Effects of oxygen-sulfur substitution on glycosaminoglycan-priming naphthoxylosides

Bioorganic & Medicinal Chemistry 15 (2007) 5283–5299

Eﬀects of oxygen–sulfur substitution on glycosaminoglycan-

priming naphthoxylosides

a

b

a,

*

˚

Marten Jacobsson, Katrin Mani and Ulf Ellervik

^aOrganic Chemistry, Lund University, PO Box 124, SE-221 00 Lund, Sweden

^bDepartment of Experimental Medical Science, Division of Neuroscience, Glycobiology Group, Lund University,

Biomedical Center A13, SE-221 84 Lund, Sweden

Received 9 March 2007; revised 20 April 2007; accepted 2 May 2007

Available online 6 May 2007

Abstract—Three series of sulfur-containing analogs to the selectively antiproliferative 2-(6-hydroxynaphthyl) b-D-xylopyranoside

were synthesized and their biological properties investigated. A short, general route to hydroxynaphthyl disulﬁdes from dihydroxy-

naphthalenes was developed to utilize the disulﬁde bond as a sulfur-selective protecting group to enable the orthogonal protection of

hydroxyls and thiols. The results indicate that hydrophobic, uncharged oxygen–sulfur substituted naphthoxylosides are taken up by

cells and initiate priming of GAG chains to a greater extent compared to the oxygen analogs. No correlation between priming ability

and antiproliferative activity was observed.

1. Introduction

treatment with this xyloside reduced the average tumor

load by 70–97% in a SCID mice model.⁴Studies into

the toxicity of the 14 isomeric xylosylated dihydroxy-

naphthalenes revealed diﬀerences in antiproliferative

activity depending on the aglycon structure.^5,6

Glycosaminoglycan (GAG) chains anchored to core

proteins form proteoglycans (PG), a class of extracellu-

lar macromolecules having functions ranging from bulk

construction material to cell–cell interactions. The bio-

synthesis of GAG chains starts with the formation of

a glycosidic bond between serine residues of the core

protein and the unique xylose residue of the resulting

To extend our investigation of the structure-activity

relationships (SAR) for naphthoxylosides we are synthe-

sizing sulfur-containing naphthoxylosides. Sulfur is a

classic isostere for oxygen and the substitution will ex-

pand the range of physical as well as chemical properties

for the naphthoxylosides. Both the O-linked naphthoxy-

losides and the S-linked naphthoxylosides are of

interest.

GAG chain.¹

A

speciﬁc linker tetrasaccharide,

GlcA(b1–3)Gal(b1–3)Gal(b1–4)Xylb, is then assembled

on which GAG chains are elongated and modiﬁed

(Fig. 1). The polysaccharide is further modiﬁed

through N-deacetylation/N-sulfation, O-sulfation, and

epimerization.

Aryl thiols are more acidic (pK_a6–8) compared to the

corresponding phenols (pK_a8–11), which could be ex-

pected to inﬂuence cellular uptake of the xyloside, redox

properties, and electron density distribution of the agly-

con. Also, the possibility of disulﬁde bond formation al-

lows the use of aryl thiol O-xylosides to create libraries

of diverse naphthoxylosides using dynamic combinato-

rial chemistry (DCC). DCC has recently been intro-

duced as a method to create dynamic libraries, that is,

collections of compounds whose constitution is adjusted

depending on the conditions.⁷Examples of DCC are the

generation and screening of a library of possible conca-

navalin A vaccines⁸and the discovery of a psammaplin

A type antibacterial agent with good activity against

Xylosides carrying hydrophobic aglycon can enter cells

and serve as primers of GAG synthesis.²Depending

on the structure of the aglycon, diﬀerent GAG chains

are formed. We have previously shown that the GAG-

priming 2-(6-hydroxynaphthyl) b-^D-xylopyranoside (1,

Fig. 1) selectively inhibits proliferation of transformed

or tumor-derived cells in vitro as well as in vivo.³Also,

Keywords: Glycosaminoglycan; Xylose; Disulﬁdes; Thio-b-D-xylopyr-

anoside; Thioether.

*

Corresponding author. Tel.: +46 46 222 8220; fax: +46 46 222

8209; e-mail: ulf.ellervik@organic.lu.se

doi:10.1016/j.bmc.2007.05.008

5284

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

Core Protein

HN

a)

OH

COOH

O

HO

O

HO

O

NH

O

AcHN

OH

n

O

HN

b)

HO

O

OH

1

OH

Figure 1. (a) Biosynthesis of glycosaminoglycan (GAG) chains start with xylosylation of a serine residue after which a linker tetrasaccharide

(GlcA(b1–3)Gal(b1–3)Gal(b1–4)Xylb) is assembled. (b) Xylosides carrying hydrophobic aglycon, such as compound 1, can enter cells and serve as

primers of GAG chains.

methicillin-resistant Staphylococcus aureus.⁹In both

cases the reversibility of the thiol-disulﬁde linkage was

used.

2. Synthesis

We envisaged obtaining the disulﬁde-protected thiols

from cleavage of an aryl–alkyl thioether bond by

dissolving metal reduction in liquid ammonia.^19–22

Aryl–alkyl thioethers are usually synthesized by

coupling of aromatic halides with alkyl thiols,²³or

nucleophilic substitution of alkyl halides with aryl thi-

ols, when the thiol is available.²⁴While the yields are

normally high, this method requires the availability of

a suitable aryl thiol or aryl halide.

Xylosides with S-glycosidic bonds are of great biological

interest since the length of the carbon–sulfur bond can

result in increased stability toward glycosidases.¹⁰Also,

it has been shown that S-xylosides can act as primers of

GAG synthesis with comparable or even higher eﬃ-

ciency than the corresponding O-xyloside and also show

activity in vivo.^11–132-Naphthyl 1-thio-b-D-xylopyrano-

side shows a higher proportion of HS priming compared

to 2-naphthyl b-^D-xylopyranoside but both these com-

pounds have poor antiproliferative properties. To infer

toxicity on these compounds, a hydroxyl group can be

added to the aglycon, thus mimicking the known

antiproliferative mono-xylosylated dihydroxynaphtha-

lenes.^3–6

Displacement of phenolic hydroxyls under the inﬂuence

of strong acids is another method that has been shown

to give excellent yields for simple compounds.^25,26How-

ever, bis-phenolic compounds were transformed into the

analogous bis-thioethers.²⁷We therefore ﬁrst set out to

ascertain whether it was possible to obtain mono-hydroxy

mono-thioethers using the same method (Scheme 1).

The six symmetric naphthalene diols 2a–f were chosen

for this study (Fig. 3) with the letters a–f indicating

the substitution pattern of the naphthalene skeleton.

After minor optimization, the mono-thiopropyl-naph-

thols 3a–f (Scheme 1) were obtained in yields from

33% to 86%.

Synthesis of these sulfur-containing xylosides requires

naphthalene moieties carrying both a hydroxyl group

and a thiol and the possibility of selective protection

of hydroxyl and thiol groups. Here we present a solution

phase synthesis of thiol-containing xylosides using a

disulﬁde linker as the thiol-protecting group. The disul-

ﬁde is a protecting group which is unique for thiols, and

it can easily be reduced/deprotected to the thiol by a

variety of reagents, both in solution and immobilized

on solid-support (e.g., aryl- and alkyl phosphines,^14,15

polystyrene-bound triphenyl phosphine (Ph₃P-PS),¹⁶

nucleophilic hydrides^17,18) as exempliﬁed in Figure 2.

HO

2a-f

OH

i

3a: 75%

HO

3b: 33%

3c: 76%

3d: 86%

S

3e: 62%

3f: 61%

ii, iii

S

HO

4a: 77%

4b: 95%

S

O₂, I₂, FeCl₃, Phosphines (e.g. n-Bu₃P,

4c: 96%

4d: 95%

4e: 83%

4f: 86%

S

Me₃SiCl,

Ph₃P, PS-Ph₃P),

DTT, NaBH₄, LiAlH₄

NaClO₂(aq.)

HO

SH

Scheme 1. Reagents and conditions: Synthesis of hydroxynaphthyl

disulﬁdes from dihydroxynaphthalenes. (i) p-TsOH, n-PrSH, toluene,

100 ꢁC; (ii) Na, NH₃, Et₂O; (iii) I₂, MeOH, H₂O. The letters a–f

indicate the substitution pattern of the aromatic residue (cf. Fig. 3).

2

Figure 2. Reversibility of the disulﬁde bond and a few examples of

reagents for the conversions.

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5285

OH

2a

2b

OH OH

HO

2c

2d

HO

OH

2e

2f

Figure 3. The letters a–f are used in the following text indicating the

substitution pattern on the naphthalene skeleton.

Cleavage of the thioether to give the thiols and, upon

oxidation, disulﬁdes 4a–f (Scheme 1), was easily per-

formed by dissolving metal reduction followed by io-

dine-mediated oxidation. Recrystallization gave the

pure products in 40–80% yield without the need for

chromatography. However, column chromatography

of the residues raised the yields to 77–96%. NMR data

for 3a–f and 4a–f are given in Table 1.

Xylosylation of the hydroxynaphthyl disulﬁdes was ﬁrst

investigated using the peracetylated xylose donor fol-

lowing the method presented by Lee et al.³⁴in which

anomerization is prevented by the addition of triethyl-

amine. Whilst this method has previously been success-

ful for the xylosylation of several naphthol derivatives,³⁵

the yields were disappointingly low in this case. As we

have noted earlier,³⁶trichloroacetimidate donors can

give good results for aromatic O-glycosylation reactions.

Using a two-fold excess of trichloroacetimidate xylopyr-

anoside donor per phenolic hydroxyl gave excellent re-

sults and at 0 ꢁC no Fries-rearrangement was detected

(Scheme 2, compounds 5a–f).

To obtain the xylosides with free aryl thiol groups,

deacetylation followed by disulﬁde cleavage was ﬁrst at-

tempted. Unfortunately, this order of deprotection

encountered diﬃculties due to the very low solubilities

of the deprotected bis-xylosylated naphthalene disulﬁdes

across a range of solvents. Therefore, an alternative

route was developed in which the disulﬁde was cleaved

using Ph₃P-PS in THF with catalytic acid. After stirring

at 80 ꢁC overnight, NaOMe–MeOH was added to re-

move the acetate groups. After 10 min the reaction

was neutralized by the addition of acidic ion exchange

resin. The solution was ﬁltered and immediately concen-

trated to give the fully deprotected O-xylosides (6a–f).

After ﬁltration only one compound was detected by

TLC, except in the case of 6c for which the cleavage

reaction was repeated using fresh Ph₃P-PS and micro-

wave heating to give one product, as judged by TLC.

NMR of these products were troublesome due to spon-

taneous oxidation to the corresponding disulﬁdes in

methanolic solution. All attempts to purify the thiols

using chromatography or crystallization gave partial

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M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

O

AcO

HO

O

AcO

i

ii, iii, iv

S

O

HO

O

HO

SH

O

AcO

HO

O

5a: 78%

5b: 88%

5c: 81%

5d: 95%

5e: 75%

5f: 86%

6a: 99%

6b: 100%

6c: 100%

6d: 93%

6e: 98%

6f: 95%

4a-f

v, vi

AcO

PivO

O

AcO

vii, viii

SH

ix

S

O

HO

S

OAc

HO

OPiv

OH

7a

8a: 67%

8b: 71%

9a: 97%

9b: 96%

9c: 34%

9d: 44%

9e: 95%

9f: 98%

7b

7c

7d

7e

7f

8c: not isolated

8d: not isolated

8e: 76%

8f: 61%

Scheme 2. Reagents and conditions: Synthesis of O- and S-xylosides from a common hydroxynaphthyl disulﬁde precursor. (i) 2,3,4-Tri-O-acetyl-b-D-

xylopyranoside trichloroacetimidate, BF₃ÆOEt₂, CH₂Cl₂, 0 ꢁC; (ii) Ph₃P-PS, HCl (aq), THF, 80 ꢁC, sealed tube; (iii) NaOMe–MeOH, MeOH; (iv)

Amberlite IR-120H⁺; (v) Piv₂O, 110 ꢁC; (vi) Ph₃P-PS, HCl (aq), THF, 110 ꢁC, sealed tube, microwave irradiation. Or 80 ꢁC thermal heating; (vii) t-

BuONa, DMF, 0 ꢁC; (viii) 2,3,4-Tri-O-acetyl-a-^D-xylopyranosyl bromide, THF. 0 ꢁC to rt; (ix) NaOMe–MeOH, MeOH, CH₂Cl₂. The letters a–f

indicate the substitution pattern of the aromatic residue (cf. Fig. 3).

oxidation to the disulﬁde and the compounds were

therefore not puriﬁed further before biological testing.

The purity was judged to be at least 90–95% by NMR

and TLC analysis.

Instead, a basic procedure using xylosyl bromide donor

was used (Scheme 3). Deprotonation of the thiol using t-

butoxide at 0 ꢁC followed by addition of the donor gave

the thio-xylosides in satisfactory yields.³⁷For 8c and 8d

puriﬁcation was troublesome and lowered the yields

substantially. Therefore these were deprotected prior

to full puriﬁcation and characterization. Deprotection

To synthesize the S-xylosides 9a–f, we envisaged a route

with protection of the phenol followed by disulﬁde bond

cleavage. Early attempts using acetate as the phenolic

protecting group gave rise to problems in the cleavage

step. Phenolic acetates are labile under acidic conditions

and the use of Ph₃P-PS requires a catalytic amount of

acid to lower the reaction times¹⁶causing deprotection

of the phenol. Therefore an alternative protecting group

was needed, preferably one which could be removed in

the same step as the deprotection of the acetates.

´

under Zemplen conditions gave the S-xylosides 9a–f

(Scheme 2). The diﬃculties in puriﬁcation of 8c and 8d

probably arose from non-quantitative conversion in

the pivaloylation step giving traces of hydroxynaphthyl

1-thio-2,3,4-tri-O-acetyl-b-^D-xylopyranoside, which was

converted into ﬁnal product upon deprotection.

Naphthyl b-^D-xylopyranosides carrying aryl O-alkyl

ethers have previously been reported to prime GAG

Aryl pivaloates are more stable toward acid compared

to the acetates, yet readily cleavable with NaOMe–

MeOH. Pivaloylation of disulﬁde 4e in hot pivaloyl

anhydride over night gave the bis-O-pivaloylated deri-

vate as the only product. The pivaloyl groups were sta-

ble upon cleavage of the disulﬁde bond using Ph₃P-PS

both at 80 ꢁC in a sealed tube overnight or at 110 ꢁC un-

der microwave irradiation for 15 min (Scheme 2) and

this gave full conversion of the pivaloylated hydroxy-

naphthyl disulﬁdes into single products with slightly

lower R_f-values.

3a-f

i

10a: 96%

O

AcO

O

10b: 96%

10c: not isolated

10d: 83%

10e: 100%

10f: 86%

OAc

S

ii

11a: 98%

11b: 95%

11c: 90%*

11d: 95%

11e: 98%

11f: 98%

O

HO

Xylosylation of the crude thiols was ﬁrst attempted

using trichloroacetimidate donor but this gave complex

mixtures as judged by TLC. Next, peracetylated xylose

was tried. Whilst the correct product was obtained, the

method gave unreliable and low yields (20–40%), as well

as low purity even after multiple chromatographic steps

with diﬀerent mobile phases and recrystallization. The

impurities probably arise from Fries-rearrangement

and could not be avoided even by using lower amounts

of promoter.

OH

Scheme 3. Reagents and conditions: Xylosylation of thiopropyl

substituted naphthols. (i) 1,2,3,4-Tetra-O-acetyl-b-D-xylopyranoside,

BF₃ÆOEt₂, Et₃N, CH₂Cl₂, rt; (ii) NaOMe–MeOH, MeOH, CH₂Cl₂.

For 10c: (i) 2,3,4-Tri-O-acetyl-b-D-xylopyranoside trichloroacetimi-

date, BF₃ÆOEt₂, CH₂Cl₂, 0 ꢁC; (ii) NaOMe–MeOH, MeOH, CH₂Cl₂.

The letters a–f indicate the substitution pattern of the aromatic residue

(cf. Fig. 3). ^*Yield over two steps.

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5287

synthesis. The ethyl ether analog¹²gave a slight increase,

while the methyl⁴and butyl derivatives¹²decreased the

proportion of HS as compared to priming by 2-naphthyl

b-^D-xylopyranoside. The synthesis of the hydroxynaph-

thyl disulﬁdes gave six thiopropyl-substituted naphthols

(3a–f), which in two steps were transformed into naph-

thoxylosides 11a–f (Scheme 3). Xylosylation of 3a–f

using peracetylated xylose in the presence of Et₃N to

minimize anomerization was successful and gave high

yields and purity. Only for 10c, the yield was a disap-

pointing 28% but the use of trichloroacetimidate donor

and one-pot deacetylation gave the desired xyloside 11c

in 90% over two steps. The remaining ﬁve xylosides were

easily deacetylated in nearly quantitative yields.

3. Results and discussion

The chemistry and biology of the three diﬀerent groups

of compounds are discussed in separate sections below

Table 2.

3.1. Hydroxynaphthyl 1-thio-b-^D-xylopyranosides

Gradient HPLC retention times, in contrary to isocratic

retention times, can be treated as linear free-energy re-

lated parameters,³⁹, that is, gradient HPLC retention

times can be used to substitute log P values in biological

evaluations. Correlations between the lipophilicity of

naphthoxylosides and their GAG-priming abilities and

toxicity have been shown previously.^6,12The gradient

HPLC retention times for the hydroxynaphthyl 1-thio-

b-^D-xylopyranosides were measured using a C-18 col-

umn with a gradient of acetonitrile in water (0.1% triﬂu-

oroacetic acid). The retention times were measured for

three separate runs per compound and the calculated

mean retention times are presented in Table 3.

Compared to the retention times for the analogous

mono-xylosylated dihydroxynaphthalenes^6,35these com-

pounds are generally less polar due to the diminished

hydrogen bonding capability of sulfur, but the trend

for retention times versus substitution pattern is similar.

For example, the compounds with vicinal substituents

(i.e., 9c and 9d) are generally less polar compared to

the other analogs since a larger hydrophobic surface is

exposed.

Earlier results have shown that some mono-xylosylated

dihydroxynaphthalenes are unstable in the culture

media used.⁶For example, 1-(4-hydroxynaphthyl) b-D-

xylopyranoside was further hydroxylated to give

4-(1,2-dihydroxynaphthyl) b-^D-xylopyranoside in less

than 48 h. In contrast, bis-xylosylated analogs were sta-

ble in the medium for more than 96 h.³⁵To evaluate the

stability of the hydroxynaphthyl 1-thio-b-^D-xylopyrano-

sides, compounds 9a–f were dissolved in DMSO–water

(1:1, 20 mM), diluted with cell growth medium (Ham’s

F-12 medium) to 0.2 mM, and incubated at 37 ꢁC. Sam-

ples were taken at 0, 1, 3, 24, 48, 72, and 96 h, and the

amounts of xyloside, compared to 4,4⁰-dihydroxybi-

phenyl used as internal standard, were measured by

HPLC. The results are shown in Figure 4.

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M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

Table 3. Gradient HPLC retention times (min), antiproliferative activity (ED₅₀, lM) of hydroxynaphthyl 1-thio-b-D-xylopyranosides toward HFL-1

cells and T24 cells, and GAG priming capability

Compound

HPLC retention time (min)

ED₅₀HFL-1 (lM)

ED₅₀T24 (lM)

GAG priming

Peak maximum (fraction)

9a

9b

9c

9d

9e

9f

22.25 0.07

20.44 0.08

25.82 0.04

25.52 0.06

20.42 0.06

21.81 0.10

130

135

120

100

145

125

160

320

225

260

355

230

40.7

29.8

32.6

43.5

44.3

37.4

44

41

42

44

The GAG-priming capacity is given as the integrated value of ³⁵S detected per minute for fractions containing soluble GAG chains divided by the

integrated value for fractions of untreated cells.

It has been shown that the uptake of xylosides in animal

cells is fast with a plateau being reached in a matter of

minutes.⁴⁰Therefore, decomposition could be of little

consequence as long as the decomposition products

are not toxic to cells.

naphthyl 1-thio-b-^D-xylopyranosides we could not ﬁnd

any correlations between HPLC retention times and

ED₅₀. However, it is interesting to note that these com-

pounds, contrary to the analogous hydroxynaphthyl b-

D-xylopyranosides, are more toxic toward normal

HFL-1 cells compared to transformed T24 cells.

For 9a two more lipophilic UV-active peaks appeared in

the HPLC trace as the xyloside decomposed, but since

the xyloside was not highly toxic to cells this was not ex-

pected to inﬂuence the priming studies. For 9c no major

UV-active decomposition products were seen.

To test the ability of the xylosides in priming GAG syn-

thesis, T24 cells were incubated with 100 lM xyloside

and [³⁵S]sulfate followed by isolation and size separation

of free GAG chains. All cells secreted alkali sensitive

proteoglycans (example shown in Fig. 5, Pool I). How-

ever, treatment with the xylosides also initiated synthesis

of free GAG chains (Fig. 5, Pool II). The proportion of

GAG-priming is given as the integrated value of ³⁵S de-

tected per minute for fractions in Pool II (Table 3) di-

vided by the integrated value for fractions of untreated

cells.

For the determination of the antiproliferative activity,

normal HFL-1 cells (human fetal lung ﬁbroblasts) and

T24 cells (human bladder carcinoma cells) were used.

The xylosides were added to the growth medium at var-

ious concentrations and cell proliferation was recorded

using the crystal violet method.³The inhibitory eﬀect

of the compounds is expressed as ED₅₀(lM) scored

after 96 h of exposure relative to untreated cells

(Table 3).

All the hydroxynaphthyl 1-thio-b-^D-xylopyranosides

gave very strong priming of GAG chains as indicated

in Table 3. The GAG priming capability could not be

correlated to the antiproliferative eﬀect. The separation

procedure of the GAG chains also indicates the relative

size of diﬀerent GAG chains, that is, longer chains are

eluted faster from the column. The GAG chains synthe-

sized on the hydroxynaphthyl 1 thio-b-^D-xylopyrano-

sides are relatively short.

We have earlier shown correlations between the ED₅₀

values for HFL-1 cells and HPLC retention times, that

is, the toxicity increased with the lipophilicity, indicating

eﬀects on cellular uptake. For T24 cells, most com-

pounds followed a similar pattern but some analogs

showed a much stronger activity.^6,35For the hydroxy-

Amount (%)

100

Radioactivity

(dpm)

Pool II

4000

80

60

40

20

0

3000

2000

1000

Pool I

0

20

40

60

80

100

0

Time (h)

10

20

30

40

50

60

Fraction

Figure 4. Stability of hydroxynaphthyl 1-thio-b-D-xylopyranosides 6a–

f in Ham’s F-12 medium at 37 ꢁC. The graph shows that whilst 9b,d–f

are stable for the ﬁrst 48 h and then decompose slightly, 9a (dashed

line) and 9c (dotted line) decompose quickly and totally.

Figure 5. Example of priming of GAG chains in T24 cells incubated

with 9e. Pool II contains xyloside-primed GAG chains. The dashed

line shows the results for untreated cells.

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5289

3.2. Thionaphthyl b-D-xylopyranosides

evaluated and the data are presented in Table 4. Gener-

ally, these compounds show approximately 2–3 times

stronger antiproliferative properties compared to the

corresponding hydroxynaphthyl 1-thio-b-^D-xylopyrano-

side whilst the GAG priming capability is lowered by 4–

6 times. The GAG chains are longer compared to the

hydroxynaphthyl 1-thio-b-^D-xylopyranosides.

The retention times for the thionaphthyl b-^D-xylopyr-

anosides were measured as described in Section 3.1

and the calculated mean retention times are presented

in Table 4. As expected the retention times for the

thionaphthyl b-^D-xylopyranosides show that these com-

pounds are less polar compared to the hydroxynaphthyl

1-thio-b-^D-xylopyranosides. Aromatic thiols are less

hydrophilic and also less prone to be protonated com-

pared to phenols. Also, the diﬀerence in lipophilicity be-

tween the most and least polar is less than that for the

hydroxynaphthyl b-^D-xylosides.

3.3. Propylthio-naphthyl b-^D-xylopyranosides

Alkylation of the aromatic thiol gives several eﬀects,

such as elimination of the possibility of disulﬁde forma-

tion and introduction of a highly hydrophobic alkylthio

group. The retention times for the propylthio naphthyl

b-^D-xylopyranosides were, as expected, higher than for

the other compounds with retention times between 34

and 38 min. The diﬀerence in retention times was smal-

ler within the series than for the previously discussed

compounds. The antiproliferative activity toward

HFL-1 cells and T24 cells, as well as their GAG priming

capability, was evaluated and the data are presented in

Table 5. All six propylthio-naphthyl b-^D-xylopyrano-

sides were stable at 37 ꢁC for 96 h in the growth medium

used in the priming and proliferation studies.

These compounds are sensitive toward air oxidation,

yielding the analogous disulﬁdes and in all cases complete

conversion to the disulﬁde was seen in less than 48 h with

only 6c and 6d having half-lives of more than about 1 h in

the weakly basic growth medium. The increased stability

of these two isomers is most probably due to steric inter-

actions upon disulﬁde formation. The disulﬁdes had sig-

niﬁcantly longer retention times as compared to their

corresponding thiols, with all disulﬁdes eluting between

36 and 40 min. We could therefore not perform quantita-

tive stability tests for these compounds although no major

decomposition was seen over 96 h. Biological evaluation

of the pure disulﬁdes was not possible due to their low sol-

ubility, hampering both their synthesis and their adminis-

tration in solution. It is reasonable tosuggest that both the

free thiol and the disulﬁde are present in the cells in vari-

ous proportions depending on the cellular redox condi-

tions. During the proliferation studies no precipitates

were seen in the medium, indicating rapid uptake of the

thiols into the cells.

The lipophilicities of the propylthio-naphthyl b-^D-xylo-

pyranosides are, as expected, higher than for either

6a–f or 9a–f. Also, as seen for 6a–f, substitution of the

highly hydrophilic phenolic hydroxyl group for a more

lipophilic group decreases the diﬀerence in retention

time between the isomers. Contrary to the results from

both 6a–f or 9a–f and from the previously presented

hydroxynaphthyl b-^D-xylopyranosides,⁶the isomers in

which the substituents on the naphthalene moiety are

placed in the 1,8- and 2,3-positions are less lipophilic.

This is probably due to ‘‘masking’’ of the highly lipo-

philic thiopropyl group by the xylose residue.

The antiproliferative activity toward HFL-1 cells and

T24 cells, as well as their GAG priming capability, were

Table 4. Gradient HPLC retention times (min), antiproliferative activity (ED₅₀, lM) of thionaphthyl b-D-xylopyranosides toward HFL-1 cells and

T24 cells, and GAG priming capability

Compound

HPLC retention time (min)

ED₅₀HFL-1 (lM)

ED₅₀T24 (lM)

GAG priming

Peak maximum (fraction)

6a

6b

6c

6d

6e

6f

29.64 0.19

28.93 0.03

29.76 0.01

29.76 0.05

28.56 0.01

28.56 0.08

70

60

55

110

45

9.6

5.8

38

33

37

38

37

38

60

5.2

6.2

145

135

360

60

100

7.6

10.2

The GAG-priming capacity is given as the integrated value of ³⁵S detected per minute for fractions containing soluble GAG chains divided by the

integrated value for fractions of untreated cells.

Table 5. Gradient HPLC retention times (min), antiproliferative activity (ED₅₀, lM) of propylthio-naphthyl b-D-xylopyranosides toward HFL-1

cells and T24 cells, and GAG priming capability

Compound

HPLC retention time (min)

ED₅₀HFL-1 (lM)

ED₅₀T24 (lM)

GAG priming

Peak maximum (fraction)

11a

11b

11c

11d

11e

11f

37.90 0.01

37.92 0.36

34.77 0.11

35.33 0.05

37.13 0.05

37.00 0.09

30

20

30

40

12.4

11.1

2.5

37

34

35

37

100

30

155

70

5.5

30

25

40

60

10.5

8.1

The GAG-priming capacity is given as the integrated value of ³⁵S detected per minute for fractions containing soluble GAG chains divided by the

integrated value for fractions of untreated cells.

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M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

Generally, these compounds showed strong antiprolifer-

ative properties with only minor discrimination for dif-

ferent cell lines. With a few exceptions the GAG

priming capability is higher compared to the thionaph-

thyl b-^D-xylopyranosides. The GAG chains are similar

in length to those primed by the thionaphthyl b-^D-

xylopyranosides.

xyloside, the biotransformation of aglycone such as

hydroxylation of the naphthalene ring and the nuclear

targeting of xyloside derived products are some of the

important factors involved in the antiproliferative

activity.

We have also demonstrated a general route for the syn-

thesis of hydroxynaphthyl disulﬁdes via mono-thioalkyl

ethers from dihydroxynaphthalenes. This route is of

general interest for the synthesis of sulfur substituted

aromatic compounds and we are currently investigating

the mechanistic aspects of the thioether formation. Also,

we have highlighted the strengths and weaknesses of dif-

ferent pyranoside donors,³⁶and demonstrated the eﬃ-

ciency of solid support bound reagents, especially for

compounds where puriﬁcation needs to be minimized.

3.4. Heparan sulfate priming

It has previously been shown that HS priming is an

important factor for the selective antiproliferative activ-

ity of xylosides.^4,6HS-deﬁcient CHO cells have been

shown to be insensitive to the antiproliferative 2-(6-

hydroxynaphthyl) b-^D-xylopyranoside (1) whilst wild

type CHO cells were highly sensitive. The tumor selec-

tive antiproliferative 1 initiated priming of HS chains

(12% of total GAG chains) in tumor derived T24 cells.

The HS chains were subsequently degraded by nitric

oxide into HS oligosaccharides and accumulated in the

nucleus. This xyloside preferentially inhibited growth

of T24 cells at low concentrations with an ED₅₀value

of 50 lM. In order to investigate the correlation between

aglycon structure, HS priming, and selective antiprolif-

erative activity we investigated the HS priming ability

of 2-(6-thionaphthyl) b-^D-xylopyranoside (6e), where

the O-glycosidic linkage in 1 has been replaced with an

S-glycosidic linkage, and 2-(6 hydroxynaphthyl) 1-

thio-b-^D-xylopyranosides (9e) in which the phenolic hy-

droxyl group has been changed to an aryl thiol group, in

tumor derived T24 cells. The amount of HS chains

primed in the cells was determined by digestion of

GAG pool with HNO₂at pH 1.5 which cleaves HS

chains. Both 6e and 9e initiated priming of HS chains

to some extent (14% and 8% of total GAGs, respec-

tively). None of these xylosides were selectively antipro-

liferative toward tumor cells. There was no correlation

between HS priming and selective antiproliferative

activity. This could however be due to the production

of diﬀerent HS or diﬀerent HS degradation in these cells.

We have also conﬁrmed our previous ﬁndings that xylo-

sides are not always stable in the cell growth medium

used for studies in vivo. Also, previous ﬁndings that

xylosides with S-glycosidic bonds can be eﬀective prim-

ers of GAG synthesis were conﬁrmed.^11,12

5. Experimental

5.1. Chemistry

CH₂Cl₂, Et₂O, and THF for reactions were dried by

passing through a column of Al₂O₃(neutral, activity

grade I). Anhydrous DMF was of commercial grade

and used without further puriﬁcation. NMR-spectra

were recorded with a Bruker DRX 400 spectrometer at

400 MHz (¹H) and 100 MHz (¹³C) and assigned using

2D-methods (COSY, HMQC, HMBC). 2D-experiments

were recorded with gradient enhancements using sine

shaped gradient pulses. The chemical shifts are reported

in ppm with the residual solvent peaks (¹H) and solvent

signals (¹³C) as reference (dH CDCl₃= 7.26 ppm, dC

CHCl₃= 77.0, dH CD₃OD = 3.31 ppm, dC CD₃OD =

49.0). High-resolution mass-spectra were recorded on a

Micromass Q-Tof (ESI) or JEOL SX-102 (FAB).

Reactions using microwave irradiation were performed

on a Smith Creator microwave reactor from Personal

Chemistry i Uppsala AB. Reactions were monitored

by TLC using alumina plates coated with silica gel and

visualized using either UV-light or by charring with an

ethanolic anisaldehyde solution. Preparative chroma-

tography was performed with silica gel (35–70 lm,

4. Conclusions

Taken together, the results from this study indicate that

hydrophobic, uncharged oxygen–sulfur substituted

naphthoxylosides are taken up by cells and initiate prim-

ing of GAG chains. No correlation between priming

ability and antiproliferative activity was observed. No

selective antiproliferative activity in beneﬁt to tumor de-

rived cells was detected. The antiproliferative eﬀect of

naphthoxylosides and their priming ability were depen-

dent on the aglycone structure and the structure of gly-

cosidic linkage between the aglycone and the xyloside.

Alterations in the substitution pattern of the aglycone

aﬀected the priming ability and the antiproliferative

activity. No correlation between HS priming and tumor

selective antiproliferative activity was observed indicat-

ing that HS priming may be necessary for growth inhi-

bition but it is not a suﬃcient requirement. Previous

attempts to determine the mechanism for selective

growth inhibition suggest that the priming of HS synthe-

sis, the ﬁne structure of HS chains synthesized by the

˚

60 A).

5.1.1. 4-(Propylthio)naphthalen-1-ol (3a). 1,4-Dihydroxy-

naphthalene (2a) (2.08 g, 13.0 mmol) and p-TsOH

(1.1 g, 6.3 mmol) were suspended in toluene (76 mL)

and propanethiol (1.4 mL, 15.8 mmol) was added. The

suspension was stirred at 75 ꢁC for 105 min and then al-

lowed to reach room temperature. The suspension was

diluted with Et₂O (40 mL) and washed three times with

NaHCO₃(satd aq). The aqueous phases were extracted

with Et₂O and the combined organic phases were dried

(Na₂SO₄), ﬁltered and concentrated. Column chroma-

tography (SiO₂, toluene–methanol 30:1) gave 3a as a

gray amorphous solid (2.13 g, 75%). Recrystallization

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5291

(heptane) gave an analytical sample as white crystals;

mp 83 ꢁC (lit. 81–82 ꢁC).²⁸¹³C NMR (MeOD): d

155.5, 136.8, 134.8, 127.8, 127.3, 126.8, 125.9, 124.1,

123.1, 108.9, 39.3, 24.0, 13.7. HRMS calcd for

C₁₃H₁₅OS (M + 1): 219.0844, found: 219.0864.

129.6, 129.2, 127.9, 119.9, 110.1, 37.2, 23.7, 13.74.

HRMS calcd for C₁₃H₁₅OS (M + 1): 219.0844, found:

219.0859. The bis-substituted compound was also iso-

lated (1.24 g, 38%).

5.1.6. 7-(Propylthio)naphthalen-2-ol (3f). 2,7-Dihydroxy-

naphthalene (2f) (1.49 g, 9.3 mmol) and p-TsOH

(801.2 mg, 4.6 mmol) were suspended in toluene

(53 mL) and propanethiol (1.18 mL, 13.0 mmol) was

added. The suspension was stirred at 100 ꢁC overnight

and then worked-up as for 3a. Column chromatography

(SiO₂, toluene–acetone 50:1 ! 10:1) gave 3f as a white

amorphous solid (1.23 g, 61%). An analytical sample

was recrystallized (heptane) to give white crystals; mp

87 ꢁC. ¹³C NMR (MeOD): d 157.2, 137.0, 136.3,

130.4, 129.2, 128.2, 125.9, 125.3, 119.0, 109.3, 36.2,

23.7, 13.8. HRMS calcd for C₁₃H₁₅OS (M + 1):

219.0844, found: 219.0863. The bis-substituted com-

pound was isolated in 24% yield (607 mg, 2.2 mmol)

and some starting material was isolated (66 mg, 4%).

5.1.2. 5-(Propylthio)naphthalen-1-ol (3b). 1,5-Dihydroxy-

naphthalene (2b) (1.92 g, 12.0 mmol) and p-TsOH

(1.0 g, 6.0 mmol) were suspended in toluene (70 mL)

and propanethiol (4.3 mL, 47.8 mmol) was added. The

suspension was stirred at 100 ꢁC overnight and then

worked-up as for 3a. Column chromatography (SiO₂,

toluene–methanol 20:1 ! 5:1) gave 3b as an oﬀ-white

amorphous solid (872 mg, 33%). An analytical sample

was recrystallized (heptane) to give oﬀ-white crystals;

mp 101–102 ꢁC. ¹³C NMR (MeOD): d 155.2, 135.7,

134.7, 129.2, 127.6, 127.0, 125.3, 122.3, 117.1, 109.4,

37.1, 23.7, 13.9. HRMS calcd for C₁₃H₁₅OS (M + 1):

219.0844, found: 219.0863. The bis-substituted com-

pound was also isolated (1.37 g, 41%) and starting mate-

rial was recovered (414 mg, 22% mmol).

5.1.7. 4-Hydroxy-1-naphthyl disulﬁde (4a). Ammonia

(35 mL) was condensed into a stirred solution of 4-(pro-

pylthio)naphthalen-1-ol (3a) (424 mg, 1.94 mmol) in dry

Et₂O (11 mL). Small pieces of sodium metal were added,

allowing decolorization of the solution between addi-

tions, until the blue color persisted for more than

30 min. Excess sodium was destroyed by the careful addi-

tion of NH₄Cl. The suspension was diluted with Et₂O

(15 mL) and the ammonia was evaporated by placing

the reaction ﬂask in a room-temperature water bath.

Water (15 mL) was added and the aqueous phase was

acidiﬁed with HCl (aq concd) and extracted with Et₂O.

The combined organic phases were dried (MgSO₄), ﬁl-

tered, and concentrated. The crude concentrate was dis-

solved in MeOH (25 mL) and H₂O (15 mL) was added.

Under vigorous stirring, I₂(10% w/v in MeOH) was added

dropwise until the dark color of iodine persisted. Excess

iodine was destroyed by the addition of aqueous NaHSO₃

(10% w/v). The MeOH was removed under vacuum and

the aqueous suspension was extracted three times with

Et₂O. The combined organic phases were dried (MgSO₄),

ﬁltered, and concentrated. Recrystallization (toluene/

heptane/MeOH) gave 1c as oﬀ-white crystals (200 mg,

59%); mp 150–152 ꢁC (lit. 152 ꢁC).²⁹The mother liquor

was chromatographed (SiO₂, toluene–methanol 10:1) giv-

ing 4a (61 mg, 18%) for a total yield of 77%. ¹³C NMR

(MeOD): d 157.4, 137.2, 136.6, 127.8, 127.3, 127.1,

125.9, 124.1, 123.8, 108.4. HRMS calcd for C₂₀H₁₄O₂S₂

(M): 350.0435, found: 350.0438.

5.1.3. 8-(Propylthio)naphthalen-1-ol (3c). 1,8-Dihydroxy-

naphthalene (2c) (930 mg, 5.8 mmol) and p-TsOH

(502 mg, 2.9 mmol) were suspended in toluene (33 mL)

and propanethiol (2.11 mL, 23.3 mmol) was added.

The suspension was stirred at 100 ꢁC overnight and then

worked-up as for 3a. Column chromatography (SiO₂,

toluene–methanol 100:1 ! 10:1) gave 3c as an oil

(964 mg, 76%). ¹³C NMR (CDCl₃): d 156.1, 138.2,

133.4, 130.8, 130.2, 128.1, 126.6, 124.4, 121.6, 112.7,

40.5, 23.1, 13.8. HRMS calcd for C₁₃H₁₅OS (M + 1):

219.0844, found: 219.0855. The bis-substituted com-

pound can be obtained using more equivalents of prop-

anethiol. In this experiment the starting material, 2c,

was recovered in 24%.

5.1.4. 3-(Propylthio)naphthalen-2-ol (3d). 2,3-Dihydroxy-

naphthalene (2d) (1.96 g, 12.3 mmol) and p-TsOH

(1.1 g, 6.3 mmol) were suspended in toluene (70 mL)

and propanethiol (4.45 mL, 49.1 mmol) was added.

The suspension was stirred at 100 ꢁC overnight and then

worked-up as for 3a. Column chromatography (SiO₂,

toluene–methanol 25:1 ! 10:1) gave 3d as a white amor-

phous solid (2.30 g, 86%). An analytical sample was

recrystallized (heptane) to give white crystals; mp 56–

57 ꢁC. ¹³C NMR (MeOD): d 154.6, 134.8, 130.2,

129.3, 127.9, 127.5, 126.9, 126.8, 124.5, 109.7, 35.3,

23.4, 13.9. HRMS calcd for C₁₃H₁₅OS (M + 1):

219.0844, found: 219.0871. The bis-substituted com-

pound was isolated (187 mg, 5.5%) and some starting

material was recovered (158 mg, 8%).

5.1.8. 5-Hydroxy-1-naphthyl disulﬁde (4b). Ammonia

(30 mL) was condensed into a stirred solution of 5-(pro-

pylthio)naphthalen-1-ol (3b) (224 mg, 1.0 mmol) in dry

Et₂O (10 mL). The reaction was performed and

worked-up as for 4a. Recrystallization (toluene/hep-

tane/MeOH) gave 4b as oﬀ-white crystals (141 mg,

79%); mp 211–213 ꢁC (lit. 212 ꢁC).³⁰The mother liquor

was chromatographed (SiO₂, toluene–methanol 5:1)

giving 4b (29 mg, 16%) for a total yield of 95%. ¹³C

NMR (MeOD): d 155.3, 135.6, 134.1, 132.1, 128.2,

127.2, 125.1, 125.0, 117.2, 109.6. HRMS calcd for

C₂₀H₁₄O₂S₂(M): 350.0435, found: 350.0431.

5.1.5. 6-(Propylthio)naphthalen-2-ol (3e). 2,6-Dihydroxy-

naphthalene (2e) (1.92 g, 12.0 mmol) and p-TsOH (1.0 g,

6.0 mmol) were suspended in toluene (70 mL) and prop-

anethiol (4.45 mL, 49.1 mmol) was added. The suspen-

sion was stirred at 100 ꢁC for 14 h and then worked-

up as for 3a. Column chromatography (SiO₂, toluene–

methanol 20:1) gave 3e as a white amorphous solid

(1.63 g, 62%). An analytical sample was recrystallized

(heptane) to give white crystals; mp 84–85 ꢁC. ¹³C

NMR (MeOD): d 156.5, 134.9, 131.7, 130.2, 129.8,

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M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

20

D

1

5.1.9. 8-Hydroxy-1-naphthyl disulﬁde (4c). Ammonia

(25 mL) was condensed into a stirred solution of 8-(pro-

pylthio)naphthalen-1-ol (3c) (231 mg, 1.1 mmol) in dry

Et₂O (8 mL). The reaction was performed and

worked-up as for 4a. Recrystallization (toluene/heptane)

gave 4c as yellow crystals (127 mg, 69%); mp 170 ꢁC (lit.

168–169 ꢁC).³¹The mother liquor was chromatographed

(SiO₂, toluene–methanol 10:1) giving 4c (50 mg, 27%)

for a total yield of 96%. ¹³C NMR (MeOD): d 155.8,

138.3, 134.0, 127.5, 126.9, 126.7, 124.3, 122.5, 120.9,

110.6. HRMS calcd for C₂₀H₁₄O₂S₂(M): 350.0435,

found: 350.0427.

78%). ½aꢀ ꢁ 50:4^ꢂ(c 0.53, CHCl₃). H NMR (CDCl₃):

d 8.28 (m, 2H, H-8), 8.18 (m, 2H, H-5), 7.51 (m, 4H,

H-6, H-7), 7.40 (d, 2H, J 8.1 Hz, H-3), 6.87 (d, 2H, J

8.1 Hz, H-2), 5.38 (m, 4H, H-1⁰, H-2⁰), 5.31 (dt, 2H, J

7.2, 0.9 Hz, H-3⁰), 5.06 (dt, 2H, J 7.4, 4.6 Hz, H-4⁰),

4.27 (dd, 2H, J 12.2, 4.5 Hz, H-5⁰), 3.62 (dd, 2H, J

12.2, 7.4 Hz, H-5⁰), 2.16, 2.12, 2.10 (s, 6H each,

–OAc). ¹³C NMR (CDCl₃): d 169.98, 169.96, 169.7,

154.0, 134.6, 133.8, 127.6, 126.6, 126.4, 126.1, 122.3,

108.1, 98.3, 70.3, 69.9, 68.4, 62.0, 21.0, 20.9, 20.9.

HRMS calcd for C₄₂H₄₂O₁₆S₂Na (M+Na): 889.1812,

found: 889.1805.

5.1.10. 3-Hydroxy-2-naphthyl disulﬁde (4d). Ammonia

(30 mL) was condensed into a stirred solution of 3-(pro-

pylthio)naphthalen-2-ol (3d) (620 mg, 2.84 mmol) in dry

Et₂O (15 mL). The reaction was performed and worked-

up as for 4a. Recrystallization (toluene/heptane/MeOH)

gave 4d as yellow crystals (198 mg, 40%); mp 160–

163 ꢁC. The mother liquor was chromatographed

(SiO₂, toluene–methanol 5:1) to give 4d (274 mg, 55%)

for a total yield of 95%. ¹³C NMR (MeOD): d 154.1,

135.5, 130.2, 128.2, 127.5, 127.2, 127.0, 126.6, 124.7,

110.1. HRMS calcd for C₂₀H₁₄O₂S₂(M): 350.0435,

found: 350.0427.

5.1.14. 5-(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-1-naph-

thyl disulﬁde (5b). 5-Hydroxy-1-naphthyl disulﬁde (4b)

(20.4 mg, 0.06 mmol) and 2,3,4-tri-O-acetyl-b-^D-xylo-

pyranoside trichloroacetimidate (97.4 mg, 0.23 mmol)

were suspended in CH₂Cl₂(1.5 mL) and cooled to

0 ꢁC. BF₃ÆOEt₂(3 lL, 0.02 mmol) was added and the

reaction was performed and worked-up as for 5a to give

5b as an oﬀ-white amorphous solid (44.2 mg, 88%).

20

D

½aꢀ ꢁ 74:2^ꢂ(c 0.88, CHCl₃). ¹H NMR (CDCl₃): d

8.13 (dt, 2H, J 7.5, 0.9 Hz, H-8), 8.05 (d, 2H, J 8.6 Hz,

H-4), 7.65 (dd, 2H, J 7.2, 1.2 Hz, H-6), 7.43 (t, 2H, J

8.2 Hz, H-3), 7.32 (dd, 2H, J 7.3, 1.2 Hz, H-7), 7.12

(dd, 2H, J 7.7, 0.7 Hz, H-2), 5.37 (m, 4H, H-1⁰, H-2⁰),

5.31 (m, 2H, H-3⁰), 5.07 (dt, 2H, J 7.4, 4.6 Hz, H-4⁰),

4.28 (dd, 2H, J 12.2, 4.6 Hz, H-5⁰), 3.62 (dd, 2H, J

12.1, 7.5 Hz, H-5⁰), 2.15, 2.12, 2.09 (s, 6H each,

–OAc). ¹³C NMR (CDCl₃): d 170.0, 169.7, 152.8,

133.9, 133.4, 130.9, 126.6, 126.6, 125.5, 123.0, 120.0,

109.4, 98.8, 70.6, 70.1, 68.5, 62.0, 20.95, 20.94, 20.92.

HRMS calcd for C₄₂H₄₂O₁₆S₂Na (M+Na): 889.1812,

found: 889.1812.

5.1.11. 6-Hydroxy-2-naphthyl disulﬁde (4e). Ammonia

(30 mL) was condensed into a stirred solution of 6-(pro-

pylthio)naphthalen-2-ol (3e) (327 mg, 1.50 mmol) in dry

Et₂O (10 mL). The reaction was performed and worked-

up as for 4a. Recrystallization (toluene/heptane/MeOH)

gave 4e as oﬀ-white crystals (156 mg, 56%); mp 222 ꢁC

(lit. 221–222 ꢁC).³²The mother liquor was chromato-

graphed (SiO₂, toluene–methanol 10:1) to give 4e

(63 mg, 24%) for a total yield of 83%. ¹³C NMR

(MeOD): d 157.5, 136.1, 132.2, 130.4, 129.89, 129.87,

128.7, 128.6, 120.3, 110.1. HRMS calcd for

C₂₀H₁₄O₂S₂(M): 350.0435, found: 350.0439.

5.1.15. 8-(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-1-naph-

thyl disulﬁde (5c). 8-Hydroxy-1-naphthyl disulﬁde (4c)

(17.9 mg, 0.05 mmol) and 2,3,4-tri-O-acetyl-b-^D-xylo-

pyranoside trichloroacetimidate (85.2 mg, 0.20 mmol)

were suspended in CH₂Cl₂(1.5 mL) and cooled to

0 ꢁC. BF₃ÆOEt₂(3 lL, 0.02 mmol) was added and the

reaction was performed and worked-up as for 5a to give

5.1.12. 7-Hydroxy-2-naphthyl disulﬁde (4f). Ammonia

(30 mL) was condensed into a stirred solution of 7-(pro-

pylthio)naphthalen-2-ol (3f) (411 mg, 1.88 mmol) in dry

Et₂O (10 mL). The reaction was performed and worked-

up as for 4a. Recrystallization (toluene/heptane/MeOH)

gave 4f as oﬀ-white crystals (266 mg, 80%); mp 208 ꢁC

(lit. 205 ꢁC).³³The mother liquor was chromatographed

(SiO₂, toluene–methanol 5:1) to give 4f (17 mg, 5%) for

a total yield of 86%. ¹³C NMR (MeOD): d 157.6, 136.7,

136.0, 130.6, 130.0, 129.1, 126.1, 123.7, 119.9, 109.7.

HRMS calcd for C₂₀H₁₄O₂S₂(M): 350.0435, found:

350.0432.

5c as an oﬀ-white amorphous solid (35.9 mg, 81%).

20

D

½aꢀ ꢁ 109:5^ꢂ(c 0.21, CHCl₃). ¹H NMR (CDCl₃): d

7.78 (dd, 2H, J 7.7, 1.0 Hz, H-2), 7.59 (dd, 2H, J 8.2,

0.8 Hz, H-4), 7.56 (dd, 2H, J 8.3, 0.8 Hz, H-5), 7.41 (t,

2H, J 8.0 Hz, H-6), 7.27 (t, 2H, J 7.9 Hz, H-3), 7.22

(dd, 2H, J 7.9, 1.0 Hz, H-7), 5.55 (m, 4H, H-1⁰, H-2⁰),

5.30 (t, 2H, J 7.1 Hz, H-3⁰), 5.09 (dt, 2H, J 7.0,

4.5 Hz, H-4⁰), 4.42 (dd, 2H, J 12.2, 4.5 Hz, H-5⁰), 3.71

(dd, 2H, J 12.2, 6.8 Hz, H-5⁰), 2.12, 2.06, 2.05 (s, 6H

each, –OAc). ¹³C NMR (CDCl₃): d 170.3, 170.0,

169.4, 153.6, 136.6, 132.1, 126.4, 126.3, 126.1, 123.83,

123.81, 123.0, 109.9, 99.2, 70.9, 70.2, 68.7, 62.1, 21.0.

HRMS calcd for C₄₂H₄₂O₁₆S₂Na (M+Na): 889.1812,

found: 889.1809.

5.1.13. 4-(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-

1-naphthyl disulﬁde (5a). 4-Hydroxy-1-naphthyl disulﬁde

(4a) (20.3 mg, 0.06 mmol) and 2,3,4-tri-O-acetyl-

trichloroacetimidate

b-^D-xylopyranoside

(96.1 mg,

0.23 mmol) were suspended in CH₂Cl₂(1.5 mL) and

cooled to 0 ꢁC. BF₃ÆOEt₂(3 lL, 0.02 mmol) was added

and the mixture was stirred for 20 min at 0 ꢁC. Et₃N

was added and the solution was concentrated and ﬁl-

tered through a short column of SiO₂(CH₂Cl₂-acetone

20:1). Column chromatography (SiO₂, heptane–EtOAc

1:1) gave 5a as an oﬀ-white amorphous solid (39.3 mg,

5.1.16. 3-(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-2-naph-

thyl disulﬁde (5d). 3-Hydroxy-2-naphthyl disulﬁde (4d)

(19.8 mg, 0.06 mmol) and 2,3,4-tri-O-acetyl-b-^D-xylo-

pyranoside trichloroacetimidate (96.4 mg, 0.23 mmol)

were suspended in CH₂Cl₂(1.5 mL) and cooled to

0 ꢁC. BF₃ÆOEt₂(3 lL, 0.02 mmol) was added and the

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5293

reaction was performed and worked-up as for 5a to give

5d as an oﬀ-white amorphous solid (46.6 mg, 95%).

0.1 M, aq) was added. The mixture was heated at

80 ꢁC in a sealed tube under careful stirring overnight.

The mixture was removed from heat and MeOH (1 mL)

was added followed by NaOMe–MeOH (0.5 mL, 1 M)

and the reaction mixture was stirred for 10 min, neu-

tralized with Amberlite IR-120H⁺then carefully ﬁl-

tered and immediately concentrated to give 6a as an

20

D

½aꢀ þ 29:5^ꢂ(c 0.98, CHCl₃). ¹H NMR (CDCl₃): d

7.88 (s, 2H, H-4), 7.72 (d, 2H, J 8.2 Hz, H-5), 7.63 (d,

2H, J 8.1 Hz, H-8), 7.48 (s, 2H, H-1), 7.40 (dt, 2H, J

8.2, 1.2 Hz, H-7), 7.33 (dt, 2H, J 8.1, 1.2 Hz, H-6),

5.50 (d, 2H, J 4.9 Hz, H-1⁰), 5.31 (m, 4H, H-2⁰, H-3⁰),

5.04 (dt, 2H, J 6.2, 4.1 Hz, H-4⁰), 4.40 (dd, 2H, J 12.4,

4.0 Hz, H-5⁰), 3.73 (dd, 2H, J 12.4, 6.4 Hz, H-5⁰), 2.17,

2.17, 2.15 (s, 6H each, –OAc). ¹³C NMR (CDCl₃): d

170.0, 169.99, 169.5, 151.6, 133.1, 130.3, 127.3, 127.0,

126.6, 126.3, 125.6, 125.4, 111.2, 98.7, 69.6, 69.2, 68.1,

61.5, 21.1, 21.0. HRMS calcd for C₄₂H₄₂O₁₆S₂Na

(M+Na): 889.1812, found: 889.1805.

oﬀ-white amorphous solid (9.9 mg, 99%). No further

20

puriﬁcation was performed. ½aꢀ ꢁ 37:2^ꢂ(c 0.18,

D

MeOD). ¹³C NMR (CD₃OD): d 152.4, 133.3, 128.8,

126.6, 126.4, 125.3, 124.8, 122.5, 120.6, 109.3, 101.9,

76.4, 73.4, 69.6, 65.6. HRMS calcd for C₁₅H₁₆O₅SNa

(M+Na): 331.0616, found: 331.0649.

5.1.20. 1-(5-Thionaphthyl) b-^D-xylopyranoside (6b). 5-

(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-1-naphthyl

disulﬁde (5b) (14.4 mg, 0.02 mmol) and Ph₃P-PS (25 mg,

3 mmol/g) were suspended in THF (1.4 mL) and HCl

(0.12 mL, 0.1 M, aq) was added and the reaction was

5.1.17. 6-(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-2-naph-

thyl disulﬁde (5e). 6-Hydroxy-2-naphthyl disulﬁde (4e)

(20.2 mg, 0.06 mmol) and 2,3,4-tri-O-acetyl-b-^D-xylo-

pyranoside trichloroacetimidate (92.2 mg, 0.22 mmol)

were suspended in CH₂Cl₂(1.5 mL) and cooled to

0 ꢁC. BF₃ÆOEt₂(3 lL, 0.02 mmol) was added and the

reaction was performed and worked-up as for 5a to give

performed as for 6a to give 6b as an oﬀ-white amor-

20

phous solid (10.2 mg, quant.). ½aꢀ ꢁ 50:2^ꢂ(c 0.28,

D

MeOD). ¹³C NMR (CD₃OD): d 153.4, 132.7, 128.6,

127.9, 126.5, 125.8, 124.7, 120.2, 118.7, 109.4, 101.8,

76.4, 73.4, 69.6, 65.6. HRMS calcd for C₁₅H₁₆O₅SNa

(M+Na): 331.0616, found: 331.0654.

5e as an oﬀ-white amorphous solid (37.4 mg, 75%).

20

D

½aꢀ ꢁ 22:7^ꢂ(c 0.72, CHCl₃). ¹H NMR (CDCl₃): d

7.91 (d, 2H, J 1.8 Hz, H-1), 7.69 (d, 2H, J 8.7 Hz, H-

3), 7.67 (d, 2H, J 8.9 Hz, H-7), 7.60 (dd, 2H, J 8.7,

1.9 Hz, H-4), 7.32 (d, 2H, J 2.5 Hz, H-5), 7.17 (dd,

2H, J 8.9, 2.4 Hz, H-8), 5.33 (d, 2H, J 5.6 Hz, H-1⁰),

5.24 (m, 4H, H-2⁰, H-3⁰), 5.03 (dt, 2H, J 7.3, 4.8 Hz,

H-4⁰), 4.27 (dd, 2H, J 12.2, 4.6 Hz, H-5⁰), 3.60 (dd,

2H, J 12.2, 7.3 Hz, H-5⁰), 2.11, 2.10, 2.10 (s, 6H each,

–OAc). ¹³C NMR (CDCl₃): d 170.0, 169.99, 169.5,

154.8, 133.6, 133.0, 130.2, 129.5, 128.4, 127.4, 127.2,

119.7, 111.4, 98.5, 70.6, 70.1, 68.6, 62.0, 20.94, 20.92,

20.88. HRMS calcd for C₄₂H₄₂O₁₆S₂Na (M+Na):

889.1812, found: 889.1817.

5.1.21. 1-(8-Thionaphthyl) b-^D-xylopyranoside (6c). 8-

(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-1-naphthyl

disulﬁde (5c) (8.2 mg, 0.01 mmol) and Ph₃P-PS (14 mg,

3 mmol/g) were suspended in THF (1.4 mL) and HCl

(0.12 mL, 0.1 M, aq) was added. Disulﬁde cleavage,

deprotection, and work-up was performed as for 6a

after which TLC-analysis showed a substantial amount

of disulﬁde. Cleavage was performed again using new

Ph₃P-PS under microwave irradiation (110 ꢁC for

15 min). Gave 6c as an oﬀ-white amorphous solid

20

D

(5.8 mg, quant.). ½aꢀ ꢁ 44:2^ꢂ(c 0.20, MeOD). ¹³C

5.1.18. 7-(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-2-naph-

thyl disulﬁde (5f). 7-Hydroxy-2-naphthyl disulﬁde (4f)

(19.8 mg, 0.06 mmol) and 2,3,4-tri-O-acetyl-b-^D-xylo-

pyranoside trichloroacetimidate (91.0 mg, 0.22 mmol)

were suspended in CH₂Cl₂and cooled to 0 ꢁC. BF₃ÆOEt₂

(3 lL, 0.02 mmol) was added and the reaction was per-

NMR (CD₃OD): d 153.6, 136.6, 131.7, 125.9, 125.6,

124.0, 122.8, 122.5, 109.6, 102.3, 76.7, 73.4, 69.9, 65.8.

HRMS calcd for C₁₅H₁₆O₅SNa (M+Na): 331.0616,

found: 331.0654.

5.1.22. 2-(3-Thionaphthyl) b-^D-xylopyranoside (6d). 3-

(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-2-naphthyl

disulﬁde (5d) (14.5 mg, 0.02 mmol) and Ph₃P-PS (25 mg,

3 mmol/g) were suspended in THF (1.4 mL) and HCl

(0.12 mL, 0.1 M, aq) was added and the reaction was

formed and worked-up as for 5a to give 5f as an oﬀ-

20

white amorphous solid (42.0 mg, 86%). ½aꢀ ꢁ 23:0^ꢂ(c

D

0.93, CHCl₃). ¹H NMR (CDCl₃): d 7.87 (d, 2H, J

1.8 Hz, H-1), 7.74 (d, 4H, J 8.8 Hz, H-3, H-5), 7.52

(dd, 2H, J 8.8, 1.8 Hz, H-4), 7.26 (d, 2H, J 2.4 Hz, H-

8), 7.15 (dd, 2H, J 9.0, 2.4 Hz, H-6), 5.31 (d, 2H, J

5.6 Hz, H-1⁰), 5.24 (m, 4H, H-2⁰, H-3⁰), 5.03 (dt, 2H, J

7.3, 4.8 Hz, H-4⁰), 4.25 (dd, 2H, J 12.1, 4.6 Hz, H-5⁰),

3.59 (dd, 2H, 12.1, 7.3 Hz, H-5⁰), 2.11, 2.10, 2.09 (s,

6H each, –OAc). ¹³C NMR (CDCl₃): d 170.0, 169.99,

169.5, 155.1, 135.3, 134.5, 129.7, 129.1, 128.8, 125.2,

124.1, 119.2, 110.9, 98.4, 70.6, 70.1, 68.5, 62.0, 20.94,

20.91, 20.87. HRMS calcd for C₄₂H₄₂O₁₆S₂Na

(M+Na): 889.1812, found: 889.1816.

performed as for 6a to give 6d as an oﬀ-white

20

amorphous solid (9.6 mg, 93%). ½aꢀ ꢁ 31:9^ꢂ(c 0.24,

D

MeOD). ¹³C NMR (CD₃OD): d 161.0, 132.3, 130.0,

126.6, 126.5, 125.8, 125.1, 124.6, 124.0, 110.8, 101.9,

76.0, 73.1, 69.5, 65.5. HRMS calcd for C₁₅H₁₆O₅SNa

(M+Na): 331.0616, found: 331.0590.

5.1.23. 2-(6-Thionaphthyl) b-^D-xylopyranoside (6e). 6-

(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-2-naphthyl

disulﬁde (5e) (13.3 mg, 0.02 mmol) and Ph₃P-PS (23 mg,

3 mmol/g) were suspended in THF (1.4 mL) and HCl

(0.12 mL, 0.1 M, aq) was added and the reaction was

5.1.19. 1-(4-Thionaphthyl) b-^D-xylopyranoside (6a). 4-

(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-1-naphthyl

disulﬁde (5a) (14.0 mg, 0.02 mmol) and polystyryldi-

phenylphosphine (Ph₃P-PS) (24 mg, 3 mmol/g) were

suspended in THF (1.4 mL) and HCl (0.12 mL,

performed as for 6a to give 6e as an oﬀ-white

20

D

amorphous solid (9.3 mg, 98%). ½aꢀ ꢁ 14:2^ꢂ(c 0.30,

MeOD). ¹³C NMR (CD₃OD): d 155.0, 132.2, 130.3,

127.9, 127.8, 127.4, 126.9, 126.2, 119.3, 110.6, 101.5,

5294

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

76.3, 73.4, 69.6, 65.6. HRMS calcd for C₁₅H₁₆O₅SNa

(M+Na): 331.0616, found: 331.0636.

tion was diluted with EtOAc and washed with NaHCO₃

(satd aq), brine, and H₂O. The solution was dried

(MgSO₄), ﬁltered, and concentrated. The residue was

chromatographed (SiO₂, Heptane–EtOAc 1:1) to give

5.1.24. 2-(7-Thionaphthyl) b-^D-xylopyranoside (6f). 7-

(2,3,4-Tetra-O-acetyl-b-^D-xylopyranoside)-2-naphthyl

disulﬁde (5f) (15.1 mg, 0.02 mmol) and Ph₃P-PS (26 mg,

3 mmol/g) were suspended in THF (1.4 mL) and HCl

(0.12 mL, 0.1 M, aq) was added and the reaction was

8b as an oﬀ-white amorphous solid (90.8 mg, 71%).

20

D

½aꢀ ꢁ 80:4^ꢂ(c 0.45, CHCl₃). ¹H NMR (CDCl₃): d

8.36 (dt, 1H, J 8.7, 0.9 Hz, H-4), 7.90 (dt, 1H, J 8.5,

0.9 Hz, H-8), 7.84 (dd, 1H, J 7.3, 1.1 Hz, H-6), 7.57

(dd, 1H, J 8.7, 7.5 Hz, H-3), 7.47 (dd, 1H, J 8.5,

7.3 Hz, H-7), 7.26 (dd, 1H, J 7.5, 0.9 Hz, H-2), 5.18 (t,

1H, J 8.1 Hz, H-3⁰), 5.08 (t, 1H, J 8.1 Hz, H-2⁰), 4.97

(dt, 1H, J 8.3, 4.9 Hz, H-4⁰), 4.83 (d, 1H, J 8.3 Hz, H-

1⁰), 4.26 (dd, 1H, J 11.7, 4.9, Hz, H-5⁰), 3.34 (dd, 1H,

J 11.7, 8.9 Hz, H-5⁰), 2.13, 2.08, 2.04 (s, 3H each, –

OAc), 1.50 (s, 9H, –OPiv). ¹³C NMR (CDCl₃): d

177.0, 169.9, 169.8, 169.4, 147.2, 135.3, 133.6, 130.3,

127.8, 126.3, 126.1, 123.5, 122.6, 118.5, 87.0, 72.0,

70.2, 68.4, 65.2, 39.5, 27.3, 20.8, 20.7. HRMS calcd for

C₂₆H₃₀O₉SNa (M+Na): 541.1508, found: 541.1497.

performed as for 6a to give 6f as an oﬀ-white amorphous

20

D

solid (10.2 mg, 95%). ½aꢀ ꢁ 16:2^ꢂ(c 0.25, MeOD). ¹³C

NMR (CD₃OD): d 155.9, 134.9, 130.0, 128.8, 127.9,

127.5, 125.4, 125.0, 117.9, 109.3, 101.3, 76.3, 73.4,

69.6, 65.6. HRMS calcd for C₁₅H₁₆O₅SNa (M+Na):

331.0616, found: 331.0633.

5.1.25. 1-(4-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acet-

yl-b-^D-xylopyranoside (8a). 4-Hydroxy-1-naphthyl disul-

ﬁde (4a) (19.3 mg, 0.06 mmol) was suspended in pivalic

anhydride (Piv₂O) (1.0 mL) and heated to 110 ꢁC over-

night. The solution was concentrated under high vac-

uum, the residue was dissolved in THF (0.7 mL), and

Ph₃P-PS (82.3 mg, 3 mmol/g) was added followed by

HCl (0.06 mL, 0.1 M, aq). The mixture was heated to

110 ꢁC for 15 min under microwave irradiation, then

carefully ﬁltered and immediately concentrated. The

residue was dissolved in DMF (0.4 mL) and the solution

was cooled to 0 ꢁC. t-BuONa (9.0 mg, 0.08 mmol)

was added and the solution was stirred at 0 ꢁC for

5 min. 2,3,4-Tri-O-acetyl-a-^D-xylopyranosyl bromide

(26.0 mg, 0.08 mmol) in THF (0.2 mL) was added and

the solution was stirred at 0 ꢁC for 30 min after which

it was ﬁltered through a short column of SiO₂(Hep-

tane–EtOAc 1:1) and concentrated. The residue was

chromatographed (SiO₂, Heptane–EtOAc 2:1) to give

5.1.27. 2-(6-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acet-

yl-b-^D-xylopyranoside (8e). 6-Hydroxy-2-naphthyl disul-

ﬁde (4e) (51.4 mg, 0.15 mmol) was pivaloylated as for

8a. The residue was dissolved in THF (1.4 mL) and

Ph₃P-PS (222.1 mg, 3 mmol/g) was added followed by

HCl (0.12 mL, 0.1 M, aq). The mixture was heated to

110 ꢁC for 15 min under microwave irradiation then

carefully ﬁltered and immediately concentrated. The res-

idue was dissolved in DMF (0.4 mL) and the solution

was cooled to 0 ꢁC. t-BuONa (6.3 mg, 0.06 mmol)

was added and the solution was stirred at 0 ꢁC for

7 min. 2,3,4-Tri-O-acetyl-a-^D-xylopyranosyl bromide

(89.5 mg, 0.26 mmol) in THF (0.2 mL) was added and

the solution was allowed to regain rt. After 3 h the

solution was worked-up as for 8b. The residue was chro-

matographed (SiO₂, Heptane–EtOAc 1:1) to give 8e as

8a as an oﬀ-white amorphous solid (26.6 mg, 67%).

20

D

½aꢀ ꢁ 59:7^ꢂ(c 0.49, CHCl₃). ¹H NMR (CDCl₃): d

8.49 (d, 1H, J 8.0 Hz, H-8), 7.91 (d, 1H, J 8.3 Hz, H-

5), 7.83 (d, 1H, J 7.9 Hz, H-3), 7.61 (m, 1H, H-7), 7.58

(m, 1H, H-6), 7.21 (d, 1H, J 7.9 Hz, H-2), 5.17 (t, 1H,

J 8.2 Hz, H-3⁰), 5.07 (t, 1H, 8.2 Hz, H-2⁰), 4.97 (dt,

1H, J 8.4, 5.0 Hz, H-4⁰), 4.77 (d, 1H, J 8.5, H-1⁰), 4.24

(dd, 1H, J 11.7, 4.9 Hz, H-5⁰), 3.31 (dd, J 11.7, 8.9 Hz,

H-5⁰), 2.14, 2.07, 2.04 (s, 3H each, –OAc), 1.50 (s, 9H,

–OPiv). ¹³C NMR (CDCl₃): d 176.7, 170.0, 169.8,

169.4, 148.2, 135.3, 133.5, 127.7, 127.3, 127.0, 126.8,

126.2, 121.7, 117.9, 87.2, 72.1, 70.3, 68.4, 65.3, 39.6,

27.3, 20.8, 20.73, 20.72. HRMS calcd for C₂₆H₃₀O₉SNa

(M+Na): 541.1508, found: 541.1528.

an oﬀ-white amorphous solid (104.2 mg, 76%).

20

D

½aꢀ ꢁ 59:9^ꢂ(c 0.34, CHCl₃). ¹H NMR (CDCl₃): d

7.99 (s, 1H, H-1), 7.81 (d, 1H, J 8.8 Hz, H-8), 7.75 (d,

I H, J 8.6 Hz, H-4), 7.56 (dd, 1H, J 8.6, 1.6 Hz, H-3),

7.52 (d, 1H, J 2.1 Hz, H-5), 7.23 (dd, 1H, J 8.8,

2.1 Hz, H-7), 5.21 (t, 1H, J 7.9, H-3⁰), 5.00 (t, 1H, J

7.9 Hz, H-2⁰), 4.94 (dt, 1H, J 8.1, 4.9 Hz, H-4⁰), 4.92

(d, H 1, J 8.1 Hz, H-1⁰), 4.32 (dd, 1H, J 11.9, 4.8 Hz,

H-5⁰), 3.46 (dd, 1H, J 11.9, 8.6 Hz, H-5⁰), 2.13, 2.07,

2.06 (s, 3H each, –OAc), 1.41 (s, 3H, –OPiv). ¹³C

NMR (CDCl₃): d 177.1, 169.9, 169.8, 169.4, 149.4,

133.2, 132.0, 131.4, 130.5, 129.3, 129.1, 128.3, 122.0,

118.4, 86.3, 71.8, 69.9, 68.4, 65.1, 39.2, 27.2, 20.8, 20.7.

HRMS calcd for C₂₆H₃₀O₉SNa (M+Na): 541.1508,

found: 541.1492.

5.1.26. 1-(5-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acet-

yl-b-^D-xylopyranoside (8b). 5-Hydroxy-1-naphthyl disul-

ﬁde (4b) (47.8 mg, 0.14 mmol) was pivaloylated as for

8a. The residue was dissolved in THF (1.4 mL) and

Ph₃P-PS (204.4 mg, 3 mmol/g) was added followed by

HCl (0.12 mL, 0.1 M, aq). The mixture was heated to

110 ꢁC for 15 min under microwave irradiation then

carefully ﬁltered and immediately concentrated. The res-

idue was dissolved in DMF (0.4 mL) and the solution

was cooled to 0 ꢁC. t-BuONa (30.0 mg, 0.27 mmol)

was added and the solution was stirred at 0 ꢁC for

10 min. 2,3,4-Tri-O-acetyl-a-^D-xylopyranosyl bromide

(83.6 mg, 0.25 mmol) in THF (0.2 mL) was added and

the solution was allowed to regain rt. After 2 h the solu-

5.1.28. 2-(7-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acet-

yl-b-^D-xylopyranoside (8f). 7-Hydroxy-2-naphthyl disul-

ﬁde (4f) (46.7 mg, 0.13 mmol) was pivaloylated as for

8a. The residue was dissolved in THF (1.4 mL) and

Ph₃P-PS (197.0 mg, 3 mmol/g) was added followed by

HCl (0.12 mL, 0.1 M, aq). The mixture was heated to

110 ꢁC for 15 min under microwave irradiation then

carefully ﬁltered and immediately concentrated. The res-

idue was dissolved in DMF (0.4 mL) and the solution

was cooled to 0 ꢁC. t-BuONa (29.3 mg, 0.26 mmol)

was added and the solution was stirred at 0 ꢁC for

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5295

15 min. 2,3,4-Tri-O-acetyl-a-D-xylopyranosyl bromide

(82.3 mg, 0.24 mmol) in THF (0.2 mL) was added and

the solution was allowed to regain rt. After 2 h the solu-

tion was worked-up as for 8b. The residue was chro-

and concentrated. Chromatography (SiO₂, CH₂Cl₂–

MeOH 5:1) gave 9c as an oﬀ-white amorphous solid

20

(13.1 mg, 34%). ½aꢀ ꢁ 89:2^ꢂ(c 0.24, MeOH). ¹³C

NMR (CD₃OD): d ^D155.7, 137.9, 133.9, 130.4, 128.5,

127.8, 126.4, 124.8, 121.5, 112.8, 90.7, 79.3, 73.9, 70.8,

70.5. HRMS calcd for C₁₅H₁₆O₅SNa (M+Na):

331.0616, found: 331.0627.

matographed (SiO₂, Heptane–EtOAc 1:1) to give 8f

20

D

(76.8 mg, 61%). ½aꢀ ꢁ 38:7^ꢂ(c 0.45, CHCl₃). ¹H

NMR (CDCl₃): d 7.93 (d, 1H, J 1.3 Hz, H-1), 7.83 (d,

1H, J 8.9 Hz, H-5), 7.79 (d, 1H, J 8.6 Hz, H-4), 7.52

(dd, 1H, J 8.5, 1.8 Hz, H-3), 7.49 (d, 1H, J 2.2 Hz, H-

8), 7.22 (dd, 1H, J 8.9, 2.3, H-6), 5.21 (t, 1H, J 7.9, H-

3⁰), 5.00 (t, 1H, J 7.9 Hz, H-2⁰), 4.94 (dt, 1H, J 8.4, 4.9

Hz, H-4⁰), 4.92 (d, H 1, J 8.2 Hz, H-1⁰), 4.32 (dd, 1H,

J 11.8, 4.8 Hz, H-5⁰), 3.46 (dd, 1H, J 11.8, 8.6 Hz,

H-5⁰), 2.13, 2.07, 2.06 (s, 3H each, –OAc), 1.41 (s, 3

H, –OPiv). ¹³C NMR (CDCl₃): d 177.2, 169.9, 169.8,

169.4, 149.4, 133.9, 131.3, 130.6, 130.6, 129.4, 129.1,

128.4, 122.0, 118.2, 86.2, 71.8, 69.9, 68.4, 65.2, 39.2,

27.2, 20.8, 20.73, 20.72. HRMS calcd for C₂₆H₃₀O₉SNa

(M+Na): 541.1508, found: 541.1525.

5.1.32. 2-(3-Hydroxynaphthyl) 1-thio-b-^D-xylopyranoside

(9d). 3-Hydroxy-2-naphthyl disulﬁde (4d) (28.5 mg,

0.08 mmol) was pivaloylated as for 8a. The residue

was dissolved in THF (0.7 mL) and Ph₃P-PS

(121.2 mg, 3 mmol/g) was added followed by HCl

(0.06 mL, 0.1 M, aq). The mixture was heated to

110 ꢁC for 15 min under microwave irradiation then

carefully ﬁltered and immediately concentrated. The res-

idue was dissolved in DMF (0.4 mL) and the solution

was cooled to 0 ꢁC. t-BuONa (15.5 mg, 0.14 mmol)

was added and the solution was stirred at 0 ꢁC for

5 min. 2,3,4-Tri-O-acetyl-a-^D-xylopyranosyl bromide

(43.3 mg, 0.13 mmol) in THF (0.2 mL) was added and

the solution was stirred at 0 ꢁC for 25 min after which

it was worked-up, deprotected, and puriﬁed as for 9a.

5.1.29. 1-(4-Hydroxynaphthyl) 1-thio-b-^D-xylopyranoside

(9a). 1-(4-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (8a) (12.7 mg, 0.02 mmol) was

dissolved in CH₂Cl₂(1 mL). MeOH (1 mL) and

NaOMe–MeOH (0.5 mL, 1 M) were added, the mixture

was stirred for 3 h, neutralized with Amberlite IR-120H⁺,

ﬁltered, and concentrated. The residue was ﬁltered

Gave 9d as an oﬀ-white amorphous solid (17.3 mg,

20

D

44%). ½aꢀ ꢁ 33:9^ꢂ(c 0.32, MeOH). ¹³C NMR

(CD₃OD): d 154.1, 134.2, 128.6, 127.0, 126.4, 125.6,

125.5, 123.2, 120.7, 109.1, 87.6, 77.7, 72.4, 69.4, 69.3.

HRMS calcd for C₁₅H₁₆O₅SNa (M+Na): 331.0616,

found: 331.0629.

(SiO₂, CH₂Cl₂–MeOH 5:1) to give 9a as an oﬀ-white

20

amorphous solid (7.3 mg, 97%). ½aꢀ ꢁ 73:7^ꢂ(c 0.41,

D

MeOH). ¹³C NMR (CD₃OD): d 156.3, 137.5, 137.2,

127.8, 127.4, 127.1, 125.8, 123.7, 119.7, 108.7, 91.4,

79.3, 74.2, 71.0, 70.5. HRMS calcd for C₁₅H₁₆O₅SNa

(M+Na): 331.0616, found: 331.0623.

5.1.33. 2-(6-Hydroxynaphthyl) 1-thio-b-^D-xylopyranoside

(9e). 2-(6-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (8e) (18.5 mg, 0.04 mmol) was

deprotected and puriﬁed as for 9a. Gave 9e as an oﬀ-

20

5.1.30. 1-(5-Hydroxynaphthyl) 1-thio-b-^D-xylopyranoside

(9b). 1-(5-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (8a) (21.8 mg, 0.04 mmol) was

white amorphous solid (10.5 mg, 95%). ½aꢀ ꢁ 35:4^ꢂ(c

D

0.58, MeOH). ¹³C NMR (CD₃OD): d 155.8, 134.4,

132.0, 130.5, 128.8, 128.5, 126.3, 126.2, 118.5, 108.4,

89.0, 77.9, 72.3, 69.5, 69.1. HRMS calcd for C₁₅H₁₆O₅S-

Na (M+Na): 331.0616, found: 331.0580.

deprotected and puriﬁed as for 9a. Gave 9b as an oﬀ-

20

white amorphous solid (7.3 mg, 97%). ½aꢀ ꢁ 100:9^ꢂ(c

D

0.66, MeOH). ¹³C NMR (CD₃OD): d 155.0, 136.5,

133.2, 131.5, 127.8, 126.9, 125.1, 124.0, 117.8, 109.3,

90.5, 79.2, 74.3, 70.9, 70.3. HRMS calcd for C₁₅H₁₆O₅S-

Na (M+Na): 331.0616, found: 331.0586.

5.1.34. 2-(7-Hydroxynaphthyl) 1-thio-b-^D-xylopyranoside

(9f). 2-(7-O-Pivaloyl-naphthyl) 1-thio-2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (8f) (22.2 mg, 0.04 mmol) was

deprotected and puriﬁed as for 9a. Gave 9f as an oﬀ-

20

D

5.1.31. 1-(8-Hydroxynaphthyl) 1-thio-b-^D-xylopyranoside

(9c). 8-Hydroxy-1-naphthyl disulﬁde (4c) (26.9 mg,

0.08 mmol) was pivaloylated as for 8a. The residue

was dissolved in THF (0.7 mL) and Ph₃P-PS

(120.0 mg, 3 mmol/g) was added followed by HCl

(0.06 mL, 0.1 M, aq). The mixture was heated to

110 ꢁC for 15 min under microwave irradiation then

carefully ﬁltered and immediately concentrated. The res-

idue was dissolved in DMF (0.4 mL) and the solution

was cooled to 0 ꢁC. t-BuONa (15.6 mg, 0.14 mmol)

was added and the solution was stirred at 0 ꢁC for

5 min. 2,3,4-Tri-O-acetyl-a-^D-xylopyranosyl bromide

(42.0 mg, 0.12 mmol) in THF (0.2 mL) was added and

the solution was stirred at 0 ꢁC for 30 min after which

it was ﬁltered through a pad of SiO₂(Heptane–EtOAc

1:1) and concentrated. The residue was dissolved in

CH₂Cl₂(2 mL) and MeOH (2 mL), and NaOMe–

MeOH (1 mL, 1 M) was added. The solution was stirred

for 2 h, neutralized with Amberlite IR-120H⁺, ﬁltered,

white amorphous solid (13.0 mg, 98%). ½aꢀ ꢁ 31:9^ꢂ(c

0.71, MeOH). ¹³C NMR (CD₃OD): d 155.7, 135.2,

131.1, 128.9, 128.8, 127.7, 127.4, 126.1, 118.3, 108.1,

88.7, 77.8, 72.4, 69.5, 69.0. HRMS calcd for C₁₅H₁₆O₅S-

Na (M+Na): 331.0616, found: 331.0630.

5.1.35. 1-(4-(Propylthio)naphth-1-yl)-2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (10a). 4-(Propylthio)naphthalen-1-

ol (3a) (545 mg, 2.5 mmol) and 1,2,3,4-tetra-O-acetyl-

b-^D-xylopyranoside (1.59 g, 5.0 mmol) were dissolved

in CH₂Cl₂(37 mL). Et₃N (350 lL, 2.5 mmol) was added

followed by BF₃ÆOEt₂(1.56 mL, 12.4 mmol) and the

solution was stirred for 2 h. Excess Et₃N was added

and the solution was concentrated. Column chromatog-

raphy (SiO₂, heptane–EtOAc 3:1) gave 10a as an oﬀ-

20

white amorphous solid (1.14 g, 96%). ½aꢀ ꢁ 62:1^ꢂ(c

D

0.58, CHCl₃). ¹H NMR (CDCl₃): d 8.46 (d, 1H, J

8.3 Hz, H-8), 8.17 (d, 1H, J 8.4 Hz, H-5), 7.60 (m, 1H,

H-7), 7.59 (d, 1H, J 8.1 Hz, H-3), 7.53 (m, 1H, H-6),

5296

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

7.02 (d, 1H, J 8.1 Hz, H-2), 5.38 (m, 1H, H-1⁰, H-2⁰),

5.31 (m, 1H, H-3⁰), 5.07 (dt, 1H, J 7.4, 4.6 Hz, H-4⁰),

4.28 (dd, 1H, J 12.2, 4.6 Hz, H-5⁰), 3.62 (dd, 1H, J

12.2, 7.4 Hz, H-5⁰), 2.85 (t, 2H, J 7.3 Hz, –SCH₂–),

2.16 (s, 3H, –OAc), 2.12 (s, 3H, –OAc), 2.09 (s, 3H,

–OAc), 1.61 (tq, 2H, J 7.4, 7.3 Hz, –CH₂Me), 1.00 (t,

3H, J 7.4 Hz, –CH₃). ¹³C NMR (CDCl₃): d 170.0,

169.7, 152.3, 134.7, 130.8, 127.3, 127.28, 126.26, 126.2,

125.8, 122.3, 108.7, 98.5, 70.4, 70.0, 68.5, 61.9, 37.7,

22.9, 20.94, 20.93, 20.91, 13.5. HRMS calcd for

C₂₄H₂₈O₈SNa (M+Na): 499.1403, found: 499.1420.

5.0 Hz, H-1⁰), 5.28 (m, 1H, H-2⁰), 5.24 (m, 1H, H-3⁰),

5.02 (dt, 1H, J 6.3, 4.1 Hz, H-4⁰), 4.35 (dd, 1H, J

12.3, 4.1 Hz, H-5⁰), 3.67 (dd, 1H, J 12.3, 6.4 Hz, H-

5⁰), 2.96 (dt, 2H, J 7.6, 2.9 Hz, –SCH₂–), 2.17, 2.15,

2.13 (s, 3H each, –OAc), 1.76 (tq, 2H, J 7.6, 7.3 Hz,

–CH₂Me), 1.10 (t, 3H, J 7.3 Hz, –CH₃). ¹³C NMR

(CDCl₃): d 169.9, 169.85, 169.4, 152.1, 131.9, 130.2,

129.0, 126.9, 126.5, 125.6, 125.4, 125.0, 110.8, 98.5,

69.5, 69.1, 68.1, 61.3, 33.5, 21.9, 20.9, 20.86, 20.8,

13.7. HRMS calcd for C₂₄H₂₈O₈SNa (M+Na):

499.1403, found: 499.1422.

5.1.36. 1-(5-(Propylthio)naphth-1-yl) 2,3,4-tri-O-acetyl-b-

D-xylopyranoside (10b). Synthesized as for 10a. 5-(Pro-

pylthio)naphthalen-1-ol (3b) (515 mg, 2.4 mmol) gave

5.1.39. 1-(6-(Propylthio)naphth-2-yl) 2,3,4-tri-O-acetyl-b-

D-xylopyranoside (10e). Synthesized as for 10a. 6-(Pro-

pylthio)naphthalen-2-ol (3e) (509 mg, 2.3 mmol) gave

10e as an oﬀ-white amorphous solid (1.11 g, quantita-

10b as oﬀ-white amorphous solid (1.08 g, 96%).

20

D

20

½aꢀ ꢁ 74:0^ꢂ(c 0.52, CHCl₃). ¹H NMR (CDCl₃): d

tive). ½aꢀ ꢁ 21:8^ꢂ(c 0.50, CHCl₃). H NMR (CDCl₃):

1

D

8.12 (d, 1H, J 8.6 Hz, H-8), 8.05 (d, 1H, J 8.6 Hz, H-

4), 7.57 (dd, I H, J 7.3, 1.1, H-2), 7.45 (dd, 1H, J 8.6,

7.8 Hz, H-7), 7.41 (dd, 1H, J 8.6, 7.3 Hz, H-3), 7.11

(d, 1 H, J 7.8 Hz, H-6), 5.38 (m, 2H, H-1⁰, H-2⁰), 5.31

(t, 1H, J 7.2 Hz, H-3⁰), 5.07 (dt, 1H, J 7.3, 4.6 Hz, H-

4⁰), 4.28 (dd, 1H, J 12.2, 4.6 Hz, H-5⁰), 3.61 (dd, 1H, J

12.2, 7.3 Hz, H-5⁰), 2.96 (t, 2H, J 7.2 Hz, –SCH₂–),

2.15 (s, 3H, –OAc), 2.11 (s, 3H, –OAc), 2.09 (s, 3H, –

OAc), 1.69 (tq, 2H, J 7.4, 7.2 Hz, –CH₂Me), 1.05 (t,

3H, J 7.4 Hz, –CH₃). ¹³C NMR (CDCl₃): d 169.9,

169.8, 169.5, 152.6, 134.0, 134.0, 128.2, 126.2, 125.9,

125.4, 120.3, 119.8, 109.1, 98.6, 70.4, 70.0, 68.4, 61.8,

36.2, 22.5, 20.78, 20.76, 13.5. HRMS calcd for

C₂₄H₂₈O₈SNa (M+Na): 499.1403, found: 499.1394.

d 7.71 (d, 1H, J 1.8 Hz, H-1), 7.68 (d, 1H, J 9.0 Hz,

H-8), 7.65 (d, 1H, J 8.6 Hz, H-4), 7.43 (d, 1H, J 8.6,

1.8 Hz, H-3), 7.32 (d, 1H, J 2.4 Hz, H-5), 7.17 (dd,

1H, J 9.0, 2.4 Hz, H-7) 5.32 (d, 1H, J 5.6 Hz, H-1⁰),

5.27 (m, 1 H, H-3⁰), 5.25 (m, 1H, H-2⁰), 5.05 (dt, 1H,

J 7.1, 4.7 Hz, H-4⁰), 4.28 (dd, 1H, J 12.2, 4.7, H-5⁰),

3.60 (dd, 1H, J 12.2, 7.1, H-5⁰), 2.98 (t, 2H, J 7.4 Hz,

–SCH₂–), 2.12, 2.11, 2.11 (s, 3H each, –OAc), 1.71 (tq,

2H, J 7.4, 7.3 Hz, –CH₂Me), 1.05 (t, 3 H, J 7.3 Hz,

–CH₃). ¹³C NMR (CDCl₃): d 169.9, 169.8, 169.4,

154.1, 132.9, 132.4, 130.4, 128.8, 128.4, 127.6, 127.0,

119.3, 111.3, 98.5, 70.5, 70.1, 68.4, 61.8, 35.9, 22.5,

20.79, 20.77, 20.7, 13.4. HRMS calcd for C₂₄H₂₈O₈SNa

(M+Na): 499.1403, found: 499.1393.

5.1.37. 1-(8-(Propylthio)naphth-1-yl) 2,3,4-tri-O-acetyl-b-

D-xylopyranoside (10c). Synthesis of 10c using the meth-

od described above for 10a gave a poor yield (28%) and

therefore synthesis of the desired product 11c (see be-

low) was performed using a trichloroacetimidate donor

and full isolation of the acetylated product was not per-

formed prior to deacetylation. Data for 10c are nonethe-

5.1.40. 1-(7-(Propylthio)naphth-2-yl) 2,3,4-tri-O-acetyl-b-

D-xylopyranoside (10f). Synthesized as for 10a. 7-(Pro-

pylthio)naphthalen-2-ol (3f) (523 mg, 2.4 mmol) gave

10f as an oﬀ-white amorphous solid (976 mg, 86%).

20

D

½aꢀ ꢁ 11:9^ꢂ(c 0.39, CHCl₃). ¹H NMR (CDCl₃): d

7.71 (d, 1H, J 8.9 Hz, H-4), 7.68 (d, 1H, J 8.5 Hz, H-

5), 7.61 (d, 1H, J 1.7 Hz, H-8), 7.32 (dd, 1H, J 8.5,

1.7, H-6), 7.26 (d, 1H, J 2.5, H-1) 7.12 (dd, 1H, J 8.9,

2.5 Hz, H-3), 5.33 (d, 1H, J 5.6, H-1⁰), 5.28 (m, 1 H,

H-3⁰), 5.25 (m, 1H, H-2⁰), 5.05 (dt, 1H, J 7.3, 4.6 Hz,

H-4⁰), 4.28 (dd, 1H, J 12.2, 4.6 Hz, H-5⁰), 3.61 (dd,

1H, J 12.2, 7.3 Hz, H-5⁰), 3.00 (t, 2H, J 7.2 Hz,

–SCH₂–), 2.12, 2.11, 2.11 (s, 3H each, –OAc), 1.73 (tq,

2H, J 7.4, 7.2 Hz, –CH₂Me), 1.07 (t, 3 H, J 7.4 Hz,

–CH₃). ¹³C NMR (CDCl₃): d 169.9, 169.9, 169.4,

154.8, 135.7, 134.6, 129.5, 128.1, 128.0, 125.8, 125.2,

118.2, 110.4, 98.4, 70.5, 70.0, 68.4, 61.8, 35.2, 22.4,

20.79, 20.76, 20.7, 13.5. HRMS calcd for C₂₄H₂₈O₈SNa

(M+Na): 499.1403, found: 499.1403.

20

1

less reported here. ½aꢀ ꢁ 105:8^ꢂ(c 0.51, CHCl₃). H

D

NMR (CDCl₃): d 7.51 (d, 1H, J 8.2 Hz, H-5), 7.49 (d,

1H, J 8.3 Hz, H-4), 7.35 (t, 1H, J 7.8 Hz, H-3), 7.34

(t, 1H, J 7.6 Hz, H-6), 7.19 (d, 1H, J 7.6 Hz, H-7),

7.10 (dd, 1H, J 7.8, 0.9 Hz, H-2), 5.57 (dd, 1H, J 8.2,

6.3 Hz, H-2⁰), 5.39 (d, 1H, J 6.3 Hz, H-1⁰), 5.29 (t, 1H,

J 8.2 Hz, H-3⁰), 5.14 (dt, 1H, J 7.9, 4.9 Hz, H-4⁰), 4.36

(dd, 1H, J 12.2, 4.9 Hz, H-5⁰), 3.63 (dd, 1H, J 12.2,

7.9 Hz, H-5⁰), 2.89 (t, 2H, J 7.7 Hz, –SCH₂–), 2.10,

2.10, 2.05 (s, 3H each, –OAc), 1.79 (tq, 2H, J 7.7,

7.7 Hz, –CH₂Me), 1.12 (t, 3H, J 7.4 Hz, –CH₃). ¹³C

NMR (CDCl₃): d 170.3, 169.9, 169.5, 154.1, 136.3,

136.2, 125.9, 125.8, 124.2, 123.6, 123.55, 121.6, 109.6,

99.2, 71.7, 70.3, 68.9, 62.3, 34.8, 21.2, 21.0, 20.9, 20.8,

14.1. HRMS calcd for C₂₄H₂₈O₈SNa (M+Na):

499.1403, found: 499.1427.

5.1.41. 1-(4-(Propylthio)naphth-1-yl) b-^D-xylopyranoside

(11a). 1-(4-(Propylthio)naphth-1-yl) 2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (10a) (828 mg, 1.74 mmol) was sus-

pended in MeOH (19 mL) and NaOMe/MeOH (1 mL,

1 M) was added. The mixture was stirred for 90 min

after which it was neutralized with Amberlite IR-120

H⁺. The mixture was ﬁltered, concentrated, and ﬁltered

5.1.38. 1-(3-(Propylthio)naphth-2-yl) 2,3,4-tri-O-acetyl b-

D-xylopyranoside (10d). Synthesized as for 10a. 3-(Pro-

pylthio)naphthalen-2-ol (3d) (520 mg, 2.4 mmol) gave

10d as oﬀ-white amorphous solid (941 mg, 83%).

through SiO₂(CH₂Cl₂–MeOH 5:1) to give 11a as a clear

20

½aꢀ ꢁ 33:3^ꢂ(c 0.69, CHCl₃). ¹H NMR (CDCl₃): d

20

oil (597 mg, 98%). ½aꢀ ꢁ 71:1^ꢂ(c 0.26, MeOH). ¹³C

D

7.70 (m, 2H, H-6, H-7), 7.57 (s, 1H, H-1), 7.42 (s,

1H, H-4), 7.39 (m, 2H, H-5, H-8), 5.38 (d, 1H, J

D

NMR (CD₃OD): d 153.0, 134.5, 130.9, 126.6, 126.0,

125.2, 124.9, 122.6, 108.8, 101.7, 76.3, 73.4, 69.6, 65.6,

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

5297

37.0, 22.3, 12.2. HRMS calcd for C₁₈H₂₂O₅SNa

(M+Na): 373.1086, found: 373.1080.

Hydrosphere C18 column (15 cm · 4.6 mm, 5 lm). The

system was controlled by the Hewlett-Packard Chem-

Station for LC software suite. The mobile phase con-

sisted of H₂O + 0.1% triﬂuoroacetic acid (TFA) with a

gradient of acetonitrile from 1 min increasing by 1.2%

per minute until all UV-absorbing (254 nm) peaks had

eluted. The mean retention times and standard devia-

tions were calculated from three separate runs per

compound.

5.1.42. 1-(5-(Propylthio)naphth-1-yl) b-^D-xylopyranoside

(11b). 1-(5-(Propylthio)naphth-1-yl) 2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (10b) (819 mg, 1.72 mmol) was

deprotected and puriﬁed as for 11a. Gave 11b as a white

20

amorphous solid (572 mg, 95%). ½aꢀ ꢁ 69:9^ꢂ(c 0.29,

D

MeOH). ¹³C NMR (CD₃OD): d 154.8, 135.2, 134.8,

129.2, 128.0, 127.1, 126.1, 122.2, 120.1, 110.9, 103.3,

77.8, 74.8, 71.0, 67.0, 37.0, 23.5, 13.7. HRMS calcd for

C₁₈H₂₂O₅SNa (M+Na): 373.1086, found: 373.1080.

5.1.48. Stability of naphthoxylosides. A solution of xylo-

side (20 mM in H₂O–DMSO 1:1) was diluted in fresh

Ham’s F-12 cell growth medium to about 0.2 mM.

4,4⁰-Dihydroxybiphenyl (0.1 mM) was used as an inter-

nal standard. The samples were incubated at 37 ꢁC and

samples were taken at t = 0, 1, 3, 24, 48, 72, and 96 h,

and analyzed by HPLC.

5.1.43. 1-(8-(Propylthio)naphth-1-yl) b-^D-xylopyranoside

(11c). 8-(Propylthio)naphthalen-1-ol (3c) (32.4 mg,

0.15 mmol) and 2,3,4-tri-O-acetyl-b-^D-xylopyranoside

trichloroacetimidate (120.4 mg, 0.29 mmol) were sus-

pended in CH₂Cl₂(4.5 mL) and cooled to 0 ꢁC.

BF₃ÆOEt₂(4 lL, 0.03 mmol) was added and the mixture

was stirred for 18 min at 0 ꢁC. Excess Et₃N was added

and the solution was concentrated and ﬁltered through

SiO₂(Toluene–EtOAc 4:3). The crude product was

5.2. Biology

The human bladder carcinoma cell-line T24 and human

lung ﬁbroblasts, HFL-1, were obtained from ATCC.

Regular cell culture media, ^L-glutamine, penicillin–

streptomycin, trypsin, and donor calf serum were ob-

tained from Life Technologies. Dulbecco’s modiﬁed Ea-

gles medium (DMEM) and Ham’s F-12 medium were

purchased from Sigma. Na₂³⁵SO₄(1310 Ci/mmol) was

obtained from Amersham International. Epidermal

growth factor was purchased from Genzyme and crystal

violet from Merck. The prepacked Superose 6 HR 10/30

and Dextran T-500 were from Pharmacia-LKB, and

DE-53 DEAE–cellulose was from Whatman. Water

for HPLC-analysis was from a Millipore Milli-Q system.

deprotected and puriﬁed as for 11a. Gave 11c as a white

20

amorphous solid (46.6 mg, 90%). ½aꢀ ꢁ 124:1^ꢂ(c 0.23,

D

MeOH). ¹³C NMR (CD₃OD–CDCl₃1:1): d 155.9,

137.5, 134.9, 127.0, 126.7, 126.0, 124.7, 124.2, 123.7,

111.0, 104.6, 76.8, 74.5, 70.7, 66.9, 35.6, 22.2, 14.2.

HRMS calcd for C₁₈H₂₂O₅SNa (M+Na): 373.1086,

found: 373.1078.

5.1.44. 1-(3-(Propylthio)naphth-2-yl) b-^D-xylopyranoside

(11d). 1-(3-(Propylthio)naphth-2-yl) 2,3,4-tri-O-acetyl-

b-^D-xylopyranoside (10d) (823 mg, 1.73 mmol) was

deprotected and puriﬁed as for 11a. Gave 11d as a white

20

amorphous solid (573 mg, 95%). ½aꢀ ꢁ 66:9^ꢂ(c 0.21,

5.2.1. Cell culture and radiolabeling. Cells were cultured

as monolayers in Modiﬁed Earles Medium (MEM) sup-

plemented with 10% (v/v) fetal bovine serum, 2 mM ^L-

glutamine, penicillin (100 U/mL), and streptomycin

(100 lg/mL) in an incubator with humidiﬁed atmo-

sphere and 5% CO₂at 37 ꢁC. Conﬂuent cells were prein-

cubated for 1 h in low-sulfate, MgCl₂-labeling medium

supplemented with 2 mM glutamine. The pre-incubation

medium was replaced by fresh medium containing

50 mCi/mL of [³⁵S]sulfate and 0.1 mM xyloside. 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).

D

MeOH). ¹³C NMR (CD₃OD): d 154.2, 133.9, 131.4,

129.5, 127.9, 127.7, 127.6, 126.8, 125.8, 111.1, 102.8,

77.6, 74.6, 71.0, 66.9, 34.7, 23.2, 13.9. HRMS calcd for

C₁₈H₂₂O₅SNa (M+Na): 373.1086, found: 373.1088.

5.1.45. 1-(6-(Propylthio)naphth-2-yl) b-^D-xylopyranoside

(11e). 1-(6-(Propylthio)naphth-2-yl) 2,3,4-tri-O-acetyl-b-

D-xylopyranoside (10e) (819 mg, 1.72 mmol) was

deprotected and puriﬁed as for 11a. Gave 11e as a white

20

amorphous solid (590 mg, 98%). ½aꢀ ꢁ 34:4^ꢂ(c 0.26,

D

MeOH). ¹³C NMR (CD₃OD): d 156.6, 134.1, 133.7,

131.6, 129.6, 129.3, 128.7, 128.1, 120.6, 111.9, 102.9,

77.7, 74.8, 71.1, 67.0, 36.6, 23.6, 13.6. HRMS calcd for

C₁₈H₂₂O₅SNa (M+Na): 373.1086, found: 373.1096.

5.2.2. Isolation of xyloside-primed radiolabeled GAG.

The procedures have been described in detail previ-

ously.³[³⁵S]Sulfate labeled polyanionic macromolecules

were isolated from the culture medium by ion exchange-

chromatography on DEAE–cellulose at 4 ꢁC. Samples

were mixed with 1.3 vol of 7 M urea, 10 mM Tris, pH

7.5, 0.1% Triton X-100, and 10 mM NEM, and passed

over a 1 mL-column of DEAE equilibrated with 6 M

urea, 0.5 M NaOAc, pH 5.8, 5 lg/mL ovalbumin, and

0.1% Triton X-100. After sample application, the col-

umns were washed successively with 10 mL-portions of

(a) equilibration buﬀer (see above), (b) 6 M urea,

10 mM Tris, pH 8.0, 5 lg/mL ovalbumin, 0.1% Triton

X-100, and (c) 50 mM Tris, pH 7.5. Bound material

5.1.46. 1-(7-(Propylthio)naphth-2-yl) b-^D-xylopyranoside

(11f). 1-(7-(Propylthio)naphth-2-yl) 2,3,4-tri-O-acetyl-b-

D-xylopyranoside (10f) (820 mg, 1.72 mmol) was

deprotected and puriﬁed as for 11a. Gave 11f as a white

20

amorphous solid (592 mg, 98%). ½aꢀ ꢁ 29:5^ꢂ(c 0.33,

D

MeOH). ¹³C NMR (CD₃OD): d 157.2, 136.7, 136.3,

130.2, 129.3, 129.0, 126.4, 126.2, 119.5, 111.0, 102.8,

77.7, 74.8, 71.1, 67.0, 35.9, 23.5, 13.7. HRMS calcd for

C₁₈H₂₂O₅SNa (M+Na): 373.1086, found: 373.1076.

5.1.47. HPLC of naphthoxylosides. High-performance li-

quid chromatography was run on a Hewlett-Packard

Series II 1090 Liquid Chromatograph and a YMC

5298

M. Jacobsson et al. / Bioorg. Med. Chem. 15 (2007) 5283–5299

was eluted with 5 · 1 mL 4 M guanidine–HCl, 50 mM

NaOAc, pH 5.8, and 5 lg/mL ovalbumin. Radioactive

fractions were pooled, precipitated with 5 vol of 95%

ethanol overnight at ꢁ20 ꢁC using 100 lg of dextran

as carrier. After centrifugation in a Beckman JS-7.5 at

4000 rpm and 4 ꢁC for 45 min material was dissolved

in 4 M guanidine–HCl, 50 mM NaOAc, pH 5.8, and free

xyloside primed GAG chains were separated from PG

by hydrophobic interaction chromatography on Octyl-

Sepharose followed by gel permeation FPLC on Supe-

rose 6. Radioactivity was determined in a b-counter.

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Article Doi

Effects of oxygen-sulfur substitution on glycosaminoglycan-priming naphthoxylosides

DOI: 10.1016/j.bmc.2007.05.008

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