NBS promoted cascade reaction of β-anhydroicaritine and alcohols

ABSTRACT An unusual NBS promoted cascade reaction of β-anhydroicaritine and alcohols was successfully developed to prepare chromane-3,4-dione 3 in a moderate yield. A mechanism was also put forward to explain this reaction. These chromane-3,4-dione derivatives showed a potent cytotoxicity against HepG2 and MCF-7 cell lines, which were expected to be a potential lead candidate for the treatment of liver cancer and breast cancer. GRAPHICAL ABSTRACT


Introduction
Icaritin (Figure 1) is one of flavonoids with many pharmacological and biological activities, such as neuroprotective effects (1), osteogenic differentiation promoting and osteoclast differentiation inhibiting activities (2), and a Growth inhibition of cancer PC-3 cells (3), induction of apoptosis in human hepatoma HepG2 cells (4), and multidrug resistance reversal activity (5).In particular, icaritin exhibits estrogenic activity and stimulates the proliferation of ER-positive breast cancer MCF-7 cells at submicromolar concentrations (6).These characteristics make icaritin a promising candidate for SERMs and an anticancer agent against human breast cancer.And many medicinal chemists have also paid much attention to its structure and activity relationship (SAR).
Halogenation is one of the classic organic chemical reactions (7)(8)(9).Halogen groups such as F, Cl, Br, etc., as a good pharmacophore, can not only effectively change the lipophilicity of the compound, but also increase the activity and improve the efficacy.Studies have found that many anticancer drugs have been shown to have significant anticancer properties by introducing bromine-containing groups (10)(11)(12)(13).
Therefore, we intend to introduce a halogen group at the C-6 position of icaritin by means of bromination reaction to improve the lipophilicity of icaritin.Herein we described our latest research findings.

Chemistry
After perusal of the literature, there is few literature to report on the introduction of halogen atoms into icaritin or icaritin derivatives, probably because these special flavonoid molecules have more halogenation reaction sites, such as site a, b and c.How to selectively introduce halogen group at the reaction site a is much more difficult (Figure 2).
To avoid addition to the double bond and to simplify the reaction, we chose β-anhydroicaritine instead of icaritin as our substrate.Even so, things are still difficult than we thought.A number of different bromination conditions were tried, all of which gave a decomposed product (containing p-methoxy phenyl moiety), but not the desired product (Table 1).
Occasionally, methanol was used as the solvent for this reaction.To our surprise, under the condition of NBS (1 equiv.)and methanol (2 ml), an unexpected novel chromane-3, 4-dione compound 3 was formed and separated by chromatography in 26% chemical yield.Thus, this reaction was further optimized by different reaction conditions, including different molar ratios, temperatures and times.All of the reactions were quenched when the starting material was disappeared.The yield was calculated as pure compound after purification by chromatography.The results are summarized in Table 2.
This intriguing finding drove us to expand different alcohol substrates.Thus, treatment of a series of alcohols with β-anhydroicaritine under NBS conditions afforded the designed products 3a-h in moderate yields, respectively.Yields ranged from 35% to 67%.All data are summarized in Table 3.
This particular reaction result prompted us to conduct extensive research on its mechanism.Considering the existence of conjugated double bond, a possible reaction mechanism was proposed to explain the formation of the product (Scheme 1).
An important step in this mechanism is the formation of the three-membered ring bromonium ion (first step).The bromonium ion intermediate is highly active and is subject to the attack of nucleophilic reagent RO -to form α-bromoalkoxy compound.In the following, dehydrohalogenation (-HBr) will afford the new chromane-3,4dione compounds in good yields.
There is one more question that bothers us.Which reaction process occurs first between the aromatic bromination reaction and the alcohol-addition reaction?To test this, NCS was used for the chlorination of aromatic rings.However, no reaction occurred even under the same conditions as NBS (Scheme 2).On the one hand, this experiment proves that the process of alcohol-addition reaction occurs before the bromination of the benzene ring; on the other hand, it also indirectly supports our mechanistic protocol that the formation of bromonium ion is a key step in the cascade reaction.

Materials
Melting points were determined in capillary and were uncorrected. 1H and 13 C NMR were recorded on a Varian Unity INOVA 400 MHz spectrometer using TMS as an internal standard.MS spectrum (TOF) was measured on an Agilent 1100 Series VS (ES, 4000 V) Mass spectra.All reagents and solvents are reagent grade and were used directly without further purification.To a round flask was added β-anhydroicaritin (50 mg, 0.135 mmol) and methanol (2 mL) under nitrogen atmosphere.The mixture was then cooled to 0°C, and NBS (48 mg, 2 equiv.)was added in portion.The reaction mixture was then warmed to room temperature and stirred for 4 h.After the consumption of the starting material (TLC detected), the solvent was evaporated completely under reduced pressure.DCM (10 mL) and water (2 mL) was added to dilute the mixture.The organic phase was separated and dried over MgSO 4 .The residue was purified by chromatography through a silica gel column using PE: EA = 6: 1 as eluent to afford the title compound (3a, 42 mg, 65%) as a pale yellow solid.M.p.149.5-150.2°C,R f = 0.34 (PE: EA = 2: 1). 1
2. Cell viability was evaluated by the MTT assay.

Table 2 .
Optimization for the NBS promoted cascade reaction.

Table 3 .
NBS promoted cascade reaction of β-anhydroicaritine with alcohols.Proposed mechanism for cascade reaction of β-anhydroicaritine with alcohols.