396 



DESCRIPTIONS OF ANTIBIOTICS 



aqueous H2SO4 , and n-pentane added to separate 

 an aqueous layer. Aqueous extract concentrated 

 at room temperature under a fan. Addition of 

 acetone precipitates impurities. II. Broth-fil- 

 trate extracted with n-butanol at pH 2.2. Extract 

 filtered, neutralized, and pentane added. Aqueous 

 layer treated as in I. III. Broth-filtrate stirred 

 with Magnesol at harvest pH, filtered, then ad- 

 sorbed on activated charcoal. Eluted with water- 

 saturated n-butanol. Butanol concentrated in 

 vacuo. Acetone added and mixture filtered. Solvent 

 mixture from filtration concentrated in vacuo with 

 the constant addition of water. Aqueous solution 

 remaining after solvents have evaporated is ly- 

 ophilized. Purified by partition chromatography 

 on Dicalite with water as solvent and 85 per cent 

 ethyl acetate-15 per cent n-butanol saturated with 

 water as developer. Active fractions concentrated, 

 decolorized with Darco, and filtered while hot. 

 Addition of petroleum ether precipitates crude 

 totomycin. Can also be chromatographed on 

 Celite, with water as solvent and ethyl acetate- 

 water-n-butanol (1:5:9) as developer. 



Chemical and physical properties: Amorphous 

 powder. Very soluble in water. Soluble in metha- 

 nol and ethanol. Less soluble in/3-methoxyethanol, 

 1,4-dioxane, n-butanol, formamide, hot pentanol, 

 and dimethylformamide. Sparingly soluble in 

 acetone, eth\'l methyl ketone, and ethyl acetate. 

 Insoluble in chloroform, ether, benzene, and pe- 

 troleum ether. Infrared spectrum given in refer- 

 ence 1. C = 53.56%; H = 6.18%; N = 2.95%. 

 C21H59NOU . Distribution coefficients and Rf 

 values in various systems given in reference 1. 

 Most stable at acid or neutral pH. Completely 

 destroyed at 100°C for 1 hour in 0.1 N HCI or 0.1 

 N NaOH. 



Biological activity: Active on Streptococcus hemo- 

 lyticus at 2 /zg per ml. Much less active on other 

 bacteria, including certain gram-negative bac- 

 teria (16 to 31 Mg per ml). Not active on B. cereus, 

 B. subtilis, Sal. gallinarum, E. coli, or Ps. aeru- 

 ginosa. Active against S. hemolyticus infections in 

 mice. 



Toxicity: LD50 (mice) 1067 to 1154 mg per kg 

 intraperitoneally, 1110 mg per kg intravenously. 



Reference: 1. British Patent 758,276, October 3, 

 1956. 



Toyocamycin 



Produced by: Streptomyces sp. resembling S. 

 albus (1), S. toyocaensis (3), Streptomyces sp. (4). 



Synonyms: Antibiotic E 212 (2), antibiotic 1037 

 (4), unamycin B. Probably the same as monilin. 

 Similar to vengicide. 



Method of extraction: I. Broth adsorbed on 

 lonex C (carbonic acid ion exchange resin, H+ 

 form) at pH 7.6 to 8.0. Diluted with 80 per cent 

 aqueous acetone (2, 3). Eluates are either: (a) 

 Evaporated in vacuo at pH 5.0, then extracted 

 with butanol at pH 8.0, and re-extracted into 

 acidic water (pH 2.0). Water-extract evaporated 

 in vacuo (pH 5.0), then extracted with ethyl ace- 

 tate at pH 8.0. E.xtract concentrated in vacuo to 

 dryness. Residue taken up in anhydrous methanol 

 at 50°C. Cooling gives crystals. Recrystallized 

 from anhydrous methanol. Or (b) evaporated to 

 dryness in vacuo. Residue taken up in methanol. 

 Ether added and the supernatant concentrated to 

 dryness in vacuo. Crystallized as above. A small 

 amount of toyocamycin can be extracted from 

 the mycelium with 80 per cent aqueous acetone 

 (2, 3). II. Extracted from broth-filtrate with 

 butanol at pH 9.0. Concentration of extract gives 

 crystals (4). 



Chemical and physical properties: Weak base (2). 

 Anhydrous form (from absolute methanol or ace- 

 tone) : colorle.ss, fine needles; m.p. 243°C. Hydrate: 

 Colorless prisms; m.p. 239-243°C (3). Soluble (3) 

 or moderately soluble (2) in methanol, ethanol, 

 n-butanol, ethyl acetate, dioxane, and acetone. 

 Sparingly soluble in water and ether. Insoluble in 

 benzene, chloroform, and petroleum ether (2, 3). 

 Ultraviolet absorption spectrum maxima at 230 

 (^11 400) and 277 niM (^Jl 548) in water; 235 

 (E]f^ 700) and 273 m^ (El'L 563) in 0.1 N HCI; 

 233 (^iJm 398) and 280 ni;. (E\:°^ 571) in 0.1 X 

 NaOH; and 230 and 279 m/u, with a weak maximum 

 at 339 ni/x (methanol) (2, 3). Infrared spectrum 

 given in references 2 to 4. [a]f = -51° (c = 0.13 

 per cent in water) (4). Conflicting reports on nin- 

 hydrin test (2, 3). Negative biuret, Sakaguchi, 

 Fehling, FeClj , Molisch, Millon, Ehrlich, and 

 Pauly tests. Relatively stable at pH 5.0, but un- 

 stable at pH 2.0, 7.0, or 9.0 (2). C = 49.63%o; H = 

 4.79%,; N = 24.14%. C12H14N5O4 (3). Toyocamy- 

 cin has the same structure as tubercidin plus a 

 nitrile group (6). 



Biological activity: Inhibits Candida at 1.0 to 

 12.5 Mg per ml. Slightly active (6.3 jug per ml) on 

 Zygosaccharomyces and Aspergillus oryzae (12.5 

 yug per ml). Also active on M. tuberculosis var. 

 hominis H37Rv at 2 ng per ml, but not on other 

 mycobacteria, bacteria, or fungi. Conflicting re- 

 ports on anii-T richomonas vaginalis activity (2, 4). 

 Slight inhibitory effect on Ehilich carcinoma 

 (ascitic) (2, 3). Active on influenza A (PR 8) in 

 vitro, probal:ily by direct inactivation of the virus 

 particle (5). 



Toxicity: Mice tolerate 5 mg per kg intrave- 



