Biological activity of bis-benzimidazole derivatives on DNA topoisomerase I and HeLa, MCF7 and A431 cells

Abstract

Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H₂ receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.

Keywords::

• Bis-benzimidazoles
• DNA topoisomerase I
• Cytotostaticity

Introduction

Benzimidazoles and their derivatives are important compounds because of their pharmacologic properties. The diverse substitutions of benzimidazole compounds give rise to antihelmintic [1,2], antifungal 3,4,5, antitumor [6], antiviral [3,4], H₂ receptor blocker and proton pump inhibitor activities [7]. Several benzimidazole derivatives are also active as inhibitors of DNA topoisomerases 8,9,10,11. DNA topoisomerases are essential enzymes that regulate conformational changes in DNA topology by catalyzing concerted breakage and rejoining of DNA strands during many genetic processes, including DNA replication, transcription, recombination and transposition [12]. Intermediates in the strand passage reaction involve either single- or double-stranded breaks, defining type I and type II enzymes, respectively. Type I topoisomerases (topo I) make a single-stranded break in a DNA duplex, mediate passage of the intact strand through the break, and then reseal it. Type II topoisomerases (topo II), on the other hand, create transient breaks in both strands of a duplex, pass an intact DNA segment through the break and then reseal the cleavage site [12,13]. Over the past years, DNA topoisomerases have been recognized as an effective approach for the development of chemotherapeutics 14,15,16,17,18,19,20. As part of our work in searching for biologically active compounds, we synthesized twelve bis-benzimidazole derivatives of which five of them were original with different substitutions at position 5- (Table I). The structures of the synthesized compounds were elucidated by melting point, IR, ¹H NMR and LC-MS analyses. The compounds were screened for their interference with topo I activity via in vitro supercoil relaxation assays. Selected derivatives were subjected to the MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay in vitro on three human cell lines; HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) [21].

Table I.  Physical constants of the synthesized compounds.

Compound	R	R1	X	m.p. (°C)	Yield (%)	Mol. formula
1	H	H	CH2	295–298	25.6	C15H12N4
2	CH3	H	CH2	283–286	7.5	C17H16N4
3	CH3	CH3	CH2	278–283	7.1	C19H20N4
4	H	H	(CH2)2	344–345	69.0	C16H14N4
5	CH3	H	(CH2)2	271	22.0	C18H18N4
6	CH3	CH3	(CH2)2	275–276	19.0	C20H22N4
7	H	H	(CH2)3	267	37.2	C17H16N4
8	CH3	H	(CH2)3	120	16.8	C19H20N4
9	CH3	CH3	(CH2)3	296	21.7	C21H24N4
10	H	H	(CH2)4	331	93.3	C18H18N4
11	CH3	H	(CH2)4	230	17.6	C20H22N4
12	CH3	CH3	(CH2)4	142	23.0	C22H26N4

Experimental

Chemistry

Melting points were determined with a Buchi 510 capillary melting point apparatus. The IR spectra of compounds were monitored as potassium bromide pellets on a Jasco FT/IR-430 spectrometer. The NMR spectra were recorded on a Varian AS 400 Mercury Plus NMR spectrometer. NMR spectra (400 MHz for ¹H) were recorded in CD₃OD. Chemical shifts were measured in parts per million (δ). J values are given in Hz. Mass spectra (LC-MS) were performed on a Waters 2695 Alliance Micromass ZQ LC-MS Spectrometer Electrosprey Ionization (ESI). Analytical thin-layer chromatography (TLC) was run on Merck silica gel plates (Kieselgel 60F₂₅₄) with detection by UV light. All starting materials and reagents were high-grade commercial products. All chemical drawings and calculations were performed by using the ChemDraw Ultra 9.0

General procedure for the synthesis of the bis-benzimidazole derivatives

The bis-benzimidazoles were synthesized by the method of acid-catalyzed condensation of a substituted o-phenylenediamine with carboxylic acid [2]. All of the bis-benzimidazoles were symmetrically substituted using the same subtituent on both benzimidazole moieties. The 12 bis-benzimidazoles with various substitutions covered in this study are given in Table I. Briefly, 0.005 moles of carboxylic acid and 0.01 moles of substituted o-phenylenediamine in 10 mL of 4N HCl were refluxed for 6 h–13 h at 135°C in an oil bath. The mixture was cooled and neutralized with 25% NH₄OH. The precipitate was filtered and recrystallized from ethanol/water.

The yield percentages and the melting points of the synthesized compounds are listed in Table I and spectral data obtained are as follows;

Di(1H-benzo[d]imidazol-2-yl)methane (1)

IR (KBr) cm^{− 1}: 3060, 2834, 1621, 1542, 1430, 1033. ¹H-NMR (CD₃OD) δ: 4.56 (2H, s, –CH₂–), 7.21 (4H, dd, J = 3.2, 5.6 Hz, H-5, H-5′, H-6, H-6′), 7.52 (4H, dd, J = 3.2, 6.4 Hz, H-4, H-4′,H-7, H-7′). ES-MS m/z Calculate (M⁺): 248.28, Found (M⁺+1): 249.31.

Bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane (2)

IR (KBr) cm^{− 1}: 3378, 2919, 1627, 1542, 1448, 1031. ¹H-NMR (CD₃OD) δ: 2.42 (6H, s, 2xAr–CH₃), 4,50 (2H, s, –CH₂–), 7.04 (2H, dd, J = 1.6, 8 Hz, H-6, H-6′), 7.31 (2H, brs, H-4, H-4′), 7.39 (2H, d, J = 8.4 Hz, H-7, H-7′). ES-MS m/z Calculate (M⁺): 276.34, Found (M⁺+1): 277.40.

Bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane (3)

IR (KBr) cm^{− 1}: 3384, 2921, 1629, 1533, 1448, 1022. ¹H-NMR (CD₃OD) δ: 2.33 (12H, s, 4xAr–CH₃), 4.46 (2H, s, –CH₂–), 7.27 (4H, s, H-4, H-4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 304.39, Found (M⁺+1): 305.44.

1,2-di(1H-benzo[d]imidazol-2-yl)ethane (4)

IR (KBr) cm^{− 1}: 3432, 2931, 1627, 1569, 1454, 1014. ¹H-NMR (CD₃OD) δ: 3.93 (4H, s, –CH₂CH₂–), 7.62 (4H, dd, J = 3.2, 6.4 Hz, H-5, H-5′, H-6, H-6′), 7.81 (4H, dd, J = 3.2, 6.4 Hz, H-4, H-4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 262.31, Found (M⁺+1):263.16.

1,2-bis(5-methyl-1H-benzo[d]imidazol-2-yl)ethane (5)

IR (KBr) cm^{− 1}: 3023, 2917, 1631, 1540, 1452, 1022. ¹H-NMR (CD₃OD) δ: 2.42 (6H, s, 2xAr–CH₃), 3.36 (4H, s, –CH₂CH₂–), 7.01 (2H, dd, J = 0.8, 8.2 Hz, H-6, H-6′), 7.27 (2H, brs, H-4, H-4′), 7.36 (2H, d, J = 8.4 Hz, H-7, H-7′). ES-MS m/z Calculate (M⁺): 290.36, Found (M⁺+1): 291.38.

1,2-bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)ethane (6)

IR (KBr) cm^{− 1}: 3363, 2842, 1627, 1565, 1465, 1006. ¹H-NMR (CD₃OD) δ: 2.43 (12H, s, 4xAr–CH₃), 3.85 (4H, s, –CH₂CH₂–), 7.54 (4H, s, H-4, H4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 318.42, Found (M⁺+1): 319.17.

1,3-di(1H-benzo[d]imidazol-2-yl)propane (7)

IR (KBr) cm⁻¹: 3357, 2917, 1627, 1562, 1459, 1006. ¹H-NMR (CD₃OD) δ: 2.36 (2H, quin., J = 7.6, –CH₂CH₂CH₂–), 2.98 (4H, t, J = 7.4, –CH₂CH₂CH₂–), 7.18 (4H, dd, J = 3.6, 6 Hz, H-5, H-5′, H-6, H-6′), 7.48 (4H, m., H-4, H4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 276.34, Found (M⁺+1): 277.39.

1,3-bis(5-methyl-1H-benzo[d]imidazol-2-yl)propane (8)

IR (KBr) cm^{− 1}: 3357, 2917, 1627, 1562, 1459, 1006. ¹H-NMR (CD₃OD) δ: 2.34 (2H, quin, J = 7.2, –CH₂CH₂CH₂–), 2.42 (6H, s, 2xAr–CH₃), 2.96 (4H, t, J = 7.2 Hz, –CH₂CH₂CH₂–), 7.03 (2H, dd, J = 0.8, 8.4 Hz, H-6, H-6′), 7.27 (2H, dd, J = 0.8, 2.4 Hz, H-4, H-4′), 7.36 (2H, d, J = 8 Hz, H-7, H-7′). ES-MS m/z Calculate (M⁺): 304.39, Found (M⁺+1): 305.45.

1,3-bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)propane (9)

IR (KBr) cm^{− 1}: 3097, 2933, 1631, 1538, 1454, 998. ¹H-NMR (CD₃OD) δ: 2.30 (2H, m., –CH₂CH₂CH₂–), 2.32 (12H, s, 4xAr–CH₃), 2.91 (4H, t, J = 7.6, –CH₂CH₂CH₂–), 7.23 (4H, s, H-4, H4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 332.44, Found (M⁺+1): 333.47.

1,4-di(1H-benzo[d]imidazol-2-yl)butane (10)

IR (KBr) cm^{− 1}: 3434, 3046, 2956, 1619, 1548, 1438, 1039. ¹H-NMR (CD₃OD) δ: 2.11 (4H, quin., J = 3.2 Hz, –CH₂CH₂CH₂CH₂), 3.35 (4H, m., –CH₂CH₂CH₂CH₂–), 7.57 (4H, dd, J = 3.2, 6.4 Hz, H-5, H-5′, H-6, H-6′), 7.76 (4H, dd, J = 3.2, 6 Hz, H-4, H4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 290.36, Found (M⁺+1): 291.43.

1,4-bis(5-methyl-1H-benzo[d]imidazol-2-yl)butane (11)

IR (KBr) cm^{− 1}: 3397, 3046, 2921, 1631, 1540, 1452, 1045. ¹H-NMR (CD₃OD) δ: 1.89 (4H, m, –CH₂CH₂CH₂CH₂), 2.41 (6H, s, 2xAr–CH₃), 2.90 (4H, m., –CH₂CH₂CH₂CH₂–), 6.99 (2H, dd, J = 1.2, 8.4 Hz, H-6, H-6′), 7.25 (2H, brs, H-4, H-4′), 7.33 (2H, d, J = 8 Hz, H-7, H-7′). ES-MS m/z Calculate (M⁺): 318.42, Found (M⁺+1): 319.49.

1,4-bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)butane (12)

IR (KBr) cm^{− 1}: 3376, 3203, 2931, 1635, 1542, 1452, 1043. ¹H-NMR (CD₃OD) δ: 1.91 (4H, m, –CH₂CH₂CH₂CH₂), 2.35 (12H, s, 4xAr–CH₃), 2.96 (4H, m., –CH₂CH₂CH₂CH₂), 7.28 (4H, s, H-4, H4′, H-7, H-7′). ES-MS m/z Calculate (M⁺): 346.47, Found (M⁺+1): 347.53 (M⁺+1).

Plasmid supercoil relaxation assays

Plasmid supercoil relaxation assays were carried out as described [22]. Briefly, 20 μL of reaction mixture contained one unit of calf thymus topoisomerase I, 0.5 mg of supercoiled (sc) pBR322 (TAKARA, Otsu-Shiga, Japan), in the presence or absence of the test compounds in 35 mM Tris-HCl (pH 8.0), 72 mM KCl, 5 mM MgCl₂, 5 mM dithio erythrole (DTT), 5 mM spermidine, and 0.1% bovine serum albumin. A stock solution of 10 mg/mL CPT in dimethl sulfoxide (DMSO) was serially diluted for comparisons. The relaxation products were analyzed on 1% agarose gels in Tris-borate EDTA (TBE buffer (45 mM Tris borate and 1 mM EDTA, pH 8.0) in a horizontal electrophoresis apparatus (5 V/cm) (Thermo EC250) and photographed under UV light after staining in ethidium bromide (EtdBr) solution (0.5 μg/mL). The relationship between the binding of EtdBr and the amount of fluorescence given by sc and relaxed DNA (rlx DNA) under UV light was carried out as described [23]. DNA bands were quantified from gel photographs using BioRad Multianalyst (ver. 1.1). One unit of the enzyme activity was defined as the activity removing the supercoils from 500 ng of sc plasmid substrate at 37°C.

Cytostatic assays

Antiproliferative effects of the test compounds were measured in vitro on three human cell lines: HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma), by using the MTT assay [21]. Briefly, cancer cells (5000/well) were seeded onto a 96-well microplate and attached to the bottom of the well overnight. On the second day, 200 μL of new medium containing the test substances was added. After incubation for 72 h, the living cells were assayed by the addition of 20 μL of 5 mg/mL MTT solution. MTT was converted by intact mitochondrial reductase and precipitated as blue crystals during a 4 h contact period. The medium was then removed, and the precipitated crystals were dissolved in 100 μL of DMSO during a 60 min period of shaking. Finally, the reduced MTT was assayed at 545 nm, using a microplate reader; wells with untreated cells were utilized as controls. All in vitro experiments were carried out on two microplates with at least five parallel wells. Doxorubicin was used as positive control. Stock solutions of the tested substances (30 mM) were prepared with DMSO. The highest DMSO concentration (0.3%) of the medium did not have any significant effect on the cell proliferation. Their antiproliferative effects were determined in the concentration range 0.1–30 mM, the dose-response curves were fitted by means of GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA) and the IC₅₀ values were calculated.

Results and discussion

Chemistry

The ¹H NMR spectrum of non-substituted compounds (1, 4, 7 and 10) having symmetrical structures, contain two 1,2-disubstituted systems. The proton signals of alkyl chains combining benzimidazol rings were observed at the expected form/region [24]. Protons of benzimidazoles were seen in a defined form of ABX systems in compounds having methyl groups at 5- position (2, 5, 8 and 11) while they were assigned as four hydrogen singlets at 4- and 7- positions of the benzimidazole rings for the derivatives bearing methyl groups at both 5- and 6- positions (3, 6, 9 and 12).

Biological activity assays

The most frequently employed method for monitoring DNA topoisomerase activity utilizes an in vitro supercoil relaxation assay using plasmid substrates [11,13]. We employed this method to identify if the synthesized derivatives interfere with topo I reactions. Our assay employs supercoiled plasmid DNA (sc DNA) and relies on the ability of the enzyme to relax sc DNA, which can be separated as discrete bands using gel electrophoresis. An inhibition of relaxation activity due to the presence of a particular inhibitor is monitored in the form of an accumulated faster-migrating sc DNA (Form I). A representative assay using 1 μg/μL concentration of the test compounds in topo I reactions is given in Figure 1. The sc DNA (Figure 1A, lane 1) was fully relaxed (Form II) (Figure 1A, lane 2) and the presence of the organic solvent DMSO did not interfere with the topo I activity (Figure 1A, lane 3). Relaxation of sc DNA substrate was inhibited upon incubation with some of the compounds to different extends (Figure 1, lanes 4-15). The 2 and 3 were found to exert strongest interference (Figure 1A, lanes 8 and 12, respectively), while the 7 and 5 showed an intermediate effect in supercoil relaxation activity of the enzyme (Figure 1A, lanes 6 and 9, respectively). The other derivatives gave rise to either neglectable (Figure 1A. lanes; 4, 11, 13 and 15 for 1, 11, 6 and 12, respectively) or no interference with topo I reaction (Figure 1A. lanes; 5, 7, 10 and 14 for the 4, 10, 8 and 9, respectively). We then quantified sc and rlx DNA band intensities and the average values were summarized in Figure 1B. The most effective derivatives 2 and 3 had average interference percentages of 98% and 87%, respectively, as revealed with the remaining sc band intensity with reference to sc pBR322 while this value varied between 2%–30% for 6, 7, 11 and 12 (Figure 1B). We next made a serial comparison of topo I reactions using decreasing concentrations of 1, 2 and 4 as well as the well-known anti-tumor drug, Camptothecin (CPT). The inhibition obtained with the test compounds decreased upon dilutions, which showed the detected sc DNA bands were concentration-dependent (data not shown).

Figure 1.  Inhibitory activity of compounds 1-12 on DNA topoisomerase I. A. A representative assay using 1 μg/μL concentration of the test compounds in topo I reactions., lane 1, pBR322 DNA without enzyme; lane 2, pBR322 with 1 unit of DNA topoisomerase I; lane 3, same as lane 2 in DMSO; lanes 4-15, same as lane 2 in the presence of the test compounds from 1 to 12, respectively. B. Quantitative assessment of the inhibition obtained with the compounds.

Many reports in recent years have shown that DNA topoisomerase inhibition by numerous natural and synthetic compounds is closely associated with the cytotoxic potentials of the inhibitory compounds [14,16,18,20]. A representative number of compounds (4/12) were selected to carry out MTT assays in vitro on HeLa, MCF7 and A431 cells to identify if their effects on topo I reactions coincided with their cytotoxicity. The selected compounds were the 3, 5, 6 and 9. The 3 and 5 represented high (87%) and intermediate (30%) interference in topo I results, respectively. The other two cpds represented either neglectable (6; 2%) or derivatives with no interference (9; 0%). Among the four cpds, only 3 manifested a considerable cytotoxicity with relatively low calculated IC₅₀ values of 3.74 μM and 3.58 μM in A431 and MCF7 cells, respectively, while this value was 13.98 μM when HeLa cells were used (Table II). These values are higher than those of the reference agent doxorubicin reflecting a lower cytotoxic potency but a compound with IC₅₀ of 5–10 μM can be considered as a lead for design and synthesis of further analogues. The other compounds did not give a considerable cytotoxicity in MTT tests (data not shown).

Table II.  Calculated IC₅₀ values in MTT tests.

	Calculated IC50 values (μM)
Test Compound	A431	HeLa	MCF7
Cpd.3	3.74	13.98	3.58
Doxorubicin	0.19	0.16	0.31

The characteristic of 3 is to have the shortest alkyl group and to bear two methyl groups, which are electron donors at both rings (Table I). It has a comparable structure to 2 as well, the other remarkably effective compound on topo I. Considering the resemblance of 2 and 3 to each other and their difference from the other tested compounds, the length of alkyl chain and methyl substitutions could be regarded as the main determinants of the biological activity results. On the other hand, given the complex mechanism of topo I reaction, which involves the enzyme binding to DNA, the breakage of DNA strands, and the religation of the DNA following the strand-passing step, our data do not accommodate a complete mechanistic interpretation of the structure-related activity. Nevertheless, the inhibitory effects obtained with some of bis-benzimidazole derivatives are a significant result as these compounds can be potential sources of anticancer agents.

Acknowledgements

The authors thank Ms. Pakize Canturk for her valuable help with the plasmid supercoil relaxation assays and gel analyses.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Notes

^† Current Address: Department of Biochemistry, Faculty of Science, Ege University, Izmir, 35100, Turkey.

^‡ These authors are equal contributors to the manuscript.