neo-Clerodane Diterpenoids and Other ... - ACS Publications

Article pubs.acs.org/jnp

neo-Clerodane Diterpenoids and Other Constituents of Salvia filipes Emma Maldonado,* Leonel Galicia, Ma. Isabel Chávez, and Simón Hernández-Ortega Instituto de Química, Universidad Nacional Autónoma de México, Circuito Exterior, Ciudad Universitaria, Coyoacán, 04510, D.F., México S Supporting Information *

ABSTRACT: A series of neo-clerodane-type diterpenoids were isolated from the aerial parts of Salvia filipes, including the new compounds 4-epi-polystachyne A (1), salvifilines A (3), C (7), and D (8), and salvifiline B, which was isolated as the 15-O-methyl derivatives 4/5. In addition, the five known diterpenoids (2, 9−12), together with ursolic, oleanolic, and betulinic acids, and the flavone eupatorin were also isolated. The structures were determined by analysis of their spectroscopic data, mainly 1D and 2D NMR. The structure of salvifiline D was confirmed by X-ray analysis. The cytotoxic activities of the diterpenoids were evaluated, but all were inactive against a panel of six human cancer cell lines.

Salvia, the most diverse genus of the Lamiaceae family, includes about 1000 species distributed in the subgenera Calosphace, Leonia, Salvia, and Sclarea. It is the second most diverse genus in Mexico, where it is represented by 292 species, most of them endemic (85−88%). With a few exceptions (15 species), the Mexican Salvia species are included in the subgenus Calosphace.1−3 Chemical investigations of Salvia species have shown that their major metabolites are terpenoids, including monoterpenoids, sesquiterpenoids, diterpenoids, sesterterpenoids, and triterpenoids. The diterpenoid group is the largest and includes abietanes and clerodanes, with normal or modified carbon skeletons, although some pimarane and labdane diterpenoids have also been isolated from these plants.4 Many of these diterpenoids exhibit interesting biological activities.4 As part of a program to discover new metabolites from Salvia,5,6 we have analyzed the aerial parts of Salvia f ilipes Benth., a perennial herb included in the section Polystachyae of the subgenus Calosphace.3 From an ethnopharmacological point of view this species, the same as other salvias of the “mirto complex” (S. coccinea, S. elegans, S. f ulgens, S. involucrata, and S. microphylla), is used in México and Central America against a variety of ailments that range from cultural illnesses (“susto”, “mal de ojo”) to digestive, hepatic, and nervous system diseases.4,7 In this paper the isolation and identification of the new neo-clerodane diterpenoids 4-epi-polystachyne A (1), salvifilines A (3), C (7), and D (8), a mixture of epimeric 15-O-methylsalvifilines B (4/5), and 11 known compounds are discussed.

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RESULTS AND DISCUSSION Compound 1 was obtained as colorless crystals whose molecular formula was established as C20H22O5 by HRFABMS. The IR spectrum showed the presence of a γ-lactone (1776 cm−1), a β-substituted furan ring (1503, 875 cm−1), and double-bond (1681 cm−1) functionalities. The 1H and 13C © 2016 American Chemical Society and American Society of Pharmacognosy

Received: June 30, 2016 Published: September 28, 2016 2667

DOI: 10.1021/acs.jnatprod.6b00605 J. Nat. Prod. 2016, 79, 2667−2673

Journal of Natural Products

Article

Figure 1. Selected NOE correlations of compounds 1 and 2.

Table 1. 1H NMR Data of Compounds 1−5 and 7−9 (500 MHz, CDCl3) position 1

1

3a

2

2

5.48, ddd (10.0, 4.5, 2.5) 5.89, m

5.47, br d (10.0) 5.92, m

5.46, br dd (10.0, 1.5) 5.85, m

3α

2.59, m

2.17, br d (17.5)

3β 4

2.22, br d (19.0) 2.35, d (6.0)

6α

2.00, dd (15.0, 4.0)

6β

1.70, dd (15.0, 1.5)

7

4.36, dd (4.0, 1.5)

2.38, br d (17.5) 2.07, m 2.33, dd (11.5, 5.0) 1.80, dd (14.0, 4.5) 1.28, br d (14.0) 4.44, d (4.5)

8 10 11α

1.83, q (7.5) 2.56, m 1.93, dd (13.5, 7.5)

11β

2.64, dd (13.5, 7.5)

12

5.20, dd (7.5, 7.5)

14

6.32, dd (2.0, 1.0)

15

7.40, dd (2.0, 1.5)

16 17 19pro‑S 19pro‑R

7.36, 1.29, 4.58, 3.89,

20 OH or OMe

5.73, s

a

m d (7.5) d (9.5) d (9.5)

1.96, q (7.0) 2.67, br s 1.88, dd (13.5, 8.0) 2.65, dd (13.5, 7.5) 5.28, dd (8.0, 7.5) 6.32, dd (2.0, 1.0) 7.40, dd (2.0, 1.5) 7.36, m 1.27, d (7.0) 4.84, d (8.0) 4.02, dd (8.0, 2.0) 5.23, s

7a

4/5 5.44, br d (10.0)

5.44, br d (10.0)

5.87, br ddd (10.0, 5.0, 1.5) 2.58, m

5.85, ddt (10.0, 5.5, 2.0) 2.17, br d (17.5)

8a

9

5.41, br d (10.5) 5.80, m

6.23, dd (10.0, 2.5)

2.28, m

6.95, d (5.0)

6.33, ddd (10.0, 5.0, 3.0)

1.95, m 2.59 dd (11.5, 4.5) 1.46 dd (14.0, 4.5

2.22, br d (19.0) 2.34, d (7.0)

1.95, m 2.57, dd (12.0, 5.0)

2.24, m 2.57, d (6.0)

2.00, dd (14.5, 4.0)

1.46, dd (14.0, 4.5)

1.26, d (14.0)

1.69, dd (14.5, 1.5)

1.26, d (14.0)

1.87, dd (15.0, 3.5) 1.66, d (15.0)

4.31, d (4.0)

4.37, dd (4.0, 1.5)

4.32, d (4.5)

4.23, d (3.5)

1.82, q (7.5) 2.77, br s 1.77, m

q (7.5) br s dd (13.5,

1.82, q (7.5) 2.75, br s 1.93, dd (13.5, 8.0)

dd (13.5,

2.61, dd (13.5, 8.5)

br dd (8.5,

4.99, br

dd (1.5,

6.10, br s

1.67, q (7.0) 2.43, br s 2.00, dd (13.5, 7.5) 2.56, dd (13.5, 8.5) 4.89, dd (8.5, 7.5) 6.12, br s

6.10, br s

1.84/1.83, 2.58/2.56, 1.89/1.87, 8.0) 2.71/2.69, 8.5) 4.98/4.96, 8.0) 7.00/6.98, 1.5) 5.80/5.77,

1.11, d (7.5) 4.15, br d (8.0) 4.52, d (8.0)

1.251/1.249, d (7.5) 4.57/4.55, d (9.5) 3.887/3.883, d (9.5)

6.13, 1.10, 4.16, 4.49,

5.25 s 7.78, br s

5.79/5.77, s 3.60/3.57, s

5.24, s 7.91, br s

2.62, m 4.91, dd (8.0, 7.5) 7.27, s

m

1.28, m 1.89, dddd (14.0, 14.0, 4.0, 2.0) 1.59, dddd (14.0, 14.0, 4.5, 4.5) 2.10, m 2.46, br d (4.5) 2.37, m 2.14, br d (15.5) 2.73, dd (15.5, 8.0) 5.75, br d (8.0) 6.42, m 7.40, dd (1.5, 1.0)

br s d (7.0) br d (7.5) d (7.5)

6.12, 1.11, 4.53, 3.90,

br s d (7.0) d (9.0) d (9.0)

5.79, s 7.88, br s

7.46 dd (2.0, 1.5) 4.04, dd (9.0, 2.0) 4.54, d (9.0) 1.28, s

Determined in DMSO-d6.

connectivities between H-7 and C-5 (δC 40.0), C-9 (δC 59.0), C-17 (δC 14.6), and C-20; between H-12 and C-9, C-11 (δC 38.4), C-13, C-14, C-16, and C-20; and between H-20 and C-7, C-8 (δC 39.8), C-9, C-10 (δC 43.5), and C-12. A double bond at C-1 was deduced from the signals at δH 5.48, δC 127.4 (CH1) and δH 5.89, δC 126.3 (CH-2), together with the HMBC connectivities between H-1 and C-3 (δC 19.7), C-5, C-9, and C10; between H-2 and C-3 and C-10; and of H-3α (δH 2.59 m), H-3β (δH 2.22 br d, J = 19.0 Hz), H-4, and H-19pro‑S (δH 4.58 d, J = 9.5 Hz) with C-18 (δC 176.6). The above data indicated that compound 1 has the same 2D structure as polystachyne A (2), a neo-clerodane diterpenoid previously isolated from S. polystachya5 and also in the present study. The relative configuration of 1 was determined by the NOESY correlations

NMR spectra confirmed the presence of the furan ring via the signals at δC 128.7 (C); δH 6.32, δC 108.3 (CH); δH 7.40, δC 143.8 (CH); and δH 7.36, δC 138.6 (CH). The signals for a lactone carbonyl carbon at δC 176.6 and an oxymethylene (δH 4.58 and 3.89, δC 79.8) were consistent with the presence of a γ-lactone functionality. These data were considered as an indication of a clerodane-type diterpenoid, since β-substituted furan rings bonded to C-12 and 18,19-γ-lactones are the most common structural features of clerodanes from Salvia.4,8 The presence of a 2,8-dioxabicyclo[3.3.0]octane moiety in 1 was deduced from the signals for an acetalic methine (δH 5.73 s, δC 109.5, CH-20) and two oxymethines (δH 4.36 dd, J = 4.0, 1.5 Hz, δC 86.2, CH-7) (δH 5.20 dd, J = 7.5, 7.5 Hz, δC 74.3, CH12). These assignments were based on the long-range HMBC 2668



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Table 2. 13C NMR Data of Compounds 1−5 and 7−9 (125 MHz, CDCl3) position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OMe a

1 127.4, 126.3, 19.7, 44.4, 40.0, 39.9, 86.2, 39.8, 59.0, 43.5, 38.4, 74.3, 128.7, 108.3, 143.8, 138.6, 14.6, 176.6, 79.8, 109.5,

2 CH CH CH2 CH C CH2 CH CH C CH CH2 CH C CH CH CH CH3 C CH2 CH

128.68, CH 127.4, CH 20.5, CH2 45.4, CH 42.0 C 30.3, CH2 86.7, CH 40.0, CH 58.6, C 50.3, CH 38.3, CH2 75.8, CH 128.73, C 108.3, CH 143.8, CH 138.5, CH 14.6, CH3 175.3, C 79.5, CH2 110.1, CH

3a 128.9, 126.6, 19.9, 43.9, 41.3, 29.9, 86.2, 39.2, 57.9, 48.3, 35.0, 74.3, 137.3, 146.2, 97.4, 169.6, 13.9, 175.3, 78.6, 109.9,

CH CH CH2 CH C CH2 CH CH C CH CH2 CH C CH CH C CH3 C CH2 CH

4/5 127.2, CH 126.4, CH 19.7, CH2 44.4, CH 39.92/39.89, C 39.83/39.82, CH2 86.51/86.49, CH 39.76, CH 58.88/58.85, C 43.15/43.09, CH 36.5/36.0, CH2 74.1, CH 140.4/140.1, C 142.0/141.4, CH 102.7, CH 169.1, C 14.3, CH3 176.5, C 79.7, CH2 110.0/109.9, CH 57.4/57.1

7a 128.8, 126.6, 19.9, 44.0, 41.3, 29.9, 86.2, 39.3, 57.7, 48.3, 34.7, 75.8, 170.6, 115.4, 170.2, 97.6, 13.9, 175.3, 78.6, 109.8,

CH CH CH2 CH C CH2 CH CH C CH CH2 CH C CH C CH CH3 C CH2 CH

8a 127.5, 126.0, 19.3, 43.2,

CH CH CH2 CH

b

38.8, 85.8, 39.0, 58.1, 42.3, 34.7, 74.2, 170.7, 115.5, 170.3, 97.9, 13.9, 176.9, 78.7, 109.1,

CH2 CH CH C CH CH2 CH C CH C CH CH3 C CH2 CH

9 133.2, 126.1, 128.7, 132.8, 39.8, 21.8, 16.7, 41.2, 35.2, 50.0, 36.4, 70.9, 127.0, 108.6, 144.5, 138.6, 172.7, 168.7, 76.9, 31.3,

CH CH CH C C CH2 CH2 CH C CH CH2 CH C CH CH CH C C CH2 CH

Determined in DMSO-d6. bNot observed.

10, and H-20 (Figure 1). These data were in agreement with structure 3, in which H-4 is α-oriented. Compound 3 was named salvifiline A. Compounds 4/5 were obtained as a mixture whose HRFABMS data exhibited a protonated molecular ion at m/z 389.16048 [M + H]+, indicating a molecular formula of C21H24O7. In the NMR spectra of the mixture, most of the proton and carbon signals were duplicated. These signals were similar to those of 4-epi-polystachyne A (1), except those related to the C-12 side chain, which in compounds 4/5 corresponded to a 15-methoxy-14-en-16,15-olide moiety. The latter was deduced from the pairs of signals for C-13 (δC 140.4/ 140.1), CH-14 (δH 7.00/6.98, δC 142.0/141.4), CH-15 (δH 5.80/5.77, δC 102.7), C-15-OCH3 (δH 3.60/3.57, δC 57.4/ 57.1), and C-16 (δC 169.1), as well as from the HMBC spectrum, which showed correlations of H-12 with C-11, C-13, C-14, C-16, and C-20; of H-14 and H-15 with C-16; and of 15OCH3 with C-15. As in compound 1, NOE cross-peaks of H-4 with both C-6 protons and H-19pro‑R established the βorientation of H-4. These NOE interactions were not evident in compounds 2 and 3, in which H-4 is α-oriented. In this manner, the structures of the constituents of this mixture were determined as 4/5. The presence of the C-15 methoxy groups in these compounds indicated that they most probably are artifacts of the extraction/isolation process; thus, the true natural product is most likely 6. Compounds 4/5 are named 15-O-methylsalvifilines B. The HRFABMS ions of compounds 7 and 8 (m/z 375.144 00 and 375.143 00, respectively) and their 13C NMR data were in agreement with the molecular formula C20H22O7. Analysis of their NMR data showed that these compounds are C-4 epimers. Both compounds contain a γ-hydroxy-α,βunsaturated-γ-lactone moiety, different from that present in compound 3. The lactone units in 7 and 8 were identified as 16-hydroxy-13-en-15,16-olides via their NMR data, which in the case of 7 showed resonances at δC 170.6 for the β-carbon (C-13), δH 6.10, δC 115.4 for the α-methine (CH-14), δC 170.2

between H-10/H-19pro‑S and H-20, which showed that these protons were α-cofacial, provided that compound 1 is also considered a neo-clerodane diterpenoid.8 The β-orientations of H-7 and H-12 were deduced from the NOESY cross-peaks between CH3-17/H-7, H-11β, and H-12, while the correlations between H-19pro‑R/H-4 and H-6α, as well as those between H4/H-3β, H-6α, and H-6β, established the β-orientation of H-4 (Figure 1). In the case of polystachyne A (2), the NOESY spectrum showed correlations between H-19pro‑S/H-4, H-10, and H-20 and between H-4/H-10, which confirmed the assigned α-orientation of H-4 (Figure 1).5 Thus, the structure of 1 was determined to be the C-4-epimer of polystachyne A and was named 4-epi-polystachyne A. It should be noted that in the original publication5 the structure of polystachyne A was elucidated as 2, and erroneously, H-4 was depicted with a βorientation, instead of the α-orientation stated in the corresponding discussion. Compound 3 was obtained as colorless crystals. Its molecular formula was defined as C20H22O7 by HRFABMS (m/z 375.1435 [M + H]+). The IR spectrum showed the presence of hydroxy (3338 cm−1), γ-lactone (1776 cm−1), α,βunsaturated-γ-lactone (1737 cm−1), and olefinic (1635 cm−1) functionalities. The 1H and 13C NMR data of 3 resemble those of 2 (Tables 1 and 2), with the exception of the signals of the C-12 side chain. These signals indicated the presence of a γhydroxy-α,β-unsaturated-γ-lactone, and they were assigned to C-13 (δC 137.3), CH-14 (δH 7.27, δC 146.2), CH-15 (δH 6.10, δC 97.4), C-15-OH (δH 7.78), and C-16 (δC 169.6). These assignments were based on the HMBC cross-peaks between H12 and C-11, C-13, C-14, and C-20. In accordance with the presence of the γ-hydroxybutenolide moiety and, therefore, with an equilibrium between the C-15-hemiacetal moieties, the 1 H and 13C NMR signals for CH2-11 to C-16 were broadened. The relative configuration of 3 was deduced to be the same as 2 from the NOESY interactions of H-10 with H-1, H-4, H-11α, H-19pro‑S, and H-20; of H-12 with H-11β and H-17; of H-17 with H-7, H-8, H-11β, and H-12; and of H-19pro‑S with H-4, H2669



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Figure 2. X-ray crystallographic displacement ellipsoid diagram of salvifiline D (8).

Figure 3. (A) 1H NMR spectrum of the mixture after subtracting the signals of compound 9. (B) 1H NMR spectrum of compound 9. (C) 1H NMR spectrum of the mixture of compounds 9 and 10.

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for the lactone carbonyl (C-15), δH 6.13, δC 97.6 for the hemiacetalic methine (CH-16), and δH 7.91 for the C-16-OH proton. Compound 8 showed similar signals for this moiety (Tables 1 and 2). The remaining NMR signals of compound 7 were similar to those of 2 and 3, while the corresponding signals of 8 resembled those of 1 and 4/5. The NOESY spectrum of 7 confirmed the α-orientation of H-4, by its interactions with H-3α, H-10, and H-19pro‑S, while the NOE cross-peaks of H-4 with H-6α, H-6β, and H-19pro‑R proved the β-orientation of H-4 in compound 8. Compounds 7 and 8 were named salvifilines C and D, respectively. A single-crystal X-ray analysis of salvifiline D (8), using Cu Kα radiation for the anomalous structure dispersion, permitted confirmation of its structure and absolute configuration. The Flack absolute structure parameter was 0.05(17) (Figure 2). In addition to the new diterpenoids, a mixture of linearolactone (9) and its 3,4-dihydro derivative 10 (≈2:3 ratio) was isolated. Although all attempts to separate this mixture failed, the different ratio of its constituents enabled elucidation of their structures. Thus, the HPLC-MS analysis showed pseudomolecular ions at m/z 341 and 343 for 9 and 10, respectively, which are in accordance with the molecular formulas C20H20O5 and C20H22O5. Analysis of the NMR spectra allowed assignment of most of the signals and identification of the less abundant compound (9) as the clerodane diterpenoid linearolactone, which was previously isolated from S. lineata9,10 and S. polystachya.5 The more abundant compound (10) exhibited 13C NMR signals for γand δ-lactone carbonyls, a trisubstituted furan ring, one double bond, one methyl, five methylenes, four methines, and two unprotonated carbons. These signals allowed the proposal of the structure of 10 as a 3,4-dihydro derivative of 9. Signal overlap between 1.0 and 3.0 ppm in the 1H NMR spectrum, however, prevented the confirmation of this proposal. Thus, considering the availability of an authentic sample of 9,5 it was decided to subtract its 1H NMR signals from the spectrum of the mixture (Figure 3C), without affecting the signals of compound 10.11 This subtraction was done using the MestReNova program, version 8.1. The resulting spectrum (Figure 3A) allowed the location of all the proton signals of 10. Thus, the position of the double bond at C-1 could be confirmed by the HMBC connectivities of H-1, H-2, H-8, H-11, and H-19pro‑S with C-10. The NOE correlations of H19pro‑S with H-4 and H-10; of H-10 with H3-17; and of H3-17 with H-8 revealed that all these protons are cofacial and, therefore, established the structure of the major component as 10, a compound previously isolated from S. haenkei.12 The NMR data (Table S1, Supporting Information) and those published for 1012 were similar, although some differences were observed for the signals of C-2 (δC 129.0; lit. δC 127.2) and H-4 (δH 2.38 dd, J = 13.0, 6.0 Hz; lit. δH 2.40, assigned to H-3) and mainly for that of H-12 (δH 5.67 ddd, J = 8.0, 1.5, 1.5 Hz; lit. δH 4.46 dt, J = 8.4, 1.5 Hz). Despite the above, we believe that the compounds are the same. On the other hand, analysis of the 1D and 2D NMR spectra of linearolactone (9) led to the reassignment of some of the published signals.9,10 Complete 1H and 13C NMR data are included in Tables 1 and 2. Other known compounds isolated from this plant are rhyacophiline (11),13 salvianduline D (12),14 a mixture of βsitosterol and stigmasterol, betulinic acid,15 a mixture of ursolic and oleanolic acids, β-sitosterol glucoside, and the flavone eupatorin (13).16 These compounds were identified by analysis

of their spectroscopic data and comparison with authentic samples and/or with published data. The cytotoxic activity of the isolated diterpenoids (except the mixture of 9 and 10) was evaluated against a panel of six human cancer cell lines. However, none of them exhibited significant inhibitory activity at the assayed concentrations (20.0 or 25.0 μM).

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points (uncorrected) were determined on a Fisher-Johns melting point apparatus (Fisher Scientific, USA). Optical rotations were measured on a PerkinElmer 343 polarimeter. IR spectra were measured on a Nicolet FTIR-Magna 750 spectrophotometer. 1H and 13C NMR spectra were recorded on a Varian Inova 500 (1H at 500 MHz; 13C at 125 MHz) spectrometer with tetramethylsilane as an internal standard. HRDARTMS were determined on a JEOL AccuTOF JMS-T100LC mass spectrometer. HRFABMS were obtained on a JEOL JMSAX505HA mass spectrometer. HPLC-MS analysis was carried out on a Bruker Esquire 6000 spectrometer fitted with an Agilent 1200 series HPLC and an ESI source. Column chromatography (CC) was performed on silica gel 60 (Merck G) and was accelerated with a vacuum. TLC was performed on precoated Alugram Sil G/UV254 plates (0.25 mm). Plant Material. The aerial parts of Salvia f ilipes Bentham were collected in Tenango de Doria, State of Hidalgo, Mexico, in November 2012 and identified by Ma. del Rosario Garciá Peña. A voucher specimen (MEXU-1 340 282) was deposited at the National Herbarium, Instituto de Biologia,́ Universidad Nacional Autónoma de México. Extraction and Isolation. Dried and ground aerial parts (1.53 kg) were successively extracted by percolation with acetone (∼12 L) and MeOH (∼8 L) to obtain, after solvent evaporation, 69.21 and 131.92 g of extracts, respectively. The combined extracts were dissolved in MeOH−H2O, 4:1 (1.0 L), and extracted with hexanes (5×, 300 mL) to give 56.93 g of extract, after solvent evaporation. The aqueous MeOH fraction was concentrated, diluted with H2O, and extracted with EtOAc (5×, 300 mL) to give 49.22 g of extract. The hexanes extract was fractionated by CC eluted with hexanes, hexanes−CHCl3 (1:1), CHCl3, acetone, and MeOH, to obtain 6.69, 14.57, 22.89, 6.40, and 5.6 g of residues, respectively. The hexanes−CHCl3-soluble residue was fractionated using CC eluted with mixtures of hexanes− EtOAc of increasing polarity as follows: fractions A1−A14 (19:1); A15−A47 (9:1); and A48−53 (4:1). Fractions A7−A18 (1.21 g) were subjected to CC eluted with hexanes−acetone, 97:3 (fr. B1); 19:1 to 17:3 (fr. B2). Fr. B1 gave a mixture of β-sitosterol and stigmasterol (999 mg). Fr. B2 was discolored with activated charcoal and crystallized from EtOAc−hexanes to give 433 mg of betulinic acid. Fractions A31−A33 were purified by CC eluted with hexanes−EtOAc (17:3) to obtain a mixture of ursolic and oleanolic acids. Fractions A34−A38 were subjected to CC eluted with hexanes−EtOAc (17:3) to afford a mixture of ursolic and oleanolic acids and 2 (82.4 mg). The mother liquors of the ursolic and oleanolic acid fractions were combined with the CHCl3- and Me2CO-soluble residues and discolored with activated charcoal. The resulting residue (27.6 g) was purified by CC (hexanes−EtOAc, 17:3) to obtain an additional amount of a mixture of the acids for a total yield of 7.64 g. Fractions A39−A52 were subjected to CC using hexanes−EtOAc (7:3) as eluent to obtain fractions C1−C27. CC (hexanes−EtOAc, 17:3) of fractions C17−C27 gave 1 (160.7 mg). The EtOAc-soluble extract was fractionated by CC eluted with mixtures of hexanes−EtOAc of increasing polarity to give fractions D1−D48 (4:1); D49−D71 (7:3); D72−D75 (3:2); D76−D84 (1:1); and D85−D96 (1:4). CC (hexanes−Me2CO 17:3) of fractions D9− D12 (3.36 g) gave 11 (672 mg). Fractions D13−D24 (6.598 g) were subjected to CC eluted with CHCl3−MeOH (99:1) to obtain fractions E1−E23. Compound 11 (411 mg) was obtained from fractions E3 and E4, and 2 (105.7 mg), from fractions E5−E7. Mother liquors of 11 and 2 (2.65 g) were subjected to CC eluted with CHCl3−MeOH (199:1) 2671



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Salvifiline C (7): colorless crystals (EtOAc−i-Pr2O−hexanes); mp 242−246 °C; [α]20D +32 (c 0.2, MeOH); IR (CHCl3) νmax 3306, 1764, 1728, 1459, 1377, 1024, 981, 938, 896 cm−1; 1H NMR and 13C NMR (DMSO-d6) see Tables 1 and 2; HRDARTMS m/z 375.14400 [M + H]+ (calcd for C20H23O7, 375.14438). Salvifiline D (8): colorless crystals (EtOAc−i-Pr2O−hexanes); mp 232−235 °C; [α]20D −45 (c 0.2, MeOH); IR (CHCl3) νmax 3583, 3350, 1769, 1711, 1469, 1428, 1363,1045, 1032, 1011, 985, 936 cm−1; 1 H NMR and 13C NMR (DMSO-d6) see Tables 1 and 2; HRFABMS m/z 375.14300 [M + H]+ (calcd for C20H23O7, 375.14438). X-ray Crystallographic Data of Salvifiline D (8). The data were collected from a colorless needle (0.239 × 0.210 × 0.124 mm) at 296(2) K on a Bruker D8 Venture κ-geometry diffractometer equipped with multilayer Cu Kα radiation (λ 1.541 78 Å): C20H22O7 (formula weight 374.37); orthorhombic, space group P212121; a = 7.1015(2) Å, b = 9.7204(2) Å, c = 26.2050(6) Å; α = β = γ = 90°, V = 1808.92(8) Å3; Z = 4; Dcalc= 1.375 Mg/m3; F(000) = 792. A total of 9940 reflections were collected in the theta range of 3.373° to 68.097°, with 3253 independent reflections [R(int) = 0.0540], completeness to θmax was 99.7%. The structure was solved by direct methods using SHELXS-2014/717 and refined by full matrix least-squares on F2 using SHELXL-2014/717 with anisotropic temperature factors for nonhydrogen atoms converging at final R indices [I > 2σ(I)], R1 = 0.0435, wR2 = 0.0955. Absolute structure parameter: 0.05(17). Hydrogen atoms, except those bonded to oxygen, were included at calculated positions and were not refined. Cytotoxicity Assay. Cytotoxicity of the isolated diterpenoids (except 9 and 10) was screened by the sulforhodamine B (SRB) method,18 against six human cancer cell lines: glioblastome (U-251), prostatic adenocarcinoma (PC-3), chronic myelogenous leukemia (K562), colorectal adenocarcinoma (HCT-15), mammary adenocarcinoma (MCF-7), and lung adenocarcinoma (SKLU-1). Cell suspensions (100 μL containing 5000−7500 cells per well) were placed into 96well microtiter plates and incubated at 37 °C for 24 h, in a 5% CO2 atmosphere. The cultures were exposed to the tested compounds for 48 h. Then, the cells were fixed and stained with SRB as described,18 and the absorption was measured at 515 nm. Crystallographic data for the structure reported in this paper have been deposited with the Cambridge Crystallographic Data Centre, CCDC 1505704. Copies of these data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336

to give 2 (total yield 374.6 mg), 11 (total yield 1.208 g), and 12 (11.3 mg). Mother liquors of 12 were purified by Si gel CC (hexanes− Me2CO, 3:1) to obtain 12 (6.0 mg) and 3 (302.6 mg). CC of fractions D25−D37 (5.54 g) using mixtures of hexanes−EtOAc as eluents gave fractions E1−E79 (17:3); E80−90 (4:1); and E91−E110 (7:3). Crystallization of fractions E42−E90 (EtOAc−hexanes) afforded 1 (47.8 mg). Mother liquors of 1 were combined with fractions E21− E41 (1.04 g) and subjected to Si gel CC eluted with hexanes−acetone (3:1) to afford 1 (31.2 mg) and 3 (87.2 mg). Fractions D38−D50 (4.86 g) were subjected to Si gel CC eluted with mixtures of hexanes− acetone of increasing polarity to give fractions F1−F23 (3:1) and F24−F45 (7:3). Fractions F10−F28 were purified by Si gel CC (hexanes−EtOAc, 3:2) to obtain 13 (14.5 mg). Crystallization of fraction D51 (EtOAc−hexanes) afforded a mixture of 9 and 10 (205.8 mg). Si gel CC of fractions D52−D101 (17.1 g) using hexanes− EtOAc (3:2 and 1:1) as eluent gave fractions G1−G36 and G37−G49, respectively. Fractions G14−G49 were further chromatographed over Sephadex LH-20 with MeOH to afford fractions H1−H39. Purification of fractions H7−H11 (8.43 g) by Si gel CC eluted with a hexanes− acetone gradient (9:1 to 0:1) gave fractions I1−I93. Further purification of fractions I56−I90 by Si gel CC (hexanes−EtOAc gradient) afforded fractions J1−J47 (3:2); J48−J61 (1:1), and J62−69 (4:1). β-Sitosterol glucoside (34 mg) was isolated from fractions J3 and J4. Fractions J11−J20 were purified by Si gel CC eluted with CHCl3−MeOH (98:2) to obtain fractions K1−K29. Further purification of fractions K4−K16 (1.91 g, Si gel CC, hexanes− EtOAc, 7:3) gave fractions L1−L30. Fractions L10−L25 were purified by Si gel CC (CHCl3−EtOAc, 7:3) to obtain fractions M1−M27. Fractions J3−10, K3, L6−L9, M5−M7, and I8−I27 were mixed (2.36 g) and subjected to Si gel CC eluted with benzene−EtOAc (17:3, fractions N1−N19; and 4:1, fractions N20−N26). Crystallization of fractions N11−N13 gave 4/5 (127.4 mg). The mother liquors of 4/5 were combined with fractions N14−N25 and purified by Si gel CC using benzene−iPrOH (97:3) as eluent, to obtain an additional amount of 4/5 (40.3 mg). Fractions J21−J69, K17−K30, and M8− M27 were mixed (11.5 g) and subjected to Si gel CC eluted with a hexanes−acetone gradient to afford fractions O1−O70 (17:3); O71− O101 (4:1); O102−O111 (3:1); O112−O117 (7:3); O118−O130 (3:2); and O131−O135 (0:1). Fractions O23−O35 gave an additional amount of 13 for a total yield of 41.8 mg. Crystallization of fractions O41−O52 gave 4/5 for a total yield of 185.8 mg. Fractions O53− O124 (2.045 g) were chromatographed over a Si gel column using CHCl3−MeOH (19:1) as eluent, to obtain fractions P1−P27. Fractions P13−P26 and O125−O130 were mixed (2.207 g) and subjected to Si gel CC eluted with a hexanes−acetone gradient to give fractions Q1−Q22 (7:3); Q23−Q30 (13:7); Q31−Q34 (3:2); and Q35−Q46 (1:1). Fractions Q10−Q39 (1.87 g) were purified by Si gel CC eluted with a benzene−EtOAc gradient (4:1 to 3:2). Fractions eluted with benzene−EtOAc (3:1 to 3:2) were combined with fractions Q35−Q46 (1.38 g) and purified by Si gel CC eluted with CHCl3−EtOAc (7:3) to obtain fractions R1−R55. Crystallization of fractions R15−R23 (EtOAc−hexanes−iPr2O) gave 7 (19.5 mg). Crystallization of fractions R24−R28 (MeOH) afforded 3 (23.2 mg) for a total yield of 413 mg. Crystallization of mother liquors of 3 gave 49.6 mg of 8. 4-epi-Polystachyne A (1): colorless crystals (EtOAc−hexanes); mp 180−182 °C; [α]20D +56 (c 0.2, CHCl3); IR (CHCl3) νmax 1776, 1681, 1503, 1466, 1363, 1048, 1025, 1011, 945, 875 cm−1; 1H NMR and 13C NMR (CDCl3) see Tables 1 and 2; HRFABMS m/z 343.1552 [M + H]+ (calcd for C20H23O5, 343.1545). Salvifiline A (3): colorless crystals (EtOAc−hexanes); mp 262−264 °C; [α]20D +21 (c 0.2, CH3OH); IR (CHCl3) νmax 3338, 1776, 1737, 1635, 1461, 1377, 1207, 1083,1000, 937, 896 cm−1; 1H NMR and 13C NMR (DMSO-d6) see Tables 1 and 2; HRFABMS m/z 375.1435 [M + H]+ (calcd for C20H23O7, 375.1444). 15-O-Methylsalvifilines B (4/5): colorless crystals (EtOAc− hexanes); mp 208−210 °C; [α]20D −20 (c 0.2, CHCl3); IR (CHCl3) νmax 1772, 1467, 1443, 1363, 1026, 988, 936, 922 cm−1; 1 H NMR and 13C NMR (CDCl3) see Tables 1 and 2; HRDARTMS m/z 389.16048 [M + H]+ (calcd for C21H25O7, 389.16003).

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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.6b00605. 1D and 2D NMR spectra of compounds 1, 3−5, 7, and 8, as well as the 1H NMR and 13C NMR data of compound 10 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: 525556224412. Fax: 525556162217. E-mail: emmaldon@ unam.mx. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We are grateful to R. Patiño for the IR and optical rotations; H. ́ R. Gaviño, A. Peña, E. Huerta, and B. Quiroz for the Rios, ́ and J. Pérez NMR spectra; and L. Velasco, C. Márquez, L. Rios, ́ for the cytotoxicity assay. for the MS. We also thank T. Ramirez 2672



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REFERENCES

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