Facile Synthesis of 1,2-Disubstituted Benzimidazoles as Potent Cytotoxic Agents

Abstract

Benzimidazoles have a broad spectrum of biological and pharmacological properties, including anticancer activity. This study reports the facile synthesis and cytotoxic evaluation of twenty-eight 1,2-disubstituted benzimidazoles (6a–β), based on condensation reactions between N-benzyl o-phenylenediamine and benzylamine. The reactions were solvent-free, with the use of Na₂S₂O₅ as an inexpensive and environmentally friendly oxidizing agent, and progressed rapidly. Cytotoxicity assessments using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay were performed against the A549, HeLa, and MCF-7 cell lines for all synthesized compounds (6a–β). Among them, 6j, 6k, 6l, and 6n displayed good activities against the A549 and MCF-7 cell lines. These compounds possessed IC₅₀ values ranging from 2.55 to 4.50 µM, corresponding to 1.4-fold to 2.4-fold stronger potencies than that of the positive control 5-fluorouracil (5-FU) (IC₅₀ = 6.08 µM) against MCF-7 cells, while 6k (IC₅₀ = 3.22 µM) was consistent with 5-FU on the A549 cell line (IC₅₀ = 3.77 µM). Structure–activity relationship analyses revealed the 3-pyridinyl moiety at C-2 and the CH₃, OCH₃, or 1,3-dioxolyl groups on the benzene ring at the N-1 position of the benzimidazole heterocycle as key structural features effectuating the observed cytotoxicities.

Introduction

Benzimidazole is an important pharmacophore and privileged structure in medicinal chemistry.^1–3) Benzimidazole derivatives are associated with a wide range of biological properties, including antihypertensive,⁴⁾ anti-inflammatory,⁵⁾ antibacterial,^6,7) antifungal,^8–10) antiallergic,^11–13) antiviral,^9,14) antioxidant,^15,16) antidiabetic, and antitumor activities.¹⁾ Optimization of the substituents around the benzimidazole nucleus resulted in the generation of many bioactive compounds, as exemplified by albendazole as antimicrobial, astemizole as antihistaminic, omeprazole as antiulcer, bendamustine as antitumor, and enviradine as antiviral, as well as many lead compounds in a wide range of other therapeutic areas (Fig. 1).

Fig. 1. Bioactive Benzimidazole-Containing Compounds

In particular, benzimidazole is one of the most prominent nitrogen containing heterocycles with cytotoxic properties against different types of cancer cell lines.^17–19) Many 1,2-disubstituted benzimidazole derivatives with antiproliferative effects towards various cancer cell lines have been reported, and compounds I,²⁰⁾ II,²⁰⁾ III,²¹⁾ and IV²²⁾ are representative examples (Fig. 2).

Fig. 2. Structures of Representative Cytotoxic Benzimidazoles

Due to their potent bioactivities in the field of medicinal chemistry, various methods have been developed for benzimidazole synthesis.²³⁾ Generally, benzimidazole can be constructed based on the condensation of o-phenylenediamines with carboxylic acid or its derivatives (acyl chlorides, esters, amides, and nitriles) under strongly acidic conditions at elevated temperatures,²⁴⁾ or with aldehydes/alcohols in the presence of various metallic or metal-free catalytic reagents.²⁵⁾ In addition, oxidative cross-coupling reactions between o-phenylenediamines and primary amines for selectively producing 2-substituted benzimidazoles have recently received keen attention.²⁶⁾ However, these methods generally require metallic catalysts and the use of organic solvents or prolonged reaction times.^27,28) Considering these issues, we have recently succeeded in developing efficient, solvent-free condensation reaction conditions between o-phenylenediamines and benzylamines, using sodium metabisulfite (Na₂S₂O₅) as the only oxidant, to construct various 2-substituted benzimidazole derivatives.²⁹⁾ Among the synthesized benzimidazoles, compounds V, VI, and VII displayed remarkably improved cytotoxicities, compared to the positive control, 5-fluorouracil (5-FU), against the human lung (A549), human cervical (HeLa) and human breast adenocarcinoma (MCF-7) cancer cell lines. Based on these findings, as well as the fact that N-substitutions of the benzimidazole have resulted in strong and selective antiproliferative activities,^30–32) we aimed to synthesize 1,2-disubstituted benzimidazole derivatives by incorporating various phenyl units bearing different substituents and testing their cytotoxicities against three human cancer cell lines: A549, HeLa, and MCF-7. By analyzing the influence of the substituents on the phenyl moiety, the structure–activity relationship (SAR) guidelines were deduced.

Results and Discussion

The desired 1,2-disubstituted benzimidazole derivatives (6a–β) were efficiently synthesized by following the previously developed synthetic procedure illustrated in Chart 1.²⁹⁾ The N-substituted o-phenylenediamines 4a–f were prepared as precursors by the S_NAr reaction of the commercially available o-fluoro-nitrobenzene 1 with substituted benzylamines 2a–f, followed by the reduction of the nitro group to the free amino moiety.³³⁾ The condensation reactions between N-substituted o-phenylenediamines 4a–f and substituted benzylamines 5 proceeded readily, using sodium metabisulfite (Na₂S₂O₅) as the only oxidant under solvent-free conditions. The reactions tolerated various substituents on the benzene rings of both the benzylamines 5 and o-phenylenediamines 4a–f, leading to the formation of twenty-eight 1,2-disubstituted benzimidazoles 6a–β in reasonable to good yields (Table 1).

Chart 1. Synthetic Procedure of 1,2-Disubstituted Benzimidazoles

Table 1. Synthesis and Cytotoxicity of the 1,2-Disubstituted Benzimidazoles (6a–β)

Compd.	R1	R2	Yielda) (%)	IC50 (µM)
				A549	HeLa	MCF-7
6a34, 41)	Bn	Ph	72	56.26 ± 4.04	61.72 ± 2.55	87.71 ± 0.48
6b42)	4-CH3-Bn	Ph	70	58.01 ± 0.83	61.07 ± 3.01	61.09 ± 1.35
6c	2-Cl-Bn	Ph	76	48.72 ± 1.26	82.44 ± 2.02	>100
6d39)	4-OCH3-Bn	Ph	71	49.32 ± 1.03	67.13 ± 1.65	46.04 ± 1.53
6eb)	1,3-Benzodioxol-5-ylmethyl	Ph	67	>100	>100	>100
6fb)	3-Pyridinylmethyl	Ph	68	>100	>100	>100
6g35)	Bn	4-CH3-Ph	59	41.75 ± 0.32	45.90 ± 0.42	46.97 ± 2.36
6h36)	Bn	4-OCH3-Ph	56	54.76 ± 1.47	43.09 ± 1.34	64.23 ± 1.00
6i37)	Bn	2-Cl-Ph	59	33.58 ± 0.70	41.32 ± 0.78	30.40 ± 0.55
6j38)	Bn	3-Pyridinyl	51	7.58 ± 0.08	67.31 ± 1.28	4.20 ± 0.45
6kb)	4-CH3-Bn	3-Pyridinyl	49	3.22 ± 0.09	59.46 ± 2.01	4.50 ± 0.07
6lb)	4-OCH3-Bn	3-Pyridinyl	46	10.63 ± 0.39	67.07 ± 1.28	3.61 ± 0.27
6mb)	2-Cl-Bn	3-Pyridinyl	49	>100	>100	>100
6n	1,3-Benzodioxol-5-ylmethyl	3-Pyridinyl	45	9.01 ± 0.56	31.47 ± 0.76	2.55 ± 0.13
6o	3-Pyridinylmethyl	3-Pyridinyl	47	>100	>100	>100
6p	3-Pyridinylmethyl	4-CH3-Ph	57	42.22 ± 1.30	54.85 ± 2.23	47.63 ± 0.84
6q	3-Pyridinylmethyl	4-OCH3-Ph	54	>100	>100	>100
6r	3-Pyridinylmethyl	2-Cl-Ph	56	>100	>100	>100
6s35)	4-CH3-Bn	4-CH3-Ph	57	38.65 ± 0.65	36.93 ± 0.60	32.33 ± 0.96
6t	1,3-Benzodioxol-5-ylmethyl	4-CH3-Ph	60	31.13 ± 0.63	34.74 ± 0.38	24.92 ± 2.67
6u40)	4-CH3-Bn	4-OCH3-Ph	52	51.70 ± 1.12	>100	48.07 ± 0.31
6v	1,3-Benzodioxol-5-ylmethyl	4-OCH3-Ph	52	83.70 ± 0.85	93.44 ± 2.63	84.24 ± 1.38
6w43)	4-OCH3-Bn	4-OCH3-Ph	56	54.26 ± 0.86	>100	90.33 ± 0.84
6x	2-Cl-Bn	4-OCH3-Ph	55	33.43 ± 1.51	51.92 ± 1.07	42.47 ± 0.17
6y	4-CH3-Bn	2-Cl-Ph	55	28.46 ± 0.72	40.76 ± 1.67	34.12 ± 0.98
6z42)	2-Cl-Bn	2-Cl-Ph	61	33.55 ± 0.64	>100	43.62 ± 1.06
6ab)	1,3-Benzodioxol-5-ylmethyl	2-Cl-Ph	55	25.98 ± 0.68	51.22 ± 0.83	22.29 ± 0.91
6β	4-OCH3-Bn	2-Cl-Ph	53	21.11 ± 0.27	49.71 ± 0.93	25.46 ± 5.24
5-FUc)	—	—	—	3.77 ± 0.07	7.21 ± 0.31	6.08 ± 0.29

a) Isolated yields. b) Commercially available. c) 5-Fluorouracil (5-FU) was used as a positive control. Data are presented as mean ± standard deviation (S.D.) (n = 3).

The cytotoxicities of the synthesized compounds 6a–β were then evaluated against the three cancer cell lines: A549, HeLa, and MCF-7. The results are presented in Table 1. Most of the compounds exerted significant cytotoxicities against the cancer cell lines at the tested concentrations. Compounds 6t and 6α were moderate activities against MCF-7, while 6y, 6α, and 6β were against A549, respectively, with submicromolar IC₅₀ values. Notably, compounds 6j, 6k, 6l, and 6n displayed good activity on two cell lines, A549 and MCF-7, with IC₅₀ values ranging from 2.55 to 10.63 µM. These compounds exhibited 1.4- to 2.4-fold stronger activities than the positive control 5-FU (IC₅₀ = 6.08 µM) on the MCF-7 cell line, while 6k (IC₅₀ = 3.22 µM) displayed comparable activity to 5-FU against the A549 cell line (IC₅₀ = 3.77 µM).

SAR analyses suggested that the single functionalizations of the benzene ring at the N-1 position with substituents such as the CH₃ (compound 6b), Cl (6c), or OCH₃ (6d) group were ineffective in augmenting the activity against the three tested cell lines, compared to the simple unsubstituted analog 6a. Complete loss of the activity was observed in the cases of 6e and 6f with the 3,4-dioxolyl and 3-pyridinyl groups, respectively, on the three tested cell lines, and 6c with the Cl group on the MCF-7 cell line. However, the introduction of the CH₃ (6g), OCH₃ (6h), or Cl (6i) group to the benzene ring at the C-2 position of the benzimidazole structure increased the activity by 1.3- to 2.8-fold against the three tested cancer cell lines, compared to that of 6a, although the activities of these compounds were still much lower than the positive control 5-FU. Notably, the introduction of the 3-pyridinyl moiety at the C-2 position (6j) dramatically increased the cytotoxicity against the A549 and MCF-7 cell lines, which were 7.4-fold and 21-fold, respectively, stronger than those of 6a. The activity of 6j was comparable to that of 5-FU against the MCF-7 cell line. Interestingly, further functionalization of the benzene ring at the N-1 position of 6j with the CH₃ group (6k) considerably enhanced the activity against the A549 cell line, which was around 2.4-fold stronger than 6j and comparable to 5-FU, while almost remained the same activity as 6j against the HeLa and MCF-7 cell lines. In contrast, the 3,4-dioxolyl group in 6n considerably increased the activity towards the MCF-7 cell line, which was 1.6- and 2.4-fold higher than those of 6j and 5-FU, respectively, while nearly maintained the same activity as 6j against the A549 cell line. Meanwhile, the OCH₃ group in 6l led to almost the same activity as 6j against all three tested cell lines.

The combination of the 3-pyridinyl group at C-2 with the 2-Cl-Bn group (6m) or the 3-pyridinylmethyl group (6o) at the N-1 position eliminated the activity against both the A549 and MCF-7 cell lines. A similar loss of activity on the three tested cell lines and the HeLa cell line was also observed with the two compounds 6q and 6r, and the three compounds 6u, 6w, and 6z, respectively. Other cases with the simultaneous presence of two substituents on the benzene rings at the N-1 and C-2 positions (6y, 6x, 6t, and 6β) led to an increase in activity, compared to 6a, but were still weaker than that of 5-FU. Importantly, the considerable loss of the activities observed for 6s, 6u, and 6y compared to 6k, for 6w and 6β compared to 6l, and for 6t, 6v, and 6α compared to 6n emphasized the key role of the 3-pyridinyl moiety at the C-2 position in inducing the desired cytotoxicities of the synthesized 1,2-disubstituted benzimidazoles.

In summary, the good cytotoxicities of the 1,2-disubstituted benzimidazoles 6j, 6k, 6l, and 6n towards the A549 and MCF-7 cell lines were crucially attributed to the 3-pyridinyl moiety at the C-2 position. The activity was significantly enhanced with the further introduction of the CH₃ to the benzene ring at the N-1 position of the benzimidazole heterocycle against the A549 cell line, while the presence of the OCH₃, or the 3,4-dioxolyl group at this position slightly increased the cytotoxicity against the MCF-7 cell line (Fig. 3). These structures provide new scaffolds as potential leads for further optimization and development of new anti-cancer compounds.

Fig. 3. Potent Cytotoxic Structures of Evaluated 1,2-Disubstituted Benzimidazoles

Conclusion

The facile synthesis of twenty-eight 1,2-disubstituted benzimidazoles (6a–β) was successfully developed, based on the condensation reaction between N-benzyl o-phenylenediamine and benzylamine, using Na₂S₂O₅ as an inexpensive and environmentally friendly oxidizing agent under solvent-free conditions and with short reaction times. Cytotoxicity assessments against the A549, HeLa, and MCF-7 cell lines revealed that 6j, 6k, 6l, and 6n displayed good activity against the A549 and MCF-7 cell lines, which were 1.4-fold to 2.4-fold stronger than the positive control 5-FU against the MCF-7 cells, while 6k displayed almost the same activity as that of 5-FU against the A549 cell line. SAR analyses indicated that the 3-pyridinyl moiety at C-2, or the CH₃, OCH₃, or 3,4-dioxolyl group on the benzene ring at the N-1 position of the benzimidazole structure were the key structural features giving rise to the observed cytotoxicities. Our findings highlighted 6j, 6k, 6l, and 6n as promising scaffolds for further optimization in the development of effective anticancer drugs.

Experimental

General Information

Reactions were monitored by TLC on 0.2 mm pre-coated silica gel 60 F₂₅₄ plates (Merck Millipore, Burlington, MA, U.S.A.). ¹H-NMR and ¹³C-NMR spectra were measured with 400 and 500 MHz JEOL ECA400II spectrometers. Chemical shifts are given in parts per million relative to tetramethylsilane (Me₄Si, δ = 0); J values are given in Hz. Mass spectrometry data were recorded on an Agilent 6530 qTOF spectrometer (Santa Clara, CA, U.S.A.). Fourier transform (FT)-IR was conducted using the KBr pellet method with a Thermo Nicolet 6700 spectrometer (Waltham, MA, U.S.A.). Silica gel 60 (0.063–0.200 mm, Merck) was used for column chromatography. The purities of the compounds that were synthesized and used for the cytotoxic assay were estimated based on their peak areas on the HPLC chromatograms, using an Agilent Infinity II 1260 HPLC system coupled with a SUPELCO Discovery C18 (4.6 × 250 mm) column (flow rate, 0.5 mL/min; mobile phase, water and MeOH, both containing 0.1% formic acid; 0–40 min: 50 to 100% MeOH, UV: 190 to 400 nm). All compounds were >95% pure according to HPLC spectrometry (see spectra in Supplementary Materials).

General Procedure for the Synthesis of Benzimidazole Derivatives 6a–β

A mixture of o-phenylenediamines 1 (0.1 mmol), sodium metabisulfite (Na₂S₂O₅) (38 mg, 0.2 mmol), and benzylamines 4a–f (0.3 mmol) in a 10 mL sealed tube was stirred at 140 °C for 1.5 h. At the end of the reaction, the mixture was cooled to room temperature and water (2 mL) was added. The water layer was extracted by ethyl acetate (3 × 3 mL). The combined organic layers were washed with water (3 × 5 mL), followed by a saturated aqueous solution of NaCl (3 × 3 mL), and dried over Na₂SO₄, and solvents were evaporated under reduced pressure. The crude products were purified by column chromatography to obtain the desired 1,2-disubstituted benzimidazole derivatives 6a–β.

All compounds were >95% pure according to HPLC spectrometry (see spectra in Supplementary Materials). Spectral data of 6a–b, 6d, 6g–j, 6s, 6u, 6w, and 6z were reported previously (Table 1). Compounds 6e–f, 6k–m, and 6α are commercially available.

Cytotoxicity Assay

The cytotoxic activities of the synthesized compounds were evaluated against three human cancer cell lines (A549, HeLa, and MCF-7), using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay with some modifications.⁴⁴⁾ All synthesized compounds were dissolved in DMSO to make 10 mM stock solutions. Serial dilutions were prepared in the culture medium. The positive control, 5-FU, was dissolved in dimethyl sulfoxide (DMSO) to make a 10 mM stock solution and then stored at −20 °C until use.

The human cancer cell lines were cultured in α-minimum essential medium (α-MEM), supplemented with 1% antibiotic antimycotic solution and 10% fetal bovine serum, at 37 °C and in a 5% CO₂ atmosphere. Cells at 80–90% confluency were harvested and centrifuged at 3000 rpm for 3 min. The supernatant was discarded and the cell pellet was resuspended in fresh medium. Aliquots (100 µL) of the cells were seeded in 96-well plates (2 × 10³ cells/well) and incubated for 24 h. The cells were then washed with phosphate-buffered saline (PBS), and five concentrations of tested compounds (6.25, 12.5 25, 50, 100 µM or 1.56, 3.12, 6.25, 12.5, 25 µM) and the positive control, 5-FU (1.56, 3.12, 6.25, 12.5, 25 µM) were added to the wells. After a 72 h incubation, the cells were washed with PBS, and 100 µL of medium containing MTT solution (5 mg/mL) was added to each well and incubated for 3 h. The absorbance was recorded using a microplate reader at 570 nm. Percent proliferation inhibition was calculated using the following formula:

  

• A_t: Absorbance of test compound, A_b: Absorbance of blank, A_c: Absorbance of control.

The concentrations (IC₅₀ values) of the compounds required to inhibit 50% of the growth of the human cancer cell lines were calculated based on the relationship between the concentrations and the percentage of inhibition, using the GraphPad Prism 10.3.0 software. Each experiment was performed three times, and all data are presented as mean ± standard deviation (S.D.).

Acknowledgments

Hieu Trong Le was funded by the Master, Ph.D. Scholarship Programme of Vingroup Innovation Foundation (VINIF), code VINIF.2023.TS.044. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (JSPS KAKENHI Grant JP22H02777 to H.M.).

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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