ITAPEJARAFURAN, (5R,5aS,9aR)-4,5,5a,6,7,9a-HEXAHYDRO-1,5,8-TRIMETHYLNAPHTHO[2,1-b]FURAN FROM BACCHARIS PUNCTULATA (ASTERACEAE) AND CYTOTOXIC EVALUATION ON MIA PaCa-2 CELL LINEAbstract
Furanocadinan-type sesquiterpene itapejarafuran has been reported as part of the essential oil of Baccharis punctulata DC. (Asteraceae) leaves. This species is widely distributed in South America and popularly known as “chilka saru saru” in Bolivia and traditionally used for treatment of luxations, contusions and asthma. Since the stereochemistry of molecules can drastically modify its biological activity, in this study we aimed to isolate and determine the complete stereochemistry of the compound. The structure of itapejarafuran and its absolute configuration were determined by nuclear magnetic resonance, optical rotation and electronic circular dichroism with high-level quantum-chemical calculations. The corresponding computational calculations indicated that this compound obtained from the essential oil of Baccharis punctulata leaves corresponds to the RSR isomer, (5R,5aS,9aR)-4,5,5a,6,7,9a-hexahydro-1,5,8-trimethylnaphtho[2,1-b]furan. This is, to the best of our knowledge, the first determination of the absolute configuration of itapejarafuran from B. punctulata. Establishing the stereochemistry for this furanosesquiterpene provides a robust framework for future structure-activity studies and chemotaxonomic comparisons within Baccharis genera, in which furanocadinane skeletons are frequent components of essential oils. Furthermore, itapejarafuran exhibited toxicity against cancer on MIA PaCa-2 cells (half-maximal inhibitory concentration (IC50): 150 µM), indicating a potential application as chemotherapeutic candidate and providing insights for future investigations.
Keywords:
Baccharis punctulata; furanosesquiterpene; itapejarafuran; absolute configuration; antitumor activity; MIA PaCa-2.
INTRODUCTION
Baccharis punctulata DC., known as “chilka saru saru”, has been traditionally used by rural communities in Bolivia for treatment of luxations, contusions, and asthma.1 It is widely distributed in Brazil, Paraguay, Uruguay, Argentina, and Bolivia. In Brazil, it occurs preferentially in the Southeast and South regions, in the Cerrado, Atlantic Forest and Pampa biomes.2
B. punctulata presents high variability in the chemical composition of its essential oil obtained from aerial parts.3-9 Ascari et al.8,9 evaluated samples of the essential oil from B. punctulata leaves collected between 2017 and 2019 in the West and Southwest regions of Paraná State, Brazil. The samples were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) and the compound verboccidentafuran 1 was present in all of the samples collected in the Southwest region, whereas it was absent in the samples from the West region. The variation of its relative proportion (%) can be attributed to the existence of more than one chemotype for this species. Other studies have reported the presence of compound 1 in B. punctulata,3,6,7 Baccharis salicifolia10-13 and Baccharis latifolia,11 as well as in several species from the genera Verbesina,14 Eupatorium,15 Raulinoreitzia and Symphyopappus,16 and Chromolaena.17 Bohlmann and Lonitz14 first identified and isolated compound 1 (Figure 1) from the aerial parts of Verbesina occidentalis (L.) Walter. Compound 1 was also isolated from the roots of Chromolaena arnottiana (Griseb.) R.M.King & H.Rob.18 and Chromolaena pseudoinsignis K. & R.,19 and from the aerial parts of Aristeguetia glutinosa (HBK) K. & R.20
Structure of verboccidentafuran 1 in the relative stereochemistry (adapted from Bohlmann et al.)19
In order to better understand the stereochemistry of compound 1, Bohlmann and Trantow21 carried out the synthesis based on an initial Diels-Alder reaction, on which the relative stereochemistry of the compound was defined as depicted in Figure 1 for (5α,5aα,9aα)-(±)-4,5α,5α,6,7,9α-hexahydro-1,5,8-trimethylnaphtho[2,1-b]furan. Their assignments were made by means of nuclear magnetic resonance (NMR) and comparison of the chemical shift signals to those from several other furan derivatives obtained from the same source, V. occidentalis.
A molecule stereochemistry plays a crucial role in the structure-activity relationship, influencing everything from biological activity to enzyme selectivity.22,23 The structural resolution of chiral centers is challenging in many natural compounds, requiring hyphenated techniques for complete elucidation. The combination of optical rotation (OR), electronic circular dichroism (ECD) and NMR techniques, associated with high-level quantum chemical computational calculations, allows for a reliable assignment to stereochemical centers.24-26 Computational methods to predict NMR spectra are constantly advancing and are considered paramount in the unequivocal structural elucidation of the stereochemistry of compounds of natural and synthetic sources.26-28 The diastereomeric parameter 4 (DP4) probability, using NMR chemical shift calculations, is a common tool for stereochemical assignment of molecules. To improve DP4 performance, a modified probability, DP4+, has been developed, with the insertion of unscaled data and the application of higher theory levels to perform the calculations.27-30
In our studies with B. punctulata, we reported anti-inflammatory activity8 and, in some cellular models, suppression of inflammatory processes that may be related to cancer.31,32 However, the available data on the anti-cancer activities remain scarce and fragmented. Pancreatic ductal adenocarcinoma (PDAC), the most frequent histological subtype of pancreatic cancer, is an extremely aggressive malignancy characterized by late diagnosis, extensive desmoplasia and profound resistance to standard chemotherapeutics.33 Therefore, the search for bioactive compounds that show a positive response against cancer cells underscores the urgent need for new therapeutic strategies.
This study reports the isolation and defines, for the first time, the absolute configuration of the compound 2 named itapejarafuran, (5R,5aS,9aR)-4,5,5a,6,7,9a-hexahydro-1,5,8-trimethylnaphtho[2,1-b]furan from B. punctulata. The determination of the stereochemistry was based on a combination of OR, ECD, NMR and computational methods. In addition, we report its cytotoxic activity in a cancer cell model, supporting the hypothesis that this compound may represent one of the key bioactive constituents contributing to the anti-inflammatory activity previously described for B. punctulata.
EXPERIMENTAL
General experimental procedures
Optical rotations were measured on a PerkinElmer polarimeter, model 343 (chloroform, concentration (c) 1.0, 589 nm, 20 °C, optical length 1 cm). ECD spectrum was obtained using a Jasco spectropolarimeter, model J-715 (ethanol, c 0.33, 350-195 nm, cell length 0.5 cm, 24 °C, band width 1.0 nm). High-resolution mass spectra were recorded on a maXis 3G Bruker Daltonics (ESI (+)-QToF) mass spectrometer (nebulizer: 2.7 bar, dry gas: 8 L min-1, temperature: 200 °C). NMR spectrum was recorded on a Bruker Avance III HD (300 MHz for 1H and 75 MHz for 13C) using tetramethylsilane as an internal standard, and thin layer chromatography (TLC) was performed with Merck Kiesegel 60 F254 plates. Silica gel 60, 0.040-0.063 mm, was used for column chromatography.
Isolation and purification
The isolation of compound 2 was accomplished from an essential oil sample (BP7♂) obtained by our research group.9 Approximately 1 g of the essential oil was submitted to a silica gel chromatographic column, eluted with hexane and monitored by TLC, resulting in the purification of compound 2 (80.3 mg), the major compound present in the essential oil.
Compound 2
Oil; [α]D20 +136° (c 1.0, CHCl3); HRMS (ESI (+)-QToF) m/z, calcd. for C15H20O [M + H]+: 216.1560; 1H NMR (300 MHz, CDCl3) d 7.0408 (1H, bs, H-2), 5.3882 (1H, bs, H-9), 3.2447 (1H, bs, H-9a), 2.6484 (1H, dd, J 5.34 and 16.11 Hz, H-4), 2.2331-2.1486 (1H, m, H-4’), 1.9708-1.904 (3H, m, H-5, H-6 and H-7), 1.6554-1.6439 (5H, m, H-5a, H-6’ and H-13), 2.0215 (3H, d, J 1.2 Hz, H-11) and 1.0567 (3H, d, J 6.66 Hz, H-12); 13C NMR (75 MHz, CDCl3) d 149.98 (C, C-3), 137.40 (CH, C-2), 133.15 (C, C-8), 123.06 (CH, C-9), 120.28 (C, C-10), 119.53 (C, C-1), 38.40 (CH, C-5a), 33.60 (CH, C-9a), 31.19 (CH2, C-4, C-4’), 27.99 (CH, C-5), 26.62 (CH2, C-7), 25.04 (CH2, C-6, C-6’), 23.73 (CH3, C-13), 19.23 (CH3, C-12), 8.87 (CH3, C-11).
Computational methods
Energy minimization of conformers
Initial information regarding the relative stability of the conformers, particularly with respect to rings B and C (Figure 2), was obtained using starting 3D structures from the Marvin Sketch34 program for all possible isomers. Given the 3 chiral carbons, the 8 possible combinations (isomers) were named combining letters R or S in the sequence for atoms 5, 5a and 9a. These isomers had their structures optimized with program Gamess35 with the 6-31G(d) basis set and the B3LYP functional. Then, all possible ring conformation combinations for each isomer were prepared, resulting in 8 isomers and 16 different combined ring conformations = 128 starting enantiomers/conformers. Each one was further optimized and had its Gibbs energy evaluated within the ECD spectra calculation step, so that the stable conformations were then analyzed for reasonable populations (according to the Boltzmann distribution), and later submitted to specific programs to calculate other properties. We found that the 128 combinations converged to only 25 different combinations of enatiomer/conformation, conformations evaluated according to Cremer and Pople ring parameters36 with the CONFORMA37 program, these combinations were submitted to a cut for a significant minimum relative population (> 5%), which decreased them to the number of 12.
Absolute stereochemistry of itapejarafuran 2 according to the measured data and analyses by quantum chemistry simulations
Calculation of the electronic circular dichroism spectra and Gibbs energy of compound 2
Each of the 12 structures was then optimized again with the Orca quantum chemistry program38 with the def2-TZVP(-f) basis set and the WB97X-D3 functional, using the cpcm solvation model for ethanol.39 Also, the Gibbs free energy was estimated in this step to allow the determination of the relative population of the conformers within each enantiomer. Then, the Orca program was employed to perform time-dependent density functional theory (DFT) calculations on verboccidentafuran conformers, set to seek for 30 roots. The resulting data were processed using the Multiwfn program40 to generate the calculated spectra. Grace41 was used to plot the calculated spectra for the 8 enantiomers against experimental data.
Calculation of the optical rotation of compound 2 at 589 nm
The 12 remaining structures were also optimized with the Dalton42 quantum chemistry program with the 6-311++G(2d,2p) basis set and the B3LYP functional, using the empirical dispersion correction DFT-D3.43 The OR was estimated at 589 nm; values were obtained for calculations with London atomic orbitals. No significant difference was observed between the conformers (that account for ca. 100% population) of any specific enantiomer.
Calculation of the nuclear magnetic resonance spectra of compound 2
The 12 remaining structures were also optimized with the Orca38 quantum chemistry program, with the PCSSEG-2 AUTOAUX basis set and the B3LYP44 functional in chloroform (cpcm model).39 The hydrogen (1H NMR) spectra were estimated with the same program and conditions. Comparisons between the estimated spectra to the experimental values were performed with diastereomeric parameter 4 plus (DP4+) probability analysis with unscaled shifts.29 Plot of the spectrum was performed with program Multiwfn 3.8.40
In vitro biological activity of compound 2
Cell culture
For the in vitro cytotoxicity assays,45 a human pancreatic carcinoma cell line (MIA PaCa-2, CRL-1420) obtained from the American Type Culture Collection (Manassas, VA, USA) was used. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% (v/v) fetal bovine serum (Life Technologies, USA), 2 mM glutamine, 100 IU mL-1 penicillin, 100 μg mL-1 streptomycin, and 25 mM of 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) (pH 7.2). Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO2.
Cytotoxicity assays
The cytotoxicity assay was performed in MIA PaCa-2 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay.46 The cells (1 × 105 cells mL-1) were seeded in 96 well plates and incubated for 24 h with increasing concentrations of compound 2 (1-1000 µg mL-1). Each concentration was analyzed in sextuplicate wells within a single independent experiment. After treatment, 10 µL of MTT solution (5 mg mL-1) was added to each well and incubated for 4 h. The medium was removed, and 100 µL of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 570 nm with background correction at 630 nm using an Asys Expert Plus Microplate Reader (Biochrom Ltd., UK). Corrected absorbance values (570-630 nm) were expressed as percentage of viable cells relative to the untreated control group. Data are presented as mean ± standard deviation (SD) of six technical replicates. The half-maximal inhibitory concentration (IC50) values were determined by nonlinear regression using a four-parameter logistic model in GraphPad Prism 8.0 software.46 The Hill slope was constrained to -1.5 to improve parameter stability during curve fitting. Model fitting was performed using least-squares regression. The goodness of fit was evaluated by the coefficient of determination (R2). The 95% confidence intervals (95% CI) for IC50 were calculated using the profile likelihood method implemented in the software.
RESULTS AND DISCUSSION
Structural determination of compound 2
Compound 2 was obtained as an optically active, colorless oil with a specific rotation value of [α]D20 +136° (c 1.0, CHCl3). The chemical formula was suggested as C15H20O based on the molecular ion peak [M + H]+ at m/z 216.1560, as determined by high-resolution mass spectrometry coupled with electrospray ionization-quadrupole time-of-flight (HRMS (ESI-(+)-QToF)) analysis. The corresponding spectrum is depicted in Figure 1S (Supplementary Material).
The NMR spectroscopic data of compound 2 were assigned based upon the analysis of the mono 1H and 13C spectra (Table 1), and two-dimensional correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), heteronuclear multiple quantum coherence (HMBC) and nuclear overhauser and exchange spectroscopy (NOESY) spectra. The corresponding spectra are provided in the Supplementary Material (Figures 2S to 7S).
The 1H NMR spectrum showed signals at dH 2.6484 (dd, J 5.34, 16.11 Hz) and at dH 2.2331-2.1486 (m) that refer to the diastereotopic hydrogens H-4 and H-4’ and three signals at dH 2.0215 (d, J 1.2 Hz), 1.0567 (d, J 6.6 Hz) and 1.6554-1.6439 (m), assigned to the methyl hydrogens H-11, H-12 and H-13. At dH 3.2447, 7.0408 and 5.3882, three broad singlet signals were observed, which refer to the methinic hydrogens H-9a and H-2 of the furanic ring and the olefinic hydrogen (H-9). The 13C NMR spectrum indicated the presence of thirteen signals associated with three methyl carbons, three methylenic carbons, five methine carbons and four non-hydrogenated carbons. At dC 38.40 and 33.60, two signals were observed for the methine carbons C-5a and C-9a, at dC 123.06 and 133.15, signals for the olefinic carbons C-8 and C-9 and at dC 137.40, for the methine carbon (C-2) of the furanic ring. These assignments are consistent with a furanocadinene-type structure.
The stereochemistry of the chiral centers could be inferred from the analysis of the 1H-1H COSY and NOESY spectra. The 1H 1H COSY spectrum showed couplings between the signals dH 1.0567 (H-12) and dH 1.9708-1.9014 (H-5), dH 1.6554-1.6439 (H-5a) and dH 1.9708-1.9014 (H-5), dH 1.6554-1.6439 (H-5a) and dH 3.2447 (H-9a), and between dH 5.3882 (H-9) and dH 3.2447 (H 9a). The relative stereochemistry of the structure can be attributed by NMR, initially based on the NOE correlations of H-9a. According to the 1Hx1H NOESY spectrum, H-9a correlates with the hydrogens 5, 5a and 9. Once the fused rings are derived from the cis-decaline, the observation of NOE between H-9a and H-5a indicates that both hydrogens are oriented in a cis relationship. Furthermore, given that the structure is sufficiently rigid to restrict conformational exchange, no alternative spatial proximity would account for these NOE interactions. Another important factor is the scalar coupling constant between H-9a and H-5a. Although the H-9a signal is not well resolved, not providing the J values, the half-line width, W, can be measured. Since W corresponds to the sum of all J couplings of the aforementioned hydrogen, W for 9a corresponds to the sum of 3JH-H of 9a with 5a and 9. A value in the magnitude of 10.6 Hz indicates, in this case, excluding the vinylic coupling, a cis-like orientation of the hydrogens 5a and 9a.47 Analysis of the NMR spectra were consistent with the structure of compound verboccidentafuran 1 previously isolated by Bohlmann and Lonitz,14 and González.6
Structural optimization and energy calculation of conformers for compound 2
According to the Gibbs energies, for any isomer, estimated by the Orca program, either one or at most two conformers accounted for nearly 100% population of the studied compound, at room temperature. Table 2 presents the Cremer and Pople parameters36 for each ring and the respective estimated population proportion in each relevant case. The three-letter acronyms for each isomer refer to the S or R conformation for atoms 5, 5a and 9a (Figure 2), respectively. Their 3D structures, superposed and drawn with the program PyMOL,48 are depicted in Figure 8S (Supplementary Material).
Cremer and Pople parameters for each ring and the respective estimated population proportion for relevant conformers of each isomer
Absolute stereochemistry of compound 2
Electronic circular dichroism (ECD)
Figure 3 compares observed and representative calculated ECD spectra for all enantiomers, considering the relative population of respective conformers. The negative peak at 209 nm could be reasonably predicted by RRR, RSR, RSS and SSR (all in shades of blue), especially the last three, while the other enantiomers (all in shades of red) lead to a positive peak at this region. Nevertheless, we were unable to reproduce the lower positive peak at 237 nm, even after trying different methods or basis sets within the quantum chemistry program. Although difficulties in precisely estimating ECD spectra have been pointed out,49 the results can nevertheless be useful to corroborate enantiomer determination, preferably with the use of a second experimental method comparing calculated values. Therefore, OR measurements were obtained.
Comparison of experimental ECD spectra (black line) with calculated ones (see inset legend)
Optical rotation at 589 nm
The measured value for [α]D20 at 589 nm was +136.0° / [dm (g cm 3)]. The Dalton program estimated this rotation to be approximately +68, -82, +78, -153, +153, -78, +82 and -67° / [dm (g cm-3)], for isomers RRR, RRS, RSR, RSS, SRR, SRS, SSR and SSS, respectively, considering the most abundant conformers (ca. 100% population when applicable). Clearly, the values calculated by OR, compared to the experimental values, confirm that the enantiomers RRR, RSR, SRR, and SSR are the correct ones for the compound; the comparison with the ECD spectrum calculations converges to RSR and SSR. NMR analyses were carried out to further enforce the determination of the enantiomer.
Nuclear magnetic resonance calculations
The simulated NMR spectra were calculated for each isomer/conformer(s) and combined between conformers when needed. The resulting data were submitted to DP4+ analysis.28Table 3 shows the estimated probability of each isomer according to the spectral data.
NMR measurements pointed the highest probability for a single isomer; this result, combined with ECD and OR analyses, indicates that compound 2 obtained from our sample of essential oil corresponds to the RSR isomer, named itapejarafuran, (5R,5aS,9aR)-4,5,5a,6,7,9a-hexahydro-1,5,8-trimethylnaphtho[2,1-b]furan. Its corresponding calculated spectrum is shown in Figure 9S (Supplementary Material).
Biological activity of compound 2
The cytotoxicity analysis of compound 2 demonstrated a dose-dependent reduction in MIA PaCa-2 cell viability (Figure 4). Nonlinear regression analysis using a four-parameter logistic model with a constrained Hill slope (-1.5) yielded an IC50 value of 150 µM, with a 95% confidence interval (86.65-258.1 µM). The goodness of fit was acceptable (R2 = 0.897), indicating an adequate model adjustment under the applied constraints. Data are presented as mean ± SD of six technical replicates. Compound 2 exhibited moderate cytotoxic activity against the MIA PaCa-2 cells line.
Dose-dependent cell viability curve of MIA PaCa-2 cells treated with itapejarafuran for 24 h. Nonlinear regression analysis using a four-parameter logistic model with a constrained Hill slope (-1.5) yielded an IC50 value of 150 µM (95% CI: 86.65-258.1 µM; R2 = 0.897). Each point represents the mean ± standard deviation of six technical replicates from a single experiment
For contextual comparison pusposes, literature data33 indicate that classical chemotherapeutic agents, such as doxorubicin and gemcitabine, typically exhibit IC50 values in the nanomolar to low micromolar range in MIA PaCa-2 cells (e.g., doxorubicin with an IC50 of 55 nM and gemcitabine with an IC50 of 5 nM). Although compound 2 shows lower potency compared to these established chemotherapeutic agents, it is important to emphasize that the present study focused on the identification and initial biological characterization of a newly isolated molecule rather than direct comparison with standard drugs. Moreover, considering the intrinsic chemoresistance and limited therapeutic responsiveness of this pancreatic cancer model, the moderate, yet clearly dose-dependent cytotoxic effect observed for compound 2 supports its potential as a lead structure and justifies further pharmacological and mechanistic investigations.33
Earlier investigations involving B. punctulata have described anti-inflammatory activity8 and, in certain cellular models, the inhibition of inflammatory pathways that may be associated with cancer.31,32 Our findings suggest that itapejarafuran may be one of the bioactive constituents contributing to these effects. In our model, the observed cytotoxic activity provides an encouraging indication that this compound merits further investigation, particularly regarding its potential as a chemotherapeutic candidate, although with moderate potency compared to reference drugs.
CONCLUSIONS
This study reports the isolation and defines, for the first time, the absolute configuration of the compound 2 named itapejarafuran, (5R,5aS,9aR)-4,5,5a,6,7,9a-hexahydro-1,5,8-trimethylnaphtho[2,1-b]furan from B. punctulata. Comparing the measured ECD spectra and OR measurement data with the simulated values, there was a strong indication that the product obtained is the enantiomer RSR. This was further confirmed with NMR data and its quantum chemistry simulations. The stereochemistry definition of such active compounds must be taken into consideration for a valid analysis.
In addition, cytotoxicity assay of itapejarafuran was evaluated and cytotoxic activity against MIA PaCa-2 cells was observed. Further studies are needed to elucidate the biological mechanisms involved and to determine how the chemical characteristics of itapejarafuran relate to its observed biological activity and potential therapeutic applications, thereby representing a promising avenue for future research in the development of potential chemotherapeutic candidates.
SUPPLEMENTARY MATERIAL
Complementary material for this work (NMR and HRMS (ESI (+) QToF) spectra) is available at http://quimicanova.sbq.org.br/, as a PDF file, with free access.
ACKNOWLEDGMENTS
The authors express their gratitude to Mr. Vicanor M. Ascari (in memoriam) for his help on Baccharis punctulata collections and CNPq and Fundação Araucária for fellowships at UTFPR.
DATA AVAILABILITY STATEMENT
The authors confirm that all data generated or analyzed is available in the text.
REFERENCES
-
1 Fernandez, E. C.; Sandi, Y. E.; Kokoska, L.; Fitoterapia 2003, 74, 407. [Crossref]
» Crossref -
2 Heiden, G.; Baumgratz, J. F. A.; Esteves, R. L.; Rodriguesia 2012, 63, 649. [Crossref]
» Crossref -
3 Schossler, P.; Schneider, G. L.; Wunsch, D.; Soares, G. L. G.; Zini, C. A.; J. Braz. Chem. Soc. 2009, 20, 277. [Crossref]
» Crossref -
4 Minteguiaga, M.; González, A.; Cassel, E.; Umpierrez, N.; Fariña, L.; Dellacassa, E.; Chem. Biodiversity 2018, 15, e1800017. [Crossref]
» Crossref -
5 Budel, J. M.; Wang, M.; Raman, V.; Zhao, J.; Khan, S. I.; Rehman, J. U.; Techen, N.; Tekwani, B.; Monteiro, L. M.; Heiden, G.; Takeda, I. J. M.; Farago, P. V.; Khan, I. A.; Molecules 2018, 23, 2620. [Crossref]
» Crossref -
6 González, M. D.; J. Essent. Oil Res. 2019, 31, 573. [Crossref]
» Crossref -
7 González, M. D.; J. Essent. Oil Res. 2022, 35, 197. [Crossref]
» Crossref -
8 Ascari, J.; de Oliveira, M. S.; Nunes, D. S.; Granato, D.; Scharf, D. R.; Simionatto, E.; Otuki, M.; Soley, B.; Heiden, G.; J. Ethnopharmacol. 2019, 234, 7. [Crossref]
» Crossref -
9 Ascari, J.; de Oliveira, M. S.; Rüdiger, A. L.; Elisabetsky, E.; Nunes, D. S.; Iulek, J.; Scharf, D. R.; Heiden, G.; Quim. Nova 2025, 48, 7. [Crossref]
» Crossref -
10 Zdero, C.; Bohlmann, F.; King, R. M.; Robinson, H.; Phytochemistry 1986, 25, 2841. [Crossref]
» Crossref -
11 Loayza, I.; Abujder, D.; Aranda, R.; Jakupovic, J.; Collin, G.; Deslauriers, H.; Jean, F. I.; Phytochemistry 1995, 38, 381. [Crossref]
» Crossref -
12 Malizia, R. A.; Cardell, D. A.; Molli, J. S.; González, S.; Guerra, P. E.; Grau, R. J.; J. Essent. Oil Res. 2005, 17, 194. [Crossref]
» Crossref -
13 Jakupovic, J.; Schuster, A.; Ganzer, U.; Bohlmann, F.; Boldt, P. E.; Phytochemistry 1990, 29, 2217. [Crossref]
» Crossref -
14 Bohlmann, F.; Lonitz, M.; Phytochemistry 1978, 17, 453. [Crossref]
» Crossref -
15 de Souza, T. J. T.; Bordignon, S. A. L.; Apel, M. A.; J. Nat. Prod. 2017, 80, 45. [Crossref]
» Crossref -
16 de Souza, T. J. T.; Bordignon, S. A. L.; Apel, M. A.; Henriques, A. T.; Phytochemistry 2021, 186, 112734. [Crossref]
» Crossref -
17 Murakami, C.; Lago, J. H. G.; Perazzo, F. F.; Ferreira, K. S.; Lima, M. E. L.; Moreno, P. R. H.; Young, M. C. M.; Chem. Biodiversity 2013, 10, 621. [Crossref]
» Crossref -
18 Bohlmann, F.; Zdero, C.; King, R. M.; Robinson, H.; Phytochemistry 1979, 18, 1177. [Crossref]
» Crossref -
19 Bohlmann, F.; Singh, P.; Jakupovic, J.; King, R. M.; Robinson, H.; Phytochemistry 1982, 21, 371. [Crossref]
» Crossref -
20 Zdero, C.; Bohlmann, F.; King, R. M.; Phytochemistry 1991, 30, 2991. [Crossref]
» Crossref -
21 Bohlmann, F.; Trantow, T.; Liebigs Ann. Chem. 1983, 1983, 1689. [Crossref]
» Crossref -
22 Galbiati, A.; Zana, A.; Borsari, C.; Persico, M.; Bova, S.; Tkachuk, O.; Corfu, A. I.; Tamborini, L.; Basilico, N.; Fattorusso, C.; Bruno, S.; Parapini, S.; Conti, P.; Molecules 2023, 28, 3172. [Crossref]
» Crossref -
23 Reddy, C. N.; Eedara, A. C.; Malik, S.; Mondhe, D. M.; Bharate, S. B.; Andugulapati, S. B.; Bioorg. Chem. 2025, 157, 108262. [Crossref]
» Crossref -
24 Huo, Z.; Zhu, F.; Zhang, X.; Zhang, X.; Liang, H.; Yao, J.; Liu, Z.; Zhang, G.; Yao, Q.; Qin, G.; Mar. Drugs 2022, 20, 333. [Crossref]
» Crossref -
25 Zhuanga, Y.; Yang, F.; Menona, A.; Song, J. M.; Espinoza, R. V.; Schultz, P. J.; Garner, A. L.; Tripathi, A.; J. Nat. Prod. 2023, 86, 1801. [Crossref]
» Crossref -
26 Zeb, M. A.; Zhou, X.; Wang, M.; Kong, Y.; Zhang, X.; Ni, D.; Tu, W.; Li, X.; Jiang, K.; Xiao, W.; J. Mol. Struct. 2025, 1328, 141291. [Crossref]
» Crossref -
27 Zanardi, M. M.; Sarotti, A. M.; J. Org. Chem. 2021, 86, 8544. [Crossref]
» Crossref -
28 Marcarino, M. O.; Cicetti, S.; Zanardi, M. M.; Sarotti, A. M.; Nat. Prod. Rep. 2022, 39, 58. [Crossref]
» Crossref -
29 Grimblat, N.; Zanardi, M. M.; Sarotti, A. M.; J. Org. Chem. 2015, 80, 12526. [Crossref]
» Crossref -
30 Ermanis, K.; Parkes, K. E. B.; Agbackb, T.; Goodman, J. M.; Org. Biomol. Chem. 2016, 14, 3943. [Crossref]
» Crossref -
31 Burgos, C.; Alfonso, L.; Ferro, E.; Langjahr, P.; Revista Paraguaya de Reumatologia 2022, 8, 45. [Crossref]
» Crossref -
32 Burgos, C.; Alvarenga, N.; Heiderich, H.; Florentín-Pavía, M.; Sotelo, P. H.; Carpinelli, M. M.; Giménez, V.; Langjahr, P.; Trop. J. Nat. Prod. Res. 2021, 5, 1055. [Crossref]
» Crossref -
33 Brugiapaglia, S.; Spagnolo, F.; Curcio, C.; Biomolecules 2025, 15, 725. [Crossref]
» Crossref - 34 Marvin Sketch, version 18.5; ChemAxon, Hungary, 1998.
-
35 Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Elbert, S. T.; Gordon, M. S.; Jensen, J. H.; Koseki, S.; Matsunaga, N.; Nguyen, K. A.; Su, S.; Windus, T. L.; Dupuis, M.; Montgomery Junior, J. A.; J. Comput. Chem. 1993, 14, 1347. [Crossref]
» Crossref -
36 Cremer, D.; People, J. A.; J. Am. Chem. Soc. 1975, 97, 1354. [Crossref]
» Crossref -
37 Iulek, J.; Zukerman-Schpector, J.; Quim. Nova 1997, 20, 433. [Crossref]
» Crossref -
38 Neese, F.; WIREs Computational Molecular Science 2018, 8, e1327 [Crossref]; Neese, F.; WIREs Computational Molecular Science 2012, 2, 73. [Crossref]
» Crossref» Crossref -
39 Barone, V.; Cossi, M.; J. Phys. Chem. A 1998, 102, 1995. [Crossref]
» Crossref -
40 Lu, T.; Chen, F.; J. Comput. Chem. 2012, 33, 580. [Crossref]
» Crossref - 41 Grace, WYSIWYG 2D plotting tool, version 5.1.22; Grace Development Team, Weizmann Institute of Science, Rehovot, 2024.
-
42 Aidas, K.; Angeli, C.; Bak, K. L.; Bakken, V.; Bast, R.; Boman, L.; Christiansen, O.; Cimiraglia, R.; Coriani, S.; Dahle, P.; Dalskov, E. K.; Ekstrom, U.; Enevoldsen, T.; Eriksen, J. J.; Ettenhuber, P.; Fernandez, B.; Ferrighi, L.; Fliegl, H.; Frediani, L.; Hald, K.; Halkier, A.; Hattig, C.; Heiberg, H.; Helgaker, T.; Hennum, A. C.; Hettema, H.; Hjertenæs, E.; Høst, S.; Høyvik, I.; Iozzi, M. F.; Jansık, B.; Jensen, H. J.; Jonsson, D.; Jørgensen, P.; Kauczor, J.; Kirpekar, S.; Kjærgaard, T.; Klopper, W.; Knecht, S.; Kobayashi, R.; Koch, H.; Kongsted, J.; Krapp, A.; Kristensen, K.; Ligabue, A.; Lutnæs, O. B.; Melo, J. I.; Mikkelsen, K. V.; Myhre, R. H.; Neiss, C.; Nielsen, C. B.; Norman, P.; Olsen, J.; Olsen, J. M. H.; Osted, A.; Packer, M. J.; Pawlowski, F.; Pedersen, T. B.; Provasi, P. F.; Reine, S.; Rinkevicius, Z.; Ruden, T. A.; Ruud, K.; Rybkin, V. V.; Sałek, P.; Samson, C. C. M.; de Meras, A. S.; Saue, T.; Sauer, S. P. A.; Schimmelpfennig, B.; Sneskov, K.; Steindal, A. H.; Sylvester-Hvid, K. O.; Taylor, P. R.; Teale A. M.; Tellgren, E. I.; Tew, D. P.; Thorvaldsen, A. J.; Thøgersen, L.; Vahtras, O.; Watson, M. A.; Wilson, D. J. D.; Ziolkowski, M.; Agren, H., WIREs Computational Molecular Science 2014, 4, 269. [Crossref]
» Crossref -
43 Grimme, S.; Antony, J.; Ehrlich, S.; Krieg, H.; J. Chem. Phys. 2010, 132, 154104. [Crossref]
» Crossref -
44 Stephens, P. J.; Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J.; J. Phys. Chem. 1994, 98, 11623. [Crossref]
» Crossref -
45 Gradiz, R.; Silva, H. C.; Carvalho, L.; Botelho, M. F.; Mota-Pinto, A.; Sci. Rep. 2016, 6, 21648. [Crossref]
» Crossref -
46 Levy, D.; Ruiz, J. L. M.; Celestino, A. T.; Silva, S. F.; Ferreira, A. K.; Isaac, C.; Bydlowski, S. P.; Biochem. Biophys. Res. Commun. 2014, 446, 720 [Crossref]; GraphPad Prism, version 8.0; GraphPad Software, San Diego, CA, USA, 2018.
» Crossref -
47 Dodziuk, H.; Jaszunski, M.; Schilf, W.; Magn. Reson. Chem. 2005, 43, 646. [Crossref]
» Crossref - 48 PyMOL, version 3.0.0; Schrödinger, LLC, USA, 2026.
-
49 Mazzeo, G.; Cimmino, A.; Masi, M.; Longhi, G.; Maddau, L.; Memo, M.; Evidente, A.; Abbate S.; J. Nat. Prod. 2017, 80, 2406. [Crossref]
» Crossref
Edited by
-
Associate Editor handled this article:
Cristiano Raminelli
