'Click chemistry' synthesis of a library of 1,2,3-triazole-substituted galactose derivatives and their evaluation against Trypanosoma cruzi and its cell surface trans-sialidase

Article abstract of DOI:10.1016/j.bmc.2010.02.053

Trypanosoma cruzi trans-sialidase (TcTS) plays a key role in the recognition and invasion of host cells and in enabling the parasite to escape the human immune response. To explore this potential drug target, we have synthesized a small library of substra

Full text of DOI:10.1016/j.bmc.2010.02.053

Bioorganic & Medicinal Chemistry 18 (2010) 2412–2427
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
‘Click chemistry’ synthesis of a library of 1,2,3-triazole-substituted
galactose derivatives and their evaluation against Trypanosoma cruzi
and its cell surface trans-sialidase
a
a
b
b
Ivone Carvalho a, , Peterson Andrade , Vanessa L. Campo , Paulo M. M. Guedes , Renata Sesti-Costa ,
*
João S. Silva b, Sergio Schenkman c, Simone Dedola d, Lionel Hill e, Martin Rejzek d, Sergey A. Nepogodiev d,
Robert A. Field d,
*
a Faculdade de Ciências Farmacêuticas de Ribeirão Preto, USP, Av. Café S/N, CEP 14040-903, Ribeirão Preto, SP, Brazil
b Faculdade de Medicina de Ribeirão Preto, USP, Av. Bandeirantes 3900, CEP 14049-900, Ribeirão Preto, SP, Brazil
c Departament of Microbiology, Immunology and Parasitology, Universidade Federal de São Paulo, Rua Botucatu 862 8 Andar 04023-062, São Paulo, SP, Brazil
d Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
e Department of Metabolic Biology, John Innes Centre, Colney Lane, Norwich, NR4 7UH, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Trypanosoma cruzi trans-sialidase (TcTS) plays a key role in the recognition and invasion of host cells and
in enabling the parasite to escape the human immune response. To explore this potential drug target, we
have synthesized a small library of substrate analogues based on 1,4-disubstituted 1,2,3-triazole
derivatives of galactose modified at either the C-1 or C-6 positions. This was achieved by coupling the
appropriate azido-sugars with a panel of 23 structurally diverse terminal alkynes by using the cop-
per(I)-catalyzed alkyne-azide cycloaddition (CuAAC) reaction, giving a library of 46 derivatives in good
to excellent yield and with complete regioselectivity. The sugar triazoles showed weak inhibition
Received 23 December 2009
Revised 23 February 2010
Accepted 25 February 2010
Available online 2 March 2010
Keywords:
Trypanosoma cruzi
Trans-sialidase
Galactose
Triazole
‘Click chemistry’
towards TcTS-catalyzed hydrolysis of 20-(4-methylumbelliferyl)-
a-D-N-acetylneuraminic acid in vitro
(<40% inhibition at 1 mM concentration); many of the compounds assessed proved to be acceptor sub-
strates for the enzyme. Despite this modest inhibitory activity, in vitro trypanocidal activity assays
against the trypomastigote form of T. cruzi Y strain revealed several compounds active in the low 100s
of
specific mode of anti-parasite action rather than a generic cytotoxic effect.
Ó 2010 Elsevier Ltd. All rights reserved.
lM range. Further assessment of these compounds against cultured mouse spleen cells suggests a
1. Introduction
for instance),5 there are very few respectable inhibitors (Ki
<500
M) of the parasite enzyme.6,7
l
Trypanosoma cruzi, the causative agent of Chagas’ disease, is a
protozoan parasite that must invade host cells in order to complete
its life cycle. The parasite utilizes a cell surface trans-sialidase en-
zyme (TcTS) to transfer sialic acid from host cells to mucin-like gly-
coproteins in order to modify its carbohydrate coat so that it is not
recognized by the human immune response and also to assist host
cell invasion.1,2 On both counts, inhibition of TcTS offers the poten-
tial for therapeutic intervention, since the enzyme represents a
unique route by which the parasite acquires an essential sialic
acid-containing coat. TcTS shares substantial sequence and
structural homology with bacterial and viral sialidases.3,4 However,
while there are numerous nanomolar inhibitors of the latter that
have made it into the clinic (anti-viral neuraminidase inhibitors,
In the search for new tools8 with which to manipulate parasite
glycosylation, potentially with therapeutic gain, we have been
investigating the chemistry of the parasite cell surface mucin gly-
coproteins,9 which are the natural acceptor substrates for TcTS.10
In particular, the b-galactopyranoside residues (b-Galp) on the mu-
cin coat of the Y and CL-Brener strains of the parasite act as accep-
tors for TcTS-catalyzed transfer of sialic acid.11,12 X-ray
crystallography of co-complexes between the enzyme and sub-
strates illustrates the contribution made by each hydroxyl group
of galactose acceptors in binding to TcTS. Galactose positions 1
and 6 are not implicated in the binding process, whilst the galact-
ose 3-OH is well positioned to interact with both the catalytic
Asp59 and the anomeric carbon of the sialic acid donor substrate.13
The selection of galactose positions 1 and 6 for modification was
also based on the higher reactivity and ease of differentiation of
these positions with respect to the other hydroxyl groups on the
sugar ring. In addition, in a systematic assessment of TcTS acceptor
substrate analogues, we have noted that modification at either the
* Corresponding authors. Tel.: +55 16 36024709; fax: +55 16 36024879 (I.C.); tel.:
+44 1603 450720; fax: +44 1603 450018 (R.A.F.).
E-mail addresses: carronal@usp.br (I. Carvalho), rob.field@bbsrc.ac.uk (R.A.
Field).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.02.053

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2413
1 or 6 positions of galactose is accommodated by the enzyme.14
A
b-D-galactopyranoside, since use of di-O-isopropylidene galactose
number of studies have also shown that TcTS has a great tolerance
for b-galactoside substrates,9,15–20 which prompted us to consider
the exploration of the TcTS active site through the application of
robust azide-alkyne 1,3-dipolar cycloaddition ‘click chemistry’
(CuAAC) based around modified carbohydrate templates.21,22 Car-
bohydrates bearing a 1,2,3-triazole group prepared by ‘click chem-
istry’23 have been explored as potential inhibitors of glycosidases24
and fucosyltransferases,25 as well as for studying the substrate
specificity of b-1,2-mannosyltransferases.26 In addition, triazole-
linked sialic acid derivatives have been investigated as inhibitors
of influenza neuraminidases,27,28 with replacement of the guani-
dino group of the commercially available anti-flu drug zanamivir
by a 1,2,3-triazole group giving a similar EC50 to the parent drug.27
The current study focused on the chemical synthesis of a new
class of 1,4-disubstituted 1,2,3-triazoles starting from galactose
derivatives that bear an azide group at either the C-1 or C-6 posi-
tions. Coupling of such azides to a panel of 23 structurally diverse
terminal alkynes using the copper(I)-catalyzed alkyne-azide cyclo-
addition (CuAAC) reaction21–23 gave 46 disubstituted triazoles dis-
playing a range of physicochemical properties. These compounds
were assessed as TcTS inhibitors, as well as anti-trypanosomal
agents; their cytotoxicity against a mammalian cell line is also
reported.
would require additional steps to cap the reducing terminus as a
glycoside.
Preparation of 6-azido-galactoside 1 comprised 6-O-tosylationof
methyl galactoside 3, followed by tri-O-acetylation and displace-
ment by azide, providing the desired product in 25% overall yield.
Theyield is compromised by a difficultseparationof theunprotected
sulfonate ester from the di-tosylate (O-3 and O-6) by-product. A
significant improvement was achieved by performing a one pot
tosylation/acetylation reaction, which afforded intermediate 4 in
reasonable yield (56%) following chromatographic purification.
The displacement of the 6-O-tosylate group of 4 with NaN3 in DMF
at 120 °C gave peracetylated methyl 6-azido-6-deoxy-b-
pyranoside (1) in near quantitative yield (Scheme 1).
D-galacto-
2.2. Triazole library synthesis
CuAAC reactions involving azido-galactoside 1 or the commer-
cially available per-O-acetylated b- -galactopyranosyl azide
D
2
were carried out with a diverse set of commercially available ter-
minal alkynes, 5–27, having different steric bulk, hydrogen-bond-
ing and hydrophobic groups, linked to flexible and rigid side
chains (Scheme 2). The 1,3-dipolar cycloaddition coupling was
performed on
a
small scale (ꢀ0.13 mmol of azide) with
a
10 mol % excess of the terminal alkynes in a sealed tube under
microwave-assisted conditions. Copper sulfate (0.03 mM) and so-
dium ascorbate (0.1 mM) were used in the experiments for the
in situ generation of Cu(I) catalyst. Typically, the reactions were
conducted at 70 °C for 10 min in DMF (0.1 mL), with the progress
of the reaction being monitored by TLC. After completion of the
reaction, solvent was removed by co-evaporation with toluene.
This approach using ester-protected sugar azides 1 and 2 allowed
the separation of catalyst residues, as well as the removal of small
impurities, by simple silica-gel filtration. Combinations of sugar
azides 1 and 2 with 23 alkynes (5–27) afforded a diverse library
of 46 derivatives 28a/b–50a/b in excellent yields (70–100%), with
two exceptions (compounds 48a and 48b were obtained in 33
and 44% yield, respectively), and with complete regioselectivity
(only formation of the 1,4-disubstituted triazoles was noted, as
judged by 1H NMR spectroscopy—see below). Finally, removal of
the acetate protecting groups with catalytic NaOMe in MeOH affor-
ded the library of triazole galactosides (compounds 28c/d–50c/d,
Fig. 1) in near quantitative yield.
The majority of the 1,4-triazole-galactoside derivatives were
obtained in one microwave cycle of 70 °C for 10 min. However,
cycloaddition reactions involving terminal acetylenes 14, 15, 16,
18 and 25 were more resistant to the initial coupling conditions
and complete conversion was only achieved after 2 or 3 heating cy-
cles of 70–100 °C for up to 30 min, which afforded products 37a/b,
38a/b, 39b, 41b and 48a/b, respectively. The electron-withdrawing
effects of functional groups at para-positions on the aromatic ring
may influence the reactivity of the terminal alkyne, as illustrated
by the presence of bromine in 15 and trifluoromethyl groups in
16. Additionally, attempts at coupling 4-ethynyl pyridine with
sugar azides 1 or 2 gave no product, even under more forcing con-
ditions. Propargyl chloride and propiolic acid also proved unsuit-
able, with starting material remaining even after extended
reaction, whilst reaction with propargyl amine resulted in notable
2. Results and discussion
2.1. Synthesis of sugar azides
Although the derivatization of monosaccharides is very well
explored, application of methods that are effective for one stereo-
isomer is not always predictable. The synthesis of protected
6-azido-6-deoxy-glycosides has previously been achieved by
conventional procedures that involve either displacement of deriv-
atives bearing a leaving group at C-6 (halogens or sulfonates) with
inorganic azide29,30 or directly from the free 6-OH compound with
NaN3 in the presence of PPh3/CBr4.31 The latter has been success-
fully applied in the
while a b- -glucopyranoside reacted to only a limited extent under
the same conditions and a b- -galactopyranoside was essentially
unreactive. Although the synthesis of methyl 6-azido-6-deoxy-
-mannopyranoside has been described in high yield from the
commercial methyl -mannopyranoside by tosylation followed
by azide displacement,32–34 the same strategy applied to phenyl
b- -glucopyranosides gave the corresponding 6-azide derivative
in only moderate yield.35 On the other hand, Maunier36 synthe-
sized methyl 6-azido-6-deoxy- -glucopyranoside in almost
a-D-gluco- and a-D-manno-pyranoside series,
D
D
a
-
D
a
-D
D
a
-D
quantitative yield by substitution of the corresponding 6-chloro-
6-deoxy derivative with sodium azide. Regarding galactopyrano-
sides, to our knowledge only fully protected 6-azido-6-deoxy
derivatives have been reported in the literature.37 Even though
two more protection/deprotection steps are required, the use of
1,2;3,4-di-O-isopropylidene-a-D-galactopyranose can provide the
corresponding free 6-azido-6-deoxy sugar by treatment with me-
syl chloride then NaN3, followed by deprotection, in good yield
(74%).38 Driven by the need to prepare per-O-acetylated methyl
6-azido-6-deoxy-b-D-galactopyranoside (1), we adapted the
route described for the
a-
D
-manno series,32 starting from methyl
1.TsCl/Py
N3
OH
OTs
O
HO
HO
AcO
AcO
AcO
2. AcCl
NaN3/DMF
O
O
OMe
OMe
OMe
AcO
98%
57%
AcO
HO
AcO
3
4
1
Scheme 1. Synthesis of methyl 6-azido-6-deoxy-b-D-galactopyranoside (1).

2414
I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
X
X
N
N
N
N
N3
O
N
AcO
AcO
N
AcO
AcO
HO
HO
ii
O
O
OMe
OMe
OMe
AcO
AcO
HO
X
(5-27)
1
28-50a
28-50c
i
OAc
O
AcO
AcO
X
N
X
N
OAc
O
OH
O
AcO
AcO
HO
HO
ii
N3
N
N
N
N
AcO
AcO
HO
2
28-50b
28-50d
Set of terminal alkynes
F3C
n
OH
n
R
R
5n=1
6 n=3
7n=4
8 n=0
9 n=1
10 n=2
11R= NH2
12 R= Br
13 R= CF3
14 R= NH2
15 R= Br
16 R= CF3
17
O
O
N
N
O
N
N
H
O
18
20
21
22
19
OH
O
COOMe
26
HO
O
24
25
27
23
Scheme 2. Synthesis of a library of sugar triazoles obtained from dipolar cycloaddition reactions of galactose derivatives substituted with azide at C-6 (1) or C-1 (2) and a
series of terminal alkynes (5–27). Reagents and conditions: (i) CuSO4, Na ascorbate, DMF, microwave heating normally at 70 °C, 10 min; (ii) NaOMe, MeOH.
R
N
N
N
N
N
N
N
N
N
N
N
N
N
N
R
OH
n
N
n
CF3
Gal
Gal
Gal
Gal
Gal
40a-d
28a-d n= 1
29a-d n= 3
30a-d n= 4
31a-d n= 0
32a-d n= 1
33a-d n= 2
34a-d R= NH2
35a-d R= Br
36a-d R= CF3
37a-d R= NH2
38a-d R= Br
39a-d R= CF3
O
O
N
OH
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Me
N
Gal
O
Gal
Gal
Gal
Gal
Gal
43a-d
41a-d
42a-d
44a-d
45a-d
O
N
Gal
HO
N
N
N
OH
N
O
N
N
N
N
N
N
O
N
OMe
N
N
N
N
N
N
Gal
Gal
Gal
Gal
46a-d
47a-d
48a-d
49a-d
50a-d
OAc
O
OH
O
AcO
AcO
HO
HO
AcO
AcO
HO
HO
O
O
OMe
OMe
Gal =
OAc
OH
OAc
OH
a series
b series
c series
d series
Figure 1. Library of sugar triazoles 28–50 (series c and d) obtained by deacetylation of adducts 28–50 (series a and b) synthesized from azides 1 or 2 and a diverse set of
alkynes 5–27 by click chemistry approach.

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2415
product decomposition. Comparing the reactivity of 6-azide 1 and
anomeric azide 2 under the CuAAC conditions with these more
resistant acetylene derivatives, the reaction proceeded slower with
azide 2 than azide 1 (Scheme 2). Furthermore, for the complete
conversion of azide 2 to triazoles 37b and 38b, the reactions re-
quired heating (100 °C) for at least 15 min longer than the corre-
sponding azide 1. This is likely attributable to a combination of
steric and electronic effects: elsewhere we have noted the impact
of anomeric stereochemistry and glycoside bulk on the reactivity
of glycosyl azides.39 The condensation of the bis-alkyne 25 was
also not straightforward, since the reaction provided the mono-tri-
azole derivative, which was only converted to the dimer 48a/b un-
der forcing conditions and in lower yield.
pyridoxal phosphate and DANA, which are reported to be weak
TcTS inhibitors.20 In general, compounds containing a triazole
group at either the C-6 (c series) or C-1 (d series) position of galact-
ose showed moderate to weak inhibition of TcTS at 0.5–1 mM con-
centration, with compound 32d being the strongest TcTS inhibitor
in these series (37% inhibition).
2.4. Sugar triazoles as acceptor substrates for Trypanosoma
cruzi trans-sialidase
The mechanism of TcTS inhibition with galactose-based com-
pounds may include direct competition with the donor substrate
which might result in inhibition of trans-sialylation per se. Alterna-
tively, a galactose-based compound might act as a TcTS substrate,
competing with, and thereby inhibiting, the TcTS-catalyzed cleav-
age of MuNANA. Considering the promiscuity of TcTS towards
substituted galactoside substrates,14,15 additional assays were per-
formed with the sugar triazole library to assess the potential of
these compounds as substrates for TcTS-catalyzed trans-sialylation.
These experiments were conducted by incubation of individual
members of the triazole library with TcTS in the presence of MuN-
ANA as a donor.7 Although the TcTS enzyme used here is a recom-
binant His-tagged protein for ease of purification,43 as noted
elsewhere, crude cleared bacterial cell lysates are suitable for syn-
thetic biotransformations.9,10e,14,44,45 On an analytical scale, incu-
bations showed near complete conversion of all members of the
triazole library to the corresponding sialylated adducts within a
few hours at room temperature, as judged by TLC and confirmed
by ESI-MS analysis of the crude reaction mixtures. Data shown in
Scheme 3 for triazoles 32c and 32d are representative of that ob-
tained with either the C-6 substituted c series or C-1 substituted
d series of sugar triazoles.
The peracetylated 6-triazolyl-galactosides(library series a, Fig. 1)
gave characteristic 1H NMR signals, which showed the presence of
one regioisomer.23 For instance, the chemical shifts of the H-6
protons of both azido precursor 1 and products 28–50a, were d
3.1–3.5 (J ꢀ12.0 Hz) and 4.3–4.6 (J ꢀ14.0 Hz), respectively. For this
library series, a minor influence of the triazole group was observed
on the galactose H-5 signal, which has a chemical shift of d 3.8 in
the precursor 1 and approximately d 4.0 in the a series library. Char-
13
acteristic 1H and C signals of the triazole ring at 7.5 ppm (H-50),
139.0 ppm (C-50) and 148.0 ppm (C-40), along with the triazole side
chain, such as methylene signals of compounds 32a and 32b, respec-
tively, at d 4.11–4.02 and d 4.21–4.10 ppm, were also observed. 1H
and 13C NMR spectroscopy of galactosyl-triazoles 28–50b showed
chemical shifts of H-1 at d 5.8–5.9 (J ꢀ9.3 Hz), downfield compared
to the precursor azide 2 at d 4.6 (J ꢀ8.6 Hz).
Furthermore, a modest downfield shift was observed for the gal-
actose H-2 signal due to the influence of the triazole ring at C-1,
changing from d 5.1 ppm in azide 2 to approximately d 5.5 ppm in
the triazole products for b series. Regarding the heteroaromatic tri-
azole ring in the b series, H-50 was observed in the range d 7.2–
7.8 ppm, while C-50 and C-40 were at d 138.0 ppm and 148.0 ppm,
respectively, whichis similarto the corresponding 6-triazolyl-galac-
toside a series. In summary, the structures and purity of each of the
acetylated derivatives 28a,b–50a,b were supported by spectro-
scopic data; following deacetylation the target deprotected com-
pounds 28c,d–50c,d were obtained in near quantitative yield.
These biotransformation experiments show that while the su-
gar triazoles reported are indeed weak TcTS hydrolysis inhibitors
they are more effective inhibitors than lactose and they are chem-
ically competent substrates for the enzyme, so confirming their
ability to bind to its active site.
2.5. In vitro trypanocidal activity and cytotoxicity towards
mammalian cells
2.3. Sugar triazoles as inhibitors of Trypanosoma cruzi trans-
sialidase
Before embarking on the optimization of the weak ‘hits’ ob-
tained in the TcTS screen, and given the lack of any clear struc-
ture–activity relationship in the inhibition data, we first assessed
the whole cell activity of the sugar triazole library. While TcTS is
key to enabling parasite evasion of the human immune response,
adhesion to and invasion of host cells,1,2 it is not known to play a
role in parasite survival in culture. Any trypanocidal activity from
the sugar triazole library might therefore reasonably be inter-
preted as being TcTS-independent. In order to deconvolute general
cytotoxicity from parasite-specific cell kill, the library was also
screened against cultured mammalian cells.
Various methods for the determination of TcTS activity have been
described.40 Amongst them fluorimetric methods40e for assessment
of the residual TcTS hydrolase activity are practical for screening
purposes. Such assays, which are routinely used to assay microbial
neuraminidases, rely on the residual hydrolase activity of TcTS, di-
rectly measuring the cleavage of 20-(4-methylumbelliferyl)-
a-D-N-
acetylneuraminic acid (MuNANA) which releases fluorescent meth-
ylumbelliferone (Mu). Structural studies on TcTS-MuNANA complex
and the ternary complex TcTS-lactose-DANA (DANA = 2,3-dehydro-
2-deoxy-N-acetylneuraminic acid) show a common binding site for
Mu and lactose,41 where the latter may be viewed as either a product
(where sialyllactose is used as a donor substrate) or an acceptor sub-
strate. Kinetic analysis supporting a ping-pong mechanism for
TcTS42 shows no net effect on Mu production from MuNANA from
the addition of lactose, which hides a compensating increase in Km
off-set by and increase in kcat (i.e., addition of lactose results in no
net change in kcat/Km). However, if a lactose (or galactose) derivative
were to exhibit significantly different association or dissociation
rates from TcTS, net inhibition of Mu release would be achieved.
With this in mind, and for ease of assay as highlighted above, a
preliminary screen of the sugar triazole library (28c,d–50c,d) was
conducted using TcTS-catalyzed hydrolysis of MuNANA. Results
from this screen are summarized in Table 1, along with data for
2.5.1. Trypanocidal activity
The trypanocidal activity46 of the entire sugar triazole library
(28c,d–50c,d) was evaluated against the host infective trypomasti-
gote form of T. cruzi Y strain using benznidazole (N-benzyl-2-nitro-
1-imidazolacetamide), the current frontline drug used to treat Cha-
gas disease as a control.47 Data for the most active compounds are
reported in Figure 2. Whilst many of the compounds displayed
rather limited or no trypanocidal activity after 24 h at the tested
concentrations, compounds 37d, 39c, 44c and 48c,d, with activities
in the low 100s of lM range (approx 75 lg/mL), represent poten-
tial leads for further exploration. It is notable that 37d and 44c
both have amine substituents attached to the triazole ring, whilst
48c and 48d contain two sugar units. The most active in vitro TcTS

2416
I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
Table 1
TcTS inhibition by sugar triazoles
R
% Inhibition at 1 mMa,b
% Inhibition at 1 mM
Compound
Compound
OH
N
N
R
R
HO
N
N
N
O
N
HO
HO
O
OH
OMe
HO
OH
OH
28c
29c
28
24
28d
29d
11
27
OH
OH
30c
27
30d
26
31c
14
31d
15
32c
33c
29
14
32d
33d
37
16
34c
35c
36d
24
29
11
34d
35d
36d
30
33
14
H2N
Br
F3C
37c
38c
39c
34
3c
37d
38d
39d
19
Nic
9c
NH2
Br
10c
CF3
40c
41c
3
40d
41d
31
25
CF3
O
11c
N
O
42c
43c
44c
14
20
17
42d
43d
44d
13
15c
22
N
N
N
O
45c
46c
47c
Ni
10
29
45d
46d
47d
10
14
13
OH
OH
OH
48c
49c
16
30
48d
49d
8
COOMe
28

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2417
Table 1 (continued)
R
% Inhibition at 1 mMa,b
% Inhibition at 1 mM
Compound
Compound
OH
N
N
R
R
HO
N
N
N
O
N
HO
HO
O
OH
OMe
HO
OH
O
50c
25
50d
27
O
Pyridoxal phosphate
DANA
65
33
Ni: no inhibition.
a
All data reported are accurate to 5%.
At 1 mM concentration, lactose shows no inhibition in this assay.
Samples dissolved in H2O containing 0.6% of DMSO and tested at 0.5 mM.
b
c
OH
N
O
HO
N
N
COOH
HO
32c
HO
O
OH
AcHN
COO-Na+
HO
OMe
HO
O
HO
HO
O
AcHN
O
O
O
TcTS
51c
HO
ESI-MS m/z: 649 [M+Na]+
Phosphate buffer pH 7.5
25ºC
OH
HO
CH3
HO
COOH
MUNANA
32d
OH
O
N
HO
O
N
N
AcHN
HO
O
OH
51d
ESI-MS m/z: 635 [M+Na]+
Scheme 3. Sialic acid transfer reactions catalyzed by TcTS using MuNANA as donor substrate and representative triazole-sugar acceptors 32c and 32d, to provide sialyl
products 51c and 51d, respectively.
Figure 2. Trypanocidal activity of the c and d series sugar triazoles evaluated against trypomastigote forms of T. cruzi Y strain. BZ is the benznidazole control. For compounds
38c, 38d, 39c, 39d, 41c and 43d the maximum tested concentration was 0.75 mM due to their lower aqueous solubility, which required the addition of DMSO (0.6%).
inhibitor, 32d, was not particularly active against the parasite (50%
parasite cell kill in the high 100s of M range) whilst whole
the 50–100 lM range. Evidently mammalian cell toxicity does
not directly mirror anti-parasite activity.
l
parasite activities are evident at concentrations well below the
level of in vitro TcTS inhibition for compounds 37d, 39c, 44c and
48c,d.
2.5.3. Comparative toxicity
Further analysis of Figures 2 and 3 identifies compounds that
are toxic to the parasite but not to mouse cells (e.g., 29c, 34d,
37d, 42d, 44c, 47c, 48c, 50d), some that are much more toxic to
mouse cells than to the parasites (e.g., 32d, 38d), and some that
are toxic to both (e.g., 33d, 38c, 39c, 39d, 43d, 48d). There is no
clear correlation between general compound structure (i.e., c or d
series, 6- vs 1-position attachment of galactose to the triazole) and
2.5.2. Mammalian cell toxicity
Moving on to mammalian cell toxicity, the trypanosome-active
compounds highlighted in Figure 2 were screened against cultured
mouse spleen cells;48 data are shown in Figure 3. In this case,
compounds 32d, 38d, 39c and 43d showed 50% mouse cell kill in

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I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
Figure 3. Percentage cell death caused by the c and d series sugar triazoles evaluated against cultured mouse spleen cells. BZ is benznidazole. For compounds 32d, 38c, 38d,
39c, 39d, 41c and 43d the maximum tested concentration was 0.25 mM.
cell selectivity, but anti-mammalian cell activity is strongest for
triazoles with hydrophobic substituents whilst anti-trypanosomal
activity shows no such preference for triazole substituent.
cated eluents. Compounds were visualized under UV light
(254 nm) and/or dipping in ethanol-sulfuric acid (95:5, v/v) or orci-
nol (2%) in aqueous sulfuric acid (10%), followed by heating the
plate for a few minutes. Column chromatography was performed
on silica gel 60 (Fluorochem, 35–70 mesh) or on a Biotage Horizon
High-Performance FLASH Chromatography system using 12 mm or
25 mm flash cartridges with the eluents indicated. The microwave-
assisted reactions were carried out in a Biotage Initiator System,
using sealed tubes. All the fluorescence measurements were per-
formed on a Synergy HT Multi-Mode Microplate Reader, using
Gen5 software. Nuclear magnetic resonance spectra were recorded
on a JEOL Lambda 400 MHz. 1H NMR spectra recorded at 400 MHz
were referenced to dH 7.27 for CDCl3, dH 3.35 for CD3OD, dH
4.63 ppm for D2O, and 13C NMR spectra recorded at 100 MHz and
were referenced to dC 77.0 for CDCl3 and dC 49.15 for CD3OD.
Assignments were made with the aid of HSQC and COSY experi-
ments. Optical rotations were measured at ambient temperature
on a Perkin–Elmer model 341 polarimeter using a sodium lamp.
Accurate mass electrospray ionization mass spectra ESI-MS and
ESI-HRMS were obtained from the John Innes Centre metabolite
analysis service on a Thermo Finningan DecaXPplus using positive
and negative ionization modes, and Thermofisher LTQ Orbitrap
XL mass spectrometers, respectively.
3. Conclusions
In summary, a library of 46 sugar 1,2,3-triazoles was synthesized
using copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) ‘click
chemistry’ from galactose derivatives containing either a C6- or C1-
azide group (compounds 1 and 2, respectively). In general, these
compounds proved to be moderate to weak TcTS inhibitors in vitro
(<40% inhibition at 1 mM concentration), while all compounds eval-
uated showed the ability to act as acceptor substrates for TcTS-cat-
alyzed trans-sialylation. Unexpectedly, some of the sugar triazoles
showed trypanocidal activity in the low 100s of lM range against
cultured trypomastigote forms of T. cruzi Y strain. These whole par-
asite activities are evident at concentrations well below the level of
in vitro TcTS inhibition by the compounds concerned, indicative of a
non-TcTS related mode of action at the cellular level. Assessment of
the most active TcTS inhibitors as cytotoxic agents against mamma-
lian cells established that some such compounds are toxic, but there
is little direct correlation between anti-parasite activity and mam-
malian cell toxicity. Taken together, these data suggest that at least
some sugar triazoles are not generic cytotoxic agent, but are capable
of selectively affecting the parasite. N-Methyl benzylamine deriva-
tive 44c, in particular, stands out as a candidate for further work
4.2. Methyl 2,3,4-tri-O-acetyl-6-O-p-toluenesulfonyl-b-D-galact-
opyranoside (4)
(50% parasite kill at ꢀ200
lM; <<10% mouse cell kill at 1 mM). Fur-
ther studies to elucidate the mode of action of such sugar triazoles
Methyl b-D-galactopyranoside 3 (1.0 g, 5.15 mmol) was dis-
solved in dry pyridine (12 mL) and cooled to 0 °C. Tosyl chloride
(2.82 g, 15.0 mmol) was slowly added and the reaction mixture
was stirred overnight at room temperature. Acetic anhydride
(13 mL) was added subsequently and the mixture was stirred for
12 h. The mixture was concentrated by co-evaporation with tolu-
ene, diluted with DCM and the organic layer was washed with
1 M HCl, satd NaHCO3 solution, dried (MgSO4) and concentrated
under reduced pressure. The crude product was purified by flash
chromatography (EtOAc/Tol, 15–30%) to give compound 5 (1.37 g,
are clearly called for.
4. Experimental section
4.1. General methods
All chemicals were purchased as reagent grade and used with-
out further purification. Solvents were dried according to standard
methods.49 2,3,4,6-Tetra-O-acetyl-b-
D
-galactopyranosyl azide
was purchased from Carbosynth and MuNANA [20-(4-methylum-
belliferyl)- -N-acetylneuramic acid sodium salt] was purchased
2
56%); mp = 47–50 °C; ½a D25
ꢁ
ꢂ8.7 (c 1.0 CHCl3); 1H NMR (CDCl3,
400 MHz), d (ppm): 7.77 (2H, d, J 8.2 Hz, Ar); 7.35 (2H, d, J
8.2 Hz, Ar); 5.36 (1H, dd, J3,4 3.3 Hz, J4,5 1.0 Hz, H-4); 5.14 (1H,
dd, J1,2 7.8 Hz, J2,3 10.4 Hz, H-2); 4.98 (1H, dd, J2,3 10.4 Hz, J3,4
a
-D
from Toronto Research Chemicals Inc. Alkynes were purchased
from Sigma–Aldrich. Benznidazole (N-benzyl-2-nitro-1-imidazo-
lacetamide), used as positive control in the trypanocidal assays,
was purchased from Roche. Reactions were monitored by thin
layer chromatography (TLC) on 0.25 mm pre-coated silica gel
plates (Whatman, AL SIL G/UV, aluminium backing) with the indi-
0
3.3 Hz, H-3); 4.36 (1H, d, J1,2 7.8 Hz, H-1); 4.13 (1H, dd, J5,6
6.6 Hz, J6,6 10.0 Hz, H-60); 4.01 (1H, dd, J5,6 6.1 Hz, J6,6 10.0 Hz,
0
0
0
H-6); 3.94 (1H, dd, J4,5 1.0 Hz, J5,6 = J5,6 6.4 Hz, H-5); 3.48 (3H, s,
OMe); 2.46 (3H, s, CH3Ar); 2.05, 2.03, 1.96 (9H, 3s, 3 ꢃ CH3). 13C
NMR (CDCl3, 100 MHz), d (ppm): 169.8, 169.3 (COCH3); 145.2

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2419
0
(C-Ar); 129.8, 127.9 (CH-Ar); 101.8 (C-1); 70.5 (C-5 or C-3); 70.4
3.7 Hz. J5.6 8.4 Hz, H-5); 3.42 (3H, s, OCH3); 2.99 (1H, br s, OH);
2.20, 2.06, 1.99 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.3, 170.1, 169.5 (COCH3); 147.8 (C-quat triazole);
123.1 (CH triazole); 102.0 (C-1); 71.8 (C-5); 70.7 (C-3); 68.6 (C-
2); 68.0 (C-4); 57.2 (OCH3); 56.3 (CH2); 50.2 (C-6); 20.8, 20.7,
(C-3 or C-5); 68.4 (C-2); 66.9 (C-4); 66.4 (C-6); 56.9 (OCH3); 21.5
(CH3Ar); 20.6–20.3 (COCH3). IR (KBr) mmax
: 2941.2; 1755.1;
1367.4; 1220.9; 1078.1 cmꢂ1. ESI-MS m/z, calcd for C20H26O11
S
[M+Na]+ 497.1, found 497.0.
20.5 (COCH3). IR (KBr)
mmax: 3400,0; 1749.3; 1371.3; 1222.8;
4.3. Methyl 2,3,4-tri-O-acetyl-6-azido-6-deoxy-b-D-galactopyrano-
1049.2 cmꢂ1. ESI-MS m/z, calcd for C16H23N3O9 [M+H]+ 402.1;
[M+Na]+ 424.1, found 402.1; 424.1. Deprotected compound 28c:
ESI-MS m/z, calcd for C10H17N3O6 [M+H]+ 276.1; [M+Na]+ 298.1,
found 276.2; 298.2.
side (1)
A suspension of compound 4 (0.78 g, 1.65 mmol) in DMF
(25 mL) containing sodium azide (1.63 g, 25.1 mmol) was heated
at 120–130 °C for approx. 6 h. The solution was allowed to cool, di-
luted with water (30 mL) and extracted with DCM. The organic ex-
tract was washed with water, sat NaHCO3 solution and water,
dried (MgSO4) and concentrated under reduced pressure to give
compound 1 as a crystalline solid (0.56 g, 1.62 mmol, 98%);
4.4.3. 3-[1-(Methyl 6-deoxy-b-
triazol-4-yl]-propan-1-ol (29c)
D-galactopyranosid-6-yl)-1H-1,2,3-
Triacetate 29a: (88% yield); 1H NMR1H NMR (CDCl3, 400 MHz), d
(ppm): 7.38 (1H, s, CH triazole); 5.40 (1H, d, J3,4 3.2 Hz, H-4); 5.21
(1H, dd, J1,2 7.8 Hz, J2,3 10.5 Hz, H-2); 5.01 (1H, dd, J2,3 10.5 Hz, J3,4
mp = 103–104 °C; ½a D25
ꢁ
ꢂ15.0 (c 1.0 CHCl3); 1H NMR (CDCl3,
3.2 Hz, H-3); 4.54 (1H, dd, J5,6 4.1 Hz, J6,6 14.1 Hz, H-6); 4.41 (1H,
0
dd, J5,6 8.3 Hz, J6,6 14.1 Hz, H-60); 4.32 (1H, d, J1,2 7.8 Hz, H-1);
0
0
400 MHz), d (ppm): 5.34 (1H, dd, J3,4 3.4 Hz, J4,5 1.0 Hz, H-4); 5.20
(1H, dd, J1,2 7.8 Hz, J2,3 10.5 Hz, H-2); 5.03 (1H, dd, J3,4 3.4 Hz, J2,3
10.5 Hz, H-3); 4.4 (1H, d, J1,2 7.8 Hz, H-1); 3.86 (1H, ddd, J4,5
0
4.13 (1H, dd, J5,6 4.1 Hz, J5,6 8.3 Hz, H-5); 3.70 (2H, t, J 5.9 Hz,
CH2); 3.41 (3H, s, OCH3); 2.83 (2H, t, J 7.3 Hz, CH2); 2.31 (1H, br
s, OH); 2.20, 2.06, 1.98 (9H, 3s, 3 ꢃ CH3); 1.90 (2H, q, J 6.7 Hz,
CH2). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.2; 170.0; 169.5
(COCH3); 147.5 (C-quat triazole); 122.4 (CH triazole); 102.1 (C-
1); 71.9 (C-5); 70.7 (C-3); 68.6 (C-2); 67.9 (C-4); 61.8 (CH2OH);
57.2 (OCH3); 50.0 (C-6); 31.9 (CH2); 22.0 (CH2); 20.8, 20.7, 20.5
(COCH3). ESI-MS m/z, calcd for C18H27N3O9 [M+H]+ 430.1;
[M+Na]+ 452.1, found 430.2; 452.2. Deprotected compound 29c:
ESI-MS m/z, calcd for C12H21N3O6 [M+H]+ 304.1; [M+Na]+ 326.1,
found 304.2; 326.2.
0
0
1.0 Hz, J5,6 4.0 Hz, J5,6‘8.4 Hz, H-5); 3.56 (1H, dd, J5,6 8.4 Hz, J6,6
13.0 Hz, H-60); 3.55 (3H, s, OMe); 3.13 (1H, dd, J5,6 4.0 Hz, J6,6
0
13.0 Hz, H-6); 2.16, 2.08, 1.91 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3,
100 MHz), d (ppm): 170.2, 170.0, 169.4 (COCH3); 102.0 (C-1);
72.9 (C-5); 70.8 (C-3); 68.7 (C-2); 68.0 (C-4); 57.0 (OCH3); 50.5
(C-6); 20.7, 20.5, 20.4 (COCH3). IR (KBr)
mmax: 2941.2; 2100.3;
1747.4; 1244.0; 1047.3 cmꢂ1. ESI-MS m/z, calcd for C13H19N3O8
[M+Na]+ 368.1, found 368.0. ESI-HRMS [M+Na]+ calcd 368.10644,
found 368.10693.
4.4. Synthesis of 1,2,3-triazole-modified galactosides by CuAAC
click reaction
4.4.4. 4-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-butan-1-ol (30c)
Triacetate 30a: (74% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.35 (1H, s, CH triazole); 5.38 (1H, d, J3,4 3.3 Hz, H-4); 5.21 (1H, dd,
J1,2 8.1 Hz, J2,3 10.5 Hz, H-2); 5.00 (1H, dd, J2,3 10.5 Hz, J3,4 3.3 Hz, H-
4.4.1. General procedure
Sugar derivative 1 or 2 containing azide group at C-6 or C-1
(0.13 mM), respectively, was dissolved in DMF (0.1 mL, solution
0.5–1.0 M) in a microwave flask (0.2 mL) equipped with a stirring
bar. After dissolution, one of the acetylene derivatives 5–27
(0.15 mM, 1.1 equiv) was added. Then, sodium ascorbate (0.1 equiv)
and CuSO4 (0.03 equiv) were added and the tube was sealed. The
mixture was stirred for 25 s and heated for 10 min at 70 °C (18 W)
in the microwave, unless stated otherwise. The reaction was fol-
lowed by TLC and after completion the reaction mixture was parti-
tioned between H2O and EtOAc, the aqueous phase was extracted
with EtOAc (3–5 times depending on the solubility of the specific
compound). The organic phase was dried over MgSO4, filtered, con-
centrated and the residual solvent was finally co-evaporated with
toluene. The peracetylated product (a compound of series a or b)
was fully characterized prior the deprotection. The acetate was dis-
solved in methanol (1.0 mL) and sodium methoxide (1.0 M in meth-
anol) was added until pH 9.0 was achieved. After stirring for 1 h at
room temperature, the mixture was neutralized with Amberlite-
IRA-400 (H+), filtered and concentrated in vacuo. The resulting prod-
uct (a compound of series c or d) was obtained in quantitative yield,
not further purified and characterized by ESI-MSonly. These data are
shown in Sections 4.4.2–4.4.46 together with complete character-
ization data for acetylated precursors (compounds of series a or b).
0
0
3); 4.54 (1H, dd, J5,6 4.4 Hz, J6,6 14.2 Hz, H-6); 4.44 (1H, dd, J5,6
8.2 Hz, J6,6 14.2 Hz, H-60); 4.32 (1H, d, J1,2 8.1 Hz, H-1); 4.13 (1H,
dd, J5,6 4.4 Hz, J5,6 8.2 Hz, H-5); 3.65 (2H, t, J 6.4 Hz, CH2); 3.41
0
0
(3H, s, OCH3); 2.75 (2H, t, J 7.3 Hz, CH2); 2.20, 2.04, 1.96 (9H, 3s,
3 ꢃ CH3); 1.89 (1H, br s, OH); 1.75 (2H, q, J 7.0 Hz, J 7.8 Hz, J
7.5 Hz, CH2); 1.60 (2H, q, J 6.3 Hz, J 6.6 Hz, J 8.2 Hz, CH2). 13C
NMR (CDCl3, 100 MHz), d (ppm): 170.3, 170.0, 169.5 (COCH3);
148.0 (C-quat triazole); 122.2 (CH triazole); 102.1 (C-1); 71.9 (C-
5); 70.8 (C-3); 68.5 (C-2); 67.8 (C-4); 62.3 (CH2OH); 57.1 (OCH3);
49.8 (C-6); 32.0 (CH2); 25.5 (CH2); 25.2 (CH2); 20.78, 20.7, 20.5
(COCH3). ESI-MS m/z, calcd for C19H29N3O9 [M+H]+ 444.2;
[M+Na]+ 466.2, found 444.2; 466.2. Deprotected compound 30c:
ESI-MS m/z, calcd for C13H23N3O6 [M+H]+ 318.1; [M+Na]+ 340.1,
found 318.2; 340.2.
4.4.5. [1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-benzene (31c)
Triacetate 31a: (97% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.87 (1H, s, CH triazole); 7.82 (2H, d, J 7.1 Hz, CH-Ar); 7.43 (2H,
pseudo t, Jꢀ7.4 Hz, CH-Ar); 7.34 (1H, t, J 7.4 Hz, CH-Ar); 5.49 (1H,
d, J3,4 3.4 Hz, J4,5 2.8 Hz, H-4); 5.23 (1H, dd, J1,2 8.0 Hz, J2,3
10.4 Hz, H-2); 5.05 (1H, dd, J2,3 10.4 Hz, J3,4 3.4 Hz, H-3); 4.65
0
0
(1H, dd, J5,6 3.5 Hz, J6,6 14.2 Hz, H-6); 4.48 (1H, dd, J5,6 8.8 Hz,
0
4.4.2. [1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
J6,6 14.2 Hz, H-60); 4.33 (1H, d, J1,2 8.0 Hz, H-1); 4.18 (1H, dd, J4,5
0
triazol-4-yl]-methanol (28c)
2.8 Hz, J5,6 3.5 Hz, J5,6 8.8 Hz, H-5); 3.40 (3H, s, OCH3); 2.20, 2.09,
Triacetate 28a: (79% yield); ½a D25
ꢁ
+7.3 (c 1.0 CHCl3); 1H NMR
2.00 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm):
170.3, 170.0, 169.5 (COCH3); 147.8 (C-quat triazole); 130.4 (C-quat
Ar); 128.9, 128.2, 125.6, 121.1 (CH-Ar); 102.1 (C-1); 71.9 (C-5);
70.7 (C-3); 68.6 (C-2); 68.0 (C-4); 57.2 (OCH3); 50.3 (C-6); 20.8,
20.7, 20.5 (COCH3). ESI-MS m/z, calcd for C21H25N3O8 [M+H]+
448.1; [M+Na]+ 470.1, found 448.1; 470.2. Deprotected compound
(CDCl3, 400 MHz), d (ppm): 7.63 (1H, s, CH triazole); 5.43 (1H, d,
J3,4 3.0 Hz, H-4); 5.21 (1H, dd, J1,2 8.0 Hz, J2,3 10.5 Hz, H-2); 5.04
(1H, dd, J2,3 10.5 Hz, J3.4 3.0 Hz, H-3); 4.77 (2H, br s, CH2); 4.60
0
0
(1H, dd, J5,6 3.7 Hz, J6,6 14.2 Hz, H-6); 4.44 (1H, dd, J5,6 8.4 Hz,
J6,6 14.2 Hz, H-60); 4.34 (1H, d, J1,2 8.0 Hz, H-1); 4.16 (1H, dd, J5,6
0

2420
I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
31c: ESI-MS m/z, calcd for C15H19N3O5 [M+H]+ 322.1; [M+Na]+
344.1, found 322.2; 344.1.
J3,4 3.3 Hz, H-3); 4.69 (1H, dd, J5,6 3.2 Hz, J6,6 14.2 Hz, H-6); 4.50
0
(1H, dd, J5,6 8.9 Hz, J6,6 14.2 Hz, H-60); 4.34 (1H, d, J1,2 7.8 Hz, H-
0
0
0
1); 4.21 (1H, dd, J5,6 3.2 Hz, J5,6 8.9 Hz, H-5); 3.42 (3H, s, OCH3).
4.4.6. [1-(Methyl 6-deoxy-b-
triazol-4-yl]-phenylmethane (32c)
D
-galactopyranosid-6-yl)-1H-1,2,3-
2.23, 2.06, 2.00 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.2, 170.0, 169.5 (COCH3); 145.3 (C-quat triazole);
133.6, 131.0 (C-quat Ar); 130.5, 129.4, 127.7, 124.4 (CH-Ar);
121.0 (CH triazole); 102.1 (C-1); 72.0 (C-5); 70.7 (C-3); 68.6 (C-
2); 68.0 (C-4); 57.3 (OCH3); 50.5 (C-6); 20.8, 20.7, 20.5 (COCH3).
ESI-MS m/z, calcd for C21H24BrN3O8 [M+H]+ 526.1; [M+Na]+
548.1, found 526.0; 548.1. Deprotected compound 35c: ESI-MS
m/z, calcd for C15H18BrN3O5 [M+H]+ 400.0; [M+Na]+ 422.0, found
400.1; 422.1.
Triacetate 32a: (94% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.28–7.18 (6H, m, CH-Ar, CH triazole); 5.41 (1H, d, J3,4 3.3 Hz, H-4);
5.16 (1H, dd, J1,2 8.0 Hz, J2,3 10.3 Hz, H-2); 4.99 (1H, dd, J2,3 10.3 Hz,
0
J3,4 3.3 Hz, H-3); 4.55 (1H, dd, J5,6 3.4 Hz, J6,6 14.1 Hz, H-6); 4.32
(1H, dd, J5,6 9.0 Hz, J6,6 14.1 Hz, H-60); 4.23 (1H, d, J1,2 8.0 Hz, H-
1); 4.11–4.02 (3H, m, H-5, CH2); 3.21 (3H, s, OCH3); 2.16, 2.04,
1.97 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm):
170.1, 170.0, 169.4 (COCH3); 147.9 (C-quat triazole); 138.9 (C-quat
Ar); 128.6, 126.5, 123.0 (CH-Ar); 101.9 (C-1); 71.8 (C-5); 70.7 (C-3);
68.6 (C-2); 67.9 (C-4); 57.0 (OCH3); 50.0 (C-6); 32.2 (CH2); 20.7,
0
0
4.4.10. 2-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-trifluoromethylbenzene (36c)
20.6, 20.5 (COCH3). IR (KBr) mmax
:
1751.2; 1454.2; 1244.0;
Triacetate 36a: (91% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.99 (1H, d, J 7.8 Hz, H-30); 7.90 (1H, s, CH triazole); 7.75 (1H, d, J
7.8 Hz, H-60); 7.64 (1H, t, J 7.8 Hz, H-50); 7.49 (1H, t, J 7.8 Hz, H-
40); 5.68 (1H, d, J3,4 3.5 Hz, H-4); 5.24 (1H, dd, J1,2 8.0 Hz, J2,3
10.5 Hz, H-2); 5.07 (1H, dd, J2,3 10.5 Hz, J3,4 3.5 Hz, H-3); 4.69
1047.3 cmꢂ1. ESI-MS m/z, calcd for C22H27N3O8 [M+H]+ 462.2;
[M+Na]+ 484.1, found 462.1; 484.2. Deprotected compound 32c:
ESI-MS m/z, calcd for C16H21N3O5 [M+H]+ 336.1; [M+Na]+ 358.1,
found 336.2; 358.2.
0
0
(1H, dd, J5,6 2.7 Hz, J6,6 14.4 Hz, H-6); 4.50 (1H, dd, J5,6 9.2 Hz,
0
4.4.7. [1-(Methyl 6-deoxy-b-
D
-galactopyranosid-6-yl)-1H-1,2,3-
J6,6 14.4 Hz H-60); 4.35 (1H, d, J1,2 8.0 Hz, H-1); 4.23 (1H, dd, J5,6
0
triazol-4-yl]-2-phenylmethane (33c)
2.7 Hz, J5,6 9.2 Hz, H-5); 3.44 (3H, s, OCH3). 2.23, 2.06, 2.00 (9H,
Triacetate 33a: (94% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.29–7.17 (6H, m, CH-Ar, CH triazole); 5.40 (1H, d, J3,4 3.4 Hz, H-4);
5.20 (1H, dd, J1,2 8.0 Hz, J2,3 10.3 Hz, H-2); 5.01 (1H, dd, J2,3 10.3 Hz,
3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.2, 170.0,
169.5 (COCH3); 144.2 (C-quat triazole); 132.1, 131.6 (C-quat Ar);
129.2, 128.3, 127.2, 126.1 (CH-Ar); 123.2 (CH triazole); 102.1 (C-
1); 72.0 (C-5); 70.7 (C-3); 68.6 (C-2); 68.1 (C-4); 57.1 (OCH3);
50.6 (C-6); 20.8, 20.7, 20.5 (COCH3). ESI-MS m/z, calcd for
C22H24F3N3O8 [M+H]+ 516.1; [M+Na]+ 538.1, found 516.1; 538.1.
Deprotected compound 36c: ESI-MS m/z, calcd for C16H18F3N3O5
[M+H]+ 390.1; [M+Na]+ 412.1, found 390.2; 412.1.
0
J3,4 3.4 Hz, H-3); 4.52 (1H, dd, J5,6 3.9 Hz, J6,6 14.1 Hz, H-6); 4.36
(1H, dd, J5,6 8.3 Hz, J6,6 14.1 Hz, H-60); 4.28 (1H, d, J1,2 8.0 Hz, H-
0
0
0
1); 4.09 (1H, dd, J5,6 3.9 Hz, J5,6 8.3 Hz, H-5); 3.35 (3H, s, OCH3);
3.07–2.96 (4H, m, CH2); 2.19, 2.05, 198 (9H, 3s, 3 ꢃ CH3). 13C
NMR (CDCl3, 100 MHz), d (ppm): 170.2, 170.0, 169.5 (COCH3);
147.3 (C-quat triazole); 140.9 (C-quat Ar); 128.4, 126.1, 122.3
(CH-Ar); 102.0 (C-1); 71.9 (C-5); 70.7 (C-3); 68.6 (C-2); 67.9 (C-
4); 57.1 (OCH3); 50.0 (C-6); 35.4 (CH2); 27.2 (CH2); 20.7, 20.6,
20.5 (COCH3). ESI-MS m/z, calcd for C23H29N3O8 [M+H]+ 476.2;
[M+Na]+ 498.2, found 476.2; 498.2. Deprotected compound 33c:
ESI-MS m/z, calcd for C17H23N3O5 [M+H]+ 350.1; [M+Na]+ 372.1,
found 350.2; 372.2.
4.4.11. 4-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-aniline (37c)
Triacetate 37a: (92% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.70 (1H, s, CH triazole); 7.59 (2H, d, J 8.5 Hz, H-30, H-50); 6.72 (2H,
d, J 8.5 Hz, H-20, H-60); 5.47 (1H, d, J3,4 3.3 Hz, H-4); 5.22 (1H, dd, J1,2
8.0 Hz, J2,3 10.5 Hz, H-2); 5.03 (1H, dd, J2,3 10.5 Hz, J3,4 3.3 Hz, H-3);
0
0
4.59 (1H, dd, J5,6 3.5 Hz, J6,6 14.1 Hz, H-6); 4.44 (1H, dd, J5,6 8.7 Hz,
0
4.4.8. 2-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
J6,6 14.1 Hz, H-60); 4.31 (1H, d, J1,2 8.0 Hz, H-1); 4.13 (1H, dd, J5,6
0
triazol-4-yl]-aniline (34c)
3.5 Hz, J5,6 8.7 Hz, H-5); 3.81 (2H, br s, NH2); 3.39 (3H, s, OCH3);
Triacetate 34a: (100% yield); 1H NMR (CDCl3, 400 MHz), d
2.20, 2.05, 1.99 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.3, 170.0, 169.5 (COCH3); 148.1 (C-40); 146.7 (C-quat tri-
azole); 126.8 (C-30, C-50); 120.7 (C-10); 119.7 (C-20, C-60); 115.2 (CH
triazole); 102.2 (C-1); 71.9 (C-5); 70.7 (C-3); 68.6 (C-2); 68.0 (C-4);
57.2 (OCH3); 50.2 (C-6); 20.8–20.5 (COCH3). ESI-MS m/z, calcd for
C21H26N4O8 [M+H]+ 463.2; [M+Na]+ 485.0, found 463.2; 485.2.
Deprotected compound 37c: ESI-MS m/z, calcd for C15H20N4O5
[M+H]+ 337.1; [M+Na]+ 359.1, found 337.2; 359.1.
(ppm): 7.85 (1H, s, CH triazole); 7.31 (1H, d, J3 ,4 7.5 Hz, H-30);
0
0
7.12 (1H, dd, J4 ,5 7.2 Hz, J5 ,6 8.0 Hz, H-50); 6.76 (1H, d, J5 ,6
0
0
0
0
0
0
8.0 Hz, H-60); 6.72 (1H, t, J 7.5, H-40); 5.48 (3H, m, H-4, NH2);
5.22 (1H, dd, J1,2 8.0 Hz, J2,3 10.5 Hz, H-2); 5.04 (1H, dd, J2,3
0
10.5 Hz, J3,4 3.4 Hz, H-3); 4.63 (1H, dd, J5,6 3.4 Hz, J6,6 14.1 Hz, H-
6); 4.48 (1H, dd, J5,6 8.8 Hz, J6,6 14.1 Hz, H-60); 4.31 (1H, d, J1,2
0
0
0
8.0 Hz, H-1); 4.14 (1H, dd, J5,6 3.4 Hz, J5,6 8.8 Hz, H-5); 3.39 (3H,
s, OCH3); 2.21, 2.06, 1.99 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3,
100 MHz), d (ppm): 170.3, 170.0, 169.5 (COCH3); 148.4 (C-quat. tri-
azole); 145.1 (C-20), 129.1 (C-50); 127.6 (C-30); 121.2 (CH triazole);
117.4 (C-40); 116.8 (C-60); 113.4 (C-10); 102.1 (C-1); 71.9 (C-5); 70.7
(C-3); 68.6 (C-2); 68.0 (C-4); 57.2 (OCH3); 50.4 (C-6); 20.8, 20.7,
20.5 (COCH3). ESI-MS m/z, calcd for C21H26N4O8 [M+H]+ 463.2;
[M+Na]+ 485.0, found 463.1; 485.1. Deprotected compound 34c:
ESI-MS m/z, calcd for C15H20N4O5 [M+H]+ 337.1; [M+Na]+ 359.1,
found 337.2; 359.1.
4.4.12. 4-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-bromobenzene (38c)
Triacetate 38a: (96% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.86 (1H, s, CH triazole); 7.68 (2H, d, J 8.5 Hz, H-20, H-60); 7.55 (2H,
d, J 8.6 Hz, H-30, H-50); 5.47 (1H, d, J3,4 3.4 Hz, H-4); 5.23 (1H, dd, J1,2
8.0 Hz, J2,3 10.5 Hz, H-2); 5.05 (1H, dd, J2,3 10.5 Hz, J3,4 3.4 Hz, H-3);
4.65 (1H, dd, J5,6 3.6 Hz, J6,6 14.4 Hz, H-6); 4.51–4.46 (1H, m, H-60);
0
0
4.33 (1H, d, J1,2 8.0 Hz, H-1); 4.17 (1H, dd, J5,6 3.6 Hz, J5,6 8.6 Hz, H-
5); 3.40 (3H, s, OCH3). 2.21, 2.06, 2.00 (9H, 3s, 3 ꢃ CH3). 13C NMR
(CDCl3, 100 MHz), d (ppm): 170.2, 170.0, 169.5 (COCH3); 146.8
(C-quat triazole); 132.0, 129.3 (C-quat Ar); 127.1 121.2 (CH Ar);
121.1 (CH triazole); 102.1 (C-1); 71.8 (C-5); 70.7 (C-3); 68.5 (C-
2); 67.9 (C-4); 57.2 (OCH3); 50.4 (C-6); 20.8, 20.7, 20.5 (COCH3).
ESI-MS m/z, calcd for C21H24BrN3O8 [M+H]+ 526.1; [M+Na]+
548.1, found 526.0; 548.1. Deprotected compound 38c: ESI-MS
m/z, calcd for C15H18BrN3O5 [M+HCOO]ꢂ 444.0, found 443.9.
4.4.9. 2-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-bromobenzene (35c)
Triacetate 35a: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.36 (1H, s, CH triazole); 8.14 (1H, d, J 7.8 Hz, H-60); 7.64
(1H, d, J 8.0 Hz, H-30); 7.41 (1H, pseudo t, J 7.6 Hz, H-40); 7.20
(1H, pseudo t, J 7.7 Hz, H-50); 5.52 (1H, d, J3,4 3.3 Hz, H-4); 5.24
(1H, dd, J1,2 7.8 Hz, J2,3 10.3 Hz, H-2); 5.06 (1H, dd, J2,3 10.3 Hz,

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2421
4.4.13. 4-[1-(Methyl 6-deoxy-b-
triazol-4-yl]-trifluoromethylbenzene (39c)
D
-galactopyranosid-6-yl)-1H-1,2,3-
170.0, 169.5 (COCH3); 150.0 (C-20), 149.52 (C-60) 148.5 (C-quat tri-
azole); 136.0 (C-40), 127.4 (C-30), 123,3 (C-50); 120.2 (CH triazole);
102.0 (C-1); 71.8 (C-5); 70.7 (C-3); 68.6 (C-2); 67.9 (C-4); 57.0
(OCH3); 50.4 (C-6); 20.8, 20.7, 20.5 (COCH3). ESI-MS m/z, calcd
for C20H24N4O8 [M+H]+ 449.1; [M+Na]+ 471.1, found 449.1; 471.2.
Deprotected compound 42c: ESI-MS m/z, calcd for C14H18N4O5
[M+H]+ 323.1; [M+Na]+ 346.1, found 323.2; 345.2.
Triacetate 39a: (98% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.95 (1H, s, CH triazole); 7.93 (2H, d, J 8.3 Hz, H-30, H-50); 7.68 (2H,
d, J 8.3 Hz, H-20, H-60); 5.68 (1H, d, J3,4 3.5 Hz, H-4); 5.23 (1H, dd, J1,2
8.0 Hz, J2,3 10.5 Hz, H-2); 5.05 (1H, dd, J2,3 10.5 Hz, J3,4 3.5 Hz, H-3);
0
0
4.68 (1H, dd, J5,6 3.6 Hz, J6,6 14.2 Hz, H-6); 4.50 (1H, dd, J5,6 8.7 Hz,
J6,6 14.2 Hz, H-60); 4.34 (1H, d, J1,2 8.0 Hz, H-1); 4.19 (1H, dd, J5,6
0
0
3.6 Hz, J5,6 8.7 Hz, H-5); 3.40 (3H, s, OCH3); 2.22, 2.05, 2.00 (9H,
4.4.17. 3-[1-(Methyl 6-deoxy-b-
triazol-4-yl]-pyridine (43c)
Triacetate 43a: (85% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
D-galactopyranosid-6-yl)-1H-1,2,3-
3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.2, 170.0,
169.5 (COCH3); 146.4 (C-quat triazole); 133.8, 130.0 (C-quat Ar);
125.9, 125.7, 124.9, 123.2 (CH Ar); 121.9 (CH triazole); 102.2 (C-
1); 71.8 (C-5); 70.7 (C-3); 68.6 (C-2); 67.9 (C-4); 57.3 (OCH3);
50.4 (C-6); 20.8, 20.7, 20.5 (COCH3). ESI-MS m/z, calcd for
C22H24F3N3O8 [M+H]+ 516.1; [M+Na]+ 538.1, found 516.1; 538.1.
Deprotected compound 39c: ESI-MS m/z, calcd for C16H18F3N3O5
[M+H]+ 390.1; [M+Na]+ 412.1, found 390.2; 412.1.
8.98 (1H, s, CHPy, H-20); 8.58 (1H, d, J5 ,6 4.8 Hz, H-60); 8.20 (1H, dd,
0
0
J4 ,5 8.0 Hz, J4 ,6 1.7 Hz H-40); 7.96 (1H, CH triazole); 7.38 (1H, dd,
0
0
0
0
J4 ,5 8.0 Hz, J5 ,6 4.8 Hz, H-50); 5.49 (1H, d, J3,4 3.4 Hz, H-4); 5.24
(1H, dd, J1,2 8.0 Hz, J2,3 10.3 Hz, H-2); 5.05 (1H, dd, J2,3 10.3 Hz,
0
0
0
0
0
J3,4 3.4 Hz, H-3); 4.68 (1H, dd, J5,6 3.6 Hz, J6,6 14.1 Hz, H-6); 4.51
(1H, dd, J5,6 8.5 Hz, J6,6 14.1 Hz, H-60); 4.34 (1H, d, J1,2 8.0 Hz, H-
0
0
0
1); 4.19 (1H, dd, J5,6 3.6 Hz, J5,6 8.5, H-5); 3.40 (3H, s, OCH3);
4.4.14. 3-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
2.22, 2.02, 2.04 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.2, 170.0, 169.5 (COCH3); 149.4 (C-60) 146.9 (C-20);
144.7 (C-quat triazole); 132.9 (C-40); 126.5 (C-30), 123.8 (C-50),
121.4 (CH triazole); 102.1 (C-1); 71.8 (C-5); 70.6 (C-3); 68.5 (C-
2); 67.9 (C-4); 57.2 (OCH3); 50.4 (C-6); 20.8, 20.7, 20.5 (COCH3).
triazol-4-yl]-trifluoromethylbenzene (40c)
Triacetate 40a: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.08 (1H, s, H-20); 7.99 (1H, d, J 7.6 Hz, H-60); 7.95 (1H, s,
CH triazole); 7.60–7.53 (2H, m, H-40, H-50); 5.68 (1H, d, J3,4
3.5 Hz, H-4); 5.24 (1H, dd, J1,2 8.0 Hz, J2,3 10.5 Hz, H-2); 5.06 (1H,
IR (KBr) m
max: 1749.3; 1369.4; 1220.9; 1047.3 cmꢂ1. ESI-MS m/z,
0
dd, J2,3 10.5 Hz, J3,4 3.5 Hz, H-3); 4.68 (1H, dd, J5,6 3.5 Hz, J6,6
calcd for C20H24N4O8 [M+H]+ 449.1; [M+Na]+ 471.1, found 449.2;
471.1. Deprotected compound 43c: ESI-MS m/z, calcd for
C14H18N4O5 [M+H]+ 323.1; [M+Na]+ 346.1, found 323.2; 345.1.
14.2 Hz, H-6); 4.50 (1H, dd, J5,6 8.7 Hz, J6,6 14.1, Hz H-60); 4.35
0
0
0
(1H, d, J1,2 8.0 Hz, H-1); 4.21 (1H, dd, J5,6 3.5 Hz, J5,6 8.7 Hz, H-5);
3.40 (3H, s, OCH3). 2.22, 2.05, 2.00 (9H, 3s, 3 ꢃ CH3). 13C NMR
(CDCl3, 100 MHz), d (ppm): 170.2, 170.0, 169.5 (COCH3); 146.5
(C-quat triazole); 131.2, 129.4 (C-quat Ar); 128.7, 124.8, 123.0,
122.4 (CH Ar); 121.6 (CH triazole); 102.1 (C-1); 71.8 (C-5); 70.7
(C-3); 68.6 (C-2); 67.9 (C-4); 57.2 (OCH3); 50.4 (C-6); 20.8, 20.7,
20.5 (COCH3). ESI-MS m/z, calcd for C22H24F3N3O8 [M+H]+ 516.1;
[M+Na]+ 538.1, found 516.1; 538.1. Deprotected compound 40c:
ESI-MS m/z, calcd for C16H18F3N3O5 [M+H]+ 390.1; [M+Na]+ 412.1,
found 390.2; 412.1.
4.4.18. N-Benzyl-N-methyl-1-[1-(methyl 6-deoxy-b-D-galac-
topyranosid-6-yl)-1H-1,2,3-triazol-4-yl]-methylamine (44c)
Triacetate 44a: (70% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7,56 (1H, s, CH triazole); 7.31–7.22 (5H, m, CH Ar); 5.44 (1H, d, J3,4
3.2 Hz, H-4); 5.21 (1H, dd, J1,2 8.0 Hz, J2,3 10.5 Hz, H-2); 5.02 (1H,
0
dd, J2,3 10.5 Hz, J3,4 3.2 Hz, H-3); 4.58 (1H, dd, J5,6 3.7 Hz, J6,6
14.1 Hz, H-6); 4.42 (1H, dd, J5,6 8.8 Hz, J6,6 14.1 Hz, H-60); 4.29
0
0
0
(1H, d, J1,2 8.0 Hz, H-1); 4.15 (1H, dd, J5,6 3.7 Hz, J5,6 8.8 Hz, H-5);
3.71 (2H, s, CH2); 3.54 (2H, s, CH2); 3.31 (3H, s, OCH3). 2.21, 2.20,
2.05, 1.99 (12H, 4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.2, 170.0, 169.5 (COCH3); 145.5 (C-quat triazole);
138.7 (C-quat Ar); 128.8, 128.2, 127.0 (CH Ar); 123.9 (CH triazole);
102.1 (C-1); 71.9 (C-5); 70.7 (C-3); 68.6 (C-2); 67.9 (C-4); 61.4
(CH2); 57.1 (OCH3); 52.0 (CH2); 50.2 (C-6); 42.1 (NCH3); 20.8,
20.6, 20.5 (COCH3). ESI-MS m/z, calcd for C24H32N4O8 [M+H]+
505.2; [M+Na]+ 527.2, found 505.2; 527.2. Deprotected compound
44c: ESI-MS m/z, calcd for C18H26N4O5 [M+H]+ 379.2; [M+Na]+
401.2, found 379.2; 401.2.
4.4.15. [1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-N-phthalimidylmethane (41c)
Triacetate 41a: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.87–7.70 (4H, m, CH Ar); 7.69 (1H, s, CH triazole); 5.44
(1H, d, J3,4 3.3 Hz, H-4); 5.18 (1H, dd, J1,2 8.0 Hz, J2,3 10.2 Hz, H-
2); 5.01 (1H, d, J 15.3 Hz, CH2); 5.00 (1H, dd, J2,3 10.2 Hz, J3,4
3.4 Hz, H-3); 4.97 (1H, d, J 15.3 Hz, CH2); 4.60 (1H, dd, J5,6 3.0 Hz,
J6,6 14.2 Hz, H-6); 4.37 (1H, dd, J5,6 9.0 Hz, J6,6 14.2 Hz, H-60);
0
0
0
0
4.26 (1H, d, J1,2 8.0 Hz, H-1); 4.07 (1H, dd, J5,6 3.0 Hz, J5,6 9.0 Hz,
H-5); 3.30 (3H, s, OCH3). d (ppm): 170.2, 169.9, 169.5 (COCH3);
167.5; 166.9 (CO); 142.9 (C-quat triazole); 134.2, 132.0, 124.3
(CH Ar); 129.9, 128.2 (C-quat Ar); 123.4 (CH triazole); 101.9 (C-
1); 71.8 (C-5); 70.6 (C-3); 68.6 (C-2); 67.9 (C-4); 57.1 (OCH3);
50.3 (C-6); 33.0 (CH2); 20.8, 20.6, 20.5 (COCH3). ESI-MS m/z, calcd
for C24H26N4O10 [M+H]+ 531.1; [M+Na]+ 553.1, found 531.2; 553.2.
Deprotected compound 41c: ESI-MS m/z, calcd for C18H20N4O7
[M+Na]+ 427.1, found 427.2.
4.4.19. 4-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
triazol-4-yl]-butanoic acid (45c)
Triacetate 45a: (82% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.41 (1H, s, CH triazole); 5.41 (1H, d, J3,4 3.2 Hz, H-4); 5.21 (1H, dd,
J1,2 8.0 Hz, J2,3 10.3 Hz, H-2); 5.02 (1H, dd, J2,3 10.3 Hz, J3,4 3.2 Hz, H-
0
0
3); 4.57 (1H, dd, J5,6 3.9 Hz, J6,6 14.2 Hz, H-6); 4.41 (1H, dd, J5,6
8.7 Hz, J6,6 14.2 Hz, H-60); 4.32 (1H, d, J1.2 8.0 Hz, H-1); 4.13 (1H,
0
0
dd, J5,6 3.9 Hz, J5,6 8.7 Hz, H-5); 3.41 (3H, s, OCH3); 2.80 (2H, t, J
4.4.16. 2-[1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
7.3 Hz, CH2); 2.41 (2H, t, J 7.3 Hz, CH2); 2.21, 2.06, 1.99 (9H, 3s,
3 ꢃ CH3); 2.06–1.99 (2H, m, CH2). 13C NMR (CDCl3, 100 MHz), d
(ppm): 177.8 (CO2H); 170.3, 170.0, 169.5 (COCH3); 147.0 (C-quat
triazole); 122.6 (CH triazole); 102.0 (C-1); 71.9 (C-5); 70.7 (C-3);
68.6 (C-2); 67.9 (C-4); 57.2 (OCH3); 50.1 (C-6); 33.1 (CH2); 24.5
triazol-4-yl]-pyridine (42c)
Triacetate 42a: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.58 (1H, d, J4 ,5 4.1 Hz, H-60); 8.23 (1H, CH triazole) 8.15
0
0
(1H, d, J2 ,3 8.0 Hz, H-30); 7.77 (1H, dt, J2 ,3 8.0 Hz, J3 ,4 1.7 Hz, H-
40); 7.23 (1H, m, H-50); 5.47 (1H, d, J4,5 2.8 Hz, H-4); 5.23 (1H, dd,
J1,2 8.0 Hz, J2,3 10.5 Hz, H-2); 5.05 (1H, dd, J2,3 10.5 Hz, J3,4 3.4 Hz,
0
0
0
0
0
0
(CH2); 24.3 (CH2); 20.8, 20.7, 20.5 (COCH3). IR (KBr) mmax: 2939.3;
0
0
1747.4; 1222.8; 1064.6 cmꢂ1. ESI-MS m/z, calcd for C19H27N3O10
[M+H]+ 458.2, found 458.2. Deprotected compound 45c: ESI-MS
m/z, calcd for C13H21N3O7 [M+H]+ 332.1; [M+Na]+ 354.1, found
332.1, 354.2.
H-3); 4.66 (1H, dd, J5,6 3.6 Hz, J6,6 14.1 Hz, H-6); 4.51 (1H, dd, J5,6
8.5 Hz, J6,6 14.1 Hz, H-60); 4.34 (1H, d, J1,2 8.0 Hz, H-1); 4.20 (1H,
0
0
dd, J4,5 2.8 Hz, J5,6 8.5, H-5); 3.54 (3H, s, OCH3); 2.22, 2.06, 1.99
(9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.2,

2422
I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
4.4.20. (2R/S)-1-[1-(Methyl 6-deoxy-b-
D
-galactopyranosid-6-yl)-
1222.8; 1070.4 cmꢂ1. ESI-MS m/z, calcd for C17H23N3O10 [M+H]+
1H-1,2,3-triazol-4-yl]-propan-2-ol (46c)
430.1; [M+Na]+ 452.1, found 430.0; 452.1. Deprotected compound
49c: ESI-MS m/z, calcd for C11H17N3O7 [M+H]+ 304.1; [M+Na]+
326.1, found 304.1; 326.1.
Triacetate 46a: (85% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.45 (1H, s, CH triazole); 5.39 (1H, dd, J3,4 3.4 Hz, J4,5 8.3 Hz, H-4);
5.21 (1H, dd, J1,2 7.8 Hz, J2,3 10.5 Hz, H-2); 5.02 (1H, dd, J2,3 10.5 Hz,
0
J3,4 3.4 Hz, H-3); 4.56 (1H, dt, J5,6 4.2 Hz, J6,6 14.1 Hz, H-6); 4.44
4.4.24. [1-(Methyl 6-deoxy-b-D-galactopyranosid-6-yl)-1H-1,2,3-
(1H, dd, J5,6 9.5 Hz, J6,6 14.1 Hz, H-60); 4.33 (1H, d, J1,2 7.8 Hz, H-
1); 4.18–4.14 (2H, m, H-5, CHOH); 3.43 (3H, s, OCH3); 2.87 (1H,
dd, J 3.5 Hz, J 15.0 Hz, CH2); 2.75 (1H, dd, J 8.3 Hz, J 15.0 Hz,
CH2); 2.20, 2.04, 1.98 (9H, 3s, 3 ꢃ COCH3); 1.27–1.24 (3H, m,
CH(OH)CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.3, 170.0,
169.5 (COCH3); 145.2 (C-quat triazole); 123.3 (CH triazole); 102.1
(C-1); 71.8 (C-5); 70.7 (C-3); 68.6 (C-2); 67.8 (C-4); 67.1, 67.0
(CHOH); 57.2 (OCH3); 50.0 (C-6); 34.8, 34.7 (CH2CHOH); 22.9
(CH(OH)CH3); 20.8, 20.7, 20.5 (COCH3). ESI-MS m/z, calcd for
C18H27N3O9 [M+H]+ 430.1; [M+Na]+ 452.1, found 430.2; 452.2.
Deprotected compound 46c: ESI-MS m/z, calcd for C12H21N3O6
[M+H]+ 304.1; [M+Na]+ 326.1, found 304.2; 326.2.
triazol-4-yl]-methanol O-(R/S)-epoxypropyl ether (50c)
Triacetate 50a: (86% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.65 (1H, s, CH triazole); 5.42 (1H, br s, H-4); 5.24–5.20 (1H, ddd,
0
0
J1,2 8.0, J
10.2 Hz, J 2.4 Hz, H-2); 5.02 (1H, dd, J2,3 10.2 Hz, J3,4
2,3
0
3.0 Hz, H-3); 4.71–4.69 (2H, m, CH2); 4.59 (1H, dd, J5,6 3.7 Hz, J6,6
14.2 Hz, H-6); 4.44 (1H, dd, J5,6 8.4 Hz, J6,6 14.2 Hz, H-60); 4.32
0
0
0
(1H, d, J1,2 8.0 Hz, H-1); 4.13 (1H, dd, J5,6 3.7 Hz, J5,6 8.4 Hz, H-5);
3.84 (1H, dt, J 2.0 Hz, J 2.44, J 11.4 Hz, CH2); 3.45–3.14 (1H, m,
CH2); 3.40 (1H, s, OCH3); 3.16–3.15 (1H, m, CH); 2.80–2.78 (1H,
m, CH2); 2.62–2.60 (1H, m, CH2). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.3; 170.0; 169.5 (COCH3); 144.9 (C-quat triazole);
124.1 (CH triazole); 102.0 (C-1); 71.8 (C-5); 71.0 (CH2); 70.7 (C-
3); 68.6 (C-2); 67.9 (C-4); 64.5 (CH2); 57.2 (OCH3); 50.6 (CH);
4.4.21. (1R/S)-1-[1-(Methyl 6-deoxy-b-
D
-galactopyranosid-6-yl)-
50.2 (C-6); 44.1 (CH2); 20.8, 20.6, 20.5 (COCH3). IR (KBr) mmax:
1H-1,2,3-triazol-4-yl]-propan-1-ol (47c)
1749.3; 1369.4; 1222.8; 1047.3 cmꢂ1
. ESI-MS m/z, calcd for
Triacetate 47a: (89% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.55 (1H, s, CH triazole); 5.43 (1H, br s, H-4); 5.21 (1H, dd, J1,2
7.8 Hz, J2,3 10.5 Hz, H-2); 5.03 (1H, dd, J2,3 10.5 Hz, J3,4 3.2 Hz, H-3);
C19H27N3O10 [M+H]+ 458.1, found 458.2. Deprotected compound
50c: ESI-MS m/z, calcd for C13H21N3O7 [M+H]+ 332.1, found 332.2.
0
4.80 (1H, m, CHOH); 4.59 (1H, dapp J6,6 14.1 Hz, H-6); 4.41 (1H, dd,
4.4.25. [1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-methanol
J5,6 8.6 Hz, J6,6 14.1 Hz, H-60); 4.33, 4.32 (1H, 2 ꢃ d, J1,2 7.8 Hz, H-
1); 4.15 (1H, m, H-5); 3.40, 3.39 (3H, s, OCH3); 2.73 (1H, br s, OH);
2.20, 2.06, 1.98 (9H, 3s, 3 ꢃ CH3); 1.88 (2H, q, J 6.4 Hz, J 7.2 Hz,
CH2); 0.96 (3H, t, J 7.2 Hz, CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.3; 170.0; 169.5 (COCH3); 151.3 (C-quat triazole);
122.0, 121.9 (CH triazole); 102.0 (C-1); 71.9 (C-5); 70.7 (C-3); 68.6
(C-2); 68.2 (CHOH); 67.9 (C-4); 57.1 (OCH3); 50.2, 50.1 (C-6); 30.3,
30.2 (CH2CH3); 20.8, 20.7, 20.5 (COCH3); 9.7 (CH2CH3). ESI-MS m/z,
calcd for C18H27N3O9 [M+H]+ 430.1; [M+Na]+ 452.1, found 430.1;
452.2. Deprotected compound 47c: ESI-MS m/z, calcd for
C12H21N3O6 [M+H]+ 304.1; [M+Na]+ 326.1, found 304.2; 326.1.
(28d)
0
0
Tetraacetate 28b: (91% yield); ½a D25
ꢁ
+4.0 (c 1.0 CHCl3); 1H NMR
(CDCl3, 400 MHz), d (ppm): 7.86 (1H, s, CH triazole); 5.86 (1H, d,
J1,2 9.3 Hz, H-1); 5.55 (1H, pseudo t, J 9.7 Hz, H-2); 5.56 (1H, d,
J3,4 3.2 Hz, H-4); 5.26 (1H, dd, J2,3 10.5 Hz, J3,4 3.2 Hz, H-3); 4.81
(2H, d, J 5.6 Hz, CH2); 4.24 (1H, pseudo t, J 6.3 Hz, H-5); 4.21 (1H,
dd, J5,6 5.6 Hz, J6,6 11.4 Hz, H-6); 4.14 (1H, dd, J5,6 7.1 Hz, J6,6
11.4 Hz, H-60); 2.72 (1H, t, J 5.6 Hz, OH); 2.22, 2.04, 2.01, 1.89
(14H, 4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4,
170.0, 169.8, 169.2 (COCH3); 148.4 (C-quat triazole); 120.2 (CH tri-
azole); 86.2 (C-1); 74.0 (C-5); 70.8 (C-3); 67.9 (C-2); 66.9 (C-4);
0
0
0
61.2 (C-6); 56.5 (CH2); 20.7, 20.5, 20.3 (COCH3). IR (KBr) mmax
:
4.4.22. 1,3-Bis{1-[(methyl 6-deoxy-b-
1H-1,2,3-triazol-6-yl}-propane (48c)
D
-galactopyranosid-6-yl)]-
3400,0, 1753.2; 1371.3; 1222.8; 1060.8 cmꢂ1. ESI-MS m/z, calcd
for C17H23N3O10 [M+H]+ 430.1; [M+Na]+ 452.1, found 430.0;
452.1. Deprotected compound 28d: ESI-MS m/z, calcd for
C9H15N3O6 [M+Na]+ 284.1, found 284.1.
Triacetate 48a: (33% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
7.39 (2H, s, CH triazole); 5.40 (2H, d, J 3,4 3.4 Hz, H-4); 5.21 (2H, dd,
J1,2 8.0 Hz, J 2,3 10.5 Hz, H-2); 5.02 (2H, J2,3 10.5 Hz, J3,4 3.4 Hz, H-3);
4.55 (2H, m, H-6); 4.40 (2H, dd, J5,6 8.3 Hz, J6,6 14.1 Hz, H-60); 4.32
(2H, d, J1,2 8.0 Hz, H-1); 4.13 (2H, m, H-5); 3.41 (6H, s, OCH3); 2.75
(4H, t, J 7.3 Hz, CH2); 2.20, 2.06, 1.98 (18H, 3s, 3 ꢃ CH3); 2.13–1.98
(2H, m, CH2). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.1, 169.8,
169.4 (COCH3); 147.3 (C-quat triazole); 122.4 (CH triazole); 102.0
(C-1); 71.8 (C-5); 70.7 (C-3); 68.7 (C-2); 67.7 (C-4); 57.0 (OCH3);
49.9 (C-6); 29.6 (CH2); 24.4 (2 ꢃ CH2); 20.7, 20.5, 20.3 (COCH3).
ESI-MS m/z, calcd for C33H46N6O16 [M+H]+ 783.3; [M+Na]+ 805.3,
found 783.4; 805.3. Deprotected compound 48c: ESI-MS m/z, calcd
for C21H34N6O10 [M+Na]+ 553.2, found 553.3.
4.4.26. 3-[1-(b-
1-ol (29d)
D
-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-propan-
0
0
Tetraacetate 29b: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.64 (1H, s, CH triazole); 5.83 (1H, d, J1,2 9.5 Hz, H-1);
5.55 (1H, d, J3,4 3.4 Hz, H-4); 5.51 (1H, pseudo t, J 9.8 Hz, H-2);
5.26 (1H, dd, J2,3 10.2 Hz, J3,4 3.4 Hz, H-3); 4.24 (1H, t, J 6.4 Hz, H-
0
0
5); 4.20 (1H, dd, J5,6 5.9 Hz, J6,6 11.4 Hz, H-6); 4.15 (1H, dd, J5,6
6.9 Hz, J6,6 11.4 Hz, H-60); 3.69 (2H, t, J 6.3 Hz, CH2); 2.87 (2H, t, J
7.3 Hz, CH2); 2.35 (1H, br s, OH); 2.22, 2.06, 2.05, 1.89 (12H, 4s,
4 ꢃ CH3); 1.95 (2H, q, J 6.3 Hz, J 7.3 Hz, CH2). 13C NMR (CDCl3,
100 MHz), d (ppm): 170.4, 170.0, 169.8, 169.2 (COCH3); 148.2 (C-
quat triazole); 119.4 (CH triazole); 86.2 (C-1); 74.0 (C-5); 70.7 (C-
3); 67.9 (C-2); 66.9 (C-4); 61.5 (CH2OH); 61.2 (C-6); 31.7 (CH2);
22.0 (CH2); 20.7, 20.5, 20.2 (COCH3). ESI-MS m/z, calcd for
C19H27N3O10 [M+H]+ 458.1; [M+Na]+ 480.1, found 458.0; 480.1.
Deprotected compound 29d: ESI-MS m/z, calcd for C11H19N3O6
[M+Na]+ 312.1, found 312.2.
0
4.4.23. Methyl [1-(methyl 6-deoxy-b-D-galactopyranosid-6-yl)-
1H-1,2,3-triazol-4-yl]-carboxylate (49c)
Triacetate 49a: (96% yield); 1H NMR (CDCl3, 400 MHz), d (ppm):
8.20 (1H, s, CH triazole); 5.49 (1H, d, J3,4 3. Hz, H-4); 5.22 (1H, dd,
J1,2 7.8 Hz, J2,3 10.5 Hz, H-2); 5.04 (1H, dd, J2,3 10.5 Hz, J3,4 3.3 Hz, H-
0
0
3); 4.69 (1H, dd, J5,6 3.4 Hz, J6,6 14.2 Hz, H-6); 4.49 (1H, dd, J5,6
8.7 Hz, J6,6 14.2 Hz, H-60); 4.32 (1H, d, J1,2 7.8 Hz, H-1); 4.17 (1H,
0
0
dd, J5,6 3.4, J5,6 8.7 Hz, H-5); 3.95 (3H, s, CO2CH3); 3.39 (3H, s,
4.4.27. 4-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-butan-
OCH3); 2.20, 2.04, 1.97 (9H, 3s, 3 ꢃ CH3). 13C NMR (CDCl3,
100 MHz), d (ppm): 170.3; 170.0; 169.5 (COCH3); 161.0 (CO2CH3);
140.0 (C-quat triazole); 128.9 (CH triazole); 102.1 (C-1); 71.5 (C-5);
70.6 (C-3); 68.5 (C-2); 67.9 (C-4); 57.2 (OCH3); 52.3 (CO2CH3); 50.7
1-ol (30d)
Tetraacetate 30b: (86% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.61 (1H, s, CH triazole); 5.82 (1H, d, J1,2 9.3 Hz, H-1);
5.55 (1H, m, H-4); 5.53 (1H, pseudo t, J 9.7 Hz, H-2); 5.25 (1H,
dd, J2,3 10.2 Hz, J3,4 3.4 Hz, H-3); 4.23 (1H, t, J 6.4 Hz, H-5); 4.19
(C-6); 20.7, 20.6, 20.5 (COCH3). IR (KBr)
mmax: 1749.3; 1371.3;

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2423
0
0
0
(1H, dd, J5,6 5.9 Hz, J6,6 11.4 Hz, H-6); 4.15 (1H, dd, J5,6 6.9 Hz, J6,6
2.7 Hz, H-4); 5.32 (NH); 5.28 (1H, dd, J2,3 10.2 Hz, J3,4 3.4 Hz, H-
3); 4.28–4.14 (3H, m, H-6, H-60, H-5); 2.25, 2.05, 1.99, 1.89 (12H,
4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4, 170.0,
169.8, 169.1 (COCH3); 148.8 (C-quat triazole); 145.1 (C-20); 129.4
(C-50); 128.1 (C-30); 118.3 (CH triazole); 117.5 (C-40); 116.7 (C-
60); 113.2 (C-10); 86.3 (C-1); 74.1 (C-5); 70.7 (C-3); 67.8 (C-2);
66.9 (C-4); 61.2 (C-6); 20.7, 20.6, 20.5, 20.2 (COCH3). ESI-MS m/z,
calcd for C22H26N4O9 [M+H]+ 491.1; [M+Na]+ 513.1, found 491.0;
513.1. Deprotected compound 34d: ESI-MS m/z, calcd for
C14H18N4O5 [M+H]+ 323.1; [M+Na]+ 345.1, found 323.0; 345.1.
11.4 Hz, H-60); 3.66 (2H, t, J 6.3 Hz, CH2OH); 2.77 (2H, t, J 7.3 Hz,
CH2); 2.26, 2.05, 2.01, 1.88 (12H, 4s, 4 ꢃ CH3); 1.92 (1H, br s,
OH); 1.80 (2H, q, J 6.8 Hz, J 7.3 Hz, J 7.5 Hz, CH2); 1.57 (2H, q, J
6.8 Hz, J 6.6 Hz, J 7.0 Hz, CH2). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.4, 170.0, 169.8, 169.3 (COCH3); 148.7 (C-quat triazole);
119.1 (CH triazole); 86.2 (C-1); 73.9 (C-5); 70.8 (C-3); 67.9 (C-2);
66.9 (C-4); 62.2 (CH2OH); 61.2 (C-6); 31.9 (CH2); 25.3 (CH2); 25.2
(CH2); 20.7, 20.6, 20.5, 20.2 (COCH3). ESI-MS m/z, calcd for
C20H29N3O10 [M+H]+ 472.1; [M+Na]+ 494.1, found 472.0; 494.2.
Deprotected compound 30d: ESI-MS m/z, calcd for C12H21N3O6
[M+Na]+ 326.1, found 326.2.
4.4.32. 2-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-bromo-
benzene (35d)
4.4.28. [1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-benzene
Tetraacetate 35b: (93% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.46 (1H, s, CH triazole); 8.05 (1H, dd, J 1.7 Hz, J 7.8 Hz,
H-60); 7.67 (1H, dd, J 1.2 Hz, J 8.0 Hz, H-30); 7.59 (1H, d, J 8.0 Hz,
H-40); 7.42 (1H, dd, J 1.2 Hz, J 7.8 Hz, H-50); 7.22 (1H, dt, J 1.7 Hz,
J 1.2 Hz, J 8.0 Hz, CH Ar); 5.93 (1H, d, J1,2 9.5 Hz, H-1); 5.69 (1H, t,
J 10.0 Hz, H-2); 5.58 (1H, d, J3,4 3.4 Hz, H-4); 5.29 (1H, dd, J2,3
(31d)
Tetraacetate 31b: (93% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8,05 (1H, s, CH triazole); 7.90 (2H, d, J 7.1 Hz, CH Ar);
7.45 (2H, pseudo t, J 7,3 Hz, CH Ar); 7.36 (1H, t, J 7.3 Hz, CH Ar);
5.90 (1H, d, J1,2 9.5 Hz, H-1); 5,64 (1H, pseudo t, J 10.0 Hz, H-2);
5.58 (1H, d, J 2.7 Hz, H-4); 5.29 (1H, dd, J2,3 10.5 Hz, J3,4 3.3 Hz,
H-3); 4.18 (3H, m, H-6, H-60, H-5); 2.25, 2.06, 2.05, 1.90 (12H, 4s,
4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4, 170.0,
169.8, 169.2 (COCH3); 148.5 (C-quat); 129.9, 128.8, 128.5, 125.9
(CH Ar); 117.8 (CH triazole); 86.3 (C-1); 74.1 (C-5); 70.9 (C-3);
67.7 (C-2); 66.9 (C-4); 61.2 (C-6); 20.7, 20.6, 20.5, 20.3 (COCH3).
ESI-MS m/z, calcd for C22H25N3O9 [M+H]+ 476.1; [M+Na]+ 498.1,
found 476.0; 498.1. Deprotected compound 31d: ESI-MS m/z, calcd
for C14H17N3O5 [M+Na]+ 330.1, found 330.1.
0
10.3 Hz, J3,4 3.4 Hz, H-3); 4.28 (1H, dd, J5,6 5.8 Hz, J5,6 6.9 Hz, H-
0
0
5); 4.23 (1H, dd, J5,6 5.8 Hz, J6,6 11.3 Hz, H-6); 4.17 (1H, dd, J5,6
6.9 Hz, J6,6 11.3 Hz H-60); 2.24, 2.05, 2.02, 1.91 (12H, 4s, 4 ꢃ CH3).
13C NMR (CDCl3, 100 MHz), d (ppm): 170.3, 170.1, 169.8, 169.0
(COCH3); 146.0 (C-quat triazole); 133.6, 127.6 (C-quat Ar) 130.8,
129.7, (CH Ar); 121.4 (CH triazole); 86.3 (C-1); 74.1 (C-5); 70.9
(C-3); 67.7 (C-2); 66.9 (C-4); 61.2 (C-6); 20.7, 20.6, 20.5, 20.3
(COCH3). ESI-MS m/z, calcd for C22H24BrN3O9 [M+Na]+ 575.8, found
576.0. Deprotected compound 35d: ESI-MS m/z, calcd for
C14H16BrN3O5 [M+Na]+ 408.0, found 408.1.
0
4.4.29. [1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-phenyl-
methane (32d)
Tetraacetate 32b: (81% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.49 (1H, CH triazole); 7.34–7.22 (5H, m, CH Ar,); 5.80
(1H, d, J1,2 9.3 Hz, H-1); 5.52 (1H, d, J3,4 3.5 Hz, H-4); 5.50 (1H,
pseudo t, J 9.7 Hz, H-2); 5.22 (1H, dd, J2,3 10.2 Hz, J3,4 3.5 Hz, H-
3); 4.21–4.10 (5H, m, H-6, H-60, H-5, CH2); 2.19, 2.03, 1.99, 1.87
(12H, 4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.3,
170.0, 169.8, 169.0 (COCH3); 148.2 (C-quat); 138.6 (C-quat);
128.7, 128.6, 126.5 (CH Ar); 119.8 (CH triazole) 86.2 (C-1); 74.0
(C-5); 70.8 (C-3); 67.8 (C-2); 66.8 (C-4); 61.2 (C-6); 32.1 (CH2);
4.4.33. 2-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-trifluoro-
methylbenzene (36d)
Tetraacetate 36b: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.01 (1H, s, CH triazole); 7.88 (1H, d, J 7.8 Hz H-30); 7.78
(1H, d, J 7.8 Hz, H-60); 7.64 (1H, t, J 7.6 Hz, H-50); 7.52 (1H, t, J
7.6 Hz, H-40); 5.93 (1H, d, J1,2 9.3 Hz, H-1); 5.68 (1H, t, J 9.8 Hz,
H-2); 5.58 (1H, d, J3,4 3.4 Hz, H-4); 5.29 (1H, dd, J2,3 10.2 Hz, J3,4
3.4 Hz, H-3); 4.27 (1H, pseudo t, J 6.4 Hz, H-5); 4.22 (1H, dd, J5,6
0
0
0
6.0 Hz, J6,6 11.5 Hz, H-6); 4.18 (1H, dd, J5,6 6.8 Hz, J6,6 11.5 Hz,
H-60); 2.23, 2.04, 2.02, 1.91 (12H, 4s, 4 ꢃ CH3). 13C NMR (CDCl3,
100 MHz), d (ppm): 170.3, 170.1, 169.8, 168.9 (COCH3); 145.0 (C-
quat triazole); 132.0, 128.9 (C-quat); 128.1, 127.5, 126.7 (CH Ar);
121.3 (CH triazole); 86.3 (C-1); 74.2 (C-5); 70.9 (C-3); 67.6 (C-2);
66.9 (C-4); 61.2 (C-6); 20.7, 20.6, 20.5, 20.1 (COCH3). ESI-MS m/z,
calcd for C23H24F3N3O9 [M+Na]+ 566.1, found 566.1. Deprotected
compound 36d: ESI-MS m/z, calcd for C15H16F3N3O5 [M+Na]+
398.1, found 398.1.
20.7, 20.6, 20.4, 20.2 (COCH3). IR (KBr)
mmax: 1751.2;1369.4;
1220.9; 1064.6 cmꢂ1. ESI-MS m/z, calcd for C23H27N3O9 [M+H]+
490.1; [M+Na]+ 512.1, found 490.0; 512.2. Deprotected compound
32d: ESI-MS m/z, calcd for C15H19N3O5 [M+Na]+ 344.1, found 344.2.
4.4.30. 2-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-phenyl-
ethane (33d)
Tetraacetate 33b: (75% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.49 (1H, CH triazole) 7.31–7.19 (5H, m, CH Ar,); 5.81
(1H, d, J1,2 9.3 Hz, H-1); 5.56–5.51 (2H, m, H-2, H-4); 5.23 (1H,
dd, J2,3 10.3, J3,4 3.4 Hz, H-3); 4.22–4.11 (3H, m, H-6, H-60, H-5)
3.07–2.98 (4H, m, CH2); 2.21, 2.05, 2.01, 1.92 (12H, 4s, 4 ꢃ CH3).
13C NMR (CDCl3, 100 MHz), d (ppm): 170.3, 170.0, 169.8, 169.0
(COCH3); 148.0 (C-quat); 141.0 (C-quat); 128.4, 126.1 (CH Ar);
119.21 (CH triazole); 86.2 (C-1); 73.9 (C-5); 70.9 (C-3); 67.7 (C-
2); 66.9 (C-4); 61.2 (C-6); 35.3 (CH2); 27.5 (CH2); 20.6, 20.5, 20.2
(COCH3). ESI-MS m/z, calcd for C24H29N3O9 [M+H]+ 504.1;
[M+Na]+ 526.2, found 504.0; 526.2. Deprotected compound 33d:
ESI-MS m/z, calcd for C16H21N3O5 [M+Na]+ 358.1, found 358.2.
4.4.34. 4-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-aniline
(37d)
Tetraacetate 37b: (88% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.91 (1H, s, CH triazole); 7.64 (2H, d, J 8.5 Hz, H-30, H-50);
7.29 (2H, d, J 8.5 Hz, H-20, H-60); 5.87 (1H, d, J1,2 9.5 Hz, H-1);
5.64 (1H, t, J 10.0 Hz, H-2); 5.56 (1H, d, J3,4 3.2 Hz, H-4); 5.26
(1H, dd, J2,3 10.0 Hz, J3,4 3.2 Hz, H-3); 4.24–4.13 (3H, m, H-6, H-60,
H-5); 3.82 (NH2); 2.24, 2.06, 1.99, 1.89 (12H, 4s, 4 ꢃ CH3). 13C
NMR (CDCl3, 100 MHz),
d (ppm): 170.4, 170.1, 169.9, 169.2
(COCH3); 148.7 (C-40); 147.0 (C-quat triazole); 127.1 (C-30, C-50);
120.4 (C-10); 116.8 (C-20, C60); 115.2 (CH triazole); 86.3 (C-1);
74.1 (C-5); 70.8 (C-3); 68.1 (C-2); 67.1 (C-4); 61.4 (C-6); 20.8,
20.5, 20.3 (COCH3). ESI-MS m/z, calcd for C22H26N4O9 [M+H]+
491.1; [M+Na]+ 513.1, found 491.1; 513.1. Deprotected compound
37d: ESI-MS m/z, calcd for C14H18N4O5 [M+H]+ 323.1; [M+Na]+
345.1, found 323.0; 345.1. ESI-HRMS [M+Na]+ found 345.1168;
calcd 345.1169.
4.4.31. 2-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-aniline
(34d)
Tetraacetate 34b: (88% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.06 (1H, s, CH triazole); 7.42 (1H, d, J3 ,4 7.5 Hz, H-30);
7.12 (1H, t, J 8.4 Hz, H-50); 6.76 (1H, m, H-40, H-60); 5.89 (1H, d,
J1,2 9.3 Hz, H-1); 5.66 (1H, t, J 10.2 Hz, H-2); 5.57 (1H, d, J4,5
0
0

2424
I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
4.4.35. 4-[1-(b-
benzene (38d)
D
-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-bromo-
581.1, found 581.1. Deprotected compound 41d: ESI-MS m/z, calcd
for C17H18N4O7 [M+Na]+ 413.1, found 413.1.
Tetraacetate 38b: (quantitative yield); 1H NMR (CDCl3,
400 MHz), d (ppm): 8.06 (1H, s, CH triazole); 7.74 (2H, d, J 8.6 Hz,
H-20, H-60); 7.57 (2H, d, J 8.6 Hz, H-30, H-50); 5.90 (1H, d, J1,2
9.3 Hz, H-1); 5.61 (1H, t, J 10 Hz, H-2); 5.58 (1H, dd, J4,5 1.0 Hz,
J3,4 3.4 Hz, H-4); 5.29 (1H, dd, J2,3 10.2 Hz, J3,4 3.4 Hz, H-3); 4.27
4.4.39. 2-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-pyridine
(42d)
Tetraacetate 42b: (95% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.61 (1H, d, J4 ,5 4.1 Hz, H-60); 8.50 (1H, CH triazole) 8.16
0
0
(1H, t, J 6.4 Hz, H-5); 4.21 (1H, dd, J5,6 5.8 Hz, J6,6 11.5 Hz, H-6);
(1H, d, J2 ,3 8.0 Hz, H-30); 7.79 (1H, dt, J2 ,3 8.0 Hz, J3 ,4 1.7 Hz, H-
40); 7.25 (1H, m, H-50); 5.92 (1H, d, J1,2 9.2 Hz, H-1); 5.62 (1H, dd,
0
0
0
0
0
0
0
4.17 (1H, dd, J5,6 7.0 Hz, J6,6 11.5 Hz, H-60); 2.24, 2.04, 2.02, 1.90
(12H, 4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.3,
170.0, 169.8, 169.2 (COCH3); 147.4 (C-quat triazole); 132.0, 127.4
(CH Ar), 128.9, 122.4 (C-quat Ar); 118.0 (CH triazole); 86.3 (C-1);
74.1 (C-5); 70.7 (C-3); 67.8 (C-2); 66.9 (C-4); 61.2 (C-6); 20.7,
20.6, 20.5, 20.3 (COCH3). ESI-MS m/z, calcd for C22H24BrN3O9
[M+Na]+ 575.8, found 576.1. Deprotected compound 38d: ESI-MS
m/z, calcd for C14H16BrN3O5 [M+HCOO]ꢂ 430.0, found 429.9.
0
0
J1,2 9.2 Hz, J
10.2 Hz, H-2); 5.57 (1H, d, J3,4 3.4 Hz, H-4); 5.29
2,3
0
(1H, dd, J2,3 10.2 Hz, J3,4 3.4 Hz, H-3); 4.28 (1H, t, J5,6 5.8 Hz, J5,6
0
6.8 Hz, H-5); 4.21 (1H, dd, J5,6 5.8 Hz, J6,6 11.5 Hz, H-6); 4.15 (1H,
dd, J5,6 6.8 Hz, J6,6 11.5 Hz, H-60); 2.24, 2.04, 2.02, 1.92 (12H, 4s,
4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.3, 170.0,
169.8, 169.0 (COCH3); 149.7 (C-20); 149.5 (C-60); 148.9 (C-quat tri-
azole); 136.9 (C-40), 123.1 (C-50), 120.6 (C-30); 120.3 (CH triazole);
86.3 (C-1); 74.0 (C-5); 70.8 (C-3); 68.0 (C-2); 66.9 (C-4); 61.3 (C-6);
20.7, 20.6, 20.5, 20.3 (COCH3). ESI-MS m/z, calcd for C21H24N4O9
[M+H]+ 477.1; [M+Na]+ 499.0, found 477.0; 499.1. Deprotected
compound 42d: ESI-MS m/z, calcd for C13H16N4O5 [M+Na]+ 331.1,
found 331.1.
0
0
4.4.36. 4-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-trifluoro-
methylbenzene (39d)
Tetraacetate 39b: (93% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.18 (1H, s, CH triazole); 7.98 (2H, d, J 8.0 Hz, H-30, H-50);
7.69 (2H, d, J 8.3 Hz, H-20, H-60); 5.92 (1H, d, J1,2 9.3 Hz, H-1);
5.62 (1H, t, J 10.0 Hz, H-2); 5.59 (1H, d, J3,4 3.2 Hz, H-4); 5.29
(1H, dd, J2,3 10.2 Hz, J3,4 3.2 Hz, H-3); 4.28 (1H, t, J 6.4 Hz, H-5);
4.4.40. 3-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-pyridine
(43d)
Tetraacetate 43b: (quantitative yield); 1H NMR (CDCl3,
400 MHz), d (ppm): 9.06 (1H, br s, H-20); 8.61 (1H, br s, H-60);
0
0
4.22 (1H, dd, J5,6 5.8 Hz, J6,6 11.5 Hz, H-6); 4.12 (1H, dd,, J5,6
7.1 Hz, J6,6 11.5 Hz, H-60); 2.25, 2.05, 2.03, 1.85 (12H, 4s,
4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.3, 169.9,
169.8, 169.3 (COCH3); 147.1 (C-quat triazole); 133.4, 130.7 (C-quat
Ar) 126.7, 126.0, (CH Ar); 118.8 (CH triazole); 86.4 (C-1); 74.2 (C-
5); 70.7 (C-3); 67.8 (C-2); 66.9 (C-4); 61.2 (C-6); 20.7, 20.6, 20.5,
20.3 (COCH3). ESI-MS m/z, calcd for C23H24F3N3O9 [M+Na]+ 566.1,
found 566.1. Deprotected compound 39d: ESI-MS m/z, calcd for
C15H16F3N3O5 [M+HCOO]ꢂ 420.1, found 420.0.
0
8.22 (1H, dd, J4 ,5 7.8 Hz, J4 ,6 1.7 Hz, H-40); 8.17 (CH triazole);
0
0
0
0
7.39 (1H, dd, J4 ,5 7.8 Hz, J5 ,6 4.8 Hz, H-5 0); 5.94 (1H, d, J1,2
9.3 Hz, H-1); 5.63 (1H, pseudo t, J 9.8 Hz, H-2); 5.59 (1H, dd, J3,4
3.4 Hz, J4,5 1.0 Hz, H-4); 5.30 (1H, d, J2,3 10.3 Hz, J3,4 3.4 Hz, H-3);
0
0
0
0
0
4.30 (1H, dd, J4,5 1.0 Hz, J5,6 5.8 Hz, J5,6 7.0 Hz, H-5); 4.23 (1H, dd,
0
0
0
J5,6 5.8 Hz, J6,6 11.4 Hz, H-6); 4.19 (1H, dd, J5,6 7.0 Hz, J6,6
11.4 Hz, H-60); 2.27, 2.05, 2.03, 1.92 (12H, 4s, 4 ꢃ CH3). 13C NMR
(CDCl3, 100 MHz), d (ppm): 170.3, 170.0, 169.8, 169.2 (COCH3);
149.6 (C-60) 147.2 (C-20); 145.4 (C-quat); 133.1 (C-40); 126.2 (C-
30); 123.7 (C-50); 118.4 (CH triazole); 86.4 (C-1); 74.2 (C-5); 70.7
(C-3); 67.9 (C-2); 66.9 (C-4); 61.2 (C-6); 20.7, 20.6, 20.5, 20.2
4.4.37. 3-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-trifluoro-
methylbenzene (40d)
Tetraacetate 40b: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.14 (1H, s, CH triazole); 8.11 (1H, s, H-20); 8.07 (1H, d, J
7.6 Hz, H-60); 7.58–7.43 (2H, m, H-40, H-50); 5.92 (1H, d, J1,2
9.3 Hz, H-1); 5.60 (1H, t, J 9.8 Hz, H-2); 5.59 (1H, d, J3,4 3.4 Hz, H-
4); 5.30 (1H, dd, J2,3 10.3 Hz, J3,4 3.4 Hz, H-3); 4.28 (1H, pseudo t,
(COCH3). IR (KBr) m
max: 1753.2; 1369.4; 1222.8; 1064.6 cmꢂ1. ESI-
MS m/z, calcd for C21H24N4O9 [M+H]+ 477.1; [M+Na]+ 499.0, found
477.0; 499.1. Deprotected compound 43d: ESI-MS m/z, calcd for
C13H16N4O5 [M+HCOO]ꢂ 353.1, found 352.9.
0
J 6.4 Hz, H-5); 4.23 (1H, dd, J5,6 5.7 Hz, J6,6 11.5 Hz, H-6); 4.19
4.4.41. N-Benzyl-N-methyl-1-[1-(b-D-galactopyranosyl)-1H-1,2,3-
(1H, dd, J5,6 7.1 Hz, J6,6 11.5 Hz, H-60); 2.26, 2.05, 2.03, 1.92 (12H,
4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4, 170.0,
169.8, 169.2 (COCH3); 147.1 (C-quat triazole); 131.6, 130.8 (C-quat
Ar); 126.6, 125.1, 124.8, 122.7 (CH Ar); 118.4 (CH triazole); 86.4 (C-
1); 74.2 (C-5); 70.7 (C-3); 67.9 (C-2); 66.9 (C-4); 61.2 (C-6); 20.7,
20.6, 20.5, 20.3 (COCH3). ESI-MS m/z, calcd for C23H24F3N3O9
[M+Na]+ 566.1, found 566.1. Deprotected compound 40d: ESI-MS
m/z, calcd for C15H16F3N3O5 [M+Na]+ 398.1, found 398.1.
0
0
triazol-4-yl]-methylamine (44d)
Tetraacetate 44b: (84% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.79 (1H, s, CH triazole); 7.37–7.24 (5H, m, CH Ar); 5.84
(1H. d. J1.2 9.3 Hz, H-1); 5.55 (2H, dd, J1,2 9.3 Hz, J2,3 10.4 Hz, H-2,
H-4); 5.25 (1H, dd, J2,3 10.4 Hz, J3,4 3.4 Hz, H-3); 4.23 (1H, pseudo
0
t, J 6.2 Hz, H-5); 4.20 (1H, dd, J5,6 5.9 Hz, J6,6 11.1 Hz, H-6); 4.15
(1H, dd, J5,6 6.7 Hz, J6,6 11.1 Hz, H-60); 3.75 (1H, d, J 14.1 Hz,
CH2); 3.72 (1H, d, J 14.1 Hz, CH2); 3.57 (1H, d, J 13.3 Hz, CH2);
3.52 (1H, d, J 13.3 Hz, CH2); 2.23, 2.04, 2.01, 1.85 (15H, 5s,
5 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.3, 170.0,
169.8, 169.0 (COCH3); 146.0 (C-quat triazole); 138.5 (C-quat Ar);
129.1, 128.2, 127.2 (CH Ar); 121.0 (CH triazole); 86.3 (C-1); 74.0
(C-5); 70.8 (C-3); 67.9 (C-2); 66.9 (C-4); 61.3 (C-6); 61.2 (CH2);
51.9 (CH2); 41.9 (NCH3); 20.7, 20.6, 20.5, 20.2 (COCH3). ESI-MS
m/z, calcd for C25H32N4O9 [M+H]+ 533.2; [M+Na]+ 555.2, found
533.1; 555.1. Deprotected compound 44d: ESI-MS m/z, calcd for
C17H24N4O5 [M+H]+ 365.1; [M+Na]+ 387.1, found 365.1; 387.2.
0
0
4.4.38. 2-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-N-
phthalimidylmethane (41d)
Tetraacetate 41b: (84% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.93 (1H, s, CH triazole); 7.88–7.85 (2H, m, CH Ar); 7.76–
7.71 (2H, m, CH Ar); 5.82 (1H, d, J1,2 9.5 Hz, H-1); 5.53 (1H, d, J3,4
3.4 Hz, H-4); 5.52 (1H, pseudo t, J 9.7 Hz, H-2); 5.23 (1H, dd, J2,3
10.3 Hz, J3.4 3.4 Hz, H-3); 5.05 (1H, d, J 15.4 Hz, CH2); 4.96 (1H, d,
J 15.4 Hz, CH2); 4.23–4.16 (2H, m, H-6, H-60); 4.09 (1H, q, J5,6 4.5,
J5,6 9.8 Hz, H-5); 2.24, 2.03, 1.99, 1.82 (12H, 4s, 4 ꢃ CH3). 13C
0
NMR (CDCl3, 100 MHz),
d (ppm): 170.3, 170.0, 169.8, 169.0
4.4.42. 4-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-butanoic
(COCH3); 167.6 (CO); 143.4 (C-quat triazole); 134.2, 132.0, 123.5
(C-quat, CH Ar); 121.5 (CH triazole); 86.2 (C-1); 74.1 (C-5); 70.8
(C-3); 67.8 (C-2); 66.9 (C-4); 61.2 (C-6); 32.9 (CH2); 20.7, 20.6,
20.4, 20.2 (COCH3). ESI-MS m/z, calcd for C25H26N4O11 [M+Na]+
acid (45d)
Tetraacetate 45b: (81% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.64 (1H, s, CH triazole); 5.83 (1H, d, J1,2 9.3 Hz, H-1);
5.56–5.51 (2H, m, H-2, H-4); 5.25 (1H, dd, J2,3 10.2 Hz, J3,4 3.4 Hz,

I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
2425
0
0
H-3); 4.24–4.20 (2H, m, H-6, H-5); 4.15 (1H, dd, J5,6 6.8 Hz, J6,6
5.57 (1H, d, J3,4 3.2 Hz, H-4); 5.50 (1H, pseudo t, J 9.7 Hz, H-2);
5.28 (1H, dd, J2,3 10.2 Hz, J3,4 3.2 Hz, H-3); 4.26 (1H, pseudo t, J
11.0 Hz, H-60); 2.82 (2H, dt, J 2.8 Hz, J 7.3 Hz, CH2); 2.42 (2H, t, J
7.3 Hz, CH2); 2.23 (3H, s, CH3); 2.05–2.01 (8H, 2s, m, 2 ꢃ CH3,
CH2); 1.89 (3H, s, CH3). 13C NMR (CDCl3, 100 MHz), d (ppm):
178.2 (CO2H); 170.4, 170.0, 169.9, 169.2 (COCH3); 147.7 (C-quat
triazole); 119.5 (CH triazole); 86.3 (C-1); 74.0 (C-5); 70.8 (C-3);
67.9 (C-2); 66.9 (C-4); 61.2 (C-6); 32.9 (CH2); 24.6 (CH2); 24.1
0
6.3 Hz, H-5); 4.21 (1H, dd, J5.6 5.8 Hz, J6,6 11.5 Hz, H-6); 4.16 (1H,
dd, J5,6 7.0 Hz, J6,6 11.5 Hz, H-60); 3.98 (3H, s, CO2CH3); 2.23,
2.05, 1.99, 1.89 (12H, 4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d
(ppm): 170.3, 169.9, 169.8, 169.2 (COCH3); 160.7 (CO2CH3); 140.6
(C-quat triazole); 126.3 (CH triazole); 86.5 (C-1); 74.3 (C-5); 70.5
(C-3); 68.0 (C-2); 66.8 (C-4); 61.2 (C-6); 52.4 (CO2CH3); 20.6,
0
0
(CH2); 20.7, 20.5, 20.3 (COCH3). IR (KBr)
mmax: 2939.3; 1751.2;
1220.9; 1062.7 cmꢂ1. ESI-MS m/z, calcd for C20H27N3O11 [M+H]+
486.1; [M+Na]+ 508.1, found 486.0; 508.2. Deprotected compound
45d: ESI-MS m/z, calcd for C12H19N3O7 [M+Na]+ 340.1; [M+2Na]+
363.0, found 340.2 363.2.
20.5, 20.2 (COCH3). IR (KBr) mmax: 1751.2; 1369.4; 1218.9;
1064.6 cmꢂ1. ESI-MS m/z, calcd for C18H23N3O11 [M+Na]+ 480.1,
found 480.1. Deprotected compound 49d: ESI-MS m/z, calcd for
C10H15N3O7 [M+Na]+ 312.1, found 312.1.
4.4.43. (2R/S)-1-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-
4.4.47. [1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-methanol
propan-2-ol (46d)
O-(R/S)-epoxypropyl ether (50d)
Tetraacetate 46b: (87% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.73 (1H, s, CH triazole); 5.83, 5.81 (1H, 2 ꢃ d, J1,2 9.3 Hz,
H-1); 5.56 (1H, d, J3,4 3.2 Hz, H-4); 5.46 (1H, dt, J1,2 9.3 Hz, J2,3
10.2 Hz, H-2); 5.27 (1H, dd, J2,3 10.2 Hz, J3,4 3.2 Hz, H-3); 4.25–
4.15 (4H, m, H-6, H-60, H-5, CHOH); 2.93–2.70 (2H, m, CH2); 2.22,
2.05, 2.01, 1.89 (12H, 4s, 4 ꢃ CH3); 1.29–1.25 (3H, m, CH3). 13C
Tetraacetate 50b: (88% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.88 (1H, s, CH triazole); 5.85 (1H, d, J1,2 9.3 Hz, H-1);
5.54 (1H, t, J 9.7 Hz, H-2); 5.53 (1H, d, J3,4 3.4 Hz, H-4); 5.25 (1H,
dd, J2,3 10.5 Hz, J3,4 3.4 Hz, H-3); 4.75 (1H, d, J 12.4 Hz, CH2); 4.70
(1H, d, J 12.4 Hz, CH2); 4.24 (1H, pseudo t, J 6.3 Hz, H-5); 4.18
0
0
(1H, dd, J5,6 5.6 Hz, J6,6 11.4 Hz, H-6); 4.14 (1H, dd, J5,6 7.0 Hz,
J6,6 11.4 Hz, H-60); 3.83 (1H, ddd, J 3.0 Hz, J 5.8 Hz, CH2); 3.45
0
NMR (CDCl3, 100 MHz),
d
(ppm): 170.3, 170.0, 169.8, 169.3
(COCH3); 146.0, 145.7 (C-quat triazole); 120.8, 120.5 (CH triazole);
86.3 (C-1); 74.0 (C-5); 70.7, 70.6 (C-3); 68.2, 68.0 (C-2); 67.1
(CHOH); 66.9, 66.8 (C-4); 61.2 (C-6); 35.2, 34.8 (CH2); 22.7 (CH3);
20.7, 20.6, 20.5, 20.2 (COCH3). ESI-MS m/z, calcd for C19H27N3O10
[M+H]+ 458.1; [M+Na]+ 480.1, found 458.0; 480.1. Deprotected
compound 46d: ESI-MS m/z, calcd for C11H19N3O6 [M+Na]+ 312.1,
found 312.2.
(1H, q, J 5.8 Hz, CH2); 3.20 (1H, sl, CH); 2.81 (1H, t, J 4.6 Hz, CH2);
2.64 (1H, dd, J 3.0 Hz, J 4.6 Hz, CH2); 2.23, 2.05, 2.01, 1.90 (12H,
4s, 4 ꢃ CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4, 170.0,
169.8, 169.1 (COCH3); 145.6 (C-quat triazole); 121.2 (CH triazole);
86.3 (C-1); 74.0 (C-5); 71.1 (CH2); 70.8 (C-3); 67.9 (C-2); 66.9 (C-4);
64.5 (CH2); 61.2 (C-6); 50.6 (CH); 44.3 (CH2); 20.7, 20.6, 20.5, 20.3
(COCH3). IR (KBr) m
max: 1753.2; 1371.3; 1220.9; 1064.6 cmꢂ1. ESI-
MS m/z, calcd for C20H27N3O11 [M+H]+ 486.1, found 486.2. Depro-
tected compound 50d: ESI-MS m/z, calcd For C12H19N3O7 [M+H]+
318.1, found 318.1.
4.4.44. (1R/S)-1-[1-(b-D-Galactopyranosyl)-1H-1,2,3-triazol-4-yl]-
propan-1-ol (47d)
Tetraacetate 47b: (100% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.79, 7.78 (1H, s, CH triazole); 5.85 (1H, d, J1,2 9.3 Hz, H-
1); 5.57–5.51 (2H, m, H-4, H-2); 5.26 (1H, dd, J2,3 10.2 Hz, J3,4
3.4 Hz, H-3); 4.85 (1H, m, CHOH); 4.24 (1H, t, J 6.6 Hz, H-5) 4.22–
4.13 (2H, m, H-6, H-60); 2.70, 2.65 (1H, 2 ꢃ s, OH); 2.23, 2.05,
2.04, 1.88 (12H, 4s, 4 ꢃ CH3, CH2); 1.93–1.86 (2H, m, CH2); 0.98
(3H, t, J 7.3 Hz, CH3). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4,
170.0, 169.8, 169.1 (COCH3); 152.0 (C-quat triazole); 119.1 (CH tri-
azole); 86.2 (C-1); 74.0 (C-5); 70.7 (C-3); 68.3 (CHOH); 67.9 (C-2);
66.9 (C-4); 61.2 (C-6); 30.3 (CH2);. 20.7, 20.6, 20.5, 20.2 (COCH3);
9.5 (CH3). ESI-MS m/z, calcd for C19H27N3O10 [M+H]+ 458.1;
[M+Na]+ 480.1, found 458.0; 480.1. Deprotected compound 47d:
ESI-MS m/z, calcd for C11H19N3O6 [M+Na]+ 312.1, found 312.1.
4.5. Biological assays
4.5.1. Fluorimetric TcTS inhibition assay
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.43 Inhibi-
tion was assessed using the continuous fluorimetric assay described
by Douglas and co-workers.40e Briefly, the assay was performed in
duplicate in 96-well plates containing phosphate buffer solution at
pH 7.4 (25
solution (25
for 10 min at 26 °C followed by addition of MuNANA (Km
0.68 mM;40e 25
L of a 0.4 mM solution giving an assay concentra-
lL), recombinant enzyme solution (25
lL) and inhibitor
l
L of a 4.0 mM solution). This mixture was incubated
=
l
4.4.45. 1,3-Bis{1-[(b-
propane (48d)
D
-galactopyranosyl)]-1H-1,2,3-triazol-6-yl}-
tion of 0.1 mM). The fluorescence of the released product (Mu)
was measured after 10 min, with excitation and emission wave-
lengths of 360 and 460 nm, respectively, and the data were analyzed
with GraphPad Prism software version 4.0 (San Diego, CA, USA).
Tetraacetate 48b: (44% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 7.65 (2H, s, CH triazole); 5.82 (2H, d, J1,2 9.2 Hz, H-1);
5.59–5.52 (4H, m, H-4, H-2); 5.24 (2H, dd, J2,3 10.2 Hz, J3,4 3.4 Hz,
H-3); 4.24–4.12 (6H, m, H-6, H-60, H-5); 2.78 (4H, dt, J 2.9 Hz, J
7.3 Hz, CH2); 2.23, 2.05, 2.01, 1.89 (24H, 4s, 4 ꢃ CH3); 2.12–2.05
(2H, m, CH2). 13C NMR (CDCl3, 100 MHz), d (ppm): 170.4, 170.0,
169.8, 169.1 (COCH3); 148.1 (C-quat triazole); 119.4 (CH triazole);
86.2 (C-1); 74.0 (C-5); 70.8 (C-3); 67.8 (C-2); 66.9 (C-4); 61.2 (C-6);
28.5 (CH2); 24.8 (CH2); 20.7, 20.5, 20.3 (COCH3). ESI-MS m/z, calcd
for C35H46N6O18 [M+H]+ 839.3; [M+Na]+ 861.3, found 839.4; 861.3.
Deprotected compound 48d: ESI-MS m/z, calcd for C19H30N6O10
[M+Na]+ 525.2, found 525.3. ESI-HRMS [M+H]+ found 503.2096;
calcd 503.2096.
Inhibition percentages were calculated by the equation:
%
I = 100 ꢃ [1 ꢂ (Vi/V0)], where Vi is the velocity in the presence of
inhibitor and V0 is the velocity in absence of inhibitor.
4.5.2. Sialic acid transfer reactions catalyzed by TcTS
A cell free extract containing TcTS was obtained from a cell pellet
of the expression strain, which was thawed on ice and re-suspended
in 30 ml of phosphate buffer saline (PBS). After 30 min incubation on
ice, the viscosity of the solution was decreased by addition of DNase
(20
l
g/mL). The cells were then sonicated on ice for 5 ꢃ 1 min cy-
cles, with 1 min intervals between bursts. The suspension was cen-
trifuged to remove cell debris and the supernatant was filtered
4.4.46. Methyl [1-(b-
D
-galactopyranosyl)-1H-1,2,3-triazol-4-yl]-
through a 0.22 lm filter. The cleared protein lysate obtained was
carboxylate (49d)
employed without further purification in biotransformations.
Tetraacetate 49b: (96% yield); 1H NMR (CDCl3, 400 MHz), d
(ppm): 8.41 (1H, s, CH triazole); 5.90 (1H, dd, J1,2 9.3 Hz, H-1);
The enzymatic reactions were conducted with 20-(4-methylum-
belliferyl)-a-D-N-acetylneuraminic acid (sodium salt) as a donor

2426
I. Carvalho et al. / Bioorg. Med. Chem. 18 (2010) 2412–2427
substrate. To a solution of donor substrate (20
0.1 M phosphate buffer pH 7.5, 5 equiv) and acceptor substrate
(20 L of a 1 mM solution in 0.1 M phosphate buffer pH 7.5,
1 equiv) was added 10 L of the crude TcTS to effect complete con-
l
L of a 5 mM in
and the BBSRC (UK Biotehcnology and Biological Sciences Research
Council). We are grateful to Dr. Rose Mary Zumstein Georgetto
Naal for the fluorimeter facilities, to Claudio Rogerio Oliveira for
preparing recombinant TcTS and to Helen Alves de Oliveira for
assistance with T. cruzi experiments.
l
l
version within 4 h at 25–30 °C. After completion of the reaction, as
judged by TLC (CH3CN/EtOAc/Isopropanol/H2O, 85:20:50:3, v/v),
protein was precipitated by addition of EtOH (50 lL) and the mix-
Supplementary data
ture was then centrifuged. The supernatant was transferred to a
new Eppendorf tube and analyzed by mass spectrometry. TLC
plates were visualized by dipping into orcinol solution and heating.
The release of umbelliferone from the donor was observed by UV
lamp only, which showed the donor as small bright spot at low
Rf and the by-product umbelliferone as a much brighter spot at
higher Rf (CH3CN/EtOAc/Isopropanol/H2O, 85:20:50:3, v/v). Lactose
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.02.053.
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