THEIR PRODUCTION PROCESS

Technical Field

This invention relates to novel aliphatic tricarboxylic acid compounds, and particularly to novel aliphatic tricarboxylic acid compounds produced by fermentation of an unidentified fungus Z-285, which has been deposited as FERM BP-5153. This invention also relates to processes for producing the aliphatic tricarboxylic acids, and a pharmaceutical composition comprising the same, which is useful in the treatment of hypercholesterolemia, fiingal infections or the like.

Background Art

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

Inhibition of cholesterol biosynthesis constitutes an important approach to the reduction of low density lipoprotein cholesterol. Lovastatin, now commercially available, is one of the potent antihypercholesterolemic agents which inhibit HMG-CoA reductase, thereby reducing the level of mevalonic acid present. However, mevalonic acid is also the obligate precursor for non¬ steroidal isoprenoids such as dolicol, ubiquinone and isopentenyl tRNA. Furthermore, mevalonic acid is required for production of farnesyl diphosphate and geranylgeranyl diphosphate, the terpenoid substrates for protein prenylation.

Squalene synthetase (SQS) occupies a key branchpoint in the isoprenoid pathway, catalyzing the first committed step in the de novo cholesterol biosynthesis. This enzyme catalyzes the reductive dimerization of two molecules of farnesyl diphosphate to form squalene via the intermediate

presqualene diphosphate. Inhibition of SQS should leave unhindered biosynthetic pathways to ubiquinone, dolichol and isopentenyl tRNA.

In previous efforts for discovery of SQS inhibitors, Corey, E. J. and R. Volante describe diphosphate-containing compounds as SQS inhibitors in J. Am. Chem. Soc. 98: 1291, 1976. Biller, S. A., et al. also describe farnesyl phosphonates as SQS inhibitors in J. Am. Chem. Soc. 113: 8522, 1991.

Recently certain nonphosphorous-containing SQS inhibitors such as zaragozic acid C (Kenneth, F. B., et al. U.S. 5,026,554, June 25, 1991) and viridiofungins (Harris, G. H., et al. Tetrahedron Lett., 34: 5253, 1993) have been isolated from microbial sources.

Brief Disclosure of the Invention The present invention provides novel aliphatic tricarboxylic acid compounds of formula tj):

) or its pharmaceutically acceptable salt, wherein Y is ethyl or ethenyl; and R ¹ , R ² and R ³ are independently H or C, _.5 alkyl.

Preferably, the compounds of formula (I) include a compound CJ-13,981, wherein Y is ethenyl; and R ¹ , R ² and R ³ are H, and a compound CJ-13,982, wherein Y is ethyl; and R ¹ , R ² and R ³ are H.

This invention also provides a culture FERM BP-5153 which is capable of producing aliphatic tricarboxylic acid compounds.

Also, this invention provides a process for producing the above aliphatic tricarboxylic acid compounds, which comprises cultivating a microorganism

having the identifying characteristics of FERM BP-5153, or a mutant or recombinant form thereof. The process may further comprise the subsequent step of isolating said aliphatic tricarboxylic acid compounds from the fermentation broth. Further, this invention provides a composition for the treatment of fungal infections and inhibition of SQS comprising the compound of formula (I) or a pharmaceutically acceptable base addition salt thereof in an amount effective in such treatments, and a pharmaceutically acceptable carrier.

Also, this invention is directed to a method for the antifungal treatment of a subject in need of such treatment which comprises administering to said subject an antifungal amount of the compound of formula (I) or a pharmaceutically acceptable base addition salt thereof.

Furthermore, this invention is directed to a method for the inhibition of

SQS or the lowering of the serum cholesterol level which comprises administering to a subject in need of such inhibition or said lowering of said serum cholesterol level an effective amount of the compound of formula (I) or a pharmaceutically acceptable base addition salt thereof.

Therefore, the present invention is useful for the treatment or prevention of hypercholesterolemia, fungal infections or the like. Detailed Description of the Invention

The microorganism used in this invention is the strain Z-285 which was isolated from roots of a plant in Chihuahuan Desert by Dr. John Zak's group of

Texas Technical University, Lubbock, Texas, U.S. It was deposited as FERM

BP-5153 in the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (located at 1-3 Higashi 1-chome,

Tsukuba, Ibaraki 305, Japan) under the Budapest Treaty on July 4, 1995.

FERM BP-5153 was inoculated from slants of yeast extract-malt extract agar onto plates of identification media and the plates were incubated at 25 °C for 1 to 5 weeks under alternative 12-hour fluorescent or black light and 12- hour darkness. The results were read at varying times but most commonly

were taken at 11 days. The colors were determined by comparisons with color chips from Color Standards and Color Nomenclature, Ridgway, R., 1912.

Media used for description of the culture and references to their formulation are as follows: 1. Cornmeal Agar: Carmichael. J. W., Mycologia 49: 820-830, 1957.

2. Czapek-Sucrose Agar: Raper, K. B. and D. I. Fennell, 1965. The Genus Aspergillus, p. 36.

3. Malt Extract Agar: ibid, p. 38.

4. Oatmeal Agar: quaker oats 30 g, agar 15 g, tap water 1 1. 5. Potato Dextrose Agar: peeled potatoes lOOg, dextrose 10 g, coconut milk 50 ml, agar 20 g, tap water 1 1.

6. V-8 Juice Agar: ATCC medium 343, ATCC Media Handbook, p. 17, 1984.

7. Potato Carrot Agar: Lechevalier, M. P., J. Lab. Clin. Med. 71 : 934-944, 1968, but use only 30 g potatoes, 2.5 g carrots and 20 g agar.

8. Yeast Extract-Soluble Starch Agar: Emerson, R., Mycologia 50: 589-621, 1958. Culture FERM BP-5153 exhibited the following characteristics: Cornmeal Agar: Attaining 8.3 cm diameter in 11 days; white to cream color (XVI); smooth, submerged to fluffy; reverse cream color (XVI) to light olive-gray (LI); no soluble pigment.

Czapek-Sucrose Agar: Attaining 5.5 cm diameter in 11 days; pale flesh color to seashell pink (XIV); smooth, submerged to fluffy; reverse safrano pink (TI) to pale salmon color (XIV); no soluble pigment.

Malt Extract Agar: Attaining 8.5 cm diameter in 11 days; pale pinkish buff to pinkish buff (XXIX); smooth, floccose to finely funiculose; reverse pinkish buff (XXIX), olive gray to olivaceous black (LI); no soluble pigment.

Oatmeal Agar: Attaining 8.5 cm diameter in 11 days; pale ochraceous- buff to light buff (XV); smooth, loosely floccose to finely funiculose; pale ochraceous-buff (XV) to pale salmon color (XIV), with or without concentric mouse gray (LI) zones; no soluble pigment.

Potato Dextrose Agar: Attaining 8.5 cm diameter in 11 days; pale ochraceous-salmon (XV); smooth, floccose to funiculose; reverse pale ochraceous-salmon to light ochraceous-salmon (XV), with concentric light olive-gray to olivaceous black (LI) zones; no soluble pigment. V-8 Juice Agar: Attaining 8.5 cm diameter in 11 days; pale cinnamon- pink to pale pinkish cinnamon (XXIX); smooth, floccose to funiculose; reverse salmon color to flesh-color (XIV), with or without contric light mouse gray to mouse gray (LI) zones; no soluble pigment.

Potato Carrot Agar: Attaining 8.0 cm diameter in 11 days; white to off- white; smooth, submerged to fluffy; reverse colorless to pale ochraceous buff (XV); no soluble pigment.

Yeast Extract-Soluble Starch Agar: Attaining 8.5 cm diameter in 11 days; white to cartridge buff (XXX); smooth, floccose to funiculose; reverse pale ochraceous-salmon to hght ochraceous-salmon (XV); no soluble pigment. Morphological Properties: Morphological properties were observed after

2, 3 and 5 weeks of incubation. The following observations were obtained after 3 weeks of incubation on malt extract agar under alternative 12-hour fluorescent light and 12-hour of darkness. Hyphae branched; septate; hyaline, brown to black; smooth to roughened; 1.5-8.0 μm diameter. Chlamydospores single or catenulate; borne terminaly, laterally or intercalarily; hyaline, brown to black; globose, subglobose, oval, elliptical, or irregular; 4-13 μm diameter or 5-17 x 4-11 μm. No sexual spores or conidia were produced on any of the media used after 5 weeks of incubation.

Temperature Relations: Growth was moderate at 20 °C, moderate to good at 28°C, good to excellent at 37°C, and none at 45°C and 50°C.

FERM BP-5153 is characterized by the pale yellowish, pale orange-pink, pale pinkish buff, light buff to tan colonies; the pale yellowish, pale pinkish orange, pale orange, pale orange-buff, pale buff, pale gray, gray to black colony reverse; and lack of soluble pigment. The hyphae were septate and branched, and hyaline initially, but became tan, pink buff or ochraceous buff.

They might become brown, gray or black after a long incubation of 4 to 5 weeks or upon exposure to fluorescent light or black light. On several media chlamydospores of various shapes were produced. No sexual spores or condia were produced on any of the media used. On the basis of the above, FERM BP-5153 can not be identified to a fungal genus because of the absence of sexual spores or condia necessary for identification and thus is considered as an unidentified fungus. It has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Japan, under the accession number FERM BP- 5153 as an unidentified fungus.

In this invention, a mutant or recombinant form of FERM BP-5153 having the ability to produce the aliphatic tricarboxylic acid compounds of formula (I), can be also used. The mutant or recombinant form may be obtained by spontaneous mutation, artificial mutation with ultraviolet radiation or treatment with mutagen such as N-methyl-N '-nitro-nitrosoguanidine or ethyl methanesulfonate, or a cell technology method such as protoplast fusion, gene manipulation or the like, according to well-known methods.

According to the present invention, the aliphatic tricarboxylic acid compounds of formula (I) may be produced by aerobic fermentation of FERM BP-5153, or a mutant or recombinant form thereof, under conditions similar to those generally employed to produce bioactive compounds by fermentation.

FERM BP-5153, or a mutant or recombinant form thereof, is usually fermented under submerged aerobic conditions with agitation at a temperature of 20 to 40°C for 1 to 10 days, which may be varied according to fermentation conditions. Cultivation of FERM BP-5153 to produce said aliphatic tricarboxylic acid compounds preferably takes place in aqueous nutrient media at a temperature of 25 to 35 °C for 1 to 3 days. The pH of medium may be adjusted in the range from 4.0 to 9.0, preferably from 5.5 to 7.0.

Nutrient media useful for fermentation include a source of assimilable carbon such as sugars, starches and glycerol; a source of organic nitrogen such

as casein, enzymatic digest of casein, soybean meal, cotton seed meal, peanut meal, wheat gluten, soy flour, meat extract and fish meal. A source of growth substances such as mineral salts, sodium chloride and calcium carbonate; and trace elements such as iron, magnesium, copper, zinc, cobalt and manganese may also be utilized with advantageous results. If excessive foaming is encountered during fermentation, antifoam agents such as polypropylene glycols or silicons may be added to the fermentation medium.

Aeration of the medium in fermenters for submerged growth is maintained at 3 to 200% , preferably at 50 to 150% volumes of sterile air per volume of the medium per minute. The rate of agitation depends on the type of agitator employed. A shake flask is usually run at 150 to 250 rpm whereas a fermenter is usually run at 300 to 2,000 rpm. Aseptic conditions must, of course, be maintained through the transfer of the organism and throughout its growth.

The aliphatic tricarboxylic acid compounds of formula (I) may be isolated by standard techniques such as extraction and various chromatographic techniques.

The aliphatic tricarboxylic acid compounds, CJ-13,981 and CJ-13,982 were isolated in a substantially pure form from the fermentation mixture, and identified by various spectroscopic techniques such as UV spectrophotometry, NMR and mass spectrometries. According to the analyses, these compounds are believed to have the following chemical formulas:

The compounds of formula (I) wherein R ¹ , R ² and R ³ are other than H, can be prepared by alkylation of CJ-13981 or CJ-13982 using a suitable alkylating agent under suitable conditions known to those skilled in the art.

The SQS inhibitory and antifungal activities of the aliphatic tricarboxylic acid compounds, CJ-13,981 and CJ-13,982, produced by the process of this invention were measured by the standard in vitro protocol described below:

Preparation of Microsomes

The livers freshly obtained from Wistar rats were rinsed in ice-cold PBS and briefly homogenized in buffer A (50 mM MOPS-NaOH; pH 7.4, 10 mM MgCl ₂ , 1 mM EDTA and 10 mM DTT) containing the protease inhibitors (500 μM PMSF, 10 μM aprotinin, 10 μM leupeptin and 10 μM chymostatin). The homogenate was centrifuged at 3,000 x g for 20 min at 4 °C. The supernatant was recentrifuged at 20,000 x g for 30 min at 4 °C. The supernatant was again centrifuged at 100,000 x g for 60 min at 4 °C. After centrifugation, the supernatant was removed and the pellet (microsomal fraction) was suspended in buffer A. This microsomal fraction has a protein concentration of about 13.2 mg/ml. The microsomal suspensions were stored at -70 °C until use. Under this condition, SQS activity was stable for at least several months.

Squalene Synthetase Assay Assay was performed in 96-well microtiter plates containing a 50-μl reaction mixture per well which consists of 50 mM MOPS-NaOH (pH 7.4), 10 mM KF, 10 mM MgCl ₂ , 2 mM CHAPS, 10 mM DTT, 0.5 mM NADPH, 50 mM ascorbate, 20 units/ml ascorbate oxidase, 2 mM glucose-6-phosphate, 20 units/ml glucose-6-phosphate dehydrogenase, rat liver microsomal protein and 6 μM [ ³ H] famesyldiphosphate. After incubation at 37°C for 30 min, reaction was terminated by the addition of 20 μl 1 -propanol containing unlabeled 1 % squalene. The reaction mixture (30 μl) was applied onto polyester-backed silica gel TLC sheets (Sigma, 10 x 10 cm) which was developed by ..-hexane - ethyl acetate (7:3). After drying, radioactivity in zones containing the squalene

(Rf = 0.8) was scraped and measured by a scintillation counter. SQS inhibitory activity is calculated by the formula:

[DPM Sample - DPM Blank] Inhibition (%) = ( 1 ) x 100

[DPM Control - DPM Blank]

Antifungal Assay

The antifungal activities of the compounds of the present invention were determined by an agar plate dilution method using a 8-mm paper disk (AD V ANTEC, trademark), an agar plate medium (Antibiotic Medium 11 , Difco) and a test organism (Candida albicans). The aliphatic tricarboxylic acid compounds, CJ-13,981 and CJ-13,982, showed antifungal activities.

The pharmaceutically acceptable acid addition salts of CJ-13,981 and CJ- 13,982 are prepared in a conventional manner by treating a solution or suspension of the free acid with about one chemical equivalent of a pharmaceutically acceptable base. Conventional concentration and recrystallization techniques are employed in isolating the salts. Illustrative of suitable bases are alkali metal hydroxides such as sodium or potassium hydroxide, alkahne earth metal hydroxides such as magnesium or calcium hydroxide, and ammonium or organic amines such as diethanolamine or N- methylglucamine .

The aliphatic tricarboxylic acid compounds of formula (I) and pharmaceutically acceptable acid addition salts thereof are useful in the treatment of hypercholesterolemia, fungal infections or the like. The aliphatic tricarboxyhc acid compounds of formula (I) may be administered alone or in combination with pharmaceutically acceptable carriers, in either single or multiple doses. Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents. The pharmaceutical compositions formed by combining the aliphatic tricarboxylic acid compounds of formula (I) and the pharmaceutically acceptable carriers are

then readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, injectable solutions and the like. These pharmaceutical compositions can, if desired, contain additional ingredients such as flavorings, binders, excipients and the like. Thus, for purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as atarch, alginic acid and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. SoUd compositions of a simillar type may also be employed as fillers in soft and hard filled gelatin capsules. Preferred materials for this include lactose or milk sugar and high molecular weight polyethylene glycols. When aqueous suspensions or elixirs are desired for oral administration, the essential active ingredients therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if desired, emulsifying or suspending agents, together with diluents such as water, ethanol, propylene glycol, glycerin and combinations thereof.

For parenteral administration, solutions of the aliphatic tricarboxylic acid compounds of formula (I) in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be wmployed. Such aqueous solutions should be suitable buffered if necessary and the hquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitioneal administration. In this connection, the sterle aqueous media employed are all readily available by standard techniques known to those skilled in the art.

Additionally, the aliphatic tricarboxylic acid compounds of formula (I) may be administered topically when treating conditions of the skin and this may be done by way of creams, jellies, gels, pastes, and ointments, in accordance with standard pharmaceutical practice.

In general, the tricarboxylic acid compounds of formula (T) are present in the above dosage forms at concentration levels ranging 5 to 70 % by weight, preferably 10 to 50% by weight.

In general, a therapeutically effective daily dose for the active compound will range from 0.01 to 100 mg/kg, generally from about 1 to about 5 mg/kg, body weight of the subject to be treated for fiingal infections and from 0.2 to 100 mg/kg, for instance 0.2 to 1 mg/kg, for the inhibition of SQS and the lowering of the serum cholesterol level. As is generally known, the effective dosage for the active compound depends on the intended route of administration and other factors such as age and weight of the patient, as generally known to a physician. The dosage also depens on the illness to be treated.

Examples The present invention is illustrated by the following examples. However, it should be understood that the invention is not limited to the specific details of these examples. Spectral and physico-chemical data were obtained by the following instruments: UV, JASCO Ubest-30; NMR, JEOL JNM-GX270 updated with a LSI-11/73 host computer, TH-5 tunable probe and version 1.6 software; and LRFAB- and HRFAB-MS, JEOL JMS-700 Mstation. All NMR spectra were measured in CD ₃ OD unless otherwise indicated and peak positions are expressed in parts per million (ppm) based on the intemal standard of the CH ₃ OH peak at 3.35 ppm for »H NMR and 49.0 ppm for ¹³ C NMR. The peak shapes are denoted as follows: s (singlet), d (doublet), t (triplet), q (quartet), (multiple.) and br (broad). All FAB-MS spectra were measured using glycerol- matrix. Example One

Fermentation of FERM BP-5153

One hundred (100) ml of Medium- 1 (potato dextrose broth 2.4% , yeast extract 0.5% and agar 0.1 %) in a 500-ml flask was inoculated with a vegitative cell suspension from a slant culture of FERM BP-5153. The flask was shaken

at 26°C for 4 days on a rotary shaker with 7-cm throw at 210 rpm, to obtain a first seed culture.

A 500-ml flask containing Medium- 1 (150 ml) was inoculated with 5 ml of the first seed culture. The flask was shaken at 26°C for 3 days on a rotary shaker, to obtain a second seed culture.

The second seed culture was used to inoculate a 6-1 fermentation vessel containing 3 1 of sterile medium (Medium-2: potato dextrose broth 2.4%). Aeration was carried out at 26°C for 9 days with 1 ,700 rpm at 3 1 per min. Extraction and Isolation The fermentation broth (3 1) was filtered after the addition of 2 1 of ethanol. The filtrate was concentrated to aqueous solution (1 1) and adjusted to pH 3 with IN HCl. Then it was extracted 3 times with each of 1 1 of ethyl acetate. The extract was dried over anhydrous Na ₂ S0 ₄ and evaporated. The extract (3.5 g) was applied to a YMC-pack ODS AM-343 column (20 x 250 mm, Yamamura trademark) and eluted with methanol-0.1 % TFA in water (85:15) at flow rate of 10 ml/min. Detection was made by UV absorbance at 220 nm. The eluted peaks were collected to yield the CJ-13,981 (198 mg) and CJ-13,982 (508 mg). HPLC Analvsis Analytical HPLC of samples containing the aliphatic tricarboxylic acid compounds of CJ-13,981 and CJ-13,982 was performed using a YMC-pack ODS AM-312 column (6.0 x 250 mm, Yamamura trademark) and eluted with methanol-0.1 % TFA in water (85:15) at a flow rate of 0.8 ml/min. The retention times of CJ-13,981 and CJ-13,982 were 7.0 and 8.7 min, respectively.

Ch aracterization

The physico-chemical properties of CJ-13,981 and CJ-13,982 are as follows:

CJ-13,981 was isolated as a white powder, and exhibited the following spectral data: Η NMR (270 MHz, CD ₃ OD) δ (ppm) 1.14-1.40 (16H, m), 1.47

(IH, m), 1.79 (IH, m), 2.03 (2H, m), 2.64 (IH, dd, 2.9, 11.7), 2.68 (IH, d, 16.5), 3.07 (IH, d, 16.5), 4.87-5.01 (2H, m) and 5.79 (IH, ddt, 10.1 , 17.0, 6.6); ¹³ C NMR (67.5 MHz, CD ₃ OD) δ (ppm) 28.89, 29.61 , 30.91, 31.01 , 31.24 (2C), 31.40 (2C), 31.46, 35.69, 43.08, 55.59, 77.60, 115.49, 140.94, 174.65, 176.97 and 177.41 ; FAB-MS (m/z) 357 (M-H) ^" ; UV max (MeOH) 215 nm; [α] ²⁶ _D (c 0.81, acetone) -18.52.

CJ-13,982 was isolated as a white powder, and exhibited the following spectral data: Η NMR (270 MHz, CD ₃ OD) δ (ppm) 0.89 (3H, t, 6.5), 1.27 (20H, m), 1.49 (IH, m), 1.79 (IH, m), 2.64 (IH, m), 2.68 (IH, d, 16.5) and 3.08 (IH, d, 16.5); ¹³ C NMR (67.5 MHz, CD ₃ OD) δ (ppm) 15.26, 24.52, 28.87, 29.60, 31.26 (3C), 31.44 (2C), 31.54 (3C), 33.86, 43.08, 55.57, 77.59, 174.68, 176.97 and 177.41; FAB-MS (m/z) 359 (M-H) ^" ; HRFAB-MS (m/z) obs. 359.2073 (calcd. for C ₁₈ H ₃₁ 0 ₇ , 359.2067); UV max (MeOH) 215 nm; [α] ²⁶ _D (c 2.6, acetone) -18.34. Example Two

Preparation of Trimethyl Ester of CT-13.981

A solution of 2 mg of CJ-13,981 in 0.5 ml of acetonitrile was treated at room temperature with 10 equivalents of (trimethylsilyl)diazomethane in n- hexane. After 2 hours, the reaction mixture was applied to a YMC-pack ODS AM-343 column (20 x 250 mm, Yamamura trademark) and eluted with methanol- water (85:15) at flow rate of 10 ml/min. Detection was made by UV absorbance at 220 nm. The eluted peak was collected to yield a trimethyl ester of CJ-13,981.

Example Three Preparation of Trimethyl Ester of C.T-13.982

A solution of 2 mg of CJ-13,982 in 0.5 ml of acetonitrile was treated at room temperature with 10 equivalents of (trimethylsilyl)diazomethane in n- hexane. After 2 hours, the reaction mixture was applied to a YMC-pack ODS

AM-343 column (20 x 250 mm, Yamamura trademark) and eluted with methanol- water (85:15) at flow rate of 10 ml/min. Detection was made by UV

absorbance at 220 nm. The eluted peak was collected to yield a trimethyl ester of CJ-13,982.