TETRALIN DERIVATIVES AS HMG-CoA REDUCTASE INHIBITORS

Abstract: This invention relates to novel tetralin derivatives having inhibitory activity against an enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase and represented by general formula (I) in which R1 is hydrogen or acetyl, R2 is carboxy and R3 is hydroxy, or R2 and R3 may form lactone, and pharmaceutical acceptable salts thereof, to processes for the preparation thereof and to a pharmaceutical composition comprising the same.

Description: TETRALIN DERIVATIVES AS HMG-CoA REDUCTASE INHIBITORS TECHNICAL FIELD This invention relates to novel tetralin derivatives and pharmaceutically acceptable salts thereof which are useful as a medicament. DISCLOSURE OF INVENTION The present invention relates to novel tetralin derivatives and pharmaceutically acceptable salts thereof which have inhibitory activity against an enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), to a process for preparation thereof, to a pharmaceutical composition containing the same, to a use of the same as a medicament, and to a method for treating hypercholesterolaemic and hyperlipoproteinaemic states and associated conditions. Accordingly, one object of this invention is to provide the tetralin derivatives and pharmaceutically acceptable salts thereof which inhibit the activity of HMG-CoA reductase and therefore are capable of lowering blood serum cholesterol levels and blood lipid levels. Another object of this invention is to provide a process for production of the tetralin derivatives and salts thereof. A further object of this invention is to provide a pharmaceutical composition containing, as an active ingredient, tetralin derivatives and salts thereof. Still further object of this invention is to provide a use of the tetralin derivatives and salts thereof as a medicament and a method for treating hypercholesterolaemic states, hyperlipoproteinaemic states and associated conditions in human beings or animals. High levels of blood cholesterol and blood lipids are conditions which are involved in the onset of arteriosclerosis. It is well known that inhibitors of HMG-CoA reductase are effective in lowering the level of blood plasma cholesterol, especially low density lipoprotein cholesterol (LDL-C). It has now been established that lowering LDL- C levels affords protection from coronary heart disease. The tetralin derivatives and salts thereof inhibit HMG-CoA reductase and so inhibit cholesterol biosynthesis. They lower concentrations of cholesterol in blood. Thus they are useful for treating hypercholesterolaemic and hyperlipoproteinaemic states (e. g. atherosclerosis) and associated conditions (e. g. angina, myocardial infarction, cerebral vascular occlusion, arterial aneurism, peripheral vascular disease, recurrent pancreatitis and xanthomas). The tetralin derivatives can be represented by the following general formula (I). in which R^ is hydrogen or acetyl, R2 is carboxy and R is hydroxy, or R2 and Rg may form lactone. Among the tetralin derivatives and salt thereof, compounds in which < Ri is acetyl and which are referred to as WF14919A substance and WF14919B substance can be produced by fermentation. WF14919A substance and WF14919B substance are referred briefly as WF14919 substances. And the fermentation can be conducted using WF14919 substances-producing strain such as Agonomycetes strain No. 14919 in a nutrient medium. It is to be understood that the production of the WF14919 substances is not limited to the use of the particular organism described herein, which is given for the illustrative purpose only. This invention also includes the use of any mutants which are capable of producing the WF14919 substances including natural mutants as well as artificial mutants which can be produced from the described organism by conventional means such as irradiation of X-ray, ultra-violet radiation, treatment which N-methyl-N'-nitro-N-nitrosoguanidine, 2- aminopurine, and the like. Particulars of Agonomycetes strain No. 14919 is as follows: Characteristics of producing Strain No. 14919 The fungus strain No. 14919 was originally isolated from a soil sample, collected at Mt. Kiyosumi, Chiba-ken, Japan. This organism grew very rapidly on various culture media, and formed white-edged and dark gray colonies. During one month, the strain formed neither teleomorph nor anamorph. The differentiated hyphal structures (e. g. sclelotia, bulbils, chlamydospores and clamp connections) were also not observed. These characteristics showed that strain No. 14919 was classified in the Mycelia Sterilia (Hyphomycetes) Its mycological characteristics were as follows. Cultural characteristics on various agar media are summarized in Table 1. Culture on malt extract agar grew spreading broadly, attaining more than 7.5 cm in diameter after two weeks at 25 °C. This colony surface was plane, cottony, dark gray and yellowish white at the edge. The reverse was greenish gray and yellowish white at the edge. Conidial structures were not observed. Colonies on potato dextrose agar grew spreading broadly, attaining 6.5 - 7.0 cm in diameter under the same conditions. The surface was plane, felty, pale gray to black and white at the edge. The reverse was black and yellowish white at the center. Conidial structures were not produced. The vegetative hyphae were smooth, septate, hyaline and branched. The hyphal cells were cylindrical and 1.5 - 5.0 μm in diameter. For inducing the strain to sporulate, we attempted several tests: (1) inoculating to a steam-sterilized flat piece of leaf affixed to a com meal agar plate (Matsushima's method); (2) throwing a piece of cultures into sterile water; etc. But we were not able to observe the characteristic morphogenesis of strain No. 14919, after all. Strain No. 14919 was able to grow at the temperature range from 9 to 33 °C with the growth optimum at 24 to 27 °C. These temperature data were determined on potato dextrose agar (made by Nissui). From above-mentioned characteristics, we named the producing strain Agonomycetes strain No. ' 14919. And it was deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology (1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki, Japan) as PERM BP-3752 (deposited date: February 17, 1992). Table 1. Cultural characteristics of strain No. 14919 Medium Cultural characteristics Malt extract agar G: Spreading broadly, >7.5 cm (Blaskeslee 1915) S: Circular, plane, cottony, formed no conidial structures, dark gray (1F1), yellowish white (3A2) at the edge R: Greenish gray (30F2), yellowish white (3A2) at the edge (to be continued) Medium Cultural characteristics Potato dextrose G: Spreading broadly, 6.5 - 7.0 cm agar (Difco 0013) S: Circular, plane, felty, formed no conidial structures, pale gray (1B1) to black, white (1A1) at the edge R: Black, yellowish white (4A2) at the center Czapek's solution G: Rapidly, 4.0 - 4.5 cm agar (Raper and S: Circular to irregular, plane, raised, felty, Thorn 1949) formed no conidial structures, yellowish gray (4B2), white (1A1) at the edge R: Dark brown (6F4), brownish orange (6C5) at the colony edge Sabouraud dextrose G: Spreading broadly, >7.5 cm agar (Difco 0190) S: Raised, felty, formed no conidial structures, brownish gray (6C2), yellowish white (4A2) at the center, produced brown exudations and brown soluble pigment R: Dark brown (6F4) Oatmeal agar G: Spreading broadly, >7.5 cm (Difco 0552) S: Felty to cottony, formed no conidial structures, dark gray (1F1) to black R: Brownish gray (5E2), orange gray (5B4) at the center Emerson Yp Ss agar G: Spreading broadly, 6.0 - 6.5 cm (Difco 0739) S: Circular, plane, felty, formed no conidial structures, black, white (1A1) at the edge R: Yellowish white (3A2) (to be continued) Medium Cultural characteristics Com meal agar G: Spreading broadly, 5.5 - 6.0 cm (Difco 0386) S: Circular, raised, cottony, formed no conidial structures, yellowish white (4A2), partly dark gray (1F1) R: Yellowish white (4A2), partly dark gray (1F1) MY20 agar* G: Spreading broadly, >7.5 cm S: Circular, raised, floccose, formed no conidial structures, yellowish white (4A2), partly medium gray (1E1) R: Light yellow (4A5) Abbreviation: G: growth, measuring colony size in diameter S: colony surface R: reverse * MY20 agar: 5 g peptone, 3 g yeast extract, 3 g malt extract, 200 g glucose and 20 g agar per liter of water These characteristics were observed after 14 days of incubation at 25 t. . The color descriptions were based on the Methuen Handbook of Colour 2 ). References: 1) Barron, G. L.: The Genera of Hyphomycetes from Soil, Williams & Wilkins Co., Baltimore, 1968. 2) Kornerup, A. and J. H. Wanscher: Methuen Handbook of Colour (3rd ed.), 525p., Methuen, London, 1978. Production of the WF14919 substances The WF14919 substances are produced when the WF14919 substances-producing strain is grown in a nutrient medium containing sources of assimilable carbon and nitrogen under aerobic conditions (e. g. shaking culture, submerged culture, etc.). The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, sucrose, starch, fructose or glycerin, or the like. The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cotton seed flour, soybean meal, com steep liquor, dried yeast, wheat germ, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e. g. ammonium nitrate, ammonium sulfate, ammonium phosphate, etc.), urea or amino acid, or the like. The carbon and nitrogen sources, though advantageously employed in combination, need not to be used in their pure form because less pure materials, which contain traces of growth factors and considerable quantities of mineral nutrients, are also suitable for use. When desired, there may be added to the medium mineral salts such as sodium or calcium carbonate, sodium or potassium phosphate, sodium or potassium chloride, sodium or potassium iodide, magnesium salts, copper salts, zinc salts, cobalt salts, or the like. If necessary, especially when the culture medium foams seriously a defoaming agent, such as liquid paraffin, fatty oil, plant oil, mineral oil or silicone, or the like may be added. Agitation and aeration of the culture mixture may be accomplished in a variety of ways, such as agitation by a propeller or similar mechanical agitation equipment, by revolving or shaking the fermenter, and the like. The fermentation is usually conducted at a temperature between about 10°C and 40°C, preferably 20°C to 30°C, for a period of about 50 hours to 150 hours, which may be varied according to fermentation conditions and scales. When the fermentation is completed, the culture broth is then subjected for recovery of the WF14919 substance to various procedures conventionally used for recovery and purification of biological active substance, for instance, solvent extraction with an appropriate solvent or a mixture of some solvents, chromatography or recrystallization from an appropriate solvent or a mixture thereof. WF14919A substance may be converted to its salt since it is acidic substance. The salt of the WF14919A substance can be prepared by a conventional manner, during or after the recovery and purification of the WF14919A substance. Suitable salts of the WF14919A substance are conventional pharmaceutically acceptable salts and include a metal salt such as an alkali metal salt (e. g. sodium salt, potassium salt, etc.) and an alkaline earth metal salt (e. g. calcium salt, magnesium salt, etc.), an ammonium salt, an organic base salt (e. g. trimethylamine salt, triethylamine salt, pyridine salt, picoline salt, dicyclohexylamine salt, N, N'- dibenzylethylenediamine salt, etc.), and the like. WF14919A substance has the following physico-chemical properties: Appearance: Colorless needles Molecular formula : Elementary analysis: Calcd for C 1 H^oO^: C 67.00, H 7.50 (%) Found: C 66.80, H 7.63 (%) Molecular weight: 376.46 [FAB-MS: m/z 399 (M + Na) + ] Melting point: 136 - 137 °C Specific rotation: [α] D (23 °C): +20 ° (c=0.68 in methanol) Ultraviolet absorption spectrum: λ max ( methanol ) : 210 > 240 > 280 nm Solubility: Soluble: methanol, ethyl acetate, acetone Insoluble: water Color reaction: Positive: eerie sulfate reaction and iodine vapor reaction Negative: ninhydrin reaction, Molisch reaction and ferric chloride reaction Thin layer chromatography (TLC): Stationary phase Developing solvent Rf value Silica Gel 60 chloroform:methanol:acetic acid 0.30 F 254* (100:10:1, v/v) HPTLC RP-18 acetonitrile:water:phosphoric acid 0.50 WF 254 s * (600:400:1, v/v) * made by E. Merck High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 60% aqueous acetonitrile - 0.1% phosphoric acid Column: YMC-AM-303** (S-5, 12θA ODS, 4.6mmID x 250mm) Flow rate: 1.0 ml/minute Detection: UV (210 nm) Retention time: 5.69 minutes ** trade name: YMC Co. Infrared absorption spectrum: v mjlY (KBr): 3300, 2950, 1710, 1670, 1440, 1380, 1270, 1250, 1090, 980 cm "1 H Nuclear magnetic resonance spectrum: (400 MHz, CD 3 OD) δ R 7.08 (IH, br d, J=8Hz), 6.93 (IH, br d, J=8Hz), 6.45 (IH, br d, J=16Hz), 6.03 (IH, m), 5.62 (IH, dd, J=16 and 6.5Hz), 4.40 (IH, m), 4.23 (IH, m), 2.84 (IH, m), 2.55 (IH, dd, J=15.5 and 5Hz), 2.46 (IH, dd, J=15.5 and 8Hz), 2.30 (IH, m), 2.23 (3H, br s), 2.08 to 1.97 (2H, m), 2.01 (3H, s), 1.83 to 1.69 (2H, m), 1.42 (IH, m), 1.03 (3H, d, J=6.5 Hz) as shown in Fig. 1 13 C Nuclear magnetic resonance spectrum: 175.3 (s), 172.3 (s), 139.5 (s), 139.2 (d), 137.0 (s), 134.8 (s), 132.1 (s), 131.3 (d), 128.8 (d), 128.0 (d), 71.8 (d), 70.3 (d), 67.7 (d), 44.6 (t), 43.2 (t), 39.2 (t), 38.6 (t), 25.1 (d), 22.1 (q), 21.5 (q), 20.8 (q) as shown in Fig. 2 Nature: Acidic substance. From the above physical and chemical properties, WF14919A substance is inferred to have the following chemical formula. (7-(8-Acetoxy-5,6,7,8-tetrahydro-2,6-dimethyl- 1 -naphthyl)-3 ,5-dihydroxy-6-heptenoic acid) WF14919B substance has the following physico-chemical properties: Appearance: Colorless powder Molecular formula : C 21 H 26°5 Molecular weight: 358.44 [FAB-MS: m/z 381 (M + Na) + ] Melting point: 167 - 168 °C Specific rotation: [α] D (23 °C): -16 ° (c=0.4 in methanol) Ultraviolet absorption spectrum: λ maχ (methanol): 210, 240, 280 nm Solubility: Soluble: methanol, ethyl acetate, acetone Insoluble: water Color reaction: Positive: eerie sulfate reaction and iodine vapor reaction Negative: ninhydrin reaction, Molisch reaction and ferric chloride reaction Thin layer chromatography (TLC): Stationary phase Developing solvent Rf value Silica Gel 60 chloroform:methanol:acetic acid 0.57 F 254* (100:10:1, v/v) hexane:acetone (1:1, v/v) 0.67 * made by E. Merck High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 60% aqueous acetonitrile - 0.1% phosphoric acid Column: YMC-AM-303** (S-5, 12θA ODS, 4.6mm_D x 250mm) Flow rate: 1.0 ml/minute Detection: UV (210 nm) Retention time: 8.70 minutes ** trade name: YMC Co. Infrared absorption spectrum: v maχ (KBr): 3300, 2960, 1730, 1720, 1370, 1250, 1240, 1090, 1050, 960 cm "1 H Nuclear magnetic resonance spectrum: [400 MHz, CDC1 3 -CD 3 0D(1:1)] δ H 7.13 (IH, br d, J=8Hz), 6.99 (IH, br d, J=8Hz), 6.58 (IH, br d, J=16Hz), 5.99 (IH, m), 5.71 (IH, dd, J=16 and 5Hz), 5.37 (IH, m), 4.37 (IH, m), 2.88 (IH, m), 2.79 (IH, dd, J=18 and 5Hz), 2.65 (IH, m), 2.34 (IH, m), 2.26 (3H, br s), 2.15 to 1.91 (4H, m), 2.02 (3H, s), 1.48 (IH, m), 1.07 (3H, d, J=6.5 Hz) as shown in Fig. 3 13 C Nuclear magnetic resonance spectrum: [100 MHz, CD 3 α3-CD 3 OD(l:l)] δ c 172.1.(s), 171.5 (s), 138.1 (s), 136.4 (s), 134.2 (s), 133.5 (d), 131.4 (s), 130.9 (d), 129.0 (d), 128.6 (d), 76.4 (d), 69.4 (d), 62.3 (d), 38.8 (t), 38.6(t), 38.0 (t), 35.8 (t), 24.4 (d), 22.0 (q), 21.5 (q), 20.6(q) as shown in Fig. 4 • Nature: neutral substance From the above physical and chemical properties, WF14919B substance is inferred to have the following chemical formula. (5-[2-(8-Acetoxy-5,6,7,8-tetrahydro-2,6-dimethyl- l-naphthyl)vinyl]-3-hydroxy-5-pentanolide) WF14919A substance and WF14919B substance can be introduced to their corresponding deacylated compound, respectively and they are referred to as FR152415 substance and FR152416 substance, respectively. FR152415 substance can be obtained from WF14919A substance by a conventional chemical deacetylation. Thus obtained FR152415 substance has the following physico- chemical properties: Appearance: white powder Molecular formula : C 19 H 26°5 Molecular weight: 334.42 [FAB-MS: m/z 357 (M + Na) + ] Melting point: 99 - 101 °C Specific rotation: [α] D (25 °C): +86.6 ° (c=0.5 in methanol) Ultraviolet absorption spectrum: λ maχ (methanol): 220, 240, 280 nm Solubility: Soluble: methanol, ethyl acetate, acetone Slightly soluble: water Color reaction: Positive: eerie sulfate reaction and iodine vapor reaction Negative: ninhydrin reaction, Molisch reaction and ferric chloride reaction Thin layer chromatography (TLC): High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 60% aqueous acetonitrile - 0.1% phosphoric acid Column: YMC-AM-303** (S-5, 12θA ODS, 4.6mm_D x 250mm) Flow rate: 1.0 ml/minute Detection: UV (210 nm) Retention time: 3.50 minutes ** trade name: YMC Co. Infrared absorption spectrum: v m ll α α J γ - (KBr): 3449, 3286, 2946, 2922, 1702, 1441, 1249, 1170, 1084, 1052, 967 cm "1 H Nuclear magnetic resonance spectrum: (400 MHz, CD 3 OD) δ jj 7.03 (IH, d, J=8Hz), 6.89 (IH, d, J=8Hz), 6.78 (IH, d, J=16Hz), 5.76 (IH, dd, J=7 and 16Hz), 4.96 (IH, t, J=3Hz), 4.48 (IH, q, J=7Hz), 4.25 (IH, m), 2.83 (IH, m), 2.54 (IH, dd, J=5 and 15Hz), 2.47 (IH, dd, J=8 and 15Hz), 2.22 to 2.32 (IH, m), 2.27 (3H, s), 2.12 to 2.25 (IH, m), 1.98 (IH, m), 1.86 (IH, m), 1.77 (IH, m), 1.41(1H, m), 1.07 (3H, d, J=6Hz) as shown in Fig. 5 13 C Nuclear magnetic resonance spectrum: (100 MHz, CD 3 OD) δ c 175.4 (s), 139.2 (s), 138.6 (d), 135.9 (s), 135.9 (s), 134.5 (s), 130.6 (d), 129.1 (d), 128.7 (d), 72.3 (d), 67.4 (d), 65.9 (d), 45.0 (t), 43.4 (t), 42.1 (t), 39.6 (t), 24.2 (d), 22.4 (q), 21.1 (q) as shown in Fig. 6 Nature: acidic substance. From the above physical and chemical properties, and further considering that FR152415 substance can be introduced from WF14919A substance, FR152415 substance is inferred to have the following chemical formula. (3,5-Dihydroxy-7-(5,6,7,8-tetrahydro-8-hydroxy-2,6-dimethyl- l-naphthyl)-6-heptenoic acid) FR152416 substance can be obtained from FR 152415 substance by a conventional lactonization. Thus obtained FR152416 substance has the following physico- chemical properties: Appearance: colorless needles Molecular formula : C 19 H 24°4 Molecular weight: 316.40 [FAB-MS: m/z 339 (M + Na) + ] Melting point: 144 - 146 °C Specific rotation: [α] D (26 °C): +107.6 ° (c=0.5 in methanol) Ultraviolet absorption spectrum: λ max ( methanol ) : 218 > 240 > 280 ∞ 11 Solubility: Soluble: methanol, ethyl acetate, acetone Insoluble: water Color reaction: Positive: eerie sulfate reaction and iodine vapor reaction Negative: ninhydrin reaction, Molisch reaction and ferric chloride reaction Thin layer chromatography (TLC): Stationary phase Developing solvent Rf value * made by E. Merck High Performance Liquid Chromatography (HPLC): Condition: Mobile phase: 60% aqueous acetonitrile - 0.1% phosphoric acid Column: YMC-AM-303** (S-5, 12θA ODS, 4.6π_mID x 250mm) How rate: 1.0 ml/minute Detection: UV (210 nm) Retention time: 4.54 minutes ** trade name: YMC Co. Infrared absorption spectrum: v max ^ B ^ : 3501> 3331 ' 2943 ' 2904 ' 1708 ' 1443 ' 1378> 1260 ' 1240, 1162, 1088, 1069, 1038, 962 cm '1 H Nuclear magnetic resonance spectrum: [400 MHz, DMSO-d 6 ] δ j 7.04 (IH, d, J=8Hz), 6.89 (IH, d, J=8Hz), 6.87 (IH, d, J=16Hz), 5.90 (IH, dd, J=7, 16Hz), 5.26 (IH, m), 4.69 (IH, m), 4.19 (IH, ), 2.77 (IH, m), 2.72 (IH, dd, J=5 and 17.5Hz), 2.46 (IH, m), 2.23 (3H, s), 2.23 to 2.10 (2H, m), 2.00 to 1.84 (3H, m), 1.32 (IH, m), 1.01 (3H, d, J=6 Hz) as shown in Fig. 7 13 C Nuclear magnetic resonance spectrum: [100 MHz, DMSO-d 6 ] δ c 170.0(s), 136.5 (s), 135.8 (s), 134.1 (s), 133.1 (d), 132.5 (s), 129.0 (d), 128.8 (d), 127.4 (d), 76.5 (d), 63.4 (d), 61.3 (d), 40.7 (t), 38.6 (t), 38.0 (t), 35.8 (t), 22.2 (d), 21.9 (q), 20.5 (q) as shown in Fig. 8 Nature: neutral substance From the above physical and chemical properties, and further considering that FR 152416 substance can be introduced from FR152415 substance, FR152416 substance is inferred to have the following chemical formula. (3-Hydroxy-5-[2-(5,6,7,8-tetrahydro-8-hydroxy- 2,6-dimethyl-l-naphthyl)vinyl]-5-pentanolide) The tetralin derivatives and salts thereof are potent inhibitors of an enzyme HMG-CoA reductase and are capable of inhibiting cholesterol biosynthesis. Therefore, they can lower blood serum cholesterol levels and blood lipid levels and are useful for the treatment of hyperlipemia and atherosclerosis. Now, in order to show the utility of the tetralin derivatives, the test data on HMG-CoA inhibitiag activity and cholesterol biosynthesis inhibiting activity of the WF14919 substances are shown in the following. Test 1 Inhibition of cholesterol synthesis in HepG2 cells Method: HepG2 cells (HB 8065, a human hepatoma cell line) were obtained from the American Type Culture Collection. Cholesterol synthesis in HepG2 cells was determined according to the method of Brown et al. (J. Biol. Chem. 253, 1121-1128, 1978) with some modifications. HepG2 cells were grown in flasks containing Eagle's modified imnimum essential medium with non-essential amino acids supplemented with pyruvate (1 mM), penicillin G (100 units/ml), streptomycin (100 units/ml), and 10% fetal bovine serum (FBS) in a humidified incubator (5% C0 2 ) at 37 °C. On day 0, the cells (3 x 10 5 cells/well) were seeded into 35 mm 6-well plastic culture dishes (2 ml/well). On day 4, the medium was replaced with 1 ml of fresh medium containing 10% human, lipoprotein-deficient serum instead of FBS and the cells were preincubated for 2 hours at 37 °C with WF14919A substance (or WF14919B substance or FR152415 substance) dissolved in 2 μl of dimethylsulfoxide (DMSO). DMSO (2 μl) was added to the control culture. Then, 1 mM [1- C]acetic acid, sodium salt (37 MBq/mmol, DuPont/NEN Research Products) was added to the medium and incubated at 37 °C for 2 hours. After incubation, the cells were washed with phosphate-buffered saline (PBS), pH 7.4 and then dissolved in 1 ml of aqueous 15% KOH. To each dissolved cell lysate was added 1 ml of 15% KOH in 95% ethanol and then samples were saponified for 1 hour at 75 °C. The non- saponifiable lipids were extracted twice with 2 ml of petroleum ether, evaporated to dryness, resuspended in 100 μl of diethyl ether, and subjected to thin layer chromatography on a silica gel plate with a solvent system containing benzene/ethyl acetate (9:1, v/v). The spot corresponding to authentic cholesterol was scraped into a vial and the radioactivity was counted with a liquid scintillation counter (TRI- CARB 1500, Packard Instrument Co.). (to be continued) Test 2 Inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) activity in detergent solubilized HepG2 cell extracts Method: HMG-CoA reductase activity was determined as described according to the method of Brown et al. (J. Biol. Chem. 249, 789-796, 1974) with some modifications. HepG2 cells (2 x 10 cells) were seeded into 75-cm flasks containing 25 ml of medium described above with 10% FBS and incubated for 4 days. The cells were washed three times with cold PBS and scraped with a policeman. After centrifugation (500 x g, 5 minutes at 4 °C), the resultant cell pellet was frozen and kept at -80 °C until use. Cell extracts were prepared by suspending the pellet in 400 μl of buffer containing 0.1 M potassium phosphate, pH 7.4, 5 mM EDTA, 0.2 M KC1 and 0.25% Emulgen 409P (Kao Chemicals Co.). After incubation for 10 minutes at 37 °C, the suspension was centrifuged at 12,000 x g for 15 minutes at 4 °C. Aliquots of the supernatant (25 μg protein) were incubated at 37 °C for 2 hours in a final volume of 50 μl containing 0.1 M potassium phosphate, pH 7.4, 20 mM glucose 6-phosphate, 2.5 mM NADP, 4 mM dithiothreitol, 0.5 unit of glucose 6-phosphate dehydrogenase and 40 μM DL-3-hydroxy- 3-methyl[3- 14 C]glutaryl-coenzyme A (1040 MBq/mmol, DuPont/NEN Research Products). WF14919A substance (or FR152415 substance) was dissolved in 1 μl of DMSO and added to the assay mixture. The reaction mixture containing DMSO (1 μl) was served as a control. The reaction was terminated by adding 10 μl of 2 N HC1 to the assay mixture. The mixture was further incubated for 15 minutes at 37 °C for lactonization of mevalonic acid. [ C]Mevalonolactone formed was isolated by thin layer chromatography on a silica gel plate with a solvent system consisting of toluene/ethanol (1:1, v/v). The spot corresponding to authentic mevalonolactone was scraped into a vial and counted with a liquid scintillation counter. Results: Substances Inhibition of HMG-CoA reductase WF14919A substance 9.5 x 10 '10 M FR152415 substance 2.3 x 10 '8 M For therapeutic administration, the tetralin derivatives or salts thereof are used in the form of conventional pharmaceutical preparation which contains said substance, as an active ingredient, in admixture with pharmaceutically acceptable carriers such as an organic or inorganic solid or liquid excipient which is suitable for oral, parenteral and external(topical) administration. The pharmaceutical preparations may be in solid form such as tablet, granule, powder, capsule, suppository, solution, suspension, syrup, emulsion, lemonade, lotion, ointment, gel, and the like. If needed, there may be included in the above preparations auxiliary substances, stabilizing agents, wetting agents and other commonly used additives such as lactose, stearic acid, magnesium stearate, terra alba, sucrose, com starch, talc, gelatin, agar, pectin, peanut oil, olive oil, cacao butter, ethylene glycol, tartaric acid, citric acid, fumaric acid, and the like. While the dosage of the tetralin derivatives may vary from and also depend upon the age, conditions of the patient, a kind of diseases, etc. In general, amount between about 0.1 mg and about 1,000 mg or even more, preferably between about 1 mg and about 200 mg per day may be administered to a patient. An average single dose of about 0.1 mg, 1 mg, 10 mg, 20 mg, 30 mg, 50 mg, 100 mg, 200 mg, 250 mg of the tetralin derivatives may be used in treating hypercholesterolaemic and hyperlipoproteinaemic states and associated conditions. The following Examples are given for the purpose of illustrating this invention in more detail. Example 1 (1) Fermentation : An aqueous seed medium (60 ml) containing sucrose 4%, cotton seed flour 2%, dried yeast 1%, peptone 1%, KH 2 P0 4 1%, Tween 80 0.1% and CaC0 3 0.2% was poured into a 250-ml Erlenmeyer flask and sterilized at 120 °C for 30 minutes. A loopful of Agonomycetes strain No. 14919 was inoculated from a slant culture into the flask. The flask was shaken on a rotary shaker (220 rpm, 5.1 cm-throw) at 25 °C for 4 days and then transferred at the rate of 2% to 160 ml of the same sterile seed medium in each of fifteen 500-ml Erlenmeyer flasks. The flasks were shaken on a rotary shaker (220 rpm, 5.1 cm- throw) at 25 °C for 3 days. The resultant seed culture was inoculated to 20 liters of sterile production medium (pH 6.5) consisting of glucose 0.5%, glycerin 0.5%, soluble starch 4%, dried yeast 0.5%, corn steep liquor 0.5%, potato protein 1.5%, Adekanol LG-109 (defoaming agent, Asahi Denka Co., Ltd.) 0.05% and Silicone KM-70 (defoaming agent, Shin-Etsu Chemical Co., Ltd.) 0.05% in a 20-liter jar fermenter. Fermentation was carried out at 25 °C for 4 days under aeration of 20 liters/min and agitation of 300 rpm. The production of active compounds in the fermentation broth was monitored by measuring its enzyme inhibitory activity and HPLC analysis. (2) Isolation : The cultured broth (70 liters) was extracted with 70 liters of acetone by intermittent mixing. The acetone extract was filtered with an aid of diatomaceous earth and 110 liters of water was added and then pH was adjusted to 3.5 with IN HC1. The mixture was passed through a column (10 liters) of Diaion HP-20 (Mitsubishi Chemical Ind. Co., Ltd.). The column was washed with 30% aqueous acetone and eluted with 60% aqueous acetone. This eluate (30 liters) was concentrated in vacuo to an aqueous solution, adjusted to pH 10.0 with 1 N NaOH and treated with an equal volume of ethyl acetate. The ethyl acetate fraction was discarded and the residual aqueous solution was adjusted to pH 2.0 with 1 N HC1 and extracted with an equal volume of ethyl acetate. The ethyl acetate extract was concentrated under reduced pressure to give an oily residue. The oily residue was mixed with twice its weight of acidic silica gel ( silicar CC-4 special, Mallinckrott Inc ), and this mixture was slurried in dichloromethane. After the solvent was evaporated, resultant dry powder was subjected to a column chromatography of the same acidic silica gel (640 ml) which was packed with dichloromethane. The column was developed with dichloromethane (3.2 Hters), mixed solutions of dichloromethane and methanol (100:1, 3.2 liters; 50:1, 3.2 liters; 25:1, 3.2 liters; 10:1, 3.2 liters). The fractions containing objective compounds were collected and concentrated under reduced pressure to give an oily residue. The oily residue was dissolved in 50% aqueous acetonitrile containing 0.1% of phosphoric acid and subjected to a column chromatography (300 ml) of YMC gel (ODS-AM 120-S50, YMC Co., Ltd) packed and developed with the same solvent system. Fractions containing objective compounds were collected and evaporated to give residual aqueous solution. This solution was adjusted to pH 4.0 with 1 N NaOH and extracted with an equal volume of ethyl acetate. The ethyl acetate layer was concentrated under reduced pressure to give an yellowish oil. This oil was dissolved in 30% aqueous acetonitrile and subjected to a column (350 ml) of YMC gel (ODS-AM 120-S50, YMC Co., Ltd.) which was packed with 30% aqueous acetonitrile containing 0.1% of phosphoric acid. After washing of the column with 30% aqueous acetonitrile containing 0.1% of phosphoric acid, WF14919A substance was eluted with 40% aqueous acetonitrile containing 0.1% of phosphoric acid and WF14919B substance was eluted with 50% aqueous acetonitrile containing 0.1% phosphoric acid. The objective compounds were finally isolated with high performance liquid chromatography using an YMC pre-packed column (D-ODS-5, S5 12θA , 30mmID x 250mm, YMC Co., Ltd.) with 50% aqueous acetonitrile containing 0.1% phosphoric acid. After all, 7.9 mg of WF14919A substance and 5.5 mg of WF14919B substance were obtained as colorless crystals. Example 2 (1) Fermentation : An aqueous seed medium (60 ml) containing sucrose 4%, cotton seed flour 2%, dried yeast 1%, peptone 1%, KH 2 P0 4 1%, Tween 80 0.1% and CaC0 3 0.2% was poured into a 250-ml Erlenmeyer flask and sterilized at 120 °C for 30 minutes. A loopful of Agonomycetes strain No. 14919 was inoculated from a slant culture into the flask. The flask was shaken on a rotary shaker (220 rpm, 5.1 cm-throw) at 25 °C for 4 days and then transferred at the rate of 2% to 160 ml of the same sterile seed medium in each of twenty 500-ml Erlenmeyer flasks. The flasks were shaken on a rotary shaker (220 rpm, 5.1 cm-throw) at 25 °C for 3 days. The resultant seed culture was inoculated to 150 liters of sterile production medium consisting of glucose 0.5%, glycerin 0.5%, soluble starch 4%, dried yeast 0.5%, corn steep hquor 0.5%, potato protein 1.5%, Adekanol LG-109 (defoaming agent, Asahi Denka Co.) 0.05% and Silicone KM-70 (defoaming agent, Shin-Etsu Chemical Co.) 0.05% in a 200-liter jar fermenter. Fermentation was carried out at 25 °C for 3 days under aeration of 150 liters/min and agitation of 200 rpm. The production of active compound in the fermentation broth was monitored by measuring its enzyme inhibitory activity and HPLC analysis. (2) Isolation : The cultured broth (155 liters) was extracted with 140 liters of acetone by intermittent mixing. The acetone extract was filtered with an aid of diatomaceous earth and 265 liters of water was added. The mixture was passed through a column (30 liters) of Diaion HP-20 (Mitsubishi Chemical Ind. Co.). The column was washed with 30% aqueous acetone and 40% aqueous acetone, and then eluted with 60% aqueous acetone. To the eluate (43 liters) was added 37 liters of water and passed through a column (4 liters) of Diaion SP-207 (Mitsubishi Chemical Ind. Co.). After washing with 30% aqueous acetonitrile containing 0.1% of phosphoric acid and 35% aqueous acetonitrile containing 0.1% of phosphoric acid, the active fractions were eluted with 40% aqueous acetonitrile containing 0.1% of phosphoric acid and 45% aqueous acetonitrile containing 0.1% of phosphoric acid. To the eluate (22.5 liters) was added an equal volume of water and subjected to a column (4 liters) of YMC gel (ODS-AM 120-S50, YMC Co.). The column was developed with 35% aqueous acetonitrile containing 0.1% of phosphoric acid and 40% aqueous acetonitrile containing 0.1% of phosphoric acid. Active fractions (11 liters) were combined, concentrated in vacuo to an aqueous solution, adjusted to pH 4.0 with 1 N NaOH and extracted with an equal volume of ethyl acetate. The extract was concentrated in vacuo to give WF14919A substance in the form of colorless crystals (2.8 g). Example 3 WF14919A substance (200mg) was suspended in a distilled water (1 liter), and agitated for 45 hours at room temperature. After incubation, this aqueous solution was subjected to a preparative HPLC column of YMC pre-packed ODS-AM (S-5, 12θA, 20mm_D x 250mm, YMC CO., Ltd.), which was packed and developed with 30% aqueous acetonitrile containing 0.1% phosphoric acid. Active fractions (116 ml) were combined, adjusted to pH 5.0 with IN NaOH and concentrated in vacuo to an aqueous solution. The concentrate was adjusted to pH 4.0 and extracted with an equal volume of ethyl acetate. The extract was concentrated in vacuo to give FR152415 substance (82.7 mg) in the form of white powder. Example 4 To the stirred solution of FR 152415 substance (232 mg) in tetrahydrofuran was added 1-hydoxybenzotriazole (47 mg) and 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (150 mg) successively at 0°C. The solution was stirred at room temperature for 5 hours. The solution was evaporated to give the residue, which was dissolved with ethyl acetate. The organic layer was washed with water, aqueous 0.01N hydrogen chloride, aqueous saturated sodium bicarbonate and water, and dried. Evaporation of the solvent followed by recrystallization from hexane-ethyl acetate afforded FR152416 substance (153 mg). BRIEF DESCRIPTION OF DRAWINGS Fig. 1 : H Nuclear magnetic resonance spectrum of WF14919A substance. Fig. 2: 13 C Nuclear magnetic resonance spectrum of WΦ14919A substance. Fig. 3: H Nuclear magnetic resonance spectrum of WF14919B substance. FFiigg.. 44:: - 1 '• 3 C Nuclear magnetic resonance spectrum of WF14919B substannceee. F Fiigg.. 55:: \ H Nuclear magnetic resonance spectrum of FR152415 substanc ee. FFiigg.. 66:: l 13 C Nuclear magnetic resonance spectrum of FR152415 substannceee.. F Fiigg.. 77:: H Nuclear magnetic resonance spectrum of FR152416 substance ~.. FFiigg.. 88:: 1 1 : 3 C Nuclear magnetic resonance spectrum of FR152416 substance

Claims: CLAIMS 1. Tetralin derivatives which have the following general formula (I). in which Ri is hydrogen or acetyl, R is carboxy and R 3 is hydroxy, or R 2 and R 3 may form lactone, or salt thereof. 2. A process for production of WF14919 substances or salts thereof, which comprises culturing a WF14919 substances-producing strain in a nutrient medium and recovering the same. 3. Biological pure culture of Agonomycetes strain No. 14919. 4. A pharmaceutical composition comprising WF14919 substances or salts thereof. 5. Use of a compound of claim 1 for the manufacture of a medicament for therapeutic treatment or prevention of hypercholesterolaemic and hyperlipoproteinaemic states and associated conditions in human beings or animals. 6. A method for treating or preventing hypercholesterolaemic and hyperlipoproteinaemic states and associated conditions which comprises administering tetralin derivatives of Claim 1 or salts thereof to human beings or animals. 7. A compound of claim 1 for use as a medicament.

Inventors: GOTO TOSHIO, HATANAKA HIROSHI, OKUHARA MASAKUNI, SAKAMOTO KAZUTOSHI, SATO TOMOKO, SHIBATA TOSHIHIRO, TAKASE SHIGEHIRO, GOTO, TOSHIO, HATANAKA, HIROSHI, OKUHARA, MASAKUNI, SAKAMOTO, KAZUTOSHI, SATO, TOMOKO, SHIBATA, TOSHIHIRO, TAKASE, SHIGEHIRO
Applicants: FUJISAWA PHARMACEUTICAL CO, FUJISAWA PHARMACEUTICAL CO., LTD.
Application year: 1993
Main classification: A61K31
IPC codes: A61K31, A61P3, C07C59, C07C69, C07D309, C12N1, C12N9, C12P7, C12P17, C12R1