Note: Descriptions are shown in the official language in which they were submitted.
99~
The present invention relates to a method for
preparing maytansinol, maytanacine and maytansinol pro-
pionate which are anti-tumor agents.
It has been known that, among the above com-
pounds, maytanacine and maytansinol propionate have strong
anti-tumor activity [Kupchan et al.: Journal of the
American Chemical Society 97, 5294 ~1975)]. On the other
hand, maytansinol itself has only weak anti-tumor activity
(See the above reference) but it is a useful inter-
mediate for easy preparation of maytanacine and maytansinol
propionate and other various derivatives.
Maytansinol and maytanacine have been obtainedby the above Kupchan et al. from bark of Putterlickia
verruscosa ~Plant belonging to the genus Maytenus) and
thus yield is extremely low, such as 0.025 mg of the
former compound and 0.36 mg of the latter from 1 kg of
dried bark of the plant. ~s to maytansinol propionate,
it has been obtained by chemical propionation of maytan-
sinol.
The present inventors have collected various
soil and other samples and investigated antibiotics
produced by microorganisms which are isolated from the
samples, and these studies have reached findings that
some of the microorganisms thus isolated can accumulate
maytansinol, maytanacine or maytansinol propionate in
the culture medium, these microorganisms belong to the
genus Nocardia, and that these compounds can also be
2~
obtalned by cultivating mutants derived from these
microorganisms in a proper nutrient medium under appro
priate conditions.
Further studies on the basis of these findings
have now reached completion of the present invention.
Thusg the present invention relates to a method
for preparing maytansinol, maytanacine or maytansinol
propionate, which comprises cultivating a microorganism,
which belongs to the genus Nocardia and is capable of
producing maytansinol, maytanacine or maytanslnol
propionate 9 in a culture medium to accumulate maytansinol,
maytanacine or maytansinol propionate in the cultured
broth and recovering the same.
According to the prior art~ the above compounds are
obtainable from plants, but the plants are limited to
specific ones3 and great expense and a long period of
time are required for the production at each stage of
growth, felling~ d~ying and pulverizing of the plants
and extractiong separation and purification. Further,
the yleld is extremely low.
On the contraryg the processes of the present
invention can be conducted easily and smoothly by cultivation
of the microorganism, and a large amount of the objective
compounds can be produced and obtained when desired.
me present invention is the first example of
obtaining these compounds as metabolites of microorganismsg
~o~z99~
and can be said as an excellent method for preparation
thereof.
As an example of the microorganism usable in the
present methodg there may be men~ioned an actinomycete
strain No. C-15003 which we isolated from soil and other
samples in our screening for antibiotic-producing
microorganisms.
me microbiological characters of Strain No. C-
15003 w2re investigated by procedures analogous to those
proposed by Schirling & Gottlieb [International Journal
of Systematic Bacteriology 16~ 313-340 (1966)]. The
results of observations at 28C over 21 days are as
~ollows.
1) Morphological characters
The ve~etative mycelium extends well and develops
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 instances9 may divide'into fragments
resembling rod bacteria or branched short lengths of
hypha~., m e strain gives good growth on various
taxonomical media~ with aerial mycelium being superimposed
on the vegetative mycelium3 although it frequently forms
coremia like bodies (50 200 x 200 - lOOO~m) on which
further aerial growth takes place. Many of the aerial
mycelia are flexuous, straight or a loosely spiral like
configuratlon being encountered on a few occasions.
~1~9Z999
Microscopic examination of aged cultures reveals that
only in few cases the conidia like cells occur in chains,
while the cell suspensions obtained from the surfaces
o~ such culturesg 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 l.C-2.0 ~m) bodies resembling
arthrospores.
Electron-microscopic examinations showed that
these bodies had smooth surfaces.
2) The constituents of cells
The strain was shake-cultured in modified ISP
No. 1 medium at 28C for 66 to 90 hours, at the end of
which time ~he cells were collected and rinsed. By
the method of B. Becker et al. [Applied Microbiology
12, l~21 (1964)] and the method of MoP ~ Lechevalier. `
[Journal of Laboratory and Clinical Medicine 71, 934
(1968)], the above whole cells were examined for
diaminopimelic acid and sugar compoSition. The former
was found to be the meso--~orma while spots were
detected, which corresponded to galactose and arabinose.
3) Characteristics on taxonomical media
The strain showed comparatively good growth on
various media~ with the vegetative mycelium being
colorless to pale yellow in initial phases of culture
and light yellowish tan to yellowish tan in later phases.
The strain produces soluble pigments~ yellow to yellowi~h
.
g~
tan9 in various taxonomical media. The aerial mycelium
is powdery and generally gives moderate growth~ being
white to yellow or light yellowish tan. The characteristics
of the strain in various taxonomical media are set
forth in Table 1.
Table 1 Cultural characteristics of Strain No. C-15003
on taxonomical media
(A) Sucrose~nit~atê agaro
Growth (G): Luxuriantg Brite Melon Yellow (3 ia)*
to Amber (3 lc~, coremia like bodies
formed
Aerial mycelium (AM): Scant, white
Soluble pigment (SP) None or pale yellowish tan
(B) Glycerol.nitrate agàro
G Moderate, Lt Ivory (2 ca)*, coremia like bodies
formed
AM: Moderateg white
SP: None
(C) Glucose~ asparàgin~ a~ar:
G Moderate 3 Brite Marigold (3 pa)~ toBrite
Yellow (2 pa~*
AM: Scant, white
SP: ~rite Yellow (2 pa)*
(D) Glycerol;aspar~gine ag~ro
G : Moderate, Lt Ivory (2 ca)*~ coremia like bodies
~ormed
-- 5 --
.
AM: Scantg white
SP: None
(~) Starch agarO
G : Moderateg Lt Ivory (2 ca)~ to Lt Wheat
(2 ea)*g coremia like bodies formed
AM: Abundant, Lt Ivory (2 ca)*
SP: None
(F) Nutrient agar:
G : Moderate, Lt Ivory (2 ca)* to Colonial Yellow
(2 ga)*, coremia like bodies formed
AM: Scant, white
SP: None
(G) Calcium malate agar:
G : Moderate~ Lt Ivory ~2 ca)* to Lt wheat (2 ea)*,
coremia like bodies formed.
AM: Moderateg white to Lt Ivory (2 ca)*
SPo None
(H) Yeast extract-malt extract agar.
G : Moderate~ Amber (3 lc)* to Brite Yellow
(3 la)*g coremia lik~ bodies formed
~M: Moderate, white to Lt Ivory (2 ca)*
SP: None
(I) Oatmeal agar:
G : Moderate, Lt Ivory (2 ca)* to Colonial Yellow
(2 ~a)*, coremia like bodies formed
AM: Scant, white to light yellow
SP: None
(J) Peptone yeas~ extract iron ag~r
G : Moderate~ Colonial Yellow (2 ga)*
AM: None
SP: Colonial Yellow (2 ga)*
(K) Tyrosine agar-
G : Moderate9 Lt Ivory ~2 ca)* to Lt Melon Yellow
(3 ea)*, coremia like bodies formed
AM: Moderate, white to Lt Ivory (2 ca)*
SP: Camel (3 ie)*
* The color codes aceording to Color Harmony Manual,
4th ed. (Container Corporation of America, 1958)
4) Physiological characters
The physiological characters of the strain are shown
in Table 2. Temperature range for growth: 12C to 38C.
me temperature ran~e in which good aerial growth occurs
on agar (ISP NoO 2) is 20 to 35Co
Table 2 The physiological characters of Strain
No. C-15003
Temperature range for growth: . 12 to 38C
Temperature range fo~ aerial growth: 20 to 35C
~iquefaction of gelatin: Positive
Hydrolysis of starch: Positive
Reduction of nitrates: Positive
Peptonization of milk: Positive
Coagulation of milk: Negative
Z~9
Decomposition of casein: Positive
Production of melanoid pigments: Ne~ative (peptone yeast
extract iron agar),
positive ~tyrosine agar)
Decomposition of tyrosine: ~ositive
Decomposition of ~nthine: Negative
Decomposition of hypoxanthine. Ne~ative
Tolerance to lysozyme: Positive
Tolerance to sodium chloride: 2%
5) Utilization of various carbon sources
The utilization of various carbon sources was
in~estigated using a medium described in Pridham and
Gottlleb [Journal of Bacteriology 56, 107 (1948)]
and a basal medium of the same composition plus 0.1%
of yeast extract. The resultant spectrum is shown
in Table 3.
Table 3 me utili2ation of carbon sources by Strain No.C-15003
Source of carbon Growth Sources of carbon Growth
D-Xylose + ++* Raffinose + +*
L= Arabinose + + Melibiose + +
D= Glucose ~ +~ i-Inositol - -
D-Galactose + + D~Sorbitol - -
D-Fructose ~+ ++ D-Mannitol +~ ~+
L-Rhamnose + + 51ycerol _ +
D-l~annose ~+ ++ Soluble starch +
Sucrose ~+ +~ Control
-- 8 --
" : .. ~
g9
Lactose - - *
Maltose +
Trehalose -~ ~
* Basal medium with 0.1% yeast extract added
Note: +~: Luxuriant growth
~: Good growth
Growth
+~ Poor growth
-~ ~o growth
6) Other characteristics
me cells were harvested by the proceaure previously
described in 2) and DNA was p~epared by a procedure
analogous to that of ~. Marmur et al. ~Journal of
Molecular Biology, 3,2083 1961]. The G-C(Guanine-
Cytosine) content of the DNA was found to be about 71 mole %.
Gram-staining of the vegetative mycelium of this
strain was positive.
me above characteristics of Strain No. C-15003
were compared with the descriptions in S. A. Waksman's
'~he Actinomycetes Vol. 2"[The Williams and Wilkins
Co., 1961]; R. E. Buchanan and N. E. Glbbons,'~ergey's
Manual of Determinative Bacteriology, 8th ed, 1974 ";
and other literatures.
Whilst this strain was thought to belong to Group
m of the genus Nocardia~ the failure to find any
species having the characters so far described among
, ~ ~
~zs~
the known strains led us to conclude that this straln
represented a novel species of microorganism.
The present Strain NoO C--15003 has been deposited
at Fermentation Research Instltute, Agency of Industrial
Science and Technology (FERM) under the receipt number
of 3992 and at Institute for Fermentation~ Osaka (IFO)
under the accession number of IFO 13726 and at The
American Type Culture Collection (ATCC), Marylandg U.S.A.
under the accession number of 31281.
~ hile Strain No. C-15003 is a novel species of the
genus Nocardia as just mentioned3 it is liable3 as are
microorganisms generally, to undergo variations and
mutations, whether spontaneously or under the influence
of a mutagen. For exampleg the many variants of the
strain which are obtainable by irradiation with X-raysg
~amma raysg ultraviolet light9 etc., by monocell isolationg
by culture on media containing various chemicals, or
by any other mutagenic treatment, as well as the mutants
spGntaneously derived from the strain, should not be
substantially considered io represent any other distinct
species butg rather~ any of such vàriants and mutants
capable of elaborating maytansinol~ maytanacine and
maytansinol propionate may be invariablY utilized for
the purposes of this invention as the strain NoO C-15003
By way of example~ subjecting Stra~n No. C-15003 to
various mutag~enic treatments yields mutants substantially
-- 10 --
l~Zgg~
lacking the ability to produce soluble pigments~ mutants
with substrate mycelia which are colorlessg yellowish
green3 reddish tan or orange red, mutants whose hyphae
are ready to fragment into bacillary elements or
branched short hyphal fragments~ and mutants with
abundant white aerial mycelia or substantially without
aerial mycelia.
The medium employed for the cultivation of strain
capable of producing maytansinol~ maytanacine or
maytansinol propionate (hereinafter~ sometimes
abbreviated as l'producible strain") may be whichever
of a liquid and a solid medium only if it contains
nutrients ~hich the strain may utilize9 although a
liquid medium is preferred for high-production runs.
The medium may comprise cArbon and nitrogen sources
~hich Strain No. C-15003 may assimilate and digest,
inorganic matter~ trace nutrients3 etcO As examples of
said carbon sources may be mentioned glucose, lactoseg
sucrose, maltose, dextring starchg glycerol, manni~ol,
sorbitol~ etc., fats and oils (eOg. soybean oila lard
oil, chicken oil, etc.) and so forth. The nitrogen
sources may for example be meat extract~ yeast extractj
dried yeast3 soybean mealg corn steep liquor~ peptone,
casein, cottonseed flour, spent molasses, ure~a
ammonium salts (e.g. ammonium sulfate, ammonium chlorideg
ammonium nitrate, ammonium acetate, etc.) and so fQrth.
g~ ~ ~ 9 ~ ~
The medium may further contain salts of sodiumy
potassium9 calcium, magnesiuma etc. 9 salts of iron,
manganese, zinc~ cobaltg nickel~ etc. 9 salts of
phosphoric acid3 boric acid~ etc and organic acid
salts such as acetates and propionates. Further, the
medium may containg as added, various ami~o acids (e.g.
glutamic acidg aspartic acida alanine, glycine3 lysine,
methionine3 proline3 etc. )3 peptides (e.g. dipeptides,
tripeptidesa etc.)~ vitamins (e.g. Bl/ B23 nicotinic
acid3 B12~ C, E, etc.), nucleic acids (e.g. pur~ne~
pyrimidine and derivatives thereof and so forth. For
the purpose of adjusting the pH of the medium~ there
may be added an inorganic or organic acid, alkali~
buffer or the like. Suitable amounts of oils, fats,
surfactants, etc. may also be added as antifoams.
The cultivation may be conducted by any of the
stationary a shake, submerged aerobic and other cultural
conditions. For hi~h production runs 3 submerged aerobic
culture is of course preferred. ~hile 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 'o 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 to 7.5. While
3~
the incubation time also is variable according to the
same factors as mentioned aboveg it is advisable to
continue the incubation until the titer of the desired
antibiotic product becomes max-Lmal~ In the case of
shake culture or aerobic submerged culture in liquid
medium9 the time required normally ranges from about
48 to 144 hours.
The potency of the antibiotic was assayed with
Tetrahymena pyriformis W as an assay or~anism. Thusg
the above microorganism was grown on a test medium
20 g of Protesse-peptone (Difco), 1 g of yeast extract
(Difco), 2 g of glucose, 1000 ml of distilled water and
10 ml of 1 M-phosphate buffer (pH 7.0)~ at 28C
for 44 to 48 hours and the potency of the antibiotic
was determined by the serial dilution method with a
monitoring of the turbidity of growth and by a thin-
layer chromatographic (briefly~ TLC) assay to be
described hereinaf~er.
Maytanacine 9 maytansinol propionate or maytansinol
is produced and accumulated in the resultant cultured
broth, both extracellularly and intracellularly.
None of these substances shows distinc~ antibiotic
activity, and thus they have been detected by TLC which
is set forth in parallel to detection by activity to
the Tetrahymlena strain. Thusg the fermentation broth
is separated into cells and filtrate by ~lltration
~2999
or centrifugation and the filtrate is extracted with
the same volume of ethyl acetate. To the cells is
added the same amount of 70% acetone-water as the
filtrate and, after an hour's stirring at 20C9 the
suspension is filtered. The acetone is removed from
the filtrate and the resultant aqueous filtrate is
extracted with ethyl acetate. Each of the extracts
is concentrated to 1/100 by volume and sub~ected to
thin-layer chromatography on a silica gel-gl~ss plate
(Merck, West Germany~ ~ieselgel* 60 F 254~ 0.25 mm,
20 x 20) (solvent system:chloroform-methanol=9 1).
The potency was determined on the basis of the
intensity of spots detected by irradiation with
ultraviolet light at 2537 Ao
Maytanacine 3 maytansinol propionate or maytansinol
which ls thus produced in the cultured broth, are
lipophyl and neutral, they can be conveniently recovered
by separation and purification procedures which are
normally emplo~ed for the harvest of such microbial
metabolites. For example~ there may be employed a
procedure which utilizes the difference in solubility
between the antibiotic and impurity9 means which utilizes
the fractionating adsorptive affinity of various
adsorbents such as activated carbong macroporous
nonionic resins, silica gel, alumlna, etc., a procedure
of removing the impuritles by means of ion exchange
*TTademark
- 14 -
,
2~
resins9 and so forth, as applied singly or in a suitable
combination or as applied in repetition.
Since, as aforesaidg maytanacine, maytansinol
propionate or maytansinol occur in both the filtrate
and cellsg the antibiotics are separated and ~urlfied
by means of such an adsorbentg if one is employed,
either directly or after a solvent extraction in the
case of the filtrate a or after a solvent extraction
in the case of microbial cells. The solvent extraction
may be performed by any of the following and other
methods e.g. (1) solvent extraction from the culture
broth prior to separation of cells and (2) solvent
extraction of the cells and the filtrate obtained by
filtration3 centrifugation or other process. To
extract the filtrate and cells independently, the follow-
ing procedure may be taken advantageously.
The solvents suitable for extraction of the
filtrate are water-immiscible organic sol~ents such
as fatty ac~d esters 3 e.g. ethyl acetate and amyl
acetate; alcohols, e.g. butanol3 halogenated hydrocarbons,
e.g. chloroform; and ketonesg e.g. methyl isobutyl
ketone. The extraction is carried out at a pH near
neutral and, preferablyg the culture fluid previously
adjusted to pH 7 is extracted with ethyl acetate.
The extract is washed with water and concentrated under
reduced pressure. Then, a nonpolar solvent such as
~z~
petroleum ether or hexane is added to the
concentrate and the crude product I containing the
active compound is recoveredO Because, on TLCg a
number of spots are detected ot;her than maytanac~ne3
maytansinol propionate or maytansinol 9 the product I
is sequentially sub~ected to the following purification
procedures. Thus, a routine purification procedure,
particularly adsorption chromatography is useful and,
for this purpose~ one of those common adsorbents such
as silica gel, alumina, macroporous nonionic adsorbent
resin, etc. may be employed. For purificatlon from
the crude product I, silica gel is most useful. And
development may be carried out, for example starting
~ith petroleum ether and hexane and elution is performed
by the addition of a polar sol~ent such as ethyl
acetate9 acetone, ethanol or methanol. In a typical
process, using silica gel (Merck, West Germany9 0.05-
O.2 mm) as a carrier, column chromatography is carried
out t~ith a serial increase in the hexane to ethyl
acetate ratio. The eluate is sampled and investigated
by TLC and the fractions containing e~fective
ingredients are pooled and concentrated under reduced
pressure. ~hen, petroleum ether or hexane is added
to the concentrate, whereby the crude product ~ is
obtained. S:ince this product still contains impurities,
it is further purified as follows. For example, the
16 -
lO~Zg~9
product ~ may be purified by means of a second
silica gel column using a different solvent system.
The developing systemfOr this purpose may conslst in
a halogenated hydrocarbon such as dichloromethane or
chloroform9with the addition of polar solvent such as
an alcohol, e.g. ethanol or methanol, a ketone 3 e.g.
acetone or methyl ethyl ketone, or the like. In this
way, maytanacir.e, maytansinol propionate or maytansinol
is isolated. The order of solvent systems for the
first and second silica gel columns may be reversed
and, in addition, ordinary organic solvents may be
used in con~unction with the above systems if necessary.
~ lhere a macroporous adsorbent resin is used as
purification means for crude product ~ elution of
maytanacineg maytansinol propionate or maytansinol
ls accomplished with a mi~ture of water with a lower
alcohol, a lower ketone or an ester. The lower
alcohol may for example be methanol, ethanol, propanol
or butanol and the lower ketone may ~or example be
acetone or methyl ethyl ketone. The ester may for
example be ethyl acetate. In a typical procedure,
the crude product ~ is dissolYed in 60% methanol-
water and adsorbed on a column of Diaion*HP-10
(Mitsubishi Kasei K~K.). The column is washed with
70% methanol-water and~ then, elution is carried ou~
with 90% methanol-water. In this way, maytanacine,
*Trademark
:~o~9~
maytansinol propionate or maytansinol is eluted from
the column.
In either of the processes described above3 the
fractions containing the object components are pooled
and concentrated under reduced pressure To the dry
product is added 5 to 8 volumes of ethyl acetate
and the mixture is allowed to standg whereupon crystals
of maytanacine, maytansinol propionate or maytansinol
separate3 respectively when the crystals contain
maytanacine and maytansinol propionateg they 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 exampleg silica gel is employed~
development is carried out with hexaneg ethyl acetate,
or chloroform-methanol~ whereby maytansinol propionate
and maytanacine emerge in that order. After detection
by TLC~ the fractions are respectively concentrated
under reduced pressure and ethyl acetate is added to
the concentrates. In this manner9 t.he re.spective
compounds can be obtained as crystals. When a
macroporous nonionic adsorbent resin.is employed3
gradient elution with a varying ratio of alcohol~
ketone or ester to water may be utilized. For example,
by the gradient elution method involving the use of
-18 -
99
60Z methanol-water and 95% methanol-water, with 5%
sodium chloride added, maytanacine and maytansinol
propionate emerge in the order mentioned. After
sampling and detection by TLC 3 each group of active
fractions is concentrated under reduced pressure and
crystallized from ethyl acetate. The isolated
crystals include ethyl acetate as a solvent of
crystallization and3 after drying over phosphorus
pentoxide at 70C for 8 hoursg show the followin~
physical and chemical properties. (Table 4)
Table 4
Maytanacine Maytansinol Maytansinol
propionate
C30H39ClN209 C31H41ClN2 9C28H37ClN208
. .
point(C)235-236 188-190 172.5-174
Specific [~]D2 -121+10 [~]D2-127+10 [~]22_313O+loo
rotation (C=0.25,CHC13) (C=0.35,CHC13) (C=0022~CHCl3)
C59.62 59.93 59.28
Found H6.93 6.82 6.38
(%) N4.28 4.32 5.02
Cl5-74 5.57 6.15
_
Analysis C59.85 59.94 59.52
Calcd. H6.48 6.65 6.60
(%) N4.61 4.51 4.96
Cl5.84 5.71 6.27
- 19 -
99~
-
Maytanacine Maytansinol Maytansinol
propionate
C30H39ClN209 C31H41 1 2 9 C28H37clN208
. I . _ .
Ultraviolet 233(30330) 1 233(30240) 232(32750)
spectrum 240(sh.28240) 240(sh.28400) 244(sh.30~50)
nm() 252(27850) 252(27650) 25~(31650)
280(5680) 2~0~5740) 281(5750)
288(5660j 288(5710) 288t5700)
....
Infrared 1740gl73091670 1740,173~il670, 1715,167091580
absorption 80 1580
spectrum 15
Mas~
s(pectrum 545,48534703450 5599485~470,450 503,485,4709
Acidgneu- lipophyl and Iipophyl and lipophyl and
tral or neutral sub- neutral sub- neutral sub
basic stance stance stance
Dragendorff Dragendorff Dragendorff
Color reaction reaction: reac-~ion:
reactions posi~ive pos~tive positive
Beilstein Beilstein Beilstein
reaction: reaction: reaction:
positive posi-tive positive
Above mentioned data of elemental analysls~
specific rotation9 UV spectra3 IR spectra9 mass spectra9
etc. are in good agreement with the data of maytanacine,
maytansinol propionate and maytansinol which are
given in the literature Kupchan et al. (The Journal
- 20 -
: . . .: . :.,. : . : . .
999
of American Chemical Society 979 5294 (1975) .
The following examples are further illustrative
but by no means limitative of the invention, wherein
"part(s)" is based on weight unless otherwise noted
and the relationship between "part(s)" and "part(s)
by volume" corresponds to that between "gram(s)"
and ''milliliter(s)''g and 'i%i' is based on "weight/volume"
unless otherwise noted.
Example 1
/~laytansinol-, maytanacine- and maytansinol
propionate-producible Nocardia No. C-15003 (IF0 13726;
FERM 3992 ~ ATCC 31281) as grown on a medium (yeast
extract-malt extract agar) was used to inoculate a
200 parts by volume fermenter containing 40 parts
by volume of a seed culture medium (2% glucose9 3%
soluble starch, l$ raw soybean meal a 1% corn steep
liquora 0.5% Polypepton*, 0.3% NaCl, 0.5% CaC03,
pH 7.0). The inoculated medium was incubated at 28C
~or 48 hours to obtain an inoculum. A 0.5 part by
volume portion of the inoculum thus obtained was
transferred to a 200 parts by volume fermenter
containing 40 parts by volume of a fermentation
medium composed of 5% dextrin3 3% corn steep l~quor,
0.1% Polypepton and 0 5% CaC03 (pH 7 ) a ~and cultivated
at 28C for 90 hours to give inoculum (seed culture?.
As determined by the serial d~lution method
*Trademark
;~:. , ... .:,. -.,
. : , ... .. , .: . : .
~2g~9
using Tetrahymena pyriformis W as an assay organism
and maytansinol propionate as the standard product,
the above culture was found to have a titer of 25 ~g/ml.
Example 2
A lO parts by volume portion of the inoculum
(seed) obtained in Example 1 was transferred to a
2000 parts by volume fermenter containing 500 parts
by volume of a seed culture medium (same as above)
and incubated at 28C for 48 hours. A 500 parts by
volume portion of the resultant culture was transferred to
a 50000 parts by volume tank of stainless steel
containing ~0000 parts by volume of seed culture
medium and cultivated at 28C under aeration (30000
parts by volume~min.), agitation [280 r.p.m. (1/2DT)]
and internal pressure (1 k~/cm2) to obtain a seed
culture. This culture was used to seed a 200000
parts by volume tank of stainless steel containing
100000 parts by volume of a fermentation medium
similar to the one used in Example 1 at an inoculation
rate of 10%. The inoculated medium was incubated at
28C under aeration (100000 parts by 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/ml.
To 900C)0 parts by volume of the culture obtained
- 22 -
above was added 2000 parts of Hyflo-Super-Cel* (Johns -
Manville, U. S. A.) and, after thorough mixing, the
mixture was filtered on a pressure filter to obtain
85000 parts by volume of filtrate and 32000 parts of
moist cells. The filtrate (85000 parts by volume)
was stirred and extracted with 30000 parts by volume
of ethyl acetate. This procedure was repeated once
again. The ethyl acetate layers were pooled, washed
t~ice with 30000 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 filtrate was -
stirred with 10 parts of silica gel (Merck, West 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 (400 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 by volume
of hexane-ethyl acetate (1:3), 500 parts by volume of
ethyl acetate and 1000 parts by volume of ethyl acetate-
*Trademark
~ 23
99
methanol (50:1)3 and 1000 parts by volume of ethyl
acetate-methanol (25~ with the eluate being
collected in 100 parts by volume fractions.
One part by volume portion of each fraction was
concentrated to drynessg and ~.1 part by volume o~
ethyl acetate was added to the concentrate. The
mixture was spotteed at 2.5 cm from the bottom edge
of a silica gel-glass plate (Merck, West Germany,
60 F 2549 0.25 mm, 20 x 20) and developed for about
17 cm with a solvent systemofethyl acetate-metXanol
(19:1). After developmentg de~ection was carried
out with ultraviolet light t2537A).
The active fractions No. 25-30 of Rf 0.58-
O.~3 and the fractlons No.38-40 of Xf 0.25-0.30 were
collected and Goncentrated under reduced pressure to
about 20 parts ~y vo-ume, respectively. To these con-
centrates were added~ each 150 parts by volume of
petroleum ether to obtain 5 parts of a crude product
and 2 parts of crude màytansinol.
In 10 parts by volume of ethyl acetate was dissolved
0.5 part of the crude product ~ obtained abo~e and the
solution was stirred well with 4 parts of silio~ gel
(Merck~ West Germany~ 0.05-0.2 mm3. The ethyl acetate
was removed under reduced pressure. The residue was
applied to the top of a column of 300 parts by volume
silica gel and the colu~n was first washed with 500 parts
~ 24 -
.. : .: ,.,
lO~Z999
by volume of chloroform and then eluted with 500 parts
by volume of chloroform-methanol (50:1), 500 parts by
volume of chloroform-methanol (20:1) and 500 parts by
volume o~ 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
concentrated under reduced pressure. To the concentrate
was added 0.05 part by volume of ethyl acetateg and the
mixture as a sample was subjected to thin layer
chromatography (developing system: chloroform-methanol=
9:1) .
The fraction Nos. 40 and 41 absorbing at 2537 A
in the ~one of Rf 0.48-0.50 were collected and concentrated
to dryness under reduced pressure. To the residue was
added 0.~ part by volume of ethyl acetate and the mixture
was allowed to stand~ whereupon 0.05 part mixed
crystals of maytanacine and maytansinol propionate
were obtained.
0.05 part of the above mixed crystals of maytanacine
and maytansinol propionate was dissolved in 5 parts by
volume of methanolg fallowed by addition of 0.1 part
of sodium chloride and 5 parts bX volume of water.
A column was packed with 200 parts b~ volume of Diaion*
HP-10 (Mitsubishi Kasei K.K.) and washed with 600 parts
by volume of 50% methanol-water containing 5~ of NaCl.
The sample solution prepared above was passed through
*Trademark
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-. : ..... ... ..
10~9~9
the column~ and gradient elution was carried out using
1500 parts by volume of 60% methanol-water containing 5%
NaCl and 1500 parts by volume of g5~ methanol-water.
The eluate was collected in 15 parts by volume fractions
and each fraction was investigated by thin layer
chromatography. The fractions 130 tQ 135 contained
maytanacine, and the fractions 138-142 contained maytansinol
propionate~
Each group of fractions was concentrated and dissolved
by the addition of 30 ml of water and 50 ml of ethyl
acetate. The solution was shaken in a separatory ~unnel
and the water layer was separated andg after washing twice
with 30ml-portions of water~ the ethyl acetate layer was
dried over anhydrous sodium sulfateg concentrated and
allowed to stand. In the above manner9 crystals were
obtained from each ~roup of fractions. The crystals were
collected by filtration and driedO Maytanacine 0.013
part. Maytansinol propionate 0O025 part.
In 3 parts by volume of ethyl acetate was dissolved
0.2 part of the crude maytansinol obtained above and the
solution was stirred well with 0.5 part of silica gel
(Merckg West Germany, 0.05-0.2 mm). The ethyl acetate
was removed under reduced pressure. The residue was
applied to the top of a column of 80 parts by volume
silica gel and the column was first washed with 150 parts
by volume of chloroform and then eluted with 150 parts
by volume of chloro~orm-methanol (50:1), 150 parts
- 26 -
3zg~9
by volume of chloroform-methanol (20:1) and 300 par~s by
volume of chloroform-methanol ~10:1). The eluate was
collected in 10 parts by volume fractions.
A 0.5 part~ by volume portion of each fraction was
concentrated under reduced pressure. To the concentrate
was added 0.05 part by volume of ethyl acetate, and
the mixture as a sample was subjected to thin layer
chromatography (developing system: chloroform-methanol=
9:~). 0
me fraction Nos. 50 to 52 ~bsorbing at 2537 A
in the zone of Rf 0.33 to ~.38 were collected and
concentrated to dryness under reduced pressure. To
the residue was added 0.5 part by volume of ethyl
acetate and the mixture was allowed to stand, whereupon
0.020 part crystals of maytansinol were obtained.
Example 3
With stirring, 32000 parts of the cells obtained in
Example 2 were extracted ~Jith 50000 parts by volume o~
70% acetone-waterfor 3 hours andg ~heng filtered on a
pressure filter. me extraction with 50000 parts by
volume of 70% acetone-water and subsequent filtration
was repeated once again. The filtrates were pooled and
the acetone was remo~ed by concentration under reduced
pressure. The resultant aqueous system T~as passed
through a column of 5000 parts by volume Diaion*HP-10
(Mitsubishi l~asei K.K.). me column was washed with
*Trademark
-- 27 --
, : . .; :...... . .
~o9z~99
20000parts by volume of waker and 50% aqueous methanol,
followed by elution with 15000 parts by volume of 90%
methanol-water. The eluate was concentrated under reduced
pressure to 3000 parts by volume and shaken with 3000
parts by volume of water and 3000 parts by volume of
ethyl acetate. The above procedure was repeated once
again. The ethyl acetate layers were combined3 washed with
water, dried by the addition of anhydrous sodium sulfate
and concentrated under reduced pressure to 200 parts by
volume. Following the addition of petroleum etherg the
precipi-tate was recovered by filtration (280 parts).
The above-obtained crude product I was purified by means
of a column of silica gel similarly to Example 1 to
recover 1.0 part of crude product ~ and 0O5 part of crude
maytansinol.
Example 4
1000 Parts by volume of the culture of Exa~ple 2
was inoculated into a 2000Q0 parts by volume tank of
stainless steel containing 100000 part~ by volume of a
seed culture medium and the lnocu~ated medium was
incubated at 28C under aeration (100000 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 2000000 parts by volume tank of stainless steel containing
1000000 parts by volume of a fermentation medium as same
as that used in Example 1 at a transplantation rate of
- 28 -
.... . . .
~ - ; ::: :
~L~9;~g~9
10%. Cultivation was carr~ed out at 28C under aera-tion
(1000000 parts by volume/min.),agitation [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/ml
as assayed by the assay procedure described in Example 1.
To 90~00 parts by volume of the above-obtained culture
was adde~ ~00000 parts by volume of acetone and, after
an hour's stirring, 20Q00 parts o~ Hyflo-Super-Cel*
(Johns -- Manvilleg U.S~A.)9 was addedO The mixture was
further stirred and filtered on a pressure filter machine.
To 1700000 parts by volume of the resultant filtrate was
added 500000 parts by volum~ of w ter and9 ln a Podb~lniak*
(Podbielniak~ Inc., U.S.A.)g the mixture was extracted
wlth 1000~00 parts by volume of ethyl acetateO The
ethyl acetate layer was washed with water, dried by the
addition of anhydrous sodium sulfate and concentrated
under reduced pressure. To the concentrate was added
petroleum ether and the resultant precipitate was recovered
by filtration and dried. By the above procedure was
obtained 680 parts of crude product I. Thereafterg as
in Examples 2 and 3 9 this crude product was purified to
obtain 1.1 parts of maytanacine,2,2 parts of maytansinol
propionate, and 0.1 part of maytansinol.
*Trademark
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