Sesquiterpenoids Isolated from Two Species of the Asteriscus Alliance

Article pubs.acs.org/jnp

Sesquiterpenoids Isolated from Two Species of the Asteriscus Alliance Jorge Triana,† José Luis Eiroa,*,† Manuel Morales,† Francisco J. Perez,† Ignacio Brouard,‡ José Quintana,§ Mercedes Ruiz-Estévez,§ Francisco Estévez,§ and Francisco León*,‡,⊥ †

Departamento de Química, Unidad Asociada al CSIC, Universidad de Las Palmas de Gran Canaria, Campus de Tafira, 35017 Las Palmas de Gran Canaria, Canary Islands, Spain ‡ Instituto de Productos Naturales y Agrobiología-Consejo Superior de Investigaciones Científicas (CSIC), Avenida Astrofísico Francisco Sánchez 3, 38206 La Laguna, Tenerife, Spain § Departamento de Bioquímica, Universidad de Las Palmas de Gran Canaria, Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Canary Islands, Spain S Supporting Information *

ABSTRACT: Investigation of the aerial parts of two Spanish members of the Asteriscus alliance, Asteriscus graveolens subsp. stenophyllus and Asteriscus schultzii, afforded four new sesquiterpene lactones containing a humulene skeleton (1−4) and one new sesquiterpene lactone of the asteriscanolide type (5). Their chemical structures were determined on the basis of the HRMS and from 1D and 2D NMR spectroscopic studies. Both species showed different profiles of sesquiterpenoid constituents. A. schultzii did not show humulene or asteriscane sesquiterpenes, suggesting a resemblance to the genus Pallenis, another member of the Asteriscus alliance. A literature review on chemical isolates from the Asteriscus alliance supported the placement of A. schultzii in the genus Pallenis. The isolated components (1−5) were assessed for cytotoxicity against the HL-60 and MOLT-3 leukemia cell lines, with compound 1 showing activity in both biological assays (IC50 value range 4.1−5.4 μM).

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The sesquiterpene lactones constitute a major group of sesquiterpenoids found principally in the family Asteraceae. Humulene is one of the fundamental skeletons involved in sesquiterpene biosynthesis. Moreover, the humulyl cation may be considered to be the biogenetic precursor of other bi- and tricyclic sesquiterpene skeletons including those of the sterporene, pentalenene, caryophyllene, asteriscane, and protoilludane types, among others.3,4 The presence of sesquiterpene lactones in the Asteriscus species is especially significant from a biogenetic point of view. The majority of the sesquiterpenes isolated from Asteriscus possess a humulene moiety, suggesting that this feature is a chemotaxonomic characteristic for the entire genus Asteriscus. Following a systematic study of plants from the Canary Islands, Spain,5 this report describes the isolation and structural elucidation of five new sesquiterpene lactones (1−5) (Figure 1) from A. graveolens subsp. stenophyllus (Link) Greuter [syn. Nauplius stenophyllus (Link) Webb] collected in Gran Canaria, as well as five known sesquiterpenoids (6−10) (Figure 1) and three additional known compounds. A. schultzii (Bolle) Pit &

major epicenter of plant diversity is observed and distributed in the Iberian Peninsula, northwestern Africa, and the Macaronesia region. These areas contain a rich flora, with at least 3000 endemic species.1 The Asteriscus alliance is a monophyletic group belonging to the Asteraceae family and comprising three distinct and closely related genera (Asteriscus, Ighermia, and Pallenis). Evidence suggests that this group originated in northwestern Africa and underwent adaptive migration, leading to colonization in the Canary and Cape Verde archipelagos.1 Historically, there was a considerable controversy regarding the taxonomic classification of the Asteriscus alliance. In 1997, the genus Nauplius Cass. was included under the genus Asteriscus, based on phylogenetic analysis using internal transcriber spacer (ITS) sequences of their nuclear rDNA.2 Approximately 50 different Asteriscus species have been described, of which only 11 are currently recognized. Some of these include various subspecies: Asteriscus sericeus, A. intermedius, and A. graveolens subsp. stenophyllus (Canary Islands), A. schultzii (Canary Islands and Morocco), A. smithii, A. daltonii subsp. daltonii, and A. daltonii subsp. vogelli (Cape Verde Islands), A. graveolens subsp. graveolens (North Africa), A. graveolens subsp. odorus (Canary Islands and North Africa), A. imbricatus (Morocco), and A. aquaticus (Mediterranean).1,2 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: November 10, 2015

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DOI: 10.1021/acs.jnatprod.5b01013 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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human cancer cell lines (HL-60 and MOLT-3) were investigated.

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RESULTS AND DISCUSSION Purification by column chromatography of an ethanol extract of the aerial parts of A. graveolens subsp. stenophyllus afforded four new humulenoid sesquiterpenes, namely, 6β,7β-epoxyasteriscunolide A (1), 2α,3α-epoxyasteriscunolide C (2), 6β-hydroxyasteriscunolide A (3), and 6β-ethoxyasteriscunolide A (4), and also a new asteriscanoid sesquiterpene, asteriscanolidenol (5), along with the known sesquiterpenoids asteriscunolide A (6),7 asteriscunolide C (7),7 8-oxo-6,7,9,10-tetrahydrohumulen-1,12olide (8),8 asteriscanolide (9),9 and 8-oxo-α-humula-6E,9Zdien-12-oic acid (10),8 as well as vanillin,10 stigmasterol,11 and tricin.12 From the ethanolic extract of A. schultzii, two known sesquiterpenes, 1α,4β,6β-trihydroxyeudesmane (11)13 and teuclatriol (12),14 and several other known substances, dehydroabietic acid,15 7-oxodehydroabietic acid,16 stigmasterol,11 scoparone,17 scopoletin,17 and 5,6,7-trimethoxycoumarin,18 were isolated. The structures of these known compounds were confirmed by comparison of their spectroscopic data (MS, and 1H and 13C NMR) with values in the literature. Compound 1 was isolated as colorless needles, mp 146−148 °C. The IR spectrum showed the presence of an absorption band at 1756 cm−1 that indicated a γ-lactone group, as well as a conjugated carbonyl group absorbing at 1640 cm−1. The molecular formula of 1 was established as C15H18O4 by HRESIMS (m/z 285.1107 [M + Na]+). The 1H NMR spectra (Table 1) showed characteristic signals for three methyl groups at δH 1.54 (3H, s, CH3-13), 1.55 (3H, s, CH3-14), and 1.43 (3H, s, CH3-15), three olefinic protons at δH 6.89 (1H, br t, J = 1.7 Hz, H-2), 6.35 (1H, d, J = 13.9 Hz, H-9), and 5.57 (1H, d, J = 13.9 Hz, H-10), and two signals at δH 4.68 (1H, br t, J = 1.7 Hz, H-1) and 2.91 (1H, dd, J = 4.2, 9.8 Hz, H-6) corresponding to the geminal protons to the oxygenated groups. The 13C NMR and DEPT spectra (Table 1) of 1 disclosed 15 carbons,

Figure 1. Structures of sesquiterpenoids identified from the aerial parts of Asteriscus graveolens subsp. stenophyllus and A. schultzii.

Proust [syn. Nauplius schultzii (Bolle) Wiklund] collected in Lanzarote, Spain, yielded seven known compounds including two sesquiterpenoids (11 and 12) (Figure 1). On the basis of previous chemical studies of the Asteriscus species, the utility of these kinds of compounds from the chemotaxonomic point of view and the significant presence of humulene lactones in the present results are discussed. A review of the literature was conducted on different sesquiterpenoids previously obtained from various species in the Asteriscus alliance. Additionally, earlier studies have shown that sesquiterpene lactones, including those containing a humulene skeleton, display cytotoxic activities against cancer cells.6 In the present study, the potential cytotoxic effects of compounds 1−5 against two Table 1. NMR Spectroscopic Data of Compounds 1−4 in CDCl3 1a position

δC

1 2 3 4a 4b 5a 5b 6 7 8 9 10 11 12 13 14 15 OCH2 CH3

89.4 150.6 132.0 18.3 25.8 65.7 63.1 197.4 128.7 146.8 41.5 173.3 26.3 21.4 32.2

δH mult. (J, Hz)

δC

4.68, br t (1.7) 6.89, br t (1.7)

83.4 59.6 58.9

2.31−2.37, m 1.68, m 2.39, m 2.91, dd (4.2, 9.8)

24.5

6.35, d (13.9) 5.57, d (13.9)

1.54, s 1.55, s 1.43, s

3c

2

27.9 130.4 138.3 202.0 130.9 152.2 40.0 171.8 19.4 24.6 20.8

δH mult. (J, Hz)

a

4.27, s 3.78, s 1.61, 2.69, 2.10, 2.31, 5.67,

δC

b

89.6 150.3 131.2

m br t (10.0) dd (10.0, 12.0) br d (10.0) br d (10.9)

6.21, d (16.3) 6.58, d (16.3)

1.91, s 1.33, s 1.31, s

19.4 31.8 73.3 48.7 195.1 131.2 143.2 41.3 170.3 10.0 26.6 32.0

δH mult. (J, Hz) 4.75, br s 7.06, br s 2.00, m 2.31, m 1.91, m 2.27, m 3.62, m 2.58 qd (2.3, 7.2) 6.14, d (13.8) 5.45, d (13.8)

1.28, d (7.2) 1.56, s 1.46, s

4a δC 90.3 149.9 134.1 21.3 29.5 78.3 51.4 202.8 132.2 147.7 42.2 173.9 7.2 25.7 31.9 64.5 15.4

δH mult. (J, Hz) 4.65, t (1.8) 6.90, t (1.4) 2.20, 2.41, 1.62, 1.67, 3.36, 2.96,

m ddd (3.8, 12.0, 16.0) m m ddd (3.1, 4.6, 6.0) qd (2.7, 6.5)

6.06 d (13.6) 5.50, d (13.6)

0.90, 1.58, 1.41, 3.45, 1.14,

d (6.6) s s m t (6.5)

a Spectra measured at 500 MHz for 1H NMR and 125 MHz for 13C NMR. bSpectra measured at 150 MHz for 13C NMR. cSpectra measured at 400 MHz for 1H NMR and 100 MHz for 13C NMR.

B



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which were indicative of an α,β-unsaturated carbonyl at δC 197.4, s, C-8; 128.7 d, C-9, and δC 146.8, d, C-10, a carbonyl ester at δC 173.3 s, C-12, and an epoxide functional group at δC 65.7, d, C-6, and δC 63.1, s, C-7. These NMR data were closely related to those of the known humulanolide astericunolide C (7)7 and suggest that compound 1 is a humulene sesquiterpene. Further assignments were carried out based on 2D NMR data. Thus, HMBC (Figure 2) correlations of H-1 with C-12, C-3,

Figure 2. Selected HMBC correlations (H to C) for compound 1. Figure 3. Selected NOESY correlations for compound 2.

and C-11; H-2 with C-1, C-12, and C-4; CH3-13 with C-6, C-8, and C-7; and CH3-14 with C-11, C-9, and C-1 were observed. The relative configuration of the epoxide group was determined from NOESY experiments by the correlation between CH3-13 and H-6, which showed clearly that these protons are on the same face of the molecule. Accordingly, the structure of 1 was proposed as 6β,7β-epoxyastericunolide A. Compound 2 was isolated as an amorphous solid. The molecular formula was determined to be C15H18O4, based in its HRESIMS data (m/z 285.1104 [M + Na]+). The 13C NMR spectrum (Table 1) gave a total of 15 separate carbon resonances (three methyls, two methylenes, five methines, and five quaternary carbons), in agreement with the molecular formula. The 1H NMR spectrum (Table 1) of compound 2 displayed signals for three methyl groups at δH 1.91 (3H, s, CH3-13), 1.33 (3H, s, CH3-14), and 1.31 (3H, s, CH3-15) and three olefinic protons at δH 5.67 (1H, br d J = 10.9 Hz, H-6), 6.21 (1H, d, J = 16.3 Hz, H-9), and 6.58 (1H, d, J = 16.3 Hz, H10). The 1H NMR spectra of 1 and 2 were similar in regard to their functional group analysis. However, the epoxide group of compound 2 was not located between C-6 and C-7. The COSY experiment showed correlations between the signals at δH 4.27 (1H, s) and 3.78 (1H, s) assigned to the H-1 and H-2 protons, respectively. This led to the observation that the double bond between C-2 and C-3 is epoxidized. Its 13C NMR spectrum (Table 1) showed signals characteristic of an epoxide ring at δC 59.6, d, C-2; 58.9, s, C-3. In the HMBC spectrum of 2, the signals corresponding to H-2 showed correlations with C-11 and C-4, and H-1 with C-14 and C-15. The geometry of the C9, C-10 double bond was determined to be E by considering the coupling constant (J9,10 = 16.3 Hz), similar to data reported for asteriscunolide C.19 The relative configuration of 2 was determined from the NOESY spectrum (Figure 3). Important correlations were observed from CH3-13 to H-6 and H-5, leading to the conclusion that the geometry of the C-6, C-7 double bond is Z. Correlations were also observed between H-1 and H-2 and between H-2 and H-9, H-4a, CH3-14, and H-5a, suggesting a cis epoxide. From these data, compound 2 was assigned as 2α,3α-epoxyasteriscunolide C, which is reported for the first time. Compound 3 was isolated as a yellow oil. The 1H NMR data (Table 1) of this compound were very similar to those of compound 1, suggesting that both structures are closely related. On the basis of its HREIMS data (m/z 264.1354 [M]+), two additional hydrogen atoms were found for 3 when compared to

1, corresponding to a molecular formula of C15H24O4, suggesting that 3 lacks an epoxide. The IR spectrum showed absorption bands at 3427, 1745, and 1663 cm−1 typical for hydroxy, γ-lactone, and ketone groups. The 13C NMR spectra of 3 (Table 1) carried out at 25 and 50 °C exhibited in each case a poor resolution due to the occurrence of a conformational equilibrium of the humulene skeleton.20 Similar cases are reported for germacrane sesquiterpenes.21 The high-field signal at δC 10 d for CH3-13 was reported previously in the humulene systems.22 The COSY experiment disclosed three partial structures, CH2CH2CHCHCH3, CHCH, and CHCH, corresponding to the C-4, C-5, C-6, C-7, C-13; C-1, C-2; and C-9, C-10 fragments. The geometry of the C-9, C-10 double bond was determined to be Z by considering the coupling constant (J9,10 = 13.8 Hz), comparable to those reported for asteriscunolide A.19 The assignments were based on the correlations shown in the HSQC and HMBC experiments. The relative configuration of 3 was determined from the ROESY spectrum (Figure 4), with important correlations observed from H-1 to H-2, H-9, H-4a, CH3-14, and CH3-15; from H-6 to H-7; and from H-7 to H-9, suggesting that the methyl group at C-7 and the hydroxy group at C-6 are in the same orientation, in agreement with the small short coupling constant J6,7 = 2.6 Hz. On the basis of these experiments, 3 was assigned as 6β-hydroxylasteriscunolide A.

Figure 4. Selected ROESY correlations for compound 3. C



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and C-7; CH3-14 with C-1, C-10, and C-11; and H-9 with C-3, C-8, and C-10 were observed. These assignments were similar to those of the known sesquiterpenoid asteriscanolide,9 with the exception of a disubstituted double bond occurring between C-5 and C-6 and the hydroxy group attached to C-7. COSY and HMBC experiments (Figure 5) showed correlations

Compound 4 was isolated as a yellow oil. Its molecular formula, C17H24O4, was determined by HRESIMS (m/z 315.1570 [M + Na]+). The 1H NMR data of 4 (Table 1) were very similar to those of 3, suggesting that the two compounds are closely related. Additional signals of an ethoxy group at δH 3.45 (2H, m, OCH2CH3) and 1.14 (3H, t, J = 6.5 Hz, OCH2CH3) revealed that the C-6 hydroxy group of 3 has been alkylated in 4. The absence of a characteristic OH group band in the IR spectrum confirmed the lack of a hydroxy group at C-6. On the basis of the above evidence, compound 4 was assigned as 6β-ethoxyasteriscunolide A. Compound 4 was obtained as a presumed extraction artifact, due to the use of hot EtOH during the purification process. Compound 4 might be the result of an intermolecular Michael addition of compound 6.23 Asteriscanolidenol (5) was isolated as an amorphous white powder with mp 177−179 °C. Its molecular formula was determined as C15H20O4 by HRESIMS at m/z 287.1261 [M + Na]+, indicating six degrees of unsaturation. Its IR spectrum showed an absorption band at 3458 cm−1, corresponding to a hydroxy group, and two bands due the presence of carbonyl groups at 1765 and 1709 cm−1, corresponding to a γ-lactone and an aliphatic ketone, respectively. The 1H NMR spectrum of 5 (Table 2) exhibited signals for three methyls at δH 1.49 (3H,

Figure 5. Selected COSY (bold lines) and HMBC (H to C) correlations for compound 5.

between H-1, H-2, H-3, and H-9 similar to asteriscanolide. An additional correlation between CH3-13 and H-9 in the NOESY experiment supports an α orientation for the hydroxy group. Therefore, the structure of compound 5 (asteriscanolidenol) was established as shown (Figure 1). The secondary metabolite profile of the genus Asteriscus is characterized predominantly by the presence of sesquiterpenoids with humulene and asteriscane skeletons, and these could be used as chemotaxonomic generic markers. In contrast, the genus Pallenis, another member of the Asteriscus alliance, has no reported humulene-astericane sesquiterpenoids. The distribution of the sesquiterpenoids (not including essential oils) isolated from the Asteriscus alliance is shown in Table S1 (Supporting Information), and the chemical structures of the above-mentioned compounds are grouped in Figures S1 and S2 (Supporting Information). The absence of humulene-astericane sesquiterpenoid derivatives in A. schultzii indicates the possible deviation of this species from the genus Asteriscus, supporting its possible placement under the closely related genus of the Asteriscus alliance Pallenis. Further phylogenetic studies, as well as the phytochemical analysis of additional A. schultzii populations, should be performed to confirm this proposal. The cytotoxic properties of compounds 1−5 were evaluated using two human leukemia cancer cell lines. Thus, compound 1 displayed cytotoxic properties against two human leukemia cell lines, HL-60 myeloid and MOLT-3 lymphoid cell lines. Treatment of this compound resulted in a concentrationdependent reduction of cell viability (Figure S3, Supporting Information), with no significant differences between either cell line. Compound 1 showed similar IC50 (concentrations inducing a 50% inhibition of cell growth) values (ca. 4−5 μM) in each leukemia cell line. The IC50 value against HL-60 cells was 5.4 ± 2.2 μM, similar to the value obtained for MOLT-3 cells (4.1 ± 1.4 μM). In contrast, the other new compounds (2−5) did not display cytotoxicity (IC50 > 10 μM) against either cell line. The antitumor agent etoposide was used as a positive control for MOLT-3 (IC50 = 0.7 ± 0.2 μM) and HL-60 (IC50 = 0.5 ± 0.1 μM) cells.

Table 2. NMR Spectroscopic Data of Compound 5a in CDCl3 position

δC

1 2 3 4α 4β 5 6 7 8 9 10β 10α 11 12 13 14 15

91.3 46.7 43.6 23.4 132.9 134.2 77.1 214.2 44.8 39.2 41.7 176.2 28.0 24.6 22.5

δH mult. (J, Hz) 4.29, 3.30, 3.08, 2.27, 2.42, 5.97, 5.86,

d (4.2) ddd (4.3, 5.3, 5.6) ddd (7.7, 8.0, 15.6) m m dt (7.0, 10.4) dd (0.9, 10.4)

3.46, dt (7.4, 11.3) 1.52, dd (7.5, 13) 2.54, dd (11.8, 13.0)

1.49, s 1.04, s 1.28, s

a

Spectra measured at 500 MHz for 1H NMR and 125 MHz for 13C NMR.

s, CH3-13), 1.28 (3H, s, CH3-15), and 1.04 (3H, s, CH3-14), two olefinic protons corresponding to a disubstituted double bond at δH 5.97 (1H, dt, J = 7.0 and 10.4 Hz, H-5) and 5.86 (1H, dd, J = 0.9 and 10.4 Hz, H-6), and one signal at δH 4.29 (1H, d, J = 4.2 Hz, H-1) corresponding to the geminal proton to the oxygenated group. The 13C NMR (Table 2) and DEPT spectra showed 15 signals, which were indicative of a carbonyl lactone at δC 176.2, s, C-12 and an aliphatic ketone at δC 214.2, s, C-8. The presence of a disubstituted double bond was indicated from signals at δC 132.9, d, C-6 and δC 134.2, d, C-5. The remaining signals corresponded to three CH3, two CH2, four CH, and two quaternary carbons. Thus, 5 was found to contain three unsaturated functionalities and three ring systems in its structure. HMBC correlations of CH3-13 with C-8, C-6,

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded with a PerkinElmer model 343 polarimeter. IR spectra were recorded using a Bruker model IFS-55 spectrophotometer. 1H and 13C NMR spectra were obtained on Bruker models AMX-400, D



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1370, 1201, 1085, 975 cm−1; 1H NMR and 13C NMR data, see Table 1; EIMS m/z 264 [M]+, 249, 247, 220, 168, 161, 150, 140, 125, 109, 107, 96, 91, 69; HREIMS m/z 264.1354 [M]+ (calcd for C15H20O4, 264.1362). 6β-Ethoxyasteriscunolide A (4): yellow oil; [α]20D +16.1 (c 0.25, CHCl3); IR (film, NaCl) νmax 3015, 2955, 2915, 2849, 1741, 1670, 1345, 1210, 1025, 973 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 315.1570 [M + Na]+ (calcd for C17H24O4Na, 315.1572). Asteriscanolidenol (5): amorphous, white powder; mp 177−179 °C; [α]25D −24.0 (c 0.01, CHCl3); IR (film NaCl) νmax 3458, 3035, 2960, 2031, 2874, 1765, 1709, 1462, 1369, 1263, 1199, 1157, 1063, 995 cm−1; 1H NMR and 13C NMR data, see Table 2; EIMS m/z 264 [M]+, 247 [M − OH]+, 221, 203, 178, 161, 147, 133, 121, 107, 95, 79; HRESIMS m/z 287.1261 [M + Na]+ (calcd for C15H20O4Na, 287.1259). Cell Cultures and Cytotoxicity Assays. The human leukemia MOLT-3 and HL-60 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and grown in RPMI 1640 containing 2 mM L-glutamine supplemented with 10% (v/v) heat-inactivated fetal bovine serum.24 The cytotoxicity of compounds 1−5 was analyzed using the colorimetric 3-(4,5dimethyl-2-thiazolyl-)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Briefly, 1 × 104 exponentially growing cells were seeded in 96-well microculture plates with various concentrations of test compounds. After the addition of MTT (0.5 mg/mL), cells were incubated at 37 °C for 4 h. Sodium dodecyl sulfate (SDS) (10% w/v) in 0.05 M HCl was added to the wells and then incubated at room temperature overnight under dark conditions. The absorbance was measured at 570 nm. Concentrations inducing a 50% inhibition of cell growth (IC50) were determined graphically for each experiment by a nonlinear regression using the curve-fitting routine of the computer software Prism 4.0 (GraphPad). Values are means ± SE from at least three independent experiments, with each performed in triplicate.25 Etoposide was used as a positive control.

-500, and -600 NMR spectrometers with standard pulse sequences operating at 400, 500, and 600 MHz for 1H and 100, 125, and 150 MHz for 13C NMR, respectively. CDCl3 was used as solvent. EIMS were taken on a Micromass model Autospec (70 eV) spectrometer. HRESIMS was performed with an LCT Premier XE Micromass Waters spectrometer in the positive-ionization mode (Waters Corporation). Column chromatography (CC) was carried out on silica gel 60 (Merck 230−400 mesh), and preparative TLC on silica gel 60 PF254+366 plates (20 × 20 cm, 1 mm thickness). Plant Material. The aerial parts of Asteriscus graveolens subsp. stenophyllus and Asteriscus schultzii were collected by J.L.E. at Temisas, Agüimes, Gran Canaria, Canary Islands, Spain, and Famara, Teguise, Lanzarote, Canary Islands, Spain, respectively. The plants were identified by Dr. Rosa Febles, Viera y Clavijo Botanical Garden, Gran Canaria, Spain, and the respective voucher specimens have been deposited with numbers LPA32708 for A. graveolens subsp. stenophyllus and LPA23622-LPA23623 for A. schultzii in the herbarium at this institution. Extraction and Isolation. Aerial parts of the two Asteriscus species were exhaustively extracted with 95% EtOH in a Soxhlet apparatus for 72 h. The solvent was concentrated under reduced pressure, and the extracts were subsequently fractionated by silica gel column chromatography using hexane and EtOAc mixtures of increasing polarity. The extraction of 4.6 kg of A. graveolens subsp. stenophyllus gave 420 g of a viscous mass, which was subjected to column chromatography on silica gel, affording five fractions. Fraction 1 (hexane−EtOAc, 4:1) was purified using preparative TLC with hexane−EtOAc (4:1) as solvent, to give stigmasterol (31 mg), asteriscunolide A (6) (25 mg), asteriscunolide C (7) (11 mg), and 6β-ethoxyasteriscunolide A (4) (12 mg). Fraction 2 (hexane−EtOAc, 3:2) gave 8-oxo-6,7,9,10tetrahydrohumulen-1,12-olide (8) (37 mg), 6β,7β-epoxyastericunolide A (1) (9.1 mg), and 2α,3α-epoxyasteriscunolide C (2) (7.2 mg), after purification using preparative TLC eluted with hexane−EtOAc (4:1) twice. Fraction 3 (hexane−EtOAc,1:1) gave tricin (45 mg) and 8-oxoα-humula-6E,9Z-dien-12-oic acid (10). Purification of fraction 4 (hexane−EtOAc, 2:3) yielded vanillin (9.2 mg) and asteriscanolide (9) (23 mg). Fraction 5 (hexane−EtOAc, 1:4) was eluted with hexane−EtOAc (1:1) using CC to give asteriscanolidenol (5) (7.3 mg) and 6β-hydroxyasteriscunolide A (3) (2.1 mg). The extraction of 1.10 kg of A. schultzii gave 85 g of a viscous material. This extract was chromatographed using CC over silica gel with hexane−EtOAc of increasing polarity, and four fractions were obtained. Fraction 1, eluted with hexane−EtOAc (4:1), gave stigmasterol (27 mg). Elution of fraction 2 with hexane−EtOAc (3:2) gave scopoletin (10 mg) and scoparone (15 mg). Fraction 3 was eluted by CC using hexane−EtOAc (3:2) and gave two subfractions (3A and 3B). Purification of subfraction 3A on CC using hexane− EtOAc (4:1) afforded 1α,4β,6β-trihydroxyeudesmane (11) (6 mg) and teuclatriol (12) (11 mg). Subfraction 3B was eluted with hexane− EtOAc (3:2) followed by preparative TLC (benzene−EtOAc, 4:1), yielding 5,6,7-trimethoxycoumarin (11 mg). Fraction 4 (hexane− EtOAc, 1:4) was purified using preparative TLC (benzene−EtOAc, 1:1), yielding dehydroabietic acid (4 mg) and 7-oxodehydroabietic acid (4 mg). 6β,7β-Epoxyasteriscunolide A (1): colorless needles (CH3OH); mp 146−148 °C; [α]20D +2.0 (c 0.01, CHCl3); IR (film, NaCl) νmax 3011, 2966, 2935, 2874, 1756, 1640, 1450, 1380, 1328, 1192, 1083, 985 cm−1; 1H NMR and 13C NMR data, see Table 1; EIMS m/z 262 [M]+, 247 [M − CH3]+, 219, 201, 191, 167, 166, 151, 140, 109, 96, 81, 67; HREIMS m/z 262.1263 [M]+ (C15H18O4, calcd for 262.1251); HRESIMS m/z 285.1107 [M + Na]+ (calcd for C15H18O4Na, 285.1103). 2α,3α-Epoxyasteriscunolide C (2): amorphous solid; IR (film, NaCl) νmax 2955, 2915, 2849, 1784, 1731, 1659, 1439, 1323, 1275, 1182, 1080, 993 cm−1; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z 285.1107 [M + Na]+ (calcd for C15H18O4Na, 285.1103). 6β-Hydroxyasteriscunolide A (3): yellow oil; [α]20D −8.9 (c 0.21, CHCl3); IR (film, NaCl) νmax 3427, 3013, 2969, 2874, 1745, 1663,

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b01013. Sesquiterpenoids previously isolated from the Asteriscus alliance and copies of 1D and 2D NMR spectra and HRMS of compounds 1−5 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 34-928-454427. Fax: 34-928-260135. E-mail: jeiroa@ dqui.ulpgc.es (J. L. Eiroa). *Tel: +1-662-915-2014. Fax: +1-662-915-5638. E-mail: jfleon@ olemiss.edu; jfl[email protected] (F. León). Present Address ⊥

Department of BioMolecular Sciences, Division of Medicinal Chemistry, School of Pharmacy, University of Mississippi, University, Mississippi 38677, United States.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by Agencia Canaria de Investigación, Innovación y Sociedad de la Información (C200801000174 to J.T.). F.L. was supported by the JAEDoc Program from the Ministerio de Ciencia e Innovación, Spain. E



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Article

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Sesquiterpenoids Isolated from Two Species of the Asteriscus Alliance

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