CA1077421A - Antibiotic sf-1540 from streptomyces - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE:
A novel derivative of the antibiotic substance SF-1540 is prepared by treating the antibiotic substance SF-1540 with methanol to form the corresponding antibiotic substance SF-1540 derivative. The antibiotic substance SF-1540 itself is obtained by culturing a substance SF-1540-producing microorganism belonging to the genus Streptomyces, such as Streptomyces hygroscopicus, in a medium and isolating from the cultured broth the substance SF-1540 thus formed. The novel derivative exerts a potent inhibitory activity against moulds.

Description

10'774~1 The present invention relates to a novel antibiotic substance and its derivative, and a process for the preparation thereof. More specifically, this invention relates to a novel antibiotic substance named SF-1540 and its derivative and to a process for preparing the same in which a substance SF-1540-producing microorganism belonging to the genus Streptomyces is --cultured in a medium under aerobic condition and a active subs-tance, substance SF-1540, which is produced in a cultured broth and exerts a potent inhibitory activity against moulds is recover-ed from said cultured broth. As a results of our fu~ther studies on nature of the active substance isolated in a pure state, it has been confirmed that the active substance is a novel antibiotic different from known substances and named substance SF-1540.
As one e~ample of the substance SF-1540-producing mi-croorganisms belonging to the genus Streptomyces, there may be mentioned the strain named by us as Streptomyces hygroscopicus SF-1540, the morphological characteristics of which are as summarized below. This strain has been deposited under an . . .
accession No.2607 with Technical Research Institute of Microbial Industry, Agency of Industrial Science & Technology, ~apan.
(ATCC No. 31257) I. ~orpholoqical characteristics A~undant aerial mycelium on starch agar medium, oatmeal a~ar medium, yeast-malt agar medium and tyrosine agar medium.
Good sporulation. Straight branches without cluster-like branches.
Aerial hyphae are termina~ed with compact closed spirals or short open spirals. Sclerotia not formed. Spore surface is warty under an electron-microscope. Spores are elliptical to short cylindri-cal in shape and 0.8 - 1.1 x 1.1 - 1.4 ~ in size. Spore chains are in 10 or more spores per spore chain.

II. Characteristic growth in various media .
- .

~077421 .
Soluble Medium Growth Aerial mycelium pigment ..... _ . . .

: Sucrose-nitrate Aqar Colorless Scant, grey None Glucose-asparagine Pale yellow Scant, qrey None agar Glycerol-aspara- Pale yellow Scant, white None gine agar to grey - Starch agar Good,greyish Abundant,grey None yellow to greyish brown, gradually becom-~ ing hygroscopic Oatmeal agar Good,greyish Grey to greyish None to . yellow brown,gradually faint becoming hy- yellow groscopic : Yeast-malt agar Good,pale Scant, grey None yellowish . brown Tyrosine agar Good,brown Abundant, grey, None gradually be-coming hygroscopic Nutrient agar Pale yellow Very scant, None white .. .. _ ..
LNot~ All culture temperatures of 28C.
III. Physiological characteristics (I) Temperature range for growth Good growth at a temperature range of 15 - ~9 C. on starch-yeast agar medium.

(2) Gelatin liquefaction: Positive at 20C. for 30 days.

(3) Hydrolysis of starch: Positive (~) Coagulation of skim milk: Negative (at 28C. and 37C.) Peptonization of skim milk: Positive (at 28C. and ; 37C.) (5) Melanin formation: Negative v Carbon source utilization pattern (Pridham-Gottlieb's agar medium) (I) Positive: D-glucose, D-fructose, D-mannitol, I-inositol, L-arabinose, rhamnose (2) Doutful : D-xylose, raffinose (3) Negative: Sucrose Summing up morphologieal eharaeteristies of the strain SF-1540 from the foregoing, aerial hyphae forms spirals and spore surface is warty. ¢Dlor of growth is pale yellow to .~......... .
yellowish brown to greyish yellow. Aerial hyphae is greyish brown and gradually becomes hygros~opic.
These characteristics of the strain SF-1540 are in fair agreement with those of Streptomyces hygroscopicus which belongs to the genus Streptomyces. More specifically, the strain SF-1540 has been observed to have the following three points which are considered as characteristics of Streptomyces ` hygroseopieus. 1) Spiral is formed, 2) Greyish brown aerial hyphae grows and 3) Aerial hyphae beeomes hygroseopie.
In comparison between the strain SF-1540 and Streptomyces hvgroseopicus which has been described by Waksman in The Actino~
mycetes, Vol. 2, 230 - 231 (1961), their characteristics are generally common each other, though some differences are observed ` about formation of soluble pigment on sucrose-nitrate agar and glucose-asparagine agar and so on.
From the above, the strain SF-1540 has been reasonably regarded to belong to the species of Streptomyces hygroscopicus "
in view of the eharaeteristies as specified in the above three points, though some differenees are to be observed from the disclosure made by Waksman and we have, aeeordingly, named the -~ 30 strain SF-1540 Streptomyees hygroseopicus SF-1540 as distingui-shed from other publiely-konwn strains.
The strain SF-15~0, a seen in other strains of the , `` ` iO774Zl ~ us Streptomyces, is apt to be varied in its characteristics and may be easily variable artificially, for example, by means of ultraviolet ray, X ray, high-frequency wave, radiant ray or chemicals. Consequently, the strains usable in this invention include all of the variants which are capable of producing the substance SF-1540.
In the present process, the above strain is cultured in a culture medium containing those nutrients ordinarily utili-zable by other microorganisms. As nutrient sources, there may be employed the well-known materials usually utilized for cultu-re of organisms belonging to the genus Streptomyces. For ins-tance, as a carbon source may be employed glucose, starch, gly-cerol, dextrin, sucrose, starch syrup, molasses, soy-bean oil and so on. Also, as a nitrogen source may be employed soy-bean meal, corn steep liquor, wheat embryo, cotton seed meal, ammonium sulfate, sodium nitrate and so on. In addition, inorganic salts such as calcium carbonate, sodium chloride, potassium chloride, a phosphate etc. as well as organic and inorganic materials which may act to promote the growth of this strain and increase the production of the substance SF-1540 may be incorporated, if neces-sary.
In carrying the cultivation into practice, liquid culture may be made in the same manner as generally used for the production of antibiotics and, in particular, submerged culture is most preferable. Cultivation may be usually effected under aerobic condition and culture temperatures of 25 - 35C are usually applied, but, in most cases, some 28C.- may be applied.
Maximum production of the substance SF-1540 is obtained in 2-5 days by both shaking and tank cultures.
The above-mentioned culture conditions may be selected and applied for the optimum ones depending upon the properties of the respective producing strains employed. The substance SF-, ' ' ~ : . ' ~077~Zl 40 is included in both mycelia and cultured broth and may be extracted from both of them, separately. Alternatively, the cultured broth was made weakly acidic without any filtration, whereupon the substance SF-1540 is adsorbed onto mycelia or precipitated in situ and then only mycelia portions may be extracted. In carrying the extraction into practice, any well-know procedures usually employed for the recovery of fat-soluble ; natural products from a cultured broth are adaptable, since the substance SF-1540 is fat-soluble as apparent from the physicoche-10 mical properties thereof mentioned below.
As an example, the cultured broth of Streptomyces hygroscopicus strain SF-1540 and the a~ueous solution obtained by removal of methanol from an aqueous methonal extract of mycelia is extracted with a water-in~iscible organic solvent, e.g. ethyl acetate, whereby the substance SF-1540 is transferred into the organic solvent layer. The extract is concentrated under reduc-ed pressure to leave a syrupy substance, from which a crude subs-; tance SF-1540 is precipitated by the addition of cyclohexane.
The crude precipitate containing the substance SF-1540 is purif-~0 ied through a column chromatography, e.q. silica gel column chro-matography using benzene-acetone (20: 1) as a developing agent Gr Sephadex LH-20 tPharmacia Co., Ltd.) using an organic solvent such as methanol, ethyl acetate and so on as a developing agent.
Physicochemical properties of the substance SF-1540, a pale yellow powder, produced according to the process of this invention are as shown below.
1) Elemental analysis Carbon 65.16~ llydrogen 8. 24 Nitrogen 2.17% Oxygen 23. 79~
2) Molecular weight (measured by a vapor pressure method) 3) Melting point :10774Zl 131 - 133C.

~`4) Optical rotation [~] ~ 31.6 (C - 1, methanol) . 'r , 5) Ultraviolet absorption sepctrum The ultraviolet absorption spectrum in a nethanolic solution i6 as shown in Fig. 1 Maximum absorption 249 nm (El% 605) lcm Shoulder 2~0 nm (~1~ 270) lcm 6) Infrared absorption spectrum The infrared absorption spectrum in a Ksr tablet method is as shown in Fig. 2.
7) Nuclear Magnetic Resonance spectrum The NMR spectrum in CDC~3, 100 MHz, is as shown in Fig. 3 -8) Solubility in solvents Soluble in methanol, ethanol, n-butanol, ethyl acetate, benzene, acetone, chloroform, carbon tetrachloride and ethyl ether.
Isoluble in water and cyclohexane 9) Stabilit~
~ntibacterial activity decreased by approximately 70% in 0.02 N HC~ and 0.02 N NaOH. Somewhat unstable to an acid and an alkali.
10) Rf values in various chromatographies Single spot having Rf value of 0.68 on a silica gel thin-layer chromatography (available from Merck & Co., Inc.) de-veloped with chloroform-methanol (5:1), Rf value of 0.35 on the chromatography developed with benzene-acetone (2:1), ~f value of 0.31 developed with ethyl acetate.
The present invention further relates to a useful and new derivative of the above mentioned antibiotic substance SF-1540 and to a process for preparing the derivative.
`:

: - . ~ .. . , :
- ..
- . . , , : - ,.. ~ . , . ~

~077421 As described above detailedly, the antibiotic substan-ce SF-1540 can be solvent-extracted and isolated from a cultur-ed broth, for example of Streptomyces hygroscopicus SF-1540 and in an antimicrobial agent effective mainly against gram-positive bacteria and various moulds.
- The substance SF-1540 itself is, however, of a relati-vely high toxicity and, in case where its aqueous suspen~ion is intraperitonieally given to mice, all animals died at a dose of not less than 5 mg./kg.
As a result of our further studies to prepare and test various derivatives thereof in order to improve such a disadvan-tage, it has been found that a new derivative can be formed by treating the substance SF-1540 with methanol and keep the anti-biotic activity of the parent compound unchanged as it is, simul-taneously with an acute toxydity reduced to at least one twentyth or less that of the parent compound.
More specifically, the substance SF-1540 in its pure state or a crude material containing said substance can be dissolv-ed in methanol or a methanol-containing mixture and the resulting solution is allowed to stand at a temperature of 5 - 60C., pre-ferably room temperature, for several hours to several days to afford the derivative of substance SF-1540 (hereinafter referr-ed to as "the present derivative"). For example, a solution of substance SF-1540 in methanol is left at room temperature for 2 days, whereby the substantially complete reaction proceeds to produce the present derivative quantitatively~ In addition, it is feasible to employ as a starting material the crude substance SF-1540 which is with different purities and obtainable during the isolation stage from a cultured broth. However, the lower a purity of the starting material is, the slower the reaction proceeds and, where the crude substance SF-1540 with a purity of 80% is employed, the reaction may proceed by approximately '' . . : - ' ~' ' ' :
, :

`I` 10774Zl even through treatment with methanol at room temperature for one day, but not further and some 20% of the starting material remain unreacted.
The present derivative obtainable from treatment of substance SF-1540 with methanol may be further purified, if re-` quired, according to the purification procedures of substance SF-1540. In order that the present derivative may be separated from unreacted substance SF-1540, it is particularly convenient to employ a column of Sephadex*LH-20 and then develop said column with methanol.
Physicochemical properties of the derivative of this invention are as shown below.
The present derivative is a pale yellow powder and melts at 114 - 116C in its amorphous form. The compound is re-latively stable under neutral condition, but unstable under aci-dic and alkaline conditions.
_,20 Optical rotation rd I t 21 (C = 1%, CHC~3) Elementary analysis :C, 66.69; H, 8.62t N, 1.68; O, 23.96 (~) Molecular weight : about 700 (measured by a vapor pressure method) Ultraviolet absorption spectrum: The ultraviolet absorption spectrum in a metha-nolic solution (10 mcg./m~) is as shown in Fig. 4. Maximum absorption in neutral methanol 249 nm (Elcm 585) Infrared absorption spectrum: The infrared absorption spectrum in a KBr tablet method is as shown in Fig. 5.

Nuclear Magnetic Resonance spectrum: The NMR spectrum in CDC~3, 100 MHz, is as shown * Trademark ; -8-i --, - - - : .

1 774Zl in Fig. 6.
Solubility in solvents: Soluble in methanol, ethanol, butanol, ethyl acetate, benzene, acetone, chloroform, carbon tetra-chloride and e-thyl ether and sparingly soluble in water.
Color reaction: Positive to potassiumpermanganate and sulfuric acid and negative ; to ninhydrin.
As particularly analogous compounds to the present derivative may be mentioned the substance SF-1540 as well as the substance SF-1540B,both of whlch show a substantially identical ultraviolet absorption spectrum. However, the present derivative can be definitely distinguished from the above two compounds, based upon sillca gel thin-layer chromatographies shown in the ~; folIowing Table 1.
` Table 1 Rf value ... .
The present derivative SF-1540 SF-1540B
Benzene-acetone (2:1) 0.35 0.23 0.05 Ethyl acetate 0.42 0.31 0.08 Chloroform-methanol (5:1) 0.67 0.57 0.17 In assay of the substance SF-1540 and the present derivative, there is used the following procedures. Potato-glucose.
agar medium is used as a test medium. Test organism is Piricularia oryzae. By the assay using the above-mentioned materials, is observed a liner relationship between logarithm of a concentration and inhibition zone in the substance SF-1540 and the present ?.. ~ .
- derivative at 32 mcg/ml, showing ccrrespondingly inhibition zones of 52.6 - 31.2 mm (Paper disc plate method). In the present _ g ,:
: .

. .

1077~Zl : ~rivative, the linear relationship is observed at a concentra-tion of 32 mcq/m~ - 2 mcg/m~ showin~ correspondinqly inhibition 20nes of 47.7 - 28.5 mm.
~ ntibacterial spectra of the substance SF-1540 and the present derivative against various microorganisms are shown - in the following Table 2.
Table 2 , _ Test organism MIC (mcg/m~) Medium Substance The present SF-1540 derivative Bacillus sublitis ATCC 6633 6.25 12.5 -Staphylococcus aureus 209p 12.5 12.5 Escherichia coli ~100 > 100 .
Mycobacterium smegmatis 607 25 25 2 Candida albicans 100 ~ 100 3 Saccharomyces cerevisiae 100 100 3 Saccharomyces chevalieri 0.78 ~ 0.39 3 Saccharomyces sake 50 ~100 3 Alternaria kikuchiana 3.12512.5 4 Botrytis cinerae 3.1256.25 4 Pellicularia sasakii 0.193.1 4 Muccor angulisporus ~100 ~ 100 4 Leptosphaeri salvinii 0.390.1 4 Trichophyton asteroides 100 100 4 Piricularia orizae 0.780.1 4 _ . _ _ _ _ . Colletotrichum lagenarium 3.125 0.1 4 - Fusarium oxysporum 100 ~100 4 [Note~ :

1: Bouillon media 2: Glycerin~bouillon media 3: Sabouraud's media , ~ .
:, ' ` ` ~0774Zl

4: Potato~glucose~agar melia As apparent from the above results, the substance SF- -1540 and the present derivative have characteristics in that it exerts little antibacterial activities against gram-negative bacteria, but does activities against ~ram-~ositive bacteria ` yeast and mould. The activities of the present derivative are substantially identical to those of the parent compound, the substance SF-1540, but it exerts a far more increased activity against certain moulds than the parent compound does.
In case where the present compound is intraperitoneally administered to mice, all animals survived at 15 mg/kg and 50 mg/kg, whereas, in case of the parent substance SF-1540, all animals died at 5 mg/kg and survived at 2 mg/kq.
Inasmuch as other known antibiotic substances are not found which show the above-defined physico-chemical and biologi-- cal properties, the present substances are to be regarded as new antibiotic substances.
This invention will be more fully illustrated by way of the following examples. However, it should be appreciated `~ 20 that various changes and modifications may be made within the ,: scope of this invention, even if not specifically shown herein.
; Example 1 Spores of Streptomyces hyqroscopicus strain SF-1540 (deposit number 2607) was inoculated to 1 ~ of a liquid medium containing 1 % starch, 3~ soy bean meal (pH 7) (using ten Sakaguchi'flasks). Shaking culture was effected at 28C. for 40 hours to obtain a seed culture. 35 k of a liquid medium con-taining 2.5~ glucose, 2.0% wheat embryo, 0.5% soluble vegetable protein, 0.25% sodium chloride (pll 7.0) was inoculated with the seed culture. Cultivation was continued at 28 C for 76 hours under aerate~ agitation (using two 50 ~-jar fermentors).

Cultured broth was then adjusted to pH 3 - 4 with 6N

`` ` ~0774Z~
~ drochloric acid, whereupon the substance SF-1540 was trans-ferred into mycelium fractions. To the broth was added a filter - aid, Hyflo Super Cel*, and mycelia were collected by filtration : and then e~tracted with 9,C of methanol. The mycelia were filter-ed off to yield 11~ of an a~ueous methanol solution.
The aqueous methanol solution thus obtained was con-centrated under reduced pressure to 2.5 ~ of an aqueous solution.
; The solution was extracted three times with ethyl acetate (2.5~), whereby the substance SF-1540 was transferred into the ethyl acetate phase, which was then dehydrated with anhydrous sodium sulfate and concentrated under reduced pressure to leave 30 g of an yellowish brown powder.
The yellowish brown powder containing the substance SF-1540 was extracted with cyclohexane to give 9.3 g of a brown powder containing the crude substance SF-1540 in cyclohexane-insoluble portions. The brown powder thus obtained was dissolv-ed in a small amount of acetone and the resulting solution was chromatoaraphed over a silica gel (~00 m ~) column packed with benzene using benzene-acetone (20:1) as a developing agent.
Those fractions of Nos. 387 - 470 were collected which showed an activity against Piricularia oryzae, each fractio~ being lS g.
The collected fractions were concentrated under reduced pressure to yield 728 m~ of the substance SF-1540 with a purity of 80~
as a pale yellow powder. 350 mg of the so obtained pale yellow powder was dissolved in a small amount of acetone and the result-inq solution was chromato~raphed over a silica gel (100 m ~) column ; packed with benzene using benzene-acetone (20:1) as a developing agent. Those fractions of Nos~ 421 - 443 were collected which showed an activity as seen abovc, each fraction being 10 g. The collected fractions were concentrated under reduced pressure to afford 65 mg of the substance SF-1540 as a pale yellow powder.
Example 2 * Trademark .'~, ' .

~77~Zl 350 mg of the substanc~ SF-1540 (728 mg) obtained as a pale yellow powder with a purity of 80~ in the above Example 1 was dissolved in a small amount of ethyl acetate and the result-ing solution was chromatographed over a Sephadex LH-20 (300 m,~) column packed with ethyl acetate using ethyl acetate as a develop-ing agent. sy each 5 g fractionation, fractions No.17 - 20 were subjected to a silica gel thin-layer chromatography (benzene:
~: acetone = 2:1), whereby a single spot of the substance SF-1540 was detected. These fractions were concentrated under reduced pressure to afford 168 mg of a pale yellow powder.
~ Example 3 '~ 500 mg of the crude substance SF-1540 (with a purity of 80%) obtained in the above Reference example was dissolved in 10 m ~ of methanol and the resulting solution was left at room temperature for 24 hours. The mixture was concentrated and passed through a Sephadex Ll~-20 (300 m Q ) column which was then developed with methanol. Effluents were in each 10 g portion collected and those fractions of Nos. 20 - 30 were recovered and concentrated to dryness to afford 184 mg of the present deri-vative. M.P. 114 - 116C.
Separately, 30 mg of unreacted substance SF-1540 was recovered from those fractions of Nos. 17 - 18.
_xample 4 500 mg of the substance SF-1540 with a purity of not less than 99% was dissolved in 10 m ~ of methanol and the result-; ing solution was left at room temperature for 2 days. The mixture ;~ was concentrated to dryness and, after addition of 20 ml~of fresh methanol, again concentrated to dryness to leave 490 mg of a powder substantially composed of the present derivative solely.

Claims (6)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for preparing a derivative of the antibiotic substance SF-1540, which comprises treating said antibiotic substance SF-1540 with methanol to form the corresponding antibiotic substance SF-1540 derivative.

2. A process according to claim 1, wherein said treatment with methanol is conducted at a temperature of from 5 to 60°C for several hours to several days.

3. A process for preparing a derivative of the antibiotic substance SF-1540, which comprises culturing a substance SF-1540-producing microorganism belonging to the genus Streptomyces in a medium, isolating from the cultured broth the substance SF-1540 thus obtained and treating said antibiotic substance SF-1540 with methanol to form the corresponding antibiotic substance SF-1540 derivative.

4. A process according to claim 3, wherein said SF-1540-producing microorganism is Streptomyces hygroscopicus SF-1540.

5. A process according to claim 3, wherein said SF-1540-producing microorganism is cultured under aerobic condition at a temperature of from 25 to 35°C for 2 - 5 days.

6. A derivative of the antibiotic substance SF-1540 which has the following physicochemical properties: an optical rotation of = + 21° in chloroform; an elementary analysis of 66.69% carbon, 8.62% hydrogen, 1.68% nitrogen and 23.96% oxygen;
an ultraviolet absorption spectrum shown in Fig. 4; an infrared absorption spectrum shown in Fig. 5 and a nuclear magnetic resonance spectrum shown in Fig. 6; a color reaction positive to potassium permanganate and sulfuric acid and negative to ninhydrin, sparingly soluble in water and soluble in methanol, ethanol, acetone, chloroform and ethyl acetate, whenever obtained by a process according to claims 1, 3 or 4, or their obvious chemical equivalents.