Synthesis and biological testing of thioalkane- and thioarene-spaced bis-β-d-glucopyranosides

Article abstract of DOI:10.1016/j.bmc.2009.01.010

A three-step synthesis of bis-β-d-glucopyranosides containing thioalkane or thioarene spacers of different length and flexibility is described. The key-step reaction allows an easy modulation of final saccharidic products so that a library of molecules wi

Full text of DOI:10.1016/j.bmc.2009.01.010

Bioorganic & Medicinal Chemistry 17 (2009) 1456–1463
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological testing of thioalkane- and thioarene-spaced
bis-b-D-glucopyranosides
b
b
Maria C. Aversa a, Anna Barattucci a, Paola Bonaccorsi a, , Francesca Marino-Merlo , Antonio Mastino ,
*
Maria T. Sciortino b
a Dipartimento di Chimica Organica e Biologica, Università degli Studi di Messina, Salita Sperone 31 (vill. S. Agata), 98166 Messina, Italy
b Dipartimento di Scienze della Vita, Sezione di Scienze Microbiologiche, Genetiche e Molecolari, Università degli Studi di Messina, Salita Sperone 31 (vill. S. Agata), 98166 Messina, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 July 2008
Revised 8 January 2009
Accepted 10 January 2009
Available online 15 January 2009
A three-step synthesis of bis-b-D-glucopyranosides containing thioalkane or thioarene spacers of different
length and flexibility is described. The key-step reaction allows an easy modulation of final saccharidic
products so that a library of molecules with different glycosidic residues and spacers can be obtained.
Two of the new thioarene-spaced bis-b-D-glucopyranosides endow with a specific cytotoxic potential.
A more detailed investigation of one of the two compounds ascertains that this effect is attributable to
induction of cell death by apoptosis.
Keywords:
Apoptosis
Ó 2009 Elsevier Ltd. All rights reserved.
Biological tests
Sulfenic acids
Thio-bis-glucopyranosides
1. Introduction
flexibility and length between terminal mannopyranoside resi-
dues.3 Recently, the synthesis of multivalent arrays of mannose
A number of biological processes are controlled by protein-car-
bohydrate interactions that function as transfer of information be-
tween cell surface and cell substrata or in cell–cell recognition
events.1 In particular, carbohydrates have been implicated in fun-
damental processes that regulate cellular growth and death, or
cause the onset of infection, for two fundamental reasons: (i) most
cells are covered by carbohydrates and (ii) carbohydrates are capa-
ble of forming many different combinatorial structures, each one
carrying a specific biological message, from relatively small num-
bers of sugar units. This fundamental and eclectic role of carbohy-
drates has inspired the synthesis of glycoconjugates to be used as
potential therapeutics or useful tools for the understanding of
binding specificity and other properties of carbohydrate recogni-
tion events. Tailored small molecules have been synthesized with
the aim of developing libraries of glycoconjugate inhibitors. A
calix[4]arene has been used as scaffold for the introduction of
sugar units with the idea of mimicking, to some extent, a small
portion of the cell surface presenting glycosylated residues on
the exterior of the lipophilic region.2 A significant investigation
has been conducted on the binding and cross-linking interactions
of Con A and Dioclea grandiflora lectin (DGL) with a series of
divalent carbohydrates that possess spacer groups with increasing
mono- and disaccharides was described as potential anti-bacteria
infective agents.4 Finally, a series of b-
D
-manno- and b- -glucopyr-
D
anosides have been synthesized that show inhibitory activity for
the HIV-1 protease, providing a basis for the development of more
potent carbohydrate based peptidomimetic inhibitors.5 These
investigations on small molecule inhibitors highlight how the con-
trol of molecular architecture and saccharide spacing can play a
significant role on the enhancement of the glycoconjugate-model
receptor binding activity.
In this work we describe a three-step synthesis of bis-b-D-gluco-
pyranosides containing thioalkane or thioarene spacers of different
lengths and flexibility and the results of biological tests conducted
on some of them. The adopted synthetic procedure allows an easy
modulation of final saccharidic products so that a library of mole-
cules with different glycosidic residues and spacers is achievable in
principle. The presence of sulfur atoms in the skeleton of glucodi-
saccharides offers the opportunity to vary the oxidation state of
sulfur atoms and to study the effect that this variation can play
on the biological profile of the corresponding thioglycoconjugates.
In order to investigate the relationship between molecular struc-
ture and biological response, we performed a preliminary screen-
ing of the effects of the newly synthesized thioalkane- and
thioarene-spaced bis-b-D-glucopyranosides on cell viability. More-
over we focused our attention on the ability of the synthesized
compounds to induce apoptotic cell death, taking into account
* Corresponding author. Tel.: +39 090 6765187; fax: +39 090 393895.
E-mail address: paola.bonaccorsi@unime.it (P. Bonaccorsi).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.010

M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
1457
the pivotal role of apoptosis regulation in both the controlled
expansion and removal of immune cells and cancer progression
and therapy.6
cially available bis-thiols.9 The later compounds were considered
as suitable starting products for the synthesis of precursors of
bis-sulfenic acids, such as 20–23 in Scheme 2, to be generated
in situ in the presence of 2-propynyl tetra-O-acetyl-b-D-glucopy-
ranoside (24).10 For instance the double syn-addition of the bis-sul-
fenic acid 20 onto the triple bond of the monosaccharide 24 should
2. Chemistry
provide the thioarene-spaced bis-b-
proach was considered attractive for entry to bis-b-
sides also because the starting products are less expensive with
respect to diynes such as 6 or 7.
D
-glucopyranoside 25. This ap-
-glucopyrano-
As part of a programme centred on the study of the syn-addi-
tion of sulfenic acids on the triple bond of suitable unsaturated
molecules, we forecast that the reaction of glycosulfenic acids,
such as 3, with terminal diynes, such as 4 and 5 (Scheme 1), could
represent an efficient procedure to obtain thioglycoconjugates
with two sugar moieties separated by alkane or arene spacers.
The possibility of obtaining the desired products in three steps,
starting from various monosaccharide derivatives and commer-
cially available, structurally different, unsaturated linkers, would
give easy access to a library of molecules to submit for biological
testing.
D
1,3-Benzenedithiol (8), 1,3-propanedithiol (10), and 1,5-pen-
tanedithiol (11) were involved in the nucleophilic addition of
the two sulfur functions to acrylonitrile in the presence of a cat-
alytic quantity of Triton B, whereas diethyl isopropylidenemalo-
nate was used as electrophile acceptor towards (1,10-biphenyl)-
4,40-dithiol (9) after having experienced that the double addition
of 9 to acrylonitrile leads to intractable sulfenic acid precursors.
Subsequent controlled oxidation of disulfides 12–15 allowed the
formation of bis-sulfoxides 16–19 that represent the precursors
of the corresponding sulfenic acids 20–23. Thermolysis of 16 in
1,2-dichloroethane (95 °C), of 18 and 19 in toluene (110 °C),
and of 17 in DCM, in the presence of monosaccharide 24, pro-
2,3,4,6-Tetra-O-acetyl-1-thio-b-D-glucopyranose (1) was used
as starting material for the preparation of epimeric sulfoxide pre-
cursors 2 (RS/SS 1:2) of sulfenic acid 3, following a well-assessed
synthetic pathway (Scheme 1).7 In a series of attempts, where re-
agent ratios and reaction times were changed, the epimeric
mixture of sulfoxides 2 was thermolyzed in dichloromethane
(DCM) at reflux in the presence of 1,6-heptadiyne (4) or 1,7-octa-
diyne (5). The access to thioalkane- and thioarene-spaced bis-b-
vided sulfurated bis-b-D-glucopyranosides 25–28. The diastereo-
meric mixture of glucosulfoxides 25 was further oxidized with
m-CPBA to the unique bis-sulfone 29 in order to investigate by
comparison if the oxidation state of sulfur atom plays any signif-
icant role in the biological behavior of the compounds under
study.
D
-glucopyranosides via this method proved problematic, and this
approach was successful only in the preparation of monosaccha-
rides 6 and 7, possessing an unsaturation qualified for a further
attack by glucosulfenic acid 3. Compounds 6 and 7 were obtained
in low yield (see experimental section) as 2:1 mixtures each of sul-
fur epimers. In both cases the column chromatography allowed the
isolation of both epimers, whose configuration was assigned taking
into account the role of the exo-anomeric effect.8 Although the
methodology did not offer a direct entry to thioalkane- and thioa-
rene-spaced bis-b-D-glucopyranosides as desired, monosaccharides
6 and 7 were subjected to a second thermolysis in the presence of
sulfoxides 2 but, even in this case, no traces of the corresponding
thioalkane- and thioarene-spaced bis-b-D-glucopyranosides were
detected.
3. Biological results and discussion
The relationship between molecular structure and biological
activity of sulfurated bis-b-D-glucopyranosides 25–29 was inves-
tigated firstly by comparing the toxic effect exerted by some of
the synthesized compounds using a conventional viability assay.
Sugars 25–29 were dissolved in DMSO (10 mM). These mother
solutions were diluted in RPMI-1640 medium supplemented
with 5% FBS, 2 mM L-glutamine and penicillin–streptomycin to
reach 10-fold increasing concentrations (1, 10 and 100
lM).
The obtained samples were added to 2 ꢀ 104 U937 monocytoid
The failure of this approach to provide the desired molecules
was overcome by applying the sulfenic acid chemistry to commer-
cells in 100
ll total volume and incubated at 37 °C in a CO2
incubator. As a control, the same cells were exposed to the vehi-
cle alone (DMSO), in amounts corresponding to those employed
for dissolving the compounds. The percentage of dead cells was
OAc
evaluated after 24
h incubation by microscopy analysis of
OAc
O
OAc
1
AcO
AcO
O
a)
b)
SO
OAc
EtO2C
stained cells using a standard trypan blue exclusion test. No sig-
nificant change of dead cell percentage was detected with any
compound at the lower concentrations (1 and 10
AcO
AcO
SH
CO2Et
lM) assayed.
2
At 100 M concentration, disaccharides 26 and 28 induced low
l
levels of toxicity (Fig. 1B and D), without modifying the absolute
number of cells. Compound 27 was slightly more cytotoxic than
compounds 26 and 28 at the same concentration (Fig. 1C). The
obtained results allowed us to associate the highest, specific
cytotoxic potential to compound 29 (Fig. 1E) and compound 25
(Fig. 1A), both characterized by a single central ring linking the
two disaccharide units. Moreover, the cytotoxic potential of com-
pounds 25, 27 and 29, sharing a similar three carbon linker
length, was associated with a decrease in the absolute number
c)
OAc
O
OAc
AcO
SOH
AcO
3
4 n = 1
5 n = 2
c)
n
of cells at 100 lM concentration. The observed biological activity
appears not significantly affected by the oxidation state of the
sulfur atoms.
OAc
O
OAc
O
We then focused our attention on the effects on cell growth and
apoptosis of sugar 25 as a model for this family of compounds. To
this purpose, the biological activity of 25 was investigated deeper
by performing further experiments at a narrow range of concentra-
6 n = 1
7 n = 2
AcO
AcO
S
n
Scheme 1. Reagents and conditions: (a) Diethyl isopropylidenemalonate, Triton B,
THF, ꢁ78 °C; (b) m-CPBA, DCM, ꢁ78 °C; (c) DCM, reflux.
tions (from 10 to 75 lM). The effects of 25 on cell metabolic activity

1458
M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
R
R
R1
R2
R
R
R
R1
R2
R1
R2
R
R1
R2
R
m-CPBA
R1
R2
R
R
R
HS Spacer
SH
SO Spacer SO
S
Spacer
S
Triton B
8-11
16-19
12-15
Δ
Spacer:
a
b
HOS Spacer
SOH
OAc
20-23
OAc
AcO
AcO
O
O
OAc
c n = 1
d n = 2
24
Δ
n
OAc
O
AcO
AcO
AcO
O
OAc
OAc
O
OAc
O
SO Spacer
SO
25, 26-28
m-CPBA
OAc
OAc
AcO
AcO
O
AcO
O
O
OAc
OAc
O
SO2
SO2
OAc
29
Thiol Spacer
R
H
Me
R1
R2
CN
CO2Et CO2Et
Sulfide Sulfoxide Sulfenic acid Glucodisaccharide (yield %)
8
a
b
c
d
25 (40)
26 (30)
27 (20)
28 (40)
12
13
14
15
16
17
18
19
20
21
22
23
H
9
10
11
H
H
H
CN
CN
H
Scheme 2.
were first measured by determining oxygen burst using the MTS
assay in the monocytic cell line U937 and in the lymphocytic cell
line Molt-3. The results, shown in Figure 2A and B, respectively,
represent the mean values of three determinations for each con-
centration tested. A highly significant inhibitory effect of 25 on cell
metabolic activity was observed in both cell lines at the highest
concentration assayed, in agreement with preliminary experi-
ments indicating a cytotoxic potential for this compound. Surpris-
ingly, lower concentrations of 25 did not inhibit the cell metabolic
activity, but rather stimulated it both in U937 and Molt-3 cells,
although in the latter ones the differences between control and
treated cells were not significant. The absolute number of viable
cells were, then, evaluated at 24 h incubation in cultures undergo-
ing treatment with different concentrations of 25, in order to ascer-
tain whether this effect could be associated to the thioarene-
Moreover the interest in evaluating the influence that different
significant moieties of 25 could exert on its cytotoxic potential
prompted us to treat U937 cells also with compounds 8, 16, and
1,2,3,4,6-penta-O-acetyl-b-D-glucopyranose (PAGP). 1,3-Benzene-
dithiol (8) corresponds to the aromatic central ring, 3,30-(m-phen-
ylenedisulfinyl)dipropionitriles 16 mimic the rigid core with
flexible arms, both constituting with the sugar moiety the molecule
of 25 in its whole. After 24 h incubation, apoptosis was evaluated by
microscopy analysis followed by staining with acridine orange. Ex-
cept at the lower concentration of 10 lM, compound 25 was found
to induce a significantly higher level of apoptosis, in a dose-response
fashion, in comparison with the vehicle alone in U937 cells as well as
in Molt-3 cells (Fig. 4A and B). Actually, the latter cells appeared to be
much more sensitive than the former ones to apoptosis induction at
the highest concentration of 25 assayed. No significant difference
was observed between apoptosis values detected in samples treated
with 8, 16 or PAGP, all of which representing the significant features
of the 25 skeleton, and vehicle treated samples (Fig. 4A). These
results suggest that apoptosis is the main form of cell death associ-
ated with the cytotoxic potential of compound 25 and that the
specific induction of apoptosis is a consequence of the interactions
of the whole molecular structure of the thiglucodisaccharide under
study with the cells rather than the effect of one of the significant
moieties that constitute compound 25.
spaced bis-b-D-glucopyranoside influence on cell growth. Results
indicate that the treatment with 25 significantly stimulated the
growth of both U937 (Fig. 3A) and Molt-3 (Fig. 3B) cells at concen-
trations ranging from 25 to 50
lM, while the concentration of
75 M caused a dramatic decrease of total viable cell number.
l
These results paralleled those obtained in the MTS assay enforcing
their reliability, due to the comprehensible association between
the effects of 25 on the conversion of MTS into the formazan prod-
uct, accomplished by dehydrogenase enzymes in metabolically
active cells and the actual quantity of viable cells.
We then investigated whethercell toxicity exerted by 25could be
attributable to its capability of inducing apoptotic death. For detec-
tion of apoptosis, U937 and Molt-3 cells were treated either with the
4. Conclusion
In conclusion, we have described an easy synthetic approach to
vehicle alone or with 25 at concentrations ranging from 10 to 75 lM.
a new class of thioalkane- and thioarene-spaced bis-b-D-glucopyr-

M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
1459
Figure 1. Effects of sugars 25-29 on cell death in U937 cells. U937 cells were treated with control vehicle (DMSO) or with increasing concentrations of the different sugars. (A)
compound 25. (B) compound 26. (C) compound 27. (D) compound 28. (E) compound 29. The percentage of dead cells was evaluated after 24 h incubation and determined by
the trypan blue exclusion test. Each data point represents the mean and standard deviation from three different determinations. Comparison of the means between treated
samples and corresponding controls, by Student’s t-test for independent samples: 100
l
M, p < 0.001 for all compounds; all other comparisons, NS.
anosides. From a chemical point of view the key-step of the process
offers wide chances of modulating the synthesis due to the possi-
bility of changing both the sugar nature of the unsaturated accep-
tors and the sulfenic acid skeleton. The obtained data on the
biological activity of this new class of compounds indicate that
their effects greatly vary according to their molecular structure.
While most of the compounds showed very low levels of toxicity
even at high concentrations, sulfurated bis-b-D-glucopyranosides
with a central aromatic ring endowed with a specific cytotoxic
potential. In particular, a more detailed investigation performed
on compound 25, demonstrated that it was a good inductor of cell
death by apoptosis. Moreover, the unexpected observation that in
the range of 10–50 lM compound 25 stimulated the metabolic
activity and the cell growth, suggests that the induction of cell
death might underline an activation-induced cell death.11 The
obtained results open new perspectives in future investigations
on the synthesis of new compounds of this family and on the com-
prehension of their biological activities.
5. Experimental
5.1. Chemistry
5.1.1. Materials and methods
Figure 2. Effect of sugar 25 on metabolic activity. (A) U937 cells were grown in
96-well microtiter plates and exposed to different concentrations of control
vehicle (DMSO) or compound 25 for 24 h, before cell metabolic activity was
quantified by the MTS assay. Results are expressed as absorbance values measured
at 490 nm. Each data point represents the mean and standard deviation from three
different determinations. Comparison of the means between treated samples and
Solvents were purified according to standard procedures. All
reactions were monitored by TLC on commercially available pre-
coated plates (Aldrich silica gel 60 F 254) and the products were
visualized with vanillin [1 g of 4-hydroxy-3-methoxybenzaldehyde
dissolved in MeOH (60 mL) and conc. H2SO4 (0.6 mL)]. Silica gel
used for column chromatography was Aldrich 60, 230–400 mesh
ASTM, 0,040–0,063 mm. Melting points were recorded on a micro-
scopic apparatus and are uncorrected. 1H and 13C NMR spectra
were recorded on a Varian Mercury 300 spectrometer at 300 and
75 MHz respectively in CDCl3 solutions. J values are given in Hz;
corresponding controls, by Student’s t-test for independent samples: 10
lM, NS;
25 M, p = 0.013; 50 M, p = 0.047; 75 M, p = 0.003. (B) Molt-3 cells were
l
l
l
processed as described in (A) for assaying the effect of 25 on metabolic activity.
Each data point represents the mean and standard deviation from three different
determinations. Comparisons of the means between treated samples and corre-
sponding controls, by Student’s t-test for independent samples: 75
all other comparisons, NS.
lM, p = 0.001;

1460
M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
Figure 4. Effect of compound 25 on apoptosis. (A) U937 cells were exposed for 24 h
to different concentrations of control vehicle (DMSO), 1,2,3,4,6-penta-O-acetyl-b-
Figure 3. Effect of sugar 25 on cell growth. (A) U937 cells were grown in 96-well
microtiter plates and exposed to different concentrations of control vehicle
(DMSO) or compound 25 for 24 h, before cell growth was assayed by evaluating
the total number of viable cells per well using the trypan blue exclusion test. Each
data point represents the mean and standard deviation from three different
determinations. Comparisons of the means between treated samples and corre-
D-
glucopyranose (PAGP), compound 8, compound 16 and compound 25. Apoptosis
was evaluated by fluorescence microscopy after staining of the cells with acridine
orange. Percentage apoptosis = number of apoptotic cells / total cells counted ꢀ
100%. Each data point represents the mean and standard deviation from three
different determinations. Multiple comparisons, by Tukey’s honestly significant
sponding controls, by Student’s t-test for independent samples: 10
lM, NS; 25 lM,
difference (HSD) test: 10 lM, NS among all groups; all other concentrations, NS
p = 0.026; 50 M, p = 0.004; 75 M, p = 0.006. (B) Molt-3 cells were processed as
l
l
among groups except for 25 versus all corresponding groups, p < 0.001. (B) Molt-3
cells were exposed for 24 h to different concentrations of control vehicle (DMSO)
and compound 25. Apoptosis was evaluated as described in (A). Each data point
represents the mean and standard deviation from three different determinations.
Comparisons of the means between treated samples and corresponding controls, by
described in (A) for assaying the effect of 25 on cell growth. Each data point
represents the mean and standard deviation from three different determinations.
Comparisons of the means between treated samples and corresponding controls,
by Student’s t-test for independent samples: 10
p = 0.001; 75 M, p = 0.003.
lM, NS; 25 lM, p < 0.001; 50 lM,
l
Student’s t-test for independent samples: 10
p = 0.013; 75 M, p = < 0.001.
lM, NS; 25 lM, p = 0.018; 50 lM,
l
the attributions are supported by Attached Proton Test (APT).
Proton and carbon nuclei, marked with (0) pertain to the monosac-
charide residues when the sugar function is stated as substituent in
the compound nomenclature. IR spectra were recorded in CHCl3
solution on a Perkin-Elmer FT-IR Spectrum BX.
(AB dd, J5 ,6 B 2.4, HB-60), 3.77 (ddd, H-50), 2.6–1.8 (m, H2-3,4,5, H-
7), 2.09, 2.03, 2.02 and 2.01 [4 s, 4 ꢀ C(O)Me]. Elemental analysis
calcd (%) for C21H28O10S (472.50): C 53.38, H 5.97; found: C
53.29, H 5.84.1H NMR of (RS)-6: d 5.90 (d, J1A,1B 1.2, HA-1), 5.78
0
0
(d, HB-1), 5.4–5.0 (m, H-20,30,40), 4.35 (d, J1 ,2 9.7, H-10), 4.3–4.1
(split AB system, H2-60), 3.8–3.7 (m, H-50), 2.6–1.8 (m, H2-3,4,5,
H-7), 2.10, 2.09, 2.04 and 2.01 [4 s, 4 ꢀ C(O)Me]. Elemental analysis
calcd (%) for C21H18O10S (472.50): C 53.38, H 5.97; found: C 53.31,
H 5.80.
0
0
5.1.2. General procedure A for the synthesis of 2-[(2,3,4,6-tetra-
O-acetyl-b-D-glucopyranosyl)sulfinyl]-1-alkenynes 6 and 7
A solution of sulfoxides 29 (1,16 g, 2,00 mmol) and commercial
ethynyl acceptor 4 or 5 (11.00 mmol) in DCM (3 mL) was main-
tained at reflux. When the reaction appeared complete by TLC (dis-
appearance of starting sulfoxides required about one night), the
solvent was removed under reduced pressure and the reaction
crude was purified by flash column chromatography on silica gel
(petrol/EtOAc 8:2).
5.1.2.2. 2-[(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)sulfinyl]-
1-octen-7-ynes 7. Epimeric mixture 7 was prepared from sulfox-
ides 2 and 1,7-octadiyne (5), following procedure A. A subsequent
column chromatography on silica gel (petrol/EtOAc 2:8) allowed
the separation of the two sulfinyl sulfur epimers 7,10 as colorless
oils (25% total yield). The more mobile (SS)-7 was obtained in 2:1
ratio with respect to the less mobile (RS)-7. 1H NMR of (SS)-7: d
5.1.2.1. 2-[(2,3,4,6-Tetra-O-acetyl-b-D-glucopyranosyl)sulfinyl]-
1-hepten-6-ynes 6. Epimeric mixture 6 was prepared from sulf-
oxides 2 and 1,6-heptadiyne (4), following procedure A. A subse-
quent column chromatography on silica gel (petrol/EtOAc 5:5)
allowed the separation of the two sulfinyl sulfur epimers 6,10 as
colorless oils (24% total yield). The more mobile (SS)-6 was ob-
tained in 2:1 ratio with respect to the less mobile (RS)-6. 1H NMR
0
0
0
0
5.83 (d, J1A,1B 0.9, HA-1), 5.72 (d, HB-1), 5.33 (dd, J1 ,2 9.3, J2 ,3 9.1,
H-20), 5.28 (dd, J3 ,4 9.4, H-30), 5.08 (dd, J4 ,5 9.8, H-40), 4.44 (d, H-
0
0
0
0
10), 4.26 (AB dd, J5 ,6 A 4.7, J6 A,6 B 12.6, HA-60), 4.17 (AB dd, J5 ,6 B
2.4, HB-60), 3.76 (ddd, H-50), 2.5–1.5 (m, H2-3,4,5,6, H-8), 2.09,
2.03, 2.01 and 2.00 [4 s, 4 ꢀ C(O)Me]. Elemental analysis calcd
(%) for C22H30O10S (486.53): C 54.31, H 6.22; found: C 54.26, H
6.12. 1H NMR of (RS)-7: d 5.87 (broad s, HA-1), 5.77 (broad s,
0
0
0
0
0
0
0
0
of (SS)-6: d 5.85 (d, J1A,1B 0.9, HA-1), 5.73 (d, HB-1), 5.34 (dd, J1 ,2
9.1, J2 ,3 8.8, H-20), 5.28 (dd, J3 ,4 9.2, H-30), 5.09 (dd, J4 ,5 9.8, H-
0
0
0
0
0
0
40), 4.45 (d, H-10), 4.26 (AB dd, J5 ,6 A 4.6, J6 A,6 B 12.6, HA-60), 4.17
HB-1), 5.40 (dd, J1 ,2 9.3, J2 ,3 9.2, H-20), 5.33 (dd, J3 ,4 9.4, H-30),
0
0
0
0
0
0
0
0
0
0

M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
1461
5.10 (dd, J4 ,5 9.5, H-40), 4.31 (d, H-10), 4.3–4.1 (split AB system,
H2-60), 3.8–3.7 (m, H-50), 2.6–1.6 (m, H2-3,4,5, H-7), 2.08, 2.07,
2.05 and 2.03 [4 s, 4 ꢀ C(O)Me]. Elemental analysis calcd (%) for
C22H30O10S (486.53): C 54.31, H 6.22; found: C 54.15, H 6.19. 13C
NMR of (SS)-7: 170.4, 170.2, 169.2 and 169.1 [4 ꢀ C(O)Me], 149.9
(C-2), 118.8 (C-1), 91.3 (C-10), 83.8 (C-7), 76.6, 73.7, 67.4 and
67.1 (C-20,30,40,50), 68.9 (C-8), 61.5 (C-60), 27.7, 27.6 and 26.8 (C-
3,4,5), 20.71, 20.68, 20.54 and 20.51 [4 ꢀ C(O)Me], 18.1 (C-6). 13C
NMR of (RS)-7: 170.43, 170.40, 169.1 and 168.8 [4 ꢀ C(O)Me],
148.5 (C-2), 118.6 (C-1), 88.3 (C-10), 83.7 (C-7), 76.8, 73.8, 67.7
and 67.3 (C-20,30,40,50), 68.9 (C-8), 61.9 (C-60), 28.0, 27.6 and 26.7
(C-3,4,5), 20.7, 20.60, 20.57 and 20.5 [4 ꢀ C(O)Me], 18.0 (C-6).
(q, Jvic 7.2, 4 ꢀ OCH2), 3.59 (s, 2 ꢀ H-2), 1.54 (s, 2 ꢀ CMe2), 1.31
(t, 4 ꢀ CH2Me). Elemental analysis calcd (%) for C32H42O8S2
(618.80): C 62.11, H 6.84; found: C 61.96, H 6.82.
0
0
5.1.5. General procedure C for the synthesis of disulfoxides 16
and 17, and disulfone 29
m-CPBA (80%) was dissolved in DCM (10 mL/m-CPBA mmol)
and added dropwise to a solution of the disulfide or disulfoxide
in the same volume of DCM at ꢁ78 °C (1 mol of m-CPBA for every
molar site to be oxidized in the substrate). When the reaction ap-
peared complete by TLC (EtOAc/petrol 8:2), a 10% water solution of
Na2S2O3 was added. Almost all experiments performed were con-
cluded just after finishing the addition of the oxidant. The sepa-
rated organic layers was washed twice with a saturated solution
of NaHCO3 and then twice with brine. Evaporation of the solvent
gave the expected sulfoxide or sulfone.
5.1.3. General procedure B for the synthesis of 3,30-dithiodipro-
pionitriles 12, 14, and 15
To a stirred solution of the bis-thiol (2.29 mmol) in anhyd THF
(6 mL) at ꢁ78 °C, in an inert atmosphere, Triton B (40 wt. % solu-
tion in MeOH, 0.15 mL, 0.33 mmol) and, after 10 min, acrylonitrile
(0.30 mL, 4.58 mmol) were added. The mixture was allowed to
reach rt spontaneously, and when the reaction appeared complete
by TLC (petrol/EtOAc 5:5), it was quenched by water addition. The
crude product was extracted twice with Et2O (10 mL). The com-
bined organic layers were washed with brine and dried over
Na2SO4. After filtration of the inorganic solid, the solvent was re-
moved under reduced pressure.
5.1.5.1. 3,30-(m-Phenylenedisulfinyl)dipropionitriles 16. Disul-
fide 12 was oxidized following the general procedure C. Disulfox-
ides 16 (meso/racemate 1:1) were obtained as a yellow oil
(0.53 g, 1.89 mmol, 95% yield) not needing any purification before
its involvement in the next reaction steps. 1H NMR of 1:1 meso/
racemate mixture: d 8.0–7.8 (m, ArH), 3.4–2.6 (m, 2 ꢀ H2-2,3). Ele-
mental analysis calcd (%) for C12H12N2O2S2 (280.53): C 51.41, H
4.31; found: C 51.37, H 4.25.
5.1.3.1. 3,30-(m-Phenylenedithio)dipropionitrile (12). Commer-
cial dithiol 8 was subjected to general procedure B. The reaction
crude was purified by flash column chromatography on silica gel
(petrol/EtOAc 9:1). Compound 12 was isolated as a pale yellow
oil (0.51 g, 2.05 mmol, 90% yield). 1H NMR: d 7.5–7.3 (m, ArH),
3.16 (t, J2,3 7.2, 2 ꢀ H2-3), 2.64 (t, 2 ꢀ H2-2). Elemental analysis
calcd (%) for C12H12N2S2 (248.36): C 58.03, H 4.87; found: C
57.89, H 4.72.
5.1.5.2. Tetraethyl 1,10-[1,10-biphenyl-4,40-diyldi(sulfinyl)]di-[(1-
methyl)ethyl]propanedioates 17. Disulfide 13 was oxidized fol-
lowing the general procedure C. Disulfoxides 17 (meso/racemate
1:1) were obtained in a quantitative yield as an oil not needing
any purification before its prompt involvement in the next reaction
step. 1H NMR of diastereomeric mixture: d 7.77 (m, ArH), 4.3–4.2
(m, 4 ꢀ OCH2), 3.78 (s, 2 ꢀ H-2), 1.4–1.3 (m, 2 ꢀ CMe2 e 4 ꢀ CH2Me).
5.1.5.3.
2,20-(m-Phenylendisulfonyl)di-2,20-propenyl bis-b-
D-
5.1.3.2. 3,30-(Propane-1,3-diyldithio)dipropionitrile (14). Com-
mercial dithiol 10 was subjected to general procedure B. Com-
pound 14 was obtained, as a yellow oil (0.48 g, 2.24 mmol), in
quantitative yield, not needing of further purification. 1H NMR: d
2.8–2.6 (m, 2 ꢀ CH2SCH2CH2CN), 1.91 (quintuplet, Jvic 7,0,
CH2CH2CH2). Elemental analysis calcd (%) for C9H14N2S2 (214.35):
C 54.31, H 6.22; found: C 54.26, H 6.12.
glucopyranoside bis-2,3,4,6-tetraacetate (29). Bis-sulfoxides 25
were oxidized following the general procedure C. Bis-sulfone 29
was purified by flash column chromatography (petrol/EtOAc 6:4
up to petrol/EtOAc 4:6) and obtained as a white solid (m.p. 65–
70 °C, 0.10 g, 0.10 mmol, 77% yield).1H NMR: d 8.36 (t, Jmeta 1.7,
H-200), 8.13 (dd, Jortho 7.7, H-400,600), 7.79 (t, H-500), 6.53 (d, Jgem 0.4,
2 ꢀ HA-30), 6.16 (d, 2 ꢀ HB-30), 5.19 (dd, J2,3 9.2, J3,4 9.5, 2 ꢀ H-3),
5.06 (dd, J4,5 9.9, 2 ꢀ H-4), 4.93 (dd, J1,2 8.2, 2 ꢀ H-2), 4.53 (d,
5.1.3.3. 3,30-(Pentane-1,5-diyldithio)dipropionitrile (15). Com-
mercial dithiol 11 was subjected to general procedure B. Com-
pound 15 was obtained, as a yellow oil (0.49 g, 2.03 mmol), in
91% yield, not needing of further purification. 1H NMR: d 2.8–2.6
(m, 2 ꢀ CH2SCH2CH2CN), 1.7–1.5 (m, CH2CH2CH2CH2CH2). 13C
NMR: 118.3 (2 ꢀ C-1), 32.0 [SCH2(CH2)3CH2S], 28.9, 27.7 and 27.6
[2 ꢀ C-3 e CH2(CH2)3CH2], 18.9 (2 ꢀ C-2). Elemental analysis calcd
(%) for C11H18N2S2 (242.40): C 54.50, H 7.48; found: C 54.32, H 7.21.
2 ꢀ H-1), 4.47 (d AB, J1 A,1 B 14.1, 2 ꢀ HA-10), 4.32 (d AB, 2 ꢀ HB-
10), 4.3–4.1 (m, 2 ꢀ H2-6), 3.69 (ddd, J5,6A 4.7, J5,6B 2.5, 2 ꢀ H-5),
2.09, 2.02 and 2.00 [3 s, 8 ꢀ C(O)Me]. 13C NMR: 170.6, 170.1,
169.4 and 169.3 [8 ꢀ C(O)Me], 145.6 (C-100,300), 141.0 (2 ꢀ C-20),
133.1 (C-400,600), 130.7 (C-500), 127.9 (2 ꢀ C-30), 127.7 (C-200), 100.0
(2 ꢀ C-1), 72.5, 72.0, 70.8 and 68.0 (2 ꢀ C-2,3,4,5), 65.1 (2 ꢀ C-10),
61.6 (2 ꢀ C-6), 20.7, 20.6 and 20.5 [8 ꢀ C(O)Me]. IR: mmax 1756
(CO) cmꢁ1. Elemental analysis calcd (%) for C0H50O24S2 (978.94):
C 49.08, H 5.15; found: C 48.97, H 5.05.
0
0
5.1.4. Tetraethyl [1,10-(1,10-biphenyl-4,40-diyldithio)]di-[(1-
methyl)ethyl]propanedioate (13)
5.1.6. General procedure D for the synthesis of disulfoxides 18
and 19
To
a stirred solution of the commercial thiol 9 (0.5 g,
2.29 mmol) in anhyd THF (10 mL) at ꢁ78 °C, in an inert atmo-
sphere, Triton B (40 wt. % solution in MeOH, 0.16 mL, 0.35 mmol))
and, after 10 min, diethyl isopropylidenemalonate (1.79 mL,
9.13 mmol) were added. The mixture was allowed to reach rt spon-
taneously, and when the reaction appeared complete by TLC
(petrol/EtOAc 5:5; disappearance of starting sulfoxides required
about one night), the solvent was removed under reduced pres-
sure. The reaction crude was purified by column chromatography
(petrol/EtOAc 9:1). Compound 13 was obtained as a yellow oil
(0.64 g, 1.03 mmol) in 45% yield. 1H NMR: d 7.70 (half A2B2 system,
Jortho 8.3, ArH-2,20,6,60), 7.60 (half A2B2 system, ArH-3,30,5,50), 4.25
m-CPBA carefully dried over P2O5 (90%, 1.17 g, 6.10 mmol) was
dissolved in DCM (40 mL) and slowly added to a solution of an
equimolar amount of the disulfide (3.05 mmol) in DCM (20 mL)
at ꢁ78 °C. The reaction mixture was allowed to reach room tem-
perature and stirred for 2 h. Anhydrous KF (1.5 g) was added and
the mixture stirred overnight. After filtration, the solvent was
evaporated under reduced pressure.
5.1.6.1. 3,30-(Propane-1,3-diyldisulfinyl)dipropionitriles
18. Disulfide 14 was oxidized following the general procedure D.
Disulfoxides 18 (meso/racemate 1:1) were obtained as a rose-colored

1462
M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
solid (0.45 g, 1.83 mmol, 60% yield) not very soluble in CHCl3. 1H NMR
of 1:1 meso/racemate mixture: d3.1–2.9 (m, 2 ꢀ CH2SCH2CH2CN), 2.5–
2.4 (m, CH2CH2CH2). Elemental analysis calcd (%) for C9H14N2O2S2
(246.35): C 43.88, H 5.73; found: C 43.62, H 5.71.
5.1.10. 2,20-[Pentane-1,5-diyldi(sulfinyl)]di-2,20-propenyl bis-b-
D-glucopyranoside bis-2,3,4,6-tetraacetates 28
A solution of bis-sulfoxides 19 (0.34 g, 1.24 mmol) and the ethy-
nyl acceptor 24 (1.45 g, 3.73 mmol) in toluene (6 mL) was main-
tained at reflux. When the reaction appeared complete by TLC
(disappearance of starting sulfoxides required about 4 h), the sol-
vent was removed under pressure and the reaction crude was puri-
fied by flash column chromatography on silica gel (petrol/EtOAc
5:5 up to EtOAc/MeOH 9:1). The mixture of the three sulfinyl dia-
stereomers 28 was obtained as a pale yellow oil (0.47 g, 0.50 mmol,
40% yield).1H NMR of diastereomeric mixture: d 6.0–5.9 (m,
2 ꢀ ˜CH2), 5.2–5.0 (m, 2 ꢀ H-2,3,4), 4.6–4.1 (m, 2 ꢀ H-1, H2-6,
CH2˜CCH2), 3.7 (m, 2 ꢀ H-5), 2.9–2.6 (m, 2 ꢀ CH2S), 2.1–2.0 [m,
8 ꢀ C(O)Me], 1.9–1.5 (m, CH2(CH2)3CH2). IR: nmax 1756 (CO),
1056 (SO) cmꢁ1. Elemental analysis calcd (%) for C39H56O22S2
(940.97): C 49.78, H 6.00; found: C 49.56, H 5.92.
5.1.6.2. 3,30-(Pentane-1,5-diyldisulfinyl)dipropionitriles 19. Disul-
fide 15 was oxidized following the general procedure D. Disulfox-
ides 19 (meso/racemate 1:1) were obtained as a white solid (0.81 g,
2.95 mmol, 97% yield) not very soluble in CHCl3. 1H NMR of 1:1
meso/racemate mixture: d 3.1–2.7 (m, 2 ꢀ CH2SCH2CH2CN), 2.0–
1.7 (m, CH2CH2CH2CH2CH2). 13C NMR of 1:1 meso/racemate mix-
ture: 117.4 (2 ꢀ C-1), 51.9 [SCH2(CH2)3CH2S], 46.5 (2 ꢀ C-3), 27.6
[CH2CH2CH2CH2CH2], 22.3 [CH2CH2CH2CH2CH2], 11.0 (2 ꢀ C-2).
Elemental analysis calcd (%) for C11H18N2O2S2 (274.40): C 48.15,
H 6.61; found: C 48.09, H 6.54.
5.1.7. 2,20-(m-Phenylendisulfinyl)di-2,20-propenyl bis-b-
D-gluco-
pyranoside bis-2,3,4,6-tetraacetates 25
5.2. Biological tests
A solution of bis-sulfoxides 16 (0.11 g, 0.39 mmol) and 2-propy-
nyl b- -glucopyranoside 2,3,4,6-tetraacetate (24) (0.61 g, 1.57
5.2.1. Cells
D
The biological tests were performed in U937 and Molt-3 cells.
The U937 cell line was established from the pleural effusion of a
patient with histiocytic lymphoma and has been characterized as
a monocytic cell line. The Molt-3 cell line was established from
the peripheral blood of a patient with acute lymphoblastic leuke-
mia and has been characterized as a T-lymphocytic cell line. The
utilized cell lines share characteristics suitable for this study,
such as rapid growth in suspension and sensitivity to cytotoxic
agents.
mmol) in 1,2-dichloroethane (4 mL) was maintained at reflux.
When the reaction appeared complete by TLC (disappearance of
starting sulfoxides required about one night), the solvent was re-
moved under pressure and the reaction crude was purified by flash
column chromatography on silica gel (petrol/EtOAc 6:4 up to
EtOAc 100%). The mixture of the three sulfinyl diastereomers 25
was obtained as a white solid (m.p. 65–68 °C, 0.14 g, 0.15 mmol,
40% yield).1H NMR of diastereomeric mixture: d 7.9–7.3 (m, ArH),
6.3–5.6 (m, 2 ꢀ ˜CH2), 5.3–3.6 (m, GluH and 2 ꢀ CH2˜CCH2), 2.1–
2.0 [m, 8 ꢀ C(O)Me]. Elemental analysis calcd (%) for C40H50O22S2
(946.94): C 50.73, H 5.32; found: C 50.66, H 5.21.
5.2.2. Evaluation of cytotoxicity and apoptosis
Cytotoxicity was evaluated by microscopy analysis, using the
trypan blue exclusion test as a standard viability assay. Apoptosis
was evaluated in the cells by their morphological analysis followed
by staining with acridine orange, as previously described.12 Over
600 cells, including those showing typical apoptotic characteris-
tics, were analyzed using a fluorescence microscope. The identifi-
cation of apoptotic cells was based on the presence of uniformly
stained nuclei showing chromatin condensation and nuclear
fragmentation.
5.1.8. 2,20-[1,10-Biphenyl-4,40-diyldi(sulfinyl)]di-2,20-propenyl
bis-b-D-glucopyranoside bis-2,3,4,6-tetraacetates 26
A solution of bis-sulfoxides 17 (0.62 g, 0.95 mmol) and the
ethynyl acceptor 24 (1.48 g, 3.81 mmol) in DCM (6 mL) was
maintained at reflux. When the reaction appeared complete by
TLC (disappearance of starting sulfoxides required about one
night), the solvent was removed under pressure and the reaction
crude was purified by flash column chromatography on silica gel
(petrol/EtOAc 6:4 up to petrol/EtOAc 2:8). The mixture of the
three sulfinyl diastereomers 26 was obtained as a pale yellow
oil (0.30 g, 0.28 mmol, 30% yield).1H NMR of diastereomeric mix-
ture: d 7.8–7.6 (m, ArH), 6.2–5.9 (m, 2 ꢀ ˜CH2), 5.2–4.9 (m, 2 ꢀ
H-2,3,4), 4.5–3.9 (m, 2 ꢀ H-1, 2 ꢀ H2-10,6), 3.7–3.6 (m, 2 ꢀ H-5),
5.2.3. Evaluation of metabolic activity and cell growth
The effects on the metabolic activity were examined by the
MTS colorimetric method, using a commercial kit (MTS, Cell Titer
96 Aqueous One Solution, Promega). The assay was performed by
2.1–1.0 [m, 8 ꢀ C(O)Me]. IR: nmax 1756 (CO), 1042 (SO) cmꢁ1
.
seeding 2 ꢀ 104 U937 cells in 100
l
L in the presence of different
Elemental analysis calcd (%) for C46H54O22S2 (1023.03): C 54.01,
H 5.32; found: C 53.96, H 5.28.
concentrations of the thioarene-spaced bis-b-D-glucopyranoside
or the vehicle, in RPMI-1640 medium supplemented with 5%
FBS, 2 mM L-glutamine and penicillin-streptomycin, for 24 h.
Twenty microliters of ‘‘CellTiter 96 Aqueous One Solution Re-
agent” were added directly to the culture wells at the end of
the culture and incubated for 4 h, after which, absorbance was
read at 490 nm.
5.1.9. 2,20-[Propane-1,3-diyldi(sulfinyl)]di-2,20-propenyl bis-b-
D-
glucopyranoside bis-2,3,4,6-tetraacetates 27
A solution of bis-sulfoxides 18 (0.30 g, 1.24 mmol) and the ethy-
nyl acceptor 24 (1.45 g, 3.73 mmol) in toluene (6 mL) was main-
tained at reflux. When the reaction appeared complete by TLC
(disappearance of starting sulfoxides required about 4 h), the
solvent was removed under pressure and the reaction crude was
purified by flash column chromatography on silica gel (petrol/
EtOAc 5:5 up to EtOAc/MeOH 9:1). The mixture of the three sulfi-
nyl diastereomers 27 was obtained as a pale yellow oil (0.23 g,
0.25 mmol, 20%).1H NMR of diastereomeric mixture: d 6.0–5.9
(m, 2 ꢀ ˜CH2), 5.2–5.0 (m, 2 ꢀ H-2,3,4), 4.6–4.1 (m, 2 ꢀ H-1, H2-
6, CH2˜CCH2), 3.7 (m, 2 ꢀ H-5), 3.0–2.7 (m, 2 ꢀ CH2S), 2.0 (m,
CH2CH2CH2), 2.1–2.0 [m, 8 ꢀ C(O)Me]. IR: nmax 1756 (CO), 1040
(SO) cmꢁ1. Elemental analysis calcd (%) for C38H54O22S2 (926.95):
C 49.24, H 5.87; found: C 49.10, H 5.65.
5.2.4. Statistical analysis
Data analysis was performed using the SPSS statistical software
system (version 12.0 for Windows, Chicago, IL). Comparison of
means were carried out using the Student’s t-test for independent
samples and the Tukey’s honestly significance test (HSD), as a mul-
tiple comparison test, where appropriate.
Acknowledgement
The authors thank the Università degli Studi di Messina for finan-
cial support (PRA).

M. C. Aversa et al. / Bioorg. Med. Chem. 17 (2009) 1456–1463
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