Novel Class of Benzimidazoles: Synthesis, Characterization and Pharmaceutical Evaluation ^†

Abstract

The wide range of biological processes and functions that benzimidazole moieties can be used for makes them very interesting synthetic molecules. A novel class of scaffolds for benzimidazole heterocycles has been successfully constructed in the present study and synthesized by using the starting material of O-phenylenediamine derivatives (1a–c). The 1-methyl-2-(methylthio)-1H-benzo[d]imidazole derivatives (3a–c) have been synthesized as intermediate compounds by treating the precursors (1a–c) with carbon disulfide followed by N- and S-methylation with iodomethane and anhydrous potassium carbonate. In the latter step, the intermediate molecules were converted into benzimidazole-containing methyl-piperazine (4a–c), piperazin-ol tethered benzimidazoles (5a–c), and phenylpiperazine holding benzimidazoles (6a–c). The structures assigned to target compounds have been analyzed and confirmed via IR, NMR, and MS analysis. The antibacterial, anthelmintic, and anticancer properties of the target compounds were examined. The biological study showed that the compounds 6b, 4c, and 5a emerge as excellent antibacterial, antifungal, and anthelmintic agents, respectively, whereas heterocycle 6a showed excellent anticancer activity against hepatocyte-derived cell line HUH7, as well as the MCF7 breast cancer cell line with IC₅₀ values of 6.41 and 9.70 µg/mL, respectively. The discovery of a novel class of hetero compounds with multiple hetero moieties that may aid in medication design is also highlighted in this study.

1. Introduction

Using antimicrobial chemicals can either remove or prevent the development of microorganisms [1]. Protozoans, viruses, bacteria, and fungi like mold and mildew are among these microorganisms. Due to COVID-19, controlling microbiological infections has become a global concern over the past two to three years. Hence, it needs urgent multisectoral action to achieve the Sustainable Development Goals (SDGs). To treat parasitic worm infections, anthelmintic medications are utilized [2]. This includes roundworms, or nematodes, as well as flatworms, including flukes and tapeworms. They play a major role in both veterinary and human tropical medicine. Two billion individuals worldwide are thought to be infected with parasites, according to estimates from the World Health Organization. Additionally, parasitic worms affect crops and cattle, which has an effect on food production and, consequently, the economy. Domestic pet infections are also significant. In fact, for animal health firms carrying out drug discovery programs, the companion animal industry is a significant economic factor.

The primary cause of death in India is cancer, a chronic illness that affects numerous cell-signaling pathways and causes disordered cell processes such as irregular cell growth and disrupted apoptosis [3,4]. Global cancer data confirmed that, of all cancer types, blood cancer, liver cancer, breast cancer, lung cancer, colon cancer, prostate cancer, cervical cancer, ovarian cancer, and brain cancer, among others, are the most important in terms of death [5]. Clinical chemotherapeutic drugs have demonstrated positive outcomes in the management of cancer. These substances have deadly side effects, such as bone marrow depression, and certain medications cause alopecia [6]. The development of anticancer drugs that have minimal side effects and are as inexpensive as feasible remains a prospective research field for the pharmaceutical industry worldwide, despite our advanced scientific understanding.

From their discovery as significant heterocycles to their current application in AIDS treatment, benzimidazoles have a rich and impressive record. An imidazole ring at the 4,5-position and benzene combine to generate a ring fusion, which is the heterocyclic compound known as benzimidazole. Benzimidazole and its derivatives are of tremendous interest because of their wide range of biological activity and clinical applications. These medications are quite strong, having a high selectivity ratio together with high inhibitory action. It is very clear that the benzimidazole system exists in a natural substance in the case of vitamin B12 (cyanocobalamin). It is extracted from liver extracts and the fungus Streptomyces griseus. It is a factor that prevents pernicious anemia. The benzimidazole nucleus is also present in a wide range of important naturally occurring substances, such as amino acids like purine and histidine, which are important for the biochemistry of living systems. Biotin (vitamin B7) also contains the reduced form of the imidazole ring. Because it is a structural isostere of a nucleotide that occurs naturally, benzimidazole also interacts easily with the biopolymers of biological systems. This character performs a multitude of biological activities. Benzimidazole and its derivatives have been used as antibacterial agents for over 20 years. Thousands of benzimidazole analogs have been produced, and their pharmacological efficacy has been tested. They are of tremendous interest because of their diverse biological activities and therapeutic applications. These heterocyclic systems can function as bactericides or bacteriostats, which gives them a variety of uses [7], in addition to fungicides, and they can be found in several antiparasitic treatments [8]. Several of them demonstrate notable antiprotozoal action [9]. They have been demonstrated to have modest in vitro anthelmintic activity [10], anti-viral activity [11], anti-asthmatic and anti-diabetic activity [12], anti-tumor activity [13], antimalarial activity [14], antiulcer activity [15], anticancer activity, and anti-HIV activity, as well as having numerous anti-convulsant, antioxidant, anti-proliferative, anti-hypertensive, anti-inflammatory, proton pump inhibitor, and anti-trichinellosis applications [16].

Benzimidazoles show promise as intestinal cystitis treatments, inhibitors of smooth muscle cell proliferation, and anticancer drugs in a variety of chemistry applications. Prominent benzimidazole derivatives have been identified as gonadotropin-releasing hormone receptor antagonists, non-nucleoside HIV-1 reverse transcriptase inhibitors, and, surprisingly, alkynyl benzimidazoles as metabotropic glutamate receptor modulators. A common component found in many pharmacologically active chemicals and natural products is the imidazole core [17].

Mice infected with Syphacia obvelata showed a 50% anthelmintic activity in response to synthetic benzimidazole piperazine derivatives [18]. Furthermore, piperazine derivatives of 5(6)-substituted-(1H-benzimidazol-2-ylthio)-acetic acids (Figure 1: A) [19] have shown good anthelmintic activity [20]. Pyrimido benzimidazole (Figure 1: B) [21] and dioxino benzimidazothiazol-9-ones (Figure 1: C) [22] have shown anti-inflammatory and analgesic properties, as determined by rat paw edema generated by carrageenan and writhing tests induced by phenyl quinone.

As seen above, benzimidazole and its derivatives containing piperazine are important for a variety of positive biological actions. To obtain fresh biodynamic leads, it is worthwhile to synthesize these molecular analogs. As a result, we established and synthesized a few unique piperazine-linked benzimidazole moieties that have antibacterial, anthelmintic, and anticancer properties in particular [23].

2. Materials and Methods/Methodology

Chemistry

An open capillary tube showed the melting point of every synthetic compound, and it was not corrected. Fourier transform infrared spectroscopy, or FT-IR, with a Bruker alpha spectrophotometer was used to record IR spectra. The Bruker AV400 was used to obtain ¹H-NMR (CDCl₃) and ¹³C-NMR (DMSO) spectra at 400 MHz and 100 MHz, respectively. Data of chemical shifts are given in δ ppm. Mass spectra of the compounds were obtained using the mass spectrometer Joel JMS-D-300 instrument at 70 eV. The progress of each reaction was checked in thin-layer chromatography (TLC) and a few compounds were purified via the column chromatography method. The entire essential solvents were AR-grade and they were purified from the distillation process.

Experimental

Synthesis of 1H-benzo[d]imidazole-2-thiole (2a)

The benzene-1,2-diamine (1a, 0.108 g, 10.00 mmol) and carbon disulfide (0.076 g, 10.00 mmol) mixture was put into a clean, round-bottomed flask and refluxed in the presence of potassium hydroxide (0.056 g, 10.00 mmol) in methanol (10 mL) for 8 h at 70 °C. TLC was used to monitor the reaction’s completion (hexane/ethyl acetate; 7/3). The reaction mixture was completed, allowed to cool to room temperature, and then added to ice-cold water. The solution was stirred for five minutes and acidified with acetic acid. The solid product obtained was filtered and washed thoroughly with ice-cold water. To obtain a pure compound, the material was dried and then recrystallized from ethanol (2a). Yield: 72%, m.p. 172–174 °C. In a similar procedure, other benzimidazole derivatives, 2b and 2c, were prepared.

5-Methoxy-1H-benzo[d]imidazole (2b)

Obtained from the mixture of 4-methoxybenzene-1-2-diamine (1b, 0.138 g, 10.00 mmol), carbon disulfide (0.076 g, 10.00 mmol), and potassium hydroxide (0.056 g, 10.00 mmol) and refluxed in methanol (10 mL) for 8 h at 70 °C. Yield: 71%, m.p. 190-192 °C.

5-Nitro-1H-benzimidazole-2-thiol (2c)

Obtained from 4-nitrobenzene-1,2-diamine (1c, 0.153 g, 10.00 mmol), carbon disulfide (0.076 g, 10.00 mmol), and potassium hydroxide (0.056 g, 10.00 mmol) and refluxed in methanol (10 mL) for 8 h at 70 °C. Yield: 71%, m.p. 209–210 °C.

Synthesis of 1-methyl-2-(methylthio)-1H-benzo[d]imidazole (3a)

A mixture of 1H-benzo[d]imidazole-2-thiol (2a, 0.150 g, 10.00 mmol), methyl iodide (0.85 mL, 10.00 mmol), and potassium carbonate (0.138 g, 10.00 mmol) was added to a clean, round-bottomed flask containing a dimethyl formamide (10 mL) solvent and the mixture was stirred at room temperature for about 6 h. The completion of the reaction was monitored via TLC (hexane/ethyl acetate 7/3). The solid obtained was filtered, washed with water, dried, and purified via recrystallization from ethanol to obtain the compound (3a). Yield: 68%, m.p. 200-203 °C. By following the same procedure, other benzimidazole derivatives, 3b and 3c, were prepared.

5-Methoxy-1-methyl-2-(methylthio)-1H-benzo[d]imidazolethiole  (3b)

Obtained from 5-methoxy-1H-benzo[d]imidazole (2b, 0.148 g, 10.00 mmol), methyl iodide (0.85 mL, 10.00 mmol), and potassium carbonate (0.138g, 10.00 mmol) in dimethyl formamide (10 mL) at room temperature for about 6 h. Yield: 71%, m.p. 209–211 °C.

1-Methyl-2-(methylthio)-5-nitro-1H-benzo[d]imidazole  (3c)

Obtained from 1-methyl-5-nitro-1H-benzo[d]imidazole-2-thiol (2c, 0.209 g, 10.00 mmol), methyl iodide (0.85 mL, 10.00 mmol), and potassium carbonate (0.138 g, 10.00 mmol) in dimethyl formamide (10 mL) at room temperature for about 6 h. Yield: 65%, m.p. 196–198 °C.

Synthesis of 1-methyl-2-(4-methylpiperazine-yl)-1H-benzo[d]imidazole (4a)

A mixture of 1-methyl-2-(methylthio)-1H-benzo[d]imidazole (3a, 0.178 g, 10.00 mmol) and 1-methyl piperazine (0.100 g, 10.00 mmol) was added to a clean, round-bottomed flask containing dry ethanol (10 mL) and refluxed for 12 h at 80 °C. The progress of the reaction was monitored using TLC (hexane/ethyl acetate; 7/3). After completion, the reaction mixture was cooled to room temperature and poured into ice-cold water. The separated product was filtered off to obtain the target compound (4a). Further, it was purified via recrystallization from ethanol. Yield: 73%, m.p. 174–176 °C, IR (ν, cm⁻¹, KBr): 2958 (C-H), 1621 (C=N), 1445 (C=C), 1293 (C-O-C), 1095 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.26–7.60 (m, 3H, Ar-H), 3.28 (s, 3H, OCH₃), 2.94–2.97 (t, 4H, 2CH₂), 2.66–2.67 (t, 4H, 2CH₂), 2.28 (s, 3H, NCH₃), 2.23 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 158.7, 156.0, 139.5, 126.2, 113.0, 111.1, 100.3, 57.2, 56.9, 55.3, 52.0, 51.9, 46.1, 33.2, MS (m/z): 230.10.

In a similar procedure, other derivatives, 4b and 4c, were prepared from 3b and 3c, respectively.

5-Methoxy-1-methyl-2-(4-methylpiperazin-1-yl)-1H-benzo[d]imidazole (4b)

Obtained from 5-methoxy-1-methyl-2-(methylthio)-1H-benzo[d]imidazolethiole (3b, 0.208 g, 10.00 mmol) and 1-methyl piperazine (0.162 g, 10.00 mmol) in dry ethanol (10 mL) at 80 °C for 12 h. Yield: 70%, m.p. 173-175 °C, IR (ν, cm⁻¹, KBr): 2970 (C-H), 1621 (C=N), 1472 (C=C), 1293 (C-O-C), 1095 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.26–7.60 (m, 3H, Ar-H), 3.28 (s, 3H, OCH₃), 2.94–2.97 (t, 4H, 2CH₂), 2.66–2.67 (t, 4H, 2CH₂), 2.28 (s, 3H, NCH₃), 2.23 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 158.7, 156.0, 139.5, 126.2, 113.0, 111.1, 100.3, 57.2, 56.9, 55.3, 52.0, 51.9, 46.1, 33.2, MS (m/z): 260.14.

1-Methyl-2-(4-methylpiperazin-1-yl)-5-nitro-1H-benzo[d]imidazole (4c)

Obtained from 1-methyl-2-(methylsulfanyl)-5-nitro-1H-benzimidazole (3c, 0.223 g, 10.00 mmol) and 1-methyl piperazine (0.162 g, 10.00 mmol) in dry ethanol (10 mL) at 80 °C for 12 h. Yield: 68%, m.p. 187-189 °C, IR (ν, cm⁻¹, KBr): 2944 (C-H), 1615 (C=N), 1463 (C=C), 1105 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.50–7.76 (m, 3H, Ar-H), 3.80–3.89 (t, 4H, 2CH₂), 3.60–3.75 (t, 4H, 2CH₂), 2.56 (s, 3H, NCH₃), 2.40 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 158.9, 144.0, 140.1, 138.3, 118.2, 112.2, 110.1, 57.1, 56.9, 52.0, 51.9, 46.1, 33.2, MS (m/z): 275.09.

Synthesis of 2-[4-(1-Methyl-1H-benzo[d]imidazol-2-yl)piperazin-1-yl]ethanol (5a)

A mixture of 1-methyl-2-(methylthio)-1H-benzo[d]imidazole (3a, 0.178 g, 10.00 mmol) and 2-(4-methylpiperazin-1-yl)ethanol (0.144 g, 10.00 mmol) was added to a clean, round-bottomed flask containing dry ethanol (10 mL) and refluxed for 12 h at 80 °C. The development of the reaction was monitored using TLC (eluent hexane/ethyl acetate 7/3). The reaction mixture was allowed to cool to room temperature once it was finished. After the solution was added to ice-cold water, the desired heterocycle was obtained by filtering out and recrystallizing the separated solid from ethanol (5a). Yield: 63%, m.p.160–162 °C, IR (ν, cm⁻¹, KBr): 3390 (O-H), 2952 (C-H), 1635 (C=N), 1488 (C=C), 1128 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.27–7.40 (m, 4H, Ar-H), 3.85 (t, 2H, OCH₂), 3.33 (s,1H, OH), 3.15 (t, 4H, 2CH₂), 3.06 (t, 2H, CH₂), 2.59 (t, 4H, 2CH₂), 2.70 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 158.7, 141.2, 134.0, 123.0, 122.7, 118.2, 109.7, 59.4, 59.1, 52.2, 52.0, 51.0, 33.8, MS (m/z): 260.16.

In a similar way, other derivatives, 5b and 5c, were prepared from 3b and 3c, respectively.

2-[4-(5-Methoxy-1-methyl-1H-benzo[d]imidazol-2-yl)piperazin-1-yl]ethanol (5b)

Obtained from 5-methoxy-1-methyl-2-(methylthio)-1H-benzo[d]imidazolethiole (3b, 0.208 g, 10.00 mmol) and 2-(4-methylpiperazin-1-yl)ethanol (0.144 g, 10.00 mmol) in dry ethanol (10 mL) at 80 °C for 12 h. Yield: 81%, m.p. 205–207 °C, IR (ν, cm⁻¹, KBr): 3394 (O-H), 2950 (C-H), 1635 (C=N), 1459 (C=C), 1260 (C-O-C), 1122 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.19–7.45 (m, 3H, Ar- H), 3.89 (s, 3H, OCH₃), 3.72 (t, 2H, OCH₂), 3.53 (t, 2H, CH₂), 3.39 (s,1H, OH), 3.09 (t, 4H, 2CH₂), 2.79 (t, 4H, 2CH₂), 2.65 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 159.0, 156.0, 139.7, 126.2, 113.0, 111.1, 100.3, 59.5, 59.0, 55.2, 52.3, 52.1, 51.0, 33.4, MS (m/z): 290.15.

2-[4-(1-Methyl-5-nitro-1H-benzo[d]imidazol-2-yl)piperazin-1-yl]ethanol (5c)

Obtained from 1-methyl-2-(methylsulfanyl)-5-nitro-1H-benzimidazole (3c, 0.223 g, 10.00 mmol) and 2-(4-methylpiperazin-1-yl)ethanol (0.144 g, 10.00 mmol) in dry ethanol (10 mL) at 80 °C for 12 h. Yield: 61%, m.p. 172-175 °C, IR (ν, cm⁻¹, KBr): 3200 (O-H), 2920 (C-H), 1635 (C=N), 1550 (N-O), 1435 (C=C), 1125 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.52–8.19 (m, 3H, Ar-H), 3.98 (t, 2H, OCH₂), 3.69 (s, 1H, OH), 3.26 (t, 2H, CH₂), 3.25 (t, 4H, 2CH₂), 2.75 (t, 4H, 2CH₂), 2.67 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 158.8, 144.0, 140.2, 139.5, 118.2, 112.5, 110.2, 59.5, 59.1, 52.2, 52.0, 51.1, 51.0, 33.7, MS (m/z): 305.14.

Synthesis of 1-methyl-2-(4-phenylpierazine-yl)-1H-benzo[d]imidazole (6a)

The 1-methyl-2-(methylthio)-1H-benzo[d]imidazole (3a, 0.178 g, 10.00 mmol) and 1-methyl-4-phenylpiperazine (0.176 g, 10.00 mmol) mixture was taken in a clean, round-bottomed flask containing dry ethanol (10 mL) and refluxed for about 14 h at 80 °C. TLC was used to check the reaction progress (hexane/ethyl acetate 7/3). The reaction mixture was finished, allowed to cool to room temperature, and then added to ice-cold water. To obtain the target chemical, the separated product was filtered and then recrystallized from methanol (6a). Yield: 58%, m.p. 169–172 °C, IR (ν, cm⁻¹, KBr): 2977 (C-H), 1625 (C=N), 1488 (C=C), 1134 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 6.59–7.27 (m, 9H, Ar-H), 3.29 (t, 4H, 2CH₂), 2.92 (t, 4H, 2CH₂), 2.60 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 159.0, 149.2, 141.0, 134.1, 129.5, 129.1, 123.0, 122.7, 121.2, 118.3, 114.1, 114.0, 110.0, 49.4, 49.2. 49.1, 48.9, 33.1, MS (m/z): 292.17.

Similarly, other derivatives, 5b and 5c, were prepared from 3b and 3c, respectively.

5-Methoxy-1-methyl-2-(4-phenyliperazine-1-yl)-1H-benzo[d]imidazole (6b)

Obtained from 5-methoxy-2-(methylthio)-1H-benzo[d]imidazole (3b, 0.208 g, 10.00 mmol) and 1-methyl-4-phenylpiperazine (0.176 g, 10.00 mmol) in dry ethanol (10 mL) at 80 °C for about 14 h. Yield: 64%, m.p. 209-210 °C, IR (ν, cm⁻¹, KBr): 2986 (C-H), 1619 (C=N), 1470 (C=C), 1239 (C-O-C), 1129 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.24–7.91 (m, 8H, Ar-H), 3.60 (s, 3H, OCH₃), 3.40 (t, 4H, 2CH₂), 3.37 (t, 4H, 2CH₂), 2.76 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 158.5, 156.0, 149.2, 139.5, 129.5, 129.1, 126.2, 121.8, 114.3, 114.0, 113.1, 111.2, 100.1, 55.3, 49.4, 49.3, 49.1, 49.0, 33.5, MS (m/z): 322.14.

1-Methyl-5-nitro-2-(4-phenylpiperazin-1-yl)-1H-benzimidazole (6c)

Obtained from 1-methyl-2-(methylsulfanyl)-5-nitro-1H-benzimidazole (3c, 0.223 g, 10.00 mmol) and 1-methyl-4-phenylpiperazine (0.176 g, 10.00 mmol) in dry ethanol (10 mL) at 80 °C for about 14 h. Yield: 54%, m.p. 195-198 °C, IR (ν, cm⁻¹, KBr): 2961 (C-H), 1620 (C=N), 1550 (N-O), 1461 (C=C), 1125 (C-N-C), ¹H-NMR (CDCl₃, 400 MHz, δ ppm): 7.25–8.05 (m, 8H, Ar-H), 3.60 (t, 4H, 2CH₂), 3.39 (t, 4H, 2CH₂), 2.65 (s, 3H, NCH₃), ¹³C-NMR (DMSO, 100 MHz, δ ppm): 159.0, 149.5, 144.1, 140.0, 139.6, 129.5, 129.3, 121.8, 118.3, 114.2, 114.0, 112.5, 110.0, 49.3, 49.1, 48.8, 33.1, MS (m/z): 337.15.

Biology

Antibacterial activity

Bacterial peptone (6 g), a pancreatic digest of casein (4 g), yeast extract (3 g), beef extract (1.5 g), dextrose (1.0 g), and agar (15.0 g) were all dissolved in distilled water to make 1 L of the medium. Then, 1M sodium hydroxide and 1M HCl were added to the solution to bring it to the desired pH (6.5–6.6). To obtain 40 g/mL, the test materials were all dissolved in the lowest amount of dimethyl formamide (DMF), and the final volume was made with distilled water. The cup–plate approach involves diffusing an antibiotic through a Petri dish’s hardened layer of agar to the point where it completely stops the development of the added microbe in a circle or zone surrounding the cavity containing the antibiotic solution. After being injected with the required amount of the microbe suspension at a temperature of 40 to 50 °C, the previously liquified medium was then poured onto Petri plates to a depth of 3 to 4 mm. Agar was removed from each Petri dish using a sterile cork borer after five identically sized cups were pierced. Using sterile pipettes, the standard and sample solutions were introduced to the bored cups at predefined concentrations. The plates were allowed to stay at room temperature for one hour in order to reduce the impact of the time differences between the applications of different solutions. These were then kept at 37 °C for the following 24 h. Then, the zone of inhibition developed was precisely measured and noted. There were three average readings for each zone of inhibition that was recorded. Separate calculations were made for the water zone of inhibition.

Anthelmintic activity

The Garg and Atal approach was used to measure the anthelmintic activity¹³, analyzed utilizing postmortem earthworm pheritima warmers (class: Annelid, order: oligochaete), which were produced at the time of the experiment in Shimoga by a local supplier. The worms were sorted for consistent size and length after being washed in water to remove any clinging debris. For acclimation, the worms were maintained in a solution of 6% dextrose. For the activity, worms with typical motility were chosen. Equal-sized Petri dishes were chosen, and 2 mL of each component (4 mg/mL) in 0.1% T-20 suspension was added to each dish before the volume was brought to 25 mL with an aqueous dextrose solution (6%). T-20 (25 mL of 0.1% solution) was made in another Petri dish and added to a 6% dextrose solution with albendazole (1 mg/mL) as the control. Another Petri plate was used to hold the T-20 suspension (0.1% in 1 mL), which was then added to make a volume of 25 mL using a dextrose (6%) solution. As a rule, albendazole was used. One worm was positioned in each Petri dish. The worm’s paralysis was evaluated by submerging it in water kept at 500 °C and noting the length of time it took to become paralyzed. The worm was assumed to have died when it ceased moving at 50 °C, and the duration of that death was noted.

Anticancer activity

Using two human cancer cell lines—liver (HUH7) and breast (MCF7)—triplicate SRB assays [24] were used to assess the newly synthesized compounds’ anticancer potential in vitro. There were 40 μM to 2.5 μM serial dilutions applied. For the cytotoxic impact, camptothecin and 5-fluorouracil (5-FU) were utilized as the standard medication and the positive control, respectively. A total of 2000–5000 cancer cells per well were seeded onto 96-well plates using 200 μL of the medium, and they were then incubated at 37 °C in an environment of 5% CO₂ and 95% air. One plate per cell line was fixed with 100 μL of ice-cold trichloroacetic acid (TCA) (10%) after 24 h. The time-zero plate is represented by this plate. The target compounds were dissolved in DMSO (dimethyl sulfoxide) to a final concentration of 40 mM in order to be tested, and they were then kept at +4 °C. The cells were subjected to drugs and then incubated for 72 h at 37 °C in the same environment and for 1 h at +4 °C in the dark. After that, TCA was eliminated by repeatedly washing with double-distilled water. Following a drying process, 100 μL of 0.4 percent sulforhodamine B (SRB) in an acetic acid solution (1%) was used to stain the plates. This was followed by a 10 min dark incubation period at room temperature. The plates were allowed to air-dry after the unbound dye was removed with 1% acetic acid. After that, 200 μL of 10 mM Tris-Base was used to dissolve the bound stain in order to determine the absorbance values. At 515 nm, the optical density (OD) values were obtained.

3. Results

The synthetic method of producing benzimidazole-containing heterocycles is revealed in Scheme 1. The compound 5-methoxy-1H-benzo[d]imidazole derivatives (2a–c) were first synthesized by treating benzene-1,2-diamine derivatives (1a–c) with carbon disulfide and a catalytical amount of potassium hydroxide in methanol. The intermediate compound 1-methyl-2-(methylthio)-1H-benzo[d]imidazole derivatives (3a–c) were synthesized by refluxing the above-synthesized compounds (2a–c) with iodomethane and anhydrous potassium carbonate in DMF. Finally, the target compounds methyl-piperazine holding benzimidazoles (4a–c), piperazin-ol-tethered benzimidazoles (5a–c), and phenyl piperazine-holding benzimidazoles (6a–c) were synthesized by refluxing the intermediate compounds (3a–c) with 1-phenylpiperazine, 2-(4-methylpiperazin-1-yl)ethanol and 1-methyl-4-phenylpiperazine, respectively, in dry ethanol. The structure of all the final compounds was confirmed via IR, NMR, and mass spectral studies.

Biology

Antibacterial activity

Table 1. Antibacterial activity of the screened compounds (zone of inhibition (in mm)) *.

Compounds	S. aureus	B. subtillis	E. coli	S. paratyphi-A	C. albicans	A. niger
4a	14	15	14	15	11	12
4b	11	10	10	11	12	11
4c	15	16	14	14	16	14
5a	15	13	13	15	11	13
5b	14	14	11	14	10	12
5c	12	15	13	14	14	10
6a	16	12	12	13	16	12
6b	18	16	15	16	15	14
6c	9	9	8	9	10	12
Procaine penicillin	20	21	-	-	-	-
Streptomycin	-	-	20	23	-	-
Fluconazole	-	-	-	-	21	21

* n = 5, p < 0.05.

summarizes the antimicrobial activity finding, and Figure 2 displays the activity comparison. The findings demonstrated that, against the majority of the examined microorganisms, every tested drug had moderate-to-excellent activity. Heterocycle 6b was the most active of the evaluated compounds; it exhibited similar activity to the conventional medications streptomycin and procaine penicillin. This could be because the piperazine ring connects the methoxy (-OCH₃) groups to the benzimidazole. Furthermore, a few additional compounds had good-to-moderate activity. The compounds known as heterocycles 4c and 6a demonstrated remarkable antifungal activity, which might be attributed to their resonance phenyl ring and nitro group, which are connected to the benzimidazole through the piperazine moiety. Moderate action was shown by the other compounds.

Anthelmintic  activity

The anthelmintic activity actual result is summarized in

Table 2. Result of anthelmintic activity of target compounds.

Compounds	Mean Paralyzing Time + S.D. * (min)	Mean Death Time + S.D. * (min)
4a	14.59 ± 0.06	33.17 ± 0.11
4b	13.11± 0.03	22.58 ± 0.14
4c	16.05 ± 0.13	22.31 ± 0.26
4d	16.88 ± 0.22	36.38 ± 0.16
4e	16.06 ± 0.05	27.67 ± 0.11
4f	17.04 ±0.65	31.56 ± 0.15
4g	15.06 ± 0.15	29.32 ± 0.91
5a	11.63 ± 0.16	21.48 ± 0.20
5b	14.11 ± 0.18	33.38 ± 0.10
5c	17.12 ± 0.11	34.05 ± 0.30
Albendazole	9.35 ± 0.12	20.74 ± 0.36

* n = 5, p < 0.05, Concentration = 4 mg/mL.

and the comparison of activity is shown in Figure 3. All of the studied drugs exhibited good-to-great activity, according to the data. Among the screened compounds, heterocycle 5a showed excellent activity, and it was also comparable with the standard drug Albendazole. This may be due to the presence of methyl (-CH₃) and ethyl alcohol (-CH₂-CH₂-OH) groups linked to the benzimidazole ring through the piperazine ring. In addition, compounds 4a and 4b showed moderated activity, which may be due to the presence of methoxy (-OCH₃) and methyl (-CH₃) groups. This result shows that the compounds with electron-donating groups have potent anthelmintic activity. Other compounds showed moderate activity.

Anticancer activity

The result of the anticancer activity is summarized in

Table 3. IC₅₀ values of tested heterocycles against human liver (HUH7) and breast (MCF7) cancer cell lines in µg/mL.

Compound	HUH7a	MCF7a
4a	NI	9.32
4b	NI	NI
4c	NI	NI
5a	11.69	NI
5b	NI	NI
5c	NI	NI
6a	6.41	9.70
6b	NI	NI
6c	NI	NI
Camptothecin	0.16	>0.01
5-Fluorouracil	31.21	3.45

All the experiments were conducted in triplicate (1 < R2 < 0.8). NI: no inhibition.

, and the comparison of activity is shown in Figure 4. The results envisioned that only compounds 5a and 6a against the human liver (HUH7) cell line and compounds 4a and 6a against the breast (MCF7) cancer cell line were found to show activity, whereas other compounds failed to possess inhibition against any of the two examined cell lines. Among these four screened compounds, heterocycle 5a, with an ethanol group in the piperazine side, demonstrated excellent activity against only the HUH7 cell line, with an IC₅₀ value of 11.69 µg/mL, and it was also comparable with the standard drugs Camptothecin and 5-Fluorouracil. On the other hand, compound 4a, containing a methyl group in the piperazine part, showed excellent activity against only the MCF7 cell line, with an IC₅₀ value of 9.32 µg/mL. Interestingly, compound 6a, with phenyl ring-bounded piperazine, showed excellent activity against both the cell lines, with IC₅₀ values of 6.41 and 9.70 µg/mL, respectively.

4. Discussion

Carbon disulfide is commonly added to N-H bonds in reactions involving N-nucleophiles and carbon disulfide. Strong bases like KOH, which are used in the synthesis of a wide variety of organosulfur compounds, are the ideal tools for preparing thiol compounds containing imidazole moieties (2a–c). In comparison, the plane compound 2a was obtained in good yield compared to the other two derivatives with either electron-donating methoxy groups or electron-receiving nitro groups. Further, compound 3b was obtained in good yield compared to the other derivatives due to the presence of an electron-donating methoxy group, which facilitated the alkylation reaction, and the same was observed in compounds 5b and 6b. To methylate secondary amines (2a–c), two equivalents of methyl iodide would be required. The amount of methyl iodide equivalents used can reveal details about the amine substitution pattern. Apart from the production of methiodide, an amine hydroiodide salt is the end result of every methylation. It is necessary to deprotonate this salt before further methylation. Usually, K₂CO₃ is used as a weak base to do this. Products 3a–c were obtained as a result of the reaction when alkali (K₂CO₃) was added to 2a–c at a low temperature or at room temperature. While electron-withdrawing substituents had no noticeable effects on product yields but did significantly shorten the reaction time, electron-releasing substituents slightly enhanced the yields and decreased the reaction times. The main challenges that we encountered during the preparation of target compounds were use of solvents and temperatures to set optimized reaction conditions, i.e., dry ethanol at 90–100 °C in a water bath, to afford the target compounds in excellent yield. The appearance of molecular ions peaks at 292.10, 260.16, and 292.17 corresponding to their molecular mass confirmed the structure assigned to heterocycles 4a, 5a, and 6a, respectively. In the IR spectra, the appearance of the absorption band at 1621 cm⁻¹ for C=N stretching; 1095 cm⁻¹ for C-N-C for 4a; 3390 cm⁻¹ due to O-H; 1635 cm⁻¹ due to C=N stretching for 5a and 1625 due to C=N; and 1134 due to C-N-C for 6a confirmed the respective structures. In the ¹H-NMR spectra, the appearance of singlets at δ 2.28 ppm due to the –N-CH₃ group and at δ 3.33 due to the -OH group, as well as multiplets at 6.59–7.27 due to Ar-H, confirmed the structure of compounds 4a, 5a, and 6a, respectively. The spectral study of all other derivatives also confirmed the structure assigned to the respective compounds.

5. Conclusions

The current study discovered a simple process for the synthesis of derivatives of benzimidazoles. The yield of all benzimidazole derivatives was discovered to be between 54 and 81%. The structures were established via IR, NMR, and MS analyses. Among the synthesized compounds, heterocycle 6b emerged as an excellent antimicrobial agent, and compounds 4c and 6a were potent antifungal agents. Heterocycle 5a was found to possess excellent anthelmintic activity, and derivatives 4a, 5a, and 6a were found to show anticancer activity. Therefore, these heterocycles can be used as a promising lead for the design of new antibacterial, anthelmintic, and anticancer agents.