Synthesis, chemical characterization and antimicrobial activity of new acylhydrazones derived from carbohydrates

Article abstract of DOI:10.1016/j.molstruc.2019.02.045

A new series of glycosylated acylhydrazones was synthesized and all the chemical structures were confirmed by High Resolution Mass Spectrometry (HRMS), ¹H and ¹³C Nuclear Magnetic Resonance (¹H-NMR; ¹³C-NMR) and Fourier Transform Infrared (FTIR) spectroscopy methods. The mass accuracy between the calculated and found values observed in HRMS analyses were near or lower than 5 ppm, which are acceptable for proposing a molecular formula using this technique. All of the synthesized compounds were screened for their antibacterial, antifungal and antiviral activities. Five compounds (12, 13, 14, 16 and 19) exerted a modest antifungal activity against the strains evaluated. Derivative 14 showed fungicidal activity against Candida glabrata at 173.8 μM and saccharide unit contributed to the increase of the antifungal potential against this strain. New chemical manipulation of derivative 14 can make it possible to obtain new potentially antimicrobial agents.

Full text of DOI:10.1016/j.molstruc.2019.02.045

Accepted Manuscript
Synthesis, chemical characterization and antimicrobial activity of new acylhydrazones
derived from carbohydrates
Fernanda Dias Guilherme, Julia Évelin Simonetti, Lais Regina Santos Folquitto,
Adriana Cotta Cardoso Reis, Josidel Conceição Oliver, Amanda Latércia Tranches
Dias, Danielle Ferreira Dias, Diogo Teixeira Carvalho, Geraldo Célio Brandão, Thiago
Belarmino de Souza
PII:
S0022-2860(19)30175-9
DOI:
https://doi.org/10.1016/j.molstruc.2019.02.045
MOLSTR 26202
Reference:
To appear in: Journal of Molecular Structure
Received Date: 16 October 2018
Revised Date: 6 February 2019
Accepted Date: 12 February 2019
Please cite this article as: F.D. Guilherme, Julia.É. Simonetti, L.R.S. Folquitto, A.C. Cardoso Reis,
Josidel.Conceiçã. Oliver, Amanda.Laté.Tranches. Dias, D.F. Dias, D.T. Carvalho, Geraldo.Cé. Brandão,
T.B.d. Souza, Synthesis, chemical characterization and antimicrobial activity of new acylhydrazones
derived from carbohydrates, Journal of Molecular Structure (2019), doi: https://doi.org/10.1016/
j.molstruc.2019.02.045.
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ACCEPTED MANUSCRIPT
Synthesis, chemical characterization and antimicrobial activity of new
acylhydrazones derived from carbohydrates
Fernanda Dias Guilherme1, Julia Évelin Simonetti1, Lais Regina Santos Folquitto2,
Adriana Cotta Cardoso Reis3, Josidel Conceição Oliver4, Amanda Latércia Tranches
Dias4, Danielle Ferreira Dias2, Diogo Teixeira Carvalho4, Geraldo Célio Brandão3,
Thiago Belarmino de Souza3*
1
Departamento de Farmácia Industrial, Universidade Federal de Santa Maria, 97105-
900, Brazil.
2 Instituto de Química, Universidade Federal de Alfenas, 37130-000, Brazil.
3 Departamento de Farmácia, Universidade Federal de Ouro Preto, 35400-000, Brazil.
4
Faculdade de Ciências Farmacêuticas, Universidade Federal de Alfenas, 37130-000,
Brazil.
* Corresponding author:
E-mail: thiagobs83@yahoo.com.br; phone: 5535998290199
Postal address: Universidade Federal de Ouro Preto (Campus Morro do Cruzeiro –
Escola de Farmácia). Zip code: 35400-000; City/State: Ouro Preto/MG; Brazil.

ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Abstract
A new series of glycosylated acylhydrazones was synthesized and all the chemical
structures were confirmed by High Resolution Mass Spectrometry (HRMS), 1H and 13C
Nuclear Magnetic Resonance (1H-NMR; 13C-NMR) and Fourier Transform Infrared
(FTIR) spectroscopy methods. The mass accuracy between the calculated and found
values observed in HRMS analyses were near or lower than 5 ppm, which are
acceptable for proposing a molecular formula using this technique. All of the
synthesized compounds were screened for their antibacterial, antifungal and antiviral
activity. Five compounds (12, 13, 14, 16 and 19) exerted a modest antifungal activity
against the strains evaluated. Derivative 14 showed fungicidal activity against Candida
glabrata at 173.8 ꢀM and saccharide unit contributed to the increase of the antifungal
potential against this strain. New chemical manipulation of derivative 14 can make it
possible to obtain new potentially antimicrobial agents.
Key words: acylhydrazones, glycosides, chemical characterization, antimicrobial
activity.

ACCEPTED MANUSCRIPT
1. Introduction
The increase in the incidence of microbial infections combined with the growth
of resistance of microorganisms against the available antimicrobial drugs has led to an
increase in the number of deaths in hospitalized patients, especially among those
immunosuppressed [1]. In the absence of an effective drug, several bacterial, fungal, or
viral infections can represent a serious threat to infected patients.
Among the organic compounds with biological potential to represent new
prototypes to antimicrobial agents, we can highlight the acylhydrazone derivatives. The
acylhydrazone units present in different substances have been related to a wide range of
biological effects, such as antibacterial [2,3], antifungal [4] and antiviral [5].
On the other hand, still in the search for new potentially bioactive substances,
the synthesis of glycosidic derivatives has also attracted the attention of synthetic
chemists, since there are several reports of synthesis and biological evaluation of
substances associated with different carbohydrates. The presence of a saccharidic unit in
these compounds can be responsible for facilitating their penetration into the cell of
microorganisms, to create new points of interaction with some action target, and to
provide a significant improvement in their properties [6,7].
Considering the experience of our research group involving the synthesis of
potentially antimicrobial glycosidic derivatives [8-11], we describe here the synthesis,
chemical characterization and antimicrobial evaluation of a series of glycosidic
derivatives containing an acylhydrazone unit in its structure, whose structural pattern is
shown in Figure 1 below (A). The proposed structural pattern was inspired by a series
of acylhydrazones (B-F) synthesized by Cachiba and coworkers [12] active against
different species of bacteria and fungi, whose chemical structures are also shown in
Figure 1. The association of carbohydrate units with substances containing the structural
pattern of compounds B-F could originate potentially new antimicrobial
acylhydrazones.

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Figure 1: Structural pattern of the new synhthesized acylhydrazones glycosylated (A)
and acylhydrazones with antimicrobial potential (B-F)
2. Results and discussion
2.1 Chemistry
Employing a convergent synthesis, initially were synthesized the key intermediates
(vanillin glycosides and hydrazides) to afford the acylhydrazones glycosylated proposed
(Figure 2). To obtain the glucoside (5) and galactoside (6), initially
D-Glucose (1) and
D
-galactose (2) were pentacetylated in the presence of acetic anhydride and the
corresponding peracetylated derivatives were converted to the acetylglycosides
bromides 3 and 4, respectively, after reaction with hydrobromic acid [6]. The reaction of
the glycosyl halides with 4-hydroxybenzaldehyde (vanillin) provided the peracetyl
glycosides of interest [13]. On the other hand, the synthesis of the hydrazides employed
was possible from the p-aminobenzoic acid which, after esterification with ethanol and
N-acylation with acetyl or benzoyl chlorides, providing the derivatives 7 and 8, which
upon reaction with hydrazine hydrate provided the hydrazides of interest 9 and 10 [12].

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OH
OAc
OAc
R2
R2
R2
O
i, ii
iii
O
O
O
R1
R1
AcO
R1
AcO
O
HO
OH
OAc
Br
OH
OAc
CHO
1: R1=OH; R2=H
2: R1=H; R2=OH
3: R1=OAc; R2=H
4: R1=H; R2=OAc
5: R1=OAc; R2=H
6: R1=H; R2=OAc
i: Ac2O, AcNa, 80oC; ii: HBr, Ac2O, r.t.; iii: LiOH aq., vanillin, acetone,
r.t.
O
O
O
iii
H2NHN
O
i, ii
EtO
O
HO
N
R1
N
H
R1
NH2
H
9: R1= CH3
10: R1= C6H5
7: R1= CH3
8: R1= C6H5
i: EtOH, HCl, 80oC; ii: Ac2O, MeOH ou benzoyl chloride, r.t.; iii: NH2NH2.H2O,
EtOH, r.t.
Figure 2: Synthesis of synthetic intermediates vanillin glycosides and hydrazides
Finally, as shown in Figure 3, the reaction between the peracetyl vanillin glycosides (5
and 6) and the previously obtained hydrazides (9 and 10) provided the peracetyl
glycosylated acylhydrazones 11-14, which after subjected to methanolysis conditions
afforded the corresponding deacetylated glycosides 15-18 [12]. To evaluate the
biological importance of the carbohydrates in derivatives synthesized, two non-
glycosylated acylhydrazones (19 and 20) were also synthesized from the reaction of the
hydrazides 9-10 with vanillin under the same conditions employed for obtaining of the
derivatives 11-14. The compound 20 were previously described [13].

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Figure 3: Synthesis of proposed acylhydrazones
The NMR signals of the compounds were assigned based on spectral data previously
described in the literature of analogous substances to those synthesized in this work
[14,15]. In 1H NMR spectra of all synthesized acylhydrazones was possible to observe a
signal corresponding to the hydrazone proton (imine double bond; -N=CH-) near 8.4
ppm for compounds 11-20, suggesting the obtaining of the E-isomer for all the
compounds, since Z-isomers usually have this signal registered near 7.9 ppm [12, 16].
The aromatic protons were observed between 8.00-6.82 ppm for all the compounds,
whose integrals values were consistent with the synthesized substances. The signals
referents to the acylhydrazone (NH) and amide (NH) groups were registered as singlets
near 11.7 and 10.5 ppm, respectively. The signals relative to the saccharidic carbons
were registered between 100.7-60.3 ppm for all glycosides and carbonyl carbons (ester
13
groups) were registered near 170 ppm for peracetylated derivatives 11-14 in C NMR
spectra of these compounds. The selective deacetylation of derivatives 11-14 to afford
the respective compounds 15-18 also could be noticed by the FTIR spectra, which
showed bands between 1744-1715 cm-1 (axial deformation of esters carbonyl carbons)
for peracetylated compounds and was not registered for the deacetylated derivatives.
The hydroxyl bands of deacetylated (compounds 15-18) and phenolic (compounds 19
1
13
and 20) were observed near 3300 cm-1. The main H and C NMR signals and FTIR
bands registered for acylhydrazones 11-20 are summarized in Table 1 below.
1
Table 1: Main H and 13C NMR signals and FTIR bands registered for acylhydrazones
11-20
13C NMR (ppm)
1H NMR (ppm)
FTIR (cm-1)
Compound
Sugar
carbons
98.3-61.6
Acylhydrazone
carbonyl
Aromatic
protons
7.83-7.15
Imine
protons
8.38
Acetyl
carbonyl
1739
Hydroxyl
groups
-
162.5
11
12
13
14
15
16
17
18
98.7-62.0
98.9-61.3
99.3-61.6
99.5-60.6
99.6-60.6
100.2-60.3
100.7-60.7
162.9
162.5
162.9
162.5
162.4
162.5
163.2
8.00-7.19
7.88-7.15
8.00-7.16
7.88-7.15
7.99-7.16
7.85-7.14
7.98-7.15
8.42
8.38
8.42
8.37
8.40
8.36
8.40
1744
-
1748
-
1742
-
-
-
-
-
3296
3286
3291
3231

ACCEPTED MANUSCRIPT
-
-
162.4
162.7
7.88-6.82
7.94-6.83
8.32
8.33
-
-
3304
3306
19
20
The new acylhydrazones synthesized were also characterized by HRMS and the
molecular picks values are shown in Table 2, where can be observed that the mass
accuracy between the calculated and found values were near or lower than 5 ppm,
which are acceptable for proposing a molecular formula using this technique [17-19].
Table 2: Data obtained from HRMS analysis and molecular formula of the synthesized
acylhydrazones 11-20.
Compound
HRMS
calculated
[M – H]-
HRMS
found
Mass accuracy Molecular
(ppm)
formula
[M – H]-
656.2097
718.2254
656.2097
718.2254
488.1675
550.1831
488.1675
550.1831
326.1146
388.1303
656.2088
718.2242
656.2077
718.2251
488.1667
550.1824
488.1674
550.1825
326.1129
388.1292
1.37
1.67
3.04
0.41
1.63
1.27
0.20
1.09
5.21
2.83
C31H34N3O13
C36H36N3O13
C31H34N3O13
C36H36N3O13
C23H26N3O9
C28H28N3O9
C23H26N3O9
C28H28N3O9
C17H16N3O4
C22H18N3O4
11
12
13
14
15
16
17
18
19
20
Another physicochemical properties of the obtained acylhydrazones were determined,
as melting point, solubility, specific optical rotation [α]D and logP, as a way of
presenting a better characterization of the compounds. These properties as well the yield
of each compound are shown in Table 3.
Table 3: Physicochemical properties and yield of the synthesized acylhydrazones 11-
20.
Compound
11
Melting point (oC) Solubility*
135-136 DMSO
[α]D
LogP
Yield (%)
-25°
0.83
56

ACCEPTED MANUSCRIPT
146-147
71-73
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
-22°
-8°
2.73
0.83
2.73
-0.08
1.81
-0.08
1.81
1.76
3.65
76
79
12
13
14
15
16
17
18
19
20
137-138
145-147
150-151
178-180
184-186
272-273
202-203
-23°
-15°
-23°
-25.2°
-54.2°
-
84
100
51
64
100
61
-
55
* The solubility of the compounds was evaluated at 0.03g.mL-1 in several solvents (water, chloroform,
methanol, ethanol, acetonitrile, pyridine, N,N-dimethylformamide), however they were soluble only in
dimethylsufoxide (DMSO).
2.2 Biological assays
The antimicrobial activity of the new acylhydrazones was evaluated against different
strains of fungi (Candida albicans, C. tropicalis, C. krusei, C. glabrata and C.
parapsilosis), bacteria (Escherichia coli, Enterococcus faecalis, Micrococcus luteus,
Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus) and
Zika virus (ZIKV). Fungi and bacteria were assayed by the microdilution method and
the results were estimated using the inhibitory concentration that was able to inhibit
microbial growth at 50% (IC50; fungistatic and bacteriostatic activities) and at 90%
(IC90; fungicide and bactericidal activities) [20,21]. The positive controls employed in
the assays were chloramphenicol (antibacterial assay) and fluconazole (antifungal
assay). The antiviral and cytotoxic activities were evaluated by the colorimetric method
of MTT [22].
The acylhydrazones 12, 13, 14, 16 and 19 showed a modest antifungal activity against
the strains evaluated in the range of 907.1-173.8 ꢀM, as related in Table 4. Among the
active compounds, the derivative 14 was the most promising as it was the only
glycoside (derived from D-galactose) that showed a fungicidal activity against Candida
parapsilosis at 173.8 ꢀM. This result also shows that the presence of the saccharide unit
contributed to the increase of the antifungal potential against this strain, since its
respective non glycosylated acylhydrazone 20 was inactive. The same result was
observed for the β-glucoside 12, active at 173.8 ꢀM against this same strain. The
increase of antifungal potential of an aglycone by the insertion of a
D-glucose unit was
already described by our research group in another study [11]. Moreover, the

ACCEPTED MANUSCRIPT
peracetylated actylhydrazones were more active when compared with the respective
deacetylated derivatives, as only compound 16 showed antifungal activity. Although
derivatives 12 and 14 exhibited moderate antifungal activity (IC50 values in the range of
695.1-173.8 ꢀM against C. albicans, C. tropicalis and C. parapsilis for 12 and IC90
value of 173.8 ꢀM for 14) Cachiba and coworkers [12] described a series of structurally
similar non-glycosylated acylhydrazones with higher antifungal potential, and the most
potent derivative described (compound D; Figure 1) was active against C. albicans, C.
krusei and C. tropicalis between 17-34 ꢀM.
Table 4: Antifungal activity (ꢀM) of the new acylhydrazones synthesized
C. albicans C. tropicalis
C. krusei
ATCC
6258
C. glabrata C. parapsilosis
Compound
ATCC
10231
ATCC 750
ATCC
90030
ATCC 22019
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
-
760.8
-
-
-
11
12
-
-
-
-
-
695.1
695.1
-
-
173.8
-
-
-
-
-
-
760.8
760.8
380.4
380.4
13
-
-
-
-
-
-
-
-
695.2
-
14
-
-
-
-
173.8
-
-
-
-
-
15
-
-
-
-
-
-
-
907.1
-
226.8
16
-
-
-
-
-
-
-
-
-
-
17
-
-
-
-
-
-
-
-
-
-
18
-
-
-
-
-
764.3
-
382.1
764.3
382.1
19
-
-
-
-
-
-
-
-
-
-
-
-
20
-
-
-
1.6
3.3
104.5
52.2
3.3
Fluc
Derivatives 11, 16, 19 and 20 were weakly active against E. faecalis, M. luteus and S.
aureus in the range of 760.8-1528.5 ꢀM and inactive against E. coli, P. aeruginosa and

ACCEPTED MANUSCRIPT
S. thyphimurium (Table 5). The acylhydrazones synthesized by Cachiba and colleagues
[12] (prototypes of this work), although not very potent, showed higher activity against
S. aureus, in the range of 380-3370 ꢀM. In addition, they also showed moderate activity
against E. coli and P. aeruginosa between 360-3370 ꢀM.
Wang and coworkers also synthesized a series of non-glycosylated and structurally
related acylhydrazones synthesized here and evaluated their antibacterial potential. Five
derivatives were strongly active against E. coli, P. aeruginosa, S. aureus and Bacillus
subtilis in the range of 0.39-12.5 ꢀg.mL-1 and it was observed that the more active
compounds carry a mono or dihydroxylated benzene ring in their structures [23].
Table 5: Antibacterial activity (ꢀM) of the new acylhydrazones synthesized
E. coli
ATCC
25922
E. faecalis M. luteus
P.
S.
S. aureus
ATCC
6538
Compound
ATCC
29212
ATCC
10240
aeruginosa
ATCC
thyphimuri
um ATCC
14028
27853
-
-
-
760.8
-
-
-
-
-
-
-
-
-
-
-
-
-
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
IC90
IC50
11
12
13
14
15
16
17
18
19
20
Chl
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
907.1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1528.5
-
-
-
1284.9
-
-
1284.9
-
-
-
-
-
-
-
-
-
-
-
3.1
6.2
1.6
99.0
3.1
6.2

ACCEPTED MANUSCRIPT
The compounds exhibit moderate to low cytotoxicity. In the normal cell line MRC-5
(lung fibroblast), compound 11 was the most cytotoxic with CC50 of 86.4 ꢀM, while the
other derivatives showed CC50 greater than 139.0 ꢀM (Table 6). Considering Vero cells
(Cercopithecus aethiops kidney normal) , CC50 varied from 31.4 to 194.1 µM. Although
all compounds showed higher cytotoxicity for this cell line, the CC50 values were higher
than 30 ꢀM showing once again the low cytotoxicity of these new acylhydrazones.
None of the compounds tested inhibited the Zika virus multiplication cycle when
challenged against viral infection in Vero cells of MOI 1.0.
Table 6: Cytotoxicity (MRC-5 and Vero, n = 3) and antiviral activity against ZIKV of
acylhydrazones 11-20
CC50 (µM)
MRC-5
IC50 (µM)
ZIKV*
Compounds
Vero
86.4 ± 2.8
> 139.0
> 152.2
> 139.3
> 204,4
> 181.4
> 204.4
> 181.4
> 305.7
186.8 ± 4.1
31.4 ± 2.8
69.6 ± 0.2
120.8 ± 1.9
101.3 ± 1.7
173.8 ± 3.9
104.3 ± 2.4
141.9 ± 3.1
95.5 ± 3.2
194.1 ± 4.8
87.2 ± 4.3
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
11
12
13
14
15
16
17
18
19
20
* Viral titer: 1.0 x 107 TCID100/mL in 72 h; NA = Not active.
3. Conclusion
In this work was synthesized a new series of glycosylated and non-glycosylated
acylhydrazones. The chemical structures of the new compounds were elucidated by
combined spectrometry and spectroscopy NMR, HRMS and FTIR methods and they
were evaluated against bacterial (Escherichia coli, Enterococcus faecalis, Micrococcus
luteus, Pseudomonas aeruginosa, Salmonella typhimurium and Staphylococcus aureus),
fungi (Candida albicans, C. tropicalis, C. krusei, C. glabrata and C. parapsilosis) and
Zika virus strains. The acylhydrazones 12, 13, 14, 16 and 19 showed a modest

ACCEPTED MANUSCRIPT
antifungal activity against the strains evaluated in the range of 907.1-173.8 ꢀM and the
presence of a saccharide unit in the compounds contributed to the increase of the
antifungal potential against Candida parapsilosis, since a respective non glycosylated
acylhydrazone (20) was inactive, as already described by our research group in another
study [11]. The compounds showed not a significant antibacterial and antiviral activity.
4. Material and methods
4.1 Physical measurements
Melting point of the compounds was determined on Microquímica MOAs 301 apparatus
and was uncorrected. FTIR spectroscopy was performed by Spectrum One, Perkin-
1
13
Elmer. H-NMR and C-NMR spectra were obtained on Bruker AC-300 spectrometer
1
13
(300 MHz for H-NMR and 75 MHz for C spectra) in deuterated 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
1
in Hertz (Hz). The following abbreviations were used for the H multiplicities: singlet
(s), doublet (d), multiplet (m) and broad signal (br s). The specific optical rotation [α]D
were measured on Perkin Elmer 341 polarimeter, at 22°C. High resolution mass spectra
were acquired using a LC/MS-ESI-QTOF (Bruker) mass spectrometer and the samples
were solubilized in MeOH, following direct injection. Reaction courses and product
mixtures were monitored by thin-layer chromatography (TLC) on commercial silica gel
60 plates.
4.2 Synthesis of vanillin glycosides 5 and 6
The β-glucoside (5) and β-galactoside (6) derived from vanillin were synthesized from
D
-glucose and D-galactose. Initially, 2.5 g (0.0126 mol) of D-glucose or D-galactose
were solubilized in 12.6 mL (0.1234 mol) of acetic anhydride and to this solution were
added 20 drops of concentrated sulfuric acid, under manual shaking. This mixture was
subjected to ultrasonic bath for 25 minutes, when noticed the complete of the reaction
by TLC. The mixture was poured into a beaker containing iced water and the solid
obtained was collected by vacuum filtration and purified by recrystallization from ethyl

ACCEPTED MANUSCRIPT
alcohol, affording the peracetylated glucose and galactose in 55% and 53% yield,
respectively.
Thereafter, the peracetylated derivatives obtained were converted to the corresponding
acetobromo glycosides: in a flash was prepared a solution containing 10 mL of acetic
anhydride and 2.5 mL of hydrobromic acid (added dropwise, under cooling, using an
addition funnel) and this solution was added to another flask containing 1 (5.04 mmol) g
of peracetylated glucose or galactose solubilized in 10 mL do dichloromethane, also
under cooling. The reaction was monitored by TLC and after 4 hours observed its end.
The mixture was poured into a beaker containing iced water, extracted with
dichloromethane (3 x 30 mL) and the organic layer obtained was washed with saturated
aqueous NaHCO3 (3 x 30 mL) and water until pH 7. The organic layer was dried by
anhydrous Na2SO4 e concentrated to afford the acetobromo glycosides (glucoside and
galactoside) as colorless oils in 85 % yield.
Finally, in a flask containing 1.5 g (9.85 mmol) of 4-hydroxy-3-methoxybenzaldehyde
(vanillin) solubilized in 5 mL of an aqueous solution of LiOH (0.21g; 8.87 mmol) was
added the corresponding acetobromo glycoside (1.34 g; 3.28 mmol) solubilized in
acetone (20 mL) and the evolution of the reaction was monitored by TLC. After 6 hours
stirring at room temperature, the acetone was removed and it was added 10 mL of water
to the mixture, which was extracted with dichloromethane (3 x 30 mL). The organic
layer obtained was washed with aqueous NaOH 10% (3 x 30 mL) and water until pH 7,
dried by anhydrous Na2SO4 e concentrated to afford the peracetylated glucoside (5) and
galactoside (6) as white solids in 55% and 53% yield, respectively, after purified by
recrystallization from isopropyl alcohol.
4.3 Synthesis of hydrazides 9 and 10
Initially, 2 g of 4-aminobenzoic acid were solubilized in 20 mL of ethyl alcohol and 10
drops of hydrochloric acid were added to this solution. The mixture was refluxed during
3 hours and the ethylic ester obtained was isolated by filtration in 82 % yield after the
mixture is poured into iced water.
Thereafter, 1 g (6.03 mmol) of the amino ester obtained was solubilized in 5 mL of
dichloromethane and to this solution were added 0.42 mL of acetyl chloride (6.03
mmol) or 0.70 mL of benzoyl chloride (6.03 mmol). The mixture was stirred at room
temperature and the reaction was monitored by TLC until the total consume of the

ACCEPTED MANUSCRIPT
amino ester. The solvent was eliminated and the acetamide (7) or benzamide (8) were
obtained in 65 % and 71 % yields, respectively.
Finally, to a solution of 6 equivalents of hydrazine hydrate in 10 mL of ethyl alcohol at
5°C was added slowly dropwise 1 g of acetamide 7 (4.82 mmol) or 1 g of benzamide 8
(3.71 mmol) solubilized in ethyl alcohol. The solid product obtained after addition was
filtered affording the hydrazides 9 and 10 in 74 % and 76% yield, respectively.
4.4 General procedure for synthesis of acylhydrazones 11-14 and 19-20
To a solution of the corresponding aldehyde (glucoside 5, galactoside 6 or vanillin; 1
equiv.) in ethyl alcohol was added the hydrazides 9 or 10 (1 equiv.) at room
temperature. The mixture was colled to 0oC when was added a catalyst amount of
hydrochloride acid to the reaction and this mixture was stirred during 24 hours at room
temperature. The reaction was monitored TLC and the solid product obtained was
collected by filtration and recrystallized by isopropyl alcohol.
N-(4-{[(2E)-2-(4-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)acetamide (11): This compound was
prepared by 1.035 mmol of 5 and 1.035 mmol of 9, according to the general procedure.
The product was obtained in 56% yield as white crystals after purified by
recrystallization from isopropyl alcohol; mp 135-136oC; [α]D -25º (c 0.004, DMSO); IR
1
(ῡ/cm–1): 3259, 3060, 2946, 1739, 1689, 1649, 1601 1537 1510. H-NMR (300 MHz,
DMSO-d6) δ: 11.73 (1H, s, NH), 10.25 (1H, s, NH), 8.38 (1H, s, -CH=N-), 7.86 (2H, d,
J3 = 8.7 Hz, Ar-H), 7.70 (2H, d, J3 = 8.7 Hz, Ar-H), 7.38 (1H, s, Ar-H), 7.24-7.15 (2H,
m, Ar-H), 5.49-5.37 (2H, m, sugar), 5.10-4.97 (2H, m , sugar), 4.23-4.20 (2H, m,
sugar), 4.09-4.05 (1H, m, sugar), 3.81 (3H, s, OCH3), 2.08-1.97 (15H, s, OCOCH3,
NHCOCH3). 13C-NMR (75 MHz, DMSO-d6) δ: 170.0-168.8 (5C, OCOCH3,
NHCOCH3), 162.5 (1C, =NNHCO-), 149.9 (1C, Ar), 147.2 (1C, Ar), 147.0 (1C, -
CH=N-), 142.4 (1C, Ar), 130.2 (1C, Ar), 128.5 (2C, Ar), 127.5 (1C, Ar), 121.0 (1C,
Ar), 118.2 (2C, Ar), 115.5 (1C, Ar), 109.6 (1C, Ar), 98.3 (1C, sugar), 71.8 (1C, sugar),
70.8 (1C, sugar), 70.6 (1C, sugar), 68.0 (1C, sugar), 61.6 (1C, sugar), 55.9 (1C, OCH3),
24.1 (1C, NHCOCH3), 20.5-20.3 (4C, OCOCH3). HRMS-ESI: m/z calcd. for
C31H34N3O13 (M-H)-: 656.2097; found: 656.2088.

ACCEPTED MANUSCRIPT
N-(4-{[(2E)-2-(4-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)benzamide (12): This compound was
prepared by 0.39 mmol of 5 and 0.39 mmol of 10, according to the general procedure.
The product was obtained in 76% yield as white crystals after purified by
recrystallization from isopropyl alcohol; mp 146-147oC; [α]D -22º (c 0.004, DMSO); IR
(ῡ/cm–1): 3350, 3280, 2963, 1744, 1650, 1599, 1505. 1H-NMR (300 MHz, DMSO-d6) δ:
11.76 (1H, s, NH), 10.52 (1H, s, NH), 8.42 (1H, s, -CH=N-), 8.00-7.94 (6H, m, Ar-H),
7.62-7.55 (3H, m, Ar-H), 7.40 (1H, s, Ar-H), 7.23-7.10 (2H, m, Ar-H), 5.49-5.37 (2H,
m, sugar), 5.11-5.01 (2H, m , sugar), 4.24-4.21 (2H, m, sugar), 4.10-4.07 (1H, m,
sugar), 3.82 (3H, s, OCH3), 2.03-1.98 (12H, s, OCOCH3). 13C-NMR (75 MHz, DMSO-
d6) δ: 170.3-169.4 (4C, OCOCH3), 166.2 (1C, NHCOPh), 162.9 (1C, =NNHCO-), 150.3
(1C, Ar), 147.6 (1C, Ar), 147.5 (1C, -CH=N-), 142.7 (1C, Ar), 135.0 (1C, Ar), 132.3
(1C, Ar), 130.6 (1C, Ar), 128.8 (2C, Ar), 128.5 (3C, Ar), 128.1 (2C, Ar), 121.3 (1C,
Ar), 119.9 (2C, Ar), 118.0 (1C, Ar), 110.2 (1C, Ar), 98.7 (1C, sugar), 72.2 (1C, sugar),
71.2 (1C, sugar), 71.0 (1C, sugar), 68.4 (1C, sugar), 62.0 (1C, sugar), 56.3 (1C, OCH3),
20.8-20.7 (4C, OCOCH3). HRMS-ESI: m/z calcd. for C36H36N3O13 (M-H)-: 718.2254;
found: 718.2242.
N-(4-{[(2E)-2-(4-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)acetamide (13): This compound was
prepared by 1.035 mmol of 6 and 1.035 mmol of 9, according to the general procedure.
The product was obtained in 79% yield as white crystals after purified by
recrystallization from ethyl alcohol; mp 71-73oC; [α]D -8º (c 0.005, DMSO); IR (ῡ/cm–
1): 3353, 2990, 1748, 1715, 1686, 1592, 1512. 1H-NMR (300 MHz, DMSO-d6) δ: 11.74
(1H, s, NH), 10.25 (1H, s, NH), 8.38 (1H, s, -CH=N-), 7.86 (2H, d, J3 = 8.7 Hz, Ar-H),
7.70 (2H, d, J3 = 8.7 Hz, Ar-H), 7.39 (1H, s, Ar-H), 7.22-7.15 (2H, m, Ar-H), 5.36-5.32
(2H, m, sugar), 5.26-5.23 (2H, m , sugar), 4.39-4.37 (1H, m, sugar), 4.12-4.09 (2H, m,
13
sugar), 3.81 (3H, s, OCH3), 2.16-1.95 (15H, s, OCOCH3, NHCOCH3). C-NMR (75
MHz, DMSO-d6) δ: 170.0-168.8 (5C, OCOCH3, NHCOCH3), 162.5 (1C, =NNHCO-),
150.0 (1C, Ar), 147.3 (1C, Ar), 147.0 (1C, -CH=N-), 142.4 (1C, Ar), 130.2 (1C, Ar),
127.5 (2C, Ar), 121.0 (1C, Ar), 117.7 (2C, Ar), 109.7 (1C, Ar), 98.9 (1C, sugar), 70.4
(1C, sugar), 70.1 (1C, sugar), 68.3 (1C, sugar), 67.2 (1C, sugar), 61.3 (1C, sugar), 55.9
(1C, OCH3), 24.1 (1C, NHCOCH3), 20.5 (4C, OCOCH3). HRMS-ESI: m/z calcd. for
C31H34N3O13 (M-H)-: 656.2097; found: 656.2077.

ACCEPTED MANUSCRIPT
N-(4-{[(2E)-2-(4-(2,3,4,6-tetra-O-acetyl-β-
D
-galactopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)benzamide (14): This compound was
prepared by 0.39 mmol of 6 and 0.39 mmol of 10, according to the general procedure.
The product was obtained in 84% yield as white crystals after purified by
recrystallization from ethyl alcohol; mp 137-138oC; [α]D -23º (c 0.002, DMSO); IR
1
(ῡ/cm–1): 3353, 3291, 2995, 1742, 1658, 1601, 1542, 1513. H-NMR (300 MHz,
DMSO-d6) δ: 11.75 (1H, s, NH), 10.51 (1H, s, NH), 8.42 (1H, s, -CH=N-), 8.00-7.94
(6H, m, Ar-H), 7.62-7.53 (3H, m, Ar-H), 7.40 (1H, s, Ar-H), 7.24-7.16 (2H, m, Ar-H),
5.39-5.33 (2H, m, sugar), 5.27-5.24 (1H, m , sugar), 4.40-4.38 (1H, m, sugar), 4.13-4.10
(3H, m, sugar), 3.83 (3H, s, OCH3), 2.16-1.95 (12H, s, OCOCH3). 13C-NMR (75 MHz,
DMSO-d6) δ: 170.3-169.5 (4C, OCOCH3), 166.2 (1C, NHCOPh), 162.9 (1C,
=NNHCO-), 150.4 (1C, Ar), 147.7 (1C, Ar), 147.5 (1C, -CH=N-), 142.7 (1C, Ar), 135.0
(1C, Ar), 132.2 (1C, Ar), 130.6 (1C, Ar), 128.8 (3C, Ar), 128.5 (1C, Ar), 128.1 (2C,
Ar), 121.3 (1C, Ar), 119.9 (2C, Ar), 118.1 (2C, Ar), 110.2 (1C, Ar), 99.3 (1C, sugar),
70.8 (1C, sugar), 71.4 (1C, sugar), 68.7 (1C, sugar), 67.6 (1C, sugar), 61.6 (1C, sugar),
56.3 (1C, OCH3), 20.8 (4C, OCOCH3). HRMS-ESI: m/z calcd. for C36H36N3O13 (M-H)-
: 718.2254; found: 718.2251.
N-(4-{[(2E)-2-(4-hydroxy-3-metoxybenzylidene)hydrazino]carbonyl}phenyl)acetamide
(19): This compound was prepared by 0.517 mmol of vanillin and 0.517 mmol of 9,
according to the general procedure. The product was obtained in 61% yield as white
crystals after purified by recrystallization from ethyl alcohol; mp 2792-273oC; IR (ῡ/cm–
1): 3304, 3219, 2931, 1666, 1634, 1609, 1511. 1H-NMR (300 MHz, DMSO-d6) δ: 11.61
(1H, s, NH), 10.24 (1H, s, NH), 9.57 (1H, s, OH), 8.32 (1H, s, -CH=N-), 7.86 (2H, d, J3
= 8.7 Hz, Ar-H), 7.70 (2H, d, J3 = 8.7 Hz, Ar-H), 7.30 (1H, s, Ar-H), 7.08-7.05 (1H, m,
Ar-H), 6.83 (1H, d, J3 = 8.1 Hz, Ar-H), 3.82 (3H, s, OCH3), 2.08 (3H, s, NHCOCH3).
13C-NMR (75 MHz, DMSO-d6) δ: 168.8 (1C, NHCOCH3), 162.4 (1C, =NNHCO-),
148.9 (1C, Ar), 148.0 (1C, Ar), 147.9 (1C, -CH=N-), 142.3 (1C, Ar), 128.5 (2C, Ar),
127.7 (1C, Ar), 125.8 (1C, Ar), 122.1 (1C, Ar), 118.2 (2C, Ar), 115.4 (1C, Ar), 108.8
(1C, Ar), 55.5 (1C, OCH3), 24.1 (1C, NHCOCH3). HRMS-ESI: m/z calcd. for
C17H16N3O4 (M-H)-: 326.1146; found: 326.1129.

ACCEPTED MANUSCRIPT
N-(4-{[(2E)-2-(4-hydroxy-3-metoxybenzylidene)hydrazino]carbonyl}phenyl)benzamide
(20): This compound was prepared by 0.39 mmol of vanillin and 0.39 mmol of 10,
according to the general procedure. The product was obtained in 55% yield as white
crystals after purified by recrystallization from ethyl alcohol; mp 202-203oC; IR (ῡ/cm–
1): 3306, 3245, 2837, 1655, 1644, 1602, 1506. 1H-NMR (300 MHz, DMSO-d6) δ: 11.69
(1H, s, NH), 10.53 (1H, s, NH), 9.54 (1H, br s, OH), 8.33 (1H, s, -CH=N-), 7.94-7.92
(6H, m, Ar-H), 7.63-7.50 (3H, m, Ar-H), 7.39-7.33 (1H, m, Ar-H), 7.09 (1H, d, J3 = 6.0
13
Hz, Ar-H), 6.84 (1H, d, J3 = 6.0 Hz, Ar-H), 3.81 (3H, s, OCH3). C-NMR (75 MHz,
DMSO-d6) δ: 166.2 (1C, NHCOPh), 162.7 (1C, =NNHCO-), 149.3 (1C, Ar), 148.4 (2C,
Ar), 142.6 (1C, -CH=N-), 135.0 (1C, Ar), 132.2 (1C, Ar), 128.8 (3C, Ar), 128.7 (2C,
Ar), 128.1 (2C, Ar), 126.2 (1C, Ar), 122.5 (1C, Ar), 119.9 (2C, Ar), 115.8 (1C, Ar),
100.3 (1C, Ar), 55.9 (1C, OCH3). HRMS-ESI: m/z calcd. for C22H18N3O4 (M-H)-:
388.1303; found: 388.1292.
4.5 General procedure for synthesis of acylhydrazones 15-18
The peracetylated acylhydrazones 11-14 (0.5 mmol) were solubilized in a solution of
potassium hydroxide in methyl alcohol (15 mL; 0.5 mol.L-1) and this mixture was
stirred during 40 minutes at 0oC, when was noticed the completed the reaction by TLC.
It was added IRA-120 resin to the mixture until the neutralization of pH, when the resin
was removed by filtration and the filtrate was concentrated under reduced pressure to
afford the deacetylated compounds.
N-(4-{[(2E)-2-(4-(β-D-glucopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)acetamide (15): This compound was
prepared by 0.243 mmol of 11, according to the general procedure. The product was
obtained in 100% yield as yellow crystals; mp 145-147oC; [α]D -15º (c 0.004, DMSO);
1
IR (ῡ/cm–1): 3501, 3296, 3218, 1666, 1637, 1610, 1551, 1508. H-NMR (300 MHz,
DMSO-d6) δ: 11.68 (1H, s, NH), 10.24 (1H, s, NH), 8.37 (1H, s, -CH=N-), 7.86 (2H, d,
J3 = 7.8 Hz, Ar-H), 7.70 (2H, d, J3 = 8.1 Hz, Ar-H), 7.37 (1H, s, Ar-H), 7.15 (2H, br s,
Ar-H), 4.99 (1H, br s, sugar), 3.69-3.16 (9H, m, sugar and OCH3), 2.07 (3H, s,
NHCOCH3). 13C-NMR (75 MHz, DMSO-d6) δ: 168.8 (1C, NHCOCH3), 162.5 (1C,
=NNHCO-), 149.2 (1C, Ar), 148.2 (1C, Ar), 147.4 (1C, -CH=N-), 142.3 (1C, Ar), 128.5
(2C, Ar), 128.1 (1C, Ar), 127.6 (1C, Ar), 121.6 (1C, Ar), 118.2 (2C, Ar), 114.8 (1C,

ACCEPTED MANUSCRIPT
Ar), 108.7 (1C, Ar), 99.5 (1C, sugar), 77.1 (1C, sugar), 76.8 (1C, sugar), 73.1 (1C,
sugar), 69.5 (1C, sugar), 60.6 (1C, sugar), 55.5 (1C, OCH3), 24.1 (1C, NHCOCH3).
HRMS-ESI: m/z calcd. for C23H26N3O9 (M-H)-: 488.1675; found: 488.1667.
N-(4-{[(2E)-2-(4-(glucopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)benzamide (16): This compound was
prepared by 0.139 mmol of 12 according to the general procedure. The product was
obtained in 51% yield as yellow crystals; mp 150-151oC; [α]D -23º (c 0.004, DMSO); IR
1
(ῡ/cm–1): 3286, 2921, 1639, 1602, 1505. H-NMR (300 MHz, DMSO-d6) δ: 11.73 (1H,
s, NH), 10.52 (1H, s, NH), 8.40 (1H, s, -CH=N-), 7.99-7.93 (6H, m, Ar-H), 7.61-7.52
(3H, m, Ar-H), 7.39 (1H, s, Ar-H), 7.16 (2H, br s, Ar-H), 5.30 (1H, br s, sugar), 5.10-
5.01 (3H, m , sugar), 4.57 (1H, br s, sugar), 3.83 (4H, br s, sugar and OCH3), 3.69-3.66
13
(1H, mm sugar). C-NMR (75 MHz, DMSO-d6) δ: 165.9 (1C, NHCOPh), 162.4 (1C,
=NNHCO-), 149.2 (1C, Ar), 148.3 (1C, Ar), 147.5 (1C, -CH=N-), 142.2 (1C, Ar), 134.6
(1C, Ar), 131.8 (1C, Ar), 128.4 (3C, Ar), 128.2 (3C, Ar), 127.7 (2C, Ar), 121.6 (1C,
Ar), 119.9 (2C, Ar), 114.8 (1C, Ar), 108.9 (1C, Ar), 99.6 (1C, sugar), 77.0 (1C, sugar),
76.8 (1C, sugar), 73.1 (1C, sugar), 69.6 (1C, sugar), 60.6 (1C, sugar), 55.6 (1C, OCH3).
HRMS-ESI: m/z calcd. for C28H28N3O9 (M-H)-: 550.1831; found: 550.1824.
N-(4-{[(2E)-2-(4-(β-D-galactopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)acetamide (17): This compound was
prepared by 0.228 mmol of 13, according to the general procedure. The product was
obtained in 64% yield as yellow crystals; mp 178-180oC; [α]D -25.2º (c 0.004, DMSO);
1
IR (ῡ/cm–1): 3451, 3291, 3203, 2910, 1665, 1633, 1609, 1508. H-NMR (300 MHz,
DMSO-d6) δ: 11.68 (1H, s, NH), 10.24 (1H, s, NH), 8.36 (1H, s, -CH=N-), 7.85-7.71
(4H, m, Ar-H), 7.36 (1H, s, Ar-H), 7.14 (2H, br s, Ar-H), 4.97 (1H, br s, sugar), 3.82-
13
3.43 (9H, m, sugar and OCH3), 2.07 (3H, s, NHCOCH3). C-NMR (75 MHz, DMSO-
d6) δ: 168.8 (1C, NHCOCH3), 162.5 (1C, =NNHCO-), 149.2 (1C, Ar), 148.3 (1C, Ar),
147.4 (1C, -CH=N-), 142.3 (1C, Ar), 128.5 (2C, Ar), 128.1 (1C, Ar), 127.6 (1C, Ar),
121.5 (1C, Ar), 118.2 (2C, Ar), 114.8 (1C, Ar), 108.8 (1C, Ar), 100.2 (1C, sugar), 75.5
(1C, sugar), 73.5 (1C, sugar), 70.1 (1C, sugar), 68.1 (1C, sugar), 60.3 (1C, sugar), 55.5
(1C, OCH3), 24.1 (1C, NHCOCH3). HRMS-ESI: m/z calcd. for C23H26N3O9 (M-H)-:
488.1675; found: 488.1674.

ACCEPTED MANUSCRIPT
N-(4-{[(2E)-2-(4-(D-galactopyranoside)-3-
metoxybenzylidene)hydrazino]carbonyl}phenyl)benzamide (18): This compound was
prepared by 0.139 mmol of 14 according to the general procedure. The product was
obtained in 100% yield as yellow crystals; mp 184-186oC; [α]D -54.2º (c 0.004, DMSO);
1
IR (ῡ/cm–1): 3292, 3231, 2924, 1639, 1602, 1505. H-NMR (300 MHz, DMSO-d6) δ:
8.40 (1H, s, -CH=N-), 7.98-7.93 (6H, m, Ar-H), 7.62-7.55 (3H, m, Ar-H), 7.39 (1H, s,
Ar-H), 7.15 (2H, br s, Ar-H), 4.95 (1H, d, J3 = 6.0 Hz, sugar), 3.83 (3H, s, OCH3), 3.72-
3.60 (6H, mm sugar). 13C-NMR (75 MHz, DMSO-d6) δ: 166.2 (1C, NHCOCH3), 163.2
(1C, =NNHCO-), 149.6 (1C, Ar), 148.6 (1C, Ar), 147.8 (1C, -CH=N-), 142.5 (1C, Ar),
135.1 (1C, Ar), 132.2 (1C, Ar), 129.2 (1C, Ar), 128.2 (3C, Ar), 128.1 (2C, Ar), 121.8
(1C, Ar), 119.9 (2C, Ar), 115.2 (2C, Ar), 109.4 (1C, Ar), 100.7 (1C, sugar), 75.9 (1C,
sugar), 73.9 (1C, sugar), 70.6 (1C, sugar), 68.4 (1C, sugar), 60.7 (1C, sugar), 56.0 (1C,
OCH3). HRMS-ESI: m/z calcd. for C28H28N3O9 (M-H)-: 550.1831; found: 550.1825.
4.6 Antifungal and antibacterial activities evaluation
The synthesized compounds were evaluated in vitro for their antibacterial and
antifungal activities and the inhibitory concentrations of 50% (IC50) and 90% (IC90) of
microbial growth were determined in µmol/mL and compared among the
microorganisms. The tests were all done in duplicates. The antifungal activity were
performed according to microdilution methodology in RPMI 1640 broth supplemented
with 2% glucose as document E.DEF 7.3.1 [20]. The determination of antibacterial
activity were performed according to microdilution methodology in Mueller Hinton
broth adjusted with cations as described by document ISO 20776-1: 2006 [20,21]. The
stock solutions of all the compounds were prepared in DMSO 1% at final concentration
and tested at concentrations (µg/mL) from 500 to 0.98 µg/mL. The standard drug
fluconazole were applied as control of fungistatic action at concentrations from 64 to
0.031 g/mL and the standard drug chloramphenicol as a control of bacteriostatic action
at concentrations from 125 to 0.06 g/mL. The negative control employed for antifungal
evaluation was composed only by the culture medium RPMI supplemented with glucose
2% and the solvent DMSO. For antibacterial activity, the negative control was
composed only by the culture medium Mueller Hinton. The microplates were incubated
at 35◦C for 24 h for bacteria and 37◦C and for 24 h for fungi. Results were visualized
and analyzed at 530 nm in an Anthos Zenyth 200rt Microplate Reader.

ACCEPTED MANUSCRIPT
4.7 Cytotoxicity and antiviral assay
Kidney cell Vero (ATCC® CCL-81™) and normal human lung fibroblast cell MRC-5
(ATCC® CCL-117™) were cultured in Dulbecco’s modified Eagle’s medium (DMEM,
Cultilab, Campinas, SP, Brazil) at 37 °C, in 5% CO2 atmosphere, supplemented with
5% fetal bovine serum, 100 ꢀg/mL streptomycin, 100 U/mL penicillin and 2.5 ꢀg/mL
amphotericin B [24]. Vero and MRC-5 cells were exposed to different concentrations
(200 to 1.56 µg/mL) of compounds for 72 h. After incubation, cell viability was
assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Merck) assay at a concentration of 2 mg/mL in phosphate buffered saline (PBS) [24,25].
ZIKV was kindly donated by Dr. E. Kroon (UFMG, Belo Horizonte, Brazil). The virus
was cultured in C6/36 cells and titrated by the plaque assay in Vero cells [25]. The titer
of virus stock was calculated in plaque-forming units (PFU) per milliliter (3.07 × 109
PFU/ml). The antiviral activity (inhibitory concentrations of 50% of viral growth - IC50)
was evaluated by the MTT assay [26]. Experiments were carried out with eight different
concentrations (100 to 0.78 µg/mL). The cytotoxic concentration against 50% of MRC-
5 normal cells growth (CC50) for each compounds were calculated from concentration-
effect-curves after no linear regression analysis [24]. Statistical calculations were
carried out with the GraphPad prism 5.0 software package (Statistical). Results are
expressed as the mean ± S.E.M. of three independent experiments. Student’s t-test was
used for statistical analyses; P values > 0.05 were considered to be significant.
Conflicts of interest
The authors have declared no conflicts of interest.
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Synthesis of a new set of glycosylated acylhydrazones;
Characterization by combined HRMS, NMR and FTIR spectroscopy;
Antimicrobial activity of the synthesized compounds.