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Gaditanone, a Diterpenoid Based on an Unprecedented Carbon Skeleton Isolated from Euphorbia gaditana M. Eugenia Flores-Giubi,† María J. Durán-Peña,† José M. Botubol-Ares,† Felipe Escobar-Montaño,† David Zorrilla,‡ Antonio J. Macías-Sánchez,† and Rosario Hernández-Galán*,† †

Departamento de Química Orgánica, Instituto de Biomoléculas, and ‡Departamento de Química Física, Facultad de Ciencias, Universidad de Cádiz, Campus Universitario Puerto Real s/n, 11510, Puerto Real, Cádiz, Spain S Supporting Information *

ABSTRACT: A novel diterpenoid, gaditanone (2), which possesses an unprecedented 5/6/4/6-fused gaditanane tetracyclic ring skeleton, and a new jatrophane (1) were isolated from the aerial parts of Euphorbia gaditana. The chemical structures and absolute configurations were determined by extensive spectroscopic NMR studies and ECD data analysis. A proposed biosynthetic pathway is presented for compound 2.

T

he genus Euphorbia is the largest in the Euphorbiaceae family, comprising more than 2000 known species and ranging from annuals to trees.1 Many plants of this genus have been used extensively in the search for natural-product-based drugs because of the extraordinary chemical diversity of their isoprenoids and the wide variety of pharmacological properties they possess.2 Therefore, for the last 40 years, Euphorbia species have attracted considerable attention. In particular, the diterpenoids, the major secondary metabolites in Euphorbia species, are represented by a number of macrocyclic and polycyclic skeletons such as jatrophane, lathyrane, tigliane, daphnane, ingenane, and myrsinane.2c,3 Of special interest are tiglianes, ingenanes, lathyranes, and jatrophanes, which are known protein kinase C (PKC) activators,4 with an ability to activate PKCs, being associated with a wide range of biological activities reported for these groups of compounds such as cell differentiation,5 skin inflammation,6 tumor promotion,7 inhibition of cell killing by HIV-1,8 cytotoxicity,9 and latent HIV-1 reactivation.2a,10 Recent reports indicate that PKCs play a role in neural precursor proliferation.11 Euphorbia species from the Iberian Peninsula are being studied as part of our ongoing research on the identification of new bioactive compounds capable of modulating PKC isoforms,12 especially in neural cell proliferation.11 Euphorbia gaditana Coss., an endangered species, is an annual plant13 distributed throughout southern Spain (western Andalusia) and northern Africa (Algeria and Tunisia). To the best of our knowledge, to date there have been no phytochemical reports on E. gaditana in the literature, and we believe that it could be an important new source of potentially bioactive diterpenoids. The petroleum ether-soluble fraction of the methanol extract of E. gaditana was fractionated and purified by chromatography to afford a new jatrophane (1) and a novel diterpenoid with an unprecedented 5/6/4/6 fused-ring skeleton (2). Terpenes containing a four-membered ring are scarce in the literature.14 Compound 1 showed a sodium adduct ion in the HRESIMS at m/z 665.2944 [M + Na]+, corresponding to the sodiated © 2017 American Chemical Society and American Society of Pharmacognosy

molecular formula C35H46O11Na. The IR spectrum of 1 showed absorption bands ascribable to hydroxy groups (3450 cm−1) and carbonyl groups (1736 cm−1). Analysis of the NMR data (Table 1) revealed that 1 is closely related structurally to other diterpenoids previously described in the literature that possess a jatrophane skeleton.15 Jatrophanes are characterized by a highly functionalized transbicyclic[10.3.0]pentadecane unit and typically form polyesters by esterification of their hydroxy groups. Five ester residues were identified and attributable to the presence of four acetate groups and one benzoate group. Moreover, three olefinic protons and one hydroxy group were also identified (Table 1). Three acetoxy groups were deduced as being attached to C-3, C-8, and C-14 from HMBC correlations, whereas the benzoate chain was located at C-9. The gCOSY spectrum enabled assignment of the free hydroxy group as attached to C-7 due to the correlations observed for protons H-7 (δH 4.07) and OH-7 (δH 4.34). Finally, the remaining acetoxy group must be located at C-15 (Figure 1). The NOESY correlations of H-4/H3-17/H-7/H-8 and H-4/ H-14/H-13/H-11 indicated that these protons are directed inward in the macrocyclic ring, whereas the correlations of H5/H-9/OH-7/and H-9/OAc-15 were used to place these protons on the opposite face of the macrocycle (Figure 2). The E geometry of the C-11/C-12 olefin linkage followed from the Received: April 16, 2017 Published: July 5, 2017 2161

DOI: 10.1021/acs.jnatprod.7b00332 J. Nat. Prod. 2017, 80, 2161−2165

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Table 1. NMR Spectroscopic Data (500 MHz, CDCl3) for Compounds 1 and 2 1

a−c

position

δC, type

1a 1b 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 OH-7 CO-3 COMe-3 CO-8 COMe-8 CO-9 COBz-9 1′ 2′, 6′ 3′, 5′ 4′ CO-14 COMe-14 CO-15 COMe-15

41.0, CH2 36.3, CH 81.0, CH 46.4, CH 122.5, CH 133.8, C 77.5, CH 69.9, CH 74.4, CH 39.1, C 134.6, CH 130.9, CH 39.2, CH 80.6, CH 93.5, C 16.7, CH3 15.8, CH3 23.6,a CH3 21.1,a CH3 20.2, CH3 171.2, C 20.8,b CH3 169.4, C 20.9, CH3 167.3, C 129.5, C 129.7, CH 128.6, CH 133.6, CH 170.9, C 20.8,b CH3 169.3, C 23.2, CH3

2 δH (J in Hz) 3.54, 1.30, 2.18, 4.62, 3.30, 5.75,

dd (14.4, dd (14.4, m t (9.2) dd (11.0, dq (11.0,

δC, type

5.6) 12.8)

9.2) 1.4)

4.07, brd (5.4) 5.28, dd (5.4, 4.4) 4.77, d (4.4) 5.11, 5.60, 2.59, 4.95,

d (15.8) dd (15.8, 8.8) m d (2.8)

1.01, 1.73, 1.12, 1.11, 0.93, 4.34,

d (6.5) d (1.4) sa sa d (7.0) d (5.0)

40.2, CH2 40.6, CH 79.5, CH 48.9, CH 33.9,a CH 50.3, C 206.6, C 74.1, CH 75.1, CH 38.2, C 54.9, CH 40.5, CH 34.0,a CH 74.9, CH 89.8, C 19.2, CH3 20.3, CH3 23.1, CH3 24.4, CH3 16.3, CH3 170.5, C 21.1,b CH3 170.0, C 22.6,b CH3 165.5, C

2.00, s 1.88, s

8.07, d (8.2) 7.48, dd (8.2, 7.2) 7.61, t (7.2) 2.11, s 2.12, s

129.3, C 129.7, CH 128.6, CH 133.5, CH 171.0, C 21.0,b CH3 169.2, C 20.5,b CH3

δH (J in Hz) 3.39, 1.24, 2.01, 5.01, 2.01, 3.03,

dd dd m dd m dd

(14.2, 7.5) (14.2, 11.4) (7.5, 3.5) (11.5, 8.5)

5.73, d (11.4) 5.57, d (11.4) 2.27, 2.19, 2.11, 5.08,

d (11.6) m m d (7.0)

1.16, 1.29, 1.33, 0.96, 1.02,

d (7.1) s s s d (7.4)

2.12, sc 1.99, sc

8.02, d (8.2) 7.46, dd (8.2, 7.5) 7.59, t (7.5) 2.11, sa 2.00, sc

Interchangeable signals.

Figure 1. Selected 1H−1H COSY and HMBC correlations for 1.

coupling constant of J11,12 = 15.8 Hz and NOESY correlations of H-11/H3-17/H-13, while the E stereochemistry of the C-5/ C-6 olefin linkage was deduced from NOESY correlations between H-4/H3-17 and H-5/H-9. Further NOESY correlations of H-3/H-4/H3-16 revealed their proximity in space. Therefore, since H-4 is α-oriented, a characteristic of all jatrophane-type diterpene derivatives isolated to date,16 the absolute configuration for 1 was suggested to be assigned as 2R,3S,4S,7S,8S,9S,13S,14S,15R. Compound 2 was assigned a molecular formula of C35H44O11 (14 unsaturations) on the basis of the observed sodium adduct

Figure 2. Selected NOESY correlations exhibited by 1.

ion in its HRESIMS (m/z 663.2780 [M + Na]+). The 13C NMR and DEPT spectra revealed the presence of 35 carbon resonances encompassing nine methyl groups, one methylene group, 15 methine groups (including four oxygenated sp3 carbons), and 10 nonprotonated carbons (including one oxygenated sp3 carbon, five ester carbons, and one ketone group). A benzoate and four acetate groups were identified 2162

DOI: 10.1021/acs.jnatprod.7b00332 J. Nat. Prod. 2017, 80, 2161−2165

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using the 1H and 13C NMR spectra (Table 1). The aforementioned functionalities accounted for 10 degrees of unsaturation; the remaining double-bond equivalents therefore required four additional rings in the molecule. Analysis of the 2D NMR data of 2 (in both CDCl3 and C6D617) led to the construction of its planar structure (Figure 3). The 1H−1H COSY spectrum indicated the presence of two

Figure 4. Selected NOESY correlations exhibited by 2. Figure 3. Key 1H−1H COSY and HMBC correlations of 2.

isolated spin systems of H2-1/H-2(H3-16)/H-3/H-4/H-5/H12(H-11)/H-13(H3-20)/H-14 and H-8/H-9. The gHMBC spectrum of 2 showed correlations from H2-1 to C-3, C-4, and C-15; from H-3 to C-15; from H3-16 to C-1, C-2, and C-3; and from H-4 to C-14, which, in combination with the chemical shifts of these proton and carbon resonances, revealed the presence of a five-membered ring (A) with a secondary methyl group at C-2. In addition, the HMBC cross-peak of the oxymethine proton H-3 and the ester carbonyl at δC 170.5 showed the attachment of an acetyl group at C-3. Further HMBC correlations from H-4 and H-13 to C-5 and from H-13 to C-14 and C-15, combined with the corresponding 1H−1H COSY correlations, revealed a six-membered ring (B) joined at positions C-4 and C-15, possessing a secondary methyl group at C-13. The correlation at δC 171.0 with the oxymethine signal at δH 5.08 (H-14) demonstrated the location of one of the acetyl groups at position C-14. Moreover, a four-membered ring (C) joined at positions C-5 and C-12 and substituted with a methyl group at C-6 was established via long-range HMBC correlations from H-11, H-4, and H3-17 to C-6; from H-13 to C-11; and from H-11 and H3-17 to C-5. The presence of another six-membered ring (D) with a gem-dimethyl group fused to ring C was derived from the HMBC correlations from H-11 to C-18, C-10, and C-9 and from H3-18 and H3-19 to C9. HMBC couplings of H-8, H-11, and H3-17 to a keto group (C-7) revealed the connection between C-6 and C-7. Lastly, long-range correlations of H-8 and H-9 protons and the ester carbonyls at δC 170.0 and 165.5, respectively, supported the locations of an acetyl group at C-8 and the benzoyl group at C9. The remaining acetyl group did not exhibit a long-range correlation with an oxymethine proton, therefore indicating its attachment to the quaternary carbon at C-15. The relative configuration of 2 was deduced from the NOESY spectrum. As depicted in Figure 4, the NOESY correlations of H3-16/H-3/ H-4/H3-17/H-8/H3-18/and of H-4/H-13/H-11/H3-17 shed light on their cofacial relationship and were therefore judged to be α-oriented. Furthermore, the NOESY cross-peaks of H-5/H12/H-9/H3-19 revealed that these protons were also cofacial and hence were considered to be β-oriented. The absolute configuration of 2 was deduced in a preliminary manner using the octant rule for the cyclohexanone ring Cotton effects. The electronic circular dichrosim (ECD) spectrum of gaditanone (2) (Figure 5) exhibited a negative Cotton effect at 290 nm (Δε −3.44) corresponding to the n−π* transition of the cyclohexanone chromophore. On the basis of the octant rule for cyclohexanones18 and the relative configuration established by the NOESY data, the absolute

Figure 5. Experimental and calculated ECD spectra for 2.

configuration of gaditanone (2) was determined to be 2R,3S,4S,5S,6S,8R,9S,11S,12S,13S,14S,15S. This absolute configuration of 2 was supported by comparison of the experimental ECD spectrum with the ECD spectrum predicted from quantum mechanical timedependent density functional theory (TDDFT) calculations.19 The theoretically calculated ECD spectrum of 2 was in good agreement with the experimental ECD spectrum. Gaditanone (2) is the first example of a natural product with a 5/6/4/6 fused-ring system, for which the skeleton was called gaditanane. From a biosynthetic point of view, compounds 1 and 2 appear to be closely related. Gaditanone (2) might be biosynthesized from jatrophane 1 by oxidation at C-7, followed by concerted intramolecular [2+2] cycloaddition. In order to check the hypothesis proposed, compound 1 was treated with pyridinium chlorochromate (PCC) to almost quantitatively give 3, which was treated with a catalytic amount of benzophenone in CH2Cl2 under UV light, producing 2 in 97% yield and thus supporting a biogenetic relationship between the two compounds (Scheme 1). Furthermore, this chemical correlation supported the stereochemical assignation given above for compound 1 and is consistent with the Scheme 1. Chemical Correlation between 1 and 2

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DOI: 10.1021/acs.jnatprod.7b00332 J. Nat. Prod. 2017, 80, 2161−2165

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Chemical Transformation of 1. Jatrophane 1 (5.0 mg, 7.8 μmol) was dissolved in CH2Cl2 (0.3 mL) and then added dropwise to a suspension of PCC (2.5 mg, 11.7 μmol) and powdered 4 Å molecular sieves (5.0 mg) in CH2Cl2 (0.5 mL) at room temperature. The reaction was stirred vigorously for 1 h, diethyl ether (4 mL) was then added, and the mixture was stirred for an additional 1 h. The suspension was filtered over a silica gel pad (petroleum ether−ethyl acetate, 80:20, 100 mL). The solvent was evaporated under reduced pressure to give compound 3. (2R,3S,4S,8R,9S,13S,14S,15R)-3,8,14,15-Tetraacetoxy-9benzoyloxyjatropha-5E,11E-dien-7-one (3): amorphous solid (4.9 mg, 98%); [α]20D −43.3 (c 0.13, CHCl3); IR (film) νmax 2968, 2935, 1748, 1664, 1443, 1372, 1268, 1229, 1027, 710 cm−1; 1H NMR (CDCl3, 500 MHz) δ 8.02 (2H, d, J = 7.5 Hz, H-2′ and H-6′), 7.59 (1H, t, J = 7.5 Hz, H-4′), 7.46 (2H, t, J = 7.5 Hz, H-3′ and H-5′), 7.02 (1H, d, J = 10.0 Hz, H-5), 5.82 (1H, dd, J = 16.2, 8.4 Hz, H-12), 5.68 (1H, d, J = 8.6 Hz, H-9), 5.58 (1H, d, J = 8.6 Hz, H-8), 5.03 (1H, d, J = 16.2 Hz, H-11), 5.02 (1H, dd, J = 8.1, 5.0 Hz, H-3), 4.98 (1H, d, J = 3.0 Hz, H-14), 3.78 (1H, dd, J = 14.5, 7.0 Hz, H-1a), 3.06 (1H, dd, J = 10.0, 8.1 Hz, H-4), 2.42 (3H, s, C3-COCH3), 2.26 (1H, m, H-13), 2.20 (1H, m, H-2), 2.12 (3H, s, C14-COCH3), 2.00 (3H, s, C15COCH3), 1.87 (3H, s, H-17), 1.85 (3H, s, C8-COCH3), 1.37 (1H, dd, J = 14.5, 11.5 Hz, H-1b), 1.24 (3H, s, H-18), 1.12 (3H, d, J = 6.9 Hz, H-16), 1.09 (3H, s, H-19), 0.90 (3H, d, J = 7.0 Hz, H-20); 13C NMR (CDCl3, 125 MHz) δ 195.1 (C, C-7), 170.7 (C, CO-14), 169.9 (C, CO-3), 169.8 (C, CO-15), 169.6 (C, CO-8), 165.7 (C, CO-9), 141.8 (CH, C-5), 139.1 (CH, C-11), 136.7 (C, C-6), 133.3 (CH, C-4′), 131.8 (CH, C-12), 129.7 (CH, C-3′ and C-5′), 129.6 (C, C-1′), 128.6 (CH, C-2′ and C-6′), 94.7 (C, C-15), 83.2 (CH, C-9), 81.9 (CH, C8), 81.6 (CH, C-3), 79.5 (CH, C-14), 49.4 (CH, C-4), 43.4 (CH2, C1), 39.9 (CH, C-13), 39.1 (C, C-10), 39.0 (CH, C-2), 24.9 (CH3, C19), 23.4 (CH3, COCH3-3), 21.1 (CH3, COCH3-15), 20.8* (CH3, COCH3-14), 20.8* (CH3, C-8), 20.7 (CH3, COCH3-8), 19.8 (CH3, C20), 18.4 (CH3, C-16), 12.9 (CH3, C-17) (*interchangeable signals); HRESIMS m/z 641.2952 [M + H]+ (calcd for C35H45O11, 641.2962). Intramolecular [2+2]-Cycloaddition of 3. Compound 3 (4.0 mg, 6.25 μmol) was dissolved in CH2Cl2 (0.2 mL), and benzophenone (0.2 mg, 1.1 μmol) was added. The mixture was irradiated at 254 nm for 4 h. Then, evaporation of the solvent gave a crude product, which was purified by silica gel column chromatography eluted with petroleum ether−EtOAc (65:35) to give a compound (3.88 mg, 97%), for which the spectroscopic data were identical to those of the natural product gaditanone (2). Computational Methods. Conformational analysis of 2 was performed by using the semiempirical method PM6. The energies, oscillator strengths, and rotational strengths of the first 20 electronic excitations were calculated using the TD-DFT-B3LYP/6-31G(d,p) level of theory. The ECD spectrum of the conformer was simulated using a Gaussian function with a half-bandwidth of 0.33 eV. The corresponding theoretical ECD spectrum of the enantiomer (ent-2) was depicted by inverting that of 2. All quantum computations were performed using the Gaussian 09 program package,20 on a highperformance computer with 256 cores located at CICA (Centro ́ ́ In the 200−400 nm region, the Informático Cientifico de Andalucia). theoretically calculated ECD spectrum of 2 was in good agreement with the experimental ECD spectrum (Figure 5). This supported the assignment made of the absolute configuration for 2.

assumption for the configuration of H-4, based on previous observations.16 In conclusion, diterpenoids 1 and 2 were isolated from E. gaditana, the latter possessing an unusual 5/6/4/6 fused-ring skeleton. The biosynthetic relationship between both compounds has also been confirmed by chemical correlation. The biological activities of these compounds in terms of PKC activation seem worthy of investigation.



EXPERIMENTAL SECTION

General Experimental Procedures. Unless otherwise noted, materials and reagents were obtained from commercial suppliers and were used without further purification. Dichloromethane was freshly distilled from CaH2. Optical rotations were determined with a digital polarimeter. Infrared spectra were recorded on a FT-IR spectrophotometer and reported as wavenumber (cm−1). ECD and UV spectra were recorded on a JASCO J-1500 CD spectrometer. 1H and 13C NMR measurements were recorded on an Agilent 500 MHz NMR spectrometer with SiMe4 as the internal reference. Chemical shifts were referenced to CDCl3 (δH 7.25, δC 77.0) and C6D6 (δH 7.16, δC 128.4). NMR assignments were made using a combination of 1D and 2D techniques. High-resolution mass spectroscopy (HRMS) was performed in a QTOF mass spectrometer in the positive-ion ESI mode. Purification by semipreparative and analytical HPLC was performed with a Hitachi/Merck L-6270 apparatus equipped with a differential refractometer detector (RI-7490). A LiChrospher Si gel 60 (5 μm) LiChroCart (250 mm × 4 mm) column and a LiChrospher Si gel 60 (10 μm) LiChroCart (250 mm × 10 mm) were used in isolation experiments. Silica gel (Merck) was used for column chromatography. TLC was performed on Merck Kiesegel 60 F254, 0.25 mm thick. Plant Material. Due to Euphorbia gaditana being an endangered species, R.H.-G. was authorized by Consejeriá de Medio Ambiente of Junta de Andalucia to collect and manipulate samples of this species in order to carry out the present phytochemical study. The whole plants of E. gaditana were collected at Los Parralejos (Vejer de la Frontera), Cádiz, Spain, in June 2011 by Dr. L. Plaza-Arregui (Consejeriá de Medio Ambiente of Junta de Andalucia) and J. Luis Rendón (Botanical Garden of San Fernando, Cádiz). Extraction and Isolation. The air-dried plant (1.8 kg) was powdered and extracted with 2.5 L of MeOH at room temperature for 3 × 24 h. The MeOH extract from the aerial parts was evaporated under reduced pressure to yield a crude extract (92 g), which was suspended in water (1 L) and then partitioned with petroleum ether (2 L), dichloromethane (1 L), and ethyl acetate (1 L), sequentially. After removing the solvent, the petroleum ether-soluble extract yielded 10.9 g of residue. A portion of this extract (1.35 g) was subjected to column chromatography over silica gel, eluting with an increasingly polar gradient of ethyl acetate (0−100%) in petroleum ether, to afford 16 fractions (A1−A16), according to TLC analysis. Fractions A6−A10 presented similar TLC profiles and were combined and further purified by analytical HPLC, using petroleum ether−EtOAc (80:20) as the mobile phase, to yield 1 (54.0 mg, tR 54 min) and 2 (1.4 mg, tR 15 min). (2R,3S,4S,7S,8S,9S,13S,14S,15R)-3,8,14,15-Tetraacetoxy-9benzoyloxyjatropha-5E,11E-dien-7-ol (1): amorphous solid; [α]20D −135 (c 0.11, CHCl3); UV (MeOH) λmax (log ε) 205 (4.09), 230 (4.97) nm; ECD (MeOH) λ (Δε) 203 (1.30), 213 (−12.23), 226 (0.23), 246 (−5.59), 275 (0.43) nm; IR (film) νmax 3450, 2968, 1736, 1368, 1276, 1235, 1117, 1026, 757, 714 cm−1; 1H and 13C NMR data (CDCl3, 500 MHz) (see Table 1); HRESIMS m/z 665.2944 [M + Na]+ (calcd for C35H46O11Na, 665.2938). Gaditanone (2): amorphous solid; [α]20D −17 (c 0.12, CHCl3); UV (MeOH) λmax (log ε) 244 (5.02) nm; ECD (MeOH) λ (Δε) 238 (0.32), 291 (−3.44), 321 (0.38) nm; IR (film) νmax 2968, 2930, 1734, 1451, 1376, 1239, 1107, 1067, 1023, 755, 714 cm−1; 1H and 13C NMR data (CDCl3, 500 MHz, see Table 1) (500 MHz, C6D6, see Table S1, p S3, Supporting Information); HRESIMS m/z 663.2780 [M + Na]+ (calcd for C35H44O11Na, 663.2781).



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00332. ECD calculations, spectroscopic data, and 1D and 2D NMR spectra of compounds 1−3 (PDF) 2164

DOI: 10.1021/acs.jnatprod.7b00332 J. Nat. Prod. 2017, 80, 2161−2165

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Corresponding Author

*E-mail: [email protected]. ORCID

Rosario Hernández-Galán: 0000-0003-1887-4796 Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This research was supported by a grant from MINECO (BFU2015-68652-R). Use of the NMR and MS facilities at the Servicio Centralizado de Ciencia y Tecnologiá of the University of Cádiz is acknowledged. The authors are grateful to Dr. M. J. Ortega (University of Cádiz) for helpful discussions on circular dichroism and to Dr. L. Plaza-Arregui (Consejeriá de Medio ́ and J. Luis Rendón (Botanical Ambiente of Junta de Andalucia) Garden of San Fernando) for collecting the plant material.



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DOI: 10.1021/acs.jnatprod.7b00332 J. Nat. Prod. 2017, 80, 2161−2165