U.S. patent application number 11/014658 was filed with the patent office on 2005-06-09 for hydroxylation of compactin to pravatatin by micromonospora.
Invention is credited to Ambrus, Gabor, Barta, Istvan, Boros, Sandor, Horvath, Gyula, Horvath, Ildiko, Ilkoy, Eva, Jekkel, Antonia, Konya, Attila, Mozes, Julia, Nagy, Zsuzsanna, Salat, Janos, Somogyi, Gyorgy, Szabo, Istvan Mihaly.
Application Number | 20050124051 11/014658 |
Document ID | / |
Family ID | 10992008 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050124051 |
Kind Code |
A1 |
Jekkel, Antonia ; et
al. |
June 9, 2005 |
Hydroxylation of compactin to pravatatin by micromonospora
Abstract
The present invention relates to a new microbial process for the
preparation of compound of formula (I) from a compound of general
formula (II) wherein R stands for an alkali metal or ammonium ion,
by the submerged culture of a strain which is able to
6.beta.-hydroxylate the compound of formula (II) in aerobic
fermentation and by the separation and purification of the product
of formula (I) formed in the course of the biocoversion. The latter
comprises the cultivation of a Micromonospora strain which is able
to 6.beta.-hydroxylate a compound of general formula (II)--wherein
R is as defined above--at 25-32.degree. C. on a nutrient medium
containing available carbon--and nitrogen sources and mineral
salts, thereafter feeding the substrate to be transformed into the
developing culture, then hydroxilating the substrate until
finishing of the bioconversion, then separating the compound of
formula (I) from the culture broth and, if desired, purifying the
same.
Inventors: |
Jekkel, Antonia; (Budapest,
HU) ; Ambrus, Gabor; (Budapest, HU) ; Ilkoy,
Eva; (Budapest, HU) ; Horvath, Ildiko;
(Budapest, HU) ; Konya, Attila; (Dunakeszi,
HU) ; Szabo, Istvan Mihaly; (Budapest, HU) ;
Nagy, Zsuzsanna; (Budapest, HU) ; Horvath, Gyula;
(Budapest, HU) ; Mozes, Julia; (Ecser, HU)
; Barta, Istvan; (Budapest, HU) ; Somogyi,
Gyorgy; (Budapest, HU) ; Salat, Janos;
(Budapest, HU) ; Boros, Sandor; (Szod,
HU) |
Correspondence
Address: |
IVAX CORPORATION
4400 BISCAYNE BOULEVARD
MIAMI
FL
33137
US
|
Family ID: |
10992008 |
Appl. No.: |
11/014658 |
Filed: |
December 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11014658 |
Dec 16, 2004 |
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10030726 |
Jun 4, 2002 |
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10030726 |
Jun 4, 2002 |
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PCT/HU00/00066 |
Jun 29, 2000 |
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Current U.S.
Class: |
435/135 ;
435/197; 435/252.3 |
Current CPC
Class: |
C12R 2001/29 20210501;
C12P 7/42 20130101; C12N 1/205 20210501; C12P 17/06 20130101 |
Class at
Publication: |
435/135 ;
435/252.3; 435/197 |
International
Class: |
C12P 017/06; C12P
007/62; C12N 009/18; C12N 001/21 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 12, 1999 |
HU |
P 9902352 |
Claims
1-7. (canceled)
8. A microbial process for the preparation of the compound of
formula (I) 5from a compound of the general formula (II) 6wherein R
stands for an alkali metal or ammonium ion, by the submerged
culturing of a suitable bacterial strain which is able to
6.beta.-hydroxylate a compound of formula (II) under aerobic
conditions and by the separation and purification of the compound
of formula (I) formed in the course of the bioconversion comprising
the steps of a) cultivating a suitable bacterial strain which is
able to 6.beta.-hydroxylate a compound of formula (II)--wherein R
is as defined above to a compound of the formula (I)--at
25-32.degree. C. on a nutrient medium containing available carbon
and nitrogen sources and mineral salts, thereafter b) feeding the
substrate to be transformed into a developing culture, then c)
hydroxylating the substrate until finishing of the bioconversion,
then d) separating the compound of formula (I) from the culture
broth and, if desired, purifying the same.
9. A process as claimed in claim 1, wherein the bacterial strain
has the 6.beta.-hydroxylate characteristics of Micrononospora.
10. A process as claimed in claim 1, wherein the bacterial strain
has the ability to 6.beta.-hydroxylate the compound of formula (II)
into Pravastatin.
Description
[0001] The present invention relates to a new microbial process for
the preparation of pravastatin.
[0002] More particularly, this invention relates to a microbial
process for the preparation of pravastatin of formula (I) 1
[0003] from a compound of the general formula (II) 2
[0004] wherein R stands for an alkali metal or ammonium ion, with a
microorganism, wherein said microorganism is a prokaryote from
genus Micromonospora, which is able to hydroxylate a compound of
the general formula (II) at the 6.beta.position.
[0005] The hypercholesterolaemia has been recognized as a major
risk factor for atherosclerotic disease, specifically for coronary
heart disease. During the past two decades
3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase
EC. 1.1.1.34) as the major rate-limiting enzyme in the cholesterol
biosynthesis, has been extensively studied. Mevinolin and related
compounds biosynthesised by selected strains of different fungal
species were found to be competitive inhibitors of this enzyme
[Endo, A. et al., J. Antibiotics 29, 1346-1348 (1976); Endo, A. et
al., FEBS Lett. 72, 323-326 (1976): Kuo, C. H. et al., J. Org.
Chem. 48, 1991-1998 (1983)].
[0006] Pravastatin is also a member of the family of HMG-CoA
reductase inhibitors. At first, pravastatin was found as a minor
urinary metabolite of compactin in dog (Tanaka, M. et al.,
unpublished) in the course of metabolic studies of compactin [Arai,
M. et al. Sankyo Kenkyusho Nempo, 40, 1-38 (1988)].
[0007] The main characteristic property of pravastatin as the
hydroxylated product of compactin is its tissue selectivity. This
drug strongly inhibits sterol synthesis in liver and in intestine,
but weakly in other organs. It is advantageous that pravastatin
possesses lower toxicity than the other HMG-CoA reductase
inhibitors.
[0008] It has been reported that microbial hydroxylation of
compactin can be accomplished in various extent by several strains
of species belonging to many different genera of fungi, and by
strains of actinomycete species belonging to the genera Nocardia,
Actinomadura and Streptomyces, among others Streptomyces
roseochromogenes and Streptomyces carbophilus (U.S. Pat. No.
5,179,013, U.S. Pat. No. 4,448,979, U.S. Pat. No. 4,346,227, U.S.
Pat. No. 4,537,859, Japanese Patent No. 58,010,572).
[0009] A problem with using fungi for the production of pravastatin
from compactin is that these organisms generally do not tolerate
higher concentrations of compactin in liquid culture media,
presumably due to its antifungal activity [Serizawa, N. et al., J.
Antibiotics 36, 887-891 (1983)]. In Streptomyces carbophilus the
cytochrome P450 system has been shown to be required for the
hydroxylation of compactin to pravastatin [Matsuoka, T. et al.,
Eur. J. Biochem. 184, 707-713 (1989)]. Difficulty of genetic
improvement of the ability of hydroxylation with the use of such an
enzyme is that it is a complex of proteins rather than a single
protein.
[0010] Our investigation were focused on finding an actinomycete
strain which would produce pravastatin from salts of acidic form of
compactin with higher yield and by applying higher substrate
concentration in the bioconversion than those known from former
patent specifications.
[0011] During the screening, covering about 6000 actinomycetes,
mostly our own isolates, but also authentic strains from
international strain collections, five Streptomyces and five
Micromonospora were selected for further studies, because they
proved to be able to hydroxylate the sodium salt of the acidic form
of compactin into pravastatin. These ten actinomycete strains, from
which eight strains have been taxonomically identified at species
level in our laboratory, were the following:
[0012] Streptomyces violaceus (according to Kmpfer et al. 1991),
strain No. 1/43.
[0013] Streptomyces rochel (Berger et al., 1949; Waksman and
Lechevalier, 1953), strain No. 1/41.
[0014] Streptomyces resistomycificus (Lindenbein, 1952), strain No.
1/44.
[0015] Streptomyces sp., strain No. 1/28.
[0016] Streptomyces lanatus, (Frommer, 1959), strain No. 1/16.
[0017] Micromonospora sp., strain No. IDR-P.sub.3.
[0018] Micromonospora purpurea (Luedemann and Brodsky, 1964),
strain No. IDR-P.sub.4.
[0019] Micromonospora echinospora (Luedemann and Brodsky, 1964),
strain No. IDR-P.sub.5.
[0020] Micromonospora megalomicea (Weinstein et al, 1969), strain
No. IDR-P.sub.6.
[0021] Micromonospora rosaria (Horan and Brodsky, 1986), strain No.
IDR-P.sub.7.
[0022] Since, up to now, there are no data in the literature on the
ability of Micromonospora to convert salts of the acidic form of
compactin into pravastatin, we have thoroughly studied not only
this particular enzymatic ability, but also the taxonomic position
of these above listed strains of Micromonospora.
[0023] Taxonomic Position of Strains IDR-P.sub.3, --P.sub.4,
--P.sub.5, --P.sub.6 and --P.sub.7 at Generic Level
[0024] All of these strains produced well developed mycelia,
composed of branched hyphae of about 0.4-0.7 .mu.m in diameter.
Aerial mycelium is absent or occurs only in traces. Nonmotile
spores are borne on sporophores singly. Hyphae of the substrate
mycelium are Gram-positive and not acid-fast. Stains Nos. IDR
P.sub.3-P.sub.7 are aerobic, chemo-organotrophic and sensitive to
pH below 6.0. Walls contain meso-diaminopimelic acid. The above
listed diagnostic properties--as key characters--clearly
demonstrate. That these monosporic actinomycete strains are typical
members of the genus Micromonospora.
[0025] Taxonomic description of Micromonospora sp., strain No.
IDR-P.sub.3
[0026] Micromorphological properties: Substrate mycelium is
composed of well developed, more curved than straight, monopodially
branching filaments. Spores on the sporophores are single,
spherical approximately 1.8 .mu.m in diameter and dispersing more
or less evenly on hyphal filaments. Spores are either sessile or on
the end of short sporophores. In broth cultures spores were not
observed on the hyphae presumably because the release of mature
spores is very quick.
[0027] Cultural-Macromorphological Properties:
[0028] Czapek-sucrose agar: Medium growth, the colonies have
reddish colour covered by point-like black sporulating areas.
[0029] Glucose-asparagine agar: The growth was recorded as point
like and elevated, reddish-brown or black colonies. Reddish
diffusible pigments.
[0030] Nutrient agar: Fair growth, elevated, reddish-brown or black
colonies. Reddish-brown exopigment in the medium.
[0031] Yeast extract-malt extract agar (ISP Med. 2): Well
developed, elevated and wrinkled, brown colonies, covered partly
with black sporulating areas or with "pseudo-aerial mycelium" (this
is appearing as a restricted whitish or greyish bloom). Brownish or
brownish-red soluble pigment.
[0032] Inorganic salts-starch agar (ISP Med. 4): Medium growth of
reddish-brown elevated and wrinkled colonies. Light reddish soluble
pigment.
[0033] Glycerol-asparagine agar (ISP Med. 5): Growth only in
traces, off-white or light orange coloured, flat colonies, light
rose soluble pigment.
[0034] Carbon source utilization: Good growth on and positive
utilization of L-arabinose, D-galactose, D-fructose, D-glucose,
D-xylose, lactose, meliblose, sucrose, D-mannitol, dulcitol,
glycerol and inositol. Growth with L-rhamnose, D-raffinose and
inulin was slightly better than on the negative control medium.
Nitrogen source utilization: Good growth with yeast extract and
NZ-Amine, no or weak utilization of NaNO.sub.3.
[0035] Other physiological-biochemical properties: Cellulose and
starch are hydrolyzed, milk is digested strongly. Nitrate reduction
test is negative. No growth on potato slices without calcium
carbonate (pH 5.8-6.0). No melanoid pigment production.
[0036] This strain No. IDR-P.sub.3 of Micomonospora sp. was
isolated from a mud sample of Lake Balaton (Hungary).
[0037] Systematic position: Further comparative systematic studies
would be necessary to clarify the exact taxonomic position of this
strain among the species of the genus Micromonospora. On the basis
of certain properties it seems to be not impossible, that strain
IDR-P.sub.3 represents a new species within the genus
Micromonospora.
[0038] Differential-Diagnostic Description and Identification of
Micromonospora Strains IDR-P.sub.4, --P.sub.5, --P.sub.6 and
P.sub.7
[0039] Strain IDR-P.sub.4
[0040] On the above listed diagnostic media, generally, good
growth, orange to orange red, red, sometimes yellowish or rose
coloured colonies. Soluble pigments and aerial mycelium are not
produced. The number of solitary spores is relatively low. They
occur on the sporophores terminally. Substrate mycelium is composed
of well branching hyphae. Aerial mycelium absent. No growth on
D-melibiose, raffinose, mannitol, glycerol, lactose, L-rhamnose but
good growth on D-arabinose, glucose, D-xylose and weak growth on
D-galactose and D-fructose. On the basis of these conventional
diagnostic properties we have identified this strain as a member of
species Micromonospora purpurea (Luedemann and Brodsky, 1964).
[0041] Strain IDR-P.sub.5
[0042] This strain produces mostly solitary sporophores and
sphaerical dark brown to black spores (0.8-1.5 .mu.m in diameter)
which adhere firmly to the sporophores until maturation. According
to our electronmicroscopic observations, on the surface of these
spores warty structures or outgrowths ("blunt spines" according to
the Vol. 4 of Bergey's Manual of Syst. Bact 1989, pages 2448) can
be observed, which is very characteristic of the spores of
Micromonospora echinosora. Otherwise, the cultural-morphological
and physiological diagnostic properties of this strain are also
very similar to those of the M. echinospora. The colour of the well
developed colonies on the standard diagnostic media is orange-brown
or dark purple. The sporulating layer is black or purplish black,
waxy. Aerial mycelium absent. Melanin pigment not produced. Milk
digested. Good growth on D-xylose, D-arabinose, D-glucose, and
sucrose, but no growth with L-rhamnose, raffinose, D-galactose,
D-fructose, D-meliblose and glycerol. We consider this strain as a
typical member of Micromonospora echinospora.
[0043] Strain IDR-P.sub.6
[0044] On the majority of diagnostic media moderate to weak growth.
The orange or orange red colonies consist of long branched
filaments (appr. 0.6 .mu.m in diameter) and a limited number of
solitary, sphaerical, dark coloured spores (0.6-1.0 .mu.m in
diameter). Does not produce aerial mycelium. In certain media weak
reddish or rose coloured soluble pigments are formed. On tyrosine
agar melanold pigments were not produced. On a basal medium the
following carbon sources have been utilized by this strain:
D-xylose and D-fructose; only weakly: D-melibiose, mannitol and
galactose, but no or sporadic growth was observed with glycerol,
L-rhamnose, lactose and raffinose (see also Kawamoto, I. et al.:
Agric. Biol. Chem., 47, 203-215, 1983). Strain No. IDR-P.sub.6
shows a considerable similarity to the species Micromonospora
megalomicea, (Weinstein, 1972) and we consider it as a member of
this species.
[0045] Strain IDR-P.sub.7
[0046] Good to moderate growth on Bennett agar, Czapek sucrose
agar, glucose-asparagine agar, nutrient agar, oatmeal agar,
potato-dextrose agar, etc. The colour of the vegetative mycelial
pigments ranges from reddish-brown to purplish-brown. On certain
media wine red diffusible pigments are formed. On the surface of
the colonies black spots are frequently produced. Vegetative hyphae
(average diameter: 0.5 .mu.m) are intensively branched. Spores
(1.4-1.7 .mu.m in diameter) are borne singly, sessile or on short
sporophores and occur along the length of the hyphae. Growth and
sporulaton are of open web type of Luedemann. The following
compounds are utilized by this strain as only source of carbon in
medium: D-glucose, lactose, D-mannitol. L-rhamnose, sucrose and
D-xylose. Dulcitol, glycerol, D-melibiose and D-raffinose are not
utilized. We have identified strain No. IDR-P.sub.7 as a typical
member of Micromonospora rosaria (Horan and Brodsky, 1986).
[0047] The above presented Micromonospora strains were deposited at
the National Collection of Agricultural and Industrial
Microorganisms (NCAIM), Budapest, Hungary, under the below given
number-designations:
1 Micromonospora sp. IDR-P.sub.3 NCAIM (P) B 001268 Micromonospora
purpurea IDR-P.sub.4 NCAIM (P) B 001271 Micromonospora echinospora
ssp. echinospora NCAIM (P) B 001272 IDR-P.sub.5. Micromonospora
megalomicea ssp. nigra NCAIM (P) B 001273 IDR-P.sub.6.
Micromonospora rosaria IDR-P.sub.7. NCAIM (P) B 001274
[0048] Based on the above the invention relates to a new microbial
process for the preparation of pravastatin of formula (I) 3
[0049] from a compound of general formula (II), 4
[0050] wherein R stands for an alkali metal or ammonium ion, by the
submerged cultivation of a strain which is able to
6.beta.-hydroxylate a compound of formula (II) in aerobic
fermentation and by the separation and purification of the compound
of formula (I) formed in the course of the bioconversion comprising
the steps of
[0051] a) cultivating a strain of a species belonging to the genus
Micromonospora which is able to 6.beta.-hydroxylate a compound of
formula (II)-- wherein R is as defined above--on a nutrient medium
containing assimilable carbon--and nitrogen sources and mineral
salts at 25-32.degree. C., thereafter
[0052] b) feeding the substrate to be transformed into the
developed culture, then
[0053] c) hydroxylating the substrate until the end of
bioconversion, then
[0054] d) separating the compound of formula (I) from the culture
broth and, if desired, purifying the same.
[0055] The scope of the invention extends to the wild strains and
any mutants of species belonging to the genus Micromonospora which
are able to hydroxylate the sodium salt of the acid form of
compactin to pravastatin.
[0056] According to a preferred embodiment of the present invention
pravastatin is produced with a Micromonospora strain selected from
the group consisting of Micromonospora sp. IDR-P.sub.3 [NCAIM (P) B
001268], Micromonospora purpurea IDR-P.sub.4 [NCAIM (P) B 001271],
Micromonospora echinospora IDR-P.sub.5 [NCAIM (P) B 001272],
Micromonospora megalomicea IDR-P.sub.6 [NCAIM (P) B 001273] and
Micromonospora rosaria IDR-P.sub.7 [NCAIM (P) B 001274].
[0057] According to the most preferred embodiment of the invention
pravastatin is produced with Micromonospora sp. strain IDR-P.sub.3
[NCAIM (P) B 001268].
[0058] The present invention can be carried out by in situ
fermentation method, that is when, hydroxylation is accomplished
with the participation of an actively growing Micromonospora
culture.
[0059] The hydroxylation may be conducted by employing agitation as
shake-flask culture or aeration and agitation in fermentors, when
the compound of the formula (II) is added to the growing cultures.
In such cases an anti-foaming agent may be employed. The adequate
density of culture of this strain could be achieved by the use of
an appropriate medium containing available carbon and nitrogen
sources, inorganic salts as well as trace elements.
[0060] E.g. glucose, glycerol, dextrin, starch, rhamnose, xylose,
sucrose and soluble starch proved to be assimilable carbon sources
while soybean meal, corn steep liquor, peptone, yeast extract, meat
extract, ammonium citrate and ammonium sulfate as good nitrogen
sources. Inorganic salts such as calcium carbonate, sodium
phosphates, potassium phosphates etc., may be added to the culture
medium. Preferred media for the growth of this selected strain are
those described in the examples.
[0061] The bioconversion of compactin to pravastatin can be done by
different fermentation techniques, e.g., batch culture, fed-batch
culture. Preferably, an agitated liquid submerged culture is used.
The preferred temperature is about 25.degree. C. to 37.degree. C.,
most preferably about 25.degree. C. to 32.degree. C.
[0062] The preferred pH is about 6.0 to 9.0, most preferably about
7.0 to 8.5. The preferred shaking condition is about 200 rpm to 400
rpm, most preferably about 250 rpm.
[0063] The invention provides a method for converting compactin
acid sodium salt to pravastatin. Compactin acid sodium salt can be
used in this invention at any concentration which will result in
production of pravastatin. Preferably, the compactin concentration
is between 0.1 and 10 g/liter, more preferably is between about 0.3
and 3.0 g/liter.
[0064] The invention is meant to cover any percentage of conversion
of compactin to pravastatin by the strains of Micromonospora spp.,
at least 30% and most preferably at least about 90%.
[0065] In the course of the fermentation the composition of the
culture broth is controlled by a high performance liquid
chromatographic (HPLC) method. According to the HPLC method the
sample of the broth is diluted twofold with methonol, centrifuged
and the supernatant is used for the analysis. Parameters of the
HPLC system used for the analysis are: Waters analytical HPLC
equipment; column packing: Waters Novapack C.sub.18 5 .mu.m;
measurement at 237 nm; injection volume 10 .mu.l; flow rate 0.6-0.9
ml/min linear gradient; gradient elution is used, eluents: solvent
A=acetonitrile-0.1 M NaH.sub.2PO.sub.4 in water (25:75), solvent
B=acetonitrile-water (pH 2 with H.sub.3PO.sub.4) (70:30).
[0066] Parameters of Gradient Elution:
2 Flow rate Time (min) (ml/min) Eluent A (%) Eluent B (%) 0 0.6 100
0 2 0.7 100 0 12 0.9 0 100 21 0.9 0 100 22 0.9 100 0 27 0.7 100
0
[0067] Retention times: pravastatin (Na salt) 10.6 min; compactin
(acid Na salt) 19.5 min; pravastatin (lactone form) 17.3 min,
compactin (lactone form) 23.5 min.
[0068] Any known method can be used for the isolation of
pravastatin, e.g., extraction-reextraction, anion exchange
chromatography, precipitation.
[0069] For the recovery of the product from the broth it is
advantageous to take into consideration the fact, that during the
bioconversion pravastatin is formed in its acidic form, thus it can
be isolated from the filtrate of the broth by its adsorption on an
anion exchange resin column. For the isolation of the product it is
advantageous to use a strongly basic anion exchange resin which is
a polystyrene-divinylbenzene polymer carrying quaternary ammonium
active groups e.g. Dowex AI 400 (OH.sup.-), Dowex 1.times.2
(OH.sup.-), Dowex 2.times.4 (OH.sup.-), Amberlite IRA 900
(OH.sup.-) resins. The product adsorbed on the ion exchange resin
can be eluted from the column by aqueous acetic acid or a sodium
chloride containing acetone-water mixture, preferably by 1% sodium
chloride containing acetone-water (1:1) mixture. Pravastatin
containing fractions are combined and the acetone being in the
eluate is distilled off in vacuum. The pH of the concentrate is
adjusted with 15% sulphuric acid into the range of 3.5-4.0 and the
acidified aqueous solution is extracted by ethyl acetate. From the
ethyl acetate extract pravastatin can be extracted by 1/10 and 1/20
volume ratio of 5% sodium hydrogen carbonate or weakly alkaline
water (pH 7.5-8.0). It was experienced, that pravastatin can be
recovered in a pure form from the above obtained alkaline aqueous
extract by column chromatography on a non-ionic adsorption resin.
An advantageous method is, that first of all the ethyl acetate
dissolved in the aqueous phase is removed by vacuum distillation
from the alkaline aqueous extract and then the aqueous extract is
loaded on a Diaion HP-20 column.
[0070] Pravastatin adsorbed on the column is purified by elution
with aqueous acetone in which the acetone content is gradually
increased, then the chromatographic fractions containing
pravastatin as a single component are combined and concentrated in
vacuum. The concentrate is clarified with charcoal and lyophilized,
then crystallized from an ethanol-ethyl acetate mixture, affording
pravastatin in a quality acceptable for pharmaceutical
application.
[0071] After finishing the bioconversion pravastatin can be
extracted either from the fermentation broth or from the filtrate
obtained after the separation of the micelium mass. The latter can
be removed either by filtration or centrifugation, however, it is
advantageous especially in an industrial scale to make a whole
broth extraction. Before extraction the pH of either the
fermentation broth or the filtrate of the broth is adjusted to
3.5-3.7 with a mineral acid preferably with diluted sulphuric acid.
The extraction is done with acetic acid ester with a 2-4 carbon
atom containing aliphatic alcohol preferably with ethyl acetate or
isobutyl acetate. The ethyl acetate extract is washed with water
and dried with anhydrous sodium sulphate. Then the lactone
derivative is prepared from pravastatin. The lactone ring closure
is carried out in dried ethyl acetate solution at room temperature,
under continuous stirring by inducing the lactone formation with
catalytic amount of trifluoro-acetic acid. The lactone ring closure
is checked by thin layer chromatographic analysis (TLC). After
finishing the lactone formation the ethyl acetate solution is
washed at first with 5% aqueous sodium hydrogen carbonate solution
and then with water, and it is dried with anhydrous sodium sulphate
and evaporated in vacuum. The residue is purified with silica gel
column chromatography used as the eluent mixtures of ethyl
acetate--n-hexane with gradually increasing ethyl acetate content.
Pravastatin is prepared from the pravastatin lactone by hydrolysis
at room temperature in acetone with equivalent quantity of sodium
hydroxide. When the pravastatin sodium salt formation has been
completed, the pravastatin is precipitated with acetone. Then the
precipitate is filtered and washed with acetone and n-hexane and
dried in vacuum, then crystallized from an ethanol-ethyl acetate
mixture.
[0072] It was found, that the chromatography on Sephadex LH-20 gel
is advantageously applicable for purifying pravastatin. By
application of this method pravastatin exceeding the purity of
99.5% (measured by HPLC) can be produced.
[0073] In the course of our experiments the following invention has
been recognized: from the organic solvent extract, preferably from
the ethyl acetate or isobutyl acetate extract of the broth or the
broth filtrate of Micromonospora sp. IDR-P.sub.3 strain which is
able to 6.beta.-hydroxylate a compound of general formula (II),
pravastatin can be precipitated as a crystalline salt with
secondary amines. Further it was found, that for the salt formation
several secondary amines containing alkyl-, cycloalkyl-, aralkyl-
or aryl-substituents are appropriate. Expediently non-toxic
secondary amines were selected among them, e.g., dioctylamine,
dicyclohexylamine, dibenzylamine. The isolation of the organic
secondary amine salt intermediates, e.g., the dibenzylamine salt
was carried out by adding dibenzylamine in 1.5 equivalent quantity
related to the pravastatin content of the extract, then the extract
is concentrated by vacuum distillation to 5% of its original
volume, then another quantity of dibenzylamine is added into the
concentrate in 0.2 equivalent ratio. The crystalline dibenzylamine
salt is precipitated from the concentrate. The crystalline crude
product is filtered and dried in vacuum, and it is clarified with
charcoal in methanol or acetone solution. Then with
recrystallization of the clarified product from acetone
chromatographically pure pravastatin dibenzylamine salt
intermediate can be obtained.
[0074] Pravastatin organic secondary amine salts can be transformed
to pravastatin by sodium hydroxide or a sodium alkoxide preferably
sodium ethoxide.
[0075] The isolation of pravastatin via a secondary amine salt
intermediate is a simpler procedure than any of the ever known
isolation procedures. During the procedure artefacts are not
formed, and the separation of pravastatin from the by-products of
the bioconversion and from the various metabolic products
biosynthesized by the hydroxylating microorganism can be
advantageously solved.
[0076] The process according to the invention is presented by the
following examples.
EXAMPLE 1
[0077] Spores were obtained from the surface of a 7-10 day old,
soluble starch agar (SM) slant culture of Micromonospora sp.
IDR-P.sub.3 [NCAIM (P) B 001268] strain and suspended in 5 ml of
sterile distilled water. This suspension was then used to inoculate
100 ml of sterile TI inoculum medium in a 500 ml Erlenmeyer
flask.
3 Composition of SM medium Soluble starch 10.0 g Na.sub.2HPO.sub.4
1.15 g KH.sub.2PO.sub.4 0.25 g KCl 0.2 g MgSO.sub.4 .times.
7H.sub.2O 0.2 g Agar 15.0 g in 1000 ml of distilled water
[0078] The pH of the medium was adjusted to 7.0 before
sterilization and the mixture was sterilized at 121.degree. C. for
25 minutes.
4 Composition of TI medium Soluble starch 20.0 g Yeast extract 10.0
g in 1000 ml of tap water
[0079] The pH was adjusted to 7.0 before sterilization and heat
treated at 121.degree. C. for 25 minutes.
[0080] The developing culture was shaken on a rotary shaker (250
r.p.m.; and amplitude: 2.5 cm) for 3 days, at 32.degree. C., then 5
ml aliquots from it were used to inoculate 10 Erlenmeyer flasks of
500 ml volume each containing 100 ml of TT medium sterilized at
121.degree. C. for 25 minutes.
5 Composition of TT medium Potato starch 30.0 g Soybean meal 30.0 g
CaCO.sub.3 5.0 g CoCl.sub.2 .times. 6H.sub.2O 2.0 mg Palm oil 2.0 g
in 1000 ml of tap water
[0081] The pH was adjusted to 7.0 before heat sterilization.
[0082] The incubation was carried out at 32.degree. C. for 72 hours
then 50 mg of compactin acid sodium salt was added to each flask in
distilled water, and the cultivation was carried out for 96 hours.
The conversion rate of compactin acid sodium salt into pravastatin
measured by HPLC was 82%.
[0083] After finishing the fermentation the cultures were united,
and from the obtained collective fermentation broth, which
contained 410 mg of pravastatin, the isolation of the latter was
carried out as follows: The fermentation broth was centrifuged at
2500 r.p.m. for 20 min. The supernatant of the broth and the
mycelial mass were separated, then the latter was resuspended in
250 ml of water and the obtained suspension was stirred for one
hour and filtered. The pH of the combined centrifuged broth and the
filtrate was adjusted by 15% sulphuric acid to 4.0, then the acidic
filtrate was extracted with 3.times.300 ml of ethyl acetate. The
combined ethyl acetate extracts were washed with 300 ml of water,
dried with anhydrous sodium sulphate and concentrated in vacuum to
100 ml volume. Then pravastatin lactone was prepared from
pravastatin by adding trifluoroacetic acid in catalytical amount at
room temperature under continuous stirring. Formation of
pravastatin lactone was controlled by TLC method: adsorbent:
Kieselgel 60 F.sub.254 DC (Merck) aluminium foil; developing
solvent: acetone-benzene-acetic acid (50:50:1.5) mixture;
detection: with phospho-molybdic acid reagent. The R.sub.f value of
pravastatin lactone was 0.7. After the completion of the lactone
formation the ethyl acetate was washed with 2.times.20 ml of 5%
aqueous sodium hydrogen carbonate then washed with 20 ml of water,
dried with anhydrous sodium sulphate and evaporated in vacuum. 0.5
g of evaporation residue was obtained, which was chromatographed on
10 g of Kieselgel 60 adsorbent containing column (diameter of the
column: 1.2 cm, height of the adsorbent bed: 17 cm). For elution
ethyl acetate-n-hexane mixtures were used in which the ethyl
acetate content was gradually increased. Pravastatin lactone was
eluted from the column with the mixture of 60% ethyl acetate-40%
n-hexane. The fractions containing pravastatin lactone were
combined and evaporated in vacuum. The residue obtained, which
contained 230 mg of pravastatin lactone, was dissolved in 5 ml of
acetone and then under stirring 110 mole % of sodium hydroxide was
added in 1M ethanolic solution. Stirring of the solution was
continued for half an hour at room temperature. Subsequently, the
solution was concentrated to 2 ml volume and 4 ml of acetone was
added to the concentrate. The mixture was kept at +5.degree. C.
overnight. The precipitate was filtered, washed with 2 ml of
acetone and then with 2 ml of n-hexane and dried in vacuum at room
temperature. The resulting crude pravastatin was dissolved in
ethanol, clarified by charcoal, then crystallized from
ethanol-ethyl acetate mixture. In this way 170 mg of pravastatin
was obtained.
[0084] Melting point 170-173.degree. C. (decomp.)
[0085] [.alpha.].sub.D.sup.2=+156.degree. (c=0,5, in water).
[0086] Ultraviolet absorption spectrum (20 .mu.g/ml, in methanol):
.lambda..sub.max=231, 237, 245 nm (log .epsilon.=4.263; 4.311;
4.136).
[0087] Infrared absorption spectrum (KBr): vOH 3415, vCH 2965, vC=0
1730, vCOO-1575 cm.sup.-1.
[0088] .sup.1H-NMR spectrum (D.sub.2O, .delta., ppm): 0.86, d, 3H
(2-CH.sub.3); 5.92, dd, J=10.0 and 5.4 Hz, 1H (3-H); 5.99, d,
J=10.0 Hz, 1H (4-H); 5.52, br, 1H (5-H); 4.24, m, 1H (6-H); 5.34,
br, 1H (8-H); 4.06, m, 1H (.beta.-H), 3.65, m, 1H (.delta.-H);
1.05, d, 3H (2'-CH.sub.3); 0.82, t, 3H (4'-H.sub.3).
[0089] .sup.13C-NMR spectrum (D.sub.2O, .delta., ppm): 15.3, q
(2-CH.sub.3); 139.5, d (C-3); 129.5, d (C-4); 138.1, s (C-4a);
127.7, d (C-5); 66.6, d (C-6); 70.1, d (C-8); 182.6, s (COO.sup.-);
72.6, d (C-.beta.); 73.0, d (C-.delta.); 182.0, s (C-1'); 18.8, q
(2'-CH.sub.3); 13.7, q (C-4').
[0090] Positive FAB mass spectrum (characteristic ions):
[M+Na].sup.+469; [M+H].sup.+447.
[0091] Negative FAB mass spectrum (characteristic ions):
[M-H].sup.-445, [M-Na].sup.-423, m/z 101 [2-methyl-butyric
acid-H].sup.-.
EXAMPLE 2
[0092] 10 Erlenmeyer flasks of 500 ml volume each containing 100 ml
of MT.sub.1 bioconversion medium were inoculated with inoculum
culture prepared as described in Example 1, then incubated at
28.degree. C. for 96 hours and 50 mg of compactin acid sodium salt
was added to each flask in distilled water, then the hydroxylation
was carried out for 72 hours when another 50-50 mg of substrate was
added to the cultures in distilled water and the fermentation was
continued for 72 hours.
6 Composition of MT.sub.1 bioconversion medium Potato starch 10.0 g
Dextrose 20.0 g Soybean meal 10.0 g Yeast extract 10.0 g CaCO.sub.3
5.0 g Sunflower oil 2.0 g in 1000 ml of tap water
[0093] The pH of the bioconversion medium was adjusted to 7.0
before sterilization. The mixture was sterilized at 121.degree. C.
for 25 minutes.
[0094] After finishing the bioconversion period the cultures were
united and the pravastatin was isolated from the collective broth
according to the following procedure:
[0095] The united broth, which contained 750 mg of pravastatin
according to the HPLC assay was centrifuged at 2500 r.p.m. for 20
min. The separated micelium mass was stirred with 250 ml of water
for an hour, then filtered. The centrifuged broth the filtrate were
combined and the pH of the resulting solution was adjusted to a
3.5-4.0 value, with 15% sulphuric acid, then the solution was
extracted with 3.times.300 ml of ethyl acetate. Then 150 mole % of
dibenzylamine--calculated for the pravastatin content--was added to
the ethyl acetate extract. The ethyl acetate extract was evaporated
to about 30 ml volume and the suspension was kept overnight at
0-5.degree. C. The precipitated pravastatin acid dibenzylamine salt
was filtered and washed on the filter with cooled ethyl acetate and
n-hexane, finally dried in vacuum. The 1.1 g of crude pravastatin
acid dibenzylamine salt was dissolved in 33 ml of acetone at
62-66.degree. C. temperature, and the solution was clarified with
0.1 g of charcoal for half an hour. Then the charcoal was removed
by filtration from the solution. Crystals precipitated from the
clarified solution were dissolved again at the above temperature,
then the solution was kept at +5.degree. C. overnight. The
precipitate was filtered, washed with cooled acetone and n-hexane
and dried in vacuum. Pravastatin acid dibenzylamine salt obtained
(0.7 g) was suspended in 10 ml of ethanol, then 110 mole % of
sodium hydroxide was added to the solution by feeding 1M aqueous
solution. Stirring of the alkaline solution was continued for half
an hour at room temperature. After the completion of the sodium
salt formation 30 ml of water was added and the pH of the solution
was neutralized, then ethanol was distilled off in vacuum. The
aqueous concentrate was chromatographed on a column filled with 50
ml of Dialon HP 20 resin (diameter of the column: 1.5 cm, height of
the resin bed: 28 cm). The column was eluted with acetone-deionized
water mixtures, where the concentration of the acetone was
increased in 5% steps. Pravastatin could be eluted from the column
by a 15% acetone containing acetone-deionized water mixture.
Fractions were analysed by TLC method given in the Example 1. The
R.sub.f value of pravastatin was 0.5. Fractions containing
pravastatin were combined and the acetone content was evaporated in
vacuum. By the lyophilization of the aqueous residue 390 mg of
chromatographically pure pravastatin was obtained.
EXAMPLE 3
[0096] 4.5 litres of TT/2 medium, in a laboratory fermentor, were
sterilized at 121.degree. C. for 45 minutes and inoculated with 500
ml of inoculum shake culture prepared as described in Example 1,
then incubated at 32.degree. C., aerated with 250 l of sterile
air/h and stirred with a flat blade stirrer at 300 r.p.m. The
incubation was continued for 72 hours and 2.5 g of compactin acid
sodium salt was added to the culture. After 48.sup.th hour of the
bioconversion period the compactin substrate was completely
consumed from the fermentation broth, then an additional 2.5 g of
compactin acid sodium salt was added again into the culture. The
second dose of compactin was consumed within 24 hours. The
conversion rate of compactin acid sodium salt into pravastatin was
about 90% in the bioconversion process.
7 Composition of TT/2 bioconversion medium Glucose 75.0 g Soluble
starch 50.0 g Soybean meal 50.0 g Yeast extract 50.0 g Pepton 5.0 g
NaNO.sub.3 20.0 g CaCO.sub.3 25.0 g in 4500 ml of tap water
EXAMPLE 4
[0097] 4.5 litres of the TT/1 fermentation medium, in a laboratory
fermentor were sterilized at 121.degree. C. for 45 minutes and
inoculated with 500 ml of the inoculum shake culture prepared as
described In Example 1, then incubated at 28.degree. C., aerated
with 200 l sterile air/h and stirred with a flat blade stirrer at
400 r.p.m.
8 Composition of TT/1 bioconversion medium Glucose 125.0 g Potato
starch 25.0 g Soybean meal 50.0 g Yeast extract (Gistex) 50.0 g
Pepton 50.0 g CoCl.sub.2 .times. 6H.sub.2O 10.0 mg Sunflower oil
10.0 g in 4500 ml of tap water
[0098] The pH of the bioconversion medium was adjusted to 7.0
before sterilization.
[0099] Cultivation was continued at 28.degree. C. for 96 hours. At
this time 2.5 g of compactin acid sodium salt was added in sterile
filtered aqueous solution to the culture. By the 5.sup.th day of
fermentation the compactin acid sodium salt was completely consumed
from the fermentation broth. Then the substrate feeding was
repeated daily for further 3 days in 2.5 g/day portions. The
compactin acid sodium salt substrate was gradually consumed during
the four days and converted completely to pravastatin. According to
the results of HPLC measurements at the end of the fermentation
period from 10 g of compactin substrate 9 g of pravastatin has been
produced.
[0100] After finishing the bioconversion the pravastatin formed in
the concentration of 1800 .mu.g/ml was isolated as follows:
[0101] 5 litres of culture broth were centrifuged at 2500 r.p.m.
for 20 min. Then 2 litres of water were added to the separated
mycelial mass and the suspension was stirred for one hour and
filtered. These two filtrates were united and passed through with a
flow rate of 500 ml/hour on a column containing 300 g (540 ml) of
Dowex AI 400 (OH.sup.-) resin (diameter of the column: 4 cm, height
of the resin bed: 43 cm), then the resin bed was washed with 1
litre of deionized water. Thereafter the column was eluted with 1
litre of acetone-water (1:1) mixture containing 10 g of sodium
chloride by collecting 50 ml fractions. The fractions were analysed
by the TLC method given in the Example 1. Fractions containing the
product were combined and the acetone was distilled off in vacuum.
The pH of the concentrate was adjusted to 3.5-4.0 value by 15%
sulphuric acid, then it was extracted 3.times.250 ml of ethyl
acetate. 40 ml of deionized water was added to the combined ethyl
acetate extract, then the pH was adjusted to 7.5-8.0 value by 1M
sodium hydroxide. After 15 min stirring the aqueous and ethyl
acetate phases were separated, then the ethyl acetate solution was
extracted with 2.times.40 ml of deionized water as it was written
before. Then the combined alkaline aqueous solution was
concentrated to 50 ml volume and chromatographed on a column filled
with 600 ml of Diaion HP20 (Mitsubishi Co., Japan) non ionic
adsorbent resin (diameter of the column: 3.8 cm, height of the
resin bed; 53 cm). The column was washed with 600 ml of deionized
water, then eluted with acetone-deionized water mixtures, where the
concentration of acetone was increased in 5% steps, collecting 50
ml fractions. The eluate was analysed by TLC method given in the
Example 1. Pravastatin was eluted from the column by an
acetone-deionized water mixture containing 15% of aceton. Fractions
containing pravastatin as single component were combined and the
solution was concentrated in vacuum to 150 ml volume. Subsequently,
0.6 g of charcoal was added to the concentrated aqueous solution
and pravastatin was clarified at room temperature for 1 hour. Then
the charcoal was filtered and the filtrate was lyophilised. The
resulting 6.5 g of lyophilised pravastatin was crystallized twice
from a mixture of ethanol and ethyl acetate. The precipitate was
filtered and washed with 20 ml of ethyl acetate and 20 ml of
n-hexane, and dried in vacuum at room temperature. Thus 4.6 g of
chromatographically pure pravastatin was obtained.
EXAMPLE 5
[0102] A spore suspension was prepared with 5 ml of sterile
distilled water from the surface of a 10 days old, soluble starch
agar slant culture, as described in Example 1, of Micromonospora
echinospora ssp. echinospora IDR-P.sub.5 [NCAIM (P) B 001272]
strain--being able for the 6.beta.-hydroxylation of compactin acid
sodium salt--and the obtained spore suspension was used to
inoculate 100 ml of inoculum medium TI sterilized in a 500 ml
Erienmeyer flask. Composition of the medium TI was also described
in Example 1. The inoculated medium was shaken on a rotary shaker
(250 r.p.m., 2.5 cm amplitude) for 3 days at 28.degree. C., then 5
ml aliquots of the developed culture were transferred into 100-100
ml of bioconversion medium TT/1 sterilized in 500 ml Erlenmeyer
flasks for 25 min at 121.degree. C. Composition of the medium TT/1
is described In Example 4. Flasks were shaken on a rotary shaker
(250 r.p.m. 2.5 cm amplitude) for 3 days at 25.degree. C., then
10-10 mg of compactin substrate (compactin acid sodium salt) was
added in sterile filtered aqueous solution into the cultures, then
the fermentation was continued for 168 hours.
[0103] At the end of the bioconversion the pravastatin content of
the fermentation broth was determined by an HPLC method. At this
time the average pravastatin concentration was 40 .mu.g/ml.
EXAMPLE 6
[0104] The fermentation, substrate feeding and bioconversion were
carried out with strain IDR-P.sub.6, [NCAIM (P) B 001273] of
Micromonospora megalomicea ssp. nigra as it was written in Example
5. The pravastatin content of the fermentation broth was determined
by an HPLC method. At the end of the bioconversion the pravastatin
content of the broth was 50 .mu.g/ml.
EXAMPLE 7
[0105] 5 ml aliquots of an inoculum culture of strain IDR-P.sub.4
[NCAIM (P) B 001271] of Micromonospora purpurea prepared as
described in Example 1 were used to seed 100-100 ml of TT/14 medium
dispensed in 500 ml Erienmeyer flasks and sterilezed for 25 min at
121.degree. C.
9 Composition of medium TT/14 Potato starch 5.0 g Glucose 25.0 g
Yeast extract (GISTEX) 15.0 g Pepton 15.0 g CaCO.sub.3 1.0 g in
1000 ml of tap water
[0106] The pH of the bioconversion medium was adjusted to 7.0
before sterilization.
[0107] Flasks were shaken on a rotary shaker (250 r.p.m., 2.5 cm
amplitude) for 3 days. The substrate feeding, the bioconversion and
determination of the pravastatin content were carried out as
described in Example 5. At the end of the bioconversion the
pravastatin content of the fermentation broth was 40 .mu.g/ml.
EXAMPLE 8
[0108] The fermentation, substrate feeding and bioconversion were
carried out with strain IDR-P.sub.7, [NCAIM (P) B 001274] of
Micromonospora rosaria as it was written in Example 1. At the end
of the bioconversion 350 .mu.g/ml pravastatin was measured in the
fermentation broth by HPLC method.
* * * * *