GB1565451A - Antibiotics containing an optionally substituted spermadine side chain - Google Patents

Description

(54) ANTIBIOTICS CONTAINING AN OPTIONALLY SUBSTITUTED SPERMADINE SIDE CHAIN (71) We, AMERICAN CYANAMID COMPANY, a corporation organised and existing under the laws of the State of Main, United States of America, of Berdan Avenue, Township of Wayne, State of New Jersey, United States of America, do hereby declare the invention for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- This invention relates to new antibiotics designated cis-BMl23v" alkylated-cis BMl23v1, cis-BMI23r2 and alkylated-cis-BMl23v2. The present invention includes within its scope the antibiotics in dilute forms, as crude concentrates, in pure crystalline form, and as mixtures of the , and Y2 forms. The effects of the new antibiotics on specific microorganisms, together with their chemical and physical properties, differentiate them from previously described antibacterial agents.

The novel antibiotics of the present invention are organic bases and thus are capable of forming acid-addition salts with a variety of organic and inorganic saltforming reagents. Thus, acid-addition salts, formed by admixture of the antibiotics 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 antibiotics of the present invention are, in general, crystalline solids relatively soluble in water, methanol and ethanol but are relatively insoluble in non-polar organic solvents such as diethyl ether, benzene, toluene, and the like. For purposes of this invention the antibiotic free bases are equivalent to their non-toxic acid-addition salts. Hereinafter, cis-BM123v refers to a mixture in any proportions of cis-BM123v, and cis-BM123v2, and trans123v refers to a mixture in any proportions of trans-BM123v, and trans-BM123v2.

The new antibiotics which have been designated cis-BM123v1 and cis-BM I 23r2 and alkylated-cis-BMI23P1 and alkylated-cis-BM123v2 may be prepared by the photochemical transformation of their corresponding trans-isomers. The photolytic conversion of alkylated-trans-BM123v, alkylated-trans-BM123v, and alkylatedtrans-BM123v2 to the corresponding alkylated-cis-BM123v, alkylated-cis-BM123v1, and alkylated-cis-BM123v, is preferably effected by dissolving or dispersing the trans-isomer starting material in water and irradiating the solution with light. The concentration of the rrans-isomer starting material in the water is not critical.

The light employed in the photolytic process of the present invention is advantageously of a wavelength not less than 2,500 Angstroms and is preferably of a wavelength from 2,500 to 4,000 Angstroms. In order to conveniently achieve this, the reaction may be carried out in a vessel constructed of a material such as quartz, which filters out substantially all the light passing through the vessel having a wavelength below about 2,500 Angstroms. The light source is conveniently a high pressure mercury arc lamp of about 450 watts.

The temperature at which the photolysis is carried out is not particularly critical for good yields of product, but is conveniently within the range from 5"C. to 50 C.; for instance, from 25 C. to about 30 C. The time required for substantial conversion of the alkylated-trans-isomer to the corresponding alkylated-cis-isomer will naturally vary within the light intensity and the temperature, and is therefore best determined by trial in the individual case. However, a period of time ranging from 20 minutes to two hours is generally sufficient.

In like manner, antibiotics trans-BMl23v, trans-BM123v1 and trans-BM123v2 may be photochemically transformed to the corresponding antibiotics cis-BMl23v, cis BM123v, and cis-BMI23v2, respectively.

After the irradiation step is complete, the product may be obtained by standard procedures. For example, the reaction mixture may be lyophilized or evaporated to dryness and the residue may be dissolved in a minimal amount of solvent such as ethanol or methanol. The resulting solution may be diluted with diethyl ether or acetone, and the resulting precipitated product may be recovered by filtration. Further purification may then be achieved by standard techniques such as crystallization or chromatography.

The starting materials designated alkylated-trans-BM123u, alkylated-trans BM123v1, and alkylated-trans-BMl23i'2 are derived by the reductive alkylation of antibiotics trans-BMl23v, trans-BM123P, or trans-BM123v2 with an aldehyde or ketone of the following general formula:

wherein R, is hydrogen, alkyl having up to 8 carbon atoms, phenyl, benzyl or alkenyl having from 2 to 8 carbon atoms; R2 is alkyl having up to 3 carbon atoms; R3 is alkyl having up to 8 carbon atoms, N,N-di(alkyl C1-C4)amino-(alkyl C1-C4), chloroalkyl having up to 4 carbon atoms, phenyl, benzyl, A-phenylethyl, alkenyl having from 2 to 8 carbon atoms or hydroxyalkyl having up to 4 carbon atoms, or R2 and R3 taken together with the associated methylidyne group is cyclobutyl, cyclopentyl, monomethylcyclopentyl, dimethylcyclopentyl, triethylcyclopentyl, cyclohexyl, monomethylcyclohexyl, dimethylcyclohexyl, trimethylcyclohexyl, cycloheptyl or a moiety of the formula:

wherein R4 is hydrogen, alkyl having up to 4 carbon atoms, phenyl or benzyl.

The reductive alkylation process whereby the alkylated-trans-isomers may be prepared is carried out as follows: Antibiotic trans-BM123v, trans-BMl23v1 or trans-B Ml 23v2 is dissolved in a suitable solvent such as water, methanol, methyl "Cellosolve" (registered Trade Mark), or mixtures thereof, in an amount in excess of an equimolar amount of the desired aldehyde or ketone is then added followed bv the addition of a reductive sufficiency of sodium cyanborohydride. The pH of the reaction mixture is maintained at 6.0-8.0 with dilute mineral acid during the course of the reaction. After one to 24 hours at ambient temperature(10 -35 C.), the reaction mixture is evaporated to dryness in vacuo and the residue is triturated with methanol and filtered. The filtrate is diluted with acetone and the solid product that precipitates is removed by filtration and dried in vacuo.

The products may also be obtained from the reductive alkylation reaction mixtures by other standard procedures such as precipitation, concentration, solvent extraction or combinations of these procedures. After isolation, the products may be purified by any of the generally known methods for purification.

These include recrystallization from various solvents and mixed solvent systems, chromatographic techniques, and counter current distribution, all of which are usually employed for this purpose.

In like manner, antibiotics cis-BM123v, cis-BM123v, and cis-BMl23v2 may be reductively alkylated to provide the alkylated-cis-BM123v, alkylated-cis-BM123l, and alkyláted-cis-BMI23v2 of the present invention.

Aldehydes and ketones which may be so employed in the above reductive alkylation process (according to the general procedure of Example 8) are set forth in Table I below. In this table, the derivative name indicates whether antibiotic trans-BM123v or antibiotic cis-BM123v is the starting material employed.

TABLE I

Carbonyl Reagent Employed Derivative Name 1-dipropylamino-2-propanone 1-methyl-2-(N,N-dipropylamino) ethyl-trans-BM123v 1-chloro-3-pentanone 1-ethyl-3-chlorpropyl-trans BM123v 4-methyl-pentanone | 1,3-dimethylbutyl-trans-BM123γ phenylacetone 1-methyl-2-phenyl ethyl-trans- BM123v 6-methyl-5-hepten-2-one | 1,5-dimethylhexen-4-yl-trans BM123v 3-methyl-2-pentanone | 1,2-dimethylbutyl-cis-BM123γ 5-methyl-2-hexanone 1,4dimethylp entyl-cis-BM 123v 3-ethyl-2-p entanon e 1-m ethyl-2-ethylbutyl- trang BM 123v 3,5-dimethyl-2-octanone | 12,4-trimethylheptyl-trans BM123v 3-octanone 1-ethylhexyl-trans-BM123v 3-methyl-2-hexanone 1,2-dimethylpentyl-trans-BM123γ 2-pentanone 1-methylbutyl-cis-BM123γ 2-butanone 1-methylpropyl-cis-BM123γ acetone isopropyl-trans-BM123v 3-dec anone t-ethyloctvl-trans-BM123v 3-undecanone 1-ethylnonyl-trans-BM123γ mesityl oxide 1,3dimethylbuten-2yl-cis-BM123γ 4methyl-2-he xanone 1 , Idim ethylp entyl-tran s-BM 123v 2,4,4-trimethyl cy clopentanone 2,4,4-trimethylcyclop entyl-trans BM123v 3,3-dimethyl-2-butanone | 1,2,2-trimethylpropyl-cis-BM123γ 3,5-dimethylcycloh exanon e 3,5-dimethyl cyclohexanone BM123v 4-hydnxy-3-methyl-2-butanone 1,2-dimethyl-3-hydroxypropyl-trans BM123v I-di ethyl amino- 3-butanone 1-methyl-3-(N,N-di ethyl amino)propyl trans-BM123γ 3-hydroxy-3-methyl-2-butanone 1,2-dimethyl-2-hydroxypropyl-trans BM123v TABLE I (Continued)

Carbonyl Reagent Employed Derivative Name 3-pentanone 1-ethylpropyl-cis-BM123v 3-methyl-2-butanone 1,2-dimethylpropyl-trans-BM123v 4-heptanone 1-propylbutyl-cis-BM123v dim ethyl amino acetone 2-methyl-2-(NiN-dime thyl amino)- ethyl-trans-BM123v 5-diethylamino-2-pentanone 1-methyl-4-(N,N-diethylamino) butyl-trans-BM123v cy clobutanone cy clobutyl-trans-EM 123v 2-octanone 1-methylheptyl-trans-BM123v 4-phenyl-2-butanone 1-methyl-3-phenylpropyl-trans BM123v, 5-chloro-2-pentanone 1-methyl-4-chlorobutyl-trans BM123v 5-hexene-2-one l-methyl-penten-4-yl-trQns-BMi23v - cyclohexanone cyclohexyl-trans-BM123v 2-hexanone 1-methylpentyl-trans-BM123v 2-heptanone 1-methylhexyl-trans-BM123v cycloheptanone cycloheptyl-trans-BM123v cyclopentanone cyclophentyl-trans-BM123v 44-dimethyl-2-pentanone 1,3,3-trimethylbutyl-cis-BM123v 2,6-dimethyl-3-heptanone 1-isopropyl-4-methylphentyl-cis BM123v 4-octanone 1-propylpentyl-trans-BM123v 3-hepta none 1-ethyip entyl-trans-BM123v 1-benzyl-4-piperidone 1-benzyl-4-piperidyl-trans-BM123v 1-methyl-4-piperidone 1-methyl-4-piperidyl-trans-BM123v 3-methylcyclopentanone 3-methylcyclopentyl-trans-BM123v 3,3-dimethyl-2-butanone 1-methyl-2,2-dimethylpropyl-cis BM123v 4-methyl-2-heptanone 1,3-dimethylhexyl-trans-BM123v 3,4-dimethyl-2-hexanone 1,2,3-trimethylpentyl-trans-BM123v 1,3,3-trimethylcyclopentanone 1,3,3-trimethylcyclopentyl-trans BM 123v TABLE I (Continued)

Carbonyl Reagent Empolyed Derivative Name 5-hexen-2-one 1-methyl-p enten-4-yl-cis-BM 123v 2-methylcyclopentanone 2-methylcy ci op entyl-trans-BM 123v 2,4-dimethylcyclop entanone 2,; dimethylcyclopentyl-trnns- BM123v 3-hexanone | 1-ethylbutyl-trans-BM123v 3-hydroxy-2-butanone 1-methyl-2-hydroxypropyl-trans- BM 123v 4-hydroxy-3-methyl-2-butanone | 1,2-dimethyl-3-hydroxypropyl-trans BM123v 2-nonanone 1-methyloctyl-cis-BM123v 5-hydroxy-2-pentanone 1-methyl-4-hydroxybutyl-trans BM123y 2-decanone 1-methylnonyl-trans-BM123v phenylacetaldehyde 2-ph enylethyl-trans-BMl23v 2-ethylhexanal 2-ethylhexyl-cis-BM123v 2,2-dimethylbutanal 2,2-dimethylbutyl-trans-BM123v 2,2-dimethylprop anol 2,2-dimethylpropyl-trans-BM123v 2-ethyl-2-butenal 2-ethyl-2-butenyl-trans-BM123v 2-methyl-2-butenal 2-methyl-2-bu teny l-cis-BM12 3v 2-methyl-2-pentenal 2-methyl-2-pentenyl-trans-BM123v formaldehyde methyl-trans-BM123v acetaldehyde ethyl-trans-BM123v benzaldehyde benzyl-trans-BM123v The antibiotics designated BM123ss1, BM123ss2, trans-BM123v, and trans BMl23v2 are formed during the cultivation under controlled conditions of a new strain of an undertermined 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. BMl23. 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, Illinois, and has been added to its permanent collection. It is freely available to the public in this depository under its accession No. NRRL 5646. Herein BM123p refers to a mixture in any proportion of BM123ss1 and BM123ss2.

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 Shining and Gottlieb, Internat. Journ. of Syst. Bacteriol. 16:213-240 (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 ed. (1948), Container Corp. of America, Chicago, Illinois. Descriptive details are recorded in Tables II through VI below.

Amount of Growth Moderate on yeast extract, asparagine dextrose, Benedict's, Bennett's, potato dextrose and Weinstrein'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.

Reverse Color Colorless to yellowish shades.

Miscellaneous Physiological Reactions No liquefaction of gelatin; nitrates reduced to nitrates in 7 days; melanoid pigments not formed on peptone-iron agar; no peptonization or curd formation in purple milk; NaCI tolerance in yeast extract agar 4% 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, I arabinose, lactose, d-mannitol, d-melibiose, d-raffinose, l-rhamnose, sucrose and d-xylose.

Chemical Composition The organism belongs to cell wall type IV, i.e., contains meso-2,6 diaminopimelic acid and has a type A whole-cell sugar pattern, i.e., contains arabinose and galactose. Methylated whole cell extracted, 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 elongated primitive spirals. The flexuous elements are irregularly segmented into short elliptical to cylindrical sections (spores?) which disarticulate readily. The spiral terminal protions are less conspicuously segmented. Segments generally range 0.8-1.7 x x 0.3--0.5 um, averaging 0.4 ,umx 1.2 um.

Diagnosis The morphological characteristics of Culture No. BM123 are difficult to observe and interpret because 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. BM 123 contains meso-2,6 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 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 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 undetermined species of Nocardia until such a diagnosis is feasible.

TABLE II Cultural Characteristics of Norcardia sp.NRRL 5646 Incubation: 14 days Temperature: 32 C

Amount of Aerial Mycelium Soluble Reverse Medium Growth and/or Spores Pigment Color Remarks Yeast Extract Agar Moderate Aerial mycelium whitish, light None Mustard Darkened areas in sub (3 i e) strate mycelium.

Coremia formed on surface mycelium Hickey and Trensner's Light No aerial mycelium None Colorless to Peripheral areas of Agar Yellowish- coloniers becoming green olive-green Asparagine dextrose Moderate Trace of whitish aerial None Amber Surface lightly Agar mycelium (3 1c) wrinkled Bendict's Agar Moderate Aerial mycelium whitish None Nude Tan Coremia abundantly light (4 gc) formed on surface mycelium Bennett's Agar Moderate Trace of whitish aerial None Camel Surface lightly mycelium (3 ie) wrinkled Inorganic Salts-strarch Trace No aerial mycelium None Colorless Agar Kuster's Oatflake Agar Trace No aerial mycelium None Colorless Czapek's Solution Agar Trace No aerial mycelium None Colorless TABLE II (Continued)

Amount of Aerial Mycelium Soluble Reverse Medium Growth and/or Spores Pigment Color Remarks Potato dextrose Agar Moderate Aerial mycelium whitish, None Camel light (3 ie) Tomator Paste Oatmeal Light No aerial mycelium None Colorless Agar Pablum Agar Light No aerial mycelium None Colorless Rice Agar Trace No aerial mycelium None Colorless Weintesin's Agar Moderate No aerial mycelium None Colorless to yellowish TABLE III Micromorphology of Nocardia sp. NRRI 5646

Medium Aerial Mycelium and/or Sporiferous Structures Yeast Extract Agar Aerial mycelium arises from substrate mycelium as sparingly branced, flexous elements that commonly terminate in elongated primitive spirals.

The flexuous elements are irregularly segmentend into short sections (spores?) which disarticulate readly. 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 m x 1.2 m.

TABLE IV Miscellaneous Physiological Reaction of Nocardia sp. NRRL 5646

Medium Incubation Period Amount of Growth Physiological Reaction 7 days Gelatin Light No liquefaction Gelatin 14 days Good No liquefaction Organic Nitrate 7 days Good Nitrates reduced to nitrites Broth Organic Nitrate 14 days Good Nitrates reduced to nitriles Broth Peptone-iron 24-48 hours Good No melanin pigments reduced Agar Purple Milk 7 days Good No peptonization or curd formation = = Yeast extract 7 days Moderate NaCl tolerance . 4% but 7% Agar plus (4, 7, 10 and 13%) NaCl TABLE V Carbon Source Utilization Pattem of Nocardia sp. NRRL 5646 Incubation: 10 days Temperature: 320C

Carbon Source Utilization* Adonitol 0 l-Arabinose 0 Glycerol 3 d-Fructose 1 i-Inositol 2 Lactose 0 d-Mannitol 0 Salicin 2 d-Melibiose 0 d-Raffinose 0 Rhamnose 0 Maltose 1 Sucrose 0 d-Trehalose 3 d-Xylose 0 Dextrose 3 Nagative Control 0 *3-Good Utilization 1-Poor Utilization 2-Fair Utilization 0-No Utilization TABLE VI Chemical Composition of Nocardia sp. NRRL 5646 Cell Wall Type Major Constituents Type IV meso-DAP, arabinose, galactose The production of BM123A and trans-BMl23v 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, trrn2s- BMI23v-producing mutants derived from this organism by various means such as exposure to X-radiation, ultraviolet radiation, nitrogen mustard. and actinophages may also be used. A viable culture of a typical 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 are substantially the same as those of NRRL 5646, it 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 (b) production of a rosewood tan mycelial pigment on Bennett's and yeast extract agars.

The novel antibacterial agents of the present invention are, in general, crystalline solids of relatively limited solubility in non-polar solvents such as diethyl ether and n-hexane, but considerably more soluble in solvents such as water and lower alkanols. Antibiotics trans-BM123v, and trans-BMl23v2 are structural isomers and may be represented by the following structural formulae:

The reductive alkylation of trans-BM123, trans-BM123z'1, trans-BMl23v2, cis BM123v, cis-BM123v or cis-BMl23s2 with ketones takes place on the spermadine side-chain to form derivatives of the formula:

wherein R is a moiety of the formulae:

and R2 and R3 are as hereinabove defined. The reductive alkylation of trans BM123v, trans-BM123v1, trans-BM123v2, cis-BM123v, cis-BM123v1 or cis-BM123v2 with aldehydes takes place on the spermadine side-chain to from mono-, di-, and tri-substituted derivatives of the formulae:

wherein R and R, are as hereinabove defined.

The potential usefulness of the cis-isomers of the alkylated derivatives of trans BM123 is demonstrated by their ability to control systemic lethal infections in mice. These new substances show high in vivo antibacterial activity in mice against Escherichia coli US3 l l when administered by a single subcutaneous dose to groups of Carworth Farms CF-l mice, weight about 20 gm., infected intraperitoneally with a lethal dose of this bacteria in a 10-3 trypticase soy broth TSP dilution of a 5 hour TSP blood culture. In Table VII below is set forth the in vivo activity of typical products of this invention against Escherichia coli US311 in mice.

TABLE VII Alive/Total Mice Dose-mg./kg. of Tested (7 days Compound body weight post infection) Cis-BMl23v 2 4/5 1/5 0.5 1/5 Cis-BM123v Oral Dose Alive/Total Mice (mg./kg. of body Tested (7 days weight) after infection) Cis-BMl23v Infected, non- 18/20 mice died treated controls within 3 days after infection Isopropyl- 2.0 5/5 cis-BMl23v 1.0 5/5 0.5 3/5 0.25 1/5 0.12 2/5 l-Propylpentyl- 2.0 5/5 cis-BMl23v 1.0 2/5 0.5 0/5 0.25 0/5 0.12 0/5 1,5-Dimethyl- 2.0 5/5 4-hexenyl-eis- 1.0 5/5 BMl23P 0.5 5/5 0.25 0/5 0.12 0/5 l-Benzyl-4- 2.0 5/5 piperidyl- 1.0 3/5 cis-BMl23v 0.5 0/5 0.25 0/5 0.12 0/5 Infected, 5/25 non-treated (3 days post controls infection) Fermentation Process Selected to Produce Primarily BM123 and trans-BMl23v Cultivation of Nocardia s

Inoculum Preparation for BMl23p and trans-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 scripings or washings of spores from an agar slant of the culture. The following medium is ordinarily used: Bacto-tryptone 5 gm.

Yeast extract 5 gm.

Beef extract 3 gm.

Glucose l0 gm.

Water to 1000 ml.

The flasks were incubated at a temperature from 25--29"C., preferably 280C. and agitated vigorously on a rotary shaker for 30 to 48 hours. 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 inocula are used to seed 12 liter batches of the same medium 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 inocula 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.6 Ammonium sulfate 3 gm.

Potassium phosphate, dibasic 3 gm.

Calcium carbonate 1 gum.

Magnesium sulfateheptahydrate 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 0.8 liters of sterile air per liter of broth per minute, and the fermenting mixture is agitated by an impeller driven at 15W300 revolutions per minute. The temperature is maintained at 25-290C., 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 BMl23p and trans-BM123P For the production of BMl23p and trans-BMl23v in tank fermentors, the following fermentation medium is preferably used: 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.

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--28"C. usually 26"C. The mash is aerated with sterile air at a rate of 0.34.5 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 is illustrated by the Examples which follow.

EXAMPLE 1.

Inocolum preparation for BM123A and trans-BM123v A typical medium used 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 5 gm.

Glucose 10 gm.

Water to 1000 ml.

Two 500 milliliter flasks each containing 100 milliliters of the above sterile medium were inoculated with 5 millilters each of a frozen vegitative 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 inoculum 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 gum.

Magnesium sulfate heptahydrate 1.5 gm.

Glucose 10 gum.

Water to 1000 ml.

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 28"C. and Hodags FD82 was used to seed a 3000 liter fermentation.

EXAMPLE 2.

Fermentation Employing Nocardia sp. NRRL 8050 and Medium Favoring the Production of BMl23 and trans-BMl23v 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 1200C. 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 260 C. using Hodage 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 7072 revolutions per minute. At the end of 67 hours of fermentation time the mash was harvested.

EXAMPLE 3.

Isolation of BM123p and trans-BMl23v 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 (a cross-linked dextran-epichlorohydrin cation exchange gel available from Pharmacia Fine Chemicals, Inc.). 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 G60 (2040 mesh) (a granular activated carbon available from Atlas Chemical Industries, Inc.). 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 in vacuo to about 7 liters of an aqueous phase and the pH adjusted from 4.5 to 6.0 with Amberlite IR--45 (OH-) resin (a weakly basic polystyrenepolyamine type anion exchange resin). The resin was removed by filtration and the filtrate was concentrated in vacuo to about 1 liter and then lyophilized to give 38 grams of material consisting primarily of BM123 along with a small amount of trans-BM123v (primarily trans-BMl23v2). 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 lyophilized to give 213 grams of material consisting of primarily of trans BM123v. 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 filtrate was concentrated in vacuo to about 1.5 liters and then lyophilized to give 56 grams of impure trans-BM123.

EXAMPLE 4.

Further Purification of trans-B Ml 23v A slurry of CM Sephadex C-25 (NH4] 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 trans-BM123v prepared as described in Example 3 was dissolved in about 10 milliliters of 2% aqueous ammonium cride and applied to the column. The column was then eluted with a gradient between 6 liters each of 2% and 4% aqueous ammonium chloride. Fractions of about 75 milliliters each were collected automatically every 15 minutes. Antibiotic trans-BM 123v was located by monitoring the column effluent in the ultra-violet and by bioautography of dipped paper disks on large agar plates seeded with Klebsiella pneumoniae strain AD. The majority of trans-BMl23v was located between fractions 71-107 inclusive.

One hundred thirty milliliters of granular Darco O60 (2040 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 chromatography were combined and passed through the granular carbon column. The charged column was washed with 600 milliliters of water and then developed with I liter of 50% aqueous acetone. The eluates, both of which contains trans-BM123v, where concentrated to aqueous phases in vacuo and lyophilized to give a total of 836 milligrams of trans-BMl23v as the hydrochloride salt. A microanalytical sample was obtained by subjecting the above material to a repeat of the above process.

Antibiotic trans-BM123v does not possess a definite melting point, but gradual decomposition starts in the vicinity of 200 C. Micronalysis 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 trans-BM123v gave a U.V. absorption maximum at 286 nm with E1%/1 cm = 250.

The position of this of this maximum did not change with pH. Trans-BM123v had a specific rotation of [α]D25 = +71 (C = 0.97 in water) and exhibited characteristic abdorption 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.

EXAMPLE 5. Isolation of trans-BM123v, 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 height of approximately 70 centimeters. The excess 2% aqueous sodium chloride was drained away and 4.11 gram of a sample containing primarily trans-BM123v1 along with some trans-BMl23v2 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 20o and 40o aqueous sodium chloride. Fractions of about 75 milliliters each were collected automatically every 15 minutes. Antibiotic trans-BMl23v 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 trans-BM123v was located between fractions 64-90 inclusive; the initial fractions (64-80) contained a mixture of trans-BM123v1 and trans-BM123v2 whereas the later fractions (81-90) contained essentially pure trans-BM123v1.

One hundred milliliters of granular Darco G-60 (20-40 mesh) was suspended in water, transferred to a glass column, allowed to settle and the escess 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 500 milliliters of water and then developed with 500 milliliters of 10% aqueous methanol followed by I liter of 50 aqueous methanol. The 50% aqueous methanol eluate, which contained the majority of trans-BM123v1, was adjusted from pH 5.9 to 6.0 with Amberlite IR-45 (OH-1) resin. The resin was removed by filatration and the filtrate was concentrated in vacuo to an aqueous phase and lyophilized to give 294 milligrams of white amorphous trans-BMl23v1 as the hydrochloride salt.

Antibiotic trans-BM123v1 does not possess a definite melting point, but gradual decomposition statrs in the vicinity of 200 C. Microanylsis of a sample equilibrated humidity gave C, 37.84%; H, 5.73; N, 15.58; Cl (ionic), 10.01% loss of dryving 10.45%. In methanol trans-BM123v1 gave a U.V. absorption maximum at 286 nm with E l cm = 225. The position of this maximum did not change with pH.

Trans-BM123v1 had a specific rotation of +55 (C=0.803 in water) and 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-'.

EXAMPLE 6.

Isolation of trans-BM123v2 A 25 grams sample containing primarily trans-BMl23v2 and BM123p, prepared as described in Example 3, was dissolved in about 120 milliliters of 20/o aqueous sodium chloride and applied to a column containing 1800 ml. of CM Sephadex C-25 INa+l in 2% aqueous sodium chloride. The column was then eluated 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 trans-BMl23v was located by monitoring the column fractions in the ultraviolet. The majority of trans-BM123P was located between fraction 7-18 inclusive; the initial fractions (7-15) contained essentially pure trans-BM123v2 and the later fractions (16-18) contained a mixture of trans BM123v1 and trans-BM123v2.

Six hundred milliliters of granular Darco G-20 (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 trans-BM123v2 as the hydrochloride salt. The 50% aqueous methanol eluate was adjusted from pH 4.6 to 6.1 with Amberlites (OH-) resin. The resin was removed by filtration and the filtrate was concentrated in vacuo to an aqueous phase and lyophilized to give 3.645 grams of slightly less pure white amorphous trans-BM123v2 as the hydrochloride salt.

Antibiotic trans-BM123v2 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 C. 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 10/ methanol trans-B Ml 123s2 gave a U.V. absorption maximum at 286 nm with E 1 'cm= 220. The position of this maximum did not change with pH. Trans-BMl23v2had a specific rotation of +60 (C=0.51 in water) and 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.

EXAMPLE 7.

Paper Partition and Thin Layer Chromatography of BM123P and trans-BM123v The BM123 antibiotics 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 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. Representative Rf values are listed in Table VII below: TABLE VIII Component RF trans- 0.85 BM123v BM 123p 0.50,0.70 The fi component was a mixture of two antibiotics using this system. BM123 was composed of a major antibiotic (Rf = 0.50) called BMl231 and a minor antibiotic (Rf - 0.70) called BMI23,B2.

The BM123 antibiotics can also be distinguished by thin layer chromatography. For this purpose precoated 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 substance to be chromatographed (about 2040 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 one hour. The antibiotics were detected by using either standard ninhydrin or Sakaguchi spray reagents. Representative Rf values are listed in Table IX below: TABLE IX Component Rf BM123v 0.17,0.23 BM123p 0.08,0.14 Both BM123p and trans-BM123v were a mixture of two components using this system. BM123p was composed of a major component (Rf = 0.08) which was BM123p, and a minor component (Rf = 0.14) which was BMI23,B2. The less polar component of trans-BM123 (Rf = 0.23) was trans-BM123v, and the more polar component (Rf = 0.17) was trans-BM123i,2.

EXAMPLE 8.

General Procedure for Reductive Alkylation of Antibiotic trans-BM123P or Antibiotic cis-BM123v To a stirred solution of 100 mg. of antibiotic trans-BM123v or antibiotic cis BM123v in 20 ml. of methanol is added 5 ml. (or 5 g.) of the appropriate aldehyde or ketone and 100 mg. of sodium cyanoborohydride. The pH of the resulting solution is maintained at about 7.0 with 0.1N methanolic hydrogen chloride over a 30 to 24 hour period. The reaction is monitored by thin layer chromatography to the disappearance of the starting antibiotic. The reaction mixture is then filtered and the filtrate is evaporated to dryness. The residue is triturated with 3 ml. of methanol and filtered. The filtrate is diluted with 50 ml. of acetone and the precipitate which forms is removed by filtration and dried. The methanol solvent. may be replaced by 20 ml. of water wherever the starting aldehyde or ketone is water soluble.

EXAMPLE 9.

Preparation of methyl-trans-BM123v To a solution of 1.0 g. of trans-BM123v and 2.5 ml. of a 37% aqueous formaldehyde solution in 50 ml. of water was added, portionwise, 400 mg. of sodium cyanoborohydride. The pH of the reaction mixture was maintained at 7.0 with IN hydrochloric acid during this addition. The reaction mixture was stirred an additional ten minutes at room temperature and then evaporated to dryness in vacuo. The residue was triturated with 20 ml. of methanol, filtered and the filtrate diluted with 250 ml. of acetone. The product which precipitated was removed by filtration and dried; yield, 667 mg.

EXAMPLE 10.

Preparation of isopropyl-trans-BM123v To a solution of 200 mg. of trans-BM123v in 30 ml. of methanol was added 5 ml. of acetone. To this solution was added 139 mg. of sodium cyanborohydride and the reaction mixture was stirred at room temperature for 30 minutes. During this time the pH of the reaction mixture was maintained between 7.4 and 7.8 by the addition of 0.1N methanolic hydrogen chloride. The small amount of precipitate which had been formed was removed by filtration and the filtrate was evaporated to dryness in vacuo. The residue was triturated with two ml. of methanol and filtered. The filtrate was diluted with 100 ml. of acetone and the solid product that separated was removed by filtration and dried; yield, 184 mg.

EXAMPLE 11.

Preparation of p-phenylethyl-trans-BM123v To a solution of 200 mg. of trans-BM123v in 15 ml. of water and 25 ml. of acetonitrile was added a solution of 2 ml. of phenylacetaldehyde in 4 ml. of ethanol.

To this was added 103 mg. of sodium cyanoborohydride. The reaction mixture was stirred at room temperature for thirty minutes during which time the pH of the mixture was maintained at 7 with 0.2N hydrochloric acid. The reaction mixture was then filtered and the filtrate was evaporated to dryness in vacuo. The residue was triturated with two ml. of methanol and filtered. The filtrate was diluted with 100 ml. of acetone and the product that separated was removed by filtration and dried; yield, 180 mg.

EXAMPLE 12.

Preparation of 1,3,3-trimethylbutyl-trans-BM123v To a solution of 200 mg. of trans-B M123v hydrochloride in 50 ml. of methanol was added 3 ml. of 4,4-dimethyl-2-pentanone and 106 mg. of sodium cyanoborohydride. The reaction solution was maintained at pH 7 by the dropwise addition of methanolic hydrogen chloride. The reaction was stirred at room temperature for 18 hours and filtered. The filtrate was evaporated to dryness in vacuo. The residue was dissolved in 3 ml. of methanol, diluted with 50 ml. of acetone and filtered, yield 125 mg.

EXAMPLE 13.

Preparation of l-methylphenethyl-trans-BM 23v To a solution of 200 mg. of trans-BM123v in 50 ml. of methanol was added 5 ml. of phenylacetone. To this solution was added 170 mg. of sodium cyanoborohydride and the reaction mixture stirred at room temperature for 3 and a half hours. During this time the pH of the reaction mixture was maintained at 7.0 with methanol saturated with hydrogen chloride gas. Reaction mixture was concentrated to about 5 ml. volume, diluted with two ml. of methanol, and filtered.

Filtrate was poured into 100 ml. of acetone and the solid product that separated was removed by filtration and dried; yield 233 mg.

EXAMPLE 14.

Preparation of 1 -methylnonyl-trans-B Ml 23v Sodium cyanoborohydride (100 mg.) was added to a solution of trans-BM123v (200 mg.) and 2-decanone (1 ml.) in 40 ml. of methanol. The pH of the solution was adjusted to 7.0 and maintained at 7.0 j 0.2 by the addition of 0.1N methanolic hydrogen chloride as necessary. After 19.5 hours the reaction mixture was filtered and the filtrate was concentrated in vacuo at 350C. The residue was slurried in 5 ml. of methanol and filtered. The filtrate was added to 50 ml. of acetone. The off white solid which precipitated was collected by filtration, washed with acetone, and dried in vacuo. The yield of crude l-methylnonyl-trans-B M123v was 167 mg.

EXAMPLE 15.

Preparation of 1,3-dimethylbutyl-trans-BM123v To a solution of 210 mg. of trans-BM123v in 50 ml. of methanol was added 5 ml. of methyl isobutyl ketone. To this solution was added 166 mg. of sodium cyanoborohydride and the reaction mixture stirred at room temperature for five hours. During this time the pH of the reaction mixture was maintained at 7.0 with methanol saturated with hydrogen chloride gas. Reaction mixture was evaporated to dryness, in vacuo. The residue was triturated with two ml. of methanol and filtered. The filtrate was diluted with 100 ml. of acetone and the solid product that separated was removed by filtration and dried; yield, 210 mg.

EXAMPLE 16.

Preparation of isopropyl-trans-BM123P, A mixture of 50 mg. of trans-BM123v1, 5 ml. of acetone and 60 mg. of sodium cyanoborohydride in 35 ml. of methanol was stirred at room temperature for 40 minutes. The pH of the solution was maintained at 7 by the dropwise addition of methanolic hydrogen chloride solution. The mixture was evaporated to dryness in vacuo. The residue was triturated with 5 ml. of methanol and the resulting solution was diluted with 50 ml. of acetone; yield, 49 mg.

By following the above procedure but substituting for acetone the carbonyl compound starting materials set forth in the following table, there is obtained the corresponding alkylated products.

Starting Material Product formaldehyde* methyl-trans-B Ml 23v1 acetaldehyde ethyl-trans-B M 123v, propionaldehyde n-propyl-trans-B M 123v, n-butyraldehyde n-butyl-trans-BM 123v, isobutyraldehyde isobutyl-trans-BM123v, 2-butanone sec-butyl-trans-BM 123v1 3-pentanone l-ethylbutyl-trans-BM 123v, methyl isobutyl ketone 1 ,3-dimethylbutyl-trans-BM 123v, * As 37% aqueous solution in water as solvent instead of methanol.

EXAMPLE 17.

Preparation of iso-propyl-trans-B Ml 23v2 A mixture of 41 mg. of trans-BMl23v2, 5 ml. of acetone and 50 mg. of sodium cyanoborohydride in 35 ml. of methanol was stirred at room temperature for 40 minutes. The pH of the solution was maintained at 7 by the dropwise addition of a methanolic hydrogen chloride solution (saturated). The mixture was filtered and evaporated to dryness in vacuo. The residue was triturated with 5 ml. of methanol and the resulting solution was diluted with 50 ml. of acetone; yield, 46 mg.

By following the above procedure but substituting for acetone the carbonyl compound starting materials set forth in the following table, there is obtained the corresponding alkylated products.

Starting Material Product formaldehyde* methyl-trans-BM123v2 acetaldehyde ethyl-trans-BM l23v2 propionaldehyde n-propyl-trans-BM123v2 n-butyraldehyde n-butyl-trans-B Ml 23v2 isobutyraldehyde isobutyl-trans-BM123v2 2-butanone sec-butyl-trans-BM 1 23v2 3-pentanone 1 -ethylbutyl-trans-B M123v2 methyl isobutyl ketone 1,3-dimethylbutyl-trans-BM123v2 * As 37% aqueous solution in water as solvent instead of methanol.

EXAMPLE 18.

Preparation of 1-methyl-2-phenyl-ethyl-trans-BM123v2 A mixture of 200 mg. of trans-BM123v2, 5 ml. of phenylacetone and 170 mg. of sodium cyanoborohydride in 50 ml. of methanol was stirred at room temperature for 3 hours and 45 minutes. During this time the pH of the reaction mixture was maintained at 7 with drop-wise addition of a methanolic hydrogen chloride solution (saturated). The mixture was evaporated to dryness in vacuo. The residue was triturated with 5 ml. of methanol and the resulting methanol solution was diluted with approximately 50 ml. of acetone, yield 233 mg.

EXAMPLE 19.

Preparation of isopropyl-cis-BM123v A solution of 200 mg. of isopropyl-trans-BM123v in 200 ml. of water is photolyzed with a "Hanovia" light in a water-jecketed, three-necked, round bottom flask for a period of one hour, during which time the maximum ultraviolet absorption of the reaction solution solution shifts from 290 m to 275 m . The product is then

EXAMPLE 20 Preparation of I-propylpentyl-cis-BM123v A solution of 175 mg. of 1-propylpentyl-trans-BM123v is 175 ml. of water and 45 ml. of methanol was irradiated with a high pressure ultraviolet lamp for one hour and 15 minutes. The solution was then evaporated to dryness in vacuo to yield 169 mg. of product.

EXAMPLE 21.

Preparation of 1,5-dimethyl-4-hexenyl-cis-BM123v A solution of 200 mg. of 1,5-dimethyl-4-hexenyl-trans-BM123v in 200 ml. of water was irradiated with a high pressure ultraviolet lamp for one hour. The solution was then freeze dried to yield 182 mg. of product.

EXAMPLE 22.

Preparation of 1 -benzyl-4-piperidyl-cis-B M I 23v A solution of 200 mg. of l-benzyl-4-piperidyl-trans-BMl23v in 200 ml. of water and 20 ml. of methanol was irradiated with a high pressure ultraviolet lamp for one hour. The solution was then freeze dried to yield 125 mg. of product.

EXAMPLE 23.

Preparation of 3,5-dimethylcyclohexyl-trans-B Ml 23v A solution of 200 mg. of trans-BM123v, 5 ml. of 3,5-dimethylcyclohexanone and 200 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored at room temperature for 1 hour. During this time the pH of the solution was maintained at 7 with the addition of a saturated solution of hydrogen chloride in methanol. The reaction was triturated with 3 ml. of methanol, filtered and the filtrate was diluted with 40 ml. of acetone, yield 200 mg.

EXAMPLE 24.

Preparation of 2,4-dimethylcyclopentyl-trans-BM l23v A solution of 206 mg. of trans-BM123v, 3 ml. of 2,4-dimethylcyclopentanone and 104 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored at room temperature for 6 hours. During this time the pH of the solution was maintained at 7 with the addition of a saturated solution of hydrogen chloride in methanol. The reaction was triturated with 3 ml. of methanol, filtered and the filtrate was diluted with 40 ml. of acetone, yield 101 mg.

EXAMPLE 25.

Preparation of 3-methylcyclohexyl-trans-8M123v A solution of 200 mg. of trans-BM123v, 1.5 ml. of 3-methylcyclohexanone and 200 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored at room temperature for 2 hours. During this time the pH of the solution was maintained at 7 with the addition of a saturated solution of hydrogen chloride in methanol. The reaction was triturated with 3 ml. of methanol, filtered and the filtrate was diluted with 40 ml. of acetone, yield 200 mg.

EXAMPLE 26.

Preparation of 2,4,4-trimethylcyclopentyl-trans-B Ml 23v A solution of 200 mg. of trans-BM123v, 5 ml. of 2,4,4-trimethylcyclopentanone and 179 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored at room temperature for 24 hours. During this time the pH of the solution was maintained at 7 with the addition of a saturated solution of hydrogen chloride in methanol. The reaction was triturated with 3 ml. of methanol, filtered and the filtrate was diluted with 40 ml. of acetone, yield 176 mg.

EXAMPLE 27.

Preparation of 2-methylcyclopentyl-trans-B M 1 23v A solution of 211 mg. of trans-BM123v, 3 ml. of 2-methylcyclopentanone and 98 mg. of sodium cyanoborohydride in 50 ml. of methanol was stored at room temperature for 3.5 hours. During this time the pH of the solution was maintained at 7 with the addition of a saturated solution of hydrogen chloride in methanol. The reaction was triturated with 3 ml. of methanol, filtered and the filtrate was diluted with 40 ml. of acetone, yield 157 mg.

EXAMPLE 28.

Preparation of cis-BM123s A solution of 200 mg. of trans-BM123v in 200 ml. of water is photolyzed with a HanoviaO light in a water-jacketed, three-necked, round bottom flask for a period of time of half an hour, during which time the maximum U.V. absorption of the reaction solution shifts from 290 me to 275 my. The reaction is best monitored by taking aliquots at various time intervals and measuring the ultraviolet absorption.

The reaction is complete when the maximum absorption shifts from 290 my to 275 mfs. The product is then recovered by lyophilization.

The above procedure is repeated but with the solution being photolyzed for 1.5 hours. Lyophilization yields 170 mg. of cis-BM123v.

Claims (15)

WHAT WE CLAIM IS:

1. Antibiotic cis-BM123vt of the formula

and the pharmacologically acceptable acid-addition salts thereof.

2. Antibiotic cis-BMI23v2 of the formula:

and the pharmacologically acceptable acid-addition salts thereof.

3. A mixture of antibiotic cis-BM123vl, as defined in Claim 1, with antibiotic cis-BM123v2, as defined in Claim .

4. A process of preparing cis-BM123v1, characterized by irradiating an aqueous solution of trans-BM123v1 with light of a wavelength of from 2,500A to 4,000A and continuing said irradiation until the trans-isomer is substantially converted to the cis-isomer.

5. A process of preparing cis-BM 123v2, characterized by irradiating an aqueous solution of trans-BMl23v2 with light of a wavelength of from 2,500A to 4,000A and continuing said irradiation until the trans-isomer is substantially converted to the cis-isomer.

6. Compounds of the formulae:

wherein R is a moiety of the formulae:

or a mixture of moieties of formulae (V) and (VI) and R, is hydrogen, alkyl having up to 8 carbon atoms, phenyl, benzyl or alkenyl having from 2 to 8 carbon atoms; and the pharmacologically acceptable acid-addition salts thereof.

7. A compound of the formula:

wherein R is as defined in Claim 6, R2 is alkyl having up to three carbon atoms; R3 is alkyl having up to 8 carbon atoms, N,N-di(alkyl C1-C4)amino(alkyl C1-C4), chloroalkyl having up to 4 carbon atoms, phenyl, benzyl, p-phenylethyl, alkenyl having from 2 to 8 carbon atoms, or hydroxyalkyl having up to 4 carbon atoms; or R2 and R3 taken together with the associated methylidyne group is cyclobutyl, cyclopentyl, monomethylcyclopentyl, dimethylcyclopentyl, trimethylcyclopentyl, cyclohexyl, monomethylcyclohexyl, dimethylcyclohexyl, trimethylcyclohexyl, cycloheptyl or a moiety of the formula:

wherein R4 is hydrogen, alkyl having up to 4 carbon atoms, phenyl or benzyl; and the pharmacologically acceptable acid-addition salts thereof.

8. A compound according to Claim 7, wherein R, R2 and R3 are as defined in Claim 7 except that R2 and R3 taken together with the associated methylidyne group cannot be a moeity of the formula:

9. Compounds of the formulae:

wherein R is as defined in Claim 6, R, is hydrogen or alkyl having up to four carbon atoms, R2 is alkyl having up to three carbon atoms and R3 is alkyl having up to 8 carbon atoms or alkenyl having from 2 to 8 carbon atoms.

10. A process of preparing compounds of the formulae:

wherein R and R, are as defined in Claim 6, characterized by irradiating an aqueous solution of compounds of the formulae:

wherein R and R are as hereinabove defined with light of a wavelength of from 2,500A to 4,oooA and continuing said irradiation until the trans-isomer is substantially converted to the cis-isomer.

11. A process of preparing compounds of the formula:

wherein R, R2 and R3 are as defined in Claim 7, characterized by irradiating an aqueous solution of compounds of the formula:

wherein R, R2 and R3 are as hereinabove defined with light of a wavelength of from 2,500A to 4,000A and continuing said irradiation until the trans-isomer is substantially converted to the cis-isomer.

12. A process of preparing compounds of the formulae:

wherein R and R, are as defined in Claim 6, or characterized by alkylating an amine of the formula:

wherein R is as hereinabove defined with an aldehyde of the formula: R1-CHO wherein R, is as hereinabove defined, in the presence of a reducing agent in a solvent inert to the reactants for a period of time sufficient for a substantial degree of reductive alkylation to take place.

13. A process of preparing compounds of the formula:

wherein R, R2 and R3 are as defined in Claim 7, characterized by alkylating an amine of the formula:

wherein R is as hereinbefore defined with a ketone of the formula:

wherein R2 and R3 are as hereinbelow defined in the presence of a reducing agent in a solvent inert to the reactants for a period of time sufficient for a substantial degree of reductive alkylation to take place.

14. A compound according to any one of Claims 6, 7 or 9 and specifically identified herein.

15. A process of preparing a compound according to any one of Claims 6, 7 or 9 and substantially as described in any one of Examples 19-27 herein.