Note: Descriptions are shown in the official language in which they were submitted.
! 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
22
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
X-5425 -5-
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.
.
-
. 1 1 7~869
X-5425 -6-
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.
.
.... . ___ ., . . . _
! 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.
X-5425 -8-
.
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
. , .
- X-5425 -9-
., .
,.
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~... ,.. ~... ' ' '
1 171869
.
.
- X-5425 -10-
:'
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
.. . ..
.
X-5425 -12-
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.
.
' 171869
L~
. ~ i
X-54Z5 -13- -
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
X-5425 -14-
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
X-5425 -15-
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
X-5425 -16-
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.
i
