A new rearranged tricyclic abietane diterpenoid from Salvia chloroleuca Rech. f. & Allen

Phytochemical investigation of the roots of Salvia chloroleuca led to the isolation and identification of a new rearranged abietane diterpenoid (1). Its structure was elucidated by interpretation of the 1D and 2D NMR spectra and completed by the analysis of the HR-ESI-MS data. Compound 1 is the secondly reported compound on a rearranged tricyclic abietane (4,5-seco-5,10-friedo-abietane) diterpenoid with a ketal functionality between C-2 and C-11. A plausible biosynthetic pathway of 1 was also proposed.


Introduction
Salvia is the largest genus of the Lamiaceae family, which is widely used in flavouring and traditional medicine throughout the world (Wu & Li 1982). From a phytochemical perspective, the main secondary metabolites of Salvia species are terpenoids and flavonoids (Topc u 2006;Rustaiyan et al. 2007). Among terpenoids, diterpenes are the largest group, classified into subgroups, such as abietane, clerodane, pimarane and labdane (Wu et al. 2012). Abietanes and rearranged abietanes comprise the largest group of known Salvia diterpenoids (Wu et al. 2012).
Abietanes from Salvia species are known to possess various biological activities such as antibacterial, antiprotozoal, antioxidant, anti-inflammatory, antitumour, antihypertensive, as well as phytotoxic and DNA damaging properties (Ryu et al. 1997;Topc u & Ahmet 2007;Nejad Ebrahimi et al. 2013;Akaberi et al. 2015). There are 58 Salvia species growing naturally in Iran, 17 of which are endemic (Rechinger 1987). Salvia chloroleuca Rech. f. & Allen is an endemic perennial herb that grows wild in the central and north-east of Iran (Rechinger 1987). A literature survey revealed that there were no reports around the chemical constituents of S. chloroleuca, with the exceptions of analysis of the essential oil (Yousefzadi et al. 2008;Jassbi et al. 2012). In our efforts to discover new and potentially bioactive secondary metabolites from Iranian Salvia species (Moridi Farimani et al. 2012Nejad Ebrahimi et al. 2014;Moridi Farimani & Mazarei 2014, Moridi Farimani & Miran 2014, we investigated the acetone extract of the roots of S. chloroleuca. Our studies led to the isolation and structural elucidation of a new rearranged abietane diterpenoid with a ketal functionality between C-2 and C-11.

Results and discussion
The acetone extract obtained from the roots of S. chloroleuca was separated by normal and reverse phase chromatography to afford a new rearranged abietane diterpenoid. Compound 1 (Figure 1) was isolated as a yellow gummy material. It had a molecular formula of C 21 H 26 O 3 as indicated by HR-ESI-MS (m/z 349.1779 [M þ Na] þ , calcd. 349.1773). The molecular formula accounted for nine indices of hydrogen deficiency. Its IR spectrum displayed a conjugated carbonyl group absorbance at 1690 cm 21 . The 13 C NMR spectrum showed 21 carbon signals, which were resolved through a DEPT experiment into 6 methyl, 1 methylene, 6 methine and 8 quaternary carbons. 13 C NMR data showed signals for a tetrasubstituted double bond (dc 133.1, 134.8), four trisubstituted double bonds (dc 125.9, 137.7; 131.7, 138.3; 127.6, 129.8; 137.8, 141.2) and one carbonyl carbon (dc 197.7). Three carbon signals at dc 94.9 (C), 66.9 (CH) and 50.6 (CH 3 ) indicated the presence of oxygen-bearing sp 3 carbons. The absence of other sp or sp 2 carbon signals implied that the structure of 1 contained three rings, including one cyclic ether, which is compatible with the molecular formula of C 21 H 26 O 3 . From the 1 H and 13 C NMR spectra, an abietane diterpenoid skeleton could be concluded. In the 1 H NMR spectrum, three olefinic methyl doublets at d H 1.80 (d, J ¼ 1.0 Hz), 1.83 (d, J ¼ 1.0 Hz) and 2.22 (d, J ¼ 1.0 Hz), and two methyl doublets at d H 1.14 (d, J ¼ 6.8 Hz) and 1.16 (d, J ¼ 6.8 Hz) were observed. The latter two doublets, together with a methine signal at d H 2.89 (sept, J ¼ 6.8 Hz), were indicative of an isopropyl moiety. Besides the isopropyl group, the presence of 3 methyl singlets in the 1 H 2 121 Natural Product Research NMR and 21 carbon signals in the 13 C NMR spectra revealed an abietane diterpenoid structure (Topcu & Ulubelen 1996). Comparison of the NMR data of 1 with those of diterpenoids previously isolated from S. sahendica indicated that 1 likely had the same carbon skeleton as sahandone (Jassbi et al. 2006). Small but diagnostic differences in the 1 H NMR spectra of 1 and sahandone were observed, such as the resonance of three methine protons at d H 7.04, 7.16 and 7.06 was paramagnetically shifted (Dd ¼ þ0.16 and þ 0.08 and þ 0.11 ppm, respectively) in comparison to those of sahandone. In the HMBC spectrum of compound 1 (Figure S1), diagnostic cross-peaks were observed between H-15 (d H 2.89, sept, J ¼ 6.8 Hz) and C-13 (d C 141.2), C-14 (d C 137.8) and C-12 (d C 197.7), and also between H-14 (d H 7.04, s) and C-12 (d C 197.7). Other key HMBC correlations were seen from H-7 (d H 7.06, d, J ¼ 7.6 Hz) to C-14 (d C 137.8), and also from H-14 (d H 7.04, s) to C-7 (d C 127.6). Furthermore, HMBC correlation between H-12 and C-11 in sahandone was not observed here. These differences were in agreement with the location of the carbonyl group on C-12. Unambiguous assignment of 1 H and 13 C NMR data was achieved by a combination of HSQC, COSY and HMBC experiments. The relative configuration of 1 was determined from a NOESY spectrum and was in accord with that of sahandone. In the NOESY spectrum, diagnostic cross-peaks between H-2, MeO and H-1a confirmed their location on the same face of the molecule. Therefore, the structure of compound 1 was elucidated to be 8-isopropyl-9a-methoxy-4-methyl-2-(2-methylprop-1-en-1-yl)-2,3dihydrobenzo[de ]chromen-9(9aH)-one, and named as sahandone B.
Several 4,5-seco-bicyclo-and tricyclo-abietanes have been isolated from Salvia species (Wu et al. 2012), both ortho-and para-quinone scaffolds could be seen among them. Four 4,5seco-bicyclo-abietanes have been obtained from the roots of Salvia prionitis, and showed significant cytotoxic activity against several cell lines (Chen et al. 2002;Chang et al. 2005). To the best of our knowledge, sahandone (Jassbi et al. 2006) and sahandone B (1) were the only structures that have a rearranged tricyclic abietane diterpenoid with a ketal functionality between C-2 and C-11. From a biogenetic point of view, this new scaffold may derive from an ortho-quinone moiety such as miltirone via the Wagner -Meerwein rearrangement as a key step ( Figure 2). Sahandone (Jassbi et al. 2006) may be initiated from a para-quinone isomer such as 12-deoxyroyleanone.

Experimental 3.1. General experimental procedures
Optical rotations were measured using a Perkin-Elmer 341 polarimeter (PerkinElmer, Waltham, MA, USA). IR spectra were recorded on a Shimadzu IR-450 instrument with KBr pellets (Shimadzu, Kyoto, Japan). 1 H and 13 C NMR, and 1 H-1 HCOSY, HSQC, NOESY and HMBC experiments were measured on a Bruker Avance TM 500 DRX (500 MHz for 1 H and 125 MHz for 13 C) spectrometer with tetramethylsilane as an internal standard (Bruker Biospin, Rheinstetten, Germany). HR-ESI-MS spectra were acquired on a Bruker microTOF ESI-MS system (Bruker, Bremen, Germany). TLC was conducted on Merck silica gel 60 F254 pre-coated plates, and spots were visualised by spraying anisaldehyde -H 2 SO 4 reagent followed by heating. Semipreparative HPLC separations were conducted on a Knauer system equipped with a PDA detector, and with a Vertex C 18 column (250 mm £ 20 mm i.d. Knauer, Berlin, Germany).

Plant material
The roots of S. chloroleuca were collected in May 2012 from the suburbs of Tehran. The plant was authenticated by Dr Y. Ajanii and a voucher specimen was deposited at the Herbarium of Complex of Academic Center for Educational and Cultural Research under number ACECR-244.

Supplementary material
Experimental details relating to this article are available online.