CA1056748A - Neothramycin antibiotics from streptomyces - Google Patents

Abstract

NEW ANTIBIOTICS
Abstract of the Disclosure There are disclosed two new antibiotics formerly denominated MC916-A and MC916-B and now called neothramycin A
and neothramycin B; and their methyl derivates which are potent inhibitors of the growth of leukemia cells, e.g.
Leukemia L-1210 cells in mice. They are produced by controlled fermentation of Streptomyces FERM-P 2452 (A.T.C.C. 31123).

Description

Cross-Reference to Related Application This application is related to copending application Serial Number 221,848 filed ~qarch 11, 1975.
Background of the Invention This invention relates to chemical compounds produced by fermentation and used to inhibit the growth of leukemia cells.

~056748 Summary of the Invention This invention relates to two new antibiotics which each exhibit a high activity inhibitory to the growth of leukemia cells and are useful as anti-tumor agents bu~ exhibit low antibacterial activity. More particularly, this invention relates to the new antibiotics formerly designated ~IC916-A
substance and MC916-B substance, respectively, and now designated neothramycin A and neothramycin B, respectively, and also to a process for the production of these new antibi-otics by cultivation of a strain of StreptomYces. This inventionalso relates to the recovery and purification of these specific new antibiotic substances and to their use for pharmaceutical purposes.
Furthermore, this invention relates to a process for the production of the antibiotic methylneothramycin which comprises reacting neothramycin with anhydrous methanol.
Hereinafter, by the term neothramycin is meant neothramycin A or neothramycin B or their mixtures and by the term methylneothramycin is meant methylneothramycin A or methylneothramycin B or their mixtures 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 from such soil samples and investigated metabolic products which are produced by the aerobic 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-ku, Tokyo, Japan, L~

105~;748 and we have designated this newly isolated micro-organism as MC916-C4 strain. It has been conflrmed that this MC916-C4 strain belongs to the genus Stre~to~vces, We have now found that two new anti- -biotics havlng a low antibacterial activity but hlgh activity inhlbitory to the growth of leukemla L-1210 cells in mice and to the growth of a certaln klnd of tumor cells are produced and accumulated in the culture broth of the MC916-C4 strain. We have now succeeded in isolating these new antiblotlcs from the culture broth and designated them as neothramycin A and neothramycin B, respectively.
An ob~ect of this invention is to provide new substances which are useful as anti-tumor agents.
Another ob~ect of this invention is to provide the neothramycin A and neothramycin B, either alone or in mlxture thereof as new and useful anti-tumor agents. A further ob~ect of this invention is to provide a process for the preparation of the neo-thramycin A and neothramycin B by cultivation of the MC916-C4 strain. Other ob~ects of this invention wlll be clear from the following descriptions.
According to one aspect of this inventlon, there ls provlded as a new antiblotic substance, neo-thramycin having an activlty lnhlbitory to the growth of leukemia L-1210 cells in mice and a low antibacterial actlvity, said substance having an acldlc function;
being soluble in methanol, ethanol, propanol, chloroform 3~

1(~56748 and dioxane and slightly soluble in water but sparingly soluble or substantially insoluble in ethyl ether and n-hexane; being positive to Rydon-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 (25:75:900 by volume) as an electrolyte solution; said substance bei.ng at least one member selected from the group consisting of neothra-mycin A and neothramycin B;
a) said neothramycin A being further charac-terized by giving C, 57.46~, H, 5.76~, N, 9084~ and the remainder oxygen upon elemental analysis thereof; giv-ing a molecular weight of 250 to 300 as measured by Barger-Akiya method; having an infrared absorption spectrum pelleted in potassium bromide corresponding to that shown in Figure 1 of the attached drawings and 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 corresponding to those shown in Figure 3 of the attached drawings characterized by absorption maxima at 223 nm (El~Cm 855), 240 nm (shoulder), 265 nm (El~Cm 290) and 318 nm (El~Cm 156) in a solution thereof in 10~ water-methanol, by ab-sorption maxima at 223 nm (El~Cm 885), 240 nm (shoulder) 265 nm (El%Cm 290) and 320 nm (E1%Cm 139) in a solu-tion thereof in N/10 HCl-methanol (1:9) and by absorp-tion maxima at 228 nm (E1%Cm 635), 254 nm (El cm 566), 291nm(E1%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 charac-terized 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 Barger-Akiya method; having an infrared absorption spectrum pelleted in potassium bromide corresponding to that shown in Figure 2 of the attached drawings and charac-terized by absorption peaks at 3400, 2960, 1630 (shoul-der), 1600, 1510, 1440, 1400, 1280, 1200, 1120, 1080, 1010, 990, 940, 870, 790 and 760 cm 1; having ultravio-let absorption spectra corresponding to those shown in Figure 4 of the a,ttached drawings and characterized by absorption maxima at 224 nm (El%Cm 935), 240 nm (shoul-der), 265 nm (shoulder) and 318 nm (El%Cm 167) in a solution thereof in 10% water-methanol (1:9), by absorp-tion 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 (E1%Cm 725), 291 nm (El cm 456) and 324 nm (El%Cm 466) in a solution thereof in N/10 NaOH-methanol (1:9); and giving an Rf va,lue 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 neothramycin B substances, either alone or in a mix-ture 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 105 CO
and decomposes at 132-147 C. with foaming and which exhibits a specific optical rotation r~]26 = +272 (C 0.52, dioxane). From the results of elemental analysis and the determination of molecular weight, it is probable that neothramycin A has an empirical formula C13H14N204 1/2 H20. This formula has been confirmed by high-resolution mass-spectrometry (Found:
m/e 262.0934, Calcd. mol. wt. for C13H14N204 262.0952).
The ultra-violet absorption spectrum of neothramycin A
in an alkaline solution exhibits a shift towards the longer wave length as shown in Figure 3. As shown in Table 1, NI~ spectrum of neothramycin A shows the presence of 14 protons. Neothramycin B is very simi-lar in its properties to neothramycin A and has been obtained as a colorless powder which has no definite melting point, commences to decompose at 144 C. with foaming and completely melts at 151 C. and which ex-hibits a specific optical rotation [~D = ~314 (C o.48, dioxane). Neothramycin B has the empirical formula: C13H14N204~1/2 H20. This formula has been confirmed by high-resolution mass-spectrometry (Found: m/e 262,09~9, Calcd. mol. wt. for CL~H14N20L~ 262-0952) -The ultraviolet absorption spectrum of neothramycin B in an alkaline solution exhibits a shift towards the longer wave length, as shown in Figure 4. As shown in Table 1, NMR spectrum of neothramycin B shows the pre-sence of 14 protons, similarly to neothramycin A. Neo-thramycin 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 22%, respectively, at room temperature for 16 hours. In 50~ aqueous ethanol of pH 6.5 or pH 8. o at room temperature for 16 hours, 80-90% activity of neo-thramycin A and 70-80~ activity of neothramycin B re-main, but an equilibrium conversion of neothramycin A
to B or B to A is shown by thin-layer chromatographic analysis. Neothramycin A or B is easily converted to a mixture of methyl-neothramycins A [Rf 0.71 on silica gel thin-layer chromatogram with chloroform-methanol (10:1 volume)]and B (Rf 0 61) in anhydrous methanol at room temperature for 16 hours. Methy~Lneothramycin A
is crystallized from a mixture of acetone and benzene, colorless microcrystals, m.p. 1~7-140 C. (dec.); [a]D6 +640 (c, 0.24, dioxane), MS, m/e 276.1089 (Calcd. mol.
wt. for C14H16N204, 276.1108). Methylneothramycin B is obtained as a colorless powder, m.p. 61-69 c. (decO );
[a]26 +778 (c, 0.22, dioxane)~ MS, m/e 276~1071. w spectra of methylneothramycins are similar to those of lOS6748 neothramycins and the PMR chemical shifts are shown in Table 1. Mild hydrolysis of methylneothramycin A or B
in O.OlN HCl-dioxane (1:1 in volume) at room temperature for one hour followed by column chromatography on silica gel gives neothramycins A and B in a good yield.
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 benzodi-azepine structurec They are distinguished from anthra~
mycin, dextrochrysin and sibiromycin by their UV spectra. The W spectra of tomamycin and neothramycins are very simi-lar but they are different in their molecular formulae and other spectra.

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V O v ~b O v t~ ~ V 1:~ V h 105674~3 By structural studies, the following struc-tures have been submitted by the inventors for neo-thramycin A (Rl = OH, R2 = H), neothramycin B (R = H, R2 = OH), methylneothramycin A (Rl = OCH3, R2 = H) and methylneothramycin B (Rl = H, R = OCH~).

~R2 Referring to the attached drawings:
Figure 1 shows a curve of the infrared ab-sorption spectrum of a sample of neothramycin A pel-leted in potassium bromide.
Figure 2 shows a curve of the infrared ab-sorption spectrum of a sample of neothramycin B p-el-leted in potassium bromide.
Figure 3 shows curves of the ultraviolet ab-sorption spectrum of a sample of neothramycin A dis-solved 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 dis-solved in 10~ water-methanol, in N/10 NaOH-methanol 9) and in N/10 HCl-methanol (1:9), respectively.
The neothramycin A and neothramycin B of this invention have low antibacterial and antifungal acti-vity as will be clear from the antibacterial spectra of these substances shown in Table 2 below. The minimum inhibitory concentrations (mcg./ml.) of neothramycin A
and B to various bacteria have been determined on nutrient agar plates which were incubated at a tempera-ture o~ 37 C. for 17 hours, The minimum inhibitory concentrations to various fungi have been determined on nutrient agar plates containing 1~ glucose after incu-bation at 27 C. ~or 40 hours.

Table 2 Minimum Inhibitory Concentration~
(Mc~./ml,) Test Qr~anis~s~ MC~16-A
Staphylococcus aureus Smith 50 100 Staphylococcus aureus 209P> 100 ~ 100 Klebsiella pneumoniae PCI 60250 100 Fscheric~ia coli NIHJ 100 100 Fscherichia coli K-12 100 100 Pseudomonas aeruginosa No. 12~ 100 ~ 100 Baclllus subtilis PCI 219 100 ~100 ~scherichia coli ~677 50 100 Fscherichia coli JR66/W677 100 ~ 100 Aeromonas salmonecida ATCC 1~174 25 5 Vibrio anguillarur.l NCBM 6 50 100 Saccharomyces cerevisiae 50 ~ 100 Candida albicans 31~7 > 100 > 100 Asper~illus niger 100 > 100 Piricularia oryzae 50 ~ 100 Xanthomonas citri > 100 ~ 100 Xanthomonas oryzae 50 100 ~L05674~

As stated hereinbefore, neothramycin A and B of this invention haye a high inhibitory activity to the growth of leukemia cells and are expected to be useful as an agent for treating therapeutically a living animal affected by leukemia.
Chemotherapeutic effects of the neothramycin A and B against leukemia L-1210 in mice were investigated in the following manner. Leukemia L-1210 cells (105 cells/mouse) were injected intraperitoneally in mice of CDF 1 strain weighing 19-22 g.
For the treatment of the leukemia so infected, administration of neothramycin A and B were commenced immediately after the tumor inoculation. The leukemic mice were used in groups each of four mice for each dose. When 300, 150, 75, 37.5 and 18.7 mcg./mouse/day of the neothramycin A and B were dosed by intraperitoneal injection once daily for 10 days, the highly favorable effects on the survival ratio (%) were observed as will be clear from the results shown in Table 3 below.

1056741!3 Average of Survival Rate (%) Dosage (mcg/mouse/day) Neothra~ycin A Neothra~ycin B

300 death death (toxic dose) (toxic dose) 37.5 154 128 18.7 122 103 The survival Ratio (~) is calculated by dividing the number of days of survival of the treated animals (e.g. 10) by the number of days of survival of the control animals (e.g. 8) and multiplying by 100, e.g. 10/8 x 100 = 125. Ratios greater than 125 are generally considered significant.
An effective prolongation in the survival rate (%) of mice inoculated with leukemia L-1210 was also observed by treatment with methylneothramycin A
or methylneothramycin B, as shown in Table 4 below.

Average of Survival Rate (%) Dosage Methylneothra-Methylneothra-(mcg/mouse/day) mycin A mycin B
200 Toxic Toxic ___ 115 12.5 ~~~ 103 ~, ,~ 3 ~056748 The neothramycin A ancl B of this invention are of a low toxicity to animal and man, as shown by the fact that the neothramycin A and B exhibit LD50 values of 20-30 mg./kg. and 20-~0 mg./kg., respectively, in mice, when a solution of 0.25-0.5~ by weight of neo-thramycin A or B in 10~ dimethylsul~oxide-water is ~n~ected lntraperitoneally in mice for the purpo~e of estlmating the acute toxicity of these sub-stances According to a second aspect of this invention, there is provided a process for the production of neothramycin A and neothramycin B, which com-prises cultivating a neothramycin-producing strain of the genus StrePtomvces under aerobic conditions in a suitable culture medium therefor contalning assimilable carbon and nitrogen sources for a period of time sufficient to produce and accumulate neothramycin A and neothramycin B in the culture mediumJ and recovering a mixture of the neothramycin A and neothramycin B from the culture, and subsequently, if required, separating the recovered mixture into the 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 Stre~tomvces may be used as long as this strain produces neothramycin. A suitable example of the strain IArhich may be employed in this lnvention for the production of neothramycin is the above-mentioned MC~16-C4 strain of Stre~tomvces.

This MC916-C4 straln was deposited on February 2, 1974 in a Japanese authorized depository "Fermentation Research Institute, Agency of Industrial Science and Technology", Inage, Chlba-Clty, Japan, under deposlt number FERM-P 2452. m ls r~C916-C4 strain wa 9 al~o deposited ln the American Type Culture Collection, Washlngton, D. C " U.S.A. under A.T.C.C. number 31123.
Cultural and taxonomic characteristics of the MC916-C4 strain are described below.
1. M~icrosco~ical mor~holo~y MC916-C4 strain has branched substrate mycelia from which aerial hyphae develops in the form of hook or open spirals. No whorl-branching is observed, Matured spore chains usually bear more than 10 conidal spores. Spores measure about 0.6-0,8 by 1.0-1.2 microns in slze and have a smooth surface.

2. Characteristics of the growth on variou~ culture media m e designation of colors in 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 [3 pc, amber] colored growth bears thln aerlal hyphae of light brownish gray to light gray color. Soluble pigment is faintly tinged with yellow.

10567~8 (2) On glucose-asparagine agar (incubated at 27 C.):
Dull yellow orange [3nc, Amber to 4pe, Orange Rust]
colored growth develops aerial hyphae of light gray to llght brownlsh gray color [2ihJ Dk covert Gray].
Soluble pigment is faintly tinged with yellow.

(3) On glycerol-asparagine agar [ISP No. 5 medlum, incubated at 27 C.): Dark yellow orange to yellowish brown [3pi, Golden Brown3 colored growth develops aerial hyphae o~ brownish gray [3ih, Belge Gray] to gray [5ih, Shadow Gray] color. Soluble pigment with yellowish tinge to yellowish brown tinge is produced.

(4) On inorganic salts-starch agar [ISP No. 4 medium, incubated at 27 C.): Pale yellowish brown to yellowish brown [3pi, Golden Brown] colored growth develops aerial hyphae of light brownish gray [3fe, Silver Gray] color. Soluble pigment is tinged with brown. The reverse side of the growth is dark yellowish brown in color.
(5~ On tyrosine agar (ISP No. 7 medium, incubated at 27 C.): Dark yellow to yellowish brown [4pg, Dark Luggage Tan] colored growth bears aerial hyphae of light brownish gray. Soluble pigment is tinged with dark yellow to yellowish brown.
(6) On nutrient agar (incubated at 27 C.):
The growth is colored pale yellowish brown to pale brown without developlng aerial hyphae. Soluble pigment is faintly tinged wlth brown.

10567'~8 (7) On yeast extract-malt extract agar (ISP No. 2 medium, incubated at 27 C.): Yellowlsh brown [4pg,, Dk Luggage Tan] to yellow orange [4pe, Orange Rust]
colored growth develops aerial hyphae of llght gray [2fe, Covert Gray to light brownish gray [21h, Dk Covert Gray]color. Soluble plgment of yellowish brown to brown color is produced, m e reverse slde of the growth is colored dark yellowish brown.
(8) On oatmeal agar (ISP No. 3 medlum, incubated at 27 C.): Reddish yellow to dark yellow orange [4pe, Orange Rust] colored growth with aerial hyphae of llght gray [5fe, Ashes] to brownish gray [31h, Beige Gray] color, Soluble pigment is tinged wlth yellow.
(9) On glycerol-nltrate agar (incubated at 27 C.):
Pale yellow to reddish yellow [3pc, Amber] colored growth bears slightly developed aerial hyphae of brownlsh white to light brownish gray color, Soluble pigment is faintly tinged with yellow.
(10) On starch agar (incubated at 27 C.): The growth is colored dull yellow to yellowish brown [2pi, Mustard Brown] without developing aerial hyphae or rarely with developing aerial hyphae of white.
Soluble pigment is falntly tinged with brown.
(ll) On calcium-malate agar (incubated at 27 C.):
The growth is colored pale yellow to pale olive with-out developing aerial hyphae or with slightly developing aerial hyphae of white. Soluble plgment is faintly tinged with yellow.

(12) On cellulo~e (lncubated at 27 C.): Colorless growth without aerlal hyphae. No soluble pigment i8 produced.
(13~ On gelatin stab: On plain gelatin medium ~incubated at 20 C.), the grow~h ls colorless to dull yellow colored without developlng aerlal hyphae, and with produclng soluble pigment o~ faintly yellow tinge.
On glucose-peptone-gelatin medium (incubated at 27 C.) J
the growth is pale yellow to dull yellow in color.
Aerial hyphae are not developed initially but ones o~
grayish white color are produced later, No production of soluble pigment is observed.
(14) On skimmed mllk (incubated at 37 C.):
The growth is colored pale yellow to pale orange without developing aerial hyphae. Soluble pigment is very ~aintly tinged wlth orange.
. PhYsiolo~ical ~ro~erties (1) Temperature ~or growth Growth on glucose asparagine agar was examined at 20 C., 24 C., 27 C., 30 C., 37C,, and 50 C. The MC916-C4 strain grew at ~11 temperatures tested, except at 50 C. Optimum temperature for good growth was observed to be in the vicinity of 30 C.
(2) Lique~action o~ gelatin Plain gelatin (15%) medium started to liquefy from the 5th day o~ incubation at 20 C. The degree of lique~action was medium. The gelatln (15%) in glucose-peptone-gelatin medium started to lique~y ~ro~ the 2nd day o~ incubation when incubated at 27 C., and the 105674~
grade of lique~action was then medlum to strong.
(3) Hydrolysis of starch Starch in inor~a~ic salts-starch-agar medium and in starch-agar medium was hydrolyzed starting from the 5th day of incubation when incubated at 27 C. The grade of hydrolysis was medium to strong.
(4) Coagulation and peptonization of skimmed mllk When incubated at 37 C., the coagulation of skimmed milk started at the 4th day of incubation and the peptonization was observed at the 5th day of incubation after the coagulation was complete. The grades of coagulation and peptonization were medium to strong.

(5) Formation of melanoid pigment No pigmentatlon was observed neither on trypton-yeast extract broth (ISP No. 1 medium)~ nor on peptone-yeast extract iron agar (ISP ~Jo. 6 medium), nor on tyroslne-agar (ISP No. 7 medium), when incubated at

(6) Utilization of carbon sources for growth Utilization of the following carbohydrates was tested in Pridham-Gottlieb agar medium (ISP No 9 medium) as incubated at 27 C.
Glucose and L-rhamnose ,rere utilized for growth.
L-Arabinose, D-fructoseJ sucrose, inositol and D-mannitol ~Jere not utilized. Utilization of ~-xylose was doubtful. Raffinose was sometimes utilized but lOS6748 not utilized other times.

(7) Llquefaction of calcium malate Calcium malate in calclum malate-agar medium was liquefied around the growth starting at the 9th day of incubation, when incubated at 27~ C, The grade of liquefaction was medium to strong,

(8) Reduction of nitrate Reduction of nltrate was observed in aqueous peptone solution containing 1,0% sodlum nltrate (ISP No. 8 mediu~), 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 Streptom~ces and that the aerial hyphae form open spirals but does not develop whorl, m e surface of spore is smooth under microscopic observation, On various media, the growth 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 hydrolysis are of medium to strong grade, On the basis of the above-mentioned properties, the MC916-C4 strain i~ compared to known analogous species of Streptom~ces with reference to descriptions of International Streptomyces Pro~ect (ISP), It ls found that the MC916-C4 strain resembles strePtomAyces naraensls (see "International Journal of Systematlc ~acteriology" Vol, 22, page 323 (1972). However, it is noted that the MC916-C4 straln is dlf~erent from strePtomvces naraensis ISP 5508 strain in respect to their utilization of carbon sources.
naraensis produces cycloheximide~ simllarly to the MC916-C4 strain. Furthermore, among cyclo-hexlmide-produclng strainsJ it is found that strains of Group C which are analogous to Stre~tom~ces ~riseolus as reported in an artlcle by T. Furumal et al. tltled "On cyclohexlmlde-producing micro-organismsl' [see the "Journal of Antlbiotics" Ser.
B,J Vol. 17, No. 4, page 181 (1964)] are very similar to the MC916-C4 strain.
The MC916-C4 straln ls well coincident wlth the above Group C stralns in ~any respects~ though the MC916-C4 strain has not been tested as to whether it has the properties o~ hemolysis, liquefactlon of serum and utlllzation of galactose and lactose which were shown by the Group C stralns. However, those Group C stralns are not avallable at present, as they are already dead. In this situatlon, comparison of the MC916-C4 straln ls now made with Stre~tomvces sp IF0 3300 which is known to produce fermicidln, an antlbiotlc analogous to cyclohexlmide, and whlch ls reported in the above artlcle b~ T. Furumai et al.
to be well coincldent wlth said Group C strains. The results of comparison are shown ln Table 5 below, with reference to the descrlptions of the "Journal of Antibiotlcs".

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10567~8 As will be seen from the above Table, the MC916-C4 strain is coincident with the Group C strain described 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 Bacteriology" Vol. 18, page 122 (1968)] which has been reported to resemble said IFO 3300 strain, in that S.
griseolus does not form open spirals in the aerial hyphae thereof and is somewhat different from the MC916-C4 strain in respect to the utilization of carbon sources. Further com-parisons 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 distinguished 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, utilization 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 its mutants. In other words, this invention includes the use of all strains of the genus Streptomyces which produce neothramycin.

105674~
Neothramycin can be obtained by aerobic cultivation of spores or mycelia of a neothramycin-producing strain of the genus Streptomyces such as Streptomyces 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 available 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 carbo-hydrates such as glucose, starch, glycerol, maltose, dextrin, saccharose, lactose, molasses and the like as well as fat or oil are useful as the carbon sources. 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.
For the production of neothramycin on a large scale, liquid cultivation is preferred. Any temperature at which the neothramycin-producing strain is able to grow and produce the 105674~3 neothramycin can be employed for the cultivation, but a preferred cultivation temperature is within a range of 25 to 35C. The cultivation is continued for a period of time sufficient to produceand accumulate a sufficient amount of neothramycin A and B in the culture medium. For instance, a culture medium comprising 2% glucose, 2% glycerol, 1.2% soybean meal, 1.0% cotton seed flour, 0.32% calcium carbonate, 0.5%
sodium chloride and 0.0005% manganese chloride tetrahydrate was prepared and sterilized at pH 6.8. This medium was then inoculated with spores or mycelia harvested from a slant culture of the MC916-C4 strain. ~Ihen it was shake-cultivated aerobically at 28C., the production and accumulation of neothramycin in the culture medium reached a maximum at the end of incubation for 3 to 5 days.
Assay of neothramycin can be made using staphylococcus aureus or Escherichia coli as the test organism according to a standard cup-plate method which has usually been employed for the assay of known antibiotics. A pure neothramycin A which was obtained from the Example 3, described later, of this invention mav be used as an authentic sample which exhibits a potency of 1000 units pèr mg. In case the other antibiotic substances such as cycloheximide are simultaneously produced in the culture broth of the MC916-C4 strain in addition to the neothramycin, the culture broth may be washed with ethyl acetate or other suitable organic solvent to remove such other antibiotic substances by extraction. The remaining aqueous phase may then be émployed for the assay of the contents of the neothramycin A
and B according to the aforesaid standard cup-plate method.
For the recovery of the neothramycin from the culture medium, the culture broth of the neothramycin-producing strain ~05674~

may either be treated with a suitable organic solvent such as n-butanol to provide an extract of the neothramycin in said solvent or may be treated with a suitable adsorbent such as active carbon to make the neothramycin adsorbed by the adsorbent. Distribution of the neothramycin A or neothramycin B between n-butanol and water was examined, and it is found that the partition coefficient of the neothramycin in n-butanol/water is greater than 5 at a pH value of 2 to 7.
Accordingly, the neothramycin can be extracted with n-butanol from the aqueous culture broth which has been adjusted to a pH value of 2 to 7 and preferably of about 6. The neothramycin is substantially insoluble in and hence is practically not extractable with ethyl acetate or chloroform from the liquid portion of the culture broth. If required, therefore, it is possible to treat the culture broth with ethyl acetate or chloroform for extraction in order to remove the soluble impurities from the culture broth. To separate the neothramycin from the culture broth it is preferred that the culture broth is treated with active carbon as the adsorbent. The neothramycin which has been adsorbed by active carbon can be eluted therefrom by means of a mixture of methanol and water, a mixture of propanol and water or a mixture of acetone and water, etc. The efficiency of the elution may be improved when the elution is done under weakly alkaline conditions. Purification of the neothramycin can be made using the above-mentioned extraction method and adsorption-elution method in a suitable combination of them or in a repeated manner. Further purification may be achieved by a usual column chromatography on Sephadex LH-20 (a commercial product sold by Pharmacia Co., Sweden) or silica gel. The known antibiotic cycloheximide which may frequently be co-existent in the culture broth of the ~C916-C4 strain can readily be separated from the neothramycin of this invention by extracting with ethyl acetate or by chromatography on Sephadex LH-20.
To isolate the neothramycin A from the neothramycin B, a mixture of the neothramycin A and neothramycin s may be subjected to a column chromatography on silica gel with chloroformmethanol (30:1 volume) as the developing solvent.
The isolated neothramycin A or the isolated neothramycin s can be purified by column chromatography on silica gel using suitable mixed organic solvents as the developing solvent.
The recovery of the neothramycin A and neothramycin B
may typically be carried out in the following way: The culture broth containing the neothramycin is at first filtered or centrifuged to remove the solid matters together with the mycelia. The broth filtrate is then treated with active carbon to adsorb the neothramycin therefrom. The active carbon carry-ing the adsorbed neothramycin is eluted with 50% acetone-water (a mixture of acetone and water at a ratio of 1:1 by volume) at pH 8Ø The eluate is collected in fractions and the active fractions are combined together and concentrated to dryness under reduced pressure at a temperature of up to 40C. or otherwise freeze-dried to give a crude powder. This crude powder is extracted with aqueous ethanol so that a greater part of the active components is separated in the resulting extract.
This extract is concentrated to dryness under reduced pressure at a temperature of up to 40C. or otherwise freeze-dried to give a second crude powder. A solution of this crude powder in methanol is passed through a column of Sephadex LH-20 (a trade mark of Pharmacia Co., Sweden) which is subsequently developed with methanol. During this chromatographic process, the possibly co-existing cYcloheximicle is eluted in such fractions running out in the first-half phase of the process, whereas the mixture of the neothramycin A and B is eluted in such fractions running out in the later-half phase of the process.
The active fractions containing the neothramycin A
and B are combined together and then concentrated to dryness under reduced pressure at a temperature of up to 40C., to afford a crude powder. This powder is taken into a small volume of methanol and the methanolic solution is uniformly admixed with an amount of neutral silica gel. The admixture was dried by evaporation and then placed on the top of a column of a further amount of said neutral silica gel which has been impregnated with a mixture of chloroform and ethanol (30:1 by volume). The silica gel column is subsequently developed with the chloroform-ethanol (30:1 by volume). During this chromato-graphy process, the neothramycin A is eluted in the active fractions running in the first-half phase of the process, while the neothramycin B is eluted in the active fractions running out in the later-half phase of the process. The active fractions containing the neothramycin A and the active fractions containing the neothramycin B are concentrated to dryness under reduced pressure at a temperature of up to 40C., respectively, to give a crude powder of the neothramycin A and a crude powder of the neothramycin B.
The crude powder of the neothramycin A so obtained is taken into an appropriate amount of chloroform and the solution is passed through a column of a neutral silica gel which has been impregnated with chloroform. This silica gel -10567~3 column is washed with chloroform and then developed at 5C.
with chloroform-ethanol (60;1 by volume). The eluate is collected in fractions, and the desired active fractions solely containing the neothramycin A are detected by referring to test results of biological assay and thin layer chromatography of each fraction. The desired active fractions so chosen are combined together and concentrated to dryness under reduced pressure at a temperature of up to 40C. to give the neothra-mycin A as a colorless powder. This powder may further be purified to a colorless powder of a pure neothramycin A by repeating the above-mentioned silica gel chromatographic process or by dissolving said powder into a small volume of chloroform, adding ethyl ether to the chloroform solution, filtering off and drying the resulting precipitate. A colorless powder of pure neothramycin B may be obtained from the aforesaid crude powder of the neothramycin B by purifying in the same manner as for the neothramycin A. It is preferred, however, that the column chromatography on silica gel is made using a mixture of chloroform and ethanol (100:1 by volume) as the developing solvent.
In view of the aforesaid properties of the neothramycin A and neothramycin B, it has been confirmed that these substances are new antibiotics which are differentiated from any of the known antibiotics. According to a third aspect of this invention, there is provided a method for therapeutically treating a living animal, including man, affected by leukemia, which comprises administering the neothramycin A and/or the neothramycin B to said animal in a dosage sufficient to reduce the affection by leukemia. According to a fourth aspect of this invention, there is further provided a pharmaceutical composition comprising the neothramycin A and/or the neothramycin s in an amount sufficient to reduce the affection by leukemia in vivo, the neothramycin A and/or the neothramycin B being in combination with a pharmaceutically acceptable carrier. It will be appreciated that the actual preferred amounts of the neothramycin used will vary according to the partlcular compound belng u~ed, the partlcular composltion formulated, the mode of application and the particular sltu~ and organlsm being treated.
Many factors that modi~y the action of the drug wlll be taken into account by the skllled in the art, for example, age, body weight, sex, diet, time o~
administration, route of adminlstratlon, rate Or excretlon, drug combinations~ reactlon ~ensitlvitles and severity of the disease. Optlmal applicati~n rates for a glven ~et o~ condltion~ can be ascertained by the skllled ln the art u~lng conventional dosage determinatlon test~ in vlew o~ the above guidelines.
It ls believed that using the precedlng descriptlon and wlthout further elaboratlonJ one skilled ln the art can utlllze the concept o~ this invention to its rullest extent. The following pre~erred specl~lc embodlment3 are, therefore, to be con~trued as merely lllustrative and not limitative o~ the re-mainder-of the dl~closure in any way.
Descrl~tion of the Prefejrred ~mbodime~t~

~056748 ~am~e 1, A loopful quantlty of stre~,to~m,Yces Bp. MC916-C4 (identified a~ A.T.C.C. 3112~) which was lncubated in slant agar medlum was lnoculated to a sterile liquid culture medlum (pH 7.0, 125 ml.) comprlslng 2.5% malto3e, 0.75% peptone, 0.75~ meat extract, 0.3% yea3t extract, 0.3% sodlum chlorlde and 0.1%
magne~lum sulfate (7H20). The lnoculated medlum was shake-cultured at 28 C. for 48 hour~ to glve a primary seed culture. This prlmary seed culture was inoculated at an inoculum size of 0.48% by volume to 50~ of a sterlllzed liquld culture medium (pH
7.0) containlng ~,5% ~tarch syrup, 0,75% peptone, 0.75% meat extract, 0.3% yeast extract, 0.3% 30dlum chlorlde and 0.1% magneslum sulfate (7H20) ln a stainless steel ~ermentor of a capaclty of 130R .
The lnoculated medlum was cultured at 28 C. for 24 hours under aeratlon and agltatlon to provlde a secondary seed culture. ~hls secondary seed culture wa~ lnoculated at an lnoculum size of 2% by volume ( 6l) to a llquld culture medlum (pH 6.8, 300l) comprlslng 2~ glucose, 2% glycerol, 1.2% soybean meal, 1.0% cotton seed powder, -32~
calclum carbonate, 0.5~ sodlum chlorlde and 0.0005%
mangane~e chlorlde (4H20) whlch had been sterlllzed at 120 C. for 30 minutes. The cultlvatlon was made at 28 C. for 92 hours under aeration and agitatlon (2~0 r.p.m.) whlle the rate of aeration was 150 Q¦ mlnute for the ~lrst 24 hours and then lncreased to 300 I/minute for the subsequent period of 24th hour to 92 hour of the cultlvatlon.
The resultlng culture broth (pH 7.3, 300~, potency 88 u./ml.) wa~ adm~xed with 24 kg. o~ a ~llter aid (dlatom2ceou~ earth commerclally available under a trade mark 'lHlfro-supercell~) and the admlxture Wa8 flltered by mean~ of a pre~-~llter to glve ~00~ o~ the broth flltrate. m e broth flltrate wa~ well admlxed with 3 Kg. of a¢tlve carbon at ambient temperature ~or 1 hour under agltation, ~o that the antibiotlcc were adsorbed on the carbon.
The active carbon portlon wa~ collected by centrl~ugatlon and then wa~hed with 150 1 o~
water. ~he wa~hed carbon was admlxed wlth 70l of 50% acetone-water (pH 8.0) for 1 hour under agltatlon, ~o that the antlbiotlc~ were extracted lnto the ~ol~ent Thl~ extractlon wa~ conducted twlce and the extract~ 80 obtalned were comblned together to a volume o~ 118¦. m e extract ~olutlon wa~ concentrated under reduced pressure at a temperature Or up to ~0 C. and the concentrated ~olutlon (2.6Q) wa~ freeze-drled to glve 565 g. o~
a brown colored powder (potency, ~5 u./mg.) whlch ¢ontalned the neothramycin A and B. Thl~
brown colored powder was extracted wlth 11.6~ o~ 80%
ethanol-water. The ln~oluble matter~ whlch had no antlbacterlal actlvlty were removed by ~lltratlon, 3~

B

105~;'748 to yield 11.2Q of an ethanolic extract. This extract was concentrated under reduced ~ressure at a temperature of up to 40C. to a volume of 800 ml., and the concentrated solution was freeze-dried to afford 218.5 g. of a crude powder (potency, 53 u./mg.). This crude powder was divided into five equal parts, and each part was dissolved in 20 ml.
of methanol. The methanolic solution was passed through a column (90 mm diameter) of 4Q of Sephadex LH-20, which was subsequently developed with methanol. The eluate was collected in 200 ml. fractions, and it was found that cyclo-heximide was eluted out in the fraction Nos. 9 to 11 while a mixture of the MC916-A and -B substances was eluted in the fraction Nos. 12 to 14. The fraction Nos. 12-14 were combined together and concentrated to dryness under reduced pressure at a temperature of up to 40C. to give 47 g. of a crude powder (potency, 160 u./mg.) comprising the mixed neothramycin A and B. Yield 28% (based on the neothramycin content of the broth3.

Example 2 The crude powder (33 g.3 comprising the mixed neothramycin A and B obtained in Example 1 was taken into a small volume of methanol, and the solution was uniformly mixed with 60 g. of a neutral silica gel, followed by drying under reZuced pressure. The dried mass so obtained was placed on the top of a column (60 mm diameter) of 660 g. of said neutral silica gel which had been impregnated with chloroform-ethanol (30:1 by volume). This silica gel column was developed at 5C. by passing a flow of chloroform-ethanol (30:1 by volume) through said column. The eluate was collected in 130 ml. fractions, and it was found that the neothramycin A was eluted in the fraction Nos. 23-31 while the neothramycin B was eluted in the fraction Nos. 35-49.
The combined active fraction Nos. 23-31 was concentrated to dryness under reduced pressure at a temperature of up to 40C. to give 1.47 g. of a yellowish crude powder of the neothramycin A (potency, 520 u./mg.). Yield 14%. The combined fraction Nos. 35-49 were concentrated to dryness in the same manner to give 1.03 g. of a yellowish crude powder of the neothramycin B ~potency, 450 u./mg.). Yield 9%.

Example 3 The yellowish crude powder of the neothramycin A (l g.) obtained in Example 2 was dissolved in 20 ml. of chloroform, and the solution was passed at 5C. through a column (13 mm diameter) of 20 g. of a neutral silica gel of the same grade as employed in Example 2 which had been impregnated with chloroform. The column was washed with 400 ml. of chloroform and subsequently developed with chloroform-ethanol (60:1 by volume). The eluate was collected in 8 ml. fractions, and it was found that the neothramycin A was eluted in the fraction Nos. 13-27. The combined fraction Nos. 13-27 was concentrated to dryness under reduced pressure at a temperature of up to 40C., giving 320 mg. of a faintly yellow colored powder. This powder was taken into a minimum volume of chloroform and to this solution was added ethyl ether until the precipitate formed was not longer deposited. The precipitate was removed by filtration and dried, affording 183 mg. of a colorless powder of pure neothramycin A (potency 1000 u/.mg.). Yield 35%.

1056741~
Example 4 The yellowish crude powder of the neothramycin B
(810 mg.) obtained in Example 2 was taken into 16 ml. of chloro-form and the solution was passed at 5C. through a column (13 mm diameter) of 16 g. of neutral silica gel of the same grade as employed in Example 2 which had been impregnated with chloroform. The column was washed with 320 ml. of chloroform and then developed with chloroform-ethanol (100:1 by volume). The eluate was collected in 6.4 ml. fractions, and it was found that the neothramycin B was eluted in the fraction Nos. 34-70. The combined fraction Nos. 34-70 was concentrated to dryness under reduced pressure at a temperature of up to 40C. to give 160 mg. of a faintly yellow colored powder. This powder was taken into a minimum volume of chloroform, and to 1~5~i748 this solution was added ethyl ether until the preci-pitate was no longer deposited. The precipitate was removed by filtration and dried, affording 85 mg. of a colorless powder of pure neothramycin B (potency, 620 u./mg.). Yield 14.6~.

Example 5 The colorless powder of neothramycin A (153 mg.) obtained in Example 3 was dissolved in a small volume of ethanol and rechromatographed on a column of a neutral silica gel (4.o7 g.) of the same grade as employed in Example 2 which was developed with water-saturated ethyl acetate at 5 C. The eluate was col-lected in o.6 ml. fractions. The fractions (Nos. 55-95 ) containing neothramycin A were combined and concen-trated to dryness under reduced pressure at a tempera-ture of up to 40 C., affording 53 mg. of a colorless powder of pure neothramycin A (potency 1000 u./mgO).

Yield 35%o Example 6 The colorless powder of neothramycin B (74 mg.) obtained in Example 4 was rechromatographed on a neutral silica gel (2.53 gO) which was developed with water-saturated ethyl acetate by the same manner as employed in Example 5. The eluate was collected in o,35 ml. fractions. The fractions (Nos. 75-157) con-taining neothramycin B were combined and concentrated to dryness under reduced pressure at a temperature of up to 40 C., affording 36 mgO of a colorless powder 3~

1056~48 of pure neothramycin B (potency 835 u./mg.). Yield 65%.

Example 7 A primary seed culture (each 0.5 ml.) of Streptomyces sp. MC916-C4, which was obtained by the similar manner as employed in Example 1 was inoculated to each 30 ml. of a sterilized liquid culture medium (pH 6.8) containing 2% glucose, 2% glycerol, 1.2% soy-bean meal~ 1.0% cottonseed powder, 0.32% calcium car-bonate, 0.5% sodium chloride and 0.0005% manganese chloride (4H20) in four Erlenmeyer flasks. The inocu-lated medium was cultured at 28 C. for 92 hours on a rotary-shaker (220 r.p.m.). me resulting culture broth (pH 6.5, 90 ml., potency 780 u./ml.) was extracted with 90 ml. of butanol under ice-cooling. The butanol-extract was concentrated to dryness under reduced pres-sure at a temperature of up to 40 C., affording 242 mg. of a brownish syrup containing neothramycins A and B (potency 100 u./mg.). Yield 44%.

Example 8 A yellowish crude powder (1.0 g., potency 756 u./mg.) containing neothramycins A and B, which was obtained by the similar way as described in Exam-ples 1 and 2 was dissolved in 20 ml. of methanol. The methanol solution was kept at 25 C. for 16 hours and concentrated to dryness under reduced pressure, afford-ing 1.0 g. of a mixture of methylneothramycins A and B.
The mixture was chromatographed on a column of silica gel (50 g., Wako-gel C-200, Wako Chemicals, Osaka) *Trade Mark ~a ., .

,,, which was developed with a mixture of benzene and methanol (20 :1 in volume). The eluate was cut into 12.5 ml. fractions. Fractions (Nos. 19-25) containing methylneothramycin A and fractions (Nos. 26~~8) con-taining a mixture of methylneothramycins A and B were obtained. The fractions Nos. 26-~8 were concentrated to dryness and the residue was rechromatographed on a column of silica gel (18 g.) by the same manner des-cribed above. Fractions containing methylneothramycin A and the above-mentioned fractions containing neo-thramycin A were combined and concentrated to dryness yeilding a colorless powder (299 mg.). The powder was crystallized with a mixture of acetone and benzene to yield 240 mg. of colorless crystals of methylneothra-mycin A. Fractions containing methylneothramycin B
were combined and concentrated to dryness, affording 175 mg. of a colorless powder of pure methylneothramy-cin B.

Example 9 ~ Crystalline methylneothramycin A (225 mg.) obtained in Example 8 was dissolved in 45 ml. of O.Ol NHCl-dioxane (1:1 in volume) and the solution was kept at room temperature (22 C. ) for one hour. The solution was adjusted to pH 6.o with 1 N NaOH and concentrated to dryness under reduced pressure, affording 216 mg. of a colorless powder containing neothramycins A and B.
The powder was chromatographed on a column of silica gel (20 g. ) by the similar manner as employed in Example 2. Pure neothramycin A (87 mg.) and neothramycin B (69 mg.) were obtained.

~1 Hydrolysis of methylneothramycin B (100 mg.) in 20 ml. of 0.01 NHCl-dioxane (1:1 volume) at room temperature for one hour by the same way as described above gave 95 mg.
of a colorless powder containing neothramycins A and B.
The Sephadex LH-20 used in the preceding examples can be replaced by other similar gel-filtration agents. e.g.

* * * *
Sephadex G25 to G200 , Sepharose 4B and 6B (Pharmacia Fine Chemicals AB, Uppsala, Sweden) and Bio-Gel Al.5m (Bio Rad Co.).
Preferred gel-filtration agents include the carboxymethyl substltuted cross-linked dextran gels described in columns 3 and 4 of U.S. patent 3,819,836.
The pharmaceutically acceptable salts of the substances of the present invention include nontoxic metallic salts such as sodium, potassium, calcium and aluminum, the ammonium salt and substituted ammonium salts, e.g. salts of such nontoxic amines as trialkylamines including triethylamine, procaine, dibenzylamine, N-benzyl-beta-phenethylamine, l-ephenamine, N,N'-dibenzylethylenediamine, dehydroabietylamine, N,N'-bis-dehydroabietylethylenediamine, N-(lower)alkyl-piperidine, e.g. N-ethylpiperidine, and other amines which have been used to form salts with benzylpenicillin.

*Trade Marks _ 42 -

Claims (10)

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

1. A process for producing the antibiotic methylneothra-mycin, which comprises reacting neothramycin with anhydrous methanol.

2. A process as in Claim 1, wherein the reaction is carried out at room temperature.

3. A process as in Claim 1, which includes the further step of concentrating methylneothramycin to a dry powder.

4. A process as in Claim 1, which comprises the further step of forming a pharmaceutically acceptable salt of methylneothramycin.

5. A process as in Claim 1 for the preparation of methyl-neothramycin A, which comprises reacting neothramycin with anhydrous methanol, concentrating the resulting methylneothra-mycin, and fractionating the concentrated methylneothramycin chromatographically.

6. A process as in Claim 1 for the preparation of methylneothramycin B, which comprises reacting neothramycin with anhydrous methanol, concentrating the resulting methylneo-thramycin, and fractionating the concentrated methylenothramycin chromatographically.

7. A process as in Claim 1 for producing the antibiotic methylneothramycin, 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 nutirent medium, recovering neothramycin from the culture medium, and reacting the resulting neothramycin with anhydrous methanol.

8. The antibiotic methylneothramycin, whenever prepared by the process of Claim 1, 2 or 7, or an obvious chemical equivalent thereof.

9. The antibiotic methylneothramycin A, whenever prepared by the process of Claim 5, or an obvious chemical equivalent thereof.

10. The antibiotic methylneothramycin B, whenever prepared by the process of Claim 6, or an obvious chemical equivalent thereof.