Abstract:
Paraherquamide and dihydroparaherquamide, known compounds, are disclosed as having antiparasitic activity. The compounds are fungal metabolites isolated from the known fungus Penicillium paraherquei, and from a newly isolated strain of Penicillium charlesii. Compositions including paraherquamide and dihydroparaherquamide as the active ingredient thereof for antiparasitic uses against endo and ecto parasites are also disclosed, as well as processes for the preparation of paraherquamide from the newly isolated fungus.

Description:
BACKGROUND OF THE INVENTION 
     Paraherquamide and dihydroparaherquamide are known compounds and are disclosed in Yamazaki et al in Tetrahedron Letters 22 135-136 (1981). Paraherquamide is indicated as being a fungal metabolite of Penicillium paraherquei. Dihydroparaherquamide is prepared from paraherquamide by catalytic hydrogenation. No uses for the compounds are suggested. 
     SUMMARY OF THE INVENTION 
     This invention is concerned with the use of paraherquamide and dihydroparaherquamide as antiparasitic agents and the preparation of paraherquamide from a newly isolated strain of the fungus Penicillium charlesii. Thus, it is an object of this invention to describe such uses. A further object is to describe compositions containing such compounds as the active ingredient thereof. A still further object is to describe the new fungus and the preparation and isolation of paraherquamide therefrom. Further objects will become apparent from a reading of the following description. 
    
    
     DESCRIPTION OF THE INVENTION 
     Paraherquamide has the following structure: ##STR1## Dihydroparaherquamide has the following structure: ##STR2## 
     Paraherquamide is isolated as a fungal metabolite of Penicillium paraherquei using standard fermentation and isolation techniques. The analytical and structural characteristics of paraherquamide are described in detail in Yamazaki et al Tetrahedron Letters 22 135-136 (1981). 
     Dihydroparaherquamide is prepared from paraherquamide by catalytic hydrogenation over palladium on a carbon support. The analytical characteristics of dihydroparaherquamide are also given in Yamazaki et al. 
     The novel strain of Penicillium charlesii is identified in the culture collection of Merck &amp; Co., as MF 5123. The morphological and cultural characteristics of MF 5123 are as follows: 
     Morphology of MF 5123 
     Conidiophores are simple or variously branched with each branch bearing a monoverticillate penicillus and conidial chains forming a long narrow compact column. Conidiophore walls are smooth or nearly so. Conidia are mostly globose, 1.8 μ to 2.4 μ, slightly roughened. 
     Cultural characteristics of MF 5123 
     Czapek Dox agar 
     Colonies are white, velvety and raised, becoming grey-green with cream colored areas. The reverse side is yellowish-brown and cream. As culture ages, aerial growth becomes grayish brown to brown and a tan soluble pigment diffuses into medium. 
     Potato dextrose agar 
     Colonies are white becoming grey-green, flat, granular. Reverse side is greenish-brown. 
     Sabouraud Maltose 
     Colonies are white becoming brownish green with vectors of Yellowish-tan, raised, velvety. Reverse side is dark brown. A brown soluble pigment diffuses into agar. 
     Yeast-extract Malt-extract agar 
     Colonies are white becoming gray-green with yellowish areas, raised, velvety. Reverse side is dark brown edged with tan. 
     Corn agar 
     Colonies are white becoming gray-green, flat, granular. Reverse side is yellowish. As culture ages, it becomes brown with greenish tones. 
     A comparison with culture descriptions in A Manual of the Penicillia by K. B. Raper and C. Thom and with known cultures show this culture to be a new strain of the known species Penicillium charlesii. 
     A sample of MF 5123, Penicillium charlesii has been deposited in the Permanent culture collection of the American Type Culture Collection at 12301 Parklawn Drive, Rockville, Md., 20852 and has been assigned the accession number ATCC 20841. 
     The above description is illustrative of a strain of Penicillium charlesii MF 5123 which can be employed in the production of the instant compound. However, the present invention also embraces mutants of the above described microorganism. For example, those mutants which are obtained by natural selection or those produced by mutating agents including ionizing radiation such as ultraviolet irradiation, or chemical mutagens such as nitrosoguanidine or the like treatments are also included within the ambit of this invention. 
     Paraherquamide may be prepared during the aerobic fermentation of a producing strain of Penicillium charlesii MF 5123 in either an agitated aqueous medium or in a static solid medium. 
     Where the nutrient medium is an aqueous medium, suitable media such as those used for the production of many antibiotic substances are suitable for use in this process for the production of paraherquamide. 
     Such nutrient media contain sources of carbon and nitrogen assimilable by the microrganism and generally low levels of inorganic salts. In addition, the fermentation media may contain traces of metals necessary for the growth of the microorganisms, and production of the desired compound. These are usually Present in sufficient concentrations in the complex sources of carbon and nitrogen, which may be used as nutrient sources, but can, of course, be added separately to the medium if desired. 
     In general, ingredients such as sugars, for example dextrose, sucrose, maltose, lactose, glycerol, corn, millet, wheat, dextran, cerelose, corn meal, oat flour, and the like, and starches are suitable sources of assimilable carbon in the nutrient media. The exact quantity of the carbon source which is utilized in the medium will depend, in part, upon the other ingredients in the medium, but it is usually found that an amount between 0.5 and 90% by weight of the medium is satisfactory. These carbon sources may be used individually or several such carbon sources may be combined in the same medium. 
     Various nitrogen sources such as yeast hydrolysates, yeast autolysates, yeast cells, tomato paste, corn meal, oat flour, soybean meal, casein hydrolysates, yeast extracts, corn steep liquors, corn, millet, wheat, distillers solubles, cottonseed meal, meat extract and the like, are readily assimilable by Penicillium charlesii MF 5123 in the production of the instant compound. The various sources of nitrogen can be used alone or in combination in amounts ranging from 0.2 to 95% by weight of the medium. 
     Among the nutrient inorganic salts, which can be incorporated in the culture media are the customary salts capable of yielding sodium, potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride, carbonate, and like ions. Also included are trace metals such as cobalt, manganese, iron, molybdenum, cadmium, zinc, copper, and the like. 
     It should be noted that the media described hereinbelow and in the Examples are merely illustrative of the wide variety of media, which may be employed, and are not intended to be limitative. 
     The following are Examples of media suitable for growing strains of Penicillium charlesii MF 5123. 
     
         ______________________________________Medium ADextrose               1.0        g.Soluble starch         10.0       g.Beef extract           3.0        g.Yeast autolysate (As ardamine pH                  5.0        g.available from Yeast Products, Inc.,Clifton, N.J.)NZ Amine-E (casein hydrolysate-                  5.0        g.available from Humko-Sheffield-Memphis, Tenn.)MgSO.sub.4.7H.sub.2 O  0.05       g.Phosphate Buffer       2.0        mlCaCO.sub.3             0.5        g.Distilled water        1000       ml.pH 7.0-7.2Phosphate BufferKH.sub.2 PO.sub.4      91.0       gNa.sub.2 HPO.sub.4     95.0       gDistilled water        1000       mlpH 7.0Medium BTomato paste           20.0       g.Primary yeast          10.0       g.Dextrin (CPC starch)   20.0       g.CoCl.sub.2.6H.sub.2 O  0.005      g.Distilled water        1000       mlpH 7.2-7.4Medium CCorn meal              20.0       g.Distillers solubles    10.0       g.Soybean meal           15.0       g.Sodium citrate         4.0        g.CaCl.sub.2.2H.sub.2 O  0.5        g.MgSO.sub.4.7H.sub.2 O  0.1        g.CoCl.sub.2.6H.sub.2 O  0.01       g.FeSO.sub.4.2H.sub.2 O  0.01       g.Polyglycol P2000 (Polypropylene glycol                  2.5        mg.mw 2000)Distilled water        1000       ml.pH 6.5Medium DLactose                20.0       g.Distillers solubles    15.0       g.Autolyzed yeast (Ardamine pH)                  5.0        g.Distilled water        q.s. to 1000                             mlpH 7.0Medium ETomato paste           40.0       g.Oat Flour              10.0       g.Distilled water        1000       mlpH 7.0Medium FCorn Steep Liquor      15.0       g.(NH.sub.4).sub.2 SO.sub.4                  4.0        g.CaCO.sub.3             6.0        g.Soluble Starch         20.0       g.Corn meal              1.0        g.Soybean meal           4.0        g.Glucose                5.0        g.KH.sub.2 PO.sub.4      0.3        g.Lard oil               2.5        g.Distilled water        1000       ml.pH 6.7Medium GDextrose               10.0       gAsparagine             1.0        gK.sub.2 HPO.sub.4      0.1        gMgSO.sub.4.7H.sub.2 O  0.5        gYeast Extract          0.5        gOat Flour              10.0       gCaCO.sub.3             3.0        gTrace Element Mix      10.0       mlDistilled water        1000       mlAdjust pH to 7.2Trace Element MixFeSO.sub.4.7H.sub.2 O  1000       mgMnSO.sub.4.4H.sub.2 O  1000       mgCuCl.sub.2.2H.sub.2 O  25         mgCaCl.sub.2.2H.sub.2 O  100        mgH.sub.3 BO.sub.3       56         mg(NH.sub.4).sub.6 MO.sub.4 O.sub.24.6H.sub.2 O                  19         mgZnSO.sub.4.7H.sub.2 O  200        mgDistilled water        1000       mlMedium HMedium G               1000       mlOat Flour              10         gpH 7.2______________________________________ 
    
     The fermentation employing Penicillium charlesii MF 5123 can be conducted at temperatures ranging from about 20° C. to about 40° C. For optimum results, it is most convenient to conduct these fermantations at a temperature in the range of from about 20° C. to about 30° C. Temperatures of about 24°-26° C. are most preferred. The pH of the nutrient medium suitable for producing the instant compounds can vary from about 3.0 to 8.5 with a preferred range of from about 4.0 to 7.0. 
     Small scale fermentations are conveniently carried out by placing suitable quantities of nutrient medium in a flask employing known sterile techniques, inoculating the flask with either spores or vegetative cellular growth of Penicillium charlesii MF 5123 loosely stoppering the flask with cotton and permitting the fermentation to proceed at a constant room temperature of about 25° C. on a rotary shaker at from 0 to 300 rpm for about 2 to 21 days. For larger scale work, it is preferable to conduct the fermentation in suitable tanks provided with an agitator and a means of aerating the fermentation medium. The nutrient medium is made up in the tank and after sterilization is inoculated with a source of vegetative cellular growth of Penicillium charlesii MF 5123. The fermentation is allowed to continue for from 5 to 20 days while agitating and/or aerating the nutrient medium at a temperature in the range of from about 20° to 28° C. The degree of aeration is dependent upon several factors such as the size of the fermentor, agitation speed, and the like. Generally the larger scale fermentations are agitated at about 95 to 300 RPM and about 2 to 20 cubic feet Per minute (CFM) of air. 
     The fermentation of Penicillium charlesii MF 5123 is also successfully carried out in a solid fermentation medium under static, that is, non-agitated, conditions. The solid phase aerobic fermentation utilizes the same sources of carbon, nitrogen and inorganic salts as are used for the above-described submerged aqueous fermentation with the primary differences in the constitution of the medium being the quantity of water present. The solid phase fermentations constitute from 30 to 80% by weight of water. Where in comparison with a submerged fermentation medium which may utilize from 10 to 100 g of solid ingredients per liter of medium 1 to 10% w/v), a solid phase medium will contain from 20 to 70% w/v of the solid ingredients. 
     The solid phase fermentation may be carried out aerobically by maintaining a large ratio of surface area to the mass of the medium. This is readily accomplished by utilizing a 0.3 to 8 cm depth of medium in a fermentation tray or flask. Since the medium is not mechanically agitated, this ensures the Presence of sufficient oxygen for growth. Alternatively the solid phase fermentation may be carried out in special trays fitted with sterile gauze for passing air through the solid medium or across the top thereof. Optionally the solid phase fermentation may be carried out with a tight fitting cover. 
     The fermentation of large scale portions of media may be carried out in stages of increasing quantities of media and it is not necessary that all of the stages be of the same type, that is aqueous or solid. It has been found to be preferable to carry out the initial stages of fermentation in aqueous media and transfer the media to larger scale solid media. 
     The compound of this invention is found primarily in the mycelium on termination of the Penicillium charlesii MF 5123 fermentation and may be removed and separated therefrom as described below. 
     The separation of the Paraherquamide from the whole fermentation broth and the recovery of said compound is carried out by solvent extraction and application of chromatographic fractionations with various chromatographic techniques and solvent systems. 
     The instant compound has slight solubility in water, but is soluble in organic solvents. This property may be conveniently employed to recover the compound from the fermentation broth. Thus, in one recovery method, the whole fermentation broth is combined with approximately an equal volume of an organic solvent. While any organic solvent may be employed, it is preferable to use a water immiscible solvent such as ethyl acetate, methylene chloride, chloroform and the like. Generally several extractions are desirable to achieve maximum recovery. The solvent removes the instant compound as well as other substances lacking the antiparasitic activity of the instant compound. If the solvent is a water immiscible one, the layers are separated and the organic solvent is evaporated under reduced pressure. The residue is placed onto a chromatography column containing Preferably, silica gel. The column retains the desired products and some impurities, but lets many of the impurities, particularly the non-polar impurities, pass through. The column is washed with a moderately polar organic solvent such as methylene chloride or chloroform to further remove impurities, and is then washed with a mixture of methylene chloride or chloroform and an organic solvent of which acetone, methanol, and ethanol and the like are preferred. The solvent is evaporated and the residue further chromatographed using column chromatography, thin layer chromatography, preparative layer chromatography, high pressure liquid chromatography and the like, with silica gel, aluminum oxide, ion exchange resins, dextrans gels and the like, as the chromatographic medium, with various solvents and combinations of solvents as the eluent. Thin layer, high pressure, liquid and preparative layer chromatography may be employed to detect the presence of, and to isolate the instant compound. The use of the foregoing techniques as well as others known to those skilled in the art, will afford purified compositions containing the instant compound. 
     It has been unexpectedly discovered that paraherquamide and dihydroparaherquamide are potent endo-and ecto-antiparasitic agents against parasites particularly helminths, ectoparasites, insects, and acarides, infecting man, animals and plants. 
     The disease or group of diseases described generally as helminthiasis is due to infection of an animal host with parasitic worms known as helminths. Helminthiasis is a prevalent and serious economic problem in domesticated animals such as swine, sheep, horses, cattle, goats, dogs, cats and poultry. Among the helminths, the group of worms described as nematodes causes widespread and often times serious infection in various species of animals. The most common genera of nematodes infecting the animals referred to above are Haemonchus, Trichostronqylus, Ostertagia, Nematodirus, Cooperia, Ascaris, Bunostomum, Oesophaqostomum, Chabertia, Trichuris, Stronqylus, Trichonema, DictYocaulus, Capillaria, Heterakis, Toxocara, Ascaridia, Oxyuris, Ancylostome, Uncinaria, Toxascaris and Parascaris. Certain of these, such as Nematodirus, Cooperia, and Oesophagostomum attack primarily the intestinal tract while others, such as Haemonchus and Ostertagia, are more prevalent in the stomach while still others such as Dictyocaulus are found in the lungs. Still other Parasites may be located in other tissues and organs of the body such as the heart and blood vessels, subcutaneous and lymphatic tissue and the like. The parasitic infections known as helminthiases lead to anemia, malnutrition, weakness, weight loss, severe damage to the walls of the intestinal tract and other tissues and organs and, if left untreated, may result in death of the infected host. The compounds of this invention have unexpectedly high activity against these parasites, and in addition are also active against Dirofilaria in dogs, Nematospiroides, Syphacia, Aspiculuris in rodents, arthropod ectoparasites of animals and birds such as ticks, mites, lice, fleas, blowfly, in sheep Lucilia sp., biting insects and such migrating diperous larvae as Hypoderma sp. cattle, Gastrophilus in horses, and Cuterebra sp. in rodents. 
     The instant compounds are also useful against parasites which infect humans. The most common genera of parasites of the gastro-intestinal tract of man are Ancylostoma, Necator, Ascaris, Strongyloides, Trichinella, Capillaria, Trichuris, and Enterobius. Other medically important genera of parasites which are found in the blood or other tissues and organs outside the gastrointestinal tract are the filiarial worms such as Wuchereria, Bruqia, Onchocerca and Loa, Dracunuculus and extra intestinal stages of the intestinal worms Strongyloides and Trichinella. The compounds are also of value against arthropods parasitizing man, biting insects and other dipterous pests causing annoyance to man. 
     The compounds are also active against household pests such as the cockroach, Blatella sp., clothes moth, Tineola sp., carpet beetle, Attagenus sp., and the housefly Musca domestica. 
     The compounds are also useful against insect pests of stored grains such as Tribolium sp., Tenebrio sp. and of agricultural plants such as aphids, (Acyrthiosiphon sp.); against migratory orthopterans such as locusts and immature stages of insects living on plant tissue. The compounds are useful as a nematocide for the control of soil nematodes and plant parasites such as Meloidogyne sp. which may be of importance in agriculture. 
     These compounds may be administered orally in a unit dosage form such as a capsule, bolus or tablet, or as a liquid drench where used as an anthelmintic in mammals. The drench is normally a solution, suspension or dispersion of the active ingredient usually in water together with a suspending agent such as bentonite and a wetting agent or like excipient. Generally, the drenches also contain an antifoaming agent. Drench formulations generally contain from about 0.001 to 0.5% by weight of the active compound. Preferred drench formulations may contain from 0.01 to 0.1% by weight. The capsules and boluses comprise the active ingredient admixed with a carrier vehicle such as starch, talc, magnesium stearate, or di-calcium phosphate. 
     Where it is desired to administer the instant compounds in a dry, solid unit dosage form, capsules, boluses or tablets containing the desired amount of active compound usually are employed. These dosage forms are prepared by intimately and uniformly mixing the active ingredient with suitable finely divided diluents, fillers, disintegrating agents, and/or binders such as starch, lactose, talc, magnesium stearate, vegetable gums and the like. Such unit dosage formulations may be varied widely with respect to their total weight and content of the antiparasitic agent depending upon factors such as the type of host animal to be treated, the severity and type of infection and the weight of the host. 
     When the active compound is to be administered via an animal feedstuff, it is intimately dispersed in the feed or used as a top dressing or in the form of pellets which may then be added to the finished feed or optionally fed separately. Alternatively, the antiparasitic compounds of this invention may be administered to animals parenterally, for example, by intraruminal, intramuscular, intratracheal, or subcutaneous injection in which the active ingredient is dissolved or dispersed in a liquid carrier vehicle. For parenteral administration, the active material is suitably admixed with an acceptable vehicle, preferably of the vegetable oil variety such as peanut oil, cotton seed oil and the like. Other parenteral vehicles such as organic preparation using solketal, glycerol formal, and aqueous parenteral formulations are also used. The active compound or compounds are dissolved or suspended in the parenteral formulation for administration; such formulations generally contain from 0.005 to 5% by weight of the active compound. 
     Although the antiparasitic agents of this invention find their primary use in the treatment and/or prevention of helminthiasis, they are also useful in the prevention and treatment of diseases caused by other parasites, for example, arthropod parasites such as ticks, lice, fleas, mites and other biting insects in domesticated animals and poultry. They are also effective in treatment of parasitic diseases that occur in other animals including humans. The optimum amount to be employed for best results will, of course, depend upon the particular compound employed, the species of animal to be treated and the type and severity of parasitic infection or infestation. Generally good results are obtained with our novel compounds by the oral administration of from about 0.001 to 10 mg per kg of animal body weight, such total dose being given at one time or in divided doses over a relatively short period of time such as 1-5 days. With the preferred compounds of the invention, excellent control of such parasites is obtained in animals by administering from about 0.025 to 0.5 mg per kg of body weight in a single dose. Repeat treatments are given as required to combat re-infections and are dependent upon the species of Parasite and the husbandry techniques being employed. The techniques for administering these materials to animals are known to those skilled in the veterinary field. 
     When the compounds described herein are administered as a component of the feed of the animals, or dissolved or suspended in the drinking water, compositions are provided in which the active compound or compounds are intimately dispersed in an inert carrier or diluent. By inert carrier is meant one that will not react with the antiparasitic agent and one that may be administered safely to animals. Preferably, a carrier for feed administration is one that is, or may be, an ingredient of the animal ration. 
     Suitable compositions include feed premixes or supplements in which the active ingredient is present in relatively large amounts and which are suitable for direct feeding to the animal or for addition to the feed either directly or after an intermediate dilution or blending step. Typical carriers or diluents suitable for such compositions include, for example, distillers&#39; dried grains, corn meal, citrus meal, fermentation residues, ground oyster shells, wheat shorts, molasses solubles, corn cob meal, edible bean mill feed, soya grits, crushed limestone and the like. The active compounds are intimately dispersed throughout the carrier by methods such as grinding, stirring, milling or tumbling. Compositions containing from about 0.005 to 2.0% weight of the active compound are particularly suitable as feed premixes. Feed supplements, which are fed directly to the animal, contain from about 0.0002 to 0.3% by weight of the active compounds. 
     Such supplements are added to the animal feed in an amount to give the finished feed the concentration of active compound desired for the treatment and control of parasitic diseases. Although the desired concentration of active compound will vary depending upon the factors previously mentioned as well as upon the particular compound employed, the compounds of this invention are usually fed at concentrations of between 0.00001 to 0.002% in the feed in order to achieve the desired antiparasitic result. 
     In using the compounds of this invention, the individual compounds may be prepared and used in that form. Alternatively, mixtures of the individual compounds may be used, or other active compounds not related to the compounds of this invention. 
     The compounds of this invention are also useful in combatting agricultural pests that inflict damage upon crops while they are growing or while in storage. The compounds are applied using known techniques as sprays, dusts, emulsions and the like, to the growing or stored crops to effect protection from such agricultural pests. 
     The following examples are provided in order that this invention might be more fully understood; they are not to be construed as limitative of the invention. 
     EXAMPLE 1 
     A 5% portion of an 18×150 MM test tube containing 3.5 g of soil and dried culture MF 5123 (ATCC 20841) was used to inoculate an unbaffled Erlenmeyer flask containing 50 ml of Medium 1. After three days of incubation of 28° C., agitated on a rotary shaker (5cm throw) at 212 rpm, a 2.0 ml aliquot of the growth was aseptically transferred to a 250 ml Erlenmeyer flask containing Medium 2. After inoculation, Medium 2 was incubated 7 days without agitation at 25° C. After 7 days of incubation 15 ml of sterile distilled water was added to each flask. Incubation was then continued at 25° C. for a further seven days with agitation on a rotary shaker at 160 rpm. 
     EXAMPLE 2 
     A 2.0 ml volume of a frozen vegetative growth in 10% glycerol of the culture MF 5123 (ATCC 20841) was used to inoculate an unbaffled Erlenmeyer flask containing 50 ml of Medium 1. After 3 days of incubation at 28° C., agitated on a rotary shaker (5cm throw) at 212 rpm, a 2.0 ml portion of the growth was aseptically transferred to a 250 ml Erlenmeyer flask containing Medium 2. After inoculation, Medium 2 was incubated 7 days without agitation at 25° C. After 7 days of incubation 15 ml of sterile distilled water was added to each flask. Incubation was then continued at 25° C. for a further 9 days with agitation on a rotary shaker at 160 rpm. 
     EXAMPLE 3 
     A 2.0 ml volume of a frozen vegetative growth in 10% glycerol of the culture MF 5123 (ATCC 20841) was used to inoculate an unbaffled Erlenmeyer flask containing 50 ml of Medium 1. After 3 days of incubation at 28° C., agitated on a rotary shaker (5 cm throw) at 212 rpm, a 2.0 ml portion of the growth was aseptically transferred to a 250 ml Erlenmeyer flask containing Medium 2. After inoculation, Medium 2 was incubated for 7 days without agitation at 25° C. 
     
         ______________________________________Medium 1Corn Steep Liquor     5.0      gTomato Paste          40.0     gOat Flour             10.0     gGlucose               10.0     gTrace Elements Mix    10.0     mlDistilled Water       q.s. 1000                          mlpH 6.8Trace Elements MixFeSO.sub.4.7H.sub.2 O 1.0      gMnSO.sub.4.4H.sub.2 O 1.0      gCuCl.sub.2.2H.sub.2 O 25.0     mgCaCl.sub.2            0.1      g(NH.sub.4).sub.6 MoO.sub.2.4H.sub.2 O                 19.0     mgZnSO.sub.4.7H.sub.2 O 0.2      gDistilled Water       q.s. 1000                          mlMedium 2Amount per 250 ml ErlenmeyerflaskCorn                  10.0     gYeast extract         0.5      gSodium tartrate       0.1      gFeSO.sub.4.7H.sub.2 O 0.01     gL-cysteine            0.1      gGlycerol              0.5      mlCoCl.sub.2.6H.sub.2 O 0.002    gDistilled Water       15.0     mlAutoclave         20 minutes, 15 pounds, 121° C.then Distilled Water  10.0     mlre-autoclave      20 minutes, 15 pounds, 121° C.______________________________________ 
    
     EXAMPLE 4 
     The contents of six seven day solid media 250 ML fermentation flasks from Example 3 were combined and extracted twice with six hundred ml. of ethyl acetate. The extracts were combined, dried with sodium sulfate and concentrated to 60 ml. 
     A three ml. aliquot of the 60 ml concentrate was further concentrated to 0.5 ml. This sample was subjected to preparative thin layer chromatography on an E. Merck Silica-Gel 60 (0.5 mm thickness) plate using a solvent system of 5:5:0.5 v/v/v hexane:methylene chloride:methanol. After chromatography nine zones were selected by U.V. quenching and iodine staining of a template section of the plate. The selected areas were scraped and each eluted with 2 ml. of methanol. The eluted samples were labeled A thru I. One hundred mcl of each sample was submitted for C. elegans assay. Sample C Rf=0.16 was active against C. elegans. Sample C was concentrated to dryness and the residue taken up in 400 mcl of methanol and subjected to preparative HPLC chromatography on a DuPont Zorbax ODS 0.94×25 cm column maintained at 60 degrees C. The chromatography was carried out using an isocratic system of 65:35 v/v methanol:water at a flow rate of 4 ml/min. for thirty minutes followed by a linear gradient to 100% methanol over 10 minutes and held at 100% methanol for 35 minutes. The effluent stream was monitored at 226 nm and a setting of 1.28 AUFS using a Laboratory Data Control Spectromonitor II detector equipped with a 1 mm path length cell, and a Spectra-Physics SP4100 Computing Integrator. Twenty five fractions were collected. Fraction nine with a retention time of 11.3 minutes was active against C. elegans. Fractions eight thru twelve were combined and concentrated to dryness. The residue was labeled J and submitted for N.M.R. and mass spectral analysis and identified as structure I Paraherquamide. Sample J contained 1.8 mg. of paraherquamide based on U.V. analysis using Published U.V. data (Tetrahedron Letters Vol. 22 pp 135-136 1980). 
     EXAMPLE 5 
     The contents of sixteen-seven day solid media 250 ml fermentation flasks from Example 3 were combined and extracted with three one liter portions of ethyl acetate. High pressure liquid chromatography (HPLC) analysis of the three extracts indicated that extracts one and two contained 430 and 130 mg respectively of paraherquamide. Extract three contained only 4 mg and was discarded. Extracts one and two were combined and dried with sodium sulfate. The dried extract was concentrated to an oily residue. The residue was taken up in methanol to a volume of 40 ml. This solution was chromatographed on a 200 ml column of LH-20, previously equilibrated with methanol, using methanol at a flow rate of 3 ml./min collecting a 40 ml fore cut followed by sixty-one seven ml fractions. HPLC analysis showed that fractions 10 thru 16 contained paraherquamide Rt=7.9 minutes. Fractions 10 thru 16 were combined and concentrated to an oily residue. The residue was taken up in 5:5:0.5 v/v/v hexane:methylene chloride:methanol to a volume of 13.5 ml. This solution was chromatographed on a 500 ml column of E. Merck Silica-Gel 60 (0.04 to 0.063mm particle size) previously equilibrated with the same solvent system at a flow rate of 10 ml/min. A 200 ml forecut was collected followed by 315 eight ml fractions. Fractions were combined as follows based on T.L.C. analysis. Fractions 150 to 179; 180 to 199; 200 to 239; 240 to 270; and 271 to 315. The combined fractions were labeled A thru E. Samples C and D were combined, concentrated to dryness and labeled F. Sample F was taken up in 2 ml of methanol and filtered. The filtrate was subjected to preparative HPLC chromatography using a Whatman Magnum 20 ODS-3 2.2×25 cm column at room temperature using an isocratic solvent system of 68/32 v/v methanol/water at a flow of 10 ml/min. The effluent stream was monitored using a Gilson model 116 U.V. detector equipped with a 0.05 mm path length cell and a setting of 1.28 AUFS, the detector signal being monitored by a Spectra-Physics SP4100 Computing Integrator. Thirty fractions were collected. Fractions 16 thru 18 were combined, based on the U.V. recording. The combined fractions were concentrated to dryness to yield 205.4 mg of pure paraherquamide, structure I. 
     EXAMPLE 6 
     Solid Phase Fermentation. The inoculant was prepared by placing the contents of one frozen vial of MF-5123 (ATCC 20841) into 54 ml of medium 1 in a 250 ml flask and agitated at 28° C. for 48 hours on a rotary shaker at 220 rpm. At the completion of the fermentation period 10 ml of the fermentation broth was transferred to 800 ml of medium 1 in a 2 liter flask and agitated at 28° C. for 48 hours on a rotary shaker at 200 rpm. 
     The production medium was prepared by dissolving all of the ingredients of medium 3 except the cracked corn in 3.5 1 of distilled water and combining this solution with 3 kg of cracked corn in a 50×75×5 cm filter tray. The tray was sterilized uncovered for 20 minutes at 121° C. The contents of the tray were stirred and another tray of the same dimension was placed over the first as a lid and taped tightly closed. The covered tray was autoclaved for an additional 20 minutes at 121° C. The tray was removed from the autoclave and allowed to cool for several hours before inoculation. After cooling, the tray was inoculated with 1 liter of the inoculant. The inoculant was distributed uniformly throughout the medium by mixing with a sterile spatula. The lid was taped securely to the tray and the solid culture fermented at 25° C. for several days without agitation. 
     The above solid phase fermentation was repeated using the same procedure with the exception that after the uncovered sterilization 2.0 liter of distilled water was added to the tray. 
     
         ______________________________________Medium 3 (Solid phase production medium)Material         concentration (g/tray)______________________________________Cracked corn (Agway)            3000.0Yeast extract    150.0Sodium tartrate  30.0FeSO.sub.4.7H.sub.2 O            3.0CoCl.sub.2.6H.sub.2 O            0.6L-cysteine       30.0Glycerol         150.0pH - No adjustment______________________________________ 
    
     EXAMPLE 7 
     A tray of 3 kg of solid media from example 6 and 20 flasks containing 80 gm of solid media (2) each from Example 3 were extracted with ethyl acetate using 2×600 ml of solvent per flask and 2×4 liters of solvent per tray. The extracts were combined, labeled A and stored at 5° C. A tray of 3 kg solid media (2) and 20 flasks containing 80 gm of solid media per flask were extracted with ethyl acetate and the extracts combined and labeled B. 
     Samples A and B were combined and concentrated, under reduced pressure at 26° C., to 800 ml. This concentrate was further concentrated, using high vacuum and a 40° C. water bath, to a volume of 300 ml. Three hundred ml of methanol was added to the oily concentrate. Precipitation was observed and the solution was stored at 5° C. overnight. The precipitate was removed by filtration and washed with 200 ml of methanol. The filtrate and the methanol wash were combined giving a volume of 800 ml which was labeled C. HPLC analysis of Sample C indicated the presence of 4.3 gm of paraherquamide. Sample C was concentrated under reduced pressure, to remove methanol to a final volume of 200 ml. To this concentrate 200 ml of acetonitrile was added and labeled D. Solution D was extracted with 1×400 ml of hexane followed by 3×300 ml hexane extractions. The acetonitrile layer, 310 ml, contained 3.9 gm of paraherquamide, by HPLC analysis. The acetonitrile solution was concentrated to dryness and labeled E. The hexane extracts were discarded. 
     EXAMPLE 8 
     Sample E from Example 7 was taken up in 1:1 methylene chloride/ethyl acetate to a volume of 180 ml. The solution was chromatographed on a 4 liter column of silica gel (Grace), which had previously been equilibrated with ethyl acetate. The chromatography was carried out with ethyl acetate at a flow rate of 200 ml/min collecting five four liter fractions followed by 24×500 ml fractions. Fractions 3 thru 9 containing 1.95 gms of paraherquamide were combined and labeled F. Fraction two, which was highly colored and contained considerable paraherquamide, was concentrated to dryness for rechromatography. The residue, which was labeled G, was taken up in 1:1 methylene chloride:ethylacetate to a volume of 180 ml and chromatographed as above. Five four liter fractions followed by 24×500 ml fractions were collected. Fractions 4 thru 17 containing paraherquamide were combined and labeled H. Samples F and H were combined. HPLC analysis of the combined fractions indicated they contained 4 gms of paraherquamide. The combined sample was concentrated to yield 9.5 grams of solids which were labeled J. 
     EXAMPLE 9 
     Sample J from Example 8 was taken up in 3:1 methanol:methylene chloride to a volume of 110 ml and applied to a 2&#34;×6&#39; Sephadex LH 20 column previously equilibrated with methanol. Chromatography was carried out with methanol at a flow rate of 20 ml/min collecting 20 ml fractions. The fractions were analyzed by T.L.C. and HPLC and paraherquamide was found to be present in fractions 155 thru 186. Fractions 155 thru 164 combined and labeled A. Fractions 165 thru 175 combined and labeled B. Fractions 176 thru 186 combined and labeled C. Samples A and B were each concentrated to dryness and their residue triturated with 20 ml of cold ethyl acetate. The triturates were filtered and the solids combined and labeled D. The filtrates were combined and labeled E. 
     EXAMPLE 10 
     Sample D from Example 9 was taken up in 80 ml of 2:1 hexane:acetone and chromatographed, in two 40 ml portions, on silica gel (Grace) previously equilibrated with 2:1 hexane acetone. Portion one was chromatographed on one liter of silica gel and portion two on 700 ml of silica gel. Chromatography in both instances was carried out with 2:1 hexane:acetone at a flow rate of 20 ml/min collecting 25 ml fractions. Fractions 153 thru 220 and 143 thru 230 from the respective columns contained a total of 3.5 gms of paraherquamide. 
     EXAMPLE 11 
     Paraherquamide (5 mg, 0.01moles) was dissolved in 2 mL of methanol. 10% palladium-on-carbon (5 mg) was added and hydrogenation carried out under 40 psig, 23° C. for 2.5 hours. Reaction was 100% completed after 2.5 hours and no side products were observed as determined by HPLC and TLC. The reaction mixture was filtered and the catalyst washed with methanol. The combined methanol solutions containing dihydroparaherquamide were taken to dryness and purified initially by HPLC on an analytical Whatman ODS-3 column using a 60:40, water:methanol 1% acetic acid and 0.1% triethylamine solvent system delivered at 1.5 mL/min. A 40% aqueous methanol solvent system was used subsequently for final purification. Identification was based on mass spectral and NMR analyses. 
     EXAMPLE 12 
     Four &#34;Parasite-free&#34; sheep were exposed to infection with infective L 3  of H. contortus, O. circumcincta, T. axei, T. colubriformis, C. curticei, C. oncophora, and Oe. columbianum. Twenty-four days after the last oral dose of infective larvae, when the infections were patent, three of the sheep were randomly assigned to treatment with either 6, 3 or 1 mg/kg body weight of paraherquamide as a single oral dose. The fourth sheep was maintained as an untreated control. All treatments were administered as solutions at a volume of 0.2 ml/kg P.O.: 6 mg/kg as a solution containing 30 mg/ml; 3 mg/kg as a solution containing 15 mg/ml; 1 mg/kg as a solution containing 5 mg/ml. The vehicle used was a mixture of dimethylsulfoxide: propylene glycol:miglyol (1.5:5:3.5). 
     Four days following treatment fecal worm egg counts of the 3 treated sheep fell from a level of 4,000-20,000 EPG to a barely detectable or undetectable level while the untreated control sheep egg count remained at ≃9,000 EPG. 
     Eight days following treatment the treated and control sheep were sacrificed and their residual worm burdens were recovered and identified using standard procedures. The worm burdens of the treated sheep were compared with that of the untreated control and the evaluations are tabulated as Table 1. 
     
                                           TABLE 1__________________________________________________________________________Evaluation of the anthelimintic activity of Paraherquamideas a single oral dose against 7 species of nematodes in experimentallyinfected sheep;                  Efficacy.sup.b                         Tricho-                     Oesoph-    Haemonchus           Ostertagia                  Ostertagia                         strong-                             Trichostonglylus        agostumNo. Dosage    contortus           circumcincta.                  circumcincta,                         lylus                             colubriformis                                     Cooperia                                          Cooperia                                                Cooperia                                                     colum-Sheep    mg/kg    (a)    L.sub.4                  Adult  axei                             (a)     curticei                                          oncophora                                                spp..                                                      bianum__________________________________________________________________________1   0    (400)  (2680) (280)  (3590)                             (4648)  (3275)                                          (2273)                                                (190)                                                     (7)1   6.0, P.O.    3      3      2      3   3       3    3     3    31   3.0, P.O.    3      3      3      3   3       3    3     3    31   1.0, P.O.    3      2      3      3   3       3    3     3    0__________________________________________________________________________ a = benzimidazole resistant isolate. b = () = Control worm burden. :0 = 0-50% effective; 1 = 51- 75% effective; 2 = 76-90% effective; 3 = ≧91% effective. 
    
     EXAMPLE 13 
     A suspension of the crude extract of paraherquamide was Prepared in 5% acetone 0.2% Triton aqueous solution. Using a standard test procedure, this material was tested in two replicates against first instar Lucilia sericata at a concentration of 1 ml whole broth equivalent (the prepared material is diluted equally with serum). All larvae in both replicates were completely immobile and presumed dead within 2 hours. 
     EXAMPLE 14 
     The same suspension used in Example 13 was used at a concentration of 2 ml whole broth equivalent to screen for activity against tick larvae. For this test Dermacentor albipictus larvae were immersed in 2 ml whole broth equivalent Paraherquamide for 10 minutes. The liquid was then removed with vacuum retaining the ticks on drug-impregnated filter paper. The compound was rapidly and completely active.