U.S. patent application number 10/966000 was filed with the patent office on 2005-03-03 for new salts of hmg-coa reductase inhibitors.
This patent application is currently assigned to LEK Pharmaceuticals d.d.. Invention is credited to Pflaum, Zlatko.
Application Number | 20050049422 10/966000 |
Document ID | / |
Family ID | 20432331 |
Filed Date | 2005-03-03 |
United States Patent
Application |
20050049422 |
Kind Code |
A1 |
Pflaum, Zlatko |
March 3, 2005 |
New salts of HMG-CoA reductase inhibitors
Abstract
Lovastatin, pravastatin, simvastatin, mevastatin, atorvastatin,
and derivatives and analogs thereof are known as HMG-CoA reductase
inhibitors and are used as antihypercholesterolemic agents. The
majority of them are produced by fermentation using microorganisms
of different species identified as species belonging to
Aspergillus, Monascus, Nocardia, Amycolatopsis, Mucor or
Penicillium genus, some are obtained by treating the fermentation
products using the methods of chemical synthesis or they are the
products of total chemical synthesis. The present invention relates
to the new amine salts of HMG-CoA reductase inhibitors, the
preparation thereof, the preparation of pure HMG-CoA reductase
inhibitors via amine salts thereof, use of the amine salts of
HMG-CoA reductase inhibitors in the process for semisynthetic
preparation of HMG-CoA reductase inhibitors, use of the amine salts
of HMG-CoA reductase inhibitors in the process for biotechnological
modification of HMG-CoA reductase inhibitors as well as the
conversion of the amine salts of HMG-CoA reductase inhibitors into
the pharmaceutically acceptable salts of the HMG-CoA reductase
inhibitors and the conversion of the amine salts of HMG-CoA
reductase inhibitors into the HMG-CoA reductase inhibitors in the
lactone form.
Inventors: |
Pflaum, Zlatko; (Domzale,
SI) |
Correspondence
Address: |
COHEN, PONTANI, LIEBERMAN & PAVANE
Suit 1210
551 Fifth Avenue
New York
NY
10176
US
|
Assignee: |
LEK Pharmaceuticals d.d.
|
Family ID: |
20432331 |
Appl. No.: |
10/966000 |
Filed: |
October 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10966000 |
Oct 15, 2004 |
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10320285 |
Dec 16, 2002 |
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6838566 |
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10320285 |
Dec 16, 2002 |
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09787387 |
Apr 25, 2001 |
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6583295 |
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09787387 |
Apr 25, 2001 |
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PCT/IB99/01554 |
Sep 17, 1999 |
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Current U.S.
Class: |
548/537 |
Current CPC
Class: |
C07D 309/30 20130101;
C07C 211/09 20130101; C07C 211/35 20130101; C07C 2601/14 20170501;
C07C 211/10 20130101; C07C 211/14 20130101; C07C 211/07
20130101 |
Class at
Publication: |
548/537 |
International
Class: |
C07D 027/36 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 18, 1998 |
SI |
P-9800240 |
Claims
1-48 (canceled)
49. The sodium salt of pravastatin in a crystalline form.
50. The sodium salt of pravastatin in solid form made by a process
comprising the steps of: (a) dissolving an amine salt of
pravastatin in an alkaline solution containing sodium cations; (b)
adding ethyl acetate to said alkaline solution; (c) cooling of said
alkaline solution; and (d) forming and isolating the solid salt of
pravastatin sodium; wherein the amine salt of pravastatin comprises
an amine selected from the group consisting of amines of formulae I
and II: 5wherein a1) R.sub.1, R.sub.2, R.sub.3 and R.sub.4
independently denote: a hydrogen atom; a straight or a branched
alkyl group having 1 to 8 carbon atoms; a cycloalkyl group having 3
to 8 carbon atoms; an arylalkyl group wherein the alkyl group is
methyl or ethyl and the aryl group is phenyl, which is optionally
substituted by an N-alkyl or N,N-dialkyl group wherein the alkyl
group is alkyl having 1 to 4 carbon atoms; an arylalkyl group which
is optionally substituted by one or more substituents; a
hydroxyalkyl group having 2 to 4 carbon atoms; or an aminoalkyl
group having 2 to 4 carbon atoms, which are optionally substituted
by an N-alkyl or N,N-dialkyl group wherein the alkyl group is alkyl
having 1 to 4 carbon atoms; X denotes a hydrogen atom, a hydroxyl
group, a halogen or a methyl group; m and n independently denote an
integer from 0 to 5; or a2) NR.sub.1R.sub.2 or NR.sub.3R.sub.4
denote a heterocyclic ring having 3 to 7 methylene groups attached
to a hydrogen atom, one of these groups being optionally
substituted by an oxygen or a sulphur atom or an imine group; and
X, m and n are the same as defined above; b) 6wherein: b1)
R'.sub.1, R'.sub.2, and R'.sub.3 are the same or different and
denote hydrogen, alkyl, alkenyl, amino- or hydroxy- or
alkoxy-substituted alkyl or alkenyl, or substituted
amino-substituted alkyl or alkenyl, provided that R'.sub.1,
R'.sub.2, and R'.sub.3 are not hydrogen at the same time; or b2)
R'.sub.1, and R'.sub.2, and optionally R'.sub.3, together with the
nitrogen atom form an optionally substituted heterocyclic ring
system including the nitrogen atom as a ring member, and optionally
including an additional hetero atom, and if R'.sub.3 is not part of
the ring system it is independently selected from hydrogen, alkyl,
alkenyl, amino- or hydroxyl- or alkoxy-substituted alkyl, or
substituted amino-substituted alkyl; or b3) R'.sub.1 is an
optionally substituted cyclic group of formula III,
R'(CHR'.sub.4).sub.m-- III wherein m is zero or an integer from 1
to 5, R' is optionally substituted aliphatic hydrocarbon cyclic
system having 3 to 8 carbon atoms in the ring, R'.sub.4 is
hydrogen, alkyl, amino- or hydroxy- or alkoxy-substituted alkyl,
substituted amino-substituted alkyl, or a group of the same formula
as R'.sub.1 as defined herein above; R'.sub.2 and R'.sub.3 are the
same as R'.sub.1 or hydrogen, alkyl, alkenyl, amino- or hydroxy- or
alkoxy-substituted alkyl, or substituted amino-substituted alkyl or
alkenyl; or b4) R'.sub.1 is an optionally substituted aryl group of
formula IV: 7wherein R'.sub.5 is hydrogen or one or more
substituents, and m is zero or an integer from 1 to 5; and R.sub.12
and R.sub.13 may be independently hydrogen, alkyl, amino- or
hydroxyl or alkoxy-substituted alkyl, or substituted
amino-substituted alkyl, or groups of the same general formula
R'.sub.1.
51. The sodium salt of pravastatin according to claim 50, wherein
the amine salt of pravastatin is the tertiarybutyl amine salt of
pravastatin.
52. The sodium salt of pravastatin according to claim 50, wherein
the amine salt of pravastatin is dissolved in an alkaline
ethanol/water mixture in step (a).
53. The sodium salt of pravastatin according to claim 50, wherein
the amine salt of pravastatin is dissolved in an ethanolic solution
and/or aqueous solution of NaOH or Na.sub.2CO.sub.3.
54. The sodium salt of pravastatin according to claim 50, wherein
the amine salt of pravastatin sodium dissolved in step a) is
prepared by dissolving pravastatin in the free acid form in ethyl
acetate and adding the respective amine into said solution, thereby
crystallizing said amine salt of pravastatin.
55. A pharmaceutical composition containing the sodium salt of
pravastatin according to claim 49.
56. A method of treating hypercholesteremia in humans, comprising
administering to a patient in need of such treatment an
antihypercholesterolemic effective amount of the sodium salt of
pravastatin according to claim 49.
57. The sodium salt of pravastatin according to claim 50, wherein
the amine salt of pravastatin is the tertiarybutyl amine salt of
pravastatin.
58. The sodium salt of pravastatin according to claim 51, wherein
the amine salt of pravastatin is dissolved in an alkaline
ethanol/water mixture in step (a)
59. The sodium salt of pravastatin according to claim 51, wherein
the amine salt of pravastatin is dissolved in an ethanolic solution
and/or aqueous solution of NaOH or Na.sub.2CO.sub.3.
60. The sodium salt of pravastatin according to claim 52, wherein
the amine salt of pravastatin is dissolved in an ethanolic solution
and/or aqueous solution of NaOH or Na.sub.2CO.sub.3.
61. The sodium salt of pravastatin according to claim 51, wherein
the amine salt of pravastatin sodium dissolved in step a) is
prepared by dissolving pravastatin in the free acid form in ethyl
acetate and adding the respective amine into said solution, thereby
crystallizing said amine salt of pravastatin.
62. The sodium salt of pravastatin according to claim 52, wherein
the amine salt of pravastatin sodium dissolved in step a) is
prepared by dissolving pravastatin in the free acid form in ethyl
acetate and adding the respective amine into said solution, thereby
crystallizing said amine salt of pravastatin.
63. The sodium salt of pravastatin according to claim 53, wherein
the amine salt of pravastatin sodium dissolved in step a) is
prepared by dissolving pravastatin in the free acid form in ethyl
acetate and adding the respective amine into said solution, thereby
crystallizing said amine salt of pravastatin.
64. A method of treating hypercholesteremia in humans, comprising
administering to a patient in need of such treatment an
antihypercholesterolemic effective amount of the sodium salt of
pravastatin according to claim 50.
65. A method of treating hypercholesteremia in humans, comprising
administering to a patient in need of such treatment an
antihypercholesterolemic effective amount of the sodium salt of
pravastatin according to claim 51.
66. A method of treating hypercholesteremia in humans, comprising
administering to a patient in need of such treatment an
antihypercholesterolemic effective amount of the sodium salt of
pravastatin according to claim 52.
67. A method of treating hypercholesteremia in humans, comprising
administering to a patient in need of such treatment an
antihypercholesterolemic effective amount of the sodium salt of
pravastatin according to claim 53.
68. A method of treating hypercholesteremia in humans, comprising
administering to a patient in need of such treatment an
antihypercholesterolemic effective amount of the sodium salt of
pravastatin according to claim 54.
Description
[0001] The present application is a continuation of U.S. patent
application Ser. No. 09/787,387, which is based on International
Application No. PCT/IB99/01554, filed Sep. 17, 1999. These
applications are incorporated herein by reference.
TECHNICAL FIELD AND BACKGROUND ART
[0002] Lovastatin, pravastatin, simvastatin, mevastatin,
atorvastatin and derivatives and analogs thereof are examples of
known as HMG-CoA reductase inhibitors which are used as
antihypercholesterolemic agents. The majority of them are produced
biotechnologically by fermentation using microorganisms of
different species identified as species belonging to Aspergillus,
Monascus, Nocardia, Amycolatopsis, Mucor or Penicillium genus, some
are obtained by treating the fermentation products using the
methods of chemical synthesis, thus leading to semi-synthetic
substances, or they are the products of total chemical
synthesis.
[0003] The present invention relates to a new industrial process
for isolation and/or purification of HMG-CoA reductase inhibitors
via salts thereof with specific amines. The invention enables a
user to obtain the pure amine salts of HMG-CoA reductase inhibitors
from the fermentation broth in case the substances are produced by
biotechnological (microbiological) processes, or from the reaction
mixture in case the substances are produced by semisynthetic or
total chemical synthesis. The step of forming salts with amine may
be one of the steps in the process for isolation and/or
purification of HMG-CoA reductase inhibitors or precursor
substances thereof. The amines described in the present
specification are very useful for the formation of salts in the
composition of media in processes for biotechnological modification
of HMG-CoA reductase inhibitors or precursors thereof. The salts
thus formed may be used as the starting substances or intermediates
for the preparation of semisynthetic derivatives and analogs
thereof, or by employing simple techniques known from the
literature, if required, to be converted into the pharmaceutically
acceptable salts and lactones, respectively.
[0004] The processes for the isolation and purification of
antihypercholesterolemic agents known from patent and technical
literature include different combinations of extraction,
chromatography, lactonization and crystallization methods. Some of
them additionally include the isolation and purification via
different salts. In U.S. Pat. Nos. 9,342,767 and 4,319,039, the
ammonium salt of lovastatin (in the carboxylate form) is isolated
directly from the organic phase which has been extracted from the
fermentation medium. In the same patent the preparation of
ethylenediamine, tetramethylammonium, potassium and
N-methylglucamine salts as well as the salts of different amino
acids such as L-lysine, L-arginine and L-ornithine is also
described. The aforementioned salts are prepared from the already
purified substance and the option for their use in the process of
isolation or purification is not mentioned. GB 2055100A also
describes the formation of the sodium and calcium salts of
lovastatin, which comprises the extraction in methanol, two steps
of preparative liquid reverse-phase chromatography, crystallization
from methanol and recrystallization from ethanol, and the
conversion into the salt using an aqueous solution of sodium or
calcium hydroxide. However, without including various
chromatography methods, the methods described do not yield a
product of the purity comparable to the product obtained by using
the present invention. U.S. Pat. No. 4,346,227 discloses a process
for the preparation of the sodium salt of pravastatin, wherein
chromatographic techniques are also used but the final product is
obtained only after lyophilization which is not an economical
process in a large scale production operations. EP 65,835 discloses
the preparation of the L-ornithine and t-octylamine salts of
tetrahydro-M-4 or tetrahydro-Iso-M-4 (wherein M4 denotes a specific
HMG-CoA reductase inhibitor, M-4 and Iso-M-4 representing the
isomers hydroxylated at 6- and 3-biphenyl ring position,
respectively, and "tetrahydro" means that the condensed biphenyl
ring system is fully hydrogenated) as final products, that is from
the respectively purified sodium salts thereof, but not as
intermediates via which the isolation would be carried out. Other
salts of tetrahydro-M-4 or IsoM-4 with ammonia, an amino acid or an
organic amine are also contemplated as final products, including
octylamine, 2-ethylhexylamine, benzylamine, a-methyl-benzylamine,
phenethylamine, dibenzylamine, N-methylbenzylamine,
N,N-dimethylbenzylamine, N,N-diethylbenzylamine,
N-ethyl-N-methylbenzylam- ine, tribenzylamine, cyclopentylamine,
cyclohexylamine cycloheptylamine, N-methylcyclopentylamine,
N-ethylcyclohexylamine, N-ethylcycloheptylamine- ,
dicyclohexylamine, N,N-dimethylcyclopentylamine,
N,N-dimethylcyclohexyla- mine, N,N-diethylcycloheptylamine,
pyrrolidine, N-methylpyrrolidine, piperidine, N-methylpiperidine
and morpholine. GB 2073199A also discloses the preparation of
different salts of HMG-CoA reductase inhibitors from the already
isolated substance in the lactone form. U.S. Pat. Nos. 5,763,653
and 5,763,646 disclose the preparation of the cyclopropylamine and
n-butylamine amides of lovastatin and their use in a process of
chemical semisynthesis of simvastatin. U.S. Pat. No. 5,403,860
discloses, as final products, amine salts of octahydronaphthalene
oxime derivatives of HMG-CoA inhibitors, the derivatives deriving
from ML-236A, ML-236B, MB-530A and MB-530B. As final amine salts,
t-octylamine, dibenzylamine, dicyclohexylamine, morpholine,
D-phenylglycine alkylester and D-glucosamine salts are
mentioned.
[0005] Technical Problem
[0006] In industry there exists a constant need for rationalization
of the production and shortening of the production processes as
well as for the use of least expensive starting raw materials or
intermediate substances. To date the isolation of the final
products in the case of HMG-CoA reductase inhibitors has been a
multi-stage process wherein each step adds its share to the losses
resulting in the final yield rarely greater than 60%. In addition,
a product in the lactone form or lactone converted into the sodium
salt is used as the starting substance in the process of
semisynthesis (e.g. in a process for preparing simvastatin) or
biochemical conversion (e.g. in a process for preparing
pravastatin). The preparation of lactone is one of the least
economical steps in the production of HMG-CoA reductase inhibitors
since losses in the course of the conversion from the acid into the
lactone form and optionally further into the salts are greater than
20%. Therefore, there is a constant need for the starting
substances and/or the intermediate substances which would be
sufficiently pure, with small losses during their conversion, low
costs, and the preparation per se should be technologically
simple.
SUMMARY OF THE INVENTION
[0007] In our developmental and research work we have surprisingly
found that HMG-CoA reductage inhibitors form the salts with certain
amines which crystallize from mother liquor once they are formed.
It has surprisingly been found that crystals of the amine salt of
the desired HMG-CoA reductase inhibitor of high purity may be
obtained from the liquors containing a large number of impurities
and undesired HMG-CoA reductase inhibitor analogs. Contrary to the
statements from U.S. Pat. No. 5,403,860 that lower yields are
obtained when using the salts of HMG-CoA reductase inhibitor as
starting or intermediate substances in a process for preparing the
substances (1a) mentioned below, we have surprisingly found that,
when using the amine salts of HMG-CoA reductase inhibitors
according to the present invention, the yields and the purity of
the prepared HMG-CoA reductase inhibitors are equal to or greater
than when using the HMG-CoA reductase inhibitors in the lactone
form. 1
[0008] Furthermore, we surprisingly discovered that in processes
for the biotechnological modification of HMG-CoA reductase
inhibitors the formation of amine salts of HMG-CoA reductase
inhibitors in the medium which derives from the fermentation liquor
provides, in comparison with the mere metal salts as described in
publicly accessible literature, an efficient means for the
isolation and/or purification of HMG-CoA reductase inhibitors by
means of simple crystallization. The amines which are described in
the present specification and which readily form salts with HMG-CoA
reductase inhibitors are thus particularly suitable as auxiliary
materials or processing aids for the isolation and/or purification
of HMG-CoA reductase inhibitors. Furthermore, they can be
excellently used as starting materials or intermediates of
semisynthetic preparation or biotechnological modification of
HMG-CoA reductase inhibitors and, furthermore, for the conversion
into pharmaceutically acceptable salts or into the lactone form of
the respective HMG-CoA reductase inhibitors. Accordingly, the novel
amine salts of HMG-CoA reductase inhibitors of the present
invention are also highly valuable as such.
[0009] The present invention provides:
[0010] a) the novel salts of HMG-Corm reductase inhibitors with
organic amines, wherein those specific salts are excluded which are
disclosed in the prior art, but in different contexts as mentioned
above,
[0011] b) a process for the preparation of salts of HMG-CoA
reductase inhibitors with amines,
[0012] c) a use of salts of HMG-CoA reductase inhibitors with
amines as processing aids or starting substances or intermediate
substances in various processes,
[0013] d) a process for the preparation of the pure HMG-CoA
reductase inhibitors from/via amine salts thereof,
[0014] e) a process for the semisynthetic preparation of HMG-CoA
reductase inhibitors, wherein the amine salts of HMG-CoA reductase
inhibitors are used as the starting substances,
[0015] f) a process for the biotechnological modification of
HMG-CoA reductase inhibitors, wherein one of the components of the
medium is the amine salt of HMG-CoA reductase inhibitors,
[0016] g) a process for the conversion of the amine salts of
HMG-CoA reductase inhibitors into the pharmaceutically acceptable
salts of HMG-CoA reductase inhibitors, and
[0017] h) a process for the conversion of the amine salts of
HMG-CoA reductase inhibitors into HMG-CoA reductase inhibitors in
the lactone form.
[0018] The amine which is used according to the present invention
for the formation of the salts with a HMG-CoA reductase inhibitor
is selected from the group consisting of organic amines of the
following-formulae I and II:
[0019] a) 2
[0020] wherein:
[0021] a1) R.sub.1, R.sub.2, R.sub.3, and R.sub.4 independently
denote
[0022] a hydrogen atom
[0023] a straight or branched alkyl group having 1 to 8 carbon
atoms, or
[0024] a cycloalkyl group having 3 to 8 carbon atoms, or
[0025] an arylalkyl group wherein the alkyl group is methyl or
ethyl and the aryl group is phenyl, which is optionally substituted
by an N-alkyl or N,N-dialkyl group wherein the alkyl group is alkyl
having 1 to 4 carbon atoms, or
[0026] an arylalkyl group which is optionally substituted by one or
more substituents,
[0027] a hydroxyalkyl group having 2 to 4 carbon atoms, or
[0028] an aminoalkyl group having 2 to 4 carbon atoms, which are
optionally substituted by an N-alkyl or N,N-dialkyl group wherein
the alkyl group is alkyl having 1 to 4 carbon atoms;
[0029] X denotes a hydrogen atom, a hydroxyl group, a halogen or a
methyl group;
[0030] m and n independently denote an integer from 0 to 5; or
[0031] a2) NR.sub.1R.sub.2 or NR.sub.3R.sub.4 denote a heterocyclic
ring having 3 to 7 methylene groups, one of these groups being
optionally substituted by an oxygen or a sulfur atom or an amine
group; X, m and n are as defined above; 3
[0032] wherein:
[0033] b1) R'.sub.1, R'.sub.2, and R'.sub.3 are the same or
different and denote hydrogen, alkyl, alkenyl, amino- or hydroxy-or
alkoxy-substituted alkyl or alkenyl, or substituted amino-alkyl or
alkenyl, provided that R'.sub.1, R'.sub.2, and R'.sub.3 are not
hydrogen at the same time; or
[0034] b2) R'.sub.1, and R'.sub.2, and optionally R'.sub.3,
together with the nitrogen atom form an optionally substituted
heterocyclic ring system including the nitrogen atom as a ring
member, and optionally including an additional hetero atom, and if,
R'.sub.3 is not part of the ring system it is independently
selected from hydrogen, alkyl, alkenyl, amino- or hydroxy- or
alkoxy-substituted alkyl, or substituted amino-alkyl; or
[0035] b3) R'.sub.1, is a group of general formula III,
R'--(CHR'.sub.4).sub.m-- III
[0036] wherein m is zero or an integer from 1 to 5, R' is
optionally substituted aliphatic hydrocarbon cyclic system having 3
to 8 carbon atoms in the ring, R'.sub.4 is hydrogen, or alkyl,
amino- or hydroxy- or alkoxy-substituted alkyl, or substituted
amino-alkyl, or a group of the same general formula as R'.sub.1, as
defined herein above; R'.sub.2 and R'.sub.3 are the same as
R'.sub.1 or hydrogen, alkyl, alkenyl, amino- or hydroxy- or
alkoxy-substituted alkyl, or substituted amino-alkyl or alkenyl;
or
[0037] b4) R'.sub.1 is an optionally substituted aryl group of
general formula IV; 4
[0038] wherein R'.sub.5 is hydrogen and one or more substituents,
and m is zero or in integer from 1 to 5; and R'.sub.2 and R'.sub.3
may be independently hydrogen, alkyl, amino- or hydroxy- or
alkoxy-substituted alkyl, or substituted amino-alky, or groups of
the same general formula R'.sub.1
[0039] Substitutions which are not explicitly specified are usual
"inert" substituents, such as halogens, a hydroxyl group, alkyl
having 1 to 4 carbon atoms, alkoxyl having 1 to 4 carbon atoms,
acyloxyl having 1 to 4 carbon atoms and esterified carboxyl having
1 to 4 carbon atoms.
[0040] Advantageous examples of amines which form the salt with
HMG-CoA reductase inhibitors are: (.+-.)-1,2-dimethylpropylamine,
3-(2-aminoethylamino)-propylamine, n-butylamine, secondary
butylamine, tertiary butylamine (TBA), dibutylamine, tertiary
amylamine, cyclopentylamine, cyclohexylamine, cycloheptylamine,
dicyclohexylamine (DCHA), N-methylcyclohexylamine,
N,N'-diisopropylethylenediamine (DIPEDA), N,N'-diethylenediamine,
N-methyl-1,3-propanediamine, N-methylethylenediamine,
N,N,N',N'-tetramethyl-1,2-diaminoethane,
N,N,N',N'-tetramethyl-1,4-diaminobutane,
N,N,N',N'-tetramethyl-1,6-diamin- ohexane, 1,2-dipiperidinethane,
dipiperidinemethane, 2-amino-3,3-dimethylbutane,
N,N-dimethylcyclohexylamine, neopentylamine, adamantylamine,
N,N-diethylcyclohexylamine, N-isopropylcyclohexylamine,
N-methyl-cyclohexylamine, cyclobutylamine and norborylamine.
Preferably in terms of crystallization efficiency, combined with
low toxicity and low costs, the amine is selected from the group
consisting of n-butylamine, secondary butylamine, TBA,
dibutylamine, tertiary amylamine, cyclohexylamine, DCHA,
N-methylcyclohexylamine and DIPEDA. The amine may particularly be
selected from the group consisting of TBA, DIPEDA, DCHA and
N-methylcyclohexylamine.
[0041] The amines specified above are advantageous over the direct
isolation via the salts with ammonia in terms of purification
efficiency. Furthermore, amines having a larger organic group, and
especially those having bulky groups, generally show a more readily
crystallization and to a lower extent form salts with unwanted side
products when compared with amines having small organic groups.
Accordingly, amines having at least one hydrocarbon residue with
secondary or tertiary carbon atoms, or with cyclic hydrocarbon
structure (either aromatic or aliphatic), and organic diamines are
particularly suitable for the present invention.
[0042] Any type of HMG-CoA reductase inhibitors can be used
according to the present invention. Those HMG-CoA reductase
inhibitors selected from the group consisting of mevastatin,
pravastatin, lovastatin, simvastatin, fluvastatin and atorvastatin
have shown good results and are particularly preferred.
[0043] For isolating and purifying the desired HMG-CoA reductase
inhibitor, the amine salt is most effectively formed directly from
the crude medium of the respective HMG-CoA reductase inhibitor,
which crude medium is usually derived from a fermentation broth as
the result of a biotechnological process or from a reaction mixture
as the result of a semisynthesis or the total synthesis and usually
contains the desired HMG-CoA reductase inhibitor together with
unwanted side products and impurities. The crude medium may
preferably contain the HMG-CoA reductase inhibitor in its acid
from, and the formation of the amine salt may be effected by simply
adding the amine to the crude medium. The crude medium may be an
organic phase or a mixture of an organic or an aqueous phase where
the impure HMG-CoA reductase inhibitor is present in an organic
solvent, such as ethyl acetate, ether or acetonitrile. Ethyl
acetate is preferred as the organic solvent. After the
biotechnological treatment, the crude medium in the organic phase
is preferably obtained from the fermentation broth by a process
including the step of extracting the HMG-CoA reductase inhibitor
into the aforementioned organic solvent. The process for the
preparation of the amine salts of HMG-CoA reductase inhibitors may
include the following steps:
[0044] a) contacting the medium containing the HMG-CoA reductase
inhibitor, which is preferably in the acid form in an organic
solvent, with at least one of the amines specified above,
[0045] b) optionally forming crystallization nuclei by known
techniques,
[0046] c) filtering the crystals crystallized out,
[0047] d) washing the crystals with an organic solvent, and
[0048] e) drying the crystals.
[0049] The term "contacting" includes the known techniques for the
preparation of the salts from substances with acid properties and
substances with alkaline properties. The crystallization is
preferably carried out at a temperature between 0 and 30.degree.
C., more preferably between 4 and 22.degree. C.
[0050] The term "organic solvent" means organic solvents which are
used in industry such as ethyl acetate, butyl acetate, ether and
acetonitrile, including their aqueous mixtures.
[0051] Since the amines specified above effectively form salts with
the HMG-CoA reductase inhibitors, they are also particularly
suitable as auxiliary materials or processing aids in a process for
preparing the HMG-CoA reductase inhibitor in a purified form.
Previously isolated HMG-COA reductase inhibitors can be thus
obtained in a higher purity. The purified form is usually prepared
by crystallization. Accordingly, the present invention provides a
process for the isolation and/or purification of a HMG-CoA
reductase inhibitor.
[0052] In further aspects of the present invention, the salt as
specified above is suitably used as the starting substance or the
intermediate substance in a process for preparing the HMG-CoA
reductase inhibitor which is in a modified form, in a
pharmaceutically active salt form or in the lactone form.
Specifically, the modified form is obtained by chemical
modification or biotechnological modification, which modifications
are known to those skilled in the art. The pharmaceutically active
salt is preferably a metal salt, such as the sodium salt or the
calcium salt.
[0053] In this connection, the term "a process for the
semi-synthetic preparation of HMG-CoA reductase inhibitors" means
the preparation of HMG-CoA reductase inhibitors using any of the
known chemical modifications of the HMG-CoA reductase inhibitors.
An example of such process is the semisynthesis of simvastatin from
lovastatin as the starting substance. Most preferably, the TBA salt
of lovastatin is used as the starting substance.
[0054] Furthermore, the term "a process for the biotechnological
modification of HMG-CoA reductase inhibitors" means the preparation
of HMG-CoA reductase inhibitors using microorganisms or enzymatic
systems thereof for modifying HMG-CoA reductase inhibitors. An
example of such process is the biotechnological conversion of
mevastatin into pravastatin. The amine salt is preferably the TBA
salt.
[0055] Furthermore, the term "a process for the conversion of the
amine salts of HMG-CoA reductase inhibitors into the
pharmaceutically acceptable salts of HMG-CQA reductase inhibitors"
includes processes for the preparation of HMG-CoA reductase
inhibitors by one of the known methods wherein the amine salts of
HMG-CoA reductase inhibitors are used as the starting substance.
Specific examples or converted salts are the sodium salts of
pravastatin and fluvastatin and the calcium salt of
atorvastatin.
[0056] Furthermore, the term "conversion of the amine salts of
HMG-CoA reductase inhibitors into HMG-CoA reductase inhibitors in
the lactone form" includes processes for the preparation of HMG-CoA
reductase inhibitors in the lactone form by one of the known
methods wherein the amine salts of HMG-CoA reductase inhibitors are
used as the starting substance. Examples of HMG-CoA reductase
inhibitors converted into the lactone form are lovastatin,
mevastatin or simvastatin.
[0057] The present invention is illustrated but in no way limited
by the following examples.
EXAMPLES
Example 1
Preparation of Free Acid of Mevastatin and Conversion Thereof into
Salt Thereof with Tertiary Butylamine (TBA)
[0058] Mevastatin (200 g) was suspended in 30% volume/volume (v/v)
aqueous acetonitrile solution (2500 ml), 3 equivalents of
triethylamine were added and the mixture was heated to 80.degree.
C. and stirred for 30 minutes. After the completed reaction,
acetonitrile was evaporated, the remaining solution was acidified
to pH 4 with phosphoric acid and extracted into ethyl acetate
(2.times.1000 ml). The pooled extracts were dried by the addition
of 30 g of sodium sulphate, the desiccant was filtered off and the
solution was concentrated (950 ml). TBA (1.5 equivalents) was added
to the solution and crystallization was carried out for 30 minutes
at 8.degree. C. The crystals formed were filtered and washed with
ethyl acetate (2.times.100 ml) and subsequently dried at 40.degree.
C. for 15 hours. The crystals obtained (the TBA salts of mevastatin
215 g) were white in color with a HPLC purity of 96.8%. The yield
of the hydrolysis and crystallization was 91%.
Example 2
Preparation of the Sodium Salt of Nevastatin from the TBA Salt of
Mevastatin
[0059] The TBA salt of mevastatin (1 g), obtained by the process
disclosed in example 1, was dissolved in 3 ml of ethanol (96% v/v)
and sodium hydroxide (40 g/L of ethanol) was added. The resulting
mixture was precipitated in the ethyl acetate (60 ml). After the
crystallization (30 min) at 8.degree. C. the crystals were filtered
off, washed with ethyl acetate and dried. The product: crystals of
the sodium salt of mevastatin (0.65 g) pale brown in color with a
HPLC purity of 98%.
Example 3
Isolation of the TBA Salts of Lovastatin from the Fermentation
Broth
[0060] A fermentation broth (160 L) obtained by the fermentation
with a microorganism Aspergillus terreus ATCC 20544 and having a
lovastatin content of 1 g/L was transferred from the fermenter into
the tank (400 L) and pH was adjusted to 10 with the addition of 1 M
aqueous sodium hydroxide 10 minutes of vigorous stirring the pH of
the broth was decreased to 9 by adding 1 M sulphuric acid solution
and the biomass was filtered off. The filtrate obtained was
acidified to a pH value of 6.5 with 1 M sulphuric acid solution and
160 L of ethyl acetate was added. The slurry was subsequently
stirred for 20 minutes. The aqueous and ethyl acetate phases were
separated by extraction centrifuge and the ethyl acetate extract
was concentrated in a rotavapor to the volume of 14 L. The
concentration of lovastatin in the form of free acid in
concentrated ethyl acetate extract was 10.1 g/L. To the obtained
lovastatin solution (HPLC purity 72.7%) in the form of free acid in
ethyl acetate (800 ml) 1.05 equivalents of TBA were added. After
crystals were crystallized out, they were filtered off, washed with
ethyl acetate (2.times.50 ml) and dried in a vacuum oven at
35.degree. C. for 24 hours. The yield: 8.06 g of crystals of the
TBA salt of lovastatin with a HPLC purity of 99.2%.
Example 4
Preparation of the Salt of Lovastatin with Tertiary Amylamine
[0061] Lovastatin (5 g) was treated according to the process for
hydrolysis disclosed in example 1 and sodium salt thereof (4.8 g)
was prepared and dissolved in 100 ml of water. pH was adjusted to 4
with phosphoric acid (10% aqueous solution) and the formed free
acid of lovastatin was extracted from the water phase into ethyl
acetate (2.times.100 ml). The pooled ethyl acetate extracts were
dried with sodium sulphate, the solution was filtered off and
concentrated to ca. 100 ml. To the solution, prepared in the above
manner (ca. 100 ml), 1.05 equivalents of tertiary amylamine were
added. After crystals were crystallized out, they were filtered
off, washed with ethyl acetate (2.times.10 ml) and dried in a
vacuum oven (35.degree. C., 24 hours). The yield: 4.8 g of crystals
of the tertiary amylamine salt with a HPLC purity of 98.6%.
Example 5
Preparation of Pravastatin in the Free Acid Form and Conversion
into the (.+-.)-1,2-Dimethylpropylamine Salt Thereof
[0062] Pravastatin (11 g, HPLC purity 97.20) in the form of Na salt
was dissolved in water (100 ml) and pH was adjusted to 4 with
phosphoric acid (10% aqueous solution).
[0063] The resulting free acid was extracted from the water phase
into ethyl acetate (2.times.100 ml).
[0064] The pooled ethyl acetate extracts were dried with sodium
sulphate, the solution was filtered off and concentrated to a
volume of ca. 100 ml.
[0065] To the concentrate (9 ml) thus obtained, 1.5 equivalents of
(.+-.)-1,2-dimethyl-propylamine were added. After crystals were
crystallized out, they were filtered off, washed with 20 ethyl
acetate (2.times.10 ml) and dried in a vacuum oven at 35.degree. C.
The yield: crystals yellow in color with a HPLC purity of
98.28%.
Examples 6-13
The conversion of the Free Acid Form of Pravastatin into the Amine
Salt Form as Described in Example 5 was Repeated with Other
Amines
[0066] The amines used and results are shown in the Table
below:
1 Ex. AMINE EFFECT HPLC purity 6 3-(2 aminoethylamino)- An oil is
formed* -- propylamine 7 N,N.sup.1-diisopropyl- Pale yellow
crystals 99.3% ethylenediamine 8 N,N.sup.1-diisopropyl- Yellow
crystals 99.3% ethylenediamine 9 N-methyl-1,3- An oil is formed* --
propanediamine 10 N-methylethylenediamine An oil is formed -- 11
Secondary-butylamine White crystals 98.9% 12 Tertiary-butylamine
(TBA) Yellowish crystals 97.4% 13 Tertiary-amylamine Off-white
crystals 97.9% *from an oil formed, crystals are crystallized out
overnight at 4.degree. C.
Example 14
Preparation of the Salt of Pravastatin with Secondary Butylamine
from the Crude Sodium Salt of Pravastatin
[0067] According to the process described in example 5, pravastatin
in the free acid form was prepared from the sodium salt of
pravastatin (HPLC purity 83.6%). Further the salt or pravastatin
with secondary butylamine was prepared by the process described in
example 5. White crystals with a HPLC purity or 97.3% were
obtained.
Example 15
Preparation of the Sodium Salt of Pravastatin from the TBA Salt of
Pravastatin (I)
[0068] 8.9 g of the TBA salt of pravastatin as obtained in example
12 were dissolved in 22 ml of ethanol (96%) and subsequently
precipitated in 450 ml of ethyl acetate. Crystallization was
carried out at 8.degree. C. for 60 minutes, the crystals formed
were filtered off, washed with two 50 ml portions of ethyl acetate
and dried at 40.degree. C. for 5 hours. 1 g of the recrystallized
TBA salt of pravastatin, obtained in the above-described manner,
was dissolved in 5 ml of ethanolic solution of 0.43N NaOH and
precipitated in 60 ml of ethanol. After 30 minutes at 8.degree. C.,
crystals formed were filtered off and dried. The formed crystals of
the sodium salt of pravastatin (0.75 g) were dark yellow in
color.
Example 16
Preparation of the Sodium Salt of Pravastatin from the TBA Salt of
Pravastatin (2)
[0069] The process described in example 15 was repeated wherein 1 g
of the recrystallized TBA salt of pravastatin was dissolved in 3 ml
of water, instead of ethanolic solution of 0.43N NaOH, and
subsequently an aqueous sodium carbonate solution (equivalent) was
added. The obtained solution was diluted with 5 ml of ethanol and
the resulting sodium salt was precipitated with ethyl acetate.
After 30 minutes at 8.degree. C., crystals formed were filtered off
and dried. The formed crystals of the sodium salt of pravastatin
(0.65 g) were yellow in color.
Example 17
Preparation of Pravastatin from the TBA Salt of Mevastatin
[0070] Preparation of Inoculum for Production
[0071] The colonies of microorganism Amycolatopsis orientalis ATCC
19795 were transferred to a sterile potter and homogenized. The
resulting colonies were transferred to agar slopes and incubated in
the thermostat at 26.degree. to 30.degree. C. for 7 to 14 days.
During that time surfaces of agar slopes were overgrown by cultures
of homogeneous, folded, smooth, white to pale greyish-blue
mycelium. Further, 10 ml of sterile water was poured onto the agar
slopes, the culture was scraped off with the pipette and the
contents transferred into the potter. A portion (0.5 to 1 ml) of
the resulting culture was then inoculated into the vegetative
medium.
[0072] Agar Medium for the Preparation of Agar Slopes and Petri
Plates.
2 Raw material Amount Dextrin 10 g Consumer's glucose 5 g Casaminic
acid 3 g Yeast extract 4 g Agar 15 g Sterile water up to 1000 ml No
pH adjustment needed.
[0073] Vegetative Phase of Fermentation
[0074] The inoculum grown on the slope at 26.degree. to 30.degree.
C. for 10 days and prepared according to the above-described method
was inoculated in a 500-ml Erlenmeyer flask containing 50 ml of the
vegetative medium. After 24 hours of shaking at 220 rpm at
28.degree. C., the culture was transferred onto the fermentation
medium.
[0075] Vegetative Medium:
3 Raw material Amount Corn starch for fermentation 20 g Soybean
flour for fermentation 14 g Glucose 10 g Yeast extract 5 g
NaH.sub.2PO.sub.4 .times. 2H.sub.20 3.3 g Tap water to 1000 ml
[0076] Conversion of the TBA Salts of Mevastatin into
Pravastatin
[0077] The contents of fifteen Erlenmeyer flasks with the culture,
prepared according to the above-described method, were used to
inoculate the fermenter (50 L) with 30 L of fermentation medium.
After 20-hour fermentation, the solution of the TBA salt of
mevastatin and glucose (1200 g of glucose and 70 g of TBA salt of
mevastatin (assay of mevastatin: 80%) dissolved in 5 L of water)
was continually added to the medium at the flow rate 2 ml/min.
During the fermentation 70% oxygen saturation of the medium was
maintained by stirring with the frequency between 300 and 600 rpm.
Analyses of the concentration of pravastatin in the fermentation
broth showed the total final concentration of pravastatin in the
fermentation broth to be 690 g/kg of broth after 76 hours of
fermentation at temperature between 24.degree. and 30.degree. C.,
indicating a 40% conversion of mevastatin into pravastatin.
[0078] Fermentation Medium:
4 Raw material Amount Corn starch for fermentation 20 g Soybean
flour for fermentation 5 g Glucose 10 g Yeast extract 5 g Antifoam
agent 10 g All raw materials were dissolved in tap water, the pH
was then adjusted to 7.4.
Example 18
Preparation of TBA Salt of Simvastatin
[0079] Simvastatin (1.95 g) was suspended in 50 ml of 30% (v/v)
acetonitrile, triethylamine (1 ml) was added and the solution was
heated for 20 minutes at 70.degree. C. After completion of reaction
acetonitrile was evaporated from the solution, the remaining
solution was acidified to pH 4 with phosphoric acid. Simvastatin in
the acid form was extracted into ethyl acetate (2.times.50 ml) and
the pooled extracts were dried with 2 g of sodium sulphate. The
desiccant was filtered off and the ethyl acetate solution was
concentrated to 20 ml. To the solution 1.05 equivalents of TBA were
added and crystallization was carried out for 1 hour at 8.degree.
C. The product was filtered off and dried at 40.degree. C. for 1
hour. The yield: 2 g of the TBA salt of simvastatin.
Example 19
Conversion of the TBA Salt of Simvastatin into Simvastatin
Lactone
[0080] The TBA salt of simvastatin (1.6 g), obtained by the process
disclosed in example 18, was dissolved in water (36 ml), the
solution was then acidified to pH 3.7 with an aqueous phosphoric
acid solution and simvastatin in the acid form was extracted into
ethyl acetate (2.times.50 ml). The pooled extracts were dried with
sodium sulphate (2 g) and the desiccant was then filtered off. The
ethyl acetate solution was concentrated to 20 ml.
[0081] To the solution trifluoroacetic acid (0.5 ml) was added and
the reaction mixture was heated for 25 minutes at 50.degree. C.
After completion of the reaction, the ethyl acetate solution was
extracted with 50% (w/w) aqueous solution of ammonium hydrogen
carbonate. The organic phase was dried with sodium sulphate (2 g),
the desiccant was filtered off and the ethyl acetate solution was
concentrated to 4 ml. Crystallization was carried out for 1 hour at
8.degree. C. The product was then filtered off and dried at
40.degree. C. for 1 hour. The yield: 0.9 g of simvastatin in the
acid form.
Example 20
Preparation of Free Acid of Simvastatin
[0082] Simvastatin (20 g) was dissolved in the mixture of water (80
ml) and 8M KOH (18 ml). The solution was stirred in nitrogen
atmosphere for two hours at room temperature.
[0083] After that 120 ml of ethyl acetate was added and pH was
adjusted to 2-3 with 5% aqueous solution of HCl. Ethyl acetate
phase was washed with mixture of water (100 ml), 5% aqueous
solution of HCl and 5% aqueous solution of NaCl (50 ml). The
obtained ethyl acetate phase was then dried for three hours with
addition of 20 g MgSO.sub.4 anhydrite and filtrated.
[0084] Preparation of Amine Salt
[0085] After that 18 ml of N-methylcyclohexylamine was added into
the ethyl acetate phase. After two days at temperature between 0
and 5 degrees C. crystals of simvastatin N-methylcyclohexylamine
salt were formed. Obtained crystals were filtered off and washed
with 20 ml of ethyl acetate, 40 ml ethyl acetate/n-heptane (1:1)
and 40 ml of pentane. The yield: 20.5 g of N-methylcyclohexylamine
salt of simvastatin. The yield calculated to the starting
simvastatin was 79%. The same results were obtained also with
cyclohexylamine (by the use of the same method).
Example 21
Preparation of Pravastatin in Purified Form
[0086] Pravastatin (30 g, HPLC purity 90.2%) in the form of Na salt
was dissolved in water (100 ml) and pH was adjusted to 3 with HCl
(10% aqueous solution). The resulting free acid was extracted from
the water phase into ethyl acetate (3.times.200 ml). The pooled
ethyl acetate extracts were dried with sodium sulphate, the
solution was filtered off and 7.604 g of N-methylcyclohexylamine
was added (drop by drop at intensive stirring). After crystals were
crystallized out, they were filtered off and recrystallized from
the mixture of ethyl acetate/methanol. The yield: 21 g of crystals
of pravastatin N-methylcyclohexylamine salt with a HPLC purity of
99.28%.
Example 22
Preparation of Free Acid of Atorvastatin
[0087] Calcium salt of atorvastatin (3 g) was suspended in 100 ml
water. The pH was adjusted with the phosphoric acid to 4 and after
that free acid of atorvastatin was extracted with ethyl acetate
(3.times.100 ml). Combined ethyl acetate extract was dried and 2.7
g of oil containing atorvastatin in the free acid form was
obtained.
[0088] Preparation of Amine Salt
[0089] 1 g of atorvastatin in the free acid form was dissolved in
acetonitrile (100 ml) and 1.1 molar equivalents of TBA was added.
The obtained mixture was concentrated to 30 ml and after four hours
at 8.degree. C. the TBA salt of atorvastatin crystallized. White
precipitate was filtered off and dried in a rotary evaporator.
Yield: 1 g of TBA salt of atorvastatin.
Example 23
Preparation of Amine Salt
[0090] 1 g of atorvastatin in the free acid form was dissolved in
methanol (20 ml) and 1.1 molar equivalents of dicyclohexylamine
(DCHA) in 50 ml of n-hexane was added. After four hours at
8.degree. C. the DCHA salt of atorvastatin crystallized. White
precipitate was filtered off and dried in a rotary evaporator.
Yield: 1.1 g of DCHA salt of atorvastatin.
* * * * *