Synthesis, HIV-RT inhibitory activity and SAR of 1-benzyl-1H-1,2,3-triazole derivatives of carbohydrates

Article abstract of DOI:10.1016/j.ejmech.2008.02.047

This paper describes the synthesis of several 1-benzyl-1H-1,2,3-triazoles attached to different carbohydrate templates and their in vitro inhibitory profile against HIV-1 reverse transcriptase. In addition a theoretical comparison of the most active compo

Full text of DOI:10.1016/j.ejmech.2008.02.047

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 373e383
http://www.elsevier.com/locate/ejmech
Original article
Synthesis, HIV-RT inhibitory activity and SAR of
1-benzyl-1H-1,2,3-triazole derivatives of carbohydrates
Fernando de C. da Silva a, Maria Cecilia B.V. de Souza a, Izabel I.P. Frugulhetti b,
Helena C. Castro b, Silmara L. de O. Souza b, Thiago Moreno L. de Souza b,
Diego Q. Rodrigues b, Alessandra M.T. Souza a, Paula A. Abreu b,
Fabiana Passamani c, Carlos R. Rodrigues c, , Vitor F. Ferreira
*
a,**
a Universidade Federal Fluminense, Departamento de Qu´ımica Orgaˆnica, Instituto de Qu´ımica, Outeiro de S~ao Jo~ao Baptista,
CEP 24020-141, Nitero´i, RJ, Brazil
b Universidade Federal Fluminense, LABioMol, Departamento de Biologia Geral e Celular, Outeiro de S~ao Jo~ao Baptista,
CEP 24020-150, Nitero´i, RJ, Brazil
c Laborato´rio de Modelagem Molecular e QSAR (ModMolQSAR), Faculdade de Farma´cia, Universidade Federal do Rio de Janeiro,
CEP 21941-590, Rio de Janeiro, RJ, Brazil
Received 26 November 2007; received in revised form 25 February 2008; accepted 29 February 2008
Available online 16 March 2008
Abstract
This paper describes the synthesis of several 1-benzyl-1H-1,2,3-triazoles attached to different carbohydrate templates and their in vitro
inhibitory profile against HIV-1 reverse transcriptase. In addition a theoretical comparison of the most active compounds with other classical
antivirals was also performed. Our results showed 2a, 2d and 2g as the most active compounds that inhibited the HIV-1 reverse transcriptase
catalytic activity with cytotoxicity lower than AZT and SI higher than DDC and DDI. The overall theoretical analysis of the molecular descrip-
tors of 2a, 2d and 2g revealed that their HOMO energy is similar to other antivirals in use (AZT, DDC, DDI and 3TC) and together with the
volume may contribute for the biological profile as they may allow new interactions with the target. In fact the 1,2,3-triazole compounds
presented more lipophilicity and higher molecular volume and weight than the antivirals studied, which suggested that these features might
not only contribute for new interactions with the HIV-RT but also influence the specificity and consequently the low cytoxicity profile of these
compounds. Thus these data point them as promising leading compounds for generating new anti-HIV-RT compounds.
Ó 2008 Published by Elsevier Masson SAS.
Keywords: HIV-RT; 1,2,3-Triazole; Anti-HIV; Antiviral
1. Introduction
treatment [3,4]. This enzyme plays an essential and multifunc-
tional role in the virus replication. It allows the transcription of
HIV-single-stranded RNA genome into a DNA double helix
capable of integration into host cell chromosomes [5e9].
The combination of HIV reverse transcriptase and protease
inhibitors in the highly active antiretroviral therapies (HAART)
against HIV infection led to a decline in morbidity and mortal-
ity [5e9]. Nonetheless, viral replication is still persisting as
lymphatic system and central nervous system acts as reservoir
for the virus [5], and wherein some antivirals, more particularly
the protease inhibitors (PIs), do not penetrate at an efficient in-
hibitory level [10]. In addition the emergence of drug-resistant
Acquired immune deficiency syndrome (AIDS) is the first
pandemic disease of the molecular biology era, which killed
over 20 million people worldwide [1,2]. The human immuno-
deficiency virus (HIV) is the AIDS causative agent and its
reverse transcriptase (HIV-TR) is the main target of AIDS
* Corresponding author.
** Corresponding author. Tel.: þ55 21 26292345.
E-mail addresses: rangel@pharma.ufrj.br (C.R. Rodrigues), cegvito@vm.
uff.br (V.F. Ferreira).
0223-5234/$ - see front matter Ó 2008 Published by Elsevier Masson SAS.
doi:10.1016/j.ejmech.2008.02.047

374
F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
virus strains due to high HIV mutation rate continues to restrain
the long-term clinical efficacy of these molecules [11e14]
whereas side effects limit the use of some of these antivirals
(i.e. AZT) demanding new options [14e21]. Therefore despite
the advances in therapy, the progression of AIDS in HIV-
positive individuals remains a major health problem in the
world today.
In order to identify a 1,2,3-triazole lead compound against
HIV-RT, our research efforts have been directed towards the
discovery of new chemical entities that are effective antivirals
with low cytotoxicity. This paper describes the synthesis and
in vitro activity against HIV-RT of several 1-benzyl-1H-1,2,3-
triazoles attached to different carbohydrate templates. A theo-
retical comparison of the most active 1,2,3-triazole compounds
and some of the current antivirals in use was also performed.
1,2,3-Triazoles are an important class of heterocyclic
compounds due to their wide range of applications as pharma-
ceutical agents [22]. Among the pharmaceutical uses, 1,2,3-
triazoles are known as antiplatelets [23], dopamine D2 receptor
ligands (related to Schizophrenia) [24], b-lactamase inhibitors
[25], anticonvulsants [26], antimicrobials [27e29], and antiin-
flammatory [30,31] agents, but the potential of this class as
antivirals against HIV is poorly described in literature. Alvarez
and coworkers described the series of [2,5-bis-O-(tert-butyldi-
methylsilyl)-b-D-ribofuranosyl]-3-spiro-50-(40-amino- and 40-
(N-acetylamino)-l0,20-oxathiole 20,20-dioxide) (TSAO) deriva-
tives in which the pyrimidinelpurine moiety of the molecule
was replaced by a 1,2,3-triazole ring with various substitutions
at C-4 or C-5 [32]. These authors showed several members of
this class of compounds with potent anti-HIV-1 activities,
pointing the promising profile of 1,2,3-triazoles for generating
leading compounds [32].
2. Chemistry
The diversity of chemical structures of the 1,2,3-triazole
family and their useful biological activities made these
compounds attractive targets in synthetic organic chemistry.
Many studies have been reported on the synthesis of the
1,2,3-triazole ring system [33e42]. The potential of this hetero-
cycle led us to develop a synthetic route for preparing glycocon-
jugate-triazoles and examine their antiviral activity (Fig. 1).
The syntheses of compounds 3aeg were started by the prepara-
tion of enamines 1aeg by the procedure previously described
by us [43]. The preparation of triazoles 2aeg was performed
by the [2N þ 1N] method, as early described by Arnold and
our group [44e47], performing the transference of the diazo
group to enamines 1aeg.
N
Ph
NH
O
N
TFA/H2O
r.t., 24h
CH3SO2N3, NaH
CH3CN, 48h, r.t.
N
N
OR2
OR1
OR1
N
N
O
O
1a-g
2a-g
3a-g
O
O
O
O
OCH3
O
R1=
O
O
O
O
2a-g
O
O
O
O
O
O
O
O
O
2d
2c
2b
2a
O
O
O
O
OBn
O
O
O
O
O
O
O
O
O
2g
2f
2e
OH
OH
R2=
O
OH
OH
O
HO
3a-g
O
O
HO
OH
OH
OH
OH
OH
OH
OH OH
OH
3d
HO
3a
3b
3c
OH
O
OH
OH
O
O
OBn
OH
HO
HO
OHOH
3e
OH
3f
3g
Fig. 1. Structures of 1H-1,2,3-Triazole derivatives 2aeg and 3aeg.

F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
375
Table 1
Biological characterization of 1H-1,2,3-triazole derivatives 2aeg and 3aeg
including anti-HIV-1-RT and cytotoxicity activities
Thus, the isopropylidene group from carbohydrate moiety was
hydrolyzed through the reaction with a solution of TFA/H2O (1:1)
at room temperature during 24 h [48] producing the correspond-
ing D-glycoconjugated 1,2,3-triazoles 3aeg containing as carbo-
hydrate units ribose, galactose, fructose, alose, glucose, xylose in
their native form, in excellent yields (Fig. 1). Triazoles 3aeg were
obtained as a mixture of a and b anomers for 3c and 3f (1,2,3-tri-
azoles from D-glucose and D-alose, respectively, were obtained in
the a and b-pyranoside forms). All the triazoles were fully char-
acterized by 1H NMR, 13C NMR, IR spectroscopy and HRMS.
Compound
R
Inhibitiona (%)
CC50 (mM)
2a
63.6
837.45
OCH3
O
O
O
O
O
O
O
2b
28.8
123.42
O
3. Pharmacology
O
O
3.1. Anti-HIV-RT activity assay
O
2c
3.0
3529.00
208.37
O
The inhibitory effect of triazoles at 50 mM or different
concentrations (0.01e50 mM) was evaluated on the HIV-RT
polymerase activity using recombinant HIV-1 enzyme. The
polymerization reactions (50 mL) contained 50 mM Tris HCl
(pH 7.8), 6 mM MgCl2, 1 mM dithiotreitol, 50 mM KCl, 20
mM dTTP, 10 mM of [3H] dTTP (47 Ci/mmol), and 150 mg
poly(rA)$oligo(dT) template primer (Pharmacia) and 1 U of
enzyme. The reaction mixture was incubated at 37 ꢀC for 30
min, and the incubation was stopped by adding ice-cold 5%
trichloroacetic acid (TCA) containing 20 mM of sodium pyro-
phosphate. The precipitates were collected on Whatman GF/C
filters and washed with sodium phosphate 0.1 M. The incorpo-
rated triphosphate was measured by assaying for 3H in a liquid
scintillation counter. AZT was used as antiviral control. The
selective indexes were calculated based on our experimental
results (1,2,3-triazole compounds and AZT) and based on
literature (didadosine-DDI and zalcitabine-DDC and 3TC).
O
O
O
O
2d
64.7
O
O
O
O
O
2e
2f
O
O
43.5
47.9
2745.47
3382.47
O
O
O
O
O
O
OBn
2g
65.4
724.06
O
O
O
OH
3.2. Cytotoxicity assays
3a
3b
3c
33.2
45.6
14.0
1172.64
25,640.55
1437.50
OH OH
Vero cells were cultured in Dulbecco modified Eagle medium
(DMEM) supplemented with 5% fetal bovine serum (FBS;
HyClone, Logan, Utah), 100 U/mL penicillin and 100 mg/mL
streptomycin, at 37 ꢀC in 5% CO2. Monolayers of about 104
Vero cells in 96-multiwell plates were treated with several
concentrations of the compounds for the 72 h. Then, 50 ml of
1 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT; Sigma) was added to evaluate cell
viability according to procedures described elsewhere [49].
The 50% cytotoxic concentration (CC50) was calculated by
linear regression analysis of the doseeresponse curves. All
experiments were performed in duplicate at least three times.
OH
OH
O
OH
HO
OH
O
HO
HO
OH
OH
OH
OH
O
3d
3e
3f
35.9
45.6
35.8
1798.60
648.90
800.45
OH
OH
OH
O
OH OH
4. Results
OH
O
OH
4.1. Anti-HIV-RT activity
HO
HO
O
Initially all compounds were tested at 50 mM and the results
are shown in Table 1. All compounds inhibited HIV-RT to
some degree and 2aeg derivatives presented an inhibitory profile
higher than compounds 3aeg. Importantly, 2a, 2d and 2g pro-
duced the highest inhibitory values (63e65%) at 50 mM (Table 1).
OH
OBn
3g
36.2
596.00
126.00
OH
AZT
e
94.25
a
Determined at 50 mM.

376
F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
The determination of the IC50 of the most active compounds
guidelines for the design of more potent antivirals comparing
them with other antivirals currently in use. The minimum
energy conformation of the most active compounds and
antivirals current issue, obtained by the AM1 semiempirical
method, were submitted to a Single-Point ab initio calculation
with a 3-21G* basis set available on SPARTAN’04 program
(Wavefunction Inc. Irvine, CA, 2000). Molecular electrostatic
potential maps (MEPs), HOMO and LUMO eigen values and
orbital coefficients, and the molecular dipole moments were
calculated. In this work, we also studied the drug score of
the compounds, which are based on topological descriptors,
fingerprints of molecular druglikeness, structural keys or other
properties as clog P and molecular weight [49]. In case of
osiris property explorer (http://www.organic-chemistry.org/),
the occurrence frequency of each fragment is determined
within the collection of traded drugs and within the suppos-
edly non-druglike collection of Fluka compounds.
against HIV-RT (2a ¼ 2.2 ꢁ 0.8 mM, 2d ¼ 5.0 ꢁ 0.5 mM and
2g ¼ 1.98 ꢁ 0.4 mM) revealed values higher than AZT, but
similar to ddC (zalcitabine) and 3TC (lamivudine) and lower
than ddI (didadosine) (Fig. 2).
4.2. Cytotoxicity profile
In this study, we also determined the cytotoxicity profile
(CC50) of the 1,2,3-triazoles (Table 1). Our experimental results
of the most active compounds (2a, 2d and 2g) revealed a better
cytotoxicity profile (2a ¼ 837.45 mM, 2d ¼ 208.37 mM and
2g ¼ 724.06 mM) compared to AZT (CC50 ¼ 126 mM) (Table
1). In fact the two series 2 and 3 presented a lower cytotoxic
profile than AZT (CC50 ¼ 126 mM) except for the 2b compound
(CC50 ¼ 123.42 mM) (Table 1). Interestingly, the selective
index of the 2a and 2g was higher than DDC and DDI whereas
2d was only higher than DDI (Fig. 2).
By comparing the three more active drugs (2a, 2d and 2g)
with other antivirals (AZT, DDC, 3TC and DDI), we observed
that they presented similar HOMO and LUMO energy values.
In case of triazoles, HOMO and LUMO energy ranges from
ꢂ8.67 to ꢂ9.57 and 2.39 to 2.76, respectively, whereas the
antivirals evaluated herein presented values from ꢂ8.37 to
4.3. Molecular modeling evaluation
We employed molecular modeling studies for the most
active triazole derivatives in the attempt of providing useful
Fig. 2. Comparison of the most active 1,2,3-triazoles compounds (2a, 2d, 2g) and some anti-HIV drugs (AZT, DDC, DDI and 3TC) currently in use. Experimental
IC50 (A), selective index (B), and theoretical toxicity risk profile and drug score calculated using Osiris program (C and D, respectively).

F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
377
ꢂ9.42 and 2.29 to 2.90, respectively (Fig. 3). In contrast, the
triazole derivatives presented more lipophilicity (clog P) and
higher molecular volume (MV) and molecular weight (MW)
(Fig. 3). Despite their structural differences, the electrostatic
potential map of 2a, 2d and 2g revealed an analogous negative
charge distribution pattern in the right of the molecule, similar
to DDC and 3TC (Fig. 3).
The three 1,2,3-triazoles (2a, 2d and 2g) were submitted to
an in silico ADMET screening (http://www.organic-chemistry.
org/prog/peo/) to analyze their overall drug score potential
compared to the commercial antiviral drugs, AZT, DDC,
3TC and DDI. Therefore we determined for the most active
compounds and antivirals currently in use: (a) all parameters
for the fulfilment of Lipinski rule of 5; (b) the predicted
toxicity risks (mutagenic, irritant, tumorigenic, and reproduc-
tive effects), and (c) the drug score that combines druglike-
ness, clog P, log S, molecular weight and toxicity risks, and
theoretically may be used to evaluate the drug potential of
a compound (Fig. 3).
Our results revealed that the lipophilicity (clog P) of these
1,2,3,triazoles is higher than that observed for commercial
drugs but is not greater than 5.0, fulfilling one of the Lipinski
rule of 5 [50]. The molecular weight of 1,2,3-triazole deriva-
tives (403 < MW > 465) is greater than other drugs (211
The comparison of the most active compound (2g) with the
less active one (2c) showed a different distribution of the
density of the orbital HOMO, which is concentrated in differ-
ent moieties of their structures. In the most active compound
(2g), HOMO is concentrated at the benzyloxy substituent
from the furanose moiety, in contrast to the less active
compound (2c) where it remains on the benzyl substituent of
the triazolic moiety (Fig. 4). In addition compound 2g also
3
˚
presented a higher volume (MV ¼ 465.66 A ) than compound
3
˚
2c (MV ¼ 417.05 A ) (not shown).
Fig. 3. Comparison of theoretical parameters of 2a, 2d and 2g compounds and of some anti-HIV compounds currently in use. The most stable conformers, elec-
trostatic potential maps, fulfilment of Lipinsky rule of 5 (number of hydrogen donors and acceptors, clog P and molecular weights e MW, molecular volumes e
MV), and molecular electronic properties (EHOMO, ELUMO, dipole moment).

378
F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
Fig. 4. Comparison of the 2D-structure, the most stable conformer, the HOMO density and the HOMO density encoded onto a van der Waals surface of 1,2,3-
triazole compounds presenting the lower (2c) and the higher (2g) inhibitory profile against HIV-RT. In HOMO density encoded onto a van der Waals surface
(isodensity 0.002 e/au3), the HOMO absolute density coefficient was mapped from deepest red (0.00) to deepest blue (0.01) (for interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
< MW > 267) but close to more than 80% of all Fluka traded
drugs (MW > 450), and the number of hydrogen bond accep-
tors (HA) and donors (HD) are also within the Lipinski rule of
5 (HA < 10 and HD < 5 respectively) (Fig. 3).
derived from acetonide carbohydrates and for analogues 3a,
3d and 3g from native carbohydrates reinforced that the
protective group is an important feature for the anti-HIV activ-
ity observed.
Interestingly the 1,2,3-triazole derivatives (2a, 2d and 2g)
showed equivalent or higher drug score values than the antivi-
rals AZT, DDC, 3TC and DDI (Fig. 2). Importantly the most
active derivative (2g) presented a higher value than that
observed for DDC, 3TC and DDI (Fig. 2). In agreement to
our cytotoxicity experimental results, our theoretical study
showed 2a, 2d and 2g with a better profile for low tumorige-
nicity, mutagenicity and reproductive effects than the antivi-
rals currently in use studied herein (Fig. 2).
In a promising way, the biological activity of 2a, 2d and 2g
was analogous to DDC, DDI and 3TC but with a higher selec-
tive index, and a safer experimental cytotoxicity profile as
their CC50 increased at least two-fold compared to AZT. In
addition, as the two 1,2,3-triazole series (2 and 3) presented
both low theoretical and experimental cytotoxic results, these
data may indicate the potential safer profile of this heterocycle
class compared to other groups currently in use.
The overall theoretical analysis of the molecular descriptors
of 2a, 2d and 2g revealed that their HOMO energy is similar to
other antivirals in use (AZT, DDC, DDI and 3TC), which
suggested that the nucleophilicity might be an important fea-
ture for the biological activity. In addition, considering the
importance of stereoelectronic complementarity to the druge
receptor interaction, the comparison between the most active
(2g) and the less active (2c) compounds revealed that features
such as HOMO density (concentrated at the benzyloxy substit-
uent of 2g) and volume (related to the benzyl substituent on
the 2g furanose moiety) may together contribute for the bio-
logical profile as they may allow new interactions with the
target (i.e. hydrophobic). In fact the 1,2,3-triazole compounds
presented more lipophilicity and higher molecular volume and
5. Discussion
Two 1,2,3-triazole series were synthesized and tested
against HIV-RT, a key enzyme for HIV treatment. Interest-
ingly, all compounds inhibited HIV-RT at some degree, which
reinforced the potential profile of 1,2,3-triazoles not only for
those biological activities already described in the literature
(i.e. antiplatelets [23]) but also for an antiviral profile.
Importantly, our biological data suggested that hydroxyl
blockage in 2 is important for interacting with HIV-RT as these
compounds were the most active molecules. The different
biological profiles observed for compounds 2a, 2d and 2g

F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
379
molecular weight than the antivirals studied, which suggested
that these features might not only contribute for new interac-
tions with the HIV-RT but also influence the specificity and
consequently the low cytoxicity profile of these compounds.
In addition the evaluation of the electrostatic potential map
of 2a, 2d and 2g revealed an analogous negative electronic
distribution to DDC and 3TC that may help to orientate
them on binding to the HIV-RT.
F254 Merck plates. Microanalyses were performed on Per-
kineElmer Model 2400 instrument and all values were within
ꢁ0.4% of the calculated compositions. Chemicals employed
were obtained from commercial supplies and used without
purifications, unless otherwise stated. High-resolution elec-
tron-impact mass spectra (70 eV) were performed on Auto
Spec M513 Waters instrument.
All active compounds (2a, 2d and 2g) fulfilled the Lipinski
rule of 5 (number of hydrogen bond acceptors < 10 and donors
< 5, clog P < 5, molecular weight < 500) despite their chem-
ical structural differences from the antivirals studied herein.
The theoretical parameters such as the clog P that according
to Lipinski rule of 5 is important for drug good absorption
and permeation are kept under the best values [50].
7.2. General procedures
7.2.1. General procedure for the preparation of
3-benzylamino-but-2-enoic ester derivatives
The preparation of enamines 1aeg was achieved as de-
scribed in our previous publications [43].
All drug score values and theoretical toxicity evaluations of
the most active compounds were better than that observed for
commercial antivirals. These data reinforced the potential
profile of these compounds but it is important to notice that
the toxicity predicted herein neither is a fully reliable toxicity
prediction, nor guarantees that these compounds are com-
pletely free of any toxic effect [51e53]. However, once again
these theoretical results help to strengthen the promising
profile of these compounds already pointed by the experimen-
tal cytotoxicity assay performed herein.
7.2.1.1. Spectral data of the new enamine esters
7.2.1.1.1. 3-Benzylamino-but-2-enoic acid 3-(1,2:5,6-di-O-
isopropylidene)-a-D-allofuranose ester (1c). Compound 1c
was obtained as a white solid. Yield: 60%; m.p. 124e124.5
ꢀC; [a]D20þ160 (c 0.4, CH2Cl2); IR (KBr) n (cmꢂ1): 1648
and 1606; 1H NMR (300.00 MHz, CDCl3) d (ppm): 1.34
(3H, s, CH3), 1.35 (3H, s, CH3), 1.43 (3H, s, CH3), 1.55
(3H, s, CH3), 5.82 (1H, d, J ¼ 3.9 Hz, H-10), 4.81 (1H, dd,
J ¼ 3.7 and 4.6 Hz, H-20), 4.91 (1H, dd, J ¼ 4.9 and 8.8 Hz,
H-30), 4.20 (1H, dd, J ¼ 3.4 and 8.8 Hz, H-40), 4.35 (1H,
ddd, J ¼ 3.4, 6.6 and 6.6 Hz, H-50), 4.04 (1H, dd, J ¼ 6.8
and 8.5 Hz, H-60), 3.92 (1H, dd, J ¼ 6.3 and 8.3 Hz, H-600),
4.61 (1H, s, H-2), 2.03 (3H, s, H-4), 4.51 (2H, d, J ¼ 6.3 Hz,
NHCH2Ph), 7.29e7.39 (2H, m, H-2a), 7.29e7.39 (2H, m,
H-3a), 7.29e7.39 (1H, m, H-4a), 8.89 (1H, t, J ¼ 6.3 Hz,
NH); 13C NMR (75.0 MHz, CDCl3) d (ppm): 25.1 (CH3),
26.0 (CH3), 26.5 (CH3), 26.6 (CH3), 103.8 (C-10), 78.2
(C-20), 70.2 (C-30), 77.1 (C-40), 74.8 (C-50), 64.9 (C-60),
112.8 (C-70), 109.6 (C-80), 46.7 (NHCH2Ph), 82.2 (C-2),
162.7 (C-3), 19.3 (C-4), 138.2 (C-1a), 127.2 (C-2a), 128.6
(C-3a), 126.6 (C-4a), 168.8 (C]O). HRMS calcd for
C23H31NO7: 433.2101, found 433.2101.
6. Conclusion
To summarize, we synthesized two new classes of triazoles
(2 and 3) that present HIV-RT inhibition activity. Importantly
the triazoles with the protected carbohydrate (2) are more ef-
fective than those containing unprotected carbohydrate groups
(3). The significant activity, low cytotoxicity, and potential
theoretical profile of triazoles 2a, 2d and 2g suggest that these
may be considered as promising lead molecules for further
synthetic and biological exploration.
7. Experimental protocol
7.2.1.1.2. 3-Benzylamino-but-2-enoic acid 5-(3-O-benzyl-
1,2-O-isopropylidene)-a-D-xylofuranose ester (1g). Com-
pound 1g was obtained as a yellow oil. Yield: 67%; [a]D20
ꢂ25 (c 1.2, CH2Cl2); IR (film, CHCl3) n (cmꢂ1): 1651 and
7.1. Chemistry
1
Melting points were determined with a Buchi Model B-545
instrument and are uncorrected. Infrared (IR) spectra were
recorded on PerkineElmer 1420 spectrophotometer in KBr
pellets. NMR spectra were recorded on a Varian Unity Plus
300 spectrometer in the indicated solvent, operating at
300 MHz (1H) and 75 MHz (13C) employing tetramethylsilane
or the solvent as the internal reference, at room temperature.
Chemical shifts (d) are expressed in ppm and the coupling
constant (J ) in hertz. Purified samples were used for measur-
ing physical constants and spectral data. The optical rotations
were recorded with a PerkineElmer 243B Polarimeter
(sodium lamp at 589 nm). Analytical grade solvents were
used. Trifluoroacetic anhydride was freshly distilled before
being used. Column chromatography was performed on silica
gel flash from Acros. Reactions were routinely monitored by
thin layer chromatography (TLC) on silica gel precoated
1606; H NMR (300.00 MHz, CDCl3) d (ppm): 1.31 (3H, s,
CH3), 1.48 (3H, s, CH3), 5.94 (1H, d, J ¼ 3.7 Hz, H-10),
4.61 (1H, d, J ¼ 3.9 Hz, H-20), 3.97 (1H, d, J ¼ 2.9 Hz,
H-30), 4.39e4.42 (1H, m, H-40), 4.33 (1H, dd, J ¼ 6.3 and
11.7 Hz, H-50), 4.39e4.42 (1H, m, H-500), 4.66 (1H, d,
J ¼ 12.0 Hz, H-60), 4.52 (1H, d, J ¼ 12.0 Hz, H-600), 4.56
(1H, s, H-2), 1.90 (3H, s, H-4), 4.42 (2H, d, J ¼ 6.0 Hz,
NHCH2Ph), 7.23e7.35 (2H, m, H-2a), 7.23e7.35 (2H, m,
H-3a), 7.23e7.35 (1H, m, H-4a), 7.23e7.35 (2H, m, H-20a),
7.23e7.35 (2H, m, H-30a), 7.23e7.35 (1H, m, H-40a), 8.92
(1H, t, J ¼ 5.8 Hz, NH); 13C NMR (75.0 MHz, CDCl3)
d (ppm): 26.1 (CH3), 26.6 (CH3), 105.0 (C-10), 82.1 (C-20),
81.5 (C-30), 78.4 (C-40), 59.9 (C-50), 71.8 (C-60), 111.5
(C-70), 46.6 (NHCH2Ph), 82.7 (C-2), 162.0 (C-3), 19.2 (C-
4), 138.4 (C-1a), 126.5 (C-2a), 128.6 (C-3a), 128.3 (C-4a),
137.2 (C-10a), 127.2 (C-20a), 127.7 (C-30a), 127.5 (C-40a),

380
F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
169.7 (C]O). HRMS calcd for C26H31NO6: 453.2151, found
453.2151.
136.7 (C-4), 138.2 (C-5), 9.1 (C5eCH3), 133.8 (C-1a),
127.1 (C-2a), 129.0 (C-3a), 128.5 (C-4a), 161.1 (C]O).
HRMS calcd for C23H29N3O7: 444.1771, found 444.1633.
7.2.2.1.3. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 3-(1,2:5,6-di-O-isopropylidene)-a-D-allofuranose
ester (2c). Compound 2c was obtained as a yellow solid.
Yield: 50%; m.p. 97e98 ꢀC; [a]D20 þ83 (c 1.5, CHCl3); IR
(KBr) n (cmꢂ1): 1733, 1714, 1380 and 1372; 1H NMR
(300.00 MHz, CDCl3) d (ppm): 1.32 (3H, s, CH3), 1.32 (3H,
s, CH3), 1.38 (3H, s, CH3), 1.54 (3H, s, CH3), 5.88 (1H, d,
J ¼ 3.9 Hz, H-10), 4.93 (1H, dd, J ¼ 3.9 and 5.1 Hz, H-20),
5.11 (1H, dd, J ¼ 5.1 and 8.3 Hz, H-30), 4.36e4.43 (1H, m,
H-40), 4.36e4.43 (1H, m, H-50), 4.03 (1H0 dd, J ¼ 6.1 and
8.8 Hz, H-60), 4.10 (1H, dd, J ¼ 6.6 and 8.8 Hz, H-600), 2.46
(3H, s, C5eCH3), 5.50 (1H, d, J ¼ 15.6 Hz, NCH2Ph), 5.56
(1H, d, J ¼ 15.6 Hz, NCH2Ph), 7.17e7.21 (2H, m, H-2a),
7.32e7.39 (2H, m, H-3a), 7.32e7.39 (1H, m, H-4a); 13C
NMR (75.0 MHz, CDCl3) d (ppm): 25.1 (CH3), 26.4 (CH3),
26.9 (CH3), 26.9 (CH3), 104.4 (C-10), 78.0 (C-20), 72.8
(C-30), 77.8 (C-40), 75.0 (C-50), 65.7 (C-60), 113.5 (C-70),
110.2 (C-80), 52.3 (NCH2Ph), 136.4 (C-4), 139.1 (C-5), 9.4
(C5eCH3), 134.0 (C-1a), 127.6 (C-2a), 129.4 (C-3a), 128.9
(C-4a), 160.8 (C]O). HRMS calcd for C23H29N3O7:
444.1771, found 444.1765.
7.2.2.1.4. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 3-(1,2:5,6-di-O-isopropylidene)-a-D-glucofuranose
(2d). Compound 2d was obtained as a yellow oil. Yield:
50%; [a]2D0 ꢂ30 (c 1.6, CHCl3); IR (film, CHCl3) n (cmꢂ1):
1726, 1383 and 1374; 1H NMR (300.00 MHz, CDCl3)
d (ppm): 1.27 (3H, s, CH3), 1.31 (3H, s, CH3), 1.41 (3H, s,
CH3), 1.54 (3H, s, CH3), 5.97 (1H, d, J ¼ 3.7 Hz, H-10),
4.64 (1H, d, J ¼ 3.7 Hz, H-20), 5.51 (1H, d, J ¼ 2.9 Hz,
H-30), 4.33 (1H, dd, J ¼ 2.9 and 7.8 Hz, H-40), 4.41 (1H,
ddd, J ¼ 5.1, 5.6 and 7.8 Hz, H-50), 4.09 (1H, dd, J ¼ 5.1
and 8.8 Hz, H-60), 4.14 (1H, dd, J ¼ 5.6 and 8.8 Hz, H-600),
2.46 (3H, s, C5eCH3), 5.54 (2H, s, NCH2Ph), 7.18e7.20
(2H, m, H-2a), 7.32e7.39 (2H, m, H-3a), 7.32e7.39 (1H,
m, H-4a); 13C NMR (75.0 MHz, CDCl3) d (ppm): 25.1
(CH3), 26.1 (CH3), 26.6 (CH3), 26.7 (CH3), 105.0 (C-10),
83.3 (C-20), 76.4 (C-30), 79.7 (C-40), 72.4 (C-50), 67.0 (C-60),
112.1 (C-70), 109.1 (C-80), 51.9 (NCH2Ph), 136.1 (C-4),
138.8 (C-5), 8.9 (C5eCH3), 133.6 (C-1a), 127.1 (C-2a),
129.0 (C-3a), 128.5 (C-4a), 160.3 (C]O). HRMS calcd for
C23H29N3O7: 444.1771, found 444.1785.
7.2.2. General procedure for the preparation of
1-benzyl-1H-1,2,3-triazole derivatives 2aeg
To a stirred mixture of sodium hydride (6.67 mmol, free of
oil) in anhydrous acetonitrile (4 mL), under nitrogen at room
temperature, was added a solution of the b-enamino ester 1
(1.85 mmol in 4 mL of anhydrous acetonitrile). The stirring
was continued for 30 min, followed by dropwise addition of
mesyl azide (5 mmol in 1 mL of anhydrous acetonitrile).
Additional stirring was kept for 48 h and the reaction was
quenched with water. The resulting solution was extracted
with methylene chloride (3 ꢃ 30 mL) and the organic layer
was washed (2 ꢃ 20 mL) with aqueous sodium hydroxide
solution (10%, w/v) and water (1 ꢃ 10 mL). After drying
over anhydrous sodium sulfate, the solvent was removed under
reduced pressure to leave the 1,2,3-triazoles (2aeg) as yellow
oils or solids.
7.2.2.1. Spectral data of the 1-benzyl-1H-1,2,3-triazole
derivatives (2aeg)
7.2.2.1.1. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 5-(1-methoxy-2,3-O-isopropylidene)-b-D-ribofura-
nose ester (2a). Compound 2a was obtained as a yellow oil.
Yield: 92%; [a]2D0 ꢂ33 (c 1.5, CHCl3); IR (film, CHCl3) n
(cmꢂ1): 1652 and 1605; 1H NMR (300.00 MHz, CDCl3)
d (ppm): 1.32 (3H, s, CH3), 1.48 (3H, s, CH3), 4.99 (1H, s,
H-10), 4.64 (1H, d, J ¼ 5.8 Hz, H-20), 4.80 (1H, dd, J ¼ 0.6
and 5.8 Hz, H-30), 4.51 (1H, dt, J ¼ 0.6 and 6.9 Hz, H-40),
4.39 (2H, dd, J ¼ 2.0 and 6.9 Hz, H-50), 3.33 (3H, s, OCH3),
2.46 (3H, s, C5eCH3), 5.54 (2H, s, NCH2Ph), 7.16e7.39
(2H, m, H-2a), 7.16e7.39 (2H, m, H-3a), 7.16e7.39 (1H,
m, H-4a); 13C NMR (75.0 MHz, CDCl3) d (ppm): 24.7
(CH3), 26.2 (CH3), 109.2 (C-10), 84.9 (C-20), 81.6 (C-30),
83.8 (C-40), 64.6 (C-50), 112.3 (C-60), 54.8 (OCH3), 51.8
(NCH2Ph), 136.3 (C-4), 138.3 (C-5), 8.9 (C5eCH3), 133.6
(C-1a), 127.0 (C-2a), 128.9 (C-3a), 128.4 (C-4a), 160.9
(C]O). HRMS calcd for C20H25N3O6: 430.1743, found
403.1705.
7.2.2.1.2. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 6-(1,2:3,4-di-O-isopropylidene)-a-D-galactopyranose
ester (2b). Compound 2b was obtained as a yellow oil. Yield:
50%; [a]2D0 - 40 (c 1.5, CHCl3); IR (film, CHCl3) n (cmꢂ1):
1732, 1382 and 1373; 1H NMR (300.00 MHz, CDCl3)
d (ppm): 1.32 (3H, s, CH3), 1.34 (3H, s, CH3), 1.46 (3H, s,
CH3), 1.49 (3H, s, CH3), 5.54 (1H, d, J ¼ 4.5 Hz, H-10),
4.33 (1H, dd, J ¼ 2.4 and 4.6 Hz, H-20), 4.63 (1H, dd,
J ¼ 2.4 and 7.8 Hz, H-30), 4.34 (1H, dd, J ¼ 1.9 and 7.8 Hz,
H-40), 4.18 (1H, dt, J ¼ 1.7 and 6.3 Hz, H-50), 4.49 (1H, d,
J ¼ 6.3 Hz, H-60), 2.45 (3H, s, C5eCH3), 5.52 (1H, d,
J ¼ 15.6 Hz, NCH2Ph), 5.58 (1H, d, J ¼ 15.6 Hz, NCH2Ph),
7.15e7.18 (2H, m, H-2a), 7.32e7.39 (2H, m, H-3a),
7.32e7.39 (1H, m, H-4a); 13C NMR (75.0 MHz, CDCl3)
d (ppm): 24.3 (CH3), 24.8 (CH3), 25.8 (CH3), 25.9 (CH3),
96.1 (C-10), 70.4 (C-20), 70.5 (C-30), 70.8 (C-40), 65.8 (C-50),
63.5 (C-60), 109.5 (C-70), 108.6 (C-80), 51.9 (NCH2Ph),
7.2.2.1.5. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 3-(1,2:4,5-di-O-isopropylidene)-b-D-fructopyranose
ester (2e). Compound 2e was obtained as a yellow solid.
Yield: 80%; m.p. 164e165 ꢀC; [a]D20 ꢂ105 (c 1.6, CHCl3);
IR (KBr) n (cmꢂ1): 1729, 1373 and 1385; 1H NMR
(300.00 MHz, CDCl3) d (ppm): 1.36 (3H, s, CH3), 1.43
(3H, s, CH3), 1.50 (3H, s, CH3), 1.59 (3H, s, CH3), 3.93
(1H, d, J ¼ 9.3 Hz, H-10), 4.01 (1H, dd, J ¼ 9.3 Hz, H-100),
5.40 (1H, d, J ¼ 8.1 Hz, H-30), 4.48 (1H, dd, J ¼ 5.4 and
8.1 Hz, H-40), 4.28 (1H, dd, J ¼ 1.8 and 5.4 Hz, H-50),
4.12 (1H, d, J ¼ 13.7 Hz, H-60), 4.20 (1H, dd, J ¼ 2.4 and
13.7 Hz, H-600), 2.46 (3H, s, C5eCH3), 5.53 (2H, s,
NCH2Ph), 7.13e7.21 (2H, m, H-2a), 7.32e7.40 (2H, m,

F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
381
H-3a), 7.32e7.40 (1H, m, H-4a); 13C NMR (75.0 MHz,
CDCl3) d (ppm): 25.9 (CH3), 26.2 (CH3), 26.3 (CH3), 27.6
(CH3), 71.5 (C-10), 103.5 (C-20), 70.3 (C-30), 74.7 (C-40),
73.6 (C-50), 60.2 (C-60), 112.1 (C-70), 109.5 (C-80), 51.8
(NCH2Ph), 136.0 (C-4), 138.4 (C-5), 9.1 (C5eCH3), 133.6
(C-1a), 127.0 (C-2a), 128.9 (C-3a), 128.4 (C-4a), 160.9
(C]O). HRMS calcd for C23H29N3O7: 444.1771, found
444.1816.
furnishing the 1,2,3-triazoles (3aeg) as brown oils, in quanti-
tative yield.
7.2.3.1. Spectral data of the 1-benzyl-1H-1,2,3-triazole
derivatives (3aeg)
7.2.3.1.1. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 5-b-D-ribofuranose ester (3a). 1H NMR (300.00
MHz, DMSO-d6) d (ppm): 5.10 (1H, s, H-10), 4.35 (1H, d,
J ¼ 11.7 Hz, H-20) and 4.33 (1H, d, J ¼ 11.7 Hz, H-20), 4.60
(1H, dd, J ¼ 3.2 and 11.7 Hz, H-30), 4.08 (1H, dd, J ¼ 3.2
and 6.6 Hz, H-40), 3.80 (1H, d, J ¼ 4.4 Hz, H-50), 4.14 (1H,
d, J ¼ 4.4 and 6.6 Hz, H-500), 2.60 (3H, s, C5-CH3), 5.77
(2H, s, NCH2Ph), 7.31e7.51 (2H, m, H-2a), 7.31e7.51 (2H,
m, H-3a), 7.31e7.51 (1H, m, H-4a); 13C NMR (75.0 MHz,
DMSO-d6) d (ppm): 102.0 (C-10), 79.1 (C-20), 71.2 (C-30),
75.4 (C-40), 66.2 (C-50), 51.0 (NCH2Ph), 136.0 (C-4), 138.8
(C-5), 9.1 (C5eCH3), 135.2 (C-1a), 127.5 (C-2a), 129.1
(C-3a), 128.3 (C-4a), 161.1 (C]O).
7.2.3.1.2. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 6-a-D-galactopyranose ester (3b). 1H NMR (300.00
MHz, DMSO-d6) d (ppm): 5.06 (1H, d, J ¼ 3.5 Hz, H-10)
and 5.05 (1H, d, J ¼ 3.0 Hz, H-10), 3.68 (1H, dd, J ¼ 3.5 and
9.5 Hz, H-20), 3.79e3.87 (1H, m, H-30), 3.93 (1H, dd,
J ¼ 2.8 and 7.0 Hz, H-40) and 3.98 (1H, dd, J ¼ 5.2 and
7.2 Hz, H-40), 4.33e4.38 (1H, m, H-50), 4.23e4.27 (1H, m,
H-60), 4.27e4.45 (1H, m, H-600), 2.57 (3H, s, C5eCH3) and
2.58 (3H, s, C5eCH3), 5.73 (2H, s, NCH2Ph), 7.30e7.55
(2H, m, H-2a), 7.30e7.55 (2H, m, H-3a), 7.30e7.55 (1H,
m, H-4a); 13C NMR (75.0 MHz, DMSO-d6) d (ppm): 97.5
and 92.8 (C-10), 69.4 and 71.9 (C-20), 73.1 and 76.0 (C-30),
67.8 and 68.8 (C-40), 81.5 and 82.7 (C-50), 64.4 and 64.3
(C-60), 50.9 (NCH2Ph), 135.9 (C-4), 138.8 and 138.7 (C-5),
8.87 and 8.92 (C5eCH3), 135.1 (C-1a), 127.4 (C-2a), 128.9
(C-3a), 128.2 (C-4a), 161.0 (C]O).
7.2.3.1.3. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 3-b-D-allopyranose ester (3c). 1H NMR (300.00
MHz, DMSO-d6) d (ppm): 5.69 (1H, d, J ¼ 3.2 Hz, H-10),
3.43 (1H, dd, J ¼ 3.2 and 8.0 Hz, H-20), 4.80 (1H, d, J ¼
8.0 Hz, H-30), 3.68e3.76 (1H, m, H-40), 3.49e3.58 (1H, m,
H-50), 3.49e3.58 (1H, m, H-60), 3.75 (1H, dd, J ¼ 6.8 and
11.0 Hz, H-600), 2.58 (3H, s, C5eCH3), 5.73 (1H, s, NCH2Ph),
7.29e7.54 (2H, m, H-2a), 7.29e7.54 (2H, m, H-3a),
7.29e7.54 (1H, m, H-4a); 13C NMR (75.0 MHz, DMSO-d6)
d (ppm): 94.1 (C-10), 74.6 (C-20), 75.3 (C-30), 65.9 (C-40),
70.1 (C-50), 61.2 (C-60), 50.9 (NCH2Ph), 136.8 (C-4), 137.8
(C-5), 9.4 (C5eCH3), 135.2 (C-1a), 127.5 (C-2a), 129.0
(C-3a), 128.2 (C-4a), 160.6 (C]O).
7.2.3.1.4. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 3-a-(or b)-D-glucofuranose ester (3d). 1H NMR
(300.00 MHz, DMSO-d6) d (ppm): 5.11 (1H, d, J ¼ 9.3 Hz,
H-10a), 4.56 (1H, d, J ¼ 7.6 Hz, H-10b), 3.27 (1H, m, H-
20a), 3.30 (1H, dd, J ¼ 7.6 and 9.5, H-20b), 3.55e3.66 (1H,
m, H-30a), 3.50 (1H, dd, J ¼ 5.8 and 9.5 Hz, H-30b), 3.34e
3.40 (1H, m, H-40a or H-40b), 3.55e3.66 (1H, m, H-50a or
H-50b), 3.55e3.66 (2H, m, H-60a or H-60b), 2.57 (3H, s,
C5eCH3), 5.73 (2H, s, NCH2Ph), 7.31e7.53 (2H, m, H-2a),
7.31e7.53 (2H, m, H-3a), 7.31e7.53 (1H, m, H-4a); 13C
7.2.2.1.6. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 1-(2,3:4,5-di-O-isopropylidene)-b-fructopyranose
ester (2f). Compound 2f was obtained as a yellow oil. Yield:
70%; [a]2D0 ꢂ14 (c 1.6, CHCl3); IR (film, CHCl3) n (cmꢂ1):
1723 and 1382; 1H NMR (300.00 MHz, CDCl3) d (ppm):
1.34 (3H, s, CH3), 1.46 (3H, s, CH3), 1.51 (3H, s, CH3),
1.54 (3H, s, CH3), 4.26 (1H, d, J ¼ 11.5 Hz, H-10), 4.69 (1H,
dd, J ¼ 11.5 Hz, H-100), 4.59 (1H, d, J ¼ 2.7 Hz, H-30), 4.65
(1H, dd, J ¼ 2.7 and 7.8 Hz, H-40), 4.24e4.28 (1H, m, H-50),
3.78 (1H, dd, J ¼ 0.5 and 12.9 Hz, H-60), 3.96 (1H, dd,
J ¼ 1.7 and 12.9 Hz, H-600), 2.47 (3H, s, C5eCH3), 5.51
(1H, d, J ¼ 15.4 Hz, NCH2Ph), 5.57 (1H, d, J ¼ 15.4 Hz,
NCH2Ph), 7.14e7.17 (2H, m, H-2a), 7.32e7.39 (2H, m,
H-3a), 7.32e7.39 (1H, m, H-4a); 13C NMR (75.0 MHz,
CDCl3) d (ppm): 23.9 (CH3), 25.2 (CH3), 25.7 (CH3), 26.4
(CH3), 64.5 (C-10), 101.4 (C-20), 70.1 (C-30), 69.9 (C-40),
70.7 (C-50), 61.1 (C-60), 109.0 (C-70), 108.9 (C-80), 51.7
(NCH2Ph), 136.3 (C-4), 138.7 (C-5), 8.8 (C5eCH3), 133.7
(C-1a), 127.0 (C-2a), 128.9 (C-3a), 128.4 (C-4a), 160.9
(C]O). HRMS calcd for C23H29N3O7: 444.1771, found
444.1752.
7.2.2.1.7. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 5-(3-O-benzyl-1,2-O-isopropylidene)-a-D-xylofura-
nose ester (2g). Compound 2g was obtained as a yellow oil.
Yield: 70%; [a]2D0 ꢂ25 (c 1.5, CHCl3); IR (film, CHCl3) n
(cmꢂ1): 1721, 1374 and 1360; 1H NMR (300.00 MHz,
CDCl3) d (ppm): 1.33 (3H, s, CH3), 1.49 (3H, s, CH3), 5.98
(1H, d, J ¼ 3.7 Hz, H-10), 4.65 (1H, d, J ¼ 3.7 Hz, H-20),
4.08 (1H, d, J ¼ 2.7 Hz, H-30), 4.58e4.63 (1H, m, H-40),
4.58e4.63 (2H, m, H-50), 4.54 (1H, d, J ¼ 12.0 Hz, H-60),
4.72 (1H, d, J ¼ 12.0 Hz, H-600), 2.42 (3H, s, C5eCH3), 5.54
(2H, s, NCH2Ph), 7.17e7.39 (2H, m, H-2a), 7.17e7.39 (2H,
m, H-3a), 7.17e7.39 (1H, m, H-4a), 7.17e7.39 (2H, m, H-
20a), 7.17e7.39 (2H, m, H-30a), 7.17e7.39 (1H, m, H-40a);
13C NMR (75.0 MHz, CDCl3) d (ppm): 26.1 (CH3), 26.6
(CH3), 105.0 (C-1), 82.0 (C-2), 81.4 (C-3), 77.8 (C-4), 62.1
(C-5), 71.7 (C-6), 111.7 (C-7), 51.8 (NCH2Ph), 137.0 (C-4),
138.3 (C-5), 8.9 (C5eCH3), 133.7 (C-1a), 128.3 (C-2a),
128.9 (C-3a), 128.4 (C-4a), 136.4 (C-10a), 127.0 (C-20a),
127.5 (C-30a), 127.7 (C-40a), 161.0 (C]O). HRMS calcd for
C26H29N3O6: 479.2056, found 479.2104.
7.2.3. General procedure for the preparation of 1-benzyl-
1H-1,2,3-triazole derivatives 3aeg
To a bottom balloon containing 1 mmol of 1-benzyl-
1H-1,2,3-triazole derivatives 2, 10 mL of 50% trifluoroacetic
acid in water was added. The stirring was continued for
48 h. Then the solvent was removed under reduced pressure

382
F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
NMR (75.0 MHz, DMSO-d6) d (ppm): 92.3 (C-10a), 96.8 (C-
10b), 70.4 (C-20a), 76.2 (C-20b), 72.8 (C-30a), 73.5 (C-30b),
60.1 (C-40a and b), 71.9 (C-50a), 78.5 (C-50b), 60.8 (C-60a
and b), 50.8 (NCH2Ph), 136.4 (C-4a), 136.6 (C-4b), 138.2
(C-5a), 138.3 (C-5b), 9.0 (C5eCH3), 135.2 (C-1a a and b),
127.4 (C-2a a and b), 128.9 (C-3a a and b), 128.1 (C-4a
a and b), 160.6 (C]O a), 160.8 (C]O b).
plasmid pUC12N. This recombinant enzyme was composed of
66 kDa protein. The bacteria containing this plasmid were
grown for 12e16 h with shaking at 37 ꢀC and were collected
by centrifugation for 10 min, at 10,000 rpm. The pellet was
washed once with cold 100 mM NaCl, 20% (vol/vol) glycerol,
1% triton X-100, 1 mM EDTA, 2 mM dithiotreitol, 25 mM
Tris chloride, pH 8.0. The lysates were kept at 4 ꢀC for
30 min, and the insoluble material was removed by centrifuga-
tion at 10,000 rpm. The supernatant was collected and passed
over Sephadex G-25 columns at 4 ꢀC pre-equilibrated with
0.2 M NaCl, 2 mM dithiotreitol, 0.2% Triton X-100, 20%
glycerol, 25 mM Tris chloride, pH 7.4. After loading, the col-
umns were washed with the same pre-equilibration buffer, and
the fractions were assayed both for reverse transcriptase activ-
ity and for protein concentration. The polymerization reactions
(50 mL) contained 50 mM Tris HCl (pH 7.8), 6 mM MgCl2,
1 mM dithiotreitol, 50 mM KCl, 20 mM dTTP, 10 mM of
[3H] dTTP (47 Ci/mmol), and 150 mg poly(rA)$oligo(dT)
template primer (Pharmacia) and 1 U of enzyme. The reaction
mixture was incubated at 37 ꢀC for 30 min, and the incubation
was stopped by adding ice-cold 5% trichloroacetic acid (TCA)
containing 20 mM of sodium pyrophosphate. The precipitates
were collected on Whatman GF/C filters and washed with
sodium phosphate 0.1 M. The incorporated triphosphate was
7.2.3.1.5. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 3-b-D-fructopyranose ester (3e). 1H NMR (300.00
MHz, DMSO-d6) d (ppm): 3.41e3.59 (2H, m, H-10), 5.43
(1H, d, J ¼ 10.0 Hz, H-30), 4.01 (1H, dd, J ¼ 3.5 and 10.0
Hz, H-40), 3.88e3.89 (1H, m, H-50), 4.00 (1H, d, J ¼
12.0 Hz, H-60), 3.64 (1H, dd, J ¼ 1.2 and 12.0 Hz, H-600),
2.60 and 2.61 (3H, s, C5eCH3), 5.77 and 5.78 (2H, s,
NCH2Ph), 7.33e7.53 (2H, m, H-2a), 7.33e7.53 (2H, m,
H-3a), 7.33e7.53 (1H, m, H-4a); 13C NMR (75.0 MHz,
DMSO-d6) d (ppm): 64.7 (C-10), 97.4 and 101.6 (C-20), 68.4
(C-30), 70.9 (C-40), 67.8 (C-50), 63.6 (C-60), 51.2 (NCH2Ph),
136.7 (C-4), 138.7 (C-5), 9.3 (C5eCH3), 135.5 (C-1a),
127.8 (C-2a), 129.3 (C-3a), 128.5 (C-4a), 161.0 (C]O).
7.2.3.1.6. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 1-b-D-fructopyranose ester (3f). 1H NMR (300.00
MHz, DMSO-d6) d (ppm): 4.22 (1H, d, J ¼ 11.5 Hz, H-10),
4.34 (1H, dd, J ¼ 11.5 Hz, H-100), 4.28e4.30 (1H, m, H-30),
4.28e4.30 (1H, m, H-40), 3.91e3.99 (1H, m, H-50),
3.91e3.99 (1H, m, H-60), 3.74e3.84 (1H, m, H-600), 2.59
and 2.60 (3H, s, C5eCH3), 5.77 and 5.78 (1H, s, NCH2Ph),
7.32e7.53 (2H, m, H-2a), 7.32e7.53 (2H, m, H-3a),
7.32e7.53 (1H, m, H-4a); 13C NMR (75.0 MHz, DMSO-d6)
d (ppm): 65.2 (C-10), 97.0 and 100.2 (C-20), 69.1 (C-30),
67.8 (C-40), 69.6 (C-50), 63.5 (C-60), 50.9 (NCH2Ph), 136.2
(C-4), 138.7 (C-5), 9.1 and 9.2 (C5eCH3), 135.2 (C-1a),
127.4 (C-2a), 129.0 (C-3a), 128.2 (C-4a), (C]O).
7.2.3.1.7. 1-Benzyl-5-methyl-1H-[1,2,3]triazole-4-carbox-
ylic acid 5-a-D-xylofuranose ester (3g). 1H NMR (300.00
MHz, DMSO-d6) d (ppm): 4.82 (1H, d, J ¼ 4.4 Hz, H-10),
4.10e4.31 (1H, m, H-20), 4.51e4.59 (1H, m, H-30),
4.59e4.70 (1H, m, H-40), 4.77 (1H, dd, J ¼ 3.4 and 12.4 Hz,
H-50), 4.82 (1H, d, J ¼ 12.4 Hz, H-500), 4.61 (1H, d, J ¼
12.0 Hz, H-60), 4.68 (1H, d, J ¼ 12.0 Hz, H-600), 2.55 and
2.56 (3H, s, C5eCH3), 5.77 (2H, s, NCH2Ph), 7.32e7.57
(2H, m, H-2a), 7.32e7.57 (2H, m, H-3a), 7.32e7.57 (1H,
m, H-4a), 7.32e7.57 (2H, m, H-20a), 7.32e7.57 (2H, m,
H-30a), 7.32e7.57 (1H, m, H-40a); 13C NMR (75.0 MHz,
DMSO-d6) d (ppm): 102.1 (C-10), 83.8 (C-20), 82.4 (C-30),
78.0 (C-40), 63.0 (C-50), 71.4 (C-60), 50.9 (NCH2Ph), 138.3
(C-4), 138.7 (C-5), 8.8 (C5eCH3), 135.1 (C-1a), 127.4
(C-2a), 128.9 (C-3a), 128.2 (C-4a), 135.1 (C-10a), 127.4
(C-20a), 128.9 (C-30a), 128.2 (C-40a), 160.9 (C]O).
3
measured by assaying for H in a liquid scintillation counter.
One unit of enzyme is defined as the amount of enzyme that
incorporates 1 pmol of dTTP in 30 min at 37 ꢀC under stan-
dard assay conditions.
7.2.5. Cytotoxicity assay
Vero cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 5% fetal bovine serum
(FBS; HyClone, Logan, Utah), 100 U/mL penicillin and 100
mg/mL streptomycin, at 37 ꢀC in 5% CO2. Monolayers of
about 104 Vero cells in 96-multiwell plates were treated
with several concentrations of the compounds for the 72 h.
Then, 50 mL of a 1 mg/mL solution of 3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma)
was added to evaluate cell viability according to procedures
described elsewhere [49]. The 50% cytotoxic concentration
(CC50) was calculated by linear regression analysis of the
doseeresponse curves. All experiments were performed in
duplicate at least three times.
7.3. Molecular modeling and SAR studies
7.3.1. Molecular modeling methods
We employed molecular modeling studies for triazole
derivatives in the attempt to elucidate a structureeactivity
relationship (SAR) to provide useful guidelines for the design
of more potent antivirals. The minimum energy conformation
of the most active and the last active compounds, obtained by
the AM1 semiempirical method, were submitted to a single-
point ab initio calculation with a 3-21G* basis set available
on SPARTAN’04 program (Wavefunction Inc. Irvine, CA,
2000). Molecular electrostatic potential maps (MEPs), HOMO
and LUMO eigen values and orbital coefficients, and the
All triazoles synthesized were then tested in the biological
assays without any other further analytical chemistry analysis.
7.2.4. Enzyme activity assays
The inhibitory effect of triazoles at a concentration of
50 mM was evaluated on the RT polymerase activity using
recombinant HIV-1 enzyme. The sequence that expressed
RT HIV-1 was introduced into an Escherichia coli expression

F.deC. da Silva et al. / European Journal of Medicinal Chemistry 44 (2009) 373e383
383
[21] E. De Clercq, J. Med. Chem. 48 (2005) 1297e1313.
[22] K. Kacprzak, Synlett (2005) 943e946.
molecular dipole moments were calculated. In this work, we
also studied the druglikeness and the drug score of the com-
pounds, which is based on topological descriptors, fingerprints
of molecular druglikeness, structural keys or other properties
as clog P and molecular weights [49]. In case of Osiris Property
Explorer (http://www.organic-chemistry.org/), the occurrence
frequency of each fragment is determined within the collection
of traded drugs and within the supposedly non-druglike collec-
tion of Fluka compounds.
[23] A.C. Cunha, J.M. Figueiredo, J.L.M. Tributino, A.L.P. Miranda,
H.C. Castro, R.B. Zingali, C.A.M. Fraga, M.C.B.V. de Souza,
V.F. Ferreira, E.J. Barreiro, Bioorg. Med. Chem. 11 (2003) 2051e2059.
[24] R. Menegatti, A.C. Cunha, V.F. Ferreira, E.F.R. Perreira, A. El-Nabawi,
A.T. Eldefrawi, E.X. Albuquerque, G. Neves, S.M.K. Rates,
C.A.M. Fraga, E.J. Barreiro, Bioorg. Med. Chem. 11 (2003) 4807e4813.
[25] R.G. Micetich, S.N. Maiti, P. Spevak, T.W. Hall, S. Yamabe, N. Ishida,
M. Tanaka, T. Yamazaki, A. Nakai, K. Ogawa, J. Med. Chem. 30
(1987) 1469e1474.
[26] J.L. Kelley, C.S. Koble, R.G. Davis, E.W. McLean, F.E. Soroko,
B.R. Cooper, J. Med. Chem. 38 (1995) 4131e4134.
Acknowledgements
[27] B.S. Holla, M. Mahalinga, M.S. Karthikeyan, B. Poojary, P.M. Akberali,
N.S. Kumari, Eur. J. Med. Chem. 40 (2005) 1173e1178.
[28] K. Dabak, O. Sezer, A. Akar, O. Anac, Eur. J. Med. Chem. 38 (2003)
215e218.
Fellowships granted to V.F.F., M.C.B.V.S, I.I.P.F., H.C.C.
and C.R.R. by CNPq (Brazil) is gratefully acknowledged.
The authors’ thanks to Dr. M.A. Dal Tasso and Professor F.
Radler for the HRMS spectrum. This work was partially sup-
ported by CNPq, FAPERJ, Farmanguinhos/Fiocruz and UFF.
[29] D. Willner, A.M. Jelenevsky, L.C. Cheney, J. Med. Chem. 15 (1972)
948e951.
[30] G. Biagi, G. Dell’Omodarme, M. Ferretti, I. Giorgi, O. Livi, V. Scartoni,
Farmaco 45 (1990) 1181e1192.
[31] G. Biagi, O. Livi, V. Scartoni, A. Lucacchini, M.R. Mazzoni, Farmaco 41
(1986) 597e610.
References
´
´
[32] R. Alvarez, S. Velazquez, A. San-Felix, S. Aquaro, E. De Clercq,
C.F. Perno, A. Karlsson, J. Balzarini, M.J. Camarasa, J. Med. Chem.
37 (1994) 4185e4194.
[1] A. Bartlett, R. Demasi, J. Quinn, C. Moxham, F. Rousseau, AIDS 15
(2001) 1369e1377.
[2] UNAIDS 2005 report, <http://www.unaids.org> (accessed, 14.11.07).
[3] F. Barre-Sinoussi, J.C. Chermann, M.T. Nugeyre, S. Chamaret, J. Gruest,
C. Dauget, C. Axler-Bin, F. Vezinet-Brun, C. Rouzioux, W. Rozenbaum,
L. Montagnier, Science 220 (1983) 868e871.
[33] W.G. Lewis, L.G. Green, F. Grynszpan, Z. Radic, P.R. Carlier, P. Taylor,
M.G. Finn, K.B. Sharpless, Angew. Chem., Int. Ed. 41 (2002) 1053e1057.
[34] Q. Wang, T.R. Chan, R. Hilgraf, V.V. Fokin, K.B. Sharpless, M.G. Finn,
J. Am. Chem. Soc. 125 (2003) 3192e3193.
[35] L.V. Lee, M.L. Mitchell, S.J. Huang, V.V. Fokin, K.B. Sharpless,
C.H. Wong, J. Am. Chem. Soc. 125 (2003) 9588e9589.
[36] A.J. Link, D.A. Tirrell, J. Am. Chem. Soc. 125 (2003) 11164e11165.
[37] A.E. Speers, G.C. Adam, B.F. Cravatt, J. Am. Chem. Soc. 125 (2003)
4686e4687.
[4] R.C. Gallo, S.Z. Salahuddin, M. Popovic, G.M. Shearer, M. Kalpana,
B.F. Haynes, T.J. Palker, R. Redfield, J. Oleske, B. Safai, G. White,
P. Foster, P.D. Markham, Science 224 (1984) 500e503.
[5] H.C. Castro, N.I.V. Loureiro, M. Pujol-Luz, A.M.T. Souza,
M.G. Albuquerque, D.O. Santos, L.M. Cabral, I.C. Frugulhetti,
C.R. Rodrigues, Curr. Med. Chem. 13 (2006) 313e324.
[38] A.E. Speers, B.F. Cravatt, Chem. Biol. 11 (2004) 535e546.
[39] T.S. Seo, Z. Li, H. Ruparel, J. Ju, J. Org. Chem. 68 (2003) 609e612.
[40] S. Lober, P. Rodriguez-Loaiza, P. Gmeiner, Org. Lett. 5 (2003) 1753e1755.
[41] C.W. Tornoe, S.J. Sanderson, J.C. Mottram, G.H. Coombs, M. Meldal, J.
Comb. Chem. 6 (2004) 312e324.
[6] A.S. Mehanna, in: A. Burger, D.J. Abraham, J. Andrako, J.H. Block,
R.T. Borchardt, A. Abraham (Eds.), Rationale of Design of Anti-HIV
Drugs, sixth ed., Burger’s Medicinal Chemistry and Drug Discovery,
vol. 5 John Wiley and Sons, Inc., New Jersey, 2003, pp. 4574e4584.
[7] O.S. Pedersen, E.B. Pedersen, Antivir. Chem. Chemother. 10 (1999)
285e314.
[42] H. Gallardo, F. Ely, A.J. Bortoluzzi, G. Conte, Liq. Cryst. 32 (2005)
667e671.
[43] F.C. Silva, M.C.B.V. de Souza, V.F. Ferreira, S.J. Sabino,
O.A.C. Antunes, Catal. Commun. 5 (2004) 151e155.
[8] E.P. Pec¸anha, O.A.C. Antunes, Quim. Nova 25 (2002) 1108e1116.
[9] M. Shimada, H. Hosaka, H. Takaku, J.S. Smith, M.J. Roth, S. Inouye,
M. Inouye, J. Biol. Chem. 269 (1994) 3925e3927.
[10] J.N. Blankson, D. Persaud, R.F. Siliciano, Annu. Rev. Med. 53 (2002)
557e593.
[44] F.M. Stojanovic, Z. Arnold, Collect. Czech.Chem. Commun. 32 (1967)
2155e2156.
¨
[45] O. Sezer, K. Dabak, O. Anac¸, A. Akar, Helv. Chim. Acta 79 (1996) 449e453.
[46] G.A. Romeiro, L.O.R. Pereira, M.C.B.V. de Souza, V.F. Ferreira,
A.C. Cunha, Tetrahedron Lett. 38 (1997) 5103e5106.
[11] H. Mitsuya, K. Maeda, D. Das, A.K. Ghosh, Adv. Pharmacol. 56 (2008)
169e197.
[47] A.C. Cunha, L.O.R. Pereira, R.O.P. de Souza, M.C.B.V. de Souza,
V.F. Ferreira, Nucleosides Nucleotides Nucleic Acids 20 (2001)
1555e1569.
[12] S.C. Kalichman, G. Di Berto, L. Eaton, Sex Transm. Dis. 35 (2008) 55e60.
[13] X. Duval, D. Descamps, G. Breton, S. Darmon, J.L. Ecobichon,
´
S. Delarue, G. Peytavin, C. Leport, J.L. Vilde, F. Brun-Vezinet, Antivir
´
Ther. 10 (2005) 197e199.
[48] K.A. Winans, C.R. Bertozzi, Chem. Biol. 9 (2002) 113e129.
[49] T. Mosman, J. Immunol. Methods 65 (1983) 55e63.
[50] I.V. Tetko, Drug Discov. Today 10 (2005) 22e36.
[51] A.C. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Adv. Drug Deliv.
Rev. 23 (1997) 3e25.
[14] R.S. Hogg, D.R. Bangsberg, V.D. Lima, C. Alexander, S. Bonner, B. Yip,
E. Wood, W.W. Dong, J.S. Montaner, P.R. Harrigan, PLoS Med. 3 (2006) 356.
[15] E. De Clercq, Med. Mal. Infect. 30 (2000) 421e430.
[16] E. De Clercq, J. Clin. Virol. 22 (2001) 73e89.
[52] M.S. Costa, N. Boechat, E.A. Rangel, F.de C. da Silva, A.M. de Souza,
C.R. Rodrigues, H.C. Castro, I.N. Junior, M.C. Lourenc¸o, S.M. Wardell,
V.F
. Ferreira, Bioorg. Med. Chem. 14 (2006) 8644e8653.
[53] L.R. Dias, M.B. Santos, S. Albuquerque, H.C. Castro, A.M. de Souza,
A.C. Freitas, M.A. DiVaio, L.M. Cabral, C.R. Rodrigues, Bioorg. Med.
Chem. 15 (2007) 211e219.
[17] X.L. Li, K. Chan, Adv. Drug Deliv. Rev. 39 (1999) 81e103.
[18] E. De Clercq, Biochim. Biophys. Acta 1587 (2002) 258e275.
[19] J.R. Huff, Bioorg. Med. Chem. 7 (1999) 2667e2669.
[20] L.M. Schultze, H.H. Chapman, J.P. Dubree, R.J. Jones, K.M. Kent,
T.T. Lee, M.S. Louie, M.J. Postich, E.J. Prisbe, J.C. Rodhloff, R.H. Yu,
Tetrahedron Lett. 39 (1998) 1853e1856.