Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
  • C07D498/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • 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
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial 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
  • 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
  • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
• • 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
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/465—Streptomyces

Definitions

• the invention relates to a novel secondary metabolite obtainable from a mutant strain of Streptomvces longisporoflavus having the formula
• salts of compound (I) or (II) are, for example, salts with suitable mineral acids, such as hydrohalic acids, sulfuric acid or phosphoric acids, e.g., hydrochlorides, hydrobromides, sulfates, hydrogen sulfates or phosphates, salts with suitable aliphatic or aromatic sulfonic acids or N-substituted sulfamic acids, e.g.
• methanesulfonates benzenesulfonates, p-toluenesulfonates or N-cyclohexylsulfamates (cyclamates), or salts with strong organic carboxylic acids, such as lower alkanecarboxylic acids or optionally unsaturated or hydroxylated aliphatic dicarboxylic acids, e.g. acetates, oxalates, malonates, maleates, fumarates, malates, tatrates or citronates.
• strong organic carboxylic acids such as lower alkanecarboxylic acids or optionally unsaturated or hydroxylated aliphatic dicarboxylic acids, e.g. acetates, oxalates, malonates, maleates, fumarates, malates, tatrates or citronates.
• the invention further relates to a process for the production of the compound of the formula (I), which process comprises cultivating Streptomvces longisporoflavus mutant strain M14 (DSM 8325) in a suitable nutrient medium and isolating said compound.
• the process comprises cultivating Streptomvces longisporoflavus mutant M14 (DSM 8325) in a culture medium containing assimilable sources of carbon, sources of nitrogen and mineral salts, separating the cells from the culture broth and extracting it with an organic water-immiscible solvent, separating the organic phase from the aqueous phase, concentrating the organic phase by evaporation and purifying the resulting crude compound of the formula (I) by means of chromatography and/or crystallization from the same or a different organic water-immiscible solvent.
• aqueous solution or suspension which contains suitable sources of carbon and nitrogen as well as mineral salts.
• suitable sources of carbon and nitrogen are complex organic substances, such as proteins and protein hydrolysates, typically casein, peptone, meat extract, yeast extract, cotton seed, soybean meal, peanut meal, sun-flower seed derived protein nutrient, etc.
• suitable mineral salts typically include chlorides, carbonates, sulfates and phosphates of alkali metals and alkaline earth metals, for example sodium, potassium, calcium and magnesium, and also commercially available trace element mixtures. It is also advantageous to add further growth-promoting substances, especially vitamins, conveniently in the form of commercially available standard vitamin solutions.
• Cultivation is carried out aerobically, i.e. conveniently in surface cultures, or preferably submerged with shaking or stirring while introducing air or oxygen into the shaking flask or fermenter. Cultivation is preferably carried out in the neutral pH range, i.e. at about pH 5-8, preferably at pH 6-7, and in the temperature range from about 25-30°C, preferably at about 28°C.
• the cultivation time is preferably from 3 to 5 days.
• the biomass is separated from the culture broth when cultivation is complete, and extracted with an organic water-immiscible solvent.
• Suitable organic solvents include esters of aliphatic acids, typically lower alkyl esters, such as methyl or ethyl esters, of lower alkanoic acids such as acetic acid, and halogenated hydrocarbons, e.g. methylene chloride or chloroform. Extraction of the aqueous culture broth can be done twice or three times. After concentrating the combined extracts by evaporation, the residue is further purified by chromatographic means, e.g. by means of column chromatography (such as HPLC or flash chromatography) or TLC. Alternatively or additionally, the product may be crystallized from an organic water-immiscible solvent such as one of those detailed above.
• the compound of the formula (I) can be conveniently crystallized e.g. from ethyl acetate.
• the novel compound has valuable, especially pharmacologically valuable, properties and can be used for the prevention and treatment of diseases in a mammal, such as a human or an animal.
• the compound of the formula (I) is a selective inhibitor of isotypes of the enzyme proteinkinase C. While staurosporine of the formula (II) shows no significant differences in the inhibition of isotypes of the enzyme proteinkinase C, the compound of the formula (I) most selectively and preferentially inhibits isotypes alpha, beta-2 and gamma whilst, e.g., isotypes beta-1 and eta are inhibited to a much lower degree (by a factor of 5-10).
• Isotypes alpha, beta-2 and gamma of proteinkinase C are inhibited by the compound of the formula (I) already at a concentration (IC 50 ) of approximately 0.04 ⁇ mol/liter.
• the compound of the formula (I) inhibits other enzymes, for example proteinkinase A, protein-phosphorylase-kinase and protein-tyrosin-kinase, only at much higher concentrations. This demonstrates the surprising selectivity of the compound of the formula (I).
• the compound of the formula I has antiproliferative properties which can be demonstrated by the inhibitory action on the growth of human T24 bladder carcinoma cells.
• the compound of the formula (I) can be used, in particular, for the prophylaxis or treatment of diseases which respond to proteinkinase C inhibition. It can also be used as an antitumor active ingredient, for example for the therapy of tumors of the bladder.
• the novel compound can be used furthermore for the treatment of multidrug resistant tumors, in particular P-glycoprotein mediated resistance, and shows synergistic effects when combined with other antitumor active compounds, particularly all those showing cross-resistance in MDR (multidrug resistance) like doxorubicin, daunorubicin, vincristine, vinblastine, etoposide, taxol, mitomycin C, actinomycin D or mitoxantrone.
• MDR multidrug resistance
• the compound of the formula (I) is a potent inhibitor of the osteoclast proton pump (OPP).
• Osteoclasts are a class of bone cells responsible for bone resorption which, during normal bone remodeling, is followed faithfully by osteoblastic bone formation. Osteopenia results from an imbalance of the opposing activities of osteoclasts and osteoblasts such that the rate of bone resorption exceeds the rate of accretion. Osteoclasts resorb bone by secreting protons and lysosomal enzymes into a sealed extracellular lacuna beneath the cell. The resulting low pH demineralizes the bone and activates the secreted lysosomal enzymes which then digest the exposed organic collagen matrix.
• OPP osteoclast proton pump
• the protons are pumped out of the osteoclasts by an ATP-dependent H + -pump (H + -ATPase proton pump, osteoclast proton pump) located in the ruffled border membrane of the cell bordering the extracellular lacuna. Inhibition of proton secretion would offer a way to control bone loss.
• the osteoclast proton pump appears to be a member of the multimeric vacuolar class of H + -ATPase enzymes present in the membrane of a variety of intracellular vacuoles (e.g. clathrin-coated vesicles, chromaffin granules, lysosomes).
• the compound according to the invention is an extremely potent inhibitor of H + - ATPase proton pump isolated from clathrin-coated vesicles from bovine brain.
• the inhibitory concentration (IC 50 ) is approximately 0.0006 ⁇ M.
• the compound according to the invention can be used for the prevention or treatment of the elevated bone loss characteristic of a variety of pathological conditions such as primary osteoporosis (juvenile, idiopathic, congenital, postmenopausal, involutional) and secondary osteopenia associated with maUgnancy, renal insufficiency, endocrinopathies, malabsorption syndromes, hepatic disease, immobilization, arthropathies and iatrogenic causes.
• pathological conditions such as primary osteoporosis (juvenile, idiopathic, congenital, postmenopausal, involutional) and secondary osteopenia associated with maUgnancy, renal insufficiency, endocrinopathies, malabsorption syndromes, hepatic disease, immobilization, arthropathies and iatrogenic causes.
• the inventive compound can be used in a method for protecting nerve cells of mammal from degeneration induced by excitatory amino acids. Examples are Alzheimer's disease, moto neuron disease, Parkinson's disease, cerebrovascular disease, AIDS dementia, epilepsy, Huntington's disease and concussive or penetrating injuries to the brain or spinal cord.
• the inventive compound can be administered in combination with a neurotrophic factor, preferably a member of the neurotrophic family, and most preferably nerve growth factor (NGF).
• a neurotrophic factor preferably a member of the neurotrophic family, and most preferably nerve growth factor (NGF).
• the invention also relates to pharmaceutical compositions comprising a therapeutically effective amount of the active ingredient, in particular an amount which is effective for the prophylaxis or therapy of one of the above-mentioned diseases, together with pharmaceutically acceptable carriers which are suitable for topical, for example in the treatment of skin tumors, enteral, for example oral or rectal, or parenteral (for example i.v., s.c. or transdermal) administration, and which can be inorganic or organic, solid or liquid.
• pharmaceutically acceptable carriers which are suitable for topical, for example in the treatment of skin tumors, enteral, for example oral or rectal, or parenteral (for example i.v., s.c. or transdermal) administration, and which can be inorganic or organic, solid or liquid.
• compositions which are used for oral administration are, in particular, tablets or gelatine capsules which comprise the active ingredient together with diluents, for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol, and/or lubricants, for example silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
• diluents for example lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycerol
• lubricants for example silica, talc, stearic acid or salts thereof, such as magnesium stearate or calcium stearate, and/or polyethylene glycol.
• Tablets can also comprise binders, for example magnesium aluminum silicate, starches such as maize, wheat or rice starch, gelatine, methylcellulose, sodium carboxylmethylcellulose and/or polyvinylpyrrolidone and, if desired, disintegrants, for example starches, agar, algic acid or a salt thereof such as sodium alginate, and/or effervescent mixtures, or adsorbants, colorants, flavourings and sweeteners.
• binders for example magnesium aluminum silicate, starches such as maize, wheat or rice starch, gelatine, methylcellulose, sodium carboxylmethylcellulose and/or polyvinylpyrrolidone and, if desired, disintegrants, for example starches, agar, algic acid or a salt thereof such as sodium alginate, and/or effervescent mixtures, or adsorbants, colorants, flavourings and sweeteners.
• the pharmacologically active compound of the present invention can furthermore be used in
• Such solutions are preferably isotonic aqueous solutions or suspensions, it being possible for those to be prepared prior to use, for example in the case of lyophilized compositions which comprise the pure active ingredient or the active ingredient together with a carrier, for example mannitol.
• the pharmaceutical compositions can be sterilized and/or comprise excipients, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating the osmotic pressure and/or buffers.
• compositions which, if desired, can comprise further pharmacologically active substances, are prepared in a manner known per se, for example by means of conventional mixing, granulating, sugar-coating, dissolving or lyophilization processes and comprise from approximately 0.01 % to 90 %, in the case of lyophilizates up to 100 %, in particular from approximately 0.1 % to approximately 50 %, of the active ingredient, a concentration of active ingredient of below 1 % being particularly suitable for compositions for topical administration.
• the invention also relates to the use of the compound of the formula I, preferably in the form of pharmaceutical compositions, for the prophylaxis or the therapeutic treatment of the human or animal body, in particular in the case of the above-mentioned diseases.
• the invention especially relates to a method for inhibiting proteinkinase C, especially the above-mentioned isotypes thereof, in a warm-blooded species requiring such a treatment, whereby an effective proteinkinase C-inhibiting dose of the compound of the formula I is administered to this representative of a warm-blooded species.
• the invention likewise especially relates to a method for inhibiting osteoclast proton pump in a warm-blooded species requiring such a treatment, whereby an effective osteoclast proton pump-inhibiting dose of the compound of the formula I is administered to such warm-blooded species.
• the dose of the active ingredient depends, inter alia, on the type and severity of the disease, the nature and size of the species to be treated, the age and condition of the species, the response to treatment and the way in which the active ingredient is administered.
• a representative of a warm-blooded species having a body weight of approximately 70 kg is administered a daily dose of 1 mg to 1500 mg, preferably 10 mg to 500 mg, of the compound of the formula I.
• This total daily dose is preferably split into 1-3 daily administrations.
• the dose in the case of oral administration is approximately two to three times higher than in the case of parenteral administration, i.e. rather in the upper range of the above-mentioned doses.
• novel compound of the formula (I) is furthermore a valuable intermediate for the production of other pharmaceutically active compounds such as compounds inhibiting subtypes of proteinkinase C or osteoclast proton pump.
• novel strain Streptomvces longisporoflavus M14 DSM 8325 is a further embodiment of the present invention.
• the examples which follow illustrate the invention without any restriction whatsoever.
• soluble starch 20 g/1 yeast extract 4 g/1 agar 20 g/1 pH before sterilization: about 7
• the cultures exhibited a white to grey color on the surface of the mycelium.
• a sterile Tween 80 solution (0.5%) was added to each plate. Spores were removed by scraping the surface of these plates with an inoculating loop by gentle pressure. The spore suspension obtained was then transferred to a glass Petri dish. The spore concentration is controlled and determined with a microscope. The suspension was irradiated under the UV lamp with constant agitation: 500m W/cm 2 during 50". The irradiated spore suspension was then diluted by factor 100 to 1000, left for 1 day at room temperature in the dark, and then plated out on complete medium plates (sporulation agar SPA). The plates were left incubated at 28°C in the dark for 5 days and then they were replica-plated to "minimal medium” (MM)
• mutant M 14 One colony appeared to have a poor and thin development on both minimal media but a normal development on the complete medium (so-called ghost colony) and was designated mutant M 14.
• the fermentation is started using 2 inoculum stages.
• Media used for the preculture and fermentation are the following ones:
• fermentation medium NL 3 (antifoam SAG 471 is added only if necessary), see above.
• the temperature used is about 28°C.
• the inoculum stages are performed at a pH from about 7.6, whereas the fermentation is performed at a pH from about 6.0 to 7.0. a. First Preculture
• Cryo-tubes, slants or agar plates are used to inoculate Erlenmeyer flasks containing the medium NL148a. The flask is left to incubate at 28°C for 48 hours at 220 rpm.
• One milliliter of fresh culture is used to inoculate 100 ml of medium NL 148a in 500 ml Erlenmeyer flasks with 1 baffle (1% inoculum). The flasks are left to incubate at 28°C for 48 hours at 250 rpm.
• IR (KBr): 3420, 3252, 3049, 2930, 1660, 1587, 1458, 1420, 1398, 1348, 1317, 1283, 1250, 1227, 1173, 1151, 1126, 1101, 1070, 1045, 1020, 955, 922, 851, 822, 789, 773, 744, 663, 631 cm "1 (the most prominent absorption bands are underlined).
• the fermentation broths obtained from different fermenter cultures are combined to yield 77 1.
• the whole broth is extracted by vigorous stirring with twice the volume of ethyl acetate.
• the filter cake is washed with an additional volume of 301 of ethyl acetate.
• the combined organic phases are separated, concentrated and evaporated to dryness.
• the crude extract is triturated with 11 of n-heptane. The evaporation of the heptane phase gives a residue of a thin oil, which according to TLC analysis is completely devoid of staurosporine related compounds.
• the heptane-insoluble residue is dissolved in dichloromethane which leads to a spontaneous crystallization of a first crop of crystals.
• the raw crystallizate is dissolved in methanol, treated with active carbon and filtered. The clear filtrate is concentrated and recrystallized from ethyl acetate.
• the mother liquor and the previously obtained solution in dichloromethane are combined and chromatographed on a 1 1 column with silicagel Si 60 (Merck, particle size 15-25 ⁇ m).
• the fractions containing the desired product are eluted with a solvent mixture consisting of 95 % dichloromethane and 5 % methanol, combined and evaporated to dryness. Rechromatography of these fractions using the same method leads to semipure material. Recrystallization from ethyl acetate yields a second crop of pure substance.
• the mother liquors are further purified by a repeated chromatography and recrystallization cycle which produces a third crop of pure substance.
• Tablets comprising 50 mg of active ingredient are prepared in the customary manner in the following composition:
• the active ingredient is mixed with some of the wheat starch, the lactose and colloidal silica, and the mixture is passed through a sieve.
• Some more wheat starch is made into a paste with 5 times the amount of water in a water bath, and the powder mixture is kneaded with this paste until a slightly kneadable composition has been formed.
• the kneadable composition is pressed through a sieve of mesh size approx. 3 mm and dried, and the dry granules obtained are passed again through a sieve.
• the remainder of the wheat starch, the talc and the magnesium stearate are thereinafter admixed, and the mixture is compressed to give 1000 notched tablets of 357 mg weight each containing 50 mg of active ingredient.
• the strain Streptomvces longisporoflavus M14 was deposited on May 27, 1993 with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM) (German Collection of Microorganisms and Cell Cultures) in Braunschweig, FRG, under No. DSM 8325.
• DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen

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  • 1994-06-09 WO PCT/EP1994/001883 patent/WO1995000520A1/en not_active Application Discontinuation
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  • 1994-06-09 JP JP7502384A patent/JPH08511785A/ja active Pending
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