Classifications

• • 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
  • C07D493/10—Spiro-condensed systems
• • 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
  • 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

Definitions

• This invention relates to novel anti-tumor, anti-allergy, and anti-inflammatory compounds, to their preparation, and to pharmaceutical compositions containing such compounds. More specifically, this invention relates to a fermentation broth, and the component parts thereof obtainable by cultivation of a fungus Nattrassia mangiferae.
• acyl means a straight or branched chain acyl, that is formyl or an alkanoyl, of 1 to 6 carbons.
• exemplary of acyl are formyl, acetyl, propionyl, butyryl, and the like.
• a broken line (m ) denotes a chemical bond below the plane of the page, while a solid line ( ⁇ ——) denotes a chemical bond above the plane of the page.
• SINEPT means Selective Insensitive Nuclei Enhanced Through Polarization Transfer
• APT means Attached Proton Test
• DEPT means Distortionless Enhancement of Polarization Transfer
• HETCOR means Heteronuclear correlation
• NOE means Nuclear Overhauser Effect
• TMS means tetramethylsilane.
• the present invention provides novel compounds A, B, C, D, E, F, G r H and acetate of A which have activity as anti-tumor agents, anti-allergy agents, and anti-inflammatory agents.
• the present invention provides a pharmaceutical composition which comprises an effective amount of a compound of the formula A, B, C, D, E, F, G, H or acetate of A. .
• the present invention provides methods for the treatment of tumors, allergy and inflammation.
• the present invention provides a biologically pure culture of the fungus Nattrassia man ⁇ iferae. said culture being capable of producing the compounds A, B, C, D, G and H as defined above, in a recoverable quantity upon fermentation under aerobic conditions in an aqueous medium containing assimilable sources of nitrogen and carbon.
• the present invention provides a process for the preparation of a novel fungal broth complex which comprises cultivating the producing fun ⁇ us Nattrassia man ⁇ iferae. in a nutrient medium under aerobic conditions until substantial activity is imparted to the medium. Specifically, we have employed the fungus Nattrassia man ⁇ iferae. ATCC 74078. CHARACTERIZATION OF THE PRODUCING MICROORGANISM
• the fungus substrate was collected in Guatemala in an arid area approximately 300 meters in elevation. The fungus itself was isolated from dead leaves about one meter above ground. The fungus was deposited in Schering Central Culture Collection as SCF 0642. A representative subculture of the fungus designated Nattrassia man ⁇ iferae has been deposited and is now a part of the permanent collection of the American Type Culture Collection, Rockville, Maryland, where it has been assigned accession number ATCC 74078.
• Nattrassia man ⁇ iferae ATCC 74078 Subcultures of Nattrassia man ⁇ iferae ATCC 74078 are available upon request to the aforementioned agency.
• this invention embraces mutants and variants thereof that exhibit the same or substantially the same taxonomical and physiological properties of the deposited strain which properties include the production of compounds A, B, C, D, G, and H.
• This fungus is an isolate of the type species of the genus Nattrassia. specifically, N. man ⁇ iferae Sutton & Dyko, 1989 (Mycol. Res. 93 (4):466-488, 1989).
• Nattrassia is in the class Coelomycetes, order Sphaeropsidales, family Sphaeriaceae.
• N.- man ⁇ iferae is the current name of this fungus, until recently, it was known by its synonym, Hendersonula toruioidea Nattrass, 1933, ("Transactions of the British Mycological Society” 18:189-197).
• the fungus is most commonly identified in laboratory culture by the presence of its synanamorph Scvtalidium dimidiatum (Penz.) comb. nov. [ident. Torula dimidiata], Sutton & Dyko (Mycol. Res. 93 (4):466-488, 1989) created this combination of genus name and species name to include isolates commonly known as the "Scvtalidium state of Hendersonula toruioidea" by which this fungus had been initially identified.
• Scvtalidium dimidiatum differs from most other Scvtalidium species which are thermophilic, acidophilic, or ectomycorrhizal on Ericaceous plants. Spore morphology distinguishes it from S. li ⁇ nicola and S.-jaDonicum.
• the fungus was isolated from a substrate collected along the road between Cuilapa and Chiquimullilla, Guatemala. The area is arid and approximately 300 meters in elevation. The fungus was isolated from dead leaves about one meter above ground.
• Nattrassia man ⁇ iferae ATCC 74078 is the ability to produce compounds A, B, C, D, G and H.
• a loopful of a culture of the fungus which was maintained on Potato Dextrose Agar (Difco) was transferred to a germination medium consisting in grms/l of: proteose peptone #3, 5.0; sodium chloride, 5.0; potassium phosphate (monobasic ), 5.0; yeast extract, 3.0: cerelose, 20.0: and soy grits, 5.0.
• the culture was incubated at 24°C for 6 days at 300 rpm.
• a 5% inoculum of this culture is transferred to the same germination and incubated in a similar manner.
• Inoculum developed in this manner was transferred to fermentation medium consisting in grams/I of neopeptone, 10.0; and cerelose, 40.0.
• a six day fermentation at 24°C produced a material with protein kinase C (PKC) inhibitory activity.
• PKC protein kinase C
• the SCF 0642 complex, containing at least six biologically active components was removed from the fermentation by ethyl acetate extraction, followed by adsorbing onto XAD-16 ( a non ionic resin) and eluting with methanol.water gradient.
• XAD-16 a non ionic resin
• Six major components A, B, C, D, G, and H were separated from the complex by reverse phase chromatography using methanol: water and acetonitrile:water as the eluting solvent system.
• the PKC inhibitors were isolated and purified by the procedure given in Fig. 1 below.
• the culture broth (40 L) was extracted with ethyl acetate at harvest pH.
• the EtOAc extract was evaporated in vacuo.
• the residue was dissolved in methanol and absorbed on a small amount of XAD-16 resin, in order to prepare a plug impregnated with. the active components.
• the plug was loaded onto an XAD-16 column and eluted with a 0-100% aqueous methanol gradient.
• the 80-100% MeOH fractions were combined as the crude active complex.
• the crude material was chromatographed on a CHP-20P column and eluted with a gradient of aqueous methanol (0-100%).
• the early PKC-active eluate, 60% MeOH fraction mainly contained the compound A based on TLC analysis (Rf value: 0.38; plate: SG60, F254, Merck; solvent system: 98:2, CH ⁇ Cla/MeOH). This fraction was further purified by chromotography on CHP-20P eluting with 20-40% aqueous acetonitrile to obtain the pure Compound A (120 mg).
• the later PKC-active eluate, 70% MeOH fraction was found as a mixture by TLC analysis (three major components, Rf values were 0.21 , 0.29, 0.31 , respectively, under the same conditions as above).
• the acetate of compound A was made as follows: Compound A was treated with acetic anhydride/pyridine to afford the compound A acetate. More specifically, the following reaction procedure was employed.
• Ci-Ce esters may be prepared.
• the compounds which may be prepared by such methods are of the following formula:
• the physico-chemical properties, spectroscopic and crystallographic data of nine active compounds of the invention are summarized in Tables 1 , 2, 3, 4, 5, 6, 7, 8 and 9 below.
• the biological activities are summarized in tables 10 and 11.
• the compounds gave a negative response to ninhydrin and Rydon tests, and a positive response to iodine vapor.
• These PKC inhibitors are soluble in chloroform, dichloromethane and dimethyl sulfoxide; partially soluble in ethyl acetate, methanol and acetone; insoluble in water, petroleum ether and hexane.
• the structures of the compounds were determined by analysis of the spectroscopic data including UV, IR, MS (FAB, Cl and El- HRMS), H and 1 3C NMR spectra and an X-ray diffraction study for
• Compound F Compounds A, A-acetate, B, C, D E, F, G, and H have the following physical and spectral data shown in Tables 1 -9. Detailed assignments of each proton and carbon for these compounds were achieved by APT, DEPT, HETCOR, and SINEPT experiments. The relative stereochemistry of all compounds was established based on x- ray crystallographic studies on compound F and NMR spectroscopic data obtained from NOE experiments.
• the crystal structure was solved by direct methods (MULTAN11/82). Approximate coordinates for all nonhydrogen atoms were derived from an E-map. Hydrogen atoms were located in a series of difference Fourier syntheses evaluated following several rounds of full-matrix least squares refinement of positional and thermal parameters of the carbon and oxygen atoms (at first isotropic, then anisotropic). In the next rounds of least squares calculations, hydrogen atom positional and isotropic thermal parameters and an extinction correction were included as variables. In the course of these iterations, the hydrogen atoms of the acetyl methyl group did not refine to physically acceptable positions, and so these atoms were incorporated at calculated positions during the final least squares cycles. A final difference Fourier synthesis contained no unusual features.
• Compounds B, C, D, E, G and H can be assigned the following relative stereochemistry based on the correlation of relative stereochemistry of F and A, and on an analysis of the NMR spectroscopic data:
• Protein kinase C is a Ca 2+ and phospholipid-dependent protein kinase involved in mediating a wide variety of cellular responses to growth factors, hormones, oncogenes and other modulators of growth control .
• PLC Protein kinase C
• the present invention provides compounds which are potent inhibitors of PKC.
• Table 10 demonstrates the inhibition of PKC in vitro with potencies in the micromolar range.
• cytotoxic activity versus T24 bladder carcinoma cells was evaluated ( results also shown in Table 10). All compounds were cytotoxic to the T24 bladder carcinoma cells with potencies in the low micromolar range. Cytotoxicity to the T24 cells was compared to cytotoxicity versus a normal, non-transformed cell line, HEPM, grown in co-culture.
• Compounds A, C , D , G and H isolated from the fungal broth demonstrated the surprising result of having greater potency versus the transformed cells as compared to the normal cells (Table 10). Taken together, these data indicate the potential antitumor therapeutic activity of these compounds.
• the ability of transformed cells to invade normal tissue and metastasize to distant sites is also a hallmark of cancer.
• the compounds which are the subject of this invention were also evaluated for ability to inhibit the invasive properties of tumor cells in vitro.
• Table 10 displays data which shows the ability of these compounds to inhibit the invasion of HT1080 fibrosarcoma cells through a reconstituted basement membrane (Matrigel). This activity supports the potential anti- invasive and anti-metastatic therapeutic activity of these compounds.
• PKC is an important regulatory enzyme that exhibits broad tissue distribution and is thought to play an important role in the regulation of cellular functions. For example, activation of PKC has been associated with stimulus-secretion coupling leading to the release of mediators such as histamine from mast cells and basophils. Furthermore, an inhibitor of PKC staurosporine can inhibit these processes (1 ). In addition to mast cells and basophils, other cells closely associated with inflammatory conditions are also activated by PKC (2). Asthma is a condition of acute respiratory distress superimposed on an underlying chronic pulmonary inflammation. The eosinophil is the major infiltrating cell in asthma that contributes to pulmonary inflammation (3). The present invention describes compounds that are inhibitors of PKC with the potential to inhibit eosinophil accumulation in allergic airways.
• guinea pigs were anesthetized with a combination of subcutaneous ketamine HCI (50mg/kg) and xylazine (10 mg/kg).
• BAL was performed by flushing the lungs once with 5 ml 0.9% saline using a tracheal cannula.
• the lavage fluid was centrifuged (150 x g for 10 minutes at 4° C) to pellet cells.
• the cell pellet was resuspended in 2.5 ml saline, and total cells were counted using a standard hemocytometer.
• Cytospin preparations were made using a Shandon cytocentrifuge (150 x g for 10 minutes at room temperature).
• the cells were fixed and stained using Leukostat ® (Fisher Scientific, San Francisco, CA). Differential counts on at least 200 cells were conducted using morphologic criteria to classify cells into neutrophils, eosinophils, and monocytes.
• TPA means 12-O-tetradecanoylphorbol- 13-acetate.
• mice Male hairless mice (3-6 mice per group) were received at 4-6 weeks of age from Charles River Laboratories.
• TREATMENT - Mice were anesthetized with metaphane and placed on their side.
• the dorsal skin was topically treated first with compounds (25 ⁇ l methanol), followed with 30 seconds by treatment with TPA (0.5 nm - 1 nm in a volume of 12.5 to 15 ⁇ l). All treatments were accomplished by m ⁇ cropipette.
• the treatment with 25 ⁇ l of compounds was spread over an average area of 2 cm 2 to 3 cm 2 .
• the treatment with 12.5 to 15 ⁇ l of TPA was spread over an average area of 2 cm 2 and was applied in the center of the area previously treated with compounds.
• Cell line M27, 2x10 5 cells were injected subcutaneously into the flank.
• mice Group B vehicle control (HP-BCD) 5 mice Group C Cytoxan 250 mg/kg x 1 5 mice
• mice Cytoxan 125 mg/kg x 1 5 mice
• Formulation and route Drugs were prepared as a fine suspension in HP-BCD and given i.p. In the data in Tables 13, 14, 15 and 16, Cytoxan is used as a positive control and vehicle only treated animals are defined as the control group at 100%.
• Cell line Murine lung carcinoma M27, 2 x 10 5 cells were injected subcutaneously into the flank.
• Drugs were prepared as a fine suspension in HP-BCD and given i.p.
• Results The experiment ended on day 36. Lung colonies from animals that died on days 30 through 36 were counted. Animals that died before day 30 were excluded because lung colonies were too small to be determined. A and A acetate significantly reduced the . number of lung colonies as compared to the control groups (Table 15).
• compounds of the invention have activity as anti-tumor agents.
• G, and H have been isolated from the fungus Natrissia. man ⁇ iferae. ATCC 74078. All compounds including A, B, C, D, E, F, G, H and the acetate of A contain a highly strained polycyclic ketal-ring system which presents a unique structural feature. This unusual class of compounds demonstrates potential utilities in tumor biology, as well as anti-allergy and anti-inflammatory therapy.
• R is H or acyl
• compositions containing compounds of the invention to treat tumors allergy, and inflammation.
• Compounds of the invention can be administered as tablets, capsules, suspensions or aerosols. They can be administered orally, subcutaneously, intravenously, or by inhalation.
• a minimal dosage required for anti- allergy activity is generally between 1 and 1000 milligrams one to four times daily.
• the compounds of this invention can be administered in any number of conventional dosage forms, e.g., topical, oral, parenteral, rectal, transdermal, and the like.
• Oral or rectal dosage forms include capsules, tablets, pills, powders, cachets, and suppositories.
• Liquid oral dosage forms include solutions and suspensions.
• Parenteral preparations include sterile solutions and suspensions.
• Topical dosage forms can be aerosols, creams, ointments, lotions, transdermal devices (e.g., of the conventional patch or matrix type) and the like.
• compositions contemplated by the above dosage forms can be prepared with conventional pharmaceutically acceptable excipients and additives, using conventional techniques.
• pharmaceutically acceptable excipients and additives are intended to include carriers, binders, flavorings, buffers, thickeners, coloring agents, stabilizing agents, emulsifying agents, dispersing agents, suspending agents, perfumes, preservatives lubricants, etc.
• Suitable pharmaceutical acceptable solid carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low melting waxes, cocoa butter and the like.
• Capsules can be made wherein the active compound is inserted into the capsules along with a pharmaceutically acceptable carrier.
• the active compounds of this invention can be mixed with pharmaceutically acceptable excipients or be used in finely divided powder form without excipients for inclusion into the capsules. Similarly, cachets are included.
• Liquid form preparations include solutions, suspensions and emulsions such as water or water-propyiene glycol solutions for parenteral injection. Liquid preparations can also be formulated in solution in polyethylene glycol and/or propyiene glycol, which may contain water.
• Aqueous solutions suitable for oral use can be prepared by adding the active component in water and adding suitable colorants, flavors, stabilizing, sweetening, solubilizing and thickening agents as desired.
• Aqueous suspensions suitable for oral use can be made by dispersing the active component in finely divided form in water with viscous material, i.e., pharmaceutically acceptable natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose and other well-known suspending agents.
• Formulations for topical application may include, the above liquid forms, as well as creams, aerosols, sprays, dusts, powders, lotions and ointments which are prepared by combining an active ingredient according to this invention with conventional pharmaceutical acceptable diluents and carriers commonly used in topical, dry, liquid, cream and aerosol formulations.
• Ointment and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
• bases may, thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil.
• Thickening agents which may be used according to the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propyiene glycol, polyethylene glycols, woolfat, hydrogenated lanolin, beeswax, etc.
• Lotions may be formulations with an aqueous or oil base and will, in general, also include one or more of pharmaceutically acceptable stabilizing agents, emulsifying agents, dispersing agents, suspending agents, thickening agents, coloring agents, perfumes and the like.
• Powders may be formed with the aid of any suitable pharmaceutically acceptable powder base, e.g., talc, lactose, starch, etc.
• Drops may be formulated with an aqueous base or non- aqueous base also comprising one or more pharmaceutically acceptable dispersing agents, suspending agents, solubilizing agents, etc.
• the topical pharmaceutical compositions may also include one or more preservatives or bacteriostatic agents, e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, etc.
• preservatives or bacteriostatic agents e.g., methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chlorides, etc.
• topical pharmaceutical compositions may also contain an active compound of this invention in combination with other active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics and antipruritic agents.
• active ingredients such as antimicrobial agents, particularly antibiotics, anesthetics, analgesics and antipruritic agents.
• solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for either oral or parenteral administration.
• liquid forms include solutions, suspensions and emulsions.
• solid form preparations are most conveniently provided in unit dose form and as such are used to provide a single liquid dosage unit.
• sufficient solid may be provided so that after conversion to liquid form, multiple individual liquid doses may be obtained by measuring predetermined volumes of the liquid form preparation as with a syringe, teaspoon or other volumetric container. When multiple liquid doses are so prepared, it is preferred to maintain the unused portion of said liquid doses under conditions which retard possible decomposition.
• the solid form preparations intended to be converted to liquid form may contain, in addition to the active material, pharmaceutically acceptable flavorants, colorants, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents and the like.
• the solvent utilized for preparing the liquid form preparation may be water, isotonic water, ethanol, glycerine, propyiene glycol and the like as well as mixtures thereof. Naturally, the solvent utilized will be chosen with regard to the route of administration, for example, liquid preparations containing large amounts of ethanol are not suitable for parenteral use.
• the compounds of this invention may also be deliverable transdermally for systemic distribution.
• the transdermal compositions can take the form of creams, lotions and/or emulsions and can be included in a transdermal patch of the matrix or reservoir type as are conventional in the art for this purpose.
• a composition of the invention comprises a therapeutically effective amount of a compound of the invention in combination with a pharmaceutically acceptable carrier material.
• the compounds of this invention may be administered by any conventional mode of administration by employing a therapeutically effective amount of a compound of this invention for such mode.
• the dosages may be varied depending upon the requirements of the patient in the judgment of the attending clinician, the severity of the condition being treated and the particular compound being employed. Determination of the proper dosage for a particular situation is within the skill of the art. Treatment can be initiated with smaller dosages which are less than the optimum dose of the compound. Thereafter, the dosage should be increased by small increments until the optimum effect under the circumstances is reached. For convenience, the total daily dosage may be divided and administered in portions during the day if desired.

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• 1993
  • 1993-04-01 WO PCT/US1993/002737 patent/WO1993020081A1/en active Application Filing
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