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Abstract

Six compounds previously isolated and reported from Macropidia fuliginosa were evaluated for antimicrobial activity against 11 different microbial strains, with all compounds displaying broad spectrum activity. In addition, the compounds were also assessed for their antiproliferative activity and cytotoxicity. Further investigation of M. fuliginosa has led to the discovery of a previously described acenaphthylenediol from the bulbs of the plant.

Keywords

HPLC-NMR biological testing antibacterial bioactivity phenolics

In recent years, the Marine and Terrestrial Natural Product (MATNAP) research group has been extensively investigating the chemistry of plants that belong to the Haemodoraceae family.^1

-4 As part of our continuing efforts to chemically profile Australian plants derived from this family, Macropidia fuliginosa (Hook.) Druce, commonly known as the Black Kangaroo paw which is an endemic species of Western Australia, was recently investigated, with 16 compounds being reported from the bulbs and flowers of the plant.⁵ Antibiotic assessment of the isolated compounds showed that some were active against both Gram-positive and Gram-negative bacteria.⁵ Among the compounds isolated in the original study of the plant were anigorufone (1), anigopreissin A (2), 2-phenylnaphthalic anhydride (3), rutin (4), fuliginosin A (5), and fuliginosone (6) (Figure 1). Four of these compounds (2, 3, 5, and 6) had previously displayed some antibiotic properties.

Figure 1

Compounds isolated from Macropidia fuliginosa.

These 6 compounds were selected for further evaluation of their antibiotic and antifungal against a selection of microorganisms, including Gram-positive and Gram-negative bacteria and against various fungi. The activity of the isolated compounds was compared to standard antibiotic and antifungal compounds. The antiproliferative effect and cytotoxicity of the compounds were also evaluated.

Further investigation of the plant has subsequently identified the presence of a previously described acenaphthylenediol derivative from an enriched fraction of the bulbs of the plant.

Results and Discussion

Antimicrobial evaluation of the compounds was carried out against 6 bacterial strains (3 Gram-positive and 3 Gram-negative), and 5 fungal strains, at a concentration of 1 mg/mL.

All 6 natural products evaluated showed a range of activity across all microorganisms tested, particularly across the bacteria. The results of the antimicrobial assays are provided in Table 1, whereby the zones of inhibition are quoted as the mean diameter in mm of the clear inhibition zone after 24 hours of incubation. The positive controls were ampicillin and ciprofloxacin (antibiotic positive controls), as well as amphotericin B and carbendazim (antifungal positive control).

Table 1

Antimicrobial Activity of Compounds (1-6) Isolated From Macropidia fuliginosa.

Compound^a	Bacteria									Fungi
Compound^a	Bacillus subtilis (6633 B1)	Staphylococcus aureus (511 B3)	Staphylococcus aureus MRSA (134/93 R9)	Escherichia coli (458 B4)	Pseudomonas aeruginosa (SG 137 B7)	Pseudomonas aeruginosa (K 799/61 B9)	Enterococcus faecalis VRE(1528 R10)	Mycobacterium vaccae (10 670 M4)	Sporobolomyces salmonicolor (549 H4)	Candida albicans (C. alb. H8)	Penicillium notatum (JP36 P1)
Anigorufone (1)	13^b	12	10/16^b	15^b	17^b	0	0	17^c	21^c	13	16^c
Anigopreissin A (2)	21	23	23	15^b	0	15/20^c	20/23^b,d	30	22^c	12/15^b	12/17^c
2-Phenylnaphthalic anhydride (3)	12^b	NT	NT	14^b	NT	NT	NT	NT	NT	NT	NT
Rutin (4)	12^b	0	0	14^b	0	0	0	0	20^c	0	14^b
Fuliginosin A (5)	13	12/15^b	11/13^b	14^b	0	0	13^d	20^c	20^c	0	0
Fuliginosone (6)	12^b	13^b	0	14^b	NT	0	0	12^c	22^c	0	15^c
Ampicillin (antibiotic positive control)	35/38^c	38/50^b	14^c	36/40^c/44^b	0	36/44^c	34/38^c	23^c	0	0	0
Carbendazim (antifungal positive control)	0	0	0	0	0	0	14^c	0/e	43^c	13^b	12/56^c
Amphotericin B (antifungal positive control)	0	0	0	0	0	0	0	0	18^c	21	18^c
Ciprofloxacin (antibiotic positive control)	31	21	0	25/36^c	28	28/37^c	18^d	24^c	^cNT	NT	NT
Solvent control—H₂O	0	0	0	0	0	0	0	0	0	0	0
Solvent control—DMSO	11^b	12^b	0	13^b	13^b	14^b	12^c	12^b	13^b	0	13^b

DMSO, dimethyl sulfoxide; MRSA, methicillin-resistant Staphylococcus aureus; NT, not tested; VRE, vancomycin-resistant Enterococci.

^aAll compounds tested at 1 mg/mL concentration (except amphotericin B and ciprofloxacin which were tested at 10 and 5 µg/mL, respectively).

^bIndicates partial inhibition (mm) with lots of resistant colonies (normal size) in the inhibition zone.

^cIndicates partial inhibition (mm) with tiny little colonies within the inhibition zone.

^dIndicates growth promotion (mm) around the inhibition zone.

^eIndicates faint microbial growth (mm).

Interestingly, 2 strains of Pseudomonas aeruginosa tested showed vastly different results for 2 of the compounds (1 and 2). In recent years, P. aeruginosa has been identified as a drug resistant bacteria, highlighting the importance of discovering new drug-leads against this microorganism.⁶ As the results differ between 2 strains tested, this suggests that the mode of action of inhibition of 2 compounds is different against 2 strains. Three of the compounds (1, 2, and 5) tested also showed activity against the methicillin-resistant Staphylococcus aureus (MRSA) strain of S. aureus, another drug resistant bacteria. The least active compound across all strands tested was rutin (4).

Anigopreissin A (2) and fuliginosin A (5) both contain the same core carbon skeleton, and show similar antimicrobial activity. However, anigopreissin A (2) shows larger zones of inhibition and also inhibits additional microorganisms. This clearly indicates that the vinyl phenol moiety present in 2 is responsible for the greater biologically activity observed in comparison to the presence of the formaldehyde moiety in 5 and provides some Structure Activity Relationship insight.

2-Phenylnaphthalic anhydride (3) and fuliginosone (6) are also structurally related analogs. The addition of an oxygen into the “A” ring does not have a large impact on the biological activity. Similarly, anigorufone (1) shares the same “B,” “C,” and “D” ring structure as 2-phenylnaphthalic anhydride (3) and fuliginosone (6), and no significant differences in activity were observed. This again suggests that the differences observed in the A ring all result in similar biological activity.

Compared to the positive controls, some compounds (1, 2, and 5) showed equal or better inhibition against S. aureus MRSA and Mycobacterium vaccae. Inhibition against Candida albicans and Penicillium notatum was shown to be equal or better by some compounds (1, 2, and 6). Among 6 compounds tested in this study, compound 2 appeared to possess the highest potency against M. vaccae, Bacillus subtilis, S. aureus (incl. MRSA), vancomycin-resistant Enterococci (VRE), the yeasts Sporobolomyces salmonicolor (Basidiomycota) and C. albicans (Ascomycota). While compound 2 was more active against P. aeruginosa strain K 799/61 B9, but not strain SG 137 B7, compound 1 was more potent against strain SG 137 B7, but lacked activity against strain K 799/61 B9.

The antiproliferative activity and cytotoxicity of the compounds is given in Table 2. The antiproliferative activity is the ability of a compound to reduce the proliferation of cells by not allowing the cells to multiply rapidly. Cytotoxicity refers to causing harm to cells and thereby killing them. These results indicated that the strongest cytotoxic compound was anigopreissin A (2). This compound also showed, together with anigorufone (1), the strongest antiproliferative activity.

Table 2

Antiproliferative Activity and Cytotoxicity of Compounds Isolated From Macropidia fuliginosa.

Compound	Antiproliferative effect		Cytotoxicity
Compound	HUVECGI₅₀ (µg/mL)	K-562GI₅₀ (µg/mL)	HeLaCC₅₀ (µg/mL)
Anigorufone (1)	10.6 (±1.0)	3.0 (±0.2)	28.6 (±0.5)
Anigopreissin A (2)	7.4 (±0.2)	7.8 (±0.2)	13.9 (±0.06)
2-Phenylnaphthalic anhydride (3)	29.1 (±1.0)	48.6 (±1.8)	>50
Rutin (4)	>50	>50	>50
Fuliginosin A (5)	11.9 (±0.8)	22.9 (±0.5)	40.8 (±1.2)
Fuliginosone (6)	14.1 (±1.8)	>50	46.2 (±1.3)
Imatinib	10.9 (±1.2)	0.1 (±6.7 × 10⁻³)	-
Doxorubicin	0.1	1.0 (±0.6)	-

Further investigation of an enriched nonpolar fraction derived from the bulbs of M. fuliginosa (see the “Experimental” section) yielded a previously described acenaphthylenediol derivative (7). The natural product 3-phenyl-1,2-dihydroacenaphthylen-1,2-diol (7) had previously been identified from another plant belonging to the Haemodoraceae family in which M. fuliginosa is also a member. In the original paper, the relative configuration of 7 was determined on the basis of the observed coupling constants of the protons at positions 1 and 2.⁷ It has been documented that in cis-substituted acenaphthenes, the protons display coupling constants of 6.2 Hz, while trans-substituted acenaphthenes display coupling constants of 1.2 Hz.⁸ Similarly, in this study, a coupling constant of 5.5 Hz was observed for 7, confirming a cis geometry for the position 1 and 2 methines. Compound 7 was not evaluated for biological activity in this study due to the lack of material.

In the original assignment of the Nuclear Magnetic Resonance (NMR) data of 7, it became evident that some mis-assignments of the chemical shifts had been made (see Supplemental Tables S1-S3). In the original reporting of 7, Heteronuclear Multiple Quantum Coherence (HMQC) NMR experiments were carried out, but none of these data were provided in the original publication.⁷ Heteronuclear Multiple Bond Coherence (HMBC) NMR experiments were also conducted on 7 and these data were provided; however, some of the 2-bond and 3-bond correlations had been mis-assigned (ie, a 2-bond correlation was reported as a 3-bond correlation, or vice versa)⁷ (see Supplemental Material S3). The total number and magnitude of NMR shifts reported for both the ¹H and ¹³C NMR assignments for 7 are consistent with the data secured herein (see Supplementary Materials S1 and S2), but the assignments of these chemical shifts to the structure of 7 are not. Subsequently, the assignments for H-1, H-2, H-7, H-8, H-3′, H-4′, and H-5′ have been revised. The assignments for the carbons at positions C-2a, C-3, C-5a, C-8, C-8a, C-1′, C-2′, C-3′, C-4′, C-5′, and C-6′ have also been revised. Comparison of the literature with data obtained in this study is provided in the supporting information file (Supplemental Tables S1-S3). The ¹H, gradient correlation spectroscopy (gCOSY), heteronuclear single-quantum correlation spectroscopy with adiabatic pulses (HSQCAD), and gradient heteronuclear multiple-bond spectroscopy with adiabatic pulses (gHMBCAD) NMR data obtained in this study are also provided in the supporting information file (Supplemental Tables S4-S7).

Conclusion

Compounds isolated from M. fuliginosa showed broad spectrum activity against both the bacteria and fungi tested. Four of the compounds showed equal or better inhibition against various microorganisms, including resistant strains of bacteria. The compounds also showed promising antiproliferative activity.

Experimental

General Experimental Conditions

¹H (500 MHz) and ¹³C (125 MHz) NMR spectra were acquired in CDCl₃ on a 500 MHz Agilent DD2 NMR spectrometer with reference to solvent signals (δ_H 7.26; δ_C 77.0). Two-dimensional NMR experiments recorded included gCOSY, HSQCAD, and gHMBCAD. Silica gel flash chromatography was carried out using Davisil LC35Å silica gel (40-60 mesh) with a 20% stepwise solvent elution from 100% petroleum spirits (60°C-80°C) to 100% CH₂Cl₂ to 100% EtOAc and finally to 100% MeOH. All analytical High Pressure Liquid Chromatography (HPLC) analyses and method development were performed on a Dionex P680 solvent delivery system equipped with a PDA100 UV detector (operated using “Chromeleon” software). Analytical HPLC analyses were carried out using either a gradient method (0-2 minutes 10% CH₃CN/H₂O; 14-24 minutes 75% CH₃CN/H₂O; 26-30 minutes 100% CH₃CN; and 32-40 minutes 10% CH₃CN/H₂O or an isocratic method (70% CH₃CN/H₂O) on an Alltech Alltima HP C18 (250 × 4.6) 5 µm column at a flow rate of 1.0 mL/min. Semipreparative HPLC was carried out on a Varian Prostar 210 solvent delivery system equipped with a Prostar 335 PDA detector (operated using “Star Workstation” software) using an isocratic method (70% CH₃CN/H₂O) using an Alltech Alltima C18 (250 × 10) 5 µm column at a flow rate of 3.5 mL/min. Mass Spectrometry (MS) for pure compounds was carried out on a Perkin Elmer AxION 2 TOF.

Previously Isolated Natural Products for Biological Evaluation

Compounds 1 to 6 that were evaluated for antimicrobial activity were previously isolated and identified in the previous study by Brkljača et al.⁵ The original specimens of M. fuliginosa (Hook.) Druce (Haeomodoracae) were purchased from the Kuranga Native Nursery (Mount Evelyn, Victoria, Australia) and from Gardenworld Nursery (Braeside, Victoria, Australia) in January 2011, October 2012, and November 2012. The bulbs of the plants purchased in 2011 and 2012 were used in this study, while the flowers were harvested from the plants purchased in 2012. The bulbs and flowers were separated and then stored at −80°C until extraction. Voucher specimens were designated the code numbers: 2011-01a (bulbs), 2011-02a (bulbs), 2012-01a (bulbs), and 2012_05a (flowers), respectively, and are deposited in the School of Applied Sciences (Discipline of Applied Chemistry), RMIT University.

Current Natural Products Isolated

The crude extract was decanted and concentrated under reduced pressure and sequentially solvent partitioned (triturated) into CH₂Cl₂ and MeOH soluble extracts, respectively. The CH₂Cl₂ extract was subjected to flash silica gel column chromatography (20% stepwise elution from petroleum ether (60°C-80°C) to CH₂Cl₂ to EtOAc and, finally, to MeOH). The 100% CH₂Cl₂ fraction was subjected to Sephadex LH-20 column chromatography (100% MeOH) to yield 4 fractions. The first fraction was subjected to Reversed Phase - High Pressure Liquid Chromatography (RP-HPLC) (70% CH₃CN/H₂O) to yield compound (7).

Antimicrobial Assays

To determine their biological activities, anigorufone (1), anigopreissin A (2), 2-phenylnaphthalic anhydride (3), rutin (4), fuliginosin A (5), and fuliginosone (6) were dissolved in dimethyl sulfoxide (DMSO) at a concentration 1 mg/mL. Antibacterial bioassays were performed as previously published⁹ using B. subtilis (strain JMRC:STI:10880), S. aureus (strains JMRC:ST:10 760 and JMRC:ST:33 793 (MRSA)), Escherichia coli (strain JMRC:ST:33699), P. aeruginosa (strains JMRC:ST:33 772 and JMRC:ST:337721), Enterococcus faecalis (strain JMRC:ST:33 700 (VRE)), and M. vaccae (strain JMRC:STI:10670). The bacteria were cultivated on standard I nutrient agar in Petri dishes at 37°C. Antifungal bioassays were conducted at 30°C using the basidiomycetous yeast S. salmonicolor (strain JMRC:ST:35974) and the filamentous ascomycete P. notatum (strain JMRC:STI:50164), which were cultivated on malt agar, and the ascomycetous yeast C. albicans (strain JMRC:STI:25000), which was cultivated on yeast morphology agar. After inoculation, a disc (9 mm in diameter) was removed from the center of the Petri dish and 50 µL (corresponds to 50 µg of pure compound) of the test solution was added to the cavity. After 18 hours of incubation at the respective temperatures, growth inhibition or promotion occurred as a halo around the inoculation hole. The diameter of the inhibition zones was measured (in mm) and documented as given in Table 1. Ampicillin (1 mg/mL in deionized water), ciprofloxacin (5 µg/mL in deionized water) and carbendazim (1 mg/mL in DMSO), amphotericin B (10 µg/mL in DMSO/MeOH 1:1) were used as the antibacterial and antifungal reference compounds, respectively.

Antiproliferative Assays

Cell proliferation and cytotoxicity assays are applied to assess the efficacy and potency of natural products. The following cell lines and cell culture mediums were used: HUVEC (ATCC CRL-1730) and Dulbecco′s Modified Eagle′s Medium (DMEM) (CAMBREX 12-614F); K-562 (DSM ACC 10) and RPMI 1640 (CAMBREX 12-167F); and HeLa (DSM ACC 57) and RPMI 1640 (CAMBREX 12-167F).

Cells were grown in the appropriate cell culture medium supplemented with 10 mL/L ultraglutamine 1 (CAMBREX 17-605E/U1), 550 µL/L (50 mg/mL) gentamicin sulfate (CAMBREX 17-518Z), and 10% heat-inactivated fetal bovine serum (SIGMA F7524) at 37°C in 5% CO₂ in high-density polyethylene flasks (NUNC 156340).

To decide if the compounds have an antiproliferative and/or a cytotoxic effect on human cells, the cytotoxicity (cell death) and the antiproliferative activity (retardation of cell proliferation) were measured. For this, different colorimetric assays for determining the number of viable cells were used. Under these experimental conditions, the optical density measured from the CellTiter-Blue reagent and methylene blue assay is proportional to the number of viable cells.

The test substances were dissolved in DMSO before being diluted in Dulbecco′s Modified Eagle′s Medium (DMEM). The adherent cells were harvested at the logarithmic growth phase after soft trypsinization using 0.25% trypsin in Phosphate Buffer Solution (PBS) containing 0.02% Ethylenediaminetetraacetic acid (EDTA) (Biochrom KG L2163). For each experiment, approximately 10 000 cells were seeded with 0.1 mL culture medium per well of the 96-well microplates (HUVEC: flat-bottomed NUNC 167008, K-562: round-bottomed NUNC 163320). To test the antiproliferative effect of the natural products on HUVEC and K-562, the cells were incubated for 72 hours in plates prepared with control and different dilutions of test substances. The GI50 values were defined as being where the inhibition of proliferation is 50% compared to the untreated control.

For the cytotoxicity assay, HeLa cells were preincubated for 48 hours without the test substances. To test the cytotoxic effect of the natural products on HeLa, the dilutions of the compounds were carried out carefully on the subconfluent monolayers of HeLa cells after the preincubation time. After incubation time, the cytolytic effect of compounds was analyzed in comparison to negative control. The 50% cytotoxicity concentration (CC₅₀) was defined as the test compound concentration required for destruction in 50% of the cell monolayer compared to the untreated control.

The cells were incubated with dilutions of the natural products in microplates for 72 hours at 37°C in a humidified atmosphere and 5% CO₂. This incubation was found to be an optimum time for the evaluation of the cytotoxicity and the inhibition of cell proliferation by finding out the number of viable cells stained with CellTiter-Blue reagent or methylene blue.

For estimating the influence of the natural products on cell proliferation of K-562, the numbers of viable cells present in multiwell plates via CellTiter-Blue assay (PROMEGA) were determined. This uses the indicator dye resazurin to measure the metabolic capacity of cells as indicator of cell viability. Viable cells of untreated control retain the ability to reduce resazurin into resorufin, whereas nonviable cells rapidly lose metabolic capacity and do not reduce the indicator dye. The absorption maximum for resazurin is 605 nm and the absorption maximum for resorufin is 573 nm. Thus, the absorbance measurements at 570 nm using 600 nm as a reference wavelength can be used to monitor the results. Values are compared to blank wells containing CellTiter-Blue reagent without cells.

The adherent HUVEC and HeLa cells were fixed by glutaraldehyde (MERCK 1.04239.0250) and stained with a 0.05% solution of methylene blue (SERVA 29198) for 15 minutes. After gently washing, the stain was eluted with 0.2 mL of 0.33 N HCl in the wells. The optical densities were measured at 660 nm (methylene blue) in SUNRISE microplate reader (TECAN).

Under these experimental conditions, the signals from the methylene blue and CellTiter-Blue reagent are proportional to the number of viable cells.

A repeat determination was conducted in all experiments and 4 replicates were assayed.

The calculations of different values of GI₅₀ and CC₅₀ were performed with the software Magellan (TECAN).

Offline (HPLC-NMR and HPLC-MS) Characterization of Compounds

3-Phenyl-1,2-dihydroacenaphthylen-1,2-diol⁷: 0.7 mg (0.002% dry weight); insufficient amount isolated to obtain a specific rotation; UV (extracted from PDA) (70% CH₃CN/D₂O) λ_max 219, 239, 261, 295 nm; ¹H NMR (500 MHz, CDCl₃) δ 7.86 (1H, d, J = 8.5 Hz, H-5), 7.78 (1H, d, J = 7.0 Hz, H-6), 7.78 (2H, d, J = 7.0 Hz, H-2′/H-6′), 7.66 (1H, d, J = 8.5 Hz, H-4), 7.60 (1H, m, H-8), 7.58 (1H, dd, J = 7.0, 8.0 Hz, H-7), 7.50 (2H, dd, J = 7.0, 8.0 Hz, H-3′/H-5′), 7.41 (1H, t, J = 7.0 Hz, H-4′), 5.62 (1H, d, J = 5.5 Hz, H-2), 5.52 (1H, d, J = 5.5 Hz, H-1); ¹³C NMR (125 MHz, CDCl₃) δ 142.9 (C, C-8a), 139.7 (C, C-1′), 138.5 (C, C-2a), 136.4 (C, C-3), 135.7 (C, C-8b), 130.4 (C, C-5a), 129.9 (CH, C-4), 128.9 (CH, C-2′/C-6′), 128.7 (CH, C-3′/C-5′), 128.3 (CH, C-7), 127.5 (CH, C-4′), 126.1 (CH, C-5), 124.7 (CH, C-6), 121.4 (CH, C-8), 74.3 (CH, C-1), 72.6 (CH, C-2); HPLC-MS m/z 245.0959 (calcd for C₁₈H₁₃O, 245.0961, [M−OH]⁺), m/z 285.0886 (calcd for C₁₈H₁₄NaO₂, 285.0886, [M+Na]⁺). HPLC-MS m/z 245[M−OH]⁺, 285 [M+Na]⁺.

Supplemental Material

Supplementary Material - Supplemental material for Antimicrobial Evaluation of the Constituents Isolated From Macropidia fuliginosa (Hook.) Druce

Supplemental material, Supplementary Material, for Antimicrobial Evaluation of the Constituents Isolated From Macropidia fuliginosa (Hook.) Druce by Robert Brkljaca, Hans-Martin Dahse, Kerstin Voigt and Sylvia Urban in Natural Product Communications

Footnotes

Acknowledgment

KV wishes to express her gratitude to Mrs Christiane Weigel for her technical assistance in the performance of the antimicrobial activity assays.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Brkljača

Urban

. HPLC-NMR and HPLC-MS profiling and bioassay-guided identification of secondary metabolites from the Australian plant Haemodorum spicatum . J Nat Prod. 2015;78(7):1486-1494.doi:10.1021/np500905g

Dias

DA.

Goble

DJ.

Silva

CA.

Urban

. Phenylphenalenones from the Australian plant Haemodorum simplex . J Nat Prod. 2009;72(6):1075-1080.doi:10.1021/np900016h

Urban

Brkljača

White

JM.

Timmers

. Phenylphenalenones and oxabenzochrysenones from the Australian plant Haemodorum simulans . Phytochemistry. 2013;95:351-359.doi:10.1016/j.phytochem.2013.07.019

Norman

EO.

Lever

Brkljača

Urban

Distribution

. Distribution, biosynthesis, and biological activity of phenylphenalenone-type compounds derived from the family of plants, Haemodoraceae. Nat Prod Rep. 2019;36(5):753-768.doi:10.1039/C8NP00067K

Brkljača

White

JM.

Urban

. Phytochemical Investigation of the Constituents Derived from the Australian Plant Macropidia fuliginosa . J Nat Prod. 2015;78(7):1600-1608.doi:10.1021/acs.jnatprod.5b00161

Master

RN.

Clark

RB.

Karlowsky

JA.

Ramirez

Bordon

. Analysis of resistance, cross-resistance and antimicrobial combinations for Pseudomonas aeruginosa isolates from 1997 to 2009. Int J Antimicrob Agents. 2011;38(4):291-295.doi:10.1016/j.ijantimicag.2011.04.022

Luis

JG.

Lahlou

EH.

Andres

LS.

Echeverri

Fletcher

WQ.

Phytoanticipins

. New Acenaphtylene and Dimeric Phenylphenalenones from the Resistant Musa Selected Hybrid SH-3481. Tetrahedron. 1997;53(24):8249-8256.

Sternhell

Westerman

. Proton nuclear magnetic resonance spectra of 1,2-disubstituted acenaphthenes. J Org Chem. 1974;39(25):3794-3796.doi:10.1021/jo00939a047

Krieg

Jortzik

Goetz

A-A

et al. Arylmethylamino steroids as antiparasitic agents. Nat Commun. 2017;8(1):14478.doi:10.1038/ncomms14478

Supplementary Material

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