IE46064B1 - Antibiotic c-15003 - Google Patents

Abstract

A novel Antibiotic C-15003 is produced by cultivating a microorganism of the genus Nocardia. The Antibiotic C-15003 is useful as an antifungal agent or an antiprotozoan agent.

Description

This invention relates to Antibiotic C-15OO3, which is a novel antibiotic, a method of producing Antibiotic C-15OO3 and a method of producing derivatives from the said antibiotic.

We collected various soil and other samples and performed a screening of the microorganisms isolated from such samples. We have found, by that screening, that certain of the microorganisms were able to produce a novel antibiotic, that such microorganisms belonged to the genus Nocardia and that, by cultivating any of those microorganisms in a suitable medium, it was possible to have the said antibiotic accumulated in the culture broth. We also found that derivatives could be obtained from the antibiotic. Further studies ensued, resulting in the development of this invention.

This invention is therefore directed to: (1) Antibiotic C-15OO3 which has the general formula (i) Cll(wherein ll represents -CO-C1I \ 7 , -00-011,,-011,.-011- or Cll3 “ J X 011-CO-CH„-CH \ J ); CHj (2) A method of producing Antibiotic C-15003 5 which method comprises cultivating an Antibiotic C-15OO3produeing strain of the genus Nocardia in a medium to cause the strain to elaborate and accumulate Antibiotic C-15OO3 in the culture broth, and recovering the said Antibiotic C-15OO3 from the said broth; and (3) A method of producing a compound of the formula (11): which method comprises hydrolysing Antibiotic C-15OO3 ol' the general formula (I): K r*·46064 (wherein It represents -CO-CH CII, CH, -C0-CH2-CHo-CI13 or -co-ch2-ch^ /CIi3 CH, )· In the context of this invention, the term Antibiotic C-15003” means, generically, the three compounds having the above general formula (i) as a group, or a mixture of two or three of said compounds or, severally, any of these same compounds. Referring, also, to the .CH, general formula (i), the compound in which R is -CO-CH J CH, is referred to herein as Antibiotic C-15003 P-3 or more briefly as C-15003 P-3; the compound in which It is -CO-CHg-CHg-CILj is referred to herein as Antibiotic C-15003 P-31 or, more briefly, as C-15003 P-3J; the .CH, compound in which R is -CO-CH„-CH' CH, is referred to herein as Antibiotic C-15003 P-4 or, more briefly, as C-15003 P-4; and the compound in which R is H(general formula (II)) is referred to herein as Antibiotic C-15003 P-0 or, more briefly, as C-15003 P-0.

As an example of the Antibiotic C-15003-producing strain of microorganism, there may be mentioned an actinomycetes Strain No. C-I5OO3 which we isolated from 6 0 6 4 soil and other samples in our screening for antibioticproducing mi croorganisms.

The microbiological characteristics of Strain No.

C-J5OO3 were investigated by procedures analogous to those proposed by Schi.riing ii Gottlieb (international Journal of Systematic Bacteriology I ό., 313-340 (1066)). The results of our observations at 2b°C over 21 days are as follows. (l) Morphological characters The vegetative mycelium extends well and develops into blanches, both on agar and in liquid media. Many of the hyphae measure 0.8 to 1.2 Jim in diameter and, in certain instances, may divide into fragments resembling rod bacteria or branched short lengths of hyphae. The strain gives good growth on various taxonomical media, with the aerial mycelium being superimposed on the vegetative mycelium, although it frequently forms corcmia-like bodies (50-200 x 200 - JOGO/irn) on which further aerial growth takes place. Many of the aerial mycelia are flextious or straight, a loosely spiral-like configuration being encountered on a few occasions. Microscopic examination of aged cultures reveals that only in a lev; cases do the conidia-like cells occur in chains, while the cell suspensions obtained from the surfaces of the cultures, as microscopically examined, contain many elongated ellipsoidal (0.8-1.2 pm x 4.8-6.8 pm) and ellipsoidal (0.8-1.2 x 1.0-2.0 pm) bodies resembling arthrospores.

Electron-microscopic examination showed that these bodies had smooth surfaces. (2) The constituents of cells The strain was shake-cultured in a modified ISP No.l medium at 28°C for from 66 to 9θ hours, at the end of which time the cells were collected and rinsed.

The above whole cells were examined for diaminopimelic acid and for sugar composition by the method of B-Becker et al. (Applied Microbiology 12, 421. (1964)) and the method of M.P. Lechevalier (Journal of Laboratory and Clinical Medicine 71, 934 (1968)). The former was found to he the meso-form, while spots were detected which correspond to galactose and arabinose. (3) Characteristics on taxonoinical media The strain showed a comparatively good growth on various media, with the vegetative mycelium being colourless to pale yellow in the initial phases of culture and light yellowish tart to yellowish tan in latex· phases. The strain produces soluble pigments, which are yellow to yellowish tan in colour in various taxonomical media.

The aerial mycelium is powdery and generally gives moderate growth, being white to yellow or light yellowish tan.

The characteristics of the strain in various taxonomical media are set forth in Table 1 just below Tab I e I Cultural characteristics ol Strain No. C-I5OO5 mi l.axonoin i eu I media (Λ) Sucrose nitrate agar: Growth (G); Moderate, Bright Melon Yellow (5 ia)* to Amber Tan (> lc)*, coremia-like bodies formed Aerial mycelium (AM): Scant, white Soluble pigment (SP): None or pale yellowish tan (B) Glycerol nitrate agar: G. Moderate, Light Ivory (2 ca)*, coremia-like bodies formed AM: Moderate, white SP: None (C) Glucose asparagine agar: G: Moderate, Bright Marigold (> pa)* to Bright Yellow (2 pa)*.

AM: Scant, white SP: Bright Yellow (2 pa)* (D) GJycei'ol asparagine agar: G: Moderate, Light Ivory (2 ca)*, coremia-Iikc bodies formed AM: Scant, white SP: None (E) Starch agar: G: Moderate, Light Ivory (2 ca)* to Light Wheat (2 ea)*, corcinia-1 ike bodies formed AM: Abundant, Light Ivory (2 ca)* SP: None (F) Nutrient agar: G: Moderate, Light Ivory (2 ea)* to Colonial Yellow (2 ga)*, corania-like bodies formed AM: Scant, white SP: None (G) Calcium malate agar: G: Moderate Light ivory (2 ca)* to Light Wheat (2 ea)*, coremia-like bodies formed.

AM: Moderate, white to Light Ivory (2 ca)* SP: None (li) Yeast extract-malt extract agar: G: Moderate, Amber (3 Ic)* to Bright Yellow (3 la)*, corcmia-1ike bodies formed AM: Moderate, white to Light Ivory (2 ca)* SP: None (i) Oatmeal agar: G: Moderate, Light Ivory (2 ca)* to Colonial Yellow (2 ga)*, coremia-like bodies formed AM: Scant, white to light yellow SP: None (J) Peptone yeast extract iron agar: 8' 6 0 6 4 G: Moderate, Colonial Yellow (2 ga)* ΛΜ: None SI’; Colonial Yellow (2 ga)* (K) Tyrosine agar G: Moderate, Light ivory (2 oa)* to Light Melon Yellow (3 ca)*, eoreini.a-J.ike bodies formed.

AM: Moderate, white to Light Ivory (2 ca)*.

SP: Camel (3 ie)* * The colour codes according to Color Harmony Manual, 4tli ed. (Container Corporation of America, 105s). (4) i'hysiologi cal character The physiological characteristics of the strain are shown in Table 2 below. Temperature range for growth: 12°C to 3y°C. Tin; temperature range in which good aerial growth occurs on agar (ISP No. 2) is 2i) to 35°C.

Table 2 The physiologieal characteristics of Strain No. c-ι5003 Temperature range for growth: 12 to 5ts°C.

Temperature range for aerial growth: 20 to 35°C Liquefaction ol' gelatin: Positive Hydrolysis of starch; Positive Reduction of nitrates: Positive Peptonization of milk: Positive Coagulation of milk: Negative 25 Decomposition of casein: Positive Production of meianoid pigments: o 4 6 0 6 4 Negative (peptone yeast extract iron agar), positive (tyrosine agar) Decomposition of tyrosine: Positive Decomposition oi xanthine: Negative Decomposition of hypoxanthine: Negative Tolerance to lysozyme: Positive Tolerance to sodium chloride: 2 °,ί ) Utilization of various carbon sources The utilization of various carbon sources was investigated using a medium described in Pridham and Gottlieb (Journal of Bacteriology 56, 107 (1948)) and a basal medium of the same composition plus 0.1 ji of yeast extract. The resulting spectrum is shown in Table 3 Table 3 The utilization of carbon sources by Strain No. C-15003 Source of carbon Growth Sources of carbon Growth D-Xylose · ++* Raffinose +* L-Arabinose + Melibiosc + + D-Glucose ++ ++ i-Inositol - - D-Galactose + + D-Sorbitoi - D-Fructose +++ ++ D-Mannitoi + + ++ L-Rhamnose + + Glycerol - + D-Mannose +++ ++ Soluble starch + + Sucrose ++ ++ Control __ 6 0 6 4 Lactose - -* Maltose j; + Trehalose + ++ * Basal medium with 0.1 % yeast extract added Note: +++: Luxuriant growth ++: Good growth +: Growth _+: Poor growth No gjowtli ()) Other characteristics The cells were harvested by the procedure previously described in (2) above and DNA was prepared by a procedure analogous to that of d. Marmur et al. (Journal of Molecular Biology 20«, .1061). The G-G (Guanine-Cytosine) content of the DNA was found to be about 71 mole Grain-staining of the vegetative mycelium of this strain was positive.

The above characteristics of Strain No, C-15003 were compared with the descriptions in S.A. Waksman's The Actinomyces Vol. 2 (The Williams and Wilkins Co., 1961); R.E, Buchanan and N.E. Gibbons, Bergey's Manual of Determinative Bacteriology, 8th ed, J97'i; and like literature references.

Whilst this strain was thought to belong to Group ill known of the genus Nocardi a, the failure to find among the/strains · 6 0 6 4 any species having the characteristics so far described led us to conclude that this strain represented a novel species of microorganism.

The present Strain No. C-15003 has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology (FERM) under the receipt number of 3992; at The Institute for Fermentation, Osaka (IFO) under the accession number of IFO 13726 and at The American Type Culture Collection (ATCC), Maryland, U.S.A. under the accession number of 31281.

Wile Strain No. C-15003 is a novel species of the genus Nocardia as just mentioned, it is liable, as are microorganisms generally, to undergo variations and mutations, either spontaneously or under the influence of a mutagen.

For example, the many variants of the strain which are obtainable by irradiation with X-rays, gamma rays and ultraviolet light, by monocell isolation, by culture on media containing various chemicals, or by any other mutagenic treatment, as well as the mutants spontaneously derived from tho strain, should not be considered to represent any other distinct species but, l-uther, any of sueli variants and mutants capable of elaborating C-15003 P-3, P-3' and/or P-4 may be invariably utilized for the purposes of this invention. Uy way of example, subjecting Strain No. C-15003 to various mutagenic treatments yields mutants which substantially lack the ability to produce soluble pigments, mutants with substrate mycelia which are colourless, yellowish green, reddish tan or orange-red, mutants whose hyphae are ready to fragment into bacillary elements or branched short hyphai fragments, and mutants with abundant white aerial mycelia or substantially without aerial mycelia.

The medium employed for the cultivation of such an Antibiotic C-15003-producing strain may be either a liquid or a solid medium provided only it contains nutrients which the strain may utilize, although a liquid medium is preferred for high-production runs. The medium may comprise carbon and nitrogen sources which Strain No. C-15003 may assimilate and digest, inorganic matter, trace nutrients, and so on. As examples of said carbon sources there may be mentioned glucose, lactose, sucrose, maltose, dextrin, starch, glycerol, mannitol and sorbitol, fats or oiis (e.g. soybean oil, lard oil or chicken oil). The nitrogen sources may be for example meat extrae.t, yeast extract, dried yeast,, soybean meal, corn steep liquor, peptone, e.ottonseeil flour, spent molasses, urea or ammonium salts (e.g. ammonium sulphate, ammonium chloride, ammonium nitrate or ammonium acetate). The medium may further contain salts of sodium, potassium, calcium or magnesium, salts of iron, manganese, zinc, cohalt or nickel, salts of phosphoric acid or boric acid, and organic acid salts such 1.3 as acetates or propionates. Further, the medium may contain, for example, as additives, various aminoaclds (e.g. glutamic acid, aspartic acid, alanine, glycine, lysine, methionine or proline), peptides (e.g. dipeptides or tripeptides), vitamins (e.g. B^, Bg, nicotinic acid, B^o, C or E) and nucleic acids (e.g. purine, pyrimidine and derivatives thereof). For the purpose of adjusting the pH of the medium, there may he added an inorganic or organic acid, alkali, buffer or the like. Suitable amounts of oils, fats or surfactants may also be added as antifoaming agents.

The cultivation may be conducted under stationary, shaking, submerged aerobic or other cultural conditions.

For high production runs, submerged aerobic culture is preferred. While the conditions of culture depend upon the condition and composition of the medium, the strain, cultural method and other factors, it is nonnalJy preferred to carry out incubation at 20 to 35°θ with an initial pH substantially of 7.0. A temperature of from 25 to 30°C is particularly desirable in an intermediate stage of cultivation, with an initial pH of 6.5 to 7.5. While the incubation time is also variable according to the same factors as those mentioned above, it is advisable to continue the incubation until the titer of the desired antibiotic product becomes a maximum.

In the ease of shaking culture or aerobic submerged culture in liquid media, the time required normally ranges from 48 to 144 hours.

The potency of the antibiotic was assayed with Te ti aliymeiia pyri formi s W as an assay organism. Thus, the above microorganism was grown on a test medium [20 g of Proteose-peptone (Dil'eo) (Trade Mark), lq of yeast extract (Difco), 2 g of glucose, 1000 >.’/ of distilled water and 10 mt· of M-pbusphate buffer ([ill 7.'·)] at 2H°C tor from T'i to 4h hours, and the potency of tin* antibiotic was determined by the serial dilution method with a monitoring of the turbidity of growth and Ute effect on ascites tumor cel ls, and by a thin-layer chromatographic (briefly TLC) assay to be described hereinafter.

The novel Antibiotic C-J5OO3 J*—5* and/or P-4 is produced and accumulated in the resulting, fermentation broth, both extraeelIularly and in trace I I ιι 1 ur I,v.

J5 These substances have also been delected by TLC.

Tints, the fermentation broth is separated into ceils and filtrate by filtration of cenl ri i'uging and the nitrate is extracted with the same volume of ethyl acetate. To the ceils is added the same amount oi 70 'i acetone-water as the filtrate and, after an hour's stirring at 20°C, the suspension is filtered. The acetone is removed from the filtrate and the resulting aqueous filtrate is extracted with ethyl acetate. Each of the extracts is concentrated to i/JOO by volume and subjected to thin25 layer chromatography on a silica gel-glass plate (Merck, West Germany, Kieselgel (trademark) 60 F 254, 0.25 mn, 20 x 20) (solvent system : chloroform-methanol = 9:1). The potency was determined on the basis of the intensity of spots detected by irradiation with ultraviolet light at 2537 A.

Because C-15003 P-3, P-3' and/or P-4, which are thus produced in the fermentation broth, are liphophylic neutral substances, they can be conveniently recovered by the separation and purification procedures which are normally employed for the harvesting of such microbial metabolites. For example, there may be employed a procedure which utilizes the difference in solubility between the antibioti and the impurity, means which utilize the adsorptive affinity of various adsorbents such as activated carbon, maeroporous nonionic resins, silica gel, alumina, etc., a procedure of removing the impurities by means of ionexchange resins, and so on, used alone or in a suitable combination or in repetition.

Since, as afozesaid, C-15003 P-3, P-3' and P-4 occur in both the filtrate and cells, the antibiotics are separated and purified by means of such an adsorbent, if one is employed, either direetly or after a solvent extraction in the ease of the filtrate, or after a solvent extraction in the case of microbial cells. The solvent extraction may be performed e.g, by any of the following methods, (i.e. (l) solvent extraction from the culture broth prior 1.o separation of Llie cells and (2) solvent extraction of the cells arid the filtrate obtained by filtration, centrifuging or other like processes. To extract tile filtrate and ceils independently, the following procedure may he used advantageously.

The solvents suitahie for extraction of the filtrate may he water-immiscible organic solvents such as fatty acid esters, e.g. ethyl acetate or amyl acetate; alcohols, e.g. butanol; halogenated hydrocarbons, e.g. chloroform; or ketones, e.g. methyl i.sobuiyl ketone. The extraction is carried out at a pH near neutral and, preferably, the culture f'Luid previously adjusted to pH 7 is extracted with ethyl acetate. The extract is washed with water and concentrated under reduced pressure. Then a non-polar solvent such as petroleum ether or hexane is added to the concentrate and the crude product (I) containing the active compound is recovered. Because, on TLC, a number of spots are detected in audition to Antibiotic C-15003, the product (I) is sequentially subjected to the following purification procedures. Thus, as a routine purification procedure, adsorption chromatography is useful and, for this purpose, one of those common adsorbents such as silica gel, alumina or a macrOporous noil-ionic adsorbent resin, may be employed. Em· purification from the crude product (l), silica gel is most useful. Development may be carried out, 460 64 i'or example starting with petroleum ether and hexane, and elution of Antibiotic C-15OO3 may he performed hy the addition of a polar solvent such as ethyl acetate, acetone, ethanol or methanol. In a typical process using silica gel (Merck, West Germany, 0.05-0.2 mm) as a carrier, column chromatography is carried out with a serial increase in the hexane to ethyl acetate ratio. The eluate is sampled and investigated by TLC and the fractions containing C-15005 are pooled and concentrated under reduced pressure. Then, petroleum ether or hexane is added to the concentrate, whereby the crude product (II) is obtained. Since this product still contains impurities, it is further purified as follows. For example, the product (IT) may be purified by means of a second siJica gel column using a different solvent system. The developing system for this purpose may consist in a halogenated hydrocarbon such as dichioromethane or chloroform, with the addition of a polar solvent such as an alcohol, e.g. methanol or ethanol, a ketone, e.g. acetone or inethyl ethyl ketone, or the like. In this way, Antibiotic C-15OO3 is isolated. The order of solvent systems .for the first and second silica gel columns may be reversed and, in addition, ordinary organic solvents may be used in conjunction with the above systems if necessary.

Where a macroporous adsorbent resin is used as a purification means for crude product (IT), elution of Antibiotic C-15003 is accomplished with a mixture of water with a lower alcohol, a lower ketone or an ester. The lower alcohol may be for example methanol, ethanol, propanol or butanol and the lower ketone may be l'er example acetone «r methyl ethyl ketone. The ester may be for example ethyl acetate. In a typical procedure, the crude product (il) is dissolved in 00 4 methanol-water· and adsorbed on a column of Di a ion (Trade Mark) IIP-IU (Mitsubishi Kasei K.K.). The column is washed with 70 4 methanol.-water and, then, elation is carried out with S>0 % methanol-water, in this way, Antibiotic C-15003 is eluted from the column.

In either of the processes described above, tile fractions containing Antibiotic C-15003 are pooled and concentrated under reduced pressure. 5 to 8 Volumes of ethyl acetate are added to the dry product and the mixture is allowed to stand, whereupon crystals of Antibiotic G-J5OO3 separate out. These crystals contain C-15003 P-3, P-3' and P-A . These compounds are then separated from each other· by means of an adsorbent such as those mentioned hereinbefore. Thus, using silica gel 01· a maeroporous non-ionic adsorbent resin and the above solvents, the desired compounds may be fractionally eluted. When, for example, silica gel is employed, development is carried out with hexane, ethyl acetate or clil oto 1 orm-metlianoi , wliereb\r C-J5O05 P-A, P-3* and P-3 emerge in that order. After detection by TLC, the fractions corresponding to C-15003 P-4, P-3' and P-3 are respectively concentrated under reduced pressure and ethyl acetate is added to the concentrates The respective compounds can be obtained as crystals in this manner. When a maeroporous non-ionic adsorbent resin is employed, gradient elution with a varying ratio of alcohol, ketone or ester to water may be utilized.

For example, the gradient elution method involving the use of 60% methanol-water and 95 % methanol-water, with cl° sodium chloride added, causes C-15003 P-3, P-3’ and P-4 to merge in the order mentioned. After sampling and detection by TLC, each group of active fractions is concentrated under reduced pressure and crystallized from ethyl acetate.

The isolated crystals include ethyl acetate as a solvent of crystallization and, after drying over phosphorus pentoxide at 70°C for 8 hours, show the following physical and chemical properties (Tabla 4): 460G4 φ σ' π—1 j >/ 1 “Ί rA '..j -J j X' Φ of, j • t 1/. if X If \ r< x if. σ' (\J •Jj V.· O CM : : - > -.'·' Hi 1 Φ 1 f-S 1 f'J O U χ ' X 4 if , Φ X Ρ' 1 r-i » Φ — • ο i +1 II ι; .1 (.- <: 1 •H n κ I (\I r<' ‘ -J N\ | r-i Φ rn η Ο u\ r-i I Ο ο •Η -Ρ ο •Η ,Ω σ' : S ! if : ιϊ\ ’ * νυ ' Νλ II σ'' I Ο ί CJ XX ! ι Ο ί JV X I rc. ι ο ί c> U\ Φ Φ1 σ' o C\J O N\ Li> rH ΙΛ CM Φ r-i ,Η- co lA ι—1 r-i ; · • ♦ • • • • t r—1 ί O o -a. i.f\ O xO -ί- u\ 1 o • ' Φ Φ r-i o \Ω r-i ίί\ m r< λ Φ 11 κ\ σ' ο I t OJ Ph ‘V rc. Ο Ο· \υ c^ Κ'.

CJ co Φ LT\ ij X Μ -ρ ο; αί (\! ίυ ο Based on the molecular formula shown above and the antimicrobial and antitumor activity delta given hereinafter, the present antibiotic was compared with the known groups of antibiotics. The literature search failed to locate any distinct, group similar to Antibiotic C-15003. However, a search for substances that might, give ultraviolet absorptions similar to those of tile present antibiotic among components of plant and other naturally-occurring organic compounds led us to the maytanacine groups and, based on the molecular formulae involved, in particular, it was assumed that the antibiotic belongs to the maytanacine group of compounds containing two nitrogen atoms, Maytanacine was obtained as a component of plants and was reported in Journal of the American Chemical Society 97, 5294 (1975). Tlie mass spectrum of maytanacinc is as follows.

M+-(a) M+-(a+b) 4S5-CH. 435-C0 545 4 «5 (a)= 11,,() < 11NC0 479 450 (b)=R-C-OH The presence of m/e 485, 470 and 450 for C-15003 P-3, P-3’ and P-4 convinced us that these compounds have a skeletal structure identical with that of maytanacine, differentiating them for maytanacine in the kind of acyl group in the 3position. It is thus clear that Antibiotic C-15003 is a v ίο novel compound. Wien C-15003 P-3, P-3' and P-4 were each degraded with alkali and analysed by gas chromatography for the liberated carboxylic acids, it was found that isobutyric acid, butyric acid and isovaleric acid were obtainable from C-15003 P-3, C-15003 P-3' and C-15003 P-4, respectively. Figure 1 shows the structures, based on the above data, of C-15003 P-3, P-3' and P-4.

R .CI1„ P-3 -CO-CH . -5 CH„ p-31 -C0-CH2-CH2-CH3 ^.CH P-4 -CO-CH -CH \ ch3 Biological activity: A) Antimicrobial activity: With trypticase-soy agar (BBL) as an assay medium, the inhibitory concentrations against the microorganisms named below were investigated by the paper disc method.

Thus, filter-paper dises (Toyo Seisakusho, thin-type, 8 mm dia.) each impregnated witli 0.02 m£. of a 300 jig/W solution of C-15003 P-3, P-3' or P-4 were placed on pLates respectively inoculated with the microoganisms named below to investigate the minimal inhibitory concentrations. The results showed that the antibiotics had no activity against the following microorganisms: Escherichia coii. Proteus vulgaris, Proteus niirabilis, Pseudomonas .aeruginosa, Staphylococcus aureus, Bacillus subtil is. Bacillus cereus, Klebsiella pneumoniae, Serratia marcescens and Mycobacterium avium.

On the other hand, with agar plates containing the assay medium (3.5 g disodiura hydrogen phosphate, 0.5 g monopotassium dihydrogen phosphate, 5 a yeast extract (Difco (trade mark)), 10 g glucose, 15 g agar and 1000 distilled water, pH 7.0), the growth inhibition against 'falaromyccs ave I I aliens was assayed, in this assay, the minimal, inhibitory concentrations were 5 ^ig/m£ for C-I5OO3 P-3 and P-3', and 1.5 pg/mi for C-15003 P-4.

Furthermore, the wild strain of Tetrahymena pyriformis W as an assay organism was cultivated on an assay medium (composed of 20 g Proteose-peptone (Difco), 1 g yeast extract, 2 g glucose, 1000 m£ distilled water and 10 ra£ i M-phosphate buffer pH 7>0) at 286 C for 44 to 48 hours, and the growth inhibitory activity of the antibiotic compounds against this particular microorganism was determined by the serial dilution method. Growth inhibition occurred at 1 jig/mt for C-15003 P-3 and P-3', and at 0.5 ;ug/md for 015003 P-4.

Anti-fungal activity is shown in Table 5 below.

As seen from Table 5, C-I5OO3 has growth inhibitory activity against microorganisms which cause plant diseases.

The filter-paper discs impregnated with 0.02 mi of a 1000 ug/m^ solution Of C-15003 were placed on plates respectively inoculated with the microorganisms of the following Table 5.

Table 5 Anti-microbial .spectra Test organisms IFO number medium hour Inhibition diameter Alternaria kikuohiana 7515 PEA* 48 38 Fusicladium levieri 6477 PSA* 90 68 Helainthosporium sipmoideum var. irrepulare 5275 PSA* 48 55 Pyrieularia cryzae - PSA* 48 53 Elginoe fawc e 1: ti 8417 PSA* 90 55 Fus a ri υ o oxy r η c rum 1. cucumerrnum - PSA* 48 20 Guignaidi u 1 ari cina 7888 PSA* 48 12 Cochlioborur; miyabeanus 527? PSA* 48 60 Dianorthe cj.tri 9170 PSA* 48 55 Gibberella zee- 8850 PSA* 48 37 Sclerotinia nclerotioi-um +595 PSA* 90 65 Venturia pirina 6189 PSA* 48 50 JPellieularia uacakii 9253 PSA* 48 50 P.ythium crlipnidernatum 7050 PSA* 48 58 Sotrytis cinerca - PSA* 48 48 Aspergillus riper 4066 PSA* 48 0 Penicillium chrycogenum 4626 PSA* 48 35 Khizopuπ niyr icans 6188 PSA* 48 25 Saceharomyce- eerevmi - >.e 48 71209 PSA* 0 Rholotorula i-alra 0907 PSA* 48 28 Trichophyton rubrum 5*&7 * * GB 48 38 « 27 Test organisms XFO number medium hour Inhibition diameter Trichophyton mantagrophytes 7522 GB** 48 38 Candida albicans 0583 GB** 48 0 Candida util is 0619 GB** 48 0 Cryptococcus neoformans 0410 GB** 48 43 * PSA: Potato sucrose agar medium ** GB : Glucose nutrient agar medium B) Antitumor activity The therapeutic effects of C-15003 P-3, P-3' and P-4 (dosed intraperitoneally for 9 consecutive days) upon P388 leukaemia in mice (l x 10^ eells/animal, mouse, intraperitoneally transplanted) were investigated. The results showed that, in terms of life span-extending ratio, these compounds had an antitumor activity as high as 200 # at the dose level of 0.00625 mg/kg/day.

C) Toxici ty In an acute toxicity test with mice as test animals, which involved intraperitoneal injections of C-i5OO3 P-3, P-3' and P-4, all of those antibiotics showed an LD^0 value of more than 0.313 mg/lcg.

As mentioned hereinbefore, the present Antibiotic C-15003 has strong inhibitory activity against fungi and protozoa and, therefore, is of value as an antifungal or nntiprotozoan agent. Furthermore, because Antibiotic C-15003 displays ii life span-extending action upon tumourbearing mammalian animals (e.g. the mouse), it is also expected that the compound will be of use as an antitumour drug.

Antibiotic C-15003, as an antifungal and antiprotozoan agent, can be used with advantage for an assessment of the bacterial ecology in the soil, active sludge, animal body fluid or the like. Thus, when valuable bacteria are to be isolated from soil samples or when the actions of bacteria are to be evaluated independently of those of fungi and protozoa in connection with the operation and analysis of an active sludge system used in the treatment of waste water, the present antibiotic may be utilized to obtain a selective growth of the bacterial flora without permitting growth of the concomi tant fungi and protozoa in the specimen. In a typical instance, the sample is added to a liquid or solid medium and 0.1 md of a 10 to 100 j.ig/m£ solution of the antibiotic in 1 ¢. methanol-water is added per m£ of the medium, which is then incubated.

The present Antibiotic C-15003 can also be used as an anti-microbial agent for the treatment of plant diseases caused by the microorganisms mentioned in the above Table 5.

In a typical application, Antibiotic C-15003 is used in the form of a 1 y aqueous methanolic solution containing 0-5 |ig/m£ -5 |ig/W of the antibiotic. For instance Antibiotic C-15003 may be used for the control of reddish-brown sheath rot, blast, Ilelminthosporium leaf spot and the sheath blight of rice plants.

Xt is thus apparent that C-15003 P-3, P-3’ and P-4 are all novel compounds having the same skeletal structure, and can also be used as intermediates for the production of other pharmaceutically useful compounds. Thus, by way of a deacylation reaction,<7+^15003 P-0 (maytansinol) with a hydroxyl group in the 3-position can be derived from the present antibiotic. In this connection, because the acyl group is in a position beta to the carbonyl group, the conventional reductive hydrolysis reaction can be employed with advantage. Thus, by means of a complex metal hydride (e.g. lithium aluminum hydride (LiAlH^)) at a low temperature (e.g. -20-0°C), the 0-ester bond in the 3-position may be hydrolytically cleaved, without affecting other functional groups, e.g. carbonyl, epoxy or carbon-carbon double bonds, to yield maytansinol. The physical and chemical data on this maytansinol sample thus obtained are in good agreement with the data given in Kupchan et al, The Journal of American Chemical Society 97, 5294-5295 (1975)).

The following examples are further illustrative but by no means limitative of the invention. References to part(s) are based on weight unless otherwise specified, and the relationship between part(s) and part(s) by volume corresponds to that between gram(s) and inilliliter(s), and 5ό Is based on weight/volume unless otherwise specified.

Example 1 Nocardia No. C-15003 (IFO 13726; FERM 3992; ATCC 31281) as grown on a medium (yeast extract-malt extract agar) was used to inoculate a 200 parts by volume fermenter containing 40 parts by volume of a seed culture medium (2;! glucose, $i soluble starch, I lo raw soybean meal, 1 % corn steep liquor, 0.5 1 Polypepton, 0.3 a NaC£ and 0.5 1 CaCO^, pH 7.0). The inoculated medium was incubated at 28°C for 48 hours to obtain an inoculum. A 0.5 part by volume portion of the inoculum thus obtained was transferred to a 200 parts by volume fermenter containing 40 parts by volume of a fermentation medium composed of 5 1 dextrin, 3 1 corn steep liquor, 0.1 1 Polypepton and 0.5 % CaCO^ (pH 7.), and was cultivated at 28°C for 90 hours to give an inoculum (seed culture).

As determined by the .serial dilution method using Tetraliymena pyriformis W as an assay organism and Antibiotic C-15003 P-5 as the standard sample, the above culture was found to have a titer of 25 jtg/mi..

Example 2 A 10 parts by volume portion of the inoculum (seed) obtained in Example 1 was transferred to a 2,000 parts by volume fermenter containing 500 parts by volume of a seed culture medium (the same as the above) and incubated at 28°C for 48 hours. A 500 parts by volume portion of the resulting culture was transferred to a 50,000 parts by volume tank of stainless steel containing 30,000 parts by volume of seed culture medium and cultivated at 28°C, under aeration (30,000 parts by volume/min), agitation (280 r.p.m. (l/2DT).) and internal pressure (l kg/cm2) to obtain a seed culture. This culture was used to seed a 200,000 parts by volume tank of stainless steel containing 100,000 parts by volume of a fermentation medium similar to that used in Example 1 at an Inoculation rate of 10 ¢.

The inoculated medium was incubated at 28°C, under aeration (100,000 parts by volume/min.), agitation (200 r.p.m.

(% DT)) and internal pressure (l kg/cm‘) for 90 hours.

As determined by the same procedure as that described in Example 1, the culture obtained above was found to have a titer of 25 jig/md.

Examp]e 3 To 95,000 parts by volume of the culture obtained in Example 2 were added 2,000 parts of Hyflo-ouporool· Super Gel Sales (trade mark, Johns Manville/Corporation, U.S.A.) and, after thorough mixing, the mixture was filtered on a pressure filter to obtain 85,000 parts by volume of fiLtrate and 32,000 parts of moist ceils. The filtrate, 85,000 parts by volume, was stirred and extracted with 30,000 parts by volume of ethyl acetate. This procedure was repeated once again. The ethyl acetate layers were pooled, washed twice with 30,000 parts by volume portions of water, dried by the addition of 500 parts of anhydrous sodium sulphate and concentrebed under reduced pressure to 200 parts by volume. Petroleum ether was added to the concentrate and the resulting precipitate was recovered by filtration (53 parts). This crude product (i) was stirred with 100 parts by volume of ethyl acetate and the insolubles were filtered, off. The filtrate was stirred with 10 parts of silica gel (Merck, West Germany, 0.05-0.2 mm) and the ethyl acetate was removed under reduced pressure. The residue was applied 1,o the top of a silica gel column (400 parts by volume). Elution was carried out witli 500 parts by volume of hexane, 500 parts by volume of hexane-ethyl acetate (3:1), 500 parts by volume of hexane-ethyl acetate (1:1 ), 500 parts by volume of hexane-ethyl acetate (1:3), 50O parts by volume of ethyl acetate and 1.,000 parts by volume of ethyl acetate-methanol (50:1), with the eluate being collected in 100 parts by volume fractions.

Eor one part by volume portion of each fraction was concentrated to dryness, and 0.1 part by volume of ethyl acetate was added to the concentrate to give a mixture.

The mixture was spotted at 2.5 cm from the bottom edge of a silica gel-glass plate (Merck, ' . he im. .. t,-.. - 25A, 0.25 mm, 20 x 20), and dcvelopid iux about --nt Witil ii solvent system ol ethyl acetate jiietin.ni.: ,1/. Alter development, detection was aoi-ried mit with ali.aviolet, light (2537A).

The active fractions No. 25-Ni. 2« ,1 iiiO.i. -o were collected and concentrated undex reduced ... -ssure- uo about 20 parts by volume. To this concentrate wer- adued 150 parts by volume oi' petioleum ethei' ro obta * pairs of a crude product 11 i).

Example A With stirring, 52,000 parts ol' the huim _-ciss obtained in Example 5 were txrracted wirh ό',,οοο ,,μζγ· by volume of 70 % acetone-water for 3 hours ai.u then 1 111 < 1 ed on a pressure filtei The extraction with 31,000 parts by volume of 70 % ae.etoiie-water and the subsequent fil tration were repeated once again. The filtrates were pooled and the acetone was removed by concentration under reduced pre-sure. The resulting aqueous system was passed througn a column of 5,000 parts by volume Diaion (Trade Mark) ΠΡ-10 (Mitsubishi Kasei K.K.). The column was washed with 20,000 parts by volume of water and 50 # aqueous methanol, followed by elution with 15,000 parts by volume of 90 % methanol-water. The eluate was concentrated under reduced pressure to 3,000 parts by volume and shaken with 5,000 parts by volume of water 3A and 3)000 parts by volume ol' ethyl acetate, the above procedure was repeated once again. The ethyl acetate layers were combined, washed with water, dried by the addition of anhydrous sodium sulphate and concentrated under reduced pressure to 200 parts by volume. Following the addition of petroleum ether, the precipitate was recovered by filtration (23 parts). The above product was purified by means of a column of silica gel to recover 8.0 parts of crude product II.

Example 5 Jn JO parts hy volume of ethyl acetate there were dissolved J.5 parts of the crude product (ii) obtained in Example 5 and the solution was thoroughly stirred with 4 parts of silica gel (Mer«di, West (Icmiany, 0.05-0.2 mm), The ethyl acetate was removed under- reduced pressure.

The residue was applied to the top of a column of 300 parts by volume silica gel, and the column was first washed with 500 parts by vo 1 uiiie or chloroform and then eluted with 500 par ts by volume or ch 10 ro form-methano1 (50:1), 20 500 parts by volume or ch1oro form-me thano1 (20:1) and 500 parts by vo fume of ch 1.0 ro form-me thano 1 (10:1).

The eluate was collected in 25 parts by volume fractions. An 0.5 part by volume portion of each fraction was concentrated under reduced pressure. To the concentrate 25 was added 0.05 part by volume of ethyl acetate, and the J? mixture as a sample was subjected to silica gel thin layer chromatography (developing system: chloroform-methanol= 9:1).

The fractions Nos. 39 and 40 absorbing at 2537 A 5 in the zone of Hf 0.50-0.60 were collected and concentrated to dryness under reduced pressure. 2 Parts by volume of ethyl acetate were added to the residue and the mixture was allowed to stand, whereupon 0.150 part crystals of Antibiotic C-15003 was obtained.

The above crystals of Antibiotic C-15003 (0.150 part) were dissolved in 15 parts by volume of methanol, followed by the addition of 0.300 part of sodium chloride and 15 parts by volume of water. A column was packed with 200 parts by volume of Diaion (Trade Mark) HP-10 (Mitsubishi Kasei K.K.j and calibrated with 600 parts by volume of 50 # methanolwater containing 5 # of NaC £·. The sample solution prepared above was passed through the column, and gradient elution was carried out using 1,500 parts by volume of 60 ?£ methanol-water containing 5 NaC^ and 1,500 parts by volume of 95 F methanol-water. The eluate was collected in 15 parts by volume fractions and each fraction was investigated by silica gel thin layer chromatography.

The fractions 145 to 153 contained C-15003 P-3, the fractions 167-180 contained C-15003 P-3' and P-4 and the fractions 185-190 contained C-15003 P-4. 46034 Each group ol’ fractions was concentrated and dissolved by the addition of 50 parts by volume of water and 100 parts by volume of ethyl acetate. The solution was shaken in a separation funnel, the water layer was separated and, after washing with two 50 parts by volume portions of water, the ethyl acetate layer was dried over anhydrous sodium sulphate, concentrated and allowed to stand. In the above manner, crystals were obtained from each group of fractions. The crystals were col 1ecteu by filtration and dried.

C-15003 P-3 0.0/0 part C-15003 P-3', P-4 lM(U part C-15003 P-4 0.015 part The mixed crystals of C-15003 P-3' and P-4 (0.018 part) were dissolved in 0.3 part by volume of ethyl, acetate and spotted in a line at a distance of 2.5 cm from the bottom edge of a silica gel glass plate (Merck, West Germany, Kleselgel 60 0.25 mm, 20 x 20), followed by development with ethyl acetate-methanol (19:1). Alter development to about 18 cm, the absorption bands at 111' 0.6s (P-4) and Rl 0.65 (P-3' ) were scraped ol'f and each was independently extracted twice with ethyl acetate containing a small amount of water. The resulting ethyl, acetate extract was washed with water, dried over anhydrous sodium sulphate, concentrated under reduced pressure and allowed to stand. 0.010 Part crystals of C-15003 P-4 and 0.003 part crystals oi C-15003 P-31 were obtained from the fractions of Rf 0.68 and Rf 0.65, respectively.

Example 6 One thousand parts by volume of the culture of Example 2 were inoculated into a 200,000 parts by volume tank of stainless steel containing 100,000 parts by volume of a seed culture medium and the inoculated medium was incubated at 28°C under aeration (100,000 parts by volume/min.) and agitation (200 r.p.m.) for 48 hours to prepare a seed culture. This seed culture was transferred to a 2,000,000 parts by volume tank of stainless steel containing 1,000,000 parts by volume of a fermentation medium similar to that used in Example 1 at a transplantation rate of 10 c/o. Cultivation was carried, out at 28°C under aeration (1,000,000 parts by volume/min.), agitation (120 r.p.m. (1/3 DT)) and internal pressure (l kg/cm2) for 90 hours.

The resulting culture was found to have a titer of 20 jig/ini as assayed by the assay procedure described in Example 1.

To 900,000 parts by volume of the above culture were added 900,000 parts by volume of acetone and, after an hour’s stirring, 20,000 parts of Ilyflo-Supercel (trade mark, Johns Manville, U.S.A.) were added. The mixture was further stirred and filtered on a pressure filter machine.

To 1,70(),000 parts hy volume of the resulting filtrate were added 500,000 parts by volume of water and, in a Podbielniak (trade mark) extracter (Podbeilniak, Inc.), the mixture was extracted with 1,00(),0()() parts by volume of ethyl, acetate. The ethyl acetate layer was washed with water, dried by the addition oi' anhydrous sodium sulphate and concentrated under reduced pressure. To the concentrate was added petroleum ether and the resulting precipitate was recovered by filtration and dried. By the above procedure were obtained 68 parts of the crude product (f). Thereafter, as in Examples 3, 4 and 5, this crude product wits purified to obtain 9.5 parts of C-15003 P-5, 0.3()() part of C-I500 P-3' awl 2.5 parts of C-15003 P 4.

Example 7 Itt I part by volume o i Letroh.yd ro In ran was dissolved ¢).015 part of the Antibiotic C-15003 crystals obtained in Example 5, and after the solution was cooled to -5°C. 0.012 part of lithium aluminum hydride was added. The mixture was allowed to stand for 2 hours. Following the addition of 0.5 part by volume of a i / aqueous solution of the reaction mixture was extracted with 2 parts by volume of ethyl acetate. The ethyl acetate layer was washed with water, dried hy the addition of anhydrous sodium sulphate and concentrated under reduced pressure.

Pj’eparat j ve TLC with silica gei was carried out on the 46664 concentrate and the zone of Rf 0.25 'to 0.3 was scraped off and extracted with ethyl acetate containing a small amount of water. The extract was washed with water, dried over anhydrous sodium sulphate and concentrated under reduced pressure, whereupon crystals separated out. The crystals were recovered by filtration and dried. By the above procedure there was obtained 0.0X0 part of P-15003 P-0, melting point 174°C.

Elemental analysis: Found C, 59.65; ΪΙ, 6.58; N, 5·02; Cf, 6.51; ealcd. for CggH^CfNgOg C, 59.52; H, 6.60, N, 4.96; C2, 6.27 IR: 1715, 1670, 1580(cm-1) UV(nm): 232(32750), 244(sh, 30850), 252(31650), 281.(5750), 288(5700) . In properties, -this product is identical with maytansinol.

Claims (8)

1. Antibiotic C-15003 which has the general formula: -di, 5 -CO-CH, -CJI, J “ cil.

2. An antibiotic as claimed in Claim 1, wherein R is -CO-CH

3. , An antibiotic as claimed in Claim 1, wherein R is -C0-CH o -CH a -CH . 10

4. An antibiotic as claimed in Claim 1, wherein B is Oil. -CO-CH -CH 'CH,

5. A method ol producing Antibiotic C-15003 which comprises cultivating a microorganism belonging to the genus Nocardia and capable of producing Antibiotic C-15003 in a culture medium containing assimilable carbon sources and digestible nitrogen sources until Antibiotic 5 C-15003 has been substantially accumulated therein; and recovering Antibiotic C-15003.

6. A method as claimed in Claim 5» wherein the microorganism is Nocardia No. C-15003 (|ATCC 91201j IFO 13726^ PERM 3992). which comprises subjecting Antibiotic 0-15003 of the CH-C0-CH o -WI J , to reductive hydrolysis. “ CH8, A method asi claimed in Claim 7, wherein the reductive hydrolysis is conducted by employing a complex metal hydride. 5 9· A method as claimed in Claim 8, wherein the complex metal hydride is lithium aluminium hydride.

7. 10. A method according to Claim > substantially as herein described with reference to any of the specific Examples 1 to 6

8. 11. A method according to Claim 7 substantially as herein 10 described with reference to Example 7.