Synthesis, biological evaluation and molecular docking studies of trans-indole-3-acrylamide derivatives, a new class of tubulin polymerization inhibitors

Abstract

In this study, we synthesized a series of trans-indole-3-acrylamide derivatives (3a–k) and investigated their activity for inhibition of cell proliferation against five human cancer cell lines (HeLa, MCF7, MDA-MB-231, Raji and HL-60) by MTT assay. Compound 3e showed significant antiproliferative activity against both the Raji and HL-60 cell lines with IC₅₀ values of 9.5 and 5.1 µM, respectively. Compound 3e also exhibited moderate inhibitory activity on tubulin polymerization (IC₅₀ = 17 µM). Flow cytometric analysis of cultured cells treated with 3e also demonstrated that the compound caused cell cycle arrest at the G2/M phase in HL-60 and HeLa cells. Moreover, 3e, the most active compound, caused an apoptotic cell death through the activation of caspase-3. Docking simulations suggested that 3e binds to the colchicine site of tubulin.

1. Introduction

Cancer remains one of the leading causes of death worldwide and requires a pressing need for the development of novel and more effective treatments. Although the chemotherapy is the major method for treatment for various cancer types, the narrow dosing window of current drugs with regard to their efficacy and safety and significant drug resistance resulting a failure of antitumor drugs to exert their effects in certain cancer types limit the use of contemporary cancer chemotherapy. Therefore, the design and discovery of more effective and safer anticancer drug candidates are of interest in contemporary medicinal chemistry.^{1, 2}

Microtubules are important in mitosis and have been recognized as an important target for the development of novel anticancer drugs.³ Agents targeting tubulin such as the vinca alkaloids and taxoids are potent chemotherapeutics currently used in the clinic. Among them, colchicine was the first tubulin-binding agent to have antivascular effects causing hemorrhagic necrosis in human tumors.⁴ Combretastatins which are isolated from the South African tree Combretum caffrum are also a group of antimitotic compounds and combretastatin A-4 (CA-4, Fig. 1) is one of the well-known natural tubulin-binding molecule affecting microtubule dynamics.⁵ CA-4 has provided researchers a simple structural template for the design of related compounds with potent activity and a large number of combretastatin analogues have been prepared as potential anticancer agents including chalcones, some of which have recently been reviewed.^6–10 Chalcones (1,3-diaryl-2-propen-1-ones) with an ionone system between two aromatic rings (Fig. 1) serve as precursors for the preparation of various flavonoids and exhibit interesting pharmacological activities^{11, 12} such as anticancer^{13– 15} and antiproliferative activities.^16–21 Their broad biological properties are reported to be due to the α,β-unsaturated ketone moiety.²² Chalcones in which both the 1,3-diaryl rings are separated by α,β-unsaturated carbonyl system with three-carbon lengths are structurally similar to indolyl heterocycles (Fig. 1). There are many indole-based compounds found to be effective as tubulin assembly inhibitors such as the recently reported 3-arylthioindoles, which induced significant apoptotic cell death.²³ However, indole-based chalcones remain largely unexplored for their anticancer potential.^24–26 Kumar et al. showed that indolyl chalcones inhibited the growth of A549, PaCa-2 and PC-3 cancer cell lines at micromolar concentrations.²⁷ Cinnamon amides having an α,β-unsaturated ketone moiety (phenylcinnamides in Fig. 1) are shown to bind to tubulin, thereby causing an inhibition of its polymerization and alteration in the tubulin-microtubule equilibrium.^28–31

Figure 1.

Chemical structures of known tubulin inhibitors and synthesized compounds

Encouraged with these results and to discover novel anticancer agents, we have synthesized a series of novel indolylacrylamide derivatives and evaluated their anticancer activities. The newly synthesized compounds structurally resemble the indolyl chalcone structure (Fig. 1).

2. Results and Discussion

2.1. Chemistry

We synthesized a series of amide derivatives of trans-indole-3-acrylic acid as illustrated in Scheme 1. First, trans-indole-3-acrylic acid 2 was generated by Knoevenagel condensation of indole-3-carbaldehyde with malonic acid in the presence of piperidine as reported previously.³² Treatment of 2 with appropriate amines in the presence of triethylamine and ethyl chloroformate, which was used as the carboxylate activator, produced trans-indole-3-acrylamide derivatives 3a–k in moderate to good yields (40%–69%). Compounds were purified by automated flash chromatography and checked for purity with UPLC before being tested in biological assays (purity was >97%). The structures of these compounds were confirmed by high resolution mass spectrometry (HRMS), IR and ¹H- and ¹³C-NMR spectral data. Final acryl amide derivatives exhibited a characteristic strong absorption peaks in the area of 1638–1733 cm⁻¹, which was attributable to the C=O of the amide moiety. In the ¹HNMR spectra of compounds 3a–k, one of the olefinic protons (CH=CH-CO) was observed as a doublet at about 7.61–7.83 ppm, while the other (CH=CH-CO) was observed as a doublet at about 6.64–7.15 ppm, with coupling constants of 15.6 or 16.0 Hz indicating the presence of the (E) isomer.

Scheme 1.

Reagents and conditions; a) Malonic acid, pyridine, piperidine, 5 h, 40 °C; b) 2, ethyl chloroformate, NEt₃, amine derivative, CH₂Cl₂, overnight, rt.

2.2. Biological Evaluations

2.2.1. Effects of the compounds on the viability of cancer cells

trans-Indolyl-3-acrylamide derivatives 3a–k were screened against five human cancer cell lines (HeLa, MCF7, MDA-MB-231, Raji and HL-60) using the 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT cell proliferation assay has been widely accepted as a reliable way to measure the cell proliferation rate when metabolic events lead to apoptosis or necrosis.^33–36 After incubation with compounds at different concentrations for 48 h, the cells were treated with MTT to measure their growth/viability (% of the untreated control) using a spectrophotometer as described previously.^{37, 38} Experiments were performed in quadruplicate. The IC₅₀ values (Table 1) were calculated from concentration– response curves by means of the PRISM 5, GraphPad Software.³⁹

Table 1.

In vitro cytotoxic activity of compounds 3a–k

			IC50 (µM)a
Compnd.	n	Ar	HeLa	MDA-MB-231	MCF7	Raji	HL-60
3a	0		>100	>100	>100	22.6±1.45	17.8±1.54
3b	0		>100	>100	>100	22.9±1.76	10.1±0.87
3c	0		31.4± 9.01	>100	>100	10.3±2.43	6.2±0.44
3d	0		>100	>100	>100	9.3±1.93	7.1±0.31
3e	0		23.7±2.46	>100	36.3±3.07	9.5±1.28	5.1±0.32
3f	0		>100	>100	>100	>100	>100
3g	1		>100	>100	>100	33.1±1.40	>100
3h	1		36.5±6.26	>100	>100	>100	>100
3i	1		>100	>100	>100	32.9±0.94	>100
3j	1		>100	>100	>100	>100	>100
3k	1		>100	>100	>100	>100	>100

^aEach experiment was independently performed four times, and data are shown as means ± SD.

In general, compounds having a phenylamidic moiety (3a–e) displayed greater antiproliferative potency than the molecules possessing a benzylamidic moiety (3g–k). Compounds having methoxy substituent at positions 3 and 4 or at 3 and 5 on the phenyl amidic moiety of the molecules (3c, 3d) exerted potency on the Raji and HL-60 cell lines (IC₅₀ values of 6.2–10.3 µM). Compound 3e showed appreciable antiproliferative activity against both Raji and HL-60 cell lines, with IC₅₀ values of 9.5 and 5.1 µM, respectively. Breast cancer cell lines MDA-MB-231 and MCF7 were not sensitive towards the newly synthesized compounds with the exception of the 3,4,5-trimethoxy-substituted phenyl amide derivative 3e, which was the most effective compound found in the phenyl amide series.

In the series of benzylamide derivatives, compound 3h, with 2,3-dimethoxy substitutions on the amide moiety, showed weak activity against HeLa cells. Derivatives 3g (3-methoxy-4-hydroxy-substituted benzylamide derivative) and 3i (2,5-dimethoxy-substituted benzylamide derivative) had weak activity against Raji cells.

2.2.2. Inhibition of tubulin polymerization and colchicine binding

Based on their structural resemblance to phenylcinnamides, we considered tubulin as a potential target³¹ for our active compounds. To investigate whether the antiproliferative activity of these compounds is due to an interaction with tubulin, we evaluated the effects of 3a–k on the polymerization of purified tubulin, using the highly potent CA-4 as reference (Table 2).⁴⁰

Table 2.

Inhibition of tubulin polymerization and colchicine binding by synthesized compounds and CA-4.

Compound	Inhibition of tubulin assemblya IC50 (µM)± SD	Inhibition of colchicine bindingb % Inhibition ± SD
		50 µM	5 µM	1 µM
3a	> 40	13 ± 5	-	-
3b	> 40	15 ± 5	-	-
3c	> 40	11 ± 2	-	-
3d	> 40	26 ± 5	-	-
3e	17 ± 1	50 ± 4	11 ± 4	-
3f	> 40	11 ± 1	-	-
3g	> 40	14 ± 5	-	-
3h	> 40	12 ± 4	-	-
3i	> 40	13 ± 4	-	-
3j	> 40	23 ± 2	-	-
3k	> 40	13 ± 0.2	-	-
CA-4	1.3 ± 0.07		98 ± 0.8	88 ± 0.3

^aTubulin was at 10 µM.

^bTubulin and colchicine were at 1 and 5 µM concentrations, respectively.

Only 3e inhibited tubulin polymerization with IC₅₀ values below 20 µM. Compounds 3e with 3,4,5-trimethoxy substituents on the amide moiety had IC₅₀ value of 17 µM. This was in agreement with 3e being the compound with the greatest antiproliferative activity. Three compounds (3b, 3c, 3d) of the five best antiproliferative agents were inactive as antitubulin agents. Therefore, the results suggest that there may be another, more dominant molecular mechanism of action for these three compounds for their antiproliferative activity.

All synthesized compounds were also examined for their inhibitory effects at 5 and/or 50 µM on the binding of 5 µM [³H]colchicine to 1 µM tubulin (Table 2).⁴¹ Significant inhibition was only observed with 3e at 50 µM, in accord with their effects on the assembly reaction.

2.2.3. Analysis of cell cycle effects

Considering the cytotoxic activity of 3e and its antitubulin properties, we further analyzed its effects on cell cycle distribution of cultured HL-60 and HeLa cells, as determined by flow cytometry (Fig. 2). After treatment of cells with 3e for 24 h, we observed a concentration dependent increase of G2/M-phase cells with a concomitant reduction in the proportion of cells in G1 and S phases with both cell lines. The increase in the G2/M phase cells occurred at a concentration as low as 10 µM, while more than 80% of the HeLa and 60% of the HL-60 cells were blocked in G2/M at higher concentrations (15–30 µM). These results were in good agreement with the inhibitory effect of 3e on tubulin polymerization and also on the proliferation of both cell lines.

Figure 2.

Percentage of cells in each phase of the cell cycle in HL-60 (Panel A) and HeLa cells (Panel B) treated with 3e at the indicated concentrations for 24 h. Cells were fixed and labeled with PI and analyzed by flow cytometry as described in the experimental section. Data are represented as means ± SEM of three independent experiments

2.2.4. Compound 3e induces apoptosis

To characterize the mode of cell death by 3e, the annexin-V/propidium iodide (PI) biparametric cytofluorimetric assay was performed on both the HeLa and HL-60 cell lines. Since PI, which stains DNA and only enters the dead cells, and annexin-V [conjugated to fluorescein isothiocyanate (FITC)], which binds to phosphatidylserine (PS) located only on the outer membrane of apoptotic cells, the assay permits quantitation of live cells (annexin-V−/PI−), early apoptotic cells (annexin-V+/PI−), late apoptotic cells (annexin-V+/PI+) and necrotic cells (annexin-V−/PI+). As shown in Fig. 3, treatment of both cell lines with compound 3e induced a concentration- and time-dependent increase in annexin-V+/PI− and annexin-V+/PI+ fractions, indicating activation of the apoptotic cell death machinery.

Figure 3.

Flow cytometric analysis of apoptotic cells after treatment of HL-60 (panels A and B) and HeLa (panels C and D) cells with 3e at the indicated concentrations after incubation for 24 (panels A and C) or 48 h (panels B and D). The cells were harvested and labeled with annexin-V-FITC and PI and analyzed by flow cytometry. Data are represented as means ± SEM of three independent experiments

2.2.5. Effects of 3e on caspase activation

To further evaluate the apoptotic process induced by 3e, we performed immunoblot analysis on HL-60 protein extracts. During apoptosis, the endogenous 113 kDa Poly-ADP-ribosyl-polymerase (PARP) protein is cleaved into 89 kDa and 24 kDa fragments, which can be shown by Western blot analysis. As shown in Fig. 4, 3e induced the activation of caspase-3 with the appearance of the cleaved fragments and the subsequent cleavage of its substrate PARP, after 24 h and 48 h treatments. This observation indicated that the 3e-induced apoptosis was caspase-dependent.

Figure 4.

Western blot analysis of caspase-3 and PARP after treatment of HL-60 cells with 3e at the indicated concentrations and for the indicated times. To confirm equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody

2.2.6. Molecular docking studies

To determine the possible binding modes of the most active compound 3e to tubulin, docking studies were carried out using the high-resolution crystal structure of the tubulin/DAMA-colchicine complex (PDB ID: 1SA0).⁴²

By following the reported docking technique^{43, 44} for 1SA0⁴², a Glide 5.8⁴⁵ run in single precision mode (GlideScore SP) was used to collect the best ranked pose of each ligand as shown in Fig. 5. Ligand 3e assumed a V-shaped conformation in the same binding region of DAMA-colchicine (X-Ray ligand, Fig. 5-A). The trimethoxyphenyl group of 3e was positioned in a similar orientation of the corresponding ring in the co-crystallized DAMA-colchicine. In both structures, the middle methoxy group formed a hydrogen bond with Cys241.

Figure 5.

3D visualization of (A) DAMA-colchicine (X-Ray Ligand)⁴² and docking results of the best ranked docking pose of 3e (B). The main interacting residues are shown and labeled. Hydrogen bonds are indicated by black dashed lines

3. Conclusions

We synthesized a series of indolylacrylamides as potential anticancer agents and determined their cytotoxic activity against five human cancer cell lines. Compound 3e, the most potent derivative in this series, displayed antiproliferative activity with IC₅₀ values in the range of 5.1 to 36.3 µM against the tested cancer cell lines. Compound 3e was also the most active inhibitor of tubulin polymerization and inhibited the binding of [³H]colchicine to tubulin. Compound 3e caused HeLa and HL-60 cells to accumulate in the G2/M phase of the cell cycle as was the case with most antitubulin agents. 3e also induced apoptotic cell death by activation of caspase-3 activity. Moreover, molecular docking studies demonstrated a potential binding mode for compound 3e in the colchicine site of tubulin resembling the co-crystallized DAMA-colchicine binding mode.

4. Experimental

4.1. Chemistry

Chemicals purchased from commercial vendors were used without purification. Thin-layer chromatography (TLC) was performed on Merck 60F254 plates. Reactions were monitored by TLC on silica gel, with detection by UV light (254 nm) or charring Dragendorff reagent.⁴⁶ Melting points were determined with an SMP-II Digital Melting Point Apparatus and are uncorrected (Schorpp Geaetetechnik, Germany). IR spectra were obtained using a Perkin Elmer Spectrum 400 FTIR/FTNIR spectrometer equipped with a Universal ATR Sampling Accessory. ¹H-NMR spectra were recorded in CDCl₃ or DMSO-d₆ on a Varian Mercury 400 MHz High Performance Digital FT-NMR spectrometer using tetramethylsilane as the internal standard at the NMR facility of the Faculty of Pharmacy, Ankara University. ¹³C-NMR spectra were recorded in CDCl₃ on a Varian Mercury 300 MHz FT-NMR spectrometer using tetramethylsilane as the internal standard at the NMR facility of FARGEM (Pharmaceutical Research and Development Center) Inc. All chemical shifts were recorded as δ (ppm). High resolution mass spectra data (HRMS) were collected using a Waters LCT Premier XE Mass Spectrometer (high sensitivity orthogonal acceleration time-of-flight instrument) using ESI (+) method. The instrument was coupled to an AQUITY Ultra Performance Liquid Chromatography system (Waters Corporation, Milford, MA, USA). Flash chromatography was performed with a Combiflash^®Rf automated flash chromatography system with RediSep columns (Teledyne-Isco, Lincoln, NE, USA) using CH₂Cl₂-methanol (0%–5%) solvent gradients.

4.2. Synthesis of trans-indole-3-acrylic acid 2

trans-Indole-3-acrylic acid was synthesized by Knoevenagel condensation between indole-3-carbaldehyde and malonic acid as previously reported.³² (Yield 91%, mp 195 °C)

4.3. General procedure for the preparation of amide derivatives of trans-indole-3-acrylic acid

To a solution of the acid derivative (1 mmol) in CH₂Cl₂ were added triethylamine (2 mmol) and ethyl chloroformate (1 mmol), followed by stirring at 0 °C for 30 min. After addition of the appropriate amine derivative (1.2 mmol), the mixture was stirred for an additional 1 h at 0 °C. Then, the reaction mixture was warmed to room temperature and stirred overnight. After the solvent was evaporated under reduced pressure, acetone was added, filtered, and evaporated. The residue was dissolved in CH₂Cl₂, and the organic phase was washed with a 1% NaHCO₃ solution and brine, dried over Na₂SO₄, and evaporated under vacuum. The final residue was purified by flash column chromatography (Combiflash® Rf) using CH₂Cl₂-MeOH (0%–5%) as eluents.

4.3.1. (E)-N-(2,4-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylamide 3a (CAS Registry Number: 953135-26-7)

Yield 40%, mp 165–167°C; IR (FTIR/FTNIR-ATR): 1644 cm⁻¹ (C=O), 3172 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.60 (1H, s), 9.15 (1H, s), 8.11 (1H, d, J=6.8 Hz), 8.04 (1H, d, J=9.2 Hz), 7.97 (1H, s), 7.72 (1H, d, J=16 Hz), 7.47 (1H, d, J=7.2 Hz), 7.20 (2H, m), 7.07 (1H, d, J=15.6 Hz), 6.64 (1H, d, J=2.8 Hz), 6.52 (1H, m), 3.87 (3H, s), 3.75 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 165.6, 156.7, 151.2, 138.1, 134.8, 131.6, 125.5, 123.1, 122.9, 121.9, 121.2, 121.0, 117.1, 113.1, 112.9, 104.6, 99.3, 56.4, 55.9; HRMS C₁₉H₁₉N₂O₃ [M+H]⁺ Calc. 323.1396, Found m/z 323.1397.

4.3.2. (E)-N-(2,5-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylamide 3b (CAS Registry Number: 953194-76-8)

Yield 63%, mp 158–160 °C; IR (FTIR/FTNIR-ATR): 1655 cm⁻¹ (C=O), 3403 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.64 (1H, s), 9.29 (1H, s), 8.15 (1H, d, J=7.2 Hz), 8.02 (1H, m), 7.81 (1H, s), 7.75 (1H, d, J=15.6 Hz), 7.47 (1H, d, J=7.6 Hz), 7.20 (2H, m), 7.15 (1H, d, J=15.6 Hz), 6.97 (1H, d, J=9.2 Hz), 6.60 (1H, m), 3.84 (3H, s), 3.70 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 166.0, 153.6, 143.7, 138.1, 135.6, 132.0, 129.6, 125.4, 123.0, 121.3, 121.0, 116.8, 113.1, 112.9, 112.1, 108.3, 107.7, 56.9, 55.9; HRMS C₁₉H₁₉N₂O₃ [M+H]⁺ Calc. 323.1396, Found m/z 323.1393.

4.3.3. (E)-N-(3,4-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylamide 3c (CAS Registry Number: 953243-47-5)

Yield 68%, mp 244–246 °C; IR (FTIR/FTNIR-ATR): 1649 cm⁻¹ (C=O), 3328 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.63 (1H, s), 9.86 (1H, s), 7.94 (1H, m), 7.81 (1H, s), 7.73 (1H, d, J=15.6 Hz), 7.48 (1H, m), 7.43 (1H, s), 7.21 (3H, m), 6.92 (1H, d, J=8.8 Hz), 6.78 (1H, d, J=15.6 Hz), 3.76 (3H, s), 3.73 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 165.3, 149.2, 145.1, 138.1, 134.9, 134.1, 131.6, 125.5, 123.0, 121.1, 120.6, 116.8, 113.0, 112.8, 112.8, 111.2, 104.5, 56.3, 55.9; HRMS C₁₉H₁₉N₂O₃ [M+H]⁺ Calc. 323.1396, Found m/z 323.1393.

4.3.4. (E)-N-(3,5-Dimethoxyphenyl)-3-(1H-indol-3-yl)acrylamide 3d

Yield 45%, mp 218–221 °C; IR (FTIR/FTNIR-ATR): 1649 cm⁻¹ (C=O), 3206 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.68 (1H, s), 9.96 (1H, s), 7.95 (1H, m), 7.84 (1H, s), 7.75 (1H, d, J=15.6 Hz), 7.49 (1H, m), 7.23 (2H, m), 6.96 (2H, s), 6.79 (1H, d, J=15.6 Hz), 6.21 (1H, m), 3.74 (6H, s). ¹³C-NMR (DMSO-d₆) δ: 165.7, 161.2, 142.1, 138.1, 135.6, 131.9, 125.5, 123.1, 121.2, 120.7, 116.5, 113.1, 112.8, 97.8, 95.5, 55.7. HRMS C₁₉H₁₉N₂O₃ [M+H]⁺ Calc. 323.1396, Found m/z 323.1391.

4.3.5. (E)-3-(1H-Indol-3-yl)-N-(3,4,5-trimethoxyphenyl)acrylamide 3e

Yield 53%, mp 246–248 °C; IR (FTIR/FTNIR-ATR): 1733 cm⁻¹ (C=O), 3304 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.68 (1H, s), 9.96 (1H, s), 7.95 (1H, m), 7.83 (1H, s), 7.74 (1H, d, J=15.6 Hz), 7.48 (1H, m), 7.23 (2H, m), 7.10 (2H, s), 6.79 (1H, d, J=15.6 Hz), 3.77 (6H, s), 3.63 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 165.5, 153.4, 138.1, 136.6, 135.2, 133.7, 131.8, 125.5, 123.0, 121.1, 120.6, 116.7, 113.1, 112.8, 97.1, 60.8, 56.3; HRMS C₂₀H₂₁N₂O₄ [M+H]⁺ Calc. 353.1501, Found m/z 353.1503.

4.3.6. (E)-3-(1H-Indol-3-yl)-N-(2,4,6-trimethoxyphenyl)acrylamide 3f

Yield 41%, mp 221–223 °C; IR (FTIR/FTNIR-ATR): 1651 cm⁻¹ (C=O), 3324 cm⁻ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.57 (1H, s), 8.68 (1H, s), 7.96 (1H, d, J=7.2 Hz), 7.77 (1H, s), 7.61 (1H, d, J=16 Hz), 7.47 (1H, d, J=7.2 Hz), 7.20 (2H, m), 6.83 (1H, d, J=15.6 Hz), 6.27 (2H, s), 3.79 (3H, s), 3.73 (6H, s). ¹³C-NMR (DMSO-d₆) δ: 165.9, 159.7, 157.2, 138.0, 134.0, 131.1, 125.5, 122.8, 121.0, 120.6, 117.0, 112.9, 112.8, 108.7, 91.6, 56.3, 56.0; HRMS C₂₀H₂₁N₂O₄ [M+H]⁺ Calc. 353.1501, Found m/z 353.1490.

4.3.7. (E)-N-(4-Hydroxy-3-methoxybenzyl)-3-(1H-indol-3-yl)acrylamide 3g

Yield 55%, mp 213–215 °C; IR (FTIR/FTNIR-ATR): 1702 cm⁻¹ (C=O), 3266 cm⁻¹ (N-H). ¹HNMR (DMSO-d₆) δ: 11.55 (1H, s), 8.87 (1H, s), 8.23 (1H, t, J=5.6 Hz), 7.89 (1H, d, J=7.2 Hz), 7.57 (1H, m), 7.63 (1H, d, J=15.6 Hz), 7.45 (1H, d, J=7.6 Hz), 7.18 (2H, m), 6.88 (1H, s), 6.72 (1H, m), 6.67 (1H, d, J=15.6 Hz), 4.29 (2H, d, J=5.2 Hz), 3.75 (3H, s). The OH-signal was not observed in the spectrum, due to solvent exchange. ¹³C-NMR (DMSO-d₆) δ: 166.8, 148.1, 146.1, 138.0, 133.8, 131.0, 130.9, 125.5, 125.2, 122.8, 120.9, 120.7, 116.9, 115.9, 112.9, 112.8, 112.6, 56.2, 42.9; HRMS C₁₉H₁₉N₂O₃ [M+H]⁺ Calc. 323.1396, Found m/z 323.1397.

4.3.8. (E)-N-(2,3-Dimethoxybenzyl)-3-(1H-indol-3-yl)acrylamide 3h

Yield 69%, mp 211–213 °C; IR (FTIR/FTNIR-ATR): 1639 cm⁻¹ (C=O), 3206 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.56 (1H, s), 8.21 (1H, t, J=5.6 Hz), 7.91 (1H, d, J=7.6 Hz), 7.75 (1H, s), 7.63 (1H, d, J=15.6 Hz), 7.45 (1H, d, J=7.6 Hz), 7.18 (2H, m), 6.88 (3H, m), 6.71 (1H, d, J=16 Hz), 4.40 (2H, d, J=6 Hz), 3.80 (3H, s), 3.76 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 166.9, 152.9, 146.9, 138.0, 133.8, 133.5, 131.0, 125.5, 124.5, 122.8, 121.1, 120.9, 120.6, 116.8, 112.9, 112.8, 112.3, 60.8, 56.3, 37.7; HRMS C₂₀H₂₁N₂O₃ [M+H]⁺ Calc. 337.1552, Found m/z 337.1544.

4.3.9. (E)-N-(2,4-Dimethoxybenzyl)-3-(1H-indol-3-yl)acrylamide 3i

Yield 48%, mp 164–166 °C; IR (FTIR/FTNIR-ATR): 1644 cm⁻¹ (C=O), 3241 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.54 (1H, s), 8.07 (1H, t, J=6 Hz), 7.92 (1H, d, J=8 Hz), 7.74 (1H, s), 7.61 (1H, d, J=15.6 Hz), 7.45 (1H, d, J=7.6 Hz), 7.18 (3H, m), 6.71 (1H, d, J=16 Hz), 6.57 (1H, s), 6.50 (1H, d, J=8 Hz), 4.28 (2H, d, J=5.6 Hz), 3.81 (3H, s), 3.74 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 166.9, 160.4, 158.4, 138.0, 133.6, 131.0, 129.8, 125.5, 122.8, 120.9, 120.7, 119.8, 116.9, 112.9, 112.8, 104.9, 98.8, 56.0, 55.8, 37.7; HRMS C₂₀H₂₁N₂O₃ [M+H]⁺ Calc. 337.1552, Found m/z 337.1539.

4.3.10. (E)-N-(2,5-Dimethoxybenzyl)-3-(1H-indol-3-yl)acrylamide 3j

Yield 57%, mp 190–193 °C; IR (FTIR/FTNIR-ATR): 1638 cm⁻¹ (C=O), 3299 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.56 (1H, s), 8.18 (1H, t, J=5.6 Hz), 7.93 (1H, d, J=8 Hz), 7.76 (1H, s), 7.63 (1H, d, J=15.6 Hz), 7.46 (1H, d, J=8 Hz), 7.18 (2H, m), 6.93 (1H, d, J=8.8 Hz), 6.79 (2H, m), 6.73 (1H, d, J=15.6 Hz), 4.34 (2H, d, J=5.6 Hz), 3.77 (3H, s), 3.68 (3H, s). ¹³C-NMR (DMSO-d₆) δ: 172.2, 168.4, 168.3, 164.1, 155.2, 155.0, 136.7, 133.2, 132.7, 125.6, 121.2, 121.0, 119.8, 115.1, 115.0, 112.5, 111.6, 68.4, 24.4; HRMS C₂₀H₂₁N₂O₃ [M+H]⁺ Calc. 337.1552, Found m/z 337.1548.

4.3.11. (E)-N-(3,4-Dimethoxybenzyl)-3-(1H-indol-3-yl)acrylamide 3k⁴⁷

Yield 68%, mp 176–178 °C; IR (FTIR/FTNIR-ATR): 1657 cm⁻¹ (C=O), 3292 cm⁻¹ (N-H). ¹H-NMR (DMSO-d₆) δ: 11.52 (1H, s), 8.24 (1H, t, J=5.6 Hz), 7.86 (1H, d, J=7.6 Hz), 7.72 (1H, s), 7.61 (1H, d, J=16 Hz), 7.42 (1H, d, J=7.6 Hz), 7.13 (2H, m), 6.89 (2H, m), 6.80 (1H, m), 6.64 (1H, d, J=16 Hz), 4.30 (2H, d, J=5.2 Hz), 3.71 (3H, s), 3.69 (3H, s). ¹³C-NMR (DMSOd₆) δ: 166.8, 149.2, 148.4, 138.0, 133.9, 132.7, 131.0, 125.5, 122.8, 120.9, 120.6, 120.2, 116.8, 112.9, 112.7, 112.3, 112.1, 56.2, 56.0, 42.8; HRMS C₂₀H₂₁N₂O₃ [M+H]⁺ Calc. 337.1552, Found m/z 337.1544.

4.4. Anticancer activity

4.4.1. Cell lines and cell culture

The human cancer cell lines, cervical carcinoma (HeLa), estrogen receptor positive breast carcinoma (MCF7), estrogen receptor negative breast carcinoma (MDA-MB-231), Burkitt’s lymphoma (Raji) and human promyelocytic leukemia (HL-60), were obtained from ATCC.

4.4.2. Cytotoxicity assay

MDA-MB-231 and MCF7 cells were cultured in DMEM whereas HeLa, HL-60 and Raji cells were grown in RPMI-1640 medium in a humidified atmosphere containing 5% CO₂ at 37 °C. Both DMEM and RPMI-1640 medium were supplemented with 10% fetal bovine serum (FBS), 200 mM L-glutamine, 100 IU/mL penicillin and 100 µg/mL streptomycin (all from Hyclone Laboratories, Logan, UT, USA). Cell viability was determined using the MTT assay (Cell Proliferation Kit I, Roche, Germany). Briefly, cells were seeded in a 96-well plate at 10,000 cells per well and cultured overnight in growth medium containing 1% FBS. Then cells were treated with test compounds at different concentrations (from 0.1 to 125 µM) for 48 h. As a solvent control, cells were also treated with dimethyl sulfoxide (DMSO) at a final concentration of 0.1%. At the end of the incubation time, MTT (final concentration, 0.5 mg/mL) was added to each well, and the plate was incubated for an additional 4 h. After formation of blue formazan crystals, medium containing MTT was discarded, and DMSO was added to the wells to dissolve the MTT crystals. The absorbance of samples was measured with a Spectra Max M3 microplate reader (Molecular Devices, Sunnyvale, CA, USA) at 570 nm. Average absorbance values from quadruplicate replicates per test compound and solvent control (DMSO) were calculated. Mean solvent control values were set to 100% viability, and then the effects of test compounds on cell viability were calculated by comparing mean values obtained from compound treated culture wells with those of the solvent controls. IC₅₀ values were calculated from concentration–response curves by means of PRISM 5, Graph Pad Software.³⁹

4.4.3. Tubulin polymerization assays

Purified bovine brain tubulin⁴⁸ was used in turbidimetric polymerization studies and in the colchicine binding assay. Tubulin and the desired concentrations of compound were preincubated at 30 °C for 15 min in a 0.24 mL reaction volume. Reaction mixtures were then placed on ice, and 10 µL of 10 mM GTP was added. All concentrations are in terms of the final reaction volume of 0.25 mL: tubulin at 1.0 mg/mL (10 µM), 0.8 M monosodium glutamate (adjusted to pH 6.6 in a 2 M stock solution with HCl), 4% (v/v) DMSO, and 0.4 mM GTP. The ice-cold reaction mixtures were transferred to cuvettes held at 0 °C in recording spectrophotometers (Beckman models DU7400 and DU7500) equipped with electronic temperature controllers. After baselines were established at 350 nm, the temperature was jumped to 30 °C over about 30 s, and the IC₅₀ is defined as the concentration of compound that inhibits turbidity development by 50% at 20 min. Detailed method description has been published previously.⁴⁰ The method is generally most reliable at compound concentrations up to 20 µM. At higher concentrations, compound precipitation and/or absorbance often leads to interference with the turbidity readings caused by tubulin assembly.

4.4.4. Inhibition of colchicine binding assays

The colchicine binding assay was performed in 0.1 mL reaction volumes. Each assay tube contained 0.1 mg/mL (1.0 µM) tubulin, 5% (v/v) DMSO, 5.0 µM [³H]colchicine (Perkin Elmer), varying concentrations of potential inhibitors, as indicated, and additional components, including 1.0 M glutamate, shown to stabilize the colchicine binding activity of tubulin^{41, 49} for prolonged times at 37 °C. Samples were incubated for 10 min at 37 °C, at which time the reaction in control samples is 40–60% complete. The reactions were stopped with water at 0 °C, and the diluted samples (total volume, about 2 mL, with several 2 mL rinses) were filtered through a stack of two DEAE-cellulose filters (Whatman) in a 12-place manifold (Millipore) under a weak vacuum (filtration time about 10 min). The filters were then rapidly washed three times (2 mL each) with ice-cold water, using a stronger vacuum. Radiolabel bound to the filters was measured in a liquid scintillation counter.

4.4.5. Flow cytometric analysis of cell cycle distribution

For flow cytometric analysis of DNA content, 5×10⁵ HeLa and HL-60 cells were treated with different concentrations of the test compounds for 24 h. After the incubation period, the cells were collected, centrifuged and fixed with ice-cold ethanol (70%). The cells were then treated with lysis buffer containing RNAse A and 0.1% Triton X-100 and then stained with PI. Samples were analyzed on a Cytomic FC500 flow cytometer (Beckman Coulter). DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems).

4.4.6. Annexin-V assay

Surface exposure of PS on apoptotic cells was measured by flow cytometry with a Coulter Cytomics FC500 (Beckman Coulter) by adding annexin-V-FITC to cells according to the manufacturer’s instructions (Annexin-V Fluos, Roche Diagnostic). Simultaneously, the cells were stained with PI. Excitation was set at 488 nm, and the emission filters were at 525 and 585 nm, respectively, for FITC and PI.

4.4.7. Western blot analysis

HL-60 cells were incubated in the presence of test compounds and, after different times, were collected, centrifuged and washed twice with ice-cold phosphate-buffered saline. The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates were centrifuged at 15000 × g at 4 °C for 10 min. The protein concentration in the supernatant was determined using the BCA protein assay reagents (Pierce, Italy). Equal amounts of protein (20 µg) were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5–15 % acrylamide gels) and transferred to a PVDF Hybond-p membrane (GE Healthcare). Membranes were blocked with 5% bovine serum albumin for 2 h. Membranes were then incubated with primary antibodies against caspase-3 (Alexis) and PARP (Cell Signalling) overnight at 4 °C. Membranes were next incubated with peroxidase-labeled secondary antibodies for 60 min. All membranes were visualized using ECL Select (GE Healthcare) and exposed to Hyperfilm MP (GE Healthcare). To ensure equal protein loading, each membrane was stripped and reprobed with anti-β-actin antibody (Sigma-Aldrich).

4.5. Docking and Modeling Studies

A structure based procedure was applied to discover and study the putative binding mode of the most potent compounds in our compound series. The PDB used (PDB ID: 1SA0⁴²) in this work was first submitted to the Protein Preparation Wizard (only Chain A and B) protocol of the Schrödinger Suite⁴⁵ by following similar procedures described previously.^{43, 44} Later, ligand preparation was applied by the LigPrep 2.5⁴⁵ run to correctly assign the protonation states and atom types of the molecule. An additional conformational search by using the Mixed torsional/Low-mode sampling method present in MacroModel 9.9⁴⁵ and setting 50 conformations as output for each compound was needed to reproduce correctly the binding pose of the X-Ray ligand (RMSD 0.877 Ǻ on heavy atoms) and was then applied to all the other chemical entities. The grid generation and all docking runs were performed by leaving all the variables at default values in the Glide 5.8⁴⁵ program used in single precision docking mode (SP) and saving up to the 10 best poses per conformation. The best ranking pose was visualized with PyMOL 1.6.⁴⁵