Antibiotics DO-248-A and B and process for preparing the same
BACKGROUND OF THE INVENTION 
     (1) Field of the Invention 
     This invention relates to antibiotics DO-248-A and DO-248-B of the following Formula I: ##STR2## wherein R is ethyl or isopropyl, and their pharmaceutically acceptable salts. Further it relates to a process for preparing these antibiotics and to anti-acid-fast organism compositions containing as an active ingredient these antibiotics and/or their pharmaceutically acceptable salts. 
     (2) Description of the Prior Art 
     Antibiotics have long been clinically used as an anti-tubercle drug and especially aminoglycosides such as streptomycin and kanamycin and semisynthetic macrolides such as rifampicin are generally used in combined drugs with other synthetic anti-tubercle drugs. Generally, it is hard to prevent occurrence of resistant microorganisms where an antibiotic has long been administered and it is a present problem that such resistant microorganisms have been occurred against the anti-tubercle drugs as above mentioned. 
     Though antibiotic pheganomycins have been known as antibiotics having α-guanidino-3,5-dihydroxyphenyl acetic acid residue {Proceeding of the 15th Symposium on Peptide Chemistry, 121 (1977)}, DO-248-A and DO-248-B are glycin derivatives and have a distinctly different chemical structure from pheganomycins which are hexa- or heptapeptide derivatives. 
     SUMMARY 
     Antibiotics DO-248-A and DO-248-B of this invention have antibacterial activity against acid-fast bacteria and kanamycin- and rifampicin-resistant bacteria and are more active against the pathogenic bacterium of atypical fast-acid bacterium disease than kanamycin. DO-248-A, DO-248-B, or their pharmaceutically acceptable salts are, as an active ingredient of anti-fast-acid drugs, orally or parenterally administered to human beings or animal. Antibiotics DO-248-A and DO-248-B are prepared by incubating an antibiotic DO-248-A-and/or DO-248-B-producing microorganisms belonging to the genus Streptoverticillium in a medium and recovering antibiotic DO-248-A and/or DO-248-B from the culture broth. 
    
    
     BRIEF DESCRIPTION OF DRAWINGS 
     FIG. 1, FIG. 2, FIG. 3 and FIG. 4 show UV absorption spectrum in an aqueous solution, IR absorption spectrum in a potassium bromide tablet,  1  H-NMR spectrum and  13  C-NMR spectrum of Do-248-A, respectively, and 
     FIG. 5, FIG. 6, FIG. 7 and FIG. 8 show UV absorption spectrum in an aqueous solution, IR absorption spectrum in a potassium bromide tablet,  1  H-NMR spectrum and  13  C-NMR spectrum of DO-248-B, respectively. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The physicochemical properties of the antibiotics DO-248-A and DO-248-B of this invention are as follows. 
     (1) 
     DO-248-A 
      ○1  Elemental analysis: Calcd. (%); for C 14  H 20  N 4  O 5 .2H 2  O, C 46.66, H 6.71, N 15.58. Found (%); C 47.01, H 6.15, N 15.44. 
      ○2  Molecular weight (by secondary ion mass spectrometry): (M+1) +  325. 
      ○3  Melting point: 194°-200° C. 
      ○4  Specific rotation: [α] 22 .5 D  +84.3±1.3 (CO.966, water). 
      ○5  UV (see FIG. 1): 
     λ max   H .sbsp.2 O  nm (E 1  cm 1% ) 276 (36); 
     λ max   H .sbsp.2 O+1NHCl  1drop nm (E 1  cm 1% ) 283 (36); 
     λ max   H .sbsp.2 O+1NNaOH  1drop nm (E 1  cm 1% ) 297 (74). 
      ○6  IR (see FIG. 2): ν max   KBr  cm -1  3350, 1660, 1605, 1433, 1390, 1305, 1280, 1130, 1080, 1015, 823, 803. 
      ○7  Solubility: soluble in water, readily soluble in methanol, ethanol and dimethylformamide, slightly soluble in chloroform, ethylacetate and acetone, insoluble in benzene, ether and hexane. 
      ○8  Color reaction: Sakaguchi&#39;s reaction; positive. 
      ○9  Appearance and distinction between base, acid and neutral 
     colorless powder, amphoteric. 
      ○10   1  H-NMR [200 MHz.D 2  O, external reference TMS (δ value from DSS; 0.65)] (see FIG. 3): δ ppm  (J=Hz) 1.23(d 6H J=7), 3.35(m 1H J=7), 3.61(AB d 1H J=17), 3.83(AB d 1H J=17), 5.10(s 1H), 6.48(s 2H). 
      ○11   13  C-NMR (δ value 67.4) (see FIG. 4): δ ppm  20.5(q×2), 25.0(d), 44.4(t), 59.3(d), 108.1(d×2), 123.8(s), 134.8(s), 156.4(s×2), 157.3(s), 171.0(s), 177.0(s). 
      ○12  Amino acid analysis: Glycine and ammonia were detected. 
     (2) 
     DO-248-B 
      ○1  Elemental analysis: Calcd. (%); for C 13  H 18  N 4  O 5 .2H 2  O, C 45.08, H 6.40, N 16.18. Found (%); C 45.26, H 6.13, N 16.13. 
      ○2  Molecular weight (by secondary ion mass spectrometry): (M+1)+311. 
      ○3  Melting point: 189°-193° C. 
      ○4  UV (see FIG. 5): 
     λ max   H .sbsp.2 O  nm (E 1  cm 1% ) 276 (36), 281.5 (36); 
     λ max   H .sbsp.2 O+1NHCl  1drop nm (E 1  cm 1% ) 276 (36), 281.5 (36); 
     λ max   H .sbsp.2 O+1NNaOH  1drop nm (E 1  cm 1% ) 297 (72). 
      ○5  IR (see FIG. 6): ν max   KBr  cm -1  3350, 1660, 1608, 1435, 1393, 1310, 1250, 1105, 1000. 
      ○6  Color reaction: Sakaguchi&#39;s reaction; positive. 
      ○7  Appearance and distinction between base, acid and neutral 
     colorless powder, amphoteric. 
      ○8   1  H-NMR [200 MHz.D 2  O, external reference TMS (δ value from DSS; 0.65)] (see FIG. 7): δ ppm  (J=Hz) 1.00(t 3H J=7.3), 2.52(q 2H J=7.3), 3.61(AB d 1H J=17), 3.83(AB d 1H J=17), 5.10(s 1H), 6.50(s 2H). 
      9○   13  C-NMR [25.2 MHz, D 2  O, internal reference dioxane (δ value 67.4)]: δ ppm  (J=Hz) 13.6(q), 16.9(t), 44.5(t), 59.4(d), 107.4(d×2), 120.1(s), 134.8(s), 156.0(s×2), 157.3 (s), 171.0(s), 177.0(s) (see FIG. 8). 
     Through the above physiochemical properties the chemical structures of DO-248-A and DO-248-B were determined to be the following Formula I. ##STR3## (wherein R is ethyl or isopropyl) Namely, DO-248-A is N-(α-guanidino-3,5-dihydroxy-4-isopropylphenylacetyl)glycine of Formula I wherein R is isopropyl and DO-248-B is N-(α-guanidino-3,5-dihydroxy-4-ethylphenylacetyl)glycine of Formula I wherein R is ethyl. Thus, DO-248-A and B are concluded to be novel antibiotics. 
     DO-248-A and DO-248-B are produced by a strain belonging to Streptoverticillium isolated from a soil sample in accordance with a usual method. The microoganism was identified to be the same species as Streptoverticillium roseoverticillatum (shinobu) Forina and Locci from an investigation for classification. 
     Taxonomical properties of the microorganism are as follows. 
     (1) Morphological property (incubated in yeast extract-malt extract agar medium at 28° C. for 14 days) 
     This microorganism grows well and forms lots of aerial mycelia, to which conidia adhere. Under a microscope the aerial mycelia branching into a whorl are observed, and the forms of spore chains are straight and the number of conidium per chain is mostly below 10. Under an electron microscope the surface of the conidium is smooth. Any sporangium, flagellated spore and sclerotium are not observed. 
     (2) Cultural characteristics 
     
                                           TABLE 1                                 
__________________________________________________________________________
                Aerial mycelium                                           
                          Substrate                                       
                               Soluble                                    
Medium     Growth                                                         
                formation                                                 
                     color                                                
                          mycelium                                        
                               pigment                                    
__________________________________________________________________________
Sucrose nitrate                                                           
           Good Good Pink None to                                         
                               None                                       
agar medium               pink                                            
Glucose asparagine                                                        
           Good Good Pink to                                              
                          Light red                                       
                               None                                       
agar medium          pale pink                                            
Glycerol asparagine                                                       
           Good Good Pink Pale red                                        
                               None                                       
agar medium                                                               
Inorganic salt                                                            
           Good Good Pinkish                                              
                          Dull red                                        
                               None                                       
starch agar medium   white to                                             
                     pale pink                                            
Tyrosine agar medium                                                      
           Good Good Pale pink                                            
                          Dull red                                        
                               Brownish                                   
                     to pink   black                                      
                               (trace)                                    
Nutrition agar                                                            
           Good None --   Grayish                                         
                               Grayish                                    
medium                    brown                                           
                               brown                                      
Yeast extract-malt                                                        
           Good Good Pale pink                                            
                          Reddish                                         
                               Yellowish                                  
extract agar medium  to pale                                              
                          orange                                          
                               brown                                      
                     brown                                                
Oatmeal agar medium                                                       
           Good Good Pale pink                                            
                          Light red                                       
                               None                                       
                     to pale                                              
                     brown                                                
Bennet&#39;s agar medium                                                      
           Good Good Pale pink                                            
                          Dark Yellowish                                  
                     to pale                                              
                          reddish                                         
                               brown                                      
                     orange                                               
                          orange                                          
                               (trace)                                    
__________________________________________________________________________
 Colors are from GUIDE TO COLOUR STANDARD (Japan Color Institute).        
 
    
     (3) Physiological properties 
     Gelatin liquifaction: negative; 
     Melanin production: positive; 
     Tyrosinase reaction: weakly positive; 
     Coagulation of milk: negative; 
     Peptonization of milk: weakly positive; 
     Starch hydrolysis: positive. 
     (4) Utilization of carbohydrates 
     
         ______________________________________                                    
Kind of carbohydrates                                                     
                   Growth                                                 
______________________________________                                    
L-arabinose        +                                                      
D-xylose           +                                                      
D-glucose          ++                                                     
D-fructose         ++                                                     
Sucrose            +                                                      
Inositol           ++                                                     
L-rhamnose         +                                                      
Raffinose          +                                                      
D-mannitol         ++                                                     
Control (without sugar)                                                   
                   +                                                      
______________________________________                                    
 ++: Good growth                                                          
 +: Fair growth                                                           
 
    
     The microorganism is confirmed to grow fairly in the control (without sugar). 
     (5) Growth temperature 
     The microorganism grows at 14°-45° C. and the optimum growth temperature is 26° C. to 32° C. 
     (6) Composition of the cell wall 
     The diaminopimelic acid in LL-type was detected. 
     This microorganism evidently belongs to the genus Streptoverticillium deduced from several properties as above mentioned. 
     The closest species to the microorganism are searched for in the following literatures. 
     (1) Waxman S. A.: The Actinomycetes, vol. 2 (1961), (Literature 1) 
     (2) Elwood B. Shirling and David Gottlieb: International Journal of Systematic Bacteriology (Literature 2), vol. 18, 69-189, 279-392 (1968), vol. 19, 391-512 (1969), vol. 22, 265-394 (1972) 
     (3) Bergey&#39;s Manual of Determinative Bacteriology, the eighth edition (1974), (Literature 3) 
     (4) Other literatures refering to new species of actinomycetes 
     As a result, the following 3 species are recognized as the closest species. 
     (1) Streptoverticillium hiroshimense (Literature 2, vol. 18, 130-134 (1968); Literature 3, 835) 
     (2) Streptoverticillium roseoverticillatum (Literature 2,  vol. 18, 168 (1968); Literature 3, 834) 
     (3) Streptoverticillium biverticillatus (Literature 2, vol. 18, 300 (1968); Literature 3, 834) 
     The microorganism is examined and compared with the above 3 species. As the result it has been found that the microorganism is similar to the above 3 species and is most similar to Streptoverticillium roseoverticillatum because both have the same properties except for coagulation of milk. Accordingly, the strain DO-248 was identified to be a strain belonging to the species and named Streptoverticillium roseoverticillatum DO-248. This strain had been deposited as FERM 7561 since Mar. 26, 1984 in the Fermentation Research Institute Agency of the Industrial Science &amp; Technology at Yatabe-machi, Tsukuba-gun, Ibaraki Pref. Japan and has been transfered to the deposit under Budapest Treaty with accession No. FERM BP-745 since Mar. 22, 1985. 
     This invention involves every process for preparing DO-248-A and/or DO-248-B by antibiotics DO-248-A- and/or DO-248-B-producing microorganisms belonging to the genus Streptoverticillium including the above DO-248 strain. 
     The process for preparing DO-248-A and/or DO-248-B by antibiotics DO-248-A- and/or DO-248-B-producing microorganisms is shown below. The process may be performed according to the general fermentation processes of antibiotics. Namely DO-248-A- and/or DO-248-B-producing microorganisms are incubated in a medium containing several nutrients under an aerobic condition. The incubating condition and composition of the medium may be the same as the one generally used in producing antibiotics. The medium contains carbon sources, nitrogen sources and inorganic salts as a general. Vitamins, precursors and so on may be further added as occasion demands. The carbon sources such as glucose, sucrose, starch, dextrin, glycerol, molasses, organic acids and the like may be employed alone or as a mixture. Nitrogen sources such as soy bean meal, corn steep liquor, meat extract, yeast extract, cottonseed powder, peptone, wheat germ, ammonium sulfate, ammonium nitrate and so on may be employed alone or as a mixture. Inorganic acids such as calcium carbonate, sodium chloride, potassium chloride, magnesium sulfate, cobalt chloride, several phosphates, etc. may be added in a medium as occasion demands. 
     The culture may be performed in accordance with the method generally used in preparing antibiotics. In this invention liquid culture is particularly preferable and in a mass production submerged aeration culture is preferable. Preferably, pH of the medium is about 5.5 to about 8.5, temperature of the fermentation is about 20° C. to about 40° C., more preferably about 25° C. to about 32° C. Cultivating time greatly depends on scale of the fermentation and is preferably about 20-80 hours in a large scale production. 
     Whenever occasion demands, an antifoamer such as vegetable oil and the like may be added before or during the fermentation. 
     After the cultivation is over, DO-248-A and DO-248-B may be isolated and recovered from the culture medium according to the general method. For example, that may be performed by filtration, centrifugation, adsorption and desorption and chromatography using several active adsorbents, extraction using several organic solvents and their combinations as occasion demands. 
     DO-248-A and B provided by this invention are effective on fast-acid bacteria including tubercle bacillus. A result of an antibacterial activity test of DO-248-A is shown as follows. 
     METHOD OF THE TEST 
     Fast-acid bacillus (0.01 mg) shown in Table 1 was inoculated into 2.0 ml of Dubos Tween albumin liquid medium and the minimal inhibitory concentration (MIC, μg/ml) of DO-248-A (Compound A), kanamycin (KM) and rifampicin (RFP) was measured by two-fold dilution method. The incubation temperature for Mycobacterium marinum was 28° C. and that for all other microorganisms was 37° C. The judgement was made generally 2 weeks later but 1 week later on M. furtuitum and M. chelonei and 5 weeks later on M. xenopi. 
     
                                           TABLE 2                                 
__________________________________________________________________________
Test Microorganism                                                        
                  MIC (μg/ml)                                          
Species     Strain                                                        
                  Compound A                                              
                         KM    RFP                                        
__________________________________________________________________________
M. tuberculosis                                                           
            H37Rv 0.05   0.025 &lt;0.001                                     
&#34;           R-15.sup.1                                                    
                  0.10   &lt;0.001                                           
                               &gt;25                                        
&#34;           R-18.sup.2                                                    
                  0.025  &gt;25   &gt;25                                        
M. bovis    BCG   0.05   0.00156                                          
                               &lt;0.001                                     
M. kansasii KMC1101                                                       
                  0.00313                                                 
                         6.25  0.0125                                     
M. marinum  KMC1202                                                       
                  12.5   3.13  1.56                                       
M. scrofulaceum                                                           
            KMC2102                                                       
                  0.2    0.0125                                           
                               0.00313                                    
M. gordonae KMC2201                                                       
                  0.00625                                                 
                         0.39  0.0125                                     
M. szulgai  KMC2401                                                       
                  3.13   1.56  0.025                                      
M. xenopi   KMC2301                                                       
                  0.39   0.10  0.00625                                    
M. simiae   KMC1302                                                       
                  6.25   12.5  &gt;25                                        
M. avium    KMC3101                                                       
                  0.78   3.13  0.20                                       
M. intracellulare                                                         
            KMC3209                                                       
                  0.025  0.05  &lt;0.001                                     
M. nonchromogenicum                                                       
            KMC3602                                                       
                  12.5   &gt;25   25                                         
M. fortuitum                                                              
            KMC4101                                                       
                  &gt;25    25    3.13                                       
M. chelonei KMC4201                                                       
                  &gt;25    &gt;25   6.25                                       
M. avium-intracellulare                                                   
            clinical                                                      
                  0.10   6.25  3.13                                       
            isolated                                                      
            strain 16                                                     
__________________________________________________________________________
 .sup.1 Rifanpicin resistant strain                                       
 .sup.2 Kanamycin and rifanpicin resistant strain                         
 
    
     As shown in Table 2, the antibiotic DO-248-A of this invention is effective against kanamycin-resistant strains and rifanpicin-resistant ones and more effective against M. aviumintracellulare, a causative microorganism of refractory atypical fast-acid disease, than kanamycin and rifanpicin. DO-248-B is as effective as DO-248-A. Accordingly, these antibiotics and their pharmaceutically acceptable salts can be used as active ingredients of drugs for fast-acid bacteria for human beings and animal. 
     DO-248-A, DO-248-B, or their pharmaceutically acceptable salts are orally or parenterally administered to human beings or animal. They can be orally administered as tablets, capsules, powders, liquids and so on formulated by common pharmaceutically acceptable excipients, stabilizers, preservatives, wetting agents, surfactants, flavors, fragrances and so on, and parenterally as injections, suppositories and the like. The dosage is considerably varied depending on condition of disease, sex, age, weight, etc. but about 0.2 to about 8 g/day are preferable for a normal adult. The anti-fast-acid drug of this invention may be administered together with other anti-fast-acid drugs in the same manner as generally practised. 
     Examples of preparing the desired materials of this invention, DO-248-A and DO-248-B, are shown as follows but it should not be understood to restrict this invention. 
     EXAMPLE 1 
     (a) 
     Fermentation step 
     S medium: 0.5% soluble starch, 0.5% glucose, 0.5% polypeptone, 0.5% meat extract, 0.25% yeast extract, 0.25% sodium chloride, deionized water (pH 7.0 before sterilization) 
     X medium: 2.0% raw starch, 2.0% glucose, 2.0% defatted soy bean powder, 0.5% yeast extract, 0.25% sodium chloride, 0.35% calcium carbonate, 0.0005% manganese dichloride tetrahydrate, 0.0005% cupper sulfate pentahydrate, 0.0005% zinc sulfate heptahydrate 
     One platinum loop of Streptoverticillium roseoverticillatum DO-248 strain (FERM BP-745) was inoculated into a 500 ml-Sakaguchi&#39;s flask in which 100 ml of the above S medium was charged and cultured at 28° C. for 48 hours with shaking. Each 4 ml portions of this medium was inoculated into 500 ml-Sakaguchi&#39;s flasks charged with 100 ml of the above X medium and cultured at 28° C. for 4 days with shaking to give 43.8 L of the cultured medium. 
     (b) 
     Isolation step 
     To 43.8 L of the cultured medium provided in the above step was added 1.2 kg of a filter aid, Hyflo Super Cel® (Jhons-Manville Sales Corp.) and the cells were filtered out. The filtrate (39 L) was passed through a column with 4.36 L of a synthetic adsorbent HP-20 (150-300 μm, Mitsubishi Chemical Industries Co., Ltd.) to absorb the desired ingredient. The column was washed with water and eluted with methanol to give active fractions, which were concentrated under reduced pressure. The active fractions in the first half contaminated with many water soluble impurities were subjected to column chromatography with 580 ml of a synthetic adsorbent CHP-20P (75-150 μm, Mitsubishi Chemical Industries Co., Ltd.), the resulting active fractions were combined and lyophilized to yield 25 g of a brown crude powder. Then, 25 g of this crude powder is adsorbed to a column with 470 g of silicagel (63-200 μm, Merck &amp; Co.) and eluted with chloroform:methanol:water (15:10:1) to remove impurities and then with methanol to elute the active ingredient. The active fractions were evaporated to dryness in vacuo. Into a small amount of water was dissolved 3.18 g of the resulting yellowish brown residue containing the active ingredient and passed through a column of 700 ml of Sephadex LH-20 (Pharmacia AB). The column was washed with water and eluted with methanol. The eluate was evaporated to dryness in vacuo. The resulting power (1.22 g) is dissolved again in a small amount of water and passed through a column with Toyopearl HW-50C (100-500 μm, Toyo Soda Mfg. Co., Ltd.) to absorb the active ingredient. The column was washed with water and then eluted with methanol. The active fractions were evaporated to dryness in vacuo (according to this purification impurities are not removed completely, but DO-248-B, the minor component, was collected in a fair amount from the first half fractions.). Each crude powders were combined and passed through reverse phase Lobar® column (Lichroprep RP-18®, 40-63 μm, 25×310 mm, Merck &amp; Co.) with methanol-water (1:1) to remove impurities and resulting active fractions were evaporated to dryness in vacuo to provide 445 mg of yellow powder. At last the resultant was separated to DO-248-A and DO-248-B by reverse phase high performance liquid chromatography with semi-collection column, Nucleosil® 10C 8  (10×250 mm, M. Nargel Co., Ltd.), (it takes 14-15 minutes and 7-8 minutes to elute DO-248-A and DO-248-B, respectively, with methanol-water (1:2) at the flow rate of 3 ml/minute. A UV 254 nm detecter was employed.). The eluates were then lyophilized to yield 116 mg and 16 mg of colorless powder of DO-248-A and DO-248-B, respectively. 
     EXAMPLE 2 
     (a) 
     Fermentation step 
     S medium: 0.5% soluble starch, 0.5% glucose, 0.5% polypeptone, 0.5% meat extract, 0.25% yeast extract, 0.25% sodium chloride, deionized water (pH 7.0 before sterilization). 
     A medium: 2% raw starch, 2% glucose, 2% defatted soy bean powder, 1% Bactosoyton (Trade name), 0.25% sodium chloride, water (pH 7.0 before sterilization). 
     To a 2 L Erlenmeyer flask charged with 800 ml of S medium consisting of the above composition was inoculated a seeding culture of Streptoverticillium roseoverticillatum DO-248 (FERM BP-745) on slant and incubated at 28° C. for 24 hours with shaking of 180 r.p.m. 
     Into 30 L jars charged with 20 L of A medium consisting of the above composition was planted each 800 ml portions of this culture medium and incubated with 20 L/minute aeration and 180-300 r.p.m. at 28° C. for 5 days. 
     (b) 
     Isolation step 
     The culture medium provided in the above step was adjusted to pH 3.0 and centrifuged by Scharples type centrifuge to give 100 L of the supernatant. The supernatant was passed through a column of 10 L of Dowex® 50×4 (NH 4   +  type) (U.S., Dow Chemical Co.) at a flow rate of 700 ml/minute. After washing with water, the column was eluted with 0.3N aqueous ammonia to give 35 L of active fractions. After ammonia was removed, the fractions were adjusted to pH 9.0 and passed through a column of 5 L of Dowex 1×2 (Cl -  type) at the flow rate of 150 ml/minute. The column was eluted with 50% methanol containing 5% sodium chloride and 13 L of the resulting active fractions were evaporated in vacuo. The residual solution was adjusted to pH 7.0 and passed through a column of 2.5 L of HP-20 (Mitsubishi Chemical Industries Co., Ltd.). After washing with water, the column was eluted with 50% methanol and 2.7 L of the active fractions were evaporated. The resultant was lyophilized to yield 2.25 g of crude powder of DO-248. 
     (b) 
     Purification step of DO-248 A 
     Detection and quantitation of DO-248-A in the following purification step were performed with high performance liquid chromatography tracing absorbance at 230 nm, employing a column (4×250 nm) of Nucleosil® 10C 8  (M. Nargel Co., Ltd.) and a mixture of 20 mM phosphate buffer (pH 7.0) and acetonitrile (5:1). Under the condition a retention volume of DO-248-A was 8.00 ml. 
     The crude powder (2.0 g) provided in the above step (containing about 105 mg of DO-248-A) was dissolved in about 40 ml of 20 mM phosphate buffer (pH 7.0). After removing a small amount of impurities, this solution was subjected on a column (200 ml) of QAE-Sephadex A-25 (Pharmacia AB). After 200 ml of 20 mM phosphate buffer (pH 7.0) passed through the column, a linear concentration gradient elution was performed with 600 ml of the same buffer and 600 ml of the same containing 1M sodium chloride. The eluting solution was traced with the above high performance liquid chromatography to give fractions containing DO-248-A. The collected eluates were applied to a column (100 ml) of CHP-20P (Mitsubishi Chemical Industries Co., Ltd.). The column was washed with water and then a linear concentration gradient elution was performed with 300 ml of water and 300 ml of 80% methanol. The eluting solution was traced with high performance liquid chromatography to give fractions containing DO-248-A. The collected eluates were evaporated in vacuo and then lyophilized to yield 100 mg of powder whose purity was about 70%. About 50 mg portions of this powder was subjected to a high performance liquid chromatography collection column (Nucleosil® 30C 18 , 20 ×250  mm) at one time and developed with a mixture of 50 mM phosphate buffer (pH 7.0) and methanol (9:1). The fractions were traced through absorbance at 230 nm and those fractions containing the desired substance in a peak were collected. This collected solution was passed through a column (40 ml) of CHP-20P and the column was washed with water and eluted with 50% methanol and the eluates containing DO-248-A were collected by detecting with the above high performance liquid chromatography, evaporated in vacuo and then lyophilized to give 60 mg of a colorless powder of DO-248-A.