Regioselective reductive openings of acetals; mechanistic details and synthesis of fluorescently labeled compounds

Article abstract of DOI:10.1021/jo0526284

Regioselective reductive openings of mixed phenolic-benzylic acetals, using BH₃·NMe₃-AlCl₃, was investigated, and a mechanism where the outcome is directed by the electrostatic potential of the two oxygen atoms is presente

Full text of DOI:10.1021/jo0526284

Regioselective Reductive Openings of Acetals; Mechanistic Details
and Synthesis of Fluorescently Labeled Compounds
Richard Johnsson,† Katrin Mani,‡ Fang Cheng,‡ and Ulf Ellervik*,†
Organic Chemistry, Lund UniVersity, P.O. Box 124, SE-221 00 Lund, Sweden, and
Department of Experimental Medical Science, DiVision of Neuroscience, Lund UniVersity,
Biomedical Center C13, SE-221 84 Lund, Sweden
ulf.ellerVik@organic.lu.se
ReceiVed December 22, 2005
Regioselective reductive openings of mixed phenolic-benzylic acetals, using BH3‚NMe3-AlCl3, was
investigated, and a mechanism where the outcome is directed by the electrostatic potential of the two
oxygen atoms is presented. The regioselective acetal opening was used in the synthesis of a fluorescently
labeled analogue to antiproliferative xylosides. The fluorescently labeled xyloside was tested for uptake,
antiproliferative activity, and glycosaminoglycan priming in different cell lines. The xyloside was taken
up by all cell lines but did not initiate glycosaminoglycan biosynthesis.
Introduction
the linker between the protein and carbohydrate in proteogly-
cans.
Proteoglycans, composed of glycosaminoglycan (GAG) chains
covalently attached to a core protein, are widely expressed in
invertebrate and vertebrate tissues. Many biological functions
of proteoglycans are due to interactions between GAG chains
and a variety of pathogens and molecules, such as prion pro-
tein, viruses, growth factors, cytokines, and factors involved in
blood coagulation.1 The first step in GAG biosynthesis is
xylosylation of a serine residue. A specific linker tetrasaccharide,
GlcA(â1-3)Gal(â1-3)Gal(â1-4)Xylâ, is then assembled and
serves as an acceptor for elongation of GAG chains (Figure 1).
Addition of GlcNAc or GalNAc to the nonreducing terminal
GlcA residue determines whether heparan sulfate or chondroitin
sulfate/dermatan sulfate is initiated. The general structure is then
modified through N-deacetylation/N-sulfation, epimerization,
and O-sulfation.
Xylosides with hydrophobic aglycons can penetrate cell
membranes and initiate GAG synthesis by serving as acceptors
in the first galactosylation step. The composition of the GAG
chains assembled on the xylosides depends on the structure of
the aglycon, which may reflect selective partitioning of primers
into different intracellular compartments or into different
branches of biosynthetic pathways.
The xyloside-primed GAG chains can be retained inside the
cells but are usually mainly secreted into the medium. Xylosides
have been used for several purposes, including inhibition of
tumor growth2 and inhibition of scrapie prion protein infectivity.3
We have previously reported that the GAG-priming 2-(6-
hydroxynaphthyl)-â-D-xylopyranoside 1 (Figure 2) selectively
inhibits growth of transformed or tumor-derived cells in vitro
as well as in vivo. Treatment with this xyloside reduced the
average tumor load by 70-97% in a SCID mice model.4
To further investigate these effects we decided to synthesize
and evaluate the fluorescently labeled analogue 2 (Figure 2).
Biosynthesis of GAG chains can also take place independently
of core protein synthesis by using xylosides as primers. Xylose
is an unusual structural component in mammalian cells, i.e., as
† Organic Chemistry.
(2) (a) Belting, M.; Borsig, L.; Fuster, M. M.; Brown, J. R.; Persson, L.;
Fransson, L.-A° .; Esko, J. D. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 371.
(b) Kolset, S. O.; Sakurai, K.; Ivhed, I.; Overvatn, A.; Suzuki, S. Biochem.
J. 1990, 265, 637.
(3) Ben-Zaken, O.; Tzaban, S.; Tal, Y.; Horonchik, L.; Esko, J. D.;
Vlodavsky, I.; Taraboulos, A. J. Biol. Chem. 2003, 278, 40041.
(4) Mani, K.; Belting, M.; Ellervik, U.; Falk, N.; Svensson, G.; Sandgren,
S.; Cheng, F.; Fransson, L.-A° . Glycobiology 2004, 14, 387.
‡ Division of Neuroscience.
(1) (a) Bernfield, M.; Go¨tte, M.; Park, P. W.; Reizes, O.; Fitzgerald, M.
L.; Lincecum, J.; Zako, M. Annu. ReV. Biochem. 1999, 68, 729. (b)
Horonchik, L.; Tzaban, S.; Ben-Zaken, O.; Yedidia, Y.; Rouvinski, A.; Papy-
Garcia, D.; Barritault, D.; Vlodavsky, I.; Taraboulos, A. J. Biol.Chem. 2005,
280, 17062. (c) Tiwari, V.; O’Donnell, C. D.; Oh, M. J.; Valyi-Nagy, T.;
Shukla, D. Biochem. Biophys. Res. Commun. 2005, 338, 930.
10.1021/jo0526284 CCC: $33.50 © 2006 American Chemical Society
Published on Web 04/05/2006
3444
J. Org. Chem. 2006, 71, 3444-3451

RegioselectiVe ReductiVe Openings of Acetals
FIGURE 1. Glycosaminoglycan chains consist of a linker tetrasaccharide unit (GlcA(â1-3)Gal(â1-3)Gal(â1-4)Xylâ) coupled to serine residues
of the protein chain. The general structure is then modified through N-deacetylation/N-sulfation, epimerization, and O-sulfation.
SCHEME 1. Synthesis of Compound 5a
a Reagents and conditions: (i) anthraldehyde dimethyl acetal, pTSA,
FIGURE 2. Compound 2 was synthesized as a fluorescent analogue
MeCN, 2 h; (ii) BH3‚NMe3, AlCl3, THF, 0 °C, 3 h; (iii) BH3‚NMe3, AlCl3,
toluene, rt, 5 min.
of the antiproliferative compound 1.
Regioselective Acetal Openings
4H-benzo[1,3]dioxine (4), which was synthesized from salicylic
alcohol by treatment with anthraldehyde dimethyl acetal.
Compound 4 was subjected to NaCNBH3-HCl (standard
conditions for regioselective opening of benzylidene acetals of
carbohydrates), which only resulted in degradation.
The results indicate that these mixed acetals are more sensitive
to hydrolysis, and instead we tried BH3‚NMe3.17 Borane
complexes in combination with Lewis acids (e.g., AlCl3) have
been used for reductive openings of acetal-protected carbohy-
drates and the regioselectivity is mainly directed by the solvent;
reaction in THF usually gives the benzylic ether in position 6
of carbohydrates whereas toluene gives 4-benzylic ethers but
also degradation.17 The mechanistic background to this selectiv-
ity is unclear.
Treatment of compound 4 with BH3‚NMe3-AlCl3 in THF
for 3 h at 0 °C gave 2-(anthracen-9-yl-methoxymethyl)-phenol
5 in an excellent 83% yield, whereas reaction in toluene for 5
min resulted in decomposition (Scheme 1). Compound 4 was
also treated with BH3‚NMe3 in THF without AlCl3, which gave
no reaction, nor did any reaction occur with AlCl3 in THF.
Interestingly, reverse addition order, i.e., first addition of AlCl3
and after 15 min addition of BH3‚NMe3, gave complete
degradation of compound 4. The more reactive BH3‚THF
complex18 gave a low yield of 10% as well as degradation of
the starting material. Treatment with AlCl3 in toluene resulted
in decomposition.
We have earlier introduced 9-anthraldehyde acetals as
fluorescent protecting groups for carbohydrates.5 The acetals
of both gluco- or galactopyranosides could be regioselectively
opened using NaBH3CN/THF/HCl6 to give 6-O-(9-anthracenyl)-
methyl ethers. We realized that compound 2 could be synthe-
sized by a reductive regioselective opening of a mixed phenolic-
benzylic acetal.
Despite the large number of methods published for reductive
regioselective openings of acetals (e.g., BH3‚THF-Cu(OTf)2,7
NaCNBH3-HCl,6,8 BH3‚NMe2H-BF3‚OEt2,9 Me2BBr-BH3,10
Bu3SnH-MgBr2‚OEt2,11 Me3SiH-BF3‚OEt2,12 Et3SiH-TFA,13
Et3SiH-TfOH,14 Et3SiH-PhBCl2,14 BH3-Bu2OTf 15), there are,
to our knowledge, no reductive openings of mixed phenolic-
benzylic acetals known, and only one observation of a regiose-
lective opening (i.e., nucleophilic attack).16 There are also still
uncertainties about the mechanism of regioselective openings
of normal aliphatic acetals.
To investigate reductive openings of mixed phenolic-benzylic
acetals, a model system was designed using 2-anthracen-9-yl-
(5) Ellervik, U. Tetrahedron Lett. 2003, 44, 2279.
(6) Garegg, P. J.; Hultberg, H.; Wallin, S. Carbohydr. Res. 1982, 108,
97.
(7) Shie, C.-R.; Tzeng, Z.-H.; Kulkarni, S. S.; Uang, B.-J.; Hsu, C.-Y.;
Hung, S.-C. Angew. Chem., Int. Ed. 2005, 44, 1665.
(8) Garegg, P. J.; Hultberg, H.Carbohydr. Res. 1981, 93, C10.
(9) Oikawa, M.; Liu, W.-C.; Nakai, Y.; Koshida, S.; Fukase, K.;
Kusumoto, S. Synlett 1996, 1179.
(10) Guindon, Y.; Girard, Y.; Berthiaume, S.; Gorys, V.; Lemieux, R.;
Yoakim, C. Can. J. Chem. 1990, 68, 897.
(11) Zheng, B.-Z.; Yamauchi, M.; Dei, H.; Kusaka, S.-I.; Matsui, K.;
Yonemitsu, O. Tetrahedron Lett. 2000, 41, 6441.
(12) Debenham, S. D.; Toone, E. J. Tetrahedron: Asymmetry 2000, 11,
385.
One advantage of studying the regioselective reductive acetal
opening using an aromatic system is that the electronic effects
of the two oxygen atoms can be altered by substituents of the
aromatic moiety.
To provide insight into the mechanism, three more substances
were synthesized carrying (i) an electron-donating group (OMe),
(13) DeNinno, M. P.; Etienne, J. B.; Duplantier, K. C. Tetrahedron Lett.
1995, 36, 669.
(14) Sakagami, M.; Hamana, H. Tetrahedron Lett. 2000, 41, 5547.
(15) Jiang, L.; Chan, T.-H. Tetrahedron Lett. 1998, 39, 355.
(16) Tsubaki, K.; Otsubo, T,; Tanaka, K.; Fujo, K.; Kinoshita, T. J. Org.
Chem. 1998, 63, 3260.
(17) Ek, M.; Garegg, P. J.; Hultberg, H.; Oscarson, S. J. Carbohydr.
Chem. 1983, 2, 305.
(18) (a) Ashby, E. C. J. Am. Chem. Soc. 1959, 81, 4791. (b) Brown, H.
C.; Subba Rao, B. C. J. Org. Chem. 1957, 22, 1136.
J. Org. Chem, Vol. 71, No. 9, 2006 3445

Johnsson et al.
SCHEME 2. Synthesis of Compound 9a
SCHEME 4. Synthesis of Compund 15a
a Reagents and conditions: (i) MeMgCl, Et2O, 4 h; (ii) anthraldehyde
dimethyl acetal, pTSA, MeCN, 18 h; (iii) BH3‚NMe3, AlCl3, THF, 0 °C, 3
h; (iv) BH3‚NMe3, AlCl3, toluene, rt, 5 min.
TABLE 1. Electrostatic Potential for Acetal Oxygen Atoms
(kJ/mol)a
a Reagents and conditions: (i) BH3‚THF, THF, 0 to 60 °C, 3.5 h; (ii)
anthraldehyde dimethyl acetal, pTSA, THF, 3.5 h; (iii) BH3‚NMe3, AlCl3,
THF, 0 °C, 3 h.
compound
phenolic oxygen
benzylic oxygen
4
8
12
15
-141
-148
-103
-146
-150
-154
-117
-151
SCHEME 3. Synthesis of Compound 12a
a The electrostatic potentials were calculated using DFT at (B3LYP/
6-31G*) level.
a Reagents and conditions: (i) BH3‚THF, THF, 0 to 60 °C, 3.5 h; (ii)
anthraldehyde dimethyl acetal, pTSA, MeCN, 18 h; (iii) BH3‚NMe3, AlCl3,
THF, 0 °C, 3 h.
(ii) an electron-withdrawing group (NO2), and (iii) sterically
demanding methyl groups in the benzylic position.
2-Anthracen-9-yl-6-methoxy-4H-benzo[1,3]dioxine (8) was
synthesized from 5-methoxy salicylic acid (6), which was first
reduced using BH3‚THF to give 2-hydroxymethyl-4-methoxy-
phenol (7) in 73% yield. Compound 7 was then transformed
into the anthraldehyde acetal (8) in a modest 32% yield and
subsequently treated with BH3‚NMe3-AlCl3 in THF for 3 h at
0 °C to give 2-(anthracen-9-yl-methoxymethyl)-4-methoxy-
phenol (9) in 73% yield (Scheme 2).
2-Anthracen-9-yl-6-nitro-4H-benzo[1,3]dioxine (12) was syn-
thesized from 5-nitro salicylic acid (10) in a similar manner.
However, treatment with BH3‚NMe3-AlCl3 in THF for 3 h at
0 °C resulted in no product and 69% recovery of compound 12
(Scheme 3).
The sterically hindered 2-anthracen-9-yl-4,4-dimethyl-4H-
benzo[1,3]dioxine (15) was synthesized from methyl salicylate
(13) by treatment with MeMgCl followed by acetal formation.
Treatment with BH3‚NMe3-AlCl3 in THF for 3 h at 0 °C
resulted in 99% recovery of 15, whereas reaction in toluene
resulted in decomposition in less than 5 min (Scheme 4).
These results indicate that the regioselectivity of the acetal
openings is directed by both electronic effects and steric effects.
To further investigate the mechanism, the electrostatic potentials
of the four substances were calculated using density functional
theory at the B3LYP/6-31G* level and default settings in
FIGURE 3. Electrostatic potential is displayed as a gradient from -160
kJ/mol in red to 130 kJ/mol in blue: (A) compound 4, (B) compound
8, (C) compound 12, and (D) compound 15.
Spartan ’02 for Macintosh.19 The results show that the benzylic
oxygen is the more basic in all four compounds, indicating that
this oxygen should react first with a Lewis acid (Table 1 and
Figure 3).
Altogether these results indicate that the Lewis acid (i.e., BH3
or AlCl3 dependent on the addition order) first binds to the more
basic oxygen (i.e., the benzylic oxygen). The reductions then
proceed, promoted by reaction with AlCl3 (Figure 4).
Probably reduction from the phenolic position (i.e., yielding
benzylic alcohols), for example, by reverse order addition, gives
decomposition by an unknown mechanism. The preorganization
is highly sensitive to the electrostatic potential of the oxygen
since the electron-deficient compound 12 does not react under
these conditions. The more reactive BH3‚THF complex gives
more decomposition, probably as a result of less discrimination
between the two oxygen atoms. The lower yield from the
(19) (a) Spartan ’02 for Macintosh; Wavefunction, Inc.: Irvine, CA. (b)
Becke, A. D. J. Chem Phys. 1993, 98, 5648. (c) Lee, C.; Yang, W.; Parr,
R. G. Phys. ReV. B 1988, 37, 785.
3446 J. Org. Chem., Vol. 71, No. 9, 2006

RegioselectiVe ReductiVe Openings of Acetals
TABLE 2. Gradient Retention Times and pKa Values for
Compounds 1 and 2
compound
retention time (min)
pKa
1
2
12.49 ( 0.01
36.86 ( 0.04
9.63 ( 0.02
10.52 ( 0.09
give 2-antracen-9-yl-4H-naphtho[2,3-d][1,3]dioxin-7-ol (18) in
76% yield.5 The acetal was xylosylated in 72% yield using 2,3,4-
tri-O-acetyl-R/â-D-xylopyranosyl trichloroacetimidate to give (2-
antracen-9-yl-4H-naphtho[2,3-d][1,3]dioxin-7-yl)
2,3,4-tri-
FIGURE 4. Mechanism proposal. The borane first binds to the more
basic benzylic oxygen, and the reduction then proceeds, promoted by
reaction with AlCl3.
O-acetyl-â-D-xylopyranoside (19).21 The xylosylation was car-
ried out in CH2Cl2 with a catalytic amount of TMSOTf and
molecular sieves in order to minimize degradation of the
acid sensitive anthracenylidene acetal.22 Regioselective opening
of the anthraldehyde acetal 19 was then performed using
BH3‚NMe3 and AlCl3 in THF for 45 min to give 6-(2-hydroxy-
3-(antracen-9-yl-methoxymethyl))-naphthyl) 2,3,4-tri-O-acetyl-
â-D-xylopyranoside (20) in an excellent 91% yield.
SCHEME 5. Synthesis of Compound 2a
Deprotection of the acetyl groups by standard Zemple´n
conditions (0.05 M NaOMe/MeOH) gave a low yield. Instead
guanidine/guanidinium nitrate, a less basic method,23 was used,
giving 6-(2-hydroxy-3-(antracen-9-yl-methoxymethyl))-naph-
thyl)-â-D-xylopyranoside (2) in 77% yield.
Gradient HPLC retention times can be used to substitute log
P values in biological evaluations.24 The gradient HPLC
retention times for compounds 1 and 2 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. The acidity constants (pKa) of phenolic compounds
are usually difficult to predict by computational methods.25 The
pKa’s of compounds 1 and 2 were instead measured using
spectroscopic methods at pH 9, 10, and 11. The results are
summarized in Table 2. The absorbance spectrum of compound
2 (in MeCN) and fluorescence spectrum (in MeCN, λEx ) 350
nm) are presented in Figure 5. The highly unpolar (9-anthra-
cenyl)methyl moiety makes compound 2 highly unpolar as
reflected by the retention time. Compound 2 is also much less
acidic as a result of the electron-donating properties of the
substituent.
a Reagents and conditions: (i) BH3‚THF, THF, 0 to 60 °C, 3.5 h; (ii)
anthraldehyde dimethyl acetal, pTSA, MeCN, 1 h; (iii) 2,3,4-tri-O-acetyl-
R/â-D-xylopyranosyl trichloroacetimidate, TMSOTf, MS 300 AW, CH2Cl2,
3 h; (iv) BH3‚NMe3, AlCl3, THF, 0 °C, 45 min; (v) guanidine-guanidinium
nitrate, 30 min.
Biological Results
As compound 2 is highly lipophilic and uncharged, it may
penetrate cell membranes, initiate GAG synthesis, and thereby
affect various cellular processes. Compound 2 was accordingly
tested for uptake, antiproliferative activity, and GAG priming
in different cell lines. To investigate the antiproliferative activity
of compound 2, 3T3 A31 cells, 3T3 SV40 cells, and T24 cells
were treated with the xyloside for 96 h, and the effect on cell
growth was studied using the crystal violet method. Treatment
reduction of the electron-rich compound 8 is probably also a
result of less discrimination between the two oxygen atoms.
There is also a steric effect as indicated by the unreactive
compound 15 (the electrostatic potential of the benzylic oxygen
in compound 15 is equal to the highly reactive compound 4).
No conclusions can be drawn from the reactions in toluene
since only degradation was observed.
(20) Yoon, N. M.; Pak, C. S.; Brown, H. C.; Krishnamurthy, S.; Stocky,
T. P. J. Org. Chem. 1973, 38, 2786.
(21) (a) Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260. (b) Mori, M.; Ito, Y.; Ogawa, T. Carbohydr. Res. 1990, 195,
199.
(22) Lichtenthaler, F. W.; Kla¨res, U.; Szurmai, Z.; Werner, B. Carbohydr.
Res. 1998, 305, 293.
Synthesis of Fluorescently Labeled Naphthoxylosides
With a well working protocol for regioselective acetal open-
ings we synthesized compound 2 from commercially available
3,7-dihydroxy-2-naphthoic acid (Scheme 5).
(23) Ellervik, U.; Magnusson, G. Tetrahedron Lett. 1997, 38, 1627.
(24) Valko´, K.; Plass, M.; Bevan, C.; Reynolds, D.; Abraham, M. H. J.
Chromatogr. A 1998, 797, 41.
(25) Hanai, T.; Koizumi, K.; Kinoshita, T.; Arora, R.; Ahmed, F. J.
Chromatogr. A 1997, 762, 55.
3,7-Dihydroxy-2-naphthoic acid (16) was reduced by BH3‚
THF to give 2,6-dihydroxy-3-(hydroxymethyl)-naphthalene (17)
in 58% yield.20 The triol was then treated with anthraldehyde
dimethyl acetal in MeCN under acidic conditions (pTSA) to
J. Org. Chem, Vol. 71, No. 9, 2006 3447

Johnsson et al.
FIGURE 5. Absorbance spectrum of compound 2 (1.15 mM in MeCN,
solid line) and fluorescence spectrum of compound 2 (1.15 mM in
MeCN, λEx ) 350 nm, dotted line).
FIGURE 6. Effect of compound 2 on growth of 3T3 A31 (squares),
3T3 SV40 (circles), and T24 (triangles) cells was studied as described
in Experimental Section. Cell numbers were determined after 96 h of
culturing and expressed as the percentage of growth in the absence of
drugs.
with compound 2 inhibited growth of normal 3T3 A31 cells
and transformed 3T3 SV40 and T24 cells in a dose-dependent
manner (Figure 6).
FIGURE 7. Uptake of compound 2 by different cell lines. The panels
show fluorescence microscopy of (A) 3T3 A31, (B) 3T3 SV40, and
(C) T24 cells treated with 100 µM of compound 2 for 16 h. Bar:
20 µm.
At a concentration between 100 and 200 µM the transformed
T24 and 3T3 SV40 cells were more growth inhibited than
normal 3T3 A31 cells. To examine to what extent the growth
inhibition was correlated with uptake of the xyloside, the cells
were treated with 100 µM xyloside for 16 h and then fixed and
studied using fluorescence microscopy. The xyloside was taken
up by all used cell lines. In 3T3 A31 cells and 3T3 SV40 cells
the xyloside was located largely inside the cytoplasm, whereas
in T24 cells the xyloside was accumulated in para- and
perinuclear compartments (Figure 7).
To study the GAG priming ability of compound 2, 3T3 A31
cells were left untreated or were treated with 100 µM xyloside
and [35S]sulfate overnight and free GAG chains in the medium
and the cell extracts were isolated. 3T3 A31 cells secreted three
size-dependent pools (I, II, and III) of radiolabeled material into
the culture medium (Figure 8).
Large-sized (I) and intermediate-sized (II) material consisted
of alkali-sensitive proteoglycans, whereas the regular-sized (III)
chains were free alkali-resistant oligosaccharides. Xyloside
treatment suppressed endogenous proteoglycan production to
some extent. A very small amount of xyloside-primed GAG
chains were recovered from the medium of xyloside-treated cells
(pool III). In the cell extracts suppression of endogenous
proteoglycan was noted. No cell-associated GAG chains primed
by compound 2 were detected, indicating that the xyloside did
not initiate GAG priming in these cells.
Conclusions
Mixed phenolic-benzylic acetals can be regioselectively
opened using BH3‚NMe3-AlCl3 in THF at 0 °C to yield a
benzylic ether and the free phenol. We are currently investigat-
ing the possibilities for inverse openings.
We propose a mechanism where the borane first associates
to the more basic oxygen (i.e., the benzylic oxygen). The
reductions then proceed, promoted by reaction with AlCl3.
We have used the regioselective acetal opening in the
synthesis of a fluorescent analogue to antiproliferative xylosides.
The fluorescently labeled compound was taken up by all cell
lines but did not initiate glucosaminoglycan biosynthesis.
Experimental Section
6-(2-Hydroxy-3-(anthracen-9-yl-methoxymethyl)-naphthyl)-
â-D-xylopyranoside (2). Guanidinium nitrate (127 mg, 1.04 mmol)
was dissolved in MeOH/CH2Cl2 (10 mL, 9:1) and methanolic
3448 J. Org. Chem., Vol. 71, No. 9, 2006

RegioselectiVe ReductiVe Openings of Acetals
128.4, 126.6, 125.4, 125.2, 124.9, 121.8, 121.4, 117.7, 98.0, 68.2.
HRMS calcd for C22H17O2 (M + H): 313.1229, found 313.1221.
2-(Anthracen-9-yl-methoxymethyl)-phenol (5). To a solution
of 4 (20 mg, 0.07 mmol) and BH3‚NMe3 (34 mg, 0.46 mmol) in
THF (2 mL) was added AlCl3 (69 mg, 0.51 mmol in 1 mL THF)
during 5 min at 0 °C. The mixture was stirred at 0 °C for 3 h, then
diluted with CH2Cl2, and washed with saturated aqueous NaHCO3.
The organic phase was dried (Na2SO4) and concentrated. The
residue was chromatographed (SiO2, heptane/EtOAc 10:1) to give
1
5 (17 mg, 83%) as a white solid. H NMR (CDCl3): δ 8.51 (s, 1
H, H-10′), 8.29 (dd, 2 H, J 8.8, 0.7 Hz, H-1′, H-8′), 8.04 (ddd, 2
H, J 8.4, 0.7, 0.6 Hz, H-4′, H-5′), 7.48-7.58 (m, 4 H, H-2′, H-3′,
H-6′, H-7′), 7.40 (s, 1 H, OH), 7.27-7.22 (m, 1 H, H-4), 7.05 (dd,
1 H, J 7.5, 1.4 Hz, H-3), 6.85-6.92 (m, 2 H, H-6, H-5), 5.60 (s, 2
H, CH2-ant), 4.86 (s, 2 H, CH2-ben). 13C NMR (CDCl3): δ 156.6,
?31.6, 131.3, 129.9, 129.4, 129.3, 128.8, 126.9, 125.3, 124.0, 122.5,
120.2, 116.9, 71.8, 64.5. HRMS calcd for C22H18O2Na (M + Na):
337.1204, found 337.1193.
2-Hydroxymethyl-4-methoxy-phenol (7). Compound 6 (296
mg, 1.76 mmol) was dissolved in THF (1.3 mL) and cooled to 0
°C under argon. BH3‚THF (1 M, 4.4 mL) was added dropwise
during 30 min. The mixture was stirred at 0 °C for 45 min and
then left to attain room temperature. After 1 h BH3‚THF (1 M, 0.9
mL) was added and the mixture was stirred at room temperature
for 1 h and then at 60 °C for 1.5 h. The mixture was then added to
a mixture of ice and HCl (0.5 M, 100 mL) and extracted with Et2O.
The organic phase was washed with saturated aqueous NaHCO3
twice, then dried (Na2SO4), and concentrated to give 7 (197 mg,
73%) as a light yellow solid. 1H NMR data was in agreement with
published data.26
FIGURE 8. Secreted proteoglycan and GAG in 3T3 A31 cells treated
with compound 2. 3T3 A31 cells grown to confluence were labeled
with [35S]sulfate for 24 h in the absence (solid line) or presence (dashed
line) of 100 µM of compound 2. Polyanionic macromolecules from
the culture medium were isolated by recovery on DEAE-cellulose
followed by hydrophobic interaction chromatography on octyl-
Sepharose and gel permeation chromatography on Superose 6. Pro-
teoglycans and heparan sulfate were determined as described in
Experimental Section.
2-Anthracen-9-yl-6-methoxy-4H-benzo[1,3]dioxine (8). To a
solution of 7 (98 mg, 0.64 mmol) and anthraldehyde dimethyl acetal
(180 mg, 0.71 mmol) in THF (7 mL) was added pTSA (5 mg).
The mixture was stirred at room temperature for 3.5 h, then diluted
with CH2Cl2, washed with saturated aqueous NaHCO3 twice, and
then dried (Na2SO4). The residue was chromatographed (SiO2,
heptane/EtOAc 10:1 f 5:1) to give 8 (70 mg, 32%) as a light
FIGURE 9. Numbering of compounds 2 and 20.
MeONa (0.20 mL, 1 M) was added to give a clear stock solution.
Compound 20 (23 mg, 0.037 mmol) was dissolved in the stock
solution (2 mL). The mixture was stirred at room temperature for
30 min, acidified with HOAc (0.05 mL), diluted with CH2Cl2 (17
mL), filtered on SiO2 and concentrated. The residue was chromato-
graphed (SiO2, CH2Cl2/MeOH 10:1) to give 2 (14.4 mg, 77%) as
1
yellow solid. H NMR (CDCl3): δ 8.64 (d, 2 H, J 8.9 Hz, H-1′,
H-8′), 8.55 (s, 1 H, H-10′), 8.03 (dd, 2 H, J 8.1, 1.7 Hz, H-4′,
H-5′), 7.45-7.53 (m, 4 H, H-2′, H-3′, H-6′, H-7′), 7.34 (s, 1 H,
H-2), 6.94 (d, 1 H, J 8.9 Hz, H-8), 6.84 (dd, 1 H, J 8.9, 3.0 Hz,
H-7), 6.71 (d, 1 H, J 2.9 Hz, H-5), 5.41, 5.19 (ABq, 1 H each, J
14.9 Hz, H-4), 3.84 (s, 3 H, CH3). 13C NMR (CDCl3): δ 154.6,
148.0, 131.8, 130.4, 130.2, 129.4, 129.3, 126.6, 125.2, 125.0, 121.9,
118.4, 114.5, 109.9, 98.0, 68.1, 56.1. HRMS calcd for C23H19O3
(M + H): 343.1334, found 343.1333.
a light yellow solid. [R]21 +7 (c 0.01, DMSO-d6). 1H NMR
D
(DMSO-d6): δ 9.75 (s, 1 H, OH), 8.66 (s, 1 H, H-10′′), 8.46 (d, 2
H, J 8.7 Hz, H-1′′, H-8′′), 8.12 (dd, 2 H, J 7.7, 1.4 Hz, H-4′′, H-5′′),
7.69 (s, 1 H, H-1′), 7.60 (d, 1 H, J 9.0 Hz, H-8′), 7.52-7.59 (m,
4 H, H-2′′, H-3′′, H-6′′, H-7′′), 7.26 (d, 1 H, J 2.3 Hz, H-5′), 7.11
(s, 1 H, H-4′), 7.09 (dd, 1 H, J 9.0, 2.4 Hz, H-7′), 5.61 (s, 2 H,
CH2-ant), 5.30 (d, 1 H, J 4.8 Hz, OH), 5.08 (d, 1 H, J 4.3 Hz,
OH), 5.03 (d, 1 H, J 4.8 Hz, OH), 4.89 (d, 1 H, J 7.4 Hz, H-1),
4.87 (s, 2 H, CH2-nap), 3.73 (dd, 1 H, J 11.0, 5.0 Hz, H-5), 3.20-
3.30 (m, 4 H, H-2, H-3, H-4, H-5). 13C NMR (DMSO-d6): δ 152.9,
152.0, 131.0, 130.5, 129.7, 129.2, 128.8, 128.3, 128.1, 128.0, 126.9,
126.3, 126.2, 125.2, 124.6, 118.8, 110.6, 108.4, 101.2, 76.5, 73.1,
69.4, 67.4, 65.7, 64.2. HRMS calcd for C31H28O7Na (M + Na):
535.1733, found 535.1721.
2-(Anthracen-9-yl-methoxymethyl)-4-methoxy-phenol (9). Ac-
cording to the same procedure as for 5: 8 (22 mg, 0.07 mmol),
BH3‚NMe3 (30 mg, 0.41 mmol), THF (2 mL), AlCl3 (62 mg, 0.47
mmol in 1 mL THF). Chromatographed (SiO2, heptane/EtOAc 10:1
f 5:1) to give 9 (16 mg, 73%) as a white solid. 1H NMR
(CDCl3): δ 8.51 (s, 1 H, H-10′), 8.29 (d, 2 H, J 8.3 Hz, H-1′,
H-8′), 8.04 (d, 2 H, J 8.4 Hz, H-4′, H-5′), 7.54-7.58 (m, 2 H,
H-2′, H-7′), 7.47-7.51 (m, 2 H, H-3′, H-6′), 6.95 (s, 1 H, OH),
6.84 (d, 1 H, J 8.8 Hz, H-6), 6.79 (dd, 1 H, J 8.8, 2.8 Hz, H-5),
6.60 (d, 1 H, J 2.8 Hz, H-3), 5.60 (s, 2 H, CH2-ant), 4.80 (s, 2 H,
CH2-ben), 3.74 (s, 3 H, CH3). 13C NMR (CDCl3): δ 153.2, 150.4,
131.6, 131.3, 129.4, 129.3, 127.4, 126.9, 125.4, 124.0, 123.2, 117.5,
115.1, 114.2, 71.6, 64.4, 56.0. HRMS calcd for C23H20O3Na (M +
Na): 367.1310, found 367.1311.
2-Anthracen-9-yl-4H-benzo[1,3]dioxine (4). To a solution of
3 (110 mg, 0.89 mmol) and anthraldehyde dimethyl acetal (247
mg, 0.98 mmol) in MeCN (10 mL) was added pTSA (5 mg). The
mixture was stirred at room temperature for 2 h, then diluted with
CH2Cl2, washed with saturated aqueous NaHCO3 twice, and then
dried (Na2SO4). The residue was chromatographed (SiO2, toluene)
2-Hydroxymethyl-4-nitro-phenol (11). According to the same
procedure as for 7: 10 (299 mg, 1.63 mmol), THF (1.3 mL), BH3‚
THF (1 M, 4.4 mL), BH3‚THF (1 M, 0.85 mL), ice and HCl (0.5
1
to give 4 (217 mg, 78%) as a yellow solid. H NMR (CDCl3): δ
1
8.65 (d, 2 H, J 8.9 Hz, H-1′, H-8′), 8.56 (2, 1 H, H-10′), 8.04 (dd,
2 H, J 8.1, 1.7 Hz, H-4′, H-5′), 7.46-7.53 (m, 4 H, H-2′, H-3′,
H-6′, H-7′), 7.41 (s, 1 H, H-2), 7.27-7.25 (m, 1 H, H-6), 7.18 (d,
1 H, J 6.9 Hz, H-5), 7.09 (dt, 1 H, J 7.5, 1.1 Hz, H-7), 7.00 (dd,
1 H, J 8.2, 0.7 Hz, H-8), 5.45, 5.23 (ABq, 1 H each, J 14.8 Hz,
H-4). 13C NMR (CDCl3): δ 154.0, 131.8, 130.5, 130.2, 129.3,
M, 85 mL) to give 11 (87 mg, 31%) as a yellow solid. H NMR
data was in agreement with published data.27
(26) Manetsch, R.; Zheng, L.; Reymond, M. T.; Woggon, W.-D.;
Reymond, J.-L. Chem. Eur. J. 2004, 10, 2487.
(27) Arenz, C.; Giannis, A. Eur. J. Org. Chem. 2001, 137.
J. Org. Chem, Vol. 71, No. 9, 2006 3449

Johnsson et al.
2-Anthracen-9-yl-6-nitro-4H-benzo[1,3]dioxine (12). To a
solution of 11 (51 mg, 0.30 mmol) and anthraldehyde dimethyl
acetal (82 mg, 0.33 mmol) in MeCN (4 mL) was added pTSA (5
mg). The mixture was stirred at room temperature for 18 h, then
diluted with CH2Cl2, washed with saturated aqueous NaHCO3 twice,
and then dried (Na2SO4). The residue was chromatographed (SiO2,
heptane/EtOAc 10:1 f 5:1) to give 12 (23 mg, 22%) as a yellow
H-2′, H-3′, H6′, H-7′), 7.24 (s, 1 H, H-10), 7.11 (d, 1 H, J 2.2 Hz,
H-6), 7.04 (dd, 1 H, J 8.8, 2.3 Hz, H-8), 5.67, 5.34 (ABq, 1 H
each, J 15.3 Hz, H-4). 13C NMR (DMSO-d6): δ 153.9, 149.3, 131.0,
130.0, 129.8, 129.4, 128.9, 128.0, 127.6, 127.0, 126.3, 125.1, 125.0,
123.0, 122.3, 119.1, 111.3, 108.3, 97.3, 67.6. HRMS calcd for
C26H19O3 (M + H): 379.1334, found 379.1320.
(2-Antracen-9-yl-4H-naphtho[2,3-d][1,3]dioxin-7-yl) 2,3,4-tri-
O-acetyl-â-D-xylopyranoside (19). A solution of 18 (99 mg, 0.263
mmol), 2,3,4-tri-O-acetyl-R/â-D-xylopyranosyl trichloroacetimidate
(244 mg, 0.581 mmol) and ground MS 300 AW (0.3 g) in CH2Cl2
(7 mL) was stirred at room temperature under argon for 1 h.
TMSOTf (0.05 M in CH2Cl2, 0.250 mL) was added dropwise to
the mixture. After 1 h TMSOTf (0.05 M in CH2Cl2, 0.050 mL)
was added and stirred for 1 h. Et3N (3 mL) was added (pH ∼8)
and the mixture was concentrated. The residue was chromato-
graphed (SiO2, heptane/EtOAc 2:1) to give 19 (120 mg, 72%) as a
light yellow solid. [R]21D -16 (c 0.89, CHCl3). 1H NMR (CDCl3):
δ 8.65 (δ, 2 Η, J 8.6 Hz, H-1′′, H-8′′), 8.57 (s, 1 H, H-10′′), 8.05
(dd, 2 H, J 7.5, 1.9 Hz, H-4′′, H-5′′), 7.66 (d, 1 H, J 9.0 Hz, H-9′),
7.56 (s, 1 H, H-5′), 7.45-7.55 (m, 5 H, H-2′, H-2′′, H-3′′, H-6′′,
H-7′′), 7.35 (d, 1 H, J 2.2 Hz, H-6′), 7.33 (s, 1 H, H-10′), 7.16 (dd,
1 H, J 8.9, 2.4 Hz, H-8′), 5.61, 5.42 (ABq, 1 H each, J 10.0 Hz,
H-4′), 5.20-5.30 (m, 3 H, H-1, H-2, H-3, JH1-H2 5.6 Hz), 5.06
(dt, 1 H, J 7.2, 4.6 Hz, H-4), 4.30 (dd, 1 H, J 12.1, 4.6 Hz, H-5),
3.62 (dd, 1 H, J 12.1, 7.5, H-5), 2.13, 2.12, 2.11 (s, 3 H each,
OAc). 13C NMR (CDCl3): δ 170.2, 170.1, 169.7, 153.5, 151.3,
131.8, 130.6, 130.4, 130.2, 129.7, 129.3, 128.8, 126.7, 126.4, 125.2,
124.8, 123.6, 123.2, 119.7, 112.7, 111.41, 111.37, 99.0, 98.9, 98.5,
70.8, 70.4, 68.70, 68.67, 62.1, 21.03, 21.00. HRMS calcd for
C37H32O10Na (M + Na): 659.1893, found 659.1900.
1
solid. H NMR (CDCl3): δ 8.60 (s, 1 H, H-10′), 8.52 (d, 2 H, J
8.9 Hz, H-1′, H-8′), 8.15-8.19 (m, 2 H, H-7, H-5), 8.06 (dd, 2 H,
J 8.0, 1.7 Hz, H-4′, H-5′), 7.48-7.56 (m, 4 H, H-2′, H-3′, H-6′,
H-7′), 7.46 (s, 1 H, H-2), 7.05 (d, 1 H, J 8.7 Hz, H-8), 5.48,
5.29 (ABq, 1 H each, J 15.1 Hz, H-4). 13C NMR (CDCl3): δ
159.2, 131.7, 131.1, 130.2, 129.5, 128.2, 128.0, 127.0, 125.3, 125.0,
124.7, 124.4, 122.0, 121.5, 118.3, 99.0, 67.9. HRMS calcd for
C22H15NO4Na (M + Na): 380.0899, found 380.0900.
2-(1-Hydroxy-1-methyl-ethyl)-phenol (14). To methylmagne-
sium chloride (12 mL, 3 M in THF) stirred at 0 °C under argon
was added 13 (0.50 mL, 3.9 mmol) dissolved in Et2O (3 mL) during
10 min. The mixture was stirred at 0 °C for 1.5 h and then left to
attain room temperature. After 2.5 h ice (14 g) was added and the
mixture was acidified with 10% aqueous HCl. The water phase
was extracted with Et2O and the combined organic phases were
washed with saturated aqueous NaHCO3, dried (Na2SO4), and
concentrated. The residue was chromatographed (SiO2, heptane/
EtOAc 2:1) to give 14 (515 mg, 88%) as a clear oil. 1H NMR was
in agreement with published data.28
2-Anthracen-9-yl-4,4-dimethyl-4H-benzo[1,3]dioxine (15). Ac-
cording to the same procedure as for 12: 14 (100 mg, 0.66 mmol),
anthraldehyde dimethyl acetal (182 mg, 0.72 mmol), MeCN (7 mL),
pTSA (5 mg). Chromatographed (SiO2, toluene) to give 15 (116
1
mg, 52%) as a light yellow solid. H NMR (DMSO-d6): δ 8.73-
6-(2-Hydroxy-3-(anthracen-9-yl-methoxymethyl)-naphthyl)
2,3,4-tri-O-acetyl-â-D-xylopyranoside (20). To a solution of 19
(70 mg, 0.1 mmol) and BH3‚NMe3 (50 mg, 0.69 mmol) in THF (3
mL) was added AlCl3 (110 mg, 0.83 mmol in 2 mL THF) during
5 min at 0 °C. The mixture was stirred at 0 °C for 45 min, then
diluted with CH2Cl2, and washed with saturated aqueous NaHCO3.
The organic phase was dried (Na2SO4) and concentrated. The
residue was chromatographed (SiO2, heptane/EtOAc 2:1 f 1:1) to
give 20 (64 mg, 91%) as a light yellow solid. [R]21D -16 (c 0.79,
8.75 (m, 3 H, H-10′, H-1′, H-8′), 8.12-8.16 (m, 2 H, H-4′, H-5′),
7.52-7.55 (m, 5 H, H-2′, H-3′, H-6′, H-7′, H-2), 7.45 (dd, 1 H, J
7.8, 1.5 Hz, H-5), 7.23 (ddd, 1 H, J 8.1, 7.4, 1.6 Hz, H-7), 7.09
(dt, 1 H, J 7.6, 1.2 Hz, H-6), 6.90 (dd, 1 H, J 8.1, 1.1 Hz, H-8),
1.86, 1.66 (s, 3 H each, CH3). 13C NMR (DMSO-d6): δ 152.2,
131.0, 130.2, 129.7, 129.4, 128.9, 127.8, 126.8, 126.4, 126.3, 125.1,
124.9, 121.5, 116.9, 92.0, 76.6, 30.6, 28.7. HRMS calcd for
C24H20O2Na (M + Na): 363.1361, found 363.1350.
1
CHCl3). H NMR (CDCl3): δ (s, 1 H, H-10′′), 8.29 (d 2 H, J 8.8
2,6-Dihydroxy-3-(hydroxymethyl)-naphthalene (17). Com-
pound 16 (395 mg, 1.93 mmol) was dissolved in THF (1.5 mL)
and cooled to 0 °C under argon. BH3‚THF (1 M, 5 mL) was added
dropwise during 10 min. The mixture was stirred at 0 °C for 35
min and then left to attain room temperature. After 1 h BH3‚THF
(1 M, 1 mL) was added and the mixture was stirred at room
temperature for 1 h and then at 60 °C for 1.5 h. The mixture was
then added to a mixture of ice and HCl (0.5 M, 100 mL) and
extracted with Et2O. The organic phase was washed with satu-
rated aqueous NaHCO3 twice, then dried (Na2SO4), and concen-
Hz, H-1′′, H-8′′), 8.05 (d, 2 H, J 8.1 Hz, H-4′′, H-5′′), 7.66 (d, 1
H, J 8.1 Hz, H-8′), 7.45-7.60 (m, 5 H, H-4′, H-2′′, H-3′′, H-6′′,
H-7′′), 7.36 (s, 1 H, H-1′), 7.27 (d, 1 H, J 2.4 Hz, H-5′), 7.23 (s,
1 H, OH), 7.14 (dd, 1 H, J 8.9, 2.4 Hz, H-7′), 5.63 (s, 2 H,
CH2-ant), 5.20-5.30 (m, 3 H, H-1, H-2, H-3), 5.04 (dt, 1 H, J
7.5, 4.7 Hz, H-4), 4.98 (s, 2 H, CH2-nap), 4.26 (dd, 1 H, J 12.1,
4.7 Hz, H-5), 3.57 (dd, 1 H, J 12.1, 7.6, H-5), 2.12, 2.11, 2.09 (s,
3 H each, OAc). 13C NMR (CDCl3): δ 170.2, 170.1, 169.6, 153.5,
153.1, 131.6, 131.3, 129.44, 129.36, 128.9, 128.3, 127.6, 127.2,
126.9, 125.9, 125.4, 123.9, 119.7, 111.9, 111.5, 99.1, 71.7, 70.9,
70.4, 68.8, 64.6, 62.1, 21.02, 21.00, 20.98. HRMS calcd for
C37H34O10Na (M + Na): 661.2050, found 661.2022.
1
trated to give 17 (213 mg, 58%) as a light yellow solid. H NMR
(CD3OD): δ 7.65 (s, 1 H, H-4), 7.46 (d, 1 H, J 8.8 Hz, H-8), 7.02
(d, 1 H, J 2.1 Hz, H-5), 7.00 (s, 1 H, H-1), 6.95, (dd, 1 H, J 8.8,
2.5 Hz, H-7), 4.73 (s, 2 H, CH2). 13C NMR (CD3OD): δ 156.6,
155.0, 134.3, 133.5, 132.8, 130.7, 128.5, 121.6, 112.6, 112.3, 64.0.
HRMS calcd for C11H10O3Na (M + Na): 213.0528, found
213.0537.
High-pressure liquid chromatography was run on a Supelco
LC-18-DB column (15 cm × 4.6 mm, 5 µm). The mobile phase
consisted of H2O + 0.1% TFA with a gradient of MeCN from 1
min increasing by 1.2% per minute. The retention times are the
mean values for three separate runs for each compound.
Acidity constants (pKa) were determined spectrophotometrically.
Compound 2 (1.4 mg) was dissolved in MeOH (1.5 mL); 0.040
mL of this solution was added to 1 mL each of HCl (aq, 0.1 M),
NaOH (aq, 0.1 M), and pH 9, 10, and 11 buffers; and the absorption
was measured at 365 nm. The pKa was calculated from at least
three measurements at each pH.
2-Antracen-9-yl-4H-naphtho[2,3-d][1,3]dioxin-7-ol (18). To a
solution of 17 (466 mg, 2.45 mmol) and anthraldehyde dimethyl
acetal (740 mg, 2.95 mmol) in MeCN (20 mL) was added pTSA
(5 mg). The mixture was stirred at room temperature for 1 h, then
neutralized by addition of Et3N, and concentrated with toluene 3
times. The residue was chromatographed (SiO2, heptane/EtOAc
1
2:1) to give 18 (704 mg, 76%) as a light yellow solid. H NMR
(DMSO-d6): δ 9.55 (s, 1 H, OH), 8.70-8.80 (m, 3 H, H-1′, H-8′,
H-10′), 8.10-8.15 (m, 2 H, H-4′, H-5′), 7.66 (s, 1 H, H-2), 7.62
(d, 1 H, J 8.5 Hz, H-9), 7.58 (s, 1 H, H-5), 7.50-7.55 (m, 4 H,
Absorbance and Fluorescence. Compound 2 (1.18 mg) was
dissolved on DMSO (0.100 mL), and 0.010 mL of this solution
was added to 2.00 mL MeCN.29
(28) Biswas, S.; Ghosh, A.; Venkateswaran, R. V. Synth. Commun. 1991,
21, 1865.
(29) (a) Corwell, E. P.; Powell, A.; Varsel, C. J. Anal. Chem. 1963, 35,
184. (b) Corwell, E. P.; Varsel, C. J. Anal. Chem. 1963, 35, 189.
3450 J. Org. Chem., Vol. 71, No. 9, 2006

RegioselectiVe ReductiVe Openings of Acetals
Cell Culture, Radiolabeling and Extraction Procedures. Cells
were cultured as monolayers in Dulbeccos Modified Earles Medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM
L-glutamine, penicillin (100 U/mL), and streptomycin (100 µg/mL)
in an incubator with humidified atmosphere and 5% CO2 at
37 °C. Confluent cells were preincubated for 1 h in low-sulfate,
MgCl2-labeling medium supplemented with 2 mM glutamine. The
preincubation medium was replaced by fresh medium containing
50 mCi/mL of [35S]sulfate and the xyloside. Dilutions were made
from 20 mM stock solutions in DMSO/water (1:1, v/v). After the
incubation 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).
In Vitro Growth Assay using Crystal Violet Method. The
procedure has been described elsewhere.30 The inhibitory effect of
the compounds is expressed as the percentage of growth in the
absence of drugs.
Fluorescence Microscopy. Cells were seeded at a concentration
of 10,000 cells/well in chamber slides with covers, allowed to
adhere overnight, and then treated with 100 µM of compound 2
for 16 h. After the treatment cells were washed with phosphate-
buffered saline three times and fixed with acetone for 2-4 min.
The fixed cells were then visualized in a microscope. Excitation
was obtained at 250 nm, and the emitted light was filtered at 461
nm. Images were digitized for annotation, and printing.
Isolation of Xyloside-Primed Radiolabeled GAG. The proce-
dures have been described in detail previously.30 Free xyloside-
primed GAG chains were separated from proteoglycans by hydro-
phobic interaction chromatography on octyl-Sepharose followed by
gel permeation FPLC on Superose 6. Radioactivity was determined
in a beta counter.
Degradation Procedures. GAG chains were released from
proteoglycans by treatment with 0.5 M NaOH, 0.1 M NaBH4 at
room temperature overnight. Samples were neutralized with HOAc.
Heparan sulfate chains were degraded by deaminative cleavage by
using the pH 1.5-HNO2 method.31 The samples were lyphilized and
redissolved for analysis by gel-permeation chromatography on
Superose 6 to determine the proportions of total heparan sulfate.
Acknowledgment. The Swedish Research Council, Associa-
tion for International Cancer Research, the Cancer Fund, the
Swedish Cancer Society, the American Cancer Society, the
Crafoord Foundation, the Thelma Zoegas Foundation, and the
Bergvall Foundation supported this work. We thank Professor
Hakon Leffler at the Department of Laboratory Medicine, Lund
Univesity for access to microscope facilities.
Supporting Information Available: Additional experimental
details; NMR spectra of compounds 2, 4, 5, 8, 9, 12, 15, and
17-20; and computational data. This material is available free of
charge via the Internet at http://pubs.acs.org.
JO0526284
(30) Mani, K.; Havsmark, B.; Persson, S.; Kaneda, Y.; Yamamoto, H.;
Sakurai, K.; Ashikari, S.; Habuchi, H.; Suzuki, S.; Kimata, K.; Malmstro¨m,
A.; Westergren-Thorsson, G.; Fransson, L.-A° . Cancer Res. 1998, 58, 1099.
(31) Shively, J. E.; Conrad, H. E. Biochemistry 1976, 15, 3932.
J. Org. Chem, Vol. 71, No. 9, 2006 3451