Acrotrione: An Oxidized Xanthene from the Roots of Acronychia

Mar 13, 2019 - Environmental Futures Research Institute, Griffith University, Gold Coast Campus, Southport 4222 , Australia. ‡ Griffith Institute fo...
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Acrotrione: An Oxidized Xanthene from the Roots of Acronychia pubescens Luke P. Robertson,†,‡ Leonardo Lucantoni,‡ Sandra Duffy,‡ Vicky M. Avery,‡ and Anthony R. Carroll*,†,‡ †

Environmental Futures Research Institute, Griffith University, Gold Coast Campus, Southport 4222, Australia Griffith Institute for Drug Discovery, Griffith University, Nathan Campus, Brisbane 4111, Australia

‡

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S Supporting Information *

ABSTRACT: A new oxidized xanthene, acrotrione (1), and two known acetophenones (2 and 3) were isolated from a methanol extract of the roots of Acronychia pubescens. The structure of 1 was elucidated on the basis of its (+)-HRESIMS, 2D NMR, and ECD data. Acritrione (1) contains an unusual oxidized furo[2,3-c]xanthene moiety that has not been previously reported. Moderate antiplasmodial activity for these natural products against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) Plasmodium falciparum was determined with IC50 values ranging from 1.7 to 4.7 μM.

T

he genus Acronychia (Rutaceae) comprises 48 species of shrubs and small trees distributed throughout South Asia, Indonesia, New Caledonia, and Australia (19 endemic).1 Historically, Acronychia species have been used in traditional medicines as antimalarial,2 antifungal, antipyretic, and antihemorrhagic agents.3 Chemical investigations on this genus have revealed an array of natural products, including alkaloids,4 acetophenones,5 terpenoids,6 lignans,7 and cinnamic acids.8 As part of our efforts to identify new antiplasmodial drug leads,9 a library of extracts from Australian endemic Rutaceae species was screened against chloroquine-sensitive (3D7) Plasmodium falciparum. The methanol extract of the roots of Acronychia pubescens F.M. Bailey displayed 80% growth inhibition at 0.4 μg/mL, warranting a detailed chemical investigation of the species. Commonly known as “Hairy Aspen” and growing up to 15 m in height, A. pubescens is a rainforest tree endemic to southeast Queensland and northeast New South Wales, Australia.1 Previous chemical studies of the species have been limited, revealing only three simple furoquinolines.10 Reported herein are the isolation, structural elucidation, and antiplasmodial activity testing of a new oxidized xanthene (1) and two known acetophenones (2 and 3) (Figure 1). Exhaustive extraction of A. pubescens roots with MeOH followed by sequential purification with H2O/MeOH gradient reversed-phase and n-hexane/CH2Cl2/MeOH gradient normal-phase HPLC led to the purification of acrotrione (1), acronyculatin A (2), and acronylin (3). The structures of the known natural products acronyculatin A (2)11 and acronylin (3)12,13 were determined by 2D NMR data analysis and comparison to literature spectroscopic values. Acrotrione (1) was isolated as a yellow amorphous solid. A protonated molecular ion peak in the (+)-HRESIMS at m/z 587.2844 allowed the elemental formula, C32H43O10+, to be © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Natural products isolated from the roots of A. pubescens: the new oxidized xanthene acrotrione (1) and the known acetophenones acronyculatin A (2) and acronylin (3).

assigned to 1. The UV spectrum gave absorption maxima at 335, 290, 269, and 228 nm, which were indicative of an acetophenone.14 The 1H NMR spectrum of 1 displayed signals associated with six aliphatic methyl groups, two methyl ketone groups, one aromatic methoxy group, three methylenes, and five methines (Table 1). Resonances that could be attributed to four hydroxy protons were also observed. The 13C NMR spectrum contained resonances associated with 13 sp2 hybridized carbons. These were three keto carbonyls, a 2,4,6-trioxygenated aromatic ring, and four olefinic carbons (Table 1). An additional 19 sp3 hybridized carbons were observed, of which four were oxygenated. Comparison of the 1H and 13C NMR data and molecular formula of 1 to literature data14 suggested that this compound could be an acetophenone dimer derivative containing three isoprene units. All previously reported Acronychia-type acetophenone (AtA) Received: November 11, 2018

A

DOI: 10.1021/acs.jnatprod.8b00956 J. Nat. Prod. XXXX, XXX, XXX−XXX

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2a) confirmed this substructure. This was also supported by comparison of the acquired 2D NMR spectroscopic data of the

Table 1. NMR Spectroscopic Data for Acrotrione (1) in DMSO-d6 acrotrione (1) position

δca,

δH (J in Hz) b

type

1 2 3 4 5 6 1′

108.8, C 158.3, C 114.3, C 155.5, C 109.8, C 160.8, C 22.1, CH2

2′ 3′ 4′ 5′ 1′′

123.2, CH 130.4, C 25.3, CH3 17.8, CH3 27.3, CH

2′′

39.8, CH2

3′′ 4′′ 5′′ 1′′′ 2′′′ 3′′′

25.1, CH 24.8, CH3 20.7, CH3 111.9, C 193.2, C 43.8, CH

4′′′ 5′′′ 6′′′ 1′′′′

97.2, C 80.5, C 178.1, C 30.9, CH2

2′′′′ 3′′′′ 4′′′′ 5′′′′ MeO-2 MeCO-1 MeCO-1 MeCO-1′′′ MeCO-1′′′ OH-3′′′′ OH-4′′′ OH-5′′′ OH-6

92.9, CH 69.1, C 25.4, CH3 25.5, CH3 62.6, CH3 31.1, CH3 203.8, C 31.6, CH3 196.7, C OH OH OH OH

HMBCc

3.11, dd (15.0, 5.8); 3.16, m 5.02, t (6.6)

2, 2′, 3, 3′, 4

1.57, s 1.67, s 3.78, ddd (11.8, 3.6, 0.8) 2.08, m 1.69, m 1.79, m 0.90, d (6.5) 0.99, d (6.5)

2′, 3′, 5′ 2′, 3′, 4′ 2′′, 2′′′, 3′′′, 4, 4′′′, 5, 6

3.19, d (0.8)

1′′, 2′′, 2′′′, 4′′′, 5, 5′′′

Figure 2. (a) Key HMBC (→) and COSY (bold bonds) correlations of 1. (b) Key ROESY correlations in the energy-minimized conformation of 1.

2.21, dd (13.1, 5.1) 2.54, dd (13.1, 10.8) 4.78, dd (10.8, 5.1)

5′′′, 6′′′ 2′′′′, 3′′′′

1.13, 1.16, 3.63, 2.61,

s s s s

2′′′′, 3′′′′, 5′′′′ 2′′′′, 3′′′′, 4′′′′ 2 1, MeCO-1

2.12, s

1′′′, MeCO-1′′′

4.81, s 7.02, s 6.57, s 13.69, s

2′′′, 3′′′, 4′′′, 5′′′ 1′′′′, 4′′′, 5′′′, 6′′′ 1, 2, 4, 5, 6, MeCO-1

coisolated compound 3 in DMSO-d6. The chemical shift of C4 (δC 155.5) suggested it is an ether. H-1′′ (δH 3.78) was assigned as benzylic to the B ring from HMBC correlations to C-4/C-5/C-6. COSY correlations between H-1′′/H-2′′/H3′′/H-4′′/H-5′′ were used to assign the isopentyl unit. HMBC correlations from H-1′′ to a keto carbonyl resonance at δC 193.2 (C-2′′′), a ketal/hemiketal carbon resonance at δC 97.2 (C-4′′′), and a methine carbon resonance at δC 43.8 (C-3′′′) established that C-1′′ is attached to either a 1-keto-3-ketal or 1-keto-3-hemiketal moiety. A small vicinal (J = 0.8 Hz) coupling between H-1′′ and H-3′′′ confirmed that C-3′′′ is alpha to both the ketone and the ketal/hemiketal. HMBC correlations from the hydroxy resonance at δH 7.02 (OH-4′′′) to C-3′′′/C-4′′′ established that C-4′′′ is a hemiketal. Both OH-4′′′ and OH-5′′′ (δH 6.57) showed HMBC correlations to oxygenated carbon resonances at δC 97.2/80.5 (C-4′′′/C-5′′′), indicating that they are arranged as a vicinal diol, while a 3JCH HMBC correlation from OH-5′′′ to C-1′′′′ revealed that C5′′′ is vicinal to C-1′′′′. COSY correlations between H2-1′′′′/ H-2′′′′ were used to assign their positions as vicinal, and the chemical shift of C-2′′′′ (δC 92.9) suggested that it is an ether carbon.5 HMBC correlations from a gem-dimethyl (δH 1.16/ 1.13) to C-2′′′′ and a quaternary oxygenated carbon resonance at δC 69.1 (C-3′′′′) was used to establish that an oxygenated isopropyl group is attached to C-2′′′′. ROESY correlations between the hydroxy resonance at δH 4.81 (OH-3′′′′) and H1′′′′/H3-5′′′′ confirmed that C-3′′′′ is hydroxylated (Figure 2b). Three-bond HMBC correlations were observed from both the methyl ketone (MeCO-1′′′, δH 2.12) and H-3′′′ to a nonprotonated olefinic carbon at δC 111.9 (C-1′′′), indicating that the keto carbonyl (C-2′′′) is linked to MeCO-1′′′ by C1′′′. HMBC correlations from OH-5′′′/H2-1′′′′ to the final unassigned carbon resonance (δC 178.1, C-6′′′) indicated that

4′, 5′

−3′′′ 2′′, 3′′, 5′′ 2′′, 3′′, 4′′

a

125 MHz. b800 MHz. cHMBC correlations are from proton(s) stated to the indicated carbon.

dimers contain two 2,4,6-trioxygenated aromatic rings with substitution of isoprene and methyl ketone moieties at C-3/C5 and C-1, respectively.14 The linkage of monomers occurs via an isopentyl group at C-3/C-5, although cyclization of C-3/C5 isoprene functionalities to adjacent oxygen atoms may also occur, resulting in the formation of furan or pyran rings. However, unlike previously reported AtA dimers, 1 only contained three oxygenated aromatic resonances between δC 170.0−140.0. Additional and unusual resonances associated with a keto carbonyl (δC 193.2) and a strongly deshielded olefinic carbon (δC 178.1) indicated that 1 possesses atypical AtA structural features. The structure of the B ring, which is the same in all known AtA dimers,15 was determined from analysis of the 2D NMR data and corroborated by comparison of resonances to literature values.5 HMBC correlations (Figure B

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cases, the calculated ECD spectrum of (1′′R,3′′′S,4′′′S,5′′′S,2′′′R)-1 showed better matches with the experimental data than its enantiomer, allowing this to be assigned as the absolute configuration of 1. Most of the currently known chiral AtAs are racemic, with evidence coming from X-ray crystallography,18 optical rotation data,5,14 and Mosher’s acid analysis.5 To the best of our knowledge, only three AtAs are reported to be optically active,11,19 although the absolute configuration of only one of these has been determined.11 Acrotrione (1) contains a highly unusual furo[2,3-c]xanthene structure that has not been reported previously. A plausible biosynthesis could start from the coisolated AtA monomer acronylin (3) (Figure 4). Prenylation of C-5 in 3

C-1′′′ and C-6′′′ must be linked (Figure 2a). The deshielded chemical shift of C-6′′′ supported it as an oxygenated olefinic carbon beta to two carbonyls,16 allowing a cyclohexenone substituted at C-1′′′ by the methyl ketone group to be defined. Logically, this indicated an ether linkage is present between C2′′′′ and C-6′′′, generating a 2-(2-hydroxypropan-2-yl)-2,3dihydro-1-furan ring at C-5′′′/C-6′′′. Comparison with literature spectroscopic data of AtA dihydrofurans confirmed this.5 A final unassigned degree of unsaturation defined by MS analysis and an unfilled valence for C-4′′′ (δC 97.2) with bonds only to C-3′′′/C-5′′′/OH-4′′′ assigned led to the conclusion that an ether linkage is present between C-4 and C-4′′′, although there were no HMBC correlations to support this. In the absence of relevant HMBC data, ROESY correlations between H-2′ and H-1′′′′/H3-5′′′′/OH-4′′′ confirmed this assignment (Figure 2b). With the 2D structure of 1 established, the relative configuration of the five stereogenic centers were determined by further analysis of ROESY data. ROESY correlations between OH-4′′′/H-3′′′ and OH-5′′′/H3′′′ indicated that each is α positioned. A correlation between OH-5′′′ and H-2′′′′ established the position of H-2′′′′ as α. Finally, the 0.8 Hz coupling between H-1′′ and H-3′′′ indicated that these are at 90 deg to each other, and thus H1′′ was assigned as β. This was confirmed by a ROESY correlation observed between OH-4′′′ and H-2′′. After the relative configuration of 1 was confirmed, its absolute configuration was determined by comparison of experimental and predicted ECD spectra of the two possible enantiomers, calculated using the time-dependent density functional theory (TDDFT) method.17 The elucidation of the absolute configuration of 1 proved challenging, with none of the calculated ECD spectra showing a perfect match (Figure 3). Spectra calculated using the CAM-B3LYP/def2-SVP//

Figure 4. Proposed biogenetic origin of 1 from the coisolated monomer acronylin (3).

followed by dimerization with another unit of 3 would result in the formation of the known AtA dimer acrovestone.18 Epoxidation at C-4′′′/C-5′′′ and subsequent nucleophilic attack of C-4′′′ by OH-4 then could cause the formation of an ether linkage between C-4 and C-4′′′. Keto-tautomerism at C2′′′ would result in a ketone moiety at C-2′′′. Finally, epoxidation of the double bond in the isoprene attached at C5′′′ and nucleophilic attack of the epoxide carbon C-2′′′′ would lead to 1. All of the A. pubescens natural products isolated (1−3) were tested for antiplasmodial activity against chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) strains of P. falciparum. Cytotoxicity was also determined using the HEK293 mammalian cell line to evaluate selectivity for the parasite (Table 2). Compounds 1 and 3 were moderately active against 3D7 P. falciparum, with IC50 values of 2.7 and 1.8 μM, respectively. Similar activity was also observed against the Dd2 strain, and the Dd2/3D7 IC50 ratio of 1.7 for both compounds suggests equal sensitivity by both the drugresistant and wild-type parasite strains. Some cytotoxicity was also observed for both compounds, with 81 and 79% growth inhibition of HEK293 cells at 40 μM for 1 and 3, respectively. Conversely, 2 showed very weak activity, reaching only 38 and 20% growth inhibition at 40 μM against P. falciparum 3D7 and Dd2 strains, respectively. Weak cytotoxicity was also observed against HEK293 cells, showing only 7% growth inhibition at 40

Figure 3. Comparison of the experimental and calculated ECD spectra of (1′′R,3′′′S,4′′′S,5′′′S,2′′′R)-1. Structures were optimized at the B3LYP/def2-SVP level. The functional/basis set combinations shown in the key are those used for the calculation of electronic transition and rational strength.

B3LYP/def2-SVP and M062X/def2-SVP//B3LYP/def2-SVP functional/basis set combinations each provided average matches. Both successfully reproduced the positive Cotton effects at 240, 280, and 320 nm and the negative Cotton effect at 230 nm. However, both calculated spectra showed lower positive molar ellipticity at ∼300 nm, while in the experimental spectrum, this manifested as a shoulder at 310 nm between the 320 nm peak and the more intense peak at 280 nm. In both C

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Table 2. Antiplasmodial Activity and Cytotoxicity of Natural Products Isolated from the Roots of A. pubescens IC50 ± SD (μM) (n = 2) compound

3D7

Dd2

HEK293

selectivity index (SI)

Dd2/3D7 ratio

1 2 3 artesunateb chloroquineb dihydroartemisininb puromycinb pyrimethamineb pyronaridineb

2.7 ± 0.7 37.5% ± 22.7a 1.8 ± 0.3 0.0021 ± 0.0016 0.0139 ± 0.0015 0.0009 ± 0.0002 0.0702 ± 0.0108 0.0077 ± 0.0017 0.0082 ± 0.0020

4.7 ± 0.7 19.8% ± 11.1a 3.1 ± 0.6 0.0033 ± 0.0022 0.1360 ± 0.0255 0.0016 ± 0.0003 0.0747 ± 0.0188 n.d. 0.0110 ± 0.0010

80.7% ± 2.6a 7.4% ± 4.0a 79.1% ± 3.8a 62.5% ± 16.5a 53.2% ± 26.4a 41.1% ± 11.5a 0.6940 ± 0.183 52.0% ± 10.2a 1.990 ± 0.263

n.d. n.d. n.d. n.d. n.d. n.d. 9.9 n.d. 246.1

1.7 n.d. 1.7 1.6 9.8 1.8 1.1 n.d. 1.3

Percentage growth inhibition at 40 μM. bReference compound. Selectivity index = HEK293/3D7. n.d. = not determined.

a

(YMC-pack diol, 5 μm, 120-NP, 21.2 × 150 mm) using a gradient from n-hexane (100%) to CH2Cl2 (100%) over 50 min, then to CH2Cl2 (90%)/MeOH (10%) from minutes 51−60. Fraction 10 contained acronyculatin A (2) (2.3 mg, 1.2 × 10−2% dry wt); fraction 34 contained acronylin (3) (0.4 mg, 2.0 × 10−3% dry wt), and fraction 60 contained acrotrione (1) (1.7 mg, 8.5 × 10−3% dry wt). Acrotrione (1); yellow amorphous solid; [αD25] + 84 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 335 (5.93), 290 (6.43), 269 (6.55), 228 (6.51) nm; ECD (c 0.001, MeOH) λmax (Δε) 293 (+2.7), 257 (−0.1) 240 (+0.8) nm; IR (film) νmax 3351, 1650, 1366, 1202, 1019 cm−1; 1H and 13C NMR data, Table 1; HRESIMS m/z [M + H]+ 587.2844 (calcd for C32H43O10+, 587.2856). Computational Methods. The lowest energy conformers of 1 were generated using Schrödinger MacroModel 2016 by following the procedure reported by Willoughby et al.20 Initial geometry optimizations were performed on each of the 19 generated conformers with first-principle calculations based on density functional theory (DFT) at the B3LYP/6-31G(d) level using Grimme’s empirical dispersion corrections (D3).21 The conformers were then reoptimized with the B3LYP/def2SVP functional/basis set combination utilizing empirical dispersion corrections (D3) and the polarizable continuum solvent model (PCM).22 Free energy calculations were performed at the same level. Electronic transition and rational strength were calculated using TDDFT using the CAMB3LYP/def2-SVP and M062X/def2-SVP combinations with consideration of the solvent effect using the PCM. Boltzmann-weighted UV and ECD spectra were calculated using the freely available software SpecDis23 using a sigma/gamma value of 0.3 eV. The experimental ECD spectrum was processed using SDAR.24 All DFT calculations were carried out using the Gaussian 16 suite of programs.25 All TDDFT protocols were based on the method described by Pescitelli and Bruhn.26 Compounds and Screening Methods. Test compounds were dissolved in 100% DMSO to obtain 10 mM stock solutions. Chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin, and pyrimethamine were used as reference compounds. Stock solutions were prepared at 2.5 mM (dihydroartemisinin, artesunate) or 10 mM (all other reference compounds) in 100% DMSO, except chloroquine and pyronaridine, which were dissolved in water. Puromycin (5 μM) and 0.4% DMSO were used as positive and negative controls, respectively. Experimental compounds were tested in 16-concentration dose−response using three concentrations per log dose against 3D7 (drug-sensitive) and Dd2 (drug-resistant) P. falciparum parasite strains and against HEK293 cells for cytotoxicity assessment. Final assay concentration ranges of 40 μM to 0.4 nM were used for experimental compounds. Reference antimalarial compounds/drugs were tested in 21 concentration dose−response range of 40 μM to 0.01 nM (10 μM to 0.003 nM for dihydroartemisinin and artesunate). In Vitro Antiplasmodial Image-Based Assay. P. falciparum parasites (3D7 and Dd2 strains) were grown in RPMI 1640 supplemented with 25 mM HEPES, 5% AB human male serum, 2.5 mg/mL Albumax II, and 0.37 mM hypoxanthine. Parasites were

μM. Structurally, 2 differs from 3 only by the presence of a formyl group at C-5; however, 3 is considerably more bioactive.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were logged on a JASCO P-1020 polarimeter and [α]D values are reported in 10−1 deg cm2 g−1. UV spectra were recorded on a Shimadzu UV1800 UV−vis spectrophotometer. ECD spectra were recorded on a JASCO J-715 spectropolarimeter. IR spectra were recorded using a ThermoFisher Scientific Nicolet iS5/iD5 ATR spectrometer. NMR spectra were recorded at 25 °C on a Bruker Avance III 500 MHz spectrometer (BBFO Smartprobe, 5 mm 31P-109Ag) and a Bruker Avance III HDX 800 MHz with a triple (TCl) resonance 5 mm cryoprobe. NMR spectra were referenced to the solvent peak for (CD3)2SO at δH 2.50 and δC 39.52. High-resolution mass measurements were acquired using mobile phase 100% CH3CN on an Agilent Technologies 6530 Accurate-Mass Q-TOF LC/MS with a 1200 Series autosampler and 1290 Infinity HPLC. The C18 silica gel used was Alltech Sample Prep C18 35−75 μm, 150 Å. HPLC columns were Betasil 5 μm, 100 Å, 21.2 × 150 mm and YMC-pack diol, 5 μm, 120NP. For HPLC, a Merck Hitachi L7100 pump in tandem with a Merck Hitachi L7455 PDA detector and a Merck Hitachi L7250 autosampler were used. A Gilson 215 liquid handler was used to collect fractions. All organic solvents used were Scharlau HPLC grade, and H2O was Millipore Milli-Q PF filtered. Trifluoroacetic acid (TFA) was spectroscopy grade from Alfa Aesar. Parasite strains 3D7 and Dd2 were obtained from BEI Resources. O+ erythrocytes were obtained from the Australian Red Cross Blood Service. CellCarrier poly-D-lysine coated imaging plates were from PerkinElmer. 4′,6Diamidino-2-phenylindole (DAPI) stain were from Invitrogen. Triton-X, saponin, chloroquine, artesunate, puromycin, pyronaridine, dihydroartemisinin, and pyrimethamine were all from Sigma-Aldrich. HEK293 cells were purchased from the American Tissue Culture Collection. The 384-well Falcon sterile tissue culture treated plates were from BD. Plant Material. A. pubescens root material was purchased from Burringbar Rainforest Nursery in August 2017. A voucher specimen (ACRUT007) is housed within the School of Environment and Science, Gold Coast Campus, Griffith University. Extraction and Isolation. Oven-dried ground A. pubescens roots (20 g) were exhaustively extracted in MeOH (1 L), yielding a brown gum (1.5 g). The extract (1 g) was adsorbed onto C18 silica gel (1 g), and the extract-impregnated gel was loaded into a HPLC precolumn cartridge (10 × 20 mm) and connected in series to a C18-bonded silica HPLC column (Betasil 5 μm, 100 Å, 21.2 × 150 mm). The column was then eluted with a gradient from H2O/0.1% TFA (100%) to MeOH/0.1% TFA (100%) over 60 min at a flow rate of 9 mL/min. The column was then eluted with MeOH for a further 10 min. Fractions were collected every min, and UV-DAD spectroscopic analysis was conducted in tandem with the separation. Fractions 53− 63 (250 mg) were recombined and further purified by diol HPLC D

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subjected to two rounds of sorbitol synchronization before undergoing compound treatment. Ring stage parasites were exposed to the test compounds in 384-well imaging CellCarrier microplates (PerkinElmer), as previously described.27 Plates were incubated for 72 h at 37 °C, 90% N2, 5% CO2, 5% O2, and then the parasites were stained with 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI) and imaged using an Opera QEHS microplate confocal imaging system (PerkinElmer). Images were analyzed as previously described.27 In Vitro Cytotoxicity Assay. Human embryonic kidney cells (HEK293) were maintained in DMEM medium supplemented with 10% FBS. HEK293 cells were exposed to the compounds in TCtreated 384-well plates (Greiner) for 72 h at 37 °C, 5% CO2, and then the medium was removed from the wells and replaced with an equal volume of 44 μM resazurin. After an additional 5−6 h incubation at standard conditions, the total fluorescence (excitation/emission: 530/ 595 nm) was measured using an Ensight plate reader (PerkinElmer). The positive control, puromycin, had an IC50 value of 0.69 ± 0.18 μM toward the HEK293 cell line. Biological Data Analysis. Raw data were normalized using the in-plate positive and negative controls to obtain normalized percent inhibition data, which were then used to calculate IC50 values through a four-parameter logistic curve fitting in Prism (GraphPad). The experiments were carried out in two biological replicates, each consisting of two technical repeats.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00956. 1D and 2D NMR spectra for acrotrione (1) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +61 7 55529187. Fax: +61 7 55529047. E-mail: a. carroll@griffith.edu.au. ORCID

Anthony R. Carroll: 0000-0001-7695-8301 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by an Australian Postgraduate Award (APA) provided by the Australian Commonwealth Government. We acknowledge Australian Research Council Grants (LP120200557 awarded to V.M.A. and LE140100119 for NMR equipment). The authors acknowledge the support of the Griffith University eResearch Services Team and the use of the High Performance Computing Cluster “Gowonda” for TDDFT calculations. We thank W. Loa-Kum-Cheung, J. Carrington, and R. Stewart for technical assistance and the Australian Red Cross Blood Service for the provision of human blood. We are grateful to G. Leiper for photograph provision.

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REFERENCES

(1) Hartley, T. G. In Flora of Australia Vol. 26Meliaceae, Rutaceae, Zygophyllaceae; Wilson, A., Ed.; ABRS/CSIRO: Melbourne, 2013; pp 104−118. (2) Hnawia, E.; Hassani, L.; Deharo, E.; Maurel, S.; Waikedre, J.; Cabalion, P.; Bourdy, G.; Valentin, A.; Jullian, V.; Fogliani, B. Pharm. Biol. 2011, 49, 369−376. (3) Epifano, F.; Fiorito, S.; Genovese, S. Phytochemistry 2013, 95, 12−18. (4) Lahey, F. N.; Thomas, W. C. Aust. J. Chem. 1949, 2, 423−426. E

DOI: 10.1021/acs.jnatprod.8b00956 J. Nat. Prod. XXXX, XXX, XXX−XXX