NO129960B - - Google Patents

Description

Microbiological method for the production of

a new antibiotic called Antibioticum 66-40,

later also termed sisomicin.

The present invention relates to a microbiological method for the production of a new antibiotic with a broad spectrum and designated Antibioticum 66-40. It is later also designated as sisomicin. This new antibiotic counteracts the growth of gram-positive and gram-negative bacteria. According to the invention, it can be prepared, isolated and used in its free form or in the form of one of the following derivatives: a solvate, a salt, a solvate of such, or a condensation product with an aldehyde.

Antibioticum 66-40 is a basic pseudo-oligosaccharide which can easily be distinguished from other pseudo-oligosaccharides by its biological, physical and chemical properties as described below.

Antibioticum 66-40 has the following general formula, which is to understand that no stereochemical instructions must be derived from this:

The characteristic main feature of the invention is that a strain of the species Micromonospora inyoensis" is cultivated in an aqueous nutrient medium under aerobic conditions, after which the said antibiotic active product is isolated in free form or in the form of a solvate and, if desired, the product thus obtained is transferred to an acid addition salt or a solvate thereof, or to a condensation product with an aldehyde.

Micromonospora inyoensis was isolated from a soil sample taken in the Inyo National Forest in the White Mountains, California.

(In what follows, it is sometimes referred to as M.inyoensis). One of the characteristics of this species is its ability to form Antibioticum 66-40. It is aerobic and grows well on the surface of various solid and liquid nutrient media. It shows particularly good growth and formation of the antibiotic active product under submerged, aerobic conditions.

A strain of M.inyoensis is deposited as living organisms in the permanent collection of The Northern Utilization and Research Division, Agricultural Research Service, US Department of Agriculture, Peoria, Illinois, where it is assigned the identification number NRRL 3292. Subcultures of M.inyoensis NRRL 3292 are available to the public on request to the above-mentioned institution.

This strain NRRL 3292 has proven to be well suited for use in the method according to the invention.

M.inyoensis can be distinguished from other species of Micromonospora by different taxonomic parameters. This is explained in more detail in the tabular compilation below. (Table A).

Regarding culture characteristics for M.inyoensis, the following is recommended in addition to what appears above: After 14 days of cultivation of M.inyoensis at 24 - 26°C in an agar medium containing 3% "NZ Amine Type A", 1% dextrose and 1 .5% agar growth is observed to be from fairly good to poor. Macroscopically, no aerial mycelium is visible. From time to time, a few well-developed colonies appear at a late stage in the inoculation area. On some plates, a faint reddish brown diffusible pigment associated with the colonies is observed.

When describing the color formation, the following reference system is used here: Color designations consist of two indicators. The first is a color name taken from the "Descriptive Color Name Dictionary" by Tayler, Knoche and Granville, published in 1950 by The Container Corporation of America, USA with a color chip number corresponding to the color name. This number is taken from "The Color Harmony Manual", 4th edition, published in 1958 by The Container Corporation of America. The second designator consists of a color name and number referring to the synonym or near synonym in the National Bureau of Standards (USA), Circular 553, 1 Nov-Ember 1955.

The color of the surface of the colonies varies from Tile Red g5ne-Strong Brown 55 to Brown Mahogany mopi. Microscopically, the mycelium is elongated and branched, regular and not transversely divided, observed by phase contrast microscopy. The diameter of the mycelium is approximately 0.5 u. The spores are borne individually on simple sporophores with diameters from 1.0 to 1.5 |x. The grooves have rough walls and are egg-shaped to spherical. M.inyoensis grows well at 28 - 37°C, no growth takes place at 50°C. Growth is poor on glucose-aspargin-agar medium. A growing colony of M.inyoensis hydrolyzes gelatin, milk, starch and reduces nitrates to nitrites when the test is carried out as described in Gordon et al. J. Bacteriology 69, 147 (1956) and 73, 15 (1957). Sucrose can be used as a source of carbon.

Further culture characteristics for M.inyoensis are set out in Table B below.

M.inyoensis is able to make use of different sources for carbon and nitrogen. In table C below, observations on the utilization of carbohydrates are shown. A visual assessment of the degree of growth is made in a medium consisting of 0.5% yeast extract, 1% carbohydrate and 1.5% agar in distilled water.

In table D below, utilization of nitrogen is shown, as determined by visual assessment of growth on agar plates in a medium consisting of 1% glucose, 1.5% agar and a nitrogen source as indicated in the table, all in distilled water.

Antibioticum 66-40 has a characteristic absorption spectrum in the infrared region, in mineral oil (Nujol) as shown in fig. 1 in the attached drawing. The more representative absorption bands are indicated in the following table E where: S =. strongly

M = medium

W = weak

br = broad VS = very strong

VW = very weak

Antibioticum 66-40 also has a characteristic nuclear magnetic resonance spectrum [NMR spectrum], thus as shown in fig. 2 in the attached drawing. The NMR spectrum was obtained using the "Varian A-60-A" spectrometer

on a solution of Antibioticum 66-40 in deuterium oxide (DgO), (approx. 0.4 ml with a concentration of approx. 20 mg/ml). The spectrum is registered in parts per parts per million (ppm) from the sodium salt of 3-(trimethylsilo)-propanesulfonic acid, the internal standard.

In the following table F, additional data regarding qualitative samples with Antibioticum 66-40 (in the form of free base) and physical constants for the same are listed.

TABLE F

a) Color reactions

b) Physical constants Absorption in the ultraviolet range: transparent in the range between 220 and 400 mp.. c) Solubility of Antibioticum 66-40 base in various solvents

+) The terminology in English is according to U.S.Pharmacopia XVIII, page 8.

Table G below shows the mass spectrum for Antibioticum 66-40 in the form of free base. In this table, m/e in the heading denotes the ratio of mass to charge, while Rel. Int. denotes relative intensity, and as the name indicates, the intensity for the peaks at the different ratios of mass to charge (m/e) is compared to the same for the peak m/e = 118.

As mentioned above, the invention also encompasses the production of derivatives of Antibioticum 66-40, namely its solvates (e.g. hydrates), salts and solvates thereof, as well as its condensation products with aldehydes. Such derivatives should, of course, be acceptable from a pharmaceutical point of view.

Some of these derivatives are described in detail below.

As Antibioticum 66-40 is basic, it does not readily form toxic salts with organic and inorganic acids such as e.g. hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, stearic acid, propionic acid, tartaric acid and benzoic acid. Partially neutralized polybasic acids can also be used, neutralized e.g. with an inorganic base such as sodium hydroxide or with an organic base. In general, the salts with mineral acids, such as those formed with hydrochloric acid, sulfuric acid, phosphoric acid and the like, are soluble in water and can be obtained by concentrating or lyophilizing aqueous solutions of the same or by precipitation with a water-miscible organic solvent, preferably a lower aliphatic alcohol or a lower aliphatic ketone. However, it should be noted that hydrochlorides of Antibioticum 66-40 show a significant solubility in methanol and are therefore atypical. The hydrochlorides can be precipitated from aqueous solutions by adding a lower alkyl ketone such as acetone. By titrating an aqueous solution of Antibioticum 66-40 with less than the stoichiometric amount of acid, it is possible to form partial acid addition salts. As used herein, the term "acid addition salts" includes all such compounds.

The non-toxic acid addition salts described above show essentially the same antibiotic spectrum as the free nitrogen base, but they differ from this with respect to solubility properties.

Antibioticum 66-40 and its acid addition salts form hydrates with water and form other solvates with organic solvents. It is therefore obvious that in the isolation processes described here, this antibiotic is normally obtained as a hydrate, while its acid addition salts are normally obtained as solvates with lower aliphatic alcohols. These hydrates and solvates are relatively stable. Therefore, the isolated products contain water or other solvent, generally in an amount of approx. 1 mole per moles of antibiotic.

Physical data for some derivatives of this type are compiled in table H below.

TABLE H

Physical constants for Antibioticum 66- 40- salts Antibioticum 66- 40- hydrochloride as methanol solvate [a]J<5> (C = 1% in H20) + 112.2°

The analysis corresponds to the gross formula<n:><C>lgH37<N>5<0>7<v5>HCl-CH30H

Ultraviolet absorption: Transparent in the range between 220 and 400 mu-.

Antibiotic 66-40-sulphate as methanol solvate

[a]p<5> (C = 1% in H 2 O) + 105.1°

The analysis corresponds to the gross formula: (C19H37N507)2•5H2S04•2CH30H

Ultraviolet absorption: Transparent in the range between 220 and 400 mu-.

As mentioned above, Antibioticum 66-40 also does not form toxic condensation products with aldehydes

by means of methods which are known per se. The preferred products which come into consideration here and which are probably Schiff bases are those which are produced by condensing Antibioticum 66-40 with aldehydes which have up to 12 carbon atoms in the molecule. Among such aldehydes are aliphatic, alicyclic, aromatic and heterocyclic compounds. Aldehydes used

with a closed ring structure can have substituents such as hydroxyl, halogen, nitro, lower alkoxy, lower aBsanoyloxy and the like. Examples of aldehydes can be mentioned: acetaldehyde, crotonaldehyde, furfural, cyclopen-

tylacetaldehyde, vanillin, veratraldehyde, benzaldehyde, salicylaldehyde and pyridoxal.

It should be noted that these condensation products are not stable in the presence of water.

As mentioned above, Antibioticum 66-40 is formed when the organism that builds it up, namely M.inyoensis, is grown in an aqueous nutrient medium under aerobic and preferably submerged conditions.

For the production of limited quantities of this antibiotic, surface cultures in bottles or shake flasks can be used instead of submerged conditions. The nutrient medium in which the organism is grown contains a source of carbon, e.g. an assimilable carbohydrate. An assimilable nitrogen compound or an assimilable proteinaceous material is also required. Among the preferred sources of carbon are glucose, maltose, mannose, sucrose, starch, corn starch and the like. Among the preferred sources of nitrogen are corn starch liquid, yeast extracts, soybean meal, meat peptones, casein hydrolysates, meat extracts and the like. Combinations of these sources for carbon and nitrogen can be used with advantage. It is generally not necessary to add trace elements, as tap water is used in the nutrient medium. However, the addition of cobalt salts has been found to be beneficial.

The production of Antibioticum 66-40 can be carried out at any temperature which leads to a satisfactory growth of microorganisms, e.g. between 20 and 40°C, preferably 25 - 35°C.

Optimum 1st production is usually achieved in the course of 3 - 7 days. The medium's pH value generally remains fairly close up to 7 during the fermentation. The final pH value is partly dependent on the possible presence of buffers, and is advantageously set at around 8.0 for sterilization. When the growth takes place in large vessels and tanks, it is desirable to prepare a vegetative inoculation material in a nutrient medium by inoculating this with a soil culture or a culture on an inclined substrate or with a lyophilized culture of the organism. When an active inoculation material has been obtained in this way, this is transferred under aseptic conditions to sterile vessels or tanks. The medium in which the vegetative inoculation material is produced can be the same as, or different from, that used for the production of the antibiotic agent in tanks, provided that good growth of the microorganism is achieved.

After completion of the fermentation, the medium can be worked up, e.g. in the following way: the entire medium's pH value is set to approx. 2

with mineral acid, preferably aqueous sulfuric acid, whereby the basic water-soluble antibiotic is released from the mycelium and dissolved in the aqueous fermentation medium. The entire mixture is filtered to remove the nutrient medium, after which the filtrate is neutralized with the subsequent addition of oxalic acid to precipitate calcium ions. After further filtration and neutrality adjustment, preferably with ammonia, the clear neutralized filtrate is passed through an ion exchange resin, preferably of the "IRC-50 Amberlite" type in the ammonium form. Examples of resins of this type which can be used here for both anionic and cationic exchange can be found in "Handbook of Chemistry and Physics", 42nd edition, Chemical Rubber Publishing Company, Cleveland, Ohio (1960). The spent nutrient medium is discarded, and the antibiotic is eluted from the resin with ammonia. The eluate is concentrated and evaporated to a residue consisting of impure Antibioticum 66-40 with a potential of approx. 5O0 micrograms per mg, determined by the method described below.

Purification of the impure antibiotic can be accomplished using an anion exchange resin, preferably "Dowex 1 X 2" or an "Amberlite IRA 401S" type. It can then be adsorbed from an aqueous solution on the column and eluted from this with distilled water. The material obtained in this way shows a potential of approximately 900 micrograms per mg. Alternatively, the impure antibiotic can be purified by e.g. column chromatography on cellulose using a solvent system consisting of chloroform, methanol, 17% ammonium hydroxide (2:1:1). The solvent system's upper phase is used to first "wet" the column, while the lower phase is used for elution purposes. The antibiotic agent can be placed in the column by adsorption from a concentrated solution in the upper phase of the above-mentioned solvent system.

To examine the different preparations for potential expressed as micrograms per mg of Antibioticum 66-40, a standard method, the "cylinder cup assay method", is generally used, using Staphylococcus aureus ATCC 6538P (ATCC = American Type Culture Collection) as the test organism. This method is completely analogous to that described by Oden et al. in "Antimicrobial Agents and Chemotherapy" (1963): A microgram of the antibiotic activity is the amount of material which produces a zone response of 16.3 + 0.9 mm under the conditions of the test method, and is expressed in micrograms per mg.

In the following, some embodiments of the method for the production of Antibioticum 66-40 by fermentation and isolation of the product are described as examples.

Example 1

Tank fermentation of Micromonospora inyoensis

Germination stage 1

Under aseptic conditions, a lyophilized culture (or cells obtained from a culture on slants) of M. inyoensis is added to a 300 ml shake flask containing 100 ml of the following sterile medium:

The flask and its contents are incubated for 4 days at 36°C on a rotary shaker (280 revolutions per minute, 50.8 mm stroke).

Germination step 2

Under aseptic conditions, 25 ml of the medium from germination stage 1 is transferred to a 2 liter shaking flask containing 500 ml of the sterile germination medium indicated above. The flask and its contents are incubated for 3 days at 28°C on a rotary shaker (280 rpm, 50.8 mm stroke).

Fermentation step

Under aseptic conditions, 500 ml of the medium obtained from germination stage 2 is transferred to a 14 liter fermentation tank containing 9.5 liters of the following sterile medium:

For sterilization of this medium, its pH value is set to 8. Fermentation takes place under aerobic conditions for 66 - 90 hours while stirring at 250 revolutions per minute. minute and with an air supply of 4.5 liters per liters per minute and at 22.5 atm. The potential of the antibiotic that had formed at the end of this period reaches a peak of 150 - 225 micrograms per ml and remains relatively constant. The fermentation medium's pH value changes quite a bit during the formation of Antibioticum 66-40, as it varies in the range between 6.8 and 7.3.

Example 2

Isolation of Antibioticum 66-40

The entire medium from example 1 is adjusted to a pH value of 2 with 6N sulfuric acid. (For the purposes of this example, amounts are given calculated on 170 liters of fermentation medium obtained by mixing acidified media from 17 loads obtained by proceeding as indicated in example 1). The acidified medium is stirred for approx. 15 minutes, after which it is filtered. The mycelium is washed with water, and the washing water is mixed with the filtrate. The pH value of the mixture is adjusted to 7 with 6N ammonium hydroxide. Oxalic acid is added to the thus neutralized filtrate in an amount sufficient to precipitate calcium and filtered. The mixture is neutralized again with ammonia. The filtrate is fed into an adsorption column for cation exchange and which contains 1500 - 2000 g of "IRC-50 Amberlite" in the ammonium form. The eluate is discarded, the resin is washed with water and eluted with 2N ammonia. There, fractions of 400 ml are collected and evaluated by disc testing with S. aureus ATCC 6538P.

The active fractions are mixed and evaporated to dryness in a vacuum, whereby approx. 28 g impure Antibioticum 66-40 with an activity of approx. 500 micrograms per mg.

Example 3

Purification of Antibiotics 66- 4Q

28 g of impure Antibioticum 66-40 thus obtained as stated in example 2 is dissolved in 10 ml of distilled water and fed to an adsorption column for anion exchange with "Dowex" 1X2: the hydroxyl form. 2CXX) g of this resin is suspended in water, and the suspension is brought into a column with a diameter of 63.5 mm and a height of 914.4 mm. The column is eluted with distilled water at a rate of approx. 23 ml per minute, as fractions of lOO ml are collected. It is checked with a conductivity meter and by disc testing against Staphylococcus aureus. The disk test produces a rough separation of antibiotic-containing eluate fractions from fractions that are free of antibiotic. To ensure that the fractions are properly combined, a portion of each fraction is subjected to paper chromatography using a chloroform:methanol:17% ammonium hydroxide system (2:1:1) as the lower phase. Each paper is coated with ninhydrin by solidification, and the eluates containing similar material are combined. They are then lyophilized, whereby you get approx. 5.7 g Antibioticum 66-40 which, when tested, shows a potential of approx. 900 micrograms per mg.

Example 4

Production of Antibioticum 66-4Q-sulphate

Dissolve 3.9 g of Antibioticum 66-40 in the form of a base prepared as described in example 3 in 60 ml of water, and the solution's pH value is adjusted to 4.5 with 6N sulfuric acid. The solution is then stirred with decolorizing charcoal for approx. half an hour and filtered; the filtrate is added to approx. 1 liter of methanol. The mixture is filtered and dried, whereby 4.8 g of Antibioticum 66-40-sulphate is obtained, which shows a potential of approx. 640 micrograms per mg.

Example 5

Alternative purification method for Antibioticum 66-40 over its sulfate

200 g of Whatman No. 1 cellulose powder is mixed with 20 ml of the upper phase of a solvent system consisting of chloroform:methanol:17% ammonium hydroxide (2:1:1) and packed in small segments in a column with an internal diameter of 6.35 mm and height 508 mm. The lower phase of the solvent system is allowed to flow through the column until a yellow band of impurities emerges. 2 g of Antibioticum 66-40-sulphate prepared as described in example 4 are dissolved in approx. 3 ml

of the upper solvent phase, is mixed with a little cellulose powder, dried in a vacuum and packed on top of the cellulose column. The lower phase is allowed to flow through the column at a rate of 1 ml per minute, collecting 15 ml fractions every 15 minutes.

Equal parts of each fraction are deposited in spots on filter paper and tested with ninhydrin reagent to determine the presence or absence of antibiotic. Paper chromatography of the antibiotic-containing fractions shows that the desired material is located between fractions 121 and 190.

Fractions 121 - 190 are mixed and evaporated to dryness, redissolved in water and passed through "IRA 401S" (an anion exchange resin) in the hydroxyl circuit. The eluate's pH value is adjusted to 4.5 with sulfuric acid. The eluate is then treated with charcoal, filtered and evaporated to a smaller volume. The concentrate is added. to an excess of methanol, and the white precipitate that is thereby formed is separated by filtration. The precipitate is dissolved in water and passed through an "IRA 401S" resin column in the hydroxyl form. The effluent is collected, evaporated and lyophilized, whereby approx. 300 mg Antibioticum 66-40 in the form of base and which shows a potential of approx. lOOO micrograms per mg.

Example 6

Production of Antibioticum 66- 4Q- hydrochloride

104.7 mg of Antibioticum 66-40 in the form of a base prepared as described in example 2 is dissolved in 4 ml of water, and the solution's pH value is adjusted to 4.5 with hydrochloric acid. The solution is then evaporated to dryness, and the residue is dissolved in methanol. The solution obtained is added to an excess of acetone and filtered. As a precipitate, you get 130 mg Antibioticum 66-40-hydrokioride with a potential of 753 micrograms per mg.

Example 7

Preparation of crystalline Antibiotic 66-4Q monohydrate A (26 x 2.5 cm) silica gel chromatography column is prepared using the lower (organic) phase of a solvent mixture consisting of isopropanol:CHC13:ammonium hydroxide in the volume ratio 1:2: 1 as development eluent. Dissolve 1.0 g of base in 5.0 ml of solvent mixture. The solution obtained is adsorbed on silica gel and chromatographed. 5.0 ml fractions are collected there, and the position of the desired fractions is determined by thin-layer chromatography on silica gel plates. The desired (44-78) fractions are combined and evaporated in vacuo, whereby a pale yellow syrup is obtained which, after azeotropic distillation with ethanol, crystallizes as pale yellow rosettes. The product obtained in this way is monohydrate which melts at approx. 185 - 190°C. The yield is approx. 650 mg.

Example 8 Preparation of benzaldehyde condensation products

of antibiotic 66- 4Q

5.0 g of Antibioticum 66-40 in 60 ml of absolute ethanol are treated with 5.9 g of benzaldehyde (a small excess of more than 5 equivalents), and the mixture is boiled under reflux for 1 hour. The resulting solution is cooled and filtered, whereby 7.0 g of a white crystalline solid is obtained, m.p. 123 - 126°C [α]^ = + 43.2°

(C = 0.3% in CHC±3). Elemental analysis indicates 5 benzaldehyde residues.

In a similar way, by using an equivalent amount of the relevant aldehyde instead of benzaldehyde, the following aldehyde condensation products such as e.g.

Antibiotic 66-40-salicylidene derivative: m.p. 159-163°C Antibiotic 66-40-pyridoxylidene derivative: " >300°C (a)p =51.1

(methanol)

Antibiotic 66-40-n-propylidene derivative: " 129-134°C Antibiotic 66-40-n-dodecylidene derivative: (a)o =+26>3(dioxane) Antibiotic 66-40-p-chlorobenzylidene-

derivative: " 208-213°C Antibiotic 66-40-5-nitrofurylidene-

derivative: " >300°C

Antibioticum 66-40 and its pharmaceutically acceptable solvates, salts and solvates thereof as well as condensation products with aldehydes have a broad antibiotic spectrum. As mentioned above, this antibiotic has the property that it counteracts the growth of gram-positive and gram-negative bacteria and it can be used alone or in combination with other antibiotics to prevent the growth of or reduce the number of bacteria in different environments. It can e.g. used for disinfection of glass laboratory equipment and equipment for dental treatment as well as medical equipment infected with Staphylococcus aureus or with other bacteria whose growth is prevented by Antibioticum 66-40. Because of its particularly effective activity against gram-negative bacteria, it is advantageous in combating infections caused by such microorganisms.

The in vitro activity of Antibioticum 66-40 against various Gram-positive and Gram-negative bacteria is shown in Table J below. The minimum inhibitory concentration (MIC) was determined using a yeast-meat extract as the test medium. A two-fold serial dilution technique was used. The MIC is the midpoint between the last clear reagent tube and the first cloudy reagent tube, determined by visual observation. The provisions were carried out under application-_3

deise of a 10 dilution of a 24 hour culture of the bacteria tested. All reagent tubes were incubated for 18 hours at 37°C. In table j, Antibioticum 66-40 was used with a potential of lOOO micrograms per mg.

The acute toxicity of Antibioticum 66-40 in the form of its sulfate was determined in a standardized way according to different routes, on mice weighing 18 - 20 g. Data for the toxicity are listed in table K below, expressed as free base.

TABLE K

Antibiotic 66-4Q's acute toxicity

Antibioticum 66-40 shows antibacterial activity against pathogenic bacterial infections produced in laboratory test animals, especially in mice. To determine in vivo the protective activity of Antibioticum 66-40 against infections in mice originating from pathogenic bacteria, the mice were twice given the antibiotic agent, once immediately before an intraperitoneal injection of the infecting bacteria and once 4 hours after such injection. The number of surviving animals was determined 48 hours after infection, and the data obtained analyzed according to standard methods to determine PDC_ values with 95% tolerance. Table L below shows the protective activity of Antibioticum 66-40 against various pathogenic bacteria.

TABLE L

Protective effect of Antibioticum 66-4Q on mice

It is clear from table L that the therapeutic index (LD5q/PD5q) for subcutaneous introduction of the antibiotic agent varies from 330 to 2400 as regards the gram-positive organisms and from 150 to 410 as regards the gram-negative organisms.

In addition, mice infected intraperitoneally with 8 doses of LD,_q Rickettsia akaria received 100% protection by subcutaneous injection of 2 mg of Antibioticum 66-40 once per dogn for 4 dogn.

Because of the above in vivo data, and especially because of the favorable therapeutic index shown by Antibiotic 66-40, it is obvious that Antibiotic 66-40 can be used to control and treat many different infections in mammals. Among such infections are those caused by organisms of genera such as Streptococcus, Staphylococcus, Escherichia, Salmonella, Klebsiella, Proteus, Pseudomonas and the like. These organisms cause or are believed to cause mastitis in cattle, urinary tract infections and diarrhoea. Genes from the same organisms are thought to cause skin diseases and diseases of the upper respiratory organs or to make pre-existing effects of such diseases more serious in mammals. Antibioticum 66-40 and its derivatives therefore provide a powerful agent for combating such organisms and disease states caused by them.

Claims (2)

1. Procedure for the production of a new antibiotic active product designated Antibioticum 66-40, later also designated "sisomicin", and with the general formula: in the free form or in the form of one of the following derivatives: a salt, a solvate of the free base, a solvate of a salt thereof, or a condensation product with an aldehyde, characterized in that a strain of the species Micromonospora inyoensis is grown in a aqueous nutrient medium under aerobic conditions, after which the said antibiotic active product is isolated in free form or in the form of a solvate and, if desired, the product thus obtained is transferred to an acid addition salt or a solvate thereof, or to a condensation ion product with an aldehyde. 2. Method according to claim 1, characterized in that Micromonospora inyoensis NRRL 3292 is used as the strain.