CA1056747A - Antibiotics neothramycin a and b from streptomyces - Google Patents

Abstract

Abstract of the Disclosure There are disclosed two new antibiotics neothramycin A having the formula:

and neothramycin B having the formula:

Description

Back~round of the Invention This invention relates to chemical compounds produced by fermen-tation and used to inhibit the growth of leukemia cells.

10~674~
Summary of the Invention This invention relates to two new antibiotics which each exhibit a high activity inhibitory to the gro~th of leukemia cells and are useful as anti-tumor agents but exhibit low antibacterial activity. More particularly, this invention relates to the new antibiotics neothramycin A and neothramycin B, (initially designated MC916-A substance and MC916-B substance), respectively, and also to a process for the production of these new antibiotics by cultivation of a strain of Streptomyces. This invention also relates to the recovery and purification of these specific new antibiotic substances and to their use for pharmaceutical purposes.
Hereinafter, by the term "neothramycin" is meant neothramycin ~. and neothramycin B or their mixture unless otherwise stated.
Some antibiotics which are useful as anti-tumor agents for the therapeutic treatment of leukemia, for example, are daunomycin, adriamycin, etc. In an attempt to obtain further new anti-tumor agents of antibiotic type, we collected various soil samples, isolated microorganism frcm such soil samples and investigated metabolic products which are produced by the aerohic cultivation of the isolated microorganisms.
We isolated a new microorganism from a soil sample collected in the grounds of Biseibutsu Kagaku Kenkyu-sho in Shinagawa-~u, Tokyo, Japan, and we have designated this newly isolated microorganism as MC916-C4 strain. It has been confirmed that this ~lC916-C4 strain belongs to the genus Streptomyces. We have now found that two new anii-biotics having a low antibacterial activity but high activity inhibitory to the growth of leukemia L-1210 cells in mice and to the growth of a certain kind of tumor cells are produced and accumulated in the culture broth of the MC916-C4 strain. T~e have now succeeded in isolating these new antibiotics from the culture broth and designated them as neothramycin A and neothramycin B, respectively.
An object of this invention is to provide new substances which are useful as anti-tumor agents. Another object of this invention is to provide neothramycin A and neothramycin B, either alone or in mixture thereof as new and useful anti-tumor agents. A further object of this inven-tion is to provide a process for the preparation of neo-thramycin A and neothramycin B by cultivation of the MC916-C4 strain. Other objects of this invention will be clear from the following descriptions.
According to one aspect of this invention, there is provided as a new antibiotic substance, neothramycin substance having an activity inhibitory to the growth of leukemia L-1210 cells in mice and a low antibacterial activity, said substance having an acidic function; being soluble in methanol, ethanol, propanol, chloroform and dioxane and slightly soluble in water but sparingly soluble or sub-stantially insoluble in ethyl ether and n-hexane; being positive to ~ydon-Smith reaction and red tetrazolium reaction, weakly positive to ninhydrin reaction but negative to Ehrlich reaction and Sakaguchi reaction; giving essentially only carbon, hydrogen, nitrogen and oxygen upon elemental analysis thereof; exhibiting a relative mobility of said substance to alanine (1.0) being 0.17 on high-voltage filter paper electrophoresis ~3500 volts, 35 minutes) using formic acid-acetic acid-water t25:75:900 by volune) as an electrolyte ;~, solution; said substance being at least one member selected from the group consisting of neothramycin A and neothramycin B;
a) said neothramycin A being further characterized by giving C, 57.46%, H, 5.76%, N, 9.84~ and the remainder oxygen upon elemental analysis thereof; giving a molecular weight of 250 to 300 as measured by ~arger - AXiya method;
having an infrared absorption spectrum pelleted in potassium bromide characterized by absorption peaks at 3450, 2750, 1630 (shoulder), 1600, 1510, 1460, 1440, 1410, 1280, 1200, 1180, 1120, 1080, 1010, 870, 790 and 760 cm 1; having ultraviolet absorption spectra characterized by absorption maxima at 223 nm (El%Cm 855), 240 nm (shoul~er), 265 nm (El%Cm 290) and 318 nm (El~Cm 156) in a solution thereof in 10~ water-methanol, by absorption maxima at 223 nm (El%Cm 885), 240 nm (shoulder), 265 nm (~l%cm 290) and 320 nm (El%Cm 139) in a solution thereof in N/10 HCl-methanol (1:9) and by absorption maxima at ~28 nm (El%Cm 635~, 254 nm (El cm 566), 291 nm (El~Cm 422) and 324 nm (El%Cm 412) in a solution thereof in N/10 NaOH-methanol (1:9); and giving an Rf value of 0.57 in thin layer chromatography on silica gel with chloroform-methanol (10:1 by volume) as the developing solvent; and b) said neothramycin B being further characterized by giving C, 57.00%, H, 5.58%, N, 9.75% and the remainder oxygen upon elemental analysis thereof; giving a molecular weight of 250 to 300 as measured by ~arger - Akiya method;
having an in rared absorption spectrum pelleted in potassium bromide characterized by absorption peaks at 3400, 2960, 1630 (shoulder), 1600, 1510, 1440, 1400, 1280, 1200, 1120, .

~056747 1080, 1010, 990, 940, 870, 790 and 760 cm 1; having ultra-violet absorption spectra characterized by absorption maxima at 224 nm (El~Cm 935), 240 nm (shoulder), 265 nm (shoulder) and 318 nm (El~Cm 167) in a solution thereof in 10~ water-methanol (1:9), by absorption maxima at 224 nm (El%Cm 1000), 240 nm (shoulder), 265 nm (shoulder) and 320 nm (El~Cm 156) in a solution thereof in N/10 HCl-methanol (1:9) and by absorption maxima at 228 nm (El%Cm 800), 254 nm (El~Cm 725), 291 nm (El~Cm 456) and 324 nm (El~Cm 466) in a solution thereof in N/10 NaOH-methanol (1:9); giving an Rf value of 0.50 in thin layer chromatography on silica gel with chloroform-methanol (10:1 by volume) as the developing solvent.
This invention embraces neothramycin A and neo-thramycin B, either alone or in a mixture of them, which may be present in a dilute solution, as a crude concentrate, as a crude solid, as a purified solid, as the free acid form and in the form of a salt thereof with a metal or an organic amine. Neothramycin A has been obtained as a colorless powder which has no definite melting point, melts gradually near 105C. and decomposes at 132-147C. with foaming and which exhibits a specific optical rotation ~O~] 26 = +272 (C 0.52, dioxane). From the results of elemental analysis and the determination of molecular weight, it is very probable that neothramycin A has an empirical formula C13Hl4N2O4-1/2H2O.
This formula has been confirmed by high-resolution mass-spectrometry (Found: m/e 262. 0934, calculated molecular weight for C13H14N2O4 262 . 0952) . The ultraviolet absorption spectrum of neothramycin A in an alkaline solution exhibits a shi't towards the longer wave length. As shown in 3 0 Table 1, NMR spectrum of neothramycin A shows the presence ~,., of 14 protons. Neothramycin B is very similar in its properties to neothramycin A and has been obtained as a colorless po~Jder which has no definite melting point, commer.ces to decompose at 144C. with foaming and completely melts at 151C. and which exhibits a specific optical rotation [~]D6 = +314 (C 0.48, dioxane). Neothramycin B has the empirical formula: C13H14N204-1/2H2O. This formula has been confirmed by high-resolution mass-spectrometry (Found:
m/e 262.0939, calculated molecular weight for C13H14N2O4, 262.0952). The ultraviolet absorption spectrum of neothramycin B in an alkaline solution exhibits a shift towards the longer wave length. As shown in Table 1, N~R spectrum of neothramycin B shows the presence of 14 protons, similarly to neothramycin A.
Neothramycin A and B are stable for a long period of time when stored in the form of a solid powder thereof in a cold and dark place.
But, neothramycin A and B are unstable in 50%
aqueous ethanol of pH 2.5 and the activities are reduced to 25% and 2Z~, respectively, at room temperature for 16 hours.
In 50~ aqueous ethanol of pH 6.5 or pH 8.0 at room temperature for 16 hours, 80-90% activity of neothramycin A and 70-80~
activity of neothramycin B remain, but an equilibrium con-version of neothramycin A to B or B to A is shown by thin-layer chromatographic analysis.
From these data, neothramycins A and B are isomers which are convertible into each other and belong to the anthramycin group of antibiotics possessing a benzodiazepine structure. They are distinguished from anthramycin, dextro-chrysin and sibiromycin by their W spectra. The W spectra of tomamycin and neothramycins are very simllar but they A~ jJ

~056747 PMR Chemical Shifts of Neothramycins Proton Neothramycin A ~eothramycin B
CH2x2 1.7-2.5 1.7-2.5 CH 3.80 m 3.78 m arom. OCH3 3.90 s 3.88 s OH 5.00 d 5.10 d CH 5.69 dd 5.7a m arom. H 6.70 s 6.69 s arom. H 7.43 s 7.40 s CH 7.62 d 7.70 d phenol OH 8.00 s 7.98 s Chemical shifts, ~(ppm) were measured in deuterodioxane using TMS as the internal reference.

are different in their molecular fo:rmulae and other spectra.
By struc~ural studies, the following structures have been submitted by the inventors for neothramycin A
(R = OH, R = H) and neothramycin B (Rl = H, R2 = OH).

CH30 ~ ~ R2 In another aspect of this invention there is provided the process for producing the antitumor antibiotic neothramycin selected from the group consisting of neothramycin A, neothramycin B and mixtures thereof, said neothramycin A
having the formula:

HO ~ N =
CH30 ~ ~ N ~
O HO H

and said neothramycin B having the formula:

CH30 ~ o~= ~ OH

which comprises culturing a neothramycin-producing strain of Streptomyces having the identifying characteristics of A.T.C.C. 31123 under submerged aerobic conditions in a nutrient medium containing a carbon source and a nitrogenous ~' nutrient until a substantial amount of neothramycin is produced by said organism in said nutrient medium, and recovering the neothramycin from the nutrient medium when the pharmaceutically acceptable salt of the neothramycin is desired, the process further comprises a step of forming said salt by methods known per se.

~eferring to the attached drawings: -Figure 1 shows a curve of the infrared absorption spectrum of a sample of neothramycin A pelleted in potassium bromide.

Figure 2 shows a curve of the infrared absorption spectrum of a sample of neothramycin B pelleted in potassium bromide.
Figure 3 shows curves of the ultraviolet absorption spectrum of a sample of neothramycin A dissolved in 10%
water-methanol, in N/10 NaOH-methanol (1:9) and in N/10 HCl-methanol ~1:9), respectively.
Figure 4 shows curves of the ultraviolet absorption spectrum of a sample of neothramycin B dissolved in 10%
water-methanol, in N/10 NaOH-methanol (1:9) and in N/10 HCl-methanol (1:9), respectively.
The neothramycin A and neothramycin B of this invention have low antibacterial and antifungal activity as will be clear from the antibacterial spectra of these sub-stances shown in Table 2 below. The minimum inhibitory concentrations (mcg./ml.) of neothramycin A and neothramycin B

to various bacteria have been determined on nutrient agar plates which were incubated at a temperature of 37C. for 17 hours. The minimum inhibitory concentrations to various fungl have been determined on nutrient agar plates containing 1% gluco~e after lncubation at 27C. for 40 hours.
_ g _ ,1~

Table 2 Minimum Inhibitory Concentratlons (Mc~./ml.) L3a~liam9 Neothramycin A Neothramycin B
Staphylococcus aureus Smith50 100 Staphylococcus aureus 209P~ 100 ~ 109 Klebslella pneumoniae PCI 602 50 100 Escherichla coli ~IHJ 100 100 Escherichia coli K-12 100 100 Pseudomonas aeruginosa No. 12 ~ lO0 > 100 Baclllus subtilis PCI 219100 ~100 E~cherichla coli W677 50 100 ~scherichia coli JR66/W677lO0 > lO0 Aeromonas salmonecida ATCC 14174 25 5 Vibrlo angulllarum NCBM 6 50 100 Saccharomyces cerevisiae 50 ~ 100 Candlda albicans 31lL7 > 100 ~ 100 Aspergillus niger 100 ~ lO0 Piricularia or~zae 50 > 109 Xanthomonas cltrl ~ 100 > 100 Xanthomonas oryzae 50 100 ~ ,~/~,, .

As stated hereinbefore, neothramycin A and neo-thramycin B of this invention have a high inhibitory activity to the growth of leukemia cell3 and are expected to be useful a~ an agent for treating therapeutically a livlng anlmal affected by leukemla, Chemotherapeutlc effects of neo-thramycin A and B a~alnst leukemia L-1210 ln mlce were inve~tigated ln the followlng manner, Leukemla L-1210 cells (105 cells/mouse) were lnJected lntra-perltoneally ln mice of CDF 1 straln welghing 19-22 g.
For the treatment of the leukemla 80 lnfected, admlni~tratlon of neothramycin A and B were ¢ommenced lmmediately after the tumor inoculatlon.
The leukemic mlce were used ln group~ each of four mlce for each dose, When 300, 150, 75, 37,5 and 18.7 mcg,/mouse/day of neothramycin A and B
were dosed by lntraperltoneal lnJection once dally for 10 days, the hlghly favorable effect~ on the ~urvlval ratlo (%) were observed as wlll be clear from the re~ults shown ln Table 3 below.

lable 3 Avera~e of Su~vlval Ratio Do~a~e ~g~mQuge/day) Neothramycin A Neothram~cin B

300 death death (toxlc dose) (toxic dose~

37.5 154 128 18.7 122 103 me Survival Ratio (%~ 1~ calculated by dlvldlng the number o~ day~ of ~urvival of the treated animals (e.g, 10) by the number of days o~ survlval o~ ~he control animal3 (e,g. 8) and multlplying by 100, e.g. ~ x 100 - 125. Ratio3 greater than 125 are generallg con~idered ~ignlflcant.
The neothramycin A and B of this invention are of a low toxlclty to anlmal and man, a~ ~hown by the fact that the neothramycin A

!, ~ ~, 12 , ~ .

and -B exhibit LD50 values of 20 - 30 mg./kg. and 20 - 30 mg./kg., respectively, in mice, when a solution of 0.25-0.5~ by weight of neothramycin A
or B in 10% dimethylsulfoxlde-water is injected intraperitoneally in mice for the purpose of esti-mating the acute toxicitv of these substances.
~ cccrding to a second aspect of this inven-tion, there is provided a process for the production of neothramycin A and neothramycin B, which compxises cultivating a neothramycin-producing strain of the genus Streptomyces under aerobic conditions in a suitable culture medium therefor containing assimilable carbon and nitrogen sources for a period of time sufficient to produce and accumulate neothramycin A
and neothramycin B in the culture medium, and recovering a mixture of neothramycin A and neothramycin B from the culture, and subsequently, if reouired, separating the recovered mixture into neothramycin A and neothramycin B in their isolated forms. For the production of neothramycin according to the process of this invention, a strain of the genus Streptomycas may be used as lon~
as this strain produces neothramycin. ~ suitable example of the strain which may be employed in this invention for the production of neothramycin is the above-mentioned MC916-C4 strain of Streptomyces.

Thl8 MC916-C4 strain was deposited on February 2, 1974 in a Japanese authorized deposltory "Fermentatlon Research Institute, Agency of Industrial Sclence and Technology", Inage, Chiba-City, Japan, under deposit number FERM-P 2452. m is MC916-C4 strain was al~o deposited in the Amerlcan Type Culture Collection, Washington, D. C , U.S.A. under A.T.C.C. number ~1123.
Cultural and taxonomic characteristics o~ the MC916-C4 strain are described below.
1. Microsco~ical mo~hQlQæ~
MC916-C4 strain has branched substrate mycelia ~rom which aerial hyphae develops in the form of hook or open spirals. No whorl-branching ls observed, Matured spore chains usually bear more than lO conidal spores. Spores measure about o.6-o.8 by 1.0-1.2 mlcrons in size and have a smooth surface.

2. Characteristics of the ~rowth on various culture media m e designatlon of colors ln brackets [ ] mentioned below follows the color standard given in the "Color Harmony Manual" published by Container Corporation of America.
(1) On sucrose-nitrate agar (incubated at 27 C.):
Pale yellow to reddish yellow [~ pc, amber] colored growth bears thin aerlal hyphae o~ llght brownlsh gray to pale gray color. Soluble plgment is faintly tinged wlth yellow.

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(2) On glucose-asparagine agar (incubated at 27 C.):
Dull yellow orange ~nc, Amber to 4pe, Orange Rust]
colored growth develops aerial hyphae o~ light gray to light brownish gray color [2ih, Dk covert Gray].
Soluble pigment is faintly tinged wlth yellow.

(3) On glycerol-asparagine agar [ISP No. 5 medium, lncubated at 27 C.): Dark yellow orange to yellowish brown ~pi, Golden Brown3 colored growth develops aerial hyphae of brownish gray [~lh, Beige Gray] to gray ~5ih, Shadow Gray] color. Soluble pigment with yellowlsh tinge to yellowish brown tinge ls produced.

(4) On inorganic salt-starch agar (ISP No. 4 medium, incubated at 27 C.): Pale yellowish brown to yellowish brown [3pl, Golden Brown] colored growth develops aerial hyphae of llght brownish gray [3fe, Silver Gray] color. Soluble plgment is tinged with bro~tn. The reverse side of the growth is dark yellowish brown in color.

(5) On tyroslne agar (ISP No. 7 medlum, incubated at 27 C.): Dark yellow to yellowlsh brown [4pg, Dark Luggage Tan] colored growth bears aerial hyphae of light brownish gray. Soluble pigment is tinged wlth dark yellow to yellowi~h brown.

(6) On nutrlent agar (incubated at 27 C.):
The growth is colored pale yellowish brown to pale brown without developing aerial hyphae. Soluble plgment is falntly tlnged with brown.

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(7) On yeast extract-malt extract agar (ISP No. 2 medium, lncubated at 27 C.): Yellowlsh brown ~4pg., Dk Luggage Tan] to yellow orange [4pe, Orange Rust]
colored growth develops aerial hyphae of light gray [2fe, Covert Gray to llght brownl~h gray [21h, Dk Covert Gray color. Soluble plgment of yellowish brown to brown color ls produced. The reverse slde of the growth ls colored dark yellowlsh brown.

(8) On oatmeal agar (ISP No. ~ medlum, lncubated at 27 C.): Reddlsh yellow to dark yellow orange [4pe, Orange Ru3t] colored growth wlth aerial hyphae of llght gray [5fe, Ashes] to brownlsh gray [31~, Beige Gray] color, Soluble pigment ls tinged wlth yellow.

(9) On glycerol-nltrate agar (incubated at 27 C,):
Pale yellow to reddlsh yellow [3pc, Amber] colored growth bears ~lightly developed aerlal hyphae of ~ brownish white to light brownlsh gray color. Soluble pigment is falntly tinged wlth yellow.

(10) On starch agar (incubated at 27 C.): me growth 1~ colored dull yellow to yellowish brown [2pi, Mustard Brown] without developing aerial hyphae or rarely with developing aerial hyphae of white.
Soluble plgmsnt is falntly tinged wlth brown~

(11) On calclum-malate agar (incubated at 27 C,):
The growth ls colored pale yellow to pale ollve with-out developlng aerial hyphae or wlth sllghtly developing aerlal hyphae of whlte~ Soluble plgment is faintly tinged with yellow.

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(12) On cellulose (incubated at 27 C,): Colorless growth without aerial hyphae. No soluble pigment is produced.

(13) On gelatin stab: On plaln gelatln medium (incubated at 20 C.), the growth i~ colorles~ to dull yellow colored wlthout developing aerial hyphae, and wlth produclng soluble pigment of ~alntly yellow tlnge.
On glucose-peptone-gelatln medium (incubated at 27 C.), the growth is pale yellow to dull yellow in color.
Aerial hyphae are not developed lnitially but ones of grayl3h white color are produced later. No production of ~oluble pigment is ob~erved.

(14) On skimmed mllk (incubated at 37 C.):
The growth is colored pale yellow to pale orange without de~eloping aerial hyphae. Soluble pigment is very faintly tinged with orange.
. Phvsioloical ~ro~erties tl) Temperature for growth Growth on gluco~e asparagine agar was examined at 20 C., 24 C,, 27 C., 30 C., 37C,, and 50 C. me MC916-C~ strain grew at all temperaturea tested, except at 50 C. Optimum temperature for good growth was ob~erved to be ln the vicinity of 30 C.
(2) Liquefaction of gelatin Plain gelatln (15%) medium ~tarted to liquefy from the 5th day of lncubation at 20 C. me degree of lique~action wa~ medium. me ~elatin (15%) in gluco~e-peptone-gelatin medium started to liquefy from the 2nd day of incubation when incubated at 27 C., and the grade of liquefactlon was then medium to strong.
(3) Hydrolysis of starch Starch in inorganic salts-starch-agar medium and ln tarch-agar medium was hydrolyzed starting from the 5th day of incubation when lncubated at 27 C. The grade of hydrolysis was medium to strong.
(4) Coagulation and peptonization of sklmmed milX
When incubated at 37 C., the coagulatlon of sklmmed milk started at the 4th day of incubation and ths peptonization was observed at the 5th day of lncubation after the coagulation was complete. The grades of coagulatlon and peptonization were medium to strong.
(5) Formation of melanoid pigment No plgmentatlon was observed neither on trypton-yeast extract broth (ISP No. 1 medium), nor on peptone-yeast extract iron agar (ISP No. 6 medium), nor on tyroslne agar (ISP No. 7 medlum), when lncubated at 27 C.
(6) Utllizatlon of carbon sources for growth Utlllzatlon o~ the followlng carbohydrates was tested ln Prldham-Gottlieb agar medium (ISP No. 9 medium) as lncubated at 27 C.
Glucose and L-rhamnose were utilized for growth.
L-Arablnose, D-fructose, sucrose, lnositol and D-mannltol were not utllized. Utilizatlon o~ D-xylose was doubtful. Rafflnose was sometlmes utilized but ,~
~f ~ , ~OS6747 not utilized other tlmes.
(7) Liquefaction of calcium malate Calclum malate ln calcium malate-agar medium was liquefied around the growth starting at the 9th day of incubation, when lncubated at 27 C. The grade of llquefaction was medium to strong.
(8) Reduction of nitrate Reduction of nitrate was observed ln aqueous peptone solution containing 1.0% sodium nitrate (ISP No. 8 medlum), when incubated at 27 C.
Summarizing the above-mentioned characteristics of the MC916-C4 strain, it is noted that this strain belongs to the genus StrePtomvce~ and that the aerial hyphae form open spirals but does not develop whorl.
me surface of spore is smooth under microscopic observation. On various media, the ~rowth has a color of yellowish orange to yellowish brown with developing aerial hyphae of light brownish gray to brownish gray color. Soluble pigment ls tinged with yellow to brown or with yellowish brown. No melanoid pigment is produced, Proteolysis and starch hydrol-Jsis are of medium to strong grade.
On the basis of the above-mentioned properties, the MC916-C4 strain i~ compared to kno~ analogous ~pecle3 of Stre~tomvces with reference to descrlptions of International Streptomyces Pro~ect (ISP). It is found that the MC916-C4 strain resembles StrePtom~ces naraen~is (see "Internatlonal Journal of Systematic A~" - 19 -Bacteriology" Vol. 22, page 32~ (19723. However, it is noted that the MC916-C4 straln is dl~ferent ~rom Stre~tomvces naraensis ISP 5~o8 strain ln respect to thelr utillzatlon of carbon sources.
S. naraensls produces cycloheximide, simllarly to the MC916-C4 straln. Furthermore, among cyclo-hexlmlde-produclng strains, it is found that stralns of Group C whlch are analogous to Stre~tom~ces ~riseolus as reported ln an article by T. Furumai et al. titled "On cycloheximlde-produclng micro-organlsma" [see the "Journal of Antibiotics" Ser.
B., Vol. 17, No. 4, page 181 (1964)] are very ~imilar to the MC916-C4 ~traln, me MC916-C4 straln ls well coincident wlth the above Group C stralns ln many re~pects, though the MC916-C4 straln has not been tested as to whether it has the properties of hemolysls, liquefactlon of serum and utilizatlon of galactose and lactose which were shown by the Group C straln~. However, those Group C stralns are not available at present, as they are already dead. In this sltuatlon, comparlson of the MC916-C4 straln ls now made wlth Stre~tomvces sp, IFO 3300 whlch ls known to produce fermlcidln, an antlblotlc analogous to cycloheximlde, and whlch ls reported in the above article by T. Furumai et al.
to be well colncldent wlth sald Group C strains, The result~ of comparlson are sho~m ir. Table 4 below, wlth reference to the descrlptlons of the "Journal of Antlblotlcs".

~'~ '' O ~ S ~h n5 ~ P' rR h t ~m I I I ~ ~
~1 9 F: ~ S ~
~:
m C~ ~8 o ~ m m mo ~ ~
a o ~ a~ ~ o ^ s ~ ~o~ *l *l * ~ I
m h ~ o ~ a~o ~S ~ ~ o ~
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mo~ m ,13 o~ ~ ~
~o ~ ~:o ~ 3 ~ o ~~ m b~ 0.4 m ,~
o ~i ~ ~m~l ~
~1o o ~o o ~ ~ ~ S
'~ ~ ~H h ~ I I I ~ +
01:4H H ~ tU ~ O ~1 0 ~ ,c ~ ~ o Q~ 3C 3 b~
L h ~ 1~ I O
D. ~ ~ ~ ~ O t~ ~ O
G~ m~ 0 ~ ~ ~ ~ ~ Z o S ~ 0 0~
~ ~OS
:~ S~ h O o ~ ~: S
V ~ O ~ ~1 ~
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~ ~ ~ O b~a~ S ~ ~I O 'I
v ~ e ~ h D~ o o ~
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gs ~ ~ e V ~1 3 ~ C~ O
0 ~3~ o O O O O

o ~ ~ bOO !~ ~Z; Z ~
m c~ a~ ~ ~ ~ m o o ~ o I ~ ~ cq m J~ ~rl al O O O ~rl ~: H H H h ~
0~ ~ O ~ O O O ~ bO
o P. o o o o a~ h O

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o ~ o ~ Ç~.
o ~ ~ V~ o o~ o C~

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C ~o S v h~ ~ ~ O c 0 ~ o~ ~
O-H ~ ~ I '8 :5 ~ E3 ~ ~I ri o~, o-l~ ~ ~ o o u~ m ~,~ ~ ~ ~ 0 ~ h S

o O
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a ~0 o~
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v ~ m ~o~ sO ~
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,~ 0 a) I q~ o "3 H E ~ ~ ~ 8 bO~: O ~ 0 o ~
O ~ J~ ~ O
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0 ~ ~ o ~ ~ o ,~ o ~1 N O ~r1 o ~ O ~ ID ~) ~ m ~ a) O S: O ~rl o o 11~ O
~ H H ~ ~ N o 0 0 H :5 0 ~ 0 q~ o a~ ~ bO~ O~ S~ O
~; K ~ o O ;~
o ~ ~ s~ ~ ~~ O ~I L ~ L
p p ~ cs ~ Q ~ u~ H ~ ~ ~ ~::

A~;,...

~056747 U~ ,, ,, C~
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C~ o N
a ta ~ ^
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z t~ 0 ,1 +1 O
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As will be seen from the above Table, the MC916-C4 strain is coincident with the Group C strain descri~ed in the above-mentioned literature but is differentiated from Streptomyces sp. IFO 3300 strain in respect to the coagulation and peptonization of milk. Furthermore, the MC916-C4 strain is different from Streptomyces griseolus [see the "International Journal of Systematic sacteriology" Vol. 18, page 12~ (1968)]
which has been reported to resemble said IFO 3300 strain, in that S. griseolus does not form open spirals in thè aerial hyphae thereof and is somewhat different from the MC916-C4 strain in respect to the utilization of carbon sources. Further comparisons of the MC916-C4 strain with Streptomyces sp.
IFO 3300, StrePtomyces griseolus ISP 5067 and Streptomyces -naraensis ISP 5508 are carried out. It has been found that the MC916-C4 strain is related to Streptomyces sp. IFO 3300 and Streptomyces naraensis ISP 5508 and most to the former strain.
The IFO 3300 strain is somewhat different from the MC916-C4 strain having a tinge of orange in the color of growth. The MC916-C4 strain is clearly dlstinguished from said ISP 5508 strain in respect to the reduction of nitrate and from said ISP 5067 strain in respect to the formation of spirals, utiliza-tion of carbon sources and reduction of nitrate.
Mutation of actinomycetes occurs frequently in either artificial or spontaneous conditions. Accordingly, this invention includes the use of the MC916-C4 strain as well as it mutants. In other words, this invention includes the use of all strains of the genus Streptomyces which produce neothramycin.
The neothramycin can be obtained by aerobic cultiva-tion of spores or mycelia of a neothramycin-producing strain .r -of the genus Streptomyces such as Strpetomyces sp. MC916-C4 strain (identified as A.T.C.C. 31123). In carrying out the process of the second aspect of this invention, an amount of spores or mycelia of a neothramycin-producing strain is inoculated to a suitable culture medium therefor comprising nutrient sources and is then incubated under aerobic conditions so that there is obtained a culture broth containing neothramycin.
Generally, constituents of culture media commonly employed for the cultivation of ordinary actinomycetes can be used for the purpose of this invention. For instance, commercially avail-able soybean meal, peanut powder, cotton seed powder, dried yeast, peptone, meat extract, casein, corn steep liquor, N-Z amine, ammonium nitrate, ammonium sulfate and the like may be useful as the nitrogen carbon sources. Commercially available carbohydrates such as glucose, starch, glycerol, maltose, dextrin, saccharose, lactose, molasses and the like as well as fat or oil are useful as the carbon source. In addition, sodium chloride, calcium carbonate, magnesium sulfate, manganese chloride, sodium phosphate or other inorganic salts can be employed for the salt-additive in the culture medium. Various heavy metal salts may also be added in trace quantities, if required. Any of the nutrient materials which are known for the cultivation of actinomycetes may be employed in the process of this invention, as long as it is assimilable by the neothramycin-producing strain for the production of neothramycin.

* Trademark.

105~;747 For the production of neothramYcin on a large scale, llquid cul~ivatlon ~ 5 preferred. Any temperature at which the neothramycin-producing strain 18 able to grow and produce the neothramycin can be employed for the cultivation, but a pre~erred cultlvation temperat.Lre i~ wlthln a range of 2~ to 35 C. The cultlvatlon 18 contlnued for a perlod of t~me ~u~flclent to produce and accumulate a sufficient amount of neothramycin A and B in the culture medium. For instance, a culture medlum comprislng 2% glucose, 2% glycerol, 1.2% soybean meal, 1.0% cotton seed flour, 0.32~
calclum carbonate, 0.5% ~odlu~ chlorlde and 0.0005%
manganese chlorlde tetrahydrate was prepared and sterilized at pH 6.8. -ml8 medlum was then lnoculated with spores or mycella harvested from a slant culture o~ the MC916-C4 strain. When it was ~hake-cultlvated aeroblcally at 28 C., the productlon and accumulation of neothramycin in the culture medium r.~ached a maxlmum at the end o~ lncubatlon ~or 3 to 5 days, Assay of neothramycin can be made using Sta~hvlococcus aure.~s or Escherlchla coli as the test organlsm accordlng to a 3tandard cup-plate method whlch has usually been employed for the assay of known antiblotics. Pure neothramvcin A which was obtalned from the Example ~, descrlbed later, Or thl~ inventlon may be used a8 an authentl¢ sample whlch exhibit~ a potency of 1000 unlts per mg. In case the other antlbiotic 3ubstances such as cyclo-hexlmlde are ~lmultaneou~ly produced ~n the culture broth of the MC916-C4 ~traln in additlon to the ~eothramycin, the culture broth may be washed wlth ethyl acetate or other 3ultable organlc 301-vent to remove such other antlblotic ~ub~tances by extractlon. The remalning aqueou~ pha~e may tben be lmployed for the a~say of the contents of neothramycin A and B according to the aforeaald ~tandard cup-plate method.
For the recovery o~ the neothramycin from the culture medlum, the culture broth of the neo-thramycin-producing ~train may elther be treated wlth a sultable organlc solvent such as n-butanol to provlde an extract of the neothramycin in sald solvent or may be treated with a suitable ad~orbent such a~ active carbon to make the neo-thramycin adsorbed by the adsorben$. Distrlbuti4n of the neothramycin A or neothramycin B
between n-butanol and water was examined, and lt is found that the partition coefflclent of the neo-thramycin in n-butanol/water ls greater than 5 at a pH value of 2 to 7. Accordingly, the neo-thramycin can be extracted with n-butanol from the aqueou~ culture broth whlch ha~ been ad~u3ted to a pH value of 2 to 7 and pre~erably of about 6.
The neothramycin is substantially insoluble in and hence 18 practically not extractable with ethyl acetate or chloroform from the liquld portion ; r;

of the culture broth. If required9 there~ore, lt 18 posslble to treat the culture broth wlth ethyl aoetate or chloro~orm for extractlon in order to remove the soluble impuritles ~rom the culture broth. To separate the neothramycin from the culture broth lt ls pre~erred that the culture broth ls treated wlth actlve carbon a~ the adsorbent. The neo-thramycin whlch has been ad~orbed by active carbon can be eluted thererrom by means o~ a mixture o~
methanol and water, a m~xture of propanol and water or a mlx$ure o~ acetone and water, etc. The efflclency of the elution may be lmproYed when the elutlon ls done under weakly alkallne condltions. Purlfication o~ the neothramycin can be made uslng the above-mentloned extractlon method and adsorption-elutlon method ln a ~ultable comblnatlon of them or ln a repeated manner. Further puriflcatlon may be achleved b~ a u~ual column chromatography on Sephadex IH-20 (a commerclal product sold by Pharm~c~a Co., Sweden) or slllca gel. The known antlblotlc cycloheximlde whlch may fr~quently be co-existent ln the culture broth of the MC916-C4 strain can readll~ be sepQrated from the neothramycin of this invention by extracting with eth~l acetate or b~ chromatogra~hy on Sephadex I~-20 To lsolate the neothramycin A from the neothramycin ~, a mixture of the neothramycin A and *Trade Mark ~ ~i neothramycin ~ may be subjected to a column chromatography on ~llica gel wlth chloroform-methanol (30:1 by volume) as the developing ~olvent.
~he isolated neothramycin A or the isolated neothramycin B can be purified by column chromatography on 3111ca gel using sultable mixed organlc solvents as the de~eloplng sol~ent~
The recovery Or the neothramycin A and neo-thramycin B may typically be carried out in the following way: ~he culture broth contalnlng the neothramycin ls at first rlltered or centrlfuged to remove the ~olld matters together with the mycella. The broth flltrate 18 then treated wlth actlve carbon to adsorb ~he neothramycin there~rom. The actlve carbon carrying the ad~orbed neothramycin is eluted with 50% acetone-water (a mlxture of acetone and water at a ratlo of 1:1 by volume) at pH 8Ø ~he eluate 18 collected ln rractlon~ and the actlve fractlon~ are combined together and concentrated to dr~ness under reduced pre~ure at a temperature Or up to 40 C. or otherwl~e ~reeze-drled to glve a crude powder.
m 1~ crude powder 18 extracted with aqueous ethanol 80 that a greater part Or the actlve component3 18 separated in the resultlng extract.
Thls extract i8 concentrated to drynes~ under reduced pre~ure at a temperature Or up to 40 C.

~,, or otherwise freeze-dried to give a second crude powder.
A solut~on of this crude powder in methanol 1~
passed through a column of Sephadex IH-20 (a product of Pharmacla Co., Sweden) whlch is subsequen~ly developed wlth methanol. Durlng thl~ chromatographlc proces~, the posslbly co-existing cyclohexlmlde 13 eluted in ~uch ~ractlons runnlng out ln the fir~t-half phase of the process, whereas the mlxture of the neothramycin A and B is eluted in such fractions runnlng out ln the later-half phase of the proce~.
The actlve ~ractions containing the neothramycin A and B are combined together and then con-centrated to dryness under reduced pres~ure at a temperature o~ up to 40 C" to a~ford a crude powder. Ihls powder i9 taken into a small volume of methanol and the methanollc solutlon is unl~ormly admixed wlth an amount o~ neutral silica gel. The admixture was drled by evaporatlon and then placed on the top of a column of a further amount of said neutral slli¢a gel which has been ~mpregnated wlth a mixture of chloroform and ethanol (30:1 by volume).
~he sillca gel column i~ sub~equently developed with the chloroform-ethanol (30:1 by volume). During thls chromatograph~ proce~s, the neothramycin A is eluted ln the actlve fractlons runnlng ln the first-half pha~e o~ the proce~s, whlle the neothramycin B
18 eluted in the actlve fractlons runnlng out ln the later-half phase of the process. The actlve fractlons ~' .

contalnlng the neothramycin A and the active ~ractions contalnlng the neothramycin B are concentrated to dryne3s under reduced pressure at a temperature o~ up to 40 C., respectively, to glve a crude powder of the neothramycin A and a crude powder o~ the neothramycin ~.
The crude powder of the MC916-A substance 80 obtained 18 taken lnto an appropriate amount of chlorororm and the solutlon 18 passed through a column of a neutral slllca gel whlch has been lmpregnated wlth chloroform. ffl 19 slllca gel column ls washed wlth chloroform and then developed at 5 C. wlth chloro~ormrethanol (60:1 by volume).
The eluate 1~ collected ln ~ractions, and the desired actlve fractlons solely contalnlng the neothramycin A are detected by referring to test results of blologlcal assay and thln layer chromatography o~ each rractlon. me deslred actlve fractlons so chosen are comblned together and concentrated to dryness under reduced pre~sure at a temperature of up to 40 C. to glve the neothramycin A as a colorless powder. This powder may ~urther be purl~led to a colorless powder of pure neothramycin A by repeating the above-mentioned silica gel chromatographlc proce~s or by dlssolvlng sald powder lnto a sm~ll volume of chloro~orm, addlng ethyl ether to the chloro~orm solutlon, fllterlng o~ and dry~ng the resultlng precipltate. A colorless powder ,;., ~
, . .
~, . ...

~056747 of pure neothramycin B may be obtained from the a~oresald crude powder o~ the neothramycln s by purlfylng in the ~ame manner a8 for the neothramycin A.
It is preferred, however, that the column chromatography on slllca gel 18 made u~lng a mlxture of chloro~orm and ethanol (lOO:l by volume) as the developlng solvent.
In view of the aforesaid propertles of the neo-thramycin A and neothramycin B, it has been con-flrmed that theqe substance~ are new antiblotic3 whlch are dlf~erentlated from any of the known antlblotlc~. Accordlng to a khlrd aqpect o~ thls lnventlonJ there 1~ pro~lded a method for thera-peutlcally treatlng a llvlng anlmal, lncluding man, a~ected by leukemla, whlch comprl~es admln-lstering the neothramycin A and/or neothramycin B
~ubstance to sa$d anlmal ln a do~age ~uf~lclent to reduce the affection by leukemia. Accordlng to a ~ourth a~pect of this ln~entl~ , there 1~ ~urther provlded a pharmaceutlcal compo~ition comp~l~tng the neothramycin A and/or the neothramycin B
ln an amount ~uffl¢ient to reduce the affectlon by leukemia ln vlvo, the neothramycin A and/or the neothramycin B being in combina~ion with a pharmaceutlcally acceptable carrler. It will be appreciated that the actual pre~erred amount~ of the neothramycin uséd will vary according to , , , .,, . ~, the partlcular compound belng used, the partlcular compositlon formulated, the mode o~ appllcatlon and the partlcular sltus and organlsm belng treated.
Many factors that modlfy the actlon of the drug wlll be taken lnto account by the skilled ln the art, ~or example, age, body weight, sex, dlet, time of adminlstratlon, route of adminlstratlon, rate Or excretlon, drug combinatlons, reactlon sen~ltlvlties and severlty of the dlsease. Optlmal applicatlan rate~ ~or a glven set of condltlons can be a~certalned by the skllled ln the art uslng conventional dosage determinatlon tests in view o~ the above g ldelines.
It ls belleved that uslng the precedlng descrlptlon and wlthout ~urther elaboratlon, one skllled ln the art can utll~ze the concept of thls inventlon to lts ~ullest extent. The ~ollowing preferred speciflc embodiments are, therefore, to be construed as merely illustratl~e and not llmltative of the re-malnder of the dl3closure ln any way.
De~crl~tion of the Preferred Embodlmen~

am~le 1 A loopful quantl~g o~ Stre~tom~ce~ sp. MC916-C4 (ldentlfied as A.T.C.C. 31123) whlch wa~ incubated in slant agar medlum ~a~ inoculated to a sterile llquid culture medlum (pH 7.0~ 125 ml.) compri~ing 2.5~ malto~e, 0.75% peptone, 0.75% meat extract, 0.3% yeast extract, 0.3~ sodlum chlorlde and 0.1%
magneslum sulfate (7H20). The inoculated medlum waa shake-cultured at 28 C, ~or 48 hours to give a primary seed culture. This prlmary seed culture was inoculated at an inoculum slze of 0.48% by volume to 50l or a ~terllized liquld culture medium (pH
7.0) oontainlng 3,5% ~tarch ~yrup, 0,75% peptone, 0.75% meat extract, 0.3% yeast extract, 0.3% ~odlum chloride and 0.1% magneslum sulfate (7H20) ln a stainless steel fermentor of a capacity of 130R .
The lnoculated medlum was cultured at 28 C. for 24 hour~ under aeratlon and agltation to provide a secondary seed culture. This secondar~ seed culture wa~ lnoculated at an inoculum size of 2% by volume ( 6l) to a liquld culture medium (pH 6.8, 300l) comprl31ng 2% glucose, 2% glycerol, 1.2% soybean meal, 1.0% cotton seed powder, 0.3$
calcium carbonate, 0.5% sodium chloride and 0.0005%
~anganese chloride (4H20) whlch had been sterlllzed at 120 C. for 30 mlnute~. The cultivation was made ak 28 C. for 92 hour~ under aeration and agltatlon (250 r.p.m.) whlle the rate Or aeration was 150 ~¦ mlnute for the flrst 24 hour~ and then lncreased 105674~.7 to 300 ~/minute ~or the ~ubsequent period o~ 24th hour to 92 hour of the cultivation.
The resulting culture broth (pH 7.3, 300R, potency 88 u./ml.) wa 8 admixed wlth 24 kg. o~ a filter ald (diatomaceous earth commerclally available under a trade mark HIFR0-SUPERCEL) and the admixture was flltered by mean~ of a press-~ilter to give 300Q of the broth ~iltrate. The broth flltrate wa~ well admixed with 3 Kg. o~
active carbon at ambient temperature for 1 hour under agitation, so that the antiblotics were ad~orbed on the carbon.
The active carbon portion was collected b~
centrlfugation and then washed with 150 l Or water. m e washed carbon was admixed with 70 of 50% acetone-water (pH 8.0) ~or 1 hour under agitation, 80 that the antib$otlcs were extracted into the ~olvent. This extractlon was conducted twice and the extracts 80 obtalned were combined together to a volume of 118~. The extract solution was concentrated under reduced pressure at a temperature Or up to ~0 C. and the concentrated solutlon (2.6Q) was freeze-dried to give 565 g. of a brown colored powder (potency, 35 u./mg.) which contained the neothramycin A and B. This brown colored powder wa~ extracted wlth 11.6~ o~ 80%
ethanol-water. The $nsoluble matters which had no antlbacterlal act$vlty were removed by flltratlon, 10567~

to yleld 11.2Q o~ an ethanollc extract. Thls extract was concentrated under reduced pre~sure at a temperature o~ up to 40 C. to a volume o~ 800 ml.9 and the concentrated solutlon was freeze-dried to a~ord 218.5 g. of a crude powder (potency, 53 u./mg.). Thi8 crude powder was dlvlded into rlve equal part~, and each part was dl~olved in 20 ml. of methanol. me methanol~¢ solutlon wa~ passed through a column (90 mm diameter) of 4~ o~ Sephadex LH-20, which was subsequently developed with methanol. lhe eluate was collected ln 200 ml. rractlon~, and lt was round that cyclohexlmide wa~ eluted out ln the rractlon Nos. 9 to 11 whlle a mixture Or the neothramycin A and B
~ubstances was eluted ln the rractlon No~. 12 to 14.
The fra¢tion Nos. 12-14 were combined together and concentrated to drynes~ under reduced pres~ure at a temperature Or up to 40 C. to give 47 g. Or a crude powder (potency, 160 u./mg.) comprl~lng the mlxed neothramycin A and B. Yield 28% (based on the neothramycin content of the culture broth).

Exam~le 2 The crude powder (3~ g.) comprlsing the mlxed neothramycin A and B obtained in Example 1 was taken lnto a small volume Or methanol, and the solutlon was unlformly mlxed wlth 60 g. of a neutral slllca gel, followed by drylng under reduced pres~ure.
The drled mas~ 80 obtalned was placed on the top o~

A~

lOS6747 a column (60 mm diameter~ Or 660 g. Or sald neutral sillca gel which had been lmpregnated with chlorororm ethanol (30:1 by ~olume). Th13 3111ca gel column was developed at 5 C. by passlng a ~low oP chloro~orm-ethanol (30:1 by volume) through sald column. The eluate was collected ln 130 ml. rractlons, and lt was found that the neothramycin A was eluted in the rractlon Nos. 2~-31 whlle the neothramycin B
was eluted ~n the fractlon Nos. ~5-49. The combined actiYe fractlon Nos. 23-31 Wa3 concentrated to dr~nes~
under reduced pressure at a temperature of up to 40 C. to glve 1.47 g. of a yellowl~h crude powder of the neothramycin A (potency, 520 u./mg.). Yield 14%. me ¢ombined rractlon Nos. 35-49 were concentrated to dryness ln the ~ame mQnner to gl~e 1.0~ g. Or a yellowi~h crude powder Or the neothramycin B
(potency, 450 u./mg.), Yleld 9%.

Exam~le ~
The yellowtsh crude powder o~ the neothramycin A
(1 g.) obtained in Example 2 was dissolved in 20 ml. of chlorororm, and the solution was pas~ed at 5 C. through a column (1~ mm dlameter) o~ 20 g, of a neutral sillca gel of the same grade as employed in Example 2 whlch had been lmpregnated wtth chloro~orm, The column was washed wlth 400 ml. of chloroform and subsequently developed wlth chloroform-ethanol (60:1 by volume). The eluate Wa8 collected ln 8 ml. fractlons, "~, ~,.,; ~ , .

10567~'7 and it was round that the neothramycin A was eluted in the ~ractlon Nos. 13-27. me combined Practlon Nos. 13-27 was concentrated to dryne~3 under reduced pressure at a temperature oP up to 40 C., glving ~20 mg. of a raintly yellow colored powder.
Thl~ powder was taken lnto a mlnlmum volume oP
chloroform and to thls solutlon was added ethyl ether until the precipltate ~ormed was not longer depo~ited. ~he preclpltate wa 8 removed by filtration and drled, a~fording 183 mg. of a colorles~ powder of pure neothramycin A (potency 1000 u./mg.). Yield 35%.
Exam~Dle 4 me yellowi~h crude powder o~ the neothramycin B (810 mg.) obtained in Example 2 was taken into 16 ml. of chloro-Porm and the ~olutlon was passed at 5 C. through a column (1~ mm dlameter) o~ 16 g. of neutral slllca gel oP the same grade as employed ln Example 2 whlch had been lmpregnated with chlorororm. The column was washed with ~20 ml. of chloroform and then developed wlth chloroPorm-ethanol (100:1 by volume).
me eluate was collected in 6.4 ml. Practlons, and it was round that the neothramycin B was eluted in the fractlon Nos. 34-70, The combined fraction Nos. ~4-70 was concentrated to drynes~ under reduced pre3~ure at a temperature of up to 40 C, to glve 160 mg. Or a faintly ~ellow colored powder, Thl~ powder was taken lnto a mlnlmum volu~e oP chloroPorm, and to 105674~

thls ~olution was added ethyl ether untll the pre-clpitate wa~ no longer deposlted. The preclpltate was removed by rlltratlon and dried, a~ording 85 mg. of a colorles~ powder o~ pure neothramycin B
(potency, 620 u./mg.). Yleld 14.6%.

The Sephadex IH-20 used ln the precsding example~ can be replaced by other slmilar gel-*

flltratlon agent~, e.g. Sephadex G25 to G200,Sepharose ~B and 6B (Pharmacia Flne Chemlcals AB, Uppsala, Sweden) and Blo-Gel Al.5m (Blo Rad Co.).
Prererred gel-~iltratlon agent~ include the carboxymethyl substltuted cross-llnked dextran gels descrlbed in ¢olumns ~ and 4 of U.S. patent 3,819,836.
The pharmaceutlcally acceptable salt3 o~ the substances of the present lnventlon include nontoxic metallic salts such a~ sodium, pota~ium, calcium and aluminum, the ammonium salt and substituted ammonlum salts, e.g. salts of such nontoxic amines as tri-alkylamlnes includlng triethylamine, procaine, dl-benzglamine, N-benzyl-beta-phenethylamlne, l-ephenamine, N,N'-dlbenzylethylenediamine, dehydroabletylamlne, N,N~-bls-dehydroabietylethylenedlamine, N-(lower)-alkyl-plperldine, e.g. N-ethglplperidine, and other amlnes whlch have been used to form ~alts wlth benzyl-penlcillln *Trade Mark~
A~ 39 _

Claims (33)

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

1. The process for producing the antitumor antibiotic neothramycin selected from the group consisting of neothramycin A, neothramycin B and mixtures thereof, said neothramycin A having the formula:

and said neothramycin B having the formula:

which comprises culturing a neothramycin-producing strain of Streptomyces having the identifying characteristics of A.T.C.C. 31123 under submerged aerobic conditions in a nutrient medium containing a carbon source and a nitrogenous nutrient until a substantial amount of neothramycin is produced by said organism in said nutrient medium, and recovering the neothramycin from the nutrient medium.

2. The process of Claim 1, further comprising forming a pharmaceutically acceptable salt of the neothramycin by methods known purpose.

3. The process of Claim 1 in which the strain of Streptomyces Is cultured in a nutrient medium at a temperature in the range of 24°C. to 35°C.

4. The process of Claim 1 in which the strain of Streptomyces is cultured in a nutrient medium at a temperature in the range of 25°C. to 29°C. with a pH from 6 to 8.

5. The process of Claim 1 in which the neothramycin produced in the cultured broth is extracted and purified by a process which includes at least one process selected from the group consisting of salting-out, solvent precipitation, butanol extraction, dialysis, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, electrofocusing and adsorption followed by elution from an ion exchange resin.

6. The process of Claim 1 in which the medium con-taining the neothramycin is stored in a cold or frozen state.

7. The process of Claim 1 in which the medium con-taining the neothramycin is freeze dried.

8. The process of Claim 3 further comprising forming a pharmaceutically acceptable salt of the neothramycin by methods known per se.

9. The process of Claim 8 in which a pharmaceutically acceptable metallic, ammonium or amine salt of the neo-thramycin is prepared.

10. The process of Claim 9 in which the metallic salt is selected from the group consisting of the sodium, potassium, calcium and aluminum salts thereof.

11. The process of Claim 9 in which the amine salt of the neothramycln is selected from the group consisting of the triethylamine, procaine, dibenzylamlne, N-benzyl-beta-phenethylamine, l-ephenamine, N,N'-dibenzylethylenediamine, dehydroabiethylamine, N,N'-dehydroabienzylethylenediamine, N-ethylpiperidine salts thereof.

12. The process for producing neothramycin A having the formula:

which comprises culturing a neothramycin A producing strain of Streptomyces having the identifying characteristics of A. T. C. C. 31123 under submerged aerobic conditions in a nutrient medium containing a carbon source and a nitrogenous nutrient until a substantial amount of neothramycin A is produced by said organism in said nutrient medium, and recovering neothramycin A from the nutrient medium.

13. -The process of Claim 12, further comprising forming a pharmaceutically acceptable salt of neothramycin A by methods known per se.

14. The process of Claim 12 in which the strain of Streptomyces is cultured in a nutrient medium at a temperature in the range of 24VC. to 35°C.

15. The process of Claim 12 in whlch the strain of Streptomyces is cultured in a nutrient medium at a temperature in the range of 25-C. to 29°C. with a pH from 6 to 8.

16. The process of Claim 12 in which neothramycin A
produced in the cultured broth is extracted and purified by a process which includes at least one process selected from the group consisting of salting-out, solvent precipitation, butanol extraction, dialysis, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, electro-focusing and adsorption followed by elution from an ion exchange resin.

17. The process of Claim 12 in which the solution containing neothramycin A is stored in a cold or frozen state.

18. The process of Claim 12 in which the solution containing neothramycin A is freeze dried.

19. The process for producing neothramycin B having the formula:

which comprises culturing a neothramycin B producing strain of Streptomyces having the identifying characteristics of A.T.C.C. 31123 under submerged aerobic conditions in a nutrient medium containing a carbon source and a nitrogenous nutrient until a substantial amount of neothramycin B is produced by said organism in said nutrient medium, and recovering neothramycin B from the nutrient medium.

20. The process of Claim 19, further comprising forming a pharmaceutically acceptable salt of neothramycin B by methods known per se.

21. The process of Claim 19 in which the strain of Streptomyces is cultured in a nutrient medium at a temperature in the range of 24°C. to 35°C.

22. The process of Claim 19 in which the strain of Streptomyces is cultured in a nutrient medium at a temperature in the range of 25°C. to 29°C. with a pH from 6 to 8.

23. The process of Claim 19 in which neothramycin B
produced in the cultured broth is extracted and purified by a process which includes at least one process selected from the group consisting of salting-out, solvent precipitation, butanol extraction, dialysis, ultrafiltration, isoelectric precipitation, gel filtration, electrophoresis, electro-focusing and adsorption followed by elution from an ion exchange resin.

24. The process of Claim 19 in which the solution containing neothramycin B is stored in a cold or frozen state.

25. The process of Claim 19 in which the solution containing neothramycin B is freeze dried.

26. Antitumor antibiotic neothramycin selected from the group consisting of neothramycin A having the formula:

neothramycin B, and mixtures thereof, whenever prepared by the process of Claim 1, 3 or 4 or by an obvious chemical equivalent thereof.

27. A pharmaceutically acceptable salt of neothramycin selected from the group consisting of neothramycin A having the formula:

neothramycin B

and mixtures thereof, whenever prepared by the process of Claim 2 or 8 or by obvious chemical equivalent thereof.

28. Neothramycin A having the formula:

whenever prepared by the process of Claim 12. 14 or 15 or by an obvious chemical equivalent thereof.

29. A pharmaceutically acceptable salt of neothramycin A
having the formula:

whenever prepared by the process of Claim 13 or by an obvious chemical equivalent thereof.

30. Neothramycin A having the formula:

1701. /8 whenever prepared by the process of Claim 16, or 19 or by an obvious chemical equivalent thereof.

31. Neothramycin B having the formula:

whenever prepared by the process of Claim 19, 21 or 22 or by an obvious chemical equivalent thereof.

32. A pharmaceutically acceptable salt of neothramycin B
having the formula:

whenever prepared by the process of Claim 20 or by an obvious chemical equivalent thereof.

33. Neothramycin B having the formula:

or a pharmaceutically acceptable salt thereof, whenever prepared by the process of Claim 23 or 24 or by an obvious chemical equivalent thereof.