Synthesis and antimicrobial activity of 6-triazolo-6-deoxy eugenol glucosides

Article abstract of DOI:10.1016/j.carres.2015.04.002

A new series of 1,2,3-triazole eugenol glucosides were synthesized. The new compound structures were confirmed by MS, ¹H NMR and ¹³C NMR. All of the synthesized compounds were screened for antimicrobial and cytotoxic activity. Five c

Full text of DOI:10.1016/j.carres.2015.04.002

Accepted Manuscript
Synthesis and antimicrobial activity of 6-triazolo-6-deoxy eugenol glucosides
Thiago Belarmino de Souza, Paulo Otávio Botelho Raimundo, Saulo Fernandes
Andrade, Taciane Maira Magalhães Hipólito, Naiara Chaves Silva, Amanda Latercia
Tranches Dias, Masaharu Ikegaki, Raissa Prado Rocha, Luiz Felipe Leomil Coelho,
Marcia Paranho Veloso, Diogo Teixeira Carvalho, Danielle Ferreira Dias
PII:
S0008-6215(15)00106-8
10.1016/j.carres.2015.04.002
CAR 6977
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 16 February 2015
Revised Date: 1 April 2015
Accepted Date: 3 April 2015
Please cite this article as: de Souza TB, Raimundo POB, Andrade SF, Hipólito TMM, Silva NC, Dias
ALT, Ikegaki M, Rocha RP, Coelho LFL, Veloso MP, Carvalho DT, Dias DF, Synthesis and antimicrobial
activity of 6-triazolo-6-deoxy eugenol glucosides, Carbohydrate Research (2015), doi: 10.1016/
j.carres.2015.04.002.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and antimicrobial activity of 6-triazolo-6-deoxy
eugenol glucosides
Thiago Belarmino de Souzaa, Paulo Otávio Botelho Raimundob, Saulo Fernandes Andradec,
Taciane Maira Magalhães Hipólitod, Naiara Chaves Silvae, Amanda Latercia Tranches Diase,
Masaharu Ikegakid, Raissa Prado Rochae, Luiz Felipe Leomil Coelhoe, Marcia Paranho Velosod,
Diogo Teixeira Carvalhod, Danielle Ferreira Diasa*
a Instituto de Química, Universidade Federal de Alfenas, 37130-000, MG, Brazil
b Centro Universitário de Lavras, 37200-000, MG, Brazil
c
Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul, Porto Alegre, 90610-000, RS,
Brazil
d Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas, 37130-000, MG, Brazil
e Instituto de Ciências Biomédicas, Departamento de Microbiologia e Imunologia, Universidade Federal
de Alfenas, 37130-000, MG, Brazil
*Corresponding autor:
E-mail adress: danielle.dias@unifal-mg.edu.br
Postal address: Instituto de Química, Universidade Federal de Alfenas-MG, 37130-000 Alfenas, MG,
Brazil. Tel.: +55 3532991261
Highlights
• First time synthesis of eugenol glucosides with different triazole moieties
• Impressive antimicrobial activities against Salmonella typhimurium and Micrococcus
luteus
• Low cytotoxicity on normal host cells
Abstract
A new series of 1,2,3-triazole eugenol glucosides were synthesized. The new compound
1
structures were confirmed by MS, H-NMR and 13C-NMR. All of the synthesized
compounds were screened for antimicrobial and cytotoxic activity. Five compounds
exerted significant activity against the Gram-negative bacteria Salmonella typhimurium
with low IC50 values (49.73-68.53 µΜ), and seven compounds were active against the
Gram-positive bacteria Micrococcus luteus (42.89-210.94 ꢀM). In vitro cytotoxicity on
mouse spleen cells was also evaluated. One compound bearing a phenyl substituent at
the triazole ring showed good activity against Salmonella typhimurium (49.73 ꢀM) and
low toxicity to normal cells (CC50 = 157.83 ꢀM). Thus, the compounds herein can be
considered for further modification for improving their antibacterial activity or
obtaining novel antibacterial drug candidates.
Keywords: eugenol glucosides; triazoles; antimicrobial activity

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1 Introduction
Microbial resistance to antibiotics has become a serious threat to human health.1 Since
the development of sulfonamides and penicillin, many new classes of antibacterial
compounds have been developed. Most antibiotics used today were discovered
approximately 70 years ago, and newer versions have mainly been generated by
chemical modifications based on these compounds. However, only two new classes of
antibiotic (daptomycin and linezolid) have been discovered and approved for use since
the early 1960s. The low level of new antibiotic class discovery and the emergence of
antibiotic resistance in several microorganisms have a significant impact on patient
management.2
The incidence of fungal infections has increased, and has been associated with high
mortality in immunocompromised patients.3 Azole antifungals have emerged as
vanguard drugs for the treatment and prophylaxis of many systemic mycoses.
Fluconazole was first introduced and serves an excellent drug for treating superficial
and invasive fungal infections. The other triazoles available for clinical use include
itraconazole, voriconazole and posaconazole.4 The mechanism of action of azoles is
based on fungal cytochrome P450 14-α-sterol demethylase and 24-methylene
dihydrolanosterol demethylase inhibition, which are key enzymes of ergosterol
biosynthesis.5
The ideal antimicrobial agent should exhibit a high biological activity (fungicidal or
bactericidal activity), reluctance to induce resistance, broad spectrum of action and low
toxicity. None of the current clinical antimicrobial agents possess all of these
requirements. Therefore, the discovery of new, more effective, less toxic and safe
antimicrobial agents with novel mechanisms of action is urgently required.6
Recently, we described the synthesis and antifungal evaluation of eugenol glycosides.7
Eugenol is a phenolic compound found in Indian clove, basil and cinnamon, which are
widely used as flavoring agents and preservatives in food, beverages and cosmetics.
Eugenol is a widespread antiseptic and anesthetic used in dentistry8 and possesses many
pharmacological activities, such as antifungal, antibacterial9 and anti-inflammatory.10
We observed that the peracetylated eugenol glucoside (Figure 1) was the most potent of
the synthesized compounds (IC50: 3.8µM against Candida glabrata). As result of our
continuous search for new antifungal agents, in particular, glycosides, we describe here
new eugenol glucosides with a triazole group at the sugar C6 position.

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The most reactive sites of a monosaccharide, such as glucose, are the C1 and C6
positions. The anomeric position is substituted in the glycoside form; thus, the C6
position is more accessible for modification, as it differs from the other positions
because it is a primary hydroxyl. The 1,2,3-triazole ring can be rapidly and selectively
synthesized from alkyne and azide compounds,11 and its introduction can enhance
eugenol glycoside antimicrobial activity, improve water solubility due to its
hydrophility and increase its basicity.12 In this context, we take advantage of active
eugenol peracetyl glucoside (1) and the remarkable antifungal activity of triazole groups
and describe the synthesis of new triazole eugenol glucoside derivatives and evaluate
their antifungal, antibacterial and cytotoxicity activities.
2 Results and discussion
2.1 Chemistry
Glucoside 2 was synthesized following a previously reported protocol,7 as shown in
Scheme 1. Briefly, this synthesis was accomplished by glycosylating eugenol with
peracetylglucosyl bromide in an alkaline acetone/water solution, followed by the
deacetylation of peracetylglucoside in a methoxide/methanol solution, an adaptation of
Zemplén’s transesterification method that originally employs sodium/methanol.13

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1,2,3-Triazole derivatives 11-15 were prepared following a seven-step synthetic route
and are described here for the first time (Scheme 2); their synthetic pathway initially
employed the selective tosylation of the less hindered, primary 6-hydroxyl group of
glucoside 2. As this hydroxyl group is more reactive than the others, monotosyl
derivative 3 was expected to be the first and major product, which is in fact what was
observed. Thus, to avoid tosylating other positions, the reaction was stopped as soon as
TLC analysis indicated product formation. Derivative 3 was obtained in high yield as a
1
colorless oil after column chromatography. Analyses of the H and 13C NMR spectra
showed a pair of doublets at
δ 7.70 and 7.17 ppm, typical of a para-disubstituted tosyl
group. The remaining hydroxyl groups of the saccharide unit of derivative 3 were then
acetylated. According to the literature,14 the formation of 3,6-anhydro derivatives is
quite common when working with unprotected glycosides with a good leaving group at
C6, especially in reactions under heating, as would be expected in the tosyl-azide
conversion step. Thus, we protected the hydroxyl groups with an acetyl group to prevent
this undesired nucleophilic displacement and also to obtain peracetylated analogues of
the final products for a biological activity comparison. Derivative 4 was obtained in an
82% yield as a pure, white and crystalline solid after reaction with 3 and acetic
anhydride in pyridine. Treatment of 4 with sodium azide in dimethylformamide at 90°C
smoothly afforded azide derivative 5 in 90% yield. This compound is a key intermediate
for 1,2,3-triazole synthesis, and its identity was confirmed by a sharp IR stretching band
at 2100 cm-1 and a typical methylene carbon-azide chemical shift at
δ 51.1 ppm. The
click reaction of 5 with different alkynes afforded peracetylated triazole glucosides 6-
10. This method, 1,3-dipolar cycloaddition between alkynes and alkyl azides in the
presence of copper acetate and sodium ascorbate, generating the reactive catalyst Cu(I)
in situ, has been widely used to obtain 1,2,3-triazole derivatives.15-16 Click reactions
were conducted under room temperature by stirring reagents in a THF/water mixture,
and after 1 h, all reactions were complete, as evidenced by TLC analyses. The five
1,2,3-triazole derivatives were obtained in good yields and high purity after column
chromatography. Compounds 6-10 were then deacetylated by being stirred in a
potassium hydroxide/methanol solution for 30 min, affording 1,2,3-triazoles 11-15.
Acetylated and deacetylated 1,2,3-triazole derivatives were characterized by infrared,
1H and 13C NMR spectroscopies and high-resolution mass spectrometry. In the 13C
NMR spectra of the peracetylated compounds, carbonyl peaks were recorded in the δ
170.0-169.2 ppm range, and these signals were not observed for corresponding

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deacetylated derivatives. The typical H-5' singlet of the 1,2,3-triazole ring was observed
in the δ 8.31-7.49 ppm range and in the C4' and C5' peaks between δ 150-139 ppm and
δ 129-121 ppm in the NMR spectra of all triazole derivatives, confirming heterocycle
formation.17 The different substituents on the C4' position of the triazole ring also
showed consistent IR and NMR signs for all compounds.
2.2 In vitro assays
All of the synthesized 1,2,3-triazoles and synthetic intermediate compounds were
inactive against the evaluated fungi species: Candida albicans (C. albicans), C.
tropicalis, C. krusei, C. parapsilosis and C. glabrata. The results were estimated using
the inhibitory concentration that was able to inhibit microbial growth at 50% (IC50).
These assays were performed in duplicate, and the results obtained from the replicates
were comparable.
However, when the antibacterial activity against different bacteria species was
evaluated, some of the synthesized compounds showed bacteriostatic action. The results
indicate that all of the synthesized compounds, with the exception of derivative 6,
showed bacteriostatic activity for one species. Optimal results were observed against
Micrococcus luteus (M. luteus) and Salmonella typhimurium (S. typhimurium).

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M. luteus is part of the normal flora of the skin or microbiota of mammals. This
bacterium is also found in various environments, such as soil, air and water, and is
resistant to severe environmental conditions, including a high salt concentration and dry
substrates. M. luteus is not considered to be the general cause of bacterial infections in
humans; however, several reports have shown that endocarditis induced by this strain
can result in death.18 Furthermore, this bacterium is a source of nosocomial infections in
immunocompromised individuals and can cause complications in pre-existing
infections, such as meningitis, pneumonia and urinary tract infections. M. luteus can
also become resistance to antibiotics, which is caused by the presence of
extrachromosomal genetic elements. In some cases, M. luteus has exhibited resistance to
multiple drugs.19
Salmonella sp. are pathogenic bacteria that commonly cause foodborne infections.
Salmonella sp. have demonstrated significant resistance to antibiotics. In addition,
treatments that increase the susceptibility of immunocompromised patients to foodborne
infections exist. According to the literature,20 systemic infection by S. typhimurium has
been described after tetracycline, ampicillin, oxacillin and gentamicin use. Cutaneous
salmonellosis has also been described in compromised patients after sulfonamide,
tetracycline, ampicillin and chloramphenicol use, with worsening clinical outcomes.20
Derivatives 3, 4, 5, 8, 11, 13 and 14 were active only against M. luteus, while
derivatives 7, 9, 10, 12 and 15 showed bacteriostatic activity only against S.
typhimurium. Structure analyses suggest that bulky aromatic (phenyl) and aliphatic
(cyclohexyl) groups linked to the triazole ring favor antimicrobial action in gram-
negative strains. An exception to this trend is compound 9, which bears an acetate group
connected to the triazole ring but has a peracetylated glucoside ring, different from its
derivative glucoside 14, which exhibits bacteriostatic activity only against M. luteus
(IC50 of 141.21 µM). Among the compounds that showed antimicrobial activity against
S. typhimurium, derived phenyl-containing compound 7 presented the best antibacterial
activity profile (IC50 of 49.73 µM).
Concerning M. luteus, peracetylated derivative 4, which has a tosylate group in its
structure, was the most active (IC50 of 42.89 µM). Azide derivative 5 was two-fold less
potent than tosylate derivative 4. Among the triazole derivatives, in general, only
deprotected glucosides were active against M. luteus, except in case of compound 8.
This finding might indicate that hydrophilic triazoles enhance activity against this

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bacterium. Moreover, compounds bearing bulky phenyl and cyclohexyl substituents
were inactive, most likely due to steric effects.
Compounds containing hydroxymethyl (11) and chloromethyl (8, 13) groups connected
to the triazole ring, which is considered a pharmacophoric group, presented
bacteriostatic action against M. luteus.
According to our results, peracetylation of glucoside rings does not generate a
significant effect on the selectivity and activity of compounds against the species of
tested bacteria. Furthermore, the new series did not demonstrate significant activity
against Escherichia coli, Enterococcus feacalis and Staphylococcus aureus.
Finally, the starting compounds (1 and 2) were inactive against all of the bacterial
species evaluated, suggesting that the 1,2,3-triazole group is essential for the observed
bacteriostatic activity of the eugenol glucosides synthesized. Further modifications of
the ring substituents may lead to the continued discovery of new agents with
antimicrobial activity.
The cytotoxic activities of all of the compounds were tested in spleen cells obtained
from Swiss-Webster mice. Compounds 3, 4, 8, 13 and 15 exhibited high toxicity in
spleen cells, with CC50 values ranging from 64.28 to 67.93 µM, and 5, 7, 9, 10, 11, 12
and 14 were less toxic, demonstrating CC50 values ranging from 118.89 to 234.94 µM
(Table 2). The more toxic compounds carry a leaving group in their structures (3 and 4
possess a tosyl group, and 8 and 13 have a chloromethylene group), except for
derivative 15. The less toxic compounds carry a 1,2,3-triazole group in their structures,
except for derivative 5, which possess an azide group in place of a triazole ring.
The selectivity indices of compounds 7, 9, 10 and 12 (especially 7) suggest their
potential as antibacterial agents to treat Salmonella sp. infections. These values also
indicated a more discrete potential of the synthesized molecules herein to treat M. luteus
infections. Among the seven compounds with antibacterial activity only against M.
luteus, molecules 4, 11 and 14 have selective indices of ≥ 1 (Table 2).

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Table 1 In vitro antibacterial activity (µM) of the synthesized compounds.
Salmonella
typhimurium
ATCC 14028
Escherichia coli
ATCC 25922
Micrococcus luteus
Enterococcus faecalis Staphylococcus aureus
ATCC 51299 ATCC 6538
ATCC 10240
Compound
IC50
-
IC90
-
IC50
-
IC90
-
IC50
-
IC90
-
IC50 IC90
IC50
-
IC90
-
a
-
-
1
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
95.67
-
-
-
-
-
3
-
-
-
-
42.89
-
-
-
-
-
4
-
-
-
-
132.92
-
-
-
-
-
5
-
-
-
-
-
-
-
-
-
-
6
49.73
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7
8
88.54
54.58
53.39
-
65.93
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9
10
11
12
13
14
15
Chl
210.94
-
104.76
144.12
-
-
68.53
2.72
5.04
2.25
5.07
1.11
2.10
5.50
9.59
6.12
9.22
a No significant activity
Chl: Chloramphenicol

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Table 2 Cytotoxic activity and selectivity index for the antibacterial compounds on tested
microorganisms
Selectivity index
Compound
CC50 (µΜ) CC90 (µΜ)
Salmonella
typhimurium
Micrococcus
luteus
ATCC 14028 ATCC 10240
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1
2
3
4
5
60.93
67.93
122.17
125.91
0.64
1.58
112.68
319.33
0.85
ND
ND
ND
6
7
8
9
10
11
12
13
14
15
ND
157.83
63.77
350.05
132.88
228.80
237.60
448.22
213.72
119.42
336.65
148.93
3.17
ND
0.72
ND
101.11
108.39
234.94
111.89
64.28
1.85
ND
2.03
ND
1.11
ND
1.70
ND
0.61
ND
166.39
65.54
1.15
ND
0.96
CC50: cytotoxic concentration for 50% of cells
CC90: cytotoxic concentration for 90% of cells
ND: not done
Selectivity indexes: CC50/IC50.
3 Conclusion
This work described the synthesis and antimicrobial activity of a series of 1,2,3-triazole
eugenol glucosides. This series was prepared in five steps from known eugenol
glucoside in good overall yields. Several compounds exhibited relevant activity against
Salmonella typhimurium and Micrococcus luteus. Compounds 7, 9, 10, 12 and 15 were
active against Salmonella typhimurium, while 3, 4, 5, 8, 11, 13 and 14 demonstrated
bacteriostatic activity against Micrococcus luteus. Structure-activity relationship studies
showed that the nature of the triazole substituent is important for the observed activity.
Concerning Salmonella typhimurium, the introduction of a bulky group (phenyl or
cyclohexyl) is generally favorable. However, small substituents (CH2Cl, CH2OH and
CO2Et) seem to enhance the activity against Micrococcus luteus. Apparently, glucoside
ring peracetylation does not generate a significant effect on the selectivity and activity
of compounds against the types of strains evaluated. Considering potency and
selectivity, we consider compound 7 to be a good candidate for improving antimicrobial
activity in future studies.

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4 Experimental
4.1 Physical measurements
Melting point of the compounds was determined on Microquímica MOAs 301 apparatus
and was uncorrected. IR spectroscopy was performed by Shimadzu a FTIR-Affinity-1
1
spectrometer. H-NMR and 13C-NMR spectra were obtained on Bruker AC-300
1
spectrometer (300 MHz for H-NMR and 75 MHz for 13C spectra) in deuterated
chloroform, methanol or dimethylsulfoxide. Chemical shifts (δ) were reported in parts
per million (ppm) with reference to tetramethylsilane (TMS) as internal standard and
coupling constants (J) were reported in Hertz (Hz). The following abbreviations were
used for the 1H multiplicities: singlet (s), doublet (d), triplet (t), quartet (qr), quintet (qt),
double doublet (dd), triple doublet (td), multiplet (m) and broad signal (br s). The
specific optical rotation [α]D were measured on Perkin Elmer 341 polarimeter, at 20°C.
High resolution mass spectra were acquired using a LCMS-IT-TOF mass spectrometer
and the samples were solubilized in MeOH + 0.1% formic acid, following manual
injection. Reaction courses and product mixtures were monitored by thin-layer
chromatography (TLC) on commercial silica gel 60 plates. For chromatography,
column grade silica gel (0.040–0.063 mm mesh size) was employed.
4.2 Synthesis of the compounds
4.2.1
Synthesis
of
4-allyl-2-methoxyphenyl-(6-O-toluenesulfonyl-β- -
D
glucopyranoside) (3)
To a solution of 2 (1.53 mmol) in pyridine (2.5 mL) was added a solution of
tosyl chloride (3.84 mmol) in pyridine (2.5 mL) at 0° C, and the reaction was stirred at
this temperature for 3 hours and monitored by TLC. 100 mL of ethyl acetate was added
to the mixture, which was washed with 4M HCl (3x30mL) and water until pH 7.
Organic phase was dried with Na2SO4 and concentrated, providing a colorless oily
crude product (70% yield) which was purified by chromatography
(dichloromethane/methanol 98:2); [α]D -44º (c 0.005, DMSO); IR (ῡ/cm–1): 3373, 2914,
1
1594, 1508, 1448, 1172. H-NMR (300 MHz, CDCl3) δ: 7.70 (2H, d, J3 = 7.8 Hz, Ar-
H), 7.17 (2H, d, J3 = 7.8, Ar-H), 6.87 (1H, d, J3 = 7.6 Hz, Ar-H), 6.67 (1H, s, Ar-H),

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6.61 (1H, d, J3 = 7.8 Hz, Ar-H), 5.98-5.84 (1H, m, allylic), 5.10-5.04 (2H, m, allylic),
4,64 (1H, br s, sugar), 4,31-4.19 (7H, m, sugar, OH), 3.74 (3H, s, OCH3), 3.57-3.51
13
(2H, br s, sugar, OH), 3.30 (2H, d, J3 = 6.1 Hz, allylic), 2.32 (3H, s, C6H5CH3); C-
NMR (75 MHz, CDCl3) δ: 149.8 (1C, ar.), 145.2 (1C, ar.), 144.7 (1C, ar.), 137.7 (1C,
allylic), 136.2 (1C, ar.), 133.0 (1C, ar.), 130.2 (2C, ar.), 128.4 (2C, ar.), 121.4 (1C, ar.),
118.8 (1C, ar.), 116.4 (1C, allylic), 112.9 (1C, ar.), 102,4 (1C, sugar), 76.3 (1C, sugar),
74.04 (1C, sugar), 73.5 (1C, sugar), 69.9 (1C, sugar), 69.4 (1C, sugar), 56.4 (1C,
OCH3), 40.3 (1C, allylic), 22.0 (1C, C6H5CH3); HRMS-ESI: m/z calcd. for C23H28O9S
(M+Na)+: 503.1346; found: 503.1278.
4.2.2 Synthesis of 4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-O-toluenesulfonyl
β- -glucopyranoside) (4)
-
D
To a solution of 3 (0.31 mmol) in pyridine (5 mL) was added dropwise acetic
anhydride (3.1 mmol) and the reaction was monitored by TLC. After 2 hours, 10 mL of
water and concentrated HCl was added to the reaction at 0oC to give pH 1. The mixture
was extracted with dichloromethane (3x20 mL) and the organic layer obtained was
washed with water until pH 7, dried and concentrated, affording the interest product as a
white solid (82% yield); m.p. 106-107oC; [α]D -16 (c 0.005, DMSO); IR (ῡ/cm–1): 2923,
1753, 1597, 1515, 1503, 1172; 1H-NMR (300 MHz, CDCl3) δ: 7.74 (2H, d, J3 = 8.1 Hz,
Ar-H), 7.29 (2H, d, J3 = 8.1, Ar-H), 6.91 (1H, d, J3 = 8.0 Hz, Ar-H), 6.71 (1H, s, Ar-H),
6.66 (1H, dd, J3 = 8.1 Hz, J4 = 1.8 Hz, Ar-H), 5.98-5.87 (1H, m, allylic), 5.23-4.98 (5H,
m, sugar, allylic), 4,85 (1H, d, J3 = 7.5 Hz,), 4,15-4.04 (2H, m, sugar), 3.78 (3H, s,
OCH3), 3.80-3.74 (1H, m, sugar), 3.34 (2H, d, J3 = 6.6 Hz, allylic), 2.41 (3H, s,
C6H5CH3), 2.05-2.00 (9H, 3s, OCOCH3); 13C-NMR (75 MHz, CDCl3) δ: 170.67-169.78
(3C, ester C=O), 150.9 (1C, ar.), 145.6 (1C, ar.), 144.6 (1C, ar.), 137.6 (1C, allylic),
137.3 (1C, ar.), 132.8 (1C, ar.), 130.3 (2C, ar.), 128.5 (2C, ar.), 121.1 (1C, ar.), 120.7
(1C, ar.), 116.4 (1C, allylic), 113.4 (1C, ar.), 101,2 (1C, sugar), 72.8 (1C, sugar), 72.1
(1C, sugar), 71.5 (1C, sugar), 69.0 (1C, sugar), 68.1 (1C, sugar), 56.4 (1C, OCH3), 40.4
(1C, allylic), 22.1 (1C, C6H5CH3), 21.0 (3C, OCOCH3); HRMS-ESI: m/z calcd. for
C29H34O12S (M+Na)+: 629.1663; found: 629.1650.

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4.2.3 Synthesis of 4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-azido-6-deoxy-β-
glucopyranoside) (5)
D-
To a solution of 4 (0.33 mmol) in 15 mL of N,N-dimethylformamide was added
sodium azide (3.3 mmol) and the mixture was heated to 90oC. After the end of reaction,
noted by TLC after 3 hours, the solvent was removed and the product obtained was
partitioned between water/ethyl acetate. The aqueous phase was extracted with ethyl
acetate (3x20 mL) and the organic layer dried with Na2SO4 and concentrated, providing
the compound as a white solid (90% yield); m.p. 129-130oC; [α]D -36 (c 0.005, DMSO);
1
IR (ῡ/cm–1): 2942, 2100, 1743, 1594, 1510, 1470; H-NMR (300 MHz, CDCl3) δ: 7.06
(1H, d, J3 = 7.8 Hz, Ar-H), 6.71-6.69 (2H, m, Ar-H), 6.00-5.87 (1H, m, allylic), 5.27-
5.24 (2H, m, allylic), 5.10-5.05 (3H, m, sugar), 4.95 (1H, d, J3 = 7.7 Hz, sugar), 3.80
(3H, s, OCH3), 3.70-3.63 (1H, m, sugar), 3.42-3.24 (4H, m, sugar, allylic), 2.08-2.02
(9H, 3s, OCOCH3); 13C-NMR (75 MHz, CDCl3) δ: 170.2-169.3 (3C, ester C=O), 150.6
(1C, ar.), 144.0 (1C, ar.), 137.2 (1C, ar.), 137.1 (1C, allylic), 120.9 (1C, ar.), 120.7 (1C,
ar.), 115.9 (1C, allylic), 112.8 (1C, ar.), 101,0 (1C, sugar), 73.3 (1C, sugar), 72.4 (1C,
sugar), 71.2 (1C, sugar), 69.5 (1C, sugar), 55.8 (1C, OCH3), 51.1 (1C, sugar), 39.9 (1C,
allylic), 20.6 (3C, OCOCH3); HRMS-ESI: m/z calcd. for C22H27O9N3 (M+Na)+:
500.1640; found: 500.1549.
4.2.4 General procedure for synthesis of 1,2,3-triazole derivatives 6-10
To a solution of 5 (0.209 mmol) in tetrahydrofuran (0.5 mL) was added the
corresponding acetylene (0.229 mmol, 1.1 equiv) and water (0.5 mL). To this mixture
was added sodium ascorbate (0.6 equiv) and copper acetate (0.5 equiv) solubilized in
0.5 ml of water. The reaction was monitored by TLC and 1 hour after its completion
was observed. Then was added 30 mL of water to the mixture, which was extracted with
dichloromethane (3x20mL), the organic layer dried with Na2SO4 and concentrated,
providing pure 6-10 derivatives after purification by CCS.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-methanol (6): this product was obtained in 78% yield as white
crystals after purified by chromatography (hexane/ethyl acetate 4:6); m.p. 160-161oC;
1
[α]D -24 (c 0.005, DMSO); IR (ῡ/cm–1): 2933, 1748, 1592, 1508, 1465, 1249; H-NMR

ACCEPTED MANUSCRIPT
(300 MHz, CDCl3) δ: 7.53 (1H, s, triazole), 6.69-6.61 (3H, m, Ar-H), 5.96-5.87 (1H, m,
allylic), 5.31-5.20 (2H, m, allylic), 5.09-4.93 (3H, m, sugar), 4.88 (1H, d, J3 = 7.6 Hz,
sugar), 4.66-4.60 (3H, m, CH2OH, sugar), 4.33 (1H, dd, J2 = 14.3 Hz, J3 = 8.9 Hz,
sugar), 3.89-3.82 (1H, m, sugar), 3.76 (3H, s, OCH3), 3.31 (2H, d, J3 = 6.7 Hz, allylic),
2.91 (1H, br s, OH), 2.09-2.02 (9H, 3s, OCOCH3); 13C-NMR (75 MHz, CDCl3) δ:
170.0-169.3 (3C, ester C=O), 150.4 (1C, ar.), 147.6 (1C, ar.), 143.5 (1C, triazole), 137.1
(1C, ar.), 137.0 (1C, allylic), 123.6 (1C, triazole) 120.4 (1C, ar.), 120.0 (1C, ar.), 116.0
(1C, allylic), 113.0 (1C, ar.), 100.3 (1C, sugar), 72.6 (1C, sugar), 72.1 (1C, sugar), 71.1
(1C, sugar), 69.6 (1C, sugar), 56.1 (1C, sugar), 55.8 (1C, OCH3), 50.8 (1C, CH2OH),
39.8 (1C, allylic), 20.6-20.5 (3C, OCOCH3); HRMS-ESI: m/z calcd. for C25H31O10N3
(M+H)+: 534.2082; found: 534.2020.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-benzene (7): this product was obtained in 64% yield as white
crystals after purified by chromatography (hexane/ethyl acetate 55:45); m.p.: 180-
181oC; [α]D -64 (c 0.005, DMSO); IR (ῡ/cm–1): 3081, 1736, 1592, 1510, 1458; 1H-NMR
(300 MHz, CDCl3) δ: 7.77 (1H, s, triazole), 7.71 (2H, dd, J3 = 7.3 Hz, J4 = 1.4 Hz, Ar-
H), 7.39-7.32 (3H, m, Ar-H), 6.64 (1H, d, J3 = 8.1 Hz, Ar-H), 6.60 (1H, d, J3 = 1.9 Hz,
Ar-H), 6.34 (1H, dd, J3 = 8.1 Hz, J4 = 2.0 Hz, Ar-H), 5.78-5.69 (1H, m, allylic), 5.31-
5.27 (2H, m, allylic), 5.06-4.87 (4H, m, sugar), 4.70 (1H, dd, J2 = 14.3 Hz, J3 = 2.3 Hz,
sugar), 4.36 (1H, dd, J2 = 14.3 Hz, J3 = 9.2 Hz, sugar), 3.95-3.87 (1H, m, sugar), 3.72
13
(3H, s, OCH3), 3.13 (2H, d, J3 = 6.7 Hz, allylic), 2.11-2.03 (9H, 3s, OCOCH3); C-
NMR (75 MHz, CDCl3) δ: 170.0-169.3 (3C, ester C=O), 150.1 (1C, ar.), 147.8 (1C, ar.),
143.5 (1C, ar.), 136.9 (1C, allylic), 130.2 (1C, ar.), 128.7 (2C, ar.), 128.1 (1C, ar.),
125.7 (2C, ar.), 121.5 (1C, triazole), 120.6 (1C, ar.), 119.4 (1C, ar.), 115.9 (1C, allylic),
113.0 (1C, ar.), 100.3 (1C, sugar), 72.8 (1C, sugar), 72.1 (1C, sugar), 71.1 (1C, sugar),
69.8 (1C, sugar), 55.8 (1C, OCH3), 50.9 (1C, sugar), 39.6 (1C, allylic), 20.6-20.5 (3C,
OCOCH3); HRMS-ESI: m/z calcd. for C30H33O9N3 (M+H)+: 580.2290; found:
580.2252.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-chloromethylene (8): this product was obtained in 70% yield as
white crystals after purified by chromatography (hexane/ethyl acetate 6:4); m.p. 162-

ACCEPTED MANUSCRIPT
163°C; [α]D -24 (c 0.005, DMSO); IR (ῡ/cm–1): 2928, 1748, 1592, 1508, 1249; 1H-NMR
(300 MHz, CDCl3) δ: 7.55 (1H, s, triazole), 6.72-6.64 (3H, m, Ar-H), 5.99-5.87 (1H, m,
allylic), 5.27-5.25 (2H, m, allylic), 5.10-4.90 (4H, m, sugar), 4.65 (1H, dd, J2 = 14.4
Hz, J3 = 1.9 Hz, sugar), 4.57 (2H, d, J4 = 2.0 Hz, CH2Cl), 4.32 (1H, dd, J2 = 14.4 Hz, J3
= 9.0 Hz, sugar), 3.88-3.83 (1H, m, sugar), 3.78 (3H, s, OCH3), 3.32 (2H, d, J3 = 6.7 Hz,
allylic), 2.10-2.03 (9H, 3s, OCOCH3); 13C-NMR (75 MHz, CDCl3) δ: 170.0-169.3 (3C,
ester C=O), 150.4 (1C, ar.), 144.6 (1C, ar.), 143.5 (1C, triazole), 137.2 (1C, ar.), 136.9
(1C, allylic), 124.5 (1C, triazole) 120.6 (1C, ar.), 120.0 (1C, ar.), 116.1 (1C, allylic),
113.1 (1C, ar.), 100.4 (1C, sugar), 72.6 (1C, sugar), 72.1 (1C, sugar), 71.1 (1C, sugar),
69.7 (1C, sugar), 55.8 (1C, OCH3), 50.9 (1C, sugar), 39.8 (1C, allylic), 35.8 (1C,
CH2Cl), 20.6-20.5 (3C, OCOCH3); HRMS-ESI: m/z calcd. for C25H30O9N3Cl (M+H)+:
552.1743; found: 552.1685.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-ethyl formate (9): this product was obtained in 68% yield as
white crystals after purified by chromatography (hexane/ethyl acetate 6:4); m.p. 165-
166°C; [α]D -44º (c 0.005, DMSO); IR (ῡ/cm–1): 2933, 1741, 1714, 1592, 1542, 1510,
1
1254; H-NMR (300 MHz, CDCl3) δ: 8.13 (1H, s, triazole), 6.69-6.56 (3H, m, Ar-H),
5.93-5.84 (1H, m, allylic), 5.29-5.25 (2H, m, allylic), 5.06-4.96 (3H, m, sugar), 4.83
(1H, d, J3 = 7.5 Hz, sugar), 4.71 (1H, dd, J2 = 14.3 Hz, J3 = 2.2 Hz, sugar), 4.43-4.34
(3H, m, CO2CH2CH3, sugar), 3.87 (1H, td, J3 = 9.5 Hz, J4 = 2.3 Hz, sugar), 3.79 (3H, s,
OCH3), 3.29 (2H, d, J3 = 6.6 Hz, allylic), 2.10-2.03 (9H, 3s, OCOCH3), 1.38 (3H, t, J3 =
7.1 Hz, CO2CH2CH3); 13C-NMR (75 MHz, CDCl3) δ: 170.0-169.2 (3C, ester C=O),
160.3 (1C, ester C=O), 150.4 (1C, ar.), 143.5 (1C, triazole), 140.3 (1C, ar.), 137.2 (1C,
ar.), 137.0 (1C, allylic), 129.3 (1C, triazole), 120.4 (1C, ar.), 120.1 (1C, ar.), 115.9 (1C,
allylic), 113.0 (1C, ar.), 100.9 (1C, sugar), 72.3 (1C, sugar), 72.0 (1C, sugar), 71.1 (1C,
sugar), 69.7 (1C, sugar), 61.2 (1C, CO2CH2CH3), 55.8 (1C, OCH3), 51.5 (1C, sugar),
39.8 (1C, allylic), 20.5 (3C, OCOCH3), 14.3 (1C, CO2CH2CH3); HRMS-ESI: m/z calcd.
for C27H33O11N3 (M+H)+: 576.2188; found: 576.2138.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-cyclohexane (10): this product was obtained in 60% yield as
white crystals after purified by chromatography (hexane/ethyl acetate 7:3); m.p. 152-
153°C; [α]D -32 (c 0.005, DMSO); IR (ῡ/cm–1): 2923, 1738, 1592, 1510, 1448, 1256;

ACCEPTED MANUSCRIPT
1H-NMR (300 MHz, CDCl3) δ: 7.26 (1H, s, triazole), 6.69-6.57 (3H, m, Ar-H), 5.96-
5.83 (1H, m, allylic), 5.29-5.25 (2H, m, allylic), 5.09-4.96 (3H, m, sugar), 4.87 (1H, d,
J3 = 7.8 Hz, sugar), 4.62 (1H, dd, J2 = 14.4 Hz, J3 = 2.3 Hz, sugar), 4.27 (1H, dd, J2 =
14.4 Hz, J3 = 9.2 Hz, sugar), 3.9 (1H, td, J3 = 9.5 Hz, J3 = 2.3 Hz sugar), 3.77 (3H, s,
OCH3), 3.30 (2H, d, J3 = 6.7 Hz, allylic), 2.71-2.63 (1H, m, CH), 2.10-2.03 (9H, 3s,
OCOCH3), 2.00-1.92 (2H, m, CH2), 1.81-1.70 (4H, m, sp3, CH2), 1.40-1.30 (4H, m, sp3,
CH2); 13C-NMR (75 MHz, CDCl3) δ: 170.0-169.3 (3C, ester C=O), 153.6 (1C, ar.),
150.3 (1C, triazole), 143.9 (1C, ar.), 136.9 (1C, allylic), 136.8 (1C, ar.), 121.2 (1C,
triazole) 120.6 (1C, ar.), 119.3 (1C, ar.), 116.0 (1C, allylic), 113.0 (1C, ar.), 100.4 (1C,
sugar), 72.8 (1C, sugar), 72.1 (1C, sugar), 71.1 (1C, sugar), 69.8 (1C, sugar), 55.8 (1C,
OCH3), 50.8 (1C, sugar), 39.8 (1C, allylic), 35.3 (1C, CH), 32.9 (1C, CH2), 32.8 (1C,
CH2), 29.6 (1C, CH2), 26.1 (1C, CH2), 25.9 (1C, CH2), 20.6-20.5 (3C, OCOCH3);
HRMS-ESI: m/z calcd. for C30H39O9N3 (M+H)+: 586.2759; found: 586.2746.
4.2.5 General procedure for synthesis of 1,2,3-triazole derivatives 11-15
The triacetylated 1,2,3-triazoles (1 mmol) were solubilized in a solution of KOH
in MeOH (20 mL, 1.0 mol/L) and stirred at 0oC for 30 min. After the completion of the
reaction, noticed by TLC, the mixture was neutralized with IRA-120 resin. The resin
was filtered off and washed with methanol. The collected filtrate was concentrated in
vaccum to afford deacetylated 1,2,3-triazoles 11-15.
{1-[4-allyl-2-methoxyphenyl-(6-deoxy-β- -glucopyranosid-6-yl)]-1H-1,2,3-triazol-4-
D
yl}-methanol (11): this product was obtained in 100% yield as white crystals; m.p.
205-206°C; [α]D -48 (c 0.005, DMSO); IR (ῡ/cm–1): 3311, 2928, 2856, 1640, 1592,
1510, 1259; 1H-NMR (300 MHz, DMSO) δ: 7.66 (1H, s, triazole), 6.76 (1H, d, J4 = 1.2
Hz, Ar-H), 6.50 (2H, br s, Ar-H), 5.99-5.86 (1H, m, allylic), 5.49 (1H, d, J3 = 5.6 Hz,
OH), 5.32 (1H, d, J3 = 4.6 Hz, OH), 5.24 (1H, d, J3 = 3.9 Hz, OH), 5.14 (1H, br s, OH),
5.08 (2H, s, CH2OH), 5.05-5.01 (2H, m, sugar), 4.74-4.69 (2H, m, allylic), 4.45-4.32
(3H, m, sugar), 3.71 (3H, s, OCH3), 3.65 (1H, dd, J2 = 9.2 Hz, J3 = 1.9 Hz, sugar), 3.27
13
(2H, d, J3 = 6.5 Hz, allylic), 3.15-3.07 (1H, m, sugar); C-NMR (75 MHz, DMSO) δ:
149.1 (1C, ar.), 147.6 (1C, ar.), 144.3 (1C, ar.), 137.8 (1C, allylic), 134.0 (1C, ar.),
123.6 (1C, triazole), 120.2 (1C, ar.), 116.3 (1C, ar.), 115.7 (1C, allylic), 112.9 (1C, ar.),

ACCEPTED MANUSCRIPT
100.6 (1C, sugar), 76.3 (1C, sugar), 74.4 (1C, sugar), 73.2 (1C, sugar), 71.2 (1C, sugar),
55.6 (1C, OCH3), 54.9 (1C, sugar), 50.7 (1C, CH2OH), 39.1 (1C, allylic); HRMS-ESI:
m/z calcd. for C19H25O7N3 (M+H)+: 408.1765; found: 408.1604.
{1-[4-allyl-2-methoxyphenyl-(6-deoxy-β- -glucopyranosid-6-yl)]-1H-1,2,3-triazol-4-
D
yl}-benzene (12): this product was obtained as white crystals in quantitative yield; m.p.
223-224°C; [α]D -56 (c 0.005, DMSO); IR (ῡ/cm–1): 3335, 2918, 2856, 1637, 1592,
1
1510, 1259; H-NMR (300 MHz, DMSO) δ: 8.25 (1H, s, triazole), 7.74-7.71 (2H, m,
Ar-H), 7.46-7.31 (3H, m, Ar-H), 6.69 (1H, s, Ar-H), 6.44 (1H, d, J3 = 8.1 Hz, Ar-H),
6.14 (1H, d, J3 = 8.1 Hz, Ar-H), 5.75-5.61 (1H, m, allylic), 5.54 (1H, d, J3 = 4.6 Hz,
OH), 5.31 (1H, br s, OH), 5.26 (1H, br s, OH), 3.80-3.74 (1H, m, sugar), 3.68 (3H, s,
OCH3), 3.37-3.19 (6H, m, sugar), 3.05 (2H, d, J3 = 5.6 Hz, sugar); 13C-NMR (75 MHz,
DMSO) δ: 148.8 (1C, ar.), 146.1 (1C, triazole), 144.1 (1C, ar.), 137.4 (1C, allylic),
133.6 (1C, ar.), 130.8 (1C, ar.), 128.8 (2C, ar.), 127.7 (1C, ar.), 125.1 (2C, ar.), 122.4
(1C, triazole), 119.9 (1C, ar.), 115.8 (1C, ar.), 115.6 (1C, allylic), 112.7 (1C, ar.), 100.1
(1C, sugar), 76.4 (1C, sugar), 74.4 (1C, sugar), 73.1 (1C, sugar), 71.4 (1C, sugar), 55.5
(1C, OCH3), 51.1 (1C, sugar), 38.9 (1C, allylic); HRMS-ESI: m/z calcd. for
C24H27O6N3 (M+H)+: 454.1973; found: 454.1888.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-chloromethylene (13): this product was obtained as white
crystals, 97% yield; m.p. 168-169°C; [α]D -44 (c 0.005, DMSO); IR (ῡ/cm–1): 3335,
1
2952, 2918, 1635, 1592, 1510, 1460; H-NMR (300 MHz, MeOH) δ: 7.69 (1H, s,
triazole), 6.74 (1H, s, Ar-H), 6.58-6.50 (2H, m, Ar-H), 5.95-5.81 (1H, m, allylic), 5.02-
4.95 (2H, m, allylic), 4.84-4.71 (5H, m, sugar), 4.54 (2H, d, J4 = 3.0 Hz, CH2Cl), 4.36
(1H, dd, J2 = 14.4 Hz, J3 = 9.2 Hz, sugar), 3.73 (3H, s, OCH3), 3.42 (2H, d, J3 = 8.1 Hz,
13
allylic), 3.63 (1H, td, J3 = 9.4 Hz, J4 = 2.0 Hz, allylic); C-NMR (75 MHz, MeOH) δ:
149.6 (1C, ar.), 144.1 (1C, ar.), 143.9 (1C, ar.), 137.4 (1C, allylic), 135.5 (1C, ar.),
124.9 (1C, triazole), 120.5 (1C, ar.), 117.2 (1C, ar.), 114.6 (1C, allylic), 112.7 (1C, ar.),
101.2 (1C, sugar), 76.0 (1C, sugar), 74.5 (1C, sugar), 73.4 (1C, sugar), 71.4 (1C, sugar),
55.2 (1C, OCH3), 51.2 (1C, sugar), 39.3 (1C, allylic), 34.9 (1C, CH2Cl); HRMS-ESI:
m/z calcd. for C19H24O6N3Cl (M-H)+: 424.1281; found: 424.1381.

ACCEPTED MANUSCRIPT
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-ethyl formate (14): this product was obtained in 94% yield,
white crystals; m.p. 130-132°C; [α]D -48 (c 0.005, DMSO); IR (ῡ/cm–1): 3464, 2923,
1
2856, 1721, 1637, 1592, 1513; H-NMR (300 MHz, DMSO) δ: 8.34 (1H, s, triazole),
6.78 (1H, s, Ar-H), 6.52-6.44 (2H, m, Ar-H), 5.97-5.88 (1H, m, allylic), 5.07-4.99 (2H,
m, allylic), 4.93-4.50 (6H, m, sugar), 4.36 (2H, qt, J3 = 6.8 Hz, CO2CH2CH3), 3.82 (3H,
s, OCH3), 3.76-3.73 (1H, m, sugar), 3.50 (2H, d, J3 = 5.6 Hz, allylic), 1.35 (3H, t, J3 =
6.5 Hz, J3 = 7.0 Hz, CO2CH2CH3); 13C-NMR (75 MHz, DMSO) δ: 160.5 (1C, ester
C=O), 149.5 (1C, ar.), 144.1 (1C, ar.), 139.1 (1C, allylic), 137.4 (1C, allylic), 135.3 (1C,
ar.), 129.7 (1C, triazole), 120.3 (1C, ar.), 116.9 (1C, ar.), 114.6 (1C, allylic), 112.6 (1C,
ar.), 101.4 (1C, sugar), 76.0 (1C, sugar), 74.1 (1C, sugar), 73.3 (1C, sugar), 71.5 (1C,
sugar), 60.8 (1C, CO2CH2CH3), 55.1 (1C, OCH3), 51.4 (1C, sugar), 39.3 (1C, allylic),
13.2 (1C, CO2CH2CH3); HRMS-ESI: m/z calcd. for C21H27O8N3 (M+H)+: 450.1871;
found: 450.1771.
{1-[4-allyl-2-methoxyphenyl-(2,3,4-tri-O-acetyl-6-deoxy-β- -glucopyranosid-6-yl)]-
D
1H-1,2,3-triazol-4-yl}-cyclohexane (15): this product was obtained in quantitative
yield as white crystals; m.p. 216-217°C; [α]D -56 (c 0.005, DMSO); IR (ῡ/cm–1): 3473,
1
2923, 2856, 1640, 1592, 1510; H-NMR (300 MHz, DMSO) δ: 7.49 (1H, s, triazole),
6.76 (1H, s, Ar-H), 6.45 (2H, m, Ar-H), 5.96-5.82 (1H, m, allylic), 5.51 (1H, br s, OH),
5.32 (1H, br s, OH), 5.25 (1H, br s, OH), 5.08-5.01 (2H, m, allylic), 4.77-4.69 (2H, m,
sugar), 4.24 (1H, dd, J2 = 14.3 Hz, J3 = 9.6 Hz, sugar), 3.70 (3H, s, OCH3), 3.72-3.28
(4H, m, sugar), 3.48 (2H, d, J3 = 6.7 Hz, allylic), 3.12 (1H, br s, CH), 1.85-1.79 (2H, m,
13
CH2), 1.35-1.15 (8H, m, CH2); C-NMR (75 MHz, DMSO) δ: 152.1 (1C, ar.), 149.0
(1C, triazole), 144.4 (1C, ar.), 137.7 (1C, allylic), 133.9 (1C, ar.), 121.4 (1C, triazole)
120.1 (1C, ar.), 115.8 (1C, allylic), 115.6 (1C, ar.), 112.9 (1C, ar.), 100.2 (1C, sugar),
76.3 (1C, sugar), 74.7 (1C, sugar), 73.2 (1C, sugar), 71.5 (1C, sugar), 55.7 (1C, OCH3),
51.0 (1C, sugar), 39.2 (1C, allylic), 34.7 (1C, CH), 32.6 (2C, CH2), 25.8 (3C, CH2);
HRMS-ESI: m/z calcd. for C24H33O6N3 (M+H)+: 460.2442; found: 460.2326.

ACCEPTED MANUSCRIPT
4.3 In vitro bioassays
4.3.1 Antifungal activity evaluation
The compounds were evaluated in vitro for their antifungal activity using Mueller
Hinton Broth and the microdilution method following methodology and interpretative
criteria proposed by document M27A3.21 The stock solutions of all the compounds were
prepared in DMSO 1% and tested at concentrations (µg/mL) 100; 60; 30; 15; 7.5; 3.75;
1.875; 0.468; 0.23; 0.06. The standard drug fluconazole was applied as control of
fungistatic action at concentrations (µg/mL) 64; 32; 16; 8; 4; 2; 1; 0.5; 0.25; 0.125. The
microplates were incubated at 37oC for 24 h. Results were visualized and analyzed at
530 nm in an Anthos Zenyth 200rt Microplate Reader. The inhibitory concentrations of
microbial growth of the compounds were determined by reduction of 50% in the
absorbance values compared to the absorbance value of maximum growth of the isolates
(IC50) in µg/mL. The tests were done in duplicates.
4.3.2 Antibacterial activity evaluation
The antibacterial activity of the products was evaluated against strains of American
Type Culture Collection, including Gram-negative (Escherichia coli ATCC 25922,
Salmonella Typhimurium ATCC 14028) and Gram-positive (Staphylococcus
aureus ATCC 6538, Enterococcus faecalis ATCC 51299, Micrococcus luteus ATCC
10240). A microdilution protocol using Mueller Hinton Broth (Imedia, Curitiba, PR,
Brazil) in 96-well colourless microplates was performed according to the standardised
method of the Clinical Laboratories Standardization Institute by the document M07-A9.
22 All analysis were done in triplicate. Chloramphenicol (positive control) and the stock
solutions of all the synthesized compounds were prepared in DMSO
(dimethylsulfoxide) and tested at concentrations (µg/mL) 100, 50, 25, 12.5, 6.25, 3.12,
1.56, 0.78, 0.39 and 0.19. The microplates were incubated at 37°C/24h for S. aureus
ATCC 6538 and 37°C/16-20h for the other strains. The microbial growth was quantified
by spectrometric measurement at 530 nm in Anthos Zenyth 200rt Microplate Reader.
The results were expressed as IC50 (concentration of the substance that was able to
inhibit 50% of bacteria growth) and IC90 (concentration of the substance that was able to
inhibit 90% of bacteria growth).

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4.3.3 Cytotoxicity assay
The cytotoxicity of the compounds (200 to 1.5 ꢀg/mL) to mouse spleen cells was
determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) method.7 The spleen cells from Swiss Webster mice were prepared by gently
disrupting spleens using forceps in 5 ml of RPMI 1640 medium with L-glutamine
(Sigma Aldrich, USA) and then isolated via centrifugation (500 g). The cell pellet was
incubated for 4 min with red blood cells lysis buffer (one part of 0.17 mol/L Tris to nine
parts of 0.16 mol/L ammonium chloride). The cells were washed again and suspended
to a concentration of 2.4×106 cells/mL in RPMI 1640 supplemented with 1% fetal calf
serum and antibiotics (100 ꢀg/mL penicillin and 100 ꢀg/mL streptomycin). The cell
suspension of spleen cells at a concentration of 2.4 × 105 cells/ml was distributed in a
96-well plate, 90 ꢀL in each well with 10 ꢀL of test compounds at different
concentrations, incubated at 37 °C in an incubator at 5% CO2 for 48 h. After, 10 ꢀL of
MTT dye (5 mg/mL) was added and the cells were incubated again for an additional 4 h
period. Then, the medium was carefully removed and added to 100 ꢀL of DMSO for
solubilization of formazan crystals. The plates were shaken for 5 min and absorbance
for each sample was measured in a spectrophotometric microplate reader at 560 nm.
The percentage of cytotoxicity was calculated as [(A-B)/Ax100)], where A and B are
the absorbances of control and treated cells, respectively. Data were analyzed using
linear regression to obtain values for CC50 and CC90 (cytotoxic concentration for 50%
and 90% of cells, respectively). Selectivity indexes were expressed as the ratio
CC50/IC50.
5. Acknowledgments
This work was supported by FAPEMIG, CNPq, CAPES and FINEP through grants and
fellowships.
6. Conflict of interest
The authors declare no conflict of interest.

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7. References
1. Norrby S. R. Clin. Microbiol. Infect. 2005, 11, 9-11.
2. Woodford, N.; Livermore, D. M. J. Infect. 2009, 59 (Suppl. 1), S4–S16.
3. Low, C-Y.; Rotstein C. F1000 Med. Rep. 2011, 3, 1-8.
4. Aperis, G.; Mylonakis, E. Expert Opin. Investig. Drugs 2006, 15, 579-602.
5. Ghannoum, M. A.; Rice, L. B. Clin. Microb. Rev. 1999, 12, 501-517.
6. Wise, R. J. Antimicrob. Chemother. 2011, 66, 1939-1940.
7. Souza, T. B.; Orlandi, M.; Coelho, L. F. L.; Malaquias, L. C. C.; Dias, A. L. T.;
Carvalho, R. R.; Silva, N. S.; Carvalho, D.T. Med. Chem. Res. 2014, 23, 496-
502.
8. Jadhav, B. K.; Khandelwal, K. R.; Ketkar, A. R.; Pisal, S. S. Drug Dev. Ind.
Pharm. 2004, 30, 195-203.
9. Laekeman, S. M.; Hoof, V. L.; Haemers, A.; Berghe, V. A. D.; Herman, A. G.;
Vlietink, A. K. Phytother. Res. 1990, 4, 90-96.
10. Daniel, A. N.; Sartoretto, S. M.; Schmidt, G.; Caparroz-Assef, S. M.; Bersani-
Amado, C. A.; Cuman, R. K. N. Rev. Bras. Farmacogn. 2009, 19, 212-217.
11. Kolb, H. C.; Finn, M. G.; Sharpless, K. B.; Angew. Chem. Int. Ed. 2001, 40,
2004-2012.
12. Song, Q.L.; Guo, B.Q.; Zhang, W.; Lan, P.; Sun, P.H.; Chen, W.M. J. Antibiot.
2011, 64, 571–581.
13. Roy, R.; Kim, J. M. Tetrahedron 2003, 59, 3881-3893.
14. Lewis, B. A.; Smith, F.; Stephen, A. M. Methods Carbohydr. Chem. 1963, 2,
172-188.
15. Freitas, L. B. O.; Ruela, F. A.; Pereira, G. R.; Alves, R. B.; Freitas, R. P.; Santos,
L. J. Quim. Nova 2011, 34, 1791-1804.
16. Deobald, A. M.; Camargo, L. R. S.; Alves, D.; Zukerman-Schpector, J.; Corrêa,
A. G.; Paixão, M. W. Synthesis 2011, 24, 4003-4010.
17. Carvalho, I.; Andrade, P.; Campo, V. L.; Guedes, P. M. M.; Sesti-Costa, R.;
Silva, J. S.; Schenkman, S.; Dedola, S.; Hill, L.; Rejzek, M.; Nepogodiev, S. A.;
Field, R. A. Bioorg. Med. Chem. 2010 18, 2412-2427.
18. Miltiadous, G.; Elisaf, M. J. Med. Case Rep. 2011, 5, 251.
19. Dib, J. R.; Liebl, W.; Wagenknecht, M.; Farías, M. E.; Meinhardt, F. Appl.
Microbiol. Biotechnol. 2013, 97, 63-75.

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20. Keeney, K. M.; Yurist-Doutsch, S.; Arrieta, M-C; Finlay, B. B. Annu. Rev.
Microbiol. 2014, 68, 217-235.
21. Clinical And Laboratory Standards Institute (CLSI). Reference method for broth
dilution antifungal susceptibility testing of yeasts; Approved Standard M27-A3,
3th ed.; National Committee for Clinical Laboratory Standards: Wayne, Pa,
2008.
22. Clinical And Laboratory Standards Institute (CLSI). Methods for dilution
antimicrobial susceptibility testes for bacteria that grow aerobically; Approved
Standard M07-A9, 9th ed.; National Committee for Clinical Laboratory
Standards: Wayne, Pa, 2012.