Synthesis, conformation and biology of naphthoxylosides

Article abstract of DOI:10.1016/j.bmc.2011.05.007

Proteoglycans (PG) are polyanionic proteins consisting of a core protein substituted with carbohydrate chains, that is, glycosaminoglycans (GAG). The biosynthesis of GAG can be manipulated by simple xylosides carrying hydrophobic aglycons, which can enter

Full text of DOI:10.1016/j.bmc.2011.05.007

Bioorganic & Medicinal Chemistry 19 (2011) 4114–4126
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, conformation and biology of naphthoxylosides
Anna Siegbahn a, Ulrika Aili a,b, Agata Ochocinska a, Martin Olofsson a, Jerk Rönnols c, Katrin Mani b,
Göran Widmalm c, Ulf Ellervik a,
⇑
a Center for Analysis and Synthesis, Chemical Center, Lund University, PO Box 124, SE-221 00 Lund, Sweden
b Department of Experimental Medical Science, Lund University, BMC A13, SE-221 84 Lund, Sweden
c Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91 Stockholm, Sweden
a r t i c l e i n f o
a b s t r a c t
Article history:
Proteoglycans (PG) are polyanionic proteins consisting of a core protein substituted with carbohydrate
chains, that is, glycosaminoglycans (GAG). The biosynthesis of GAG can be manipulated by simple xylo-
sides carrying hydrophobic aglycons, which can enter the cell and initiate the biosynthesis. While the
importance of the aglycon is well investigated, there is far less information on the effect of modifications
in the xylose residue.
Received 29 March 2011
Revised 29 April 2011
Accepted 4 May 2011
Available online 10 May 2011
We have developed a new synthetic protocol, based on acetal protection and selective benzylation, for
modification of the three hydroxyl groups in xylose. Thus we have synthesized twelve analogs of 2-naph-
thyl b-D-xylopyranoside (XylNap), where each hydroxyl group has been epimerized or replaced by meth-
Keywords:
Naphthoxylosides
Synthesis
Conformational analysis
Antiproliferative activity
Tumor cells
oxy, fluoro, or hydrogen.
To gain more information about the properties of xylose, conformational studies were made on some of
the analogs. It was found that the 4C1 conformation is highly predominant, accompanied by a nonnegli-
gible population of the 2S0 conformation. However, deoxygenation at C3 results in a large portion of the
1C4 conformation.
Glycosaminoglycan
The GAG priming ability and proliferation activity of the twelve analogs, were investigated using a
matched pair of human breast fibroblasts and human breast carcinoma cells. None of the analogs initi-
ated the biosynthesis of GAG, but an inhibitory effect on endogenous PG production was observed for
analogs fluorinated or deoxygenated at C4. From our data it seems reasonable that all three hydroxyl
groups in XylNap are essential for the priming of GAG chains and for selective toxicity for tumor cells.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
chain is later on modified by epimerization, deacetylation and
sulfation reactions, resulting in extensive structural diversity (
Outer surfaces of tumor cells are covered by a variety of gly-
cosaminoglycan chains (GAG) that are involved in the pathobiol-
ogy of all stages of cancer progression. Thereby, they can affect
cancer cell proliferation, tumor invasion and metastasis, and can-
cer stem cell differentiation.1–3 These long unbranched polyanion-
ic carbohydrate chains are covalently attached to proteins as
proteoglycans (PG). Two classes of GAG chains, namely condroitin
sulfate/dermatan sulfate (CS/DS) and heparan sulfate (HS) are
coupled to the protein by xylose, a highly unusual carbohydrate
in mammalian cells. The biosynthesis of GAG starts by coupling
of xylose to a serine residue of the protein followed by the
Fig. 1).
The knowledge on structure recognition of the antiproliferative
GAG is still scarce due to a lack of effective molecular tools to iden-
tify and correlate specific structures with functions. The structure
of PG/GAG, as well as the expression of enzymes involved in their
biosynthesis and degradation varies in normal and tumor cells and
there is ample evidence indicating an importance of GAG chain
structure for the control of cell proliferation and on aggressive tu-
mor phenotype. For example, Sasisekharan and co-workers previ-
ously showed that GAG fragments, released from tumor cell
surface, inhibit tumor growth and colonization in vitro as well as
in vivo by affecting tumor cell proliferation, apoptosis and
neovascularization.4
A problem with using GAGs as anti-tumor substances is the lim-
ited uptake of these highly polyanionic macromolecules. However,
simple xylosides carrying hydrophobic and uncharged aglycons
can enter cells and serve as primers for GAG formation, indepen-
dently of core protein synthesis.5,6 The xylosides initiate GAG syn-
thesis by serving as acceptors in the first galactosylation step. The
formation of
a linker tetrasaccharide (i.e., GlcAb1–3Galb1–
3Galb1–4Xylb–). The linker tetrasaccharide is further elongated
by addition of repeating disaccharides, for example, –4GlcAb1–
4GlcNAca1– for heparan sulfate (HS) or –4GlcAb1–3GalNAcb1–
for chondroitin sulfate/dermatan sulfate (CS/DS). The growing
⇑
Corresponding author.
E-mail address: ulf.ellervik@organic.lu.se (U. Ellervik).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.05.007

A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
4115
Figure 1. Proteoglycans are composed of glycosaminoglycan chains (GAG) linked to a core protein by the highly unusual carbohydrate xylose.
composition of GAG assembled on the xyloside primers depend
mainly on the structure of the aglycon, which may reflect selective
partitioning of primers into different intracellular compartments
or into different branches of biosynthetic pathways. In most cases,
priming of CS/DS dominates, and synthesis of free HS chains is low
or undetectable. Increased yields of HS, which has proven to be
important for anti-tumor therapy, can be obtained when the agly-
con of the xylosides comprises aromatic, polycyclic structures,
such as naphthol derivatives. These xyloside-primed GAG chains
can be retained inside the cells, but are usually secreted into the
medium, and show interesting biological properties, such as acti-
vation of basic fibroblast growth factor, antithrombotic effects,
and growth inhibition of transformed cells.7–11 We have previously
reported that the GAG priming XylNapOH (1a, Fig. 2) selectively
inhibits growth of tumor cells in vitro as well as in vivo.12 Treat-
ment with this xyloside at a pharmacologically relevant dose re-
duced the average tumor load by up to 97% in a SCID mice model.10
In a recent publication we used radiolabeled xylosides to
investigate the mechanism of these tumor selective xylosides.13
We could thus show that tumor cells treated with XylNapOH, se-
crete specific GAG chains. These GAG chains are then re-internal-
ized by both normal and cancer cells, and further on transported
to the nuclei where they induce apoptosis. We also showed that
XylNapOH treatment lowers the level of histone H3 acetylation
selectively in bladder and breast carcinoma cells without affect-
ing the expression of histone H3. The down-regulation of histone
H3 acetylation makes the transcription of DNA impossible and
the cells are forced into apoptosis, which is an explanation for
the toxic effects shown by these molecules. In contrary, XylNap-
OH-primed GAG chains from normal cells, or GAG chains from
the analogous XylNap (1b, Fig. 2), are not internalized and do
not cause growth retardation. We propose that, in vivo, the anti-
proliferative XylNapOH-primed GAG chains produced by tumor
cells inhibit tumor growth in an autocrine fashion by formation
of antiproliferative GAG chains on the xyloside pro-drug, while
surrounding normal cells produce no antiproliferative GAG
chains.
O
O
R
HO
HO
HO
HO
O
O
OH
R
1a: R = OH, XylNapOH
1b: R = H, XylNap
2a: R = OMe
2b: R = F
While the importance of the aglycon has been investigated in a
number of publications,12,14–22 there is far less information on the
effect of modifications in the xylose residue. In an article from
2c: R = epi-OH
2d: R = H
O
O
HO
R
1996, Esko et al. investigated a series of modified benzyl b-D-xylo-
O
O
R
HO
sides. These compounds were tested for priming of GAG chains in
Chinese hamster ovary (CHO) cells. Apart from the 3-fluoro- as
well as the 2- and 3-methoxy analogs, all compounds failed to ini-
tiate priming.
Being a pentose, rather than a hexose like glucose or galactose,
xylose poses unusual difficulties in selective protection of the three
hydroxyl groups and there is a need for uniform methods for the
OH
OH
3a: R = OMe
3b: R = F
4a: R = OMe
4b: R = F
3c: R = epi-OH
3d: R = H
4c: R = epi-OH
4d: R = H
Figure 2. Structures of XylNapOH, XylNap and analogs.

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A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
synthesis of analogs. The three hydroxyl groups are all secondary,
equatorial and thus of very similar reactivity. In the 70’s, Chalk
et al. reported the order of reactivity of hydroxyl groups in methyl
three different functionalities (OMe, F, deoxy) while others
remained unchanged. In addition, we synthesized the epimerized
products, thus altogether twelve new compounds (Fig. 2).
b-
OH > 2-OH.23 Contradictory results were published by Kondo a
decade later, who stated that tosylation of methyl b- -xylopyrano-
D-xylopyranoside on sulfonylation in pyridine as 4-OH > 3-
2. Results and discussion
2.1. Synthesis
D
side gave the order of reactivity 4-OH > 2-OH > 3-OH.24 Kishimoto
et al. observed the same trend while studying the reactivity to-
wards a b-4-O-type quinone methide.25 Later on, Helm and Ralph
described a method to introduce ester-type protecting groups in
xylose in a selective manner using Bu2SnO to direct the reactiv-
ity.26 They obtained mainly the 4-O-protected compounds of the
Three different strategies were evaluated for the introduction of
protective groups in xylose: BDA, partial benzylation and bulky si-
lyl groups (i.e., TBDPS).
Standard conditions for introduction of the BDA group, that is,
butane-2,3-dione, a catalytic amount of CSA, and CH(OMe)3 in
methanol, gave two inseparable mixtures of free 2-OH and 4-OH
with different stereochemistry of the BDA.33 The conditions were
changed to 2,2,3,3-tetramethoxybutane34 and a catalytic amount
of BF3ꢀOEt2 in acetonitrile, which gave the two BDA-protected
derivatives 5a and 5b in a 3:2 ratio in quantitative yield, without
mixed stereochemistry in the acetal (Scheme 1).
b-anomer while the
The stannylidene complexes of b-
a
-anomer gave the 2-O-protected species.
-xylopyranosides can also be
D
used to obtain selective chloroacetylation of 4-OH.27 Another
way to selectively protect the 4-OH is to use 2,3-anhydro-ribopy-
ranoside derivatives. Derivatization of the 4-position, and
subsequent opening of the 2,3-anhydro ring provides xylosides
with 2- and 3-position unprotected.28
Direct, regioselective phase transfer benzylation using BnBr,
QI and KOH (10%) in CH2Cl2, resulted in a separable mixture of
mainly di-protected compounds. 2-Naphthyl 2,4-di-O-benzyl-b-
It is also possible to use cyclic acetals such as isopropylidene ke-
tal26 or butanediacetal (BDA)29, which have been used to protect
xylosides, leading to two major products: 2,3- and 3,4-protected
sugars. Acetals are widely used as protecting groups but their
acid-lability might cause compatibility problems in some reac-
tions. Silicon protecting groups could alternatively be used, but
they are known to migrate under acidic conditions, leading to
undesired products.30,31 In addition, silicon groups are not stable
towards fluoride ions. The tert-butyl-diphenyl silyl (TBDPS) was
successfully employed in monoprotection of a trimethylsilylethyl
xyloside, giving mainly the 4-O-protected compound.32 However,
a general protective group strategy for all positions of xylose is
not presently available.
The aims of this work are: (i) to pin-point the requirements of
the xylose moiety for specific cell toxicity and GAG priming in a
matched normal and cancer cell pair, that is, CCD1095-SK (human
breast fibroblasts), HCC70 (human breast carcinoma cells); (ii) to
form a baseline for comparison of effects shown by naphthoxylo-
sides modified either in the sugar residue or the aromatic moiety;
(iii) to develop methodology for selective manipulations on xylose
and (iv) to investigate how modifications in the sugar residue af-
fect the conformation, and thereby properties, of naphthoxylo-
sides. Thus, each of the hydroxyl groups in 1b was replaced by
D
-xylopyranoside (6a) was obtained in 66%, 2-naphthyl 2,3-O-
di-benzyl-b- -xylopyranoside (6b) in 14% yield and 2-naphthyl
3,4-di-O-benzyl-b- -xylopyranoside (6c) in 6% yield.
D
D
Finally, TBDPS protection, using TBDPSCl, Et3N, and DMAP in
CH2Cl2, gave mainly compound 7c with the TBDPS group in posi-
tion 4 along with compound 7a and only trace amounts of com-
pound 7b.
Since the silyl protective group strategy was not very successful,
this route was abandoned. The BDA group is acid labile and in some
cases a loss of final product was observed in the deprotection step
due to cleavage of the naphthyl moiety. Because of the low yield of
6c in the benzylation reaction, we, therefore, decided to use the
benzyl group strategy for position 3 (Scheme 3) and 4 (Scheme
4) and to use the BDA route for position 2 (Scheme 2).
Subsequently, chemical transformations leading to target mole-
cules were performed. The most straightforward was the synthesis
of the methoxy derivatives and a similar procedure was employed
for each of the positions. The unprotected hydroxyl group was
deprotonated using sodium hydride and methyl iodide was intro-
duced to give the 2-OMe (8), 3-OMe (15) and 4-OMe compounds
MeO
OMe
O
Si
O
TBDPS =
HO
O
a
ONap
ONap
O
O
OH
O
OMe
5a (61%)
Bn =
OMe
5b (39%)
Nap =
b
c
O
O
O
BnO
HO
HO
BnO
BnO
1b
ONap
ONap
ONap
ONap
BnO
OBn
6a (66%)
OBn
OH
6b (14%)
6c (6%)
O
O
O
HO
HO
HO
TBDPSO
ONap
ONap
HO
TBDPSO
TBDPSO
7a (20%)
OH
7b (not isolated)
OH
7c (46%)
Scheme 1. Protective group strategies for xylose. Reagents and conditions: (a) 2,2,3,3-Tetramethoxybutane (2.2 equiv), BF3ꢀOEt2 (cat.), CH3CN, o.n.; (b) BnBr (2.2 equiv), QI
(2 equiv), KOH (10% aq), CH2Cl2, rt, 16 h; (c) TBDPSCl (2 equiv), Et3N (2 equiv), DMAP (1 equiv), CH2Cl2, 65 h.

A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
4117
OMe
R2
O
O
ONap
O
R1
OMe
5a: R1 = OH, R2 = H
ix
i
iii
9: R1 = OTf, R2 = H
8: R1 = OMe, R2 = H
13: R1 = OCS2CH3, R2 = H
iv
10: R1 = H, R2 = OAc
11: R1 = H, R2 = OH
12: R1 = F, R2 = H
v
x
O
vi
HO
HO
ONap
OCS2CH3
14
ii
vii
O
viii
xi
HO
O
O
O
HO
ONap
HO
HO
HO
HO
ONap
ONap
ONap
HO
HO
HO
F
OMe
2a
2b
2c
2d
Scheme 2. Transformation of position 2. Reagents and conditions: (i) MeI (2 equiv), NaH (2 equiv), DMF, 0 °C, N2, 16 h, 8 (89%); (ii) 95% TFA/CH2Cl2 1:1, 15 min, 2a (quant.);
(iii) Tf2O (2 equiv), pyridine (4 equiv), CH2Cl2, ꢁ78 to 0 °C, N2, 2.5 h; (iv) CsOAc (5 equiv), DMF, 50 °C, 3 d, 10 (82% over two steps); (v) 0.05 M NaOMe, 2.5 h, 11 (90%); (vi)
DAST (2 equiv), CH2Cl2, 40 °C, N2, 2 h, 12 (42%); (vii) 95% TFA/CH2Cl2 1:7, 15 min, 2b (90%); (viii) 95% TFA/CH2Cl2 1:7, 15 min, 2c (21%); (ix) (a) NaH (2 equiv), imidazole (cat.),
THF, 0 °C to rt, 45 min; (b) CS2 (9 equiv), N2, rt, 1 h; (c) MeI (3 equiv), rt, N2, 21 h, 13 (80%); (x) 95% TFA/CH2Cl2 1:1, 15 min, 14 (86%);(xi) AIBN (cat.), n-Bu3SnH (1.5 equiv),
toluene, 1 h, 2d (30%).
Bn =
O
BnO
R1
ONap
OBn
R2
Nap =
6a: R1 = OH, R2 = H
viii
i
iii
16: R1 = OTf, R2 = H
15: R1 = OMe, R2 = H
19: R1 = H, R2 =H
iv
17: R1 = H, R2 = OH
18: R1 = F, R2 = H
vi
v
ii
vii
ix
O
O
O
O
HO
HO
MeO
HO
HO
ONap
ONap
ONap
ONap
F
OH
OH
OH
OH
3c
OH
3a
3b
3d
Scheme 3. Transformation of position 3. Reagents and conditions: (i) MeI (2 equiv), NaH (2 equiv), DMF, 0 °C, N2, 16 h 15 (98%); (ii) 10% Pd/C, HCl (21.7 equiv), DMF, H2, rt, 3a
(94%) (iii) Tf2O (1.5 equiv), pyridine (3 equiv), CH2Cl2, ꢁ10 °C, N2, 2 h; (iv) QNO2 (4 equiv), DMF, 50 °C, N2, 16 h, 17 (54%); (v) DAST (2.5 equiv), CH2Cl2, ꢁ40 °C, N2, 1 h; MeOH,
20 min, 18 (47%); (vi) 10% Pd/C, HCl (21.7 equiv), DMF, H2, rt, 3b (94%); (vii) 10% Pd/C (HCl (21.7 equiv), DMF, H2, rt, 3c (73%); (viii) (a) NaH (2 equiv), imidazole, THF, 0 °C–rt,
45 min; (b) CS2 (10 equiv), 1 h, N2, rt, 1 h; (c) MeI (3 equiv), rt, N2, 16 h; (d) AIBN (0.8 equiv), n-Bu3SnH (15 equiv), toluene,
D, 40 min, 19 (68%); (ix) 10% Pd/C, HCl (21.7 equiv),
DMF, H2, rt, 3d (85%).
(20).35 The yields of these one-step procedures were almost
quantitative.
to be modified. The 3-F compound was obtained after reacting at
ꢁ40 °C, whereas formation of the 2-F and 4-F required heating to
+40 °C. This diversity suggests different reactivity for each of the
groups, which may be caused by conformation.
Deoxygenation reactions were performed using a two-step
method, that is, xanthate formation followed by Barton–McCombie
radical deoxygenation.35 Xanthates were synthesized from carbon
disulfide and methyl iodide that were reacted with deprotonated
hydroxide in positions 2, 3 or 4. Yields varied from modest to good.
These intermediates were then deoxygenated using AIBN and n-
Bu3SnH in toluene at reflux.
The fluoro- and epi-analogs were obtained from analogous reac-
tion sequences. First, 6a and 6b were reacted with trifluorome-
thane sulfonic anhydride. The resulting triflates (16 and 21) were
inverted to hydroxyl groups in the presence of quaternary nitrite
salt in DMF at 50 °C36 to give 17 and 22 in 54% and 50% yield,
respectively. For position 2, a reaction using QNO2 gave only 24%
of the desired product 11. Thus the inversion was performed using
CsOAc instead of the quaternary nitrite salt, to give the acetate 10
in 82% over two steps.37 The acetate was then hydrolyzed using
Zemplén conditions to give compound 11. To form the fluorinated
derivatives 12 (42%), 18 (47%), and 23 (41%), the above reaction se-
quence was extended by the addition of DAST.38 Interestingly,
depending on the position, the temperature of the reaction had
For position 2, the xanthate was removed after the deprotection
since the regular order led to degradation of the material, indicat-
ing that the xylopyranoside is more acid-labile when the hydroxyl
group in position 2 is removed.

4118
A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
R2
Bn =
R1
O
ONap
BnO
OBn
Nap =
6b: R1 = OH, R2 = H
viii
i
iii
21: R1 = OTf, R2 = H
20: R1 = OMe, R2 = H
24: R1 = H, R2 = H
iv
22: R1 = H, R2 = OH
23: R1 = F, R2 = H
vi
v
ii
vii
O
ix
OH
HO
O
O
O
F
MeO
HO
ONap
ONap
ONap
ONap
HO
HO
OH
OH
OH
OH
4a
4c
4b
4d
Scheme 4. Transformation of position 4. Reagents and conditions: (i) MeI (2 equiv), NaH (2 equiv), DMF, 0 °C, N2, 16 h; (ii) 10% Pd/C, HCl (21.7 equiv), DMF, H2, rt, 4a (66%);
(iii) Tf2O (1.5 equiv), pyridine (3 equiv), CH2Cl2, ꢁ10 °C, N2, 2 h; (iv) QNO2 (4 equiv), DMF, 50 °C, N2, 16 h 22 (50%); (v) DAST (2.5 equiv), CH2Cl2, ꢁ40 °C, N2, 1 h; MeOH, 20 min,
23 (41%); (vi) 10% Pd/C, HCl (21.7 equiv), DMF, H2, rt, 4b (61%); (vii) 10% Pd/C, HCl (21.7 equiv), DMF, H2, rt, 4c (33%); (viii) (a) NaH (2 equiv), imidazole, THF, 0 °C to rt, 45 min;
(b) CS2 (10 equiv), 1 h, N2, rt, 1 h; (c) MeI (3 equiv), rt, N2, 16 h; (d) AIBN (0.8 equiv), n-Bu3SnH (15 equiv), toluene,
D
, 40 min, 24 (93%); (ix) 10% Pd/C, HCl (21.7 equiv), DMF,
H2, rt, 4d (45%).
n
The BDA protecting group was removed by treatment with 95%
TFA in CH2Cl2 for a short time and gave compounds 2a–d in modest
to quantitative yields. The benzylated compounds were deprotec-
ted using 10% Pd/C (Degussa type) under atmospheric pressure of
hydrogen39 to give 3a–d and 4a–d in modest to high yields.
refined together with JHH coupling constants using a total line-
shape analysis as implemented in the PERCH NMR spin simulation
software.40 The 1H and 13C chemical shifts of the xylose residues in
1a, 1a (Na), 1b, 2a, 3a, 3b, 3d and 4a are compiled in Table 1. The
chemical shifts are similar but changes due to substituents, atomic
replacement or deoxygenation lead to conspicuous differences. The
nJHH coupling constants differ suggesting conformational changes,
both small and large, between the seven xylosides.
2.2. Conformation
1D 1H and 13C experiments together with a number of 2D 1H,
1H- and 1H, 13C-correlated experiments were used to assign 1H
and 13C NMR chemical shifts of xylosides 1a, 1b, 2a, 3a, 3b, 3d,
4a and the monodeprotonated form of 1a, 1a (Na), in methanol
as a solvent. The 1H NMR chemical shifts were subsequently
Analysis of the conformational space available to the xylosides
is herein based on three-bond 1H,1H spin–spin coupling constants.
To this end molecular mechanics models were built in canonical
ring conformations and subsequently energy minimized. A hexo-
pyranose residue may be described by a pseudorotational wheel
Table 1
dH,
nJHH, dC of xylosides in methanol-d4 at 37 °C and sugar ring populations. For methylene groups the 1H chemical shift and 3JHH of the pro-R proton is given prior to that of the
pro-S proton
Compound
1
2
3
4
5
Me
4C1
2S0
5.3
1C4
0
1a
4.942
7.530
103.51
4.865
7.419
104.08
5.029
7.492
103.05
5.079
7.451
103.07
5.041
7.387
103.03
5.064
7.530
102.50
5.271
4.513
101.60
5.024
7.325
102.93
3.479
9.082
74.87
3.456
9.032
74.96
3.508
9.104
74.83
3.199
9.067
84.70
3.575
8.902
74.38
3.742
8.805
73.25
3.802
7.081, 4.046
68.40
3.527
8.933
74.84
3.450
8.805
77.81
3.437
8.704
77.87
3.471
8.803
77.78
3.483
8.876
77.36
3.199
8.611
87.27
4.346
8.553
97.81
1.828, 2.353
6.873, 3.860
35.47
3.545
8.030
76.79
3.596
5.356, 10.180
71.14
3.585
5.332, 10.128
71.22
3.611
5.355, 10.125
71.11
3.616
5.391, 10.100
71.12
3.668
5.320, 9.966
70.72
3.865
5.719, 10.274
69.41
3.856
3.132, 5.652
66.05
3.307
5.161, 9.765
80.78
3.959, 3.404
ꢁ11.477
66.97
3.948, 3.363
ꢁ11.443
66.94
3.977, 3.447
ꢁ11.454
66.99
3.959, 3.416
ꢁ11.451
66.93
3.955, 3.444
ꢁ11.485
66.86
3.999, 3.467
ꢁ11.667
65.76
4.002, 3.545
ꢁ11.608
68.26
4.154, 3.421
ꢁ11.565
64.39
94.7
1a (Na)
1b
94.0
93.7
94.4
92.4
95.9
48.0
90.6
6.0
6.3
5.6
7.6
4.1
0
0
0
2a
3.695
0
61.10
3.683
3a
0
60.91
3ba
3db
4a
0
52.0
0
3.519
59.02
9.4
a
3
3
1
2JF,H3 = 52.427 Hz, JF,H2 = 13.391 Hz, JF,H4 = 13.275 Hz, JF,C3 = 184.1 Hz.
b
4
4
2JH3(pro-R),H3(pro-S) = ꢁ13.538 Hz, JH3(pro-R),H5(pro-S) = ꢁ1.174 Hz, JH3(pro-S),H5(pro-R) = ꢁ1.162 Hz.

A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
4119
Table 2
and the itinerary between different conformations in describing
the conformational space available to it.41 Since xylopyranose is
lacking the primary hydroxymethyl group present in glucose one
may anticipate enhanced ring flexibility and several conformations
need to be considered, in particular the 4C1 conformation, various
skew conformations and the 1C4 conformation. However, the boat,
half-chair and envelope conformations are higher in potential en-
ergy and will not be considered in the present analysis.42 The
three-state equilibrium between the two chair conformers and
one of the skew conformers for each of the six skew conformers
Antiproliferative activity and retention times for compounds 1b–4d. The antiprolif-
erative activity was recorded towards CCD1095-SK cells and HCC70 cells and
expressed as ED50-values (
lM)
Compound Modification
Antiproliferative
Retention time (min)
activity (ED50
, lM)
CCD1095-SK
HCC70
1b
2a
2b
2c
2d
3a
3b
3c
3d
4a
4b
4c
4d
—
OMe
F
243
55
91
127
22
24.66 0.022
31.13 0.030
32.57 0.023
23.61 0.004
29.57 0.002
27.22 0.016
28.66 0.009
27.95 0.015
30.06 0.052
29.20 0.007
28.64 0.075
22.85 0.011
26.97 0.058
12
50
19
17
Epi
363
228
145
94
3
was analyzed using the five JHH coupling constants in the pyra-
Deoxy
OMe
F
nose ring. For the xylosides 1a, 1b, 2a, 3a, 3b and 4a the 4C1 con-
formation is highly predominant and of the skew conformations
only a contribution of the 2S0 conformation gave a better agree-
72
Epi
182
79
178
179
135
37
Deoxy
OMe
F
Epi
Deoxy
n
ment between the JHH coupling constants from the molecular
93
32
48
mechanics models and the experimentally determined ones, sug-
gesting its presence in a conformational equilibrium, which has
also been proposed for b-D-xylopyranose based on modeling with
371
284
285
the MM3 program.43 Notably, there exists a nonnegligible popula-
tion of the 2S0 conformation in 1a, 1a (Na), 1b, 2a, 3a, 3b and 4a,
that is, >4% up to almost 10%, with an evident increase in popula-
tion as the methoxy group substitution is shifted away from the
anomeric center. We conclude that the 2S0 conformation is present
in equilibrium with the 4C1 conformation (Scheme 5) in the seven
studied xylosides (Table 1). The conformational equilibrium of the
3
3-deoxy-analogue 3d was also investigated, using seven JHH cou-
pling constants in the pyranose ring. Interestingly, a two-state
equilibrium between 4C1 and 1C4 is then present in which the
two conformations are equally populated (Scheme 5 and Table 1).
For conformational analysis the xylopyranosides were dissolved
in methanol, which like the hydroxyl groups of the solute mole-
cules can act as both hydrogen bond donors and acceptors. Except
for the deoxygenations the structural modifications retain hydro-
gen-bonding abilities. In methyl a- and b-D-xylopyranoside the sol-
vent structure and hydroxyl group conformational preferences in
methanol have been investigated by molecular dynamics simula-
tions.44 The simulations showed solvent structuring around the
solute molecule with the hydroxyl groups of methanol donating
hydrogen bonds to the hydroxyl groups of the methyl xylopyrano-
sides. The hydroxyl groups of the solute molecules are arranged in
a counter-clockwise orientation, that is, towards the glycosidic
oxygen. This structural network will upon substitution or epimer-
ization of the xyloside hydroxyl groups be perturbed and can be al-
tered to different extent. The relative importance of the anomeric
effect, 1,3-diaxial interactions of electronegative groups and poten-
tial intraresidue hydrogen bonds may subsequently be ascertained
by future molecular dynamics simulations. The present investiga-
tion has shown that conformational equilibria need to be consid-
Figure 3. Relative gradient HPLC retention times of compounds 2a–4d compared to
the unmodified 1b (the retention time for 1b, 24.66 min, has been subtracted from
the actual retention times).
Gradient HPLC retention times can be used to substitute log P
values.45 The gradient HPLC retention times for the compounds
were measured using a C-18 column and a mobile phase of water
(0.1% TFA) with a gradient of MeCN from 1 min increasing by 1.2%
per minute. The retention times were measured for three separate
runs per compound, and the calculated mean retention times are
presented in Table 2. To visualize the polarities of the compounds,
the gradient HPLC retention times relative to the unmodified 1b
were plotted (Fig. 3). In general, the exchange of a hydroxyl group
for methoxy, fluorine or hydrogen, results in less polar compounds.
However, the analogs epimerized in position 2 or 4 are more polar,
which indicates that the hydroxyl groups are more available for
interaction with water. In contrary, modifications in position 3
do not give large changes in polarity and the epimerized analog
3c is actually less polar than the methoxy analog.
ered for the b-D-xylopyranosides and analogs thereof.
ONap
HO
HO
O
HO
HO
ONap
ONap
2.3. Biology
O
OH
4C1
OH
2SO
For determination of GAG priming and proliferation activity, a
matched pair of normal breast fibroblasts (CCD-1095Sk) and hu-
man breast carcinoma cells (HCC70 cells) were used. Untreated
breast carcinoma cells secreted large sized PG and medium sized
ONap
O
HO
O
GAG into the culture medium. Treatment with 2-naphthyl b-
D-
OH
xylopyranoside (1b) or 2-(6-hydroxynaphthyl) b- -xylopyranoside
D
OH
OH
(1a) initiated biosynthesis of free, regular-size GAG chains that
were secreted into the medium as expected. Furthermore, the size
of GAG chains produced by 1a was smaller compared to the size of
4C1
1C4
Scheme 5. Conformational behavior of xylose analogs 1b (top) and 3d (bottom).

4120
A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
GAG chains produced by 1b to some extent. In contrary, the twelve
analogous compounds (2a–4d) used in this study did not induce
priming of GAG chains.
As previously reported b-D-xylosides with hydrophobic agly-
cones can initiate synthesis of GAG chains in most cell types, and
at sufficiently high xyloside concentration endogenous PG synthe-
sis is competitively inhibited. We observed a small inhibitory effect
on endogenous PG production for compounds 4b and 4d (33% and
24%, respectively, Fig. 4).
During the formation of the linker tetrasaccharide, the xylose
residue is galactosylated at C4 (3Galb1–4Xylb). If the acceptor hy-
droxyl group at C4 is missing, as in the case of 4b and 4d, the GAG
chain elongation can be anticipated to decline. Kuberan and co-
workers have published a series of 4-deoxy-4-fluoro-xylosides
with various triazole-based aglycons with the aim of finding a
selective GAG inhibitor.46,47 By comparing the elution profile of
PG/GAG extracts from untreated CHO cells with the elution profiles
of PG/GAG extracts from CHO cells that were treated with 4-deoxy-
4-fluoro-xylosides, it was found that some of the xylosides were
GAG inhibitors, showing inhibition by up to ꢂ90% after treatment
at 1 mM of the xylosides.
To further investigate the inhibitory effect shown by naphtho-
xylosides fluorinated in position 4 we incubated HCC70 cells
with the GAG primer 1a, which gave a strong priming of GAG
chains ( Fig. 5, solid black line). With the addition of 1 equiv
(0.1 mM) of the 4-fluoro analog 4b, the amount of GAG chains
decreased by 23% (Fig. 5, dotted red line).
To establish the antiproliferative effects of these compounds,
2a–4d were added to the growth medium at increasing concentra-
tions, and cell proliferation was recorded using a crystal violet
method.9,10 The inhibitory effect of the compounds are expressed
as ED50 (M) scored after 96 h of exposure (Table 2).
Figure 6. Relative antiproliferative activity of compounds 2a–4d in (a) CCD1095-SK
(normal) cells and (b) HCC70 (tumor) cells.
To compare the antiproliferative effects of these compounds in
the two cell lines, the ED50 value for the reference compound (Xyl-
Nap, 1b) was subtracted from the ED50 value for each compound
and the relative ED50 values were plotted (Fig. 6). Thus, negative
values indicate a stronger antiproliferative effect compared to the
reference 1b.
In general, the normal cells showed a higher sensitivity to-
wards these compounds, compared to the reference 1b but no
structure-activity relationships could be deduced from these
data. In contrary, tumor cells were less sensitive and only the
three fluoro analogs showed toxicity comparable to 1b.
We have earlier observed a correlation between toxicity of var-
ious xylosylated dihydroxynaphthalenes and the polarity, mea-
sured as gradient HPLC retention times. However, we could not
find any correlations between the ED50 values found for these com-
pounds and their retention times.
Figure 4. Priming of PG and GAG in HCC70 cells incubated with 0.1 mM 4b (dotted
blue line) or 0.1 mM 4d (dotted red line). The solid line shows the result for
untreated cells.
3. Conclusions
We have developed a new synthetic protocol, based on acetal
protection and selective benzylation, for modification of all three
positions in xylose. Using this protocol we have synthesized ana-
logs where each hydroxyl group has been epimerized or replaced
with methoxy, fluoro or hydrogen.
We have investigated the conformational behavior of some of
these compounds and found that they usually adopt a normal
4C1-conformation, accompanied by a nonnegligible population of
the 2S0 conformation. However, deoxygenation at position 3 results
in a significant amount of the 1C4-conformation. The conforma-
tional flexibility of the compounds epimerized at C2 and at C3, that
is, 2c and 3c, may be even more complex as suggested by a preli-
minary analysis of NMR data and these conformational equilibria
will be addressed in a future study.
Figure 5. Priming of GAG in HCC70 cells incubated with 1a (solid black line) and
equimolar amounts of 1a and 4b (dotted red line).

A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
4121
We have earlier shown that some monoxylosylated dihydroxy-
naphthalenes, especially 2-(6-hydroxynaphthyl) b- -xylopyrano-
1H, J = 7.4, 3.5, C1–H), 4.29 (ddd, 1H, J = 51.2, 8.5, 7.5, C2–H),
3.98 (ddd, 1H, J = 11.3, 5.2, 1.2, C5–H), 3.72 (dt, 1H, J = 15.4, 8.8,
C3–H), 3.67 (dt, 1H, J = 10.0, 5.2, C4–H), 3.49 (dd, 1H, J = 11.3,
10.1, C5–H); 13C NMR (MeOH-d4): d 156.1, 135.8, 131.4, 130.5,
128.6, 128.2, 127.5, 125.4, 119.8, 112.0, 100.3 (d, J = 23.5 Hz),
D
side (XylNapOH, 1a), show selective toxicity versus tumor cells
and that the selective activity is dependent on priming of soluble
GAG chains, especially heparan sulfate. With this investigation
we conclude that it seems difficult to reestablish the tumor selec-
tive antiproliferative activity by modifying the xylose residue in
naphthoxylosides. None of the tested analogs 2a–4d, showed prim-
ing of soluble GAG chains and consequently no selective toxicity.
Furthermore, our data indicate that normal and tumor cells re-
act differently to these compounds, with the normal cells being
more sensitive. The only compounds that showed increased toxic-
ity in tumor cells were the fluorinated analogs. Some of these fluo-
rinated analogs were shown to be inhibitors of the biosynthesis of
PG as well as of the biosynthesis of soluble GAG chains primed
from XylNap.
93.4 (d, J = 186.9 Hz), 75.2 (d, J = 18.2 Hz), 70.7 (d, J = 7.8 Hz),
+
66.9; HRMS calcd for
279.1066.
C
15H16FO4 [M+H]: 279.1033; found:
4.1.3. 2-Naphthyl b-D-lyxopyranoside (2c)
This was prepared from 11 as described for 2a but with 1 mL
TFA instead of 8 mL (21%). ½a D20
ꢃ
ꢁ99° (c 0.6, CH3OH); 1H NMR
(MeOH-d4): d 7.81–7.73 (m, 3H, ArH), 7.46 (d, 1H, J = 2.4, ArH),
7.45–7.32 (m, 2H, ArH), 5.45 (d, 1H, J = 2.5, C1–H), 4.14–4.08 (m,
2H, C2–H and C5–H), 3.87 (ddd, 1H, J = 9.7, 6.1, 3.5, C4–H), 3.78
(dd, 1H, J = 6.5, 3.5, C3–H), 3.44 (dd, 1H, J = 11.7, 5.9, C5–H); 13C
NMR (MeOH-d4): d 156.5, 135.9, 131.2, 130.3, 128.6, 128.1, 127.4,
125.2, 120.1, 111.9, 99.9, 73.5, 69.6, 69.3, 63.9; HRMS calcd for
4. Experimental section
4.1. Synthesis
+
C15H16NaO5 [M+Na]: 299.0895; found: 299.0882.
4.1.4. 2-Naphthyl 2-deoxy-b-D-threopyranoside (2d)
n-Bu3SnH (0.061 mL, 0.23 mmol) in toluene (3 mL) was heated
to reflux. 14 (55 mg, 0.15 mmol) in toluene (7 mL) was then added
dropwise, followed by AIBN (2.5 mg, 0.015 mmol). The mixture
was then stirred for 1 h, after which it was evaporated and chro-
NMR spectra were recorded with a Bruker Avance II operating
at 294 K. 1H NMR spectra were assigned using COSY (2D homonu-
clear shift correlation) with a gradient selection. Chemical shifts
are given in ppm downfield from Me4Si, with reference to residual
CHCl3 (7.26) or MeOH-d4 (3.31). Coupling constant values are given
matographed (CH2Cl2/MeOH 20:1) to give 2d (11.5 mg, 30%). ½a D20
ꢃ
ꢁ62° (c 0.28, CH3OH); 1H NMR (MeOH-d4): d 7.80–7.72 (m, 3H,
ArH), 7.45–7.38 (m, 1H, ArH), 7.36–7.31 (m, 2H, ArH), 7.21 (dd,
1H, J = 8.9, 2.4, ArH), 5.44 (dd, 1H, J = 7.8, 2.6, C1–H), 4.05 (dd,
1H, J = 11.4, 4.3, C5–H), 3.76–3.68 (m, 1H, C3–H), 3.57–3.48 (m,
1H, C4–H), 3.42 (dd, 1H, J = 11.4, 8.5, C5–H), 2.37 (ddd, 1H,
J = 13.0, 4.7, 2.7, C2–H), 1.82 (ddd, 1H, J = 13.0, 9.8, 7.9 Hz, C2–H);
13C NMR (MeOH-d4): d 156.2, 135.9, 131.2, 130.3, 128.6, 128.0,
127.4, 125.2, 119.9, 111.6, 98.8, 71.7, 71.2, 66.0, 38.3; HRMS calcd
in Hz. Mass spectra were recorded on Micromass Q-Tof microTM
.
Optical rotations were measured on Perkin Elmer instruments,
Model 341 polarimeter. Agilent 1100 Series HPLC with Thermo Sci-
entific ODS-2 HYPERSIL C18 column was used to record retention
times for log P correlation. Reactions were monitored by TLC using
alumina plates coated with silica gel and visualized using UV light
or by charring with para-anisaldehyde. Preparative chromatogra-
phy was performed with silica gel (35–70 lm, 60 Å). All the sol-
+
for C15H16NaO4 [M+Na]: 283.0946; found: 283.0976.
vents were dried prior to use unless otherwise stated. The
purchased reagents were used without further purification.
Synthesis and physical characterization of compound 1b has
been described.17 A simplified synthetic route was used that
comprised treatment of a mixture of 1,2,3,4-tetra-O-acetyl-b-
4.1.5. 2-Naphthyl 3-methoxy-b-D-xylopyranoside (3a)
10% Pd/C (Degussa type) (14 mol %) (91 mg, 0.04 mmol) was
suspended in DMF (8 mL) and purged with H2 vacuum-pumped
into the reaction vessel. HCl (37%) (21.7 equiv, 0.206 mL,
6.73 mmol) in DMF (8 mL) was added and H2 was re-introduced.
The resulting mixture was pre-conditioned for 10 min after which
time the substrate (144 mg, 0.31 mmol) was added in DMF (15 mL)
followed by H2. The mixture was then stirred for 4 h. The reaction
was then quenched with Et3N (0.288 mL), filtered through Celite
washing with EtOAc. The crude mixture was washed twice with
water, extracted from EtOAc. Combined organic layers were
washed with brine and dried over MgSO4, evaporated and purified
by flash chromatography (heptane/EtOAc, 3:1 to 1:1) yielding 3a
D
-xylopyranose and 2-naphthol in CH2Cl2 with BF3ꢀOEt2 followed
by de-O-acetylation. The analyses of the products were in accor-
dance with published data.
4.1.1. 2-Naphthyl 2-methoxy-b-D-xylopyranoside (2a)
Compound 8 (150 mg, 0.37 mmol) was dissolved in CH2Cl2
(8 mL) and 95% TFA (8 mL) was added. After 15 min, the red solu-
tion was evaporated and the remaining crude was dissolved in
Et2O (10 mL) and washed twice with NaHCO3 (sat. aq). The aque-
ous layers were extracted with Et2O and the combined organic lay-
ers were dried over MgSO4, filtered, concentrated, and
(94%). ½a 2D0
ꢃ
ꢁ37° (c 0.2, MeOH); 1H NMR (MeOH-d4) d 7.84–7.52
chromatographed (CH2Cl2/MeOH 20:1) yielding 2a (quant.). ½a D20
ꢃ
(m, 3H, ArH), 7.51–7.24 (m, 4H, ArH), 5.05 (d, 1H, J = 7.5, C1–H),
3.96 (dd, 1H, J = 11.4, 5.4, C5–H), 3.69 (s, 3H, C3–OCH3), 3.63–
3.68 (m, 1H, C4–H), 3.57 (dd, 1H, J = 8.9, 7.5, C2–H), 3.46 (dd, 1H,
J = 11.4, 10.1, C5–H), 3.20 (t, 1H, J = 8.9, C3–H); 13C NMR (MeOH-
d4) 156.6, 131.3, 130.4, 128.6, 128.1, 127.4, 125.3, 120.0, 111.9,
102.9, 87.3, 74.4, 70.7, 66.9, 61.1; HRMS calcd for C16H18O5Na+
[M+Na]: 313.1046; found: 313.1210.
ꢁ28° (c 0.78, CH3OH); 1H NMR (MeOH-d4): d 7.79 (m, 3H, ArH),
7.40 (m, 3H, ArH), 7.23 (dd, 1H, J = 8.9, 2.5, ArH), 5.08 (d, 1H,
J = 7.5, C1–H), 3.96 (dd, 1H, J = 11.3, 5.2, C5–H), 3.70 (s, 3H,
OCH3), 3.62 (ddd, 1H, J = 10.1, 8.9, 5.3, C4–H), 3.48 (t, 1H, J = 9.0,
C3–H), 3.42 (dd, 1H, J = 11.3,10.2, C5–H), 3.18 (dd, 1H, J = 9.0, 7.5,
C2–H); 13C NMR (MeOH-d4) d 156.4, 135.9, 131.3, 130.5, 128.6,
128.1, 127.5, 125.3, 119.8, 111.8, 102.9, 84.7, 77.3, 71.1, 66.9,
+
4.1.6. 2-Naphthyl 3-deoxy-3-fluoro-b-D-xylopyranoside (3b)
61.2; HRMS calcd for C16H18NaO5 [M+Na]: 313.1052; found:
This was prepared from 18 as described for 3a (94%). ½a D20
ꢁ23°
ꢃ
313.2138.
(c 0.3, MeOH); 1H NMR (MeOH-d4) d 7.87–7.75 (m, 3H, ArH), 7.50–
7.24 (m, 4H, ArH), 5.07 (d, 1H, J = 7.6, C1–H), 4.35 (dt, 1H, J = 52.5,
8.7, C3–H), 4.00 (ddd, 1H, J = 11.4, 6.1, 5.6, C5–H), 3.82–3.92 (m,
1H, C4–H), 3.74 (ddd, 1H, J = 16.4, 8.7, 7.6, C2–H), 3.48 (ddd, 1H,
J = 11.4, 10.3, 1.0, C5–H); 13C NMR (MeOH-d4) d 135.8, 131.4,
130.4, 128.6, 128.1, 127.4, 125.4, 120.0, 112.0, 102.3 (J = 11.3),
4.1.2. 2-Naphthyl 2-deoxy-2-fluoro-b-D-xylopyranoside (2b)
This was prepared from 12 as described for 2a but with 1 mL
TFA instead of 8 mL (90%). ½a D20
ꢃ
ꢁ23° (c 0.56, CH3OH); 1H NMR
(MeOH-d4): d 7.82–7.75 (m, 3H, ArH), 7.47–7.41 (m, 1H, ArH),
7.39–7.33 (m, 2H, ArH), 7.23 (dd, 1H, J = 8.9, 2.5, ArH), 5.29 (dd,

4122
A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
97.8 (J = 183.4), 73.2 (J = 18.3), 69.4 (J = 18.0), 65.7 (J = 9.4); HRMS
4.1.13. (1R, 3S, 4S, 6R, 9S, 10R)-10-Hydroxy-3,4-dimethoxy-3,4-
dimethyl-9-(2-naphthyl)oxy-2,5,8-trioxabicyclo[4.4.0]decane
(5a) and(1S, 3R, 4R, 6R, 7S, 10R)-10-hydroxy-3,4-dimethoxy-3,4-
dimethyl-7-(2-naphthyl)oxy-2,5,8-trioxabicyclo[4.4.0]decane
(5b)
calcd for C15H15FO4Na+ [M+Na]: 301.0847; found: 301.1021.
4.1.7. 2-Naphthyl b-D-ribopyranoside (3c)
This was prepared from 17 as described for 3a (73%). ½a D20
ꢁ124°
ꢃ
(c 1.1, MeOH); 1H NMR (MeOH-d4) d 7.80–7.74 (m, 3H, ArH), 7.46–
7.41 (m, 2H, ArH), 7.37–7.33 (m, 1H, ArH), 7.24 (dd, 1H, J = 9.0, 2.5,
ArH), 5.59 (d, 1H, J = 4.0, C1–H), 4.08 (app. t,1H, J = 3.2, C3–H),
3.79–3.91 (m, 4H, C2–H, C4–H and C5–H); 13C NMR (MeOH-d4) d
155.9, 135.9, 131.2, 130.4, 128.6, 128.1, 127.4, 125.2, 119.8,
111.8, 100.5, 72.4, 70.4, 68.3, 65.6; HRMS calcd for C15H16O5Na+
[M+Na]: 299.0895; found: 299.1111.
Compound 1b (800 mg, 2.90 mmol) was dissolved in dry CH3CN
(40 mL) under N2 atmosphere. 2,2,3,3-Tetramethoxybutane
(1.010 mL, 5.79 mmol) was added to the solution, followed by
addition of BF3ꢀOEt2 (0.10 mL, 0.84 mmol) and the solution got
dark brown. After 18 h, Et3N (4 mL, 29 mmol) was added and the
dark brown color partly disappeared. The mixture was then con-
centrated and purified by flash chromatography (heptane/EtOAc
2:1). 5a and 5b were obtained as white crystals in 61% and 39%,
4.1.8. 2-Naphthyl 3-deoxy-b-
D
-erythropyranoside (3d)
respectively. 5a: ½a D20
ꢃ
81° (c 1.2, CHCl3); 1H NMR (CDCl3): d 7.82–
This was prepared from 19 as described for 3a (85%). ½a D20
ꢃ
ꢁ116°
7.74 (m, 3H, ArH), 7.49–7.43 (m, 1H, ArH), 7.42–7.36 (m, 2H,
ArH), 7.28 (dd, 1H, J = 9.0, 2.5, ArH), 5.04 (d, 1H, J = 7.3, C1–H),
4.01 (dd, 1H, J = 10.8, 4.9, C5–H), 3.95–3.86 (m, 2H, C4–H and
C2–H), 3.79 (t, 1H, J = 9.7, C3–H), 3.60 (t, 1H, J = 10.6, C5–H), 3.34
(s, 3H, OCH3), 3.30 (s, 3H, OCH3), 2.49 (d, 1H, J = 2.4, C2–OH),
1.40 (s, 3H, CH3), 1.32 (s, 3H, CH3); 13C NMR (CDCl3): d 154.7,
134.3, 130.3, 129.8, 127.8, 127.4, 126.6, 124.7, 119.1, 112.0,
(c 1.7, MeOH); 1H NMR (MeOH-d4) d 7.83–7.74 (m, 3H, ArH), 7.48–
7.24 (m, 4H, ArH), 5.26 (d, 1H, J = 4.8, C1–H), 3.85 (m, 1H, C4–H),
4.00 (ddd, 1H, J = 11.5, 3.2, 1.0, C5–H), 3.80 (ddd, 1H, J = 8.0, 4.8,
3.9, C2–H), 3.54 (ddd, 1H, J = 11.5, 5.9, 1.1, C5–H), 2.35 (dtd, 1H,
J = 13.4, 3.9, 1.2, C3–H), 1.81 (dtd, 1H, J = 13.4, 8.0, 0.8, C3–H);
13C NMR (MeOH-d4) d 156.1, 135.9, 131.2, 130.4, 128.6, 128.1,
127.4, 125.2, 120.0, 111.7, 101.6, 68.4, 68.3, 66.0, 35.7; HRMS calcd
for C15H16O4Na+ [M+Na]: 283.1310; found: 283.1357.
102.6, 100.0, 99.7, 72.3, 71.4, 65.7, 64.6, 48.2, 48.2, 17.9, 17.8;
+
HRMS calcd for
C
21H26NaO7
[M+Na]: 413.1576; found:
413.2185. 5b: ½a D20
ꢃ
ꢁ103° (c 0.98, CHCl3); 1H NMR (CDCl3): d
4.1.9. 2-Naphthyl 4-methoxy-b-
D
-xylopyranoside (4a)
7.79–7.73 (m, 3H, ArH), 7.47–7.42 (m, 1H, ArH), 7.40–7.34 (m,
2H, ArH), 7.24 (dd, 1H, J = 8.9, 2.4, ArH), 5.28 (d, 1H, J = 7.2, C1–
H), 4.16 (dd, 1H, J = 11.6, 5.3, C5–H), 4.04–3.96 (m, 1H, C4–H),
3.87–3.77 (m, 2H, C2–H and C3–H), 3.50 (dd, 1H, J = 11.7, 9.1,
C5–H), 3.39 (s, 3H, OCH3), 3.33 (s, 3H, OCH3), 2.28 (d, 1H, J = 2.9,
C4-OH), 1.48 (s, 3H, CH3), 1.43 (s, 3H, CH3); 13C NMR (CDCl3): d
154.9, 134.4, 130.2, 129.5, 127.8, 127.3, 126.5, 124.5, 119.4,
111.8, 99.9, 99.8, 99.5, 73.0, 69.1, 67.7, 66.6, 48.2, 48,2, 17.8,
This was prepared from 20 as described for 3a (66%). ½a D20
ꢁ54°
ꢃ
(c 1.5, MeOH) 1H NMR (MeOH-d4) d 7.79–7.75 (m, 3H, ArH), 7.45–
7.25 (m, 4H, ArH), 5.02 (d, 1H, J = 7.4, C1–H), 4.16 (dd, 1H, J = 11.4,
5.0, C5–H), 3.52 (s, 1H, OCH3), 3.50–3.56 (m, 2H, C2–H and C3–H),
3.42 (dd, 1H, J = 11.4, 9.9, C5–H), 3.27–3.33 (m, 1H, C4–H); 13C
NMR (MeOH-d4) 156.6, 135.8, 130.3, 128.6, 128.1, 127.4, 125.3,
120.0, 111.9, 102.8, 80.8, 76.8, 74.8, 64.4, 59.1; HRMS calcd for
16H18O5Na+ [M+Na]: 313.1052; found: 313.1026.
17.8; HRMS calcd for C21H26NaO7 [M+Na]: 413.1576; found:
+
C
413.2185.
4.1.10. 2-Naphthyl 4-deoxy-4-fluoro-b-D-xylopyranoside (4b)
This was prepared from 23 as described for 3a (61%). ½a D20
ꢃ
ꢁ35°
4.1.14. 2-Naphthyl 2,4-di-O-benzyl-b-D-xylopyranoside (6a), 2-
(c 0.3, MeOH); 1H NMR (MeOH-d4) d 7.80–7.76 (m, 3H, ArH), 7.46–
7.25 (m, 4H, ArH), 5.12 (d, 1H, J = 7.3, C1–H), 4.49 (dddd, 1H,
J = 50.3, 13.6, 8.1, 5.4, C4–H), 4.17 (dt, 1H, J = 11.7, 5.4, C5–H),
3.80–3.66 (m, 2H, C5–H and C3–H), 3.56 (ddd, 1H, J = 9.4, 7.3,
0.6, C2–H); 13C NMR (MeOH-d4) d 156.5, 135.8, 131.3, 130.4,
128.6, 128.1, 127.4, 125.3, 120.0, 112.0, 102.6, 91.0 (d, J = 180.1),
75.6 (d, J = 18.8), 74.2 (d, J = 9.1), 63.9 (d, J = 28.6); HRMS calcd
for C15H15FO4Na+ [M+Na]: 301.0847; found: 301.0865
naphthyl 2,3-di-O-benzyl-b-
naphthyl 3,4-di-O-benzyl-b-
D
-xylopyranoside (6b), and 2-
-xylopyranoside (6c)
D
Compound 1b (3.00 g, 0.011 mol) was dissolved in CH2Cl2
(150 mL) while stirring and BnBr (2.84 mL, 0.024 mol), QI (8.02 g,
0.022 mol) and KOH (10%) (150 mL) were added to this solution.
Stirring continued for 22 h after which time the reaction mixture
was transferred into a separating funnel and extracted from CH2Cl2
(2 ꢄ 100 mL). The organic layers were washed with brine (200 mL)
and dried over MgSO4. The solvent was removed under reduced
pressure and the crude mixture was purified by column chroma-
tography eluting with heptane/EtOAc gradient from 16:1 to 4:1.
Di-protected compounds were obtained in following yields: 3-OH
(6a) (66%), 4-OH (6b) (14%), and 2-OH (6c) (6%). Compound 6a:
4.1.11. 2-Naphthyl a-L-arabinopyranoside (4c)
This was prepared from 22 as described for 3a (33%). ½a D20
ꢁ4°
ꢃ
(c 0.6, MeOH); 1H NMR (MeOH-d4) d 7.80–7.74 (m, 3H, ArH),
7.45–7.27 (m, 4H, ArH), 5.02 (d, 1H, J = 7.1, C1–H), 3.99 (dd, 1H,
J = 12.4, 2.8, C5–H), 3.91–3.93 (m, 1H, C4–H), 3.88 (dd, 1H,
J = 9.1, 7.1, C2–H), 3.80 (dd, 1H, J = 12.4, 1.5, C5–H), 3.68 (dd,
1H, J = 9,1, 3.5, C3–H); 13C NMR (MeOH-d4) 156.7, 135.8, 131.2,
130.3, 128.6, 128.1, 127.4, 125.2, 120.1, 111.9, 102.8, 74.2, 72.3,
½
a 2D0
ꢃ
ꢁ30° (c 0.5, CHCl3); 1H NMR (CDCl3) d 7.8–7.7 (m, 3H, ArH),
7.5–7.2 (m, 14H, ArH), 5.15 (d, 1H, J = 7.4, C1–H), 5.05 (d, 1H,
J = 11.4, CH2Ph), 4.83 (d, 1H, J = 11.4, CH2Ph), 4.80 (d, 1H, J = 11.8,
CH2Ph), 4.67 (d, 1H, J = 11.8, CH2Ph), 4.05 (dd, 1H, J = 11.6, 5.1,
C5–H), 3.82 (td, 1H, J = 9.2, 2.2, C3–H), 3.66–3.60 (m, 1H, C4–H),
3.59 (dd, 1H, J = 9.2, 7.4, C2–H), 3.43 (dd, 1H, J = 11.6, 9.5, C5–H);
13C NMR (CDCl3) d 154.9, 138.2, 134.4, 129.8, 128.7, 128.3, 128.1,
128.0, 127.80, 127.3, 124.6, 111.5, 102.0, 81.0, 75.8, 74.9, 73.4,
64.3; HRMS calcd for C29H28O5Na+ [M+Na]: 479.1834; found:
69.7, 67.2; HRMS calcd for
found: 299.0900.
C
15H16O5Na+ [M+Na]: 299.0890;
4.1.12. 2-Naphthyl a-L-threopyranoside (4d)
This was prepared from 24 as described for 3a (45%). ½a D20
ꢁ16°
ꢃ
(c 0.5, MeOH); 1H NMR (MeOH-d4) d 7.79–7.74 (m, 3H, ArH), 7.45–
7.26 (m, 4H, ArH), 4.98 (d, 1H, J = 7.3, C1–H), 4.01 (ddd, 1H, J = 12.0,
4.9, 2.3, C5–H), 3.67–3.75 (m, 2H, C3–H and C5–H), 3.43 (dd, 1H,
J = 8.7, 7.3, C2–H), 2.00 (app. dp, 1H, J = 13.1, 2.3, C4–H), 1.70
(dddd, 1H, J = 17.0, 13.1, 10.9, 4.9, C4–H); 13C NMR (MeOH-d4)
156.8, 135.9, 131.3, 130.7, 130.4, 128.7, 128.1, 127.4, 125.3,
120.1, 111.9, 102.9, 76.4, 72.2, 62.6, 34.2; HRMS calcd for
C15H16O4Na+ [M+Na]: 283.0941; found: 283.0933.
479.2127. Compound 6b: ½a D20
ꢃ
ꢁ11° (c 1.5, CHCl3); 1H NMR (CDCl3)
d 7.81–7.75 (m, 3H, ArH), 7.48–7.23 (m, 14H, ArH), 5.32 (d, 1H,
J = 5.9, C1–H), 4.98 (d, 1H, J = 11.2, CH–Ph), 4.94 (d, 1H, J = 11.6,
CH–Ph), 4.81 (d, 1H, J = 11.2, CH–Ph), 4.70 (d, 1H, J = 11.6, CH–
Ph), 4.16 (dd, 1H, J = 11.6, 4.2, C5–H), 3.84–3.77 (m, 2H, C2–H
and C4–H), 3.62 (app. t, 1H, J = 7.3, C3–H), 3.49 (dd, 1H, J = 11.6,
7.9, C5–H), 2.54 (d, 1H, J = 5.2, C4-OH); 13C NMR (CDCl3) d 129.8,
128.8, 128.7, 128.3, 128.2, 128.0, 127.8, 127.3, 126.6, 124.5,

A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
4123
119.0, 111.3, 100.9, 81.2 79.4, 74.5, 74.5, 68.9, 64.5; HRMS calcd for
ArH), 7.27 (dd, 1H, J = 9.0, 2.4, ArH), 5.23 (d, 1H, J = 0.9, C1–H), 4.34
(dt, 1H, J = 10.4, J = 5.1, C4–H), 4.29 (s, 1H, C2–H), 4.08 (dd, 1H,
J = 10.9, 5.2, C5–H), 3.79 (dd, 1H, J = 10.1, 2.8, C3–H), 3.53 (t, 1H,
J = 10.8, C5–H), 3.32 (s, 3H, OCH3), 3.29 (s, 3H, OCH3), 2.59 (d, 1H,
J = 2.0, C2–OH), 1.40 (s, 3H, CH3), 1.32 (s, 3H, CH3); 13C NMR
(CDCl3): d 154.5, 134.3, 130.2, 129.7, 127.8, 127.3, 126.6, 124.7,
119.0, 111.5, 100.6, 99.9, 99.4, 70.7, 69.9, 64.7, 62.5, 48.3, 48.1,
17.9, 17.9; HRMS calcd for C21H26NaO7+ [M+Na]: 413.1576; found:
413.2404.
C
½
29H28O5Na+ [M+Na]: 479.1834; found: 479.1845. Compound 6c:
a 2D0
ꢃ
ꢁ69° (c 1.0, CHCl3); 1H NMR (CDCl3) d 7.80–7.72 (m, 3H,
ArH), 7.46–7.30 (m, 13H, ArH), 7.23 (dd, 1H, J =8.9, 2.5, ArH), 5.31
(d, 1H, J = 5.1, C1–H), 4.90, 4.82 (ABq, 1H each, J = 11.5, CH2–Ph),
4.70 (s, 2H, CH2–Ph), 4.15 (dd, 1H, J = 12.0, 3.5, C5–H), 3.93 (m,
1H, C2–H), 3.77 (t, 1H, J = 6.4, C3–H), 3.70 (dt, 1H, J = 6.4, 3.5, C4–
H), 3.58 (dd, 1H, J = 12.0, 6.5, C5–H), 3.07 (d, 1H, J = 6.2, C2–OH);
13C NMR (CDCl3) d 154.8, 138.5, 137.7, 134.4, 130.0, 129.6, 128.7,
128.7, 128.3, 128.0, 128.0, 128.0, 127.8, 127.3, 126.6, 124.5,
119.1, 111.2, 100.4, 79.1, 76.2, 73.8, 72.7, 70.7, 61.7; HRMS calcd
for C29H28O5Na+ [M+Na]: 479.1834; found: 479.1740.
4.1.18. (1R, 3S, 4S, 6R, 9R, 10S)-10-fluoro-3,4-dimethoxy-3,4-
dimethyl-9-(2-naphthyl)oxy-2,5,8-trioxabicyclo[4.4.0]decane
(12)
4.1.15. (1R, 3S, 4S, 6R, 9R, 10S)-10-methoxy-3,4-dimethoxy-3,4-
dimethyl-9-(2-naphthyl)oxy-2,5,8-trioxabicyclo[4.4.0]decane
(8)
Compound 12 was obtained from 11 in 42% using analogous
method as for compound 18. For this compound, the reaction mix-
ture was heated to +40 °C instead of being cooled. ½a D20
ꢃ
104° (c 0.78,
Compound 8 was obtained from 5a using the same method as
CHCl3); 1H NMR (CDCl3): d 7.82ꢁ7.75 m, 3H, ArH), 7.49–7.36 (m,
3H, ArH), 7.28 (dd, 1H, J = 8.9, 2.4, ArH), 5.21 (dd, 1H, J = 7.0, 4.8,
C1–H), 4.61 (ddd, 1H, J = 52.1, 9.2, 7.0, C2–H), 4.05–3.89 (m, 3H,
C3–H, C4–H and C5–H), 3.61 (dd, 1H, J = 10.8, 9.9, C5–H), 3.34 (s,
3H, OCH3), 3.31 (s, 3H, OCH3), 1.34 (s, 3H, CH3), 1.32 (s, 3H, CH3);
13C NMR (CDCl3): d 154.7, 134.3, 133.1, 129.8, 127.8, 127.4,
126.3, 119.3, 112.1, 100.6 (d, J = 24.4), 100.0, 99.8, 89.2 (d,
J = 189.9), 71.2 (d, J = 18.3), 65.2 (d, J = 8.0), 64.2, 48.3, 48.2, 17.8,
for 15 (89%). ½a 2D0
ꢃ
89° (c 1.2, CHCl3); 1H NMR (CDCl3): d 7.83–
7.77 (m, 3H, ArH), 7.50–7.45 (m, 1H, ArH), 7.42–7.37 (m, 2H,
ArH), 7.28 (dd, 1H, J = 8.9, 2.5, ArH), 5.06 (d, 1H, J = 7.1, C1–H),
4.02–3.89 (m, 2H, C5–H and C4–H), 3.80 (t, 1H, J = 9.7, C3–H),
3.72 (s, 3H, C2-OCH3), 3.57 (t, 1H, J = 10.3, C5–H), 3.51 (dd, 1H,
J = 9.2, 7.6, C2–H), 3.36 (s, 3H, OCH3), 3.33 (s, 3H, OCH3), 1.41 (s,
3H, CH3), 1.32 (s, 3H, CH3); 13C NMR (CDCl3) d155.1, 134.4, 130.2,
129.7, 127.8, 127.3, 126.5, 124.6, 119.2, 111.8, 102.8, 99.7, 99.7,
+
17.7; HRMS calcd for C21H25FNaO6 [M+Na]: 415.1533; found:
80.2, 72.9, 65.8, 64.2, 60.9, 48.2, 48.1, 18.0, 17.7; HRMS calcd for
415.1556.
+
C22H28NaO7 [M+Na]: 427.1733, found: 427.1716.
4.1.19. (1R, 3S, 4S, 6R, 9R, 10S)-3,4-dimethoxy-3,4-dimethyl-10-
(mehtylthio)thiocarbonyloxy-9-(2-naphthyl)oxy-2,5,8-
trioxabicyclo[4.4.0]decane (13)
NaH (42 mg, 1.06 mmol) and imidazole (2 cristals) were added
to 5a (206 mg, 0.53 mmol) in THF (8 mL) under N2 atmosphere. The
mixture was stirred for 1 h, and CS2 (0.285 mL, 4.75 mmol) was
introduced. After 18 h, MeI (0.082 mL, 1.32 mmol) was added. An
hour later, the mixture was diluted with CH2Cl2, and washed with
water, HCl (0.2 M), NaHCO3 (sat. aq), and water. The organic phase
was then dried over MgSO4, filtered, concentrated and chromato-
4.1.16. (1R, 3S, 4S, 6R, 9R, 10R)-10-acethoxy-3,4-dimethoxy-3,4-
dimethyl-9-(2-naphthyl)oxy-2,5,8-trioxabicyclo[4.4.0]decane
(10)
Compound 5a (150 mg, 0.38 mmol) was dissolved in CH2Cl2
(5 mL) and the solution was cooled to ꢁ78 °C under N2, while stir-
ring. Pyridine (0.125 mL, 1.5 mmol) was added, followed by drop-
wise addition of Tf2O (0.130 mL, 0.77 mmol). The temperature
was raised to 0 °C. After 3 h, the reaction mixture was diluted with
CH2Cl2, washed with NaHCO3 (sat. aq), and dried over Na2SO4.
Once filtered, solvents were evaporated and the remaining crude
triflate was submitted to subsequent reaction. The crude was dis-
solved in DMF (8 mL) and CsOAc (185 mg, 0.96 mmol) was intro-
duced under nitrogen atmosphere, while stirring. The resulting
mixture was heated to 50 °C for three days. After that time it
was allowed to cool to rt and solvents were evaporated. The crude
was purified by flash chromatography eluting with heptane/EtOAc
graphed (heptane/EtOAc 2:1) to give 13 (203 mg, 80%). ½a D20
ꢃ
120°
(c 1.3, CHCl3); 1H NMR (CDCl3): d 7.80–7.73 (m, 3H, ArH), 7.48–
7.42 (m, 1H, ArH), 7.40–7.35 (m, 2H, ArH), 7.19 (dd, 1H, J = 8.9,
2.4, ArH), 6.21–6.14 (m, 1 H, C2–H), 5.35 (d, 1H, J = 6.4, C1–H),
4.13–4.02 (m, 3H, C3–H, C4–H and C5–H), 3.60–3.69 (m, 1H, C5–
H), 3.31 (s, 3H, OCH3), 3.27 (s, 3H, OCH3), 2.60 (s, 1H, SCH3), 1.35
(s, 6H, CH3); 13C NMR (CDCl3): d: 215.3, 155.0, 134.3, 130.3,
129.7, 127.8, 127.3, 126.6, 124.7, 119.4, 112.0, 101.2, 100.0, 99.8,
(4:1), which gave 10 in 82% yield over the two steps. ½a D20
ꢃ
73° (c 1.7,
CHCl3); 1H NMR (CDCl3): d 7.80–7.72 (m, 3H, ArH), 7.45 (dt, 1H,
J = 6.9, J = 1.3, ArH), 7.39–7.34 (m, 1H, ArH), 7.33 (d, 1H, J = 2.4,
ArH), 7.18 (dd, 1H, J = 8.9, 2.5, ArH), 5.65 (dd, 1H, J = 3.1, 1.1, C2–
H), 5.28 (d, 1H, J = 1.1, C1–H), 4.24 (dt, 1H, J = 10.4, 5.1, C4–H),
4.12 (dd, 1H, J = 11.0, 5.1, C5–H), 3.87 (dd, 1H, J = 10.2, 3.1, C3–
H), 3.58 (t, 1H, J = 10.9, C5–H), 3.29 (s, 3H, OCH3), 3.27 (s, 3H,
OCH3), 2.28 (s, 3H, OAc), 1.31 (s, 3H, CH3), 1.29 (s, 3H, CH3); 13C
NMR (CDCl3, 100.62 MHz): d 170.7, 154.6, 134.3, 130.2, 129.7,
127.8, 127.3, 126.6, 124.6, 119.0, 111.4, 100.4, 99.9, 98.5, 69.4,
79.1, 70.8, 65.4, 64.2, 48.2, 48.0, 19.6, 17.8, 17.7; HRMS calcd for
+
C
23H28NaO7S2 [M+Na]: 503.1174; found: 503.1158.
4.1.20. 2-Naphthyl2-O-(methylthio)thiocarbonyl-b-D-threopyra-
noside (14)
This compound was prepared from 12 as described for 2a (86%).
½ ꢃ
a 2D0
16° (c 1.1, CHCl3); 1H NMR (CDCl3): d 7.81–7.74 (m, 3H, ArH),
7.49–7.44 (m, 1H, ArH), 7.43–7.37 (m, 2H, ArH), 7.21 (dd, 1H,
J = 8.8, 2.5, ArH), 5.99 (dd, 1H, J = 6.9, 5.4, C2–H), 5.45 (d, 1H,
J = 5.3, C1–H), 4.27 (dd, 1H, J = 12.0, 4.0, C5–H), 4.02–3.97 (m, 1H,
C3–H), 3.93–3.88 (m, 1H, C4–H), 3.61 (dd, 1H, J = 12.0, J = 7.1,
C5–H), 2.88 (d, 1H, J = 5.2, C2–H), 2.63 (s, 3H, SCH3); 13C NMR
(CDCl3): d 216.6, 154.3, 134.3, 130.3, 129.9, 127.8, 127.4, 126.8,
124.9, 119.0, 111.8, 98.8, 79.5, 73.1, 69.5, 63.8, 19.8; HRMS calcd
69.1, 64.8, 63.0, 48.3, 48.1, 21.3, 17.9, 17.8; HRMS calcd for
+
C
23H28NaO8 [M+Na]: 455.1682; found: 455.2603.
4.1.17. (1R, 3S, 4S, 6R, 9R, 10R)-10-hydroxy-3,4-dimethoxy-3,4-
dimethyl-9-(2-naphthyl)oxy-2,5,8-trioxabicyclo[4.4.0]decane
(11)
+
for C17H18NaO5S2 [M+Na]: 389.0493; found: 389.0494.
Compound 10 (58 mg, 0.13 mmol) was dissolved in 0.05 M
NaOMe (5 mL). The solution was stirred for 2.5 h, after which
Amberlite IR 120 H+ was added until pH was neutral. The mixture
was then filtered and concentrated under reduced pressure to give
4.1.21. 2-Naphthyl 2,4-di-O-benzyl-3-methoxy-b-D-xylopyra-
oside (15)
6a (200 mg, 0.44 mmol) was dissolved in freshly distilled DMF
(5 mL) under N2 atmosphere and cooled to 0 °C while stirring.
NaH (35 mg, 0.88 mmol) was then added portion-wise and stirring
11 (47 mg) in 90% yield. ½a D20
ꢃ
109° (c 0.86, CHCl3); 1H NMR (CDCl3):
d7.81–7.74 (m, 3H, ArH), 7.48–7.43 (m, 1H, ArH), 7.41–7.36 (m, 2H,

4124
A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
continued for further 15 min at sub-ambient temperatures. Subse-
quently, MeI (0.055 mL, 0.88 mmol) was introduced and the mix-
ture was left to stir for 18 h warming to rt. The reaction mixture
was then quenched using NH4Cl sat. and extracted from CH2Cl2
(2 ꢄ 10 mL). Combined organic layers were washed with brine
(15 mL) and dried over MgSO4, filtered and concentrated under re-
duced pressure. Compound 15 was obtained in 98% yield after flash
126.7, 119.0, 111.6, 101.6 (d, J = 11.7), 96.7 (d, J = 186.4), 79.5 (d,
J = 18.2), 75.4 (d, J = 17.2), 74.8, 73.4 (d, J = 1.7), 63.2 (d, J = 9.6);
HRMS calcd for C29H27FO4Na+ [M+Na]: 481.1786; found: 481.1812.
4.1.24. 2-Naphthyl 2,4-di-O-benzyl-3-deoxy-b-D-erythropyra-
noside (19)
Compound 6a (198 mg, 0.43 mmol) and imidazole (3 cristals)
were dissolved in THF (10 mL) under N2 atmosphere. The resulting
solution was cooled to 0 °C while stirring and NaH (21 mg,
0.87 mmol) was added. The mixture was stirred for 1 h, warming
to rt, and CS2 (0.259 mL, 4.30 mmol) was introduced. After another
hour MeI (0.080 mL, 1.29 mmol) was injected. The reaction pro-
gress was monitored by TLC (heptane/EtOAc, 3:1) and it indicated
its completion after one more hour. The solvents were removed
under reduced pressure and the crude was submitted to further
transformations without purification. The resulting crude was dis-
solved in toluene (10 mL) and AIBN (57 mg, 0.35 mmol) and n-
Bu3SnH (1.74 mL, 6.45 mmol) were added. The mixture was
brought to reflux for 30 min and evaporated. The resulting crude
was purified by flash chromatography eluting with heptane/EtOAc
chromatography (heptane/EtOAc 5:1). ½a D20
ꢃ
ꢁ14° (c 0.6, CHCl3); 1H
NMR (CDCl3) d 7.83–7.72 (m, 3H, ArH), 7.5–7.2 (m, 14H, ArH), 5.12
(d, 1H, J = 7.5, C1–H), 5.01 (d, 1H, J = 11.1, CH2–Ph), 5.01 (d, 1H,
J = 11.1, CH2–Ph), 4.85 (d, 1H, J = 11.1, CH2–Ph), 4.79 (d, 1H,
J = 11.7, CH2–Ph), 4.67 (d, 1H, J = 11.7, CH2–Ph), 4.02 (dd, 1H,
J = 11.6, 5.3, C5–H), 3.71 (s, 3H, C3–OCH3), 3.67–3.60 (m, 2H, C2–
H and C4–H), 3.44 (dd, 1H, J = 8.9, 7.9, C3–H), 3.39 (dd, 1H,
J = 11.7, 9.8, C5–H); 13C NMR (CDCl3) d 154.9, 138.3, 138.2, 134.3
130.0, 129.6, 128.5, 128.4, 128.2, 127.9, 127.8, 127.8, 127.7,
127.2, 126.5, 124.4, 119.0, 111.4, 102.1, 85.5, 81.5, 77.5, 75.1,
73.4, 64.1, 61.3, HRMS calcd for C30H30O5Na+ [M+Na]: 493.1991;
found: 493.2007.
4.1.22. 2-Naphthyl 2,4-di-O-benzyl-b-
D
-ribopyranoside (17)
gradient 14:1 to 10:1, allowing 19 in 68% yield. ½a D20
ꢁ32° (c 2.2,
ꢃ
6a (100 mg, 0.22 mmol) was dissolved in CH2Cl2 (5 mL) and the
solution was cooled to ꢁ10 °C under N2, while stirring. Tf2O
(0.055 mL, 0.33 mmol) was then added dropwise followed by pyr-
idine (0.053 mL, 0.66 mmol). After 2 h the reaction mixture was
quenched with HCl (0.5 N) (2 mL), washed with NaHCO3 (sat.)
(7 mL), brine (10 mL) and dried over Na2SO4. Once filtered, solvents
were evaporated and the remaining crude triflate was submitted to
subsequent reaction. The crude (116 mg, 0.22 mmol) was dissolved
in DMF (8 mL) and QNO2 (254 mg, 0.88 mmol) was introduced un-
der nitrogen atmosphere, while stirring. The resulting mixture was
heated to 50 °C for 16 h. After that time it was allowed to cool to rt
and H2O (20 mL) was added. The solution was extracted from
EtOAc (3 ꢄ 30 mL), dried over MgSO4 and evaporated. The crude
was purified by flash chromatography eluting with heptane/EtOAc
CHCl3); 1H NMR (CDCl3) d 7.82–7.77 (m, 3H, ArH), 7.50–7.27 (m,
14H, ArH), 5.32 (d, 1H, J = 5.9, C1–H), 4.89 (d, 1H, J = 11.8, CH–
Ph), 4.80 (d, 1H, J = 11.8, CH–Ph), 4.66 (d, 1H, J = 11.8, CH–Ph),
4.59 (d, 1H, J = 11.8, CH–Ph), 4.10 (ddd, 1H, J = 11.3, 4.0, 1.7, C5–
H), 3.70–3.65 (m, 2H, C2–H and C4–H), 3.59 (dd, 1H, J = 11.3, 7.4,
C5–H), 2.50 (dtd, 1H, J = 13.1, 4.6, 1.7, C3–H), 1.90 (dt, 1H,
J = 13.1, 9.3, C3–H); 13C NMR (CDCl3) d 154.8, 138.5, 138.4, 134.5,
129.7, 128.7, 128.6, 128.0, 128.0, 127.9, 127.8, 127.4, 126.6,
124.5, 119.1, 111.2, 101.6, 74.2, 72.5, 71.3, 71.2, 66.2, 33.2; HRMS
calcd for C29H28O4Na+ [M+Na]: 463.1885; found: 463.0302.
4.1.25. 2-Naphthyl 2,3-di-O-benzyl-4-methoxy-b-D-xylopyra-
noside (20)
This was prepared from 6b as described for 15 (quant.). ½a D20
ꢃ
9°
(3:1), which gave 17 in 54% yield over the two steps. ½a D20
ꢃ
ꢁ29° (c
(c 1.0, CHCl3); 1H NMR (CDCl3) d7.82–7.76 (m, 3H, ArH), 7.50–7.25
(m, 14H, ArH), 5.19 (d, 1H, J = 7.3, C1–H), 5.04 (d, 1H, J = 11.0, CH–
Ph), 4.87–4.93 (m, 2H, CH–Ph), 4.87 (d, 1H, J = 11.0, CH–Ph), 4.17
(dd, 1H, J = 11.5, 5.0, C5–H), 3.75 (dd, 1H, J = 9.0, 7.3, C2–H), 3.65
(dd, 1H, J = 9.0, 8.4, C3–H), 3.55 (s, 3H, OCH3), 3.50–3.56 (m, 1H,
C4–H), 3.41 (dd, 1H, J = 11.5, 9.5, C5–H); 13C NMR (CDCl3) d
155.2, 135.8, 131.3, 130.4, 128.6, 128.1, 127.4, 125.3, 120.0,
111.6, 102.4, 81.5, 83.6, 80.0, 75.6, 75.4, 63.8, 59.2; HRMS calcd
for C30H30O5Na+ [M+Na]: 493.1991, found: 493.2011.
0.8, CHCl3); 1H NMR (CDCl3) d 7.80–7.74 (m, 3H, ArH), 7.47–7.30
(m, 13H, ArH), 7.22 (dd, 1H, J = 8.9, 2.5, ArH), 5.63 (d, 1H, J = 5.6,
C1–H), 4.93 (d, 1H, J = 11.9, CH–Ph), 4.79 (d, 1H, J = 11.9, CH–Ph),
4.69 (d, 1H, J = 12.00, CH–Ph), 4.62 (d, 1H, J = 12.0, CH–Ph), 4.32
(dd, 1H, J = 7.8, 3.4, C3–H), 3.96 (dd, 1H, J = 11.7, 7.5, C5–H), 3.86
(dd, 1H, J = 11.7, 3.9, C5–H), 3.69–3.65 (m, 2H, C2–H and C4–H),
2.79 (d, 1H, J = 4.7, C3-OH); 13C NMR (CDCl3) d 154.8, 138.0,
137.9, 134.5, 130.0, 129.7, 128.7, 128.7, 128.6, 128.6, 128.1,
128.0, 127.9, 127.9, 127.8, 127.3, 126.6, 124.5, 119.0, 111.1, 98.2,
77.0, 74.3, 73.5, 71.9, 67.4, 61.3; HRMS calcd for C29H28O5Na+
[M+Na]: 479.1834; found: 479.2127.
4.1.26. 2-Naphthyl 2,3-di-O-benzyl-a-L-arabinopyranoside (22)
This was prepared from 6b as described for 17 (50%.). ½a D20
ꢁ25°
ꢃ
(c 0.28, CHCl3); 1H NMR (CDCl3) d 7.83–7.76 (m, 3H, ArH), 7.50–
7.27 (m, 14H, ArH), 5.20 (d, 1H, J = 6.5, C1–H), 5.02 (d, 1H,
J = 11.2, CH–Ph), 4.86 (d, 1H, J = 11.2, CH–Ph), 4.83 (d, 1H,
J = 11.7, CH–Ph), 4.74 (d, 1H, J = 11.7, CH–Ph), 4.14 (dd, 1H,
J = 12.4, 3.8, C5–H), 4.07–4.02 (m, 2H, C2–H and C4–H), 3.73 (dd,
1H, J = 8.1, 3.6, C3–H), 3.63 (dd, 1H, J = 12.4, 2.0, C5–H); 13C NMR
(CDCl3) d 155.1, 138.3, 137.9, 134.4, 129.6, 128.7, 128.6, 128.3,
128.2, 128.0, 128.0, 127.8, 127.3, 126.5, 124.5, 119.3, 111.5,
101.09, 79.3, 77.8, 75.1, 72.7, 66.2, 64.7; HRMS calcd for
4.1.23. 2-Naphthyl 2,4-di-O-benzyl-3-deoxy-3-fluoro-b-D-
xylopyranoside (18)
Compound 17 (108 mg, 0.24 mmol) was dissolved in CH2Cl2
(4 mL) and cooled to ꢁ40 °C, under N2, while stirring. DAST
(0.078 mL, 0.59 mmol) was then added. The reaction was moni-
tored by TLC (heptane/EtOAc, 3: 1) which indicated its completion
after 1 h. It was quenched with MeOH (2 mL) and stirred for addi-
tional 10 min. The crude mixture was washed with NaHCO3 (sat.)
(10 mL), brine (20 mL) and dried over MgSO4. Purification by flash
chromatography eluting with heptane/EtOAc (8:1) allowed 18 in
C
29H28O5Na+ [M+Na]: 479.1829; found: 479.1849.
47% yield. ½a 2D0
ꢃ
4° (c 1.1, CHCl3); 1H NMR (CDCl3) d 7.81–7.74 (m,
4.1.27. 2-Naphthyl 2,3-di-O-benzyl-4-deoxy-4-fluoro-b-D-xylopy
ranoside (23)
3H, ArH), 7.48–7.21 (m, 14H, ArH), 5.12 (d, 1H, J = 7.4, C1–H),
4.97 (d, 1H, J = 11.4, CH–Ph), 4.89 (d, 1H, J = 11.4, CH–Ph), 4.84
(d, 1H, J = 11.8, CH–Ph), 4.72 (dt, 1H, J = 51.8, 8.5, C3–H), 4.67 (d,
1H, J = 11.8, CH–Ph), 4.06 (dt, 1H, J = 11.9, 5.6, C5–H), 3.87–3.75
(m, 2H, C2–H and C4–H), 3.41 (ddd, 1H, J = 11.9, 9.9, 0.7, C5–H);
13C NMR (CDCl3, 100.62 MHz) d 154.8, 138.0, 137.8, 134.4, 130.2,
129.8, 128.7, 128.7, 128.5, 128.2, 128.2, 128.0, 127.8, 127.3,
Compound 23 was obtained using the method described for 12.
The compound was purified using flash chromatography, but some
impurities remained and it was not possible to eliminate them.
½
a 2D0 +2° (c 2.0, CHCl3) 1H NMR (CDCl3): d 7.82–7.75 (m, 3H, ArH),
7.50–7.30 (m, 14H, ArH), 7.25–7.22 (m, 1H, ArH), 5.27 (d, 1H,
ꢃ
J = 6.7, C1–H), 4.98 (d, 1H, J = 11.2, CH2–Ph), 4.93–4.67 (m, 4H,

A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
4125
CH2–Ph and C4–H), 4.26–4.18 (m, 1H, C5–H), 3.89–3.73 (m, 2H,
C3–H and C2–H), 3.71–3.59 (m, 1H, C5–H); 13C NMR (CDCl3): d
154.8, 138.2, 138.1, 134.4, 130.1, 129.8, 128.6, 128.5, 128.3,
128.3, 128.3, 128.1, 128.0, 128.0, 127.8, 127.3, 126.7, 124.6,
119.0, 101.8, 90.4 (J = 182.4), 81.4 (J = 19.0), 80.1 (J = 9.4), 63.1
(J = 35.7). HRMS calcd for C29H27FO4Na+ [M+Na]: 481.1786; found:
481.1810.
from Bionuclear Scandinavia AB, Bromma, Sweden. Crystal violet
was obtained from Merck, Germany. The prepacked Superose 6
HR 10/30m, Dextran T-500 and octyl-Sepharose CL-4B were from
Pharmacia-LKB, Sweden, and DE-53 DEAE-cellulose was from
Whatman. Water for HPLC-analysis was from a Millipore Milli-Q
system.
4.3.2. Cell culture, radiolabeling and extraction procedures
Cells were cultured as monolayers according to manufacturer’s
instructions. Confluent cells were incubated in low-sulfate, MgCl2-
labeling medium supplemented with 2 mM glutamine, 20 mCi/mL
of [35S] sulfate and different xylosides. Dilutions were made from
20 mM stock solutions in Me2SO/water (1:1, v/v). After the incuba-
tion period, culture medium was collected and pooled with two
washings of ice-cold PBS (0.137 M NaCl, 3 mM KCl, 8 mM Na2HPO4,
2 mM KH2PO4, pH 7.5). Cells were extracted with 0.1–0.2 mL/Cm2
dish of 0.15 M NaCl, 10 mM EDTA, 2%(v/v) Triton X-100, 10 mM
4.1.28. 2-Naphthyl 2,3-di-O-benzyl-4-deoxy-
a-L-threopyra-
noside (24)
This was prepared from 6b as described for 19 (93%.). ½a D20
ꢃ
17° (c
1.1, CHCl3); 1H NMR (MeOH-d4) d 7.73–7.70 (m, 3H, ArH), 7.45–
7.21 (m, 14H, ArH), 5.14 (d, 1H, J = 7.0, C1–H), 4.96 (d, 1H,
J = 11.3, CH -Ph), 4.84 (d, 1H, J = 11.3, CH -Ph), 4.72 (d, 1H,
J = 11.8, CH–Ph), 4.67 (d, 1H, J = 11.8, CH–Ph), 4.02 (ddd, 1H,
J = 11.9, 4.7, 2.8, C5–H), 3.73 (ddd, 1H, J = 13.3, 8.4, 5.1, C3–H),
3.62 (td, 1H, J = 11.9, 2.5, C5–H), 3.56 (dd, 1H, J = 8.4, 7.0, C2–H),
2.16 (dp, 1H, J = 13.3, 2.5, C4–H), 1.74–1.64 (m, 1H, C4–H); 13C
NMR (MeOH-d4) d 155.1, 135.9, 131.3, 130.5, 128.9, 128.7, 128.6,
128.1, 127.4, 125.3, 119.9, 110.4, 101.4, 81.6, 77.9, 74.6, 71.5,
60.8, 27.8; HRMS calcd for C29H28O4Na+ [M+Na]: 463.1880; found:
463.1888.
KH2PO4, pH 7.5, 5 lg/ml ovalbumin containg 10 mM N-ethylmalei-
mide, and 1 mM diisopropylphosphoro-fluoridate on a slow shaker
at 4 °C for 10 min.
4.3.3. Isolation of xyloside-primed radiolabeled GAG
The procedures have been described in detail previously.9 35S]
[
4.2. NMR spectroscopy and molecular modeling
Sulfate labeled polyanionic macromolecules were isolated from the
culture medium by ion exchange chromatography on DEAE–cellu-
lose at 4 °C. Samples were passed over a 0.2 mL-column of DEAE
equilibrated with 6 M urea, 0.5 M NaOAc, pH 5.8, 5 lg/mL ovalbu-
min, 0.1% Triton X-100. After sample application, the columns were
washed successively with (a) equilibration buffer, (5 mL), (b) 6 M
urea, 10 mM Tris, pH 8.0, 5 lg/mL ovalbumin, 0.1% Triton X-100
NMR experiments for conformational analysis were performed
on three different spectrometers: a Bruker Avance III 600 MHz
spectrometer equipped with a 5 mm PFG triple resonance probe,
a Bruker AVANCE 500 MHz spectrometer and a Bruker AVANCE
III 700 MHz spectrometer; the latter two equipped with 5 mm
TCI Z-Gradient CryoProbes. Chemical shifts are reported in ppm
using residual methanol (dH 3.31) and methanol-d4 (dC 49.0) as ref-
erences. NMR samples were prepared by dissolving 1–2 mg of the
xylosides in 0.5 mL of methanol-d4 (99.9%, Aldrich). 1H and 13C
NMR chemical shift assignments were performed at 37 °C using
2D 1H,1H-TOCSY,48 1H,13C-HSQC,49 1H,13C-H2BC50 and 1H,13C-
(5 mL) and (c) 50 mM Tris, pH 7.5 (10 mL). Bound material was
eluted with 2 x 0.5 mL of 4 M guanidine–HCl, 50 mM NaOAc, pH
5.8. The eluted free xyloside-primed GAG chains were separated
from PG by hydrophobic interaction chromatography on Octyl-Se-
pharose column equilibrated with 4 M guanidine–HCl, 50 mM
NaOAc, pH 5.8. After equilibration, BSA were added and columns
washed again with 2.5 mL of 4 M guanidine–HCl, 50 mM NaOAc,
pH 5.8 to avoid unspecific binding. After sample application the
unbound xyloside-primed GAG were washed with 3 ꢄ 0.2 mL of
4 M guanidine–HCl, 50 mM NaOAc, pH 5.8. Radioactive samples
were precipitated with 5 vol of 95% ethanol overnight at ꢁ20 °C
n
HMBC51 experiments. 1H chemical shifts and JHH coupling con-
stant of compounds 1a, 1a (Na), 1b, 2a, 3a, 3b, 3d and 4a were
determined with aid of the PERCH NMR spin simulation software
(PERCH Solutions Ltd., Kuopio, Finland). Chemical shifts and cou-
pling constants were altered iteratively until the simulated and
experimental spectra appeared highly similar according to visual
inspection and the total root-mean-square value was close to or
using 100 lg of dextran as carrier. After centrifugation in a Beck-
man JS-7.5 at 4000 rpm for 30 min, material was dissolved in
4 M guanidine–HCl, 50 mM NaOAc, 0.2% Triton X-100, pH 5.8 fol-
lowed by gel permeation FPLC on Superose 6. Radioactivity was
determined in a b-counter.
2
4
below 0.1%. JHH and JHH were assumed to have negative signs
2
whereas JFH have a positive sign.52,53 3D models of compounds
1a, 1b, 2a, 3a, 3b, 3d and 4a were built using the VEGA ZZ software
(release 2.3.1.2).54 The molecular structures were energy mini-
mized, using algorithms included in the program, in consecutive
order (i) steepest descent; (ii) conjugate gradient and (iii) trun-
cated Newton. Calculated nJHH coupling constants for the different
ring conformations of compounds 1a, 1b, 2a, 3a, 3b, 3d and 4a
were based on the Karplus-type relationships proposed by Haasnot
et al.55 as implemented in the JANOCCHIO software.56
4.3.4. In vitro growth assay using crystal violet method
Cells were harvested by trypsinization and seeded into 96-well
microculture plates at 5000 cells/well in MEM supplemented with
10% fetal calf serum, 1%
streptomycin (100 g/mL) or RPMI-1640 supplemented with 10%
fetal calf serum, penicillin (100 U/mL), and streptomycin (100 g/
L-glutamine, penicillin (100 U/mL), and
l
l
mL). After 24 h of plating the cells were allowed to proliferate in
4.3. Biological testing
serum-free Ham’s F-12 medium supplemented with insulin
(10 lg/mL) and transferrin (25 lg/mL) in the presence of increas-
4.3.1. Materials
ing concentrations of xyloside. Controls without xylosides were in-
cluded. The total exposure-time was 96 h. Cells were then fixed in
1% glutaraldehyde dissolved in Hanks’ balanced salt solution (NaCl
80 g/L, KCl 4 g/L, glucose 10 g/L, KH2PO4 600 mg/L, NaHPO4
475 mg/L) for 20 min, then the cells nuclei were stained with
0.1% crystal violet. After washing and cell lysis for 24 h in 1% Triton
X-100, the amount of bound dye was measured at A600nm in a
microplate photometer (Titertek multiscan). The inhibitory effect
of the compounds is expressed as the percentage of growth in
the absence of drugs.
Breast cancer cells from patients with infiltrative ductal cancer
(HCC70 cells) and normal fibroblasts from breast tissue of patients
with infiltrative ductal cancer (CCD-1095Sk cells) were obtained
from ATCC, LGC Promochem AB, Borås, Sweden. Regular cell cul-
ture media, L-glutamine, penicillin–streptomycin, trypsin, and do-
nor calf serum were obtained from Life Technologies. Minimal
essential Medium (MEM) and Ham’s F-12 medium were purchased
from Sigma. Na235SO4 (1310 Ci/mmol) was obtained from Perkin
Elmer Sverige AB. D-[1-3H]galactose (20 Ci/mmol) was obtained

4126
A. Siegbahn et al. / Bioorg. Med. Chem. 19 (2011) 4114–4126
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The Swedish Research Council, The Crafoord Foundation, FLÄK
(The Research School in Pharmaceutical Science), The Gunnar
Nilssons Foundation, The Jeansson Foundation, The Kock founda-
tion, The Knut and Alice Wallenberg Foundation, The Lennander
Foundation, The Royal Physiographic Society in Lund, The Swedish
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