Synthesis and antimicrobial activity of glycosylated 2-Aryl?5?amidinobenzimidazoles

Article abstract of DOI:10.21577/0103-5053.20170227

A series of new glycosylated 2-aryl-5-amidinobenzimidazoles derived from four different carbohydrates (D-glucose, D-galactose, N-acetyl-D-glucosamine and lactose) were synthesized by the condensation of the appropriate 4-formyl-3-methoxyphenyl glycoside with 4-amidino- or 4-N-isopropylamidino-ortho-phenylenediamine hydrochloride. All the compounds were properly characterized by high resolution mass spectrometry, uni- and bidimensional¹H and¹³C nuclear magnetic resonance and then were evaluated for their antibacterial and antifungal potential. Considering the antifungal potential of them, two derivatives were active against Candida parapsilosis at 96.4 μmol L^-1 and another was active against this same strain at 83.5 μmol L^-1. In addition, one benzamidine showed activity against Candida glabrata at 97 μmol L^-1. Considering the antibacterial potential of these compounds, six of them showed better activity against three different stains: three of them with IC₅₀ of 96.4, 97 and 83.5 μmol L^-1 against Gram-positive Micrococcus luteus, the other two with IC₅₀ 96.5 and 96.4 μmol L^-1 against Gram-positive Enterococcus faecalis and one against Gram-negative Escherichia coli at 90.5 μmol L^-1. These findings suggest this structural pattern can be employed for design of more potent agents for discovery of new antimicrobial drug candidates.

Full text of DOI:10.21577/0103-5053.20170227

http://dx.doi.org/10.21577/0103-5053.20170227
J. Braz. Chem. Soc., Vol. 29, No. 6, 1304-1317, 2018
Printed in Brazil - ©2018 Sociedade Brasileira de Química
Article
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl‑5‑amidinobenzimidazoles
Thiago B. de Souza,a Josidel C. Oliver,b Ana Paula B. Gomes,c Cícero Flávio S. Aragão,c
Leandro S. Ferreira,c Fernando Henrique A. Nogueira,c Amanda Latércia T. Diasb and
Ricardo J. Alves*,a
aDepartamento de Produtos Farmacêuticos, Faculdade de Farmácia,
Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil
bDepartamento de Microbiologia e Imunologia, Instituto de Ciências Biomédicas,
Universidade Federal de Alfenas, 37130-000 Alfenas-MG, Brazil
cDepartamento de Farmácia, Centro de Ciências da Saúde,
Universidade Federal do Rio Grande do Norte, 59012-570 Natal-RN, Brazil
A series of new glycosylated 2-aryl-5-amidinobenzimidazoles derived from four different
carbohydrates (D-glucose, D-galactose, N-acetyl-D-glucosamine and lactose) were synthesized
by the condensation of the appropriate 4-formyl-3-methoxyphenyl glycoside with 4-amidino- or
4-N-isopropylamidino-ortho-phenylenediamine hydrochloride. All the compounds were properly
characterized by high resolution mass spectrometry, uni- and bidimensional 1H and 13C nuclear
magnetic resonance and then were evaluated for their antibacterial and antifungal potential.
Considering the antifungal potential of them, two derivatives were active against Candida
parapsilosis at 96.4 µmol L-1 and another was active against this same strain at 83.5 µmol L-1. In
addition, one benzamidine showed activity against Candida glabrata at 97 µmol L-1. Considering the
antibacterial potential of these compounds, six of them showed better activity against three different
stains: three of them with IC50 of 96.4, 97 and 83.5 µmol L-1 against Gram-positive Micrococcus
luteus, the other two with IC50 96.5 and 96.4 µmol L-1 against Gram-positive Enterococcus
faecalis and one against Gram-negative Escherichia coli at 90.5 µmol L-1. These findings suggest
this structural pattern can be employed for design of more potent agents for discovery of new
antimicrobial drug candidates.
Keywords: 2-aryl-5-amidinobenzimidazoles, glycosides, antibacterial, antifungal
Introduction
Among the various known potentially antimicrobial
heterocyclic compounds, benzimidazoles occupy a
prominent position and there are several reports of
synthesis of benzimidazoles with antibacterial5 and
antifungal6 potential. The benzimidazole nucleus is
considered an important pharmacophore since it can act
as a bioisostere group of different molecular constituents
of the microorganisms.7
On the other hand, amidine derivatives have also
occupied an important position among the biologically
active compounds, since this strongly basic group is
easily protonated in physiological conditions, allowing
important interactions with several targets of different
microorganisms.8 Göker et al.9 reported the synthesis
and antimicrobial activity of a series of benzamidines
derived from flavones and benzopyranones. Some
benzamidines were active against methicillin-resistant
Microbial infections represent a serious public health
problem and are associated with a large number of deaths
worldwide. The microbial resistance has increased these
numbers and made the available antimicrobial therapy
poorly effective.1 Although a small number of Candida
species are pathogenic to humans, candidiasis is considered
the main cause of hospital infections, mainly due to use of
immunosuppressive drugs or modern medical procedures
that leave the patient more susceptible to contamination by
these microorganisms.2,3 In addition, few antibacterial drugs
have been discovered in the last 50 years, making infections
caused by Gram-positive or Gram-negative bacteria also a
serious health threat.4
*e-mail: ricardodylan@farmacia.ufmg.br

Vol. 29, No. 6, 2018
de Souza et al.
1305
Staphyloccocus aureus and Staphylococcus epidermidis,
Escherichia coli and Enterococcus faecalis in the range of
1.56-50 µg mL-1. Other benzamidines synthesized in this
study showed antifungal potential against Candida albicans
and Candida krusei in the range of 3.12-50 µg mL-1.
In this context, we report here the synthesis and
antimicrobial potential of new glycosylated 2-aryl-
5-amidinobenzimidazole derivatives. Four different
carbohydrates (D-glucose, D-galactose, N-acetyl-
D-glucosamine and lactose) were used, aimed to investigate
the influence of the carbohydrate moiety on the biological
activity of the compounds. There are several reports of
biologically active glycosylated compounds, especially
against microorganisms, and the presence of the saccharide
unit has proved essential for their activity.4,10 We envisaged
the synthesis of the target molecules according to the
retrosynthetic shown in Scheme 1. The aryl glycosides
and the substituted ortho-phenylenediamines need for the
synthesis can be easily obtained by procedures described
in the literature.9
benzamidines in combination with four glycosylated
aldehydes, followed by deacetylation of peracetylated
derivatives initially obtained.
Melting points of synthesized compounds were
determined on Microquímica MOAs 301 apparatus and
are uncorrected. Infrared spectroscopy was performed
1
on Spectrum One, PerkinElmer spectrophotometer. H
and 13C nuclear magnetic resonance (NMR) spectra were
obtained on BrukerAvance DRX-200 (200 MHz FT NMR)
and DRX-400 (400 MHz FT NMR) spectrometers in
deuterated chloroform or dimethyl sulfoxide (DMSO).
Chemical shifts (d) 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 (q), multiplet (m) and broad signal (br s).
The specific optical rotations [α]D were measured on
PerkinElmer 341 polarimeter, at 20 °C. High resolution
mass spectra were acquired using a liquid chromatography
mass spectrometry-ion trap-time of flight (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 silica gel-G TLC
plates (Merck) and column grade silica gel (0.063-0.200 mm
mesh size) was employed for chromatography.
Experimental
Chemistry
The proposed 2-aryl-5-amidinobenzimidazoles
were synthesized from the reaction of two 3,4-diamino-
Scheme 1. Retrosynthetic analysis for the target compounds.

1306
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles
J. Braz. Chem. Soc.
Synthesis of acetamidobenzonitrile (1)
for 30 min. After 4 days stirring at room temperature, the
suspension was filtrated and the solid was washed with
120 mL of ethyl ether. The yellow solid obtained was
dissolved in dry methanol and to this solution was added
1 mL of isopropylamine; the mixture was heated under
reflux for 3 h. The solvent was removed and the solid
resulting was washed with 150 mL of ethyl ether and
120 mL of ethyl acetate.
4-Aminobenzonitrile (8.46 mmol) was added, slowly,
to 8 mL of acetic anhydride and the temperature was
maintained between 35 and 40 °C. After the complete
addition, the suspension was poured into a bath of ice/water
and the yellow solid obtained was collected by filtration and
washed with water, affording the desired product.
Synthesis of 4-amino-3-nitrobenzonitrile (2)
4-Amino-3-nitro-N-isopropylbenzamidine hydrochloride (5)
This product was obtained as a yellow solid (63% yield);
mp 259.5-261.6 °C; IR (ATR) ν / cm-1 3456, 3161 (NH2),
Potassium nitrate (15.6 mmol) was dissolved in 8 mL of
concentrated H2SO4 and the mixture was cooled to below
0 °C. To this solution, it was added, slowly, 7.8 mmol of 1
and the temperature was maintained at 0 °C for 3.5 h. The
mixture was poured into a bath of ice/water and the yellow
solid obtained was collected by filtration and washed with a
small amount of water. The obtained product was suspended
in 30 mL of 2 mol L-1 H2SO4 and heated under reflux for
3 h. The suspension was cooled to room temperature and
the yellow solid was collected by filtration and washed with
small amount of cold water, to afford the desired product
in 74% yield.
1
3047, 2942 (NH.HCl amidine), 1685 (C=N); H NMR
(200 MHz, DMSO-d6) d 8.44 (br s, 1H, H arom.), 8.08
(br s, 2H, NH2), 7.76 (br s, 1H, H arom.), 7.20 (br s, 1H,
H arom.), 4.07 (m, 1H, CH), 1.23 (d, J 5.6 Hz, 6H, CH3);
13C NMR (50 MHz, DMSO-d6) d 159.5, 148.6, 134.2,
129.3, 127.2, 119.2, 115.5, 44.9, 21.5.
General procedure for the synthesis of diaminobenzamidines
(4 and 6)
A solution of 3 (4.6 mmol) or 5 (3.81 mmol) in
60 mL of ethanol and 10% Pd-C was hydrogenated until
the required quantity of H2 was taken up. The Pd-C was
removed by filtration and the ethanol was concentrated
affording the desired products, which were used without
previous purification.
Synthesis of 3-nitrobenzamidine (3)
A sample of 2 (6.13 mmol) was suspended in 55 mL of
dry methanol and the suspension was saturated with HCl(g)
for 30 min. After 4 days stirring at room temperature, the
suspension was filtrated and the solid was washed with
120 mL of ethyl ether. The yellow solid obtained was
dissolved in dry methanol and saturated with NH3 during
6 h. The solution was concentrated to half and to the
resulting solution was added 60 mL of ethyl ether. The
resulting precipitated was collected by filtration and washed
with 50 mL of ethyl acetate, affording the interest product.
3,4-Diaminobenzamidine hydrochloride (4)
This product was obtained in 93% yield as a yellow
solid; mp 230-235 °C; IR (ATR) ν / cm-1 3466, 3420
(NH2), 3200, 3115, 3031 (NH.HCl amidine), 1641 (C=N);
1H NMR (200 MHz, DMSO-d6) d 8.80 (s, 2H, H amidine),
8.69 (s, 2H, H amidine), 7.00 (d, J 8.4 Hz, 1H, H arom.),
6.95 (s, 1H, H arom.), 6.59 (d, J 8.2 Hz, 1H, H arom.),
5.44 (br s, 2H, NH2); 3.43 (sl, 2H, NH2); 13C NMR
(50 MHz, DMSO-d6) d 165.5, 141.7, 134.0, 118.9,
114.0, 112.7.
4-Amino-3-nitrobenzamidine hydrochloride (3)
This product was obtained as a yellow solid (60%
yield); mp > 300 °C; IR (ATR) ν / cm-1 3453, 3420 (NH2),
3134, 3052 (NH.HCl amidine), 1685 (C=N), 1628 (NH2);
1H NMR (200 MHz, DMSO-d6) d 9.38 (br s, 2H, H
amidine), 9.12 (br s, 2H, H amidine), 8.62 (s, 1H, H arom.),
8.17 (s, 2H, NH2), 7.87 (d, J 9.0 Hz, 1H, H arom.), 7.19 (d,
J 9.0 Hz, 1H, H arom.); 13C NMR (50 MHz, DMSO-d6) d
163.5, 149.1, 133.6, 129.6, 127.6, 119.4, 113.2.
3,4-Diamino-N-isopropylbenzamidine hydrochloride (6)
This product was obtained in 98% yield as a yellow
solid; mp 227-229 °C; IR (ATR) ν / cm-1 3379, 3207
(NH2), 3082, 2972, 2941 (NH.HCl amidine), 1689 (C=N);
1H NMR (200 MHz, DMSO-d6) d 8.71 (s, 2H, H amidine),
6.85 (br s, 2H, H arom.), 6.56 (d, J 8.2 Hz, 1H, H arom.),
5.53 (s, 2H, NH2), 4.92 (s, 2H, NH2), 4.04 (m, 1H,
CH), 1.20 (d, J 5.6 Hz, 6H, CH3); 13C NMR (50 MHz,
DMSO-d6) d 162.0, 140.7, 134.3, 118.3, 116.0, 112.8,
112.6, 44.4, 21.6.
Synthesis of 3-nitrobenzamidine (5)
A sample of 2 (6.13 mmol) was suspended in 55 mL of
dry methanol and the suspension was saturated with HCl(g)

Vol. 29, No. 6, 2018
de Souza et al.
1307
General procedure for the synthesis of peracetylated
mp 88.6-90.1 °C; [α]D –12.5° (c 0.48 CH2Cl2); IR (ATR)
ν / cm-1 1741 (C=O ester), 1687 (C=O aldehyde); 1H NMR
(400 MHz, CDCl3) d 9.88 (s, 1H, CHO), 7.42-7.39 (m,
2H, H-11 and H-9), 7.17 (d, J 8.0 Hz, 1H, H-8), 5.35 (d,
J 2.8 Hz, 1H, H-4’), 5.32 (t, J 8.8 Hz, 1H, H-3), 5.22 (t,
J 8.8 Hz, 1H, H-2), 5.14-5.08 (m, 2H, H-1 and H-2’), 4.97
(dd, J 12.0 Hz, J 3.2 Hz, 1H, H-3’), 4.53-4.51 (m, 2H,
H-1’ and H-6), 4.17-4.06 (m, 3H, H-6’, H-6’’ and H-6’’’),
3.93-3.89 (m, 3H, H-4 and H-5’), 3.88 (s, 1H, OCH3),
3.79-3.75 (m, 1H, H-5), 2.15-1.97 (s, 21H, COCH3);
13C NMR (100 MHz, CDCl3) d 190.8, 170.3-169.1, 151.1,
150.9, 132.7, 125.3, 117.8, 110.7, 101.13, 99.3, 76.0,
73.0, 72.4, 71.3, 70.9, 70.7, 69.1, 66.6, 61.8, 60.8, 56.1,
20.7-20.5.
glycosides (11-13)
A solution of the corresponding glycosyl bromide
(1 equiv.) in acetone (20 mL) was added to a solution of
4-hydroxy-3-methoxybenzaldehyde (vanillin) (3 equiv.)
in 1.0 mol L-1 lithium hydroxide (10 mL) and the solution
was stirred for 2 h at room temperature. The completion
of reaction was monitored by TLC, when acetone was
removed, and the resulting suspension was extracted with
dichloromethane (3 × 50 mL). The crude product was
washed with 10% sodium hydroxide (3 × 30 mL), water
and dried over anhydrous sodium sulfate. After filtration
and removal of the solvent under reduced pressure, the
crude product was recrystalized from isopropyl alcohol,
affording the title compounds.
2
3
Synthesis of peracetylated glycoside (14)
4-Formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-
β-D-glucopyranoside (11)
2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-gluco-
pyranosyl chloride (5.44 mmol) was solubilized in
acetonitrile (50 mL). To this solution were added
4-hydroxy-3-methoxybenzaldehyde (10.8 mmol), K2CO3
(23.7 mmol), polyethyleneglycol 4000 (0.27 mmol) and
the mixture was stirred for 3 h at room temperature,
when the completion of reaction was observed by TLC.
The suspension obtained was filtered and the filtrated
was concentrated to residue. The crude product was
solubilized in chloroform (60 mL) and the organic
layer was washed with 10% NaOH and water and
dried over anhydrous sodium sulfate. After filtration
and removal of the solvent under reduced pressure,
the crude product was recrystallized from isopropyl
alcohol.
This product was obtained in 57% yield as a white
solid; mp 136.1-137.3 °C; [α]D –39.2° (c 0.51 CH2Cl2);
IR (ATR) ν / cm-1 1753, 1737 (C=O ester), 1694 (C=O
1
aldehyde); H NMR (400 MHz, CDCl3) d 9.89 (s, 1H,
CHO), 7.43-7.40 (m, 2H, H-9 and H-11), 7.21 (d, J 8.0 Hz,
1H, H-8), 5.34-5.28 (m, 2H, H-2 and H-3), 5.13 (t, J 6.8 Hz,
1H, H-4), 5.09 (d, J 6.4 Hz, 1H, H-1), 4.27 (dd, 1H, H-6,
3
2
3
2J 12.4 Hz, J 5.2 Hz), 4.18 (dd, J 12.4 Hz, J 2.4 Hz,
1H, H-6’), 3.86 (s, 3H, OCH3), 3.85-3.70 (m, 1H, H-5),
2.07-2.04 (4s, 12H, COCH3); 13C NMR (100 MHz, CDCl3)
d 190.8, 170.5-169.2, 151.1, 151.0, 132.8, 125.3, 118.2,
110.8, 99.7, 72.4, 72.2, 71.0, 68.2, 61.9, 56.1, 20.6-20.5.
4-Formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-
β-D-galactopyranoside (12)
4-Formyl-2-methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-glucopyranoside (14)
This product was obtained in 60% yield as a white solid;
mp 123.1-123.8 °C; [α]D −8.1° (c 0.49 CH2Cl2); IR (ATR)
ν / cm-1 1752, 1740 (C=O ester), 1693 (C=O aldehyde);
1H NMR (400 MHz, CDCl3) d 9.89 (s, 1H, CHO), 7.43-7.40
(m, 2H, H-11 and H-9), 7.25 (d, J 8.4 Hz, 1H, H-8), 5.55 (t,
J 9.2 Hz, 1H, H-2), 5.46 (d, J 2.8 Hz, 1H, H-4), 5.12 (dd,
J(ax-ax) 10.4 Hz, J(ax-eq) 3.6 Hz, 1H, H-3), 5.05 (d, J 8.0 Hz,
1H, H-1), 4.23 (dd, 2J 11.8 Hz, 3J 6.8 Hz, 1H, H-6), 4.16
This product was obtained in 54% yield as a white
solid; mp 197.1-198.2 °C; [α]D –15.0° (c 0.53 CH2Cl2); IR
(ATR) ν / cm-1 1736 (C=O ester), 1690 (C=O aldehyde),
1671 (C=O amide); 1H NMR (400 MHz, CDCl3) d 9.88
3
(s, 1H, CHO), 7.42 (s, 1H, H-11), 7.41 (dd, J 10.4 Hz,
4J 1.6 Hz, 1H, H-9), 7.22 (d, J 8.0 Hz, 1H, H-8), 5.86 (d,
J 8.0 Hz, 1H, NH), 5.50 (t, J 9.6 Hz, 1H, H-3), 5.40 (d,
J 8.0 Hz, 1H, H-1), 5.13 (t, J 9.6 Hz, 1H, H-4), 4.27 (dd,
2
3
(dd, J 11.2 Hz, J 6.4 Hz, 1H, H-6’), 4.07-4.03 (m, 1H,
H-5), 3.90 (s, 3H, OCH3), 2.17-2.02 (4s, 12H, COCH3);
13C NMR (100 MHz, CDCl3) d 190.8, 170.3-169.3, 151.2,
150.9, 132.7, 125.3, 117.9, 110.7, 100.35, 71.2, 70.6, 68.4,
66.8, 61.3, 56.1, 20.6-20.5.
3
2
2J 12.0 Hz, J 5.4 Hz, 1H, H-6), 4.16 (dd, J 12.0 Hz,
3J 2.4 Hz, 1H, H-6’), 4.06 (qr, J 8.4 Hz, 1H, H-2), 3.90
(s, 3H, OCH3) 3.88-3.84 (m, 3H, H-5), 2.06-2.04 (s, 9H,
OCOCH3), 1.96 (s, 3H, NHCOCH3); 13C NMR (100 MHz,
CDCl3) d 190.8, 170.6-170.5, 169.4, 151.0, 150.8, 132.6,
125.5, 118.3, 110.6, 98.9, 72.2, 71.6, 68.5, 62.0, 56.1,
55.0, 23.3, 20.6-20.6.
4-Formyl-2-methoxyphenyl 2,3,6,2’,3’,4’,6’-hepta-O-acetyl-
β-D-lactoside (13)
This product was obtained in 52% yield as a white solid;

1308
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles
J. Braz. Chem. Soc.
General procedure for the synthesis of peracetylated
2-[4-(2,3,6,2’,3’,4’,6’-Hepta-O-acetyl-β-D-lactosyloxy)-
3-methoxyphenyl]benzimidazole-5-carboxamidine
hydrochloride (17)
benzamidines (15-22)
To a solution of the corresponding glycoside (11-14)
(1 equiv.) in ethanol (35 mL), it was added the
3,4-diaminobenzamidine 4 or 6 (1 equiv.) and
p-benzoquinone (1 equiv.). The mixture was heated at
70 ºC for 4 h, when the completion of reaction was observed
by TLC. The solution was concentrated to residue and the
pure product was obtained by column chromatography on
silica gel.
This product was obtained in 63% yield as a brown
solid after purified by chromatography (dichloromethane/
methyl alcohol 95:5 to 85:15); mp 197.2-199.5 °C;
[α]D −47.6° (c 0.42 MeOH); IR (ATR) ν / cm-1 3348, 3119
(NH.HCl amidine), 1740 (C=O ester), 1675 (C=N);
1H NMR (400 MHz, DMSO-d6) d 13.70 (br s, 1H, H
benzimidazole), 9.17 (br s, 4H, H amidine), 8.15 (br s,
1H, H-18), 7.95 (s, 1H, H-11), 7.84 (d, J 7.0 Hz, 1H, H-9),
7.77 (br s, 1H, H-15), 7.67 (d, J 7.0 Hz, 1H, H-16), 7.26
(d, J 8.4 Hz, 1H, H-8), 5.48 (d, J 8.0 Hz, 1H, H-1), 5.30 (t,
J 8.0 Hz, 1H, H-3), 5.25 (d, J 3.2 Hz, 1H, H-4’), 5.18 (dd,
2J 10.0 Hz, 3J 3.6 Hz, 1H, H-3’), 5.02 (t, J 9.0 Hz, 1H, H-2),
4.88 (t, J 9.2 Hz, 1H, H-2’), 4.80 (d, J 8.0 Hz, 1H, H-1’),
2-[4-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy)-
3-methoxyphenyl]benzimidazole-5-carboxamidine
hydrochloride (15)
This product was obtained in 64% yield as a brown solid
after purified by chromatography (dichloromethane/methyl
alcohol 87:13); mp 196.1-199.3 °C; [α]D −50.0° (c 0.40
MeOH); IR (ATR) ν / cm-1 3340, 3119 (NH.HCl amidine),
2
4.38 (d, J 10.4 Hz, 1H, H-6), 4.30-4.25 (m, 1H, H-5’),
4.23-4.03 (m, 4H, H-5, H-6’, H-6’’, H-6’’’), 3.94-3.89 (m,
1H, H-4), 3.89 (s, 3H, OCH3), 2.10-1.91 (s, 21H, COCH3);
13C NMR (100 MHz, DMSO-d6) d 170.13-169.04, 166.0,
149.7, 147.6, 124.6, 119.6, 117.2, 111.3, 99.8, 97.8,
76.1, 72.1, 71.9, 70.9, 70.2, 69.7, 68.8, 67.0, 62.0, 60.8,
56.1, 20.6-20.2; HRMS (ESI) m/z, calcd. for C41H49O19N4
[M + H]+: 901.2986, found: 901.3029.
1
1740 (C=O ester), 1675 (C=N); H NMR (400 MHz,
DMSO-d6) d 13.75 (br s, 1H, NH benzimidazole), 9.31 (br
s, 2H, H amidine), 9.04 (br s, 2H, H amidine), 8.20-7.68 (m,
5H, H-9, H-11, H-15, H-16 and H-18), 7.29 (d , J 8.8 Hz,
1H, H-8), 5.53 (d, J 8 Hz, 1H, H-1), 5.41 (t, J 9.6 Hz, 1H,
H-3), 5.11 (t, J 8.8 Hz, 1H, H-2), 5.03 (t, J 9.6 Hz, 1H, H-4),
4.27-4.21 (m, 2H, H-6 and H-5), 4.11 (d, J 10.4 Hz, 1H,
H-6’), 3.90 (s, 3H, OCH3), 2.05-1.98 (4s, 12H, COCH3);
13C NMR (100 MHz, DMSO-d6) d 169.9-168.9, 166.0,
149.8, 147.5, 124.8, 119.6, 117.6, 111.3, 98.1, 71.8, 70.9,
70.6, 68.0, 61.6, 56.1, 20.4-20.2; HRMS (ESI) m/z, calcd.
for C29H33O11N4 [M + H]+: 613.2140, found: 613.2174.
2-[4-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-
β-D-glucopyranosyloxy)-3-methoxyphenyl]benzimidazole-
5-carboxamidine hydrochloride (18)
This product was obtained in 65% yield as a brown
solid after purified by chromatography (dichloromethane/
methyl alcohol 88:12); mp 218.4.1-220.2 °C; [α]D −47.3°
(c 0.38 MeOH); IR (ATR) ν / cm-1 3110 (NH.HCl amidine),
1740 (C=O ester), 1663 (C=O amide); 1H NMR (400 MHz,
DMSO-d6) d 13.78 (br s, 1H, H benzimidazole), 9.20 (br s,
3H, H amidine), 8.14-7.66 (m, 6H, NH, H-9, H-11, H-15,
H-16 and H-18), 7.32 (d, J 8.4 Hz, 1H, H-8), 5.47 (d,
J 8.0 Hz, 1H, H-1), 5.26 (t, J 10.0 Hz, 1H, H-3), 4.94 (t,
J 9.6 Hz, 1H, H-4), 4.22 (dd, 2J 11.6 Hz, 3J 4.4 Hz, 1H, H-6),
4.15-4.98 (m, 3H, H-2, H-5 and H-6’), 3.89 (s, 3H, OCH3),
2.06-2.00 (3s, 9H, OCOCH3) 1.86 (s, 3H, NHCOCH3);
13C NMR (100 MHz, DMSO-d6)d169.9, 169.6-169.3, 166.1,
149.6, 147.9, 124.2, 119.1, 116.7, 111.5, 97.9, 72.3, 70.9,
68.3, 61.6, 56.2, 53.3, 22.6, 20.5-20.3; HRMS (ESI) m/z,
calcd. for C29H34O10N5 [M + H]+: 612.2300, found: 612.2322.
2-[4-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-
3-methoxyphenyl]benzimidazole-5-carboxamidine
hydrochloride (16)
This product was obtained in 61% yield as a brown
solid after purified by chromatography (dichloromethane/
methyl alcohol 87:13); mp 191.5-194.1 °C; [α]D –13.6° (c
0.44 MeOH); IR (ATR) ν / cm-1 3117 (NH.HCl amidine),
1
1741(C=O ester), 1675 (C=N); H NMR (400 MHz,
DMSO-d6) d 13.80-13.71 (br s, 1H, NH benzimidazole),
9.32 (br s, 2H, H amidine), 9.09 (br s, 2H, H amidine),
8.22 (m, 5H, H-9, H-11, H-15, H-16 and H-18), 7.29 (d,
J 8.4 Hz, 1H, H-8), 5.44 (d, J 6.4 Hz, 1H, H-1), 5.35 (s,
1H, H-4), 5.31-5.24 (m, 2H, H-2 and H-3), 4.45-4.42 (m,
1H, H-5), 4.18-4.09 (m, 2H, H-6 and H-6’), 3.91 (s, 3H,
OCH3), 2.17-1.96 (4s, 12H, COCH3); 13C NMR (100 MHz,
DMSO-d6) d 169.3-169.0, 166.7, 149.3, 147.6, 119.1, 98.8,
70.4, 70.1, 68.3, 67.2, 62.7, 56.2, 20.5-20.3; HRMS (ESI)
m/z, calcd. for C29H33O11N4 [M + H]+: 613.2140, found:
613.2162.
2-[4-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyloxy)-
3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)
carboxamidine] hydrochloride (19)
This product was obtained in 60% yield as a brown
solid after purified by chromatography (dichloromethane/
methyl alcohol 92:8); mp 187.5-190.0 °C; [α]D –33.3°

Vol. 29, No. 6, 2018
de Souza et al.
1309
(c 0.42 MeOH); IR (ATR) ν / cm-1 3072 (NH.HCl amidine),
1743 (C=O ester), 1660 (C=N); H NMR (400 MHz,
5.22 (d, J 3.2 Hz, 1H, H-4’), 5.15 (dd, 2J 10.0 Hz, 3J 3.4 Hz,
1H, H-3’), 4.99 (t, J 9.0 Hz, 1H, H-2), 4.84 (t, J 9.0 Hz,
1H, H-2’), 4.76 (d, J 8.0 Hz, 1H, H-1’), 4.35 (d, 2J 10.4 Hz,
1H, H-6), 4.23-4.20 (m, 1H, H-5’), 4.14-3.97 (m, 5H, H-5,
H-6’, H-6’’, H-6’’’, H-21), 3.91-3.86 (m, 1H, H-4), 3.82 (s,
3H, OCH3), 2.04-1.88 (s, 21H, COCH3), 1.28 (d, J 6.4 Hz,
6H, H-22); 13C NMR (100 MHz, DMSO-d6) d 170.1-169.0,
162.4, 149.7, 147.6, 124.8, 119.3, 117.2, 109.4, 99.8, 97.8,
76.1, 72.1, 71.9, 70.9, 70.2, 69.6, 68.9, 67.0, 62.0, 60.8,
56.1, 44.9, 21.2, 20.6-20.2; HRMS (ESI) m/z, calcd. for
C44H55O19N4 [M + H]+: 943.3455, found: 943.3474.
1
DMSO-d6) d 13.88 (br s, 1H, NH benzimidazole), 9.50 (br
s, 3H, H amidine), 7.97 (m, 2H, H-15 and H-18), 7.87 (d,
J 8.4 Hz, 1H, H-16), 7.77 (br s, 1H, H-11), 7.55 (d, J 8.4 Hz,
1H, H-9), 7.29 (d, J 8.8 Hz, 1H, H-8), 5.53 (d, J 8 Hz, 1H,
H-1), 5.42 (t, J 9.6 Hz, 1H, H-3), 5.11 (t, J 8.8 Hz, 1H,
H-2), 5.03 (t, J 9.6 Hz, 1H, H-4), 4.26-4.23 (m, 2H, H-6
and H-5), 4.12-4.10 (m, 2H, H-6’ and H-21), 3.91 (s, 3H,
OCH3), 2.05-1.98 (4s, 12H, COCH3), 1.32 (d, J 6.4 Hz,
1H, H-22); 13C NMR (100 MHz, DMSO-d6) d 169.9-168.9,
167.0, 149.8, 147.4, 124.9, 119.6, 117.7, 111.3, 98.2, 71.8,
70.9, 70.6, 68.0, 61.6, 56.1, 44.9, 21.3, 20.4-20.2; HRMS
(ESI) m/z, calcd. for C32H39O11N4 [M + H]+: 655.2610,
found: 655.2611.
2-[4-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-gluco-
pyranosyloxy)-3-methoxyphenyl]benzimidazole-
5-[N-(isopropyl)carboxamidine] hydrochloride (22)
This product was obtained in 80% yield as a brown solid
after purified by chromatography (dichloromethane/methyl
alcohol 87:13); mp 221.3-223.0 °C; [α]D –19.0° (c 0.42
MeOH); IR (ATR) ν / cm-1 3296, 3098 (NH.HCl amidine),
2-[4-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-
3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)
carboxamidine] hydrochloride (20)
1
This product was obtained in 61% yield as a brown solid
after purified by chromatography (dichloromethane/methyl
alcohol 91:9); mp 208.5-210.1 °C; [α]D –27.2° (c 0.44
MeOH); IR (ATR) ν / cm-1 2979 (NH.HCl amidine), 1744
(C=O ester), 1669 (C=N); 1H NMR (400 MHz, DMSO-d6)
d 13.85 (br s, 1H, NH benzimidazole), 9.62 (br s, 3H,
H amidine), 8.01 (s, 1H, H-18), 7.98 (s, 1H, H-11), 7.87
(d, J 8.0 Hz, 1H, H-15), 7.77 (d, J 8.0 Hz, 1H, H-16), 7.55
(d, J 8.4 Hz, 1H, H-9), 7.29 (d, J 8.4 Hz, 1H, H-8), 5.43
(d, J 6.8 Hz, 1H, H-1), 5.35 (s, 1H, H-4), 5.31-5.24 (m,
2H, H-2 and H-3), 4.45-4.41 (m, 1H, H-5), 4.18-4.12 (m,
3H, H-6, H-6’ and H-21), 3.91 (s, 3H, OCH3), 2.17-1.96
(4s, 12H, COCH3), 1.31 (d, 6H, H-22, J 6.4 Hz); 13C NMR
(100 MHz, DMSO-d6) d 169.9-169.0, 162.4, 153.8, 149.9,
147.5, 124.9, 122.5, 119.6, 117.8, 111.4, 98.8, 70.4, 70.0,
68.2, 67.2, 61.2, 56.1, 44.9, 21.2, 20.4-20.3; HRMS (ESI)
m/z, calcd. for C32H39O11N4 [M + H]+: 655.2610, found:
655.2619.
1729 (C=O ester), 1663 (C=N); H NMR (400 MHz,
DMSO-d6) d 13.76 (br s, 1H, NH benzimidazole), 9.46
(br s, 3H, H amidine), 8.13 (d, 1H, NH amide, J 8.8 Hz),
8.01 (s, 1H, H-18), 7.94 (s, 1H, H-11), 7.86 (d, J 8.4 Hz,
1H, H-9), 7.75 (s, 1H, H-15), 7.55 (d, J 8.0 Hz, 1H, H-16),
7.32 (d, J 8.4 Hz, 1H, H-8), 5.47 (d, J 8.4 Hz, 1H, H-1),
5.27 (t, J 9.8 Hz, 1H, H-3), 4.95 (t, J 9.6 Hz, 1H, H-4),
2
3
4.23 (dd, J 12.0 Hz, J 4.8 Hz, 1H, H-6), 4.15-3.98 (m,
4H, H-6’, H-5, H-2, H-21), 3.89 (s, 3H, OCH3), 2.03-1.90
(3s, 9H, OCOCH3), 1.86 (s, 3H, NHCOCH3), 1.28 (d,
J 6.4 Hz, 6H, H-22); 13C NMR (100 MHz, DMSO-d6)
d 169.9, 169.6-169.3, 162.4, 149.6, 147.8, 124.3, 119.7,
116.8, 97.9, 72.3, 70.9, 68.3, 61.6, 56.2, 53.3, 44.9, 22.6,
21.3, 20.5-20.3; HRMS (ESI) m/z, calcd. for C32H34O10N5
[M + H]+: 654.2770, found: 654.2799.
General procedure for the synthesis of deacetylated
benzamidines (23-30)
2-[4-(2,3,6,2’,3’,4’,6’-Hepta-O-acetyl-β-D-lactosyloxy)-
3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)
carboxamidine] hydrochloride (21)
The peracetylated benzamidine (15-22; 0.5 mmol)
was solubilized in a solution of KOH in MeOH (20 mL,
1.0 mol L-1) and the solution was stirred at 0 °C for 30 min.
After the completion of the reaction, as observed by TLC,
the mixture was neutralized with IRA-120 resin at 0 °C.
The resin was filtered off and washed with methanol. The
collected filtrate was concentrated in vacuum to afford the
deacetylated derivatives.
This product was obtained in 52% yield as a yellow
solid after purified by chromatography (dichloromethane/
methyl alcohol 93:7); mp 195.5-198.3 °C; [α]D –25.0°
(c 0.40 MeOH); IR (ATR) ν / cm-1 3366, 3083 (NH.HCl
1
amidine), 1742 (C=O ester), 1672 (C=N); H NMR
(400 MHz, DMSO-d6) d 13.76 (br s, 1H, H benzimidazole),
9.45 (br s, 3H, H amidine), 8.08 (s, 1H, H-18), 7.91 (s, 1H,
H-11), 7.81 (d, J 8.4 Hz, 1H, H-9), 7.73 (br s, 1H, H-15),
7.51 (d, J 7.6 Hz, 1H, H-16), 7.22 (d, J 8.4 Hz, 1H, H-8),
5.44 (d, J 8.0 Hz, 1H, H-1), 5.26 (t, J 9.6 Hz, 1H, H-3),
2-[4-(β-D-Glucopyranosyloxy)-3-methoxyphenyl]
benzimidazole-5-carboxamidine (23)
This product was obtained in 100% yield as a brown
solid; mp 214.5-216.8 °C; [α]D –15.3° (c 0.52 MeOH);

1310
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles
J. Braz. Chem. Soc.
IR (ATR) ν / cm-1 3132 (OH), 1675 (C=N); H NMR
(400MHz, DMSO-d6)d13.66(brs, 1H, NHbenzimidazole),
9.31 (s, 2H, H amidine), 9.04 (s, 2H, H amidine), 8.11 (s,
1H, H-18), 7.89 (s, 1H, H-11), 7.85 (d, J 8.4 Hz, 1H, H-9),
7.74 (d, J 8.0 Hz, 1H, H-15), 7.64 (d, J 8.0 Hz, 1H, H-16),
7.24 (d, J 8.8 Hz, 1H, H-8), 5.03 (d, J 6.4 Hz, 1H, H-1), 3.82
(s, 1H, OCH3), 3.66 (d, J 11.2 Hz, 1H, H-6), 3.60-3.17 (m,
5H, H-2, H-3, H-4, H-5 and H-6’); 13C NMR (100 MHz,
DMSO-d6) d 166.1, 149.1, 148.6, 122.7, 121.8, 121.0,
119.9, 115.2, 110.8, 99.6, 77.0, 76.8, 73.1, 69.6, 60.6,
55.8; HRMS (ESI) m/z, calcd. for C21H25O7N4 [M + H]+:
445.1718, found: 445.1797.
IR (ATR) ν / cm-1 3241 (OH), 1681 (C=N); H NMR
1
1
(400 MHz, DMSO-d6) d 9.41 (s, 2H, H amidine), 9.10 (s,
2H, H amidine), 8.17 (s, 1H, H-18), 8.01 (s, 1H, H-11),
7.91-7.82 (m, 3H, H-9, H-15 and NH), 7.73 (d, J 8.4 Hz,
1H, H-16), 7.33 (d, J 8.8 Hz, 1H, H-8), 5.17 (d, J 8.4 Hz,
1H, H-1), 3.89 (s, 3H, OCH3), 3.74-3.20 (m, 6H, H-2,
H-3, H-4, H-5, H-6 and H-6’), 1.81 (s, 1H, NHCOCH3);
13C NMR (100 MHz, DMSO-d6) d 169.2, 165.8, 149.7,
123.3, 121.1, 117.2, 112.4, 99.6, 77.9, 74.5, 70.7, 61.2,
57.1, 56.1, 23.6; HRMS (ESI) m/z, calcd. for C23H28O7N5
[M + H]+: 486.1983, found: 486.1987.
2-[4-(β-D-Glucopyranosyloxy)-3-methoxyphenyl]
benzimidazole-5-[N-(isopropyl)carboxamidine] (27)
2-[4-(β-D-Galactopyranosyloxy)-3-methoxyphenyl]
benzimidazole-5-carboxamidine (24)
This product was obtained in 100% yield as a brown
solid; mp 206.5-209.0 °C; [α]D −63.6° (c 0.44 MeOH); IR
(ATR) ν / cm-1 3123 (OH), 1669 (C=N); 1H NMR (400 MHz,
DMSO-d6) d 7.99 (s, 1H, H-18), 7.92 (s, 1H, H-11), 7.82
(d, J 8.8 Hz, 1H, H-9), 7.74 (d, J 8.4 Hz, 1H, H-15), 7.53
(d, J 8.4 Hz, 1H, H-16), 7.26 (d, J 8.4 Hz, 1H, H-8), 5.31
(br s, 1H, OH), 5.14 (br s, 1H, OH), 5.05 (d, J 7.2 Hz, 1H,
H-1), 4.58 (br s, 1H, OH), 4.11-4.08 (m, 1H, H-21), 3.91 (s,
1H, OCH3), 3.68 (d, J 10.8 Hz, 1H, H-6), 3.48-3.16 (m, 5H,
H-2, H-3, H-4, H-5 and H-6’), 1.30 (d, J 6.4 Hz, 6H, H-22);
13C NMR (100 MHz, DMSO-d6) d 162.2, 154.1, 148.9,
148.2, 122.8, 122.2, 121.6, 119.6, 115.0, 110.6, 99.4, 76.8,
76.6, 72.9, 69.4, 60.4, 55.6, 44.7, 21.1; HRMS (ESI) m/z,
calcd. for C24H31O7N4 [M + H]+: 487.2187, found: 487.2229.
This product was obtained in 100% yield as a brown
solid; mp 216.5-219.0 °C; [α]D −55.0° (c 0.40 MeOH); IR
(ATR) ν/ cm-1 3156 (OH), 1673 (C=N); 1H NMR (400 MHz,
DMSO-d6) d 13.58 (br s, 1H, NH benzimidazole), 9.30 (s,
2H, H amidine), 8.99 (s, 2H, H amidine), 8.11 (br s, 1H,
H-18), 7.87 (s, 1H, H-11), 7.78 (d, J 8.4 Hz, 1H, H-9), 7.73
(br s, 1H, H-15), 7.63 (d, J 8.0 Hz, 1H, H-16), 7.24 (d,
J 8.8 Hz, 1H, H-8), 5.12 (br s, 1H, OH), 4.99 (d, J 7.6 Hz,
1H, H-1), 4.65 (br s, 1H, OH), 4.54 (br s, 1H, OH), 3.89
(s, 1H, OCH3), 3.71-3.33 (m, 6H, H-2, H-3, H-4, H-5,
H-6 and H-6’); 13C NMR (100 MHz, DMSO-d6) d 166.1,
149.2, 148.6, 122.7, 121.8, 119.8, 115.2, 110.8, 100.2, 75.5,
73.5, 70.1, 68.0, 60.2, 55.8; HRMS (ESI) m/z, calcd. for
C21H25O7N4 [M + H]+: 445.1718, found: 445.1763.
2-[4-(β-D-Galactopyranosyloxy)-3-methoxyphenyl]
benzimidazole-5-[N-(isopropyl)carboxamidine] (28)
This product was obtained in 96% yield as a brown
solid; mp 203.7-205.8 °C; [α]D −47.6° (c 0.42 MeOH);
2-[4-(β-D-Lactosyloxy)-3-methoxyphenyl]benzimidazole-
5-carboxamidine (25)
This product was obtained in 100% yield as a brown
solid; mp 225.6-228.0 °C; [α]D −68.1° (c 0.44 MeOH);
1
IR (ATR) ν / cm-1 3122 (OH), 1669 (C=N); H NMR
1
IR (ATR) ν / cm-1 3198 (OH), 1674 (C=N); H NMR
(400 MHz, DMSO-d6) d 13.72 (s, 1H, H benzimidazole),
9.51 (s, 1H, H amidine), 9.38 (s, 1H, H amidine), 8.99 (s,
1H, H amidine), 8.00 (s, 1H, H-18), 7.93 (s, 1H, H-11),
7.82 (d, J 8.4 Hz, 1H, H-9), 7.74 (br s, 1H, H-15), 7.54 (d,
J 8.4 Hz, 1H, H-16), 7.27 (d, J 9.2 Hz, 1H, H-8), 5.11 (br s,
1H, OH), 5.02 (d, J 8.0 Hz, 1H, H-1), 4.68 (br s, 2H, OH),
4.10-4.09 (m, 1H, H-21), 3.98 (s, 1H, OCH3), 3.66-3.54 (m,
6H, H-2, H-3, H-4, H-5, H-6 and H-6’), 1.31 (d, J 6.4 Hz,
6H, H-22); 13C NMR (100 MHz, DMSO-d6) d 162.3, 149.0,
148.5, 122.6, 121.7, 119.7, 115.1, 110.7, 100.0, 75.4, 73.4,
70.0, 67.9, 60.1, 55.7, 44.8, 21.2; HRMS (ESI) m/z, calcd.
for C24H31O7N4 [M + H]+: 487.2187, found: 487.2194.
(400 MHz, DMSO-d6) d 9.33 (s, 2H, H amidine), 9.00 (s,
2H, H amidine), 8.13 (s, 1H, H-18), 7.91 (s, 1H, H-11),
7.82 (d, J 8.4 Hz, 1H, H-9), 7.77 (d, J 8.0 Hz, 1H, H-15),
7.66 (d, J 8.4 Hz, 1H, H-16), 7.28 (d, J 8.4 Hz, 1H, H-8),
5.41 (d, J 7.6 Hz, 1H, H-1), 4.26 (d, J 7.6 Hz, 1H, H-1’),
3.91 (s, 1H, OCH3), 3.64-3.38 (m, 12H, H-2, H-2’, H-3,
H-3’, H-4, H-4’, H-5, H-5’, H-6, H-6’, H-6’’ and H-6’’’);
13C NMR (100 MHz, DMSO-d6) d 166.2, 149.3, 148.7,
122.1, 120.3, 115.5, 111.2, 104.0, 99.2, 80.3-68.5, 60.5-
60.2, 56.0; HRMS (ESI) m/z, calcd. for C27H35O12N4
[M + H]+: 607.2246, found: 607.2265.
2-[4-(2-Acetamido-2-deoxy-β-D-glucopyranosyloxy)-
3-methoxyphenyl]benzimidazole-5-carboxamidine (26)
This product was obtained in 100% yield as a brown
solid; mp 211.7-213.7 °C; [α]D −9.5° (c 0.42 MeOH);
2-[4-(β-D-Lactosyloxy)-3-methoxyphenyl]benzimidazole-
5-[N-(isopropyl)carboxamidine] (29)
This product was obtained in 100% yield as a yellow
solid; mp 213.4-215.8 °C; [α]D −80.0° (c 0.40 MeOH);

Vol. 29, No. 6, 2018
de Souza et al.
1311
IR (ATR) ν / cm-1 3232 (OH), 1668 (C=N); H NMR
(400 MHz, DMSO-d6) d 9.33 (br s, 2H, H amidine), 9.00
(s, 2H, H amidine), 7.99 (s, 1H, H-18), 7.92 (s, 1H, H-11),
7.82 (d, J 8.4 Hz, 1H, H-9), 7.74 (d, J 8.4 Hz, 1H, H-15),
7.52 (d, J 8.0 Hz, 1H, H-16), 7.27 (d, J 8.4 Hz, 1H, H-8),
5.48 (br s, 1H, OH), 5.32 (d, J 7.6 Hz, 1H, H-1), 5.11 (br
s, 1H, OH), 4.82-4.55 (m, 5H, OH), 4.26 (d, J 6.4 Hz,
1H, H-1’), 4.10-4.07 (m, 1H, H-21), 3.91 (s, 1H, OCH3),
3.74-3.34 (m, 12H, H-2, H-2’, H-3, H-3’, H-4, H-4’, H-5,
H-5’, H-6, H-6’, H-6’’ and H-6’’’), 1.30 (d, J 6.0 Hz, 6H,
H-22); 13C NMR (100 MHz, DMSO-d6) d 162.4, 154.2,
149.1, 148.2, 123.1, 122.4, 121.8, 119.7, 115.1, 110.8,
103.8, 99.1, 80.1-68.1, 60.3-60.0, 55.8, 44.9, 21.3; HRMS
(ESI) m/z, calcd. for C30H41O12N4 [M + H]+: 649.2715,
found: 649.2742.
DMSO-d6) d 13.78 (s, 1H, NH benzimidazole), 9.39, 9.33
(s, 2H, H amidine), 9.12 (s, 2H, H amidine), 8.04 (s, 1H,
H-9), 7.93-7.80 (m, 2H, H-11, H-12), 7.70-7.63 (m, 2H,
H-3, H-5), 7.16 (d, J 8.4 Hz, 1H, H-6), 3.91 (s, 3H, OCH3),
3.85 (s, 3H, OCH3); 13C NMR (100 MHz, DMSO-d6) d
166.2, 154.2, 150.8, 148.9, 143.3, 139.0, 121.8, 121.4,
120.9, 111.8, 111.5, 55.7, 55.6; HRMS (ESI) m/z, calcd.
for C16H17O2N4 [M + H]+: 297.1346, found: 297.1353.
1
2-(3,4-Dimethoxyphenyl)-N-isopropyl-1H-benzimidazole-
5-carboxamidine hydrochloride (32)
This product was obtained in 70% yield as a brown solid,
mp165.2-168.0°C,IR(ATR)ν/cm-1 3074(NH.HClamidine),
1668 (C=N); 1H NMR (400 MHz, DMSO-d6) d 13.91 (s,
1H, NH benzimidazole), 9.55-9.39 (m, 2H, H amidine),
9.05 (s, 1H, H amidine), 8.06 (s, 1H, H-9), 7.96-7.87
(m, 2H, H-3, H-11), 7.69 (d, J 8.4 Hz, 1H, H-12), 7.54
2-[4-(2-Acetamido-2-deoxy-β-D-glycopyranosyloxy)-
3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)
carboxamidine] (30)
3
4
(dd, J 19.2 Hz, J 8 Hz, 1H, H-5), 7.15 (d, J 8.8 Hz,
1H, H-6), 4.16-4.11 (m, 1H, H-15), 3.91 (s, 3H, OCH3),
3.85 (s, 3H, OCH3), 1.31 (d, J 6.4 Hz, H-16); 13C NMR
(100 MHz, DMSO-d6) d 162.4, 154.0, 150.8, 148.9, 143.2,
138.5, 122.3, 122.0, 121.6, 111.8, 111.5, 55.7, 55.6, 44.9,
21.3; HRMS (ESI) m/z, calcd. for C19H23O2N4 [M + H]+:
339.1816, found: 339.1833.
This product was obtained in 95% yield as a yellow
solid; mp 201.3-203.5 °C; [α]D –23.8° (c 0.42 MeOH);
1
IR (ATR) ν / cm-1 3234 (OH), 1668 (C=N); H NMR
(400 MHz, DMSO-d6) d 7.99 (s, 1H, H-18), 7.91-7.87 (m,
2H, H-11 and NH), 7.82 (d, J 8.4 Hz, 1H, H-9), 7.73 (d,
J 8.4 Hz, 1H, H-15), 7.52 (d, J 8.4 Hz, 1H, H-16), 7.28
(d, J 8.4 Hz, 1H, H-8), 5.13-5.11 (m, 2H, H-1 and OH),
4.66 (br s, 1H, OH), 4.10-4.05 (m, 1H, H-21), 3.74 (s,
1H, OCH3), 3.71-3.20 (m, 6H, H-2, H-3, H-4, H-5, H-6
and H-6’), 1.82 (s, 1H, COCH3), 1.30 (d, J 6.0 Hz, 6H,
H-22); 13C NMR (100 MHz, DMSO-d6) d 169.2, 162.4,
154.3, 149.6, 148.8, 124.0, 122.5, 121.7, 119.9, 116.7,
116.6, 99.3, 77.4, 73.9, 70.3, 60.7, 56.3, 55.6, 44.9, 23.0,
21.4; HRMS (ESI) m/z, calcd. for C26H34O7N5 [M + H]+:
528.2453, found: 528.2458.
Antifungal and antibacterial activities evaluation
The benzamidines and starting glycosides were
evaluated in vitro for their antibacterial and antifungal
activities and the inhibitory concentrations of microbial
growth were determined at 50% (IC50) and 90% (IC90)
in µmol mL-1 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 7.3.1.11 The
determination of antibacterial activity were performed
according to microdilution methodology in Mueller
Hinton broth adjusted with cations as described by
document ISO 20776-1:2006.12-14 The stock solutions of
all the compounds were prepared in DMSO 1% at final
concentration and tested at concentrations from 500 to
0.98 µg mL-1. The standard drug fluconazole was applied
as control of fungistatic action at concentrations from
64 to 0.031 g mL-1 and the standard drug chloramphenicol
as a control of bacteriostatic action at concentrations from
125 to 0.06 g mL-1. The microplates were incubated at
35 ºC for 24 h for bacteria and 37 ºC and for 24 h for
fungi. Results were visualized and analyzed at 530 nm in
an Anthos Zenyth 200rt Microplate Reader.
General procedure for the synthesis of benzamidines
(31-32)
To a solution of 3,4-dimethoxybenzaldehyde (1 equiv.)
in ethanol (35 mL), it was added the corresponding
3,4-diaminobenzamidine (4 or 6) (1 equiv.) and
p-benzoquinone (1 equiv.). The mixture was heated at
70 ºC for 4 h, when the completion of reaction was observed
by TLC. The solution was concentrated to residue and the
pure product was obtained by CCS.
2-(3,4-Dimethoxyphenyl)-1H-benzimidazole-
5-carboxamidine hydrochloride (31)
This product was obtained in 69% yield as a brown
solid; mp 170.5-173.3 °C; IR (ATR) ν / cm-1 3297, 3060
1
(NH.HCl amidine), 1686 (C=N); H NMR (400 MHz,

1312
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles
J. Braz. Chem. Soc.
Scheme 2. Synthesis of 3,4-diaminobenzamidines 4 and 6.
Results and Discussion
Chemistry
The preparation of 3,4-diaminobenzamidines (4 and 6)
was performed in good yields from the nitrobenzamidines 3
or 5 as described by Fairley et al.15 and reproduced by
Göker et al.9 The 4-amino-3-nitrobenzonitrile (2) was
converted to the corresponding imidate by reaction with
anhydrous methanol in the presence of HCl(g). The reaction
of this imidate with ammonia or isopropylamine afforded
the nitroamidines 3 (60% yield) and 5 (63% yield),
which upon catalytic hydrogenation, provided the
3,4-diaminobenzamidines 4 (93% yield) and 6 (98%
yield), respectively, as hydrochloride salts (Scheme 2).
The already described glycosides 11-13 were
prepared from the reaction between the corresponding
glycosyl bromide (7, 8 or 9) with 4-hydroxy-
3-methoxybenzaldehyde (vanillin) in acetone and LiOH
according to the method described by Conchie et al.16 and
reproduced by Souza et al.10 The reaction of 10, prepared
as described by Horton17 with vanillin in acetonitrile,
in the presence of potassium carbonate and PEG 4000,
afforded the glycoside 14 (Scheme 3).All glycosides were
obtained in yields higher than 52% after purification as
β-anomers, as confirmed by the H-1 coupling constants
Scheme 3. Synthesis of glycosides 11-14.
The reaction of 3,4-diaminobenzamidines (4 or 6)
with glycosides 11-14 furnished the peracetylated 2-aryl-
5-amidinobenzimidazoles 15-22 which upon deacetylation
conditions afforded the derivatives 23-30, as depicted in
Scheme 4. The derivatives 15-30 were synthesized for the
first time.
There are several methods to synthesize benzimidazoles
described and most of them employ the condensation
of an 3,4-diaminobenzene with carboxylic acids, esters,
nitriles, acyl chlorides or aldehydes in presence of oxidizing
agents such as nitric acid, nitrobenzene and quinones.18 As
shown is Scheme 4, in the present work the peracetylated
benzimidazoles15-22wereobtainedingoodyields(52-80%)
from the reaction of 3,4-diaminobenzamidines 4 or 6 with
glycosylated aldehydes 11-14, using para-benzoquinone as
oxidizing agent. Deacetylation of peracetylated derivatives
in methanolic solution of potassium hydroxide10 provided
1
around 8 Hz in the corresponding H NMR spectra.
Besides, one observes a singlet at around 9.8 ppm of
each compound corresponding to the aldehyde proton.
Their infrared spectra showed bands relatives to the ester
and aldehyde carbonyl groups near 1750 and 1690 cm-1,
respectively.

Vol. 29, No. 6, 2018
de Souza et al.
1313
Scheme 4. Synthesis of glycosylated 2-aril-5-amidinobenzimidazoles 15-30.
the deacetylated benzamidines 23-30 in yields higher than
95%. In the proton NMR spectra of compounds 23-26 it was
observed two signals between 9.0-9.4 ppm corresponding to
the three amidine protons. For derivatives 27-30 the signals
of the two amidine protons are observed around 9.4 ppm.
The imidazole protons of all benzamidines were registered
as broad signals near 13 ppm.
The assignment of the signals to the protons in
1H NMR spectra of the compounds was possible by
using heteronuclear multiple-bond correlation (HMBC)
and correlation spectroscopy (COSY) experiments. As
exemplified for peracetylated benzamidine 20, a correlation
was observed between C-20 and aromatic H-16 in the
HMBC experiment, which unequivocally confirmed the
identity of H-16, registered as a doublet (Figure 1a). From
the assignment of H-16, its correlation with H-15 can
be observed in the COSY experiment (Figure 1b). The
correlation between C-13 and H-11 also confirmed the
identity of this aromatic proton as a singlet (Figure 1a).
These observations are in agreement with the identity of
the 2-aryl-5-amidinobenzimidazole system.19
(Candida albicans, C. tropicalis, C. krusei, C. glabrata
and C. parapsilosis) and bacteria (Escherichia coli,
Enterococcus faecalis, Micrococcus luteus,
Pseudomonas aeruginosa, Salmonella typhimurium and
Staphylococcus aureus) 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).11-14
Regarding the antifungal potential observed for the
compounds, the peracetylated glucoside 15 and galactoside
16 derivatives were active against C. parapsilosis at
96.4 µmol L-1, suggesting that the benzamidine group is
important for the activity, since the starting peracetylated
glucoside 11 and galactoside 12 were inactive at the highest
concentration evaluated, as shown in Table 1. In addition,
the presence of the peracetylated saccharide units in 15
or 16 also contributed to the antifungal potential of these
compounds, since the corresponding derivative 31 (devoid of
a saccharide moiety) was also inactive against this strain. The
benzamidine derivative 15 also showed a moderate activity
against other Candida spp. evaluated at 192.8 µmol L-1
(Table 1). Considering C. glabrata, this trend was not
observed, since derivative 31 was two-fold more active than
benzamidine derived from D-glucose (15).Among isopropyl
benzamidines, any saccharide unit contributed negatively for
antifungal activity of these derivatives, since only derivative
32 was active against C. parapsilosis (IC50 83.5 µmol L-1)
and C. tropicalis (IC50 167 µmol L-1).
Finally, for comparative purposes, the reaction of
3,4-diaminobenzamidines 4 or 6 with 3,4-dimethoxy-
benzaldehyde (in the conditions previously shown) afforded
the corresponding derivatives devoid of the saccharide
units, as shown in Scheme 5 below.
In vitro assays
All benzamidines, as well as the starting glycosides,
were evaluated against different species of fungi
The benzamidine nucleus also contributed to the
antibacterial activity of the synthesized series, and the

1314
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles
J. Braz. Chem. Soc.
Figure 1. Correlations between H-16 and C-20 by HMBC (a) and between H-15 and H-16 by COSY (b) for compound 20.
Scheme 5. Synthesis of 2-aryl-5-amidinobenzimidazoles 31 and 32.
compounds that showed the best potential were peracetylated
galactoside 16 (IC50 96.4 µmol L-1 against M. luteus),
N-acetylglucosamine glycoside 18 (IC50 96.5 µmol L-1
against E. faecalis), glucoside 19 (IC50 90.5 µmol L-1
against Gram-negative E. coli) and deacetylated lactoside
29 (IC50 96.4 µmol L-1 against E. faecalis), as shown in
Table 2. Interestingly, all starting glycosides (11-14) were
inactive against the evaluated bacterial strains, suggesting
the importance of the benzamidine nucleus for the activity
of these compounds. Regarding E. coli and E. faecalis, the
presence of a saccharide moiety attached to the benzamidine
nucleus was essential for the activity observed for the

Vol. 29, No. 6, 2018
de Souza et al.
1315

1316
Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles
J. Braz. Chem. Soc.

Vol. 29, No. 6, 2018
de Souza et al.
1317
compounds mentioned, since the derivatives having no sugar
units (31 and 32) were inactive against these two strains.
On the other hand, considering M. luteus, the presence of
carbohydrates did not contribute to the biological activity,
since benzamidines 31 and 32, devoid of a saccharide moiety,
were the most active compounds, showing antibacterial action
against this species at 97.0 and 83.5 µmol L-1, respectively.
In view of these findings, the derivatives 15, 16, 18,
19, 29, 31 and 32 can be considered for further molecular
modifications for design of new agents with antimicrobial
potential.
4. Souza, T. B.; Raimundo, P. O. B.; Andrade, S. F.; Hipólito, T.
M. M.; Silva, N. C.; Dias, A. L. T.; Ikegaki, M.; Rocha, R. P.;
Coelho, L. F. L.; Veloso, M. P.; Carvalho, D. T.; Dias, D. F.;
Carbohydr. Res. 2015, 410, 1.
5. Tuncbilek, M.; Kiper, T.;Altanlar, N.; Eur. J. Med. Chem. 2009,
44, 1024.
6. Ansari, K. F.; Lal, C.; Eur. J. Med. Chem. 2009, 44, 2294.
7. Ansari, K. F.; Lal, C.; Eur. J. Med. Chem. 2009, 44, 4028.
8. Özden, S.;Atabey, D.;Yildiz, S.; Göker, H.; Bioorg. Med. Chem.
2005, 13, 1587.
9. Göker, H.; Özden, S.;Yıldız, S.; Boykin, D.; Eur. J. Med. Chem.
2005, 40, 1062.
Conclusions
10. Souza, T. B.; Orlandi, M.; Coelho, L. F. L.; Malaquias, L. C.
C.; Dias, A. L. T.; Carvalho, R. R.; Silva, N. C.; Carvalho, D.
T.; Med. Chem. Res. 2014, 23, 496.
We described herein the synthesis of a new series
of glycosylated 2-aryl-5-amidinebenzimidazoles. The
compounds were obtained in good yields by coupling
vanillin glycosides with 3,4-diaminobenzamidines and
subsequent deacetylation. Two N-unsubstituted amidines
derived from D-glucose (15) and D-galactose (16)
showed antifungal activity against C. parapsilosis at
96.4 µmol L-1 and the non-glycosylated amidines 31 and
32 showed activity against C. glabrata (IC50 97 µmol L-1)
and C. parapsilosis (IC50 83.5 µmol L-1), respectively. In
addition, derivatives 16, 31 and 32 were actives against
M. luteus in the range of 83-97 µmol L-1, derivatives 18 and
29 showed activity against E. faecalis at 96 µmol L-1 and
compound 19 inhibited growth of E. coli at 90.5 µmol L-1.
11. European Committee on Antimicrobial Susceptibility Testing
(EUCAST); Antimicrobial Susceptibility Testing, EUCAST
Disk Diffusion Method, version 6.0, EUCAST, 2017. Available
at http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_
files/Disk_test_documents/Version_5/Manual_v_6.0_EUCAST_
Disk_Test_final.pdf, accessed in December 2017.
12. Clinical and Laboratory Standards Institute (CLSI); Performance
Standards for Antimicrobial Susceptibility Testing; Twenty-
Fourth Informational Supplement, CLSI document M100-S24;
Clinical Laboratory Standards Institute: Wayne, USA, 2014.
13. European Committee on Antimicrobial Susceptibility Testing
(EUCAST); Method for the Determination of Broth Dilution
Minimum Inhibitory Concentrations of Antifungal Agents for
Yeasts; Document E. Def 7.3.1; 2017. Available at http://www.
eucast.org/fileadmin/src/media/PDFs/EUCAST_files/AFST/
Files/EUCAST_E_Def_7_3_1_Yeast_testing__definitive.pdf,
accessed in December 2017.
Supplementary Information
Supplementary information (1H, 13C NMR and HRMS
spectra of the synthesized compounds) is available free of
charge at http://jbcs.org.br as PDF file.
14. International Organization for Standardization (ISO); ISO
20776-1: Clinical Laboratory Testing and In Vitro Diagnostic
Test Systems-Susceptibility Testing of Infectious Agents and
Evaluation of Performance of Antimicrobial Susceptibility Test
Devices; ISO: Geneva, Switzerland, 2006.
Acknowledgments
This work was supported by FAPEMIG, CNPq and
CAPES through grants and fellowships.
15. Fairley, T. A.; Tidwell, R. R.; Donkor, I.; Naiman, N. A.;
Ohemeng, K. A.; Lombardy, R. J.; Bentley, J. A.; Cory, M.;
J. Med. Chem. 1993, 36, 1746.
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Submitted: September 18, 2017
Published online: December 12, 2017
This is an open-access article distributed under the terms of the Creative Commons Attribution License.