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WO1992011257A1 - Phenoxanes, leur procede de production, et compositions contenant lesdites phenoxanes - Google Patents

Phenoxanes, leur procede de production, et compositions contenant lesdites phenoxanes Download PDF

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Publication number
WO1992011257A1
WO1992011257A1 PCT/EP1991/002440 EP9102440W WO9211257A1 WO 1992011257 A1 WO1992011257 A1 WO 1992011257A1 EP 9102440 W EP9102440 W EP 9102440W WO 9211257 A1 WO9211257 A1 WO 9211257A1
Authority
WO
WIPO (PCT)
Prior art keywords
phenoxane
methanol
concentrating
production
organic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP1991/002440
Other languages
English (en)
Inventor
Gerhard Höfle
Norbert Bedorf
Edgar Forche
Klaus Gerth
Herbert Irschik
Rolf Jansen
Brigitte Kunze
Hans Reichenbach
Florenz Sasse
Heinrich Steinmetz
Wolfram Trowitzsch-Kienast
Sefik Alkan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novartis AG
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Original Assignee
Ciba Geigy AG
Helmholtz Zentrum fuer Infektionsforschung HZI GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ciba Geigy AG, Helmholtz Zentrum fuer Infektionsforschung HZI GmbH filed Critical Ciba Geigy AG
Publication of WO1992011257A1 publication Critical patent/WO1992011257A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to phenoxane of formula
  • the invention relates to phenoxane which is characterised by the following parameters:
  • UV (Methanol): ⁇ ( ⁇ /lg ⁇ ) 204 (75420/4.877), 229 (53850/4.731), 244 (sh), 252 (sh), 262 (sh), 274 nm (sh).
  • the 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.
  • the process comprises
  • the phenoxane can be conveniently crystallised from tert-butylmethyl ether/petroleum ether
  • aqueous solution or suspension which contains suitable sources of carbon and nitrogen as well as mineral salts.
  • 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 preferably 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.
  • 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.
  • C r C 4 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(C j -C 4 )ether, such as diethyl ether or, preferably, tert-butylmethyl ether and, if necessary, filtered over silica gel.
  • 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-l-propen-l-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.
  • 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.
  • the compositions contain the active ingredient together with a pharmaceutically acceptable carrier.
  • 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 drag ⁇ es, tablets or capsules contain from about 0.01 g to 1.0 g of active ingredient
  • 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 drag ⁇ e 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.
  • 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 star
  • 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.
  • Drag ⁇ e 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 drag ⁇ e coatings, for example to identify or indicate different doses of active ingredient
  • 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 starches, and/or glidants such as talcum or magnesium stearate, and with or without stabilisers.
  • 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.
  • 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.
  • 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.
  • 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.
  • Production strain bacterium Polyangium spec, strain PI V019, family of the Polyangiaceae, suborder of the Sorangineae. order of the Mvxobacterales. 2. Origin of the production strain: isolated in March 1988 from a soil sample taken from Ephesus, Turkey.
  • the vegetative cells are cylindrical with flattened ends, 0.6-0.7 x 2 ⁇ 5 ⁇ m.
  • yeast agar e.g. VY/2 Agar (bakers' yeast 0.5 %, based on fresh weight; CaCl 2 -2H 2 00.1 %. 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.
  • 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 ⁇ m diameter, which are densely packed in flat heaps. Within are found myxospores, which are morphologically very similar to the vegetative cells, but are physio ⁇ logically drying-resistant resting cells.
  • a suitable culture medium is Poll medium: Probion PS (unicellular protein from Methylomonas clarae; Hoechst, Frankfurt) 0.4 %; starch 0.3 %; MgS0 4 - 7H 2 0
  • the production strain PI V019 was deposited on 11th December 1990 with the Deutsche Sammlung von Mikroorganismen (DSM) (German Collection of Micro-organisms) in Braunschweig under No. DSM 6270.
  • DSM Deutsche Sammlung von Mikroorganismen
  • 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 character ⁇ isation are effected in accordance with Example 3.
  • Example 2 Fermentation in a bioreactor
  • UV (methanol): ⁇ ( ⁇ g ⁇ ) 204 (75420/4.877), 229 (53850/4.731), 244 (sh), 252 (sh), 262 (sh), 274 nm (sh).
  • 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 the 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.
  • 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.
  • NADH ubiquinone oxidoreductase
  • 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 RPMI 1640 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 x 10 6 cells/ml. After adding 0.5 ⁇ g/ml PPD * the cells (150 ⁇ l per well) are distributed in 96 well flat bottom plates (Costar Nr. 3596). Wells are then supplemented with 50 ⁇ l volumes of test compounds diluted in RPMI.
  • Stimulated cultures are pulsed after 5 days (37°C, 7 % C0 2 in air) by adding 1.0 ⁇ Ci 3 H-Thymidine/well in 10 ⁇ l 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:
  • mice (Balb/c or any other) are injected at the base of the tail s.c. with 100 ⁇ g of antigen in
  • the cells (2 xl0 7 -4 x 10 7 per mouse) are washed 3 times in BSS and the viable cell number (using FDA) is ajusted to 2.2 x 10 6 cells/ml in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 4 mM L-glutamin, Penicillin (100 ⁇ g/ml) Streptomycin (100 ⁇ g/ml), 2-mercaptoethanol (50 ⁇ M, Gibco) and 5 % heat inactivated horse serum (Gibco).
  • DMEM Dulbecco's Modified Eagle's Medium
  • Penicillin 100 ⁇ g/ml
  • Streptomycin 100 ⁇ g/ml
  • 2-mercaptoethanol 50 ⁇ M
  • 5 % heat inactivated horse serum Gibco
  • Ex vivo assay 20 ⁇ l/well ovalbumin (in PBS 300 ⁇ g/ml stock solution), 20 ⁇ l/well test compounds (in DMEM) and 160 ⁇ l well cell suspension are pipetted into 96 well flat bottom microtiter plates (Nunclon, Delta 167008). The final concentration of a) antigen: 30 ⁇ g/ml; b) of compounds 25 to 0.09 ⁇ M; c) of cells 3.6 x 10 5 /well. Cyclosporin A (CSA) is included in each assay as reference compound.
  • CSA Cyclosporin A
  • Cultures are pulsed after 48h incubation (37°C, 7 % CO 2 in air) by adding 1.0 ⁇ Ci 3 H-Thymidine/welI (Amersham: spec. act. 5 Ci/mmol) in 10 ⁇ l 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:
  • the inhibition curve is plotted and the IC 50 is reported in ⁇ mol/liter.
  • Phenoxane exhibits a better potency (IC 50 :0.002 ⁇ M) than CSA (IC 50 :0.03 ⁇ M) 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 ⁇ M levels. Also the compound was not toxic to Swiss 3T3 fibroblasts at 10 ⁇ M levels.
  • Example 5 Pharmaceutical composition for oral administration
  • Capsules containing 25 mg of active ingredient, i.e. phenoxane, are prepared as follows:
  • 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.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Saccharide Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

L'invention concerne une phénoxane de formule (I), son procédé de production, et des compositions thérapeutiques contenant ladite phénoxane.
PCT/EP1991/002440 1990-12-21 1991-12-18 Phenoxanes, leur procede de production, et compositions contenant lesdites phenoxanes Ceased WO1992011257A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE4041282A DE4041282A1 (de) 1990-12-21 1990-12-21 Phenoxane, herstellungsverfahren und mittel
DEP4041282.2 1990-12-21

Publications (1)

Publication Number Publication Date
WO1992011257A1 true WO1992011257A1 (fr) 1992-07-09

Family

ID=6421096

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1991/002440 Ceased WO1992011257A1 (fr) 1990-12-21 1991-12-18 Phenoxanes, leur procede de production, et compositions contenant lesdites phenoxanes

Country Status (8)

Country Link
AU (1) AU9068891A (fr)
DE (1) DE4041282A1 (fr)
IE (1) IE914520A1 (fr)
IL (1) IL100453A0 (fr)
MX (1) MX9102729A (fr)
PT (1) PT99908A (fr)
WO (1) WO1992011257A1 (fr)
ZA (1) ZA9110053B (fr)

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Liebigs Annalen der Chemie, no. 7, VCH Verlagsgesellschaft mbH, Weinheim (DE) R. Jansen et al.: "Phenoxan: a novel inhibitor of HIV-1 infection in cell cultures from polyangium sp., strain pl VO19 (Myxobacteria), pages 707,708, see the whole article *
Tetrahedron (Incl. Tetrahedron Reports), vol. 14, 1961, Pergamon Press Ltd, Oxford (GB) Y. Hirata et al.: "The structure of aureothin, a nitro compound obtained rom streptomyces thioluteus", pages 252-274, see pages 252-262 *

Also Published As

Publication number Publication date
IL100453A0 (en) 1992-09-06
DE4041282A1 (de) 1992-07-02
PT99908A (pt) 1992-12-31
MX9102729A (es) 1992-06-01
AU9068891A (en) 1992-07-22
ZA9110053B (en) 1992-08-26
IE914520A1 (en) 1992-07-01

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