Note: Descriptions are shown in the official language in which they were submitted.
~ 17a423
METHOD OF PREPARING 23-DEXOCY-5-O-MYCAMINOSYLTYLONOLIDE
Summary of the Invention
This invention relates to a process for producing
23-deoxy-5-O-mycaminosyltylonolide and its 20-dihydro
derivative from the new macrolide antibiotic, 23-de(mycino-
syloxy)tylosin, and to its 20-dihydro derivative and is a
divisional of Canadian application Serial No. 379,332
filed June 9, 1981. 23-De(mycinosyloxy)tylosin, which
will be called de(mycinosyloxy)tylosin or DMOT for
convenience herein, has structure 1:
~-/o \~-CH3
/1' 7-\ ~H
CH3-il~ 2 ~ ~ 6T-CH32-2o
z3 j C~
\O ~ /1 2 ~ ~ ~H
0~ 5, - -CH3,~2- 3~3
\ 4;/ o ~ -OH
CH3 rr - _~
~ ~H~
Although no stereochemical assignments are indicated in
the structures given herein, the stereochemistry of the
compounds is identical to that of tylosin. The neutral
sugar in structure 1 is mycarose, and the amino-sugar in
1 is mycaminose. The dihydro-derivative of DMOT, i.e.
20-dihydro-23-de(mycinosyloxy)tylosin, will be called
dihydro-DMOT for convenience herein.
1 ~75~23
X-5141 -2-
Dihydro-DMOT has structure 2:
CH3-0 \t-CH2-CHzOH
C~3
~3-t/ ~T/ \
10~-CH2-~ 3
~ ~ - -CH3 f - \CH3
t- ~
~H3 ~__s
~Hs
DMOT and dihydro-DMOT inhibit the growth of
organisms which are pathogenic to animals. More speci-
fically, they are antibacterial agents whicn are espe-
cially active against gram-positive microorganisms and
Mycoplasma species.
The hydroxyl groups of DMOT and dihydro-DMOT
can be esterified on the 2', 4~, 3" and 3-hydroxy~ groups
to form useful acyl ester derivatives. In addition,
dihydro-DMOT can be esterified on the 20-hydroxyl group.
Esterification of the 2'-hydroxyl group is most facile.
Typical esters are those of a monocarboxylic acid or
hemi-esters of a dicarboxylic acid having from 2 to 18
carbon atoms.
DMOT, dihydro-DMOT and their acyl este.
~ 175423
derivatives are basic compounds which, when treated with
acids, are converted to acid addition salts. These
addition salts are also part of this invention. To
simplify discussions of utility, the term "DMOT compound"
is used and refers to DMOT, dihydro-DMOT, a specified acyl
ester derivative of these compounds, or a pharmaceutically
acceptable acid addition salt of DMOT, dihydro-DMOT or of
their acyl ester derivatives.
Also described is a new strain of Streptomyces
fradiae, NRRL 11271, and the method of producing DMOT or
dihydro-DMOT by culturing this strain under submerged
aerobic fermentation conditions until a substantial level
of antibiotic activity is produced. DMOT or dihydro-DMOT
can be extracted from basified broth filtrate with polar
organic solvents and can be further purified by adsorptive
or extractive procedures.
This invention relates to a new method of
preparing 23-deoxy-5-O-mycaminosyltylonolide (abbreviated
herein as DOMT) and 20-dihydro-23-deoxy-5-O-mycaminosyl-
tylonolide (dihydro-DOMT) by mild acid hydrolysis of DMOT
or dihydro-DMOT, respectively. DOMT has structure 3:
11~5~23
,,
X-5141 -4-
t~
\O ~ C~3
t ~ ~H
~3
Des¢ription of the Drawin~
The infrared absorption spectrum of DMOT
(free base) in chloroform is presented in the accompa-
nying drawing.
Detailed Descrip~ion
The following paragraphs describe the proper-
ties of DMOT.
DMOT
The structure of DMOT is shown in formula 1.
DMOT is a white amorphous solid which softens at about
158 and melts at about 165-167C. Elemental analysis
indicates that it has the followins approximate per~ent-
age composition: carbon, 62~; hydrogen, 8%; nitrogen,
2%; oxygen, 27%. It has an empirical formula of
C38H63NO12 and a molecular weight of about 126 ~725 as
determined by mass spectrom2try).
,
,
.
4 2 3
X-5141 -5-
The in~rared absorption spectrum of DMOT (free
base) in chloroform is shown in the accompanying drawing.
Observable absorption maxima occur at the following fre-
quencies (cm 1): 3653 (small), 3588 (shoulder), 3470
(broad), 3026 (shoulder), 2998 (shoulder), 2969 (intense~,
2932 (intense), 2873 (shoulder), 1709 (intense), 1669
(medium), 1616 (v. small), 1583 (intense), 1447 (medium),
1400 (medium), 1364 (medium), 1309 (medium), 1278
(small), 1175 (medium), 1151 (medium), 1106 (small), 1066
(shoulder), 1036 (intense), 1001 (medium), 982 (medium),
972 (shoulder), 946 (small), 913 (v. small), 891 (v.
small), 853 (v. small), 826 (small).
The ultraviolet absorption spectrum of DMOT
in neutral ethanol exhibits an absorption maximum at
283 nm (~ 21,500).
DMOT (free base) has the following specific
rotation:
talD -62.75 (c 1, CH30H).
Electrometric titration of DMOT in 66~
aqueous dimethylformamide indicates the presence of a
titratable group with a PKa value of about 7.3.
DMOT free base is sparingly soluble in water,
but is soluble in most polar organic solvents, such as
acetone, methanol, ethanol, dimethylformamlde, chloro-
form and dimethyl sulfoxide. DMOT acid addition salts
are more soluble in water than is DMOT base.
DMOT can be distinguished from tylosin and
from DOMT by paper and thin-layer chromatography. The
approximate Rf and RY values of these antibiotics are
3 summarized in Tables 1 and 2. In Table 2 Rx value is
the ratio of movement expressed relative to that of
tylosin, which was given a value of 1Ø Bioautography
with Bacillus subtilis was used for detection.
1 175423
X-5141 -6-
Table 1
Thin-Layer Chromatography of DMOT
_Rf Value
Compound Ab B C
Tylosin 0.530.53 0.67
DMOT 0.700.56 0.67
DOMT 0.480.17 0.24
aMedium: Merck, Darmstadt - Silica Gel 60
bSolvent: A = ethyl acetate:diethylamine (96:4)
B = acetone:ethanol (2:1)
C = chloroform:methanol (3:1)
Table 2
Paper Chromatography of DMOTa
RX
Compound Db E
Tylosin 1.00 1.00
DklOT 1.50 1.09
DOMT 0.50 0.97
25 aPaper: Whatman No. 1 treated with 0.75 M
RH2PO4 buffer at pH 4.0 and dried
bSolvent: D = ethyl acetate saturated with
water
E = n-butanol saturated with water
~ 175423
X-5141 ~7~
Dihydro-DMOT
Dihydro-DMOT can be obtained by chemical
reduction or by fermentation. When preparing dihydro-
DMOT by chemical reduction, known procedures such as,
for example, treatment with an approximately stoichio-
metric amount of sodium borohydride in an alcoholic
solvent, may be used. Dihydro-D~OT is also produced by
the S. fradiae NRRL 11271 of this invention under
controlled fermentation conditions.
Ester Derivatives
_
DMOT and dihydro-DMOT can be esterified at
the 2', 4", 3" and 3-positions to give acyl ester
derivatives by treatment with acylating agents using
methods known in the art. In addition, dihydro-DMOT
can be esterified at the 20-position. Esterification
of the 2'-hydroxyl group is most facile. Typical
acylating agent~s include anhydrides, halides (usually
in combination with a base or other acid scavenger) and
active esters of organic acids. Acylation can also be
achieved by using a mixture of an organic acid and a
dehydrating agent such as N,N'-dicyclohexylcarbodiimide.
~cylations can also be carried out en~ymatically as
described by Okamoto et al. in U.S. 4,092,-173. Once
formed, the acyl derivatives can be separatQd and
purified by known techniques.
The 2'-monoester derivatives can be prepared
by selective esterification techniques generally known
in the art, such as, for example, treatment of the
antibiotic with a stoichiometric quantity (or a slight
1 175423
(
X-5141 -8-
excess) of an acylating agent, such as an acyl anhydride,
at about room temperature for from about 1 to about 24
hours until esterification is substantially complete.
The 2'-monoester can be isolated from the reaction
mixture by standard procedures such as extraction,
chromatography and crystallization.
Useful esters are those of organic acids
including aliphatic, cycloaliphatic, aryl, aralkyl,
heterocyclic carboxylic, sulfonic and alkoxycarbonic
acids of from 2 to 18 carbon atoms, and of inorganic
acids, such as sulfuric and phosphoric acids.
Representative suitable esters include those
derived from acids such as acetic, chloroacetic, pro-
pionic, butyric, isovaleric, alkoxycarbonic, stearic,
cyclopropanecarboxylic r cyclohexanecarboxylic, ~-
cyclohexylpropionic, l-adamantanecarboxylic, benzoic,
phenylacetic, phenoxyacetic, mandelic and 2-thienyl-
acetic acids, and alkyl-, aryl-, and aralkyl-sulfonic
acids, the aryl- and aralkyl- acids optionally bearing
substituents such as halogen, nitro, lower alkoxy and
the like on the aromatic moiety. Suitable esters also
include hemi-esters derived from dicarboxylic acids
such as succinic, maleic, fumaric, malonic and phthalic
acids.
Pharmaceutically acceptable ester derivatives
are a preferred group. Other ester derivatives are
useful, however, as intermediates.
, ~ O
1 175423
X-5141 -9-
Salts
DMOT, dihydro-DMOT and their specified acyl
derivatives form acid addition salts. The acid addit-
ion salts of DMOT, dihydro-DMOT and of their acyl
derivatives are also part of this invention. Such salts
are useful, for example, ~or separating and purifying
DMOT, dihydro-DMOT and their acyl derivatives. In addi-
tion the salts have an improved solubility in water.
Representative suitable salts include those
salts formed by standard reactions with both organic
and inorganic acids such as, for example, sulfuric,
hydrochloric, phosphoric, acetic, succinic, citric,
lactic, maleic, fumaric, palmitic, cholic, pamoic,
mucic, D-glutamic, d-camphoric, glutaric, glycolic,
phthalic, tartaric, formic, lauric, stearic, salicylic,
methanesulfonic, benzenesulfonic, sorbic, picric,
benzoic, cinnamic and li~e acids.
Pharmaceutically acceptable acid addition
salts are an ~specially preferred group of salts of
this invention. "Pharmaceutically acceptable" salts
are salts in which the toxicity of the compound as a
whole toward warm-blooded animals is not increased
relative to the non-salt form.
Preparaticn of DO~T and Dihydro-DOM~
This invention relates to new methods
of preparing 23-deoxy-5-0-.~ycaminosyltyionolide (3
(DOMT) and di'nydro-~O~T by mild acid ~ydrolysis of DMO~
and dihydro-DMOT, respactively. ~ild acid hydrolysis
conditions are known in the art. Appropriate solutior.s
~ ~L7542~
X-5141 -10-
having a pH of about four or below can be used to
accomplish the hydrolysis. Temperatures of about 20
to about 100C can be used in this method. The reaction
time needed to carry out the hydrolysis varies, depend-
ing upon the pH of the reaction mixture and the tempera-
ture used. At higher pH levels the reaction rate is
slower, and at higher temperatures the reaction rate is
faster. The reaction is carried out by treating either
DMOT or dihydro-DMOT with a mild acid solution for a
time sufficient to effect removal of the mycarosyl
~roup to give DOMT or dihydro-DOMT, respectively.
Alternatively, and sometimes preferably,
DOMT or dihydro-DOMT can be prepared by treating DMOT
or dihydro-DMOT in the fermentation broth in which it
is produced, using mild acidic conditions as above
described for a time sufficient to convert the DMOT or
dihydro-DMOT to DOMT or dihydro-DOMT, respectively.
DOMT or dihydro-DOMT thus prepared can be isolated from
the fermentation broth using techniquss known in the art.
DOMT is identical to depoxycirramycin Al
(de-epoxycirramycin Al). The preparation and activity
of depoxycirramycir. Al are described by H. Tsukiura et
al. in J. Antibiotics _ (3), 89-99, and 100-105 (1969).
Tsukiura et al. prepare depoxycirramycin A1 by treating
cirramycin Al with potassium iodide in acetic acid.
Another potential method of making ~O~T is
suggested by T. Suzuki et a3. in Chemistry Letters 1973,
793-798. This method involves treating antibiotic
B-58941 with potassium iodide in acetic acid to obtain
a product which "may be identical with depoxycirramycin
A "
1- ~
.
~ 175~2~
X-5141 -ll-
DOMT is also related to M-4365 G2 (repromicin)
and rosamicin, being 4'-hydroxy-M-4365 G2 or de-epoxy-
4'-hydroxy-rosamicin, respectively [see A. Kinumaki et
al., J. Antibiotics 30 (6), 450-454 (1977)]. Prepara-
tion of DOMT from either M-4365 G2 or rosamicin, how-
ever, would be impractical.
Preparation of DMOT and Dihydro-~MOT by S. fradiae.
DMOT and dihydro-DMOT are prepared by cultur-
ing a strain of Streptomyces fradiae which producesthese compounds under submerged aerobic conditions in a
suitable culture medium until substantial antibiotic
activity is produced. As wiil be appreciated by those
s~illed in the art, DMOT is produced first in the
fermentation process. Dihydro-DMOT is produced when
the fermentation is carried out for a longer time, thus
permitting the DMOT present to be reduced enzymatically.
- The culture medium used to grow Streptomyces
fradiae NRRL 11271 can be any one of a number of media.
For economy in production, optimal yield, and ease of
product isolation, however, certain culture media are
preferred. Thus, for example, preferred carbon sources
in large-scale fermentation include carbohydrates such
as dextrin, slucose, starch, and corn meal and oils
such as soybean oil. Preferred nitrogen sources include
corn meal, soybean meal, fish meal, amino acids and the
like. Among the nutrient inorganic salts which can be
incorporated in the culture media are the customary
soluble salts capable of yielding iron, potassium,
sodium, magnesium, calcium, ammonium, chloride, carbonate,
sulfate, nitrate, and like ions.
1 17 ~423
X-5141 -12-
Essential trace elements necessary for the
growth and development of the organism should also be
included in the culture medium. Such trace elements
commonly occur as impurities in other constituents of
the medium in amounts sufficient to meet the growth
requirements of the organism. It may be necessary to
add small amounts (i.e. 0.2 ml/L) of an antifoam agent
such as polypropylene glycol (M.W. about 2000) to
large-scale fermentation media if foaming becomes a
problem.
For production of substantial quantities of
DMOT or dihydro-DMOT, submerged aerobic f ermentation in
tanks is preferred. Small quantities of DMOT or
dihydro-DMOT may be obtained by shake-flask culture.
Because of the time lag in antibiotic production commonly
associated with inoculation of large tanks with the
spore form of the organism, it is preferable to use a
vegetative inoculum. The vegetative inoculum is prepared
by inoculating a small volume of culture medium with
the spore form or mycelial fragments of the organism to
obtain a fresh, actively growing culture of the orga~ism.
The vegetative inoculum is then transferred to a larger
tank. The medium used for the vesetative inoculum can
be the same as that used for larger fermentations, but
other media can also be used.
S. fradiae NRRL 11271 can be grown at tem-
peratures between about 10 and about 37C. Optimum
antibiotic production appears to occu at temperatures
of about 28C.
~ :17~4~3
X-5141 -13-
As is customary in aerobic submerged culture
processes, sterile air is bubbled through the culture
medium. For efficient antibiotic production the percent
of air saturation for tan~ production should be about
30% or above (at 28C and one atmosphere of pressure).
Antibiotic production can be followed during
the fermentation by testing samples of the broth against
organisms known to be sensitive to these antibiotics.
One useful assay organism is Staphylococcus aureus ATCC
9144. The bioassay is conveniently performed by an auto-
mated turbidometric method. In addition, antibiotic
production can be readily monitored by high-performance
liquid chromatography with W detection.
Following its production under submerged
aerobic fermentation conditions, D~OT or dihydro-DMOT
can be recovered from the fermentation medium by methods
used in the fermentation art. Reco~ery of DMOT or
dihydro-DMOT is accomplished by an initial filtration
of the fermentation broth. The filtered broth can then
be further purified to give the desired antib.otic. A
variety of techniques may be used in this purification.
A preferred technique for purification of the filtered
broth involves adjusting the broth to about pH 9;
extracting the broth with a suitable solvent such as
ethyl acetate, amyl acetate or methyl isohutyl ketone;
extracting the organic phase with an aqueous acidic
solution; and precipitating the antibiotic by making
the aqueous extract basic. Further purification involves
the use of extraction, adsorption and/or precipitation
techniques.
.
1 ~75423
(
X-5141 -14-
The Microorganism
~ he new microorganism of this invention was
obtained by chemical mutagenesis of a Streptomyces
fradiae strain which produced tylosin. The micro-
J oxganism obtained by mutagenesis produces only minimalamounts of tylosin, but produces DMOT as a major
component.
For characterization purposes, the new
organism was compared with Streptomyces fradiae strain
M48-E 2724.1, a tylosin-producing strain derived from
S. fradiae NRRI 2702. S. fradiae NRRL 2702 was dis-
_
closed by Hamill et al. in U.S. Patent 3,178,341,
issued April 13, 1965. In the discussions herein the
tylosin-producing S. fradiae M48-E 2724.1 culture will
be called "E2724.1".
The new strain which produces DMOT and
dihydro-DMOT, NRRL 11271, is also classified as a strain
of Streptomyces fradiae. In characterizing this organ-
ism, the methods recommended for the International
Streptomyces Project for the characterization of
Streptomyces species have been followed [E. B. Shirlingand D. Gottlieb, "Methods For Characterization of
Streptomyces S~ecies," Internal. Journal of Systematic
..
Bacteriology, 16 (3), 313-340 (1966~] along with certain
supplementary tests. The following references to S.
fradiae in the literature were consulted: 1) R. E.
Buchanan and N. E. Gibbons, "Bergey's ~anuai of Detex-
minative Bacteriology," 8th ed., The Williams and
Wilkins Co., Baltimore, Md., 1974, p. 815; and 2) E. B.
Sllirling and D. Gottlieb, "Cooperative Description of
1 ~7~2~
X-5141 -15-
Streptomyces. II. Species Description from FirstStudy," Internal. Journal of Systematic Bacteriology,
8 (2), 118, (1968).
The following description of the s~rain which
produces DMOT compares its characteristics with those
of the tylosin-producing S. fradiae strain "E2724.1".
Characterization of the Microorqanism
The spore-chain morphology of the new strain
and of the E2724.1 strain is in the Retinaculum-
Apertum (RA) section. Hooks, loops, and irregular
coils are short and generally not of a wide diameter.
This is best observed on ~SP#2 (yeast-malt extract
agar) for strain E2724.1 and on Czapek's solution agar
for the new strain. The spore surface is smooth; the
spore shape is spherical with an average size of
0.65 ~M in diameter. The diameter range is from 0.61
to 0.71 ~M.
The most obvious differences between these
strains are seen in their cultural characteristics~
The E2724.1 strain produces aerial mycelia fairly well
on most media and is in the White color series. The
new strain of this invention produces very little if
any aerial mycelia. When present, it is in the White
2S to Gray color series. The reverse sides of these
colonies have no distinctive pigments produced. They
are light to moderate yellow in color. Melanoid pig-
ment production is negativel.
lMelanoid-pigment pr~duction was tested using ISP~
(tryptone-yeast extract broth), ISPit6 (peptone
yeast extract-iron agar), ISPit7 (tyrosine agar),
and IS~#7 agar without tyrosine.
~ 175~23
X-5141 -16-
A summary of the important similarities and
differences between the E2724.1 strain and the new
strain of this invention is given in Table 3.
~ .
1 ~75~23
X-5141 -17-
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1 17$~23
( X-5141 -18-
The morphology and growth characteristics of
the S. fradiae E2724.1 and NRRL 11271 strains are com-
pared in Table 4. In the tables which follow the anti-
biotic sensitivities (Table 5), carbon utilization
S (Table 6) and miscellaneous physiological character-
istics (Table 7) are compared.
~ 175~ 2 3
X-5141 -19-
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X-5141 -20-
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1 17542~
X-5141 -21-
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~ ~75423
~-5141 -22-
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X-5141 -23-
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~ 175423
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1 175423
X-5141 -25-
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1 175423
( X-5141 -26-
Based on the foregoing characteristics ~he
organism which produces DMOT and dihydro-DMOT, NRRL 11271,
is classified as a new strain of Streptomyces fradiae.
This new culture has been deposited and made part of
S the stock culture collection of the Northern Regional
Research Center, Agricultural Research, North Central
Region, 1815 North University Street, Peoria, Illinois,
61604, from which it is available to the public under
the accession number NRRL 11271.
As is the case with other organisms, the
characteristics of Streptomyces fradiae NRRL 11271 are
subject to variation. For example, recombinants, mu-
tants or artificial variants of the NRRL 11271 strain
may be obtained by treatment with various known physical
and chemical mutagens, such as ultraviolet light, X-rays,
gamma rays, and N-methyl-N'-nitro-N-nitrosoguanidine.
All natural and artificial variants, mutants and recom-
binants of Streptomyces fradiae NRRL 11271 which retain
the characteristic of DMOT production may be used in
this invention.
Activity of The DMOT Compounds
The DMOT compounds inhibit the growth of
pathogenic bacteria, especial1y gram-positive bacteria
and Mycoplasma species. For example, Table ~ summarizes
ths minimal inhibitory concentrations (MIC), as measured
by standard agar-dilution assays, at whlch DMOT (free
base) inhibits certain bacteria.
I
~ 175~23
X-5141 -27-
Table 8
In Vitro Activity of DMOT Free Base
.
Organism MIC (~g/ml)
Streptococcus pyogenes C203 0.25
Streptococcus pneumoniae Park I 0.13
Streptococcus sp. (Group D) 282 0.5
Staphylococcus aureus 3055 1.0
Pasteurella multocida 6.25
10 Pasteurella hemolytica 25.00
Mycoplasma gallisepticum 0.097
Mycoplasma hyopneumoniae 0.39
Mycoplasma h~orhinis 0.78
15 The DMOT compounds have shown ln vivo anti-
microbial activity against experimental bacteria~
infections. When two doses of test compound were
administered to mice in experimental infections, the
activity observed was measured as an ED50 value ~effec-
tive dose in mg/kg to protect 50~ of the test animais:
see Warren Wick, et al., J. Bacteriol. 81, 233-235
(1961)]. An ED50 value observed for DMOT is
given in Table 9.
,
- ~ 175423
X-~141 -28-
Table 9
ED50 Value of DMOT
-
Test Streptococcus
Com~ound
DMOT Free Base 6.3
Bacterial
Challenge (X LD50) 268
aSubcutaneous; mg/kg x 2
For the prevention or treatment of Mycoplasma
infections in poultry, an effective non-toxic amount of
a DMOT compound is administered to birds orally or
parenterally. DMOT compounds are most conveniently
administered with a pharmaceutically acceptable carrier,
such as the water ingested by the birds.
In order to illustrate more f~lly the operation
of this invention, the following examples are provided:
Example 1
~o A Shake-flask FPrmentation of DMOT
.
A lyophilized pellet of Streptomyces fradiae
NRRL 11271 is dispersed in 1-2 ml of sterilized water.
A portion of this solution (0.5 ml) is used to inoculate
a vegetative medium ~150 ml) having the following
composition:
Ingredient Amount (%)
Corn steep liquor 1.0
Yeast extract 0.5
Soybean grits 0 5
CaCO3 0-3
Soybean oil (crude) 0.45
Deionized water 97.25
1 175~23
( X-5141 -29-
Alternatively, a vegetative culture of S.
fradiae NRRL 11271 preserved, in l-ml volumes, in liquid
nitrogen is rapidly thawed and used to inoculate the
vegetative medium. The inoculated vegetative medium is
incubated in a 500-ml Erlenmeyer flask at 29C. for
about 48 hours on a closed-box shaker at about 300 rpm.
This incubated vegetative medium (0.5 ml) is
used to inoculate 7 ml of a production medium having the
following composition:
IngredientAmount (%)
Beet molasses 2.0
Corn meal 1.5
Fish meal 0.9
Corn gluten 0 9
NaCl ~ 0.1
( H4)2~P4 0~04
CaCO3 0.2
Soybean oil (crude)3 0
Deioni~ed water 91.36
The inoculated fermentation medium is incu-
bated in a 50-ml bottle at 29C. for about 6 days on a
closed-box shaker at 300 rpm.
B. Tank Fermentation of DMOT
In order to provide a larger voiume of lnocu-
lum, 60 ml of incubated vegetative m~dium, prepared in
a manner similar to that described in section A, is
used to inoculate 38 L of a secor.d-stage vegetative
growth medium having the following composition:
1 17~42 ~
X-5141 -30-
Ingredient Amount (~)
Corn steep liquor 1.0
Soybean oil meal 0.5
Yeast extract 0.5
CaCO3 0.3
Soybean oil (crude) 0.5
Lecithin (crude) 0.015
Water 97.185
Adjust pH-to 8.5 with 50% NaOH solution.
This second-stage vegetative medium is incu-
bated in a 68-liter tank for about 47 hours at 29C.
Incubated second-stage medium (4 L) thus pre-
pared is used to inoculate 40 liters of sterile produc-
i5 tion medium having the following composition:
Ingredient Amount (~)
Fish meal 0.9i88
Corn meal 1.575
Corn gluten 0.9188
CaCO3 0.210
NaCl 0.105
(NH4)2HP4 0.042
Beet molasses 2.10
Soybean oil (crude) 3.15
Lecithln 0,0945
Water 90.8859
Adjust pH to 7.2 with 50~ ~aOH solution.
1175423
X-5141 -31-
The inoculated production medium is allowed
to ferment in a 68-liter tank for about 5 days at a
temperature of 28C. The fermentation medium is aerated
with sterile air to keep the dissolved oxygen level be-
tween about 30-~ and so% and is stirred with conventional
agitators at about 300 rpm.
Example 2
Isolation of DMOT
Fermentation broth, obtained as described in
Example 1, and having a pH of 7.2, is filtered usins a
filter aid. Ethyl acetate (400 ml) is added to the
filtrate (1450 ml). The pH of the solution is adjusted
to 9.1 by the addition of sodium hydroxide. The solu-
tion is stirred 10 minutes, and the ethyl acetate is
separated (filtering through a filter aid to clear any
emulsion which fcrms). The filtrate is again extracted
with ethyl acetate (200 ml). Water (200 ml) is added
to the combined ethyl acetate extracts; the pH of this
solution is adjusted to 4.1 with phosphoric acid. After
extraction, the aqueous phase is separated, and the
organic phase is discarded. The aqueous phase is
adjusted to pH 9.1 with sodium hydroxide and ~hen
concentrated to a volume of about 100 ml under vacuum.
An amorphous precipitate fcrms. After permitting the
precipitate to stand overnight, it is separated by
filtration. The precipitate is dissolved in acetone
(20 ml); water (75 ml) is added. The solution is
concentrated under vacuum to remove acetone. The
precipitate which forms is separated by filt-ation ar:d
1 ~75423
X-5141 -32-
washed with water to give about 500 mg of DMOT (1). An
additional 260 mg is obtained in a similar manner from
the filtrate.
Example 3
Preparation of DOMT
DMOT (11 g), prepared as described in Example 2,
is dissolved in a dilute hydrochloric acid solution
(HCl added to water until the pH of the solution is
1.8). The resulting solution is allowed to stand for
24 hours at room temperature and then is adjusted to pH
9.0 by addition of sodium hydroxide. This basic solu-
tion is extracted with chloroform. The chloroform
extract is dried under vacuum to give 9.65 g of DOMT (3).
Example 4
Preparation of Dihydro-DMOT
DMOT (50 mg), prepared as described in Example 2,
is dissolved in an aqueous isopropyl alcohol solution
(approximately 40~; 25 ml). Sodium borohydride (20 mg)
is dissolved in a 30% aqueous isopropyl alcohol solu-
tion (10 ml). The NaBH4 solution (1 ml) is added to
the sGlution containing DMOT. The resulting mi~ture is
stirred for 5 minutes, is ad,usted to pH 7.5 with
phosphoric acid, and is concentrated under vacuum to
remove 'he isopropyl alcohol. Chloroform (50 ml) is
added. The pH of the aqueous phase is adjusted to 7.~.
After extraction, the chlo~oform is separated and
evaForated .o dryness under vacuum to give dihydro-DMOT.
~ 175~23
X-5141 -33-
Example 5
Preparation of Dihydro-DOMT
Dihydro-DMOT, prepared as described in
Example 4, is treated in the manner described in
Example 3 to give dihydro-DOMT.
Example 6
Alternative Preparation of DOMT
DOMT is prepared from DMOT by treating DMOT
in the fermentation broth in which it is produced with
mild acid as described in Example 3. Isolation of DOMT
is accomplished by a procedure similar to that described
for DMCT in Example 2.
Example 7
2'-O-Propionyl-DMOT
DMOT is dissol~ed in acetone and treated witn
1.2 equivalents of propionic anhydride at room tempera-
ture for about six hours to give 2'-O-propionyl-DMOT.
Fxamples 8-10
2'-O-Isovaleryl-DMOT, prepared according to
the procedure of Example 7, but using isovaleric anhy-
dride.
2'-O-Benzoyl-DMOT, prepared according to ~he
procedure of E~ample 7, but using benzoic anhydride.
2'-O-(n-Butyryl)DMOT, prepared accordin~ to
~he procedure of Example 7, but using n-butyric anhy-
dride.
