Lignans from the Australian Endemic Plant ... - ACS Publications

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Lignans from the Australian Endemic Plant Austrobaileya scandens Trong D. Tran,† Ngoc B. Pham,† Ron Booth,‡ Paul I. Forster,‡ and Ronald J. Quinn*,† †

Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland 4111, Australia Queensland Herbarium, DSITI, Brisbane Botanic Gardens, Mt. Coot-tha Road, Brisbane, Queensland 4066, Australia

‡

S Supporting Information *

ABSTRACT: The sole species of the vascular plant family Austrobaileyaceae, Austrobaileya scandens, is endemic to the tropical rainforest of northeastern Queensland, Australia. A single lead-like enhanced fraction of A. scandens showed potent inhibition against human prostate cancer PC3 cells. Chemical investigation of this plant resulted in the isolation of two new aryltetralin lignans, austrobailignans 8 and 9 (1 and 2), and the synthetic compound nicotlactone B (3), newly identified as a natural product together with nine known lignans (4−12). Their structures were established on the basis of spectroscopic analyses. Absolute configurations of the new compounds were determined by quantum chemical electronic circular dichroism (ECD) calculations employing time-dependent density functional theory. The ECD calculations were also used to assign the absolute configuration of marphenol K (4) and revise the absolute configuration of kadsurindutin C (20). Ten out of the 12 isolated compounds inhibited the growth of PC3 cells with IC50 values ranging from micromolar to nanomolar. Marphenol A (5) was found for the first time to induce apoptosis and arrest the S cell cycle phase of PC3 cells.

T

he vascular plant Austrobaileya scandens is a climbing shrub and is the sole representative of the family Austrobaileyaceae, which is found exclusively in the wet tropical rainforests of northeastern Queensland, Australia.1 Austrobaileya and its family are grouped in the so-called “basal angiosperms”, that is, those flowering plant lineages that diverged over 125 million years ago.2 It was first discovered by S.F. Kajewski in 19293 and later described by C.T. White in 1933.4 Initially, this genus was classified into the two species A. scandens and A. maculata.5 A. maculata was then determined to be conspecific with A. scandens on detailed taxonomic analysis.3 So far, chemical investigation of A. scandens has been limited to two studies. A first study on A. scandens aerial parts led to the isolation of 11 compounds, including eight lignans, two sterols, and a sesquiterpene.6 A second study on A. scandens leaf essential oil resulted in the detection of 65 compounds of which the major components were sesquiterpenes.7 As a part of our efforts to discover bioactive natural products with antiprostate cancer activity,8−10 a subset of lead-like enhanced fractions in the Nature Bank library, which was generated from two steps of chromatography to capture constituents having log P less than 5,11 was screened against the human prostate cancer PC3 cells. One fraction from A. scandens potently inhibited the growth of PC3 cells. Subsequent isolation led to the identification of two new compounds, austrobailignans 8 and 9 (1 and 2), and a new natural product, nicotlactone B (3), together with nine known lignans including marphenol K (4),12 marphenol A (5),13 β-peltatin-A-methyl ether (6),14 austrobailignan 1 (7),6 deoxypodophyllotoxin (8),15 and austrobailignans 3 (9), 4 (10), 5 (11), and 7 (12)6 (Figure 1). © XXXX American Chemical Society and American Society of Pharmacognosy

Lignans are characterized by the coupling of two phenylpropanoid units16 and show varied structures containing one to several stereogenic centers.17 Due to the presence of UV chromophores in a molecule, the absolute configurations of optically active lignans used to be determined by electronic circular dichroism (ECD) spectroscopic analysis based on some empirical, semiempirical, or exciton coupling rules.18 However, these approaches are limited in scope due to the need for structural prerequisites.19 Another option, which has presently become an acceptable means for straightforward and nondestructive assignment of absolute configuration, is to predict the ECD spectrum by quantum chemical calculations employing time-dependent density functional theory (TDDFT) and compare it with the experimental data.19−26 Recently, several studies have reported the successful application of the TDDFT ECD calculations for assigning absolute configurations of lignans belonging to some structure subclasses, such as dibenzocyclooctadiene,19 diaryltetrahydrofuran,25 dibenzylbutane,25 seco-lignan,26 and tetrahydrofuran dineolignan.27 This paper reports the isolation and structure elucidation of the new lignans 1 and 2 and the lignan 3 analogue lacking one of the aromatic moieties. Their absolute configurations were determined by quantum chemical TDDFT ECD calculations. The quantum chemical calculations were also employed to assign the absolute configuration of the known compound marphenol K (4) and to revise the absolute configuration of kadsurindutin C (20). The antiproliferative activity of all isolated compounds against the PC3 cells was also evaluated. Received: November 2, 2015

A

DOI: 10.1021/acs.jnatprod.5b00988 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. NMR Spectroscopic Data for 1 and 2 Recorded at 30 °C 1 position

Figure 1. Lignans isolated from the Australian plant A. scandens.

This is the first time that the mode of antiproliferation of a seco-lignan, marphenol A (5), has been reported.

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RESULTS AND DISCUSSION The dried ground samples of A. scandens were extracted exhaustively with n-hexane, CH2Cl2, and MeOH. The CH2Cl2 and MeOH extracts were combined and chromatographed on a C18 HPLC column (MeOH/H2O/0.1% TFA) and subsequently on Diol HPLC columns (n-hexane/isopropyl alcohol) to yield two new lignans (1 and 2) and the lignan 3 analogue, lacking one of the aromatic moieties, along with the nine known compounds 4−12 (Figure 1). Compound 1 was obtained as a white powder. The (−)-HRESIMS spectrum displayed a deprotonated molecule [M − H]− at m/z 383.1131, consistent with the molecular formula C21H20O7. The 1H NMR and gHSQCAD data of 1 (Tables 1 and S1, Supporting Information) showed the presence of four aromatic signals H-5 (δH 6.31), H-2 (δH 6.57), H-2′ (δH 6.27), and H-6′ (δH 6.10), two methylenedioxy groups 3-OCH2O-4 (δH 5.83 and δH 5.82) and 4′-OCH2O-5′ (δH 5.81 and δH 5.80), one methoxy 3′-OCH3 (δH 3.80), three methines H-8 (δH 2.16), H-7′ (δH 4.30), and H-8′ (δH 2.51), one methylene H-7 (δH 2.92 and δH 2.35), and one methyl H-9 (δH 1.06) (Table 1). Two tetrasubstituted aromatic rings were assigned based on HMBC correlations of H-5/C-1, C-3; H-2/ C-6, C-4; H-2′/C-3′, C-4′, C-6′; and H-6′/C-2′, C-4′, C-5′ (Figure 2A). HMBC correlations from two sets of methylenedioxy protons indicated that they were located at C-3 and C-4 and C-4′ and C-5′. The position of a methoxy group (δH 3.80) was assigned to be at C-3′ due to its HMBC correlation to C-3′. COSY correlations led to the establishment of an aliphatic spin system in ring B (Figure 2A). HMBC correlations facilitated the connections from C-7 to C-1 and from C-7′ to C-6 and C-1′. A carboxylic acid group was located at C-8′ based on an HMBC correlation from H-7′ to the C-9′ carbonyl carbon (δC 176.6). Thus, the 2D structure of 1 was established (Figure 2A).

δCa,c

2

δH (J in Hz)a

1 2 3 4 5 6 7

130.4 108.1 147.5 147.3 110.0 132.8 38.8

8

26.2

Hα 2.92, dd (5.4, 16.8) Hβ 2.35, dd (11.4, 16.8) 2.16, m

9

21.1

1.06, d (6.0)

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 3-OCH2O-4

139.6 111.1 144.0 134.9 149.5 105.0 49.2 56.3 176.6 101.5

4′-OCH2O-5′

101.7

6-OCH3 3′-OCH3

56.8

6.57, s

6.31, s

6.27, d (1.2)

6.10, d (1.2) 4.30, d (5.4) 2.51, dd (5.4, 11.4) Hα Hβ Hα Hβ

5.83, 5.82, 5.81, 5.80,

3.80, s

d d d d

(1.2) (1.2) (1.2) (1.2)

δCb,c 121.4 140.2 134.7 147.4 103.8 131.5 26.1

31.6 71.5

135.6 110.5 142.1 133.4 147.7 103.8 42.4 45.5 174.8 100.5 100.6 58.9 56.1

δH (J in Hz)b

6.25, s Hα 3.05, dd (4.8, 16.2) Hβ 2.45, dd (11.4, 16.2) 2.51 (obscured by DMSO) Hα 4.42, t (7.8) Hβ 3.96, dd (8.4, 10.2) 6.39, d (1.2)

6.07, d (1.2) 4.47, d (4.8) 2.91, dd (4.8, 13.8) Hα 5.95, Hβ 5.96, Hα 5.91, Hβ 5.92, 3.97, s 3.75, s

d d d d

(0.6) (0.6) (0.6) (0.6)

a1 H NMR at 600 MHz referenced to residual MeOH-d4 solvent (δH 3.31) and 13C NMR at 150 MHz referenced to residual MeOH-d4 solvent (δC 49.00). b1H NMR at 600 MHz referenced to residual DMSO-d6 solvent (δH 2.50) and 13C NMR at 150 MHz referenced to residual DMSO-d6 solvent (δC 39.52). c13C chemical shifts obtained from correlations observed in gHSQCAD and gHMBCAD spectra.

Figure 2. (A) COSY and key HMBC correlations of 1. (B) Key ROESY correlations of 1.

The relative configuration of 1 was elucidated by the analyses of the 3JHH values and ROESY correlations. A syn relative configuration between H-7′ and H-8′ was deduced by their axial−equatorial relationship via a 3JHH coupling constant of 5.4 Hz. An anti relative configuration of H7′ and H-8′ to H-8 was assigned by a large 3J8,8′ axial−axial coupling constant of 11.4 Hz and confirmed by the ROESY correlation between H-7′ and H-8′ and the absence of a ROESY correlation between H-7′ and H-8 (Figure 2B).28 Therefore, the relative configuration of 1, austrobailignan 8, was determined as (7′R*,8R*,8′S*). Compound 2 was purified as a white powder. A sodium adduct ion [M + Na]+ at m/z 435.1063 observed in the B



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its ROESY correlations to H-7β supported its location at C-2. COSY correlations from H-9α and H-9β to H-8 and HMBC correlations from H-9α to C-8′ and C-9′ allowed the establishment of a five-membered lactone moiety. The 3J7β,8 value of 11.4 Hz, 3J8,8′ value of 13.8 Hz, and 3J8′,7′ value of 4.8 Hz indicated the axial−axial−axial−equatorial relationships of H-7β, H-8, H-8′, and H-7′, hence defining the relative configuration of 2, austrobailignan 9, as (7′R*,8R*,8′R*). A review of ECD data by Loike and Ayres18 indicated that the (7′R)-aryltetralin lignans displayed a first positive Cotton effect (CE) near 290 nm and a second negative CE near 270 nm, while the (7′S)-configuration showed an opposite pattern. Experimental ECD spectra of 1 and 2 (Figure 4A,B) showed similar CE patterns (Experimental Section) to those of (7′R)-aryltetralin lignans, indicating they had (7′R)-configurations. However, the survey on the CEs of some aryltetralin lignans (Figure 5) showed that kadsurindutin C (20) did not follow this rule. Therefore, the assignment of the absolute configuration for the two aryltetralin lignans 1 and 2 required additional consideration. Here, TDDFT ECD calculations were

(+)-HRESIMS spectrum indicated 2 had a molecular formula of C22H20O8. The 1H NMR data of 2 were similar to those of 1 (Tables 1 and S2, Supporting Information). The major differences between the 1H NMR spectra of 2 and 1 were that the former had one extra methoxy signal at δH 3.97 and two additional methylene protons at δH 4.42 and 3.96 but showed one less aromatic proton signal and no methyl doublet. Following 1D and 2D NMR analysis, the same aryltetralin lignan skeleton as 1 was confirmed (Figure 3). An HMBC correlation from the methoxy signal (δH 3.97) to C-2 as well as

Figure 3. COSY and key HMBC and ROESY correlations of 2.

Figure 4. Comparisons of the experimental (blue) and calculated (red) ECD spectra of 1 (A) and 2 (B).

Figure 5. Survey on the Cotton effects of some aryltetralin lignans. Blue: absolute configurations of compounds comply with the Loike and Ayres review. Red: absolute configuration of kadsurindutin C (20) disobeys the Loike and Ayres review. C



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275 nm (Figure 6), the opposite of the experimental data (Figure 5). The absolute configuration of compound 20 was thus revised as (7′R,8S,8′R). Compound 3 was isolated as a white amorphous powder. The (+)-HRESIMS data showed a sodium adduct ion [M + Na]+ at m/z 257.0785, suggesting the molecular formula C13H14O4. Combined 1D and 2D NMR data (Tables 2 and S3, Supporting Information) indicated the presence of one carbonyl carbon (δC 178.0), six aromatic carbons (δC 147.5, 147.5, 131.2, 121.1, 108.0, and 107.0), one methylenedioxy (δC 101.1 ppm), three methines (δC 85.2, 46.2, and 42.3), and two methyl carbons (δC 13.5 and 12.4). A 1,2,4-trisubstituted benzene ring was deduced on the basis of 3J values of H-2 (δH 7.03, d, J = 1.0 Hz), H-5 (δH 6.92, d, J = 8.0 Hz), and H-6 (δH 6.89, dd, J = 1.0, 8.0 Hz) as well as their HMBC correlations to appropriate aromatic carbons. A methylenedioxy group was located at C-3 and C-4 of the aromatic ring according to HMBC correlations. COSY correlations permitted the establishment of an aliphatic system in ring B. An ester carbonyl (δC 178.0) was assigned due to its HMBC correlations with H-8′ and H-9′. An HMBC correlation from H-7 to C-7′ as well as characteristic chemical shifts of an oxygenated methine C-7 indicated a connection from C-7 to C-7′ via an oxygen atom. The assignment of a γ-lactone moiety was further confirmed by a diagnostic IR absorption of the γ-lactone carbonyl at 1769 cm−1.35 HMBC correlations from H-7 to C-2 and C-6 indicated the linkage from C-7 to C-1, establishing the 2D structure of 3 (Figure 7A). This is the first time that compound 3, nicotlactone B, has been isolated from a natural source. Previously, it was reported as a synthetic intermediate for the synthesis of galcatine36 and (+)-galbacin.37 The ROESY data indicated that H-7 had correlations with H-9 and H-8′, while H-8 displayed a correlation with H-9′ (Figure 7A). This evidence suggested that 3 had the same relative configuration as the synthetic compound.37 However, the absolute configuration of synthetic nicotlactone B was defined as (7R,8R,8′R) with a specific rotation of +16.6,37 while the natural product 3 showed a specific rotation of −18, indicating they are enantiomers. The natural product 3 displayed a positive CE at 201 nm and two negative CEs at 217 and 236 nm in the ECD spectrum. The calculated ECD spectrum of (7S,8S,8′S)-nicotlactone B using the TDDFT method showed good agreement with the experimental data (Figure 7B), confirming the absolute configuration of the natural product 3 as (7S,8S,8′S). The structure of compound 4 was defined as 4,4-bis(4hydroxy-3-methoxyphenyl)-2,3-dimethylbutanol (marphenol K) based on NMR (Table S4, Supporting Information) and MS data analyses. This compound was previously isolated from Schisandra propinqua.12 Due to the rotatable bonds at C-8 and C-8′, its relative configuration could not be determined on the basis of a ROESY experiment. Therefore, two stereoisomers 4-I

used to support the determination of the absolution configurations of 1 and 2. The absolute configuration of kadsurindutin C (20) was also re-examined using experimental and calculated ECD spectroscopic data. The calculated ECD spectrum of the (7′R,8R,8′S)-stereoisomer of 1 showed good agreement with the experimental ECD spectrum (Figure 4A), indicating the absolute configuration of austrobailignan 8 (1) as (7′R,8R,8′S). The calculated ECD spectra of (7′R,8R,8′R)-2 also showed a pattern similar to the experimental one (Figure 4B), supporting the assignment of the absolute configuration for 2 as (7′R,8R,8′R)-austrobailignan 9. The TDDFT ECD calculations for 1 and 2 resulted in a positive CE near 290 nm and a negative CE near 270 nm, in agreement with those reported by Ayres and Loike.18 However, the calculated ECD spectrum of kadsurindutin C (20), previously assigned the (7′S,8R,8′S) absolute configuration,34 showed a negative CE at 289 nm and a positive CE at

Figure 6. Calculated ECD spectra of two enantiomers of kadsurindutin C (20).

Table 2. NMR Spectroscopic Data for 3 in DMSO-d6 Recorded at 30 °Ca position

δC

1 2 3 4 5 6 7 8 9 7′ 8′ 9′ 3-OCH2O-4

131.2 107.0 147.5 147.5 108.0 121.1 85.2 46.2 13.5 178.0 42.3 12.4 101.1

δH (J in Hz)b 7.03, d (1.0)

6.92, 6.89, 4.85, 2.03, 0.99,

d (8.0) dd (1.0, 8.0) d (10.0) m d (6.5)

2.45, m 1.16, d (7.0) Hα 6.03, d (1.0) Hβ 6.04, d (1.0)

a1

H NMR at 500 MHz referenced to residual DMSO solvent (δH 2.50) and 13C NMR at 125 MHz referenced to residual DMSO solvent (δC 39.52). b13C chemical shifts obtained from correlations observed in gHSQCAD and gHMBCAD spectra

Figure 7. (A) COSY and key HMBC and ROESY correlations of 3. (B) Comparison of the experimental (blue) and calculated (red) ECD spectra of 3. D



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Figure 8. (A) Two possible stereoisomers (4-I and 4-II) of 4. (B) Comparison of the experimental (blue) ECD spectrum of 4 and the calculated (red, yellow and green) ECD spectra of 4-I and 4-II.

spectroscopic data comparison (NMR, MS, and ECD) with the literature. Antiproliferative activity of the isolated compounds against human prostate cancer PC3 cells was evaluated using the Alamar Blue assay. Ten out of the 12 compounds inhibited the growth of PC3 cells with IC50 values in the nanomolar to micromolar range (Table 4). Compared to the aryltetralin lignans (1 and 2 and 6−10), of which IC50 values were from 0.016 to 2.062 μM, a 2,5-diaryltetrahydrofuran lignan (12) and two seco-lignans (4 and 5) showed less activity with IC50 values of 5.109, 6.363, and 1.907 μM, respectively, while a norlignan (3) and a dibenzylbutane lignan (11) displayed no antiproliferative activity. In particular, the aryltetralin lignans with a lactone ring (2 and 6−8) showed potent inhibition against PC3 cells with IC50 values ranging from 0.016 to 0.050 μM. The inhibition of aryltetralin lignans without a lactone ring (1, 9, and 10) decreased significantly from 16- (7 versus 10) to 129-fold (6 versus 9) potency. This indicated that opening the lactone ring in the aryltetralin lignans resulted in the loss of activity. A comparison of the inhibitory activity of three pairs 2/6, 7/8, and 9/10 showed that compounds with a 4′,5′-dimethoxy group were approximately 3-fold more effective than those with a 4′,5′methylenedioxy group. In addition, antiproliferative activity of the pairs 1/9, 2/7, and 6/8 were comparable, suggesting the replacement of a carboxylic group with a methyl group at C-8′ (1-9) or the presence of a methoxy group at C-2 (2/7 and 6/8) did not have an important effect on antiproliferation. Compounds 1, 2, and 6−10 share a core structure of an aryltetralin lignan with a potently cytotoxic natural product, podophyllotoxin, and two anticancer drugs, etoposide and teniposide. Structure− activity relationship of the aryltetralin core structure was previously under intensive investigations.45 However, this is the first time that the seco moiety at C-8 and C-8′, the presence of a 4′,5′-methylenedioxy group, and a methoxy group at C-2 in the aryltetralin lignans have been discussed. The aryltetralin lignans are well-known to cause apoptosis by inducing the G2/M phase arrest, disrupt microtubules, and

and 4-II were possible for 4 (Figure 8A). Density functional theory NMR calculations together with the DP4 probability analysis, which have recently emerged as powerful tools to assign the relative and absolute configurations of natural products,38−43 were used to examine the relative configuration of 4. 13C and 1H NMR chemical shifts were calculated at the mPW1PW91/6-31G(d)//B3lyp/6-31G(d) levels using multistandard references with benzene for sp2-hybridized carbons and methanol for sp3-hybridized carbons to provide the calculated chemical shifts for each conformer.44 The calculated data were compared with the experimental data based on the statistical parameters, including the correlation coefficient (R2), the corrected mean absolute error (CMAE), and the DP4 probability to determine which of the two stereoisomers fit with the experimental NMR data of 4. The results indicated that both calculated 13C and 1H NMR chemical shifts of the isomer 4-I correlated closely with the experimental data (Figure S24, Supporting Information). They also showed CMAE values (Table 3) lower than those of isomer 4-II. Significant differences were observed in DP4 probabilities. In particular, 4-I had 100% probability for 13C NMR chemical shifts and 97.6% probability for 1H NMR chemical shifts, while 4-II had 0.0% probability for 13C NMR chemical shifts and 2.4% probability for 1H NMR chemical shifts (Table 3). These probabilities indicated that the assignment of the isomer 4-I as the structure of 4 was at a high level of confidence.38 This information suggested that compound 4 had (8S*,8′R*) relative configuration. The absolute configuration of 4 was also determined by comparing its experimental and calculated ECD spectra. As shown in Figure 8B, the calculated ECD spectrum of the (8R,8′S)-isomer agreed with the experimental data. Therefore, the structure of 4, marphenol K, was established as shown. Other known lignans, marphenol A (5),13 β-peltatin-A-methyl ether (6),14 austrobailignan 1 (7),6 deoxypodophyllotoxin (8),15 austrobailignan 3 (9),6 austrobailignan 4 (10),6 austrobailignan 5 (11),6 and austrobailignan 7 (12),6 were assigned by E



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Table 3. Comparison of Experimental and Calculated 13C and 1H NMR Chemical Shifts in DMSO-d6 for 4-I and 4-II and Their Statistical Parameters 4-I

a

4-II

4-I

4-II

δC (exptl)

δCa (calcd)

δCb (scaled)

δCa (calcd)

δCb (scaled)

δH (exptl)

δHa (calcd)

δHb (scaled)

δHa (calcd)

δHb (scaled)

1 2 3 4 5 6 7

119.6 136.4 111.7 147.1 144.3 115.0 65.0

120.6 137.4 111.0 144.9 143.3 113.8 63.8

121.4 138.1 111.8 145.6 144.0 114.5 64.7

120.9 137.6 110.2 145.0 143.3 113.8 61.8

121.6 138.6 110.7 146.1 144.4 114.4 61.6

6.71

6.86

6.90

6.83

6.86

6.89

6.54

6.59

6.64

6.67

8 9 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 4-OCH3 3′-OCH3 CMAEb DP4

35.1 9.2 136.4 111.7 147.2 144.2 115 119.6 54.7 35.0 11.5 55.4 55.4

36.0 10.2 137.3 111.8 145.8 144.0 114.6 121.8 55.1 35.3 12.3 52.5 52.6

34.3 15.7 137.5 110.9 145.2 143.4 114.2 120.6 55.2 40.1 11.5 51.6 51.7

33.6 14.7 138.4 111.4 146.3 144.4 114.8 121.2 54.9 39.5 10.4 51.2 51.3

6.62 3.17 3.27 1.58 0.65

6.58 3.25 3.31 1.51 0.46

6.62 3.33 3.38 1.61 0.57

6.56 3.16 3.56 1.52 0.61

6.59 3.22 3.59 1.55 0.64

6.86

6.69

6.74

6.64

6.67

6.63 6.91 3.48 2.66 0.47 3.57 3.61

6.61 6.90 3.75 2.25 0.49 3.53 3.54

6.64 6.93 3.78 2.28 0.52 3.56 3.57 0.15 ± 0.11 2.4%

no.

35.0 9.1 136.6 111.0 145.1 143.3 113.8 121.1 54.1 34.3 11.2 51.6 51.7 1.0 ± 0.9 100.0%

1.8 ± 1.7 0.0%

6.62 6.71 3.43 2.54 0.55 3.73 3.74

6.59 6.87 3.40 2.57 0.36 3.49 3.54 0.12 ± 0.08 97.6%

Calculated chemical shifts using benzene and MeOH as standard references. bValues obtained after systematic errors were removed.

The increase in nuclear and mitochondrial intensities, which displayed brighter blue-whitish and brighter red-whitish fluorescent appearance, respectively, in the images (Figure 9B), indicated that the nuclei and mitochondria had more packed masses.54 The brighter blue-whitish fluorescence also demonstrated the condensation of chromatins in the cell nucleus, which is considered as a hallmark feature of the apoptotic pathway of programmed cell death.55−57 Treatment with 5 also caused the mitochondrial texture in PC3 cells to become more aggregated compared to the typical elongated shape in the vehicle DMSO.54 The cellular phenotypes of chromatin condensation, mitochondrial aggregation, and cell roundness have been known to be associated with the induction of apoptosis.54,55 In order to explore whether the growth inhibition induced by 5 was associated with the regulation of the PC3 cell cycle, the cell cycle distribution in the presence of 5 was analyzed by flow cytometry (Figure 10). Treatment of PC3 cells with 5 at the concentrations of 0.1, 0.3, and 1 μM for 24 h caused an increase of cells in the S phase from 27.5 to 35.1% compared to that of a control (15.8%), while there was a concomitant reduction in the number of cells in the G0/G1 and G2/M phases in each case. This information suggested that compound 5 induced S cell cycle arrest in PC3 cells. In conclusion, three out of 12 compounds, austrobailignans 8 (1) and 9 (2) and nicotlactone B (3), isolated from the Australian endemic plant A. scandens were new naturally occurring lignans. Quantum chemical TDDFT ECD calculations supported the assignment of absolute configurations for the new compounds and the two known compounds, marphenol K (4) and kadsurindutin C (20). Antiproliferative evaluation of isolated compounds against PC3 cells indicated that compounds 1, 2, 4−10, and 12 isolated from the active

Table 4. Antiproliferative Evaluation of Compounds 1−12 Against PC3 Cells compound

IC50 (μM) or % inhibitiona

1 2 3 4 5 6 7 8 9 10 11 12 taxol doxorubicin

1.63 0.047 15% 6.363 1.907 0.016 0.050 0.018 2.062 0.823 27% 5.109 0.002 0.360

Each IC50 (μM) or % inhibition at 10 μM was determined as the mean of two independent experiments with triplicate determinations for each concentration. a

inhibit the enzyme topoisomerase II.45 However, a mode of antiproliferation of the 7,8-seco-lignans with 15 natural products found up to now13,46−53 has not been reported. Therefore, in this study, compound 5 was chosen to further examine the mechanism of its antiproliferation. An immunofluorescence assay with three markers for cell membrane, nuclei, and mitochondria indicated that compound 5 had effects on cell morphology, nuclei, and mitochondria of PC3 cells in a dose-dependent manner (Figure 9A). In particular, cells treated with 5 increased the cell size and induced cells to become round, with an increase in cell and mitochondrial area. F



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Figure 9. (A) Phenotypic profile for cell proliferation study of 5 (results were normalized by a vehicle DMSO). (B) Representative images of the PC3 cells treated with 5, 1.7, and 0.5 μM of 5 and DMSO in three channels: Hoechst 33342 (blue), MitoTracker (red), and CellMask (yellow) (scale bar: 50 μm); chromatin condensation (white arrow), aggregated mitochondrial texture (green arrow), and round and larger cell size (cyan arrow) compared with vehicle DMSO. spectra, which included gCOSY, gHSQCAD (1JCH = 140 Hz), gHMBCAD (nJCH = 8 Hz), and ROESY. LRMS data were acquired using a Mariner TOF mass spectrometer (Applied Biosystems Pty Ltd.). HRMS data 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 were used. A Betasil C18 column (5 μm, 21.2 × 150 mm) and Hypersil BDS C18 column (5 μm, 10 × 250 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 H2O (Arium proVF) from Sartorius Stedim Biotech. Plant Material. A specimen of the plant of A. scandens (order Austrobaileyales, family Austrobaileyaceae) was collected at the

fraction fully accounted for its antiproliferation and provided additional structure−activity relationship information. This is the first time that the mode of antiproliferation of marphenol A (5) has been reported.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 polarimeter (10 cm cell). Electronic circular dichroism spectra were measured 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. The 1H and 13C NMR chemical shifts were referenced to the DMSO-d6 solvent peaks at δH 2.50 and δC 39.52. Standard parameters were used for the 2D NMR G



Article

Figure 10. Effect of 5 on PC3 cell cycle progression. from 100% H2O (0.1% TFA) to 100% MeOH (0.1% TFA) was performed over 60 min at a flow rate of 9.0 mL/min, and 60 fractions (1.0 min each) were collected. Fractions 30−32 were combined and chromatographed on the Hypersil BDS C18 HPLC column (5 μm, 10 × 250 mm) with a linear gradient from 15% MeOH (0.1% TFA)/85% H2O (0.1% TFA) to 60% MeOH (0.1% TFA)/40% H2O (0.1% TFA) at a flow rate of 4 mL/min in 60 min to yield 3 (0.5 mg, 0.0015% dry weight). Fractions 35−39 were combined and loaded on the Betasil C18 preparative HPLC column (5 μm, 21.2 × 150 mm) with a linear gradient from 25% MeOH (0.1% TFA)/75% H2O (0.1% TFA) to 100% MeOH (0.1% TFA) at a flow rate of 9 mL/min in 60 min, yielding 4 (0.8 mg, 0.0024% dry weight), 5 (0.8 mg, 0.0024% dry weight), 2 (0.8 mg, 0.0023% dry weight), 6 (2.1 mg, 0.0062% dry weight), 7 (4.5 mg, 0.0132% dry weight), and 8 (5.8 mg, 0.0171% dry weight). Fractions 44−48 were combined and subjected to the Betasil C18 preparative HPLC column (5 μm, 21.2 × 150 mm) with a linear gradient from 50% MeOH (0.1% TFA)/50% H2O (0.1% TFA) to 100% MeOH (0.1% TFA) at a flow rate of 9 mL/min in 60 min/ yielding 12 (3.4 mg, 0.0100% dry weight), 9 (3.8 mg, 0.0112% dry weight), and 10 (2.9 mg, 0.0085% dry weight). (7′R,8R,8′S)-Austrobailignan 8 (1): white amorphous powder, [α]D25 +33 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 214 (4.3), 245 (3.2), and 282 (3.0) nm; ECD (MeOH) λmax (Δε) 217 (−0.71), 240 (+0.23), 275 (−0.40), and 291 (+0.06) nm; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table 1; (−) HRESIMS m/z 383.1131 [M − H]− (calcd for [C21H19O7]− 383.1136, Δ −1.3 ppm). (7′R,8R,8′R)-Austrobailignan 9 (2): white amorphous powder, [α]D25 −62 (c 0.05, MeOH); UV (MeOH) λmax (log ε) 208 (4.2) and 285 (3.1) nm; ECD (MeOH) λmax (Δε) 212 (−3.59), 247 (+0.87), 274 (−0.53), and 288 (+0.16) nm; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table 1; (+) HRESIMS m/z 435.1063 [M + Na]+ (calcd for [C22H20O8Na]+ 435.1050, Δ 3.0 ppm). (7S,8S,8′S)-Nicotlactone B (3): white amorphous powder, [α]D25 −18 (c 0.03, MeOH); UV (MeOH) λmax (log ε) 205 (4.3), 236 (3.4), and 280 (2.1) nm; ECD (MeOH) λmax (Δε) 201 (+2.84), 217 (−0.44), and 236 (−0.25) nm; IR (film) νmax 2918, 2849, 1769, 1208, 1141, and 1033 cm−1; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table 2; (+) HRESIMS m/z 257.0785 [M + Na]+ (calcd for [C13H14O4Na]+ 257.0784, Δ 0.4 ppm). (8R,8′S)-Marphenol K (4): white amorphous powder, [α]D25 −28 (c 0.02, MeOH); UV (MeOH) λmax (log ε) 212 (4.1) and 286 (3.1) nm;

Wooroonooran National Park, Queensland, Australia in 1998. A voucher specimen (BRIAQ0605720) has been deposited at the Queensland Herbarium, Queensland, Australia. A second specimen of the plant of A. scandens (order Austrobaileyales, family Austrobaileyaceae) was collected at the State Forest 756 Mount Father Clancy, Queensland, Australia in 2000. A voucher specimen (BRIAQ0607146) has also been stored at Queensland Herbarium, Queensland, Australia. Extraction and Isolation. The first dried ground sample of A. scandens (8.2 g) was extracted exhaustively with n-hexane (2 × 100 mL), CH2Cl2 (3 × 100 mL), and MeOH (3 × 100 mL). The CH2Cl2 and MeOH extracts were combined and filtered through polyamide gel to remove tannins, and then the solvents were evaporated to yield a yellow residue (0.1 g). This crude extract was preadsorbed onto C18 (1.0 g) and packed dry into a small cartridge, which was connected to a Betasil 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 9.0 mL/min, and 60 fractions (1.0 min each) were collected. The pure compound 7 (2 mg, 0.0244% dry weight) was obtained from fractions 34−35, and the pure compound 8 (2.2 mg, 0.0268% dry weight) was obtained from fraction 37. Fractions 31−32 were combined and chromatographed on a Hypersil BDS C18 HPLC column (5 μm, 10 × 250 mm) with a linear gradient from 15% MeOH (0.1% TFA)/85% H2O (0.1% TFA) to 60% MeOH (0.1% TFA)/40% H2O (0.1% TFA) at a flow rate of 4 mL/min in 60 min to yield 4 (0.2 mg, 0.0024% dry weight). Fractions 40−41 were combined and chromatographed on a Diol HPLC column (5 μm, 150 × 10 mm) from 100% n-hexane to 50% n-hexane/50% n-hexane/isopropyl alcohol (2:8) at a flow rate of 4 mL/min in 30 min to afford 1 (0.5 mg, 0.0061% dry weight) and 12 (1.1 mg, 0.0134% dry weight). Fractions 49−50 were also combined and separated on the Diol HPLC column (5 μm, 150 × 10 mm) from 100% n-hexane to 80% n-hexane/ 20% n-hexane/isopropyl alcohol (2:8) at a flow rate of 4 mL/min in 30 min to yield 9 (3.2 mg, 0.0390% dry weight) and 11 (4.1 mg, 0.0500% dry weight), respectively. The second dried ground sample of A. scandens (34.0 g) was extracted exhaustively with n-hexane (3 × 150 mL), CH2Cl2 (4 × 150 mL), and MeOH (4 × 150 mL). The CH2Cl2 and MeOH extracts were combined, and the solvents were evaporated to yield a yellow residue (0.9 g). This crude 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 H



Article

ECD (MeOH) λmax (Δε) 202 (−4.54), 223 (+1.55), and 273 (−0.48) nm; 1H (600 MHz) and 13C (150 MHz) NMR data are summarized in Table S4 in the Supporting Information; (+) HRESIMS m/z 369.1678 [M + Na]+ (calcd for [C20H26O5Na]+ 369.1672, Δ 1.6 ppm). Computational Details. TDDFT ECD Calculations for 1−4. All molecular mechanics and quantum chemical calculations were performed using Macromodel and Jaguar interfaced to the Maestro program (version 2012, Schrödinger). All conformational searches used the MMFF force field. The searches were done in the solution phase (water). All conformers having internal relative energies within 2 kcal/mol were subjected to geometry optimization at the density functional theory level using the B3LYP functional and the 6-31G(d,p) basis set. Optimized conformers obtained in the former calculations were subjected to TDDFT calculations using the B3LYP functional and the 6-31G(d) basis set in MeOH (SCRF) on Gaussian 09.58 For each conformer, all of the resultant rotational strengths were converted into Gaussian distributions with a 0.15−0.25 eV half bandwidth and summed to give the final calculated ECD spectrum based on the Boltzmann weighted average.59,60 Calculated ECD spectra were generated using the SpecDis program.61 DFT NMR Calculations for 4-I and 4-II. Molecular mechanics and quantum chemical calculations for 4-I and 4-II were performed in DMSO using the same procedure as above. Single-point calculations in DMSO with the mPW1PW91 functional and the 6-31G(d) basis set were employed to provide the shielding constant of 13C and 1H nuclei. Meanwhile, the same procedure was applied on benzene and MeOH. Final 13C and 1H NMR chemical shifts were obtained as the results of the Boltzmann weighted average. The chemical shifts were calculated according to the equation

535 nm and an emission of 590 nm on the PerkinElmer EnVision Multilabel Reader 2104. Eight-point concentration response curves were analyzed using nonlinear regression, and IC50 values were determined by using GraphPad Prism 5. Paclitaxel and doxorubicin were used during each screening as positive controls. Cell Cycle Analysis. Cell cycle was assessed using the PI staining assay. PC3 cells were treated with 5 at various concentrations including 0 (DMSO only), 0.1, 0.3, and 1 μM for 24 h. The cells were collected, washed with ice-cold PBS buffer, and fixed with 70% EtOH at −20 °C for 16 h. Fixed cells were washed with ice-cold 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, California, USA). Data analysis was performed using the ModFit LT 4.0 software (Verity Software House, Maine, USA). Immunofluorescence Staining. Cells (2000 cells/well) were allowed to seed in a 96-well plate (CellCarrier, PerkinElmer, part number 6005558) containing 0.4 μL of a compound at room temperature for 30 min and placed under 5% CO2 in a humidified environment at 37 °C. After 72 h incubation, 100 μL of a solution containing 1/1000 Hoechst 33342 (Molecular Probes, Invitrogen, USA), 1 μM MitoTracker Deep Red (Molecular Probes, Invitrogen, USA), and 1/1000 CellMask plasma membrane (Molecular Probes, Invitrogen, USA) was added to each well and incubated for 30 min at 37 °C. Stained cells were washed once with PBS to remove excess stains and then replaced with 100 μL of PBS before being imaged. Automated Microscopy and Image Analysis. Plates were imaged with an Operetta high-content wide-field fluorescence imaging system coupled to Harmony software (PerkinElmer). Wells in a 96-well plate were captured at 25 locations per well at 20× magnification at three wavelengths (350 nm, Hoechst 33342; 554 nm, CellMask plasma membrane; 644 nm, MitoTracker Deep Red). The three images were combined and analyzed using the Harmony software. The analysis protocol involved the following steps: (1) each cell nucleus was identified using Hoechst stain; (2) the cell cytoplasm as defined from CellMask fluorescence; and (3) cells that overlapped the border of the image were excluded from the analysis. Each concentration was repeated in at least two separate experiments. To obviate observer bias, the image analysis was automated using the same parameters for every image.

x δcalcd = σref − σ x + δref

where δxcalcd is the calculated shift for nucleus x (in ppm); σx is the shielding constant for nucleus x; σref and δref are the shielding constant and chemical shift of the reference compound computed at the same level of theory, respectively. Calculated shielding constant of reference standards in DMSO are σCbenzene = 70.6178, σCMeOH = 143.1737 ppm, σHbenzene = 24.5876, and σHMeOH = 28.6513 ppm. Chemical shifts of reference standards in DMSO are δCbenzene = 128.30 and δCMeOH = 48.59 ppm and δHbenzene = 7.37 and δHMeOH = 3.16 ppm.62 Statistical parameters were used to quantify the agreement between experimental and calculated data: • The slope (a), the intercept (b), and the correlation coefficient (R2) were determined from a plot of δcalcd against δexptl for each particular compound. • Systematic errors during the shift calculation were removed by empirical scaling according to δscaled = (δcalcd) − b)/a. • The corrected mean absolute error was defined as ∑i n= 1|δscaled − δexptl|/n. • DP4 parameters were calculated by using the online applet at http://www-jmg.ch.cam.ac.uk/tools/nmr/. Antiproliferative Assay. Human prostate adenocarcinoma cells (PC3) and human neonatal foreskin fibroblast (noncancer cells) were grown in media RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum. Cells were grown under 5% CO2 in a 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 10 μM to 3.3 nM (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. Cell proliferation was measured with the addition of 10 μL of a 60% Alamar Blue (Invitrogen) solution in media to each well of the microtiter plate to give a final concentration of 10% Alamar Blue. The plates were incubated at 37 °C, 5% CO2, and 80% humidity within 24 h. The fluorescence of each well was read at an excitation of

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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.5b00988. 1 H NMR, HSQC, and HMBC spectra of 1−3 and DFT calculations of 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: r.quinn@griffith.edu.au. Phone: +61-7-3735-6009. Fax: +61-7-3735-6001. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS 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 the HRESIMS measurements. We acknowledge the Australian Research Council for support toward NMR and MS equipment (ARC LE0668477 and ARC LE0237908) and funding (ARC Discovery DP130102400). I



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Article

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