β-Elemene derivatives produced from SeO2-mediated oxidation reaction

Herein, we report the first access of β-elemene derivatives through the SeO2-mediated oxidation reaction. Several new compounds were isolated through such a one-step reaction, and their structures were elucidated using various 2D-NMR techniques. This method provides easy access to multiple oxidative β-elemene derivatives in one single step and represents the first modifications on cyclohexyl ring of β-elemene. It is expected to open up the opportunity for future derivatization on cyclohexyl ring of β-elemene. The new compounds obtained above showed better anti-proliferation activities than β-elemene itself on several cancer cell lines. Among them, compound 17 shows the best activity in antiproliferation assays of A549 and U-87MG cell lines.


Introduction
The plant Curcuma wenyujin Y. H. Chen et C. Ling belongs to one of the important traditional Chinese medicines (TCM), and has been used to treat cancer and various diseases for nearly a thousand years [1][2][3][4]. The essential oil obtained from this plant is called elemene extracts, which contain at least four sesquiterpene isomers, namely α-elemene, β-elemene (1), practically more hindered Figure 1. (a) The structure of β-elemene with carbon atoms numbering; (b) the ground-state chair conformation of β-elemene [18]. The two hydrogen atoms on C-2 and C-4 are theoretically accessible to SeO 2 -mediated allylic oxidation besides C-13 and C- 14. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 7: 200038

Results
β-Elemene raw material is the gift from Holley Kingkong Pharmaceutical Co., Ltd. GC-MS analysis 1 suggests that it contains only about 78% of β-isomer, plus the other three isomers. Since the four elemene isomers possess very similar structure and physical properties, to purify them in the laboratory represents a challenge. Therefore, the material was used as-is. Understandably, the presence of other isomers will generally give lower yield as well as complicate the isolation process. β-Elemene raw material was subjected to a SeO 2 -mediated oxidation reaction in CH 2 Cl 2 , with 5 equivalent of TBHP at 0 o C for 6 h. After standard work-up process, the crude product was purified in silica gel chromatography ( petroleum ether (PE)/ethyl acetate (EA)) to yield four fractions, with polarity from the least to the most: fraction I (R f = 0.9, PE/EA = 4:1, 7.4% yield), fraction II (R f = 0.7, PE/EA = 4:1, 2.8% yield), fraction III (R f = 0.2, PE/EA = 4:1, 8.6% yield) and fraction IV (R f = 0.15, PE/ EA = 4:1, 21.6% yield) (Scheme 1).
The above four fractions were analysed by HPLC. The results revealed that only fraction IV contains a single compound, the other three fractions were all mixtures of two compounds.
Fraction I appears to be a single spot in TLC (petroleum-ethyl acetate system). After screening with several mix solvent systems for TLC, petroleum ether/acetone system was found to be the best solvent to resolve the two compounds (Scheme 2). The structure of compound 2 was established by NMR in comparison with references [35]. The structure of compound 3 was elucidated through various 2D NMR techniques (see electronic supplementary material for details). It should be noted that compound 2 was synthesized in the literature involving three steps and a tedious HPLC purification process [35].
The 1 H NMR of fraction II reveals two sets of signals in about 1 to 1 ratio, containing both aldehyde proton and allylic protons connecting to a hydroxyl group, presumably from compounds 6 and 7. Attempt to resolve them in TLC using a variety of mix solvent systems (similar to fraction I) was proven to be unsuccessful. We then turned to protecting group strategy. Thus, 50 mg of fraction II was treated with TBDMS-Cl/imidazole to install TBDMS group on hydroxyl groups, resulting in two compounds 4 and 5, which were carefully separated in silica gel chromatography. The isolation yield OH OH ref. [37] 3-step total yield: 12% ref. [24,35] 3-step total yield: 6-8% Figure 2. Summary of the allylic oxidation of β-elemene. 1 GC-MS analysis was performed in Agilent Technologies 7890B (GC system) and 5977A MSD (Mass unit) under the following conditions: Agilent gas chromatograph and gas work station, FID detector, capillary chromatography (Agilent 19091S-433UI, HP-5 ms Ultra Inert, 60-325°C, 30 m × 250 mm × 0.25 mm); injection temperature: 250°C, detector temperature: 230°C, rise range: starting temperature: 50°C, maintain for 2 min, rise to 80°C at the rate of 20°C per minute, maintained for 2 min, then increased to 150°C at the rate of 30°C per minute, maintained for 5 min; carrier gas: helium, flow rate: 24.2 ml min −1 , chromatographic column flow rate: 1.2 ml min −1 , pressure: 9.8 psi, tail gas flow rate: 3 ml min −1 , injection volume: 1 ml, split ratio: 100 : 1. The retention time of β-elemene is 9.05 min under the above conditions. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 7: 200038 is low because these two compounds are close to each other and the majority material stays as a mixture. If necessary, the mixture can be subjected to chromatography repeatedly to give a better yield of pure 4 and 5. After standard deprotection of tert-butyldimethylsilyl group, compounds 6 and 7 were obtained respectively. The structures of 6 and 7 were established by NMR. Alternatively, compounds 6 and 7 might be separated using a suitable column in the preparative HPLC system (Scheme 3).
The 1 H NMR of fraction III did not show the aldehyde proton signal. Therefore, the mixture presumably contains allylic alcohols. After screening a set of mixed solvent systems for TLC, dichloromethane/acetone (1:1 v/v) appears to give the best resolution. Thus compounds 8 and 9 were obtained in about 3 to 2 ratio, and their structures were established via various 2D NMR techniques (Scheme 4) [36].
The analysis by HPLC and 1 H NMR of fraction IV indicates it is a single compound, whose structure was established as compound 10 by comparing its 1 H NMR with known compound 13,14-bis(hydroxyl)β-elemene [9,38,39]. Compound 10 is a known compound reported in the literature. It required a threestep synthesis using the literature method (reaction sequence: bis-allylic chlorination, nucleophilic displacement with OAc − and hydrolysis of ester), with a total yield of 19% [38]. Our method provides alternative access to this key intermediate in a single step.
To further expand the usefulness of SeO 2 -mediated allylic oxidation reaction in β-elemene analogue synthesis, compound 12 2 was subjected to a milder condition to see if we can obtain a higher yield and better regioselectivity of the oxidation product. To our delight, with less catalyst and oxidant (0.4 equivalent of SeO 2 and 0.8 equivalence of TBHP), the selective installation of the hydroxyl group was achieved in moderate to good yield. Compound 13 can serve as a key intermediate for further functionalize the C-13 and C-14 position of β-elemene (Scheme 5).
Huang reported that β-elemenal (11) (figure 3) showed better anti-proliferation activity against several tumour cell lines than β-elemene (1) itself [13]. Apparently the aldehyde functional group contributes to the biological activity. Thus, compounds 2, 8, 9 and 10 were converted to the  β-Elemene was subjected to allylic chlorination in NaOCl/acetic acid to obtain a mixture of compound 11 and its regioisomer (i.e. Clgroup in 14-position of β-elemene). After repeated chromatography in preparative HPLC, compound 11 was obtained in pure form.
All new compounds and β-elemene were subjected to cell proliferation inhibition assay against two tumour cell lines: A549 and U-87MG. The results will be discussed below. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 7: 200038 The β-elemene derivatives obtained above were assessed in anti-proliferation assay against A549 and U-87MG cell lines. The activity of some representative compounds is shown in table 1. Compounds 6, 7, 11, 15 and 17 showed improved inhibitory activities than β-elemene. It is interesting to note that all the compounds showing better biological activities possess an aldehyde functional group. The reason for such phenomenon is currently being investigated in our laboratory.

Methods
The suspension of specific tumour cells was adjusted to 5 × 10 4 ml −1 or 2 × 10 4 ml −1 with DMEM + 2 mm glutamine + 10% FBS medium. Add 100 µl cell suspension to 96-well cell culture plate, and the final cell concentration is 5000 cells well −1 (72 h). DMSO was used to dissolve the compound to be tested as 100 mM storage solution. The final concentration of 200× compound was prepared with storage solution and DMSO, and the gradient dilutions of 3× series were prepared, and then diluted 20 times with culture medium respectively. Finally, 10 µl corresponding 10-fold solution was added to each cell hole, and each drug concentration was in duplicate holes. The final concentrations of each compound were 300 µM, 100 µM, 33.33 µM, 11.11 µM, 3.704 µM, 1.235 µM and 0.412 µM, and the final concentration of DMSO per pore was 0.5%. Incubate in a 37°C, 5% CO 2 incubator for 72 h. After 72 h of drug treatment, add 100 µl celltiter glo detection reagent into each hole according to the CTG operation instructions, melt and balance the CTG solution to room temperature in advance, mix it with microporous plate shaker for 2 min, place it at room temperature for 10 min, and the luminescence is recorded with a luminometer. The cell survival rate was calculated by the formula: (V sample -V blank )/(V vehicle control -V blank ) × 100%. V sample is the reading of the drug treatment group, V vehicle control is the average of the solvent control group, and V blank is the average of the blank control hole. By using graphpad prism 5.0 software, a nonlinear regression model was used to draw the Stype dose survival curve and calculate the IC 50 value.

Conclusion
SeO 2 -mediated allylic oxidation reaction was first applied to β-elemene, a substrate bearing three carboncarbon double bonds and several allylic hydrogen atoms, and was discovered to produce seven derivatives of β-elemene in a single step. Several additional analogues of β-elemene were further synthesized and found to display better inhibitory activities against A549 and U-87 cell proliferation. All compounds in this article can serve as key intermediates for further derivatization of β-elemene. Our approaches represent the first success to introduce functional groups onto the cyclohexyl ring, a challenging task unexplored before.