Abstract
A series of novel 5-(4-methylpiperazin-1-yl)-2-phenyl-1H-benzimidazoles (5–14) were synthesized and evaluated for their in vitro antiproliferative activities against the human leukemia cell line HL-60. Compounds 5–7 and 10–12 exhibited potent antiproliferative activities against this cell line. The quantitative analysis of apoptosis by flow cytometry demonstrated that the percentages of apoptotic HL-60 cells treated with compounds 5 and 10–12 were significantly higher than in the control.
1 Introduction
Acute myeloid leukemia (AML) is a malignant disorder characterized by the accumulation of clonal leukemic hematopoietic precursor cells arrested at various stages of normal myeloid development [1]. The development and progression of AML is highly related to dysregulated apoptotic pathways. The current treatment option for AML is chemotherapy with drugs such as cytarabine, daunorubicin, or idarubicin, which target the eradication of cancer cells. For the last 40 years, serious efforts have been made to design and synthesize potential chemotherapeutic agents for anticancer therapy [2, 3].
Benzimidazole derivatives have been reported to exhibit antibacterial, antiviral, antiparasitic, anthelmintic, antitumor, anti-inflammatory, antioxidant, antiulcer, antihypertensive, anticoagulant, antidepressant, anticonvulsant, antihistaminic, antiasthmatic, and antidiabetic activities [4–7]. Recently, some benzimidazoles have also been found to have remarkable antileukemic activities [8–13].
It is known that Hoechst 33342 and 33258 (Figure 1) are adenine-thymine-specific dyes that stain DNA by binding to its minor groove. It was reported that Hoechst 33342, bearing an ethoxy group, induced apoptosis and caused cell death in HL-60 cells; however, this effect could not be seen with Hoechst 33258 containing a hydroxy group [14]. In our previous communication, we reported the antileukemic activities of the two 2-(4-phenoxyphenyl)-1H-benzimidazoles I (IC50 20.65±0.06 μM) and II (IC50 27.83±0.04 μM) against the human chronic myelogenous leukemia cell line K562 (Figure 1) [13]. These findings prompted us to investigate a series of novel hydroxy, alkoxy, or aryloxy substituted monobenzimidazole derivatives of the Hoechst compounds for their antiproliferative activity against the AML cell line HL-60.
Chemical structures of Hoechst 33342 and 33258 and 2-(4-phenoxyphenyl)-1H-benzimidazoles I and II.
2 Materials and methods
2.1 Analytical methods
Uncorrected melting points were measured on a Büchi B-540 capillary melting point apparatus (Büchi Labortechnik, Flawil, Switzerland). Infrared (IR) spectra were obtained using an Agilent Cary 630 Fourier transform IR (FTIR) with Diamond ATR accessory (Agilent Technologies, Santa Clara, CA, USA). 1H-nuclear magnetic resonance (NMR) and 13C-NMR spectra were recorded employing a Varian Mercury 400 MHz FT spectrometer (Varian, Palo Alto, CA, USA). Chemical shifts (δ) are given in ppm relative to tetramethylsilane, and coupling constants (J) are reported in Hz. The 1H-NMR spectra of some compounds are not sufficiently resolved under standard conditions because of the tautomeric effect of the imidazole moiety. To prevent the tautomeric effects, the compounds were dissolved in dimethylsulfoxide (DMSO)-d6 followed by a tiny amount of dry NaH. After the addition of two to three drops of D2O to the NMR tube and thorough stirring, very clear NMR spectra were obtained [15]. The IR and NMR spectra of the compounds are provided in the Supplementary Material. Mass spectra were taken on a Waters Micromass ZQ (Micromass UK, Manchester, UK) in the positive-ion mode, and high-performance liquid chromatography was performed using a C18 column in a Waters Alliance instrument. Elemental analyses were performed on a Leco CHNS-932 analyzer (Leco, St. Joseph, MI, USA). All reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.2 Chemistry
The synthetic pathways for the preparation of the targeted benzimidazoles (5–14) are shown in Scheme 1. 4-Phenoxybenzaldehyde (1) [16] and 4-(3,4-dimethoxyphenoxy)benzaldehyde (2) [17] were synthesized by the reaction between phenol/3,4-dimethoxyphenol and 4-fluorobenzaldehyde in the presence of anhydrous K2CO3 in dimethylacetamide (DMAC). The nucleophilic displacement of the chloro group of 5-chloro-2-nitroaniline by the reaction with 1-methylpiperazine in dimethylformamide (DMF) in the presence of anhydrous K2CO3 gave compound 3 [18]. The reduction of the nitro group yielded 4-(4-methylpiperazin-1-yl)-1,2-diaminobenzene (4) [18]. The targeted benzimidazoles (5–14) were prepared from the condensation reaction between compound 4 and various Na2S2O5 adducts of compounds 1, 2, or commercial aldehydes in DMF. The respective aldehyde (compounds 1, 2, or commercial aldehydes; 7.5 mmol) was dissolved in 30 mL ethanol. Sodium bisulfite (0.8 g) in water (5 mL) was added to this solution and stirred vigorously. Afterwards, ethanol was added and the mixture was cooled. The resulting precipitate was filtered off and dried. A mixture of this salt (2 mmol) and 4-(4-methylpiperazin-1-yl)-1,2-diaminobenzene (4; 2 mmol) in DMF (2–3 mL) was heated at 135 °C for 4 h [19]. The reaction mixture was cooled and poured into water; the solid was collected and purified by column chromatography using 25% methanol in EtAc.
General synthesis of novel 5-(4-methylpiperazin-1-yl)-2-phenyl-1H-benzimidazoles.
Reagents: (a) anhydrous K2CO3, DMAC; (b) anhydrous K2CO3, DMF; (c) H2/Pd-C; and (d) Na2S2O5 adducts of compounds 1, 2, and commercial aldehydes.
2.2.1 2-[4-(Benzyloxy)phenyl]-5-(4-methylpiperazin-1-yl)-1H-benzimidazole (5):
Yield 25%, mp 189–190 °C, IR (FTIR/Diamond ATR) νmax (cm−1): 2967, 2873, 2816, (C-H), 1610–1451 (aromatic C=C, C=N), 1319–1127 (C-N, C-O). 1H-NMR (DMSO-d6+D2O+NaH) δ: 2.22 (s, 3H), 2.49 (piperazinyl methylenes, overlapped DMSO-d6), 3.01 (t, 4H, piperazinyl methylenes), 5.13 (s, 2H), 6.52 (dd, 1H, J=8.4 Hz, J=2.4 Hz), 6.94–6.96 (m, 3H), 7.22 (d, 1H, J=8.8 Hz), 7.32–7.43 (m, 3H), 7.48 (d, 2H, J=7.2), 8.13 (dd, 2H, J=8.8 Hz, J=2.0 Hz). 13C-NMR (DMSO-d6+D2O+NaH) δ: 159.9, 157.5, 148.0, 144.6, 143.0, 137.8, 131.3, 128.9, 128.2, 128.1, 127.9, 115.6, 114.5, 110.7, 104.1, 69.6, 55.6, 51.9, 46.2. MS (ESI+) m/z (rel. intensity): 399 (M+H, 100). Anal. for C25H26N4O·1.75H2O, Calcd. C 69.82, H 6.91, N 13.02. Found C 69.58, H 7.16, N 12.75.
2.2.2 5-(4-Methylpiperazin-1-yl)-2-(4-phenoxyphenyl)-1H-benzimidazole (6):
Yield 32%, mp 185–186 °C, IR (FTIR/Diamond ATR) νmax (cm−1): 2965, 2937, 2812 (C-H), 1612–1451 (aromatic C=C, C=N), 1284–1144 (C-N, C-O). 1H-NMR (DMSO-d6+D2O+NaH) δ: 2.18 (s, 3H), 2.48 (piperazinyl methylenes, overlapped DMSO-d6), 2.99 (t, 4H, piperazinyl methylenes), 6.54 (dd, 1H, J=8.8 Hz, J=2.4 Hz), 6.90–7.01 (m, 5H), 7.10 (t, 1H, J=7.6 Hz), 7.22 (d, 1H, J=8.4 Hz), 7.37 (td, 1H, J=8.8 Hz, J=2.0 Hz), 8.17 (dd, 2H, J=8.4 Hz, J=2.0 Hz). 13C-NMR (DMSO-d6+D2O+NaH) δ: 159.5, 157.7, 155.7, 147.9, 145.1, 142.9, 133.8, 130.7, 128.5, 123.9, 118.9, 116.2, 111.5, 104.4, 55.6, 51.9, 46.2. MS (ESI+) m/z (rel. intensity): 385 (M+H, 30), 214 (100). Anal. for C24H24N4O·1.75H2O, Calcd. C 69.29, H 6.66, N 13.46. Found C 69.60, H 6.33, N 13.09.
2.2.3 2-[4-(3,4-Dimethoxyphenoxy)phenyl]-5-(4-methylpiperazin-1-yl)-1H-benzimidazole (7):
Yield 20%, mp 150–152 °C (bubbling), IR (FTIR/Diamond ATR) νmax (cm−1): 2946, 2927, 2812 (C-H), 1634–1436 (aromatic C=C, C=N), 1272–1146 (C-N, C-O). MS (ESI+) m/z (rel. intensity): 445 (M+H, 100). Anal. for C26H28N4O3·1.5H2O, Calcd. C 66.22, H 6.62, N 11.88. Found C 66.57, H 6.76, N 11.49. Detailed NMR data are given Table 1.
1H-NMR, 13C-NMR, COSY, HSQC, and HMBC data of compound 7 in DMSO-d6+D2O+NaH.
 | ||||
|---|---|---|---|---|
| Atom no. | 13C (δC) | 1H (δH) | COSY | HMBC |
| a | 46.0 | 2.20 (s, 3H) | Ca/H-3′″ | |
| b | 56.0 | 3.70 (s, 3H) | ||
| b′ | 56.3 | 3.72 (s, 3H) | ||
| 2 | 159.4 | C2/H2′ and H6′ | ||
| 3a | 144.8 | C-3a/H-7 | ||
| 4 | 104.2 | 6.97 (d, 1H, J=2.4 Hz) | H4/H6 | C-4/H-6 |
| 5 | 147.8 | C-5/H-7 | ||
| 6 | 111.2 | 6.52–656 (m, 2H, overlapped H-6″) | H6/H4, H6/H7 | C-6/H-4 |
| 7 | 115.9 | 7.24 (d, 1H, J=8.4 Hz) | H7/H6 | |
| 7a | 142.8 | C-7a/H-6, H-4 | ||
| 1′ | 132.9 | C-1′/H3′ and 5′ | ||
| 2′ and 6′ | 128.2 | 8.15 (dd, 2H, J=8.4, 2.0 Hz) | H2′ and 6′/H3′ and 5′ | |
| 3′ and 5′ | 117.6 | 6.88 (dd, 2H, J=8.4, 2.0 Hz) | H3′ and 5′/H2′ and 6′ | |
| 4′ | 156.8 | C4′/H3′ and 5′, C4′/H2′ and 6′ | ||
| 1″ | 150.7 | C1″/H6″ | ||
| 2″ | 104.8 | 6.72 (d, 1H, J=2.8 Hz) | H2″/H6″ | C2″/H6″ |
| 3″ | 150.1 | C3″/Hb, H5″ | ||
| 4″ | 145.5 | C4″/Hb′, H2″, H5″ | ||
| 5″ | 112.9 | 6.94 (d, 1H, J=8.8 Hz) | H5″/H6″ | C5″/H6″ |
| 6″ | 110.6 | 6.52–6.56 (m, 2H, overlapped H-6) | H6″/H2″, H6″/H5″ | C6″/H2″ |
| 2′″ | 51 | 3.01 (t, 4H) | H2′″/H3′″ | C2′″/H2′″, H3′″ |
| 3′″ | 55 | 2.49 (overlapped DMSO-d6) | H3′″/H2′″ | C3′″/Ha, H2′″, H3′″ |
COSY, correlation spectroscopy; HMBC, heteronuclear multiple-bond correlation; HSQC, heteronuclear single-quantum correlation.
2.2.4 2-Methoxy-4-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]phenol (8):
Yield 15%, mp 235–236 °C, IR (FTIR/Diamond ATR) νmax (cm−1): 3477 (O-H), 2831, 2804 (C-H), 1628–1449 (aromatic C=C, C=N), 1284–1138 (C-N, C-O). 1H-NMR (CD3 OD) δ: 2.40 (s, 3H), 2.71 (t, 4H, piperazinyl methylenes), 3.22(t, 4H piperazinyl methylenes), 3.97 (s, 3H), 6.91 (d, 1H, J=8.0 Hz), 7.02 (dd, 1H, J=8.4 Hz, J=2.0 Hz), 7.10 (s, 1H), 7.45 (d, 1H, J=8.8 Hz), 7.50 (dd, 1H, J=8.4 Hz, J=2.0 Hz), 7.67 (d, 1H, J=2.0 Hz). MS (ESI+) m/z (rel. intensity): 339 (M+H, 100). Anal. for C26H28N4O3·1.5H2O, Calcd. C 66.22, H 6.62, N 11.88. Found C 66.57, H 6.76, N 11.49.
2.2.5 2-Methoxy-5-[5-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]phenol (9):
Yield 25%, mp 165–167 °C (bubbling), IR (FTIR/Diamond ATR) νmax (cm−1): 3550 (O-H), 2937, 2814 (C-H), 1630–1438 (aromatic C=C, C=N), 1287–1142 (C-N, C-O). MS (ESI+) m/z (rel. intensity): 339 (M+H, 100). Anal. for C19H22N4O2·1.25H2O, Calcd. C 63.23, H 6.84, N 15.52. Found C 63.05, H 6.98, N 15.13. Detailed NMR data are given Table 2.
1H-NMR, 13C-NMR, COSY, HSQC, and HMBC data of compound 9 in DMSO-d6+D2O+NaH.
 | ||||
|---|---|---|---|---|
| Atom no. | 13C (δC) | 1H (δH) | COSY | HMBC |
| a | 45.9 | 2.19 (s, 3H) | Ca/H3″ | |
| b | 55.5 | 3.67 (s, 3H) | ||
| 2 | 154.3 | C2/H2′, H6′ | ||
| 3a | 140.1 | C3a/H7 | ||
| 4 | 101.1 | 6.95 (d, 1H, J=2.0 Hz) | H4/H6 | C4/H6 |
| 5 | 147.1 | C5/H4, H7 | ||
| 6 | 113.1 | 6.78 (dd, 1H, J=8.4, 2.0 Hz) | H6/H4, H6/H7 | C6/H4 |
| 7 | 115.5 | 7.32 (d, 1H, J=8.4 Hz) | H7/H6 | |
| 7a | 135.7 | C7a/H4, H6 | ||
| 1′ | 124.5 | C1′/H5′ | ||
| 2′ | 115.9 | 7.14 (d, 1H, J=2.4 Hz) | H2′/H6′ | C2′/H6′ |
| 3′ | 159.0 | C3′/H5′ | ||
| 4′ | 152.7 | C4′/H2′, H5′, H6′ | ||
| 5′ | 111.0 | 6.65 (d, 1H, J=8.0 Hz) | H5′/H6′ | |
| 6′ | 109.2 | 6.94 (dd, 1H, J=8.0, 2.4 Hz) | H6′/H2′, H6′/H5′ | C6′/H2′ |
| 2″ | 50.6 | 3.04 (t, 4H) | H2″/H3″ | C2″/H3″, H2″ |
| 3″ | 55.1 | 2.46 (t, 4H) | H3″/H2″ | C3″/Ha, H2″, H3″ |
2.2.6 2-[3-(Benzyloxy)-4-methoxyphenyl]-5-(4-methylpiperazin-1-yl)-1H-benzimidazole (10):
Yield 28%, mp 125–127 °C (dec.), IR (FTIR/Diamond ATR) νmax (cm−1): 2957, 2937, 2877, 2808 (C-H), 1630–1448 (aromatic C=C, C=N), 1285–1149 (C-N, C-O). 1H-NMR (DMSO-d6) δ: 2.17 (s, 3H), 2.46 (piperazinyl methylenes, overlapped DMSO-d6), 2.96 (t, 4H, piperazinyl methylenes), 3.72 (s, 3H), 5.10 (s, 2H), 6.49 (dd, 1H, J=8.8 Hz, J=2.4 Hz), 6.88 (d, 1H, J=8.8 Hz), 6.91 (d, 1H, J=2.4 Hz), 7.18 (d, 1H, J=8.4 Hz), 7.29–7.38 (m, 3H), 7.48 (d, 2H, J=8.0 Hz), 7.72 (dd, 1H, J=8.4 Hz, J=2.0 Hz), 7.95 (d, 1H, J=2.0 Hz). 13C-NMR (DMSO-d6) δ: 160.1, 148.5, 148.0, 147.9, 144.9, 143.0, 138.0, 131.4, 129.0, 128.8, 125.5, 19.6, 112.4, 112.3, 111.1, 104.3, 70.3, 56.2, 55.7, 52.0, 46.3. MS (ESI+) m/z (rel. intensity): 429 (M+H, 40), 236 (100). Anal. for C26H28N4O2·1.25H2O, Calcd. C 69.23, H 6.82, N 12.42. Found C 69.43, H 6.87, N 12.30.
2.2.7 2-[4-(Benzyloxy)-3-methoxyphenyl]-5-(4-methylpiperazin-1-yl)-1H-benzimidazole (11):
Yield 32%, mp 169–170 °C, IR (FTIR/Diamond ATR) νmax (cm−1): 2935, 2844, 2808 (C-H), 1602–1438 (aromatic C=C, C=N), 1269–1146 (C-N, C-O). 1H-NMR (DMSO-d6+D2O+NaH) δ: 2.19 (s, 3H), 2.48 (piperazinyl methylenes, overlapped DMSO-d6), 2.98(t, 4H, piperazinyl methylenes), 3.82 (s, 3H), 5.05 (s, 2H), 6.51 (dd, 1H, J=8.4 Hz, J=2.4 Hz), 6.92 (d, 2H, J=8.0 Hz), 7.20 (d, 1H, J=8.4 Hz), 7.28-7.44 (m, 5H), 7.67 (dd, 1H, J=8.0 Hz, J=2.4 Hz), 7.88 (d, 1H, J=2.0 Hz). 13C-NMR (DMSO-d6+D2O+NaH) δ: 160.1, 149.2, 148.0, 147.1, 144.9, 143.0, 138.0, 131.8, 129.1, 128.5, 128.4, 119.1, 115.9, 114.1, 111.2, 111.1, 104.3, 70.6, 56.0, 55.7, 52.0, 46.3. MS (ESI+) m/z (rel. intensity): 429 (M+H, 80), 130 (100). Anal. for C26H28N4O2·1.25H2O, Calcd. C 69.23, H 6.82, N 12.42. Found C 69.51, H 6.47, N 12.36.
2.2.8 2-[3,4-Bis(benzyloxy)phenyl]-5-(4-methylpiperazin-1-yl)-1H-benzimidazole HCl (12):
Yield 30%, mp 267–267 °C (bubbling), IR (FTIR/Diamond ATR) νmax (cm−1): 3408, 3347 (N+-H), 2838 (C-H), 1636–1453 (aromatic C=C, C=N), 1276–1157 (C-N, C-O). 1H-NMR (DMSO-d6+D2O+NaH) δ: 2.53 (s, CH3-N, overlapped DMSO-d6), 2.92 (4H, piperazinyl methylenes), 3.26 (4H, piperazinyl methylenes), 5.21 and 5.24 (s+s, 4H), 6.97 (d, 1H, J=8.4 Hz), 7.07 (s, 1H), 7.21 (d, 1H, J=8.8 Hz), 7.32–7.52 (m, 11H), 7.67 (dd, 1H, J=8.8 Hz, J=2.0 Hz), 7.87 (d, 1H, J=2.0 Hz). MS (ESI+) m/z (rel. intensity): 505 (M+H, 50), 274 (100). Anal. for C32H32N4O2·3HCl·2H2O, Calcd. C 59.12, H 6.04, N 8.61. Found C 59.14, H 6.22, N 8.76.
2.2.9 2-(3,5-Dimethoxyphenyl)-5-(4-methylpiperazin- 1-yl)-1H-benzimidazole (13):
Yield 16%, mp 196–198 °C, IR (FTIR/Diamond ATR) νmax (cm−1): 2939, 2808 (C-H), 1593–1436 (aromatic C=C, C=N), 1287–1157 (C-N, C-O). 1H-NMR (DMSO-d6+D2O+NaH) δ: 2.16 (s, 3H), 2.46 (piperazinyl methylenes, overlapped DMSO-d6), 2.96 (4H, piperazinyl methylenes), 3.72 (s, 6H), 6.24 (dd, 1H), 6.54 (dd, 1H, J=8.8 Hz, J=2.4 Hz), 6.93 (d, 1H, J=2.4), 7.22 (d, 1H, J=8.4 Hz), 7.39 (d, 2H, J=2.0 Hz). 13C-NMR (DMSO-d6+D2O+NaH) δ: 160.7, 159.8, 147.7, 145.2, 142.7, 140.0, 116.4, 111.9, 104.6, 104.4, 99.7, 55.7, 55.5, 51.9, 46.2. MS (ESI+) m/z (rel. intensity): 353 (M+H, 65), 218 (100). Anal. for C20H24N4O2·H2O·0.1C4H8O2, Calcd. C 64.60, H 7.12, N 14.77. Found C 64.69, H 7.42, N 14.35.
2.2.10 5-(4-Methylpiperazin-1-yl)-2-(pyridin-3-yl)-1H-benzimidazole (14):
Yield 20%, mp 162–164 °C (dec.), IR (FTIR/Diamond ATR) νmax (cm−1): 2939, 2810 (C-H), 1630–1442 (aromatic C=C, C=N). 1H-NMR (DMSO-d6+D2O+NaH) δ: 2.16 (s, 3H), 2.46 (piperazinyl methylenes, overlapped DMSO-d6), 2.96 (t, 4H, piperazinyl methylenes), 3.72 (s, 6H), 6.57 (dd, 1H, J=8.8 Hz, J=2.0 Hz), 6.95 (d, 1H, J=2.4), 7.25 (d, 1H, J=8.4 Hz), 7.31 (dd, 1H), 8.33 (dd, 1H), 8.44 (dt, 1H, J=8.4 Hz, J=2.0 Hz), 9.31 (d, 1H, J=2.0 Hz). 13C-NMR (DMSO-d6+D2O+NaH) δ: 157.2, 148.1, 147.4, 145.4, 143.0, 133.7, 124.0, 116.6, 112.0, 110.0, 104.4, 55.7, 51.9, 46.4. MS (ESI+) m/z (rel. intensity): 353 (M+H, 65), 218 (100). Anal. for C17H19N5·H2O, Calcd. C 65.57, H 6.79, N 22.49. Found C 65.67, H 6.60, N 22.13.
2.3 Biological tests
2.3.1 Cell culture:
Human promyelocytic leukemia cells (HL-60) were grown in RPMI 1640 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 mg/mL; Sigma-Aldrich) in a 5% CO2 atmosphere at 37 °C. Sterile bovine serum was inactivated at 60 °C for 30 min before preparing the medium. The cells were stained with trypan blue and counted to seed an equal number of cells to each well of 6- and 96-well plates.
2.3.2 Preparation of compounds:
The compounds were dissolved in DMSO and kept as 10 mM stock solutions at −20 °C, protected from light. In none of the experiments did the DMSO concentration exceed 0.5%, which did not interfere with cell growth. Cisplatin was used as a positive control in all viability experiments.
2.3.3 Cell viability test:
The MTT test was used to determine cell viability. Briefly, the cells (4×104 per well) were seeded into 96-well plates and their proliferation was determined with the Cell Proliferation Kit I (MTT; Roche, Mannheim, Germany) as described by the manufacturer. The cells were treated at various concentrations (0.01, 0.1, 1, 5, 10, 20, 30, 40, and 50 μM) of the newly synthesized benzimidazoles (compounds 5–14) for 48 h. Mitochondrial succinate dehydrogenase of living cells reduces the tetrazolium dye, and the resulting formazan crystals, correlating with the number of viable cells, can be dissolved and measured colorimetrically [20]. The spectrophotometric absorbance was measured using a microplate reader (Biotek, Winooski, VT, USA) at 550 nm with a reference wavelength of 690 nm. Data were expressed as percent cell viability of the untreated control. All experiments were conducted in triplicate, and DMSO was used as negative control in the corresponding concentrations.
2.3.4 Detection of apoptosis by flow cytometry:
HL-60 cells (4×105 per well) were seeded into 6-well plates. The cells were incubated with compounds at 5 μM concentration for 48 h. The cells were then collected, washed with phosphate-buffered saline twice, and resuspended in binding buffer according to the supplier’s instructions (BD Biosciences, Erembodegem, Belgium). Then, 5 μL Annexin V-PE and 5 μL 7-AAD were added to 100 μL of cell suspension. After vortexing briefly, the cells were incubated for 15 min at room temperature. Then, 1× binding buffer (400 μL) was added to the cells that were then subjected to flow cytometry (Accuri C6, Accuri Cytometers, Ann Arbor, MI, USA). The dual parametric dot-plots combining Annexin V-PE and 7-AAD fluorescence (Figure 2) show the viable cell population in the lower left quadrant (Annexin V−7-AAD−), the early apoptotic cells in the lower right quadrant (Annexin V+7-AAD−), and the late apoptotic/dead cells in the upper right quadrant (Annexin V+7-AAD+).
Flow cytometric analysis of PS externalization (Annexin V binding) and cell membrane integrity (7-AAD staining) in HL-60 cells treated with compounds 5 and 10–12 at 5 μM concentration.
The dual parametric dot-plots combining Annexin V-PE and 7-AAD fluorescence show the viable cell population in the lower left quadrant (Annexin V−7-AAD−), the early apoptotic cells in the lower right quadrant (Annexin V+7-AAD−), and the late apoptotic/dead cells in the upper right quadrant (Annexin V+7-AAD+).
3 Results and discussion
Some novel 5-(4-methylpiperazin-1-yl)-2-phenyl- 1H-benzimidazoles (5–14) were synthesized and investigated for their in vitro antiproliferative activities in the leukemia cell line HL-60 using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. The results of the cell viability test are reported in Table 3.
Chemical structures and in vitro antiproliferative activities of compounds 5–14 against HL-60 cells.
 | |||||
|---|---|---|---|---|---|
| Compound | R1 | R2 | R3 | X | IC50 (μM) |
| 5 | H |  | H | C | 1.86±0.09 |
| 6 | H |  | H | C | 1.65±0.15 |
| 7 | H |  | H | C | 1.80±0.15 |
| 8 | OCH3 | OH | H | C | >100 |
| 9 | OH | OCH3 | H | C | >100 |
| 10 |  | OCH3 | H | C | 1.86±0.20 |
| 11 | OCH3 |  | H | C | 1.70±0.11 |
| 12 |  |  | H | C | 1.56±0.09 |
| 13 | OCH3 | H | OCH3 | C | 9.07±0.63 |
| 14 | – | H | H | N | >100 |
| Cisplatin | 1.41±0.06 | ||||
Benzimidazole derivatives containing a benzyloxy (5), phenoxy (6), 3,4-dimethoxyphenoxy (7), 3-benzyloxy-4-methoxy (10), 4-benzyloxy-3-methoxy (11), or 3,4-dibenzyloxy (12) group exhibited potent antiproliferative activity against the HL-60 cell line. The IC50 values for these compounds were determined as 1.86±0.09, 1.65±0.15, 1.80±0.15, 1.86±0.20, 1.70±0.11, and 1.56±0.09 μM, respectively. These values are close to the IC50 of the positive control, cisplatin (1.41±0.06 μM). The replacement of the benzyloxy group with the phenoxy, 3,4-dimethoxyphenoxy, or methoxy group, respectively, did not result in a significant change in activity.
Compounds 8 and 9, bearing hydroxy groups at positions 3 and 4 of the phenyl ring, respectively, did not exhibit antiproliferative activity, their IC50 being >100 μM. The substitution of the hydroxy group with benzyl enhanced the antiproliferative activity significantly. Compounds 10–12 with benzyloxy groups are more lipophilic and therefore may possess greater cell membrane permeability compared with compounds 8 and 9. The 3,5-dimethoxy substituted derivative, compound 13, displayed moderate antiproliferative activity, with an IC50 of 9.07±0.63 μM. The replacement of the substituted phenyl ring with the 3-pyridinyl group (compound 14) led to loss of the activity against HL-60 cell (IC50 >100 μM).
One of the plasma membrane alterations in the early stages of apoptosis or programmed cell death is the translocation of phosphatidylserine (PS) from the inner leaflet of the plasma membrane to the outer leaflet; thus, Annexin V binding assay is performed to detect the surface exposure of PS [21]. In this study, flow cytometric analysis with Annexin V-PE/7-amino-actinomycin D (7-AAD) staining was carried out to explore the effects of the novel benzimidazoles on the apoptosis of HL-60 cells (Figures 2 and 3). The percentage of early apoptotic HL-60 cells (in the lower right quadrant, Annexin V+) treated with compounds 5 and 10–12 containing the benzyloxy group was significantly higher (59.9±2.37%, 48.4±0.63%, 60.8±1.86%, and 54.7±3.72%, respectively) than for the control cells (32.3±1.57%) at 48 h. The percentage of late apoptotic/dead HL-60 cells (in the upper right quadrant, Annexin V+7-AAD+) treated with compounds 5 and 10–12 were significantly higher (27.9±2.39%, 27.1±0.54%, 29±2.6%, and 35.6±3.25%, respectively) than for the control cells (5.4±0.6%) at 48 h.
Histogram representing the percentage of apoptotic HL-60 cells after treatment with compounds 5 and 10–12 at 5 μM concentration. Results are representative of three independent experiments. Error bars indicate±SD. *p<0.05, significantly different from control.
4 Conclusions
In this study, 10 new benzimidazole derivatives (5–14) were prepared and evaluated for their in vitro antiproliferative and apoptosis-inducing activities against AML HL-60 cells. Compounds 5–7 and 10–12 exhibited remarkable activities comparable to those of cisplatin. If these activities and specificities are confirmed in in vivo models and clinical studies, these compounds may serve as chemopreventive agents or adjuvants to the conventional therapeutics for the treatment of leukemia.
Acknowledgments
The Ankara University Research Fund (Project No. 14A0237001) provided support for the acquisition of IR data, and the Central Instrumental Analysis Laboratory of the Faculty of Pharmacy, Ankara University, supported the acquisition of the NMR, mass spectrometry, IR, and elemental analysis data.
Declaration of interest: The authors report no declarations of interest.
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The online version of this article (DOI: 10.1515/znc-2014-4189) offers supplementary material, available to authorized users.
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