Comment

Zanthoxylum alatum (Rutaceae) is an evergreen shrub growing at up to 2000 m above sea level in the hot valleys of the Himalayan region. It has a variety of applications in traditional medicine, including anthelmintic and painkilling activities. Extracts of leaves are also used as disinfectants and against scabies and house flies. The chemical constituents of the plant's stem wood (Ishii, Hosoya, Ishikawa & Haginiwa, 1974), root wood (Ishii, Hosoya, Ishikawa, Hueda & Haginiwa 1974), and stem and root bark (Ishii et al., 1977), have been reported, and include coumarins, furoquinoline alkaloids, benzo[c]phenanthridines, lignans, steroids and terpenoids. However, studies of the bioactivity of these compounds from Z. alatum have not been reported to date.

A chloroform extract of the leaves of Z. alatum showed antifeedant activity against the larvae of Spodoptera litoralis, and also antiproliferative activity against multiresistant cell lines. Asarinin was identified as one of the major constituents of the chloroform extract. The asarinin sample obtained from the plant crystallized as a racemic compound, although a positive []_D²⁰ of 43° was observed. Pure (+)-asarinin has an []_D²⁰ of about 120° (Takahashi & Nakagawa, 1966). The observed []_D²⁰ implies that the two enantiomers occur in the plant in different proportions and that racemic asarinin crystallizes first. To the best of our knowledge, the structure of racemic asarinin, the title compound, (I), has not been reported previously, so we present it here.

The furofuran asarinin, (I), has four asymmetric centres (labelled C1, C2, C5 and C6 in this study, with relative configurations 1R, 2R, 5R and 6S). The (-)-enantiomer of asarinin seems to be the prevalent form in nature (as judged from citations in Chemical Abstracts, but we have not counted these exactly). The naturally occurring stereoisomers epiasarinin (inverted at C6) and sesamin (inverted at C5) are also known; for a brief overview, see Aldous et al. (2006). A search of the Cambridge Structural Database (CSD, Version 5.33; Allen, 2002; see also Table 1) reveals that several crystal structures have been reported, but in some cases confusion may have arisen. For epiasarinin, the structure of the synthetic racemate was determined by Aldous et al. (2006; we adopt an analogous atom-numering scheme for the racemate of asarinin), but no enantiomerically pure structure has yet been determined. Three closely similar structures of sesamin have been reported, by Baures et al. (1992), Hsieh et al. (2005) and Li et al. (2005). However, the sign of the optical rotation is given as (+) by Hsieh et al. (2005), as (-) by Li et al. (2005) and is not quoted by Baures et al. (1992). In no case could the absolute configuration be determined crystallographically because the anomalous dispersion effects were insignificant. For asarinin, there are no fewer than five reported structures in the CSD, but they are given three different families of refcodes. Again, the unit cells are closely similar. The references are: Li et al. (2005), optical rotation (-) (the space group is reported as P1 with two independent molecules, but the cell constants are close to those of the other determinations, and inspection of the structure suggests that the true space group is indeed P2₁); Macías et al. (1992), (-); Parmar et al. (1998), optical rotation given as (-) in the CSD but (+) in the publication; Il'in et al. (1994), (-); and Mata et al. (1998). For this last determination, the absolute configuration was determined crystallographically by exploiting the stronger anomalous dispersion effects of copper radiation, but the optical rotation was not given, so frustratingly no correlation has been established between the absolute configuration and the optical rotation for this compound despite five determinations of its crystal structure. The determination of the absolute configuration of (+)-asarinin by the `optical shift rule' (Freudenberg & Sidhu, 1961) has not been universally accepted [see e.g. Gunatilaka et al. (1982)].

The molecule of asarinin in the racemate is shown in Fig. 1. Molecular dimensions may be regarded as normal. A least-squares fit to the (inverted) molecule of VUKBUY gives an r.m.s. deviation of 0.08 Å for all atoms except C21-O28, since this ring is oriented differently with respect to the furofuran system [torsion angle O7-C6-C20-C21 = -2.96 (14)°, cf. -30.9° for the corresponding angle of VUKBUY].

In the packing pattern of (I), four short intermolecular HO contacts might be interpreted as `weak' hydrogen bonds (Table 2). The shortest two of these link the molecules via inversion centres to form molecular ribbons parallel to [101] (Fig. 2).

Figure 1 The molecular structure of (I), showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 50% probability level. This enantiomer has the configurations S, S, S and R at atoms C1, C2, C5 and C6, respectively. The crystal structure, however, contains both entantiomers.

Figure 2 A packing diagram for (I), viewed perpendicular to (11). Dashed lines indicate hydrogen bonds. H atoms not involved in hydrogen bonds have been omitted for clarity.

Experimental

The plant material was collected from Rudraprayag Uttarakhand, India (29° 43' 19'' North, 78° 31' 6'' East). The plant was identified by the Botanical Survey of India, Dehradun, and a voucher specimen has been deposited (BSD Accession No. 112749).

The shade-dried leaves of Z. alatum were extracted exhaustively with 90% ethanol at room temperature. The combined ethanolic extracts were concentrated under reduced pressure at 723 K to a dark viscous mass. Water was added (10% by weight) and the mixture was extracted with hexane (3 × 500 ml). The combined organic phases were washed with water and dried by evaporation of the solvent under reduced pressure to yield 35.4 g of solid residue. The water phase was extracted again, this time with chloroform (3 × 500 ml), and the combined chloroform phases were washed with water and dried with MgSO₄. Evaporation of the solvent under reduced pressure yielded another 6.5 g of solid residue. The extracts were tested against the polyphagous pest Spodoptera litoralis for antifeedant activity and against K526/Adr cells for cytotoxic activity.

The biologically active chloroform extract was subjected to column chromatography over silica gel (60-120 mesh) using chloroform-methanol of increasing polarity. Eluates were collected in fractions of 50 ml and each was evaporated to dryness under reduced pressure. Fractions exhibiting similar thin-layer chromatography behaviour were combined. Three fractions collected upon elution with chloroform-methanol, viz. A (solvent ratios 39-46:100), B (50-68:100) and C (70-100:100), afforded solid material after evaporation of the solvents. Fraction B was separated again by chromatography with chloroform-methanol (9:1 v/v) to yield asarinine (12 mg).

To obtain single crystals of asarinin, the compound (1 mg) was dissolved in pentane (2 ml). The resulting solution was concentrated by evaporation in a slightly permeable screw-capped vial. Single crystals of (I) in the form of colourless laths formed over a period of 96 h.

Crystal data

C20H18O6 Mr = 354.34 Triclinic, a = 6.5002 (4) Å b = 10.8931 (7) Å c = 11.8005 (8) Å = 103.793 (6)° = 102.111 (6)° = 95.836 (6)° V = 783.22 (9) Å3 Z = 2 Cu K radiation = 0.93 mm-1 T = 100 K 0.25 × 0.20 × 0.10 mm

Data collection

Oxford Xcalibur diffractometer with Nova source and Atlas detector Absorption correction: multi-scan (CrysAlis PRO; Agilent, 2012) Tmin = 0.770, Tmax = 1.000 41535 measured reflections 3254 independent reflections 3200 reflections with I > 2(I) Rint = 0.027

Refinement

R[F2 > 2(F2)] = 0.035 wR(F2) = 0.086 S = 1.05 3254 reflections 235 parameters H-atom parameters constrained max = 0.31 e Å-3 min = -0.21 e Å-3

Table 1 X-ray structure determinations of asarinin and its stereoisomers from the CSD (Allen, 2002) Compound Reference CSD refcode Optical rotation Space group Notes Synthetic epiasarinin Aldous et al. (2006) CERWOM (±) P21/n   Sesamin Baures et al. (1992) TAPWAI Not given P21   Sesamin Hsieh et al. (2005) TAPWAI01 (+) P21 a Sesamin Li et al. (2005) TAPWAI02 (-) P21 a Asarinin Li et al. (2005) MAKJIT (-) P1 (probably P21) b, e Asarinin Macías et al. (1992) VUKBUY (-) P21 b Asarinin Parmar et al. (1998) VUKBUY03 (+) P21 c Asarinin Il'in et al. (1994) YODLEI (-) P21 b Asarinin Mata et al. (1998)) YODLEI01 Not given P21 d Asarinin This work   (±) P   Notes: (a) no coordinates were deposited; (b) no H-atom coordinates were deposited; (c) given in CSD as (-) (probably erroneous); (d) absolute configuration determined by anomalous dispersion; (e) given in CSD as `unknown solvate', but may be solvent-free.

Table 2 Hydrogen-bond geometry (Å, °) D-HA D-H HA DA D-HA C4-H4AO17i 0.99 2.41 3.3501 (14) 159 C15-H15O26ii 0.95 2.45 3.3093 (14) 151 C16-H16O28iii 0.95 2.67 3.5503 (14) 154 C18-H18BO28iv 0.99 2.60 3.2600 (13) 124 Symmetry codes: (i) -x+1, -y+1, -z+1; (ii) -x, -y+1, -z; (iii) -x+1, -y+1, -z; (iv) x-1, y+1, z+1.

H atoms were placed in calculated positions and refined using a riding model, with aromatic C-H = 0.95 Å, methylene C-H = 0.99 Å and methine C-H = 1.00 Å, and with U_iso(H) = 1.2U_eq(C).

Data collection: CrysAlis PRO (Agilent, 2012); cell refinement: CrysAlis PRO; data reduction: CrysAlis PRO; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: XP (Siemens, 1994); software used to prepare material for publication: SHELXL97.

Supplementary data for this paper are available from the IUCr electronic archives (Reference: FA3296 ). Services for accessing these data are described at the back of the journal.