Design, synthesis, and evaluation of the multidrug resistance-reversing activity of D-glucose mimetics of hapalosin

Article abstract of DOI:10.1021/jm970709p

When five substituents of hapalosin were placed on D-glucose, molecular modeling revealed that the substituents on mimetics 2 and 3 occupy similar spatial positions as the corresponding substituents on hapalosin. Mimetic 3 and all the glucopyranoside inte

Full text of DOI:10.1021/jm970709p

981
J . Med. Chem. 1998, 41, 981-987
Design , Syn th esis, a n d Eva lu a tion of th e Mu ltid r u g Resista n ce-Rever sin g
Activity of D-Glu cose Mim etics of Ha p a losin
Tam Q. Dinh,† Charles D. Smith,§ Xiaohui Du,† and Robert W. Armstrong*,†
Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, and Fox Chase Cancer
Center, Philadelphia, Pennsylvania 19111
Received October 17, 1997
When five substituents of hapalosin were placed on D-glucose, molecular modeling revealed
that the substituents on mimetics 2 and 3 occupy similar spatial positions as the corresponding
substituents on hapalosin. Mimetic 3 and all the glucopyranoside intermediates generated in
its synthesis were assessed for their ability to reverse multidrug resistance (MDR) mediated
by P-glycoprotein (P-gp) or the multidrug resistance-associated protein (MRP). None of the
sugar compounds were as effective as hapalosin in inhibiting P-gp in cytotoxicity and drug
accumulation assays using MCF-7/ADR cells. By contrast, four D-glucose compounds exhibited
similar efficacy as hapalosin in antagonizing MRP in cytotoxicity assays with HL-60/ADR cells.
In tr od u ction
useful because of their intrinsic toxicity or their adverse
effects on the toxicity of accompanying anticancer drugs.
It would clearly be advantageous to develop new MDR
modulators with superior therapeutic profiles.
Many cancer chemotherapy programs often provide
temporary clinical improvement. Unfortunately, the
emergence of less easily treated tumor cells frequently
results in the ultimate failure of chemotherapy. Tumor
cells which survive the initial therapeutic attack often
recover with increased resistance to both the original
therapeutic agent and other structurally unrelated
drugs. This phenomenon is known as multiple drug
resistance (MDR).1
The most common mechanism for acquired MDR
involves overexpression of drug transporters which act
as ATP-dependent drug-efflux pumps, actively removing
a broad spectrum of structurally diverse compounds.
Enhanced efflux of these compounds reduces their
intracellular accumulation and, hence, their cytotoxicity.
The best-studied member of this family of ATPases is
P-glycoprotein (P-gp), a transmembrane protein which
extrudes many natural product drugs, including adria-
mycin, Vinca alkaloids, etoposide, gramicidin D, acti-
nomycin D, and taxol. A related drug transporter that
can confer drug resistance is the multidrug resistance-
associated protein (MRP),2 although its profiles of
substrates and antagonists are not identical to those of
P-gp.3 However, virtually all currently described MDR-
reversing agents inhibit the activity of both P-gp and
MRP.
Hapalosin (1), a cyclic depsipeptide isolated from the
cyanobacterium Hapalosiphon welwitschii, reverses
MDR by inhibiting P-gp.5 The relationship between the
structure and anti-MDR activity of hapalosin6 and its
analogues has been the focus of our research. We have
examined the non-N-Me analogue,6d,7 analogues incor-
porating trans-4-hydroxy-L-proline,7 and the triamide
analogue of hapalosin.8 The elegant work of Hirsch-
mann et al. on nonpeptidal mimetics of the cyclic
hexapeptide L-363,301 intrigued us.9 They created
peptidomimetics containing D-glucose as the scaffold
which were agonists of the somatostatin receptor. Mo-
lecular modeling suggested that D-glucose might also
be a good scaffold for mimetics of hapalosin (2 and 3).
We have prepared a series of D-glucose mimetics of
hapalosin and assessed their ability to antagonize P-gp
and MRP.
Since each of these drug transporters can be ex-
pressed in human tumor cells, a significant amount of
effort has been directed toward the identification and
development of compounds which inhibit the drug-efflux
property of P-gp and/or MRP. A number of compounds
have been shown to reverse MDR in vitro, usually by
competing with cytotoxic drugs for binding to P-gp.4
However, the majority of compounds shown to have
anti-MDR activity are synthetic derivatives of a small
number of chemical families, e.g., dihydropyridines,
thioxanthenes, and phenothiazines. Importantly, none
of these compounds have yet proven to be clinically
Resu lts a n d Discu ssion
Design . The substituents of hapalosin (1) which
might be important for its anti-MDR activity were taken
as the isopropyl group (represented by atom a), the
† University of California.
§ Fox Chase Cancer Center.
S0022-2623(97)00709-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/14/1998

982 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Ta ble 1. Distances (Å) between Atomsa
Dinh et al.
compound
a-b
a-c
a-d
a-e
b-c
b-d
b-e
c-d
c-e
d-e
hapalosin (1)
mimetic (2)
mimetic (3)
4.77
4.43
5.49
6.79
6.50
6.41
6.38
7.14
7.38
5.01
5.25
5.33
4.33
4.38
4.24
6.00
6.64
7.08
6.92
7.10
8.13
6.87
3.66
3.20
7.71
5.66
5.68
3.00
3.59
4.10
a
For all three compounds, the n-heptyl group was replaced with a methyl group to simplify the computation.
benzyl group (atom b), the hydroxyl group (atom c), the
n-heptyl group (atom d), and the R-methyl group (atom
e). Maintaining the same clockwise order of the groups,
the groups were placed at each of the five sites of
functionalization on D-glucose in order to determine the
optimum positioning of the substituents. Furthermore,
ether or aliphatic linkages at C-1 and C-6 of D-glucose
were examined.
Molecular modeling studies were performed with
Macromodel (v4.5)10 using AMBER* force field and GB/
SA chloroform solvation11 as previously described.6d,8 To
reduce the number of conformations found and compu-
tational time, the n-heptyl chain was replaced with a
methyl group in hapalosin and the sugar compounds.
For hapalosin (the major conformer), distance con-
straints between protons exhibiting NOESY cross-peaks
were applied.6d,8 The lowest-energy conformations of
hapalosin and mimetics 2 and 3 are considered in this
paper.
The D-glucose compounds which best resemble hapa-
losin are mimetics 2 and 3. The only difference between
the two mimetics is that 2 is a C-glycoside whereas 3 is
F igu r e 1. Superimposition of hapalosin and mimetics 2 and
3: (a) top view with C-Bn 2 on the left and O-Bn 3 on the
right; (b) angular view with 2 on the left and 3 on the right.
The n-heptyl group of the three compounds was replaced with
a methyl group to simplify the computation.
an O-glycoside. Table 1 lists the distances between the
substituents of the three compounds. The distances for
the two mimetics are similar to those for hapalosin
except for the c-d and c-e distances. In hapalosin, the
hydroxyl group is substantially farther from the n-
heptyl group and the methyl group than it is in the two
sugar mimetics. Nearly all the distances between
substituents for C-glycoside 2 are closer to those for
hapalosin than the distances for O-glycoside 3.
Ta ble 2. Distances (Å) between Corresponding Atoms of
Superimposed Hapalosin and Mimetica
mimetic
a-a
b-b
c-c
d-d
e-e
2
3
0.57
0.64
1.04
1.59
2.19
2.77
1.47
1.69
0.32
0.32
Hapalosin is superimposed on mimetics 2 and 3 in
Figure 1. Figure 1a offers a top view and Figure 1b an
angular view. The fit in the overlay of the five substit-
uents is good, particularly for the C-Bn glycoside 2. It
is interesting that although the ring skeleton of hapal-
osin is clearly larger than the pyranose ring of the
mimetics, the corresponding isopropyl, phenyl, n-heptyl
(replaced by a methyl group to simplify computation),
and methyl groups of the superimposed structures
occupy similar spatial positions. Only the hydroxyl
group of the two mimetics does not overlap well with
that of hapalosin. The distances between the corre-
sponding substituents of superimposed hapalosin and
mimetics are presented in Table 2. On the basis of the
molecular modeling results, D-glucose compounds 2 and
3 seem to mimic hapalosin well, with C-Bn 2 being the
more analogous mimetic.
Syn th esis. Introduction of an R-C-Bn group at C-1
of several tetra-O-benzyl-D-glucopyranosides was at-
tempted with BnTMS. It was hoped that C-glycosida-
tion could be accomplished with BnTMS as it could
routinely be with allylsilanes. However, reactions of the
methyl glycoside with TMSOTf/BnTMS,12 of the p-
nitrobenzoyl glycoside with BF3‚OEt2/BnTMS,13 and of
the glycosyl fluoride with BF3‚OEt2/BnTMS14 were
unsuccessful. Electrophilic aromatic substitution of
a
For hapalosin and the mimetics, the n-heptyl group was
replaced with a methyl group to simplify the computation.
BnTMS, promoted by the positive charge being stabi-
lized by a â C-Si bond, was not a problem in all three
reactions. Considering that an R-O-Bn glycoside could
be much more easily made than an R-C-Bn glycoside
and that the O-Bn glycoside 3 also appeared to be a good
mimetic of hapalosin, we pursued the synthesis of 3.
The synthesis of mimetic 3 is illustrated in Scheme
1. It commenced with the alkylation of diacetone
D-glucose15 to produce n-heptyl ether 4. The diacetonide
was hydrolyzed back to the lactol form,9 and the lactol
was converted to the R-O-Bn glycoside 5. The C-4 and
C-6 hydroxyl groups were protected as the p-methoxy-
benzylidene acetal 6. Benzylation of the hydroxyl group
afforded the dibenzyl ether 7. Regioselective cleavage
of the p-methoxybenzylidene acetal was achieved with
DIBAH in toluene.16 The acetal did not react with
DIBAH in CH2Cl2.
Transformation of activated derivatives of alcohol 8
i
to isobutyl 11 was attempted with PrMgCl. However,
the tosylate, the bromide,17 and the triflate18 derivatives
i
did not react with PrMgCl. Therefore, alcohol 8 was
oxidized to aldehyde 9 which underwent a Wittig
reaction.19 Most of olefin 10 did not undergo diimide

D-Glucose Mimetics of Hapalosin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 983
Sch em e 1a
a
(a) n-Heptyl-I, NaH, DMF (100%); (b) (i) 60% aq AcOH, 90 °C, (ii) AcCl, BnOH, 80 °C (71% for 2 steps, R:â ) 2.4:1); (c) p-anisaldehyde,
cat. PTSA, PhH, 110 °C, Dean-Stark (88%); (d) NaH, BnBr, DMF (100%); (e) DIBAH, PhMe, 0 °C; (f) Swern [O] (67% for 2 steps); (g)
iPrPPh3I, nBuLi, THF, 0 °C (95%); (h) PhS(O)2NHNH2, aq NaOH, 2-MeOEtOH, reflux, 3 cycles (77%); (i) 10% Pd/C, H2 (1 atm), EtOH, 25
min (29% 12, 52% 13); (j) DDQ, H2O, CH2Cl2 (87%); (k) NaH, MeI, DMF (88%); (l) 10% Pd/C, H2 (1 atm), EtOH, 25 min (77%).
reduction with H2NNH2/CuSO4/AcOH/NaIO4,20 with H2-
NNH2/CuSO4/H2O2,21 or with HONH3HCl/NaOAc/
EtOAc.22 Diimide reduction of olefin 10 was effected
with benzenesulfonyl hydrazide and NaOH.23
alone (the PBS curves) or in the presence of 25 nM
actinomycin D, which is transported by P-gp. In drug
accumulation assays, MCF-7/ADR cells were treated
with one of eight doses of a compound and then
incubated with [3H]vinblastine, which is also trans-
ported by P-gp. For MRP-mediated MDR, cytotoxicity
assays (Figure 3) were conducted with HL-60/ADR
cells26 which express MRP but not P-gp. Cells were
exposed to varying amounts of a compound alone (the
PBS curves) or in the presence of 2 nM vincristine, an
MRP-transported drug.
None of the sugar compounds reversed P-gp-mediated
MDR as well as hapalosin and verapamil. Compounds
5, 7, 10, 11, and 15 exhibited very weak activity in
cytotoxicity assays with MCF-7/ADR cells. The intrinsic
cytotoxicity of aldehyde 9 was the greatest of all the
compounds. The MDR-reversing profiles of compounds
3, 6, 8, and 12-14 are presented in Figure 2. All of
these compounds, particularly mimetic 3, were at least
significantly less potent than hapalosin and verapamil.
In addition, all of the sugar compounds exerted very
little effect on the accumulation of [3H]vinblastine in
MCF-7/ADR cells. Only diol 13 had a modest impact.
On the other hand, compounds 8 and 12-14 showed
similar efficacy in reversing MRP-mediated MDR as
hapalosin and verapamil (Figure 3). Mimetic 3 surpris-
ingly displayed the weakest activity in Figure 3. Dibenz-
yl ether 7 has the same profile as alcohol 6. The
dramatic rise in the intrinsic cytotoxicity of hapalosin
and compound 8 from 5 to 10 µg/mL is curious. As it
Hydrogenolysis of compound 11 over 10% Pd/C in 25
min yielded 29% of monool 12 and 52% of diol 13. In a
shorter reaction time, more monool 12 could be formed
than diol 13. The two products did not contain a
deprotected C-1 benzyl ether group since only their
R-anomer was detected. During overhydrogenolysis of
compounds 10, 11, and 15, anomers resulting from
deblocking of the C-1 benzyl ether group were observed.
Hydrogenolysis of compound 11 over W-2 Raney nickel24
was slow during which the formation of diols and the
triol competed with that of monool 12.
Completion of the synthesis of mimetic 3 involved
DDQ deprotection of the PMB ether 11. Methylation
of alcohol 14 furnished dibenzyl ether 15 which was
hydrogenolyzed to mimetic 3. Only the R-anomer of the
hydrogenolysis product was observed. Hydrolysis of
compound 15 under acidic conditions generated anomers
of the lactol which differed from the hydrogenolysis
product of 15.
Mu ltid r u g Resista n ce-Rever sin g Activity. The
anti-MDR activities of synthetic hapalosin (1), mimetic
3, compounds 5-15, and verapamil (as a positive
control) were evaluated. For P-gp-mediated MDR,
cytotoxicity assays (Figure 2) were conducted with MCF-
7/ADR cells25 which overexpress P-gp but not MRP.
Cells were exposed to one of eight doses of a compound

984 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Dinh et al.
F igu r e 2. Reversal of P-gp-mediated MDR. MCF-7/ADR cells were incubated with the indicated concentrations of a compound
in the presence of phosphate-buffered saline (as a control) (O) or 25 nM actinomycin D (b). Cell survival after 48 h was determined
as indicated in the Experimental Section. Values represent the mean ( SD of triplicate samples.
F igu r e 3. Reversal of MRP-mediated MDR. HL-60/ADR cells were incubated with the indicated concentrations of a compound
in the presence of phosphate-buffered saline (as a control) (O) or 2 nM vincristine (b). Cell survival after 48 h was determined
as indicated in the Experimental Section. Values represent the mean ( SD of triplicate samples.
was to MCF-7/ADR cells, aldehyde 9 was the most
inherently cytotoxic to HL-60/ADR cells. Compounds
5, 10, 11, and 15 were much less effective in potentiat-
ing the cytotoxicity of vincristine than compounds 12-
14.
centration of a compound which inhibits cell prolifera-
tion by 20% in the absence of a cytotoxic drug and is
usually considered the maximum dose appropriate for
combination with a cytotoxic drug. EC50 is the ap-
proximate concentration of a compound which enhances
the toxicity of actinomycin D or vincristine to the point
where 50% of the cells are killed.
The results of the in vitro cytotoxicity assays are
summarized in Table 3. IC20 is the approximate con-

D-Glucose Mimetics of Hapalosin
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6 985
Ta ble 3. Reversal of P-gp- and MRP-Mediated MDR
Reversal of MDR is defined as the ability of a compound to
potentiate the cytotoxicity of actinomycin D toward the MCF-
7/ADR cells.
MCF-7/ADR cellsa
HL-60/ADR cellsb
c
d
c
d
compound
IC20
EC50
IC20
EC50
To test for reversal of MRP-mediated MDR, human pro-
myelocytic leukemia HL-60/ADR cells26 were treated with
varying concentrations of a compound alone or in the presence
of 2 nM vincristine for 48 h. The number of surviving cells
was then determined using the CellTiter AQueous assay
system from Promega. The percentage of cells killed was
calculated as the percentage decrease in MTS tetrazolium
metabolism compared with control cultures. Reversal of MDR
is defined as the ability of a compound to potentiate the
cytotoxicity of vincristine toward the HL-60/ADR cells.
[3H]Dr u g Accu m u la tion Assa y. MCF-7/ADR cells were
plated into 24-well tissue culture dishes and allowed to grow
to 90% confluency. The cells were washed with phosphate-
buffered saline (PBS) and then incubated in 0.5 mL of RPMI
1640 medium containing a compound and 20 nM [3H]vinblas-
tine sulfate (Amersham Corp.) for 60 min at 37 °C. The
cultures were rapidly washed three times with ice-cold PBS.
Intracellular [3H]drug was solubilized with 0.3 mL of 1%
sodium dodecyl sulfate in water and quantified by liquid
scintillation counting.
Gen er a l P r oced u r es. 1H and 13C NMR spectra were
obtained at room temperature with a Bruker ARX-400 or ARX-
500 spectrometer. 1H NMR chemical shifts are referenced to
TMS (0.00 ppm) and those for 13C NMR to CDCl3 (77.0 ppm).
IR spectra were recorded with a Nicolet 510P FT-IR spectrom-
eter. Optical rotations were measured with a Perkin-Elmer
241MC polarimeter, and concentrations (c) are reported in
g/mL. High-resolution mass spectroscopy (HRMS) was per-
formed with a VG Autospec instrument for the EI and CI
methods and a VG ZAB-SE instrument for the FAB method.
All water-sensitive reactions were conducted in oven- or
flame-dried glassware under a nitrogen atmosphere. The
starting materials were azeotroped two or three times with
benzene before the reactions. Solvents were distilled im-
mediately prior to use: CH2Cl2 from P2O5, PhMe from CaH2,
MeOH from magnesium metal, and THF from sodium metal/
benzophenone ketyl. Anhydrous DMF was purchased from the
Aldrich Chemical Co. and utilized without further purification.
Most commercially available reagents were distilled before use.
Thin-layer chromatography (TLC) was performed on silica gel-
coated plates (Merck, Kieselgel 60 F254, 0.25 mm thickness for
analytical and 0.5 mm for preparative TLC) and visualized
by UV light and/or p-anisaldehyde staining. After all aqueous
extractions of crude reaction products, the combined organic
layers were dried over MgSO4 and concentrated in vacuo before
further treatment.
3-O-Neoh ep t yl-1,2:5,6-d i-O-isop r op ylid en e-r-D-glu co-
fu r a n ose (4). To a solution of diacetone D-glucose (5.00 g,
19.2 mmol) and 1-iodoheptane (4.70 mL, 28.8 mmol) in DMF
(50 mL) at 0 °C was added NaH (deoiled, 691 mg, 28.8 mmol)
in three portions. After the mixture stirred at 0 °C for 15 min
and then at 25 °C for 2 h, the reaction was quenched with
water. Extraction in CH2Cl2 (3 × 100 mL) with water (100
mL) resulted in pure heptyl ether 4 (6.91 g, 100% yield) as a
colorless oil.
Ben zyl 3-O-Neoh ep tyl-r-D-glu cop yr a n osid e (5). A solu-
tion of diacetonide 4 (6.80 g, 19.0 mmol) in 60% aqueous acetic
acid (35 mL) was stirred at 90 °C for 15 h. The solvent was
removed in vacuo, and the crude product was azeotroped with
benzene (3 × 15 mL). Addition of acetyl chloride (1.35 mL,
19.0 mmol) to a mixture of the crude lactol in benzyl alcohol
(50 mL) quickly dissolved the sugar. After the solution was
stirred at 80 °C for 15 h, BnOH was removed under high
vacuum. Flash chromatography with silica gel (gradient from
10% to 90% EtOAc/hexanes) furnished R-O-benzyl glycoside
5 (3.51 g, 50% yield for two steps) and the â-anomer (1.47 g,
21% yield for two steps), both as white solids.
hapalosin
verapamil
3
5
6
7
8
9
10
11
12
13
14
15
6
>20
20
.20
12
1
1
7
6
.20
.20
.20
.20
.20
6
1.8
.20
>20
.20
.20
.20
.20
0.7
1.8
10
10
2.5
2.5
1.7
1.5
>20
2.5
.20
.20
6
5
3
4
.20
.20
12
12
10
.20
.20
4
6
6
>20
>20
>20
1.8
3.5
2.5
.20
18
a
P-gp-mediated MDR assay using 25 nM actinomycin D.
MRP-mediated MDR assay using 2 nM vincristine. c The ap-
b
proximate concentration (µg/mL) of a compound which inhibits cell
proliferation by 20% in the absence of the particular cytotoxic drug.
d
The approximate concentration (µg/mL) of a compound which
results in 50% of the cells being killed in the presence of the
particular cytotoxic drug.
Con clu sion
None of the D-glucose compounds in Scheme 1 possess
an effective capacity to reverse P-gp-mediated MDR.
However, the ability of compounds 8 and 12-14 to
antagonize MRP-mediated MDR is similar to that of
hapalosin. In both types of cytotoxicity assays, the
sugar compounds with no free hydroxyl group have
much weaker activities than the compounds with a free
hydroxyl group (except for triol 5).
Mimetic 3 exhibits poor anti-MDR activity even
though its five substituents seem to occupy similar
spatial positions as the corresponding substituents of
hapalosin. This may be due to a couple of reasons.
First, the computed lowest-energy, distance-constrained
conformation of the major conformer of hapalosin in
chloroform may not be the bioactive conformation.
Hapalosin is flexible and can adopt many other confor-
mations, one or some of which being responsible for
inhibition of drug transporters. By contrast, the D-
glucose compounds are most likely constrained to the
chair conformation in which four of the substituents are
equatorial. Second, the amide-diester ring skeleton of
hapalosin may be important in its interaction with and
antagonism of P-gp and MRP.
Exp er im en ta l Section
Cytotoxicity Assa ys. MCF-7 breast carcinoma cells and
MCF-7/ADR cells, an MDR subline,25 were obtained from the
Division of Cancer Treatment of the National Cancer Institute
and were grown in RPMI 1640 containing 10% fetal bovine
serum and 50 µg/mL gentamycin sulfate.
To test for reversal of P-gp-mediated MDR, MCF-7/ADR
cells were seeded in 96-well tissue culture dishes at ap-
proximately 15% confluency and were allowed to attach and
recover for 24 h. The cells were then treated with varying
concentrations of a compound alone or in the presence of 25
nM actinomycin D for 48 h according to previously described
procedures.27 The number of surviving cells was then deter-
mined by staining with sulforhodamine B.28 The percentage
of cells killed was calculated as the percentage decrease in
sulforhodamine B binding compared with control cultures.
Control cultures included equivalent amounts of ethanol (as
the solvent control), which did not modulate the growth or drug
sensitivity of these cells at doses utilized in these studies.
Ben zyl 3-O-Neoh ep tyl-4,6-(p-m eth oxyben zylid en e)-r-
D-glu cop yr a n osid e (6). A solution of triol 5 (3.45 g, 9.36
mmol), p-anisaldehyde (2.30 mL, 18.7 mmol), and p-toluene-
sulfonic acid monohydrate (89 mg, 0.47 mmol) in benzene (60

986 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 6
Dinh et al.
mL) was refluxed at 110 °C for 15 h with a Dean-Stark trap
attached. PTSA was quenched with triethylamine (7 drops
from a Pasteur pipet), and benzene was removed in vacuo.
Flash chromatography with silica gel (gradient to 40% EtOAc/
hexanes) afforded p-methoxybenzylidene acetal 6 (4.02 g, 88%
yield) as a white solid.
Ben zyl 2-O-Ben zyl-3-O-n eoh ep tyl-4,6-(p-m eth oxyben -
zylid en e)-r-D-glu cop yr a n osid e (7). To a solution of alcohol
6 (3.67 g, 7.54 mmol) and benzyl bromide (1.08 mL, 9.05 mmol)
in DMF (35 mL) at 0 °C was added NaH (80% oil dispersion,
339 mg, 11.3 mmol) in two portions. The mixture was stirred
at 0 °C for 10 min and then at 25 °C for 14 h. The reaction
was quenched with water and the mixture extracted in CHCl3
(2 × 100 mL) with water (70 mL). Flash chromatography with
silica gel (gradient from 100% CHCl3 to 0.6% EtOAc/CHCl3)
produced dibenzyl ether 7 (4.36 g, 100% yield) as a white solid.
Ben zyl 2-O-Ben zyl-3-O-n eoh eptyl-4-(p-m eth oxyben zyl)-
r-D-glu cop yr a n osid e (8). DIBAH (4.60 mL, 4.60 mmol in
hexanes) was added slowly to a solution of p-methoxy-
benzylidene acetal 7 (1.32 g, 2.29 mmol) in toluene (10 mL) at
0 °C (the solution solidified at -78 °C). After the mixture
stirred at 0 °C for 1 h, the reaction was carefully quenched
with cold MeOH (5 mL), and the mixture was stirred at 0 °C
for a few minutes and extracted in CHCl3 (2 × 50 mL) with 1
M HCl (50 mL) followed by water (30 mL). The crude product
(1.33 g of white solid, 100% crude yield) contained mostly the
C4-OPMB, C6-OH regioisomer 8.
benzenesulfonyl hydrazide and 70 equiv of NaOH for 3 days
resulted in only 11:10 ) 1:1.2.
Ben zyl 3-O-Neoh ep tyl-4-O-(p-m eth oxyben zyl)-6-d eoxy-
6-isop r op yl-r-D-glu cop yr a n osid e (12) a n d Ben zyl 3-O-
Neoh ep tyl-6-d eoxy-6-isop r op yl-r-D-glu cop yr a n osid e (13).
Ten percent palladium on activated carbon (Degussa type, 15
mg) was added to a solution of 11 (41 mg, 0.068 mmol) in EtOH
(1.3 mL), and the flask was purged with N2 followed by H2.
After being stirred at 25 °C for 25 min under H2 (1 atm), the
mixture was filtered through a plug of Celite, and the plug
was washed well with MeOH and EtOAc. Preparative TLC
with silica gel (20% EtOAc/hexanes eluent) provided monool
12 (Rf ) 0.43, 10 mg, 29% yield) and diol 13 (Rf ) 0.37, 14 mg,
52% yield), both as colorless oils.
Ben zyl 2-O-Ben zyl-3-O-n eoh ep tyl-6-d eoxy-6-isop r op yl-
r-D-glu cop yr a n osid e (14). To a mixture of PMB ether 11
(139 mg, 0.230 mmol) and water (190 µL) in CH2Cl2 (3.4 mL)
was added DDQ (111 mg, 0.491 mmol). The flask was sealed,
and the mixture was stirred vigorously at 25 °C for 1.5 h.
Extraction in CH2Cl2 (2 × 25 mL) with saturated NaHCO3 (2
× 20 mL) and flash chromatography with silica gel (gradient
to 12% EtOAc/hexanes) furnished alcohol 14 (97 mg, 87% yield)
as a colorless oil.
Ben zyl 2-O-Ben zyl-3-O-n eoh ep tyl-4-O-m eth yl-6-d eoxy-
6-isop r op yl-r-D-glu cop yr a n osid e (15). To a solution of
alcohol 14 (97 mg, 0.20 mmol) and MeI (62 µL, 1.0 mmol) in
DMF (1.7 mL) at 0 °C was added NaH (deoiled, 17 mg, 0.70
mmol). The mixture was stirred at 0 °C for 5 min and then at
25 °C for 10 h. After the reaction was quenched with water,
extraction in CH2Cl2 (2 × 20 mL) with water (15 mL) and
preparative TLC with silica gel (8% EtOAc/hexanes eluent)
generated methyl ether 15 (Rf ) 0.42, 88 mg, 88% yield) as a
colorless oil.
Ben zyl 3-O-Neoh eptyl-4-O-m eth yl-6-deoxy-6-isopr opyl-
r-D-glu cop yr a n osid e (3). A suspension of dibenzyl ether
compound 15 (30 mg, 0.060 mmol) and 10% Pd/C (Degussa
type, 10 mg) in EtOH (1.0 mL) was stirred at 25 °C for 25 min
under H2 (1 atm) and then filtered through a plug of Celite.
(The longer the reaction transpires, the more diol anomers are
formed!) Preparative TLC with silica gel (15% EtOAc/hexanes
eluent) produced C-2 alcohol 3 (Rf ) 0.41, 19 mg, 77% yield)
as a colorless oil.
Ben zyl
2-O-Ben zyl-3-O-n eoh ep t yl-4-O-(p -m et h oxy-
ben zyl)-5-(d eh yd r oxym eth yl)-5-for m yl-r-D-glu cop yr a n o-
sid e (9). DMSO (839 µL, 11.8 mmol) was added dropwise to
a solution of oxalyl chloride (516 µL, 5.91 mmol) in CH2Cl2
(16 mL) at -78 °C. The solution was stirred at -78 °C for 20
min, and then a solution of crude alcohol 8 (1.14 g, 1.97 mmol)
in CH2Cl2 (23 mL) was added at -78 °C at a moderate rate.
After the mixture stirred at -78 °C for 40 min, triethylamine
(2.47 mL, 17.7 mmol) was added slowly at -78 °C and then
the dry ice/acetone bath was removed. The mixture was
stirred at 0 °C for 30 min, the reaction quenched with 0.1 M
NaHSO4, and the mixture extracted in CH2Cl2 (2 × 50 mL)
with 0.1 M NaHSO4 (2 × 40 mL). Flash chromatography with
silica gel (gradient to 50% EtOAc/hexanes) gave aldehyde 9
(766 mg, 67% yield for two steps) as a colorless oil.
Ben zyl
2-O-Ben zyl-3-O-n eoh ep t yl-4-O-(p -m et h oxy-
Ack n ow led gm en t. T.D., X.D., and R.A. thank NIH
(Grant GM69674) and UCLA for financial support.
Computational resources from Professors Y. Rubin’s and
K. Houk’s groups and the Office of Academic Computing
at UCLA are greatly appreciated. C.S. thanks NIH
(Grant CA64983) for supporting the biological testing
and Staci N. Smith for conducting the assays.
ben zyl)-5-(d eh yd r oxym eth yl)-5-(2-m eth ylp r op -1-en yl)-r-
D-glu cop yr a n osid e (10). To a suspension of isopropyltri-
phenylphosphonium iodide (heated at 100 °C under high
vacuum overnight; 1.19 g, 2.75 mmol) in THF (25 mL) at 0 °C
was added nBuLi (1.31 mL, 2.54 mmol in pentane) dropwise.
The dark-blood-red mixture was stirred at 0 °C for 5 min and
then at 25 °C for 30 min. The mixture was recooled to 0 °C,
a solution of aldehyde 9 (730 mg, 1.27 mmol) in THF (15 mL)
was added slowly, and the pretty dark-red mixture was stirred
at 0 °C for 3 h. After the reaction was quenched with water,
much THF was removed in vacuo. Extraction in CH2Cl2 (2 ×
40 mL) with brine (40 mL) and flash chromatography with
silica gel (gradient to 14% EtOAc/hexanes) afforded olefin 10
(724 mg, 95% yield) as a colorless oil. Note: Do not expose
the phosphonium reagent or the ylide to air since the ylide is
easily quenched by air!
Ben zyl 2-O-Ben zyl-3-O-n eoh eptyl-4-(p-m eth oxyben zyl)-
6-d eoxy-6-isop r op yl-r-D- glu cop yr a n osid e (11). To a solu-
tion of olefin 10 (280 mg, 0.465 mmol) and benzenesulfonyl
hydrazide (1.60 g, 9.29 mmol) in 2-methoxyethanol (13 mL)
was slowly added 3 M NaOH (4.65 mL, 14.0 mmol). The
solution was refluxed for 15 h and then extracted in CH2Cl2
(2 × 40 mL) with brine (30 mL) followed by 0.1 M NaHSO4 (2
× 30 mL). 1H NMR of the crude product showed 10:11 ) 2.3:
1. After the crude product was submitted to a second cycle of
diimide reduction with the same amount of reagents, the 11:
10 ratio increased to 2.2:1. A third cycle of diimide reduction
using the same amount of reagents resulted in 11:10 ) 5.5:1.
Flash chromatography with silica gel (gradient to 10% EtOAc/
hexanes) provided isobutyl 11 (215 mg, 77% yield) as a
colorless oil. Note: Reaction of olefin 10 with 60 equiv of
Su p p or tin g In for m a tion Ava ila ble: Complete charac-
terization data of compounds 3-15, including specific rotation,
1
IR, HRMS, and H and 13C NMR (6 pages). Ordering informa-
tion is given on any current masthead page.
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