April 9, 1968 D. LEFENMINE ETAL NEUTRAMYCIN AND A METHOD FOR PRODUCING 3,377,242. BY US ING STREPTOMYCES RIMO SUS Filed Oct. 9, 1964 2. Sheets-Sheet l

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INVENTORS DONALD W. LEFEMNE MURRAY DANN SAMUELOWEN THOMAS 46/ VOE April 9, 1968 D. LEFEMINE ETAL 3,377,242 NEUTRAMYCIN AND A METHOD FOR PRODUCING BY USING STREPTOMYCES RIMO SUS Filed Oct. 9, 1964 2 Sheets-Sheet 2

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l l s ???? s INVENTORS - ALSNBN AV2 DONALD FEM NE MURRAY DAN N N SAMUELOWEN THOMAS STANLEY EU O E O R ? 2 - A770A7WAY 3,377,242 United States Patent Office Patented Apr. 9, 1968

2 positive bacteria. The effects of the new antibiotic on NEUTRAMYCIN- AND3,377,242 A METHOD FOR PRODUC specific microorganisms together with the chemical and ING BY USING STREPTOMYCES RIMOSUS physical properties of the antibiotic differentiate it from Donald Lefemine, West Nyack, and Murray Dann, Samuel previously described antibiotics. Owen Thomas, and Stanley Eugene De Voe, Pearl 5 The new antibiotic which we have termed neutramycin River, N.Y., assignors to American Cyanamid Com (formerly designated AE-705-W) is formed during the pany, Stamford, Conn., a corporation of Maine cultivation under controlled conditions of a new strain Continuation-in-part of abandoned application Ser. No. of Streptomyces rimosus which was isolated from a soil 303,312, Aug. 20, 1963. This application Oct. 9, 1964, sample collected in Berhampore, Orissa, India. The fol Ser. No. 404,559 0. lowing is a general description of the organism based 7 Claims. (C. 167-65) on the diagnostic characteristics observed. The under scored descriptive colors are those of Ridgway (1912) "Color Standards and Color Nomenclature.” ABSTRACT OF THE DESCLOSURE Amount of growth.-Moderate to good on most media; The invention relates to a new antibiotic of unknown colony surfaces generally convoluted or cracked. configuration produced by fermentation of a new strain Aerial mycelium and/or spore color.-Aerial mycelium of Streptomyces rimosus. The novel antibiotic, designated and spores white on all media supporting sporulation. neutramycin, is effective in inhibiting the growth of gram Soluble pigment.-Moderate to abundant, in various positive bacteria. The chemical, physical and biological shades of brown on most media. properties of neutramycin are described in detail herein 20 Reverse color-In various shades of dark brown on after. most media. Miscellaneous physiological reactions.--Reduces nitrate This application is a continuation-in-part of our co to nitrate; liquefies gelatin; and is achromogenic on pep pending application Ser. No. 303,312, filed Aug. 20, 1963, tone-iron agar (Difico). Utilizes dextrose, d-xylose, 1-ara now abandoned, which application, in turn, is a continua binose and d-melezitose well, but shows poor utilization tion-in-part of our copending application Ser. No. of adonitol, dextran, d-fructose, i-inositol, lactose, d-man 228,157, filed Oct. 3, 1962, now abandoned. nitol, d-raffinose, 1-rhamnose, sucrose, and d-trehalose, This invention relates to a new antibiotic and to its and non-utilization of salicin and di-melibiose. production by fermentation, to methods for its recovery Morphology.--Sporophores arising as long open spirals and concentration from crude solutions, and to processes 30 from aerial mycelium; commonly branched. Spores for its purification. Smooth-walled, globose to elliptical, 0.6-0.8 x 0.7-1.0a. The present invention includes within its scope the The cultural, morphological, and physiological charac antibiotic in dilute forms, as crude concentrates, and teristics of the new strain of Streptomyces rinosus are in pure crystalline forms. These novel products are active set forth in the following tables. The descriptive colors against a variety of microorganisms including Gram ” taken from Ridgway are set in bold type. TABLE I-CULTURAL CHIARACTERISTICS OF NEW STRAIN OF S. RIMO SUS Incubation: 14 days; Temperature: 28°C. Medium AmountGrowth of Aerial Mucelium and Spores Soluble Pigment Reverse Color Remarks Czapek's Solution Agar------Good------Aerial mycelium white, scanty. Hessian Brown; Hay's Maroon.-- Colonies raised, wrinkled. Tomato Paste Agar------do------AerialSporulation mycelium very and poor. spores, abundant. Abundantexudate. colorless to pinkish white, scanty. Claret Brown; Maroon.------Colonies raised, wrinkled. Bennett's Agar------do------AerialSporulation mycelium poor and spores Auburn;abundant. Auburn------AbundantDo. colorless exudate. white, sparse. Sporulation moderate. poor. Asparagine Dextrose Agar------do------do------Claret Brown; Claret Brown---- Colonies raised, wrinkled. abundant. Abundant colorless to yellow Hickey and Tresner's Agar------do------Aerial mycelium and en masse Tawny; Tawny------Coloniesish exudate. wrinkled and furrowed good.???? white. Sporulation ??? ?era?e. Abundant coloriess exudate. Carvajal’s Oatmeal Agar------Moderate.----- Aerial mycelium and spores None.------Pinkish white. Sporulation scanty. Cinnamon. Potato Dextrose Agar------do------Aerial mycelium and spores Claret Brown; Maroon. ------Colonies raised, wrinkled. Tomato Paste Oatmeal Agar. Good------Aerialwhitish. mycelium Sporulation and en light.masse. Brownish;abundant. light Chestnut- ? ColoniesModerate raised, brownish wrinkled exudate. and sporulation white. Sporula- Brown. cracked. s tion moderate. Yeast Extract Agar------. do------Aerial Laycelium and spores Auburn; Auburn.------Colonies raised, wrinkled. whitish. Sporulation light. moderate. ??????? colorless to yellow (norganic Salts Starch Agar ------do------Aerial cycelium and en masse None------Cinnamon- Coloniesish exudate. raised, wrinkled and spores white. Sporulation Buff. cracked. moderate. Oat Flake Agar------Moderate.----- Aerial mycelium and spores Hazel; Inoderate. Eazel.------?to light. Sporulation moderate TABLE II.-MORPHOLOGICAL CEARACTERISTICS Medium. Aerial Mycelium and Sporiferous Structure Spore Shape Spore Size Inorganic Salts Starch Agar--- Sporophores arising as long open spirals from aerial Iny celium. Sporophores commonly branched. Globose to elliptical; smooth-walled 0.6-0.8i x 0.7-1.0,

3,377,242 7 8 The flasks are placed on a reciprocating shaker and agi acetic anhydride plus 7.2 ml. of dry pyridine, and kept at tated vigorously for 48 hours, at 28° C. The flask inocula room temperature for 20 hours. The mixture is then con are transferred to 9 liter bottles which contain 6 liters of centrated in vacuo to a yellow oil. The oil is dissolved the above liquid medium. These bottles are aerated for in chloroform and added to an aluminum oxide column 24 hours to encourage further growth. At the end of this (30 grams aluminum oxide) and developed with chloro time the 9 liter bottles are used to seed fermentor tanks. form. The acetate effluent is collected and concentrated to an oily residue. The oil containing acetate is then crystal Example 2 lized three times using acetone and petroleum ether (30 75 C.), yielding 250 milligrams of the white, needle-like A fermentation medium is prepared according to the crystalline acetate. The sample was dried for 4 hours at following formula: O 60° C. over POs in high vacuum prior to analysis. Grams Elemental analysis: C, 58.60; H, 7.88; O, (direct) Bactopeptone ------5 32.40. Melting point 123-125 C. Optical rotation Glucose ------10 ox25-9.3 (c., 0.862 in ethanol). Ultraviolet maxima MolaSSeS ------20 15 at 216 mu Calcium carbonate ------Water to 1000 milliliters. (EE-300) The fermentation medium is sterilized at 120° C. with in methanol, shoulder at 240 mu. steam at 15 pounds pressure for 60 minutes. The pH of (E = 168) the medium before and after sterilization is between 7.0 20 in methanol. and 7.8. 1500 liters of the sterile medium in 1000 gallon We claim: fermentors are inoculated with 12 liters of the bottle 1. Neutramycin, a substance effective in inhibiting the inoculum described above and the fermentation is car growth of gram-positive bacteria and being characterized ried out at 28° C. for 90 hours. The medium is agitated by the following properties: by an impeller operating at 100 revolutions per minute. (a) a melting point of 222-223 C., At the end of the fermentation the mash is assayed. (b) an optical rotation a D25=-34.5 (c.- 0.987% in ethanol), Example 3.-Isolation (c) containing the elements carbon, hydrogen, and Sixteen hundred liters of fermented mash, to which oxygen in substantially the following percentages diatomaceous earth is added in the proportion 2% 30 by weight when dried under normal conditions: weight/volume, is filtered. The filter pad is washed with Ao volume of water and the pad then discarded. The Carbon ------58.63 pooled water wash and filtrate (volume 1600 liters) is Hydrogen ------8.09 then adjusted to pH 7.0, saturated with ethyl acetate and Oxygen (direct) ------32.42 extracted in a three-stage countercurrent extractor with (d) having the following elemental analysis when ethyl acetate using 0.75 volume of solvent per volume dried for one week over POs at 100 C. under re of aqueous phase. The pooled ethyl acetate extract duced pressure: (volume 1350 liters) is concentrated in vacuo to ap Carbon ------59.17 proximately 600 of the volume. The ethyl acetate con 40 Hydrogen ------8.22 centrate is then poured onto an aluminum oxide (chro matographic grade) column. The adsorption column is (e) a molecular weight determination of 513-686, then developed with ethyl acetate to elute the desired (f) characteristic absorption in the ultraviolet region antibiotic activity. of the spectrum at 216 mu, The effluents from the column are pooled using the (E3=340) following criteria: (1) In vitro data-Agar diffusion plate method of in methanol and a shoulder at 240 mp. assay using Bacillus cereus, Corynebacterium xerosis and Staphylococcus aureus as the test organisms. (E??=205) (2) Paper chromatography.-Using Whatman No. 1 50 in methanol, paper and a solvent system consisting of n-amyl acetate, (g) characteristic absorption in the infrared region as dibutyl ether, glacial acetic acid and water. shown in FIGURE 1 and, (3) Microscopic observation of concentrated aliquots (h) a proton resonance spectrum as shown in FIG of effluents. URE 2. V- Based upon these criteria similar fractions are pooled 2. The acetyl derivative of the substance defined in and concentrated in vacuo to a dark, brownish-yellow claim 1. oily residue. The concentrate is dissolved in a minimal 3. A process for the production of neutramycin which volume of ethyl acetate and then petroleum ether (30 comprises cultivating a neutramycin producing strain of 75 C.) is added slowly, precipitating the product as an Streptomyces rimosus NIRRIL No. 3016 in an aqueous off-white crystal. The crystals are collected via filtration 60 nutrient medium containing assimilable sources of car and washed using petroleum ether (30-75 C.). Yield bohydrate, nitrogen, and inorganic salts under submerged 12.5 grams. The product is recrystallized using ethyl ace aeroboic conditions until substantially antibacterial ac tate plus petroleum ether (30-75° C.) and dried in vacuo tivity due to neutramycin is imparted to said medium. over phosphorous pentoxide at 60° C. Alternative crystal 4. A process for the production of neutramycin which lization solvents are ethyl alcohol plus petroleum ether comprises cultivating Streptomyces rimosus NRRL No. (30-75° C.), chloroform plus petroleum ether (30-75° 3016 in an aqueous nutrient medium containing as C.), methyl alcohol plus diethyl ether, hot water, hot semilable sources of carbohydrate, nitrogen and inor ethyl alcohol, or hot ethyl acetate. The chemical analysis ganic salts under submerged aerobic conditions for a of this product and its other chemical, physical, and bio period of from 24 to 240 hours, and at a temperature logical properties have already been described. 70 of from 20 to 35 C., and recovering the antibiotic so produced. EXAMPLE 4-Preparation of acetyl derivative of the 5. A process as in claim 4 in which the antibiotic is new antibiotic recovered from the fermentation broth by filtering the Five hundred milligrams of the product prepared ac broth, extracting the antibiotic activity from the beer cording to Example 3 is added to 7.2 milliliter of dry 75 filtrate with a solvent, passing the antibiotic activity 3,377,242 9 10 through a chromatographic column, and separating the References Cited antibiotic activity therefrom. D PATEN 6. A composition of matter consisting of the sub- UNITED STATES TENTS stance according to claim 1 in the crystalline state. 2,516,080 7/1950 Sobin et al. ------167-65 7. A therapeutic composition of matter containing as 5 an essential antibacterial agent a compound as character- ALBERT T. MYERS, Primary Examiner. ized in claim 1, and a pharmaceutical carrier. D. M. STEPHENS, Assistant Examiner.