IE914520A1 - Phenoxanes, process for the preparation thereof and¹compositions - Google Patents

Abstract

The invention relates to phenoxane of formula (I), to a process for the production thereof, and to therapeutic compositions containing phenoxane.

Description

Phenoxanes, process for the production thereof and compositions The present invention relates to phenoxane of formula CH3 OCHg In particular, the invention relates to phenoxane which is characterised by the following parameters: Analytical HPLC: column (0 · length) 4 · 250 mm, packed with HD-Sil 18-5-100 (ex Kronwald), detection by UV absorption at 210-250 nm, eluant methanol/water(80:20), flow 1.5 ml/min, tR = 7,9 min; mp 92-94°C.

UV (Methanol): Xmax (ε/lg ε) = 204 (75420/4.877), 229 (53850/4.731), 244 (sh), 252 (sh), 262 (sh), 274 nm (sh).

^-NMR ([D4] methanol, 600,127 MHz): δ = 8.39 (s, IH), 7.30 (m, 2H), 7.19 (m, 3H), 6.33 [s, IH (br.)], 4.10 (s, 3H), 3.15 (t, IH, J = 7.4 Hz), 2.88 (q, 3H, J = 7,4 Hz), 2.72 [t, IH, J = 7.4 Hz (br.)], 1,93 (d, 3H,J= 1.2 Hz), 1.88 (s, 3H), 1.11 ppm (t, 3H,J = 7.4 Hz); 13C-NMR ([D4] methanol, 75.5 MHz): δ = 182.65 s, 166.89 s, 164.46 s, 149.75 s 140.70 d, 139.58 s, 137.80 s, 135.31 s, 130.00 d (2C), 129.22 d (2C), 127.84 d, 127.42 d, 126.37 s, 101.11s, 56.85 q, 38.82 t, 27.85 t, 18.58 t, 17.76 q, 14.00 q, 7.30 q.

(+)FAB Mass spectroscopy (xenon, matrix glycerol), m/z = 380 [M+H]+: EI-MS (70 eV, 160 C, m/z > 90): m/z (%) = 379 (100), 365 (22), 364 (88), 336 (5), 323 (14), 308 (13), 234 (49), 207 (10), 194 (18), 192 (12), 171 (14), 156 (16), 143 (11), 131 (69), 129 (37), 128 (17), 116 (26), 115 (31), 91 (92).

High resolution : C23H25NO4 calcd. 379.1783 found 379.1781 (EI-MS). -2The invention further relates to a process for the production of phenoxane, which comprises cultivating Polyangium spec. DSM 6270 in a suitable nutrient medium and isolating phenoxane.

In particular, the process comprises - cultivating Polyangium spec. DSM 6270 in a medium containing sources of carbon, sources of nitrogen and mineral salts, - separating the biomass from the culture and - extracting it with an organic, dipolar aprotic solvent, - concentrating the extract, - taking up the residue in water and a water-immiscible organic, dipolar aprotic solvent, - concentrating the organic solvent phase, - taking up the concentrate in a lower alkanol and a lower hydrocarbon which is immiscible with said alkanol, - concentrating the alkanolic phase, - taking up the residue in a lower aliphatic ether and, if necessary, filtering the liquid phase over silica gel, and - isolating phenoxane in a manner known per se from the liquid phase.

The phenoxane can be conveniently crystallised from tert-butylmethyl ether/petroleum ether As nutrient medium it is preferred to use an aqueous solution or suspension which contains suitable sources of carbon and nitrogen as well as mineral salts. Particularly suitable sources of carbon and nitrogen are complex organic substances, such as proteins and protein hydrolysates, typically casein and unicellular protein (as from Methylomonas clarae), and also yeast agar. Suitable mineral salts typically include chlorides, carbonates, sulfates and phosphates of alkali metals and, more particularly, alkaline earth metals, for example sodium, potassium and, preferably, calcium and magnesium, and also commercially available trace element mixtures. It is also advantageous to add further growth-promoting substances, especially vitamins, conveniently in the form of commercially available standard vitamin solutions. It is preferred to use the nutrient media employed in the Examples herein.

Cultivation is carried out aerobically, i.e. conveniently in resting surface cultures, or -3prcferably submerged with shaking or stirring while introducing air or oxygen into the shaking flask or fermenter. Cultivation is preferably carried out in the neutral pH range, i.e. at about pH 6-8, preferably at pH 7, and in the temperature range from 25-35°C, preferably at about 30°C. The cultivation time is preferably from 3 to 5 days.

To isolate the phenoxane, the biomass is separated from the culture broth when cultivation is complete, and extracted with an organic, dipolar aprotic solvent. Suitable dipolar aprotic solvents include esters of aliphatic acids, typically lower alkyl esters, such as methyl or ethyl esters, of acetic acid, and ethers, such as tetrahydrofuran and dioxane, and aliphatic ketones, such as acetone. After concentrating the extract, the residue is partitioned between water and a water-immiscible organic, dipolar aprotic solvent, conveniently one of the esters mentioned above. After concentrating the organic phase, the residue is partitioned between a lower (i.e. CrC4) alkanol, such as ethanol or, preferably, methanol, and a lower hydrocarbon, preferably an aliphatic hydrocarbon, which is immiscible with said alkanol, typically hexane or heptane. The alkanolic phase is isolated and concentrated, and the residue is taken up in a lower aliphatic ether, preferably a di(Cj-C4)ether, such as diethyl ether or, preferably, tert-butylmethyl ether and, if necessary, filtered over silica gel. To obtain the pure phenoxane, the ethereal solution is diluted with petroleum ether and cooled. The crystallised phenoxane can be subjected to a further crystallisation from tert-butylmethyl ether.

The phenoxane of the indicated formula can be cis-trans-isomerised by UV radiation with respect to the 1-phenyl-1-propen-1-yl grouping. The invention accordingly also relates to the pure isomers as well as mixtures thereof. The mixtures of isomers can be separated by conventional methods, as by chromatographic methods, into the individual isomers.

The novel compounds have useful, especially pharmacologically useful, properties and can be used for the prevention and treatment of diseases in a mammal, such as a human or an animal. They are, for example, suitable for the treatment of autoimmune diseases such as rheumatic arthritis, multiple sclerosis, insulin dependent diabetes mellitus, psoriasis and systemic lupus erythematosus, as well as allergic diseases such as IgE mediated allergic diseases and asthma. Moreover, they inhibit graft rejection by T cells.

The invention also relates to a pharmaceutical composition which contains phenoxane together with customary carriers and/or diluents. Compositions for enteral, especially oral, as well as parenteral, administration are preferred. Preferably, the compositions contain -4the active ingredient together with a pharmaceutically acceptable carrier.

The pharmaceutical compositions contain from about 5 % to 95 % of the active ingredient, formulations in single dosage unit form containing preferably about 20 % to 90 % of active ingredient, and formulations not in single dosage unit form preferably containing about 5 % to 20 % of active ingredient. Dosage unit forms such as dragdes, tablets or capsules contain from about 0.01 g to 1.0 g of active ingredient.

The pharmaceutical compositions of the present invention are prepared in a manner known per se, for example by conventional mixing, granulating, confectioning, dissolving or lyophilising methods. Pharmaceutical compositions for oral administration may conveniently be obtained by combining the active ingredient with solid carriers, optionally granulating a resulting mixture and processing the mixture or granulate, if desired or necessary after the addition of suitable excipients, to tablets or dragde cores.

Suitable carriers are in particular fillers such as sugars, conveniently lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, typically tricalcium phosphate or calcium biphosphate, and also binders such as starch pastes, typically maize, corn, rice or potato starch, gelatin, tragacanth, methyl cellulose and/or polyvinylpyrrolidone, and/or, if desired, disintegrators, such as the above-mentioned starches, also carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate. Excipients are in particular glidants and lubricants, conveniently silica, talcum, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol. Dragde cores can be provided with suitable non-enteric or enteric coatings, typically using concentrated sugar solutions which may contain gum arabic, talcum, polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide, shellac solutions in suitable organic solvents or mixtures of solvents or, for the preparation of enteric coatings, solutions of suitable cellulose preparations such as acetyl cellulose phthalate or hydroxypropyl methyl cellulose phthalate. Dyes or pigments can be added to the tablets or dragde coatings, for example to identify or indicate different doses of active ingredient.

Further pharmaceutical compositions for oral administration are dry-filled capsules made of gelatin and also soft-sealed capsules consisting of gelatin and a plasticiser such as glycerol or sorbitol. The dry-filled capsules can contain the active ingredient in the form of granules, conveniently in admixture with fillers such as lactose, binders such as -5starches, and/or glidants such as talcum or magnesium stearate, and with or without stabilisers. In soft capsules, the active ingredient is preferably dissolved or suspended in a suitable liquid, typically a fatty oil, paraffin oil or a liquid polyethylene glycol, to which a stabiliser can also be added.

Suitable pharmaceutical compositions for rectal administration are typically suppositories, which consist of a combination of the active ingredient with a suppository base. Examples of suitable suppository bases are natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols and higher alkanols.

Particularly suitable dosage forms for parenteral administration are typically aqueous injection suspensions which may contain substances which increase viscosity, conveniently sodium carboxymethyl cellulose, sorbitol and/or dextran, and optionally stabilisers. In addition, the active ingredient, with or without adjuvants, can also be in lyophilised form and suspended prior to parenteral administration by addition of water and, optionally, the additional substances mentioned.

The invention also relates to a method of treating any of the specified diseases in a mammal, which method comprises administering to said mammal a therapeutically effective amount of phenoxane together with pharmaceutically acceptable carriers. The dosage of active ingredient, i.e. phenoxane, will depend on the disease to be treated and the state of the disease, as well as on the species, age, weight and individual condition of the patient, and also on the mode of application, and will thus be selected by the physician in attendance. For example, autoimmune diseases are usually treated by one daily dose over a few days, i.e. 1 to 4 days. The daily dosage when administered to a mammal, especially a human, of approximately 70 kg body weight, is from about 0.3 to about 30 mg/kg. Administration is made in the form of pharmaceutical compositions as specified above.

The invention is illustrated by the following non-limitative Examples.

Example 1: Production conditions 1.

Production strain: bacterium Polyangium spec., strain Pl VO19, family of the Polyangiaceae, suborder of the Sorangineae, order of the Myxobacterales. -62. Origin of the production strain: isolated in March 1988 from a soil sample taken from Ephesus, Turkey. 3. Description of the production strain: the vegetative cells are cylindrical with flattened ends, 0.6-0.7 x 2-5 pm. On a solid nutrient medium the organism grows well on yeast agar, e.g. VY/2 Agar (bakers’ yeast 0.5 %, based on fresh weight; CaCl2-2H2O 0.1 %, cyanocobalamine 0.5 mg/1, agar 1.5 %; pH 7.2). No growth is observed on pure glucose-mineral salt media. Proteins are rapidly hydrolysed, e.g. casein or unicellular protein. Chitin is not attacked. The strain preferably grows at 30°C in the neutral pH range and aerobically. On suitable nutrient media the colony spreads gradually over the culture dish and penetrates deeply into the agar. Short, broad radial fissures form on the surface. If yeast cells are present in the nutrient medium, they are substantially degraded. After some days fruiting bodies may form. These consist of small grey-brown sporangioles of about 15-30 pm diameter, which are densely packed in flat heaps. Within are found myxospores, which are morphologically very similar to the vegetative cells, but are physiologically drying-resistant resting cells. In liquid media the strain grows in small cell clusters, both in a shaking bottle at 160 rpm (100 ml of medium in a 250 ml Erlenmeyer flask and 500 ml of medium in a 1000 ml Erlenmeyer flask, respectively) as well as in bioreactors (tested up to a scale of 601). A suitable culture medium is Poll medium: Probion PS (unicellular protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; starch 0.3 %; MgSO4- 7H2O 0.1 %; CaCl2-2H2O 0,05 %; 1 ml/1 of standard trace element solution (Schlegel, Allgemeine Mikrobiologie, 56th ed., p. 174, Thieme-Verlag, Stuttgart 1985), and ml/1 of standard vitamin solution (Schlegel, loc.cit. p. 174) pH 7.0. The cultures are kept at 30°C for 3-4 days. The strain Pl VO19 can be preserved, conveniently by freezing at -80°C or with liquid nitrogen. 4. Efficacy of the production strain: in cell extracts, and also in some XAD eluates (see below) of Pl VO19, an anti-HIV activity is detectable in the cell test. Inhibition of reverse transcriptase can be detected in vitro in a HIV-lysate as well as in isolates of M-MuLV virus. The pyrone-containing substance was called phenoxane.

. Accessibility of the strain: the production strain Pl VO19 was deposited on 11th December 1990 with the Deutsche Sammlung von Mikroorganismen (DSM) -7(German Collection of Micro-organisms) in Braunschweig under No. DSM 6270. 6. Detection of phenoxane: The qualitative detection of phenoxane is made by extracting the biomass with acetone. Alternatively, the cultures can also be stirred with XAD 1180 (ex Rohm & Haas, Frankfurt) and the XAD eluted after sieving with methanol and acetone. Aliquots of the concentrated extracts are tested for inhibition of reverse transcriptase from the Moloney Murine Leukemia Virus (M-MuLV). In the thin-layer chromatography test, phenoxane is identified in comparison with isolated pure substance. Working up, isolation and characterisation are effected in accordance with Example 3. 7. Production conditons of phenoxane: In a shaking flask, phenoxane is formed during growth and reaches maximum activity at the end of the logarithmic to early stationary phase after 3-4 days Example 2: Fermentation in a bioreactor 1 bioreaktor (b50) ex Giavanola Frferes, Monthey, Switzerland, with blade stirrer. Medium: Probion PS (unicellular protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; MgSO4-7H2O 0,1 %; CaCl2-2HjO 0,05 %; 1 ml/1 each of standard trace element and vitamin solution (Schlegel, loc.cit.), pH 7.2. 60 1 of medium are inoculated with 5 1 of a culture from well-cultured shaking flasks. The aeration rate is adjusted to 200 Nl/hour, the speed to 200 rpm. The pO2 value, which is 90 % saturation at the start of fermentation, falls continuously to 50 % until the completion of fermentation after 100 hours. The pH increases over the course of the fermentation from 7.0 to 7.5.

In cell extracts, also in some XAD eluates (see above) of Pl VO19, an antibiotic activity against some fungi (e.g. Botrytis cinerea) is detectable. Aliquots of the concentrated extracts are further tested against fungi in the agar diffusion test. In the thin-layer chromatography test, phenoxane is identified in comparison with isolated pure substance.

Example 3: Isolation and characterisation of phenoxane 615 g of moist biomass are obained from a 601 fermentation of the strain Pl VO19 and extracted 4 times with about 150 ml of acetone. The combined extracts are concentrated under vacuum and the residue is partitioned between ethyl acetate and water. In the event - 8of poor phase separation, saturated NaCl solution is added. The organic phase is concentrated (7.7 g) and the residue is partitioned between methanol and heptane. 3.9 g of crude product from the methanol phase are taken up in tert-butylmethyl ether and filtered over 50 ml of silica gel. The crude product (3.2 g) eluted with tert-butylmethyl ether is subsequently filtered in butylmethyl ether over 100 ml of Florisil. The eluate containing phenoxane is stored for crystallisation from tert-butylmethyl ether/petroleum ether. 185 mg of crystalline phenoxane are separated. A further 68 mg are obtained after crystallisation of the combined mother liquors.

Physical and chemical properties of phenoxane Analytical HPLC: Column (0 · length) 4 · 250 mm, packed with HD-Sil 18-5-100 (ex Kronwald), Detection by UV absorption at 210-250 nm, eluant methanol/water(80:20), flow 1.5 ml/min, tR = 7,9 min; mp 92-94°C.

UV (methanol): Xmax (ε/lg ε) = 204 (75420/4.877), 229 (53850/4.731), 244 (sh), 252 (sh), 262 (sh), 274 nm (sh).

^-NMR ([D4] methanol, 600.127 MHz): δ = 8.39 (s, IH), 7.30 (m, 2H), 7.19 (m, 3H), 6.33 [s, IH (br.)], 4.10 (s, 3H), 3.15 (t, IH, J = 7.4 Hz), 2.88 (q, 3H, J = 7.4 Hz), 2.72 [t, IH, J = 7,4 Hz (br.)], 1.93 (d, 3H, J = 1.2 Hz), 1.88 (s, 3H), 1.11 ppm (t, 3H, J = 7.4 Hz); 13C-NMR ([D4] methanol, 75.5 MHz): δ = 182.65 s, 166.89 s, 164.46 s, 149.75 s 140.70 d, 139.58 s, 137.80 s, 135.31 s, 130.00 d (2C), 129.22 d (2C), 127.84 d, 127.42 d, 126.37 s, 101.11 s, 56.85 q, 38.82 t, 27.85 t, 18.58 t, 17.76 q, 14.00 q, 7.30 q.

(+)FAB Mass spectroscopy (xenon, matrix glycerol), m/z = 380 [M+H]+: EI-MS (70 eV, 160 C, m/z > 90): m/z (%) = 379 (100), 365 (22), 364 (88), 336 (5), 323 (14), 308(13), 234 (49), 207 (10), 194(18), 192(12), 171 (14), 156(16), 143 (11), 131 (69), 129 (37), 128 (17), 116 (26), 115 (31),91 (92).

High resolution: C23H25NO4 calcd. 379.1783 found 379.1781 (EI-MS).

Example 4: Biological characterisation of phenoxane Antibiotic activity The antibiotic activity is determined in the agar diffusion test from the diameter of the inhibiting zone, or in the dilution series test from the culture density. Phenoxane inhibits -9the growth of some fungi, e.g. Mucor hiemalis, Botrytis cinerea, Gibberella fujikuroi, Rhizopus arrhizus, Pythium debaryanum. The MIC for Ustilago maydis is 19 ng/ml.

Inhibition of NADH oxidation Phenoxane inhibits the NADH oxidation of submitochondrial particles of bovine hearts. Investigations by differential spectroscopy indicate as site of activity for phenoxane the complex I (NADH: ubiquinone oxidoreductase) of the eucaryotic respiratory chain.

Inhibition of purified protein derivate (PPD) induced human ficoll separated T lymphocyte proliferation Method: Heparinized venous blood (10 U heparin/ml, liquemine, Roche) is diluted 1:2 in PBS and overlayed on half volume of Ficoll-Hypaque (Pharmacia) (eg: 15 ml ficoll + 30 ml blood/PBS in 50 ml conical tube). Tubes are centrifuged at 2000 g for 10 min (or 500 g for 45 min) at room temperature without brake. The interface is gently collected and washed 2-3 times with PBS or BSS and resuspended in RPMI1640 supplemented with either 5 % pooled human serum or with 10 % fetal calf serum.

Viability is determined and cell density is adjusted to 0.7-1 χ 106 cells/ml. After adding 0.5 pg/ml PPD* the cells (150 μΐ per well) are distributed in 96 well flat bottom plates (Costar Nr. 3596). Wells are then supplemented with 50 pi volumes of test compounds diluted in RPMI.

Stimulated cultures are pulsed after 5 days (37°C, 7 % CO2 in air) by adding 1.0 pCi 3H-Thymidine/well in 10 μΐ RPMI (Amersham: spec. act. 5 Ci/ mmol). After an additional 20 hours the cells are harvested with a 96-well harvester (LKB) on glass fibre filters (LKB). The filters are dried and the retained radioactivity is counted in Optiscint (LKB) in a Betaplate (LKB) scintillation counter (LKB). The results are expressed as the mean cpm of triplicate wells, in the presence and absence of drugs. The effect of drugs is reported as percentage inhibition as follows: Exp. cpm % inhibition = 100- - x 100 control cpm Exp. cpm = T lym. + PPD + compound control cpm = T lym. + PPD (100 % incorp.) - 10The inhibition curve is plotted and the IC50 is reported in micromolar. In this assay phenoxane had an IC50 of 0.018 μΜ.

Inhibition of antigen (ovalbumin) induced mouse T cell proliferation Method (as described in Eur. J. Immunol. 8; 112,1978): Mice (Balb/c or any other) are injected at the base of the tail s.c. with 100 pg of antigen in 50 μΐ (e.g. ovalbumin, Miles, fraction V, 2 mg/ml in PBS, mixed 1:1 with CFA, Difco). Seven to eight days later the draining lymph nodes are collected, pooled and a single cell suspension in cold BSS (balanced salt solution) is made using a homogenisor (FORTUNA, Nr. 1.2440.33, Huber + Co. Reinach).

The cells (2 xl07-4 χ 107 per mouse) are washed 3 times in BSS and the viable cell number (using FDA) is ajusted to 2.2 χ 106 cells/ml in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) containing 4 mM L-glutamin, Penicillin (100 pg/ml) Streptomycin (100 pg/ml), 2-mercaptoethanol (50 μΜ, Gibco) and 5 % heat inactivated horse serum (Gibco).

Ex vivo assay: 20 μΙ/well ovalbumin (in PBS 300 pg/ml stock solution), 20 μΙ/well test compounds (in DMEM) and 160 μΙ/well cell suspension are pipetted into 96 well flat bottom microtiter plates (Nunclon, Delta 167008). The final concentration of a) antigen: pg/ml; b) of compounds 25 to 0.09 μΜ; c) of cells 3.6 x 105/well. Cyclosporin A (CSA) is included in each assay as reference compound.

Cultures are pulsed after 48h incubation (37°C, 7 % CO2 in air) by adding 1.0 pCi 3H-Thymidine/well (Amersham: spec. act. 5 Ci/mmol) in 10 μΐ DMEM. After additional 20 hours the cells are harvested with a 96-well harvester (LKB) on to glass fibre filters (LKB). The filters are dried and the retained radioactivity is counted in Optiscint (LKB) in a Betaplate (LKB) scintillation counter (LKB). The results are expressed as the mean cpm of triplicate wells, in the presence and absence of drugs. The effect of drugs is reported as percentage inhibition as follows: Exp. cpm % inhibition = 100- - x 100 control cpm - 11 Exp. cpm = T lym. + ovalbumin + compound control cpm = T lym. + ovalbumin (100 % incorp.) The inhibition curve is plotted and the IC50 is reported in pmol/liter. Phenoxane exhibits a better potency (IC5o:0.002 μΜ) than CSA (IC5O:0.03 μΜ) in inhibiting the activation/proliferation of antigen specific T cells. This effect of the drug was not due to toxicity because, when it was added to the culture not at Oh but at 48h (after proliferation occured), it did not effect the viability of T cells at 10 μΜ levels. Also the compound was not toxic to Swiss 3T3 fibroblasts at 10 μΜ levels.

Example 5: Pharmaceutical composition for oral administration Capsules containing 25 mg of active ingredient, i.e. phenoxane, are prepared as follows: Composition (for 1000 capsules): active ingredient 25 g talcum 3.6 g corn starch 2.4 g magnesium stearate 1.6 g lactose 0.4 g g The powdered substances are passed through a 0.6 mm sieve and mixed. Portions of 33 mg of the mixture are filled into gelatin capsules in a capsule filling machine.

Claims (12)

Claims:

1. Phenoxane of formula CH 3 och 3

2. Phenoxane characterised by the following parameters: Analytical HPLC: column (0 · length) 4 · 250 mm, packed with HD-Sil 18-5-100 (ex Kronwald), detection by UV absorption at 210-250 nm, eluant methanol/water(80:20), flow 1.5 ml/min, t R = 7,9 min; mp 92-94°C. UV (Methanol): X max (ε/lg ε) = 204 (75420/4,877), 229 (53850/4,731), 244 (sh), 252 (sh), 262 (sh), 274 nm (sh). ’H-NMR ([D 4 ] methanol, 600,127 MHz): δ = 8.39 (s, 1H), 7.30 (m, 2H), 7.19 (m, 3H), 6.33 [s, 1H (br.)], 4.10 (s, 3H), 3.15 (t, 1H, J = 7.4 Hz), 2.88 (q, 3H, J = 7,4 Hz), 2.72 [t, 1H, J = 7.4 Hz (br.)], 1,93 (d, 3H, J = 1.2 Hz), 1.88 (s, 3H), 1.11 ppm (t, 3H, J = 7.4 Hz); 13 C-NMR ([D 4 ] methanol, 75.5 MHz): δ = 182.65 s, 166.89 s, 164.46 s, 149.75 s 140.70 d, 139.58 s, 137.80 s, 135.31 s, 130.00 d (2C), 129.22 d (2C), 127.84 d, 127.42 d, 126.37 s, 101.11 s, 56.85 q, 38.82 t, 27.85 t, 18.58 t, 17.76 q, 14.00 q, 7.30 q. (+)FAB Mass spectroscopy (xenon, matrix glycerin), m/z = 380 [M+H] + : EI-MS (70 eV, 160 C, m/z > 90): m/z (%) = 379 (100), 365 (22), 364 (88), 336 (5), 323 (14), 308 (13), 234 (49), 207 (10), 194 (18), 192 (12), 171 (14), 156 (16), 143 (11), 131 (69), 129 (37), 128 (17), 116(26), 115(31),91 (92). High resolution : C 23 H 25 NO 4 calcd. 379.1783 found 379.1781 (EI-MS).

3. A pharmaceutical composition comprising phenoxane as claimed in any of claims 1 or 2, together with pharmaceutically acceptable carriers and/or diluents. - 13

4. A compound as claimed in any of claims 1 or 2 for use in a method for the therapeutic or prophylactic treatment of the human or animal body.

5. Use of a compound as claimed in any of claims 1 or 2 for the production of a pharmaceutical composition.

6. A process for the production of phenoxane as claimed in any of claims 1 or 2, which comprises cultivating Polyangium spec. DSM 6270 in a suitable nuitrient medium and isolating phenoxane.

7. A process according to claim 6, which comprises - cultivating Polyangium spec. DSM 6270 in a medium containing sources of carbon, sources of nitrogen and mineral salts, - separating the biomass from the culture and - extracting it with an organic, dipolar aprotic solvent, - concentrating the extract, - taking up the residue in water and a water-immiscible organic, dipolar aprotic solvent, - concentrating the organic solvent phase, - taking up the concentrate in a lower alkanol and a lower hydrocarbon which is immiscible with said alkanol, - concentrating the alkanolic phase, - taking up the residue in a lower aliphatic ether and, if necessary, filtering the liquid phase over silica gel, and - isolating phenoxane in a manner known per se from the liquid phase.

8. A process according to claim 7, which comprises crystallising phenoxane from tert-butylmethyl ether/petroleum ether. FD 4.4/UL/hdr* -149. A pharmaceutical composition according to claim 3, substantially as hereinbefore described.

9. 10. Use according to claim 5, substantially as hereinbefore described .

10. 11. A process for the production of phenoxane as claimed in claim 1 or 2, substantially as hereinbefore described and exemplified.

11.

12. Phenoxane as claimed in claim 1 or 2, whenever produced by a process claimed in a preceding claim.