Diterpenoids and Lignans from Cephalotaxus fortunei - Journal of

Article pubs.acs.org/jnp

Diterpenoids and Lignans from Cephalotaxus fortunei Jin-Xin Zhao,† Yao-Yue Fan,† Jin-Biao Xu,† Li-She Gan,‡ Cheng-Hui Xu,† Jian Ding,† and Jian-Min Yue*,† †

State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zuchongzhi Road, Shanghai 201203, People’s Republic of China ‡ College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, People’s Republic of China S Supporting Information *

ABSTRACT: Five new diterpenoids including two Cephalotaxus troponoids (1 and 2), two 17-nor-cephalotane-type diterpenoids (3 and 4), and an abietane-type diterpenoid (5), two new lignans (6 and 7), and a new trisnorneoligan (8) along with eight known compounds were identified from the twigs and leaves of Cephalotaxus fortunei. The structure of 11-hydroxyhainanolidol was revised as 10-hydroxyhainanolidol (9) by X-ray crystallographic data. Compounds 3 and 4 are the first examples of 17-nor-cephalotane-type diterpenoids that are likely the biosynthesis precursors of the co-occurring troponoids (e.g., 1, 2, and 9). Compound 1 exhibited cytotoxic activities against HL-60 and A-549 cells with IC50 values of 0.77 ± 0.05 and 1.129 ± 0.057 μM, respectively.

T

he genus Cephalotaxus, the sole member of the Cephalotaxaceae family, has only nine species worldwide, of which seven species and three varieties are native to China.1 The plants in this genus were extensively studied for the antitumor Cephalotaxus alkaloids and troponoids.2 Although a large array of alkaloids2 were identified in the past half a century, only five Cephalotaxus troponoids2,3 were discovered. Recently,4 three diterpenoids with a C20 intact carbon skeleton (named cephalotane) and two additional troponoids were isolated from C. mannii, and four related norditerpenoids were identified from C. sinensis. It is particularly noted that cephalotane-type diterpenoids were believed to be the biosynthesis precursors of the degraded 17-nor-diterpenoids, e.g., troponoids, and a more rational biosynthesis pathway for this compound class has thus been put forward.4a Cephalotaxus fortunei Hook. f. is a major species of China.1 Previously, a number of alkaloids were isolated from the plants,5 and seven abietane-type diterpenoids were identified from its cell cultures.6 As part of a continuing study of the structurally interesting and/or bioactive cephalotane-type diterpenoids and their degraded products from Cephalotaxaceae plants, two new Cephalotaxus troponoids (1 and 2), two new 17-norcephalotane-type diterpenoids (3 and 4), a new abietane-type diterpenoid (5), two new lignans (6 and 7), and a new trisnorneoligan (8) along with eight known compounds were identified from C. fortunei. Compounds 3 and 4 represent the first examples of 17-nor-cephalotane-type diterpenoids featuring an unprecedented cycloheptene unit. The structure of 11-hydroxyhainanolidol7 was revised as 10-hydroxyhainanolidol (9) by X-ray crystallographic data and biosynthesis considerations.4,8 Cytotoxic evaluation showed that compound 1 exhibited significant activities against HL-60 and A-549 tumor © 2017 American Chemical Society and American Society of Pharmacognosy

cell lines. Based on the cytotoxic results, the structure−activity relationships for the cephalotane-type related diterpenoids are briefly discussed.

■

RESULTS AND DISCUSSION Compound 1 was acquired as a pale yellow, amorphous powder with a specific rotation of [α]27D = −9 (c 0.3, MeOH). Its (−)-HRESIMS data displayed a deprotonated ion [M − H]− at m/z 325.1067 (calcd 325.1076) for a molecular formula of C19H18O5 (11 indices of hydrogen deficiency) with the aid of its 13C NMR data. Its NMR data (Tables 1 and 2) showed resonances assignable to two methyls (δH 0.99, d, J = 7.1 Hz; 2.60, s), two methylenes, five methines (two oxygenated), two trisubstituted and one persubstituted double bond, one quaternary Received: September 2, 2016 Published: January 31, 2017 356

DOI: 10.1021/acs.jnatprod.6b00802 J. Nat. Prod. 2017, 80, 356−362

Journal of Natural Products

Article

Table 1. 1H NMR (400 MHz) Data of 1−5 position

1a

1

3.44, dd (8.2, 4.7)

2.83, dd (9.9, 4.4)

2 3 4 5 6α 6β 7α 7β 8 10 11 12 13

5.19, t (4.7) 3.98, m 1.81, qd (7.1, 3.4)

4c

5a

2.92, dd (7.9,5.3)

2.96, dd (7.7, 5.3)

4.74, t (4.4) 3.59, td (4.1, 1.5) 1.28, qd (7.1, 4.1)

4.85, dd (5.3, 4.1) 3.71, t (4.1) 1.94, m

4.85, dd (5.3, 3.9) 3.73, t (3.9) 1.84, m

α 1.64, m β 2.17, dt (12.8, 3.4) 1.95, m 3.49, dd (10.3, 5.5)

2.59, m 1.96, m 2.86, m 2.59, m

2.35, ddd (14.2, 9.9, 4.0) 1.91, m 2.94, m 2.74, ddd (16.9, 8.0, 4.0)

3.53, d (8.2)

3.62, d (9.9)

1.83, td (14.0, 5.2) 2.05, m 2.02, m 1.40, m 2.57, m 2.81, d (7.9)

1.86, m 2.07, m 2.00, m 1.39, m 2.86, m 2.86, d (7.7)

2.82, m β 1.79, dt (14.9, 5.4) α 2.20, dt (14.9, 5.4) 3.98, m α 1.98, m β 2.06, m 1.28, d (7.2)

2.85, m β 1.71, m α 2.16, m 4.28, m α 1.91, m β 2.13, m 1.19, d (7.2)

3c

0.96, d (7.2)

0.98, d (7.2)

6.99, s

14 15

7.12, s

6.96, s

16

2.60, s

2.46, s

0.99, d (7.1)

0.92, d (7.1) 5.65, s

8.52, s 3.88, m a 4.09, dd (10.3, 6.6) b 4.30, dd (10.3, 5.9) 1.59, d (7.0) 1.20, s 1.11, s 1.18, s

Measured in pyridine-d5. bMeasured in methanol-d4. cMeasured in CDCl3.

Table 2. 13C NMR (125 MHz) Data of 1−5

a

1.87, dd (12.8, 4.8) 2.85, m 2.85, m

7.15, s 6.99, s

17 18 19 20 a

2b

position

1a

2b

3c

4c

5a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

46.3 80.2 74.2 38.5 45.1 22.6 29.6 144.2 163.0 44.3 140.3 143.6 141.7 186.5 142.6 23.6

48.0 79.1 75.2 38.5 45.3 22.8 31.1 149.4 147.2 50.1 149.2 149.6 141.3 188.4 139.3 24.0

44.7 78.6 75.0 33.8 46.6 25.6 29.8 36.7 169.1 47.4 146.3 26.7 42.9 69.6 42.0 21.5

45.0 78.5 75.1 34.1 46.4 25.8 29.8 33.6 171.0 47.5 147.5 26.9 41.1 67.3 42.0 18.3

175.2 17.5 202.4

177.2 17.2 79.6

175.2 16.1 205.1

175.1 16.1 204.6

36.3 28.2 77.1 39.2 49.0 36.0 197.2 124.0 156.2 37.7 110.5 162.1 130.6 128.0 36.5 66.9 17.2 27.8 15.5 23.2

b

confirmed by the key HMBC cross-peaks (Figure 1A) of H-1 and H-10/C-20 (δC 202.4).

Figure 1. Selected HMBC (A) and ROESY (B) cross-peaks of 1.

The relative configuration of 1 was assigned as the same as hainanolidol by their similar NMR and ROESY data (Figure 1B). The H-6β and H-7β were assigned by the ROESY correlations of H-6β/H-7β and H-6β/H-19. The ROESY correlations of H-6α/H-10 and H-7α/H-10 revealed that these protons were α-oriented. The structure of 1, 20-oxohainanolidol, was thereby assigned as shown. Compound 2 gave a molecular formula of C19H18O5 according to the 13C NMR data and the (−)-HRESIMS ion at m/z 373.1292 [M + HCO2]− (calcd 373.1287). Analysis of the NMR data of 2 (Tables 1 and 2) suggested that its structure was closely related to 1, and the only difference between the two compounds

c

Measured in pyridine-d5. Measured in methanol-d4. Measured in CDCl3.

carbon, and three carbonyl carbons, as defined via the DEPT and HSQC data. The double bonds and carbonyls accounted for six indices of hydrogen deficiency, and the remaining five thus required the presence of five rings in 1. These data revealed that compound 1 was a C19 Cephalotaxus troponoid and structurally resembled the coexisting hainanolidol.8 The only difference between the two compounds was a C-20 carbonyl group in 1 replacing the C-20 methylene motif in hainanolidol, which was 357



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Figure 2. ECD spectra of compounds 1, 2, and 9 (A) and compounds 3 and 4 (B).

involved a hydroxy group at C-20 (δC 79.6) replacing the C-20 carbonyl group of 1, which was confirmed via the key HMBCs (Figure S1, Supporting Information) from H-20 (δH 5.65) to C-2, C-9, C-10, and C-11. This was in accord with the molecular formula of 2 (two more mass units than 1). Although there were no ROESY correlations (Figure S22, Supporting Information) to define the orientation of H-20, its β-orientation was assigned by the H-20 singlet resulting from a near 90° dihedral angle between H-20 and H-1. For analogues with a H-20α, it showed a triplet (δH 5.67, br t, J = 7.1 Hz),9 supporting the assignment of a H-20β for 2. In addition, comparing the 13C NMR data of 2 with those of hainanolidol,8b the C-10 resonance was shielded by ΔδC −1.2 due to the γ-gauche effect from OH-20 (Figure S2, Supporting Information), which reaffirmed the above assignment. Therefore, the structure of compound 2, 20α-hydroxyhainanolidol, was characterized as shown. The absolute configurations of compounds 1 and 2 were proposed as depicted by the similarity of their electronic circular dichroism (ECD) curves to that of compound 9 in the 200− 300 nm range (Figure 2A) and biosynthesis considerations. The molecular formula C19H24O5 was assigned to 3 with eight indices of hydrogen deficiency by the 13C NMR data and the (−)-HRESIMS ion at m/z 331.1536 [M − H]− (calcd 331.1545). The NMR data (Tables 1 and 2) of 3 showed many similarities to those of 1 except for the absence of the proton and carbon signals for the tropone moiety. The 1H−1H COSY correlations (Figure 3A) revealed the presence of two protonbearing sequences of C(10)−C(1)−C(2)−C(3)−C(4)−C(19) and C(6)−C(7)−C(8)−C(15)−C(14)−C(13)−C(12)− C(16) in 3. The HMBC cross-peak networks (Figure 3A) of H-8/C-9, C-11, and C-14 (δC 69.6); H-10/C-6, C-9, C-11, and C-20; H-12/C-9, C-11, and C-20; H-6/C-5 and C-18; H-4/C18; H-19/C-5; and H-2/C-18 delineated the framework with an unprecedented cycloheptene moiety in 3 replacing the tropone rings in compounds 1 and 2. The relative configuration of 3 was defined by the ROESY data (Figure 3B). The ROESY cross-peaks of H-19/H-6β, H-6β/ H-7β, H-7β/H-15β, H-13β/H-15β, and H-13β/H-16 revealed that all of these protons were cofacial and were arbitrarily assigned β-orientations. The ROESY correlations of H-6α/H-10, H-8/H-10, H-7α/H-8, H-8/H-14, H-13α/H-14, and H-14/H-15α showed that they were α-oriented. Finally, an X-ray diffraction study (Figure 4) using the anomalous dispersion of Cu Kα radiation defined its absolute configuration as (1S, 2R, 3R, 4S, 5S,

Figure 3. 1H−1H COSY (A), key HMBC (A), and ROESY (B) crosspeaks of 3.

Figure 4. ORTEP drawing of compound 3.

8R, 10S, 12S, 14R) [absolute structure parameter: 0.04(4)].10 The structure of 3, cephafortoid A, was thus assigned and is the first example of a 17-nor-cephalotane-type diterpenoid; it is believed to be the biosynthesis precursor for Cephalotaxus troponoids.4a Compound 4 had the same molecular formula as 3 as assigned by the 13C NMR data and the (−)-HRESIMS ion at m/z 331.1538 [M − H]− (calcd 331.1545). Their NMR data 358



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Table 3. 1H NMR (400 MHz) and 13C NMR (125 MHz) Data of 6−8 in CDCl3

(Tables 1 and 2) were highly similar to slight variations apparent at the cycloheptene ring. By comparing with 3, H-14 was deshielded by ΔδH 0.30 and C-14 was shielded by ΔδC 2.3, suggesting that compound 4 was the 14-epimer of 3. The key ROESY correlations of H-14 with H-16, H-13, and H2-15 (Figure S3, Supporting Information) supported this assignment. The absolute configuration of 4 was assigned as depicted by the similar ECD spectrum to that of 3 (Figure 2B). The structure of 4, 14-epicephafortoid A, was thereby defined as shown. Compound 5 was obtained as a pale yellow, amorphous powder. Its (−)-HRESIMS gave a deprotonated molecular ion peak at m/z 331.1904 [M − H]− (calcd 331.1909), indicating a molecular formula of C20H28O4 in conjunction with the 13C NMR data. The IR data suggested the presence of hydroxy (3406 cm−1) and conjugated carbonyl (1657 cm−1) groups. The 1H NMR spectrum of 5 displayed two aromatic singlets at δ 7.15 and 8.52 (Table 1). Its 13C NMR data further demonstrated the presence of a benzene ring (Table 2) and a conjugated carbonyl group (δC 197.2). The NMR data showed many similarities to those of crossogumerin C,11 margocinin,12 and margoclin,12 suggesting that it is an abietane-type diterpenoid. Two hydroxy groups were located at C-3 and C-16 by the chemical shifts of H-3/16 and C-3/16. The HMBC cross-peaks (Figure 5A)

6 position 1 2

126.3 113.0

3 4 5

146.7 145.1 114.6

6

123.6

7

42.9

8 9

79.3 177.2,

1′ 2′

129.5 107.8

3′ 4′ 5′

147.0 146.4 114.3

6′

120.0

7′

80.6

8′ 9′

50.1 58.5

3-OMe 4-OMe 1′-OMe 2′-OMe 3′-OMe 4′-OMe 4-OH 8-OH 9-OH

56.2

4′-OH 9′-OH


a

supported the above assignment, and especially the locations of the hydroxy groups at C-3 and C-16 were confirmed by the crosspeaks from H-3 (δH 3.49, dd, J = 10.3, 5.5 Hz) to C-4, C-5, C-18, and C-19 and from H2-16 (δH 4.09, dd, J = 10.3, 6.6 Hz and 4.30, dd, J = 10.3, 5.9 Hz) to C-13, C-15, and C-17. Its relative configuration was established by comparing the NMR data with the known analogues and analyzing the ROESY data (Figure 5B). The ROESY correlations of H-1α/H-3, H-3/ H-18, and H-5/H-18 suggested that H-3 was α-oriented. However, the relative configuration of C-15, just like those of crossogumerin C11 and margocinin,12 was left unassigned. The structure of 5, cephafortoid B, was thereby characterized as shown. The (−)-HRESIMS ion at m/z 389.1231 [M − H]− (calcd 389.1236) and 13C NMR data of compound 6 revealed the molecular formula C20H22O8 with 10 indices of hydrogen deficiency. Inspection of the 1D NMR data (Table 3) showed the presence of two 1,3,4-trisubstituted benzene rings, a tertiary carbon (δC 79.3), two methines, two methylenes, one ester carbonyl

δC

56.0

7 δH

6.78, d (1.8)

6.83, d (8.1) 6.67, dd (8.1, 1.8) a 3.03, d (13.5) b 3.23, d (13.5)

δC 126.9 113.4 149.1 148.5 111.3 123.0 42.8

8 δH

6.79, d (1.9)

6.77, d (8.2) 6.71, dd (8.2, 1.9) a 3.05, d (13.5) b 3.24, d (13.5)

79.2 177.2

6.31, d (2.0)

6.83, d (8.1) 6.63, dd (8.1, 2.0) 5.45, d (8.7) 2.37, m a 3.75, m b 3.89, m 3.85, s

3.72, s 5.57, br s 3.40, br s

130.1 108.4 149.6 149.5 110.9 119.1 80.4 50.2 58.5 55.9 56.1

55.9 56.1

6.37, d (2.0)

6.77, d (8.2) 6.68, dd (8.2, 2.0) 5.49, d (8.6) 2.34, m a 3.76, m b 3.90, m 3.84, s 3.84, s

δC

δH

133.7 119.2

6.89, s

146.8 145.7 114.5

6.89, s

108.5

6.89, s

87.6

5.49, d (6.7)

53.8 64.6

3.54, m 3.90, m (2H)

57.1 150.4 95.2 154.4 116.2

6.54, s

108.5

6.78, s

56.1,a

3.87, s

57.1 56.2,a

3.84, s 3.86, s

3.70, s 3.85, s 5.66, s 3.33, br s 1.59, t (5.1)

5.65, br s 2.49, br s

2.49, br s

Interchangeable assignments.

(δC 177.2), and two methoxy groups (δH 3.72 and 3.85, each 3H, s). These data revealed a lignan-type structure for 6 that structurally resembled 5-(3″,4″-dimethoxypheny)-3-hydroxy-3(4′-hydroxy-3′-methoxybenzyl)-4-(hydroxymethyl)dihydrofuran2-one.13 The difference was an OH-4′ in 6 instead of OCH3-4′ in the latter. This was confirmed by the HMBC (Figure S4A, Supporting Information) correlations from OH-4′ to C-3′, C-4′, and C-5′. The relative configuration of 6 was deduced based on the ROESY data and the NMR data comparison with the known lignan.13 In the ROESY spectrum (Figure S4B, Supporting Information), the cross-peaks of H-7′/H-9′ and H-8′/H-7 allowed the tentative assignments of α-orientations for H-7′ and OH-8 and a β-orientation for H-8′.13 In addition, the attachment of the methoxy groups at C-3 and C-3′ was confirmed by the ROESY correlations of H-2/OCH3-3 and H-2′/OCH3-3′, respectively. Thus, the structure of 6, cephafortin A, was defined as depicted. 359



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The sodiated HRESIMS ion at m/z 441.1522 [M + Na]+ and the 13C NMR data revealed a molecular formula of C22H26O8 (calcd 441.1525) for 7. Inspection of the NMR data (Table 3) showed that it was structurally related to 6 and had four aromatic methoxy groups (δH 3.70, 3.84, 3.84, and 3.85, each 3H, s), suggesting that the OH-4 and OH-4′ in 6 were likely replaced by two methoxy groups in 7. This was confirmed by the key HMBCs of OCH3-4/C-4 and OCH3-4′/C-4′ (Figure S5, Supporting Information). The relative configuration of 7 was identified as the same of 6 by the similar coupling constants in the 1H NMR spectra. The structure of 7, 4,4′-di-O-methylcephafortin A, was thereby elucidated as shown. The absolute configurations of 6 and 7 were assigned by comparison of their experimental and quantum chemical TDDFTcalculated ECD spectra (for details see the ECD calculations for compounds 6 and 7 in the Supporting Information). In the region of 200−350 nm, the experimental ECD curves of compounds 6 and 7 matched well with those of the calculated ones, respectively (Figure 6). For compound 6, the calculated ECD


the HMBC cross-peaks of OH-4/C-3 (δC 146.8), C-4 (δC 145.7), and C-5 (δC 114.5); OCH3-3/C-3; and H-7/C-2 and C-6. When the 1H NMR spectrum was recorded in acetone-d6 (Figure S71, Supporting Information), the couplings of H-2, H-5, and H-6 were clearly observed (δH 6.99, d, J = 1.9 Hz, H-2; δH 6.79, d, J = 8.1 Hz, H-5; and δH 6.84, dd, J = 8.1, 1.9 Hz, H-6), which supported the assignment of the 1,3,4-trisubstituted benzene moiety. Thus, the structure of compound 8 with a dihydrobenzofuran trisnorneolignan skeleton was defined as shown. The coupling constant (J7,8 = 6.7 Hz) showed that the H-7 and H-8 were trans-oriented,14,16 and this was supported by the ROESY correlation networks (Figure 7B). In addition, the ECD spectrum (Figure S8, Supporting Information) showed a negative Cotton effect at 241 nm, suggesting that compound 8 (cephafortin B) was (7R,8S)-configured.14,16 The 1H and 13C NMR spectra (Figures S79 and S80, Supporting Information) of compound 9 were identical to those of the known compound 11-hydroxyhainanolidol, which was structurally assigned by NMR analysis and calculating the minimum energy with SYBYL software.7 However, comparison of its NMR data with those of 1−4, and especially the known compound 10-hydroxyharringtonolide,4a obviously shows that the assigned configurations of C-3 and C-10 in 11-hydroxyhainanolidol are incorrect. Thus, a single-crystal X-ray study on compound 9 using the anomalous dispersion of Cu Kα radiation not only permitted revision of its relative configuration but also established the absolute configuration as (1R, 2R, 3R, 4S, 5R, 10R) [absolute structure parameter: −0.01(6)]10 (Figure 8). Compound 9 was renamed as 10-hydroxyhainanolidol.4,8 Five known Cephalotaxus troponoids, hainanolidol,8 6-en-harringtonolide,4a harringtonolide,8,17 10-hydroxyharringtonolide,4a and fortunolide B,18 a known cephalotane-type diterpenoid, mannolide A,4a and a known lignan, 5-(3″,4″-dimethoxypheny)-3hydroxy-3-(4′-hydroxy-3′-methoxybenzyl)-4-(hydroxymethyl)dihydrofuran-2-one13 were also isolated and identified by comparing the observed and reported NMR and MS data. All the new isolates and 9 were assessed for their cytotoxicity against the human tumor cell lines HL-60 and A549 by using MTT19 and the SRB20 methods, respectively. Doxorubicin21 was the positive control (IC50 = 0.031 ± 0.002 and 0.124 ± 0.001 μM against HL-60 and A549, respectively). Only compound 1 showed activities against HL-60 and A-549 cells, with IC50 values of 0.77 ± 0.05 and 1.129 ± 0.057 μM, respectively. The other compounds were inactive (the inhibition rates were lower than

Figure 6. Experimental (black lines) and B3LYP/6-311++G(2d,2p)// B3LYP/6-311++G(2d,2p)-calculated (red lines) ECD spectra of 6 (A) and 7 (B).

spectrum showed about 10 nm red-shift, but the tendency was fully consistent with the experimental ECD curves. The HRESIMS ion peak at m/z 687.2401 [2 M + Na]+ (calcd 687.2417) and the 13C NMR data suggested a molecular formula of C18H20O6 for compound 8. The 1H NMR data (Table 3) indicated the presence of a typical dihydrobenzofuran moiety (δH 5.49, d, J = 6.7 Hz, H-7; 3.54, m, H-8; and 3.90, 2H, m, H2-9), which was consistent with its IR absorptions (1612, 1498, and 1454 cm−1) and the UV maxima (233 and 299 nm).14 Furthermore, two substituted benzene rings, three methoxy groups (δH 3.84, 3.86, and 3.87, each 3H, s), and a hydroxy group (δH 5.66, s) were evident by inspection of its NMR data (Table 3). These structural fragments were then connected to furnish the framework of 8 by the HMBC data (Figure 7A). A 1′,2′,4′,5′tetrasubstituted benzene moiety was first recognized by the HMBC cross-peaks from H-3′ (δH 6.54, s) to C-1′, C-2′, C-4′, and C-5′ and from H-6′ (δH 6.78, s) to C-8. Although H-2, H-5, and H-6 exhibited an unexpected three-proton singlet (δH 6.89, 3H, s),15 the 1,3,4-trisubstituted benzene moiety was revealed by 360



Article

20-Oxohainanolidol (1): pale yellow, amorphous powder; [α]27D −9 (c 0.3, MeOH); UV (MeOH) λmax (log ε) 249 (4.33), 313 (3.66) nm; IR (KBr) νmax 3365, 2964, 2924, 2854, 1762, 1724, 1614, 1593m 1541, 1427, 1352, 1039, 891 cm−1; 1H NMR (pyridine-d5), see Table 1; 13C NMR (pyridine-d5), see Table 2; (+)-ESIMS m/z 327.2 [M + H]+, 675.3 [2 M + Na]+; (−)-ESIMS m/z 325.0 [M − H]−; (−)-HRESIMS m/z 325.1067 [M − H]− (calcd for C19H17O5, 325.1076). 20α-Hydroxyhainanolidol (2): white, amorphous powder; [α]27D +95 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 241 (4.29), 317 (3.79) nm; IR (KBr) νmax 3593, 3363, 2924, 2852, 1753, 1660, 1630, 1539, 1468, 1429, 1358, 1286, 1051, 991 cm−1; 1H NMR (methanol-d4), see Table 1; 13C NMR (methanol-d4), see Table 2; (+)-ESIMS m/z 329.2 [M + H]+, 679.2 [2 M + Na]+; (−)-ESIMS m/z 373.3 [M + HCO2]−; (−)-HRESIMS m/z 373.1292 [M + HCO2]− (calcd for C20H21O7, 373.1287). Cephafortoid A (3): colorless crystals (MeOH/H2O, 50:1); mp 100− 102 °C; [α]25D −121 (c 0.7, MeOH); UV (MeOH) λmax (log ε) 242 (4.10) nm; IR (KBr) νmax 3357, 2925, 2868, 1736, 1684, 1630, 1452, 1362, 1267, 1238, 1045, 839, 715, 646 cm−1; 1H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 333.2 [M + H]+, 687.3 [2 M + Na]+; (−)-ESIMS m/z 331.1 [M − H]−; (−)-HRESIMS m/z 331.1536 [M − H]− (calcd for C19H23O5, 331.1545). 14-epi-Cephafortoid A (4): colorless gum; [α]25D −149 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 241 (4.00) nm; IR (KBr) νmax 3456, 3321, 2933, 2871, 1747, 1670, 1624, 1448, 1360, 1213, 1047, 1018 cm−1; 1 H NMR (CDCl3), see Table 1; 13C NMR (CDCl3), see Table 2; (+)-ESIMS m/z 333.2 [M + H]+, 687.3 [2 M + Na]+; (−)-ESIMS m/z 331.1 [M − H]−; (−)-HRESIMS m/z 331.1538 [M − H]− (calcd for C19H23O5, 331.1545). Cephafortoid B (5): yellowish, amorphous powder; [α]26D +6 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 232 (4.07), 281 (3.94) nm; IR (KBr) νmax 3406, 3263, 2960, 2877, 1753, 1657, 1600, 1574, 1473, 1308, 1263, 1045, 1016 cm−1; 1H NMR (pyridine-d5), see Table 1; 13C NMR (pyridine-d5), see Table 2; (+)-ESIMS m/z 333.3 [M + H]+; (−)-ESIMS m/z 331.3 [M − H]−, 663.5 [2 M − H]−; (−)-HRESIMS m/z 331.1904 [M − H]− (calcd for C20H27O4, 331.1909). Cephafortin A (6): colorless gum; [α]27D +15 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 232 (4.01), 282 (3.63) nm; IR (KBr) νmax 3363, 2956, 2924, 2852, 1761, 1604, 1518, 1275, 1242, 1128, 1034 cm−1; 1H and 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 413.1 [M + Na]+; (−)-ESIMS m/z 801.7 [2 M + Na − 2H]−; (−)-HRESIMS m/z 389.1231 [M − H]− (calcd for C20H21O8, 389.1236). 4,4′-Di-O-methylcephafortin A (7): colorless gum; [α]27D +16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 233 (4.08), 279 (3.70) nm; IR (KBr) νmax 3406, 2956, 2924, 2854, 1770, 1516, 1464, 1263, 1238, 1142, 1026 cm−1; 1H and 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 441.0 [M + Na]+; (−)-ESIMS m/z 463.3 [M + HCO2]−; (+)-HRESIMS m/z 441.1522 [M + Na]+ (calcd for C22H26O8Na, 441.1525). Cephafortin B (8): colorless gum; [α]27D +1 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 233 (4.34), 290 (4.05), 299 (4.03) nm; IR (KBr) νmax 3477, 2958, 2927, 2854, 1612, 1516, 1498, 1454, 1282, 1215, 1190, 1169, 1113, 1032, 862 cm−1; 1H and 13C NMR (CDCl3), see Table 3; (+)-ESIMS m/z 355.1 [M + Na]+, 687.2 [2 M + Na]+; (+)-HRESIMS m/z 687.2401 [2 M + Na]+ (calcd for C36H40O12Na, 687.2417). 10-Hydroxyhainanolidol (9): colorless crystals (MeOH/H2O, 50:1); mp 312−314 °C; 1H and 13C NMR (methanol-d4), see reported data for 11-hydroxyhainanolidol;7 (+)-ESIMS m/z 329.2 [M + Na]+, 679.2 [2 M + Na]+; (−)-ESIMS m/z 327.1 [M − H]−. X-ray Crystallographic Analysis. The crystals of compounds 3 and 9 were obtained from the recrystallization in a mixture of solvents (MeOH/H2O, 50:1) at room temperature. The X-ray crystallography studies of the two compounds were completed according to the usual procedure (for details see X-ray Crystallographic Analysis, Table S1, and Table S2 in the Supporting Information). Cytotoxicity Assays. All the new isolates and compound 9 were tested for cytotoxicity against the HL-60 and A-549 cells by the MTT19 and SRB20 method, respectively. In these tests, doxorubicin was used as the positive control.

Figure 8. ORTEP drawing of compound 9.

50% at 10 μM). Compounds 3 and 4, featuring a cycloheptene unit, were inactive, further suggesting that the tropone motif is essential for cytotoxic activity.4a,8 Comparison with compound 1 and the known cytotoxic Cephalotaxus troponoids,4a,8 which all possessed a 3,20-ether bridge, indicated that a conjugated C-20 carbonyl group or a 3,20-ether bridge is also crucial for the cytotoxic activities of Cephalotaxus troponoids.

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EXPERIMENTAL SECTION

General Experimental Procedures. The experiments have been accomplished according to a standard procedure with minor modifications (General Experimental Procedures, Supporting Information). Plant Material. The identification of the plant material of Cephalotaxus fortunei Hook. f. and the deposition of the voucher specimen were included in the Plant Material part of Supporting Information. Extraction and Isolation. The powder of the plant sample (7 kg) was extracted with 95% EtOH at room temperature to obtain a brown syrup (550 g), which was suspended in water and partitioned with EtOAc. The EtOAc part (185 g) was subjected to a column of D101 macroporous absorption resin washed with 30%, 80%, and 90% ethanol in H2O, sequentially. The main part (130 g, from the elution of 80% EtOH in H2O) was chromatographed over an MCI gel column eluted with gradients of MeOH/H2O (50 to 100%) to obtain five fractions, A−E. Fraction A (49 g) was fractionated into five fractions (A1−A5) on a silica gel column eluted with petroleum ether/EtOH (5:1 to 1:1, v/v). Fraction A4 (11 g) was separated by column chromatography (CC) with C18 reversed-phase silica gel (MeOH/H2O, 10% to 35%) to give three subfractions (A4a−A4c). Fraction A4b was chromatographed over a silica gel column (CHCl3/MeOH, 200:1 to 10:1, v/v) to give two major parts, A4b1 and A4b2. Both were further separated by semipreparative HPLC with a mobile phase of 25% MeCN in H2O. Part A4b1 yielded compounds 2 (3 mg), 3 (20 mg), and 4 (3 mg), and part A4b2 gave 6 (3 mg) and 9 (3 mg). Fraction A4c afforded hainanolidol (3 mg) by a process similar to that described for A4b. Fraction B (11 g) was fractionated into four parts (B1−B4) by silica gel CC eluted with petroleum ether/acetone (5:1 to 1:1, v/v). Fraction B1 (280 mg) was subjected to a column of C18 reversed-phase silica gel eluted with gradients of MeOH/H2O (45% to 60%) to obtain one major component, which was further purified by semipreparative HPLC (40% MeCN in H2O as the mobile phase) to afford mannolide A (3 mg). By similar procedures, fraction B2 (1.2 g) yielded compounds 7 (3 mg) and 8 (9 mg); fraction B3 (1.1 g) yielded compounds 1 (4 mg), 5 (3 mg), and 5-(3″,4″-dimethoxypheny)-3hydroxy-3-(4′-hydroxy-3′-methoxybenzyl)-4-(hydroxymethyl)dihydrofuran-2-one (7 mg); and fraction B4 (915 mg) yielded 10-hydroxyharringtonolide (2 mg) and fortunolide B (2 mg). RP-18 silica gel CC of fraction C (10 g) afforded two major components, and both were purified by semipreparative HPLC to yield 6-en-harringtonolide (2 mg) and harringtonolide (103 mg), respectively. 361



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Article

(16) Matsuda, N.; Sato, H.; Yaoita, Y.; Kikuchi, M. Chem. Pharm. Bull. 1996, 44, 1122−1123. (17) Buta, J. G.; Flippen, J. L.; Lusby, W. R. J. Org. Chem. 1978, 43, 1002−1003. (18) Du, J.; Chiu, M. H.; Nie, R. L. J. Nat. Prod. 1999, 62, 1664−1665. (19) 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. (20) Xiao, D.; Zhu, S. P.; Gu, Z. L. Acta Pharmacol. Sin. 1997, 18, 280− 283. (21) Laginha, K. M.; Verwoert, S.; Charrois, G. J. R.; Allen, T. M. Clin. Cancer Res. 2005, 11, 6944−6949.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00802. IR, ESIMS, HRESIMS, 1D and 2D NMR spectra of 1−9; ECD calculations for 6 and 7; and the ECD spectrum of 8 (PDF) X-ray crystallographic data of 3 (CIF) X-ray crystallographic data of 9 (CIF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (J.-M. Yue): +86-21-50806718. Fax: +86-21-50806718. E-mail: [email protected]. ORCID

Jian-Min Yue: 0000-0002-4053-4870 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation (Nos. 21532007, U1302222, and 81321092) of the People’s Republic of China and the project “Personalized MedicinesMolecular Signature-based Drug Discovery and Development” (No. XDA12020321), Strategic Priority Research Program of the Chinese Academy of Sciences. We thank Prof. S.-Q. Tang of Guangxi Normal University for the identification of the plant material.

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REFERENCES

(1) Zheng, W. J.; Fu, L. G. In Zhongguo Zhiwu Zhi; Science Press: Beijing, 1978; Vol. 7, pp 423−432. (2) Abdelkafi, H.; Nay, B. Nat. Prod. Rep. 2012, 29, 845−869. (3) Evanno, L.; Jossang, A.; Nguyen-Pouplin, J.; Delaroche, D.; Herson, P.; Seuleiman, M.; Bodo, B.; Nay, B. Planta Med. 2008, 74, 870−872. (4) (a) Ni, G.; Zhang, H.; Fan, Y. Y.; Liu, H. C.; Ding, J.; Yue, J. M. Org. Lett. 2016, 18, 1880−1883. (b) Xu, J. B.; Fan, Y. Y.; Gan, L. S.; Zhou, Y. B.; Li, J.; Yue, J. M. Chem. - Eur. J. 2016, 22, 14648−14654. (5) (a) Paudler, W. W.; Kerley, G. I.; McKay, J. J. Org. Chem. 1963, 28, 2194−2197. (b) Paudler, W. W.; McKay, J. J. Org. Chem. 1973, 38, 2110−2112. (c) Bocar, M.; Jossang, A.; Bodo, B. J. Nat. Prod. 2003, 66, 152−154. (6) Xu, X. H.; Zhang, W.; Cao, X. P.; Xue, S. Phytochem. Lett. 2011, 4, 52−55. (7) Yoon, K. D.; Jeong, D. G.; Hwang, Y. H.; Ryu, J. M.; Kim, J. J. Nat. Prod. 2007, 70, 2029−2032. (8) (a) Sun, N. J.; Xue, Z.; Liang, X. T.; Huang, L. Acta Pharm. Sin. 1979, 14, 39−44. (b) Zhang, M.; Liu, N.; Tang, W. J. Am. Chem. Soc. 2013, 135, 12434−12438. (9) Rogers, D. H.; Frey, B.; Roden, F. S.; Russkamp, F. W.; Willis, A. C.; Mander, L. N. Aust. J. Chem. 1999, 52, 1053−1108. (10) Flack, H. D. Acta Crystallogr., Sect. A: Found. Crystallogr. 1983, A39, 876−881. (11) Gumersindo, M. L.; Isabel, L. B.; Ignacio, A. J. D.; Laila, M. M.; Ramiro, Q. Q.; Leovigildo, Q.; Gonzalo, J. M. R. Bioorg. Med. Chem. Lett. 2014, 24, 2105−2109. (12) Ara, I.; Siddiqui, B. S.; Faizi, S.; Siddiqui, S. Phytochemistry 1990, 29, 911−914. (13) Li, L.; Seeram, N. P. J. Agric. Food Chem. 2011, 59, 7708−7716. (14) Huang, X. X.; Zhou, C. C.; Li, L. Z.; Peng, Y.; Lou, L. L.; Liu, S.; Li, D. M.; Ikejima, T.; Song, S. J. Fitoterapia 2013, 91, 217−223. (15) Yang, Y. N.; Zhu, H.; Chen, Z.; Liu, F.; An, Y. W.; Feng, Z. M.; Jiang, J. S.; Zhang, P. C. J. Nat. Prod. 2015, 78, 705−711. 362


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