2-[(6''-CHLORO-1''H-BENZIMIDAZOLE-2''-YL)THIO]-N-(3-CHLOROPHENYL)ACETAMIDE; 2-[(6''-CHLORO-1''H-BENZIMIDAZOLE-2''-YL)THIO]-N-(4-CHLOROBENZYL)ACETAMIDE AND 6-CHLORO-2-(NONYLTHIUM)-1H-BENZIMIDAZOLE AND THEIR USE AS QUORUM SENSING INHIBITORS.

Abstract

This invention relates to the synthesis of three benzimidazoles, designed using bioisosterism, synthesized by a two-step methodology: first, the synthesis of a thione, and in the second step, the alkylation of the thione to obtain the benzimidazoles 5a-5c. These compounds exhibit quorum sensing inhibitor activity, which was evaluated using a biosensor. Compounds 5a and 5b show excellent and good quorum sensing inhibitor activity, respectively; compound 5c exhibits low activity. To understand the reasons for this behavior, molecular docking calculations were performed. These calculations revealed that this behavior is due not to point interactions, atom by atom, but to supramolecular interactions, in which point-plane interactions play a crucial role.

Description

2-[(6”-CHLORO-1”H-BENZIMIDAZOLE-2”-YL)THIO]- / V-(3-CHLOROPHENYL)ACETAMIDE; 2-[(6”CHLORO-1”H-BENZIMIDAZOLE-2”-YL)THIO]-N-(4-CHLOROBENZYL)ACETAMIDE AND 6-CHLORO-2(NONYLTHIUM)-I / 7-BENZIMIDAZOLE AND THEIR USE AS QUORUM SENSING INHIBITORS. FIELD OF INVENTION The present invention falls within the field of organic chemistry and pharmaceuticals, specifically concerning benzimidazole compounds with antimicrobial biological activity and quorum sensing inhibitor activity. It relates to the process of obtaining three benzimidazole compounds and their use in combating drug-resistant bacteria by inhibiting quorum sensing. BACKGROUND OF THE INVENTION Drug-resistant bacteria exist, primarily in hospitals, and are opportunistic, infecting immunocompromised individuals. This problem exists in Mexico and throughout the world, even in developed countries. One way to eliminate or minimize this problem is through quorum sensing (QS) inhibition. This approach is attractive because QS inhibitors do not generate drug resistance since they do not harm the bacteria; they only disrupt bacterial communication, a phenomenon that triggers virulence factors. Having different QS inhibitor compounds available is important because it broadens the range of options, in addition to the intrinsic properties of the compounds themselves, which are used as drugs or disinfectants. In the prior art we find patent US7790904B2 called in English “Substituted benzimidazole derivatives” and US8143422B2 with the title “Benzimidazole derivatives” in which some compounds with benzimidazole acetamide structure are shown and their potential use in medicines is mentioned, however, no reference is made to the diseases or the mechanism of action. Furthermore, the literature has reported several antiparasitic benzimidazole compounds and modulators of metabolic disorders, some even acting as quorum sensing inhibitors in Pseudomonas aeruginosa. However, the synthesis method for the compounds described in this invention, the compounds themselves, and their evaluation in QS inhibition have not been previously reported. Testing with Chromobacterium violaceum provides an approach to studying QS, in addition to its antimicrobial and antipathogenic activity, by using this bacterium as a biosensor. The compounds mentioned in the present invention open a new window of possibilities in quorum sensing research. To our knowledge, there is currently no drug or medication, such as benzimidazoles, that includes a compound with quorum sensing inhibitory activity, which is essential to minimize or eliminate hospital-acquired infections. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to the method of synthesizing benzimidazole compounds (5a-5c) with the structure shown in Figure 4 and their use as quorum sensing inhibitors, designed by bioisosterism (Figure 1). The three benzimidazoles (5a-5c) synthesized using the method described in this invention are novel compounds and have not been previously tested as quorum sensing inhibitors. These compounds were synthesized in two steps: the formation of a thione (39), whose structure is shown in Figure 2 and its IR spectrum in Figure 3; and the subsequent alkylation of the thione to obtain the benzimidazoles. The thione was alkylated, and the benzimidazoles (5a-5c) were obtained. Once synthesized, the compounds were evaluated in vitro using the Chromobacterium violaceum CviR biosensor. Finally, to understand the reason for the activity of these benzimidazoles, a molecular docking analysis was performed. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Design of benzimidazoles as bioisosteres of acyl homoserine lactones. Figure 2. Intermediate in the synthesis of benzimidazoles. Figure 3. 13C NMR spectrum of thione (39). Figure 4. Structure of the benzimidazoles 5a-5c. Figure 5. Alkylation of thione (39) to obtain the benzimidazoles 5a-5c. Figure 6. Structure of 2-[(6”-chloro-7”H-benzimidazol-2”-1l)t1o]-A / -(3-chlorophenyl)acetamide (5a). Figure 7. Structure of 2-[(6”-chloro-77- / -benzimidazol-2”-yl)thio]-N-(4-chlorobenzyl)acetamide (5b). Figure 8. Structure of 6-chloro-2-(nonylthio)-7H-benzimidazole (5c). Figure 9. Interactions of the ligand-protein complex for compound (5a), showing μ-μ interactions. Figure 10. Reaction conditions for obtaining the intermediate (39). Figure 11. Evaluation of 5-chloro-2-mercaptobenzimidazoles in Chromobacterium violaceum. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to certain benzimidazole compounds (5a, 5b and 5c) with a general structure shown in Figure 4, to the process of synthesizing the compounds, as well as their antimicrobial and quorum sensing inhibitory activity. The two-stage process for obtaining the compounds comprises: 1st stage. Synthesis of 5-chloro-1,3-dihydro-2H-benzimidazol-2-thione Table 1. Reaction conditions for obtaining the intermediate (39). Test Energy Source Time (h) Efficiency (%) 1 Conventional 3 52 2 MW 3 24 3 Conventional 5 88 Conditions: phenylenediamine (1 Eq), carbon disulfide (1.3 Eq), potassium hydroxide (3 Eq), EtOH / H2O 7:3 (5 mL), 80 °C. In an ACE pressure vessel, preferably equipped with magnetic stirring, 4-chlorophenylenediamine (5.61 mmol, 1 Eq), carbon disulfide (7.23 mmol, 1.3 Eq), potassium hydroxide (16.83 mmol, 3 Eq), and 5 mL of an ethanol / water mixture (7:3) are placed. The reaction mixture is heated to 80 °C for 5 h. After the reaction time has elapsed, the solvent is evaporated under reduced pressure, and 15 mL of a 5% aqueous HCl solution are added to the residue. The suspension is stirred for 15 min and then vacuum filtered. The solid retained on the filter paper is washed with 40 mL of distilled water in two 20 mL portions, and the solid is dried at 40 °C in an oven. The compound is thus obtained. An advantage of this method is that the thione (39) is synthesized without a catalyst. The thione was characterized and the following data were presented: Efficiency: 88%. Brown solid. PF 303 °C 1H NMR (300 MHz, Acetona-d6) δ: 7.17 (dd, J = 1.86, 0.86 Hz, 1H, H-6), 7.14 (d, J = 0.86 Hz, 1H, H-4), 7.13 (d, J = 1.86 Hz, 1 Η, H-7). 13C NMR (75 MHz, Acetona-d6) δ 170.4 (C-2), 133.7 (C-4a), 131.7 (C-7a), 127.1 (C-5), 122.1 (C-6), 110.4 (C-7), 109.3 (C-4). DIP-MS (ESI; MH) m / z (calculated): 182.97, m / z (encontrado): 182.88. The 13C MRI spectroscopy coincides with the report. 2a. Etapa-Alquilación de la 5-chloro-1,3-dihidro-2H-benzimidazol-2-tiona. In a container, such as a 100 mL round-bottom flask equipped with magnetic stirring, the intermediate, thione (39) (1.35 mmol, 1 Eq), the alkyl halide (1.35 mmol, 1 Eq), and 15 mL of ethanol are placed. The reaction mixture is stirred for 16 hours at 60 °C. After this time, the solvent is evaporated from the reaction mixture, and the solid is suspended in a 20% aqueous potassium carbonate solution (30 mL). The solution is then vacuum filtered, and the solid retained on the filter paper is washed with 40 mL of distilled water in two 20 mL portions and dried at 40 °C in an oven. The three compounds (see Figure 6-8) synthesized using the preferred method were spectroscopically characterized and yielded the following data: • 2-[(6”-chloro-7''H-benzimidazol-2”-yl)thio]- / V-(3-chlorophenyl)acetamide (5a) Efficiency: 80%. Solid construction. PF 228-231 °C. Rf = 0.28 (hexane / AcOEt 7:3); IR (film) ü = 1652.2, 1586.4, 1423.7 cm1. NMR Ή (300 MHz, DMSO-d6) δ: 10.76 (s, 1H, NH), 7.77 (s, 1H, H2'), 7.64 (d, J = 1.64 Hz, 1 Η, H-7”), 7.56 (d, J = 8.63 Hz, 1 Η, H-4”), 7.42 (d, J = 8.36 Hz, 1 Η, H-6'), 7.31 (m, 2H, H-5',5), 7.09 (d, J = 7.84 Hz, 1 Η, H-4'), 4.41 (s, 2H, H-2). RMN13C (75 MHz, DMSOd6) δ 166.0 (C-1), 152.3 (C-2”), 140.5 (C-1'), 137.1 (C-7a”), 134.9 (C-4a”), 133.5 (C-3 ), 131.0 (C5'), 128.3 (C-6 ), 124.1 (C-5”), 123.8 (C-4 ), 119.0 (C-2'), 117.9 (C-6'), 115.1 (C-4”), 113.8 (C-7”), 36.9 (C-2). DIP-MS (ESI; MH) m / z (calculated): 349.99, m / z (encontrado): 349.93. • 2 - [(6”-chloro-· / ”H-benzim¡dazol-2”-¡l)thio] -N- (4-chlorobencyl) acetamida (5b) Efficiency: 83%. Solid blue. PF 115-118 °C. Rf = 0.15 (hexane / AcOEt 7:3); IR (film) ú = 3735.7, 3649.3, 3283.2, 1640.0, 1557.9, 1393.9 cm1. NMR Ή (300 MHz, DMSO-d6) δ: 8.85 (t, J = 5.95 Hz, 1H, NH), 7.53 (d, J = 1.98 Hz, 1H, H-7”), 7.4 (d, J = 8.64 Hz, 1H, H-4”), 7.23 (m, 5H, H3',4',5”), 4.25 (d, J= 5.95 Hz, 2H, H-1'), 4.11 (s, 2H, H-2). RMN13C (75 MHz, DMSO-de) δ 167.4 (C-1), 152.1 (C-2), 139.3 (C-7a), 138.4 (C-2'), 136.9 (C-4a”), 131.7 (C-5'), 129.3 (C-3'), 128.5 (C4'), 127.1 (C-6”), 122.9 (C-5”), 115.1 (C-4”), 114.0 (C-7”), 42.2 (C-Γ), 35.4 (C-2). DIP-MS (ESI; ΜΗ) m / z (calculated): 364.00, m / z (encountered): 363.95. • 6-chloro-2-(nonylthio)-íH-benzimidazol (5c) Yield: 70%. Pink solid. PF 73-76 °C. Rf = 0.66 (hexane / AcOEt 7:3); IR (film) ü = 3032.6, 1389.6 cm-1. NMR Ή (300 MHz, CDCI3) δ: 9.89 (s, 1H, NH), 7.51 (d, J= 1.97 Hz, 1H, H-7), 7.42 (d, J = 8.56 Hz, 1H, H-4), 7.16 (dd, J= 8.56, 1.97 Hz, 1H, H-5), 3.28 (m, 2H, Η-Γ), 1.71 (m, 2H, H-2'), 1.27 (m, 12H, H-3',4',5',6',7',8'), 0.84 (t, J = 6.83 Hz, 3H, H-9'). RMN13C (75 MHz, CDCI3) δ 152.6 (C-2), 139.8 (C-7a), 137.8 (C-4a), 127.9 (C-6), 122.7 (C-5), 114.6 (C-4), 113.8 (C 7), 32.7 (C-1'), 31.8, 29.5 (C-2'), 29.4, 29.2, 29.1 (C-3 ), 28.7 (C-4'), 22.6, 14.1 (C-9'). DIP-MS (ESI; MH) m / z (calculated): 309.11, m / z (encontrado): 309.05. To demonstrate the use of the calculations, below, some of the evaluations carried out will be described. Antimicrobial and QS inhibiting effect. Evaluation of compounds synthesized in Chromobacterium violaceum CV026 To evaluate the synthesized compounds as potential quorum sensing inhibitors, the bacterium Chromobacterium violaceum CV026 was chosen. This strain has been reported as a biosensor for quorum sensing inhibition studies because it produces only the dye violacein when quorum sensing occurs. The dye is easily detectable and quantifiable. Furthermore, the CV026 strain does not synthesize its own AHL10, allowing researchers to control the amount of violacein produced by the bacterium according to the concentration of AHL added to the culture medium. This is crucial, as high concentrations of violacein hinder the observation of the inhibitory effect.The compounds in question yielded the following results when evaluated as QS inhibitors, and the results for the 5-chloro-2-mercaptobenzimidazole series are shown in Figure 11. It was observed that, at a concentration of 1000 μM, compound 5a exhibited an antimicrobial effect, while at concentrations of 100 pM and 10 pM it caused inhibition of 85% and 27%, respectively, with an IC50 value of 36.67 pM. Benzimidazole 5b showed inhibition at concentrations of 1000 pM and 100 pM, with inhibitions of 95% and 31%, respectively. The IC50 was 376.92 pM. Compound 5c exhibited QS inhibition of 23% at 1000 pM, 24% at 100 pM, and 17% at 10 pM. In order to find explanations for the activity of these benzimidazoles as bioisosteres of AHL, molecular docking studies were carried out, observing that the benzimidazoles have supramolecular bonds with the amino acids of the CviR protein. Evaluation by molecular docking. Modeling and optimization of the ligands to be evaluated. The 2D chemical structures of the evaluated ligands were generated in the ACD / ChemSketch program, subsequently the structures were modeled in the Gaussview 5.0 program and optimized to a semi-empirical calculation level AM-1 using the Gaussian 09 program. Protein preparation The PDB files corresponding to the three-dimensional structure of the proteins used were downloaded from the Protein Data Bank. These PDB files were subjected to Ramachandran analysis on the RAMPAGE server at the University of Cambridge. Proteins were included if the amino acid residue values ​​in favorable regions were at least 90%. Subsequently, water molecules and the crystallized ligand were removed from the three-dimensional structure using the PyMOL program. The activity of these compounds is due to the total supramolecular interactions of the entire molecule with the receptor, although the relevant role that the aromatic rings play in the complexation of the bioisosteres with the receptor protein CviR can be noted due to the ττ-π interactions they present, which were observed by molecular docking (Figure 9). In addition, it was verified by docking that the excellent and good activity as quorum sensing inhibitors of compounds 5a and 5b is due to the amide groups and the aromatic rings, elements that benzimidazole 5c does not have, thus explaining its low activity since even at 1000 μM it did not reach 50% activity.

Claims

1. Compounds derived from benzimidazole, characterized by having the chemical formulas: a) 2-[(6”-chloro-7”H benzimidazole-2”-yl)thio]- / V-(3-chlorophenyl)acetamide, (5a) b) 2-[(6”-chloro-f ”H-benzimidazole-2’,-yl)thio]-N-(4-chlorobenzyl)acetamide, (5b) c) 6-chloro-2-(nonylthio)-1 H-benzimidazole, (5c) 2. The compounds according to claim 1, characterized by having antimicrobial and anti-quorum sensing activity.

3. The compounds according to claim 1, for use in the preparation of drugs for the treatment of nosocomial infections resulting from the quorum sensing phenomenon.

4. The compounds according to claim 1, characterized in that (5a) is a purple solid with a melting point between 228-231 °C.

5. The compounds according to claim 1, characterized in that (5a) has Rf = 0.28 (hexane / AcOEt 7:3); IR (film) 0 = 1652.2, 1586.4, 1423.7 crrr1. NMR Ή (300 MHz, DMSO-de) δ. 10.76 (s, 1H, NH), 7.77 (s, 1H, H-2'), 7.64 (d, J= 1.64 Hz, 1H, H-7”), 7.56 (d, J = 8.63 Hz, 1H, H-4), 7.42 (d, J = 8.36 Hz, 1H, H-6'), 7.31 (m, 2H, H-5'.5”), 7.09 (d, J = 7.84 Hz, 1H, H-4'), 4.41 (s, 2H, H-2). 13C NMR (75 MHz, DMSO-cfe) δ 166.0 (C-1), 152.3 (C2”), 140.5 (C-1'), 137.1 (C-7a”), 134.9 (C-4a), 133.5 (C-3'), 131.0 (C-5 ), 128.3 (C-6'), 124.1 (C-5”), 123.8 (C-4'), 119.0 (C-2), 117.9 (C-6'), 115.1 (C-4”), 113.8 (C-7), 36.9 (C-2). DIP-MS (ESI; MH) m / z (calculated): 349.99, m / z (found): 349.

93.

6. The compounds according to claim 1, characterized in that 5a exhibits antimicrobial and anti-quorum sensing activity at a concentration from 10 μM- 1000 μM.

7. The compounds according to claim 1, characterized in that 5a has quorum sensing inhibitory activity of 27-85%.

8. The compounds according to claim 1, characterized in that 5b is a blue solid, with a melting point between 115-118 °C.

9. The compounds according to claim 1, characterized in that 5b has Rf = 0.15 (hexane / AcOEt 7:3); IR (film) 0 = 3735.7, 3649.3, 3283.2, 1640.0, 1557.9, 1393.9 cnr 1. 1H NMR (300 MHz, DMSO-d6) δ: 8.85 (t, J= 5.95 Hz, 1H, NH), 7.53 (d, J = 1.98 Hz, 1H, H7"), 7.4 (d, J = 8.64 Hz, 1H, H-4"), 7.23 (m, 5H, H-3',4',5"), 4.25 (d, J= 5.95 Hz, 2H, H-1'), 4.11 (s, 2H, H-2). 13C NMR (75 MHz, DMSO-d6) δ 167.4 (C-1), 152.1 (C-2”), 139.3 (C-7a”), 138.4 (C-2'), 136.9 (C-4a”), 131.7 (C-5 ), 129.3 (C-3’), 128.5 (C-4 ), 127.1 (06”), 122.9 (05”), 115.1 (04”), 114.0 (07”), 42.2 (C-1'), 35.4 (C-2).

10. The compounds according to claim 1, characterized in that 5b exhibits antimicrobial and anti-quorum sensing activity at a preferred concentration between 100 μM and 1000 μM.

11. The compounds according to claim 1, characterized in that 5b has quorum sensing inhibitory activity of 31-95% 12. The compounds according to claim 1, characterized in that 5c is a pink solid, with a melting point of 73-76 °C.

13. The compounds according to claim 1, characterized in that 5c has Rf = 0.66 (hexane / AcOEt 7:3); IR (film) ü = 3032.6, 1389.6 cm1. 1H NMR (300 MHz, CDCh) δ: 9.89 (s, 1H, NH), 7.51 (d, J= 1.97 Hz, 1H, H-7), 7.42 (d, J= 8.56 Hz, 1H, H-4), 7.16 (dd, J = 8.56, 1.97 Hz, 1H, H-5), 3.28 (m, 2H, H-1'), 1.71 (m, 2H, H-2'), 1.27 (m, 12H, H3',4',5',6',7',8'), 0.84 (t, J = 6.83 Hz, 3H, H-9'). 13C NMR (75 MHz, CDCI3) δ 152.6 (C-2), 139.8 (C-7a), 137.8 (C-4a), 127.9 (C-6), 122.7 (C-5), 114.6 (C-4), 113.8 (C-7), 32.7 (C-1'), 31.8, 29.5 (C-2'), 29.4, 29.2, 29.1 (C-3'), 28.7 (C-4'), 22.6, 14.1 (C-9). DIP-MS (ESI; MH) m / z (calculated): 309.11, m / z (found): 309.

05.

14. The compounds according to claim 1, characterized in that 5c exhibits antimicrobial and antiquorum sensing activity at a concentration between 10 μM- 1000 μM 15. The compounds according to claim 1, characterized in that 5c has quorum sensing inhibitory activity of 17-24% 16. A synthetic method for obtaining certain benzimidazole compounds, characterized by using biosysosterism from the catalyst-free alkylation of a thione, comprising the following two steps: a) Synthesis of 5-chloro-1,3-dihydro-2H-benzimidazole-2-thione. In a pressure vessel, such as an ACE pressure tube equipped with magnetic stirring, 4-chlorophenylenediamine (5.61 mmol, 1 Eq), carbon disulfide (7.23 mmol, 1.3 Eq), potassium hydroxide (16.83 mmol, 3 Eq) and 5 mL of an ethanol / water (7:3) mixture are placed. The reaction mixture is heated to 80 °C for 5 h. Once the reaction time has elapsed, the solvent is evaporated under reduced pressure and 15 mL of a 5% aqueous HCl solution is added to the residue. The suspension is stirred for 15 min and then vacuum filtered. The solid retained on the filter paper is washed with 40 mL of distilled water in two 20 mL portions. The solid is then dried at 40 °C in an oven.The result is a thione. b) Alkylation of 5-chloro-1,3-dihydro-2H-benzimidazol-2-thione. In a container such as a 100 mL round-bottom flask equipped with magnetic stirring, the thione obtained in the previous step (1.35 mmol, 1 Eq), the alkyl halide (1.35 mmol, 1 Eq), and 15 mL of ethanol are placed. The reaction mixture is kept under stirring for 16 hours at 60 °C. After this time, the solvent is evaporated from the reaction mixture, and the solid is suspended in a 20% aqueous potassium carbonate solution (30 mL). Subsequently, it is vacuum filtered. The solid retained on the filter paper is washed with 40 mL of distilled water in two 20 mL portions and dried at 40 °C in an oven.

17. The method of claim 16, characterized by having an 80% yield in the synthesis of the compound 2-[(6”-chloro-7’7- / benzimidazol-2”-yl)thio]-A / -(3-chlorophenyl)acetamide.

18. The method of claim 16, characterized by having an 83% yield in the synthesis of compound 2-[(6”-chloro-777-benzimidazol-2”-1l)thio]-N-(4-chlorobenzyl)acetamide.

19. The method of claim 16, characterized by having a 70% yield in the synthesis of the compound 6-chloro-2-(nonylthio)-1 H-benzimidazole.