GB2261374A - 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 (Gibbs, 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-translational modifications are involved with Ras membrane localization, and all 3 modifications occur at the C-terminus of Ras. The Ras C-terminus contains a sequence motif termed a "CAAX" or "Cys-Aaa1-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 al., 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., ibid; 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 al., 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-Aaal 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 zero awl., Proc.

Natl. Acad. Sci. USA 86:6630-6634(1989)). Cytosollocalized forms of Ras with normal GTPase activity do not act as inhibitors. Gibbs et al., ibid, 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 blodks 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., acid; Reiss et. al., ibid; Reiss et 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) Cl~5alkyl; c) c,5alkyl 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 of formula (I) in which at least one of Z1, Z2 and Z3 is hydrogen.

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-dioxabicyclot3.2.1]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 or farnesyl-protein transferase containing the compounds in which one or more of Z1, Z2 or Z3 is Cl~5alkyl or C15alkyl substituted with phenyl or phenyl substituted with 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 culture, MF5453, observed as a sterile mycelium.

Mutants of MF5453 are also capable of producing compounds of this invention.

The culture MF5453 is that of a fungus isolated from a water sample obtained from the Jalon river, Zaragoza, Spain. This culture has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, MD 20852 as ATCC 20986.

The microorganism MF5453 exhibits the following morphological characteristics: Colonies 22-24 mm in diameter in two weeks on potato-dextrose agar (Difco) at 25"C in continuous fluorescent light, 40-45 mm diameter in two weeks on deer dung-extract agar (Mycology Guidebook, media M-ll, p. 660) at 25"C. Colonies on potato-dextrose agar consisting of moderately thick, 1-2 mm deep, mycelium, cottony at the center, becoming felty to velutinous towards the margin, forming obscure concentric ridges, occasionally forming sectors of differing mycelial texture and color, margin entire, without a fringe of submerged leading hyphae. Colony color at first white or some white aerial hyphae, soon becoming cream, Cream Color, Cartridge Buff, (capitalized color names from Ridgway, R.Color Standards and Nomenclature, Washington, D.C. 1912), finally light gray to gray, Light Olive-Gray, Gray, Pallid Neutral Gray, Neutral Gray, Gull Gray, Deep Gull Gray. Colony reverse grayish brown to yellow brown or cream, Cinnamon-Drab, Cinnamon, Ochraceous-Buff, Antimony Yellow, Warm Buff, Cartridge Buff. Odors and exudates absent.

Hyphae ascomycetous in morphology, undifferentiated, septate, branched 1.5-3.5 Fm in diameter, hyaline in water and KOH. No reproductive structures formed on any of cultural conditions surveyed, including incubation in both continuous light and 12/12 hour dark cycles on either-cornmeal agar, hay extract agar, oatmeal agar, dung extract agar, and malt extract agar.

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, autolysates), 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 salt 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 condition 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 40"C.

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 3.55 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.

The pH of the aqueous mycelial fermentation is adjusted to between 1 and 9 (preferably between 3 and 5) preferably mixed with a water miscible solvent such as 50% methanol and the mycelia filtered. The active compound may then be isolated from the aqueous filtrate by several methods including: 1. Liquid-liquid extraction of the aqueous filtrate into a water immiscible solvent such as methyl ethyl ketone, ethyl acetate, diethyl ether, or dichloromethane preferably after having adjusted the pH to between 3 and 5.

2. Solid-liquid extraction of the aqueous filtrate onto an organic matrix such as SP207 or HP-20 and elution with an organic solvent (aqueous or non aqueous) such as 90/10 methanol/water or 90/10 acetone/water.

3. Adsorption of the active compound from the aqueous filtrate onto an ionic exchange resin such as Dowex l(Cl-) or Dowex 50 (Ca2+) and elution with a high ionic strength organic/aqueous solvent such as 90/10 methanol/aqueous 30% NH4Cl. This material could then be desalted by employing either method 1 or 2 above. Each of these three methods may also be used in the further purification of the active compound.

The fraction containing active compound from the above methods could then be dried in vacuo leaving the crude active compound. The crude active compound is then generally subjected to several separation steps such as adsorption and partition chromatography, and precipitation. For each separation step, fractions are collected and combined based on results from an assay and/or HPLC analysis.

The chromatographic separations may be carried out by employing conventional column chromatography with ionic or nonionic adsorbent.

When silica gel is the adsorbent, an alcohol/chlorohydrocarbon/organic acid mixture such as methanol/chloroform/acetic acid/ water is useful as an eluant. For reverse phase chromatography, the preferred adsorbent 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 active compound can be precipitated out of a non-polar solvent as the quinine salt. The preferred solvent for precipitation is diethyl ether.

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'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)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 is described below: RASIT ASSAY Farnesyl-protein transferase (Ftase) from bovine brain was chromatographed on DEAE-Sephacel (Pharmacia, 0-0.8 M NaCI gradient elution), N-octyl agarose (Sigma, 0-0.6 M NaCl gradient elution), and a mono Q HPLC column (Pharmacia, 0-0.3 M NaCl gradient). Ras-CVLS (Cys-Val-Leu-Ser) at 3.5 WM, 0.25 SM [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 IC50(M) A 0.140 Fm (1S,3S,4S,5R,6R,7R)-1-[(4S)-acetoxy-3-methylene-5(R)- methyl-6-phenyl]hexyl-4,6,7-trihydroxy-6-0-(4(S), 6(S)dimethyl-2-octenoyl)-2,8-dioxabicycloL3.2.l)- octane-3.4-5-tricarboxvlic 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 Elements Mix per liter Corn Steep Liquor 5 g FeSO47H20 1.0 Tomato Paste 40 g MnSO4-4H2O 1.0 Oat Flour 10 g CuC12.2H2O 0.025 Glucose 10 g CaCl2'2H2O 0.1 Trace Element Mix 10 ml H3BO3 0.056 pH adjusted to 6.8 (presterile) (NH4)6Mo7O24.4H2O 0.019 50 mls/nonbaffled 250 mls ZnS04.7H20 0.2 Erlenmeyer flask autoclave 20 minutes (121 C, dissolved in 1L 0.6 N HC1 15 psi) MBM Production Medium gifl Malt extract (Difco) 5.0 Glucose 15.0 Peptone 1.0 KH2P04 1.0 MgS04 0.5 distilled H2O 1000.0 mls (no pH adjustment) 45 mls/nonbaffled 250 mls Erlenmeyer flask autoclave 15 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 MF5453 Culture MF5453 was inoculated into KF seed medium using one glass scoop of the original soil tube. The KF seed flask was incubated for 73 hours at 25"C, 220 rpm, 85% humidity. At the end of this incubation, 2.0 mls aliquots were aseptically transferred to each of 75 MBM production medium flasks. These production flasks were then incubated at 25"C, 220 rpm, 85% humidity, with a fermentation cycle of 14 days. Flasks were harvested as follows: mycelial growth was homogenized for 20 seconds at high speed using a Biohomogenizer/mixer (Biospec Products Inc. Bartlesville, Ok); and then 45 mls methanol was added to each flask (final methanol concentration was approximately 50%). Flasks were then returned to the shaker and agitated at 220 rpm for 30 minutes.Subsequently, the contents of the flasks were pooled.

B. Isolation of Compound A A 6 liter 50% methanol homogenized fungal extract exhibiting a pH of 4.5 was employed in the following isolation procedure. The mycelia was filtered through celite and the recovered mycelia was extracted again by stirring overnight with 3 L of 50% methanol and again filtered.

The combined extract (9 L) of 50% methanol was diluted to 25% methanol with water (total volume 18 L) and applied to a Mitsubishi HP-20 column (750 ml) at a flow rate of 80 ml/minute. The column was washed with water (1 L) and eluted with a stepwise gradient of methanol consisting of 50/50 methanol/H20 (1 L), 60/40, methanol/H20 (1 L), 80/20 methanol/H2O (2 L,) 90/10 methanol/H2O (1 L), 100% methanol (2 L), and 100% acetone (1 L). The fractions from 50/50 to 90/10 methanol/H20 were combined and diluted with water to 35/65 methanol/H2O (total volume 10 L).

The 10 L of 35/65 methanol/H20 was acidified with 1.0 N HC1 (20 ml) to pH 3.0 and extracted into EtOAc (4 L). The EtOAc layer was separated and the solvent removed in vacuo to yield 260 mg of an orange oil.

A portion (10%) of the orange oil was dissolved in 1 ml methanol and diluted with 0.8 ml 10 mM potassium phosphate (pH 6.5) with some precipitation. The suspension was applied to a preparative HPLC column (Whatman Magnum 20 C18, 22 mm ID X 25 cm, 8 ml/minute. The initial mobile phase was 60/40 methanol/10 mM potassium P04, pH 6.5, and after 20 minutes the mobile phase was changed to 80/20 methanol/10 mM potassium phosphate, pH 6.5.

Fractions of 8 ml each were collected, and the fractions from 31 to 33 minutes (2) were combined, diluted with water to 35% methanol, acidified with 10% HC1 to pH 3, and extracted into EtOAc. The solvent was removed in vacuo and a clear slightly yellow oil identified as the titled compound was obtained.

EXAMPLE 2 Preparation of Compound A A. Culturing MF5453 A 250 mL Erlenmeyer flask containing 54 mL of the seed medium (KF) was inoculated with MF5453 and incubated at 25"C and 85% humidity at 220 rpm for 72 hours. Ten mL of the culture was transferred to each of three 2-liter Erlenmeyer flasks containing 500 mL of the KF seed medium. These flasks were cultivated for 44 hours at 25"C, 85% humidity at 220 rpm. Seven hundred fifty mL of the resulting culture were used to inoculate each of 2 22-Liter fermentors containing 15 Liters of MBM production medium.

Sterilization conditions were as follows: 122"C, 15 psi for 25 minutes. The pH of the medium after sterilization was 5.0. The fermentation conditions were as follows: 25CC, at an agitation rate of 300 rpm, and airflow rate of 4.5 liters per minute, and a back pressure of 5 psi. After 357 hours of cultivation under these conditions the batches were harvested.

B. Isolation of Compound A Three independent fermentations (3 L), (10 L), and (10 L) were employed in the following isolation procedure. The 3 L fermentation was cultured following Example IA and each 10 L fermentation was cultured following the procedure of Example 2A. Each fermentation was filtered through celite and the mycelia extracted individually overnight (2 X) with 1 L, 3 L, and 3 L 50% methanol respectively.

The filtrates and extracts were all combined and diluted with water to 25% methanol (total volume 45 L). This was applied to a Mitsubishi HP-20 column (1.5 L) at a flow rate of 100 ml/minute. The column was then washed with water (4 L), and 40% methanol (6 L). The column was then eluted with 100% methanol (6 L).

The 100 % methanol HP-20 eluate was diluted with 10 mM H3P04 (6 L, total volume 12 L) and extracted into CH2C12 (4 L). The solvent was removed in vacuo to yield 21 g of a yellow oil.

The above concentrate was dissolved in methanol (500 ml) and diluted with 20 mM potassium PO4, pH 7.0 (500 ml). This solution was applied to a Dowex 1 X 2 (C1-) column (350 ml) at a rate of 25 ml/minute. The column was washed with 50/50 methanol/water (2 L), 50/50 methanol/3% NaCl solution (1 L), and the product was eluted with 90/10 methanol/30% aqueous NH4Cl- solution (2 L). The methanol/NH4Cl eluate was diluted with 20 mM H3P04 (2 L) and extracted with CH2C12 (2 L). The solvent was removed in vacuo to yield a thick orange oil.

A portion of the orange oil (24 mg) was dissolved in 1 ml methanol and 0.4 ml of 10 mM H3P04 was added with some clouding . This suspension was applied to a preparative HPLC column (Whatman Magnum 20 C18, 22 mm ID x 25 cm, 8 ml/minute, mobile phase 80/20 methanol/10 mM H3P04 pH 2.5). The fractions eluting between 16 and 20 minutes were combined, diluted with 50 mM H3PO4 (50 ml), and extracted with CH2C12 (2 x 75 ml). The CH2C12 was removed in vacuo to yield a slightly yellowed oil. This material exhibited identical 1H-NMR and UV spectra with that of Compound A isolated in Example 1.

EXAMPLE 3 As a specific embodiment of an oral composition of a compound of this invention, 20 mg of the compound from Example 1 is formulated with sufficicent finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

EXAMPLE 4 Preparation of an Ammonium Salt A 0.1 mmol sample of the free acid of Compound (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 5 Preparation of Potassium Salt A solution of 0.1 mmol of the free acid of Compound (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 of Compound (I) can be formed.

EXAMPLE 6 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 7 Preparation of an Ethvlenediamine 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 8 Preparation of a Tris(hydroxvmethyl)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(hydroxymethyl)aminomethane dissolved in 10 ml of methanol.

Evaporation of the solvent gives a corresponding salt form of Compound (I), the exact composition of which is determined by the molar ratio of amine added.

The method can also be applied to other amines such as, but not limited to: diethanolamine and diethylamine.

EXAMPLE 9 The preparation of a L-arginine salt To a solution of 0.1 mmol of the free acid of a compound of formula (I) in 10 ml of 6:4 methanol/ water is treated with an aqueous-solution of 0.1-0.3 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 Compound (I).

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

EXAMPLE 10 Preparation of Compound B (Method 1) A solution of 90 mg of Compound A in 5 ml of ethyl acetate was treated with a slight excess of ethereal diazomethane. After 5 minutes excess diazomethane was removed and the solvent was evaporated to give Compound B.

EXAMPLE 11 Preparation of Compound B A solution of 2 mg of Compound A in 0.5 ml of acetonitrile-was treated at room temperature with 10 equivalents of DBU and 10 equivalents of MeI.

After 2 hours the reaction was diluted with 10 ml of dichloromethane and washed successively with 10 ml of 0.1 M phosphoric acid, 10 ml of water1 10 ml of saturated sodium bicarbonate and 10 ml of water.

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

The method of Example 11 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.

1 NMR Data The 13C NMR spectrum of Compound A was recorded in CD30D at 100 MHz on a Varian XL400 spectrometer. Chemical shifts are given in ppm relative to tetramethylsilane (TMS) at zero ppm using the solvent peak at 49.0 ppm as internal standard.

The 13C NMR spectrum of Compound B was recorded at 75 MHz (ambient temperature) on a Varian XL300 spectrometer in C6D6 using the solvent peak at 128.0 ppm as internal standard.

1H NMR Spectra The 1H NMR spectra were recorded at 400 MHz in CD30D and C6D6 on a Varian XL400 spectrometer.

Chemical shifts are shown in ppm relative to TMS at zero ppm using the solvent peaks at 3.30 ppm (CD30D) and 7.15 ppm (C6D6) 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 690 by FAB-MS (observed [M+H]+ at m/z 691, and with addition of lithium acetate tMLi3+Li]+ (ie., the lithium adduct of the trilithium salt) at m/z 715).

The molecular formula C35H46014 was determined by HR-EI measurement of the penta-trimethysilyl derivative (calc for C35H46O14+(SiC3H8)5 1050.4864, found 1050.4829). Other critical fragment ions were observed in the silyl spectrum as follows: [M-AcOH]+, calc for C33E44013+(SiC3H8)5 990.4597, found 990.4625; C23H25O10+(SiC3H8)4, calc 749.3015, found 749.3022; [M-(CO2H+SiC3H8))+, calc for C34H45012+(SiC3H8)4 933.4458, found 933.4450; C16H2308+(SiC3H8)4, calc 631.2974, found 631.2944.

1H NMR Spectrum (CD30D,40"C): 13C NMR Chemical Shifts (CD30D. 40 C): 11.5, 14.3, 19.4, 20.6, 21.0, 26.7, 30.8, 33.3, 35.2, 35.6, 37.9, 41.0, 44.5, 75.8, 76.8, 80.4, 81.3, 82.7, 91.3, 106.9, 111.7, 120.0, 127.0, 129.4 (2x), 130.3(2x), 141.7, 147.9, 157.6, 166.8, 168.7, 170.3, 172.2, 172.7 ppm.

W(MeOH) X max 209 nm ( = 36,000) IR(as free acid: film on ZnSe): 3400-2500 br, 2960, 2930, 2875, 1745-1700, 1650, 1450, 1375, 1280-1225, 1185, 1135, 1020, 985, 750, and 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 732 by EI-MS and forms a di-(trimethylsilyl) derivative.

The molecular formula C38H52014 was determined directly by HR-EI measurement (calc 732.3357, found 732.3329).

1H.NMR Spectrum(C6D6, 22 C): 13C NMR Chemical Shifts (C6D6. 75 MHz): 11.3, 14.0, 18.9, 20.2, 20.6, 26.3, 30.0, 32.1, 34.6, 34.7, 37.1, 40.2, 43.3, 51.9, 52.1, 53.3, 75.3, 76.2, 78.8, 82.0, 82.8, 89.8, 106.2, 111.8, 118.8, 126.2, 128.6(2x), 129.6(2x), 140.9, 146.6, 157.2, 166.0, 166.5, 167.2, 169.5, 170.3 ppm.

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 (Cl, Br, I, F) or hydroxy; or a pharmaceutically acceptable salt of a compound of formula (I) in which at least one of Z1, Z2 and Z3 is hydrogen; 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 a compound of Claim 1 wherein the relative configuration within 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 7 and a pharmaceuticallyacceptable 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 (lS,3S,4S,5R,6R,7R)-l-((4S)-acetoxy-3-methylene-5(R)- methyl-6-phenyl]hexyl-4,6,7-trihydroxy-6-0-(4(S),6(S)- dimethyl-2-octenoyl)-2 , 8-dioxabicyclo[3 .2. 1]-octane- 3,4-5-tricarboxylic acid or a pharmaceutically acceptable carrier.

8. A pharmaceutical composition for use in treating cancer, comprising a therapeutically effective amount of (lS,3S,4S,5R,6R,7R)-l-E(4S)- acetoxy-3-methylene-5(R)-methyl-6-phenyl]hexyl-4,6,7- trihydroxy-6-0-(4(S),6(S)-dimethyl-2-octenoyl)-2,8- dioxabicycloE3.2.l)-octane-3,4-5-tricarboxylic acid or a pharmaceutically acceptable carrier.

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 through 8.