U.S. patent application number 10/379136 was filed with the patent office on 2003-08-21 for ansamitocins.
This patent application is currently assigned to SmithKline Beecham plc. Invention is credited to Fulston, Mark.
Application Number | 20030157669 10/379136 |
Document ID | / |
Family ID | 22725075 |
Filed Date | 2003-08-21 |
United States Patent
Application |
20030157669 |
Kind Code |
A1 |
Fulston, Mark |
August 21, 2003 |
Ansamitocins
Abstract
Improved purification methods for ansamitocins are
disclosed.
Inventors: |
Fulston, Mark; (Redhill,
GB) |
Correspondence
Address: |
GLAXOSMITHKLINE
Corporate Intellectual Property - UW2220
P.O. Box 1539
King of Prussia
PA
19406-0939
US
|
Assignee: |
SmithKline Beecham plc
|
Family ID: |
22725075 |
Appl. No.: |
10/379136 |
Filed: |
March 4, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10379136 |
Mar 4, 2003 |
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09828758 |
Apr 9, 2001 |
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6573074 |
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60196361 |
Apr 12, 2000 |
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Current U.S.
Class: |
435/119 ;
540/460 |
Current CPC
Class: |
C12P 17/181 20130101;
A61P 35/00 20180101; C12P 17/188 20130101 |
Class at
Publication: |
435/119 ;
540/460 |
International
Class: |
C12P 017/18 |
Claims
1. A method for preparing purified ansamitocins comprising the
steps of: a. culturing an ansamitocin-producing microorganism in a
liquid culture medium; b. treating the culture medium to facilitate
solvent extraction of ansamitocins; c. extracting ansamitocins from
the culture medium with an aromatic hydrocarbon solvent; d.
concentrating the extracted ansamitocins; and e. purifying the
ansamitocins by crystallization.
2. A method for preparing purified ansamitocins comprising the
steps of: a. culturing an ansamitocin-producing microorganism in a
liquid culture medium; b. extracting ansamitocins from the culture
medium with an aromatic hydrocarbon solvent; c. concentrating the
extracted ansamitocins; and d. purifying the ansamitocins by
crystallization.
3. The method of claim 1 or 2 wherein the ansamitocin producing
microorganism is Actinosynnema spp.
4. The method of claim 3 wherein the Actinosynnema spp. is
Actinosynnema pretiosum ATCC 31565.
5. The method of claim 3 wherein the Actinosynnema spp. is
Actinosynnema pretiosum ATCC 31281.
6. The method of claim 1 wherein the treatment is heating at about
75.degree. C.
7. The method of claim 1 or 2 wherein the solvent is toluene.
8. The method of claim 7 wherein the ratio of toluene to
heat-treated culture medium is about 1:1.
9. The method of claim 8 wherein the extraction is at about
45.degree. C.
10. The method of claim 1 or 2 wherein the solvent is xylene.
11. The method of claim 10 wherein the ratio of xylene to
heat-treated culture medium is about 1:1.
12. The method of claim 11 wherein the extraction is at about
45.degree. C.
13. The method of claim 1 or 2 wherein the ansamitocins comprise
acylated ansamitocins that can undergo reductive cleavage to form
maytansinol.
14. Ansamitocins prepared by the process of claim 1 or 2.
15. A cell-binding agent maytansinoid complex prepared by
converting ansamitocins prepared by the process of claim 1 or 2
into the cell-binding agent maytansinoid complex.
16. The cell-binding agent maytansinoid complex of claim 15 wherein
the cell-binding agent is an antibody.
Description
FIELD OF THE INVENTION
[0001] This invention relates to processes for the preparation of
ansamitocins, in particular ansamitocins which can be converted to
maytansinol.
BACKGROUND OF THE INVENTION
[0002] Highly cytotoxic maytansinoid drugs and their therapeutic
use have been described in U.S. Pat. No. 5,208,020. These drugs can
be prepared from ansamitocin precursors produced by fermentation of
microorganisms such as Actinosynnema.
[0003] Under defined culture conditions, Actinosynnema spp. such as
Actinosynnema pretiosum produce a number of related ansamitocins.
The major product is ansamitocin P-3 with an isobutyryl moiety at
the C-3 position. Other ansamitocins differing only in the C-3 acyl
side chain are produced as minor components, P-1 (ethionyl moiety),
P-2 (propionyl moiety), P-3' (butyryl moiety), P-4 (isovaleryl
moiety) and P-4' (valeryl moiety). All these compounds may undergo
reductive cleavage to produce a. common product, maytansinol (form
P-0). In addition, a number of other ansamitocins are produced at
low levels, which are modified at other sites in the molecule
(hydroxylated or n-demethylated). These do not produce the desired
P-0 on deacylation.
[0004] Processes for ansamitocin P-3 production from Actinosynemma
spp. have been described in U.S. Pat. Nos. 4,162,940; 4,228,239;
4,356,265; and 4,450,234. In general, these methods require adding
a filter aid and a water-miscible solvent to whole fermentation
broth, removing solids and extracting the aqueous fraction with a
water-immiscible solvent, concentrating and precipitating with
petroleum ether, purifying the precipitate using silica
chromatography, and crystallizing followed by further
chromatography or recrystallization. Alternative processes utilize
Diaion HP-10 adsorption instead of silica chromatography followed
by further solvent extraction and crystallization.
[0005] These processes can be used to gain acceptable yields of
ansamitocin P-3, but the methods involve a large number of stages
which introduce limitations in large-scale production operations,
particularly because of the extremely toxic nature of the
ansamitocin compounds and the necessity of ensuring the safety of
the human operators of the processes. Thus, a need exists to have a
safer alternative procedure available, utilizing fewer and more
contained stages.
SUMMARY OF THE INVENTION
[0006] One aspect of the present invention is a method for
preparing purified ansamitocins comprising the steps of:
[0007] a. culturing an ansamitocin-producing microorganism in a
liquid culture medium;
[0008] b. treating the culture medium to facilitate solvent
extaction of ansamitocins;
[0009] c. extracting ansamitocins from the culture medium with an
aromatic hydrocarbon solvent;
[0010] e. concentrating the extracted ansamitocins; and
[0011] f. purifying the ansamitocins by crystallization.
[0012] Another aspect of the present invention is a method for
preparing purified ansamitocins comprising the steps of:
[0013] a. culturing an ansamitocin-producing microorganism in a
liquid culture medium;
[0014] b. extracting ansamitocins from the culture medium with an
aromatic hydrocarbon solvent;
[0015] c. concentrating the extracted ansamitocins; and
[0016] d. purifying the ansamitocins by crystallization.
DETAILED DESCRIPTION OF THE INVENTION
[0017] All publications, including but not limited to patents and
patent applications, cited in this specification are herein
incorporated by reference as though fully set forth.
[0018] Methods are provided for preparing purified ansamitocins
without a filtration step. The methods comprise the steps of
culturing an ansamitocin-producing microorganism in a liquid
culture medium, treating the culture medium to release ansamitocins
from the microorganism into the culture medium thereby facilitating
solvent extraction, extracting ansamitocins from the treated
culture medium with an aromatic hydrocarbon solvent, concentrating
the extracted ansamitocins, and purifying the ansamitocins by
crystallization. Alternatively, the treatment step can be
omitted.
[0019] The purified ansamitocins may be reduced to maytansinol and
include ansamitocin P-3, P-1, P-2, P-3', P-4 and P-4'. The purified
ansamitocins contain only very low levels of undesirable
ansamitocins with modifications at other sites in the molecule.
Preferably, the ansamitocin-producing microorganism is
Actinosynnema spp. Particularly preferred is Actinosynnema
pretiosum ATCC 31565. Also particularly preferred is Actinosynnema
pretiosum ATCC 31281. The microorganisms can be grown by
fermentation culture techniques well-known to those skilled in the
art such as those disclosed in U.S. Pat. No. 4,450,234.
[0020] One embodiment of the method of the invention uses treatment
of the microorganism to aid release of intracellular ansamitocins
and render cell-associated ansamitocins more amenable to solvent
extraction. Exemplary treatment methods include sonication,
increased pressure or increased temperature. Preferably, the
treatment is a heat treatment. The heat treatment can be conducted
at about 60.degree. C. to about 80.degree. C. Preferably, the heat
treatment step is conducted at about 75.degree. C. which kills the
microorganism and facilitates solvent extraction of ansamitocins.
In an alternative embodiment, no treatment step is used prior to
extraction since approximately 60% of total ansamitocins can be
found in the culture medium.
[0021] Ansamitocins can be extracted from culture medium with the
use of aromatic hydrocarbon solvents. The aromatic hydrocarbons
have a particular selectivity for the ansamitocins over other broth
constituents, which ensures only simple processes are required
downstream to isolate pure product. Preferably, the aromatic
hydrocarbon solvent is toluene or xylene. Particularly preferred is
toluene as this is more amenable to low temperature evaporation.
The properties of toluene and xylene are such that the solvent and
aqueous layers are readily separated under gravity without the need
for mechanical separation and thus greater containment of the
process and increased operator safety are achieved. The extraction
can be conducted at about 20.degree. C. to about 60.degree. C.
Preferably, the extraction is conducted at about 45.degree. C. The
pH of the aqueous solution prior to extraction should be in the
range 3 to 9. Preferably, the pH is near neutrality, i.e., pH 6 to
8.
[0022] In general, one equivalent volume of solvent to whole broth
(1:1) is preferred for the extraction. Alternative ratio ranges of
2:1 to 1:4 can also be used. The solutions are generally mixed
slowly with stirring and the mixture stirred until about >80% of
the ansamitocins have been extracted into the organic layer.
Preferably the mixture is left to settle under gravity at a
temperature between 15.degree. C. and 50.degree. C. The preferred
temperature for settling is about 45.degree. C.
[0023] The organic layer is removed, and concentration of the
extract by volume reduction of the solvent may be carried out in
vacuo or by other methods well-known to those skilled in the art.
After volume reduction, the concentrated extract can be optionally
dissolved in a polar solvent such as methanol, and clarified using
a membrane filter such as PTFE, or a depth filter such as silica or
alumina.
[0024] Crystallization is used to purify the desired ansamitocins,
particularly P-3, by reducing the levels of unwanted ansamitocins.
A preferred solvent mixture for crystallization is ethyl acetate
and heptane. Such crystallization is performed in a conventional
manner. To aid dissolution of the solid for crystallization, a
small volume of methanol or similar polar solvent may be added
prior to addition of ethyl acetate and then treatment with larger
volumes of heptane, optionally with stirring and cooling to afford
the crystallized product. The product may be recrystallized
following the same procedure.
[0025] Alternatively, prior to crystallization, the impure
ansamitocins extracted from the fermentation broth may be purified
using silica gel, e.g., by passing a solution of the solvent
extract through a bed of silica. Solvents used for the
chromatography may be toluene and toluene-methanol mixes. Other
solvents known to those skilled in the art can also be used. The
fractions containing ansamitocin P-3 are pooled and concentrated
under reduced pressure.
[0026] Methods are also provided for analysis of ansamitocins by
HPLC. Quantitation of ansamitocin P-3 and analysis of ansamitocin
ratios are achieved by the methods. These methods were employed in
the Examples set forth below.
[0027] Quantitation of ansamitocin P-3 in broth and extraction
samples can be determined on a C18 Waters Q Spherisorb S5 ODS2
column, 4.6.times.250 mm, with a 10 mm guard column. UV detection
is at 252 nm and 205 nm. An isocratic mobile phase of 1 ml/min 60%
MeCN (0.05% TFA) in water (0.05% TFA) and a 20 .mu.l injection
volume are used.
[0028] Analysis of ansamitocin ratios in downstream process samples
can be determined on a C8 Waters Symmetry Shield column,
3.9.times.150 mm, with no guard column. UV detection is at 252 nm
and 205 nm. A gradient mobile phase of 1 ml/min 35-45% MeCN (0.05%
TFA) in water (0.05% TFA) over 30 minutes with a 10 minute
re-equilibration at 40.degree. C. and a 10 .mu.l injection volume
are used.
[0029] Analysis of ansamitocin ratios in chromatography fractions
and final product can be determined on a C8 Waters Symmetry Shield
column, 3.9.times.150 mm, with no guard column with an LC-MS
detection system, atmospheric pressure electrospray ionisation, +ve
ion mode. 30V cone voltage Mass detection to positively identify
the peaks is achieved by full scan MS (scan from 600-700 amu with
quad 1). MS-MS fragmentation to determine class of ansamitocin is
carried out using the same gradient system. A gradient mobile phase
of 1 ml/min 35-45% MeCN (0.05% TFA) in water (0.05% TFA) over 30
minutes with a 10 minute re-equilibration at 40.degree. C. and a 10
.mu.l injection volume are used.
[0030] Quantitation of ansamitocin P-3 in waste streams and other
low-level samples requiring high sensitivity can be determined on a
C18 Waters Spherisorb S5 ODS2 column, 4.6.times.250 mm, with a 10
mm guard column and LC-MS-MS atmospheric pressure electrospray
ionisation detection system, +ve ion mode, 30V cone voltage. For
determination of structural type, the molecular ions of the major
species were selected and the fragmentation patterns observed were
a predominant 547 ion indicating N-methylated and a 533 indicating
N-demethylated. An isocratic mobile phase of 1 ml/min 60% MeCN
(0.05% TFA) in water (0.05% TFA) and a 20 .mu.l injection volume
are used.
[0031] The process of the invention can be used to make
cell-binding agent/maytansinoid complexes which are useful as
tumor-activated pro-drugs. Ansamitocins prepared by the process of
the invention can undergo reductive cleavage to maytansinol which
can be used as described in U.S. Pat. No. 5,208,020 to produce
N-methyl-L-alanine containing maytansinoid derivatives. These
derivatives are then conjugated to cell-binding agents, preferably
antibodies, via various linkers such as a disulfide link.
[0032] An exemplary cell-binding agent/maytansinoid complex can be
prepared by a process comprising the following steps:
[0033] (1) reducing ansamitocins prepared by the process of the
invention to maytansinol;
[0034] (2) esterifying maytansinol with N-methyl-L-alanine
derivatives to form a disulfide-containing maytansinoid ester;
[0035] (3) reducing the disulfide-containing maytansinoid ester
prepared by step (2) to a thiol-containing maytansinoid;
[0036] (4) introducing dithiopyridyl groups into a cell-binding
agent; and
[0037] (5) linking the thiol-containing maytansinoid produced by
step (3) to the dithiopyridyl cell-binding agent of step (4) by a
disulfide link.
[0038] The present invention will now be described with reference
to the following specific, non-limiting examples.
EXAMPLE 1
Extraction of Actinosynnema pretiosum Culture Broth and Ansamitocin
Purification
[0039] 37L of whole broth containing the producing strain
Actinosynnema pretiosum ATCC 31565 (ansamitocin P-3 titer 86.3
mg/L) were heat-treated in situ at 75.degree. C. for 60 mins to
kill the micro-organism and facilitate solvent extraction of the
ansamitocins. 40L of toluene were added and the mixture warmed to
45.degree. C. Phases were agitated such that a vortex of upper
toluene phase was drawn into the lower broth phase but without
emulsification or complete homogenisation of the phases. Extraction
was completed within 16 hours, and separation under gravity within
2 hours.
[0040] 39L of toluene containing 80 mg/L P-3 was recovered by
siphon, and evaporated using a 20L rotary evaporator (bath
temperature 40-45.degree. C., rate .about.9L/hr). 11.2 g of mobile
oil, containing 3.1 g of P-3; 27.6% w/w) was generated after
evaporation. The resulting extract was transferred to a flask by
dissolution in toluene and re-evaporation. (Extraction stage
yield=97%)
[0041] The extract was taken up in 120 ml toluene and loaded onto a
silica column (Kieselgel 60, 125 ml bed volume packed in toluene, 4
cm diam..times.10 cm) in 375 ml toluene (3 bv). The column was
washed with 2 bv toluene and then eluted with 4.times.2 bv of 2%
MeOH in toluene, followed by 12.times.1 bv of 4% MeOH in toluene.
The column was eluted at 40 ml/min and produced tight bands of
color. Fractions 7-10 containing ansamitocin P-3 were bulked and
evaporated to produce 3.2 g of oily solid containing 2.5 g of P-3.
This material was analyzed by LC-MS and MS-MS.
[0042] At this stage the product contained 85.1% ansamitocin P-3,
and a total of 93.9% of the desired ansamitocins. (Column stage
yield=80.6%)
[0043] The product from the silica column was taken up in 200 mL
EtOAc warmed to 40.degree. C. Heptane (200 mL) was added and the
solution allowed to cool. The solution was seeded with 1 mg pure
P-3 crystals (crystallization also spontaneously occurred at other
sites in the flask). After 4 hours at ambient temperature the
supernatant was analyzed by HPLC and 0.8 g P-3 (30%) was determined
to still be in solution. Further heptane was added (150 ml) and the
flask left for a further 3 hours and re-analysed. This indicated
that 0.4 g of P-3 (16%) remained in solution. The flask was left
overnight at 4.degree. C. Subsequent analysis indicated that only
70 mg of P-3 (3%) remained in solution. The mother liquors were
removed by aspiration, using a sintered filter line assembly. The
white needle crystals were washed with 2.times.15 mL 1:3
EtOAc:heptane. The crystals were dried in situ under vacuum on a
rotary evaporator at 30.degree. C. for 10 hours. 2.5 g of crystals
were obtained. (Crystallization yield=86%)
[0044] The final product contained 86% ansamitocin P-3, and a total
of 98.4% of the desired acylated ansamitocins (P-1, P-2, P-3, P-3',
P-4, P-4').
EXAMPLE 2
Extraction of Actinosynnema pretiosum Culture Broth and Ansamitocin
Purification
[0045] 1,100L of whole broth containing the producing strain
Actinosynnema pretiosum ATCC 31565. (ansamitocin P-3 titer 75.1
mg/L, 82.6 g P-3) were heat-treated in situ at 75.degree. C. for 60
mins to kill the microorganism and facilitate solvent extraction of
the ansamitocins. 77.6 g of P-3 remained after the heat kill
process. An equal volume of toluene pre-warmed to 45.degree. C. was
added and the mixture was maintained at 45.degree. C. Phases were
agitated such that a vortex of upper toluene phase was drawn into
the lower broth phase but without emulsification or complete
homogenization of the phases. Extraction was carried out for 45
hours, followed by separation under gravity which occurred within
30 min. (Extraction stage yield 90.3%.)
[0046] 1,127L of the toluene extract, containing the ansamitocins,
were concentrated to 22L using a falling film evaporator (FFE). The
concentrate was transferred to a 50L rotary evaporator and
evaporated to low volume (evaporation rate 14.6L/hr). The FFE was
rinsed with 2.times.20L toluene and the rinsings passed to the
evaporator to ensure complete transfer of product. The concentrate
was evaporated to dryness.
[0047] The dry extract was taken up in 7.2L of 4% methanol in
toluene and loaded onto a silica column (Kieselgel 60, 4.8L bed
volume packed in 4% methanol in toluene, 15.0 cm diam..times.27.0
cm, loading rate 120 mL/min). The column was eluted isocratically
using 4% methanol in toluene at a flow rate of 227-384 mL/min and
produced tight bands of color. Initial fractions were one bed
volume; fractions 3 to 6 were collected as half bed volumes.
Fractions were monitored by TLC (Kieselgel 60 F.sub.254 plates, run
in 5% methanol in dichloromethane, visualized by UV at 254 nm) and
those containing ansamitocins were monitored by HPLC and LC-MS.
Fraction selection for crystallization was based on HPLC and LC-MS
analysis of the fractions to optimize ansamitocin P-3 recovery and
minimize undesired ansamitocins. Fractions 5 to 10 containing
ansamitocin P-3 were bulked and evaporated to produce a solid
containing 54.6 g of P-3. At this stage the product contained 77.1%
ansamitocin P-3, and a total of 96.1% of the desired ansamitocins.
(Column stage yield=92.5%)
[0048] The product from the silica column was taken up in 91 mL
methanol previously warmed to 46.degree. C., followed by 546 mL
ethyl acetate warmed to the same temperature. Further aliquots of
methanol were added to aid dissolution of the solid. A total of 166
mL of methanol was added to the mixture. Heptane (100 mL) warmed to
50.degree. C. was added to the first sign of cloudiness and then
the solution was allowed to cool to ambient as crystallization
commenced. After 4 hours, a further 1,324 mL of heptane (at ambient
temperature) was added. The supernatant was analyzed by HPLC and
6.6 g P-3 was determined to still be in solution. The mixture was
cooled on ice and further aliquots of heptane (200 and 400 mL) were
added until 3.9 g of P-3 (6.8%) remained in the mother liquor.
[0049] The mother liquors were removed by aspiration, using a
sintered filter line assembly. The crystals were washed with
2.times.150 mL 1:3 ethyl acetate:heptane. The crystals were dried
in situ on a rotary evaporator, initially under low vacuum,
followed by high vacuum (1.0-1.3 mbar) at 30.degree. C. for 88.5
hours. 76.4 g of crystals were obtained.
[0050] The final product contained 74.5% ansamitocin P-3 (56.9 g)
and a total of 97.8% of the desired acylated ansamitocins. (Overall
yield=69%)
EXAMPLE 3
Silica Chromatography and Crystallization of the Ansamitocins
[0051] 1,008L of Actinosynnema pretiosum whole broth with a titer
of 64.8 mg/L ansamitocin P-3 were heat treated, extracted with
toluene and evaporated essentially as described in Example 1.
[0052] The concentrate containing 34.5 g ansamitocin P-3 was taken
up in 3 L toluene and loaded onto a silica column (Kieselgel 60,
3.0 L bed volume, packed in toluene, 13.8 cm diam..times.16.6 cm).
The column was topped with a 4 cm bed of sand. The column was
washed with 5L of toluene, followed by 20L of 2% MeOH in toluene,
which was collected as 5 L fractions. The column was then eluted
with 20 L of 4% MeOH in toluene, collected as 2.5 L fractions. The
ansamitocins were eluted in fractions 9 through to 15. Fraction
selection for crystallization was based on HPLC and LC-MS analysis
of the fractions to optimize ansamitocin P-3 recovery and minimize
undesired ansamitocins. Fractions 10 to 12 were bulked and
evaporated to dryness to yield an oil containing 32.5 g of
ansamitocin P-3.
[0053] At this stage the product contained 91.8% ansamitocin P-3,
and a total of 95.3% of the desired acylated ansamitocins. (Column
stage yield=94.2%)
[0054] The concentrate from the bulked silica fractions, was warmed
in a water bath at 50.degree. C. and dissolved in a minimum volume
of warm methanol/ethyl acetate (50.degree. C.). 60 mL of methanol
was added initially, followed by slow addition of 300 mL of ethyl
acetate. A further 20 mL of warm methanol was added at which point
the concentrate was completely dissolved. 200 mL of warm heptane
(50.degree. C.) was added and the crystallization solution removed
from the water bath. Crystallization commenced, and further heptane
was added in 200 mL aliquots until a total volume of 800 mL had
been added. The mixture was cooled in an ice bath for 18 hours. The
crystallization was monitored by HPLC analysis of the mother
liquors. At 18 hours 6.3% ansamitocin P-3 remained in the mother
liquors. A further 200 mL of heptane was added and the mixture
cooled for a further five hours. HPLC analysis indicated 4.0%
ansamitocins remained in the mother liquors.
[0055] The crystals were recovered by aspiration of the dark brown
mother liquors. The crystals were washed three times with 50 ml of
heptane:ethyl acetate, 3:1 and were dried under vacuum (0.8 mBar)
overnight. The crystals were uniform, fine, off-white needles
containing 28.2 g of ansamitocin P-3. (Crystallization stage
recovery=86.9%)
[0056] The final product contained 93.1% ansamitocin P-3 and a
total of 98.4% desired acylated ansamitocins.
EXAMPLE 4
Purification and Crystallization of Ansamitocins from Toluene
Extract
[0057] 1,001L of Actinosynnema pretiosum whole broth with a titer
of 74.5 mg/L ansamitocin P-3 were heat treated, extracted with
toluene and evaporated essentially as described in Example 1 to
give 9.5 L of concentrated extract containing 54.7 g of ansamitocin
P-3.
[0058] The concentrate was heated to 45.degree. C. and 14.5 L of
heptane was added over 33 min to precipitate the ansamitocins. The
mixture was cooled on ice. A further 5 L of heptane was added and
the mixture allowed to settle overnight. The mother liquor was
removed by aspiration and the precipitate was washed with 10 L of
toluene:heptane, 1:1. HPLC analysis indicated the mother liquor
contained 6.5% of ansamitocin P-3, and the wash contained 1.8% P-3.
The precipitate was dissolved in 2 L of methanol and filtered
through a 0.2 micron filter.
[0059] The methanol solution was evaporated to dryness and
re-dissolved in 85 mL of methanol at 50.degree. C. 510 mL of ethyl
acetate was added to the solution followed by 200 mL of heptane.
The mixture was cooled to ambient and a further 990 mL of heptane
was slowly added as crystallisation commenced. 4.14 g of
ansamitocin P-3 remained in the mother liquor. A further 400 mL of
heptane was added and the mixture cooled on ice. 2.7 g of
ansamitocin P-3 remained in the mother liquor.
[0060] The mother liquor was removed by aspiration and the crystals
were dissolved in 110 mL of methanol and 520 mL ethyl acetate at
50.degree. C. Heptane was added in two 75 mL aliquots, the mixture
was cooled to ambient and another 900 mL added. A further 400 mL of
heptane was added and the mixture cooled on ice and left overnight
to crystallize. HPLC analysis indicated 2.3 g of ansamitocin P-3
remained in the mother liquor. The mother liquor was removed by
aspiration and the crystals were washed with heptane:ethyl acetate,
3:1 and were dried under vacuum (0.6 mbar) overnight. The 47.8 g of
crystals contained 84.3% ansamitocin P-3, and a total of 97.0%
desired acylated ansamitocins.
EXAMPLE 5
Extraction of Actinosynnema pretosium Culture Broth with Xylene and
Ansamitocin Purification
[0061] 220 mL of heat treated Actinosynnema pretiosum, with a titre
of 44 ug/ml ansamitocin P-3, were placed in a 45.degree. C. water
bath and an equal volume of xylene was added. The phases were mixed
such that a vortex of xylene was drawn in to the lower broth phase
but without forming an emulsion. After 20 hours, 64% of the
ansamitocin P-3 had extracted into the xylene phase.
[0062] The mixture was separated under gravity and 170 mL of xylene
were removed. The xylene extract was evaporated to dryness. The
xylene extract was purified using silica chromatography. The
extract was dissolved in 2 mL of 4% methanol in toluene and loaded
and eluted using the same solvent mix. Fractions (0.5 mL) were
collected and assayed by TLC (Kieselgel 60 F.sub.254 plates run in
dichloromethane:methanol 20:1). Fractions containing ansamitocin
P-3 were crystallized using the procedure described in previous
examples. The crystalline product was of comparable quality in
terms of colour and purity with that produced by toluene extraction
of fermentation broth.
[0063] The present invention may be embodied in other specific
forms without departing from the spirit or essential attributes
thereof, and, accordingly, reference should be made to the appended
claims, rather than to the foregoing specification, as indicating
the scope of the invention.
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