Current Medicinal Chemistry, 2005, 12, 657-666
Betulinic Acid and Its Derivatives: A Review on their Biological Properties
Perumal Yogeeswari* and Dharmarajan Sriram Pharmacy Department, Birla Institute of Technology & Science, Pilani-333031, INDIA Abstract: Betulinic acid is a naturally occurring pentacyclic triterpenoid and has been shown to exhibit a
variety of biological activities including inhibition of human immunodeficiency virus (HIV), antibacterial,
antimalarial, antiinflammatory, anthelmintic and antioxidant properties. This article reports a survey of the
literature dealing with betulinic acid related biological properties that has appeared from the 1990’s to the
beginning of 2003. A broad range of medical and pharmaceutical disciplines are covered, including a brief
introduction about discovery, phytochemical aspects, organic synthesis, anti-HIV and cytotoxic mechanisms of
action. Various structural modifications carried out and their biological and pharmacokinetic profiles are also
Keywords: Betulinic acid, Anti-HIV derivatives, Anti-HIV mechanism, Anticancer derivatives, Anticancer mechanism;
Antiinflammatory, Antimalarial, biotransformation.
Natural products are the organic molecules which are Betulinic acid is a triterpene of natural origin isolated elaborated by living tissues derived from higher plants, from various plants. It can be isolated from the methanolic fungi, microbes, marine organisms and animals and exhibit a extract of Quisqualis fructus [7], the dichloromethane- remarkably wide range of chemical diversity and a methanol extract of the twigs of Coussarea paniculata [8], multiplicity of biological properties. Thousands of year’s the dichloromethane-methanol (1:1 v/v) extract of natural resources have been in use for combating human Argentinean legume Caesalpinia paraguariensis [9], the ailments. Over the last fifteen years interest in drugs of plant methanolic extract of the leaves of Vitex negundo [10], twigs origin has been reviving and growing steadily, and the drug of Ilex macropoda [11], the ethanolic extract of the roots of researchers are exploring the potential of natural products for Anemone raddeana [12], leaves and wood of Doliocarpus the cures of still unsurmountable diseases like cancer and schottianus [13], ethanolic extract of Tovomita krukovii [14], fruits of Chaenomeles lagenaria [15], methanol, hexane andethyl acetate extracts of stem bark of Berlinia grandiflora Betulinic acid (1), 3β-hydroxy-lup-20(29)-en-28-oic acid,
[16], methanolic extract of the aerial parts of Vietnamese is a widely distributed pentacyclic lupane-type triterpene in Orthosiphon stamineus [17], leaves of Eucalyptus the plant kingdom. Considerable amounts of betulinic acid camaldulensis [18], stem barks of Physocarpus intermedium (up to 2.5%) are available in the outer bark of a variety of [19] and Tetracentron sinense [20], chloroform extract of tree species that are valuable for timber purposes [1]. A barks of Syncarpa glomulifera [21], methanolic extract of closely related compound, betulin (1a), is a major
leaves of Combretum quadrangulare [22], methyl ethyl constituent of white-barked birch trees (Betula species) with ketone extract of Tetracera boiviniana [23], dichloromethane yields up to 22% (dry weight) [2, 3]. Compound 1a can be
extract of stem bark of Brazilian medicinal plant Zizyphus easily converted to 1 in high yields synthetically [4].
joazeiro [24], ethanolic extract of the root barks of the White birch bark, Betula alba (which contains betulinic Tanzanian tree Uapaca nidida [25], Ipomea pescaprae [26], acid) has been used by Native Americans as a folk remedy.
Ancistrocladus heyneanus [27], Diospyros leucomelas [28], They used it in tea and other beverages to treat stomach and and from the leaves of Syzrgium claviforum [5].
intestinal problems such as diarrhea and dysentry. In Russia,it has been reportedly used since 1834. In 1994, scientists at During the isolation of betulinic acid from the above the University of North Carolina reported that chemicals mentioned plant sources, a closely related compound betulin found in white birch bark slowed the growth of human (1a) was also isolated from many plants in high yield (up to
immunodeficiency virus (HIV) [5]. The following year, a 22%). Son et al. [29] developed a method for obtaining researcher at the University of Illinois reported that betulinic betulinic acid from betulin. Betulin was oxidized with acid killed melanoma cells in mice [6]. Since then, a number chromium oxide (VI) into betulonic acid (2) which was
of researchers have conducted laboratory tests on betulinic reduced with sodium borohydride to yield a mixture of 3- acid to determine antitumor properties, especially with hydroxy epimers containing 85% of natural beta-epimer.
respect to melanoma cells with some promising resultswhich may warrant future study. Betulinic acid has recently ANTI-HIV ACTIVITY OF BETULINIC ACID
been selected by the National Cancer Institute for addition DERIVATIVES
into the RAID (Rapid Access to Intervention inDevelopment) program.
Betulinic acid has been shown to inhibit HIV-1 replication [30]. Based on its chemical structure, betulinic *Address correspondence to this author at the Pharmacy Department, acid derivatives have been reported as inhibitors of HIV-1 Birla Institute of Technology & Science, Pilani-333031, INDIA; E-mail: entry [31], HIV-protease [32] or of reverse transcriptase (RT) 0929-8673/05 $50.00+.00
2005 Bentham Science Publishers Ltd.
658 Current Medicinal Chemistry, 2005, Vol. 12, No. 6
Yogeeswari and Sriram
1a = CH2OH
Fig. (1). Structures of betulinic acid, betulin, dihydrobetuline acid and amide derivatives of betulink acid.
[33]. Since a number of betulinic acid derivatives have been betulinic acid, with an overall yield of 31% [44]. Among shown to inhibit HIV-1 at a very early stage of the viral life them, the compound N’-{N-[36-hydroxyl-20(29)ene-28-oyl]- cycle, these compounds have the potential to become useful 8-amino octanoyl}-1-statin (4c) was found to be the most
additions to current anti-HIV therapy, which relies primarily potent anti-HIV compound particularly against HIV-1 strain on combination of RT and protease inhibitors.
IIIB/LAI with a SI of 200. The compounds were not activeor were much less active against two isolates of Zairianorigin NDK and ELI. However, no significant activity could STRUCTURE-ACTIVITY RELATIONSHIP STUDIES
be found against HIV-2 (ROD and EHO isolates).
Compound 4c was also examined for a possible inhibition of
Fujoka et al. [5] isolated betulinic acid from the leaves of the in vitro activity of several purified HIV-1 enzymes. No Syzrgium claviflorum and on random screening it was found inhibition of RT, integrase, or protease was detected at a that betulinic acid exhibited inhibitory activity against HIV- concentration of the derivatives compatible with cellular 1 replication in H9 lymphocyte cells with an EC50 value of activity, suggesting that the corresponding steps were not 1.4 µM and a selectivity index (SI) value of 9.8.
involved in the inhibitory mechanism of these compounds.
Hydrogenation of betulinic acid yielded dihydrobetulinic
acid (3) which showed slightly more potent anti-HIV
Kashiwada et al. [34] reported syntheses and anti-HIV activity with an EC50 of 0.9 µM and a SI of 14.
activities of some derivatives by modifying the C-3 hydroxyl
group in betulinic acid and dihydro betulinic acid. 1 and 3
Mayaux et al. [31] synthesized certain amide derivatives were treated with 3, 3-dimethylglutaric anhydride and of betulinic acid (4a-c) by a five step procedure starting from
Fig. (2). C3 modified derivatives of betullinic acid and dihydrobetulinic acid.
Betulinic Acid and Its Derivatives
Current Medicinal Chemistry, 2005, Vol. 12, No. 6 659
diglycolic anhydride in pyridine in the presence of 4- the 3 position, preferentially occurs in the 3β-position. The (dimethylamino) pyridine to furnish the corresponding 3-O- 3β-methoxy (7d) and 3-amino (7h) derivatives were found
acyl derivatives (5c, 5d, 6c and 6d). In contrast, similar
inactive. The inactivity of 7d might be due to a steric
treatment of 1 and 3 with dimethylsuccinic anhydride
hindrance by the methyl group. The introduction of a second afforded a mixture of 3-O-(2’, 2’-dimethylsuccinyl) and 3-O- hydroxyl group led to a complete loss of activity. Thus, (3’, 3’-dimethylsuccinyl) betulinic acid derivatives (5a and
almost all chemical modifications in ring A led to 5b) and dihydro betulinic acid derivatives (6a and 6b),
respectively. The anti-HIV assay indicated that compounds The antiviral properties of 30-(hydroxyethyl)thio- (8a),
5b and 6b were extremely potent in acutely infected H9
30-[2-(diethylamino)ethyl]thio- (8b), 30-(1-pyrrolidinyl)-
lymphocytes with EC50 values less than 3.5 x 10-4 µM and (8e), and 3, 30-dihydroxy- (8g) derivatives remained high,
SI values > 20,000 and > 14,000, respectively. In contrast, but were not better than that of the unsubstituted derivative compounds 5a and 5c showed anti-HIV activities with EC50
7a. This illustrated a lack of steric requirements by the HIV-
values of 2.7 and 0.56 µM, respectively, and SI values of 1 molecular target for groups at position C 5.9 and 13.8, respectively. Compounds 5c, 5d, 6c and 6d
acidic substitution such as 30-(carboxymethyl) thio (8c) was
also exhibited anti-HIV activities with EC50 values ranging clearly detrimental to potency. A similar drop in activity from 0.01 to 2.3 x 10-3 µM and SI values from 1017 to was observed when a secondary nitrogen was directly 2344. None these compounds inhibited the HIV-RT in the concentration range of 167-219 µM. In the HIV-induced 30 (8d and 8i). The combination of a
free carboxyl and a secondary amine as well as an membrane fusion inhibition assay compounds 5a-d and 6a-d
unsubstituted amino moiety (8f and 8h) led to a significant
inhibited syncytia formation in the concentration range of 33- drop in activity. In conclusion, the isopropylidene group seems important for optimal activity, probably due to Evers et al. [35] synthesized a series of ω -undecanoic binding to a hydrophobic pocket. A certain lack of steric acid and amides of lup-20(29)-en-28-oic acid derivatives by hindrance accommodates a variety of substituents without improvement of activity. However, favorable interactions betulinic acid and evaluated for activity in CEM4 and MT-4 were observed for primary and secondary amines as well as cell cultures against HIV-1 strain IIIB/LAI. Structural for the free carboxylic acid moiety.
variations in ring A of the triterpene highlighted the importance of the 3β-hydroxy substituent. Epimerization of methylation of the amide moiety in 7f, replacement of the
the hydroxyl group at C-3 from 3β (7a) to 3α (7b) led to a
amide by an ester in 7e, and replacement of the carbonyl by a
10-fold drop in activity. The 3-keto derivative (7c) was
methylene as in 9c led to a complete loss of activity. The
found to show intermediate activity, whereas the 3-deoxy importance of the hydrogen donating NH group was derivative (7g) displayed no activity at all. These point to a
highlighted by the fact that the corresponding ester 7e was
critical hydrogen bond interaction involving the oxygen at completely inactive. This was further corroborated by the 10 (IC 9564)
Fig. (3). C17 modified betulinic acid derivatives.
660 Current Medicinal Chemistry, 2005, Vol. 12, No. 6
Yogeeswari and Sriram
Fig. (4). O-acyl derivattives of betulinic acid and dihydrobetulinic acid.
lack of biological activity of the reversed amide 9a and the
which showed slightly lesser activity (EC50 = 29.2 µM; SI urea derivative 9b in which the NH group occupied a
= 2.9) than the corresponding nonketone derivative 11b.
different special position. The dramatic loss of activity for The triacylated compound 11d displayed potent anti-HIV
most of the modification on the triterpene skeleton suggests activity with an EC50 value of 0.045 µM and a SI of 389.
a stringent specificity for the compounds. All the compounds The 3-epi derivative of 11a (11e), which displayed lower
were inactive against HIV-2 ROD. All these compounds inhibition of HIV. Bioisosteric replacement of 11a to an
were found to interfere with HIV-1 entry in the cells at a amide derivative (11f) resulted in reduction of anti-HIV
activity (EC50 = 0.5 µM; SI = 36.6). The above resultsshowed that acylation only at the C28 position did not result Soler et al. [36] synthesized a novel series of ω - in significant increase or decrease of activity. However, aminoalkanoic acid derivatives of betulinic acid and compounds with acyl side chains at both C3 and C28 evaluated for their activity against HIV-1. The anti-HIV-1 positions reached optimal activity. An addition of a third activities of several members of this new series were found to chain at C30 led to increased potency. Activity was affected be in the nanomolar range in CEM-4 and MT-4 cell by the type of side chain linkage (ester) and the 3β cultures. Among them, compound 10 was found to display
configuration resulted in the most impressive EC50 as well the best overall activity with an EC50 of 50 ± 26 nM (CEM4) and 40 ±19 nM (MT-4) with a SI of >100.
Nevirapine tested under the same conditions, displayed an Kashiwada et al. [38] prepared four isomeric 3, 28-di-O- (dimethylsuccinyl) betulin derivatives and evaluated their 50 value of 84 ± 21 nM (CEM4). No inhibition was observed with 10 against RT, integrase or protease at
anti-HIV potency. Among these derivatives, 12c
demonstrated the highest activity in acutely infected H9 cells with an EC50 value of 0.87 nM and inhibited uninfected H9cell growth with an IC50 value of 36.9 µM. Its calculated SI Sun et al. [37] synthesized various O-acyl betulinic and value (42,400) was comparable to that of zidovudine dihydro betulin derivatives. Among them, the most potent (41,622). Compound 12a was also extremely potent with an
compound 11a with two 3’, 3’-dimethylglutaryl groups
EC50 value of 0.02 µM and SI of 1680. Compound 12b
displayed anti-HIV activity with an EC50 value of 0. 66 nM displayed fair activity (EC50 = 0.4 βM; SI = 96.5), while and SI of 21,515. The dihydro betulin derivative of 11a
12d was toxic.
showed a SI of 2253. Monoacylbetulin (11b), containing a
substituted glutaryl group only at C28 position, had an EC50
Sun et al. [39] synthesized compound 10 analogues and
value of 3.6 µM and a SI of 7.8. Conversion of the 3β- compounds 13a and 13b were the most promising
hydroxy group of 11b to the monoketo derivative led to 11c,
compounds against HIV infection with EC50 values of 0.33 Betulinic Acid and Its Derivatives
Current Medicinal Chemistry, 2005, Vol. 12, No. 6 661
and 0.46 µM respectively. Both compounds inhibited terminal sequence (“fusion peptide”), thought to insert in the syncytium formation with EC50 value of 0.40 and 0.33 µM, target cell membrane, and two domains with a predicted α- respectively. The structure activity relationship data also helix conformation separated by a region containing a indicated that the double bond in 10 can be eliminated and
conserved dicystein motif, representing a highly the statin moiety can be replaced with L-leucine while immunogenic determinant [44]. Several residues in the proximal helix and the loop region of gp41 seem to beinvolved in interaction with gp120 [45]. Peptidescorresponding to the proximal (N) and distal α-helix domains of HIV-1 gp41 spontaneously from highly stablecoiled-coil structures with an inner core of three parallel N helices on which are stacked three C helices placed in an antiparallel orientation [46]. Structural analysis of the gp41 ectodomain of the HIV-2 related similar immunodeficiencyvirus revealed the same organization [47]. Whether the formation of this structure is the motive force driving the viral and target membranes into a closer position [46], or whether this structure is already present in the native form ofgp41 is not known [47]. Very few compounds targetinggp41 have been described to date. Among them, betulinic acid derivatives were found to block cell-cell fusion and HIV- 1 infection at a post binding step [31] by preventing gp41 from attaining its fusion-active conformation [46]. In fact,
evidence was obtained from two different experiments that 4c
acts at an early stage in the infection cycle. First, scientists
observed a clear dose-dependent inhibitory effect of
compounds at micromolar concentration on the synthesis of
proviral cytoplasmic DNA, as measured by PCR, only 2h after the onset of infection. Secondly, they studied the Fig. (5). C17 derivatives of betulinic acid with potent anti-
influence by adding the compounds at various times soon after the exposure of MT-4 cells to HIV-1. Such an
experiment showed that postponing the addition of 4c for 1h
was enough to cancel the inhibitory potency of this
compound on the subsequent production of viral antigens.
Compound 4c blocked virus infection at a post binding step
The HIV-1 and HIV-2 envelope glycoproteins (Env) necessary for virus membrane fusion and that target of this consist of noncovalent complexes of surface (gp120) and compound is contained within, or interacts with the HIV-1 transmembrane (gp41) subunits, both derived from a gp160 envelop gp120/gp41. This was the first report of a precursor which is oligomerized and cleaved during its nonpeptidic compound having this potential, since until transport to the cell surface [40]. The function of these now only monoclonal antibodies or peptides have been proteins is to mediate virus entry by allowing binding of shown to selectively affect the HIV-1 membrane fusion step virions to the cell surface and fusion of their lipidic [48, 49]. “Time of addition” experiments suggested that compound 10 interacted with an early step of HIV-1
The initial step of virus entry (binding) is mediated by replication. As syncytium formation but not virus-cell gp120, while gp41 is responsible for the membrane fusion binding, seem to be affected, these derivatives were assumed process itself. These events seem to be usually triggered by to interact with the post binding virus-cell fusion process the interaction of gp120 with two classes of cell surface molecules, CD4 and chemokine receptors, in particular Kanamoto et al. [50] examined the mechanism of anti- CCR5 or CXCR4, often viewed as HIV co-receptors [41]. In HIV action of the novel compound YK-FH312 (5b). To
vivo, strains using CXCR4 (termed X4 strains) or both determine the step(s) of HIV replication affected by 5b, a
CXCR4 and CCR5 (R5X4) are isolated at later stages of syncytium formation inhibition assay in MOLT-4/HIV-1 IIB infection, while strains using CCR5 (R5) are predominant at and MOLT-4 co-culture [51], a multinuclear activation of the early stages. The X4 strains, in particular, when adapted galactosidase indicator (MAGI) assay in MAGI-CCR5 cells to replication in T-cell lines, are characterized by a relatively [52], an electron microscopic observation [53] and a time of labile gp120-gp41 association, evidenced by the shedding of addition assay [54] were performed. In neither the syncytium gp120 spontaneously or upon contact with soluble CD4 or formation inhibition assay nor in the MAGI assay for de anti-gp120 antibodies [42], while the gp120-gp41 complex nova infection, did the compound show inhibitory effects of R5 strains seems comparatively stable [43].
against HIV replication. Conversely, no virions were Like other retroviral transmembrane proteins, gp41 detected in HIV-1 infected cell cultures treated with YK- comprises an N-terminal extracellular domain (ectodomain), FH312 either by electron microscopic observation or by viral a membrane-spanning domain, and C-terminal cytoplasmic yield in the supernatant. In accordance with a p24 enzyme domain, apparently dispensable for the fusion process [40].
linked immunosorbant assay of culture supernatant in the The main features of the ectodomain are a hydrophobic N- time of addition assay, 1/K-FH312 inhibited virus 662 Current Medicinal Chemistry, 2005, Vol. 12, No. 6
Yogeeswari and Sriram
expression in the supernatant when it was added 18h post in human melanoma cells as demonstrated by AnnexinV infection. However, western blot analysis of the cells in the binding and by the emergence of cells with apoptotic time of addition assay revealed that the production of viral characteristics and was more pronounced in human proteins in the cell was not inhibited completely by YK- melanoma cell lines than in normal human melanocytes.
FH312. These results suggest that YF-FH312 might affect Zuco et al. [60] studied the in vitro cytotoxicity of HIV at the step(s) of virion assembly and/or budding of betulinic acid in melanoma and non-melanoma tumor cell lines and compared with that of doxorubicin. It was also Holz-Smith et al. [55] studied the role of HIV-1 envelope tested on cell lines expressing a different p53 status.
in the anti-HIV activity of the derivative IC9564 (10).
Betulinic acid proved active in vitro against a panel of Compound 10 inhibited replication of both HIV-1 primary
neoplastic cell lines, including melanomas, small and non- isolates (DH 012, 89.6 and QZ 4734) and laboratory-adapted small cell lung carcinomas, ovarian and cervical carcinomas.
strains (NL4-3). DH 012 and 89.6 are dualtropic viruses that It exerted its antiproliferative activity on all the tested lines can use both CCR5 and CXCR4. In the virus infectivity in a very narrow range of doses (1.5-4.5 µg/ml), and was effective against wild-type p53 and mutant p53 neoplastic 90 of 10 for NL4-3 was 0.22 ± 0.05
cell lines derived from cancers clinically resistant to Zidovudine against NL4-3 in the same assay is 0.045 µM.
conventional antineoplastic drugs. In contrast, doxorubicin showed its cytotoxic activity in a larger range of 90s for DH012, QZ4734, and HIV-1 89.6 are >5, 2.65 and 1.84 µM, respectively. To test the antifusion concentrations (0.014-0.34 µg/ml). The growing colony activity, 10 was tested in a MOLT4/CEM-IIB fusion assay
inhibition assay indicated that betulinic acid exerted a system. The concentration of 10 required to completely
cytotoxic effect on two wild-type p53 cells (IGROV-1 and inhibit syncytium formation was 0.33 µM. The compound A2780) and one mutant p53 cell (ME665/2/60/), while it 10 did not significantly affect simian immunodeficiency virus
had a cytostatic effect on the mutant p53 cell line (SIV) or respiratory syncytial virus (RSV) replication at ME665/2/21. In the in vivo experiments on IGROV-1 concentrations up to 30 µM. The lack of activity against ovarian carcinoma xenografts, survival times of mice both SIV and RSV suggests that compound 10 specifically
receiving betulinic acid (100mg/kg intraperitoneally (i.p.) disrupts HIV-1 entry rather than a nonspecific charge-charge every 3-4 days for a total of six treatments) were significantly interaction or hydrophobic binding. Analysis of a chimeric higher (p<0.01) than those of controls; the survival time virus derived from exchanging envelope regions between increased from 16 ± 1.03 days in control mice to 22 ± 2.59 compound 10-sensitive and compound 10-resistant viruses
days in animals receiving betulinic acid.
indicated that regions within gp120 and the 25-amino acids Fulda et al. [61, 62] identified betulinic acid as a new at the N-terminus (fusion domain) of gp41 are key cytotoxic agent against neuroectodermal tumor cells determinants for the drug sensitivity. By developing a drug including neuroblastoma, medulloblastoma, glioblastoma resistant mutant from the NL4-3 virus, two mutations were and Ewing’s sarcoma cells, which represent the most found within the gp120 region (G237R and R252K) and one common solid tumors of childhood. Neuroblastoma cells was found within the fusion domain of gp41 region resistant to CD95- or doxorubicin-triggered apoptosis (R533A). The mutations were reintroduced into the NL4-3 remained sensitive to treatment with betulinic acid, and envelope and analyzed for their role in the resistance of betulinic acid exhibited potent antitumor activity on primary compound 10. Both of the gp120 mutations contributed to
tumor cell cultures from all neuroblastoma (4/4), all the drug sensitivity. On the contrary, the gp41 mutation (RS33A) did not appear to affect the compound 10
most glioblastoma patients (20/24) with an ED sensitivity. These results suggest that HIV-1 gp120 plays a µg/ml ex vivo. These findings suggest that betulinic acid key role in the anti-HIV-1 activity of compound 10.
may be a promising new lead in the treatment ofneuroectodermal tumors in vivo.
Jeong et al. [63] coupled the betulinic acid with a series of amino acids at the C-28 carboxylic acid portion and Betulinic acid was identified as a highly selective growth evaluated the cytotoxicity of the derivatives against cultured inhibitor of human melanoma, neuroectodermal and human melanoma (MEL-2) and human epidermal carcinoma malignant tumor cells and was reported to induce apoptosis of the mouth (KB) cell lines. Among the derivatives, the free in these cells. Anticancer agents with different modes of acid of the alanine (14b) and valine (14c) analogues showed
action have been reported to trigger apoptosis in toxicity against KB (ED50 = 4.6 and 9 µg/ml, respectively).
chemoselective cells [56]. Alterations of mitochondrial Methyl ester of 14b and 14c conjugates and the free acid of
functions such as permeability transition (PT) have been the glycine (14a) conjugate showed toxicity against MEL-2
found to play a major role in the apoptosis process including comparable to betulinic acid (ED50 = 3.5, 2.1, 4.2 and 4.2 cell death induced by chemotherapeutic agents [57, 58].
µg/ml respectively). The free acid of the 14b conjugate
showed the best toxicity profile (ED
Selzer et al. [59] studied the effect of betulinic acid alone MEL-2; however it also showed toxicity against KB (ED and in combination with irradiation in human melanoma cells. Betulinic acid strongly and consistently suppressed the µg/ml). Meanwhile the methyl ester of 14a, and
methionine (14f), tryptophan (14h), and alanine (14b)
growth and colony forming ability of all human melanoma analogues showed improved cytotoxicity against MEL-2 cell lines. In combination with ionizing radiation, the effect when converted to the corresponding free acid conjugates of betulinic acid on growth inhibition was additive in colony-forming assays. Betulinic acid also induced apoptosis 50 4.2-10.2 µg/ml, 9-12.9 µg/ml, 8.6->20 µg/ml and Betulinic Acid and Its Derivatives
Current Medicinal Chemistry, 2005, Vol. 12, No. 6 663
1.5-3.5 µg/ml, respectively). However, the methyl ester of prototype cytotoxic agent that triggers apoptosis by a direct the phenyl alanine (14g), leucine (14d), glutamic acid (14e)
effect on mitochondria [66]. In isolated mitochondria, and 14c analogues showed the loss of cytotoxicity against
betulinic acid directly induces a loss of transmembrane MEL-2 when converted to the corresponding free acid potential independent of a benzyloxycarbonyl-Val-Ala-Asp- conjugates (ED50 = 9-20 µg/ml, 6.2-9 µg/ml, 15.3->20 fluoromethyl ketone inhibitable caspase. This is inhibited by µg/ml and 2.1-9 µg/ml, respectively).
bongkrekic acid, an agent that stabilizes the PT porecomplex. Mitochondria undergo betulinic acid induced PTmediated cleavage of caspase-8 and caspase-3 in a cell-freesystem. Soluble factors such as cytochromeC or AIF(apoptosis-inducing factor) released from betulinic acid treated mitochondria are sufficient for cleavage of caspasesand nuclear fragmentation. Addition of cytochrome C to cytosolic extracts results in the cleavage of caspase-3, but notof caspase-8. However, supernatants of mitochondria, which have undergone PT, as well as partially purified AIF,activate both caspase-8 and caspase-3 in cytosolic extracts and suffice to activate recombinant caspase-8. These findingshow that the induction of mitochondrial PT alone is sufficient to trigger the full apoptosis program and that betulinic acid may induce apoptosis via a direct effect on ANTIBACTERIAL ACTIVITY
Fig. (6). C28 peptide derivatives of betulinic acid.
Betulinic acid extracted from the leaves of Vitex negundo Kim et al. [64] modified the C-20 alkene functional demonstrated antibacterial activity against Bacillus subtilis group of betulinic acid. The chemical modification at this at a concentration of 1000 µg/disc with a zone of inhibition position was initiated by converting the double bond to a of 18.8 mm2 [10]. Similarly betulinic acid and its three new ketone (15a) using a OsO
functionality was readily transformed to oximes (15b and
7β-(4-hydroxy-3’-methoxybenzoyloxy) betulinic acid and 15c). The compounds were evaluated for their cytotoxicity
27-(4-hydroxy-3’-methylbenzoyloxy) betulinic acid, which against the human colon carcinoma cell line HCY-116, and were isolated from the stem bark of Brazilian medicinal plant human melanoma cell lines M14-MEL, SK-MEL-2, and Zizyphus jaazerio [24], showed considerable activity against UACC-257. The results showed that when the double bond was oxidized to a ketone (15a), loss of cytotoxicity was
observed, suggesting that the presence of highly
electronegative oxygen atom may change the electrostatic
property of betulinic acid, rendering it less toxic. Converting
to oximes (15b and 15c) also appeared to result in the loss
The in vitro antiplasmodial activity (IC50) of betulinic of cytotoxicity, probably due to the same reason described acid isolated from the root bark of the Tanzanian tree against above. These results suggest that the cytotoxicity profile of Chloroquine-resistant (K1) and –sensitive (T9-96) betulinic acid derivatives may be sensitive to both the size of Plasmodium falciparum were found to be 19.6 µg/ml and substituent at the C-20 position and its electrostatic 25.9 µg/ml, respectively. The in vitro activity of the related triterpene betulin demonstrated no activity up to 500 mg/mlfor both K1 and T9-96 strains. When betulinic acid was tested for in vivo activity in a murine malaria model (P. berghi), the top dosage employed (250 mg/kg/day) wasineffective at reducing parasitemia and exhibited some Betulinic acid isolated from Triphyophyllum peltatum and Ancistrocladus heyneanus also exhibited moderate to good in vitro antimalarial activity against asexual erythrocytic stages of the human malaria parasite Fig. (7). C20 modified betulinic acid derivatives.

Betulinic acid isolated from Diospyros leucomelas Betulinic acid is a novel anticancer drug and induces showed anti-inflammatory activity in the Carrageenan and apoptosis and hence differs from “Classical” anticancer serotonin paw edema tests and TPA and EPP ear edema agents such as doxorubicin [65]. Betulinic acid is a tests [28]. Betulinic acid isolated form Ipomoea pes-caprae 664 Current Medicinal Chemistry, 2005, Vol. 12, No. 6
Yogeeswari and Sriram
showed pronounced antinociceptive properties in the decreased cell viability by 6%, which could be prevented by writhing test and formalin test in mice [26].
pretreatment with 2 µM U73122. Together, the resultssuggest that betulinic acid induced significant [Ca2+]iincreases in MDCK cells in a concentration-dependent ANTHELMINTIC ACTIVITY
manner, and also induced mild cell death.
Enwerem et al. [16] examined the anthelmintic activity Betulin and betulinic acid modified at the C-3 and C-28 of methanol, hexane and ethyl acetate extracts of Berlina positions have been evaluated in vitro for antiviral activity.
grandiflora, which contain betulinic acid as the major It was found that simple modifications of the parent structure component. Caenorhabditis elegans, a free living soil of lupane triterpenes produced highly effective agents against nematode, was used as an in vitro model in the study. A influenza A and herpes simplex type 1 viruses [68, 69].
suspension of worms was treated with the extracts. After Betulinic acid isolated from the ethanol extract of Tovomita seven days of incubation, activity was assessed in terms of krukovii was studied for its effect on aspartic proteases by number of worms exhibiting motility. The results showed Zhang et al. [14]. Specifically, it showed inhibitory effects that the crude extracts (500 ppm) showed antihelmintic against Candida albicans secreted aspartic protease with an activity in the order ethyl acetate > methanol > hexane.
Betulinic acid isolated from the ethyl acetate fraction showedstrong anthelmintic activity at 100 ppm comparable to METABOLIC TRANSFORMATION OF BETULINIC
Betulinic acid is currently undergoing preclinical development for the treatment or prevention of malignant Yamashita et al. [12] investigated the effect of betulinic melanomas. An important factor in the evaluation of the acid and betulin on the stimulus induced superoxide efficacy and safety of a drug is the study of its mammalian generation and tyrosyl phosphorylation of proteins in human metabolism. A prospective approach was undertaken to neutrophils. The various stimuli employed were N-formyl- study the metabolism of betulinic acid utilizing methionyl-leucyl-phenylalanine (fMLP), phorbal-12- microorganisms (particularly fungi) as in vitro model myristate-13-acetate (PMA) and arachidonic acid (AA). The systems to mimic and predict the metabolic fate and other fMLP-induced superoxide generation was remarkably xenobiotics in mammalian systems [70].
suppressed by betulin, while betulinic acid showed no effect.
Chatterjee et al. [71] reported the isolation and structural The PMA-induced superoxide generation was suppressed by elucidation of 28-O-β-D-glucopyranosy-3β-hydroxy-lup- betulin in a concentration-dependent manner, while the 20(29)-en-28-oate (16), a conjugate fungal metabolite of
efficiency was lower than that of the fMLP-induced betulinic acid, from resting cell suspensions of superoxide generation. The AA-induced superoxide Cunninghamella species NRRL5695. A total of 13 fungal generation was weakly enhanced by betulinic acid. The effect cultures were screened for the ability to catalyze the of these triterpenoids on the tyrosyl phosphorylation of bioconversion of betulinic acid. Cunninghamella species protein in fMLP-treated human neutrophils showed that NRRL569T was the only culture capable of reproducibly betulin suppressed the tyrosyl phosphorylation, but it was bioconverting 1 to the more polar metabolite 16. The in
vitro cytotoxicity assay of 16 revealed no activity against
Chou et al. [67] examined the effect of betulinic acid on intracellular free Ca2+ levels in Madin Darby canine kidney Kouzi et al. [72] studied the microbial transformation of cells (MDCK). It caused significant increase in [Ca2+]i betulinic acid utilizing three microorganisms. Bioconversion concentration dependently between 25 and 500 nM with an of betulinic acid (1) with resting-cell suspensions of
EC50 of 100 nM. The [Ca2+]i signal was composed of an Phenobarbital-induced Bacillus megaterium ATCC14581 initial gradual rise and a plateau. The response was decreased resulted in the production of the known betulinic acid (2)
by removal of extracellular Ca2+ by 45% ± 10%. In a Ca2+- and two new metabolites: 3β, 7β-dihydroxy-lup-20(29)-en- free medium, pretreatment with 1 µM thapsigargin (an 28-oic acid (17a) and 3β, 6α, 7β-trihydroxy-lup-20(29)-en-
endoplasmic reticulum Ca2+ pump inhibitor) abolished the 28-oic acid (17b). Biotransformation of 1 with the growing
betulinic acid –induced (250 µM) [Ca2+]i increase.
culture of Cunninghamella elegans ATCC9244 produced Conversely, pretreatment with betulinic acid only partly one new metabolite characterized as 1β, 3β, 7β-trihydroxy- inhibited the thapsigargin-induced [Ca2+]i increase. Addition lup-20(29)-en-28-oic acid (17c). Incubation of 1 with
of 3mM Ca2+ induced a [Ca2+]i increase after pretreatment growing cultures of Mucor mucedo UI-4605 afforded with 250 nM betulinic acid in a Ca2+-free medium for 5 metabolite 17a. The in vitro cytotoxicity of all the
min. This [Ca2+]i increase was not altered by the addition of compounds was evaluated against two human melanoma cell 20 µM of SKF96365 and 10 µM econazole, two drugs that lines, Mel-1 (lymph node) and Mel-2 (pleural fluid).
have been shown to inhibit capatative Ca2+ entry. Inhibiting Compared to 1 (ED
inositol 1,4,5-triphosphate formation with a phospholipase 50 = 3.3 and 1.0 µg/mL), metabolite 17c
C inhibitor U73122 (2 µM) abolished the betulinic acid- induced (250 nM) Ca2+ release. Pretreatment with 10 µM 50 = 17.1 and 7.2 µg/mL) and 17b (ED 50 = 10.9,
and 16.8 µg/mL) were less active against both Mel1 and La3+, inhibited betulinic acid-induced (250 nM) [Ca2+]i increases by 85%, whereas 10 µM of verapamil, nifedipineand diltriazem had no effect. Tryphan blue exclusion revealed Chatterjee et al. [73] studied the biotransformation of that acute exposure to betulinic acid (250 nM) for 2-3 min betulinic acid with a resting cell suspension of Bacillus Betulinic Acid and Its Derivatives
Current Medicinal Chemistry, 2005, Vol. 12, No. 6 665
Fig. (8). Microbial biotransformation products of betulinic acid.
megaterium ATCC13368, which resulted in the production Cole, B.J.W.; Bently, M.D. Holzforsch., 1991, 45, 265.
of four metabolites, which were identified as 3-oxo-lup- Kim, D.S.H.L.; Chen, Z.; Nguyen, V.T.; Pezzuto, J.M.; Qiu, S.;
Lu, Z. Synth. Commun., 1997, 27, 1607.
20(29)-en-28-oic acid (2), 3-oxo-11α-hydroxy-lup-20(29)-en-
Fujioka, T.; Kashiwada, Y.; Kilkuskie, R.E.; Consentino, L.M.; 28-oic acid (18a), and 3β, 7 β, 15α-trihydroxy-lup-20(29)-en-
Ballas, L.M.; Jiang, J.B.; Janzen, W.P.; Chen, I.S.; Lee, K.H. J. 28-oic acid (18b). Metabolites 2, 18a and 18b were more
Nat. Prod., 1994, 57, 243.
cytotoxic than betulinic acid against Mel-2 cell line.
Pisha, E.; Chai, H.; Lee, I.S.; Chagwedera, T.E.; Farnsworth,
N.R.; Cordell, G.A.; Beecher, C.W.; Fong, H.H.; Kinghorn, A.D.;
Brown, D.H. Nat. Med., 1995, 10, 1046.
Kwon, H.C.; Min, Y.D.; Kim, K.R.; Bang, E.J.; Lee, C.S.; Lee, DISTRIBUTION OF BETULINIC ACID
K.R. Arch. Pharm. Res., 2003, 26, 275.
Prakash, C.V.S.; Schilling, J.K.; Johnson, R.K.; Kingston, D.G. J.
Nat. Prod.
, 2003, 66, 419.
Udeani et al. [74] studied the pharmacokinetics and Woldemichael, G.M.; Singh, M.P.; Maiese, W.M.; Timmermann, tissue distribution of betulinic acid in CD-1 mice. The B.N. Z. Naturforsch., 2003, 58, 70.
results showed that after ip 250 and 500 mg/Kg dose, the Chandramu, C.; Manohar, R.D.; Krupadanam, D.G.; serum concentrations reached peaks at 0.15 and 0.23h, Dashavantha, R.V. Phytother. Res. , 2003, 17, 129.
Kim, D.K.; Nam, I.Y.; Kim, J.W.; Shin, T.Y.; Lim, J.P. Arch. respectively. The 250 and 500 mg/Kg betulinic acid i.p.
Pharm. Res., 2002, 25, 617.
doses were found to have elimination half-lives of 11.5 and Yamashita, K.; Lu, H.; Lu, J.; Chen, G.; Yokoyama, T.; Sagara, 11.8h and total clearances of 13.6 and 13.5 l/Kg/h, Y.; Manabe, M.; Kodama, H. Clin. Chim. Acta, 2002, 325, 91.
respectively. The pharmacokinetic parameters observed for De Oliveira, B.H.; Santos, C.A.; Espindola, A.P. Phytochem. i.p. betulinic acid 500 µg/Kg in the skin of mice were as Anal., 2002, 13, 95.
Zhang, Z.; Elsohly, H.N.; Jacob, M.R.; Pasco, D.S.; Walker, L.A.; follows: Ka (h-1) 0.257, K10 (h-1) 0.234, t1/2 (α) (h) 2.63, Clark, A.M. Planta Med., 2002, 68, 49.
t1/2 (β) (h) 20.2, V (l/Kg) 0.61, AUC (µg/mL) 3504, Tmax Guo, X.; Zhang, L.; Quan, S.; Hong, Y.; Sun, L.; Liu, M. Zhogguo (h) 3.90 and Cmax (µg/mL) 300.9. The distribution of Zhong Yao Za Zhi, 1998, 23, 546.
betulinic acid in tissues at 24h post i.p. administration in a Enwerem, N.M.; Okogun, J.I.; Wambebe, C.O.; okorie, D.A.;
Akah, P.A. Phytomed., 2001, 8, 112.
descending order was as follows: perirenal fat (2260 µg/g), Tezuka, Y.; Stampoulis, P.; Banskota, A.H.; Awale, S.; Tran, ovary (1998 µg/g), spleen (1287 µg/g), mammary gland K.Q.; Saiki, I.; Kadota, S. Chem. Pharm. Bull., 2000, 48, 1711.
(1184 µg), uterus (980 µg/g), and bladder, lymph node, Siddiqui, B.S.; Sultana, I.; Begum, S. Phytochem., 2000, 54, 861.
liver, small intestine, caecum, lung, thymus, colon, kidney, Kim, Y.K.; Yoon, S.K.; Ryu, S.Y. Planta Med., 2000, 66, 485.
Yi, J.H.; Zhang, G.L.; Li, B.G.; Chen, Y.Z. Phytochem., 2000, 53,
Recently, Cheng et al. [75] developed a robust assay Setzer, W.N.; Setzer, M.C.; Bates, R.B.; Jackes, B.R. Planta based on liquid chromatography/mass spectrometry to Med. , 2000, 66, 176.
Banskota, A.H.; Tezuka, Y.; Adnyana, I.K.; Xiong, Q.; Hase, K.; conduct a quantitative analysis of betulinic acid in mouse, Tran, K.Q.; Tanaka, K.; Saiki, I.; Kadota, S. Biol. Pharm. Bull., rat and dog plasma. At 15 and 25 µg/mL in mouse, rat or 2000, 23, 456.
dog plasma, betulinic acid was 99.99% bound to serum Ma, J.; Starck, S.R.; Hecht, S.M. J. Nat. Prod., 1999, 62, 1660.
proteins, and, at 5 µg/mL, betulinic acid was > or =99.97% Schuhly, W.; Heilmann, J.; Callis, I.; Sticher, O. Planta Med.,
1999, 65, 740.
bound following i.p.l or intravenous administration in vivo.
Steele, J.C.; Warhurst, D.C.; Kirby, G.C.; Simmonds, M.S.
Phytother. Res. , 1999, 13, 115.
Krogh, R.; Kroth, R.; Berti, C.; Madeira, A.O.; Sourza, M.M.;
Cechinel-Felho, V.; Delle-Monache, F.; Yunes, R.A. Pharmazie,
1999, 54, 464.
Maurya, S.K.; Devi, S.; Pandey, V.B. Fitother., 1989, 60, 468.
Bringmann, G.; Saeb, W.; Assi, L.A.; Francois, G.; Narayanan, O’Connel, M.M.; Bently, M.D.; Campbell, C.S. Phytochem., A.S.S.; Peters, K.; Peters, E.M. Planta Med., 1997, 63, 255.
1998, 27, 2175.
666 Current Medicinal Chemistry, 2005, Vol. 12, No. 6
Yogeeswari and Sriram
Recio, M.C.; Giner, R.M.; Manez, S.; Gueho, J.; Julien,H.R.; Kimpton, J.; Emerman, M. J. Virol., 1992, 66, 2232.
Hostettmann, K.; Rios, J.L. Planta Med., 1995, 61, 9.
Kohno, T.; Fujioka, Y.; Goto, T.; Morimatsu, S.; Morita, C.; Son, L.B.; Kaplan, A.P.; Spilevskii, A.A.; Andiia-Pravdivyi, I.; Nakano, T.; Sano, K. J. Virol. Methods, 1998, 72, 137.
Alekseeva, S.B.; Griborev, V.B.; Shvets, V.I. Bioorg. Khim., Nakashima, H.; Ichiyama, K.; Inaazawa, K.; Ito, M.; Hayashi, 1998, 24, 787.
H.; Nishihara, Y.; Tsujii, E.; Kino, T. Biol. Pharm. Bull., 1996, 19,
Fujoka, T.; Kashiwada, Y.; Kilkushi, R.E.; Consentino, L.M.; Ballas, L.M.; Jiang, J.B.; Janzen, W.P.; Chen, I.S.; Lee, K.H. J. Holz-Smith, S.L.; Sun, I.C.; Jin, L.; Matthews, T.J.; Lee, K.H.; Nat. Prod., 1994, 57, 243.
Chen, C.H. Antimicrob. Agents Chemother., 2001, 45, 60.
Mayaux, J.F.; Bousseau, A.; Pauwels, R.; Huet, T.; Henin, Y.; Fischer, D.E. Cell, 1994, 78, 539.
Dereu, N.; Evers, M.; Soler, F.; Poujado, C.; De Clercq, E.; Le Kroemer, G.; Zamzami, N.; Susin, S.A. Immunol. Today, 1997,
Pecq, J.B. Proc. Natl. Acad. Sci., 1994, 91, 3564.
Xu, H.X.; Zeng, F.Q.; Wan, M.; Sim, K.Y. J. Nat. Prod., 1996,
Decaudin, D.; Geley, S.; Hisch, T.; Castedo, M.; Marchelti, P.; Macho, A.; Kofler, R.; Kroemer, G. Cancer Res. , 1997, 57, 62.
Pengsuparp, T.; Cai, L.; Fong, H.H.S.; Kinghorn, A.D.; Pezzuto, Setzer, E.; Pimental, E.; Wacheck, V.; Schlegal, W.; J.M.; Wani, M.; Wall, M.E. J. Nat. Prod., 1994, 57, 415.
Pehamberger, H.; Jansen, B.; Kodym, R. J. Invest. Dermatol., Kashiwada, Y.; Hashimota, F.; Cosentina, L.M.; Chen, C.H.; 2000, 114, 935.
Garren, P.E.; Lee, K.H. J. Med. Chem., 1996, 39, 1016.
Zuco, V.; Supino, R.; Righeltis, S.C.; Cleris, L.; Marchesi, E.; Evers, M.; Poujade, C.; Soler, F.; Ribeill, Y.; James, C.; Lelievre, Passerini, C.G.; Formelli, F. Cancer Lett., 2002, 175, 17.
Y.; Gueguen, J.C.; Reisdorf, D.; Morize, I.; Pauwels, R.; De Fulda, S.; Jeremias, I.; Pietsh, T.; Debatin, K.M. Klin. Padiatr. , Clercq, E.; Henin, Y.; Bousseau, A.; Mayaux, J.F.; Le Pecq, J.B.; 1999, 211, 319.
Dereu, N. J. Med. Chem., 1996, 39, 1056.
Fulda, S.; Jeremias, I.; Steiner, H.H.; Pietsch, T.; Debatin, K.M.
Soler, F.; Poujade, C.; Evers, M.; Carry, J.C.; Henin, Y.; Int. J. Cancer, 1999, 82, 435.
Bousseau, A.; Huet, H.; Pauwels, R.; De Clercq, E.; Mayaux, J.F.; Jeong, H.J.; Chai, H.B.; Park, S.Y.; Kim, D.S.H.L. Bioorg. Med. Le Pecq, J.B.; Dereu, N. J. Med. Chem., 1996, 39, 1069.
Chem. Lett., 1999, 9, 1201.
Sun, I.C.; Wang, H.K.; Kashiwada, Y.; Shen, J.K.; Cosentino, Kim, J.Y.; Koo, H.M.; Kim, D.S.H.L. Bioorg. Med. Chem. Lett., L.M.; Chen, C.H.; Yang, L.M.; Le, K.H. J. Med. Chem., 1998,
2001, 11, 2405.
Fulda, S.; Friesen, C.; Los, M.; Scaffidi, C.; Mier, W.; Benedict, Kashiwada, Y.; Chiyo, J.; Ikeshino, Y.; Nagao, T.; Okabe, H.; M.; Nunez, G.; Krammer, P.H.; Peter, M.E.; Debatin, K.M.
Cosentino, L.M.; Fowke, K.; Lee, K.H. Bioorg. Med. Chem. Lett. , Cancer Res. , 1997, 57, 4956.
2001, 11, 183.
Fulda, S.; Scaffidi, C.; Susin, S.A.; Krammer, P.H.; Kroemer, G.; Sun, I.C.; Chen, C.H.; Kashiwada, Y.; wu, J.H.; Wang, H.K.; Peters, M.E.; Debatin, K.M. J. Biol. Chem., 1998, 273, 33942.
Lee, K.H. J. Med. Chem., 2002, 45, 4271.
Chou, K.J.; Fang, H.C.; Chung, H.M.; Cheng, J.S.; Lee, K.C.; Freed, E.O.; Martin, M.A. J. Biol. Chem., 1995, 270, 23883.
Tseng, L.L.; Tang, K.Y.; Jan, C.R. Eur. J. Pharmacol. , 2000, 408,
Berger, E.A.; Murphy, P.M.; Farber, J.M. Annu. Rev. Immunol., 1999, 17, 657.
Baltina, L.A.; Flekhter, O.B.; Nigmatullina, L.R.; Boreko, E.I.; Sattentau, Q.J.; Moore, J.P. J. Exp. Med., 1991, 174, 407.
Pavlova, N.I.; Nikolaeva, S.N.; Savinova, O.V.; Tolstikov, G.A.
Moore, J.P.; McKeating, J.H.; Huang, Y.; Ashkenazi, A.; Ho, Bioorg Med. Chem. Lett., 2003, 13, 3549.
D.D. J. Virol., 1992, 66, 235.
Pavlova, N.I.; Savinova, O.V.; Nikolaeva, S.N.; Boreko, E.I.; Gallaher, W.R.; Ball, J.M.; Garry, R.T.; Grittin, M.C.; Montelaro, Flekhter, O.B. Fitother., 2003, 74, 489.
R.C. AIDS Res. Hum. Retroviruses, 1989, 67, 5939.
Clark, A.M.; Hufford, C.D. Med. Res. Rev., 1991, 11, 473.
Helseth, E.; Olshevsky, U.; Furman, C.; Sodroski, J. J. Virol., Chatterjee, P.; Pezzuto, J.M.; Kouzi, S.A. J. Nat. Prod., 1999, 62,
1991, 65, 2119.
Chan, D.C.; Fass, D.; Berger, J.M.; Kim, P.S. Cell, 1997, 89, 263.
Kouzi, S.A.; Chatterjee, P.; Pezzuto, J.M.; Hamann, M.T. J. Nat. Caffrey, M.; Cai, M.; Kaufman, J.; Stahl, S.J.; Wingfield, P.T.; Prod., 2000, 63, 1653.
Covell, D.G.; Gronenborn, A.M.; Clore, G.M. EMBOJ, 1998, 17,
Chatterjee, P.; Kouzi, S.A.; Pezzuto, J.M.; Hamann, M.T. Appl. Environ. Microbiol., 2000, 66, 3850.
Freed, E.O.; Myers, D.J.; Risser, R. J. Virol., 1991, 63, 190.
Udeani, G.O.; Zhao, G.M.; Shin, Y.G.; Cooke, B.P.; Graham, J.; Wild, C.; Oas, T.; Mc Danal, C.; Bolognesi, D.; Matthews, T.
Beecher, C.W.W.; Kinghorn, A.D.; Pezzuto, J.M. Biopharm. Proc. Natl. Acad. Sci. USA , 1992, 89, 10537.
Drug Dispos., 1999, 20, 379.
Kanamoto, T.; Kashiwada, Y.; Kanbara, K.; Gotoh, K.; Cheng, X.; Shin, Y.G.; Levine, B.S.; Smith, A.C.; Tomaszewski, Yoshimori, M.; Goto, T.; Sano, K.; Nakashima, H. Antimicrob. J.E.; Van Breemen, R.B. Rapid Commun. Mass Spectrum., 2003,
Agents Chemother., 2001, 45, 1225.
Tochikura, T.S.; Nakashima, H.; Tanabe, A.; Yamamoto, N.
Virol., 1988, 164, 542.


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