Synthesis and anti-breast cancer activity of novel indibulin related diarylpyrrole derivatives

Abstract

Background

During recent years, a number of anti-tubulin agents were introduced for treatment of diverse types of cancer. Despite their potential in the treatment of cancer, drug resistance and adverse toxicity, such as peripheral neuropathy, are some of the negative effects of anti-tubulin agents. Among anti-tubulin agents, indibulin was found to cause minimal peripheral neuropathy. Thus far, however, indibulin has not entered clinical usage, caused in part by its poor aqueous solubility and other developmental problems in preclinical evaluation.

Objectives

With respect to need for finding potent and safe anticancer agents, in our current research work, we synthesized several indibulin-related diarylpyrrole derivatives and investigated their anti-cancer activity.

Methods

Cell cultur studies were perfomred using the MTT cell viability assay on the breast cancer cell lines MCF-7, T47-D, and MDA-MB231 and also NIH-3 T3 cells as representative of a normal cell line. The activity of some of the synthesized compounds for tubulin interaction was studied using colchicine binding and tubulin polymerization assays. The annexin V-FITC/PI method and flow cytometric analysis were used for studying apoptosis induction and cell cycle distribution.

Results and conclusion

Two of the synthesized compounds, 4f and 4 g, showed high activity on the MDA-MB231 cell line (IC₅₀ = 11.82 and 13.33 μM, (respectively) and low toxicity on the normal fibroblast cells (IC₅₀ > 100 μM). All of the tested compounds were more potent on T47-D cancer cells and less toxic on NIH-3 T3 normal cells in comparison to reference compound, indibulin. The tubulin polymerization inhibition assay and [³H]colchicine binding assay showed that the main mechanism of cell death by the potent synthesized compounds was not related to an interaction with tubulin. In the annexin V/PI staining assay, the induction of apoptosis in the MCF-7 and MDA-MB231 cell lines was observed. Cell cycle analysis illustrated an increased percentage of sub-G-1 cells in the MDA-MB231 cell line as a further indication of cell death through induction of apoptosis.

Graphical abstract.

Novel Indibulin analogous as anti-breast cancer agents

Introduction

Breast cancer is the most common invasive cancer in women all over the world. Approximately one third (32%) of cancers diagnosed in female patients are breast cancers. It is the fifth most common cause of death related to cancer and the leading cause of cancer death in women. Women have a 1 in 8 lifetime risk of developing breast cancer and 1 in 35 risk of breast cancer causing death in the US [1–4]. The rates of breast cancers are increasing, but these rates differ by race and ethnicity. The disease displays a high level of complexity because of more than 18 sub-types of breast cancer. The stages of tumor progression are not well understood yet in breast cancer. Among diverse risk factors concerning developing breast cancer are life style, genetic, environmental and biological factors. There are several strategies to overcome breast cancer including surgery, radiation therapy, chemotherapy, hormonal therapy and targeted therapy, but the morbidity and mortality caused by breast cancer remains high [1, 3–5]. Improving the treatment of breast cancer is thus an important scientific and medical goal.

The microtubule system is an important component of eukaryote cells. It has critical roles in various basic cellular functions, such as formation and maintaining cell morphology, important roles in intracellular and extracellular motility, cell division through formation of the mitotic spindle, organelle transport and cell signaling [6]. During the last decade, attention to anti-tubulin agents and their usage in the treatment of a large number of cancers has been expanded. Microtubule-targeting compounds by affecting microtubule dynamics can cause both the stabilization or destabilization of microtubules, depending on their exact binding site in tubulin, and this disruption of microtubule dynamics invariable lead to cell death through apoptosis. Taxoids and related compounds act in the taxol binding site of tubulin and speed up tubulin polymerization. With a different mechanism of action, inhibition of microtubule assembly, a large number of anti-cancer agents interact with the colchicine site of tubulin. These colchicine site compounds include combrestastatin A-4, nocodazole and indibulin. In both cases, interaction with tubulin disrupts the normal function of cells in mitotic processes and controls the high cell division in cancerous cells [7]. Peripheral nephropathy emerged as a major side effect of paclitaxel and of some anti-mitotic agents that target the colchicine site. Indibulin, representing a new generation of anti-tubulin agent, exerted an interesting anticancer activity without leading to peripheral nephropathy in animal models. In fact, indibulin discriminates between mature neuronal and non-neuronal tubulin [8]. There are reports about efforts for introducing new tubulin polymerization inhibitor inspired by the structure of indibulin, several series of related structures were synthesized and evaluated for anti-cancer activity [9–11] (Fig. 1a). A significant tumor growth inhibition in a mouse xenograft model of head and neck cancer was observed for one of these new indibulin-related structures [9] (Fig. 1b).

Fig. 1.

a Diverse structural changes reported on indibulin structure. b Compound A with significant head and neck tumor growth inhibition

Pyrrole is one of the most useful heterocycles in the synthesis of new biologically active molecules. Many compounds that include a pyrrole ring have been synthesized in drug discovery laboratories. Some of these compounds have exerted various biologic effects, including anticancer activity, and these compounds display diverse mechanisms of action [12–14]. From the medicinal chemist’s viewpoint, the design and synthesis of new compounds for breast cancer treatment is necessary. We decided that indibulin, with its reported lack of neurotoxicity, was a worthy target, in particular because synthesis of analogues was related to our previous efforts [15–21] in the design and synthesis of new anticancer agents. We therefore synthesized a novel series of indibulin related diarylpyrrole derivatives and evaluated their anticancer activity on breast cancer cell lines as well as a mouse fibroblast normal cell line to represent noncancer cells, using the 3-(4,5-dimethylthiazoyl-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) cell viability assay. Design of the target compounds was carried out based on development of an indibulin family, in which the fused ring in the indole moiety was converted to a flexible phenyl-pyrrole ring system (Fig. 2). To determine the mechanism of cytotoxic activity of potent compounds, we also investigated the effect of synthesized compounds on tubulin polymerization, colchicine binding, cell cycle progression and apoptosis induction.

Fig. 2.

Structural changes on indibulin in current work

Materials and methods

General

All Chemical reagents and solvents were bought from Merck (Darmstadt, Germany). Melting points were determined on a Kofler hot-stage instrument (Reichert, Vienna, Austria). The ¹H NMR spectra were obtained on a Bruker FT-500 MHz spectrometer (Bruker, Darmstadt, Germany) and CDCl₃ was as solvent. ¹³C NMR spectra were also taken at 125 MHz. The spin multiplicities are written as s (singlet), d (doublet), t (triplet), and m (multiplet) and the Coupling constant (J) values are given in Hertz (Hz). Mass spectra were done on an Agilent 5975B instrument (Agilent Technologies, Santa Clara, CA, USA) with triple-axis detector. IR spectra were obtained by a Nicolet Magna 550-FT spectrometer (Nicolet Instrument Corporation, Madison, WI, USA). Elemental analyses were determined using a Perkin Elmer Model 240-C instrument (PerkinElmer, Hopkinton, MA, USA).

Chemistry

1-(4-chlorophenylpentane)-1,4-dione (2a) and 1-phenylpentane-1,4-dione (2b) were Synthesized according to the methods reported in the literature [22, 23].

General procedure for the preparation of 2-methyl-1,5-diaryl-1H-pyrrole derivatives 3a-j

1,4-Diketone derivatives 2a-b (1 mmol) and the appropriate aniline derivative (1.1 mmol) and p-tuloenesulfonic acid (PTSA), catalytic amount, were dissolved and reacted in refluxing ethanol for 6 h. while the reaction is completed, as indicated by thin layer chromatography (TLC), the solvent is evaporated and the remained residue was purified using column chromatography (hexane/ ethyl acetate 10:1 as eluent) to obtain pyrrole derivative 3a-j.

General procedure for the preparation of 2-(2-methyl-1,5-diaryl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide derivatives 4a-j

After dissolving appropriate pyrrole 3a-j (1 mmol) and trimethylamine (TEA) (1.2 mmol) in dichloromethane (DCM) (10 mL), the solution was treated with oxalyl chloride (1.1 mmol) dropwise at 0 °C. After the addition was completed, the mixture was stirred at room temperature (RT) for 4 h, then the mixture was concentrated to remove residual oxalyl chloride under reduced pressure. The crude was dissolved in DCM and 4-aminopyridine (1 mmol), TEA (1.2 mmol) and 4-N,N-dimethylaminopyridine (DMAP) as a catalyst were added to the reaction mixture. The reaction mixture was stirred in RT for 12 h and after completion of the reaction (TLC), the solvent was removed using rotary evaporator and the residue was subjected to flash chromatography (hexane/ethyl acetate 3:1, as eluent). Final products 4a-j were obtained in a pure crystal form.

2-(5-(4-chlorophenyl)-2-methyl-1-phenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (4a)

Yellow solid; yield 40%; mp 234–236 °C.

IR (KBr, cm⁻¹): 3343, 3150, 3031, 1702, 1638, 1593, 1479, 1415, 1327, 1146, 875, 749, 693; ¹H NMR (500 MHz, CDCl₃): δ 2.49 (s, 3H, CH₃), 7.00 (d, J = 8 Hz, 2H, H-Ar), 7.12–7.18 (m, 4H, H-Ar), 7.44 (br s, 3H, H-Ar), 7.61 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine), 9.37 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.8, 112.8, 113.6, 117.0, 128.1, 128.3, 129.0, 129.4, 129.5, 130.1, 133.0, 134.2, 137.0, 143.8, 144.2, 151.0, 160.9, 180.5; Mass, m/z (%): 415 (M+, 10), 294 (35), 133 (16), 103 (50), 73 (100), 51 (5); Anal. Calcd for C₂₄H₁₈ClN₃O₂: C, 69.31; H, 4.36; N, 10.10. Found: C, 69.21; H, 4.31; N, 10.13.

2-(5-(4-chlorophenyl)-1-(4-fluorophenyl)-2-methyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (4b)

Yellow solid; yield 43%; mp 204–206 °C.

IR (KBr, cm⁻¹): 3343, 3152, 3034, 1707, 1642, 1595, 1505, 1415, 1326, 1227, 1145, 832, 754, 693; ¹H NMR (500 MHz, CDCl₃): δ 2.47 (s, 3H, CH₃), 6.99 (d, J = 8 Hz, 2H, H-Ar), 7.13–7.17 (m, 6H, H-Ar), 7.61 (s, 1H, H-Pyrrole), 7.65 (d, J = 5.5 Hz, 2H, H-Pyridine), 8.58 (d, J = 5.5 Hz, 2H, H-Pyridine), 9.41 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.7, 112.7, 113.6, 116.6 (d, J = 22.5 Hz), 116.8, 128.4, 129.4, 129.8 (d, J = 8.75 Hz), 132.9, 133.2, 133.9, 143.8, 144.1, 150.8, 160.8, 162.3 (d, J = 248.75 Hz), 180.6; Mass, m/z (%): 433 (M+, 12), 312 (100), 248 (7), 128 (8), 51 (5); Anal. Calcd for C₂₄H₁₇ClFN₃O₂: C, 66.44; H, 3.95; N, 9.69. Found: C, 66.58; H, 3.93; N, 9.68.

2-(1,5-bis(4-chlorophenyl)-2-methyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (4c)

Yellow solid; yield 44%; mp 213–215 °C.

IR (KBr, cm⁻¹): 3342, 2925, 2854, 1695, 1644, 1592, 1491, 1415, 1326, 1161, 1093, 830, 753, 669; ¹H NMR (500 MHz, CDCl₃): δ 2.48 (s, 3H, CH₃), 7.00 (d, J = 8 Hz, 2H, H-Ar), 7.09 (d, J = 8 Hz, 2H, H-Ar), 7.17 (d, J = 8 Hz, 2H, H-Ar), 7.42 (d, J = 8 Hz, 2H, H-Ar), 7.61 (s, 1H, H-Pyrrole), 7.64 (d, J = 5 Hz, 2H, H-Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine), 9.36 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.7, 112.9, 113.6, 116.9, 128.5, 129.4, 129.5, 129.8, 131.8, 133.2, 134.0, 135.1, 135.4, 144.0, 144.1, 151.0, 160.9, 180.6; Mass, m/z (%): 449 (M+, 7), 328 (60), 149 (25), 100 (90), 72 (100); Anal. Calcd for C₂₄H₁₇Cl₂N₃O₂: C, 64.01; H, 3.81; N, 9.33. Found: C, 64.19; H, 3.84; N, 9.35.

2-(5-(4-chlorophenyl)-2-methyl-1-(p-tolyl)-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4d)

Yellow solid; yield 48%; mp 156–158 °C.

IR (KBr, cm⁻¹): 3341, 3144, 3037, 2954, 2852, 1700, 1638, 1593, 1503, 1417, 1326, 1146, 829, 754, 691; ¹H NMR (500 MHz, CDCl₃): δ 2.40 (s, 3H, CH₃), 2.45 (s, 3H, CH₃), 7.00–7.03 (m, 4H, H-Ar), 7.13 (d, J = 7.5 Hz, 2H, H-Ar), 7.22 (d, J = 7.5 Hz, 2H, H-Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J = 5.5 Hz, 2H, H-Pyridine), 8.57 (d, J = 5.5 Hz, 2H, H-Pyridine), 9.45 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.7, 21.1, 112.67, 113.67, 116.65, 127.8, 128.3, 129.3, 130.1, 130.2, 132.8, 133.9, 134.3, 139.0, 143.9, 144.3, 150.9, 161.0, 180.5; Mass, m/z (%): 429 (M+, 16), 308 (100), 280 (12), 244 (10), 121 (19), 78 (17), 55 (15); Anal. Calcd for C₂₅H₂₀ClN₃O₂: C, 69.85; H, 4.69; N, 9.77. Found: C, 69.76; H, 4.65; N, 9.79.

2-(5-(4-chlorophenyl)-1-(4-methoxyphenyl)-2-methyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4e)

Yellow solid; yield 51%; mp 162–164 °C.

IR (KBr, cm⁻¹): 3338, 3037, 2962, 2840, 1701, 1638, 1587, 1507, 1416, 1327, 1249, 1030, 832, 755; ¹H NMR (500 MHz, CDCl₃): δ 2.46 (s, 3H, CH₃), 3.84 (s, 3H, OCH₃), 6.93 (d, J = 8 Hz, 2H, H-Ar), 7.02 (d, J = 8 Hz, 2H, H-Ar), 7.05 (d, J = 8 Hz, 2H, H-Ar), 7.15 (d, J = 8 Hz, 2H, H-Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine), 9.41 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.7, 55.4, 112.57, 113.6, 114.8, 116.5, 128.3, 129.1, 129.4, 129.5, 130.2, 132.9, 134.0, 143.9, 144.5, 150.9, 159.7, 161.0, 180.4; Mass, m/z (%): 445 (M+, 17), 324 (90),135 (31), 84 (100), 51 (34); Anal. Calcd for C₂₅H₂₀ClN₃O₃: C, 67.34; H, 4.52; N, 9.42. Found: C, 67.45; H, 4.50; N, 9.43.

2-(2-methyl-1,5-diphenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4f)

Yellow solid; yield 54%; mp 216–218 °C.

IR (KBr, cm⁻¹): 3335, 3063, 2855, 1697, 1634, 1582, 1497, 1410, 1328, 1146, 876, 766, 695; ¹H NMR (500 MHz, CDCl₃): δ 2.49 (s, 3H, CH₃), 7.00–7.15 (m, 7H, H-Ar), 7.41 (br s, 3H, H-Ar), 7.64 (br s, 3H, H-Pyrrole & H-Pyridine), 8.58 (d, J = 5 Hz, 2H, H-Pyridine), 9.38 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.8, 112.4, 113.6, 116.7, 127.0, 128.1, 128.2, 128.3, 128.8, 129.4, 131.6, 135.2, 137.3, 143.9, 144.1, 150.8, 161.0, 180.6; Mass, m/z (%): 381 (M+, 20), 324 (12), 260 (100), 217 (10), 128 (8), 77 (9); Anal. Calcd for C₂₄H₁₉N₃O₂: C, 75.57; H, 5.02; N, 11.02. Found: C, 75.66; H, 5.05; N, 11.01.

2-(1-(4-fluorophenyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl) acetamide (4 g)

Yellow solid; yield 49%; mp 205–207 °C.

IR (KBr, cm⁻¹): 3336, 3043, 2915, 1701, 1633, 1500, 1477, 1411, 1329, 1145, 966, 875, 766, 695; ¹H NMR (500 MHz, CDCl₃): δ 2.46 (s, 3H, CH₃), 7.06 (d, J = 7 Hz, 2H, H-Ar), 7.08–7.19 (m, 7H, H-Ar), 7.59 (s, 1H, H-Pyrrole), 7.65 (d, J = 5.5 Hz, 2H, H-Pyridine), 8.56 (d, J = 5.5 Hz, 2H, H-Pyridine), 9.49 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.7, 112.4, 113.6, 116.4 (d, J = 22.5 Hz), 116.7, 127.1, 128.1, 128.3, 129.8 (d, J = 8.75 Hz), 131.4, 133.1, 135.2, 143.7, 143.9, 150.9, 161.0, 162.2 (d, J = 248.5 Hz), 180.7; Mass, m/z (%): 399 (M+, 17), 278 (100), 129 (12), 95 (10), 55 (6); Anal. Calcd for C₂₄H₁₈FN₃O₂: C, 72.17; H, 4.54; N, 10.52. Found: C, 72.10; H, 4.55; N, 10.45.

2-(1-(4-chlorophenyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (4 h)

Yellow solid; yield 46%; mp 215–217 °C.

IR (KBr, cm⁻¹): 3258, 3144, 3058, 2923, 1708, 1648, 1583, 1503, 1412, 1329, 1149, 829, 744, 697; ¹H NMR (500 MHz, CDCl₃): δ 2.47 (s, 3H, CH₃), 7.06–7.12 (m, 4H, H-Ar), 7.18–7.22 (m, 3H, H-Ar), 7.38 (d, J = 7.5 Hz, 2H, H-Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-Pyridine), 8.57 (d, J = 5 Hz, 2H, H-Pyridine), 9.46 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.7, 112.60, 113.64, 116.9, 127.2, 128.2, 128.3, 129.4, 129.6, 131.3, 134.7, 135.1, 135.7, 143.6, 143.9, 150.9, 160.9, 180.7; Mass, m/z (%): 415 (M+, 13), 294 (100), 230 (9), 128 (12), 78 (5); Anal. Calcd for C₂₄H₁₈ClN₃O₂: C, 69.31; H, 4.36; N, 10.10. Found: C, 69.39; H, 4.38; N, 10.14.

2-(2-methyl-5-phenyl-1-(p-tolyl)-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4-yl)acetamide (4i)

Yellow solid; yield 39%; mp 212–214 °C.

IR (KBr, cm⁻¹): 3347, 3153, 3034, 2919, 2864, 1708, 1637, 1593, 1500, 1409, 1325, 1144, 823, 764, 698; ¹H NMR (500 MHz, CDCl₃): δ 2.38 (s, 3H, CH₃), 2.46 (s, 3H, CH₃), 7.02 (d, J = 7.5, 2H, H-Ar), 7.07–7.17 (m, 5H, H-Ar), 7. 20 (d, J = 7.5, 2H, H-Ar), 7.60 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-Pyridine), 8.57 (d, J = 5 Hz, 2H, H-Pyridine), 9.47 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.8, 21.1, 112.2, 113.6, 116.6, 126.9, 127.8, 128.0, 128.2, 129.9, 131.7, 134.5, 135.2, 138.7, 143.9, 144.1, 150.9, 161.1, 180.5; Mass, m/z (%): 395 (M+, 20), 274 (100), 231 (8), 128 (7), 91 (5); Anal. Calcd for C₂₅H₂₁N₃O₂: C, 75.93; H, 5.35; N, 10.63. Found: C, 75.99; H, 5.38; N, 10.61.

2-(1-(4-methoxyphenyl)-2-methyl-5-phenyl-1H-pyrrol-3-yl)-2-oxo-N-(pyridin-4 yl)acetamide (4j)

Yellow solid; yield 52%; mp 134–136 °C.

IR (KBr, cm⁻¹): 3338, 3039, 2927, 2837, 1700, 1636, 1594, 1503, 1478, 1411, 833, 754, 694; ¹H NMR (500 MHz, CDCl₃): δ 2.47 (s, 3H, CH₃), 3.83 (s, 3H, OCH₃), 6.91 (d, J = 8 Hz, 2H, H-Ar), 7.06 (d, J = 8 Hz, 2H, H-Ar), 7.10 (d, J = 6 Hz, 2H, H-Ar), 7.12–7.19 (m, 3H, H-Ar), 7.61 (s, 1H, H-Pyrrole), 7.65 (d, J = 5 Hz, 2H, H-Pyridine), 8.57 (d, J = 5 Hz, 2H, H-Pyridine), 9.41 (s, 1H, NH); ¹³C NMR (125 MHz, CDCl₃): δ 13.8, 55.4, 112.2, 113.6, 114.2, 116.5, 126.9, 128.0, 128.3, 129.1, 129.8, 131.7, 135.3, 143.9, 144.3, 150.9, 159.5, 161.1, 180.5; Mass, m/z (%): 411 (M+, 19), 290 (100), 236 (25), 152 (15), 111 (30), 83 (51), 57 (60); Anal. Calcd for C₂₅H₂₁N₃O₃: C, 72.98; H, 5.14; N, 10.21. Found: C, 72.87; H, 5.10; N, 10.23.

Biological evaluation

Cell culture

For cell culture, appropriate cell lines, MCF-7, MDA-MB231, T47-D (human breast cancer cell lines) and NIH-3 T3 (mouse fibroblast cell line) were purchased from the Pasteur Institute (Tehran, Iran). RPMI-1640 medium (Sigma-Aldrich, St. Louis, MO, USA) was used for cell culturing, supplemented with penicillin (100 U/mL), 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA), and (100 μg/mL) streptomycin (Roche, Mannheim, Germany) in a humidified incubator with 5% CO₂ at 37 °C.

Cytotoxicity evaluation by the MTT assay

MTT assay for all compounds was done according to method reported in our previous work [24].

Colchicine binding inhibition and in vitro inhibitory tubulin polymerization assay

In the present study, purified bovine brain tubulin was used in the tubulin polymerization and colchicine binding assays, prepared as reported previously [25]. The experimental method for measuring [³H]colchicine binding inhibition to tubulin was reported in detail previously [26]. Briefly, the ligand binding assay was performed in 0.1 mL reaction volumes. Each reaction mixture contained 0.1 mg/mL (1.0 μM) tubulin, 5.0 μM [³H]colchicine and 5.0 μM of potential inhibitory compound. Combretastatin-A4 (CA-4), a highly potent inhibitor of colchicine binding to tubulin, was used as a positive standard [27]. Reaction mixtures were incubated for 10 min at 37 °C, the time that the reaction in control reaction mixtures is about 40–60% complete. At this time, reaction mixtures were diluted and stopped with water at 0 °C, and the mixtures were filtered through Whatman DEAE-cellulose filters. The filters then washed with ice-cold water. Radiolabel bound to the filters was measured using a liquid scintillation counter. For investigation of tubulin polymerization inhibitory potential of selected compounds, 1.0 mg/mL (10 μM) purified tubulin was treated without Guanosine-5′-triphosphate (GTP) with various concentrations of synthesized compound at 30 °C for 15 min. Then reaction mixtures were placed on ice, and GTP (10 μL of 10 μM) was added. Reaction mixtures were transferred to cuvettes held at 0 °C in recording spectrophotometers. After baselines were recorded at 350 nm at 0 °C, the temperature was increased to 30 °C rapidly (over about 30 s) and change in turbidity was followed over 20 min. Compound concentrations that resulted in a 50% decrease in turbidity (IC₅₀) were then determined [28].

Annexin V-FITC/PI assay

To quantify the cell death modality, the annexin V/propidium iodide (PI) staining assay was used. Briefly, 3 × 10⁵ cells (MCF-7 for 4b and MDA-MB231 for 4e) were plated and then the cells were treated with 4b or 4e (at the IC₅₀ concentration for 24 h). After treatment, cells were washed twice with phosphate buffered saline (PBS), mixed with 500 μL of binding buffer, and stained with 5 μL of annexin V-fluorescein isothiocyanate (FITC) and 5 μL of PI (PI 50 μg/mL) for 10 min at RT in the dark. Apoptotic cells were quantified by a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA); 1 × 10⁴ cells were counted for each sample. Both early apoptotic (annexin V-positive, PI-negative) and late apoptotic (double positive of annexin V and PI) cells were detected. The percentages of cells in each quadrant were analyzed using the Flowing Software version 2.4.1 (Turku Centre for Biotechnology University of Turku, Finland). The experiments were repeated at least three times.

Flow cytometric analysis of cell cycle distribution

For analysis of DNA content using flow cytometry, 1 × 10⁶ MCF-7 cells, in a 6-well plate, were treated with compound 4e (at the IC₅₀ concentration) for 24 h. For cell fixation, cells were centrifuged and 0.7 ml of cold 75% ethanol was added to samples, and the samples were placed on ice for at least 3 h. After washing with PBS, cells were resuspended in 0.25 mL of PBS, with 5 μL of 10 mg/mL RNase A and Triton X-100 (0.1%). Then, cells were incubated at 37 °C for 1 h, and 10 μL of 50 μg/mL PI was added. The fluorescence of the cells was determined with a FACS-Calibur flow cytometer (BDBiosciences, San Jose, CA) [29].

Results and discussion

Chemistry

The synthetic pathway of novel target compounds is outlined in Scheme 1. In the first step, appropriate derivatives of benzaldehyde, 1a or 1b, were treated with methyl vinyl ketone in the presence of sodium cyanide as catalyst and N,N-dimethylformamide (DMF) as solvent to yield 1 aryl-1,4-pentanediones 2a-b. Then, diones 2a-b were refluxed with aniline derivatives using a catalytic amount of PTSA in ethanol for 6 h to give corresponding 1,5-diarylpyrrole derivatives 3a-j. The compounds 4a-j were prepared through two continuous reactions. In the first step, reaction of oxalyl chloride with pyrrole derivatives in the presence of dry TEA in DCM at RT gave the related acyl chloride. Then, the mixture was concentrated under reduced pressure to remove excess oxalyl chloride. The residue was dissolved in dry DCM and TEA, a catalytic amount of DMAP and 3-aminopyridine were added and stirred at RT for 12 h. The mixture was concentrated and the final products 4a-j were purified using column chromatography (hexane/ethyl acetate 3:1, as eluent). Altogether, ten target compounds were synthesized in good to moderate yields. Chemical structure of all the final compounds was confirmed by ¹H NMR, ¹³C NMR, MS, IR and elemental analysis.

Scheme 1.

Synthetic pathway of desired compounds 4a-j

Biological evaluation

Anticancer activity

In the first step of biological investigation, our target compounds 4a-j were tested for their cytotoxicity against three breast cancer cell lines (MCF-7, T47-D and MDA-MB231) as well as a mouse embryonic fibroblast cell line (NIH-3 T3) as a normal cell line, using the MTT assay. Indibulin was used as positive control. In the MCF-7 cell line, except compound 4b, none of the target compounds showed an IC₅₀ < 10 μM. 4b had an IC₅₀ of around 7.5 ± 0.5 μM. In the case of the T47-D cell line, target compounds exerted good to moderate cytotoxicity, with IC50 values between 12.19 and 45.49 μM. Compounds 4i (IC₅₀:12.19 μM), 4 h (IC₅₀:13.49 μM), 4 g (IC₅₀:15.61 μM) and 4f (IC₅₀:16.60 μM) showed the best cytotoxic activity. For the MDA-MB231 cell line, results of the cytotoxicity assay were similar to those obtained with the T47-D cell line. In this cell line, the range of cytotoxic activity (IC₅₀) was between 10.55 and 37.11 μM. Compounds 4e, 4f and 4 g were the most potent and showed IC₅₀ values of 10.55, 11.82 and 13.33 μM, respectively. Compounds 4f and 4 g had no cytotoxicity even at 100 μM concentration against the normal cell line, and their selectivity towards the breast cancer cells make these two derivatives especially interesting for future investigations. In addition we should note that none of the target compounds had any cytotoxic effect on the normal NIH-3 T3 cell line, while indibulin did. Results of the MTT assay are shown in Table 1.

Table 1.

In vitro cytotoxic activities (IC₅₀)^{a, b} of synthesized compounds 4a-j

Compounds	MCF-7	T47-D	MDA-MB231	NIH-3 T3
4a	>10	34.04 ± 2.9	33.79 ± 1.8	99.1 ± 1.4
4b	7.5 ± 0.5	41.95 ± 2.1	33.63 ± 3.2	21.0 ± 2.3
4c	>10	40.56 ± 3.6	37.11 ± 2.8	76.2 ± 3.5
4d	>10	43.28 ± 1.7	28.69 ± 1.6	53.5 ± 1.7
4e	>10	45.49 ± 2.3	10.55 ± 1.3	47.4 ± 1.9
4f	>10	16.6 ± 1.5	11.82 ± 2.2	>100
4 g	>10	15.61 ± 2.1	13.33 ± 1.5	>100
4 h	>10	13.49 ± 2.7	26.02 ± 3.1	35.5 ± 2.8
4i	>10	12.19 ± 1.4	28.06 ± 3.3	37.9 ± 1.8
4j	>10	23.17 ± 1.9	32.52 ± 2.4	49.3 ± 2.1
Indibulin	>100	>100	9.0 ± 0.6	11.2 ± 1.6

^aIC₅₀ values are in μM

^bIC₅₀, compound concentration required to inhibit cell proliferation by 50%

Colchicine binding and tubulin polymerization inhibition

Tubulin protein dynamics (polymerization and depolymerization) is one of the most important and attractive targets for designing anticancer agents. To investigate the ability of compounds 4a-j to bind to the colchicine site of tubulin, we tested their inhibition potential at 5 μM on the binding of 5 μM [³H]colchicine to 1 μM tubulin. None of the tested compounds had a significant inhibitory effect in this assay (results not shown), while the potent colchicine site inhibitor CA-4 was strongly active (98 ± 0.2% inhibition of colchicine binding, with an IC₅₀ of 0.73 ± 0.04 μM for inhibition of tubulin assembly). Furthermore, we evaluated the effects of 4b and 4 g on the polymerization of purified tubulin, using the highly potent CA-4 as a reference standard. Inhibition was not observed at 20 μM, the highest concentration examined. Tubulin polymerization inhibitory effect of indibulin (IC50 = 0.30 μM) was also reported in the literature [30]. These data indicate that an interaction with tubulin is not the main cause of anticancer activity of the effective compounds in this series. Thus, we investigated other cell death mechanisms.

Annexin V/ PI apoptosis assay

In order to examine whether the anticancer effects of drugs on the cells were associated with the induction of apoptosis or not, FITC conjugated annexin V and PI staining was used to distinguish apoptotic cells. MCF-7 and MDA-MB231 cells were incubated with 4b and 4e at their respective IC₅₀ concentration for 24 h, and the percentages of apoptotic cells were measured. Control cells were negative for both annexin V-FITC and PI. Treatment of both the MCF-7 and MDA-MB231 cell lines with either 4b or 4e increased the percentage of both early (very significantly) and late apoptotic cells (Fig. 3). With respect to these results, it seems that early apoptosis induction is the major cause of cell death with these compounds.

Fig. 3.

4b induced cell death modes in the MCF-7 cell line. a4b (7.5 μM) treated MCF-7 cells for 24 h. b After 24 h of incubation with 4b, both the percentages of early apoptotic cells and late apoptotic cells (stained with both annexin V and PI) increased significantly. Data are representative of three independent experiments. * P < 0.05 denotes a mean significantly different from untreated cells. 4e induced cell death modalities in the MDA-MB-231 cell line. c4e (10.55 μM) treated MDA-MB-231 for 24 h. d After 24 h of incubation with 4e, the percentages of early and late apoptotic cells (stained with both annexin V and PI) increased significantly. Data are representative of three independent experiments. * P < 0.05 denotes a mean significantly different from untreated cells

Cell cycle analysis

With regard to cytotoxic activity and apoptosis induction ability of 4e, we evaluated the effect of 4e on cell cycle distribution of cultured MDA-MB231 cell line using flow cytometric cell cycle analysis. After treatment of cells with compound 4e at the IC₅₀ concentration for 24 h, there was an increased percentage of cells in the sub-G1 region, representing an increase in cell death consistent with the induction of apoptosis shown in Fig. 4.

Fig. 4.

4e induces cell cycle arrest in the MDA-MB-231 cells. a Typical DNA content histograms of cells treated with 4e (10.55 μM) for 24 h

Docking study

With respect to the results of tubulin polymerization inhibition, we decided to study the possible mode of interactions of 4f and 4 g with tubulin protein using molecular docking software to understand the reason of inactivity of synthesized compounds in inhibiting the tubulin polymerization. Indibulin was also docked in tubulin active site for comparison as a reference. Conversion of rigid and flat indole part of indibulin to flexible 2-arylpyrrole and substituting the N-4-chlorobenzyl part with N-aryl moiety, drastically decreased the effect of synthesized compounds on tubulin polymerization in comparison with indibulin, and the interaction of these parts with active site of tubulin is the key cause in showing activity or inactivity of tested compounds on tubulin polymerization. in both 4f and 4 g, phenyl ring attached to C-5 of pyrrole ring showed interactions with VAL176, SER177 and TYR223 residues. These interactions exactly repeated between the above resides and six-membered ring of indole of indibulin. SER177 has another interaction with five-membered ring of indole of indibulin, which this interaction is absent between SER177 and pyrrole ring of 4f and 4 g. Because of the ability of phenyl ring attached to C-5 of pyrrole ring in 4f and 4 g, it seems the interactions of the phenyl ring in these compounds is not as strong as the interactions in indole of indibulin with the mentioned residues. The N-4-chlorobenzyl moiety of indibulin has interactions with ALA787, LEU681 and CYS674, but these interactions are completely absent for the N-aryl part of the 4f and 4 g. there is also some other differences in mode of interactions between the residues in active site and structures of 4f, 4 g and indibulin. Altogether, it seems these differences in interactions, especially presented for N-4-chlorobenzyl moiety of indibulin, are the cause of inactivity of 4f and 4 g in tubulin polymerization inhibition (Fig. 5).

Fig. 5.

Possible mode of interaction between tubulin active site and 4f, 4 g and indibulin structures

Conclusions

We synthesized a novel series of indibulin-related 1,5-diarylpyrroles as potential antiproliferative agents and investigated their cytotoxic activity against four diverse cell lines. Compounds 4b and 4e showed the highest cytotoxic activity on the MCF-7 and MDA-MB231 cell lines, respectively. Both of the compounds induced apoptosis in tested cell lines. Compounds 4f and 4 g not only showed high toxicity on the MDA-MB231 cancer cell line but also showed very low toxicity on normal cells (IC₅₀ above 100 μM). This is an ideal choice to continue further pharmacological investigation on these two derivatives for finding their exact mechanism of action. A ligand binding assay plus a tubulin polymerization inhibitory assay showed that interaction of active compounds with tubulin was not the cause of the anticancer activity by the synthesized compounds and that other important factors may trigger apoptosis induction and finally cell death. Our data indicate that it will be beneficial to investigate other derivatives with the 1,5-diaryl pyrrole scaffold in a search for more potent and safer anticancer agents.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.