IE44567B1 - Antibiotic bm123 and production thereof - Google Patents

Abstract

Reductive alkylation with aldehydes R1-CHO or ketones R2-CO-R3 results in alkylation of the antibiotics BM 123 gamma 1, gamma 2 or mixtures thereof at the terminal primary amino group. With ketones there is single N-alkylation, while with aldehydes the primary amino group is substituted once or twice, depending on the amount of reagents, and the proximal secondary amino group on the side chain can likewise be alkylated. The meanings of R1, R2 and R3 are to be found in Claim 1. The resulting derivatives have a potent antibacterial action; they can be used for the treatment of infectious diseases.

Description

This invention relates to new antibacterial agents designated BM123 Antibacterials BM123T^ and BM123)^ are structural isomers and each may be represented by the following structural formulae (I and II respectively): 445S7 (II, ΒΜ123ίζ) 44867 The novel antibacterial agents of the present invention are organic bases and thus are capable of forming acid-addition salts with a variety of organic and inorganic salt-forming reagents. Thus, acid-addition salts, formed by admixture of the antibacterial free base with up to three equivalents of an acid, suitably in a neutral solvent, are formed with such acids as sulfuric, phosphoric, hydrochloric, hydrobromic, sulfamic, citric, maleic, fumaric, tartaric, acetic, benzoic, gluconic, ascorbic, and related acids. The acid-addition salts of the antibacterial agents of the present invention are, in general, crystalline solids relatively soluble in water, methanol and ethanol but are relatively insoluble in non-polar solvents such as diethyl ether, benzene, toluene, and the like. For purposes of this invention, the anti-bacterial free bases are equivalent to their non-toxic acid-addition salts. BM123'/ refers to a mixture in any proportions of BM123/1 and BMI23/2· The new antibacterial agents which'we have designated BM123'/1 and BM123'/, are formed curing the cultivation under controlled conditions of a new strain of an undetermined species of Nocardia. This new antibiotic producing strain was isolated from a garden soil sample collected at Oceola, Iowa, and is maintained in the culture collection of the Lederle Laboratories Division, American Cyanamid Company,Pearl River, New York, as Culture No. BM123. A viable culture of the new microorganism has been deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, - 5 44567 Illinois, and has been added to its permanent collect ion. It is freely available to the public in this depository under its accession number NRRL 5645.

The following is a general description of the microorganism Nocardia sp. NRRL 5646, based on diagnostic characteristics observed. Observations were made of the cultural, physiological, and morphological features of the organism in accordance with the methods detailed by Shirling and Gottlieb,Internat. Journ. of Syst. Bacteriol. 16:313-340 (1966). The chemical composition of the culture was determined by the procedures given by Lechevalier et al., Advan. Appl. Microbiol. 14:47-72 (1971). The underscored descriptive colors and color chip designations are taken from Jacobson et al., Color Harmony Manual, 3rd. edit. (1948), Container Corp, of America, Chicago, Illinois. Descriptive details are recorded in Tables I through V below.

Amount of Growth Moderate on yeast extract, asparagine dextrose, Benedict's, Bennett's, potato dextrose and Weinstein's agars; light on Hickey and Tresner's, tomato paste, oatmeal and pablum agars and a trace of growth on inorganic salts-starch, Kuster's oatflake, Czapek's solution and rice agars.

Aerial Mycelium Aerial mycelium whitish when present; produced only on yeast extract, asparagine dextrose, Benedict's, Bennett's and potato dextrose agars.

Soluble Pigments No soluble pigments produced. <1 4 fit (»7 Reverse Color Colorless to yellowish shades.

Miscellaneous Physiological Reactions No liquefaction of gelatin; nitrates reduced to nitrites in 7 days; melanoid pigments not formed on peptone-iron agar; no peptonization or curd formation in purple milk; NaCl tolerance in yeast extract agar ii but <^7%; optimal growth temperature 32°C. Carbon source utilization, according to the Pridham and Gottlieb method [J. Bacteriol. 56;107-114 (1948)] as follows; Good utilization of glycerol, salicin, d-trehalose and dextrose; fair utilization of i-inositol; and poor to non-utilization of d-fructose, maltose, adonitol, 1-arabinose, lactose, d-mannitol, d-melibiose, d-raffinose, 1-rhamnose, sucrose and d-xylose.

Chemical Composition The organism belongs to cell wall'. ,IV, i.e., contains meso-2,6-diaminopimellc acid and has a type A whole-cell sugar pattern, i.'e., contains arabinose and galactose. Methylated whole cell extracts, when subjected to gas chromatography, showed fatty acid patterns similar to those produced by Nocardia asteroides ATCC 3308.

Micromorphology Aerial mycelium arises from substrate mycelium as sparingly branched moderately long flexous elements that commonly terminate in elongated primitive spirals. The flexous elements are - 7 445G7 irregularly segmented into short elliptical to cylindrical sections (spores?) which disaet ieulate readily. The spiral terminal portions are less conspicuously segmented. Segments gener5 ally range 0.8-1.7 pm x 0.3-0.5 pm, averaging 0.4 pm x 1.2 pm.

Diagnosis The morphological characteristics of Culture No. BM123 are difficult to observe and interpret be10 cause of the poor development of aerial mycelium on most media. Hence, considerable importance is attached, out of necessity, to the chemical analysis in determining the generic relationship of the organism. On the basis of the system proposed by Lechevalier et al., Culture No. BM123 contains meso-2,S-diaminopimelic acid in its whole cells and sugar analysis shows arabinose and galactose to be present. Therefore, the culture belongs to cell wall type IV. A comparison of the gas chromatography pattern of Culture No. BM123 with that of Nocardia aster/ides ATCC 3308 showed the two to be remarkably similar. Other characteristics of Culture No. BM123 that are in keeping with the Nocardia concept, are its fragmenting aerial . growth on some media and the total absence of aerial growth on most media. In view of the lack of adequate criteria for the characterization of Nocardia to the species level, no attempt has been made to make this determination. Therefore, Culture No. BM123 will be considered an undeter- 8 44567 mined species of Nocardia until such is feasible. diagnosis ω co IfU Ό ¢4 cd a o & O ω o *rt •P w •rt k ω p o s JC o H s s a H a u o o Li P CL § E-t >s cd •a j* rrt ·· C o •rt P cd £3 a o c H “Ί- P · Π- (4 3 3 W (/] o GJ c c 63 «-Ι >P · o v> c P a S Π d .rt >» d * w d Π3 » GJ U d k| l I rt »H § 3 t. o 4-Ϊ Π3 k P ia£ C> rt ri o A s 3 s. β; u a it .rt o a &o ω tsO < d u 0 rt rt p o •r» •rl s? ϊ-t CJ d o d c · H • 2 nj 2 o k W k0 § CJ *ϋ ft» a ri >i GJ GJ fcj) GJ GJ d •fi *rt D fi GJ ,fi .rt l Ο rt •H GJ H & P c Pl β CJ d 3d GJ i-K d Λ1 d GJ 0 ‘rt 0 > P fi GJ ΰ GJ Ch fi >4 k it o k -rt· k k O k •rt d ρ O d C O rt 3 k 0 ,° & 3 k Q w υ ¢4 (LOO 01 is υ Ol ft* - , O •P o I w 32 M υ V) (A c .—*» V) GJ »rt o d a GJ UJ k CJ rt > fi · &4 uo •rt »-< e? d rt MO O Ο · k, frt k UJ p <*i Ο P rt 4) aj|m GJ at O »> w p •0 Se* i= *—* H UJ cc fi O 0J ω Έ 3 cd O ό S 51 O u 1- • z JU o tL. GJ Ό) CJ GJ ®ί .0 tu fi fi ’ cr 2 fi . fi o o o 1 o o P £S & & is s ta « □ P O V» , vt UJ QZ O « 0. tn ja Ui 3 •s 3 a rt •rt •rt n p k •K k X »H G) •rt GJ o < £ »» d 32 d 34 § •rt g rt ω § ΙΛ »rt Cr 3 GJ fi •rt GJ 2 •H GJ P P μ P s •d ri •rt b tu a s •3· GJ 32 5 U 0 >♦ s. rt >1 • irt g « e e Grt 3 d j •ri O 3 0 3 •rt p rt · k •rt ri •rt k d P GJ GJ H d +? GJ * rt GJ e* •rl 32 d ο ο •rt 32 O LJ d UJ k ω d A> , k M W J <. α -η 0 k & G) k s O *s rt s E4 B r4 E4 B Z rt , 6 a a> GJ CJ GJ o p P P P Ή GJ u. d Λ d a! d O O k fcj k k k S’ rt GJ •3 •rt »4 GJ GJ *ϋ 0) •d is 5 o Mo o t- a 2 < V) GJ k tn GJ O , fi k· 1 w . •K tn GJ Ϊ fe k • SUM P o d & GJ *d . Aga H d rt to « < a k nJ GJ UJ P fi fi w GJ 32 s X d +: e •rt GJ w s k n u •rt P P .s s P 50 a? ω k GO τ GJ . .60 p V) q} ' , 34 < ,-. d < a fi k (Λ al a ' · • Λ q fi O GJ •rt a GJ fi ΪΗ tc « « H I k o r4 O V) W ·Η W > 4) 0 Η H 0 4) Vl V] o i—i O nJ 4i •H 33 k ω & 4) P (4 k £ 4» •a ia 4) I •>M k 4) & S k Table I continued 4h <3 β O ” k •X nJ a 0 Λ) M *o , W o iu P X «Ο 0 ii +j Λ F CM k Λ 4) ω 43 < W (0 H (k «3 4> ti P 43 nJ nJ U O a 3? O •a $. fk k KJ ω u rt ¢3 - 11 44S67 TABLE II Micromorphology of Nocardia sd NRRL 5646 Medium Aerial Mycelium and/or Sporix'erous Structures 10 Yeast Extract • Agar Aerial mycelium arises from substrate, mycelium as sparingly branched, flexuous elements that commonly terminate in elongated primitive spirals. The flexuous elements are irregularly segmented into short sections (spores?) which disarticulate’readily. The spiral terminal portions are less conspicuously segmented. Segments generally range 0.8-1.7/un x 0.3-0.5 jam, averaging 0.4/an x 1.2 /an.

TABLE III Miscellaneous Physiological Reaction of Nocardia sp. NRRL 5646 I PHYSIOLOGICAL REACTION · | No liquefaction No liquefaction Nitrates reduced, to nitrites Nitrates reduced to nitrites No melanin pigments produced No peptonization or curd formation t- V P ,5 · -4 I1A o O S h «J 0 P d a ft I AMOUNT OF GI2OWTH | Light Good t? Good Good Good Moderate | INCUBATION PERIOD | 7 days w & «0 <4* H & *0 fr- u & Ό 3 fl H 3 0 Λ CO #sr 1 •a CM 7 days · (A & *o fr- s 3 o Ul a Gelatin a •PI 40 «ί $ · Organic Nitrate Broth V ί h •P •rt ft· •3$ · s s MW h O C 0 fc . •rt 1 . · V • § a P M $ •M K a 4) £ P * ¢3 O ~ p « m X JS M 4> rH • P A a u rt rt rt 0 tie >< «; *rt 44867 TABLE IV Carbon Source Utilization Pattern of Nocardia sp. NRRL 5046 Incubation; 10 days Temperature: 32°C.

Carbon Source • Utilization Adonitol 0 1-Arabinose 0 Glycerol 3 d-Fructose 1 i-Inositol 2 Lactose 0 <3-Mannitol 0 Salicin 2 <3-Melibiose 0 tl-Raffinose 0 Shamnose. 0 Maltose 1 Sucrose 0 id-Trehalose 3 di-Xylose 0 Dextrose 3 Negative Control 0 - * 3-Good Utilization 1-Poor Utilization 2-Pair Utilization 0-No Utilization 4567 1 TABLE V Chemii'.il Composition o( Noc;it~d in np. NRRL 5<>4fe Cell Wall Type Major constituents Type IV meso-DAP, arabinose, galactose It is to be understood that for the production of these new antibacterial agents the present invention is not limited to this particular organism or to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes only. In fact, it is desired and intended to include the use of mutants produced from this organism by various means such as exposure to x-radiation, ultraviolet radiation, nitrogen mustard, actinophages, and :· the like. Viable cultures of one such mutant strain has been deposited with the Culture collection Laboratory, Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Illinois, and has been added to its permanent collection under its accession number NRRL 8050. Although the cultural, physiological, and morphological- features of NRRL 8050 is * substantially the same as those of NRRL 5646; NRRL 8050 produces enhanced amounts of BM1231/ during aerobic fermentation. Also, NRRL 8050 varies from the parent NRRL 5646 as follows: (a) slower reduction of nitrates to nitrites; and (b) production of a rosewood tan mycelial pigment on Bennett's and yeast extract agars.

The antibacterial agents were compared in vitro - 15 44567 using a variety of gram positive and, gram negative bacteria as well as M. smegmatis by the standard agar dilution procedure. The results are reported as minimal inhibitory concentrations (meg./ml.) in Table VI. Genta5 micin sulfate was run as a comparison.

HI Hi Hl rf!l E*l β •P ϋ Ρ φ «ρ φ φ Ρ m ΰ «ρ Φ 3 ο ω ιη CM ο Minimal Inhibitory Concentration (meg./ml.

V η (Μ rP V Μ CM tn ιη ιη ιη ιη CM οι CM ιη ιη ι—1 rp CM <Μ ιη •Ρ Η • • • * • » • • • • • ο ο Ο ο ο Ο Ο Ο Ο CM rp Ο Ο ιη ιη ιη tn ιη Η (Μ rp CM CM Η Η CM CM ιη ιη Η Η • t 9 Ο ο Ο Ο Ο Ο Ο ο Ο <Μ ο Ο Ο η m m ιη m ιη ιη ιη CM <Μ CM CM <Μ CM Μ ιη CM Η • • * • • • m ο ο ο ο Η Ο ο ο ο ΙΛ CM tn CM ιη σν ο Γ* ω ω ιο t*» r* ά « s ο vo υ'· § η »Ρ •Ρ U ϋ S Φ η Β XJ Ρ •Η CM CM Η «'· Ο ιη ιΡ ω I ω >1 Η W β Φ •Ρ w r*· ον ω Η I η Η % ζ On Ο W ω Μ* ΙΟ I—1 rp ω Η σν •Ρ ω Ρ< (0 to ω η • • co •Ρ σ> φ β β β Ο 0 0 0 rd ον β φ Φ Φ y 55 55 Ρ Φ φ ρ Ρ Ρ •Η φ ϋ Ο βΐ 3 β β •Η ω Μ X Φ y 0 (0 Φ φ βι •Ρ •Ρ Φ § Ρ ω •Η rP rp s <Ρ «ί φ 10 co ω β X) •Ρ •Ρ β φ β β β φ 0 Ρ «Ρ •Ρ co φ 0 ϋ ϋ ρ rP X) XI Ρ β φ Ρ 0 ϋ υ Φ -OT β β φ ϋ •Ρ φ 0 0 0 0 co co ψί ϋ β -Ρ 0 0 ϋ co ϋ 0 rp □ 0 0 0 (0 β co CO φ □ φ rp rp ιΡ β Η β β Λ 0 Φ Λ Ν >i rP rp Η γΡ Φ •Ρ β 0 X! Χ3 Λ rP Η Η rp β α •Ρ Ρ Λ α α •Η ϋ ‘Ρ •Ρ >1 φ ϋ ?! (0 φ φ 0 ϋ U ϋ Ρ ρ Ρ Ρ •Ρ •Ρ Ρ , Φ Φ Φ Φ ρ •Ρ φ β ω ω (0 Η Η « Η ο W W ττε-; Ό φ 3 . d •ri μ d υ >! Ml >41 «I <1 c •id υ •ri φ Ε ·Ρ (Ο GJ •Ρ W-I C π-i ω 2 ο ω σι η rH § CM ιη ο « ο ιη ο ο ιη ο ο ιη CM Η • · © Ο ιη cm Η ό ο ιη Η ιη ο CM ιη ιη rri ο CM • β * Ο ο ο ιη ιη η ο οι ιη ο ο ο V col tn in m tn in CMI CM rri CM CM CM «ri H CM rril a • • « • o t * • 2 pal o O © o o in l-i O O o ιη *ί· rri Ο Η ςη η υ co σ» ri< u r- in to «ri co & in to Q *a· © kp < rri © o 2 co σ* © u CM O id υ CD u O υ to V) g a EH υ u 0 0 3 o •ri «flj E-i EH c d ri is d < •ri •ri id 0 oi tn tr ΰ w •ri •ri •ri to 3 3 •ri 0 «ri p iri •ri •ri ri • ri rri x; 0 Φ •ri d ri Φ Φ rri ft 0 d Λ <0 Φ to > tr ft d tn tn w •Pl •ri (0 ri ri «ri ω 01 Λ •ri nJ ri 0 3 nJ id id (d 01 Λ «—( £ E > d d rri «ri g 0 r-{ g 0 iri ι—l id m •ri φ ω 01 03 6 E Φ Φ rri •ri ri •ri 3 3 3 p 0 d d H c GJ (0 Φ Φ Φ »3 0 0 Φ Π3 Λ Λ •P •μ •P 3 3 E 6 tr» tr u 0 0 0 Φ Φ tri Tri •ri M ω «ri ri ri ri ω 01 d (d X3 O w ¢4 ft ft . ft ft ft ω w W The antibacterial agent BMI23/ is also active in vivo against a variety of organisms. This new antibacterial is thereby potentially useful as a therapeutic agent in treating bacterial infections in mammals. The new antibacterial can be expected to be usefully employed for treating or controlling bacterial infections by parenteral administration.

The usefulness of the new antibacterial agent is demonstrated by its ability to control systemic lethal infections in mice. The new substance shows high in vivo antibacterial activity in mice against Proteus mirabilis ATCC 4671, Klebsiella pneumoniae AD, and Escherichia coli US311 when aministered by a single subcutaneous dose to groups of Carworth Farms CF-1 mice, weight about 20 gm., infected intraperitoneally with a lethal dose of these bacteria in 10-^·^, 10-^ and 10 3 trypticase soy broth TSP dilutions, respectively, of a 5 hour TSP blood culture.

Table VII, below, illustrates the in vivo antibacterial activity of BM123/” against these three bacteria.

TABLE VII Single Subcutaneous Dose mg./kg.

Infected, non-treated 10 _Controls__ 0.5 Infected, non-treated Controls Single Subcutaneous Dose mg./kg. 0.5 0.25 0.12 Infected, non-treated 25 _Controls Alive/Total Mice Tested, 7 Days After Infection BM123 Proteus Mirabilis 20/20 9/25 2/20 68/70 Mice Died within 1 day after infection /5 8/10 4/10 0/10 /20 Mice died within 2 days after infection Alive/Total Mice Tested, 7 Days After Infection /10 9/10 /10 4/10 3/10 1/5 18/20 Mice died within 3 days after infection Cultivation of Nocardia sp. NRRL 8050 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of the novel antibiotics include as assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolyzate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations, such as potassium, magnesium, calcium, ammonium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles is provided by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanicl impeller. An antifoaming agent, such as Hodag FD82 may be added as needed.

Inoculum Preparation for BM123<)/ Primary shaker flask inoculum of Nocardia sp.

NRRL 8050 is prepared by inoculating 100 milliliters of sterile liquid medium in 500 milliliter flasks with scrapings or washings of spores from an agar slant of the culture. The following medium is ordinarily used: Bacto-tryptone 5 mg.

Yeast extract 5 gm.

Beef extract 3 gm.

Glucose 10 gm.

Water to 1000 ml.

The flasks were incubated at a temperature from -29°C., preferably 28°C. and agitated vigorously on a rotary shaker for 30 to 48 nours. The inocula are then transferred into sterile screw cap culture tubes and stored at below 0°F, This bank of vegetative inoculum is used instead of slant scrapings for inoculation of additional shaker flasks in preparation of this first stage of inoculum.

These first stage flask inoeula are used to seed 12 liter batches of the same media in 20 liter glass fermentors. The inoculum mash is aerated with sterile air while growth is continued for 30 to 48 hours.

The 12 liter batches of second stage inoeula are used to seed tank fermentors containing 300 liters of the following sterile liquid medium to produce the third and final stage of inoculum: Meat solubles 15 gm. Ammonium sulfate 3 gm. Potassium phosphate, dibasic 3 gm. Calcium carbonate 1 gm. Magnesium sulfate heptahydrate 1.5 gm. Glucose 10 gm. Water to 1000 ml.

The glucose is sterilized separately.

The third stage inoculum is aerated at 0.4 to 15 0.8 liters of sterile air per liter of broth per minute, and the fermenting mixture is agitated by an impeller driven at 150-300 revolutions per minute. The temperature is maintained at 25-29°C., usually 28°C. The growth is continued for 48 to 72 hours, at which time the inoculum is used to Seed a 3000 liter tank fermentation .

Tank Fermentation for BMllS1’/ For the production of BM1237/ £n tank fermentors, the following fermentation medium is preferably used Meat solubles 30 gm.

Ammonium sulfate 6 girt. Potassium phosphate, dibasic 6 gm. Calcium carbonate 2 gm. Magnesium sulfate heptahydrate 3 gm. Glucose 20 gm. Water to 1000 ml.

The glucose is sterilized separately.

Each tank is inoculated with 5 to 10% of third - 22 44567 stage inoculum made as described under inoculum preparation. The fermenting mash is maintained at a temperature of 25-28°C., usually 26°C. The mash is aerated with sterile air at a rate of 0.3-0.5 liters of sterile air per liter of mash per minute and agitated by an impeller driven at 70 to 100 revolutions per minute. The fermentation is allowed to continue from 65-90 hours and the mash is harvested.

The invention will be described in greater detail in conjunction with the following specific examples.

Example 1 Inoculum Preparation for BM123'/ A typical medium used to grow the first and second stages of inoculum was prepared according to the following formula: Bacto-tryptone Yeast extract Beef extract Glucose Water to gm. 5 gm. 3 gm. gm. 1000 ml.

Two 500 milliliter flasks each containing 100 milliliters of the above sterile medium were inoculated with 5 milliliters each of a frozen vege**.ive inoculum from Nocardia sp. NRRL 8050. The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28°C. The resulting flask inocurum was transferred to a 5 gallon glass fermentor containing 12 liters of the above sterile medium. The mash was aerated with sterile air while growth was carried out for about 48 hours, after which the contents were used to seed a 100 gallon tank fermentor containing 300 liters of the following sterile liquid medium: Meat solubles 15 gm. Ammonium sulfate 3 gm. Potassium phosphate, dibasic 3 gm. Calcium carbonate 1 gm. Magnesium sulfate heptahydrate 1.5 gm. Glucose 10 gm. Water to 1000 ml.

The glucose is sterilized separately.

The third stage of inoculum mash was aerated with sterile air sparged into the fermentor at 0.4 liters of air per liter of mash per minute. Agitation was supplied by a driven impeller at 240 revolutions per minute.

The mash was maintained at 28eC. and Hodag FD82 was used as a defoaming agent. After 48 hours of growing time the inoculum mash was used to seed a 3000 liter fermentation.

Example 2 Fermentation Employing Nocardia sp. NRRL 8050 for the Production of BM123*/ A fermentation medium was prepared according to the following formula: Meat solubles 30 gm.

Ammonium sulfate 6 gm.

Potassium phosphate, dibasic 6 gm.

Calcium carbonate 2 gm.

Magnesium sulfate heptahydrate 3 gm.

Glucose 20 gm.

Water to 1000 ml.

The glucose is sterilized separately.

The fermentation medium was sterilized at 120°C. 25 with steam at 20 pounds pressure for 60 minutes. The pH of the medium after sterilization was 6.9. Three thousand liters of sterile medium in a 4000 liter tank fermentor was inoculated with 300 liters of inoculum such as described in Example 1, and the fermentation was carried out at 26°C. using Hodag'- FD82 as a defoaming agent. Aeration 4456? was supplied at the rate of 0.35 liter of sterile air per liter of mash per minute. The mash was agitated by an impeller driven at 70-72 revolutions per minute. At the end of 67 hours of fermentation time the mash was harvested.

Example 3 isolation of BM1231/ A 3000 liter portion of fermentation mash prepared as described in Example 2, pH 4.3, was adjusted to pH 7.0 with sodium hydroxide and filtered using 5% diatomaceous earth as a filter aid. The cake was washed with about 100 liters of water and discarded. The combined filtrate and wash was pumped upward through three parallel 8 1/4 x 48 stainless steel columns each containing 15 liters of CM Sephadex c-25 [Na+] resin. The charged columns were washed with a total of about 390 liters of water and then developed with 200 liters of 1% aqueous sodium chloride followed by 560 liters of 5% aqueous sodium chloride. The 5% aqueous sodium chloride eluate was clarified by filtration through diatomaceous earth and the clarified filtrate passed through a 9 x 60 glass column containing 25 liters of granular Darco activated carbon (20-40 mesh). The charged column was washed with 120 liters of water and then developed with 120 liters of 15% aqueous methanol followed by 340 liters of 50% aqueous methanol and then 120 liters of 50% aqueous acetone. The 50% aqueous methanol eluate was adjusted from pH 4.65 to 6.0 with Amberlite® IR-45 (OH~) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to about 6.3 liters and then lyo- 25 phylized to give 213 grains Of material consisting primarily of BM123y,. The 50% aqueous acetone eluate was adjusted from pH 4.0 to 6.0 with Amberlite® IR-45 (OH-) resin. The resin was removed by filtration and the £il5 trate was concentrated in vacuo to about 1.5 liters and then lyophylized to give 56 grams of impure BM1231/.

Example 4 Further Purification of BMI23,Z A slurry of CM Sephadex C-25 [NH^J in 2% aqueous ammonium chloride was poured into a 2.6 centimeter diameter glass column to a resin height of approximately 62 centimeters. The excess 2% aqueous ammonium chloride was drained away and a 5.0 gram sample of ΒΜΪ23 prepared as described in Example 3 was dissolved in about milliliters of 2% aqueous ammonium chloride and applied to the column. The column was then eluted with a gradient between 6 liters each and 2% and 4% aqueous ammonium chloride. Fractions of about 75 milliliters each were collected automatically every 15 minutes. Antibiotic BM123 was located by monitoring the column effluent in the ultraviolet and by bioautography of dipped paper disks on large agar plates seeded with Klebsiella pneumoniae strain AD. The majority of BM123/ was.located between fractions 71-107 inclusive.

One hundred thirty milliliters of granular Darco' activated carbon (20-40 mesh) was suspended in water, transferred to a glass column, allowed to settle and the excess water was allowed to drain away. Fractions 84-96 inclusive from the above CM Sephadex chroma30 tography were combined and passed through the granular carbon column. The charged column was washed with 600 milliliters of water and then developed with 1 liter of 20% aqueous methanol followed by 1 liter of 50% aqueous acetone. These eluates, both of which contained HMU'l'X*, were concentrated to aqueous phases in vacuo and lyophilized to give a total of 886 milligrams of BM1239^ as the hydrochloride salt. A microanalytioal sample was obtained by subjecting the above material to a repeat of the above process.

Antibiotic BM1231/ does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200eC. Microanalysis of a sample equilibrated for 24 hours in a 72°F. atmosphere containing 23% relative humidity gave C, 39.44%; H, 6.10%; N, 16.19%; Cl(ionic), 11.54%; loss on drying, 8.19%. In water BM123X*Z gave a U.V. absorption maximum at 286 nm with 1% Elcm “ 250· position of this maximum did not change ° with pH. BM123 had a specific rotation of (a]D = +71° (C ·» 0.97 in water).

Antibiotic BM123^ exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths; 770, 830, 870, 930, 980, 1035, 1105, 1175, 1225, 1300, 1340, 1370, 1460, 1510, 1555, 1605, 1660, 1740, 2950 and 3350 cm-1. A standard infrared absorption spectrum of BM123 prepared in a KBr pellet is shown in Figure 1 of the accompanying drawings.

Example 5 Isolation of BM1239^ A slurry of CM Sephadex1 C-25 [Na+] in 2% aque- 27 44S67 ous sodium chloride was poured into a centimeter diameter glass column to a resin height ot approximately 70 centimeters. The excess 2% aqueous sodium chloride was drained away and 4.11 gram of a sample containing primarily 31-11237^ along with some BM123’/2 and other impurities, prepared as described in Example 3, was dissolved in about 10 milliliters of 2% aqueous sodium chloride and applied to the column. The column was then eluted with a gradient between 4 liters each of 2% and 4% aqueous sodium chloride. Fractions of about 75 milliliters each were collected automatically every 15 minutes. Antibiotic BM1231/ was located by monitoring the column effluent in the ultraviolet and by bioautography of dipped paper disks on large agar plates seeded with Klebsiella pneumoniae strain AD. The majority of BM123/ was located between fractions 64-90 inclusive; the initial fractions (64-80) contained a mixture of BM123T^ and BM123j^ wereas the later fractions (81-90) contained essentially pure BM123/^.

One hundred milliliters of granular Darco- activated carbon (20-40 mesh) was suspended in water, transferred to a glass column, allowed to settle and the excess water was allowed to drain away. Fractions 81-90 inclusive from the above CM Sephadex chromatography were combined and passed through the granular carbon column.

The charged column was washed with 50u mxxxiliters of water and then developed with 500 milliliters of 10% aqueous methanol followed by 1 liter of 50% aqueous methanol. The 50% aqueous methanol eluate, which contained the majority of BM123)^, was adjusted from pH 5.9 to 6.0 4456? with Amberlite® IR-45 (OH-1) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to an aqueous phase and lyophilized to give 294 milligrams of white amorphous BM1231^ as the hydrochloride salt.

Antibiotic BM123/1 does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200°C. Microanalysis of a sample equilibrated for 24 hours in a 70°F. atmosphere containing 60% relative humidity gave C, 37.84%; H, 5.73%; N, 15.58%; Cl(ionic), 10.01%; loss on drying 10.45%. In methanol BM1239^ gave a U.V. absorption maximum at 286 nm with Elcm = 225. The position of this maximum did not change with pH. BM123?y had a specific rotation of +55° (C = 0.803 in water).

Antibiotic BM123)^ exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 770, 830, 870, 930, 980, 1045, 1080, 1110, 1125, 1175, 1225, 1305, 1345, 1380, 1465, 1515, 1560, 1605, 1660, 1730, 2950 and 3350 cm-1. A standard infrared absorption spectrum of BMl23i^ prepared in a KBr pellet is shown in Figure 2 of the accompanying drawings. A standard proton magnetic resonance spectrum of BM123/j determined on a DjO solution in a 100 megacycle spectrometer is shown in Figure 4 of the accompanying drawings.

Example 6 Isolation of ΒΜ123?ζ A 25 gram sample containing primarily BM123^, prepared as described in Example 3, was dissolved in about 120 liull'.liteis oi 2s aqueous sodium ehlottde and applied to a column containing 1800 ml. of CM Sephadex^0 C-25 [Na+] in 2¾ aqueous sodium chloride. The column was then eluted with a gradient between 20 liters each of 2% and 4% aqueous sodium chloride. The initial 12 liters of eluate was collected in a large bottle and discarded. Thereafter fractions of about 800 milliliters each were collected automatically every 40 minutes. Antibiotic BM123'/ was located by monitoring the column fractions in the ultraviolet. The majority of BM123J^ was located between fractions 7-18 inclusive; the initial fractions (7-15) contained essentially pure BM123)^ and the later fractions (16-18) contained a mixture of BM123'/^ and BMI23/, Six hundred milliliters of granular Darco activated carbon (20-40 mesh) was suspended in water, transferred to a glass column, allowed to settle and the excess water was allowed to drain away. Fractions 7-15 inclusive from the above CM Sephadex chromatography were combined and passed through the granular carbon column. The charged column was washed with 3 liters of water and then developed with 3 liters of 10% aqueous methanol followed by 6 liters of 50% aqueous methanol. The 10% aqueous methanol eluate was adjusted from pH 5.8 to 6.0 with Amberlite® IR-45 (OH-) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to an aqueous phase and lyophilized to give 595 milligrams of white amorphous BM123?^ as the hydrochloride salt. The 50% aqueous methanol eluate was adjusted from pH 4.6 to 6.1 with Amberlite® IR-45 (OH-) resin. 44S67 The resin was removed by filtration and the filtrate was concentrated in vacuo to an aqueous phase and lyophilized to give 3.645 grains of slightly less pure white amorphous BMI23/2 as the hydrochloride salt.

Antibiotic BM123)2 does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200°C. Microanalysis of a sample equilibrated for 24 hours in a 70°F. atmosphere containing 60% relative humidity gave C, 36.14%; H, 5.67%; N, 15.1%; Cl(ionic) 11.11%; loss on drying 10.87%. In methanol BM123i>^ gave a U.V. absorption maximum at 286 nm with = 220. The position of this maximum does not change with pH. BM123?2 had a specific rotation of +60° (C = 0.851 in water).

Antibiotic BM123?2 exhibited characteristic absorption in the infrared region of the spectrum at the following wavelengths: 770, 830, 870, 950, 980, 1035, 1110, 1175, 1225, 1285, 1345, 1380, 1470, 1515, 1560, 1605, 1660, 1755, 2950 and 3350 cm-1. A standard infrared absorption spectrum of BM123/^> prepared in a KBr pellet is shown in Figure 3 of the accompanying drawings.

A standard proton magnetic resonance spectrum of BM123^ determined, on a D2O solution in a 100 megacycle spectrometer is shown in Figure 5 of the accompanying drawings.

Example 7 Paper Partition and Thin Layer Chromatography of BM123^/>' The antibacterial agents can be distinguished by paper chromatography. For this purpose Whatman No. 1 strips were spotted with a water or methanol solution of the substances and equilibrated for 1 to 2 hours in the , presence of both upper and lower phases. The strips were 1 developed overnight with the lower (organic) phase obtained from mixing 90% phenol:m-cresol:acetic acid:pyridine:water (100:25:4:4:75 by volume). The developed strips were removed from the chromatographic chamber, air dried for 1 to 2 hours, washed with ether to remove residual phenol and bioautographed on large agar plates seeded with Klebsiella pneumoniae strain AD. BM123/^ Rf 0.85.

Claims (1)

1. A compound of antibacterial BM1 4 4 ft tt ? 23/ of the formula: and the pharmacologically acceptable acid-addition salts thereof. and the pharmacologically acceptable acid-addition salts thereof.