Note pubs.acs.org/jnp
Antitrypanosomal Triterpenoid with an ε‑Lactone E‑Ring from Salvia urmiensis Mahdi Moridi Farimani,*,† Samad Nejad Ebrahimi,†,‡ Peyman Salehi,† Mir Babak Bahadori,† Ali Sonboli,§ Hamid Reza Khavasi,⊥ Stefanie Zimmermann,‡,∥ Marcel Kaiser,∥,▽ and Matthias Hamburger‡ †
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran Department of Pharmaceutical Sciences, Division of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland § Department of Biology, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran ⊥ Department of Chemistry, Shahid Beheshti University, G. C., Evin, Tehran, Iran ∥ Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland ▽ University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland ‡
S Supporting Information *
ABSTRACT: A new triterpenoid, urmiensolide (1), was isolated from Salvia urmiensis. The structure was elucidated by a combination of 1D and 2D NMR, HRESIMS, and X-ray crystallographic analyses. The absolute configuration was established by comparison of experimental and simulated ECD spectra. Urmiensolide is the first pentacyclic triterpenoid bearing a ε-lactone E-ring. The compound showed in vitro antitrypanosoal activity with an IC50 value of 5.6 μM against the Trypanosoma brucei rhodesiense STIB 900 strain and a selectivity index of 33. A possible biosynthetic pathway of 1 from α-amyrin is proposed.
Salvia is the largest genus of the Lamiaceae family and comprises over 900 species found throughout the world. Salvia species are a source of structurally diverse terpenoids1 with a broad spectrum of bioactivities, such as cardiotonic, antibacterial, antioxidant, antitumor, and antimalarial properties.2 One of the most significance features of Salvia species is the number of triterpenoid derivatives produced with diverse carbocyclic skeletons,3 of which most can be related biogenetically to ursane and oleane precursors, although some diterpenoid−monoterpenoid conjugated isoprenoids have also been isolated.4 The genus Salvia is represented in the Iranian flora by 61 species, of which 17 are endemic.5 Salvia urmiensis Bunge is an endemic- and range-restricted species that grows in the West Azerbaijan province of northwestern Iran.6 A literature survey showed that there has been no phytochemical study on S. urmiensis, with the exception of an analysis of the essential oil.7 In our efforts to discover new and potentially bioactive secondary metabolites from Iranian Salvia species,1b,2b,d,4c we investigated the acetone extract of the aerial parts of S. urmiensis. Here we report the isolation, structure elucidation, and biological activity of urmiensolide (1), a triterpenoid with a unique seven-membered lactone E-ring. Urmiensolide (1) was isolated from an acetone extract of S. urmiensis as colorless needles (CHCl3). It had a molecular © XXXX American Chemical Society and American Society of Pharmacognosy
formula of C30H46O5, as deduced from HR-ESI-TOFMS (m/z 509.3243 [M + Na]+, calcd 486.3345). The molecular formula accounted for eight indices of hydrogen deficiency. The 13C NMR spectrum (Table 1) showed 30 carbon resonances, which were identified with the aid of HSQC and DEPTQ spectra as eight methyl, six methylene, eight methine, and eight quaternary carbons. The 13C NMR spectrum showed signals Received: April 28, 2013
A
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Table 1. 1H and 13C NMR Data of Compound 1 (CDCl3/ methanol-d4, 500 MHz for δH; pyridine-d5, 125 MHz for δC)a
a
position
δH (J in Hz)
δC
1 2α 2β 3 4 5 6 7α 7β 8 9 10 11 12 13 14 15α 15β 16α 16β 17 18 19 20 21α 21β 22 23 24 25 26 27 28 29 30
4.34, dd (4.8, 7.9) 3.19, dd (7.9, 14.5) 2.78, dd (4.8, 14.5)
79.6, d 44.5, t
1.57, 1.56, 1.55, 1.43,
m m m m
2.03, d (9.2) 4.54, br d (9.2) 5.75, d (2.9)
1.59, 1.16, 1.69, 2.13,
m t (3.5) m dt (3.5, 13.8)
2.75, 1.65, 1.48, 2.36, 3.01,
br d (9.8) m m dd (2.2, 14.0) dd (6.1, 14.0)
1.19, 1.23, 1.30, 1.07, 1.22, 1.45, 1.01, 1.13,
s s s s s s d (6.6) d (6.8)
216.1, 47.7, 51.4, 19.8, 34.1,
s s d t t
43.2, 55.2, 44.5, 67.3, 133.5, 139.8, 42.6, 27.6,
s d s d s s s t
33.2, t 85.0, 56.0, 37.8, 40.5, 40.4,
s d d d t
172.4, 28.4, 21.3, 13.8, 19.1, 22.6, 26.8, 21.0, 20.3,
s q q q q q q q q
Figure 1. (A) Key HMBC connectivities and (B) NOESY correlations of 1.
group was located at C-3. HMBC connectivities of H-1 (δH 4.34) with C-2 (δC 44.5), C-3(δC 216.1), C-10 (δC 44.5), and C-25 (δC 13.8) corroborated the constitution of ring A. A second hydroxy group at C-11 was confirmed by HMBC correlations from H-11 to C-9 (δC 55.2), C-12 (δC 133.5), and C-13 (δC 139.8). The coupling constants of H-1 (dd, J = 7.9, 4.8 Hz) and H-11 (br d, J = 9.2 Hz) indicated that the two hydroxy groups were in equatorial position. HMBC connectivities of CH3-30 (δH 1.13) with C-20 (δC 40.5) and C-21 (δC 40.4) and of H-20 (δH 1.48) and H-21 (δH 2.36, 3.01) with a carbonyl group (δC 172.4) indicated a lactone carbonyl at C22. A quaternary carbon at δC 85.0 (C-17) correlated with CH328 (δH 1.45), H-18 (δH 2.75), H-16 (δH 1.69, 2.13), and H-15 (δH 1.15, 1.59), thereby supporting the E-ring lactone moiety. The relative configuration of 1 was corroborated by a NOESY spectrum (Figure 1B). Diagnostic cross-peaks between H3-23 (δH 1.19) and H-2α (δH 3.19), between H-1 (δH 4.34) and H-5 (δH 1.57), and between H-9 (δH 2.03) and H3-27 (δH 1.22) confirmed their cofacial orientation. Similarly, NOESY cross-peaks between H3-24 (δH 1.23), H-2β (δH 2.78), H3-25 (δH 1.30), H-11 (δH 4.54), H3-29 (δH 1.01), H-18 (δH 2.75), and H3-28 (δH 1.45) confirmed their occupation of the same face of the molecule. Conclusive evidence for the structure of urmiensolide (1) (3-oxo-1β,11α-dihydroxy-17,22-seco-urs-12en-22,17-lactone) was obtained from single-crystal X-ray diffraction (Figure 2). The absolute configuration of 1 was established by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. The overall pattern of the calculated ECD spectrum matched well with the experimental data (Figure 3). In particular, two positive Cotton effects (CE) were observed at 202 and 295 nm. The positive CE at 295 nm in the experimental spectrum was due to the n → π* transition, and the intense positive CE around 202 nm belongs to the π → π* transitions of the carbonyl and double-bond groups. On the
δ values were established from HMBC, COSY, and HSQC.
indicative of a trisubstituted double bond (δC 133.5, 139.8) and of two carbonyl carbons (δC 172.4, 216.1). Three carbon signals at δC 67.3 (CH), 79.6 (CH), and 85.0 (C) indicated the presence of oxygen-bearing sp3 carbons. The absence of other sp or sp2 carbon signals implied that 1 contained five rings, including one heterocycle, in order to satisfy its indices of hydrogen deficiency. The 1H NMR spectrum (Table 1) displayed the characteristic signals of six methyl singlets (δH 1.07, 1.19, 1.22, 1.23, 1.30, and 1.45), two methyl doublets [δH 1.01 (J = 6.6 Hz) and 1.13 (J = 6.8 Hz)], two oxygenated methines (δH 4.54, br d, J = 9.2 Hz, and δH 4.34, dd, J = 4.8, 7.9 Hz), and an olefinic proton (δH 5.75, d, J = 2.9 Hz). Therefore, the structural features were reminiscent of an ursane-type triterpenoid containing one double bond and two carbonyl groups. The partial structure of 1 was solved by the HMBC correlations of the eight methyl groups. Analysis of other HMBC correlations (Figure 1A) confirmed the location of substituents. The signals of CH3-23 (δH 1.19) and CH3-24 (δH 1.23) were correlated with the resonances of a carbonyl group (C-3, δC 216.1), an aliphatic methine (C-5, δC 51.4), and a quaternary carbon (C-4, δC 47.7), suggesting that the carbonyl B
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exhibited moderate activity with good selectivity index (IC50 = 5.6 ± 0.8 μM). In the cytotoxic assays, 1 showed an IC50 value of >185 ± 0.27 μM against rat skeletal myoblasts (L6 cells) and thus had a selectivity index of 33.1 (IC50 for L6 cells divided by IC50 for T. b. rhodesiense). The positive control, melarsoprol, had an IC50 of 4.0 nM. Pentacyclic triterpenoids with a seco lactone moiety are rare in nature. Only a few A-ring seco lactone triterpenoids derived from oleane and fridelane skeletons9 and two triterpenoids derived from lupane with a δ-lactone E-ring have been reported.10 Urmiensolide (1) is the first example of a pentacyclic triterpenoid lactone with the E-ring expanded to a seven-membered ring. From a biogenetic point of view, this new scaffold may derive from α-amyrin, via Baeyer−Villiger oxidation as a key step (Scheme 1).
Figure 2. Single-crystal X-ray structure of 1.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured using a JASCO P-2000 automatic digital polarimeter. 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 a zgradient was used for 1H-detected experiments; 13C NMR spectra were recorded with a 5 mm BBO probe head with a z-gradient. Spectra were analyzed using Bruker TopSpin 2.1 software. CDCl3, methanold4, and pyridine-d5 for NMR was purchased from Armar Chemicals. HRESIMS spectra in positive mode were recorded on a Bruker microTOF ESIMS system with a scan range of m/z 150−1500. ECD spectra were recorded in MeOH (400 μg/mL) on an AVIV CD spectrometer model 62ADS and analyzed with the AVIV 60DS V4.1 software. Plant Material. The aerial parts of Salvia urmiensis were collected at full flowering stage in May 2010 in Takab, West Azarbaijan province. A voucher specimen (MPH-1220) has been deposited in the herbarium of Medicinal Plants and Drugs Research Institute (MPH) of Shahid Beheshti University, Tehran, Iran. Extraction and Isolation. The dried plant material (2.0 kg) was extracted successively with n-hexane (3 × 15 L), acetone (3 × 15 L), and MeOH (3 × 15 L) by maceration at room temperature. Fractionation of the acetone extract (50 g) by column chromatography on silica gel (n-hexane−EtOAc step gradient) afforded 12 fractions. The less polar fractions were composed of waxes and carotenoids and
Figure 3. ECD spectra of 1; experimental ECD (blue) and calculated ECD (TDDFT/B3LYP/6-31G**/SCRF-CPCM) in MeOH (black).
basis of these data the absolute configuration of 1 was established as 1R, 5R, 8R, 9S, 10S, 11R, 14S, 18S, 19S, 20R, 17R. The antitrypanosomal activity of triterpenoids was described by Hoet et al.8 Urmiensolide (1) was tested for in vitro antitrypanosomal activity against the Trypanosoma brucei rhodesiense STIB 900 strain and for cytotoxicity assessment against L6 cells (rat myoblast cell line). The compound Scheme 1. Plausible Biogenetic Pathway of 1
C
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(8) Hoet, S.; Pieters, L.; Muccioli, G. G.; Habib-Jiwan, J. L.; Opperdoes, F. R.; Quetin-Leclercq, J. J. Nat. Prod. 2007, 70, 1360− 1363. (9) (a) Chai, X.-Y.; Xu, Z.-R.; Bai, C.-C.; Zhou, F.-R.; Tu, P.-F. Fitoterapia 2009, 80, 408−410. (b) Michalet, S.; Payen-Fattaccioli, L.; Beney, C.; Cegiela, P.; Bayet, C.; Cartier, G.; Noungoue-Tchamo, D.; Tsamo, E.; Mariotte, A.-M.; Dijoux-Franca, M. G. Helv. Chim. Acta 2008, 91, 1106−1117. (c) Ku, Y. L.; Rao, G. V.; Chen, C.-H.; Wu, C.; Guh, J.-H.; Lee, S.-S. Helv. Chim. Acta 2003, 86, 697−702. (10) Maldonado, E.; Díaz-Arumir, H.; Toscano, R. A.; Martínez, M. J. Nat. Prod. 2010, 73, 1969−1972.
were not further investigated. From fraction 6 [3 g, eluted with hexane−EtOAc (70:30)] crude crystals were obtained, which were recrystallized from acetone to afford β-sitosterol (200 mg). Fraction 8 [2.1 g, eluted with n-hexane−EtOAc (50:50)] was subjected to silica gel column chromatography and eluted with a gradient of CHCl3− MeOH (100:0, 98:2, 95:5) to afford four fractions, 8a−8d. Fraction 8a (100 mg) was recrystallized from acetone to afford 2α-hydroxyursolic acid (15 mg). Fraction 10 [1 g, eluted with n-hexane−EtOAc (30:70)] was triturated with acetone to give an insoluble solid, which was recrystallized from MeOH to afford luteolin (80 mg). Fraction 11 [1.1 g, eluted with n-hexane−EtOAc (20:80)] was separated on a silica gel column [CHCl3−acetone (9:1)] into seven subfractions (11a−11g). Subfraction 11c was recrystallized from MeOH to afford 1 (15 mg). Urmiensolide (1): colorless crystals; [α]25 D = +51.9 (c 0.8, CHCl3); for 1H and 13C NMR data, see Table 1; ECD (MeOH, c = 9.0 mM, 0.1 cm) [θ]202 = +68 306, [θ]295 = +7362; HR-ESI-TOFMS m/z 509.3243 [M + Na]+, calcd for C30H46O5, 486.3345).
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ASSOCIATED CONTENT
S Supporting Information *
1D and 2D NMR spectra, X-ray crystal analysis data, calculated ECD spectra of conformers of 1, and details for the bioassay can be found as Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +98 21 29904043. Fax: +98 21 22431783. E-mail: m_
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS Financial support by the Shahid Beheshti University Research Council and Iran National Science Foundation (INSF; Grant No. 86023.07) is gratefully acknowledged. Thanks are due to Dr. T. Sharpe (Biozentrum, University of Basel) for technical assistance with measurement of UV and CD spectra.
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REFERENCES
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