Asymmetric total synthesis of (1S,2S,4S)-β-elemene

Elemenes are sesquiterpene natural products extracted from Chinese medicinal herbs and have been used as an important antitumor drug in China. Here, we report the first stereoselective total synthesis of (1S,2S,4S)-β-elemene using (R)-carvone as a chiral pool starting material. The isopropenyl moiety was achieved in a highly stereoselective manner through 1,4-Michael conjugate addition. The following transformations like regio- and stereoselective aldol condensation, Wittig olefination have been employed as the key steps, resulting in a concise total synthesis of (1S,2S,4S)-β-elemene. Our accomplishment will allow further biological investigations of this natural product and open opportunities for developing a new potentially promising antitumor drug.


Introduction
Traditional Chinese medicine (TCM) has been used to prevent and treat diseases for thousands of years, and now plays a more and more important role in the small molecule drug discovery. 1 One of the most signicant and recent developments is the use of elemenes in the treatment of various malignant tumors. 2 Elemenes are a family of sesquiterpenes extracted from the Chinese medicinal herb Curcumae Rhizoma, and are classied into a-elemene (A), b-elemene (B), g-elemene (C), d-elemene (D) according to the position of the double bond (Fig. 1). 3 Owing to their extensive and potent in vitro and in vivo antitumor activities, elemene oral emulsion and elemene injection have been approved by the China National Medical Products Administration (NMPA) as noncytotoxic broad-spectrum antitumor drugs, 2a,e and are currently under consideration for clinical studies in the United States. 4 (1S,2S,4R)-b-Elemene (1, Fig. 1) is the predominant active ingredient of clinically administrated elemene antitumor drugs, with a recommended content of not less than 85%. 2e Previous studies have indicated that 1 not only inhibits the growth of tumor but also enhances the activity of immune system, 5 and even can reverse multiple-drug resistance, 6 making it unique to the conventional chemotherapeutic drugs. Interestingly, (1R,2R,4S)-b-elemene (2), the enantiomer of 1, was isolated from lower plants or microorganisms, such as the gorgonian Eunicea mammosa, 7 while 1 is commonly widespread in higher plants.
Very recently, a new isomer of b-elemene, namely (1S,2S,4S)-belemene (3), was isolated and characterized by Shi and coworkers from the essential oil of Curcuma wenyujin. 8 As compared to 1, 3 was found to possess higher antitumor activities for all cancer cell lines tested and lower toxicity in mice, 8 demonstrating its potential importance in the development of a new promising anticancer agent. However, the exceptionally low content of 3 in the Chinese medicinal herb greatly restricted its systematic pharmacological evaluation. Therefore, development of an efficient and practical approach to synthesize 3 using readily available reagents under mild reaction conditions is highly desirable.
The intriguing pharmacological properties and structural diversities of b-elemene have attracted signicant attention from the synthetic community. 9 Indeed, several elegant asymmetric total syntheses of b-elemene have been described in the literature or patents. 4a, [10][11][12] In 1967, Rao and co-workers described the rst asymmetric total synthesis of 1 in 9 steps using ozonolysis of a-santonin 4 as the key transformation (Scheme 1a). 10 In 1995, Corey and co-workers reported the rst asymmetric total synthesis of 2 in 7 steps via enantioselective Ireland-Claisen rearrangement of b,g-unsaturated ester from geraniol 6 (Scheme 1b). 11 To the best of our knowledge, the total synthesis of (1S,2S,4S)-b-elemene (3) has yet to be reported. Herein, we present the rst and asymmetric total synthesis of 3 in ve or eight steps from (R)-carvone 8, a commercially available chiral pool starting material (Scheme 1c).

Results and discussion
Intrigued by the fascinating pharmacological properties of (1S,2S,4S)-b-elemene (3), we embarked on the rst total synthesis of this sesquiterpene natural product. Our conceptual route to 3 is shown in Scheme 2. Retrosynthetically, 3 could be traced back to primary alcohol 9 via Wittig olenation and common oxidation reaction. We then envisioned that 9 could be generated from the chiral pool starting materials 8 by employing chemoselective and diastereoselective 1,4-Michael conjugate addition of Grignard reagent, aldol condensation and simple reduction of ketone.
Chiral pool synthesis represents a useful approach to synthesize enantiopure organic molecules from readily available enantiomerically pure compounds, which features low cost, high abundance, and general renewability. 13 Therefore, our synthesis began with the preparation of optically pure intermediate 12 from the chiral pool building block (R)-carvone 8 ($0.39 per g, Merck), as shown in Scheme 3. Stimulated by Knölker's pioneering work, 14a it was anticipated that the new generated stereogenic center in cyclohexanone 11 could be secured by diastereoselective 1,4-Michael addition of isopropenyl Grignard reagent 10 to 8. 14b To our delight, we were able to isolate desired product 11 exclusively in 70% yield. Notably, the steric and electronic inuence in the diastereoselective transformations of chiral enolates have been systematically studied by Krause's group. 15 Treatment of 11 with formaldehyde in the presence of KOH afforded primary alcohol 12 as a single diastereomer in 50% isolated yield for three cycles. 14b, 16 Subsequently, oxidation of 12 with Dess-Martin periodinane (DMP) 17 gave aldehyde 13 in 70% yield. In an attempt to obtain advanced intermediate 14 via Wittig olenation, several reaction conditions (e.g., PPh 3 PCH 2 Br/n-BuLi, TMSCH 2 MgBr) 18 were screened, but none afforded the desired product. Instead, the undesired retro-aldol reaction would occur preferentially to yield 11 in quantitative yield.
To avoid this undesired side reaction, we next turned our attention to the protection of alcohol 12 at an early stage (Scheme 4). First, compound 12 was treated with Ac 2 O 19 in the presence of triethylamine (TEA) in DCM afforded 15 in 90% yield. Subsequent treatment of 15 with sodium borohydride (NaBH 4 ) 20 in MeOH gave alcohol 16 in 90% yield (d.r. ¼ 3 : 2). As planned ahead, removal of the hydroxyl group in 16 using Barton-McCombie radical deoxygenation 21 and a subsequent KOH (dissolved in MeOH solution) workup delivered desired product 9 in 50% yield for two steps. Next, the oxidation of primary hydroxyl group in 9 using pyridinium chlorochromate (PCC) 22 in DCM afforded desired aldehyde 17 in 75% yield. With 17 in hand, we continued toward the nal stage of the total synthesis of (1S,2S,4S)-b-elemene (3) via Wittig olenation. Gratifyingly, treatment of 17 with PPh 3 PCH 2 Br and n-BuLi in THF, 18 gave 3 in 82% yield, completing the rst and asymmetric total synthesis of 3. The 1 H and 13 C NMR spectra of synthetic 3 and its optical rotation (synthetic, [a] 20 D ¼ +24.5 (c 1.0, EtOH); Scheme 1 Asymmetric total syntheses of b-elemene 1-3.
Scheme 3 Attempts to synthesize advanced intermediate 14.
natural, [a] 20 D ¼ +36.2 (c 1.4, EtOH)) were in good agreement with those of the isolated natural product. 8 Taken together, an eight-step asymmetric total synthesis of 3 was accomplished in 8.7% overall yield from readily available starting material 8. Moreover, the structure and absolute conguration of naturally occurring 3 were unambiguously conrmed by our asymmetric total synthesis.
Although (1S,2S,4S)-b-elemene (3) has been accomplished in a linear sequence of eight steps from 8, however, the yield of this protocol was relatively low (8.7% overall yield), and the use of stannyl reagent would put off its acceptability in the pharmaceutical industry. Consequently, we next sought to develop a more practical route to access 3, which would be more attractive for pharmaceutical manufacturing.
With this target in mind, our new protocol for the total synthesis of (1S,2S,4S)-b-elemene (3) is depicted in Scheme 5. In an attempt to the reduction of ketone 12 under standard conditions of Wolff-Kishner-Huang reduction (NH 2 NH 2 , KOH), 23 decomposition of starting material was observed at the high temperature. Inspired by the elegant work of Kim's group, 24 a zinc-modied cyanoborohydride (TsNHNH 2 /ZnCl 2 / NaBH 3 CN) was developed to be a versatile reducing agent under milder reaction conditions. Pleasingly, the desired product 9 could in fact be achieved, but along with another over-reduced byproduct 18 (9 : 18 ¼ 1 : 3). We attributed this result to the possible coordination of hydroxyl group and neighbouring double bond with the electrophilic zinc chloride. It was noticed that separation of 9 and 18 by column purication was proved to be difficult. Therefore, we continued to optimize this reaction. Encouraged by Kappe's remarkable work, 25 by using zinc powder (particle size < 45 mm) in acetic acid/hydrochloric acid, ketone 12 was successfully reduced to desired product 9, which was subsequently subjected to DMP oxidation without further chromatographic purications, affording aldehyde 17 in 54% yield for two steps. Finally, the Wittig olenation was employed to provide the targeted (1S,2S,4S)-b-elemene (3) in 82% yield, completing a ve-step asymmetric total synthesis of 3 in 15.5% overall yield. We believed that such a process would be commercially relevant and potentially more practical for pharmaceutical manufacturing.

Conclusions
In summary, the rst total synthesis of (1S,2S,4S)-b-elemene (3) was achieved in ve steps (15.5% overall yield) or eight steps (8.7% overall yield) from the chiral pool starting material 8. The stereocenters in 3 were constructed in simple and diastereoselective manners based on the chiral pool strategy. Notably, the transformations involved in our current synthetic routes, including 1,4-Michael conjugate addition, aldol condensation and Wittig olenation are well-developed and viable. Moreover, this work will allow the diverse syntheses of other b-elemene and their analogs to enable further biological research, which is underway and will be reported in due course.

General methods
Reagents were purchased at the highest commercial quality (>95%) and used without further purication, unless otherwise stated. Anhydrous tetrahydrofuran (THF), toluene (PhMe) were distilled from sodium-benzophenone, dichloromethane (DCM), methanol (MeOH) were distilled from calcium hydride. All reactions were carried out with magnetic stirring, and if moisture or air sensitive, under an argon atmosphere with dry solvents. External bath temperatures were used to record all reaction temperatures. Low temperature reactions were carried out in a Dewar vessel lled with acetone/dry ice (À78 C) or distilled water/ice (0 C). Reactions were monitored by thinlayer chromatography (TLC) carried out on 0.25 mm Tsingdao silica gel glass-backed plates (60F-254) and visualized under UV light at 254 nm. Staining was performed with an ethanolic solution of phosphomolybdic acid and heat as developing agents. Tsingdao silica gel (60, particle size 0.040-0.063 mm) was used for ash column chromatography. Yields refer to chromatographically, unless otherwise specied. NMR spectra were recorded on Brüker Advance 500 ( 1 H: 500 MHz, 13 C 125 MHz) and Brüker Advance 400 ( 1 H: 400 MHz, 13 C 100 MHz). The spectra were reported as followed: chemical shi d in ppm (multiplicity, coupling constant J in Hz, number of protons) for 1 H NMR spectra and chemical shi d in ppm for 13 C NMR spectra. The following abbreviations were used to explain the multiplicities: s ¼ singlet, d ¼ doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet, or combinations thereof. Residual solvent peaks of CDCl 3 (d H ¼ 7.26 ppm, d C ¼ 77.16 ppm) were used as an internal reference, unless otherwise stated. High resolution mass spectrometric (HRMS) data were recorded on a Brüker Apex IV RTMS instrument and a VG Auto Spec-3000 spectrometer, respectively. Optical rotation values were measured with a Jasco P-1020 polarimeter. The specic rotation was calculated as followed: [a] 20 D ¼ (a Â 100)/(c Â d). Synthesis of compound 11. Isopropenyl Grignard reagent 10 (1.0 M in THF, 133.0 mL, 133.0 mmol, 2.0 equiv.) was added dropwise to a suspension of copper(I) iodide (12.6 g, 66.2 mmol, 1.1 equiv.) in dry THF (300.0 mL) at À78 C and the resulting mixture was stirred for 1 h at the same temperature. A solution of (R)-carvone 8 (10 g, 66.67 mmol, 1.0 equiv.) in dry THF (50.0 mL) was added to the above solution at À78 C. The reaction was allowed to warm to room temperature and stirred for 3 h, and then quenched with a saturated solution of NH 4 Cl (100 mL). The resultant aqueous layer was extracted with ethyl acetate (3 Â 150 mL), and the combined organic layers were dried over Na 2 SO 4 . The extracts were ltered off and then evaporated under vacuum. The residue was puried by ash chromatography on silica gel (EtOAc/hexanes ¼ 1 : 20 / 1 : 15) to give compound 11 (8.9 g) as a pale yellow oil in 70% yield. Synthesis of compound 12. KOH (4.6 g, 82.1 mmol, 2.0 equiv.) was dissolved in MeOH (200 mL) and a solution of 11 in MeOH (10.0 mL) was slowly added at 0 C. The resulting mixture was stirred for 30 min and a solution of formalin (39% in H 2 O, 9.4 mL, 125.1 mmol, 3.0 equiv.) was added in a dropwise manner. The reaction mixture was stirred for 1.5 h at the same temperature and then quenched with a saturated solution of NH 4 Cl (30.0 mL). Aer removal of methanol under vacuum, the mixture was then extracted with CH 2 Cl 2 (3 Â 50.0 mL), and the combined extracts were dried over Na 2 SO 4 . The extracts were ltered off and then evaporated under vacuum. The residue was puried by ash chromatography on silica gel (EtOAc/hexanes ¼ 1 : 20 / 1 : 10) to give compound 12 (4.6 g) as a pale yellow oil in 50% cumulative yield (the recovery of starting material 11 was ran for three cycles Synthesis of compound 13. To a solution of 12 (500 mg, 2.25 mmol, 1.0 equiv.) in dry DCM (20.0 mL) was added DMP (1.15 g, 2.7 mmol, 1.2 equiv.) at 0 C. The reaction was allowed to warm to room temperature and stirred for 1 h, then quenched with sat. aq. Na 2 SO 3 (10.0 mL). The resultant aqueous layer was extracted with CH 2 Cl 2 (3 Â 20.0 mL), and the combined organic layers were dried over Na 2 SO 4 . The extracts were ltered off and then evaporated under vacuum. The residue was puried by ash chromatography on silica gel (EtOAc/hexanes ¼ Synthesis of compound 15. To a stirred solution of 12 (4.6 g, 20.7 mmol, 1.0 equiv.) and TEA (8.6 mL, 62.2 mmol, 3.0 equiv.) in DCM (100 mL) was added acetic anhydride (4.0 mL, 41.4 mmol, 2.0 equiv.) at 0 C. The resulting mixture was allowed to warm to room temperature and stirred overnight. Then the resultant reaction mixture was diluted by sat. aq. NaHCO 3 (50.0 mL) and subsequently extracted with CH 2 Cl 2 (3 Â 50.0 mL). The combined organic layers were dried over Na 2 SO 4 , ltered, and concentrated under reduced pressure. The residue was puried using ash column chromatography on silica gel (EtOAc/hexanes ¼ 1 : 50 / 1 : 30) to give compounds 15 (4.9 g) as a colorless oil in 90% yield. 4.36 (d, J ¼ 11.4 Hz, 1H), 4.20 (d, J ¼ 10.9 Hz, 0.7H), 4.00 (d, J ¼ 10.9 Hz, 0.7H), 3.76 (dd, J ¼ 6.5, 3.9 Hz, 0.7H), 3.55 (dd, J ¼ 12.  145.48, 114.1, 114.0, 110.7, 109.5, 73.4, 69.0, 67.4, 67.3,  43.5, 42.5, 41.9, 41.3, 38.3, 38.1, 31.6, 30.96, 29.7, 28.6 Synthesis of compound 9. To a solution of compound 16 (1.5 g, 5.64 mmol, 1.0 equiv.) in DCM (30.0 mL) was added DMAP (1.37 g, 11.28 mmol, 2.0 equiv.), phenyl chlorothionocarbonate (1.94 g, 11.28 mmol, 2.0 equiv.) at room temperature. The resulting mixture was stirred overnight and ltered through a pad of silica gel (eluent : EtOAc). The organic solvent was removed under reduced pressure and the resultant residue was dissolved in toluene (20.0 mL). Then the reaction mixture was heated to reux and tributyltin hydride (3.30 g, 11.28 mmol, 2.0 equiv.) and AIBN (370 mg, 11.28 mmol, 0.2 equiv.) was added. Aer being stirring for 8 h, a solution of KOH (1.0 g) in MeOH (20.0 mL) was added and stirred for 2 h. The reaction mixture was diluted by water (20.0 mL) and the aqueous layer was extracted with ethyl acetate (3 Â 30.0 mL), and the combined organic layers were dried over Na 2 SO 4 . The extracts were ltered off and then evaporated under vacuum. The residue was puried by ash chromatography on silica gel (EtOAc/hexanes ¼ 1 : 50 / 1 : 20) to give compound 9 (500 mg) as a white solid in 50% yield for two steps. Synthesis of compound 17 (method B). To a solution of 12 (400 mg, 1.8 mmol, 1.0 equiv.) in ethanol (20.0 mL), glacial acetic acid (10.0 mL) and conc. hydrochloric acid (1.0 mL). The mixture was heated to 80 C and under stirring zinc powder (2.0 g, particle size < 45 mm) was added in small portion (each about 0.1 g). The overall time of the zinc addition is about 2 h. When the addition was nished, the reaction mixture was heated under reux for 10 min and ltered still hot from excess of zinc. The ltrate was concentrated and the resultant residue was dissolved in DCM (20.0 mL), then DMP (916 mg, 2.16 mmol, 1.2 equiv.) was added at 0 C. The reaction mixture was allowed to warm to room temperature and stirred for 1 h, then quenched with sat. aq. Na 2 SO 3 (10.0 mL). The resultant aqueous layer was extracted with CH 2 Cl 2 (3 Â 20.0 mL), and the combined organic layers were dried over Na 2 SO 4 . The extracts were ltered off and then evaporated under vacuum. The residue was puried by ash chromatography on silica gel (EtOAc/hexanes ¼ 1 : 80 / 1 : 50) to give compound 17 (200 mg) as a white solid in 54% yield for two steps.
Compound 18 was isolated for characterization purpose by taking an aliquot from the combined organic layers, followed by concentration under reduced pressure and the residue was puried by PTLC (silica gel, EtOAc/hexanes ¼ 1 : 10 Synthesis of compound (1S,2S,4S)-b-elemene (3). To a solution of methyltriphenylphosphonium bromide (571 mg, 1.60 mmol, 2.2 equiv.) in THF (10.0 mL) was added n-BuLi (50.0 mL of 2.5 M in hexanes, 125 mmol, 1.25 equiv.) dropwise over 5 min at À78 C. Aer 30 min stirring at this temperature, the reaction was allowed to warm to room temperature and keep stirring for another 20 min. Then the reaction was cooled to À78 C again and a solution of 17 (9.40 mL, 150 mmol, 1.50 equiv.) in dry THF (2.0 mL) was added. The mixture was allowed warm to room temperature again over 1 h and quenched with sat. aq. NH 4 Cl (5.0 mL). The resultant aqueous layer was extracted with ethyl acetate (3 Â 15.0 mL), and the combined organic layers were dried over Na 2 SO 4 . The extracts were ltered off and then evaporated under vacuum. The residue was puri-ed by ash chromatography on silica gel (100% hexanes) to give (1S,2S,4S)-b-elemene (121 mg) as a colorless liquid in 82% yield.

Conflicts of interest
There are no conicts to declare.