Design, Synthesis and in Vitro Antibacterial Activity of 2-thiomethyl-benzimidazole Derivatives

Ablo Evrard¹, Coulibali Siomenan^1*, Camara Tchambaga Etienne¹, Toure Daouda², Coulibaly Souleymane¹, Sissouma Drissa¹, Adjou Ané¹

¹Laboratoire de Constitution et Réaction de la Matière, UFR Sciences des Structures de la Matière et Technologie, Université Félix Houphouët Boigny de Cocody, Abidjan, Côte d’Ivoire.
²Département de Biologie, UFR des Sciences Biologiques, Université Peleforo Gon Coulibaly, Korhogo, Côte d’Ivoire.

DOI: 10.4236/abc.2021.114012                

Abstract

A series of novel substituted benzimidazole (7a - n) derivatives were synthesized and characterized by ¹H, ¹³C Nuclear Magnetic Resonance (NMR) spectra and High Resolution Mass Spectrometry (HRMS). The substitution was done in position -1 and -2 by appropriate groups. These compounds are obtained by N-alkylation reaction with thiomethyl-1H-benzimidazole intermediates (5a - g). Design of intermediates (5a - g) was made by condensation reaction between 2-methylbenzimidazole thiourunium salt (3) and functionalized halides (4) in the presence of sodium hydroxide (NaOH). Among the twenty-one compounds synthesized, ten were evaluated for their antimicrobial activity on three bacterial strains namely: Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa ATCC 27853. Only E. coli ATTC 25922 was susceptible to the synthesized derivatives 5g, 7f and 7h with a significant antibacterial activity (CMI is between 250 and 500 μg/mL).

Keywords

Antibacterial Activity, Benzimidazole, 2-Methylbenzimidazole Thiouronium, Halide

1. Introduction

The benzimidazole scaffold is a significant pharmacophore with great interest due to its broad spectrum of biological activities [1] [2]. Its presence in a vast list of drugs such as etomidate, cimetidine, omeprazole, lansoprazole, azomycin, flumazenil, thyroliberin, methimazole, pilocarpine and etomidate in played the role of pharmacophore or as a substituent group [3]. A large number of works done towards benzimidazole scaffold became patents for a majority of biological properties, ranging from antitumor [4], anti-inflammatory [5] [6], anticancer [7] [8] and antifungal activities [9] [10] [11]. Among the structural variations on this ring, those affecting 1, 2 and 5 positions were very important for their pharmacological effect. Indeed, the optimization of the biological properties depends on the nature of the substituents on these positions [12] [13]. Recent studies showed that presence of a thiol group in 2 position enhanced biological activities such as antimicrobial [14], inflammatory [15], antiviral [16], antibacterial [17], antioxidant [18] [19], anticancer [20] and anti-proliferative [21]. It should be noted that, despite the large therapeutic arsenal, there is still an efficiency limit effects proved by the increase in strains resistant to bacteria. In this case, the design and synthesis of new antibacterial agents based on the flexibility of the benzimidazole scaffold became essential. Therefore, in this article, we designed new thiomethylbenzimidazole derivatives by introducing diverse substituents on 1 and 2 positions. The originality of our work lied in the introduction of the methylene group between the C-2 carbon of benzimidazole and the sulfur atom. The in vitro antimicrobial evaluation of the obtained compounds was conducted as well. Moreover, a detailed analysis of the structure of compounds would provide an opportunity to understand the structure-activity relationship and to identify a more advantageous option. Obtained results may be used for purposeful search of chemotherapeutic agents among compounds finding promising objects for studies aimed at developing compounds with other types of pharmacological activity.

2. Materials and Methods

2.1. Materials

2.1.1. Materials of Chemistry

All reagent-grade chemicals were obtained from commercial suppliers and were used as received. Unless otherwise indicated, ¹H and ¹³C (NMR) spectra were recorded on a Bruker Advance III spectrometer at ¹H (300 MHz), ¹³C (75 MHz) or ¹H (400 MHz), ¹³C (101 MHz) or ¹H (600 MHz), ¹³C (400 MHz), respectively, in CDCl₃, DMSO-d6 and Acetone-d6 solutions. For ¹H NMR assignments, the chemical shifts are reported in ppm on the δ scale. The following notation is used for the ¹H NMR spectral splitting patterns: s (singlet), d (doublet), dd (doublet of doublet), t (triplet), q (quadruplet), m (multiplet) and further qualified as app (apparent), br (broad) coupling constants, J are reported in Hz. (HRMS) were measured in the electrospray (ESI) mode on a LC-MSD TOF mass analyzer.

2.1.2. Biological Materials

Bacterial strains and inoculum preparation

The antibacterial tests were carried out on three bacterial mice: E. coli ATCC 25922, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 all from the Bacteriology laboratory of the Pasteur Institute of Côte d’Ivoire and isolated from the gastric fluid of a patient hospitalized at the University Hospital Center of Cocody (Abidjan). The standardization of the bacterial inoculun was carried out on colonies of young culture in 0.9% NaCl and measurement of the optical density with DENSIMAT made it possible to obtain an imoculum corresponding to approximately 10 CFU/mL.

2.2. Methods

2.2.1. Methods of Synthesis

Synthesis of benzimidazole derivatives

Synthesis methods of 2 methyl-1H-benzamidazole thiourunium chloride salt (3)

To a solution of 2-(chloromethyl)-1H-benzimidazole (1 eq, 57.2 mmol) in 50 mL of acetonitrile, thiourea (1 eq, 57.2 mmol) was added. The mixture was brought to reflux for 2 hours. After cooling to room temperature, a precipitate was formed, filtered, washed several times with ethyl acetate and then dried in the open air to afford brown crystals, yield = 92%, m.p = 192˚C.

General procedure for the synthesis of 2-((thioalkyl)methyl)-1H-benzimidazoles derivatives (5a - g)

To a solution of 2-methylbenzimidazole thiourunium chloride salt (1 eq, 2.61 mmol) in 10 mL of absolute ethanol was added 10 mL of sodium hydroxide solution. The mixture was stirred under reflux, and then an appropriate alkylating agent (1.2 eq, 3.14 mmol) was added. The reaction stayed like this for one more hour. After cooling to room temperature, the mixture was diluted in dichloromethane and washed several times with water. The organic phase was dried over anhydrous Na₂SO₄. And the solvent was evaporated in vacuo. The residue obtained after evaporation of solvent was purified by silica column chromatography (hexane/ethyl acetate: 7/3) to give compounds 5a - g.

General procedure for the synthesis of N-Alkyl 2-((thioalkyl)méthyl)-1H-benzimidazoles derivatives (7a - n)

To a solution of 2-((thioAlkyl)methyl)-1H-benzimidazole (1 eq, 10 mmol) in 8 mL of DMF, potassium carbonate (6 eq, 60 mmol) was added and the mixture was stirred at 50˚C for 1 hour. Ethyl or benzyl chloride (4 eq, 40 mmol) was added and the mixture was stirred for 3 hours at 50˚C. The reaction mixture was cooled to room temperature and the organic phase was extracted with dichloromethane, dried over anhydrous MgSO₄ and evaporated in vacuo. The residue obtained was purified by silica column chromatography (hexane/ethyl acetate: 7/3) to give compounds 7a - n.

Products characterizations

2-((methylthio)methyl)-1H-benzimidazole 5a

Yellow crystals, yield = 96%, m.p = 148˚C - 150˚C. ¹H NMR (600 MHz, Acetone-d₆) δ 7.55 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 7.18 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 4.00 (s, 2H, CH₂S), 2.57 (s, 3H, SCH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 152.27 (C=N), 121.74 (CH-Ar), 28.32 (CH₂-S), 25.40 (S-CH₃). HRMS (ESI) Calc. for C₉H₁₀N₂SNa (M + Na⁺) = 201.0251 Found = 201.0254.

2-((isobutylthio)methyl)-1H-benzimidazole 5b

Yellow crystals, yield = 68%, m.p = 126˚C - 126˚C. ¹H NMR (600 MHz, Acetone-d₆) δ 7.55 (t, J = 4.7 Hz, 2H, H-Ar), 7.18 (dd, J = 6.0, 3.1 Hz, 2H, H-Ar), 3.97 (s, 2H, CH₂S), 2.48 (d, J = 6.9 Hz, 2H, SCH₂), 1.80 (m, 1H, CH), 0.93 (d, J = 6.7 Hz, 6H, 2CH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 152.21 (C=N), 121.71 (CH-Ar), 40.56 (CH), 28.01 (CH₂S), 21.23 (CH₃). HRMS (ESI) Calc. for C₁₂H₁₇N₂S (M + H⁺) = 221.1308 Found = 221.1313.

3-(((1H-benzimidazol-2-yl)methyl)thio)propanoate d’éthyle 5c

Yellow crystals, yield = 25%, m.p = 104˚C - 106˚C. ¹H NMR (600 MHz, Acetone-d₆) δ 7.56 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 7.19 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 4.08 (q, J = 7.1 Hz, 2H, OCH₂), 4.04 (s, 2H, CH₂S), 2.85 (t, J = 7.2 Hz, 2H, CH₂C=O), 2.62 (t, J = 7.2 Hz, 2H, SCH₂), 1.19 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 171.23 (C=O), 151.99 (C=N), 121.82 (CH-Ar), 59.99 (OCH₂), 34.05 (CH₂CO), 28.60 (CH₂S), 26.62 (SCH₂), 13.61 (CH₃). HRMS (ESI) Calc. for C₁₅H₁₇O₂N₂S (M + H⁺) = 261.1054 Found = 261.1058.

2-((ethylthio)methyl)-1H-benzimidazole 5d

Yellow crystals, yield = 62%, m.p = 132˚C - 136˚C. ¹H NMR (600 MHz, Acetone-d₆) δ 7.56 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 7.19 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 4.01 (s, 2H, CH₂S), 2.59 (q, J = 7.4 Hz, 2H, SCH₂), 1.21 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 152.29 (C=N), 121.76 (CH-Ar), 28.34 (CH₂S), 25.42 (SCH₂), 13.82 (CH₃). HRMS (ESI) Calc. for C₁₀H₁₃N₂S (M + H⁺) = 193.0929 Found = 193.0925.

2-((butylthio)methyl)-1H-benzimidazole 5e

Yellow crystals, yield = 57%, m.p = 144˚C - 146˚C. ¹H NMR (300 MHz, CDCl₃) δ 7.54 (t, J = 4.7 Hz, 2H, H-Ar), 7.15 (dd, J = 6.0, 3.1 Hz, 2H, H-Ar), 3.95 (s, 2H, CH₂S), 2.44 (t, J = 6.9 Hz, 2H, SCH₂), 1.61-1.52 (m, 2H, CH₂), 1.41-1.26 (m, 2H, CH₂) 0.90 (t, J = 6.7 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 150.57 (C=N), 142.25, 135.01 (Cq-Ar), 122.39, 121.81, 119.47, 109.27 (CH-Ar), 31.33 (CH₂S), 31.02 (SCH₂), 27.87 (CH₂), 21.76 (CH₂), 13.51 (CH₃). HRMS (ESI) Calc. for C₁₂H₁₆N₂SNa (M + Na⁺) = 243.2319 Found = 243.2315.

2-(((1H-benzimidazol-2-yl)méthyl)thio)acétate d’éthyle 5f

Yellow crystals, yield = 96%, m.p = 68˚C - 72˚C. ¹H NMR (600 MHz, Acetone-d₆) δ 7.56 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 7.19 (dd, J = 6.0, 3.1 Hz, 2H, H-Ar), 4.13 (s, 2H, SCH₂C=O), 4.10 (q, J = 7.1 Hz, 2H, OCH₂), 3.45 (s, 2H, CH₂S), 1.21 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 169.66 (C=O), 151.28 (C=N), 121.83 (Cq-Ar), 60.78 (OCH₂), 32.97 (CH₂), 13.50 (CH₃). HRMS (ESI) Calc. for C₁₂H₁₅N₂O₂S (M + H⁺) = 251.0909 Found = 251.0913.

2-(((1H-benzimidazol-2-yl)méthyl)thio)propanoate d’éthyle 5g

Yellow crystals, yield = 38%, m.p = 100˚C - 102˚C. ¹H NMR (600 MHz, Acetone-d₆) δ 7.60 (dt, J = 6.7, 3.3 Hz, 2H, H-Ar), 7.23 (dd, J = 6.1, 3.1 Hz, 2H, H-Ar), 4.26 (d, J = 14.7 Hz, 1H, CH₂S), 4.17 (d, J = 14.7 Hz, 1H, CH₂S), 4.12 (q, J = 7.1 Hz, 2H, OCH₂), 3.72 (q, J = 7.2 Hz, 1H, CH), 1.42 (d, J = 7.2 Hz, 3H, CH₃), 1.23 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 172.28 (C=O), 151.47 (C=N), 121.87 (C-Ar), 60.71 (OCH2), 41.03 (CH), 28.60 (CH₂S), 16.63 (CH₂), 13.48 (CH₃). HRMS (ESI) Calc. for C₁₅H₁₇O₂N₂S (M + H⁺) = 265.0909 Found = 265.0905.

N-éthyl-2-((methylthio)methyl)-1H-benzimidazole 7a

Yellow oil, yield = 62%. ¹H NMR (300 MHz, CDCl₃) δ 7.36 - 7.32 (m, 1H, H-Ar), 7.28 (d, J = 1.9 Hz, 1H, H-Ar), 7.24 (d, J = 1.8 Hz, 1H, H-Ar), 4.25 (q, J = 7.3 Hz, 2H, CH₂N), 3.92 (s, 2H, CH₂S), 2.13 (s, 3H, CH₃), 1.47 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 150.09 (C=N), 142.08, 134.96 (Cq-Ar), 122.45, 121.85, 119.41, 109.28 (CH-Ar), 38.62 (CH₂N), 29.41 (CH₂S), 14.93 (CH₃S), 14.75 (CH₃CH₂). HRMS (ESI) Calc. for C₁₁H₁₅N₂S (M + H⁺) = 207.0949 Found = 207.0953.

N-éthyl-2-((méthyllthio)butyl)-1H-benzimidazole 7b

Yellow oil, yield = 67%. ¹H NMR (300 MHz, CDCl₃) δ 7.76 - 7.71 (m, 1H, H-Ar), 7.35 (ddd, J = 4.5, 2.3, 0.5 Hz, 1H, H-Ar), 7.28 (d, J = 1.9 Hz, 1H, H-Ar), 7.25 (d, J = 1.8 Hz, 1H, H-Ar), 4.27 (q, J = 7.3 Hz, 2H, CH₂N ), 3.94 (s, 2H, CH₂S), 1.61 - 1.52 (m, 2H, CH₂ ), 1.48 (t, J = 7.3 Hz, 3H, CH₃), 1.41 - 1.26 (m, 2H, CH₂), 0.87 (t, J = 7.3 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 150.57 (C2), 142.25 (C9), 135.01 (C4), 122.39 (C6), 121.81 (C7), 119.47 (C5), 109.27 (C8), 38.68 (CH₂N), 31.33 (CH₂S), 31.02 (CH₂S), 27.87 (CH₂), 21.76 (CH₂), 14.80 (CH₃), 13.51 (CH₃). HRMS (ESI) Calc. for C₁₄H₂₀N₂SNa (M + Na⁺) = 271.1439 Found = 271.1442.

N-éthyl-2-((méthylthio)éthyl)-1H-benzimidazole 7c

Yellow oil, yield = 79%. ¹H NMR (300 MHz,) δ 7.87 - 7.62 (m, 1H, H-Ar), 7.37 - 7.33 (m, 1H, H-Ar), 7.28 (d, J = 2.0 Hz, 1H, H-Ar), 7.25 (d, J = 1.9 Hz, 1H, H-Ar), 4.27 (q, J = 7.3 Hz, 2H, CH₂N), 3.96 (s, 2H, CH₂S), 2.59 (q, J = 7.4 Hz, 2H, CH₂CH₃), 1.48 (t, J = 7.3 Hz, 3H, CH₃), 1.25 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 150.59 (CN), 142.26, 135.03 (Cq-Ar), 122.46, 121.88, 119.52, 109.32 (CH-Ar), 38.74 (CH₂N), 27.55 (CH₂S), 25.64 (CH₂), 14.85 (CH₃), 14.28 (CH₃). HRMS (ESI) Calc. for C₁₆H₁₇N₂S (M + H⁺) = 221.1019 Found = 221.1021.

N-éthyl-2-((méthylthio)isobutyl)-1H-benzimidazole 7d

Red oil, yield = 75%. ¹H NMR (300 MHz, CDCl₃) δ 7.77 - 7.68 (m, 1H, H-Ar), 7.38 - 7.31 (m, 1H, H-Ar), 7.28 (d, J = 1.9 Hz, 1H, H-Ar), 7.25 (d, J = 1.9 Hz, 1H, H-Ar), 4.27 (q, J = 7.3 Hz, 2H, CH₂N), 3.94 (s, 2H, CH₂S), 2.47 (d, J = 6.8 Hz, 2H, CH₂), 1.79 (dt, J = 13.4, 6.7 Hz, 1H, CH), 1.48 (t, J = 7.3 Hz, 3H, CH₃), 0.93 (d, J = 6.6 Hz, 6H, 2CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 150.62 (C=N), 142.26, 135.03 (Cq-Ar), 122.41, 121.82, 119.51, 109.30 (CH-Ar), 40.61 (CH₂N), 38.72 (CH₂S), 28.32 (CH₂), 28.10 (CH), 21.81 (2CH₃), 14.82 (CH₃).

HRMS (ESI) Calc. for C₁₄H₂₀N₂SNa (M + Na⁺) = 271.1310 Found = 271.1306

N-benzyl-2-((méthylthio)méthyl)-1H-benzimidazole 7e

Red oil, yield = 82%. ¹H NMR (300 MHz, CDCl₃) δ 7.79 (dt, J = 7.6, 1.3 Hz, 1H, H-Ar), 7.37 - 7.15 (m, 6H, H-Ar), 7.07 (dd, J = 7.3, 2.2 Hz, 2H, H-Ar), 5.47 (s, 2H, CH₂N), 3.84 (s, 2H, CH₂S), 2.13 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 150.55 (C=N), 141.91, 135.71, 135.57 (Cq-Ar), 128.74, 127.69, 126.10, 122.72, 122.03, 119.40, 109.55 (CH-Ar), 46.92 (CH₂N), 29.48 (CH₂S), 14.85 (CH₃). HRMS (ESI) Calc. for C₁₆H₁₇N₂S (M + H⁺) = 269.1019 Found = 269.1015.

N-benzyl-2-((méthylthio)éthyl)-1H-benzimidazole 7f

Yellow crystals, yield = 72%, m.p = 96˚C - 98˚C. ¹H NMR (300 MHz, CDCl₃) δ 7.80 (m, J = 7.5, 1.4 Hz, 1H, H-Ar), 7.39 - 7.18 (m, 6H, H-Ar), 7.15 - 7.03 (m, 2H, H-Ar), 5.52 (s, 2H, CH₂N), 3.91 (s, 2H, CH₂S), 2.61 (q, J = 7.4 Hz, 2H, CH₂), 1.26 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 151.08 (C=N), 142.19, 135.86 (Cq-Ar), 135.76, 128.85, 127.79, 126.23, 122.76, 122.09, 119.58, 109.61 (CH-Ar), 47.08 (CH₂N), 27.78 (CH₂S), 25.60 (CH₂), 14.25 (CH₃). HRMS (ESI) Calc. for C₁₇H₁₈N₂SNa (M + Na⁺) = 306.1231 Found = 306.1235.

N-benzyl-2-((méthylthio)isobutyl)-1H-benzimidazole 7g

Yellow oil, yield = 69%. ¹H NMR (300 MHz, CDCl₃) δ 7.96 - 7.71 (m, 1H, H-Ar), 7.44 - 7.18 (m, 6H, H-Ar), 7.17 - 7.03 (m, 2H, H-Ar), 5.53 (s, 2H, CH₂N), 3.88 (s, 2H, CH₂S), 2.49 (d, J = 6.9 Hz, 2H, CH₂), 1.98 - 1.55 (m, 1H, CH), 0.96 (d, J = 6.6 Hz, 6H, 2CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 151.15 (C=N), 142.17, 135.87 (Cq-Ar), 135.79, 128.87, 127.81, 126.28, 122.77, 122.09, 119.59, 109.63 (CH-Ar), 47.09 (CH₂N), 40.59 (CH₂S), 28.56 (CH₂), 28.10 (CH), 21.82 (2 CH₃). HRMS (ESI) Calc. for C₁₉H₂₂N₂SNa (M + Na⁺) = 333.1525 Found = 333.1522.

N-benzyl-2-((méthylthio)butyl)-1H-benzimidazole 7h

Yellow crystals, yield = 60%, m.p = 78˚C - 80˚C. ¹H NMR (600 MHz, DMSO-d₆) δ 7.62 (dd, J = 7.5, 1.6 Hz, 1H, H-Ar), 7.38 - 7.33 (m, 1H, H-Ar), 7.31 (dd, J = 8.3, 6.7 Hz, 2H, H-Ar), 7.28 - 7.22 (m, 1H, H-Ar), 7.16 (ddt, J = 7.0, 3.0, 1.8 Hz, 4H, H-Ar), 5.55 (s, 2H, CH₂N), 4.01 (s, 2H, CH₂S), 2.58 - 2.52 (m, 2H, CH₂), 1.52 - 1.43 (m, 2H, CH₂), 1.30 (dt, J = 14.8, 7.4 Hz, 2H, CH₂), 0.82 (t, J = 7.4 Hz, 3H, CH₃). ¹³C NMR (151 MHz, DMSO-d₆) δ 152.06 (C=N), 142.59, 137.17 (Cq-Ar), 135.88, 129.06, 127.92, 127.15, 122.68, 122.09, 119.30, 110.99 (CH-Ar), 47.03 (CH₂N), 31.12 (CH₂S), 31.10 (SCH₂), 27.29 (CH₂), 21.69 (CH₂), 13.89 (CH₃). HRMS (ESI) Calc. for C₁₉H₂₂N₂SNa (M + Na⁺) = 333.1551 Found = 333.1552.

N-éthyl-2-(((1H-benzimidazol-2-yl)méthyl)thio)acétate d’éthyle 7i

Yellow oil, yield = 64%. ¹H NMR (300 MHz, CDCl₃) δ 7.86 - 7.67 (m, 1H, H-Ar), 7.41 - 7.34 (m, 1H, H-Ar), 7.34 - 7.20 (m, 2H, H-Ar), 4.29 (q, J = 7.3 Hz, 2H, OCH₂), 4.11 (s, 2H, SCH₂CO), 4.10 (q, J = 7.3 Hz, 2H, CH₂N), 3.39 (s, 2H, CH₂S), 1.50 (t, J = 7.3 Hz, 3H, CH₃), 1.23 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 169.89 (C=O), 149.57 (C=N), 142.38, 135.05 (Cq-Ar), 122.68, 122.02, 119.69, 109.41 (CH-Ar), 61.46 (OCH₂), 38.77 (SCH₂C=O), 33.16 (CH₂N), 28.14 (CH₂S), 14.96 (CH₃), 14.01 (CH₃). HRMS (ESI) Calc. for C₁₄H₁₈O₂N₂SNa (M + Na⁺) = 301.1009 Found = 301.1011.

N-benzyl-2-(((1H-benzimidazol-2-yl)méthyl)thio)acétate d’éthyle 7j

Red oil, yield = 62%. ¹H NMR (300 MHz, CDCl₃) δ 7.90 - 7.72 (m, 1H, H-Ar), 7.51 - 7.22 (m, 6H, H-Ar), 7.22 - 6.98 (m, 2H, H-Ar), 5.52 (s, 2H, CH₂N), 4.10 (q, J = 7.3 Hz, 2H, OCH₂) 4.03 (s, 2H, SCH₂C=O), 3.39 (s, 2H, CH₂S), 1.20 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 169.82 (C=O), 150.17 (C=N), 142.29, 135.93, 135.74 (Cq-Ar), 129.00, 127.97, 126.34, 123.06, 122.29, 119.76, 109.74 (CH-Ar), 61.49 (OCH₂), 47.19 (CH₂N), 33.21(SCH₂C=O), 28.47 (CH₂S), 14.00 (CH₃). HRMS (ESI) Calc. for C₁₉H₂₀O₂N₂SNa (M + Na⁺) = 363.1180 Found = 363.1178.

N-éthyl-2-(((1H-benzimidazol-2-yl)méthyl)thio)propanoate d’éthyle 7k

Red oil, yield = 60%. ¹H NMR (300 MHz, CDCl₃) δ 7.74 - 7.65 (m, 1H, H-Ar), 7.36 - 7.10 (m, 3H, H-Ar), 4.17 (q, J = 9 Hz, 2H, OCH₂), 4.14 - 4.00 (m, 4H, CH₂N, CH₂S), 3.57 (q, J = 7.2 Hz, 1H, CH), 1.46 - 1.35 (m, 6H, 2CH₃), 1.17 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 172.69 (C=O), 149.86 (C=N), 142.37, 134.97 (Cq-Ar), 122.69, 122.07, 119.59, 109.50 (CH-Ar), 61.31 (OCH₂), 41.34 (CH₂N), 38.82 (CH), 27.63 (CH₂S), 17.22 (CH₃), 14.97 (CH₃), 14.04 (CH₃). HRMS (ESI) Calc. for C₁₅H₂₁O₂N₂S (M + H⁺) = 293.1147 Found = 293.1150.

N-benzyl-2-(((1H-benzimidazol-2-yl)méthyl)thio)propanoate d’éthyle 7l

Red oil, yield = 63%. ¹H NMR (300 MHz, CDCl₃) δ 7.92 - 7.67 (m, 1H, H-Ar), 7.38 - 7.21 (m, 6H, H-Ar), 7.15 - 7.04 (m, 2H, H-Ar), 5.51 (d, J = 3.6 Hz, 2H, CH₂N), 4.08 (q, J = 12 Hz, 2H, OCH₂), 4.07 (d, J = 3.6 Hz, 1H, CH₂S), 4.06 (d, J= 3.6 Hz, 1H, CH₂S), 3.61 (q, J = 7.2 Hz, 1H, CH), 1.46 (d, J = 7.3 Hz, 3H, CH₃), 1.19 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 172.64 (C=0), 150.41 (C=N), 142.31, 135.71, 128.97 (Cq-Ar), 127.94, 126.30, 123.01, 122.29, 119.77, 109.73 (CH-Ar), 61.32 (OCH₂), 47.21 (CH₂N), 41.39 (CH), 27.97 (CH₂S), 17.23 (CH₃), 14.00 (CH₃). HRMS (ESI) Calc. for C₂₀H₂₃O₂N₂S (M + H⁺) = 355.1341 Found = 355.1338.

N-éthyl-3-(((1H-benzimidazol-2-yl)méthyl)thio)propanoate d’éthyle 7m

Red oil, yield = 63%. ¹H NMR (600 MHz, Acetone-d₆) δ 7.56 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 7.19 (dd, J = 6.0, 3.2 Hz, 2H, H-Ar), 4.10 (q, J = 7.3 Hz, 2H, CH₂N), 4.08 (q, J = 7.1 Hz, 2H, OCH₂), 4.04 (s, 2H, CH₂S), 2.85 (t, J = 7.2 Hz, 2H, CH₂C=O), 2.62 (t, J = 7.2 Hz, 2H, SCH₂), 1.50 (t, J = 7.3 Hz, 3H, CH₃), 1.19 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (400 MHz, Acetone-d₆) δ 171.23 (C=O), 151.99 (C=N), 142.33, 135.73 (Cq-Ar), 122.70, 122.10, 119.61, 109.52 (CH-Ar), 59.99 (OCH₂), 47.21 (CH₂N), 34.05 (CH₂CO), 28.60 (CH₂S), 26.62 (SCH₂), 13.61 (CH₃). HRMS (ESI) Calc. for C₁₅H₂₁O₂N₂S (M + H⁺) = 293.1279 Found = 293.1282.

N-benzyl-3-(((1H-benzimidazol-2-yl)méthyl)thio)propanoate d’éthyle 7n

Yellow crystals, yield = 61%, m.p = 134˚C - 136˚C. ¹H NMR (300 MHz, CDCl₃) δ 7.92 - 7.67 (m, 1H, H-Ar), 7.34 - 7.17 (m, 6H, H-Ar), 7.12 - 7.02 (m, 2H, H-Ar), 5.49 (d, J = 3.6 Hz, 2H, CH₂N), 4.04 (q, J = 7.1 Hz, 2H, OCH₂), 4.01 (s, 2H, CH₂S), 2.82 (t, J = 7.0 Hz, 2H, CH₂C=O), 2.58 (t, J = 7.0 Hz, 2H, SCH₂), 1.17 (t, J = 7.1 Hz, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃) δ 172.61 (C=0), 150.38 (C=N), 142.27, 135.67, 128.95 (Cq-Ar), 127.91, 126.26, 123.01, 122.28, 119.78, 109.74 (CH-Ar), 61.30 (OCH₂), 47.23 (CH₂N), 34.06 (CH₂CO), 28.59 (CH₂S), 26.61 (SCH₂), 13.63 (CH₃). HRMS (ESI) Calc. for C₂₀H₂₃O₂N₂S (M + H⁺) = 355.1452 Found = 355.1448.

2.2.2. Biological Methods

Preparation of stock solutions

The synthetic compounds were dissolved in dimethyl sulfoxide (DMSO) with the aim to get a concentration of 1000 µg/mL. This stock solution was used for the antibacterial tests. At this concentration, DMSO has no effect on the growth of the bacteria tested (negative control). Stock solutions were sterilized at 121˚C/15minutes. Sterility tests showed no microbial contamination.

Preparation of agar diffusion test

First, the antibacterial activity of the compounds was evaluated by the diffusion method described by the National Committee for Clinical Laboraty Standards (NCCLS) [22] with some modifications. The previously prepared bacterial inoculum was inoculated by swabbing onto each plate of Mueller-Hinton agar. A volume of 80 μL of each compound was placed in the wells made using a Pasteur pipette and after incubation at 37˚C for 18 - 24 hours. The diameters (including wells) of the inhibition zones around the wells was measured (in mm) by using a caliper. The compound was named active if the diameter of inhibition is ≥8 mm. A Ciprofloxacin disc (5 μg) was used as a reference antibiotic (positive control). Each test was repeated three times.

Methods to determine the Minimum Inhibitory Concentration (MIC)

The macro dilution technique in liquid medium reported by Okou et al. [23], with some modifications made it possible to determine the MIC. Double dilution series of each compound was distributed in 10 hemolysis tubes, followed by addition of bacterial inoculum at 10⁶ CFU/mL. A final concentration range of 0.98 to 500 μg/mL was obtained. The whole was incubated at 37˚C for 18 - 24 hours. The MIC is defined as the lowest concentration for which there is no visible growth.

3. Results and Discussions

3.1. Chemistry

The synthesis of new benzimidazole derivatives (7a - n) was carried out by interaction between 2-(substitutedthio)methyl)-1H-benzimidazole (5a - g) with alkyl or benzyl halides (Scheme 1). These new derivatives were obtained using the method described by Lopes et al. [24]. This method consists to heat at 60˚C in dimethylformamide (DMF), the mixture of compounds (5a - g) and alkyl or benzyl halides 6 in the presence of potassium carbonate (K₂CO₃). The reaction

Scheme 1. Synthesis route of compounds 7a - n.

generated an amide ion which further reacts with the electrophile to give the N-alkylated benzimidazoles. Compounds (7a - n) were isolated and purified by silica column chromatography. About the compounds (5a - g) synthesis, they were carried out by nucleophilic substitution reaction (S-alkylation) between 2-methylbenzimidazole thiourunium chloride salt (3) and functionalized alkyl halides (4). The reaction was set up in the presence of sodium hydroxide (NaOH) in a water-ethanol mixture for 1 hour. The benzimidazole thiouronium chloride salt (3) used to synthesize compounds (5a - g) was obtained via an intermediate reaction, by mixing compound 1 with thiourea 2 under reflux for 1 hour. Analysis of ¹H and ¹³C NMR spectra of compounds (5a - g) showed the presence of peaks corresponding to different alkyl groups. In the NMR spectra of compounds (7a - n) we noted the disappearance of pyrrolic nitrogen proton around 12 ppm and the appearance of peaks corresponding to ethyl and benzyl groups. The presence of N-ethyl groups of compounds (7a - n) was characterized by the presence of a quadruplet around 4.2 ppm with a coupling constant of 7.3 Hz. We could speculate on the fact that, quadruplet corresponds to two protons directly linked to nitrogen atom. Formation of N-benzyl compounds was also confirmed by the presence of a singlet around 5.5 ppm corresponding to protons of methylene group (CH₂N). For compounds 7l and 7n, the two protons was observed in form of doublet. Structures of both compounds were also confirmed by the ¹³C NMR spectrum.

3.2. Biology

3.2.1. Agar Diffusion Test

Among the synthesized benzimidazole derivatives, ten of them were in vitro evaluated by diffusion on agar method and macrodilution in liquid medium on E. coli ATCC 25922, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853. The results of agar diffusion test are reported in Table 1.

Compounds 5c, 5g, 7f and 7h showed good antibacterial activity on E. coli ATTC 25922 with inhibition diameters in a range between 15 ± 0.04 mm and 18 ± 0.01 mm, meaning that they are very sensitive to this bacterial strain. They also showed good antibacterial activity against S. aureus ATCC 25923 with inhibition diameters ranging from 11 ± 0.10 to 18 ± 0.02 mm. On the other hand, they had no effect on P. aeruginosa ATCC 27853 at a concentration of 1000 µg/mL. Compounds 7i, 7j, 7m, 7n, 7k and 7l, did not reveal antibacterial activity on E. coli ATTC 25922 and S. aureus ATCC 25923 at concentration of 1000 µg/mL with inhibition diameters less than 8 mm. While, they showed an efficient antibacterial activity on P. aeruginosa ATCC 27853 with inhibition diameters between 12 ± 0.02 mm and 16 ± 0.04 mm.

3.2.2. Minimun Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (CMB) Determination

The MIC was determined only for the most active molecules observed during sensitivity tests with induction of a diameter in the zone of inhibition equal or greater than 15 mm [25]. The results of MIC and CMB are reported in Table 2.

Table 1. Zone of inhibition diameters in mm (mean ± standard deviation).

Table 2. Minimum inhibitory concentration (MIC in µg/mL) and bactericidal concentration (CMB in µg/mL). NB: - is not determined.

Compounds 5g, 7f and 7h showed significant antibacterial activity with MIC ranging from 250 to 500 µg/mL on E. Coli ATTC 25922. Among these three molecules, only 5g inhibited efficiently S. aureus ATCC 25923 with a MIC = 250 µg/mL. As for 7n, it exhibited good antibacterial activity on P. aeruginosa ATCC 27853 with a MIC = 500 μg/mL. All compounds determined with the CMB, data revealed that the values were greater than 500 µg/mL. Among them, 5g presented the best Minimum Inhibitory Concentration (MIC = 250 µg/mL). 7k and 7l obtained respectively by introduction of an ethyl and benzyl group on pyrrolic nitrogen of compound 5g were inactive. Therefore, we conclude that N-alkylation was not improved the inhibitory activity of compound 5g.

4. Conclusion

We have prepared a series of heterocyclic compounds 5a - g and 7a - n from 2-chloromethyl-1H-benzimidazole 1. The antibacterial activity was evaluated in vitro for ten compounds against three bacterial mice: E. coli ATCC 25922, S. aureus ATCC 25923 and P. aeruginosa ATCC 27853 using the diffusion on agar method and macrodilution in liquid medium and Ciprofloxacin as a reference. Some have shown good antibacterial activity on the three bacterial mice. The MIC was performed only for the most active compounds and some showed significant antibacterial activity on E. Coli ATTC 25922. Moreover among these compounds, 5g exhibited the best minimum inhibitory concentration.

Acknowledgements

We wish to thank the laboratory (Laboratoire de Méthodologie et Synthèse de Produits Naturels) of the University of Quebec in Montreal (Canada) and the Laboratory LG2A of Jules Verne Picardic University (France) for providing us the chemical reagents and material for the spectroscopic analyzes.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References

[1]	Vinodkumar, R., Vaidya, S.D., Kumar, B.V.S., Bhise, U.N., Bhirud, S.B. and Mashelkar, U.C. (2008) Synthesis, Anti-Bacterial, Anti-Asthmatic and Anti-Diabetic Activities of Novel N-Substituted 2-(4-styrylphenyl)-1H-benzimidazole and N-substituted-3[4-(1H-benzimidazole-2-yl)-phenyl]-acrylic Acid Tertbutyl Ester. Arkivoc, 2008, 37-49. https://doi.org/10.3998/ark.5550190.0009.e05
[2]	Hazelton, J.C., Iddon, B., Suschitzky, H. and Woolley, L.H. (1995) 2H-Benzimidazoles (Isobenzimidazoles). Part 10. Synthesis of Polysubstituted O-Phenylenediamines and Their Conversion into Heterocycles, Particularly 2-Substituted Benzimidazoles with Known or Potential Anthelminthic Activity. Tetrahedron, 51, 10771-10794. https://doi.org/10.1016/0040-4020(95)00642-L
[3]	Sharma, S., Gangal, S. and Rauf, A. (2009) Convenient One-Pot Synthesis of Novel 2-Substituted Benzimidazoles, Tetrahydrobenzimidazoles and Imidazoles and Evaluation of Their in Vitro Antibacterial and Antifungal Activities. European Journal of Medicinal Chemistry, 44, 1751-1757. https://doi.org/10.1016/j.ejmech.2008.03.026
[4]	Wells, G., Lowe, P.R. and Stevens, M.F.G. (2000) Antitumor Benzothiazoles. 13. (Diacetoxy)Iodobenzene (DAIB) Oxidation of 2-(4-hydroxy-3-methoxyphenyl)benzothiazole and Related Compounds in the Presence of Dienophiles. Arkivoc, 2000, 779-797. https://doi.org/10.3998/ark.5550190.0001.511
[5]	Achar, K.C.S., Hosamani, K.M. and Seetharamareddy, H.R. (2010) In-Vivo Analgesic and Anti-Inflammatory Activities of Newly Synthesized Benzimidazole Derivatives. European Journal of Medicinal Chemistry, 45, 2048-2054. https://doi.org/10.1016/j.ejmech.2010.01.029
[6]	Khan, S.A. and Nandan, A.M. (1997) 2-Substituted Benzimidazoles as Antiinflammatory and Analgesic Agents. Indian Journal of Heterocyclic Chemistry, 7, 55-58.
[7]	Repicky, A., Jantova, S. and Cipak, L. (2009) Apoptosis Induced by 2-acetyl-3-(6-methoxybenzothiazo)-2-yl-amino-acrylonitrile in Human Leukemia Cells Involves ROS-Mitochondrial Mediated Death Signaling and Activation of p38 MAPK. Cancer Letters, 277, 55-63. https://doi.org/10.1016/j.canlet.2008.11.026
[8]	Devmurari, V.P., Shivanand, P., Goyani, M.B., Nandanwar, R.R., Jivani, N.P. and Perumal, P. (2010) Synthesis and Anticancer Activity of Some Novel 2-Substituted Benzothiazole Derivatives. International Journal of Chemistry and Technology Research, 2, 681-689.
[9]	Ansari, K.F. and Lal, C. (2009) Synthesis, Physicochemical Properties and Antimicrobial Activity of Some New Benzimidazole Derivatives. European Journal of Medicinal Chemistry, 44, 4028-4033. https://doi.org/10.1016/j.ejmech.2009.04.037
[10]	Vinodkumar, R., Vaidya, S.D., Kumar, B.V.S., Bhise, U.N., Bhirud, S.B. and Mashelkar, U.C. (2008) Synthesis, Anti-Bacterial, Anti-Asthmatic and Anti-Diabetic Activities of Novel N-substituted-2-(4-phenylethynyl-phenyl)-1H-benzimidazoles and N-Substituted 2[4-(4,4-dimethyl-thiochroman-6-yl-ethynyl)-phenyl)-1H-benzimidazoles. European Journal of Medicinal Chemistry, 43, 986-995. https://doi.org/10.1016/j.ejmech.2007.06.013
[11]	Vaidya, S.D., Kumar, B.V.S., Kumar, R.V., Bhise, U.N. and Mashelkar, U.C. (2007) Synthesis, Anti-Bacterial, Anti-Asthmatic and Anti-Diabetic Activities of Novel N-substituted-2-(benzo[d]isoxazol-3-ylmethyl)-1H-benzimidazoles. Journal of Heterocyclic Chemistry, 44, 685-691. https://doi.org/10.1002/jhet.5570440327
[12]	Mariappan, G., Hazarika, R., Alam, F., Karki, R., Patangia, U. and Nath, S. (2015) Synthesis and Biological Evaluation of 2-Substituted Benzimidazole Derivatives. Arabian Journal of Chemistry, 8, 715-719. https://doi.org/10.1016/j.arabjc.2011.11.008
[13]	Boggu, P., Venkateswararao, E., Manickam, M., Kwak, D., Kim, Y. and Jung, S.-H. (2016) Exploration of 2-Benzylbenzimidazole Scaffold as Novel Inhibitor of NF-κB. Bioorganic & Medicinal Chemistry, 24, 1872-1878. https://doi.org/10.1016/j.bmc.2016.03.012
[14]	Gund, D.R., Tripathi, A.P. and Vaidya, S.D. (2017) Synthesis and Antimicrobial Activity of Some Novel N-Substituted Benzimidazoles. European Journal of Chemistry, 8, 149-154. https://doi.org/10.5155/eurjchem.8.2.149-154.1563
[15]	Shafiee, A., Ebrahimzadeh, M.A., Zarghi, A. and Dehpour, A.R. (1998) Synthesis and Anti-Inflammatory Activity of 1-benzyl-2-(X-thio) pyrrolo [2,3-d]imidazole-5-carboxylates. Pharmacy and Pharmacology Communications, 4, 99-101.
[16]	Wang, Y., Inguaggiato, G., Jasamai, M., Shah, M., Hughes, D., Slater, M. and Simons, C. (1999) Synthesis and Biological Evaluation of Novel 2’-deoxy-4’-thio-imidazole Nucleosides. Bioorganic & Medicinal Chemistry, 7, 481-487. https://doi.org/10.1016/S0968-0896(98)00255-7
[17]	Kidwai, M., Misra, P., Dave, B., Bhushan, K.R., Saxena, R.K. and Singh, M. (2000) Microwave Activated Solid Support Synthesis of New Antibacterial Quinolones. Monatshefte fuer Chemie, 131, 1207-1212. https://doi.org/10.1007/s007060070029
[18]	Maddila, S., Palakondu, L. and Chunduri, V.R. (2010) Synthesis, Antibacterial, Antifungal and Antioxidant Activity Studies on 2-benzylthio- and 2-benzylsulfonyl-1H-imidazoles. Der Pharmacia Lettre, 2, 393-402.
[19]	Al-Zamil, N.O., Al-Sadhan, K.A., Isab, A.A., Wazeer, M.I. and Al-Arfaj, A.R.A. (2007) Silver (I) Complexes of Imidazolidine-2-thione and Triphenylphosphines: Solid-State, Solution NMR and Antimicrobial Activity Studies. Spectroscopy, 21, Article ID: 473914. https://doi.org/10.1155/2007/473914
[20]	Balewski, L., Saczewski, F., Bednarski, P.J., Gdaniec, M., Borys, E. and Makowska, A. (2014) Structural Diversity of Copper (II) Complexes with N-(2-Pyridyl)Imidazolidin-2-Ones(Thiones) and Their in Vitro Antitumor Activity. Molecules, 19, 17026-17051. https://doi.org/10.3390/molecules191017026
[21]	Cesarini, S., Spallarossa, A., Ranise, A., Schenone, S., Rosano, C., La Colla, P., Sanna, G., Busonera, B. and Loddo, R. (2009) N-Acylated and N,N’-diacylated imidazolidine-2-thione derivatives and N,N’-diacylated tetrahydropyrimidine-2 (1H)-thione Analogues: Synthesis and Antiproliferative Activity. European Journal of Medicinal Chemistry, 44, 1106-1118. https://doi.org/10.1016/j.ejmech.2008.06.010
[22]	National Commitee for Clinical Laboratory Standards (2000) NCCLS Approval Standard Document M2-A7. National Commitee for Clinical Laboratory Standards, Vilanova.
[23]	Okou, O.C., Yapo, S.E.-S., Kporou, K.E., Baibo, G.L., Monthaut, S. and Djaman, A.J. (2018) évaluation de l’activité antibactérienne des extraits de feuilles de Solanum torvum Swartz (Solanaceae) sur la croissance in Vitro de 3 souches d’entérobactéries. Journal of Applied Biosciences, 122, 12287-12295. https://doi.org/10.4314/jab.v122i1.8
[24]	Lopes, A.B., Wagner, P. and Gulea, M. (2019) Synthesis of Benzimidazole-Fused Medium-Sized N,S-Heterocycles via Palladium-Catalyzed Cyclizations: Synthesis of Benzimidazole-Fused Medium-Sized N,S-Heterocycles via Palladium-Catalyzed Cyclizations. European Journal of Organic Chemistry, 2019, 1361-1370. https://doi.org/10.1002/ejoc.201801686
[25]	Fertout-Mouri, N., Latreche, A., Mehdadi, Z. and Bengherraz, Z. (2016) Activité antibactérienne de quatre extraits de Teucrium polium L. du mont de Tessala (Algérie occidentale) [Antibacterial Activity of Four Extracts of Teucrium polium L. of Tessala mount (western Algeria)]. Bulletin de la Société Royale des Sciences de Liège, 85, 253-262.