Article Cite This: J. Nat. Prod. 2017, 80, 3143−3150
pubs.acs.org/jnp
Bibenzyl-Based Meroterpenoid Enantiomers from the Chinese Liverwort Radula sumatrana Xiao Wang,†,§ Lin Li,†,§ Rongxiu Zhu,‡ Jiaozhen Zhang,† Jinchuan Zhou,† and Hongxiang Lou*,† †
Department of Natural Products Chemistry, Key Lab of Chemical Biology of Ministry of Education, School of Pharmaceutical Sciences, Shandong University, Jinan 250012, People’s Republic of China ‡ School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, People’s Republic of China S Supporting Information *
ABSTRACT: Six new pairs of bibenzyl-based meroterpenoid enantiomers, (±)-rasumatranin A−D (1−4) and (±)-radulanin M and N (5 and 6), and six known compounds were isolated from the adnascent Chinese liverwort, Radula sumatrana. Their structures were elucidated based on spectroscopic data and chiral phase HPLC-ECD analyses. The structures of 1 and 7 were also confirmed by single-crystal X-ray diffraction analysis. Cytotoxicity tests of the isolated compounds showed that 6-hydroxy-3-methyl-8phenylethylbenzo[b]oxepin-5-one (8) showed activity against the human cancer cell lines MCF-7, PC-3, and SMMC-7721, with IC50 values of 3.86, 6.60, and 3.58 μM, respectively, and induced MCF-7 cell death through a mitochondria-mediated apoptosis pathway.
M
cytotoxic activities against MCF-7 human breast cancer, PC-3 human prostate cancer, and SMMC-7721 hepatic cancer cell lines. The isolation, structural elucidation, and cytotoxicity evaluation of the isolated compounds and the apoptosis of MCF-7 cells induced by 8 are reported herein.
ultiple terpenoids, bibenzyls, and other aromatic compounds from liverworts (Hepaticae) show fascinating biological activities, including cytotoxic, antifungal, antimicrobial, and antioxidant properties.1 Chemical investigations of Radula Dum., the only genus in the family Radulaceae, 2,3 which comprises approximately 150−200 species, showed a characteristic series of linear and cyclic prenyl bibenzyls.4−10 Furthermore, some Radula species metabolize bibenzyl cannabinoids, which can be divided into three types: o-cannabichromene-, o-cannabicyclol-, and tetrahydrocannabinol-type compounds, the first type being the most prevalent.9 Naturally occurring bibenzyl cannabinoids are rare, and almost all of them are distributed in Radula species, suggesting that they could be chemosystematic markers of Radulaceae, which belongs to the order Jungermanniales.7 As part of an ongoing search for new bioactive substances from Chinese liverworts,11−13 the chemical constituents from the adnascent liverwort R. sumatrana Steph., collected on Mount Fanjing (2300 m), Guizhou Province, People’s Republic of China, in May 2014, were investigated. Accordingly, four new pairs of bibenzyl/monoterpenoid hybrid enantiomers, (±)-rasumatranins A−D (1−4); two new pairs of prenyl bibenzyl enantiomers, (±)-radulanins M and N (5 and 6); and the known (±)-bibenzyl/o-cannabicyclol hybrid (7),14 6hydroxy-3-methyl-8-phenylethylbenzo[b]oxepin-5-one (8),15 radulanin A (9),16 (±)-6-phenethyl-2-(prop-1-en-2-yl)-2,3dihydrobenzofuran-4-ol (10),16 (±)-radulanin I (11),17 and (+)-radulanin J (12)17 were isolated. This represents the first isolation of bibenzyl iso-tetrahydrocannabinoids from Radula species.5−7 The isolated compounds were evaluated for their © 2017 American Chemical Society and American Society of Pharmacognosy
Received: May 4, 2017 Published: December 7, 2017 3143
DOI: 10.1021/acs.jnatprod.7b00394 J. Nat. Prod. 2017, 80, 3143−3150
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Table 2. 13C NMR Spectroscopic Data for Compounds 1−4a
RESULTS AND DISCUSSION (±)-Rasumatranin A (1) was obtained as colorless needles following crystallization from MeOH. Its molecular formula of C24H30O4 was assigned based on the (+)-HRESIMS protonated molecule at m/z 383.2222 [M + H]+ (calcd 383.2217, C24H31O4) and 13C NMR spectroscopic data (Table 2), Table 1. 1H NMR Spectroscopic Data for Compounds 1−4a
position
1
2
3
4
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
δH, mult. (J in Hz)
3.86, dd (6.0, 2.7) 3.40, t (2.7)
3.75, dd (6.6, 3.1) 3.86, br s
2.44, dt (10.5, 2.7) 1.46, dd (13.1, 4.5) 1.23, m
1.83, dt (12.9, 3.1) 1.40, m
2.26, dd (14.0, 3.0) 3.36, ddd (14.0, 12.8, 6.0) 1.73, m
1.93, dq (13.8, 2.1) 1.63, td (13.8, 4.5) 1.36, s 4.68, t (1.8) 4.37,s 1.68, s 6.12, d (2.4)
1.94, m
2.48, dt (14.4, 8.7) 2.39, ddd (14.4, 9.9, 1.5) 1.28, s
1.63, td (13.8, 4.8) 1.35, s 0.81, s
4.71, d (3.0) 1.13, s
6.24, d (2.4) 2.83, m
6.25, d (2.5) 3.98, m
2.49, m
2.58, m
2.83, m
2.93, m
2.70, m
2.65, m
7.27, m 7.27, m 7.18, m
7.28, m 7.24, m 7.14, t (7.3)
3.50, d (6.0)
3.24, d (6.6) 3.66, s 7.96, s
2
3.63, t (3.6)
3
3.66, br s
4
2.37, dt (12.6, 3.5) 1.36, m
5a 5b 6a 6b 7 9a 9b 10 1′ 3′ 5′ 1″a
1.30, s 0.80, s 1.21, s 6.10, d (2.4)
4″, 8″ 5″, 7″ 6″
6.25, d (2.4) 3.96, ddd (13.0, 11.2, 5.5) 2.57, ddd (13.0, 11.5. 5.5) 2.74, ddd (13.5, 11.5, 5.5) 2.65, ddd (13.5, 11.2, 5.5) 7.24, m 7.24, m 7.16, m
HO-2 HO-8 HO-6′
4.38, d (3.5) 3.47, s 8.02, s
1″b 2″a 2″b
a
2.01, dd (13.2, 5.4) 1.64, d (13.2)
8.10, s
1.26, m
1.21, s 6.10, d (2.5)
a
position
1
2
3
4
1 2 3 4 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1″ 2″ 3″ 4″, 8″ 5″, 7″ 6″
77.1 73.4 37.2 46.2 21.8 35.7 26.7 72.4 24.3 30.4 100.5 158.1 114.7 144.1 109.1 157.6 36.0 39.2 143.3 129.4 128.9 126.4
77.1 72.0 41.6 50.2 23.6 39.8 24.3 147.8 111.7 23.3 100.4 158.5 109.6 144.8 108.4 157.3 35.8 38.2 143.4 129.1 129.1 126.6
77.4 73.1 38.1 55.1 21.8 40.5 24.1 72.3 24.0 33.0 100.5 158.7 111.1 145.8 109.3 157.3 36.0 39.0 143.7 128.9 129.3 126.3
81.6 64.7 53.0 21.8 48.0 24.8 97.5 79.7 23.0 30.4 138.7 154.7 110.6 137.8 112.6 154.4 33.2 37.7 143.0 129.1 129.3 126.6
Spectra recorded at 150 MHz in acetone-d6.
24.3). The 1D NMR data in the aromatic region and the two methylenes (δC 39.2, δH 3.96/2.57 and δC 36.0, δH 2.74/2.65) represented the typical characteristics of a bibenzyl skeleton,10,14 as affirmed by the 1H−1H COSY correlations between H-6″ and H-4″/5″/7″/8″ and the HMBC crosspeaks from H-5′ to C-1″ and from H-4″/8″ to C-2′ (Figure 1),
1.43, s 6.32, d (2.9) 6.47, d (2.9) 2.91, m
2.80, m
7.27, m 7.22, m 7.17, tt (7.2, 1.3)
8.07, s
Spectra recorded at 600 MHz in acetone-d6.
Figure 1. Selected HMBC (H → C) and 1H−1H COSY (bold H−H) correlations of 1, 4, and 5.
accounting for 10 indices of hydrogen deficiency. The IR and UV spectra of 1 showed traits of the presence of aromatic moieties. The 1H NMR spectroscopic data of 1 (Table 1) in acetone-d6 displayed a phenolic group [δH 8.02 (s)], seven aromatic protons [δH 7.24 (m, 4H), 7.15 (m), 6.25 (d, J = 2.5 Hz), and 6.10 (d, J = 2.5 Hz)], four methylenes [δH 3.96 (ddd, J = 13.0, 11.2, 5.5 Hz), 2.57 (ddd, J = 13.0, 11.5. 5.5 Hz); 2.74 (ddd, J = 13.5, 11.5, 5.5 Hz), 2.65 (ddd, J = 13.5, 11.2, 5.5 Hz); 2.01 (dd, J = 13.2, 5.4 Hz), 1.64 (d, J = 13.2 Hz); and 1.36 (m, 2H)], three methines [δH 3.63 (t, J = 3.6 Hz), 3.66 (br s), and 2.37, dt (J = 12.6, 3.5 Hz)], and three methyl singlets (δH 0.80, 1.21, and 1.30). The 13C NMR and HSQC data showed 12 aromatic carbons at δC 158.1−100.5; four methylenes (δC 39.2, 36.0, 35.7, and 21.8); three methines, including one oxygenated (δC 73.4, 46.2, and 37.2); two oxygenated tertiary carbons (δC 77.1 and 72.4); and three methyl carbons (δC 30.4, 26.7, and
leaving two indices of hydrogen deficiency for two additional rings. The aliphatic NMR signals indicated the presence of a monoterpenoid moiety. Thus, 1 could be a bibenzyl-based terpenoid. The monoterpenoid moiety was further elaborated via the HMBC correlations from H3-7 to C-1, C-2, and C-6; H3-9/10 to C-4 and C-8; and H-3 to C-2, C-4, and C-5. In conjunction with the spin system, C-2(H)−C-3(H)−C-4(H)−C-5(H2)−C6(H2), identified via the 1H−1H COSY spectrum, these data were reminiscent of the presence of a menthane structural unit (Figure 1). The HMBC correlations from HO-6′ to C-1′, C-5′, and C-6′ confirmed the location of the phenolic group. The HMBC correlations from H3-7 to C-2′ and from H-3 to C-3′ confirmed the fusion of the bibenzyl moiety and the menthane moiety via C-3 and C-3′ and an ether linkage between C-1 and 3144
DOI: 10.1021/acs.jnatprod.7b00394 J. Nat. Prod. 2017, 80, 3143−3150
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Figure 2. Selected NOESY correlations (H↔H) of 1, 2, and 4.
C-2′. Thus, the 2D structure of 1 as a bibenzyl/isotetrahydrocannabinol,18−20 a new type of bibenzyl cannabinoid, was established. The relative configuration of 1 was defined via NOESY data (Figure 2). The connection of the monoterpenoid and bibenzyl moieties through the pyran ring implied that H-3 and Me-1 were equatorial.21 Generally, the chair conformation of cyclohexane is more stable when the more bulky groups occupy equatorial positions; thus H-4 was axially oriented. The proton of the 2-hydroxy group correlated with H-4 and H3-7, while H-2 exhibited a correlation with H3-7, suggesting that HO-2 was axially oriented. This conclusion was validated by a single-crystal X-ray diffraction analysis using Cu Kα radiation (Figure 3, CCDC 1465205), which revealed that 1 was a pair of enantiomers. The mixture, with a specific rotation approaching zero, was further analyzed by HPLC-ECD using a CHIRALPAK AD-H analytical column, presenting two peaks in a ratio of approximately 1:1 with inverse electronic circular dichroism (ECD) spectra (Figures S10, S11, Supporting Information). The matched calculated and experimental ECD spectra of 1a (Figure S11, Supporting Information) indicated (1S,2R,3S,4R) and (1R,2S,3R,4S) absolute configurations for 1a and 1b, respectively. (±)-Rasumatranin B (2) was acquired as a yellowish, amorphous solid and assigned a molecular formula of C 24 H 28 O 3 based on the protonated molecule at m/z 365.2118 [M + H]+ (calcd 365.2111), as revealed by (+)-HRESIMS and 1D NMR data (Tables 1 and 2). The IR and UV spectra closely resembled those of 1. The aromatic protons in the 1H NMR spectrum of 2 were similar to those of 1, indicating the presence of the bibenzyl unit. The major differences between the 1H NMR spectra of the two compounds involved the replacement of one of the methyl groups of the gem-dimethyl moiety in 1 by an olefinic methylene group (δH 4.68, 4.37). The 13C NMR and HSQC spectra confirmed the olefinic methylene moiety (δC 111.7). The 2D structure of 2 was determined from the HMBC correlations from H-9a/H-9b/H3-10 to C-4 and C-8. The relative configuration of 2 was defined by the NOE correlations (Figure 2) of H-2 with H-4 and H3-7, H-4 with H3-10, and H-3 with H3-10, similar to those of gambogellic acid A.22
Figure 3. X-ray ORTEP drawings of 1a and 1b.
Additionally, because H-3 was equatorial, the coupling constant of H-3 (t, J = 2.7 Hz) implied that H-2 (dd, J = 6.0, 2.7 Hz) and H-4 (dt, J = 10.5, 2.7 Hz) were both axial. The specific rotation of 2 was nearly zero. The chiral phase HPLC-ECD analysis showed that 2a/2b were in a ratio of 1:1, and the mirror imagetype ECD spectra of 2a/2b revealed their enantiomeric relationship (Figures S21, S22, Supporting Information). The calculated and experimental ECD spectra of 2a were in agreement, thus permitting assignment of the absolute configurations of 2a and 2b as (1S,2S,3S,4R) and (1R,2R,3R,4S), respectively. 3145
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was racemic (Figures S43, S44, Supporting Information). The absolute configurations of 4a and 4b were deduced by comparison of the experimental and calculated ECD spectra as (1S,2R,3S,7S) and (1R,2S,3R,7R), respectively. Radulanin M (5) was acquired as a colorless solid. The molecular formula C19H20O3 was ascertained from the sodium adduct ion at m/z 319.1306 [M + Na]+ (calcd 319.1305) in the (+)-HRESIMS data. The UV spectrum showed three absorption bands at 301.6, 265.4, and 220.2 nm. The 1D NMR (Table 3) and HSQC data revealed a bibenzyl core, a
(±)-Rasumatranin C (3), obtained as a whitish solid, possessed the same molecular formula as 1, C24H30O4, as determined from the protonated molecule at m/z 383.2218 [M + H]+ (calcd 383.2217) in the (+)-HRESIMS spectrum. The 1D NMR spectroscopic data (Tables 1 and 2) were similar to those of 1. The HSQC spectrum contained the following aliphatic signals: three methyl; four methylene; three methine, including one oxygenated (δC 73.1); and two oxygenated tertiary carbons (δC 77.5, 72.3). The cross-peaks in the 1H−1H COSY and HMBC spectra suggested that 3 had the same 2D structure as 1; thus, they were configurational isomers. The relative configuration of 3 was established from the NOESY data (Figure S28, Supporting Information), which were similar to those of gambogellic acid A: H-2 correlated with H-4, H-6b, and H3-7.22 A correlation of H-4 with H-6b was also observed, which supported the axial orientation of H-4. Thus, 3 was a C-2 epimer of 1. The orientation of C-8 has almost no effect on the 13 C NMR chemical shift of C-4 when C-2 is a methylene.23 Therefore, the chemical shift differences of C-4 and C-6 in 1 and 3 (Table 2) might be ascribed to the different orientation of HO-2.24 Compound 3 was also found to be a pair of enantiomers based on the two chiral phase HPLC peaks in a ratio of approximately 1:1 with mirror image-type ECD curves (Figures S32, S33, Supporting Information). This suggested that the absolute configurations of 3a and 3b were (1S,2S,3S,4R) and (1R,2R,3R,4S), respectively. Rasumatranin D (4) was isolated as a yellowish oil. The molecular formula C24H28O4 was assigned according to the ammonium adduct ion at m/z 398.2327 [M + NH4]+ (calcd 398.2326) in the (+)-HRESIMS data, implying 11 indices of hydrogen deficiency. The 1H NMR data (Table 1) showed aromatic proton resonances analogous to those of 1. Twelve aromatic carbon resonances belonging to the bibenzyl moiety appeared in the 13C NMR data (Table 2), entailing a tricyclic substituent. Together with the HSQC data, the aliphatic substituents were determined to include three methyl (δC 23.0, 24.8, and 30.4); two methylene (δC 21.8 and 48.0); three methine, including one dioxygenated (δC 53.0, 64.7, and 97.5); and two oxygenated tertiary carbons (δC 81.6, and 79.7). The HMBC correlations from H3-9/10 to C-3 and C-8; H3-6 to C1, C-2, and C-5; and H-7 to C-2, C-3, and C-8 and the 1H−1H COSY spin system of C-5(H2)−C-4(H2)−C-3(H)−C-2(H)− C-7(H) indicated that the substituent had a carbon skeleton of an iridane-type monoterpenoid (Figure 1). The chemical shifts of C-1 (δC 81.6), C-7 (δC 97.5), C-1′ (δC 138.7), and C-2′ (δC 154.7) indicated that the monoterpenoid and bibenzyl moieties were connected across two oxygen atoms. Similar to the patterns of tetrasubstituted bibenzyls and 3′,4′,5′-trisubstituted flavonoids,25−28 the oxy-substituents were located at C-1′, C-2′, and C-6′. The HMBC correlation from H-1″ to C-5′ and the coupling constants of H-3′ and H-5′ (4J = 2.9 Hz) indicated that the monoterpenoid substituent was connected with the bibenzyl moiety at C-1′ and C-2′. However, no HMBC correlations were observed between the monoterpenoid and bibenzyl moieties. An NOE correlation between H3-6 and H-3′ (Figure 2) indicated that they were spatially close.14 Thus, the 2D structure of 4 was elucidated as shown. The relative configuration was determined based on the NOESY correlations (Figure 2) of H-2 with H3-6 and H3-6 with H-7, suggesting that they had the same orientation. NOESY data analysis also showed that both H-3 and H3-6 correlated with H5a, implying that they had the same orientation. The specific rotation and chiral phase HPLC-ECD analysis suggested that 4
Table 3. 1H and 13C NMR Spectroscopic Data for Compounds 5 and 6a 5 position 2 3 4 5 6 7 8 9 10 11 1′ 1″ 2″ 3″ 4″ 5″ 6″ 7″ 8″ HO-3 HO-6 HO-2″
δH, mult. (J in Hz) 3.94, s 5.79, d (12.4) 6.69, d (12.4) 6.49, d (1.4) 6.40, d (1.4)
1.29, s 2.78, m 2.87, m 7.25, 7.25, 7.17, 7.25, 7.25, 3.90, 8.63,
m m tt (7.0, 1.6) m m s s
6 δC 79.0 72.4 135.7 118.2 156.7 110.2 143.6 111.7 161.0 113.0 26.0 37.9 37.7 142.4 128.9 129.0 126.5 129.0 128.9
δH, mult. (J in Hz) 3.94, s 5.79, d (12.3) 6.69, d (12.3) 6.52, s 6.41, s
1.29, s 2.76, m 2.86, m
6.84, 7.02, 6.74, 7.09, 3.88, 8.60, 8.28,
d (7.7) t (7.7) t (7.4) d (7.4) s s s
δC 79.2 72.7 135.8 118.5 157.0 110.4 144.6 111.9 161.3 113.1 26.3 36.5 32.7 128.9 156.0 115.8 127.9 120.3 130.8
a1
H and 13C NMR data recorded in acetone-d6 at 400 and 100 MHz, respectively.
methyl (δH 1.29, δC 26.0), an oxygenated methylene (δH 3.94, δC 79.0), an oxygenated tertiary carbon (δC 72.4), and two olefinic protons (δH 6.69, δC 118.2 and δH 5.79, δC 135.7). Based on the HMBC correlations (Figure 1) from H3-1′ to C-2, C-3, and C-4; H2-2 to C-10; H-5 to C-6 and C-10; and HO-6 to C-11, 5 was deduced as a bibenzyl with a dihydrooxepin skeleton, similar to radulanin A (9).16 Subsequently, 5 was also determined to be a pair of enantiomers based on the HPLCECD data (Figures S54, S55, Supporting Information). Radulanin N (6) was obtained as a white powder. (+)-HRESIMS analysis indicated a molecular formula of C19H20O4 based on the sodium adduct ion at m/z 335.1256 [M + Na]+ (calcd 335.1254). The main difference in the 1D NMR spectra (Table 3) of 5 and 6 was that 6 had one additional phenolic group (δH 8.28). Two triplets [δH 6.74 (t, J = 7.4, H-7″) and 7.02 (t, J = 7.7, H-6″)] and two doublets [δH 7.09 (t, J = 7.4, H-8″) and 6.84 (t, J = 7.7, H-5″)] indicated that the Ar-OH group was ortho substituted. The proposed structure was consistent with the HMBC correlations from HO-4″ to C3″ and C-4″. The chiral phase HPLC-ECD data suggested that 6 was racemic (Figures S65, S66, Supporting Information). 3146
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the tested compounds, 8 exhibited the strongest inhibition of cell viability in several cancer cell lines. MCF-7 cells might be insensitive to many chemotherapeutics in apoptosis induction due to its characteristic caspase-3 deficiency.32 The underlying mechanism of the cytotoxic activity of 8 in MCF-7 cells was investigated. The exposure of 8 to MCF-7 cells decreased cell viability in a dose- and time-dependent manner (Figure 5A). The apoptotic morphologies of MCF-7 cells were altered, including nucleus shrinkage, when exposed to 8 (2 μM) for 24 h, and granular apoptotic bodies formed after treatment with 4 μM 8 for at least 6 h, shown by 4′,6-diamidino-2-phenylindole (DAPI) staining (Figure 5B). Cells were stained with annexin V-FITC and propidium iodide (PI) and subsequently analyzed by flow cytometry, revealing that the proportion of cells stained with annexin V increased in 8-treated cells in a dose-dependent manner. Treatment with 8 (2 μM) for 24 h induced an accumulation of early (annexin V+/PI−) and late (annexin V+/ PI +) apoptotic cells compared with the control. The percentages of apoptotic cells were 47.37% and 58.59% at 2 and 4 μM, respectively (Figure 5C). Mitochondria are a vital organelle involved in the intrinsic mitochondria-mediated apoptosis pathway.33 To investigate the effect of 8 on the mitochondrial membrane potential (MMP, ΔΨ) in MCF-7 cells, the MMP was examined using JC-1 dye. Compound 8 caused a loss of ΔΨ in a dose-dependent manner for MCF-7 cells (Figure 5D). After exposure to concentrations of 1, 2, and 4 μM 8 for 24 h, the ΔΨ was reduced to 82.1%, 53.2%, and 31.8% of the value of the 0 μM control, respectively (for 2 and 4 μM, P < 0.01 vs the 0 μM control). Western blotting was used to analyze the responses of apoptosis-related protein to 8. Pro-apoptotic Bax increased slightly when exposed to 8, while the expression of anti-apoptotic Bcl-2 decreased significantly (Figure 5E). Cleaved caspase-9 and poly ADPribose polymerase (PARP) were also observed. In conclusion, the induction of apoptosis by 8 activates the mitochondriamediated apoptosis pathway in MCF-7 cells.34,35
Compound 7 was previously synthesized through a photochemical reaction,14 but its relative configuration was not discussed. Single-crystal X-ray diffraction analysis with Cu Kα radiation (CCDC 1499110) revealed that 7 was a racemate ([α]20D = 0 (c 0.1, MeOH)), as shown in Figure 4. The chiral
Figure 4. X-ray ORTEP drawings of 7a and 7b.
phase HPLC-ECD spectra showed that the ECD spectrum of 7a had Cotton effects similar to those of cannabiorcicyclolic acid (Figures S67, S68, Supporting Information).29 Thus, the absolute configurations of 7a and 7b were (1S,2S,3R,7S) and (1R,2R,3S,7R), respectively. Chiral phase HPLC-ECD analyses of 10−12 showed that 10 and 11 were racemic mixtures (Figures S69−S71, Supporting Information). Asakawa et al. showed that R. javanica biosynthesized (−)-radulanin I and (+)-radulanin J and that the methylation of (−)-radulanin I led to (−)-radulanin J.17 In the present study, it was observed that R. sumatrana produced (±)-radulanin I (11) and only (+)-radulanin J (12) ([α]20D = +31 (c 0.1, MeOH)). In conclusion, R. sumatrana biosynthesizes various bibenzylbased meroterpenoid enantiomers, with 1−4 and 7 as bibenzyl/ monoterpenoid hybrids, and 5/6 and 8−12 as bibenzyl/ hemiterpenoid hybrids. As reported,30 monoterpenoids are biosynthetically derived from geranyl diphosphate (GPP, C10), and the monoterpenoid and bibenzyl moieties are conjugated through C−O and C−C oxidative coupling, respectively. The putative biosynthetic pathways of the isolated bibenzyl/ menthane and bibenzyl/iridane hybrids are proposed in Scheme S1 in the Supporting Information. The cytotoxic activities of all compounds were evaluated against MCF-7 breast cancer, PC-3 human prostate cancer, and SMMC-7721 human hepatoma cell lines using the MTT assay,31 with adriamycin as a positive control (Table 4). Among
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General Experimental Procedures. Melting points were measured with an X-6 micromelting point apparatus. Optical rotations were obtained on a GYROMAT-HP polarimeter. UV data were recorded on a Shimadzu UV-2450 spectrophotometer. ECD spectra were obtained on a Chirascan spectropolarimeter. IR spectra were recorded on a Nicolet iN 10 micro FTIR spectrometer. NMR spectra were recorded on a Bruker Avance DRX-600 spectrometer operating at 600 (1H) and 150 (13C) MHz and a Bruker AV 400 spectrometer operating at 400 (1H) and 100 (13C) MHz in acetone-d6 or CDCl3 with tetramethylsilane as the internal standard. HRESIMS experiments were carried out on an LTQ-Orbitrap XL instrument. HPLC was performed on a system consisting of an Agilent 1200 G1311A quaternary pump equipped with an Agilent 1200 G1322A degasser, an Agilent 1200 G1329B 1260ALS, an Agilent 1200 G1315D DAD detector, and an Eclipse XDB-C18 5 μm column (4.6 × 250 mm and 9.4 × 250 mm). HPLC-ECD spectra were recorded on a JASCO LCNetII/ADC system equipped with a JASCO PU-2089 Plus quaternary gradient pump, JASCO CD-2095 Plus chiral detector, and a CHIRALPAK AD-H 5 μm column (4.6 × 250 mm), while the preliminary HPLC analyses were carried out with a Waters Delta 600 with a Waters 996 photo diode array detector and the same column. All solvents used were of analytical grade. Silica gel (200−300 mesh; Qingdao Haiyang Chemical Co. Ltd., Qingdao, P. R. China), RP C18 silica gel (40−63 μm, FuJi), and Sephadex LH-20 (25−100 μm; Pharmacia Biotek, Denmark) were used for column chromatography (CC). Thin-layer chromatography (TLC) was carried out with silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.). Compounds
Table 4. Cytotoxicity of Compounds 2, 7, 8, and 11 against Human Cancer Cellsa compound
MCF-7
PC-3
SMMC-7721
2 7 8 11 adriamycin
38.23 13.99 3.86 24.69 0.70
>40 19.59 6.60 >40 0.42
>40 14.50 3.58 34.21 0.66
EXPERIMENTAL SECTION
Results are expressed as the mean IC50 values in μM from triplicate measurements. a
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Figure 5. Compound 8-induced apoptosis in MCF-7 cells. (A) MCF-7 cells were treated with 8 (2.5−20 μM) for 12−48 h. The cell viability rate denoted as a percentage of the control rate (8, 0 μM) at the concurrent time point was estimated using the MTT assay. All data are expressed as the mean ± SD obtained of triplicate experiments. (B) Fluorescence micrographs (DAPI staining) of MCF-7 cells treated or untreated with 8 for the indicated time. (C) Representative apoptotic profile of MCF-7 cells treated with or without 8 from flow cytometric analysis. The corresponding histograms quantified by densitometry are expressed on the right side. All data are expressed as the mean ± SD obtained of triplicate experiments, **p < 0.01 vs 0 μM control group. (D) Compound 8 induced the loss of ΔΨ. After MCF-7 cells were treated with 8 (1−4 μM) for 24 h, ΔΨ was measured by flow cytometry. **P < 0.01, vs 0 μM control group. (E) Compound 8 induced intrinsic mitochondria-mediated apoptosis based on Western blot analysis of apoptosis-related protein expression. were visualized under UV light and by spraying with H2SO4−EtOH (1:9, v/v) followed by heating. Plant Material. The liverwort R. sumatrana was collected from the Fanjing Mountains, Guizhou Province, People’s Republic of China, in May 2014, and authenticated by Prof. Yuan-Xin Xiong, College of Life Sciences, Guizhou University, People’s Republic of China. A voucher specimen (No. 20140517003) has been deposited at the Department of Natural Products Chemistry, School of Pharmaceutical Sciences, Shandong University, People’s Republic of China. Extraction and Isolation. Air-dried and smashed plant material of R. sumatrana (33.2 g) was extracted with 95% EtOH. The crude extract (3.8 g) was chromatographed on a silica gel column (200−300 mesh) and eluted with a petroleum ether/acetone gradient (200:1 to 5:1) to give four fractions (Fr. 1−4). Fr. 1−3 were subjected to RP-18 silica gel CC (MeOH/H2O, 5:5 to 9:1), yielding subfractions 1A, 1B, 2A, 2B, 3A, and 3B. Fr. 1A was purified by HPLC (MeOH/H2O, 80:20, 1.8 mL/min) to afford 11 (4.5 mg, tR = 29.8 min), and Fr. 1B was purified by HPLC (MeOH/H2O, 87:13, 1.8 mL/min) to give 8 (2.7 mg, tR = 29.8 min). Separation of Fr. 2A by HPLC (MeOH/H2O, 70:30, 1.5 mL/min) gave 12 (3.0 mg, tR = 72.2 min), 9 (10.3 mg, tR = 75.3 min), and 10 (2.1 mg, tR = 80.6 min), while Fr. 2B gave 7 (20.9 mg, tR = 38.6 min) after HPLC separation (MeOH/H2O, 83:17, 1.8 mL/min). Fr. 3A was purified by HPLC (MeOH/H2O, 63:37, 1.8 mL/min) to yield 5 (4.3 mg, tR = 48.4 min). Fr. 3B was purified by HPLC (MeOH/H2O, 73:27, 1.8 mL/min) to afford 2 (2.5 mg, tR = 32.4 min) and 4 (1.1 mg, tR = 33.9 min). Fr. 4 was separated on
Sephadex LH-20 (CH2Cl2/MeOH, 5:5) to yield Fr. 4A and Fr. 4B. Fr. 4A was purified by HPLC (MeOH/H2O, 52:48, 1.8 mL/min), affording 3 (1.2 mg, tR = 42.9 min) and 1 (1.6 mg, tR = 51.9 min). Purification of Fr. 4B via HPLC (MeOH/H2O, 60:40, 1.8 mL/min) afforded 6 (3.7 mg, tR = 25.9 min). (±)-Rasumatranin A (1): pink needles (MeOH); mp 230−232 °C; UV (MeOH) λmax (log ε) 207 (4.47) nm; IR νmax 3386, 2927, 1612, 1594 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (acetone-d6), see Tables 1 and 2; HRESIMS m/z 383.2222 [M + H]+ (calcd 383.2217). (±)-Rasumatranin B (2): orange, amorphous solid (MeOH); UV (MeOH) λmax (log ε) 206 (4.55) nm; IR νmax 3372, 2936, 1612 cm−1; 1 H NMR (600 MHz) and 13C NMR (150 MHz) data (acetone-d6), see Tables 1 and 2; HRESIMS m/z 365.2118 [M + H]+ (calcd 365.2111). (±)-Rasumatranin C (3): pink, amorphous powder (MeOH); UV (MeOH) λmax (log ε) 208 (4.42) nm; IR νmax 3351, 2932, 1615 cm−1; 1 H NMR (600 MHz) and 13C NMR (150 MHz) data (acetone-d6), see Tables 1 and 2; HRESIMS m/z 383.2218 [M + H]+ (calcd 383.2217). (±)-Rasumatranin D (4): yellowish oil (MeOH); UV (MeOH) λmax (log ε) 203 (4.49) nm; IR νmax 3355, 2927, 1601, 1456 cm−1; 1H NMR (600 MHz) and 13C NMR (150 MHz) data (acetone-d6), see Tables 1 and 2; HRESIMS m/z 398.2327 [M + NH4]+ (calcd 398.2326). 3148
DOI: 10.1021/acs.jnatprod.7b00394 J. Nat. Prod. 2017, 80, 3143−3150
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TX (10 mL of PBS + 10 μL of 10% Tritonx-100) was used to wash the coverslips three times, which were quickly mounted using mounting medium PBS/glycerol = 1:1 (v/v) and analyzed by fluorescence microscopy. Analysis of Apoptosis by Flow Cytometry. Cells were seeded at a density of 1 × 105 cells/mL onto six-well plates overnight and then exposed to 5% serum medium containing different concentrations of 7 for 24 h. The cells were harvested and washed with cold PBS. After centrifuging at 200g for 5 min, the supernatant was removed, and the cells were resuspended in 400 μL of binding buffer and incubated at room temperature in the dark for 15 min with 5 μL of annexin VFITC, followed by 5 μL of PI (50 mg/L) for another 5 min. According to the manufacturer’s instructions, the apoptotic ratio was analyzed by flow cytometry (Becton Dickinson, USA) using WinMDI 2.9 software. Measurement of MMP. Cells (1 × 105 cells/well) were seeded in six-well plates, incubated for 12 h, and then treated with various concentrations of 8 at 37 °C for 24 h. The cells were harvested, washed once with PBS, and stained with 10 μg/mL JC-1 for 20 min at 37 °C. Next, the cells were washed once with PBS and kept at 4 °C during the measurements. The MMP (ΔΨ) was measured by flow cytometry. The green emission was analyzed in fluorescence channel 1 (FL-1), and the orange-red emission in channel 2 (FL-2). ΔΨ was expressed as the ratio of FL-2/FL-1. Western Blotting Assay. Cells exposed to the compounds were harvested, washed twice with PBS, collected, and lysed with RIPA buffer containing a fresh protease inhibitor mixture (50 μg/mL aprotinin, 0.5 mM PMSF, 1 mM sodium orthovanadate, 10 mM NaF, and 10 mM glycerolphosphate). Protein concentrations were quantified using the BCA assay. Equal amounts of protein extracts were separated via 9−12% SDS/polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat milk in TBST buffer (20 mM Tris-buffered saline and 0.5% Tween 20) for 1 h at room temperature prior to incubation with specific antibodies at 4 °C. Following washing with TBST and incubating with peroxidase-conjugated secondary antibodies, the immunoblot proteins were visualized in an enhanced chemiluminescence detection system (Millipore, Germany) and exposed to X-ray films. Statistical Analysis. Data are presented as the mean (SD) of all replicates and were analyzed by one-way ANOVA followed by Tukey’s t test. A P-value < 0.05 was considered statistically significant. Statistical analysis was performed using the SPSS 14.0 statistical software program (SPSS, Inc., Chicago, IL, USA).
(±)-Radulanin M (5): colorless crystals (MeOH); UV (MeOH) λmax (log ε) 220 (4.41) nm; IR νmax 3218, 2923, 1572, 1065 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 3; HRESIMS m/z 319.1306 [M + Na]+ (calcd 319.1305). (±)-Radulanin N (6): white solid (MeOH); UV (MeOH) λmax (log ε) 220 (4.40) nm; IR νmax 3314, 1618, 1454 cm−1; 1H NMR (400 MHz) and 13C NMR (100 MHz) data (CDCl3), see Table 3; HRESIMS m/z 335.1256 [M + Na]+, calcd 335.1254. X-ray Crystal Structure Analysis.36 Colorless needles of 1 and 7 were obtained from a MeOH solution. All crystallographic data were collected on a Bruker D8 venture diffractometer equipped with an APEXII CCD using Cu Kα radiation (λ = 1.541 78 Å) at 293(2) K. The APEX2 Software Suite was used for cell refinement and data reduction. The structure was refined with full-matrix least-squares calculations on F2 using SHELXL-2014/7. Crystal Data for Compound 1. C24H30O4, M = 382.48, monoclinic system, space group P21, a = 16.1803(7) Å, b = 16.2266(6) Å, c = 16.6650(6) Å, α = 90.00°, β = 106.243(2)°, γ = 90.00°, V = 4200.8(3) Å3, Z = 8, μ(Cu Kα) = 0.646 mm−1, F(000) = 1648. A crystal of dimensions 0.23 × 0.20 × 0.15 mm3 was selected for measurements. A total of 32 276 reflections, collected in the θ range of 3.939° to 68.479°, yielded 3860 unique reflections (Rint = 0.0706). The final stage converged to R1 = 0.0617 (wR2 = 0.1624) for 3083 observed reflections [with I > 2σ(I)] and 262 variable parameters, R1 = 0.0754 (wR2 = 0.1753) for all unique reflections, and a goodness-of-fit = 1.045. The Flack parameter is 0.1(4). Crystal Data for Compound 7. C24H28O2, M = 348.46, monoclinic system, space group P21, a = 14.8049(12) Å, b = 6.0967(5) Å, c = 23.0240(18) Å, α = 90.00°, β = 107.414(3)°, γ = 90.00°, V = 1982.9(3) Å3, Z = 4, μ(Cu Kα) = 0.562 mm−1, F(000) = 752. A crystal of dimensions 0.23 × 0.21 × 0.14 mm3 was selected for measurements. A total of 25 083 reflections, collected in the θ range of 3.173° to 68.211°, yielded 3624 unique reflections (Rint = 0.0458). The final stage converged to R1 = 0.0443 (wR2 = 0.1081) for 2936 observed reflections [with I > 2σ(I)] and 240 variable parameters, R1 = 0.0560 (wR2 = 0.1166) for all unique reflections, and a goodness-of-fit = 1.048. The Flack parameter is −0.1(6). Cell Lines and Cell Culture. Human hepatocellular carcinoma cell line SMMC-7721 and human breast adenocarcinoma cell line MCF-7 were purchased from the Shanghai Institute for Biological Sciences (SIBS), China Academy of Sciences (China). Human prostate cancer cell line PC-3 was obtained from the American Type Culture Collection (ATCC). The cell lines were cultured in RPMI-1640 (HyClone) medium containing 10% fetal bovine serum (Sijiqing Company Ltd.), 100 units/mL penicillin G, and 100 μg/mL streptomycin in a stable environment at 37 °C and 5% CO2. MTT Assay. Cells were seeded into 96-well plates at (3−5) × 103 cells/well in advance. After adherence overnight, various concentrations of all compounds were added, followed by incubation for the indicated time. Then, 12 μL of MTT (5.0 mg/mL) solution was added, and the plates were incubated for another 4 h at 37 °C. After removing the medium with MTT, 150 μL/well DMSO was added to the purple formazan crystals. The optical density at 570 nm was determined using a microplate reader (Bio-Rad 680) to assess cell viability. The IC50 values were calculated using Graphpad Prism 5. All experiments were performed in triplicate experiments. The cell viability inhibitory ratio was calculated through comparison to the vehicle control using the following formula:
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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.7b00394. Calculation details, proposed potential biosynthetic pathways of 1, 4 and 7; 1D and 2D NMR, HRESIMS, IR, UV, and ECD spectra for compounds 1−6; and chiral phase HPLC analysis of compounds 1−6 and 9−12 (PDF)
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AUTHOR INFORMATION
Corresponding Author
Cell viability ratio (%)
*Tel: +86-531-8838-2012. Fax: +86-531-8838-2019. E-mail:
[email protected].
= (A570 sample − A570 blank)/(A570 control − A570 blank) × 100%
ORCID
Hongxiang Lou: 0000-0003-3300-1811
DAPI Staining. Cells were seeded on 12 mm round glass coverslips in 24-well plates overnight and treated with various concentrations of 7 at 37 °C for the indicated time. After the cells were washed with cold phosphate-buffered saline (PBS) and fixed using a 1:1 mixture of cold MeOH/acetone (v/v), the cells were washed with PBS and stained with DAPI (4 μg/mL) for 10 min at room temperature. Then, PBS-
Author Contributions §
X. Wang and L. Li contributed equally to this paper.
Notes
The authors declare no competing financial interest. 3149
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(30) Dewick, P. M. Medicinal Natural Products: A Biosynthetic Approach; John Wiley & Sons: New York, 2009; p 550. (31) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589−601. (32) Jänicke, R. U. Breast Cancer Res. Treat. 2009, 117, 219−221. (33) Kamal, A.; Tamboli, J. R.; Ramaiah, M. J.; Adil, S. F.; Pushpavalli, S. N. C. V. L; Ganesh, R.; Sarma, P.; Bhadra, U.; PalBhadra, M. Bioorg. Med. Chem. 2013, 21 (21), 6414−6426. (34) Robbins, D.; Zhao, Y. J. Signal Transduction 2012, 2012, 101465. (35) Chipuk, J. E.; Kuwana, T.; Bouchier-Hayes, L.; Droin, N. M.; Newmeyer, D. D.; Schuler, M.; Green, D. R. Science 2004, 303, 1010− 1014. (36) Detailed crystallographic data of compounds 1 and 7 have been deposited in the Cambridge Crystallographic Data Centre as CCDC 1465205 and 1499110, respectively. The data can be obtained free of charge via http://www.ccdc.cam.ac.uk/data_request/cif (or from the CCDC, 12 Union Road, Cambridge CB21EZ, U.K.; fax: + 44-1223336−033; e-mail:
[email protected]).
ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China (nos. 81630093 and 81473107).
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