Artabolide, a novel polar auxin transport inhibitor isolated from Artemisia absinthium

A new sesquiterpene with an a-methylene-g-lactone moiety, artabolide (1), and three known derivatives 2e4 were isolated from Artemisia absinthium. The structure of 1 was elucidated by 1D and 2D NMR analyses, and the absolute configuration was determined using the modified Mosher’s method and X-ray crystal structural analysis. Approximately 2.5 mg/plant treatment of artabolide (1) exhibited ca. 50% inhibition of polar auxin transport in radish hypocotyls, while 2e4 showed no significant inhibitory activity. Therefore, these results support the importance of the a-methylene-g-lactone moiety for the inhibition of polar auxin transport. 2013 The Authors. Published by Elsevier Ltd. All rights reserved.


Introduction
The plant hormone auxin (indole-3-acetic acid; IAA) plays an important role in plant growth and development. First, IAA is synthesized in the apical part of the shoot and young leaf and is then transported basipetally in a cell-to-cell system. This system, named polar auxin transport, is regulated by the influx carriers AUXIN RESISTANT1/LIKE AUXIN RESISTANT (AUX1/LAX) and efflux carriers PIN-FORMED (PIN) and ATP-binding cassette subfamily B (ABCB) located on the plasma membrane. 1e3 Over many decades, the mechanism of polar auxin transport has been revealed using synthetic polar auxin transport inhibitors, such as N-(1-naphthyl)phthalamic acid (NPA), 2,3,5-triiodobenzoic acid, 9-hydroxyfluorene-9-carboxylic acid, and morphactin. NPA, the most frequently used inhibitor of auxin efflux, has been shown to noncompetitively inhibit PIN and ABCB auxin efflux activities. 4 Furthermore, a number of flavonoids have been reported to be some of the few naturally occurring inhibitors of polar auxin transport. 5 Flavonoids have also been shown to displace NPA from the plasma membrane binding site. 6 However, except in the case of flavonoids, few studies on natural polar auxin transport inhibitors have been reported.
In our exploratory study of naturally occurring inhibitors of polar auxin transport, we isolated and identified physiologically 4hydroxy-b-thujone and dehydrocostus lactone as naturally occurring inhibitors of polar auxin transport from some Asteraceae plants. 7 We recently found that the EtOAc-soluble portion of an acetone extract of Artemisia absinthium has potent inhibitory activity against polar auxin transport. We successfully isolated a new germacranolide-type sesquiterpene, artabolide (1), as the bioactive substance, and also isolated three known pelenolides 2e4.
Here we report the isolation, structural elucidation, and the structureeactivity relationship of 1e4 for polar auxin transport activity.

Extraction and separation of A. absinthium
The aerial parts of A. absinthium (750 gFW) were extracted with 80% acetone/H 2 O. The crude extract was partitioned between EtOAc and H 2 O. The EtOAc-soluble portion was subjected to repeated silica-gel column chromatography on a Sep-Pak ODS cartridge to afford a mixture of compounds 1 and 2. The mixture was purified by reversed-phase high-performance liquid chromatography (HPLC) to yield artabolide (1, 0.00025%) and hydroxypelenolide (2, 0.0022%). The other two fractions shown as brown spots on thinlayer chromatography (TLC) visualized by 10% H 2 SO 4 /H 2 O and heat treatment were subjected to Sep-Pak ODS cartridges and subsequently purified by reversed-phase HPLC to yield ketopelenolide a (3, 0.00027%) and ketopelenolide b (4, 0.000093%), respectively. The structures of 2e4 were identified by comparison with spectral data in the literature (Fig. 1). 8,9

Structural elucidation
The molecular formula of 1 was established as C 15  that the degree of unsaturation was five, three of these signals could be assigned to two olefins and one carbonyl group. Consequently, 1 was shown to be a bicyclic compound consisting of a 10membered ring and g-lactone. and C-12. Therefore, the connection points between the 10membered ring and a-methylene-g-lactone were determined as the C-6 and C-7 positions. Artabolide (1) was determined to be a germacranolide-type sesquiterpene lactone, 3-hydroxy-4,6,7(H)germa-cra-1(10),11(13)-dien-6,12-olide.
The relative structure of 1 was deduced from a nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of 1. To determine the absolute configuration, the modified Mosher's method was applied to 1. 10 The (R)-and (S)-MTPA esters (1a and 1b) were prepared by the reaction of 1 with (S)-and (R)-MTPA chloride, respectively. Each proton signal of 1a and 1b was assigned by 1 He 1 H COSY, and Dd (d S Àd R ) values were obtained (Fig. 4). Judging from the obtained values, the configuration of 1 at C-3 was denoted R. Thus, the absolute configuration of 1 was determined to be 3R, 4S, 6R, 7S.    Artabolide (1) was successfully crystallized from an n-hexane/ EtOAc mixed solution in the orthorhombic space group, P2 1 2 1 2 1 (#19). The ORTEP drawing of 1 is shown in Fig. 5. X-ray crystal structural analysis defined the absolute configuration of 1, which was in agreement with that described above, and also indicated that the 10-membered ring adopted a chairechair conformation.

Inhibitory effects of isolated compounds
The polar auxin transport regulatory activity of isolated compounds 1e4 was tested using a suitable radish hypocotyl bioassay system. 11e15 As a result, 1 showed the most potent inhibition of polar auxin transport (Fig. 6). Approximately 2.5 mg/plant treatment of 1 showed ca. 50% inhibition of polar auxin transport compared with that of control. In addition, hydroxypelenolide (2) also exhibited the inhibition of polar auxin transport, but the effect was much weaker than that of 1. Ketopelenolide a (3) and ketopelenolide b (4) did not show any such activity. These results suggest that the a-methylene-g-lactone moiety is important for the inhibition of polar auxin transport and that the hydroxyl group of the 10-membered ring may affect the intensity of the activity.

Conclusion
A new germacranolide-type sesquiterpene with an a-exomethylene-g-lactone moiety, artabolide (1), and three known compounds 2e4 were isolated from A. absinthium. From the results of spectroscopic analysis, the structure of 1 was determined as 3hydroxy-4,6,7(H)-germacra-1(10),11(13)-dien-6,12-olide. The absol ute configuration and the spatial conformation of 1 were also determined as 3R, 4S, 6R, 7S, and chairechair form by X-ray crystallography and the modified Mosher's method, respectively. By using the radish hypocotyl assay system method, 1 was shown to potently inhibit the polar transport of the plant hormone, IAA, although the other isolated compounds showed weak or no activity. This result indicated that the a-methylene-g-lactone moiety is important for the inhibition of polar auxin transport. To the best of our knowledge, this is the first report in which a sesquiterpene with an a-methylene-g-lactone moiety isolated from A. absinthium possessed the inhibition of polar auxin transport.

General experimental procedure
Optical rotations were recorded on a JASCO DIP-370 spectrometer. Ultraviolet (UV) spectra were recorded on a HITACHI U-2000A spectrometer. IR spectra were recorded on a JASCO FT/IR-300 spectrometer. 1 H and 13 C NMR spectra were measured and recorded on a BRUKER Avance 500 spectrometer in CDCl 3 . The resonances of CDCl 3 at d H 7.26 and d C 77.0 were used as internal references for the 1 H and 13 C NMR spectra. ESI-MS was recorded on a WATERS SYNAPT G2 mass spectrometer.

Bioassay for polar auxin transport
Measurement of polar auxin transport was performed according to the method previously reported with suitable modification. 11e15 Briefly, 2-cm hypocotyl segments were excised from 6-day-old radish seedlings and charged into 1.5-mL Eppendorf plastic tubes in inverted orientation. Twenty microliters of 1% agar containing 14 Clabeled IAA (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA.) with or without the test compound was supplied at the bottom of the tubes. After incubation at room temperature for 9e18 h, a 2-mm piece of the opposite side of the segment was cut and directly put into a vial of liquid scintillation cocktails. Radioactivity of the small slices was counted by a liquid scintillation counter.