CA1175423A - Method of preparing 23-deoxy-5-0- mycaminosyltylonolide - Google Patents

Abstract

ABSTRACT

A method is described for producing 23-deoxy-5-0-mycaminosyltylonolide of formula by mild acid hydrolysis of 23-de(mycinosyloxy)tylosin.
It is an antibacterial agent.

Description

~ 17a423 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 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 ,, 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).

, , .

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.

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 ( 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

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~

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 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 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 ( 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~

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 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.

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r~ Z ~ E~ Z ~ a ( 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 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 ~

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.

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 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 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.

Claims (5)

Claims:

1. The method of preparing 23-deoxy-5-0-mycamino-syltylonolide or its 20-dihydro derivative which comprises treating 23-de(mycinosyloxy)tylosin or its 20-dihydro derivative with a mild acid solution for a time and at a temperature sufficient to cleave the mycarosyl group from the de(mycinosyloxy)tylosin.

2. The method of preparing 23-deoxy-5-O-mycamino-syltylonolide which comprises treating 23-de(mycinosyloxy) tylosin with a mild acid solution for a time and at a temperature sufficient to cleave the mycarosyl group from 23-de(mycinosyloxy)tylosin.

3. The method of claim 2 wherein the 23-de-(mycinosyloxy)tylosin is present in the fermentation broth in which it is produced.

4. The method of preparing 20-dihydro-23-deoxy-5-0-mycaminosyltylonolide which comprises treating 20-dihydro-23-de(mycinosyloxy)tylosin with a mild acid solution for a time and at a temperature sufficient to cleave the mycarosyl group from the 20-dihydro-23-de (mycinosyloxy)tylosin.

5. The method of claim 4 wherein the 20-dihydro-23-de(mycinosyloxy)tylosin is present in the fermentation broth in which it is produced.