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Patent 2474688 Summary

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(12) Patent: (11) CA 2474688
(54) English Title: IMPROVED PROCESS FOR OBTAINING BETULINIC ACID
(54) French Title: PROCEDE DE PRODUCTION AMELIORE D'ACIDE BETULINIQUE
Status: Deemed expired
Bibliographic Data
(51) International Patent Classification (IPC):
  • C07J 63/00 (2006.01)
  • A61K 31/56 (2006.01)
  • C07J 53/00 (2006.01)
(72) Inventors :
  • SAUTER, MARKUS (Germany)
(73) Owners :
  • BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG (Germany)
(71) Applicants :
  • BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG (Germany)
(74) Agent: FETHERSTONHAUGH & CO.
(74) Associate agent:
(45) Issued: 2012-03-20
(86) PCT Filing Date: 2003-01-30
(87) Open to Public Inspection: 2003-08-14
Examination requested: 2008-01-28
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/EP2003/000905
(87) International Publication Number: WO2003/066659
(85) National Entry: 2004-07-27

(30) Application Priority Data:
Application No. Country/Territory Date
102 04 271.3 Germany 2002-02-02

Abstracts

English Abstract




The invention relates to an improved method for obtaining highly pure,
crystalline betulinic acid from a methanolic extract of ground plane bark
and/or plane rind. The invention also relates to a highly pure, crystalline
betulinic acid, which is solvated with an equivalent of ethanol.


French Abstract

L'invention concerne un procédé de production amélioré d'acide bétulinique cristallin présentant un degré de pureté élevé, à partir d'un extrait méthanolique d'écorce de platane et/ou de rhytidome de platane moulu(e)(s). L'invention se rapporte en outre à cet acide bétulinique cristallin extrêmement pur, qui est solvaté au moyen d'un équivalent d'éthanol.

Claims

Note: Claims are shown in the official language in which they were submitted.



CLAIMS:
1. Process for obtaining highly pure, crystalline betulinic acid from a
methanolic extract of ground plane cortex and/or plane bark by concentrating
the
extract and crystallising it from an alcohol, wherein

a) the ground plane cortex is heated to boiling once with 2 to 5 times
the amount of methanol for 0.5 to 5.0 hours, extracted, the extract obtained
is filtered
at a temperature between 40 and 60°C, evaporated down to 30 to 70% of
its volume
and crystallised at a temperature between -10 and +10°C; and

b) the following steps are carried out successively for washing and
isolating the residue of the methanolic extract:

(i) heating the residue in a nonpolar diluent;
(ii) separating the residue from the diluent;

(iii) heating the residue in ethanol, clarifying the ethanolic phase by the
addition of activated charcoal and separating the activated charcoal from the
ethanolic phase at a temperature between 60 and 95°C; and

(iv) crystallising and separating the betulinic acid from the ethanolic
phase.

2. Process according to claim 1, wherein the residue of the methanolic
extract is taken up in 2 to 8 times the amount of aliphatic hydrocarbon and
the
mixture is heated to boiling.

3. Process according to claim 2, wherein the aliphatic hydrocarbon is
.eta.-hexane.

4. Process according to any one of claims 1 to 3, wherein in step (ii) the
residue is separated from the diluent by centrifuging.

7


5. Process according to any one of claims 1 to 4, wherein in step (iii) the
residue is taken up in 20 to 100 times the amount of ethanol and heated to
boiling.
6. Process according to any one of claims 1 to 5, wherein in step (iv) the
ethanolic phase is cooled to temperatures of -10 to +10°C and the
betulinic acid
precipitated is separated off by centrifuging and dried.

7. Process according to claim 6, wherein in step (iv) the ethanolic phase is
cooled to temperatures of 0 to +5°C.

8. Process according to any one of claims 1 to 7, wherein the betulinic
acid thus obtained is present as the monoethoxide.

8

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02474688 2004-07-27
WO 03/066659 PCT/EP03/00905
80006pct.210

Improved process for obtaining betulinic acid
Description
The invention relates to an improved process for obtaining highly pure,
crystalline betulinic
acid from a methanolic extract of ground plane bark.

Background to the invention
Betulinic acid is 3 (3-hydroxy-lup-20(29)-ene-28-acid of formula
CH2
H3C

CH3 CH3 COON
H CH3
HO
H3C CH3

It is known that betulinic acid is effective against the growth of melanoma
cells (e.g. Pisha et
at., Nature Medicine 1, 1995, 1046 ff) as well as against other cancer cells
(e.g. Sunder et al.,
US Patent US 6,048,847). In addition its amides are supposed to be suitable
for use against
HIV (e.g. Evers et al., J. Med. Chem. 39, 1996, 1056 ff, or Soler et at., J.
Med. Chem. 39,
1996, 1069 ff). Consequently betulinic acid is in great demand.

Apart from being produced synthetically (e.g. L. Ruzicka et al., Helv. Chim.
Acta 21, 1938,
1076 ff) betulinic acid may also be obtained from various plants, particularly
trees, such as for
example from the outer bark or cortex of Picramniapentandra (e.g. Herz et al.,
Phytochemistry 11, 1972, 3061 ff), from the cortex of Arbutus menziesii
(Robinson et al.,
Phytochemistry 9, 1970, 907 ff) and from the cortex of Ziziphus mauritiana
(e.g. Pisha et al.,
Nature Medicine 1, 1995, 1046 ff).


CA 02474688 2011-01-21
25771-927

It is difficult to isolate betulinic acid from these starting materials. By
contrast it would appear
more promising to obtain it from the cortex and/or bark of the plane tree
(Platanus acerifolia).
DE 197 13 768 proposes a process for obtaining betulinic acid, in which a
powder obtained
from plane bark is extracted with a medium polarity solvent such as for
example
dichloromethane, chloroform or diethyl ether.

However, this process is unsuitable for the industrial recovery of large
amounts of betulinic
acid, as very large volumes of the medium polarity solvent have to be used to
extract the
betulinic acid (7 litres of dichloromethane are used to 150 g of powdered
plane bark).
Bruckner et al., J. Chem. Soc. 1948, 948-951 describe a process for obtaining
betulinic acid
from plane bark in which the ground bark is extracted with methanol, the
extract obtained is
evaporated down, and the concentrate is repeatedly recrystallised from
methanol in the
presence of charcoal. However, the betulinic acid thus obtained still contains
a large number
15. of impurities.

The aim of the present invention was therefore to provide an improved process
for the large-
scale recovery of highly pure, crystalline betulinic acid from plane bark
and/or plane cortex
which avoids the disadvantages of the known methods.

Detailed description of the invention
Surprisingly it has now been found that highly pure crystalline betulinic acid
can be obtained
from a methanolic extract of ground plane bark and/or plane cortex by
evaporating the extract
down and crystallising it from an alcohol if the residue from the methanolie
extraction is
washed with a nonpolar diluent before the crystallisation.

25,
The present invention thus relates to a process for obtaining highly pure,
crystalline betulinic
acid from a methanolic extract of ground plane bark and/or plane cortex by
evaporation of the
extract and crystallisation from an alcohol, characterised in that before the
crystallisation the
residue is washed with a nonpolar diluent.

2


CA 02474688 2004-07-27

The invention further relates to highly pure crystalline betulinic acid which
is solvated with
one equivalent of ethanol.

The Western plane (Platanus occidentalis) has proved particularly suitable as
the plane tree in
question.

Suitable alcohols for recrystallising the crude betulinic acid are generally
aliphatic alcohols
with 1 to 4 C-atoms, particularly methanol, ethanol or isopropanol or mixtures
thereof, most
preferably ethanol.

Suitable nonpolar diluents are generally aliphatic, cycloaliphatic or aromatic
hydrocarbons or
mixtures thereof, preferably aliphatic hydrocarbons with 5 to 8 carbon atoms,
particularly
pentane, hexane or heptane, most preferably n-hexane, cycloaliphatic
hydrocarbons with 5 to
8 carbon atoms, particularly cyclopentane, cyclohexane, methylcyclohexane, or
aromatic
hydrocarbons with 6 to 9 carbon atoms, particularly toluene or xylene.

Where, in the interests of simplicity, the ratio of two substances is given
hereinbefore or
hereinafter in the form "x to y-fold amount", where x and y denote the lower
and upper limits,
respectively, of the amount specified, this refers to "x to y" parts by weight
of one substance
in relation to one (1) part by weight of the other substance.

Preferred embodiments of the invention are:

(A) processes in which the following steps for washing and isolating the
residue of the
methanolic extract are carried out successively:
(i) heating the residue in a nonpolar diluent;
(ii) separating the residue from the diluent;
(iii) heating the residue in ethanol, clarifying the ethanolic phase by the
addition of
activated charcoal and separating the activated charcoal from the ethanolic
phase at a
temperature between 60 and 95 C; and
(iv) crystallising and separating the betulinic acid from the ethanolic phase.

(B) processes in which the ground plane cortex is heated to boiling with 2 to
5 times the
amount of methanol for 0.5 to 5.0 hours, extracted, the extract obtained is
filtered at a
temperature between 40 and 60 C, evaporated down to 30 to 70 % of its volume
and
crystallised at a temperature between - 10 and + 10 C.

3


CA 02474688 2011-01-21
25771-927

In an embodiment, the present invention provides a process for
obtaining highly pure, crystalline betulinic acid from a methanolic extract of
ground
plane cortex and/or plane bark by concentrating the extract and crystallising
it
from an alcohol, wherein a) the ground plane cortex is heated to boiling once
with
2 to 5 times the amount of methanol for 0.5 to 5.0 hours, extracted, the
extract
obtained is filtered at a temperature between 40 and 60 C, evaporated down to
30 to 70% of its volume and crystallised at a temperature between -10 and +10
C
and b) the following steps are carried out successively for washing and
isolating
the residue of the methanolic extract; (i) heating the residue in a nonpolar
diluent;
(ii) separating the residue from the diluent; (iii) heating the residue in
ethanol,
clarifying the ethanolic phase by the addition of activated charcoal and
separating
the activated charcoal from the ethanolic phase at a temperature between
60 and 95 C; and (iv) crystallising and separating the betulinic acid from the
ethanolic phase.

The term "betulinic acid" as used above and hereinafter includes
both betulinic acid as such and the hydrates and solvates thereof, preferably
betulinic acid which is solvated with 1 to 2 equivalents of an alcohol,
particularly
one equivalent of ethanol.

2a


CA 02474688 2004-07-27

(C) processes in which the residue of the methanolic extract is taken up in 2
to 8 times the
amount of an aliphatic hydrocarbon, preferably cyclohexane, methylcyclohexane
or n-hexane,
particularly n-hexane, and the mixture is heated to boiling.

(D) processes in which in step (ii) the residue is separated from the diluent
by centrifuging
or suction filtering.

(E) processes in which in step (iii) the residue is taken up in 20 to 100
times, preferably
30 to 80 times, particularly 40 to 60 times the amount of ethanol and heated
to boiling.

(F) processes in which in step (iv) the ethanolic phase is cooled to
temperatures of -10 to
+ 10 C, preferably 0 to + 5 C, the betulinic acid precipitated is separated
off by centrifuging
or suction filtering and dried.

(G) processes in which the betulinic acid obtained is in the form of the
monoethoxide.
Highly pure crystalline betulinic acid which is solvated with one equivalent
of ethanol is
particularly preferred, particularly the betulinic acid ethoxide of formula

CH2
H3C

SH3: CH3 COOH

H CH3 CAM
HO
H3C CH3

In a most particularly preferred embodiment of the process according to the
invention the
powdered plane cortex is suspended in 2 to 5 times, preferably 3 to 4 times as
much methanol
and heated for 1 to 5 hours, particularly about 2 hours, preferably by
refluxing. The
suspension is filtered at 40 to 60 C, particularly at about 50 C, the methanol
extract is
evaporated down to 60 to 40 %, particularly about 50 % of its volume. The
concentrated
extract is cooled to 0 to 10 C, particularly about 5 C, filtered off and
washed with cold
methanol. The residue is preferably dried in vacuo at 40 to 70 C,
particularly at about 50 C.
In this way, the crude betulinic acid is obtained as a colourless powder
(about 2.0 to 2.5 %,
based on plane cortex). This is suspended in 4 to 8 times, particularly 5 to 6
times as much
n-hexane, cyclohexane or methylcyclohexane and heated for 0.5 to 2 hours,
particularly I
hour, particularly by refluxing. The suspension obtained is centrifuged or
suction filtered,
It


CA 02474688 2004-07-27

washed with 1.5 to 4.5 times, particularly about 3 times as much hot n-hexane,
cyclohexane or
methylcyclohexane, and the residue is dried overnight at about 40 C,
preferably in vacuo.

The betulinic acid thus obtained is dissolved in 30 to 50 times as much
ethanol, with heating.
Activated charcoal is added and the mixture is filtered while hot. As it
cools, crystallisation
begins. The mixture is cooled to 0 to 10 C, particularly 5 C, suction
filtered or centrifuged
and washed with cold ethanol.
Highly pure crystalline betulinic acid is obtained, which is solvated with one
mol of ethanol.
The following Example serves to illustrate a process for obtaining betulinic
acid which is
carried out by way of example. It is intended solely as a possible procedure
provided as an
illustration, without restricting the invention to its contents.

5


CA 02474688 2004-07-27
EE ample

1A Extraction of the plane cortex
4500 g of plane cortex powder (ground in a cutter mill) are suspended in 15 1
of methanol and
refluxed for 2 h. The suspension is filtered at about 50 C, the methanol
extract is evaporated
down to 50% of its volume (7.5 1). The concentrated extract is cooled to 5 C,
filtered off and
washed with 2 1 of cold methanol. The residue is dried in vacuo at 50 C.
104 g of crude betulinic acid are obtained as a colourless powder (2.3 %,
based on plane
cortex). HPLC: 90.6 %

1B Stirring out from nonpolar solvent
93 g of betulinic acid obtained according to Example 1A are suspended in 600
ml of n -hexane
and refluxed for 1 hour. The suspension is suction filtered through a nutsche,
washed with
300 ml of hot n-hexane, and the residue is dried overnight at 40 C in vacuo.
89.5 g (96.2 %) of betulinic acid are obtained as a colourless powder. HPLC:
94.75 %
Cyclohexane, methylcyclohexane or mixtures of these solvents may also be used
analogously.
1C Recrystallisation from ethanol
89 g of betulinic acid obtained according to Example 1B are refluxed and
dissolved in 4 1 of
ethanol. 40 g of activated charcoal are added and the mixture is filtered hot
through a
nutsche. After cooling to ambient temperature, crystallisation sets in. The
mixture is cooled to
5 C, suction filtered and washed with 500 ml of cold ethanol. 63.5 g (71.3 %)
of crystalline
betulinic acid is obtained, which is solvated with 1 mol of ethanol. HPLC:
93.8 % (percentage
area 100%); melting point = 296-298 C, [a]D = +8, c= 0.9 in pyridine.

1D Working up the mother liquor
4 1 of the ethanolic mother and washing liquor from Example 1C are evaporated
down to 1
litre in vacuo, suction filtered at 5 C and washed with cold ethanol.
20 g (22.5 %) of betulinic acid are obtained. HPLC: 96.5 %

6

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Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date 2012-03-20
(86) PCT Filing Date 2003-01-30
(87) PCT Publication Date 2003-08-14
(85) National Entry 2004-07-27
Examination Requested 2008-01-28
(45) Issued 2012-03-20
Deemed Expired 2016-02-01

Abandonment History

Abandonment Date Reason Reinstatement Date
2006-01-30 FAILURE TO PAY APPLICATION MAINTENANCE FEE 2006-02-10

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Registration of a document - section 124 $100.00 2004-07-27
Application Fee $400.00 2004-07-27
Maintenance Fee - Application - New Act 2 2005-01-31 $100.00 2004-07-27
Reinstatement: Failure to Pay Application Maintenance Fees $200.00 2006-02-10
Maintenance Fee - Application - New Act 3 2006-01-30 $100.00 2006-02-10
Maintenance Fee - Application - New Act 4 2007-01-30 $100.00 2006-12-15
Maintenance Fee - Application - New Act 5 2008-01-30 $200.00 2007-12-19
Request for Examination $800.00 2008-01-28
Maintenance Fee - Application - New Act 6 2009-01-30 $200.00 2008-12-23
Maintenance Fee - Application - New Act 7 2010-02-01 $200.00 2009-12-22
Maintenance Fee - Application - New Act 8 2011-01-31 $200.00 2010-12-22
Maintenance Fee - Application - New Act 9 2012-01-30 $200.00 2011-12-23
Final Fee $300.00 2012-01-09
Maintenance Fee - Patent - New Act 10 2013-01-30 $250.00 2013-01-14
Maintenance Fee - Patent - New Act 11 2014-01-30 $250.00 2014-01-17
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
BOEHRINGER INGELHEIM PHARMA GMBH & CO. KG
Past Owners on Record
SAUTER, MARKUS
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Document
Description 
Date
(yyyy-mm-dd) 
Number of pages   Size of Image (KB) 
Abstract 2004-07-27 1 9
Claims 2004-07-27 2 61
Description 2004-07-27 6 257
Cover Page 2004-10-01 1 26
Claims 2011-07-22 2 50
Claims 2011-01-21 2 59
Description 2011-01-21 7 282
Cover Page 2012-02-21 1 27
Prosecution-Amendment 2011-02-17 2 88
PCT 2004-07-27 8 306
Assignment 2004-07-27 4 124
Prosecution-Amendment 2008-01-28 1 45
Prosecution-Amendment 2011-07-22 4 138
Prosecution-Amendment 2010-07-21 2 51
Prosecution-Amendment 2011-01-21 8 291
Correspondence 2012-01-09 2 60