GB2261373A - Inhibitors of farnesyl protein transferase as anti-cancer agents - Google Patents

Abstract

Pharmaceutical compositions containing the compounds of structural formula (I): <IMAGE> wherein Z1, Z2 and Z3 are each H, or C1-5 alkyl optionally substituted by phenyl or phenyl substituted with methyl, methoxy, halogen or hydroxy, and methods of treatment utilizing these compositions for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras, of utility in treating cancer.

Description

TITLE OF THE INVENTION INHIBITORS OF FARNESYL PROTEIN TRANSFERASE BACKGROUND OF THE INVENTION The Ras gene is found activated in many human cancers, including colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias.

Biological and biochemical studies of Ras action indicate that Ras functions like a G-regulatory protein, since Ras must be localized in the plasma membrane'and must bind with GTP in order to transform cells CGibbs, J. et al., Microbiol. Rev. 53:171-286 (1989). Forms of Ras in cancer cells have mutations that distinguish the protein from Ras in normal cells.

At least 3 post-translationai modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or "Cys-Aaal-Aaa2-Xaa" box (Aaa is an aliphatic amino acid, the Xaa is any amino acid) (Willumsen et al., Nature 310:583-586 (1984)). Other proteins having this motif include the Ras-related GTP-binding proteins such as Rho, fungal mating factors, the nuclear lamins, and the gamma subunit of transducin.

Farnesylation of Ras by the isoprenoid farnesyl pyrophosphate (FPP) occurs in vivo on Cys to form a thioether linkage (Hancock et al., Cell 57:1167 (1989); Casey et al., Proc. Natl. Acad. Sci.

USA 86:8323 (1989)). In addition, Ha-Ras and N-Ras are palmitoylated via formation of a thioester on a Cys residue near a C-terminal Cys farnesyl acceptor (Gutierrez et al., EMBO J. 8:1093-1098 (1989); Hancock et al., Cell 57: 1167-1177 (1989)). Ki-Ras lacks the palmitate acceptor Cys. The last 3 amino acids at the Ras C-terminal end are removed proteolytically, and methyl esterification occurs at the new C-terminus (Hancock et al., ibid). Fungal mating factor and mammalian nuclear lamins undergo identical modification steps (Anderegg et al., J.Biol. Chem. 263:18236 (1988); Farnsworth et ii., J.

Biol. Chem. 264:20422 (1989)).

Inhibition of Ras farnesylation in vivo has been demonstrated with lovastatin (Merck & Co., Rahway, NJ) and compactin (Hancock et al., acid; Casey et al., acid; Schafer et al., Science 245:379 (1989)). These drugs inhibit HMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids and the farnesyl pyrophosphate precursor.

It has been shown that a farnesyl-protein transferase using farnesyl pyrophosphate as-a precursor is responsible for Ras farnesylation. (Reiss et al., Cell, 62: 81-88 (1990); Schaber et al., J. Biol.

Chem;, 265:14701-14704 (1990); Schafer et Science, 249: 1133-1139 (1990); Manne et al., Proc.

Natl. Acad. Sci USA, 87: 7541-7545 (1990)).

Inhibition of farnesyl-protein transferase and, thereby, of farnesylation of the Ras protein, blocks the ability of Ras to transform normal cells to cancer cells. The compounds of the invention inhibit Ras farnesylation and, thereby, generate soluble Ras which, as indicated infra, can act as a dominant negative inhibitor of Ras function. While soluble Ras in cancer cells can become a dominant negative inhibitor, soluble Ras in normal cells would not be an inhibitor.

A cytosol-localized (no Cys-Aaa1- Aaa2-Xaa box membrane domain present) and activated (impaired GTPase activity, staying bound to GTP) form of Ras acts as a dominant negative Ras inhibitor of membrane-bound Ras function (Gibbs et al., Proc.

Natal. Acad. Sci. USA 86:6630-6634(1989)). Cytosollocalized forms of Ras with normal GTPase activity do not act as inhibitors. Gibbs çt al., acid, showed this effect in Xenopus oocytes and in mammalian cells.

Administration of compounds of the invention to block Ras farnesylation not only decreases the amount of Ras in the membrane but also generates a cytosolic pool of Ras. In tumor cells having activated Ras, the cytosolic pool acts as another antagonist of membrane-bound Ras function. In normal cells having normal Ras, the cytosolic pool of Ras does not act as an antagonist. In the absence of complete inhibition of farnesylation, other farnesylated proteins are able to continue with their functions.

Farnesyl-protein transferase activity may be reduced or completely inhibited by adjusting the compound dose. Reduction of farnesyl-protein transferase enzyme activity by adjusting the compound dose would be useful for avoiding possible undesirable side effects such as interference with other metabolic processes which utilize the enzyme.

These compounds and their analogs are inhibitors of farnesyl-protein transferase.

Farnesyl-protein transferase utilizes farnesyl pyrophosphate-to covalently modify the Cys thiol group of the Ras CAAX box with a farnesyl group.

Inhibition of farnesyl pyrophosphate biosynthesis by inhibiting HMG-CoA reductase blocks Ras membrane localization in vivo and inhibits Ras function.

Inhibition of farnesyl-protein transferase is more specific and is attended by fewer side effects than is the case for a general inhibitor of isoprene biosynthesis.

Previously, it has been demonstrated that tetrapeptides with the CAAX sequence inhibit Ras farnesylation (Schaber et al., ibid; Reiss et. al., ibid; Reiss zero al., PNAS, 88:732-736 (1991)).

However, the reported inhibitors of farnesyltransferase are metabolically unstable or inactive in cells.

It is, therefore, an object of this invention to develop pharmaceutical compositions containing the compounds of this invention and methods of treatment utilizing these compositions for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras.

SUMMARY OF THE INVENTION The present invention relates to pharmaceutical compositions containing the compounds of structural formula (I):

and methods of treatment utilizing these compositions for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras.

DETAILED DESCRIPTION OF THE INVENTION The present invention is directed to pharmaceutical compositions containing the compounds of structural formula (I) which are inhibitors of farnesyl-protein transferase and the oncogene protein Ras:

wherein Z1, Z2 and Z3 are each independently selected from; a) H; b) C15alkyl; c) C15alkyl substituted with a member of the group consisting of: i) phenyl, ii) phenyl substituted with methyl, methoxy, halogen (C1, Br, F, I) or hydroxy.; or a pharmaceutically acceptable salt of a compound of of formula (I).

In one embodiment of the present invention are those pharmaceutical compositions which are inhibitors of farnesyl-protein transferase containing the compounds of formula (I) wherein the relative stereochemical configuration of the 2,8-dioxabicyclo(3.2.1]octane ring is as shown below:

Throughout this specification and claims where stereochemistry is described for the dioxabicyclo(3.2.l] octane ring, the configuration implied is relative.

The actual configuration may be as shown or that of its enantiomer.

Further illustrating this embodiment are those pharmaceutical compositions which are inhibitors of farnesyl-protein transferase containing the compounds of structural formula (I) wherein the relative configuration at positions 3,6 and 7 is as shown below:

In one class of this embodiment are those pharmaceutical compositions which are inhibitors of farnesyl-protein transferase containing the compounds of structure (I) wherein the relative configuration at the 4-position is as shown below:

Exemplifying this class is the pharmaceutical composition which is an inhibitor of farnesyl-protein transferase containing the compound wherein Z1, Z2 and Z3 are each hydrogen or a pharmaceutically acceptable salt thereof. The compound wherein Z1, Z2 and Z3 are each hydrogen is hereafter referred to as Compound A.

Further illustrating this class are those pharmaceutical compositions which are inhibitors of farnesyl-protein transferase containing the compounds in which one or more of Z1, Z2 or Z3 is C15alkyl or C15alkyl substituted with phenyl or substituted phenyl wherein the substituent is methyl, methoxy, halogen or hydroxy. In a specific illustration, Z1, Z2 and Z3 are each methyl. This compound is hereafter referred to as Compound B.

The compounds of formula (I) are prepared in an aerobic fermentation procedure employing a novel fungal culture, MF5465, identified as Leptodontium elatius. Although the use of this organism is specifically described herein, other species of the genus Leptodontium including mutants of the above described organism are also capable of producing compounds of this invention.

The culture MF5465 is that of a fungus, a lignicolous Hyphomycete, Leptodontium elatius, isolated from wood in the Joyce Kilmer Memorial Forest in North Carolina. This culture has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, MD 20852 as ATCC 74011.

The culture MF5465, identified as Leptodontium elatius exhibits the following morphological features.

Colonies attaining 12-15 mm in 7 days on oatmeal agar (Difco), with both aerial and submerged mycelium. Colony surface flat to appressed in side view, minutely velvety with a metallic sheen towards the margins, dull towards the center, hyaline at the margin, but soon becoming pale to dark gray, finally black, often developing olivaceous colors in age, Pallid Neutral Gray, Light Gull Gray, Deep Gull Gray, Dark Gull Gray, Slate-Gray, Deep Olive-Gray, Olive-Gray, (capitalized color names from Ridgway, R.

1912. Color Standards and Nomenclature, Washington, D.C.), with similar reverse pigmentation, without exudates diffusible pigments or odors.

Conidiogenous cells holoblastic, arising as the terminal cells of relatively undifferentiated conidiophores, with tapered, subulate apices, with the conidiogenous loci confined to the extreme apex.

Occasionally with undifferentiated conidiogenous loci directly on vegetative hyphae. Developing conidia adhere to comidiophore terminus in a thin, irregular to ladder-like rachis in groups of up to 4-15 conidia. Conidiophores originating as undifferentiated branches at right or subacute angles from vegetative hyphae, gradually elongating, remaining simple or forming l-3-branch points, usually at right to subacute angles, usually clustered in small groups when viewed from above, 1-3 septate, cylindrical to conical with tapered apices hyaline when young but developing olivaceous to olivaceous gray pigments from the base upward in age, with walls slightly thicker than those of vegetative hyphae, 20-65 X 3-5 pin. Conidia formed abundantly on common media such as oatmeal, malt extract, or corn meal agar, 3.5-5 Fm X 1-2 pin, aseptate, smooth, thin-walled, allantoid, suballantoid, to short cylindrical, or narrowly elliptical, often with a small proximal scar or apiculus, without visible slime or gelatinous materials. Hyphae septate, branched, cylindrical or occasionally inflated, up to Spin in diameter.

Compounds of this invention can be obtained by culturing the above noted microorganism in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under aerobic conditions. Nutrient media may also contain mineral salts and defoaming agents.

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, glycerin, starch, dextrin, and the like. Other sources which may be included are maltose, mannose, sucrose, and the like. In addition, complex nutrient sources such as oat flour, corn meal, millet, corn and the like may supply utilizable carbon. The exact quantity of the carbon source which is used in the medium will depend, in part, upon the other ingredients in the medium, but is usually found in an amount ranging between 0.5 and 5 percent by weight.

These carbon sources can be used individually in a given medium or several sources in combination in the same medium.

The preferred sources of nitrogen are amino acids such as glycine, methionine, proline, threonine and the like, as well as complex sources such as yeast extracts (hydrolysates, autolyates), dried yeast, tomato paste, soybean meal, peptone, corn steep liquor, distillers solubles, malt extracts and the like. Inorganic nitrogen sources such as ammonium salts (eg. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.) can also be used.

The various sources of nitrogen can be used alone or in combination in amounts ranging between 0.2 to 90 percent by weight of the medium.

The carbon and nitrogen sources are generally employed in combination, but need not be in pure form. Less pure materials which contain traces of growth factors, vitamins, and mineral nutrients may also be used. Mineral salts may also be added to the medium such as (but not limited to) calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, magnesium salts, copper salts, cobalt salts and the like. Also included are trace metals such as manganese, iron, molybdenum, zinc, and the like. In addition, if necessary, a defoaming agent such as polyethylene glycol or silicone may be added, especially if the culture medium foams seriously.

The preferred process for production of compounds of this invention consists of inoculating spores or mycelia of the producing organism into a suitable medium and then cultivating under aerobic condition.

The fermentation procedure generally is to first inoculate a preserved source of culture into a nutrient seed medium and to obtain, sometimes through a two step process, growth of the organisms which serve as seeds in the production of the active compounds. After inoculation, the flasks are incubated with agitation at temperatures ranging from 20 to 30"C, preferably 25 to 28"C. Agitation rates may range up to 400 rpm, preferably 200 to 220 rpm.

Seed flasks are incubated over a period of 2 to 10 days, preferably 2 to 4 days. When growth is plentiful, usually 2 to 4 days, the culture may be used to inoculate production medium flasks. A second stage seed growth may be employed, particularly when going into larger vessels. When this is done, a portion of the culture growth is used to inoculate a second seed flask incubated under similar conditions but employing shorter time.

After inoculation, the fermentation production medium is incubated for 3 to 30 days, preferably 4 to 14 days, with or without agitation (depending on whether liquid or solid fermentation media are employed). The fermentation is conducted aerobically at temperatures ranging from 20 to 4000.

If used, agitation may be at a rate of 200 to 400 rpm. To obtain optimum results, the temperatures are in the range of 22 to 28"C, most preferably 24 to 26"C. The pH of the nutrient medium suitable for producing the active compounds is in the range of 3.5 to 8.5, most preferably 5.0 to 7.5. After the appropriate period for production of the desired compound, fermentation flasks are harvested and the active compound isolated.

An alcoholic solvent is employed to extract a compound of this invention from the solid fermentation medium. The preferred solvent for extraction of the solid fermentation is methanol.

The mixture of alcoholic solvent and fermentation broth is vigorously stirred and filtered, and water added to the filtrate. The aqueous methanol extract is then adsorbed on an anion exchange resin. The preferred resin is Dowex-l (C1-). The active compound can be eluted from Dowex-l using a high salt eluant; the preferred eluant is 3% ammonium chloride in 90% methanol/water. After elution from the ion exchange resin, the active compound may be recovered from the eluate by diluting the eluate with water, lowering the pH to 2.5, and extracting into an organic solvent; the preferred solvent for extraction is dichloromethane. The organic extract is then evaporated to afford partially purified active compound.

The active compound is further purified by chromatographic separation which may be carried out by employing reverse phase chromatagraphy. The preferred adsorbent for this chromatography is a C8 bonded phase silica gel. The preferred eluant for reverse phase chromatography is a mixture of acetonitrile and water buffered at a low pH, such as 0.1% phosphoric acid, or trifluoroacetic acid.

The pharmaceutically acceptable salts of the compounds of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene- diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine., diethylamine, piperazine, trishydroxymethyl)aminomethane, and tetramethylammonium hydroxide. The salts included herein encompass those wherein one, two-or all three of the carboxyl groups are in the salt form.

The intrinsic farnesyl-protein transferase (FTase) activity of representative compounds of this invention was measured by the assay as described below: RASIT ASSAY Farnesyl-protein transferase (Ftase) from bovine brain was chromatographed on DEAE-Sephacel (Pharmacia, 0-0.8 M NaCl gradient elution), N-octyl agarose (Sigma, 0-0.6 M Nacl gradient elution), and a mono Q HPLC column (Pharmacia, 0-0.3 M NaCI gradient). Ras-CVLS (Cys-Val-Leu-Ser) at 3.5 CLAM, 0.25 WM (3H]FPP, and the indicated compounds were incubated with this partially purified enzyme preparation. The Ftase data presented below is a measurement of the ability of the test compound to inhibit RAS farnesylation in vitro.

TABLE 1 Inhibition of RAS farnesylation by compounds of this invention Compound IC5o(M) A 0.032 WM (lS,3S,4S,5R,6R,7R)-l-L(4)-acetoxy- 5-methyl-6-phenyl]hexyl-4, 6 , 7-tri- hydroxy-6-O-(6-methyl-9-phenyl-4- nonenoyl)-2 , 8-dioxabicylco[3 .2.1]- octane-3,4,5-tricarboxylic acid The pharmaceutical compositions containing the compounds of structural formula I inhibit farnesyl-protein transferase and the farnesylation of the oncogene protein Ras. These compounds are useful as pharmaceutical agents for mammals, especially for humans. These compounds may be administered to patients for use in the treatment of cancer.

Examples of the type of cancer which may be treated with the compounds of this invention include, but are not limited to, colorectal carcinoma, exocrine pancreatic carcinoma, and myeloid leukemias.

The compounds of this invention may be administered to mammals, preferably humans, either alone or, preferably, in combination with pharmaceutically-acceptable carriers or diluents, optionally with known adjuvants, such as alum, in a pharmaceutical composition, according to standard pharmaceutical practice. The compounds can be administered orally or parenterally, including intravenous, intramuscular, intraperitoneal, subcutaneous and topical administration.

For oral use of a chemotherapeutic compound according to this invention, the selected compounds may be administered, for example, in the form of tablets or capsules, or as an aqueous solution or suspension. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch, and lubricating agents, such as magnesium stearate, are commonly added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents may be added. For intramuscular, intraperitoneal, subcutaneous and.

intravenous use, sterile solutions of the active ingredient are usually prepared, and the pH of the solutions should be suitably adjusted and buffered.

For intravenous use, the total concentration of solutes should be controlled in order to render the preparation isotonic.

The present invention also encompasses a pharmaceutical composition useful in the treatment of cancer, comprising the administration of a therapeutically effective amount of the compounds of this invention, with or without pharmaceutically acceptable carriers or diluents Suitable compositions of this invention include aqueous solutions comprising compounds of this invention and pharmacologically acceptable carriers, e.g. saline, at a pH level, e.g., 7.4. The solutions may be introduced into a patient's intramuscular blood-stream by local bolus injection.

When a compound according to this invention is administered into a human subject, the daily dosage will normally be determined by the prescribing physician with the dosage generally varying according to the age, weight, and response of the individual patient, as well as the severity of the patient's symptoms.

In one exemplary application, a suitable amount of compound is administered to a human patient undergoing treatment for cancer. Administration occurs in an amount between about 0.1 mg/kg of body weight to about 20 mg/kg of body weight of a mammal per day, preferably of between 0.5 mg/kg of body weight to about 10 mg/kg of body weight of a mammal per day.

The composition of media employed in the following Examples are listed below: KF SEED MEDIUM Trace Element Mix 2 per liter gilt Corn Steep Liquor 5 g FeSO4.7H20 1.0 Tomato Paste 40 g MnSO4'4H20 1.0 Oat Flour 10 g CuC12b2H2O 0.025 Cerelose 10 g CaCl2.2H20 0.1 Trace Element H3BO3 0.056 Mix &num;2 10 ml (NH4)6No7O24.4H20 0.019 Distilled Water 1000 ml ZnSO4S7E2O 0.2 pH adjusted to 6.8 (presterile) 50 mls/nonbaffled 250 mls dissolved in 1L 0.6 N HC1 Erlenmeyer flask autoclave 20 minutes (121 C, 15 psi) Production Media BRF Cracked corn 10.0 g/flask Brown rice 5.0 g/flask Base liquid #3 10.0 mls/flask Base liquid &num;;2 20.0 mls/flask Base liquid #3 Base liquid #2 gilt gilt Ardamine PH 0.2 Yeast extract 1.0 KH2P04 0.1 Sodium tartrate 0.5 MgS04X7H2O 0.1 KH2PO4 0.5 Sodium tartrate 0.1 distilled water 1000.0 mls FeSO47H20 0.01 ZnSO4.7E2O 0.01 (no pH adjustment) distilled H2O 1000.0 mls (no pH adjustment) autoclave 15 minutes (121 C, 15 psi) add 15.0 mls distilled H2O/flask autoclave 15 minutes (121 C, autoclave 20 minutes (121 C, 15 psi) 15 psi) add 15.0 mls distilled H20/flask autoclave 20 minutes (121 C, 15 psi) EXAMPLES Examples provided are intended to assist in a further understanding of the invention.Particular materials employed, species and conditions are intended to be further illustrative of the invention and not limitative of the reasonable scope thereof.

EXAMPLE 1 Preparation of Compound A A. Culturing MF5465 Culture MF5465, inoculated from a soil tube using one glass scoop of soil, was grown in 3 KF seed medium flasks for 74 hours at 25or, 220 rpm, 85% humidity. The flasks were then pooled, and sterile glycerol added to obtain a final concentration of 10%. The contents were mixed and 2.0 ml aliquots were dispensed aseptically into sterile cryotubes.

The vials were frozen and maintained at -80 C.

Three vails containing frozen vegetative mycelia were defrosted and transfered, one to each of three KF seed medium flasks. These seed flasks were incubated for 71 hours at 25"C, 220 rpm, 85% humidity. At completion of the incubation, the three KF flasks were pooled and the seed was used to inoculate 56 F1 production medium flasks. Care was taken to manually distribute seed growth throughout the solid production medium. Production flasks were incubated statically at 25"C for 21 days. Flasks were harvested as follows: 45 mls 75% methanol was added to each production flask; growth was manually broken apart into small fragments by use of a glass pipette; flasks were re-stoppered and placed onto a gyrotory shaker and agitated for 30 minutes at 220 rpm while the extraction proceeded.After shaking, the contents of the individual flasks were pooled by pouring the solvent-extract off the mycelial covered corn and into a 2 liter Erlenmeyer flask. Contents of each flask were then subjected to a second extraction with another 45 mls 75% methanol.

Extraction proceeded as above with the resultant extracts being pooled into a second 2 liter Erlenmeyer flask.

B. Isolation of Compound A The extracts from above (4800 mL) were loaded onto a DOWEX-1 column (500 mL resin) at a rate of 20 mL/min. The column was then washed with 50% methanol/water (300 mL), and 90% methanol/water (500 mL), and then eluted with 3% ammonium chloride in 90% methanol/water. Six fractions (500 mL) were collected. The first 3 fractions were combined, diluted with water (1 L), and adjusted to pH 2.5 with conc. hydrochloric acid. The acidified eluate was extracted with dichloromethane (2 x 500 mL).

Evaporation of the dichloromethane extract afforded an oily residue (402 mg). The residue was dissolved in methanol (1.2 ml) and loaded on a prep HPLC column (Dynamax 60A, 8 um C8, 21.6 x 250 mm with guard column). The column was eluted with 727.

acetonitrile/28% (0.1% phosphoric acid in water) with a 10 mL/min flow rate. Collecting 5 mL fractions, the desired compound eluted in fractions 29-34.

Fractions 29-34 were combined and ethyl acetate (30 mL) was added. After washing with water (10 mL), the organic layer was evaporated to give Compound A as an oil.

EXAMPLE 2 Preparation of an Ammonium Salt A 0.1 mmol sample of the free acid of a compound of formula (I) is dissolved in 10 ml of ethyl acetate. The resulting solution is saturated with gaseous ammonia upon which the ammonium salt precipitates from solution.

EXAMPLE 3 Preparation of a Potassium Salt A solution of 0.1 mmol of the free acid of a compound of formula (I) in 10 ml of methanol is treated with an aqueous or methanolic solution containing 0.3 mmol of potassium hydroxide.

Evaporation of the solvent affords the tri-potassium salt. Addition of between 0.1 and 0.3 mmol of potassium hydroxide yields analogously mixtures of the mono-potassium, di-potassium and tri-potassium salts whose composition depends upon the exact amount of potassium hydroxide added.

In a similar fashion the sodium and lithium salts can be formed..

EXAMPLE 4 Preparation of a Calcium Salt A solution of 0.1 mmol of the free acid of a compound of formula (I) in 20 ml of 6:4 methanol/ water is treated with an aqueous solution of 0.1 mmol of calcium hydroxide. The solvents are evaporated to give the corresponding calcium salt.

EXAMPLE 5 Preparation of an Ethylenediamine Salt A solution of 0.1 mmol of the free acid.of a compound of formula (I) in 10 ml of methanol is treated with 0.1 mmol of ethylenediamine. Evaporation of the solvent affords the ethylenediamine salt.

The procedure can also be applied to the preparation of the N,N"-dibenzylethylenediamine salt.

EXAMPLE 6 Preparation of a Tris(hydroxymethyl)aminomethane Salt To a solution of 0.1 mmol of the free acid of a compound of formula (I) in 10 ml of methanol is added from 0.1 to 0.3 mmol of tris(hydroxy methyl)aminomethane dissolved in 10 ml of methanol.

Evaporation of the solvent gives a corresponding salt form, the exact composition of which is determined by the molar ratio of amine added. Similarly prepared are the salts of L-ornithine, L-lysine, and N-methylgluacamine.

EXAMPLE 7 Preparation of an L-arginine Salt A solution of 0.1 mmol of the free acid of a compound of formula (I) in 20 ml of 6:4 methanol/ water is treated with an aqueous solution of 0.1 to 0.3 mmol of L-arginine. Evaporation of the solvent affords the title salt, the exact composition of which is determined by the molar ratio of amino acid to the free acid of formula (I) used.

Similarly prepared are the salts of L-ornithine, L-lysine and N-methylglucamine.

EXAMPLE 8 Preparation of a Compound B (Method 1) To 5 mg of L-697,350 in methanol (5 ml) was added 2 ml of freshly distilled diazomethane in ether (2.05 M). After 5 minutes the solvent was removed to afford trimethyl ester (Compound B) as an oil.

EXAMPLE 9 Preparation of Compound B (Method 2) A solution of 2 mg of Compound A in 0.5 ml of acetonitrile is treated at room temperature with 10 equivalents of DBU and 10 equivalents of MeI.

After 2 hours the reaction is diluted with 10 ml of dichloromethane and washed successively with 10 ml of 0.1 M phosphoric acid, 10 ml of water, 10 ml of saturated sodium bicarbonate and 10 ml of water.

After drying over sodium sulfate, the organic layer is concentrated and the residue is chromatographed on silica gel using mixtures of hexane and ethyl acetate to give Compound B.

The method of Example 9 is also suitable for the preparation of other ester derivatives such as 1) ethyl and other lower alkyl esters and 2) benzyl and substituted benzyl esters.

Mass Spectral Data Mass spectra were recorded on Finnigan-MAT model MAT212 (electron impact, EI, 90 eV), MAT 90 (Fast Atom Bombardment, FAB), and TSQ70B (FAB, EI) mass spectrometers. Exact mass measurements were performed at high resolution (HR-EI) using perfluorokerosene (PFK) or perfluoropolypropylene oxide (Ultramark U1600F) as internal standard.

Trimethylsilyl derivatives were prepared with a 1:1 mixture of BSTFA-pyridine at room temperature.

13C NMR-Data 13C NMR spectra were recorded in CD30D at 75 MHz on a Varian XL-300 spectrometer. Chemical shifts are given in ppm relative to TMS at zero ppm using the solvent peak at 49.0 ppm (:D30D) as internal standard.

1H NMR Spectra la NMR spectra were recorded at 300 MHz on a Varian XL-300 spectrometer. Chemical shifts are shown in ppm relative to TMS at zero ppm using the solvent peaks at 3.30 ppm (CD3OD)as internal standards.

Phvsical Properties of the compounds of Structure I: Compound A-the compound of structure (I) wherein Z1 Z2 and Z3 are each hydrogen.

Mass Spectral Data: This compound has the molecular weight 754.

The molecular formula C40H50014 was determined by HR-MS measurement of the penta-trimethylsilyl derivative (calc for C40H50014 754.3198, found 754.3154.

1H NMR spectrum (300 MHz) (CD30D, 22"C): 7.22 (m, 4H), 7.13 (m, 6H), 6.23 (d, 1.8), 5.36 (m, 2H), 5.24 (s), 4.88 (q, 3.9), 4.03 (d, 1.8), 2.73 (dd, 13.3, 5.6 Hz), 2.54 (t, 7.6, 2H), 2.3 (m, 5H), 2.04 (s, 3H), 2.04 (m, 2H), 1.89 (br t, 7.0, 2H), 1.6 (m, 5H), 1.27 (m, 2H), 0.93 (d, 6.8, 3H), 0.86 (d, 6.8, 3H), ppm.

13C NMR (CD30D): 173.06, 173.04, 172.43, 170.14, 168.46, 143.84, 141.88, 138.78, 130.15 (2x), 129.36 (2x), 129.24 (4x), 127.53, 126.89, 126.61, 107.17, 90.94, 82.15, 81.11, 78.10, 76.57, 75.56, 40.46, 39.62, 37.77, 37.56, 36.87, 36.21, 35.34, 32.43, 30.44, 28.75, 21.25, 21.13, 20.08, 14.32 ppm.

IR (as a free acid; film on ZnSe): 3200 br, 2936, 1733, 1496, 1454, 1437, 1375, 1250, 1180, 1148, 1026, 972, 898, 831, 746, 700 cm-l Compound B-the trimethyl ester of Compound A, i.e.

the compound of structure (I) wherein Z1, Z2 and Z3 are each methyl.

Mass Spectral Data: This compound has the molecular weight 796 by FAB-MS (observed EM+Cs]+ at m/z 929.

NMR Spectrum (300 MHz) (CD3OD6,22 C): 7.22 (m, 6H), 7.15 (m, 4H), 6.16 (d, 1.9), 5.32 (m, 2H), 5.24 (s), 4.9 (m), 4.00 (d, 1.9), 3.81 (s, 3H), 3.70 (s, 3H), 3.68 (s, 3H), 2.73 (dd, 13.3, 5.7 Hz), 2.56 (dt, 2.5, 7.6, 2H), 2.35 (m, 3H), 2.25 (m, 2H), 2.08 (m), 2.05 (s, 3H), 2.01 (m), 1.88 (br t, 7.4, 2H), 1.68 (m, 2H), 1.56 (m, 3H), 1.29 (m, 2H), 0.94 (d, 6.8, 3H), 0.86 (d, 6.8, 3H) ppm.

13C NMR Chemical Shifts 13C NMR (CD30D): 173.04, 172.85, 171.16, 168;75, 167.39, 143.94, 141.98, 138.96, 130.21 (2x), 129.43 (2x), 129.30 (4x), 127.49, 126.96, 126.66, 107.49, 91.12, 81.89, 81.17, 78.04, 76.83, 76.20, 53.62, 53.03, 52.77, 40.52, 39.75, 37.87, 37.61, 36.93, 36.09, 35.19, 32.46, 30.53, 28.78, 21.27, 21.13, 20.13, 14.35 ppm.

IR (film on ZnSe ): 3200 br, 2917, 2848, 1738, 1603, 1441, 1371, 1246, 1149, 1124, 1030, 968, 748, 702 cm-l

Claims (9)

WHAT IS CLAIMED IS:

1. A pharmaceutical composition for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras, comprising a therapeutically effective amount of a compound of structural formula (I)

wherein Z1 Z2 and Z3 are each independently selected from a) H; b) C15 alkyl; c) C15 alkyl substituted with a member of the group consisting of i) phenyl, ii) phenyl substituted with methyl, methoxy, halogen (C1, Br, I, F) or hydroxy; or a pharmaceutically acceptable salt of a compound of formula (I); or a pharmaceutically acceptable carrier.

2. A pharmaceutical composition for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras, comprising a therapeutically effective amount of compound of Claim 1 wherein the relative configuration of structural formula (I) is:

or a pharmaceutically acceptable carrier.

3. A pharmaceutical composition for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras, comprising a therapeutically effective amount of a compound of Claim 2 wherein the relative configuration within structural formula (I) is:

or a pharmaceutically acceptable carrier.

4. A pharmaceutical composition for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras, comprising a therapeutically effective amount of a compound of Claim 3 wherein the relative configuration of structural formula (I) is:

or a pharmaceutically acceptable carrier.

5. A pharmaceutical composition for use in inhibiting farnesyl-protein transferase and farnesylation of the oncogene protein Ras, comprising a therapeutically effective amount of a compound of Claim 1 which is:

or a pharmaceutically acceptable carrier.

6. A pharmaceutical composition for use in treating cancer, comprising a therapeutically effective amount of a compound according to Claims 1 through 5 or a pharmaceutically acceptable carrier.

7. A pharmaceutical composition for use in inhibiting farnesyl protein transferase and farnesylation of the oncogene protein Ras, comprising a therapeutically effective amount of a compound of (lS,3S,4S,5R,6R,7R)-1-[(4)-acetoxy-5-methyl-6-phenyl]- hexyl-4,6,7-trihydroxy-6-0-(6-methyl-9-phenyl-4-nonenoyl)-2,8-dioxabicylco[3.2.1]octane-3,4,5-tricarb- oxylic acid.

8. A pharmaceutical composition for use in treating cancer, comprising a therapeutically effective amount of (lS,3S,4S,SR,6R,7R)-l-((4)- acetoxy-5-methyl-6-phenyl]hexyl-4, 6, 7-trihydroxy-6- 0-(6-methyl-9-phenyl-4-nonenoyl)-2,8-dioxabicylco- [3.2.1]octane-3,4,5-tricarboxylic acid.

9. A method of treating cancer, comprising the administration to a subject in need of such treatment a therapeutically effective amount of a composition according to Claims 1-8.