PROTEIN KINASE C INHIBITION AND NOVEL COMPOUND BALANOL

Abstract: Balanol, which is derived from fungi of the genus Verticillium, especially the fungus Verticillium balanoides, is provided. Balanol, when substantially pure, is soluble in dimethyl sulfoxide, water, and methanol; insoluble in ethyl acetate and chloroform; gives a positive color reaction with polymolybdic acid, ninhydrin reagent, and ferric chloride; is negative with Dragendorff's reagent and Iodoplatinate spray; has an Rf 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. Compounds of the invention are useful for inhibiting protein kinase C and treating conditions related to, or affected by inhibition of protein kinase C, particularly cancer tumors, inflammatory disease, reperfusion injury, and cardiac dysfunctions related to reperfusion injury.

Description: PROTEIN KINASE C INHIBITION AND NOVEL COMPOUND BALANOL FIELD OF THE INVENTION 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. BACKGROUND OF THE INVENTION 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). The mechanisms of protein kinase C action have been described in U.S. Patent 4,816,450 issued March 28, 1989 to Bell et al., the disclosure of which is specifically incorporated as fully set forth herein. Protein kinase C is activated by diacylglycerol (DAG) , a neutral lipid, and when activated will transfer the γ- phosphate of MgATP to a serine or threonine residue on a substrate protein. 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. In addition, it has been suggested that 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 . Cancer Cells 2 : 95-102 (1990). Certain protein kinase C inhibitors have been demonstrated to block platelet aggregation and release of neutrophil activating agents such as platelet activating factor, PAF. See Schachtele et al., Biochem . Biophy. Res . Commun . 151:542 (1988); Hannun et al., J. Biol . Chem. 262 : 13620 (1987); and Yamada et al. , Biochem . Pharmacol . 37 : 1161 (1988). Protein kinase C inhibitors have also been shown to inhibit neutrophil activation, and chemotactic migration. See Mclntyre et al., J. Biol Chem . 262 : 15730 (1987); Lambreth et al., J. Biol . Chem . 263 : 3818 (1988); Pittet et al., J. Biol . Chem . 252:10072 (1987); and Gaudry et al.. Immunology 63 : 715 (1988). Neutrophil degranulation and release of proteolytic enzymes and reactive oxygen intermediates has also been shown. See Wilson et al., J. Biol . Chem . 261 : 12616 (1986); Fujita et al. , Biochem . Pharmacol . 35:4555 (1986); Berkow et al., J. Leukoc , Biol . 41:441 (1987); Salzer et al., Bioc en. Biophys . Res . Commun . 148 : 747 (1987); Kramer et al., J. Biol . Chem . 262 : 5876 (1988); and Dewald et al., Biochejn. J " . 264:879 (1989). It is apparent that 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. Natural products that inhibit protein kinase C have been reported. See Tamaoki et al., Bio /Technology 8 : 732 (1988). For example, saturosporine, Nakano et al., J. Antibiotics 40:706-708 (1987), an alkaloid metabolite, and its 7-hydroxy congener, Talςahashi et al., J " . Antibiotics , 42 : 571- 576 (1989) , isolated from Streptomyces staurosporeus , have shown protein kinase C inhibitory activity at nanomolar concentrations. Hypericin and pseudohypericin, both from plants of Hypericum, are also reported as protein kinase C inhibitors. See Lavie et al., Proc. Natl . Acad. Sci . USA 86:5963-5967 (1989). These two compounds also have potent anti-retroviral activity. 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). Thus, 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. SUMMARY OF THE INVENTION The present invention provides novel protein kinase C inhibitor 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. 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. Further, 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. This invention is more particularly pointed out in the appended claims and is described in its preferred embodiments in the following description. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 i .llustrates 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. < DETAILED DESCRIPTION OF THE INVENTION 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. 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. 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. Moreover, it is believed that modifications of the Balanol nucleus will also be likely to find utility in therapeutics and as a protein kinase C inhibitor. Applicants have performed proton nuclear resonance spectroscopy and carbon nuclear resonance spectroscopy on samples of Balanol; the data are reported in Table 4 and Table 5. This data suggest to the inventors that Balanol can be represented by the following formula: It is to be understood that the foregoing structure is not yet a certainty and that Balanol is to be identified in the alternative by the structure and but its physical properties set forth above. It can be appreciated by one having ordinary skill in the art that optical isomers of this structure are possible and that some optical isomers may have more activity than others. Furthermore, while the above formula is believed to an accurate representation of the structure of the compound of the present invention, other isomers are possible. 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. Accordingly, 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 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. Cells transformed with the oncogenes ras , sis , erbB , abl , and src have been shown to contain elevated levels of DAG which is then believed to activate protein kinase C. Indeed, studies on ras transformed cells have shown protein kinase C activation to be concomitant with elevation of DAG. Phorbol esters, such as phorbol myristate acetate (PMA) , have complex effects on cells including effects on membrane function, mitogenesis, differentiation, and gene expression. Synthetic diacylglycerols mimic many of the effects of PMA in vitro and inhibitors of protein kinase C have been shown to block PMA-induced effects on cells. Thus, 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. In addition, 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. Several studies have shown increased expression of protein kinase C in certain tumor types such as breast and lung carcinomas. Activated protein kinase C has also been detected in human colon carcinomas although increased expression on the gene level was not seen. Topoisomerases are directly modulated by protein kinase C as substrates for the enzyme and protein kinase C inhibitors have been shown to potentiate the action of chemotherapy drugs such as cis-platin. Other compounds which have been identified specifically as inhibitors of protein kinase C have shown early promise as therapeutic agents in inhibiting tumor growth in animal models. Animal studies have shown that perhaps 50% or more of 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) . 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. It is accepted that the damage by neutrophils to myocardial tissue proceeds through a cascade of events, one of the earliest being the bonding of activated neutrophils to damaged vascular endothelium. However, the binding of the neutrophils is significantly enhanced by their activation and this an even earlier event is the generation of molecules, such as cytokines, and chemotactic factors, which can function as activation stimuli. These molecules probably originate from damaged and aggregated platelets, from damaged vascular endothelium, or from the oxidation of plasma proteins or lipids by endothelial-derived oxidants. Strategies for overcoming the deleterious effects of reactive oxygen intermediates have centered on the development of scavengers for the molecules. Superoxide dis utase (SOD) has been shown to be a particularly effective scavenger of superoxide, but suffers from a very short half-life in the blood. 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. However, none of the strategies designed to scavenge reactive oxygen intermediates will prevent the aggregation of platelets, the release of chemotactic molecules, the activation and adherence of neutrophils to vascular endothelium, or the release of proteolytic enzymes from activated neutrophils. An advantage of protein kinase C inhibitors as therapeutics for reperfusion injury is that they have been demonstrated to 1) block platelet aggregation and release of neutrophil activating agents such as PAF, 2) block neutrophil activation, chemotactic migration, and adherence to activated or damaged endothelium, and 3) block neutrophil release of proteolytic enzymes and reactive oxygen intermediates. Thus these agents have the capability of blocking all three of the most significant mechanisms of pathogenesis associated with reperfusion injury and should thus have a decided therapeutic advantage. 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. The 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. It is contemplated that the daily dosage of the compound will be in the range of from about 1 μg to about 100 mg per kg of body weight, preferably from about 1 μg to about 1 mg per kg body weight, and more preferably from about 10 μg 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. For example, 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. 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. For parenteral administration, 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. 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 chlorbutanol. The following examples are illustrative of the present invention and are not intended to limit the scope of the invention. It should be noted that a known compound, όphiocordin, is an antifungal antibiotic which, like Balanol, has the formula C 21 H 22 N 2 0 8 . However, Ophiocordin is a natural product isolated from Cordyceps ophioglossoides which is known to have the structure: SUBSTITUTESHEET As is apparent, Ophiocordin shares structural groups with Balanol, but they are arranged completely differently. In accord with the different structures, NMR data generated by Balanol and Ophiocordin are different. EXAMPLES EXAMPLE 1 Growth of Verticillium balanoides fungus. 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. On Cornmeal, the colonies measured approximately 3.4 mm in diameter having a white color, no reverse color and a felted morphology. Colonies grown on YePD were approximately 5.0 mm in diameter having a white color with no reverse color and with minutely tomentose and slightly furrowed morphology. Growth on PDA yielded colonies of approximately 3.4 mm in diameter having a white to tawny color, a white to tawny color on the reverse and slightly furrowed morphology. Growth on Czapek's medium yielded colonies of approximately 1.4 mm in diameter having a white to primuline yellow color, a primuline yellow reverse and minutely 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 μm in diameter having frequent swellings to approximately 7 μm 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 μm by 0.8 to 2.5 (5.0) μm in diameter. These cells further comprised a swollen base of the phialides of approximately 7 to 15 μm in length that tapered to approximately 0.8 μm in diameter. Conidia were cuneiform, hyaline, smooth-walled and aseptate being approximately 2.5 to 3.5 μm long and 1.8 to 2.2 μm wide at the apex; further, being 1.0 to 1.3 μm 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. For the preparation of the 10 1 size fermentations, multiple 250 ml Erlenmeyer flasks were employed. The 6 ml culture was first transferred to 75 ml YePD broths and incubated for 7 days. 10 ml of such seed cultures were used to inoculate 75 ml cornmeal broth. After cultivation, the cultures were combined and filtered to separate the mycelia and the filtrate. EXAMPLE 3 Isolation Crude methanol extracts of both mycelia and freeze- dried filtrate showed protein kinase C inhibitory activity (IC 50 ) of less than approximately 5 μg/ml in the protein kinase C inhibition assay explained in detail in Example 5. Fractionation to isolate the protein kinase C inhibitor of the invention was guided by such enzyme assays. All fractions at each purification step were evaluated and only those which showed improved protein kinase C inhibitory activity were further fractionated. 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 μg/ml. The combined 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 μg/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. 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. The desired protein kinase C inhibitor was present in the last fractions eluted from the column with 100% methanol. The most active fraction from the above chromatography was further fractionated by reverse phase HPLC using a C 18 column (octadecyldimethylsilyl bonded phase silica gel, Waters microBondapak) under gradient conditions with acetonitrile (CH 3 CN) and MeOH. The elution of active component was monitored by UV at 254 um. Lyophilization of HPLC fractions yielded pure light yellow solid material. « EXAMPLE 4 Physico-chemical Characteristics. 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) . 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. 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. The ultraviolet spectrum (Shimadzu) of the compound when neutral (H 2 0) and acidic (0.1 N HC1) has a maximum at approximately 256 nm and an extinction coefficient of approximately .22,807; and when basic (0.1 N NaOH) has a maximum at approximately 289 nm and an extinction coefficient of approximately 37,368. 1 H NMR using a 300 MHz Varian Gemini dual proton-carbon probe 5 mm showed peaks at approximately (MeOH-d 4 ) , δ 7.40 (1H, d, 8.7 Hz), 7.06 (1H, d, 8.1 Hz), 6.7 (1H, t, 7.9 Hz), 6.70 (1H, s) , 6.5 (2H, dd, 7.9 Hz, 8.0 Hz), 5.08 (1H, dt, 3.4, 5.0 Hz) , 4.38 (1H, m) , 4.13 (1H, m) , 2.7- 3.05 (4H, m) , and 1.55-1.95 (4H, m) . An example of the instrument settings for the 300 MHz Varian Gemini dual proton-carbon probe of 5 mm in a proton NMR analysis is illustrated in Table 1. The analysis utilized pulse sequence S2Pul, and C0300 as a solvent. The H NMR spectrum of the compound of the invention using a 300 MHz dual proton-carbon probe of 5mm is depicted in Figure 1. TABLE 1 An example of the instrument settings for the 300 MHz 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. EXAMPLE 5 Protein Kinase C Inhibition. 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) is used in the assay as the phosphorylation acceptor protein because it is readily available and serves as a good substrate for protein kinase C. 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. In the screening assay, phosphatidylserine (PS) and DAG are co-sonicated to form unilamellar and multilamellar vesicles. The 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 μM, respectively. 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) . 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. TABLE 3 Assay Component Concentration HEPES pH 7.5 20 μM MgCl 2 20 μM CaCl 2 100 μM EGTA 95 μM Histone HI 200 μg/ml Phosphatidylserine 40 μg/ml Diacylglycerol 1.8 μg/ml Protein Kinase C 0.6 μg/ml γ- P-ATP 20 μM 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) . TABLE 4 Proton Nuclear Magnetic Resonance Spectroscopy Data for Balanol 10 15 20 TABLE 5 Carbon Nuclear Magnetic Resonance Spectroscopy Data For Balanol Carbon Chemical Shift Multiplicity Integration (ppm) 2 CH 2 1 3 CH 1 4 CH 1 5 CH 2 1 10 6 CH 2 1 7 CH 2 1 1 ' - C 1 2 ' C 1 3', T CH 2 15 4', 6 " CH 2 5 ' C 1 1" C 1 2" C 1 3", 7" CH 2 20 4", 6' C 2 5" C 1 8" C 1 9" C 1 10" C 1 25 11" C 1 12" CH 1 13" CH 1 14" C 1 15" C 1

Claims: What is claimed is: 1. A compound isolatable from Verticillium balanoides having protein kinase C inhibitory activity which compound is soluble in dimethyl sulfoxide, water, and methanol; insoluble in ethyl acetate and chloroform; gives a positive color reaction with polymolybdic 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; has a molecular weight of approximately 550; the ultraviolet spectrum of said compound when neutral (water) and acidic (0.1 N HC1) has a maximum at approximately 256 nm and an extinction coefficient of approximately 22,807; and when basic (0.1 N NaOH) has a maximum at approximately 289 nm and an extinction coefficient of approximately 37,368; or a pharmaceutically acceptable salt thereof. 2. A composition comprising: a pharmaceutically acceptable carrier or diluent and, a therapeutically effective amount of the compound of claim 1 or a pharmaceutically acceptable salt thereof. 3. A method of inhibiting protein kinase C comprising contacting a protein kinase C with an inhibitory amount of the compound of claim 1. 4. A method of inhibiting protein kinase C comprising contacting a protein kinase C with an inhibitory amount of the compound of claim 2. 5. The use of the composition of claim l in the manufacture of a medicament for inhibiting protein kinase C. 6. The use of the composition of claim 1 in the manufacture of a medicament for inhibiting growth of mammalian tumor cells. A compound using the formula: or a pharmaceutically acceptable salt thereof. 8. A composition comprising: a pharmaceutically acceptable carrier or diluent and, a therapeutically effective amount of the compound- of claim 6 or.a pharmaceutically acceptable salt thereof. 9. A method of inhibiting protein kinase C comprising contacting a protein kinase C with an inhibitory amount of the compound of claim 8. 10. A method of inhibiting protein kinase C comprising contacting a protein kinase C with an inhibitory amount of the compound of claim 8. 11. The use of the composition of claim 6 in the manufacture of a medicament for inhibiting protein kinase C. 12. The use of the composition of claim 6 in the manufacture of a medicament for inhibiting growth of mammalian tumor cells.

Inventors: BALLAS LAWRENCE M, BOROS CHRISTIE, HALLOCK YALI F, JANZEN WILLIAM P, KULANTHIVEL PALANIAPPAN, BALLAS, LAWRENCE, M., BOROS, CHRISTIE, HALLOCK, YALI, F., JANZEN, WILLIAM, P., KULANTHIVEL, PALANIAPPAN
Applicants: SPHINX PHARMA CORP, SPHINX PHARMACEUTICALS CORPORATION
Application year: 1992
Main classification: A61K31
IPC codes: A61K31, A61K35, A61P9, A61P29, A61P35, A61P43, C07D223, C12N9, C12P17, C12R1