US3671628A - Tirandamycin and process for making same - Google Patents

Abstract

Antibiotic tirandamycin producible by culturing Streptomyces tirandis var. tirandis in an aqueous nutrient medium. Tirandamycin is active against Bacillus subtiles and can be used to minimize or prevent odor in fish and fish crates caused by this organism.

Description

United States Patent Sebek et al. 1 June 20, 1972 s41 TIRANDAMYCIN AND PROCESS FOR [56] References Cited I MAKING SAME OTHER PUBLICATIONS 72] inventors: Oldrich K. Sebek, Kalamazoo; Curtis E. Sugawara et al., .I. of Antibiotics. Vol. 10, set. A. 1957, pages Meyer, Galesburg, both of Mich. i38- I42.

[73] Assignee: The Upjohn Company, Kalamazoo, Mich. I Primary mminer kmme Goldberg [221 Filed: Sept. 8, 1969 Anomey-George T. .lohannesen and John Kekich Antibiotic tirandamycin producibie by culturing Srreplor'nyces il 424/221,; u-mndis van tirandis in an aqueous nutrient medium Tiranda [58] Field of Search ..424/120; 195/80 and be used minimize or prevent odor in fish and fish crates caused by this organism.

9 Claim, 1 Drawing Figure PKTENTEDJUHO m2 illilllll lllllll Jlllll IllllJllll NOISSIWSNVHJ.

CURTIS E MEYER OLDRICH K. SEBEK ATTORNEY TIRANDAMYCIN AND PROCESS FOR MAKING SAME BRIEF SUMMARY OF THE INVENTION Tirandamycin (U-29,275) is an amphoteric phenolic compound which is producible by culturing a tirandamycinproducing actinomycete in an aqueous nutrient medium. It has the property of adversely affecting the growth of Grampositive bacteria, for example, Staphylococcus aureus, Streptococcus hemolyticus, Streptococcus faecalis, Bacillus subtilis, and Diplococcus pneumoniac. Accordingly, tirandamycin can be used alone or in combination with other antibiotic agents to prevent the growth of or reduce the number of bacteria, as disclosed above, in various environments.

DETAILED DESCRIPTION OF THE INVENTION CHARACTERIZATION OF TIRANDAMYCIN AS THE SODIUM SALT Elemental Analyses Calcd. for C H,,NO-,Na:

C, 60.1 I; H, 5.96; 0, 25.55; N, 3.18; Na, 5.24

Found:

C, 58.1 I; H, 6.28; N, 2,86; Na, 4.74 Ultraviolet Spectrum Tirandamycin sodium salt has a characteristic ultraviolet absorption spectrum as follows:

The infrared absorption spectrum of tirandamycin sodium salt suspended in mineral oil mull is reproduced in the drawing. Tirandamycin shows peaks at the following wave lengths expressed in reciprocal centimeters:

Band Frequency Band Frequency (cm") Intensity (cm') Intensity 3720 M I210 S 3600 M I155 S 3360 S 1130 S 3300 sh S I120 S 3130 M I087 S 3000 (oil) S I068 S 2960 (oil) S 1055 S 2900 (oil) S 1045 M 2760 W I020 S 1730 S I000 M I670 S 995 M I640 S 960 M I625" l 590 S 955 M (Broad) I530 S 930 W I485 S 920 W 1470 (oil) S 907 M 1415 S 900 M 1395 S 893 M I383 S 865 S I355 (oil) M 840 W I345 M 8I5 W I320 M 805 W I305 M 795 M I295 M 780 W I290 M 763 M I260 S 745 S I255 S 725 S Band intensifies are indicated as S"M", and W", respectively, and are approximated in terms of the backgrounds in the vicinity of the bands. An S band is of the same order of intensity as the strongest in the spectrum; M bands are between one-third and two-thirds as intense as the strongest band, and W bands are less than one-third as intense as the strongest band. These estimates are made on the basis of a percent transmission scale. The designation SH" denotes a shoulder.

Ferric chloride test Positive Molish Negative Ninhydrin Sakaguchi Negative Millon Biuret Negative Solubilities Soluble in moist chloroform and in ethylene chloride.

CHARACTERIZATION OF TIRANDAMYCIN AS THE BROMOBENZENE SOLVATE Elemental Analyses Calcd for: C H NqBrz C, 58.49; H, 5.61; N, 2.43; O, 19.49; Br, 13.9l Found: C, 58.68; H, 5.44; N, 2,35; 0, 19.92; Br, 13.25

Molecular Weight High resolution mass spectrometry gave a measured mass of 417.1792 which corresponds to C l-ENC having a theoretical mass of417.1786.

Tirandamycin as the amorphous free phenol is very soluble in diethyl ether, ethyl acetate, acetone (and does not seem to solvate in acetone whereas sodium salt gives solvate insoluble in acetone). It is also soluble in chloroform, ethylene chloride, and lower alcohols, for example, methanol and ethanol. The amorphous free phenol of tirandamycin is insoluable in water and petroleum ether. It is transitorily quite soluble in benzene but rapidly forms a solvate which crystallizes therefrom.

THE ANTIBACTERIAL PROPERTIES OF TIRANDAMYCIN Minimum Inhibitory Test Organism Concentration (NS/ml.) S taphylococcur aureu: 250 Streptococcus hemolyticus 0.5 Streptococcus faecalis 0.5 Bacillus .subtilis 8 Diplococcus pneumoniae 0.5

THE MICROORGANISM The actinomycete used according to this invention for the production of tirandamycin is Streptomyces tirandis var. tirandis. One of its strain characteristics is the production of tirandamycin. A sub-culture of the living organism was deposited without restriction and can be'obtained from the permanent collection of the Northern Utilization and Research Division, Agricultural Research Service, US. Department of Agriculture, Peoria, Illinois, USA. Its accession number in this repository is NRRL 3689.

(1962)]Microscopic Characteristics 3 The microorganism of this invention was studied and characterized by Alma Diet: of the Upjohn Research Laboratories.

DESCRIPTION OF THE MICROORGANISM sections of Pridharn et al. [Applied Microbiol.6:52-79 Tlm l t me!.hseeuasdwithenxet tbs. is s ted. in he e. ssstio s. on. th ba is. 92.2 3219: teristics noted in the literature citations or by actual comparison as was possible for most of the organisms listed.

The new organism might be placed in the Aureus, Dia-' stations, or Antibioticus Series of Baldacci [Giomale di Microbiologia 6:10-27 (1958)] or the Cinereus-color group of l-liitter [Systematik der Streptomyceten unter bcsondere Bs i qksish i uasdsr. vqmihnes .sb. ld sn...AL ib iL-.L l(arger,.Basel (1967)]. However, the single characteristic of aerialcolor mass is insufficient to relate the new organism to the cultures included in these color groups. The new culture differs significantlyin pigment characteristics, growth on carboncompounds in synthetic media and antibiotic production from organisms assigned to those color groups. I S. tirandishad some similarity to Streptomyces melanogenes [The Journal of Antibiotics, Ser. A 10:138-142 (1957)] on Ektachrome. S. melanogenes was differentiated by trace gray aerial growthon Oil percent tyrosine agar and casein starch agar and red-tan and yellow-tan reverses on these media. S. inelanagenes did not solubilize casein but did solubilize xanthine. It also produces the antitumor antibiotic melanomycrn. I

S. rirandis can be differentiated from named species in the Upjohn culture collectionand from those described in the literature. It is readily identified by its color pattern, its sporophore and spore type and antibiotic production pattern. Therefore, it is proposed that the new soil isolate be designated Streptomyces n'randis Dietz sp. n. and that this type species be designated the type variety Srrepromyces tirandis var. tirandis in accordance with Rule 911(2) of the lntemational Code of Nomenclature of Bacteria llntem. J. System.

Bacteriol. l6 :459490 1966)]. Srreptomyces u'randis Dietz sp. n. Color Characteristics Aerial growth gray (gray-white, gray-cream, gray-tan). Melanin positive. Appearance on Ektachrome [Ann. NY.

Acad. Sci. 60:152-154 (1954)]is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the White (W), Yellow (Y), and Gray (G) color series of Tresner and Backus [Applied Microbioi. 11:335-338 Sporophores small, straight, to open spiral to spiral (RF, RA, 8) in the sense of Pridham et al. (supra). Spores smooth, long, many angled at ends (appearing octangular) by direct electron microscopic examination. Spores ridged with much Zsurt'a-ce detail when examined by the carbon replication 'method of I Diet: and Mathews [Applied Microbiol. 10:258-263 (1962); 16:935-941 (1968)].

Cultural and Biochemical Characteristics I See Table 3.

Carbon Utilization Growth of the culture on carbon compounds was determinedin' the synthetic medium of Pridham and Gottlieb [J Bacteriol. 56:107-114 (1948)] and in their modified medium [International Journal of Systemic Bacteriology 16:313-340 (1966)]. .In the former, the culture showed slightgr gwth on the control; good growth on D-xylose, L-arabino se, rhamnose, D-frustose, D-galactose, D-glucose, D-mannose, maltose, sucrose, lactose, cellobiose, raffinose, dextrin, inulin, soluble starch, glycerol, D-mannitol, D-sorbitol, inositol, and sodium acetate; moderate growth on salicin, sodium oxalate, sodium tartrate, sodium citrate, sodium succinate; slight growth on dulcitol, phenol, and cresol. There was no growth on sodium salicylate. Inv the modified medium there was no growth on the negative control. There was good growth on the positive control (glucose). Growth was equal to or better than on the glucose control on L-arabinose, sucrose, D-xylose, inositol, D- mannitol, D-fructose; rhamnose, and ratfinose. Growth was doubtful on cellulose. Temperature Growth was fair at 1 8 C., good at 24-37 C. on Bennetts and maltose-tryptone agars. 0n Czapeks sucrose agar, growth was fair at 18 C. and slight at 24-37 C. On all media there was trace vegetative growth in 24 hours at 45 C. and C.

The characteristics of Streptomyces tirandis Dietz sp. n.,

NRRL 3689, are given in the following tables:

Table 1 Appearance of S. tirandis on Ektachrome Table 2 Reference Color Characteristics of S. lirandis Table 3 Cultural and Biochemical Characteristics of S. tirandis Appearance of S. tirandis on Ektachrome' Agar Medium Surface I Reverse Bennett's Gray Brown Czapek'a sucrose Trace gray Colorless Maltoae-tryptone Trace gray Brown Peptone-iron No aerial growth Brown 0.1% tyrosine Fair gray Brown Casein starch Fair gray Tan-brown 'Dietz, A., Ektachrome Transparencies as Aids in Actinomycete Classification," Annals of the New York Academy of Sciences, Q!l-.l$1l2-....

.. TABLE 2 Reference Color Characteristics of S. iirandis ISCC-NBS method of designating color and a dictionary of color names,

Agar medium Color harmony manual 3rd ed.,'1948 Z circular 553 a S 301) 1 sand a 91gm dark grayish yellow. Bennett's R 210 light mustard tan ggg lllghlttolilve brown.

- g g 0 we. P 2ge covert tan, gr'lege .{94m light olive brown.

. 109gm light grayish olive.

' S 2cb ivory tlnt- 92m yellowish white. v 93gm yellowish gray. 'Czapek's sucrose R 1 5 ec putty gggm grahyish lyellow. Y gm ye OWlS gray. P S 3il1 beige gray, mouse; oliraii gray.

* m 111 um gra Maltose-tryptone R Spl clove brown, deep brown uiggmdmiiierzife yelgotwish brown.

, g er ye owrs rown. 5 gig ifiiobe brown, cinnamon brown, light brown gg'm giggeato yelllpwish brown.

es ver gray gm g rowms gray. 1 R 3ie camel, maple sugar, tan to, "ggm liggt ytellowlilsh brlrlwgn. t

gmo eraeye OWlS rown 0.... Yeast extract malt .extract (ISP 2) 41g light spice brown, sandalwood, toast tan lighi'abrogvrig gm mo era e rown. P

3ge beige, camel {79m light grayish yellowish brown.

TABLE 2-Continucd Agar medium Color harmony manual 3rd ed., 1048 2 S 3fe silver gray ti3gm light brownish 'ra Oatmeal (ISP-3) .{R c biscuit, ectu, oatmeal. sand.. DOgm grayish yellow. 3

l" Zea light ivory, eggshell.. Xllgni yellow pale. is lde natural, string. h3gni yellowish gray. Inorganic-salts starch USP-4) R 31g adobe browmcinnamon brown, light brown TTgni moderate yellowish ln'own.

l 2ge covert tan, grlege H ilOgm grayish yellow. s 3el) sand I fllgnl dark grayish yellow. Glycerol-asparagine USP-5) R 2ie light mustard tan ..{'.Hg light olive hrown.

P 106g light olive.

NoTE.S=Surfaee. P=Pig ment. R=Reverse.

1 Matte surface used for all chips.

1 Jacobson, E., \\.C. Granville, and C.E. Foss. 1048. Color Ilarmouy Manual, 3rd Ed. (ontainer (.orporation of America. (.hieago. lllinois. 3 Kelly, l-..L.. and D.B. Judd. 1955. The ISCC-NBS Method of Designating Colors and a Dictionary of Color Names. Dept. (.onnn. Fire. 553.

TABLE 3 ultural and Biochemical Charaeteristies of S. tirandis Surface diu Reverse Other Agar media:

leptone-iroiL. No aerial growth to very slight trace gray aerial Brown.. llrowu pigment.

growth.

Calcium iiialate.'.. Gray-cream to gray-tan aerial growth (lray .[No pigment.

(Malate not solnhilized. Glueose-asparagine (tray-cream aerial growth. Yellow-olive No pigment. Skim milk Trace to good gray-cream aerial growth Yellow-tan. l Yellow-tan pigment.

(Casein soluhilized around growth. Tyrosine Gray-cream aerial growth. Brown Brown pigment.

Tyrosine soluhilized.

Xanthiue Gray aerial growth. Yellow lale yellmr-tan pigment.

'lXanthine not soluhilized. Nutrient starch Gray-white aerial growth. Yellow. .j'Yt'llOW pigment.

[Stareh hydrolyzed. Yeast extract-malt extract Gray-cream aerial growth Yellow-tan. Yellow-tan pigment. Beuuetts Good gray aerial growth. (lream-tan.... lale yellow pigment. (zapek's sum-050.. Traee gray-whit e aerial growth.. Gray. No pigment. .\laltose-tryptone. (lood gray aerial growth. Tan. lale tan pigment. leptone-yeast extract-iron USP-6).. .\'o aerial growth Brown. Brown. Tyrosine (ISP-T) (ream-gray aerial growth. Brown.. .i Very slight traee brown pigment.

(i elatin media:

Traee gray aerial llaiu Traee gray aerial growth... Nutrient Broth media:

Synthetic nitrate Nutrient nitrate Litmus milk Trace gray aerial growth on brown surface ring Traee white. to gray aerial growth on surface ring .lT-an pigment in upper of medium.

lLiquef-aetion complete. lTan pigment in upper of medium. (Liquefaction complete.

.lFloceulent bottom growth.

[Nitrate redueed to nitrite. {Tan pigment.

. Floeeulent bottom growth.

N'trate reduced to nitrite.

plI 6.2-7.2.

The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. lt is to be understood also that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, for example, an assimilable carbohydrate, and a nitrogen source, for example, an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include com steep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers' solids, animal peptonc liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron, and the like, need not be added to the fermentation media since tap water and unpurified ingredients are used as media components. 7

Production of the compound of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example, between about 18 and C., and preferably between about 20 and 32 C. Ordinarily, optimum production of the compound is obtained in about 2 to ID days. The medium normally remains basic during the fers present, if any, and in part on the initial pH of the culture medium.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new comound, as long as it is such that a good growth of the microorganism is obtained.

The new compound of the invention is an amphoteric chemical compound. In the form of the free phenol it is very soluble in diethyl ether, ethyl acetate, acetone, chloroform, ethylene chloride, methanol, and ethanol. It is relatively insoluble in water and petroleum ether. Tirandamycin is transitorily quite soluble in benzene but rapidly fonns a solvate which crystallizes therefrom. In this regard, tirandamycin as the sodium salt readily forms a solvate with acetone, and in the free phenol form forms solvates with benzene and bromobenzene. These solvates are stable compounds and usefermentation. The final pH is dependent, in part, on the bufful to characterize the free phenol form of tirandamycin.

nan

lOlMl mm A variety of procedures can be employed in the isolation and purification of tirandamycin, for example, solvent extraction, partition chromatography, silica gel chromatography, liquid-liquid distribution in a Craig apparatus, and crystallization from solvents. Solvent extraction procedures are preferred for commercial recovery inasmuch as they are less time consuming and less expensive.

In a preferred recovery process, tirandamycin is recovered from its culture medium by separation of the mycelia and undissolved solids by conventional means, such as by filtration or centrifugation. The antibiotic is then removed from the filtered or centrifuged broth by extraction. For the extraction of tirandamycin from the filter broth, water-immiscible organic solvents in which it is soluble, for example, l-butanol, methyl ethyl ketone, benzene, and methylene chloride (preferred) can be used. Advantageously, the extraction is carried on after the filtered beer is adjusted to a pH of about 2 to 4 with a mineral acid. When the filtered beer is extracted at a pH of 6 or above, the base salt, for example sodium, is extracted by chlorinated hydrocarbons, for example, methylene chloride. When such extracts are concentrated, the solvated crystalline salt of the antibiotic can be precipitated with solvents such as acetone, benzene, or bromobenzene.

The extract obtained from an extraction of the filtered beer with methylene chloride at a pH of about 2 to 4 can be converted to a salt by adjusting the pH to a basic pH with a base, for example, sodium hydroxide, and the preparation freeze dried. This preparation can be used in environments where higher purity of the antibiotic is not essential.

Tirandamycin can be purified from a crude preparation, as described above, by subjecting the preparation to chromatography on a silica gel column which is developed with a solvent system consisting of 5% methanol in methylene chloride. Active fractions, as determined by bioactivity against the microorganism B. cereus, the assay which is described infra, are pooled and concentrated to dryness.

Also, tirandamycin can be purified by successive transfers from protonated to non-protonated forms and vice versa, especially with other types of treatments intervening as, for example, solvent extractions and washings, chromatography, and fractional liquidliquid extraction. In this manner, salts of tirandamycin can be employed to isolate or upgrade the antibiotic.

Tirandamycin forms salts with alkaline metals, alkaline earth metals, and amines. Metal salts can be prepared by dissolving tirandamycin in methanol, adding a dilute metal base until the pH of the solution is about 7 to 8, and freeze drying the solution to provide a dried residue consisting of the tirandamycin metal salt. Tirandamycin metal salts include the sodium, potassium, and calcium salts. Amine salts of tirandamycin, including those with organic bases, such as primary, secondary, and tertiary, mono-, di-, and polyamines also can be formed using the above-described rather commonly employed procedures. Other salts are obtained with therapeutically efiective bases which impart additional therapeutic effects thereto. Such bases are, for example, the purine bases such as theophyllin, theobromin, caffeine, or derivatives of such purine bases; antihistaminic bases which are capable of forming salts with weak acids; pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenaline, ephedrine, and the like; choline, and others.

Tirandamycin, its salts and solvates are active against Bacillus subtilis and can be used for treating breeding places of silk worms to prevent or minimize infections caused by this organism. The novel compounds of the invention also are active against Streptococcus faecalis and can be used to disinfect washed and stacked food utensils contaminated with this bacteria.

Hereinafier are described non-limiting examples of the process and products of the present invention. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 Part A Fermentation A soil stock of Streptomyces tirandis var. tirandis NRRL 3689, is used to inoculate a series of 500-ml. Erlenmeyer flasks, each containing ml. of sterile preseed medium consisting of the following ingredients:

Glucose monohydrate 25 g.lliter Pharmarnedia' 25 g.lliter Tap water q.s. Balance 'Pharmamedia is an industrial grade of cottonseed flour produced by Trader's Oil Mill Company, Fort Worth, Texas.

The flasks are grown for 3 days at 28 C. on a reciprocating shaker.

Preseed inoculum, described above, is used to inoculate a 20-liter seed tank containing sterile medium consisting of the Wilson's Peptone Liquor No. 159 is a preparation of hydrolyzed proteins of animal origin.

The seed I tank is inoculated with 5 percent (volume/volume) of the preseed inoculum. The seed tank is incubated at 28 C. for 2 days with agitation at the rate of 400 rpm and aeration at the rate of 10 standard liters/minute. The medium is adjusted to pH 7.2 with aqueous sodium hydroxide before sterilization.

The seed inoculum, described above, is used to inoculate a 400-liter fermentation tank containing 250 liters of sterile fermentation medium consisting of the following ingredients:

Bacto Peptone (Difco) 5 g.lliter Sodium chloride 5 g.lliter Cornsteep Liquor 0.1 g.lliter Tap water Balance The fermentation medium is inoculated with 5 percent (volume/volume) of the seed inoculum, described above. The fermentation proceeds for 5 days during which time the fermentation medium is agitated at a rate of 280 rpm, and aeration provided at the rate of 200 liters/minute. The temperature in the fermentation tank is maintained at 28 C. When foaming occurs, lard oil is used as antifoam.

A typical tirandamycin fermentation, as described above, can be illustrated by the following assay profile:

Assay Hours (biounits/ml.) 0 69 l 1.4 92 16.0 1 l4 1 2.6

The assay is a disc plate assay using the microorganism Bacillus cereus. The assay is conducted at a pH of 7.0. A standardized spore suspension of B. cereus (0.5 ml.) is inoculated into 1 liter of melted BBL seed agar (30.5 g.lliter), supplied by Baltimore Biological Laboratories. Agar plates are then poured, lfi-inch' paper discs saturated with testing material are placed on the agar, and the plates are incubated overnight at 32 C.

One biounit (BU) is defined as that amount of the active material which gives a 20 mm. diameter zone of inhibition. 1 Mg. (1,000 mcg.) of tirandamycin assays approximately 270 BU. Thus, a preparation assaying 4.0 mcg. tirandamycin per ml. is converted to 1.08 BU.

Par B-Recovery Whole fermentation beer I ,000 ml. assaying 1.08 BU against B. cereus), prepared as described above, is filtered with the aid of about percent diatomaceous earth. The filter cake is washed with l/ l 0 beer-volume of water. The combined clear beer and wash is adjusted to pH 2.0 with sulfuric acid and then extracted with one-half volume of methylene chloride. The methylene chloride extract is concentrated in vacuo. Additional water is added and the remainder of the methylene chloride is removed by distillation. The remaining aqueous suspension is adjusted to a pH of 7.5 with 50 percent aqueous sodium hydroxide and freeze dried; yield, 62 mg. of crude tirandamycin assaying 34.0 mcg./mg. against B. cereus. Part C--Purification To 750 ml. of a methylene chloride concentrate, obtained as described above in Part B, is added 2 liters of water, and while the mixture is stirred, the pH is raised to with 5 N sodium hydroxide. The aqueous extract is back-extracted into 500 ml. of methylene chloride at a pH of 6.3. After drying and concentrating the extract, it is chromatographed on a silica gel column developed with 5% methanol in methylene chloride. Fractions from the column which are bioactive against B. cereus are pooled and concentrated to dryness in vacuo. The dried solid is dissolved in chloroform and 5 percent methanol is added. Addition of more methanol and refrigeration results in crystallization of the sodium salt of tirandamycin solvated with chloroform. A portion of this sodium salt of tirandamycin is converted to the free phenol by dissolving l g. in 200 ml. methylene chloride and ml. methanol. The pH is adjusted to 2.0 with 4.5 N alcoholic hydrogen chloride. The solution is washed free of sodium chloride, dried with anhydrous sodium sulfate, and concentrated to dryness in vacuo; yield, 800 mg. of tirandamycin in the free phenol form.

EXAMPLE 2 By reacting tirandamycin sodium salt, as prepared in Example l, with acetone, there is obtained the acetone solvate of tirandamycin sodium salt. This compound is active against various Gram-positivie bacteria.

EXAMPLE 3 By reacting tirandamycin free phenol form, as prepared in Example 1, with bromobenzene, there is obtained the bromobenzene solvate of tirandamycin. This compound is active against various Gram-positive bacteria.

EXAMPLE 4 By substituting benzene for bromobenzene in Example 3 there is obtained the benzene solvate of tirandamycin. This compound is active against various Gram-positive bacteria.

We claim:

1. A composition of matter selected from the group consisting of tirandamycin, a compound which a. is effective in inhibiting the growth of various Gram-positive bacteria; and, as the sodium salt;

' ing Streptomyces tirandis var. tirandis in an aqueous nutrient b. has an optical rotation [01],, +5 1 (c, 1 percent in d. has a characteristic infrared absorption spectrum as shown in the accompanying drawing, and as the bromobenzene solvate;

e. has the following calculated empirical formula and elemental analyses: C H NO-Br C, 58.68; H, 5.44; N, 2.35; O, 19.92; Br, 13.25; and

f. has a measured mass of 417.1792 as determined by high resolution mass spectrometry; and non-toxic base addition salts thereof.

2. Composition of matter in dry form comprising tirandamycin, the compound defined in claim 1, said composition of matter assaying about 34.0 meg/mg. of tirandamycin on the B. cereus assay.

3. The compound, tirandamycin, according to claim 1, in its essentially pure free phenol form.

4. Sodium salt of tirandamycin according to claim 1.

5. The compound, according to claim 4, in its essentially pure crystalline form.

6. The bromobenzene solvate of tirandamycin, the compound defined in claim 1.

7. A process for making tirandamycin, the compound defined in claim 1, which comprises cultivating Streptomyces tirandis var. tirandis in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of tirandamycin.

8. A process according to claim 7 which comprises cultivatmedium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of tirandamycin and isolating the tirandamycin so produced.

9. A process according to claim 8 in which the isolation comprises filtering the fermentation medium, extracting the filtrate with a water-immiscible solvent for tirandamycin, and recovering tirandamycin from the solvent extract.

nines no-

Claims (8)

1. 2. Composition of matter in dry form comprising tirandamycin, the compound defined in claim 1, said composition of matter assaying about 34.0 mcg./mg. of tirandamycin on the B. cereus assay.
2. 3. The compound, tirandamycin, according to claim 1, in its essentially pure free phenol form.
3. 4. Sodium salt of tirandamycin according to claim 1.
4. 5. The compound, according to claim 4, in its essentially pure crystalline form.
5. 6. The bromobenzene solvate of tirandamycin, the compound defined in claim 1.
6. 7. A process for making tirandamycin, the compound defined in claim 1, which comprises cultivating Streptomyces tirandis var. tirandis in an aqueous nutrient medium under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of tirandamycin.
7. 8. A process according to claim 7 which comprises cultivating Streptomyces tirandis var. tirandis in an aqueous nutrient medium containing a source of assimilable carbohydrate and assimilable nitrogen under aerobic conditions until substantial antibiotic activity is imparted to said medium by the production of tirandamycin and isolating the tirandamycin so produced.
8. 9. A process according to claim 8 in which the isolation comprises filtering the fermentation medium, extracting the filtrate with a water-immiscible solvent for tirandamycin, and recovering tirandamycin from the solvent extract.

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