Synthesis of Scutellarein Derivatives with a Long Aliphatic Chain and Their Biological Evaluation against Human Cancer Cells

Scutellarin is the major active flavonoid extracted from the traditional Chinese herbal medicine Erigeron breviscapus (Vant.) Hand-Mazz., which is widely used in China. Recently, accumulating evidence has highlighted the potential role of scutellarin and its main metabolite scutellarein in the treatment of cancer. To explore novel anticancer agents with high efficiency, a series of new scutellarein derivatives with a long aliphatic chain were synthesized, and the antiproliferative activities against Jurkat, HCT-116 and MDA-MB-231 cancer cell lines were assessed. Among them, compound 6a exhibited the strongest antiproliferative effects on Jurkat (IC50 = 1.80 μM), HCT-116 (IC50 = 11.50 μM) and MDA-MB-231 (IC50 = 53.91 μM). In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO2 on Jurkat and HCT-116 cell lines. The results showed that a proper long aliphatic chain enhanced the antiproliferative activity of scutellarein.


Introduction
Cancer is the most threatening and difficult-to-treat disease. Traditional Chinese herbal medicines have been applied for the treatment of cancers for many years [1]. Compounds isolated from traditional Chinese herbs exhibit pharmacological activities [2] and have been recognized as valuable resources for the development of novel anti-cancer drugs [3]. Scutellarin (1, Figure 1) is the major active flavonoid extracted from the traditional Chinese herbal medicine Erigeron breviscapus (Vant.) Hand-Mazz., which is used clinically in cardiovascular diseases [4]. Recently, accumulating evidence has highlighted the potential role of scutellarin and its main metabolite scutellarein (2, Figure 1) [5] in the treatment of cancer [6][7][8]. Nevertheless, low stability and poor lipophilicity of scutellarin and scutellarein limited their further application [9]. Derivatives have been synthesized to overcome these disadvantages and increase biological activity [10][11][12]. We report herein the synthesis of a series of scutellarein derivatives with a long aliphatic chain and their evaluation as anti-cancer agents.

Results and Discussion
The methodologies for preparing the target scutellarein derivatives are outlined in Scheme 1. Scutellarin (1) was reacted with acetic anhydride and catalytic 4-N,N-dimethylaminopyridine (DMAP) in pyridine under reflux to afford 5,6,7,4′-O-tetraacetyl scutellarein (3) in 54% yield and 6,7,4′-O-triacetyl scutellarein (4) in 23% yield. 6,7,4′-O-triacetyl scutellarein (4) is assigned its structure based on 1 H-NMR analysis. Following literature protocols [13,14], the assignment of the single free phenol hydroxyl group utilized the narrow peak shape and high chemical shift (12.90 ppm, CDCl3) of the hydroxyl proton. The single free phenol hydroxyl group is at the C-5 position, and can form a hydrogen bond with the carbonyl group at C-4 position [15]. 3 and 4 are converted to 7-O-aliphatic-5,6,4′-O-triacetylscutellarein (5) and 7-O-aliphatic-6,4′-Odiacetylscutellarein (6) by reacting with RBr using K2CO3 in N,N-dimethylformamide (DMF). The position of aliphatic chain connected with scutellarein was clarified by using Hetero-nuclear Multiple-Bond Connectivity (HMBC). The triplet at 4.08 ppm in the spectrum of 5d ( Figure 2) belongs to the OCH2 protons of the aliphatic chain. A cross peak is observed at 155.68 ppm, and belongs to C-7. Similarly, the triplet at 4.06 ppm in the spectrum of 6a (Figure 3) belongs to the OCH2 protons of the aliphatic chain. A cross peak is observed at 157.30 ppm, and belongs to C-7. The OCH2 protons of the aliphatic chain were correlated with C-7 as shown in Figure 4 indicated that the aliphatic chain is bonded to the C-7 position [16].
The biological activities of 7-O-aliphatic scutellarein derivatives 5a-d and 6a-d in inhibiting cancer cell line proliferation were assayed in vitro using CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) according to the previously reported method [17]. Anticancer drug sodium arsenite (NaAsO2) [18,19] was used as a positive control. Unexpectedly, most of the compounds were not able to inhibit the cancer cell proliferation at concentrations ≤100 μM, as shown in Table 1. However, the compounds 5a, 5b, 6a and 6b displayed anti-proliferation activity at concentrations ≤100 μM, and their activities were higher than both scutellarin and scutellarein. In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO2 on Jurkat and HCT-116 cell lines. The results showed that the aliphatic chain enhanced the anti-proliferation activity. However, the anti-proliferation activity is not stronger when the aliphatic chain is longer. A proper long aliphatic chain is beneficial to enhancing the anti-proliferative activity of scutellarein. 6a exhibited the strongest antiproliferative effects on Jurkat (IC50 = 1.80 μM),

Results and Discussion
The methodologies for preparing the target scutellarein derivatives are outlined in Scheme 1. Scutellarin (1) was reacted with acetic anhydride and catalytic 4-N,N-dimethylaminopyridine (DMAP) in pyridine under reflux to afford 5,6,7,4′-O-tetraacetyl scutellarein (3) in 54% yield and 6,7,4′-O-triacetyl scutellarein (4) in 23% yield. 6,7,4′-O-triacetyl scutellarein (4) is assigned its structure based on 1 H-NMR analysis. Following literature protocols [13,14], the assignment of the single free phenol hydroxyl group utilized the narrow peak shape and high chemical shift (12.90 ppm, CDCl3) of the hydroxyl proton. The single free phenol hydroxyl group is at the C-5 position, and can form a hydrogen bond with the carbonyl group at C-4 position [15]. 3 and 4 are converted to 7-O-aliphatic-5,6,4′-O-triacetylscutellarein (5) and 7-O-aliphatic-6,4′-Odiacetylscutellarein (6) by reacting with RBr using K2CO3 in N,N-dimethylformamide (DMF). The position of aliphatic chain connected with scutellarein was clarified by using Hetero-nuclear Multiple-Bond Connectivity (HMBC). The triplet at 4.08 ppm in the spectrum of 5d ( Figure 2) belongs to the OCH2 protons of the aliphatic chain. A cross peak is observed at 155.68 ppm, and belongs to C-7. Similarly, the triplet at 4.06 ppm in the spectrum of 6a ( Figure 3) belongs to the OCH2 protons of the aliphatic chain. A cross peak is observed at 157.30 ppm, and belongs to C-7. The OCH2 protons of the aliphatic chain were correlated with C-7 as shown in Figure 4 indicated that the aliphatic chain is bonded to the C-7 position [16].
The biological activities of 7-O-aliphatic scutellarein derivatives 5a-d and 6a-d in inhibiting cancer cell line proliferation were assayed in vitro using CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) according to the previously reported method [17]. Anticancer drug sodium arsenite (NaAsO2) [18,19] was used as a positive control. Unexpectedly, most of the compounds were not able to inhibit the cancer cell proliferation at concentrations ≤100 μM, as shown in Table 1. However, the compounds 5a, 5b, 6a and 6b displayed anti-proliferation activity at concentrations ≤100 μM, and their activities were higher than both scutellarin and scutellarein. In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO2 on Jurkat and HCT-116 cell lines. The results showed that the aliphatic chain enhanced the anti-proliferation activity. However, the anti-proliferation activity is not stronger when the aliphatic chain is longer. A proper long aliphatic chain is beneficial to enhancing the anti-proliferative activity of scutellarein. 6a exhibited the strongest antiproliferative effects on Jurkat (IC50 = 1.80 μM), The position of aliphatic chain connected with scutellarein was clarified by using Hetero-nuclear Multiple-Bond Connectivity (HMBC). The triplet at 4.08 ppm in the spectrum of 5d ( Figure 2) belongs to the OCH 2 protons of the aliphatic chain. A cross peak is observed at 155.68 ppm, and belongs to C-7. Similarly, the triplet at 4.06 ppm in the spectrum of 6a (Figure 3) belongs to the OCH 2 protons of the aliphatic chain. A cross peak is observed at 157.30 ppm, and belongs to C-7. The OCH 2 protons of the aliphatic chain were correlated with C-7 as shown in Figure 4 indicated that the aliphatic chain is bonded to the C-7 position [16].
The biological activities of 7-O-aliphatic scutellarein derivatives 5a-d and 6a-d in inhibiting cancer cell line proliferation were assayed in vitro using CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (MTS) according to the previously reported method [17]. Anticancer drug sodium arsenite (NaAsO 2 ) [18,19] was used as a positive control. Unexpectedly, most of the compounds were not able to inhibit the cancer cell proliferation at concentrations ≤100 µM, as shown in Table 1. However, the compounds 5a, 5b, 6a and 6b displayed anti-proliferation activity at concentrations ≤100 µM, and their activities were higher than both scutellarin and scutellarein. In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO 2 on Jurkat and HCT-116 cell lines. The results showed that the aliphatic chain enhanced the anti-proliferation activity. However, the anti-proliferation activity is not stronger when the aliphatic chain is longer. A proper long aliphatic chain is beneficial to enhancing the anti-proliferative activity of scutellarein. 6a exhibited the strongest antiproliferative effects on Jurkat (IC 50 = 1.80 µM), HCT-116 (IC 50 = 11.50 µM) and MDA-MB-231 (IC 50 = 53.91 µM). In addition, 6b with a free phenol hydroxyl group at C-5 position exhibited stronger anti-proliferative effects than 5b. These results indicate that the hydroxyl group at C-5 of scutellarein derivatives may be important anti-proliferative activity. HCT-116 (IC50 = 11.50 μM) and MDA-MB-231 (IC50 = 53.91 μM). In addition, 6b with a free phenol hydroxyl group at C-5 position exhibited stronger anti-proliferative effects than 5b. These results indicate that the hydroxyl group at C-5 of scutellarein derivatives may be important anti-proliferative activity.   HCT-116 (IC50 = 11.50 μM) and MDA-MB-231 (IC50 = 53.91 μM). In addition, 6b with a free phenol hydroxyl group at C-5 position exhibited stronger anti-proliferative effects than 5b. These results indicate that the hydroxyl group at C-5 of scutellarein derivatives may be important anti-proliferative activity.

Materials and Methods
Reagents and solvents were obtained from commercial suppliers and used without further purification unless stated otherwise. Pyridine was dried by boiling with CaH2 prior to distillation and stored over KOH, DMF (N,N-dimethylformamide) was stored over 4 Å molecular sieves. Reactions were monitored by thin-layer chromatography (TLC), and performed on 0.2 mm silica gel GF254 plates using UV light as the visualizing agent. Chromatographic purification of products was accomplished using forced-flow chromatography on 300-400 mesh silica gel. The 1 H-NMR (400 MHz, CDCl3) and 13 C-NMR (100 MHz, CDCl3) spectra were measured on a Bruker AV 400 MHz (Bruker, Karlsruhe, Germany) in CDCl3 as solvent, using (TMS) as an internal standard, and chemical shifts are expressed as δ ppm. (3) and 6,7,4′-O-triacetyl scutellarein (4) Scutellarin (10 g, 21.6 mmol) was dissolved into the Ac2O/Pyridine(50 mL, 3:2, v/v) solution and reacted reflux for 4 h. After the reaction was completed (monitored by TLC), the solvent was removed, and the resulting residue was then purified by column chromatography on silica gel using 50% ethyl acetate in petroleum ether as eluent to afford 3 (5.3 g, 11.7 mmol, 54%) and 4 (2.1 g, 5.1 mmol, 23%).