Enhancing the divergent activities of betulinic acid via neoglycosylation

Article abstract of DOI:10.1021/ol8025704

(Chemical Equation Presented) Using neoglycosylation, the impact of differential glycosylation upon the divergent anticancer and anti-HIV properties of the triterpenoid betulinic acid (BA) was examined. Each member from a library of 37 differentially glycosylated BA variants was tested for anticancer and anti-HIV activities. Enhanced analogs for both desired activities were discovered with the corresponding antitumor or antiviral enhancements diverging, on the basis of the appended sugar, into two distinct compound subsets.

Full text of DOI:10.1021/ol8025704

ORGANIC
LETTERS
2009
Vol. 11, No. 2
461-464
Enhancing the Divergent Activities of
Betulinic Acid via Neoglycosylation
Randal D. Goff and Jon S. Thorson*
UniVersity of Wisconsin National CooperatiVe Drug DiscoVery Group, Pharmaceutical
Sciences DiVision, School of Pharmacy, UniVersity of Wisconsin-Madison, 777
Highland AVe., Madison, Wisconsin 53705
jsthorson@pharmacy.wisc.edu
Received November 7, 2008
ABSTRACT
Using neoglycosylation, the impact of differential glycosylation upon the divergent anticancer and anti-HIV properties of the triterpenoid
betulinic acid (BA) was examined. Each member from a library of 37 differentially glycosylated BA variants was tested for anticancer and
anti-HIV activities. Enhanced analogs for both desired activities were discovered with the corresponding antitumor or antiviral enhancements
diverging, on the basis of the appended sugar, into two distinct compound subsets.
The sugars attached to pharmaceutically important natural
products often dictate key pharmacological properties and/
or molecular mechanisms of action.1 While there is also
precedent for improving nonglycosylated natural product-
based therapeutics via glycoconjugation, including colchi-
cine,2 mitomycin,3 podophyllotoxin,4 rapamycin,5 or taxol,6
studies designed to systematically understand and/or exploit
the role of carbohydrates in drug discovery are often limited
by the availability of practical synthetic and/or biosynthetic
tools.7 Neoglycosylation takes advantage of a chemoselective
reaction between free reducing sugars and N-methoxyamino-
substituted acceptors.8 This reaction has enabled the process
of “neoglycorandomization” wherein alkoxyamine-appended
natural-product-based drugs are differentially glycosylated
with a wide array of natural and unnatural reducing sugars.2,9
(7) (a) Salas, J. A.; Me´ndez, C. Trends Microbiol. 2007, 15, 119–232.
(b) Thibodeaux, C. J.; Melanc¸on, C. E.; Liu, H. Nature 2007, 446, 1008–
1016. (c) Blanchard, S.; Thorson, J. S. Curr. Opin. Chem. Biol. 2006, 10,
263–271. (d) Griffith, B. R.; Langenhan, J. M.; Thorson, J. S. Curr. Opin.
Biotechnol. 2005, 16, 622–630. (e) Langenhan, J. M.; Griffith, B. R.;
Thorson, J. S. J. Nat. Prod. 2005, 68, 1696–1711.
ˇ
(1) (a) Krˇen, V.; Rezanka, T. FEMS Microbiol. ReV. 2008, 32, 858–
889. (b) Thorson, J. S.; Hosted, T. J., Jr.; Jiang, J.; Biggins, J. B.; Ahlert,
J. Curr. Org. Chem. 2001, 5, 139–167. (c) Weymouth-Wilson, A. C. Nat.
Prod. Rep. 1997, 14, 99–110.
(8) (a) Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269–
12278. (b) Peri, F.; Deutman, A.; La Ferla, B.; Nicotra, F. Chem. Commun.
2002, 1504–1505. (c) Carrasco, M. R.; Nguyen, M. J.; Burnell, D. R.;
MacLaren, M. D.; Hengel, S. M. Tetrahedron Lett. 2002, 43, 5727–5729.
(d) Carrasco, M. R.; Brown, R. T. J. Org. Chem. 2003, 68, 8853–8858. (e)
Carrasco, M. R.; Brown, R. T.; Serafimova, I. M.; Silva, O. J. Org. Chem.
2003, 68, 195–197. (f) Peri, F. MinireV. Med. Chem. 2003, 3, 658–665. (g)
Peri, F.; Jime´nez-Barbero, J.; Garc´ıa-Aparicio, V.; Tvaroˇska, I.; Nicotra,
F. Chem. Eur. J. 2004, 10, 1433–1444. (h) Peri, F.; Nicotra, F. Chem.
Commun. 2004, 623–627. (i) Langenhan, J. M.; Thorson, J. S. Curr. Org.
Synth. 2005, 2, 59–81. (j) Carrasco, M. R.; Brown, R. T.; Doan, V. H.;
Kandel, S. M.; Lee, F. C. Biopolymers 2006, 84, 414–420. (k) Nicotra, F.;
Cipolla, L.; Peri, F.; La Ferla, B.; Redaelli, C. AdV. Carb. Chem. Biochem.
2008, 61, 363–410.
(2) Ahmed, A.; Peters, N. R.; Fitzgerald, M. K.; Watson, J. A., Jr.;
Hoffmann, F. M.; Thorson, J. S. J. Am. Chem. Soc. 2006, 128, 14224–
14225.
(3) Ghiorghis, A.; Talebian, A.; Clarke, R. Cancer Chemother. Phar-
macol. 1992, 29, 290–296.
(4) Imbert, T. F. Biochimie 1998, 80, 207–222.
(5) Abel, M.; Szweda, R.; Trepanier, D.; Yatscoff, R. W.; Foster,
R. T.Rapamycin carbohydrate derivatives. U.S. Patent 7,160,867, January
9, 2007.
(6) Liu, D.; Sinchaikeul, S.; Reddy, P. V. G.; Chang, M.; Chen, S.
Bioorg. Med. Chem. Lett. 2007, 17, 617–620.
10.1021/ol8025704 CCC: $40.75
Published on Web 12/22/2008
2009 American Chemical Society

Neoglycorandomization has led to increases in anticancer
efficacy of the cardenolide digitoxin,9a,c mechanistic alter-
ation and improvements in the synergistic effects of the
nonglycosylated alkaloid colchicine,2 and enhancements in
the potency of the glycopeptide vancomycin against antibiotic-
resistant organisms.9b
Pichette and coworkers revealed that the attachment of
saccharides at C3 moderately improved the antiproliferative
activity (up to 4-fold) and selectivity of BA in a sugar-
dependent manner.15 However, only D-Ara, D-Gal, D-Glc,
D-Man, L-Rha, and D-Xyl were employed in this initial study,
and antiviral activity was not assessed. To more systemati-
cally assess the impact of BA glycosylation upon both
anticancer activity/selectivity and antiviral activity in parallel,
herein we report the synthesis and anticancer/antiviral
activities of a 37-member library of BA C3-neoglycosides.
Our findings indicate that groups of BA derivatives with
improved antitumor or antiviral properties are divergent and
sugar-dependent.
The initial strategy for methoxyamine handle installation
at C3 involved reduction of imine 4 (created from 2)14a using
BH3·t-BuNH2 to give a 3:1 ratio of desired (5) to undesired
diastereomers (see Scheme 1). However, neoglycosylation
Figure 1. Structures of betulinic acid (1), betulin (2), and
3-aminobetulinic acid (3).
Scheme 1. Attempted Direct Neoglycosylation of Betulinic Acid
Many natural products are known to exhibit multiple, diverse
biological activities.10 To assess the impact of differential
glycosylation upon a natural product with known multiple
activities, we selected the lupane-type triterpernoid betulinic acid
(BA, 1) as a model. BA and its reduced form (betulin, 2) exhibit
a wide variety of biological functions, the most prevalent of
which are anticancer and anti-HIV activities.11 In cancer cells,
BA induces apoptosis through multiple mechanisms, including
disruption of the mitochondrial membrane potential and sup-
pression of vascular endothelial growth factor and survivin
proteins.12 Although the exact mechanism of BA anti-HIV
activity has yet to be elucidated, many BA analogs disrupt viral
fusion to host cells through interference with the gp41 viral
glycoprotein or function as inhibitors of the late stage of capsid
protein maturation.13
Although BA derivatization (primarily at C3 and/or C28)
has yielded anti-HIV or antitumor enhancements,11,14 few
glycosylated BAs have been pursued or studied. Studies by
of 5 failed possibly because of the hindered adjacent C4
dimethyl substitution. Consistent with this, aglycon 5 was
also resistant to acetylation conditions (i.e., Ac2O, DMAP,
refluxing pyridine).
(9) (a) Langenhan, J. M.; Peters, N. R.; Guzei, I. A.; Hoffman, M. A.;
Thorson, J. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 12305–12310. (b)
Griffith, B. R.; Krepel, C.; Fu, X.; Blanchard, S.; Ahmed, A.; Edmiston,
C. E.; Thorson, J. S. J. Am. Chem. Soc. 2007, 129, 8150–8155. (c)
Langenhan, J. M.; Engle, J. M.; Slevin, L. K.; Fay, L. R.; Lucker, R. W.;
Smith, K. R.; Endo, M. M. Bioorg. Med. Chem. Lett. 2008, 18, 670–673.
(10) (a) Deorukhkar, A.; Krishnan, S.; Sethi, G.; Aggarwal, B. B. Exp.
Opin. InVest. Drugs 2007, 16, 1753–1773. (b) Butler, M. S. Nat. Prod.
Rep. 2008, 25, 475–516. (c) Udenigwe, C. C.; Ramprasath, V. R.; Aluko,
R. E.; Jones, P. J. H. Nutr. ReV. 2008, 8, 445–454. (d) Saladino, R.;
Gualandi, G.; Farina, A.; Crestini, C.; Nencioni, L.; Palamara, A. T. Curr.
Med. Chem. 2008, 15, 1500–1519.
Previously, colchicine neoglycosylation was enabled by
replacing the natural colchicine N-acetyl group with N-(N′-
methoxyglycine).2 While not a direct neoglycosylation of
BA, we postulated that a similar methoxyglycine handle
would distance the hindered BA C4 quaternary center from
the requisite neoglycosylation alkoxyamine. Toward this
goal, 1 (prepared in three steps from 2)16 was esterified at
the C3 hydroxyl group using chloroacetyl chloride in the
presence of DMAP. Under Finklestein conditions, the
chloride (6) was exchanged with iodide to facilitate the SN2
(11) For reviews, see: (a) Cichewicz, R. H.; Kouzi, S. A. Med. Res.
ReV. 2004, 24, 90–114. (b) Yogeeswari, P.; Sriram, D. Curr. Med. Chem.
2005, 12, 657–666. (c) Sami, A.; Taru, M.; Salme, K.; Jari, Y.-K. Eur.
J. Pharm. Sci. 2006, 29, 1–13. (d) Fulda, S. Int. J. Mol. Sci. 2008, 9, 1096–
1107.
(12) (a) Schmidt, M. L.; Kuzmanoff, K. L.; Ling-Indeck, L.; Pezzuto,
J. M. Eur. J. Cancer 1997, 33, 2007–2010. (b) Fulda, S.; Jeremias, I.;
Steiner, H. H.; Pietsch, T.; Debatin, K.-M. Int. J. Cancer 1999, 82, 435–
441. (c) Chintharlapalli, S.; Papineni, S.; Ramaiah, S. K.; Safe, S. Cancer
Res. 2007, 67, 2816–2823.
(14) (a) Kim, D. S. H. L.; Pezzuto, J. M.; Pisha, E. Bioorg. Med. Chem.
Lett. 1998, 8, 1707–1712. (b) Kashiwada, Y.; Chiyo, J.; Ikeshiro, Y.; Nagao,
T.; Okabe, H.; Cosentino, L. M.; Fowke, K.; Lee, K. H. Bioorg. Med. Chem.
Lett. 2001, 11, 183–185.
(15) (a) Gauthier, C.; Legault, J.; Lebrun, M.; Dufour, P.; Pichette, A.
Bioorg. Med. Chem. 2006, 14, 6713–6725. (b) Thibeault, D.; Gauthier, C.;
Legault, J.; Bouchard, J.; Dufour, P.; Pichette, A. Bioorg. Med. Chem. 2007,
15, 6144–6157.
(13) (a) Li, F.; Goila-Gaur, R.; Salzwedel, K.; Kilgore, N. R.; Reddick,
M.; Matallana, C.; Castillo, A.; Zoumplis, D.; Martin, D. E.; Orenstein,
J. M.; Allaway, G. P.; Freed, E. O.; Wild, C. T. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 13555–13560. (b) Aiken, C.; Chen, C. H. Trends Mol.
Med. 2005, 11, 31–36.
(16) Kim, D. S. H. L.; Chen, Z.; Nguyen, V. T.; Pezzuto, J. M.; Qiu,
S.; Lu, Z.-Z. Synth. Commun. 1997, 27, 1607–1612.
462
Org. Lett., Vol. 11, No. 2, 2009

displacement by methoxyamine in the same reaction vessel
(see Scheme 2). This three-step procedure provided aglycon
Scheme 2. Synthesis of Betulinic Acid Neoglycosides
7 in good yield (58%), a marked improvement over the
previous colchicine N′-methoxyglycine incorporation strategy
(eight steps, 40% yield).2
Optimal neoglycosylation conditions of 7 were identified
using L-ribose (see Table S1, Supporting Information),
validating for the first time an ester-linked neoglycosylation
handle. In contrast to the typical DMF/acetic acid (3:1)
neoglycosylation solvent system,2,8,9 we found 6:1 MeOH/
CH2Cl2 to be optimal. Notably distinct from prior neogly-
cosylation applications,2,8,9 an external proton source was
also unnecessary, likely because of the intrinsic carboxylic
acid of 7. Production of the corresponding neoglycoside
library (BA1-32, see Figure S1, Supporting Information)
employed similar conditions (90 µM aglycon, 3 equiv of
sugar, 40 °C, 48 h), with an average isolated yield of 33%.
Unlike previously reported libraries that revealed a predomi-
nance of ꢀ-anomers,8,9 the anomeric bias in the context of
BA neoglycosylation was not as strong (see Table S2,
Supporting Information).
Figure 2. (a) Divergent activity of betulinic acid neoglycosides
against HIV-1-infected CEM-SS cells and A549 cancer cells
compared to that of parent 1. (b) Structures of most-active
neoglycosides and their anomeric ratios (R:ꢀ).
The cytoxicity of the library members was assessed in seven
human cancer cell lines representing a broad range of carcino-
mas including breast, colorectal, CNS, lung, and prostate. Two
standards, 1 (the parent natural product) and 2 (betulin), were
also examined. Eleven library members displayed IC50 values
below a threshold of 25 µM (∼2-3-fold the activity of 1) in at
least one cell line, four of which (D-alloside BA1, D-altroside
BA3, L-fucoside BA9, and L-xyloside BA32) were equipotent
to the parent in one or more cell lines (see Figure 2 and Table
S3, Supporting Information).
To assess the impact of the ester linker on this activity,
a subset of representative amide-linked neoglycosides was
subsequently synthesized. This group was designed to
represent diverse sugar structures and a range of potencies
(as defined by the ester-linked series), specifically, one
equipotent hexose (D-altrose), a “lower threshold” (IC50
∼10-20 µM) pentose (D-xylose), an “upper threshold”
(IC50 ∼15-25 µM) hexosuronic acid (D-glucuronate), and
a representative threshold (IC50 g 25 µM) pentose (L-
ribose) and deoxyhexose (D-fucose). To circumvent the
need for BA C28 acid protection during C3 acylation,16
N-hydroxysuccinimidyl ester 8 was reacted with 3 in
pyridine (see Scheme 3) and the resulting imine (9) was
reduced with BH3·Et3N complex in the presence of
ethanolic HCl to provide aglycon 10 in good yield (85%).
Neoglycosylation of 10 was performed as described above
for ester 7, employing identical conditions to produce
ABA1-5 (see Figure S1, Supporting Information) with an
average isolated yield of 40%. These five compounds had
a notably decreased bias toward the ꢀ-anomer and as
Org. Lett., Vol. 11, No. 2, 2009
463

To assess the corresponding impact of differential glyco-
sylation upon antiviral activity, the entire set of compounds
was subsequently tested in a single-dose (10 µM) anti-HIV-1
assay against CEM-SS cells (i.e., CD4+ T lymphocytes)
infected with the IIIB strain of HIV-1. Compound efficacy
was determined by the percent increase of cytoprotective
effect (CPE), which is prevention of intercellular virus
replication, over untreated HIV-1-infected cells. Under these
conditions, 19 of 32 ester-linked compounds displayed at
least a 2-fold improvement over parent 1 with seven
(D-alloside BA1, L-alloside BA2, D-fucoside BA8, L-fucoside
BA9, 3- and 6-deoxy-D-glucoside BA17 and BA18 respec-
tively, and L-xyloside BA32) displaying g10-fold enhance-
ments (the most active being ∼20-fold better than parent 1,
see Figure 2 and Table S4, Supporting Information). It is
important to note that at 10 µM host cell cytotoxicity of the
ester series was not a contributing factor as only 4 of the 32
esters led to g10% reduction of host cell viability. In
contrast, antiviral activity could not be achieved without
significant host cell cytotoxicity with amides ABA1-5 or
aglycon 10. Dilution of the amide series to 1 µM abated
host cell cytotoxicity, yet only D-glucuronide ABA3 dem-
onstrated any significant CPE (32% increase in CPE), while
10 had no noticeable activity at 1 µM (see Table S4,
Supporting Information).
In summary, neoglycosylation has enabled the study of
the influence of glycodiversification upon the divergent
activities of BA. This study revealed distinct sets of sugars
to discretely augment either the anticancer or anti-HIV
activity of BA, the more dramatic of the two being the latter.
Whereas the anticancer or anti-HIV activites of BA neogly-
cosides were predominately dictated by the appended sugar,
the nature of the alkoxyamine handle connection to the
scaffold (i.e., ester versus amide) also appeared to contribute
to the divergence of the mode of action. As a first application
of neoglycosylation toward a triterpenoid and the first
installation of the methoxyamine handle via a linker strategy,
this study also significantly extends the utility of neoglyco-
sylation as a tool for natural product glycodiversification.
Scheme 3. Synthesis of 3-Aminobetulinic Acid Neoglycosides
expected displayed the same anomeric ratios as their ester
analogs (see Table S2, Supporting Information).
The cytoxicity of ABA1-5 was evaluated in five human
cell lines of breast, colorectal, lung, and prostate carci-
nomas. Interestingly, although aglycons 7 and 10 displayed
potencies relatively similar to that of parent 1, the
activities of four (D-altroside ABA1, D-fucoside ABA2,
L-riboside ABA4, and D-xyloside ABA5) of the five
glycosylated ABA subset were slightly improved over the
parent natural product (∼2-fold). A comparison of the
ester-linked and amide-linked series also revealed clear
improvements (2- to g5-fold) with these same four sugars
in the ABA group (see Figure 2 and Table S3, Supporting
Information). While it is tempting to attribute this amide-
versus-ester trend simply to the potential stability differ-
ences of the neoglycosylation linkers, the differences in
magnitude of potency improvement (e.g., e2-fold for
D-xylosides BA31 versus ABA5 compared to g6-fold for
L-ribosides BA29 versus ABA4) or cell line selectivity
(e.g., a reversal in potency trend between amide series
ABA1/ABA3/ABA5 and ester series BA3/BA20/BA31 in
HT-29) may contradict this straightforward explanation.
It is also important to note that there appears to be no
correlation between the current study and the previous
Pichette O-glycoside study. For example, the best sugar
in the context of direct C3-O-glycosylation, L-rhamnose,15
was inactive in the context of an ester-linked neoglycoside
(BA27, g25 µM). In a similar manner, one of the best
sugars in the context of either neoglycosylation approach,
D-xylose (BA31 and ABA5), led to a slight decrease in
potency as the C3-O-glycoside.15
Acknowledgment. We thank the School of Pharmacy
Analytical Instrumentation Center for analytical support and
the Keck UWCCC Small Molecule Screening Facility for
cancer cell line cytotoxicity screening. This work was
supported by the NIH (U19 CA113297 and AI52218).
Supporting Information Available: Experimental pro-
cedures, figures and characterization of BA1-32 and
ABA1-5, comprehensive assay data, and NMR spectra. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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