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A new germacranolide from Ageratina vernalis Miguel Á. Fuentes-Figueroa , Neively Tlapale-Lara , Beatriz HernándezCarlos , Pedro Joseph-Nathan & Eleuterio Burgueño-Tapia To cite this article: Miguel Á. Fuentes-Figueroa , Neively Tlapale-Lara , Beatriz HernándezCarlos , Pedro Joseph-Nathan & Eleuterio Burgueño-Tapia (2020): A new germacranolide from Ageratina�vernalis , Natural Product Research, DOI: 10.1080/14786419.2020.1827400 To link to this article: https://doi.org/10.1080/14786419.2020.1827400
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NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1827400
A new germacranolide from Ageratina vernalis Fuentes-Figueroaa, Neively Tlapale-Laraa, Beatriz Hernandez-Carlosb, Miguel A. ~o-Tapiaa Pedro Joseph-Nathanc and Eleuterio Burguen Departamento de Quımica Organica, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prolongacion de Carpio y Plan de Ayala, Mexico City, Mexico; bInstituto de Agroindustrias, Universidad Tecnologica de la Mixteca, Huajuapan de Leon, Mexico; cDepartamento de Quımica, Centro de Investigacion y de Estudios Avanzados, Instituto Politecnico Nacional, Mexico City, Mexico a
ABSTRACT
ARTICLE HISTORY
The aerial parts of Ageratina vernalis provided the new germacranolide 1,10-epoxydeltoidin A (3), together with the known pentacyclic triterpenoid hopane-6a,22-diol (1), and the also known germacranolides deltoidin A (2) and 15-hydroxydeltoidin A (4). In addition, pTsOH catalyzed cyclization of 2 afforded the new guaianolide 5. The absolute configuration of 2, 4, and 5 was assigned by vibrational circular dichroism spectroscopy, while the complete 1H and 13C NMR data assignments of 2-5 followed from 1 D- and 2 D-NMR experiments.
Received 29 April 2020 Accepted 5 September 2020 KEYWORDS
Ageratina vernalis; germacranolides; vibrational circular dichroism
1. Introduction The genus Ageratina, contains 334 species (Plant List 2013. Version 1.1), including some that were segregated from the genus Eupatorium (Clewell and Wooten 1971). Typical isolates from Ageratina species are flavonoids (del Barrio et al. 2011), thymols mez et al. 1982; Aguilar-Guadarrama et al. (Bustos-Brito et al. 2016), chromenes (Go 2009), benzofurans (Aguilar-Guadarrama et al. 2009; Lee et al. 2009), and germacranolides (Quijano et al. 1980; Tamayo-Castillo et al. 1988). Some sesquiterpene lactones isolated from this genus have shown interesting activities as antimicrobial (Arciniegas ~o-Tapia CONTACT Eleuterio Burguen [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1827400. ß 2020 Informa UK Limited, trading as Taylor & Francis Group
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Figure 1. Formulas of natural products 1-4 from A. vernalis and of 5 obtained by cyclization of 2.
et al. 2018) and anticancer (Orofino-Kreuger et al. 2012; Eiroa et al. 2018) agents. Recently, some thymol derivatives isolated from A. glabrata (Arreaga-Gonzalez et al. 2018) were used to develop a methodology for the determination of the enantiomeric excesses and the absolute configuration (AC) of epoxythymols. A. vernalis is endemic to Mexico and lacks of reported chemical studies. We now isolated the new germacranolide 3, the hopane triterpenoid 1, and the known germacranolides 2 and 4 (Figure 1) from the hexanes extract of the aerial parts of A. vernalis. Furthermore, the new guaianolide 5 was obtained by p-TsOH catalyzed cyclization of compound 2. The AC assignment of 2, 4, and 5 was achieved by vibrational circular dichroism.
2. Results and discussion The hexanes extracts of the aerial parts of Ageratina vernalis collected from the state of Oaxaca, Mexico were subjected to Si-gel column chromatography to afford 1–4. The first isolated compound, which turned out to be 1, was obtained by successive column chromatographies from the less polar fractions. Its 1H NMR spectrum (Figure S1) showed eight singlet methyl group signals at d 1.21, 1.18, 1.15, 1.04, 1.01, 0.97, 0.87, and 0.76, and the signal of a methine geminal to a hydroxy group at d 3.96 (td, J ¼ 10.6, 4.2 Hz). These signals, along with the 30 sp3 carbon signals observed in
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the 13C NMR spectrum (Figure S2) suggested a pentacyclic triterpene structure for compound 1. Comparison of the data with those of hopane-6a,22-diol (Elix et al. 1982) allowed the identification of 1. This compound has typically been described from cryptogams, as lichens (Elix et al. 1982) and ferns (Shiojima and Ageta 1994), although it has also been found in higher plants like Iris missourensis (Wong et al. 1986). This is the first time that 1 is described from an Ageratina species. The second isolate was 2. It showed the characteristic 1H NMR signals of an angeloyloxy group at d 6.14 (1H, qq, J ¼ 7.3, 1.5 Hz, H-30 ), 1.98 (3H, dq, J ¼ 7.3, 1.5 Hz, H-40 ), and 1.84 (3H, quint, J ¼ 1.5 Hz, H-50 ), typical signals of an exocyclic methylene group at 6.40 (1H, bd, J ¼ 3.5 Hz, H-13a), 5.75 (1H, bd, J ¼ 3.1 Hz, H-13b), and three methine groups, each geminal to an oxygen atom, at d 2.85 (1H, d, J ¼ 8.8 Hz, H-5), 4.46 (1H, dd, J ¼ 8.8, 8.4 Hz, H-6), and 5.77 (1H, bdd, J ¼ 5.2, 5.0 Hz, H-8) (Figure S3). These signals and the 13C NMR chemical shifts (Figure S4), suggested a germacranolide sesquiterpene lactone. Additional 2 D NMR experiments allowed identifying it as deltoidin A (2). This compound was described 40 years ago (Quijano et al. 1980) and only partial 1H NMR data were provided at that time. Thus, complete 1H and 13C NMR data assignments, derived from 1 D- and 2 D-NMR experiments, are summarized in the supplementary material (Table S1). Rechromatography of a fraction containing 2 gave additional 12.1 mg of deltoidin A and 2.1 mg of the new germacranolide 3. The 1H NMR spectrum of 3 showed signals very similar with those of 2, except for a signal at d 2.90 (1H, dd, J ¼ 10.9, 1.7 Hz, H-1) instead of the H-1 vinyl proton signal. Additionally, the 13C NMR spectrum showed two new oxygen bearing carbon signals at d 64.4 (C-1) and 58.8 (C-10), instead of the C-1 C-10 double bond signals (Figure S5–S10). These evidences revealed the structure of 1,10-epoxydeltoidin A (3). The trans configuration of 1,10-oxirane was assigned through NOESY experiments where correlations of Me-14 (d 1.37) with Me-15 (d 1.44), and of H-1 (d 2.90) with H-2a (d 2.23), H-3a (d 1.41), and H-5 (d 2.96) were observed (Figure S11). The lactone and angeloyloxy group configurations were deduced by comparing the observed and calculated J5,6, J6,7, and J7,8 values of the minimum energy conformer, which were 8.6, 8.5, and 1.0, versus 9.5, 9.8, 0.6 Hz, respectively. Thus, considering that 2 and 3 are biogenetically related, the configuration depicted for 3 was assumed. Compound 4 showed similar 1H NMR signals than 2, although two doublets at d 3.94 (1H, d, J ¼ 12.4 Hz, H-15a) and d 3.79 (1H, d, J ¼ 12.4 Hz, H-15b) appear instead of the Me-15 group signal of 2 (Figure S12). In turn, the 13C NMR spectrum showed a signal at d 60.8 corresponding to the C-15 hydroxymethylene group, thus allowing to propose the structure of 15-hydroxydeltoidin A (4) (Figure S13). This compound was reported (Bohlmann et al. 1985) as part of an inseparable mixture of angelate and tiglate esters for which only scarce 1H NMR data was provided. 1H and 13C NMR data assignments were achieved using 1 D- and 2 D-NMR experiments, and are shown in the supplementary material (Table S1). The AC of 2 was correctly assumed (Quijano et al. 1980) considering biogenetic aspects and with the aid of coupling constants values for H-5, H-6, H-7, H-8, and H-13. Herein, we confirmed the AC using vibrational circular dichroism (VCD) spectroscopy ~o-Tapia and Joseph-Nathan 2015, 2017; Joseph-Nathan and Gordillo-Roman (Burguen
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Table 1. Comparison of experimental and DFT B3LYP/DGDZVP calculated IR and VCD spectra of 2, 4, and 5. Comp.
anHa
SIRb
SEc
SEd
ESIe
Cf
2 4 5
0.980 0.980 0.980
96.7 94.9 92.1
77.4 82.7 76.1
10.9 13.6 6.0
66.5 69.1 70.1
99 99 99
a
Anharmonicity factor. IR spectral similarity (%). c VCD spectral similarity for the correct enantiomer (%). d VCD spectral similarity for the incorrect enantiomer (%). e Enantiomer similarity index calculated as (SESE). f Confidence level for the AC determination (%). b
2015). Thus, a molecular model based on the relative configuration deduced from the 1 ~o-Tapia H NMR data was constructed. Following the described methodology (Burguen and Joseph-Nathan 2015; Joseph-Nathan and Gordillo-Roman 2015), a conformational search of this model, using the Monte Carlo protocol and MMFF94 molecular mechanics, was performed. The results were submitted to single point energy calculation using density functional theory (DFT) and the B3LYP/6-31G (d) functional and basis set, followed by complete geometry optimization at the DFT B3LYP/DGDZVP level of theory. Final calculation of the IR and VCD frequencies at the same level of theory was then achieved. The conformational search provided 13 conformers within DE ¼ 9.53 kcal/mol, which after single point energy calculations left only three conformers in a 0.79 kcal/mol energy gap, the fourth conformer, being 4.86 kcal/mol above the global minimum, is not contributing to the overall conformational picture. The complete geometry optimization matched the ring geometry of conformers 2a and 2 b (Figure S14) which is essentially the same, since the conformers differing in the angeloyloxy group orientation, for which the H-8 C-8 O C1’, C-8OC1’CO, and O C1’C2’C3’ dihedral angles are 26.6, 2.1, and 168.4, and 31.3, 2.2, and 30.7 degrees, respectively. After frequencies calculation, these two conformers were observed with a DG ¼ 1.24 kcal/mol. The thermochemical parameters are shown in the supplementary material (Table S2). The weighted IR and VCD curves were compared with the corresponding experimental spectra (Figure S15) using the CompareVOA software. The confidence level (C) (Debie et al. 2011) for the 4S,5R,6S,7R,8R AC assignment was 99% (Table 1). The AC of 4 was also assigned by VCD spectroscopy as above. The Monte Carlo search gave 24 conformers in a DE ¼ 9.89 kcal/mol gap. After single point energy calculations only three conformers within a 2.42 kcal/mol energy gap, contributing with 93.3% of the conformation population were further considered. Complete geometry optimizations and frequencies calculations showed these three conformers (Figure S14) in a DG ¼ 1.56 kcal/mol gap as shown in Table S2. The main conformational difference between the two most stable conformers lies in the orientation of the hydroxy group (Figure S14) as evidenced by the C-3 C-4 C-15 O and C-4 C-15 OH dihedral angles for 4a and 4 b which are 168.8 and 40.5, and 70.7 and 58.9 degrees, respectively. For compound 4, a great agreement was observed between the experimental IR and VCD spectra with the respective calculated spectra (Figure S16) and the numerical data obtained from the CompareVOA software, including the C value of 99% for the
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4S,5R,6S,7R,8R AC assignment, are shown in Table 1. The scarce isolated amount (2.1 mg) of 3 precluded obtaining a reliable VCD spectrum. Inspection of the VCD spectra of 2 and 4 shows a strong positive band at 1135 and 1137 cm1, respectively, which is related to the lactone ring stereogenic centers. These bands were attributed, according to a GausView software evaluation, to strong C11 C-12 and C-12 O-6 asymmetric stretching which are accompanied by CH-6 and CH-7 asymmetric bendings. It is biogenetically accepted that germacranolides are the precursors of eudesmanolides and guaianolides through C-5 C-10 or C-1 C-5, respectively, cyclizations (Fischer et al. 1979). It is also known that acid-catalyzed cyclization of 1,10-epoxides leads to eusdemanolides, while if the oxyrane function appears at C-4 C-5 it leads to guaiano~eda-Acosta et al. 1993). Consequently, compound 2 was treated with a catalides (Castan lytic amount of pTsOH at room temperature to generate the new guaianolide 5 in 63.9% yield. The structure of 5 was deduced by 1 D- and 2 D-NMR experiments (Figure S18–S23), including gHMBC, in which the correlations observed between Me-14 (d 1.65) and C-9 (d 39.5), and of H-2 (d 2.26, 2.42), H-8 (d 5.69), and H-9 (d 2.52, 2.61) with C-10 (d 125.8). The complete NMR data assignments are shown in the experimental part. The 4 R,5S,6S,7R,8R AC assignment shown in 5 was done by VCD as above. The conformers search using the Monte Carlo protocol gave 10 conformers within a 10 kcal/mol energy window, which after single point energy analysis using DFT B3LYP/6-31G(d) were reduced to three within a DG ¼ 4.71 kcal/mol gap. Complete energy optimization at the DFT B3LYP/DGDZVP level left only two conformers within a DG ¼ 1.04 kcal/mol gap. The main difference for conformers 5a and 5 b is the angeloyloxy group orientation, for which the H-8 C-8 O C1’, C-8OC1’CO, and O C1’C2’C3’ dihedral angles are 25.0, 0.1, and 174.6, and 2.4, 2.02, and 17.4 degrees, respectively. After IR and VCD frequencies calculations at the same level of theory, these two conformers showed DG ¼ 2.09 kcal/mol from which the most stable conformer (Figure S14) contributed with 99% of the conformational population. The calculated and experimental IR and VCD spectra are shown in Figure S17 and the CompareVOA comparison showed a 99% reliability (Table 1) for the 4 R,5S,6S,7R,8R AC assignment. The band associated with the lactone ring stereogenic centers was observed at 1151 cm1. The 1H and 13C NMR data are summarized in the experimental section. The purity of the isolated compounds followed from evaluation of their signals in the NMR spectra, as can be observed in the supplementary material. Compound 1 showed inhibitory activity of a-glucosidase at concentration seven times lower than acarbose, a drug used in the treatment of diabetes type II (Karunaratne et al. 2014); while compound 2 has shown selective activity against gram negative bacteria (Arciniegas et al. 2018). No additional biological activity tests for 1–5 were performed during the present work.
3. Experimental 3.1. General experimental procedures The melting point was done on an Electrothermal IA9100X1 apparatus and is uncorrected. Optical rotations were recorded in CHCl3 using a JASCO DIP-370 polarimeter.
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1
H, 13C and 2 D NMR experiments were done using a Varian System 500 (125 MHz for 13 C) spectrometer, CDCl3 was used as solvent and TMS as internal reference. Column chromatography separations were achieved using Natland silica gel 230-400 Mesh.
3.2. Plant material Ageratina vernalis (Vatke & Kurtz) R.M.King & H.Rob is well documented in the literature (Turner 2014). It is a 2-4 m tall shrub with rounded stems 3-5 mm wide, the leaves are 10–20 cm long and 4–8 cm wide, and has 3–5 cm long petioles. The International Compositae Alliance (TICA) (Compositae Working Group (CWG)), 2020) indicates that A. vernalis is the accepted name of species previously known as Ageratina chiapensis, Ageratina subcoriacea, Conoclinium grandiflorum, Eupatorium chiapense, and Eupatorium vernale. For the present study the aerial parts were collected from near the town Rıo Grande, Oaxaca, Mexico (N 16 060 0.496”, W 96 290 0.245”) in May 2016. A plant specimen, authenticated by M. C. Ernestina Cedillo-Portugal, is deposited noma de Chapingo, Texcoco, at the Herbarium ‘Jorge Salas Espinosa’, Universidad Auto Mexico under voucher number 25770.
3.3. Isolation of natural products Powdered dried aerial parts (1.3 kg) were extracted with hexanes at reflux (3 4 L) for 4 h, filtered, and the solvent evaporated under reduced pressure. The residue (4.4 g), dissolved in acetone, was kept at 4 C for 14 h and filtrated to remove fatty materials. The filtrate was evaporated and the residue (3.1 g) was column chromatographed. Elution with hexanes-EtOAc mixtures (19:1, 9:1, 4:1, 7:3, and 3:2) collecting 100 mL fractions afforded, after thin layer chromatography evaluation, the five main fractions A-E. The 1H NMR spectrum of fraction A (514 mg) revealed the presence of fatty material and was discarded. Fraction B (315 mg) was rechromatographed using hexanes-EtOAc mixtures. Those fractions eluted with 7:3 mixtures afforded 6.0 mg of 1. Fraction C (303 mg) was column rechromatographed using hexanes-EtOAc mixtures of increasing polarity (19:1 to 2:3) to afford 30.3 mg of deltoidin A (2). A fraction (56 mg) of this chromatography containing 2 was column rechromatographed to give additional 12.1 mg of 2 and 2.1 mg of 3. Rechromatography of fraction D, using mixtures as in the previous cases gave 9.5 mg of 4. Rechromatography of fraction E gave no useful compound.
3.3.1. Characterization of natural products Compound 1: White solid; mp 222–225 C; 1H and 13C NMR data were the same as those reported. lit. colorless prism, mp 221–223 C (Elix et al. 1982). Compound 2: White semi-solid; [a]D 132.8 (c 0.42, CHCl3); lit. (Quijano et al. 1980) white solid, mp 159–161 C; [a]D: 145.1, CHCl3; 1H and 13C NMR data in Table S1. Compound 3: Oil; [a]D 22.4(c 0.42, CHCl3); IR ¼ 2,929; 2,925; 2,856; 2,258; 1,770; 1,718; 1,456; 1,390; 1,292; 1,238; 1,139 cm1; 1H NMR (CDCl3, 500 MHz) d: 2.90 (dd, J1,2b ¼ 10.9 Hz, J1,2a ¼ 1.7 Hz, H-1), 2.23 (m, H-2a), 1.54 (bddd, J2b,2a ¼ 13.4 Hz, J2b,1 ¼ 10.9 Hz, J2b,3b ¼ 4.4 Hz, H-2b), 1.41 (m, H-3a), 2.29 (ddd, J3b,3a ¼ 13.2 Hz,
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J3b,2b ¼ 4.4 Hz, J3b,2a ¼ 2.2 Hz, H-3b), 2.96 (d, J5,6 ¼ 8.6 Hz, H-5), 4.54 (dd, J6,5 ¼ 8.5 Hz, J6,7 ¼ 8.5 Hz, H-6), 3.11 (ddt, J7,6 ¼ 8.5 Hz, J7,13 ¼ 3.4 Hz, J7,8 ¼ 1.0 Hz, H-7), 5.76 (ddd, J8,9b ¼ 6.0 Hz, J8,9a ¼ 2.1 Hz, J8,7 ¼ 1.2 Hz, H-8), 1.41 (m, H-9a), 2.97 (dd J9b,9a ¼ 15.4 Hz, J9b,8 ¼ 6.0 Hz, H-9b), 5.73 (dd, J13a,7 ¼ 3.1 Hz, J13a,13b ¼ 0.9 Hz, H-13a), 6.41 (dd, J13b,7 ¼ 3.5 Hz, J13b,13a ¼ 0.9 Hz, H-13b), 1.37 (s, H-14), 1.44 (s, H-15), 6.16 (qq, J3’,40 ¼ 7.3 Hz, J3’,50 ¼ 1.5 Hz, H30 ), 1.98 (dq, J4’,30 ¼ 7.3 Hz, J4’,50 ¼ 1.5 Hz, H-40 ), 1.85 (quint, J5’40 ¼ 1.5 Hz, H-50 ); 13C NMR (CDCl3, 125 MHz): 64.4 (C-1), 24.4 (C-2), 34.7 (C-3), 61.0 (C-4), 64.6 (C-5), 74.7 (C-6), 50.2 (C7), 67.4 (C-8), 43.3 (C-9), 58.8 (C-10), 135.5 (C-11), 168.0 (C-12), 122.9 (C-13), 20.0 (C-14), 17.0 (C-15), 165.9 (C-10 ), 126.1 (C-20 ), 141.3 (C-30 ), 15.9 (C-40 ), 20.5 (C-50 ). Compound 4: Oil; [a]D 109.2 (c 0.42, CHCl3); IR ¼ 3,579; 2,933; 2,256; 1,768; 1,716; 1,456; 1,288; 1,228; 1,147; 1,137 cm1. 1H and 13C NMR data in Table S1.
3.4. Cyclization reaction To a solution of 21.6 mg (62.4 mmol) of deltoidin A (2) in 1 mL of CH2Cl2 was added 1 mg of pTsOH (5.8 mmol). The reaction mixture was stirred at room temperature for 4.5 h, followed by addition of 5 mL of water and 5 mL of CH2Cl2. The phases were separated and the organic layer was washed with aqueous NaHCO3, H2O, dried over anhydrous Na2SO4, filtered, and evaporated. The residue was column chromatographed using hexanes-EtOAc mixtures (from 19:1 to 7:3) to afford 13.8 mg (63.9%) of guaianolide 5. Compound 5: Oil; [a]D 53.4 (c 0.42, CHCl3); IR ¼ 3,589; 2,927; 2,854; 1,768; 1,710; 1,234; 1,151 cm1; 1H NMR (CDCl3, 500 MHz) d: 2.42 (bdd, J2a,2b ¼ 16.9 Hz, J2a,3a ¼ 8.8 Hz, H-2a), 2.26 (m), H-2b), 1.85 (J3a,2b ¼ 12.2 Hz, J3a,3b ¼ 12.2 Hz, ddd, J3a,2a ¼ 8.8 Hz, H-3a), 1.80 (ddd, J3b,3a ¼ 12.2 Hz, J3b,2b ¼ 7.9 Hz, J3b,2a ¼ 1.1 Hz, H-3b), 2.81 (bd, J5,6 ¼ 10.9 Hz, H-5), 4.44 (dd, J6,5 ¼ 10.9 Hz, J6,7 ¼ 10.4 Hz, H-6), 3.02 (dddd, J7,6 ¼ 10.4 Hz, J7,13b ¼ 3.3 Hz, J7,13a ¼ 3.1 Hz, J7,8 ¼ 1.5 Hz, H-7), 5.69 (ddd, J8,9b ¼ 4.7 Hz, J8,9a ¼ 2.5 Hz, J8,7 ¼ 1.5 Hz, H-8), 2.52 (bd, J9a,9b ¼ 16.7 Hz, H-9a), 2.61 (bdd, J9b,9a ¼ 16.7 Hz, J9b,8 ¼ 4.7 Hz, 9b), 5.62 (bd, J13a,7 ¼ 3.1 Hz, 13a), 6.27 (bd, J13a,7 ¼ 3.3 Hz, 13 b), 1.65 (bs, H-14), 1.33 (s, H-15), 6.08 (qq, J3’,40 ¼ 7.3 Hz, J3’,50 ¼ 1.5 Hz, H-30 ), 1.90 (dq, J4’,30 ¼ 7.3 Hz, J4’,50 ¼ 1.5 Hz, H-40 ), 1.80 (quint, J5’40 ¼ 1.5 Hz, H-50 ); 13C NMR (CDCl3, 125 MHz):.d: 133.0 (C-1), 30.2 (C-2), 38.3 (C-3), 80.4 (C-4), 58.7 (C-5), 78.0 (C-6), 52.3 (C-7), 64.8 (C-8), 39.5 (C-9), 125.8 (C-10), 134.9 (C-11), 168.8 (C-12), 121.0 (C-13), 24.3 (C-14), 22.7 (C-15), 167.0 (C-10 ), 127.4 (C-20 ), 138.5 (C-30 ), 15.8 (C-40 ), 20.6 (C-50 ).
3.5. Ir and VCD measurements The spectra were obtained on a dual PEM BioTools ChiralIR2X FT-VCD spectrophotometer operated at a resolution of 4 cm1 in chloroform-d 100% atom-D, using 0.17, 0.13, and 0.12 M solutions of 2, 4, and 5, respectively, in a BaF2 cell with a path length of 101 lm. Five 1 h data-blocks were added for each sample. The baseline was improved by subtracting the spectrum of the solvent acquired under the same conditions.
3.6. Computational methods Monte Carlo searches of 2, 4, and 5 were achieved using the MMFF94 level of calculation as implemented in the Spartan’04 software (Wavefunction Inc., Irvine, CA). The
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M. Á. FUENTES-FIGUEROA ET AL.
single-point energy of each conformer was calculated using DFT with the B3LYP functional and the 6-31 G(d) basis set. The selected conformers were submitted to further conformational optimization by DFT calculations at the B3LYP/DGDZVP level of theory with the Gaussian’03 software (Gaussian Inc., Wallingford, CT). The IR and VCD frequencies calculations were carried out at the same level of theory. All minimum energy geometries were tested for the absence of imaginary frequencies, and their relative free energies were used to calculate their Boltzmann distributions. The Boltzmann-weighted IR and VCD spectra were calculated with Lorentzian functions and a bandwidth of 6 cm1. Calculated and experimental spectra were compared using the CompareVOA software (BioTools) (Debie et al. 2011).
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported by SIP-IPN (Grants 20195548 and 20201306) and CONACYT (Grant A1S-17910). MAFF thanks to CONACYT for doctoral scholarship 737291. We thank to M. C. Ernestina Cedillo-Portugal for authentication of the botanical specimen.
ORCID Pedro Joseph-Nathan
http://orcid.org/0000-0003-3347-3990
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Clewell AF, Wooten JW. 1971. A revision of Ageratina (Compositae: Eupatorieae) from eastern North America. Brittonia. 23(2):123–143. Compositae Working Group (CWG). 2020. Global compositae database. http://www.compositae. orgon2020-05-20. Debie E, de Gussem E, Dukor RK, Herrebout W, Nafie LA, Bultinck PA. 2011. A confidence level algorithm for the determination of absolute configuration using vibrational circular dichroism or Raman optical activity. Chemphyschem. 12(8):1542–1549. n JS, Parra F. 2011. Antiviral activdel Barrio G, Spengler I, Garcıa T, Roque A, Alvarez AL, Caldero ity of Ageratina havanensis and major chemical compounds from the most active fraction. Rev Bras Farmacogn. 21(5):915–920. n F. 2018. Secondary Eiroa JL, Triana J, Perez FJ, Castillo QA, Brouard I, Quintana J, Estevez F, Leo metabolites from two Hispaniola Ageratina species and their cytotoxic activity. Med Chem Res. 27(7):1792–1799. Elix JA, Whitton AA, Jones AJ. 1982. Triterpenes from the lichen genus Physcia. Aust J Chem. 35(3):641–647. Fischer NH, Olivier EJ, Fischer HD. 1979. The biogenesis and chemistry of sesquiterpene lactones. In: Herz W, Grisebach H, Kirby GW, editors. Progress in the chemistry of organic natural products. Vol. 38. Vienna: Springer-Verlag; p. 47–390. mez F, Quijano L, Caldero n JS, Perales A, Rıos T. 1982. 2,2-Dimethylchromenes from Go Eupatorium aschembornianum. Phytochemistry. 21(8):2095–2097. Joseph-Nathan P, Gordillo-Roman B. 2015. Vibrational circular dichroism absolute configuration determination of natural products. In: Kinghorn AD, Falk H, Kobayashi J, editors. Progress in the chemistry of organic natural products. Vol. 100. Switzerland: Springer International Publishing; p. 311–451. Karunaratne V, Thadhani VM, Khan SN, Choudhary MI. 2014. Potent a-glucosidase inhibitors from the lichen Cladonia species from Sri Lanka. J Natn Sci Foundation Sri Lanka. 42(1):95–98. Kreuger MRO, Grootjans S, Biavatti MW, Vandenabeele P, D’Herde K. 2012. Sesquiterpene lactones as drugs with multiple targets in cancer treatment: focus on parthenolide. Anticancer Drugs. 23(9):883–896. Lee ST, Davis TZ, Gardner DR, Stegelmeier BL, Evans TJ. 2009. Quantitative method for the measurement of three benzofuran ketones in rayless goldenrod (Isocoma pluriflora) and white snakeroot (Ageratina altissima) by high-performance liquid chromatography (HPLC). J Agric Food Chem. 57(12):5639–5643. Plant List. 2013. Version 1.1. Published online March 20, 2020. http://www.theplantlist.org. n JS, Go mez F, Gardun ~o JT, Rıos T. 1980. Deltoidin A and B, two new germaQuijano L, Caldero cranolides from Eupatorium deltoideum. Phytochemistry. 19(9):1975–1977. Shiojima K, Ageta H. 1994. Fern constituents: Triterpenoids isolated from the leaves of Adiantum edgeworthii. Structures of 19a-hydroxydiantone and fern-9(11)-en-25-oic acid. Chem Pharm Bull. 42(1):45–47. Tamayo-Castillo G, Jakupovic J, Bohlmann F, Rojas A, Castro V, King RM. 1988. Germacranolides and other constituents from Ageratina species. Phytochemistry. 27(9):2893–2897. Turner BL. 2014. Taxonomy of the Ageratina vernalis (Asteraceae: Eupatorieae) complex. Phytologia. 96:12–18. Wong SM, Oshima Y, Pezzuto JM, Fong HH, Farnsworth NR. 1986. Plant anticancer agents XXXIX: Triterpenes from Iris missouriensis (Iridaceae). J Pharm Sci. 75(3):317–320.
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A new germacranolide from Ageratina vernalis Miguel Á. Fuentes-Figueroa , Neively Tlapale-Lara , Beatriz HernándezCarlos , Pedro Joseph-Nathan & Eleuterio Burgueño-Tapia To cite this article: Miguel Á. Fuentes-Figueroa , Neively Tlapale-Lara , Beatriz HernándezCarlos , Pedro Joseph-Nathan & Eleuterio Burgueño-Tapia (2020): A new germacranolide from Ageratina�vernalis , Natural Product Research, DOI: 10.1080/14786419.2020.1827400 To link to this article: https://doi.org/10.1080/14786419.2020.1827400
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NATURAL PRODUCT RESEARCH https://doi.org/10.1080/14786419.2020.1827400
A new germacranolide from Ageratina vernalis Fuentes-Figueroaa, Neively Tlapale-Laraa, Beatriz Hernandez-Carlosb, Miguel A. ~o-Tapiaa Pedro Joseph-Nathanc and Eleuterio Burguen Departamento de Quımica Organica, Escuela Nacional de Ciencias Biologicas, Instituto Politecnico Nacional, Prolongacion de Carpio y Plan de Ayala, Mexico City, Mexico; bInstituto de Agroindustrias, Universidad Tecnologica de la Mixteca, Huajuapan de Leon, Mexico; cDepartamento de Quımica, Centro de Investigacion y de Estudios Avanzados, Instituto Politecnico Nacional, Mexico City, Mexico a
ABSTRACT
ARTICLE HISTORY
The aerial parts of Ageratina vernalis provided the new germacranolide 1,10-epoxydeltoidin A (3), together with the known pentacyclic triterpenoid hopane-6a,22-diol (1), and the also known germacranolides deltoidin A (2) and 15-hydroxydeltoidin A (4). In addition, pTsOH catalyzed cyclization of 2 afforded the new guaianolide 5. The absolute configuration of 2, 4, and 5 was assigned by vibrational circular dichroism spectroscopy, while the complete 1H and 13C NMR data assignments of 2-5 followed from 1 D- and 2 D-NMR experiments.
Received 29 April 2020 Accepted 5 September 2020 KEYWORDS
Ageratina vernalis; germacranolides; vibrational circular dichroism
1. Introduction The genus Ageratina, contains 334 species (Plant List 2013. Version 1.1), including some that were segregated from the genus Eupatorium (Clewell and Wooten 1971). Typical isolates from Ageratina species are flavonoids (del Barrio et al. 2011), thymols mez et al. 1982; Aguilar-Guadarrama et al. (Bustos-Brito et al. 2016), chromenes (Go 2009), benzofurans (Aguilar-Guadarrama et al. 2009; Lee et al. 2009), and germacranolides (Quijano et al. 1980; Tamayo-Castillo et al. 1988). Some sesquiterpene lactones isolated from this genus have shown interesting activities as antimicrobial (Arciniegas ~o-Tapia CONTACT Eleuterio Burguen [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2020.1827400. ß 2020 Informa UK Limited, trading as Taylor & Francis Group
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M. Á. FUENTES-FIGUEROA ET AL.
Figure 1. Formulas of natural products 1-4 from A. vernalis and of 5 obtained by cyclization of 2.
et al. 2018) and anticancer (Orofino-Kreuger et al. 2012; Eiroa et al. 2018) agents. Recently, some thymol derivatives isolated from A. glabrata (Arreaga-Gonzalez et al. 2018) were used to develop a methodology for the determination of the enantiomeric excesses and the absolute configuration (AC) of epoxythymols. A. vernalis is endemic to Mexico and lacks of reported chemical studies. We now isolated the new germacranolide 3, the hopane triterpenoid 1, and the known germacranolides 2 and 4 (Figure 1) from the hexanes extract of the aerial parts of A. vernalis. Furthermore, the new guaianolide 5 was obtained by p-TsOH catalyzed cyclization of compound 2. The AC assignment of 2, 4, and 5 was achieved by vibrational circular dichroism.
2. Results and discussion The hexanes extracts of the aerial parts of Ageratina vernalis collected from the state of Oaxaca, Mexico were subjected to Si-gel column chromatography to afford 1–4. The first isolated compound, which turned out to be 1, was obtained by successive column chromatographies from the less polar fractions. Its 1H NMR spectrum (Figure S1) showed eight singlet methyl group signals at d 1.21, 1.18, 1.15, 1.04, 1.01, 0.97, 0.87, and 0.76, and the signal of a methine geminal to a hydroxy group at d 3.96 (td, J ¼ 10.6, 4.2 Hz). These signals, along with the 30 sp3 carbon signals observed in
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the 13C NMR spectrum (Figure S2) suggested a pentacyclic triterpene structure for compound 1. Comparison of the data with those of hopane-6a,22-diol (Elix et al. 1982) allowed the identification of 1. This compound has typically been described from cryptogams, as lichens (Elix et al. 1982) and ferns (Shiojima and Ageta 1994), although it has also been found in higher plants like Iris missourensis (Wong et al. 1986). This is the first time that 1 is described from an Ageratina species. The second isolate was 2. It showed the characteristic 1H NMR signals of an angeloyloxy group at d 6.14 (1H, qq, J ¼ 7.3, 1.5 Hz, H-30 ), 1.98 (3H, dq, J ¼ 7.3, 1.5 Hz, H-40 ), and 1.84 (3H, quint, J ¼ 1.5 Hz, H-50 ), typical signals of an exocyclic methylene group at 6.40 (1H, bd, J ¼ 3.5 Hz, H-13a), 5.75 (1H, bd, J ¼ 3.1 Hz, H-13b), and three methine groups, each geminal to an oxygen atom, at d 2.85 (1H, d, J ¼ 8.8 Hz, H-5), 4.46 (1H, dd, J ¼ 8.8, 8.4 Hz, H-6), and 5.77 (1H, bdd, J ¼ 5.2, 5.0 Hz, H-8) (Figure S3). These signals and the 13C NMR chemical shifts (Figure S4), suggested a germacranolide sesquiterpene lactone. Additional 2 D NMR experiments allowed identifying it as deltoidin A (2). This compound was described 40 years ago (Quijano et al. 1980) and only partial 1H NMR data were provided at that time. Thus, complete 1H and 13C NMR data assignments, derived from 1 D- and 2 D-NMR experiments, are summarized in the supplementary material (Table S1). Rechromatography of a fraction containing 2 gave additional 12.1 mg of deltoidin A and 2.1 mg of the new germacranolide 3. The 1H NMR spectrum of 3 showed signals very similar with those of 2, except for a signal at d 2.90 (1H, dd, J ¼ 10.9, 1.7 Hz, H-1) instead of the H-1 vinyl proton signal. Additionally, the 13C NMR spectrum showed two new oxygen bearing carbon signals at d 64.4 (C-1) and 58.8 (C-10), instead of the C-1 C-10 double bond signals (Figure S5–S10). These evidences revealed the structure of 1,10-epoxydeltoidin A (3). The trans configuration of 1,10-oxirane was assigned through NOESY experiments where correlations of Me-14 (d 1.37) with Me-15 (d 1.44), and of H-1 (d 2.90) with H-2a (d 2.23), H-3a (d 1.41), and H-5 (d 2.96) were observed (Figure S11). The lactone and angeloyloxy group configurations were deduced by comparing the observed and calculated J5,6, J6,7, and J7,8 values of the minimum energy conformer, which were 8.6, 8.5, and 1.0, versus 9.5, 9.8, 0.6 Hz, respectively. Thus, considering that 2 and 3 are biogenetically related, the configuration depicted for 3 was assumed. Compound 4 showed similar 1H NMR signals than 2, although two doublets at d 3.94 (1H, d, J ¼ 12.4 Hz, H-15a) and d 3.79 (1H, d, J ¼ 12.4 Hz, H-15b) appear instead of the Me-15 group signal of 2 (Figure S12). In turn, the 13C NMR spectrum showed a signal at d 60.8 corresponding to the C-15 hydroxymethylene group, thus allowing to propose the structure of 15-hydroxydeltoidin A (4) (Figure S13). This compound was reported (Bohlmann et al. 1985) as part of an inseparable mixture of angelate and tiglate esters for which only scarce 1H NMR data was provided. 1H and 13C NMR data assignments were achieved using 1 D- and 2 D-NMR experiments, and are shown in the supplementary material (Table S1). The AC of 2 was correctly assumed (Quijano et al. 1980) considering biogenetic aspects and with the aid of coupling constants values for H-5, H-6, H-7, H-8, and H-13. Herein, we confirmed the AC using vibrational circular dichroism (VCD) spectroscopy ~o-Tapia and Joseph-Nathan 2015, 2017; Joseph-Nathan and Gordillo-Roman (Burguen
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M. Á. FUENTES-FIGUEROA ET AL.
Table 1. Comparison of experimental and DFT B3LYP/DGDZVP calculated IR and VCD spectra of 2, 4, and 5. Comp.
anHa
SIRb
SEc
SEd
ESIe
Cf
2 4 5
0.980 0.980 0.980
96.7 94.9 92.1
77.4 82.7 76.1
10.9 13.6 6.0
66.5 69.1 70.1
99 99 99
a
Anharmonicity factor. IR spectral similarity (%). c VCD spectral similarity for the correct enantiomer (%). d VCD spectral similarity for the incorrect enantiomer (%). e Enantiomer similarity index calculated as (SESE). f Confidence level for the AC determination (%). b
2015). Thus, a molecular model based on the relative configuration deduced from the 1 ~o-Tapia H NMR data was constructed. Following the described methodology (Burguen and Joseph-Nathan 2015; Joseph-Nathan and Gordillo-Roman 2015), a conformational search of this model, using the Monte Carlo protocol and MMFF94 molecular mechanics, was performed. The results were submitted to single point energy calculation using density functional theory (DFT) and the B3LYP/6-31G (d) functional and basis set, followed by complete geometry optimization at the DFT B3LYP/DGDZVP level of theory. Final calculation of the IR and VCD frequencies at the same level of theory was then achieved. The conformational search provided 13 conformers within DE ¼ 9.53 kcal/mol, which after single point energy calculations left only three conformers in a 0.79 kcal/mol energy gap, the fourth conformer, being 4.86 kcal/mol above the global minimum, is not contributing to the overall conformational picture. The complete geometry optimization matched the ring geometry of conformers 2a and 2 b (Figure S14) which is essentially the same, since the conformers differing in the angeloyloxy group orientation, for which the H-8 C-8 O C1’, C-8OC1’CO, and O C1’C2’C3’ dihedral angles are 26.6, 2.1, and 168.4, and 31.3, 2.2, and 30.7 degrees, respectively. After frequencies calculation, these two conformers were observed with a DG ¼ 1.24 kcal/mol. The thermochemical parameters are shown in the supplementary material (Table S2). The weighted IR and VCD curves were compared with the corresponding experimental spectra (Figure S15) using the CompareVOA software. The confidence level (C) (Debie et al. 2011) for the 4S,5R,6S,7R,8R AC assignment was 99% (Table 1). The AC of 4 was also assigned by VCD spectroscopy as above. The Monte Carlo search gave 24 conformers in a DE ¼ 9.89 kcal/mol gap. After single point energy calculations only three conformers within a 2.42 kcal/mol energy gap, contributing with 93.3% of the conformation population were further considered. Complete geometry optimizations and frequencies calculations showed these three conformers (Figure S14) in a DG ¼ 1.56 kcal/mol gap as shown in Table S2. The main conformational difference between the two most stable conformers lies in the orientation of the hydroxy group (Figure S14) as evidenced by the C-3 C-4 C-15 O and C-4 C-15 OH dihedral angles for 4a and 4 b which are 168.8 and 40.5, and 70.7 and 58.9 degrees, respectively. For compound 4, a great agreement was observed between the experimental IR and VCD spectra with the respective calculated spectra (Figure S16) and the numerical data obtained from the CompareVOA software, including the C value of 99% for the
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4S,5R,6S,7R,8R AC assignment, are shown in Table 1. The scarce isolated amount (2.1 mg) of 3 precluded obtaining a reliable VCD spectrum. Inspection of the VCD spectra of 2 and 4 shows a strong positive band at 1135 and 1137 cm1, respectively, which is related to the lactone ring stereogenic centers. These bands were attributed, according to a GausView software evaluation, to strong C11 C-12 and C-12 O-6 asymmetric stretching which are accompanied by CH-6 and CH-7 asymmetric bendings. It is biogenetically accepted that germacranolides are the precursors of eudesmanolides and guaianolides through C-5 C-10 or C-1 C-5, respectively, cyclizations (Fischer et al. 1979). It is also known that acid-catalyzed cyclization of 1,10-epoxides leads to eusdemanolides, while if the oxyrane function appears at C-4 C-5 it leads to guaiano~eda-Acosta et al. 1993). Consequently, compound 2 was treated with a catalides (Castan lytic amount of pTsOH at room temperature to generate the new guaianolide 5 in 63.9% yield. The structure of 5 was deduced by 1 D- and 2 D-NMR experiments (Figure S18–S23), including gHMBC, in which the correlations observed between Me-14 (d 1.65) and C-9 (d 39.5), and of H-2 (d 2.26, 2.42), H-8 (d 5.69), and H-9 (d 2.52, 2.61) with C-10 (d 125.8). The complete NMR data assignments are shown in the experimental part. The 4 R,5S,6S,7R,8R AC assignment shown in 5 was done by VCD as above. The conformers search using the Monte Carlo protocol gave 10 conformers within a 10 kcal/mol energy window, which after single point energy analysis using DFT B3LYP/6-31G(d) were reduced to three within a DG ¼ 4.71 kcal/mol gap. Complete energy optimization at the DFT B3LYP/DGDZVP level left only two conformers within a DG ¼ 1.04 kcal/mol gap. The main difference for conformers 5a and 5 b is the angeloyloxy group orientation, for which the H-8 C-8 O C1’, C-8OC1’CO, and O C1’C2’C3’ dihedral angles are 25.0, 0.1, and 174.6, and 2.4, 2.02, and 17.4 degrees, respectively. After IR and VCD frequencies calculations at the same level of theory, these two conformers showed DG ¼ 2.09 kcal/mol from which the most stable conformer (Figure S14) contributed with 99% of the conformational population. The calculated and experimental IR and VCD spectra are shown in Figure S17 and the CompareVOA comparison showed a 99% reliability (Table 1) for the 4 R,5S,6S,7R,8R AC assignment. The band associated with the lactone ring stereogenic centers was observed at 1151 cm1. The 1H and 13C NMR data are summarized in the experimental section. The purity of the isolated compounds followed from evaluation of their signals in the NMR spectra, as can be observed in the supplementary material. Compound 1 showed inhibitory activity of a-glucosidase at concentration seven times lower than acarbose, a drug used in the treatment of diabetes type II (Karunaratne et al. 2014); while compound 2 has shown selective activity against gram negative bacteria (Arciniegas et al. 2018). No additional biological activity tests for 1–5 were performed during the present work.
3. Experimental 3.1. General experimental procedures The melting point was done on an Electrothermal IA9100X1 apparatus and is uncorrected. Optical rotations were recorded in CHCl3 using a JASCO DIP-370 polarimeter.
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1
H, 13C and 2 D NMR experiments were done using a Varian System 500 (125 MHz for 13 C) spectrometer, CDCl3 was used as solvent and TMS as internal reference. Column chromatography separations were achieved using Natland silica gel 230-400 Mesh.
3.2. Plant material Ageratina vernalis (Vatke & Kurtz) R.M.King & H.Rob is well documented in the literature (Turner 2014). It is a 2-4 m tall shrub with rounded stems 3-5 mm wide, the leaves are 10–20 cm long and 4–8 cm wide, and has 3–5 cm long petioles. The International Compositae Alliance (TICA) (Compositae Working Group (CWG)), 2020) indicates that A. vernalis is the accepted name of species previously known as Ageratina chiapensis, Ageratina subcoriacea, Conoclinium grandiflorum, Eupatorium chiapense, and Eupatorium vernale. For the present study the aerial parts were collected from near the town Rıo Grande, Oaxaca, Mexico (N 16 060 0.496”, W 96 290 0.245”) in May 2016. A plant specimen, authenticated by M. C. Ernestina Cedillo-Portugal, is deposited noma de Chapingo, Texcoco, at the Herbarium ‘Jorge Salas Espinosa’, Universidad Auto Mexico under voucher number 25770.
3.3. Isolation of natural products Powdered dried aerial parts (1.3 kg) were extracted with hexanes at reflux (3 4 L) for 4 h, filtered, and the solvent evaporated under reduced pressure. The residue (4.4 g), dissolved in acetone, was kept at 4 C for 14 h and filtrated to remove fatty materials. The filtrate was evaporated and the residue (3.1 g) was column chromatographed. Elution with hexanes-EtOAc mixtures (19:1, 9:1, 4:1, 7:3, and 3:2) collecting 100 mL fractions afforded, after thin layer chromatography evaluation, the five main fractions A-E. The 1H NMR spectrum of fraction A (514 mg) revealed the presence of fatty material and was discarded. Fraction B (315 mg) was rechromatographed using hexanes-EtOAc mixtures. Those fractions eluted with 7:3 mixtures afforded 6.0 mg of 1. Fraction C (303 mg) was column rechromatographed using hexanes-EtOAc mixtures of increasing polarity (19:1 to 2:3) to afford 30.3 mg of deltoidin A (2). A fraction (56 mg) of this chromatography containing 2 was column rechromatographed to give additional 12.1 mg of 2 and 2.1 mg of 3. Rechromatography of fraction D, using mixtures as in the previous cases gave 9.5 mg of 4. Rechromatography of fraction E gave no useful compound.
3.3.1. Characterization of natural products Compound 1: White solid; mp 222–225 C; 1H and 13C NMR data were the same as those reported. lit. colorless prism, mp 221–223 C (Elix et al. 1982). Compound 2: White semi-solid; [a]D 132.8 (c 0.42, CHCl3); lit. (Quijano et al. 1980) white solid, mp 159–161 C; [a]D: 145.1, CHCl3; 1H and 13C NMR data in Table S1. Compound 3: Oil; [a]D 22.4(c 0.42, CHCl3); IR ¼ 2,929; 2,925; 2,856; 2,258; 1,770; 1,718; 1,456; 1,390; 1,292; 1,238; 1,139 cm1; 1H NMR (CDCl3, 500 MHz) d: 2.90 (dd, J1,2b ¼ 10.9 Hz, J1,2a ¼ 1.7 Hz, H-1), 2.23 (m, H-2a), 1.54 (bddd, J2b,2a ¼ 13.4 Hz, J2b,1 ¼ 10.9 Hz, J2b,3b ¼ 4.4 Hz, H-2b), 1.41 (m, H-3a), 2.29 (ddd, J3b,3a ¼ 13.2 Hz,
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J3b,2b ¼ 4.4 Hz, J3b,2a ¼ 2.2 Hz, H-3b), 2.96 (d, J5,6 ¼ 8.6 Hz, H-5), 4.54 (dd, J6,5 ¼ 8.5 Hz, J6,7 ¼ 8.5 Hz, H-6), 3.11 (ddt, J7,6 ¼ 8.5 Hz, J7,13 ¼ 3.4 Hz, J7,8 ¼ 1.0 Hz, H-7), 5.76 (ddd, J8,9b ¼ 6.0 Hz, J8,9a ¼ 2.1 Hz, J8,7 ¼ 1.2 Hz, H-8), 1.41 (m, H-9a), 2.97 (dd J9b,9a ¼ 15.4 Hz, J9b,8 ¼ 6.0 Hz, H-9b), 5.73 (dd, J13a,7 ¼ 3.1 Hz, J13a,13b ¼ 0.9 Hz, H-13a), 6.41 (dd, J13b,7 ¼ 3.5 Hz, J13b,13a ¼ 0.9 Hz, H-13b), 1.37 (s, H-14), 1.44 (s, H-15), 6.16 (qq, J3’,40 ¼ 7.3 Hz, J3’,50 ¼ 1.5 Hz, H30 ), 1.98 (dq, J4’,30 ¼ 7.3 Hz, J4’,50 ¼ 1.5 Hz, H-40 ), 1.85 (quint, J5’40 ¼ 1.5 Hz, H-50 ); 13C NMR (CDCl3, 125 MHz): 64.4 (C-1), 24.4 (C-2), 34.7 (C-3), 61.0 (C-4), 64.6 (C-5), 74.7 (C-6), 50.2 (C7), 67.4 (C-8), 43.3 (C-9), 58.8 (C-10), 135.5 (C-11), 168.0 (C-12), 122.9 (C-13), 20.0 (C-14), 17.0 (C-15), 165.9 (C-10 ), 126.1 (C-20 ), 141.3 (C-30 ), 15.9 (C-40 ), 20.5 (C-50 ). Compound 4: Oil; [a]D 109.2 (c 0.42, CHCl3); IR ¼ 3,579; 2,933; 2,256; 1,768; 1,716; 1,456; 1,288; 1,228; 1,147; 1,137 cm1. 1H and 13C NMR data in Table S1.
3.4. Cyclization reaction To a solution of 21.6 mg (62.4 mmol) of deltoidin A (2) in 1 mL of CH2Cl2 was added 1 mg of pTsOH (5.8 mmol). The reaction mixture was stirred at room temperature for 4.5 h, followed by addition of 5 mL of water and 5 mL of CH2Cl2. The phases were separated and the organic layer was washed with aqueous NaHCO3, H2O, dried over anhydrous Na2SO4, filtered, and evaporated. The residue was column chromatographed using hexanes-EtOAc mixtures (from 19:1 to 7:3) to afford 13.8 mg (63.9%) of guaianolide 5. Compound 5: Oil; [a]D 53.4 (c 0.42, CHCl3); IR ¼ 3,589; 2,927; 2,854; 1,768; 1,710; 1,234; 1,151 cm1; 1H NMR (CDCl3, 500 MHz) d: 2.42 (bdd, J2a,2b ¼ 16.9 Hz, J2a,3a ¼ 8.8 Hz, H-2a), 2.26 (m), H-2b), 1.85 (J3a,2b ¼ 12.2 Hz, J3a,3b ¼ 12.2 Hz, ddd, J3a,2a ¼ 8.8 Hz, H-3a), 1.80 (ddd, J3b,3a ¼ 12.2 Hz, J3b,2b ¼ 7.9 Hz, J3b,2a ¼ 1.1 Hz, H-3b), 2.81 (bd, J5,6 ¼ 10.9 Hz, H-5), 4.44 (dd, J6,5 ¼ 10.9 Hz, J6,7 ¼ 10.4 Hz, H-6), 3.02 (dddd, J7,6 ¼ 10.4 Hz, J7,13b ¼ 3.3 Hz, J7,13a ¼ 3.1 Hz, J7,8 ¼ 1.5 Hz, H-7), 5.69 (ddd, J8,9b ¼ 4.7 Hz, J8,9a ¼ 2.5 Hz, J8,7 ¼ 1.5 Hz, H-8), 2.52 (bd, J9a,9b ¼ 16.7 Hz, H-9a), 2.61 (bdd, J9b,9a ¼ 16.7 Hz, J9b,8 ¼ 4.7 Hz, 9b), 5.62 (bd, J13a,7 ¼ 3.1 Hz, 13a), 6.27 (bd, J13a,7 ¼ 3.3 Hz, 13 b), 1.65 (bs, H-14), 1.33 (s, H-15), 6.08 (qq, J3’,40 ¼ 7.3 Hz, J3’,50 ¼ 1.5 Hz, H-30 ), 1.90 (dq, J4’,30 ¼ 7.3 Hz, J4’,50 ¼ 1.5 Hz, H-40 ), 1.80 (quint, J5’40 ¼ 1.5 Hz, H-50 ); 13C NMR (CDCl3, 125 MHz):.d: 133.0 (C-1), 30.2 (C-2), 38.3 (C-3), 80.4 (C-4), 58.7 (C-5), 78.0 (C-6), 52.3 (C-7), 64.8 (C-8), 39.5 (C-9), 125.8 (C-10), 134.9 (C-11), 168.8 (C-12), 121.0 (C-13), 24.3 (C-14), 22.7 (C-15), 167.0 (C-10 ), 127.4 (C-20 ), 138.5 (C-30 ), 15.8 (C-40 ), 20.6 (C-50 ).
3.5. Ir and VCD measurements The spectra were obtained on a dual PEM BioTools ChiralIR2X FT-VCD spectrophotometer operated at a resolution of 4 cm1 in chloroform-d 100% atom-D, using 0.17, 0.13, and 0.12 M solutions of 2, 4, and 5, respectively, in a BaF2 cell with a path length of 101 lm. Five 1 h data-blocks were added for each sample. The baseline was improved by subtracting the spectrum of the solvent acquired under the same conditions.
3.6. Computational methods Monte Carlo searches of 2, 4, and 5 were achieved using the MMFF94 level of calculation as implemented in the Spartan’04 software (Wavefunction Inc., Irvine, CA). The
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M. Á. FUENTES-FIGUEROA ET AL.
single-point energy of each conformer was calculated using DFT with the B3LYP functional and the 6-31 G(d) basis set. The selected conformers were submitted to further conformational optimization by DFT calculations at the B3LYP/DGDZVP level of theory with the Gaussian’03 software (Gaussian Inc., Wallingford, CT). The IR and VCD frequencies calculations were carried out at the same level of theory. All minimum energy geometries were tested for the absence of imaginary frequencies, and their relative free energies were used to calculate their Boltzmann distributions. The Boltzmann-weighted IR and VCD spectra were calculated with Lorentzian functions and a bandwidth of 6 cm1. Calculated and experimental spectra were compared using the CompareVOA software (BioTools) (Debie et al. 2011).
Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was supported by SIP-IPN (Grants 20195548 and 20201306) and CONACYT (Grant A1S-17910). MAFF thanks to CONACYT for doctoral scholarship 737291. We thank to M. C. Ernestina Cedillo-Portugal for authentication of the botanical specimen.
ORCID Pedro Joseph-Nathan
http://orcid.org/0000-0003-3347-3990
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