Article Cite This: J. Nat. Prod. 2018, 81, 1384−1390
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Phytochemical Study of Salvia leriifolia Roots: Rearranged Abietane Diterpenoids with Antiprotozoal Activity Mahdi M. Farimani,*,† Bahareh Khodaei,† Hanzaleh Moradi,† Abbas Aliabadi,‡ Samad N. Ebrahimi,† Maria De Mieri,§ Marcel Kaiser,⊥,∥ and Matthias Hamburger§ †
Department of Phytochemistry, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, G. C., Evin, Tehran, Iran Khorasan Razavi Agricultural and Natural Resources Research Center, Sabzevar Branch, Sabzevar, Khorasan Razavi, Iran § Division of Pharmaceutical Biology, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland ⊥ Swiss Tropical and Public Health Institute, Socinstrasse 57, CH-4002 Basel, Switzerland ∥ University of Basel, Petersplatz 1, CH-4003 Basel, Switzerland
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‡
S Supporting Information *
ABSTRACT: Phytochemical investigation of the lipophilic extract of the roots of Salvia leriifolia resulted in the isolation of the new rearranged abietane diterpenoids leriifoliol (1) and leriifolione (2), together with 10 known diterpenoids. Structure elucidations were performed via extensive NMR and HRESIMS data, and the absolute configurations of compounds 1 and 3−5 were established by evaluation of experimental and calculated ECD spectra. The antiplasmodial activity of the new isolates was assayed against Trypanosoma brucei rhodesiense, T. cruzi, Plasmodium falciparum, and Leishmania donovani and also toxicity against rat myoblast (L6) cells. Compound 1 displayed antimalarial and low cytotoxic activity with IC50 values of 0.4 and 33.6 μM, respectively, and a selectivity index of 84. Compound 2 displayed activity against T. brucei, T. cruzi, and L. donovani, with IC50 values of 1.0, 4.6, and 1.0 μM, respectively. Putative biosynthetic pathways toward the formation of 1, 2, and 3 are proposed. Leriifoliol (1) is the first 20-nor-9,10-seco-abietane, while 2 exhibits an uncommon 6−6−5 fused-ring system.
H
The genus is characterized by the presence of a broad range of isoprenoids including sesterterpenoids6,7 and di- and triterpenoids with uncommon carbon frameworks.8−10 Salvia leriifolia Benth., locally known as Nuruozak and Jobleh, grows exclusively in the Semnan and southern regions of the Khorasan Razavi Provinces of Iran.11 It has been used in folkloric medicine, and analgesic, hypoglycemic, anti-inflammatory, and anticonvulsant activities have been reported for extracts prepared from different parts of the plant.12−15 In an earlier phytochemical study on the aerial portions of the plant, a
uman protozoan parasites are a significant health threat in subtropical and tropical countries around the world, leading to over one million deaths annually.1 They pose a health risk for hundreds of millions worldwide that results in significant morbidity and socioeconomic loss. Malaria, trypanosomiasis, and leishmaniasis have disability-adjusted life years in the millions.2,3 The emergence of parasite strains that are resistant to current chemotherapy and the successful track record of natural-product-derived antiprotozoal drug discovery highlight the importance of natural sources as a valuable approach for the development of new drugs. The genus Salvia L. (family Lamiaceae) is widely distributed in Iran, with 61 species, and 17 of the plants are endemic.4,5 © 2018 American Chemical Society and American Society of Pharmacognosy
Received: December 4, 2017 Published: June 13, 2018 1384
DOI: 10.1021/acs.jnatprod.7b01019 J. Nat. Prod. 2018, 81, 1384−1390
Journal of Natural Products
Article
series of labdane-type diterpenoids were identified.16 Here the isolation, structure determination, and antiparasitic potential of the stilbene-like abietane diterpenoid leriifoliol (1) and leriifolione (2), an unprecedented five-membered C-ring abietane diterpenoid from the roots, are discussed.
Table 1. 1H and 13C NMR Data of Compounds 1 (in C5D5N) and 2 (in CDCl3) (500 MHz for δH, 125 MHz for δC)a 1 position
RESULTS AND DISCUSSION A lipophilic extract was prepared from the shade-dried powdered material of S. leriifolia roots. The residue was separated using repeated column chromatography over silica gel and preparative TLC, affording two new and 10 known diterpenoids. Compound 1 possessed a molecular formula of C19H28O3, as inferred from the sodium adduct molecular ion [M + Na]+ at m/z 327.1942 (calcd 327.1936) in the HRESI-TOFMS, accounting for six indices of hydrogen deficiency. Nineteen carbon signals were observed in the 13C NMR spectrum, which were divided into four CH3, five CH2, four CH, two oxygenated tertiary and three quaternary carbons, and a carbonyl carbon, according to the APT and HSQC spectra (Table 1). These carbons carried 26 protons; thus, the remaining hydrogens formed part of two hydroxy residues. The 13C NMR data showed resonances for a tetrasubstituted benzene moiety [δC 104.2 (CH), 120.4 (C), 126.5 (C), 128.0 (CH), 154.3 (C), and 155.3 (C)] and a carbonyl carbon (δC 213.5). To satisfy the molecular formula of C19H28O3, the structure of 1 needs to include two rings. The 1H NMR data of 1 (Table 1) showed two methyl singlets at δH 0.79 and 1.02. Two methyl doublets resonating at δH 1.46 (d, J = 6.9 Hz) and 1.48 (d, J = 6.9 Hz) and coupled with a septet at δH 3.68 (J = 6.9 Hz) showed the presence of an aromatic isopropyl moiety. Signals at δH 6.90 (s) and 7.24 (s) were indicative of a 1,2,4,5-tetrasubstituted aromatic ring. These structural characteristics resembled a seco-nor-abietane skeleton. In the COSY spectrum, three distinct spin sets of C-1−C-2−C-3, C-5−C-6−C-7, and C15−C-16−C-17 were observed. A complete assignment of all protons and carbons and the establishment of their connectivities were obtained using the HMBC (Figure 1A) and HSQC data. Downfield shifts of H-1β (δH 2.36, m), H-1α (δH 2.46, m), and H-5 (δH 2.43, m), together with the HMBC connectivities between a carbonyl carbon at δC 213.5 (C-1) and H-1α, H-1β, H2-2 (δH 1.78, 2H, m), H-5, H-6a (δH 1.95, m), and H-6b (δH 2.29, m), corroborated the location of the carbonyl moiety at C-10. HMBC connections of H-15 (δH 3.68, sept, J = 6.9 Hz) with C-12 (δC 155.3), C-13 (δC 126.5), and C-
δC
position
41.2
29.5
1α 1β 2α 2β 3α 3β 4 5 6α 6β 7
120.4 154.3 213.5 104.2 155.3 126.5 128.0 27.1 23.9 24.0 29.3 22.5
8 9 10 11 12 13 14 15 16 17 18 19
1α 1β 2
2.46, m 2.36, m 1.78, m
23.3
3
1.53, m
38.9
4 5 6a 6b 7a 7b 8 9 10 11 12 13 14 15 16 17 18 19
■
δH (J in Hz)
2
2.43, 1.95, 2.29, 2.86, 2.96,
m m m m m
6.90, s
7.24, s 3.68, sept (6.9) 1.48, d (6.9) 1.46, d (6.9) 1.02, s 0.79, s
39.5 60.8 26.1
δH (J in Hz) 1.26, 2.67, 1.56, 1.73, 1.48, 1.22,
m br d (14.5) br d (13.5) m br d (12.0) m
1.80, dd (3.0, 12.0) 2.55, dd (3.0, 14.5) 2.43, t (13.0)
δC 33.3 17.7 40.5 33.0 50.2 36.9 196.2
2.48, d (4.0) 2.28, 0.95, 0.90, 0.88, 0.92, 1.31,
m d (6.8) d (6.8) s s s
140.0 175.2 38.5 204.2 57.2 197.2 29.2 19.4 18.9 32.5 20.9 17.4
δ values were established from HMBC, COSY, and HSQC experiments.
a
14 (δC 128.0), of H-11 (δH 6.90, s) with C-8 (δC 120.4), C-9 (δC 154.3), C-12, and C-13, and of H-14 (δH 7.24, s) with C-7 (δC 29.5), C-9, C-8, C-13, C-12, and C-15, respectively, established the substitution pattern of the aromatic ring. Finally, HMBC connectivities from H-7a (δH 2.86, m) and H-7b (δH 2.96, m) to C-5 (δC 60.8), C-6 (δC 26.1), C-14, C-9, and C-8, in combination with fragment −CH−CH2−CH2− deduced from the HMQC and 1H−1H COSY spectra defined the connectivity of rings B and A through the C7−C6 chain. The 3D structure of ring A was established with the aid of the NOESY data (Figure 1B). Cross-peaks between H3-18 (δH 1.02), H-5, and H-1α (δH 2.46) corroborated their cofacial orientation. The experimental electronic circular dichroism (ECD) spectrum of 1 showed two positive Cotton effects (CEs) at 287 and 210 nm attributable to the n → π* and π → π* transitions of the carbonyl group and the aromatic ring, respectively. The computed ECD spectrum agreed with the experimental spectrum of the (5S) enantiomer with two positive CEs at 280 and 210 nm. Hence, leriifoliol (1) was established as 20-nor-9,10-seco-abieta-9,12-dihydroxy-8,11,13trien-10-one. Abietane and rearranged abietane diterpenoids have been reported from various plant species. However, stilbene-like diterpenoids are rare. Only five 9,10-seco-abietanes have been previously reported,17−21 and leriifoliol (1) is the first 20-nor9,10-seco-abietane diterpenoid. Biosynthetically 1 could be derived from ferruginol, via formation and cleavage of a dioxetane ring as key steps (Scheme 1). Compound 2 had a molecular formula of C19H26O3, as indicated by the HRESI-TOFMS data (found [M + Na]+ 1385
DOI: 10.1021/acs.jnatprod.7b01019 J. Nat. Prod. 2018, 81, 1384−1390
Journal of Natural Products
Article
Figure 1. (A) Key HMBC connectivities and (B) NOESY correlations of 1.
Scheme 1. Putative Biosynthetic Pathway to Leriifoliol (1)
Figure 2. (A) Key HMBC connectivities and (B) NOESY correlations of 2.
three CH, and seven nonprotonated carbons, based on the APT and HSQC spectra (Table 1). Thus, all 26 hydrogen atoms were accounted for, indicating the absence of a hydroxy
325.1790; calcd 325.1780), implying seven indices of hydrogen deficiency. The 13C NMR data revealed the presence of 19 carbon signals, which were divided into five CH3, four CH2, 1386
DOI: 10.1021/acs.jnatprod.7b01019 J. Nat. Prod. 2018, 81, 1384−1390
Journal of Natural Products
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Scheme 2. Putative Biosynthetic Pathway to Leriifolione (2)
Figure 3. Comparison of experimental and calculated ECD spectra for the possible stereoisomers of 1 and 3−5.
structure of 2 contained three rings. The 1H NMR data of 2 revealed signals of two secondary CH3 at δH 0.9 (3H, d, J = 6.8 Hz) and 0.95 (3H, d, J = 6.8 Hz), both coupled with a CH resonance at δH 2.28 (1H, m) (Table 1). In addition, three
group in the structure. Resonances for a tetrasubstituted double bond (δC 140.0, 175.2) and three carbonyl carbons (δC 196.2, 197.2, and 204.2) were identified in the 13C NMR spectrum. Hence the molecular formula of C19H26O3 required that the 1387
DOI: 10.1021/acs.jnatprod.7b01019 J. Nat. Prod. 2018, 81, 1384−1390
Journal of Natural Products
Article
Table 2. In Vitro Activity of Compounds 1 and 2 against P. falciparum, T. b. rhodesiense, T. cruzi, and L. donovani and Cytotoxicity in L6 Cells [IC50 (μM)]
a
compound
P. falciparuma
T. brhodesiense
T. cruzi
L. donovani
L6 cells
leriifoliol (1) leriifolione (2) choroquine melarsoprol benzonidazole miltefosine podophyllotoxin
0.4 ± 0.1 (84)b 3.6 ± 0.2 (0.7)b 0.01 ± 0.01
19.7 ± 0.1 (1.7)b 1.0 ± 0.1 (2.6)b
27.6 ± 0.2 (1.2)b 4.6 ± 0.2 (0.6)b
NDc 1.0 ± 0.1 (2.6)b
33.6 ± 0.1 2.6 ± 0.1
0.01 ± 0.01 2.2 ± 0.2 0.12 ± 0.01 0.02 ± 0.01
Average of three independent assays. bSelectivity index (SI): IC50 in L6 cells divided by IC50 in the title parasite strain. cND: not determined.
example of a naturally occurring nor-abietane isoprenoid possessing a rare five-membered C-ring. It is reasonable to assume that the biosynthetic pathway of 2 differs from other ent-abietane diterpenoids.22 A putative pathway is proposed in Scheme 2, whereby an 11,12-seco-abietane, generated via an oxidative cleavage at Δ11(12), would be the precursor to 2. The 6,6,5-tricyclic scaffold would be obtained via decarboxylation at C-12 and subsequent cyclization to establish the C-11−C-13 bond. We also propose a biosynthetic pathway for martiusane (3), a diterpenoid with a rare scaffold that has been previously reported from Hyptis martiusii Benth.23 (Supporting Information). The absolute configurations of martiusane (3), deacetylnemorone (4),24 and 11-hydroxyabieta-8,11,13-trien-7-one (5)25 were established via the ECD data. Based on the NMR data and considering a putative biosynthetic pathway, (5S,9S,10S) and (5S,9R,10S) configurations were anticipated for 3. The ECD spectrum of 3 showed two negative CEs at 380 and 245 nm and two positive CEs at 270 and 210 nm, which were due to a π−π* transition of the dihydro para-quinone moiety. The simulated ECD spectrum for the (5S,9R,10S) diastereoisomer (Figure 3B) agreed with the recorded data and thus confirmed the absolute configuration of 3. The NMR data of deacetylnemorone (4) indicated a relative configuration of (5S*,7R*,10R*). Three positive CEs at 325, 270, and 220 nm and a negative CE at 290 nm were observed in the experimental ECD spectrum. The simulated spectrum of the (5S,7R,10R) diastereoisomer was in good accordance with the experimental data (Figure 3C), and the absolute configuration of 4 was thus established as (5S,7R,10R). The experimental ECD spectrum of 11-hydroxyabieta8,11,13-trien-7-one (5), with two positive CEs at 345 and 215 nm and two negative CEs at 305 and 275 nm, matched the simulated data of the (5S,10S) diastereoisomer (Figure 3D). The antiprotozoal activity of leriifoliol (1) and leriifolione (2) was tested against Trypanosoma brucei rhodesiense, T. cruzi, Plasmodium falciparum, and Leishmania donovani parasites. The cytotoxicity of the compounds to rat myoblast L6 cells was determined for an initial assessment of the selectivity of the compounds (Table 2). Leriifoliol (1) showed an IC50 of 0.4 μM against P. falciparum and a selectivity index of 84. Compound 2 was active against T. brucei, T cruzi, and L. donovani, with IC50 values 1.0, 4.6, and 1.0 μM, respectively, but the SI values of
