CA1082626A - Antibiotics sf-1771 from streptomyces - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A new antibiotic SF-1771 substance is produced by cultivating a new strain, Streptomvces tovocaensis SF-1771 in a liquid culture medium under aerobic conditions, and this antibiotic may be isolated from the fermentation broth by treating the broth filtrate with a synthetic adsorbent resin for adsorption of the active compound and eluting the resin with an aqueous alcohol or aqueous acetone, followed by chromatographic purification. SF-1771 substance is a copper-containing `
antibiotic which shows antibacterial activity to Escherichia coli, Salmonella typhi, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis.
Removal of the copper component from SF-1771 substance by treatment with hydrogen sulfide, an alkali metal sulfide or a copper-chelating agent gives SF-1771-B
substance containing no copper component which shows antibacterial activity as high as but a lower toxicity than SF-1771 substance.

Description

SU~IARY OF TH~ NTION
__ _ This invention relates to two new and u~eful antibiotics desi~nated as SF-1771 substance and SF-1771-B
substance. -This invention further relates to the ferrnen-tative production of SF-1771 substance and the production of SF-1771-B substance by chernical treatment of SF-1771 substance as well as to the uses of S -1771 and S~-1771~B
substances.
BACKGROUND OF T}~ IIi~5NTION
A ~reat variety of pathogenic microorganisms such as bacteria and fungi are causative agents in producing dlseases in man, animals and plants. Although a number ~ -of antibiotics have been developed, some of w~,ich possess usefully high antimicrobial activity a~ainst one or more pathogenic microorganisms, there remains a need for more e~fective ~gents to combat the many diseases caused by these microorganisms in man~ animals and plants.
An object of this inventlon is -to provide new antibiotics which are useful as antibacterial agent for therapeutic treatment of bacterial infections in man and animals and/or for sterilization of surgical materials and instruments. A further obJect of this invention is to provide processes for the production of these new antibiotics. Other objects of this invention will be clear from the following descriptions.
We have made extensive research in an attempt .~ to produce new and useful antibiotics. As a result, we -have now ~ound that when a new strain of the ~enus Strcptom~ces which was isolated from a soil sample . : .

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collected in ~ada-u-mi-cho, ~akehara Clty, Hiroshima Prefecture, Japan, is cultivated in a culture rnedium under aerobic conditions, a substance exhibiting an antibacterial activity &gainst gram-negative and ~ram-~ositive bacteria is produced and accumulatecl in the culture. ~Je have now succeeded in isolating this antibacterial substance from the culture and purifying it. As result o~ studies of the chernical, physical and microbiological properties of this isolated substance, it has been confirmed that this antibi.otical :.
substance is a new antibiotic which is distinguishable: .
~rom any of the known ~ntlbiotics. Thus 9 we have ~
desi~nated this new ar.tibiotic as S~-1771 substance. .
E~AI~ED ~E~CRIPTION O~.THE I.~7FNTION
According to a ~irst aspect of this invention, therefore, there are provided as new and useful anti~
biotic~ S~-1771 substance, a blue-colored compound as an amorphous powder which is basic and soluble in water and shows an a.ntibacterial activity, of which the ~ 20 hydrochloride ~iscoloroE- slowly into brown at 185C
: and decomposes at 208~a with foaming, of which the hydrochloride is readily soluble in waterS soluble in rnethanol, sparingly.soluble in ethanol and butanol but insoluble in the other organic sol~7~nts, of which~the hydrochloride is positive in the reactions with nin-hydrin reagent, Ehrlich's reagent, potassium per-mang~.nate reagent, Greig-Leiback's reagent and i.s negative in the reaction with Sakaguchi's reagent, of .. . . .
which the hydrochloride shows: a molecular weight of about 1600 as determined by Barger method and grives : ~ 3 ~

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an elementary analysis C ~8.70i~, H 5,42~o, N 13.0~, 0 29.62~o~ S 3.62~o~ Cl 6.07~ and Cu 3.53% (as measured by atomic absorption spectroscopy), of which the hydrochloride exhibits characteristic absorption peaks at 248 nm (ElC~ = 146) and at 282-284 nm (El%m = 106) in the ultra-violet absorption spectrum when dissolved in water and exhibits cha.racteristic absorption ban~s as shown in ~igure 2 of the accompan~ing drawing~ in the infra-red absorption spec-trum when pell~ted in potassium bromi~e, and a pharmaceutically acceptable acid-addition salt of said S~-1771 substance.
Examples o~ the pharmaceutically acceptable 56~S
acid-a~dition ~ of the S~-1771 substance according to this invention include the hydrochloride, sulfate, ~:
nitrate, phosphate, acetate, maleate, fumarate, succinate, tartrate, oxalate, citrate, methanesulfonate, .:
ethanesulfonate and the like. ~;
It has further been found that when the S~-1771 substance is treated with hydrogen sulfide or an alkali metal sulfide such as sodium sulfide in so.lution in water, the copper component present in the SF-1771 substance molecule is re oved there~rom in the form of copper sulfide, so that there is produced a new further - subst~nce which does not contain the copper component but show an antibacterial activity as high as but a . ~. .
lower toxicity than the SF-1771 substance itsel~
It has been found that the SF-1771-B substance also exhibits an anti-tumor activity. Accordingly, this new : :
further substance has been designated as SF-1771-B
substance. :
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According to ~ second aspect of this invention, therefore, there are ~rovided as new and useful anti-biotic, SF-1771-B substance, a colorless or faintly yellow-colored compound in an amorphous powder which is basic and soluble in water and sho~s an antibacterial activity, and an anti-tumor activity, of which the brt~wn hydrochloride discolors into fro~ slowly at 180~C
and decomposes at 212C with foaming, o-~ which the hydrochloride is readily soluble in water, soluble in methc~nol, sparln~ly soluble in ethanol and butanol but insoluble in the other org~nic solvents, of which the hydrochloride is positive in the reactions with nin-hydr.in rsagent, Ehrlich's reagent, potassium per-manganate rea~ent and Greig-Leiback's reagent and is negative in the reaction with Sakaguchi's reagent, of which the hydrochloride shows a molecular weight of about 1600 as measured by Barger method and a specific optical rotation [a]20 -1~.4 (c 1, H20), of which the hydrochloride shows an elementary analysis a 41~36~o~ H 5.53~, N 14~.~19~, S 3.64~o and 0 28.93%, of which the hydrochloride exhibits characteristic absorptlon peak at 288-290 nm (~1~cm=84) in the ultra-violet absorption spectrum when dissolved in water and exhibits characteristic absorption bands as shown`in Figure 5 o~ the accompanying drawings in the infra-red absorption spectrum when pelleted in potassium bromide and further exhibits a proton magnetic resonance absorption spectrum as shown in Fi~ure 6 of the accompanying drawings when dissolved in deutero-`water, and a pharmaceutically acceptable acid-addition , ' ' . : .

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salt of the SF-1771-~ sub~tance.
Examples of the pharmaceutioally acc~ptable acid-addition salt of the SF-1771-B substance include those same as men~ioned herelnbefore for the S~-1771 ~ubstance of this invention.
These acld-addition salts of the S~-1771-B
substance as well as of the SF-1771 substance may be prepared by r`eactin~ the ~ree base form of the S~-1771-B substance or the S~-1771 3ubstance with a pharmaceutically acceptable ac1d such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid or acetic acid in solution in water at ambient tem-peraturs in a known manner, Referring to the accompanying dral~ings: ;
Figu~e 1 shows an ultra-violet absorption spectrum Gf the SF-1771 substance hydrochloride in -"
water.
Figure 2 shows an infra-red absorption spectrum of the SF-1771 substance hydrochloride pelleted in potassium bromide.
~ igure 3 shows patterns of ~radient elution of the S~-1771 substance and its related antibiotics when chromatographed on a ion-exchange gel-filtration ` a~ept CM-Sephadex C-25 (H ), as compared to the elution pattern of ammonium formate.
~ igure 4 shows an ultra-violet absorption spectrum of the S~-1771-B substance hydrochloride in wat~r.
Figure 5 shows an infra-red absorption spectrum - -o~ the SF-1771-B substance hydrochloride pelleted in - , . , potassium ~romideO
Figure 6 shows a proton ma~netic resonance absorption spectr~ of the SE~1771-~ substance hy~ro-chloride in deutero-~ater as measured at 100 M Hz.
The S~-1771 substance hydrochloride sho~,Js the ~.
following physical and chemical propertieso (1) Appearance: Blue-colored amorphous powder.
(2) Decompositio~ tem-perature: Discoloration into bro~ln takes place slowly at 185C and the decomposition occurs at 20~C with foaming.

(3) Elementary analysis: The SF-1771 substance hydrochloride contai.ns the elements, carbon, hydrogen, nitrogen, oxygen, sulfur, copper and chlorine and gives an analysis result:
C 38.70~o, H 5.42~, N 13.03~o, 0 29 62^~, S ~.62~, Cl 6.07~o and Cu 3.53~o (as measured : by atomic absorption spectroscopy).

(4) Ultra-violet absorption spectrumo ~he U.V.
spectrum of the SF-1771 substance hydro- :
., chloride as measured for its aqueous solution :~
is as shown in Fi~v.re 1 and exhibits absorption peaks at 248 m~ (El~m = 146~ and at 282-284 m~
(Elcm = 106),

(5) Infra-red absorption spectrum: The I.R. spectrum o~ the S~`-1771 substance hydrochloride as pelleted in potassium bromide is as shown in Figure 2.

(6) Molecular weight: The molecular wei~ht is about 1 1600, as measured by Barger me-thod and as ¦ ~ assumed that a single copper atom is present ' , ,;'' ~ - 7 , .
.
., . : , ;. -per molecule of the SF-1771 substance.

(7) Solubility: Soluble readily in ~"ater, soluble in met~anol, sparingly soluble in ethanol and butanol but insoluble in the other or~anic solvents.
(~) Color reaction: Positive to ninhydrin, ~hrlich's9 potassium perManganate and Greig-Teiback's reagents. Negative to Sakaguchi's reagent.
(9) Stability: Stable in neutral znd acidic media, ~f6.1~e but re].atively ~ e~ n alkaline media.
(10) Rf value: In a silica gel thin layer chromato-graphy(on "Kieselgel 60 ~254"' a product of I~Ierck Co., Germany), the ~F-1771 substance hydrochloride ~ives a single spot at Rf-values as shown in the following table.
~abl e 1 Develo~ment solvents Rf-value Propylalcohol-pyridine-acetic 0.76 acid-water (15~10:3:12 by volume) Methanol-lO~o aqueous ammonium ncetate-lO~o aqueous ammo~ia 0.57 (10:9:1 by volume) lOfo Aqueous am~onium acetate- 0 68 methanol (1:1 by volume) 10% Aqueous ammonium acetate- 0 54 methanol (1:2 by volume) . ~
(11) Rf value: In a ~aper chromato~raphy (the upward method), the single~spot appears at ~ `
Rf-values as shown in the ~ollowing table.
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Table 2 , Develo~ment solvents Rf-volue Water-s~turated n-butanol O
3% Aqueou~ ammonium chloride (by weight) 0.71 ~h.enol-water (~:1 by volume) 0.69 Acetone-water (1:1 by volume) 0.09 n-Butanol-methanol-water (4:1:2 by volume) ,0.06 Benzene-methanol (4:1 by volume) , 0.02 Water 0.07 (12) Optical rotation: Determin~tion of the specific optical rotation is impossible with the D-ray, :
owing to the blue color of the solution of :.' the S~-1771 substance hydrochloride. `.
(13) Elution p~ttern: ,Figure 3 shows the gradient '' . elution ~atterns of the SF-1771 subst~nce :~
hydrochloride and its rele~ted antibioti.cs . .'~
when eluted from CM-Sephade~ C-25 (H+) ( an ion-exch~nge ~el-filtr.~ltion agent " .
produced by Pharmacia Co., Sweden) developed ,:, with from O to 1.0 M ammonium formate as the eluent. Each aluate w~s collected in '.
5 ml-fractions, and optical density o~ each ', fraction was determinedO The difference between the determi.ned values of th,e optical density of each fraction and the determined value of the optical de~sity of the pure -eluent were plot,ted as ~OD ~long the ordinate, in rel~tion with the numbe~ oE the tubo for _ g _ .~

.: ' ., ' each fraction plotted in the abscissa of the coordinate as shown in ~igure 3. As the related antibiotics were tested bleomycins, victomycin and platomycin A, B for the com-parison ~ur~ose. ~rom these elution patt.erns, it is seen that the S~-1771 substance hydro-chloride is eluted later than bleomycin B2 but somewhat faster than bleomycin ~4, victo-mycin ~nd platomycin A.
The SF-]771 substance of this invention exhi.bits : . .
an antibacterial spectrum as shown in Table 3.below.
The ~inim~ inhibitory concentrations (M.I.C.) of the S~-'771 substance ~ainst various microorganisms were~
determined according to a known broth dilution method ~:
in such a manner that the determination was conducted ; after the incubation was effected~at 37C for~18 hours using Heart Infusion Agar as the incubation medium.
: Table 3 Antibacterial s~ectrum of S~1771_substance Test Microor~nisms ~scherichia coli ::o~.~g Escherichia coli K-12-R . o,~g~ :
Salmonella typhi ~ : <0.2 Pseudomonas aeruginosa ?5 Staphylocoocus aureus 209P 3.12 :
Bacillus subtilis PCI-219 ~ . 1.56~
As ~ill be seen from the results of Table 3, the . SF-1771 substance o~.this invention exhibits a high ~ antibacterial activity against gram-positive and~gram~
- 30 negative bacteria, and it shows a remarkably high ~ . .

~ . ' ' , antibacterial ~ctivity to the gram-negative bacteria.
Moreover, it has been found that the SF-1771 substance also exhibits an antifungal activity against various fungi. The minimum inhibitory concentrations of the S~-1771 substance to v~rious fungi were determlned according to a kno~n broth dilution method in such a manner that the determination was conducted after the incubation was effected at 2~C for 3 days using Sabouraud ~lucose a~ar as the incubation medium. As exception, the incubation time was 5 days for ~ri~ :
choph~ton. asteroides and As~er~illus fumi~atus. ~he antifungal spectrum of the S~-1771 substance is shown in Ta~le 4 below.
! ~able 4 Antifungal s~ectrum of_S~-1771~substance Test or~anisms ~ ~ M.I.C. ¦~/m~
Candida albicans > 100 Candida krusei 12.5 Candida parapsilosis 50 Candida stellatoidea 100 Candida tropical1s >100 Cryptococcus albidus 0,7 Cryptococcus laurentii ~ 6,25 Cryptococcus neo~ormans ~01 0.78 aryptococcus terreus ~ ~ 0.78 Cryptococcus uniguttulatus 3.13 Pichia spartinae ~ 12.5 ~ ~ -Saccharomyces cerevisi~e ~12.5 . Trichophyton asteroides 6.25 Aspergillus fumigatus 1.56 ,-. , ~ , . , ,, : ' ~urthermore, it has been found th~t the 3F-1771 substance is effective to -treat the infections of Tricho~hytons in animals. Thus, six ~roups of test ani!nals, each group consisti.n~ of 10 guinea pigs of Hartley-strain (male adult, wei~hing 310 to 400 g.), were inoculated wi.th Tricho~h,vton steroides in such a way that the fur was cut off from four ~reas of the skin at the back of each animal, these skin areas were rubbed with sand-paper and th.en a~plied thereon with an aqueous suspension of the mycelia o. ~richo~h,~ton asteroides. 2 Days after the inoculation, a solution of 1~ by wei~ht of the S~1771 substance in eth&nol was applied onto th.e ino^ulated skin areas once a~day for 9 days for the therapeutic treatment of the , Trichoph~ton infection. For the control group of t~st animals, only aqueous 70~ ethanol was applied to the inoculated skin areas in the same way as above.
After this treatment, the animals were sacrificed, ::
and from the skin areas so treated were cut out three ~ieces (3 x 3 mm) of the skin which were subsequently incubated on a plate of agar culture medium at 27 C for .,'3 days. ~licr3scopic observation showed that no growth of the fungus could be observed on all the skin pieces , ..
which were cut out from the sLin areas treated with the SF-1771 substance of this invention, whereas the growth of` the fungus was observed on substantially all the skin pieces which were cut out from the skin areas of' the control animals treated merely with the aqueous ethanol.
, ~or determination of acute toxicity o~ the .
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S~-1771 substance, solutions containing 6,25 m~ -50 mg of the S~-1771 substance per ~.2 ml of isotonic sodium chloride solution tJere intravenously injected at a dose of 0.2 ml per mouse into several groups of mice, each group consisting of five mice of ICR-strain (male adult weighing 20 g in average).
7 Days after this intravenous injection, it was estimated that percentages of the n~ber of dead mice based on the wl~ole number of ~ice tes-ted were lOO'~o, lOOY~, 60~ and 20% at the dosages of 50 mg/kg, 25 mg/kg, 12.5 mg/k~ and 6.25 mg/kg of the SF-1771 substance per mouse, respectively. Accordingly, the S~-1771 substance cannot be said to be non-toxic but is still useful ~or the sterilization o~ surgic~l materials ~nd instruments ~ r ~x as well as ~ devices and places which arQ desirably kept sterile. ~he SF-1771 su~tance mpy also be used as an antibacterial a~ent for the external application, ~or these purposes, the S~-1771 substance may be formu-lated into an aqueous solution containing 0. Ol~o to 0. l~o by weight of the SF-1771 substance or an acid addition salt thereof, Compari~on is ~ow made between the SF-1771 sub~tance and known water-soluble basic antibiotics.
As the water-soluble and basic antibiotie containing copper constituent, there are known phleomycin tT. Ikekawa et al: "Journal of Antibiotics" Ser. ~, Vol. 17, page 194 (1964)~, bleomycins (H. Umeæawa et al:
- "Journal of Antibiotics" Ser. A. Vol. 19, page 200 (1966~)), zorbamycin and zorbonomycin ~,C (A.D.Argoudeli~
et al: "Journal o~ Antibiotics" Vol. 24, page 543 (1971)), , .
' . :

YA-56-X, YA-56-Y substances ~Y. I~o et al: "Journal of Antibiotics" Vol. 24, ?age 727 (1971)), victomycin (T. Nara et al: "Journal of Antibiotics" 'Joi. 28, page 366 (1975) and platomycin A,B (T. Nara et al: "Journal of Antibiotics"
Vol. 28, page 662 (1975) ) . All of these known copper-containing antibiotics show two absorption peaks, that is, the first one at 243-2~6 nm and the second one at 290-303 nm in their ultra-violet absorption spectra. However, bleomycins and phleomycin can be dif~erentiat~d from each other and further can be differentiated from the other, known ~ ;
copper-containing antibiotics. In contrast, the SF-1771 substance of this invention shows the first absorption peak at 2~8 nm and the second one at 282-284 nm in its ultra-violet absorption spectrum, indicating that the position of the absorption peaks of the SF-1771 substance is clearly different from the positions of the absorption peaks of the aforesaid known, water-soluble and basic copper-containin~
antibiotics. In this respect, the SF-1771 substance of this invention is a water-soluble and basic antibiotic of peptide type but is differentiated from all the known water-soluble and basic copper-containing antibiotics as mentioned above.
Therefore, it is evident that the SF-1771 substance is a new antibiotic compound which is distinct from any of the known antibiotics of the bleomycin and phleomycin type.

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, The ~-1771 substance OI this invention ~ay be produced by cultivatint~ a strain of the genus Stre~tom~ces which the present inventors firstly isolated from a soil sam~le collected in Tada-u~mi-cho, ~akehara City, Hiroshlm~-Pre ecture, Japan9 as stated hereinbefore and which has now been designated as S~-1771 strain. The cultivation may be carried out under aerobic conditions in the same manner as in the production of known antibiotics by culturing known strains of the genus Stre~tom.~Jces.
The S~-1771 strain has tbe following ch~rac-teristics:
(I) Morpholo~ical observation Aerial mycelia are &bundantly produced on glycerineasparagine-agar, starch-agar, oatmeal-agar, yeast-malt-agar and tyrosine-agar, and the formation of spores is abundant, The mycelium pro~uces mono-podially branches but does not produce whorl.
The aerial mycelium bears spirals at the tip thereo~. No form~tion of such special structure as sclerotium is observed. Electron-microscopic obser-vation shows that the surface structure of the spore is spiny. The spores are of elliptical shape -to o~al shape and are normally measurin~ 0.8 - 1.1 microns by 1,2 - I.4 microns in slze. Mature s ore chains with more thlan 10 spores per chain are usually produced.
.
(IT) Cultural characteristics on different culture media ~ ~he cultural chsracterlstlcs are shown in , .

~able 5 below. In the fol:Lo-v/ing table, the des-cri tions of colors gi.ven in a backe-t [ ] are b2.sed on the ~tarldard of Color Harmony Mannu~l of Container Corpor~tion of America.
Table Growth, color of reverse Aerial Coluble Culture medium side mycelium ~i~ment Sucrose nitrate Thi~ growth, Poor,whiteNone agar colorless to grayish white __~____________________________________________________ Glucose Faintl.y Brownish None asparag.ine agar yellow ~ray [3ge] ~ -_______________________________________________________ Glyceri.ne Good growth, Abundant, - None asparagine agar faintly gray [2fe]
yellow __________________ ____________________________ _______ Starch ag~r Good growth, Abundant, None ..
faintly light gr~yish yellowish yellow brown[4ig]
______~____________________ __________________ ________ . .
Oatmeal a~ar Good growth, A4undant, None grayish light yellow `yeilowish brown[4ig]
________ __ ________ ______________ _________ ________ , Yeast malt agar Good grow-th, : Cot-tony None _ . pale yellow abundant, to yellow faintly brownish gray. `
__~___________________ ____~______ ___________________ ~yrosine agar Good growth, pale gray None . ~...................... greyish l2dc]
. yellow :
.. . .. ... ~ , . , _ _ , , Note: The incubation tempera.ture was 28 C in .:
general, unless stated otherwise. . .
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~2~26 (III) Phisiological properties (1) Growth ~emperature rangeG The SF-1771 strain grows in a temperature range of 20-~8C on yeast-malt-agar medium.
(2) Li~ue~action of gelatineO Gelatine is slowly lique~ied by the incubation at 20C ~or 21 or more daysO
(3) Hydrolysis of starch. Positive (at 28C)o (4) Coagulation of s~immed milk~ Negative (at 28C and 37C)o t5) Peptonization of skimmed milk~ Positive (at 28C and 37C)o (6) Chromogenicity: NegativeO
(IV) Utilization of carbon sources (estimated in Pridham-Gottlieb's agar medium incubated a-t 28C) (1) UtilizableO D-glucose9 D-~ructose9 D-mannitol9 I-inositolO :
(2) Not utilizable~ Sucrose9 rhamnose, raf~inose9 L-arabinose9 D-xylose The above-mentioned characteristics o~ the SF~1771 strain may be summarised as ~ollows: The SF-1771 strain belongs -to~the genus ~3~E~Y~9 ~ .
and the aerial mycelium produces spirals at the tip thereo~ and~the sur~ace structure of the spore is splnyO The SF-1771 strain shows good growth on various culture media, and the color o~ the reverse side of the growth is pale yellow to grayish yellow and is not tinged distinctivelyO The aerial mycelium is gray to brown in color 9 and no soluble ~26Z6 pigment is produced on any culture medium, The SF-1771 strain was designated as Streptomyces spO SF-1771 and ha,s been depo6ited in a Japanese public depository "Fermentation Research Institute"9 Chiba city9 Japan9 under deposi-t number FERM-P
N0~ 3253 (a culture sampleo~ this strain was received by Fermentation Research Institute on September 183 1975) and also in the "American Type Cul-ture Collection"9 Washington DoC.9 UoS,Ao9 under ATCC
number ~1248.
As kno~l strains similar to the SF-1771 strain9 there may be mentioned _treptom~ces ~y~ 3~_9 Streptomyces natalensis 9 _tr~ y~ fasiculatus and Stree_omyces alulusO
In view of the descriptions o~ IoSoPo (International Streptomyces Project) in which reference is made to the articles of the ~'International Journal of Systematic Bacteriology" VolO 189 pages 108-1109 - : .
pages 174-176 (1968) and VolO 229 pages 271-2739 ~ ~
pages 323-326 (1972))9 these known four strains ~ `
cannot be distinguished ~rom -the SF-1771 strain in ~ :
their morphological properti~sand cultural character-isticso Thus9 immediate comparison was made between the type cultures of these known ~our strains and .
the SF-1771 strainO As a result9 it has been found that the SF-1771 strain is mos-t closes-t to ~E~ Y~
toyocaensis amongst the above-mentioned known four strains and is distinguishable from -the remaining ~ :
known three strainsO More close comparison reveals ~0 that the SF-1771 strain is well coincident with the , . , - 18 - ~

iO~ 6 type culture o~ y__s t~ _ s in many properties but is distinguishable from the l~tter in respect o~ the features shown in Table 6 belowO

Table 6 Streptomyces SF 1771 toyocaensis Features strain ~ culture o- oa~ _ medlum"
Color of reverse side Grayish Yellow to of growth yellow brownish yellow Soluble pigment None Pale yellow ~ O O O O ~ ~ O O O ~ O O O O O ~ O O O O O O O O O O O O O O O O ~ O O O O O ~ O O O O O O O O O O O O O O
m Cottony 9 Aerial mycelium pale gray Poor (scant) , SF-1771 Antibiotic produced substance Toyocamycin ' :

. , Thus9 the SF-1771 strain well coincides with __m~ caensis ln their principal properties9 though distinction is observed in rainor poin-tsO It appears reasonable9 therefore9 that the SF-1771 strain is identi~ied to be a new strain o~ the known species ,Streptom,yce~s ~ ocaen~s~O Accordingly9 the SF-1771 :~
strain is now de,signated as S r~ m~
S~-1771. '' The SF-1771 strain has properties which are:
liable to vary9 as may usually be observed wi-th the other species o~ ~3~tomxcesO Thus~ for ins-tance9 the SF-1771 strain may produce a variant or mu-tant ,~
when it is treated with various mutagens such as ultra-violet radiations 9 X rays9 redio-active rays 9 ' .
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high-frequency electromagnetic waves and chemicalsO
Any natural or artificial variant or mutant of the SF-1771 strain may be used for the production of the SF-1771 substance according to the concept of this invention, provided that it has the ability to produce the SF-1771 substance of this inventionO
According to the third aspect of this invention9 therefore9 there is provided a process for the production of the SF-1771 substance9 which comprises cultivating an SF-1771 substance-producing strai.n of the genus StreEEz3~y~ in a culture medium containing assimilable carbon and nitrogen sources under aerobic conditions for a sufficient time to ~
produce and accumulate the SF-1771 substance in the : .
culture medium and then recovering this antibiotic substance from the cultureO According to a preferred embodiment of this third aspect of this invention9 there is provided a process for the production of the SF-1771 substance 9 which comprises cultivating Strepto~XQ~ Y~ aa~ SF-1771 identified as FERM-P .. :
No~ 3253 or ATCCo NoO 31248 in a culture medium contai.ning assimilable carbon and nitrogen sources under aerobic conditions ~or a su~icient time to produce and accu~ulate the SF 1771 substance in the culture medium and then recovering the SF-1771 substance from the culture~
In the process according to the third aspec-t of this invention for the production of the SF-1771 substance 9 the SF-1771 subs-tance-producing s~rain and particularly ~ E~Y9~ Ç~ SF-1771 s-train - 20 ~

lO~ Z6 may be eultivated in a known mann~r under aerobie eonditions in a eulture me~ium containing nutrients, namely such carbon and nitrogen sources which are assimilable by ordinary microorganismsO As the nutrients may be employed any of the known nu~ritive substances which have commonly been employed in the .eultivation of the known strains of S~ m~cesO
~ For`instance9 glucose9 suerose9 starch, glycerine9 starch s~Jrup, molasses, soybean oll and the like are ~ :.
useful as the carbon sourceO Soybean meal9 wheat- :
embryo9 meat extract~ peptone9 dried yeast9 corn s-teep :
liquor, ammonium sulfate9 sodium nitrate and the like is useful as the nitrogen sourceO If required 9 . .
inorganic salts such as calcium carbonate9 sodium ehloride9 potassium chloride 9 phosphates and the like may be added to the culture mediumO Furthermore9 .
to the culture medium may be added such organic and inorganic material which aid the gro-~h of the SF-1771 substance-producing strain and promote the production of the SF-1771 substance~ Copper sulfate . may be added to the culture medium9 but addition of a eopper eompound to the eulture medium is not neeessarily required beeause a trace of copper naturally occurring in the nutrient substanees of naturalorigin employed in the culture medium is ;: ::
utilized by the S~-1771 strain as the eopper source to produce the SF-lf71 substance which is the copper-containing antibiotic.
A~ the method of cultivating the SF-1771 substance-producing strain, particularly th~ SF-1771 - 21 - ~
- : - : ' .
~ ,':'''.,:

strain9 liquid cultivation method and particularly submerged liquid cultivation method are most suitable9 similarly to the general processes of producing the known antibioticsO The cultivation may suitably be effected under aerobic conditions and suti~blc incubation temperature is in a range of 25C to 35C, For the commercial or laboratory production of the SF-1771 substance9 it is of-ten preferred to carry out the cultivation at a temperature in the vicinity o~ -28Co Under these cultivation conditions9 the ~ :
concentration of the SF-1771 substance in the culture broth reaches a maximum at the end of 2 to

8 days o~ fermentation9 either in shake-cultivation method or in tank-cultivation methodO ~ : :
For assay of SF-1771 substance of this invention9 the following assay method may be employed: The assaying culture medium comprising 005%
polypeptone9 0~3% meat extract and 105% agar (pH 700) :
is usedO As the assaying strain is used Escherichia coli NIHJo In this assay method9 at a concentration o~ 25 to 5 mcg/ml of the SF-1771 substance9 the relation between the logarithm o~ the concentration o~ the SF-1771 substance and the diameter o~ the :
inhibition zone can be plotted linearly9 giving the ~w_ inhibition zone of 2200 to 160 0 mm in diameter as determined by the paper-disc method, As the SF-1771 substance of this invention is a water-soluble and basic substance of a peptide type :
containing the copper compo~ent as stated hereinbe~ore9 it may usually be recovered from -the culture broth by ' ' ' ~ 2 6 adsorbing on an inorganic adsorber such as active carbon and ~lumina or a synthetic adsorbent resin such as Amberlite XAD-2, D~aion HP-20, a weakly acidic ion-exchange resin, ion-exchange g~l-filtration agent or gel-Piltration agent 9 and then eluting from the adsorber with aid of a suitable eluen-tO
For the recovery and purifica-tion of the SF~1771 substanceO however9 -the foll~wLng method is most ef~iciento. The culture broth is ~iltrated with aid of a suitable filtration-aid such as diatomaceous earth to remove the mycelium and the o-ther solid matter9 and the broth filtrate so obtained is passed -through a column of Amberli-te XAD-2 (a synthetic adsorbent resin consisting of a microporous copolymer of styrene and divinylbenzene9 a product of Rohm & Haas COo ~ UoSoAo ) 9 for the adsorption of the S~-1771 substance. The resin is then washed with water and eluted with an eluent consistin~ of an aqueous -.
ethanol or aqueous acetone which may be adjusted to an acidic pH (pH 3O0 to 3O5) by addition of a mineral acid such as hydrochloric acid or an organic acid such as acetic acid~ The eluate is collected in ~ractions, a~ld the active ~ractions are combined together and, i~ necessary, neutralizedO The active fractions are then concentrated under reduced pressure by distilling out the organic solvent(the ethanol or acetone)O The - resulting concentrated solution is passed through a : .
column of CM-SePhadeX C-25, (an ion-exchange gel- :
filtration agent consisting o~ a carboxymethyl-substituted cross~linked dextran gel, a product o~
,' ~ .
- 23 - : :

.

, .. ..

~ 2 ~
., .

- Pharmacia Co. 9 Sweden) The colum~i is washed with ~ .
water and then eluted with an aqueous solution of a suitable salt such as 0.2 M.aqueous sodium chloride solutionO The eluate is collecied in fractions, and the active fractions are combi.ned toge-ther and .
de-salted by trea-ting with Amberlite XAD-20 The de-salted solu-tion is concentrated un~er reduced pressure and freeze-dried to give a crud.e powder containing :~
the SF-1771 substanceO For the purification of thls crude powder9 said crude powder is taken up into a .
small volume o~ water and the ~queous solution is subJect`ed to a column chromatography on Sephadex LH-20 (a product of Pharmacia Co.9 Sweden, a gel .~.
consisting of a derivative dextran sulfate) developed with methanol or a mixed solvent of butanol-me-thanol-water (4:1:2 by volume) as t~e eluentO The eluen-t i3 .
again collected in fractions9 and the active fractions which give the single spot of the SF-1771 substance .
on paper chromatography are combined to~ether and concentrated to dryness 9 affording a pure product o~
the SF-1771 substance as a blue-colored amorphous powder. `
The SF-1771-B substance according to the second aspect of this invention may ~e produced by treating the SF-1771 substance with a suitable agent which is able to remove the copper component from the .
SF-1771 substance molecule~ According to the fourth . aspect of this invention, theréfore, there is provided ~:
- a process for the production of the SF~1771-B
, ~; 30 substance~ which comprises subjecting the SF-17?1 ~;
:`'` . ' :

. , . ~ . . . . ~ . . .

~ 2 ~

substance to a treatment for the removal of the copper component from the SF-1771 substance molueculeO The treatment for the removal of the copper component from the SF-1771 substance moleculemay be ef~ected either in sucha manner that the SF-1771 substance in solution in meth~nol or other suitable inert organic solvent is reacted with hydrogen sulfide or an alkali metal sulfide such as sodium sul~ide and potassium sulfide for a su~ficient time to remove the copper component from the SF-1771 substance molecule9 or in such a manner that the SF-1771 substance is reacted with a copper-chelating agent such as 8-hydroxyquinoline ~
and dithizone for a sufficient time to remove the -copper component from the SF-1771 substanceO
When the SF-1771 substance is reacted with the copper-chelating agent for the removal o~ the copper component therefrom9 it is pre~erred that the SF-1771 substance in solution in an acidified water is reacted wi.th a solution of dithizone in chloro~ormO
These reactions for the removal of the copper constitutent from -the SF-1771 substance may be f ~Lr carried out at ambient ~e~YJ~Y~ using as the ~:~
reaction medium water.or an organic solvent in which ~.
the reagents are soluble and which is inert to the reactionO According to a pre~erred embodiment o~ the ~ourth aspect o~ this invention9 there is provided a process ~o.r the production o~ the SF-1771~B substance 9 which comprises subjecting the SF-1771 substance to - a tr~eatment with hydrogen sulfide 9 an al~ali metal sul~ide or a copper-chelating agent ~or a su~ficien-t .
' ' time to removc the copper componcn~ from the SF 1771 substance molucule. :
The fourth aspect .~rocess of this inventionfor the production o~ the SF~1771-B substance may be ef.fected not only using the SF-1771 substance which has been isolated in the ~orm of a crude powder or a pure product as stated above 9 as the starting material which is -to be treated with an agent for the removal of the copper component 9 but also using as the raw material the fermenta-tion brothg the broth filtrate or a crude solution containing the SF-1771 substance d.iss$)1ved therein9 in such a way -that the SF-1771 substarlce is reacted ~ th hydrogen sulfide9 an alkali metal sulfide or a copper-chelating agen-t while it is still present in the fermentation broth, the broth filtrate or the crude solutionO
To isolate the SF-1771-B subs-tance from the reaction.
mixture in which the SF-1771 substance has been treated with the agent for the removal o~ the copper component the~e~rom9 there may he employed such methods which are similar to those adoptable for the ~`
recovery of the S~-1771 substance from the cul-ture of the SF-1771 substance-producing microorganlsm~ `
Thus, the reaction mixture coming from the treatment of the SF-1771 substance ~or the removal ol -the .
copper component there~rom ~ay be treated with Amberlite XAD-2 ~or the adsorption of the SF-1771~B
substance, and the adsorbent resin may then be elut~d9 - followed by chromatography o~ the active fractions :
of the eluate to give a crude powder containing the ' ,, - ~6 -'~
; ' "

SF-1771-B substanceO ~'or further puri~ication9 this crude powder is chromatographed.on.a column o~
Sephadex L~I-20 or Diaion~P-20 so that a pure product o~ the SF~1771~ substance is affordedO The SF-1771~B
substallce as well as the SF-1771 substance are water-soluble and basic antibio-tics o~ peptide type 9 and hence the SF-1771-B substanoe may generally be purified by a purification method which lS analogous to such one for the purification of the SF-1771 substanceO .
The SF-1771-B substance hydrochloride shows the following physical and chemical properties 9 although some of these properties are set ou-t hereinbeforeO
(1) AppearanceO Colorless (white-colored) to slightly yellow co~ored amorphous powderO :`~
(2) Decomposition temperatureo Discoloration into bro~.~ takes place slowly at 180C and the ..
decompositi.on occurs at 212C with foamingO
(3) Elementary analy,siso The SF-1771-B substance hydrochloride contains the elements9 carbon9 .
hydrogen, nitrogen 9 oxygen 9 sulfur and chlorine and gives an analysis ~esult~
G 41~36%~ H 5053%9 N 14~19,o~ O 28093% and S 3 ~ 64/o It was noted that, upon the elementary :
analysis 9 the content of chlorine was 6~12%, . . `
although the determination of the chlorine : content wa~s difficult to carry out in an .
exact way owi.n~ to the presence o~ the ~ulfur ~.

..

component in the molecule.
(4) Ultra-violet absorption spectrumO The U~V0 spectrum of the SF-1771~B substance hydro-chloride as measured ~or its aqueous solu-tion is as shown in Figure 4 and exhibits absorption peak at 288-290 nm (E1 cm - 84)o (5) In~ra~red absorption spectrumO The IoRo spec-trum o~ the SF-1771~B substance hydro-chloride as pelleted in potassium bromide is as shown in Figure 5~
(6) Proton magnetic resonance absorption spectrum, The PoMoRo spectrum of the SF-1771-B
substance hydrochloride in deu-terium oxide is shown in Figure 6 as determined at 100 M Hzo (7) Molecular weightD About 1600 as measured by Barger methodO
(8) Solubilityo Soluble readily in water9 soluble ~.
in methanol9 sparingly soluble in e-thanol and butanol but insoluble in the other organic solvents,

(9) Color reaction . Positive to ninhydrin9 :
Ehrlich's9 potassium permanganate and Greig-Leiback's reagents, ~ .
Negative to Sakaguchi's reagentO

(10) Stability. Stable in neutral and acidic media ~ s,~,/e : -: ~:
: but relatively ~ in alkaline mediaO

(11) Rf valueo In a silica gel -thin layer chromato- . -graphy (on "K1eselgel 60 F254'l9 a product o~ Merck Co~ 9 Germany) 9 the SF-1771-B
substance hydrochloride gives a single spot at '~
- 28 - ;
.

.. ~ . . , - ~ .

Rf~values as ~hown in the -following table.
~or comparison, the Rf-values for the SF-1771 ~ubstance hydrochloride are also shown in . Table 7 belowO
Table Z
~L~ ~.
Develo~ment solvents SF-1771-B SF-1771 Propylalcohol-pyridine-acetic acid-water (15:10:3:12 by volume) 0.76 0.76 ...
Mkthanol-10% aqueous ammonium acetate-10/O aqueous ammonia (10:9:1 by volume) .0050 0057 10% Aqueous ammonium acetate-methanol ~1:1 by volume) ` 0048 0068 10% Aqueous ammonium acetate-methanol (1:2 by volume) 0038 0054 O ~ . :. ..

(12) Optical rotatlon: ~)D = -1804~ (c 19 H20), :~
The SF-1771-B substance according to the second aspect of this invention exhibits an anti-bacterial spectrum as tabulated in Table 8 below. . 7 The minimum inhibitory concen-trations (MDIoCo) of ~his substance against various microorganisms were determined according to a known bro-th.dilution : method in such a way that the determination was conducted after the incubation was effected at ~7C ..
for 18 hours using Heart Infusion Agar as -the . ~ :
incubation medium. .
'' ' ~''~"'.
. , .
' . .
~. ' ' " ~'.",.

~ . 2.9 -.

~B~26 Table 8 5~ ~

Escherichia coli 0039 Escherichia coli K-12-R 0u39 Salmonella typhi 0~19 Pseudomonas aeruginosa 50 Staphylococcus aureus 209P 6,25 Bacillus subtilis PCI--219 1.56 Salmonella pullorum 0039 Pasturella spO 001 3012 " " 015 10 56 :
" " 020 1056 As will be seen from the result of Table 8, the SF-1771-B substance of this invention also exhibits an antibacterial activity against the ~:
gram-negative and gram-positive bacteria 9 substantially as high as that of -the SF-1771 substanceO ~ :
For estimation of acute toxicity of the SF-1771-B substance 9 solutions containing 6025mg-50mg o~ the SF-1771-B substance per 002ml of isotonic sodium chloride solution were in-travenously injected Se~rc,~O./
at a dose of 002ml per mouse into ~ w*~ groups of mice, each group consisting of five mice of ICR-strain (male adult weighing 20g in average)0 7 Da~s after thisintravenous injection9 it was determined that percentages of the number of dead mice based on -the whole number of mice tested were 100%9 60%9 0/C and OY
at the dosages of 50 mg/kg, 25 mg/kg9 1205 mg/kg and 6025 mg/kg of the SF~1771 B substance per mouse 9 respectively, At the dose of 12,5 mg/kg of the SF-1771-B substance 9 all of the mice tested survived after the test9 whereas at -the dose of 1205 mg/kg the number of mice corresponding to 60% of the whole number of the test mice died9 and this reveals that the SF-1771-B substance has a significantly reduced acute toxicity as compared -to the SF-1771 substanceO
The SF-1771-B substance of this invention is considered to belong to the antibiot.ics of bleomycin phleomycin typeO It is known that bleomycin can be inactivated by a crude enzyme solution consisting of a supernatant liquid which is obtained by centrifuging rat liver homogenate at 1009000 Go In order -to estimate to how much extent the activity of the SF 1771-B substance of this invention ~
can be inactivated in vivo i.n animal as compared to :
bleomycin A29 the following -test was madeO-Thus9 lOmg of the SF-1771-B substance as subs~rate was reacted at 37C with 2 ml of a crude enzyme solution (pH 7~2) which has been prepared from the rat liver homogenate in the above-mentioned wayO At the ends of 1 hour9 3 hours9 6 hours9 10 hours and 24 hours after the beginning of the enzymatic reaction9 samples were taken out from the reaction mixture and assayed for their antibacterial potency against the assaying organism9 Escherichia coli NIHJo Percentages of the remaining antibacterial potency of -these samples based on the initial an-tibac-terial potency determined at the begirming of the reaction (the - 3~. -~ ~ 8 2 ~ ~ ~

reaction timeo 0 hour) were calcula-tedO Degree of inactivation (%) was calculated by the ~ollowing equation~
(1 Remaining Antibacterial Potency ) 100 Initial Antibacterial Potency The results obtained are tabulated in Table 9 belowO

~ ' Substrate 1 3 6 10 24 SF-1771-B 0 o 20 48 72 BleomyCin A2 (decoppered 24 36 60 66 ~94 derivative ?

From the results of the above table 9 it is presumed that the SF~1771-B substance is much more dif~icult to be inactivated than bleomycin A2 by the enzymatic reactions9 so that the SF-1771~B substance is able to show a higher activlty than bleomycin A2 in vivo in animalsO
Moreover9 it has been found that the SF-1771-B
substance of this invention exhibits an inhibitory activity to the growth of Hella S3 cells and shows a minimum degeneration concentration o~ 3~9 ~glml against these cells under microscopic observation when incubated at 37C for 48 hoursO Furthermore9 ~ .
the~SF-1771-B substance exhiblts an anti-tumor activity against Salcoma 180 tumor cellsO Thus9 an aqueous suspension (0~05 ml) of Sarcoma 180 tumor cells '.

~ 32 ~-i216 was intraperitoneally inoculated into several groups of mice 9 each group consisting o~ five mice of ICR-strain (male9 about 8-weeks~aged) at a dosage of 10~4 x 106 cells per mouseO Twenty-four hours a~ter the tumor inoculation (i.opo ) 9 16 mg/kg9 8 mg/kg9 4 mg/kg9 2 mg/kg and 1 mg/kg of the SF-1771-B
substance were dosed per mouse by intraperitoneal injection once daily for ~ daysO For comparison9 40 mg/kg9 20 mg/kg, 10 mg/kg and 5 mg/kg of bleomycin were dosed into the comparative groups o~ mice in the :
same manner as the SF-1771-B substanceO The 5 tumor-inoculated mice were used in each groupO The mean :~
number of survival days in the treated mice9 the ~:
~ ev~af~
percentage increase in li~e span (-abbcrviatcd as IoLoSo %) to the control mice9 and the ascites volume were measured to estimate the anti-tumor activity of the SF-1771-B substance and bleomycinO .
IoLoSo (%) was calcula-ted by the following equation: . :
f Mean survival days of the treated mice ..... ~ .. ~ -- -- 1 x 100 Means survival days o~ the control mice .
The results are summarized in Table 10 belowO

- - - :- . . . . : : . ... . . . ... .

~32~iZ6 Table 10 Mean number of Ascites Test Dose survival IlL~So volume 60-Days S~-1771-B 16 808 -50 0 0/5 substance ~ 31~4 7804 Ool 0/5 4 3608 lO9ol 1300 0/5 : 2 3702 11104 1902 0/5 1 29 o 0 6~o 8 2108 0/5 O~OOOO.OOOOOO,OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO
Bleomycin 40 45 o ~ 160 o 2 13 o 0 2/5 .
(com~ara- 20 3608 lO9ol 2002 0!5 .
10 350 9~09 210 4 0/5 . O O . O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O ~ O ~ O O O O O O O ~ O O O O O O O
Control 0 1706 2308 0/5 As shown in Table 109 it is seen that the dose o~ 2 mg/kg of SF-1771-B gives the maximum I~LoSo (11104%) 9 suggesting that an optimal e~fective dose of the SF-1771-B substance would be in a range of 2 to 4 mg/kg for therapeutic treatment of Sarcoma 180 tumor (ascites)-bearing mice9 and that the SF-1771-B substance woulA be e~fectlve at lower doses than bleomycinO
AccordinglyO the SF-1771-B substance of this invention is useful as an antl-tumor agent similarl~
to the bleomycin and ma~ be formulated into the known pharmaceutical form and administered in the same manner as~the bleomyclnO :
In view of the antibac-terial activity of the SF-1771 substance and the SF~1771-B substance9 there is provided according to a further aspect of this : - 34 -. .
:

~ 32 ~

inven-tion an antibacterial agent comprising the ;`
SF-1771 substance or the S~ 7].-B substance9 or an acid-addition salt -thereof, in ~bination with a carrier or vehicle thereforO According to another aspect of this in~ention9 there is provided a pharmaceu-tical composition for`therapeutic trea-tment of a living animal9 includi.ng man9 a~fected by a tumor9 which comprises as the active ingredient -the SF-1771~B
substance or a pharmaceutically acceptable acid-addition salt thereof9 in an amoun-t sufficient to reduce the i-~^ection by the tumor cell in vivo9 the active ingredient compound being in combina-tion with a pharmaceutically acceptable carrierO
It will be appreciated that the actual preferred amounts of the SF-1771 substance and the SF-1771-B
substance used will vary acc(?rding to the particular ; composit1on formulated9 the mode o~ application and the particular situs and organism being treatedO
Many ~actors that modify the action of the drug will be taken into account by the skilled in the art9 for ;
example, age9 bo1y weight9 sex9 diet9 time o~
administration9 route of administration9 rate of excret.ion, drug combinations9 reaction se~sitivities and severity of the diseaseO Optimal application rates ~or a given set of conditions can be ascertained by the skilled in the art using conventional dosage determination tests in view of the above guidelines. ~:
It is believed that usin~ the preceding descriptions and without ~urther elaboration, one skilled in the art can utilize the concept o~ this .

- 35 ~

,,'' ~ ',.' invention to its full extentO The following preferred specific embodiments are 9 therefore 9 to be cons-trued as merely illustrative and not limitative of the remainder of the disclosure in any wayO `
DESC ~ ~ ~ ODIMENTS
Exa~
A culture medium (pH 7009 35 lo ) compri,sing 400% sucrOse9 laO% so~bean oil9 300% soybean meal9 2.0% wheat-embryo9 006% sodium chloride and 00003%
copper sulfate was charged into a jar-fermentor of 50 lo capacity and sterilized by heatingO To this sterilized culture medium was inoculated a seed culture of ~ E~EY~ 5a~ ai~ SF-1771 strain (identified as FERM-P NoO 3253 and as ATCCo 31248) which was previously incubated in slant agar mediumO
The inoculated culture medium was incubated at 28C
for 114 hours under aeration and agitation (the speed o~ agitator 300 rOpOmD)~
The fermentation broth so obtained was filtrated using diatomaceous earth as the filtration-aid~ so that the broth filtrate (about 20 lo ) was collectedO This broth filtrate was passed through a column of 2 lo of adsorbent resin9 Amberlite XAD-2 (a synthetic adsorbent resi.n consisting of a microporous copolymer o~ styrene and divinylbenzene 9 a product produced by Rohm & Haas Coo9 UoSoA~) for adsorption of the active substance by the resinO
The resin was washed with 20 1D Of water and then washed with 10 lo of 50% aqueous acetone (a mixture of acetone-water 1~1 by volume) and subsequently -- ,.
, . , :', . : , ' . ` : : .

~ ~ ~ Z ~Z 6 eluted with 50% aqueous acetone (pH 39 acidi~ied by addition of hydrochloric acid) to effect the desorption of the active substanceO
The eluate was col.lected in 500 ml-fractions 9 and the ac-tive fractions were combined together and neutralized by addition of 1 N aqueous sodium hydroxide9 followed by concentration under reduced pressure to remove the acetoneO The concentrated solution was then passed through a column of 250 ml of a gel-filtration agent CM-Sephadex C-25 (H+) ...
(a carboxymethyl-substituted cross~linked dextran gel9 a product of Pharmacia CoO Sweden) for adsorption of the active substanceO The gel column was washed : with 215 lo of water and then with 005 lo of Ool M
aqueous sodium chloride9 followed by d = t with 002 M aqueous sodium chloride. The eluate was collected in 18 ml-fractions9 and it was found that the fractions NoO 124 to No~ lg8 con-tained the active substance (the total volume of these active fractions was about 10 to 12 folds the volume of the CM-Sephadex gel)O These active fractions were passed through a column of 100 ml of a synthetic adsorbent resin Diaion HP~20 (a microporous copolymer of styrene and divinylbenzene~ a product of Mltubishi. `.
Kasei CoO9 Japan~ for adsorption of -the active `
substanoeO The resin was washed with 200 lo of ; water9 followed by elution with 400 ml o~ 15%
aqueous methanol (15% methanol in water) for the :.
de-salting and by elution with 500 mlO o~ 30% ~;
aqueous methanol (30% methanol in water) for desorption .~ ' ' ~ 37 -`~, -L08i~;26 of the active substance~ The eluate was collec-ted in 100 ml-fractionsO The fractions NoO 1 to NoO 5 containing -t.he active substance were combined together and concentratecl to dryness under reduced pressure to give a green-colored crude powder of the SF-17?1 substanceO
This crude powder (320 mg) was taken up in~o 10 ml of water 9 and the resultant aqueous solution was passed through a column of 50 ml of CM-Sephadex C-25 (H ) (a product o.~ Pharmacia CoO 9 Sweden) for adsorption of the active substanceO The CM~Sephadex gel was washed with 500 ml o~ water and then developed with 0~15 M aqueous sodium chloride solutionO The eluate was collected in 18 ml-fractions 9 and the active fractions (the fraction.s NoO 82 to NoO 122) were combined together (the -total volume o~ these active fractions was about 30-folds -the volume of the CM-Sephadex gel) anl then passed again through a column of 50 ml of a synthetic adsorbent resin Diaion HP-20 (the product o~ Mitsubishi Kasei Co. 9 Japan) for adsorption o~ the SF-1771 substanceO ~
After washin~ with 100 of wa-ter9 the resin was .
eluted with 100 ml of water and then with 100 ml of 15% aqueous methanol for the de-salting and finally eluted with 30% aqueous methnol for desorption of the SF-1771 substanceO The eluate was collected in :
35 ml-fractions 9 and the active fractions freed from the sodium chloride content (the ~ractions NoO 2 to NoO 7? were combined -together and concentrated to dryness -to give 42 mg of a greenish blue colored .:
_ 38 - :

~ 6 crude powder of the SF~1771 substance (about 60%
purity)~
This crude powder (42 mg) o~ the SF-1771 substance was taken up into 2 ml o~ 90% aqueous .. -methanol (90% meth~ol in water) and the aqueous solution obtained was chromatographed on a column of 350 ml of a gel-filtration agent Sephadex LH-20 (a commerical product of Pharmacia CoO 9 Sweden~ a gel consisting of a derivative of dextrun sulfate) developed with 90% aqueous methanol9 so that 12 ml o~ the blue-colored acti~e frac-tion was afforded~
This blue-colored fraction was concentrated to dryness under reduced pres.sure to yield a blue-colored and pure product o~ the SF~1771 substance hydrochloride (containing the chelated copper component)O Yield 24 mgO
Example 2 ..
(a) A culture medium (pH 7009 300 lo ) comprising .`.
400% sucrose9 100% soybean oil9 300~ soybean meal9 2.0% wheat-em~.. ryo9 006% sodium chloride and 0,003%
copper sulfate was placed into a jar ~ermentor of 570 lo capacity and then sterilized by heatingO
To this ~s-terilized culture medium was inoculated a . :
.
culture of the SF-1771 strain (FERM-P NoO 3253 and ATCCo NoO 31248) which was previousl~ cultured on 0050 1, jar-fermenterO The incubation was carried out at 28C for 144 hou~s under aeration and agitationO :-The fermentation broth was ~iltrated using dia-tomaceous earth as ~iltra-tion-aid, The broth filtrate (abou-t .
190 1,) obtained was passed through a column of 18 lo ,` ' .

;`

- of a synthetio adsorbent resin Amberlite XAD-2 (a produc~ of Rohm & Haas .C009 U~S~Ao) for adsorption of the active substanceO The resin was washed with 180 1. o~ water and then with 40 lo Of 50/0 aqueous acetone (ace-ton~.-water, 1 1 by volume), ~ollowe~ by elution with 40 lo of 50% aqueous acetone (pH 39 acidi~ied by addition of hydrochlo~ric acid30 The eluate was collected in 5 10-fractions, and the active fractions (the fractions NoO 3 to NoO 9) - 10 were combined together and neutralized by addition o~ 1 N aqueous sodium hydroxide and concentrated under red~ced pressure by e~aporation of the ~ .
acetone.
The concentrated solution was passed through.
a column of 1 lo of a gel~filtration agent CM-Sephadex . .
C-25 (a product of Pharmacia CoO~ U~SoAo ) for adsorption of the active substanceO The column .~ .
was washed with 5 lo of water and then with 2 lo of 0.1 M aqueous sodium chloride solution, ~ollowed by development with 002 M aqueous sodium chloride solution so that -the active substance was eluted ou-tO .~
The eluate was collected in 18 ml-fractions and . .::
the active ~ractions (the fractions No~ 82 to No. 240) were passed through a column of 1 lo o~ a . ~-synthetic adsorbent resin Diaion HP-20 (a product o~ Mitsubishi Kasei CoO 7 Japan) for adsorption of the : ; .
active substance, and this resin colum~l was then -.
washed with 2 1. o~ water for the de-salting `.
purpose and then elut~d with 1 1, of 30~o aqueous methanol to effect th~ desorption of the SF-1771 . 4 ~ 8 ~

substanceD The eluate was collected in 500 ml-fractions 5 and the active fractions (the ~ractions NoO 2 to NoO 5) was again passed through a column o~
200 ml of a gel-filtration agent CM~Sephadex C-25 (a product of Pharmacia CoO 9 Sweden) for adsorpti~n of the active substanceO The gel column was washed with water and then eluted with 002 M aqueous sodium chloride solution to effect the desorption o~
the SF-1771 substanceO The eluate was collected in 18 ml-fractions and the active ~ractions (the fractions NoO 98 to No J 256) was passed through a column of 600 ml of Diaion HP-20 resin (a product of Mitsubishi Kasei CoO 9 Japan) which was subsequently washed with 2 lo 0~ water for the de salting and then eluted with 1 lo of 30% aqueous methanol to desorb the SF-1771 substanceO The eluate was collected in 500 ml fractions and the active fractions (the fractions No~ 3 to NoO 5) were combined together9 `
followed by concentration to dryness to give about 3 g. of a green-colored crude powder of the SF~1771 substance (48% purity)O
This crude powder (3 g,) of the SF-1771 :
substance was taken up into 7 ml of 90% aqueous methanol9 andthe resul-ting methanolic solution was partition~chromatographed on a column o~ 105 lo of Sephadex LH-20 (a product of Pharmacia CoO 9 Sweden) using a mixed solvent of butanol-methanol-wa-ter ~ de~e/ap~"f (4:1~Z by volume) as the ~r~Ya~b~=~ solventO
The eluate was collected in 15 ml-frac-tions 9 and the blue-c~lored active ~ractions (the fractions . ~ ..

".
.

~ 6 NoO 32 to No. 44) were ~fforded. These active fractions combined together were concentrated to dryness in vacuo to give 1~2 gO of a par-tially purified powder of the S~-1771 substance (90% purity). This crude product o~ the SF-1771 substance (1.2 g.) was taken up into 6 ml of 90% aqueous methanol and the solution so obtained was ch~omatographed on a column of 102 1.
of 5ephadex.LH-20 using 90% aqueous methanol as the, . , . developm~nt solventO The eluate'was collected in 16 ml-frac-tions and the blue colored active f`ractions . :.
(the fractions NoO 26 to NoO 31) were obtained to a total ~olume of 98 mlO These active fractions were :
combined together and concen-trated to dryness in vacuo to give a pure product of the SF-1771 substance as a blue-colored amorphous powder~ Y~.eld 1 gO
(b) This SF-1771 substance (150 mg) was dissolved in 15 ml of methanol, and into the resultant ` ''".
methanolic solution was passed gaseous hydrogen '~
sul~ide untll the solution did no longer show the ' :' ':
blue-color (for about 4 minutes)~ During this, :. ,.. ~
precipitate of copper sulfide deposited. The " : :
precipitate was filtered off and the filtrate was conc.entrated to a volume of about 4 ml under.reduced ,', ' ' pressure~ The'concentrated solution so obtained was chromatographed on a column of l 1. of Sephadex LH-20 using 90SS aqueous methanol as the development solvent. The eluate was collected in 18 ml-fractlons, .
and the active fractions (the f~actions No. 22 to . No. 27) was obtained to a total volume of 112 ml. . .~"
:, The ccmbined active fractions were co,ncentrated to ;, ,:.
- 42 - '''~,'~'; `~

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

~ ~ 8 ~ ~ 6 dryness in vacuo9 a~fording a pure product of the SF-1771-B substance as a white~colored amorphous powderO Yield 107 mgO
Exa~
A culture medium (35 lo 7 pH 700) comprising 400% sucrose, 100% soybean oilg 300% soybean meal9 2O00/o wheat-embryo 9 0 o 6% sodium chloride was placed in a jar-fermentor o~ 50 lo capacityO A~ter sterilization o~ the culture medium by heating 9 the sterilized culture medium was inoculated with a seed culture o~ the SF-1771 strain (FERM-P NoO 325~ ~ :
and ATCCo 31248) which was previously incubatedD
The cultivation was made at 28C for 114 hours `
under aeration and agitationO The ~ermentation broth was ~iltered using diatomaceous earth as :
filtration-aid, so that about 20 lo of the broth filtrate was obtainedO
Gaseous hydrogen sulfide was passed through the broth filtrate for about 40 minutes at ambient temperature 9 and thereafter the reaction solution was purged by blowing air thereinto to strip off the e~cess o~ hydrogen sul~id.e from the reaction solutionO The solution was then passed through a column o~ 2 lo of Amberlite XAD-2 (a product of Rohm & Haas CoO 9 UoSoAo ) ~or adsorption o~ the SF-1771-B sub~tanceO The resin column was washe~
with 20 lo 0~ water and -then with 10 lo 0~ 50%
aqueous acetone and f mally eluted with 50% aqueous acetone (pH 39 acidified by addition of hydrochloric 30 acid)O The eluate was collected in 500 ml-f`ractions9 _ 43 _ .
': ':..
., :

; : - , - , , ~ . . -and the active fractions (the fractions No O ~ to NoO 12 ! were combined together a~d neutralized by addition of 1 N aqueous sodium hydroxlde~ The neutralized solution was concen-trated under reduced pressure by dis-tilling off the ace-tone~ The concen-trated solution was passed through a column o~
250 ml of CM-Sephadex C-25 (H~) (a product of Pharmacia CoO 9 Sweden) ~or adsorption of the active substanceO The column was washed with water and then eluted with 0~2 M aqueous sodium chloride solution for desorption of the active substanceO
The eluate was collected in 18 ml-fractions9 and the active fractions (the fractions NoO 68 to NoO 126) were combined together9 followed by the treatment with 100 ml of a synthetic adsorbent ; :
resin Diaion HP-20 (a product of Mitsubishi Kasei CoO 9 Japan) for adsorpt.ion o~ the SF-1771-B substanceO
The resin column was washed with 300 ml of water and then eluted with 500 ml o~ 60% aqueous methanol for desorption of the active substanceO The active fractions o~ the eluate were com~ined together and concentrated to dryness to give 520 mg of a crude powder of the SF-1771 B substanceO This crude product was further purified in the same manner as in Example 29 a~fording a pure product of the .
SF-1771-B substanceO Yield 86 mg The SF-1771 substance hydrochloride (200 mg) was dissolved in 20 ml of 0O2% sodium sulfide solution in water and s-tirred for 10 minute until the blue :.
, .

--: 44 _ - , - - - ~ - .. , . ~ -~8~

color had been changed into dark brown color of the precipitated copper sulfideO The precipitated material was removed by fi].tration and discardedO
The filtrate was neutralized with 1 N
hydrochloric acid and concentrated to a volume of 10 mlO The concentrated solution was desalted with Diaion HP-20 resinO
The eluate fractions were combined toge-ther 9 followed b~ concentration to dryness to give about 188 mgO o~ a colorless crude powder o~ the SF 1771-B
substanceO
~ ;.
The SF-1771 substance hydrochlori.de (350 mg) was dissolved in 30 ml o~ Ool N hydrochlori.c acid and extracted repeatedly with chlorofGrm containing ~ fe~7sa~
0O5% dithizon until no reddish violet color rc~aindO
After the chloro~orm phase was removed9 the remaining a~ueous solution was washed repeatedly with chloroformO The aqueous solution was neutralized with Amberlite IR-45 (OH )(a.product o~ Rohm and Haas CoO 9 UoSoAo ) and concen-trated to dryness in vacuo, giving a crude yellowish powder (342 mg) o~ SF-1771-B substanceO This product of SF-1771-B
substance (342 mg) was taken up into 4 ml o~ 90% ~.
aqueous methanol and the solution so obtained was ~:
chromatographed on a column of 700 ml of Sephadex LH-20 using 90% aqueous me-thanol as the development solventO `
The eluate was collected in 10 ml-~ractions 9 and the active ~ractions (the ~ractions NoO 30 to :~

... , , ., , .. , ~ ,, .

~8~

NoO 36) were combined to a -total volume o~ 72 ml and concentrated to dryness in vacuo to give a pure product of the SF-1771-B substance as a colorless amorphous powderO Yield 287 mg, .': ' .

, ;,... ...
'.

~ , ,~

Claims (7)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the production of a SF-1771 substance and its pharmaceutically acceptable acid-addition salts thereof, said substances being a blue-colored amorphous powder which is basic, soluble in water and shows an antibacterial activity, the hydrochloride of which discolors to brown at 185°C and decomposes at 208°C with foaming; is soluble in water, soluble in methanol, sparingly soluble in ethanol and butanol; is positive in reactions with ninhydrin reagent, Ehrlich's reagent, potassium permanganate reagent, Greig-Leiback's reagent; is negative in reaction with Sakaguchi's reagent;
in reaction with Sakaguchi's reagent; shows a molecular weight of about 1600 as determined by the Barger method;
gives an elementary analysis C 38.70%, H 5.42%, N 13.03%, O 29.62%, S 3.62%, Cl 6.07% and Cu 3.53% (as measured by atomic absorption spectroscopy); exhibits characteristic absorption peaks at 248 nm (E??m = 146) and at 282-2B4 nm (E??m = 106) in the ultra-violet absorption spectrum when dissolved in water; and exhibits characteristic absorption bands as shown in Figure 2 of the accompanying drawings in the infra-red absorption spectrum when pelleted in potassium bromide, which comprises cultivating Streptomyces toyocaensis in a culture medium containing assimilable carbon and nitrogen sources under aerobic conditions for a sufficient time to produce and accumulate the SF-1771 substance in the culture medium, and then recovering this antibiotic substance from the culture in its basic form or as a pharmaceutically acceptable acid-addition salt thereof.

2. A process according to claim 1, including the step of treating the SF-1771 substance thus obtained with hydrogen sulfide, an alkali metal sulfide or an agent that chelates copper for a sufficient time to remove the copper component from the SF-1771 substance molecule and thus provide the SF-1771-B substance as a colorless or faintly colored, amorphous powder, which is basic, soluble in water and shows antibacterial and anti-tumor activity, the hydrochloride of which discolors to brown at 180°C; decomposes at 212°C
with foaming; is soluble in water, soluble in methanol, sparingly soluble in ethanol and butanol; is positive in reactions with ninhydrin reagent, Ehrlich's reagent, potassium permanganate reagent and Greig-Leiback's reagent; is negative in reaction with Sakaguchi's reagent; shows a molecular weight of about 1600 as measured by the Barger method and a specific optical rotation {a}?0- 18.4° (c l, H2O); shows an elementary analysis C 41.36%, H 5.53%, N 14.19%, S 3.64% and O 28.93%;
exhibits a characteristic absorption peak at 288-290 nm (E??m = 84) in the ultra-violet absorption spectrum when dissolved in water; exhibits characteristic absorption bands as shown in Figure 5 of the accompanying drawings in the infra-red absorption spectrum when-pelleted in potassium bromide;
and further exhibits a proton magnetic resonance absorption spectrum as shown in Figure 6 of the accompanying drawings when dissolved in deuterium oxide.

3. The process defined in claim 2, including the step of preparing a pharmaceutically acceptable acid-addition salt of the product thus obtained.

4. A process according to claim 2 in which the SF-1771 substance in solution in methanol is reacted with hydrogen sulfide by passing gaseous hydrogen sulfide into the solution.

5. A process according to claim 2 in which the SF-1771 substance still present in the filtrate of the fermentation broth of the SF-1771 substance-producing microorganism is reacted with hydrogen sulfide by passing gaseous hydrogen sulfide into the fermentation broth filtrate containing the SF-1771 substance.

6. An antibiotic selected from the group consisting of SF-1171 substance, SF-1171B substance and pharmaceutically acceptable acid addition salts thereof, said SF-1171 and SF-1171B
substances being as characterized in claims 1 and 2, whenever prepared or produced by the process defined in claim 1, 2 or 3 or by the obvious chemical equivalent.

7. The antibiotic SF-1771B substance as characterized in claim 2, whenever prepared or produced by the process defined in claim 4 or 5 or by the obvious chemical equivalent.