EP0664706A1 - Inhibition de proteine-kinase c et nouveau compose appele balanol - Google Patents

Inhibition de proteine-kinase c et nouveau compose appele balanol

Info

Publication number
EP0664706A1
EP0664706A1 EP92919060A EP92919060A EP0664706A1 EP 0664706 A1 EP0664706 A1 EP 0664706A1 EP 92919060 A EP92919060 A EP 92919060A EP 92919060 A EP92919060 A EP 92919060A EP 0664706 A1 EP0664706 A1 EP 0664706A1
Authority
EP
European Patent Office
Prior art keywords
protein kinase
compound
approximately
pharmaceutically acceptable
balanol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP92919060A
Other languages
German (de)
English (en)
Other versions
EP0664706A4 (fr
Inventor
Yali F. Hallock
William P. Janzen
Lawrence M. Ballas
Palaniappan Kulanthivel
Christie Boros
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Eli Lilly and Co
Original Assignee
Sphinx Pharmaceuticals Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sphinx Pharmaceuticals Corp filed Critical Sphinx Pharmaceuticals Corp
Publication of EP0664706A4 publication Critical patent/EP0664706A4/fr
Publication of EP0664706A1 publication Critical patent/EP0664706A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D223/00—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom
    • C07D223/02—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings
    • C07D223/06—Heterocyclic compounds containing seven-membered rings having one nitrogen atom as the only ring hetero atom not condensed with other rings with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D223/12—Nitrogen atoms not forming part of a nitro radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to diagnosis and treatment for inflammatory, cardiovascular and neoplastic diseases. More particularly, the present invention relates to novel compounds derived from fungi of the genus Verticillium, especially the fungus Verticillium balanoides (Dreschler) Dqwsett et al . , useful for inhibiting activity of the enzyme protein kinase C.
  • a preferred composition has been normal Balanol.
  • Protein kinase C is a family of calcium stimulant and phospholipid-dependent serine/threonine-specific protein kinases which play an important role in cellular growth control, regulation, and differentiation. Protein kinase C is also fundamental to the processes involved in tumorigenicity, since it is the major high-affinity receptor for several classes tumor promoters as well as for endogenous cellular diacylglycerols. These tumor promoters also stimulate protein kinase C catalysis. Direct activation of protein kinase C by tumor promoting phorbol esters has been reported. See Castagna et al. J. Biol . Chem . , 257:7847 (1982).
  • Protein kinase C is activated by diacylglycerol (DAG) , a neutral lipid, and when activated will transfer the ⁇ - phosphate of MgATP to a serine or threonine residue on a substrate protein.
  • DAG diacylglycerol
  • protein kinase C Since the activation of protein kinase C has been implicated in several human disease processes, including cancer tumors, inflammation, and reperfusion injury, inhibition of protein kinase C should be of great therapeutic value in treating these conditions.
  • Certain protein kinase C inhibitors have been reported to potentiate the antitumor activity of cis- platin both in vitro and in vivo, Grunicke et al., Adv. Enzyme Regul . 28 : 201 (1989); and German Offenlegungsschrift DE 3827974.
  • protein kinase C would be a potential target for therapeutic design because of its central role in cell growth. See Tritton, T.R. and Hickman, J.A .
  • inhibitors of protein kinase C have the potential for blocking all three of the most significant mechanisms of pathogenesis associated with myocardial reperfusion injury, and should thus have a decided therapeutic advantage. Additionally, the inhibitory effect of protein kinase C inhibitors on keratinocytes, and on the oxidative burst . in neutrophils will lead to an anti- inflammatory effect. Inflammation and reperfusion injury, particularly pertaining to cardiac injury, are common conditions for which there exists no definitive treatment despite extensive research, and appropriate treatments for these conditions are needed.
  • German Offenlegungsschrift DE 3827974 Al discloses preparations comprising a protein kinase C inhibitor such as in combination with a lipid, a lipid analog, a cytostatic agent or phospholipase inhibitor useful for cancer therapy.
  • Calphostins A to I secondary metabolites isolated from Caldosporium cladosporides , represent another group of protein kinase C inhibitors, with Calphostin C as the most active compound in this series. See Kobayashi et al., J. Antibiotics 42:1470-1474 (1989).
  • protein kinase C has been implicated in several human disease processes, including cancer tumors, inflammation, and reperfusion injury. There remains, however, a long-felt need for efficacious inhibitors of protein kinase C for therapeutic use.
  • novel compound of the invention isolated from fungi of the genus Verticillium, preferably the fungus Verticillium balanoides .
  • a preferred, novel compound of the invention has been named Balanol.
  • the novel compounds of the invention are potent protein kinase C inhibitors, inhibiting protein kinase C activity at nanomolar concentrations.
  • Balanol When substantially pure, the preferred compound of the invention, Balanol, is soluble in dimethyl sulfoxide, water, and methanol; insoluble in ethyl acetate and chloroform; gives a positive color reaction with polymolydbic acid, ninhydrin reagent, and ferric chloride; is negative with Dragendorff's reagent and Iodoplatinate spray; has an R f value of approximately 0.58 with silica gel thin layer chromatography with n-butanol/acetic acid/water at a ratio of 4:1:1 respectively; and has a molecular weight of approximately 550. Balanol is believed to have the following formula:
  • the compounds of the present invention are further useful for treating conditions related to or affected by inhibition of protein kinase C activity, particularly hyperproligenative diseases such as cancers, inflammatory disease, myocardial reperfusion injury, and cardiac dysfunctions related to reperfusion injury.
  • Inhibition of protein kinase C activity can lead to inhibition of growth of tumor cells and can thereby produce an anti-tumor effect.
  • inhibition of protein kinase C activity can also lead to inhibition of the oxidative burst in neutrophils, platelet aggregation, and keratinocyte proliferation, leading to an anti-inflammatory, effect.
  • the inhibitory activities of the compounds of the invention against platelet aggregation, neutrophil activation, and neutrophil release demonstrate its usefulness in treating reperfusion injury, particularly myocardial reperfusion injury.
  • Figure 1 i Illustrates an 1H NMR spectrum of Balanol using a 300 MHz dual proton-carbon probe of 5mm.
  • Figure 2 illustrates a C-13 NMR spectrum of Balanol using a 300 MHz dual proton-carbon probe of 5mm.
  • the present invention provides a novel protein kinase C inhibitors especially Balanol isolated from fungi of the genus Verticillium, preferably from the fungus Verticillium balanoides .
  • Verticillium balanoides has been described by Dowsett et al. (1982) Mycologia 74 : 687-690.
  • the fungus used for the isolation of the compound of the invention was recovered from a yellow rhizomorph in Pinus palustris needle litter located in a U.S. Forest Service P. palustris forest near Hoffman, North Carolina.
  • the compound of the invention may also be present in other Verticillium species, or variants, mutants, or recombinants of Verticillium balanoides , or in other organisms, however, at the present time the preferred source of the compounds of the invention is Verticillium balanoides .
  • Verticillium balanoides was deposited in the American Type Culture Collection, Rockville, Maryland, 20852 on July 19, 1991 and has accession number 74082.
  • Balanol can be isolated from the aforementioned fungus using the method described herein in the Examples. Balanol has been characterized; it is believed that the compound has the following physico-chemical characteristics. .
  • the compound generally comprises a light yellow amorphous solid that is soluble in dimethyl sulfoxide (DMSO) , water, and methanol, but is insoluble in ethyl acetate and chloroform.
  • DMSO dimethyl sulfoxide
  • the compound gives positive color reactivities with phosphomolybdic acid (PMA) reagent (20% by weight solution in ethyl alcohol) , ninhydrin reagent, and ferric chloride, but is negative with Dragendorff's reagent and Iodoplatinate spray reagent.
  • PMA phosphomolybdic acid
  • the R f value of the compound is approximately 0.58 using 0.25 mm E Merck silica gel 60F 254 thin layer chromatography (TLC) plate developed with n-butanol/acetic acid/water in a ratio of 4:1:1 respectively.
  • the molecular weight of the compound is approximately 550.
  • Other physical characteristics of Balanol are disclosed in Example 3.
  • the invention relates to the compound having the properties described above together with pharmaceutically acceptable salts of such a compound.
  • the invention relates to pharmaceutical preparations which comprise a pharmaceutically acceptable carrier in combination with either a compound having the properties described above or pharmaceutically acceptable salts of such a compound.
  • modifications of the Balanol nucleus will also be likely to find utility in therapeutics and as a protein kinase C inhibitor.
  • Balanol can be represented by the following formula:
  • Balanol is useful for treating conditions related to, or affected by inhibition of protein kinase C activity, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.
  • the invention provides methods and pharmaceutical compositions for inhibiting protein kinase C activity. Preferred methods comprise contacting mammalian tissues and/or fluids with an inhibitory amount of the compound of the invention.
  • the pharmaceutical compositions of the invention comprise the compound of the invention preferably in a pharmaceutically acceptable carrier or diluent and in a protein kinase C inhibitory amount.
  • the invention also provides methods of inhibiting an oxidative burst in neutrophils which comprise contacting a neutrophil with a protein kinase C inhibitory amount of the compound of the invention, or contacting the neutrophil with an amount of the compound of the invention effective to inhibit such oxidative burst.
  • the invention further provides methods for treating inflammation which comprise administering to a mammal suffering from inflammation a protein kinase C inhibitory amount of the compound of the invention, or administering to the mammal an amount of the compound of the invention effective to inhibit inflammation.
  • This invention additionally provides methods for inhibiting growth of mammalian tumor cells which comprise contacting a mammalian tumor with a protein kinase C inhibitory amount of the compound of the invention, or contacting the tumor cell with an amount of the compound of the invention effective to inhibit growth of the tumor.
  • Another embodiment of the invention provides methods of inhibiting mammalian keratinocyte cell proliferation which comprise administering to a mammalian keratinocyte a protein kinase C inhibitory amount of the compound of the invention, or administering to the keratinocyte an amount of the compound effective to inhibit proliferation of the keratinocyte.
  • Cancer is a disease characterized in part by uncontrolled cell growth. Protein kinase C is directly involved in cellular growth control and is believed to be involved in tumor formation. Protein kinase C is the major, if not exclusive, intracellular receptor of phorbol esters which are very potent tumor promoters. Phorbol esters and other tumor promoters bind to and activate protein kinase C. Since diacylglycerol (DAG) and phorbol esters interact at the same site, DAG's have been suggested to be the "endogenous phorbol esters" by analogy with the opiate receptor where the conservation of a high affinity receptor implied the existence of an endogenous analogue.
  • DAG diacylglycerol
  • DAG has been shown to increase the affinity of protein kinase C for Ca + and phospholipid and thus activates protein kinase C at cellular levels of these essential cofactors.
  • Extracellular signals including hormones, growth factors, and neurotransmitters are known to stimulate phosphatidylinositol turnover resulting in the generation of IP 3 and DAG.
  • Structures of 40 distinct oncogenes of viral and cellular origin have revealed that oncogenes encode altered forms of normal cellular proteins.
  • Several of the gene products appear related to growth factors or other elements involved in transmembrane signalling. These oncogene products appear to function by altering the level of critical second messengers.
  • PMA protein kinase C
  • oncogenes such as ras
  • ras oncogenes
  • protein kinase C may mediate the actions of certain oncogenes, such as ras , which cause intracellular increases in DAG and concomitant increases in protein kinase C.
  • activation of protein kinase C leads to the expression of c- myc, c-fos , c-cis , c-fms , nuclear protooncogenes important in cell transformation.
  • Overexpression of protein kinase C in NIH 3T3 cells causes altered growth regulation and enhanced tumorigenicity and in rat fibroblasts leads to anchorage- independent growth in soft agar. In these experiments, overexpression of protein kinase C in these cells resulted in tumor formation in animals receiving transplanted cells.
  • ischemic-related myocardial damage can be attributed to polymorphonuclear leukocytes (neutrophils) which accumulate at the site of occlusion. Damage from the accumulated neutrophils may be due to the release of proteolytic enzymes from the activated neutrophils or the release of reactive oxygen intermediates (ROI) .
  • ROI reactive oxygen intermediates
  • Much of the "no reflow" phenomenon associated with myocardial ischemia is attributed to myocardial capillary plugging. The plugging of capillaries has been attributed to both aggregated platelets and aggregated neutrophils. Although both cell types are aggregated during the ischemic event, the relative contribution of each to capillary plugging has not yet been established.
  • Superoxide dis utase has been shown to be a particularly effective scavenger of superoxide, but suffers from a very short half-life in the blood.
  • SOD Superoxide dis utase
  • Several companies have tackled this problem by creating versions of this enzyme with increased half-lives by techniques such as liposome encapsulation or polyethylene glycol conjugation. Reports on the effectiveness of these new version are mixed.
  • Catalase, a scavenger of hydrogen peroxide, and hydroxyl radical scavengers have also been tested and found to be effective to varying degrees.
  • the compound of the invention may be administered by any method that produces contact of the active ingredient with the agent's site of action in the body of a mammal or in a body fluid or tissue including but not limited to oral, topical, hypodermal, intravenous, intramuscular and intraparenteral.
  • the compound may be administered singly or in combination with other compounds, other pharmaceutical compounds, such as chemotherapeutic compounds, or in conjunction with therapies, such as radiation treatment.
  • the compound of the invention is preferably administered as a composition comprising the compound of the invention and a pharmaceutically acceptable carrier or diluent selected on the basis of the selected route of administration and standard pharmaceutical, practice.
  • novel compound of the invention is administered to mammals, preferably humans, in therapeutically effective amounts which are effective to inhibit protein kinase C, or to inhibit tumor cell growth, inhibit inflammation of tissue. inhibit keratinocyte cell proliferation, inhibit oxidative burst from neutrophils or inhibit platelet aggregation.
  • the dosage administered in any particular instance will depend upon factors such as the pharmacodynamic characteristics of the compound of the invention, its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment, frequency of treatment, and the effect desired.
  • the daily dosage of the compound will be in the range of from about 1 ⁇ g to about 100 mg per kg of body weight, preferably from about 1 ⁇ g to about 1 mg per kg body weight, and more preferably from about 10 ⁇ g to about 1 mg per kg per day, and preferably administered in a single dosage or in divided dosages.
  • Persons of ordinary skill will be able to determine dosage forms and amounts with only routine experimentation based upon the considerations of this invention.
  • Pharmaceutically acceptable salts of the compound of the invention are also within the scope of the invention.
  • the compound of the invention may be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions.
  • the compound may also be administered parenterally in sterile liquid dosage forms or topically in a carrier.
  • the compound of the invention may be formulated into dosage forms according to standard practices in the field of pharmaceutical preparations. See Remington 's Pharmaceutical Sciences , A. Osol, Mack Publishing Company, Easton, Pennsylvania.
  • the compound of the invention may be mixed with powdered carriers, such as lactose, sucrose, annitol, starch, cellulose derivatives, magnesium stearate, and stearic acid for insertion into gelatin capsules, or for forming into tablets.
  • powdered carriers such as lactose, sucrose, annitol, starch, cellulose derivatives, magnesium stearate, and stearic acid
  • Both tablets and capsules may be manufactured as sustained release products for continuous release of medication over a period of hours.
  • Compressed tablets can be sugar or film coated to mask any unpleasant taste and protect the tablet from the atmosphere or enteric coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration may contain coloring and flavoring to increase patient acceptance, in addition to a pharmaceutically acceptable diluent such as water, buffer or saline solution.
  • a pharmaceutically acceptable diluent such as water, buffer or saline solution.
  • the compound of the invention may be mixed with a suitable carrier or diluent such as water, a oil, saline solution, aqueous dextrose (glucose) , and related sugar solutions, and glycols such as propylene glycol or polyethylene glycols.
  • a suitable carrier or diluent such as water, a oil, saline solution, aqueous dextrose (glucose) , and related sugar solutions, and glycols such as propylene glycol or polyethylene glycols.
  • Solutions for parenteral administration contain preferably a water soluble salt of the compound of the invention.
  • Stabilizing agents, antioxidizing agents and preservatives may also be added.
  • Suitable antioxidizing agents include sodium bisulfite, sodium sulfite, and ascorbic acid, citric acid and its salts, and sodium EDTA.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben, and
  • ⁇ phiocordin is an antifungal antibiotic which, like Balanol, has the formula C 21 H 22 N 2 0 8 .
  • Ophiocordin is a natural product isolated from Cordyceps ophioglossoides which is known to have the structure:
  • the fungus Verticillium balanoides (MYCOsearch, Durham, North Carolina, accession number 25901) was grown on Yeast Extract Peptone Dextrose (YePD) , Malt, Cornmeal, Potato Dextrose (PDA), and Czapek's agar media for 7 days at 21°C and 37°C. Fungal colonies of approximately 2.5 mm in diameter were grown on Malt at 21°C and exhibited a white to Dresden Brown color according to Color Standard and Color Nomenclature , R.Ridgeway, Washington, D.C. 19122. In reverse, the colonies were Dresden Brown to Cinnamon in color. These colonies had an appressed to tomentose morphology.
  • the fungi exhibited substantially no growth in any media at 37°C.
  • Fungal colonies had substantially no odor.
  • Mycelium comprised straight, septate, branching hyphae of approximately 1 to 5 ⁇ m in diameter having frequent swellings to approximately 7 ⁇ m in diameter.
  • Conidiogenous cells were phialidic and micronematous; arose substantially directly from the arial mycelium; were solitary, lateral or terminal; and had dimensions of approximately 10 to 30 ⁇ m by 0.8 to 2.5 (5.0) ⁇ m in diameter. These cells further comprised a swollen base of the phialides of approximately 7 to 15 ⁇ m in length that tapered to approximately 0.8 ⁇ m in diameter. Conidia were cuneiform, hyaline, smooth-walled and aseptate being approximately 2.5 to 3.5 ⁇ m long and 1.8 to 2.2 ⁇ m wide at the apex; further, being 1.0 to 1.3 ⁇ m wide at the base.
  • Fungi were recovered by Barry Katz of MYCOsearch Inc. from a yellow rhizomorph in Pinus palustris needle litter located in a U. S. Forest Service P. palustris forest near Hoffman, North Carolina. Verticillium balanoides has MYCOsearch accession no. 25901.
  • EXAMPLE 2 Fermentation of Verticillium balanoides fungus The fungus Verticillium balanoides (which was deposited in the American Type Culture Collection, Rockville, Maryland 20854 on July 19, 1991 and has accession number 74082) as described in Example 1 was maintained on malt extract agar which contained 1% malt extract and 1.8% agar. The organism was transferred into a Corning 50 ml conical centrifuge tube containing 6 ml of YePD broth which consists of 1.0% Difco yeast extract, 2.0% Sigma peptone, and 2.0% dextrose (6 ml culture) . The 6 ml culture was then incubated for 7 days at 21°C while shaking at 200 rpm.
  • the 6 ml culture was then added to a 250 ml Erlenmeyer flask containing 75 ml cornmeal broth.
  • the cornmeal broth used consisted of 2.0% organically grown cornmeal, 2.0% tomato paste and 1.0% yeast extract.
  • the culture was incubated for 12 days at 21°C under shaking conditions at 200 rpm. Flask contents were then filtered to separate the filtrate from the mycelia mass.
  • the mycelia or freeze-dried filtrate from 101 culture was stirred in 1.5 1 of methanol at room temperature and was filtered under vacuum. Such extraction procedures were repeated 4-5 times. The filtrates were combined and concentrated in vacuo to give a dark brown residue which was then dissolved in 200 ml water and extracted with 200 ml n- butanol (n-BuOH) 3 times. In the case of mycelia, it was necessary to wash the water and crude extract mixture with ethyl acetate prior to extraction by n-BuOH. The extraction with n-BuOH was repeated until protein kinase C activity of the water layer dropped to an IC 50 of less than approximately 30 ⁇ g/ml.
  • n-BuOH extracts were concentrated in vacuo at room temperature yielding a dark brown residue which had protein kinase C inhibitory activity with an IC 50 of 5 ⁇ g/ml.
  • the crude n-BuOH extracts were then fractionated on a gel permeation column (cross-linked dextran, Sephadex LH-20, Pharmacia, Inc., New Jersey) equilibrated with chloroform/methanol (CHCl 3 /MeOH) at a ratio of 3:1.
  • CHCl 3 /MeOH chloroform/methanol
  • the column was eluted with a gradient composition of CHCl 3 /MeOH (3:1 followed by 2:1, 1:1 then finally by 100% methanol (MeOH) ) .
  • the fractions were pooled according to thin layer chromatography (TLC) patterns on Silica gel (E Merck) developed with n-BuOH/HOAc/H 2 0 at a ratio of approximately 4:1:1 respectively and visualized by UV lamp and ninhydrin spray reagent.
  • TLC thin layer chromatography
  • the desired protein kinase C inhibitor was present in the last fractions eluted from the column with 100% methanol.
  • the physico-chemical properties of the compound of Example 3 are as follows.
  • the compound appears as a light yellow amorphous solid that is soluble in dimethyl sulfoxide (DMSO) , H 2 0, and methanol (MeOH) and insoluble in ethyl acetate (EtOAc) .
  • DMSO dimethyl sulfoxide
  • MeOH methanol
  • EtOAc ethyl acetate
  • the compound gives a positive color reaction with phosphomolybdic acid reagent (PMA) (20% by weight solution in ethyl alcohol) , ninhydrin, and ferric chloride (FeCl 3 ) , but is negative with Dragendorff's reagent and Iodoplatinate spray reagent.
  • PMA phosphomolybdic acid reagent
  • FeCl 3 ferric chloride
  • the R f value of the compound is approximately 0.58 using E Merck silica gel 60F 254 thin layer chromatography (TLC) plates with n-butanol/acetic acid/water (n-BuOH/HOAc/H 2 0) at a ratio of 4:1:1 respectively in a thin layer chromatographic analysis. Further, the molecular weight of the compound is approximately 550. Mass spectrum analysis using a FAB, VG-Analytical ZAB 2-SF high field mass spectrometer showed (M+H) ion 551.
  • Varian Gemini dual proton-carbon probe of 5 mm in a C-13 NMR analysis is illustrated in Table 2.
  • the analysis utilized C0300 as a solvent.
  • the C-13 NMR spectrum of the compound of the invention using a 300 MHz dual proton-carbon probe of 5mm is depicted in Figure 2.
  • the protein kinase C assay is designed to duplicate the in vivo conditions required for protein kinase C function. Therefore, pH, salt and cofactor concentrations are similar to physiological levels.
  • Histone HI lysine rich
  • the enzyme is prepared from rat brain and is purified to apparent homogeneity as determined by a single band on silver stained sodium dodecyl sulfate (SDS)-polyacrylamide.
  • PS phosphatidylserine
  • DAG are co-sonicated to form unilamellar and multilamellar vesicles.
  • concentration of lipids in the assay are
  • SUBSTITUTESHEET suboptimal to maximize the detection potential of the assay for inhibitors.
  • Potential inhibitor compounds are added to the assay in dimethylsulfoxide (DMSO) at three concentrations to give final inhibitor concentrations of 4.3, 43 and 218 ⁇ M, respectively.
  • DMSO dimethylsulfoxide
  • the assay is started with the addition of enzyme and stopped after 10 min by the addition of 25% trichloroacetic acid (TCA) and 1.0 mg/ l bovine serum albumin (BSA) .
  • TCA trichloroacetic acid
  • BSA bovine serum albumin
  • the radioactive histone product is retained and washed on glass fiber filters that allow the unreacted 32 P-ATP to pass through.
  • the amount of phosphorylation is determined by the radioactivity measured in a scintillation counter.
  • Controls are included in every assay to measure background activity in the absence of enzyme, activity in the absence of lipids and the maximum enzyme activity with saturating levels of the activator lipids.
  • Table 3 shows the components and their concentration used in the protein kinase C assay.
  • HEPES is N-[2-hydroxyethyl] piperazine-N'-[2- ethanesulfonic acid] and EGTA is Ethylene- bis(oxyethylenenitrilo) tetraacetic acid.
  • Protein kinase C assay results are measured as an IC 50 value, which is the concentration of test compounds needed to inhibit 50% of the protein kinase C activity as compared with levels of protein kinase C activity in controls.
  • the compound of the invention was able to effectively inhibit protein kinase activity.
  • the compound of the present invention exhibited potent protein kinase C inhibitory activity with an IC 50 of 5 ng/ l (9 nM) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Pain & Pain Management (AREA)
  • Rheumatology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

Le Balanol, qui est dérivé des champignons de l'espèce Verticillium, en particulier du champignon Verticillium balanoides, est décrit. Le Balanol, lorsqu'il est pratiquement pur, est soluble dans le sulfoxyde diméthylique, l'eau et le méthanol; il est insoluble dans l'acétate d'éthyle et le chloroforme; il produit une réaction colorée positive avec l'acide polymolybdique, un réactif à base de ninhydrine, et le chlorure ferrique; il est négatif avec un réactif de Dragondorff et un jet d'iodoplatinate; il présente une valeur Rf de 0,58 environ déterminée par chromatygraphie en couche mince sur gel de silice avec n-butanol/acide acétique/eau en un rapport respectif de 4:1:1; et présente un poids moléculaire de 550 environ. Des composés de l'invention peuvent être utilisés pour inhiber la protéine-kinase C et traiter des états associés à l'inhibition de protéine-kinase C ou affectés par cette inhibition, en particulier des tumeurs cancéreuses, des maladies inflammatoires, des lésions dues à la reperfusion ainsi que des dysfonctionnement cardiaques associés à ces lésions.
EP92919060A 1991-08-22 1992-08-21 Inhibition de proteine-kinase c et nouveau compose appele balanol Withdrawn EP0664706A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US74863291A 1991-08-22 1991-08-22
US748632 1991-08-22
PCT/US1992/007124 WO1993003730A1 (fr) 1991-08-22 1992-08-21 Inhibition de proteine-kinase c et nouveau compose appele balanol

Publications (2)

Publication Number Publication Date
EP0664706A4 EP0664706A4 (fr) 1995-06-14
EP0664706A1 true EP0664706A1 (fr) 1995-08-02

Family

ID=25010269

Family Applications (1)

Application Number Title Priority Date Filing Date
EP92919060A Withdrawn EP0664706A1 (fr) 1991-08-22 1992-08-21 Inhibition de proteine-kinase c et nouveau compose appele balanol

Country Status (5)

Country Link
EP (1) EP0664706A1 (fr)
JP (1) JPH06510280A (fr)
AU (1) AU2504192A (fr)
CA (1) CA2115994A1 (fr)
WO (1) WO1993003730A1 (fr)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2157412A1 (fr) * 1993-03-03 1994-09-15 Julia Marie Heerding Balanoides
AU686691B2 (en) * 1994-01-12 1998-02-12 F. Hoffmann-La Roche Ag Novel azepanes and homologs thereof
US5583221A (en) * 1994-05-04 1996-12-10 Eli Lilly And Company Substituted fused and bridged bicyclic compounds as therapeutic agents
TW339325B (en) * 1995-07-05 1998-09-01 Hoffmann La Roche Novel azepane derivatives, process for the preparation thereof and pharmaceutical composition containing the same
WO1997029091A1 (fr) * 1996-02-09 1997-08-14 Phytera Symbion Aps Analogues de balanol
US6914140B1 (en) 1996-04-17 2005-07-05 Hoffmann-La Roche Inc. Asymmetric synthesis process
US5902882A (en) * 1996-04-17 1999-05-11 Hoffmann-La Roche Inc. Assymetric synthesis of azepines
US6232299B1 (en) * 1996-05-01 2001-05-15 Eli Lilly And Company Use of protein kinase C inhibitors to enhance the clinical efficacy of oncolytic agents and radiation therapy
JP2008537739A (ja) 2005-03-21 2008-09-25 ザ トラスティース オブ コロンビア ユニバーシティ イン ザ シティ オブ ニューヨーク 新規神経疼痛経路
US8846742B2 (en) 2006-02-14 2014-09-30 The Trustees Of Columbia University In The City Of New York Neuronal pain pathway modulators

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEM.BER., vol. 113,no. 6, 1980 pages 2221-26, W.A.K\NIG ET AL. 'Konstitution von Oghiocordin, einem Antibioticum mit antifungischen Eigenschaften' *
J.ANTIBIOT., vol. 47,no. 9, 1994 pages 1010-16, C.BOROS ET AL. 'Comparision of Balanol from Verticillium balanoides and Ophiocordin from Cordyceps ophioglossoides' *
See also references of WO9303730A1 *

Also Published As

Publication number Publication date
WO1993003730A1 (fr) 1993-03-04
EP0664706A4 (fr) 1995-06-14
JPH06510280A (ja) 1994-11-17
AU2504192A (en) 1993-03-16
CA2115994A1 (fr) 1993-03-04

Similar Documents

Publication Publication Date Title
US5302604A (en) Cholesterol lowering compounds produced by directed biosynthesis
US7335679B2 (en) Wortmannin analogs and methods of using same
WO1993003730A1 (fr) Inhibition de proteine-kinase c et nouveau compose appele balanol
CA2458318C (fr) Analogues de wortmannine et leurs methodes d'utilisation
US5216014A (en) Furo-coumarinsulfonamides as protein kinase C inhibitors
AU2002330029A1 (en) Wortmannin analogs and methods of using same
KR0135600B1 (ko) 항암 항생물질 mi43-37f11, 그 제조방법 및 그 용도
JPH05123179A (ja) 新規化合物ロイストロダクシン
JPH09176083A (ja) 新規化合物ｆ−１２５０９ａ
US8586626B2 (en) Metabolites of wortmannin analogs and methods of using the same
US5994412A (en) Bis-aryl ethers, compositions containing such compounds and methods of treatment
US5712306A (en) Physiologically active substances PF1092A, PF1092B and PF1092C, and contraceptives and anticancer drugs containing the same as active ingredients
JPH10182627A (ja) 新規化合物Ｓ−１５１８３ａ及びＳ−１５１８３ｂ
US7528169B2 (en) Substance having antitumor/anti-inflammatory activity
WO1993017557A1 (fr) Composes abaissant le taux de cholesterol et produits par biosynthese dirigee
KR100327043B1 (ko) 지알비2-에스에이치씨 결합 저해 화합물, 미생물로부터이의 분리 방법, 및 이를 함유하는 지알비2-에스에이치씨결합 저해용 조성물
JPH0327316A (ja) 高脂血症治療剤
AU2006202635B2 (en) Wortmannin analogs and methods of using same
JP2005272357A (ja) 抗癌剤
WO2005003076A1 (fr) Derive de cetone bicyclique substitue par de l'acide heptatrienoique et composition pharmaceutique le contenant
JP2002114741A (ja) 新規化合物ｆ−１６０５３ａ
JP2004269456A (ja) 新規発酵生産物
JP2002060392A (ja) クルブラロール及びその製造方法
JPH09143118A (ja) ポリケチド類およびその製造方法並びにそれを有効成分とするｉｃａｍ−１発現抑制剤
JP2005035948A (ja) 新規生理活性物質

Legal Events

Date Code Title Description
A4 Supplementary search report drawn up and despatched
AK Designated contracting states

Kind code of ref document: A4

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL SE

PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19940228

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LI LU MC NL SE

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19970206