Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory activities against Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmcl.2013.04.030

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(β-d-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC₅₀ values ranging from 4 to 11 μM at 37 C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4 C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.

Full text of DOI:10.1016/j.bmcl.2013.04.030

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3576–3579
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory
activities against Trypanosoma cruzi
Bessy Gutiérrez a, Christian Muñoz a, Luis Osorio a, Krisztina Fehér b, Tünde-Zita Illyés c, Zsuzsa Papp c,
Ambati Ashok Kumar c, Katalin E. Kövér c, Hernán Sagua a, Jorge E. Araya a, Patricio Morales d,
a,
László Szilágyi c, , Jorge González
⇑
⇑
a Department of Medical Technology, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile
b Department of Organic Chemistry, University of Ghent, 9000 Ghent, Belgium
c Department of Chemistry, University of Debrecen, P.O. Box 20, H-4010 Debrecen, Hungary
d Biomedical Department, Faculty of Health Sciences, University of Antofagasta, P.O. Box 170, Antofagasta, Chile
a r t i c l e i n f o
a b s t r a c t
Article history:
Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three differ-
Received 24 January 2013
Revised 10 April 2013
Accepted 12 April 2013
Available online 22 April 2013
ent Trypanosoma cruzi strains. Di-(b-
be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50
values ranging from 4 to 11 M at 37 °C. The inhibitory activities were maintained, although somewhat
lowered, at a temperature of 4 °C as well. Three further derivatives displayed similar activities against at
least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero
and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that
of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited
the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular
development of T. cruzi amastigotes as well.
D-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to
l
Keywords:
Trypanosoma cruzi
Chagas disease
Disulfide
Carbohydrate
Ó 2013 Elsevier Ltd. All rights reserved.
Trypanosoma cruzi is the etiologic agent of Chagas’s disease, an
endemic parasitosis in Latin America with 12–14 million people in-
fected.1 Acute infections are usually asymptomatic, but the ensu-
ing chronic T. cruzi infections have been associated with high
rates of morbidity and mortality. Recently, Chagas’s disease has
also been recognized as an opportunistic disease in HIV-infected
individuals2,3 and reports appeared about worldwide spreading
due to international migration.4,5
A number of drugs were reported to be effective against T. cruzi
in vitro or in animal models, but none proved to be completely sat-
isfactory for clinical use. Similarly, there are no adequate chemo-
prophylactic drugs to be used to eliminate the parasite from the
blood of serologically positive donors in order to prevent transfu-
sion-associated Chagas’s disease. Currently only two drugs, nifurti-
mox (a nitrofuran derivative, Nif) and benznidazole (a nitro-
imidazole derivative, Bz) introduced in the 1960s and 1970s, are
available for the treatment of chagasic patients. These drugs are
effective for acute infections, but their use for chronic patients re-
mains controversial.6 Furthermore, studies of the mechanisms of
action indicated that their antiparasitic activity is inextricably
linked to mammalian host toxicity.7 The efficacy of these drugs
also depends on the susceptibility of T. cruzi strains and resistance
to benznidazol has been reported.8,9 Due to these inadequacies re-
search programs were initiated to find alternative drugs for the
treatment of chagasic patients.7,10
A large variety of synthetic compounds have recently been
tested for antitrypanosomal activity in high-throughput screening
campaigns, encompassing 10000011 and more than 300000 struc-
tures10, respectively. Targets and patented drugs have been re-
viewed.12 Posaconazole recently emerged as
a
promising
candidate in mice13 and humans.14 However, new compounds
showing higher potency and selectivity in both the acute and
chronic stages of Chagas’s disease and/or better tolerability are still
needed.15,16 Of the large number of the molecules tested so far for
antitrypanosomal activity just a few contained disulfide bonds.17,18
Glycosyl disulfides were shown to exert inhibitory activities
against endogenous lectins in vivo on human tumor cell lines
and it was suggested that ‘glycosyl disulfides have potential as
platform for inhibitor design’.19,20 We have recently described spe-
cific binding of oligovalent aromatic mannosyl disulfide derivatives
to a lectin21 and explored the conformational features of diglycosyl
⇑
Corresponding authors. Tel.: +36 52 512900x22589; fax: +36 52 512
747x23744 (L.S.); tel.: +56 55 637376; fax: +56 55 637108 (J.G.).
E-mail addresses: lszilagyi@unideb.hu (L. Szilágyi), jgonzalez@uantof.cl
(J. González).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.030

B. Gutiérrez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3576–3579
3577
disulfides.22,23 Cytotoxic activities of symmetrical 1,10-glycosyl
disulfides against human tumor cell lines have been recorded.24,25
In view of these findings we have envisioned that it might be worth
to test the anti-trypanosomal activities of such structures.
The compounds selected for the tests are characterized by the
attachment of one or more monosaccharide sugars to an aromatic
core (1b, 2b, 3, 4b, 5, 6, 7b, 8) or to another monosaccharide (10
and 11) by disulfide linkages. Compound 9, of similar structure
but containing a simple sulfide bond, rather than disulfide, was
added for comparison. The chemical structures of the compounds
tested in this study are shown in Scheme 1.
somewhat lower level, against the Y strain at 4 °C in the presence
of blood constituents, this is promising with regard to the potential
use in blood banks. Further molecules, 1b, 3, 5 and 7b displayed
moderate antitrypanosomal activities against one of the strains
at least (Table 1).
Cytotoxicities of all compounds were evaluated after 18 h of
incubation with confluent HeLa, Vero and peritoneal macrophage
cell cultures. All compounds, displayed low cytotoxicities with
cytotoxic concentrations (CC50
)
ranging from moderate
(ꢀ240 M) to high (>2000 M) levels (Table 1).
l
l
Currently marketed drugs display limited efficacy against the
chronic form of the Chagas disease which is often diagnosed late
when the disease has already progressed and caused irreversible
damage in the heart and in other organs.7 It is therefore important
to find compounds that are effective against the intracellular forms
of T. cruzi. Therefore, we evaluated the effects of the glycosyl disul-
fides on parasite release from T. cruzi infected HeLa cell cultures
after 18 h of treatment at 37 °C. Several molecules of the panel
we tested, such as 1b, 3, 4b, 5, 6 and 11, were able to inhibit intra-
cellular proliferation or parasite differentiation when the number
of parasites released by T. cruzi infected HeLa cells was measured,
see Figure 1. This effect is comparable to that observed for ben-
znidazol (Fig. 1 and Refs. 30,31). Although no significant correla-
tion could be observed between activity against cell derived
trypomastigotes and amastigotes, it should be taken into account
that amastigotes and trypomastigotes are two morphologically dif-
ferent cells that live in two different environments, the blood-
stream in the case of the trypomastigotes, and the cell cytoplasm
in the case of the amastigotes.
Aromatic glycosyl disulfides 3, 5, 6, 821 and sulfide 926 have
been previously described. Diglycosyl disulfides 10 and 11 were
also prepared as published.27,28 1b, 2b, 4b, and 7b were synthe-
sized as shown in Scheme 2.
Aromatic methanethiosulfonates I, II, III, and IV21 react
smoothly with 1-thio-2,3,4,6-tetra-O-acetyl-b-
at low temperatures to provide per-O-acetylated (b-
D
-galactopyranose29
D
-galactopyr-
anosyldithiomethylene)benzenes 1a, 2a, 4a, and 7a. Deacetylation
with lithium hydroxide (LiOHxH2O) in methanol furnished 1b, 2b,
4b, and 7b, with free sugar hydroxyls. The structures of the new
compounds have been established by standard NMR and MS spec-
troscopy. Synthetic procedures are described in the Supplementary
material.
Since various strains of T. cruzi exhibit different levels of resis-
tance to Nif and Bz, and this variability may partially explain the
observed differences in effectiveness of chemotherapy, in vitro
tests were conducted to compare the activities of our molecules
against Bz-susceptible (CL), -moderate (Y), and resistant (H510 a
Colombian clone) T. cruzi strains. Furthermore, in order to assess
the potential use of these compounds for the prophylaxis of
banked blood, assays evaluating the effect of the drugs against try-
pomastigotes were performed at 4 °C in the presence of blood. The
results are summarized in Table 1 and Fig. S1.
Inspecting the results of our inhibition tests listed in Table 1
(see also Fig. S1), the following observations can be made. First,
sizeable inhibitory activities, on one T. cruzi strain at least, were
observed only for aromatic disulfide derivatives (1b, 2b, 3, 4b, 5,
7b). Second, the presence of both the aromatic ring and the disul-
fide linkage appears to be essential. This is clearly demonstrated by
the much lower activities of 10 and 11 (no aromatic rings) on the
one hand, and by the comparison of 1b to 9, on the other. While the
disulfide 1b is a moderate inhibitor of T. cruzi growth (at 37 °C), 9,
lacking just one S-atom in comparison with 1b, is much less active
The direct effect of the glycosyl disulfides on tissue culture de-
rived trypomastigotes was evaluated after 18 h of treatment at
37 °C. 2b and 4b were found to be the most active compounds with
IC50 values ranging from 4.2 1.2 to 10.9 1.2 lM against the
tested strains. These compounds also retained their activities, at
S
S
S
Glyc
Glyc
S
S
S
Glyc
S
Glyc
S
Glyc
S
S
S
1b
2b, 3
4b, 5
S
S
Glyc
Glyc
S
S
Glyc
S
S
Glyc
S
S
Glyc
Glyc
S
S
7b, 8
6
9
Glyc = β-D-galacto-
OH
O
Glyc = α-D-manno-
HO
HO
HO
O
S
S
OH
OH
HO
OH
OH
OH
O
O
OH
OH
HO
OH
HO
1b, 2b, 4b, 7b, 9
OH
10
3, 5, 6, 8
OAc
O
O
AcO
AcO
AcO
S
S
OAc
OAc
O
AcO
OAc
11
Scheme 1. Chemical structures of the molecules tested.

3578
B. Gutiérrez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3576–3579
SSO2CH3
SSO2CH3
SSO2CH3
CH2SSGal(OR)4
CH2SSGal(OR)4
OAc
OAc
O
I.
II., III.
CH2SSGal(OR)4
SH
AcO
i)
i)
OAc
1a R = Ac
1b
2a, 4a
R =Ac
2b, 4b
R = H
ii)
ii)
SSO2CH3
R = H
i)
SSO2CH3
CH3SO2S
IV.
CH2SSGal(OR)4
CH2SSGal(OR)4
(RO)4GalSSH2C
7a
R = Ac
ii)
7b R = H
i) -50°C, DMF, ii) LiOHxH2O / CH3OH
Scheme 2. Syntheses of the compounds tested.
Table 1
Trypanocidal activities and cytotoxic effects of the tested compounds
Compounds
Activity against different T. cruzi tripomastigote strains: IC50
(
l
M)* S.D.
Cytotoxicity CC50
Cell lines
(l
M)** S.D.
4 °C
37 °C
Y
Y
H510
CL
40.5 1.6
10.9 1.2
26.4 1.3
4.4 1.2
VERO
HeLA
Macrophage
1b
2b
3
4b
5
6
7b
8
139.9 2.3
30.5 1.2
38.8 1.1
10.4 1.2
116.8 1.3
225.1 3.2
210.9 1.7
260.6 1.8
247.0 2.8
243.7 1.6
153.4 1.3
P500
52.2 1.2
4.8 1.2
14.2 1.2
8.7 1.2
49.7 1.2
4.2 1.2
19.7 1.5
5.7 2.7
11.9 1.9
109.0 1.3
57.3 1.2
263.4 1.4
247.2 1.4
271.5 1.4
123.7 1.3
ND
>2000
247.0 1.4
>2000
251.3 1.4
500.8 1.5
>2000
>2000
>2000
>2000
>2000
>2000
238.9 1.5
>2000
242.3 1.2
487.7 1.4
>2000
>2000
>2000
>2000
>2000
>2000
241.4 1.4
>2000
248.4 1.2
495.7 1.3
>2000
>2000
>2000
>2000
>2000
42.8 1.3
98.4 1.2
14.5 1.2
233.3 1.4
238.2 1.4
241.4 1.4
111.8 1.3
25.6 1.5
58.1 1.5
120.0 1.4
73.5 1.6
276.2 1.4
283.1 1.4
287.2 1.4
138.5 1.3
ND
9
10
11
256.0 1.4
ND
248.6 1.3
ND
252.1 1.5
ND
Benzni-dazol
*
The IC50 is defined as the concentration required to reduce the fluorescence of the control (without the test compound) by 50%.
The CC50 is defined as the concentration needed to reduce the viability of HeLa cells by 50%.
**
Fig. 1. Effects on the intracellular development of T. cruzi. Infected HeLa cell cultures were incubated during 18 h with 25 lM solutions of the compounds listed on the x-axis.
The activity against intracellular development was evaluated by counting the number of released trypomastigotes per mL. Experiments were performed in triplicate and
values are expressed as the media 1S.D. Testing procedures are described in the Supplementary material.
(Table 1). Another correlation bears evidence for the importance of
the sugar configurations attached to the aromatic ring: in tests
conducted at 37 °C the b-galactosyl derivative 7b is significantly
more active than
b-galactosyl derivative) is more active than 5 (an
derivative). In terms of the IC50 values molecules 2b and 4b clearly
a
-mannosyl-substituted 8 and, similarly, 4b (a
a-mannosyl

B. Gutiérrez et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3576–3579
3579
stand out as the most potent inhibitors of the tested panel being
Supplementary data
active against all three of the tested T. cruzi strains. The antitrypan-
osoma activities of both compounds exceed that of benznidazol in
these in vitro tests against the Y strain (Table 1).
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
04.030. These data include MOL files and InChiKeys of the most
important compounds described in this article.
In recent years it has been recognised that the binding energy
per heteroatom or ligand efficiency (LE)32,33 is a useful parameter
to characterise the potency of an inhibitor molecule with respect
to its molecular weight. We have calculated LE values and another
score, the size-compensated LE or Fit Quality (FQ) proposed by
Reynolds et al.34 (see Table S2 and Fig. S2, Supplementary mate-
rial). In general, comparison based on ligand efficiencies allows
highlighting smaller, weaker affinity compounds. Indeed, the
monosubstitued disulfide derivative, 1b displays the highest LE
values (0.19–0.22) comparable to that of benznidazole (0.24). On
the other hand, the highest size-compensated FQ scores were ob-
tained for 2b and 4b (ꢀ0.50) again similar to that of benznidazole
(0.50). Lipophilic ligand efficiency35,36 is another parameter* link-
ing activity and lipophilicity in an attempt to assess druglike-
ness.35–37 Calculated LLE values listed in Table S2 (Supplementary
material) are close to five for 2b and 4b which is promising as a value
of five or above is considered optimal for a drug candidate.35,36
Remarkably, 2b and 4b display similar high values for all three T. cru-
zi strains; this is exceeding the values for the reference drug Bz espe-
cially at 4 °C (Table S2). Further improvement of the activity in the
series is therefore conceivable by attachment of lipophilic moieties
during a follow-up optimisation process.
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The skillful technical assistance of Sára Balla (Debrecen) is
greatly appreciated. We thank TÁMOP-4.2.2.A-11/1/KONV-2012-
0025 and OTKA K 105459 Grants (to K.E.K.) in Hungary as well
as CODEI Grant No. 5381 from Antofagasta University for financial
support.
⁄
We thank a referee to draw our attention to this point.