Methylation of 2-Aryl-2-(3-indolyl)acetohydroxamic Acids and Evaluation of Cytotoxic Activity of the Products

Abstract

2-Aryl-2-(3-indolyl)acetohydroxamic acids demonstrate promising antitumor activity, but quickly metabolize in vivo via glucuronidation of hydroxamic acid residue. In an attempt to improve their pharmacokinetics, methyl esters were synthesized via a newly developed protocol for chemoselective mono-methylation of hydroxamic acids. The cytotoxicity of these derivatives against the HeLa cell line was evaluated and found to be inferior compared to the parent lead compounds.

1. Introduction

The treatment of apoptosis-resistant tumors represents a major challenge, as they often respond poorly to traditional chemotherapeutics and tend to produce metastases, typically resulting in about a 90% death toll worldwide [1,2,3,4,5]. For this reason, the development of novel anti-cancer drugs with alternative modes of action, suitable for treatment of such resistant cell lines, can be easily justified. Recently, we communicated a concise and highly efficient synthetic method allowing for facile preparation of 2-aryl-2-(3-indolyl)acetohydroxamic acids 3 via reaction of indole derivatives 1 with β-nitrostyrenes 2 in polyphosphoric acid (PPA) (Scheme 1) [6]. It was also demonstrated that these readily available compounds are active against apoptosis- and multidrug-resistant cancer cells in vitro [7]. Initial animal studies demonstrated, however, that these compounds had no effect on cancerous tumors in mice [8]. This was linked to a very unfavorable pharmacokinetic profile, as it was shown that the concentration of compound 3 in plasma was reduced below the active threshold within an hour, presumably due to facile glucuronidation of the hydroxamic acid functionality [8]. This result prompted us to search for analogs that may be more resistant to this metabolic pathway. Herein, we wish to report an attempted synthesis of 2-(1H-indol-3-yl)-N-methoxy-2-phenylacetamides 4 via methylation of the parent 2-aryl-2-(3-indolyl)acetohydroxamic acids 3 and initial evaluation of their biologic activity against the HeLa cell line.

2. Results and Discussion

Initial attempts at methylation involved the treatment of hydroxamic acid AKS-7 (3a) with dimethyl sulfate (1.0 equiv.) in aqueous KOH according to the published procedure [9]. This method, however, did not produce any methylation, as 3a remained unchanged. We speculated that the lack of reactivity might be due to a poor solubility of the starting material in aqueous media. To address this issue, we decided to treat 3a with excess dimethyl sulfate (2.0 equiv.) in a biphasic system consisting of aqueous KOH and non-polar organic solvents such as benzene. This modification proved more productive, affording the desired 2-(1H-indol-3-yl)-N-methoxyacetamide 4a in moderate yield, along with a comparable amount of methyl 2-(1H-indol-3-yl)-N-methoxyacetimidate 5a resulting from double-fold methylation (Scheme 2).

In order to improve the chemoselectivity towards mono-methylation, the next set of optimization test reactions was performed in the presence of 1.2 equiv. of dimethyl sulfate. Optimal results were obtained when the reaction of 3a was carried out at room temperature for 12–15 h. This allowed the isolation of 4a with 60% yield (Scheme 3). The compound AKS-63 (3b) also reacted smoothly, affording the corresponding O-methylation product 4b with 63% yield. We did not detect the formation of N-methylated indole derivatives [10].

The evaluation of the anti-cancer activity of the synthesized compounds 4a,b was performed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay against the human HeLa cervical adenocarcinoma cell line. The results are presented in Figure 1, along with data obtained for the parent compounds 3a,b. It should be pointed out that in both cases, methoxy-protected hydroxamic acids 4 demonstrated inferior cytotoxic activities, as their IC₅₀ values were about an order of magnitude higher than those obtained for non-protected analogs. Evidently, hydrophilic interaction at the hydroxamic acid functionality is important for the bioactivity, and therefore the featured protection cannot be used in the design of better anti-cancer drugs based on this scaffold.

3. Materials and Methods

Human cervical adenocarcinoma HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultured in DMEM supplemented with 10% fetal bovine serum (FBS). The antiproliferative properties of the synthesized compounds were evaluated using the MTT assay [11]. All compounds were dissolved in DMSO at a concentration of either 100 or 50 mM prior to cell treatment. The cells were trypsinized and seeded at 4 × 10³ cells per well into 96-well plates. The cells were grown for 24 h, treated with compounds at concentrations ranging from 0.001 to 100 μM, and incubated for 48 h in 200 μL of medium. Then, 20 μL of MTT reagent in serum-free medium (5 mg/mL) was added to each well, and the cells were incubated for a further 2 h. The medium was removed, and the resulting formazan crystals were resolubilized in 200 μL of DMSO. A490 was measured using a Molecular Devices THERMOmax plate reader. The experiments were performed in quadruplicate and repeated at least twice for each compound. Cells treated with 0.1% DMSO were used as a negative control; 1 μM phenyl arsine oxide (PAO) was used as a positive control.

Reagents, solvents, and catalysts were purchased from commercial sources (Acros Organics and Sigma-Aldrich) and used without purification. All reactions were performed in oven-dried flasks open to the atmosphere and monitored by thin layer chromatography on TLC precoated (250 μm) silica gel 60 F254 glass-backed plates (EMD Chemicals Inc., Gibbstown, NJ, USA). Visualization was accomplished with UV light. Flash chromatography was performed using silica gel (32–63 μm, 60 Å pore size). ¹H and ¹³C NMR spectra were recorded on Bruker DRX-400 and DRX-500 spectrometers. Chemical shifts (δ) are reported in ppm relative to the TMS internal standard. Abbreviations are as follows: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), and br. (broad). See Supporting Information for ¹H and ¹³C NMR spectral charts.

N-Hydroxy-2-phenyl-2-(2-(5,6,7,8-tetrahydronaphthalen-2-yl)-1H-indol-3-yl)acetamide (AKS-63, 3b): A mixture of phenylhydrazine (108 mg, 1.00 mmol), 1-(5,6,7,8-tetrahydronaphthalen-2-yl)ethan-1-one (174 mg, 1.00 mmol), and 80% PPA (2.0 g) was vigorously stirred at 100–110 °C. The reaction progress was monitored via TLC. After complete consumption of the starting materials, the mixture was cooled to 65–70 °C, and 2-nitrostyrene (179 mg, 1.20 mmol) was added in a single portion. The mixture was stirred for 1 h, while the reaction progress was again monitored via TLC. When all intermediate indole was consumed, the mixture was quenched with cold water (50 mL) and neutralized with aqueous ammonia to pH 8. The formed precipitate was collected and recrystallized from benzene/petroleum ether. The title compound was obtained as colorless crystals, mp 131–134 °C (benzene/petroleum ether). The yield was 297 mg (0.75 mmol, 75%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.22 (s, 1H), 10.73 (s, 1H), 8.81 (s, 1H), 7.70 (d, J = 8.1 Hz, 1H), 7.37 (s, 1H), 7.32 (d, J = 8.1 Hz, 1H), 7.29–7.12 (m, 8H), 7.03 (t, J = 7.5 Hz, 1H), 6.85 (t, J = 7.5 Hz, 1H), 5.07 (s, 1H), 2.76 (s, 4H), 1.77 (s, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.7, 141.0, 136.8, 136.4, 136.3, 136.1, 129.7, 129.2, 129.1, 128.3, 128.0 (4C), 127.9, 126.2, 125.7, 122.2, 121.0, 118.4, 110.8, 108.9, 46.2, 28.8, 28.6, 22.7, 22.7; HRMS (ESI-TOF, m/z): Calculated for C₂₆H₂₄N₂NaO₂ (M + Na)⁺, 419.1730; found 419.1731.

N-Methoxy-2-(2-(naphthalen-2-yl)-1H-indol-3-yl)-2-phenylacetamide (4a): N-Hydroxy-2-(2-(naphthalen-2-yl)-1H-indol-3-yl)-2-phenylacetamide (AKS-7, 3a) [7] (392 mg, 1.00 mmol) was dissolved in aqueous potassium hydroxide (78 mg, 2.00 mmol in 0.78 mL of water). This solution was cooled in an ice bath and crushed ice was added (0.15 g) followed by dimethyl sulfate (163 mg, 1.20 mmol) and benzene (7.5 mL). The mixture was stirred for 12 h at room temperature, and the reaction progress was monitored via TLC. After complete consumption of the starting material, the mixture was quenched with water (10 mL) and basified with aqueous ammonia to pH 8–9. The formed solution was extracted with ethyl acetate (3 × 50 mL). The combined organic phases were concentrated under vacuum and the residual solid was purified by flash column chromatography on silica gel, eluting with a mixture of benzene and ethyl acetate, 6:1. The title compound was obtained as yellowish crystalline solid, mp 204–207 °C (EtOAc), Rf 0.29 (C₆H₆/EtOAc, 6:1). Yield 244 mg (0.60 mmol, 60%). ¹H NMR (400 MHz, DMSO-d₆) δ 11.53 (s, 1H), 11.36 (s, 1H), 8.04–7.97 (m, 4H), 7.89 (d, J = 6.4 Hz, 1H), 7.69 (t, J = 9.7 Hz, 2H), 7.58–7.56 (m, 2H), 7.41 (d, J = 7.8 Hz, 1H), 7.31–7.20 (m, 6H), 7.10 (t, J = 7.4 Hz, 1H), 6.93 (t, J = 7.6 Hz, 1H), 5.14 (s, 1H), 3.58 (s, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ 168.5, 140.4, 136.4, 136.2, 132.8, 132.3, 129.9, 128.2 (3C), 128.1 (4C), 128.0, 127.7, 127.6, 127.5, 126.6, 126.4, 121.7, 121.4, 118.8, 111.1, 109.4, 63.1, 46.2; FT IR, cm⁻¹: 3200, 3362 (NH), 1643 (CONH). HRMS (ESI-TOF, m/z): Calculated for C₂₇H₂₂N₂NaO₂ (M + Na)⁺, 429.1573; found 429.1576.

N-Methoxy-2-phenyl-2-(2-(5,6,7,8-tetrahydronaphthalen-2-yl)-1H-indol-3-yl)acetamide (4b): This material was obtained from N-hydroxy-2-phenyl-2-(2-(5,6,7,8-tetrahydronaphthalen-2-yl)-1H-indol-3-yl)acetamide (AKS-63, 3b) (396 mg, 1.00 mmol) according to the procedure described above for preparation of compound 4a. The title compound was obtained as light grey crystalline solid, mp 181–184 °C, Rf 0.37 (C₆H₆/EtOAc, 6:1). ¹H NMR (400 MHz, DMSO-d₆) δ 11.30 (br. s, 1H), 11.29 (br. s, 1H), 7.64 (d, J = 8.0 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.31–7.23 (m, 2H), 7.23–7.13 (m, 6H), 7.05 (t, J = 7.3 Hz, 1H), 6.88 (t, J = 7.5 Hz, 1H), 5.01 (s, 1H), 3.56 (s, 3H), 2.76 (m, 4H), 1.77 (m, 4H); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.6, 140.6, 136.8, 136.6, 136.5, 136.1, 129.6, 129.2 (2C), 128.2 (2C), 128.1 (2C), 127.7, 126.3, 125.7, 121.6, 121.1, 118.6, 111.0, 108.4, 63.1, 46.3, 28.8, 28.6, 22.7, 22.6; FT IR, cm⁻¹: 3351 (NH), 1678 (CONH); HRMS (ESI-TOF, m/z): Calculated for C₂₇H₂₆N₂NaO₂ (M + Na)⁺, 433.1886; found 433.1883.

4. Conclusions

A chemoselective method for mono-methylation of 2-aryl-2-(3-indolyl)acetohydroxamic acids 3 was developed and employed to prepare samples of 2-(1H-indol-3-yl)-N-methoxy-2-phenylacetamides 4. These were used to assess the importance of the OH group for the cytotoxic activity of indole-containing derivatives of hydroxamic acid. Protection of this group would potentially allow the improvement of the unfavorable pharmacokinetics of acetohydroxamic acids 3 used as anti-cancer agents in vivo. It was found, however, that the free OH group is critically important for the high cytotoxicity of hydroxamic acid derivatives, and the protection approach should not be considered in further SAR investigations.