U.S. patent application number 14/489834 was filed with the patent office on 2015-01-01 for anxiolytic marcgraviaceae compositions containing betulinic acid, betulinic acid derivatives, and methods.
The applicant listed for this patent is UNIVERSITY OF OTTAWA. Invention is credited to John T. Arnason, Tony Durst, Zulifiquar Merali, Luis J. Poveda-Alvarez, E. Pablo Sanchez-Vindas.
Application Number | 20150005267 14/489834 |
Document ID | / |
Family ID | 26965971 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150005267 |
Kind Code |
A1 |
Durst; Tony ; et
al. |
January 1, 2015 |
ANXIOLYTIC MARCGRAVIACEAE COMPOSITIONS CONTAINING BETULINIC ACID,
BETULINIC ACID DERIVATIVES, AND METHODS
Abstract
Pharmaceutical compositions for preventing or treating anxiety,
comprising betulinic acid or derivatives thereof are provided.
Methods for preventing or treating anxiety with betulinic acid or
derivatives thereof are also provided. The invention further
provides betulinic-acid containing preparations of plants of the
family Marcgraviaceae having anxiolytic activity and methods for
making such extracts and using them to prevent or treat anxiety in
a subject.
Inventors: |
Durst; Tony; (Ottawa,
CA) ; Merali; Zulifiquar; (Ottawa, CA) ;
Arnason; John T.; (Ottawa, CA) ; Sanchez-Vindas; E.
Pablo; (Heredia, CR) ; Poveda-Alvarez; Luis J.;
(Alejuela, CR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
UNIVERSITY OF OTTAWA |
Ottawa |
|
CA |
|
|
Family ID: |
26965971 |
Appl. No.: |
14/489834 |
Filed: |
September 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13672578 |
Nov 8, 2012 |
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14489834 |
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|
12366837 |
Feb 6, 2009 |
8623848 |
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13672578 |
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10476716 |
May 17, 2004 |
7488722 |
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PCT/CA02/00695 |
May 17, 2004 |
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12366837 |
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60290022 |
May 11, 2001 |
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60290035 |
May 11, 2001 |
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Current U.S.
Class: |
514/169 ;
552/510 |
Current CPC
Class: |
A61K 31/565 20130101;
A61K 31/16 20130101; A61K 36/45 20130101; A61K 31/40 20130101; A61K
31/215 20130101; C07J 53/002 20130101; A61K 31/58 20130101; A61K
36/185 20130101; A61K 36/185 20130101; A61K 2300/00 20130101; A61K
31/19 20130101; A61P 25/22 20180101; A61K 31/56 20130101 |
Class at
Publication: |
514/169 ;
552/510 |
International
Class: |
C07J 53/00 20060101
C07J053/00; A61K 36/185 20060101 A61K036/185; A61K 31/565 20060101
A61K031/565 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 2, 2002 |
CA |
PCT/CA02/00695 |
Claims
1. A betulinic acid-containing composition obtained from of a plant
of the family Marcgraviaceae.
2. The composition of claim 1, wherein the composition is an
extract of Marcgraviaceae, prepared by a method comprising: (a)
contacting a Marcgraviaceae plant, or part thereof, with a first
solvent in which betulinic acid is soluble, to form a betulinic
acid-containing extract; and (b) recovering the betulinic
acid-containing extract from the first solvent, thereby preparing a
betulinic acid-containing composition.
3. The composition of claim 2, wherein (b) further comprises: (a)
filtering the betulinic acid-containing extract from (a); and (b)
evaporating the first solvent.
4. The composition of claim 2, wherein (b) further comprises: (a)
filtering the betulinic acid-containing extract from (a), (b)
evaporating the solvent, (c) extracting by stirring with a second
solvent; and (d) evaporating the second solvent.
5. The composition of claim 1, comprising at least about 0.1% to
90% by weight betulinic acid.
6. The composition of claim 1, comprising at least about 0.5% by
weight betulinic acid.
7. The composition of claim 1, comprising at least about 1% by
weight betulinic acid.
8. The composition of claim 1, comprising at least about 5% by
weight betulinic acid.
9. The composition of claim 1, wherein the plant is of a genus
selected from the group consisting of Marcgraviastrum, Norantea,
Ruyschia, Sarcopera, Marcgravia, Pseudosarcopera, Souroubea, or
Schwartzia.
10. The composition of claim 1, wherein the plant is a Souroubea
species.
11. The composition of claim 11, wherein the Souroubea species is
selected from the group consisting of Souroubea gilgii, Souroubea
sympetala, Souroubea loczyi, Souroubea venosa, and Souroubea
vallicola.
12. The composition of claim 1, wherein the plant is selected from
the group consisting of Marcgraviastrum subsessile, Marcgravia
schipii, Marcgravia nepanthoides, Marcgravia browneii, Sarcopera
sessiflora, Sarcopera resulata, Schwartzia tarrazuentis, Schwarzia
costaricensis, and Schwarzia brazilenses.
13. The composition of claim 3, wherein the first solvent is water,
ethyl acetate, dichloromethane, methanol, ethanol, propanol, or
butanol.
14. The composition of claim 3, wherein the first solvent is
ethanol and the second solvent is ethyl acetate.
15. A pharmaceutical composition or a supplement comprising the
composition of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 13/672,578, filed Nov. 8, 2012 now pending, which is a
continuation application of U.S. application Ser. No. 12/366,837,
filed Feb. 6, 2009, now issued as U.S. Pat. No. 8,623,848, which is
a divisional application of U.S. application Ser. No. 10/476,716
filed May 17, 2004, now issued as U.S. Pat. No. 7,488,722; which is
a 35 USC .sctn.371 National Stage application of International
Application No. PCT/CA02/00695 filed May 10, 2002, now expired;
which claims the benefit under 35 USC .sctn.119(e) to U.S.
Application Ser. No. 60/290,035, filed May 11, 2001, now expired
and to U.S. Application Ser. No. 60/290,022, filed May 11, 2001,
now expired. The disclosure of each of the prior applications is
considered part of and is incorporated by reference in the
disclosure of this application.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to betulinic-acid containing extracts
of the plant family Marcgraviaceae, compositions containing
betulinic acid or betulinic acid derivatives, and methods for
preventing or treating anxiety using same.
[0004] 2. Background Information
[0005] There is ever-increasing interest in herbal or
natural-source remedies or medications. Many individuals would
rather use such products than conventional pharmaceutical
preparations. Additionally, medicinal substances derived from
natural products can provide commercial or industrial opportunities
for local populations in areas where medicinal plants grow or are
cultivated. Moreover, compounds identified as the active
ingredients in natural products form an important basis for
pharmaceutical research. disorders and anxiety disorders due to
medical conditions, substance abuse and not otherwise specified
anxiety (American Psychiatric Association. (1994). Diagnostic and
statistical manual of mental disorders, 4th Edn. (DSM-IV).
Washington, D.C.). Anxiety disorders are characterized by three
basic components; subjective psychological reports, behavioural
responses and physiological responses. A person usually reports
subjective feelings of tension, apprehension, dread and
expectations of an inability to cope (Alloy, L. B., Jacobson, N. S,
& Acocella, J., (1999). Abnormal Psychology: Current
Perspectives (pp. 150-172.). McGraw-Hill, Boston Mass.). These
feelings can lead the person to behavioural responses as coping
mechanisms, such as avoidance of the feared situation, impaired
speech and motor functioning, and impaired performance on complex
cognitive tasks. Physiological changes are often manifested as
well; these include muscle tension, increased heart rate and blood
pressure, dry mouth, nausea and dizziness (Weiss, S. R. B, &
Uhde, T. W. (1990). Animal models of anxiety. In Neurobiology of
Panic Disorder (pp. 3-27). Maryland: Alan R. Liss, Inc.). A natural
product, or a pharmaceutical preparation derived from a natural
product, would be of great interest in the alleviation of
anxiety.
[0006] Marcgraviaceae is a plant family common in Costa Rica.
Although the use of herbal or plant-based remedies is common
throughout South America, to our knowledge, anxiolytic activity of
plants of the family Marcgraviaceae, or of extracts or compounds
obtained therefrom, has not been reported.
[0007] A variety of natural or herbal remedies containing betulinic
acid have been described as being useful for treating depression or
stress, disorders that differ from anxiety.
[0008] For instance, U.S. Pat. No. 5,589,182 to Pater and Tashiro
describes a pharmaceutical composition in unit dosage form for
treating various diseases including depression. The composition
comprises a mixture of aqueous extracts of a number of plants,
including extracts of dried seeds of Zizyphus jujuba containing
betulinic acid. The compositions can be in the form of a health
drink, in which the dried powder or concentrated aqueous solution
is mixed with a syrup formula, and carbonated water is added.
[0009] The WPI abstract for Japanese Patent No. 57031620 indicates
that the patent is directed to the preparation of a sweet to reduce
stress when stopping smoking. The sweet is prepared by boiling
seeds of gardenia in water, boiling persimmon leaves in water, and
then mixing the two solutions with corn syrup. The abstract
indicates that the solution of boiled persimmon leaves contains,
among other things, betulinic acid. The abstract provides that a
dose of about 10 grains per day of the sweet can alleviate stress
associated with smoking cessation.
[0010] The WPI abstract for Japanese Patent No. 69031593 appears to
be directed to a method for preparation of "betulin acid" involving
extracting the seeds of Zizyphus vulgaris var. spinosus. The
abstract provides that betulin acid is a narcotic with no side
effects, and is usually obtained from Betula alba.
[0011] Betulinic acid and various derivatives thereof are known to
have pharmacological activity, and the patent literature describes
the use of such compounds for the treatment of a range of
conditions. However, these generally relate to disorders other than
anxiety.
[0012] For instance, the use of betulinic acid and derivatives
thereof for cancer chemoprevention and chemotherapy is described in
U.S. Pat. No. 6,048,847 to Jaggi et al., U.S. Pat. No. 6,046,231 to
Kosmeder et al., U.S. Pat. Nos. 5,869,535 and 5,658,947 to Dasgupta
et al., among others.
[0013] Betulinic acid and various derivatives have also been
identified as being useful as anti-viral agents. German patent
application 19713768 to Draeger et al. discloses a preparation of
betulinic acid for use as an anti-cancer and anti-HIV agent. U.S.
Pat. No. 5,679,828 also discloses betulinic acid derivatives having
anti-retroviral activity, particularly anti-HIV activity.
[0014] U.S. Pat. No. 6,124,362 to Bailey et al. identifies
betulinic acid as a preferred agent in a composition for regulating
hair growth, when applied topically to the scalp.
[0015] U.S. Pat. No. 5,529,769 to Cho et al. describes compositions
containing betulinic acid said to be useful for the treatment of
skin conditions such as wrinkling and photodamage.
[0016] Japanese Patent No. 2000-247993 appears to describe a class
of triterpenoid compounds including oleanonic acid, oleanic acid,
3-epi-oleanolic acid, betulonic acid and 3-epi-betulinic acid, and
indicates that some of these compounds are sigma receptor agonists.
The patent appears to conclude that these compounds are therefore
useful for the treatment of a broad range of disorders said to be
related to the sigma receptor, such as schizophrenia, depression,
worry, cerebrovascular disorder, senile activity, Alzheimer's
disease, Parkinson's disease, Huntington's disease, drug addiction,
stress, anxiety, depression, etc. But no data is presented that the
mentioned triterpenoid compounds have any of the recited utilities,
let alone anxiolytic activity.
SUMMARY OF THE INVENTION
[0017] It has now been discovered that preparations obtained from
plants of the family Marcgraviaceae, particularly of the genus
Souroubea or Schwartzia have potent anxiolytic activity. Betulinic
acid has been determined to be the active ingredient in these
preparations having anxiolytic activity.
[0018] Betulinic acid is a colorless crystalline solid forming
plate-like or needle-like crystals. It has a molecular weight of
456.71, a melting point of 316-318.degree. C., and an empirical
formula of C.sub.30H.sub.50O.sub.2. Betulinic acid is soluble in
water, ethanol, ether, acetone, benzene, and chloroform. Betulic
acid is a synonym for betulinic acid. The structural formula of
betulinic acid is:
##STR00001##
[0019] Phytochemical principles are conserved in closely related
species, and all members of the family Marcgraviaceae are useful in
the context of the invention. Preferred members of the family
Marcgraviaceae include plants of genera Souroubea, Schwartzia,
Marcgraviastrum, Norantea, Ruyschia, and Sarcopera. Particularly
preferred are plants of the genera Souroubea and Schwartzia.
Especially preferred are plants of the genus Souroubea. A preferred
Souroubea species is Souroubea sympelata Gigli (synonyms Souroubea
guianensis Aubl.; Ruyschia guianensis (Aubl.) Sw.). S. sympetala is
a Neotropical vine that is indigenous to Guatemala, Belize,
Nicaragua, Costa Rica, Panama, Colombia, Venezuela, and Peru.
Another exemplary Souroubea species is S. gilgii V. A. Richt
(synonym Souroubea belizensis Lundell), a Mesoamerican vine
indigenous to Guatemala, Belize, Nicaragua, Costa Rica, and Panama.
Other preferred Souroubea species include, without limitation, S.
loczyi de Roon, S. venosa Shery, and S. vallicola Woodson.
[0020] Thus, in one aspect, the invention provides a betulinic
acid-containing preparation obtained from a plant of the family
Marcgraviaceae. As used herein and in the claims, a "preparation"
obtained from Marcgraviaceae means a non-naturally occurring
composition of matter that contains less than the entire complement
of biological materials found in the entire plant or plant part. In
this respect, an intact or ground-up fruit of Marcgraviaceae would
not constitute a "preparation" as defined herein, but a composition
from which some or all of the moisture, fibre or carbohydrates are
separated, would constitute a "preparation." A wide range of
preparations is contemplated. For instance, a betulinic-acid
containing tea-like beverage, made by steeping Souroubea leaves in
hot water, and then removing the leaves, would constitute a
preparation as defined herein. Similarly, a dried, finely ground
powder of Marcgraviaceae leaves would constitute a "preparation,"
as the powder would have a moisture content far below that found in
nature.
[0021] Preferably, the Marcgraviaceae preparation is an extract
obtained by contacting Marcgraviaceae plant material, such as
fruit, leaves, other plant parts, or a mixture thereof, with a
solvent in which betulinic acid is soluble, to form a betulinic
acid-containing extract, and then recovering the betulinic
acid-containing extract, as exemplified in the Examples herein.
Preferably, the solvent used is other than water.
[0022] As used herein and in the claims, the term "plant"
encompasses whole plants as well as plant parts, including, without
limitation, plant cells, tissues, seeds, embryos, roots, leaves,
stems, et cetera.
[0023] The preparation may take the form of a pharmaceutical
composition for preventing or treating anxiety, comprising a
preparation as described above together with one or more
pharmaceutically acceptable carriers, diluents, or excipients, as
are known in the art.
[0024] In another aspect, the invention provides a commercial
package comprising a betulinic acid-containing preparation obtained
from a plant of the family Marcgraviaceae, and instructions for use
of said preparation for preventing or treating anxiety in a
subject.
[0025] The invention also provides a method for making an
anxiolytic extract of Marcgraviaceae, comprising the steps of:
[0026] (a) contacting a Marcgraviaceae plant or a part thereof,
with a solvent in which betulinic acid is soluble, to form a
betulinic acid-containing extract; and,
[0027] (b) recovering said extract.
[0028] As discussed above, the solvent used is preferably other
than water.
[0029] The invention further provides a method for preventing or
treating anxiety in a subject comprising administering to a subject
in need thereof a therapeutically effective amount of a betulinic
acid-containing preparation obtained from Marcgraviaceae.
[0030] In another aspect, the invention extends to the use of a
plant of the family Marcgraviaceae for the prevention or treatment
of anxiety in a subject.
[0031] The invention further extends to the use of a plant of the
family Marcgraviaceae for the manufacture of an anxiolytic
preparation.
[0032] In another aspect, the invention provides a method for
preventing or treating anxiety in a subject comprising
administering to a subject in need thereof a therapeutically
effective amount of betulinic acid, or a pharmaceutically
acceptable salt thereof.
[0033] In another aspect, the invention provides a pharmaceutical
composition for preventing or treating anxiety in a subject
comprising a therapeutically effective amount of betulinic acid, or
a pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier, diluent, or excipient.
[0034] In another aspect, the invention contemplates use of
betulinic acid, or a pharmaceutically acceptable salt thereof for
the prevention or treatment of anxiety in a subject.
[0035] In another aspect, the invention contemplates use of
betulinic acid, or a pharmaceutically acceptable salt thereof, for
the manufacture of a medicament for the prevention or treatment of
anxiety.
[0036] In yet another aspect, the invention provides a commercial
package comprising betulinic acid, or a pharmaceutically acceptable
salt thereof, and instructions for use for preventing or treating
anxiety.
[0037] It has further been discovered that, surprisingly, a range
of derivatives of betulinic acid have potent anxiolytic activity.
These compounds are of the formula:
##STR00002##
or pharmaceutically acceptable salts thereof, wherein R is H, OH,
unsubstituted or substituted C.sub.1-C.sub.6 alkoxy, unsubstituted
or substituted C.sub.2-C.sub.6 alkenyloxy, unsubstituted or
substituted C.sub.2-C.sub.6 alkynyloxy, unsubstituted or
substituted C.sub.1-C.sub.3 alkyloxy(aryl), unsubstituted or
substituted C.sub.1-C.sub.3 alkyloxy(heteroaryl) containing up to
two heteroatoms selected from the group consisting of N, O and S,
unsubstituted or substituted aryloxy, an unsubstituted or
substituted 5- or 6-membered heteroaryloxy ring system containing
up to two heteroatoms selected from the group consisting of N, O
and S; or
##STR00003##
wherein R.sub.1 and R.sub.2 are, independently, H, OH,
unsubstituted or substituted C.sub.1-C.sub.6 alkyl, unsubstituted
or substituted C.sub.1-C.sub.6 alkenyl, unsubstituted or
substituted C.sub.1-C.sub.6 alkynyl, unsubstituted or substituted
C.sub.1-C.sub.3 alkyl(aryl), unsubstituted or substituted aryl,
or
[0038] wherein R.sub.1 and R.sub.2, together with the nitrogen atom
to which they are bonded, form a residue of an amino acid or an
alkyl ester of a residue of an amino acid, or
[0039] wherein R.sub.1 and R.sub.2, together with the nitrogen atom
to which they are bonded form an unsubstituted or substituted three
to seven membered ring possibly containing an additional heteroatom
selected from the group consisting of N, O and S;
[0040] R.sub.3 and R.sub.4 taken together form .dbd.CH.sub.1,
.dbd.CHR.sub.8, .dbd.O, --OCH.sub.1--, or R.sub.3 is H or OH and
R.sub.4 is H, OH, CH.sub.2OH, unsubstituted or substituted
C.sub.1-C.sub.5 alkyl, unsubstituted or substituted aryl,
unsubstituted or substituted heteroaryl containing up to two
heteroatoms selected from the group consisting of N, O and S, CH,
--R.sub.7 or NR.sub.1R.sub.2,
[0041] wherein R.sub.7 is a ketone, sulfoxide, sulfone, ester or
nitrile, and wherein R.sub.1 and R.sub.2 are as defined above,
and
[0042] wherein R.sub.8 is defined as for R.sub.4 except that
R.sub.8 is not H or OH [0043] R.sub.5 and R.sub.6 taken together
form .dbd.O, or, [0044] R.sub.5 is H and R.sub.6 is OH,
##STR00004##
[0044] (C.sub.1-C.sub.6 alkyl), unsubstituted or substituted
C.sub.1-C.sub.6 alkoxy, or
##STR00005##
where R.sub.1 and R.sub.2 are as defined above; R.sub.9 and
R.sub.10 are, independently, H, OH, CH.sub.2--R.sub.11, a halogen,
SCH.sub.3, S(O)CH.sub.3, SO.sub.2CH.sub.3, unsubstituted or
substituted S-aryl, unsubstituted or substituted S(O)-aryl,
unsubstituted or substituted SO.sub.2-aryl, unsubstituted or
substituted S-heteroaryl containing up to two heteroatoms selected
from the group consisting of N, 0 and S, unsubstituted or
substituted S(O)-heteroaryl containing up to two heteroatoms
selected from the group consisting of N, 0 and S, unsubstituted or
substituted SO.sub.2-heteroaryl containing up to two heteroatoms
selected from the group consisting of N, O and S, with the proviso
that R.sub.9 and R.sub.10 cannot both be OH, wherein R.sub.11 is H,
unsubstituted or substituted C.sub.1-C.sub.6 alkyl, unsubstituted
or substituted C.sub.2-C.sub.6 alkenyl, unsubstituted or
substituted C.sub.2-C.sub.6 alkynyl, unsubstituted or substituted
aryl or unsubstituted or substituted heteroaryl containing up to
two heteroatoms selected from the group consisting of N, O and
S;
R.sub.12 is H, or
[0045] R.sub.9 or R.sub.10, together with R.sub.12 form a bond.
[0046] Therefore, in a further aspect, the invention provides a
method for preventing or treating anxiety in a subject comprising
administering to a subject in need thereof a therapeutically
effective amount of a compound of formula II as defined above.
[0047] In another aspect, the invention also contemplates use of a
compound of formula II as defined above for preventing or treating
anxiety, as well as use of such compounds for the manufacture of a
medicament for the treatment or prevention of anxiety.
[0048] In yet another aspect, the invention provides a commercial
package comprising a compound of formula II as defined above, and
instructions for use of the compound for treating or preventing
anxiety in a subject.
[0049] When used in accordance with the invention, the active
ingredient is preferably provided in the form of a pharmaceutical
composition comprising a compound as described above together with
one or more pharmaceutically acceptable carriers, diluents, or
excipients, as are known in the art. Additional active ingredients,
such as additional anxiolytic agents, as are known in the art, may
also be present.
BRIEF DESCRIPTION OF THE DRAWINGS
[0050] FIG. 1 is a bar graph depicting the time spent (seconds) in
the open arms of an elevated plus-maze by animals administered
sweetened milk (control), 95% ethanol crude Marcgraviaceae plant
extract (marc.), ethyl acetate fraction (f1) and aqueous fraction
(f2) (**Significantly different from control at p<0.01).
[0051] FIG. 2 is a bar graph depicting the difference in startle
response (relative units) to tone presented in the absence or
presence of a fear cue. Rats were administered peanut oil
(control), 95% ethanol crude Marcgraviaceae plant extract (marc.),
or ethyl acetate fraction (f1), prior to test (**Significantly
different from control at p<0.01).
[0052] FIG. 3 is a bar graph depicting the effect of betulinic acid
(1 mg/kg by gavage) on the time spent (seconds) on the open arms of
the plus-maze (**Significantly different from control at
p<0.01).
[0053] FIG. 4 is a bar graph depicting the effect of betulinic acid
on fear-potentiated startle. The score reflects the difference in
startle amplitude noted in the presence and absence of the fear-cue
(**Significantly different from control at p<0.01).
[0054] FIGS. 5a and 5b are bar graphs depicting the effect of
betulinic acid (at 0.25 mg/kg and 2.5 mg/kg) on performance on the
elevated plus maze (**Significantly different from control at
p<0.01).
[0055] FIGS. 6a and 6b are bar graphs depicting the effect of
betulinic acid (at 0.25 mg/kg and 2.5 mg/kg) on performance on the
elevated plus maze in BALB/c mice (**Significantly different from
control at p<0.01).
[0056] FIG. 7 is a bar graph depicting the effect of betulinic
acid, beta-amyrin+betulinic acid, on performance on the elevated
plus maze (**Significantly different from control at
p<0.01).
[0057] FIG. 8 is a bar graph comparing the effect of betulinic acid
and its methyl ester on time spent (in seconds) relative to a
control by pairs of rats in social interaction (*,**Significantly
different from control at p<0.05 and 0.01, respectively).
[0058] FIGS. 9a, 9b and 9c are bar graphs illustrating the effects
of various derivatives of betulinic acid on punished drinking
(Vogel test) relative to diazepam.
[0059] FIG. 10 is a bar graph illustrating the effect of acute and
chronic treatments of betulinic acid and its methyl ester, relative
to a control, on the time spent in the open arms of an elevated
plus maze.
[0060] FIG. 11 is a graph illustrating the effect of chronic
treatment with betulinic acid and its methyl ester, relative to a
control, on locomotor activity.
[0061] FIG. 12 is a graph illustrating the effect of chronic
treatment with betulinic acid and its methyl ester, relative to a
control, on weight gain.
DETAILED DESCRIPTION OF THE INVENTION
Marcgraviaceae Preparations
[0062] Betulinic acid-containing preparations of Marcgraviaceae may
be prepared by, for instance, blending or macerating Marcgraviaceae
leaves, fruit, or other plant parts in a solvent in which betulinic
acid is soluble, filtering the blended material, and then
evaporating the solvent. Suitable solvents include, without
limitation, water, ethyl acetate, dichloromethane, or low molecular
weight alcohols such as methanol, ethanol, propanol, or butanol. A
solvent other than water is generally preferred. The resulting
preparation may take the form of a solid (such as a powder), a
liquid, or other forms. Alternatively, a preparation of
Marcgraviaceae can be prepared by drying Marcgraviaceae plants or
plant parts, and then reducing the dried materials to a powder. In
an exemplified case, the extract is a dark, viscous oil, which
contains betulinic acid.
[0063] The preparation can be concentrated to varying degrees,
limited principally by the amount of plant material a patient can
conveniently ingest. Generally, at least the fibrous plant material
and naturally-occurring plant carbohydrates are separated.
Typically, the preparation will contain betulinic acid in an amount
of at least 0.1% to 90% by weight, based on the
Marcgraviaceae-derived portion of the preparation, and preferably
contains at least 0.5%, more preferably at least 1%, even more
preferably at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%,
60%, 70% or 80% by weight betulinic acid, based on the
Marcgraviaceae derived portion of the preparation.
[0064] The Marcgraviaceae preparation may be incorporated into a
pharmaceutical composition (as discussed herein), or into a
supplement, such as a nutritional supplement, a food product, a
beverage, or the like, as known in the art.
Betulinic Acid and Betulinic Acid Derivatives
[0065] The invention also provides pharmaceutical compositions for
treating or preventing anxiety, comprising betulinic acids or
betulinic acid derivatives as defined herein.
[0066] As employed herein, the singular forms "a," "an," and "the"
include plural reference unless the context clearly dictates
otherwise.
[0067] As employed herein, "alkyl" refers to straight or branched,
cyclic or non-cyclic chain alkyl; "substituted alkyl" refers to
alkyl radicals further bearing one or more substituents such as
hydroxy, alkoxy (of an alkyl group), mercapto (of an alkyl group),
aryl, heteroaryl, heterocyclic, halogen, trifluoromethyl, cyano,
nitro, amino, carboxyl, carboxyalkyl, carbamate, sulfonyl,
sulfonamide, and the like;
[0068] "alkenyl" refers to straight or branched, cyclic or
non-cyclic hydrocarbyl chain radicals having at least one
carbon-carbon double bond; "substituted alkenyl" refers to alkenyl
radicals further bearing one or more substituents as set forth
above;
[0069] "alkynyl" refers to straight or branched, cyclic or
non-cyclic hydrocarbyl chain radicals having at least one
carbon-carbon triple bond; "substituted alkynyl" refers to alkynyl
radicals further bearing one or more substituents as set forth
above;
[0070] "aryl" refers to aromatic radicals having in the range of 6
to 14 carbon atoms; "substituted aryl" refers to aryl radicals
further bearing one or more substituents as set forth above;
[0071] "heteroaryl" refers to aromatic radicals having in the range
of 6 to 14 carbon atoms containing one or more heteroatoms (e.g.,
N, O, S, or the like) as part of the ring structure; "substituted
heteroaryl" refers to heteroaryl radicals further bearing one or
more substituents as set forth above;
[0072] "alkoxy" refers to straight or branched, cyclic or
non-cyclic, alkyl chains comprising an oxy radical group;
"substituted alkoxy" refers to alkoxy radicals further bearing one
or more substituents as set forth above;
[0073] "alkenyloxy" refers to straight or branched, cyclic or
non-cyclic, hydrocarbyl chains having at least one carbon-carbon
double bond and comprising an oxy radical group; "substituted
alkenyloxy" refers to alkenyloxy radicals further bearing one or
more substituents as set forth above;
[0074] "alkynyloxy" refers to straight or branched, cyclic or
non-cyclic, hydrocarbyl chains having at least one carbon-carbon
triple bond and comprising an oxy radical group; "substituted
alkynyloxy" refers to alkynyloxy radicals further bearing one or
more substituents as set forth above;
[0075] "aryloxy" refers to aromatic hydrocarbyls having in the
range of 6 to 14 carbon atoms and comprising an oxy radical group;
"substituted aryloxy" refers to aryloxy radicals further bearing
one or more substituents as set forth above;
[0076] "heteroaryloxy" refers to aromatic hydrocarbyls having in
the range of 6 to 14 carbon atoms containing one or more
heteroatoms (e.g., N, O, S, or the like) as part of the ring
structure and comprising an oxy radical group; "substituted
heteroaryloxy" refers to heteroaryloxy radicals further bearing one
or more substituents as set forth above;
[0077] "alkyl(aryl)" refers to aryl-substituted alkyl radicals;
"substituted alkyl(aryl)" refers to alkyl(aryl) radicals further
bearing one or more substituents as set forth above;
[0078] "alkyl(heteroaryl)" refers to heteroaryl-substituted alkyl
radicals; "substituted alkyl(heteroarylaryl)" refers to alkyl
(heteroaryl) radicals further bearing one or more substituents as
set forth above;
[0079] "alkyloxy(aryl)" refers to aryl-substituted alkyl chains
comprising an oxy radical group; "substituted alkyloxy(aryl)"
refers to alkyloxy(aryl) radicals further bearing one or more
substituents as set forth above;
[0080] "alkyloxy(heteroaryl)" refers to heteroaryl-substituted
alkyl chains comprising an oxy radical group; "substituted
alkyloxy(heteroaryl)" refers to alkyloxy(heteroaryl) radicals
further bearing one or more substituents as set forth above.
[0081] Preferred compounds of the Formula II are those in which R
is unsubstituted or substituted C.sub.1-C.sub.6 alkoxy or
NR.sub.1R.sub.2. Preferred for R, and R.sub.2 are H, unsubstituted
or substituted C.sub.1-C.sub.6 alkyl, unsubstituted or substituted
C1-C3 alkyl(aryl), unsubstituted or substituted aryl, an
unsubstituted or substituted C.sub.3 to C.sub.7 ring when taken
together with the nitrogen atom to which they are bonded, or an
amino acid residue such as glycine. Preferred values for R.sub.3
and R.sub.4 are H or, when taken together, is Preferred for R.sub.5
is H; for R.sub.6 is OH or O--C(O)--(C.sub.1-C.sub.6 alkyl); or
R.sub.5 and R.sub.6 taken together are .dbd.O. Preferred values for
R.sub.9, R.sub.10, and R.sub.12 are H. Betulonic acid may be
excluded from the list of preferred derivatives. The compound of
the Formula II wherein R is OH, R.sub.3 and R.sub.4 when taken
together are .dbd.CH.sub.1, R.sub.5 is H, R.sub.6 is OH and
R.sub.9, R.sub.10 and R.sub.12 are H represents betulinic acid
itself, and is also excluded from the list of preferred
derivatives.
[0082] Derivatives of betulinic acid that are also encompassed by
the Formula II may be synthesized using procedures generally known
in the art. Thus, for example, compounds of the Formula II wherein
R is H may be prepared by the reduction of the corresponding acid
to the aldehyde. Compounds of the Formula II in which R is alkoxy,
alkenyloxy, alkynyloxy, aryloxy or heteroaryloxy may be prepared,
for example, by esterification of the acid moiety of an appropriate
betulinic acid derivative. For example esters are produced by
reacting an alkali metal salt of the acid with alkyl chloride or
bromide in THF in the presence of a catalytic amount of
tetrabutylammonium iodide. Alkyl tosylates may also be
employed.
##STR00006##
[0083] The methyl ester of betulinic acid derivatives can be
obtained from the corresponding acid by reaction with diazomethane
in diethyl ether. Surprisingly, the well-known conversion of
carboxylic acids into esters by reaction of the acid with an
alcohol in the presence of a mineral acid does not work well for
the conversion of betulinic acid into its alkyl, alkyl(aryl) or
alkyl(heteroaryl) esters.
[0084] Compounds of the Formula II in which R is NR.sub.1R.sub.2
may be prepared, for example, by the amidation of the acid moiety
of an appropriate betulonic acid derivative. An example is the
conversion of betulonic acid to N-benzyl betulinic acid amide.
Betulonic acid is converted to its acid chloride upon reaction with
SOCl.sub.2 or POCl.sub.3, reacted with benzylamine to generate the
amide function. Finally, the 3-keto function is reduced to the
3-.beta.-ol with NaBH.sub.4 in methanol. This approach can also be
used to prepare ester simply by replacing the amine with an alcohol
and an equivalent of triethylamine.
##STR00007##
[0085] Derivatives in which R.dbd.OH and R.sub.6 is alkoxy or
acyloxy can also be converted into the corresponding esters,
R.dbd.OR, and amides, R.dbd.NR.sub.1R.sub.2, by reaction conversion
to the acid chloride (R.dbd.C1) followed by treatment with alcohols
and triethylamine and prepared by reacting the compound having
R.dbd.OR and R.dbd.NR.sub.1R.sub.2 with the appropriate acyl
derivatives in the presence of pyridine and DMAP.
[0086] Compounds of the Formula II in which R.sub.3 and R.sub.4 are
hydrogen may be prepared, for example, from the hydrogenation of
the alkene moiety of an appropriate betulinic acid derivative.
Compounds of the Formula II in which R.sub.3 and R.sub.4 when taken
together are .dbd.O may be prepared, for example, from the
ozonolysis of the corresponding alkene. Reduction of this
ozonolysis product would yield compounds of the Formula II in which
R.sub.3 is hydrogen and R.sub.4 is OH. Reaction of the ozonolysis
product with an appropriate Grignard or organolithium reagent could
provide compounds of the Formula II in which R.sub.3 is OH and
R.sub.4 is unsubstituted or substituted alkyl, cycloalkyl, aryl
(such as phenyl) or heteroaryl (such as thiophenyl or furanyl).
Acid catalyzed dehydration of compounds wherein R.sub.3 is OH and
R.sub.4 is a group other than H, OH or CH.sub.2OH would result in
compounds in which R.sub.4 is .dbd.CHR.sub.8. The reaction of
LiCH.sub.2EWG (wherein EWG is an electron withdrawing group such as
a ketone, sulfoxide, sulfone, ester or nitrile) with a betulinic
acid derivative in which R.sub.3 and R.sub.4 when taken together
are .dbd.O may be used to yield compounds of the Formula II in
which R.sub.3 is hydrogen and R.sub.4 is CH.sub.2EWG. The reaction
of a betulinic acid derivative in which R.sub.3 and R.sub.4 when
taken together are =0 with ammonia, or with an appropriate primary
or secondary amine or with an appropriate amino acid under slightly
acidic conditions and reduction of the resulting immonium ion or
imine with a suitable reducing agent such as NaBH.sub.3CN leads to
compound in which R.sub.3 is H and R.sub.4 is NH.sub.2, or the
substituents on the amine employed in the reaction minus a H bonded
to N.
[0087] Appropriate betulonic acid derivatives can be converted into
the 3-.beta.-amino derivatives, R.sub.5.dbd.H,
R.sub.5.dbd.NR.sub.1R.sub.2, via the reductive amination technique
in which the 3-keto function is reacted with a primary or secondary
amine under mildly acidic conditions in the presence of
NaBH.sub.3CN.
##STR00008##
[0088] The alkene moiety of an appropriate betulinic acid
derivative may be epoxidized with peracids to form compounds in
which R.sub.3 and R.sub.4 form --O(CH.sub.2)--, or, may be
subjected to hydroboration to form compounds in which R.sub.3 is H
and R.sub.4 is CH.sub.2OH.
[0089] Compounds of the Formula II in which R.sub.6 is an ester may
be formed by esterifying the 3-OH moiety of an appropriate
betulinic acid derivative. The 3-OH moiety may also be etherified
by known methods. Derivatives in which the C2 substituents have
been modified to give the designated R.sub.9, and/or R.sub.10, can
be accessed by reacting the appropriate betulonic acid derivative
with lithium di-isopropylamide in RHF at low temperature and
quenching the intermediate enolate with a variety of electrophiles.
Potential electrophiles include alkyl or substituted alkyl
bromides, iodides or sulfonates esters, dialkyl disulfides, diaryl
disulfides, halogens or pseudohalogens, electrophilic oxygen
species such as oxaziridines. The 2-thioether derivatives can be
oxidized to the corresponding sulfoxides or sulfones with one or
two equivalents or oxidizing agents such as meta-chloroperbenzoic
acid.
##STR00009##
[0090] .beta.-Keto sufoxides can be converted into
.alpha.,.beta.-unsaturated ketones by heating to approximately
110.degree. C. This leads to compounds in which R.sub.12 and
R.sub.9 or R.sub.10 are taken together to form a bond or, in other
words, compounds that have a double bond between C1 and C2.
[0091] Table 1 illustrates some betulinic acid derivatives that may
be prepared in accordance with the invention.
TABLE-US-00001 TABLE 1 # Name R R.sub.1 R.sub.2 R.sub.3 R.sub.4
R.sub.5 R.sub.6 1 Betulinic acid (BA) OH -- -- .dbd.CH.sub.2 H OH 2
Betulonic acid OH -- -- .dbd.CH.sub.2 .dbd.O 3 Methyl OCH.sub.3 --
-- H H H OH dihydrobetulinate 4 3-acetoxyBA OH -- -- .dbd.CH.sub.2
H CO.sub.2CH.sub.3 5 Methyl betulinate OCH.sub.3 -- --
.dbd.CH.sub.2 H OH 6 Methyl OCH.sub.3 -- -- .dbd.CH.sub.2 H
CO.sub.2CH.sub.3 3-acetoxybetulinate 7 BA amide NR.sub.1R.sub.2 H H
.dbd.CH.sub.2 H OH 8 BA benzylamide NR.sub.1R.sub.2 H CH.sub.2Ph
.dbd.CH.sub.2 H OH 9 BA anilide NR.sub.1R.sub.2 H Ph .dbd.CH.sub.2
H OH 10 BA pyrrolidine NR.sub.1R.sub.2 --(CH.sub.2).sub.4--
.dbd.CH.sub.2 H OH amide 11 Betulonic acid NR.sub.1R.sub.2 H
CH.sub.2CH(CH.sub.3).sub.2 .dbd.CH.sub.2 .dbd.O isobutyl amide 12
BA isobutyl amide NR.sub.1R.sub.2 H CH.sub.2CH(CH.sub.3).sub.2
.dbd.CH.sub.2 H OH 13 BA glycine methyl NR.sub.1R.sub.2 H
CH.sub.2CO.sub.2CH.sub.3 .dbd.CH.sub.2 H OH ester amide 14 BA
glycine amide NR.sub.1R.sub.2 H CH.sub.2CO.sub.2H .dbd.CH.sub.2 H
OH 15 DihydroBA OH -- -- H H H OH 16 Ethyl betulinate
OCH.sub.2CH.sub.3 -- -- .dbd.CH.sub.2 H OH 17 3-acetoxy betulinic
NR.sub.1R.sub.2 H OH .dbd.CH.sub.2 H O (CO) CH.sub.3 acid
hydroxylamine 18 See table II OH -- -- .dbd.O H OH 19 See table II
OH -- -- .dbd.O H O (CO)CH.sub.3 20 See table II --OCH.sub.3 -- --
H OH H OH 21 See table II NR.sub.1R.sub.2 H CH.sub.2Ph .dbd.O H O
(CO) CH.sub.3 22 See table II --OCH.sub.3 -- -- .dbd.O H 0 (CO)
CH.sub.3 23 See table II --OCH.sub.3 -- -- H CH.sub.2OH H OH
TABLE-US-00002 TABLE 2 Formula of compounds 18-23 from Table 1
##STR00010## 18 ##STR00011## 19 ##STR00012## 20 ##STR00013## 21
##STR00014## 22 ##STR00015## 23
Pharmaceutical Compositions
[0092] Pharmaceutical compositions can be prepared that comprise as
active ingredients: a preparation of Marcgraviaceae; betulinic acid
in free form or in the form of a pharmaceutically acceptable salt;
or a compound of formula II in free form or in the form of a
pharmaceutically acceptable salt.
[0093] Such pharmaceutically acceptable salts are known to those
skilled in the art and include, but are not limited to, sodium,
potassium, lithium, calcium, magnesium, zinc and iron salts.
Exemplary, but non-limiting, salts include alkali metal salts, such
as sodium or potassium salts; alkaline earth metal salts, such as
calcium or magnesium salts; ammonium or alkylammonium salts,
wherein the alkylammonium cation has one to three alkyl groups and
each alkyl group independently has one to four carbon atoms; or
transition metal salts.
[0094] These pharmaceutical compositions are compositions for
enteral (e.g. oral) administration, and also rectal or parenteral
administration, also for topical administration to warm-blooded
animals (particularly humans), the pharmacological active
ingredient being present alone or together with customary
pharmaceutical excipients.
[0095] The pharmaceutical compositions comprise, for example,
approximately from 0.1% to 100%, preferably from approximately 1%
to approximately 60%, of the active ingredient. Pharmaceutical
compositions for enteral or parenteral administration are, for
example, in unit dose forms, such as dragees, tablets, capsules or
suppositories, and also ampoules. They are prepared in a manner
known per se, for example by means of conventional mixing,
granulating, confectioning, dissolving or lyophilising processes.
For example, pharmaceutical compositions for oral administration
can be obtained by combining the active ingredient with solid
carriers, optionally granulating a resulting mixture and processing
the mixture of granules, if desired or necessary after the addition
of suitable excipients, into tablets or dragee cores.
[0096] Suitable carriers include, especially, fillers, such as.
sugars, for example lactose, saccharose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example
tricalcium phosphate or calcium hydrogen phosphate, also binders,
such as starch pastes, using, for example, corn, wheat, rice or
potato starch, gelatin, gum tragacanth, methyl-cellulose and/or
polyvinylpyrrolidone, and, if desired, disintegrators, such as the
above-mentioned starches, also carboxymethyl starch, cross-linked
polyvinylpyrrolidone, agar or alginic acid or a salt thereof, such
as sodium alginate.
[0097] Excipients include, especially, flow conditioners and
lubricants, for example silicic acid, talc, stearic acid or salts
thereof, such as magnesium or calcium stearate, and/or polyethylene
glycol. Dragee cores are provided with suitable coatings that may
be resistant to gastric juices, there being used, inter alia,
concentrated sugar solutions which may contain gum arabic, talc,
polyvinylpyrrolidone, polyethylene glycol and/or titanium dioxide,
coating solutions in suitable organic solvents or solvent mixtures,
or, for the preparation of enteric coatings, solutions of suitable
cellulose preparations, such as acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate. Colourings or pigments may
be added to the tablets or dragee coatings, for example for
identification purposes or to indicate different doses of active
ingredient.
[0098] Further orally administrable pharmaceutical compositions
include dry-filled capsules consisting of gelatin, and also soft
sealed capsules consisting of gelatin and a plasticizer, such as
glycerol or sorbitol. The dry-filled capsules may contain the
active ingredient in the form of granules, for example in admixture
with fillers, such as lactose, binders, such as starches, and/or
glidants, such as talc or magnesium stearate, and optionally
stabilisers. In soft capsules, the active ingredient is preferably
dissolved or suspended in suitable liquids, such as fatty oils,
paraffin oil or liquid polyethylene glycols, to which stabilisers
may also be added.
[0099] Rectally administrable pharmaceutical compositions, for
example, suppositories that comprise a combination of the active
ingredient and a suppository base are also provided. Suitable as
suppository bases are, for example, natural or synthetic
triglycerides, paraffin hydrocarbons, polyethylene glycols and
higher alkanols. It is also possible to use gelatin rectal capsules
that comprise a combination of the active ingredient and a base
material. Suitable base materials are, for example, liquid
triglycerides, polyethylene glycols and paraffin hydrocarbons.
[0100] Aqueous solutions of an active ingredient in water-soluble
form, for example a water-soluble salt, are particularly suitable
for parenteral administration. Also suitable for parenteral
administration are suspensions of the active ingredient, such as
corresponding oily injection suspensions, these being used suitable
lipophilic solvents or vehicles, such as fatty oils, for example
sesame oil, or synthetic fatty acid esters, for example ethyl
oleate or triglycerides, or aqueous injection suspensions that
contain viscosity-increasing substances, for example sodium
carboxymethylcellulose, sorbitol and/or dextran and optionally also
stabilisers.
[0101] The dose of the active ingredient may depend on various
factors, such as the method of administration, species of
warm-blooded animal, age and/or individual condition. The preferred
route of administration is oral administration. The active
ingredient may be administered in a range of about 0.1 to about 10
mg/kg body weight per dose, more preferably about 0.1 to about 2.5
mg/kg body weight per dose, even more preferably about 0.1 to about
0.25 mg/kg body weight per dose.
[0102] The anxiolytic compounds of the present invention are fast
acting (effective within 45-60 minutes after oral administration).
Further, development of tolerance or desensitization to the
anxiolytic compounds is low, and cessation of chronic treatment
fails to elicit overt withdrawal effects. Hence, the anxiolytic
compounds may be used to treat either or both of acute or chronic
anxiety. For instance, treatment of a subject may involve daily or
near-daily administration of the anxiolytic compounds for at least
21 days, 30 days, two months, three months, six months, one year or
more.
[0103] In a normal case, the approximate estimated daily dose for a
human patient weighing approximately 75 kg is, in the case of oral
administration, from about 7.5 to about 750 mg.
[0104] Pharmaceutical compositions of the invention may contain
betulinic acid or a betulinic acid derivative as the sole active
(i.e. anxiolytic) ingredient, or may contain an additional active
ingredient, e.g. an additional anxiolytic drug such as a
benzodiazepine or buspirone.
[0105] The invention is further illustrated by the following
non-limiting Examples.
Example 1
[0106] This example illustrates the preparation of a Souroubea
extract from leaves.
[0107] Souroubea sympetala leaves were collected at Bioforesta,
Costa Rica. The leaves were immediately preserved in 95% ethanol
and stored either at room temperature or in a refrigerator.
[0108] The leaves and. the 95% ethanol were blended. Additional 95%
ethanol was added to facilitate the blending process. The blended
material was kept for 2 days at room temperature, then filtered.
The dried filter cake, which is leaf fiber, weighed 178 g.
[0109] The filtrate was evaporated under reduced pressure to afford
40.1 g of a dark powder. This powder was extracted 3 times by
stirring rapidly with 150 ml of ethyl acetate. The ethyl acetate
extracts were combined and the solvent was evaporated to afford 8.0
g of a dark viscous oil. This oil was used in rat anti-anxiety
bio-assays.
Example 2
[0110] This example illustrates the preparation of a Souroubea
extract from fruit.
[0111] Souroubea sympetala fruit was collected at Bioforesta, Costa
Rica, and preserved in 95% ethanol, and was processed similarly to
the S. sympetala leaves. The results were as follows: Dried,
insoluble in 95% ethanol, 68.5 g fiber; ethanol soluble material
8.5 g; dried ethyl acetate soluble extracts 0.37 g.
Example 3
[0112] This example illustrates the chromatographic separation of
components from leaves of Souroubea.
[0113] The ethyl acetate soluble extracts [5.70 g] were
chromatographed on 250 g of silica gel using a solvent gradient
from 100:0 hexane:ethyl acetate to 0:100 hexane:ethyl acetate. One
hundred fractions, each containing approximately 2 0 ml of eluent,
were collected.
[0114] The major components were found in fractions 15-25 (1.2 g).
This material was shown by spectroscopics methods to be a mixture
of .beta.-amyrin, and germanicol.
[0115] Fractions 32-50 yielded 560 mg of a solid that was
rechromatographed. Two pure substances were subsequently isolated.
The less polar product, 26 mg, was identified as chondrillasterol
and the more polar material as betulinic acid by comparison of
melting points and spectroscopic data (MS and .sup.1H NMR) with
known literature data. The betulinic acid content in the ethyl
acetate soluble extracts of S. sympetala is at least 0.44%.
Example 4
[0116] This example illustrates the chromatographic separation of
components from fruit of Souroubea.
[0117] The ethyl acetate soluble portion of the S. sympetala fruit
was processed as above. Betulinic acid (20 mg) was isolated from
370 mg of extracts. The betulinic acid content in this fraction is
5.4%.
Example 5
[0118] This example illustrates that a 95% ethanol extract of
leaves from Souroubea alleviates anxiety in the elevated plus-maze
test.
[0119] The majority of tests described in the following examples
were conducted on adult; male Sprague-Dawley rats (typically
weighing .about.300-450 gm) were obtained from Charles River
Canada, St. Constant, Quebec. They were individually housed in
standard clear Plexiglas cages (24.times.30.times.18 cm) and
maintained on a 12 hr light/dark cycle (7:00 A.M.-7:00 P.M. light
phase) in a temperature and humidity controlled room. Unless
indicated otherwise, animals had free access to water and
maintained on a restricted diet of 5 pellets a day of Purina Lab
Chow. All experimental procedures complied with the guidelines of
the Canadian Council on Animal Care and were approved by the
Research Ethics Committee at the University of Ottawa.
[0120] In the elevated plus-maze (EPM) paradigm, conflict is
generated by the innate drive to explore the new environment (to
locate food) and the fear or aversion to open spaces and heights
(vulnerable or dangerous zones). Typically rodents spend more time
exploring the closed portions of the maze than the open zones of
the maze. Increase in the number of entries into the open arms
(anxiety generating) as compared to the closed arms (safe zones),
as well as any increase the time spent in the open arms in
comparison to closed arms is believed to reflect reduced anxiety
(Pellow, S., et al., (1985). Validation of open:closed arm entries
in an elevated plus-maze as a measure of anxiety of the rat, In
Current Protocols in Neuroscience (pp. 8.3.6-8.3.7), John-Wiley
& Sons). Animals treated with classical anxiolytic drugs, such
as benzodiazepines (e.g. Valium), venture out more onto the open
arms of the maze.
[0121] The EPM constitutes of a plus-shaped maze with two enclosed
arms (or alleys; 50.times.10.times.40 cm) and two open arms (or
planks; 50.times.10 cm), with a 10.times.10 cm central zone. The
EPM was positioned at a height of 50 cm off the floor. All parts of
the apparatus were made from wood, with the closed arms walled in
by opaque Plexiglas. The floor was lined with coarse, black
rubberized material. To avoid distractions, the EPM was surrounded
by black curtain and the subjects were monitored remotely, using a
closed circuit camera mounted above the set-up.
[0122] The rats were randomly assigned to each of the drug
conditions. The drugs were administered intraperitoneally (i.p.) or
orally (by gavage) 60 min prior to testing. Prior to placement onto
the EPM, the rats were placed in an open-field box for a 5-min
habituation. They were then placed in the centre of the EPM, facing
one of the enclosed arms and observed remotely (via video-link
relay) for 5 min. Behaviours scored included time spent in the open
arms of the maze, time spent in the closed arms of the maze, number
of entries into the open-arms and number of entries into the closed
arm of the maze. In addition, the `risk assessment` behaviours that
included the number of unprotected head-dips (protruding the head
over the edge of an open-arm while the body was on the open arm of
the maze), and protected head-dips (protruding the head over the
edge of the maze while the hind legs are still within the closed
arms) were also scored.
[0123] In this example, rats received one of: (a) sweetened milk
(control); (b) 50 mg/kg of a 95% ethanol extract of leaves from
Souroubea; (c) 50 mg/kg of an ethanol acetate fraction of leaves
from Souroubea; or (d) 50 mg/kg of an aqueous fraction of leaves
from Souroubea; orally (by gavage) and were tested in the elevated
plus-maze test 45-60 minutes after treatment.
[0124] As can be seen in FIG. 1, rats treated with the 95% ethanol
extract (marc.) or ethanol acetate fraction (f1) of Souroubea spent
significantly more time on the open arms of the maze (as compared
to controls), indicating that this plant extract imparts
anxiolytic-like effects.
[0125] Bioassay guided fractionation revealed that the `anxiolytic`
activity was contained within the ethyl acetate fraction (f1).
Example 6
[0126] This example illustrates that a crude extract of leaves from
Souroubea alleviates anxiety in the fear-potentiated startle
paradigm.
[0127] In this anxiety/fear paradigm, rats are first trained to
associate a neutral stimulus (a light) with an aversive stimulus
(an electric shock to the foot). In response to a burst of an
auditory stimulus (110 dB white noise burst of 500 m sec duration),
rats demonstrate a reflexive startle response. However, when the
acoustic stimulus is preceded by a fear cue (the light in this
case), there is potentiation of their natural startle response.
This potentiation of startle responses is thought to result from a
classically conditioned increase in fear. Anxiolysis is inferred
when there is a reduction in the magnitude of the fear-potentiated
startle response.
[0128] The fear potentiated startle equipment (Med-Associates,
St-Albans, Vt.) constituted (1) a pressure-sensitive platform upon
which the animal is placed (in an enclosure with shock-grid floor),
(2) Speakers and amplifier, to deliver acoustic stimulus, (3)
Computer controlled light cue, and (4) a computerized interface to
deliver the auditory and/or visual stimuli, and to record the
magnitude of startle detected by the platform. The whole set up is
housed in a sound attenuated and darkened chamber.
[0129] The experiment involved three phases; a training phase, a
screening phase and the testing phase. In the 2-day screening
phase, the rats were conditioned to associate a light cue to an
electric foot-shock. Specifically, a 5 sec light cue was followed
by 0.5 sec foot shock (0.67 mA) at a random inter-trial intervals
ranging from 30-60 sec. Each animal received 10 trials per day, for
two days. The second or screening phase involved identifying the
rats that learnt to associate the light cue with the shock, which
was assessed by measuring their startle response in the absence and
the presence of the fear cue. Only animals that showed the
fear-potentiated startle response (at least a 40% increase in the
startle amplitude) were used in the next phase. In the final or
test phase, rats were treated 60 min prior to testing. Animals were
placed in the test chambers and presented with 10 trials of 110 dB
sound bursts (inter-trial interval of 30 sec) in the absence of the
light cue, and the startle response recorded. This was followed by
additional 5 trials where a 5 sec light-cue preceded the 110 dB
sound burst. The startle amplitudes were averaged over each of the
5 trial sessions. For statistical analyses, the second 5 trial
session average (without the cue) was subtracted from the last
5-trial session (with the light cue), and this difference score
analyzed using ANOVA procedure.
[0130] Rats received one of: (a) 50 mg/kg of a 95% ethanol extract
of leaves from Souroubea; (b) 50 mg/kg of an ethanol acetate
fraction of leaves from Souroubea; or (c) peanut oil (control);
orally (by gavage) and were tested in the fear-potentiated startle
paradigm 45-60 minutes after treatment.
[0131] As can be seen in FIG. 2, control rats show the expected
potentiation of the startle response when the light comes on,
however, this response is markedly attenuated in rats pretreated
with 95% ethanol extract of Souroubea (marc.) or the ethanol
acetate fraction (F1).
[0132] Thus, the anxiolytic potential of this plant product is
confirmed in two distinct and validated tests of anxiety.
Example 7
[0133] This example illustrates that betulinic acid is the active
anxiolytic ingredient of Souroubea as evidenced by the elevated
plus-maze test.
[0134] As indicated earlier, the active ingredient(s) responsible
for the anti-anxiety effects of the Souroubea plant, appeared to be
in fraction 1 (the ethanol acetate extract).
[0135] Within this fraction, betulinic acid was identified as being
present.
[0136] Rats received 1 mg/kg of betulinic acid suspended in peanut
oil orally (by gavage) and were tested in the elevated plus-maze
test 45-60 minutes after treatment. Control rats received only
peanut oil.
[0137] As shown in FIG. 3, rats treated with betulinic acid spent
significantly more time on the open arms of the plus-maze,
confirming that betulinic acid was indeed able to alleviate
anxiety.
Example 8
[0138] This example illustrates that betulinic acid has anxiolytic
effects in the fear-potentiated test of anxiety.
[0139] As indicated earlier, drugs that alleviate anxiety (e.g.
benzodiazepines) reduce the startle amplitude when the sound (110
dB) is presented in the presence of a cue previously paired with an
aversive event (foot shock).
[0140] Rats received either: (a) 1 mg/kg betulinic acid suspended
in peanut oil; or (b) peanut oil only (control); orally (by gavage)
and were tested in the fear-potentiated paradigm 45-60 minutes
after treatment.
[0141] As shown in FIG. 4, betulinic acid significantly attenuated
the startle response in the presence of the fear-cue (but not in
its absence). The scores depicted in FIG. 4 represent the
difference in startle amplitude (calculated as startle response in
the presence of the fear cue--startle response in the absence of
the fear cue). This score was much lower in the rats pretreated
with betulinic acid, suggesting that it has anti-anxiety
effects.
[0142] Since the startle scores were not altered in the absence of
the fear cue, one can surmise that attenuation of the
fear-potentiated response is not due to non-specific motor
effects.
Example 9
[0143] This example illustrates that betulinic acid has anxiolytic
effects in mice (CD-I strain) as well.
[0144] Previous experiments were all conducted in experimental
rats. Here we tested the effect of betulinic acid on CD-1 strain of
mice.
[0145] Mice received 0.25 or 2.5 mg/kg of betulinic acid, or peanut
oil only (control), intra peritoneally ("i.p."), and were tested
45-60 minutes later in the elevated plus maze test.
[0146] As can be seen in FIGS. 5a and 5b, mice treated with
betulinic acid at either the 0.25 or 2.5 mg/kg i.p. dosage entered
the open (anxiogenic) arm of the elevated plus maze more often than
controls. The proportion of time spent on the open arm of the maze
was also increased significantly.
[0147] It appears that for mice the 0.25 mg/kg dose may be the
maximally effective dose (as a higher dose did not increase the
anxiolytic effects further).
[0148] These data demonstrate that the anxiolytic effect of
betulinic acid is not restricted to rats, as it can be observed in
mice as well.
Example 10
[0149] This example illustrates that betulinic acid reduces
anxiety-like behaviours in BALB/c mice.
[0150] BALB/c mice represent a strain of mice that is very
stress-reactive. It has been suggested that this strain may
represent an animal model of trait anxiety.
[0151] Mice received dosages of betulinic acid in accordance with
the previous example.
[0152] Betulinic acid had a pronounced effect on this strain of
mice. As reflected in FIGS. 6a and 6b, both doses of betulinic acid
(0.25 or 2.5 mg/kg; i.p.) significantly increased the proportion of
time spent as well as the number of entries on the open arm of the
elevated plus maze relative to the peanut oil only control.
[0153] Thus the anxiolytic effect of betulinic acid is evident in
rats as well as mice. Furthermore, it seems effective in
alleviating anxiety in a genetically anxious strain of mice.
[0154] These data also suggest that betulinic acid may potentially
be effective in alleviating state as well as trait anxiety.
Example 11
[0155] This example compares the anxiolytic activity of betulinic
acid and some other structurally related compounds. Results are
illustrated in FIG. 7.
[0156] As expected, betulinic acid (0.5 mg/kg) increased the
proportion of time spent in the open arm of the elevated plus
maze.
[0157] However neither beta-amyrin (5 mg/kg) nor oleanolic acid
(0.5 mg/kg), two compounds structurally related to betulinic acid,
displayed any anxiolytic activity on the elevated plus maze
paradigm. When betulinic acid and amyrin were administered
together, no additive effect was seen.
Example 12
[0158] This example compares the effect of betulinic acid and
betulinic acid methyl ester on social interaction in rats.
[0159] The rat social interaction paradigm takes advantage of rats'
natural behaviour in novel situations. The more `anxious` the
animals are, the less likely they are engage in social interaction
with the cohort. Anxiolytic drugs typically increase the amount of
time rodents spend socially interacting with one another. (Pile, S.
E., (1980). The use of social interaction as a method for detecting
anxiolytic activity of chlor-diazepoxide-like drugs. In Current
Protocols in Neuroscience (pp. 8.3.3-8.3.4), John-Wiley &
Sons).
[0160] The social-interaction (SI) test arena constituted of a
60.times.60 cm white Plexiglas floor, enclosed by 35-cm high walls
of the same material. The arena was surrounded by black curtain,
and a closed circuit camera was positioned above the setup, to feed
live video signals to the experimenter seated in a separate room,
for behavioral assessment and recording.
[0161] Each rat was placed alone in the SI box for 7.5 min daily
for 2 days, in order to familiarize the animals with the test
environment. Animals were paired according to their body weights
(less than 10 g difference) and each pair was randomly assigned to
one of the treatment groups. Rats received either betulinic acid
0.5 mg/kg i.p., betulinic acid methyl ester (methyl betulinate) 0.5
mg/kg i.p., or peanut oil only (control). Drugs were injected
(i.p.) 60 min prior to testing. Tests were carried out at three
intervals, 1 h, 2.5 h and 4 h after injection. Each pair was tested
at the 1-h interval, then new pairs from within the same drug group
were formed for the 2.5-h interval and then again at the 4-h
interval (new pairs were formed for each test interval, to control
for the potential confounding effect partner familiarity). At each
of the three test intervals, animals were observed for 7.5 min. The
behaviors scored included the amount of time the pairs spent
interacting together (sniffing, grooming and chasing) as well as
the number of times the animals initiated contact.
[0162] Data analysis of interaction time using ANOVA, indicated a
significant main Treatment effect (F (2, 79)=16.577, p<0.0001)
but no Time effect (F (2, 79)=0.882, p>0.4). Thus the data were
collapsed across time and, as shown in FIG. 8, the analyses
revealed that both betulinic acid (p<0.001) and its methyl ester
derivative (p<0.0001) significantly increased the amount of
social interaction when compared to the control group. Furthermore,
betulinic acid methyl ester was also significantly more effective
at increasing social interaction than betulinic acid (p<0.05).
The frequency of initiated contact was found to be not
significant.
Example 13
[0163] This example compares the anxiolytic activity of betulonic
acid and some synthesized derivatives of betulinic acid to the
activity of diazepam, as tested in the Vogel (punished drinking)
paradigm.
[0164] In the Vogel test, rats are water deprived overnight, prior
to testing, and then given the opportunity to drink for 10 min in
the test chamber. Every 5th lick is `punished` with the delivery of
a mild shock delivered through the drinking spout. This generates
`anxiety` due to the conflict between the desire to drink (due to
the thirst) and the discomfort of the occasional shocks delivered
through the waterspout. Thus the rats drink less when they are
periodically shocked, than when they are not shocked. The number of
licks made (or the shocks accepted) by the thirsty rat is known to
be increased by drugs with anti-anxiety properties, and this test
is one of the `gold standards` accepted by drug companies. An
anxiolytic effect is inferred if an animal persists in the drinking
more than the matched controls, in the presence of waterspout
shocks. (Vogel, J. R., Beer, B., & Cloudy, D. E., (1971). A
simple and reliable conflict procedure for testing anti-anxiety
agents, In Current Protocols in Neuroscience (pp 8.3.10-8.3.12),
John-Wiley & Sons).
[0165] The Vogel setup (Coulbourn Instruments, Allentown, Pa.)
comprised a clear Plexiglas cage (30.times.25.times.3 0 cm), which
housed the animal during the test session. An external shock source
was attached to the metal spout of the water bottle. An optical
beam located at the base of the waterspout tracked the number of
licks performed by monitoring the number of beam interruptions
caused by the animal's tongue.
[0166] Animals maintained on a water deprivation schedule were
allowed access to water only during a 1-h period once a day, and
maintained on a restricted diet of 5 pellets a day of Purina Lab
Chow. Drug dosages and route of administration were as in Example
12. Prior to the testing, the animals were familiarized with the
test cages and waterspouts by allowing them to explore the cage and
have shock-free access to the water for 10 min/day for 3 days.
After each test session, the rats were given ad libitum access to
water for 60 min. The rats were then placed into the apparatus for
a 10 min test session. They were initially allowed shock-free
access to water (for the first 5 s), after which the shock circuit
was activated such that every 5th lick was accompanied by a shock
(0.4 mA).
[0167] The results are illustrated in FIGS. 9a-9c. The treatment
numbers correspond to the compounds listed in Table 1. Compounds
which showed activity at a level above the horizontal bar are
deemed to be reasonably active anxiolytics.
Example 14
[0168] Many anxiolytics lose their efficacy as the subject quickly
becomes tolerant to the drug. This example shows the effect of
chronic administration of betulinic acid and betulinic acid methyl
ester.
[0169] In an elevated-plus maze test as described in the preceding
examples, adult male Sprague Dawley rats (350-375 g) were
administered one of: (a) peanut oil vehicle (control); (b)
betulinic acid dissolved in peanut oil (SS-01); or (c) methyl ester
of betulinic acid dissolved in peanut oil (SS-DIME). The drugs were
administered orally at a daily dose of 0.5 mg/kg. via gavage for
.about.30 days. Behavioural tests were conducted between days 21-30
of chronic exposure. Animals were administered the respective
compounds one-hour prior to testing on the elevated plus maze.
"Acute administration" constituted a single dose of the test
compound in chronically-treated rats, one-hour before testing.
[0170] FIG. 10 shows the effects of chronic and acute drug
administration on the time spent (seconds) in the open arms of the
elevated plus maze. Acute administration of the drug to chronically
treated rats maintained its anti-anxiety effects, as reflected by
increased time spent on the open arms of the elevated plus maze.
This indicates no tolerance or desensitisation development upon
chronic treatment.
Example 15
[0171] Many anxiolytics cause impairment of locomotor activity
after chronic use. This example shows the effects of chronic
administration of betulinic acid (SS01) and methyl ester of
betulinic acid (SS01ME) on locomotor activity in rats.
[0172] Test animals and drug treatments were as in Example 14.
Locomotor activity was recorded for one night (5 p.m. to 7 a.m. the
following morning) in the control as well as in the betulinic acid
and methyl ester of betulinic acid exposed groups. Locomotor
activity was monitored using infrared sensors located on the roof
of the animals' cage. Eight independent sensors monitored activity
of animals according to the number of quadrants traversed. FIG. 11
shows the effects of chronic drug administration on locomotor
activity for rats treated with the control, with betulinic acid
(SS01), or with methyl ester of betulinic acid (SS01ME). The
results show that neither betulinic acid nor the methyl ester of
betulinic acid had an adverse effect on locomotor activity or
pattern. Non-alteration of the locomotor activity or patterns
suggest no adverse locomotor effect and/or sleep disturbances.
Example 16
[0173] The weight gain over the period of treatment was observed
for rats treated with the control as well as rats treated with
betulinic acid or its methyl ester (test animals and treatments as
in Example 14). FIG. 12 shows the effects of chronic drug treatment
on weight gain for rats treated with the control, with betulinic
acid (CSS01) and with the methyl ester of betulinic acid (CSS01ME).
Chronic drug treatment did not seem to affect weight gain in a
significant manner.
Example 17
[0174] This examples studied the results of cessation of chronic
treatment of rats treated with betulinic acid or its methyl ester
as in Example 14.
[0175] Upon cessation of chronic treatment, no overt withdrawal
effects were observed over the following 48 hours. Behaviour
monitoring was made every hour using time-sampling methods, for 48
consecutive hours.
Example 18
[0176] Betulinic acid showed no signs of acute or chronic toxicity,
even at repeated doses of up to 500 mg/kg. This is a dose 1000
times higher than that needed to reduce anxiety. The observed lack
of toxicity is further corroborated by work that shows betulinic
acid to be non-toxic in a Hippocratic screen at doses of 200 and
400 mg/kg.
Example 19
[0177] Tests were conducted by MDS Panlabs to determine whether any
of the classical neurotransmitter systems or the peptidergic
systems thought to play a role in anxiety are affected by betulinic
acid. The results showed that none of the following
neurotransmitters or proteins were affected by betulinic acid:
[0178] Catecholamines: adrenergic .alpha.1, .alpha.2, and .beta.;
MAO A and B; [0179] COMT: NE transporter; domaine D1 and D2, DA
transporter; [0180] Gabaergic: GABA transporter; GABA.sub.A,
agonist site; GABA.sub.A, BZP; GABA C1-channel; GABA.sub.B;
Glutamate non-selective; [0181] Histamine: Histamine H1, H2, and H3
[0182] Serotonin (5-HT): 5-HT.sub.1; 5-HT.sub.1A; 5-HT.sub.2;
5-HT.sub.3; serotonin transporter [0183] Acetylcholine: muscarinic
non-selective; nicototinic; [0184] Sigma: Sigma non-selective
[0185] Peptides: bombesin; cannabinoid CB.sub.1 and CB.sub.2;
cholecystokinin (CCK); CCK.sub.A; EGF; galanin Gal.sub.R1 and
GAL.sub.R2; GLP-1; melanocortin MC4; neuropeptide Y.sub.1 and
Y.sub.2; neurotensin; opiate non-selective; somatostatin;
tachykinin NK.sub.1, NK.sub.2, and NK.sub.3; TNF non-selective;
VIP1; cyclooxygenase; COX-1 and COX-2; interleukin IL-1.alpha.
Example 20
[0186] This example illustrates the preparation of methyl
betulinate (compound #5).
[0187] An ether solution containing diazomethane was added to
betulinic acid (100 mg, 0.22 mmole) dissolved in dichloromethane
(50 mL) containing a few drops of methanol at 0.degree. C. until
the yellow colour of diazomethane persisted. The reaction mixture
was stirred at room temperature overnight in the fume-hood to allow
the excess diazomethane to evaporate. The solvent was removed in
vacuo. The residue was re-dissolved in ethyl acetate (30 ml),
washed successively with water (10 mL), brine (10.0 mL) and water
(10 mL), dried over anhydrous magnesium sulfate, filtered then
concentrated in vacuo. The crude product (100 mg) was
recrystallized from hexane and chloroform to give 80 mg, 78% of a
white solid, mp: 220-221.degree. C., HRMS: Calculated for
C.sub.31H.sub.50O.sub.3, 470.3762; found: 470.37663.
Example 21
[0188] This example illustrates the preparation of
3-acetoxybetulinic acid (compound #4).
[0189] A mixture of betulinic acid (1.12 g, 2.45 mmole),
triethylamine (500 mg, 0.7 mL) and a catalytic amount of DMAP in 50
mL of dichloromethane was stirred for 10 minutes. Acetic anhydride
(500 mg, 0.5 mL) was added and stirring was continued overnight at
room temperature. The reaction mixture was washed successively with
water (20 mL), 5% HCl (20 mL), water (20 mL), dried over anhydrous
magnesium sulfate, filtered and concentrated under reduced
pressure. The crude product was purified by silica gel
chromatography to afford a white solid (810 mg, 66%), mp:
275-277.degree. C. (from methanol), HRMS: Calculated for
C.sub.37H.sub.50O.sub.4: 498.3711; found: 498.37201.
Example 22
[0190] This example illustrates the preparation of betulinic acid
isobutyl amide (compound #12).
[0191] The procedure for the preparation of this compound was
adapted from the method of Evers, et al. J. Med. Chem., (1996),
39:1056-1068. Other amides may be prepared similarly.
[0192] To a solution of 3-acetoxybetulinic acid (100 mg, 0.20
mmole) in dichloromethane (10.0 mL) was added first oxalyl chloride
(38.2 mg, 0.03 mL), and then a few drops of DMF. The mixture was
stirred at room temperature for 6 hours, concentrated in vacuo and
re-dissolved in 2 mL of dichloro-methane. The solution thus
prepared was added drop-wise to a solution of isobutylamine (16.2
mg, 0.02 mL) and triethylamine (22.4 mg, 0.03 mL) in 8 mL of
dichloromethane at 0.degree. C. Stirring was continued for 1 hour.
The reaction mixture was washed successively with water (5 mL), 1%
HCl (5 mL) and water (5 mL), dried over anhydrous magnesium
sulfate, filtered and concentrated in vacuo. The crude material was
stirred overnight in 20 mL of methanol with excess potassium
carbonate at 25-30.degree. C. Methanol was removed in vacuo and the
reaction mixture was re-dissolved in ethyl acetate (25 mL), washed
with water (2.times.10 mL), dried over anhydrous magnesium sulfate
then concentrated in vacuo. The crude product was purified by
silica gel chromatography to afford a white solid (100 mg, 97%),
mp: 216-217.degree. C. (from methanol), HRMS: Calculated for
C.sub.34H.sub.57NO.sub.1: 511.43918; found: 511.44049.
Example 23
[0193] This example illustrates the preparation of methyl
dihydrobetulinate (compound #3).
[0194] A mixture of methyl betulinate (100 mg, 0.21 mmole) and 2 0
mg of 30% Pd on carbon in 25 mL of methanol was stirred under
hydrogen overnight at room temperature. The reaction mixture was
filtered and the residue was washed with methanol (2.times.10 mL)
and then concentrated under reduced pressure. The crude product
(100 mg) was recrystallized from methanol to give a white solid (80
mg, 80%), mp: 236-238.degree. C., HEMS: Calculated for
C.sub.31H.sub.52O.sub.3: 472.39186; found: 472.39230.
Example 24
[0195] This example illustrates the preparation of dihydrobetulinic
acid (compound #15).
[0196] Betulinic acid (100 mg, 0.20 mmole) and 20 mg of Pd/C in 25
mL of methanol were stirred under hydrogen gas overnight at room
temperature. The reaction mixture was filtered, washed with
methanol (2.times.10 mL) and concentrated in vacuo. The crude
product was recrystallized from methanol to give 79 mg of a white
solid, mp: 297-299.degree. C., HEMS: Calculated for
C.sub.30H.sub.50O.sub.3: 458.3762; found: 458.37656.
Example 25
[0197] This example illustrates the preparation of betulinic acid
esters (R.dbd.CH.sub.2Ph, R.dbd.CH.sub.2CO.sub.2C.sub.2H.sub.5,
R.dbd.C.sub.6H.sub.13, R.dbd.CH.sub.2CH.dbd.CH.sub.2,
R.dbd.C.sub.2H.sub.5).
[0198] Sodium hydride (10 mol equivalent) was added to a solution
of betulinic acid (100 mg) in THF (5 mL) at room temperature. The
mixture was stirred for 30 minutes prior to the addition of
appropriate alkyl halide (5 mol equivalent) and a catalytic amount
of tetrabutylammonium iodide (10 mol %). The solution was stirred
overnight at room temperature. The reaction mixture was quenched
with water (20 mL) and THF was removed by rotary evaporation. The
resulting mixture was extracted with ethyl acetate (3.times.20 mL).
The combined organic extracts was washed with water (20 mL), dried
(MgSO.sub.4), filtered and concentrated under reduced pressure. The
residue was purified by silica gel chromatography using
hexane-ethylacetate as eluant to give the desired ester in 33-91%
yields. The assignment of the proton NMR peaks are base on analogy
with spectral data reported by Siddiqui et. al., for betulinic acid
and methyl betulinate. [J. Nat. Prod. 1988, Vol. 51, No. 2,
229]
[0199] Benzyl betulinate:[R.dbd.CH.sub.2Ph in Formula II]
[0200] Yield: 75%, white solid, mp 187-189.degree. C.
[0201] .sup.1H NMR (200 MHz, CDCl3): .delta.: 7.32 (m, 5H, Ph),
5.14 (d, J=3.8 Hz, 2H, --CH.sub.2-Ph), 4.70 (s, 1H, H-29a), 4.57
(s, 1H, H-29b), 3.15 (dd, J=10.3 Hz, J=5.2 Hz, 1H, H-3d), 3.00
(ddd, J=11.0 Hz, J=4.9 Hz, 1H, H-19), 1.68 (s, 3H, H-30), 0.93 (s,
3H, H-27), 0.92 (s, 3H, H-26), 0.77 (s, 3H, H-23), 0.73 (s, 3H,
H-25), 0.72 (s, 3H, H-24), [only readily assignable peaks are
reported]; .sup.13C NMR (200 MHz, CDCl3): .delta.: 175.7, 150.5,
136.4, 128.4, 128.2, 128.0, 109.5, 78.9, 65.7, 56.5, 55.3, 50.5,
49.4, 46.9, 42.3, 40.6, 38.8, 38.6, 38.1, 37.1, 36.9, 34.2, 32.0,
30.5, 29.5, 27.9, 27.3, 25.5, 20.8, 19.3, 18.2, 16.1, 15.8, 15.3,
14.6; MS (EI): 546 [M]+; HRMS: Calculated for
C.sub.37H.sub.54O.sub.3: 546.40720, found: 546.40621.
[0202] Ethyl acetoxy betulinate
{R.dbd.CH.sub.2--CO.sub.2C.sub.2H.sub.5 in Formula II]
[0203] Yield: 76%, white fluffy solid, m. p. 66-68.degree. C.
[0204] .sup.1H NMR (200 MHz, CDCl3): .delta.: 4.70 (d, J=2.0 Hz,
1H, H-29a), 4.55 (s, 3H, H-29b and CO2-CH2-CO2), 4.19 (q, J=7.2 Hz,
2H, CH2-CO2) 3.15 (dd, J=10.4 Hz, J=5.4 Hz, 1H, H-3a), 2.95 (ddd,
J=10.8 Hz, J=4.5 Hz, 1H, H-19), 1.66 (s, 3H, H-30), 0.94 (s, 3H,
H-27), 0.93 (s, 3H, H-26), 0.90 (s, 3H, H-23), 0.79 (s, 3H, H-25),
0.72 (s, 3H, H-24) [only readily assignable peaks are reported];
.sup.13C NMR (200 MHz, CDCl3): .delta.: 175.4, 168.0, 150.5, 109.6,
78.9, 61.3, 60.2, 56.5, 55.3, 50.5, 49.3, 46.7, 42.4, 40.7, 38.8,
38.7, 38.0, 37.1, 36.9, 34.2, 31.9, 30.4, 29.5, 27.9, 27.3, 25.5,
20.8, 19.3, 18.2, 16.1, 15.9, 15.3, 14.6, 14.1; MS (EI): 542 [M]+;
HRMS: Calculated for C.sub.34H.sub.54O.sub.5: 542.39712, found:
542.39682.
[0205] Hexyl betulinate [R.dbd.CH.sub.2CH.dbd.CH.sub.2 in formula
II]
[0206] Yield: 91%, white fluffy solid, m.p. 54-56.degree. C.
[0207] .sup.1H NMR (200 MHZ, CDCl3): .delta.: 4.70 (d, J=1.6 Hz,
1H, H-29a), 4.57 (s, 1H, H-29b), 4.04 (m, 2H, CH2-CO2) 3.16 (dd,
J=9.9 Hz, J=5.5 Hz, 1H, H-3a), 3.00 (ddd, J=10.8 Hz, J=4.8 Hz, 1H,
H-19), 1.66 (s, 3H, H-30), 0.94 (s, 3H, H-27), 0.89 (s, 3H, H-26),
0.86 (s, 3H, H-23), 0.79 (s, 3H, H-25), 0.73 (s, 3H, H-24) [only
readily assignable peaks are reported]; .sup.13C NMR (200 MHz,
CDCl3): .delta.: 176.3, 150.7, 109.5, 78.9, 64.0, 56.5, 55.3, 50.5,
49.4, 47.0, 42.4, 40.7, 38.8, 38.7, 38.3, 37.1, 37.0, 34.3, 32.2,
31.8, 30.6, 29.6, 28.9, 28.7, 28.0, 27.4, 26.1, 25.5, 22.6, 20.9,
19.3, 18.3, 16.1, 16.0, 15.3, 14.7; MS (EI): 554 [M]+; HRMS:
Calculated for C.sub.37H.sub.62O.sub.3: 554.46990, found:
554.47083
[0208] Allyl betulinate. [R.dbd.CH.sub.2CH.dbd.CH.sub.2 in Formula
II]
[0209] Yield: 89%, white fluffy solid, m. p. 65-68.degree. C.
[0210] .sup.1H NMR (200 MHz, CDCl3): .delta.: 5.90 (m, 1H,
CH.dbd.), 5.26, (ddd, J=17.2 HZ, J=10.3 Hz, J=1.4 Hz, 2H, CH2=)
4.70 (s, 1H, H-29a), 4.54 (br s, 3H, H-29b and CH2-CO2), 3.15 (dd,
J=10.2 Hz, J=5.5 Hz, 1H, H-3a), 3.00 (ddd, J=11.1 Hz, J=3.9 Hz, 1H,
H-19), 1.65 (s, 3H, H-30), 0.93 (s, 6H, H-27 and H-26), 0.88 (s,
3H, H-23), 0.79 (s, 3H, H-25), 0.72 (s, 3H, H-24).) [only readily
assignable peaks are reported]; .sup.13C NMR (200 MHz, CDCl3)
.delta.: 175.7, 150.6, 132.5, 118.1, 109.6, 78.9, 64.6, 56.5, 55.3,
50.5, 49.4, 46.9, 42.3, 40.7, 38.8, 38.7, 38.1, 37.1, 37.0, 34.3,
32.1, 30.5, 29.6, 27.9, 27.4, 25.5, 20.8, 19.3, 18.2, 16.1, 15.9,
15.3, 14.7; MS (EI): 496.4 [M; HRMS: Calculated for
C.sub.33H.sub.52O.sub.3: 496.39165, found: 496.39220.
[0211] Ethyl betulinate [R.dbd.C.sub.2H.sub.5 in Formula II]
[0212] Yield: 33%, white solid, m. p. 193-195.degree. C.
[0213] .sup.1H NMR (200 MHz, CDCl3): .delta.: 4.70 (s, 1H, H-29a),
4.57 (s, 1H, H-2 9b), 4.11 (m, 2H, --CH2-CO2), 3.16 (dd, J=10.3 Hz,
J=5.4 Hz, 1H, H-3a), 3.00 (ddd, J=10.7 Hz, J=4.6 Hz, 1H, H-19),
1.65 (s, 3H, H-30), 0.94 (s, 6H, H-27 and H-26), 0.89 (s, 3H,
H-23), 0.79 (s, 3H, H-25), 0.73 (s, 3H, H-24) [only readily
assignable peaks are reported]; .sup.13C NMR (200 MHz, CDCl3)
.delta.: 171.1, 150.7, 109.5, 78.9, 59.8, 56.4, 55.3, 50.5, 49.4,
47.0, 42.4, 40.7, 38.8, 38.7, 38.2, 37.1, 37.0, 34.3, 32.1, 30.6,
29.6, 28.0, 27.4, 25.5, 20.9, 19.4, 18.3, 16.1, 15.9, 15.3, 14.7,
14.3; MS (EI): 484. [M]+; HRMS. Calculated for
C.sub.32H.sub.52O.sub.3: 484.39165, found: 484.38990
Example 26
[0214] This example illustrates the preparation of betulinic acid
amides. Oxalyl chloride (1.5 mol equiv.) and 1 drop of DMF were
added to a solution of 3-acetoxybetulinic acid (100 mg) in
CH.sub.2Cl.sub.2 (10 mL). The solution was stirred for 6 h at room
temperature. The solvent and the DMF were removed in vacuo. The
remaining material was re-dissolved in CH.sub.2Cl.sub.2 (1-2 mL)
and added dropwise to a solution containing the appropriate amine
(1.1 mol equivalent) and triethyl amine (1.1 mol equivalent) in
CH.sub.2Cl.sub.2 (8 mL) at 0.degree. C. Stirring was continued for
1 h. The reaction mixture was then washed with water (5.0 mL), 1%
HCl (5 mL), water (5 mL), dried (MgSO.sub.4), filtered and
concentrated under reduced pressure. Deprotection of the hydroxyl
group was achieved by stirring the resulting 3-acetoxy amide of
betulinic acid in methanol (20 mL) containing excess
K.sub.2CO.sub.3 at 30-35.degree. C. overnight. The reaction mixture
was filtered and concentrated under reduced pressure. The crude
product was subjected to silica gel chromatography using
hexane-ethyl acetate as eluant to afford the desired amide.
Betulinic acid N-benzyl amide
[0215] Yield: 64%, white solid, m.p. 239-242.degree. C.
[0216] .sup.1H NMR (500 MHZ, CDCl3) .delta.: 7.29 (m, 5H, Ph), 5.85
(t, 1H, J=5.7 Hz, N--H), 4.72 (d, 1H, J=2.0 Hz, H-29a), 4.58 (d,
1H, J=2.1 Hz, H-2 9b), 4.47 (dd, 1H, J=14.7 Hz, J=5.8 Hz, CH-Ph),
4.35 (dd, 1H, J=14.0 Hz, J=5.6 Hz, CH-Ph), 3.16 (m, 2H, H-3a and
H-19), 1.67 (s, 3H, H-30), 0.94 (s, 6H, H-27 and H-26), 0.89 (s,
3H, H-23), 0.80 (S, 3H, H-25), 0.74 (s, 3H, H-24) [Only readily
assignable hydrogens are reported]; .sup.13C NMR (500 MHz,
CDCl.sub.3) .delta.: 175.9, 150.9, 139.2, 128.7, 127.8, 127.3,
109.3, 76.7, 55.7, 55.4, 50.7, 50.2, 46.7, 43.3, 42.5, 40.8, 38.9,
38.8, 38.4, 37.8, 37.2, 34.5, 33.8, 30.9, 29.9, 29.5, 27.9, 27.4,
25.7, 20.8, 19.5, 18.3, 16.2, 15.3, 14.7; IR (CHCl.sub.3,
cm.sup.-1): 3447.6, 2942.4, 2867.9, 2360.2, 2342.3, 1638.0, 1522.3,
1454.5, 1375.6, 1189.4, 982.4, 884.1, 756.7, 698.5, 668.4; MS (EI):
545 [M]+; HRMS. Calculated for C.sub.37H.sub.55NO.sub.2: 545.42329.
Found: 545.42218.
Betulinic acid N-isobutyl amide
[0217] Yield: 97%, white solid, m. p. 216-217.degree. C.
[0218] .sup.1H NMR (500 MHz, CDCl3) .delta.: 5.61 (t, 1H, J=5.8 Hz,
N--H), 4.71 (d, 1H, J=2.2 Hz, H-29a), 4.56 (d, 1H, J=2.3 Hz,
H-29b), 3.12 (m, 13H, CH2-N), 2.98 (m, 1H, H-19), 1.65 (s, 3H,
H-30), 0.91 (s, 3H, H-27), 0.89 (s, 1H, H-26), 0.88 (s, 3H, H-23),
0.79 (s, 3H, H-25), 0.73 (s, 3H, H-24) [Only readily assignable
hydrogens are reported]; .sup.13C NMR (500 MHz, CDCl.sub.3)
.delta.: 175.9, 150.9, 109.2, 78.9, 55.6, 55.4, 50.6, 50.1, 46.7,
46.6, 42.5, 40.7, 38.8, 38.7, 38.5, 37.7, 37.2, 34.4, 33.9, 30.9,
29.4, 28.7, 27.9, 27.4, 25.6, 20.9, 20.2, 20.1, 19.4, 18.3, 16.1,
16.0, 15.3, 14.6; IR (CHCl.sub.3, Cm.sup.-1): 3449, 2949, 2869,
2361, 2343, 1638, 1509, 1388, 1195, 1044, 983, 909, 882, 733; MS
(EI): 511. [M]+; HRMS. Calculated for C.sub.34H.sub.57NO.sub.2:
511.43918. Found: 511.44049.
Betulinic acid N-pyrrolidine
[0219] Yield: 78%, white solid, m.p. 223-226.degree. C.
[0220] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 4.69 (d, 1H,
J=2.2 Hz, H-29a), 4.54 (s, 1H, H-29b), 3.40 (m, 4H, 4.times.CH2-N),
3.09 (m, 2H, Ha and H-19), 1.65 (s, 3H, H-30), 0.93 (s, 6H, H-27
and H-26), 0.91 (s, 3H, H-23), 0.79 (s, 3H, H-25), 0.72 (s, 3H,
H-24) [Only readily assignable hydrogens are reported]; .sup.13C
NMR (500 MHz, CDCl.sub.3) .delta.: 173.6, 151.6, 108.9, 78.9, 55.4,
52.4, 50.8, 46.3, 42.0, 40.6, 38.8, 38.7, 38.6, 37.2, 37.1, 35.2,
34.4, 31.2, 30.8, 29.6, 27.9, 27.4, 25.6, 21.1, 19.6, 18.3, 16.2,
15.3, 14.7; IR (CHCl.sub.3, cm.sup.-1): 3424, 3070, 2942, 2868,
2362, 1607, 1451, 1406, 1390, 1375, 1246, 1214, 1187, 1168, 1137,
1108, 1045, 982, 917, 881, 754, 665; MS (EI): 5109. [M]+; HRMS.
Calculated for C.sub.34H.sub.55NO.sub.2: 509.42329. Found:
509.42312.
Example 27
[0221] This example illustrates the preparation of the sodium salt
of betulinic acid.
[0222] Betulinic acid (370 mg) was then dissolved in methanol (200
mL) with warming before and 2 mL of 0.41 M sodium methoxide
solution (2 mL) was added. The solvent was removed via a rotary
evaporation and a white solid was obtained (370 mg), m.p.
291-295.degree. C. Its solubility was greater in water and in
methanol than pure betulinic acid.
Example 28
[0223] This example illustrates the epoxidation of the methyl
betulinate.
[0224] To a stirred solution of methyl ester of betulinic acid (100
mg, 0.213 mmol) in CH.sub.2Cl.sub.2 (10 mL) was added sodium
acetate (100 mg, 1.22 mmol). The solution was cooled to 0.degree.
C. and peracetic acid (0.2 mL, 2.97 mmol) was added. The ice bath
was then removed and the reaction mixture was stirred for 3 h
before the reaction was quenched with 15% solution of
Na.sub.2SO.sub.3 (20 mL). The solvent was removed in vacuo and then
extracted with ethyl acetate (3.times.20 mL). The combined organic
extracts were washed successively with a saturated solution of
K.sub.2CO.sub.3 (2.times.20 mL) and brine (20 mL) before it was
dried (MgSO.sub.4) filtered and concentrated under reduced
pressure. The crude product was purified by silica gel
chromatography using hexane-ethyl acetate as eluant to give the
desired product as a white solid (80 mg, 77%).
[0225] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 3.60 (s, 3H,
O--CH3), 3.15 (dd, J=10.4 Hz, J=5.5 Hz, 1H, H-3a), 2.60 (d, J=2.1
Hz, 2H, H-29), 1.20 (s, 3H, H-30), 0.92 (s, 3H, H-27), 0.91 (s, 3H,
H-26), 0.86 (s, 3H, H-23), 0.79 (s, 3H, H-25), 0.72 (s, 3H, H-24)
[only the readily assignable peaks are reported]; .sup.13C NMR (200
MHz, CDCl.sub.3) .delta.: 176.4, 78.8, 60.1, 56.8, 56.5, 55.2,
51.3, 50.3, 50.1, 45.4, 42.3, 40.6, 38.8, 38.7, 37.4, 37.1, 36.8,
34.2, 32.0, 29.3, 27.9, 27.3, 27.0, 26.8, 20.9, 18.3, 18.2, 16.0,
15.8, 15.3, 14.5; MS (EI): 486.4 [M]+
Example 29
[0226] This example illustrates the epoxidation of 3-.beta.-acetoxy
betulinic acid.
[0227] To a solution of 3-acetoxy betulinic acid (530 mg, 1.06
mmol) in CH.sub.2Cl.sub.2 (20 mL) at 0.degree. C. was added mCPBA
(280 mg, 1.60 mmol). The ice-bath was removed and the solution was
stirred at room temperature overnight. The reaction mixture was
then washed successively with 10% solution of NaHSO3 (2.times.10
mL), saturated solution of Na2CO3 (2.times.10 mL) and brine
(2.times.10 mL) then dried (MgSO4), filtered and concentrated under
reduced pressure. The crude product was purified by silica gel
chromatography using hexane-ethyl acetate as the eluant yielding
the target epoxide as a white solid (280 mg, 51%), m.p.
290-293.degree. C.
[0228] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 4.44 (dd, 1H,
J=9.3 Hz, J=6.4 Hz, 1H, H-3a), 2.64 (s, 2H, H-29), 2.02 (s, 3H,
CH.sub.3--CO2), 1.22 (s, 3H, H-30), 0.93 (s, 3H, H-27), 0.89 (s,
3H, H-26), 0.83 (s, 3H, H-23), 0.82 (s, 3H, H-25), 0.81 (s, 3H,
H-24) [only the readily assignable peaks are reported]; .sup.13C
NMR (200 MHz, CDCl.sub.3) .delta.: 182.2, 171.1, 80.9, 60.2, 56.6,
55.3, 50.2, 49.9, 45.4, 42.3, 40.7, 40.6, 38.3, 37.7, 37.5, 37.0,
36.8, 34.1, 31.9, 29.3, 27.9, 26.9, 26.7, 23.6, 21.3, 20.8, 18.1,
18.0, 16.4, 16.1, 16.0, 14.5.
Example 30
[0229] This example illustrates the hydroboration of methyl
betulinate.
[0230] To a solution of methyl betulinate (200 mg, 0.43 mmol) in
dry THF (15 mL) at room temperature was added borane-methyl sulfide
(0.08 mL, 0.85 mmol). After 7 h, 3N NaOH (0.43 mL) was added
followed by 30% w/w H.sub.2O.sub.2 while keeping the temperature
between 30-35.degree. C. and then slowly warmed to 50.degree. C.
The reaction mixture was then stirred overnight at 50-60.degree.
C., then diluted with diethyl ether (20 mL), washed with brine (10
mL), dried (MgSO.sub.4), filtered and concentrated in vacuo. The
crude material was purified by silica gel chromatography using
hexane-ethyl acetate as eluant to yield the desired product (140
mg, 67%), m.p. 220.degree. C.-223.degree. C.
[0231] .sup.1H NMR (200 MHZ, CDCl.sub.3) .delta.: 3.76 (dd, 1H,
J=10.3 Hz, J=4.6 Hz, 1H, H-29), 3.62 (s, 3H, O--CH3), 3.39 (dd,
J=10.5 Hz, J=8.0 Hz, 1H, H-29), 3.17 (dd, J=10.1 Hz, J=5.6 Hz, 1H,
H-3a), 1.32 (s, 3H, H-30), 0.94 (s, 3H, H-27), 0.92 (s, 3H, H-26),
0.88 (s, 3H, H-23), 0.80 (s, 3H, H-25), 0.73 (s, 3H, H-24) [only
the readily assignable peaks are reported];
[0232] .sup.13C NMR (200 MHz, CDCl.sub.3) .delta.: 176.6, 78.9,
64.2, 56.8, 55.2, 51.2, 50.2, 48.7, 43.1, 42.5, 40.6, 38.8, 38.6,
38.3, 38.1, 37.1, 37.0, 34.3, 32.0, 29.6, 27.9, 27.3, 27.2, 23.8,
20.9, 18.2, 18.1, 16.0, 15.9, 15.4, 14.6; IR (CHCl.sub.3,
cm.sup.-1): 3386, 2948, 2870, 1716, 1455, 1390, 1377, 1319, 1290,
1272, 1217, 1189.2, 1167.1, 1136.6, 1105.9, 1040.7, 982.5, 945.8,
757.1, 666.6.
[0233] MS (EI): 488.4 [M]+
[0234] HRMS: Calculated for C.sub.31H.sub.52O.sub.4, 488.38656,
found 488.38549.
Example 31
[0235] This example illustrates the ozonolysis of betulinic acid
and preparation of platanic acid.
[0236] Ozone was passed through a solution of betulinic acid (200
mg, 0.44 mmol) in 5% solution of MeOH/CH.sub.2Cl.sub.2 at
78.degree. C. for 30 minutes. The reaction was then quenched with
dimethyl sulfide (2 mL) and the solvent was removed by rotary
evaporation. The reaction mixture was re-dissolved in ethyl acetate
(100 mL) and washed with water (2.times.10 mL), dried (MgSO.sub.4),
filtered and concentrated to dryness in vacuo. The crude product
was purified by silica gel chromatography using hexane-ethyl
acetate as eluant to afford the desired compound (120 mg, 60%),
m.p. 278-282.degree. C., lit. 279-282.degree. C. (J. Nat. Prod.
1994, Vol. 57, 249).
[0237] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 3.20 (m, 2H,
H-3a, H-19), 2.15 (s, 3H, H-30), 0.98 (s, 3H, H-27), 0.94 (s, 3H,
H-26), 0.89 (s, 3H, H-23), 0.80 (s, 3H, H-25), 0.73 (s, 3H, H-24)
[only the readily assignable peaks are reported]; .sup.13C NMR (200
MHz, CDCl.sub.3) .delta.: 212.2, 181.3, 78.9, 56.2, 55.3, 51.2,
50.3, 49.2, 42.2, 40.6, 38.8, 38.6, 37.5, 37.2, 36.7, 34.2, 31.4,
30.1, 29.7, 28.3, 28.0, 27.3, 27.2, 20.8, 18.2, 16.1, 15.9, 15.3,
14.7; IR (CHCl.sub.3, cm.sup.-1): 3467, 2944, 2871, 1702, 1561,
1452, 1388, 1378, 1357, 1279, 1239, 1189, 1170.4, 1137.6, 1108.2,
1074, 734, 647; MS (EI): 458 [M]+; HRMS. Calculated for
C.sub.29H.sub.46O.sub.4: 458.33963. Found: 458.33860.
Example 32
[0238] This example illustrates the preparation of methyl
platanate.
[0239] Diazomethane was added to a solution of platanic acid in
CH.sub.2Cl.sub.2 (50 mL) containing a few drops of methanol until
the reaction mixture remained permanently yellow. Excess
diazomethane was allowed to evaporate in the fumehood at room
temperature overnight before the solvent was removed in vacuo. The
crude product obtained after evaporation of the solvents was
purified by silica gel chromatography using hexane-ethyl acetate as
eluant to yield the desired compound (50 mg, 60%), m.p.
250-251.degree. C., lit. 250-251.degree. C. (J. Chem. Soc., 1963,
3269).
[0240] .sup.1H NMR (200 MHZ, CDCl3) .delta.: 3.65 (s, 3H, O--CH3),
3.20 (m, 2H, H-3a, H-19), 2.15 (s, 3H, H-30), 0.98 (s, 3H, H-27),
0.94 (s, 3H, H-26), 0.89 (s, 3H, H-23), 0.80 (s, 3H, H-25), 0.73
(s, 3H, H-24) [only the readily assignable peaks are reported];
.sup.13C NMR (200 MHz, CDCl.sub.3) .delta.: 212.4, 176.5, 78.8,
56.4, 55.2, 51.4, 51.1, 50.3, 49.4, 42.1, 40.5, 38.8, 38.6, 37.3,
37.1, 36.6, 34.1, 31.3, 30.1, 29.7, 28.2, 27.9, 27.3, 27.2, 20.8,
18.2, 16.0, 15.8, 15.3, 14.7; MS (EI): 472. [M]+; HRMS: Calculated
for C.sub.30H.sub.48O.sub.4: 472.35528. Found: 472.35078.
Example 33
[0241] This example illustrates the preparation of methyl 3-acetoxy
platanate.
[0242] Diazomethane was added to a solution of 3-acetoxy platanic
acid (100 mg, 0.2 mmol) in CH/Cl.sub.2 (50 mL) containing a few
drops of methanol until the reaction mixture remained permanently
yellow. Excess CH2N2 was allowed to evaporate in the fumehood at
room temperature overnight before the solvent was removed in vacuo.
The crude product was purified by silica gel chromatography using
hexane-ethyl acetate as eluant to yield the desired compound (80
mg, 78%), m.p. 204-206.degree. C., lit. 205-207.degree. C. (Coll.
Czech. Chem. Comm., 1970, Vol. 35, 298).
[0243] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 4.44 (dd, 1H,
J=9.8 Hz, J=6.2 Hz, H-3a), 3.64 (s, 3H, O--CH3), 3.22 (t, 1H,
J=10.7 Hz, H-19), 2.15 (s, 3H, H-30), 2.01 (s, CH3-CO2), 0.96 (s,
3H, H-27), 0.86 (s, 3H, H-26), 0.81 (S, 3H, H-23), 0.80 (s, 6H,
H-25, H-24) [only the readily assignable peaks are reported];
.sup.13C NMR (200 MHz, CDCl.sub.3) .delta.: 212.3, 176.5, 171.0,
80.8, 56.3, 55.3, 51.4, 51.1, 50.2, 49.3, 42.1, 40.5, 38.2, 37.7,
37.2, 37.0, 36.5, 34.0, 31.4, 30.1, 29.6, 28.2, 27.8, 27.1, 25.6,
21.3, 20.8, 18.1, 16.4, 16.1, 15.8, 14.6; IR (CHCl.sub.3,
cm.sup.-1): 2947, 2871, 1728, 1453, 1432, 1391, 1368, 1353, 1330,
1318, 1222, 1189, 1167, 1154, 1136, 1108, 1027, 980, 900, 757, 668;
MS (EI): 514 [M]+; HRMS. Calculated: for C.sub.32H.sub.50O.sub.5,
514.36586, found: 514.36725.
Example 34
[0244] This example illustrates preparation of 3-Acetoxy platanic
acid N-benzylamide.
[0245] Oxalyl chloride (0.03 mL, 0.30 mmol) and a few drops of DMF
were added to a solution of 3-Acetoxy platanic acid (100 mg, 0.20
mmol) in CH.sub.2Cl.sub.2 (5 mL) at room temperature.
[0246] The solution was stirred for 6 h and the solvent and DMF
were then removed by evaporation under reduced pressure. The
reaction mixture was re-dissolved in CH.sub.2Cl.sub.2 (5 mL) and
then added dropwise to a solution containing benzylamine (0.02 mL,
0.22 mmol) and triethylamine (0.03 mL, 0.22 mmol) in
CH.sub.2Cl.sub.2 (5 mL) at 0.degree. C. and stirring continued for
1 h. The reaction mixture was then washed with water (5 mL), 1% HCl
(5 mL), water (5 mL), dried (MgSO4), filtered and concentrated in
vacuo. Purification of the product was achieved by silica gel
column chromatography using hexane-ethyl acetate as eluant to yield
a white solid (60 mg, 51%).
[0247] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 7.30 (m. 5H, Ph),
5.91 (t, 1H, 5.7 Hz, N--H), 4.44 (dt, 2H, J=913.6 Hz, J=5.7 Hz,
CH-Ph and H-3a), 4.32 (dd, 1H, J=14.7 Hz, J=5.6 Hz, CH-Ph), 3.46
(dt, 1H, J=11.3, J=4.4 HZ, H-19), 2.15 (s, 3H, H-30), 2.01 (s,
CH3-CO2), 0.96 (s, 3H, H-27), 0.86 (s, 3H, H-26), 0.82 (s, 3H,
H-23), 0.81 (s, 3H, H-25), 0.80 (s, 3H, H-24) [only the readily
assignable peaks are reported]; .sup.13C NMR (500 MHz, CDCl.sub.3)
.delta.: 212.9, 175.7, 170.9, 139.0, 128.7, 127.8, 127.4, 80.9,
55.5, 55.4, 51.0, 50.4, 50.0, 43.3, 42.3, 40.7, 38.4, 38.0, 37.8,
37.1, 36.8, 34.3, 33.0, 29.5, 28.6, 27.9, 27.2, 23.7, 21.3, 20.9,
18.2, 16.5, 16.2, 16.1, 14.7; IR (CHCl.sub.3, cm.sup.-1): 3376,
2946, 2869, 1733, 1713, 1647, 1522, 1467, 1454, 1422, 1391, 1369,
1317, 1248, 1195, 1162, 1139, 1108, 1080, 916, 732, 699, 668, 647;
MS (EI): 589 [M]+; HRMS: Calculated for C.sub.38H.sub.55NO.sub.4:
589.41332. Found: 589.4140.
Example 35
[0248] This example illustrates the preparation of methyl
dihdroplatanate.
[0249] To a solution of methyl platanate (50 mg, 0.11 mmole) in
methanol/THF (3:1 mL) at 0.degree. C. was added NaBH.sub.4 (40 mg,
1.1 mmol) and the reaction was stirred at room temperature
overnight. The reaction was quenched with NH.sub.4Cl solution (5
mL) and the solvent removed in vacuo. The residue was extracted
with ethyl-acetate (2.times.10 mL) and the combined organic
extracts was then washed with water (5 mL), brine (5 mL), water (5
mL) before it was dried (MgSO.sub.4), filtered and concentrated
under reduced pressure. The crude product was purified by silica
gel chromatography using hexane-ethyl acetate as eluant yielding a
white solid as the major product and isomer (30 mg, 60%), m.p.
194-196.degree. C.
[0250] .sup.1H NMR (200 MHz, CDCl.sub.3) .delta.: 3.84 (q, 1H,
J=6.3 Hz, H-20), 3.63 (s, 3H, O--CH3), 3.17 (dd, 1H, J=10.4 Hz,
J=5.6 Hz, H-3a), 0.94 (s, 6H, H-27, and H-26), 0.88 (s, 3H, H-23),
0.80 (s, 3H, H-25), 0.73 (s, 3H, H-24) [only the readily assignable
peaks are reported];
[0251] .sup.13C NMR (200 MHZ, CDCl.sub.3) .delta.: 176.9, 78.9,
68.9, 56.9, 55.2, 51.2, 50.2, 47.9, 45.6, 42.4, 40.6, 38.8, 38.6,
37.9, 37.1, 37.0, 34.3, 31.7, 29.6, 27.9, 27.3, 27.0, 23.3, 22.2,
20.8, 18.3, 16.1, 15.9, 15.4, 14.7; IR (CHCl.sub.3, cm.sup.-1):
3407, 2945, 2869, 1714, 1655, 1561, 1454, 1390, 1376, 1320, 1275,
1217, 1189, 1166, 1135, 1106, 1090, 1045, 1034, 1002, 983, 944,
919, 811, 757; MS (EI): 474 [M]+; HRMS. Calculated for
C.sub.30H.sub.50O.sub.4: 474.37090: Found 474.37130.
Example 36
[0252] This example illustrates the preparation of methyl
3-acetoxydihydroplatanate.
[0253] To a solution of 3-acetoxy methylplatanate (110 mg, 0.21
mmole) in methanol/THF (9:3 mL) at 0.degree. C. was added
NaBH.sub.4 (81 mg, 2.10 mmol) and the reaction was stirred at room
temperature overnight. The reaction was quenched with NH.sub.4Cl
solution (10 mL) and the solvent removed in vacuo. The residue was
extracted with ethyl-acetate (2.times.20 mL) and the combined
organic extracts was then washed with water (10 mL), brine (10 mL),
water (10 mL) before it was dried (MgSO.sub.4), filtered and
concentrated under reduced pressure. The crude product was purified
by silica gel chromatography using hexane-ethyl acetate as eluant
yielding a white solid as the major product and isomer (60 mg,
54%), m.p. 255-259.degree. C.
[0254] .sup.1H NMR (500 MHz, CDCl.sub.3) .delta.: 4.45 (dd, 1H,
J=10.8, J=5.5 Hz, H-3a), 3.88 (q, J=6.4 Hz, 1H, H-20), 3.63 (s, 3H,
O--CH.sub.3), 2.02 (s, CH.sub.3--CO.sub.2), 0.94 (s, 3H, H-27),
0.88 (s, 3H, H-26), 0.83 (s, 3H, H-23), 0.82 (s, 3H, H-25), 0.81
(s, 3H, H-24) [only the readily assignable peaks are reported];
.sup.13C NMR (200 MHz, CDCl.sub.3) .delta.: 176.9, 171.1, 81.0,
68.9, 56.9, 55.3, 51.2, 50.1, 47.9, 45.6, 42.4, 40.6, 38.3, 38.0,
37.7, 37.0, 34.3, 31.7, 29.6, 27.9, 27.0, 23.6, 23.3, 22.2, 21.3,
20.8, 18.3, 16.4, 16.1, 15.9, 14.6; IR (CHCl.sub.3, cm.sup.-1):
3538, 2947, 2871, 1720, 1655, 1561, 1458, 1392, 1370, 1318, 1248,
1189, 1135, 1107, 1030, 980, 945, 901. 857, 756, 665; MS (EI): 516
[M]+; HRMS: Calculated for C.sub.30H.sub.50O.sub.4: 516.38166,
Found: 516.38129.
[0255] Unless defined otherwise all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this invention belongs.
[0256] All publications and patent applications cited in this
specification are herein incorporated by reference as if each
individual publication or patent application were specifically and
individually indicated to be incorporated by reference.
[0257] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
[0258] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it is readily apparent to those of ordinary skill
in the art in light of the teachings of this invention that certain
changes and modifications may be made thereto without departing
from the spirit or scope of the appended claims.
* * * * *