The cephalostatins. 21. Synthesis of bis-steroidal pyrazine rhamnosides (1)

Article abstract of DOI:10.1021/np200411p

The synthesis of bis-steroidal pyrazines derived from 3-oxo-11,21- dihydroxypregna-4,17(20)-diene (4) and glycosylation of a D-ring side chain with α-L-rhamnose have been summarized. Rearrangement of steroidal pyrazine 10 to 14 was found to occur with boron triflouride etherate. Glycosylation of pyrazine 10 using 2,3,4-tri-O-acetyl-α-L-rhamnose iodide led to 1,2-orthoester-α-L-rhamnose pyrazine 17b. By use of a persilylated α-L-rhamnose iodide as donor, formation of the orthoester was avoided. Bis-steroidal pyrazine 10 and rhamnosides 17b and 21c were found to significantly inhibit cancer cell growth in a murine and human cancer cell line panel. Pyrazine 9 inhibited growth of the nosocomial pathogen Enterococcus faecalis.

Full text of DOI:10.1021/np200411p

ARTICLE
pubs.acs.org/jnp
The Cephalostatins. 21. Synthesis of Bis-steroidal Pyrazine
Rhamnosides1
George R. Pettit,* Ricardo F. Mendonca, John C. Knight, and Robin K. Pettit
)
Department of Chemistry and Biochemistry, Arizona State University, PO Box 871604, Tempe, Arizona 85287-1604, United States
S Supporting Information
b
ABSTRACT: The synthesis of bis-steroidal pyrazines derived
from 3-oxo-11,21-dihydroxypregna-4,17(20)-diene (4) and
glycosylation of a D-ring side chain with R-L-rhamnose have
been summarized. Rearrangement of steroidal pyrazine 10
to 14 was found to occur with boron triflouride etherate.
Glycosylation of pyrazine 10 using 2,3,4-tri-O-acetyl-R-L-
rhamnose iodide led to 1,2-orthoester-R-L-rhamnose pyrazine
17b. By use of a persilylated R-L-rhamnose iodide as donor,
formation of the orthoester was avoided. Bis-steroidal pyrazine 10 and rhamnosides 17b and 21c were found to significantly
inhibit cancer cell growth in a murine and human cancer cell line panel. Pyrazine 9 inhibited growth of the nosocomial pathogen
Enterococcus faecalis.
n 1988, we reported the isolation and X-ray crystal structure
determination of cephalostatin 1 (1), a complex tridecacyclic
low isoelectric point), leading to shrunken mitochondria and
apoptosis. In contrast, certain other anticancer drugs, such as
staurosporine, that affect the mitochondria cause swelling and
apoptosis.1b
I
pyrazine from the South African marine tube worm Cephalodiscus
gilchristi.2a Cephalostatin 1 was found to exhibit an extraordina-
rily powerful cytostatic activity (GI50 1.2ꢀ4.2 nM) against the U.
S. National Cancer Institute (NCI) human cancer cell line
panel.3,4 To date, we have isolated and determined the structures
of 19 cephalostatins,2b which were later augmented by 25
structurally related ritterazines from the Japanese tunicate Ritter-
ella tokioka that were isolated and characterized by the Fusetani
group.5 A new total synthesis of cephalostatin 1,4a the 2009
review by Fuchs,4b and important summaries by Vollmar,4c
Moser,4d Winterfeldt,4e and Morzycki4f provide a very com-
prehensive view of the chemical and biological advances
inspired by these remarkably powerful anticancer marine natural
products.
The low bioavailability and biological importance (including a
unique apoptotic/mitochondria mechanism) of the cephalosta-
tins and ritterazines, in combination with the challenging mo-
lecular architecture, has stimulated a broad spectrum of organic
synthesis efforts in a number of laboratories. These include the
classic (65 steps) first total synthesis of cephalostatin 1 by Fuchs,6
a second enantioselective synthesis by Shair,4a and a variety of
simpler structural modifications.4,7 Many of the analogues
synthesized have been shown to be only weakly cytotoxic, even
the ones that closely resemble the cephalostatins by incorporat-
ing a spiroketal unit. Important insights into the mechanism of
action of cephalostatin 1 and other members of the series were
not revealed until Vollmar1b,4c and colleagues discovered a new
mode of apoptosis events. Cephalostatin 1 was found to induce a
novel pathway of mitochondrial activation that selectively re-
leases Smac/DIABLO (second mitochondria-derived activator
of caspases/direct inhibitor of apoptosis-binding protein with a
The cephalostatins, ritterazine B,5b and schweinfurthin A8a
share some structural features and cancer cell line inhibitory
activity with certain polyhydroxylated steroids found in nature,
particularly the steroidal glycoside OSW-1 (2),8bꢀe a saponin
that belongs to a large family of glycoconjugates with a wide
spectrum of biological and pharmacological activities.9aꢀc OSW-1
(2) has been considered structurally analogous to a masked
spiroketal, and it displays a profile and potency similar to
cephalostatin 1 (1): an NCI COMPARE analysis of 2 showed
a reasonable correlation with 1 of 0.6ꢀ0.83.8d,10a The structure
of OSW-1 (2) is related to that of cephalostatin 1 (1) through an
intermediate formed by loss of the disaccharide unit and
water.10b However, SAR studies imply that the sugar is impor-
tant for cytotoxic activity.10c While the OSW-1 disaccharide unit is
composed of L-arabinose and D-xylose, other steroid/triterpene-
type cancer cell line growth inhibitors such as solamargine (3), a
steroidal alkaloid glycoside,11 and the pentacyclic triterpene
12
hederacolchiside A1 are R-L-rhamnopyranosides. A selection
of recent examples include R-L-rhamnopyanose glycosides of the
steroidal sapogenin diosgenin13a and the lupane pentacyclic tri-
terpene betulinic acid.13b Because of the increasing evidence that
the sugar groups of active steroidal glycosides play a key role14 in
their biology and as part of our investigation into structural
modifications of compound 1 with cancer cell growth inhibitory
activity, we undertook the syntheses of bis-steroidal pyrazines
related to cephalostatin 1 that have a C-17 side chain designed for
coupling with an R-L-rhamnose donor.
Received: May 13, 2011
Published: September 07, 2011
r
Copyright
2011 American Chemical Society and
1922
dx.doi.org/10.1021/np200411p J. Nat. Prod. 2011, 74, 1922–1930
American Society of Pharmacognosy
|

Journal of Natural Products
ARTICLE
Scheme 1
’ RESULTS AND DISCUSSION
Selective acetylation of the C-21 alcohol group of 3-oxo-11,21-
dihydroxypregna-4,17(20)-diene (4) was accomplished with
acetic anhydride/imidazole to afford acetate 5 (74% yield).
Regioselective reduction of the Δ4,5 bond with sodium dithio-
nite using phase transfer catalysis afforded a mixture of 5R/
β-isomers (75% yield, R-isomer in slight excess). The 5R-isomer
(6) was isolated (45% yield) by fractional recrystallization from
acetone (Scheme 1).
Methods for the synthesis of symmetrical bis-steroidal pyr-
azines are now well established and generally involve reduction of
a 2-azido-3-oxo steroid.7e,g Therefore, bromination of 3-ketone 6
with phenyltrimethylammonium perbromide (PTAP) to give the
2-bromo-3-oxo steroid 7 in quantitative yield was first carried
out. Conversion of bromide 7 to 2-azido-3-oxo 8 was accom-
plished by reaction with sodium azide in DMF, acetic acid being
added to the reaction medium to inhibit decomposition of azide
8 to a 2-enamino-3-ketone.7h
The reduction of 2-azido-3-oxo steroids commonly used in the
synthesis of bis-steroidal pyrazines has generally utilized direct
hydrogenation or triphenylphosphine (Staudinger reaction)
for conversion to a 2-amino-3-oxo intermediate, which in turn
undergoes self-condensation to a dihydropyrazine. Subsequent
oxidation of the dihydropyrazine affords the desired pyrazine.
The Staudinger reaction was chosen for the reduction of azide 8
in order to avoid hydrogenation of the exocyclic double bond at
C-17, which had been found to be readily reduced during a
small-scale hydrogenation of azide 8. Thus, treatment (20 h) of
azide 8 (in THF) with triphenylphosphine in THF, followed by
addition of water and exposure to air, gave the C-2-symmetrical
pyrazine 9 in 77% yield. In previous reports of triphenylpho-
sphine-induced dimerization of 3-azido-2-oxo steroids,7fꢀh
the azide and triphenylphosphine were first mixed, and several
minutes later tetrahydrofuran was added. When we repeated
those conditions, the yields of the pyrazine were consistently lower
(23ꢀ40%) than when we used the THF solutions of the reactants.
Attempts to crystallize pyrazine 9 were unsuccessful and
instead gave precipitates or moist glassy foams. However, the
pyrazine was easily identified by NMR spectroscopy, which
shows the disappearance of the H-3 signals and two new signals
at δ 148 in the carbon spectrum, typical of pyrazine carbon
atoms. Deacetylation of pyrazine 9 with potassium carbonate in
aqueous methanol gave diol 10 in low yield (10ꢀ32%). How-
ever, use of KOH in methanol considerably increased the yield
(80%). Because of its low solubility in some organic solvents, the
extraction of 10 from the aqueous layer was monitored by TLC.
Pyrazine 10 was considered ideally suited for glycosylation
reactions, with four available OH groups. Since the 11β-alcohol
groups were expected to be considerably hindered by the C-18
1923
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
Scheme 2
Scheme 3
angular methyl groups, it was assumed that they would provide
regioselectivity in a glycosylation step.
Next, we converted R-L-rhamnose to a trichloroacetimidate
derivative. This type of reagent is widely used in carbohydrate
syntheses and in glycosylation of steroids such as OSW-1.14ꢀ16
Peracetylated R-L-rhamnose (11)17 was obtained in 95% yield
from R-L-rhamnose, and the anomeric acetate was selectively
hydrolyzed with KOH18 to give hemiacetal 12.19 Reaction of 12
with trichloroacetonitrile and 1,8-diazabicyclo[5.4.0]undec-7-
ene afforded 2,3,4-tri-O-acetyl-R-L-rhamnopyranosyl trichloro-
acetimidate (13)19 in 59% overall yield (Scheme 2).
Scheme 4
Boron trifluoride-catalyzed coupling of trichloroacetimidate
13 with pyrazine 10 gave a very complex mixture. Initially, this
was thought to arise from some lack of selectivity between the
allylic and 11β alcohols. The reaction mixture NMR spectrum
showed the disappearance of the signal for the allylic proton
(H-20) and a new set of signals at low field (δ ∼5.8). That
analysis suggested possible rearrangement of the steroid skele-
ton. Steroids with a (17)20 exocyclic double bond or possessing
a good leaving group at C-21 are prone to WagnerꢀMeerwein
rearrangement under acidic conditions with concomitant loss
of water20,21 and migration of the C-18 angular methyl group to
C-17.21 The main component isolated from the glycosylation
reaction mixture was analyzed. The 13C NMR spectrum showed
two extra sp2-hybridized carbons at δ 135.8 and 138.3, and a
1
low-field signal appeared at δ 5.79 in the H NMR spectrum.
The NMR analyses augmented by HRMS data allowed assign-
ment of structure 14. Presumably, the rearrangement proceeds
through a BF3-mediated loss of the C-21 OH followed by
migration of the 18-methyl group (Scheme 3). The nature of
this concerted rearrangement indicates that the migrating
methyl group should reside on the β-face of the steroid
skeleton. The relative configuration of olefin 14 was established
by comparison with the product from an analogous BF3 Et2O-
3
promoted rearrangement of dienediol 4. Glycosylation of pyr-
azine 10 with trichloroacetimidate 13 was repeated using tri-
methylsilyl trifluoromethanesulfonate (TMSOTf) as the acti-
vator, but the only product isolated was again the product of
rearrangement (14). That result clearly pointed to the need for a
glycosylation method employing neutral or basic conditions.
Carbohydrate iodides have been used in glycosylations and
found to be efficient glycosyl donors under basic catalysis in the
presence of diisopropylethylamine (DIPEA) and tetrabutylammo-
nium iodide (TBAI).22 Therefore, we prepared 2,3,4-O-acetyl-R-L-
rhamnopyranosyl iodide 1523 from R-L-rhamnose peracetate (11)
(Scheme 4). That was easily accomplished by selective substitution
of the anomeric acetate by iodide using iodotrimethylsilylane
(TMSI).22 Next, dienediol 4 was used as a model compound for
coupling rhamnose iodide 15 with pyrazine 10. Condensation of
15 with alcohol 4 in the presence of DIPEA and TBAI gave
rhamnoside 16a in 92% yield. The appearance of clear 1H NMR
signals from the H-20 and H-21 protons confirmed that rearrange-
ment of the steroid nucleus had not occurred. Another important
feature of the 1H NMR spectrum was a shift to higher field of one
of the rhamnoside acetate signals, from their normal values of δ
2.0ꢀ2.5 to δ 1.74. Such a chemical shift is characteristic of methyl
1924
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
orthoesters, which are sometimes formed between acceptors with
low reactivity and carbohydrate donors with ester-protecting
groups at C-2. Identification of the site of glycosylation was
inferred from a shift of the H-21 protons to higher field, while
the signal for H-11R remained similar to that in alcohol 4.
Orthoester 16a slowly decomposed to dienediol 4 and 2,3,4-tri-
O-acetyl-R-L-rhamnopyranoside even when kept at 0 °C, possibly
through the presence of small traces of acid or moisture that
catalyze the reverse reaction.
Scheme 5
Treatment of 1,2-orthoesters of type 16a with a catalytic
amount of TMSOTf is known to promote rearrangement of the
orthoester to a 2,3,4-glycoside.24 The major drawback in cleavage
of 16a was clearly the potential sensitivity of this steroid skeleton
to acidic conditions. As predicted, when orthoester 16a was
treated with a catalytic amount of TMSOTf only, a complex
mixture was obtained, and the 1H NMR spectrum showed signals
resulting from the C-18 rearrangement. Other Lewis acids such
as Cu(OTf)225 gave results similar to TMSOTf, and by contrast
Yb(OTf)3 promoted no reaction even at 0.5 equivalents. When
the orthoester (16a) was deacetylated with NaOMe in MeOH,
triol 16b was obtained in only 12% recovery. However, with use
of ethanolic ammonia, triol 16b was realized in 46% yield.
Interestingly, 16b proved to be more stable than its parent
of established anomeric configuration allowed a good correspon-
1
dence of the H and 13C NMR signals of 20b to an R-con-
1
(16a), as noted from a H NMR spectrum recorded following
several months of storage at ambient temperatures.
figuration, and the assignment was confirmed by synthesis of the
R- and β-rhamnosylated pyrazines.
The lability of orthoester 16a suggested the possibility that a
similar treatment of pyrazine 10 could provide a useful orthoester
prodrug modification. Coupling of 10 with rhamnose iodide 15
under the same conditions used for dienediol 4 gave, following a
three-day reaction period, orthoester pyrazine 17a in 87% yield
(Scheme 4). TLC showed the formation of an initial intermediate
(the monorhamnosylated pyrazine) that slowly disappeared to
give rise to the new, less polar 17a. Deacetylation of orthoester 17a
was realized in ethanolic ammonia solution by simple evaporation
of the solvent to yield deprotected pyrazine 17b in 77% yield. The
pyrazine orthoesters 17a and 17b were found to be quite stable, in
contrast to their monosteroidal counterpart 16. The increased
stability is probably due to the moderate pyrazine basic properties,
which inhibit acid-catalyzed orthoester decomposition.
A useful approach to avoid orthoester formation in glycosyla-
tions is to use an ether protecting group at C-2. Benzyl22,26 and
trimethylsilyl ether27 hexose iodides have been reported to par-
ticipate successfully in glycosylation reactions. The O-trimethyl-
silyl-protected carbohydrate iodides are the simplest and more
advantageous to employ, as the TMS group can be easily cleaved
by heating at reflux in methanol. Thus, persilylation of anhydrous
R-L-rhamnose with chlorotrimethylsilane in the presence of
triethylamine in DMF gave persilyl R-L-rhamnose 1828 as a clear
oil in 87% yield. Reaction with TMSI afforded 2,3,4-O-trimethyl-
silyl-R-L-rhamnopyranosyl iodide (19), which was used imme-
diately (Scheme 5).
The configuration of the substituent at C-1 has been reported
to have a characteristic effect upon the chemical shift of C-5 in
rhamnose derivatives.30 With an R-OAc at C-1, the C-5 signal
appears in the range δ 68.5 to 71.0. When C-1 bears a free R-OH,
R-OME, or R-OBn, the C-5 signal appears below δ 69.0. If C-1
bears a β-OH or β-OAc, the resonance line of C-5 is found to
appear above δ 71.4. These relationships were further supported
by spectroscopic data from a pair of rhamnose-containing R- and
β- disaccharides, of which the configuration at C-1 was confirmed by
their anomeric 1JCH coupling constants (172 Hz for the R- and
156 Hz for the β-rhamnoside); the resonance line for C-5 in the
R-rhamnoside appears at δ 66.5 and that of the β-rhamnoside at
δ 71.0.31 Rhamnoside 20b displays a signal for C-50 at δ 66.5
(H-50 appears at δ 3.89ꢀ3.93 as a multiplet), confirming the pres-
ence of the R-rhamnoside in the assigned structure. Compound
20b was deacetylated with ethanolic ammonia to produce diene
20c. The 13C NMR signal of C-50 in rhamnoside 20c appears at
δ 69.8, also consistent with the R-rhamnoside assignment.
Once acceptable reaction conditions for glycosylation of the
alcohol 4 side chain were in hand, pyrazine 10 was coupled with
persilylated rhamnose iodide 19 (Scheme 6). The glycosylation
of pyrazine 10 was slow at room temperature, probably owing to
the low solubility of the pyrazine in CH2Cl2, and consumption of
all of the starting material required heating at reflux for four days.
After the crude product was heated in refluxing MeOH in order
to cleave the TMS groups, attempts were made to isolate the
deprotected pyrazine rhamnoside directly, but separation of the
mixture was not fully achieved using a variety of chromatographic
techniques. Analysis of the mixture by HPLC showed the
presence of two major components, but their retention times
were almost identical. Acetylation of the mixture was performed
with acetic anhydride/pyridine to simplify purification. While the
acetylated mixture was barely resolved by silica gel TLC, on
neutral alumina two spots were clearly distinguished although
still with little separation. Following partial separation of the
products by column chromatography, complete resolution by
When dienediol 4 was allowed to react with iodide 19, fol-
lowed by cleavage of TMS from the product (20a) and acetyla-
tion, glycoside 20b was formed in 23% overall yield. Because of
the axially configured 2-position, assignment of the glycoside as
R or β using 3J1,2 coupling constants (0 to e3 Hz in 1H NMR)
proved difficult. Although the anomeric configuration can be
1
1
determined by use of H-coupled 13C NMR, with the JCH
constants ranging from 170 (for R) to 160 Hz (β),29 inspection
of the coupled spectrum of rhamnoside 20b did not provide
unequivocal evidence. However, as described below, a compar-
ison of the spectroscopic data with those from known substances
1925
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
HPLC yielded three pyrazine rhamnosides. The major compo-
nent isolated in 19% yield (from 10) was easily identified as the
R,R0-dirhamnoside acetate by comparison with 20b and assigned
side and for a β-rhamnoside, respectively.31 The H-10 signals for
the R-rhamnoside appear at δ 4.79 and for the β-rhamnoside at
δ 4.67.
1
structure (R,R)-21b. The H and 13C NMR spectra of this
The third reaction product obtained in low yield (6%) was
identified as β,β0-dirhamnoside (β,β)-21b. Analysis of the NMR
spectra of this isomer provided confirmation of the structure and
proton assignment for the unsymmetrical (R,β)-21b. In order to
complete this phase of our cephalostatin 1 structureꢀactivity
(SAR) research, the hexaacetate (R,R)-21b was deacetylated
with ethanolic ammonia to give, following recrystallization from
MeOH, deprotected (R,R)-21c in 56% yield. Research continues
in our laboratory to further study the potential use of tetraol 10 as
an intermediate for obtaining more potent structural modifica-
tions of cephalostatin 1.
Biological Activity. The cancer cell growth inhibitory proper-
ties were examined using the murine P388 lymphocytic leukemia
cell line (Table 1), and selected compounds were tested against a
range of human cancer cell lines (Table 2). With the P388 assay,
pyrazine 10 displayed the most significant cancer cell growth
inhibitory activity, followed by orthoester 16b and 2-bromo-
steroid 7. The biological activity of orthoester 16b is consistent
with expectations based on such functional groups in naturally
occurring substances, e.g., the marine steroidal orthoester desig-
nated orthoesterol B (active against feline leukemia virus and
other viruses).32 Interestingly, bis-steroidal pyrazine orthoester
17b was less active than the related monosteroid orthoester 16b.
Three of the SAR targets (10, 17b, and R,R-21c) were selected
for more cancer cell line evaluations employing a representative
human cancer cell line minipanel. All three bis-steroidal pyra-
zines showed significant inhibition of cancer cell growth.
Pyrazines 9, 10, and 17b were evaluated against a panel of 10
bacteria and fungi. Pyrazine 9 selectively inhibited the growth
of Enterococcus faecalis (minimum inhibitory concentration =
32 μg/mL), an important nosocomial pathogen that frequently
causes urinary tract infections, wound infections, endocarditis,
and bacteremia. At 64 μg/mL, pyrazines 10 and 17b were not
active in broth microdilution assays.
product were almost superimposable on those of 20b, the
resonance line of C-50 appearing at δ 66.5 and that of H-50 as a
multiplet at δ 3.90ꢀ3.95.
A second component obtained in 15% yield was considered to
be an unsymmetrical pyrazine because of the appearance in the
1H NMR spectrum of two sets of doublets for the H-1β protons.
Also, the rhamnose and steroidal H-20 and H-21 protons
appeared duplicated. On the symmetrical pyrazine these H-1β
protons resonate at the same frequency. After careful spectro-
scopic analyses this product was identified as the R,β-dirhamnose
(R,β)-21b. Again, the resonance signals of H-50 and C-50 were
diagnostic. Two multiplets for two different H-50 signals corre-
sponding to different rhamnose residues, one at δ 3.90ꢀ3.95 and
another at δ 3.46ꢀ3.52, were correlated to two signals in the
carbon spectrum at δ 66.4 and δ 70.4, respectively. As noted
above, these chemical shifts agree with those for an R-rhamno-
Scheme 6
’ EXPERIMENTAL SECTION
General Experimental Procedures. Unless otherwise noted, all
reagents were obtained from commercial suppliers. Except for THF,
which was distilled under argon from sodium/benzophenone immedi-
ately prior to use, other anhydrous solvents were obtained from
commercial suppliers. Triethylamine was distilled under argon from
CaH2. All reactions involving air- or moisture-sensitive reagents were
conducted under an atmosphere of argon in septum-sealed flasks.
Transfer of reagents was accomplished by standard syringe techniques.
Organic extracts of aqueous solutions were dried over anhydrous
Na2SO4 and concentrated under reduced pressure using a rotary
evaporator. Column chromatography (CC) was carried out using Merck
60 silica gel. Reaction mixtures and chromatography fractions were
analyzed employing Analtech 250 μm silica gel GHLF plates and
developed with phosphomolybdic acid (10% in EtOH). Semipreparative
HPLC was performed with a Gibson model 805 HPLC coupled with a
Table 1. Murine P388 Lymphocytic Leukemia Cell Line
Incubation Results
compound
ED50 (μg/mL)
compound
ED50 (μg/mL)
5
>100
16.6
6.6
17a
>10
>10
>10
>100
>10
>10
>10
>10
6
17b
7
20b
8
12.0
77.2
1.5
20c
9
(R,R)-21b
(R,β)-21b
(β,β)-21b
(R,R)-21c
10
14
16a
16b
>10
9.5
5.2
Table 2. Human Cancer Cell Line Evaluation Results (GI50, μg/mL)
compound
BXPC-3 (pancreas)
MCF-7 (breast)
SF-268 (CNS)
NCI-H460 (lung)
KM20L2 (colon)
DU-145 (prostate)
10
1.3
2.1
1.8
1.3
2.8
1.8
1.7
3.0
1.8
2.2
3.8
1.8
1.7
3.3
1.8
1.8
3.0
2.0
17b
(R,R)-21c
1926
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
Gibson model 117 UV detector. Analytical HPLC was conducted with a
Hewlett-Packard model 1050 HPLC coupled with a HꢀP diode-array
detector.
rapidly to a pale yellow color, which gradually disappeared with stirring at
0 °C for 20 min. The reaction was terminated with brine (20 mL) and
extracted with EtOAc. The organic layer was washed with brine (50 mL),
dried, concentrated, and filtered through a pad of silica gel (2:1
hexaneꢀEtOAc as eluent) to provide bromoketone 7 (3.08 g, 99%) as
a colorless powder. The analytical sample was obtained by crystallization
from hexaneꢀether (87% yield): mp 155ꢀ156 °C; [R]24D +53.3 (c 0.12,
Melting points were determined using an Olympus electrothermal
melting point unit and are uncorrected. The optical rotation data were
recorded with a Perkin-Elmer 241 polarimeter. IR spectra were recorded
using a Thermo Nicolet Avatar 360 infrared spectrometer as thin films or
solid samples. NMR spectra were measured at 300 (Varian XL-300),
400, or 500 MHz (Varian UNITY INOVA) for 1H and at 100 or 125
MHz for 13C using TMS as an internal reference (J values are given in
Hz). NMR assignments are based on 2D spectra (COSY and HMQC)
obtained using standard software.
Elemental analyses were performed by Galbraith Laboratories. High-
resolution mass spectra were obtained with a JEOL LCMate magnetic
sector instrument either in FAB mode with a glycerol matrix or by APCI
with a polyethylene glycol reference. Fast-atom bombardment mass
spectrometry was provided by the Washington University Mass Spectro-
metry Resource with support from the NIH National Center for
Research Resources (Grant No. P41RR0954).
1
CHCl3); IR (film) νmax 3531, 2924, 1725, 1236, 1022, 734 cmꢀ1; H
NMR (CDCl3, 400 MHz) δ 0.78ꢀ2.49 (17H, m), 1.14 (3H, s), 1.34
(3H, s), 2.06 (3H, s), 2.82 (1H, dd, J 6.0, 12.4), 4.35 (1H, br d, J 3.2),
4.58ꢀ4.71 (2H, m), 4.83 (1H, dd, J 6.0, 13.6), 5.23 (1H, tt, J 2.0, 7.6); 13
C
NMR (CDCl3, 100 MHz) δ 14.7, 19.9, 21.1, 24.0, 27.8, 30.3, 31.1, 31.7,
39.1, 43.4, 44.0, 46.4, 48.5, 51.2, 54.4, 56.8, 57.2, 60.8, 68.3, 113.3, 156.2,
171.1, 201.2; MS (APCI) m/z 453/455 [M + H]+; anal. C 60.71, H
7.70%, calcd for C23H33O4Br, C 60.93, H 7.34%.
2R-Azido-3-oxo-11β-hydroxy-21-acetoxy-5R-pregna-17(20)-ene
(8). To a solution of bromoketone 7 (0.99 g, 2.19 mmol) in DMF
(20 mL) were added acetic acid (0.13 mL) and sodium azide (0.543 g,
8.36 mmol). The mixture was stirred at room temperature (rt) for 2.5 h
and then poured onto iceꢀwater. The precipitate was collected by
filtration, washed with H2O, and dried in a desiccator over P2O5. The
crude solid was purified further by dry-column chromatography
(eluent: 5ꢀ15% EtOAcꢀtoluene) to give pure azidoketone 8 as a
colorless foam (0.75 g, 82%): Rf 0.36 (9:1 tolueneꢀEtOAc); mp
58ꢀ60 °C; [R]24D +201.6 (c 0.06, CHCl3); IR (film) νmax 3504, 2924,
3-Oxo-11β-hydroxy-21-acetoxypregna-4,17(20)-diene (5). A mix-
ture of dienediol 4 (Upjohn Co., 8.0 g, 24.20 mmol), acetic anhydride
(115 mL), and imidazole (3.3 g, 48.4 mmol) in CHCl3 (120 mL) was
heated at reflux for 3 h. After cooling, the reaction mixture was diluted
with CHCl3 (100 mL), washed with HCl (10%), saturated aqueous
NaHCO3, and brine, and dried. The solvent was removed to yield a
yellow solid that was crystallized from EtOAc to afford acetate 5 as pale
1
3103, 1725, 1236, 1022, 734 cmꢀ1; H NMR (CDCl3, 400 MHz)
yellow crystals (6.65 g, 74%): mp 190 °C (EtOAc); [R]24 +148.2
D
δ 0.82ꢀ2.52 (17H, m), 1.11 (3H, s), 1.30 (3H, s), 2.03 (3H, s), 4.04
(1H, dd, J 5.8, 13.4), 4.33 (1H, dd, J 3.3, 6.0), 4.58ꢀ4.68 (2H, m), 5.21
(1H, tt, J 2.4, 7.6); 13C NMR (CDCl3, 100 MHz) δ 15.1, 19.9, 21.1,
24.0, 27.8, 30.2, 31.1, 31.7, 37.1, 43.2, 44.1, 44.9, 46.4, 48.5, 56.8, 57.3,
60.8, 63.8, 68.4, 113.3, 156.2, 171.1, 205.2. Azide 8 was immediately
used in the next reaction.
(c 1.0, CHCl3); IR (film) νmax 3409, 1727, 1652, 1245, and 1231 cmꢀ1
;
1H NMR (CDCl3, 300 MHz) δ 1.17 (3H, s, H-18), 1.45 (3H, s, H-19),
2.06 (3H, s, Ac), 0.98ꢀ2.55 (17H, m), 4.41 (1H, br d, J 3.3 Hz, H-11R),
4.59ꢀ4.72 (2H, m, H-21), 5.23 (1H, tt, J 1.8, 7.3 Hz, H20), 5.68 (1H, d,
J 1.8 Hz, H4); 13C NMR (CDCl3, 125 MHz) δ 19.9, 21.0, 21.1, 23.9,
30.9, 31.0, 32.0, 32.4, 33.8, 34.0, 39.2, 43.9, 46.5, 56.3, 56.7, 60.8, 68.3,
113.3, 122.3, 156.0, 171.1, 172.0, 199.5; MS (APCI) m/z 373 [M + H]+;
anal. C 73.89, H 8.76%, calcd for C23H32O4, C 74.19, H 8.60%.
Bis-steroidal Pyrazine 9. To a solution of azidoketone 8 (1.45 g, 3.49
mmol) in dry THF (40 mL) was added (via cannula) triphenylpho-
sphine (2.75 g, 10.5 mmol) in dry THF (15 mL) under argon, and the
mixture was stirred at rt for 20 h. Water was added (1.4 mL) and stirring
was continued for another 24 h at rt, with exposure to air. The reaction
mixture was then concentrated and co-evaporated twice with toluene to
remove H2O. The crude residue was separated by flash chromatography
on silica gel [eluent: 3:2 tolueneꢀEtOAc (Rf 0.35)] to give pyrazine 9
(0.99 g, 77%) as a colorless glassy foam: mp 185ꢀ190 °C (EtOAc);
[R]24D +75.6 (c 0.80, CHCl3); IR (film) νmax 3316, 2925, 1737, 1443,
1401, 1240, 1023, 733 cmꢀ1; 1H NMR (CDCl3, 400 MHz) δ 0.85ꢀ2.52
(28H, m), 1.10 (6H, s, H-18), 1.16 (6H, s, H-19), 2.06 (6H, s, Ac),
2.58ꢀ2.66 (4H, m, H-4β, H-1R), 2.81 (2H, dd, J 5.4, 17.8, H-4R), 3.11
(2H, d, J 16.0, H-1β), 4.50 (2H, br s, H-11), 4.63ꢀ4.72 (4H, m, H-21),
5.22 (2H, t, J 7.2, H-20); 13C NMR (CDCl3, 100 MHz) δ 14.4, 19.9,
21.1, 24.0, 27.9, 30.7, 31.2, 32.0 35.1, 35.9, 42.5, 44.0, 45.2, 46.5, 57.2,
57.4, 60.9, 68.4, 113.1, 148.4, 148.7, 156.6, 171.1; HRMS (APCI+) m/z
741.4880 [M + H]+ (calcd for C46H65N2O6, 741.4843); anal. C 72.51, H
3-Oxo-11β-hydroxy-21-acetoxy-5R-pregna-47(20)-ene (6). To a
solution of enone 5 (6.65 g, 17.85 mmol) in toluene (120 mL) was
added a solution of Na2S2O4 (18.65 g, 107.1 mmol)33 and NaHCO3
(9.00 g, 107.1 mmol) in H2O (12 mL), followed by Adogen 464
(2.2 mL), and the mixture was vigorously stirred under reflux until most
of the starting material was consumed (1ꢀ2 h). Longer reaction times at
full consumption of the starting material caused partial reduction of the
C-3 ketone. The organic layer was separated, and the aqueous layer was
extracted with ethyl acetate. The organic phase was washed with brine
and dried, and the solvent was removed. Fractional crystallization from
acetone gave the 5R-isomer 6 as colorless needles (3.03 g, 45%): mp
218ꢀ219 °C (acetone); Rf 0.31 (17:3 DCMꢀEtOAc); [R]24 +56.2
D
(c 1.27, CHCl3); IR (film) νmax 3449, 2912, 1721, 1697, 1656,
1
1233 cmꢀ1; H NMR (CDCl3, 300 MHz) δ 0.78ꢀ2.53 (19H, m),
1.14 (3H, s), 1.27 (3H, s), 2.06 (3H, s), 4.37 (1H, br t, J 3.3), 4.59ꢀ4.72
(2H, m), 5.22 (1H, tt, J 1.9, 7.5); 13C NMR (CDCl3, 125 MHz) δ 14.2,
20.0, 21.1, 24.0, 28.3, 30.7, 31.1, 31.9, 35.8, 38.0, 38.2, 44.10, 44.14, 46.4,
47.7, 57.0, 57.5, 60.8, 68.5, 113.1, 156.4, 171.1, 212.0; MS (FAB) m/z
381 [M + Li]+, 375 [M + H]+; anal. C 73.65, H 9.47%, calcd for
C23H34O4 C 73.76, H 9.15%.
8.76, N 3.74%, calcd for C46H64N2O6 H2O, C 72.79, H 8.76, N 3.69%.
3
Bis-steroidal Pyrazine 10. To a solution of pyrazine 9 (165 mg, 0.222
mmol) in CH3OHꢀDCM (15 mL, 1:1) was added powdered KOH
(0.040 g, 0.713 mmol), and the mixture was stirred at reflux for 2 h
(monitored by TLC). The reaction mixture was concentrated, and the
residue was partitioned between brine (20 mL) and EtOAc (20 mL).
The organic layer was separated, and the aqueous layer was extracted
with EtOAc until TLC showed complete extraction. The combined
organic phase was successively washed with 1% HCl, saturated NaH-
CO3, and brine and dried. Removal of solvent gave pyrazine 10 (0.116 g,
80%) as a colorless solid. The analytical sample was obtained by
crystallization from MeOH: Rf 0.30 (2:3 tolueneꢀacetone); mp
The 5β-isomer (2.69 g, 40%) was obtained as a colorless crystalline
powder and identified by its proton spectrum: Rf 0.4 (17:3 DCMꢀ
1
EtOAc); H NMR (CDCl3, 300 MHz) δ 1.03ꢀ2.56 (19H, m), 1.15
(3H, s), 1.27 (3H, s), 2.06 (3H, s), 2.64 (1H, t, J 14.5, H-5β), 4.34 (1H,
br t, J 3.3), 4.59ꢀ4.73 (2H, m), 5.23 (1H, tt, J 1.9, 7.5).
2R-Bromo-3-oxo-11β-hydroxy-21-acetoxy-5R-pregna-17(20)-ene
(7). To a solution of ketone 6 (2.57 g, 6.86 mmol) in dry THF (50 mL)
under argon that was cooled to 0 °C was added rapidly phenyltrimethy-
lammonium perbromide via cannula. The resulting orange solution faded
350 °C (dec); [R]24 +71.0 (c 0.10, CH3OH); IR (solid) νmax 3383,
D
2917, 1401 cmꢀ1; 1H NMR (C5D5N, 400 MHz) δ 0.94ꢀ2.01 (22H, m),
1927
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
1.36 (6H, s), 2.32 (2H, dd, J 9.0, 16.6), 2.55 (2H, dd, J 9.0, 16.6), 2.74
(2H, d, J 13.6), 2.82 (2H, dd, J 4.8, 12.4, H-4β), 2.89 (2H, d, J 16.4,
H-1R), 3.03 (2H, dd, J 5.6, 18.0, H-4R), 3.62 (2H, d, J 16.0, H-1β),
4.60ꢀ4.70 (6H, m, H-11, H-21), 5.64 (1H, s, OH, exch. with D2O), 5.65
(1H, s, OH, exch. with D2O), 5.67 (2H, t, J 6.8, H-20), 6.01 (2H, br s,
OH, exch. with D2O); 13C NMR (C5D5N, 100 MHz) δ 14.5, 20.1, 24.5,
28.5, 31.3, 31.5, 32.5, 35.9, 36.3, 42.8, 44.4, 45.6, 57.8 (2C), 58.4, 67.7,
121.4, 148.7, 149.3, 151.4; HRMS (APCI+) m/z 657.46035 [M + H]+
(calcd for C42H61N2O6, 657.46314); anal. C 73.06, H 9.23, N 4.00%,
MHz) δ 0.88ꢀ2.51 (17H, m), 1.18 (3H, s, H-18), 1.34 (3H, d, J 6.0,
H-60), 1.46 (3H, s, H-19), 1.72 (3H, s, CH3CO3), 3.15 (1H, br, exch.
D2O), 3.28ꢀ3.37 (1H, m, H-50), 3.50 (1H, pseudo-t, J 9.3, H-40), 3.69
(1H, br, exch. D2O), 3.75 (1H, dd, J 3.9, 9.3, H-30), 4.02ꢀ4.23 (2H, m,
H-21), 4.40 (1H, br d, J 2.7, H-11), 4.56 (1H, dd, J 2.4, 4.2, H-20), 5.27
(1H, t, J 7.8, H-20), 5.41 (1H, d, J 2.4, H-10), 5.69 (1H, s, H-4); 13C
NMR (CDCl3, 125 MHz) δ 17.6, 20.2, 21.1, 23.9, 30.8, 31.2, 32.0, 32.4,
33.8, 35.0, 39.2, 40.9, 44.3, 46.5, 56.3, 56.6, 68.0, 68.5, 69.9, 73.5, 92.3,
115.6, 122.4, 138.2, 156.6, 172.0, 199.5; HRMS (APCI+) m/z 331.22453
[M ꢀ Rha]+ (calcd for C21H31O3, 331.22733).
calcd for C42H61N2O6 2CH3OH, C 73.29, H 9.51, N 3.89%.
3
Bis-steroidal Pyrazine 14. A mixture of pyrazine 10 (98 mg, 0.149
mmol), trichloroacetimidate 1314,19 (143 mg, 0.328 mmol), and freshly
activated molecular sieves (4 Å, powder, 0.4 g) in anhydrous CH2Cl2
Bis-steroidal Pyrazine R-L-Rhamnoside 17a. Pyrazine 17a was pre-
pared from iodide 14 (0.363 g, 0.91 mmol) and pyrazine 10 (44 mg,
0.067 mmol) by following the procedure for synthesis of 1,2-orthoester
16a. Isolation by CC on silica gel (eluent: 2:3 tolueneꢀEtOAc, Rf 0.22)
gave orthoester 17a (70 mg, 87%) as a film: IR (film) νmax 3404, 2920,
(20 mL) was stirred under argon for 15 min. Next, BF3 OEt2 (8.0 μL,
3
0.05 mmol) was added, and the mixture stirred at 0 °C for 2 h. After 24 h
1
at rt, the starting material was still the main component (by TLC) of the
1750, 1455, 1375, 1228, 1055 cmꢀ1; H NMR (CDCl3, 300 MHz)
mixture, and more BF3 OEt2 (8.0 μL, 0.05 mmol) was added. Further
δ 0.87ꢀ2.29 (30H, m), 1.06 (6H, s, H-18), 1.11 (6H, s, H-19), 1.23 (6H,
d, J 6.6 Hz, H-60), 1.75 (6H, s, CH3CO3), 2.08 (6H, s, Ac), 2.13 (6H, s, Ac),
2.46 (2H, dd, J 8.7, 17.7, H-4β), 2.58 (2H, d, J 16.0, H-1R), 2.76 (2H, dd,
J 5.3, 17.7, H-4R), 3.09 (2H, d, J 16.0, H-1β), 3.45ꢀ3.54 (2H, m, H-50),
4.06ꢀ4.16 (4H, m, H-21), 4.42 (2H, br s, H-11), 4.69 (2H, dd, J 2.7, 3.9,
H-20), 4.97ꢀ5.12 (4H, m, H-30, H-40), 5.21 (2H, t, J 7.2, H-20), 5.38 (2H,
d, J 2.7, H-10); 13C NMR (CDCl3, 100 MHz) 14.5, 17.6, 20.2, 20.9, 21.0,
24.5, 25.3, 28.4, 31.2, 31.6, 32.5, 35.3, 36.2, 42.8, 44.2, 45.2, 46.8, 57.7,
57.8, 59.0, 68.8, 69.2, 70.6, 71.8, 76.9, 97.6, 115.1, 124.3, 148.7, 149.1,
155.4, 170.2, 171.2; HRMS (APCI+) m/z 1201.6468 [M + H]+ (calcd for
C66H93N2O18, 1201.6424).
3
addition of BF3 OEt2 (33 μL, 0.26 mmol), totaling 1.1 equiv in relation
3
to imidate and 2.4 equiv to pyrazine, was required for the starting
material to be completely consumed. The mixture was separated from
the molecular sieves, diluted with CHCl3, and washed with aqueous
NaHCO3 to pH 8 and then with brine to neutrality. The organic phase
was dried, filtered, and evaporated. The residue was separated by CC on
silica gel using 2:3 tolueneꢀEtOAc as eluent to give pyrazine 14 (36%)
as a film: IR (film) νmax 3377, 2923, 1446, 1399, 1370, 1244, 1078,
1054 cmꢀ1; 1H NMR (C5D5N, 500 MHz) δ 0.86ꢀ2.41 (22H, m), 1.05
(6H, s), 1.09 (6H, s), 2.57 (2H, d, J 16.0, H-1R), 2.60ꢀ2.66 (2H, m),
2.77 (2H, dd, J 5.5, 17.5, H-4R), 3.12 (2H, d, J 17.0, H-1β), 4.45 (2H, s),
4.89ꢀ4.93 (4H, m), 5.79 (2H, dd, J 10.0, 17.5); 13C NMR (C5D5N, 100
MHz) δ 13.4, 22.9, 28.7, 29.5, 30.3, 31.1, 31.3, 33.8, 35.2, 35.9, 38.6, 42.3,
46.1, 51.7, 55.0, 65.3, 110.7, 135.8, 138.3, 145.7, 148.7, 148.8; HRMS
(APCI+) m/z 621.44219 [M + H]+ (calcd for C42H57N2O2, 621.44201).
1,2-Orthoester-3,4-di-O-acetyl-R-L-rhamnoside (16a). To a solu-
tion of tetra-O-acetyl-R-L-rhamnose (11, 0.84 g, 2.53 mmol) in DCM
(20 mL) cooled to 0 °C under a flow of argon was added TMSI (0.4 mL,
2.81 mmol). The reaction was allowed to proceed for 2 h at 0 °C. The
solvent was removed and the residue azeotroped with anhydrous
toluene until a colorless distillate persisted, in order to remove the
trimethylsilyl acetate product. The resulting pale yellow oil identified by
1H NMR as iodide 1523 (0.99 g, 98%) was redissolved in anhydrous
DCM (10 mL) and added via cannula to a stirred solution of steroid 4
(0.41 g, 1.25 mmol), DIPEA (0.43 mL, 2.50 mmol), TBAI (0.92 g, 2.50
mmol), and freshly activated molecular sieves (4 Å, 0.2 g) in anhydrous
DCM (50 mL). The solution was stirred for 21 h at reflux, and the
solvent was evaporated. The resulting oil was subjected to flash
chromatography (eluent: 1:1 tolueneꢀEtOAc, Rf 0.39) to afford ortho-
ester 16a (0.693 g, 92%): IR (film) νmax 3507, 2941, 1751, 1665, 1230,
1055 cmꢀ1; 1H NMR (CDCl3, 300 MHz) δ 0.96ꢀ2.54 (14H, m), 1.15
(3H, s, H-18), 1.23 (3H, d, J 6.0, H-60), 1.46 (3H, s, H-19), 1.74 (3H, s,
CH3CO3), 2.08 (3H, s, Ac), 2.13 (3H, s, Ac), 3.45ꢀ3.54 (1H, m, H-50),
4.08 (2H, dd, J 3.0, 7.5, H-21), 4.36 (1H, br t, J 3.0, H-11), 4.71 (1H, dd,
J 2.5, 4.0, H-20), 4.95ꢀ5.10 (2H, m, H-30,40), 5.20 (1H, t, J 7.5, H-20),
5.41 (1H, d, J 2.1, H-10), 5.68 (1H, s, H-4); 13C NMR (CD2Cl2, 125
MHz) 17.6, 20.3, 21.0, 21.2, 21.5, 24.4, 25.4, 31.3, 31.5, 32.4, 32.9, 34.2,
35.3, 39.6, 44.2, 46.6, 56.7, 57.2, 58.8, 68.7, 69.2, 70.5, 72.0, 77.0, 97.7,
115.2, 122.4, 138.3, 155.1, 170.1, 172.5, 199.3; HRMS (APCI+) m/z
603.31506 [M + H]+ (calcd for C33H47O10, 603.31688).
Bis-steroidal Pyrazine 1,2-Orthoester R-L-Rhamnoside 17b. Deace-
tylation of 17a (70 mg, 0.058 mmol) in ethanolic ammonia, according to
the procedure for alcohol 16b, gave pyrazine 17b (46 mg, 77%) as a
colorless solid following evaporation of the solvent: mp 205ꢀ208 °C
(dec); [R]24D +3.0 (c 0.1, pyridine); IR (film) νmax 3354, 2909, 1446,
1
1399, 1082, 1007 cmꢀ1; H NMR (C5D5N, 400 MHz) δ 0.92ꢀ1.98
(22H, m), 1.35 (6H, s, H-18), 1.44 (6H, s, H-19), 1.61 (6H, d, J 6.4,
H-60), 1.99 (6H, s, CH3CO3), 2.27 (2H, dd, J 8.7, 16.7), 2.50 (2H, dd,
J 8.7, 16.7), 2.75ꢀ2.84 (4H, m, includes H-4β), 2.88 (2H, d, J 16.4,
H-1R), 3.03 (2H, dd, J 5.6, 18.0, H-4R), 3.58, 4.05 (2H, pseudo-t, J 9.2,
H-40), 4.20 (2H, dd, J 4.0, 9.2, H-30), 4.54ꢀ4.61 (4H, m, H-21, 4.66 (2H,
br s, H-11), 4.89 (2H, dd, J 2.6, 3.8, H-20), 5.45 (2H, t, J 6.8, H-20), 5.59
(2H, br s, OH, exch. D2O), 5.62 (2H, d, J 1.6, H-10); 13C NMR (C5D5N,
100 MHz) δ 14.5, 18.4, 20.1, 24.4, 26.3, 28.4, 31.3, 31.4, 32.5, 35.9, 36.3,
42.8, 44.6, 45.6, 46.5, 57.5, 57.6, 58.8, 67.6, 72.0, 73.1, 73.2, 81.4, 98.2,
115.9, 124.1, 148.7, 149.4, 154.4; HRMS (Q-TOF, ESI) m/z 1033.5983
[M + H]+ (calcd for C58H85N2O14, 1033.6001).
3-Oxo-11β-hydroxy-21-O-(20,3040-tri-O-acetyl-R-L-rhamnosyl)-
pregna-4,17(20)-diene (20b). TMSI (0.5 mL, 3.5 mmol) was added to a
0.1 M solution of 1,2,3,4-tetra-O-trimethylsilyl-R-L-rhamnose (18, 1.4 g)30
in anhydrous DCM at 0 °C. The solution was stirred under argon at rt
for 10 min and then concentrated in vacuo. The residual water was
azeotroped with anhydrous toluene until the distillate color remained
pale yellow. The crude product (iodide 19, 1.51 g) was dissolved in dry
DCM (5 mL) and cannulated into a stirred mixture of dienediol 4
(0.51 g, 1.54 mmol), TBAI (1.15 g, 3.11 mmol), DIPEA (0.54 mL, 3.11
mmol), molecular sieves (4 Å, 0.5 g), and anhydrous DCM (30 mL).
The mixture was stirred at rt under argon for 24 h, and the solvent was
evaporated in vacuo. EtOAc was added to the crude residue, and the
mixture was cooled in an ice bath to precipitate TBAI, which was
removed by filtration. The filtrate was concentrated, and the residue
was dissolved in CH3OH (50 mL). The solution was heated at reflux
for 4 h, cooled, and concentrated. The methanolic mixture was made
neutral with triethylamine, the solvent was evaporated in vacuo, and
the residue was dissolved in pyridine (20 mL). Acetic anhydride
(4 mL) and DMAP (catalytic) were added, and the reaction mixture
was stirred for 10 h. Pyridine was removed by azeotroping with
1,2-Orthoester R-L-Rhamnoside 16b. A solution of acetate 16a (100
mg, 0.166 mmol) in ethanolic ammonia (20 mL, saturated at ꢀ10 °C)
was left standing overnight at 0 °C and then evaporated to dryness. The
crude residue was separated by CC on silica gel (eluent: EtOAc, Rf 0.38)
to yield alcohol 16b (40 mg, 46%) as a colorless powder: mp
124ꢀ126 °C (CHCl3); [R]24D +64.3 (c 0.60, CHCl3); IR (film) νmax
3444, 2930, 1656, 1104, 1071, 1050, 1013 cmꢀ1; 1H NMR (CDCl3, 300
1928
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
toluene, and the residue was purified by flash chromatography (eluent:
7:3 DCMꢀEtOAc, Rf 0.30) to give rhamnosylated pregnene 20b as a
foam (0.213 g, 23%): mp 108ꢀ110 °C; [R]24D +57.1 (c 0.14, CHCl3);
H-4β), 2.80 (2H, dd, J 4.5, 17.5, H-4R), 3.12 (1H, d, J 16.5, H-1β0), 3.14
(1H, d, J 16.5, H-1β00), 3.46ꢀ3.52 (1H, m, H-500), 3.90ꢀ3.95 (2H, m,
H-50), 4.08 (1H, dd, J 7.5, 11.0, H-210), 4.25ꢀ4.29 (2H, m, H-210, 2100),
4.39 (1H, d, J 8.0, 12.0, H-2100), 4.49 (2H, br s, H-11), 4.67 (1H, s, H-100),
4.79 (1H, d, J 1.5, H-10), 5.01 (1H, dd, J 3.0, 10.5, H-300), 5.01ꢀ5.10 (2H,
m, H-40,400), 5.15 (1H, t, J 7.0, H-2000), 5.22 (1H, t, J 7.0, H-200), 5.24
(1H, dd, J 1.5, 3.5, H-20), 5.33 (1H, dd, J 3.5, 10.0, H-30), 5.43 (1H, d,
J 3.0, H-200); 13C NMR (CDCl3, 125 MHz) δ 14.42, 14.49, 17.4, 17.6,
20.0, 20.2, 20.6, 20.76, 20.82, 20.85, 21.0, 24.0, 28.0, 30.9, 31.2, 32.1, 35.2,
35.9, 42.6, 43.9, 44.1, 45.3, 45.8, 46.1, 57.2, 57.3, 57.4, 57.6, 64.0, 64.6,
66.4, 68.6, 68.7, 69.1, 69.4, 70.3, 70.5, 70.8, 71.2, 71.3, 96.1, 96.8, 114.1,
114.5, 148.4, 148.9, 156.1, 156.2, 169.8, 170.0, 170.2, 170.4, 170.6.
The last substance to elute was the R,R0-dirhamnoside (R,R)-21b (35
mg, 19%): mp 188ꢀ190 °C; [R]24D +15.7 (c 0.70, CHCl3); IR (film)
1
IR (film) νmax 3469, 2936, 1750, 1667, 1372, 1225, 1050 cmꢀ1; H
NMR (CDCl3, 400 MHz) δ 0.99ꢀ2.56 (17H, m), 1.19 (3H, s, H-18),
1.22 (3H, d, J 6.4 Hz, H-60), 1.46 (3H, s, H-19), 1.99 (3H, s, Ac), 2.05
(3H, s, Ac), 2.16 (3H, s, Ac), 3.89ꢀ3.93 (1H, m, H-50), 4.10 (1H, dd,
J 7.0, 11.6, H-21), 4.18 (1H, dd, J 7.0, 11.6, H-21), 4.41 (1H, br s, H-11),
4.77 (1H, d, J 0.8, H-10), 5.08 (1H, t, J 10.0 Hz, H-40), 5.22ꢀ5.24 (2H, m,
H-20, H-20 obscured by H-20), 5.32 (1H, dd, J 3.6, 10.0, H-30), 5.68 (1H,
H-4); 13C NMR (CDCl3, 100 MHz) δ 21.01, 24.0, 31.0, 32.0, 32.5, 33.8,
34.9, 39.2, 44.0, 46.0, 56.3, 56.6, 63.8, 66.5, 68.4, 69.1, 70.2, 71.1, 96.7,
114.2, 122.3, 155.9, 170.0, 170.1, 170.5, 172.1, 199.5; HRMS (FAB) m/z
609.3236 [M + Li]+ (calcd for C33H46LiO10, 609.3251).
νmax 3360, 2912, 1751, 1223, 1051 cmꢀ1 1H NMR (500 MHz)
;
3-Oxo-11β-hydroxy-21-O-(R-L-rhamnosyl)pregna-4,17(20)-diene
(20c). Deacetylation of rhamnoside 20b (100 mg, 0.166 mmol) in
ethanolic ammonia followed the procedure for obtaining triol 16b.
Isolation of product by CC on silica gel (eluent: 9:1 DCMꢀCH3OH,
Rf 0.31) led to tetraol 20c (55 mg, 69%) as a foam: mp 100ꢀ105 °C
(CH3OH); [R]24D +42.0 (c 0.35, CH3OH); IR (film) νmax 3424, 2929,
1664, 1070, 1042; 1H NMR (C5D5N, 500 MHz) 0.86ꢀ2.69 (17H, m),
1.40 (3H, s, H-18), 1.57 (3H, s, H-19), 1.62 (3H, d, J 6.0, H-60),
4.24ꢀ4.29 (3H, m, H-50, H-40, H-21), 4.51 (2H, dd, J 3.5, 9.0, H-30,
H-11), 4.55 (1H, dd, J 1.5, 3.5, H-20), 4.66 (1H, dd, J 7.2, 11.7, H-21),
5.33 (1H, d, J 1.0, H-10), 5.40 (1H, t, J 7.0, H-20), 5.68 (1H, br s, OH,
exch. D2O), 5.68 (1H, s, H-4), 6.80 (2H, br, OH, exch. D2O); 13C
NMR (C5D5N, 125 MHz) 18.6, 20.2, 21.0, 22.8, 24.3, 31.2, 31.4, 32.1,
32.9, 34.3, 34.9, 39.6, 44.4, 46.8, 56.5, 57.1, 63.4, 67.5, 69.8, 72.4, 72.8,
74.0, 100.7, 116.4, 122.4, 154.1, 172.1, 198.5; HRMS (APCI+) m/z
477.2845 [M + H]+ (calcd for C27H41O7 477.2852).
Bis-steroidal Pyrazine R-L-Rhamnoside 21b. A solution of 2,3,4-tri-
O-trimethylsilyl-R-L-rhamnopyranoside iodide (19, 0.325 g, 0.66 mmol)
in anhydrous DCM (10 mL) was added via cannula to a stirred mixture
of pyrazine 10 (98 mg, 0.15 mmol), TBAI (0.244 g, 0.66 mmol), DIPEA
(115 μL, 0.66 mmol), and molecular sieves (4 Å, 0.2 g) in anhydrous
DCM (60 mL). The mixture was stirred under argon for 24 h at rt and at
reflux for 4 days. Desilylation of the crude residue was followed by an
unsuccessful attempt to isolate the deprotected product (21c). There-
fore, the octaol-containing residue was acetylated as described for
preparation of triacetate 20b. The crude product was separated on a
silica gel plug by elution with 7:3 DCMꢀEtOAc to give a mixture of
acetylated pyrazines (85 mg, 47%). Further separation was performed by
reversed-phase HPLC (C18; eluent: 93:7 CH3OHꢀH2O) to give three
bis-steroidal pyrazines.
δ 0.93ꢀ2.54 (28H, m), 1.10 (6H, s, H-18), 1.16 (3H, s, H-19), 1.23 (6H,
d, J 6.5, H-50), 1.99 (6H, s, Ac), 2.05 (6H, s, Ac), 2.16 (6H, s, Ac), 2.62
(2H, d, J 12.5 Hz, 18.0, H-4β), 2.65 (2-H, d, J 16.5, H-1R), 2.80 (2H, dd,
J 5.5, 18.0, H-4R), 3.14 (2H, d, J 16.5, H-1β), 3.90ꢀ3.95 (2H, m, H-50),
4.08 (2H, dd, J 7.5, 11.0, H-21), 4.27 (2H, dd, J 7.5, 11.0, H-21), 4.49
(2H, br s, H-11), 4.78 (1H, d, J 1.5, H-10), 5.08 (1H, pseudo-t, J 9.5, 10.5,
H-40), 5.22 (2H, t, J 7.5, H-20), 5.24 (2H, dd, J 1.5, 3.5, H-20), 5.33 (2H,
dd, J 3.5, 10.0, H-30); 13C NMR (CDCl3, 125 MHz) δ 14.5, 17.4, 20.0,
20.7, 20.8, 21.0, 24.0, 28.0, 29.7, 30.9, 31.2, 32.1, 35.2, 35.9, 42.6, 44.1,
45.2, 46.1, 57.2, 57.4, 64.0 (C-21), 66.5 (C-50), 68.6 (C-11), 69.1 (C-30),
70.3 (C-40), 71.2 (C-20), 96.8 (C-10), 114.1, 148.4, 148.8, 156.2, 170.0
(2C), 170.4; HRMS (APCI+) m/z 1201.638 [M + H]+ (calcd for
C66H93N2O18, 1201.642); anal. C 62.59, H 7.52, N 2.20%, calcd for
C66H92N2O18.4H2O, C 62.25, H 7.91, N 2.20%.
Bis-steroidal Pyrazine R-L-Rhamnoside Dimer (R,R)-21c. Deacetyla-
tion of R,R-21b (22 mg, 0.018 mmol) in ethanolic ammonia by the
procedure for obtaining triol 16b and isolation of product by crystallization
from MeOH led to octaol 21c (10 mg, 56%) as colorless needles: mp
280 °C (dec) (CH3OH); [R]24D ꢀ22.0 (c 0.05, pyridine); IR (film) νmax
1
3406, 2927, 1645, 1404, 1036 cmꢀ1; H NMR (C5D5N, 400 MHz)
δ 0.91ꢀ2.50 (28H, m), 1.34 (6H, s, H-18), 1.41 (6H, s, H-19), 1.63 (6H, d,
J5.6, H-60), 2.75(2H, dd, J5.6, 18.0, H-1R), 2.87 (2H, d, J16.4, H-4β), 3.01
(2H, dd, J 5.6, 18.0, H-1β), 3.59 (2H, d, J 16.4, H-4R), 4.27ꢀ4.31 (6H, m,
H-40, H-50, H-21), 4.25ꢀ4.54 (2H, d, J 3.2, 8.4, H-30), 4.56 (2H, s, H-2),
4.68ꢀ4.71 (4H, m, H-11, H-21), 5.35 (2H, s, H-10), 5.42 (2H, t, J 6.8,
H-20), 5.71 (2H, d, J 2.8, OH, exch. D2O), 6.63 (3H, br, OH, exch. D2O),
6.81 (2H, br, OH, exch. D2O); 13C NMR (C5D5N, 100 MHz) δ 14.6, 18.6,
20.2, 24.5, 28.5, 31.3, 31.6, 32.5, 35.9, 36.4, 42.8, 44.5, 45.6, 47.0, 57.6 (2),
63.5, 67.7, 69.8, 72.5, 72.9, 74.1, 100.8, 116.3, 148.8, 149.4, 154.5; HRMS
(APCI+) m/z 949.5773 [M + H]+ (calcd for C54H81N2O12, 949.5789).
Antimicrobial Susceptibility Testing. Pyrazines 9, 10, and 17b
were evaluated against the bacteria Stenotrophomonas maltophilia ATCC
13637, Micrococcus luteus Presque Isle 456, Staphylococcus aureus ATCC
29213, Escherichia coli ATCC 25922, Enterobacter cloacae ATCC 13047,
Enterococcus faecalis ATCC 29212, Streptococcus pneumoniae ATCC
6303, and Neisseria gonorrheae ATCC 49226 and the fungi Candida
albicans ATCC 90028 and Cryptococcus neoformans ATCC 90112,
according to established broth microdilution susceptibility assays.34,35
Each substance was reconstituted in a small volume of sterile DMSO and
diluted in the appropriate medium immediately prior to susceptibility
experiments. The minimum inhibitory concentration was defined as the
lowest concentration of compound that inhibited all visible growth of the
test organism (optically clear). Assays were repeated on separate days.
The first compound to elute was the β,β0-dirhamnoside (β,β)-21b
(11 mg, 6%): mp 167ꢀ169 °C; [R]24D +60.0 (c 0.10, CHCl3); 1H NMR
(CDCl3, 500 MHz) δ 0.92ꢀ2.51 (28H, m), 1.09 (6H, s, H-19), 1.11 (6H,
s, H-18), 1.29 (6H, d, J 6.5, H-60), 1.99 (6H, s, Ac), 2.06 (6H, s, Ac), 2.18
(6H, s, Ac), 2.63 (2H, dd, J 12.5, 17.5, H-4β), 2.65 (2H, d, J 17.5, H-1R),
2.80 (2H, dd, J 5.5, 18.0, H-4R), 3.12 (2H, d, J 16.5, H-1β), 3.46ꢀ3.52 (2H,
m, H-50), 4.27 (2H, dd, J 6.0, 12.0, H-21), 4.39 (2H, dd, J 8.0, 12.0, H-21),
4.49 (2H, br s, H-11), 4.67 (2H, d, J 1.0, H-10), 5.01 (2H, dd, J 3.5, 10.0,
H-30), 5.07 (2H, pseudo-t, J 9.5, 10.0, H-40, 5.15 (2H, t, J 7.0, H-20); 5.43
(2H, dd, J 1.0, 3.5, H-20); 13C NMR (CDCl3, 125 MHz) δ 14.5, 17.6, 20.2,
20.7, 20.8, 21.0, 24.0, 28.0, 30.9, 31.3, 32.1, 35.2, 35.9, 42.6, 43.9, 45.3, 45.8,
57.3, 57.6, 64.5 (C-21), 68.7 (C-11), 69.4 (C-20), 70.5 (C-5), 70.8 (C-40),
71.3 (C-30), 96.1 (C-10), 114.5, 148.4, 148.7, 156.1, 169.8, 170.2, 170.6.
The second product to elute was the unsymmetrical isomer (R,β)-
21b (27 mg, 15%): mp 175ꢀ178 °C; [R]24D +26.2 (c 0.08, CHCl3); IR
(film) νmax 3356, 2925, 1748, 1219, 1077, 1047 cmꢀ1 1H NMR
;
’ ASSOCIATED CONTENT
(CDCl3, 500 MHz) δ 0.92ꢀ2.54 (28H, m), 1.09 (3H, s, H-1900), 1.10
(3H, H-190), 1.11 (3H, s, H-1800), 1.16 (3H, s, H-180), 1.23 (3H, d, J 6.5,
H-60), 1.29 (3H, d, J 6.5, H-600), 1.99 (6H, s, Ac), 2.05 (3H, s, Ac), 2.06
(3H, s, Ac), 2.16 (3H, s, Ac), 2.18 (3H, s, Ac), 2.59ꢀ2.67 (4H, m, H-1R,
Supporting Information. 1H NMR spectra of com-
S
b
pounds 10, 17b, and 21c. This material is available free of charge
via the Internet at http://pubs.acs.org.
1929
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930

Journal of Natural Products
ARTICLE
’ AUTHOR INFORMATION
Chem. Soc. 2002, 124, 6576–6583. (e) Kubo, A.; Mimaki, Y.; Terao, M.;
Sashida, Y.; Hirano, T.; Nikaido, T.; Ohmoto, T. Phytochemistry 1992,
31, 3969.
(9) (a) Deng, L.; Wu, H.; Yu, B.; Jiang, M.; Wu, J. Bioorg. Med. Chem.
Lett. 2004, 14, 2781–2785. (b) Morzycki, J. W.; Wojtkielewicz, A.;
Wozczyꢀnski, S. Bioorg. Med. Chem. Lett. 2004, 14, 3323–3326.
(c) Hostettmann, K.; Marston, A. Saponins; Cambridge University
Press: Cambridge, UK, 1995. (d) Waller, G. R. In Advances in Experi-
mental Medicine and Biology; Waller, G. R., Yamaskai, K., Eds.; Plenum
Press: New York, 1996; Vol. 404.
(10) (a) Mimaki, Y.; Kuroda, M.; Kameyama, A.; Sashida, Y.;
Hirano, T.; Oka, K.; Maekawa, R.; Wada, T.; Sugita, K.; Beutler, J. A.
Bioorg. Med. Chem. Lett. 1997, 7, 633–636. (b) Guo, C.; LaCour, T. G.;
Fuchs, P. L. Bioorg. Med. Chem. Lett. 1999, 9, 419–424. (c) Wojtkielewicz,
A.; Dzugosz, M.; Maj, J.; Morzycki, J. W.; Nowakowski, M.; Renkiewicz, J.;
Strnad, M.; Swaczynovꢀa, J.; Wilczewska, A.; Wꢀojcik, J. J. Med. Chem.
2007, 50, 3667–3673.
Corresponding Author
*Tel: (480) 965-3351. Fax: (480) 965-2747. E-mail: bpettit@asu.edu.
’ ACKNOWLEDGMENT
We appreciate the very necessary financial support provided
by grants RO1CA90441-02-05 and 5R01CA90441-07 from the
Division of Cancer Treatment, Diagnosis and Centers, National
Cancer Institute, DHHS; the Arizona Biomedical Research
Commission; the Robert B. Dalton Endowment Fund; Dr. Alec
D. Keith; the J. W. Kieckhefer Foundation; the Margaret T.
Morris Foundation; and the Fannie E. Rippel Foundation. For
other assistance, we are pleased to thank Drs. J.-C. Chapuis and F.
Hogan, as well as M. Dodson, C. Weber, and L. Williams.
(11) Puri, R.; Wong, T. C.; Puri, R. K. J. Nat. Prod. 1994, 57,
587–596.
’ DEDICATION
(12) Yan, M.-C.; Liu, Y.; Lu, W.-X.; Wang, H.; Sha, Y.; Cheng, M.-S.
Carbohydr. Res. 2008, 343, 780–784.
This paper is dedicated to the memory of Professor Georgy B.
Elyakov (1929ꢀ2005), a pioneering explorer of the oceans and of
the organic chemistry of bioactive marine organism constituents.
(13) (a) Yokosuka, A.; Mimaki, Y. Phytochemistry 2008,
69, 2724–2730. (b) Gauthier, C.; Legault, J.; Lavoie, S.; Rondeau, S.;
Tremblay, S.; Pichette, A. Tetrahedron 2008, 64, 7386–7399.
(14) Pellissier, H. Tetrahedron 2004, 60, 5123–5162.
(15) van Steijn, A. M. P.; Kamerling, J. P.; Vliegenthart, J. F. G.
Carbohydr. Res. 1991, 211, 261–277.
(16) (a) Yu, W.; Jin, Z. J. Am. Chem. Soc. 2001, 123, 3369–3370.
(b) Deng, S.; Yu, B.; Lou, Y.; Hui, Y. J. Org. Chem. 1999, 64, 202–208.
(17) Bebault, G. M.; Dutton, G. G. S.; Warfield, C. K. Carbohydr. Res.
1974, 34, 174–179.
(18) Watanabe, K.; Itoh, K.; Araki, Y.; Ishido, Y. Carbohydr. Res.
1986, 154, 165–176.
(19) Larson, D. P.; Heathcock, C. H. J. Org. Chem. 1997, 62,
8406–8418.
(20) (a) Kohen, F.; Mallory, R. A.; Scheer, I. J. Org. Chem. 1971,
36, 716–718. (b) Ouannes, C.; Dvolaitzky, M.; Jacques, J. Bull. Soc. Chim.
Fr. 1964, 776.
’ REFERENCES
(1) (a) Antineoplastic Agents series part 562. For the preceding
contribution refer to Pettit, G. R.; Smith, T. H.; Feng, S.; Knight, J. C.;
Tan, R.; Pettit, R. K.; Hinrichs, P. A. J. Nat. Prod. 2007, 70, 1073–1083.
(b) For “The Cephalostatins 20” see: Dirsch, V. M.; Muller, I. M.;
Eichhorst, S. T.; Pettit, G. R.; Kamano; Inoue, M.; Xu, J. P.; Ichihara, Y.;
Wanner, G.; Vollmar, A. M. Cancer Res. 2003, 63, 8869–8876.
(2) (a) Pettit, G. R.; Inoue, M.; Kamano, Y.; Herald, D. L.; Arm, C.;
Dufresne, C.; Christie, N. D.; Schmidt, J. M.; Doubek, D. L.; Krupa, T. S.
J. Am. Chem. Soc. 1988, 110, 2006–2007. (b) Pettit, G. R.; Tan, R.; Xu,
J. P.; Ichihara, Y.; Williams, M. D.; Boyd, M. R. J. Nat. Prod. 1998, 61,
955–958.
(3) Chang, L.-C.; Tsai, T. R.; Wang, J.-J.; Lin, C.-N.; Kuo, K.-W.
(21) Loughhead, D. G. J. Org. Chem. 1985, 50, 3931–3934.
(22) Hadd, M. J.; Gervay, J. Carbohydr. Res. 1999, 320, 61–69.
(23) Mukhopadhyay, B.; Kartha, K. P. R.; Russel, D. A.; Field, R. A.
J. Org. Chem. 2004, 22, 7758–7760.
(24) Wang, W.; Kong, F. J. Org. Chem. 1999, 64, 5091–5095.
(25) Pleuss, N.;Kunz, H. Angew. Chem., Int. Ed. 2003, 42, 3174–3176.
(26) Lam, S. N.;Gervay-Hague, J.Carbohydr. Res. 2002, 337, 1953–1965.
(27) (a) Ferriꢃeres, V.; Roussel, M.; Gelin, M.; Plusquellec, D.
J. Carbohydr. Chem. 2001, 20, 855–865. (b) Bhat, A. S.; Gervay-Hague,
J. Org. Lett. 2001, 3, 2081–2084.
(28) Streefkerk, D. G.; de Bie, M. J. A.; Vliegenthart, J. F. G.
Carbohydr. Res. 1974, 38, 47–59.
(29) (a) Perlin, A. S.; Casu, B. Tetrahedron Lett. 1969, 34,
2921–2924. (b) Bock, K.; Pedersen, C. J. Chem. Soc., Perkin Trans. 2
1974, 293–297. (c) Pozsgay, V.; Jennings, H. J. J. Org. Chem. 1988,
53, 4042–4052.
Biochem. Biophys. Res. Commun. 1998, 242, 21–25.
(4) (a) Fortner, K. C.; Kato, D.; Tanaka, Y.; Shair, M. D. J. Am. Chem.
Soc. 2010, 132, 275–280. (b) Lee, S.; LaCour, T. G.; Fuchs, P. L. Chem.
Rev. 2009, 109, 2275–2314. (c) Rudy, A.; Lꢀopez-Antꢀon, N.; Dirsch,
V. M.; Vollmar, A. M. J. Nat. Prod. 2008, 71, 482–486. (d) Moser, B. R.
J. Nat. Prod. 2008, 71, 487–491.(e) Flessner, T.; Jautelat, R.; Scholz, U.;
Winterfeldt, E. In Progress in the Chemistry of Organic Natural Products;
Herz, W., Falk, H., Kirby, G. W., Eds.; Springer: Vienna, 2004; Vol. 87,
pp 1ꢀ80. (f) Gryszkiewicz-Wojtkielewicz, A.; Jastrzebska, I.; Morzycki,
J. W. Curr. Org. Chem. 2003, 7, 1257–1277.
(5) (a) Fukuzawa, S.; Matsunaga, S.; Fusetani, N. J. Org. Chem. 1994,
59, 6164–6166. (b) Fukuzawa, S.; Matsunaga, S.; Fusetani, N. J. Org.
Chem. 1995, 60, 608–614. (c) Fukuzawa, S.; Matsunaga, S.; Fusetani, N.
J. Org. Chem. 1997, 62, 4484–4491.
(6) LaCour, T. G.; Guo, C.; Bhandaru, S.; Boyd, M. R.; Fuchs, P. L.
J. Am. Chem. Soc. 1998, 120, 692–707.
(30) Pozsgay, V.; Neszmꢀelyi, A. Carbohydr. Res. 1980, 80, 196–202.
(31) Chauvin, A. L.; Nepogodiev, S. A.; Field, R. A. Carbohydr. Res.
2004, 339, 21–27.
(32) Giner, J.-L.; Faraldos, J. A. J. Org. Chem. 2002, 67, 2717–2720.
(33) Akamanchi, K. G.; Patel, H. C.; Meenakshi, R. Synth. Commun.
1992, 22, 1655–1660.
(34) NCCLS. Methods for Dilution Antimicrobial Susceptibility Tests
for Bacteria That Grow Aerobically: Approved Standard, 5th ed.; NCCLS
document M7-A5; NCCLS: Wayne, PA, 2000.
(35) NCCLS. Reference Method for Broth Dilution Antifungal Suscept-
ibility Testing of Yeasts: Approved Standard, 2nd ed.; NCCLS document
M27-A2; NCCLS: Wayne, PA, 2002.
(7) (a) Poza, J. J.; Rodríguez, J.; Jimꢀenez, C. Bioorg. Med. Chem.
2010, 18, 58–63. (b) Lee, S.; Jamieson, D.; Fuchs, P. L. Org. Lett. 2009,
11, 5–8. (c) Banerjee, A.; Sergienko, E.; Vasile, S.; Gupta, V.; Vuori, K.;
Wipf, P. Org. Lett. 2009, 11, 65–68. (d) Nawasreh, M. Bioorg. Med. Chem.
2008, 16, 255–265. (e) Shawakfeh, K. Q.; Al-Said, N. H.; Al-Zoubi, R. M.
Steroids 2008, 73, 579–584. (f) Kramer, A.; Ullmann, U.; Winterfeldt, E.
J. Chem. Soc., Perkin Trans. 1 1993, 2865–2867. (g) Heathcock, C. H.;
Smith, S. C. J. Org. Chem. 1994, 59, 6828–6839. (h) Cernꢀy, I.; Pouzar, V.;
Budꢂeꢁsnskꢀy, M.; Draꢁsar, P. Collect. Czech. Chem. Commun. 2000,
65, 1597–1608.
ꢁ
(8) (a) Beutler, J. A.; Shoemaker, R. H.; Johnson, T.; Boyd, M. R.
J. Nat. Prod. 1998, 61, 1509–1512. (b) Tsubuki, M.; Matsuo, S.; Honda,
T. Tetrahedron Lett. 2008, 49, 229–232. (c) Shi, B.; Wu, H.; Yu, B.; Wu, J.
Angew. Chem., Int. Ed. 2004, 43, 4324–4327. (d) Yu, W.; Jin, Z. J. Am.
1930
dx.doi.org/10.1021/np200411p |J. Nat. Prod. 2011, 74, 1922–1930