Synthesis of new 1,2,3-triazole linked benzimidazolidinone: Single crystal X-ray structure, biological activities evaluation and molecular docking studies

2.2.1 Crystal data

C₂₀H₁₉N₅O₂, M_r = 361.40, monoclinic, I2/a (No. 15), a = 16.4233(16) Å, b = 5.2305(5) Å, c = 40.413(6) Å, b = 93.973(11)^°, a = g = 90^°, V = 3463.3(7) ų, T = 150.00(10) K, Z = 8, Z' = 1, m(Cu K_a) = 0.758, 15,059 reflections measured, 3076 unique (R_int = 0.0494) which were used in all calculations. The final wR₂ was 0.1231 (all data) and R₁ was 0.0451 (I ≥ 2 s(I)).

2.3.1 Antibacterial activity

All the newly synthesized 1,2,3-triazole linked benzimidazolidinones derivatives 5a-f were assessed in vitro against a variety of two Gram-positive bacteria (S. aureus and B. subtilis) and two Gram-negative bacteria (S. typhi and E. coli). By measuring the inhibition zone using well agar difusion assay the activity of all the investigated compounds was compared to that of Ampicillin, which was utilized as a standard reference antibiotic (in mm). The inhibiting properties against these four strains are given in Table 2. It was found that the newly generated compounds 5a-f have exerted globally moderate to good inhibitory activity against all of tested bacterial strains (IZ = 8 mm to 23 mm) compared to the reference antibiotic. In detail and starting with the inhibition effect towards gram positive bacteria; against S. aureus, based on zones of inhibition results and a part from the nature of the substituents in the para- and/or ortho-positions of the terminal phenyl, derivative 5f by its ortho-methoxy group showed the highest activity (IZ = 19 mm) compared to the other compounds and Ampicillin (IZ = 17 mm) followed by compound 5b with a methoxy group in the para position which displayed a zone of inhibition value equal to that of Ampicillin while 5c (R = 2,6-diCH₃) and 5e (R = 2CH₃) showed the lowest antibacterial effects (IZ = 9 mm, IZ = 8 mm respectively). This observation indicates the importance of methoxy groups in addition to triazole and benzimidazolidinone moieties for the S. aureus inhibition and the weak effect of methyl groups. Towards B. subtilis, derivatives 5b (IZ = 19 mm) with its 4-OCH₃, 5a (IZ = 18 mm) by its 4-Cl and 5f (IZ = 17 mm) through its 2-OCH₃ groups exhibited the highest antibacterial potentials than the reference antibiotic (IZ = 14 mm) and other analogues. Furthermore, with regard to the Gram-negative bacteria: against S. typhi 5b (IZ = 22 mm) was found to be the most active compound compared to its analogs followed by compounds 5a (IZ = 21 mm) and 5f (IZ = 20 mm). So, another time these three compounds showed greater inhibition potential than Ampicillin (IZ = 15 mm). In the same context towards E. coli, derivative 5b besides to 5a showed the highest antibacterial values followed by compound 5f as Table 2 showed. Thus, based on the above findings, we can be inferred that the hybrid molecules combining the two fragments: 1-aryl-1H-1,2,3-triazole and benzimidazolidinone are considered to be a main skeleton for the design of new antibacterial compounds.

2.3.2 Molecular docking for antibacterial activity

A Molecular docking test is the finest approach to study the binding affinity of ligands against target protein to more explain the antibacterial activity (Horchani et al, 2020, Horchani et al, 2022a). To estimate the optimal conformational position within the active site of the targeted protein, all of the produced compounds 5a-f were docked against: a crystal structure of a Gram-positive bacteria « Threonine synthase from B. subtilis ATCC 6633 » and a crystal structure of Gram-negative bacteria « MenB-1,4-dihydroxy-2-naphthoyl-CoA synthase from E. coli ATCC 25922 ». All the generated docked complexes were performed and analyzed on the basis of minimum energy values (kcal/mol) and hydrogen/hydrophobic interactions. All the created docked complexes were used to perform and analyze on the basis of minimum energy values (kcal/mol) and hydrogen/hydrophobic interactions.

From the docking results, as depicted in Table 3and Fig. 3, against B. subtilis,compound 5b having an interesting binding energy value is involved in two hydrogen bonds through its benzimidazolidinone and triazole fragment with THR83 and THR86 respectively. Additionally, a Pi-cation interaction is postulated with LYS59 by the para-methoxyphenyl group besides the appearance of some hydrophobic interactions with residues: PHE132 and ILE240. Thus, from the theoretical point of view, based on studies of molecular docking, the complex Threonine synthase-Ligand becomes stable by the formation of interactions between the amino acids forming the active site and the tested molecule, this is the case of our study by noticing the interactions formed and especially the intermolecular hydrogen bonds.

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Fig. 3

A) Top view of the accessible surface of Threonine synthase from B. subtilis ATCC 6633 (PDB: 6CGQ) active site. B) Schematic representation of different interactions of 5bwithin the catalytic cavity.

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Concerning the complex: E. coli-ligand, docking studies predicted that compound 5b would be the most active, with the lowest energy value (7.80 kcal/mol) when compared to other derivatives. 5b through its triazole pharmacophore showed H-bonding interaction with the side chain of GLN88 and SER161. The 1-isopropenyl-2-benzimidazolidinone fragment in turn displayed a Pi-Sigma interaction withVal108, Alkyl and Pi-Alkyl with LEU106 and Pi-Donor hydrogen bond with TYR97. 5b via its 4-methoxyphenyltriazole moiety exhibited a Pi-Alkyl interaction with VAL159 and Pi-Donor hydrogen bonds with amino acids: THR155 and SER161. Fig. 4. The results of the antibacterial assays are in accordance with the results from the molecular modeling calculations excellently. Thus, the 1,2,3-triazole linked benzimidazolidinone derivative is able to enter the enzyme pocket by showing significant binding interactions.

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Fig. 4

C) Top view of the accessible surface of Escherichia coli (PDB: 3 T88) active site. D) Schematic representation of different interactions of 5bwithin the catalytic cavity.

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2.3.3 Antifungal activity

Using the diffusion method, all newly synthesized compounds were inspected for antifungal activity against two fungi (A. fumigatus and A. brasiliensis) and two yeast strains (C. albicans and C. neoformans) (Murray et al., 1995). Antifungal activity of compounds was compared with the nystatin reference and the results of this study were presented in Table 4. The inhibition zone of strains ranged from 9 mm to 27 mm for fungi and 11 mm to 20 mm for yeast. These results show that more compounds show stronger inhibition against fungal strains. More specifically, the compound 5b with methoxy-groups at the para position of phenyl ring of benzimidazolone exhibit the strongest inhibition compared to other products against A. brasiliensis and A. fumigatus (between 27 mm and 26 mm), which make them even more efficient than the standard nystatin (Table 4). Moreover, the results of the biological activity showed also that compound 5a bearing the p-chloro substituted benzene moiety significantly increase the activity followed by compound 5f having 2-methoxyphenyl group while derivatives 5c, 5d and 5e methyl-substituted did not show good inhibition value against all strains used. In summary, may account for the observed antifungal activity may be due to the chemical structure of the produced compounds having methoxy group or halogen such as chlorine atom in addition to the 1,2,3-triazole linked benzimidazolidinone core.

2.3.4 In vitro anti-Inflammatory activity

Protein denaturation is a well-known source of inflammation. When a protein is denatured, it loses its secondary and tertiary structures due to external stresses such heat, strong acids or bases, concentrations of inorganic salts, or organic solvents. The majority of biological proteins also lose their biological activity (Mizushima et al., 1968, Grant et al., 1970). In the current study, the inhibition of albumin denaturation method was used to assess the in vitro anti-inflammatory activity of all derivatives 5a-f and the reference drug Aspirin. As shown in Table 5, the results clearly indicate that some derivatives have significantly good inhibition. Besides to the tirazole tethered with benzimidazolidinone, the substituents introduced into the phenyl ring directly related to the triazole heterocycle have an effect on the activity. Indeed, compound 5a with chlorine atom displayed better anti-inflammatory potential at different concentrations compared to its analogues and with values very close to those of Aspirin. This observation demonstrates the importance of halogens in the anti-inflammatory potency in particular the chlorine atom. On the other hand, the 4-methxoy derivative 5b displayed good inhibitory values followed by 2-methxoy derivative 5f while the compounds 5c, 5d and 5e with methyl group showed moderate activity. We can conclude from the above findings that the mesomeric electron donor effect can enhance the anti-inflammation, this is the case of methoxy group and chlorine atom whereas the methyl group exerted just an inductive donor effect that's why did not show significant activity.

2.3.5 Molecular docking for anti-inflammatory activity

Our attention was next directed to assess the selectivity of our developed compounds 5a-f based on molecular docking by comprehending their docking with the inflammatory target receptor (PDB: 3IAZ). Table 6 demonstrates that derivative 5a has the lowest energy value (−7.90 kcal/mol) in comparison with its analogues even compared to the reference ligand Aspirin. According to Fig. 5, it was observed that the most active compound 5a bounded to the active via several interesting interactions. The triazole ring was in interaction with THR430, PRO593 and TYR660 conventional Hydrogen bond, Pi-Donor hydrogen bond, Pi-Alkyl and Pi-Pi T-shaped interactions respectively. Moreover, 5a through its benzimidazolidinone pharmacophore is involved in Alkyl and Pi-Alkyl interactions with VAL591. In addition, the 4-chlorophenyl moiety interacted with PRO655 by an Alkyl interaction. Inspired by the previous observations, molecular modeling agrees well with in vitro results of anti-inflammatory activity.

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Fig. 5

E) Top view of the accessible surface of inflammatory protein (PDB: 3IAZ) active site. F) Schematic representation of different interactions of 5awithin the catalytic cavity.

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2.3.6 ADMET and predicted physicochemical properties

ADMET properties are known as key steps and appropriate methodologies used in drug development and discovery processes (Horchani et al., 2022b, Ye et al., 2019). Furthermore, the physicochemical properties are important parameters of a compound that influence efficacy, safety, or metabolism and can be predicted using Lipinski’s rule of five (RO5), as summarized in Table 7. The rule of five (RO5) deals with the reliance of active compounds and defines the eight pharmacokinetics parameters named as the molecular weight, H‐bond donors, H‐bond acceptors, log P, number of rotatable bonds, topological polar surface area, molecular volume, percentage of absorption. So, these pharmacokinetic parameters are associated with the acceptable aqueous solubility. For more details, the predicted values of many properties categorized as molecular weight describe the size of the compound. In addition, the value of log P corresponds to the lipophilicity of the molecules which is matched to the solubility of the drug molecule in the aqueous medium. Accordingly, the higher the solubility, the higher the activity of the therapeutic agents. Moreover, the parameters of the H‐bond donor and H‐bond acceptor signalize the quantification of all atoms (N, O) besides to their efficiency in the formation of the hydrogen bond. Thus, the results obtained from the ADMET analysis with ADMETlab 2.0 software unveiled that the predicted compounds 5a-f fully obeyed Lipinski’s rule of five. These findings suggest that these synthesized analogues are feasible in order to serve as a future biological candidates.

4.1.1 General information

The purity of products was confirmed by TLC using aluminium sheets (silica gel 60 F254) purchased from Merck. ¹H and ¹³C NMR spectra were recorded on Bruker AC-400 in deuteriochloroform or DMSO‑d₆ solution. Chemical shifts are taken in ppm using TMS as an internal standard. High resolution mass spectra (HRMS) were recorded using Micromass LCT mass spectrometers (ESI technique, positive mode). Melting temperatures were determined in open glass capillaries and are not corrected. All chemical shifts values were reported in ppm and coupling canstants are taken in Hz. Different signals multiplicities are described as following: s (singlet), d (doublet), t (triplet), m (multiplet) etc. All reaction mixture were monitored by TLC and purified by column chromatography using silica gell (40–63 μm).

Reagent and commercial products were purchased from Sigma Aldrich without prior purification. The starting material (2) was formed according to the littérature. (Li et al., 2010).

4.1.2 General procedure a for the synthesis of 1-(prop-1-en-2-yl)-3-(prop-2-yn-1-yl)-1H-benzo[d]imidazol-2(3H)-one 3

To a solution of 1-(prop-1-en-2-yl)-1H-benzo[d]imidazol-2(3H)-one (2) (4 g, 0.023 mol) in acetonitrile (70 mL) was added potassium carbonate (3.87 g, 0.028 mol). The reaction mixture was refluxed at 70 °C for 30 min before addition 1.2 equiv. of propargyl bromide (3.33 g, 0.028 mol). Then, the residue was extracted with ethyl acetate (120 mL × 3). The organic extracts were dried and filtred. The crude was evaporated under vacum and recrystallized from cyclohexane to give 3 (4.75 g, 80 %).

4.1.3 General procedure B for the synthesis of 1,2,3-triazole derivatives (5a-f)

To a mixture of alkyne 3 (400 mg, 1.88 mmol) in DCM (50 mL) and an equimolar amount of aryl azideswas added NEt₃ (0.38 mL, 2.82 mmol) and CuI (0.5 mol%). The reaction mixture was stirred at rt for 24 h. The solution was extracted with dichloromethane (60 mL × 3) and dried using Na₂SO₄. After evaporation of the solvent under vacuum The crude was evaporated under vacuum and recrystallized from cyclohexane to give 5a-f.

4.1.4 1-((1-(2,6-dimethylphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3-(prop-1-en-2-yl)-1H-benzo[d]imidazol-2(3H)-one (5c)

Prepared, according to general procedure B, Yield; 69 %, off-white solid; mp 122–124 °C. R_f = 0.28 [EtOAc/cyclohexane 3:7]. ¹H NMR (CDCl₃, 400 MHz): δ 1.94 (s, 6H, 2CH₃), 2.23 (s, 3H, CH₃), 5.21 (s, 1H, =CH), 5.31 (s, 2H, CH₂), 5.35 (d, 1H, J = 1,2 Hz, =CH), 7.10 (m, 3H, H_arom), 7.13(d,2H, J = 8.4 Hz, H_arom),7.24 (m, 2H, H_arom), 7.29 (m, 2H, H_arom), 7.63 (s, 1H, H_triazole). ¹³C NMR (CDCl₃, 75 MHz): δ 17.4, 20.2, 36.7, 108.7, 109.1, 113.1, 121.6, 121.8, 128.4, 128.9, 129.1, 130.1, 135.3, 135.7, 137.9, 152.6. HRMS, (ESI) calcd C21H21N5NaO [M + Na]+: 382.1637, found 382.1638.

4.2.1 Antibacterial screening

By using the Agar Well Diffusion Assay technique, the antibacterial properties of the synthesized compounds were assessed against two Gram-positive bacteria, i.e., S. aureus and B. subtilis, and two Gram-negative bacteria, i.e., S. typhi and E. coli. These pathogenic strains were grown for 24 h at 37 °C on nutrient agar. In 10 mL of physiological medium, colonies were suspended, and mixed for 5 min, and the suspensions were calibrated to 0.5 McFarland standard turbidity.Muller Hinton Agar medium plates were covered with one millilitre of bacterial suspension and incubated for 30 min at 37 °C. The seeded agar plates were punched with wells that were approximately 6 mm in diameter, and each test substance was then put to the wells after being reconstituted in DMSO. For all of the testsubstances, DMSO served as the control. The plates were kept at room temperature for two hours to allow the chemicals to diffuse into the agar before the palates were incubated at 37 °C for 24 h. By using a digital calliper, the diameters of the clear zones of inhibition surrounding the sample were measured after 24 h.

4.2.2 Antifungal activity

Yeasts Strains: two yeast strains, C. albicans and C. neoformans, were cultivated on Sabouraud agar for 48 h at 37 °C (the ideal temperature for bacterial growth). The suspensions of pure colonies were then adjusted to 0.5 McFarland standard turbidity after being suspended in 10 mL of physiological medium and well stirred for 5 min. Plates of Sabouraud agar medium received one milliliter of yeast suspension, which was then incubated at 37 °C for 30 min. Then, using a sterile glassy borer, 6 mm diameter wells were drilled into the agar medium.The synthesized compounds were produced in DMSO (1 mg/mL) and added to the appropriate wells; as a control, DMSO was added to one of the wells. For 48 h, these plates were kept in an incubator at 37 °C to promote yeast growth. By using a digital calliper, the diameters of the clear zones of inhibition surrounding the sample were measured after 48 h.

Fungal Strains: two fungi strains, A. brasiliensis and A. fumigatus were cultured on inclined Sabouraud agar in Falcon tube 15 mL at 25 °C for 7 days (optimal temperature for their growing). The fungus strain's spore was measured at 106 spores/mL after being suspended in peptone water. After that, Sabouraud agar medium plates were covered with one millilitre of the fungal suspension and incubated at 25 °C for 30 min. Then, using a sterile glassy borer, 6 mm diameter wells were drilled in agar media. The synthesized compounds were produced in DMSO (1 mg/mL) and added to the appropriate wells; as a control, DMSO was added to one of the wells. For five days, these plates were kept in an incubator at 25 °C to promote fungi development. After five days, a digital caliper was used to measure the diameters of the clear zone of inhibition surrounding the sample.

4.2.3 In vitro anti-inflammatory activity

The anti-inflammatory activity of benzimidazolone derivatives 5a–f was determined as below:

Various concentrations of 2 mL of each compound was mixed individually with 3 mL of sodium phosphate buffer saline (pH 6.4) containing 0.2 mL of eggs albumin yielding a final volume of 5 mL. The pure blank does not contain the substrate, but will be added 3 mL of buffer solution. All compounds were re-suspended in the DMSO followed by dilution in the buffer in order that the DMSO does not exceed 1 %. Control was given a comparable volume of double-distilled water. The mixture was then heated at 70 °C for 5 min after being incubated at 37 °C for about 15 min and the absorbance was determined at 660 nm after cooling. To determine absorbance, aspirin (a standard medication) was used as positive control and as a reference medication and handled accordingly. Using the formula listed below, the % inhibition of protein denaturation was determined: $$\mathrm{P}\mathrm{e}\mathrm{r}\mathrm{c}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}=\left(\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{C}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}-\mathrm{a}\mathrm{b}\mathrm{s}\mathrm{S}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)\mathrm{x}100/\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}$$