GB2279071A - Xanthone derivative - Google Patents

Abstract

The novel compound of formula (I): <IMAGE> has activity as an inhibitor of cell proliferation and can be used to treat disorders such as atherosclerosis and cancer. The compound is produced by (i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain X08/33/298 (IMI 355998) or a mutant thereof which produces the compound of formula (I); and (ii) isolating the said compound from the fermentation medium.

Description

PHARMACEUTICAL COMPOUND The present invention relates to a compound which can inhibit cell proliferation and therefore has utility in treating disorders that are the result of uncontrolled cell proliferation, for example cancer. The invention further relates to the preparation of the said compound, to pharmaceutical and veterinary compositions containing it and to the microorganism from which it is obtained.

Many disease states have been described that are the result of uncontrolled cell proliferation. This proliferation can occur in a range of cell types and can result in disorders such as atherosclerosis, restenosis, psoriasis and cancer. An agent which can inhibit cell proliferation will therefore be useful in the control and treatment of such disorders.

It has now been found that fermentation of a nutrient medium with a strain of the fungus Asperaillus fumiaatus (Xenova organism X08/33/298, IMI 355998) produces a novel xanthone derivative which inhibits cell proliferation.

Accordingly, the present invention provides the xanthone derivative of formula (I):

The xanthone derivative (I) is methyl l-hydroxy, 7,8epoxy, 3-hydroxymethylxanthone-8-carboxylate. It is hereafter referred to as the present compound.

The present compound has been isolated from a microorganism which we have designated strain X08/33/298 and which has been identified as a strain of the fungus Asperaillus fumiaatus on the basis of the morphological data which follow.

Fungal strain X08/33/298 was incubated at 25 C on Czapek Dox agar. Colonies reached 25-35mm diameter after 7 days growth and produced a dull yellow soluble pigment. In reverse, colonies were uncoloured to dull yellow. Aerial mycelium was floccose and white without exudation but conidial heads rendered the more central regions of the colony dark turquoise to dark green or dull green.

Conidial heads were radiate to columnar and uniseriate comprising globose conidia (2-3 mm diameter) which were finely roughened to spinose. Conidiophores consisted of an uncoloured smooth walled stipe (200 - 400 mm X 5 - 11 mm) expanding into a spathulate vesicle (15 - 30 mm diameter) which was pigmented green in the upper half. The phialides (5-9 mm X 2 - 3 mm) were similarly pigmented, covered the green portion of the vesicle and sometimes extended to cover the upper two thirds.

The above described morphological and microscopic characteristics of strain X08/33/298 are almost identical with those described for Aspergillus fumigatus Fres. by Klich M.A. and Pitt J.I. (A Laboratory Guide to Aspergillus Species and their Telemorphs, CSIRO, 1988). Strain X08/33/298 is distinctive in its very floccose texture, poor sporulation and lower than average radial extension rate.

The strain X08/33/298 was isolated from a soil sample and was deposited at the International Mycological Institute, Egham, Surrey, UK on 6th February 1993 under accession number IMI 355998.

The above description is illustrative of a strain of AsDeraillus fumiqatus which can be employed in the production of the present compound. However, the present invention also embraces mutants of the above described microorganism. For example, those 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, are also included within the ambit of this invention.

The present invention further provides a process for the preparation of the present compound, which process comprises (i) fermenting, in a source of carbon, nitrogen and inorganic salts, fungal strain X08/33/298 (IMI 355998) or a mutant thereof which produces the present compound and (ii) isolating the present compound from the fermentation medium.

The present compound is typically produced during the aerobic fermentation of an aqueous nutrient medium under conditions described hereinafter, with a producing strain of Asperaillus fumiaatus X08/33/298, or a producing mutant strain of X08/33/298. 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. A biologically pure culture of the fungal strain X08/33/298 or of a mutant which produces the present compound is a further aspect of the invention.

Such cultures are substantially free from other microorganisms.

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 anti-foaming agent may be present to control excessive foaming and added at intervals as required.

Cultivation of Asneraillus fumigatus X08/33/298 can be generally effected at temperatures ranging from 20"C to 30"C and especially around 25"C. It is desirable that the organism is cultivated with aeration and agitation e.g. by shaking or stirring. The medium may be initially inoculated with a small quantity of the sporulated microorganism but in order to avoid a growth lag a larger vegetative inoculum may be used, prepared from a seed culture.Seed cultures may be prepared by inoculating a small quantity of culture medium with the spore form of the organism, and the vegetative inoculum obtained may be transferred to the fermentation medium, or, more preferably to one or more intermediate seed stages where further growth takes place before transfer to the principal fermentation medium. The fermentation may be generally carried out in the pH range 5.0 - 7.5, preferably 5.5 6.5, for a period of 2 - 10 days, e.g. about 5 days.

The present compound is found primarily in the mycelium on termination of the fermentation of strain X08/33/298 and may be removed and separated. Its separation from the mycelium and its recovery is typically achieved by solvent extraction followed by application of chromatographic fractionations with various chromatographic techniques and solvent systems. The present compound has thus been isolated in pure form in this way.

The present compound is soluble in organic solvents such as dimethylsulphoxide, ethyl acetate and methanol/dichloromethane. Thus, in one recovery method, the present compound is extracted from the culture filtrate with ethyl acetate or by using a hydrophobic resin such as Diaion HP20. In cases where the compound is found associated with the mycelium, it may be extracted with solvents such as methanol/dichloromethane.

Further purification may be achieved by chromatography (flash chromatography or HPLC). Flash chromatography is suitably performed using silica as adsorbent and various proportions of chloroform/methanol as eluent. HPLC is suitably performed using octadecyl silica as adsorbent and water and acetonitrile in various proportions as eluent. Analytical high pressure liquid chromatography (HPLC) or thin layer chromatography (TLC) may be used to monitor the presence of the desired compound.

The use of the foregoing and other known techniques will afford purified compositions containing the present compound, the presence of which is determined by analysing the various chromatographic fractions for physicochemical characteristics.

Cell proliferation is regulated primarily by the cellular environment and state of differentiation. The environment provides both positive, growth stimulatory and negative, growth inhibitory signals. Proliferation is regulated by the balance between these signals. The state of differentiation also dictates whether and how a cell will respond to particular environmental signals.

All cells possess receptors containing an extracellular ligand binding domain and an intracellular catalytic domain having tyrosine kinase activity, the so called tyrosine kinase growth factor receptors. The expression of a specific receptor gene is dependent on the cell type. The ligand for these receptors is normally specific. It is believed that the binding of the ligand to the receptor leads to an activation of the tyrosine kinase catalytic domain, which in turn leads to activation of the intracellular signalling pathway and eventually to DNA synthesis and cell proliferation.

It has been established that many human malignancies are correlated with the presence and expression of oncogenes in the human genome. The oncogenes are derived from normal cellular genes, termed proto-oncogenes, by mutation, inappropriate expression or amplification. These changes generally lead to signals producing uncontrolled proliferation or signals leading to cellular dedifferentiation. A number of proto-oncogenes are related to either a tyrosine kinase growth factor receptor or its ligand.

The epidermal growth factor (EGF) receptor, a tyrosine kinase receptor, is frequently over expressed in a wide variety of human cancers and other proliferative disorders, such as psoriasis. The ligand for this receptor, TGFa, is also often over expressed in transformed cells. Cancers of the breast, oesophagus, brain and pancreas contain cells which over-express the EGF receptor.

It has been demonstrated that inhibition of the receptor activation using a monoclonal antibody can inhibit the growth of tumours over-expressing the EGF receptor (Modjtahhedi H., Styles, J.N. and Dean, C.J.: The human EGF receptor as a target for cancer therapy: Six new rat MAbs against the receptor on the breast carcinoma NBA-MB 468, British J. Cancer, 1993, in press).

The present compound can inhibit cell proliferation mediated by activation of the EGF receptor by TGFa (and other receptor tyrosine kinases), as shown in Examples 4 and 5. It therefore has utility in controlling and treating disease states and disorders which are the result of uncontrolled cell proliferation, such as atherosclerosis, restenosis, psoriasis and cancer.

Furthermore, the toxicity of the present compound is displayed at a concentration much higher than that required to inhibit the epidermal growth factor receptor tyrosine kinase. This is because, owing to the multitude of growth stimulatory pathways, inhibition through this receptor tyrosine kinase does not seem significantly to affect other rapidly dividing cells.

A human or animal, e.g., a mammal can thus be treated by a method comprising administration thereto of a therapeutically effective amount of the present compound.

In this way, the condition of the human or animal may be improved. Amelioration of the disease state or disorder from which the human or animal is suffering can be achieved. Specific examples of cancers which the present compound may be used to treat include leukaemias and common epithelial cancers exemplified by carcinoma of the lung, colon and breast.

The present compound 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 compound may therefore be given by an 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 from 0.3 mg/kg to 30 mg/kg, most commonly in the range of 2 to 10 mg/kg, body weight. Such a dosage may be given from 1 to 5 times daily.

The present compound is 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 methods and are administered in a pharmaceutically or 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 filmcoating 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, the present compound may be encapsulated within liposomes.

The following examples illustrate the invention: EXAMPLE 1: Culture of Aspergillus fumigatus (strain X08/33/298) in liquid medium.

Starting material of Aspergillus fumigatus (strain X08/33/298) was generated by suspending a mature slant culture grown on PDA (2%D-(+)-glucose, 0.4% potato extract, 1.5% agar) in 5 ml 10% aqueous glycerol. 1 ml of this suspension, in a 1.5 ml cryovial, comprises the starting material which was retrieved from storage at -135 C. A static seed culture was produced by aseptically placing 0.5 ml starting material in 10 ml nutrient solution S(2% malt extract, 2% D-(+)-glucose, 0.1% peptone), which was incubated for 7 days at 25"C. An intermediate culture was produced by aseptically transferring the static seed culture into a 250 ml flask containing 40 ml of nutrient solution I (1% D-(+)-glucose, 1.5% glycerol, 1.5% soya peptone, 0.3% NaC1, 0.1% CaCO3, 0.5% malt extract, 0.1% Junlon PW110, 0.1% Tween 80, pH 6.0) which was incubated at 25"C on an orbital shaker (50 mm throw) at 240 rpm for 4 days. The culture was then macerated by the addition of sterile broken glass pieces followed by vigorous agitation on a vortex mixer.

A production culture was generated by aseptically transferring a macerated intermediate culture, into a 31 stirred fermenter, containing 21 of nutrient solution P (2.5% sucrose, 0.59% glutamic acid, 0.1% K2HPO4, 0.98% MES, 0.01% FeSO4.7H2O, 0.05% MgSO4.7H2O, 0.05% KC1, 20 ml vitamin mixture, 5 ml trace element mixture I). The fermentation was carried out at 25"C with an agitation rate of 500 rpm and an aeration rate of 0.25 vvm. After 5-7 days the production culture was harvested for extraction.

Aseraillus fumiaatus (X08/33/298) also grew well as intermediate cultures incubated in 25 mm X 200 mm inclined tubes shaken at 240 rpm for 8 days at 25"C in nutrient solution P.

Vitamin mix consists of (mgl1): thiamine HC1, 25; riboflavin, 25; sodium pantothenate, 25; nicotinic acid, 25; pyridoxine HC1, 25; thioctic acid, 25; folic acid, 2.5; biotin, 2.5; cyanocobalamin, 2.5; potassium paminobenzoate, 2.5; vitamin K1, 50 Ctl.

Trace elements mixture I consists of (g 11): 1M H2SO4, (lml); ZnSO4.7H2O, 0.278; MnSO4.4H2O, 0.223; H3BO3, 0.062; CuSO4.5H2O, 0.125; NazoO4.H2O, 0.048; CoCl2.6H2O, 0.048; KI, 0.083.

EXAMPLE 2: Extraction and purification of (I) from fermentation of strain X08/33/298 A 2 litre fermentation of organism X08/33/298 was centrifuged to remove the mycelium. The aqueous supernatant (1.11) was extracted with ethyl acetate (2 x 500 ml) and the ethyl acetate extract was concentrated to dryness under reduced pressure to give a red gum containing (I). Half of this red gum was extracted with dichloromethane (2 x 50 ml; ultrasonication) and the dichloromethane extract evaporated to dryness. The dry dichloromethane extract was washed repeatedly with acetonitrile until the washings became colourless. The residual grey powder (35 mg) was purified by flash chromatography on silica (2.5 x 20 cm), purified xanthone derivative (I) eluting with 10 % methanol in chloroform.

The above procedures were repeated on the remaining ethyl acetate extract to yield a yellow-green powder (43 mg) of the xanthone derivative (I).

(I) has the following ultraviolet (uv) infrared (IR), nuclear magnetic resonance (NMR) and mass spectral (MS) characteristics: UV: tmax MeCN nm: 200, 215 (sh), 278, 350 IR: Vmax KBr (diffuse reflectance) cm1: 3300 (br), 1755, 1660, 1600, 1020.

H NMR: 6 (400 MHz, (CD3)2SO) ppm: 12.1 (1H, s), 7.23 (lH,dd, J = 1.5, 9.8 Hz), 7.03 (1H, d, J = 1.2 Hz), 6.95 (1H, dd, J = 1.5, 9.8 Hz), 6.81 (1H, d, J = 1.2 Hz) 5.50 (1H, t, J = 5.4 Hz), 4.60 (2H, d, J = 4.9 Hz), 4.32 (1H, dd, J = 1.5, 3.8 Hz), 3.80 (3H, s).

13C NMR: 6 (100 NHz, (CD3)2SO)ppm: 179.8, 166.4, 159.6, 159.3, 154.8, 153.1, 138.3, 125.5, 112.2, 108.8, 108.1, 104.4, 62.1, 56.6, 55.9, 52.5.

NS m/z (DCI, NH3) : 317 (MH+) m/z (EI) 316 (M+; 20%), 284 (M-CH3OH, 80%), 141 (100%).

EXAMPLE 3: Pharmaceutical composition Tablets, each weighing 0.15g and containing 25 mg of the present compound, can be manufactured as follows: Composition for 10 000 tablets Present compound (250 g) lactose (800 g) corn starch (415 g) talc powder (30 g) magnesium stearate (5 g) The present compound, lactose and half the corn starch are mixed. The mixture is then forced through a sieve 0.5 mm mesh size. Corn starch (10 g) is suspended in warm water (90 ml). The resulting paste is granulated to a powder. The granulate is dried and comminuted on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium stearate is added, carefully mixed and processed into tablets.

EXAMPLE 4: Activity of (I) in an EGF receptor tyrosine kinase assay in vitro Materials The epidermoid carcinoma cell line A-431, which expresses high levels of the EGF receptor, was grown in DMEM supplemented with 10% foetal calf serum, glutamine and bicarbonate and incubated at 37 C in 5% CO2. Cell membranes were prepared by washing A-431 cells in phosphate buffered saline (PBS) before scraping off into PBS. After centrifugation at 800 g for 3 minutes the pellet was resuspended in lysis buffer (5mM Tris-HCl pH 7.4, lmM magnesium chloride, 2mM disodium ethylenediaminetetraacetate and 2pg/ml soya bean trypsin inhibitor, 50 g/ml phenylmethylsulphonyl fluoride) and incubated at O"C for 20-30 minutes.

The suspension was homogenised in a Potter homogeniser followed by centrifugation at 5000g for 5 minutes. The supernatant was centrifuged at 100,000 g for 1 hour to pellet the membrane material. The pellet was resuspended in solubilization buffer (25mM HEPES pH 7.4, 20% v/v glycerol, 1% v/v Triton X100 lmg/ml lipid free bovine serum albumin and 0.05% w/v sodium azide), at lml per 175cm2 flask, followed by incubation at 00C for 30 minutes with occasional gentle agitation. Undissolved material was removed by centrifugation at 100,000 g for 30 minutes. The resulting preparation was stored in aliquots at -70iC until required.

Method To 6 1 of reaction buffer (50 mM HEPES buffer at pH 7.4) was added 4 l of a solution of the present compound is dimethyl sulphoxide at various concentrations, 10O1 enzyme substrate mixture (Poly [Glu6:Lys3:Tyr] at 8.2 mg/ml and EGF at 6g/ml), 10p1 of the membrane prep and 10 l [#-32P) ATP (0.04mCi). The reaction was incubated at 37 C for 40 minutes before halting by the addition of 20 l acid stop buffer. 30p1 of the reaction mix was spotted onto a charged filter mat (Amersham, NK8932) which was washed for 30 minutes in 75mM orthophosphoric acid (500 ml per sheet).

Once dry the samples were counted, with scintillant in a ss counter.

The results, expressed as IC50 values, are set out in Table 1: TABLE 1 Activitv of (I) Against EGF Preceptor in vitro

EXPERIMENT ICw ILK 1 253 2 443 EXAMPLE 5: Activity of (I) against cultured cell lines Cell culture experiments were performed with NR6-HER (murine fibroblasts normally deficient in the expression of EGF receptor transfected with human EGF receptor cDNA), MDA MB 468 (human breast adenocarcinoma expressing moderate levels of the EGF receptor) and EMT6 (mouse mammary tumour) cells. Stock cultures were grown in 75cm2 flasks containing DMEM medium, 10% foetal calf serum, glutamine and bicarbonate in 5% CO2 gassed incubators.Cells were dissociated on passage with 0.25% trypsin and 3mM EDTA in saline. Experimental cultures were plated in 96 well microtitre plates containing 200 l of growth medium per well at a density of 10,000 cells per well. The present compound (I) was added by doubling dilutions after the cells had been allowed to attach. After incubation for 3 to 4 days the cells were fixed and stained according to the method of Skehan et al (Journal of the National Cancer Institute, 1990, 82, 1107-1112). In this method, cells were fixed in 50ill 50% cold trichloroacetic acid, layered onto the growth medium to produce a final TCA concentration of 10%. After one hour at 40C the plates were washed in tap water. After drying the cells were stained in 0.4% (wt/vol) sulphorhodamine B dissolved in 1% acetic acid for 30 minutes. The plates were then rinsed quickly four times in 1% acetic acid. After air drying the dye was solubilised in 10mM unbuffered Tris base (pH 10.5) for 5 minutes on a shaker. The optical density was read at 540nm for maximum sensitivity or between 490-530 nm so all experimental points remained in the linear range.

The results, expressed as IC50 values, are shown in Table 2: TABLE 2 Activitv of (I) aaainst Cultured Cell lines

Cell Line IC50 in nM NR6-HER 950 NDA MB 468 1400 EMT6 EMT6 790

Claims (8)

1. CLAIMS 1. The xanthone derivative of formula (I):
2. 2. 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 X08/33/298 (IMI 355998) or a mutant thereof which produces the xanthone derivative (I) of claim 1; and (ii) isolating the said derivative from the fermentation mycel ium.
3. 3. A compound as claimed in claim 1 for use in a method of treatment of the human or animal body by therapy.
4. 4. A compound according to claim 3 for use as an inhibitor of cell proliferation.
5. 5. A compound according to claim 4 for use in the treatment of atherosclerosis, restenosis, psoriasis or as an anti-tumour agent.
6. 6. A pharmaceutical or veterinary composition comprising a pharmaceutically or veterinarily acceptable carrier or diluent and, as active ingredient, the compound as defined in claim 1.
7. 7. A biologically pure culture of the fungal strain X08/33/298 (IMI 355998) or a mutant thereof which produces the xanthone derivative of formula (I) as defined in claim 1.
8. 8. A process for fermenting fungal strain X08/33/298 (IMI 355998) or a mutant thereof which produces the xanthone derivative of formula (I) defined in claim 1, which process comprises fermenting strain X08/33/298 or a mutant thereof in a source of carbon, nitrogen and inorganic salts.