GB2261375A - 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., acid). 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., ibid; Casey et al., ibid; Schafer ft al., Science 245:379 (1989)). These drugs inhibit EMG-CoA reductase, the rate limiting enzyme for the production of polyisoprenoids and the farnesyl pyrophosphate precursor.

It has been shown that a farnesyl-protein transf erase using farnesyl pyrophosphate as a precursor is responsible for Ras farnesylation. (Reiss gt 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-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.

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 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 eft. 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 compost ions for use in inhibiting farensyl-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 formuala (I):

and methods of treatment utilizing these compostions 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 compost ions 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) C1-Salkyl 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 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-dioxa bicyclo(3.2.l]octane ring is as shown below:

Throughout this specification and claims where stereochemistry is described for the dioxabicycloE3.2.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 compost ions 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 compost ions 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 compost ions which are inhibitors of farnesyl-protein transferase containing the compounds in which one or more of Z1, Z2 or Z3 is Cl~5alkyl or Cl~5alkyl 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 double bonds within the C14 dienoic side chain may both be in a trans configuration or one of the two may be in a cis configuration or both may be in a cis configuration.

The compounds of formula (I) are prepared in an aerobic fermentation procedure employing a novel culture, MF5447, identified as Sporormiella intermedia. Compounds of formula (I) may also be obtained in a fermentation procedure employing a novel culture MF5466, identified as a bitunicate ascomycete. Although the use of these organisms is specifically described herein, other organisms of the genera Sporormiella and Preussia including mutants of the above described organisms are also capable of producing compounds of this invention.

The culture MF5447 is that of a coprophilous fungus, Sporormiella intermedia, isolated from cottontail rabbit dung (Arizona). This culture has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, MD 20852 as ATCC 20985. Culture MF5466 is that of a coprophilous fungus, a bitunicate ascomycete, isolated from big horn sheep dung (Tucson, Arizona).

This culture has been deposited as ATCC 20989.

The culture MF5447, identified as Sporormiella intermedia exhibits the following morphological features.

Pseudothecia maturing in 4-5 weeks on either inoculted deer dung or on oatmeal agar (Difco) at 25"C in continous fluorescent light. Pseudothecia on surface of inoculated deer dung single to densely gregarious, embedded, with upper 10-50% protruding above the surface, 200-300 Fm in diameter, globose to subglobose, nonostiolate, glabrous, dull, uniformly black. Peridium thin, 1-2 cells thick, a textura angularis. Peridial cells isodiametric, 4-8 Fm in diameter, gray to dark olivaceous gray in KOH.

Asci abundant, arising from a common basal area, bitunicate, 8-spored, cylindrical, straight to slightly curved, with broad rounded apex, 120-180 Fm X 20-35, with a distinct basal stalk, with basal stalk 7-11 pin long. Paraphyses abundant, intermixed with asci, filamentous, septate, approximately equal in length with asci. Ascospores biseriate within the ascus, 45-53 X 10-12 pin, 4-celled, deeply constricted at the septa, end cells with rounded or tapered aspices, middle cells oblong to doliform, each cells with an obscure lateral germ slit, surrounded by a thin, refractive, hyaline sheath, with cells often easily separating, dark olivaceous gray in KOH.

Colonies on potato-dextrose (Difco) agar 10-12 mm in diameter in 7 days at room temperature, slightly raised, about 0.5-1 mm deep, with submerged margin, with surface felty to velutinous, cream when young, soon pale gray to dark gray, or finally dark olivaceous gray to almost black, Cartridge Buff (capitalized color names from Ridgway, R. Color Standards and Nomenclature, Washington, D.C. 1912), Marguerite Yellow, Olive Buff, Light Grayish Olive, Grayish olive, Deep Grayish Olive, Iron Gray, Olivaceous Black. In reverse dull yellowish olive to olivaceous gray to dark olivaceous gray. Odors and exudates absent. Often extensive black stromatic regions develop in colonies older than 2-3 weeks.

Stromatic regions may contain many embedded, confluent to gregarious pseudothecia.

The culture MF5466, an unidentified bitunicate ascomycete exhibits the following morphological features: Colonies 10-12 mm in diameter on potato-dextrose agar (Difco) at room temperature, felty, velutinous, smooth to slightly irregular in side view, up to 1 mm deep, with submerged margin, often sectoring into different colony colors, tough to rubbery in texture. Colony margins hyaline to pale, soon pale gray to olivaceous gray, finally dark gray to olivaceous gray, Cream Color, Pale Smoke Gray, Smoke Gray, Light Grayish Olive, Deep Olive Gray, Iron Gray, Olivaceous Black. In some sectors of old cultures, black stomatic tissues with rudimentary pseudothecia or pseudothecia-like structures are formed. Reverse pigmentation similar. Odors and exudates absent.Pigmentation and colony differentiation reduced on nutrient poor media, e.g. cornmeal agar, malt extract agar, dung extract agar, or hay extract agar.

Mycelium septate, highly branched, flexuous, often contorted to nodulose, with elements up to 8 Fm in diameter, hyaline to olive or olivaceous gray in KOH. Developing a basal stromatic zone of isodiametic cells in older regions of colonies.

Pseudothecia-like structures up to 400 pin in diameter, dull, black, composed of thin-walled, isodiametric cells and filamentous hyphae, a textura angularis or a combination of textura angularis and textura intricata, with isodiametric cells up to 8 pin in diameter. Immature bitunicate asci have been observed in some of these rudimentary pseudothecia after 4-6 weeks on oatmeal agar, but cultures become moribund before asci mature.

Compounds of this invention can be obtained by culturing an 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 use.

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 temperature 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 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 28CC, 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.

A mixture of an alcoholic solvent and an oxygenated solvent, such as an ester or a ketone, is employed to extract a compound of this invention from the solid fermentation medium.

The mixture is vigorously stirred and filtered, and the filtrate is concentrated under reduced pressure. Water is added to the concentrate and the pH is adjusted to about 3 with a mineral acid. The aqueous concentrate is then repeatedly extracted with a water immiscible oxygenated solvent. The water immiscible organic layer is removed and evaporated to dryness. The residue 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/TLC analysis.

The preferred solvent for extraction of the solid fermentation is a 1:1 mixture of methanol and 2-butanone. After concentrating the initial extract and diluting with water, the preferred partitioning solvent is dichloromethane.

The chromatographic separations may be carried out by employing conventional column chromatography with ionic or nonionic resin. Silica gel, such as that available from E. Merck, is the preferred 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. Ionic resins such as Dowex-l (C1-) or Dowex-50 (Ca++) are also useful in the purification. 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 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 NaCl gradient). Ras-CVLS (Cys-Val-Leu-Ser) at 3.5 WM, 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 rest compound to inhibit RAS farnesylation in vitro.

TABLE 1 Inhibition of RAS farnesylation by compounds of this invention Compound IC50( M) A 0.050 WM (lS,3S,4S,5R,6R,7R)-1-[(4)-hydroxy-3,5-dimethyl-8- phenyl)oct-7-enyl-4,6-7-trihydroxy-6-0-(tetradeca- 6,12-dienoyl)-2,8-dioxabicyclo[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: YME Plating Medium Component Amount Yeast Extract 4.0 g Malt Extract 10.0 g Glucose 4.0 g Distilled H20 1000 ml Agar 25.0 g KF SEED MEDIUM Trace Element Mix per liter gLk Corn Steep Liquor 5 g FeS04o7H20 1.0 Tomato Paste 40 g MnSO44H20 1;0 Oat Flour 10 g CuC122H20 0.025 Glucose 10 g CaC12-2H2O 0.1 Trace Element Mix 10 ml H3B03 0.056 (NH4)6Mo7024O4H2O 0.019 pH adjusted to 6.8 (presterile) ZnSO4O7H2O 0.2 50 mls/nonbaffled 250 mls Erlenmeyer flask dissolved in 1L 0.6 N HC1 autoclave 20 minutes (121"C, 15 psi) Production Media F204 BRF Millet 15.0 g/flask Brown rice 5.0 g/flask Base liquid &num;;1 10.0 mls/flask Base liquid &num;2 20.0 mls/flask Base liquid & um;1 Base liquid #2 g/L g/L Yeast extract 50.0 Yeast extract 1.0 Monosodium glutamate 10.0 Sodium tartrate 0.5 Corn oil 10.0 mls KH2P04 0.5 Sodium tartrate 10.0 distilled water 1000.0 mls FeS 4ss7H2 1.0 distilled water 1000.0 mls (no pH adjustment) (no pH adjustment) autoclave 15 minutes (121"C, 15 psi) add 15.0 mls distilled H20/flask autoclave 15 minutes (121"C, autoclave 20 minutes (121"C, 15 psi) 15 psi) add 15.0 mls distilled H2O/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 MF5447 Culture MF5447, inoculated from a soil tube using one glass scoop of soil, was grown in KF seed medium for 72 hours at 25"C, 220 rpm, 85% humidity.

At the end of this incubation period, 2.0 mls aliquots were aseptically transferred to each of 45 250 ml Erlenmeyer flasks containing F204 production medium. Production flasks were incubated at 25"C statically for 21 days and then harvested. At harvest 40 mls of methyl ethyl ketone were added to each flask and the solid growth was manually broken apart into smaller pieces. Flasks were then placed onto a gyrotory shaker and shaken at 220 rpm for 30 minutes in order to further break up the mycelial mass as well as to improve contact of the solvent with the cells. After shaking, the contents of the individual flasks were pooled by pouring the entire contents of the flasks solids and all) into a 4 L beaker.

B. Isolation of Compound A The methyl ethyl ketone liquid from approximately 2 liters of fermentation extract, cultured for 21 days as described in Example 1A was filtered off. A mixture of ethyl acetate and methanol (1:1, 2 L) was then added to the solid residue. This was stirred for 18 hours using a mechanical stirrer. The mixture was filtered and the filtrate concentrated CRotovap; 40"C) to approximately 700 mL. Ethyl acetate (700 mL) was added followed by 500 mL of 5% sodium chloride/water. After stirring for 15 minutes, the aqueous layer was removed and discarded. The ethyl acetate layer was concentrated (Rotovap; 40"C) to approximately 150 mL. Rexane (500 mL) and methanol (500 mL) were added and the mixture stirred for 15 minutes. The hexane layer was removed and discarded.The methanol layer was concentrated (Rotovap; 40"C) to afford a crude extract.

The crude extract (1.4 g) was dissolved in 25 mL of 3:1:1 hexane/toluene/methanol and applied to a Sephadex LH-20 chromatography column (1 L resin) eluting with the same solvent mixture and with a flow rate approximately 3 mL/minute. The first 1600 mL of eluant was discarded. The following 3600 mL eluant was concentrated to dryness to afford LH-20 eluate.

Approximately 310 mg of the LH-20 Eluate was dissolved in 5 mL of 5% methanol/chloroform. This was applied to a silica gel chromatography column (50 mL of E. Merck Kieselgel 40 - 63 um). The column was eluted stepwise as shown in Table la below.

Fractions 4-6 were combined and dried to afford an oily residue. The residue (115 mg) was dissolved in 4 mL of tetrahydrofuran and 5 mL of 0.005 N hydrochloric acid was added. The resulting suspension was centrifuged (10,000 rpm; ZO minutes).

The supernatant was removed and discarded to yield a precipitate.

Twenty-four milligram of this precipitate was dissolved in 0.2 mL of tetrahydrofuran and 0.2 mL of dimethylsulfoxide was added. This was then adsorbed on the resin bed of an open RP-18 chromatography column (30 mL of Bakerbond 40 pin RP-18), equilibrated with 10% tetrahydrofuran/water.

Stepwise elution (Table lb below) was followed by HPLC analysis and bioassay of the fractions.

Fractions 2-7 were combined and concentrated to approximately 50 mL. The aqueous solution was extracted with 50 mL of ethyl acetate. The ethyl acetate extract was dried and dissolved in 0.3 mL of methanol, followed by the addition of 0.4 mL dimethylsulfoxide, 0.1 mL water, and 0.05 mL 43% acetonitrile/l0 mM potassium phosphate buffer (pH 7). This solution was injected on an HPLC column (Amicon Matrex Silica MC-100A C8 15 um, 4.6 mm ID x 25 cm) eluting with 43% acetonitrile/ 10 mM potassium phosphate buffer CpH 7) at 1 mL/ minute. Fractions 9-12 were combined and 0.05 mL of 0.5 N hydrochloric acid was added, followed by 10 mL of ethyl' acetate. The ethyl acetate layer was dried to afford Compound A.

Table lea Solvent composition for silica gel chromatography of the LH-20 Eluate.

Fraction Solvent Volume Z(methanol/water/acetic acid 10:1:1) in chloroform 1 5 50 mL/fraction 2-3 10 50 mL/fraction 4-5 20 50 mL/fraction 6-7 30 50 mL/fraction 8-9 50 50 mL/fraction 10-11 75 50 mL/fraction 12 100 50 mL/fraction Table lib Solvent composition for chromatography of the precipitate on Bakerbond RP-18.

Fraction Solvent Volume % tetrahydrofuran in water 1 10 25 mL/fraction 2-3 25 25 mL/fraction 4-5 50 25 mL/fraction 6-7 75 25 mL/fraction 8 100 25 mL/fraction EXAMPLE 2 Preparation of Compound A A. Culturing MF5447 Fermentation.procedures were identical to those in Example 1A except that 23 F204 flasks were inoculated and the fermentation time was 14 days. 50 mls of methanol was added to each flask, then growth was manually broken up and flasks shaken as stated in Example 1A. Contents of the flask were pooled by pouring the entire contents into a 3 L beaker.

B. Isolation of Compound A Approximately 2 liters of a fermentation extract cultured for 14 days as described in Example 2A and containing an additional one liter of methanol was vigorously stirred for 16 hours and filtered. A second 1 L portion of methanol was added to the spent solids, stirred for 4 hours and filtered. The methanol extracts were combined and concentrated (rotovap, 47"C) to approximatley 200 mL. Water (300 mL) was added to the concentrate and the pH was adjusted to 3 with hydrochloric acid. n-Butanol (500 mL) was added and stirred for 20 minutes. The organic layer was removed and evaporated to dryness to give a black tar. This was chromatographed (2.9 g) on silica gel in a fashion similar to that described in Examples 1 and 3.Fractions containing Compound A (from HPLC analysis results) were combined and evaporated to dryness (rotovap, 40"C). The oily residue (230 mg) was dissolved in 1 mL of dimethyl sulfoxide and injected on an HPLC column (DYNAMAX 60A 8 micron C8; 21.4 mm ID x 25 cm with guard module) eluting with the solvent gradient shown in Table 2a with a flow rate of 10 mL/minute. Collecting 5 mL fractions, fractions 37-42 were combined. Ethyl acetate (50 mL) and water (50 mL) were added. After extraction, the ethyl acetate layer was separated and dried (Rotovap, 40"C) to afford Compound A.

Table 2a Solvent Gradient for HPLC Preparative Purification of Silica Gel Rich Cut.

Time(min) Solvent % acetonitrile in 0.1% H3PO4/water 0-10 70%, isocratic 10-20 70-90%, linear gradient 20-30 90%, isocratic Analytical HPLC system: column: Dynamax-60A 8 micron C8, 4.6 mm ID x 25 cm with guard (1.5 cm L) eluent: 80:20 acetonitrile/0.l% phosphoric acid in water flow rate: 1.0 mL/minutes detection: W at 250nm retention time of Compound A: 5.52 minutes EXAMPLE 3 Preparation of Compound A A. Culturing MF5466 Culture MF5466 was grown on a PME slant at 25"C for at least two weeks. One-fifth of the growth on the slant was transferred to a seed flask containing 54 mL of KF medium in a 250 mL unbaffled Erlenmeyer flask. The seed flask was then incubated for 72 hours at 24"C, 220 rpm.At the end of this incubation, 2 mL aliquots were transferred into multiple 250 ml Erlenmeyer production flasks containing F204 medium. Production flasks were then incubated stationary at 24"C for 21 days. At harvest, 50 mL of 70% methanol was added to the production flasks and mycelial growth was broken apart manually with a pipes The flasks were shaken at 220 rpm for 30 minutes. The methanol extracts were pooled, filtered and the residues were combined.

B. Isolation of Compound A The combined solids formed as described in Example 3A, were extracted vigorously with 1 L of methanol for 15 hours. The 1 L extract was concentrated to 250 ml, diluted with an equal volume of water, adjusted to-pH 8.5 and extracted with 500 ml of isopropyl acetate. The aqueous solution was then acidified to pH 4.0 and successively extracted twice with ethyl acetate and once with l-butanol.

Each extract was individually concentrated to 40 ml (Ethyl Acetate Extract 1, Ethyl Acetate Extract 2, Butanol Extract 1).

A 20 ml aliquot of Ethyl Acetate Extract 1 (250 mg total solids) was concentrated to dryness and reconstituted in 5 ml of 93/4/1/2 chloroform-methanolwater-acetic acid. The sample was charged to a 10 ml column of silica gel 60 which had been equilibrated in the same solvent. The column was then eluted stepwise (see Table 3a).

Table 3s Solvent Composition (by volume) for Silica Gel Chromatography of Crude EtOAc Extract.

Fraction CHIC13 CH30H H2 1-8 93 4 1 2 9-16 89 8 1 2 17-24 85 12 1 2 25-32 78 18 2 2 33-38 59 33 4 4 The volume of all fractions was 5 ml each.

Fractions 18-28 were combined and concentrated to dryness. The residue (40 mg total solids) was dissolved in 4 ml of tetrahydrofuran, to which was added with stirring 5 ml of water. The resulting cloudy mixture was centrifuged for 20 minutes at 15000 rpm. An oily precipitate (ppt 1) was formed and collected.

Ethyl Acetate Extract 2 (40 mL) and the remainder of Ethyl Acetate Extract 1 (20 mL) were processed in a similar fashion through the silica gel and precipitation steps to give an oily precipitate (ppt 2). The two precipitates were combined (43 mg) and dissolved in 0.5 mL of dimethyl sulfoxide and 0.5 mL of methanol. The solution was injected on an HPLC column (DYNAMAX 60A 8 micron C8; 21.4 mm ID x 25 cm with guard module) eluting with the solvent gradient shown in Table 3b and with a flow rate of 10 mL/minute. Collecting 5 mL fractions, fractions 21-36 were combined. Ethyl acetate (100 mL) was added and the pH of the aqueous phase was adjusted to 3 with hydrochloric acid. After extraction, the ethyl acetate layer was dried (Rotovap, 40"C) to afford Compound A.

Table 3b Solvent Composition of Gradient for HPLC Purification of Precipitate.

Time(min) Solvent % acetonitrile in 10 mM Potassium phosphate buffer (pH 7) 0-15 42%, isocratic 15-20 42-70%, linear gradient 20-30 70%, isocratic EXAMPLE 4 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 sufficient finely divided lactose to provide a total amount of 580 to 590 mg to fill a size 0 hard gelatin capsule.

EXAMPLE 5 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 6 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 7 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 8 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 9 Preparation of a Tris(hvdroxvmethvl)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.

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

EXAMPLE 10 Preparation of an L-arginine Salt 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 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 11 Preparation of a Compound B (Method 1) A solution of 2 mg of Compound A in 5 ml of methanol/ether (1:1) 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 12 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 12 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 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.

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 730 by FAB-MS (observed (M+H]+ at m/z 731, and with addition of lithium acetate (M'Li3+Li)+ (ie., the lithium adduct of the trilithium salt at m/z 755).

The molecular formula C39H54013 was determined by HR-EI measurement of the hexa-trimethylsilyl derivative (calc for C39H54013+(SiC3H8)6-C'H3 1147.5701, found 1147.5751).

1H NMR spectrum (400 MHz) (CD30D, 22"C) 13C NMR Chemical Shifts (CD30D, 22"C): 14.8, 15.2, 18.1, 25.3, 27.5, 30.1, 30.3 (2x), 33.2, 33.5 (2x), 33.9, 34.8, 36.7, 36.9, 38.6, 75.7, 76.7, 78.4, 81.0, 82.0, 91.1, 107.5, 125.7, 127.0 (2x), 127.9, 129.5(2x), 130.2, 131.0, 131.9, 132.57, 132.63, 139.3, 168.6, 170.3, 172.6, 173.6 ppm.

UV(MeOH): Smax is 250 nm (e 23,000) IR (as free acid: film on ZnSe): 3220 br, 2924, 2855, 2489 br, 1741, 1436-, 1417, 1266, 1152, 1122, 1004, 967, 950, 804, 744, 695 cm 1 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 772 by FAB-MS (observed EM+Li)+ at m/z 779 in the lithiated spectrum). The molecular formula C42H60013 was determined by HR-EI measurement of the tris-trimethylsilyl 'derivative (calm for C42H60013+ (SiC3H8)3 988.5220, found 988.5172).

1H NMR Spectrum (400 MHz) (C6D6,22 C)

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) 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 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:

and 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 and 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 (lS,3S,4S,5R,6R,7R)-l-EC4)-hydroxy-3,5-dimethyl-8- phenyl]oct-7-enyl-4, 6, -7-trihydroxy-6-Q-(tetradeca- 6,12-dienoyl)-2,8-dioxabicyclo[3.2.1]-octane-3,4,5- tricarboxylic acid.

8. A pharmaceutical composition for use in treating cancer comprising a therapeutically effective amount of (lS,3S,4S,5R,6R,7R)-1-(C4)- hydroxy-3 , 5-dimethyl-8-phenyl] oct-7-enyl-4, 6, -7- trihydroxy-6-0-(tetradeca-6,12-dienoyl)-2,8-dioxabi cyclo(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 through 8.