NOVEL CHOLESTEROL LOWERING COMPOUND

Abstract: A compound (I) having antihypercholesterolemic utility is produced by culturing a Streptomyces sp. in a nutrient medium in the presence of a substrate of a previously prepared solution of a compound of structural formula (II) in DMSO.

Description: TITLE OF THE INVENTION NOVEL CHOLESTEROL LOWERING COMPOUND BACKGROUND OF THE INVENTION This is a continuation-in-part of application Serial No. 07/715,518, filed June 14, 1991. Hypercholesterolemia is known to be one of the prime risk factors for ischemic cardiovascular disease, such as arteriosclerosis. Biie 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 synthetase is the enzyme involved in the first committed step of the d_e_ 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 synthetase have employed pyrophosphate or pyrophosphate analogs containing compounds such as those described in P. Ortiz de Montellano e± ai, 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 synthetase. Recently certain nonphosphorus containing inhibitors of squalene synthetase have been isolated as natural products. These natural product inhibitors are described in copending patent applications S.N. 496,734 filed March 21, 1990, S.N. 496,742 filed March 21, 1990, now issued as U.S. Patent No. 5,096,923, and S.N. 582,452 filed September 13, 1990, now issued as U.S. Patent No. 5,102,907. Semisynthetic analogs of these naturally occurring compounds have been reported in copending application S.N. 698,766 filed May 10, 1991. A need still remains for a more effective squalene synthetase inhibitor, i.e., one that provides a greater antihypercholesterolemic effect and exhibits a good safety profile. The present invention is directed to a bio- transformed analog of the above-noted natural products. DETAILED DESCRIPTION OF THE INVENTION This invention is concerned with a Compound I or a pharmaceutically acceptable salt thereof which is useful as a cholesterol lowering agent. The Compound I is prepared by a biotransformation of a solution of the compound of structural formula II in DMSO: COOH II by incubation of a Streptomyces sp. r in a nutrient medium in the presence of the solution. More particularly, Compound I is formed from Compound II via a precursor, Compound III, which is spontaneously formed upon dissolving II in DMSO and allowing the solution to stand at room temperature for about 4 to 12 months. When Compound III or a previously prepared DMSO solution of Compound II is incubated with Streptomyces sp. in a nutrient medium, Compound I is formed. The preferred Streptomyces species is Streptomyces rimosus and the preferred strain of the Streptomyces sp. is deposited in the culture collection of Merck & Co. Inc., Rahway, New Jersey as MA6817. A sample of this microorganism has been deposited under the terms of the Budapest Treaty with the American Type Culture Collection at 1230, Parklawn Drive, Rockville Maryland 20852 and has been # assigned accession number ATCC 55189. Thus, Compound I is prepared in an aerobic fermentation procedure employing the novel culture, MA6817. Mutants of MA6817 are also capable of producing the compounds of this invention and are included within the scope of this invention. The culture employed and described herein is isolated from and substantially free from its soil contaminants and free of deleterious viable contaminating microorganisms. That is, the culture is free of contaminating microorganisms that are deleterious on the reaction which produces Compound I. Source The organism was isolated from a soil sample 20 km east of Mount Olga Montana. Cell Wall Composition Cell wall analysis revealed a peptidoglycan containing L-diaminopimelic acid, and whole cell sugar analysis revealed glucose and ribose. General Growth Characteristics The organism MA6817 grows moderately well on Yeast Malt extract amd glycerol asparagine and well on inorganic salts-starch, oatmeal and trypticase soy agars. It grows at 27 β C and 37°C and grows well in liquid media such as yeast dextrose broth. Colony Morphology Substrate mycelium is light orange yellow (70.1 OY) and colonies are opaque, raised, lobate and rubbery. The colony surface is rough. Aerial mycelia appear after 5 days incubation and are white (263 White) Spore mass, when present, is white to pale yellow (263 White - 89 p.Y). Micromorphology Aerial mycelium (0.76 μm dia) radiate from the substrate mycelium and is straight and highly branched. In mature cultures, aerial mycelia terminate in chains of spores that are borne in tightly coiled spirals. Sclerotia are observed on other media especially Sigma water agar. Miscellaneous Physiological Reactions Culture does not produce melanoid pigments. Starch is hydrolyzed. Hydrogen disulfide is not produced. Carbon source utilization pattern is as follows: good utilization of α-D-lactose, β-D-lactose; moderate utilization of L-arabinose, cellobiose, D-fructose, inositol, D-maltose, D-mannitol, D-mannose, D-raffinose, D-xylose; no utilization of D-arabinose, L-rhamnose, sucrose, L-xylose. Phenotypically, this culture exhibits some similarity with strains currently identified as Streptomyces rimosus. Carbohydrate utilization patterns are quite similar with the type strain. MA6817 does, however, exhibit morphological characteristics (production of sclerotia and nest-like structures) that are not reported for strains currently clustered within this group. Placement within this species is tentative. Compound I is produced during the aerobic fermentation of a suitable aqueous nutrient media in the presence of Compound III under conditions described hereinafter, with a producing strain of the Streptomyces sp. Such nutrient media contain sources of carbon and nitrogen assimilable by the microorganism and generally low levels of inorganic salts. In addition, the fermentation media may contain traces of metals necessary for the growth of the microorganisms. These are usually present in sufficient concentration in the complex sources of carbon and nitrogen which may be used as nutrient sources, but can, of course, be added separately to the medium if desired. In general, carbohydrates such as sugars, for example dextrose, maltose, lactose, dextran, cerelose and the like, and starches are suitable sources of assimilable carbon in the nutrient media. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium but it is usually found that an amount of carbohydrate between about 0.5 and 5% by weight of the medium is satisfactory. These carbon sources can be used individually or several such carbon sources may be combined in the same medium. Various nitrogen sources such as yeast hydrolys tes, yeast autoysate, soybean meal, casein hydrolysates, yeast extracts, corn steep liquors, distillers solubles, cottonseed meal, meat extract and the like, are readily assimilable by Streptomyces sp. in the production of Compound I. The various sources of nitrogen can be used alone or in combination in amounts ranging from about 0.2 to 6% by weight of the medium. Among the nutrient inorganic salts which can be incorporated in the culture media are the customary salts capable of yielding sodium potassium, magnesium, ammonium, calicum, phosphate, sulfate, chloride, carbonate and like ions. Also included are trace metals such as cobalt, manganese, iron and the like. It should be noted that the media described hereinbelow and in the Examples are merely illustrative of the wide variety of media which may be employed, and are not intended to be limitative. Example 1 Preparation of Compound I Step A: Preparation of (1S,3S,4S,5R,6R,7R)-1-[(4S)- acetoxy-3-methylene-5-methyl-6-phenyl]- hexyl-4,6,7-trihydroxy-6-0-(4,6-dimethyl-2- octenoyl)-2,8-dioxabicyclo[3.2.1]octane-3,4, 5-tricarboxylic acid (II) 1. Culturing MF5453 Culture MF5453 (ATCC 20986) was inoculated into KF seed medium using one glass scoop of the original soil tube. The KF seed flask was incubated for 73 hours at 25 β C, 220 rpm, 85% humidity. At the end of this incubation, 2.0 mis aliquots were aseptically transferred to each of 75 MBM production medium flasks. These production flasks were then incubated at 25 C C, 220 rpm, 85% humidity, with a fermentation cycle of 14 days. Flasks were harvested as follows: mycelial growth was homogenized for 20 seconds at high speed using Biohomogenizer/ mixer (Biospec Products Inc. Bartlesville, Ok); and then 45 mis methanol was added to each flask (final methanol concentration was approximately 50%) . Flasks were then returned to the shaker and agitated at 220 rpm for 30 minutes. Subsequently, the contents of the flasks were pooled. 2. Isolation of Compound II A 6 liter 50% methanol homogenized fungal extract exhibiting a pH of 4.5 was employed in the following isolation procedure. The mycelia was filtered through celite and the recovered mycelia was extracted again by stirring overnight with 3 L of 50% methanol and again filtered. The combined extract (9 L) of 50% methanol was diluted to 25% methanol with water (total volume 18 L) and applied to a Mitsubishi HP-20 column (750 ml) at a flow rate of 80 ml/minute. The column was washed with water (1 L) and eluted with a stepwise gradient of methanol consisting of 50/50 methanol/H 2 0 (1 L) , 60/40, methanol/H 2 0 (1 L), 80/20 methanol/H 2 0 (2 L,) 90/10 methanol/H 2 0 (1 L), 100% methanol (2 L), and 100% acetone (1 L) . The fractions from 50/50 to 90/10 methanol/H 2 0 were combined and diluted with water to 35/65 methanol/H 0 (total volume 10 L) . The 10 L of 35/65 methanol/H 2 0 was acidified with 1.0 N HC1 (20 ml) to pH 3.0 and extracted into EtOAc (4 L) . The EtOAc layer was separated and the solvent removed in vacuo to yield 260 mg of an orange oil. A portion (10%) of the orange oil was dissolved in 1 ml methanol and diluted with 0.8 ml 10 mM potassium phosphate (pH 6.5) with some precipitation. The suspension was applied to a preparative HPLC column (Whatman Magnum 20 C^g, 22 mm ID X 25 cm, 8 ml/minute. The initial mobile phase was 60/40 methanol/10 mM K3PO4, pH 6.5, and after 20 minutes the mobile phase was changed to 80/20 methanol/10 mM potassium phosphate, pH 6.5. Fractions of 8 ml each were collected, and the fractions from 31 to 33 minutes were combined, diluted with water to 35% methanol, acidified with 10% HC1 to pH 3, and extracted into EtOAc. The solvent was removed in vacuo and a clear slightly yellow oil identified as Compound (II) was obtained. KF SEED MEDIUM Corn Steep Li Tomato Paste Oat Flour Glucose Trace Element pH adjusted to 6.8 (presterile) (NH 4 ) 6 Mθ 7 0 2 «4H 2 0 0.019 50 mis/nonbaffled 250 mis ZnS04«7H 2 0 0.2 Erlenmeyer flask autoclave 20 minutes (121 β C, dissolved in 1L 0.6 N HC1 15 psi) £i 4.0 LO.O 4.0 4.0 pH 7.0 (no pH adjustment) 45 mis/nonbaffled 250 mis Erlenmeyer flask autoclave 15 minutes (121 C C, 15 psi) Step B: Preparation of Compound I 1. Formation of Compound III Compound II is dissolved in DMSO (2.5 mg/ml) and the solution allowed to stand at room temperature for a period of 4 to 12 months. In a slow process (e.g., 15% conversion in eleven months), Compound III is formed from Compound II. 2. Isolation of Compound III Compound III is isolated by means of a preparative HPLC on a Beckman Ultrasphere Octyl column (10 x 250 mm) with gradient elution. The gradient program is 30% B/70% A for 0-3 minutes, linear gradient to reach 80% B/20% A from 3-33 minutes, then 100% B from 33-35 minutes and remains there for the final 10 minutes. The flow rate is constant at 3.00 ml/minute. Solvent A was 0.1% HCOOH in water and B was 0.1% HCOOH in acetonitrile-water (17:3). Evaporation of the selected fractions (retention time 38.0 min.) under a stream of nitrogen provided Compound III, having the following physical characteristics: J-H NMR spectrum (400 mHz) (CD3OD, 22 β C): 7.24 (t, 2H), 7.17 (d, 2H), 7.14 (t, 1H) , 6.87 (dd, 15.7, 8.4, 1H), 5.87 (dd, 15.7, 1.0, 1H), 5.51 (d, 2.4, 1H) , 5.05 (d, 5.0, 1H), 4.96 (s, 1H), 4.93 (s, 1H) , 3.99 (d, 2.4, 1H), 3.98 (d, 12.7, 1H), 3.83 (d, 12.7, 1H) , 2.68 (dd, 13.4, 5.9, 1H), 2.39 (dd, 13.4, 9.0, 1H), 2.2-2.43 (m, 4H), 2.08 (s, 3H) , 1.91 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.82 (d, 3H) ppm. Figure 2 shows the *H NMR spectrum, Analytical HPLC on a Beckman Ultrasphere Octyl column (4.6 x 250 mm) wherein the elution was performed in the gradient mode according to the following program: had a retention time of 18.5 minutes and exhibited an ultraviolet absorption maximum at 213 nm. 3. Seed Growth Medium #2 (50 ml) in 250 ml baffled flasks were inoculated with MA6817 and shaken in a rotary shaker at 220 rpm and 27 β C. The seed was grown for 48 hours. 4. Fermentation Production flasks (medium #2, 50 ml in 250 ml baffled flasks) were inoculated with 2 ml of the seed medium and shaken at 27 β C and 220 rpm on a rotary shaker. After 24 hours, a substrate containing the crude mixture of Compounds II and III, prepared above (Step Bl), was added to each flask. Thus, a DMSO solution of the substrate containing Compound II (1.217 mg, 76.1%) and Compound III (0.382 mg, 23.9%) was used for each of two shakeflasks. Incubation continued for 72 hours. 3. Extraction: The harvested biotransformation samples were acidified with formic acid (2 ml 88% for each flask) then extracted with ethyl acetate. The organic phase was dried over Na S0 4 then filtered and evaporated to dryness. The dry residue was redissolved in the smallest possible volume of DMSO-water (2:1 v/v) for preparative HPLC. 4. Chromatography Two different gradient methods were used in succession. The first separation was accomplished on a Beckman Ultrasphere Cyano column (10x250mm) in a gradient from 20% solvent B/80% solvent A to 65% solvent B/35% solvent A in 35 minutes at a flow rate of 3.00 ml/min. Fractions were collected every 3 minutes or according to peaks detected at 213 nm, as appropriate. The selected fractions (22.5-24.0 min) were evaporated to dryness and chromatographed again on a Beckman Ultrasphere Octyl column (10x250mm) in a gradient from 30% B/70% A to 80% B/20% A in 35 minutes then at 100% solvent B for an additional 10 minutes. The remaining conditions were the same as in the first separation. Solvent A was 20 mM HCOOH and B was acetonitrile-water (17:3 v/v) containing the same amount of HCOOH as solvent A. Evaporation of the selected fractions (retention time 33.6 min.) provided the product, Compound I, with the following physical characteristics: JH NMR spectrum (400 Hz) (CD 3 0D } 22 °C): 7.23 (t, 2H), 7.19 (d, 2H), 7.12 (t, 1H), 6.87 (dd, 15.7, 8.4, 1H), 5.86 (dd, 15.7, 1.0, 1H), 5.50 (d, 2.4, 1H), 5.06 (s, 1H), 4.93 (s, 1H), 4.01 (d, 2.4, 1H), 3.98 (d, 12.7, 1H), 3.89 (d, 5.0, 1H), 3.83 (d, 12.7, 1H), 2.75 (dd, 13.4, 5.9, 1H), 2.36 (dd, 13.4, 9.0, 1H), 2.0-2.45 (m, 4H), 1.93 ( , 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm. Figure 1 shows the ^H NMR spectrum. Analytical HPLC on a Beckman Ultrasphere Octyl column (4.6 x 250 mm) wherein the elution was performed in the gradient mode according to the following program: Solvent A: 10 mM H 3 P0 4 in water Solvent B: Acetonitrile-water (85:15 v/v) Time (min.) percent B 0 30 2 30 18 80 20 100 24 100 25 30 Flow: 0.900 ml/min Temp. : Ambient had a retention time of 19.9 minutes and exhibited an ultraviolet absorption maximum at 213 nm. In a control experiment with sterile medium #2 (i.e., no MA 6817 present in the medium) and a DMSO solution containing a mixture of compounds II and III, no detectable amount of compound I was found. Example 2 As a specific embodiment of an oral composition of a compound of this invention, 20 mg of Compound I 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 gel capsule. The present invention is also concerned with a process for making Compound I by incubation of a Streptomyces sp. f in a nutrient medium in the presence of a Compound III. Compound III can be produced by allowing the compound of structural formula II: COOH II to stand in a suitable polar organic solvent such as DMSO or acetonitrile. In a specific embodiment of the process, Compound II which has been previously left standing in DMSO for a minimum of four months such that a detectable amount of Compound III is produced, is incubated with Streptomyces sp. in a nutrient medium to form Compound I. The present invention is also concerned with a method of treating hypercholesterolemia which comprises the administration to a subject in need of such treatment of a nontoxic therapeutically effective amount of a Compound I or a Compound III or a pharmaceutically acceptable salt thereof. Specifically, the compounds of this invention are useful as antihypercholesterolemic agents for the treatment of arteriosclerosis, hyperlipidemia, familial hypercholesterolemia and the like diseases in humans. They may be administered orally or parenterally in the form of a capsule, a tablet, an injectable preparation or the like. It is usually desirable to use the oral route. Doses may be varied, depending on the age, severity, body weight and other conditions of human patients, but a daily dosage for adults is within a range of from about 20 mg to 2000 mg (preferably 20 to 100 mg) which may be given in two to four divided doses. Higher doses may be favorably employed as required. The present invention is also concerned with a method of inhibiting squalene synthetase which comprises the administration to a subject in need of such treatment of a nontoxic therapeutically effective amount of a Compound I or a Compound III or a pharmaceutically acceptable salt thereof. Specifically, the compounds of this invention are useful in treating disease conditions such as, but not limited to, hypercholesterolemia conditions which require the action of the enzyme squalene synthetase. They may be administered by the same routes in the same dosages as described for the method of treating hypercholesterolemia. The pharmaceutically acceptable salts of the compounds of this invention include those formed from cations such as sodium, potassium, aluminum, calcium, lithium, magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methylglutamine, lysine, arginine, ornithine, choline, N,N'-dibenzylethylene- diamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane, and tetra ethyl- ammonium hydroxide. The salts included herein encompass those wherein one, or two of the carboxyl groups are in the salt form. The compounds of this invention may also be administered in combination with other cholesterol lowering agents such as those which inhibit an enzymatic pathway in the biosynthesis of cholesterol. Example 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 inhibitors are lovastatin, simvastatin, pravastatin and fluvastatin. Other cholesterol lowering agents that may be administered include niacin, probucol, and the fibric acids, clofibrate and gemfibrozil. Appropriate daily dosages for adults are niacin (2-8 gm), probucol (up to 1000 mg), clofibrate (up to 2 gm) and gemfibrozil (800-1500 mg). The compounds of this invention may also be coadministered with pharmaceutically acceptable nontoxic cationic polymers capable of binding bile acids in a non-reabsorbable form in the gastro¬ intestinal tract. Examples of such polymers include cholestyramine, colestipol and poly[methyl-(3-tri- methylaminopropyl)imino-trimethylene dihalide] . The relative amounts of the compounds of this invention and these polymers is between 1:100 and 1:15,000. The intrinsic squalene synthetase inhibitory activity of the compounds of this invention was measured by the standard in vitro protocol described below: PREPARATION OF HUMAN HepG2 cell ENZYME 1. SOURCE: HEPG2 CELL LINE(Liver, hepatoblastoma, Human) ATCC No. HB 8065 2. CELL GROWTH AND MAINTENANCE Culture Medium: Minimum essential medium (MEM) with non-essential amino acids, sodium pyruvate, and 10% fetal bovine serum. The medium was changed twice weekly. A confluent monolayer was achieved in 1 week. The growth medium is prepared as listed below. So ution Volume (ml) 1. MEM (Gibco #320-1090AK) 1000 With Earle's salts and L-glutamine 2. Penicillin (10,000 units/ml), streptomycin ( 10,000 mg/ml), Gibco#600-5140 PG 10 3. MEM sodium pyruvate, 10 mM ( 100X) Gibco# 320-1140 10 4. MEM nonessential amino acids, 10 mM(lOOX) Gibco#320-1140AG 10 5. L-glutamine, 200 mM ( 100X), Gibco#320-5030AG 10 6. Hyclone fetal bovine serum, defined, Hyclone #A-111-L 100 Subculture Procedure: Remove medium, wash with PBS, add fresh trypsin (0.25%)-EDTA (0.02%) with Hank's Balanced Salt solution and let flask stand for a minute and remove the trypsin solution . Incubate flask at 37 β C until cells detached. Add fresh medium, disperse and dispense cells into new flasks. Subcultivation ratio: 1:6. PREPARATION of Delipidated Serum: Fetal calf serum(100 ml) and CAB-0-Sil (2 grams) stir overnight at 4 β C and centrifuge at 16,000 rpm for 5 hrs. Filter supernatant and store at 4 β C. 48 hrs. prior to harvest, switch cells grown in MEM with 10% Fetal Calf serum to MEM with 10% delipidated serum. 3. Harvest: Remove medium, wash with PBS, add fresh trypsin (0.25%)-EDTA (0.02%) with Hank's Balanced Salt solution, rinse and remove. Incubate flask at 37°C until cells detach. Add 6 ml of MEM medium per flask to suspend cells and combine into centrifuge tube. Spin cells at 1,000 rpm for 5 mins. Wash by resuspending cell pellet in PBS and repeat centrifuging. Count cells (2.5 x 10^ yield from 18 flasks (75 cm 2 ). Resuspend in 10 mis of 50mM HEPES (N-[2-Hydroxyethyl]ρiperazine-N'-[2-ethane- sulfonic acid]) containing 5mM MgCl 2mM MnCl lOmM DTT, pH 7.5 (enzyme suspension buffer). 4. Cell extracts: Sonicate (probe sonicator setting #60, pulse) the cell suspension on ice for 2 min. After a 1 min. cooling on ice, the sonication is repeated until greater than 90% of the cells are broken as observed microscopically. Centrifuge cell suspension for 10 mins. at 10,000 rpm. Transfer supernatant to clean tube and centrifuge at 20,000 rpm for 20 mins. The HepG2 enzyme preparation was centrifuged at 34,000 rpm to separate the cytosol and icrosomal enzymes. The resulting pellet from the 34,000 rpm centrifugation, containing the squalene synthetase, was resuspended in 5 ml of enzyme suspension buffer. The enzyme suspension was diluted 1 to 1,536 and used to perform the squalene synthetase assay using 3μM - -farnesyl pyrophosphate as the substrate. Squalene Synthetase Assay Reactions were performed in 1.2 ml polypropylene tube strips of 8. Buffer mixture and subtrate mixture for the assay were prepared from the following solution: Buffer mixture contains 270 mM HEPES, pH 7.5, 20 mM Potassium fluoride and 5.4 mM Dithiothreitol(DTT). 55 μl of this mixture was used per assay. The final concentrations of HEPES, KF and DTT in the assay are 150 mM, 11 mM and 3 mM respectively. Substrate mixture: For each reaction, 55 μl of buffer mixture was taken with 5 μl of an inhibitor solution in MeOH and 10 μl of diluted enzyme (1 to 1536 as described in the enzyme preparation, the final protein concentration of enzyme in the assay is 1.2 μg per ml.). The reaction was initiated by the addition of 30 μl of substrate solution and the mixture was incubated at 30 β C for 20 minutes. The reactions were stopped by the addition of 100 μl of 95 % EtOH, vortexed, and 100 μl of a suspension of 1 gram per ml of Bio-Rad AG 1 X 8 resin(400 mesh, Chloride form) was then added, vortexed. 800 μl of heptane was added to each tube strip and the strips were capped and vortexed for 10 minutes. 400 μl of heptane layer was then removed into a minivial and mixed with 2.5 ml of scintillation fluid and the radioactivity was determined by liquid scintillation counting. The controls were run with 5 μl of MeOH and blanks were run with the addition of 100 μl of 95% EtOH to denature the enzyme before the addition of the substrate mixture to the assay tube. Percent inhibition is calculated by the formula: (Control - Sample) X 100 Control - Blank IC50 values were determined by plotting the log of the concentration of the test compound versus the percentage inhibition. The IC50 is the concentration of inhibitor that give 50% inhibition as determined from these plots. The IC5 Q of the compound of this invention (i.e., Compound I) against squalene synthetase is estimated to be 0.67 nM. The heptane extractable products of the enzyme control and the enzyme reaction in the presence of 30 nM of the compound of invention were spiked with 5 μl of 0.5% squalene and then were analyzed by reverse phase high performance liquid chromatography. One hundred μl of heptane extract containing squalene spike was injected into a 4.6 mm X 10 cm Whatman RAC II Partisil 5 ODS-3 column, and the active peak was then eluted with a gradient of acetonitrile and water. Fractions were collected and mixed with scintillation fluid and counted in a liquid scintillation counter. The squalene peak was also monitored at 210 nm. The UV absorption peak of squalene was found to coincide with the radioactivity and was completely inhibited by 30 nM of the compound of the invention. This further confirms that the compound of the invention is a potent inhibitor of human squalene synthetase. The IC50 of Compound III against squalene synthetase is estimated to be 2.6 nM.

Claims: WHAT IS CLAIMED IS: 1. A biologically pure culture of Streptomyces sp. ATCC 55189 or an active mutant thereof, wherein the mutant has essentially the same characteristics as ATCC 55189, capable of producing Compound I in a recoverable amount in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, inorganic substances and a substrate of Compound III, wherein Compound I is characterized by the (400 MHz) ^H NMR chemical shifts, as measured in CD3OD at 22 β C, 7.23 (t, 2H), 7.19 (d, 2H), 7.12 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.86 (dd, 15.7, 1.0, IH), 5.50 (d, 2.4, IH), 5.06 (s, IH), 4.93 (s, IH), 4.01 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.89 (d, 5.0, IH), 3.83 (d, 12.7, IH), 2.75 (dd, 13.4, 5.9, IH), 2.36 (dd, 13.4, 9.0, IH), 2.0-2.45 ( , 4H), 1.93 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm. 2. The biologically pure culture of Claim 1 which has accession number ATCC 55189. 3. A Compound I characterized by the (400 MHz) ^H NMR chemical shifts, as measured in CD3OD at 22 β C, 7.23 (t, 2H), 7.19 (d, 2H), 7.12 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.86 (dd, 15.7, 1.0, IH), 5.50 (d, 2.4, IH), 5.06 (s, IH), 4.93 (s, IH), 4.01 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.89 (d, 5.0, IH), 3.83 (d, 12.7, IH), 2.75 (dd, 13.4, 5.9, IH), 2.36 (dd, 13.4, 9.0, IH), 2.0-2.45 (m, 4H), 1.93 (m, 2H), 1.1-1.4 ( , 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm, or a pharmaceutically acceptable salt thereof. 4. A compound produced by the aerobic fermentation of ATCC 55189 in an aqueous medium of assimilable carbon, nitrogen and inorganic substances in the presence of a substrate of Compound III. 5. A compound produced by the aerobic fermentation of ATCC 55189 in an aqueous medium of assimilable carbon, nitrogen and inorganic sources in the presence of a substrate of a previously prepared solution of Compound II in DMSO. 6. A Compound III characterized by the (400 mHz) 1-H NMR chemical shifts, as measured in CD 3 0D at 22 β C, 7.24 (t, 2H), 7.17 (d, 2H), 7.14 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.87 (dd, 15.7, 1.0, IH), 5.51 (d, 2.4, IH), 5.05 (d, 5.0, IH), 4.96 (s, IH), 4.93 (s, IH), 3.99 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.83 (d, 12.7, IH), 2.68 (dd, 13.4, 5.9, IH), 2.39 (dd, 13.4, 9.0, IH), 2.2-2.43 (m, 4H), 2.08 (s, 3H), 1.91 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.82 (d, 3H) ppm, or a pharmaceutically acceptable salt thereof. 7. A pharmaceutical antihypercholestero- lemic composition comprising a pharmaceutically acceptable carrier and a nontoxic therapeutically effective amount of a compound of Claim 3. 8. A method of treatment of hyperchole¬ sterolemia comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound of Claim 3. 9. A method of inhibiting squalene synthetase comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound of Claim 3. 10. A pharmaceutical antihypercholestero¬ lemic composition comprising a pharmaceutically acceptable carrier and a nontoxic therapeutically effective amount of a compound of Claim 6. 11. A method of treatment of hyperchole¬ sterolemia comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound of Claim 6. 12. A method of inhibiting squalene synthetase comprising the administration to a subject in need of such treatment a nontoxic therapeutically effective amount of a compound of Claim 6. 13. A biologically pure culture of Streptomyces sp. ATCC 55189 or an active mutant thereof, wherein the mutant has essentially the same characteristics as ATCC 55189, capable of producing Compound I in a recoverable amount in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen, inorganic substances and a substrate of a previously prepared solution of Compound II: COOH II in DMSO, wherein Compound I is characterized by the (400 MHz) 1H NMR chemical shifts, as measured in CD 3 0D at 22 β C, 7.23 (t, 2H), 7.19 (d, 2H), 7.12 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.86 (dd, 15.7, 1.0, IH), 5.50 (d, 2.4, IH), 5.06 (s, IH), 4.93 (s, IH), 4.01 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.89 (d, 5.0, IH), 3.83 (d, 12.7, IH), 2.75 (dd, 13.4, 5.9, IH), 2.36 (dd, 13.4, 9.0, IH), 2.0-2.45 (m, 4H), 1.93 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm. 14. The biologically pure culture of Claim 13 which has accession number ATCC 55189. 15. A process of making Compound III which is characterized by the (400 mHz) ^-H NMR chemical shifts, as measured in CD3OD at 22 β C, 7.24 (t, 2H), 7.17 (d, 2H), 7.14 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.87 (dd, 15.7, 1.0, IH), 5.51 (d, 2.4, IH), 5.05 (d, 5.0, IH), 4.96 (s, IH), 4.93 (s, IH), 3.99 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.83 (d, 12.7, IH), 2.68 (dd, 13.4, 5.9, IH), 2.39 (dd, 13.4, 9.0, IH), 2.2-2.43 ( , 4H), 2.08 (s, 3H), 1.91 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.82 (d, 3H) ppm, comprising dissolving a compound of stuctural formula II: COOH II in DMSO to form a solution, allowing said solution to stand for a period of at least 4 months, and recovering said Compound III. 16. A process of making Compound I which is characterized by the (400 MHz) H NMR chemical shifts, as measured in CD 3 0D at 22 β C, 7.23 (t, 2H), 7.19 (d, 2H), 7.12 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.86 (dd, 15.7, 1.0, IH), 5.50 (d, 2.4, IH), 5.06 (s, IH), 4.93 (s, IH), 4.01 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.89 (d, 5.0, IH), 3.83 (d, 12.7, IH), 2.75 (dd, 13.4, 5.9, IH), 2.36 (dd, 13.4, 9.0, IH), 2.0-2.45 (m, 4H), 1.93 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H), 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm, comprising cultivating MA6817 (ATCC 55189) or an active mutant thereof, wherein said mutant has essentially the same characteristics as MA6817, with Compound III under conditions suitable for the formation of said compound and recovering said compound. 17. A process according to Claim 16, wherein said Compound III is produced prior to cultivating by putting a compound of structural formula II: COOH II in DMSO, 18. A process of making Compound I which is characterized by the (400 MHz) ^Ε. NMR chemical shifts, as measured in CD 3 0D at 22 β C, 7.23 (t, 2H), 7.19 (d, 2H), 7.12 (t, IH), 6.87 (dd, 15.7, 8.4, IH), 5.86 (dd, 15.7, 1.0, IH), 5.50 (d, 2.4, IH), 5.06 (s, IH), 4.93 (s, IH), 4.01 (d, 2.4, IH), 3.98 (d, 12.7, IH), 3.89 (d, 5.0, IH), 3.83 (d, 12.7, IH), 2.75 (dd, 13.4, 5.9, IH), 2.36 (dd, 13.4, 9.0, IH), 2.0-2.45 (m, 4H), 1.93 (m, 2H), 1.1-1.4 (m, 5H), 1.03 (d, 3H) , 0.86 (t, 3H), 0.85 (d, 3H), 0.79 (d, 3H) ppm, comprising cultivating MA6817 (ATCC 55189) or an active mutant thereof, wherein said mutant has essentially the same characteristics as MA6817, with a solution of a compound II: COOH II 10 in DMSO which has been prepared prior to cultivating, 15 20 25 30

Inventors: ARISON BYRON H, CHEN SHIEH-SHUNG TOM, HUANG LEEYUAN, TREIBER LASZLO R, ARISON, BYRON, H., CHEN, SHIEH-SHUNG, TOM, HUANG, LEEYUAN, TREIBER, LASZLO, R.
Applicants: MERCK & CO INC, MERCK & CO., INC.
Application year: 1992
Main classification: A01N43
IPC codes: C07D493, C12P1, C12P17