Synthesis and Cytotoxicity of Novel 10-Substituted Dihydroartemisinin Derivatives Containing N-Arylphenyl-ethenesulfonamide Groups

The manuscript describes the synthesis of 10-substituted dihydroartemisinin derivatives containing N-aryl phenylethenesulfonamide groups and their in vitro anti-tumor activities against the HT-29, MDA-MB-231, U87MG, H460, A549 and HL-60 cancer cell lines and the normal WI-38 cell line. Most tested compounds showed enhanced cytotoxic activities and good selectivity toward the MDA-MB-231, HT-29 and HL-60 cell lines, with IC50 values in the single-digit μM range as compared with dihydroartemisinin (DHA), and all of them displayed less toxicity towards WI-38 cells. Among them, compounds 3c and 6c with trifluoromethoxy groups on the N-phenyl ring were found to be most active compounds against the six tested cancer cell lines.


Introduction
Artemisinin is a sesquiterpene lactone isolated from the plant Artemesia annua L. Artemisinin and its derivatives, such as dihydroartemisinin (DHA) [1], artemether, arteether, and artesunate [2], are widely used currently as front-line antimalarials [3,4]. Despite the reported neurotoxic and embryotoxic effects in animals occurring at higher doses, application of artemisinins in humans seems to be relatively safe [5]. In addition to their well-known antimalarial activity, artemisinin derivatives possess cytotoxic activity against cancer cells by inducing apoptosis [6], but high concentrations are required [7].

OPEN ACCESS
Therefore, the synthesis of new, structurally modified derivatives of artemisinin is essential. The high chemical sensitivity of the artemisinin molecule restricts broad derivatization for library synthesis for further clinical development. So far the majority of the artemisinin derivatizations were carried out on the C-10 acetal and to a lesser extent on the C-13 carbon [8]. The observation that dihydroartemisinin C-10 ester, ether or amide derivatives ( Figure 1) possess significant antitumor activity prompted previous efforts, both within our group [9,10] and by others [11][12][13]. Hybrid drugs are formed by covalently linking two distinct chemical moieties with different biological modes of action with the aim of creating binary therapies with improved biological activity and less susceptible to the development of drug resistance [14,15]. Some N-aryl-2-arylethenesulfonamides, a new class of small molecule antitubulin agents, have exhibited potent activity against a wide spectrum of cancer cell lines, especially including some drug-resistant cell lines (e.g., MES-SA, HCT116, MDA-MB, etc.). Among them, ON 24160 (5, Figure 2) was the most promising compound, with significant cytotoxicity against multiple cancer cell lines and a suitable pharmacokinetic profile [16,17]. Therefore, we have now synthesized a short series of dihydroartemisinin derivatives 3a-h in which the C-10 hydroxy was covalently linked to the N-aryl-2-arylethenesulfonamide moiety with a two-carbon chain ( Figure 2). To expand the search for new antitumor compounds, the N-methylbenzamide scaffold was next inserted into the two-carbon chain linker to obtain compounds 6a to 6j ( Figure 2). In this study, the synthesis of novel N-aryl phenylethenesulfonamides dihydroartemisinin derivatives (compounds 3a-h and 6a-j, Figure 2) was reported. Most of them exhibited potent cytotoxic activity against six human cancer cell lines, including the HT-29, MDA-MB-231, U87MG, H460, A549 and HL-60 cell lines.

Chemistry
The syntheses of target compounds 3a-h and 6a-j are outlined in Scheme 1. Reduction of artemisinin (1) with NaBH 4 gave dihydroartemisinin (2) as mixture of 10α and 10β epimers which reacted with 2-bromoethanol directly using the Lewis acid BF3·Et 2 O as catalyst to give 10-bromoethoxydihydroartemisinin (3) [18]. In our previous report, the stereochemistry of compound 3 had already been determined as 10β by the 1 H-NMR coupling constant (J = 3.4 Hz) between 9-H and 10-H and the chemical shift of 10-H (4.77 ppm) [9]. Subsequently, the target compounds 3a-h were prepared in reasonable yield by potassium iodide-catalyzed substitution of bromo compound 3 with N-aryl-4hydroxyphenylethane-sulfonamides 10 in the presence of potassium carbonate. Regarding the side chain, N-aryl-2-arylethenesulfonamide derivatives 10 were prepared from ethyl bromoacetate according to Scheme 2. Reaction of ethyl bromoacetate with Na 2 SO 3 afforded sodium 2-ethoxy-2-oxoethanesulfonate (7), which was converted to the sulfonyl chloride 8 via chlorination using POCl 3 . Then condensation of compound 8 with the corresponding substituted anilines in toluene gave the ethyl 2-(N-arylsulfamonyl)acetates 9. Finally, reaction of compounds 9 with 4-hydroxybenzaldehyde afforded the desired side chains 10 in high yield. The target compounds 6a-j with the extend side chain were prepared from intermediate 3. Firstly, treatment of 3 with sodium azide under sodium iodide catalysis in DMF according to a modified Haynes' method [19] gave 10β-azidoethoxydihydroartemisinin (4). The Staudinger reduction was used in the transformation of azido compound 4 to 10β-aminoethoxydihydroartemisinin (5). Acylation of the amino group of 5 yielded 10β-arylamidoethoxydihydroartemisinin (6). Compounds 6a-j were prepared using a procedure similar to that used to synthesize compounds 3a-h.
The formation of the four key intermediates 3-6 was confirmed by 1 H-NMR and LC-MS data, The formation of the title compounds 3a-h and 6a-j was evidenced by their 1 H-NMR, IR and LC-MS spectra, explained in the Experimental section.

Biological Activity
The cytotoxic activities of target compounds were evaluated against HT-29 (human colon cancer), MDA-MB-231 (human breast cancer), U87MG (human glioblastoma), H460 (human lung cancer), A549 (non-small-cell lung adenocarcinoma), HL-60 (human leukemic) cancer cell lines and one normal cell line WI-38 (human fetal lung fibroblasts) together with references DHA and cisplatin by a MTT assay. The results, expressed as averaged IC 50 values from at least three independent experiments, are summarized in Table 1.
As illustrated in Table 1, all of the target compounds showed moderate to excellent cytotoxic activities against the different cancer cell lines in the single-digit μM range, and displayed less toxicity against normal WI-38 cells. In general, most of the compounds displayed significant selectivity toward MDA-MB-231, HT-29 and HL-60 cancer cells. It was worth noting that compounds 3c and 6c exhibited excellent anti-tumor activities against MDA-MB-231, HT-29 and HL-60 cells with IC 50 values in the tenths of a digit nM range. Moreover, the selectivity index (IC 50 normal cell/IC 50 cancer cell) of compound 3c for HL-60 was 750, which demonstrated that the tumor cells were more sensitive than the normal cells. Analysis of Table 1 clearly reveals that the cytotoxicities of benzamide derivatives 6a-j were lower than those of the corresponding compounds 3a-h. For example, the activity of compound 3c was 2-, 3and 2-fold higher than that of benzamide analog 6c against U87MG, H460 and HL-60, respectively. In addition, it is worth mentioning that different biological properties were observed when a variety of groups were introduced into the N-phenyl moieties. The activities of compounds 3b, 3d, 3f-h, 6b, 6d and 6f-h, with the electron-withdrawing halide atoms on the N-phenyl moiety, were superior to those with electron-donating groups such as methoxy, or methyl groups or no substitutents (6i, 3e, 6e, 3a and 6a). In the first series of compounds (3a-h), the 4-chloro derivative (3f) was superior to the fluoro-substituted (3d), disubstituted (3h) or compounds substituted at other positions (3b,g). Nevertheless, the 2-chloro and 2,6-dichloro derivatives 6g,h were better than others in the second series of compounds (6a-j). Excitingly, the compounds with a trifluoromethoxy group at the 4-position of the N-phenyl exhibited exceptionally high potency in two series of compounds, as exemplified by compounds 3c and 6c.

General
Melting points were measured with a Büchi Melting Point B-540 apparatus (Büchi Labortechnik, Flawil, Switzerland) and are uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent, Palo Alto, CA, USA). 1 H-NMR spectra were performed using Bruker 300 MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an internal standard. IR spectra (KBr disks) were recorded with a Bruker IFS 55 instrument (Bruker). Elemental analysis was determined on a Carlo-Erba 1106 Elemental analysis instrument (Carlo Erba, Milan, Italy). Unless otherwise noted, all the materials were obtained from commercially available sources and were used without further purification. (2). NaBH 4 (12 g, 0.32 mol) was added slowly to a stirred solution of artemisinin (30 g, 0.11 mol) in methanol (300 mL) at −5~0 °C. The reaction mixture was stirred at 0 °C for 2 h, adjusted to pH 6 to 7 with acetic acid, and then concentrated under reduced pressure. The residue was poured into water (400 mL), and the solid product formed was collected by filtration, washed with water, and dried to yield compound 2 (25 g, 84%). m.p. Preparation of 2-(10β-Dihydroartemisinoxy)ethylamine (5). A 100 mL round-bottomed flask was charged with compound 4 (6.0 g, 17 mmol) and THF (60 mL). To this solution, triphenylphosphine (7.2 g, 27 mmol) was added slowly, and the reaction mixture was stirred for 2 h at 60 °C. Several drops of water (1.0 mL, 56 mmol) were added, and the resulting suspension was stirred for 6 h. The mixture was concentrated under reduced pressure. The crude mixture was purified by flash column chromatography (silica gel, dichloromethane/methanol, 200:1) to afford the desired compound 5  (7). Na 2 SO 3 (15 g, 0.12 mol) was dissolved in water (50 mL) and a solution of ethyl bromoacetate (20 g, 0.12 mol) in ethanol (25 mL) was added dropwise at 5-10 °C. The reaction mixture was heated to 50 °C for 1 h. After then, the solution was evaporated until dryness. Acetic acid (200 mL) and ethyl acetate (100 mL) were added to the residue, and the mixture was heated to 100 °C for 1 h. The hot mixture was filtered, and another 1,000 mL ethyl acetate was poured into the filtrate. The white crystals were filtered, washed with ethyl acetate, and dried to yield white solid 7 (23 g, 99%). m.p.: 158 °C (dec.); MS (ESI, m/z): 190.2 (M+Na) + . (8) A mixture of compound 7 (10 g, 0.053 mol) and POCl 3 (100 mL, 1.1 mol) was stirred at 80 °C for 2 h and then cooled to room temperature. The solvent was removed under vacuum, 100 mL of toluene was added, and stirred for 10 min. The reaction solution was filtered, and the solvent was evaporated under reduced pressure to obtain 8 (9.0 g, 92%) as a red oil.

General Procedure for the Preparation of Ethyl 2-(N-Arylsulfamoyl)acetate (9)
A solution of 8 (8.0 g, 0.043 mol) in toluene (100 mL) was cooled to −5~0 °C, a mixture of substituted aniline (0.086 mol), triethylamine (0.065 mol) in toluene (50 mL) was added dropwise. The reaction mixture was stirred at room temperature for 0.5-1 h, and then washed with water, 5% HCl, saturated NaHCO 3 solution, saturated NaCl solution and water. The organic phase was dried with Na 2 SO 4 and evaporated to yield 9 as a brown oil.

General Procedure for the Preparation of Target Compounds 6a-j
A mixture of compound 10 (0.024 mol), K 2 CO 3 (4.2 g, 0.03 mol) and acetone (100 mL) was stirred at room temperature for 10 min, and then compound 6 (9.6 g, 0.02 mol) and KI (0.4 g, 2.6 mmol) was added while the reaction refluxed for 7-9 h. The acetone was removed under vacuum, and CH 2 Cl 2 (100 mL) was poured into the residue. The solution was washed with water, dried with Na 2 SO 4 . The solvent was evaporated and the residue was purified by column chromatography (silica-gel, 1%-5% petroleum ether/ethyl acetate) to afford pure compounds 6a-j.