Synthesis and Antimicrobial Activity of Glycosylated 2-Aryl-5-amidinobenzimidazoles

Abstract

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

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

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 Staphyloccocus aureus and Staphylococcusepidermidis, 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

Scheme 1
Retrosynthetic analysis for the target compounds.

Experimental

Chemistry

The proposed 2-aryl-5-amidinobenzimidazoles were synthesized from the reaction of two 3,4-diaminobenzamidines 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 on Spectrum One, PerkinElmer spectrophotometer. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were obtained on Bruker Avance 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.

Synthesis of acetamidobenzonitrile (1)

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)

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.

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.

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, J9.0 Hz, 1H, H arom.), 7.19 (d, J9.0 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.

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) 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-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), 3047, 2942 (NH.HCl amidine), 1685 (C=N); ¹H 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, J5.6 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.

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, J8.4 Hz, 1H, H arom.), 6.95 (s, 1H, H arom.), 6.59 (d, J8.2 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.

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, J8.2 Hz, 1H, H arom.), 5.53 (s, 2H, NH₂), 4.92 (s, 2H, NH₂), 4.04 (m, 1H, CH), 1.20 (d, J5.6 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.

General procedure for the synthesis of peracetylated 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.

4-Formyl-2-methoxyphenyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (11)

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 aldehyde); ¹H NMR (400 MHz, CDCl₃) d 9.89 (s, 1H, CHO), 7.43-7.40 (m, 2H, H-9 and H-11), 7.21 (d, J8.0 Hz, 1H, H-8), 5.34-5.28 (m, 2H, H-2 and H-3), 5.13 (t, J6.8 Hz, 1H, H-4), 5.09 (d, J6.4 Hz, 1H, H-1), 4.27 (dd, 1H, H-6, ²J 12.4 Hz, ³J5.2 Hz), 4.18 (dd, ²J 12.4 Hz, ³J 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)

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, J8.4 Hz, 1H, H-8), 5.55 (t, J9.2 Hz, 1H, H-2), 5.46 (d, J2.8 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, J8.0 Hz, 1H, H-1), 4.23 (dd, ²J 11.8 Hz, ³J 6.8 Hz, 1H, H-6), 4.16 (dd, ²J 11.2 Hz, ³J 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.

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; 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, J8.0 Hz, 1H, H-8), 5.35 (d, J2.8 Hz, 1H, H-4'), 5.32 (t, J8.8 Hz, 1H, H-3), 5.22 (t, J8.8 Hz, 1H, H-2), 5.14-5.08 (m, 2H, H-1 and H-2'), 4.97 (dd, ²J 12.0 Hz, ³J 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.

Synthesis of peracetylated glycoside (14)

2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl 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.

4-Formyl-2-methoxyphenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β -D-glucopyranoside (14)

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 (s, 1H, CHO), 7.42 (s, 1H, H-11), 7.41 (dd, ³J 10.4 Hz, ⁴J 1.6 Hz, 1H, H-9), 7.22 (d, J8.0 Hz, 1H, H-8), 5.86 (d, J8.0 Hz, 1H, NH), 5.50 (t, J9.6 Hz, 1H, H-3), 5.40 (d, J8.0 Hz, 1H, H-1), 5.13 (t, J9.6 Hz, 1H, H-4), 4.27 (dd, ²J 12.0 Hz, ³J5.4 Hz, 1H, H-6), 4.16 (dd, ²J 12.0 Hz, ³J2.4 Hz, 1H, H-6'), 4.06 (qr, J8.4 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.

General procedure for the synthesis of peracetylated 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.

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), 1740 (C=O ester), 1675 (C=N); ¹H 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, J8.8 Hz, 1H, H-8), 5.53 (d, J8 Hz, 1H, H-1), 5.41 (t, J9.6 Hz, 1H, H-3), 5.11 (t, J8.8 Hz, 1H, H-2), 5.03 (t, J9.6 Hz, 1H, H-4), 4.27-4.21 (m, 2H, H-6 and H-5), 4.11 (d, J10.4 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-(2,3,4,6-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), 1741(C=O ester), 1675 (C=N); ¹H 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, J8.4 Hz, 1H, H-8), 5.44 (d, J6.4 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-(2,3,6,2',3',4',6'-Hepta-O-acetyl-β-D-lactosyloxy)-3-methoxyphenyl]benzimidazole-5-carboxamidine hydrochloride (17)

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, J7.0 Hz, 1H, H-9), 7.77 (br s, 1H, H-15), 7.67 (d, J7.0 Hz, 1H, H-16), 7.26 (d, J8.4 Hz, 1H, H-8), 5.48 (d, J8.0 Hz, 1H, H-1), 5.30 (t, J8.0 Hz, 1H, H-3), 5.25 (d, J3.2 Hz, 1H, H-4'), 5.18 (dd, ²J 10.0 Hz, ³J 3.6 Hz, 1H, H-3'), 5.02 (t, J9.0 Hz, 1H, H-2), 4.88 (t, J9.2 Hz, 1H, H-2'), 4.80 (d, J8.0 Hz, 1H, H-1'), 4.38 (d, ²J 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.

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, J8.4 Hz, 1H, H-8), 5.47 (d, J8.0 Hz, 1H, H-1), 5.26 (t, J10.0 Hz, 1H, H-3), 4.94 (t, J9.6 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) d 169.9, 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-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° (c0.42 MeOH); IR (ATR) ν / cm^-1 3072 (NH.HCl amidine), 1743 (C=O ester), 1660 (C=N); ¹H NMR (400 MHz, 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, J8.4 Hz, 1H, H-16), 7.77 (br s, 1H, H-11), 7.55 (d, J8.4 Hz, 1H, H-9), 7.29 (d, J8.8 Hz, 1H, H-8), 5.53 (d, J8 Hz, 1H, H-1), 5.42 (t, J9.6 Hz, 1H, H-3), 5.11 (t, J8.8 Hz, 1H, H-2), 5.03 (t, J9.6 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, J6.4 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-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)carboxamidine] hydrochloride (20)

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° (c0.44 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, J8.0 Hz, 1H, H-15), 7.77 (d, J8.0 Hz, 1H, H-16), 7.55 (d, J8.4 Hz, 1H, H-9), 7.29 (d, J8.4 Hz, 1H, H-8), 5.43 (d, J6.8 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, J6.4 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.

2-[4-(2,3,6,2',3',4',6'-Hepta-O-acetyl-β-D-lactosyloxy)-3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)carboxamidine] hydrochloride (21)

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° (c0.40 MeOH); IR (ATR) ν / cm^-1 3366, 3083 (NH.HCl amidine), 1742 (C=O ester), 1672 (C=N); ¹H 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, J8.4 Hz, 1H, H-9), 7.73 (br s, 1H, H-15), 7.51 (d, J7.6 Hz, 1H, H-16), 7.22 (d, J8.4 Hz, 1H, H-8), 5.44 (d, J8.0 Hz, 1H, H-1), 5.26 (t, J9.6 Hz, 1H, H-3), 5.22 (d, J3.2 Hz, 1H, H-4'), 5.15 (dd, ²J 10.0 Hz, ³J 3.4 Hz, 1H, H-3'), 4.99 (t, J9.0 Hz, 1H, H-2), 4.84 (t, J9.0 Hz, 1H, H-2'), 4.76 (d, J8.0 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, J6.4 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.

2-[4-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyloxy)-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° (c0.42 MeOH); IR (ATR) ν / cm^-1 3296, 3098 (NH.HCl amidine), 1729 (C=O ester), 1663 (C=N); ¹H 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, J8.8 Hz), 8.01 (s, 1H, H-18), 7.94 (s, 1H, H-11), 7.86 (d, J8.4 Hz, 1H, H-9), 7.75 (s, 1H, H-15), 7.55 (d, J8.0 Hz, 1H, H-16), 7.32 (d, J8.4 Hz, 1H, H-8), 5.47 (d, J8.4 Hz, 1H, H-1), 5.27 (t, J9.8 Hz, 1H, H-3), 4.95 (t, J9.6 Hz, 1H, H-4), 4.23 (dd, ²J 12.0 Hz, ³J 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, J6.4 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)

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.

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); IR (ATR) ν / cm^-1 3132 (OH), 1675 (C=N); ¹H NMR (400 MHz, DMSO-d₆) d 13.66 (br s, 1H, NH benzimidazole), 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, J8.4 Hz, 1H, H-9), 7.74 (d, J8.0 Hz, 1H, H-15), 7.64 (d, J8.0 Hz, 1H, H-16), 7.24 (d, J8.8 Hz, 1H, H-8), 5.03 (d, J6.4 Hz, 1H, H-1), 3.82 (s, 1H, OCH₃), 3.66 (d, J11.2 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.

2-[4-(β-D-Galactopyranosyloxy)-3-methoxyphenyl]benzimidazole-5-carboxamidine (24)

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, J8.4 Hz, 1H, H-9), 7.73 (br s, 1H, H-15), 7.63 (d, J8.0 Hz, 1H, H-16), 7.24 (d, J8.8 Hz, 1H, H-8), 5.12 (br s, 1H, OH), 4.99 (d, J7.6 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-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); IR (ATR) ν / cm^-1 3198 (OH), 1674 (C=N); ¹H NMR (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, J8.4 Hz, 1H, H-9), 7.77 (d, J8.0 Hz, 1H, H-15), 7.66 (d, J8.4 Hz, 1H, H-16), 7.28 (d, J8.4 Hz, 1H, H-8), 5.41 (d, J7.6 Hz, 1H, H-1), 4.26 (d, J7.6 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); IR (ATR) ν / cm^-1 3241 (OH), 1681 (C=N); ¹H NMR (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, J8.4 Hz, 1H, H-16), 7.33 (d, J8.8 Hz, 1H, H-8), 5.17 (d, J8.4 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)

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, J8.8 Hz, 1H, H-9), 7.74 (d, J8.4 Hz, 1H, H-15), 7.53 (d, J8.4 Hz, 1H, H-16), 7.26 (d, J8.4 Hz, 1H, H-8), 5.31 (br s, 1H, OH), 5.14 (br s, 1H, OH), 5.05 (d, J7.2 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, J10.8 Hz, 1H, H-6), 3.48-3.16 (m, 5H, H-2, H-3, H-4, H-5 and H-6'), 1.30 (d, J6.4 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.

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); IR (ATR) ν / cm^-1 3122 (OH), 1669 (C=N); ¹H 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, J8.4 Hz, 1H, H-9), 7.74 (br s, 1H, H-15), 7.54 (d, J8.4 Hz, 1H, H-16), 7.27 (d, J9.2 Hz, 1H, H-8), 5.11 (br s, 1H, OH), 5.02 (d, J8.0 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, J6.4 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.

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); IR (ATR) ν / cm^-1 3232 (OH), 1668 (C=N); ¹H 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, J8.4 Hz, 1H, H-9), 7.74 (d, J8.4 Hz, 1H, H-15), 7.52 (d, J8.0 Hz, 1H, H-16), 7.27 (d, J8.4 Hz, 1H, H-8), 5.48 (br s, 1H, OH), 5.32 (d, J7.6 Hz, 1H, H-1), 5.11 (br s, 1H, OH), 4.82-4.55 (m, 5H, OH), 4.26 (d, J6.4 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, J6.0 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.

2-[4-(2-Acetamido-2-deoxy-β-D-glycopyranosyloxy)-3-methoxyphenyl]benzimidazole-5-[N-(isopropyl)carboxamidine] (30)

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); IR (ATR) ν / cm^-1 3234 (OH), 1668 (C=N); ¹H 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, J8.4 Hz, 1H, H-9), 7.73 (d, J8.4 Hz, 1H, H-15), 7.52 (d, J8.4 Hz, 1H, H-16), 7.28 (d, J8.4 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, J6.0 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.

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 (NH.HCl amidine), 1686 (C=N); ¹H NMR (400 MHz, 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, J8.4 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.

2-(3,4-Dimethoxyphenyl)-N-isopropyl-1H-benzimidazole-5-carboxamidine hydrochloride (32)

This product was obtained in 70% yield as a brown solid, mp 165.2-168.0 °C, IR (ATR) ν / cm^-1 3074 (NH.HCl amidine), 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, J8.4 Hz, 1H, H-12), 7.54 (dd, ³J 19.2 Hz, ⁴J 8 Hz, 1H, H-5), 7.15 (d, J8.8 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, J6.4 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.

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.

Results and Discussion

Chemistry

The preparation of 3,4-diaminobenzamidines (4 and 6) was performed in good yields from the nitrobenzamidines3 or 5 as described by Fairleyet 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).

Scheme 2
Synthesis of 3,4-diaminobenzamidines 4 and 6.

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 Souzaet 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 around 8 Hz in the corresponding ¹H 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.

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.

Scheme 4
Synthesis of glycosylated 2-aril-5-amidinobenzimidazoles 15-30.

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 benzimidazoles 15-22 were obtained in good yields (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 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.

Scheme 5
Synthesis of 2-aryl-5-amidinobenzimidazoles 31 and 32.

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

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.

Finally, for comparative purposes, the reaction of 3,4-diaminobenzamidines 4 or 6 with 3,4-dimethoxybenzaldehyde (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 (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).

Table 1
In vitro antifungal activity of the synthesized compounds

Compound	IC50 (in mass / (µg mL-1)) / (µmol L-1)						IC90 (in mass / (µg mL-1)) / (µmol L-1)
	C. albicans ATCC 10231	C. tropicalis ATCC 750	C. krusei ATCC 6258	C. glabrata ATCC 90030	C. parapsilosis ATCC 22019		C. albicans ATCC 10231	C. tropicalis ATCC 750	C. krusei ATCC 6258	C. glabrata ATCC 90030	C. parapsilosis ATCC 22019
11	-	-	-	-	-		-	-	-	-	-
12	-	-	-	-	-		-	-	-	-	-
13	-	-	-	-	-		-	-	-	-	-
14	-	-	-	-	-		-	-	-	-	-
15	192.8 (125)	192.8 (125)	192.8 (125)	192.8 (125)	96.4 (62.5)		-	-	-	-	192.8 (125)
16	-	-	-	-	96.4 (62.5)		-	-	-	-	-
17	-	-	-	-	-		-	-	-	-	-
18	-	-	-	-	-		-	-	-	-	-
19	-	-	-	-	-		-	-	-	-	-
20	-	-	-	-	-		-	-	-	-	-
21	-	-	-	-	-		-	-	-	-	-
22	-	-	-	-	-		-	-	-	-	-
23	-	-	-	-	-		-	-	-	-	-
24	-	-	-	-	-		-	-	-	-	-
25	-	-	-	-	-		-	-	-	-	-
26	-	-	-	-	-		-	-	-	-	-
27	-	-	-	-	-		-	-	-	-	-
28	-	-	-	-	-		-	-	-	-	-
29	-	-	-	-	-		-	-	-	-	-
30	-	-	-	-	-		-	-	-	-	-
31	-	-	-	97.0 (31.25)	-		-	-	-	-	-
32	-	167.0 (62.5)	-	-	83.5 (31.25)		-	-	-	-	-
Fluc	1.6 (0.5)	3.2 (1.0)	104.4 (16.0)	52.2 (32.0)	3.2 (1.0)		-	-	-	-	-

• IC₅₀: inhibitory concentrations of microbial growth at 50%; IC₉₀: inhibitory concentrations of microbial growth at 90%; -: no significant activity; fluc: fluconazole.

The benzamidine nucleus also contributed to the antibacterial activity of the synthesized series, and the 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 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.

Table 2
In vitro antibacterial activity of the synthesized compounds

Compound	IC50 (in mass / (µg mL-1)) / (µmol L-1)							IC90 (in mass / (µg mL-1)) / (µmol L-1)
	Escherichia coli ATCC 25922	Enterococcus faecalis ATCC 51299	Micrococcus luteus ATCC10240	Pseudomonas aeruginosa ATCC 27853	Salmonella typhimurium ATCC 14028	Staphylococcus aureusATCC 6538		Escherichia coli ATCC 25922	Enterococcus faecalis ATCC 51299	Micrococcus luteus ATCC10240	Pseudomonas aeruginosa ATCC 27853	Salmonella typhimurium ATCC 14028	Staphylococcus aureus ATCC 6538
11	-	-	-	-	-	-		-	-	-	-	-	-
12	-	-	-	-	-	-		-	-	-	-	-	-
13	-	-	-	-	-	-		-	-	-	-	-	-
14	-	-	-	-	-	-		-	-	-	-	-	-
15	-	-	-	-	-	-		-	-	-	-	-	-
16	192.8 (125)	-	96.4 (62.5)	-	-	-		-	-	-	-	-	-
17	-	-	-	-	-	-		-	-	-	-	-	-
18	-	96.5 (62.5)	-	-	-	-		-	-	-	-	-	-
19	90.5 (62.5)	-	-	-	-	-		-	-	181.0 (125)	-	-	-
20	-	-	-	-	-	-		-	-	-	-	-	-
21	-	-	-	-	-	-		-	-	127.7 (125)	-	-	-
22	-	-	-	-	-	-		-	-	-	-	-	-
23	-	-	-	-	-	-		-	-	-	-	-	-
24	-	140.7 (62.5)	-	-	-	-		-	-	-	-	-	-
25	-	-	-	-	-	-		-	-	-	-	-	-
26	-	-	-	-	-	-		-	-	-	-	-	-
27	-	-	-	-	-	-		-	-	-	-	-	-
28	-	-	-	-	-	-		-	-	-	-	-	-
29	-	96.4 (62.5)	-	-	-	-		-	-	-	-	-	-
30	-	-	-	-	-	-		-	-	-	-	-	-
31	-	-	97.0 (31.25)	-	-	194.0 (62.5)		-	-	-	-	-	-
32	-	-	83.5 (31.25)	-	-	-		-	-	-	-	-	167.0 (62.5)
Chl	3.1 (1.0)	6.2 (2.0)	1.6 (0.5)	99.0 (32.0)	3.1 (1.0)	6.2 (2.0)		12.3 (4.0)	24.7 (98.0)	3.1 (1.0)	198.0 (64.0)	6.2 (2.0)	12.3 (4.0)

• IC₅₀: inhibitory concentrations of microbial growth at 50%; IC₉₀: inhibitory concentrations of microbial growth at 90%; -: no significant activity; chl: chloramphenicol.

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.

Conclusions

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.

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.

https://minio.scielo.br/documentstore/1678-4790/ZdSYWNbnvmtkzg6qZt4Kgnw/c357d6b5750814abd9ca1e63e8a63a90c0c54a47.pdf

Acknowledgments

This work was supported by FAPEMIG, CNPq and CAPES through grants and fellowships.

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Publication Dates

• Publication in this collection
  June 2018

History

• Received
  18 Sept 2017
• Accepted
  12 Dec 2017