A NON-CENTROSYMMETRIC POLYMORPH OF 5-HYDROXY-7-METHOXY-2-PHENYLCHROMAN-4-ONE

The bioactive compound 5-hydroxy-7-methoxy-2-phenylchroman-4-one (pinostrobin, I), was isolated from aerial parts of Nolana ramossisima I.M. Johnst., a Chilean endemic species, using a combination of medium pressure column chromatography and high speed countercurrent liquid-liquid chromatography (HSCCC). The compound crystallized as a non-centrosymmetric polymorph in the orthorhombic chiral space group P212121. Its structure had been previously reported by Shoja (Acta Cryst. C45, 828, 1989) and Yamovoi et al. (Khim. Prir. Soedin (5), 361, 2001) in the centrosymmetric space groups Pbca and P21/c, respectively.


INTRODUCTION
High speed countercurrent chromatography (HSCCC) is a support-free liquid-liquid partition chromatographic technique which eliminates the problem of the adsorption of a sample onto a solid support and has been widely used for the purification of natural products, including flavanones 1 . In this work this fast technique has been applied in combination with medium pressure column chromatography for the purification of the phenolic constituents in an organic extract of aerial parts of Nolana ramossisima I.M. Johnst. (Solanaceae), an endemic species of Paposo Valley, Northern Chile.
The main compound isolated, 5-hydroxy-7-methoxy-2-phenylchroman-4-one, synonym: pinostrobin is an important biologically active flavanone previously isolated from different plant materials. This flavanone is a constituent of Pinus strobus and many other pine species and, inter alia, Artemisia campestris, Boesenbergia pandurata, Boesenbergia rotunda, Sarcandra glabra, and Polygonum lapathifolium, and has been identified also in honey 2 , propolis 3 , and trees frequently visited by bees such as Alnus spp. and Populus spp. Pinostrobin has shown antileukemic 4 , antioxidant 5 and antibacterial activity 3 and inhibits TNF-α and IL-1β production 6 . Pinostrobin is an inducer of mammalian phase 2 chemoprotective and antioxidant enzymes 2 and an inhibitor of DNA topoisomerase I activity 7 . In our hands the title compound crystallized as a new non-centrosymmetric polymorph. The existence of polymorphic forms provides a unique opportunity for the investigation of structure-property relationships, since by definition the only variable among polymorphs is that of crystal structure, and one of the most effective strategies for studying structure-property relationships has been to follow the behavior of a physical property through a polymorphic phase change.

EXPERIMENTAL General
Nolana ramosissima was collected in Paposo, Antofagasta, Chile in September 2011. A voucher herbarium specimen is deposited in the laboratory of Natural Products, University of Antofagasta, with the number Nr 110403. Dried and finely powdered aerial parts of N. ramossisima (1562 g) were extracted with petroleum ether (2 L, 3 times in the dark, 24 h each time). After evaporation of the solvent in vacuo at 35 o C, a dark gummy extract (35 g) was obtained. The extract (20 g) was submitted to medium pressure column chromatography (Silica Gel 60 H, 7 x 40 cm) using n-hexane:ethyl acetate 9:1 as solvent system and 41 fractions of 50 mL each were collected. The fractions were combined according to their TLC analysis and 6 combined fractions were obtained. Fraction 4 (483 mg) was re-chromatographed using a Quattro MK7 HSCCC instrument 8 equipped with three preparative stainless steel coils of 115 mL each (2.10 mm I.D) and one SSI model Series II HPLC pump, using n-hexane:ethyl acetate:acetonitrile 5:3:5 v/v/v as solvent system. The filtered sample solution (5 mL) was introduced into the preparative (115 mL) column through a manual low-pressure sample injection valve using a 5 mL sample loop. The lower phase was used as the mobile phase and was pumped at a flow rate of 4.0 mL/min in the 'head-to-tail' mode. After the mobile phase front Crystal structure determination Colorless block crystals of pinostrobin of approximate dimensions 0.1x0.08x0.4 mm were obtained by slow evaporation of a MeOH solution. A well-shaped and suitably-sized single crystal was selected for the X-ray diffraction structure determination experiment. The X-ray intensity data were collected at 293 K on an Enraf Nonius CAD4-MACH3 diffractometer, using MoKα graphite monochromatic radiation ((λ = 0.71073 Å). CAD4 software was used for data collection and cell refinement, and HELENA software of the WINGX program was used for data reduction. The structure was solved by direct methods using the SHELXS2013 program 12 and the refinement was carried out using SHELXL 2013 12 . All non-hydrogen atoms of the molecule were clearly solved and full-matrix least-square refinement of these atoms with anisotropic thermal parameters was carried out. Hydrogen atoms on carbon and oxygen atom were positioned stereochemically and were refined with fixed individual displacement parameters [Uiso (H) = 1.5Ueq (C or O) for methyl and hydroxyl groups or 1.2 Ueq (C) for methine and methylene groups and the aromatic ring hydrogen atom] using a riding and rotating group model (for methyl and hydroxyl groups), with C-H bond lengths of 0.96, 0.97, 0.98 and 0.82 Å for methyl, methine, methylene and hydroxyl groups, respectively. The hydrogen atom of the hydroxyl group was located by difference Fourier synthesis and was kept rigid together with its respective oxygen atom during the subsequent refinements. WINGX software was used to analyze and prepare the data for publication 13 . Molecular graphics were prepared using OLEX2 14 and Mercury 16 . Since the most electron-rich atom is the oxygen atom, which does not have a sufficiently large anomalous scattering (using MoKα radiation), it was not possible to determine the enantiomer present using the Flack method 17 . Table 1 summarizes the results.

RESULTS AND DISCUSSION
The title compound (I) is a new polymorph of structures II and III reported by Shoja 10

CONCLUSIONS
The main conclusion of this work is that a new, non-centrosymmetric polymorph of 5-hydroxy-7-methoxy-2-phenylchroman-4-one (pinostrobin), was determined by X-ray methods, was isolated from an organic extract of aerial parts of Nolana ramossisima Johnst., an endemic species of the Paposo Valley, Northern Chile, using high speed countercurrent chromatography (HSCCC) and slow evaporation of a methanol solution.