CA1171869A - Tylactone - Google Patents

Abstract

Abstract of the Disclosure Acyl esters of tylactone (20-dihydro-20,23-dideoxytylonolide), which have the formula:

Description

! 171869 :

TYLACTONE
Summary of_the Invention This invention relates to a new macrolide derivative from which useul antibiotics, such as tylosin and tylosin derivatives, can be prepared. This new derivative is an acyl ester of 20-dihydro-20,23-dideoxy-tylonolide (referred to hereinafter as tylactone).
Tylactone has structure 1:

/\
~
~8 CH3-~1z ~t-CHz-CH~

2;~ -OH
CH~-t14 1 t4 C7H~-CHz-~ ~ ~ \ / -OH

The acyl ester derivatives of this invention have structure 2: ~ .
~ ~!~

~11 7 I

CH3-il12 1e ~t CH2-Co 23 / ~ / s~ 1 CH~-t1 4 1 t4 CH~ CH2 ~ -OR

. 2 wherein R and Rl = an acyl moiety. Each of the esters is an ester of a monocarboxylic acid or a hemi-ester of a dicarboxylic acid, each of 1 to 18 carbon atoms.
''' ;
:

~k ~ t718~9 The novel compounds of structure 2 are useful intermediates from which 16-membered macrolide anti-biotics can be prepared. Although no stereochemical assignments are indicated in the structures given herein, the stereochemistry of the compounds is identical to that of tylosin.
Description of the Drawing The infrared absorption spectrum of tylactone in chloroform is presented in the accompanying drawing.
Detailed Description The following paragraphs describe the proper-ties of tylactone.
TYlactone The structure of tylactone is shown in formula 1. Tylactone is a white solid which crystallizes from hexane or ethyl acetate-hexane and which melts at about 162-163C. It has the following approximate percentage elemental composition: carbon, 70%; hydrogen, 9.7%;
oxygen, 20.3%. It has an empirical formula of C23H38O5 and a molecular weight of about 394.
The infrared absorption spectrum of tylactone in chloroform is shown in the accompanying drawing.
Observable absorption maxima occur at the following frequencies ~cm 1): 3534 (medium), 2924 (strong), 2398 (weak), 2353 (weak), 1709 (very strong), 1678 (very strong), 1626 (small), 1592 (very strong), 1458 (strong), 1441 (shoulder), 1404 (strong), 1379 (small), 1316 (strong), 1284 (medium), 1181 (very strong), 1143 ~7~869 X-5425 _3_ .
(#trong), 1103 (medium), 1078 (medium), 1049 ~very small), 1025 (medium), 984 (very strong), 958 (strong), 923 ~medium), 911 (shoulder), 859 (small), 868 (medium) r 840 (medium), 820 (very small) and 661 (small).
The ultraviolet (W) absorption spectrum of tylactone in neutral ethanol exhibits an absorption maximum at about 282 nm (ElCm = 560).
Tylactone has the following specific rotation:
1 ]D 55.23 (c 1, CH3OH).
~ 10 Electrometric titration of tylactone in 66%
,~ ~ aqueous dimethylformamide indicates it has no titrata-ble groups.
Tylactone is nearly insoluble in water, j but ~s soluble in organic solvents such as acetone, methanol, ethanol, dimethylformamide, chloroform, diethyl ether, petroleum ether, benzene and dimethyl ~ulfoxide.
~ Tylactone can be distinguished from tylosin i by silica-gel thin-layer chromatography. Sulfuric acid spray, either concentrated or dilute ~50%), may be used for detection. With this detection system tylactone appears initially as a yellow-to-brown spot. If silica-gel plates with a fluorescent background are ; used in the chromatography, W detection is convenient.
The approximate R~ values of tylactone are summarized in Table 1.
: ., , ~ 17~8~9 X-5425 _4_ Table 1 Thin-Layer Chromatography of Tylactonea Rf Value ._ Compound A~ B
Tylactone 0.50 0.62 Tylosin 0.0 0.0 a~edium: Silica gel bSolvent: A = benzene: ethyl acetate (4:1) B = benzene: ethyl acetate (3:2) i Ester Derivatives Tylactone can be esterified at the 3- and 5-hydroxyl groups to give acyl ester derivatives by treatment with acylating agents using methods known in the art. The acyl ester derivatives of tylactone are u~eful as intermediates in the preparation of new macrolide antibiotics.

Typical acylating agents 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. Acylations can also be carried out enzymatically using procedures such as those described b~ Okamoto et al. in U.S. 4,092,473.

Once formed, the acyl derivatives can be separated and purified by known techniques.

The derivatives can be prepared by esteri-fication techniques generally known in the art, such , .

, ~ t71869 as, for example, treatment of the compound with a sto$chiometric quantity (or a slight excess) of an acylating agent, such as an acyl anhydride, in an organic solvent (for example, pyridine) at about 0C to a~out room temperature for from about 1 to about 24 hours until esterification is substantially complete.
The ester derivative can be isolated from the reaction m~xture by standard procedures such as extraction, chromato~raphy and crystallization.
10Useful esters are those of organic acids including aliphatic, cycloaliphatic, aryl, aralkyl, heterocyclic carboxylic, sulfonic and alkoxycarbonic ac~ds of from 1 to 18 carbon atoms, and of inorganic aclds, such as sulfuric and phosphoric acids.
lS Representative suitable esters include those d-rived from acids such as formic, acetic, chloro-acetlc, propionic, butyric, isovaleric, glucuronic, alkoxycarbonic, stearic, cyclopropanecarboxylic, cyclohexanecarboxylic, ~-cyclohexylpropionic, 1-20 adamantanecarboxylic, benzoic, phenylacetic, phenoxy- ¦
acet~c, mandelic and 2-thienylacetic 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-sters derived from dicarboxylic acids such as succinic,maleic, fumaric, malonic and phthalic acids.

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-. 1 1 7~869 Preparation of Tylactone Tylactone is prepared by culturing a strain of Streptomyces fradiae which produces this compound under submerged aerobic conditions in a suitable culture medium until a substantial amount of the compound is produced.
- The culture medium used to grow the Strepto-myces fradiae can be any one of a number of media For , . I
economy in production, optimal yield, and ease of -; 10 product isolation, however, certain culture media are ipreferred. Thus, for example, preferred carbon sources in large-scale fermentation include carbohydrates such as dextrin, glucose, starch, and corn meal and oils such as soybean oil. Preferred nitrogen sources include corn meal, soybean meal, fish meal, amino acids ~nd the like. Among the nutrient inorganic salts which can be ~ncorporated in the culture media are the customary soluble salts capable of yielding iron, potas~ium, sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, and like ions.
,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 ~uch as polypropylene glycol (M.W. about 2000) to large-scale fermentation media if foaming becomes a problem.

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.... . ___ ., . . . _ ! 171869 For production of substantial quantities of tylactone submerged aerobic fermentation in tanks s is preferred. Small quantities of tylactone may be obtained by shake-flask culture. Because of the time lag in production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vege-tative 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 organism. The vegetative inocu-lum is then transferred to a larger tank. The medium used for the vegetative inoculum can be the same as that used for larger fermentations, but other media can also be used.
A preferred method of preparing tylactone is . that disclosed by Richard H. Baltz and Eugene T. Seno ln a co-pending Canadian Patent application Serial No.
380,946 entitled PROCES FOR PREPARING TYLACTONE. That ~0 method comprises culturing a new microorganism which was obtained by chemical mutagenesis of a streptomYces fradiae strain which produced tylosin. The micro-organism obtained by mutagenesis produces only minimal amounts of tylosin, but produces tylactone as a major component.
The new microorganism which produces tylactone is classified as a strain of Streptomyces fradiae. A
culture of this microrganism has been deposited and made part of the stock culture collection of the Northern Regional Research Center, Agricultural Research, ~ 1718B9 o , L.

.
North Central Region, 1815 North University Street, : Peoria, Illinois, 61604, from which it is available to the public under the accession number NRRL 12188.
As is the case with other organisms, the S characteristics of Streptomyces fxadiae NRRL 12188 are subject to variation. For example, recombinants, mu-tants or variants of the NRRL 12188 strain may be obtained by treatment with various known physical and chemical . mutagens, such as ultraviolet light, X-rays, gamma .. 10 rays, and N-methyl-N'-nitro-N-nitrosoguanidine. All natural and induced variants, mutants and recombinants of Streptomyces fradiae NRRL 12188 which retain the characteristic of tylactone production may be used to - prepare the compounds of this invention.
S. fradiae NRRL 12188 can be grown at tem-. _ peratures between about 10 and about 40C. Optimum production of tylactone appears to occur at temper-~tures of about 28 C.
As is customary in aerobic submerged culture processes, sterile air is bubbled through the culture medium. For efficient antibiotic production the per-cent of air saturation for tank production should be about 30% or above (at 28C and one atmosphere of pressure).
Production of tylactone can be followed during the fermentation by testing samples of the broth, using high-performance liquid chromatography with a W detection system lsee, for example, J.H. Kennedy in J. Chromatographic Science, 16, 492-495 (1978)~.

, , ' 171869 . , .

., .
,.
Following its production under submerged , aerobic fermentation conditions, tylactone can be recovered from the fermentation medium by methods used in the fermentation art. Because of the limited soluhility of tylactone in water, it may not be altogether soluble in the medium in which it is produced. Re-covery of tylactone, therefore, can be accomplished by l) extraction of the fermentation broth or 2) fil-tration of the fermentation broth and extraction of both the filtered broth and the mycelial cake. A
variety of techniques may be used in the extraction processes. A preferred technique for purification of the filtered broth involves extracting the broth (gen-erally without pH adjustment) with a suitable solvent ! 15 such as amyl acetate or petroleum ether, concentrating the organic phase under vacuum to give crystals or an oil. If an oil is obtained, it may be purified by j adsorption chromatography.
The compounds of structures 1 and 2 are useful intermediates from which 16-membered macrolide antibiotics can be prepared. For example, tylactone ~1) can be bioconverted to tylosin by adding it to a growing culture of a bioconverting microorganism~ The bioconverting microorganism can be a Streptomyces strain which either produces tylosin itself or is capable of producing tylosin except that it is blocked in tylactone formation.
A strain which is capable of producing tylosin except that it is blocked in tylactone for-mation can be obtained by treating a tylosin-producing ;, , ' , ''',1~... ,.. ~... ' ' ' .

.

:' strain with a mutagen and screening survivors for those which are unable to produce tylosin. Those survivors which are unable to produce tylosin are further screened to determine which strains are also unable to produce tylactone. These strains are identified by adding tylactone to small shake-flask cultures of the selected ~urvivors to determine if they produce tylosin.
Streptomyces fradiae strains NRRL 2702 and NRRL 2703 àre examples of Streptomyces strains which 10 are capable of producing tylosin. A typical mutagen i which may be used to obtain the selected strains is N-methyl-N'-nitro-nitrosoguanidine.
~, The compound of structure 1 is especially - useful in the preparation of labeled compounds for !, 15 metabolic studies. By labeling either the tylactone F
portion or the added sugar moieties, the metabolic pathway of tylosin can be ascertained.
j In order to illustrate more fully the opera-tton of this invention, the following examples are 20 provided:
Example 1 A. Shake-flask Fermentation of Tylactone A lyophilized pellet of Streptomyces fradiae 25 N~RL 12188 is dispersed in 1-2 ml of sterilized water.
A portion of this solution (0.5 ml) is used to in-oculate a vegetative medium ~150 ml) having the following composition:

:; , .

! 171869 .
.

- x-5425 . .

Ingredient Amount (~) Corn steep liquor 1.0 Yeast extract 0.5 Soybean grits o.5 . 5 CaCO3 0.3 Soybean oil (crude) 0.45 Deionized water 97.25 Alternatively, a vegetative culture of S.
~radiae NRRL 12188 preserved, in l-ml volumes, in li~uid 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 ~ ~out 300 rpm.
t lS This incubated vegetative medium (0.5 ml) i5 u8ed to inoculate 7 ml of a production medium having the follcwing composition:
lngredient Amount (%) Beet molasses 2.0 Corn meal 1.5 Fish meal 0.9 ; Corn gluten o.g NaCl 0.1 ~NH4)2HPO4 0~04 : CaCO3 0.2 - Soybean oil (crude) 3.0 Deionized water 91.36 . 30 :' .

!.1 71 869 - - L
.. . ..

.

The inoculated fermentation me~ium 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 Tylactone In order to provide a larger volume of inocu-lum, 60 ml of incubated vegetative medium, prepared in a manner similar to that described in section A, is , used to inoculate 38 L of a second-stage vegetative : 10 growth medium having the following composition:
i Ingredient Amount (%) __ .._ ... . ......
Corn steep liquor 1.0 ybean meal 0.5 Yeast extract 0~5 CaCO3 , 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.

25.

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' 171869 L~

. ~ i Incubated second-stage medium (4 L) thus pre-- pared is used to inoculate 40 liters of sterile produc- tion medium having the following composition:
Ingredient Amount (%) Fish meal 0.92 Corn meal 1.57 Corn gluten 0.92 CaCO3 0.21 NaCl 0.10 i (NH4)2HP04 O.04 Beet molasses 2.10 Soybean oil ~crude) 3.15 Lecithin o.og ; lS Water go.go Ad~ust pH to 7.2 with 50% NaOH 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 between about 30% and 50% and is stirred with conventional agitators at about 300 rpm.
. Example 2 Isolation of Tylactone Fermentation broth (1600 L), obtained as ,.
- described in Example-l, is filtered using a filter aid ; (3% Hyflo Supercel, a diatomaceous earth, Johns Manville ; Corp.). The pH of the filtrate is adjusted to about 9 by the addition of 2% sodium hydroxide. The filtrate , , !171~Bg L

is extracted with amyl acetate (400 L). The amyl acetate extract (which has a high optical density reading at 282 nm but no antimicrobial activity) is concentrated under vacuum to ~ive an oil. The oil is dissolved in benzene (5 L). The benzene solution is chromatographed over a 5.25- x 36-in. silica-gel ~Grace, grade 62, Davison Chemical Co.) column, packed with benzene. Elution is monitored by silica-gel thin-layer chromatography, using a benzene:ethyl acetate (3:2) solvent system and conc. sulfuric acid spray for detection. The column is first eluted with benzene to remove lipid substances, then with benzene:ethyl , acetate (9:1) to separate and isolate tylactone.
! Fractions containing tylactone are combined and evap-i 15 orated under vacuum. Tylactone is crystallized from benzene-hexane or hot hexane to give about 2 g, m.p.
16Z-163C.
Example 3

3,5-Di-O-acetyltylactone Tylactone (200 mg), prepared as described in Example 2, is dissolved in pyridine (4 ml). Acetic anhydride (4 ml) is added. The resulting mixture is allowed to stand at room temperature for 16 hours and then is concentrated to dryness under vacuum. Methanol (5 ml) is added to the residue; the solution is heated at 60 for 1/2 hour and then is concentrated under ; vacuum to give 3,5-di-O-acetyltylactone. This compound has an Rf value of about 0.59 on silica-gel thin-layer chromatography in a benzene:ethyl acetate ~4:1) solvent system. The Rf of tylactone in this system is about 0.3.

.1 "

~ 1718B9 L

Examples 4-7 3,S-Di-O-Propionyltylactone, prepared according to the procedure of Example 3, but using propionic anhydride.
3,5-Di-O-isovaleryltylactone, prepared according to the procedure of Example 3, but using ~sovaleric anhydride.
3,5-Di-O-benzoyltylactone, prepared according to the procedure of Example 3, but using benzoic ; 10 anhydride.
3,5-Di-O-(n-butyryl)tylactone, prepared ; according to the procedure of Example 3, but using n-butyric anhydride.
Example 8 ration of Tylosin from Tylactone A Streptomyces fradiae strain which formerly produced tylosin but which is blocked in macrolide ring closure is fermented according to the procedure de-Jcribed in Example 1, Section A, except that a tem-perature of 28C is used. Tylactone is added to ; the fermentation 48 hours after inoculation. The fermentation is then continued until a substantial amount of tylosin i9 produced, i.e. about three ad-ditional days. The presence of tylosin is determined by testing samples of the broth against organisms known to be sensitive to tylosin. One useful assay organism i8 Sta~ lococcus aureus ATCC 9144. Bioassay is conveniently performed by an automated turbidometric method, by thin-layer chromatography or by high-performance liquid chromatography with W detection.

:' ` i .

- ! 17185g Example 9 Tylactone is prepared by the method of ~.
Example 1 except that a labeled acetate, propionate~
or butyrate is incorporated into the fermentation medium. Labeled tylactone thus produced is used to prepare tylosin according to the procedure of Example 8. ~.
Tylosin labeled on the macrolide ring is thereby provided.
Example 10 Tylactone, prepared by the method of Example 1, is used to prepare tylosin according to the method of Example 8 except that a labeled sugar moiety such as glucose is added to the second fermentation to provide tylosin which is labeled on the sugar moiety.

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Claims (12)

Claims:

1. A process for preparing an acyl ester derivative of tylactone which has the structure:

wherein each of said esters is an ester of a mono-carboxylic acid or a hemi-ester of a dicarboxylic acid, each of 1 to 18 carbon atoms, by reacting tylactone with a suitable acylating agent.

2. An acyl ester of tylactone as defined in claim 1 whenever prepared by the process of claim 1, or by an obvious chemical equivalent thereof.

3. The process of claim 1 wherein the acylating agent is acetic anhydride.

4. The compound 3,5-di-0-acetyltylactone, whenever prepared by the process of claim 3, or by an obvious chemical equivalent thereof.

5. The process of claim 1 wherein the acylating agent is propionic anhydride.

6. The compound 3,5-di-0-propionyltylactone, when-ever prepared by the process of claim 5, or by an obvious chemical equivalent thereof.

7. The process of claim 1 wherein the acylating agent is benzoic anhydride.

8. The compound 3,5-di-0-benzoyltylactone, when-ever prepared by the process of claim 7, or by an obvious chemical equivalent thereof.

9. The process of claim 1 wherein the acylating agent is isovaleric anhydride.

10. The compound 3,5-di-0-isovaleryltylactone, whenever prepared by the process of claim 9, or by an obvious chemical equivalent thereof.

11. The process of claim 1 wherein the acylating agent is n-butyric anhydride.

12. The compound 3,5-di-0-(n-butyryl)tylactone, whenever prepared by the process of claim 11, or by an obvious chemical equivalent thereof.