Isoleojaponin, a New Halimane Diterpene Isolated from Leonurus japonicus

Leojaponin (2), a labdane diterpene, was isolated from the EtOH extract of the herb of Leonurus japonicus together with a new halimane diterpene named isoleojaponin (1). Isoleojaponin has a new diterpene skeleton with a unique cross-conjugated α,β-unsaturated ketone system, Their structures were elucidated by physical and spectroscopic analysis, and the relative configuration of the chiral C-9 carbon was determined by a computational method, and analysis of its possible biogenesis pathways.


Introduction
Leonurus japonics Houtt. (Lamiaceae) is an annual or biennial herbaceous plant widely distributed and cultivated in China. The dried herb is used in Traditional Chinese Medicine for the treatment of various diseases, especially menstrual disturbances, dysmenorrhea, and amenorrhea [1]. Recently, phytochemical studies on this plant have been reported [2][3][4][5][6][7][8][9][10]. Our previous investigation on two plants of the family of Lamiaceae resulted in the isolation of a number of new labdane diterpenes [11,12]. In OPEN ACCESS our research on psychoactive natural products from L. japonicus, two diterpenoids were isolated and identified. One of them was identified as leojaponin by comparison of the NMR data with those reported [2]. The other one was elucidated as its isomer and named isoleojaponin (1), it has transformed structure skeleton compared with a labdane-type compound such as leojaponin, the methyl group (Me-20) connected to C-10 has shifted to link with C-9, and the double bonds of ring-B were rearranged correspondingly. This paper describes the isolation and structure elucidation of these isolates (Figure 1).
Considering the stereochemistry of 1, several similar structures ( Figure 3) have been reported [13]: 9α-cyano-15,16-epoxy-7-hydroxylabda-7,13(16),14-trien-6-one (3) and 7-hydroxyhedychenone (4) have the same configuration at C-9, and an X-ray structural analysis of 3 has confirmed the C-9α position of the nitrile group. However, the labdane-type diterpeniods 3 and 4 have no similar cross-conjugated systems as 1, so the stereochemistry of 1 could not be decided by simple comparison with the reference pattern. In order to investigate the relative configuration of C-9, computation of specific optical rotation was used in this study, the model of both α and β Me-20 epimers were built, and the optical rotation values were obtained at the B3LYP/6-31+g (d) level in methanol via PCM model with Gaussian 09. The results were shown (Figure 4). We found that the computational specific optical rotation value for   The relative stereochemistry of compound 1 could not be completely established by application of NOE experiments, because the NOESY correlations between H2-1 and H3-20 could not determine α or β-orientation for Me-20, although the cross peak was observed ( Figure 3). However, it would be reasonable to deduce from the biogenesis pathways of the co-isolation of compound 1 and 2 (with definitely β-orientation for Me-20), a stereospecific 1,2-Me migration might result in the formation of 1 as shown (Figure 5), and this type of reaction was observed by biomimetic rearrangements of simplified labdane diterpenoids [14]. Therefore, compound 1 was identified as (S)-4-(2-(furan-3-yl)ethyl)-2hydroxy-3,4,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-1(4H)-one, and named isoleojaponin. Compound 2 was isolated as a yellowish oil. The 1 H-NMR and 13 C-NMR spectra of 2 were different from those of 1 (Table 1) and leoleorin C [11], but almost identical to those of leojaponin [2], so 2 was identified as leojaponin.

General
NMR spectra were recorded on a Bruker Avance III spectrometer operating at 400 MHz for 1 H-and 100 MHz for 13 C-HRESIMS were measured with Waters UPLC-LCT Premier XE and was controlled by MassLynx 4.1 software. Optical rotations were acquired with a WZZ-2ss automatic polarimeter (Shanghai Shenguang High Strength Bolts Co., Ltd, Shanghai, China). UV spectra were recorded on a Quawell Q5000 UV/Vis spectrophotometer. IR spectra were recorded on a Varian 800 FT-IR spectrophotometer. Column chromatography was performed with silica gel (200-300 mesh, Yantai Institute of Chemical Technology, Yantai, China), Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). HPLC separation was performed on an instrument (LC-3000, Beijing Chuangxintongheng Science & Technology Co., Ltd, Beijing, China) consisting of two pumps and a UV/Vis detector with an YMC-ODS-A (250 × 10 mm) semi-preparative column packed with C18 (5 μm).

Plant Material
The herb of Leonurus japonicus was purchased from Zhangye, Gansu Province of China in March, 2014. It was identified by one of the authors (Dr. H. Wu). A voucher specimen (Code: hkwu-aynu-20140301) was deposited at the Lab of Pharmaceutical Research, Anyang Normal University.

Conclusions
The EtOH extract of the herb of L. leonurus was purified by multiple chromatographic methods. From the oily mass of one fraction two diterpeniods were isolated and identified. One was leojaponin, which can protect primary cultured rat cortical cells from glutamate-induced toxicity [6]. The new compound is isoleojaponin, bearing a furanyl group and a unique cross-conjugated á,â-unsaturated ketone system with -OH near the carbonyl group. To the best of our knowledge, diterpenes with these constructs are unprecedented and the formation of isoleojaponin from leojaponin might be caused by a cascade carbocation rearrangement whereby leojaponin underwent a 1,2-Me shift of Me-20, and two double bonds migrated and the positions of the carbonyl and hydroxyl groups exchanged correspondingly ( Figure 5).