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

: 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. unfavorable pharmacoki-netic 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-(1 H -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. 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.


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-(3indolyl)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.

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.

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).

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 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].

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).

Scheme 2. Non-selective methylation of 2-phenyl-2-(3-indolyl)acetohydroxamic acid 3a.
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 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 IC 50 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.
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.

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 × 10 3 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). 1 H and 13 C 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 1 H and 13 C NMR spectral charts.

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 × 10 3 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). 1 H and 13 C 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 1 H and 13 C NMR spectral charts.