INTRODUCTION

Following our program to isolate interesting metabolites from the Atacama Desert Flora, Northern Chile^10,12–17. Now we are dedicated to the study of Haplopappus rigidus Phil., one of the species known in Chile by the popular name “baylahuen”, is an endemic plant found at a height of over 3000 m in the Andes mountains and used as medicinal by the local population and Aymara aboriginals since precolombian times¹. All the related Chilean Haplopappus “baylahuen” species produce a resinous exudate with interesting biologically active compounds including, several clerodane and labdane diterpenoids and flavonoids with biological significance². Indeed, the Mapuche medicinal plants Haplopappus multifolius, H. taeda, H. baylahuen and remyanus showed the presence of flavonoids and labdane terpenoids with known antioxidant, choleretic, anti-inflammatory and cholagogue properties^3,4,5. In the Aymara region, the infusion of H. rigidus has been used to cure or prevent liver diseases, gastro-intestinal disorders, cough and has also been employed as a sexual stimulant attributed to the presence of diterpenoids and flavonoids^1,6,7. Moreover, from the resinous exudate of H. incinatus the antibacterial potent clerodane diterpenoid: 18-acetoxy-cis-cleroda-3-en-15-oic acid was isolated^2,8, while from the aerial parts of H. rigidus Phil, the related main clerodane rigidusol (13-hydroxy-18-acetoxy-cis-cleroda-3,14-diene) exhibited moderate cytotoxic activity against human breast adenocarcinoma cell line MCF-7⁹. In this work we report the absolute configuration of the rigidusol derivative 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid by X-ray diffraction methods, which is consistent with the previously reported for this compound^2,8 (Figure 1).

Figure 1 Absolute configuration of 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid. 

EXPERIMENTAL

The title compound C₂₂H₃₄O₄, is a clerodane diterpenoid firstly isolated from aerial parts of Croton chilensis (Muell. Arg) and identified as 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid by spectral data and relative configuration by X-ray diffraction methods¹⁰. The later compound was also isolated and reported from Haplopappus desertícola¹¹. Following our program to isolate interesting metabolites from the Atacama Desert Flora, Northern Chile^12–18, dried aerial parts of Haplopappus rigidus (1050 g) collected in april 2015 in “El Tatio”, Andean mountain range of the of Atacama Desert, II Region, Northern Chile, were defatted with hexane (3 liters, 3 times in the dark, 24 hours each time) and 58.88 g were obtained after evaporation of a ethyl acetate solution. A portion of the extract (30.30 g) was filtered and submitted to a medium pressure column chromatography system composed of an 2.5 cm × 48 cm medium pressure column (Aceglass inc, Vineland, NY, USA) packed with silicagel (Kieselgel 60 H, Merck, Darmstadt, Germany) using an isocratic solvent system of n-hexane-ethyl acetate (9.5:0.5 v:v) pumped with a medium pressure pump (FMI lab pump, Syosset, NY, USA) with a flow rate of 10 mL-minute. The collected fractions (226) were combined according to TLC analysis (Kieselgel F254 plates, developed with Hexane: EtOAc 7:3 v/v and spots visualized by spraying with vanillin: sulfuric acid 2 % in ethanol and heating). From fractions 24-37, the known compound: 13-hydroxy-18- acetoxy-cis-cleroda-3,14-diene⁹ (2.42 g) was isolated and from fractions 94135 the known compound 1 (18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid, 1.53 g) was isolated previously from the botanic specie Croton chilensis¹⁰

Recrystallization from ethyl acetate/n-hexane a room temperature yielded pure white crystals of the last compound, suitable for X-ray diffraction analysis.

The relative stereochemistry of the title compound was assigned by spectroscopic data and by chemical transformations⁸. In this paper we report the absolute configuration of the title compound which has been determined from the refinement of the Flack parameter¹⁹, x = 0.0(5), which indicate that the correct configuration had been assigned against 1353 (99%) CuKα Bijvoet pairs. On this basis the absolute configuration was assigned as C5R, C8S, C9R and C10S.

White crystals, m.p. 100-102 °C. The molecular weight was determined by Q-orbitrap HESI-MS/MS with a mass spectrometer (Q-exactive Focus, Bremen, Germany) [M-H]^–: 361.23825 calcd. for C₂₂H₃₃O₄^–: 361.23843 (Figure 2). ¹H NMR (Bruker Avance 300 MHz, CDCl₃) δ ppm: 5.70 (2H, s, H-3/H-14), 4.59 (2H, s, H-18), 2.19 (3H, s, Me-16), 2.16 (2H, m, H-2), 2.07 (3H, s, COCH₃), 2.03 (1H, m, H-1), 2.01 (2H, m, H-12), 1.95 (1H, m, H-6), 1.81 (1H, m, H-1’), 1.62 (1H, m, H-11), 1.42 (2H, m, H-8/H-11’), 1.36 (2H, m, H-7/H-10), 1.29 (2H, m, H-6’/H-7’), 1.19 (3H, s, Me-19), 0.79 (3H, s, Me-20), 0.77 (3H, s, Me-17). ¹³C NMR (¹³C NMR Bruker Avance 75 MHz, DMSO-d6) δ ppm: 17.2 (C-1), 23.9 (C-2), 129.2 (C-3), 138.2 (C-4), 40.20 (C-5), 37.1 (C-6), 28.7 (C-7), 37.5 (C-8), 36.3 (C-9), 45.1 (C-10), 36.1 (C-11), 34.7 (C- 12), 164.6 (C-13), 114.8 (C-14), 170.9 (C-15), 19.5 (C-16), 15.9 (C-17), 66.6 (C-18), 34.6 (19), 17.2 (20), 171.0 (21), 21.2 (C-22). These data, together with ESI-MS/MS and correlations observed in the HSQC and HMBC spectra, are consistent with the structure of 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid (Figures 1 and 3) confirmed by comparison of spectroscopic data with those reported in the literature for this structure^8–11.

Figure 2 Full Q-Orbitrap HESI (-) spectra of compound 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid. 

Figure 3 ORTEP Diagram of 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid. The ellipsoids are shown at the 30% probability level. 

Data collection, structural determination and refinement was performed with a Bruker AXS D8-Venture, Triumph-μI-Cu with graphite-monochromated CuKα radiation (1.54178 Å). The structure was solved by direct method, and was refined against F² by full-matrix least-squares methods using SHELXL²⁰, All of the non-hydrogen atoms were refined anisotropically. The hydrogen atoms was located from a difference Fourier map and allowed to ride on their parent C and O atoms, with isotropic displacement parameters related to the refined values of the corresponding parent atoms. The final Fourier maps, the electron-density residuals were not significant. Crystallographic data, details of data collection and structure refinement parameters for the title compound is summarized in Table 1. Program used to solve structure: SHELXS-2013^20, program used to refine structure: SHELXL-2013²⁰, molecular graphics²¹.

Table 1 Crystallographic data, details of data collection and structure refinement parameters for the title compound. 

RESULTS AND DISCUSSION

The X-ray crystal-structure determination of the title compound was undertaken in order to establish its absolute configuration. The structure of 18-acetoxy-cis-cleroda-3,13E-dien-15-oic acid consists of a clerodane skeleton and the corresponding methyl groups are α-oriented (C8, C9) while C5 is β-oriented. The acidic lateral chain is β-oriented and the double bond between C13 and C14 has a E isomeric configuration.

The Cremer and Pople parameters²² for the ciclohexene and ciclohexane rings are:

Q_T = 0.455(10) Å, θ = 134.4(13)°, φ = 115.2(17)° and Q_T = 0.560(9) Å, θ = 12.7(9)°, φ = 179.4(4)° so the onformation is sofa and chair respectively. The ciclohexene, ciclohexane rings are cis fused. In the crystal the molecules are linked by one intermolecular O—H^…O hydrogen bond forming 1D-dimensional chain with graph-set notation (15)²³ (O2—H2^…O4ⁱ 149°; O2^…O4ⁱ 2.797(9) Å; H2^…O4ⁱ 2.06 Å (symmetry code (i) x,-1+y,1+z), (Figure 4). The absolute configuration of the title compound which has been determined from the refinement of the Flack parameter¹⁹, x = 0.0(5), which indicate that the correct configuration had been assigned against 1353 (99%) CuKα Bijvoet pairs. On this basis the absolute configuration was assigned as C5R, C8S, C9R and C10S. All distances and angles are normal.

Figure 4 A view of the one-dimensional supramolecular aggregate, showing the formation of chain with set-graph motif (15)²³. [Symmetry codes: (i) x, −1+y, 1+z; (ii) x, 1+y, −1+z]. 

CONCLUSIONS

In the title compound the molecular structure and its relative configuration were established by spectroscopic and crystallographic methods twenty three year ago¹⁰, but not of the absolute configuration, due to the experiment condition used (Mo, radiation). In order to establish its absolute configuration, a single-crystal X-ray diffraction analysis of was undertaken, using Cu radiation. The X-ray molecular model of the title compound (Figure 3), confirmed all the above deductions on its structure and established the absolute configuration as C5R, C8S, C9R and C10S.