α-Selective glycosylation affords mucin-related GalNAc amino acids and diketopiperazines active on Trypanosoma cruzi

Article abstract of DOI:10.1016/j.bmc.2013.01.027

This work addresses the synthesis and biological evaluation of glycosyl diketopiperazines (DKPs) cyclo[Asp-(αGalNAc)Ser] 3 and cyclo[Asp-(αGalNAc)Thr] 4 for the development of novel anti-trypanosomal agents and Trypanosoma cruzi trans-sialidase (TcTS) inhibitors. The target compounds were synthetized by coupling reactions between glycosyl amino acids αGalNAc-Ser 7 or αGalNAc-Thr 8 and the amino acid (O-tBu)-Asp 17, followed by one-pot deprotection-cyclisation reaction in the presence of 20% piperidine in DMF. The protected glycosyl amino acid intermediates 7 and 8 were, in turn, obtained by α-selective, HgBr₂-catalysed glycosylation reactions of Fmoc-Ser/Thr benzyl esters 12/14 with αGalN₃Cl 11, being, subsequently, fully deprotected for comparative biological assays. The DKPs 3 and 4 showed relevant anti-trypanosomal effects (IC₅₀ 282-124 μM), whereas glycosyl amino acids 1 and 2 showed better TcTS inhibition (57-79%) than the corresponding DKPs (13-25%).

Full text of DOI:10.1016/j.bmc.2013.01.027

Bioorganic & Medicinal Chemistry 21 (2013) 1978–1987
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
a-Selective glycosylation affords mucin-related GalNAc amino acids
and diketopiperazines active on Trypanosoma cruzi
Maristela B. Martins-Teixeira a, , Vanessa L. Campo a, , Monica Biondo a, Renata Sesti-Costa b,
Zumira A. Carneiro b, João S. Silva b, Ivone Carvalho a,
⇑
a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. Café s/n, Ribeirão Preto, SP 14040-930, Brazil
b Faculdade de Medicina Ribeirão Preto, Universidade de São Paulo, Av. Bandeirantes 3900, Ribeirão Preto, SP 14049-900, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
This work addresses the synthesis and biological evaluation of glycosyl diketopiperazines (DKPs)
Received 2 November 2012
Revised 3 January 2013
Accepted 11 January 2013
Available online 8 February 2013
cyclo[Asp-(
mal agents and Trypanosoma cruzi trans-sialidase (TcTS) inhibitors. The target compounds were synthe-
tized by coupling reactions between glycosyl amino acids GalNAc-Ser 7 or GalNAc-Thr 8 and the
aGalNAc)Ser] 3 and cyclo[Asp-(aGalNAc)Thr] 4 for the development of novel anti-trypanoso-
a
a
amino acid (O-tBu)-Asp 17, followed by one-pot deprotection-cyclisation reaction in the presence of
20% piperidine in DMF. The protected glycosyl amino acid intermediates 7 and 8 were, in turn, obtained
Keywords:
Mucin
Trypanosoma cruzi
trans-Sialidase
Glycosylation
Glycosyl diketopiperazine
by
GalN3Cl 11, being, subsequently, fully deprotected for comparative biological assays. The DKPs 3 and
4 showed relevant anti-trypanosomal effects (IC50 282–124 M), whereas glycosyl amino acids 1 and 2
showed better TcTS inhibition (57–79%) than the corresponding DKPs (13–25%).
Ó 2013 Elsevier Ltd. All rights reserved.
a-selective, HgBr2-catalysed glycosylation reactions of Fmoc-Ser/Thr benzyl esters 12/14 with
a
l
1. Introduction
a role in the host cell invasion and in the ability to induce secretion
of pro-inflammatory cytokines IL-12, TNF-
a and nitric oxide in
Among parasitic illnesses, Chagas’ disease, caused by the proto-
zoa Trypanosoma cruzi, accounts for 10,000 deaths annually, 30% of
the patients in chronic condition develop severe cardiac disorders
and 10% suffer from intestinal illnesses, both involving clinical man-
ifestations that can lead to death after ten years. Thus, the long-term
disability and mortality induced by Chagas’ disease have been
resulting in significant economic and social impacts in many South
and Central American countries.1–3 Despite the attempts to develop
vaccines, the current treatment and control of Chagas’ disease de-
pend on chemotherapeutic agents such as benznidazol (RochaganÒ)
and nifurtimox (LampitÒ), which are almost 100% effective in the
acute infection treatment, however, they have limited efficacy in
the chronic infection phase and produce severe side effects due to
their high toxicity.4,5 In this context, posaconazole, an antifungal tri-
azole interfering with sterols biosynthesis, has been poised as a new
effective treatment for the acute and chronic Chagas’ disease pa-
tients and is entering a phase II clinical trial in the near future.5–7
Trypanosoma cruzi mucins found in insect epimastigote stage
and in the infective trypomastigotes are GPI (glycosylphosphat-
idylinositol)-anchored plasma membrane glycoproteins that play
activated macrophages.8,9 Comparative studies involving O-gly-
cans of T. cruzi mucins and those found in mammalian systems
have revealed major differences between both species, such as
the peptide backbone linked through an
GlcNAc) residue in T. cruzi, which are further substituted by gal-
actose at O-4 and O-6 positions, rather than the -N-acetylgalac-
a-N-acetylglucosamine
(a
a
tosamine (aGalNAc) substituted at O-3 and O-6 positions in
mammalian mucins (Fig. 1).10,11
The interaction between parasite mucins (TcMUC) and a unique sur-
face trans-sialidase enzyme (TcTS) is the most important mechanism
by which parasite invades cells and causes infections since TcMUC
must be sialylated by the action of TcTS before entering the cells. This
process involves the transfer of sialic acid from host glycoconjugates
to b-galactopyranosyl acceptor groups on T. cruzi mucins, forming an
a-2,3 linkage catalysed by TcTS, that does not require CMP-sialic acid
as the monosaccharide donor (Fig. 1). As a result, the parasite acquires
about 1 ꢀ 107 sialic acid residues, increasing the parasite virulence and
protecting it against complement-independent lyses induced by hu-
man anti-a-galactosyl antibodies because of the strong negative charge
on the surface.8,9,11–13 However, although it is primarily a transferase,
TcTS does have some residual hydrolase activity.14
All these features contribute to consider TcTS as a potential tar-
get, especially for searching a drug that could display potent and
selective activity against the parasite during the infection process.
⇑
Corresponding author. Tel.: +55 16 3602 4709; fax: +55 16 3602 4879.
E-mail address: carronal@usp.br (I. Carvalho).
These authors contributed equally to this manuscript.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.01.027

M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
1979
Figure 1. Model of recognition and sialylation of parasite surface mucins by T. cruzi trans-sialidase.
In this regard, a panel of TcTS inhibitors described so far are able to
occupy both donor (sialic acid) and acceptor (b-galactose) binding
sites, so that the sialic acid transfer reaction is hindered or even
blocked.15 Thus, a remarkable activity against TcTS was achieved
by assaying sulfonamide chalcones (IC50 0.9–2.5
lM) and quinoli-
nones (IC50 0.5–0.6
l
M), obtained by chemical synthesis.16 Alterna-
tively, the identification of potential inhibitors by molecular docking
studies on TcTS active site, such as the recently described benzothi-
azole derivatives (IC50 0.12–0.15 lM), were accomplished by in sil-
ico screening,17 exploring the crystal structure of TcTS in complex
with different substrates (donor/acceptor) or inhibitors,18–21 and
more recently, with Fab fragment of a neutralizing monoclonal IgG
antibody.22 At last, the biological screening using a wide natural
product library led to the identification of a series of potent flavo-
noids (IC50 17 lM) and anthraquinones (IC50 0.58 l
M).23
Accordingly, we assumed that hybrid molecules comprising an
a
GalNAc residue linked by a rigid diketopiperazine bridge to a car-
Figure 2. Structures of the target glycosyl amino acids 1 and 2, and corresponding
glycosyl diketopiperazines 3 and 4.
boxylic function, mimicking the charged sialic acid that interacts
with the enzyme, could at same time occupy and block, respec-
tively, both acceptor and donor TcTS binding sites, impairing the
enzyme activity and then parasite cell invasion. Thus, the purpose
FmocAsp-(
(6). Their synthesis involved the previous preparation of protected
glycosyl amino acids GalNAc-FmocSerOBn (7) and GalNAc-
aGalNAc)SerOBn (5) and FmocAsp-(aGalNAc)ThrOBn
of using
aGalNAc in the place of bGal TcMUC core is supported by
a
a
the fact that it does not act as a sialic acid acceptor due to the
configuration, but it still has C-3 and C-4 required configurations
to interact with the acceptor site, besides the resemblance with
a
-
FmocThrOBn (8) as source of the mucin fundamental scaffold,
which is supposed to be critical for the intended biological activity.
the natural
aGlcNAc core. In addition, the 2,5-diketopiperazine
2.1.1. Synthesis of the glycosyl amino acids
(DKP) linker represents a peculiar rigid heterocyclic system with
defined substituents around a proteolysis-resistant peptide-mim-
icking scaffold, which can be straightforwardly obtained by a con-
ventional synthetic method, such as the cross-link between two
amino acid residues via a pair of complementary amide bonds.24,25
In the context of the preparation of glycosyl diketopiperazines 3
and 4 containing
to obtain the key precursors
FmocThrOBn (10) were designed to favour selectively the forma-
tion of the -anomer during glycosylation. For this purpose, the
azide-derived glycosyl donor
GalN3Cl (11),26 unable to exhibit
a-linked glycoside, the proposed synthetic routes
a
GalN3-FmocSerOBn (9) and GalN3-
a
a
To investigate the influence of
boxylic group in the TcTS activity, we accomplished the synthesis
of glycosyl amino acids GalNAcSer (1) and GalNAcThr (2), as
aGalNAc on the DKP-containing car-
a
neighbouring group effect and straightforwardly converted to acet-
amide group, was employed in glycosylation reactions with suit-
ably protected serine and threonine amino acids.
a
a
well as their protected forms as building blocks to obtain target
glycosyl diketopiperazines containing Ser/Thr and Asp amino acids,
represented by cyclo[Asp-(aGalNAc)Ser] (3) and cyclo[Asp-(aGal-
NAc)Thr] (4) (Fig. 2). Therefore, this work focuses on the synthesis
of compounds 1–4 and their biological evaluation as TcTS inhibi-
tors and anti-trypanosomal agents.
Anomeric fluorides, bromides and iodides have also been used
in similar synthetic approaches, as well as several glycosyl donors
with leaving groups other than halides, such as trichloroacetimi-
date, pentenyl ether, phenylthiol or phenylseleno derivatives. Gly-
cosylation catalysts are commonly Lewis acids, notably silver
perchlorate or triflate, in the presence of inorganic or alkylpyridine
bases.27–31 Nonetheless, the most employed glycosylation method
to obtain building blocks such as 9 and 10 relies on Köenigs–Knorr
strategy, particularly using silver salts as catalysts, since they are
effective to assist leaving groups displacement by complexation
with anomeric halides.32
2. Results and discussion
2.1. Synthesis
The target glycosyl diketopiperazines are represented by
cyclo[Asp-(
a
GalNAc)Ser] 3 and cyclo[Asp-(aGalNAc)Thr] 4, and
In this regard, the glycosylation of FmocSer benzyl ester (12)
originated from the head-to-tail cyclisation of dipeptides
with
aGalN3Cl 11 was firstly attempted utilizing Ag2CO3 and

1980
M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
OAc
AcO
AcO
NHFmoc
OBn
OAc
AcO
AcO
O
OAc
AcO
AcO
HO
O
NHFmoc
OBn
NHFmoc
OBn
+
i
R'
O
+
O
O
N3
Cl
R
O
R'
13 R'= N3
OH
O
R
O
11
12 R= H
9
R= H, R'= N3
10
14 R=CH3
R=CH3, R'= N3
ii
HO
HO
iii
7
8
R= H, R'= NHAc
O
R=CH3, R'= NHAc
NH2
O
AcHN
OH
O
1
2
R= H
R=CH3
R
Scheme 1. Synthesis of GalNAc-derived glycosyl amino acids. Reagents and conditions: (i) HgBr2/1,2-DCE; (ii) Zn, CuSO4, Ac2O/AcOH/THF; (iii) H2/Pd-C 10%; NaOMe/MeOH.
OtBu
OAc
OAc
AcO
AcO
AcO
AcO
O
O
O
O
ii
i
NHR'
O
HN
NHFmoc
AcHN
AcHN
(hours)
O
O
OBn
O
OtBu
OBn
HO
FmocHN
17
R
R
O
O
7
8
R= H, R'= Fmoc
R= CH3, R'= Fmoc
i
15
16
5
6
R= H, R'= H
R= H
R= CH3, R'= H
R= CH3
(minutes)
OAc
OH
AcO
AcO
OH
HO
O
O
O
O
O
O
OtBu
OH
iii
iv
HN
HN
AcHN
AcHN
O
NH
O
O
NH
O
R
R
18
3
4
R= H
R= H
R= CH3
19
R= CH3
Scheme 2. Synthesis of glycosyl diketopiperazines. Reagents and conditions: (i) 20% piperidine/DMF; (ii) PyBOP, HOBt, DIEA/DMF; (iii) NaOMe/MeOH; (iv) TFA/DCM (1:1).
AgClO4 as catalytic system, in toluene/DCM mixture. Despite of the
extended reaction times and catalyst excess additions, the ex-
acceptor nature, as already verified by Lemieux26 earliest studies
with the azido chloride donor.à
pected
a
-glycoside 9 was isolated in a very low yield (10%), leading
Subsequently, the sugar azido groups of the glycosyl amino
acids 9 and 10 were reductively acetylated using zinc powder in
THF–acetic anhydride–acetic acid,34 which afforded the corre-
us to exploit the use of an alternative Lewis acid catalyst, specifi-
cally the mercuric bromide, as previously described by Carvalho
et al.33,34 Therefore, the glycosylation performed under HgBr2 cat-
sponding
aGalNAc-FmocSerOBn 7 and aGalNAc-FmocThrOBn 8,
alyst gave the
a-glycoside 9 in 48% yield, besides the bGalN3-
in yields varying from 62% to 67% (Scheme 1). 1H NMR analysis
FmocSerOBn (13), the unexpected corresponding b-linked isomer,
of 7 and 8 showed deshielded H-2 signals in comparison to the pre-
in 4% yield (Scheme 1). Even though nonselective, this reaction
cursors 9 and 10, besides the presence of NHAc. The a-anomers 7
represents a direct approach to obtain both
which can be isolated by chromatography column under gradient
conditions (EtOAc/toluene). The configuration of the formed
and b anomers 9 and 13 was assigned by their distinct coupling
constants for H-1 doublet in 1H NMR spectra, respectively with
J1,2 3.2 and 8.0 Hz.
a
and b anomers,
and 8 were then further utilised in the synthesis of the glycodipep-
tides 5 and 6.
In parallel, small amounts of compounds 7 and 8 were fully
deprotected (removal of OBn, NFmoc and OAc groups) in order to
assess their biological activities, considering that in this form they
a
represent an analog of the elemental T. cruzi mucin core aGlcNAc-
Regarding the glycosylation of FmocThr benzyl ester (14) with
Ser/Thr. Thus, standard hydrogenation reactions (10% Pd-C/H2) fol-
lowed by Zemplen deacetylations afforded the final deprotected
glycosyl amino acids 1 (80%) and 2 (90%),34 whose structures were
confirmed by the 1H NMR and ESI-MS analysis (Scheme 1).
11, the
a anomer 10 was obtained in 54%, whereas only trace
amounts of the b anomer might have been formed, as judged by
TLC, and could not be isolated (Scheme 1). Similarly to compound
9, the spectrum of the a-glycoside 10 showed characteristic signal
of H-1 with J1,2 3.4 Hz. According to the different results observed
for the glycosylation of serine and threonine amino acids, the latter
bearing an additional chiral centre in b carbon, stereoselectivity
under the described conditions appears to be dependent on the
à
Lemieux (1979) have described Hg(CN)2 catalysed glycosylation with azido
chloride donor and benzyl or tert-butyl alcohols as acceptors, obtaining glycoside
products with stereoselectivity of around 5 and 10 a/b ratio, respectively.

M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
1981
perazines cyclo[Asp-(
a
GalNAc)Ser] (18) and cyclo[Asp-(
a
GalNAc)
Thr] (19). Compounds 18 and 19 were obtained in the correspond-
ing yields of 48% and 52% after purification by chromatography
column, and their structures were confirmed by 1H NMR spectros-
copy, which showed the absence of aromatic and alkylic hydrogens
of both NFmoc and particularly OBn protecting groups. Finally, full
deprotection of glycosyl diketopiperazines 18 and 19 by means of
Zemplen deacetylations, followed by treatment with TFA/DCM
(1:1) for cleavage of O-tBu group, afforded the final deprotected
glycosyl diketopiperazines 3 (90%) and 4 (84%), confirmed by 1H
NMR and ESI-MS analysis.
2.2. Biological assays
2.2.1. Trypanosoma cruzi trans-sialidase inhibition assays
involving compounds 1-4
The enzymatic inhibition assays involving glycosyl amino acids
1 and 2, and glycosyl diketopiperazines 3 and 4 were performed
using the continuous fluorimetric method, which is based on
TcTS-catalyzed MuNANA hydrolysis.35 Compounds were tested at
1.0 mM concentration, along with pyridoxal phosphate (Py) as con-
trol, which is reported to be a weak TcTS inhibitor (60%).17
As a result, compound 2 showed high TcTS inhibition (79%) at
1.0 mM, followed by compound 1, for which satisfactory inhibitory
activity (57%) was also verified (Fig. 3). Considering its significant
TcTS inhibitory activity at 1.0 mM, compound 2 was further tested
at 0.5 and 0.25 mM, resulting in an IC50 value of 0.32 mM. Con-
versely, compounds 3 (25%) and 4 (13%) presented very weak
inhibitory activity at 1.0 mM (Fig. 3).
Figure 3. TcTS inhibition of glycosyl amino acids 1 and 2 and glycosyl diketopi-
perazines 3 and 4. The results are based on three independent experiments, each
one performed in triplicate.
2.1.2. Synthesis of glycosyl diketopiperazines
The synthesis of the glycosyl diketopiperazines cyclo[Asp-
(aGalNAc)Ser] 3 and cyclo[Asp-(aGalNAc)Thr] 4 was accomplished
in four steps, as outlined in Scheme 2. Firstly, the N-Fmoc deprotec-
tion of the glycosyl amino acids 7 and 8 with 20% piperidine in
DMF resulted in the free amino products
aGalNAc-SerOBn (15)
(78%) and GalNAc-ThrOBn (16) (69%), which were then con-
a
densed with the commercially available amino acid Fmoc-
(O-tBu)-Asp (17) to form a peptide bond, in the presence of the
coupling reagents PyBOP, HOBt, and the basic catalyst DIEA in
Therefore, this noteworthy difference of TcTS inhibition ob-
served for glycosyl amino acids 1 and 2, and glycosyl diketopiper-
azines 3 and 4 points out that the Asp-containing DKP heterocyclic
system did not exert a relevant role in TcTS inhibition in compar-
ison to the precursors 1 and 2 (see Fig. S14 in Supplementary data).
DMF. The glycodipeptides FmocAsp-(aGalNAc)SerOBn 5 and Fmo-
cAsp-( GalNAc)ThrOBn 6 were isolated in 93% and 86% yields,
a
respectively,25 and their structures were confirmed by 1H NMR
spectra, with anomeric doublets (J1,2 3.4 and 3.5 Hz), and signals
of characteristic methylenic hydrogens of the aspartic acid unit, to-
gether with ESI-MS confirmatory adducts. Subsequently, the N-
Fmoc deprotection of the glycodipeptides 5 and 6 by treatment
with 20% piperidine in DMF25 led to the intramolecular nucleo-
philic attack of the free amino groups (in basic conditions) of 5
and 6 toward their terminal carboxylate function, with displace-
ment of the benzyloxy group, giving the stable glycosyl diketopi-
2.2.2. In vitro anti-trypanosomal activities of compounds 1–4
and cytotoxicity towards mammalian cells
The glycosyl amino acids 1 and 2 and glycosyl diketopiperazines
3 and 4 were evaluated against trypomastigote and amastigote
forms of T. cruzi Tulahuen strain cultured with LLC-MK2 cells using
benznidazole (N-benzyl-2-nitro-1-imidazolacetamide, Bz), the
current frontline drug used to treat Chagas’ disease, as control.36,37
Figure 4. Percentage of anti-trypanosomal activities of compounds 1–4 and reference compound benznidazole (Bz) against Trypanosoma cruzi Tulahuen strain cultured with
LLC-MK2 cells. The results are based on three independent experiments, each one performed in duplicate.

1982
M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
Table 1
Asp had a relevant anti-trypanosomal effect if compared to the sin-
gle GalNAc-Ser/Thr 1 and 2.
In order to verify the ability of compound 4, which displayed
Anti-trypanosomal activities of compounds 1–4 and
benznidazole (Bz) expressed as IC50
a
Compound
IC50 (mM)
higher tripanocidal activity, to enter the cell and kill the intracellu-
lar parasite, Vero cells were infected with T. cruzi and after 12 h of
incubation, parasites on the supernatant were removed and the
cells were treated with compound 4 to additional 24 h. Survival
and replication of intracellular amastigotes were evaluated. As
shown in the Figure 5, compound 4 was able to enter the cell
and was very efficient in eliminating the intracellular parasite in
a dose-dependent manner, comparable to benznidazole. Hence,
the significant capability to kill T. cruzi trypomastigotes and
amastigotes forms highlights compound 4 as a prototype for fur-
ther design of new anti-trypanosomal agents based on DKP
scaffold.
To verify whether the toxicity of the compounds was either spe-
cific to the parasite or extended to mammalian cell, compounds 1–
4 were screened against cultured mouse spleen cells. According to
Figure 6, none of the compounds were cytotoxic in the range of
500–7.8 lM, the tested concentrations for which anti-trypanoso-
mal activity was observed, showing, thus, the specific role of the
1
2
3
4
>0.5
>0.5
0.282 0.008
0.124 0.007
0.030 0.001
Bz
The values were calculated based on results from Fig-
ure that were obtained from three independent
experiments, each one performed in duplicate.
4
Results of parasite viability, measured based on a colorimetric
reaction with Chlorophenol red-b- -galactoside (CPRG) as sub-
strate for T. cruzi b-galactosidase, are summarised in Figure 4.
The concentrations of compounds corresponding to 50% anti-try-
panosomal activity are expressed as IC50 (Table 1).
As shown in Figure 4 and Table 1, amongst the tested com-
pounds, 4 displayed higher anti-trypanosomal activity against T.
cruzi Tulahuen strain, presenting an IC50 value of 124 lM. In fact,
D
compounds 3 and 4 on the parasites.
in spite of presenting lower activity than Bz in the concentrations
The fact that TcTS inhibition and anti-trypanosomal activities
verified for compounds 1–4 were not directly correlated suggest
that these compounds may act against T. cruzi by a mechanism
of action different from the inhibition of the TcTS enzyme, which
enables the adhesion and invasion of parasites to host cells, but
it is not supposed to have a role in parasite survival in culture.38
Moreover, considering the absence of cytotoxicity of compounds
1–4 against mouse spleen cells, a specific mode of anti-parasite ac-
tion rather than a generic cytotoxic effect may be proposed.
range from 0.125 to 0.015 mM, it displayed anti-trypanosomal
activity in the lower concentrations of 7.00 and 3.50
was not observed for Bz. Regarding the remaining compounds,
the glycosyl diketopiperazine 3 showed IC50 of 282 M, while very
lM, which
l
low activity was observed for compounds 1 and 2. Thus, these con-
siderable distinct anti-trypanosomal activities observed for glyco-
syl amino acids 1 and 2, and the glycosyl diketopiperazines 3 and
4 indicate that, differently from what was observed for TcTS inhi-
bition, the additional 2,5-DKPs heterocyclic system containing
Figure 5. Anti-trypanosomal activity of compounds 4 and Bz against amastigote forms of T. cruzi. (A) Representative images of Vero cells infected with T. cruzi and treated
with Bz, compound 4 or Medium. ⁄The arrows indicate parasites inside the cells. (B) Quantitative analysis of parasites per cell after treatment. Values represent the results
from two independent experiments, each one performed in triplicate.

M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
1983
Figure 6. Percentage of cell death caused by compounds 1–4 and Bz, evaluated against cultured mouse spleen cells. The data are representative of three independent
experiments, each one performed in duplicate.
Performance FLASH Chromatography system using 12 or 25 mm
flash cartridges with the indicated eluents. Nuclear magnetic reso-
nance spectra were recorded on Bruker Advance DRX 300
(300 MHz), DPX 400 (400 MHz) or DPX 500 (500 MHz) spectrome-
ters. Chemical shifts (d) are given in parts per million downfield
from tetramethylsilane. Assignments were made with the aid of
HMQC and COSY experiments. Accurate mass electrospray ioniza-
tion mass spectra (ESI-HRMS) were obtained using positive or neg-
ative ionization modes on a Bruker Daltonics MicroOTOF II ESI-TOF
mass spectrometer.
3. Conclusion
In summary, the aGalNAc-Ser/Thr have been successfully enclosed
into diketopiperazine scaffolds containing a carboxylic side chain, by
means of amino acid coupling/intramolecular cyclisation approach,
providing twofold functionalised compounds 3 and 4. In addition, we
have broadened the application of HgBr2 as a catalyst for glycosylation
reactions in distinct glycosyl donor and acceptor combinations, repre-
senting a comprehensive and effective method for synthesising glyco-
syl amino acids. In particular, the synthesis of aGalNAc core 1 and 2, as
analogues of T. cruzi mucins, was accomplished, as well as of bGalN3
serine glycoside 13 as an unpredicted result.
4.2. Synthesis of glycosyl amino acids
Mucin-related
aGalNAc amino acids and diketopiperazines
4.2.1. General procedure for glycosylation reactions
showed different profiles in the biological assays. In the TcTS inhi-
bition assay, 1 and 2 displayed higher activity (57% and 79%) com-
pared to cyclic analogues, whilst 3 and 4 were much more active in
A mixture of 2-azido-3,4,6-tri-O-acetyl-2-deoxy-a-D-galacto-
pyranosyl chloride 11 and N-(fluoren-9-ylmethoxycarbonyl)-ser-
ine/threonine benzyl ester 12/14 (prepared from commercially
available amino acid Fmoc-serine/threonine by treatment with
caesium carbonate and benzyl bromide in DMF)41 in 1,2-dichloro-
ethane (2.0 mL) was refluxed for 20 or 24 h with mercuric bromide
(2.25 equiv) and the consumption of starting materials was fol-
lowed by TLC (EtOAc/toluene 2:8, v/v; EtOAc/hexane 7:3, v/v).
The resulting amber mixture was concentrated in vacuo and the
residue was purified by a silica gel chromatography column
(EtOAc/toluene 2:8, v/v; EtOAc/hexane 7:3, v/v).
anti-trypanosomal experiments (IC50 282 and 124 lM) than single
glycosyl amino acids. Although anti-trypanosomal activity appears
to be independent of TcTS inhibition mechanism, it is also not re-
lated to a generic cytotoxic effect, so that these findings point
out the
aGalNAc diketopiperazines as prototypes for designing
new anti-trypanosomal agents.
4. Experimental
4.1. General
4.2.1.1. N-(Fluoren-9-ylmethoxycarbonyl)-(2-azido-2-deoxy-3,
All chemicals were purchased as reagent grade and used with-
out further purification. Solvents were dried according to standard
4,6-tri-O-acetyl-
and N-(fluoren-9-ylmethoxycarbonyl)-(2-azido-2-deoxy-3,
4,6-tri-O-acetyl-b- -galactopyranosyl)- -serine benzyl ester
a-D-galactopyranosyl)-L-serine benzyl ester 9
methods.39 MuNANA (20-(4-methylumbelliferyl)-
a-D-N-acetylneu-
D
L
raminic acid sodium salt) was acquired from Toronto Research
Chemicals Inc. The trans-sialidase used in this study was a His-
tagged 70 kDa recombinant material truncated to remove C-termi-
nal repeats but retaining the catalytic N-terminal domain of the
enzyme.40 Reactions were monitored by thin layer chromatogra-
phy (TLC) on 0.25 nm precoated silica gel plates (Whatman, AL
SIL G/UV, aluminium backing) with the indicated eluents. Com-
pounds were visualized under UV light (254 nm) and/or dipping
in ethanol/sulfuric acid (95:5, v/v), followed by heating the plate
for a few minutes. Column chromatography was performed on sil-
ica gel 60 (Fluorochem, 35–70 mesh) or on a Biotage Horizon High-
13. According to the general procedure described in Section 4.2.1, the
reaction mixture of 11 (204 mg, 0.58 mmol) and 12 (132 mg,
0.32 mmol), in 1,2-dichloroethane (2.0 mL) was refluxed for 20 h with
mercuric bromide (258 mg, 0.71 mmol), affording both anomers (a/b
12:1) as amorphous solids (52%) after purification by silica gel chroma-
tography column (EtOAc/toluene 2:8, v/v).
a
9 (112 mg, 0.153 mmol, 48%). dH (CDCl3, 300 MHz) 7.69 (2H,
d, J 7.4 Hz, CH Fmoc arom.), 7.55 (2H, d, J 7.2 Hz, CH Fmoc arom.),
7.35–7.22 (9H, m, CH Bn arom., CH Fmoc arom.), 5.91 (1H, d, J
8.0 Hz, NH Ser), 5.31 (1H, d, J3,4 2.6 Hz, H-4), 5.22 (1H, d, J3,4
2.7 Hz, H-3), 5.17 (2H, s, CH2 Bn), 4.79 (1H, d, J1,2 3.2 Hz, H-1),

1984
M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
4.54 (1H, m, CH Ser), 4.33 (2H, d, J 7.1 Hz, CH2 Fmoc), 4.17 (1H, t, J
7.1 Hz, CH Fmoc), 4.09 (1H, dd, J 2.6 Hz, 10.8 Hz, H-5), 4.02–3.85
(4H, m, H-6a, H-6b, CH2 Ser), 3.51 (1H, dd, J1,2 3.3 Hz, J2,3 11.1 Hz,
H-2), 2.07, 2.00, 1.89 (9H, 3s, COCH3).
the glycosyl amino acid 10 (67 mg, 0.090 mmol) was dissolved in a
mixture of THF/Ac2O/AcOH (4.5 mL, 3:2:1), followed by addition of
zinc dust (76.5 mg, 1.17 mmol, 12 equiv) and saturated aqueous 10%
CuSO4 solution (100 lL). The reaction mixture was then stirred for
b 13 (9 mg, 0.012 mmol, 4%). dH (CDCl3, 300 MHz) 7.77 (2H, d, J
7.5 Hz, CH Fmoc arom.), 7.61 (2H, m, CH Fmoc arom.), 7.43–7.28
(9H, m, CH Bn arom., CH Fmoc arom.), 5.85 (1H, d, J 8.4 Hz, NH
Ser), 5.32 (1H, d, J3,4 3.3 Hz, H-4), 5.25 (2H, AB, JAB 12.3 Hz, CH2
Bn), 4.76 (1H, dd, J3,4 3.3 Hz, J2,3 10.7 Hz, H-3), 4.61 (1H, m, CH Ser),
4.46–4.37 (3H, m, CH2a Ser, CH2 Fmoc), 4.32 (1H, d, J1,2 8.0 Hz, H-
1), 4.23–4.20 (1H, m, CH Fmoc), 4.12–4.07 (2H, m, H-6a, H-6b),
3.93 (1H, dd, J 3.3 Hz, 10.3 Hz, CH2b Ser), 3.77 (1H, t, J 6.7 Hz, H-5),
3.67 (1H, dd, J1,2 8.0 Hz J2,3 10.7 Hz, H-2), 2.16, 2.06, 2.02 (9H, 3s,
COCH3). dC (CDCl3, 75 MHz) 170.3, 170.0, 169.7, 169.4 (COCH3,
COCH2Bn), 155.9 (CO Fmoc), 143.8, 143.7, 141.3, 141.3 (Cquat.
Fmoc), 135.2 (Cquat. Bn), 128.6, 128.5, 127.7, 127.1, 125.1, 120.0
(CH Fmoc arom., CH Bn arom.), 102.6 (C-1), 70.9 (C-5), 70.8 (C-3),
70.1 (CH2 Ser), 67.6 (CH2Bn), 67.3 (CH2 Fmoc), 66.2 (C-4), 61.1 (C-
6), 60.8 (C-2), 54.3 (CH Ser), 47.1 (CH Fmoc), 20.6 (COCH3). ESI-
HRMS: calcd for C37H39N4O12 [M+H]+ 731.2559, found 731.2576.
2 h at room temperature and was, subsequently, filtered through Celite
and co-evaporated with toluene. Purification by chromatography col-
umn (EtOAc/hexane 3:2, v/v) afforded the product 8 as a pale viscous
oil (42.5 mg, 0.056 mmol, 62%). dH (CDCl3, 300 MHz) 7.71 (2H, d, J
7.5 Hz, CH Fmoc arom.), 7.57 (2H, d, J 7.3 Hz, CH Fmoc arom.), 7.37–
7.24 (9H, m, CH Bn arom. e CH Fmoc arom.), 5.76 (1H, d, J 9.7 Hz,
NH Thr), 5.62 (1H, d, J 9.4 Hz, NHCO), 5.31 (1H, d, J3,4 2.2 Hz, H-4),
5.12 (1H, d, J 11.9 Hz, CH2a Bn), 5.03–4.98 (2H, m, CH2b Bn, H-3),
4.73 (1H, d, J 3.6 Hz, H-1), 4.50–4.37 (4H, m, CH2 Fmoc,
aCH Thr,
bCH Thr), 4.22–4.10 (3H, m, CH Fmoc, H-5, H-2), 4.02–3.95 (2H, m,
H-6a, H-6b), 2.10 (3H, s, COCH3), 1.96, 1.94, 1.91 (9H, 3s, COCH3),
1.23 (3H, d, J 6.4 Hz, CH3 Thr).
4.2.1.5. 2-Acetamido-2-deoxy-
1. A solution of N-(fluoren-9-ylmethoxycarbonyl)-(2-acetamido-
2-deoxy-3,4,6-tri-O-acetyl- -galactopyranosyl)- -serine benzyl
ester 7 (25 mg, 0.033 mmol) in MeOH (1.0 mL) was treated with
glacial AcOH (100 L) and 10% Pd/C (11 mg) for removal of O-Bn
a-D-galactopyranosyl-L-serine
a
-D
L
l
4.2.1.2. N-(Fluoren-9-ylmethoxycarbonyl)-(2-azido-2-deoxy-3,4,
and N-Fmoc groups. The reaction mixture was stirred and kept un-
der H2 (ꢁ1.5 atm) for 18 h and then, filtered through Celite, con-
centrated in vacuo and purified by chromatography column
(DCM/MeOH 8:2 v/v). The obtained product (9.6 mg, 0.022 mmol,
66%) was dissolved in MeOH (0.5 mL) and the pH was raised by
addition of 1 M NaOMe in MeOH. The reaction mixture was stirred
for 3 h, and then neutralized with Dowex 50WX8–200 resin. Filtra-
tion and concentration of the reaction mixture gave the product 1
as a colourless amorphous solid (5.6 mg, 0.017 mmol, 80%). dH
(D2O, 300 MHz) 4.78 (1H, d, J1,2 3.8 Hz, H-1), 4.06–3.96 (3H, m,
H-3, H-2, CH2a Ser), 3.85–3.73 (4H, m, CH2b Ser, H-4, H-5, CH
Ser), 3.63–3.60 (2H, m, H-6a, H-6b), 1.88 (3H, s, NHCH3CO). dC
(D2O, 75 MHz) 98.9 (C-1), 72.0 (C-5), 69.1, 68.2, 67.8, 66.0 (C-4,
CH Ser, C-3, CH2 Ser), 61.6 (C-6), 49.9 (C-2), 21.4 (NHCOCH3). ESI-
HRMS: calcd for C11H21N2O8 [M+H]+ 309.1292, found 309.1293.
6-tri-O-acetyl-a-D-galactopyranosyl)-L-threonine benzyl ester
10. According to the general procedure described in Section 4.2.1,
the reaction mixture of 11 (100 mg, 0.286 mmol) and 14 (62 mg,
0.143 mmol), in 1,2-dichloroethane (2.0 mL) was refluxed for
24 h with mercuric bromide (115 mg, 0.319 mmol), affording the
a
anomer 10 as an amorphous solid (56 mg, 0.075 mmol, 54%) after
purification by silica gel chromatography column (EtOAc/toluene
2:8, v/v). dH (CDCl3, 500 MHz) 7.70 (2H, d, J 7.5 Hz, CH Fmoc arom.),
7.55 (2H, d, J 7.3 Hz, CH Fmoc arom.), 7.34–7.22 (9H, m, CH Bn
arom, CH Fmoc arom.), 5.65 (1H, d, J 9.3 Hz, NH Thr), 5.37 (1H, d,
J3,4 2.9 Hz, H-4), 5.22–5.10 (3H, m, J2,3 11.1 Hz, J3,4 3.0 Hz, JA,B
12.2 Hz, H-3 e CH2 Bn), 4.83 (1H, d, J1,2 3.4 Hz, H-1), 4.40 (2H, t, J
7.0 Hz, CH2 Fmoc), 4.35 (1H, dd, J 7.5 Hz, 10.4 Hz,
aCH Thr), 4.27
(1H, dd, J 7.3 Hz, 10.6 Hz, bCH Thr), 4.21–4.13 (2H, m, CH Fmoc,
H-5), 4.00 (2H, d, J 6.5 Hz, H-6a, H-6b), 3.52 (1H, dd, J1,2 3.4 Hz,
J2,3 11.1 Hz, H-2), 2.08, 2.00, 1.96 (9H, 3s, COCH3), 1.27 (3H, d, J
6.3 Hz, CH3 Thr).
4.2.1.6. 2-Acetamido-2-deoxy-
2. N-(Fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-deoxy-
3,4,6-tri-O-acetyl- -galactopyranosyl)- -threonine benzyl ester
8 (25 mg, 0.033 mmol) was dissolved in MeOH (1.0 mL), followed
by treatment with glacial AcOH (100 L) and 10% Pd/C (10 mg)
a-D-galactopyranosyl-L-threonine
a-D
L
4.2.1.3. N-(Fluoren-9-ylmethoxycarbonyl)-(2-acetamido–deoxy-
3,4,6-tri-O-acetyl-
7. N-(Fluoren-9-ylmethoxycarbonyl)-(2-azido-2-deoxy-3,4,6-tri-
O-acetyl- -galactopyranosyl)- -serine benzyl ester (92 mg,
a-D-galactopyranosyl)-L-serine benzyl ester
l
for removal of O-Bn and N-Fmoc groups. The reaction mixture
was stirred and kept under H2 (ꢁ1.5 atm) for 15 h and then, fil-
tered through Celite, concentrated in vacuo and purified by chro-
matography column (DCM/MeOH 8:2 v/v). The obtained product
(5.0 mg, 0.011 mmol, 35.4%) was dissolved in MeOH (0.5 mL) and
the pH was raised by addition of 1 M NaOMe in MeOH. The reac-
tion mixture was stirred for 3 h, and then neutralized with Dowex
50WX8–200 resin. Filtration and concentration of the reaction
mixture gave the product 2 as a colourless amorphous solid
(3.4 mg, 0.010 mmol, 90%). dH (D2O, 500 MHz) 4.82 (1H, m, H-1),
a-
D
L
9
0.126 mmol, 1 equiv), previously washed with aqueous 5% EDTA
solution to remove mercuric remnants, was dissolved in a mixture
of THF/Ac2O/AcOH (5 mL, 3:2:1), followed by addition of zinc dust
(102 mg, 1,55 mmol, 12 equiv). The reaction mixture was treated
with saturated aqueous 10% CuSO4 solution (100 lL) and stirred
for 2 h at room temperature. The mixture was filtered through Cel-
ite and co-evaporated with toluene. Purification by chromatogra-
phy column (EtOAc/hexane 3:2, v/v) afforded the product 7 as a
pale viscous oil (63 mg, 0.084 mmol, 67%). dH (CDCl3, 500 MHz)
7.71 (2H, d, J 7.5 Hz, CH Fmoc arom.), 7.55 (2H, d, J 7.0 Hz, CH Fmoc
arom.), 7.35–7.24 (9H, m, CH Bn arom., CH Fmoc arom.), 5.86 (1H,
d, J 7.8 Hz, NH Ser), 5.60 (1H, d, J 9.1 Hz, NHCO), 5.24 (1H, d, J3,4
2.1 Hz, H-4), 5.13 (2H, AB, JAB 12.0 Hz, CH2 Bn), 4.98 (1H, dd, J2,3
11.1 Hz, J3,4 2.1 Hz, H-3), 4.70 (1H, apparent s, H-1), 4.53 (1H, m,
CH Ser), 4.46 (1H, apparent t, J2,3 11.2 Hz, H-2), 4.38 (2H, d, J
6.5 Hz, CH2 Fmoc), 4.17 (1H, t, J 6.6 Hz, CH Fmoc), 4.07–3.85 (5H,
m, H-5, H-6a, H-6b, CH2 Ser), 2.09, 1.94, 1.91, 1.84 (12H, 4 s,
COCH3).
4.07–3.82 (4H, m, H-4, H-3, H-2, CHa Thr), 3.62–3.57 (3H, m, H-
5, H-6a, H-6b), 3.50–3.48 (1H, m, CHb Thr), 1.90 (3H, s, NHCO),
1.25 (3H, d, J 6,8 Hz, CH3 Thr). dC (D2O, 75 MHz) 99.8 (C-1), 72.2
(bCH Thr), 71.6 (C-5), 69.2, 68.7 (C-4, C-3), 61.4 (C-6,
50.0 (C-2), 21.8 (NCOCH3), 17.9 (CH3 Thr). ESI-HRMS: calcd for
12H23N2O8 [M+H]+ 323.1449, found 323.1445.
aCH Thr),
C
4.3. Synthesis of glycodipeptides
4.3.1. N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-(O-tBu)-L-aspartil
-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
yl)- -serine benzyl ester 5
N-(Fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-deoxy-3,4,6
-tri-O-acetyl- -galactopyranosyl)- -serine benzyl ester 7 (60 mg,
a-D-galactopyranos-
L
4.2.1.4. N-(Fluoren-9-ylmethoxycarbonyl)-(2-acetamido-2-de-
oxy-3,4,6-tri-O-acetyl-a-D-galactopyranosyl)-L-threonine benzyl
a
-D
L
ester 8. Following the same procedure described for compound 9,

M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
1985
0.080 mmol) was treated with 20% piperidine in DMF (0.5 mL) for
4.4. Synthesis of glycosyl diketopiperazines
removal of the N-Fmoc group. The reaction mixture was stirred for
1 h at room temperature and then purified by chromatography col-
umn (EtOAc; MeOH/DCM 2:8 v/v) to afford the product 15 as a pale
oil (33.5 mg, 0.063 mmol, 78%). Subsequently, a solution of 15
(33.5 mg, 0.063 mmol) in DMF (1.5 mL) was treated with N-[(9H-
Fluoren-9-ylmethoxy)carbonyl]-(O-tBu)-L-aspartate 17 (32.7 mg,
0.079 mmol, 1.25 equiv), PyBOP (41.4 mg, 0.079 mmol, 1.25 equiv)
and HOBt (10.7 mg, 0.079 mmol, 1.25 equiv), and then with the
4.4.1. c[(O-tBu)-
L
-aspartil-O-(2-acetamido-2-deoxy-3,4,6-tri-O-
-seryl)] 18
acetyl- -galactopyranosyl)-L
a
-
D
Compound 5 (54 mg, 0.058 mmol) was treated with 20% piper-
idine in DMF (0.5 mL) and the reaction mixture was stirred for
14 h, being the consumption of the starting material followed by
TLC (EtOAc/hexane, 7:3 v/v). The mixture was then concentrated
in vacuo and the residue was purified by chromatography column
(EtOAc; MeOH/CH2Cl2 1:9 v/v). The product 18 was obtained as a
pale oil in 48% yield (16.5 mg, 0.028 mmol). dH (CDCl3, 300 MHz)
6.95 (1H, d, J 9.5 Hz, NHCO), 6.60 (1H, apparent s, NH Asp), 6.50
(1H, d, J 8.9 Hz, NH Ser), 5.29 (1H, d, J3,4 3.1 Hz, H-4), 5.13 (1H,
dd, J3,4 3.1 Hz, J2,3 11.3 Hz, H-3), 4.87 (1H, d, J1,2 3.8 Hz, H-1), 4.57
(1H, dd, J1,2 3.6 Hz, J2,3 11.4 Hz, H-2), 4.31 (1H, apparent d, J
9.1 Hz, CH Asp), 4.24–4.20 (1H, m, J 9.2 Hz, CH Ser), 4.16–3.99
(4H, m, CH2a Ser, H-5, H-6a, H-6b), 3.61 (1H, t, J 9.2 Hz, CH2b Ser),
2.89 (1H, dd, J 3.1 Hz, 17.3 Hz, CH2a Asp), 2.59 (1H, dd, 9.2 Hz,
17.5 Hz, CH2b Asp), 2.10, 2.01, 1.93, 1.85 (12H, 4s, COCH3), 1.39
(9 H, s, CH3 tBu). dC (CDCl3, 75 MHz) 98.9 (C-1), 69.9 (CH2 Ser),
68.3 (C-3), 67.2, 66.5 (C-4, C-5), 62.4 (C-6), 51.0 (CH Ser, CH Asp),
46.8 (C-2), 39.7 (CH2 Asp), 28.6 (CH3 tBu), 23.5 (NCOCH3), 21.2
(OCOCH3). ESI-HRMS: calcd for C25H38N3O13 [M+H]+ 588.2399,
found 588.2395.
DIEA (25 lL, 20.16 mg, 0.16 mmol, 2.5 equiv) to initiate the reac-
tion. The mixture was allowed to stir for 16 h at room temperature.
Purification of the crude mixture by chromatography column
(EtOAc/hexane, 7:3 v/v) gave the product 5 as an amorphous solid
(54 mg, 0.058 mmol, 93%). dH (CDCl3, 300 MHz) 7.69 (2H, d, J
7.5 Hz, CH Fmoc arom.), 7.53 (2H, d, J 6.9 Hz, CH Fmoc arom.),
7.36–7.22 (9H, m, CH Bn arom., CH Fmoc arom.), 6.03–5.97 (2H,
m, NH Asp, NH Ser), 5.23 (1H, d, J 2.7 Hz, H-4), 5.10 (2H, s, CH2
Bn), 5.02 (1H, dd, J3,4 3.0 Hz, J2,3 11.2 Hz, H-3), 4.75 (1H, d, J1,2
3.4 Hz, H-1), 4.73–4.67 (1H, m, CH Ser), 4.53–4.41 (2H, m, CH
Asp, H-2), 4.36 (2H, d, J 7.3 Hz, CH2 Fmoc), 4.16 (1H, t, J 7.0 Hz,
CH Fmoc), 4.06–3.95 (3H, m, H-5, H-6a, H-6b), 3.87–3.83 (2H, m,
CH2 Ser), 2.80 (1H, dd, J 4.8 Hz, J 16.9 Hz, CH2a Asp), 2.60 (1H, dd,
J 6.1 Hz, 16.9 Hz, CH2b Asp), 2.03, 1.91, 1.86, 1.83 (12H, 4s, COCH3),
1.37 (9H, s, CH3 tBu). dC (CDCl3, 75 MHz) 128.2, 127.8, 127.2, 126.5,
124.5, 119.4 (CH Fmoc arom., CH Bn arom.), 98.0 (C-1), 67.5 (CH2
Ser), 67.4 (C-3), 67.1 (CH2Bn), 66.8 (CH2 Fmoc), 66.6 (C-5, C-4),
61.4 (C-6), 52.2 (CH Ser), 50.6 (C-2), 50.5 (CH Asp), 47.0 (CH Fmoc),
36.7 (CH2 Asp), 27.4 (CH3 tBu), 22.4 (NCOCH3), 20.0 (OCOCH3). ESI-
HRMS: calcd for C47H56N3O16 [M+H]+ 918.3655, found 918.3655.
4.4.2. c[(O-tBu)-
L
-aspartil-O-(2-acetamido-2-deoxy-3,4,6-tri-O-
-treonyl)] 19
acetyl- -galactopyranosyl)-L
a
-
D
Compound 6 (35 mg, 0.038 mmol) was treated with 20% piper-
idine in DMF (0.5 mL) and the reaction mixture was stirred for
15 h, being the consumption of the starting material followed by
TLC (EtOAc/hexane, 7:3 v/v). The mixture was then concentrated
in vacuo and the residue was purified by chromatography column
(EtOAc; MeOH/CH2Cl2 1:9 v/v). The product 19 was obtained as a
pale oil in 52% yield (12 mg, 0.023 mmol). dH (CDCl3, 300 MHz)
7.09 (1H, d, J 1.2 Hz, NH Thr), 6.97 (1H, d, J 1.7 Hz, NH Asp), 6.65
(1H, d, J 9.2 Hz, NHCO), 5.32 (1H, dd, J4,5 1.0 Hz, J3,4 3.1 Hz, H-4),
5.07 (1H, dd, J3,4 3.1 Hz, J2,3 11.5 Hz, H-3), 4.96 (1H, d, J1,2 3.6 Hz,
H-1), 4.48 (1H, ddd, J1,2 3.6 Hz, J2,3 11.5 Hz, J 9.4 Hz, H-2), 4.29
(1H, apparent d, J 10.1 Hz, CH Asp), 4.22–4.15 (2H, m, H-5, bCH
4.3.2. N-[(9H-Fluoren-9-ylmethoxy)carbonyl]-(O-tBu)-
-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy- -galactopyranos-
yl)- -threonine benzyl ester 6
L-aspartil
a-D
L
Following the same procedure described for compound 5, the
glycosyl amino acid 8 (53.2 mg, 0.070 mmol) was treated with
20% piperidine in DMF (0.5 mL) and the reaction mixture was stir-
red for 1 h at room temperature. Purification of the mixture by col-
umn chromatography (EtOAc; MeOH/DCM 2:8 v/v) afforded the
product 16 as a pale oil (26.2 mg, 0.0486 mmol, 69%). Subse-
quently, a solution of 16 (26.2 mg, 0.0486 mmol) in DMF (1.0 mL)
was treated with N-[(9H-fluoren-9-ylmethoxy)carbonyl]-
Thr), 4.02 (2H, d, J5,6 6.5 Hz, H-6a, H-6b), 3.94 (1H, m,
aCH Thr),
3.00 (1H, dd, J 2.8 Hz, 17.2 Hz, CH2a Asp), 2.62 (1H, dd, 10.1 Hz,
17.2 Hz, CH2b Asp), 2.09, 1.98, 1.90, 1.88 (12H, 4s, CH3CO), 1.40
(9H, s, CH3 tBu), 1.35 (3H, d, J 6.6 Hz, CH3 Thr). dC (CDCl3,
75 MHz) 170.9, 170.6, 170.5, 170.4 (COCH3, bCO Asp), 167.0,
165.3 (CO Thr, CO Asp), 99.5 (C-1), 82.6 (Cquat. tBu), 73.9 (bCH
(O-tBu)-
(31.2 mg, 0.060 mmol, 1.25 equiv) and HOBt (8.10 mg, 0.060 mmol,
1.25 equiv) and then, with the DIEA (22 L, 15 mg, 0.12 mmol,
L-aspartate 17 (24.7 mg, 0.060 mmol, 1.25 equiv), PyBOP
l
2.5 equiv) to initiate the reaction. The mixture was allowed to stir
for 15 h at room temperature and the purification of the crude mix-
ture by chromatography column (EtOAc/hexane, 7:3 v/v) gave the
product 6 as an amorphous solid (38.9 mg, 0.0417 mmol, 86%). dH
(CDCl3, 300 MHz) 7.69 (2H, d, J 7.5 Hz, CH Fmoc arom.), 7.53 (2H,
t, J 6.4 Hz, CH Fmoc arom.), 7.36–7.22 (9H, m, CH Bn arom., CH
Fmoc arom.), 6.17 (1H, dd, J 1.4 Hz, 9.5 Hz, NH Thr), 6.06 (1H, d, J
7.5 Hz, NH Asp), 5.21 (1H, d, J 2.3 Hz, H-4), 5.10–4.96 (3H, m, JAB
12.0 Hz, CH2 Bn, H-3), 4.72 (1 H, d, J1,2 3.5 Hz, H-1), 4.56–4.53
Thr), 68.1 (C-3), 67.5 (C-4), 67.4 (C-5), 62.0 (C-6), 58.9 (aCH Thr),
51.5 (CH Asp), 48.0 (C-2), 39.1 (CH2 Asp), 28.0 (CH3 tBu), 22.9
(NCOCH3), 20.7 (OCOCH3), 17.0 (CH3 Thr). ESI-HRMS: calcd for
C
26H40N3O13 [M+H]+ 602.2556, found 602.2558.
4.4.3. c(
l)- -seryl)] 3
Compound 18 (16 mg, 0.027 mmol) was dissolved in MeOH
L-Aspartil-O-(2-acetamido-2-deoxy-a-D-galactopyranosy
L
(0.5 mL) and the pH was raised by addition of 1 M NaOMe in
MeOH. The reaction mixture was stirred for 3 h, and then neutral-
ized with Dowex 50WX8–200 resin. Filtration and concentration of
the reaction mixture gave an intermediate (11.5 mg, 0.025 mmol,
91.5%), which was subsequently treated with TFA/DCM (1:1 v/v)
solution (0.5 mL) and stirred for 24 h. After neutralization with
Dowex 50WX8–200 resin, the final product 3 was obtained as a
pale amorphous solid (9 mg, 0.022 mmol, 90%). dH (D2O,
300 MHz) 5.10 (1H, d, J1,2 3.6 Hz, H-1), 4.51 (1H, d, J 8.4 Hz, CH
Ser), 4.43 (1H, dd, J 4.6 Hz, J 9.2 Hz, CH Asp), 3.98 (1H, dd, J1,2
3.7 Hz, J2,3 10.9 Hz, H-2), 3.85 (1H, d, J3,4 3.1 Hz, H-4), 3.79 (1H,
dd, J3,4 3.4 Hz, J2,3 11.0 Hz, H-3), 3.74–3.54 (5H, m, CH2 Ser, H-5,
H-6a, H-6b), 2.98 (1H, dd, J 4.6 Hz, J 17.6 Hz, CH2a Asp), 2.77 (1H,
(2H, m, CH Asp,
aCH Thr), 4.47–4.37 (3H, m, H-2, CH2 Fmoc),
4.23 (1H, dd, J 1.8 Hz, 6.6 Hz, bCH Thr), 4.18 (1H, t, J 7.2 Hz, CH
Fmoc), 4.06–4.04 (1H, m, H-5), 3.92–3.89 (2H, m, H-6a, H-6b),
2.77 (1H, dd, J 4.7 Hz, 16.9 Hz, CH2a Asp), 2.61 (1H, dd, J 6.4 Hz,
16.9 Hz, CH2b Asp), 2.06, 1.89, 1.88, 1.85 (12H, 4s, COCH3), 1.38
(9H, s, CH3 tBu), 1.17 (3H, d, J 6.4 Hz, CH3 Thr). dC (CDCl3,
75 MHz) 128.8, 128.6, 127.7, 127.1, 125.0, 120.0 (CH Fmoc arom.,
CH Bn arom.), 98.9 (C-1), 76.0 (bCH Thr), 68.2 (C-3), 68.0 (CH2Bn),
67.7 (CH2 Fmoc), 67.5 (C-4), 67.2 (C-5), 62.2 (C-6), 58.5 (aCH Thr),
51.5, 51.2 (C-2, CH Asp), 47.0 (CH Fmoc), 37.4 (CH2 Asp), 28.1 (CH3
tBu), 23.3 (NCOCH3), 20.7 (OCOCH3), 18.5 (CH3Thr). ESI-HRMS:
calcd for C48H58N3O16 [M+H]+ 932.3812, found 932.3831.

1986
M. B. Martins-Teixeira et al. / Bioorg. Med. Chem. 21 (2013) 1978–1987
dd, J 4.3 Hz, J 17.6 Hz, CH2b Asp), 1.91 (3H, s, NHCOCH3). dC (D2O,
75 MHz) 91.0 (C-1), 68.6, 67.9 (C-4, C-3, CH2 Ser), 61.2 (C-5, C-6)
52.0 (CH Ser, CH Asp), 50.0 (C-2), 36.5 (CH2 Asp), 21.8 (NCOCH3).
ESI-HRMS: calcd for C15H21N3O10Cl [Mꢂ2H+Cl]ꢂ 438.0921, found
438.1462.
4.5.3. In vitro anti-trypanosomal assays
To evaluate the anti-trypanosomal activity in both trypomasti-
gote and amastigote forms of T. cruzi, LLC-MK2 cell strain (ATCC)
were resuspended in RPMI medium without phenol red at
2 ꢀ 103 cells/well and were cultured in 96-well plates for 24 h.
The cells were infected with 1 ꢀ 104 trypomastigotes forms of T.
cruzi Tulahuen strain stably expressing the b-galactosidase gene
from Escherichia coli (Tulahuen-lacZ), and after 24 h, compounds
1–4 or benznidazole were added at concentrations of 0.5, 0.25,
0.125, 0.06, 0.03, 0.015, 0.007 and 0.0035 mM. After 4 days of cul-
4.4.4. c(
l)- -treonyl)] 4
Compound 19 (12 mg, 0.023 mmol) was dissolved in MeOH
L-Aspartil-O-(2-acetamido-2-deoxy-a-D-galactopyranosy
L
(0.5 mL) and the pH was raised by addition of 1 M NaOMe in
MeOH. The reaction mixture was stirred for 3 h, and then neutral-
ized with Dowex 50WX8-200 resin. Filtration and concentration of
the reaction mixture gave an intermediate (9.5 mg, 0.02 mmol,
quantitative), which was subsequently treated with TFA/DCM
(1:1 v/v) solution (0.5 mL) and stirred for 24 h. After neutralization
with Dowex 50WX8-200 resin, the final product 4 was obtained as
ture, 50
ll of PBS containing 0.5% of Triton X-100 and 100
lM
Chlorophenol Red-b-
D-galactoside (CPRG—Sigma) were added.
Plates were incubated at 37 °C for 4 h and absorbance was read
at 570 nm.43 Results of parasite viability were measured based
on the catalysis of CPRG by b-galactosidase.
Some of the same concentrations described above were also
used to evaluate anti-trypanosomal activity of compound 4 on
amastigote forms. To this purpose, Vero cells strains (ATCC) were
cultured at 2 ꢀ 104/well on 8-well camber slides. After 12 h, the
cells were infected with 2 ꢀ 105/well trypomastigote forms of T.
cruzi Tulahuen strain for additional 12 h. The cells were washed
with PBS to remove parasites on the supernatant and incubated
with benznidazole (Bz) or compound 4 for 24 h at 37 °C. Cultures
were stained by Giemsa dye and evaluated by optical microscopy.
Anti-trypanosomal activity was determined by counting the
amount of parasites/cell in at least 200 cells/well. The experiment
was performed in triplicates.
a
pale amorphous solid (7 mg, 0.016 mmol, 84%). dH (D2O,
300 MHz) 5.07 (1H, d, J1,2 3.6 Hz, H-1), 4.37 (1H, dd, J 4.3 Hz, J
8.8 Hz, CH Asp), 4.18 (1H, m, CH Thr), 4.08 (1H, m, bCH Thr),
a
4.02 (1H, m, H-4), 3.93 (1H, dd, J1,2 3.7 Hz, J2,3 7.0 Hz, H-2), 3.78
(1H, dd, J3,4 3.7 Hz, J2,3 7.4 Hz, H-3), 3.66–3.53 (3H, m, H-5, H-6a,
H-6b), 2.94 (1H, dd, J 4.6 Hz, J 17.4 Hz, CH2a Asp), 2.75 (1H, dd, J
4.5 Hz, J 17.4 Hz, CH2b Asp), 1.89 (3H, s, NHCOCH3), 1.14 (3H, d, J
6.3 Hz, CH3 Thr). dC (D2O, 75 MHz) 90.9 (C-1), 76.1 (bCH Thr),
70.6 (C-5), 69.2 (C-4), 67.7 (C-3), 61.1 (C-6), 57.2 (aCH Thr), 50.6
(CH Asp), 49.7 (C-2), 36.4 (CH2 Asp), 21.6 (NCOCH3), 15.1 (CH3
Thr). ESI-HRMS: calcd for C16H24N3O10 [MꢂH]ꢂ 418.1467, found
418.1497.
Acknowledgments
4.5. Biological assays
We acknowledge the financial support and fellowships from FA-
PESP (Fundação de Amparo à Pesquisa do Estado de São Paulo—
Brazil) and CAPES (Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior). We are grateful to Peterson de Andrade and
Marcelo Rodrigues de Carvalho for TcTS inhibition assays, Professor
Carlos H.T.P. Silva and Jonathan Almeida for docking calculations,
Vinícius Palaretti for NMR analyses, José Carlos Tomaz for ESI-MS
analyses, Professor Rose Mary Z.G. Naal for fluorimeter facilities
and Professor Sergio Schenkman for recombinant TcTS.
4.5.1. Fluorimetric TcTS inhibition assays
trans-Sialidase used in this study was a His-tagged 70 kDa re-
combinant material truncated to remove C-terminal repeats but
retaining the catalytic N-terminal domain of the enzyme.40 Inhibi-
tion was assessed using the continuous fluorimetric assay de-
scribed by Douglas and co-workers.35 Briefly, the assays were
performed in triplicate in 96-well plates containing phosphate buf-
fer solution at pH 7.4 (25
(25 L) and inhibitor solution (25
ture was incubated for 10 min at 26 °C followed by addition of
MuNANA (Km = 0.68 mM35 25
L of a 0.4 mM solution giving an as-
l
L), recombinant enzyme solution
l
lL of 4.0 mM solution). This mix-
Supplementary data
l
say concentration of 0.1 mM). The fluorescence of the released
product (Mu) was measured after 10 min, with excitation and
emission wavelengths of 360 and 460 nm, respectively, and the
data were analysed with GraphPad Prism software version 4.0
(San Diego, CA, USA). Inhibition percentages were calculated by
the equation: % I = 100 ꢀ [1 ꢂ (Vi/V0)], where Vi is the velocity in
the presence of inhibitor and V0 is the velocity in absence of
inhibitor.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.01.027.
These data include MOL files and InChiKeys of the most important
compounds described in this article.
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