Synthesis of an ent-Halimanolide from ent-Halimic Acid

An efficient synthesis of ent-halimanolide 2 (15,16-epoxy-12-oxo-ent-halima-5(10),13(16),14-trien-18,2β-olide), from ent-halimic acid has been achieved, corroborating the structure of the natural compound and establishing its absolute configuration.


Introduction
Euphorbiaceae plants are a rich source of bioactive substances [1][2] and certain genera of this family have attracted much interest, since they contain a group of antitumor compounds [3]. Cladogynos orientalis Zipp. ex Span. (syn. Adenochlaena siamensis Ridl.) (Euphorbiaceae), known in Thailand as "Chettaphangki," is the only member of the genus Cladogynos and the roots are used as a carminative in Thai folk medicine. Chettaphanin I [4][5] and II [6][7], are the main components from their roots of this plant and the first to be known. Recently, in addition to chettaphanin I and II,

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isolation from the root extract of a series of furan diterpenes 2-4 with ent-halimane skeletons has been described [8]. Our group has transformed ent-halimic acid 1, a diterpene of known structure and absolute configuration, into chettaphanin I and II, which confirmed their structure and absolute configuration [4,6]. In this paper, we report the synthesis of ent-halimanolide 2, in order to confirm the structure of the natural compound and do SAR studies. ent-Halimanolide is a furan diterpene like chettaphanin I and II, but in this case the carboxylic acid at C-18 has formed a γ-butanolide with the hydroxyl at C-2.

Results and Discussion
In order to synthesise compound 2, from ent-halimic acid 1 it is necessary to functionalize C-2 and C-12, add a furan group in the side chain, isomerize the double bond to the more stable tetrasubstituted position and to form the lactone ring.
Two synthetic routes have been explored for the synthesis of 2: Route A and Route B, which differ in the strategy followed for the preparation of the γ-lactone, before or after of the introduction of the furan ring.
In Route A (Scheme 1) three fundamental parts can be differentiated: elaboration of the adequate ent-halimic acid tetranorderivative 9; γ-lactone formation as 12, necessary for the final introduction of the furan fragment and to prepare the functional groups required to achieve the natural product 2. The ent-halimic acid tetranorderivative 6 was obtained in excellent yield as described in the synthesis of chettaphanin I and II [4] [6]. Carbonyl protection of 6 with ethylene glycol in acid media gives the tetranorderivative 7, that already contains the tetrasubstituted double bond in the required position. The carbonyl deprotection should be done very carefully (controlling the acid and time) and in this manner ketone 8 can be obtained, which by NaBH 4 reduction gives a 1:1 mixture of hydroxyderivative 9 and γ-lactone 10.
The required aldehyde 12 was obtained by hydrolysis of 10 followed by TPAP [9] oxidation of the hydroxy derivative 11. Addition of 3-furyl lithium [10][11][12] to 12 gives a mixture of the hydroxy derivatives 13 and 14. The C-12 configuration in 13 and 14 was established by comparation of their physical properties with the ones of similar compounds [13][14] Starting from 15, previously obtained from ent-halimic acid, 1 [4], by chemoselective Wittig reaction [15][16][17], we obtained 16. Treatment of 16 under acidic conditions allows the isomerization of the side chain terminal double bond into the more stable trisusbstituted position in quantitative yield, to give compound 17. The protection of the carbonyl group gives the dioxolane 18, with concomitant isomerization of the double bond in the bicyclic system to the tetrasusbsituted position. Chemoselective oxidation of the side chain of 18 was achieved by treatment with OsO 4 [18][19][20][21], followed by cleavage of the resulting diol with Pb(AcO) 4 to give aldehyde 19 in excellent overall yield (84%) from ent-halimic acid 1. In this manner we have opened a new and versatile route to intermediate 19, a key compound for the synthesis of many natural products. Once this compound was available in large quantities in a reliable fashion it was decided to synthesize compound 2.

Conclusions
The synthesis of the natural ent-halimanolide 2 has been achieved starting from ent-halimic acid 1, confirming in this way its structure and establishing its absolute configuration. A new and versatile route to the key intermediate 19 [84% from 1] for the synthesis of natural ent-halimanolides is described. Other biological tests for 2 and several intermediates are in progress and will be reported in due course.

General
Unless otherwise stated, all chemicals were purchased were of the highest purity commercially available and were used without further purification. IR spectra were recorded on a BOMEM 100 FT-IR or an AVATAR 370 FT-IR Thermo Nicolet spectrophotometers. 1 H-and 13 C-NMR spectra were recorded for CDCl 3 solutions and referenced to the residual peak of CHCl 3 at δ 7.26 ppm and δ 77.0 ppm, for 1 H and 13 C, respectively, using Varian 200 VX and Bruker DRX 400 instruments. Chemical shifts are reported in δ ppm and coupling constants (J) are given in Hz. MS were performed at a VG-TS 250 spectrometer at 70 eV ionising voltage. Mass spectra are presented as m/z (% rel. int.). HRMS were recorded on a VG Platform (Fisons) spectrometer using chemical ionization (ammonia as gas) or Fast Atom Bombardment (FAB) technique. For some of the samples, QSTAR XL spectrometer was employed for electrospray ionization (ESI). Optical rotations were determined on a Perkin-Elmer 241 polarimeter in 1 dm cells. Diethyl ether and THF were distilled from sodium, and dichloromethane was distilled from calcium hydride under Ar atmosphere.

Methyl 12-acetoxy-2-ethylenedioxy-13,14,15,16-tetranor-ent-halim-5(10)-en-18-oate (7)
Acetate 6 (1.02 g, 3.28 mmol) dissolved in benzene (33 mL), was refluxed in the presence of p-toluenesulfonic acid (17 mg, 0.10 mmol) and ethylene glycol (2.0 mL, excess) at 138 ºC for 16 h, then the solution was diluted with AcOEt, washed with 6% aqueous NaHCO 3 and water and dried over Na 2   To an ice cooled solution of 8 (0.15 g, 0.43 mmol) in EtOH (4.3 mL), NaBH 4 (16.2 mg, 0.43 mmol) was added. After being stirred at room temperature for 3h, the reaction mixture was recooled to 0ºC and quenched with a few drops of 2 M aqueous HCl solution, diluted with EtOAc and water and extracted with EtOAc. The organic layer was washed with water. Evaporation of the dried extract gave a residue which was chromatographed on silica gel (hex/EtOAc 9:1) to afford 10 (73 mg, 49%) and 9 (70 mg, 47%).  (14) A solution of 3-bromofuran in THF 1M (0.14 mL, 0.14 mmol), was treated dropwise with n-BuLi (1.6 M in hexane, 0.09 mL, 0.14 mmol) at -78 o C under Ar atmosphere. After the reaction mixture was stirred for 30 min. at this temperature, a solution of 12 (30 mg, 0.11 mmol) in dry THF (1.1 mL) was added and stirred for an additional 30 minutes at the same temperature. The reaction mixture was treated with 10% aqueous NH 4 Cl solution, warmed to room temperature and extracted with EtOAc. The organic layer was washed with 6% NaHCO 3 , brine and dried over Na 2 SO 4 . The solvent was evaporated to afford a residue which was purified by chromatography (Hex/AcOEt 9/1) to yield 13 (20 mg, 54%) and 14 (13 mg, 36%).    N-oxide (NMO, 1.18 g, 8.70 mmol) and a solution of OsO 4 2.5% (0.3 ml, 0.01mmol) in t-BuOH. The reaction mixture was stirred at room temperature for 20 h and a saturated aqueous solution of Na 2 SO 3 (30 mL) was added. The mixture was extracted with AcOEt, and the organic layer was washed with 10% aqueous Na 2 S 2 O 3 , 2N aqueous HCl, water and brine and dried over Na 2 SO 4 . The solvent was evaporated to yield the expected mixture of hydroxy derivatives. To a solution of the hydroxy derivatives (1.15g, 3.06 mmol) in benzene (16 ml) was added LTA (3.0 g, 6.68 mmol). The reaction mixture was stirred at room temperature for 30 min and then filtered off through Celite. The solution was diluted with EtOAc and washed with 6% aqueous NaHCO 3 , water and brine and then dried and evaporated to yield 19 (980 mg, 96%) as a colourless oil; To a solution of 20 (118 mg, 0.29 mmol) in dry pyridine (1.0 mL), Ac 2 O (1.0 mL) was added and the mixture was stirred at room temperature overnight, then the reaction mixture was poured into icewater and extracted with EtOAc. The organic layer was washed successively with 2M aqueous HCl, 6% aqueous NaHCO 3 and brine. It was dried over Na 2 SO 4   To a solution of 21 (22 mg, 0.05 mmol) in dry pyridine (0.5 mL), Ac 2 O (0.5 mL) was added and the mixture was stirred at room temperature overnight. The reaction mixture was then poured into icewater and extracted with EtOAc. The organic layer was washed successively with 2M aqueous HCl, 6% aqueous NaHCO 3 and brine. It was dried over Na 2 SO 4 and the solvent was evaporated to afford 23 (128 mg, 99%) as a colourless oil; To an ice cooled solution of 26 (22.0 mg, 0.07 mmol) in EtOH (0.7 mL), NaBH 4 (13 mg, 0.33 mmol) was added. After being stirred at room temperature for 3h, the reaction mixture was recooled to 0ºC and quenched with a few drops of 2 M aqueous HCl solution, diluted with EtOAc and water and extracted with EtOAc. The organic layer was washed with water. Evaporation of the dried extract gave a residue, which was chromatographed on silica gel (hex/EtOAc 9/1) to afford 13 (8 mg, 38%), and 27 (9 mg, 43%). To a mixture of 13 (3 mg, 0.01 mmol) N-methylmorpholine-N-oxide (NMO) (4 mg, 0.03 mmol) and molecular sieves (15 mg) in anhydrous CH 2 Cl 2 (0.3 mL) under an Ar atmosphere and at room temperature, TPAP (3.0 mg, 3x10 -3 mmol) was added. The reaction mixture was stirred for 30 min. and then filtered on silica gel and Celite (DCM and EtOAc). Evaporation of the solvent yielded 2 (3.0 mg, 92%).