of the active peptides are deduced from the corresponding genomic DNA sequence.
  • the first encoding of synthetic combinatorial libraries employed DNA as the code.
  • a variety of other forms of encoding have been reported, including encoding with sequenceable bio-oligomers (e.g., oligonucleotides and peptides), and binary encoding with additional non-sequenceable tags.
  • a combinatorial library of nominally 7 7 ( 823,543) peptides composed of all combinations of Arg, Gin, Phe, Lys, Val, D-Val and Thr (three-letter amino acid code), each of which was encoded by a specific dinucleotide (TA, TC, CT, AT, TT, CA and AC, respectively), was prepared by a series of alternating rounds of peptide and oligonucleotide synthesis on solid support.
  • the amine linking functionality on the bead was specifically differentiated toward peptide or oligonucleotide synthesis by simultaneously preincubating the beads with reagents that generate protected OH groups for oligonucleotide synthesis and protected NH2 groups for peptide synthesis (here, in a ratio of 1 :20).
  • the tags each consisted of 69-mers, 14 units of which carried the code.
  • the bead-bound library was incubated with a fluorescently labeled antibody, and beads containing bound antibody that fluoresced strongly were harvested by fluorescence- activated cell sorting (FACS).
  • FACS fluorescence- activated cell sorting
  • compound libraries can be derived for use in the subject method, where the oligonucleotide sequence of the tag identifies the sequential combinatorial reactions that a particular bead underwent, and therefore provides the identity of the compound on the bead.
  • oligonucleotide tags permits extremelyly sensitive tag analysis. Even so, the method requires careful choice of orthogonal sets of protecting groups required for alternating co-synthesis of the tag and the library member. Furthermore, the chemical lability of the tag, particularly the phosphate and sugar anomeric linkages, may limit the choice of reagents and conditions that can be employed for the synthesis of non-oligomeric libraries.
  • the libraries employ linkers permitting selective detachment of the test compound library member for assay.
  • Peptides have also been employed as tagging molecules for combinatorial libraries. Two exemplary approaches are described in the art, both of which employ branched linkers to solid phase upon which coding and ligand strands are alternately elaborated. In the first approach (Kerr et al. (1993) JACS 115:2529-2531), orthogonality in synthesis is achieved by employing acid-labile protection for the coding strand and base-labile protection for the compound strand.
  • branched linkers are employed so that the coding unit and the test compound can both be attached to the same functional group on the resin.
  • a cleavable linker can be placed between the branch point and the bead so that cleavage releases a molecule containing both code and the compound (Ptek et al. (1991) Tetrahedron Lett 32:3891-3894).
  • the cleavable linker can be placed so that the test compound can be selectively separated from the bead, leaving the code behind. This last construct is particularly valuable because it permits screening of the test compound without potential interference of the coding groups. Examples in the art of independent cleavage and sequencing of peptide library members and their corresponding tags has confirmed that the tags can accurately predict the peptide structure.
  • An alternative form of encoding the test compound library employs a set of non-sequencable electrophoric tagging molecules that are used as a binary code (Ohlmeyer et al (1993) PNAS 90:10922-10926).
  • Exemplary tags are haloaromatic alkyl ethers that are detectable as their trimethylsilyl ethers at less than femtomolar levels by electron capture gas chromatography (ECGC). Variations in the length of the alkyl chain, as well as the nature and position of the aromatic halide substituents, permit the synthesis of at least 40 such tags, which in principle can encode 2 40 (e.g., upwards of 10 12 ) different molecules.
  • Both libraries were constructed using an orthogonal attachment strategy in which the library member was linked to the solid support by a photolabile linker and the tags were attached through a linker cleavable only by vigorous oxidation. Because the library members can be repetitively partially photoeluted from the solid support, library members can be utilized in multiple assays. Successive photoelution also permits a very high throughput iterative screening strategy: first, multiple beads are placed in 96-well microtiter plates; second, compounds are partially detached and transferred to assay plates; third, a metal binding assay identifies the active wells; fourth, the corresponding beads are rearrayed singly into new microtiter plates; fifth, single active compounds are identified; and sixth, the structures are decoded.
  • the structures of the compounds useful in the present invention lend themselves readily to efficient synthesis.
  • the nature of the structures, as generally described by formula I, allows the combinatorial assembly of subject inhibitors as shown in an exemplary scheme below. Many suitable reactions, including those depicted below, are both mild and reliable, and are thus well suited for combinatorial chemistry.
  • the nature of such a combinatorial approach towards the generation of a library of test compounds is apparent in the exemplary scheme below, wherein main subunits are linked combinatorially (e.g., using one of the methods described above), with potential combinatorial functionalization of the subunits bestowing additional diversity on the library.
  • the isocyanate or chloroformate could be replaced by an isothiocyanate, R 3 XS(O)Cl, R 3 XS(O) 2 Cl, R 3 XCH 2 Br or another electrophilic reagent.
  • X is N
  • a second R group may be attached, e.g., by deprotonating the amide and treating the resulting anion with a 'soft' electrophile, e.g., benzyl iodide, benzyl bromide, etc.
  • the piperazine ring may be treated with phosgene or an equivalent thereof to form a formamyl chloride (NCOC1), which can react with a thiol, amine, or alcohol to form the corresponding urea, urethane, or thiocarbamate.
  • NOC1 formamyl chloride
  • QM q X (where X represents a leaving group) may, for example, be a sulfonyl chloride, an acyl chloride, an isocyanate, in isothiocyanate, a chloroformate, an alkyl bromide, or another electrophilic reagent.
  • the group QM q may be attached by a reductive animation reaction, a palladium-mediated aryl (or heteroaryl) amine coupling, or any other suitable reaction.
  • a cysteine-like residue, or a protected variant thereof may be employed to access derivatives of Formulas I and III.
  • Analogous strategies may be employed to prepare compounds of the other structures, which differ primarily in the structure of the starting core.
  • the reaction was partitioned between ethyl acetate (30 mL) and saturated aqueous sodium bicarbonate (40 mL). A second ethyl acetate extraction was carried out on the aqueous layer. The ethyl acetate layers were combined and washed with brine (2 x 20 mL). The ethyl acetate layers were dried with MgSO 4 , filtered and concentrated. Purification was carried out by flash chromatography (100% CH 2 C1 2 , followed by 5% MeOH:CH 2 Cl 2 then 10% MeOH: CH 2 C1 2 ).
  • Trimethylsulfoxonium iodide (0.3 g, 0.00134 mol) was dissolved in anhydrous dimethyl sulfoxide (9.0 mL) and kept under a nitrogen atmosphere.
  • Sodium hydride (60% in mineral oil, 0.05 g, 0.00134 mol) was added to the DMSO solution.
  • the ylide was made over a period of two hours at room temperature.
  • Compound 5 was dissolved in a minimum of anhydrous DMSO and added via syringe to the ylide solution. The mixture was stirred at room temperature for 18 hours.
  • the DMSO solution was partitioned between ethyl acetate (30 mL) and 10%) citric acid (50 mL).
  • Compound 12 Compound 11 (2.32 g, 0.006 mol) was dissolved in 1,2- dichloroethane (60 mL). 1 -Methyl- l-H-imidazole-5-carboxaldehyde (0.68 g, 0.0062 mol) was added followed by glacial acetic acid (0.69 mL). Sodium triacetoxyborohydride (2.54 g, 0.0062 mol) was added last. The suspension was stirred for 18 hours. The reaction mixture was stripped of volatile substances and the residue was partitioned between ethyl acetate (50 mL) and 1.0 N NaOH (100 mL).
  • GGPTase I inhibitors To look at the effect of GGPTase I inhibitors in vivo, a recombinant C albicans strain engineered to express a Myc tagged CaRHOl under the control of the C. albicans PCK1 promoter is used. This promoter is repressed by glucose and derepressed by gluconeogenic carbon sources such as succinate. It should also be possible to be look at the endogenous substrates of the GGPTase I. Cells are treated with a sublethal dose of compound for a period of time which has been established from a kill curve analysis in the appropriate media.
  • MycCaRHOl In mock treated cells, MycCaRHOl should be absent from the cytosolic fraction, whereas in GGPTase I inhibitor treated cells, some MycCaRHOl should be apparent in the cytosolic fraction indicating that a proportion of the newly synthesized MycCaRHOl has not been geranylgeranylated. Figure 32 shows that this prediction is borne out.
  • the 5' and 3' non-coding regions of CaRHOl were generated by PCR and cloned into pBluescript KS- in which the CaRHOl ORF was exactly replaced with a BamHI site.
  • pSCaRHO1.5c23 a PCKl.CaURA3 cassette was inserted from pSCaPCK1.3cl to generate pSCaRHO1.19cl.
  • This vector was mutagenised to destroy one of the two BamHI sites (pSCaRHO1.22c22) into which the Myc tagged CaRHOl ORF (from pSCaRHO1.20c58) was inserted.
  • the sequence of the oligos used to generate the Myc tagged CaRHOl ORF are:
  • the sequence of the Myc tag is underlined and corresponds to the amino acid sequence EQKLISEEDL.
  • This epitope is recognized by the commercially available 9E10 monoclonal antibody.
  • the final vector designated pSCaRHO1.23c21 harbours of the 5' non-coding region of CaRHOl, the CaURA3 selectable marker, the C. albicans PCK1 promoter directing the expression of the Myc-tagged CaRHOl and the 3' untranslated region of CaRHOl.
  • the presence of the CaRHOl 5' and 3' regions should direct this cassette to one of the 2 WT alleles of CaRHOl by homologous recombination.
  • the PCKl -MycCaRHOl replacement construct was excised by a BssHII digest from the parent plasmid pSCaRHO1.23c21.
  • the desired fragment was gel purified prior to being transformed into the C. albicans strain CAF3-1.
  • the method used for CAF3-1 transformation is a lithium acetate protocol (from U. of Minnesota C. ablicans web site: http://alces.med.umn.edu/ candida/liac.html).
  • the transformation mixture is then plated onto selective (-Ura glucose) plates and incubated at 30 °C for 3 days. Individual transformants that appear are restreaked for singles and then preserved as a glycerol stock. To ensure that the correct integrative event has occurred, southern analysis was carried out on several colonies. Those colonies that exhibited the correct genotype were retained.
  • the strain used for the work described here is referred to as DIY-BL2-O58.
  • Cells of strain DIY-BL2-058 were grown overnight in YNB supplemented with 1 ⁇ g/ml- histidine, 2 ⁇ g/ml methionine, 2 ⁇ g/ml tryptophan, 200 ⁇ g/ml glutamine and 2% glucose at 220 ⁇ m at 31 °C . The cell number was then determined, cells were pelleted by centrifugation and resuspended in fresh media at a density of lxl 0 7 cells/ml and incubated as above. Cells were either treated with 14 ⁇ l DMSO alone or 14 ⁇ l of a 25.6 mg/ml stock of inhibitor in DMSO (3 ⁇ g/ml final concentration).
  • lOx TE supplemented with a protease inhibitors cocktail was added at 3-4 volumes of the pellet size (about 200 ⁇ l) and glass beads (425-600 microns; Sigma) were added to the meniscus. This mixture was then subjected to 5 1' pulses in a bead beater with 2' on ice between pulses. The mixture was then centrifuged at 3000 ⁇ m to pellet cellular debris and the supernatent removed. The beads were washed with an equal volume of buffer and the supernatent added to the initial sample. This whole cell extract (WCE) was again centrifuged at 3000 ⁇ m and the supernatent removed into a fresh tube.
  • WCE whole cell extract
  • Fractions were thawed on ice. The protein concentration was determined using the standard Bradford method for the WCEs and cytosolic fraction. 30 ⁇ g of protein were loaded for both the WCE and cytosolic fractions. For the membrane fraction, a volume equal to that loaded for the cytosolic fraction was loaded. Prior to loading, all fractions were boiled for 3' with loading dye. Standard procedures were employed for the SDS. PAGE and Western blotting.
  • the blot was pre-blocked with 4% fat free milk in PBST.
  • the 9E10 monoclonal anti-myc epitope antibody (available from Calbiochem) was incubated with the blot overnight at 4 °C at a concentration recommended by the manufacturers. The primary antibody was removed and the blot was washed 3x 15' with PBST. The blot is then incubated with 2° antibody which was goat anti-mouse HRP conjugated antibody for 1 hr at room temperature. The 2° antibody is removed and the blot washed again with 3x 15' with PBST and developed using the Pierce luminescent kit according to the manufacturers instructions.
  • the dilution series for each of the compounds may now be prepared in sequence: For each compound - start with highest dilution. Add 10 ⁇ L compound in DMSO to the 490 ⁇ L of appropriate media. Immediately vortex and add 100 ⁇ L to the appropriate row of cells on the 96-well plate. Repeat this process for the next and subsequent concentrations of this compound before starting on the dilution series for additional compounds.
  • the minimum fungicidal concentration can then be determined by plating out the entire contents of the well of the microtitre plates onto YPD or Sabourand plates. These plates are then incubated at 35 °C for 24-48 hrs.
  • the MFC corresponds to the concentration of compound where no cellular growth is observed on the plate.
  • (D) Cells are exposed to drug for 7 days for the IMR90 Cell Line, and a period of 3 days for the H460 Cell Line.
  • IMR90 Cells have 3- ⁇ 4,5-Dimethylthiazol-2-yl ⁇ -2,5- diphenyltetrazolium bromide (MTT) added to them for three hours prior to final read out. After the three hours, media and MTT are removed and
  • MTT crystals are solubilized in 100% DMSO for final OD read. All of the references and publications cited herein and U.S. Application No. 09/182,845 are hereby inco ⁇ orated by reference.

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Abstract

La présente invention concerne en partie des compositions et des méthodes permettant d'inhiber des prényltransférases.
PCT/US2002/038511 2001-12-03 2002-12-03 Compositions et methodes permettant d'inhiber des prenyltransferases Ceased WO2003047569A1 (fr)

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US7261742B2 (en) 2005-10-13 2007-08-28 S.C. Johnson & Son, Inc. Method of deodorizing a textile
US7598249B2 (en) 2004-12-30 2009-10-06 Janssen Pharmaceutica N.V. Piperazinyl and piperidinyl ureas as modulators of fatty acid amide hydrolase
WO2010068453A1 (fr) 2008-11-25 2010-06-17 Janssen Pharmaceutica Nv Modulateurs d'urée substitués par hétéroaryle d'amide d'acide gras hydrolase
WO2010141809A1 (fr) 2009-06-05 2010-12-09 Janssen Pharmaceutica Nv Modulateurs heterocycliques a base d'uree a substitution aryle de l'hydrolase des amides d'acides gras (faah)
WO2010141817A1 (fr) 2009-06-05 2010-12-09 Janssen Pharmaceutica Nv Modulateurs d'amide d'acide gras hydrolase de type diamine urée spirocyclique substituée par un groupe hétéroaryle
JP2011505405A (ja) * 2007-12-03 2011-02-24 ノバルティス アーゲー 高脂血症または動脈硬化症のような疾患の処置に有用なｃｅｔｐ阻害剤としての１，２−二置換−４−ベンジルアミノ−ピロリジン誘導体
US8598356B2 (en) 2008-11-25 2013-12-03 Janssen Pharmaceutica Nv Heteroaryl-substituted urea modulators of fatty acid amide hydrolase
US8940745B2 (en) 2010-05-03 2015-01-27 Janssen Pharmaceutica Nv Modulators of fatty acid amide hydrolase

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WO2000051611A1 (fr) * 1999-03-03 2000-09-08 Merck & Co., Inc. Inhibiteurs de la prenyle-proteine transferase

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WO2000051611A1 (fr) * 1999-03-03 2000-09-08 Merck & Co., Inc. Inhibiteurs de la prenyle-proteine transferase
US6358956B1 (en) * 1999-03-03 2002-03-19 Merck & Co., Inc. Inhibitors of prenyl-protein transferase

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7598249B2 (en) 2004-12-30 2009-10-06 Janssen Pharmaceutica N.V. Piperazinyl and piperidinyl ureas as modulators of fatty acid amide hydrolase
US9169224B2 (en) 2004-12-30 2015-10-27 Janssen Pharmaceutica Nv Piperazinyl and piperidinyl ureas as modulators of fatty acid amide hydrolase
US8530476B2 (en) 2004-12-30 2013-09-10 Janssen Pharmaceutica Nv Piperazinyl and piperidinyl ureas as modulators of fatty acid amide hydrolase
US7407515B2 (en) 2005-10-13 2008-08-05 S.C. Johnson & Son, Inc. Method of deodorizing a textile
US7261742B2 (en) 2005-10-13 2007-08-28 S.C. Johnson & Son, Inc. Method of deodorizing a textile
JP2011505405A (ja) * 2007-12-03 2011-02-24 ノバルティス アーゲー 高脂血症または動脈硬化症のような疾患の処置に有用なｃｅｔｐ阻害剤としての１，２−二置換−４−ベンジルアミノ−ピロリジン誘導体
US8759365B2 (en) 2007-12-03 2014-06-24 Novartis Ag Organic compounds
US8877769B2 (en) 2008-11-25 2014-11-04 Janseen Pharmaceutica Nv Heteroaryl-substituted urea modulators of fatty acid amide hydrolase
WO2010068453A1 (fr) 2008-11-25 2010-06-17 Janssen Pharmaceutica Nv Modulateurs d'urée substitués par hétéroaryle d'amide d'acide gras hydrolase
US8461159B2 (en) 2008-11-25 2013-06-11 Jannsen Pharmaceutica BV Heteroaryl-substituted urea modulators of fatty acid amide hydrolase
US8598356B2 (en) 2008-11-25 2013-12-03 Janssen Pharmaceutica Nv Heteroaryl-substituted urea modulators of fatty acid amide hydrolase
WO2010141809A1 (fr) 2009-06-05 2010-12-09 Janssen Pharmaceutica Nv Modulateurs heterocycliques a base d'uree a substitution aryle de l'hydrolase des amides d'acides gras (faah)
US8901111B2 (en) 2009-06-05 2014-12-02 Janssen Pharmaceutica Nv Aryl-substituted heterocyclic urea modulators of fatty acid amide hydrolase
WO2010141817A1 (fr) 2009-06-05 2010-12-09 Janssen Pharmaceutica Nv Modulateurs d'amide d'acide gras hydrolase de type diamine urée spirocyclique substituée par un groupe hétéroaryle
US8940745B2 (en) 2010-05-03 2015-01-27 Janssen Pharmaceutica Nv Modulators of fatty acid amide hydrolase
US9688664B2 (en) 2010-05-03 2017-06-27 Janssen Pharmaceutica Nv Modulators of fatty acid amide hydrolase

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