IE45212B1 - New viridogrisein antibiotics - Google Patents

Abstract

The neoviridogriseins I, II and III are obtained by aerobic fermentation of the new Streptomyces sp. P 8648 (FERM-P 3562) microorganism. These are antibiotics of the viridogrisein subgroup belonging to the mikamycinvernamycin family. They are very active against gram-positive bacteria and against the mycoplasmas.

Description

This invention relates to new dapsipeptide antibiotics vzhich can be used therapeutically and as animal feed additives.

The so-called mikamycin-vernamyein (or streptogramin) family antibiotics are classified into too major groups. One major group (Group A according to D. Vazquez, Antibiotics IIIs Kechanism of action of antimicrobial anu antitumor agents·'1, Springer-Verlag Berlin Heidelberg New York, 1375, pp. 521-534) are macrocyclic-lactone compounds, and include griseoviridin, ostreogrycin A (also known as, for example, mikaisycin A, virginiamycin Hs staphylomycinil, pristinamycin Ila, vemanycin A, streptogramin A, Synergistin A-l, Compound PA-114A1 and Compound E-l25 Factor A), ostreogrycin G (also known as, for example, virginiamycin MIX, staphylomycin Mil, pristinamycin IIB, dihydrostreogrycin A, Compound E-129 Factor B and Compound ft.P.-13920) and Compounds A-2315 A,B and C. Though, bacteriostatic, this group of antibiotics is act-'ve against Gram-positive bacteria and mycoplasmas.

It is known to produce griseoviridin together with viridogrisein using Streptomycetes. The production of griseoviridin is described in U.S. Patent Specifications Nos. 3,023,204 and 3,174,902.

The other major group (Group B according to 0. Varquez) is a macrocyclic depsipeptide compound and divided into two sub-groups, that is, the viridogrisein sub-group and the vernamycin B sub-group.

The antibiotics in this major group are mainly effective in suppressing the growth of Gram-positive bacteria such as Staphylococcus aureus and Bacillus subtilis. The vernamycin B sub-group contains 12 homologues.

Because the synonym relationships among the published names of antibiotics in this group are so complicated, it is practically impossible to list all the compound names of this sub-group. Some representative, and scientifically important, compounds of this sub-group are as follows: 1. Vernamycin.Ba = ostreogrycin B = pristinamyein IA = mikamyein B = streptogrsmin B = synergistin B-l = compound E-129 factor 2 = compound PA i!4Bl. 2. Vernamycin By = ostreogrycin BI - pristinamycin IC = compound E-129 Factor 21 3. Vernamycin Bg = ostreogrycin B2 = pristinamycin IB = compound E-129 Factor 22 = compound R.P. -13919 4. Ostreogrycin B3 = compound E-129 Factor Z3 . Patricir. A 6. Patricir? B IS 1. Vernamycin Βό 8. Vernamycin C - doricin 9. Virginiamycin (Staphylcmycin)S . Virginiamycin (Staphyloiiiycin)32 11. Virginiamycin (Staphylomycin)S3 12. Virginiamycin (Staphylomycir.)S4 (or SI) The compounds of this sub-group have the common feature that they are composed of ‘even constituents sharing the same four constituents of 3-hydroxypicolinic acid, L-threonine, L-proline and L-C-phenylglycine. In the viridogrisein sub-group there has been known only one compound, viridogrisein, qnd the identity with viridogriseih of etamycin, compound K-179 and compound F-1370A is well established. In contrast to the above vernamycin B sub-group, viridogrisein is composed of constituents, that is, 3-hydroxypicolinic acid, L-threonine, D-leucine, 4-hydroxy-D-proline, sarcosine, g-N-N-dimethyl-L-leucine, L-alanine and L-C-phenylsarcosine. As will be apparent in the following detailed description of the invention, the neoviridogrisein antibiotics of this invention belong to this viridogrisein sub-group, and one of the compounds, neoviridogrisein IV, has been identified as viridogrisein. Preparation of viridogrisein is disclosed in U.S. Patent Specification No. 3,023,204. The synergism between Group A and Group B is well known on various microorganisms, and profitably utilized to potentiate the therepeutic efficiency of pharmaceutical drugs containing the antibiotic of this family as active components. This is also true of neoviridogriseins.

The novel antibiotics of this invention have been named neoviridogrisein I, neoviridogrisein II and neoviridogrisein III.

Ihese novel antibiotics can be prepared, together with viridogrisein (neoviridogrisein IV) and griseoviridin, by cultivating Streptomyces sp. P8648 (FERM-P3562), or a mutant, capable of producing the same novel products, thereof, under aerobic conditions, at a temperature of from 18 to 37°C, in an aqueous nutrient medium containing an assimilable source of cwbon, an assimilable source of nitrogen and essential mineral salts; recovering the fermentation product from the medium; and, if desired isolating the individual antibiotic products.

The production of neoviridogriseins I and II can be enhanced by the addition of proline to the medium, during or before fermentation. The production of neoviridogriseins I and 111 can be enhanced by the addition of α-amino-n-butyric acid to the medium, during or before fermentation.

The novel antibiotics of this invention are active against various pathogenic microorganisms including Gram-positive bacteria and mycoplasm such as Staphylococcus aureus, Streptococcus pyogenes, Diplococcus pneumoniae, Mycoplasma gallisepticum, Mycoplasma fermentans and Mycoplasma agalactiae. The novel antibiotics can be used in dilute, concentrated or pure form, with or without viridogrisein and/or griseoviridin.

An antibiotic composition according to the invention comprises neoviridogrisein I, neoviridogrisein II, neoviridogrisein III, viridogrisein and griseoviridin. A pharmaceutical composition according to the invention comprises an antibiotic or antibiotics selected from neoviridci/ijciiis I, II and III in association with a pharmaceutically acceptable carrier. An animal feed composition according to the invention comprises a compound or compounds selected from neoviridogriseins I, II and III, preferably neoviridogrisein II. The pharmaceutical and animal feed compositions of the invention may additionally comprise viridogrisein and/or griseoviridin.

In this specification, the unqualified term neoviridogrisein is used generically to designate depsipeptide antibiotics called neoviridogriseins; that is, it means not only a member in the group of neoviridogriseins I, II and III and viridogrisein (= neoviridogrisein IV), but also a mixture of any two or more members selected from the said group.

V The new strain of Streptomyces called Streptomyces sp. P8648 (FERM-P 3562) has been isolated from a soil sample collected near Kuzuryu dam in Fukui-ken, Japan.

Generally speaking, the microorganism of this invention 5 elongates colourless, short aerial mycelia from well-branched (single branching) substrate mycelia. Spore chains with smooth surface are formed in a loose loop on top of aerial mycelia. Neither whirl nor ascospore is observed. The cultural characteristics of this microorganism on various agar media are described as follows: (1) Sucrose-nitrage agar Growth : Aerial mycelium : Reverse : Soluble pigment : (2) Glucose-asparagine agar Growth : Aerial mycelium : Reverse : Soluble pigment ; (3) Glycin-asparagine agar Growth : Aerial mycelium : Reverse : Soluble pigment : Poor Thin, white aerial mycelia occasionally formed Colourless to grayish white None.

Abundant Little or none. When formed, white.

Pale yellowish white to light yellow. None.

Moderate Little or none, When formed, white Pale yellowish to grayish yellow None. 213 {4) Yeast extract-malt extract agar Growth Abundant Aerial mycelium White tc white with grayish tinge Reverse Light yellow, later turning to brownish gray Soluble pigment None or rarely slight brown. (5) Starch agar Growth Moderate Aerial mycelium None or little. When formed, white Reverse Pale yellow with light grayish tan in the centre of colonies Soluble pigment None Starch hydrolysis Poor (6) Tyrosine agar Growth : Moderate Aerial mycelium None or a few spots of white aerial mycelium occasionally observed Reverse Grayish yellow to light yellowish tan Soluble pigment Initially pale purple to light reddish brown, 10 days later turning to pale brown. Little melanoid pigment formed. (7) Nutrient agar Growth Aerial mycelium Reverse Soluble pigment Good Thin, white Pale yellow None (3) Oatmeal agar Growth Aerial mycelium Reverse Good White to grayish white Grayish yellow to light reddish brown with grayish tinge Soluble pigment None The optimum growth temperature range from fhe microorganism of this invention is from 25 to 35¾. Though the growth is very poor, the microbe can grow even at a temperaure beyond the said temperature range, e.g. I0°C or 45°C, but it canndt grow at a temperature of 52°C. ; This actinomycete- liquefies gelatin in glucose-peptone-gelatin medium; weakly hydrolyzes starch in starchinorganic salts agar; and peptonizes skimmed milk without coagulation.

Production of melanoid pigment is occasionally observed in tyrosine agar, but not in peptone-yeast extract-iron agar and tyrosine-yeast extract broth.

Carbon source assimilation pattern of this microorganism is as follows (in Pridham-Gottlieb's medium): Positive Slightly positive Negative D-Xylose, D-glucose, D-fructose L-rhamnose, D-mannitol.

Sucrose L-Arabinose, i-inositol, raffinose.

In relation to the production of known, peptide and non-peptide macrolide antibiotics like mikamycins A and B, virginiamycins, ostreogrycins, etaraycin, ve many ci ns, viridogrisein, griseovirdin and pristinanycins, the following microorganisms should be compared with ί 1 2 Streptomyces sp. P8648; griseus NRRL 2426 Streptomyces griseoviridus NRRL 2427 Streptomyces sp., etamycin producer Streptomyces conganensis Streptomyces ostreegKseus Streptomyces mitakaensis Streptomyces loidensis Ihe available information or the cultural and physiological characteristic of the said microorganisms shows clearly differences between ths streptomycete claimed in this invention and the abovementioned ones. for example, Streptomyces griseus NRRL 2426 differs in that it belongs to the Section Rectiflexibiles with straight or slightly wavy spore chains while the microorganisms of this invention is included in the Section Spirales; that the former produces gray to yellowish gray aerial mycelia on yeast extract-malt extract agar while the latter produces white to grayish white aerial mycelia; and that the former utilizes L-arabinose while the latter does not. Streptomyces sp., etamycin producer which has been specified in Antibiotics Annual 1954-1955, pp 728-732, can be differentiated from the microorganism of this invention in the assimilation pattern of carbon sources and the cultural characteristics of Czapek agar, glucose-aspargine agar and nutrient agar, Streptomyces conganensis shows clear differences in the morphological characteristic of spores. Among the above-listed microorganisms, Streptomyces griseoviridus NRRL 2427 looks most similar to the streptonycetes of this invention. The results of the taxonomical comparison between the said two type cultures are summarized in the following table: Streptomyces Streptomyces sp. P 8648 griseoviridus NRRL 2427 Colour of Pale orangish to yellow White to grayish white aerial aerial mycelium. to yellowish pink with gray tinge on yeast extract-malt extract agar, oatmeal agar. mycelium poorly formed on most ISP media. White aerial mycelium abundantly formed on yeast extract-malt starch agar and glycerin- extract agar. asparagireagar. Colour of Grayish yellow to Pale yellow or light yellow substrate olivish brown or to grayish brown on most mycelium blackish brown on yeast extract-malt extract agar, oatmeal agar, starch agar and glyceri n-asparagi ne agar. ISP madia. Soluble No melanoid pigment No melanoid pigment formed. pigment formed. No other No other pigment usually pigment usually observed, but rarely brown : observed, but rarely yellow pigment poorly formed. pigroant slightly formed. Utilization of L-arabinose +++ L-arabinose - carbon sources D-fructose + sucrose D-fructose +++ sucrose + *82 12 As apparent from the preceding table, clear differences have been confi roied between Streptomyces griseoviridus NRRL 2427 and the streptomycete of this invention in the morphological and cultural characteristics and the utilization pattern of carbon sources. In addition, when both are fermented under identical conditions, the microorganism of this invention can produce neoviridogriseins I, II and III as well as viridogrisein (neoviridogrisein IV) and griseoviridin, while the type culture of Streptomyces griseoviridus NRRL 2427 produces only viridogrisein and griseoviridin, but not neoviridogriseins I, II and III. from the above described results, the microorganisms employed in this invention has bsen concluded to be a new species of Streptomyces and named Streptomyces sp. P 8648. This type culture of this microorganism has been deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, with the deposition number of FERM-F ϋο. 3562. It may be well understood to those skilled in th·.- arc that this invention is not limited to the particular microorganism which has been specified above and filed as FERM-P No. 3562 with Fermentation Research Institute, but includes all those spontaneous and artificial mutants derived from the said microorganism which are capable of producing the new antibiotics, neoviridogriseins I, II and III.

The new antibiotics of this invention are produced by inoculating and propagating Streptomyces s£. P8648 in an aqueous nutrient medium under aerobic conditions as defined above, preferably for a period of 2-14 days. The medium may be any kind of medium which has been well known for cultivating Streptomyces. For example, preferably carbon sources for the medium are glucose, glycerin, starch, dextrin, oatmeal, molasses, fat and oil. Suitable nitrogen source for the purposes of this invention are soybean meal, cotton seed meal, meat extract, peptone, dry yeast, corn steep liquor, yeast extract, casein and its hydrolysate and inorganic salts such as ammonium sulfate and ammonium nitrate. If desired, minor growth factors may be added to the medium. They include vitamins, aminoacids, organic and inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, sodium phosphate, potassium phosphate and magnesium sulfate.

The new antibiotics of this invention can be produced by fermentation in traditional vessels such as shake flasks, jar fermentors and tank fermentors but, from the economical viewpoint, the submerged cultivation under forced aeration will be most advantageous in an industrial scale.

The fermentation is desirably carried out at a temperature in the range of 25-35°C. When a shake flask or a tank fermenter is employed, the production of neoviridogriseins reaches a peak usually in 2-10 days. The pH value during fermentation may change beyond the physiological range, depending on the kind of medium employed. The pH is preferably adjusted and maintained during fermentation at from 5 to 9. Usually the pH of the medium is adjusted to 6.5 - 8.5 before inoculation.

As described above, the microorganism of this invention produces a mixture of neoviridogriseins I, II, III and IV and griseoviridin.

It is possible to change the composition of neoviridogriseins in the 4331.2 fermentation broth by a suitable combination of carbon and nitrogen sources in the medium without the specific addition of either a free aminoacid or an organic acid. But it will be more profitable from the viewpoint of industrial production to adjust the content of neoviridogriseins I, II and/or III in the fermentation broth by adding to the medium the pertinent constituent amino acid(s) in free form during fermentation.· depending on the circumstances and the demand. It goes without saying that the composition of neoviridogriseins in the fermentation broth may be varied appropriately by selecting spontaneous or artificial mutants derived from the type culture of the streptomycete of this invention; by adjusting fermentation conditions like temperature, pH and aeration; and/or by adding to the medium physiologically active agents such as enzyme inhibitors and promoters. One of the preferred embodiments of methods for selective production of particular neoviridogrisein components consists of feeding the pertinent constituent amino acid(s), alpha-amiw-n-butyric acid and/or proline during fermentation. Hors particularly, the addition of proline during fermentation increases the proportion of neoviridogriseins I and II which are more potent in antimicrobial activities than neoviridogriseins III and IV. The amount of neoviridogriseins I and III can be selectively increased by feeding alpha-amino-n-butyric acid to the medium before inoculation or during fermentation. As the microorganism of this invention produces protease during growth, proteinaceus material which contains the said pertinent constituent amino acid(s) may be added instead of the free amino acid(s). For example, proline can be substituted by casein or corresponding hydrolysates from acid hydrolysis such as, for instance, casamino acids.

The neoviridogriseins I, II and III and virfdogrisein can be isolated from the fermentation broth by conventional methods based on their physico-chemical properties as depsipeptide antibiotics. If necessary, neoviridogriseins may be recovered from the fermentation broth together with griseoviridin as a neoviridogriseins-griseoviridin mixture. When they are orepared for feed additive or veterinary drug use, a crude mixture of neoviridogriseins and griseoviridin will be more advantageous frcm the economical viewpoint.

Neoviridogriseins and griseoviridin in the fermentation broth can be extracted with a water-immiscible organic solvent. For example, ethyl acetate, butyl acetate, n-butanol, methylene chloride and chloroform are suitable for extraction of neoviridogriseins and griseoviridin at One time. When it is more desirable to selectively extract neoviridogriseins without griseoviridin, preferred organic solvents are methylisobutyl ketone, benzene, toluene and other aromatic hydrocarbons. As the mycelium substantially contains no neoviridogriseins and the extractable lipid in cells may often interfere with subsequent purification steps, it is more profitable to extract the said antibiotics with an organic solvent from the filtered broth or centrifuged broth together with the water wash.

The solvent extract of neoviridogriseins and/or griseoviridin can be further isolated and purified in a number of different ways. For example, adsorption and elution processes with active carbon, Amberlite XAD-4 and 7 (Rohm and Haas Co.), ion exchange resins such as Amberlite IR-120 (Rohm and Haas Co.) and Dowex 50W-X2 (The Dow Chemical Co.); gel filtration with Sephadex LH-20 (Pharmacia Fine Chemicals AB) and its equivalents; and/or adsorption chromatography ΰ.*? on alumina and silica gel, can be conveniently combined for isolation and purification. In addition, counter-current distribution with a suitable solvent, system may work well for the said purposes. (“Amberlite, Dowex and Sephadex are trade marks).

Neoviridogriseins I, II and III as well as viridogrisein are amorphous white solids and are soluble in methanol, ethanol, n-propanol, isopropanol, n-butanol, dioxane, ethyl acetate, n-butyl acetate, acetone, methyl ethyl ketone, methyl isobutyl ketone, benzene, toluene, methylene chloride, chloroform, carbon tetraIQ chloride, dimethylformamide and dimethyl sulfoxide; poorly soluble in water and diethyl ether; and almost insoluble in petroleum ether and n-hexane. they are stable in aqueous solution for at least one month at 23 to 37°C and for 30 minute? at 60uC, at a pH of 2 to 9. ib The melting points of neoviridogriseins II, III and viridogrisein were determined in -. Sof-er apparatus: Nec-viriesgruein II : 93°C in : 115°C Viridogrisein : 140°C; The optial rotations of the antibiotics were determined as 1% w/w solutions in methanol at 23°C using sodium D light: Neoviridogrisein I : + 13° II : - 39.3° III : + 73.7° 25 Viridogrisein : 43.7° The ultraviolet absorption spectra of neoviridogriseins I, II, III and viridogrisein, respectively, are shown in Figures 1 to 4 (in methanol) and Figures 5 to 8 (in 0.1N NaOH-methanol). The E,c<, value of the antibiotics at their maxima is as follows: 5 In neutral methanol, Neoviridogrisein I : 305 nm (65) Π : 305 nm (88) III : 305 nm (90) viridogrisein : 305 nm (90) 10 In 0.7 N NaOH-methanol, Neoviridogrisein I : 340 nm (70) - II : 340 nm (84) III : 340 nm (90) Viridogrisein : 340 nm (96) 15 The infrared absorption spectra of neoviridogriseins I, II, III and viridogrisein, each in a KBr tablet, are shown in Figures 9-12 respectively. The characteristic peaks and shoulders are observed in the following wave numbers: Neoviridogrisein I (KBr tablet) 3370, 2910, 2850, 1735, 1670 (sh.), 1460 (sh.), 1445, 1405, 1375, 1290, 1160, 1125, 1100 and 1080 cm-1. 1635, 1590 (sh), 1515, 1280, 1250 (sh.) 1200, 1190, Neovi ridnnrisein 3320, 2950, 2920 1575, 1515, 1460 1275, 1240, 1200 II (KBr tablet) 2820, 2800, 1745, 1670 (sh), 1630, 1600 (sh.), sh.) 1445, 1405, 1390, 1365, 1330 (sh.), 1295, 1195, 1160, 1130, 1095 and 1065 cm'1.

Neoviridogrisein III (KBr tablet) 3335, 2960, 2940, 2870, 1750, 1670 (sh.), 1660 (sh.) 1635, 1590 (sh.) 1515, 1450, 1405, 1390 (sh.), 1370, 1340 (sh.), 1300, 1245, 1200, 1169 (sh.), 1130, 1100 and 1065 cm'1.

In the thin layer chromatography systems indicated below, neoviridogriseins I, II arid III vired-grisein and griseoviridin have the following Rf values: (1) TLC plate Solvent Neoviridogrisein I Neoviridogrisein II Neoviridogrisein III Viri dogrisein Griseoviridin Pre-coated TLC plate SILICA GEL 60F-254, E. Merck, Darmstadt.

Benzene:methanol = 5:1 (v/v) Rf = 0.66 0.62 Rf = 0.59 0.55 O.20 (2) TLC plate : Same as (1) Solvent : Chloroform:methanol = 30;l (v/v) Neoviridogrisein I : Rf = 0.39 Neoviridogrisein II : 0.32 Neoviridogrisein III 0.19 Viridogrisein : 0.18 Griseoviridin : 0.02 For the analysis of the constituent amino acids, each neoviridogrisein component was hydrolysed in 6N HCl overnight at 110°C and the resulting hydrolysate was evaporated to dryness. After even*1/ trace of hydrochloric acid was removed by repeated evaporations, amino acids in the hydrolysate were determined by thin layer chromatography (Eastmann Chromagram sheet 13254 cellulose with fluoroescent indicator, Eastmann Kodak Co.; solvent system: n-butanol/acetic acid/water 4:1:1 v/v/υ), nigh voltage paper electrophoresis (Toyo Filter paper No. 51A, Toyo, Rohi Kaisha, Ltd.; buffer system: formic acid/acetic acid/water 25:75:900, pH = 1.8; 60V/cm at 0°C for 30 minutes) and auto-amino acid analysis {Hitachi auto-amino acid analyser KLA-5, Hitachi, Ltd.). The presence of the following amino acids was confirmed: Neoviridogrisein I : threonine leucine proline alpha-amino-n-butyric acid sarcosine C-phenylsarcosine β-Ν-Ν-di methy11eucine Neoviridogrisein II threonine 1 I'll ρ Neoviridogrisein ΙΠ Viridogrisein leucine proline alanine sarcosine C-phenylsarcosine e-N,N-dimethylleucine threonine leucine hydroxyproline alpha-amino-n-butyric acid sarcosine C-phenylsarcosine β-Ν ,N-di methyl1euci ne threoni ne leucine hydroxyproline alanine sarcosine C-phenylsarcosine β-Ν ,N-dimethyl1euci ne The presence of 3-hydroxy-picolinic acid was confirmed by mass spectrometry and thin layer chromatography as follows: Authentic samples of viridogrisein and each of neoviridogriseins Is II, III and IV were hydrolysed overnight in 6N HCl at 110°C to give 2; the hydrolysates as described above. Each hydrolysate showed only one UV-absorbing spot with the same Rf value under the indicated conditions. (1) Silica gel TLC, TLC plate : Pre-coated TLC plate SILICA GEL 60 F-254, E. Merck, Darmstadt. Solvent : Chloroforanmethanol = 2:1 (v/v) Rf : 0.46 (2) Cellulose TLC TLC plate ί Eastmann Chromsgram sheet 13254 cellulose with fluoroscent indicator, Eastman Kodak Co.

Solvent : n-Butanol:acetic acid:water = 4:1:1 (v/v) Rf : 0.62 .

The molecular weight of these antibiotics was determined by direct insertion into a mass spectrometer.

Neoviridogrisein I : 876 II : 862 III · : 892 IS Viridogrisein : 878 For the study on the chemical structure of neoviridogriseins I, II and III, these three new antibiotics and viridogrisein were hydrolysed overnight in 0.1 N NaOH at room temperature and then methylated with diazomethane before mass spectrometry according to the method of Coiiipernolle et al.. (Organic Mass Spectrometry, Vol. 6, pp 151-166, 1972). The structure of neoviridogriseins I, II and III is concluded from the available information described above to be as follows: Neoviridogrisein I CH3 z ch3 CH OH CHg CO - NH —CH — CO - NH— CH - CO Z ch2 CH CO CH—CH, N—CH. ch3 CH, / 3 Neoviridogrisein III CH, CH i CH, OH I CH / \ CH, CO —“NH—CH—CO—NH—CH — CO —N CO—CH—NH— CO—CH I I jH2 CH — CO I N — CH, I CH, ι 2 CO I •N •CH, CH CH, CH· ! CH' I CH, •CHCH, As can be seen from the preceding structures of neoviridogriseins I, II and III, the antibiotics of the invention are a group of new depsipeptide antibiotics homologous to viridogrisein. The indentity of neoviridogrisein IV with viridogrisein and etanycin was confirmed by mass spectrometry, thin layer chromatography, UV and IR spectrometry and amino acid analysis, using authentic samples of viridogrisein and etanycin.

Neoviridogrisein I, II and III have a broad antimicrobial spectrum against bacteria, mycoplasmas, antinonycetes and rickettsiae in labotarory test. More exactly, they display a remarkable activity in vitro against the usual and resistant strains of Staphylococcus aureus, as well as strains of Streptococcus pyogenes, Diplococcus pneumoniae, Sarcina iutea, Baccilus subtilis, Mycoplasma gallisepticum, Mycoplasma pulmonis, Mycoplasma fermentans and Mycoplasma agalactiae. The minimal inhibitory concentrations of the new depsipeptide antibiotics of this invention were determined individually and in admixture with viridogrisein and griseoviridiii on various microorganisms by the tube dilution method. The results are shown in Table I.

In Table I, the following abbreviations apply: NV neoviridogrisein; VG. - viridogrisein; GV -- griseoviridin; EM = erythromycin; TC = tetracycline; STii = streptothricin; PC = penicillin; LM = Icjconycin; CP = chloramphenicol; SM - streptomycin; Kiri - kanamycin; NM = neomycin; OM = oleandomycin; BHI = brain-heart infusion broth; BHI + HB = brair.-heart infusion broth containing 10% w/w horse blood; and MY = malt extract - yeast extract medium, r as a superscript indicates that the relevant microorganism is resistant to the drug or drugs in parentheses.

TABLE 1 =>s As shown in the above table of MIC values, neoviridogrisein II is more active than viridogrisein. This MIC experiment was based on the two-fold dilutions. To differentiate neoviridogrisein II and viridogrisein in their antiboitic activity, the MIC determination was repeated with a far lower dilution rate. The subsequent Table indicates that neoviridogrisein II is 2-3 times more active than viridogrisein.

TABLE 2 MIC(mcg/ml) Microorganism NV II VG 10 k^PbyJfijoccu^j^ujreus 209 P u.078 0.133 ra. sm, pc, tc/ 0.125 0.334 (TC, CP, PC)r 0.094 0.334 0.-.-1633 (PC)r 0.125 0.267 Russell (PC)r 0.125 0.267 15 Smith 0.125 0.267 Medium: brain infusion broth. Abbreviations: as listed above.

When the minimal inhibitory concentrations of neoviridogriseins I, II and III were tested in the presence of griseoviridin, the synergism was observed between the neoviridogrisein member and griseoviridin, as in the case of viridogrisein. Therefore, the synergism of the new antibiotics of this invention with griseoviridin was studied in more detail with varied ratios of the neoviridogriseins mixture to griseoviridin. The obtained results are shown in the following Table. 415212 TABLE 3 Weight ratio of NV mix : GV MIC*(mcg/ml) 100 : 0 0.313 90 : 10 0.156 BO : 20 0.125 70 : 30 0.125 60 : 40 0.094 50 : 50 0.078 40 : 60 0.094 30 : 70 0.125 20 : 80 0.250 1C : 90 0.250 0 :100 0.250 -Test microorganism : Sarcina lutea Tube dilution method with brain heart infusion broth.

From the above table it can be seen that the synergic action of the mixture of neoviridogriseins with griseoviridin was most significant at the ratio of 50:50 w/w; i.e. a 1:1 w/w mixture of neoviridogriseins and griseoviridin is 3-4 times more active than neoviridogriseins or griseoviridin alone.

The following Table 4, reports the high in vitro activity of neoviridogrisein II against various itycoplasraa strains as well as the superior synergism shown by a mixture of neoviridogrisein II and griseoviridin in comparison with a mixture of viridogrisein and griseoviridin (all mixtures 1:1 w/w). The MIC were determined by the dilution method. />8 212 (ABLE 4 Microorganism Medium NV II VG MIC values (mcg/ml) GV NV II+GV VG+VG Mycoplasma gallisepticum KP13 (1) 0.025 0.10 0.10 0.0063 0.025 Mycoplasma pulmonis PG 22 (2) 0.78 1.56 6.25 0.20 0.39 Mycoplasma ferroentans (2) <0.05 <0.05 <0.05 <0.05 <0.05 Mycoplasma agalactiae (2) 0.39 0.78 3.13 <0.05 0.39 Medium (1) : PPLO enrichment broth (Eiken, Japan) (2) : PPLO broth (Chanock's medium; Difco) As illustrated abf’ve, cne novel neoviridogriseins I-III of the present W invention display a remarkable activity against gram-positive bacteria and mycoplasma strains both alone or in various admixtures with viridogrisein (neoviridogrisein IV) and griseoviridin. It has been found that the mutual weight proportions of neoviridogriseins I-III, viridogrisein (neoviridogrisein IV) and griseoviridin in a mixture may vary within very wide limits, but the surprising antimicrobial and anti-mycoplasma activities are still retained. As a representative, though not limitive example, a mixture having the following per cant composition (by weight): . r ο 1 il 4 Ο <* *· Neoviridogrisein II 27% Neoviridogrisein IV (viridogrisein) 23% Griseovirdin 50% were tested in vitro against Streptococcus mutans, a microorganism associated with dental caries and peridontal diseases. The test was carried out in Todd Hewitt Broth (Difco) with 0.5% TC Lactalbumin hydrolysate (Difco). organisms per ml.

C for 48 hours.

Initial organism count approximately 3 x 10 Culture tubes were incubated anaerobically at The minimum inhibitory concentration (MIC) and TO minimum bactericidal concentration were both found at a neoviridogrisein concentration of 1.0 part per million (ppm).

In another representative example, the same mixture was tested in vitro against Treponema hyodysenteriae, a swine dysentry organism.

The mixture was tested as dilutions in blood agar at concentrations of 15 100, 50, 10, 5, 1, 0.5 and 0.1 ppm. Plates were inoculated with a swab and incubated 4 days at 42°C. The MIC was determined to be 0.5 ppm. In a further representative example, a mixture having the following percent composition (by weight): Neoviridogrisein III 25% Neoviridogrisein IV (viridogrisein) 25% Griseoviridin 50% was tested in vitro against several Mycoplasma strains. The minimum inhibitory concentrations (MIC) were found to be as follows: Strain MIC Mycoplasma gallisepticum KP 13 0.07 Mycoplasma pulmonis 0.20 Mycoplasma fermentans 0.05 Mycoplasma agalactiae 0.05 3212 These types of neoviridogriseins-griseoviridin mixtures are also useful in the treatment of animals suffering from infectious diseases caused by the above pathogenic bacteria.

As mikamycins A and B have been known to be very effective as feed additive, the new antibiotic compounds of this invention were subjected to the animal feed test. Neoviridogriseins as mixture were added to the chicken feed at a rate of 2 - 20 ppm and fed to male chickens for 10 we ,s. compared with the control group of chicken which received the came feed without neoviridogriseins, the neoviridogrisein-fed chickens were superior in the increase rate of body weight and the feed efficiency. Thus neoviridogriseins of this invention have been proved very useful as feed additive.

Also the compositions containing one or more of the neoviridogriseins Ι-'Π, optionally in admixture with viridogriseins (neoviridogrisein IV) and griseoviridin, wherein the mutal weight proportions of the components may vary within very wide limits, proved to be very useful as feed additive.

As a representative, though not limitative example, a mixture having the following per cent composition (by weight): Neoviridogrisein II 27% Neoviridogrisein IV (viridogrisein) 23% Griseoviridin 50% was tested in vivo by incorporation in the diet of growing chicks.

One-day old cockerels, 15 birds per treatment were used. The chicks were maintained for 11 days on a feed containing about 55% w/w rye grain, supplemented with vitamins, minerals, fat and protein sources. The high content of rye grain normally gives poor to moderate growth, and this diet is a standard one used for screening growth promoters and antibiotic feed additives. Penicillin (TOO ppm) was used as a positive control. The data is tabulated below. Feed/Grain ratio is grams feed consumed per gram of weight gained. Body Weight ratio is the ratio of chick body weight at the end of the 11 day study to the initial body weight.

The last column is the average gain per bird (in grams).

Treatment Diet Cone (ppm) Feed/Grain Ratio Body Weight Ratio (Gms) Avg B.W. Gain Rye Control - 1.334 4.696 156.47 Penicillin 100 1.197 5.012 163.20 Tested mixture 100 1.256 4.789 161.66 II 50 1.283 4.892 163.47 II 25 1.204 5.226 174.67 The following Examples illustrate this invention.

All percentages are by weight and all ratios are by volume.

EXAMPLE 1 Seed culture medium consisting of soybean meal 0.5%, Pharmamedia (Traders Oil Mill Co.) 0.5« oatmeal 0.5% dry yeast 0.5% and beet molasses 0.5% was adjusted to pH S.5 and distributed in a 50 ml. amount in a 250 ml. Erlenmeyer flask. After autoclaving at 120°C for 15 minutes, a loopful amount of Streptomyces sp. P 8648 on an ISP-2 agar slant was inoculated and the flask incubated at 28°C for 48 hours on a rotary 43312 shaker. Two milliliter of the said seed culture was transferred into a 500 ml. Erlenmeyer flask containing 100 ml. of the following fermentation medium: Soybean meal Peanut meal Oatmeal Dry yeast Ceet «0l-.ises 0.5% 0.5% 0.5% (pH 5.5 prior to autoclaving) 0.5% 0.5% and cultivated at 28°C for 96 hours on a rotary shaker at 200 r.p.m. (radius of circle 3.5 cm.). this culture broth was collected from 12 flasks and filtered to give a clear broth filtrate. The obtained cake on the filter was washed with 100 ml. of water. The water washing and the broth filtrate were ccraoinad. The antibiotic activity of this solution (pH 8.3) was determined to be 23.0 mm on a nutrient agar assay plate of Sarcina jutes when the standardized disc assay was carried out with a 8 mm paper disc, Eight hundred milliliter of the said aqueous solution was twice extracted with 200 ml. each of n-butanol, and the butanol extracts were combined and evaporated to dryness under reduced pressure to yield 90 mg. of crude powder of neoviridogriseins and griseoviridin. ’his crude powder was mixed with a small amount of silica gel and applied on a silica gel column (Wako-Gel C-1G0, Wako Pure Chemical Industries, Ltd.; 1.5 x 25 cm.). The silica gel column was eluted first with 300 ml. of a benzene-acetone mixture (5:1), and then with a benzene-acetone mixture (2:1). Ten gram fractions were collected on an automatic fraction collector. Active fractions of Nos. 25 to 35 were combined and evaporated to dryness to provide 30 mg. of neoviridogrisein 45313 mixture (consisted of neoviridogriseins I, II and III and viridogrisein).

In addition, evaporation of active fractions of Nos. 45-54 to dryness gave a crude powder, the antibiotic activity of which corresponded to griseoviridin by TLC. These two preparations were subjected to thin layer chromatography under the indicated conditions. The antimicrobial activity was detected oh a nutrient agar assay plate of Sarcina lutea.

TLC plate: Pre-coated TLC plate SILICA GEL 60 F-254, E. Merck, Darmstadt. (1) Solvent: Chloroforftnmethanol = 20 : 1 Neoviridogriseins Rf = 0.45 Griseoviridin 0.05 (2) Solvent : Benzene : acetone = 1:1 Neoviridogriseins· Rf = 0.55 Griseoviridin 0.13 EXAMPLE 2 Two hundred milliliter of the 48 hour-old culture of Streptomyces sp. P8648 in the same culture medium as in Example 1 was inoculated into a 15 liter stainless steel jar fermenter containing 10 liters of the same seed culture medium'as in Example 1 and cultivated at 27-28°C for 96 hours under forced aeration of 5 liter/min. of sterile air. The agitation during cultivation was performed at 200 r.p.m. with an impeller, the radius of which is about a fourth of the diamster of the jar fermenter. At the end of fermentation, the mycelia and solids were removed by filtration. The obtained broth filtrate was adjusted to pH 6.0 and extracted four times with 2 liters each of ethyl acetate. The ethyl acetate extracts were combined, dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure to yield 700 mg. of crude neoviridogriseins and griseoviridin. The recovered crude powder of neoviridogriseins and griseoviridin was dissolved in a small amount of methanol and charged on a Sephadex LH-20 column (3 X 50 cm.). Ten milliliter fractions were collected with methanol as an eluting solvent. Neoviridogrisein·; were collected in fraction Nos. 22-29. These fractions were collected, concentrated to dryness and further purified by silica gel column chromatography (SILICAR CC-7 Special; Mallinckrodt Chemical Works; 1.5 X 20 cm.). The elution was run with a mixture of chloroform and methanol (30 : 1). Eight 5 gram fractions from fraction No. 5 to fraction No. 12 were combined and evaporated to dryness under reduced pressure to yield 25 mg. of white powder of neoviridogriseins. The oercent composition of neoviridogriseins ϊ, II and III and viridogrisein in this powder was as follows: Neoviridogrisein I : 15% Neoviridogrisen II : 20% Neoviridogrisen III : 20% Viridogrisein : 45% Griseoviridin was found by TLC in fractions Nos. 30-34 of the said Sephadex LH-20. These active fractions were combined, evaporated to dryness under reduced pressure and crystallized in warm methanol to give 30 mg. of needle crystals of griseoviridin. The identity of these crystals with griseoviridin was proved by TLC and other physico-chemical determinations. <Λ·' EXAMPLE 3 lhe same fermentation as described in Example 1 was carried out for 96 hours except that the fermentation medium was composed of soybean meal 0.5%, Pharmamedia 0.5%, oatmeal 0.5%, dry yeast 0.5%, beet molasses 0.5% and DL-alpha-amino-n-butyric acid 0.1% (pH 6.5). The fermentation broth was collected from 13 flasks and filtered. The filtered liquid was extracted with n-butanol (300 ml. each twice).

The removal of n-butanol from the extracts left about 70 mg. of crude powder of neoviridogriseins and griseoviridin. The crude powder was analysed by silica gel thin layer chromatography followed by bioautography on Sarcina lutea as the test organism. The TLC plate employed in this assay was a product of E. Merck, Darmstadt (Pre-coated TLC plate SILICA GEL 60 F-254). The obtained Rf values and the solvent systems were as follows: Chlorofonrnmethanol 20:1 30:1 40:1 Neoviridogrisein I Rf= 0.60 0.39 0.20 II 0.56 0.32 0.16 III 0.50 0.19 0.13 Viridogrisein 0.43 0.18 0.10 20 Griseoviridin 0.50 0.02 0.00 EXAMPLE 4 Fifty milliliters of the seed culture medium containing 0.3% beef extract, 0.5% tryptone (Difco Laboratories), 0.1% glucose, 2.4% soluble starch, 0.5% yeast extract, 0.4% calcium carbonate and 0.5% soybean meal (pH 7.0) was distributed in a 250 ml. Erlenmeyer flask and autoclaved at I20°C for 15 minutes. Spores of 4&3l3 Streptomyces sp. P 8648 on an agar slant ware seeded in the said flask and shake-cultured at 25°C for 3 days to supply the seed culture. The fermentation medium was composed of 0.5% soluble starch, 2.0% glucose, 1.0% Pharmamedia, 0.5% oatmeal, 0.5% corn steep liquor, 0.05% dipotassium phosphate and 0.05% magnesium sulfate (pH 6.5). Fifty milliliter of this fermentation medium was placed in a 250 ml. conical flask and autoclaved at 120°C for 15 minutes. The size of the inoculum was 2/. (v/v). The fermentation flask was inoculated with the above described seed culture and incubated at 25°C on a rotary shaker. Twenty four or 48 hours after inoculation, a sterilized solution of casamino acid (Difco Laboratories) or proline (pH 7.0) was added to a final concentration of 0.4% and the fermentation was continued. Four days after inoculation, the fermentation broth was filtered and the broth filtrate was extracted twice with an equal volume of ethyl acetate. The ethyl acetate extracts were combined and evaporated to dry nofider under reduced pressure. This crude powder was taken in L;,, acetate and an aliquot amount of the solution was quantitatively spotted on a silica gel TLC plate. The TLC plate was developed in a solvent system of chloroform and methanol (50:1) and the solvent was evaporated off in the air. The same TLC plate was again developed in the said solvent system of chloroform and methanol (50:1). Each component of neoviridogrisens was located under ultra-violet light (3650 A), scraped off from the TLC plate and suspended in a known volume of methanol. After the silica gel was removed by decantation, the amount of the antibiotics in the extracts was determined by the UV-assay method, knowing that the epsilon value at 305 nm is 8,000. The results of the UV-assay are as follows: A S 3 '13 None Neoviridogrisein I 2% II 9 III 4 Viridogrisein 85 Amino acid added Casamino acid Proline 2% 2% 60 3 EXAMPLE 5 About 10 liters of the 23 hour-old seed culture was prepared in a jar fermenter under the same conditions as described in Example 2. The fermentation medium (600 liter) comprising 0.5% soybean meal, 0.5% Pharmamadia, 0.5% oatnteal, 0.5% dry yeast, 0.5% beet molasses and 0.1% DL-alpha-amino-n-butyric acid (pH adjusted to 6.5 prior to autoclaving) Vias steam-sterilized at 120°C for 15 minutes in a 1400 liter stainless steel tank fermenter and cooled down to 28°C. To this tank fermenter, the above-mentioned seed culture (10 liter) was added and cultivated at 28°C for 75 hours under forced aeration with stirring at 180 r.p.m. (by means of a double impeller; radius of circle, 1/4 of the diameter of the tank fermenter), the sterile air being fed at 300 liter/minute through a sparger from the bottom of the tank. At the end pf fermentation, the broth was filtered through a filter press. The broth filtrate was extracted twice with 150 liter each of n-butanol. The n-butanol extracts ware combined, washed with a small volume of saturated NaCl solution, and concentrated to 2 liters in a rotary evaporator. At this stage, 20 grams of silica gel (WAKO-GEL F-100, Wako Pure Chemical Industries, Ltd.) was added and the concentration in a rotary evaporator was further continued to complete dryness. The obtained antibiotics-silica --8212 gel mixture was suspended in a small amount of chloroform and put on top of a silica gel column (WAKQ-GEL C-100; 6.5 x 75 cm.). The elution of the antibiotic activities was carried out stepwise first with 7 liters of chloroform, then with 10 liters of a mixture of chloroform and methanol (50:1); and finally with rnethanoi. Fractions Nos. 16-29 (300 g/fraction) which were found bio-e.:tive on Sarcina lutea (neoviridogriseins) were collected and concent ated to dryness under reduced pressure to yield a crude powder of neoviridogriseins. The crude powder was dissolved in a small volume of methanol and passed through a Sephadex LH-20 column (7.0 x 45 cm.), each fraction (100 g.) being eluted with methanol. About grams of crude pcwder of neoviridogrisein mixture was recovered from fractions Nos. 6-15 after the solvent was removed by evaporation. Fractions Nos. 50-60 of the above described silica gel column contained griseoviridin.

A similar purification procedure with Sephadex LH-20 (column size 7.0 x 45 cm.) !5 as used for the neovi in mixture was repeated to provide 4 grams of crude griseoviridin EXAMPLE 6 For final purification, preparative thin layer chromatography with a silica gel TLC plate was employed. One gram of the crude neoviridogrisein mixture prepared in Example 5 was dissolved in 2 ml. of ethyl acetate and applied in bandwise fashion on ten silica gel TLC plates (Pre-coated TEC plate SILICA GEL 60 F-254). These TLC plates were first developed with a solvent system of chloroform and methanol (50:1). After the solvent was evaporated off in the air, the said TLC plates were subjected λΰ3ΐ 3 to a second development with a solvent system of chloroform and methanol (25:1). Neoviridogriseins I, II and III and viridogrisein were marked on the TLC plates under ultra-violet light (3650 A: BLAK-RAY UVL-22, Ultra-violet products, Inc.) and scraped off for elution. Each neoviridogrisein component was eluted with a small amount of methanol and evaporated to dryness. The recovered amount of each neoviridogrisein component in pure state was ss fellows: Neoviridogrisein 1:16.7 mg. (less pure, oily) 11:11.1 mg. (white powder) 111:15.2 mg. (white powder) Viridogrisein : 30.0 mg. (white powder) About 5 mg. each of the neoviridogriseins was hydrolyzed in 6N HCl at 110°C for 36 hours in a sealed tube and subjected to thin layer chromatography, paper chromatography, high voltage paper electrophoresis and autoamino acid analysis. The presence of the amino acids given above for neoviridogriseins ϊ, II and III and viridogrisein was found. The identity of neoviridogrisein IV with viridogrisein was confirmed by IR, UV, NMR and mass spectrometry, thin layer chromatography, hydrolysate analysis and antimicrobial spectrometry.

The griseoviridin perparation obtained in Example 5 was crystallized in warm methanol to yield needle crystals. Then a part of the needle crystals were compared and identified with an authentic preparation of griseoviridin by IR, UV, NMR and mass spectrometry, thin layer chromatography, elementary analysis and other physico-chemical properties.

Claims (10)

1. , A process for preoaring the three depsipeptide antibiotics (i) neoviridogrisein I of the formula CH J Z CH 3 CH .CH, r..... fi s' !L r whose Taj CH, CK, ck 2 0--Nh -CH—-CO-NH—CH — CQ- N I CH— CH, CH —CO I N—CH, CH, Oil CO I ί hr sas '··CO — ti 3—CH—_nh_c0—. -CH — | — N . CH, 1 CH- I CH 3 1 lh 3 1 CH — 1 ch 3 CH 3 in methanol) = + 13°; II of the formula CH, -CH, CH I CH, X CH 2 CH, CO —NH—CH-CO—NH-CH—CO-N CH— CH, \ CH, I 2 - CH —CO I N -CH, I CH, CO I CO —CH — NH — CO — CH-N— CH ι CH, . 3 CH-CH, I CH-CH 3 ch 3 ’ whose [a] D (1% by weight in methanol) = -39.3°; and (iii) neoviridogriesin III of the formula whose Jjxjp 3 (1% by weight in methanol = + 73.7°; which comprises cultivating Streptomyces sp. P8548 (FERM-P3562), ora mutant, capable of producing the same products, theraof, under aerobic 5 conditions, at a temperature of from 18 to 37°C in an aqueous nutrient medium containing an assimilable source of carbon, an assimilable source of nitrogen and essential mineral salts; recovering the fermentation product from the medium; and if desired, isolating neoviridogrisein I, neoviridogrisein H and/or neoviridogrisein III. 10

2. A process according to claim 1 in which viridogrisein, containing the amino acids threonine, leucine, hydroxypro!ine, alanine, sarcosine, C-phenylsarcosine and Β-Ν,Ν-dimethylleucine, and griseoviridin are also produced. 4 y 31 3

3. A process according to claim I or claim 2 in which the cultivation is carried out in the presence of alpha-amino-n-butyric acid.

4. A process according to claim 1 or claim 2 in which the cultivation is carried out in the presence of proline.

5. 5. A process according to claim 1 substantially as herein described with reference to any of the Examples. a. The antibiotic sac-stance neoviridogrisein I of the formula and optical rotation defiib-a in claim 1.

6. 7. The antibiotic substance neoviridogrisein II of the formula and 10 optical rotation defined in claim i.

7. 8. The antibiotic substance neoviridogrisein III of the formula and optical rotation defined in claim 1.

8. 9. An antibiotic composition comprising necviridogrisein I, neoviridogrisein II, neoviridogrisein III, viridogrisein and griseoviridin. IS 10. A pharmaceutical composition comprising an antibiotic or antibiotics selected from neoviridogrisein I, neoviridogrisein II and neoviridogrisein III in association with a pharmaceutically acceptable carrier. II. An animal feed composition comprising a compound or compounds selected from neoviridogrisein I, neoviridogrisein II and neoviridogrisein III. 20 12. A composition as claimed in claim 11 in which the compound is neovi ri dogri sei η I ί.

9. 13. A composition as claimed in any of claims 10 to 12 additionally comprising viridogrisein.

10. 14. A composition as claimed in any of claims 10 to 13 additionally 25 comprising griseoviridin.