WO1993017557A1 - Cholesterol lowering compounds produced by directed biosynthesis - Google Patents

Abstract

Compounds of structural formulae (I) and (II) are produced by directed biosynthesis. These compounds are squalene synthase inhibitors and thus useful as cholesterol lowering agents, antifungal agents and cancer treatment agents.

Description

TITLE OF THE INVENTION

CHOLESTEROL LOWERING COMPOUNDS PRODUCED BY DIRECTED BIOSYNTHESIS

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), now commercially available, is one 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. Patents 5,053,425; 5,055,487 and 5,026,554.

U.S. Patent 5,053,425 discloses a Zaragozic Acid compound of structure

hereafter referred to as Zaragozic Acid A.

U.S. Patent 5,026,554 discloses a Zaragozic Acid compound of structure

hereafter referred to as Zaragozic Acid C.

Applicants have now found that providing certain aryl, heteroaryl, aralkyl or heteroaralkyl carboxylic acids to cultures that produce Zaragozic Acid A or Zaragozic Acid C leads to the incorporation of an aryl or heteroaryl moiety into the C-1 side chain of the Zaragozic Acid. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to compounds of structural formulae (I) and (II)

wherein R₁ is

,

,

,

,

, or

;

wherein X is:

(a) H,

(b) halogen (F, Cl, Br, I),

(c) OH, or

(d) CH₃;

Y is:

(a) halogen (F, Cl, Br, I),

(b) OH, or

(c) CH₃;

and wherein Z¹, Z² and Z³ are each independently:

(a) H,

(b) C_1-5alkyl,

(c) C_1-5alkyl substituted with:

(i) phenyl,

(ii) phenyl substituted with methyl, methoxy, halogen (Cl, Br, I, F) or hydrosxy,

(iii) C_1-5alkylcarbonyloxy,

(iv) C_6-10arylcarbonyloxy ,

(v) C_1-5alkoxycarbonyloxy,

(vi) C_6-10aryloxycarbonyloxy,

(ix) or the groups (iii) to (vi) form a 5 to

10 membered mono- or bicyclic ring with C_1-5alkyl, or

a pharmaceutically acceptable salt thereof;

pharmaceutical compositions thereof, and their use as squalene synthase inhibitors and their use as

cholesterol lowering agents, antifungal agents and cancer treatment agents. This invention is also directed at the process of making the compounds of this invention by directed biosynthesis.

In one class of this embodiment the compound is of structural formula (I). In a particular subclass R₁ is

Exemplifying this sub-class are the compounds wherein X is H and Y is F or OH. In another sub-class of this embodiment of structural formula (I) R₁ is

or

Exemplifying this sub-class are the compounds wherein X is H or F.

In another class of this invention the compound is of structural formula (II). In a

particular sub-class, R₁ is

Exemplifying this sub-class is the compound wherein X is hydrogen.

The invention is also directed to the process of forming the compounds of Formulae (I) and (II) in a directed biosynthesis which comprises the addition of a compound of Formula (III) selected from the group consisting of:

(a) R₁-CO₂H; and

(b) R₁-CH₂-CHNH₂CO₂H;

wherein R₁ is as defined above, to a Zaragozic Acid A producing culture and or to a

Zaragozic C producing culture isolating the product (I) or (II) from the culture broth.

Known Zaragozic Acid A producing cultures suitable for producing the compounds of the present invention include:

(a) MF5453 (ATCC 20986),

(b) MF5565 (ATCC 74068),

(c) MF5599 (ATCC 74065),

(d) MF5572 (ATCC 74066), and

(e) MF5573 (ATCC 74067).

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, its morphological characteristics and a fermentation procedure using this microorganism have been described in U.S. Patent

5,053,425.

The culture MF5565 is a strain of Exserohilum rostratum, which was isolated from bark of Theobroma cacao (Philippines). The culture has been deposited with the ATCC as ATCC 74068. The microorganism MF5565, its morphological characteristics and a fermentation procedure using this microorganism have been described in U.S. Patent Application Serial No. 722,049 filed June 27, 1991.

This strain, MF5565, was recovered from the bark of Theobroma cacao, collected in Los Banos, Laguna Province, Philippines. Bark discs were removed with a leather punch (no. 245, C.S. Osborne & Co., Harrison, NJ). Discs were approximately 1 cm in diameter and 0.3-1.0 cm thick depending on the thickness of the bark and amount of force used to hammer the punch into the tree. Discs included an entire bark cross-section along with the vascular cambium, and sometimes a veneer of the outer xylem. Discs from each tree were placed in manila coin envelopes for transport to the

laboratory. Discs were soaked in 10% household bleach for 3 minutes, rinsed with sterile distilled water and briefly flamed with an alcohol lamp prior to

application to isolation media. Bark discs were applied outer side down to an agar medium (10 g malt extract, 2 g yeast extract, 1 g sodium propionate, 5 g dehydrated bovine bile, 1 mg benomyl, 50 mg

streptomycin sulfate, 50 mg chlorotetracycline, 20 g agar in 1 L distilled water) in 100 mm diameter plastic Petri dishes. Petri dishes were incubated at 24ºC, and inspected more or less daily for up to one month for the development of fungal colonies on bark discs and the agar.

Strain MF5565 exhibits the following morphological characteristics.

Colonies relatively fast-growing, in 1 week attaining a diameter of: 50 mm on cornmeal agar (Difco Laboratories); 50-52 mm on yeast-malt extract agar (10 g malt extract, 2 g yeast extract, 20 g agar in 1 L distilled water); 60 mm on V8 juice agar (200 mL V8 juice, Campbell Soup Co., 3 g CaCO₃, 20 g agar diluted to 1 L distilled water). On yeast-malt agar with both submerged and aerial mucelium, with submerged mycelium sometimes forming radial strands, floccose to cottony or lanose in age, with margin appressed, minutely fimbriate to even, hyaline to pale gray at the margin but soom darkening to dark gray or dark olive-gray, or black in age, Dark Olive-Gray, Iron Gray, Dark Mouse Gray, Dusky Green-Gray, Blackish Green-Gray, Olivaceous Black (capitalized color names from Ridgway, R. 1912. Color Standards and Nomenclature, Washington, D.C.), similar in reverse, often with patches or tufts of hyaline to pale gray aerial hyphae developing in older portions. Odors, sclerotia, stromata, or pseudothecia absent. Conidiophores arising from uppermost aerial mycelium, up to 600 μm long, 3-4.5 μm wide, straight or flexuous, with geniculate apices, with walls smooth, or occasionally finely incrusted, usually bearing 2-10 conidia, pale olive-gray to olive-gray. Conidiogenous cells polytretic, integrated, sympodial, indeterminate, terminal or intercalary, with slightly raised, darkened scars surrounding a minute pore at the conidiogenous locus. Conidia 45-250 × 7-20 μm, mostly 75-180 μm long, variable in shape, broadly ellipsoidal, fusoid, obclavate, or tapered cylindrical, straight to curved, or rarely sigmoid, with broadly rounded apices, smooth, 5-22 septate, with basal septum most thickened and darkened, with terminal septum often also darker than septa delimiting central cells, with a distinct

cylindrical hilar appendix protruding 1-2.5 μm,

pedicel-like extensions absent, initially germinating from apical and basal cells pale gray to olive-gray in 3% KOH. Hyphae septate, branched, pale olive-gray to olive-brown, usually smooth, but occasionally with fine incrustations.

Strain MF5565 belongs to the genus Exserohilum rostratum based on the combination of polytretic conidiogenous cells that give rise to predominately multiseptate, dematiaceous

phragmoconidia. The basal cell of the conidium is delimited by a thick, darkened septum, and has a protruding hilar appendix. Strain MF5565 is identified as Exserohilum rostratum based on the predominance of straight and curved conidia, darkened septa delimiting both the basal and terminal cells, and relatively long conidia (A. Sivanesan. 1987 Graminicolous species of Bipolaris, Curvularia, Drechslera, Exserohilum and their telemorphs. CMI Mycological Paper No. 158).

The culture MF5599, is a strain of Curvularia lunata var. aeria isolated from bark of Ficus elastica (Diliman, Quezon City, Philippines). The culture has been deposited with the ATCC as ATCC 74065. The microorganism MF5599 has the same morphological

characteristics as MF5572, described below.

The culture MF5572 is a strain of Curvularia lunata var. aeria, isolated from tree bark

(Philippines). This culture has been deposited with the ATCC as ATCC 74066. The microorganism MF5572, its morphological characteristics and a fermentation procedure using this microorganism have been described in U.S. Patent Application Serial No. 715,535 filed June 14, 1991.

Strain MF5572, was recovered from the bark of an unidentified tree, collected in Diliman, Quezon City, Philippines. Bark discs were removed with a leather punch (no. 245, C.S. Osborne & Co., Harrison, NJ). Discs were approximately 1 cm in diameter and 0.3-1.0 cm thick depending on the thickness of the bark and amount of force used to hammer the punch into the tree. Discs included an entire bark cross-section along with the vascular cambium, and sometimes a veneer of the outer xylem. Discs from each tree were placed in manila coin envelopes for transport to the

laboratory. Discs were soaked in 10% household bleach for 3 minutes, rinsed with sterile distilled water and briefly flamed with an alcohol lamp prior to application to isolation media. Bark discs were applied outer side down to an agar media (10 g malt extract, 2 g yeast extract, 1 g sodium propionate, 5 g dehydrated bovine bile, 1 mg benomyl, 50 mg streptomycin sulfate, 50 mg chlorotetracycline, 20 g agar in 1 L distilled water) in 100 mm diameter plastic Petri dishes. Petri dishes were incubated at 24ºC, and inspected more or less daily for up to two weeks for the development of fungal colonies on bark discs and the agar.

Strain MF5572 has been identified as Curvularia lunata var. aeria and exhibits the following morphological characteristics.

Colonies are relatively fast-growing, in 1 week attaining a diameter of: 30-35 mm on cornmeal agar (Difco Laboratories); 30-35 mm on yeast-malt extract agar (10 g malt extract, 2 g yeast extract, 20 g agar in 1 L distilled water); 40-55 mm on V8 juice agar (200 mL V8 juice, Campbell Soup Co., 3 g CaCO₃, 20 g agar diluted to 1 L distilled water). On yeast-malt agar both submerged and aerial mycelia form, are slightly raised in side view, velvety to floccose when young, cottony or lanose in age, with margin slightly raised, even to wavy, hyaline to pale gray at the margin but soon darkening to grayish olive, gray, to dark olive-gray, Smoke Gray, Light Grayish Olive, Deep Olive-Gray, Dark Olive-Gray, Iron Gray, Castor Gray (capitalized color names from Ridgway, R. 1912. Color Standards and Nomenclature, Washington, D.C.), in reverse yellowish gray towards the margin but soon olivaceous gray, in age developing dark olive-black spots and patches in the agar, often with patches or tufts of hyaline to pale gray aerial hyphae developing in older portions, odors and pseudothecia absent. The surface of cultures in excess of 3 weeks old, generally develop straight to curved, cylindrical, finger-like stromata, 0.5-1 mm tall, which project upward from the oldest regions of the colony surface. Stromata

formation is best on nutrient-rich media, e.g.

potato-dextrose agar, oatmeal agar, or glucose-yeast-malt extract agar.

Conidiophores arising from aerial hyphae, 30-200 × 3-5 μm, septate, straight or flexuous,

sometimes branched in age, with apices straight, curved or geniculate, smooth, thin- to slightly thick-walled, olive-brown to olive-gray in 3% KOH, bearing 2-10 conidia. Conidiogenous cells polytretic, integrated, indeterminate, sympodial, usually terminal on the conidiophore, sometimes intercalary in age, with slightly darkened scars surrounding a minute pore at the conidiogenous locus. Conidia 18-28 × 9-14 μm, consistently 3-septate, broadly elliptical, with penultimate, distal cell curved and distinctly swollen, with slightly flattened scar at base, without hilar appendix, smooth, pale olive-brown to olive-gray, usually with two central cells slightly darker. Hyphae pale olive-gray to dark olive-gray or olive-brown in 3% KOH, septate, branched. Stromatic tissue a textura intricata, with cells hyaline in 3% KOH.

The culture MF5573, Curvularia lunata var. lunata, was isolated from Ficυs elastica tree bark (Philippines). The culture has been deposited with the ATCC as ATCC 74067. The microorganism MF5573, its morphological characteristics and a fermentation procedure using this microorganism have been described in U.S. Patent Application Serial No. 715,535 filed June 14, 1991.

Curvularia lunata var. lunata MF5573 was recovered from the bark of Ficus elastica collected in Diliman, Quezon City, Philippines. Bark discs were removed with a leather punch (no. 245, C.S. Osborne &. Co., Harrison, NJ). Discs were approximately 1 cm in diameter and 0.3 - 1.0 cm thick depending on the thickness of the bark and amount of force used to hammer the punch into the tree. Discs included an entire bark cross-section along with the vascular cambium, and sometimes a veneer of the outer xylem. Discs from each tree were placed in manila coin

envelopes for transport to the laboratory. Discs were soaked in 10% household bleach for 3 minutes, rinsed with sterile distilled water and briefly flamed with an alcohol lamp prior to application to isolation media. Bark discs were applied outer side down to an agar media (10 g malt extract, 2 g yeast extract, 1 g sodium propionate, 5 g dehydrated bovine bile, 1 mg benomyl, 50 mg streptomycin sulfate, 50 mg chlorotetracycline, 20 g agar in 1 L distilled water) in 100 mm diameter plastic Petri dishes. Petri dishes were incubated at 24°C, and inspected more or less daily for up to two weeks for the development of fungal colonies on bark discs and the agar.

Strain MF5573 has been identified as Curvularia lunata var. lunata and exhibits the

following morphological characteristics. Colonies are relatively fast-growing, in 1 week attaining a diameter of: 35-40 mm on cornmeal agar (Difco Laboratories); 40 mm on yeast-malt extract agar (10 g malt extract, 2 g yeast extract, 20 g agar in 1 L distilled water); 45-50 mm on V8 juice agar (200 mL V8 juice, Campbell Soup Co., 3 g CaCO₃, 20 g agar diluted to 1 L distilled water). On yeast-malt agar both submerged and aerial mycelia form, with aerial mycelia sometimes forming radial strands, floccose to cottony or lanose in age, with margin appressed, minutely fimbriate, hyaline to pale gray at the margin but soon darkening to dark gray or dark olive-gray, Castor Gray, Dark Olive-Gray, Iron Gray, Dusky

Green-Gray, Blackish Green-Gray, Olivaceous Black

(capitalized color names from Ridgway, R. 1912. Color Standards and Nomenclature, Washington, D.C.), similar in reverse, often with patches or tufts of hyaline to pale gray aerial hyphae developing in older portions, occasionally forming pale gray to hyaline sectors, odors, sclerotia, stromata, or pseudothecia absent.

Conidiophores arising from surface or aerial hyphae, 15-250 × 3-5 μm, septate, straight or flexuous, sometimes branched in age, with apices straight, curved or geniculate, smooth, thin- to slightly thick-walled, olive-brown to olive-gray in 3% KOH, bearing 4-15 conidia. Conidiogenous cells polytretic, integrated, indeterminate, sympodial, usually terminal on the conidiophore, sometimes intercalary in age, with slightly darkened scars surrounding a minute pore at the conidiogenous locus. Conidia 21-30 × 9-13.5 μm, usually 3-septate, infrequently 4-septate, broadly elliptical, with penultimate, distal cell curved and often obliquely swollen, with slightly flattened scar at base, without hilar appendix, smooth, pale

olive-brown to olive-gray, usually with two central cells slightly darker. Hyphae pale olive-gray to dark olive-gray or olive-brown in 3% KOH, septate, branched.

Known Zaragozic Acid C producing cultures suitable for producing the compounds of the present invention include:

(a) MF5465 (ATCC 74011),

(b) MF5701 (ATCC 74165), and

(c) MF5703 (ATCC 74166).

The compounds of formula (II) are prepared in an aerobic fermentation procedure employing strains of Leptodontium elatius. More particularly, the strains employed may be selected from strains MF5465 (ATCC 74011), MF5701 (ATCC 74165), and MF5703 (ATCC 74166), or mutants thereof. These mutants have essentially the same characteristics of the strains (i.e., MF5465 (ATCC 74011), MF5701 (ATCC 74165), and MF5703 (ATCC 74166).) The term "mutant" refers to an MF5465 (ATCC 74011), MF5701 (ATCC 74165), or MF5703 (ATCC 74166) organism in which some gene of the genome is modified, leaving the gene or genes responsible for the organism's ability to produce a compound of formula (II) through the process of the present invention functional and heritable.

A biologically pure culture of Leptodontium elatius as claimed herein is defined as being

originally isolated from the natural environment and free of viable contaminating microorganisms. A culture of Leptodontium elatius as claimed herein is defined as being originally isolated from the natural environment and free of viable contaminating microorganisms that would be deleterious to the formation of a compound of formula (II) through the process of the present

invention.

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

The culture MF5465, identified as

Leptodontium elatius exhibits the following

morphological features.

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

Olive-Gray, (capitalized color names from Ridgway, R. 1912. Color Standards and Nomenclature, Washington, D.C.), with similar reverse pigmentation, without exudates diffusible pigments or odors.

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

Occasionally with undifferentiated conidiogenous loci directly on vegetative hyphae. Developing conidia adhere to conidiophore terminus in a thin, irregular to ladder-like rachis in groups of up to 4-15 conidia. Conidiophores originating as undifferentiated branches at right or subacute angles from vegetative hyphae, gradually elongating, remaining simple or forming 1-3 branch points, usually at right to subacute angles, usually clustered in small groups when viewed from above, 1-3 septate, cylindrical to conical with tapered apices hyaline when young but developing olivaceous to olivaceous gray pigments from the base upward in age, with walls slightly thicker than those of vegetative hyphae, 20-65 X 3-5 μm. Conidia formed abundantly on common media such as oatmeal, malt extract, or corn meal agar, 3.5-5 μm X 1-2 μm, aseptate, smooth, thin-walled, allantoid, suballantoid, to short

cylindrical, or narrowly elliptical, often with a small proximal scar or apiculus, without visible slime or gelatinous materials. Hyphae septate, branched, cylindrical or occasionally inflated, up to 5 μm in diameter.

The culture MF5701 has been identified as

Leptodontium elatius var. elatius and exhibits all the essential morphological characteristics of that

species. It was isolated from the internal tissues of a basidioma of a wood decay basidiomycete, Phellinus robiniae, which was growing parasitically on Robinia pseudoacacia (black locust) in Sussex County, New

Jersey. This culture has been deposited with the

American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852 as ATCC 74165 under conditions of the Budapest Treaty. The culture MF5703 has been identified as

Leptodontium elatius var. elatius and exhibits all the essential morphological characteristics of that

species. It was isolated from wood chip mulch at

Califon, NJ (Hunterdon County). This culture has been deposited with the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852 as ATCC 74166 under conditions of the Budapest Treaty.

Strains MF5701 and MF5703 of Leptodontium elatius var. elatius exhibit the following diagnostic morphological characteristics:

Colonies 16-22 mm in diameter after two weeks on yeast-malt extract agar (YM agar, DIFCO) at 25ºC, 12 hour photoperiod. Colonies raised, downy, wooly or floccose, developing suberect hyphal bundles in older portions of colonies, dull, obscurely zonate, with an even, submerged margin, hyaline at margin but soon, white, pale gray, gray to dark gray in age, Pearl Gray, Pale Olive-Gray, Dawn Gray, Storm Gray, Olive-Gray (capitalized color names from Ridgway, R., Color

Standards and Nomenclature. Washington, D.C. 1912). Reverse dull pale olivaceous yellow to grayish olive, Light Grayish Olive, Grayish Olive, Deep Grayish

Olive. Odors and exudates absent.

Colonies 14-16 mm in diameter after two weeks on Emerson Yp Ss (DIFCO) agar at 25°C, 12 hour

photoperiod. Colonies appressed toward margin, slightly raised toward center, obscurely radially εtriate, velvety, with margin even to minutely

fimbriate, submerged, hyaline at margin, but soon dark olivaceous gray to olivaceous black or black, Castor Gray, Iron Gray, Olivaceous Black, Blackish Green-Gray. Reverse similar in color. Odors and exudates absent.

Colonies 18-20 mm in diameter after two weeks on corn meal agar (DIFCO) at 25ºC, 12 hour

photoperiod. Colonies appressed, faintly radially striate, translucent to pale translucent gray. Reverse translucent. Odors and exudates absent.

No growth occurred on yeast-malt agar after two weeks at 37ºC.

Conidiophores absent or up to 100 μm tall, indeterminate, unbranched or with 1-4 simple branches from a main hyphal axis, straight or slightly

geniculate, often tapering to finely pointed,

geniculate apex, with wall slightly thickened at the base and becoming thinner distally, smooth-walled, septate or not, olivaceous gray to blackish gray at the base, becoming hyaline towards the apex, with a

conidial rachis accumulating at the apex or

occasionally with conidial adhering to intercalary regions, with conidial rachis up to 25 μm tall, with or without minute hyaline scars. Conidiogenous cells arising directly from hyphae, terminal or intercalary, holoblastic, sympodial, with conidia accumulating in a ladder-like rachis at a 90° to 45° angle with respect to main conidiophore axis, when on vegetative hyphae producing conidia in yeast-like masses. Conidia hyaline, ellipsoidal to allantoid, thin-walled, smooth, 4.5-6 × 1-2 μm. Vegetative hyphae septate, branched.

Vegetative cells of a culture capable of producing Zaragozic Acid A, such as: MF5453 (ATCC 20986); MF5565 (ATCC 74068); MF5599 (ATCC 74065);

MF5572 (ATCC 74066); or MF5573 (ATCC 74067) and vegetative cells of a culture capable of producing Zaragozic Acid C such as MF 5465 (ATCC 20986), MF 5701 (ATCC 74165), or MF 5703 (ATCC 74166), can be obtained by culturing the microorganism in an aqueous nutrient medium containing sources of assimilable carbon and nitrogen, preferably under aerobic conditions.

Nutrient media may also optionally contain mineral salts, high molecular weight polyanions (CARBOPOL^®, JUNLON^®), and/or defoaming agents.

The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, glycerin, and the like. Other sources which may be included are maltose, fructose, sucrose, and the like. In addition, complex nutrient sources such as oat flour, 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, and the like, as well as complex sources such as yeast extracts (hydrolysates, autolysates), dried yeast, tomato paste, peptone, corn steep liquor, 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 20 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.

The preferred process for production of these vegetative cells consists of inoculating spores or mycelia of the producing organism into a suitable medium and then cultivating under aerobic conditions. After inoculation, the flasks are incubated with agitation at temperature ranging from 20 to 30ºC, preferably 24 to 28ºC. Agitation rates may range up to 400 rpm, preferably 200 to 240 rpm. Flasks are

incubated over a period of 2 to 10 days, preferably 2 to 4 days.

To form the compounds structural formula (I), when growth of the Zaragozic Acid A producing culture is plentiful, the culture is ready to be washed, homogenized and used in directed biosynthetic studies.

In addition, the compounds structural formula (I) invention may be more selectively synthesized by inhibiting the enzyme phenylalanine ammonia lyase (PAL) which is the first step in the degradation of

L-phenylalanine to form benzoic acid. Benzoic acid has been shown to be the direct precursor of the aromatic ring system on the C-1 side chain of Zaragozic Acid A. Inhibitors of PAL include phenylpropiolic acid, D-phenylalanine, aminooxyacetic acid, p-coumaric acid, caffeic acid, D,L-ß-phenylserine and D,L-2-hydroxyphenylalanine.

Furthermore, the process of biosynthetic production of compounds of structural formula (I) may be carried out using a mutant for the parent Zaragozic Acid A producing strain that is lacking the PAL enzyme, resulting in a culture whose synthesis of the Zaragozic Acid A is dependent on an exogenous source of benzoic acid. This culture more readily incorporates the compounds of Formula (III) selected from:

(a) R₁-CO₂H, and

(b) R₁-CH₂CHNH₂CO₂H;

because these compounds are not competing with an endogenous source of benzoic acid.

After growth, cells of the Zaragozic Acid A producing culture are harvested by filtration or centrifugation. To obtain a uniform suspension, the cell mixture may optionally be homogenized using a homogenizer such as a hand-held BIOHOMOGENIZER™

(Bartlesville, OK) until no clumps or mycelial balls are visible (about 20 to 60 seconds).

Alternatively, the vegetative cells of the Zaragozic Acid A producing culture may be grown in media containing polyanions to give more beaded and grainy growth, which may eliminate the benefits of the homogenization step which transforms large balled growth to more disperse hyphal fragments.

After growth or the optional homogenization step, the Zaragozic Acid A producing cells are

harvested by filtration or centrifugation, and washed with distilled water or an aqueous buffer and

resuspended in a medium consisting of 1 to 5 % of a carbon/energy source such as glucose, glycerol, sucrose or the like and an appropriate buffer such as 5-10 mM PIPES (piperazine-N,N'-bis[2-ethanesulfonic acid]), MOPS (3-[N-morpholino]propanesulfonic acid), MES

(2-[N-morpholino]ethanesulfonic acid), MOPSO

(3-[N-morpholino]-2-hydroxypropanesulfonic acid ) , ACES (N-[2-acetamido]-2-aminoethanesulfonic acid), ADA

(N-[2-acetamido]-2-iminodiacetic acid), BES

(N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid), phosphate or the like to keep the pH less than 8, preferably pH 6 to pH 6.5. In order to guarantee uniform suspension of the cells, the container holding the cells is shaken vigorously.

Aliquots of the suspended Zaragozic Acid A producing cells are removed and are incubated at 20 to 30ºC for 24 to 144 hours with or without agitation, preferably at 25ºC for 120 hours with agitation. After this initial incubation, a compound of Formula (ill), selected from R₁-CO₂H and R₁-CH₂-CHNH₂CO₂H wherein R₁ is as noted above is added, either as a free acid or as a biologically acceptable salt form such as sodium to a final concentration of 0.01 mM to 100 mM, preferably 0.25 to 0.5 mM, followed by additional incubation of 48 to 120 hours. After the additional incubation, the biosynthesis is terminated by the addition of a solvent such as methanol or acetonitrile, preferably methanol, and the broth is clarified. In order to make the cells more permeable to the uptake of the compounds of Formula (III), the cells may be treated with toluene vapors by adding 1-2 drops of toluene to the aliquot of cells after the initial incubation. The suspension is vigorously shaken at ambient temperature for 30 seconds, followed by the addition of a compound of Formula (III) to the

suspension of cells and the additional incubation as described above.

The desired compounds of Formula (I) are extracted with solvent and purified by various

chromatographic techniques such as silica gel, reverse phase and ion exchange. Preferably the compounds of Formula (I) are isolated by anion exchange

chromatography followed by preparative reverse-phase high pressure liquid chromatography.

To produce the compounds of structural formula (II) one employs a Zaragozic Acid C producing culture. When growth is plentiful, usually 2 to 4 days, the Zaragozic Acid C producing 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, preferably a liquid production medium, is incubated 3 to 30 days, preferably 4 to 14 days, with or without agitation (depending on whether liquid or solid production media are employed). The fermentation is conducted aerobically at temperatures ranging from 20-40°C. If used, agitation may be at a rate of 200 to 400 rpm. To obtain optimum results, the temperature is in the range of 22° to 28ºC, most preferably 24° to 26ºC. The pH of the nutrient medium suitable for the process of producing compounds of structural formula (II) is in the range of 3.5 to 8.5, most preferably 5.0 to 7.5.

After initial incubation, the compound of structural formula (III) selected from R₁CO₂H and

R₁-CH₂CHNH₂CO₂H wherein R₁ is as noted above is added, either as a free acid or as a biologically acceptable salt form, such as sodium, to a final concentration of 2 to 20 mM preferably 5 to 10 mM, and the incubation is continued for another 5 to 14 days, preferably 7 to 10 days. After the additional incubation, the

biosynthesis is terminated by the addition of a solvent such as methanol or acetonitrile or by lowering the pH to about 2 by the addition of an acid such as HCl, and the compound of structural formula (II) is isolated.

After the biosynthesis is complete and the fermentation is terminated, the desired compounds of Formula (II) are extracted with solvent and purified by various chromatographic techniques such as silica gel, reverse phase and ion exchange. Preferably the

compounds of Formula (II) are isolated by anion

exchange chromatography followed by preparative

reverse-phase high pressure liquid chromatography.

Esters of the compounds of Formulae (I) and (II) may be prepared by dissolving the compound of Formula (I) or (II) in a dry organic solvent,

preferably tetrahydrofuran (THF) at 0-30°C and treating with the appropriately substituted isourea for 8-24 hours, cooling to -15°C and filtering the urea. The mono-, di- and tri- esters may be prepared by varying the number of equivalents of isourea used. The

filtrate is concentrated under reduced pressure to yield the desired ester. Esters may also be prepared by treating a compound of formula (I) or (II) with an organic halide (chloride, bromide or iodide) in a standard organic solvent in the presence of a base such as triethylamine, pyridine or DBU. Mono, di and triesters may be formed by using the appropriate number of equivalents of alkylating agent. Mixtures may be separated by HPLC.

The present invention is also directed to a method of inhibiting cholesterol. biosynthesis which comprises the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound represented by structural formula (I) or (II) and pharmaceutically acceptable salts 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 daily dosage for adults is within a range of from about 20 mg to 2000 mg (preferably 20 to 100 mg) which may be given in two to four divided doses . Higher doses aay be favorably employed as required. In addition, the present invention is

directed to a method of inhibiting the enzyme squalene synthase which comprises the administration to a subject in need of such treatment a nontoxic

therapeutically effective amount of a compound

represented by structural formula (I) or (II) and pharmaceutically acceptable salts 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 daily dosage for adults is within a range of from about 20 mg to 2000 mg (preferably 20 to 100 mg) which may be given in two to four divided doses. Higher doses may be favorably employed as required.

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-glutamine, 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. 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 another enzyme in the biosynthetic pathway in the synthesis 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. Patents 4,806,564; 4,816,477; 4,847,271; and 4,751,237; the beta-lactam derivatives disclosed in U.S. 4,983,597 and U.S.S.N. 07/540,992 filed June 20, 1990; and the substituted oxacyclopropane analogues disclosed in European Patent Publication EP 0411 703. Illustrative examples of squalene epoxidase inhibitors are disclosed in European Patent Publication EP 0318 860 and in

Japanese Patent Publication J02 169-571A. LDL-receptor gene inducer molecules are disclosed in U.S. Patent Application S.N. 07/670,640 filed March 18, 1991.

Other cholesterol lowering agents that may be

administered include niacin, probucol, the fibric acids: clofibrate and gemfibrozil, and LDL-receptor gene inducers. Representative of such combinations are those containing about 10-400 mg of a compound of formula (I) or (II) in combination with about 20-100 mg of an HMG-CoA reductase inhibitor, 20 to 200mg of a

HMG-CoA synthase inhibitor, or 2 to 200mg of a squalene epoxidase inhibitor, or 250 to 1000 mg of probucol, or 600 to 1200 mg of gemfibrozil, or 1 to 2 g of clofibrate, or 3 to 6 g of niacin, or 20 to 300 mg of an LDL-receptor gene inducer.

The compounds of this invention may also be co-administered with pharmaceutically acceptable non-toxic cationic polymers capable of binding bile acids in a non-resorbable form in the gastrointestinal tract. Examples of such polymers include

cholestyramine, colestipol and poly[methyl-(3-trimethyl)aminopropyl]imino-trimethylene dihalide. The relative amounts for co-administration of the compounds of this invention and these polymers is between 1:100 and 1:15,000 (w/w).

The intrinsic squalene synthase inhibitory activity of representative compounds of this invention was measured by the standard in vitro protocol

described below:

Preparation of Rat Liver Microsomes:

Male, Charles River CD rats (120 to 150 g) were 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-hydroxyethyl)-1-piperazine-ethanesulfonic acid), 5 mM EDTA (ethylenediaminetetraacetic acid) pH 7.5 with a Potter-Elvehjem type tissue grinder. The homogenate was centrifuged twice at 20,000 × g for 15 min. at 4ºC, discarding the pellet each time. The supernatant was then centrifuged at 100,000 × g for 1 hr at 4°C. The resulting microsomal pellet was resuspended in a volume of the above homogenizing buffer equal to one-fifth the volume of the original homogenate. This microsomal preparation has a protein concentration of about 7 mg/mL. The microsomal suspensions were stored in aliquots at -70°C. squalene synthase activity in these aliquots is stable for a least several months.

Partial Purification of Prenyl Transferase

Prenyl transferase was purified to use in the enzymatic synthesis of radiolabelled farnesyl

pyrophosphate. Prenyl transferase was 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 farnesyl

pyrophosphate per minute at 30°C in the standard assay.

The livers of 23 forty-day old male rats that had been fed 5% cholestyramine plus 0.1% lovastatin were homogenized in a WARING™ blender in 1 liter of 10 mM mercaptoethanol, 2 mM EDTA, 25 mM leupeptin, 0.005% phenylmethylsulfonyl fluoride, pH 7.0 containing 0.1 trypsin inhibitor units of aprotinin/mL. The

homogenate was centrifuged at 20,000 × g for 20 min. The supernatant was adjusted to pH 5.5. with 6 N HOAc and centrifuged at 100,000 × g for 1 hour. This supernatant was adjusted to pH 7.0 with 3 N KOH and a 35-60% ammonium sulfate fraction was taken. The 60% pellet was 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 was applied to a 12.5 × 5 cm column of DEAE-sepharose 4B equilibrated with Buffer A. The column was 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 were combined, solid ammonium sulfate was added to bring to 60% saturation and pelleted. The pellet was dissolved in 8 mL of 10 mM Tris, 10 mM β-mercaptoethanol pH 7.0 (Buffer B). The redissolved pellet was taken to 60% saturation with ammonium sulfate by adding 1.5 volumes of saturated ammonium sulfate in Buffer B. This ammonium sulfate suspension contained 3.5 units/mL with specific

activity of 0.23 units/mg and was free of isopentenyl pyrophosphate isomerase activity. This ammonium sulfate suspension was used for the synthesis of

[4-¹⁴C]farnesyl-pyrophosphate and its activity was stable stored at 4ºC for a least 6 months.

Enzymatic Synthesis of [4-¹⁴C]farnesyl-pyrophosphate

The solvent (ethanol: 0.15 N NH₄OH, 1:1),was removed from 55 mCi of [4-¹⁴C]isopentenyl pyrophosphate (47.9 mCi/mmole) by rotary evaporation. Six hundred microliters of 100 mM Tris, 10 mM MgCl₂, 4 mM

dithiothreitol pH 7.5 was added and the solution was transferred to a 1.5 mL Eppendorf centrifuge tube.

Geranyl-pyrophosphate, 250 microliters of a 20 mM solution, and 50 microliters of the ammonium sulfate suspension of prenyl transferase were added to initiate the reaction. This incubation contained 5 micromoles of geranyl pyrophosphate, 1.15 micromoles of

isopentenyl pyrophosphate, 6 micromoles of MgCl₂ and 0.18 units of prenyl transferase in a volume of 900 microliters. The incubation was conducted at 37ºC. During the incubation, the mix turned cloudy white as the newly formed magnesium complex of farnesyl pyrophosphate precipitated out of solution. The

[4-¹⁴C]farnesyl pyrophosphate was collected by

centrifugation for 3 minutes at 14,000 rpm in an

Eppendorf centrifuge tube, the supernatant removed, and the pellet was dissolved in 1.0 mL of 50 mM HEPES, 5 mM EDTA, pH 7.5. The yield was 50.7 μCi (92%) of

[4-¹⁴C]farnesyl pyrophosphate. The [4-¹⁴C]farnesyl pyrophosphate was stored in aliquots at -70°C.

Squalene Svnthase Assay

Reactions were performed in 16 × 125 mm screw cap test tubes. A batch assay mix was prepared from the following solution: mL volume for per assay 50 assays

1. 250 mM HEPES pH 7.5 20 1000

2. NaF 110 mM 10 500

3. MgCl₂ 55 mM 10 500

4. Dithiothreitol 30 mM 10 500

5. NADPH 10 mM (made fresh) 10 500

6. [4-¹⁴C]farnesyl- pyrophosphate 47.9 μCi/μmole,

and 0.025 μCi/3.0 μL 3.0 150

7. H₂O 24 1200 This assay mix was degassed under a vacuum and flushed with N₂. Solutions of the squalene

synthase inhibitors were prepared either in DMSO or MeOH and a 1:120 dilution of the microsomal protein was made with the original homogenizing buffer. For each reaction, 87 μL of the assay mix was taken with 3 μmL of an inhibitor solution (DMSO or MeOH in the

controls), warmed to 30ºC in a water bath and then the reaction was initiated by the addition of 10 mL of the 1:120 dilution of microsomal protein (0.6 μg protein total in the assay). The reactions were stopped after 20 minutes by the addition of 100 μL of a 1:1 mix of 40% KOH with 95% EtOH. The stopped mix was heated at 65ºC for 30 min., and cooled. Ten mL of heptane was added and the mix was vortexed. Two g of activated alumina was then added, the mix vortexed again, the alumina allowed to settle and 5 mL of the heptane layer was removed. Ten mL of scintillation fluid was added to the heptane solution and radioactivity was

determined by liquid scintillation counting.

Percent inhibition is calculated by the formula:

Representative of the activity of the

compounds of the present invention is that below: Compound squalene synthase

IC₅₀

Formula (I) wherein 0.14 ng/mL

Z¹, Z², and Z³ are

each hydrogen and

R₁ is 3-thiophene

Formula (II) wherein less than 100 ng/mL

Z¹, Z² and Z³ are

each hydrogen and

R₁ is 2-thiophene

The present compounds also demonstrate broad spectrum antifungal activity. Thus the present

invention is also directed to a method of treating fungus infections which comprises the administration to an organism in need of such treatment a nontoxic therapeutically effective amount of a compound

represented by the structural formula (I) and (II) 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 inhibit farnesyl-protein transferase and thereby inhibit the farnesylation 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 assays 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 [³H]FPP, and the indicated compounds are incubated with this

partially purified enzyme preparation.

RASIT ASSAY II 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 at 1.0 μM, 0.5 μM [³H]FPP, and the indicated compounds are incubated with this partially purified enzyme preparation. The FTase data is a measurement of the ability of the test compound to inhibit Ras farnesylation in vitro.

The pharmaceutical compositions containing the compounds of structural Formula (I) and (II) 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 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 between 0.5 mg/kg of body weight to about 10 mg/kg of body weight of a mammal a day.

The following examples illustrate the

formation of a compound of formula (I) and (II). EXAMPLE 1

A Compound of Formula (I) wherein R₁ is 3-thiophene and Z¹, Z² and Z³ are each hydrogen

I. Directed Biosynthesis:

Culture MF5453 was grown for 72 hours at 25°C in KF medium (U.S. Patent 5,053,425) and the cells harvested by centrifugation. The cells were washed (X2) with distilled water and resuspended to the original broth volume in 20 mM piperazine-N,N'-bis[2-ethanesulfonic acid] (PIPES) buffer (pH 6.1) containing 3% sucrose. Five mL aliquots of this suspension were transferred to each of four 50 mL

Erlenmeyer flasks and these flasks incubated at 25ºC with agitation. After 24 hours incubation,

3-thiophene-carboxylic acid (Na-salt) was added to, a final concentration of 0.25 mM, 0.5 mM and 1.0 mM to each of three of the flasks respectively, and the fourth remained a control. After an additional 96 hours incubation the biosynthesis was terminated with the addition of two volumes of methanol and the broths clarified. The broth-methanol mixture was adjusted to pH 4.5 with formic acid. The contents of the three flasks to which the 3-thiophene carboxylic acid had been added were combined. The resulting mixture was applied to a 1 mL column of BIO-RAD^® AG4X4 ion exchange resin in the formate cycle. The column was washed successively with 15 mL of MeOH-formate buffer (1/1 v/v; 50 mM formate adjusted to pH 4.5) and 15 mL of 60/40 MeCN/water (v/v). The column was then eluted with 15 mL of 60/40 MeCN/water containing 1 mL

concentrated formic acid. II. Isolation and Purification:

Fifteen mL of AG4X4 eluate was reduced under nitrogen to 8 mL. The crude extract was filtered and 2 mL injected onto a BECKMAN^® preparative HPLC (9.6 mm X 250 mm) ODS column. The column was developed at 3.0 mL/min using a 35 minute linear gradient of 40% to 80% acetonitrile in water containing 0.1% H₃PO₄. Detection was at 215 nm. Peaks with an elution time of 28.9 minutes were collected and pooled. The pooled material was diluted with four volumes of deionized water and applied to a water-equilibrated C₁₈ SPE column. After washing with five volumes of deionized water, the column was dried with nitrogen, then eluted with methanol. The eluate was evaporated to dryness to yield a substance identified as the title compound. ¹H NMR (400 MHz) (CD₃OD) 7.29 (dd, J = 4.9, 30, 1H), 7.03 (dd, 2.9, 1.4, 1H), 6.98 (dd, 4.9, 1.4, 1H), 6.86 (dd, 15.7, 8.4, 1H), 6.43 (d, 2.1, 1H), 5.83 (dd, 15.7, 1.0, 1H), 5.11 (d, 5, 1H), 5.07 (s, 1H), 5.02 (s, 1H), 4.94 (s, 1H), 4.04 (d, 2.1, 1H), 2.69 (dd, 14, 6, 1H), 2.49 (dd, 14, 8.7, 1H), 2.42 (m, 1H), 2.26 (m, 2H), 2.08 (s, 3H), 2.05 (m, 1H), 1.26-1.41 (m, approximately 3H), 1.15 (m, 2H), 1.02 (d, 6.5, 3H), 0.86 (d, ca 6.5, 3H), 0.85 (t, ca 7, 3H), 0.84 (d, ca 6.5, 3H). EXAMPLE 2

Compound of Formula (I) wherein R₁ is 3-fluorophenyl and Z¹, Z² and Z³ are each hydrogen

I. PIRECTED BIOSYNTHESIS:

Culture MF 5453 was grown for 72 hours at 25ºC in KF medium and the cells harvested by

centrifugation. The cells were washed twice with distilled water and resuspended to the original broth volume in 20 mM PIPES buffer (pH 6.1) containing 3% sucrose. Five mL aliquots of this suspension were transferred to 50 mL Erlenmeyer flasks and these flasks incubated at 25ºC with agitation. After 24 hours' incubation, 3-fluorobenzoic acid (Na-salt) was added to a final concentration of 0.25 mM. After 24 hours incubation, additional 3-fluorobenzoic acid was added to a final concentration of 0.50 mM. After an

additional 96 hours incubation, the biosynthesis was terminated with the addition of two volumes of methanol and the broths clarified.

II. HPLC ANALYSIS

HPLC analysis was performed using a ES

Industries Chromegabond FD column (5 urn particle size; 4.6 mm ID by 25 cm length) at room temperature.

UV detection was at 215 nm. The solvent system

consisted of a gradient from 30% to 60% MeCN in HPLC-grade water containing 0.1% phosphoric acid (v/v) over a thirty minute period; the 90% MeCN was held an additional 5 minutes before a return to the starting solvent. The chromatograms obtained from the control (no added fluorobenzoic acids) and the experimental broth extracts were overlaid on the same time scale to compare results and a new peak identified as a

3-fluorobenzoic analog of zaragozic acid A, was

detected.

III. ISOLATION AND PURIFICATION

Twenty mL of whole broth was combined with twenty mL methanol. The crude extract was filtered to remove cells, diluted with forty mL of DI water and applied to a water equilibrated C₁₈ Speed cartridge. After washing with water, the cartridge was eluted with twenty mL methanol. The eluate was reduced to dryness, then dissolved in two mL of 70% MeOH/H₂O. One mL was injected onto a Chromegabond FD column (4.6 mm X 250 mm). The column was developed as above. Detection was at 215 nm. Peaks with an elution time at 24.8 and, 29/1 minutes were collected. Each fraction was diluted with four volumes of DI water, then applied to water-equilibrated C₁₈ SPE columns. After washing with five volumes of DI water, the columns were dried with nitrogen, then eluted with methanol. The eluates at 29.1 minutes were evaporated to dryness to yield the 3-fluorophenyl Zaragozic Acid A analog.

¹H NMR (400 MHz) (CD₃OD) 7.27 (dt, 8.1, 8.1, 6.2, 1H), 7.03 (dt, 7.1, 1.1, 1H), 6.93 (ddd, 10.2, 2.5, 1.7, 1H), 6.87 (m, 1H). EXAMPLE 3

Compound of Formula (I) wherein R₁ is 4-fluorophenyl and Z¹, Z² and Z³ are each hydrogen

This compound was prepared following the procedure of Example 2 except that an equivalent amount of 4-fluorobenzoic acid (Na-salt) was employed. The retention time of the 4-fluorophenyl zaragozic acid A was identical' with that of the 3-fluorophenyl

derivative (29.1 minutes).

EXAMPLE 4 Compound of Formula (I) wherein R₁ is 2-fluorophenyl and Z¹, Z² and Z³ are each hydrogen

This compound was prepared following the procedure of Example 2 except that an equivalent amount of 2-fluorobenzoic acid (Na-salt) was employed. The retention time of the 2-fluorophenyl zaragozic acid A was 25.3 minutes.

¹H NMR (400 MHz) (CD₃OD) 7.28 (td, 7.6, 7.6, 1.7, 1H), 7.17 (m, 1H), 7.09 (td, 7.5, 7.5, 1.2, 1H), 6.99 (ddd, 10.3, 8.2, 1.3, 1H).

EXAMPLE 5

Compound of Formula (I) wherein R₁ is 2-furyl and Z¹, Z² and Z³ are each hydrogen

I. DIRECTED BIOSYNTHESIS:

Culture MF 5453 was grown for 72 hours at 25ºC in KF medium and the cells harvested by

centrifugation. The cells were washed twice with distilled water and resuspended to the original broth volume in 20 mM PIPES buffer (pH 6.1) containing 3% sucrose. Five mL aliquots of this suspension were transferred to 50 mL Erlenmeyer flasks and these flasks incubated at 25°C with agitation. After 24 hours' incubation, 2-furoic acid (Na-salt) was added to a final concentration of 0.25, 0.5 and 1.0 mM. After an additional 96 hours incubation, the biosynthesis was terminated with the addition of two volumes of methanol and the broths clarified.

II. HPLC ANALYSIS

HPLC analysis was performed using a Beckman Ultrasphere ODS column at room temperature (5 urn particle size; 4.6 mm ID by 25 cm length). UV

detection was at 215 nm. The solvent system consisted of a gradient from 30% to 60% MeCN (plus HPLC-grade water containing 0.1% phosphoric acid by volume) over a thirty minute period; the 90% MeCN was held an

additional 5 minutes before a 9 minute return to the starting solvent. The chromatograms obtained from the control (no added 2-furoic acids) and the experimental broth extracts were overlaid on the same time scale to compare results and a new peak identified as the

2-furyl analog of zaragozic acid A, was detected. III. ISOLATION AND PURIFICATION

Six hundred thirty mL of whole broth was combined with an equal volume of methanol. The crude extract was filtered to remove cells, diluted with 1.5 L of DI water and applied to a 15 mm X 300 mm column packed with HP-20 (Mitsubishi Chemical, 220 mm bed height water equilibrated). After washing with water, the column was eluted with 200 mL methanol. The eluate was reduced to dryness, then dissolved in twenty mL of 60% MeOH in water. One mL was injected onto a Beckman Ultrasphere ODS column (10 mm X 250 mm). The column was developed at 3.0 mL/min using a gradient from 40% to 75% MeCN in HPLC-grade water containing 0.1%

phosphoric acid (v/v) over a thirty minute period.

Detection was at 215 nm. A peak eluting at 25.2 minutes was collected. The peak fraction was diluted with an equal volume of DI water, then applied to water equilibrated C₁₈ SPE column. After washing with DI water, the column was dried with nitrogen, then eluted with methanol. The eluate was evaporated to dryness to yield the titled compound.

¹H NMR (400 MHz) (CD₃OD) 7.34 (dd, 2.1, 0.9, 1H), 6.26 (dd, 3.1, 1.9, 1H), 6.05 (d, 3.0, 1H). EXAMPLE 6

Compound of Formula (I) wherein R₁ is 2-thiophene and Z¹, Z² and Z³ are each hydrogen

I. DIRECTED BIOSYNTHESIS:

Culture (MF-5453) was grown for 72 hours at 25°C in KF medium and the cells harvested by

centrifugation. The cells were washed (X2) with distilled water and resuspended to the original broth volume in 20 mM PIPES buffer (pH 6.1) containing 3% sucrose. Five mL aliquots of this suspension were transferred to 50 mL Erlenmeyer flasks and these flasks incubated at 25ºC with agitation. After 24 hours' incubation, thiophene-2-carboxylic acid (Na-salt) was added to a final concentration of 0.25, 0.5 and 1.0 mM. After an additional 96 hours incubation, the biosynthesis was terminated with the addition of two volumes of methanol and the broths clarified.

II. HPLC Analysis

HPLC analysis was performed using a Beckman Ultrasphere ODS column at room temperature (5 um particle size; 4.6 mm ID by 25 cm length). UV

detection was at 215 nm. The solvent system consisted of a gradient from 30% to 90% MeCN (plus HPLC-grade water containing 0.1% phosphoric acid by volume) over a thirty minute period; the 90% MeCN was held an

additional 5 minutes before a 9 minute return to the starting solvent. The chromatograms obtained from the control (no added thiophene-2-carboxylic acid) and the experimental broth extracts were overlaid on the same time scale to compare results and a new peak identified as the 2-thiophene analog of zaragozic acid A, was detected.

III. ISOLATION AND PURIFICATION:

Two hundred mL of whole broth was combined with an equal volume of methanol. The crude extract was filtered to remove cells, diluted with 400 mL of DI water and applied to a 15 mm X 300 mm column packed with HP-20 (Mitsubishi Chemical, 220 mm bed height water equilibrated). After washing with water, the column was eluted with 200 mL methanol. The eluate was reduced to dryness, then dissolved in twenty mL of 60% MeOH in water. Two mL injections were made onto a

Beckman preparative HPLC (9.6 mm X 250 mm) ODS column. The column was developed at 3.0 mL/min using a 35 minute linear gradient of 40% to 80% acetonitrile in water containing 0.1% H₃PO₄. Detection was at 215 nm. Peaks with an elution time at 28.9 and 30.4 minutes were collected and pooled. The pooled materials were diluted with four volumes of DI water then each applied to water-equilibrated C₁₈ SPE columns. After washing with five volumes of DI water, the columns were dried with nitrogen, then eluted with methanol. The eluate was evaporated to dryness to yield the titled compound.

¹H NMR (400 MHz) CD₃OD) 7.15 (dd, 5.0, 1.2, 1H), 6.90 (dd, 5.1, 3.4, 1H), 6.83 (dQ, 3.5, 1.0, 1H). EXAHPLE 7 The Compound of Formula (II) wherein R₁ is 2-thiophene and Z¹, Z² and Z³ are each H

I. Directed Biosynthesis:

Culture MF5465 was grown for 48 hours at 25ºC in KF medium (U.S. Patent 5,026,554). A frozen

vegetative mycelia of MF5465, Leptodontium elatius

(ATCC 74011) was used to inoculate a starch seed flask (40 mL per 250 mL Erlenmeyer), then incubated at 25°C for 48 h. One mL of this seed was used to inoculate a 1.5 X modified GPT medium production flask (40 mL per 250 mL Erlenmeyer).

Starch Seed Medium g/L 1.5 X modified GPT Medium g/L starch (AMIDEX) 30.0 Peptone (PRIMATONE) 22.5 Cottonseed flour

(PHARMAMEDIA) 10.0 Glycerol 100.0 KH₂PO₄ 9.0 Yeast Extract (DIFCO) 7.5

Yeast Extract (FIDCO)5.0 Sodium Citrate 11.0 Cerelose 10.0 Lactose 50.0

MgSO₄·7H₂O 0.2 MgSO₄·7H₂O 0 .5

Adj. pH to 6.0 Adj . pH to 6 .0

The production flasks were incubated at 25°C with agitation (220 rpm) for fourteen days, followed by addition of 2-thiophene carboxylic acid (1.25 mg/mL) and continued incubation for seven additional days. The fermentation was terminated by adjusting the whole broth pH to 2.0 and extracting with methylethyl ketone (MEK). The MEK extracts were evaporated to dryness.

II. Isolation and Purification:

Extracts from two production flasks were dissolved in 20 mL of 60% MeCN in water and insoluble material filtered. A 2 mL sample was injected onto an Beckman Ultrasphere ODS column (10 mm X 250 mm). The column was developed at 3 . 0 mL/min using a gradient from 40% to 85% MeCN in HPLC-grade water containing 0.1% phosphoric acid (v/v) over a forty-two minute period. Detection was at 205 nm. A peak eluting at 33 minutes was collected. The peak fraction was diluted with an equal volume of distilled water, then applied to a water equilibrated C₁₈ SPE column. After washing with distilled water, the column was dried with

nitrogen, then eluted with acetonitrile. The eluate was evaporated to dryness to yield a substance

identified as the title compound. The ¹H NMR of the title compound exhibits the following characteristic resonances:

7.22 (t, 2H), 7.13 (m, 4H), 6.88 (dd, J=5.1, 3.4, 1H), 6.79 (dd, J=3.3, ca 1, 1H), 6.23 (d, J= ca 1.5, 1H), 5.38 (dt, J=15.4, 5.9, 1H), 5.32 (dd, J= 15.4, 7.5, 1H), 5.16 (s, 1H), ca 4.9 (m, 1H), 3.95 (d, J=1.7, 1H), 2.90 (dd, J = 14.4, 6.1, 1H), 2.63 (dd, J = 14.4, 8.6, 1H), 2.56 (m, 2H) 2.34 (m, 2H), 2.27 (m, 2H), 2.08 (m, 1H), 2.05 (s, 3H), 2.03 (m, 1H) , 1.87 (m, 2H),

1.52-1.69 (m, approx. 6H), 1.24-1.34 (m, approx. 3H), 0.93 (d, J = 6.8, 6H). EXAMPLE 8

Preparation of an Ammonium Salt

A 0.1 mmol sample of the free acid of a compound of formula (I) or (II) is dissolved in 10 mL ethyl acetate. The resulting solution is saturated with gaseous ammonia and the ammonium salt precipitates from solution.

EXAMPLE 9

Preparation of a Potassium Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) or (II) in 10 mL 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 10

Preparation of a Calcium Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) or (II) in 20 mL 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 11 Preparation of an Ethylenediamine Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) or (II) 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 12

Preparation of a Tris(hydroxymethyl)aminomethane Salt

To a solution of 0.1 mmol of the free acid of a compound of formula (I) or (II) 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, 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-methylglutamine.

EXAMPLE 13

Preparation of an L-arginine Salt

A solution of 0.1 mmol of the free acid of a compound of formula (I) or (II) in 20 ml of 6:4

methanol: water is treated with an aqueous solution of 0.1 to 0.3 mmol of L-arginine. Evaporation of the solvent affords the title salt, the exact composition of which is determined by the molar ratio of amino acid to the free acid of formula (I) or (II) used.

Similarly prepared are the salts of L-ornithine,

L-lysine and N-methylglutamine.

EXAMPLE 14

Preparation of the trimethyl ester of a Compound of Formula (I) or (II) (Method I )

To 5 mg of the free acid of a compound of formula (I) or (II) in methanol (5 mL) is added 2 mL of freshly distilled diazomethane in ether (2.05 M).

After 5 minutes the solvent is removed to afford trimethyl ester as an oil. EXAMPLE 15

Preparation of the trimethyl ester of a Compound of Formula (I) or (II) (Method II)

To 0.6 mg of the free acid of a compound of formula (I) or (II) in 1 mL diethyl ether at 0 C is added etheral cyanamide dropwise until the solution remains yellow. The solution is evaporated under a stream of nitrogen to yield the trimethyl ester.

EXAMPLE 16 Preparation of the tribenzyl ester of a Compound of Formula (I) or (II)

To a solution of 5 mg of the free acid of a compound of formula (I) or (II) in 0.5 mL tetrahydrofuran (THF) is treated at room temperature with 3 equivalents of N,N'-diisopropyl-O-benzyl isourea for 18 hours. The reaction mixture is then chilled to -15°C, and filtered to remove the urea. The filtrate is concentrated under reduced pressure to yield the tribenzyl ester.

The method of Example 15 is also suitable for the preparation of other ester derivatives such as 1) ethyl and the other lower alkyls, and 2) substituted benzyl esters, using the appropriately substituted isourea. By varying the number of equivalents of the substituted isourea used, the mono-, di-, and

tri-substituted esters may be selectively prepared. EXAMPLE 17

Preparation of the mono-, di- and tri-Kanebo Esters

To a stirred solution of the free acid of a compound of Formula (I) or (II) (100 mg) in 2 mL THF at 0°C under N₂ is added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 27.4 mL, 0.183 mmol) dropwise. After stirring at 0°C for 10 min, followed by an additional 10 minutes at room temperature, 4-bromomethyl-5-methyl-1,3-dioxolen-2-one (Kanebo, 58.95 mg, 0.305 mmol) is added dropwise, stirred for 10 minutes, then heated at 60°C from 2 days. Products are isolated by evaporation, prep HPLC on a reverse phase column to give mixtures of the mono, di, and tri Kanebo esters. By varying the number of equivalents of DBU and the bromo- compound used, a more selective preparation of mono-, di- or tri- esters may be accomplished. EXAMPLE 18

Preparation of the tri-pivaloyl ester

To 100 mg of the free acid of a compound of Formula (I) or (II) in 3 mL refluxing acetonitrile, 75 microliters of DBU and 72 microliters of chloromethyl pivalate is added and refluxed until completion of the reaction. The tri-pivaloyl ester is separated from the mono- and di- pivaloyl esters by reverse phase HPLC, eluted with acetonitrile-water. By varying the number of equivalents of DBU and the chloromethyl pivalate used, the mono and di esters may be selectively

prepared. EXAMPLE 19

Oral Composition

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.

Claims

WHAT IS CLAIMED IS:

1. A compound selected from structural formulae (I) and (II)

wherein R₁ is selected from the group consisting of

,

,

,

,

, and

;

wherein X is selected from:

(a) H,

(b) halogen (F, Cl, Br, I),

(c) OH, and

(d) CH₃;

Y is selected from:

(a) halogen (F, Cl, Br, I),

(b) OH, and

(c) CH₃;

and wherein Z¹, Z² and Z³ are each independently selected from:

(a) H,

(b) C_1-5alkyl,

(c) C_1-5alkyl substituted with:

(i) phenyl,

(ϋ) phenyl substituted with a substituent selected from methyl, methoxy, halogen

(Cl, Br, I, F) and hydroxy,

(iϋ) C_1-5alkylcarbonyloxy,

(iv) C_6-10arylcarbonyloxy,

(v) C_1-5alkoxycarbonyloxy,

(vi) C_6-10aryloxycarbonyloxy,

(vii)

and

(ix) or the groups (iii) to (vi) form a 5 to 10 membered mono- or bicyclic ring with C_1-5alkyl; and

a pharmaceutically acceptable salt thereof.

2. The compound of Claim 1 wherein Z¹, Z² and Z³ are each independently selected from:

(a) H,

(b) C_1-5alkyl ,

(c) C_1-5alkyl substituted with phenyl , or

(d ) pivaloyl ,

(e) sodium, potassium, aluminum, calcium,

lithium, magnesium or zinc, (f) ammonia, N,N'-dibenzylethylenediamine, diethanolamine, N-benzylphenylethylamine, or diethylamine,

(g) N-methyl-glutamine, lysine, arginine, or

ofnithine,

(h) choline,

(i) chloroprocaine or procaine,

(j) piperazine,

(k) tetramethylammonium hydroxide, and

(l) tris(hydroxymethyl)aminomethane.

3. The compound of Claim 2 wherein the compound is of structural formula (I) and R₁ is

selected from:

,

, and

.

4. The compound of Claim 3 wherein X is selected from H and F; and Y is selected from F and OH,

5. The compound of Claim 3 wherein Z¹, Z² and Z³ are each hydrogen.

6. The compound of Claim 5 wherein R₁ is selected from:

(a) 3-thiophene,

(b) 3-fluorophenyl, (c) 4-fluoroρhenyl,

(d) 2-fluorophenyl,

(e) 2-furyl, and

(f) 2-thiophene.

7. The compound of Claim 2 wherein the compound is of structural formula (II) and R₁ is

8. A pharmaceutical composition comprising a nontoxic therapeutically effective amount of a compound of Claim 1 and a pharmaceutically acceptaible carrier.

9. A pharmaceutical composition comprising a nontoxic therapeutically effective amount of a compound of Claim 1 in combination with a nontoxic therapeutically effective amount of a cholesterol lowering agent selected from the group consisting of: a) HMG-CoA reductase inhibitor,

b) HMG-CoA synthase inhibitor,

c) squalene epoxidase inhibitor,

d) probucol,

e) niacin, f) gemfibrozil,

g) clofibrate,

h) LDL-receptor gene inducer, and

i) a pharmaceutically acceptable non-toxic

cationic polymer capable of binding bile acids in a non-resorbable form in the

gastrointestinal tract.

10. A pharmaceutical composition comprising a unit dose of a compound of Claim 1 and a nontoxic therapeutically effective amount of an HMG-CoA

reductase inhibitor.

11. A method of treating hypercholesterolemia comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound of Claim 1.

12. A method of inhibiting squalene synthase comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound of Claim 1.

13. A method for inhibiting fungal growth comprising applying to the area where growth is to be controlled an antifungally effective amount of a compound of Claim 1.

14. A method for treating cancer comprising administration to a subject in need of such treatment a non-toxic therapeutically effective amount of a

compound of Claim 1.

15. A method of treating hypercholesterolemia comprising the administration to a subject in need of such treatment 20 to 100 mg of a compound of Claim 1.

16. A method for inhibiting fungal growth in a living organism in need of such treatment comprising the oral, systemic or parenteral administration of a non-toxic antifungally effective amount of a compound of Claim 1.

17. A process for the formation of a

compound of Claim 1 of structural formula (I) wherein Z¹, Z², and Z³ are each H, comprising the addition of a compound of Formula (III) selected from the group consisting of:

(a) R₁-CO₂H; and

(b) R₁-CH₂-CHNH₂CO₂H: at a concentration of 0.01 mM to 100 mM, wherein R₁ is selected from

;

;

;

;

; and

;

and wherein X is selected from:

(a) H,

(b) halogen (F, Cl, Br, I),

(c) OH, and

(d) CH₃; and Y is selected from:

(a) halogen (F, Cl, Br, I ) ,

(b) OH, and

(c) CH₃; to a Zaragozic Acid A producing culture and incubating for 48 to 120 hours at 20 to 30ºC at a pH less than 8 and isolating the product of Formula (I) from the culture broth.

18. The process of Claim 17 wherein the Zaragozic Acid A producing culture is:

(a) MF5453,

(b) MF5565,

(c) MF5599,

(d) MF5572,

(e) MF5573,

or a mutant having essentially the same characteristics as one of the above.

19. The process of Claim 18 wherein R₁ is selected from:

,

, and

.

20. The process of Claim 19 wherein X is selected from H and F and Y is selected from F and OH.

21. The process of Claim 18 wherein an inhibitor of phenylalanine ammonium lyase is added to the culture broth.

22. The process of Claim 18 wherein the cells of the Zaragozic Acid A producing culture are homogenized before the addition of the compound of Formula (III).

23. The process of Claim 18 wherein the cells of the Zaragozic Acid A producing culture are treated with toluene before the addition of the compound of Formula (III).

24. The process of Claim 18 wherein the cells of the Zaragozic Acid A producing culture are washed after growth and resuspended in an aqueous medium before the addition of the compound of Formula (III).

25. A process for the formation of a compound of Claim 1 of structural formula (II) wherein Z¹, Z² and Z³ are each hydrogen, comprising the.

addition of a compound of formula (III) selected from the group consisting of:

(a) R₁-CO₂H, and

(b) R₁-CH₂-CHNH₂CO₂H;

wherein R₁ is selected from:

;

;

;

;

; and

;

wherein X is selected from the group consisting of:

(a) hydrogen,

(b) halogen, wherein halogen is selected from the group:

(i) F,

(ϋ) Cl,

(iii) Br, and

(iv) I,

(c) hydroxy, and

(d) methyl;

and wherein Y is selected from the group consisting of:

(a) halogen, wherein halogen is selected from the group:

(i) F,

(ϋ) Cl,

(iii) Br, and

(iv) I,

(b) hydroxy, and

(c) methyl;

to a Zaragozic Acid C producing culture and isolating the product compound of Claim 1 from the culture broth.

26. The process according to Claim 25 wherein the Zaragozic Acid C producing culture is:

(a) MF5465 (ATCC 74011),

(b) MF5701 (ATCC 74165),

(c) MF5703 (ATCC 74166),

or a mutant having essentially the same characteristics as one of the above.

27. The process according to Claim 25 wherein R₁ is:

28. The process according to Claim 27 wherein R₁ is: