WO1996021456A1 - Inhibiteurs des prenyle transferases - Google Patents

Inhibiteurs des prenyle transferases Download PDF

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Publication number
WO1996021456A1
WO1996021456A1 PCT/US1996/001559 US9601559W WO9621456A1 WO 1996021456 A1 WO1996021456 A1 WO 1996021456A1 US 9601559 W US9601559 W US 9601559W WO 9621456 A1 WO9621456 A1 WO 9621456A1
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Prior art keywords
acid
group
amino
compound
ras
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PCT/US1996/001559
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English (en)
Inventor
Said Sebti
Andrew Hamilton
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University Of Pittsburgh
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Priority claimed from US08/371,682 external-priority patent/US5705686A/en
Priority claimed from US08/451,839 external-priority patent/US5834434A/en
Priority claimed from US08/582,076 external-priority patent/US6011175A/en
Application filed by University Of Pittsburgh filed Critical University Of Pittsburgh
Priority to JP52188496A priority Critical patent/JP3929069B2/ja
Priority to CA2207252A priority patent/CA2207252C/fr
Priority to EP96905380A priority patent/EP0794789A4/fr
Priority to AU49157/96A priority patent/AU4915796A/en
Publication of WO1996021456A1 publication Critical patent/WO1996021456A1/fr

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    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/57—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups
    • C07C323/58—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton
    • C07C323/59—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being further substituted by nitrogen atoms, not being part of nitro or nitroso groups with amino groups bound to the carbon skeleton with acylated amino groups bound to the carbon skeleton
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    • C07C323/50—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton
    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/60—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton with the carbon atom of at least one of the carboxyl groups bound to nitrogen atoms
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    • C07D207/30—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 two double bonds between ring members or between ring members and non-ring members
    • C07D207/34—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 two 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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    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms 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
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    • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
    • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
    • C07D263/30—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D263/34—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having two or three 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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    • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
    • C07D277/20—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D277/32—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having two or three 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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    • C07D307/00—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
    • C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three 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
    • C07D307/66—Nitrogen atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel peptidomimetics and other compounds which are useful as inhibitors of protein isoprenyl
  • Ras proteins are plasma membrane-associated GTPases that function as relay switches that transduce biological information from
  • src-homology 2 SH2 domains of several signaling proteins.
  • SH2 src-homology 2
  • m-SOS-1 a cytosolic complex of GRB-2 and a ras exchanger
  • Raf mitogen-activated protein
  • ERK extracellular-regulated protein kinase
  • MEK extracellular-regulated protein kinase
  • Ras In addition to its inability to hydrolyze GTP, oncogenic Ras must be plasma membrane-bound to cause malignant transformation (13). Ras is posttranslationally modified by a lipid group, farnesyl, which mediates its association with the plasma membrane (10-14).
  • the p21ras proteins are first made as pro-p21ras in the cytosol where they are modified on cysteine 186 of their carboxyl
  • p21Ras farnesyltransferase the enzyme responsible for catalyzing the transfer of
  • farnesyl a 15-carbon isoprenoid, from FPP to the cysteine of the CA 1 A 2 X carboxyl terminus of p21ras
  • the enzyme is a heterodimer composed of ⁇ and ⁇ subunits of molecular weights 49 and 46 kDa, respectively (17).
  • the ⁇ subunit has been shown to bind p21ras (17). Because p21ras farnesylation and subsequent membrane association are required for p21ras transforming activity (13), it has been proposed that p21ras
  • CA 1 A 2 X peptides with the greatest inhibitory activity are those where A 1 and A 2 are hydrophobic peptides with charged or hydrophilic residues in the central positions demonstrating very little inhibitory activity (18,21,23).
  • EPA 0520823 A2 discloses compounds which are useful in the inhibition of farnesyl-protein transferase and the farnesylation of the oncogene protein ras.
  • the compounds of EPA discloses compounds which are useful in the inhibition of farnesyl-protein transferase and the farnesylation of the oncogene protein ras.
  • the compounds of EPA discloses compounds which are useful in the inhibition of farnesyl-protein transferase and the farnesylation of the oncogene protein ras.
  • Cys is a cysteine amino acid
  • Xaa 1 is an amino acid in natural L-isomer form
  • dXaa2 is an amino acid in unnatural D-isomer form
  • Xaa 3 is an amino acid in natural L-isomer form.
  • EPA 0523873 A1 discloses a modification of the compounds of EPA 0520823 A2 wherein Xaa 3 is phenylalanine or p-fluorophenylalanine.
  • EPA 0461869 describes compounds which inhibit farnesylation of Ras protein of the formula:
  • Aaa 1 and Aaa 2 are aliphatic amino acids and Xaa is an amino acid.
  • the aliphatic amino acids which are disclosed are Ala, Val, Leu and lie.
  • Preferred compounds are those where Aaa 1 is Val, Aaa 2 is Leu, Ile or Val and Xaa is Ser or Met.
  • Preferred specific compounds are:
  • WO 92/18465 discloses certain farnesyl compounds which inhibit the enzymatic methylation of proteins including ras proteins.
  • EPA 0456180 A1 is directed to a
  • farnesylprotein transferase assay which can be used to identify substances that block
  • GGTase I farnesyltransferase
  • FTase I geranylgeranyltransferase I
  • FTase I attaches the lipid geranylgeranyl to the cysteine of the CAAX box of proteins where X is leucine (49,69).
  • FTase and GGTase I are ⁇ / ⁇ heterodimers that share the ⁇ subunit (61,62).
  • Cross-linking experiments suggested that both substrates (FPP and Ras CAAX) interact with the ⁇ submit of FTase (17,63).
  • GGTase I prefers leucine at the X position, its substrate specificity was shown to overlap with that of FTase in vi tro (64).
  • GGTase I is also able to transfer farnesyl to a leucine terminating peptide (65).
  • CAAX peptides are potent competitive inhibitors of FTase, rapid degradation and low cellular uptake limit their use as therapeutic agents.
  • the stragegy of the present invention to develop superior compounds for inhibiting FTase and GGTase has been to replace several amino acids in the CAAX motif by peptidemimics.
  • the rationale behind this strategy is based on the existance of a hydrophobic pocket at the enzyme active site that interacts with the hydrophobic "AA" dipeptide of the carboxyl termini CAAX of Ras molecules.
  • two very potent inhibitors of FTase i.e. Cys-3AMBA-Met and Cys-4ABA-Met
  • the peptidomimetic Cys-4ABA-Met incorporates a hydrophobic/aromatic spacer (i.e. 4-aminobenzoic acid) between Cys and Met.
  • the present application discloses several derivatives of Cys-4ABA-Met where positions 2 and 3 of 4-amino benzoic acid were modified by several alkyl, and/or aromatic groups, compounds that show great promise of ability to selectively antagonize RAS-dependent signaling and to selectively inhibit the growth of human tumors with aberrant Ras function.
  • K4B-Ras (also called K-Ras4B) is the most important Ras proteins expressed by mammalian cells.
  • K-Ras4B can be geranylgeranylated in vitro, but with relatively low efficiency; its K m for GGTase I is 7 times higher than its K m for FTase (67).
  • GGTase I CAAX-based inhibitors that can block
  • K-Ras4B may be geranylgeranylated, and that therefore inhibitors targeted at GGTase I would be effective in
  • aryl-L 2 - wherein L 2 is absent, -CH 2 -, -CH 2 CH 2 -, -CH(CH 3 )-, -O-, -S(O) q wherein q is 0, 1, or 2, -N(R')- wherein R' is hydrogen or lower alkyl, or -C(O)- and aryl is selected from the group consisting of phenyl, naphthyl,
  • heterocyclic is a monocyclic heterocyclic wherein the heterocyclic is unsubstituted or substituted with one, two, or three substituents independently selected from the group consisting of loweralkyl, hydroxy,
  • R 1a is hydrogen or lower alkyl
  • R 2 ' is
  • R 12a is hydrogen, loweralkyl or -C(O)O-R 13 , wherein R 13 is hydrogen or a carboxy-protecting group and R 12b is hydrogen or loweralkyl, with the proviso that R 12a and R 12b are not both hydrogen, ii) -C(O)NH-CH(R 14 )-C(O)OR 15 wherein R 14 is
  • R 15 is hydrogen or a carboxy-protect ing group
  • R 3 ' is
  • A represents O or 2H
  • R 0 represents SH, NH 2 , or C x H y -SO 2 -NH-, wherein C x H y is a straight chain saturated or unsaturated hydrocarbon, with x being between 1 and 20 and y between 3 and 41, inclusive; and L 1 is -NH-;
  • An important embodiment of the present invention is based on the finding that a novel group of peptidomimetics as represented by Formula (I) have a high inhibitory potency against human tumor p21ras farnesyltransferase and inhibit tumor growth of human carcinomas: C ⁇ X (I) where
  • C stands for the cysteine radical, or for the reduced form of the cysteine radical (R-2-amino-3-mercaptopropyl amine); ⁇ is the radical of a non- peptide aminoalkyl- or amino-substituted phenyl carboxylic acid; and X is the radical of an amino acid, preferably Met. Any other natural or synthetic amino acid can also be used at this position.
  • the invention also includes
  • a particularly preferred compound in this regard is:
  • cysteine radical is in the reduced form and the spacer group is 2-phenyl-4-aminobenzoic acid.
  • Another preferred compound of the invention is:
  • the compounds of Formula (I) are different from the prior art farnesyltransferase inhibitors in that they do not include separate peptide amino acids A 1 , A 2 as in prior art inhibitors represented by the formula CA 1 A 2 X.
  • the present compounds are consequently free from peptidic amide bonds.
  • the present compounds are not farnesylated by the enzyme.
  • a further important feature of the invention is the provision of the compounds of Formula (I) in the form of pro-drugs. Broadly speaking, this is accomplished by functionalizing the terminal end groups (amino, cysteine sulfur and carboxy groups) of the compounds with hydrophobic, enzyme-sensitive moieties which serve to increase the plasma membrane permeability and cellular uptake of the compounds and consequently their efficiency in inhibiting tumor cell growth.
  • prodrugs for amino and cysteine sulfur groups can include loweralkycarbonyl, arylcarbony,
  • arylalkylcarbony alkoxycarbonyl, aryloxycarbonyl, cycloalkylcarbonyk, cycloalkoxycarbonyl, and other groups well known to those skilled in the art.
  • a particularly preferred compound of the invention is the methylester form of FTI-276, which is illustrated in Figure 1A.
  • inhibitors CA 1 A 2 X as well as any other peptide with potential for biological uses for the purpose of improving the overall effectiveness of such
  • a further modification involves the provision of CA 1 A 2 X tetrapeptides or C ⁇ X peptidomimetics which have been modified by functionalizing the sulfhydryl group of the cysteine C with an alkyl phosphonate substituent, as hereinafter described.
  • Another important embodiment of the invention contemplates replacing the A 1 A 2 X portion of the CA 1 A 2 X tetrapeptide inhibitors with a non-amino acid component while retaining the desired
  • C cysteine or reduced cysteine and ⁇ represents an aryl or heterocyclic substituent such as 3-aminomethyl-biphenyl-3'-carboxylic acid, which does not include a peptide amino acid but corresponds essentially in size with A 1 A 2 X, as hereinafter described.
  • ⁇ represents an aryl or heterocyclic substituent such as 3-aminomethyl-biphenyl-3'-carboxylic acid, which does not include a peptide amino acid but corresponds essentially in size with A 1 A 2 X, as hereinafter described.
  • the invention includes pharmaceutically acceptable salts and prodrugs of Formula (II).
  • the invention also includes compounds in which further substitutions have been made at the cysteine position. These compounds comprise free cysteine thiol and/or terminal amino groups at one end and include a carboxylic acid or carboxylate group at the other end, the carboxylic acid or carboxylate group being separated from the
  • cysteine thiol and/or terminal amino group by a hydrophobic spacer moiety which is free from any linking amido group as in prior CAAX mimetics.
  • these compounds are not subject to proteolytic
  • the compounds selectively inhibit FTase both in vitro and in vivo and offer a number of other advantages over prior CAAX peptide mimetics.
  • A represents O or 2H
  • R 0 represents SH, NH 2 , or C x H y -SO 2 -NH-, wherein C x H y is a straight chain saturated or unsaturated hydrocarbon, with x being between 1 and 20 and y between 3 and 41, inclusive; and
  • B stands for -NHR, where R is an aryl group.
  • the invention also includes pharmaceutically
  • R is a biphenyl substituted with one or more -COOH groups and/or lower alkyl, e.g., methyl, as represented by the formula:
  • R 1 and R 3 represent H or COOH;
  • R 2 represents H, COOH, CH 3 , or COOCH 3 ;
  • R 4 represents H or OCH 3 ;
  • A represents 2H or O.
  • This formula represents a series of 4-amino-3'-carboxybiphenyl derivatives which mimic the Val-Ile-Met tripeptide but have restricted conformational flexibility. Reduction of the cysteine amide bond (where A is H,H) provides a completely non-peptidic Ras CAAX mimetic.
  • R is a biphenyl group with a -COOH substitution in the 3'- or 4'-position, most preferably the 3'-position, with respect to the NH-aryl group.
  • the -COOH substituent may appear as such or in pharmaceutically acceptable salt or ester form, e.g., as the alkali metal salt or methyl ester.
  • CAAX tetrapeptide known as CVIM (see EP 0461869 and U.S. Patent 5,141,851) and C-4ABA-M. These compounds are, respectively, Cys-Val-Ile-Met and Cys-4 aminobenzoic acid-Met where Cys is the cysteine radical and Met is the methionine radical.
  • a preferred non-peptide CAAX mimetic of the invention is reduced cys-4-amino-3'-biphenylcarboxylate identified as 4 in Figure 12, which is also designated FTI-265. This derivative contains no amide bonds and thus is a true non-peptide mimic of the CAAX tetrapeptide.
  • the compounds of the invention may be used in the carboxylic acid form or as pharmaceutically acceptable salts or esters thereof.
  • Lower alkyl esters are preferred although other ester forms, e.g., phenyl esters, may also be used.
  • cysteine moiety may be replaced by reduced cysteine, or by other functional groups as hereinafter disclosed.
  • An important embodiment of the present invention is based on the finding that a novel group of peptidomimetics as represented by Formula (IV) have a high inhibitory potency against geranylgeranyl transferase and disrupt oncogenic K-Ras4B processing and signalling:
  • C stands for the cysteine radical, or for the reduced form of the cysteine radical (R-2-amino-3-mercaptopropyl amine); ⁇ is the radical of a non- peptide aminoalkyl- or amino-substituted phenyl carboxylic acid; and L is the radical of leucine or isoleucine.
  • the invention also includes pharmaceutically acceptable salts and prodrugs of the compounds of Formula (IV).
  • Preferred compounds of this embodiment are derivatives of Cys-4ABA-Leu which are substituted at the 2 and/or 3 positions of the phenyl ring of 4-aminobenzoic acid (4ABA).
  • the substitutions at these positions include, but are not limited to alkyl, alkoxy and aryl (particularly to straight chain or branched groups of 1-10 carbons of the aforementioned) and naphthyl, heterocyclic rings and heteroaromatic rings.
  • GGTI-287 A particularly preferred compound of this aspect of the invention, GGTI-287, is illustrated in Figure 17.
  • the cysteine radical is in the reduced form and the spacer group is 2-phenyl-4-aminobenzoic acid.
  • Another preferred compound, also shown in Figure 17, is GGTI-297, which contains the spacer group 2-naphthyl-4-aminobenzoic acid.
  • Other spacer groups which will be readily evident as useful are described herein in connection with farnesyltransferase inhibitors.
  • pro-drug is meant a compound to which in vivo modification occurs to produce the active compound. Such compounds may, for example, be more readily delivered to their sites of action as pro-drugs.
  • the pro-drugs of the instant invention are produced by functionalizing the terminal end groups (amino, cysteine sulfur and carboxy groups) of the compounds with hydrophobic, enzyme-sensitive moieties which serve to increase the plasma membrane permeability and cellular uptake of the compounds and consequently their efficiency in inhibiting tumor cell growth.
  • a particularly preferred compound of the invention is the methylester form of GGTI-287, GGTI-286, also illustrated in Figure 17.
  • the compounds of the invention may be used in the same manner as prior CAAX tetrapeptide
  • compounds which inhibit geranylgeranyl transferase may be used in the treatment of cancer which is related to K-Ras4B.
  • the compounds of the invention may be used in pharmaceutical compositions of conventional form suitable for oral, subcutaneous, intravenous, intraperitoneal or intramuscular administration to a mammal or host.
  • pharmaceutical compositions of conventional form suitable for oral, subcutaneous, intravenous, intraperitoneal or intramuscular administration to a mammal or host This includes, for example, tablets or capsules, sterile solutions or
  • suspensions comprising one or more compounds of the invention with a pharmaceutically acceptable carrier and with or without other additives.
  • aqueous suspensions may be used with conventional suspending agents, flavoring agents and the like.
  • the amount of inhibitor administered to obtain the desired inhibitory effect will vary but can be readily determined. It is expected that the compounds of the present invention will be administered to humans or other mammals as
  • compositions may be administered via methods well known in the pharmaceutical and medical arts, which include, but are not limited to oral, parenteral, topical, and respiratory (inhalation) routes.
  • Pharmaceutical preparations may contain suitable carriers or diluents.
  • carboxy protecting group refers to a carboxylic acid protecting ester group employed to block or protect the carboxylic acid functionally while the reactions involving other functional sites of the compound are carried out.
  • Carboxy protecting groups are disclosed in Greene, "Protective Groups in Organic Synthesis", pp. 152-186 (1981), which is hereby incorporated herein by reference.
  • a carboxy protecting group can be used as a prodrug whereby the carboxy protecting group can be readily cleaved in vivo, for example by enzymatic hydrolysis, to release the biologically active parent.
  • a comprehensive discussion of the prodrug concept is provided by T. Higuchi and V. Stella in "Prodrugs as Novel Delivery Systems", vol. 14 of the ACS Symposium Series, American Chemical
  • cephalosporin fields as described in U.S. Pat. No. 3,840,556 and 3,719,667, the disclosures of which are hereby incorporated by reference.
  • esters useful as prodrugs for example, esters useful as prodrugs for
  • carboxy protecting groups are C1 to C8 loweralkyl (e.g. methyl, ethyl or tertiary butyl and the like); arylalkyl, for example, phenethyl or benzyl and substituted drivatives thereof, for example 5-indanyl and the like; dialkylaminoalkyl (e.g.
  • alkanoyloxyalkyl groups such as acetoxymethol, butyryloxymethyl, valeryloxymethyl, isobutyryloxymethyl,
  • cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl
  • arylalkylcarbonyloxyalkyl such as
  • alkoxycarbonyloxyalkyl or cycloalkyloxycarbonylalkyl such as
  • aryloxycarbonyloxyalkyl such as 2- (phenoxycarbonyloxy)ethyl, 2 -(5-indanyloxycarbonyloxy)ethyl and the like;
  • alkoxyalkylcarbonyloxyalkyl such as 2-(1-methoxy-2-methylpropan-2-oyloxy) ethyl and the like;
  • arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl and the like;
  • arylalkenyloxycarbonyloxyalkyl such as 2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like
  • alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and the like
  • alkylaminocarbonylaminoalkyl such as
  • alkanoylaminoalkyl such as acetylaminomethyl and the like
  • heterocycliccarbonyloxyalkyl such as 4-methylpiperazinylcarbonyloxymethyl and the like
  • dialkylaminocarbonylalkyl such as
  • (5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as (5-t-butyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like
  • (5-phenyl-2-oxo-1, 3-dioxolen-4-yl)alkyl such as (5-phenyl-2-oxo-1,3-dioxolen-4-yl)methyl and the like.
  • Preferred carboxy-protected compounds of the invention are compounds wherein the protected carboxy group is a loweralkyl, cycloalkyl or arylalkyl ester, for example, methyl ester, ethyl ester, propyl ester, isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester and the like or an
  • alkanoyloxyalkyl cycloalkanoyloxyalkyl
  • N-protecting group or “N-protected” as used herein refers to those groups intended to protect the N-terminus of an amino acid or peptide or to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” (John Wiley & Sons, New York (1981)), which is hereby incorporated herein by reference.
  • alkanoyl refers to R 29 C(O)-O- wherein R 29 is a loweralkyl group.
  • alkanoylaminoalkyl refers to a loweralkyl radical to which is
  • R 71 is an alkanoyl group.
  • alkanoyloxy refers to R 29 C(O)-O- wherein R 29 is a loweralkyl group.
  • alkanoyloxyalkyl refers to a loweralkyl radical to which is
  • alkenyl refers to a straight or branched chain hydrocarbon containing from 2 to 10 carbon atoms and also containing at least one carbon-carbon double bond.
  • alkenylene refers to a divalent group derived from a straight or branched chain hydrocarbon containing from 2 to 10 carbon atoms and also containing at least one carbon-carbon double bond. Examples of alkenylene include
  • alkoxy refers to R 30 O- wherein R 30 is loweralkyl as defined above.
  • Representative examples of alkoxy groups include methoxy, ethoxy, t-butoxy and the like.
  • alkoxyalkoxy refers to R 31 O-R 32 O- wherein R 31 is loweralkyl as defined above and R 32 is an alkylene radical.
  • alkoxyalkyl refers to an alkoxy group as previously defined appended to an alkyl group as previously defined.
  • alkoxyalkyl include, but are not limited to, methoxymethyl , methoxyethyl , isopropoxymethyl and the like.
  • alkoxyalkylcarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 66 -C(O)-O- wherein R 66 is an alkoxyalkyl group.
  • alkoxycarbonyl refers to an alkoxy group as previously defined appended to the parent molecular moiety through a carbonyl group.
  • alkoxycarbonyl include methoxycarbonyl, ethoxycarbonyl,
  • alkoxycarbonylaklyl refers to an alkoxylcarbonyl group as previously defined appended to a loweralkyl radical. Examples of alkoxycarbonylaklyl include
  • alkoxycarbonylaminoalkyl refers to a loweralkyl radical to which is appended R 69 -NH- wherein R 69 is an alkoxycarbonyl group.
  • alkoxycarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 63 -O- wherein R 63 is an alkoxycarbonyl group.
  • alkylamino refers to R 35 NH- wherein R 35 is a loweralkyl group, for example, methylamino, ethylamino, butylamino, and the like.
  • alkylaminoalkyl refers a loweralkyl radical to which is appended an alkylamino group.
  • alkylaminocarbonylaminoalkyl refers to a loweralkyl radical to which is appended R 70 -C(O)-NH- wherein R 70 is an alkylamino group.
  • alkylene refers to a divalent group derived from a straight or branched saturated hydrocarbon having from 1 to 10 carbon atoms by the removal of two hydrogen atoms, for example methylene, 1,2-ethylene, 1,1-ethylene, 1,3-propylene, 2,2-dimethylpropylene, and the like.
  • alkylsulfinyl refers to R 33 S(O)- wherein R 33 is a loweralkyl group.
  • alkylsulfonyl refers to R 34 S(O) 2 - wherein R 34 is a loweralkyl group.
  • alkylsulfonylalkyl refers to a loweralkyl radical to which is
  • alkynylene refers to a divalent group derived from a straight or branched chain hydrocarbon containing from 2 to 10 carbon atoms and also containing at least one
  • alkynylene examples include
  • amino refers to -NH 2 .
  • aminoalkyl refers to a loweralkyl radical to which is appended an amino group.
  • R 61 is appended an aroyloxy group (i.e., R 61 -C(O)O-wherein R 61 is an aryl group).
  • aryl refers to a mono- or bicyclic carbocyclic ring system having one or two aromatic rings including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyl and the like.
  • Aryl groups including
  • bicyclic aryl groups can be unsubstituted or
  • substituted aryl groups include tetrafluorophenyl and pentafluorophenyl .
  • arylalkenyl refers to an alkenyl radical to which is appended an aryl group.
  • arylalkenyloxycarbonyloxyalkyl refers to a loweralkyl radical
  • R 68 which is appended R 68 -O-C(O)-O- wherein R 68 is an arylalkenyl group.
  • arylalkyl refers to. a loweralkyl radical to which is appended an aryl group.
  • Representative arylalkyl groups include benzyl, phenylethyl, hydroxybenzyl, fluorobenzyl, fluorophenylethyl and the like.
  • arylalkylcarbonyloxyalkyl refers to a loweralkyl radical to which is appended an arylalkylcarbonyloxy group (i.e., R 62 C(O)O- wherein R 62 is an arylalkyl group).
  • R 67 is appended R 67 -O-C(O)-O- wherein R 67 is an arylalkyl group.
  • aryloxyalkyl refers to a loweralkyl radical to which is appended R 65 -O- wherein R 65 is an aryl group.
  • R 75 is an aryloxyalkyl
  • aryloxycarbonyalkyl refers to a loweralkyl radical to which is
  • R 65 is an aryl group.
  • arylsulfonyl refers to R 36 S(O) 2 - wherein R 36 is an aryl group.
  • R 37 refers to R 37 S(O) 2 O- wherein R 37 is an aryl group.
  • carboxyalkyl refers to a loweralkyl radical to which is appended a carboxy (-COOH) group.
  • carboxydehyde refers to the group -C(O)H.
  • carboxyamide refers to the group -C(O)NH 2 .
  • cyanoalkyl refers to a loweralkyl radical to which is appended a cyano (-CN) group.
  • cycloalkanoylalkyl refers to a loweralkyl radical to which is
  • a cycloalkanoyl group i.e., R 60 -C(O)- wherein R 60 is a cycloalkyl group.
  • cycloalkanoyloxyalkyl refers to a loweralkyl radical to which is appended a cycloalkanoyloxy group (i.e., R 60 -C(O)O- wherein R 60 is a cycloalkyl group).
  • cycloalkenyl refers to an alicyclic group comprising from 3 to 10 carbon atoms and containing a carbon-carbon double bond including, but not limited to, cyclopentenyl, cyclohexenyl and the like.
  • cycloalkyl refers to an alicyclic group comprising from 3 to 10 carbon atoms including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, adamantyl and the like.
  • cycloalkylalkyl refers to a loweralkyl radical to which is
  • cycloalkylalkyl include
  • cycloalkyloxycarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 64 -O-C(O)-O- wherein R 64 is a cycloalkyl group.
  • dialkoxyalkyl refers to a loweralkyl radical to which is
  • dialkylamino refers to R 38 R 39 N- wherein R 38 and R 39 are independently selected from loweralkyl, for example,
  • dialkylaminoalkyl refers to a loweralkyl radical to which is
  • dialkyaminocarbonylalkyl refers to a loweralkyl radical to which is appended R 73 -C(O)- wherein R 73 is a dialkylamino group.
  • dithioalkoxyalkyl refers to a loweralkyl radical to which is
  • halogen or halo as used herein refers to I, Br, Cl or F.
  • haloalkenyl refers to an alkenyl radical, as defined above, bearing at least one halogen substituent.
  • haloalkyl refers to a lower alkyl radical, as defined above, bearing at least one halogen substituent, for example, chloromethyl, fluoroethyl or trifluoromethyl and the like.
  • heterocyclic ring or
  • heterocyclic or “heterocycle” as used herein refers to a 5-, 6- or 7-membered ring containing one, two or three heteroatoms independently selected from the group consisting of nitrogen, oxygen and sulfur or a 5-membered ring containing 4 nitrogen atoms; and includes a 5-, 6- or 7- membered ring containing one, two or three
  • the 5-membered ring has 0-2 double bonds and the 6- and 7-membered rings have 0-3 double bonds.
  • heterocyclic also includes bicyclic, tricyclic and tetracyclic groups in which any of the above heterocyclic rings is fused to one or two rings independently selected from the group consisting of an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a
  • heterocyclic ring for example, indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl or benzothienyl and the like.
  • pyrrolyl include: pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl,
  • benzimidazolyl benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl, thiadiazolyl, pyrimidyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, tetrahydroquinolyl, tetrahydroisoquinolyl,
  • Heterocyclics also include bridged bicyclic groups wherein a monocyclic heterocyclic group is bridged by an alkylene group, for example,
  • Heterocyclics also include compounds of the formula
  • X* is -CH 2 -, -CH 2 O- or -O- and Y* is -C(O)- or - (C(R") 2 ) v - wherein R" is hydrogen or C 1 -C 4 -alkyl and v is 1, 2 or 3 such as 1, 3-benzodioxolyl, 1,4-benzodioxanyl and the like.
  • Heterocyclics can be unsubstituted or
  • heterocyclic alkyl refers to a heterocyclic group as defined above appended to a loweralkyl radical as defined above.
  • heterocycliccarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 72 -C(O)-O- wherein R 72 is a heterocyclic group.
  • hydroxyalkyl refers to a loweralkyl radical to which is appended an hydroxy group.
  • hydroxythioalkoxy refers to R 51 S- wherein R 51 is a hydroxyalkyl group.
  • loweralkyl refers to branched or straight chain alkyl groups
  • N-protected alkylaminoalkyl refers to an alkylaminoalkyl group wherein the nitrogen is N-protected.
  • spiroalkyl refers to an alkylene diradical, both ends of which are bonded to the same carbon atom of the parent group to form a spirocyclic group.
  • thioalkoxy refers to R 52 S- wherein R 52 is loweralkyl.
  • examples of thioalkoxy include, but are not limited to, methylthio, ethylthio and the like.
  • thioalkoxyalkyl refers to a thioalkoxy group as previously defined appended to a loweralkyl group as previously defined.
  • thioalkoxyalkyl include thiomethoxymethyl, 2-thiomethoxyethyl and the like.
  • the present invention also relates to
  • compositions which comprise a compound of the present invention in combination with a pharmaceutically acceptable carrier.
  • compositions which comprise a compound of the present invention in combination with another chemotherapeutic agent and a pharmaceutically acceptable carrier.
  • protein isoprenyl transferases i.e., protein farnesyltransferase and/or
  • geranylgeranyltransferase in a human or lower mammal, comprising administering to the patient a therapeutically effective amount of a compound of the invention.
  • invention is disclosed a method for inhibiting post-translational modification of the oncogenic Ras protein by protein farnesyltransferase, protein geranylgeranyltransferase or both.
  • invention is disclosed a method for treating or preventing restenosis in a human or lower mammal, comprising administering to the patient a therapeutically effective amount of a compound of the invention.
  • the compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids.
  • These salts include but are not limited to the following: acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate,
  • the basic nitrogen-containing groups can be quaternized with such agents as loweralky halides (such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides), dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides like benzyl and phenethyl bromides, and others. Water or oil-soluble or disperisble products are thereby obtained.
  • loweralky halides such as methyl, ethyl, propyl, and butyl chloride, bromides, and iodides
  • dialkyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates
  • long chain halides such as
  • Basic addition salts can be prepared in situ during the final isolation and purification of the compounds of formulas A-L, or separately by reacting the carboxylic acid function with a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine.
  • a suitable base such as the hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation or with ammonia, or an organic primary, secondary or tertiary amine.
  • pharmaceutically acceptable salts include, but are not limited to, cations based on the alkali and alkaline earth metals, such as sodium, lithium, potassium, calcium, magnesium, aluminum salts and the likes, as well as nontoxic ammonium,
  • quaternary ammonium, and amine cations including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
  • Other representative organic amines useful for the formation of base addition salts include diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.
  • H-RasF cells were treated with various concentrations of FTI-277, lysed and the lysates immunoblotted with anti-Ras or anti-Rap1A
  • pZIPneo, H-RasF, H-RasGG, Raf and S186 cells were treated with vehicle or FTI-277 (5 ⁇ M), lysed and lysates immunoblotted by anti-Ras antibody. Data is representative of 5 different experiments. The cells were obtained from Dr. Channing Der,
  • H-RasF cells were treated with vehicle or FTI-277, lysed and the lysates immunoprecipitated with anti-Ras antibody. The GTP and GDP were then released from Ras and separated by TLC as
  • RafF farnesylated Ras
  • RasGG geranylgeranylated Ras
  • a transforming mutant of human Raf-1 were obtained from Channing Der and Adrienne Cox (University of North Carolina, Chapel Hill, NC, USA) (26,27).
  • the cells were plated in DMEM/10% CS (Dubelco's Modified Eagles Medium, 10% calf serum) on day one and treated with vehicle or FTI-270 (20 ⁇ M) on days 2 and 3.
  • DMEM/10% CS Dubelco's Modified Eagles Medium, 10% calf serum
  • the cells were then harvested on day 4 and lysed in lysis buffer (30 mM HEPES, pH 7.5, 1% TX-100, 10% glycerol, 10 mM NaCl, 5 mM MgCl 2 , 25 mM NaF, 1 mM EGTA, 2 mM
  • mice 8 week old female nude mice (10 7 cells/flank). Nude mice (Harlan Sprague Dawley, Indianapolis, Indiana) were maintained in accordance with the Institutional Animal Care and Use Committee (IACUC) procedures and guidelines.
  • IACUC Institutional Animal Care and Use Committee
  • animals were dosed i.p. with 0.2 ml once daily for 36 days. Control animals (filled circles) received a saline vehicle whereas treated animals (open triangles) were injected with FTI-276 (50 mg/kg).
  • FIG. 11 Dose response: Antitumor efficacy and Ras processing correlations.
  • FIG. 1 Silica gel TLC relating to Ras CAAX peptide and peptidomimetic farnesylation.
  • NIH 3T3 cells that overexpress oncogenic K-Ras4B were treated with FTI-277 or GGTI-286 (0-30 ⁇ M). The cells were lysed and the lysates were
  • Figure 20 Inhibition of oncogenic activation of MAP Kinase.
  • NIH 3T3 cells that overexpress either oncogenic H-Ras or K-Ras4B were treated with either FTI-277 or GGTI-286 (0-30 ⁇ M). The cells were lysed and the lysates were
  • P-MARK designates hyperphosphorylated MAP kinase.
  • the data are representative of three independent experiments.
  • FTase farnesyltransferase
  • GGTase geranylgeranyltransferase
  • PBS phosphate-buffered saline
  • CAAX tetrapeptide where C is cysteine, A is an aliphatic amino acid and X is an amino acid
  • BSA bovine serum albumin
  • GGTase I geranylgeranyl transferase I
  • PAGE polyacrylamide gel electrophoresis
  • MAPK mitogen activated protein kinase
  • FTI farnesyltransferase inhibitor
  • GGTI geranylgeranyltransferase inhibitor
  • PMSF phenylmethylsulfonyl fluoride
  • peptidomimetics of Formula (I), one of the preferred embodiments of the invention may be made using procedures which are conventional in the art.
  • ⁇ is 2-phenyl-4-aminobenzoic acid although constrained derivatives such as tetrahydroisoquinoline-7-carboxylic acid, 2- aminomethyl pyridine- 6-carboxylic acid or other heterocyclic derivatives, may also be used.
  • the amino group of ⁇ is reacted with a suitably protected cysteinal.
  • the amino acid represented by X preferably Met, is then reacted through its amino group with the deprotected and activated carboxyl group of spacer compound ⁇ . Following deprotection by removal of other protecting groups, the compound of Formula (I) is obtained.
  • ⁇ may first be reacted with the X amino acid followed by reaction with the cysteine or cysteinal component using
  • the invention also includes the
  • Acid salts include the reaction products obtained with, for example, toluene sulfonic acid, acetic acid, propionic acid or the like as conventionally used in the art.
  • the compounds of the invention may be used to inhibit p21ras farnesyltransferase in any host containing the same. This includes both in vitro and in vivo use. Because the compounds inhibit farnesyltransferase, notably human tumor p21ras farnesyltransferase, and consequently inhibit the farnesylation of the oncogene protein ras, they may be used in the treatment of cancer or cancer cells. It is noted that many human cancers have activated ras and, as typical of such cancers, there may be mentioned colorectal carcinoma, myeloid leukemias, exocrine pancreatic carcinoma and the like.
  • the compounds of the invention may be used in pharmaceutical compositions of conventional form suitable for oral, subcutaneous, intravenous, intraperitoneal or intramuscular administration to a mammal or host.
  • pharmaceutical compositions of conventional form suitable for oral, subcutaneous, intravenous, intraperitoneal or intramuscular administration to a mammal or host This includes, for example, tablets or capsules, sterile solutions or
  • suspensions comprising one or more compounds of the invention with a pharmaceutically acceptable carrier and with or without other additives.
  • aqueous suspensions may be used with conventional suspending agents, flavoring agents and the like.
  • the amount of inhibitor administered to obtain the desired inhibitory effect will vary but can be readily determined.
  • daily dosages are dependent on the circumstances, e.g. age and weight. However, daily dosages of from 0.1 to 20 mg per kg body weight may be mentioned for purposes of illustration.
  • the ⁇ component is, in general, any non-peptide amino acid combination or other hydrophobic spacer element that produces a compound which mimics the structure and
  • hydrophobic spacers have been used as the ⁇ component according to this aspect of the invention. This includes, for example, 3-aminobenzoic acid, 4-aminobenzoic acid and 5-aminopentanoic acid as well as heterocyclic carboxylic acids such as tetrahydroiso-quinoline-7-carboxylic acid, 2-aminomethyl pyridine-6-carboxylic acid or the like as mentioned earlier, as replacements for the ⁇ component of the Formula (I) compounds.
  • the peptidomimetics of the invention include variants for Formula (I) where ⁇ stands for the radical of a non-peptide aminoalkyl or amino-substituted aliphatic or aromatic carboxylic acid or a
  • heterocyclic monocarboxylic acid for example, 3-aminobenzoic acid (3-ABA), 4-aminobenzoic acid (4-ABA) or 5-aminopentanoic acid (5-APA).
  • the ⁇ substituent may be derived from any hydrophobic, non-peptidic aminoalkyl- or amino-substituted aliphatic, aromatic or
  • other effective inhibitors for farnesyltransferase may be provided by incorporating a negatively charged residue onto the compounds of Formula (I).
  • This feature of the invention is based on a consideration of the transition state of the farnesylation reaction and the recognition that the functional enzyme complex must involve a farnesyl pyrophosphate binding site close to the peptide binding region.
  • Compounds representative of this embodiment include peptides prepared with a phosphonate residue linked at different distances to the cysteine sulfur.
  • ⁇ 1 is a phosphonate group joined to cysteine through the cysteine sulphur atom.
  • an important further feature of the invention is the modification of the compounds of the invention, as well as the tetrapeptide p21ras farnesyl transferase
  • inhibitors of the formula CA 1 A 2 X to provide prodrugs. This involves forming lipophilic enzyme-sensitive derivatives from the compounds by appropriately functionalizing the terminal groups. For example, the terminal amino groups and the cysteine sulfur can be reacted with benzyl
  • alkyl or aryl ester e.g. the methyl ester.
  • alkyl esters from 1 to 10 carbons in length
  • activated esters such as cyanomethyl or trifluoromethyl, cholesterol, cholate or
  • lipophilic when used in this context, is meant to include, inter alia, methoxycarbonyl and other long chain or carbamate groups. Examples of such groups are well known to the ordinarily skilled practitioner.
  • cholesterolyl aryl or aralkyl such as benzyl, phenylethyl, phenylpropyl or naphthyl, or alkyl, typically methyl or other alkyl of, for example, up to 8 carbon atoms or more. It is contemplated that such functional groups would be attached to the cysteine sulfur and the terminal amino and carboxy groups.
  • the functionalized pro-drug embodiment of the invention may be structurally illustrated as follows:
  • the "BBM” used in the formulas represents a shorthand reference to the bis-(carboxybenzyloxy)methyl esters of C ⁇ M and CVIM.
  • BMMM methyl methyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-methyl methyloxy substitution and the three methyl groups in the methylated phosphoric and carboxylic acid end groups.
  • the purpose of the functional groups added to the parent compounds is to improve entry of the compounds into tumor cells. Once in the cells, the functional groups are removed to liberate the active compound to function in its inhibitory capacity.
  • the functionalized pro-drugs of the invention can be prepared using conventional and well-known procedures for esterifying amino, SH and carboxylic acid groups. Hence, details of such procedures are not essential for the preparation of the present pro-drugs.
  • N-BOC-4-aminobenzoic acid 8.77 g, 36.97 mmol
  • methionine methyl ester hydrochloride 8.12 g, 40.66 mmol
  • This solution was cooled in an ice bath and triethylamine (6.7 ml), EDCI (7.80 g, 40.66 mmol) and hydroxybenzotriazole (HOBT, 5.50 g, 40.66 mmol) were added.
  • EDCI 7.80 g, 40.66 mmol
  • HOBT hydroxybenzotriazole
  • N-BOC-4-aminobenzoyl methionine methyl ester (3.53 g, 9.59 mmol) was placed into CH 2 Cl 2 (30-35 ml) and to it was added 3M HCl/ Et 2 O (38.4 ml).
  • N-BOC-S-trityl-Cys (2.86 g, 6.54 mmol) and triethylamine (1.2 ml) were placed into a dried, N 2 filled flask containing dry THF (104 ml). This was cooled to -10°C using an ice/ salt bath and isobutyl chloroformate (0.9 ml), IBCF, was added. The solution was stirred at -10°C for 40 minutes and HCl-4-aminobenzoyl methionine methyl ester (2.08 g, 6.54 mmol) in dry CH 2 Cl 2 (34.1 ml) with triethylamine (1.2 ml, 1.3 eq) was added. The solution warmed to room temperature and was
  • N-BOC-S-trityl-cysteine-4-aminobenzoyl methionine methyl ester (1 g, 1.37 mmol) was placed into a flask and taken up in CH 3 OH (13.7 ml). To this solution was added a solution of mercuric chloride (0.75 g, 2.74 mmol) in CH 3 OH (13.7 ml). Upon addition of the mercuric
  • HCl-cysteine-4-aminobenzoyl methionine methyl ester (0.51 g, 0.7 mmol) was taken up in THF (4.1 ml) and to this solution was added 0.5 M LiOH (2.9 ml) at 0°C. The heterogeneous solution was stirred at 0°C for 35-40 minutes and then the THF was removed in vacuo. The residue was taken up in CH 2 Cl 2 and was washed three times with 1M C1 followed by brine. The organic solution was dried over Na 2 SO 4 and the solvent was removed in vacuo. The pale yellow solid was taken up in 3 ml of CH 2 Cl 2 and the product was precipitated with 3-4 M HCl/ Et 2 O. The solid was collected by
  • N-BOC-4-amino-3-methylbenzoic acid (2.00 g, 7.96 mmol) was reacted with methionine methyl ester hydrochloride (1.75 g, 8.76 mmol), EDCI (1.68 g, 8.76 mmol), HOBT (1.18 g, 8.76 mmol) and Et 3 N (1.4 ml) in dry CH 2 Cl 2 (31.8 ml) according to the procedure described for N-BOC-4-aminobenzoyl methionine methyl ester in Example 1.
  • N-BOC-4-amino-3-methylbenzoyl methionine methyl ester (0.99 g, 2.59 mmol) was dissolved in CH 2 Cl 2 (15-20 ml) and precipitated with 3M HCl/Et 2 O (20.7 ml). 0.83 g (96.6%) of pale orange precipitate was obtained after drying overnight on the vacuum pump. mp 157-159°C; 1 H NMR (CD 3 OD) 2.04 (3H,s), 2.11-2.25 (1H, m), 2.47 (3H, s), 2.52-2.68 (3H.
  • N-BOC-S-trityl-cysteine (0.55 g, 1.25 mmol) in dry THF (25 ml) was reacted with Et 3 N (0.19 ml), IBCF (0.16 ml, 1.25 mmol) at -10 °C as described above.
  • HCl-4-amino-3-methylbenzoyl methionine methyl ester (0.42 g, 1.25 mmol) in dry CH 2 Cl 2 (6.5 ml) with Et 3 N (0.26 ml) was added at -10°C and the reaction mixture was allowed to stir overnight under nitrogen. Workup was carried out as
  • N-BOC-S-trityl-cysteine-4-amino-3-methylbenzoyl methionine methyl ester (0.27 g, 0.37 mmol) in THF (2.1 ml) was deprotected with 0.5M LiOH (2.9 ml) over 1.5 h at room temperature. The solvent was removed in vacuo and the residue was taken up in CH 2 Cl 2 and extracted 3 times with 1N HCl followed by extraction with brine. The organic solution was dried over Na 2 SO 4 and the solvent was removed in vacuo to give 0.19 g (73.5 %) of the free acid. The free acid was then taken up in CH 2 Cl 2 (1.4 ml) and Et 3 SiH (0.04 ml) was added followed by trifluoroacetic acid, TFA (1.4 ml). The reaction mixture was stirred at room
  • N-BOC-4-amino-3-methoxybenzoic acid (0.35 g, 1.31 mmol) was reacted with methionine methyl ester hydrochloride (0.9 g, 1.43 mmol) using EDCI as in N-BOC-4 -aminobenzoyl methionine methyl ester. After recrystallization from ethyl acetate and hexanes, 0.36 g (57.2 %) of pure product was obtained.
  • N-BOC-4-amino-3-methoxybenzoyl methionine methyl ester (0.71 g, 1.79 mmol) was taken up in CH 2 Cl 2 (4 ml) and precipitated with 3-4M HCl/ Et 2 O (12 ml). The precipitate was washed as usual with Et 2 O and dried overnight under vacuum to result in 0.55 g (88.3%) of reddish material.
  • N-BOC-S-trityl-cysteine (0.76 g, 1.74 mmol) in dry THF (27.5 ml) was reacted with Et 3 N (0.24 ml), IBCF (0.23 ml, 1.74 mmol) at -10°C as
  • N-BOC-S-trityl-cysteine-4-amino-3-methoxybenzoyl methionine methyl ester (0.18 g, 0.24 mmol) was deprotected with LiOH at room temperature as described above to give the free acid. The free acid was then further deprotected in CH 2 Cl 2 (1.2 ml) with Et 3 SiH (0.04 ml, 0.24 mmol) and TFA (1.2 ml). The product was worked up as described for HCl-cysteine-4-aminobenzoyl
  • N-BOC-S-trityl-cysteine (0.31 g, 0.66 mmol) in dry THF (11 ml) was reacted with Et 3 N (0.10 ml), IBCF (0.09 ml, 0.73 mmol) at -10 °C as described for N-BOC-S-trityl-cysteine-4-aminobenzoyl
  • N-BOC-S-trityl-cysteine-4-amino-2-phenylbenzoyl methionine methyl ester (84.70 mg, 0.11 mmol) of was taken up in THF (0.62 ml) and to this was added 0.5 M LiOH (0.32 ml) at 0 °C. The mixture was stirred at 0 °C for 35 minutes. The solvent was removed in vacuo using a cold water bath on the rotovap. The residue was worked up as described for HCl-cysteine-4-aminobenzoyl
  • Example 2 methionine in Example 1, and 60 mg of the free acid was obtained. This was then dissolved into CH 2 Cl 2 (0.8 ml) and Et 3 SiH (0.01 ml) was added followed by TFA (0.8 ml). The reaction mixture was stirred at room temperature for 1 h and worked up as described for TFA-cysteine-4-amino-3-methylbenzoyl methionine in Example 2. After lyophilization, 0.0387 g (84.0%) was obtained.
  • HPLC revealed that no epimerization had occurred, however the material was purified on the HPLC to eliminate baseline impurities. mp 150-154°C;
  • methionine methyl ester (0.11 g, 0.26 mmol) was taken up in ethyl acetate (3.0 ml). To this mixture was added SnCl 2 ⁇ 2H 2 O (0.30 g, 1.30 mmol) and the reaction was heated under nitrogen at reflux for 6h. The mixture was worked up as described for 4-amino-2-phenylbenzoyl methionine methyl ester in Example 2, to give 0.15 g of a yellow film that was wet with solvent. The material was otherwise pure by NMR and was used without further purification.
  • methionine methyl ester (0.10g, 0.26 mmol) was dissolved into dry CH 2 Cl 2 (1.4 ml) and it was allowed to stand.
  • N-BOC-S-trityl-Cys (0.12 g, 0.26 mmol) was dissolved into THF (4.4 ml) and was reacted with IBCF (0.03 ml) and Et 3 N (0.04 ml) as described above.
  • N-BOC-S-trityl-cysteine-4-amino-2-(3,5-dimethylphenyl)benzoyl methionine methyl ester (0.12 g, 0.15 mmol) was placed into THF (0.9 ml) and was reacted with 0.5 M of LiOH (0.6 ml) at 0 °C as described above, followed by deprotection with TFA (1.5 ml) and Et 3 SiH (0.24 ml). Addition of excess scavenger does not appear to affect the result.
  • the product was purified by preparative HPLC to give 23.8 mg (27.3%).
  • the deep red solution was cooled and poured over 200 ml of water. The solution was then filtered and the acid was precipitated with concentrated HCl. The red crystals were filtered and the filtrate was refiltered to give pink crystals. The first fraction was treated with activated carbon to remove some of the red color. 1.51 g (90.6%) of product was obtained.
  • N-BOC-4-amino-1-naphthoyl methionine methyl ester N-BOC-4-amino-1-naphthoic acid (0.46 g, 1.60 mmol), methionine methyl ester hydrochloride (0.35 g, 1.76 mmol), EDCI (0.43 g, 1.76 mmol), HOBT (0.24 g, 1.76 mmol) and Et 3 N ( 0.27 ml) in CH 2 Cl 2 (6.4 ml) were reacted as described for N-BOC-4-aminobenzoyl methionine methyl ester in Example 1.
  • N-BOC-4-amino-1-naphthoyl methionine methyl ester (0.57 g, 1.31 mmol) was deprotected with HCl/ ether to yield 0.31 g (64.1%) of white solid.
  • N-BOC-S-trityl-Cys (0.31 g, 0.67 mmol) in dry THF (11.2 ml) was reacted with Et 3 N (0.10 ml) and IBCF (0.10 ml, 0.74 mmol) at -10 °C as described above.
  • HCl ⁇ 4-amino-1-naphthoyl methionine methyl ester (0.25 g, 0.67 mmol) in dry CH 2 Cl 2 (3.5 ml) was added and the mixture was stirred overnight under nitrogen.
  • N-BOC-S-trityl-cysteine-4-amino-1-naphthoyl methionine methyl ester 83.3 mg, 0.11 mmol was taken up in THF (0.7 ml) and to this mixture was added 0.5 M LiOH (0.43 ml) at 0°C. The mixture was stirred at 0°C for 35 minutes. The solvent was removed in vacuo using a cold water bath. The residue was worked up as described for
  • TFA ⁇ cysteine-4-amino-1-naphthoyl methionine (0.12 g, 0.15 mmol) was dissolved in CH 3 OH (4.3 ml).
  • a solution of HgCl 2 (0.23 g, 0.86 mmol) in CH 3 OH (4.3 ml). The procedure was continued as described above and after HCl/ Et 2 O precipitation and several
  • Triethylamine (2.22 mL, 16 mmoL) and N,O-dimethylhydroxylamine hydrochloride (1.57 g, 16.1 mmol) were added to a solution of N-Boc-S-trityl cysteine (7.44 g, 16 mmol) in 85 mL of methylene chloride.
  • This mixture was cooled in an ice bath and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 3.08 g, 16.0 mmol) and HOBT (2.17 g, 16 mmol) was added.
  • the mixture was stirred at 0°C for 1 hr and at room temperature for a further 10 hr.
  • the mixture was extracted with methylene chloride and 0.5 N HCl.
  • N-Boc-S-trityl cysteinal in 10 mL of methanol was added to a solution of 4-aminobenzoyl methionine methyl ester hydrochloride (1.7836 g, 5.6 mmol) in 60 mL of methanol and 4 mL of glacial acetic acid.
  • Sodium cyanoboronhydride (0.528 g, 8.40 mmol) was added to this deep colored solution at 0 °C. The mixture was stirred at room temperature for 15 hr. After the
  • Triethylsilane was added dropwise until the deep brown color disappeared. The mixture was kept at rt for 1 hr. The solvents were evaporated and the residue was dried. This residue was dissolved in l mL of 1.7N HCl in acetic acid followed by the addition of 20 mL of 3N HCl in ether. The white precipitate was filtered and dried to give a hydrochloride salt of the desired product (159 mg, 46%). Analytical HPLC showed purity over 98%. 1 H NMR (CD 3 OD) ⁇ 7.74 (d, 8.7 Hz, 2H), 6.75 (d, 8.7 Hz, 2H), 4.73 (dd, 4.5 Hz and 4.7 Hz, 1H, Met ⁇
  • FTase and GGTase I activities from 60,000 X g supernatants of human Burkitt lymphoma (Daudi) cells were assayed as described previously for FTase (41). Briefly, 100 ⁇ g of the supernatant was incubated in 50 mM Tris, pH 7.5, 50 ⁇ M ZnCl 2 , 20 mM KCl and 1 mM
  • H-RasF cells (45) were seeded on day 0 in 100 mm Dishes (costar) in Dulbecco's modified Eagles medium (GIBCO) and allowed to grow to 40-60% confluency. On days 1 and 2, cells were fed with 4 ml of medium per plate plus various concentrations of FTI-277 or vehicle. On day 3, cells were washed one time with ice cold PBS and were
  • H-RasF cells were seeded and treated as above for Ras/Raf interaction and Ras and Rap1A
  • cytosol and membrane fractions were lysed on ice for 60 min in 50 mM Tris, pH 7.5, 5 mM MgCl 2 , 1% Triton X-100 (TX-100), 0.5% DOC, 0.05% SDS, 500 mM NaCl, 1 mM EGTA, 10 ⁇ g/ml aprotinin, 10 ⁇ g/ml soybean trypsin
  • Immunoprecipitates were washed 6 times in 50 mM HEPES, pH 7.5, 0.5 M NaCl, 0.1% TX-100, 0.0005 SDS, 5 mM MgCl 2 , drained using a syringe and bound nucleotide eluted in 12 ⁇ l of 5 mM DTT, 5 mM EDTA, 0.2% SDS, 0.5 mM GDP and 0.5 mM GTP at 68°C for 20 min.
  • Nucleotides were separated by chromatography in 78 g/linter ammonium formate, 9.6% (v/v) concentrated HCl. Plates were analyzed by autoradiogram.
  • Immune complex kinase assays were performed by incubating immunoprecipitaes from membrane and cytosol fractions in 96 ⁇ l of kinase buffer with 20 ⁇ Ci of [ ⁇ - 32 P]ATP (10 mCi/ml, Amersham) and 2 ⁇ l of the Raf-1 substrate peptide (1 mg/ml,
  • Fig. 1B shows that FTI-276 inhibited the transfer of farnesyl from [ 3 H] FPP to recombinant H-Ras-CVLS with an IC 50 of 500 pM.
  • FTI-249 the parent compound of FTI-276, inhibited FTase with an IC 50 of 200,000 pM.
  • a phenyl ring at the 2 position of the benzoic acid spacer increased inhibition potency of FTase by 400 fold confirming our prediction of a significant hydrophobic pocket within the CAAX binding site of FTase .
  • This extremely potent inhibitor was also highly
  • FTI-277 the methylester of FTI -276
  • H-RasF cells NIH 3T3 cells transformed with oncogenic (61 leucine) H-Ras-CVLS (45) were treated with FTI-277 (0-50 ⁇ M) and the lysates blotted with anti-Ras or anti-Rap1A antibodies.
  • concentrations as low as 10 nN inhibited Ras processing but concentrations as high as 10 ⁇ M did not inhbit processing of the
  • FTI-277 inhibited Ras processing with an IC 50 of 100 nM.
  • the IC 50 of FTI-249 is 100 ⁇ M, and the most potent CAAX peptidomimetics previously reported inhibited Ras processing at concentrations of 10 ⁇ M or higher (44).
  • H-RasGG cells NIH 3T3 cells transformed with oncogenic (61 leucine) H- Ras-CVLL (45) were treated with FTI-277.
  • RasGG was not affected, whereas that of RasF was completely blocked.
  • the processing of endogenous Ras is also blocked in pZIPneo cells (NIH 3T3 cells transfected with the same plasmid as H-RasF and H Ras FF except the vector contained no oncogenic Ras sequences) and Raf cells (NIH 3T3 cells transformed by an activated viral Raf (48) ) .
  • pZIPneo cells NIH 3T3 cells transfected with the same plasmid as H-RasF and H Ras FF except the vector contained no oncogenic Ras sequences
  • Raf cells NIH 3T3 cells transformed by an activated viral Raf (48)
  • Ras relays biological information from tyrosine kinase receptors to the nucleus by activation of a cacade of MAPKs (reviewed in 29-31).
  • Ras Upon growth factor stimulation, Ras becomes GTP bound and is then able to recruit the ser/thr kinase c-Raf-1 to the plasma membrane where it is activated.
  • c-Raf-1 then phosphorylates and activates MEK, a dual thr/tyr kinase, which activates MAPK.
  • epidermal growth factor has been shown to induce association of Raf with Ras (46).
  • NIH 3T3 cells were transfected with activated (GTP-locked) Ras and the effects of FTI-277 on the interaction of Ras with its immediate effector, Raf, were investigated.
  • Various NIH 3T3 cell transfectants pZIPneo, H-RasF, and H-RasGG were treated with vehicle or FTI-277, membrane and cytosolic
  • Ras/Raf complexes as described above.
  • Ras-F cells only membrane fractions contained GTP- locked Ras, as shown in Fig 4A.
  • FTI-277 Upon treatment with FTI-277, however, the non-farnesylated cytosolic Ras was found to be GTP bound.
  • binding of GTP to 61 leucine Ras does not require Ras processing and subsequent plasma membrane association.
  • the ser/thr kinase activity of Raf in Ras/Raf complexes was then determined by immunoprecipitating Raf and assaying for its ability to phosphorylate a 19-mer
  • Ras/Raf complexes in the cytoplasm where Ras is GTP-bound but Raf kinase is not activated.
  • Raf kinase is not activated when bound to Ras in a non-membranous environment is consistent with recent reports that indicate that Raf activation requires an as yet to be determined activating factor at the plasma membrane (47) .
  • Fig. 5A shows that NIH 3T3 cells transfected with pZIPneo contain only inactive MAPK but that upon transformation with oncogenic H-Ras, MAPK is activated (Fig. 5A).
  • Fig. 5B shows that FTI -277 was able to block H-RasF but not H-RasGG activation of MAPK. This is
  • Fig. 5B shows that oncogenic Raf activation of MAPK is not blocked by FTI -277 even though processing of endogenous Ras was inhibited in these cells.
  • FTI-277 is an extremely potent and highly selective FTase inhibitor. This compound inhibited Ras processing with concentrations as low as 10 nM and processing was blocked at 1 ⁇ M. The most potent inhibitor previously reported BZA-5B, blocked Ras processing only at 150 ⁇ M (44).
  • Figure 9 shows that a once daily injection of FTI- 276 or FTI-277 (50 mg/kg) inhibited tumor growth of H-RasF transformed NIH 3T3 cells.
  • an identical treatment regimen with FTI-276 and FTI -277 had no effect on the growth of Raf-transformed NIH 3T3 cells (Fig. 10), further confirming the conclusion from the results of Figures 7 and 8 that FTI-276 and FTI-277 are selective for Ras-dependent tumors.
  • H-RasF cells were treated with various doses of FTI-276 (0, 10, 50 and 100 mg/kg) and tumor size and Ras processing in the HRasF tumors in vivo were examined.
  • Figure 11A shows that throughout the 11 day treatment period, FTI-276 inhibited tumor growth in a dose dependent fashion.
  • the tumor sizes at the end of 17 days were 2490 mm 3 for saline, 1793 mm 3 for 10 mg/kg, 1226 mm 3 for 50 mg/kg and 624 mm 3 for 100 mg/kg treated animals.
  • the animals were sacrificed 5 hrs after the last injection, the tumors were excised and processed for immunoblotting with anti-Ras antibody as described in legend to Fig. 11.
  • (2a) was prepared through the reductive amination of 4 -aminobenzoyl methionine methyl ester and N-Boc-S-trityl cysteinal in methanol solution containing NaBH 3 CN and 5% acetic acid. This reaction gave the N-Boc-S-trityl, methyl ester of (2a) with a yield of 65%.
  • the protected peptidomimetic was deesterified by LiOH in THF and then deprotected by trifluoroacetic acid in methylene chloride with two equivalents of triethylsilane to give crude (2a) which was purified by reverse phase HPLC.
  • the biphenyl-based peptidomimetic (8) was prepared by the reductive amination of 4-amino-3'-methyl biphenyl with N-Boc-S-trityl cysteinal, to give the N-Boc-S-trityl derivatives of (8), which was then deprotected by trifluoroacetic acid and purified by reverse phase HPLC.
  • the peptidomimetics (4) and (5) were prepared from the reductive amination of 4-amino-3'-tert.butoxycarbonyl biphenyl and 4-amino-4'-tert.butoxycarbonylbiphenyl,
  • Elemental analyses were performed by Atlantic Microlab Inc., Georgia. Optical rotations were measured on a Perkin-Elmer 241 polarimeter. Concentrations are expressed in g/mL. Flash column chromatography was performed on silica gel (40-63 ⁇ m) under a pressure about 4 psi. Solvents were obtained from commercial suppliers and purified as following: tetrahydrofuran and ether were distilled from sodium benzophenone ketyl, methylene chloride was distilled over lithium aluminum hydride. Preparative HPLC was performed using a Waters 600 E controller and a Waters 490 E Multi-Wavelength UV detector with a 25x10 cm
  • Delta-Pak C-18 300 A cartridge column inside a Waters 25x10 cm Radial Compression Module.
  • High resolution mass spectra (HRMS) and low resolution mass spectra (LRMS) were performed on a Varian MAT CH-5 and VG 7070 mass spectrometer.
  • the 1 H NMR spectrum of this compound was complex.
  • the percentage of the aldehyde was about 65-70%, which was calculated according to the integration of the sharp singlet ( ⁇ 9.17) and the trityl peak ( ⁇ 7.40, m, 6H;7.28,m, 9H).
  • Triethylsilane was added dropwise to the deep brown mixture until the brown color had
  • the azide 20 (900 mg, 2.91 mmol) was
  • the reaction was also incubated for 30 minutes at 37°C but with recombinant H-Ras-CVLL (5 ⁇ M) and [ 3 H] GGPP (525 nM; 19.0 Ci/mmol). The reaction was stopped and passed through glass fiber filters to separate free and incorporated label.
  • the peptidomimetics were premixed with FTase or GGTase I prior to adding the remainder of the reaction mixture.
  • peptidomimetic in 50 mM Tris, pH 7.5, 50 ⁇ M ZnCl 2 , 20 mM KCl, 3 mM MgCl 2 , 1 mM DTT and 0.2% octylß-D- glucoside was incubated for 30 minutes at 37°C, then spotted onto silica gel G TLC sheets (20 x 20 cm, Brinkmann Instruments), and developed with n-propanol/5 N ammonium hydroxide/water (6:1:1).
  • the dried sheets were sprayed with En 3 Hance (DuPont NEN) and exposed to x-ray film for detection of [ 3 H] farnesylated products.
  • EJ3 cells were treated with peptidomimetics or vehicle for 20-24 h.
  • Cells were lysed in lysis buffer (10 mM Na 2 HPO 4 , pH 7.25, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 12 mM sodium deoxycholate, 1 mM NaF, 0.2% NaN 3 , 2 mM PMSF, 25 ⁇ g/ml leupeptin) and the lysates were cleared by spinning at 13,000 rpm for 15 minutes. Ras protein was immunoprecipitated overnight at 4°C with 50 ⁇ g of anti-Ras antibody (Y13-259;
  • Immunoprecipitates were washed 4 times with lysis buffer and the bound proteins were released by heating for 5 minutes in 40 ⁇ l SDS-PAGE sample buffer and subsequently electrophoresed on a 12.5% SDS-PAGE. Proteins were transferred onto
  • Rap1A processing assays 50 ⁇ g of cell lysates were electrophoresed as described above for Ras processing and transferred to
  • IC 50 values given in Table 4 represent inhibition of FTase and GGTase I in vitro by the listed compounds.
  • the results obtained showed that compound 2, i.e. Cys-4ABA-Met (1-10 ⁇ M) inhibited FTase in a concentration-dependent manner with an IC 50 of 150 nM (FTI-232, Table 4).
  • This value is similar to the previously reported IC 50 values for CVIM and Cys-4ABA-Met (35).
  • Reduction of the amide bond between cysteine and aminobenzoic acid gave the red-Cys-4ABA-Met (2a, FTI-249) which had an IC 50 of 300 nM.
  • Figures 14A and 14B graphically illustrate the results of FTase and GGTase I inhibition studies.
  • partially purified FTase and GGTase I were incubated with the peptidomimetics to be tested and their ability to transfer [ 3 H] farnesyl to H-Ras-CVLS (FTase) and [ 3 H] geranylgeranyl to H-Ras CVLL (CCTase I) was determined as described.
  • Figure 14A shows FTase inhibition by: D, (4) and ⁇ , (5) while Figure 14B plots FTase (D) and GGTase I ( ⁇ ) inhibition by (4).
  • Each curve is representative of at least four independent experiments.
  • Geranylgeranylation is a more common protein prenylation than farnesylation (49). It is, therefore, advantageous for CAAX peptidomimetics targeting farnesylation to have high selectivity towards inhibiting FTase compared to GGTase.
  • the X position determines whether the cysteine thiol will be farnesylated by FTase or geranylgeranylated by GGTase I. Those proteins or peptides with Leu or Ile at the X position are geranylgeranylated. As shown in Table 4, the present compounds do not
  • Figure 14B shows that compound 4, which is a potent FTase inhibitor, is a very poor GGTase I inhibitor.
  • FIG. 15 shows that the natural peptide CVLS (carboxyl terminal CAAX of H-Ras) is farnesylated by FTase from Burkitt lymphoma cells. Replacing the tripeptide VLS with 4-amino-3'-hydroxycarbonylbiphenyl, as in 4 did not affect potency towards FTase inhibition (Table 4) but prevented farnesylation of the cysteine thiol ( Figure 15). None of the peptidomimetics of the invention is metabolically-inactivated by FTase ( Figure 15). Thus, although AAX tripeptides are not necessary for potent FTase inhibition, they appear to be required for farnesylation.
  • FPP, F-peptide, and ORIGIN designate farnesyl pyrophosphate, farnesylated peptide and origin, respectively.
  • Figure 15 shows: Lane 1, FPP only; lane 2, FPP and CVLS but no FTase; lane 3, FPP and FTase but not peptide. Lanes 4-9 all contained FTase and FPP with lane 4, CVIM; lane 5, CVLS;
  • FIG. 16 illustrates Ras and Rap1A processing.
  • Ras transformed 3T3 cells were treated with inhibitors, lysed and the lysate A) immunoprecipitated with anti-Ras antibody or B) separated by SDS-PAGE. Immunoprecipitates from A) were separated by SDS-PAGE and blotted with anti- Ras antibody whereas samples from B) were blotted with anti-Rap1A antibody as described hereafter.
  • Figure 16 shows: Lane 1, control; lane 2,
  • lovastatin lane 3, reduced 2a (200 ⁇ M); lane 4, 4 (100 ⁇ M); lane 5, 4 (50 ⁇ M); lane 6, 4 (25 ⁇ M); lane 7, 5; lane 8, 8.
  • Figure 16A (lane 1) shows that cells treated with vehicle contain only processed Ras whereas cells treated with lovastatin (lane 2) contained both processed and unprocessed Ras indicating that lovastatin inhibited Ras
  • Lovastatin an HMG-CoA reductase inhibitor which inhibits the biosynthesis of farnesylpyrophosphate and
  • geranylgeranylpyrophosphate is used routinely as a positive control for inhibition of processing of both geranylgeranylated and farnesylated proteins (36, 37, 39, 40, 51).
  • Cells treated with reduced Cys-4ABA-Met 3 in its free carboxylate forms did not inhibit Ras processing.
  • the corresponding methyl ester of 2a (200 ⁇ M) inhibited FTase ( Figure 16A, lane 3). This is consistent with previous work that showed that neutralization of the carboxylate of CAAX peptides enhances their ability to inhibit Ras processing (37, 40, 51).
  • compound 4 has a free carboxylate negative charge, it was able to enter cells and potently inhibit Ras processing (lane 4, 100 ⁇ M compound 4).
  • Rap1A a small G-protein that is geranylgeranylated (49, 50).
  • Cells were treated with lovastatin or peptidomimetics exactly as described for Ras processing experiments.
  • Control cells contained only the geranylgeranylated Rap1A ( Figure 16B, lane 1) whereas lovastatin-treated cells contained both processed and unprocessed forms of Rap1A

Abstract

L'invention a pour objet des composés inhibiteurs des prényle transférases, particulièrement, des farnysyltransférases et des géranylgéranyle transférases I. L'invention traite également de procédés de préparations de ces composés, de compositions pharmaceutiques contenant ces composés et de leurs procédés d'utilisation.
PCT/US1996/001559 1995-01-12 1996-01-11 Inhibiteurs des prenyle transferases WO1996021456A1 (fr)

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CA2207252A CA2207252C (fr) 1995-01-12 1996-01-11 Inhibiteurs des prenyle transferases
EP96905380A EP0794789A4 (fr) 1995-01-12 1996-01-11 Inhibiteurs des prenyle transferases
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US08/371,682 1995-01-12
US08/371,682 US5705686A (en) 1993-05-18 1995-01-12 Inhibition of farnesyl transferase
US08/451,839 1995-05-30
US08/451,839 US5834434A (en) 1993-05-18 1995-05-30 Inhibitors of farnesyltransferase
US55255495A 1995-11-03 1995-11-03
US08/552,554 1995-11-03
US08/582,076 1996-01-02
US08/582,076 US6011175A (en) 1993-05-18 1996-01-02 Inhibition of farnesyltransferase

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WO1998045266A1 (fr) * 1997-04-04 1998-10-15 Ferring B.V. Derives de 3-aminopyridine pour le traitement de maladies inflammatoires et d'affections malignes
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WO2000003743A2 (fr) * 1998-07-15 2000-01-27 Mitotix, Inc. Compositions et procedes inhibant la proliferation fongique
WO2000027803A1 (fr) * 1998-11-05 2000-05-18 Jomaa, Hassan Amides de la cysteine comme inhibiteurs de la farnesyltransferase
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US8003342B1 (en) 1990-04-18 2011-08-23 Board Of Regents, The University Of Texas System Method for identifying farnesyl transferase inhibitors
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EP0794789A4 (fr) 1999-05-26
CA2207252A1 (fr) 1996-07-18
JP4138826B2 (ja) 2008-08-27
AU4915796A (en) 1996-07-31
JPH10512266A (ja) 1998-11-24
MX9705273A (es) 1998-06-30
JP3929069B2 (ja) 2007-06-13
JP2007016035A (ja) 2007-01-25
EP0794789A1 (fr) 1997-09-17
CA2207252C (fr) 2014-02-25

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