Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Labdane diterpenoids from Salvia reuterana Mahdi Moridi Farimani ⇑, Mansour Miran Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, , Tehran, Iran
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 8 August 2014 Available online xxxx Keywords: Salvia reuterana Labiatae Halogenated labdane diterpenoid SAR
a b s t r a c t Three labdane diterpenoids, 14a-hydroxy-15-chlorosclareol (1), 14a-hydroxy-15-acetoxysclareol (2), and 6b-hydroxy-14a-epoxysclareol (3), together with the known diterpenoids sclareol (4), 6b-hydroxysclareol (5), and 14a-epoxysclareol (6), as well as other common plant constituents were isolated from the n-hexane extract of aerial parts of Salvia reuterana. The structures of the new compounds were established by extensive 1D and 2D NMR spectroscopic techniques. Compound 1 is the first example of a halogenated terpenoid in the genus Salvia. Compounds 1–6 were also tested for their inhibitory activity toward HeLa and MCF-7 cell lines. Preliminary structure–activity relationship studies indicated that double bond moiety in sclareol is an essential feature for activity and modification of this moiety significantly decreased the cytotoxic activity of the resulting compounds. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The genus Salvia is a rich source of structurally diverse terpenoids (Chang et al., 1990; Rodriguez-Hahn et al., 1988, 1990). Among these, numerous diterpenoids with promising bioactivities, such as anticancer, cardiotonic and topoisomerase inhibitor properties, have been reported from Salvia species (Meng et al., 2001; Wang et al., 2004; Munro et al., 2005). The most abundant diterpenoids in the genus are abietanes and rearranged abietanes (Wu et al., 2012). Labdane diterpenoids are rather rare in Salvia species, although they are frequently found in other genera of the Lamiaceae (Kintzios, 2000). Up to now, only 18 labdane diterpenoids have been reported from Salvia (Wu et al., 2012). Salvia reuterana Boiss is an endemic species which grows in the highlands of central Iran (Rechinger, 1987). Its common name in Persian is ‘‘Mariam Goli Esfahani’’ (Mozaffarian, 1990), and the aerial parts of the plant have been used in Iranian folk medicine as sedative and anxiolyticn (Zargari, 1990). With exception of an analysis of the essential oil (Mirza and Sefidkon, 1999), S. reutereana has not been studied from a phytochemical viewpoint. In continuing our effort on discovery of novel and potentially bioactive secondary metabolites from Iranian Salvia species (Farimani et al., 2011, 2012, 2013; Moghaddam et al., 2010), we investigated the n-hexane extract from aerial parts of S. reuterana. We here report on the isolation, structure elucidation and biological activity of three new labdane diterpenoids, including a chlorinated compound.
The phytochemical investigation of n-hexane extract from S. reuterana afforded three new labdane diterpenoids (1–3) (Fig. 1), together with six known compounds namely: sclareol (4), 6b-hydroxysclareol (5), 14a-epoxysclareol (6), b-sitosterol, oleanolic acid, 5-hydroxy-7,4’-dimethoxyflavone. Compound 1 had a molecular formula C20H37O3Cl (HR-ESI-TOFMS (m/z 383.2330 [M+Na]+, calcd 383.2323) and EIMS(m/z 360 [M]+ and 362 [M+2]+ in the ratio 3:1) accounting for two degrees of hydrogen deficiency. Its IR spectrum showed the presence of hydroxyl (3423 cm1) and alkyl halide (732 cm1) groups. It did not show conjugation in its UV spectrum. The 13C NMR spectrum (Table 1) showed 20 carbon resonances which were attributed with the aid of HSQC and DEPTQ spectra to five methyl, eight methylene, three methine and four quaternary carbons. Two carbon signals at dc 75.4 and 75.7 indicated the presence of oxygen bearing sp3 carbons. Another oxygenated carbon resonance at dc 79.0 accounted for the carbon carrying a hydroxyl group with the carbinolic methine proton coming at dH 3.61 (1H, dd, J = 1.6, 9.7 Hz). The signal at dc 47.7 accounted for a chloromethylene carbon with the corresponding methylene protons resonating at dH 3.50 and 3.91, respectively (Anjaneyulu and Rao, 2000; Konishi et al., 1999). The molecule thus was bicyclic and appeared to be a labdane diterpenoid. The 13C chemical shifts of 1 were assigned with the aid of a HMQC spectrum, and by comparison with spectral data of sclareol, and location of the functional groups was confirmed by HMBC connectivities (Fig. 2). The resonance of C-13 (dc 75.7) showed correlations with H-14 (dH 3.61), CH3-16 (dH 1.13) and CH2-12 (dH 1.59 and 1.76). Other key HMBC correlations were
⇑ Corresponding author. Tel.: +98 21 29904043; fax: +98 21 22431783. E-mail address: [email protected] (M. Moridi Farimani). http://dx.doi.org/10.1016/j.phytochem.2014.08.024 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
2
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
20
17
1 8
OH
16
O
R
11
10
H
OH
16
1
OH
OH
4
H
H 18
1 R = Cl 2 R = OAc
H
9
8
5
15
Cl
O
OH
OH
OH H
19
R
3 R = OH 6 R=H
OH
Fig. 2. Selected HMBC correlations of compounds 1 (left) and 3 (right).
OH OH
H
17 OH
OH
4 19
14
20
15
OH
12
R
4 R=H 5 R = OH Fig. 1. Compounds 1–6.
observed from C-14 (dc 79.0) to CH2-15 (dH 3.50 and 3.91), CH3-16 and CH2-12. On the other hand, the chloromethylene carbon (dc 47.7) exhibited a long-range correlation with H-14. Similarly, the methyl protons at dH 0.84, 0.87, 0.90, 1.13 and 1.18 showed the
respective correlations as noted in Fig. 2. A b-orientation of CH3-17 and CH3-20 and CH3-16 was deduced from the NOESY spectrum which showed NOE contacts between CH3-20 (dH 0.87), CH3-17 (dH 1.18) and H-11b (dH 1.39), between H-11b, CH3-16 (dH 1.13) and CH3-17, and between CH3-17 and H-7b (dH 1.84) (Fig. 3). Diagnostic cross peaks between H-5a (axial) and H-9a confirmed their cofacial orientation. The stereochemistry at C-14 was also determined from the NOESY data: H-14 (dH 3.61) showed correlations with CH3-16b (dH 1.13), H-12a (dH 1.76), H-12b (dH 1.59), as well as with H-11b (dH 1.39). On the other hand both of the chloromethylene protons (dH 3.50 and dH 3.91) showed strong cross peaks with CH3-16b, H-12a (dH 1.76), and H-12b (dH 1.59). Overall, the data indicated that H-14 had to be in b–position and, assuming the R configuration at C-13 as for sclareol (Soucek and Vlad, 1963), the configuration at C-14 had to be R. To confirm this, the absolute configuration of the secondary hydroxyl group at C-14 was determined by the application of a modification of Mosher’s method (Kusumi, 1993; Ohtani et al., 1991) to 1. Treatment of 1 with (R)and (S)-MTPA chloride in the presence of pyridine afforded the
Table 1 H and 13C NMR Data of Compounds 1–3a (500 MHz for dH; 125 MHz for dC).
1
Position
1a 1b 2a 2b 3a 3b 4 5a 6a 6b 7a 7b 8 9a 10 11a 11b 12a 12b 13 14b 15a 15b 16 17 18 19 20 CH3CO CH3CO a b
1
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
41.1
1.01 dt (12.1, 3.1) 1.72b 1.65b 1.45b 1.22b 1.42b
39.6
0.88b 1.66 dt (13.5, 5.0) 1.52b 1.36b 1.07b 1.31b
41.6
0.87b 1.60b 1.62b 1.37b 1.06b 1.26b
19.5 43.2 34.2 57.6 21.5 44.8 75.4 62.9 40.5 19.4 43.6 75.7 79.0 47.7 21.6 24.1 33.9 22.0 16.1
0.96 dd (12.2, 3.0) 1.67b 1.34b 1.47b 1.84 dt (12.2, 4.2) 1.09b 1.56b 1.39b 1.76b 1.59b 3.61 3.50 3.91 1.13 1.18 0.90 0.84 0.87
dd (9.7, 1.6) dd (11.2, 9.7) dd (11.2, 1.6) s s s s s
18.3 41.9 33.2 56.0 20.4 44.0 75.5 62.0 39.1 18.0 39.3 75.4 76.3 65.8 22.8 24.1 33.3 21.5 15.4 20.4 171.3
0.86 d (11.6) 1.57b 1.19b 1.35b 1.76 dt (12.8, 4.3) 1.05b 1.48b 1.28b 1.53b 1.37b 3.60 3.99 4.21 1.12 1.11 0.80 0.72 0.73 2.03
br d (6.8) dd (11.5, 8.6) dd (11.5, 2.5) s s s s s s
18.3 44.0 33.3 57.0 68.1 50.8 73.3 62.1 39.0 17.9 41.0 70.2 58.2 43.6 24.6 25.5 33.0 23.1 16.4
0.86b 4.40 br d (2.3) 1.53b 1.91 br d (13.8) 1.08b 1.54b 1.40b 1.55b 1.55b 2.83 2.63 2.74 1.18 1.32 0.90 1.11 1.10
dd (5.0, 3.6) dd (7.0, 3.6) dd (7.0, 5.0) s s s s s
Compound 1 in CD3OD; compound 2 in CDCl3/CD3OD; compound 3 in CDCl3. d values were extracted from COSY, HSQC and HMBC experiments. Overlapping signals.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
3
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
16
H
H
H
H
H
H
5
H
H
H
H 7
Cl
HO
11
H
15 13
17
20
H
H H
H
OH
H
OH
H H
H
Fig. 3. Key NOESY correlations of 1.
-0.08 -0.04 -0.03 -0.04 -0.02 -0.01
-0.11 OMTPA
Cl
-0.10
OH
+0.12 +0.12
OH -0.03 -0.03
-0.02
Fig. 4. 1H NMR chemical shift difference values for the MTPA esters of 1 [Dd: dS(1a) dR(1b)].
(S)-MTPA ester (1a) and (R)-MTPA ester (1b), respectively. The observed chemical shift differences (DdS–R, Fig. 4) unambiguously indicated the absolute configurations of C-14 to be R. Hence, compound 1 was identified as 14R-hydroxy-15-chlorosclareol. Given that chlorohydrine 1 could be possibly formed during the extraction and isolation process from the corresponding epoxide, we repeated extraction of plant material with hexane and then subjected the extract to HPLC-ESI TOF analysis (Fig. S10 Supporting Information). The chromatogram showed a peak at Rt 19.1 min with m/z 383. Rt and MS data corresponded with 1 which was analyzed separately under identical conditions. Thus, we conclude that 1 was a genuine plant metabolite. Compound 2 had an elemental formula C22H40O5 (HR-ESI-TOFMS m/z 407.2761 [M+Na]+; calcd 407.2773), which accounted for three degrees of hydrogen deficiency. Its IR spectrum exhibited absorption bands for the hydroxyl (3447 cm1) and carbonyl (1732 cm1) functions. The 1H and 13C NMR spectrum displayed features similar to those of 1. However, in the 13C NMR spectrum of 2, resonances of an additional methyl (dC 20.4 ppm) and a quaternary carbon (dC 171.3 ppm) were observed, and the methylene signal observed in 1 at dC 47.7 ppm appeared at higher field in 2 (dC 65.8 ppm). The resonances of C-14 and C-12 were observed at dC 76.3, and 39.3 respectively (upfield shifts of ca. 2.7 and 4.3 ppm compared to 1), while the resonance of CH3-16 appeared at dC 22.8 (downfield shift of 1.2 ppm compared to 1). In the 1H NMR spectrum of 2, an additional methyl singlet appeared at dH 2.03, while the CH2-15 resonances appeared downfield (dH 3.99 and 4.21 ppm, respectively). These differences in the NMR data, together with the absence of chlorine in the molecular formula, suggested that compound 2 had an acetoxy group at C-15 instead of the chlorine observed in 1. HMBC correlations between the carbonyl resonance at dC 171.3 ppm and the carboxymethylene protons (dH 3.99 and 4.21), and the methyl protons (dH 2.03 ppm)
confirmed the location of the acetoxy group. Complete assignments of NMR spectral data were obtained from 1H–1H COSY, HMQC, and HMBC spectra, while the relative configuration was established from the NOESY spectrum. Compound 2 was therefore identified as 14a-hydroxy-15-acetoxysclareol. The HR-ESI-TOFMS of 3 showed a molecular ion peak at m/z 363.2516 [M+Na]+, consistent with a molecular formula C20H36O4 (calcd 363.2507). In the IR spectrum the absorption bands at mmax 1273 and 867 cm1 besides the characteristic band of the hydroxyl group at 3430 cm1 was consistence with the presence of the epoxide functional group in the molecule. In the 1H NMR spectrum of 3, the signals at dH 2.83 (dd, J = 3.6, 5.0 Hz), 2.74 (dd, J = 5.0, 7.0 Hz), and 2.63 (dd, J = 3.6, 7.0 Hz) were indicative of a terminal epoxide moiety in a side chain. The 13C NMR spectrum (Table 1) showed 20 carbon signals which were assigned through a DEPT experiment to five methyl, seven methylene, four methine, and four quaternary carbons. When comparing the 13C NMR data of 3 with those of sclareol, the methine carbon (dC 58.2) and a terminal methylene (dC 43.6) instead of olefinic signals in sclareol (dC 147.5 and 110.7) (Kouzj and McChesney, 1991) corroborated the presence of an epoxide at C-14/C-15. 1H and 13C NMR data strongly resembled to those of 14R-epoxysclareol, a diterpenoid from Astragalus brachystachys (Jassbi et al., 2002). However, the 13C NMR spectrum of 3 showed the presence of an additional methine at dC 68.1 instead of the methylene group (C-6). Hence, the methylene was replaced by an oxygenated methine. The signals of C-7 (dC 50.8) and C-5 (dC 57.0) were paramagnetically shifted (Dd + 6.7 and +0.9 ppm, respectively) in comparison to those of 14R-epoxysclareol. Also the resonances of neighboring H-7a (dH 1.53), H-7b (dH 1.91), and H-5a (dH 0.86) were shifted (Dd = +0.15, +0.10, and 0.04 ppm, respectively). HMBC correlations between H-5a, H-7a with C-6, and between H-6, C-8, and C-10 (Fig. 2) confirmed the location of the hydroxyl group. Diagnostic COSY correlations were observed between H-6 and H-7a and H-7b, and between H-6 and H-5. The relative configuration of the hydroxyl group at C-6 was determined as b on the basis of the magnitude of the vicinal coupling constants of the H-6 resonance (dH 4.40, brd, J = 2.3 Hz). NOESY contacts of H-6 with H-5 and H-7ax were observed. The 1H chemical shifts of CH3-17, CH3-19 and CH3-20 (dH 1.32, 1.11 and 1.10, respectively) appeared downfield (dH 0.19, 0.27, and 0.34) relative to those of 14R-epoxysclareol. These differences were in agreement with an axial orientation of the hydroxyl group at C-6 (Jassbi et al., 2002). Thus, the structure of 3 was established as 6b-hydroxy-14a-epoxysclareol. As for 1, compounds 2 and 3 were also detected in the n-hexane extract of the dried whole plants of S. reuterana obtained under mild conditions (Fig. S28 Supporting Information), indicating that
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
4
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
Table 2 Cytotoxic activities of compounds 1–6 against two human tumor cell lines.
a b
Compound
HeLa (IC50 lg/mL)a
MCF-7 (IC50 lg/mL)
1 2 3 4 5 6 Paclitaxelb
37.17 ± 0.76 57.16 ± 1.44 58.72 ± 1.51 16.60 ± 0.49 53.23 ± 0.73 59.41 ± 1.33 0.003
42.16 ± 0.95 46.74 ± 1.03 37.29 ± 1.06 32.65 ± 1.15 36.98 ± 1.12 62.07 ± 2.01 0.028
Data are expressed as mean ± standard deviation (n = 3). Positive control.
they must be natural products rather than artifacts from the isolation procedure. Halogenated diterpenoids are mainly found in marine algae and invertebrates (Dembitsky et al., 2002; Wang et al., 2013), but are rare in terrestrial plants. Four chlorine-containing labdane diterpenoids were isolated from Excoecaria agallocha, a seashore plant of the Euphorbiaceae family (Anjaneyulu and Rao, 2000; Konishi et al., 1999), while a chloroditerpene lactone was reported from Gutierrezia dracunculoides (Asteraceae) (Cruse et al., 1971). From the Lamiceae family six chlorine-containing diterpenes have been reported in Teucrium and Ajuga species (Dembitsky et al., 2002; Wang et al., 2013; Xie et al., 1992; Beauchamp et al., 1996; Bruno et al., 1995). However, compound 1 is the first example of a chlorinated diterpene from the genus Salvia. Sclareol has demonstrated a significant cytotoxic activity against human cell lines (Dimas et al., 1999; Dimas et al., 2006). Compounds 1–6 were evaluated for their in vitro cytotoxic activity against HeLa (human epitheloid cervix carcinoma) and MCF-7 (human breast adeno-carcinoma) cell lines. The results of the cytotoxicity studies are presented in Table 2. A preliminary SAR study was carried out against this short series of labdane diterpenoids. As demonstrated in Table 2, it can be stated that all of analogues displayed weaker cytotoxic activity compared to parent compound, sclareol (4). Drastic decrease or lack in cytotoxicity of compounds 1–3 and 5, 6 against tested cell lines, implying that double bond is necessary for the activity. However, replacement of this moiety with an epoxide (compounds 3 and 6) or others substituent (compounds 1 and 2) decreased the activity against two cell lines. Furthermore, the sharp decrease in cytotoxic activity of compound 5 (especially in HeLa cell line) indicated that the bioactivity is very sensitive to change in 6-position. Nevertheless, among the compounds 3 and 6, the presence of a hydroxyl group at the 6-position seemed to increase cytotoxic activity of compound 3 (especially against MCF-7 cell line). It became clear from these results that the double bond moiety in these compounds is an essential feature for activity; however, the effect of 6-hydroxyl group on the cytotoxic activity depends on the functional groups at C14–C15 bond. 3. Conclusions Three new labdane diterpenoids were isolated from the aerial parts of S. reuterana, including one which contained a chlorohydrine moiety in its structure. HPLC-ESI TOF analysis of the fresh crude extract of the plant showed that 1 was a genuine plant metabolite. Halogenated diterpenoids are mainly found in marine algae and invertebrates (Dembitsky et al., 2002; Wang et al., 2013), but are rare in terrestrial plants. Compound 1 is the first example of a chlorinated diterpene from the genus Salvia. Preliminary structure–activity relationship studies indicated that double bond moiety in sclareol is an essential feature for activity and modification of this moiety significantly decreased the cytotoxic activity of the resulting compounds.
4. Experimental 4.1. General Experimental Procedures Optical rotations were measured using a JASCO P-2000 automatic digital polarimeter. IR spectra were recorded on a Bruker Tensor 27 spectrometer. UV spectra were recorded using a Shimadzu UV-2501PC spectrophotometer. NMR spectra were recorded at a target temperature of 18 °C on a Bruker Avance III 500 MHz spectrometer operating at 500.13 MHz for 1H, and 125.77 MHz for 13C. A 1 mm TXI-microprobe with z-gradient was used for 1H-detected experiments. 13C NMR spectra were recorded with a 5 mm BBOprobe head with z-gradient. Spectra were analyzed using Bruker TopSpin 3.1 software. Deuterated solvents for NMR (100 at% D) were purchased from Armar Chemicals. HPLC separations were carried out on an series 1100 system consisting of degasser, binary high pressure mixing pump, column thermostat and photodiode array (PDA) detector (Agilent Technologies; Waldbronn, Germany). Analysis was performed on a Waters SunFireÒ C18 column (3.5 lm, 3.0 150 mm), using a gradient of MeCN/H2O 30:70 ? 0:100 in 30 min, and a flow rate of 0.4 mL/min. High resuloution MS spectra were recorded using a Bruker microTOF ESI-MS system in the range of m/z 150–1500 in positive mode. Nitrogen was used as nebulizing gas at a pressure of 2.0 bar, and as drying gas at a flow rate of 9.0 l/min (drying gas temperature 240 °C). Capillary voltage was at 4500 V, endplate offset at 500 V, hexapole at 250.0 V, skimmer 1 at 40 V and skimmer 2 at 22.5 V. Instrument calibration was performed using a reference solution of sodium formate 0.1% in isopropanol/water (1:1) containing 5 mM sodium hydroxide. Data acquisition and processing were performed using HyStar™ 3.0 and EsquireControl™ 5.2 (Bruker Daltonics). 4.2. Plant material The aerial parts of Salvia reuterana were collected in July 2011 from the northern hilly areas of Tehran, Iran. A voucher specimen (MPH-1321) has been deposited in the herbarium of the Medicinal Plant and Drug Research Institute (MPH) of Shahid Beheshti University, Tehran, Iran. 4.3. Extraction and isolation The air-dried aerial parts of S. ruterana (3.0 kg) was crushed and extracted with n-hexane (3 15 L) by maceration at room temperature. Extract was concentrated in vacuo, to afford 130 g of a dark gummy residue. The residue was separated on a silica gel column (230–400 mesh, 850 g) with a gradient of n-hexane–EtOAc (100/ 0 to 0/100) as eluent, followed by increasing concentration of MeOH (up to 25%) in EtOAc. On the basis of TLC analysis, fractions with similar composition were pooled to yield 27 combined fractions. From fraction 5 [eluted with n-hexane–EtOAc (85:15)], crude crystals were obtained, which were recrystallized from n-hexane to afford b-sitosterol (1 g). Fractions 7 and 8 [4.0 g, eluted with n-hexane–EtOAc (75:25)] were combined and subjected to silica gel column chromatography (70–230, 220 g), eluted with CHCl3 followed by a gradient of CHCl3–Me2CO (up to 16%), to give four subfractions (7a–7e). Subfraction 7a was recrystallized from Me2CO to afford 5-hydroxy-7,40 -dimethoxyflavone (150 mg). Subfraction 7b, also contained a crude solid, which was triturated with EtOAc to yield oleanolic acid (28 mg). Fraction 9 [eluted with n-hexane–EtOAc (70:30)], was triturated with CHCl3 to yield an insoluble solid, which was recrystallized from CHCl3 to afford sclareol (2 g). Fraction 13 [0.8 g, eluted with n-hexane–EtOAc (50:50)] was applied to a silica gel column (70–230 mesh), with CHCl3-Me2CO-MeOH (88:8:4) as eluent, and then was purified by
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
recrystallization from Me2CO to afford compound 1 (10 mg). Fraction 16 [1.2 g, eluted with n-hexane–EtOAc (35:65)], was chromatographed on a silica gel column (70–230 mesh), eluted with CHCl3-Me2CO (75:25, 70:30, 65:35), to give 14a-epoxysclareol (25 mg). Fraction 17 [2.0 g, eluted with n-hexane–EtOAc (20:80)], was subjected to column chromatography over a silica gel column, eluted with CHCl3 followed by increasing amounts of MeOH (up to 15%), and then was purified by recrystallization from Me2CO to afford 6b-hydroxysclareol (20 mg). Fraction 21 [0.9 g, eluted with n-hexane–EtOAc (5:95)] was separated on a silica gel column with CHCl3–Me2CO–MeOH (90:5:5), to afford seven subfractions (21a21g). Subfraction 21b was recrystallized from MeOH to yield compound 2 (21 mg). Subfraction 21f was further purified on a small silica gel column, eluted with CHCl3–Me2CO (50:50), to afford compound 3 (8 mg). 4.4. Spectroscopic data of new compounds 4.4.1. 14R-hydroxy-15-chlorosclareol (1) White powder; mp 152–153 °C; ½a25 D ¼ 19:6 (c 0.2, CHCl3); UV (MeOH) kmax (log e) 223 (2.57); IR (KBr) mmax 3423, 2926, 1461, 1383, 1261, 1090, 732 cm1; for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z 383.2330 [M+Na]+, calcd for 383.2323. 4.4.2. 14a-hydroxy-15-acetoxysclareol (2) White powder; mp 136–138 °C; ½a25 D ¼ 12:5 (c 0.2, CHCl3); UV (MeOH) kmax (log e) 225 (2.82); IR (KBr) mmax 3447, 2927, 1732, 1460, 1380, 1259, 1099, cm1; for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z 407.2761 [M+Na]+,calcd for 407.2773. 4.4.3. 6b-Hydroxy-14a-epoxysclareol (3) White powder; mp 126–128 °C; ½a25 D ¼ 8:5 (c 0.2, CHCl3); UV (MeOH) kmax (log e) 228 (2.58); IR (KBr) mmax 3430, 2924, 1460, 1379, 1273, 1146, 1079, 867 cm1; for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z 363.2516 [M+Na]+ calcd for 363.2507. 4.5. Cytotoxicity assay The human epithelioid cervix carcinoma (HeLa) and human breast adenocarcinoma (MCF-7) cell lines were purchased from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran (Tehran, Iran), and maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 lg/ml streptomycin. These cells were kept at 37 °C in a humidified atmosphere containing 5% CO2. Compounds 1–6 were dissolved in DMSO to make a stock of 1 mg/mL and further diluted to final concentrations of 10–100 lg/mL with the serum free culture medium. 4.6. Cell viability assay Cell viability was determined using the MTT assay. Briefly, 2.5 104 cells were seeded in 96-well plates at 37 °C with 5% CO2 for overnight incubation and treated with appropriate concentrations of compounds 1–6 for 24 h. The cells were then incubated with a serum-free medium containing MTT at a final concentration of 0.5 mg/mL for 4 h. The dark formazan crystals formed were dissolved in DMSO and the absorbance was measured at 570 nm. Acknowledgments Financial support by the Shahid Beheshti University Research Council is gratefully acknowledged. All spectra were performed at the Department of Pharmaceutical Sciences, Division of Pharmaceutical Biology, University of Basel. The kind assistance of Prof. M. Hamburger, Dr. S.N. Ebrahimi, and all other staff is gratefully
5
appreciated. The authors also express thanks to Dr. A. Sonboli for taxonomic identification of the plant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 08.024. References Anjaneyulu, A.S.R., Rao, V.L., 2000. Five diterpenoids (agallochins A-E) from the mangrove plant Excoecaria agallocha Linn. Phytochemistry 55, 891–901. Beauchamp, P.S., Bottini, A.T., Caselles, M.C., Hope, H., 1996. Neo-clerodane diterpenoids from Ajuga parviflora. Phytochemistry 43, 827–834. Bruno, M., Fazio, C., Piozzi, F., Savona, G., Rodriguez, B., Torre, M.C., 1995. Neoclerodane diterpenoids from Teucrium racemosum. Phytochemistry 40, 505–507. Chang, H.M., Cheng, K.P., Choang, T.F., Chow, H.F., Chui, K.Y., Hon, P.M., Lau, F.W., Yang, Y., Zhong, Z.P., 1990. Structure elucidation and total synthesis of new tanshinones isolated from Salvia miltiorrhiza Bunge (Danshen). J. Org. Chem. 55, 3537–3543. Cruse, W.B.T., James, M.N.G., Al-Shamma, A.A., Beal, J.K., Doskotch, R.W., 1971. The molecular structure of gutierolide, a novel chloro-diterpenoid lactone.. J. Chem. Soc., Chem. Comm., 1278–1280. Dembitsky, V.M., Tolstikov, A.G., Tolstikov, G.A., 2002. Natural halogenated diterpenoids. Chem. Sust. Develop. 10, 253–264. Dimas, K., Kokkinopoulos, D., Demetzos, C., Vaos, B., Marselos, M., Malamas, M., Theodoros, T., 1999. The effect of sclareol on growth and cell cycle progression of human leukemic cell lines. Leuk. Res. 23, 217–234. Dimas, K., Papadaki, M., Tsimplouli, C., Hatziantoniou, S., Alevizopoulos, K., Pantazis, P., Demetzos, C., 2006. Labd-14-ene-8,13-diol (sclareol) induces cell cycle arrest and apoptosis in human breast cancer cells and enhances the activity of anticancer drugs. Biomed. Pharmacother. 60, 127–133. Farimani, M.M., Bahadori, M.B., Taheri, S., Ebrahimi, S.N., Zimmermann, S., Brun, R., Amin, G., Hamburger, M., 2011. Triterpenoids with rare carbon skeletons from Salvia hydrangea: antiprotozoal activity and absolute configurations. J. Nat. Prod. 74, 2200–2205. Farimani, M.M., Taheri, S., Ebrahimi, S.N., Bahadori, M.B., Khavasi, H.R., Zimmermann, S., Brun, R., Hamburger, M., 2012. Hydrangenone, a new isoprenoid with an unprecedented skeleton from Salvia hydrangea. Org. Lett. 14, 166–169. Farimani, M.M., Ebrahimi, S.N., Salehi, P., Bahadori, M.B., Sonboli, A., Khavasi, H.R., Zimmermann, S., Kaiser, M., Hamburger, M., 2013. Antitrypanosomal triterpenoid with an e-lactone e-ring from Salvia urmiensis. J. Nat. Prod. 76, 1806–1809. Jassbi, A.R., Zamanizadehnajari, S., Aberoomand, P., Tahara, S.Z., 2002. Antibacterial diterpenoids from Astragalus brachystachys. Naturforsch 57c, 1016–1021. Kintzios, S.E., 2000. Sage: the genus Salvia. Harwood Academic Publishers, Amsterdam (pp. 55–68). Konishi, T., Konoshima, T., Fujiwara, Y., Kiyosawa, S., Miyahara, K., Nishi, M., 1999. Stereostructures of new labdane-type diterpenes, excoecarins F, G1, and G2 from the wood of Excoecaria agallocha. Chem. Pharm. Bull. 47, 456–458. Kouzj, S.A., McChesney, J.D., 1991. Microbial models of mammalian metabolism: fungal metabolism of the diterpene sclareol by Cunninghamella species. J. Nat. Prod. 54, 483–490. Kusumi, T., 1993. Determination of the absolute configuration of organic compounds by means of NMR spectroscopy, modified Mosher’s method. Yuki Gosei Kagaku Kyokaishi 51, 462–470. Meng, L.H., Zhang, J.S., Ding, J., 2001. Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes. Biochem. Pharmacol. 62, 733–741. Mirza, M., Sefidkon, F., 1999. Essential oil composition of two Salvia species from Iran, Salvia nemorosa L. and Salvia reuterana Boiss. Flav. Fragr. J. 14, 230–232. Moghaddam, F.M., Farimani, M.M., Seirafi, M., Taheri, S., Khavasi, H.R., Sendker, J., Proksch, P., Wray, V., Edrada, R.A., 2010. Sesterterpenoids and other constituents of Salvia sahendica. J. Nat. Prod. 71, 1601–1605. Mozaffarian, V., 1990. A Dictionary of Iranian Plant Names. Farhang Mansor, Tehran, 477. Munro, T.A., Rizzacasa, M.A., Roth, B.L., Toth, B.A., Yan, F., 2005. Studies toward the pharmacophore of salvinorin A, a Potent j Opioid receptor agonist. J. Med. Chem. 48, 345–348. Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 113, 4092–4096. Rechinger, K.H., 1987. Flora Iranica: akademische druck und verlagsanstalt. Graz, Austria 150, 403–476. Rodriguez-Hahn, L., Esquivel, B., Sanchez, A.A., Cardenas, J., Tovar, O.G., SorianoGarcia, M., Toscano, A., 1988. Puberulin and isopuberulin, benzonorcaradiene and benzocyclo-heptatriene diterpenoids of clerodanic origin from Salvia puberula. J. Org. Chem. 53, 3933–3936. Rodriguez-Hahn, L., O’Reilly, R., Esquivel, B., Maldonado, E., Ortega, A., Cardenas, J., Toscano, R.A., 1990. Tilifodiolide, tetraline-type diterpenoid of clerodanic origin from Salvia tiliaefolia. J. Org. Chem. 55, 3522–3525.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
6
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
Soucek, M., Vlad, P., 1963. On terpenes. CLII. Absolute configuration on C(13) in sclareol Collect. Czech. Chem. Commun. 28, 1211–1214. Wang, X., Bastow, K.F., Sun, C.M., Lin, Y.L., Yu, H.J., Don, M., Wu, T.S., Nakamura, S., Lee, K.H., 2004. Antitumor agents. 239. Isolation, structure elucidation, total synthesis, and anti-breast cancer activity of neo-tanshinlactone from Salvia miltiorrhiza. J. Med. Chem. 47, 5816–5819. Wang, B.G., Gloer, J.B., Ji, J.Y., Zhao, J.C., 2013. Halogenated organic molecules of Rhodomelaceae origin: chemistry and biology. Chem. Rev. 113, 3632–3685.
Wu, Y.-B., Ni, Z.-Y., Shi, Q.-W., Dong, M., Kiyota, H., Gu, Y.-C., Cong, B., 2012. Constituents from Salvia species and their biological activities. Chem. Rev. 112, 5967–6026. Xie, N., Min, Z., Zhao, S., Lu, Y., Zheng, Q., Wang, C., Mizunu, M., Linuma, M., Tanaka, T., 1992. A chlorine-containing neo-clerodane diterpene from Teucrum pernyi. Chem. Pharm. Bull. 40, 2193–2195. Zargari, A., 1990. Medicinal Plants. Tehran University Press, Tehran, 56.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
Contents lists available at ScienceDirect
Phytochemistry journal homepage: www.elsevier.com/locate/phytochem
Labdane diterpenoids from Salvia reuterana Mahdi Moridi Farimani ⇑, Mansour Miran Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G.C., Evin, , Tehran, Iran
a r t i c l e
i n f o
Article history: Received 25 June 2014 Received in revised form 8 August 2014 Available online xxxx Keywords: Salvia reuterana Labiatae Halogenated labdane diterpenoid SAR
a b s t r a c t Three labdane diterpenoids, 14a-hydroxy-15-chlorosclareol (1), 14a-hydroxy-15-acetoxysclareol (2), and 6b-hydroxy-14a-epoxysclareol (3), together with the known diterpenoids sclareol (4), 6b-hydroxysclareol (5), and 14a-epoxysclareol (6), as well as other common plant constituents were isolated from the n-hexane extract of aerial parts of Salvia reuterana. The structures of the new compounds were established by extensive 1D and 2D NMR spectroscopic techniques. Compound 1 is the first example of a halogenated terpenoid in the genus Salvia. Compounds 1–6 were also tested for their inhibitory activity toward HeLa and MCF-7 cell lines. Preliminary structure–activity relationship studies indicated that double bond moiety in sclareol is an essential feature for activity and modification of this moiety significantly decreased the cytotoxic activity of the resulting compounds. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2. Results and discussion
The genus Salvia is a rich source of structurally diverse terpenoids (Chang et al., 1990; Rodriguez-Hahn et al., 1988, 1990). Among these, numerous diterpenoids with promising bioactivities, such as anticancer, cardiotonic and topoisomerase inhibitor properties, have been reported from Salvia species (Meng et al., 2001; Wang et al., 2004; Munro et al., 2005). The most abundant diterpenoids in the genus are abietanes and rearranged abietanes (Wu et al., 2012). Labdane diterpenoids are rather rare in Salvia species, although they are frequently found in other genera of the Lamiaceae (Kintzios, 2000). Up to now, only 18 labdane diterpenoids have been reported from Salvia (Wu et al., 2012). Salvia reuterana Boiss is an endemic species which grows in the highlands of central Iran (Rechinger, 1987). Its common name in Persian is ‘‘Mariam Goli Esfahani’’ (Mozaffarian, 1990), and the aerial parts of the plant have been used in Iranian folk medicine as sedative and anxiolyticn (Zargari, 1990). With exception of an analysis of the essential oil (Mirza and Sefidkon, 1999), S. reutereana has not been studied from a phytochemical viewpoint. In continuing our effort on discovery of novel and potentially bioactive secondary metabolites from Iranian Salvia species (Farimani et al., 2011, 2012, 2013; Moghaddam et al., 2010), we investigated the n-hexane extract from aerial parts of S. reuterana. We here report on the isolation, structure elucidation and biological activity of three new labdane diterpenoids, including a chlorinated compound.
The phytochemical investigation of n-hexane extract from S. reuterana afforded three new labdane diterpenoids (1–3) (Fig. 1), together with six known compounds namely: sclareol (4), 6b-hydroxysclareol (5), 14a-epoxysclareol (6), b-sitosterol, oleanolic acid, 5-hydroxy-7,4’-dimethoxyflavone. Compound 1 had a molecular formula C20H37O3Cl (HR-ESI-TOFMS (m/z 383.2330 [M+Na]+, calcd 383.2323) and EIMS(m/z 360 [M]+ and 362 [M+2]+ in the ratio 3:1) accounting for two degrees of hydrogen deficiency. Its IR spectrum showed the presence of hydroxyl (3423 cm1) and alkyl halide (732 cm1) groups. It did not show conjugation in its UV spectrum. The 13C NMR spectrum (Table 1) showed 20 carbon resonances which were attributed with the aid of HSQC and DEPTQ spectra to five methyl, eight methylene, three methine and four quaternary carbons. Two carbon signals at dc 75.4 and 75.7 indicated the presence of oxygen bearing sp3 carbons. Another oxygenated carbon resonance at dc 79.0 accounted for the carbon carrying a hydroxyl group with the carbinolic methine proton coming at dH 3.61 (1H, dd, J = 1.6, 9.7 Hz). The signal at dc 47.7 accounted for a chloromethylene carbon with the corresponding methylene protons resonating at dH 3.50 and 3.91, respectively (Anjaneyulu and Rao, 2000; Konishi et al., 1999). The molecule thus was bicyclic and appeared to be a labdane diterpenoid. The 13C chemical shifts of 1 were assigned with the aid of a HMQC spectrum, and by comparison with spectral data of sclareol, and location of the functional groups was confirmed by HMBC connectivities (Fig. 2). The resonance of C-13 (dc 75.7) showed correlations with H-14 (dH 3.61), CH3-16 (dH 1.13) and CH2-12 (dH 1.59 and 1.76). Other key HMBC correlations were
⇑ Corresponding author. Tel.: +98 21 29904043; fax: +98 21 22431783. E-mail address: [email protected] (M. Moridi Farimani). http://dx.doi.org/10.1016/j.phytochem.2014.08.024 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
2
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
20
17
1 8
OH
16
O
R
11
10
H
OH
16
1
OH
OH
4
H
H 18
1 R = Cl 2 R = OAc
H
9
8
5
15
Cl
O
OH
OH
OH H
19
R
3 R = OH 6 R=H
OH
Fig. 2. Selected HMBC correlations of compounds 1 (left) and 3 (right).
OH OH
H
17 OH
OH
4 19
14
20
15
OH
12
R
4 R=H 5 R = OH Fig. 1. Compounds 1–6.
observed from C-14 (dc 79.0) to CH2-15 (dH 3.50 and 3.91), CH3-16 and CH2-12. On the other hand, the chloromethylene carbon (dc 47.7) exhibited a long-range correlation with H-14. Similarly, the methyl protons at dH 0.84, 0.87, 0.90, 1.13 and 1.18 showed the
respective correlations as noted in Fig. 2. A b-orientation of CH3-17 and CH3-20 and CH3-16 was deduced from the NOESY spectrum which showed NOE contacts between CH3-20 (dH 0.87), CH3-17 (dH 1.18) and H-11b (dH 1.39), between H-11b, CH3-16 (dH 1.13) and CH3-17, and between CH3-17 and H-7b (dH 1.84) (Fig. 3). Diagnostic cross peaks between H-5a (axial) and H-9a confirmed their cofacial orientation. The stereochemistry at C-14 was also determined from the NOESY data: H-14 (dH 3.61) showed correlations with CH3-16b (dH 1.13), H-12a (dH 1.76), H-12b (dH 1.59), as well as with H-11b (dH 1.39). On the other hand both of the chloromethylene protons (dH 3.50 and dH 3.91) showed strong cross peaks with CH3-16b, H-12a (dH 1.76), and H-12b (dH 1.59). Overall, the data indicated that H-14 had to be in b–position and, assuming the R configuration at C-13 as for sclareol (Soucek and Vlad, 1963), the configuration at C-14 had to be R. To confirm this, the absolute configuration of the secondary hydroxyl group at C-14 was determined by the application of a modification of Mosher’s method (Kusumi, 1993; Ohtani et al., 1991) to 1. Treatment of 1 with (R)and (S)-MTPA chloride in the presence of pyridine afforded the
Table 1 H and 13C NMR Data of Compounds 1–3a (500 MHz for dH; 125 MHz for dC).
1
Position
1a 1b 2a 2b 3a 3b 4 5a 6a 6b 7a 7b 8 9a 10 11a 11b 12a 12b 13 14b 15a 15b 16 17 18 19 20 CH3CO CH3CO a b
1
2
3
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
41.1
1.01 dt (12.1, 3.1) 1.72b 1.65b 1.45b 1.22b 1.42b
39.6
0.88b 1.66 dt (13.5, 5.0) 1.52b 1.36b 1.07b 1.31b
41.6
0.87b 1.60b 1.62b 1.37b 1.06b 1.26b
19.5 43.2 34.2 57.6 21.5 44.8 75.4 62.9 40.5 19.4 43.6 75.7 79.0 47.7 21.6 24.1 33.9 22.0 16.1
0.96 dd (12.2, 3.0) 1.67b 1.34b 1.47b 1.84 dt (12.2, 4.2) 1.09b 1.56b 1.39b 1.76b 1.59b 3.61 3.50 3.91 1.13 1.18 0.90 0.84 0.87
dd (9.7, 1.6) dd (11.2, 9.7) dd (11.2, 1.6) s s s s s
18.3 41.9 33.2 56.0 20.4 44.0 75.5 62.0 39.1 18.0 39.3 75.4 76.3 65.8 22.8 24.1 33.3 21.5 15.4 20.4 171.3
0.86 d (11.6) 1.57b 1.19b 1.35b 1.76 dt (12.8, 4.3) 1.05b 1.48b 1.28b 1.53b 1.37b 3.60 3.99 4.21 1.12 1.11 0.80 0.72 0.73 2.03
br d (6.8) dd (11.5, 8.6) dd (11.5, 2.5) s s s s s s
18.3 44.0 33.3 57.0 68.1 50.8 73.3 62.1 39.0 17.9 41.0 70.2 58.2 43.6 24.6 25.5 33.0 23.1 16.4
0.86b 4.40 br d (2.3) 1.53b 1.91 br d (13.8) 1.08b 1.54b 1.40b 1.55b 1.55b 2.83 2.63 2.74 1.18 1.32 0.90 1.11 1.10
dd (5.0, 3.6) dd (7.0, 3.6) dd (7.0, 5.0) s s s s s
Compound 1 in CD3OD; compound 2 in CDCl3/CD3OD; compound 3 in CDCl3. d values were extracted from COSY, HSQC and HMBC experiments. Overlapping signals.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
3
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
16
H
H
H
H
H
H
5
H
H
H
H 7
Cl
HO
11
H
15 13
17
20
H
H H
H
OH
H
OH
H H
H
Fig. 3. Key NOESY correlations of 1.
-0.08 -0.04 -0.03 -0.04 -0.02 -0.01
-0.11 OMTPA
Cl
-0.10
OH
+0.12 +0.12
OH -0.03 -0.03
-0.02
Fig. 4. 1H NMR chemical shift difference values for the MTPA esters of 1 [Dd: dS(1a) dR(1b)].
(S)-MTPA ester (1a) and (R)-MTPA ester (1b), respectively. The observed chemical shift differences (DdS–R, Fig. 4) unambiguously indicated the absolute configurations of C-14 to be R. Hence, compound 1 was identified as 14R-hydroxy-15-chlorosclareol. Given that chlorohydrine 1 could be possibly formed during the extraction and isolation process from the corresponding epoxide, we repeated extraction of plant material with hexane and then subjected the extract to HPLC-ESI TOF analysis (Fig. S10 Supporting Information). The chromatogram showed a peak at Rt 19.1 min with m/z 383. Rt and MS data corresponded with 1 which was analyzed separately under identical conditions. Thus, we conclude that 1 was a genuine plant metabolite. Compound 2 had an elemental formula C22H40O5 (HR-ESI-TOFMS m/z 407.2761 [M+Na]+; calcd 407.2773), which accounted for three degrees of hydrogen deficiency. Its IR spectrum exhibited absorption bands for the hydroxyl (3447 cm1) and carbonyl (1732 cm1) functions. The 1H and 13C NMR spectrum displayed features similar to those of 1. However, in the 13C NMR spectrum of 2, resonances of an additional methyl (dC 20.4 ppm) and a quaternary carbon (dC 171.3 ppm) were observed, and the methylene signal observed in 1 at dC 47.7 ppm appeared at higher field in 2 (dC 65.8 ppm). The resonances of C-14 and C-12 were observed at dC 76.3, and 39.3 respectively (upfield shifts of ca. 2.7 and 4.3 ppm compared to 1), while the resonance of CH3-16 appeared at dC 22.8 (downfield shift of 1.2 ppm compared to 1). In the 1H NMR spectrum of 2, an additional methyl singlet appeared at dH 2.03, while the CH2-15 resonances appeared downfield (dH 3.99 and 4.21 ppm, respectively). These differences in the NMR data, together with the absence of chlorine in the molecular formula, suggested that compound 2 had an acetoxy group at C-15 instead of the chlorine observed in 1. HMBC correlations between the carbonyl resonance at dC 171.3 ppm and the carboxymethylene protons (dH 3.99 and 4.21), and the methyl protons (dH 2.03 ppm)
confirmed the location of the acetoxy group. Complete assignments of NMR spectral data were obtained from 1H–1H COSY, HMQC, and HMBC spectra, while the relative configuration was established from the NOESY spectrum. Compound 2 was therefore identified as 14a-hydroxy-15-acetoxysclareol. The HR-ESI-TOFMS of 3 showed a molecular ion peak at m/z 363.2516 [M+Na]+, consistent with a molecular formula C20H36O4 (calcd 363.2507). In the IR spectrum the absorption bands at mmax 1273 and 867 cm1 besides the characteristic band of the hydroxyl group at 3430 cm1 was consistence with the presence of the epoxide functional group in the molecule. In the 1H NMR spectrum of 3, the signals at dH 2.83 (dd, J = 3.6, 5.0 Hz), 2.74 (dd, J = 5.0, 7.0 Hz), and 2.63 (dd, J = 3.6, 7.0 Hz) were indicative of a terminal epoxide moiety in a side chain. The 13C NMR spectrum (Table 1) showed 20 carbon signals which were assigned through a DEPT experiment to five methyl, seven methylene, four methine, and four quaternary carbons. When comparing the 13C NMR data of 3 with those of sclareol, the methine carbon (dC 58.2) and a terminal methylene (dC 43.6) instead of olefinic signals in sclareol (dC 147.5 and 110.7) (Kouzj and McChesney, 1991) corroborated the presence of an epoxide at C-14/C-15. 1H and 13C NMR data strongly resembled to those of 14R-epoxysclareol, a diterpenoid from Astragalus brachystachys (Jassbi et al., 2002). However, the 13C NMR spectrum of 3 showed the presence of an additional methine at dC 68.1 instead of the methylene group (C-6). Hence, the methylene was replaced by an oxygenated methine. The signals of C-7 (dC 50.8) and C-5 (dC 57.0) were paramagnetically shifted (Dd + 6.7 and +0.9 ppm, respectively) in comparison to those of 14R-epoxysclareol. Also the resonances of neighboring H-7a (dH 1.53), H-7b (dH 1.91), and H-5a (dH 0.86) were shifted (Dd = +0.15, +0.10, and 0.04 ppm, respectively). HMBC correlations between H-5a, H-7a with C-6, and between H-6, C-8, and C-10 (Fig. 2) confirmed the location of the hydroxyl group. Diagnostic COSY correlations were observed between H-6 and H-7a and H-7b, and between H-6 and H-5. The relative configuration of the hydroxyl group at C-6 was determined as b on the basis of the magnitude of the vicinal coupling constants of the H-6 resonance (dH 4.40, brd, J = 2.3 Hz). NOESY contacts of H-6 with H-5 and H-7ax were observed. The 1H chemical shifts of CH3-17, CH3-19 and CH3-20 (dH 1.32, 1.11 and 1.10, respectively) appeared downfield (dH 0.19, 0.27, and 0.34) relative to those of 14R-epoxysclareol. These differences were in agreement with an axial orientation of the hydroxyl group at C-6 (Jassbi et al., 2002). Thus, the structure of 3 was established as 6b-hydroxy-14a-epoxysclareol. As for 1, compounds 2 and 3 were also detected in the n-hexane extract of the dried whole plants of S. reuterana obtained under mild conditions (Fig. S28 Supporting Information), indicating that
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
4
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
Table 2 Cytotoxic activities of compounds 1–6 against two human tumor cell lines.
a b
Compound
HeLa (IC50 lg/mL)a
MCF-7 (IC50 lg/mL)
1 2 3 4 5 6 Paclitaxelb
37.17 ± 0.76 57.16 ± 1.44 58.72 ± 1.51 16.60 ± 0.49 53.23 ± 0.73 59.41 ± 1.33 0.003
42.16 ± 0.95 46.74 ± 1.03 37.29 ± 1.06 32.65 ± 1.15 36.98 ± 1.12 62.07 ± 2.01 0.028
Data are expressed as mean ± standard deviation (n = 3). Positive control.
they must be natural products rather than artifacts from the isolation procedure. Halogenated diterpenoids are mainly found in marine algae and invertebrates (Dembitsky et al., 2002; Wang et al., 2013), but are rare in terrestrial plants. Four chlorine-containing labdane diterpenoids were isolated from Excoecaria agallocha, a seashore plant of the Euphorbiaceae family (Anjaneyulu and Rao, 2000; Konishi et al., 1999), while a chloroditerpene lactone was reported from Gutierrezia dracunculoides (Asteraceae) (Cruse et al., 1971). From the Lamiceae family six chlorine-containing diterpenes have been reported in Teucrium and Ajuga species (Dembitsky et al., 2002; Wang et al., 2013; Xie et al., 1992; Beauchamp et al., 1996; Bruno et al., 1995). However, compound 1 is the first example of a chlorinated diterpene from the genus Salvia. Sclareol has demonstrated a significant cytotoxic activity against human cell lines (Dimas et al., 1999; Dimas et al., 2006). Compounds 1–6 were evaluated for their in vitro cytotoxic activity against HeLa (human epitheloid cervix carcinoma) and MCF-7 (human breast adeno-carcinoma) cell lines. The results of the cytotoxicity studies are presented in Table 2. A preliminary SAR study was carried out against this short series of labdane diterpenoids. As demonstrated in Table 2, it can be stated that all of analogues displayed weaker cytotoxic activity compared to parent compound, sclareol (4). Drastic decrease or lack in cytotoxicity of compounds 1–3 and 5, 6 against tested cell lines, implying that double bond is necessary for the activity. However, replacement of this moiety with an epoxide (compounds 3 and 6) or others substituent (compounds 1 and 2) decreased the activity against two cell lines. Furthermore, the sharp decrease in cytotoxic activity of compound 5 (especially in HeLa cell line) indicated that the bioactivity is very sensitive to change in 6-position. Nevertheless, among the compounds 3 and 6, the presence of a hydroxyl group at the 6-position seemed to increase cytotoxic activity of compound 3 (especially against MCF-7 cell line). It became clear from these results that the double bond moiety in these compounds is an essential feature for activity; however, the effect of 6-hydroxyl group on the cytotoxic activity depends on the functional groups at C14–C15 bond. 3. Conclusions Three new labdane diterpenoids were isolated from the aerial parts of S. reuterana, including one which contained a chlorohydrine moiety in its structure. HPLC-ESI TOF analysis of the fresh crude extract of the plant showed that 1 was a genuine plant metabolite. Halogenated diterpenoids are mainly found in marine algae and invertebrates (Dembitsky et al., 2002; Wang et al., 2013), but are rare in terrestrial plants. Compound 1 is the first example of a chlorinated diterpene from the genus Salvia. Preliminary structure–activity relationship studies indicated that double bond moiety in sclareol is an essential feature for activity and modification of this moiety significantly decreased the cytotoxic activity of the resulting compounds.
4. Experimental 4.1. General Experimental Procedures Optical rotations were measured using a JASCO P-2000 automatic digital polarimeter. IR spectra were recorded on a Bruker Tensor 27 spectrometer. UV spectra were recorded using a Shimadzu UV-2501PC spectrophotometer. NMR spectra were recorded at a target temperature of 18 °C on a Bruker Avance III 500 MHz spectrometer operating at 500.13 MHz for 1H, and 125.77 MHz for 13C. A 1 mm TXI-microprobe with z-gradient was used for 1H-detected experiments. 13C NMR spectra were recorded with a 5 mm BBOprobe head with z-gradient. Spectra were analyzed using Bruker TopSpin 3.1 software. Deuterated solvents for NMR (100 at% D) were purchased from Armar Chemicals. HPLC separations were carried out on an series 1100 system consisting of degasser, binary high pressure mixing pump, column thermostat and photodiode array (PDA) detector (Agilent Technologies; Waldbronn, Germany). Analysis was performed on a Waters SunFireÒ C18 column (3.5 lm, 3.0 150 mm), using a gradient of MeCN/H2O 30:70 ? 0:100 in 30 min, and a flow rate of 0.4 mL/min. High resuloution MS spectra were recorded using a Bruker microTOF ESI-MS system in the range of m/z 150–1500 in positive mode. Nitrogen was used as nebulizing gas at a pressure of 2.0 bar, and as drying gas at a flow rate of 9.0 l/min (drying gas temperature 240 °C). Capillary voltage was at 4500 V, endplate offset at 500 V, hexapole at 250.0 V, skimmer 1 at 40 V and skimmer 2 at 22.5 V. Instrument calibration was performed using a reference solution of sodium formate 0.1% in isopropanol/water (1:1) containing 5 mM sodium hydroxide. Data acquisition and processing were performed using HyStar™ 3.0 and EsquireControl™ 5.2 (Bruker Daltonics). 4.2. Plant material The aerial parts of Salvia reuterana were collected in July 2011 from the northern hilly areas of Tehran, Iran. A voucher specimen (MPH-1321) has been deposited in the herbarium of the Medicinal Plant and Drug Research Institute (MPH) of Shahid Beheshti University, Tehran, Iran. 4.3. Extraction and isolation The air-dried aerial parts of S. ruterana (3.0 kg) was crushed and extracted with n-hexane (3 15 L) by maceration at room temperature. Extract was concentrated in vacuo, to afford 130 g of a dark gummy residue. The residue was separated on a silica gel column (230–400 mesh, 850 g) with a gradient of n-hexane–EtOAc (100/ 0 to 0/100) as eluent, followed by increasing concentration of MeOH (up to 25%) in EtOAc. On the basis of TLC analysis, fractions with similar composition were pooled to yield 27 combined fractions. From fraction 5 [eluted with n-hexane–EtOAc (85:15)], crude crystals were obtained, which were recrystallized from n-hexane to afford b-sitosterol (1 g). Fractions 7 and 8 [4.0 g, eluted with n-hexane–EtOAc (75:25)] were combined and subjected to silica gel column chromatography (70–230, 220 g), eluted with CHCl3 followed by a gradient of CHCl3–Me2CO (up to 16%), to give four subfractions (7a–7e). Subfraction 7a was recrystallized from Me2CO to afford 5-hydroxy-7,40 -dimethoxyflavone (150 mg). Subfraction 7b, also contained a crude solid, which was triturated with EtOAc to yield oleanolic acid (28 mg). Fraction 9 [eluted with n-hexane–EtOAc (70:30)], was triturated with CHCl3 to yield an insoluble solid, which was recrystallized from CHCl3 to afford sclareol (2 g). Fraction 13 [0.8 g, eluted with n-hexane–EtOAc (50:50)] was applied to a silica gel column (70–230 mesh), with CHCl3-Me2CO-MeOH (88:8:4) as eluent, and then was purified by
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
recrystallization from Me2CO to afford compound 1 (10 mg). Fraction 16 [1.2 g, eluted with n-hexane–EtOAc (35:65)], was chromatographed on a silica gel column (70–230 mesh), eluted with CHCl3-Me2CO (75:25, 70:30, 65:35), to give 14a-epoxysclareol (25 mg). Fraction 17 [2.0 g, eluted with n-hexane–EtOAc (20:80)], was subjected to column chromatography over a silica gel column, eluted with CHCl3 followed by increasing amounts of MeOH (up to 15%), and then was purified by recrystallization from Me2CO to afford 6b-hydroxysclareol (20 mg). Fraction 21 [0.9 g, eluted with n-hexane–EtOAc (5:95)] was separated on a silica gel column with CHCl3–Me2CO–MeOH (90:5:5), to afford seven subfractions (21a21g). Subfraction 21b was recrystallized from MeOH to yield compound 2 (21 mg). Subfraction 21f was further purified on a small silica gel column, eluted with CHCl3–Me2CO (50:50), to afford compound 3 (8 mg). 4.4. Spectroscopic data of new compounds 4.4.1. 14R-hydroxy-15-chlorosclareol (1) White powder; mp 152–153 °C; ½a25 D ¼ 19:6 (c 0.2, CHCl3); UV (MeOH) kmax (log e) 223 (2.57); IR (KBr) mmax 3423, 2926, 1461, 1383, 1261, 1090, 732 cm1; for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z 383.2330 [M+Na]+, calcd for 383.2323. 4.4.2. 14a-hydroxy-15-acetoxysclareol (2) White powder; mp 136–138 °C; ½a25 D ¼ 12:5 (c 0.2, CHCl3); UV (MeOH) kmax (log e) 225 (2.82); IR (KBr) mmax 3447, 2927, 1732, 1460, 1380, 1259, 1099, cm1; for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z 407.2761 [M+Na]+,calcd for 407.2773. 4.4.3. 6b-Hydroxy-14a-epoxysclareol (3) White powder; mp 126–128 °C; ½a25 D ¼ 8:5 (c 0.2, CHCl3); UV (MeOH) kmax (log e) 228 (2.58); IR (KBr) mmax 3430, 2924, 1460, 1379, 1273, 1146, 1079, 867 cm1; for 1H and 13C NMR data, see Table 1; HR-ESI-TOFMS m/z 363.2516 [M+Na]+ calcd for 363.2507. 4.5. Cytotoxicity assay The human epithelioid cervix carcinoma (HeLa) and human breast adenocarcinoma (MCF-7) cell lines were purchased from National Cell Bank of Iran (NCBI), Pasteur Institute of Iran (Tehran, Iran), and maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin and 100 lg/ml streptomycin. These cells were kept at 37 °C in a humidified atmosphere containing 5% CO2. Compounds 1–6 were dissolved in DMSO to make a stock of 1 mg/mL and further diluted to final concentrations of 10–100 lg/mL with the serum free culture medium. 4.6. Cell viability assay Cell viability was determined using the MTT assay. Briefly, 2.5 104 cells were seeded in 96-well plates at 37 °C with 5% CO2 for overnight incubation and treated with appropriate concentrations of compounds 1–6 for 24 h. The cells were then incubated with a serum-free medium containing MTT at a final concentration of 0.5 mg/mL for 4 h. The dark formazan crystals formed were dissolved in DMSO and the absorbance was measured at 570 nm. Acknowledgments Financial support by the Shahid Beheshti University Research Council is gratefully acknowledged. All spectra were performed at the Department of Pharmaceutical Sciences, Division of Pharmaceutical Biology, University of Basel. The kind assistance of Prof. M. Hamburger, Dr. S.N. Ebrahimi, and all other staff is gratefully
5
appreciated. The authors also express thanks to Dr. A. Sonboli for taxonomic identification of the plant. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytochem.2014. 08.024. References Anjaneyulu, A.S.R., Rao, V.L., 2000. Five diterpenoids (agallochins A-E) from the mangrove plant Excoecaria agallocha Linn. Phytochemistry 55, 891–901. Beauchamp, P.S., Bottini, A.T., Caselles, M.C., Hope, H., 1996. Neo-clerodane diterpenoids from Ajuga parviflora. Phytochemistry 43, 827–834. Bruno, M., Fazio, C., Piozzi, F., Savona, G., Rodriguez, B., Torre, M.C., 1995. Neoclerodane diterpenoids from Teucrium racemosum. Phytochemistry 40, 505–507. Chang, H.M., Cheng, K.P., Choang, T.F., Chow, H.F., Chui, K.Y., Hon, P.M., Lau, F.W., Yang, Y., Zhong, Z.P., 1990. Structure elucidation and total synthesis of new tanshinones isolated from Salvia miltiorrhiza Bunge (Danshen). J. Org. Chem. 55, 3537–3543. Cruse, W.B.T., James, M.N.G., Al-Shamma, A.A., Beal, J.K., Doskotch, R.W., 1971. The molecular structure of gutierolide, a novel chloro-diterpenoid lactone.. J. Chem. Soc., Chem. Comm., 1278–1280. Dembitsky, V.M., Tolstikov, A.G., Tolstikov, G.A., 2002. Natural halogenated diterpenoids. Chem. Sust. Develop. 10, 253–264. Dimas, K., Kokkinopoulos, D., Demetzos, C., Vaos, B., Marselos, M., Malamas, M., Theodoros, T., 1999. The effect of sclareol on growth and cell cycle progression of human leukemic cell lines. Leuk. Res. 23, 217–234. Dimas, K., Papadaki, M., Tsimplouli, C., Hatziantoniou, S., Alevizopoulos, K., Pantazis, P., Demetzos, C., 2006. Labd-14-ene-8,13-diol (sclareol) induces cell cycle arrest and apoptosis in human breast cancer cells and enhances the activity of anticancer drugs. Biomed. Pharmacother. 60, 127–133. Farimani, M.M., Bahadori, M.B., Taheri, S., Ebrahimi, S.N., Zimmermann, S., Brun, R., Amin, G., Hamburger, M., 2011. Triterpenoids with rare carbon skeletons from Salvia hydrangea: antiprotozoal activity and absolute configurations. J. Nat. Prod. 74, 2200–2205. Farimani, M.M., Taheri, S., Ebrahimi, S.N., Bahadori, M.B., Khavasi, H.R., Zimmermann, S., Brun, R., Hamburger, M., 2012. Hydrangenone, a new isoprenoid with an unprecedented skeleton from Salvia hydrangea. Org. Lett. 14, 166–169. Farimani, M.M., Ebrahimi, S.N., Salehi, P., Bahadori, M.B., Sonboli, A., Khavasi, H.R., Zimmermann, S., Kaiser, M., Hamburger, M., 2013. Antitrypanosomal triterpenoid with an e-lactone e-ring from Salvia urmiensis. J. Nat. Prod. 76, 1806–1809. Jassbi, A.R., Zamanizadehnajari, S., Aberoomand, P., Tahara, S.Z., 2002. Antibacterial diterpenoids from Astragalus brachystachys. Naturforsch 57c, 1016–1021. Kintzios, S.E., 2000. Sage: the genus Salvia. Harwood Academic Publishers, Amsterdam (pp. 55–68). Konishi, T., Konoshima, T., Fujiwara, Y., Kiyosawa, S., Miyahara, K., Nishi, M., 1999. Stereostructures of new labdane-type diterpenes, excoecarins F, G1, and G2 from the wood of Excoecaria agallocha. Chem. Pharm. Bull. 47, 456–458. Kouzj, S.A., McChesney, J.D., 1991. Microbial models of mammalian metabolism: fungal metabolism of the diterpene sclareol by Cunninghamella species. J. Nat. Prod. 54, 483–490. Kusumi, T., 1993. Determination of the absolute configuration of organic compounds by means of NMR spectroscopy, modified Mosher’s method. Yuki Gosei Kagaku Kyokaishi 51, 462–470. Meng, L.H., Zhang, J.S., Ding, J., 2001. Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes. Biochem. Pharmacol. 62, 733–741. Mirza, M., Sefidkon, F., 1999. Essential oil composition of two Salvia species from Iran, Salvia nemorosa L. and Salvia reuterana Boiss. Flav. Fragr. J. 14, 230–232. Moghaddam, F.M., Farimani, M.M., Seirafi, M., Taheri, S., Khavasi, H.R., Sendker, J., Proksch, P., Wray, V., Edrada, R.A., 2010. Sesterterpenoids and other constituents of Salvia sahendica. J. Nat. Prod. 71, 1601–1605. Mozaffarian, V., 1990. A Dictionary of Iranian Plant Names. Farhang Mansor, Tehran, 477. Munro, T.A., Rizzacasa, M.A., Roth, B.L., Toth, B.A., Yan, F., 2005. Studies toward the pharmacophore of salvinorin A, a Potent j Opioid receptor agonist. J. Med. Chem. 48, 345–348. Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field FT NMR application of Mosher’s method. The absolute configurations of marine terpenoids. J. Am. Chem. Soc. 113, 4092–4096. Rechinger, K.H., 1987. Flora Iranica: akademische druck und verlagsanstalt. Graz, Austria 150, 403–476. Rodriguez-Hahn, L., Esquivel, B., Sanchez, A.A., Cardenas, J., Tovar, O.G., SorianoGarcia, M., Toscano, A., 1988. Puberulin and isopuberulin, benzonorcaradiene and benzocyclo-heptatriene diterpenoids of clerodanic origin from Salvia puberula. J. Org. Chem. 53, 3933–3936. Rodriguez-Hahn, L., O’Reilly, R., Esquivel, B., Maldonado, E., Ortega, A., Cardenas, J., Toscano, R.A., 1990. Tilifodiolide, tetraline-type diterpenoid of clerodanic origin from Salvia tiliaefolia. J. Org. Chem. 55, 3522–3525.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
6
M. Moridi Farimani, M. Miran / Phytochemistry xxx (2014) xxx–xxx
Soucek, M., Vlad, P., 1963. On terpenes. CLII. Absolute configuration on C(13) in sclareol Collect. Czech. Chem. Commun. 28, 1211–1214. Wang, X., Bastow, K.F., Sun, C.M., Lin, Y.L., Yu, H.J., Don, M., Wu, T.S., Nakamura, S., Lee, K.H., 2004. Antitumor agents. 239. Isolation, structure elucidation, total synthesis, and anti-breast cancer activity of neo-tanshinlactone from Salvia miltiorrhiza. J. Med. Chem. 47, 5816–5819. Wang, B.G., Gloer, J.B., Ji, J.Y., Zhao, J.C., 2013. Halogenated organic molecules of Rhodomelaceae origin: chemistry and biology. Chem. Rev. 113, 3632–3685.
Wu, Y.-B., Ni, Z.-Y., Shi, Q.-W., Dong, M., Kiyota, H., Gu, Y.-C., Cong, B., 2012. Constituents from Salvia species and their biological activities. Chem. Rev. 112, 5967–6026. Xie, N., Min, Z., Zhao, S., Lu, Y., Zheng, Q., Wang, C., Mizunu, M., Linuma, M., Tanaka, T., 1992. A chlorine-containing neo-clerodane diterpene from Teucrum pernyi. Chem. Pharm. Bull. 40, 2193–2195. Zargari, A., 1990. Medicinal Plants. Tehran University Press, Tehran, 56.
Please cite this article in press as: Moridi Farimani, M., Miran, M. Labdane diterpenoids from Salvia reuterana. Phytochemistry (2014), http://dx.doi.org/ 10.1016/j.phytochem.2014.08.024
