The novel antitumor antibiotics designated stephacidin A and stephacidin B are produced by fermentation of Aspergillus ochraceus ATCC-74432. The antibiotics inhibit the growth of mammalian tumors, including particularly prostate carcinoma.

BACKGROUND OF THE INVENTION
 1. Field of the Invention
 This invention relates to novel antitumor antibiotics, designated by the
 present inventors as stephacidin A and stephacidin B, which may be
 obtained by cultivation of a strain of Aspergillus ochraceus. The
 antibiotics provided by the present invention are useful in inhibiting
 tumors in mammals.
 2. Background Art
 The present inventors are not aware of any literature disclosing
 stephacidin A or B or any compounds closely related in structure.
 SUMMARY OF THE INVENTION
 The present invention provides the novel antibiotics designated by the
 present inventors as stephacidin A and stephacidin B and a fermentation
 process for production of these antibiotics using a novel strain of
 Aspergillus ochraceus designated herein as Aspergillus ochraceus WC76466
 (ATCC-74432). The antibiotics of the present invention have been found to
 be useful for the inhibition of tumors, particularly prostate carcinoma,
 in mammals.
 Also provided are pharmaceutical compositions of stephacidin A and B,
 methods for the inhibition of mammalian tumors using the antibiotics of
 the present invention and processes for obtaining the antibiotics,
 including substantially purified forms thereof.

DETAILED DESCRIPTION OF THE INVENTION
 The preferred producing strain for production of stephacidins A and B is a
 mitosporic fungus, Aspergillus ochraceus, isolated from light brown clay
 collected from Sirsaganj, Uttar Pradesh, India.
 In agar culture, colonies of the fungus exhibit the following cultural
 morphological characteristics:
 Taxonomy of the Microorganism
 Colonies on Cornmeal agar (Difco Laboratory) growing moderately fast,
 attaining 45-55 mm in diameter after 14 days at 25.degree. C., 12 hr.
 photoperiod. Colonies effuse, immerse, translucent, and zonate.
 Sporulation relatively sparse. Spore mass appears Warm-Buff (XX) to Pale
 Orange Yellow (III) (capitalized color names from Ridgway, R. 1912. Color
 Standards and Nomenclature, Washington, D.C.). Reverse uncolored.
 Diffusible pigment none. Colorless exudates present. Odor present but not
 distinct.
 Colonies on YMEA (malt extract 1%, yeast extract 0.2%, w/w) growing fast,
 attaining 60-73 mm in diameter after 14 days at 25.degree. C., 12 hr.
 photoperiod. Colonies plane, effuse to lanose, and zonate. Mycelium
 hyaline. Sporulation moderate. Spore mass appear Pale Orange Yellow (III),
 Warm-Buff (XV), Colonial Buff (XXX) to Deep Colonial Buff (XXX). Reverse
 Chamois (XXX), Cream-Buff (XXX), Light-Buff (XV) to Pale Ochraceous-Buff
 (XV). Diffusible pigment none. Colorless exudates present. Odor present
 but not distinct.
 Colonies on PDA (Difco Laboratory) growing fast, attaining 70-80 mm in
 diameter after 14 days at 25.degree. C., 12 hr. photoperiod. Colonies
 effuse, and zonate. Mycelium white to Sea-foam Yellow (XXXI). Sporulation
 heavy. Spore mass appear Warm-Buff (XV), Cream-Buff (XXX), Chamois (XXX),
 Olive-Ocher (XXX), to Deep Colonial Buff (XXX). Reverse Cream Buff (XXX),
 Isabella Color (XXX), Natal Brown (XL), Light Buff (XV) to Light Seal
 Brown (XXXIX). Diffusible pigment none. Colorless exudates present. Odor
 present but not distinct.
 Colonies on oatmeal agar growing very fast, attaining over 85 mm in
 diameter 25.degree. C., 12 hr. photoperiod after 14 days at 25.degree. C.,
 12 hr. photoperiod. Sporulation heavy. Spore mass appear Honey Yellow
 (XXX). Reverse color center Deep Olive-Buff (XL) to edge Dark Olive-Buff
 (XL). Diffusible pigment and odor none. Colorless exudates present.
 Colonies grow very fast on DG18 (Samson, R. A. et al. 1995. Introduction To
 Food-Borne Fungi. Centraalbureau voor Schimmelcultures, Baarn, The
 Netherlands. pp.308-312.), attaining over 85 mm in diameter after 14 days
 at 25.degree. C., 12 hr. photoperiod. Colonies lanate. Sporulation heavy.
 Spore mass appear Honey Yellow (XXX). Reverse Isabella Color (XXX).
 Diffusible pigment, exudates and odor none.
 The producing culture WC76466 has globose conidial head when young, spilt
 into 2-3 divergent compact columns in age. Conidiophores rise from
 substrate mycelium, commonly 650-1300 .mu.m in length, occasionally to
 2000 .mu.m, by 10-15 .mu.m in diameter, occasionally to 20 .mu.m,
 thick-walled (1-2 .mu.m), dull yellow to yellowish-brown shades, upper two
 third coarsely roughened, appearing bumpy, lower portion smooth-walled,
 not constricted beneath the vesicle. Vesicles globose, occasionally
 subglobose, thick-walled, 35-50 .mu.m in diameter, occasionally to 60
 .mu.m. Sterigmata covering entire vesicle, crowded, predominant biseriate,
 occasionally uniseriate; metula mostly wedge-shaped 12-20 .mu.m by 5-7
 .mu.m but occasionally less than 10 .mu.m in length, occasionally becoming
 septate; phialides 8-10 .mu.m by 2-3 .mu.m, 5-6 in a whirl on metula.
 Conidia globose to subglobose, hyaline to very light brown, thin-walled,
 smooth to finely roughened, 2-3.5 .mu.m in diameter mostly 2.5-3.0 .mu.m.
 Sclerotia not present.
 A biologically pure culture of Aspergillus ochraceus strain WC76466 has
 been deposited with the American Type Culture Collection, 10801 University
 Boulevard, Manassas, Va. 20108-1549, under the accession number
 ATCC-74432.
 As in the case of other producing microorganisms, the characteristics of
 the new stephacidin A and B-producing culture of the present invention,
 Aspergillus ochraceus ATCC-74432, are subject to variation. Recombinants,
 variants and mutants of the ATCC-74432 strain may be obtained by treatment
 with various known mutagens such as ultraviolet rays, X-rays, high
 frequency waves, radioactive rays, and chemicals. Natural and induced
 variants, mutants and recombinants of Aspergillus ochraceus ATCC-74432
 which retain the characteristic of producing stephacidin A and B are
 intended to be encompassed by the present invention.
 The stephacidin A and B antibiotics may be produced by cultivating a
 stephacidin A and B-producing strain of Aspergillus ochraceus, preferably
 Aspergillus ochraceus ATCC-74432 or a mutant or variant thereof, under
 submerged aerobic conditions in an aqueous nutrient medium. The
 fermentation is carried out until a substantial amount of stephacidin A
 and B are detected in the broth and then the desired antibiotics are
 harvested by extracting the active components from the mycelial growth
 with a suitable solvent. The solution containing the desired component(s)
 is concentrated and then the concentrated material subjected to
 chromatographic separation to isolate the component(s) in purified form
 substantially free of other co-produced materials.
 The producing organism is grown in a nutrient medium containing an
 assimilable carbon source, for example an assimilable carbohydrate.
 Examples of suitable carbon sources include glucose, fructose, mannose,
 maltose, galactose, mannitol, glycerol, other sugars and sugar alcohols,
 starches and other carbohydrates, or carbohydrate derivatives such as
 dextran or cerulose, as well as complex nutrients such as oat flour, corn
 meal, millet, and the like. The exact quantity of 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 of carbohydrate between
 0.5 to 10 percent by weight of the medium is satisfactory. The carbon
 sources may be used individually or several such carbon sources may be
 combined in the same medium.
 The nutrient medium should also contain an assimilable nitrogen source such
 as amino acids (e.g. glycine, arginine, threonine, methionine and the
 like), ammonium salts, or complex nitrogen sources such as yeast extracts,
 corn steep liquors, distiller solubles, soybean meal, cottonseed meal,
 fish meal, and the like. The nitrogen source may be used alone or in
 combination in amounts ranging from 0.05 to 5 percent by weight of the
 medium.
 Nutrient inorganic salts may also be incorporated in the medium and such
 salts may comprise any of the usual salts capable of providing sodium,
 potassium, magnesium, calcium, phosphate, sulfate, chloride, carbonate,
 and like ions. Trace metals such as cobalt, manganese, iron, molybdenum,
 zinc, cadmium, and the like may also be used if desired.
 Production of the stephacidin antibiotics may be effected at any
 temperature conducive to satisfactory growth of the organism, i.e.
 approximately 18-45.degree. C., and is conveniently carried out at a
 temperature of about 28.degree. C. The fermentation may be carried out in
 flasks or in laboratory or industrial fermentors of various capacities.
 When tank fermentation is to be carried out, it is desirable to produce a
 vegetative inoculum in a nutrient broth by inoculating the broth culture
 with a slant or soil culture or a lyophilized culture of the organism.
 After obtaining an active inoculum in this manner, it is transferred
 aseptically to the fermentation tank medium for large scale production of
 the desired antibiotic. The medium in which the vegetative inoculum is
 produced can be the same as, or different from, that utilized in the tank
 for the production of the new antibiotic as long as it is such that a good
 growth of the microrganism is obtained.
 When fermentation is complete, stephacidin A and B are recovered from the
 fermentation broth and separated from co-produced substances and other
 impurities by art-recognized techniques. A typical isolation procedure is
 shown in FIG. 9 and in the example which follows.
 The physico-chemical properties of stephacidin A and B are as follows:
 TABLE 1
 Physico-Chemical Properties of Stephacidin B
 Description: Off-white amorphous solid
 Solubility: Soluble in methanol, chloroform, acetone,
 acetonitrile, dimethyl sulfoxide, and the solvent
 mixture ot acetonitrile/methanol (1:1), but
 insoluble in hexane and water.
 Molecular Formula: C.sub.52 H.sub.54 O.sub.8 N.sub.6
 Molecular Weight: 890
 Mass Spectrum: HR-ESIMS ion: 891.4085 [M + H].sup.+
 Positive ESI-MS ions: 891 [M + H].sup.+, 1781 [2M +
 H].sup.+
 Negative ESI-MS ions: 889 [M - H].sup.-,
 1779 [2M - H].sup.-
 Ultraviolet Spectrum: .lambda. max.sub.MeOH (log .epsilon.) 209 (4.73), 240
 (4.54), 268
 (sh. 4.27) 301 (4.26), 346 (sh. 3.79) nm (FIG. 1).
 Sample dissolved in methanol at concentration of
 0.0011 g/L.
 Infrared Spectrum: Major Bands (cm.sup.-1) 3429, 2972, 1713, 1683,
 1671, 1638, 1520, 1459, 1386, 1337, 1276,
 1210, 1190, 1162, 1115, 1025, 1004, 826, 757,
 563, 510 cm.sup.-1 (FIG. 2)
 .sup.1 H-NMR FIG. 3, in DMSO-CD.sub.3 CN (1:1)
 .sup.13 C-NMR .delta..sub.C (DMSO--CD.sub.3 CN 1:1) in ppm (FIG. 4)
 175.1,
 173.8, 167.5, 167.0, 153.0, 152.2, 148.8, 140.9,
 140.2, 132.2, 130.4, 130.2, 128.6, 122.9, 119.5,
 118.6, 116.2, 116.1, 115.3, 111.7, 110.3, 104.5,
 98.0, 76.3, 75.2, 70.2, 68.9, 65.7, 64.7, 61.9,
 58.5, 46.5, 44.3, 44.0, 43.3, 43.2, 38.2, 35.0,
 29.8, 29.4,28.6, 28.0, 27.7, 27.2, 27.0, 26.7,
 26.6, 26.3, 24.9, 24.5, 19.4, 16.3
 TABLE 2
 Physico-Chemical Properties of Stephacidin A
 Description: white amorphous solid
 Molecular Formula: C.sub.26 H.sub.29 O.sub.3 N.sub.3
 Molecular Weight: 431
 Mass Spectrum: HR-ESIMS ion: 432.2292 [M + H].sup.+
 Positive ESI-MS ions: 449 [M + NH.sub.4 ].sup.+
 Negative ESI-MS ions: 430[M - H].sup.-
 Ultraviolet .lambda. max .sub.MeOH (log .epsilon.) 211 (4.52), 242
 (4.54), 309
 Spectrum (3.96), 335 (sh. 3.64) nm (FIG. 5). Sample
 dissolved in methanol at concentration of 0.0010
 g/L.
 Infrared Spectrum: Major Bands (cm.sup.-1) 3442, 2973, 2481, 1691,
 1673, 1638, 1439, 1384, 1201, 1158,1120, 1075,
 913, 808, 734 cm.sup.-1 (FIG. 6)
 .sup.1 H-NMR .lambda..sub.H (CDCl.sub.3 /CH.sub.3 OD) in ppm, J in Hz
 (FIG. 7) 7.10
 (1H, d, J = 8.4), 6.63 (1H, d, J = 9.7), 6.50 (1H, d,
 J = 8.4), 5.52 (1H, d, J = 9.7), 3.56 (H, d, J = 15.4),
 3.38 (1H, m), 3.27 (1H, m), 2.65 (1H, m), 2.58
 (1H, d, J = 15.4), 2.45 (1H, dd, J = 10.3, 4.7), 2.09
 (1H, dd, J = 13.5, 10.3), 1.92 (2H, m), 1.84 (1H,
 dd, J = 13.5, 4.7), 1.78, (1H, m), 1.32 (6H, s), 1.21
 (3H, s), 0.97 (3H, s)
 .sup.13 C-NMR .lambda..sub.C (CDCl.sub.3 /MeOD) in ppm (FIG. 8) 174.0,
 169.3,
 148.1, 138.6, 133.0, 129.1, 121.6, 117.9, 117.5,
 109.4, 105.1, 104.0, 75.4, 66.6, 60.3, 49.4, 43.9,
 34.6, 30.7, 29.1, 27.9, 27.0, 27.0, 24.3, 24.3,
 21.6
 Based on the characterizing properties for the antibiotics, the structures
 of stephacidin A and B have been determined to be as follows:
 ##STR1##
 Both the stephacidin A (monomer) and stephacidin B (dimer) can form
 pharmaceutically acceptable salts with nontoxic organic or inorganic acids
 and such salts are encompassed within the term "stephacidin A" and
 "stephacidin B" as used herein. For example, stephacidin A can be
 converted to a hydrochloride salt by treatment with hydrochloric acid to
 yield a water-soluble hydrochloride salt at an amino group such as 9NH.
 Similarly, stephacidin B can form acid addition salts at amino groups.
 Other examples of suitable acids for salt formation include hydrobromic,
 sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic,
 lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric,
 methanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic and
 benzenesulfonic.
 The stephacidin A and B compounds may also form pharmaceutically acceptable
 amide derivatives by treatment with standard acylation reagents such as
 acetic acid anhydride to convert amino groups to amide groups. Such
 acylation reaction may be carried out in the presence of an organic base
 in an inert organic solvent, e.g. 4-dimethylaminopyridine (DMAP) and
 triethylamine in methylene chloride. It is intended that the term
 "stephacidin A and B" as used herein includes such pharmaceutically
 acceptable amide derivatives within its scope.
 Biological Properties:
 In Vitro Cytotoxicity
 Stephacidin A demonstrated potent in vitro cytotoxicity against several
 human tumor cell lines (see Table 3 below). Stephacidin A was 10-fold less
 potent but within our in vivo criteria for acceptable activity. Good
 selectivity was observed in the testosterone dependent LNCaP cells,
 especially with stephacidin B. The effects of this compound are not
 mediated by p53, mdr or bcl2, and it is not tubulin or topoisomerase II
 mediated, indicating a novel mechanism of action.
 TABLE 3
 In vitro cytotoxicity of Stephacidin A and Stephacidin B
 IC.sub.50 (.mu.M) IC.sub.50
 (.mu.M)
 Cell Line Histotype Characteristic Stephacidin B Stephacidin A
 A2780 Ovarian Parental .33 4.0
 A2780/DDP Ovarian mutp53/bcl2+ .43 6.8
 A2780/Tax Ovarian Taxol resistant .26 3.6
 PC3 Prostate Testosterone .37 2.1
 independent
 LNCaP Prostate Testosterone .06 1.0
 sensitive
 HCT116 Colon Parental .46 2.1
 HCT116/mdr+ Colon Overexpresses .46 6.7
 Mdr+
 HCT116/Topo Colon Resistant to to .42 13.1
 etoposide
 MCF-7 Breast Estradiol
 sensitive .27 4.2
 SKBR3 Breast Estradiol- .32 2.15
 independent
 LX-1 Lung Sensitive .38 4.22
 The in vitro cytoxicity assay used for the data above was carried out as
 follows:
 In Vitro Cytotoxicity Assay
 In vitro cytotoxicity was assessed in human carcinoma cells by the MTS
 (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphenyl)-2
 H-tetrazolium, inner salt) assay (5). Cells were plated at 4,000 cell/well
 in 96 well microtiter plates and 24 hours later drugs were added and
 serial diluted. The cells were incubated at 37.degree. form 72 hours at
 which time the tetrazolium dye, MTS at 333 .mu.g/ml (final concentration),
 in combination with the electron coupling agent phenazine methosulfate at
 25 .mu.M (final concentration) was added. A dehydrogenase enzyme in live
 cells reduces the MTS to a form that absorbs light at 492 nm which can be
 quantitated spectrophotometrically. The greater the absorbance the greater
 the number of live cells. The results are expressed as an IC.sub.50, which
 is the drug concentration required to inhibit cell proliferation (i.e.
 absorbance at 492 nm) to 50% of that of untreated control cells.
 Apoptosis Induction
 Table 4 below shows that both stephacidin B and stephacidin A are potent
 inducers of apoptosis in both testosterone sensitive (LNCaP) and
 independent cells (A2780). These results suggest two independent
 mechanisms of action or one mechanism that is more essential to cells when
 testosterone sensitivity is expressed.
 TABLE 4
 Effect of Stephacidin B and Stephacidin A
 Compounds Control 0.1 x 1 x 5 x 10 x 20 x
 % of LNCaP Cells in Apoptosis
 Multiples of 72 hour cytotoxic IC50
 Stephacidin A 0.06 0.02 0.00 0.15 5.75 11.81
 Stephacidin B 0.00 0.02 0.00 0.05 4.52 11.13
 % of A2780 Cells in Apopotosis
 Multiples of 72 hour cytotoxic IC50
 Stephacidin A 4 1 .8 21 14 15
 Stephacidin B .8 .5 .2 1.0 33 5
 The apoptosis assay used for the data above was carried out as follows:
 Apoptosis Determination
 Cells were plated, incubated overnight, treated with compound for 24 hours
 then harvested by trypsinization. For cell cycle analysis cells were
 trypsinized, permeated, stained with 50 ug/ml propidium iodide and
 analyzed by FACS. For apoptosis evaluation (TUNEL assay) cells were
 permeated and reacted with TdT and fluoresceinated dUTP for 3 hours
 following the procedure recommended by the APO-Direct kit (Pharmingen).
 As indicated above, stephacidin A and B demonstrate inhibitory activity
 against mammalian tumors, particularly prostate carcinoma. Thus, in
 another aspect of the present invention, there is provided a method for
 therapeutically treating a mammalian host affected by a malignant tumor
 sensitive to stephacidin A and/or B which comprises administering to said
 host an effective tumor-inhibiting dose of stephacidin A or stephacidin B.
 In yet another aspect of the present invention, a pharmaceutical
 composition is provided which comprises an effective tumor-inhibiting dose
 of stephacidin A or B in combination with an inert pharmaceutically
 acceptable carrier or diluent.
 The pharmaceutical compositions may contain other active antitumor agents
 and may be made up in any pharmaceutical form appropriate for the desired
 route of administration. Examples of such compositions include solid
 compositions for oral administration such as tablets, capsules, pills,
 powders and granules, liquid compositions for oral administration such as
 solutions, suspensions, syrups or elixers and preparations for parenteral
 administration such as sterile solutions, suspensions or emulsions. They
 may also be manufactured in the form of sterile solid compositions which
 can be dissolved in sterile water, physiological saline or some other
 suitable sterile injectable medium immediately before use.
 For use as an antitumor agent, optimal dosages and regimens of the
 stephacidin A and B antibiotics can be readily ascertained by those
 skilled in the art. It will, of course, be appreciated that the actual
 dose of compound used will vary according to the particular composition
 formulated, the mode of application and the particular situs, host and
 disease being treated. Many factors that modify the action of the drug
 will be taken into account including age, weight, sex, diet, time of
 administration, route of administration, rate of excretion, condition of
 the patient, drug combinations, reaction sensitivities and nature and
 severity of the disease.
 The following example is provided for illustrative purposes only and is not
 intended to limit the scope of the invention. Volume ratios used in the
 present application, unless otherwise indicated, are volume/volume. The
 following abbreviations are used in the specification and drawings:
 MeOH=methonol
 EtOAc=ethyl acetate
 General Methods:
 Analytical Thin Layer Chromatography (TLC)
 Silica gel precoated thin layer chromatography plates, Kieselgel 60 F254 on
 aluminum sheet, 5.times.20 cm, 0.2 mm, were purchased from EM Separations,
 Gibbstown, N.J. The plates were developed in a tank equilibrated with
 methylene chloride/methanol (49:4 v/v). The components of the resulting
 chromatogram were detected under a UV light, and visualized by
 phosphomolybidic acid followed by prolonged heating.
 Preparative TLC
 Silica gel precoated Kieselgel 60 F254 plates on glass, 20.times.20 cm, 2
 mm, purchased from EM Separations, were used for preparative purification.
 The plates were developed in a tank equilibrated with methylene
 chloride/methanol (49:4 v/v). The components of the resulting chromatogram
 were detected under a UV light. The silica bands containing the components
 were scraped and pressed to a fine powder, followed by elution with
 chloroform/methanol (3:1, v/v). The eluant was then evaporated in vacuo to
 dryness.
 Analytical HPLC
 The purification of stephacidin B and stephacidin A was monitored by HPLC
 analysis on a Microsorb-MV 5 .mu.C-18 column, 4.6 mm i.d..times.25 cm l.
 (Rainin Instrumnet Company, Inc., Woburn, Mass.). Analyses were done on a
 Hewlett Packard 1090 Liquid Chromatograph, equipped with a model
 photodiode array spectrophotometer set at 254 and 280 nm, and HPLC.sup.3D
 ChemStation operating software. A gradient solvent system and 0.01 M
 potassium phosphate buffer (PH 3.5) was used, according to the method of
 D. J. Hook et al (J. Chromatogr. 385, 99, 1987). The eluant was pumped at
 a flow rate of 1.2 ml/min.
 Preparative HPLC
 The following components were used to construct a preparative HPLC system:
 Dynamax SD-200 pumps, Dynamax dual wavelength spectrophotometer UV-D11 and
 Dynamax method manager software, a Microsorb-MV 5 .mu.C-18 column, 10 mm
 i.d..times.25 cm l, plus 10 mm i.d..times.5 cm l. guard column (Rainin
 Instrumnet Company, Inc., Woburn, Mass.). A gradient solvent system
 consisting of acetonitrile and water, were used at a flow rate of 5 ml/min
 with run time of 32 minutes. The compounds were detected by monitoring the
 eluate stream at 254 nm.
 Analytical Instrumentation
 Low resolution MS measurements were performed with a Finnigan MAT 900
 magnetic sector mass spectrometer, using the positive electrospray
 ionization mode. MS/MS measurements were conducted with Finnigan LCQ (ion
 trap MS) with electrospray as ionization mode. High resolution MS data
 were determined with a Finnigan MAT 900 magnetic sector mass spectrometer,
 positive electrospray ionization mode, ppg reference. The UV spectra were
 obtained using a Hewlett-Packard 8452A diode array spectrophotometer. IR
 measurements were taken on a Perkin Elmer 2000 Fourier Transform
 spectrometer. .sup.1 H-NMR and .sup.13 C-NMR spectra were obtained on a
 Bruker AM-500 500 MHz instrument operating at 500.13 and 125.76 MHz,
 respectively, using a 5-mm broad-banded probe.
 EXAMPLE 1
 Preparation of Stephacidin A and B
 A. Fermentation of the antibiotics
 Fungal cultures of Aspergillus ochraceus ATCC-74432 were grown on
 potato-dextrose agar slants containing the following ingredients per liter
 of deionized water: potato infusion, 200 g; dextrose, 20 g; agar, 15 g.
 The slant cultures were allowed to grow for 7 days at 28.degree. C.
 Glycerol/water solvate (15%, w/v) was added and spore suspensions were
 prepared, divided into aliquots, and frozen in a dry ice-acetone bath. The
 frozen spore suspensions so obtained were then stored at -80.degree. C.
 From the frozen spore suspensions, 0.1 ml was used to inoculate a
 potato-dextrose slant which was inoculated at 28.degree. C. for 7 days. A
 spore suspension was made using 0.85% saline and the spore suspension was
 transferred onto a 24.5.times.24.5 cm. Nunc plate containing 250 ml of
 medium containing the following ingredients per liter of deionized water:
 soluble starch, 20 g;,dextrose, 5 g.; soybean meal, 10 g; corn steep
 liquor, 10 g; NZ-amine type A, 3 g.; sodium chloride, 3 g; calcium
 carbonate, 3 g; agar, 10 g. The culture was incubated at 28.degree. C.
 Maximum production of the desired antibiotics was achieved after 7 days of
 incubation.
 B. Isolation and Purification
 Preparation of Crude Extract A
 Each of 20 fermentation Nunc plates containing the culture grown on solid
 media was soaked with 200 ml of methanol and stayed at room temperature
 for two hours. The liquid layer was combined and concentrated to around
 800 ml under a nitrogen stream to remove most of the methanol. Water was
 then added to bring the volume to approximately 1 liter. The aqueous
 solution was then partitioned three times with an equal volume of ethyl
 acetate in a separatory funnel. Stephacidin B and stephacidin A were
 concentrated in the ethyl acetate layer. The aqueous layer was removed.
 The organic layer was combined and then evaporated to dryness in vacuo in
 a rotary evaporator to 1.1 g of residue A.
 Sephadex LH-20 Chromatography of Residue A
 Residue A (1.1 g) was dissolved in 4 ml metanol and applied to a
 3.times.100 cm Spectrum column packed with 200 g Sephadex LH-20 in
 metanol. The column was eluted with methanol. Fractions measuring 8-10 ml
 each were collected at a flow rate of 2-3 ml/min. Fractions were
 consolidated on the basis of silica TLC profiles (chloroform/methanol 9:1,
 phosphormolybdic acid spray). In this manner, seven groups of fractions
 were obtained. Analytical HPLC analyses indicated that the second group of
 fractions (Residue B, 460 mg) and the third fraction (Residue C, 250 mg)
 contained stephacidin B and stephacidin A, respectively.
 Preparative HPLC of Residue B (Isolation of Stephacidin B)
 Final purification of stephacidin B from Residue B could be achieved by
 using the specified Rainin Dynamax preparative HPLC system. A typical
 injection sample size was 5 mg/0.2 ml methanol. Elution flow rate was 5
 ml/min. Detection (UV) was at 254 nm. One hundred mg of Residue B was
 purified with the following solvent gradient:

Time (min) acetonitrile (%) water (%)
 0.00 30 70
 32.00 85 15
 37.00 30 70
 The peak at 21.4 was collected and the solvent removed in vacuo to yield 10
 mg pure stephacidin B.
 Preparative TLC of Residue B (Isolation of Stephacidin B)
 Preparative TLC was proven to be an alternative and more efficient way to
 purify stephacidin B from Residue B. 300 mg of Residue B was dissolved in
 10 ml of methylene chloride-methanol 4:1. The solution was concentrated
 under a nitrogen stream to a final volume of 2 ml, and then applied to
 four Silica gel precoated Kieselgel 60 F254 plates on glass, 20.times.20
 cm, 2 mm, EM Separations. The plates were developed in a tank equilibrated
 with methylene chloride/methanol (49:4 v/v). The components of the
 resulting chromatogram were detected under a UV light. The silica bands
 containing the components (Rf 0.84) were scraped and pressed to a fine
 powder, followed by elution with chloroform/methanol (3:1, v/v). The
 eluant was then evaporated in vacuo to give 75 mg of stephacidin B.
 Sephadex LH-20 Chromatography of Residue C
 Residue C (250 mg) was further purified again using Sephadex LH-20 column
 chromatography (3.times.100 cm Spectrum column packed with 200 g Sephadex
 LH-20 in metanol, eluted with methanol) Fractions measuring 8-10 ml each
 were collected at a flow rate of 2-3 ml/min. Fractions were consolidated
 on the basis of silica TLC profiles (chloroform/methanol 9:1,
 phosphormolybdic acid spray). In this manner, seven groups of fractions
 were obtained. Analytical HPLC analyses indicated that the second group of
 fractions (Residue D, 95 mg) contained stephacidin A.
 Preparative TLC of Residue D (Isolation of Stephacidin A)
 The final purification of stephacidin A from Residue C was achieved by
 preparative TLC. Ninty five mg of Residue D was dissolved in 5 ml of
 methylene chloride-methanol 4:1. The solution was concentrated under a
 nitrogen stram to a final volume of 1 ml, and then applied to two Silica
 gel precoated Kieselgel 60 F254 plates on glass, 20.times.20 cm, 2 mm, EM
 Separations. The plates were developed in a tank equilibrated with
 methylene chloride/methanol (49:4 v/v). The components of the resulting
 chromatogram were detected under a UV light. The silica bands containing
 the components (Rf 0.64) were scraped and pressed to a fine powder,
 followed by elution with chloroform/methanol (3:1, v/v). The eluant was
 then evaporated in vacuo to give 15 mg of stephacidin A.