WO1993018173A2 - Cd4 binding agents and inhibitors of collagenase and protein kinase c - Google Patents

Cd4 binding agents and inhibitors of collagenase and protein kinase c

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Publication number
WO1993018173A2
WO1993018173A2 PCT/GB1993/000511 GB9300511W WO9318173A2 WO 1993018173 A2 WO1993018173 A2 WO 1993018173A2 GB 9300511 W GB9300511 W GB 9300511W WO 9318173 A2 WO9318173 A2 WO 9318173A2
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WO
WIPO (PCT)
Prior art keywords
compound
molecular formula
pharmaceutically
ethers
inhibitor
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PCT/GB1993/000511
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French (fr)
Other versions
WO1993018173A3 (en
Inventor
Peter John Hylands
Stephen Keith Wrigley
Glenda Louise Chandler
Mark Anthony David Collins
Frances Fox
Michael Moore
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Xenova Limited
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Priority claimed from GB929205311A external-priority patent/GB9205311D0/en
Priority claimed from GB929207166A external-priority patent/GB9207166D0/en
Priority claimed from GB929207290A external-priority patent/GB9207290D0/en
Priority claimed from GB929208189A external-priority patent/GB9208189D0/en
Application filed by Xenova Limited filed Critical Xenova Limited
Priority to GB9418110A priority Critical patent/GB2280439B/en
Publication of WO1993018173A2 publication Critical patent/WO1993018173A2/en
Publication of WO1993018173A3 publication Critical patent/WO1993018173A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
    • C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/80—Penicillium

Definitions

  • the present invention relates to compounds useful as CD4 binding agents, as inhibitors of collagenase and in certain cases also as inhibitors of the enzyme Protein Kinase C (PKC).
  • PKC Protein Kinase C
  • the invention also relates to the
  • CD4 is a cell surface glycoprotein expressed on those T lymphocytes which recognise antigen bound to class II MHC molecules. Inhibition of class II MHC-CD4 interactions will block antibody responses, mixed lymphocyte reactions and other immune responses involving CD4 + T lymphocytes. These responses are important in pathological conditions including autoimmunity, organ graft rejection, allergy and graft versus host disease. CD4 is also the cellular receptor for human immunodeficiency virus (HIV).
  • HIV human immunodeficiency virus
  • Collagenase is a member of the connective tissue metalloproteinase family of enzymes. Collagenases control the turnover, remodelling and degradation of collagen in tissue. Collagen breakdown occurs in several pathological conditions including arthritis, tumour metastasis,
  • T cell antigen receptor Interaction of the T cell antigen receptor with its ligand causes increased turnover of phosphatidylinositol lipids and the generation of diacylglycerol (DAG) which activates PKC.
  • DAG diacylglycerol
  • the present invention provides a compound selected from:
  • UV ultraviolet
  • ir infrared
  • UV ultraviolet
  • ir infrared
  • nmr nuclear magnetic resonance
  • MS mass
  • citromycetin and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof; for use in a method of treatment of the human or animal body by therapy.
  • the compounds (a) and (b) are also inhibitors of PKC.
  • the invention therefore also provides use of a compound as defined above in the manufacture of a medicament for use as a CD4 binding agent, a collagenase inhibitor or, in the case of a compound (a) or (b), a PKC inhibitor.
  • the present compounds have been isolated from a microorganism which we have designated strain X8063 and which has been identified as a strain of the fungus
  • Penicillium glabrum on the basis of the following
  • Fungal strain X8063 was incubated for 7 days at each of 5oC, 25oC and 30oC in the following growth media
  • Czapek's agar with yeast extract (CzYa; composition per litre of distilled water: sucrose, 30g;
  • yeast extract 5g; agar 20g; KH 2 PO 4 , lg; NaNO 3 , 0.3g;
  • Bacto-peptone lg; agar, 20g
  • glycerol nitrate agar G25N; composition per 750 ml of distilled water: yeast extract,
  • Verticils borne terminally (never subterminal), monoverticillate, composed of 10 to 16 appressed phialides. Phialides ampulliform (10) -12-13 ⁇ 4-5 ⁇ m. Conidia spheroidal, 3.5-4.5 ⁇ m diameter, with faintly warted/rugulose walls. Conidia borne in long well defined columns.
  • CzYa at 25o Colonies, 31 to 40 mm diameter, plane with radial sulcation. Texture velutinous, low. Margin immersed, entire and extending approximately 1 mm.
  • Mycelium white to light beige (366) with a plane, non-sporulating, 2-4 mm periphery. Central region white, but showing in places a slight greenish tint, conidiogenesis poor. Exudate and sclerotia absent. Pale clear yellow diffusing pigment produced. Diffusion zone approximately 50mm in diameter. Reverse concolourous with surface mycelium, a yellowish pale cream (352).
  • MEA at 25o Colonies, 30 to 31 mm diameter, plane, lacking radial sulcation. Texture velutinous, low. Margin not immersed, entire. Aerial mycelium very sparse, colours due to maturing spore mass; outer region (5 mm margin) approximating pale cream (352) to light straw (384) but slightly more yellowish, central region approximating light grey (631) to aircraft grey (693) but slightly more bluish. Conidiogenesis moderate. Exudate and sclerotia absent.
  • Diffusing pigment produced in a zone approximately 45 mm in diameter, coloured a clear bright pale yellow. Reverse darkening towards the centre ranging from pale cream (352) to dark earth (450).
  • G25N at 25o Colonies, 12 to 13 mm diameter, plane with radial sulcation. Texture velutinous, low. Margin immersed and entire. Outer region (1-2 mm) colourless to white. Central region white to very pale yellow.
  • Conidiogenesis very poor and lacking distinct spore mass colour.
  • Exudate and sclerotia absent.
  • Reverse ranging from white to pale bright yellow (slightly paler than canary yellow, 309).
  • CzYa at 5o Slight growth, colonies 3 to 5 mm diameter.
  • MEA at 5o Micro-colonies to slight growth, 3 to 4 mm diameter.
  • G25N at 5o No growth.
  • strain X8063 in the genus Penicillium. From the other microscopic and gross morphological features of strain X8063 described above, and following the taxonomic scheme in Pitt, J.I., 1979 - The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromvces: London, Academic Press, X8063 may be best classified as a strain of Penicillium glabrum (Wehmer) Westling (a widely used synonym is P. frequentans Westling).
  • strain X8063 was isolated from a soil sample collected from Femes, Lanzarote in 1987 and was deposited under the Budapest Treaty at the Commonwealth Mycological Institute, Kew, Richmond, Surrey, UK on 13 December 1989 under accession number CMI 336456.
  • the present invention also embraces the use of mutants of strain X8063 which produce the present compounds.
  • mutants which are obtained by natural selection or those produced by mutating agents including ionising radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments can also be used.
  • the present invention further provides a process for the preparation of a compound as defined above, which process comprises (i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain X8063 (CMI 336456) or a mutant thereof which produces the said
  • the present compounds are typically produced during the aerobic fermentation of an aqueous nutrient medium under conditions described hereinafter, with a producing strain of Penicillium glabrum. X8063, or a producing mutant strain of X8063.
  • Aqueous media such as those used for the production of many antibiotic substances are suitable.
  • Such nutrient media contain sources of carbon and nitrogen assimilable by the microorganism. If desired inorganic salts may be added, generally at low levels. In addition, the fermentation media may contain traces of metals
  • Sources of carbon nitrogen and minerals may be provided by either simple or complex nutrients.
  • Sources of carbon will generally include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils.
  • Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium.
  • Sources of nitrogen will generally include soya bean meal, corn steep liquors, distillers' solubles, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino. acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides may also be used. Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation medium.
  • Nutrient mineral salts which may be incorporated into the culture medium include the generally used salts capable of yielding sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions.
  • An antifoam may be present to control excessive foaming and added at intervals as required.
  • the fermentation using Penicillium glabrum can be conducted at temperatures ranging from 20oC to 30oC, preferably 24-28oC. For optimal results, it is most convenient to conduct these fermentations at a temperature in the range 24-26oC.
  • the starting pH of the nutrient medium suitable for producing the compounds can vary from 5.0 to 8.5 with a preferred range of from 5.5 to 7.5.
  • Penicillium glabrum loosely stoppering the flask with cotton wool, and permitting the fermentation to proceed in a constant room temperature of about 25oC on a rotary shaker at from 95 to 300 rpm for 2 to 10 days.
  • fermentation may also be conducted in static culture on liquid or semi-solid medium.
  • the nutrient medium is made up in the tank after
  • the fermentation is allowed to continue for from 1 to 8 days while agitating and/or aerating the nutrient medium at a temperature in the range 24oC to 28oC.
  • the degree of aeration is dependent upon several factors such as the size of the fermenter and agitation speed. Generally the larger scale fermentations are agitated at about 95 to 500 rpm and aerations of about 0.5 to 1.5 VVM (volumes of air per volume of medium per minute) are used.
  • the present compounds are found primarily in the liquor of the fermentation of strain X8063.
  • the separation of the present compounds from the whole fermentation broth and their recovery is carried out by solvent extraction followed by application of chromatographic fractionations with various chromatographic techniques and solvent
  • the present compounds are acidic and are soluble in neutral and alkaline aqueous solvents. In their unionised forms the compounds are soluble in polar organic solvents such as dimethyl sulphoxide and, except in the case of the compound having the molecular formula C 27 H 16 O 12 , methanol. When impure, the uncharged form of the compounds are soluble in a wider range of organic solvents such as dichloromethane, ethyl acetate and, in the case of the compound having the molecular formula C 27 H 16 O 12 , methanol.
  • polar organic solvents such as dimethyl sulphoxide and, except in the case of the compound having the molecular formula C 27 H 16 O 12 , methanol.
  • organic solvents such as dichloromethane, ethyl acetate and, in the case of the compound having the molecular formula C 27 H 16 O 12 , methanol.
  • the whole fermentation broth is acidified to pH 3 and combined with a water-immiscible organic solvent such as ethyl acetate.
  • a water-immiscible organic solvent such as ethyl acetate.
  • the solvent removes the desired compounds, but also other substances.
  • the desired compounds are acids they can be purified further by back-extracting the water-immiscible solvent extract with a buffered alkaline aqueous solution of a salt such as ammonium acetate or sodium phosphate.
  • a salt such as ammonium acetate or sodium phosphate.
  • the present compounds are ionised and are extracted into the aqueous layer.
  • the present compounds and other acidic compounds can then by recovered from the aqueous layer by acidifying it to pH 3 and re-extracting it with fresh water-immiscible organic solvent. This solvent extract is then concentrated under reduced pressure.
  • An alternative method of recovery of the desired compounds from the liquor, particularly useful for large scale fermentations is to acidify the liquor to pH 3 and pass the acidified liquor through a chromatography column filled with a porous hydrophobic resin such as Diaion HP20 (Mitsubishi Kasei Corp). The resin is then washed with an acidic aqueous solution such as an acetic acid solution and then eluted with a water-miscible organic solvent such as methanol. The methanol eluate is concentrated under reduced pressure before diluting it with ethyl acetate or another water-immiscible organic solvent and purifying the organic solvent solution by back-extraction with an
  • the residue produced by either of these two extraction methods is then further purified by reverse phase high-pressure liquid chromatography (hplc) under acidic conditions on a column containing an adsorbent such as octadecyl silica.
  • the column retains the desired compounds and other acidic impurities. It is eluted with a mixture of an acidic aqueous solution, such as acetic acid solution, and a water-miscible organic solvent, such as tetrahydrofuran (THF). As the proportion of the organic solvent in the mixture is increased the desired compounds are eluted.
  • the eluate containing a desired compound is extracted with a water-immiscible organic solvent such as ethyl acetate and the desired compound recovered by evaporation of the extract.
  • a water-immiscible organic solvent such as ethyl acetate
  • the present compounds are further purified by reverse phase hplc on an octadecyl silica column eluted isocratically with mixtures of the same solvents used in the last hplc purification stage and the desired compound recovered from the eluate by
  • Anhydrofulvic acid is in fact a known compound which has been derived chemically from the Penicillium sp. metabolite fulvic acid (Dean, F.M. Eade, R.A., Moubasher, R. and Robertson A. (1957) J. Chem. Soc. 3497-3510) and has been prepared as an intermediate in the synthesis of fulvic acid (Yamauchi, M., Katayama, S., Todoroki, T. and
  • Anhydrofulvic acid has the following formula 1:
  • Citromycetin is a known metabolite of Penicillium frequentans (Westling) (Demetriadou, A.K., Laue, E.D.
  • Citromycetin has the following formula 2 :
  • the present compounds may be converted into a pharmaceutically or veterinarily acceptable salt, ester or ether thereof.
  • Suitable salts include salts with alkali metals such as sodium and potassium, and ammonium salts.
  • Suitable ethers are branched or unbranched, saturated or unsaturated, substituted or unsubstituted C 1 to C 6 aliphatic ethers, typically C 1 to C 6 alkyl ethers.
  • Preferred alkyl ethers are C 1 to C 4 alkyl ethers such as the methyl and ethyl ethers. Typically the ethers are peralkyl ethers.
  • Such ethers may be prepared by treatment of the xanthone derivative with a diazoalkane such as diazomethane in a suitable inert solvent or by treatment with an appropriate alkyl halide, sulphonate ester or dialkyl sulphate in the presence of base.
  • Suitable bases include alkali and alkaline earth metal hydroxides,
  • Suitable esters include esters formed with branched or unbranched, saturated or unsaturated, substituted or unsubstituted C 1 -C 6 alcohols. Methyl, ethyl and vinyl esters are typical examples. Such esters may be prepared in the same way as the ethers above or by treatment of the xanthone derivative with the appropriate alcohol in the presence of a suitable acid catalyst or activating agent.
  • the present compounds and their salts, esters and ethers have utility as CD4 binding agents. They can block the physiological function of CD4 by inhibiting its interaction with MHC class II molecules. They also have utility as collagenase inhibitors and, in the case of the compound having the molecular formula C 27 H 16 O 12 and the compound having the molecular formula C 14 H 10 O 6 as PKC
  • a human or animal eg mammal, can therefore be treated by a method comprising administration of a
  • a CD4 binding agent can selectively inhibit MHC class II-restricted responses.
  • the present compounds and their salts, esters and ethers can therefore be used as immunosuppressants, especially in the treatment of
  • autoimmune disease such as rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, multiple sclerosis and primary biliary cirrhosis, graft versus host (GVH) disease and organ rejection. They can also be used in the treatment of allergic diseases such as asthma and
  • a CD4 binding agent may be effective in preventing the entry of HIV into cells and hence of therapeutic value as an anti-HIV agent.
  • the present compounds are active in an ELISA assay based on the interaction between soluble recombinant CD4 (sCD4 (V1 + V2); from E. coli) and the monoclonal antibody Leu3a (Becton-Dickinson, Oxford, England). Leu3a binds to an epitope on the VI domain of CD4 as described by
  • Collagenase plays a wide-ranging role in the pathology of disease.
  • a present compound or a salt, ester or ether thereof can be employed to alleviate conditions including rheumatoid arthritis and osteoarthritis, tumour metastasis, in periodontal disease, corneal ulceration, excessive skin or bone collagen degradation and other disorders.
  • An assay for collagenase inhibiting activity is based on the degradation of rat type I collagen gels by human collagenase as described by Harris and Vater (1982)
  • the present compounds inhibit the action of human collagenase on rat type I collagen when tested in this assay.
  • the compound having the molecular formula C 27 H 16 O 12 caused 63% inhibition of the collagenase activity at a concentration of 25 ⁇ M.
  • the compound having the molecular formula C 14 H 10 O 6 inhibits collagenase when present in
  • the compound having the molecular formula C 27 H 16 O 12 , the compound having the molecular formula C 14 H 10 O 6 and their salts, esters and ethers can be employed to alleviate a broad range of cancerous conditions. They therefore have utility as antitumour agents. They can be used in a method of treating tumours such as breast, thyroid, colon, lung, skin and brain tumours, in particular tumours in which PKC is implicated as a causative agent. The compounds can be used to improve the condition of a patient having such a tumour.
  • the compound having the molecular formula C 27 H 16 O 12 , the compound having the molecular formula C 14 H 10 O 6 and their salts, esters and ethers can also be employed to alleviate inflammatory conditions and therefore have utility as anti- inflammatory agents.
  • the compounds can be used in a method of treating inflammatory conditions, for example asthma.
  • An inhibitor of PKC also inhibits T cell activation and can be used in the therapy of autoimmune diseases.
  • the compound having the molecular formula C 27 H 16 O 12 , the compound having the molecular formula C 14 H 10 O 6 and their salts, esters and ethers therefore have utility in the therapy of
  • autoimmune disease and can be used in a method of treating autoimmune disease such as systemic lupus erythematosus, myasthenia gravis and diabetes.
  • a PKC inhibitor also acts as an immunosuppressive agent.
  • the compound having the molecular formula C 27 H 16 O 12 , the compound having the molecular formula C 14 H 10 O 6 and their salts, esters and ethers therefore have utility as
  • immunosuppressive agents and can be used in a method of suppressing an immune response in a human or animal.
  • the compounds can be used in a method of suppressing an immune response in a human or animal.
  • the compounds can be used in a method of suppressing an immune response in a human or animal.
  • the compounds can be used in a method of suppressing an immune response in a human or animal.
  • the compounds can be used in a method of suppressing an immune response in a human or animal.
  • the compounds can be used in a method of
  • the compound having the molecular formula C 27 H 16 O 12 and the compound having the molecular formula C 14 H 10 O 6 are active in a PKC inhibition assay.
  • PKC-inhibitory activity was quantified by the ability of a compound to decrease
  • the assay employed PKC purified from bovine brain
  • phospholipid and phorbol ester a peptide substrate derived from a naturally-occurring PKC phosphorylation site in the EGF receptor and the donor substrate [ ⁇ 32 P]ATP.
  • the wash reagent was replaced with a similar volume of fresh wash reagent. Following a further 20 minute wash period, the papers were placed into individual scintillation vials and counted for 32 P in a scintillation counter.
  • Table 3 shows the degree of inhibition observed at various concentrations.
  • present compounds and their salts, esters and ethers can be administered in a variety of dosage forms, for example orally such as in the form of tablets,
  • capsules sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example
  • the present compounds and their salts, esters and ethers may therefore be given by injection or infusion.
  • the dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Typically, however, the dosage adopted for each route of administration to adult humans is 0.001 to 10mg/kg, most commonly in the range of 0.01 to 5 mg/kg, body weight. Such a dosage may be given from 1 to 5 times daily.
  • the present compounds and their salts, esters and ethers are non-toxic at therapeutic doses.
  • compositions are typically prepared following conventional veterinarily suitable form.
  • the solid oral forms may contain,
  • lactose dextrose, saccharose, cellulose, corn starch or potato starch
  • lubricants such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols
  • binding agents such as starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose, or polyvinyl pyrrolidone
  • disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dye-stuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates.
  • Such preparations may be manufactured in known manner, for example by means of mixing, granulating, tabletting, sugar coating, or film-coating processes.
  • Liquid dispersions for oral administration may be syrups, emulsions and suspensions.
  • the syrups may contain as carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol.
  • a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose.
  • the suspensions and the emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or
  • Suspensions or solutions for intramuscular injections may contain, together with the active compound, a
  • lidocaine such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if desired, a suitable amount of lidocaine
  • Solutions for intravenous injection or infusion may contain a carrier, for example, sterile water which is generally Water for Injection.
  • a carrier for example, sterile water which is generally Water for Injection.
  • they may take the form of a sterile, aqueous, isotonic saline solution.
  • a compound may be encapsulated within liposomes.
  • 1 ml of this suspension, in a 1.5 ml cryovial, comprises starting material, which was retrieved from storage at -135oC.
  • a preculture was produced by aseptically placing 0.5 ml starting material in 20 ml nutrient solution S(1.5% glycerol, 1.5% soya bean peptone, 1% glucose, 0.5% malt extract, 0.3% NaCl, 0.1% CaCO 3 , 0.1% Tween 80, 0.1% Junlon PW110, pH6) in an Erlenmeyer flask shaken at 240 rpm for 2 days at 25oC. After this period a further 20 ml of nutrient solution S was added and this secondary preculture was incubated at 25oC for 3 days in a rotary shaker at 240 rpm.
  • a production culture was generated by aseptically transferring an intermediate culture to a 751 stirred fermenter containing 501 of nutrient solution P (6.04% molasses. 0.339% casein enzymatic hydrolysate, 0.1% CaCO 3 , 0.1% Tween 80, 0.006% sodium phytate, pH6) and incubating at 25oC. Aeration was achieved by stirring the culture at an impeller speed of 350 rpm and injecting air at a rate of 0.5 WM. The pH of the fermentation was uncontrolled.
  • P 6.04% molasses. 0.339% casein enzymatic hydrolysate, 0.1% CaCO 3 , 0.1% Tween 80, 0.006% sodium phytate, pH6
  • strain X8063 also grew well in cultures incubated in tubes shaken at 240 rpm for 8 days at 25oC in nutrient solution A (2.35% sucrose, 0.97% MES buffer, 0.967%
  • Vitamin mixture consists of (mg L -1 ): thiamine 25, riboflavin 25, pantothenic acid 25, niacin 25, pyridoxine 25, thiacetic acid 25, folic acid 2.5, biotin 2.5,
  • Trace Element Mixture II consists of (per litre): 1 ml 1M H 2 SO 4 , 287 mg ZnSO 4 .7H 2 O, 223 mg MnSO 4 .4H 2 O, 125 mg
  • a 501 fermentation of the strain X8063 prepared as in Example 1 was centrifuged and the mycelium discarded.
  • the pH of the aqueous supernatant (401) was adjusted from 7.0 to 3.0 with glacial acetic acid and Diaion HP20 resin (4.01 by volume, Mitsubishi Kasei Corporation) was added with stirring and the mixture left to stand overnight. The resin was collected by filtration, washed with
  • This ethyl acetate extract was concentrated to give a brown solid (1.02g). This was redissolved in methanol/0.05 M aqueous acetic acid (1:1, 6 ml) and then further purified by preparative reverse phase hplc on the same PrepPak octadecylsilica column eluted isocratically with THF/0,05 M aqueous acetic acid (3:7) at a flow rate of 16 ml/minute. The eluate was monitored at 320 nm and that containing the peak at 10-14 minutes was collected and extracted with ethyl acetate.
  • 1 ml of this suspension, in a 1.5 ml cryovial, comprises starting material, which was retrieved from storage at -135oC.
  • a preculture was produced by aseptically placing 0.5 ml starting material in 20 ml nutrient solutions (1.5% glycerol, 1.5% soya bean peptone, 1% glucose, 0.5% malt extract, 0.3% NaCl, 0.1% CaCO 3 , 0.1% Tween 80, 0.1% Junlon PW110, pH6) in a test tube shaken at 240 rpm for 2 days at 25oC.
  • a production culture was generated by aseptically transferring a tertiary culture to a 141 stirred fermenter containing 101 of nutrient solution P (6.04% molasses.
  • Aeration was achieved by stirring the culture at an
  • Example 5 Extraction of the anhydrofulvic acid and citromycetin from fermentation of the strain X8063
  • a 101 fermentation of the strain X8063 prepared as in Example 4 was centrifuged and the mycelium discarded.
  • the pH of the aqueous supernatant was adjusted from 6.5 to 3.0 with glacial acetic acid and then the acidified supernatant was extracted with ethyl acetate (2 ⁇ 4.51).
  • the ethyl acetate extract (6.51) was then back-extracted with 0.1M aqueous ammonium acetate (3 ⁇ 1.61).
  • the pH of the first, aqueous back extract was adjusted from 4.9 to 2.6 by adding concentrated orthophosphoric acid and the acidified back extract was then extracted with ethyl acetate (2 ⁇ 1.01).
  • This ethyl acetate extract was concentrated to dryness (3.8g) and redissolved in methanol (19ml).
  • the second aqueous back extract (pH5.5) was acidified, extracted and concentrated in the same way to give more material (1.5g) containing a little more of the compound of interest.
  • the methanolic solution of the concentrated first back extract material prepared in Example 5 was purified by preparative reverse phase hplc on a PrepPak cartridge column (ID 25 mm ⁇ length 100 mm, packed with Nova-Pak HR octadecylsilica 60A pore size, 6 ⁇ m particle size.
  • Waters contained in a Waters Radial Compression Module eluted with a linear tetrahydrofuran (THF)/0.05M aqueous acetic acid gradient increasing from 15% THF to 30% THF over a period of forty minutes at a flow rate of 15 ml/minute.
  • THF linear tetrahydrofuran
  • the eluate was monitored at 350 nm, collected and treated as follows:
  • citromycetin as a pale yellow solid (18mg).
  • Photodiode array detector ( ⁇ max under acidic conditions: 366 ⁇ 3nm, 320 ⁇ 3nm, 207 nm). It appeared as a peak typically having a retention time in the range 2.5-3.5 minutes.
  • Citromycetin was identified by its UV/visible spectrum using the photodiode array detector ( ⁇ max under acidic conditions: 368 ⁇ 4nm, 302 ⁇ 4nm, 254 ⁇ 4nm, 222 ⁇ 4nm). It appeared as a peak typically having a retention time in the range 2.5-3 minutes. Its spectral
  • Tablets each weighing 0.15g and containing 25mg of the present compound can be manufactured as follows: Composition for 10,000 tablets
  • the present compound, lactose and half the corn starch are mixed. The mixture is then forced through a sieve 0.5mm mesh size. Corn starch (10g) is suspended in warm water (90ml). The resulting paste is used to granulate the powder. The granulate is dried and

Abstract

A compound which is obtainable from Penicillium glabrum X8063 (CMI 336456) and which has a molecular formula of C27H16O12, a molecular weight of 532 and defined spectral characteristics; a compound which is obtainable from Penicillium glabrum X8063 (CMI 336456) and which has a molecular formula of C14H10O6, a molecular weight of 274 and defined spectral characteristics; anhydrofulvic acid; and citromycetin; and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof; are useful as CD4 binding agents, inhibitors of collagenase and in the case of the compound of molecular formula C27H16O12 and the compound having the molecular formula C14H10O6 inhibitors of protein kinase C.

Description

CD4 BINDING AGENTS AND INHIBITORS
OF COLLAGENASE AND PROTEIN KINASE C
The present invention relates to compounds useful as CD4 binding agents, as inhibitors of collagenase and in certain cases also as inhibitors of the enzyme Protein Kinase C (PKC). The invention also relates to the
preparation of the compounds and to pharmaceutical and veterinary compositions containing them.
CD4 is a cell surface glycoprotein expressed on those T lymphocytes which recognise antigen bound to class II MHC molecules. Inhibition of class II MHC-CD4 interactions will block antibody responses, mixed lymphocyte reactions and other immune responses involving CD4+ T lymphocytes. These responses are important in pathological conditions including autoimmunity, organ graft rejection, allergy and graft versus host disease. CD4 is also the cellular receptor for human immunodeficiency virus (HIV).
Collagenase is a member of the connective tissue metalloproteinase family of enzymes. Collagenases control the turnover, remodelling and degradation of collagen in tissue. Collagen breakdown occurs in several pathological conditions including arthritis, tumour metastasis,
periodontal disease, corneal ulceration and excessive bone or skin collagen degradation.
PKC is critically involved in activation of T
lymphocytes. Interaction of the T cell antigen receptor with its ligand causes increased turnover of phosphatidylinositol lipids and the generation of diacylglycerol (DAG) which activates PKC.
We have now discovered that fermentation of a
nutrient medium with a strain of the fungus Penicillium glabrum produces compounds which are CD4 binding agents, inhibitors of collagenase and in some cases also inhibitors of PKC. Accordingly, the present invention provides a compound selected from:
(a) a compound which has a molecular formula of C27H16O12, a molecular weight of 532 and the following
ultraviolet (UV), infrared (ir), nuclear magnetic resonance
(NMR) and mass spectral (MS) characteristics:
UV: λ MeOH (log10E) nm: 432(4.28); 365sh(4.10);
max 282sh(4.10); 233(4.54);
211(4.56)
UV: λ MeOH-0.1M HCl (aqueous)
max (log10E) nm: 366(4.13); 320(4.22);
207(4.57)
UV: λ MeOH-0.1M NaOH (aqueous)
max (log10E) nm: 464 (4.14); 371(4.20);
236(4 54)
Figure imgf000004_0001
1H-NMR (400MHz): δ/ppm in (CD3)2SO :
8.69 (1H, d, J=2.2Hz), 8.04 (1H, d, J=2.2Hz), 7.04 (1H, s), 6.87 (1H, s), 6.38 (1H, s), 2.83 (3H,s), 2.29 (3H,s)
13C-NMR (100MHz): δ/ppm in (CD3)2SO :
199.3, 196.3, 172.7, 167.4, 158.7, 154.6, 152.9, 152.0,
150.6, 150.3, 148.3, 143.3, 141.0, 134.2, 129.6, 128.7,
128.0, 123.4, 121.5, 120.3, 111.5, 110.7, 109.7, 102.9, 102.3, 31.5, 31.1
MS: m/z [Fast atom bombardment (FAB), MNBA]:
533 (MH+), 515[(MH-H2O)+]
m/z [FAB, MNBA(-ve ion)]: 531 [(M-H)-1, 513
([M-H-H2O]-)
m/z[DCI, NH3]: 533 (MH+), 489 ([M-CO2]+); and pharmaceutically and veterinarily acceptable salts and esters and ethers thereof;
(b) a compound which has a molecular formula of C14H10O6, a molecular weight of 274 and the following
ultraviolet (UV), infrared (ir), nuclear magnetic resonance (nmr) and mass (MS) spectra:
Figure imgf000004_0002
Figure imgf000005_0001
,
1H-nmr (400 MHz): δ/ppm in (CD3)2SO:
12.90 (1H,s), 11.3 (1H, br s), 8.62 (1H,s), 7.13 (1H,d, J=2.4 Hz), 6.57 (1H,d, J=2.4Hz), 5.69 (1H, br s), 5.40 (1H, g, J=6.7 Hz), 1.46 (3H, d, J=6.7 HZ)
13C-nmr (100 MHz): δ/ppm in (CD3)2SO: 183.2, 178.3, 165.0,
164.9, 164.0, 144.9, 137.3, 122.0, 114.7,
110.4, 107.9, 107.2, 60.5, 20.9
MS: m/z [electron impact (EI)]: 274 [M+] , 259 [(M-CH3)+],
231 [(M-CH3CO)+]
m/z [Desorption chemical ionisation
(DCI), NH3]: 275[MH+] and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof;
(c) anhydrofulvic acid and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof; and
(d) citromycetin and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof; for use in a method of treatment of the human or animal body by therapy.
These compounds act as CD4 binding agents and as collagenase inhibitors. The compounds (a) and (b) are also inhibitors of PKC. The invention therefore also provides use of a compound as defined above in the manufacture of a medicament for use as a CD4 binding agent, a collagenase inhibitor or, in the case of a compound (a) or (b), a PKC inhibitor.
The compound (a) having the molecular formula
C27H16O12, the compound (b) having the molecular formula C14H10O6, anhydrofulvic acid and citromycetin are hereinafter referred to as the present compounds.
The present compounds have been isolated from a microorganism which we have designated strain X8063 and which has been identified as a strain of the fungus
Penicillium glabrum on the basis of the following
morphological data:
Fungal strain X8063 was incubated for 7 days at each of 5ºC, 25ºC and 30ºC in the following growth media
(from PITT, J.I., 1979; The Genus Penicillium and its
Teleomorphic States Eupenicillium and Talaromyces. London:
Academic Press.): Czapek's agar with yeast extract (CzYa; composition per litre of distilled water: sucrose, 30g;
yeast extract, 5g; agar 20g; KH2PO4, lg; NaNO3, 0.3g;
MgSO4.7H2O, 0.05g; KC1, 0.05g; FeSO4.7H2O, O.OOlg); malt extract agar with glucose and peptone (MEA; composition per litre of distilled water: malt extract; 20g; glucose, 20g;
Bacto-peptone, lg; agar, 20g); glycerol nitrate agar (G25N; composition per 750 ml of distilled water: yeast extract,
3.7g; glycerol, 250g; KH2PO4, 0.75g; NaNO3, 0.225g;
MgSO4.7H2O, 37.5mg; KC1, 37.5mg; FeSO4.7H2O, 0.75mg).
The microscopic characteristics were as follows:
Conidiophores borne from surface hyphae. Stipes of the conidiophores up to 200 μm long and 4-5 μm wide, vesiculate
(vesicles 6-7 μm wide) with smooth walls. Verticils borne terminally (never subterminal), monoverticillate, composed of 10 to 16 appressed phialides. Phialides ampulliform (10) -12-13 × 4-5 μm. Conidia spheroidal, 3.5-4.5 μm diameter, with faintly warted/rugulose walls. Conidia borne in long well defined columns.
The gross colony morphology on each growth medium was as follows (colours are coded according to the British Standards Institute publication BS381C:1980; Colours for identification, coding and special purposes):
CzYa at 25º: Colonies, 31 to 40 mm diameter, plane with radial sulcation. Texture velutinous, low. Margin immersed, entire and extending approximately 1 mm.
Mycelium white to light beige (366) with a plane, non-sporulating, 2-4 mm periphery. Central region white, but showing in places a slight greenish tint, conidiogenesis poor. Exudate and sclerotia absent. Pale clear yellow diffusing pigment produced. Diffusion zone approximately 50mm in diameter. Reverse concolourous with surface mycelium, a yellowish pale cream (352).
MEA at 25º: Colonies, 30 to 31 mm diameter, plane, lacking radial sulcation. Texture velutinous, low. Margin not immersed, entire. Aerial mycelium very sparse, colours due to maturing spore mass; outer region (5 mm margin) approximating pale cream (352) to light straw (384) but slightly more yellowish, central region approximating light grey (631) to aircraft grey (693) but slightly more bluish. Conidiogenesis moderate. Exudate and sclerotia absent.
Diffusing pigment produced in a zone approximately 45 mm in diameter, coloured a clear bright pale yellow. Reverse darkening towards the centre ranging from pale cream (352) to dark earth (450).
G25N at 25º: Colonies, 12 to 13 mm diameter, plane with radial sulcation. Texture velutinous, low. Margin immersed and entire. Outer region (1-2 mm) colourless to white. Central region white to very pale yellow.
Conidiogenesis very poor and lacking distinct spore mass colour. Exudate and sclerotia absent. Diffusing pigment absent. Reverse ranging from white to pale bright yellow (slightly paler than canary yellow, 309).
CzYa at 5º: Slight growth, colonies 3 to 5 mm diameter.
MEA at 5º: Micro-colonies to slight growth, 3 to 4 mm diameter. G25N at 5º: No growth.
CzYa, MEA and G25N at 30º: No growth.
The production of penicillate conidophores clearly places strain X8063 in the genus Penicillium. From the other microscopic and gross morphological features of strain X8063 described above, and following the taxonomic scheme in Pitt, J.I., 1979 - The Genus Penicillium and its Teleomorphic States Eupenicillium and Talaromvces: London, Academic Press, X8063 may be best classified as a strain of Penicillium glabrum (Wehmer) Westling (a widely used synonym is P. frequentans Westling).
The strain X8063 was isolated from a soil sample collected from Femes, Lanzarote in 1987 and was deposited under the Budapest Treaty at the Commonwealth Mycological Institute, Kew, Richmond, Surrey, UK on 13 December 1989 under accession number CMI 336456.
The above description is illustrative of a strain of Penicillium glabrum which can be employed in the
production of the present compounds. However, the present invention also embraces the use of mutants of strain X8063 which produce the present compounds. For example, those mutants which are obtained by natural selection or those produced by mutating agents including ionising radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments, can also be used.
The present invention further provides a process for the preparation of a compound as defined above, which process comprises (i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain X8063 (CMI 336456) or a mutant thereof which produces the said
compound having the molecular formula C27H16O12, the said compound having the molecular formula C14H10O6, anhydrofulvic acid or citromycetin; (ii) isolating the said compound having the molecular formula C27H16O12, the said compound having the molecular formula C14H10O6, anhydrofulvic acid or citromycetin from the fermentation medium; and (iii) if desired, converting the isolated compound into a
pharmaceutically or veterinarily acceptable salt, ester or ether thereof.
The present compounds are typically produced during the aerobic fermentation of an aqueous nutrient medium under conditions described hereinafter, with a producing strain of Penicillium glabrum. X8063, or a producing mutant strain of X8063. Aqueous media such as those used for the production of many antibiotic substances are suitable.
Such nutrient media contain sources of carbon and nitrogen assimilable by the microorganism. If desired inorganic salts may be added, generally at low levels. In addition, the fermentation media may contain traces of metals
necessary for the growth of the microorganisms, and
production of the desired compound. These are usually present in sufficient concentrations 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.
Assimilable sources of carbon, nitrogen and minerals may be provided by either simple or complex nutrients. Sources of carbon will generally include glucose, maltose, starch, glycerol, molasses, dextrin, lactose, sucrose, fructose, carboxylic acids, amino acids, glycerides, alcohols, alkanes and vegetable oils. Sources of carbon will generally comprise from 0.5 to 10% by weight of the fermentation medium.
Sources of nitrogen will generally include soya bean meal, corn steep liquors, distillers' solubles, yeast extracts, cottonseed meal, peptones, ground nut meal, malt extract, molasses, casein, amino. acid mixtures, ammonia (gas or solution), ammonium salts or nitrates. Urea and other amides may also be used. Sources of nitrogen will generally comprise from 0.1 to 10% by weight of the fermentation medium. Nutrient mineral salts which may be incorporated into the culture medium include the generally used salts capable of yielding sodium, potassium, ammonium, iron, magnesium, zinc, nickel, cobalt, manganese, vanadium, chromium, calcium, copper, molybdenum, boron, phosphate, sulphate, chloride and carbonate ions.
An antifoam may be present to control excessive foaming and added at intervals as required.
The fermentation using Penicillium glabrum can be conducted at temperatures ranging from 20ºC to 30ºC, preferably 24-28ºC. For optimal results, it is most convenient to conduct these fermentations at a temperature in the range 24-26ºC. The starting pH of the nutrient medium suitable for producing the compounds can vary from 5.0 to 8.5 with a preferred range of from 5.5 to 7.5.
Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in a flask by known sterile techniques, inoculating the flask with either spores or vegetative cellular growth of
Penicillium glabrum. loosely stoppering the flask with cotton wool, and permitting the fermentation to proceed in a constant room temperature of about 25ºC on a rotary shaker at from 95 to 300 rpm for 2 to 10 days. The
fermentation may also be conducted in static culture on liquid or semi-solid medium.
For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an
agitator and a means of aerating the fermentation medium. The nutrient medium is made up in the tank after
sterilization and is inoculated with a, source of vegetative cellular growth of Penicillium glabrum. The fermentation is allowed to continue for from 1 to 8 days while agitating and/or aerating the nutrient medium at a temperature in the range 24ºC to 28ºC. The degree of aeration is dependent upon several factors such as the size of the fermenter and agitation speed. Generally the larger scale fermentations are agitated at about 95 to 500 rpm and aerations of about 0.5 to 1.5 VVM (volumes of air per volume of medium per minute) are used.
The present compounds are found primarily in the liquor of the fermentation of strain X8063. The separation of the present compounds from the whole fermentation broth and their recovery is carried out by solvent extraction followed by application of chromatographic fractionations with various chromatographic techniques and solvent
systems. The present compounds have thus been isolated in this way. They can be obtained in pure form.
The present compounds are acidic and are soluble in neutral and alkaline aqueous solvents. In their unionised forms the compounds are soluble in polar organic solvents such as dimethyl sulphoxide and, except in the case of the compound having the molecular formula C27H16O12, methanol. When impure, the uncharged form of the compounds are soluble in a wider range of organic solvents such as dichloromethane, ethyl acetate and, in the case of the compound having the molecular formula C27H16O12, methanol.
Thus, in one recovery method, the whole fermentation broth is acidified to pH 3 and combined with a water-immiscible organic solvent such as ethyl acetate. Generally, several extractions are required to achieve maximal recovery. The solvent removes the desired compounds, but also other substances.
As the desired compounds are acids they can be purified further by back-extracting the water-immiscible solvent extract with a buffered alkaline aqueous solution of a salt such as ammonium acetate or sodium phosphate. In this process the present compounds are ionised and are extracted into the aqueous layer. The present compounds and other acidic compounds can then by recovered from the aqueous layer by acidifying it to pH 3 and re-extracting it with fresh water-immiscible organic solvent. This solvent extract is then concentrated under reduced pressure. An alternative method of recovery of the desired compounds from the liquor, particularly useful for large scale fermentations, is to acidify the liquor to pH 3 and pass the acidified liquor through a chromatography column filled with a porous hydrophobic resin such as Diaion HP20 (Mitsubishi Kasei Corp). The resin is then washed with an acidic aqueous solution such as an acetic acid solution and then eluted with a water-miscible organic solvent such as methanol. The methanol eluate is concentrated under reduced pressure before diluting it with ethyl acetate or another water-immiscible organic solvent and purifying the organic solvent solution by back-extraction with an
alkaline aqueous buffer solution, re-acidification of the aqueous back extract, re-extraction into organic solvents and concentration, as described in the last paragraph.
The residue produced by either of these two extraction methods is then further purified by reverse phase high-pressure liquid chromatography (hplc) under acidic conditions on a column containing an adsorbent such as octadecyl silica. The column retains the desired compounds and other acidic impurities. It is eluted with a mixture of an acidic aqueous solution, such as acetic acid solution, and a water-miscible organic solvent, such as tetrahydrofuran (THF). As the proportion of the organic solvent in the mixture is increased the desired compounds are eluted. The compound having the molecular formula C27H16O12 and the compound having the molecular formula
C14H10O6 are eluted after most of the impurities. Fractions of eluate are then typically concentrated and examined for the presence of the desired compounds by analytical hplc with photodiode array detection, as described in Example 7. The compounds of the invention can then be identified from their uv/visible spectra and retention times.
The eluate containing a desired compound is extracted with a water-immiscible organic solvent such as ethyl acetate and the desired compound recovered by evaporation of the extract. The present compounds are further purified by reverse phase hplc on an octadecyl silica column eluted isocratically with mixtures of the same solvents used in the last hplc purification stage and the desired compound recovered from the eluate by
extraction in the same way.
The use of hplc or other known techniques will afford purified compositions containing the desired
compound, the presence of which is determined by analysing the various chromatographic fractions for CD4 binding activity, for collagenase inhibitory activity, for PKC inhibitory activity in the case of the compound having the molecular formula C27H16O12 and the compound having the molecular formula C14H10O6 or for physicochemical
characteristics.
Anhydrofulvic acid is in fact a known compound which has been derived chemically from the Penicillium sp. metabolite fulvic acid (Dean, F.M. Eade, R.A., Moubasher, R. and Robertson A. (1957) J. Chem. Soc. 3497-3510) and has been prepared as an intermediate in the synthesis of fulvic acid (Yamauchi, M., Katayama, S., Todoroki, T. and
Watanabe, T. (1987) J. Chem. Soc. Perkin Trans I 389-394). Anhydrofulvic acid has the following formula 1:
Figure imgf000013_0001
Citromycetin is a known metabolite of Penicillium frequentans (Westling) (Demetriadou, A.K., Laue, E.D.
Leeper, F.J. and Staunton, J (1988) J. Chem. Soc. Perkin Trans I 763-768; Evans, G.E. and Staunton, J. (1988) J. Chem. Soc. Perkin Trans I 755-761). Citromycetin has the following formula 2 :
Figure imgf000014_0001
The present compounds may be converted into a pharmaceutically or veterinarily acceptable salt, ester or ether thereof. Suitable salts include salts with alkali metals such as sodium and potassium, and ammonium salts.
Suitable ethers are branched or unbranched, saturated or unsaturated, substituted or unsubstituted C1 to C6 aliphatic ethers, typically C1 to C6 alkyl ethers.
Preferred alkyl ethers are C1 to C4 alkyl ethers such as the methyl and ethyl ethers. Typically the ethers are peralkyl ethers.
Such ethers may be prepared by treatment of the xanthone derivative with a diazoalkane such as diazomethane in a suitable inert solvent or by treatment with an appropriate alkyl halide, sulphonate ester or dialkyl sulphate in the presence of base. Suitable bases include alkali and alkaline earth metal hydroxides,
tetraalkylammonium hydroxides and alkali and alkaline earth metal carbonates.
Suitable esters include esters formed with branched or unbranched, saturated or unsaturated, substituted or unsubstituted C1-C6 alcohols. Methyl, ethyl and vinyl esters are typical examples. Such esters may be prepared in the same way as the ethers above or by treatment of the xanthone derivative with the appropriate alcohol in the presence of a suitable acid catalyst or activating agent.
The present compounds and their salts, esters and ethers have utility as CD4 binding agents. They can block the physiological function of CD4 by inhibiting its interaction with MHC class II molecules. They also have utility as collagenase inhibitors and, in the case of the compound having the molecular formula C27H16O12 and the compound having the molecular formula C14H10O6 as PKC
inhibitors. A human or animal, eg mammal, can therefore be treated by a method comprising administration of a
therapeutically effective amount of a present compound or a pharmaceutically or veterinarily acceptable salt, ester or ether thereof.
A CD4 binding agent can selectively inhibit MHC class II-restricted responses. The present compounds and their salts, esters and ethers can therefore be used as immunosuppressants, especially in the treatment of
autoimmune disease such as rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, multiple sclerosis and primary biliary cirrhosis, graft versus host (GVH) disease and organ rejection. They can also be used in the treatment of allergic diseases such as asthma and
inflammatory diseases such as inflammatory bowel disease. A CD4 binding agent may be effective in preventing the entry of HIV into cells and hence of therapeutic value as an anti-HIV agent.
The present compounds are active in an ELISA assay based on the interaction between soluble recombinant CD4 (sCD4 (V1 + V2); from E. coli) and the monoclonal antibody Leu3a (Becton-Dickinson, Oxford, England). Leu3a binds to an epitope on the VI domain of CD4 as described by
Merkenschlager et al (1990) (Merkenschlager, M., Buck, D., Beverley, P.C.L. and Sattentau, Q.J. (1990) "Functional epitope analysis of the CD4 molecule" J. Immunology 145 2839-2845). Table 1 shows the degree of inhibition observed at various concentrations of the present
compounds.
Figure imgf000016_0001
Collagenase plays a wide-ranging role in the pathology of disease. In its role as a collagenase inhibitor a present compound or a salt, ester or ether thereof can be employed to alleviate conditions including rheumatoid arthritis and osteoarthritis, tumour metastasis, in periodontal disease, corneal ulceration, excessive skin or bone collagen degradation and other disorders.
An assay for collagenase inhibiting activity is based on the degradation of rat type I collagen gels by human collagenase as described by Harris and Vater (1982)
(Harris, E.D. and Vater, CA. (1982) in Methods in Enzymology (Cunningham, L.W. and Frederiksen, O.W., eds), Vol 82 part A, pp 423-452, Academic Press, New York). The degree of lysis is estimated after staining residual substrate with Coomassie Brilliant Blue. Rat tail tendon type I collagen is partially purified by the method
described by Miller and Rhodes (1982) (Miller, E.J. and Rhodes, R.K. (1982) in Methods in Enzymology (Cunningham, L.W. and Frederiksen, O.W., eds), Vol 82 part A, pp 33-64, Academic Press, New York) and human collagenase is derived from the conditioned medium of a human histiocytic lymphoma cell line (U937). Enzyme production is stimulated by the addition of PMA (phorbol myristate acetate) and the latent enzyme activated with trypsin.
The present compounds inhibit the action of human collagenase on rat type I collagen when tested in this assay. The compound having the molecular formula C27H16O12 caused 63% inhibition of the collagenase activity at a concentration of 25μM. The compound having the molecular formula C14H10O6 inhibits collagenase when present in
concentrations of at least 100 μM. Table 2 shows the degree of inhibition observed at various concentrations of anhydrofulvic acid and citromycetin:
Figure imgf000017_0001
Figure imgf000018_0001
In their role as PKC inhibitors the compound having the molecular formula C27H16O12, the compound having the molecular formula C14H10O6 and their salts, esters and ethers can be employed to alleviate a broad range of cancerous conditions. They therefore have utility as antitumour agents. They can be used in a method of treating tumours such as breast, thyroid, colon, lung, skin and brain tumours, in particular tumours in which PKC is implicated as a causative agent. The compounds can be used to improve the condition of a patient having such a tumour.
The compound having the molecular formula C27H16O12, the compound having the molecular formula C14H10O6 and their salts, esters and ethers can also be employed to alleviate inflammatory conditions and therefore have utility as anti- inflammatory agents. The compounds can be used in a method of treating inflammatory conditions, for example asthma.
An inhibitor of PKC also inhibits T cell activation and can be used in the therapy of autoimmune diseases. The compound having the molecular formula C27H16O12, the compound having the molecular formula C14H10O6 and their salts, esters and ethers therefore have utility in the therapy of
autoimmune disease and can be used in a method of treating autoimmune disease such as systemic lupus erythematosus, myasthenia gravis and diabetes.
A PKC inhibitor also acts as an immunosuppressive agent. The compound having the molecular formula C27H16O12, the compound having the molecular formula C14H10O6 and their salts, esters and ethers therefore have utility as
immunosuppressive agents and can be used in a method of suppressing an immune response in a human or animal. For example, the compounds can be used in a method of
preventing organ graft rejections.
The compound having the molecular formula C27H16O12 and the compound having the molecular formula C14H10O6 are active in a PKC inhibition assay. PKC-inhibitory activity was quantified by the ability of a compound to decrease
phosphorylation of a PKC-specific substrate by the enzyme. The assay employed PKC purified from bovine brain,
necessary cofactors for enzyme activity (calcium,
phospholipid and phorbol ester), a peptide substrate derived from a naturally-occurring PKC phosphorylation site in the EGF receptor and the donor substrate [γ32P]ATP.
An incubation mixture of 75μl containing Ca2+,
micelles of phosphatidylserine/phorbol ester, peptide substrate and inhibitor was preincubated at 25º C for 5 minutes before the reaction was initiated by the addition of 10μl [γ32P]ATP mixture (0.04 μCi/assay). Following 2h incubation at 25ºC, the reaction was terminated by the addition of 50μl dilute orthophosphoric acid. Aliquots of 100μl were spotted onto phosphocellulose paper squares (1 cm × 1 cm) and the binding papers placed in 75 mM
orthophosphoric acid, at least 10 ml of this wash reagent being allowed per paper. After 20 minutes with
intermittent gentle mixing, the wash reagent was replaced with a similar volume of fresh wash reagent. Following a further 20 minute wash period, the papers were placed into individual scintillation vials and counted for 32P in a scintillation counter.
The 32P incorporated into peptide was quantitatively measured by the binding papers. The results obtained were corrected for any non-specific effect using appropriate blanks (no enzyme, no substrate). The inhibition of PKC activity was determined using the following equation:
% inhibition = Non-inhibited control cpm - sample cpm × 100%
Non-inhibited control cpm
Table 3 shows the degree of inhibition observed at various concentrations.
Figure imgf000020_0001
The present compounds and their salts, esters and ethers can be administered in a variety of dosage forms, for example orally such as in the form of tablets,
capsules, sugar- or film-coated tablets, liquid solutions or suspensions or parenterally, for example
intramuscularly, intravenously or subcutaneously. The present compounds and their salts, esters and ethers may therefore be given by injection or infusion.
The dosage depends on a variety of factors including the age, weight and condition of the patient and the route of administration. Typically, however, the dosage adopted for each route of administration to adult humans is 0.001 to 10mg/kg, most commonly in the range of 0.01 to 5 mg/kg, body weight. Such a dosage may be given from 1 to 5 times daily. The present compounds and their salts, esters and ethers are non-toxic at therapeutic doses.
The present compounds and their salts, esters and ethers are formulated for use as a pharmaceutical or veterinary composition also comprising a pharmaceutically or veterinarily acceptable carrier or diluent. The compositions are typically prepared following conventional veterinarily suitable form.
For example, the solid oral forms may contain,
together with the active compound, diluents such as
lactose, dextrose, saccharose, cellulose, corn starch or potato starch; lubricants such as silica, talc, stearic acid, magnesium or calcium stearate and/or polyethylene glycols; binding agents such as starches, arabic gums, gelatin, methylcellulose, carboxymethylcellulose, or polyvinyl pyrrolidone; disintegrating agents such as starch, alginic acid, alginates or sodium starch glycolate; effervescing mixtures; dye-stuffs; sweeteners; wetting agents such as lecithin, polysorbates, laurylsulphates.
Such preparations may be manufactured in known manner, for example by means of mixing, granulating, tabletting, sugar coating, or film-coating processes.
Liquid dispersions for oral administration may be syrups, emulsions and suspensions. The syrups may contain as carrier, for example, saccharose or saccharose with glycerol and/or mannitol and/or sorbitol. In particular a syrup for diabetic patients can contain as carriers only products, for example sorbitol, which do not metabolise to glucose or which only metabolise a very small amount to glucose. The suspensions and the emulsions may contain as carrier, for example a natural gum, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or
polyvinyl alcohol.
Suspensions or solutions for intramuscular injections may contain, together with the active compound, a
pharmaceutically acceptable carrier such as sterile water, olive oil, ethyl oleate, glycols such as propylene glycol, and, if desired, a suitable amount of lidocaine
hydrochloride. Solutions for intravenous injection or infusion may contain a carrier, for example, sterile water which is generally Water for Injection. Preferably, however, they may take the form of a sterile, aqueous, isotonic saline solution. Alternatively, a compound may be encapsulated within liposomes.
The following Examples illustrate the invention.
Example 1 - Culture of strain X8063 in Liquid Media
Starting material of the strain X8063 was generated by suspending a mature slant culture, grown on PDA (2%
dextrose, 15% agar, 0.4% potato extract), in 5 ml 10% aqueous glycerol. 1 ml of this suspension, in a 1.5 ml cryovial, comprises starting material, which was retrieved from storage at -135ºC. A preculture was produced by aseptically placing 0.5 ml starting material in 20 ml nutrient solution S(1.5% glycerol, 1.5% soya bean peptone, 1% glucose, 0.5% malt extract, 0.3% NaCl, 0.1% CaCO3, 0.1% Tween 80, 0.1% Junlon PW110, pH6) in an Erlenmeyer flask shaken at 240 rpm for 2 days at 25ºC. After this period a further 20 ml of nutrient solution S was added and this secondary preculture was incubated at 25ºC for 3 days in a rotary shaker at 240 rpm.
An intermediate culture was then generated by
aseptically transferring a secondary preculture to 2.01 of nutrient solution S in a 31 fermenter. A 3 day
fermentation was carried out with a stirring rate of 500 rpm and an air flow rate of 0.5 WM. The pH was
uncontrolled and the temperature was maintained at 25ºC.
A production culture was generated by aseptically transferring an intermediate culture to a 751 stirred fermenter containing 501 of nutrient solution P (6.04% molasses. 0.339% casein enzymatic hydrolysate, 0.1% CaCO3, 0.1% Tween 80, 0.006% sodium phytate, pH6) and incubating at 25ºC. Aeration was achieved by stirring the culture at an impeller speed of 350 rpm and injecting air at a rate of 0.5 WM. The pH of the fermentation was uncontrolled.
After 5 days the liquor was harvested for extraction of the product.
The strain X8063, also grew well in cultures incubated in tubes shaken at 240 rpm for 8 days at 25ºC in nutrient solution A (2.35% sucrose, 0.97% MES buffer, 0.967%
monosodium glutamate, 0.05% MgSO4.7H2O, 0.05% KCl, 0.0037% K2HPO4, 0.002% CaCl2.2H2O, 20 ml L-1 Vitamin mixture I, 5 ml L-1 trace element mixture II, 1 ml L-1 Tween 80).
Vitamin mixture consists of (mg L-1): thiamine 25, riboflavin 25, pantothenic acid 25, niacin 25, pyridoxine 25, thiacetic acid 25, folic acid 2.5, biotin 2.5,
cyanocobalamin 2.5, p-aminobenzoic acid 2.5, vitamin K1 2.5.
Trace Element Mixture II consists of (per litre): 1 ml 1M H2SO4, 287 mg ZnSO4.7H2O, 223 mg MnSO4.4H2O, 125 mg
CuSO4.5H2O, 83 mg KI, 62 mg H3BO3, 48 mg NaMoO4.2H2O, 48 mg CoCl2.6H2O.
Example 2 - Extraction of CD4-binding activity from
fermentation of the strain X8063
A 501 fermentation of the strain X8063 prepared as in Example 1 was centrifuged and the mycelium discarded. The pH of the aqueous supernatant (401) was adjusted from 7.0 to 3.0 with glacial acetic acid and Diaion HP20 resin (4.01 by volume, Mitsubishi Kasei Corporation) was added with stirring and the mixture left to stand overnight. The resin was collected by filtration, washed with
methanol/0,05 M aqueous acetic acid (1:1, 5.0 1) and then eluted with methanol (101). The methanol eluate was concentrated to a volume of approximately one litre by rotary evaporation and then diluted with 0.05 M aqueous acetic acid (51). This aqueous mixture was extracted with ethyl acetate (2×51) and the ethyl acetate extract
concentrated to dryness to yield a brown gum (20.2g) which contained the CD4-binding compound of interest.
Example 3 - Purification of the compound having the
molecular formula C27H16O12 and the compound having the molecular formula C14H10O6 A portion (8.4g) of the ethyl acetate extract prepared in Example 2 was dissolved in methanol (40ml) and diluted with 0.05 M aqueous acetic acid (40ml). This solution was purified by preparative reverse phase hplc on a PrepPak cartridge column (ID 25 mm × length 100 mm, packed with
Nova-Pak HR octadecylsilica 60 A pore size, 6 μM particle size. Waters) contained in a Waters Radial Compression Module eluted with a linear tetrahydrofuran (THF)/0,05 M aqueous acetic acid gradient increasing from 15% THF to 30% THF over a period of forty minutes at a flow rate of 15 ml/minute. The eluate was monitored at 360 nm and the fractions containing the peak at retention time 31-37 minutes were collected and extracted with ethyl acetate.
This ethyl acetate extract was concentrated to give a brown solid (1.02g). This was redissolved in methanol/0.05 M aqueous acetic acid (1:1, 6 ml) and then further purified by preparative reverse phase hplc on the same PrepPak octadecylsilica column eluted isocratically with THF/0,05 M aqueous acetic acid (3:7) at a flow rate of 16 ml/minute. The eluate was monitored at 320 nm and that containing the peak at 10-14 minutes was collected and extracted with ethyl acetate. The ethyl acetate extract was concentrated to dryness to give a yellow-brown solid (252mg) which was redissolved in methanol/0.05 M aqueous acetic acid (1:1, 15 ml). This solution was further purified by a reverse phase hplc step using the same PrepPak column eluted with a methanol-0.05M aqueous acetic acid gradient, starting at 45% methanol for the first eight minutes and then
increasing linearly from 45 to 55% methanol over the next two minutes, at a flow rate of 18 ml/minute. The eluate was monitored at 320nm, collected and treated as follows: (I) The eluate from the centre of the peak at retention time 6.5-9 minutes was collected, diluted 1:1 with 0.05M aqueous acetic acid and extracted with ethyl acetate. The ethyl acetate extract was concentrated to dryness to yield a yellow brown solid (35mg). This was redissolved in methanol-0.05M aqueous acetic acid (1:1, 7 ml) and purified further by isocratic reverse phase hplc using the same PrepPak column eluted with methanol-0.05M aqueous acetic acid (45:55) at a flow rate of 18 ml/minute. The eluate was monitored at 325nm and that containing the peak at retention time 6.5 to 7.7 minutes was collected, diluted 1:1 with 0.05M aqueous acetic acid and extracted with ethyl acetate. The ethyl acetate extract was concentrated to dryness and redissolved in methanol-0.05M aqueous acetic acid (1:1, 6 ml). A precipitate developed in this sample on standing. It was collected to yield the compound having the molecular formula C27H16O12 as a bright yellow solid (6mg).
(II) The eluate from the centre of the peak at retention time 12-15 minutes was collected, diluted 1:1 with 0.05M aqueous acetic acid and extracted with ethyl acetate. The ethyl acetate extract was concentrated to dryness to yield a brown solid (31mg).
This was redissolved in methanol-0.05M aqueous acetic acid (1:1, 10 ml) and further purified by isocratic reverse phase hplc using the same PrepPak column eluted with methanol-0.05M aqueous acetic acid (1:1) at a flow rate of 18 ml/minute. The eluate was monitored at 280nm and that containing the peak at retention time 8.5 to 10 minutes was collected, diluted 1:1 with 0.05M aqueous acetic acid and extracted with ethyl acetate. The ethyl acetate extract was concentrated to dryness to yield the compound having the molecular formula C14H10O6 as a red-brown solid (11mg).
Example 4 - Culture of strain X8063 in Liquid Media
Starting material of the strain X8063 was generated by suspending a mature slant culture, grown on PDA (2%
dextrose, 15% agar, 0.4% potato extract), in 5 ml 10% aqueous glycerol. 1 ml of this suspension, in a 1.5 ml cryovial, comprises starting material, which was retrieved from storage at -135ºC. A preculture was produced by aseptically placing 0.5 ml starting material in 20 ml nutrient solutions (1.5% glycerol, 1.5% soya bean peptone, 1% glucose, 0.5% malt extract, 0.3% NaCl, 0.1% CaCO3, 0.1% Tween 80, 0.1% Junlon PW110, pH6) in a test tube shaken at 240 rpm for 2 days at 25ºC.
Following this incubation period, 20 ml of the above nutrient solution S was aseptically added to the above preculture and the new mixture was incubated for a further 2 days at 25ºC, forming the secondary growth stage. A tertiary growth stage was then produced by aseptically transferring the secondary preculture to 250ml of nutrient solution S in a 21 aspirator and stirring at 500 rpm for 3 days at 25ºC.
A production culture was generated by aseptically transferring a tertiary culture to a 141 stirred fermenter containing 101 of nutrient solution P (6.04% molasses.
0.339% casein enzymatic hydrolysate, 0.1% CaCO3, 0.1% Tween 80, 0.006% sodium phytate, pH6) and incubating at 25ºC.
Aeration was achieved by stirring the culture at an
impeller speed of 350 rpm and injecting air at a rate of 0.5 WM. The pH of the fermentation was uncontrolled.
After 5 days the liquor was harvested for extraction of the product.
Example 5 - Extraction of the anhydrofulvic acid and citromycetin from fermentation of the strain X8063
A 101 fermentation of the strain X8063 prepared as in Example 4 was centrifuged and the mycelium discarded. The pH of the aqueous supernatant was adjusted from 6.5 to 3.0 with glacial acetic acid and then the acidified supernatant was extracted with ethyl acetate (2×4.51). The ethyl acetate extract (6.51) was then back-extracted with 0.1M aqueous ammonium acetate (3×1.61). The pH of the first, aqueous back extract was adjusted from 4.9 to 2.6 by adding concentrated orthophosphoric acid and the acidified back extract was then extracted with ethyl acetate (2×1.01). This ethyl acetate extract was concentrated to dryness (3.8g) and redissolved in methanol (19ml). The second aqueous back extract (pH5.5) was acidified, extracted and concentrated in the same way to give more material (1.5g) containing a little more of the compound of interest.
Example 6 - Purification of the anhydrofulvic acid and citromycetin
The methanolic solution of the concentrated first back extract material prepared in Example 5 was purified by preparative reverse phase hplc on a PrepPak cartridge column (ID 25 mm × length 100 mm, packed with Nova-Pak HR octadecylsilica 60A pore size, 6 μm particle size. Waters ) contained in a Waters Radial Compression Module eluted with a linear tetrahydrofuran (THF)/0.05M aqueous acetic acid gradient increasing from 15% THF to 30% THF over a period of forty minutes at a flow rate of 15 ml/minute. The eluate was monitored at 350 nm, collected and treated as follows:
(I) The fractions containing the peak at retention time 36-40 minutes were collected, extracted with ethyl acetate and the ethyl acetate extract concentrated to give crude anhydrofulvic acid (267 mg). A column (internal diameter 2 cm, length 25 cm) was slurry packed with normal phase silica gel in chloroform/methanol/acetic acid (90:9:1) to a depth of 15 cm. The crude anhydrofulvic acid was dissolved in this solvent mixture and applied to this column. The column was eluted isocratically with the same solvent mixture.
The eluate containing the anhydrofulvic acid (as shown by tic analysis on silica gel plates, eluted with
CHCl3/MeOH/CH3CO2H (99:9:1) giving a yellow spot at Rf = 0.45) was concentrated to dryness to give a yellow solid (45 mg). This was dissolved in THF-0.05 aqueous acetic acid (1:1, 8 ml) and purified by preparative reverse phase hplc on the same NovaPak octadecylsilica column used in Example 3 eluted isocratically with THF/0.05M aqueous acetic acid (3:7) at a flow rate of 10 ml/min. The eluate was monitored at 370 nm and that containing the peak at retention time 20-21 minutes was collected and extracted with ethyl acetate. The ethyl acetate extract was
concentrated to dryness to yield pure anhydrofulvic acid as a yellow solid (16 mg).
(II) The fractions containing the peak at retention time 3-8 minutes were collected, extracted with ethyl acetate and the ethyl acetate extract concentrated to give crude citromycetin. The crude citromycetin was dissolved in THF-0.05M aqueous acetic acid (1:9, 5 ml) and purified by preparative reverse phase hplc on the same PrepPak column eluted isocratically with THF/0.05M aqueous acetic acid (1:9) at a flow rate of 15 ml/minute. The eluate was monitored at 300 nm and that containing the peak at
retention time 6-9 minutes was collected and extracted with ethyl acetate. The ethyl acetate extract was concentrated to near dryness and dissolved in THF-0.05M aqueous acetic acid (1:9, 2ml). On standing a precipitate formed in this sample. This was collected and dried to give pure
citromycetin as a pale yellow solid (18mg).
Example 7 - Detection of Compounds bv Analytical High
Performance Licruid Chromatography (hplc)
Partially purified fractions from the preparative reverse phase fractions described in Examples 3 and 6 were examined for the presence of the desired compound by analytical reverse phase hplc. The analysis was carried out on an hplc system comprising a Waters 600E Multisolvent Delivery System, a Waters U6K Injector and a Waters 990 Photodiode array detector using a Waters Radial Pak
cartridge (8mm ID × 100mm length, packed with Nova-Pak octadecylsilica, 60 A pore size, 4μM particle size. Waters) contained in a Waters Radial Compression Module eluted isocratically with methanol-0.05M aqueous acetic acid (1:1) at a flow rate of 2 ml/minute.
(a) The compound having the molecular formula C27H16O12 was identified by its UV/visible spectrum using the
Photodiode array detector (λ max under acidic conditions: 366 ± 3nm, 320 ± 3nm, 207 nm). It appeared as a peak typically having a retention time in the range 2.5-3.5 minutes.
(b) The compound having the molecular formula C14H10O6 was identified by its UV/visible spectrum using the Photodiode array detector (λ max under acidic conditions: 390 + 4 nm, 316sh ± 5 nm, 256 ± 5 nm, 216 ± 5 nm). It appeared as a peak typically having a retention time in the range 7.5-9 minutes.
(c) Anhydrofulvic acid was identified by its UV/visible spectrum using the photodiode array detector (λ max under acidic conditions : 385 ± 4 nm, 343sh ± 5 nm, 230 ± 5 nm, 210 ± 3 nm). It appeared as a peak typically having a retention time in the range 7-9 minutes using a mobile phase composition of methanol: 0.5M aqueous acetic acid (55:45) at a flow rate of 2 ml/min. Its spectral
characteristics are as follows:
Figure imgf000029_0001
103.2 , 101.2 , 94.4 ,
64.1, 19.9
MS: m/z [Desorption chemical ionisation (DCI), NH3]:
308 [(MNH4)+], 291 [MH+], 273 [(MH-H2O)+], 247 [(MH- CO2)+]
(d) Citromycetin was identified by its UV/visible spectrum using the photodiode array detector (λ max under acidic conditions: 368 ± 4nm, 302 ± 4nm, 254 ± 4nm, 222 ± 4nm). It appeared as a peak typically having a retention time in the range 2.5-3 minutes. Its spectral
characteristics are as follows:
Figure imgf000030_0001
920, 855, 795
1H nmr: δ (500 MHz, (CD3)2SO) ppm: 6.48 (1H,s), 6.22
(1H,s), 4.92 (2H,s), 2.25 (3H,s)
MS: m/z [Desorption chemical ionisation (DCI), NH3] :
308 [(MNH4)+], 291 [MH+], 273 [(MH-H2O)+],
247 [(MH-CO2)+]
Example 8 - Pharmaceutical composition
Tablets, each weighing 0.15g and containing 25mg of the present compound can be manufactured as follows: Composition for 10,000 tablets
Present compound (250g)
lactose (800g)
corn starch (415g)
talc powder (30g)
magnesium stearate (5g)
The present compound, lactose and half the corn starch are mixed. The mixture is then forced through a sieve 0.5mm mesh size. Corn starch (10g) is suspended in warm water (90ml). The resulting paste is used to granulate the powder. The granulate is dried and
comminuted on a sieve of 1.4mm mesh size. The remaining quantity of starch, talc and magnesium stearate is added, carefully mixed and processed into tablets.

Claims

1. A compound selected from:
(a) a compound which has a molecular formula of C27H16O12, a molecular weight of 532 and the following ultraviolet (UV), infrared (ir), nuclear magnetic resonance
(NMR) and mass spectral (MS) characteristics:
UV: λ MeOH (log10E) nm: 432(4.28); 365sh(4.10);
max 282sh(4.10); 233(4.54);
211(4.56)
UV: λ MeOH-0.1M HCl (aqueous)
max (log10E) nm: 366(4.13); 320(4.22);
207(4.57)
UV: λ MeOH-0.1M NaOH (aqueous)
max (log10E) nm: 464(4.14); 371(4.20);
236(4.54)
ir: ʋ KBr cm-1: 3416-3040, 1710sh, 1694,
max 1650sh, 1617, 1601, 1553,
1462, 1410sh, 1401, 1359, 1305, 1186, 1159, 1045,
1024, 986, 960
1H-NMR (400MHz): δ/ppm in (CD3)2SO :
8.69 (1H, d, J=2.2HZ), 8.04 (1H, d, J=2.2Hz), 7.04 (1H, s), 6.87 (1H, s), 6.38 (1H, s), 2.83 (3H,s), 2.29 (3H,s)
13C-NMR (100MHz): δ/PPm in (CD3)2SO :
199.3, 196.3, 172.7, 167.4, 158.7, 154.6, 152.9, 152.0,
150.6, 150.3, 148.3, 143.3, 141.0, 134.2, 129.6, 128.7,
128.0, 123.4, 121.5, 120.3, 111.5, 110.7, 109.7, 102.9,
102.3, 31.5, 31.1
MS: m/z [Fast atom bombardment (FAB), MNBA]:
533 (MH+), 515[(MH-H2O)+]
m/z [FAB, MNBA(-ve ion)]: 531 [(M-H)-3, 513
([M-H-H2O]-3
m/Z[DCI, NH3]: 533 (MH+), 489 ([M-CO2]+);
and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof;
(b) a compound which has a molecular formula of C14H10O6, a molecular weight of 274 and the following
ultraviolet (UV), infrared (ir), nuclear magnetic resonance (nmr) and mass (MS) spectra:
Figure imgf000033_0001
1H-nmr (400 MHz): δ/ppm in (CD3)2SO:
12.90 (1H,s), 11.3 (1H, br s), 8.62 (1H,s), 7.13 (1H,d, J=2.4 Hz), 6.57 (1H,d, J=2.4Hz), 5.69 (1H, br s), 5.40 (1H, g, J=6.7 Hz), 1.46 (3H, d, J=6.7 Hz)
13C-nmr (100 MHz): δ/ppm in (CD3)2SO: 183.2, 178.3, 165.0,
164.9, 164.0, 144.9, 137.3, 122.0, 114.7,
110.4, 107.9, 107.2, 60.5, 20.9
MS: m/z [electron impact (El)]: 274 [M+], 259 [(M-CH3)+],
231 [(M-CH3CO)+]
m/z [Desorption chemical ionisation
(DCI), NH3]: 275[MH+] and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof.
(c) anhydrofulvic acid and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof; and
(d) citromycetin and pharmaceutically and veterinarily acceptable salts, esters and ethers thereof; for use in a method of treatment of the human or animal body by therapy.
2. A compound according to claim 1 for use as a CD4 binding agent.
3. A compound according to claim 1 for use as a collagenase inhibitor.
4. A compound according to claim 1 which is a said compound (a) or (b) for use as an inhibitor of Protein Kinase c.
5. Use of a compound as defined in claim 1 in the manufacture of a medicament for use as a CD4 binding agent, a collagenase inhibitor or, in the case of a said compound (a) or (b), an inhibitor of Protein Kinase C.
6. A pharmaceutical or veterinary composition comprising a pharmaceutically or veterinarily acceptable carrier or diluent and, as active ingredient, a compound as claimed in claim 1.
7. A said compound (a) as defined in claim 1.
8. A said compound (b) as defined in claim 1.
9. A process for the preparation of a compound as defined in claim 1, which process comprises (i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain X8063 (CMI 336456) or a mutant thereof which
produces the said compound having the molecular formula C27H16O12, the said compound having the molecular formula C14H10O6, anhydrofulvic acid or citromycetin; (ii) isolating the said compound having the molecular formula C27H16O12, the said compound having the molecular formula C14H10O6,
anhydrofulvic acid or citromycetin from the fermentation medium; and (iii) if desired, converting the isolated compound into a pharmaceutically or veterinarily acceptable salt, ester or ether thereof.
10. A CD4 binding agent or a collagenase inhibitor comprising a compound as defined in claim 1.
11. A Protein Kinase C inhibitor comprising a said compound (a) or (b) as defined in claim 1.
12. A method of treatment a human or animal with a CD4 binding agent or a collagenase inhibitor, which method comprises administering to a human or animal in need of such treatment an effective amount of a compound defined in claim 1.
13. A method of treating a human or animal with a Protein Kinase C inhibitor, which method comprises
administering to a human or animal in need of such
treatment an effective amount of a said compound (a) or (b) defined in claim 1.
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WO1993018173A3 (en) 1993-10-28
GB2280439B (en) 1995-11-22

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