Note: Descriptions are shown in the official language in which they were submitted.
~o~
This invention relates to Antibiotic C-15003, which
is a novel antibiotic, a method of producing the same and
a method of producing derivatives from said antibiotic,
We collected many soil and other samples and
performed a screening of the microorganisms isolated
from such samples. It was found, by that screening,
that certain of the microorganisms were able to produce
a novel antlbiotic, that such microorganisms belonged
to the genus Nocardia and that by cultivating any of
those microorganisms in a suitable medium, it was
possible to have said antibiotic accumulated in the
cultured broth. It was also found that derivatives
could be obtained from said antib~otic. ~urther studies
ensued, resulting i~ ~e d~elopment of this invention,
This invention is ther~fore directed to:
(1) Antibiotic C-l5003 which ha~ the general formula
(I):
C~ CH7 0 0 0
CH ~ N ~ " ~ ~ CEI3
O (I)
N ~ O
CH3 OCH3
CH
~wherein R represents -CO-CH < 3, -CO-CH2-CH2-CH3 or
CH7 3
-CO-CH2-CH < ~ );
~, ~
Vl13
(2) A method of producing Antibiotic C-15003
-- 1 - ~.,~
12
which comprises cultivating in a suitable culture medium containing assimilable
carbon sources and digestible nitrogen sources, a microorganism belonging to
the genus Nocardia and capable of producing Antibiotic C-15003 until Antibiotic
C-15003 is substantially accumulated therein, and recovering Antibiotic C-15003.
Our copending Application Serial No. 373,582, which is divided out of
this application, relates to a method of producing a compound of the formula
(II)
CH3 O CH3
~II)
N
CH3 OCH3
characterized in that said method comprises hydrolyzing Antibiotic C-15003 of
the general formula (I): R
3 ~CH 5 ~ 1 )
H ~
C~13 OCH3
(wherein R represents -CO-CH ~ 3, -CO-CH2-CH2-CH3 or -CO-CH2-CH\ ).
In the context of this invention, the term "Antibiotic C-15003" means,
generically, the three compounds having the above general formula (I) as a
group,
C - 2 -
.
~)72lZ
or a mixture of two or three of said compounds or,
severally, any of the same compounds. Referring, also,
to the general formula (I), the compound irl which R is
f CH
-C0-CH 3 is referred to herein as "Antibiotic C-15003
~ CH3
P-3" or more briefly as "C-15003 P-3"; the compound in
which R is -C0-CH2-CH2-CH3 is referred to herein as
"Antibiotic C-15003 P-~"' or,more briefly~ as "C-15003
P-3 "';the compound in which R is ~ CH~
-C0-CH -CH
2 ~ CH
is referred to herein as "Antibiotic C-15003 P-4" or,
more briefly, as "C-15003 P-4"; and the compound in which
R is H(general formula (II)) is referred to herein as
"Antibiotic C-15003 P-0" or, more briePly, as "C-15003
P-O".
As an example of the Antibiotic C-15003-producing
strain of microorganism, there may be mentioned an
actinomycete Strain No.C-15003 which we isolated from
soil and other samples in our screening for antibiotic-
producing microorganism~s,
~ he microbiological characters of Strain No.C-15003
were investigated by procedures ahal-ogous ~o those
proposed by Schirling & Gottlieb ~Inter~ational Journal
of Systematic Bacteriology 16, 313-340 (~.966~ he
results of observations at 28C over 21 days are as
follows .
1) Morphological characters
~ he vegetative mycelium extends well and develops
-
11~)72~Z
into branches, both on agar and in liquid medium. Many
of the hyphae measure 0~8 to 1.2 ~m in diameter and, in
certain in~tances, may divide into fragments resemb~ing
rod bacteria or branched short lengths of hyphae, The
strain gives good growth on various taxonomical media,
with aerial mycelium being superimposed on the vegetative
mycelium, although it frequently forms coremia like
bodies (50-200 x 200 - 1000 ~m) on which further aerial
growth takes place, Many of the aerial mycelia are
flexuous,straight or a loo~ely spiral like configuration
being encountered on a few occasions, Microscopic
examination of aged cultures reveals that on~y in few
cases the conidia like cells occur in chains, while the
cell suspensions obtained from the surfaces of such
cultures, as microscopically examined, contained many
elongated ellipsoidal (0,8-1.2 ~m x 4.8-6.8 ~m) and
ellipsoidal (0.8-1,2 x 1.0-2.0 ~m) bodies resembling
arthrospores,
Electron-microscopic examination3 showed that these
bodies had smooth surfaces,
2) ~he constituents of cells
~ he strain was shake-cultured in modified ISP No.l
medium at 28bC for 66 to 90 hours, at the end of which
time the cells were collected and rinsed, By the method
of B.Becker et al.~Applied Microbiology 12, 421 (1964~)
and the method of M,P, ~echevalier.
(Journal of Laboratory and Clinical Medicine 71, 934
(1968)), the above whole cells were examined for diamino-
pimelic acid and sugar composition. The former was
- 4 -
11`~ Z
found to be the meso-form, while spots were detected
which corresponded to galactose and arabinose.
3) Characteristics on taxonomical media
~ he strain showed comparatively good growth on
various media, with the vegetative mycelium being
colorless to pale yellow in initial phases of culture and
light yellowlsh tan to yellowish tan in later phases.
The strain produces soluble pigments, yellow to yellowish
tan, in various taxonomical media. ~he aerial mycelium
is powdery and generally gives moderate growth, being
white to yellow or light yellowish tan. ~he characteristics
of the strain in various taxonomical media are set forth
in Table 1.
; ~able 1 Cultural characteristics of Strain No.C-15003
on taxonomical media
(A) Sucrose nitrate agar:
Growth (G): Modera-te, B~ite Melon Yellow (3 ia)~
to Amber tan (3 lc)~, coremia like bodies
formed
Aerial mycelium (AM): Scant, white
Soluble pigment (SP): None or pale yellowish tan
(B) Glycerol nitrate agar:
G: Moderate, ~t Ivor~ (2 ca)*, coremia like bodies
formed
AM: Moderate, white
SP: None
(C) Glucose asparagine agar:
G: Moderate, Brite Marigold (3 pa)* to Brite Yellow
(2 pa)*.
- 5 -
li`'~7~1Z
AM: ~cant, white
SP: Brite Yellow (2 pa)~
(D) Glycerol asparagine agar:
G: Moderate, Lt Ivory (2 ca)~ coremia like bodies
formed
AM: Scant, white
SP: None
(E) Starch agar:
: G: Mode~ate, Lt Ivory (2 ca~ to Lt Wheat (2 ea)*,
coremia like bodies formed
AM: Abundant, Lt Ivory (2 ca)*
~P: None
(~) Nutrient agar:
G: Modexate, ~t Ivory (2 ca)~ to Colonial Yellow
(2 ga)~, coremia like kodies formed
AM: sGant 1 white
SP: None
(G) C~lcium malate agar:
G: Moderate ~t Ivory (2 ca)* to Lt Wheat (2 ea)*,
coremia like bodies formed,
AM: Moderate, white to Lt Ivory (2 ca)~
SP: None
(H) Yeast extract-malt extract agar:
G: Moderate, Amber (3 lc)~ to Brite Yellow (3 laj~
coremia like bodies formed
AM: Moderate, white to ~t Ivory (2 ca)*
SP: None
(I) Oatmeal agar:
:
~ - 6 -
~1~372~;~
G: Moderate, ~t Ivory (2 ca)~ to Goloni~l Yellow
(2 ga)~, coremia like bodies fo~med
AM: Scant, white to light yellow
SP: None
(J) Peptone yeast extract iron agar:
G: Moderate, Colonial Yellow (2 ga)~
AM: None
SP: Colonial Yellow (2 ga)*
(K~ ~yrosine agar
G: Moderate, ~t Ivory (2 ca)~ to It Melon Yellow
(3 ea)~, coremia like bodies formed.
AM: Moderate, white to Lt Ivory (2 ca)~.
SP: Camel (3 ie)~
~he color codes according to Color Xarmony Manual,
4th ed. (Container Corpo~ation of America, 1958).
4) Physiological characters
~ he physiological cha~ac~ers of the strain are
shown in ~able 2 ~emperature range for growth: 12C
to 38C. ~he temperatur~ range in which good aerial
growth occurs on agar (ISP No. 2) is 20 to 35C.
Table 2 The physiological characters of Strain ~o.
C-15003.
~emperature range for growth: 12 to 38C.
Temperature range for aerial growth: 20 to 35C
~iquefaction of gelatin: Positive
Hydrolysis of starch: Positive
Reduction of nitrates: Positive
Peptonization of milk: Positive
ZlZ
Coagulation of milk: Negative
Decomposition of casein: Positive
Production of melanoid pigments:
Ne~ative (peptone yeast extract iron agar),
positive (tyrosine agar)
Decomposition of tyrosine: Positive
Decomposition of xanthine~ Negative
Decomposition of hypoxanthine: Negative
~olerance to lysozyme: Positive
Tolerance to sodium chloride: 2 %
5) Utilization of various carbon sources
~ he utilization of various carbon sources was
investigated using a medium described in Pridham and
Gottlieb (Journal of Bacteriology 56, 107 (1948)) and
a basal medium of the same composition plus 0.1 % of
yeast extract, ~he resultant spectrum is shown in ~able
3.
~able 3 ~he utilizat~o~ of carbon sources b~ Strain
No,C-15003
Source of carbon Growth Sources of carbon Growth
~-~ylose + ~+~ Raffinose + +*
L-Arabinose + + Melibiose + +
D-Glucose +~ ++ i-Inositol
D-Galactose + + D-Sorbitol - -
D-~ructose +++ ~+ D-~annitol +* +~
L~Rha~nose + + Glycerol - +
D-Mannose +++ +~ Soluble starch ~ +
Sucrose ++ ++ Control
: ,,
ii~7~i2
Lactose - -*
Maltose + +
Trehalose -~ ~+
~ Basal medium with 0.1 % yeast extract added
Note: +++: ~uxuria~t growth
+*: Good growth
Growth
~ Poor growth
- ~o growth
6) Other characteristics
~ he cells were harvested by the procedure previously
described in 2) and DN~ was prepared by a procedure
analogous to that of J, M~rmur et al.~Journal of
Molecular Biology 3~ 208, 1961), ~he G-C (Guanine-
Cytosine)content o~ ~e ~NA was found to be about 71
mole /0. .,
Gram-staining of the ~egetative mycelium of this
strain was positive,
~ h~ above characteristics of:Strain No.C-15Q03 were
compared with the descriptions in S,A, Waksman's "The
Actinomycetes Vol,2"~he Williams and Wilkins Co " 1961);
R,E, Buchanan and N,E, Gibbons,"Bergey's M~nual o~
Determinative Bacteriology~ 8th ed, 197~'; and other
literatures.
~ hilst this strain was thought to belong to Group
III o~ the genus Nocardia, the failure to ~ind any
species having the characters so far described among
the known strains led us to conclude that this strain
represented a novel species of microorganism.
The present Strain No C-15003 has been deposited
at Fermentation Research Institute, Agency of Industrial
Science and ~echnology (F~RM) under the receipt number
of 3992; at The Ins~itute ~or ~ermentation, Osaka (I~O)
under the accession number of IFO 1~726 and at ~he
American ~ype Culture Collection (A~CC)~ Maryland, U ~.A.
under the accession number of 31281.
While Strain No.C-15003 is a novel species of the
genus Nocardia as just mentioned, it is liable, as are
microorganisms generally, to u~dergo variations and
mutations, either spontaneously or under the influence
of a mutagen. For e~ample, the many variants of the
strain which are obtainable by irradiation with X-rays,
gamma rays, ultraviolet light, etc., by monocell
isolation, by culture on media containing various chemicals,
or by any other mutagenic treatment, as well as the
mutants spontaneously deri~ed fro~ the strai~, should
not be considered to represent- ang other distinct
species but, rather, any of such ~a~ants ~nd mutants
capable of elaborating C-15003 P-3, P-3~` and/or P-4 may
be invariably utilized for the purposes o~ this ~nvention.
By way of example, subjecting Strain No~C-15003 to
various mutagenic treatments yields mutants substantially
lacking the ability to produce soluble pigments, mutant~
with substrate mycelia which are colorless, ~ellowish
green, reddish tan or orange-red, mutants whose hyphae
are ready to fragment into baciIlary elements or branched
short hyphal fragments~ and mutants with abundant white
-- 10 --
- - .
l~`U72~Z
aerial mycelia or substantially without aerial mycelia
The medium employed for the cultivation of such
an Antibiotic C-15003-producing strain may ~e whichever
of a liquid and a solid medium only if it contains
nutrients which the strain may utilize, although a liquid
medium is preferred for high-production run~. ~he
medium may comprise carbon and nitrogen sources which
Strain No C-15~03 may assimilate and digest, inorganic
matter, trace nutrients, etc As examples of said
carbon sources may be mentioned glucose, lactose,
sucrose, maltose, dextrin, starch, glycerol, mannitol,
sorbitol, etc , fats and oils (e.g. soybean oil, lard
oil, chicken oil, etc ) and so forth. The nitrogen
sources may for example be meat extract, yeast extract,
dried yeast, soybean mealt corn steep liquor, pepto~e,
cottonseed flour, spent molasses, urea, ammonium salts
(e g ammonium sulfate, ammonium chloride, ammonium
nitrate, ammonium acetate, etc.) and 50 forth. The
medium may further contain salts of sodium, potassium,
calcium, magnesium, etc, salts of iroIl, manganese, zinc,
cobalt, nickel, etc,, salts of phosphoric acid, boric
acid, etc., and organic acid salts such as acetates and
propionates. ~urther, the medium may contain, as added,
various amino acids (e,g, glutamic acid, aspartic acid,
alanine, glycine, lysine, methionine, proline, etc.),
peptides (e.g. dipeptides9 tripeptides, etc,), vitamins
(e.g Bl, B2, nicotinic acid, B12, C, E, etc,), nucleic
acids, (e.g purine, pyrimidine and derivatives thereof)
1Z
and so forth. For the purpose of adjusting the pH of
the mediwn, there may be added an inorganic or organic
acid, alkali~ buffer or the like. Suitable amounts of
oils, fats, surfactants, atc. may also be added as
antiioams.
The cultivation may be conducted by any of the
stationary, shake, submerged aerobic and other cultural
conditions, ~or high production I~ns, submerged aerobic
culture is of course preferred, While the conditions
of culture, of course, depends upon the condition and
composition of medium, the strain, cultural method and
other factors, it is normally preferred to carry out
incubation at 20 to 35C with an initial pH of about 7.0
or thereabouts. Particularly desirable is a temperature
from 23 to 30C in an intermediate stage of cultivation,
with an initial pH of 6.5 ~o 7,5. While the incubation
time also is variable accor~lng to the same factors as
mentioned above, it is advisable to con~inue the incubation
~ntil the titer of the desired antibiotic product
becomes maximal, In the case of shake culture or aerobic
submerged culture in liquid medium, the time required
normally ranges from about 48 to 144 hours,
~ he potency of the antibiotic was assayed with
Tetrah2~ eyriformis W as an assay organism, Thus,
the above microorganism was grown on a test medium C20 g
Of Proteose-PePtone (Di~co), 1 g of yeast extract (Difco*) 9
*Trademark
w 12 -
J7:~Z
2 g of glucose, 1000 m~ of distilled water and 10 m~ of
1 M-phosphate buffer (pH 7,0)) at 28C for 44 to 48
hours and the potency of the antibiotic was determine~
by the serial dilution method with a mo~itering of the
turbidity of growth, effect on ascites tumor cells and
by a thin-layer chromatographic (briefly T~C) assay to
be described hereinafter
The novel Antibiotic C-15003 P-3, P-3' and/or P /l
is produced and accumulated in the resultant fermentation
broth, both extracellularly and intracellularly,
These substances have also been detected by ~LC.
~hus, the fermentation broth is separated into cells and
filtrate by filtration or centrifugation and the filtrate
is extracted with the same volume of ethyl acetate. ~o
the cells is added the same amount of 70 % acetone-
water as the filtrate and, after an hour's stirring at
20C, the suspension is filtered, ~he acetone is removed
from the filtrate and the resultant aqueous filtrate is
extracted with ethyl acetate. ~ach of the extracts is
concentrated to 1/100 by volume ~nd subjected to thin-
layer chromatography on a silica gel~ s plate (Merck,
West Germany, Kieselgel*60 ~ 254,0,25 mm, 20 x 20)
(solvent system : chloroform-methanol = 9:1)~ ~he
potency was determined on the basis of the intensity of
SpotQ detected by irradiation with ultraviolet light at
2537 A.
Because C-15003 P-3, P-3' and/or P-4, which are
thus produced in the fermentation broth, are lipophyl
*Trademark
-- 13 -
11`~7ZlZ
neutral substances, they can be conveniently recovered
by separation and purification procedure~ which are
normally employed for the harvest of such microbial
metabolites. For example, there may be employed a
procedure ~hich utili~es the difference in solubility
between the antibiotic and impurity, means which
utilizes the adsorptive affinity of various adsorbents
such as activated carbon~ macroporous nonionic resins,
silica gel, alumina, etc.~ a procedure of removing the
impurities by means of ion exchange resins9 and so
forth, as applied singly or in a suitable combination or
as applied in repetition.
Since, as aforesaid, C-15~)03 P-3, P-3' and P-4
occur in both the filtrate and ~ells, the antibiotics
are separated and purified by means of such an adsorbent,
i$ one is employed, either directly or after a solvent
extraction in the case of the filtrate, or after a
solvent extraction in the case of microbial cells, The
solvent extraction ma~ be performed by an~ of the
following and other methods e g. (1) solvent extraction
from the culture broth pri~ to separation of cells and
(2) solvent extraction of the cells and the filtrate
obtained by filtration, centrifugation or other process.
To extract the filtrate and celIs independently, the
following procedure may be taken advan~ageously.
~ he solvents suitable for extraction of the filtrate
are water-immiscible organic solvents such as fatty acid
esters, e.g. ethyl acetate and amyl acetate; alcohols,
- 14 -
e g butanol; halogena-ted hydrocarbons, e.g. chloroform;
and ketones, e g methyl isobutyl ketone. ~he extraction
is carried out at a pH near neutral and~ preferably, the
culture fluid previou~ly adjusted to pH 7 is extracted
with ethyl acetate ~he extract is washed with water and
concentrated under reduced pressure Then, a nonpolar
solvent such as petro].eum ether or hexane is added to
the concentrate and the crude product I containing the
active compound is recovered Because, on ~LC, a number
of spots are detected in addition to Antibiotic C-15003,
the product I is seque~tially subjected to the following
purification procedures. ~hus, as a routine purification
procedure, adsorption chromatography is useful and, for
this purpose, one of those common adsorbents æuch as
silica gel, alumina, macroporous nonionic adsorbent
resin, etc. may be employed. ~or purification from the
crude product I, silica gel is most useful, And development
may be carried out, for example starting with petroleum
ether and hexane and elutio~ of Antibiotic C-15003 is
perfo~med by the additio~ of a p~lar solvent such as
ethyl acetate, acetone, ethanol or methanol, In a
typical process, using silica gel (Merck7 West Germany,
0.05-0.2 mm~ as a carrier, column chromatography is
carried out with a serial increase in the hexane to
ethyl acetate ratio, ~he eluate is sampled and investi-
gated by ~LC and the ~ractions containing C-15003 are
pooled and concentrated under reduced preæsure. ~hen,
petroleum ether or hexane is added to the concentrate,
- 15 -
1~`07Z~LZ
whereby the crude product II is obtained. Since this
product still con-tains impurities, it is further purified
as follows. For example, the product II may be purified
by means o~ a second silica gel column using a different
solvent system. ~he developing system for this purpose
may consist in a halogenated hydrocarbon such as
dichloromethane or chloro~orm, with the addition of a
polar solvent such as an alcohol, e ~. methanol or
ethanol, a ketone, e.g, acetone or methyl ethyl ketone,
or the like In this ~ay, Antibiotic C-15003 is isolated.
~he order o~ solven-t systems for the first and second
silica gel columns may be reversed and, in addition,
ordinary organic solvents may be used in conjunction
with the above systems if necessary.
Where a macroporous adsorbent resin is used as
purification means for crude product II, elution of
Antibiotic C-1500~ i6 accomplished with a mixture of
water with a lower alcohol, a lower ketone or ~n ester,
~he lower alcohol may for example be methanol, ethanol,
propanol or butanol and the lower ketone may for example
be acetone or methyl ethyl ketone ~he ester may for
example be ethyl acetate In a typical procedure, the
crude product II is dissolved in 60 /0 methanol-water and
adsorbed on a column of Diaion HP-10 ~Mitsubishi Kasei
K.E.). The column is washed with 70 % methanol-water
and, then, elution is carried out with 90 % methanol-
water. In this way, Antibiotic C-15003 is eluted from
the column
*Trademark
-- 16 --
:
1Z
In either of the pro~esses described above, the
fractions containing Antibiotic C-15003 are pooled and
concentrated under reduced pressure. ~o the dry product
is added 5 to 8 volumes of ethyl acetate and the mixture
is allowed to stand, whereupon crystals of Antibiotic
C-15003 separate. These crystals contain C-15003 P-3,
P-3' and P-4 These compounds are then separated from
each other by means of an adsorbent such as those mentioned
hereinbefore. Thus, using silica gel or a macroporous
nonionic adsorbent resin and the above solvents, the
desired compounds may be fractionally eluted. When, for
example, silica gel is employed, development is carried
out with hexane, ethyl acetate, or chloroform-methanol~
whereby C-15003 P-4, P-3' and P-3 emerge in that orderO
After detection by ~C~ the fractions corresponding to
C-15003 P-4, P-3' and P-3 are respectively concentrated
under reduced pressure and ethyl acetate is added to the
concentrates. In this manner, the respective compounds
can be obtained as crystals. When a macroporous nonionic
adsorbent resin is employed, gradient elution with a
varying ratio of alcohol, ketone or ester to water may
be utilized. For ~xample, by the gradient elution method
involving the use of 60 % methanol-~water and 95 %
methanol-water, with 5 /0 sodium chloride added, C-15003
P-3, P-3' and P-4 merge in the order mentioned. After
sampling and detection by ~LC, each group of active
fractions is concentrated under reduced pressure and
crystallized from ethyl acetate. ~he isolated cry~tals
- 17 -
include ethyl acetate as a solvent of crystallization
and, after drying over phosphorus pentoxide at 70C for
8 hours, show the following physical and chemical
properties, (Table 4).
- 18 -
11~7212
_ ~ ~ ~ ' ~ ~ r
~ o(~ a:) O 01 ~9 o (~ O Cr~ ~ J,
1~ r~ o u~ o c~
01^ __ ~ .__ ... _
O ~ o P~ .
O 10~ ~ O O ~ ~ 0.
U ~ ~N ol o o, C' ~ u~ O ~D ~ LJ~
~1 'o ~; a:) +I v
~ ~ __ ~ ._ ._
~D, o ~I~ .
~0~ ~ o C~ ~ ~ .
~1 o~ ~ ~
0,~ O ~ ~ o~
C~ l C~ ~ 5zi u c~ i v
._ O ~ _ .._ .._
3 ~ ~o
. ~ ~ ~ ~ ~ V
~ ~ ....
- -- 19 -
~lV~12
. ~O O ~ ~ R ~ P
.d ~ ô ô ô o a~ P h o ~ o o rl
O O ~ N~ ~1 ~ ~ h ~ h 4~ ~ u~
~ 1~ ~ ~ ~ ~ ~ o ~
P-l O O A ~ S o 3; ~ 3 3, 3
ô .. -. ~ ^
~D N O O u~ O .cl si ~
V ~V ~, ~ ~ ~ 9 ~ ~ U
I ~ I I~ t
~ ~ q) h ~ ~:1
~ ~ O ~
h o ~ h o ~ ~ o ~1 o~ ~ o
. ~ ~ ~ ~ , ~o~p,o . .. ..... _ ~
-- 20 --
'
~, :
,!
``` li~72~Z
Based on the above molecular formula shown above and the
antimicrobial and antitumor activity data given hereinafter,
the present antibi.otic was compared with the known groups
of antibiotics. ~he literature search failed to locate
any distinct group similar to Antibiotic C-15003. However,
a search for substances that might give ultraviolet
absorptions similar to those of the present antibiotic among
component of plant and other naturally-occurring organic
compounds led us to the maytanacine groups and, based on
the molecular ~ormulas involved, in particular, it was
assumed that the antibiotic belongs to the maytanacine
group of compounds containing two nitrogen atoms,
Maytanacine ~as obtained as a component of plant
and was reported in Journal of
American Chemical Society 97, 5294(1975). ~he mass spectrum
of mayta~acine is as follows.
M+-(a) M~-(a+b) 485-CH3 485-C~
545 485 470 ~50
(a)=H20 + ~CO O
(b)=R-C-OH
~he presence of m/e 485, 470 and 450 for C-15003 P-3, P-3'
and P-4 convinces us at once that the~e compounds have a
skeletal structure identical with that of maytanacine,
differentiating them from maytanacine in the kind of acyl
group in ~-position. It is thus clear that Antibiotic
C-15003 is a novel compound, When C-15003 P-3, P-3' and
P~ were each degradated with alkali and analyzed by gas
chromatography for the liberated carboxylic acids, it was
-- 21 --
1~721Z
found that isobutyric acid, butyric acid a~d isovaleric
acid were obtainable from C-15003 P-3, C-15003 P-3' and
C-15003 P-4,resp~ctively. Fig.l shows the structures,
based on the above data, of C-15003 P-3, P-3' and P-4.
Fig.l ~ R
, CH~
C~ C~ o o 0 P-3 -C0-CH <
3 ~ ~ ~ CH3
3 ~ P-3' -CO-CH2-~H2-CH3
0 H P-4 -CO-CH2-CH <
CH3 OCH3
Biolog;~cal activity:
A) Antimicrobial acti~ity:
With trypticase~soy agar (BBL) as an assa~ medium,
the inhibitory concentr~tions against the microorganisms
named below were investigated by the paper disc method.
Thus, filter-paper disGs (Toyo Seisakusho, thin-type, 8 mm
dia.) each impre~nated wibh Q,02 m~ o~ a 300 ~g/ml solution
o~ C-15003 P-3, P-31 or P-4 were placed on plates respec-
tively inoculated with the microorgani~s named below to
investigate the minimal inhibitory co~ae~trations, ~he
results showed that the a~tibiotics had no ~ctivity against
the following microorganisms.
so ~ coli, Proteus ~ul~aris, ~ mirabilis,
Pseudomonas aeru~inosa, Staph~lococcus aureus, Bacillus
subtills, Bacillus cereus, Klebsiella ~ a~,
Serratia marc _cens, M~cobacterium avium
.
- 22 -
.: - .
~lV~lZ
On the other hand, with agar plates containing the
assay medium ~3.5 g disodium hydrogen phosphate, 0 5 g
monopotassium dihydrogen phosphate, 5 g yeast extract
(Difco~, 10 g glucose, 15 g agar, 1000 m~ distilled water,
pH 7 0), the growth inhibition against ala~o~myces
was assayed. In this assay, the minimal inhibitory con-
centrations were 3 ~g/m~ for C-15003 P-3 and P-3', and 1 5
~g/m~ for C-15003 P-4, Furthermore, the wild strain of
Tetrah~mena p~riformis W as an assay organism was cultivated
on an assay medium ~composed of 20 g Proteose-peptone
(Difco), 1 g yeast extract, 2 g glucose, 1000 m~ distilled
water and 10 m~ 1 M-phosphate bu~fer pH 7.0~ at 28C for
44 to 48 hours and the growth inhibitory activity of the
antibiotic compounds against this particular microorganism
was determined by the serial dilution method. Growth
inhibition occurred at 1 ~g/m~ for C-15003 P-3 and P-3',
and at O.5 ~g/m~ for ~-15003 P-4.
Anti-fungal actl~ity is shown in Table 5, As seen
from Table 5, C-15003 has growth inhibitory activity against
microorganisms which cause plant diseases. The filter-
paper discs impregnated with 0.02 m~ of a 1000 ~g/m~
solution of C-15003 were placed on plates re~ectively
inoculated with the microorganisms as ~ollowing ~able 5.
*Trademark
- 23 -
~)7~1Z
Table 5 Anti-microbial spectra
Test organisms I~0 mediumhourInhibition
~umber diameter
Alternaria 7515 PSA* 48 38
kikuc~na
Fusicladium levieri 6477 PSA* 9 68
Helm.nthosporium
si~noideum var. 5273 PSA* 48 55
irregulare
Pyricularia oryzae - PSA* 48 53
Elsinoe fawcetti 8417 PSA* 90 55
~usarium oxysporum _ 48 20
f, cucumerinum PSA*
Guignardia laricina 7888 PSA* 48 12
Cochlioborus miyabea~us PSA*
5277 48 60
Diapor~he citri 9170 PSA* 48 55
Gibberella zeae 8850 PSA* 48 37
Sclerotinia sclerotiorum PSA*
9395 90 65
Ve~turia pirina 6189 PSA* 48 50
Pellicularia sas~ii 9253 PSA* 48 50
P;5rthium apha~idermatum 7030 PSA* 48 58
Botrytis cinerea - PSA* 48 48
Aspergillus niger 4066 PSA* 48 0
Pe~icillium chrysogen:um
4626 P~A* ~8 35
Rhizopus nigricans 6188 PSA* 48 25
Saccharomyces cerevisiae
0209 PSA* 48
Rhodotorula rubra 0907 PSA* 48 28
Trichophyton rubrum 5467 GB 48 38
-- 24 _
.
~lZ
Test organisms I~0 medium hour Inhibition
n~lmber diameter
~richophyton ~
mantagrophytes 7522 GB 48 38
Candida albicans 0583 GB** 48 0
Candida u~ilis 0619 G~** 48 0
Cryptococcus 0410 GB** 48 43
neof orman6
* PSA: Potato sucrose agar medium
~ GB : Glucose nutrient agar medium
B) Antitumour activity
~ he therapeutic effects of C-15003 P-3, P-3' and P-4
(dosed intraperitoneally for 9 consecutive days) upon P388
leukemia in mice (1 x 106 cells/aniimal, mouse, intra-
peritoneaIly transplanted) were investigated. ~he results
showed that, in terms of life span-extending ratio, these
compounds had an antitumour activity as high as 200 % at
the dose level of 0.00625 ~g/kg/day
C) ~oxicity
In an acute toxicity test with mice as test animals,
which involved intxaperito~eal inàections of C-15003 P-3,
P-3' and P-'l, all of`these antibiotics showed a ~D50
value more than 0.313 mg/kg.
As mentioned hereinbefore, the present Antibiotic
C-15003 has strong inhibitory activity against fungi and
protozoa and, therefore, is of value as an antifungal or
antiprotozoan agent. ~urthermore, because Antibiotic
C-15003 displays a life span-exte~ding action upon tumour-
bearing mammalian animals (e.g. mouse), it is also expected
- 25 -
7Zl~Z
that the compound will be of use as an antitumour drug.
Antibiotic C-15003, as an antifungal and antiproto~oan
agent, can ~e used with advantage for an assessment of the
bacterial ecology in the soil, active sludge, animal body
fluid or the like, ~hus, when valuable bacteria are to be
isolated from soil samples or when the actions of bacteria
are to be evaluated independently of those of fungi and
protozoa in connection with the operation and analysis of
an active sludge system used in the treatment of waste
water, as the present antibiotic may be utilized to obtain
a selective growth of the bacterial flora without permitting
growth of the concomitant fungi and pro~ozoa in the
specimen. In a typical instance, the sample i9 added to
a liquid or solid medium and 0.1 m~ of a 10 to 100 ~g/m~
solution of the antibiotic in 1 % methanol-water is added
per m~ of the medium, which is then incubatedO
~ he present Antibiotic C-15003 can also be used as
an anti-microbial agerlt ~or the treatment of ~lant diseases
caused by the microorganisms me~ioned in the above Table 5
In the typical application, Antiblotia C-15003 is
used in a form of 1 % methanolic aqueous sol~tion containing
0.5 ~g/m~ - 5 ~g/m~ of the antibiotic. ~or lnstance
Antibiotic C-15003 may be used for the control of the
reddish brown sheath rot, the blast, the Helminthosporium
leaf spot and the sheat blight of rice plants,
It i8 thus apparent that C-15003 P-3, P-3' and P-4
are all novel compounds having the same skeletal structure
and can be used also as intermediates for the production
- 26 -
'~ ' . '
''. ~
~721Z
of other pharmaceutically useful compounds. Thus, by wa~
C /S~o3
of deacylation reaction, ~ 1,00, P-0 ~maytansinol~ with
a hydroxyl group in 3-position can be derived from the
present antibiotic. In this connection, becau~e the acyl
group is in position beta to carbonyl, the conventional
reductive hydrolysis reaction can be employed with
advantage. ~hus, by means of a complex metal hydride (e g.
li-thium aluminum hydride (~iA1~4)~ at a low temperature
(e.g. -20-0C), the 0-ester bond in 3-position may be
hydrolytically cleaved, without affecting other functional
groups, e.g. carbonyl, epox~, carbon-carbon double bonds,
etc,, to yield maytansinol. ~he physical and chemical
data on this maytansinol sample thus obtained is in ~ood
agreement with the data given in Kupchan et al ~he Journal
of American Chemical ~ociety 97, 5294-52a5(1975~).
~ he following e~ples are further illustra~ive but
by no means limitative o~ the invention, wherein "part(s)"
is based on weight uLless otherwise noted ahd the relation-
ship between "part(s)" and "part(s) by vol~me" corresponds
to that between "gram(s)" and "milli~ter~s)", and "%" is
based on "weight/volume" unless otherw~e no~ed.
Example
~ ocardia No C-15003 (IF0 13726; ~ERM 3992; A~CC 31281)
as grown on a medium (yeast extract-malt extra~t agarj was
used to inoculate a 200 parts by volume fermenter contai!n-
ing 40 parts by volume of a seed culture medium (2%
glucose, 3 % soluble starch, 1 % raw so~bean meal, 1 % corn
steep liquor, 0.5 % Polypepton*, 0.3 % ~aC~, 0.5 /0 CaC03,
*Trademark
-- 27 --
~1~7~2
pH 7. O) . The inoculated medium was incubated at 28C
for 48 hours to obtain an inoculum, A 0.5 part by volume
portion of the inoculum thus obt~ined was transferred to a
200 parts by volume fermenter containing 40 par~s by
volume of a fermentation medium composed of 5 % dextrin,
3 % corn steep liquor, 0.1 % Polypepton and 0.5 % CaC03
(pH 7.0), and cultivated at 28C for 90 hours to give
inoculum (seed culture).
As determined by the serial dilution method using
etrah~mena ~3~ W as an assay organism and Anti-
biotic C-15003 P-3 as the standard sample, the above
culture was found to have a titer of 25 ~g/m~,
ampl,e 2
A lO parts by volume portion of the inocul~m (seed)
obtainçd in Example 1 was transferred to a 2000 par~s by
volume fermenter containing 500 parts by volume of a seed
culture medium (same as above) and incubated at 28C for
48 hours. A 50~ parts by volume portion o~ the resultant
culture was transfe~red to a 50000 parts by volume tank of
stainless steel containing 30000 parts by volume of seed
culture medium and cultivated at 2~C, under aeration (30000
parts by volume/min.), agitation ~280 r~p~m. (l/2D~)~
and internal pressure (1 kg/cm2) to obta~n a seed culture.
This culture was ~sed to seed a 200000 parts by v~lume tank
of stainless steel containing lO0,000 parts by vo~me of a
fermentation m~dium si~ilar to the one used in ~xa~le 1
at an inoculation rate of l~ %. The inoculated medium
was incubated at 28C, under aeration (lO0,000 parts by
- 28 -
-
volume/min.), agitation [200 r.p.m. (1/2 DT]) and internal
pressure ~1 kg/cm2) for 90 hours. As determined by the
same procedure as that described in Example 1, the culture
obtained above was found to have a titer of 25 ~g/mQ.
Example 3
To 95,000 parts by volume of the culture obtained
in Example 2 was added 2,000 parts of Hyflo-Super-Cel
(Johns ManvilleJ U.S.A.) and, after thorough mixing, the
mixture was filtered on a pressure filter to obtain 85,000
parts by volume of filtrate and 32,000 parts of moist
cells. The filtrate 85,000 parts by volume was stirred
and extracted with 30,000 parts by volume of ethyl acetate.
This procedure was repeated once again. The ethyl acetate
; layers are pooled, washed twice with 30,000 parts by
volume portions of water, dried by the addition of 500
parts of anhydrous sodium sulfate and concentrated under
reduced pressure to 200 parts by volume. Petroleum
ether was added to the concentrate and the resultant
precipitate was recovered by filtration ~53 parts). This
crude product I was stirred with 100 parts by volume of
ethyl acetate and the insolubles were filtered off. The
~iltrate was stirred with 10 parts of silica gel (Merck,
~est Germany, 0.05-0.2 mm~ and the ethyl acetate was
removed under reduced pressure. The residue was applied
to the top of a silica gel column C4ao parts by volume).
Elution was carried out with 500 parts by volume of hexane,
500 parts by volume of hexane-ethyl acetate (3:1), 500
parts by volume of hexane-ethyl acetate (1:1), 500 parts
- 29 -
! .' .
.
;)7Z~z
by volume of hexane- ethyl acetate (1:3), 500 parts by
volume of ethyl acetate and 1,000 parts by volume of ethyl
acetate-methanol (50:1), with the eluate being collected
in 100 parts by volu~e fractions.
One part by volu~e portion of each fraction was
concentrated to dryness, and 0 1 part by volume of ethyl
acetate was added to the concentrate to give a mixture.
~he mixture was spotted at 2.5 cm from the bottom edge of
a silica gel-glass plate (Merck, West Germany9 60 F 2
0.25 mm, 20 x 20) and developed for about 17 cm with a
solvent system pf ethyl acetate-methanol (19:1). After
development, detection was carried out with ultraviolet
light (2537A).
The active fractions No.23-No.28 of RfO,6-0.65 were
collected and concentrated under reduced pressure to
about 20 parts by volume. To this concentrate WAS added
150 parts by volume of petroleum ether to obtain 15 parts
of a crude product Il.
E~ample 4
With stir:ri~g, 32,000 parts of .~he moist cells
obtained in Example 3 were extr.acted with 50,000 parts by
volume of 70 % acetone-water for 3 hours and,.then, filtered
on a pressure filter, ~he extraction-with 50,000 parts by
volume of 70 % acetone-water and subsequent filtration was
repeated once again. ~he filtrates were pooled and the
acetone was removed by concentration under reduced
pressure. ~he resultant aqueous system was passed through
a column of 5,000 parts by volume Diaion*XP-10 (Mitsubishi
*Trademark
~;~ ~ 3 -
12
Kasei K.~.). The column was washed with 20,000 parts by
volume Or water and 50 % aqueous methanol, followed by
elution ~ith 15,000 parts by volume of 90 % methanol-water.
~he elua~e was concentrated under reduced pressUre to
3,000 paxts by volume and shaken with 3,000 parts by volume
of water and 3,000 parts by volume of ethyl acetate. The
above procedure was repeated once again. ~he ethyl acetate
layers we~e combined9 washed with water, dried by the
addition of anhydrous sodium sulfate and concentrated
u~der redu~ed pressure to 200 parts by volume. Following
the addition of petroleum ether, the precipitate was
recovered by filtration (28 parts). The above product was
purified by means of a ¢olumn of silica gel to recover
3.0 parts of crude product II.
In 10 parts by volume of ethyl acetate was dissolved
1.5 parts of the crude product II obtained in Example 3
and the solution was stlrred well with 4 parts of silica
gel (Merck, West Germany, 0,05-0,2 mm), ~he ethyl acetate
was removed under reduced pressure~ ~he residue was
applied to the top of a column of 300 parts by volume ;
silica gel and the column was first washed with 500 parts
by volume of chloroform and then eluted with 500 parts by
volume of chloroform-methanol (50:1), 500 parts by vol~me
of chloroform-methanol (20:1) and 500 parts by volum~e of
chloroform-methanol (10:1). The eluate was collected in
25 parts by volume fractions.
A 0.5 part by volume portion of each fraction was
- 31 -
concentrated under reduced pressure. ~o the concentrate
was added 0 05 part by volume of ethyl acetate, and the
mixture as a sample was subjected to silica gel thin layer
chromatography (de~relopirLg system: chlorofoxm-methanol=
9:1).
~ he fractions Nos, 39 and 40 absorbing at 2537 A
in the zone of PLf 0.50-0.60 were collected and concentrated
to dryness under reduced pressure. To the residue was
added 2 parts by ~rolume o~ ethyl acetate and the mixture
was allowed to stand, whereupon 0 150 part crystals of
Antibiotic C-15003 were obtained.
~ he above cr~ystals of Antibiotic C--15003 (0.150
part) were dissolved in 15 paxts by volume of methanol,
followed by addition of 0~300 part of sodium chloride and
15 parts by volume of water. A column was packed with
200 parts by volume of Diaion*XP-10 (Mitæubishi Kasei K K.)
and calibrated with 600 parts by volume of 50 % methanol-
water containing 5 ~ o~ ~aC~ ~he sample solution prepared
above was passed through the oolumn, and gradient elution
was carried out using 1,500 parts by volume of 60 %
methanol-water containing 5 % NaC~ and 1,500 parts by
volume of 95 % methanol-water. I'he eluate Was, collected
in 15 parts by volume fractions and each frRction was
investigated by silica ~;el thin layer chromatography.
The fractions 145 to i53 contained C-15003 P-3, the
fractions 167-180 conta:ined C-15003 P-3' and P-4 and the
fractions 185-190 contained C-15003 P-4.
Each group of fractions was concentrate~ and dissolved
*Trademark
:, ~ 32
:llV72~
by the addition of 50 parts by volume of water and 100
parts by volume of ethyl acetate. ~he solution was shaken
in a separatory funnel and the water layer was separated
and1 after washi~g with two 50 parts by volume-portions
of water, the ethyl acetate layer was dried over anhydrous
sodium sulfate, concentrated and allowed to sta~d. In
the above manner, crystals were obtained from each group
of fractions. ~he crystals were collected by filtration
and dried.
C-15003 P-3 0.070 part
C-15003 P-3', P~
0,018 part
C-15003 P~ 0,015 part
The mixed crystals of C-15003 P-3' and P-4(0,018
part) were dissolved in 0.3 part by v~lume of ethyl
acetate and spotted in a line at a distanCff of 2,5 cm from
the bottom edge of a silica gel glass plate (Merc~, West
Germany, Kieselgel 60 ~254 0 25 mm, 20 x 20~, followed by
de~relopment with ethyl acetate-methanol (19:1), After
development to about ~8 cm, the absorption band ~t Rf 0,68
(P~) and Rf 0 65 (P-3')~ were bcrapped o~f end e~ch was
irLdependently extracted twice ~ith e~hyl a;cetate containing
a small amount of water. ~he resultant ethyl acetate
extract was washed with water, dried o~er anhydrous sodium
sulfate, concentrated under reduced presslLre and allowéd
to stand.
0.010 Part crystals of C-15003 P-4- and 0.003 part
crystals of C-15003 P-3' were obtained from the fractions
of Rf 0.68 and Rf 0,65, respectively.
- 33 -
ExamPl~ 6
One thousand parts by volume of the culture o~ Example 2
was inoculated into a 200,000 parts by volume tank o~
stainless steel contai~ing 100,000 parts by volume of a
seed culture medium and the inoculated medium was incubated
at 28C under ae~ation (100,000 parts by volume~min.) and
agitation (200 r.p.m.) for 48 hours to prepare a seed
culture, This seed culture was transferred to a 2,000,000
parts by volume tank of stainles~ steel containing
1,000,000 parts by volume of a fe~mentation medium similar
to that used in Example 1 at a transplantation ~ate of
10 %, Cultivation was carried out at 28C u~der aeration
(1,000,000 parts by volume/min.), a~itation ~120 r.p.m.
(1/3 DT)) and internal pressure (1 kg/cm2) for 90 hours.
The resultant culture was found to have a titer of 20 ~g/m~
as assayed by the assay procedure described in Example 1, --
To 900,000 parts by volume of the above culture was
added 900,000 parts by volume of acetone and, after an
hour's stirring, 20~000 pa~ts of Hyflo-Super-Cel (Johns
Manville, U.S.A,) ~a~ added, ~he mixture wa~ further
stirred and filtered on a pressure filter machine.
To 1,700,000 parts by volume of the resultant fi-trate
was added 500,000 parts by volume of water and, in a Pod- -
bielniak*(Podbielniak, Inc.), the mixture was extracted
with 1,000,000 parts by volume of ethyl acetate. ~e
ethyl acetate layer was washed with water, dried by the
addition of anhydrous sodium sulfate and concentrated under
reduced pressure. ~o the concentrate was added petroleum
*Trademark
~ 34 -
.
~10~
ether and the resultant precipitate was recovered by
filtration and dried. By the above procedure was obtained
68 parts of crude product I. Thereafter, as in Examples
3, 4 and 5, this crude product was p~rified to cbtain 9.5
parts of C-15003 P-3, 0.300 part of C-15003 P-3' and 2 5
parts of C-15003 P-4.
Exa ple 7
In 1 part by volume of tetrahydrofuran was dissolved
O.015 part of the Antibiotic C-15003 crystals obtained in
3xample 5 and after the solution was cooled to -5C,
O.012 part of lithium aluminum hydrIde was added. ~he
mixture was allowed to stand for 2 hours, Following the
addition of 0.5 part by volume of a 1 yO aqueous solution
of H2S04, the reaction mixture was extracted with 2 parts
by volume of ethyl acetate. ~he ethyl acetate layer was
washed with water, dried by the addition of a~hydrous
sodium sulfate and concentrated under reduced pressure.
Preparative TI~ with si~ica gel was carried out on the
concentrate and the ~one of Rf 0 25 to 0.3 was scraped off
a~d extracted with ethyl acet~te containing a ~mall amount
of water. ~he extract was washed with water, dried over
a~hydrous sodium sulfate and concentrated u~der reduced
pressure, whereupon crystals separate~. ~he cr~stals were
recovered by filtration and dried. By the ab~re pro~edure
33
E3 wa~ obtained 0.010 part of P 1~i40, P-0, melting point 174C.
Elemental analysis: Found C,59.65; H, 6,58; N, 5~02;
C~, 6 51; calcd. ~or C28E37C~N208 C, 59.52; H, 6~60
N, 4.96; C~, 6.27
11~)~2
IR: 1715, 1670, 1580(cm 1)
W (nm): 232(32750~, 244(sh, 30850), 252(31650), 281(5750),
288(5700)
In pr~perties, this product is identical with may-
tansinol ..
-- 36 --
