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Title:
ZARAGOZIC ACID DERIVATIVES AND METHODS OF TREATING HYPERCHOLESTEROLEMIA, FUNGAL GROWTH, AND CANCER THEREWITH
Document Type and Number:
WIPO Patent Application WO/1994/007485
Kind Code:
A1
Abstract:
The present invention relates to new compounds and compositions formed from the photochemical treatment of zaragozic acids. The compounds are effective at treating hypercholesterolemia as squalene synthase inhibitors, fungal growth, and cancer as farnesyl protein transferase inhibitors.

Inventors:
TREIBER LASZLO R (US)
ARISON BYRON H (US)
CHEN SHIEH-SHUNG TOM (US)
DOSS GEORGE A (US)
HUANG LEEYUAN (US)
MACCONNELL JOHN G (US)
MONAGHAN RICHARD L (US)
Application Number:
PCT/US1993/009144
Publication Date:
April 14, 1994
Filing Date:
September 27, 1993
Export Citation:
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Assignee:
MERCK & CO INC (US)
TREIBER LASZLO R (US)
ARISON BYRON H (US)
CHEN SHIEH SHUNG TOM (US)
DOSS GEORGE A (US)
HUANG LEEYUAN (US)
MACCONNELL JOHN G (US)
MONAGHAN RICHARD L (US)
International Classes:
A61K31/335; A61K31/355; A61K31/365; A61K31/455; C07D493/08; C07H19/01; (IPC1-7): A61K31/34
Foreign References:
EP0494622A11992-07-15
US5055487A1991-10-08
US5053425A1991-10-01
US5096923A1992-03-17
US5102907A1992-04-07
US5132320A1992-07-21
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Claims:
WHAT IS CLAIMED IS:
1. A composition comprising a compound selected from the compound of stractural formula (I), the compound of structural formula (II) and the compound of structural formula (HI): OH (ID (I) OH (HI) wherein: R is selected from: Rj is selected from (b) CH3CH=CH(CH2)4CH=CH(CH2)4C0 ; and R2 is selected from a) H, and b) CH3 R3 is Cj_5alkyl; r5alkyl; (g) or the groups (a) through (d) form a 5 to 10 membered mono or bicyclic ring with Cj_5 alkyl; (iv) C3.6 cycloalkyl; or a pharmaceutically acceptable salt thereof, provided that when R is then Rj is and when R is then Rj is when R is O then Rχ is CH3CH=CH(CH2)4CH=CH(CH2)4t!:0.
2. The composition of Claim 1, wherein Z is selected from (a) H; (b) C ^alkyl; and (c) C^alkyl substituted with a member of the group consisting of: (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (CI, Br, I, F) or hydroxy; or (iii) C i _5alky lcarbonyloxy .
3. The composition of Claim 2, wherein Z is methyl, ethyl or pivaloyloxymethyl.
4. The composition of Claim 2 wherein R3 is methyl.
5. The composition of Claim 1 wherein: R3 is CH3.
6. The composition of Claim 5 where Z is selected from: (a) H; (b) Cι _5alkyl; and (c) Cj_5alkyl substituted with a member of the group consisting of: (i) phenyl, (ii) phenyl substituted with methyl, methoxy, halogen (CI, Br, I, F) or hydroxy; and (iii) C i _5alky lcarbonyloxy .
7. The composition of Claim 5, wherein Z is methyl, ethyl or pivaloyloxymethyl.
8. A pharmaceutical composition comprising a nontoxic therapeutically effective amount of the composition of Claim 1, and a pharmaceutically acceptable carrier.
9. A pharmaceutical composition comprising a nontoxic therapeutically effective amount of the composition of Claim 1, in combination with a pharmaceutically acceptable nontoxic cationic polymer capable of binding bile acids in a nonreabsorbable form in the gastrointestinal tract and pharmaceutically acceptable carrier.
10. A pharmaceutical composition comprising a nontoxic therapeutically effective amount of the composition of Claim 1, in combination with a nontoxic therapeutically effective amount of a cholesterol lowering agent selected from the group consisting of: (a) HMGCoA reductase inhibitor; (b) HMGCoA synthase inhibitor; (c) Squalene epoxidase inhibitor; (d) Probucol; (e) Niacin; (f) Gemfibrozil; and (g) Clofibrate.
11. The composition of Claim 10 wherein the composition comprises the composition of Claim 1 and an HMGCoA reductase inhibitor selected from lovastatin, simvastatin, pravastatin and fluvastatin.
12. A method of treating hypercholesterolemia comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of the composition of Claim 1.
13. A method of inhibiting squalene synthase comprising the administration to a subject in need of such treatment of a nontoxic therapeutically effective amount of the composition of Claim 1.
14. A method for inhibiting fungal growth comprising applying to the area where growth is to be controlled an antifungally effective amount of the composition of Claim 1.
15. A method for treating cancer comprising the administration to a subject in need of such treatment of a therapeutically effective amount of the composition of Claim 1.
16. A method of inhibiting farnesylprotein transferase and famesylation of the oncogene protein Ras, comprising the administration to a subject in need of such treatment of a therapeutically effective amount of .the composition of Claim 1.
Description:
TITLE OF THE INVENTION

ZARAGOZ1C ACID DERIVATIVES AND METHODS OF TREATING HYPERCHOLES¬ TEROLEMIA. FUNGAL GROWTH. AND CANCER THEREWITH

BACKGROUND OF THE INVENTION

Hypercholesterolemia is known to be one of the prime risk factors for ischemic cardiovascular disease, such as arteriosclerosis. Bile acid sequestrants have been used to treat this condition; they seem to be moderately effective but they must be consumed in large quantities, i.e. several grams at a time, and they are not very palatable.

MEVACOR® (lovastatin) and ZOCOR® (simvastatin), now commercially available, members of a group of very active antihypercholesterolemic agents that function by limiting cholesterol biosynthesis by inhibiting the enzyme, HMG-CoA reductase.

Squalene synthase (also known as squalene synthetase) is the enzyme involved in the first committed step of the de novo cholesterol biosynthetic pathway. This enzyme catalyzes the reductive dimerization of two molecules of farnesyl pyrophosphate to form squalene. The inhibition of this committed step to cholesterol should leave unhindered biosynthetic pathways to ubiquinone, dolichol and isopentenyl t-RNA.

Previous efforts at inhibiting squalene synthase have employed pyrophosphate or pyrophosphate analog containing compounds such as those described in P. Ortiz de Montellano et al., J. Med Chem. 20, 243 (1977) and E.J. Corey and R. Volante, J. Am. Chem. Soc, 98, 1291 (1976). S. Biller (U.S. Patent 4,871,721) describes isoprenoid (phosphinylmethyl) phosphonates as inhibitors of squalene synthase.

Recently certain nonphosphorus containing inhibitors of squalene synthase have been isolated as natural products. These natural product inhibitors are described in U.S. Patent Nos. 5,132,320, 5,096,923, and 5,102,907. Semisynthetic analogs of these naturally occurring compounds have been reported in European Patent Publication EP O 512 865. However, a need still remains for a more

effective squalene synthase inhibitor, i.e., one that provides a greater antihypercholesterolemic effect and exhibits a good safety profile.

The natural product inhibitors are tricarboxylic acids. The present applicants have now found that these natural products known as Zaragozic Acid A, Zaragozic Acid B and Zaragozic Acid C undergo a photochemical reaction yielding monocarboxylic derivatives of the Zaragozic Acids, which are potent cholesterol lowering agents. The present invention discloses esters of these monocarboxylic acids.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to compounds of structural formulas (I), (II) or (III) provided below and compositions comprising such compounds. Compounds of formulas (I), (II) and (III) undergo equilibrium and all three equilibrium structures (for a given Rl , R and Z) may be present.

(ID (I)

(m) wherein:

R is selected from:

R j is selected from:

O

(b) CH 3 -CH-=CH-(CH 2 ) 4 -CH=CH-(CH 2 ) 4 -C-0- ; and

R2 is selected from:

a) H, and

R3 is C j _5alkyl;

Z is selected from

(i) H;

(ii) C^alkyl;

(iii) C j _5alkyl substituted with

(a) C^alkylcarbonyloxy;

(b) arylcarbonyloxy; (c) C 1 _5 alkoxy carbony loxy ;

( ) aryloxycarbonyloxy;

(g) or the groups a) through d) form a 5 to 10 membered mono or bicyclic ring with C j _5 alkyl;

(iv) C3. cycloalkyl; or a pharmaceutically acceptable salt thereof, provided that when R is

then Ri is

and when R is

then Rj is

when R is

One embodiment of this invention are compounds wherein Z is selected from

(a) H;

(b) C^alkyl;

(c) Cj^alkyl substituted with a member of the group consisting of: (i) phenyl,

(ii) phenyl substituted with methyl, methoxy, halogen

(CI, Br, I, F) or hydroxy; or (iii) C j ^alkylcarbonyloxy.

A second embodiment are compounds wherein Z is methyl, ethyl or pivaloyloxymethyl.

Yet a third embodiment are compounds wherein Z is hydrogen or a pharmaceutically acceptable salt thereof.

The compounds of the present invention may have asymmetric centers and may occur, except when specifically noted, as racemates, racemic mixtures or as individual diastereomers, or enantiomers, with all isomeric forms being included in the present invention.

When any variable (e.g., Rl, R2, etc.) occurs more than one time in any constituent, its definition on each occurrence is independent of its definition at every other occurrence. Also, combinations of substituents and/or variables are permissible only if such combinations result in stable compounds.

As used herein, except where noted, "alkyl" is intended to include both branched- and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms.

"Halogen" or "halo" as used herein, means fluoro, chloro, bromo and iodo. "Aryl" is intended to mean phenyl (Ph) or naphthyl.

Compounds of the present invention are active as squalene synthase inhibitors and are useful as cholesterol lowering agents and anti-fungal agents. The compounds of the present invention also inhibit farnesyl protein transferase and farnesylation of the oncogene protein Ras and are useful in treating cancer.

Compounds of formula (II) can be formed from Zaragozic Acid A (U.S. Patent 5,096,963), Zaragozic Acid B (U.S. Patent 5,132,320), and Zaragozic Acid C (U.S. Patent 5,102,907) by photochemical treatment of the parent natural product, under exposure to air, followed by esterification of the carboxyl group attached at the 5

position. Compounds of formula (II) wherein R2 is acetate can be converted to compounds wherein R2 is H by a biotransformation. A culture of MF6817 (ATCC 55189) has been employed in this transformation.

Compounds of formula (II) undergo a facile equilibrium to yield compounds of formula (I), in a C|_5 alcohol such as methanol. In the presence of water, they equilibrate to give compounds of formula (HI). This equilibrium is established rapidly when the appropriate solvent is added to the material (II). However, the equilibrium may be shifted to predominantly structure (II) in acetone solution. The equilibrium may be shifted to predominantly structure (III) by the addition of water to the alcohol solution. Structure (I) is most predominant in the pure alcohol solution. Thus, each equilibrium structure I, II, or III may be obtained substantially free of the other equilibrium structures. "Substantially free" should be understood to mean in a ratio of 80:20 or higher and more particularly with respect to (I) or (II) it may mean 90:10 or higher. Thus, the compound (I) substantially free of (II) and (HI) should be taken to mean that there is 10% or less of (II) and (HI) present.

Generally, esters are generated from an acid and an alcohol in the presence of an acid catalyst. It may be necessary to remove water (or in some cases, the ester) from the reaction mixture to drive the reaction to completion. However, strongly acidic conditions may cause undesired reactions with other parts of the acid to be esterified. This is true with the Zaragozic Acids. The following methods avoid strong acids and typically do not cause reaction with other functional groups.

SCHEME 1 Diazomethane (and substituted diazomethanes):

Q 1 C0 2 H + Q 2 CHN 2 > Q 1 C0 2 Z

Q*C0 2 H = Zaragozic Acids A, B, or C with at least one intact carboxy group. Z = -CH 2 Q 2

SCHEME 2

DBU Qi ^H + Q 3 I > Q-C0 2 Q ~

Q 3 = Ci _5alkyl, C3_6cycloalkyl.

SCHEME 3

"POM" reagent "POM" ester halomethylpivalate

X = CI, Br, I.

Methyl esters are conveniently formed by methylation of the carboxylic acid group with diazomethane, e.g., Example 2. Other diazo derivatives of the formula (Q 2 CHN2), e.g., diazoethane, react readily also to provide the appropriate ester according to Scheme 1. Treatment of primary or secondary halides in THF or benzene in the presence of 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU) will give the esters of the present invention, as illustrated in Scheme 2 and Example 3. Alternatively, pivaloyloxymethyl (POM) esters of Scheme 3 are synthesized by reaction with DBU and halomethylpivalate, as also exemplified by Example 4. Other methods of esterification of the Zaragozic Acids of this invention will readily occur to the skilled artisan. Compounds of formulae (I), (II), and (HI) are also useful for preparing prodrugs or other derivatives which are useful as squalene synthase inhibitors and which undergo a slower hemiketal- ketone equilibrium or do not exhibit this equilibrium at all. Specifically for compounds (II) and (III), the ketone functionality may be converted

to a cyclic ketal or mercaptal derivative using ethanediol or ethanedithiol; compounds of formula (III) may be converted to derivatives wherein the hydroxyls at C4 are etherified using two equivalents of an alkyl orthoformate ester. These derivatives and prodrugs are separately isolable and useful as cholesterol lowering agents.

The present invention is also concerned with a method of treating hypercholesterolemia which comprises the administration to a subject in need of such treatment of a nontoxic therapeutically effective amount of a compound of structural formula (I), (II), or (III) of the present invention, or mixture thereof, or a pharmaceutically acceptable salt thereof. Specifically, the compounds of this invention are useful as antihypercholesterolemic agents for the treatment of arteriosclerosis, hyperlipidemia, familial hypercholesterolemia and the like diseases in humans. They may be administered orally or parenterally in the form of a capsule, a tablet, an injectable preparation or the like. It is usually desirable to use the oral route. Doses may be varied, depending on the age, severity, body weight and other conditions of human patients, but a daily dosage for adults is within a range of from about 20 mg to 2000 mg (preferably 20 to 200 mg) which may be given in two to four divided doses. Higher doses may be favorably employed as required.

The present invention is also concerned with a method of inhibiting squalene synthase which comprises the administration to a subject in need of such treatment of a nontoxic therapeutically effective amount of a compound of structural formula (I), (II), or (III) of the present invention, or mixture thereof, or a pharmaceutically acceptable salt thereof. Specifically, the compounds of this invention are useful in treating disease conditions such as, but not limited to, hypercholesterol¬ emia; conditions which require the action of the enzyme squalene synthase. They may be administered by the same routes in the same dosages as described for the method of treating hypercholesterolemia.

The pharmaceutically acceptable salts of the compounds of this invention may include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from

bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene-diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethy laimine, piperazine, tris(hydroxymethyl)aminomethane, and tetramethylammonium hydroxide. These salts may be prepared by standard procedures.

The compounds of this invention may also be administered in combination with other cholesterol lowering agents such as those which inhibit an enzymatic pathway in the biosynthesis of cholesterol. Examples of such agents would include but are not limited to HMG-CoA reductase inhibitors, HMG-CoA synthase inhibitors, and squalene epoxidase inhibitors. Illustrative of such HMG-CoA reductase inhibitors are lovastatin, simvastatin, pravastatin and fluvastatin. Examples of HMG-CoA synthase inhibitors are: the beta-lactone derivatives disclosed in U.S. Patent Nos. 4,806,564, 4,816,477, 4,847,271, and 4,751,237; the beta lactam derivaties disclosed in U.S. 4,983,597 and the substituted oxacyclopropane analogues disclosed in European Patent Publication EP O 411 703. Illustrative examples of squalene epoxidase inhibitors are disclosed in European Patent Publication EP 0 318 860 and in Japanese Patent Publication Jo2 169- 571 A. LDL-receptor gene inducer molecules are disclosed in U.S. Patent Application Serial No. 07/670,640 filed March 18, 1991. Other cholesterol lowering agents that may be administered include niacin, probucol, and the fibric acids, clofibrate and gemfibrozil. Representative of such combinations are those containing about 10-400 mg of a compound of formula (I) in combination with about 20-100 mg of an HMG-CoA reductase inhibitor, 20 to 200 mg of a HMG-CoA synthase inhibitor, or 2 to 200 mg of a squalene epoxidase inhibitor, or up to 1000 mg of probucol, up to 2 g of clofibrate, 2 to 8 g of niacin, and 800 to 1500 mg gemfibrozil or 20 to 300 mg of an LDL-receptor gene inducer.

The compounds of this invention may also be coadministered with pharmaceutically acceptable nontoxic cationic

polymers capable of binding bile acids in a non-reabsorbable form in the gastrointestinal tract. Examples of such polymers include cholestyramine, colestipol and poly[methy-(3-trimethylaminopropyl) imino-trimethylene dihalide]. The relative amounts of the compounds of this invention and these polymers is between 1:100 and 1 :15,000. The intrinsic squalene synthase inhibitory activity of the compounds of this invention is measured by the standard in vitro protocols described below. The first assay measures activity against human squalene synthase, the second against rat squalene synthase.

HUMAN SQUALENE SYNTHASE ACTIVITY PREPARATION OF HUMAN HepG2 cell ENZYME

SOURCE: HEPG2 CELL LINE (Liver, hepatoblastoma, Human) ATCC No. HB 8065

CELL GROWTH AND MAINTENANCE

Culture Medium: Minimum essential medium (MEM) with non-essential amino acids, sodium pyruvate, and 10% fetal bovine serum. The medium was changed twice weekly. A confluent monolayer was achieved in 1 week. The growth medium is prepared as listed below.

Solution Volume CmL

1. MEM (Gibco #320-1090AK) 1000 With Earle's salts and L-glutamine

2. Penicillin (10,000 units/mL), streptomycin (10,000 mg/ml), Gibco#600-5140 PG 10

3. MEM sodium pyruvate, lOrnM

(100X) Gibco#320-1140 10

4. MEM nonessential amino acids,

10 mM(100X) Gibco#320-1140AG 10

5. L-glutamine, 200 mM (100X), Gibco#320-5030AG 10

Hyclone fetal bovine serum, defined, Hyclone #A-111 -L 100

Subculture Procedure: Remove medium, wash with PBS, add fresh trypsin (0.25%)-EDTA (0.02%) with Hank's Balanced Salt solution and let flask stand for a minute and remove the trypsin • solution. Incubate flask at 37°C until cells detached. Add fresh medium, disperse and dispense cells into new flasks. Subcultivation ratio: 1 :6.

Preparation of Delipidated Serum: Fetal calf serum (100 mL) and CAB-O-Sil (2 grams) stir overnight at 4°C and centrifuge at 16,000 rpm for 5 hrs. Filter supernatant and store at 4°C.

48 hrs. prior to harvest, switch cells grown in MEM with 10% Fetal Calf serum to MEM with 10% delipidated serum.

Squalene Svnthase Assay

Reactions were performed in 1.2 mL polypropylene tube strips of 8. Buffer mixture and subtrate mixture for the assay were prepared from the following solution:

Buffer mixture contains 270 mM HEPES, pH 7.5, 20 mM Potassium fluoride and 5.4 mM Dithiothreitol(DTT). 55 μL of this mixture was used per assay. The final concentrations of HEPES, KF and DTT in the assay are 150 mM, 11 mM and 3 mM respectively.

l mM

1 μg per mL

0.6 μM

2.4 μM

Harvest: Remove medium, wash with PBS, add fresh trypsin (0.25%)-EDTA (0.02%) with Hank's Balanced Salt solution, rinse and remove. Incubate flask at 37°C until cells detach. Add 6 mL of MEM medium per flask to suspend cells and combine into centrifuge tube. Spin cells at 1,000 rpm for 5 mins. Wash by resuspending cell pellet in PBS and repeat centrifuging. Count cells (2.5 x 10^ yield from 18 flasks (75 cm 2 ). Resuspend in 10 mL of 50mM HEPES (N-[2- Hydroxyethyl]piperazine-N'-[2-ethane-sulfonic acid]) containing 5mM MgCl2, 2mM MnCl2, lOmM DTT, pH 7.5 (enzyme suspension buffer).

Cell Extracts: Sonicate (probe sonicator setting #60, pulse) the cell suspension on ice for 2 min. After a 1 min. cooling on ice, the sonication is repeated until greater than 90% of the cells are broken as observed microscopically. Centrifuge cell suspension for 10 mins. at 10,000 rpm. Transfer supernatant to clean tube and centrifuge at 20,000 rpm for 20 mins. The HepG2 enzyme preparation was centrifuged at 34,000 rpm to separate the cytosol and microsomal enzymes. The enzyme suspension was diluted 1 to 1,536 and used to perform the squalene synthase assay using 3 μM 3 H-farnesyl pyrophosphate as the substrate.

For each reaction, 55 mL of buffer mixture was taken with 5 μL of an inhibitor solution in MeOH and 10 μL of diluted enzyme (1 to 1536 as described in the enzyme preparation; the final protein concentration of enzyme in the assay is 1.2 μg per mL.). The reaction was initiated by the addition of 30 mL of substrate solution and the mixture was incubated at 30°C for 20 minutes. The reactions were stopped by the addition of 100 μL of 95% EtOH, vortexed, and 100 μL of a suspension of 1 gram per mL of Bio-Rad AG 1 X 8 resin (400 mesh, Chloride form) was then added, vortexed. 800 μL of heptane was added to each tube strip and the strips were capped and vortexed for 10 minutes.

RAT SQUALENE SYNTHASE ACTIVITY Preparation of Microsomes:

Male Charles River CD rats (120 to 150 g) are fed a diet containing 0.1 % lovastatin for 4 days. The livers from these rats were homogenized in 5 volumes (mL/g) of ice cold 50 mM HEPES (4-(2- hydroxy-ethyl)-l-piperazine-ethanesulfonic acid), 5 mM EDTA (ethylenediaminetetraacetic acid) pH 7.5 with a Potter-Elvehjem type tissue grinder. The homogenate is centrifuged twice at 20,000 x g for 15 minutes at 4°C, discarding the pellet each time. The supernatant is then centrifuged at 100,000 x g for 1 hour at 4°C. The resulting micro somal pellet is resuspended in a volume of the above homogenizing buffer equal to one-fifth the volume of the original homogenate. This microsomal preparation typically has a protein concentration of about 7 mg/mL. The microsomal suspensions are stored in aliquots at -70°C. Squalene synthase activity in these aliquots is stable for at least several months.

Partial Purification of Prenyl Transferase

Prenyl transferase is purified to use in the enzymatic synthesis of radiolabelled famesyl pyrophosphate. Prenyl transferase is assayed by the method of Rilling (Methods in Enzymology 110. 125-129 (1985)) and a unit of activity is defined as the amount of enzyme that

will produce 1 μmole of famesyl pyrophosphate per minute at 30°C in the standard assay.

The livers of 23 forty-day old male rats that have been fed 5% cholestyramine plus 0.1% lovastatin are homogenized in a WARING blender in 1 liter of 10 mM mercaptoethanol, 2 mM EDTA, 25 μM leupeptin, 0.005% phenylmethyl sulfonyl fluoride pH 7.0 containing 0.1 trypsin inhibitor units of aprotinin/mL. The homogenate is centrifuged at 20,000 x g for 20 minutes. The supernatant is adjusted to pH 5.5 with 6N HO Ac and centrifuged at 100,000 x g for 1 hour. This supernatant is adjusted to pH 7.0 with 3N KOH and a 35-60% ammonium sulfate fraction taken. The 60% pellet is redissolved in 60 mL of 10 mM potassium phosphate, 10 mM mercaptoethanol, 1 mM EDTA pH 7.0 (Buffer A) and dialyzed against two 1 liter changes of Buffer A. This dialyzed fraction is applied to a 12.5 x 5 cm column of DEAE-sepharose 4B equilibrated with Buffer A. The column is washed with 700 mL of Buffer A and a 1 liter gradient from Buffer A to 100 mM potassium phosphate, 10 mM mercaptoethanol, 1 mM EDTA pH 7.0. Fractions having a specific activity greater than 0.20 units/mg are combined, solid ammonium sulfate is added to bring to 60% saturation and pelleted. The pellet is dissolved in 8 mL of 10 mM Tris, 10 mM β- mercaptoethanol pH 7.0 (Buffer B). The redissolved pellet is taken to 60% saturation with ammonium sulfate by adding 1.5 volumes of saturated ammonium sulfate in Buffer B. This ammonium sulfate suspension typically contains 3.5 units/mL with a specific activity of 0.23 units/mg and is free of isopentenyl pyrophosphate isomerase

at least 6 months.

The solvent (ethanol: 0.15 N NH4OH, 1 :1) is removed from 55 mCi of [4-14c]isopentenyl pyrophosphate (47.9 μCi/mmole) by rotary evaporation. Following addition of 600 μL of 100 mM Tris, 10 mM MgCl2, 4 mM dithiothreitol pH 7.5, the solution is transferred to a

1.5 mL Eppendorf centrifuge tube. Geranyl-pyrophosphate, 250 μL of a 20 mM solution, and 50 μL of the ammonium sulfate suspension of prenyl transferase are added to initiate the reaction. This incubation contains 5 μmoles of geranyl pyrophosphate, 1.15 μmoles of isopentenyl pyrophosphate, 6 μmoles of MgC_2 of 0.18 units of prenyl transferase in a volume of 900 μL. The incubation is conducted at 37°C. During the incubation, the mix typically turns cloudy white as the newly formed magnesium complex of famesyl pyrophoshate precipitates out of solution. The [4-14c]farnesyl pyrophosphate is collected by centrifugation for 3 minutes at 14,000 rpm in an centrifuge tube, the supernatant removed, and the pellet is dissolved in 1.0 mL of 50 mM HEPES, 5 mM EDTA, pH 7.5 The yield is typically about 50 μCi (90%) of [4- 14 C]farnesyl pyrophosphate. The [4- 14 C]farnesyl pyrophosphate is stored in aliquots at -70°C.

Squalene Synthase Assay

Reactions are performed in 16 x 125 mm screw cap test tubes. A batch assay mix is prepared from the following solution:

This assay mix is degassed under vacuum and flushed with N 2 . Solutions of the squalene synthase inhibitors are prepared either in

DMSO or MeOH and a 1:120 dilution of the microsomal protein is made with the original homogenizing buffer. For each reaction, 87 μL

of the assay mix is taken with 3 μL of an inhibitor solution (DMSO or MeOH in the controls), warmed to 30°C in a water bath and then the reaction is initiated by the addition of 10 μL of the 1 :120 dilution of microsomal protein (0.6 μg protein total in the assay). The reactions are stopped after 20 minutes by the addition of 100 μL of a 1:1 mix of 40% KOH with 95% EtOH. The stopped mix is heated at 65°C for 30 minutes, cooled, 10 mL of heptane is added and the mix is vortexed. Activated alumina (2 g) is then added, the mix vortexed again, the alumina allowed to settle and 5 mL of the heptane layer is removed. Ten mL of scintillation fluid is added to the heptane solution and radioactivity is determined by liquid scintillation counting. Percent inhibition is calculated by the formula:

r [Sample - Blgnk] ) L 1 [Control - Blank] /J X 1 UU

IC50 values are determined by plotting the log of the concentration of the test compound versus the percentage inhibition. The IC50 is the concentration of inhibitor that gives 50% inhibition as determined from these plots. The present compounds are also useful as broad spectrum antifungal agents as determined by broth and agar dilution methods. Thus the present invention is also directed to a method of treating fungal infections which comprises the administration to an organism in need of such treatment of a nontoxic therapeutically effective amount of a compound represented by the structural formulas (I), (II) or (III), and pharmaceutically acceptable salts thereof. Generally from 2 to about 20 mg/kg should be employed as a unit dosage in an antifungal treatment. Furthermore, the compounds of the present invention are useful as inhibitors of farnesyl-protein transferase and thereby of famesylation of the RAS protein and thus block the ability of RAS to transform normal cells to cancer cells. Farnesyl-protein transferase activity may be reduced or completely inhibited by adjusting the compound dose.

The intrinsic farnesyl-protein transferase (FTase) activity of representative compounds of this invention is measured by the assay described below:

RASIT ASSAY I

Farnesyl-protein transferase (Ftase) from bovine brain is 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 at 3.5 μM, 0.25 μM [ 3 H]FPP, and the compounds to be assayed are incubated with this partially purified enzyme preparation.

The pharmaceutical compositions containing the compounds of structural formula (I), (II) or (III) inhibit farnesyl-protein transferase and the famesylation 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 present invention also encompasses a method of the treatment of cancer, comprising the administration of a pharmaceutical composition comprising 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 a day.

EXAMPLE 1

Preparation of a composition of (I), (II) and (III) wherein:

R is

Ri is

R3 is methyl j

Z is H (Composition A).

Step A: Preparation of Zaragozic Acid A 1. Culruring MF5453

Culture MF5453 (ATCC 20986) 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 φm, 85% humidity. At the end of this incubation, 2.0 mL aliquots were aseptically

transferred to each of 75 MBM production medium flasks. These production flasks were then incubated at 25°C, 220 φm, 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 Biohomogenizer/mixer (Biospec Products Inc. Bartlesville, OK); and then 45 mL methanol was added to each flask (final methanol concentration was approximately 50%). Flasks were then retumed to the shaker and agitated at 220 φm for 30 minutes. Subsequently, the contents of the flasks were pooled.

(no pH adjustment) pH 7.0

45 mL/nonbaffled 250 mL Erlenmeyer flask autoclave 15 minutes (121 C, 15 psi)

2. Isolation of Zaragozic Acid A

A 6 liter 50% methanol homogenized fungal extract exhibiting a pH of 4.5 was employed in the following isolation procedure. The mycelia were filtered through CELITE and the recovered mycelial cake 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 step wise gradient of methanol consisting of 50/50 methanol/H 2 0 (1 L), 60/40, methanol/H2θ (1 L), 80/20 methanol/H2θ (2 L), 90/10 methanol/H2θ (1 L), 100% methanol (2 L), and 100% acetone (1 L). The fractions from 50/50 to 90/10 methanol/H2θ were combined and diluted with water to 35/65 methanol/H2θ (total volume 10 L).

The 10 L of 35/65 methanol/H2θ 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 Ci8, 22 mm ID X 25 cm, 8 mL/minute. The initial mobile phase was 60/40 methanol/10 mM K3PO4, pH 6.5, and after 20 minutes the mobile phase was changed to 80/20 methanol/lOmM potassium phosphate, pH 6.5. Fractions of 8 mL each were collected, and the fractions from 31 to 33 minutes 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 Zaragozic Acid A was obtained.

Step B: Preparation of Composition A

A solution of 2.5 mg/mL of Zaragozic Acid A in DMSO was prepared and 30 mL aliquots of this solution were placed in each of five 250 mL Erlenmeyer flasks. The samples were placed in a 37°C incubator at a distance of 25-30 cm from a fluorescent light and were exposed to air. The reaction was allowed to continue for 6-8 days prior to isolation.

Step C: Isolation of Composition A

The five samples from Step B were combined and mixed with water (200 mL). The solution was acidified with citric acid and extracted twice with 100 mL portions of CH2CI2. The combined methylene chloride phases were evaporated to an oily residue that was transferred to test tubes and evaporated further to dryness in a stream of nitrogen at 40-50°C. The dry residues were redissolved in small volumes of methanol and combined. The methanol solution (10-15 mL) was mixed with formic acid (0.5 mL) immediately before being loaded onto a HP-20 column (2.5 x 23 cm, 113 mL) equilibrated with 0.5% formic acid in water. The chromatogram was developed in a stepwise gradient made using 200 mL of each solvent mixture of water- acetonitrile beginning with volume ratios of 100:0, 90:10, etc in 10% increments up to 0:100. Each mixture contained 7 mL of formic acid. The flow ratio was 3 mL/minute. The bulk of product was found in the range of 1500-1615 mL of eluate. The samples were evaporated to dryness and then redissolved in methanol to 20 mg/mL concentration used in preparative HPLC. The final purification was by preparative HPLC carried out on a BECKMAN ULTRASPHERE ODS (10 x 250 mm) column using 0.1% formic acid in acetonitrile - water (60:40 v/v) as eluant at a flow rate of 4.00 mL/min and a detector setting of 213 nm. The retention time of compound IA was found to be 15.8 minutes; for comparison the retention time of Zaragozic Acid A was 10.4 minutes.

-U NMR (400 MHz) (Acetone)

HA 4.67 (d, 17.1, 1H), 4.42 (d, 17.1, 1H) 3-CH 2

IA 4.16 (d, J=12.1, 1H), 3.71 (d, 12.1, 1H) 3-CH 2

IflA 4.11 (d, 13.4, 1H), 3.91 (d, 13.4, 1H) 3-CH 2

EXAMPLE 2

Preparation of the (C-5 methyl ester of Composition A.

wherein:

R3 is -CH3, and Z is -CH3.

Into a 50 mL receiver flask was placed about 1.5 mg (2.4 micromoles) of Composition A which was dissolved in about 5 mL diethyl ether. The flask was then placed at the receiver end of a diazomethane generator apparatus and cooled by immersion into an ice bath. Into the reaction vessel was placed a solution of 5 g potassium hydroxide dissolved in 8 mL water, with 10 mL ethanol added thereafter. The mixture was heated in a water bath to about 65°C. The cold-finger condenser was filled with a dry-ice-isopropanol mixture (and kept filled during diazomethane generation). From an addition funnel equipped with a TEFLON stop-cock mounted above the reaction vessel, dropwise addition was made of a solution of 0.5 g N-methyl-N- nitroso-p-toluenesulfonamide (2.3 millimoles, a large excess) in approximately 5 mL diethyl ether. The addition lasted about 20

minutes. Diazomethane dissolved in ether slowly distilled from the reaction vessel and dripped off the cold finger into the receiver containing the solution of Composition A. An additional 10 mL ether added to the addition funnel flushed virtually all diazomethane from the reaction vessel into the receiver and left the contents of the vessel colorless and those of the receiver distinctly yellow. The receiver flask was removed from the apparatus and glacial acetic acid was added dropwise to consume the excess diazomethane, as indicated by the disappearance of the yellow color. The ether and any excess acetic acid were removed under a stream of nitrogen and the residue was redissolved in a small volume of methanol- water (1 to 1, by volume) for HPLC clean-up.

For preparative HPLC, a C^ reversed-phase column was used, with a linear gradient from 30% acetonitrile/70% water to 90% acetonitrile/10% water over 30 minutes, with a 5 minute hold at 90% acetonitrile before return to initial conditions.

For HPLC cleanup, an analytical (4.6 mmID x 250 mm) BECKMAN ULTRASPHERE ODS column was used at room temperature. The solvent system was a gradient from 30% acetonitrile/70% water to 90% acetonitrile/10% water over 30 min. with a 5 min. hold at 90% acetonitrile before return to initial conditions. Flow was 1 mL/min. UV was monitored at 215 nm. The material to be chromatographed was dissolved in a small volume of methanol-water (1 to 1) and injected 50 μL at a time. Under these conditions five peaks were collected. The second peak was the peak of interest and contained predominantly the product compound. The second peak had a retention time of 29.7 minutes. A fifth peak contained an epoxide; retention time = 33.8 minutes.

NMR of Second Peak: 7.25 (t, J=7.5, 2H), 7.12 (m, 3H), 6.81 (dd, J=15.8, 8.8, 1H), 5.80 (d, J=15.8, 1H), 5.59 (d, J=2.3, 1H), 5.05 (d, J=4.7, 1H), 4.96 (s, 1H), 4.94 (s, 1H), 4.08 (d, J=13.4, 1H), 3.98 (d, J=2.3, 1H), 3.86 (d, J=13.4, 1H), 3.76 (s, 3H), 2.66 (m, 1H), 2.45 (m,

2H), 2.30 (m, IH), 2.17 (m, IH), 2.09 (s, 3H), 1.88 (m, 2H), 1.25-1.45 (m, 3H), 1.15 (m, 2H), 1.04 (d, J=6.6, 3H), 0.86 (m, 9H).

EXAMPLE 3 Preparation of the CC-5) ethyl ester of Composition A

A solution of Compound IIA (1 equiv.), iodoethane (2 equivs.) and l,8-diazabicyclo[5.4.0]-undec-7-ene (DBU, 2 equivs.) in tetrahydrofuran (THF) is heated at 60°C with stirring for 48 hours or until the reaction is complete as monitored by TLC or HPLC. The product ethyl ester is purified by HPLC.

EXAMPLE 4 Preparation of the (C-5 POM ester of Composition A

To one equivalent of Compound IIA in refluxing acetonitrile is added one equivalent of l,8-diazabicyclo-[5.4.0]-undec-7- ene (DBU) and 2 equivalents of chloromethyl pivalate, followed by a few crystals of sodium iodide. The reaction mixture is stirred overnight at reflux and reaction progress is followed by HPLC. The POM ester can then be purified by HPLC.

EXAMPLE 5 Preparation of an unesterified formula (ϊ) compound of the structure

This compound (IA) was prepared by evaporating to dryness a sample of composition A from Example 1 and then dissolving the dried material in anhydrous methanol. A solution of (IA) was obtained substantially free of other equilibrium structures.

*NMR (400 MHz) (CD 3 OD) 4.06 (d, 13.1, IH), 3.86 (d, 13.1, IH) 3- CH 2 13 C NMR 170.8, 106.4, 93.8, 89.4, 82.8, 81.8, 63.4.

EXAMPLE 6 Preparation of an unesterified formula (II) compound of the structure

This compound (IIA) was prepared by evaporating to dryness a sample of Composition A from Example 1 and then dissolving the dried material in pure acetone. A solution of (HA) was obtained substantially free of other equilibrium structures. -E. NMR (Acetone) 4.67 (d, 17.1, IH), 4.42 (d, 17.1, IH) 3-CH 2 13 C NMR (Acetone) 197.2, 163.5, 106.8, 91.9, 82.9, 80.8, 70.3.

EXAMPLE 7

Preparation of an unesterified formula (IIP compound of the structure

( A)

This compound (IIIA) was prepared by adding to the acetone solution of Example 6 an amount of water to bring the water concentration to about 10%. A solution of HI A was obtained substantially free of other equilibrium structures. 13 C NMR (Acetone + D 2 Q) 169.7, 106.1, 90.3, 88.7, 82.5, 81.4, 70.0

EXAMPLE 8 Preparation of an unesterified formula (II) compound of the structure

(DAI)

1. Seed Growth

Aliquots of medium #2 (50 mL) in 250 mL baffled flasks were inoculated with MA6817 and shaken in a rotary shaker at 220 φm and 27°C. The seed was grown for 48 hours.

2. Fermentation

Production flasks (medium #2, 50 mL in 250 mL baffled flasks) were inoculated with 2 mL of the seed medium and shaken at 27 °C and 220 φm on a rotary shaker. After 24 hours, a substrate containing a crude mixture of Zaragozic Acid A and Composition A, prepared above (Example 1), was added to each flask. Thus, a DMSO solution of the substrate containing Zaragozic Acid A (1.217 mg, 76.1%) and Composition A (0.382 mg, 23.9%) was used for each of two shake flasks. Incubation continued for 72 hours.

3. Extraction:

The harvested biotransformation samples were acidified with formic acid (2 ml 88% for each flask) then extracted with ethyl acetate. The organic phase was dried over Na2S04 then filtered and evaporated to dryness. The dry residue was redissolved in the smallest possible volume of DMSO-water (2:1 v/v) for preparative HPLC.

4. Chromatography

Two different gradient methods were used in succession. The first separation was accomplished on a BECKMAN ULTRASPHERE CYANO column (10x250mm) in a gradient from 20% solvent B/80% solvent A to 65% solvent B/35% solvent A in 35 minutes at a flow rate of 3.00 mL/min. Fractions were collected every 3 minutes or according to peaks detected at 213 nm, as appropriate. The selected fractions (22.5-24.0 min) were evaporated to dryness and chromatographed again on a BECKMAN ULTRASPHERE OCTYL column (10x250mm) in a gradient from 30% B/70% A to 80% B/20% A in 35 minutes then at 100% solvent B for an additional 10 minutes. The remaining conditions were the same as in the first separation. Solvent A was 20 mM HCOOH and B was acetonitrile-water (17:3 v/v) containing the same amount of HCOOH as solvent A.

Evaporation of the selected fractions (retention time 33.6 min.) provided the product, Compound (HAl), with the following physical characteristics:

JH NMR spectrum (400 mHz) (CD 3 OD, 22°C): 7.23 (t, 2H), 7.19 (d,

2H), 7.12 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.86 (dd, 15.7, 1.0, IH), 5.50 (d, 2.4, IH), 5.06 (s, IH), 4.93 (s, IH), 4.01 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.89 (d, 5.0, IH), 3.83 (d, 12.7, IH), 2.75 (dd, 13.4, 5.9, IH), 2.36 (dd, 13.4, 9.0, IH), 2.0-2.45 (m, 4H), 1.93 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm.

Analytical HPLC on a BECKMAN ULTRASPHERE OCTYL column (4.6 x 250 mm) wherein the elution was performed in the gradient mode according to the following program:

Solvent A: 10 mM H3PO4 in water Solvent B: Acetonitrile- water (85:15 v/v)

Time (min.) percent B

0 30

2 30

18 80

20 100

24 100

25 30

Flow: 0.900 mL/min Temp.: Ambient

Retention time of compound IIAl = 19.9 minutes, UV max at 213 nm.

While the foregoing specification teaches the principles of the present invention, with examples provided for the puφose of illustration, it will be understood that the practice of the invention encompasses all of the usual variations, adaptations, modifications, deletions or additions of procedures and protocols described herein, as come within the scope of the following claims and its equivalents.