Note: Descriptions are shown in the official language in which they were submitted.
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)
1056748
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
1056748
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
1OS6 7 4 8
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.
1056'^~48
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S~ N (:~ N O ~ OC) ~ ~ ~
'd O I N ~ ~ L~ ~ ~ ~ O ::;
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c~ I ~ ~ ~ C~
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q~ ~0
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f~ S-~ N O o~ o G~ ~ ~ ~o
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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
--10--
1056748
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.
- 12 -
1056741!3
TABIE 3
Average of Survival Rate (%)
Dosage
(mcg/mouse/day) Neothra~ycin A Neothra~ycin B
300 death death
(toxic dose) (toxic dose)
150 200 192
167 154
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.
TABLE 4
Average of Survival Rate (%)
Dosage Methylneothra-Methylneothra-
(mcg/mouse/day) mycin A mycin B
200 Toxic Toxic
100 128 154
5 103 147
___ 115
12.5 ~~~ 103
-13-
~,
,~ 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.
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1056748
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.
-16-
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.
-17-
1056748
(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
-18-
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
27 C
(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
-19-
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
-20-
1056748
~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".
1056748
3* P,
In $~ C~
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h h ~ rl ~ C
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L~ ~ ~ O~ 3 S~ O !~ h
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--22--
1056748
~*0 .o, ~, 0
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-23-
11~56~9t8
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--24--
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.
- 25 -
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
- 26 -
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
1056748
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
- 29 -
1056748
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
- 30 -
-
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
- 31 -
1056748
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
1056748
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~
33
~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
1056748
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
1056748
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%.
- 37 -
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
- 38 -
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)
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~a
., .
,,,
1056748
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
1056748
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.
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