Description:Background of Invention
Benzimidazole boasts a broad spectrum of utility within the realms of synthetic [1] and medicinal chemistry [2]. It finds application in diverse domains, including but not limited to antifungal, anti-inflammatory, antibacterial, antioxidant, antiallergic, enzyme inhibition, anti-HIV, herbicidal, antidiabetic, anticancer, and insecticidal activities [3-14]. This renowned heterocyclic core, benzimidazole, serves as a fundamental building block in numerous widely-recognized pharmaceuticals, such as omeprazole and pimobendan (Fig. 1).

Tuberculosis stands as a formidable threat among lethal diseases, arising from infection with Mycobacterium tuberculosis [15]. Mycobacterium smegmatis, due to its genetic resemblance and shared biological traits with Mycobacterium tuberculosis, serves as a valuable model in research. Notably, its non-pathogenic nature to humans, coupled with its rapid growth rate, renders it an ideal laboratory organism. Despite the availability of antibiotics for tuberculosis treatment, the demand persists for superior, more discerning, and less toxic antibacterial agents. In our quest to design benzimidazole derivatives, we aimed to synthesize long chain alkyl-substituted benzimidazoles, endowed with antibacterial properties against Mycobacterium smegmatis. This invention relies on incorporating a long alkyl group at the 1-position of the benzimidazole ring, augmenting the molecule's hydrophobicity. This feature is pivotal, given the hydrophobic composition of both Mycobacterium smegmatis and Mycobacterium tuberculosis cell walls, characterized by complex lipids and mycolic acids. Furthermore, the heightened lipophilicity of these 1-longchain alkyl-substituted benzimidazoles facilitates their penetration of the hydrophobic mycobacterial cell wall, which is renowned for its resistance to many antibacterial agents. By surmounting this barrier, these benzimidazoles access the bacterial cytoplasm, where they may selectively target vital biomolecules or biochemical pathways essential for Mycobacterium smegmatis's survival and growth. The specific molecular target can vary but typically involves crucial components of fundamental cellular processes. Consequently, these compounds hold considerable potential for disrupting pivotal functions within Mycobacterium smegmatis, positioning them as promising candidates for further development as antibacterial agents.

Detailed description of the invention
The field of this invention pertains to the design and synthesis of a new class of organic compounds, (Z)-4-((11-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)undecyl)oxy)-4-oxobut-2-enoic acid (Scheme 1). This compound has been specifically designed and synthesized for their exceptional and selective antibacterial properties against Mycobacterium smegmatis, a non-pathogenic bacterium widely used as a model organism for studying mycobacterial infections, including tuberculosis.

Compound 5 was subjected to testing to evaluate its efficacy against Mycobacterium smegmatis mc2155 and Escherichia coli, aiming to identify potential candidates with anti-mycobacterial activity. The compound was assessed using the disc diffusion method, demonstrating antibacterial effectiveness against M. smegmatis while showing no impact on E. coli.

Results and discussion

General
All the reagents and solvents were procured from local vendors. The substrates were prepared according to the protocol outlined below. Reagents and solvents were employed without additional purification. Prior to use, EtOAc and hexane underwent distillation. Reaction conditions included the specified temperature and normal atmospheric pressure, with oversight via thin-layer chromatography (TLC) using Merck 60 F254 silica gel plates, and product visualization via UV detection. Synthesized crude benzimidazole derivative underwent purification using column chromatography, employing silica gel (100-200 mesh) as the stationary phase. Authentication of purified products relied on NMR spectra acquired on a Bruker Avance 400 and 500 Spectrometer in CDCl3 and DMSO-d6 solvents, referencing tetramethyl silane as an internal standard. Chemical shift values are reported in parts per million (ppm) on the d scale. Residual solvent peaks of CDCl3 and DMSO-d6 were identified at 7.26 and 2.50 ppm, respectively.
Synthesis of (Z)-4-((11-(2-(4-chlorophenyl)-1H-benzo[d]imidazol-1-yl)undecyl)oxy)-4-oxobut-2-enoic acid (5)
A solution was prepared by dissolving 2.0 mmol (1 equiv.) of 2-(4-chlorophenyl)-1H-benzo[d]imidazole (3) in 10 mL of dimethylformamide (DMF). Following this, 5-6 drops of triethylamine were added, and 11-bromoundecan-1-ol (2.0 mmol, 1 equiv.) was introduced. The reaction mixture was then heated to 80 °C for 12 hours with stirring at 450 rpm using a magnetic stirrer. The progress of the reaction was monitored by thin-layer chromatography (TLC). Upon completion, the crude mixture underwent extraction with water and dichloromethane (3 x 10 mL). The organic phase was concentrated under reduced pressure, yielding a crude residue (4). This residue underwent purification via flash silica gel column chromatography with EtOAC-hexane (40%) as the eluent. The outcome was the isolation of the desired product as an off-white solid. Yield = 71%; m.p. = 93 ºC; IR (KBr): ? 3209, 2924, 2854, 1603, 1452, 1410, 1089, 1014, 832, 745, 728, 588 cm-1; 1H NMR (500 MHz, CDCl3): d 7.84 – 7.78 (m, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.50 (d, J = 8.3 Hz, 2H), 7.43 – 7.38 (m, 1H), 7.35 – 7.25 (m, 2H), 4.23 – 4.16 (m 2H), 3.62 (t, J = 6.7 Hz, 2H), 1.83 – 1.68 (m, 3H), 1.54 (p, J = 6.8 Hz, 2H), 1.36 – 1.15 (m, 13H); 13C NMR (126 MHz, CDCl3): d 152.3, 142.8, 135.8, 135.4, 130.5, 129.0, 128.9, 122.8, 122.4, 119.9, 110.0, 62.8, 44.6, 32.6, 29.6, 29.3, 29.2, 28.8, 26.5; HRMS m/z: [M+H]+ calcd for C24H32ClNO2+, 399.2198; found, 399.2196.
Compound 4 (1 mmol) was dissolved in 10 mL of CH2Cl2 at 25 °C, and maleic anhydride (1 mmol) was added. The reaction mixture was stirred at 25 °C for 24 hours at 450 rpm using a magnetic stirrer, and TLC was employed to monitor the reaction progress. Once the reaction was complete, as indicated by TLC, the mixture was concentrated under reduced pressure. Further purification was achieved through silica gel column chromatography using EtOAC-hexane (30%) as the eluent, resulting in the desired product as an off-white solid (82%).
Yield = 57% (over two steps); m.p. = 138 ºC; IR (KBr): ? 2933, 2849, 1721, 1467, 1413, 1228, 1171, 1009, 842, 816, 755, 732, 561 cm-1; 1H NMR (500 MHz, CDCl3): d 7.92 – 7.86 (m, 1H), 7.70 – 7.64 (m, 2H), 7.55 – 7.49 (m, 2H), 7.42 (dt, J = 8.1, 2.9 Hz, 1H), 7.37 – 7.29 (m, 2H), 6.41 (d, J = 12.6 Hz, 1H), 6.27 (d, J = 12.6 Hz, 1H), 4.25 (td, J = 6.9, 2.5 Hz, 4H), 1.76 (p, J = 7.0 Hz, 2H), 1.68 (p, J = 6.8 Hz, 2H), 1.38 – 1.29 (m, 2H), 1.24 (d, J = 10.4 Hz, 4H), 1.11 (s, 8H); 13C NMR (126 MHz, CDCl3): d 166.1, 151.6, 142.2, 130.3, 128.4, 122.2, 121.7, 118.9, 64.2, 28.8, 28.6, 28.5, 28.4, 28.1, 27.7, 25.7, 25.1; HRMS m/z: [M+H]+ calcd for C28H34ClN2O4+, 497.2202; found, 497.2198.

Anti-mycobacterial assessment
Antibacterial tests were performed against Mycobacterium smegmatis mc2155 strain aiming to assess the efficacy of compound 5 with anti-mycobacterial activity. The disc diffusion method was employed according to CLSI guidelines with specific modifications for evaluation [16]. Initial testing against M. smegmatis utilized sterile Luria Bertani (LB) agar medium plates from Hi-Media Ltd. A single colony of M. smegmatis was used to inoculate LB broth, which was then incubated at 37 °C until the optical density (O.D) reached 0.3. Petri plates were inoculated from the broth culture using sterile cotton swabs and allowed to absorb for 15 minutes. Subsequently, sterile filter paper discs (6 mm) from Hi-Media Ltd were placed on the agar petri plates, loaded with 20 µL of a 2 mM concentration of compound 5, and allowed to diffuse for 30 minutes before incubation at 37 °C. Kanamycin served as the positive control, while DMSO, the solvent, acted as the negative control. The entire procedure was conducted under laminar airflow conditions to maintain sterility, and the experiment was independently performed three times. The zone of inhibition was observed, and results were compared to the positive control. The minimum inhibitory concentration (MIC) of the susceptible derivative was determined using the Resazurin assay, with specific modifications based on Lelovic et al., 2020 [17]. A 96-well plate was used to assess the MIC, involving two-fold serial dilutions of derivatives ranging from 400 µM to 3.12 µM in LB broth. The M. smegmatis inoculum was adjusted to approximately 5 x 105 cfu/mL. After incubation at 37 °C for 48 hours, 30 µL of 0.015% resazurin dye was added to the tube and kept at 37 °C for 4 hours. The MIC was defined as the lowest concentration with no color change. This experiment was performed in triplicate. The results revealed a substantial zone of inhibition (ZOI) and a minimum inhibitory concentration (MIC) of 12.5 µM (Fig. 2 and Table 1). Following MIC determination, a time-kill assay of compound 5 was conducted at 2x MIC. A treated mixture of 10 mL was inoculated with overnight culture, with the initial optical density set at 0.05. At 3, 6, 9, 12, 24, 36, and 48 hours, 500 µL aliquots were withdrawn, and optical density at 600 nm was monitored. A time-kill curve was constructed to depict the relationship between time and optical density (O.D600). Compound 5 demonstrated a >10-fold reduction, exhibiting comparable outcomes to kanamycin, with a continuous reduction observed up to 48 hours (Fig. 3).

Fig. 2 The graph depicts comparison of the diameter of zone of inhibition (mm) of 5 with kanamycin against Mycobacterium smegmatis

Table 1: Antibacterial activity (MIC) of the benzimidazole derivative (5) against M. smegmatis

Compound MIC (µM)
5 12.5
Kanamycin 3.12

Fig. 3 Time-kill curve analysis of the benzimidazole derivative (5) and kanamycin against M. smegmatis
, Claims:I/We claim:
1. The synthesized compound (5) with structural formula-(I).

2. A process for preparing (5) with structural formula-I as claimed in claim 1 includes:
i. Add 2.0 mmol of 2-(4-Chlorophenyl)-1H-benzo[d]imidazole (3) in 10 mL of anhydrous dimethylformamide. To this solution, add 6 drops of triethylamine and stirr the mixture on a magnetic stirrer bar for 10 minutes at 25 °C with a rotation speed of 450 rpm.
ii. Cool the reaction to 5 °C using an ice-water bath, then add 2.0 mmol of 11-bromoundecan-1-ol drop by drop.
iii. Heat the mixture in a silicon oil bath at 80 °C for 12 hours with stirring at 450 rpm using a hot plate magnetic stirrer.
iv. Extract the crude mixture with 10 mL of water and dichloromethane (3 x 10 mL).
v. Concentrate the organic phase under reduced pressure, resulting in a crude residue (4), which undergoes purification through flash silica gel column chromatography with EtOAC-hexane (40%) as the eluent.
vi. Dissolve 1 mmol of 4 in 10 mL of CH2Cl2 at 25 °C, then add 1 mmol of maleic anhydride to the solution.
vii. Stir the reaction mixture at 25 °C for 24 hours at 450 rpm using a magnetic stirrer.
viii. Concentrate the mixture under reduced pressure.
ix. Purify the product through silica gel column chromatography using EtOAC-hexane (30%) as the eluent.
3. The compound (5) with structural formula-(I) as claimed in claim 1 exhibit anti-mycobacterial properties against M. smegmatis.