Note: Descriptions are shown in the official language in which they were submitted.
CA 02705902 2010-05-13
A Method for Producing Betulinic Acid
FIELD OF THE INVENTION
The present invention relates to a semi-synthesis method for producing
natural compound, and in particular, to a semi-synthesis method for producing
betulinic acid from betulin.
BACKGROUND OF THE INVENTION
Betulinic acid (I), also known as R-betulinic acid, is a plant secondary
metabolite of pentacyclic triterpene, which is a white needle crystal with
Molecular formula of C30H4803, molecular weight of 456.71; and structural
formula:
Jr Ji
jeV
H H
21Ot-I H OH
0
F1 R
3
HO HO
I II
Betulinic acid has antimalarial, anti-inflammatory and anti-HIV activity, and
displays cytotoxic activities to melanoma cells, which makes it as an
anticancer and anti-AIDS compound with broad application and developmental
prospect. The content of betulinic acid in plants is limited, while the
content of
betulin (i.e. betulinol, II), a betulinic acid analogue, in white birch bark
is very
high, reaching 1035%. Betulinic acid may be produced through
semi-synthesis from betulin.
The United States Patent and Trademark Office disclosed a series of
invention patents entitled "Methods for manufacturing betulinic acid" on May
15,
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2001, August 7, 2001, June 18, 2002 and March 15, 2005, respectively, which
provide a five-step method for synthesizing betulinic acid from betulin:
firstly,
acylating betulin into betulin 3,28- dicarboxylic ester; secondly, converting
betulin 3,28- dicarboxylic ester into betulin-3- acetic ester through
alcoholysis;
thirdly, oxidizing betulin-3-acetic ester into betulinic aldehyde-3-acetic
ester,
and then betulinic acid-3-acetic ester; finally, deprotecting to generate
betulinic
acid. This method has long reaction route, complex reaction conditions and
high producing cost.
On September 8, 1998, The United States Patent and Trademark Office
disclosed a patent of invention entitled "Methods of Manufacturing betulinic
acid", which provides a process for synthesizing betulinic acid by a two-step
reaction: firstly, forming betulonic acid intermediate through Jones
oxidation,
and then generating betulinic acid through a selective reduction reaction.
This
method is simple, but what is generated after the reduction process is a
mixture of betulinic acid and a-betulinic acid, a non-bioactive betulinic acid
isomer. Through recrystallization, this product still contains a-betulinic
acid.
SUMMARY OF THE INVENTION
The present invention is designed to provide a method for producing
betulinic acid, with simple routing and easily purified product.
In order to achieve the object of the present invention, in an aspect, the
present invention provides a method for generating betulinic acid from betulin
through twice selective oxidations.
Wherein, the selective oxidation reaction is to oxidize C-28 hydroxyl group
of betulin.
In particular, the selective oxidation reaction comprises: step (1)
Preparation of betulinic aldehyde through selective oxidation of betulin; and
step (2) Preparation of betulinic acid through oxidation reaction of betulinic
aldehyde.
Wherein, the oxidizer for the selective oxidation reaction in step (1) is
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trichloroiminocyanuric acid or N-chlorosuccinimide; and the oxidizer for the
selective oxidation reaction in step (2) is silver oxide.
In particular, the selective oxidation reaction also comprises the
purification of betulinic acid by recrystallizing the crude betulinic acid
prepared
through oxidation reaction.
In a further aspect, the present invention provides a method for twice
selective oxidization of betulin, wherein, the oxidization reaction comprises:
oxidizing betulin into betulinic aldehyde first; then oxidizing betulinic
aldehyde
into crude betulinic acid; and finally, recrystallizing the crude betulinic
acid to
obtain betulinic acid.
In still a further aspect, the present invention provides a method for
producing betulinic acid, comprising the following steps:
(1) Preparation of betulinic aldehyde through oxidation of betulin:
Firstly, dissolve betulin into an organic solvent, and add
2,2,6,6-tetramethyl-1- piperidone, alkaline solution and oxidizer into the
mixture one by one; then make the mixture react for 0.5-12 hours at 2575 C
under stirring and produce betulinic aldehyde;
(2) Preparation of betulinic acid through oxidation of betulinic aldehyde:
Firstly, dissolve betulinic aldehyde into an organic solvent, and add sodium
hydroxide solution and silver oxide into the mixture one by one; then make the
mixture react for 3-72 hours at 2585 C under stirring, and prepare crude
betulinic acid through acidification.
Wherein, the molar ratio of betulin and 2,2,6,6-tetramethyl-1-piperidone
mentioned in step (1) is 1:0.01-0.05; and the molar ratio of betulin and the
oxidizer is 1: 0.8-1.5; the organic solvent dosage is 100-200ml per gram of
betulin; and the alkaline solution dosage is 100-200ml per gram of betulin.
In particular, the organic solvent mentioned in step (1) is selected from
one of methylene dichloride, chloroform or ethyl acetate; the alkaline
solution
is selected from one of sodium hydroxide solution, sodium carbonate solution
or sodium bicarbonate solution; and the oxidizer is trichloroiminocyanuric
acid
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or N-chlorosuccinimide.
Wherein, the pH value of the alkaline solution is 8.3-12.5;
In particular, the step (1) also comprises: standing to layer the reactant,
separating the organic layer and evaporating the organic solvent by vacuum so
as to prepare the betulinic aldehyde.
Wherein, mentioned in step (2) the molar ratio of betulinic aldehyde and
silver oxide is 1: 0.8-1.5. The organic solvent is selected from one of
tetrahydrofuran, dioxane, ethanol or acetone; and the mass concentration of
sodium hydroxide solution is 10-40%, 30% being preferable.
In particular, the organic solvent dosage is 5-20m1 per gram of betulinic
aldehyde; sodium hydroxide solution dosage is 0.5 - 1.5 ml per gram of
betulinic aldehyde, and silver oxide is prepared freshly.
In particular, the step (2) also comprises: standing to precipitate the
reaction solution of crude betulinic acid upon reaction first, filtering the
reactant
and then recrystallizing the sediment obtained by filtering so as to provide
the
betulinic acid.
Preferably, adding the acidified solution into ice water, standing to
precipitate the mixture, and then conducting the above-mentioned filtration.
In particular, step (2) also comprises: extracting the reaction solution of
crude betulinic acid upon reaction first, and then concentrating and
recrystallizing the sediment so as to obtain betulinic acid.
Preferably, adding the acidified solution into ice water, and then
conducting the above-mentioned extraction.
Wherein, a concentrated hydrochloric acid is adopted for the acidification,
and the pH value of the acidified solution is 2.0-3.0, and 2.5 being
preferable;
the mass concentration of the concentrated hydrochloric acid is 20-37%; the
methylene dichloride are adopted for the extraction of the acidified solution;
one of ethanol, methanol or acetic acid is adopted for the recrystallization.
In particular, betulinic acid is prepared by concentrating organic layer upon
extraction.
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Advantages of the present invention:
1. The present invention is to generate crude betulinic aldehyde by
oxidizing betulin and then oxidize crude betulinic aldehyde, so that, in the
oxidation product, only bioactive betulinic acid is generated, without
non-bioactive a-betulinic acid.
2. The present invention adopts a step-by-step oxidization and
highly-selective oxidizer which has no effect on C-3 hydroxyl group, thus
functional group protection is not necessary.
3. The present invention has the superiorities of short routing, simple
processing method, easily purified product and low production cost, thus
industrialized production and popularization is easily achieved.
DESCRIPTION OF DRAWINGS
Fig.1 Schematic diagram for synthesizing betulinic acid through
oxidization of betulin
DETAILED DESCRIPTION OF THE INVENTION
Further details of the invention are described by combining the Examples
of the present invention, as follows:
A method for producing betulinic acid, with the betulin as the raw material,
comprising the following steps:
(1) Preparation of crude betulinic aldehyde through oxidation of betulin:
dissolve betulin into an organic solvent, and add 2,2,6,6-tetramethyl-
1-piperidone, inorganic alkaline solution with a pH value of 8.012.5 and
oxidizer into the mixture; stir to make the mixture react at 2575 C for 0.5-12
hours; upon completion of reaction, keep the mixture standing to layer,
separate the organic layer and make the organic phase concentrated so as to
provide the grey white crude betulinic aldehyde; the molar ratio of betulin
and
2,2,6,6-tetramethyl-1-piperidone is 1:0.01-0.05, and the molar ratio of
betulin
and the oxidizer is 1: 0.8-1.5; the organic solvent dosage is 100-200ml per
CA 02705902 2010-05-13
gram of betulin; and the inorganic alkaline solution dosage is 100-200ml per
gram of betulin;
(2) Preparation of crude betulinic acid through oxidation of crude betulinic
aldehyde: dissolve crude betulinic aldehyde generated in step (1) into an
organic solvent, and add sodium hydroxide solution and the freshly prepared
silver oxide into the mixture; after stirring to make the mixture react at
2585 C
for 3-72 hours, make the reactant cool to room temperature, and filter the
reactant; The filtrate is generated betulinic acid through acidification,
filtering,
washing and drying; the organic solvent dosage is 5-20m1 per gram of crude
betulinic aldehyde;
(3) Preparation of betulinic acid through recrystallization of crude betulinic
acid: recrystallizing the crude betulinic acid obtained in step (2) by using
ethanol, methanol or acetic acid as the solvent to obtain betulinic acid.
The method for producing betulinic acid, wherein, the organic solvent
mentioned in step (1) is methylene dichloride, chloroform or ethyl acetate.
The method for producing betulinic acid, wherein, the inorganic alkaline
solution mentioned in step (1) is sodium hydroxide solution, sodium phosphate,
sodium carbonate solution or sodium bicarbonate solution.
The method for producing betulinic acid, wherein, the oxidizer mentioned
in step (1) is trichloroiminocyanuric acid or N-chlorosuccinimide.
The method for producing betulinic acid, wherein, the organic solvent
mentioned in step (2) is tetrahydrofuran, dioxane, ethanol or acetone.
Embodiment 1
Dissolve 44.3g (0.1 moL) betulin (I) into 5000m1 methylene dichloride, and
add 0.156g (0.001 moL) 2,2,6,6-tetramethyl-1-piperidone, 5000m1 sodium
bicarbonate solution with a pH value of 8.3, and 20g (0.15moL)
N-chlorosuccinimide one by one while stirring; after reacting at 25 C for 10
hours under stirring, stand to layer the mixture, separate the organic layer,
and
evaporate methylene dichloride under vacuum so as to yield 45.5g crude
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betulinic aldehyde (III); dissolve the obtained crude betulinic aldehyde into
910m1 ethanol, add 50ml sodium hydroxide solution with a mass concentration
of 30%, and then add 24g freshly prepared silver oxide into the mixture; after
react at 70 C under stirring for 5 hours, filter the mixture, acidify the
mixture
through dropwise adding concentrated hydrochloric acid (mass concentration
of 36%) into the filtrate until the pH value is 2.5, and then pour the mixture
into
5000m1 ice water, extract with methylene dichloride and concentrate so as to
provide crude betulinic acid; and recrystallize crude betulinic acid with
methanol so as to yield 23.6g betulinic acid (II) with a purity of 92.9% and
yield
of 48%.
Betulinic aldehyde is white crystal (methanol); melting point: 190-192 C
(192-193 C); El-MS m/z: 440([M+]), 207, 189, 135; 'H-NMR(300MHz ,
CDCI3);60.76 (s, 3H, 23-Me) , 0.83(s, 3H, 24-Me), 0.92 (s, 3H, 25-Me), 0.97(s,
3H, 26-Me), 0.98(s,3H,27-Me),1.70(s, 3H, 30-Me),3.19 (q, 3-H), 4.76 (s,
29a-H), 4.63(s, 29b-H), 9.68(s, 28-H);13C-NMR(300MHz, CDCI3038.9(C-1),
28.0(C-2), 79.0(C-3), 40.8(C-4), 59.4(C-5), 18.3(C-6), 34.3(C-7), 40.8(C-8),
50.5(C-9), 37.2(C-10), 20.8(C-11), 25.6(C-12), 38.7(C-13),42.6 (C-14), 29.9
(C-15), 34.3 (C-16), 55.3 (C-17), 47.6 (C-18), 48.1 (C-19), 149.8(C-20),
29.9(C-21), 38.7(C-22), 27.4 (C-23), 15.9(C-24), 16.2(C-25), 16.2(C-26),
14.3(C-27), 206.7(C-28), 110.2 (C-29), 19.0 (C-30).
The betulinic acid is white crystal (methanol); melting point:
284-287 C(285-287 C); El-MS m/z: 456([M+]), 248, 220, 207, 189, 175;
1H-NMR(300MHz,CDCI3) :O0.76(s, 3H, 23-Me), 0.83(s, 3H, 24-Me), 0.94(s, 3H,
25-Me), 0.97(s, 3H, 26-Me), 0.98(s, 3H, 27-Me), 1.69(s, 3H, 30-Me), 3.17(q,
3-H), 4.74(s, 29a-H), 4.62(s, 29b-H); 13C-NMR(300MHz, CDC13): 638.4(C-1),
28.0(C-2), 79.0(C-3), 40.7(C-4), 55.4(C-5), 18.3(C-6), 34.3(C-7), 38.4(C-8),
50.5(C-9), 37.0(C-10), 19.3(C-11), 27.4(C-12), 38.7(C-13), 42.5(C-14),
29.7(C-15), 34.3(C-16), 56.3(C-17), 49.3(C-18), 46.9(C-19), 150.4(C-20),
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29.7(C-21), 37.2(C-22), 28.0(C-23), 15.4(C-24), 16.0(C-25), 16.1(C-26),
14.7(C-27), 180.7(C-28),109.7(C-29), 19.4(C-30).
Embodiment 2
Dissolve 44.3g (0.1 moL) betulin (I) into 4430ml chloroform, and add 0.78g
(0.005 moL) 2,2,6,6-tetramethyl-1-piperidone, 4430m1 sodium phosphate
solution with a pH value of 11.5, and 26g (0.11 moL) trichloroiminocyanuric
acid
one by one while stirring; after reacting at 50 C for 0.5 hours under
stirring,
stand to layer the mixture and separate the organic layer, evaporate
chloroform under vacuum so as to yield 44.Og crude betulinic aldehyde (III).
Dissolve crude betulinic aldehyde into 220m1 tetrahydrofuran, add 50m1
sodium hydroxide solution with a mass concentration of 30%, and then add
24g freshly prepared silver oxide into the mixture; after reacting at 40 C
under
stirring for 30 hours, filter the mixture, acidify the mixture through
dropwise
adding concentrated hydrochloric acid (mass concentration of 30%) into the
filtrate until the pH value is 2.5, and then pour the mixture into 5000m1 ice
water,
extract with methylene dichloride and concentrate so as to provide crude
betulinic acid; and recrystallize crude betulinic acid with ethanol so as to
yield
22.6g betulinic acid (II) with a purity of 93.1% and yield of 46%.
Embodiment 3
Dissolve 44.3g (0.1 moL) betulin (I) into 5000m1 methylene dichloride, and
add 0.25g (0.0016 moL) 2,2,6,6-tetramethyl-1-piperidone, 5000m1 sodium
hydroxide solution with a pH value of 12.5, and 18.6g (0.08moL)
trichloroiminocyanuric acid one by one while stirring; after reacting at 30 C
under stirring for 3 hours, stand to layer the mixture and separate the
organic
layer, evaporate the methylene dichloride under vacuum so as to yield 45.Og
crude betulinic aldehyde (III); dissolve crude betulinic aldehyde into 450m1
dioxane, add 50ml sodium hydroxide solution with a mass concentration of
30%, and then add 24g silver oxide freshly prepared into the mixture; after
reacting at 40 C under stirring for 30 hours, filter the mixture, acidify the
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mixture through dropwise adding concentrated hydrochloric acid (mass
concentration of 20%) into the filtrate until the pH value is 2.5, and then
pour
the mixture into 5000m1 ice water, extract with methylene dichloride and
concentrate so as to provide crude betulinic acid; and recrystallize crude
betulinic acid with ethanol so as to yield 23.3g betulinic acid (II) with a
purity of
94.0% and yield of 48%.
Embodiment 4
Dissolve 22.2g (0.05moL) betulin (I) into 4440m1 ethyl acetate, and add
0.30g (0.0019 moL) 2,2,6,6-tetramethyl-1-piperidone, 4440ml sodium
carbonate solution with a pH value of 9.5, and 8g (0.06moL)
N-chlorosuccinimid one by one while stirring; reacting at 75 C under stirring
for
12 hours, stand to layer the mixture and separate the organic layer, evaporate
ethyl acetate under vacuum so as to yield 25.0 g crude betulinic aldehyde
(III);
dissolve the crude betulinic aldehyde into 150m1 acetone, add 30ml sodium
hydroxide solution with a mass concentration of 30%, and then add 13g silver
oxide prepared freshly into the mixture; after reacting at 25 C under stirring
for
72 hours, filter the mixture, acidify the mixture through dropwise adding
concentrated hydrochloric acid (mass concentration of 36%) into the filtrate
until the pH value is 2.5, and then pour the mixture into 2500ml ice water,
extract with methylene dichloride and concentrate so as to provide crude
betulinic acid; and recrystallize crude betulinic acid with acetic acid so as
to
yield 12.1 g betulinic acid (11) with a purity of 92.1 % and yield of 49%.
Embodiment 5
Dissolve 44.3g (0.1 moL) betulin (I) into 5000m1 methylene dichloride, and
add 0.35g (0.0022 moL) 2,2,6,6-tetramethyl-1-piperidone, 5000m1 sodium
hydroxide solution with a pH value of 12.0, and 26g (0.11 moL)
trichloroiminocyanuric acid one by one while stirring; after reacting at 35 C
under stirring for 2 hours, stand to layer the mixture and separate the
organic
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layer, evaporate methylene dichloride under vacuum so as to yield 44.8g crude
betulinic aldehyde (III); dissolve crude betulinic aldehyde into 900m1
ethanol,
add 50ml sodium hydroxide solution with a mass concentration of 30%, and
then add 24g freshly prepared silver oxide into the mixture; after reacting at
75 C for 3 hours under stirring, filter the mixture, acidify the mixture
through
dropwise adding concentrated hydrochloric acid (mass concentration of 25%)
into the filtrate until the pH value is 2.5, and then pour the mixture into
5000ml
ice water, extract with methylene dichloride and concentrate so as to provide
crude betulinic acid; and recrystallize crude betulinic acid with ethanol so
as to
yield 23.7g betulinic acid (II) with a purity of 91.2% and yield of 47%.
Embodiment 6
Dissolve 44.3g (0.1 moL) betulin (I) into 5000m1 chloroform, and add 0.16g
(0.001moL) 2,2,6,6-tetramethyl -1-piperidone, 5000m1 sodium hydroxide
solution with a pH value of 12.0, and 26g (0.11 moL) trichloroiminocyanuric
acid
one by one under stirring; after reacting at 50 C under stirring for 2 hours,
stand to layer the mixture and separate the organic layer, evaporate
chloroform under vacuum so as to yield 44.Og crude betulinic aldehyde (III);
dissolve crude betulinic aldehyde into 250m1 propanetriol, add 50ml sodium
hydroxide solution with a mass concentration of 10%, and then add 24g silver
oxide into the mixture; after reacting at 20 C for 3 hours under stirring,
filter the
mixture, acidify the mixture through dropwise adding concentrated
hydrochloric acid (mass concentration of 36%) into the filtrate until the pH
value is 2.5, and then pour the mixture into 5000ml ice water; keep the
mixture
standing for 24 hours at 0-5 C, and then filter the mixture so as to provide
the
crude betulinic acid; and recrystallize crude betulinic acid with ethanol so
as to
yield 22.6g betulinic acid (II) with a purity of 91.0% and yield of 45%.
