De novo asymmetric synthesis and biological evaluation of the trisaccharide portion of PI-080 and vineomycin B2

Article abstract of DOI:10.1021/ol801817f

(Chemical Equation Presented) A highly enantio- and diastereoselective synthesis of an α-L-aculose, α-L-rhodinose, and β-D-olivose trisaccharide is described. The key transformations include the palladium-catalyzed glycosylation, Myers' reductive rearrang

Full text of DOI:10.1021/ol801817f

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4529-4532
De Novo Asymmetric Synthesis and
Biological Evaluation of the Trisaccharide
Portion of PI-080 and Vineomycin B2
Xiaomei Yu and George A. O’Doherty*
Department of Chemistry, West Virginia UniVersity, Morgantown, West Virginia 26506
george.odoherty@mail.wVu.edu
Received August 5, 2008
ABSTRACT
A highly enantio- and diastereoselective synthesis of an r-L-aculose, r-L-rhodinose, and ꢀ-D-olivose trisaccharide is described. The key
transformations include the palladium-catalyzed glycosylation, Myers’ reductive rearrangement, diastereoselective dihydroxylation, and
regioselective Mitsunobu inversion. Significant apoptotic antitumor activity was found for this trisaccharide, which has implication for vineomycin
B2 and PI-080 structure-activity relationship.
Of the anthracycline antibiotics, the angucycline family of
antibiotics has been of particular interest to both the synthetic
and biological communities due to their unique structures
and potent antitumor and antibacterial activities.1 The
structural diversity of this class of natural products is typified
by two different C-glycoside natural products: PI-080 (1)2
and vineomycin B2 (2).3 Both PI-080 and vineomycin B2
share a ꢀ-C-glycoside linked R-L-aculose, R-L-rhodinose (4),
and ꢀ-D-olivose trisaccharide subunit, whereas PI-080 has
an additional R-L-aculose-bis-R-L-rhodinose trisaccharide (3).
In addition to representing a range of structures (Figure 1),
the angucycline family of antibiotics possesses a range of
biological activities. The antibiotic vineomycin B2 displays
(1) (a) Rohr, J.; Thiericke, R. Nat. Prod. Rep. 1992, 9, 103–137. (b)
Krohn, K.; Rohr, J. Top. Curr. Chem. 1997, 188, 127–195. (c) Andriy, L.;
Andreas, V.; Andreas, B. Mol. BioSyst. 2005, 1, 117–126.
Figure 1. Structures of PI-080 and vineomycin B2.
(2) Two natural products have been isolated with the same structure as
PI-080. For PI-080, see: (a) Kawashima, A.; Kishimura, Y.; Tamai, M.;
Hanada, K. Chem. Pharm. Bull. 1989, 37, 3429–3431. For PI-6621, see:
(b) Kawashima, A.; Yoshimura, Y.; Kamigori, K.; Yagishi, M.; Mizutani,
T. Chem. Abstr. 1989, 114, 22464d.
potent antitumor activity,3,4 whereas PI-080 strongly inhibits
blood coagulation.2
(3) For the isolation of vineomycin B2, see: (a) Omura, S.; Tanaka, H.;
Oiwa, R.; Awaya, L.; Masuma, R.; Tanaka, K. J. Antibiot. 1977, 30, 908–
916. (b) Imamura, N.; Nakinuma, K.; Ikekawa, N.; Tanaka, H.; Omura, S.
J. Antibiot. 1981, 34, 1517–1518.
While the antitumor activity of the angucyclines has been
attributed to the anthraquinone portion of natural products,4 the
10.1021/ol801817f CCC: $40.75
Published on Web 09/12/2008
2008 American Chemical Society

anticoagulant activity of PI-080 appears to be associated with
one of the trisaccharide portions of the molecule (i.e., the
trisaccharide 3).2 As part of a larger effort to use the de novo
synthesis of carbohydrates for medicinal chemistry, we became
interested in elucidating the structural origins of the anticancer
activity of vineomycin B2 (Scheme 1). In particular, we were
interested in determining whether any of the antitumor activity
associated with vineomycin B2 could be assigned to the
trisaccharide subunit 4 (R ) OPMB).
Scheme 2
.
Retrosynthetic Analysis of the Vineomycin B2
Trisaccharide
There are no reports of the total synthesis of PI-080 and
vineomycin B2,5 however the synthesis of the trisaccharide
portion 3 has been completed by Sulikowski.6 While the
trisaccharide 3 could be obtained from natural sources, access
to the trisaccharide subunit 4 (R ) OPMB) required synthesis.
Recently, we had some success using our Pd-catalyzed glyco-
sylation and post-glycosylation reactions for the synthesis of
several naturally occurring rare sugar structural motifs.7 Herein
we describe the de novo synthesis of trisaccharide subunit 4
from commercially available acylfuran. In addition, we report
its antiproliferative activity toward several tumor cells lines and
show this activity occurs via apoptosis at 50 µM and necrosis
at higher concentration.
4 as coming from ꢀ-D-pyranone 6 (Scheme 2). The two
pyranones could be prepared from the corresponding enantio-
meric furyl alcohols, which in turn could be prepared by a
Noyori reduction of 2-acylfuran (Scheme 2).
Previously, we have shown that the required Pd glycosyl
donors R-L-pyranones 5 and ꢀ-D-pyranones 6, as well as their
enantiomers, could be prepared from acyl furan 8 (Scheme 3).
The stereodivergent route employed an enantioselective Noyori
reduction (8 to 7/ent-7),8,9 an Achmatowicz oxidation, and
diastereoselective tert-butyl carbonate formation (Scheme
3).7,10 The ꢀ-pyranone 6 could be isolated in 50% when the
Boc protection was performed at elevated temperature
((Boc)2O/NaOAc in benzene at 80 °C). Alternatively,
R-pyranone 5 could be selectively prepared in 62% (R:ꢀ )
3:1) at low temperature (-78 °C).
Scheme 1
.
Structures of PI-080 and Vineomycin B2 and Their
Trisaccharide Motifs 3 and 4
Scheme 3. Approach to D/L-, R/ꢀ-Boc-Pyranones 5 and 6
With a practical route to the desired pyranone glycosyl
donors, we next turned our attention toward the preparation of
2-deoxy-ꢀ-L-olivose ring of the trisaccharide 4. Previously, we
reported an approach to 2-deoxy-ꢀ-glycoside associated with
allo- and gluco-stereochemistry.7,11 Key to the successful
implementation of this strategy is the selective Mitsunobu-
like inversion of the allo-stereochemistry of the digitoxose
sugar 12 into olivose sugar 13 (Scheme 4).
Retrosynthetically, we envisioned the R-L-aculose and R-L-
rhodinose subunits of trisaccharide 4 as coming from two R-L-
pyranones (5). Similarly, we conceived the ꢀ-D-olivose ring of
(4) Both vineomycin B2 and the related monosaccharide angucycline,
vineomycinone B2 (2 sans R-L-aculose-R-L-rhodinose), possess antitumor
activity against S-180 solid tumors in mice, thus associating the antitumor
activity with the common anthraquinone portion of the molecules; see ref 3.
(5) For the synthesis of vineomycinone B2, see: (a) Danishefsky, S. J.;
Uang, B. J.; Quallich, G. J. Am. Chem. Soc. 1985, 107, 1285–1293. (b)
Bolitt, V.; Mioskowski, C.; Kollah, R. O.; Manna, S.; Rajapaksa, D.; Falck,
J. R. J. Am. Chem. Soc. 1991, 113, 6320–6321. (c) Tius, M. A.; Gomez-
Galeno, J.; Gu, X.-q.; Zaidi, J. H. J. Am. Chem. Soc. 1991, 113, 5775–
5783. (d) Matsumoto, T.; Katsuki, M.; Jona, H.; Suzuki, K. J. Am. Chem.
Soc. 1991, 113, 6982–6992.
In practice, we began with the palladium-catalyzed glyco-
sylation (5 mol % of Pd2(dba)3·CHCl3/10% PPh3) of PMBOH
(8) Fujii, A.; Hashiguchi, S.; Uematsu, N.; Ikariya, T.; Noyori, R. J. Am.
Chem. Soc. 1996, 118, 2521–2522
.
(9) (a) Li, M.; Scott, J. G.; O’Doherty, G. A. Tetrahedron Lett. 2004,
45, 1005–1009. (b) Li, M.; O’Doherty, G. A. Tetrahedron Lett. 2004, 45,
(6) (a) Sobti, A.; Kyungjin Kim, K.; Sulikowski, G. A. J. Org. Chem.
1996, 61, 6–7. For related approaches, see: (b) Sasaki, K.; Matsumura, S.;
Toshima, K. Tetrahedron Lett. 2007, 48, 6982–6986.
6407–6411
.
(10) (a) Babu, R. S.; O’Doherty, G. A. J. Carbohydr. Chem. 2005, 24,
(7) (a) Zhou, M.; O’Doherty, G. A. Org. Lett. 2008, 10, 2283–2286.
(b) Guo, H.; O’Doherty, G. A. J. Org. Chem. 2008, 73, 5211–5220.
169–177. (b) Guo, H.; O’Doherty, G. A. Org. Lett. 2005, 7, 3921–3924
.
.
(11) Zhou, M.; O’Doherty, G. A. J. Org. Chem. 2007, 72, 2485–2493
4530
Org. Lett., Vol. 10, No. 20, 2008

Scheme 4
.
Synthesis of a Protected ꢀ-D-Olivose 13
Scheme 5. Synthesis of a ꢀ-D-Olivose/R-L-Rhodinose Disaccharide
with the ꢀ-D-pyranone 6 to form the ꢀ-benzyloxy pyranone 9
in 95% yield as a single diastereomer (Scheme 4). Ketone
reduction of pyranone 9 with NaBH4/CeCl3 gave a mixture of
allylic alcohols 10a/b in 99% yield (dr ∼ 1.5 to 1). Fortunately,
both diastereomers 10a/b could be used in the next reaction.
Applying the Myers’ reductive rearrangement conditions (NBSH,
PPh3/DEAD, NMM, -30 °C to rt)12 to the mixture of allylic
alcohols 10a/b cleanly provided olefin 11 in 95% yield. Finally,
exposing olefin 11 to the Upjohn conditions (OsO4/NMO)13
gave exclusively the diol 12 with allo-stereochemistry in 85%
yield. We then turned to our selective diol inversion. Thus,
subjecting12tothetypicalMitsunobuconditions(p-NO2PhCO2H/
PPh3/DIAD) cleanly converted it into the protected ꢀ-D-olivose
13 in 70% yield. A nice feature of this approach is that the
nitrobenzoate group also serves as a temporary protecting group.
With a viable route to the protected ꢀ-D-oliVo-sugar 13 in
hand, our efforts turned to the installation of the R-L-rhodinose
portion (Scheme 5). Thus, the oliVo-sugar 13 and R-L-pyranone
5 were coupled using our typical palladium-catalyzed glyco-
sylation conditions (5 mol % of Pd2(dba)3·CHCl3/10% PPh3,
98% yield) to form 14 with complete stereocontrol at the
anomeric center. Luche reduction of the keto group in pyranone
14 stereoselectively provided allylic alcohol 15 (97%). At this
stage, a Mitsunobu reaction (PPh3/DIAD/p-NO2PhCO2H, 74%)
was used to afford bis-nitrobenzoate 16 with inverted stereo-
chemistry at C-4 of the newly introduced sugar. Unfortunately,
all efforts to differentiate the two p-nitrobenzoate groups in 16
were unsuccessful. As a result, we opted for hydrolyses of both
benzoates with excess LiOH which afford diol 17 (80%) and
then turned to the selective introduction of the R-L-aculose at
C-4 of the rhodinose sugar.
This was most conveniently performed at low conversion using
a slight excess of R-L-pyranone 5 under dilute conditions to
provide 35% yield of the desired trisaccharide 19, along with
61% recovered starting material 18 (89% yield, based on
recovered starting material). To confirm the regioselectivity of
glycosylation, 19 was acylated with Ac2O/DMAP to form 20
(82%). Analysis of the one- and two-dimensional NMR data
of acetate 20 indicated that the glycosylation of 18 occurred
selectively at the C-4 position of the R-L-rhodinose sugar.
Overall, the trisaccharide 19 was prepared from the achiral
acylfuran 8 in 14 steps and 17% overall yield.
Scheme 6
.
Synthesis of the Vineomycin B2/PI-080
Trisaccharide 19
Our investigations of the selective introduction of the R-L-
aculose at C-4 were performed on both diol 17 and diol 18
(Scheme 6), which could easily be prepared by hydrogenation
with excess diimide (NBSH/Et3N, 80%). The Pd-catalyzed
glycosylation (5 mol % of Pd2(dba)3·CHCl3/10% PPh3) of
alcohol 18 with R-L-pyranone 5 proved to be the most selective.
With significant quantities of trisaccharide 19 in hand, we
decided to screen it against two types of cancer cell lines from
nine different tissue types (Table 1). While we were motivated
to test 19 primarily as a control for future studies, we were
delighted to find it had wide ranging cytotoxicity, displaying
significant growth inhibition (GI50 from 0.1 to 11 µM) and
cytotoxicity (LC50 from 5.1 to 100 µM) against these various
cell lines. In fact, at 50 µM, the trisaccharide 19 showed greater
(12) (a) Myers, A. G.; Zheng, B. J. Am. Chem. Soc. 1996, 118, 4492–
4493. (b) Myers, A. G.; Zheng, B.; Movassaghi, M. J. Org. Chem. 1997,
62, 7507–7507. (c) Haukaas, M. H.; O’Doherty, G. A. Org. Lett. 2002, 4,
1771–1774.
(13) VanRheenen, V.; Kelly, R. C.; Cha, D. Y. Tetrahedron Lett. 1976,
17, 1973–1976.
Org. Lett., Vol. 10, No. 20, 2008
4531

mechanism of cell death (Figure 3), the samples were then
stained with propidium iodide (PI, 30 µg/mL) and 1 µg/mL
Hoechst (HT, 1 µg/mL). Apoptotic cells appeared shrunken with
condensed nuclei and stained only for Hoechst, whereas cells
that have underwent apoptosis-dependent or apoptosis-inde-
pendent necrosis stained for both PI and Hoechst.
Table 1. Cytotoxicity Evaluation of Trisaccharide 19
19 (µM)
TGIb
2.80 >100
panel/cell
line
a
c
cell type
leukemia
GI50
LC50
RPMI-8226
SR
non-small cell lung NCI-H23
NCI-H460
0.12
0.60 19.0 >100
1.98 4.59
11.0 23.7
3.02
12.0
51.0
5.50
5.13
31.5
colon
COLO-205
SW-620
SF-268
1.66
1.35
3.09
1.82
1.53
1.82
1.62
1.60
2.32
1.49
1.74
1.81
1.63
1.42
2.64
9.54
4.10
2.87
3.22
3.32
2.95
6.46
2.81
3.99
3.20
3.38
2.77
CNS
U251
9.26
5.40
5.71
6.81
5.43
melanoma
ovarian
renal
LOX IMVI
SK-MEL-28
IGROV1
OVCAR-3
TK-10
23.1
UO-31
PC-3
DU-145
MCF7
T-47D
5.30
9.16
5.66
7.04
prostate
breast
54.1
Figure 3. Mechanism of cell death apoptosis/necrosis. H460 lung
a GI50: the drug concentration resulting in a 50% reduction in the net
protein increase in control cells. b TGI: the drug concentration resulting in
no increase in the measured protein as compared to control cells. c LC50:
the drug concentration resulting in a 50% reduction in the measured protein
as compared to control cells.
epithelial cancer cells were treated with either cisplatin (300 µM) or
19 (50, 100, and 200 µM) for 8 h in serum-free medium, following
which each well was incubated in 30 µg/mL propidium iodide (PI)
and 1 µg/mL Hoechst (HT) for 30 min. Images show cells stained for
Hoechst alone (top panel, blue), PI alone (middle panel, red), or both.
cytotoxicity than cisplatin at 300 µM (Figure 2). Interestingly,
this level of cytotoxicity is in the range of that found for other
glycosylated angucycline antibiotics.14
At 50 µM of trisaccharide 19, the H460 cells stained mainly
for Hoechst and displayed condensed nuclear bodies without
significant uptake of propidium iodide, suggesting that cell death
was due primarily to apoptosis. However at higher concentra-
tions of 19, cell death predominantly occurred via necrosis,
which was indicated by PI staining without nuclear bodies.
In conclusion, a highly enantioselective de novo route to
vineomycin B2/PI-080 trisaccharide 19 has been developed.
The trisaccharide portion of these natural products was
demonstrated to suppress the proliferation of several human
cancer cell lines via an apoptotic mechanism at 50 µM.
However, when 19 was tested at higher concentrations (100
and 200 µM), a significant amount of necrosis was also
detected. This synthetic/biological study establishes for the
first time the importance of the trisaccharide portion to the
SAR for these molecules. The preparation and further
biological investigation of other analogues are ongoing.
Figure 2. Cytotoxicity of 19/cisplatin toward H460. Graph denotes
Acknowledgment. We are grateful to Kung Wang for his
support of this project, Anand Krishnan V. Iyer and Yon
Rojanasakul (WVU) for their assistance with the cell viability
studies, the NCI Developmental Therapeutics Program for the
cytotoxicity studies, and the NSF (CHE-0749451) and the ACS-
PRF (47094-AC1) for the support of our research program and
NSF-EPSCoR (0314742) for a 600 MHz NMR at WVU.
total cell death (apoptosis + necrosis) observed upon treatment of H460
cells with either no drug (NTX), 300 µM cisplatin (CP300), 50 µM
of 19 (PI-50), 100 µM of 19 (PI-100), 200 µM of 19 (PI-200).
We decided to further study the mechanism of cytotoxicity
of 19 toward human H460 lung epithelial cancer cells (Figure
2). Thus, H460 human lung epithelial cells were treated with
trisaccharide 19 at a range of concentrations (50, 100, and 200
µM) and cisplatin (300 µM) for comparison. To ascertain the
Supporting Information Available: Complete experimen-
tal procedures and spectral data for all new compounds can be
found in the Supporting Information. This material is available
free of charge via the Internet at http://pubs.acs.org.
(14) Abdelfattah, M. S.; Kharel, M. K.; Hitron, J. A.; Baig, I.; Rohr, J.
J. Nat. Prod. 2008, ASAP 10.1021/np800281f.
OL801817F
4532
Org. Lett., Vol. 10, No. 20, 2008