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  • 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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Definitions

• the present invention relates to novel peptidomimetics and other compounds which are useful as inhibitors of protein isoprenyl transferases (particularly protein farnesyltransferase and geranylgeranyltransferase) and as anti-cancer drugs, to compositions containing such compounds and to methods of use.
• Ras proteins are plasma membrane-associated GTPases that function as relay switches that transduce biological information from extracellular signals to the nucleus (29-31) .
• Ras proteins cycle between the GDP- (inactive) and GTP- (active) bound forms to regulate proliferation and differentiation.
• extracellular signals such as epidermal and platelet derived growth factor (EGF and PDGF)
• EGF and PDGF epidermal and platelet derived growth factor
• binding of the growth factors to tyrosine kinase receptors results in autophosphorylation of various tyrosines which then bind src-homology 2 (SH2) domains of several signaling proteins.
• a cytosolic complex of GRB-2 and a ras exchanger (m-SOS-1) , is recruited by the tyrosine phosphorylated receptor where mSOS-1 catalyzes the exchange of GDP for GTP on Ras, hence activating it.
• GTP-bound Ras recruits Raf, a serine/threonine kinase, to the plasma membrane where it is activated.
• Raf triggers a kinase cascade by phosphorylating mitogen-activated protein (MAP) kinase/extracellular-regulated protein kinase (ERK) kinase (MEK) which in turn phosphorylates MAP Kinase on threonine and tyrosine residues.
• MAP mitogen-activated protein
• ERK extracellular-regulated protein kinase
• MEK MAP Kinase
• Activated MAP Kinase translocates to the nucleus where it phosphorylates transcription factors
• Ras oncogenes are the most frequently identified activated oncogenes in human tumors (1- 3) . In a large number of human cancers, Ras is
• 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) .
• p21Ras farnesyltransferase the enzyme responsible for catalyzing the transfer of farnesyl, a 15-carbon isoprenoid, from FPP to the cysteine of the CA- ⁇ X carboxyl terminus of p2lras, has been purified to homogeneity from rat brain (15,16) .
• 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) .
• p21ras farnesylation and subsequent membrane association are required for p21ras transforming activity (13) .
• p21ras farnesyltransferase would be a useful anticancer therapy target. Accordingly, an intensive search for inhibitors of the enzyme is underway (18-24, 33-44) .
• Potential inhibitor candidates are CA ⁇ X tetrapeptides which have been shown to be farnesylated by p21ras farnesyltransferase and appear to be potent inhibitors of this enzyme in vitro (15,18,21-24) .
• CA X A 2 X peptides with the greatest inhibitory activity are those where A_ and A 2 are hydrophobic peptides with charged or hydrophilic residues in the central positions demonstrating very little inhibitory activity (18,21,23).
• a major drawback with the use of peptides as therapeutic agents is their low cellular uptake and their rapid inactivation by proteases.
• the research efforts directed towards farnesyltransferase and the inhibition of its activity are further illustrated by the following patents or published patent applications: U.S. 5,141,851 WO 91/16340 WO 92/18465 EPA 0456180 Al EPA 0461869 A2
• 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.
• 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.
• the preferred compounds are said to be CV(D1)S and CV(Df)M, the amino acids being identified by conventional 3 letter and single letter abbreviations as follows:
• Valine Val V EPA 0523873 Al 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:
• Cys-Aaa 1 -Aaa 2 -Xaa where Aaa 1 and Aaa 2 are aliphatic amino acids and Xaa is an a ino 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, lie or Val and Xaa is Ser or Met.
• Preferred specific compounds are:
• U.S. patent 5,141,851 and WO 91/16340 disclose the purified farnesyl protein transferase and certain peptide inhibitors therefor, including, for example, CVIM, TKCVIM and KKSKTKCVIM.
• WO 92/18465 discloses certain farnesyl compounds which inhibit the enzymatic methylation of proteins including ras proteins.
• EPA 0456180 Al is directed to a farnesylprotein transferase assay which can be used to identify substances that block farnesylation of ras oncogene gene products while EPA 0512865 A2 discloses certain cyclic compounds that are useful for lowering cholesterol and inhibiting farnesylprotein transferase.
• geranylgeranyltransferase I An enzyme closely related to farnesyltransferase, geranylgeranyltransferase I (GGTase 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 ct/ ⁇ heterodimers that share the ⁇ subunit (61,62) .
• Cross-linking experiments suggested that both substrates (FPP and Ras CAAX) interact with the j ⁇ 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) . Furthermore, 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.
• a hydrophobic/aromatic spacer i.e. 4-aminobenzoic acid
• 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 frequently mutated form of Ras in human cancers (1,3).
• K-Ras4B is the most frequently mutated form of Ras in human cancers (1,3).
• K-Ras4B can be geranylgeranylated in vi tro, but with relatively low efficiency; its K., for GGTase I is 7 times higher than its YL_ for FTase (67) .
• GGTase I CAAX- based inhibitors that can block geranylgeranylation processing have not been reported.
• L wherein R- is i) hydrogen; ii) lower alkyl; iii) alkenyl; iv) alkoxy; v) thioalkoxy; vi) halo; vii) haloalkyl; viii) aryl-L 2 -, wherein L 2 is absent, -CH 2 -, - CH 2 CH 2 -, -CH(CH 3 )-, -0-, -S(0) q wherein q is 0, 1, or 2, -N(R')- wherein R' is hydrogen or lower alkyl, or -C(0)- and aryl is selected from the group consisting of phenyl, naphthyl, tetrahydronaphthyl, indanyl and indenyl and the aryl group is unsubstituted or substituted; or ix) heterocyclic-L 3 - wherein L 3 is absent, -CH 2 -, -CH 2 CH 2 -,
• R la is hydrogen or lower alkyl
• R 12a is hydrogen, loweralkyl or -C(0)0-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,
• R, 4 is loweralkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, thioalkoxyalkyl, hydroxyalkyl, aminoalkyl, carboxyalkyl, alkoxycarbonylalkyl, arylalkyl or alkylsulfonylalkyl and
• R 15 is hydrogen or a carboxy-protecting group
• A represents O or 2H
• R 0 represents SH, NH 2 , or C jj H y -SOj-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 or pharmaceutically acceptable salts or prodrugs thereof.
• 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 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 pharmaceutically acceptable salts and prodrugs of Formula (I) .
• 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 l t A 2 as in prior art inhibitors represented by the formula CA j A 2 X.
• the present compounds are consequently free from peptidic amide bonds. It is also to be noted that the present compounds are not farnesylated by the enzyme. They are, therefore, true inhibitors, not just alternative substrates. This may explain the high inhibitory action of the present compounds relative to their parent compounds which are farnesylated.
• a further important feature of the invention is the provision of the compounds of Formula (I) in the form of pro-drugs .
• terminal end groups amino, cysteine sulfur and carboxy groups
• 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.
• pro-drug aspect of the invention is applicable not only to the compounds of the invention but also to prior peptide inhibitors CA.A 2 X as well as any other peptide with potential for biological uses for the purpose of improving the overall effectiveness of such compounds, as hereinafter described.
• a further modification involves the provision of CA ⁇ X tetrapeptides or CSX 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 X A 2 X portion of the CAiA-jX tetrapeptide inhibitors with a non-amino acid component while retaining the desired farnesyltransferase inhibiting activity.
• These compounds may be illustrated by Formula (II) :
• C is 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 ⁇ X, as hereinafter described.
• the invention also 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 degradation inside cells while retaining the structural features required for FTase inhibition.
• 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 0 or 2H
• R 0 represents SH, H 2 , or C x H y -SOj-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
• R is a biphenyl substituted with one or more -COOH groups and/or lower alkyl, e.g., methyl, as represented by the formula:
• R_ 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 0.
• This formula represents a series of 4-amino-3 ' -carboxybiphenyl derivatives which mimic the Val-lie-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.
• CVIM 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.
• 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 farnesyl ⁇ transferase inhibitors.
• a further important feature of the invention is the provision of the compounds of the invention in the form of pro-drugs.
• pro-drug is meant a compound to which in vivo modification occurs to produce the active compound.
• 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 inhibitors to inhibit p21ras farnesyltransferase or geranylgeranyl transferase in any host containing these enzymes. This includes both in vitro and in vivo use.
• Compounds which inhibit farnesyltransferase notably human tumor p21ras farnesyltransferase, and consequently inhibit the farnesylation of the oncogene protein Ras, 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.
• 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.
• 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.
• Typical carriers for tablet or capsule use include, for example, lactose or corn starch.
• 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 pharmaceutical or chemotherapeutic agents in dosages of .1 to 1000 mg/kg body weight, preferably 1 to 500 mg/kg body weight and most preferably 10-50 mg/kg body weight. The required dose for a given individual or disease will vary, but can be determined by ordinary skilled practitioners using routine methods.
• the compounds 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. Means of determining suitable carriers and diluents are well known in the pharmaceutical arts.
• 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.
• carboxy protecting groups are will known to those skilled in the art, having been extensively used in the protection of carboxyl groups in the penicillin and 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 compounds containing carboxyl groups can be found on pages 14-21 of "Bioreversible Carriers in Drug Design: Theory and Application", edited by E.B. Roche, Permagon Press, New York, (1987) which is hereby incorporated by reference.
• Representative carboxy protecting groups are Cl to C8 loweralkyl (e.g.
• 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, isovaleryloxymethyl, 1- (propionyloxy) -1-ethyl, 1- (pivaloyloxyl) -1-ethyl, 1-methyl-1- (propionyloxy) - 1-ethyl, pivaloyloxymethyl, propionyloxymethyl and the like; cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl, such as benzoyloxymethyl, benzoyloxyethyl and the like; arylalkylcarbonyloxyalkyl, such as benzylcarbonyloxyalkyloxy
• 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 , aroyloxyalkyl or an arylalkylcarbonyloxyalkyl ester.
• the protected carboxy group is a loweralkyl, cycloalkyl or arylalkyl ester, for example, methyl ester, ethyl ester, propyl ester, isopropyl ester
• 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(0)-0- wherein R 29 is a loweralkyl group.
• alkanoylaminoalkyl refers to a loweralkyl radical to which is appended R 71 -NH- wherein R 71 is an alkanoyl group.
• alkanoyloxy refers to R 29 C(0)-0- wherein R 29 is a loweralkyl group.
• alkanoyloxyalkyl refers to a loweralkyl radical to which is appended an alkanoyloxy group.
• 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 3o ° ⁇ 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 0-R 32 0- wherein R 31 is loweralkyl as defined above and R 32 is an alkylene radical.
• alkoxyalkoxy groups include methoxymethoxy, ethoxymethoxy, t- butoxymethoxy and the like.
• 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(0)-0- 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, isopropoxycarbonyl and the like.
• alkoxycarbonylaklyl refers to an alkoxylcarbonyl group as previously defined appended to a loweralkyl radical. Examples of alkoxycarbonylaklyl include methoxycarbonylmethyl, 2-ethoxycarbonylethyl and the like.
• 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 -0- 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) -J-JH- 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(0) 2 - wherein R 34 is a loweralkyl group.
• alkylsulfonylalkyl refers to a loweralkyl radical to which is appended an alkylsulfonyl group.
• alkynyl refers to a straight or branched chain hydrocarbon containing from 2 to 10 carbon atoms and also containing at least one carbon-carbon triple bond.
• 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 carbon-carbon triple bond. Examples of alkynylene include
• amino refers to - NH 2 .
• aminoalkyl refers to a loweralkyl radical to which is appended an amino group.
• aroyloxyalkyl refers to a loweralkyl radical to which is appended an aroyloxy group (i.e., R 61 -C(0)0- 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 can be unsubstituted or substituted with one, two or three substituents independently selected from loweralkyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino, hydroxy, halo, mercapto, nitro, cyano, carboxaldehyde, carboxy, alkoxycarbonyl, haloalkyl-C(O) -NH-, haloalkenyl-C(0) -NH- and carboxamide .
• 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 to which is appended R 68 -0-C(0) -0- 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(0)0- wherein R 62 is an arylalkyl group) .
• arylalkyloxycarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 67 -0-C (0) -0- wherein R 67 is an arylalkyl group.
• aryloxyalkyl refers to a loweralkyl radical to which is appended R 65 -0- wherein R SB is an aryl group.
• aryloxthioalkoxyalkyl refers to a loweralkyl radical to which is appended R 75 -S- wherein R 75 is an aryloxyalkyl group.
• aryloxycarbonyalkyl refers to a loweralkyl radical to which is appended R 65 -0-C (0) -O- wherein R 65 is an aryl group.
• arylsulfonyl refers to R 3 € S(0) 2 - wherein R 3 € is an aryl group.
• arylsulfonyloxy refers to R 37 S(0) 2 0- 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(0)H.
• carboxylate refers to the group -C(0)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 appended a cycloalkanoyl group (i.e., R ⁇ 0 -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 appended a cycloalkyl group.
• Representative examples of cycloalkylalkyl include cyclopropylmethyl, cyclohexylmethyl, 2- (cyclopropyl)ethyl, adamantylmethyl and the like.
• cycloalkyloxycarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 64 -0-C(0) -O- wherein R 64 is a cycloalkyl group.
• dialkoxyalkyl refers to a loweralkyl radical to which is appended two alkoxy groups.
• dialkylamino refers to R 3 ⁇ R 39 N- wherein R 38 and R 39 are independently selected from loweralkyl, for example, dimethylamino, diethylamino, methyl propylamino, and the like.
• dialkylaminoalkyl refers to a loweralkyl radical to which is appended a dialkylamino group.
• dialkyaminocarbonylalkyl refers to a loweralkyl radical to which is appended R 73 -C(0)- wherein R 73 is a dialkylamino group.
• dithioalkoxyalkyl refers to a loweralkyl radical to which is appended two thioalkoxy groups.
• 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 nitrogen atoms; one oxygen atom; one sulfur atom; one nitrogen and one sulfur atom; one nitrogen and one oxygen atom; two oxygen atoms in non-adjacent positions; one oxygen and one sulfur atom in non- adjacent positions; two sulfur atoms in non- adjacent positions; two sulfur atoms in adjacent positions and one nitrogen atom; two adjacent nitrogen atoms and one sulfur atom; two non- adjacent nitrogen atoms and one sulfur atom; two non-adjacent nitrogen atoms and one oxygen atom.
• 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 cyclopenene ring and another monocyclic heterocyclic ring (for example, indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl or benzothienyl and the like) .
• Heterocyclics include: pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, furyl, thienyl, thiazolidin
• Heterocyclics also include compounds of the formula jr-*
• X* is -CH 2 -, -CH 2 0- or -O- and Y* is -C(0)- or - (C(R") 2 ) V - wherein R" is hydrogen or Ci-d-alkyl and v is 1, 2 or 3 such as 1, 3-benzodioxolyl, 1,4- benzodioxanyl and the like.
• heterocyclic alkyl refers to a heterocyclic group as defined above appended to a loweralkyl radical as defined above.
• heterocyclic alkyl examples include 2- pyridylmethyl, 4-pyridylmethyl, 4-quinolinylmethyl and the like.
• heterocycliccarbonyloxyalkyl refers to a loweralkyl radical to which is appended R 72 -C(0)-0- 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 comprising one to ten carbon atoms, including methyl, ethyl, propyl, isopropyl, n-butyl, neopentyl and the like.
• 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 processes for preparing the compounds of formula (l)-(Xll) and to the synthetic intermediates useful in such processes.
• compositions which comprise a compound of the present invention in combination with a pharmaceutically acceptable carrier.
• pharmaceutical compositions which comprise a compound of the present invention in combination with another chemotherapeutic agent and a pharmaceutically acceptable carrier.
• a method for inhibiting protein isoprenyl transferases i.e., protein farnesyltransferase and/or geranylgeranyltransferase
• a method- for treatment of conditions mediated by farnesylated or geranylgeranylated proteins for example, treatment of Ras associated tumors in humans and other mammals.
• a method for inhibiting or treating cancer in a human or lower mammal comprising administering to the patient a therapeutically effective amount of a compound of the invention alone or in combination with another chemotherapeutic agent.
• 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, digluconate, cyclopentanepropionate, dodeoylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate, maleate, methanesulfonate, nicotinate, 2- naphthalenesulfonate, oxalate, pamoate, pectinate, pers
• 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
• Basic addition salts can be prepared in si tu 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.
• Such 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. Other features of the invention will also be hereinafter apparent.
• FIG. 1 Ras CAAX peptidomimetics and FTase/GGTase I activities
• B. FTase and GGTase I inhibition assays were carried out as described in Example 12 by determining the ability of FTI-276 to inhibit the transfer of farnesyl and geranylgeranyl to recombinant H-Ras-CVLS and H- Ras-CVLL, respectively. The data are representative of at least three different experiments.
• H-RasF cells were treated with various concentrations of FTI-277, lysed and the lysates immunoblotted with anti-Ras or anti-RaplA antibodies as described in Example 13.
• B. 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,
• FIG. 3 Effects of FTI-277 on Ras/Raf Association.
• pZIPneo, H-RasF, H-RasGG and S186 cells were treated with vehicle or FTI-277 (5 ⁇ M) , homogenized and the membrane (A) and cytosolic (B) fractions were separated and immunoprecipitated by an anti-Raf antibody. The immunoprecipitates were then separated by SDS-PAGE and immunoblotted with anti-RAS antibody as described in Example 14. Data is representative of three different experiments.
• FIG. 4 Effects of FTI-277 on Ras Nucleotide Binding and Raf Kinase Activity
• A 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 described in Example 15.
• B pZIPneo and H-RasF cells were treated with vehicle or FTI-277, lysed and cells lysates immunoprecipitated with an anti- Raf antibody.
• Raf kinase was assayed by using a 19-mer autophosphorylation peptide as substrate as described in Example 16. Data are representative of three different experiments.
• Figure 5 Effect of FTI-277 on Oncogenic Activation of MAPK
• H-RasF cells were treated with various concentrations of FTI-277, cells lysed and lysates run on SDS-PAGE and immunoblotted with anti-MAPK antibody.
• B pZIPneo, H-RasF, H-RasGG, Raf, and S186 cells were treated with vehicle of FTI-277 (5 ⁇ M) , lysed and cells lysates processed as for A. Data are representative of two different experiments.
• FTI-276 inhibits selectively Ras processing and oncogenic Ras activation of MAP Kinase.
• NIH 3T3 cells transfected with empty vector (pZIPneo) , oncogenic (GTP-locked) farnesylated Ras (RasF) , geranylgeranylated Ras (RasGG) or 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 Na 3 V0 4 , 10 ⁇ g/ml Trypsin inhibitor, 25 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 2 mM PMSF) .
• 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 Na 3 V0 4 , 10 ⁇ g/ml Trypsin inhibitor, 25 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 2 mM
• mice The cells were harvested, resuspended in PBS and injected s.c. into the right and left flank of 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. On day 32 after s.c. implantation of tumors, 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) .
• FTI-276 50 mg/kg
• FIG. 9 Inhibition of Tumor Growth in Ras transformed cells by FTI-276 and FTI-277.
• Ras- transformed NIH 3T3 cells were implanted subcutaneously into nude mice, and daily intraperitoneal injections with FTI-276 and FTI- 277 (50 mg/kg) were started when the tumors reached 50 mm 3 .
• FIG. 10 Inhibition of Tumor Growth in Raf transformed cells by FTI-276 and FTI-277.
• Raf- transformed NIH 3T3 cells were implanted subcutaneously into nude mice, and daily intraperitoneal injections with FTI-276 and FTI- 277 (50 mg/kg) were started when the tumors reached 50 mm 3 .
• Lysates (25 ⁇ g) were electrophoresed on a 12.5% SDS-PAGE and immunoblotted with anti-Ras antibody Y13-238 as described previously. The blots were then reprobed with anti-RaplA antibody (Santa Cruze Biotechnologies, Santa Cruz, California) .
• FIG. 13 Energy-minimized structural conformations for CVIM and farnesyltransferase inhibitor FTI-265.
• Figures 14A and B Comparison of FTase and GGTase I inhibition by FTI-265 .and FTI-271.
• FIG. 18 Disruption of H-Ras and RaplA processing.
• NIH 3T3 cells that overexpress oncogenic H-Ras were treated with various concentrations of FTI-277 (0-50 ⁇ M) or GGTI-286 (0-30 ⁇ M) .
• the cells were lysed and the lysates were electrophoresed on SDS-PAGE and immunoblotted with either anti-Ras or anti-RaplA antibodies as described in Example 3.
• U and P designate unprocessed and processed forms of the proteins. Data are representative of three independent experiments.
• 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 electrophoresed on SDS-PAGE and immunoblotted with anti-Ras antibodies as described in Example 3.
• U and P designate unprocessed and processed forms of Ras. The data are representative of three independent experiments.
• FIG. 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 electrophoresed on SDS-PAGE and immunoblotted with an anti-MAP kinase antibody.
• 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
• DTT dithiothreitol
• 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) 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.
• ⁇ is an aminomethylbenzoic acid (particularly 3-aminomethylbenzoic acid) are disclosed in U.S. Patent application No. 08/062,287, which is hereby incorporated herein by reference.
• the acid component of ⁇ is conveniently reacted with cysteine so that the amino group of ⁇ and the cysteine carboxyl group react to form an amido group, other reactive substituents in the reactants being suitably protected against undesired reaction.
• the amino group of ⁇ is reacted with a suitably protected cysteinal.
• the amino acid represented by X 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 conventional reaction conditions.
• the invention also includes the pharmaceutically acceptable salts of the compounds of Formula (I) .
• These may be obtained by reacting the free base or acid with the appropriate amount of inorganic or organic acid or base, e.g. an alkali metal hydroxide or carbonate, such as sodium hydroxide, an organic amine, e.g. trimethylamine or the like.
• 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 vi tro 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.
• 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.
• Typical carriers for tablet or capsule use include, for example, lactose or corn starch.
• 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. For human use, daily dosages are dependent on the circumstances, e.g. age and weight.
• 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 conformation of CVIM or like tetrapeptides C ⁇ X.
• 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) .
• ⁇ substituents which may be mentioned include those obtained by using aminomethyl- or aminocarboxylic acid derivatives of other cyclic hydrophobic compounds such as furan, quinoline, pyrrole, oxazole, imidazole, pyridine and thiazole. Generally speaking, therefore, the ⁇ substituent may be derived from any hydrophobic, non-peptidic aminoalkyl- or amino-substituted aliphatic, aromatic or heterocyclic monocarboxylic acid.
• 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.
• C, X, ⁇ and ⁇ are as previously described and ⁇ _ 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 CAjA-jX, to provide pro ⁇ drugs.
• the terminal amino groups and the cysteine sulfur can be reacted with benzyl chloroformate to provide carbobenzyloxy ester end groups while the terminal carboxy group at the other end of the compound is converted to an alkyl or aryl ester, e.g. the methyl ester.
• alkyl esters from 1 to 10 carbons in length include activated esters such as cyanomethyl or trifluoromethyl, cholesterol, cholate or carbohydrate derivatives.
• 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. Derivatization of the prior peptides C ⁇ X and the peptidomimetics described herein with lipophilic or hydrophobic, enzyme-sensitive moieties increases the plasma membrane permeability and cellular uptake of the compounds and consequently their efficiency in inhibiting tumor cell growth.
• carbobenzyloxy derivatives have been referred to as one way of functionalizing the peptides and peptidomimetics to improve efficiency, it will be appreciated that a variety of other groups may also be used for the purposes noted. Typical alternatives include 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 C3M 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 methyl-N-methylethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoe
• 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.
• the mixture was stirred overnight, diluted with more CH 2 C1 2 and was extracted 3 times each with 1M HC1, 1M NaHC0 3 and water.
• N-BOC-4-aminobenzoyl methionine methyl ester (3.53 g, 9.59 mmol) was placed into CH 2 C1 2 (30-35 ml) and to it was added 3M HCl/ Et 2 0 (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.
• 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) .
• a solution of mercuric chloride (0.75 g, 2.74 mmol) in CH 3 OH (13.7 ml) .
• a white precipitate began to form.
• the mixture was heated on a steam bath at 65°C for 35 minutes and then it was cooled and the precipitate was filtered and washed sparingly with cold CH 3 OH.
• the solid After drying for several minutes on the filter, the solid was placed into a 50 ml 3-neck flask fitted with a gas inlet and outlet. Approximately 20-30 ml of CH 3 OH was added and H 2 S gas was bubbled through the heterogeneous solution for 30 minutes. Upon addition of the gas, the white solution turned orange and then black. The solution was centrifuged and the clear, colorless liquid was dried to give a white foam. This solid was placed on the vacuum pump for a short period and then was taken up in CH 2 C1 2 (10 ml) and the product was precipitated with a 3-4M HC1/ Et 2 0 solution. The precipitate was collected by centrifugation and was washed with ether until pH was neutral.
• 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 C1 2 and was washed three times with 1M HC1 followed by brine. The organic solution was dried over Na 2 S0 4 and the solvent was removed in vacuo. The pale yellow solid was taken up in 3 ml of CH 2 C1 2 and the product was precipitated with 3-4 M HC1/ Et 2 0.
• N-B0C-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 C1 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 C1 2 (15-20 ml) and precipitated with 3M HC1/ Et 2 0 (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; 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 C1 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.
• 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 C1 2 (4 ml) and precipitated with 3-4M HCl/ Et 2 0 (12 ml) . The precipitate was washed as usual with Et 2 0 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 described above.
• HCl-4-amino-3-methoxybenzoyl methionine methyl ester (0.55 g, 1.58 mmol) in dry CH 2 C1 2 ( 8.7 ml) with Et 3 N (0.30 ml) was added to the mixture and was allowed to stir overnight under nitrogen.
• 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 C1 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 methionine in Example 1, and HPLC revealed that the product was impure.
• 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 methionine methyl ester in Example 1.
• 4-amino-2- phenylbenzoyl methionine methyl ester (0.24g, 0.66 mmol) in dry CH 2 C1 2 (3.5 ml) was added and the mixture was allowed to stir overnight under nitrogen.
• N-BOC-S-trityl-cysteine-4-amino-2- (3,5- di ethylphenyl)benzoyl methionine methyl ester
• 4-amino-2- (3 , 5-di ethylphenyl)benzoyl methionine methyl ester (0.10g, 0.26 mmol) was dissolved into dry CH 2 C1 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.
• the product was worked up as described for N-BOC-S-trityl- cysteine-4-aminobenzoyl methionine methyl ester in Example 1 and chromatographed on silica gel using a 1:1 hexanes and ethyl acetate elution mixture to give 0.12 g (56.0%) of pure material.
• N-BOC-4-amino-1-naphthoic acid 4-amino-l-naphthoic acid (0.86 g, 4.61 mmol) was dissolved into dioxane (9.2 ml) and 0.5 M NaOH (9.2 ml) .
• 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 C1 2 (6.4 ml) were reacted as described for N- BOC-4-aminobenzoyl methionine methyl ester in Example 1.
• N-BOC-4-amino-l-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-l-naphthoyl methionine methyl ester (0.25 g, 0.67 mmol) in dry CH 2 C1 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-3-methylbenzoyl methionine in Example 2, and 74.1 mg of the free acid was obtained.
• TFA-cysteine-4-amino-l-naphthoyl methionine (0.12 g, 0.15 mmol) was dissolved in CH 3 0H (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 0 precipitation and several reprecipitations 31.0 mg (18.3 %) of pure white material was obtained.
• Triethylamine (2.22 mL, 16 mmoL) and N,0- 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 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) . Lowering the temperature to -45°C did not improve the aldehyde percentage.
• 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 evaporation of solvents, the residue was extracted with ethyl acetate and concentrated sodium bicarbonate. The organic phase was dried and the solvents were evaporated.
• 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%.
• the precursor to the 2- naphthyl-, 2-thiophene-, 2-pyrrole-, and 2- pyridyl-4-aminobenzoic acid spacers can be prepared by reaction of 4-nitro-2-bromotoluene with naphthalene-2-boronic acid, thiophene-2- boronic acid, pyrrole-2-boronic acid, pyridine- 2,3- or 4-boronic acid.
• EXAMPLE 12 FTase and GGTase I Activity Assay
• 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 dithiothreitol (DTT) .
• the reaction was incubated at 30°C for 30 min with recombinant Ha-Ras-CVLS (11 ⁇ M) and [ 3 H] FPP (625 nM; 16.3 Ci/mmol) for FTase, and recombinant Ha-Ras-CVLL (5 ⁇ M) and [ 3 H] geranylgeranylpyrophosphate (525 nM; 19.0 Ci/mmol) for GGTase I.
• the peptidomimetics were mixed with FTase and GGTase before adding to the reaction mixture.
• 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 concentra ⁇ tions of FTI-277 or vehicle. On day 3, cells were washed one time with ice cold PBS and were collected and lysed by incubation for 30-60 min on ice in lysis buffer (41) . Lysates were cleared (14,000 rpm, 4°C, 15 min) and supernatants collected.
• Equal amounts of lysate were separated on a 12.5% SDS-PAGE, transfered to nitrocellulose, and a western blot performed using a anti-Ras antibody (Y13-238, ATCC) or anti-RaplA antibody (Santa Cruz Biotechnology, Santa Cruz, CA) .
• Antibody reactions were visualized using peroxidase-conjugated goat anti-rat IgG for Y13-
• EXAMPLE 14 Co-immunoprecipitation of Raf and Ras Cells were seeded on day 0 in 100 mm dishes in 10 ml Dulbecco's Modified Eagles Medium (GIBCO) supplemented with 10% calf serum (Hyclone) and 1% pen/strep (GIBCO) . On days 1 and 2 cells were treated with FTI-277 (5 ⁇ M) or vehicle (confluency of cells 40-60%) . On day 3, cells were collected by centrifugation in ice cold PBS.
• GEBCO Dulbecco's Modified Eagles Medium
• FTI-277 FTI-277
• vehicle confluency of cells 40-60%
• Cell pellets were then resuspended in ice cold hypotonic buffer (10 mM Tris, pH 7.5, 5 mM MgCl 2 , 1 mM DTT, 1 mM PMSF) and cells were sonicated to break up cell pellet to promote separation of cytosol and membrane.
• the cell suspension was then centrifuged at 2,000 rpm for 10 min to clear debris after which the supernatant was loaded in ultrocentrifuge tubes and spun for 30 min at 100,000 X g to SW Ti55 Rotor to separate membrane and cytosol fractions.
• the cytosol and membrane fractions were lysed on ice for 60 min in buffer containing 30 mM HEPES, pH 7.5, 1% TX-100, 10% glycerol, 10 mM NaCl, 5 mM MgCl2, 2 mM Na 3 V0 4 , 25 mM NaF, 1 mM EGTA, 10 ⁇ M soybean trypsin inhibitor, 25 ⁇ g/ml leupeptin, 10 ⁇ g/ml aprotinin, 2 mM PMSF) .
• the lysates were clarified by centrifugation.
• Equal amounts of cytosol and membrane fractions were immunoprecipitated using 50 ⁇ l of a 25% Protein-A Sepharose C1-4B suspension (Sigma) with 1 ⁇ f/ l anti-c-Raf-1 (SC133, Santa Cruz Biotechnology, Santa Cruz, CA) .
• the samples were tumbled at 4°C for 60 min and then washed 5 times in 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , 0.1% TX-100, 10% glycerol, 20 mM NaF.
• the final pellets were run on 12.5% SDS- PAGE, transferred to nitrocellulose, and immunoblotted for the presence of Ras using anti- Ras antibody (Y13-238) and immunoblotted for the presence of Raf (c-Raf-1, SC133, Santa Cruz Biotechnology, Santa Cruz, CA) . Detection was the same as above for Ras and RaplA processing.
• H-RasF cells were seeded and treated as above for Ras/Raf interaction and Ras and RaplA processing.
• cells were labeled overnight with [ 32 P] orthophosphate at 100 ⁇ Ci/mo (Amersham PBS13) in 10 ml DMEM-phosphate supplemented with 10% calf serum, 1 mg/ml BSA and 20 mM HEPES, pH 7.5.
• the medium was removed and cells were washed one time in ice-cold PBS, scraped from the plate with a cell scraper, collected and centrifuged.
• the cell pellet was resuspended in ice-cold hypotonic buffer listed above and the cytosol and membrane fractions were separated according to the above description for Ra/Raf association.
• the 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 inhibitor, 25 ⁇ g/ml leupeptin, 1 mM DTT, 1 mg/ml BSA.
• Lysates were cleared and equal amounts of protein were immunoprecipitated using anti-Ras antibody (Y13-259) along with 30 ⁇ l Protein A- Agarose goat anti-rat IgG complex (Oncogene Science) for 60 min at 4°C. 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.
• Immune complexes were spun down quickly and 6 ⁇ l of the supernatent was loaded onto PEI cellulose thin layer chromatography plates (20 cm X 20 cm) . Nucleotides were separated by chromatography in 78 g/linter ammonium formate, 9.6% (v/v) concentrated HCl. Plates were analyzed by autoradiogram.
• Raf-1 kinase was assayed by determining the ability of Raf to transfer phosphate from [ ⁇ - 32 P] ATP to a 19-mer peptide containing an autophosphorylation site.
• Membrane and cytosol fraction isolation and Raf immunoprecipitates were washed three times with cold HEPES buffer and twice with kinase buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 12 mM MnCl 2 , 1 mM DTT, 0.2% Tween 20.
• 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, Promega) for 30 min at 25°C.
• the sequence of the Raf-1 substrate peptide is IVQQFGFQRRASDDGKLTD.
• the phosphorylation reaction was terminated by spotting 50 ⁇ l aliquots of the assay mixture onto Whatman P81 for 40 min in 0.5% orthophosphoric acid and air dried. The amount of 32 P incorporated was determined by liquid scintillation counting.
• Fig. IB 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 S0 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 selective (100-fold) for FTase over the closely related GGTase I (Fig. IB) .
• FTI-276 inhibited the transfer of geranylgeranyl from [ 3 H]GG-PP to recombinant H-Ras-CVll with an IC S0 of 50 nM (Fig. IB) .
• This 100-fold selectivity is superior to the 15-fold selectivity of the parent compound, FTI- 249.
• Data for a number of other compounds of interest are shown in Table 1.
• FTI-277 the methylester of FTI-276, was used in experiments to measure inhibition of Ras processing.
• 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-RaplA antibodies.
• concentrations as low as 10 nN inhibited Ras processing but concentrations as high as 10 ⁇ M did not inhbit processing of the geranylgeranylated RaplA.
• 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. Processing of 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) ) .
• 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
• pZIPneo, H-RasF, and H-RasGG were treated with vehicle or FTI-277, membrane and cytosolic fractions were isolated and immunoprecipitated with anti-Raf antibody as described above.
• Raf did not associate with Ras in pZIPneo cells which did not contain GTP-locked Ras, as shown in Fig. 3.
• H-RasF and H-RasGG cells contain Ras/Raf complexes in the membrane, but not in the cytosolic fractions, as shown in Fig. 3.
• Treatment of these cells with FTI-277 resulted in the accumulation of Ras/Raf complexes in the cytosolic but not membrane fractions of H-RasF cells, but not in the H-RasGG cells (Fig 3) .
• non-processed Ras can associate with Raf in a non-membranous cytoplasmic environment was confirmed by transfecting NIH 3T3 cells with a GTP-locked Ras that lacks a farnesylated site and, therefore, remains in the cytoplasm (Ras mutant with a 61 leucine oncogenic mutation and a 186 serine mutation) and showing that these cells contained only cytoplasmic Ras/Raf complexes when immunoprecipitated with Raf and blotted with antiRas antibodies (Fig. 3) . In short, farnesylation is not required for Ras to bind to Raf.
• Ras/Raf complexes as described above.
• Ras-F cells only membrane fractions contained GTP- locked Ras, as shown in Fig 4A.
• 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 autophosphorylated peptide.
• Fig. 4B shows that oncogenic Ras-F induced activation of Raf in the plasma membrane and that treatment with FTI-277 suppressed this activation.
• cytoplasmic Ras/Raf complexes that were induced by FTI-277 had basal levels of Raf kinase activity that were comparable to those of the parental NIH 3T3 cell line pZIPneo (Fig. 4B) .
• Figures 3 and 4 demonstrate that oncogenic transformation with GTP-locked Ras results in the constitutive recruitment to the plasma membrane and subsequent activation of Raf.
• FTase inhibition by FTI-277 suppresses this activation by inducing the accumulation of Ras/Raf complexes in the cytoplasm where Ras is GTP-bound but Raf kinase is not activated.
• the fact that 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) .
• Pretreatment with FTI-277 results in a concentration dependent inhibition of oncogenic Ras activation of MAPK. Concentrations as low as 300 nM were effective and the block was complete at 1 ⁇ M. Taken together, Figs.
• FIG. 3 and 5 demonstrate that at least 50% inhibition of Ras processing is required for complete suppression of MAPK activation but that less than a 100% inhibition of Ras processing is required for complete suppression of MAPK activation by Ras.
• a series of NIH 3T3 cell lines transformed with various oncogenes was used to determine whether the inhibition of MAPK activation is due to selectively antagonizing Ras function.
• Fig. 5B shows that FTI-277 was able to block H-RasF but not H-RasGG activation of MAPK. This is consistent with its ability to inhibit H-RasF but not H-RasGG processing (Fig. 2) .
• 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) .
• a nude mouse xenograft model was used.
• tumors from two human lung carcinoma cell lines are implanted subcutaneously.
• One of these (Calu-1) harbors a K-Ras oncogenic mutation and has a deletion of the tumor suppressor gene p53.
• the other human lung carcinoma (NCI-H180) has no Ras mutations.
• Thirty two days after s.c. implantation when the tumors reached sizes of 60 to 80 mm 3 the mice were randomly separated into control and treated groups (4 animals per group; each animal had a tumor on both the right and the left flank) .
• Figure 7A shows that tumors from control animals treated with saline once daily starting on day 36 grew to an average size of 566 mm 3 over a period of 64 days from tumor implantation.
• tumors treated once daily with FTI-276 50 mg/kg
• FTI-277 the methylester of FTI-276
• Figure 8) the animals were treated once daily with 50 mg/kg for 36 days (total cumulative does of 1.8 g/kg)
• no weight loss was observed and the animals appeared normal with no evidence of gross toxicity. This lack of toxicity was also observed in separate experiments where the dose was escalated to 180 mg/kg once daily.
• FTI-276 and FTI-277 essentially blocked tumor growth of Calu-I carcinoma with no evidence of gross toxicity.
• 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, respectively, with N-Boc-S-trityl cysteinal, to give the N-Boc-S-trityl, tert-butyl ester of (4) and (5) .
• Deprotection by trifluoroacetic acid and purification by reverse phase HPLC gave pure (4) and (5) .
• Delta-Pak C-18 300 A cartridge column inside a Waters 25x10 cm Radial Compression Module.
• Analytical HPLC was performed using a Rainin HP Controller and a Rainin UV-C detector with a Rainin 250x4.6 mm 5 ⁇ m Microsorb C-18 column.
• 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 purity of all the synthesized inhibitors was more than 98% as indicated by analytical HPLC.
• the azide 20 (900 mg, 2.91 mmol) was dissolved in 20 mL of methanol. A catalytic amount of 5% Pd on barium sulfate (90 mg) was added. This mixture was hydrogenated at 1 atm overnight. The catalyst was removed and the methanol was evaporated. The remaining residue was dissolved in a mixture of 0.5 N HCl (20 mL) and ether (20 mL) . The aqueous phase was neutralized with 1 N NaOH and extracted into methylene chloride. After the evaporation of solvents, a viscous oil was obtained (600 mg, 73%) .
• FTI-289 red.Cys-4-amino-2-phenyl-3' -carboxybiphenyl
• FTI-291 4- (3-aminoalanyl) -amino-3' -carboxybiphenyl
• FTI-295 4- (Ethylsulfonyl-3-aminoalanyl) -amino-3' -carboxy ⁇ biphenyl
• FTI-296 4- (Vinylsulfonyl-3-aminoalanyl) -amino-3' - carboxybipheny1
• 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.
• Recombinant H-Ras-CVLS was prepared as described previously (26) from bacteria (31) .
• Recombinant H-Ras-CVLL was prepared from bacteria (32) .
• EXAMPLE 23 Peptidomimetics Farnesylation Assay
• EJ3 cells were treated with peptidomimetics or vehicle for 20-24 h.
• Cells were lysed in lysis buffer (10 mM Na 2 HP0 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.
• lysis buffer 10 mM Na 2 HP0 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
• Ras protein was immunoprecipitated overnight at 4°C with 50 ⁇ g of anti-Ras antibody (Y13-259; hybridoma from ATCC, Rockville, MD) along with 30 ⁇ l Protein A-agarose goat anti-rat IgG complex (Oncogene Science, Uniondale, NY) .
• 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 nitrocellulose and subsequently blocked with 5% non-fat dry milk in PBS (containing 1% Tween 20 (PBS-T) and probed with Y13-259 (50 ⁇ g/ml in 3% non-fat dry milk in PBS-T) . Positive antibody reactions were visualized using peroxidase- conjugated goat anti-rat IgG (Oncogene Science, Uniondale, NY) and an enhanced chemiluminescence detection system (ECL; Amersham) .
• ECL enhanced chemiluminescence detection system
• RaplA processing assays 50 ⁇ g of cell lysates were electrophoresed as described above for Ras processing and transferred to nitrocellulose. These membranes were then blocked with 5% milk in Tris-buffered saline, pH 8.0, containing 0.5% Tween-20 and probed with anti- RaplA (l ⁇ g/ml in 5% milk/TBS-T; Santa Cruz Biotechnology, Santa Cruz, CA) . Antibody reactions were visualized using peroxidase- conjugated goat anti-rabbit IgG (Oncogene) and ECL chemiluminescence as described above.
• Table 4 indicates the IC 50 s obtained for FTase activity and GGTase-I activity, and the selectivity for a number of peptidomimetics of the invention.
• the IC S0 values given in Table 4 represent inhibition of FTase and GGTase I in vitro by the listed compounds.
• Numbers in parentheses indicate number of determinations. Where no number is given, at least two determinations were made.
• 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 lie at the X position are geranylgeranylated. As shown in Table 4, the present compounds do not significantly inhibit GGTase I and demonstrate much greater selectivity for FTase.
• Figure 14B shows that compound 4, which is a potent FTase inhibitor, is a very poor GGTase I inhibitor.
• This selectivity was much more pronounced than in the peptidomimetics 2 and 2a which were more selective for FTase relative to GGTase I by only 10 and 15- fold, respectively.
• 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) .
• 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; lane 6, compound 2a; lane 7, compound 4; lane 8, compound 5; lane 9, compound 8. The results shown indicate that the compounds of the invention are not farnesylated in contrast to the CAAX compounds. Data given are representative of two independent experiments.
• Figure 16A 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 processing.
• 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.
• Compound 4 was as potent as the methylester of its parent compound (2a) ( Figure 16A, lane 3) . Furthermore, 4 appears to be the first CAAX peptidomimetic that effectively inhibits Ras processing in whole cells directly without relying on cellular enzymes for activation. The hydrophobic character of the biphenyl group apparently compensates for the free carboxylate negative charge thus allowing the peptidomimetic to penetrate membranes and promoting its cellular uptake.
• Ras farnesylation inhibitors have also been investigated by determining their ability to inhibit processing of RaplA, a small G-protein that is geranylgeranylated (49, 50) .
• RaplA a small G-protein that is geranylgeranylated
• Cells were treated with lovastatin or peptidomimetics exactly as described for Ras processing experiments. Lysates were then separated by SDS-PAGE and immunoblotted with anti-RaplA antibody as described below. Control cells contained only the geranylgeranylated RaplA (Figure 16B, lane 1) whereas lovastatin-treated cells contained both processed and unprocessed forms of RaplA indicating, as expected, that lovastatin inhibited the processing of RaplA ( Figure 16B, lane 2) .
• AAX is completely replaced by a simple hydrophobic moiety.
• the most potent inhibitor in the CAAX series is Cys-Ile-Phe-Met (18, 22) with an IC 50 value of 30 nM.
• Peptidomimetic inhibitor 10 is as potent as CIFM despite the large difference between their structures.
• CA.A 2 X such as CIFM
• Table 4 shows that the designed non-peptide CAAX mimetics (such as compound 4) are not substrates for farnesylation. This lack of farnesylation by FTase may be due to the inhibitor binding to the enzyme in a conformation that does not permit farnesyl transfer to the thiol group.
• Geranylgeranyl tranferase Inhibitors The carboxyl terminal CAAX tetrapeptide of
• Ras is a substrate for FTase and serves as a target for designing inhibitors of this enzyme with potential anticancer activity (33) .
• Its cell-permeable methyl ester FTI-277 inhibits H-Ras processing in whole cells with an IC 50 of 100 nM (66) .
• FTI-276 is highly selective (100-fold) for FTase over GGTase I (Table 5) .
• FTI-276 and FTI-277 were prepared as described above.
• GGTase I inhibitors GGTI-287 and GGTI-286 were prepared from 2-phenyl- 4-nitrobenzoic acid (66) by reaction with L- leucine methyl ester followed by reduction with stannous chloride.
• the 4-nitro-2-phenylbenzoyl- (L) -methionine methyl ester (3.04 g, 7.83 mmol) was dissolved into 100 mL of ethyl acetate followed by the addition of stannous chloride hydrate (8.84 g, 39 mmol) .
• the mixture was refluxed for 2 hr and then extracted with a mixture of ethyl acetate and concentrated sodium bicarbonate. After the evaporation of solvents, the residue was dissolved in methylene chloride followed by addition of 3N hydrogen chloride in ether.
• This compound was prepared using the same method as for the preparation of methionine derivative (See Example 26, section B) , using 4- amino-2-phenylbenzoyl- (S) -leucine methyl ester and N-Boc-S-trityl- (L) -cysteinal as starting materials.
• This compound was prepared with the same method as for the preparation of methionine derivative (see Example 26, section C) , using 4- [2 (R) -tert-butoxycarbonyl-3- triphenylmethylthiopropyl] amino-2-phenylbenzoyl-
• This compound was prepared from the N-Boc-S-trityl protected form (section K) by saponification followed with acidic cleavage by trifluoroacetic acid. The pure compound was obtained through preparative HPLC. H NMR showed complicated diastereomers caused by the restricted rotation of aryl-aryl bond.
• FTase and GGTase I activities from 60,000 x g supernatants of human Burkitt lymphoma (Daudi) cells (ATCC, Rockville, MD) were assayed exactly as described previously for FTase (41) .
• Inhibition studies were performed by determining the ability of Ras CAAX peptidomimetics to inhibit the transfer of [ 3 H] -farnesyl and [ 3 H] - geranylgeranyl from [ 3 H] FPP and [ 3 H]GGPP to H-ras- CVLS and H-Ras-CVLL, respectively (41) .
• H-Ras cells (45) and K-Ras4B Cells (32) were kind gifts from Dr. Channing Der and Dr. Adrienne Cox (University of North Carolina, Chapel Hill) . Means of obtaining these cell lines will be easily recognized by the skilled practitioner.
• Cells were seeded on day 0 in 100 mm dishes in Dulbecco's modified Eagles medium supplemented with 10% calf serum and 1% penicillin- streptomycin. On days 1 and 2, cells were refed with medium containing various concentrations of FTI-277, GGTI-286 or vehicle (10 mM DTT in DMSO) .
• Lysates were cleared (14,000 rpm, 4°C, 15 min) and equal amounts of protein were separated on a 12.5% SDS- PAGE, transferred to nitrocellulose, and immunoblotted using an anti-Ras antibody (Y13-259, ATCC) or an anti-RapIA antibody (SC-65, Santa Cruz Biotechnology, Santa Cruz, CA) .
• Antibody reactions were visualized using either peroxidase- conjugated goat anti-rat IgG (for Y13-259) , or peroxidase-conjugated goat anti-rabbit IgG (for RaplA) and an enhanced chemiluminescence detection (ECL, Amersham Corp.) , as described previously (41) .
• the substitution of methionine in FTI-276 by a leucine in GGTI-287 (Fig. 17) increased the potency towards GGTase I by approximately 10-fold (Table 5) .
• GGTI-287 the cell-permeable methyl ester derivative of GGTI-287, GGTI-286 (Fig. 17) , was synthesized and used to treat NIH 3T3 cells which overexpress oncogenic H-Ras-CVLS (31) .
• Cell lysates were electrophoresed on SDS-PAGE and immunoblotted with an anti-Ras antibody as described in Example 28.
• Figure 18 shows that accumulation of unprocessed H-Ras did not occur at concentrations lower than 30 ⁇ M GGTI-286. Therefore, GGTI-286 is not a good inhibitor of H- Ras processing in whole cells.
• GGTI-286 is more than 15-fold selective for inhibition of geranylgeranylation over farnesylation processing (Table 5) .
• This data is in direct contrast to the FTase specific inhibitor FTI-277 which inhibited H-Ras and RaplA processing with IC 50 s of 100 nM and 50 ⁇ M, respectively (Fig. 18) .
• GGTI-286 is 25-fold more potent than FTI-277 at inhibiting geranylgeranylation in whole cells (Table 5) .
• EXAMPLE 32 JnhiJ ition of K-Ras4B Function by GGTI-286 The ability of GGTI-286 to inhibit the processing and signaling of oncogenic K-Ras4B was then evaluated.
• NIH 3T3 cells which overexpress oncogenic K-Ras4B (32) were treated either GGTI- 286 (0-30 ⁇ M) or FTI-277 (0-30 ⁇ M) and the lysates were immunoblotted with an anti-Ras antibody as described under Example 28.
• Figure 19 shows that GGTI-286 inhibited potently the processing of K- Ras4B with an IC 50 of 2 ⁇ M.
• GGTI- 286 inhibited K- Ras4B processing at concentrations (1-3 ⁇ M) (Fig. 19) that had no effect on the processing of farnesylated H-Ras (Fig. 18) .
• GGTI-286 To determine whether inhibition of K-Ras4B processing by GGTI-286 results in disruption of oncogenic signaling, the ability of GGTI-286 to antagonize oncogenic K-Ras 4B constitutive activation of MAP kinase was examined.
• Activated MAP kinase is hyperphosphorylated and migrates slower than hypophosphorylated (inactive) MAP kinase on SDS-PAGE (43, 66) .
• Figure 20 shows that K-Ras4B transformed cells contained mainly activated MAP kinase. Treatment of these cells with the FTase-specific inhibitor FTI-277 (0-30 ⁇ M) did not inhibit MAP kinase activation until 30 ⁇ M (Fig. 20) .
• GGTI-286 inhibited MAP kinase activation with an IC 50 of 1 ⁇ M and the block was complete at 10 ⁇ M.
• GGTI-286 blocked oncogenic K-Ras4B MAP kinase activation at a concentration (10 ⁇ M) where FTI-277 had no effect.
• oncogenic H-Ras activation of MAP kinase was inhibited only slightly by GGTI- 286 whereas FTI-277 completely blocked this activation at 3 ⁇ M (Fig. 20) .
• GGTI- 286 blocked K-Ras4B activation of MAP kinase at a concentration (10 ⁇ M) that had little effect on H- Ras activation of MAP kinase (Fig. 20) .
• GGTI-286 is a potent and highly selective inhibitor of K- Ras4B processing and activation of oncogenic signalling.
• K-Ras4B transformed NIH-3T3 cells were implanted subcutaneously in nude mice. When the tumors reached sizes of 50-100 mm 3 , the mice were randomly separated into control and treated groups (5 animals per group, each animal had a tumor on both the right and the left flank) .
• Figure 22 shows that tumors from control animals treated with saline once daily grew to an average size of 2900 mm 3 over a period of two weeks.
• GGTI- 286 In contrast, tumors from animals treated once daily with GGTI- 286 (25 mg/kg or 50 mg/kg) grew to a size of 1600 mm 3 or 900 mm 3 , respectively (Fig. 22) . Thus, GGTI-286 inhibited tumor growth by 50% and 70%, respectively.
• GGTI- 286 not only as a potent antagonist of K-Ras4B oncogenic signaling in cultured cells, but also as an inhibitor of tumor growth in whole animals.

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