Unique Polybrominated Hydrocarbons from the Australian Endemic

Article pubs.acs.org/jnp

Unique Polybrominated Hydrocarbons from the Australian Endemic Red Alga Ptilonia australasica Trong D. Tran, Ngoc B. Pham, and Ronald J. Quinn* Eskitis Institute for Drug Discovery, Griffith University, Brisbane, QLD 4111, Australia S Supporting Information *

ABSTRACT: The red alga Ptilonia australasica is endemic to Australian temperate waters. Chemical investigation of P. australasica led to the identification of four new polybrominated compounds, ptilones A−C (1−3) and australasol A (4). Their planar structures were established by extensive NMR and MS analyses. The low H/C ratio and the presence of a large number of heteroatoms made the structure elucidation challenging. The absolute configurations of 1, 2, and 4 were determined by quantum chemical ECD calculations employing time-dependent density functional theory. Ptilones A−C (1−3) show unique 4-ethyl-5-methylenecyclopent-2-enone (1 and 2) and 2-methyl-6-vinyl-4H-pyran-4-one (3) skeletons not previously reported in algal metabolites. Ptilone A displayed the most potent cytotoxicity against the human prostate cancer PC3 cells with an IC50 value of 0.44 μM and induced the PC3 cell cycle arrest in the G0/G1 phase. challenging by NMR. Although linear and cyclic C1−C9 hydrocarbon compounds are common in marine algae, especially in the family Bonnemaisoniaceae,12 this is the first time that the hydrocarbons with the 4-ethyl-5-methylenecyclopent-2-enone (1 and 2) and 2-methyl-6-vinyl-4H-pyran-4-one (3) skeletons have been found. Cytotoxicities of 1 and 3 and their effects on the PC3 cell cycle are also reported.

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lgae are represented by at least 30 000 species worldwide, supplying oxygen to the biosphere, food for fish and man, medicine, and fertilizers.1 More than 1300 new natural products consisting of peptides, polyketides, indoles, terpenes, acetogenins, phenols, and volatile halogenated hydrocarbons2 were isolated from algae in the period between 1985 and 2008.3 Among the three most studied types of algae (brown, green, and red algae), the red algae produce 90% halogenated compounds2 in which chlorine and bromine appear to be dominant.4 These compounds possess a wide spectrum of biological activities such as antibacterial, anticancer, antifungal, antiviral, neuron protection, heart prevention, and protection against vascular diseases.5 The alga genus Ptilonia belonging to the family Bonnemaisoniaceae consists of six species, namely, P. australasica, P. magellanica, P. mooreana, P. okadae, P. subulifera, and P. willana.6−9 The two species P. australasica and P. subulifera are endemic in Australian flora.6 The red alga P. australasica was first reported in 197510 with the isolation of seven compounds including two polybromo-γ-pyrones and five 1,1,2-tribromoalkl-en-3-one-type structures. Since then, no chemical investigation of this alga has been reported. In the search for new natural products with potential antiprostate cancer activity, a subset of lead-like enhanced fractions derived from rare or restricted marine biota in our Nature Bank library database11 was screened in cytotoxicity cell-based assays. One fraction from the Australian red alga P. australasica was found to potently inhibit the growth of the human prostate cancer PC3 cells. This paper describes the isolation and structure elucidation of four new polybrominated compounds, ptilones A−C (1−3) and australasol A (4), from P. australasica. The low H/C ratio and the presence of a large number of heteroatoms made the structure elucidation highly © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The dried, ground samples of P. australasica were extracted exhaustively with n-hexane, dichloromethane (DCM), and MeOH, respectively. The DCM and MeOH extracts were combined and chromatographed on a C18 HPLC column (MeOH/H2O/0.1% trifluoroacetic acid (TFA)) and subsequently on a Diol HPLC column (n-hexane/2-propanol) to yield four new polybrominated compounds (1−4).

Compound 1 was isolated as a colorless, amorphous solid. The (+)-HRESIMS spectrum displayed an isotopic cluster of Special Issue: Special Issue in Honor of John Blunt and Murray Munro Received: November 2, 2015

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DOI: 10.1021/acs.jnatprod.5b00989 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. NMR Data for 1 and 2 1 position 1 2 3 4 5 6 7 8 3-OCH3 4-OH

δ Ca 180.9, 129.3, 162.2, 84.6, 137.4, 101.8, 27.2,

C C C C C C CH2

7.8, CH3

δH (J in Hz)a

2.42, dq (7.2, 14.4) 1.79, dq (7.2, 14.4) 0.51, t (7.2) 6.51, s

2 gCOSYa

8 8 7

gHMBCADa

3, 4, 5,c 2c 3, 4, 5 4, 7 4, 7, 5weak

δCb 181.6, 96.5, 178.3, 82.3, 137.8, 99.0, 26.4,

C C C C C C CH2

8.4, CH3 60.5, CH3

δH (J in Hz)b

2.36, 1.87, 0.59, 4.36, 6.35,

dq (7.2, 14.4) dq (7.2, 14.4) t (7.2) s s

gCOSYa

8 8 7

gHMBCADa

3, 3, 4, 3 4,

4, 8 4, 5, 8 7 7

Referenced to DMSO-d6 solvent peaks at δC 39.52 ppm and δH 2.50 ppm, recorded at 600 MHz at 30 °C. bReferenced to DMSO-d6 solvent peaks at δC 39.52 ppm and δH 2.50 ppm, recorded at 900 MHz at 25 °C. cObserved when gHMBCAD was performed with nJCH = 3 Hz.

a

ions [M + H]+ at m/z 450.7159 in the ratio 1:4:6:4:1, corresponding to a molecular formula of C8H6Br4O2 with four double-bond equivalents. The 1H NMR spectrum recorded in DMSO-d6 (Table 1) showed the presence of four signals, corresponding to an exchangeable hydroxy (δH 6.51 ppm), two nonequivalent methylene protons (δH 2.42 and 1.79 ppm), and one methyl (δH 0.51 ppm). The 13C NMR spectrum of 1 in DMSO-d6 (Table 1) displayed eight signals including one carbonyl carbon (δC 180.9 ppm), four nonprotonated olefinic carbons (δC 162.2, 137.4, 129.3, and 101.8 ppm), one nonprotonated oxygenated carbon (δC 84.6 ppm), one methylene (δC 27.2 ppm), and one methyl (δC 7.8 ppm). The location of a hydroxy (δH 6.51 ppm) was deduced due to its HMBC correlation to C-4 (δC 84.6 ppm). COSY correlations from methylene protons H-7 (δH 2.42 and 1.79 ppm) to methyl H-8 (δH 0.51 ppm) together with HMBC correlations from H-8 to C-4 and from H-7 to C-3 (δC 162.2 ppm), C-4, and C-5 (δC 137.4 ppm) facilitated the establishment of a 1,1-diolefinic-1-hydroxy propyl fragment. The positions of three carbons including one carbonyl (δC 180.9 ppm) and two nonprotonated olefinic carbons (δC 129.3 and 101.8 ppm) were still unassigned, as no correlations were observed in the gHMBCAD spectrum. This indicated that these carbons were placed at least four bonds away from any proton. On the basis of this evidence, 11 structures (1a−1k) were possible for 1 (Figure 1A). Structures 1a and 1e were symmetric and would be expected to have six carbon resonances, which was inconsistent with eight resonances observed in the 13C NMR spectrum.13,14 The carbonyl carbon at δC 180.9 ppm cannot be adjacent to a bromine atom, as the high shielding effect of bromine would cause it to have a lower chemical shift.15,16 This evidence resulted in the disqualification of the structures 1c, 1d, 1f, 1g, 1h, 1j, and 1k. The chemical shift of the oxygenated carbon C-4 (δC 84.6 ppm) in 1b was comparable to that value (δC 84.0 ppm) of mahorone, a cyclopentenone with bromines at C-2 and C-3, previously reported,17 while its chemical shift in the three-membered ring 1i would be expected to reside at less than 80 ppm.18 As a result, the planar structure of 1b was the most likely candidate. The optimization of nJHC coupling constants (nJHC = 3, 8, and 12 Hz) in the gHMBCAD experiments was then performed to detect longer range correlations. With a nJHC coupling constant of 3 Hz, a clear four-bond correlation signal from a proton at δH 2.42 ppm to a nonprotonated carbon at δC 129.3 ppm was

Figure 1. (A) Possible planar structures of 1. (B) Key HMBC correlations observed (nJHC = 3 Hz).

observed (Figure 1B). This information confirmed that 1b was the planar structure of 1. The absolute configuration of 1 was determined by the comparison of experimental electron circular dichroism (ECD) and calculated ECD spectra. Quantum chemical ECD calculations employing time-dependent density functional theory (TDDFT) have offered a convenient and useful method for solving absolute configurations of natural products.19,20 A conformational analysis of the (4S)-stereoisomer using the Merck Molecular Force Fields (MMFFs) method found three stable conformers. These conformers were then subjected to geometrical optimization and energy calculations using the density functional theory (DFT) with the B3LYP functional and the psCSDZ basis set in MeOH (Figure 2). Optimized conformers were subsequently computed using the TDDFT B3LYP/6-31G(d) level in MeOH with the “self-consistent reaction field”. The ECD spectrum of each conformer was B



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correlations from an exchangeable hydroxy signal (δH 9.21 ppm) to C-2 (δC 149.7 ppm), C-3 (δC 142.7 ppm), and C-4 (δC 172.3 ppm), from a methyl H-9 (δH 2.29 ppm) to C-2 and C-3, and from an olefinic proton H-5 (δH 6.68 ppm) to C-3 (δC 142.7 ppm), C-4 (δC 172.3 ppm), C-6 (δC 157.8 ppm), and C-7 (δC 114.8 ppm). This information facilitated the establishment of an α,β-unsaturated carbonyl system for 3 (Figure 4). The last nonprotonated carbon at δC 99.4 ppm showed no correlation with any proton in the molecule, suggesting that this carbon was located at least four bonds away from them. In addition, the more shielded chemical shift of this carbon compared to that of other olefinic carbons suggested this carbon was adjacent to two bromine atoms, as in the case of structures 1 and 2. Two nonprotonated carbons at δC 157.8 and 149.7 ppm were linked through oxygen atom O-1 based on their typical deshielded resonances of olefinic carbons bearing an oxygen atom.15 As the positions of carbons at δC 157.8 and 114.8 ppm could not be unambiguously assigned, two possible structures were suggested for compound 3 (Figure 4A and B). Compounds 3a and 3b share core structures of six- and seven-membered rings with maltol21,22 and cinariolid,23 respectively (Figure 4). NMR comparisons between postulated structures 3a and 3b with maltol and cinariolid in CDCl3 showed that chemical shifts of 3a were aligned to those of maltol, suggesting that the structure of 3 favored the sixmembered ring system. In order to confirm the assignment, theoretical 13C chemical shifts for the two possible isomers (3a and 3b) were calculated (Table 3). The errors between the calculated chemical shifts of the isomer 3a with experimental data were lower than those of the isomer 3b. The key chemical shift for C-7 in the isomer 3b was clearly different from the observed value in 3 and confirmed isomer 3a as the structure. Furthermore, significant differences were observed in DP4 probabilities of 13C chemical shifts, of which the isomer 3a accounted for 100%, while isomer 3b had 0%. The probabilities indicated that the assignment of 3a to 3 was at a high level of confidence.24 Together all information allowed the isomer 3a to be assigned to 3. Therefore, the structure of 3, ptilone C, was determined to be a new polybrominated γ-pyrone. Compound 4 was purified as a colorless gum. The molecular formula of 4 was determined to be C6H5Br5O2 by an isotopic cluster of ions [M + Na]+ at m/z 526.6125 in the ratio 1:5:10:10:5:1 observed in the (+)-HRESIMS spectrum. The 1H NMR spectrum of 4 showed two coupling resonances at H-1 (δH 5.72, d, J = 9.0 Hz) and H-2 (δH 6.27, d, J = 9.0 Hz) and an isolated methyl signal H-2′ (δH 2.17 ppm) (Table 4). An acetate group was assigned due to an HMBC correlation from H-2′ to carbonyl C-1′ (δC 168.4 ppm). Further HMBC analysis indicated correlations from H-1 to carbon C-3 (δC 123.1 ppm)

Figure 2. Comparison of the experimental (blue) and calculated (red) ECD spectra of 1.

simulated from rotatory strengths using Gaussian distribution, and the final calculated ECD spectrum obtained based on the Boltzmann distribution of individual conformers. The calculated theoretical ECD spectrum of the (4S)-isomer predicted accurately the two negative Cotton effects (CEs) observed in the experimental ECD spectrum (Figure 2). Therefore, the absolute configuration of 1 was determined as (4S)-ptilone A. Compound 2 was isolated as a colorless, amorphous solid. The molecular formula of 2 was deduced as C9H9Br3O3 on the basis of the (+)-HRESIMS spectrum, exhibiting an isotopic cluster of adduct ions [M + Na]+ at m/z 424.7980 in the ratio 1:3:3:1. The 1H NMR data of 2 were similar to those of 1 (Table 1), the major difference being that 2 had one extra methoxy signal at δH 4.36 ppm. Following 1D and 2D NMR analysis, 2 was found to share the same 5-dibromomethylene-4ethyl-4-hydroxycyclopentenone skeleton with 1 (Figure 3A). The methoxy group was assigned at C-3 based on its HMBC correlation to a nonprotonated carbon C-3 (δC 178.3 ppm) (Figure 3A). Due to an electronic effect of the methoxy group, C-3 (δC 178.3 ppm) in 2 showed a more deshielded shift compared to that in 1 (δC 162.2 ppm). The theoretical ECD spectrum (Figure 3B) calculated for the (4S)-stereoisomer of 2 by TDDFT (B3LYP/6-31G(d)//B3LYP/psCSDZ) was in agreement with the experimental ECD. Therefore, compound 2 was established as (4S)-ptilone B. Compound 3 was purified as a colorless, amorphous solid. An isotopic cluster of ions [M + H]+ in the ratio 1:3:3:1 at m/z 386.7851 in the (+)-HRESIMS spectrum allowed the molecular formula C8H5Br3O3 with five double-bond equivalents to be assigned for 3. The 1H NMR spectrum of 3 in DMSO-d6 (Table 2) displayed three singlet resonances at δH 9.21, 6.68, and 2.29 ppm. The 13C NMR and gHSQCAD spectra (Table 2) confirmed the presence of one carbonyl (δC 172.3 ppm), five nonprotonated olefinic carbons (δC 157.8, 149.7, 142.7, 114.8, and 99.4 ppm), one olefinic tertiary carbon (δC 114.2 ppm), and one methyl carbon (δC 14.0 ppm). HMBC data showed

Figure 3. (A) Key HMBC correlations of 2. (B) Comparison of the experimental (blue) and calculated (red) ECD spectra of 2. C



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Table 2. NMR Data for 3 δ Ca

position 2 3 4 5 6 7 8 9 OH

149.7, 142.7, 172.3, 114.2, 157.8, 114.8, 99.4, 14.0,

δH (J in Hz)a

C C C CH C C C CH3

δ Cb

gHMBCADa

6.68, s

3, 4, 6, 7

2.29, s 9.21, s

2, 3 2, 3weak, 4weak

148.9, 142.9, 172.8, 113.8, 159.2, 115.0, 98.2, 14.6,

C C C CH C C C CH3

δH (J in Hz)b

gHMBCADb

6.60, s

6, 7

2.41, s

2, 3

c

a Referenced to DMSO-d6 solvent peaks at δC 39.52 ppm and δH 2.50 ppm, recorded at 600 MHz at 30 °C. bReferenced to CDCl3 solvent peaks at δC 77.16 ppm and δH 7.26 ppm, recorded at 500 MHz at 30 °C. cNot detected

Table 4. NMR Data for 4 in CDCl3a δ Cb

position 1 2 3 4 1′ 2′

41.9, 77.5, 123.1, 97.8, 168.4, 20.6,

CH CH C C C CH3

δH (J in Hz)

gCOSY

gHMBCAD

5.72, d (9.0) 6.27, d (9.0)

2 1

2, 3 1, 4, 1′

2.17, s

1′

Referenced to CDCl3 solvent peaks at δC 77.16 ppm and δH 7.26 ppm, recorded at 600 MHz at 30 °C. b13C chemical shifts obtained from correlations observed in gHSQCAD and gHMBCAD spectra. a

Figure 4. NMR data comparison of postulated structures 3a and 3b with maltol and cinariolid (blue color: chemical shifts were swapped to be consistent with each isomer).

Figure 5. Key HMBC correlations of 4.

B3LYP/psCSDZ) (Figure 6). Therefore, the absolute configuration of 4 was determined as (2S)-australasol A. Brominated C4-hydrocarbons have been found previously from the red algae in the family Bonnemaisoniaceae.12,25,26 By analogy to the biosynthesis of mahorone originating from two brominated acetones,17 compounds 1 and 2 could be produced by the condensation of two brominated butan-2-ones by nucleophilic substitution followed by aldol cyclization (Figure S2, Supporting Information). The 4-ethyl-5-methylenecyclopent-2-enone in compounds 1 and 2 is present as a scaffold in sesquiterpenes isolated from plants and marine fungi27 including gochnatiolides A−C,28,29 ainsliatrimers A and B,30

and from H-2 to carbon C-4 (δC 97.8 ppm) and an acetate carbonyl carbon C-1′, establishing the skeleton for 4 (Figure 5). Bromine atoms were placed at C-1 (2 × Br), C-3 (1 × Br), and C-4 (2 × Br) to fulfill molecular formula requirements based on the HRESIMS data, C6H5Br5O2Na. The planar structure of 4 was thus established. An experimental ECD spectrum of 4 displayed a positive CE at 237 nm and two negative CEs at 203 and 299 nm, which compared well to the theoretical ECD spectrum of the (2S)stereoisomer calculated by TDDFT (B3LYP/6-31G(d)//

Table 3. Comparison of Experimental and Calculated 13C Chemical Shifts for 3 in DMSO-d6 and CDCl3 DMSO-d6 3

a

no.

δC (expt)

2 3 4 5 6 7 8 9 DP4

149.7 142.7 172.3 114.2 157.8 114.8 99.4 14.0

CDCl3

3a

3b

3

δC (calcd)

|Δδ|

δC (calcd)

|Δδ|

δC (expt)

143.1 132.4 176.6 106.5 165.7 117.7 97.0 14.8 100%

6.6 10.3 4.3 7.7 7.9 2.9 2.4 0.8

143.1 129.6 175.0 126.0 172.2a 143.4a 89.5 15.0

6.6 13.1 2.7 11.8 14.4 28.6 9.9 1.0 0%

148.9 142.9 172.8 113.8 159.2 115.0 98.2 14.6

3a

3b

δC (calcd)

|Δδ|

δC (calcd)

|Δδ|

143.1 133.6 178.0 109.0 165.7 119.3 98.2 15.3 100%

5.8 9.3 5.2 4.8 6.5 4.3 0 0.7

143.1 130.9 176.4 127.9 172.2a 143.4a 91.1 15.2

5.8 12.0 3.6 14.1 13.0 28.4 7.1 0.6 0%

Chemical shifts were swapped to be consistent with those in 3a. D



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CDCl3 solvent peaks at δH 7.26 and δC 77.16 ppm. Standard parameters were used for the 2D NMR spectra obtained, which included gCOSY, gHSQCAD (1JCH = 140 Hz), gHMBCAD (nJCH = 8 Hz), and ROESY. Low-resolution mass spectra were acquired using a Mariner TOF mass spectrometer (Applied Biosystems Pty Ltd.). High-resolution mass measurements were acquired on a Bruker Solarix 12 T Fourier transform mass spectrometer, fitted with an Apollo API source. For the HPLC isolation, a Waters 600 pump equipped with a Water 966 PDA detector and Gilson 715 liquid handler was used. A Betasil C18 column (5 μm, 21.2 × 150 mm) and YMC Diol column (5 μm, 150 × 10 mm) were used for semipreparative HPLC. A Phenomenex Luna C18 column (3 μm, 4.6 × 50 mm) was used for LC/MS controlled by MassLynx 4.1 software. All solvents used for extraction and chromatography were HPLC grade from RCI Labscan or Burdick & Jackson, and the H2O used was ultrapure water (Arium proVF) from Sartorius Stedim Biotech. Algal Material. A specimen of Ptilonia australasica (phylum Rhodophyta, class Florideophyceae, order Bonnemaisoniales, family Bonnemaisoniaceae) was collected at Pearsons Point, Tasmania, Australia, on August 29, 2002. A voucher specimen (LRB3) has been deposited at Aquenal Pty Ltd., Hobart, Tasmania, Australia. Extraction and Isolation. A freeze-dried sample of P. australasica (20 g) was extracted exhaustively with n-hexane (250 mL), DCM (250 mL), and MeOH (2 × 250 mL), respectively. The DCM and MeOH extracts were combined, and solvents evaporated to yield a yellow residue (0.75 g). This extract was preadsorbed onto C18 (1.0 g) and packed dry into a small cartridge, which was connected to a C18 preparative HPLC column (5 μm, 21.2 × 150 mm). A linear gradient from 100% H2O (0.1% TFA) to 100% MeOH (0.1% TFA) was performed over 60 min at a flow rate of 9 mL/min, and 60 fractions (1.0 min each) were collected. Fraction 28 was obtained as pure compound 2 (0.2 mg, 0.001% dry wt). Fractions 32 and 33 were combined and chromatographed on a Diol HPLC column (5 μm, 150 × 10 mm) from 100% n-hexane to 80% n-hexane/20% 2-propanol at a flow rate of 4 mL/min in 60 min, yielding compound 3 (2 mg, 0.01% dry wt) in fraction 19 and compound 1 (1.8 mg, 0.009% dry wt) in fraction 21. Fraction 37 was chromatographed on a Diol HPLC column (5 μm, 150 × 10 mm) with a linear gradient from 100% nhexane to 80% n-hexane/20% 2-propanol at a flow rate of 4 mL/min in 60 min to obtain 4 (0.8 mg, 0.004% dry wt) eluted at 14 min. (4S)-Ptilone A (1): colorless, amorphous solid; [α]24D +2.6 (c 0.06, MeOH); UV (MeOH) λmax (log ε) 211 (4.0) and 303 (3.9) nm; ECD (c 5.5 × 10−4 M, MeOH) λmax (Δε) 204 (−0.58) and 290 (−0.16) nm; 1H and 13C NMR data, Table 1; (+) HRESIMS m/z 450.7159 [M + H]+ (calcd for C8H779Br4O2+, 450.7174, Δ −3.3 ppm). (4S)-Ptilone B (2): colorless, amorphous solid; UV (MeOH) λmax (log ε) 204 (3.9) and 302 (3.7) nm; ECD (c 4.9 × 10−4 M, MeOH) λmax (Δε) 214 (+0.41) and 292 (−0.74) nm; 1H and 13C NMR data, Table 1; (+) HRESIMS 424.7980 [M + Na] + (calcd for C9H979Br3O3Na+, 424.7994, Δ −3.3 ppm). Ptilone C (3): colorless, amorphous solid; UV (MeOH) λmax (log ε) 225 (4.1) and 290 (3.8) nm; 1H and 13C NMR data, Table 2; (+) HRESIMS m/z 386.7851 [M + H]+ (calcd for C8H679Br3O3+, 386.7862, Δ −2.8 ppm). (2S)-Australasol A (4): colorless gum; UV (MeOH) λmax (log ε) 215 (3.5) nm; ECD (c 9.8 × 10−4 M, MeOH) λmax (Δε) 203 (−1.92), 237 (+1.49), and 299 (−0.09) nm; 1H and 13C NMR data, Table 4; (+) HRESIMS m/z 526.6125 [M + Na]+ (calcd for C6H579Br5O2Na+, 526.6099, Δ 4.9 ppm). Computational Details. ECD calculations of 1, 2, and 4 were performed at 298 K using Maestro and Gaussian 09.37 Molecular mechanics calculations were performed using Macromodel interfaced to the Maestro program (version 2012, Schrödinger). All conformational searches used the MMFFs force field. Conformers having internal relative energies within 3 kcal/mol were subjected to geometry optimization using Jaguar interfaced to the Maestro program at the DFT level using the B3LYP functional and the psCSDZ basis set in MeOH. Optimized conformers were then subjected to TDDFT calculations using the B3LYP functional and the 6-31G(d) basis set in MeOH on Gaussian 09. For each conformer, all of the resultant

Figure 6. Comparison of the experimental (blue) and calculated (red) ECD spectra of 4.

and hirssutanols A and B.31 The 2-methyl-6-vinyl-4H-pyran-4one in compound 3 occurs as a scaffold in some fungal metabolites27 such as mycochromone32 and pyranonigrins.33−36 However, this is the first time that the occurrences of the 4ethyl-5-methylenecyclopent-2-enone and 2-methyl-6-vinyl-4Hpyran-4-one skeletons have been found in algal metabolites. Compounds 1, 3, and 4 were evaluated for cytotoxicity against PC3 cells (Table 5). Compounds 1 and 3 were Table 5. Cytotoxic Evaluation for Compounds 1, 3, and 4 compound

IC50 (μM)a

1 2 3 4 Taxol doxorubicin

0.44 b

10.0 nac 0.002 0.360

a

Each IC50 (μM) was determined as the mean of two independent experiments with triplicate determinations for each concentration. b Not tested due to insufficient quantity. cNot active at 80 μM.

cytotoxic, with IC50 values of 0.44 and 10.0 μM, respectively, while compound 4 was inactive at concentrations up to 80 μM. To investigate whether the growth inhibition induced by 1 and 3 was associated with the regulation of the PC3 cell cycle, the cell cycle distribution in the presence of 1 and 3 was determined (Figure 7). The results showed that 1 induced an increase of cells in the G0/G1 phase and 3 caused an increase of cells in both G0/G1 and S phases. This information suggested that 1 arrested the G0/G1 phase, while 3 induced cell cycle arrest in the both G0/G1 and S phases. In summary, chemical investigation of the red alga P. australasica resulted in four new natural products, ptilones A−C (1−3) and australasol A (4). Ptilones A−C (1−3) possess unique 4-ethyl-5-methylenecyclopent-2-enone and 2-methyl-6vinyl-4H-pyran-4-one skeletons, which have not been previously reported in algal metabolites. Compounds 1 and 3 inhibited the growth of PC3 cells. Compound 1 arrested the G0/G1 phase, while compound 3 arrested both G0/G1 and S phases of the PC3 cell cycle progression.

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EXPERIMENTAL SECTION

General Experimental Procedures. Specific rotations were measured on a JASCO P-1020 polarimeter (10 cm cell). Circular dichroism spectra were recorded on a JASCO J-715 spectropolarimeter. UV spectra were recorded on a CAMSPEC M501 UV/vis spectrophotometer. NMR spectra were recorded at 30 °C on Varian Inova 500 and 600 MHz spectrometers and at 25 °C on a Bruker 900 MHz spectrometer. The 1H and 13C chemical shifts were referenced to the DMSO-d6 solvent peaks at δH 2.50 and δC 39.52 ppm and the E



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Figure 7. Effects of 1 (A) and 3 (B) on PC3 cell cycle progression. humidified environment at 37 °C. Fifty microliters of media containing 500 cells was added to a 384-well microtiter plate (PerkinElmer, part number: 6007660) containing 0.2 μL of a compound. Final compound concentration range tested was 80 μM to 26 nM (80, 26, 8, 2.6, 0.8, 0.26, 0.08, and 0.026 μM) with a final DMSO concentration of 0.4%. Each concentration in media was tested in triplicate and in two independent experiments. Cells and compounds were then incubated for 72 h at 37 °C, 5% CO2, and 80% humidity. The addition to each microtiter well of 60% AlamarBlue solution in media (10 μL; final concentration 10% AlamarBlue) enabled measurement of cell proliferation. The plates were incubated at 37 °C, 5% CO2, and 80% humidity for 24 h, after which the fluorescence of each well was read at excitation 535 nm and emission 590 nm on the PerkinElmer EnVision Multilabel Reader 2104. Eight-point concentration response curves were then analyzed using nonlinear regression, and IC50 values

rotational strengths were converted into Gaussian distributions and summed to give the final calculated ECD spectrum based on the Boltzmann distribution of each conformer. ECD spectra were generated using the SpecDis program.38 Molecular mechanics calculations for 3a and 3b were performed using Macromodel interfaced to the Maestro program. All conformational searches used the MMFFs force field, resulting in one conformer for each compound having internal relative energies within 3 kcal/mol. Stable conformers were then optimized at the DFT level using the B3LYP functional and the psCSDZ basis set in DMSO (Jaguar interfaced to the Maestro program). Optimized structures were submitted to PERCH NMR software (v.2009.1, PERCH Solutions Ltd.) to calculate the theoretical 13C NMR chemical shifts. Cytotoxicity Assay. Human prostate adenocarcinoma cells (PC3) were grown in media RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum. Cells were grown under 5% CO2 in a F



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determined by using GraphPad Prism 5. Taxol and doxorubicin were used during each screening as positive control compounds. Cell Cycle Analysis. Regulation of the cell cycle was assessed using the propidium iodide (PI) DNA staining assay.39 PC3 cells were treated with 1 and 3 at various concentrations for 24 h. Then cells were collected, washed with ice-cold PBS buffer, and fixed with 70% alcohol at −20 °C for 16 h. Fixed cells were further washed with icecold PBS and suspended in 1 mL of solution containing 0.1% Triton X-100, 0.2 mg of RNaseA, and 2 μL of PI (1 mg/mL) at room temperature for 20 min before they were analyzed by flow cytometry FACS CyAn ADP (Beckman Coulter). Data analysis was performed using the ModFit LT 4.0 software (Verity Software House).

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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.5b00989. 1 H NMR, 13C NMR, HSQC, and HMBC spectra of compounds 1−4; proposed biosynthesis of compounds 1−2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +61-7-3735-6000. Fax: +61-7-3735-6001. E-mail: r. quinn@griffith.edu.au. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS One of the authors (T.D.T.) acknowledges Griffith University for the provision of the “Griffith University International Postgraduate Research Scholarship and Griffith University Postgraduate Research Scholarship”. We thank Dr. H. T. Vu at Eskitis Institute, Griffith University, for acquiring the HRESIMS measurements, Ms. M. Nguyen at Eskitis Institute, Griffith University, for the FACS measurements, and Dr. G. Pierens at Center for Advanced Imaging, University of Queensland, for acquiring the 900 MHz NMR measurements. We acknowledge the Australian Research Council for support toward NMR and MS equipment (ARC LE0668477 and ARC LE0237908) and funding (ARC Discovery DP130102400).

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DEDICATION Dedicated to Professors John Blunt and Murray Munro, of the University of Canterbury, for their pioneering work on bioactive marine natural products.

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REFERENCES

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