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This review will detail progress made in the previous decade on the chemistry and bioactivity of birch bark extractive products. Current and future applications of birch bark natural products in pharmaceuticals, cosmetics, and dietary supplements for the prevention and treatment of cancer, HIV,and other human pathogens are reviewed. Current developments in the technology of birch bark processing are discussed. New approaches for the synthesis of potentially valuable birch bark triterpenoid derivatives are also reviewed.
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REVIEW www.rsc.org/npr | Natural Product Reports
Birch bark research and development
PavelA.Krasutsky*
Received (in Cambridge, UK) 15th May 2006
First published as an Advance Article on the web 18th September 2006
DOI: 10.1039/b606816b
Covering: 1995 to 2006
This review will detail progress made in the previous decade on the chemistry and bioactivity of birch
bark extractive products. Current and future applications of birch bark natural products in
pharmaceuticals, cosmetics, and dietary supplements for the prevention and treatment of cancer, HIV,
and other human pathogens are reviewed. Current developments in the technology of birch bark
processing are discussed. New approaches for the synthesis of potentially valuable birch bark
triterpenoid derivatives are also reviewed.
1 Introduction
2 Bioactivities of birch bark products
University of Minnesota–Duluth, Natural Resources Research Institute, 5013
Miller Trunk Highway, Duluth, Minnesota, 55811-1442, USA. E-mail:
pkrasuts@nrri.umn.edu; Fax: +1-218-720-43-29; Tel: +1-218-720-43-34
Pavel Krasutsky is the Director of the Chemical Extractives
Program of the University of Minnesota. He received his M.S. and
Ph.D. in chemistry in 1969 (Kiev Polytechnic Institute, Ukraine),
and his Doctor of Science in chemistry in 1986 (Institute of Organic
Chemistry, Ukraine). In 1997 he received the National Award
of Ukraine in Science and Technology. Pavel has over 40 years
of experience in organic chemistry research, which includes the
following areas of organic chemistry: mechanisms of organic reac-
tions, organic chemistry of cage structures, synthesis of biologically
active compounds and drug design, synthesis and technology of
insecticides, chemistry of natural products and chemistry of chemical
extractives. He has authored nearly 160 publications in national and
international chemistry journals, including nearly 20 patents. He
has been working at the Natural Resources Research Institute at the
University of Minnesota, Duluth, since 1996. The work conducted in
his laboratory has led to the foundation of NaturNorth Technlogies,
LLC, and partnerships with other companies, institutes, agencies,
colleges, and universities.
Pavel Krasutsky
2.1 Birch bark extractive
2.2 Birch bark triterpenoids
3 Birch bark processing
3.1 Refining and formulation of outer birch bark
3.2 Manufacturing of birch bark NPs
3.3 Chemistry of birch bark NPs
4 Summary
5 Acknowledgements
6 References
1 Introduction
The bark of the birch tree has been the subject of respect and
admiration throughout prehistory
1,
and history,
3,4
as well as the
subject of curiosity of science and industry in the modern world.
5–14
The twentieth century has been a time of deep, fundamental study
into the chemistry of birch bark products,
5–14
although during this
time the application of t his work was largely limited to traditional
uses of NPs in the cosmetics industry.
15
The symbiosis of the
birch tree and civilisation should now be reconsidered through the
scientific vision of a new century. The last review on this subject
was published in 1994;
8
this review addresses the achievements of
the last decade, which have revealed remarkable biological and
medical aspects of birch bark triterpenes and their derivatives.
The most interesting of these are birch bark triterpenoids, which
represent a new class of anti-cancer and anti-HIV
16
bioactives
with a novel mechanism of action. The study of these NPs
and their derivatives has already been developed beyond the
framework of f undamental science, and ongoing clinical tests are
currently approaching the level of new drug creation.
16,17
These
developments in the area of birch bark products have stimulated
parallel development of the technology of birch bark processing,
as well as in the chemistry of triterpenoids, their synthesis and
Among the possessions of the 5300-year-old iceman (found in 1991 in
the Tyrolean Alps) were two birch bark bags and two walnut-sized birch
fungi (Piptoporus betulinus), presumed to be a “medical kit”. This fungus
contains triterpenes with anti-bacterial and anti-cancer properties.
2
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selective derivatisation.‡ Birch bark extracts have found broad use
in modern cosmetics and have the potential to be used as dietary
supplements. Additionally, with the ever-increasing efficiency of
birch bark processing and refinement technologies, outer birch
bark and inner birch bark can now be regarded as another valuable
source of natural chemicals (suberinic acids and tannins) with
multiple uses. Although the chemistry of birch bark extracts is
well established,
5–14
significant differences between the chemistry
of Eurasian and North American birches are just now being
recognised. Most notable are the chemical specifics of Alaskan
birch trees that make them a source of unique composition. Recent
research and development on birch bark processing makes all birch
natural products accessible by quality and volume to any field that
might find use for them. The goal of this review is to show that
birch bark value-added products have a great potential in addition
to the traditional uses of birch wood in the paper or forestry indus-
tries.
2 Bioactivities of birch bark products
2.1 Birch bark extractive
The chemistry of outer birch bark can be subdivided into the
chemistry of the extractive and the chemistry of the natural poly-
mer suberin.
8,9
The extractive includes a mixture of pentacyclic
triterpenoids, lupanes (major) and oleananes (minor),
9
which are
perhaps the most interesting for use as bioactive compounds
(drugs, cosmetics, dietary supplements, biocides, bactericides,
etc.). The chemical content of birch bark extracts from the 38
scientifically recognised Betula species
18
is similarly varied.
6–14
This variety makes it more practical to consider only extracts
from the industrially and commercially managed Betula species
Efforts on birch bark product research and development by the
Laboratory of Chemical Extractives (Natural Resources Research
Institute, University of Minnesota–Duluth, USA, http://www.nrri.
umn.edu/cartd/lce/) encouraged the creation of this review.
Table 1 Average chemical content (%) of birch bark extractive
a
B. pendula
9d,21
B. papyrifera B. neoalaskana
Betulin (1)78.172.4 68.1
Betulinic acid (2)4.35.4 12.5
Betulinic aldehyde (3)1.21.31.4
Lupeol (4)7.95.92.1
Oleanolic acid (5)2.00.32.2
Oleanolic acid
3-acetate (6)
—1.63.8
Betulin 3-caffeate (7)0.56.26.1
Erithrodiol (8)2.8—
Other (minor) 3.26.93.8
a
Samples of outer birch bark of Betula neoalaskana were kindly transferred
for extraction and GC/MS, NMR and HPLC analyses by the Professor
of Forest Management, Edmond C. Packee (SNRAS Forest Science
Department, University of Alaska, Fairbanks).
(B. pendula and B. pubescens, Eurasia; B. papyrifera, Northern
US and Canada) and the potentially interesting Alaskan birch,
B. neoalaskana.
19
The average chemical content of extractives
and formulas of triterpenoids for these three birch species, are
presented in Table 1 and Scheme 1. The triterpenoid chemical
content of species listed in Table 1 can also possess some
variability
9c,20
within a specific species, and depends on the age
of the tree and climatological conditions. Different methods of
analysis and the lack of standard calibration procedures
6–14,20
may
also be the reason for the reported variencies. Though minor
components (see “Other” in Table 1), the following NPs should still
be mentioned (Scheme 1): betulone (9); betulonic aldehyde (10);
lupenone (11); betulonic acid (12); oleanolic aldehyde (13)and
b-amyrin (14).
9d
The increased amount of betulinic acid (2)and
betulin 3-caffeate (7) in N. American birch bark is an important
difference, because these NPs are significant anti-cancer and anti-
HIV ingredients. By reviewing the progress on the bioactivity of
birch bark triterpenes and their derivatives over the past decade,
the importance of these differences becomes more understandable.
Scheme 1 Triterpenes of the outer birch bark of Betula pendula, B. papyrifera,andB. neoalaskana.
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Table 2 Anti-HIV activity for betulin derivatives 2, 15–18 and AZT
Compound EC
50
/lMIC
50
/lM Therapeutic index
Betulinic acid (2)1.412.99.2
3-O-(3
,3
-Dimethylsuccinyl)betulinic acid (15) <0.00035 7.0 >20 000
3,28-O-(Di-3
,3
-dimethylglutaryl)betulin (16) 0.00066 14.2 21 515
3-O-(3
,3
-Dimethylsuccinyl)-28-O-(2
,2
-dimethylsuccinyl)betulin (17) 0.00087 36.9 42 400
3-O-Glutaryldihydrobetulin (18) 0.00002 23.59 1 120 000
AZT 0.045 1873 41 622
The total average yield of B. pendula and B. pubescens extractive
(27%)
9c
is higher than that of N. American B. papyrifera and
B. neoalaskana (22%).
20,22
2.2 Birch bark triterpenoids
Triterpenoids are the most ubiquitous class of natural secondary
metabolites (there are more than 4000 compounds in the terrestrial
and marine flora). They have been widely studied, have been
previously reviewed in this journal,
23
and have high potential as
bioactive NPs.
24,25
Outer birch bark from boreal forests contains
the highest quantity of triterpenoids of all plants (20–35%). It
is generally believed that the physiological function of these
NPs is defence (a plant’s acquired resistance) against plant-
pathogens.
26–28
This has led to the expectation that triterpenoids
could also act against pathogens that cause human and animal
diseases. However, the use of these NPs, including birch bark
triterpenoids, has remained quite limited, in part because of their
low solubility (<1mgL
1
in water), high log P value (>9); and
high molecular weight (>500 Daltons). This reduces their attrac-
tiveness as promising drug candidates through the formal concept
of the rule of five,
29
rational drug discovery
29–31
and combinatorial
chemistry.
32
In spite of the fact that these qualities have limited
the interest in these NPs by the drug industry,
33–35
21 drugs
based on NPs have been launched on world markets between
1998 and 2004.
34
The three following triterpenoids are currently
undergoing clinical trials at the US National Institute of Health:
betulinic acid (2) as an anti-cancer compound,
17
a semi-synthetic
derivative of betulinic acid (PA-457) as anti-HIV compound,
16
and the natural water-soluble triterpene glycoside (QS-21, an
oleonolic acid derivative) as an adjuvant for vaccines.
34
The
primary preference for the use of triterpenoids as bioactives is their
established low toxicity. Native Americans and native Siberians
used birch bark (B. papyrifera and B. pendula respectively) as a
source of folk medicine. This historically recognised internal use
of birch bark extractive,
3,4
coupled with the scientifically measured
low toxicity of triterpenoids,
36
support the use of birch bark
chemicals not only in drugs, but as dietary supplements, cosmetics,
biocides, washing materials, agrichemicals, etc. The major birch
bark NPs 1, 2, 4, 5 and 7 merit special attention as potentially
promising bioactive compounds or precursors to drug ingredients.
Betulin (1) is one of the oldest NPs, first isolated from birch bark
and scientifically described in 1788.
37
Previous reviews
9,38,39
have
cited betulin’s moderate anti-cancer, anti-bacterial, anti-fungal,
and anti-viral activity. Betulin and birch bark extractive have been
proposed for use in cosmetics as an additive to shampoo,
40
skin-
care,
41
dental-care
42
and hair-care
43
products. For these purposes,
however, pure betulin in not usually used, but rather birch bark
extract.
15
Fundamental research into the bioactivity of betulin (1)
and betulin derivatives are ongoing. In most cases it has been
shown that betulin and dihydrobetulin derivatives are usually
more active than pure betulin as anti-cancer compounds
39,44–48
or anti-HIV compounds.
49–51
It has also been reported that
3- and 28-acylbetulin and 3,28-diacylbetulin derivatives have a
fairly high level of anti-HIV activity in vitro. In particular, 3-O-
glutaryldihydrobetulin (18)
52–56
wasmoreactiveinanin vitro assay
than the anti-HIV drug zidovudin (AZT) and all other triterpenoid
derivatives studied (Table 2, Scheme 2),
53
including PA-457 [3-O-
(3
,3
-dimethylsuccinyl)betulinic acid (15)].
16
Scheme 2 Structures of the most active in vitro anti-HIV betulin
derivatives (see Table 2).
The cytotoxicity of 3-O-phthalic betulin esters have been
tested on tumour cell lines in MTT tests.
47
It was reported that
hemiphthalic esters exhibited greater cytotoxicity than betulinic
acid (2) or relatively inactive betulin (1). Betulin and other natural
triterpenoids have been reported as selective catalytic inhibitors
of human DNA topoisomerases with IC
50
values in the range 10–
39 lM.
57
DNA topoisomerases play important roles in replication,
transcription, recombination, and chromosome segregation at
mitosis.
Recio et al.
44
reported structural requirements for the anti-
inflammatory activity of betulin and other natural triterpenoids of
the lupane, oleanane, and ursane series. All triterpenoids displayed
remarkable bioactivity against the oedema produced by phorbol
12-myristate 13-acetate (TPA). It was concluded that the basic
hydrocarbon skeleton has no critical influence on activity, but the
presence of polar anchors at C28 (hydroxylic or carboxylic) is of
the highest importance.
Structural/functional properties of betulin and dihydrobetulin
derivatives and their glycosides have been studied on Ehrlich
tumour cells.
45
Hydrogenation of betulin and adding glucose to
C3 both increased cytotoxic activity, but the presence of the two
glucose residues at C3 and C28 significantly decreases anti-cancer
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activity. Betulin 3-glycoside (19), which was isolated from the herb
extract Pulsatillae Radix,
58
inhibits the rate of solid tumour growth
in healthy male mice (S-180 cells) by 87–96% at dosages of 0.1–
1.5 mg kg
1
.
Flekhter et al.
59
and Baltina et al.
60
reported that a number
of betulin 3,28-esters have a relevant hepatoprotective effect and
influenza inhibition. Betulin has been modified at the C3 and
C28 positions and the antiviral activity has been evaluated in
in vitro assays. It was found that simple modifications to the
parent structure of lupane triterpenoids produced agents that are
effective against influenza-A and herpes simplex type-1 viruses.
Betulin has also been proposed for the treatment of viral hepatitis-
C.
61,62
These patents also claim that betulin has antiviral and
immunomodulating properties. Hepatoprotective, anti-ulcer, anti-
inflammatory, reparative, and anti-HIV activities were found
for 3-O,28-O-dinicotinoylbetulin.
63
This betulin derivative also
exhibits immunomodulatory activity. Herpes and Epstein–Barr
virus inhibition by betulin and its derivatives has been reported
by Amjad et al.
64,65
The anti-herpes activity of betulin and its
derivatives have been a subject of numerous patents.
66–68
The anti-
viral activity of some enveloped and non-enveloped viruses was
reported for betulin (1), betulinic acid (2), and betulonic acid
(12).
69
In addition to anti-viral activity, early research conducted
by plant physiologists
28,70–72
indicated that either triterpene gly-
cosides or saponins playing the major role in the self-protection
of plants against fungi. In both cases the saponins themselves
were not active against fungi or erythrocytes. The sugar portion
of the glycoside molecule is merely the hydrophilic transporting
functional group. The anti-fungal activity of these active forms was
observed at concentrations 30 lgml
1
. This is not a higher level
of activity than commercial fungicides, but it is likely to be good
enough for a plant’s self-resistance against pathogenic fungi. This
research hinted that betulin and its derivatives must display some
level of anti-microbial activity. This activity was reported against
Fusarium oxysporum,
73
Staphylococcus aureus (2–5 lgml
1
),
74
the
human pathogenic fungi Microsporum canis and Trichophyton
rubrum (12.5 lgml
1
),
75
plant fungi pathogens,
76
and other selected
bacteria and fungi.
77
The anti-microbial properties of birch bark
extract and betulin can be used in low-irritation cosmetics
78
and
anti-pathogenic fungi cosmetics.
79
The anti-fungal use of betulin
and some of its derivatives against plant pathogens have also been
patented.
80–83
The potential bioactivity of triterpene glycosides
led to two phases of research activity on their synthesis.
84,85
All
of these efforts were limited to fundamental studies, probably
because the industrial availability of betulin was limited. The
methods for the synthesis of betulin glycosides are also rather
complicated.
84,85
Analysis of the structural/functional properties
of betulin derivatives led to the idea that the more amphiphilic
characteristics that triterpenoids possess, the higher level of
general bioactivity could be observed. Bioactive triterpenoids
usually have a relatively polar structural fragment located at
the ends of a non-polar triterpenoid nucleus. A review on a
water-soluble triterpene glycoside adjuvant for vaccines supports
this idea.
86
Selective inhibition of the catalytic sub-unit of rat
liver cyclic AMP-dependent protein kinase (cAK) by amphiphilic
triterpenoids, including betulin structures, show that it is necessary
to include the lipophilic non-flexible triterpenoid fragment in the
design of amphiphilic bioactives.
87
Such a notion is also supported
by the recent synthesis (by Krasutsky et al.
88
) of betulin-based
quaternary ammonium salts as bioactive cationic surfactants. The
high level of anti-bacterial and anti-fungal activity of some water-
soluble betulin-based quaternary salts makes them promising
bioactive surfactants. Scheme 3 shows the general formulae of
these betulin-based molecules.
Scheme 3 Betulin-based amphiphilic water-soluble quaternary salts.
Betulin and birch bark extract are already used as a dietary
supplement, Betual
R
, for active liver protection, prevention and
treatment of acute alcoholic intoxication,
89
andasanadditive
to alcoholic beverages.
90
Clinical studies indicate that Betual
R
may reduce both alcoholic intoxication and hangover intensity.
89,90
Hepatoprotective effects of betulin and betulinic acid against
ethanol-induced cytotoxicity in hepG2 cells have also been re-
ported by Szuster-Ciesielska et al.
91
It is very important to note
that B. verucosa birch bark extract (betulin 70%, betulinic acid 6%
and lupeol 5%) did not display toxicity, either during clinical tests
or in the three years since Betual
R
commercialisation.
92,93
Recently,
betulin and birch bark extract have been patented as adapto-
genic remedies,
94
interferon inducers,
95
antihypoxitic products,
96
hepatitis-C preventatives and treatments,
97
anti-influenza
98
and
tuberculosis prophylactics,
99
and as additives in cosmetics, pet
foods, lipase inhibitors, and foods containing triterpenes.
100
Betulin 3-caffeate (7) and other natural triterpene caffeates are
among the less studied birch bark extract components. Ekman
et al.
21
reported levels of compound 7 of up to 0.5% in European
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B. verucosa through an indirect experimental approach. Table 1
includes direct analytical HPLC measurements for B. papyrifera
and B. neoalaskana.
20
This work shows a higher content (6%+) of
7 in the N. American bark of B. papyrifera and B. neoalaskana.
Kolomitsyn et al.
20
have reported the isolation of natural 7,aswell
as a new method of its synthesis from betulin (1) (See Section 3.3).
This work also reported the inhibition by 7 of the growth of P19,
NT2/D1 and K1735-M2 cells, compared with a number of other
birchbarktriterpenes.Notably,caffeate7 showed the highest
level of anti-proliferative activity in vitro among all birch bark
triterpenes, including betulinic acid (2).
20
This was the only expla-
nation for the inhibitory activity of N. American birch bark extract
being equivalent and higher to the level of anti-cancer activity of
betulinic acid (2). The anti-cancer and anti-inflammatory activities
of non-triterpene caffeates
101
as well of some extracts containing
triterpene caffeates
102,103
has previously been reported.
The anti-HIV activity of non-triterpene caffeic esters and their
immune modulation effect in vivo have also been reported.
104
The
presence of betulin 3-caffeates also makes birch bark extract a
good sun-block ingredient for cosmetics because of its good UV-
absorption.
20
Thus, anti-melanoma bioactivity combined with
UV-protective characteristics may lead to the creation of new
cosmeceuticals from N. American birch bark extracts or betulin 3-
caffeate (7) and other triterpene caffeates. Some birch bark extracts
include oleanolic acid 3-caffeate (23)and3b,23-dihydroxyolean-
12-en-28-oic acid 3b-caffeate (21)fromBetula davurica,
105
3b,23-
dihydroxyolean-12-en-28-oic acid 23-caffeate (22)and3b,23-
dihydroxylup-20(29)-en-28-oic acid 3b-caffeate (23)fromBetula
pubescens,
106
and betulinic acid 3-caffeate (32)fromBetula platy-
phylla Sukatchev var. japonica Hara.
107
Oleanolic acid (5),
108,109
betulinic acid (2),
82
and ursolic acid
(24)
108,110
have been reported as anti-cancer, anti-inflammatory,
anti-bacterial, and anti-viral bioactives previous to this review
period. These findings triggered a surge of drug design activity
focused on these commonly available NPs. The popularity of these
compounds is reflected by the recent publication of six reviews
of triterpenoid acid 2, 5,and24.
111–116
The moderate, but well
recognised, level of anti-cancer bioactivity of acids 5 and 24
108–110
stimulated drug design efforts to use these structures as a basis for
the creation of highly efficient synthetic anti-cancer candidates.
As a result, the design and synthesis of a highly active inhibitor
of nitric oxide production in mouse macrophages, 2-cyano-3,12-
dioxoolean-1,9-dien-28-oic acid (25), was achieved.
117
This syn-
thetic oleanane triterpenoid (CDDO) has highly potent differen-
tiating, antiproliferative, and anti-inflammatory activities,
118
and
induces apoptosis of human myeloid leukaemia cells by a caspase-
8-dependent mechanism.
119,120
Further development of different
CDDO derivatives has led to new bioactive compounds that
might be used for the prevention and treatment of certain cancers,
arthritis, multiple sclerosis, Alzheimer’s disease, and Parkinsons
disease.
121
The low content of oleanolic acid (5) and oleanolic acid acetate
(6) (see Table 1) in birch bark extract does not make birch bark a
good source for manufacturing these natural chemicals. However,
the acidic fraction of birch bark (containing betulin 3-caffeate (7),
betulinic acid (2), and oleanolic acid (5)), if separated, could be
used as an anti-cancer composition.
It has been reported that plant extracts containing oleanolic
(5) and betulinic (2) acids from Pterocarya tonkinesis (Franch.)
Dode
122
and Nerium oleander,
123
and epi-oleanolic acid (26)from
Korean mistletoe
124
manifest a high level of anti-carcinogenic
activity.
From the experience of complementary medicine, it has been
noticed that the presence of oleanoic acid (5) in plant extractives is
often accompanied by anti-bacterial properties. Such bioactivity
was reported for extractives from Syzygium guineense (against
Bacillus subtilis, Escherichia coli, Staphylococcus aureus)
125
and
Lythrum salicaria (against Proteus mirabilis and Microccocus
luteus).
126
The anti-bacterial properties of pure oleanoic acid have
also been reported (Streptococcus mutans assay).
127
The anti-caries
activity of oleanolic acid (5) with b-cyclodextrin has potential use
as a corresponding extractive for dental care products.
128
Oleanolic acid (5) has been found to be an active anti-HIV
component in the following plant extractives: Rosa woodsii,
Prosopis glandulosa, Phoradendron juniperinum, Syzygium claviflo-
rum, Hyptis capitata,andTernstromia gymnanthera.
129
Mengoni
et al.
130
reported that oleanolic acid (5) inhibits the replication
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of HIV-1 in all the cellular systems studied (EC
50
range 22.7–
57.4 lM). It was suggested that triterpenoid 5 inhibits HIV-1
protease activity. These results trigger the idea that oleanolic acid
(5) may be a good basis for anti-HIV drug design. Intensive study
of anti-HIV activity of oleanolic acid derivatives supports this
possibility.
131–133
The following pharmacological activities of oleanolic acid
(5) and derivatives also merit special mention: prevention and
treatment of anxiety and depression in mammals;
134
treatment
of hyper-sensitivity and/or hyper-reactivity,
135
and treatment of
non-insulin-dependent diabetes mellitus.
136
In addition, they have
been found to promote antibody generation
137
and immuno-
modulatory activity,
138
function as vasodilators and restorative
agents for endothelial dysfunction,
139
and exert gastroprotective
effects.
140
The use of oleanoic acid (5) as a component of comple-
mentary and conventional medicines is common in China.
110a,141
Oleanolic acid derivatives, such as QS-21,
34,86
derived from
the bark of the S. American tree Quillaja saponaria (Rosaceae),
have been found to be very efficient water-soluble triterpene
glycoside adjuvants. QS-21 is an experimental adjuvant to vac-
cines (melanoma, malaria, HIV, breast cancer, prostate cancer,
streptococcal pneumonia, influenza, herpes, hepatitis-B) being
examined in Phase II and Phase III US-NIH trials. GP1-0100
(Saponimmune) is a derivative of Quillaja saponaria saponine,
which was developed by Galenica Pharmaceuticals
142
as an
adjuvant for vaccines.
143,144
Betulinic acid (2) and its derivatives have been the most inten-
sively studied group of birch bark triterpenoids during the previous
decade because of the discovery of their unique anti-cancer and
anti-HIV activities. Although recent reviews
111,112,145
cover the
literature up to the end of 2003, it should now be worthwhile to
consider betulinic acid from the perspective of advanced birch bark
research and development. This is because birch bark seems to
be the best source, industrially and commercially, for natural and
semi-synthetic betulinic acid manufacturing (see Section 3) among
all other possible and previously reported natural sources, which
number more than 20.
111
Almost all natural extracts that contain
betulinic acid have been historically known as complementary
medicines and have been reported in fundamental studies as
being active against tumours,
145,146
cancers,
147–149
inflammation,
150
bacterial pathogens,
151,152
and viruses.
153
The period of biological
screening of natural extracts (the phytotherapy period) in the
1980s was transformed into a thorough fundamental study of
the bioactivity of pure natural triterpenoid ingredients of extracts
as possible chemotherapeutics. Yasukawa et al.
154
reported the
relevant inhibitory effects (at 5 lM concentration) of pure
betulinic acid (2) on TPA-induced inflammation as being roughly
similar to its inhibitory activities against tumour promotion in
vitro. The ensuing report of Pisha et al.
155
on betulinic acid
as a selective inhibitor of human melanoma that functions by
induction of apoptosis stimulated both fundamental resarch (into
melanomas,
154,156–158
leukaemia,
159–161
brain-tumours,
162,163
human
gliomas,
164
colon and prostate cancers,
165
the Ewing’s sarcoma
family,
166
and head and neck cancers
167
) and applied efforts (into
the prevention and treatment of melanomas,
165,168–171
tumour-
associated angiogenesis,
172
cancer and HIV,
173,174
liver, lung, colon,
prostate, and breast cancers,
175
neuroectodermal tumours,
176
leukaemia, lymphomas, and lung, prostate and ovarian cancers.
177
The triggering of apoptotic activity by betulinic acid through a
direct effect on mitochondria was reported by Fulda et al.
156,162
Galton et al.
178,179
reported that betulinic acid is an apoptosis
inducer in skin cancer cells and causes differentiation in normal
human keratinocytes. This research supports the application of
betulinic acid not only for drugs but also for cosmetics. Cosmetics
developers believe that betulinic acid (at 50–500 mg per gram
of cosmetic) may prevent and help to treat UV-induced skin
cancer,
171
reduce signs of cellulite and stimulate collagen synthesis
for skin-care products,
180
prevent sunlight-caused signs of aging,
wrinkles, and blotches,
181
and improve skin homogeneity and
pigmentations.
182
Clinical tests of betulinic acid as a treatment
for melanoma began in 1999.
17
Hata et al.
183
studied cytotoxicities
of 11 lupane group triterpenoids (Table 3) against three human
leukaemia cell lines, two melanoma cell lines, two neuroblastoma
cell lines, and normal fibroblast cells. It was reported that only
lupane triterpenes with a C28 carbonyl group (2, 3, 10, 29,
30) exhibited an inhibitory effect for cancer cell growth, in the
concentration range 0.48–11.1 lM. The most active triterpenoids,
betulinic acid (2), betulinic aldehyde (3) and betulonic aldehyde
(10) markedly inhibited eukaryotic human topoisomerase-I at an
IC
50
level of 5 lM.
Chowdhury et al.
184
reported that betulinic acid and its deriva-
tives inhibit the catalytic activity of rat liver DNA topoisomerase-
I in a dose-dependent manner with an efficacy as good as
camptothecin. In a manner different from camptothecin, betulinic
acid (2) and its derivatives interact directly with the enzyme
Table 3 IC
50
values (lM) of lupane triterpenes against human cancer cell growth
183
Leukaemia Melanoma Neuroblastoma Normal cells
Compound HL60 U937 K562 G361 SK-MEL-28 GOTO NB-1 WI38
Lupeol (4) 19.9 16.8 >20 >20 >20 >20 19.7 >20
Lupenone (11) 15.8 11.9 18.2 >20 >20 >20 >20 >20
Lupeol 3-acetate (27) >20 >20 >20 >20 >20 >20 >20 >20
Betulin (1) 14.7 14.4 14.5 12.4 16.2 17.1 16.5 15.2
Betulone (9) 18.9 16.8 18.7 10.6 >20 >20 >20 16.4
Betulin 3,28-diacetate (28) 19.2 >20 >20 >20 >20 >20 >20 >20
Betulinic aldehyde (3) 1.1 3.7 4.9 9.6 10.6 7.5 8.8 18.5
Betulonic aldehyde (10) 0.48 1.5 1.8 9.4 9.3 5.2 5.8 17.3
Betulinic acid (2) 6.6 10.0 9.8 5.2 6.5 7.9 9.5 >20
Methyl betulinate (29) 10.8 11.1 8.8 8.7 4.8 6.8 6.3 19.3
Methyl betulonate (30) 7.8 8.8 10.9 8.5 7.4 9.4 9.6 >20
924 | Nat. Prod. Rep., 2006, 23, 919–942 This journal is
©
The Royal Society of Chemistry 2006
and inhibit the formation of the topoisomerase-I complex with
the DNA. Notably, dihydrobetulinic acid (31) inhibits enzyme
activity at a concentration of 1 lM, which is ten times more
efficient than the activity of betulinic acid (2) at a concentration of
10 lM. Many efforts have been directed at the design of more
efficient anti-cancer betulinic acid derivatives.
185–196
Shentsova
et al.
185
reported that adding glucose to the C3-position of betulinic
acid and dihydrobetulinic acid increases the cytotoxic activity.
Symon et al.
186
revealed the high cytotoxicity of a betulinic acid
cyclopropane derivative against human melanomas of the Colo
38 and Bro lines, and a human ovarian carcinoma of the CaOv
line (IC
50
10 lM). It was discovered that the hemiphthalic ester of
betulinic acid is more active than betulinic acid (2).
187
The activity
of betulinic acid amides has been demonstrated against melanoma
(at 0.25 lgml
1
) and liposarcoma (at 0.3 lgml
1
).
189
A number
of betulinic acid derivatives (3-O-acyl, 3-hydrazine, 2-bromo, and
20,29-dibromo) have shown IC
50
values <1 lgml
1
on human
cancer cell lines MOLT-4, JurkatE6.1, CEM.CM3, BRISTOL8,
U937, DU145, PA-1, A549, and L132.
190
Ring A seco derivatives
of betulinic acid manifested significant cytotoxic activity against
the T-lymphoblastic leukaemia cell line CEM (4–6 lM).
191
Sarek
et al.
197,198
and Urban et al.
191,199
reported broad efforts towards
synthesis of ring A seco and E seco betulinines with cytotoxic pro-
apoptotic activity on a wide diversity of cancer cells. Research into
the structure–activity relationships for betulinic acid derivatives
are underway, but natural betulinic acid (2) still remains a fairly
plausible anti-cancer chemopreventive and chemotherapeutic can-
didate. This is due to its low toxicity, which has been accepted
through its long-term use in complementary medicinal history,
as well as its favourable therapeutic index, even at doses up to
500 mg kg
1
body weight.
200
Systemic side effects are not observed
for betulinic acid at any dose.
145
However, the low solubility of
betulinic acid in water and high hydrophobicity (log P)
201
does
not portend good delivery of this chemical to targets. Therefore,
research into the pharmacokinetics of betulinic acid,
145,200
creation
of new pro-drugs
174,202,203
and formulation
204
seems appropriate.
The efficient combined treatment and synergistic cytotoxicity of
different chemotherapeutics with betulinic acid (2) have been
observed
204
and claimed by patent.
175
Betulinic acid (2)and
its derivatives induce growth inhibition in proliferative diseases
other than cancer, such as inflammation.
44,205–207
Bernard et al.
208
suggested that betulinic acid from plant extracts is responsible for
this activity by binding and inhibiting phospholipase A
2
,witha
binding energy of 90 kcal mol
1
. These ideas have been sup-
ported by other studies.
209,210
Among natural anti-inflammatory
derivatives of betulinic acid, betulinic acid 3-caffeate (32,or
pyracrenic acid), is worth special mention. All plant extracts which
contain these triterpenoids, as well as oleanolic acid 3-caffeate (33)
manifest marked anti-inflammatory properties.
Caffeate 32 has been found in the bark of Betula platyphylla
Sukatchev var. japonica Hara,
107
and 33 in the bark of B. ermanii,
211
B. maximowicziana,
212
B. davuric,
213
and B. pubescens.
214
The anti-HIV activity of betulinic acid and its derivatives has
been reported independently by Fujioka et al.
215
and Mayaux
et al.
216
It was also reported independently that some plant
extracts that contained betulinic acid or related triterpenoid acids
showed anti-HIV activity.
217,218
This suggests that all triterpenoid
structural analogues of betulinic acid (ursolic acid, oleanolic acid,
platanic acid, moronic acid, etc.) have potential as anti-HIV
chemotherapeutics. These NPs (betulinic acid (2),
215,219
oleanolic
acid (5),
220
ursolic acid (25),
220
dihydrobetulinic acid (31)
215,219
)
inhibit HIV-1 replication in acutely infected H9 cells and inhibit
H9 cell growth at approximately the same level of bioactivity
(Table 2 and Table 4). This level of bioactivity and toxicity
was improved by studying the structure–activity relationship for
betulinic acid derivatives.
219
It was observed that derivatives with
C3 acyl groups are more active, especially if they have dimethyl
groups in the C3
position (see Table 2 and Scheme 4).
The anti-HIV parameter EC
50
for betulinic acid (see Table 3)
was improved 4000-fold for betulinic acid derivative 3-O-(3
,3
-
dimethylsuccinyl)betulinic acid (15) or DBS (PA-457)
16
.Atthe
Table 4 Anti-HIV activities for triterpenoid acids
215,220
and their derivatives
219
8–11 and AZT
Compound EC
50
/lMIC
50
/lM Therapeutic index
Betulinic acid (2) 1.4 12.9 9.3
Oleanolic acid (5)
a
3.7 47 12.7
Ursolic acid (25)
a
4.3 14.2 3.3
Dihydrobetulinic acid (31) 0.9 12.6 14
3-O-(3
,3
-Dimethylsuccinyl)betulinic acid (15) <3.5 × 10
4
7 >20 000
3-O-(3
,3
-Dimethylsuccinyl)dihydrobetulinic acid (34) <3.5 × 10
4
4.9 >14 000
3-O-(3
,3
-Dimethylglutaryl)betulinic acid (35)2.3× 10
3
4.5 1974
3-O-(3
,3
-Dimethylglutaryl)dihydrobetulinic acid (36)5.7× 10
3
5.8 1017
AZT 0.15 1875 12 500
a
IC
50
and EC
50
data in lgml
1
(from ref. 220) were recalculated to lMml
1
.
This journal is
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The Royal Society of Chemistry 2006 Nat. Prod. Rep., 2006, 23, 919–942 | 925
Scheme 4 Structures of the most active anti-HIV betulinic acid
derivatives.
same time, the toxicity parameter IC
50
was increased by only a
factor of two. Thus, the average improvement in the therapeutic
index is 2000-fold.
Similar levels of improvements in the in vitro bioactivity through
modification of triterpenes were also successfully shown with
derivatives of betulin,
52–55
moronic acid,
221
oleanolic acid,
222
and
ursolic acid.
223
It was shown that minor, but specific, changes in
structures may lead to changes in activity. For example, dihydro-
betulinic acid (31) is slightly more potent against HIV than be-
tulinic acid (2);
215
3a-epimers are less potent than 3b-triterpenoid
acids;
222,224,225
3-oxo derivatives, 3-amines and 3-ethers are less ac-
tive than 3b-h ydroxy derivatives;
216,224,226,227
and dehydration at C3
leads to non-active compounds.
224
Derivatisation of the C30 posi-
tion also leads to less active compounds. Research on a series of be-
tulinic acid amides
216,224,226,227
revealed structures (37, RPR103611)
and (38, IC9564) which were active against HIV in vitro at a
submicromolar level in a wide range of cell cultures (Scheme 5).
Scheme 5 Structures of the most active anti-HIV betulinic acid amides
(3S,4S)-N
-(N-(3b-hydroxylup-20(29)-ene-28-oyl)-8-aminooctanoyl)-4-
amino-3-hydroxy-6-methylheptanoic acid (37, RPR 103611) and (3R,4S)-
N
-(N-(3b-hydroxylup-20(29)-ene-28-oyl)-8-aminooctanoyl)-4-amino-3-
hydroxy-6-methylheptanoic acid (38, IC9564).
The mechanism of anti-HIV action is a very important factor
for the introduction of anti-retroviral therapy and the prevention
of disease development. An anti-HIV drug must be highly active
against “wild” and mutant HIV, because resistance to new drugs
can sometimes develop within days of treatment.
Of 20 anti-HIV drugs approved for use in the US, 11 are RT (re-
verse transcriptase) inhibitors (e.g., Zidovudine, Azidothymidine,
and AZT
227
), eight are PR (protease) inhibitors (e.g. Abacavir
228
),
and one is a viral fusion inhibitor (Fuseon
229
).
230
Betulinic acid
and other natural triterpenoids do manifest a moderate inhibitory
effect on HIV-1 reverse transcriptase,
231–233
as well as on HIV-
1protease.
234–236
Akihisa et al.
232
reported the inhibitory effect
of 55 triterpenoids, including birch bark lupane and oleanane
groups, on a purified HIV-1 reverse transcriptase. The best
inhibitory effect has been found for betulin 3,28-diacetate (1.3
lM), lupenone (11)(2.1lM) and betulonic aldehyde (10)(3.4
lM). The inhibition activity of betulinic acid (2) was 7.9 lM. Quere
et al.
236
provided a computational analysis for betulinic acid and
other triterpenoids as potential dimerisation inhibitors of HIV-1
protease. This theoretical work was supported by experimental
observations for natural triterpenoids.
235,237
Mayaux et al.
216
and
Soler et al.
227
reported that betulinic acid amide (37, RPR103611)
did not inhibit the in vitro activity of HIV-1 protease, reverse
transcriptase and integrase, or the binding of gp120/CD4. These
derivatives appeared to stop entry of HIV-1 at a post-binding,
envelope-dependent virus–cell fusion process. Holz-Smith et al.
238
conducted tests of analogues of compound (37, RPR103611)
betulinic acid derivative (38, IC9564) (Scheme 5). Results from
a syncytium formation assay indicated that IC9564 blocked HIV
type 1 (HIV-1) envelope-mediated membrane fusion. This research
suggested that HIV-1 gp120 plays a key role in the anti-HIV-
1 activity of IC9564. Sun et al.
226
reported that among a series
of IC9564 derivatives the
L-leucine derivative (EC
50
0.46 lM)
is equally as promising as compound 38 itself (EC
50
0.33 lM)
against HIV infection. The structure–activity relationship data
also indicated that a double bond in IC9564 can be eliminated.
Yua n et al.
239
confirmed that the HIV-1 envelope glycoprotein
gp120 is the key determinant for the anti-HIV-1 entry activ-
ity of IC9564. To date, very few fusion inhibitors have been
described.
230,240
Another new mechanism for anti-HIV action was revealed for
betulin and acylated derivatives of betulinic acid (see compounds
from Tables 2 and 4). The most promising anti-HIV drug candidate
DSB (3-O-(3
,3
-dimethylsuccinyl)betulinic acid, 15) was synthe-
sised by Kashiwada et al.
219
It was shown that neither HIV-RT
inhibition (in a concentration range 167–219 lM, IC
50
= 18 lM),
nor inhibitory activity against HIV-induced membrane fusion (in
a concentration range 33–70 lM) could explain such a high level
of HIV inhibition (EC
50
< 3.5 × 10
4
lM). Kanamoto et al.
241
reported an unusual mechanism for such anti-HIV activity. In a
p24 immunosorbent assay of culture supernatants, DBS inhibited
virus expression 18 hours after infection. This suggests that DBS
affects virion assembly step and/or budding of virions.
241
Further
study of this mechanism by Li et al.
242
demonstrated that DBS
(PA-457)
16
disrupts a late step in Gag processing. This blocks
the conversion of the capsid protein (p25) to a mature capsid
protein (p24). It has been shown that in vitro mutations of the
DSB-resistant virus map to the p25 to p24 cleavage site. The
resulting virions from DBS-treated cultures were non-infectious.
Thus, the mechanism of the anti-HIV action of DBS (PA-457)
16
and other acylated triterpenoids suggests new drug targets for
926 | Nat. Prod. Rep., 2006, 23, 919–942 This journal is
©
The Royal Society of Chemistry 2006
AIDS suppression. This new class of HIV bioactives are termed
maturation inhibitors.
It seems that there is no connection between this described
mechanism and the mechanism of the HIV budding process.
243,244
The budding and maturation processes are the last events in the
HIV infection cycle. During these events, the HIV-1 assembly
forms enveloped particles in a cell membrane that will bud
from the cell. The Gag protein is incapable of breaking a cell’s
membrane, and therefore, through its p6 domain, the Gag protein
uses cellular proteins Tsg101 for membrane cleavage. Triterpenoid
molecules, like 15–18 (see Table 2) or 15, 34–36 (see Table 4),
could interfere with the interaction between Tsg 101 and p6,
and ubiquitin, through a tetrapeptide motif (PTAP) within the
p6 domain. However, it has been reported that DBS (PA-457)
did not disrupt the Gag–Tsg101 interaction.
242
Nevertheless, this
target still seems very attractive for drug design on the basis of
triterpenoid structures for HIV therapies.
Huang et al.
245
combined the idea of complex modification
of betulinic acid at C3 (for inhibiting HIV-1 maturation) and
at C28 (for blocking HIV-1 entry). As a result the most po-
tent compound ([(N-[3b-O-(3
,3
-dimethylsuccinyl)lup-20(29)-en-
28-oyl]-7-aminoheptyl)carbamoyl]methane) inhibited HIV-1 at an
EC
50
of 0.0026 lM and was at least 20 times more efficient than
either the anti-maturation lead compound 15 (DSB, PA-457) or the
anti-entry lead compound 38 (IC9564). This bifunctional betulinic
acid derivative is active against both HIV entry and maturation.
The anti-HIV activity of triterpenoids can be summarised thus:
a) natural triterpenoids, especially of the lupane and oleanane
groups, function as moderately active anti-HIV agents at mi-
cromolar concentrations through inhibition of HIV reverse tran-
scriptase and protease and/or inhibiting the maturation process;
b) specific derivatisation of triterpenoids (C3 or C3,C28-acylation
or C17–COOH amidation) lead to significant increases in anti-
HIV activity to submicromolar concentrations and encompass
new types of virus inhibitory mechanisms; c) acylation of betulin,
betulinic acid, oleanolic acid, ursolic acid, moronic acid and
platanic acid leads to compounds blocking viral maturation
at nanomolar concentrations (maturation inhibitors); d) C17–
COOH amidation of betulinic acid leads to compounds that block
entry of HIV into cells (fusion inhibitors); and e) polyfunction-
alisation of triterpenoid molecules with certain pharmacophoric
groups could lead to the design of anti-HIV bioactives with
combined mechanisms of action (inhibiting maturation, blocking
virus entry, fusion inhibition, etc.). Fundamental research into the
anti-HIV action of betulinic acid and its derivatives has resulted in
an intensive patenting process in this direction.
174,246–253
Other than
anti-cancer or anti-HIV bioactivity the following directions of
potential betulinic acid use should be mentioned: food additives to
control obesity,
254
immunomodulatory activity,
255–257
anti-malarial
activity,
258
anti-aging cosmetics,
259
anti-wrinkle cosmetics,
260
and
anthelmintic activity.
261
Lupeol (4) is a well documented fruit-, vegetable-, and bark-
based NP found in olives, figs, mangoes, and other fruits and
medicinal herbs.
46,262,263
It is also the most lipophilic triterpenoid
component from outer birch bark extract. Lupeol (4) and its
derivatives have been found as the principal active ingredient
in the following folk medicine plants: Pimenta racemosa var.
ozua (Myrtaceae),
264
Alstonia boonei root bark,
265
Crateva nurvala
(Hindi: Varuna),
266
the leaves of Ixora coccinea L.,
267
C. religiosa
bark,
268
Dendropanax sf. querceti,
269
the leaves of Teclea nobilis,
270
the bark of Bombax ceiba,
271
the roots of Strobilanthus callosus and
Strobilanthus ixiocephala,
272
Vernonia scorpioides (Asteraceae),
273
Lactuca indica,
274
Holarrhena floribunda,
275
and the birch bark ex-
tractive of almost all Betula species.
6–14,18
The spectrum of lupeol (4)
bioactivity is rather broad, but different from the above-reviewed
birch bark triterpenoids. Anti-proliferative (anti-inflammatory
and anti-arthritis) activity for lupeol (4) is reported more fre-
quently than for betulin (1) or betulinic acid (2). Fernandez et al.
264
reported that the extract of Pimenta racemosa var. osua containing
lupeol (4) has a high level of activity against t wo experimental
models of acute inflammation (paw oedema in rats and ear oedema
in mice). The reduction of myeloperoxidase activity suggested that
the mechanism is likely related to the neutrophil migration. A
patent application
276
has claimed lupeol and its fatty acid esters
to be useful anti-inflammatory and anti-arthritis agents. Kweifio-
Okai et al.
265
reported the anti-arthritic effect of lupeol acetate.
Isolated from Alstonia boonei, this NP was studied for its anti-
arthrititic effect in CFA-induced arthritic rats. Oral treatment
resulted in an increase in spleen weight and the reduction in
serum alkaline phosphatase return to non-arthritic control values.
Anti-arthritic mechanisms of lupeol derivatives
277
were studied
with tests on the release of collagenase by rat osteosarcoma cells,
the release of five lipoxygenase inflammatory products by human
neutrophils, and on CCl
4
-induced hepatotoxicity in rats. These
tests explained the relative anti-arthritic action of triterpenoids (lu-
peol 3-linoleate > lupeol 3-palmitate > lupeol). The triterpenoids
studied equally reduced LDL release and accelerated hepatic
cell regeneration. Significant anti-inflammatory and anti-arthritis
effects were revealed for lupeol and 19aH-lupeol isolated from
Strobilanthus callosus and Strobilanthus ixiocephala roots.
272
Singh
et al.
268
reported that lupeol had a significant dose-dependent effect
on an acute and chronic inflammatory processes (LD
50
> 2gkg
1
in rats), but did not show any analgesic or anti-pyretic properties.
A similar result was reported by Geetha et al.
278
on lupeol and
lupeol 3-linoleate anti-inflammatory activity in comparison with
the non-steroidal drug Indomethacin. Latha et al.
279
have reported
the bioactivity of lupeol 3-eicosapentaenoate against adjuvant-
induced arthritis in rats. The activation of glycoproteins and
lysosomal enzymes and related inhibition of collagen in arthritic
animals were significantly changed, nearly reaching the control
level. A review on the inflammatory activity of plants and plant
extracts, listing the principal chemical ingredients that cause this
bioactivity, was published in 2003.
280
Lupeol is included in the list
of bioactives responsible for the potency of these plant extracts.
Lupeol (4) exhibits moderate but specific anti-cancer ac-
tivity against androgen-sensitive prostate cancer cells,
281
B16
2F2 melanoma cells (inhibition of the migration of malignant
melanoma cells by disassembling the actin cytosleleton),
282,283
and
pancreatic adenocarcinoma cells (inhibition of the Ras signaling
pathway),
284
and possesses anti-tumour-promoting effects in a
mouse skin tumourigenesis model (modulates NF-jBandPI3
K/Akt pathways and inhibits skin cancer in CD-1 mice),
285
and
is cytoprotective against free radical toxicity.
266
The potential
use of lupeol as a preventive anti-cancer component of dietary
supplements is an important aspect of the above-referenced
studies. Lupeol (4) and its derivatives have been suggested to
be of use for the prevention and treatment of skin disorders,
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The Royal Society of Chemistry 2006 Nat. Prod. Rep., 2006, 23, 919–942 | 927
skin cancer, prostate cancer and pancreatic cancer.
286
The nearest
NP derivative to lupeol (4), lupenone (11), exhibits a fairly
high inhibitory effect on a purified HIV-1 reverse transcriptase
(IC
50
= 2.1 lM).
232
Lupeol (4) has also been reported as an anti-oxaluric and anti-
calciuric NP in several studies.
266,287–289
The anti-urolithiatic activity
of lupeol was assessed in rats by observing the weight of stones, by
biochemical analysis of serum and urine, and by histopathology
of the bladder and kidney. Lupeol prevented the formation
of vesical calculi and reduced the size of preformed stones.
266
Malini et al.
288,289
studied the effect of lupeol (4) on urinary
enzymes in hyperoxaluric rats. Lupeol treatment (25 mg kg
1
body-weight day
1
) significantly reduced the renal excretion of
oxalate. Renal tubular damage was also reduced, as made evident
by the decreased level of the urinary marker enzymes. Lac-
tate dehydrogenase, inorganic pyrophosphatase, alkaline phos-
phatase, c-glutamyl transferase, b-glucuronidase and N-acetyl-b-
D-glucosaminidase were found to be elevated. This process lowers
the stone-forming constituents in the kidney. The protective effect
of triterpenoids on calcium oxalate crystal-inducing peroxidative
changes in experimental urolithiasis was studied by Malini et al.
289
Lupeol (4) and betulin (1) have been found to be efficient at
reducing the risk of stone formation in animals through preventing
crystal-induced tissue damage and dilution of urinary stone-
forming constituents. It is believed
288,289
that the mechanism
of this activity may involve the inhibition of calcium oxalate
crystal aggregation and enhancement of the animal’s defence
systems.
Anti-hypercholesterolemia action may be another potential
use of lupeol (4). Sudhahar et al.
290
reported the role of lupeol
and lupeol linoleate on lipemic-oxidative stress in experimental
hypercholesterolemia. The oxidative tissue damage in hyperc-
holesterolemic rats was manifested through elevation of the
cardiac marker, serum CPK, and a decline in its action in the
heart. Lupeol (4) and lupeol linoleate treatment reduces the
LPO levels and increases enzymic and non-enzymic antioxidants.
These observations emphasise the positive effects of lupeol and
its linoleate derivative for reducing the lipidemic-oxidative abnor-
malities in the early stage of hypercholesterolemic atherosclerosis.
The anti-hypercholesterolemic action of betulinic acid
291
and other
triterpenoids
292
have been reported and claimed for use in food and
beverages for vascular disorders or diseases.
The widespread availability of lupeol (4) in natural sources
that have been broadly used as human food products throughout
history makes this NP especially promising as an additive to
food and cosmetics (e.g . shea butter,
263
stimulation of the syn-
thesis of stress proteins,
293
compositions that promote melanin
formation,
294
melanogenesis regulators for hair care products,
295
low-irritation cosmetics,
296
and cosmetics containing lupeol that
prevent skin aging
297
).
Section 2 can be summarised as follows: in recent studies the
extractives of outer birch bark and birch bark triterpenoids, as
well as separated pure NPs, have shown a remarkably broad range
of positive biological activities against the most dangerous human
viral, bacterial and proliferative pathogens. The historically and
scientifically understood low toxicity of these NPs gives them
high potential in drug design. Birch bark extract, with its natural
complement of triterpenoids, has still not exhausted its potential
in dietary supplements or cosmetics.
3 Birch bark processing
Processing birch bark into the variety of NPs reviewed here
has never been achieved on an industrial scale. At the same
time, the wood processing industry (paper mills, veneer mills,
and lumber mills) can be considered to be the best high-volume
source of birch bark. This is probably because currently only
low volumes of birch bark extracts are used as ingredients for
cosmetics, shampoos,
15
and rarely as dietary supplements.
89,90,92,93
For these uses, it is sufficient to have low-volume sources of
birch bark and pilot-scale extraction equipment, and to follow the
extraction methods in the literature,
8,9,298
as a substantial market
for pure birch bark triterpenoids has not yet been developed.
Their uses are still rather limited, and do not require high-
scale manufacturing or serious R & D support. The market for
triterpenoids (betulin, betulinic acid, lupeol, oleanolic acid) as fine
chemicals
299
is usually satisfied by small-scale chemical extractive
laboratories (0.1–10 kg per year). At the same time, the necessity to
provide research and development on processing birch bark into
the corresponding NPs is growing in parallel with the growing
potential for use of these products. Satisfying the use/needs for
specific industrial R & D efforts is proceeding in the following
directions: 1) Refining and formulation of outer birch bark
to an appropriate standardised level from raw material after
debarking; 2) Optimisation of the technology of processing outer
birch bark into NPs; 3) Development of industrially viable ways
of synthesising potentially interesting products. The Chemical
Extractive Program of the University of Minnesota (US) and the
Laboratory of Chemical Extractives (UMD, NRRI) is currently
accomplishing this mission in cooperation with an industrial
partner, NaturNorth Technologies, LLC (US).
300
3.1 Refining and formulation of outer birch bark
The average paper mill or veneer plant that uses boreal birch
wood (Canada, US commercial birch tree Betula papyrifera;
Finland, Russia (Karelia, Siberia) and China B. pendula and
B. pubescens) produces 40 tons of crude birch bark (outer
birch bark 15%) daily, which represents 12% of birch wood
biomass.
7
Unfortunately, the only current high-scale usage of
this bark is as a cheap fuel ($5.0–7.0 ton
1
, 7–11 MJ kg
1
)
used by the manufacturers to save energy.
301
This means that
average plant burns 6 tons of outer birch bark daily, including
the following major birch bark NPs: 5 tons of betulin (1),
250 kg of betulinic acid (2)and470 kg of lupeol (4) + minor
components (see Table 1, data on B. pendula extract and Scheme 1).
Theoretically, this also means that from an average manufacturer
it would be possible to produce annually 1800 tons of betulin (1),
75 tons of betulinic acid (1)and150 tons of lupeol (4). These
quantities of triterpenes exceed the current demands of the drug or
cosmetics industry, but could satisfy the requirements for biocides,
fungicides, insecticides, emulsifiers, adhesives, washing materials,
shampoos, etc. These quantities of triterpenoids would also exceed
demand by as yet undeveloped markets. Essentially, natural birch
bark sources are virtually unlimited, and any prospective market
could be satisfied. It is clear, however, that yields of triterpenoids
produced from birch bark depend very much on the industrial
R & D of outer birch bark extract processing (see Section 3.2).
The crude birch bark resulting from birch wood debarking
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Fig. 1 Processing inner and outer birch bark.
(see Fig. 1) is not an appropriate natural material for high-scale
outer birch bark processing, for two main reasons: 1) Outer birch
bark must be separated from inner birch bark, woody material and
soil. A specially designed process of bark shredding and s creening
gives outer birch bark of good quality
302
; 2) The low bulk density
of outer birch bark (0.1gml
1
) makes this raw material expensive
to ship and inefficient for extraction. The process of outer birch
bark extrusion designed by Krasutsky et al.
300,302
produces birch
bark pellets with an appropriate bulk density (0.5–0.7 g ml
1
)for
transportation and easy loading into an extraction apparatus. The
world’s first facility for manufacturing outer birch bark pellets is
being launched in Two Harbors, Minnesota
303–305
in accordance
with patented technology.
302
This could be considered to be the
beginning of an industrial period of birch bark processing.
3.2 Manufacturing of birch bark NPs
This review will not discuss the numerous studies on solvents
and methods of birch bark extraction conducted prior to 1994;
a broad analysis of that period has been done by Kislitsyn.
8
All previous efforts were focused mainly on different methods
for manufacturing birch bark extract. The use of different (polar
and non-polar) solvents and methods depends very much on the
market and customers’ demands.
At this point it is useful to formulate current goals for
industrial processing, which are somewhat different from just
birch bark extract manufacturing. Pure birch bark triterpenoids
obviously have higher value as active ingredients or precursors for
drugs, special health care cosmetics, and dietary supplements. In
accordance with this imperative, it is easy to identify commercially
viable NPs from industrially available sources of outer birch bark
(see Table 1). Three major triterpenoids, betulin (1), betulinic
acid (2) and lupeol (4), seem to be the most plausible targets
for research and technology development, but it is necessary to
find the most appropriate solvent that could fulfil the multiple
roles of a solvent for extraction and for the separation of these
major components. However, recent research in this direction has
been focused mostly on the separation of one product (betulin).
Kuznetsov et al.
306–308
have found that the process of betulin
extraction and separation can be improved by hot steam activation
of birch bark. The preliminary activation of birch bark by an
auto-hydrolysis method increased the yield of betulin and suberin
by 25–40% compared to conventional extraction procedures. In
order to intensify the extraction process and to increase the
betulin yield, it was suggested to use s hort-time activation of bark
by superheated steam in the presence of NaOH.
309
Kuznetsov
et al.
310
improved betulin production by using acoustic pulses
with alkali hydrolysis and extraction of wood-processing waste
products. It was also shown that treatment of plant material with
ultrasound improves the process of triterpene dissolution and
extraction.
311
Pakdel et al.
312
reported a method for the separation
of betulin from the outer bark of B. papyrifera by sublimation
in a batch vacuum pyrolysis reactor. This process, which was
studied in the temperature range 250–300
C and under a total
pressure of 0.7 kPa, gave a yield of betulin of 9.5% on the
basis of the anhydrous bark used. A vacuum and atmospheric
sublimation technique was also proposed by Guidoin et al.
313
Roshchin et al.
314
claimed a method of preparing betulin from
the bark of B. pendula by extraction with petroleum ether. The
yield of the extract, which had a 90–95% content o f betulin (1),
was 16–25% of absolutely dry outer birch bark. Polar solvents
have also been used for the extraction of betulin.
315
Zhang et al.
316
reported extraction of betulin (1) from the bark of B. platyphylla
by extraction with supercritical CO
2
. Levdanskii et al.
317
proposed
a rather complicated sequence of solvents (hexane, ethyl acetate,
isopropyl alcohol, and water) for birch bark extraction. A claim
was made on a process for obtaining highly pure crystalline
betulin by extraction from birch bark with a high-boiling, water-
immiscible solvent.
318,319
According to this method, the water-
immiscible solvent extract is dissolved washed with dilute aqueous
base, and the aqueous phase separated off. The yield of pure
betulin (essentially free of betulinic acid) by this method did not
exceed 4% with respect to crude birch bark. The extraction of
lupeol or betulinic acid was not proposed by this method. Another
invention
320
claims the separation of betulin and lupeol by boiling
with hexane the product obtained by extraction of birch bark with
methyl tert-butyl ether and treatment with alkali solution. After
removing the solvent, the hexane-soluble lupeol is recrystallised
once from ethyl acetate. The hexane-insoluble residue is practically
pure (95%) betulin.
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Methods invented by Krasutsky et al.
321–327
have described
the separation and purification of all three major birch bark
triterpenoids (betulin (1), betulinic acid (2), and lupeol (4)). A
process of selective extraction was claimed with supercritical CO
2
(at a pressure between 3000 psi and 10 000 psi and a temperature
between about 50
C and 100
C) to provide lupeol (4), betulin (1),
and betulinic acid (2). These inventions also describe a method
for the hydrolysis and separation of the birch bark suberinic
acids 9R,10S-epoxy-18-hydroxyoctadecanoic acid (39), and threo-
9,10,18-trihydroxyoctadecanoic acid/or phloionolic acid (40). The
key benefit of these procedures is that the mild conditions of
hydrolysis and separation allow the preservation of the oxirane
ring in x-hydroxy acid 39.
Major suberinic acids that can be separated from birch bark
are presented in Scheme 6. There are no principal technical
limits for the industrial separat ion of these very important birch
bark chemicals. The total yield of these acids from the bark of
B. paperifera is 25–30%, which means that an average facility
using boreal birch wood could produce 1.5–2.0 tons of suberinic
acids daily. Comparable amounts of suberinic acids in the bark
of B. verrucosa were previously reported by Ekman.
9b
Natural
x-hydroxy fatty acids (C
18
-cutin monomers) are very interesting
NPs for their possible use as plant protectants (by inducing plant
resistance),
328,329
for the selective synthesis of the E and Z isomers
of ambrettolide,
330
as precursors of skin-protecting ceramides,
331,332
as anti-cancer agents and perfumes, for their use in consumables
containing x-hydroxy fatty acids,
333
and for film-forming materials
and polyesters.
334–336
The high application potential of birch
bark suberinic fatty acids seems still undervalued by industry
and the marketplace. The selective process for extracting acidic
and non-acidic NPs from plants has been claimed by patent.
337
The main idea of this invention (by Krasutsky et al.)isthe
binding of acidic components of plant tissue (birch bark) by
treatment with aluminum alcoholates (specifically with aluminum
isopropoxide) or another basic reagent. Consequently, any traces
of fatty acids or betulinic acid will remain stuck to the plant tissue
or precipitate, while other neutral components (betulin, lupeol,
etc., see Scheme 7) could be selectively extracted with non-polar
solvents. Acidic components can be extracted from the bark after
extraction of all neutral components with any slightly acidified
polar solvents. This approach enables the selective separation of
lupeol (4), betulin (1), and betulinic acid (2) from birch bark.
Scheme 6 Birch bark suberinic acids.
It is likely that the presence of slightly acidic aromatic hydroxy-
groups on the matrix plant polymer suberin supports the process
of betulinic acid adherence to the plant tissue.
336,338
The general
Scheme 7 Binding of acids to birch bark suberin with Al(OiPr)
3
.
character of the invention claims the use of such a procedure
337
for any extraction process when it is necessary to separate acidic
components from neutral components.
The invention of Krasutsky et al.
339
is suitable for obtaining all
major birch bark triterpenoids, betulin (1), lupeol (4), and betulinic
acid (2), at yields of about 10–12%, about 2.5% and about 2%,
respectively. By employing this method, commercial quantities
(i.e. tons) of the above triterpenoids can be obtained from birch
bark. When birch bark extract is boiled with a water-immiscible
solvent that is capable of forming an azeotropic mixture with
water and with an aqueous base, the following important processes
take place (Scheme 8): a) hydrolysis of betulin 3-caffeate (7)into
betulin and the corresponding caffeic acid salt; b) formation of
salts of betulinic acid and other fatty acids (on average, birch bark
extracts contain 5% fatty acids); c) formation of alcoholates of
polyphenols and tannins. The discoloration of the neutral fraction
(Scheme 8) is a very important feature of this process for producing
white crystalline NPs. Thus, pure white betulin (1) and lupeol
(4) can be obtained after removing water through azeotropic
distillation and subsequent filtering and crystallisation. Natural
betulinic acid (2) is obtained after separation and treatment of
solids (mixture of salts). Removing water by azeotropic distillation
with non-polar solvents (xylenes) is a very important approach.
This process results in the ultimate precipitation of all organic
fatty acid salts and their separation from the neutral triterpenoid
fraction. Other extractive processes using basic aqueous solutions
cannot provide such an efficient separation and good yield.
It is worth noting that birch bark is not the only possible large-
scale natural source of betulin. It is well-known that the presence
of betulin in birch wood pulp causes harmful pitch deposits
on papermaking machines.
340
Nikulenkova et al.
341
reported that
betulin may be isolated in large amounts from the crude sulfate
soap fraction from pulp mill manufacturing plants. Hamunen
342,343
proposed a method for betulin isolation from the crude soap of the
sulfate process at paper mill manufacturing plants that use birch
wood. A betulin content of 5–22% in four species of birch trees
can also be considered as a good source of this NP.
344
The above observations of different methods and approaches
to birch bark processing show that the contemporary state of
research and development is ready to meet high-scale commercial
interests for the manufacture of such NPs as betulin (1), lupeol
(4), betulinic acid (2), and birch bark suberinic acids (39–43). It
is also clear that birch bark extracts may be manufactured on any
scale.
3.3 Chemistry of birch bark NPs
During the reviewed ten year period the chemistry of triter-
penoids has been intensively developed, thanks to the efforts in
design of new drugs, as well as in the novel approach of the
cosmeceuticals
345–348
and neutraceuticals industries.
349–351
In the
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Scheme 8 Chemical processes during birch bark extract treatment with xylenes–NaOH–water and azeotropic distillation.
previous section the high potential for manufacture of birch bark
NPs 1, 2 and 4 is described. In this section it will be shown that
on the basis of these three burgeoning NPs, almost all other birch
bark triterpenoids and their derivatives can be synthesised.
A lot of research effort has been devoted to different methods of
synthesis of betulinic acid (2) from betulin (1), because the latter
is the most commonly available triterpenoid, and betulinic acid
(2) and its derivatives have high potential for use as anti-cancer
drugs and as precursors for anti-HIV drugs. Some independent ap-
proaches have recommended straight oxidation of betulin (1)into
betulonic acid (12) with the Jones reagent (CrO
3
/H
2
SO
4
/acetone),
and subsequent relatively stereoselective NaBH
4
/THF reduction
to 3a-and3b-betulinic acids (5 : 95 by weight) (Scheme 9).
352–355
One study
352
(Scheme 10) claimed a five-step process involving
selectively protecting the group at the C28-position of betulin (1)
(to give DHP ether 44), protecting the group at the C3-position
(to give acetyl ester 45), removing the C28-protection (to give ester
46), carrying out Jones oxidation, and hydrolysing the resulting
betulinic acid 3-acetate (47)to3b-betulinic acid (2), identical to
the natural compound. The overall yield was 55%.
Levdanskii et al.
356
invented an improved process for the
preparation of betulinic acid by oxidation of betulin to betulonic
acid with CrO
3
/AcOH and subsequent reduction with sodium
borohydride, without the isolation of free betulonic acid. The
overall yield of betulinic acid from betulin was 65% using this
process. Kogai et al.
357
invented a process for the preparation of
betulinic acid (2) by the oxidation of betulin with CrO
3
/AcOH and
subsequent sodium borohydride reduction of sodium betulonate
in water. Pichette et al.
358
reported the mild selective oxidation of
betulin (2) into betulinic aldehyde (3) on specially designed solid-
phase chromic oxide adsorbed on silica gel. Betulinic aldehyde
(3) can then be almost quantitatively oxidised to betulinic acid
Scheme 9 Two-step synthesis of 3a-and3b-betulinic acids (2).
with potassium permanganate. Roshchin et al.
359
invented another
modification of betulin (1) oxidation into betulonic acid (12)by
pyridine dichromate complex with DMF–acetic anhydride (2.5 :
3.0). Betulonic acid (12) was then reduced to a mixture of 3a-and
3b-betulinic acid (2). The ratio of isomers (5 : 95) was identical
to ratios previously reported.
352–354
All of these methods
351–358
of oxidation using common chromium oxide reagents can be
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Scheme 10 Five-step synthesis of betulinic acid (2) from betulin (1).
regarded as an improvement on the first publication on this subject
by Ruzicka et al.
360
Another approach, by Krasutsky et al.,
361–363
claims a five-step
betulinic acid (2) synthesis (Scheme 11), involving diacetylation
of betulin (1) to betulin 3,28-diacetate (48), followed by selective
alcoholysis with Al(OiPr)
3
in i PrOH to give betulin 3-acetate (45),
Swern oxidation to betulinic aldehyde 3-acetate (49), oxidation
with sodium or potassium chlorite to give betulinic acid 3-acetate
(50) and final hydrolysis of ester 50 to provide betulinic acid (2).
A recent patent
364
claimed a different sequence of operations to
provide betulinic acid 3-acetate (50) (Scheme 12): regioselective
silylation of betulin (2) with tBuMe
2
SiCl to give ether 51;
acetylation with Ac
2
O into the ether-ester 52; desilylation with
TBAF to give betulin 3-acetate (46); Oppenauer oxidation with
Al(OtBu)
3
with 1,4-quinone to give betulinic aldehyde 3-acetate
(49); and oxidation of 49 with NaClO
2
/NaH
2
PO
4
and 2-methyl-
2-butene to give 50.
All three methods shown in Schemes 10–12 use different
regioselective reactions with betulin (1) as the key steps: Scheme 10
the regioselective formation of DHP-ether 44, Scheme 11 the
regioselective alcoholysis of 3,28-diacetate 48, and Scheme 12 a
regioselective C28-silylation process.
Mitrofanov et al.
365
reported selective oxidation of betulin (1)
into betulinic acid (2) by micro-organisms in chloroform. It was
shown that the dormant cells of Mycobacterium perform this
reaction with a 36% yield.
The reviewed period has brought many interesting synthetic
approaches to the chemistry of birch bark triterpenoids through
intensive efforts in drug design and research into structure–activity
relationships. A number of methods for betulin and betulinic
Scheme 11 Five-step synthesis of betulinic acid (2).
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Scheme 12 Five-step synthesis of betulinic acid 3-acetate (50).
acid derivatisation at the C3-, C28-, C29- and C30-positions
(Scheme 13) have been developed and reported in publications
and patents.
Scheme 13 Four methods for modification of betulin and betulinic acid.
Schemes 10–12 manifest the different methods of C3-protection
of betulin (1) for betulinic acid synthesis (2).
351–353,359–362
Kim
et al.
352
have reported a modification of the C28-position of betulin
(1) through the selective formation of tetrahydropyranyl ether
44. Selective C28-silylation of betulin (1) has been claimed by
patent.
364
Tietze et al.
366
reported the selective C28-acetylation of
betulin into betulin 28-acetate and its oxidation to betulone 3-
acetate. In this work, betulone 3-acetate was a precursor to the
synthesis of [
13
C]- and [D]-betulin for biological transformations.
A method for the synthesis of betulin and dihydrobetulin 3-
acylated anti-HIV derivatives used a rather different approach
(Scheme 14) involving selective C28-tritylation of betulin (1) with
Ph
3
CCl/DMAP in DMF.
54,56
After esterification with RCOOH
in pyridine/DMAP, betulin 28-O-trityl ether (53)gave3-O-
acylated betulin derivatives 54. The following detritylation with
catalytic pyridinium tosylate in EtOH–CH
2
Cl
2
results in esters
55, and hydrogenation with catalytic Pd/C yields dihydrobetulin
3-acylated products 56.
Activation of the C30-position (Scheme 13) in betulin or other
lupane triterpenoids can be achieved through bromination with
Scheme 14 Synthesis of betulin and dihydrobetulin 3-acyl derivatives.
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NBS in CCl
4
(Scheme 15).
52,224,367
This reaction is actually directed
at the C29-unsaturated carbon atom, with consequent double
bond isomerisation leading to functionalisation with bromine at
the C30-position. Nucleophilic substitution of this active C30-
brominated compound with Y
then leads to the corresponding
C30-Y triterpenoid derivatives. These processes are quite selective,
and have been reported in numerous publications.
52,224,367
Scheme 15 Activation of the C30-position in lupane triterpenoids
through bromination with NBS.
The chemical s pecifics of lupane triterpenoids appear in their
resistance to nucleophilic substitution near the C3- and C28-
atoms. Such behaviour is quite understandable from a theoretical
point of view, because these positions are especially prone to
carbocationic Wagner–Meerwein rearrangement, similar to those
in neopentyl or norbornyl systems (Scheme 16).
368,369
For example,
the reaction of lupane triterpenoids with POCl
3
/pyridine did not
yield the corresponding chlorides, but a complicated mixture of
Wagner–Meerwein rearrangement products.
370
The “neopentyl”-
type carbocationic fragments of the betulin structure involved are
showninScheme16.
Scheme 16 Fragments of the “neopentyl”-type carbocations at C3
(A-ring, prone to elimination and rearrangement) and C28 (E-ring, prone
to rearrangement through enlargement) of betulin (1). The dashed lines
indicate the main direction of the elimination or rearrangement process.
Attempts to accomplish S
N
2 nucleophilic substitution near the
C3- and C28-atoms have not been very succesful so far. Even such
a well-known S
N
2-process as the Mitsunobu reaction,
371
which
inverts the configuration at the C3-atom of the unhindered sterol
57, providing 58,
372
led to olefinisation at the C3-atom of betulin
(1), giving 3-deoxy-2,3-dihydrobetulin (59)
52
(Scheme 17).
Symon et al.
225
reported that attempts at bimolecular substitu-
tion in lupeol 3-tosylate resulted only in D
2,3
-elimination products,
with none of the expected products of S
N
2 substitution. Attempted
Mitsunobu reaction of betulin (1) in dry THF with benzoic acid
led to the formation of 3-deoxy-2,3-dehydrobetulin (59) (44%)
and 3-deoxyhydrobetulin 28-benzoate (60) (16%) (Scheme 18).
The for mation of 60 proves the possibility of an S
N
2processat
the C28-position, but such reactions have not yet been reported
for the C3 positions of triterpenoids. These results demonstrate
conditions where the process of carbocation olefinisation at the
Scheme 17 Mitsunobu reaction for ()-3b-cholesterol (57) and betulin
(1).
Scheme 18 Mitsunobu reaction for betulin (1)withDEAD.
C3-position of lupane structure prevailed over the process of
carbocation rearrangement. The carbocation at C28 (Scheme 16,
E-ring fragment) has the option for E-ring enlargement and
subsequent olefinisation.
370,373,374
Since birch bark triterpenoids are 3b-isomers, research into
their conversion into their 3a-isomers is also worth observation.
3a-Epimers of triterpenoids (such as epi-lupeol or epi-betulinic
acid) are less common in nature,
375–377
and therefore their synthesis
from available birch bark triterpenoids is quite interesting. As
mentioned above, the C3-position is sterically restricted with
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respect to S
N
2 reactions. This problem was bypassed by the
design of various stereoselective methods for the reduction of
triterpen-3-one derivatives. Das
378
provided the first report on
the stereochemistry of the Meerwein–Ponndorf–Verley reduction
(with Al(OiPr)
3
) of betulonic acid into 3b-betulinic acid (80%)
and 3a-betulinic acid (20%). It was later shown that reduction of
betulonic acid with NaBH
4
/THF yielded a mixture of 3b-and3a-
betulinic acid (95 : 5 by weight).
352–355
This level of selectivity may
be used for the synthesis of 3b-betulinic acid from betulonic acid
(44) (Scheme 9). The best selectivity for the 3a-isomers synthesis
(78%) was achieved by Sun et al.
52
by reducing betulonic acid with
L-Selectride in THF at 78
C (Scheme 19). A similar study
225
reported lower selectivities for the reduction of betulonic acid (12)
with L-Selectride and with Raney nickel (3a/3b ratio = 60 : 40).
Scheme 19 Synthesis of 3a-betulinic acid (2) from betulonic acid (12).
Synthesis of 20-oxo-30-norlupane derivatives (involving modifi-
cation at C29, Scheme 20) can be accomplished by transformation
of the corresponding lupane derivatives with ozone.
379
For exam-
ple, synthesis of platanic acid 3-acetate (61) from betulinic acid
3-acetate (47) can be accomplished in 66% yield (Scheme 20).
224
Ashavina et al.
380
have reported the stereoselective epoxidation
of 20(29)-lupene triterpenoids with dimethyldioxirane. Another
interesting modification, at C29 of betulin 3,28-diacetate (48), was
Scheme 20 Synthesis of platanic acid 3-acetate (61).
based on a 1975 study by Suokas et al.
381
It was found that specific
acidic conditions (HBr/AcOH/Ac
2
O/PhMe) lead not to allobe-
tulin formation, as with HCl/EtOH/CHCl
3
,
382
but to the migra-
tion of the double bond from C20 to C18. This reaction was the
starting point for a five-step transformation (Scheme 21) of betulin
3,28-diacetate (48)to3b,28-diacetoxy-18-oxo-19,20,21,29,30-
pentanorlupan-22-oic acid (62), which is a promising compound
for the treatment of proliferative disorders such as cancer and
leukaemias.
383
Interestingly, compound 62 forms a fairly stable
solvate with methanol (1 : 1.5). Similar solvates of betulinic acid (2)
with ethanol (1 : 1) have been described in a patent application.
384
Sarek et al.
197,198,385
have developed syntheses for numerous
18-lupene, 18,19-secolupane, des-E lupane, and other oxidised
triterpenoids as potential anti-tumour and anti-cancer chemother-
apeutics.
The development of different methods of A-ring modification
and cleavage has also been a matter of research and synthesis of
new bioactives. Urban et al.
191,199
have reported the synthesis of A-
seco derivatives of betulinic acid with relevant cytotoxic activity.
Deng et al.
386
have described a new route to the synthesis of 24-
nortriterpene derivatives with a modified A-ring (2-hydroxy-D
1,4
-
cyclohexadiene-3-one) starting from of betulin (1) and betulinic
acid (2). The principal steps of these transformations were a Suarez
cleavage
387
of the A-ring in dihydrobetulin 28-acetate (63)andan
Scheme 21 Synthesis of 3b,28-diacetoxy-18-oxo-19,20,21,29,30-pentanorlupan-22-oic acid (62).
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Scheme 22 Synthesis of methyl 2-hydroxy-3-oxo-24-norlup-1,4-dien-28-oate (65).
SmI
2
-mediated pinacol coupling to re-close the A-ring following
removal of the C24 carbon by oxidative cleavage (Scheme 22).
387
It is possible that compound 64 could be oxidised to 65
directly with air in BuOH/BuOK. Such an A-ring oxidation
(Scheme 23) for lupeol was first reported by Ganguly et al.,
388
followed by a method for oleanolic acid by Chattopadhyay et al.,
389
for betulinic acid by Evers et al.,
224
for lupane and ursane
triterpenoids by Korovin et al.,
390
and for numerous betulinic
acid derivatives by Urban et al.
191,199
This simple process led
Scheme 23 A-Ring oxidation and cleavage of triterpenes, and synthesis
of quinoxalines.
to the corresponding keto-enol derivatives 66 in 90–97% yield.
The reaction of these keto-enols 66 with 1,2-diaminobenzene
yielded the corresponding quinoxalines 67 in 85–95% yield
(Scheme 23).
390
A-ring cleavage of the keto-enols 66 into acids 68
may be accomplished in one step with H
2
O
2
and KOH in MeOH
(Scheme 23).
199
Fluorination of the E-ring of betulin with diethylaminosulfur
trifluoride (DAST) was reported by Biedermann et al.,
391
but these
fluorinated betulinines failed to demonstrate significant in vitro
anti-cancer activity.
As previously mentioned (Section 2.1), different (triter-
penoid and non-triterpenoid) caffeates are interesting NPs be-
cause of their anti-cancer, imunomodulatory and UV-protection
activity.
20,101–104
Therefore, synthesis of these NPs from triter-
penoids is of special practical interest. It has been shown
20
that
a previously described procedure for the synthesis of caffeates
392
from caffeic acid, and of the corresponding alcohols by using
thionyl chloride, do not work well for triterpenoids. This result
triggered efforts on the development of a new method for
synthesis of triterpenoid caffeates, as shown with Scheme 24.
20
The selective alcoholysis of betulin 3,28-dibromoacetate (69)
led to betulin 3-bromoacetate (70), which was then converted
to its triphenylphosphonium salt 71. Reaction of 71 with 3,4-
dihydroxybenzaldehyde yielded betulin 3-caffeate (7). This ap-
proach may be used to synthesise any natural triterpenoid
caffeate.
Several recent and miscellaneous synthetic approaches to
the modification of birch bark triterpenoids should also be
mentioned. Tolmacheva et al.
393
obtained new 30-thio- and
936 | Nat. Prod. Rep., 2006, 23, 919–942 This journal is
©
The Royal Society of Chemistry 2006
Scheme 24 Synthesis of betulin 3-caffeate (7).
30-sulfinylbenzimidazole derivatives of betulin, which exhib-
ited anti-inflammatory activity comparable to that of sodium
diclofenac. New 3-amino derivatives of betulin and betulinic
acid have been synthesised by Uzenkova et al.
394
and Flekhter
et al.
395
Cyclopropane derivatives of betulin were synthesised by
the attachment of dichlorocarbenes or dibromocarbenes to the
double bond of betulin diacetate, followed by the deprotection
of the hydroxyl groups.
186
Yo u et al.
396
reported the synthesis
of 3-aminoacetyl derivatives with increased water solubility and
potential cytotoxic activity. Petrenko et al.
397
have synthesised
new C28 amino acid derivatives of betulonic acid as potential
bioactives, and Miskiniene et al.
398
have described the synthesis
of nitroaromatic derivatives of betulin [betulin-(28)-5
-(aziridin-
1-yl)-2
,4
-dinitrobenzoate and betulin-(28)-5
-nitro-2
-furoate] as
redoxcyclingreagents.
Addendum
It is worth mentioning the most recent research and develope-
ment efforts that have been published during the writing of this
review. These new reviews have been about the chemistry and
bioactivity of triterpenes and their derivatives.
399–402
Gauthier
403
and Mao-Cai
404
have reported the synthesis of cytotoxic and
anti-tumor triterpene glycosides and saponins. Several new triter-
penoids and their derivatives with anti-HIV activity have also been
reported.
405–408
The possible medicinal use of lupeol derivatives has
also been described,
409–411
and there have been studies on the anti-
proliferative activity of triterpenoids.
412,413
4 Summary
The previous ten years of research and development have enabled
us to reach a point when birch bark triterpenes or their derivatives
will very soon appear in the marketplace. The development of
technology for processing birch bark is also ready to meet the
demands of industry and relevant markets. The quantity of natural
products of the bark from commercially managed birch trees
(B. papyrifera, B. pendula, B. pubescens and B. neoalaskana)are
large enough to satisfy any high-volume need for birch extracts,
betulin, betulinic acid or lupeol. These NPs can be also considered
precursors for a broad range of synthetic lupane and oleanane
derivatives. Birch bark suberinic acids, which can in principle be
isolated individually, should also be worthy of attention, but the
volume of suberinic acids manufactured will depend very much
on the success of the commercialisation of birch bark extract and
birch bark triterpenoids.
5 Acknowledgements
The author of this review would like to acknowledge the assistance
and support of NaturNorth Technologies, LLC (Duluth, MN,
US). Additionally, the efforts and contributions of Dr Jon Holy
regarding biological matters (School of Anatomy and Cell Biology,
University of Minnesota, Duluth). Finally, the technical support
and assistance of Jonathan Lee (Natural Resources Research
Institute, University of Minnesota, Duluth) is acknowledged.
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