Abstract:
SY-1 substance has the following formula: ##STR1## The SY-1 substance and esters and salts thereof are antibiotics which have antibacterial, antifungal, antiviral and anticoccidial activity and also increase feed-utilization efficiency in ruminants.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     This invention relates to SY-1 substance and esters and salts thereof. 
     2. Disclosure of the Prior Art 
     Antibiotic SY-1 substance is a new member of polyether antibiotics. Especially, it resembles Salinomycin in chemical structure. 
     The inventors of this application have reported that SY-1 substance is produced by fermentation of Salinomycin producing strain belonging to Streptomyces in (Japan-Kokai 76-86191 published on July 28, 1976). 
     SUMMARY OF THE INVENTION 
     One object of this invention is to provide SY-1 substance and esters and salts thereof. 
     Another object of this invention is to provide a new production process of SY-1 substance and esters and salts thereof. 
     The SY-1 substance and esters and salts thereof have antibacterial, antifungal, antiviral and anticoccidal activity, and also increase feed-utilization efficiency in ruminants. 
     DETAILED DESCRIPTION OF THE INVENTION 
     SY-1 substance has the following formula: ##STR2## 
     SY-1 substance can also occur in the form of pharmaceutically acceptable salts and esters particularly the sodium, potassium and calcium salts and lower alkyl esters such as methyl, ethyl, propyl and butyl esters. 
     SY-1 substance can be produced by submerged aerobic fermentation of Streptomyces albus in the manner usually used for Actinomycetes culture, and extracted from at least one of the mycelial mass and the filtered broth. SY-1 substance is preferably produced by fermentation of Streptomyces albus 80614 (ATCC21838, FERM-P 419) or its mutants. 
     SY-1 substance can be produced by the processes commonly employed for culturing Actinomycetes. For industrial scale production, however, submerged aerobic fermentation is preferred. Culture temperature is preferably from 25° C. to 30° C. The culture medium can be one usually used for culturing Actinomycetes, which comprises carbon sources, nitrogen sources, inorganic salts, a small amount of organic substances and antiforming agent. Maximum production of SY-1 substance usually occurs after 72-120 hours from the start of fermentation. 
     SY-1 substance can be isolated by utilizing its physicochemical properties. SY-1 substance is soluble in various organic solvents and accordingly, it can be isolated by a solvent extraction process. Because SY-1 substance exists in both of mycerial mass and the filtered broth, a suitable amount of an organic solvent, such as ethyl acetate, butyl acetate, n-hexane or chloroform, is preferably added to the whole fermentation broth with stirring, so that the strain is autolyzed and SY-1 substance in the strain is readily extracted by the organic solvent. 
     The solvent phase is separated from the aqueous and solid phases, concentrated under vacuum and purified by column chromatography on almina or the like. Preferred solution for development is ethyl acetate, benzene, n-hexane, methanol, acetone or a mixture thereof. It is preferable to use a mixture of ethyl acetate and methanol at a ratio of 100 : 1 to 3. The eluate is fractionated and each fraction is checked by thin layer chromatography to identify the spot of SY-1 substance. The fractions showing single spot of SY-1 substance were combined, concentrated under vacuum and chilled at -5° C. to crystallize SY-1 substance. 
     Coproduced salinomycin is separated from SY-1 substance by chromatography on almina. 
     Crude crystalline SY-1 substance is then separated by filtration. The cyrstal is dissolved in the solvent such as ethyl acetate, hexane and benzene, concentrated under vacuum, and chilled for crystallization to occur. The recrystallization is repeated to give more purified cyrstalline SY-1 substance. 
     Physicochemical and biological properties of SY-1 substance (Na salt) are as follows: 
     
         ______________________________________(1)  Mp. 120-122° C(2)  [α].sub.D.sup.25 = -13° C (C = 1, MeOH)(3)  Solubility: soluble in alcohols, acetates, chloroform,ether, carbon tetrachloride, benzene and n-hexane;insoluble in water.(4)  Stability: stable in pH 7-9; unstable in pH lower than 6.(5)  Color reaction: negative for Lemieux reaction, ninhydrinreaction, ferric chloride reaction, vanillin reaction andFehling&#39;s reaction; positive for iodine reaction to showreddish brown.(6)  Elemental analysis:C:66.35; H:9.19; O:21.42(7)  Molecular formula:C.sub.42 H.sub.69 O.sub.10 Na(8)  Molecular weight: 756 (M.sup.+ m/e)(9)  UV spectrum: λ.sub.max.sup.MeOH = 285 nm(ε75)(10) IR spectrum (KBr) Na salt3300, 2950, 2925, 2875, 1710, 1560,1450, 1400, 1375, 1330, 1320, 1295,1250, 1240sh, 1220, 1210, 1175,1155, 1135sh, 1110, 1075, 1040,1020, 980, 960, 955, 925, 900, 875,850, 830, 790 (broad), 765, 750,720, 690, 660, 635, 610 (broad),590, 560, 530 (broad), 480, 440,425 (broad) cm.sup.-1.(11) Mass spectrum (75 eV) m/e,(relative intensity)756(M.sup.+)(8), 712(45), 697(3), 683(3),669(10), 641(25), 603(7), 601(72),572(19), 543(90), 514(72), 492(50),474(15), 457(11), 447(15), 415(31),393(40), 375(12), 368(15), 363(14),349(95), 333(28), 331(30), 322(41),310(45), 293(100), 275(75), 265(28),250(55), 249(77), 240(20), 236(25),225(23), 221(34), 209(44),, 207(46).(12) NMR spectrum (CDCl.sub.3)0.65, 0.72, 0.75, 0.80, 0.85, 0.88,0.89, 0.91, 0.97, 1.05, 1.17, 1.25,1.34, 1.42, 1.45, 1.47, 1.49, 1.60,1.62, 1.70, 1.80, 1.86, 1.92, 1.98,2.08, 2.34, 2.54, 2.64, 2.72, 2.85,2.95, 2.97, 3.32, 3.42, 3.50, 3.60,3.68, 3.78, 3.85, 3.88, 3.95, 3.98,4.20, 4.30, 4.36, 4.42, 6.09 (δ)(13) .sup.13 C-NMR spectrum (CDCl.sub.3)213.79, 177.10, 125.64, 121.80,105.04, 99.06, 88.45, 78.39,71.11, 76.38, 75.83, 75.59,74.73, 74.06, 71.56, 71.26,68.52, 56.26, 49.56, 48.22, 41.02,40.05, 38.77, 36.21, 33.10,32.73, 31.94, 30.05, 29.68,28.10, 26.57, 25.78, 22.67,22.19, 20.05, 17.86, 16.64,15.66, 14.38, 13.35, 13.16,11.94, 11.21, 6.95, 6.52 (ppm)(14) Toxicity: LD.sub.50 = 51 mg/Kg (mice, orally).(15) Antimicrobial spectra:______________________________________ *The data in less than 200 of m/e are not included. 
    
     Minimum inhibitory concentration (mcg/ml) is shown in Table 1. In the Table, N indicates Nutrient agar medium, GN Glycerine nutrient medium and PS Potato-sucrose agar medium. 
     
                       Table 1______________________________________Bacillus subtilis  N         100Bacillus circulans N         12.5Bacillus megaterium              N         50Staphylococcus aureus              N         100Staphylococcus epidermidis              N         50Micrococcus flavus N         100Micrococcus luteus N         100Mycobacterium smegmatis              GN        &gt;100Mycobacterium phlei              GN        &gt;100Mycobacterium avium              GN        &gt;100Escherichia coli   N         &gt;100Klebsiella pneumoniae              N         &gt;100Proteus vulgaris   N         &gt;100Psendomonas aeruginosa              N         &gt;100Piricularia oryzae PS        &gt;100Alternaria kikuchiana              PS        &gt;100______________________________________ N: nutrient agar GN: 4% glycerol nutrient agar PS: potato-sucrose agar 
    
     The Streptomyces albus 80,614 has a long hypha which is not separated into bacillary or coccoid. The aerial mycelium of Streptomyces albus 80,614 is substantially straight and sometimes diverged to have sporephores having 2-3 turns in spiral form. The surface of the spore is smooth and has no spine and the shape of the spore is a long ellipsoid or cylindrical shape having 0.5-1.0μ × 1.0-1.5μ in size. The characteristics of Streptomyces albus 80,614 are as follows: 
     
         ______________________________________Physiological reaction of Streptomyces albus 80,614Test             Response______________________________________Milk coagulation NegativeMilk peptonization            NegativeMelanin formation            NegativeTyrosinase reaction            NegativeNitrate reduction            Positive sometimes NegativeStarch hydrolysis            PositiveLiquefaction of gelation            PositiveDecomposition of cellulose            NegativeChromogenicity   NegativeOxygen requirement            AerobicOptimum growth conditions            pH 6.8 - temperature 28° CRange for growth pH 5.5-8.2            temperature 21-37° C______________________________________ 
    
     
         ______________________________________Utilization pattern of carbon sources by Streptomyces albus80,614(Pridham &amp; Gottlieb&#39;s basal medium)______________________________________++:    glucose, fructose, galactose, mannitol, xylose,  cellobiose mebibiose, inulin+:     lactose, trehalose, starch±:  maltose, mannose, sucrose, salicin, arabinose-:     melezitose, inositol, dulcitol, sorbitol, raffinose,  adonitol, rhamnose______________________________________ ++: good growth +: fair growth ±: faint growth -: no growth 
    
     
         __________________________________________________________________________Cultural characteristics of strain No. 80614                              SolubleMedium      Growth      Aerial mycelium                              pigment__________________________________________________________________________CZAPEK&#39;s agar       Good,raised,white                   Poor,white None       to pale yellowStarch inorganic       Poor or moderate,                   Moderate, powdery,                              Nonesalt agar   white to tan                   white to whitish                   grayGlucose asparagine       Poor,thin,white to                   Moderate to poor,                              Noneagar        tan         velvety,white to                   whitish grayGlycerine asparagi-       Poor to moderate,                   Poor to moderate,                              Nonene agar     tan         whiteCalcium malate       Good,raised,white                   None or poor,                              Noneagar        to pale tan whiteTyrosine agar       Poor,thin,white to                   None       None or faint       pale brown             brownNutrient agar       Poor,thin,golden                   None or poor,                              None       yellow      whiteYeast malt extract       Good,yellowish                   Good,white to                              Pale brownagar        brown       yellowish whiteOatmeal agar       Poor,colorless                   Poor to moderate,                              None or pale                   white to whitish                              brown                   grayGlucose nutrient       Poor,thin,yellow-                   None or poor,                              Noneagar        ish white   whiteGlucose peptone       Poor,thin,yellow-                   None or poor,                              None or paleagar        ish white   white      brownGlycerol CZAPEK&#39;s       Good,raised,white                   Poor or none,                              None or paleagar        to pale tan white      brownPotato plug Poor,thin,brown                   None or scanty,                              None                   white to whitish                   grayCellulose   Scant,thin,colorless                   None       NoneLitmus milk Ring growth in                   None       Faint red       medium,colorlessEgg         Poor,thin,yellowish                   None       None       white to whiteLOEFFLER&#39;s serum       Good,raised,                   None       Nonemedium      brownish yellow(27° C 10 days)__________________________________________________________________________ 
    
     Anticoccidal activity is an important property of SY-1 substance. For the prevention or treatment of coccidiosis in poultry, a non-toxic anticoccidal amount of SY-1 substance is administered to birds, preferably orally on a daily basis. SY-1 substance can be given in many ways, but it is most conveniently given with a physiologically acceptable carrier, preferably the feed. Although a variety of factors must be considered in determining an appropriate concentration of SY-1 substance, the rate of administration will be generally in the range of 0.003 to 0.06 percent by weight of unmedicated feed, and preferably in the range of 0.005 to 0.03 percent. 
     The ability to improve feed-utilization efficiency in animals is another important property of SY-1 substance. For example, SY-1 substance improves feed-utilization efficiency in ruminants when administered orally at rates of from about 0.05 mg/Kg/day to about 5.0 mg/Kg/day. Most beneficial results are achieved at rates of from about 0.1 mg/Kg/day to about 2.5 mg/Kg/day. A preferred method of administration of SY-1 bustance is by mixing it with the animals&#39; feed, however, it can be administered in other ways, for example, tablets, drenches, boluses or capsuls. 
     EXAMPLE 
     Streptomyces albus 80614 (ATCC 21838) (FERM-P No.419) was inoculated in 100 liter of liquid medium consisting of 2% glucose, 1% starch, 4% corn steep liquor, 0.2% meat extract and 0.2% sodium chloride, in a 200 liter stainless steel tank at pH 7.0, and cultured at 30° C. for 120 hours in aerobic condition. Air was passed at the rate of 100 liter/min. and the medium stirred at the rate of 250 r.p.m. After culturing, whole fermentation broth was adjusted to pH 8 with NaOH, admixed with 2% by weight of diatomaceous earth and filtered. The filtrate was extracted twice with 50 liter of butyl acetate. 
     The mycelical mass was extracted twice with 30 liter of methanol, concentrated under vacuum, and further extracted twice with 10 liter of butyl acetate after evaporating off the methanol. Both ethyl acetate extracts were combined and concentrated under vacuum to 0.2 liter. The concentrate was extracted three times with 0.4 liter of n-hexane and concentrated under vacuum to 0.1 liter. The concentrate was applied to the column of 5 liter active almina charged with butyl acetate. The column was eluted with a mixture of ethyl acetate and methanol (50:1). Each fraction was checked by thin layer chromatography (developer: ethyl acetate, detector: 10% aqueous solution of surfuric acid) to identify SY-1 substance. Fractions containing single spot of SY-1 substance were combined and concentrated under vacuum. 
     The concentrated solution was allowed to stand in refrigerator (-5° C.) overnight for crystallization to occur. The crystals thus formed were separated by filtration and dried under vacuum to give 62.3 g of crude crystalline product. The crude crystals were dissolved in 800 ml of n-hexane, and concentrated under vacuum to 200 ml. Then a small amount of water was added to this solution, and the solution was allowed to stand in refrigerator overnight to crystallize crude SY-1 substance. The recrystallization was repeated twice to give 16.5 g of pure SY-1 substance Na salt. The product had the formula (I) wherein--COOH was converted to--COONa.