Note: Descriptions are shown in the official language in which they were submitted.
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
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.
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
1077421
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
,
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~ 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-
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~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)
600
3) Melting point
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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.
`:
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- . . , ,
: - ,.. ~ . ,
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~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
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, :
`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
,:
: .
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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
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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
--11--
`` ` ~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
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~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.
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