CA1087537A - Antibiotic bm123 and production thereof - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
This disclosure describes 5 new antibacterial agents designated BM123.alpha., BM123.beta.1, BM123.beta.2, BM123.gamma.1, and BM123.gamma.2 produced in a microbiological fermentation under controlled conditions using a new strain of an undetermined species of Nocardia and mutants thereof. The new antibacterial agents are active against a variety of microorganisms and thus are useful in inhibiting the growth of such bacteria wherever the may be found.

Description

10~7S137 BRIEF SUMMP~RY OF TH~ INVI~.NTION ;~
~: rrhis inven-tion relates to 5 new antibacterial ayents designated BM123a, BM123~1, BM123~21, BMl23Y~l, and BMI.23y'2;
to their production by fermen~ation, to methods for their re- :
covery and concentration from crude solutions, and to pro~
;,` 5 cesses for their purifica-tion. The present invention in- .~
cludes within its scope the antlbacterial agents in dilute ~;
forms, as crude concentrates, and in pure crysta].line forms.
. The effec-ts of the new antibacteria:L agents on speciEic : :~microorganisms, together with their chemical and physical .
~ 10 properties, differentia-te them ~rom previously described ; antibacterial agents.
Antibacterial BM123a In.ly bc repres~ntecl by thei fol- :.
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Antibacterial BM123~1 may be represented by the 1 following structural formula (II)~
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Antibacterials BM123~1 and BM123~2 are structural ~ ;
j isomers and each may be represen~ed by the following structural formulae (III and IV respectively~

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~ 7S37 ,~ The novel an-tibacterial agents of the present in-s vention are oryanic bases and thus are capable of forming acid-addition salts with a variety of organic and inorganic s salt-forming reagents. Thus, acid-addition salts, formed by s 5 admixture of the antibacterial free base with up to three ~ equivalents oE an acid, suitably in a neutral solvent, are ,~ formed with such acids as sulfuric, phosphoric, hydrochloric, hydrobromic, sulfamic, ci-tric, 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 in-soluble in non-polar organic solvents such as diethyl et~er, benzene, toluene, and -the lilce. For purposes oE this inventioll, the antibacterial free bases are equivalent to their non-toxic acid-addition salts. Hereinafter BM123~ refers to a mixture in any proportions of BM123~1 and BM123~2, and BM123r refers to a mixture in any proportions of ~M123 ~ and BM123~2.
DETAILED DESCRIPTION OF THE INVENTION ;
The new antibacterial agents which we have desig~
nated B~1123a, BM123fll, B~123~2, BM123~1, and BM123~2 are ;
formed during the cultivation under controlled conditions oE
a new strain o~ 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, N.Y. 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, Unitecl States Department of Agriculture, Peoria, Illinois, and has been added to its permanent collec-tion. It is freely available to the public in this depository under its accession number NRRL 5646.

', The following is a general descrip-tion of the microorganism Mocardia sp. NRRL 5646, based on diagnostic ;~
characteristics observed. Observa-tions were made of the cul~
tural, physiological, and morphological features of the or~
ganism in accordance with the me-thods 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 color~
and color chip designations are taken from Jacobson et al., Color Harmony Manual, 3 rd. edit. (1948), Container Corp. of ;~
America, Chicago, Illinois. Descriptive details are recorded in Tables I through V below.
Amount oE Grow-th Moderate on yeast extract, asparagine dextrose, Benedict's, Bennett's, potato dextrose and Weinsteln'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.
~erial 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. ~;
Reverse Color Colorless to yellowish shades.
~ Miscellaneous Physiological Reactions 30 No liquefaction of gelatin; nitrates reduced to nitrites ;~
in 7 days; melanoid pigments not formed on peptone--iron agar; no peptonization or curd formatlon in purple milk; NaCl tolerance in yeast extract agar g _ . .

3L~1!37537 .

4~ but C76; optimal growth temperature 32C.
Carbon source utilization, according -to -the Pridham and Gottlieb method [J. Bacteriol. 56:107-114 (19483] ' as follows: Good utilization of glycerol, salicin, d-trehalose and dextrose; fair utilization of -inositol; and poor to non-utilization of d-fructose, maltose, adonitol, l-arabinose, lac-tose, d-mannitol, d-melibiose, d-raffinose, l-rhamnose, sucrose and d-xylose.
Chemical Composition The organism belongs to cell ~all type IV, i.e., con-tains meso-2,6-diaminopimelic 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 flexuous elements that commonly terminate :in elonga-ted primitive spirals.
The flexuous elements are irregularly segmented into short elliptical to cyclindrical sections (spores?) -;
which disarticulate readily. The spiral terminal portions are less conspicuously segmented. Segments generally range 0.8-1.7 ~m x 0.3-0.5 ~m, averaging 0.4 ym x 1.2 ~m.
Diagnosis The morphological characteristics of Culture No. BM123 - `~
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are difficult to observe and interpret because of the poor development of aerial mycelium on most media.
~ence, considerable importance is attached, out of necessity, to the chemical analysis in determining the ; .

~ 753,7 generic relationship of the organism. On the basis of the sys-tem proposed by Leche~alier et aI., Culture No. BM123 contains meso-2,6-diaminopimelic acid in ~ i :
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 asteroides 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 grow~h on most media. In view of the lack of adequate criteria for the characteristization of Nocardia to the species level, no attempt has been made to make this deter-mination. ~here~ore, Culture No. BM123 w:Lll be con- ;
sidered an undetermined species of ~ocardia until ;~
such a diagnosis is feas.ible~

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TABLE II
Micromorphology of Nocardia sp NRRL 5646 , ,~ .,,`'~
. Aerial Mycelium and/or Sporiferous ;~
Medium Structures _ ~ .~
Yeast Extract Aerial mycelium arises from substrate Agar mycelium as sparingly branched, flexuous elements that commonly term-inate 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,um x 0.3-0.5,um~ averaging 0.4Jum x 1.2 ~m.

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10875i~7 TABLE IV
Carbon Source Utilization Pattern of Nocardia s~. N~RL~5646~ r,.., ~

Incubation: 10 daysTemDera ture: 32 C . ~ " ~ '`i'.' ` ;

_ I _ _ Carbon SourceUtilization . Adonitol 0 l-Arabinose 0 : :~
Glycerol . 3 d-Fructose . . ~ 1 . i-Inositol 2 : :~
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2-~air UtLlization 0-No Utilization.

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TABLE V
Chemical Composition of Nocardia ~. NRRL 5646 Cell Wall Type Major Constituents _ Type IV meso-DAP, arabinose, galactose . . __ .. .___ " ~ ~:
It is to be understood that for the produc-tion of ~ i these new antibacterial agents the present invention is no-t - ~
limited to this particular organism or to organisms fully ~ ~ -answering the above growth and microscopic characterist:ics ~ ~-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, actino-phages, and the like. Viable cultures oE -two such mutant `
strains have been deposited with the Culture Collection Lab-oratory, Northern Utilization Research and Development Division, United States Department of Agriculture, Peoria, Illinois, and have been added to its pçrmanent collection under their accession numbers NRRL 8050 and NRRL 8103. Although the cultur-al, physiological, and morphological features of NRRL 8050 and NRRL 8103 are substantially the same as those of NRRL 5646;
NRRL 8103produces enhanced amounts of BM123~ during aerobic fermentation whereas NRRL 8050 produces enhanced amounts of BM123~ during aerobic fermentation. Also, NRRL 8050 varies from :, the parent NRRL 5646 as follows~
(a) slower reduction of nitrates to nitrites; and -. .
(b3 production of a rosewood tan mycelial pigment on Bennett!s and yeast extract agars.
Preliminary isolation, thin layer chromatography, -~
and paper chromatography experiments have shown that five anti-biotics are produced during the aerobic fermentation of - 1? ~

Nocardia sp. NRRL 5646 as heretofore designated. Nutrient media studies resulted in two types of mashes: an alpha type mash which produces primarily BM123~; and a gamma type mash which produces primarily BM123~ and BM123 ~ along with lesser amounts of BM123~, BM123~1 and BM123~2.
The antibacterial agents were compared ln vitro using a varie-ty of gram positive and gram negative bac-teria as well as M. smegmatis by the standard agar dilution pro- ;
cedure. The results are reported as minimal inhibitory con~
centrations (mcg./ml.) in Table VI. Gentamicin sulfate was run as a comparison.

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37S3rd The antibacterial ayents BM123a, ~M123~, and sM123~ are also active ln vivo against a variety of organisms.
These new antibac-terials are thereby potentially useful as therapeutic agents in treating bacterial infections in mammals.
These new antibac-terials can be expected to be usefully employed ~or treating or controlling bacterial infections by parenteral administration.
The usefulness of these new antibacterial agents is demonstrated by their ability to control systemic lethal infections in mice. These new substances show high ln vlvo antibac-terial activity in mice against Proteus mirabilis ATCC 4671, Klebsiella pneumoniae AD, and Escherichia co:Li US311 when administered by a sinyle subcutaneous dose to groups oE Carworth Farms CF-l mice, weight about 20 ym., inEected intraperitoneally with a letha:L dose of -these bacteria in 10 10 4 and 10 3 trypticase soy broth TSP dilutions, respectively, of a 5 hour TSP blood culture.
Table VII, below, illustrates the in vivo anti-bacterial activity of BMl23a~ BM123~, and BM123~ against these three bacteria.

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TABL~ VII
.. _ Single Subcutaneous Alive/To-tal Mice Tes-ted, 7 Days `
After Infection Dose mg./kg.
BM123a BM123~ BM123~
_ __ _ _ _ Proteus Mirabilis ~ ` :
512 5/5 ; ~, 16 5/5 ~; ;

2 . 0/5 9/25 1 . 2/20 `

15 0.5 0/25 : `:
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Infected, nontreated 68/70 Mice Died within 1 day controls after infection _ Klebsiella pneumoniae ;~

512 ` 4/5 _ ;
- 128 0/5.`
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32 0/57/10 ~ .
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8 .8/10 4 0/10 5/5 ~ :

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. ._ ., Infected, nontreated 20/20 Mice died within 2 days 30 . controls after infection ::

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~37S37 ., ;

Table VII Continued .

Single Subcutaneous Alive/Total Mice Tes-ted, 7 Days ;
After Infection Dose mg./kg. 1-~M123al ~IM123~ I BM123 ~ , _ Escherichia coli ~
512 5/5 ~ -l2a 1/5 9/.~0 4 5/10 10/10 ;~

0.5 0/5 ~/10 0.25 3/10 0.12 _ _ 1/5 1 `

18/20 Mice died within 3 days Infected, nontreated aEter infection controls Fermentation Process Selected to Produce Primarily BM123a Cultivation oE Nocardia sp. ~RRL 8103 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of this novel antibacterial agent include an assimilable source of carbon such as starch, sugar, ~ -25 molasses, glyceroI, etc.; an assimilable source of nitrogen -- .
such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc.; and inorganic anions and cations, such as potassium, sodium, ammonium, calcium, sulfate, carbonate, phosphate, chloride, etc. Trace elements such as 30 boron, molybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles i5 provided by forcing sterile air through or onto the surface of the fermenting medium. Fur;ther agitation in tanks is pro-.. . . , , , - - ,- :

vided by a mechanical impeller. An an-tifoaming agent such ~' as lard oil may be added as needed.
~.
Inoculum Preparation for BMl23a -Shaker flask inoculum of Nocardia sp. NRRL 8103 is prepared by inoculating 100 ml~. of sterile liquid medium . ,; .
in 500 ml. flasks with scrapings or washings of spores from an agar slant of -the culture. The following is an example ~,,i , , of a suitable medium~
Beef extract......................... 3.0 gm.
Bacto-tryptone....................... 5.0 gm. ~ ' -Yeast extract........................ 5.0 gm. ' ,';
Starch.............................. 24.0 gm.
Dextrose............................. 1.0 gm.
Water to .......................... 1,000 ml. ', ~
Adjust medium pH to 7.0 with NaOH ~,' ' The flasks are incubated at a temperature from 25-29C., preferably 28C., and agLtatecl vigorously on a'`,~
rotary shaker for 30-48 hours. These 100 ml. portions oE
inoculum are then used to inoculate one liter and 12 liter ,~
batches of the'same medium in 2 liter and 20 liter glass ferment'ors. The inoculated mash is aerated with sterile air , while'growth is continued for 40-55 hours. These batches ~ -- ~ .
of inoculum are used to inoculate tank fermentors. ; '', Tank Fermentation for BM123a , .
For the production o~ BMl23a in tank Eermentors the Eollowing medium is regularly used:
Bacto-peptone....................... 10.0 gm.
, Dextrose............................ 20.0 gm.
' Molasses............................ 20.0 gm.
L-Histidine.......................... 0.4 gm.
Ferric ammonium citrate 0.1 gm.
Calcium carbonate.................... l.0 gm.
Water to.~......................... l,000 ml.
Ad~ust medium to pH 7.2,wi*h NaOH
Each tank is inoculated with 3 to 10~ of inoculum '~
made as described above. Aeration is supplied at the rate ' of 0.2-0.8 liter of sterile air per liter of broth per ' , - minute and the fermenting mixture is agita`ted by an impeller driven at 200-400 r.p.m. The temperature is maintained at ;~
25-29C., usually at 28C. The fermentation is orcLinarily ' .... . . .

101~7~

continued for 120-190 hours, at which -time the mash is harvested.
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Fermentation Process Selected to Produce Primarily BMl23 and BMl23~
Cultivation of Nocardia sp. NRRL 8050 may be carrled 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; polypeotides, 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, molyb-denum, copper, etc.; are supplied as impurities of other con-stituents of the media. Aeration in tanks and bottles is provided by o~cing sterile air throuyh or onto the sur~ace of the ermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent, such as Hodag ; -FD82 may be added as needed.
Inoculum Preparation for BM123~ and BMl23 Primary shaker 1ask inoculum of Nocardia sp. NRRL
- 8050 is prepared by inoculating lOQ milliliters o sterile liquid medium in S00 milliliter 1asks with scrapings or washings of spores from an agar slant of the culture. The . I
following medium is ordinarily used:
Bacto-tryptone."...~.......... 5 gm.
Yeast extract.............. ... 5 gm.
Beef extract............... ... 3 gm.
~lucose.................... .. 10 gm.
Water to .................. 1000 ml.
The flasks were incubated at a temperature from 25--29C., prefe,rably 28C. and agitated vigorously on a rotary `
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shaker for 30 to 48 hours. The inocula are then transferred into sterile screw cap culture tubes and stored at below 0F.
This bank of vegetative inoculum is used instead of slant scrapings for inoculation of additional shaker flasks in pre-- 25 - ~ i ;: .

~ 75~7 :::

para~ion of ~his first stage of inoculum.
These first stage Elask inocula are usecl to seed 12 liter batches of the same medium in 20 liter glass fermentors.
The inoculum mash is aerated wi-th s-terile air while growth is continued for 30 to 48 hours.
The 12 liter batches of second stage inocula are used to seed tank fermentors containiny 300 liters of the following sterile liquid medium to produce the third and final stage of inoculum~

Meat solubles.............. ~........................ l5 gm. ~ ;
Ammonium sulfate.............. ....................... 3 gm.
Potassium phosphate, dibasic....3 gm.
Calcium carbonate............. ....................... l gm.
Magnesium sulfate hepta-........
hydrate................. ..................... l.5 gm.
Glucose....................... ...................... 10 gm.Water to...................... ................... 1000 ~nl.
The glucose is sterilized separately.
The third staye inoculum is aerated at 0.4 to 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 25-29C., usually 28C. 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_BM123~ and BM123 For the production of BM123~ and BM123~ in tank fermentors, the following fermentation medium is preferably ; ;;~
used~

Meat solubles....................................... 30 gm.
Ammonium sul'fate.................................... 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.

Each tank is inoculated with 5 to 10~ of third stage inoculum made as described under inoculum preparation. The fermenting mash is maintained at a temperature of 25-28C.

, ~ 7S~7 :, usually 26C The mash is aerated with sterile air at a rate of 0.3-0.5 liters of sterile air per li-ter of mash per minute ~ ;
and agitàted by an impeller driven at 70 to 100 revolutions per minute. The fermentation is allowed to continue rom 65-90 hours and the mash is harvested. ~-The invention will be described in greater detail in conjunction with the following specific examples.

Inoculum Preparation_for BM123a 1 0 :~
A typical medium used to grow the primary inoculum was prepared according to the following formula:
Beef ex-tract.................................... 3.0 gm.
Bacto-tryptone................................... 5.0 gm.
Yeast extract.................................... S.0 gm.
Starch.......................................... 24.0 gm.
Dextrose......................................... 1.0 gm.
Water to....................................... l,000 ml. ;
Adjust rnedium -to pll 7.0 with NaOH
Washed or scraped spores from an agar slant of Nocardia sp. NRRL 8103 were used to inoculate two 500 ml. ~ ~
flasks containing 100 ml. each of the above sterile medium. ~ -The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28C. The resulting Elask inoculum was transferred to a 5 gallon glass fermentor containing 12 Iiters oE sterlle medium. The mash was aerated with sterile air while growth was carried out or about 48 hours, after which the contents were used to seed a .
100 gallon tank fermentor containing 280 liters of sterile fermentation medium.
EXAMPI,E 2 ~ ;
.
Fermentation Employing Nocardia sp. NRRL 8103 and Medium -Favcring the Production of BM123a , A fermentation medium was prepared according to the ~ ;
following formula:

,~

1~137S~'7 Bac-to-peptone........................................ l0.0 gm. i~ ` -Dextrose.............................................. 20.0 gm.
Molasses...........O.................................. 20.0 gm. ~ -' L-Histidine................... ........................ 0,.4 gm.
Ferric ammonium citrate................................ 0.l gm. `~
Calcium carbonate........~............................ l,.0 gm.
Water to.....~............................... l,0()0 ml.
Adjust medium to pH 7.2 with NaOH
-The fermentation medium was sterilized at 120C.
with steam at 20 lbs. pressure for 60 minutes. The pH of the medium after sterilization was 6.6. Five hundred and sixty ; ~!~
liters of sterile medium in two l00 gallon tank fermentors were inoculated wlth 12 liters each of inoculum prepared as described in Example l. The fermentation was carried out at 28C. using Hodag ~ FD82 as a defoaming agent. Aeration was supplied at the rate of 0.4 liter of sterile air per liter of mash per minu-te. The mash was agitated by impellers lS driven at 200-290 revolutions per minute. At the end of approximately 139 hours of fermentation time the mash was harvested.
EXAMPLE 3 ~ ~.
, Inoculum preparation for BMl23~ and BMl23 A typical medium usecl to grow the first and second stages of inoculum was prepared according to the following ` ~
formula: ~ `

Bacto-tryptone.......... ,........ 5 gm.
Yeast extract................. ... 5 gm.
Beef extract.................. ... 3 gm.
Glucose....................... .. l0 gm.
Water to...................... l000 ml.
Two 500 milliliter flaskseach containing l00 milli~
liters of the above sterile medium were inocuLated with 5 milllliters each of a frozen vege-tative inoculum ~rom Nocardia sp. NRRL 8050. The flasks were placed on a rotary shaker and ~ ;
, agitated vigorously for 48 hours at 28C. The resulting flask - inoculum was transferred to a 5 gallon glass ~ermentor contain-ing 12 liters of the ahove sterile medium. The mash ~was aerated with sterile air while growth was carried out for about . . ;~ ; :
- 2~ -: .. : . . ' . , ` "

7S37 ~ ~

48 hours, af-ter which the contents were used to seed a 100 yallon tank fermentor containing 300 liters of the following sterile liquid medium~

Meat solubles..........O.......................... 15 gmO - , Ammonium ,sulfate......~........................... 3 gm. '~
Potassium phosphate, dibasic............... 3 gm.
Calcium carbonate........................ ~.l gm.
Magnesium sulfate'heptahydrate........... l.5 gm. ' ~ , Glucose................................... 10 gm.
Water to................................ l000 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 28C. and Hodag~ E~D82 was used as a defoaming agent. AEter 48 hours oE growing time the inoculum mash was used to seed a 3000 liter Eermentation.
~XAMPLE 4 Fermentation Employing Nocardia sp. NRRL 8050 and Medium Favor~
, .
ing_the Production of BML23~ and 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................................ l000 ml.
, The glucose is sterilized separately. '~
The fermentation medium was sterilized at 120C. 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 describe,d in Example 3, and the fermentation was carried out at 26C. using Hodag~ FD82 as a defoaming agent. Aeration 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 ~' , - 29 - ~
: ' ,, -. , , - . . ..

per minute. ~t the end of 67 hours of fermentation time the mash was harvested.
E~AMPLE 5 ,,~
Isolation of BM123~ ~ -,, :.
A fermentation was carried out as described in , ,~
Example 2. Five hundred liters of ferment.ed mash having a pH
of 6.8 was filtered using 5 kg. of diatomaceQus earth as a '' filter aid. The cake was washed with about 75 liters of water and discarded. Sodium fluoride (1 kg.) was added to the com~
bined filtrate and wash and the mixture was stirred for one hour. The resulting suspension was filtered using about 5.75 ~ -kg. of diatomaceous earth as a filter aid and the cake was washed with about 5 liters of water and discarded. The com-bined filtra-te and wash (580 liters, pH 6.7) was allowed to percolake through a column of 10 liters oE Amberlite ~ IRC-50 ;, (a methacrylic acid-divinyl benzene ion exchange resin~,~
avaiIable from Rohm & Haas Co.) (N/a+; 16-50 mesh) in a 6" x ~"
60" glass column. The charged column was washed with 40 liters of water. The BM123a was eluted by passing 120 liters of 0.3N
H2SO4 through the column. The initial 9 liters of eluate was discarded. The remaining eluate was adjusted to pH 6.0 with solid Ba(OH)2 and the precipitated barium sulfate was removed by Eiltration. The clear iltrate was concentrated n vacuo to about 20 liters which was then slowly percolated through a 12 liter bed volume column of granular Darco ~ act vated carbon (a granular activated carbon available from ICI
'of U.S.) (20-40 mesh). The charged column was washed with 40 liters of water and eluted with 60 liters of 50% aqueous meth-anol. The column eluate was concentrated to about 4 liters of aqueous solution and lyophilized to give 104.1 gms. of white solid BM123a as the sulfate salt.
The BM123a so prepared does not possess a definite melting po,int, gradual decomposition starting in the v:icinity ~: :
- 30 - `

~875137 of 200C. Microanalysis of a sample cluilibrated for 24 hours in a 73F. atmosphere containing 60~ relative humidity gave C, 32.10%; H, 5.64%; N, 10.80%; S, 4.45%; loss on drying 12.52%.
The BM123a was transparent to light in the region 220 ~o 340 nm ;~
when run-in 90% methanol at 200 mcg./ml. BM123a had a specific rotation of ~a]D5 = +40 (C=l.l in H2O). The BM123a exhibit-ed characteristic absorption in the infrared region of the spectrum at the following wavelengths: 780, 815, 950, 1050, 1110, 1250, 1340, 1395, 1560, 1670, 1705, 2950, and 3330 cm~l.
A standard infrared absorption spectrum of BM123a prepared in a ~Br pellet is shown in Figure 1 of the accompanying draw-ings.
EX~MPL~ 6 Isolation of ~M123~ and BM123 ~
A 3000 liter portion of Permentat:ion mash prepared as described in Example 4, pH 4.3, was adjusted to p}l 7.0 with sodium hydroxide and filtered using 5% diatomaceous earth as a filter aid The cake was washed with about 100 liters of i~
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 ;~
[Wa~] 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%
25 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 ~ ac-tivated 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 15% aqueous methanol eluate was concentrated ln vacuo to about 7 liters 10~7537 ~

of an aqueous phase and the p~l adjusted from 4.5 -to 6.0 with Amberlite ~ IR-45 (OH ) resin (a weakly basic polystyrene~
-polyamine type anion exchange resin). The resin was removed . ~
by filtration and the filtrate was concentrated ln vacuo to about l liter and then lyophilized to give 38 grams of material consisting primarily o BM123~ along with a small amount of BM123~ (primarily BMl23~2). 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 fil~
trate was concentrated ln vacuo to about 6. 3 liters and then lyophylized to give 213 grams of material consisting prlmarily of BM123~ The 50% aqueous acetone eluate was adjusted from ~ ;;
pH 4.0 -to 6.0 with Amberlite ~ IR-45 (O~l~) resin. The resin was ~;
removed by filkration and the ~iltra-te was concentrated ln vacuo to about 1. 5 liters and then lyophylized to give 56 grams of impure BM123~

Further purification of BM123~; Isolation of BM123 A slurry of CM Sephadex ~ C-25 ENH4] in 3% aqueous ~ '~
ammonium chloride was poured into a 2. 6 centimeter diameter glass column to a resin heigh of approximately 57 centimeters.
The excess of 3~ aqueous ammonium chloride was drained away and a 5.0 gram sample o~ BM123~ prepared as described in Ex~
, ample 6 was dissolved in about 10 milliliters of 3-~ aqueous ammonium chloride and applied to the column. The column was ;~
then eluted with a gradient between ~ liters each of 3% and 6~ aqueous ammonium chloride. Fractions of about 70 milliliters each were collected automatically every 15 minutes. Anti-biotic BMl23~ was located by monitoring the column effluent in `~
, .
the ultraviolet and by bioautography of dipped paper disks . . , : .
on large agar plates seeded with Klebsiella pneumcniae strain ~, AD. The majority of BM123~ was located between fractions 51-67 ~ .
inclusive; the initial fractions (51-58) conta:ined essentially ' .. . .. ... . . .
. . .

~ 7537 pure BM123~ hereas the later fractions ~59-67) contained a mixture of BM123~1 and BM123~2. `~
Two hundred ~ifty milli-ters of granular Darco ~acti-vated carbon (20-40 mesh) was suspended in water, transferred S to a glass column, allowed to se-ttle and the excess water wa~
allowed to drain away. Fractions 51-58 inclusive from the above ~`
CM Sephadex chromatography were combined and passed through the granular carbon column. The charged column was washed with ;
1 liter of watèr and then developed with 1.5 liters of 20~
aqueous methanol. The aqueous methanol eluate was concentrated to an aqueous phase ln vacuo and lyophilized to give 1.24 grams of white amorphous BM123~1 as the hydrochloride salt.
Antibiotic BM123~1 does not possess a def:Lnite melting point, but gradual decomposition starts in the uicinity oE 200C. Microanalysis of a sample equilibrated for 24 hours in a 72F. atmosphere containing 23~ relative humidity gave C, 39.29%; H, 6.33~; N, 16.58%; Cl(ionic), 13.28%; loss on `
drying, 6.90~. In methanol BM123~1 gave a U.V. absorption maximum at 286 nm with El% = 260. The position of this max-20 imum did not change with pH. BM123~1 had a speci~ic rotation of; ;~
[a]25 = ~67 (C=1.0 in water)-Antibiotic BM123~1 exhibited characteristic ab-sorption in the infrared region of the spectrum at the follow-ing wavelengths: 830, 870, 930, 975, 1040, 1070, 1105, 1175, ~-2S 1220, 1360, 1460, 1505, 1550, 1600, 1650, 2900, 3050 and 3350 ;~
cm 1. A standard infrared absorption spectrum of BM123~
prepared in a KBr pellet is shown in Figure 4 of the accompany-ing drawings. A standard proton magnetic resonance spectrum o BM123~1 determined on a D20 solution in a 100 megacycle : ~
spectrometer is shown in Figure 7 of the accompanyiny drawings.
..
Fractions 59-67 inclusive from the above CM Sepha-dex chromatography were combined and passed through a 200 milli--liter bed volume granular carbon column. The charged column ~ -1087537 ; ~ ~

was then washed with 1 liter of water and then developed with ~
1.5 liters of 20~ aqueous methanol. The aqueous methanol eluate was concentrated to an aqueous phase 1n vacuo and lyophilized~
to give 0.83 grams of white amorphous BM123~ as the hydrochloride~
salt.
Antibiotic BM123~ does not possess a definite melt~
ing point, but gradual decomposition start:s in the vicinlty o;f 200C. Microanalysis of a sample equilibrated for 24 hours in a 72F. atmosphere containing 23% relative. humidity gave C,~
38.81%; H, 6.19~; N, 16.52~; Cl(ionic), 13.41%; loss on drying, ;
7.32%. In methanol BM123~ gave a U.V. absorption maximum at 286 nm with El% = 255. The position of this maximum did not~
lcm change with pH. BM123~ had a speciic rotation of ;
[a]~5 = ~63 (C = 1.06 in water~
Antibiotic BM123~ exhibited characteristic ab- ;
sorption in the inErared region oE khe spectrum at the follow~
ing wa~elengths: 830, 870, 930, 975, 1040, 1070, 1105, 1175, 1220, 1360, 1460, 1505, 1550, 1600, 1650, 2925, 3050 and 3025 cm~l. A standard infrared absorption spectrum of BM123 prepared in a KBr pellet is shown in Figure 2 of the accompany~
ing drawings.
EXAMPLE 8 ;~
Further Purification oE BM123 .
A slurry o CM Sephadex ~ C-25 [NHt] in 2~ aqueous ammonium chloride was poured into a 2.6 centimeter diameter `
glass column to a resin height of approximately 62~cent~
meters.` The excess 2% aqueous ammonium chloride was~drained away and a 5.0 gram sample of ~M123~ prepared as descrlb d~
in Example 9 was dissolved in ahout 10 milliters of 2%
.: :, . :::
30~ aqueous ammonium chloride and applied to the column.; The column was then eluted wit~. a gradient between 6 liters each o 2% and 4% aqueous ammonium chloride. Fractions o~ about ,~ 75 milliliters each were co].leated automatically every 15 , ~ 7S3!7 minutes. Antibio-tic BM1~3~ ~as located by monitoring the column efflu~nt in the ul-traviolet and by bioautography of dipped paper disks on large agar plates seeded with Klebsiella pneumoniae strain AD. The majority of B~123~ was located between fractions 71-107 inclusive.
One hundred -thirty milliliters of granular Darco activated carbon (20-40 mesh) was suspended in water, transfer-red 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 chromatography were combined and passed through the granular carbon column. The charged column was washed with 600 milliliters of wa-ter and then developed with 1 liter oE 20~ aqueous methanol Eollowed by 1 li-ter of 50~ ~ ;
_ aqueous acetone.~ l'hese eluates, both oE which contained BM123~, were concentrat~d to a~ueous phases ln vacuo and lyophilized to give a tota~ of 886 milligrams of BM123 ~as the hydrochloride salt. A microanalytical sample was obtained by subjecting the above material to a repeat of the above process.
Antibiotic BM123 4does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200C. Microanalysis of a sample equilibrated for 24 hours in a 72F. a-tmosphere containing 23% relative humidity gave C, 39.44~; H, 6.10~; N, 16.19~o; Cl(ionic), 11.54%; loss on drying, 8.19%. In water BM123~ gave a U.V. absorption maximum -~
at 286 nm with ElCm = 250. The position of this maximum did ~;

not change with pH. BM123~ had a specific rotation of 2~
[a]D' = ~71 (C = 0.97 in water). ;
Antibiotic BM123 ~ exhibited characteristic absorp-tion 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 3 of 1~75i3~7 ~

the accompanying drawings.

Isolation of BM123~]
. .
A slurry of CM Sephadex ~ C-25 [Na+] in 2~ aqueous~
sodium chloride was poured into a 2.6 centimeter diameter glass column to a resin heigh of approximately 70 centimeters. The excess 2% aqueous sodium chloride was drained away and 4.11 gram of a sample containing primarily BM123~1 along with some BM123~2 and other impurities, prepared as described in Example 6, 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 sodi.um chloride. Fractions of about 75 milliliters each were collected automatically every 15 minutes. Anti-biotic ~M123~ was located by moni~oriny the column e~fluent ln the ultraviolet and by bioautography of dipped paper disks on large agar plates seeded with Klebsiella pneumoniae strain ~D. The majority of BM123~ was located between fractions ~ -64-90 inclusive; the initial fractions (64-80) contained a mixture of BM123~1 and BM123~2 whereas 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 qlass column, allowed to settle and the excess watex was allowed ~ ~;
to drain away. Fractions 81-90 inclusive from the above CM
Sephadex chromatography were comblned and passed through the granular carbon column. The charged column was washed with 500 milliliters 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 BM123~1, was adjusted from pH 5.9 to 6.0 with ~mberllte ~
IR-45(0H-l) resin. The resin was removed by f:iltration and the - ' : - .

~ . ..

..
.

7537 ~;:

filtrate was concentrated in vacuo to an aqueous phase and -lyophilized to give 294 milligrams of whit:e amorphous BM123 as the hydrochloride salt.
Antibiotic BM123~1 does not posC;ess a definite melting point, but gradual decomposition s,tarts in the vicinity of 200C. Microanalysis of a sample equilibrated ~or 24 hours in a 70F. a-tmosphere 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 BM123~ gave a U.V. absorption maximum at 286 nm with El% = 225. The position of this max~
imum did not change with pH. BM123~1 had a specific rotation of +55 (C=0.803 in water).
Antibiotic BM123~1 exhibited characteristic ab-sorption in the infrared region oE the spectrum at the follow- `
ing 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~l. A standard infrared absorption spectrum ~f BM123rl prepared in a KBr pellet is shown in Figure 5 of the accompanying drawings. A standard proton magnetic resonance spectrum of BM123~ determined on a D20 solution in a 100 megacycle spectrometer is shown in Figure 8 of the accompanying drawings.

IsolatiOn o~ BM123~2 A 25 gram sample containing primarily BM123~2 and BM123 ~, prepared as described in Example 6, was dissolved in about 120 milliliters of 2% aqueous sodium chloride and applied to a column containing 1800 ml. of CM Sephadex ~ C-25 ENa+] , ;~
in 2% 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 - 37 - ~ `

column fractions in the ultraviolet. The majority of s~l23r was located be-tween fractions 7-18 inclusive; the initial fractions (7-15) contained essentially pure BMl23~2 and the later Eractions (16-18) contained a mixture of BM123rl and BM123~ .
Six hundred milliliters of granu:Lar Darco ~ activated carbon (20-~0 mesh) was suspended in water, transferrecl 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 oE 10%
a~u~ous methanol Eollowed by 6 l:iters o~ 50~ aqueous methanol.
The 10~ aqueous methanol eluate was adjusted from pEI 5.8 to 6.0 with ~mberlite ~ 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 BM123~2 as the hydrochloride salt. The 50%
aqueous methanol eluate was adjusted from pH 4.6 to 6.1 with Amberlite ~ IR 45 (OH ) resin. The resin was removed by filtra-tion and the filtrate was concentra-ted ln vacuo to an aqueous phase and lyophilized to give 3.645 grams oE slightly less pure white amorphous BM123~2 as the hydrochloride salt.
Antibiotic BM123~2 does not possess a definite melting point, but gradual decomposition starts in the vicinity -~
, of 200C. Microanalysis of a sample equilibrated for 24 hours in a 70F. 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 BMl237~2 gave a U.V. absorption maximum at 286 nm with ElCm = 220. The position oE this maximum did not change with pH. BMl23~2 had a specific rotation of +60 (C=0.851 in water).

Antibiotic BMl23 ~ exhibited characteristic absorp-- 38 - ! ..... -~0~7~37 ;
tion in the infrared reyion oE the spec-trum at the following -wavelenyths: 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 absorp~tion spectrum of BM123~2 prepared in a KBr pellet is shown in Figure 6 of -the accompanying drawings. A standard proton magnetic resonance spectrum of BM123~2 determined on a D2O solution in a 100 mega-cycle spectrometer is shown in Fiyure 9 of the accompanying drawings. i ; !

Paper Partition and Thin Layer Chromato~raph~ o~ BM123a,~ and r The antibacter.ial agents can be di~t:inguished by p~p~r chromatocJraphy. E'or thi.s purpose Whatman No. 1 strips were spotted with a water or methanol solution oE the ~ub-stances and equilibrated for 1 to 2 hours in the presence of both upper and lower phases. The strips were developed over- ;
night with the lower (organic) phase obtained ~rom 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. Representative Rf values are listed in Table VIII below: ;~
:: . ., TABLE VIII - ;
. .. . .

Component Rf BM123 ~ 0.85 BM123!3 0.50, 0.701 ~ ~;

BM123~ 0.20 _ BM123~ was composed o~ a major antibiotic (Rf = 0.50) called BM123~1 and a minor antibio-tic ~Rf = 0.70) called BM123~2.

~ 75~q ~~ The BM123 antibiotics can also be distinguished by thin layer chromatography. For this purpose pre-coa-ted Cellulose F ~ plates (0.10 millimeters -thick), a form of thick layer cellulose supplied by EM Laboratories Inc., Elmsford, N.Y. were spotted with a water solution of the sub stance to be chromatographed (about 20-40 micrograms per spot). The plates were developed overnight with the solvent obtained by mixing l-butanol:water:pyridine:acetic acid (15:12:10:1 by volume). The developed plates were removed from the chromatographic chamber and air dried for about 1 hour. The antibiotics were detected by using either standard ninhydrin or Sakaguchi spray reagents. Representative Rf values are listed in Table IX below:
T~BLE IX

Component Rf BM123~ 0.17, 0.23 BM123~ 0.08, 0.14 BM123a 0!05 -_ .
Both BM123~ and ~ were a mixture of two components using this system. BM123~ was composed of a major component (Rf = 0.08) which was BM123~1 and a minor component (Rf =
0.14) which was BM123~2. The less polar component of BM123~ (Rf = 0.23) was named BM123~1 and the more polar component (Rf = 0.17) was named BM123~2.

.

~.' .
,' "' ~''.
- ~0 - , . .
'

Claims (10)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a compound of the following formulae:

BM123.alpha.

BM123.beta.1 BM123?1 BM123?2 which comprises cultivating Norcardia sp NRRL 8103, NRRL 8050 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibiotic activity is imparted to said medium, and recovering the antibiotic thus produced.

2. A compound of the formula:

BM123.alpha.

BM123.beta.1 BM123?1 BM123?2 and the pharmaceutically acceptable acid-addition salts thereof, whenever prepared according to the process of Claim 1 or by a chemical equivalent thereof.

3. A process for the preparation of a compound of the following formula:

BM123.alpha.

which comprises cultivating Norcardia sp NRRL 8103, NRRL
8050 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibiotic activity is imparted to said medium, and recovering the antibiotic thus produced.

4. A compound of the formula:

BM123.alpha.

and the pharmacologically acceptable acid-addition salts thereof, whenever prepared according to the process of Claim 3, or a chemical equivalent thereof.

5. A process for the preparation of a compound of the following formula:

BM123.beta.1 which comprises cultivating Norcardia sp NRRL 8103, NRRL
8050 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibiotic activity is imparted to said medium, and recovering the antibiotic thus produced.

6. A compound of the formula:

BM123.beta.1 and the pharmaceutically acceptable acid-addition salts thereof, whenever prepared according to the process of Claim 5 or by a chemical equivalent thereof.

7. A process for the preparation of a compound of the following formula:

BM123?1 which comprises cultivating Norcardia sp NRRL 8103, NRRL
8050 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibiotic activity is imparted to said medium, and recovering the antibiotic thus produced.

8. A compound of the formula:

BM123?1 and the pharmaceutically acceptable acid addition salts thereof, whenever prepared according to the process of Claim 7 or by a chemical equivalent thereof.

9. A process for the preparation of a compound of the following formula:

BM123?2 which comprises cultivating Norcardia sp NRRL 8103, NRRL 8050 or mutants thereof in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts under submerged aerobic conditions until substantial antibiotic activity is imparted to said medium, and recover-ing the antibiotic thus produced.

10. A compound of the formula:

and the pharmaceutically acceptable acid-addition salts thereof, whenever prepared according to the process of Claim 9 or by a chemical equivalent thereof.