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Journal of the Chilean Chemical Society

On-line version ISSN 0717-9707

J. Chil. Chem. Soc. vol.62 no.3 Concepción Sept. 2017

http://dx.doi.org/10.4067/s0717-97072017000303658 

Article

ABSOLUTE CONFIGURATION OF MULINOLIC ACID

IVÁN BRITO1  * 

JORGE BORQUEZ1 

EMILY HUAROTE1 

LUIS LOYOLA1 

ALEJANDRO CÁRDENAS2 

MARIO SIMIRGIOTIS3 

1Departamento de Química, Facultad de Ciencias Básicas, Universidad de Antofagasta, Casilla 170, Antofagasta, 1240000, Chile

2Departamento de Física, Facultad de Ciencias Básicas, Universidad de Antofagasta, Casilla 170, Antofagasta, 1240000, Chile

3Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, casilla 567, Valdivia 5090000, Chile

ABSTRACT

The structure of this mulinolic acid consists of a mulinane skeleton and the corresponding isopropyl, methyl, carboxyl and methyl groups at C3, C8, C5, C13, respectively, which are β-oriented, whereas the hydroxyl group at C13 is α-oriented. The cyclopentane (A), ciclohexane (B) and cicloheptene (C) rings are trans (A/B) and (B/C) cis fused, and in an envelope, chair, and twist conformation respectively.

In the crystal the molecules are linked by two intermolecular O—H⋅⋅⋅O hydrogen bond forming bidimensional supramolecular structures with graph-set notation R44(12), R44(40), R66(46) and C44(46). The absolute configuration of the title compound has been determined from the refinement of the Flack parameter16. On this basis the absolute configuration was assigned as C3R, C5S, C8S, C9S, C10S and C13R.

Keywords: absolute configuration; diterpenoid; X-ray diffraction; crystal and supramolecular structure

INTRODUCTION

Mulinum crassifolium is small cushion shrub growing up to 15 cm commonly known as “chuquicán”, “susurco” o “espinilla” which grows in the north of Chile at altitudes above 3500m. Bitter tasting infusions of the whole plant are used widely in folk medicine, principally in the treatment of diabetes, and bronchial and intestinal disorders1. This plant is well recognized as an important source of diterpenes bearing the mulinane skeleton with interesting biological activities2. The relative stereochemistry of the title compound was assigned by spectroscopic data and by chemical transformations3. In this paper we report the absolute configuration of the title compound which has been determined from the refinement of the Flack parameter15, × = 0.05(11), which indicate that the correct configuration had been assigned against 925, CuKα Bijvoet pairs. On this basis the absolute configuration was assigned as C3R, C5S, C8S, C9S, C10S and C13R.

EXPERIMENTAL

The title compound C20H32O3 was isolated using medium pressure column chromatography (MPCC). In a continuation of our program for the chemical study of the Atacama Desert Flora26, dried aerial parts of Mulinum crassifolium (750 g) collected in April 2014 in the pre-Andean mountain range of Atacama Desert, precordillera de la II Region, Northern Chile were extracted with n-hexane (1 liter, 3 times in the dark, 24 h each time) and 22 g of a yellow gum were obtained after evaporation of the solvent. A portion of the extract (10.0 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 (9.0:1.0 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 (80) were combined according to TLC analysis (Kieselgel F254 plates, developed with Hexane: EtOAc 8:2 v/v, and spots visualized by spraying with vanillin: sulfuric acid 2 % in ethanol and heating) and fifteen combined fractions were obtained. Fraction 5 (350 mg) was re-chromatographed using the same chromatographic system and the known compounds: mulin-11,13-dien-20-oic acid6 (120 mg) and mulinolic acid7 (60 mg). Recrystallization de mulinolic acid from ethyl acetate a room temperature yielded white crystals, which were suitable for X-ray diffraction analysis. The spectroscopic data showed resemblance with those similar mulinane structures reported in the literature411. The relative stereochemistry of mulinolic acid was assigned based on careful analysis of the ROESY experiment6, and in this work we have assigned the absolute configuration of this diterpene using X-ray crystal analysis.

Mulinolic acid: white crystals, m.p. 178-180 °C. The molecular weight was determined by orbitrap HR-ESI-MS/MS with a mass spectrometer. [M-H]: required: 319. 22752, found: 319.22766 for C20H31O3 (Fig. 1). 1H NMR (Bruker Avance 300 MHz, CDCl3) δ ppm: 0.86 (3H, d, J = 5.8 Hz, Me-18), 0.94 (3H, s, Me-17), 1.04 (3H, d, J = 5.8 Hz, Me-19), 1.10 (1H, dd, J = 4.6, 15.0 Hz, H-15β), 1.32 (3H, s, Me-16), 1.38 (1H, m, H7β), 1.47 (4H, m, H-6α, H-4α, H-7α, H-2β), 1.51 (1H, m, H-3α), 1.55 (1H, m, H-1α), 1.63 (1H, dd, J = 9.3/12.5 Hz, H-14β), 1.84 (1H, dd, J=4.6/12.5 Hz, H-14α), 1.91 (1H, m, H-2α), 1.93 (1H, m, H-1β), 2.08 (1H, m, dd, J = 6.0/10.8, H-9β), 2.16 (1H, br d = 10.8 Hz, H-10α), 2.42 (1H, dd, J = 3.0/9.4 Hz, H-6β), 2.61 (1H, dt, J = 15.0 Hz, H-15α), 5.58 (1H, d, J = 12.8 Hz, H-11), 5.58 (1H, d, J=12.9 Hz, H-12). 13C NMR (13C NMR Bruker Avance 75 MHz, CDCl3) δ ppm: 180.2 (COOH), 133.8 (C-11), 136.5 (C-12), 71.4 (C-13), 58.4 (C-5), 57.7 (C-3), 51.6 (C-10), 48.7 (C-9), 42.2 (C-7), 36.2 (C-14), 35.9 (C-8), 32.4 (C-6), 32.0 (C-4), 30.5 (C-15), 28.9 (C-2), 33.7 (Me-16), 27.5 (Me-17), 25.2 (C-1), 22.6 (Me-19), 22.9 (Me-18). These data, together with HR-MS data and correlations observed in the HSQC and HMBC spectra, are consistent with the structure of mulinolic acid (Figure 1).

Fig. 1 Full Orbitrap HESI (-) spectra of compound mulinolic acid. 

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 SHELXL12. 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-201312, program used to refine structure: SHELXL-201312, molecular graphics13. The absolute configuration of the title compound which has been determined from the refinement of the Flack parameter15, × = 0.05(11), indicated that correct configuration had been assigned, against 925, CuKα Bijvoet pairs.

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

Crystal data
C20H32O3 V = 4151.3(9) Å3
Mr = 320.45 Z = 8
Orthorhombic, C2221 CuKα (λ = 1.54178 Å)
a = 14.7039(19) Å α = 90° μ = 0.526 mm−1
b = 22.772(3) Å β = 90° T = 296(2) K
c = 12.3979(15) Å γ = 90° Colourless, Block, 0.200 × 0.150 × 0.150 mm
Density (calculated)/Mg/m3 1.025 F(000) = 1408.0
Data Collection
Diffractometer Bruker AXS D8-Venture, Triumph-Iμ-Cu
2621 reflections with I>2σ(I)
22363 measured reflections Rint = 0.069
2298 independent reflections
2θ range for data collection 7.156 to 111.436°
Index ranges -15 ≤ h ≤ 15, −23 ≤ k ≤ 24, −13 ≤ l ≤ 13
Refinement
R[F2>2σ(F2)] = 0.072 215 parameters
wR[F2] = 0.201 S = 1.14
Δρmax = 0.53 e⋅Å−3
Δρmin = −0.23 e⋅Å−3
Flack parameter 15 0.05(11) Goodness-of-fit on F2 1.139

RESULTS AND DISCUSSION

The molecular structure and the absolute configuration of the title compound was confirmed by x-ray diffraction analysis of suitable single crystals (Fig. 2). Colorless block crystals of title compound with approximate dimensions 0.200 × 0.150 × 0.150 mm was obtained by slow evaporation of a ethyl acetate solution. The molecular structure consists of a mulinolic acid skeleton with the required isopropyl, methyl, carboxyl and methyl group at C3, C8, C5, C13 respectively, which are β-oriented, as in mulinolic acid, whereas the hydroxyl group at C13 is α-oriented, (Fig.1). The Cremer and Pople parameters15 for the cyclopentane (A), ciclohexane (B) and cicloheptene (C) rings are: Q2 = 0.412(6) Å, φ2 =115.6(9)°; QT = 0.595(6) Å, θ = 174.3(6)°, φ = 107(6)° and Q2 = 0.207(6) Å, φ2 =104.4(18)°, Q3 = 0.592(6) Å, φ3 = 334.9(6)°, so the conformation for A, B and C rings are: envelope, chair, and twist chair respectively. The A/B and B/C rings are trans and cis fused respectively. The OH groups are involved in hydrogen bonding interactions with the average H⋅⋅⋅O distance of 1.955 Å and O−H⋅⋅⋅O angles of 149° (Table 2), so in the crystal the molecules are linked by two intermolecular O—H⋅⋅⋅O hydrogen bond forming bidimensional supramolecular structures with graph-set notation R44(12), R44(40), R66(46) and C44(46).16, (Fig. 3 and Fig. 4). All bond distances and angles are normal.

Fig.2 ORTEP Diagram of mulinolic acid. The ellipsoids are shown at the 30% probability level. 

Table 2 Hydrogen-bonding geometry (Å, °). 

D—H⋅⋅⋅A D—H H⋅⋅⋅A D⋅⋅⋅A D—H⋅⋅⋅A
O2—H2⋅⋅⋅O3i 0.82 1.83 2.600(6) 156
O3—H3A⋅⋅⋅O1ii 0.82 2.08 2.776(6) 142

symmetry code: (i) 3/2-x, 3/2-y,1/2+z; (ii) 1/2+x, 3/2-y, 1-z

Fig.3 A view of the bi-dimensional supramolecular aggregate, showing the formation of ring with set-graph motif R44(12) as representative example. For the sake of clarity, H-atoms bonded to C atoms have been omitted. 

CONCLUSIONS

In order to establish its absolute configuration, a single-crystal X-ray diffraction analysis of mulinolic acid was undertaken. The X-ray molecular model of the title compound (Fig.2), confirmed all the above deductions on its structure and established the absolute configuration as C3R, C5S, C8S, C9S, C10S and C13R.

Supplementary material

CCDC- 1548215 contains the supplementary crystallographic data for this article. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

ACKNOWLEDGEMENTS

IB acknowledgment Fondequip Program (Grant EQM130021) for the funding to purchase Single Crystal X- ray Diffractometer. JB & MS thanks FONDECYT (Chile) (Grant 1140178) and fondequip (EQM140002) for financial support. We also thank CONAF (Corporación Nacional Forestal) Chile for the permission and support in the collection of the flora of protected areas of northern Chile.

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