Eucalypglobulusals A–J, Formyl-Phloroglucinol ... - ACS Publications

Article pubs.acs.org/jnp

Cite This: J. Nat. Prod. 2018, 81, 2638−2646

Eucalypglobulusals A−J, Formyl-Phloroglucinol−Terpene Meroterpenoids from Eucalyptus globulus Fruits Xu-Jie Qin,†,# Ling-Yu Jin,†,‡,# Qian Yu,§,# Hui Liu,† Afsar Khan,⊥ Huan Yan,† Xiao-Jiang Hao,† Lin-Kun An,*,§ and Hai-Yang Liu*,†

Downloaded via IOWA STATE UNIV on January 11, 2019 at 19:43:52 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

†

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡ School of Traditional Dai-Thai Medicine, West Yunnan University of Applied Sciences, Jinghong 666100, People’s Republic of China § Institute of Medicinal Chemistry and Chemical Biology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China ⊥ Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-22060, Pakistan S Supporting Information *

ABSTRACT: Ten new formyl-phloroglucinol−terpene meroterpenoids, eucalypglobulusals A−J (1−10), and ten known analogues were isolated from Eucalyptus globulus fruits. The structures of 1−10 were determined by spectroscopic analysis, while their absolute configurations were established using calculated and experimental electronic circular dichroism (ECD) spectra. Eucalypglobulusal A was assigned as a new formyl-phloroglucinol−terpene meroterpenoid with a rearranged sesquiterpene skeleton, and an aldol condensation between C-3 and C-5 of the germacrene C moiety was proposed to be a key step in its putative biosynthetic pathway. Eucalypglobulusal F exhibited cytotoxicity against the human acute lymphoblastic cell line (CCRF-CEM) with an IC50 value of 3.3 μM, while eucalypglobulusal A, eucarobustol C, macrocarpal A, macrocarpal B, and macrocarpal D exhibited DNA topoisomerase I (Top1) inhibition. The compounds eucalypglobulusal A and macrocarpal A act as Top1 catalytic inhibitors and delay Top1 poison-mediated DNA double-strand damage.

D

Thus, it seems worthwhile to search for other Top1 catalytic inhibitors that may better overcome multidrug resistance. In a continuation of our search for acylphloroglucinol derivatives with diversified bioactivities from plants of the Myrtaceae family,5 ten new formyl-phloroglucinol−terpene meroterpenoids (1−10) and ten previously reported analogues were purified from the fruits of Eucalyptus globulus. The ten previously described compounds were identified as eucalrobusone C,6 eucarobustol C,7 macrocarpal A,8 macrocarpal B,9 macrocarpals D and E,9 eucalyptin A,10 eucalyptone,11 macrocarpal L,12 and eucalyptal B13 (Figure S1, Supporting Information), respectively. The compounds obtained were

NA topoisomerase I (Top1) is a validated molecular target in anticancer drug discovery, and it is an essential human enzyme responsible for DNA replication, transcription, and recombination.1 Based on a catalytic mechanism,2 Top1 inhibitors include “Top1 poisons” and “Top I catalytic inhibitors”. Top1 poisons may form a DNA covalent complex, which is called the Top1−DNA cleavage complex (Top1cc). In this way, they prevent DNA re-ligation and lead further to irreversible double-strand breakage, while “Top1 catalytic inhibitors” mainly inhibit the cleavage of DNA. Topotecan and irinotecan are U.S. FDA-approved Top1 catalytic inhibitors that are used widely for diverse tumor treatments. However, these camptothecin derivatives exhibit several drawbacks, including severe gastrointestinal tract toxicity,3 chemical instability, and drug efflux-mediated resistance.4 © 2018 American Chemical Society and American Society of Pharmacognosy

Received: May 29, 2018 Published: December 13, 2018 2638

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

Article

Chart 1

correlations between H3-14 and H-8b/H-3/H-2a showed a cofacial arrangement of these protons, and their α-orientations were randomly assigned (Figure 1), whereas the observed correlations between H-2b and H-1 as well as between H-8a and H3-15 in the ROESY spectrum supported β-orientations for H-1 and the C-5-acetyl group. Although the correlation of H-9′ with H3-14 was also observed, being in a flexible chain it was not considered useful to assign the C-9′ configuration. A negative Cotton effect at 239 nm and a positive Cotton effect at 305 nm in the measured ECD spectrum supported an S configuration of C-1 in 1. To further determine its absolute configuration, the ECD curves were simulated for the two epimers of 1, (1S,3R,5S,10S,9′R)-1 and (1S,3R,5S,10S,9′S)-1, as well as their respective enantiomers, (1R,3S,5R,10R,9′S)-1 and (1R,3S,5R,10R,9′R)-1. The experimental and calculated ECD curves of (1S,3R,5S,10S,9′R)-1 matched well (Figure 2). Accordingly, the structure of eucalypglobulusal A was elucidated as depicted. Eucalypglobulusal A (1) represents a novel formylphloroglucinol−terpene meroterpenoid with a rearranged sesquiterpene skeleton, and a plausible biosynthetic pathway was proposed (Figure 3). Doubly formylated phloroglucinol i and isovaleraldehyde ii could yield o-quinone methide iii via a Knoevenagel-type condensation, and carbocation iv can be generated by protonation of iii.14 Carbocation iv, as a cationic initiator, couples with germacrene C to yield carbocation v in Markovnikov fashion, which further forms the key precursor iv. The oxidative cleavage of the double bond at C-3 and C-4 in the intermediate vi would generate the intermediate vii, which may undergo an aldol condensation that constructs the new linkage between C-3 and C-5 to yield eucalypglobulusal A. A deprotonated molecular ion peak (m/z 487.2712 [M − H]−) in the HRESIMS of eucalypglobulusal B (2) established the molecular formula C28H40O7 with nine IHDs. The 1D NMR data suggested that 2 and 1 share the same 3,5-diformylphloroglucinol unit but different sesquiterpene moieties (Tables 1 and 3). The 1H−1H COSY correlations for the sesquiterpene moiety revealed two partial structures (H2-5−H6−H-7−H2-8−H2-9 and H2-2−H2-3). In the HMBC spec-

screened for their cytotoxic and Top1 inhibitory activities. In this paper, the extraction, purification, structural identification, and bioactivity evaluation of these meroterpenoids are detailed.

■

RESULTS AND DISCUSSION

Eucalypglobulusal A (1) exhibited a molecular formula of C28H38O7, as assigned by HRESIMS (m/z 485.2558 [M − H]− (calcd for C28H37O7, 485.2545), implying 10 indices of hydrogen deficiency (IHD). The characteristic information for a 3,5-diformyl-phloroglucinol unit was clearly observed in the 1D NMR spectra (Tables 1 and 3), particularly those ascribed to two formyl group protons (δH 10.06, 10.07, and δC 193.2 × 2). The signals for two secondary methyls at δH 0.82 (d, J = 6.5 Hz, H3-12′) and 0.86 (d, J = 6.5 Hz, H3-13′) along with the HMBC correlations from H3-12′/H3-13′ to C-10′/C-11′ and from H-11′ to C-9′ indicated the presence of an isopentyl group attached to a 3,5-diformylphloroglucinol moiety. The presence of a sesquiterpene unit in 1 was indicated by the 15 remaining carbons. The 1H−1H COSY spectrum of 1 gave three spin systems (Figure 1). The HMBC spectrum gave correlations from both H3-12 and H3-13 to C-7, indicating the attachment of the isopropyl group at the olefinic C-7. Similarly, the correlations from H3-15 to C-4/C-5 in the HMBC spectrum confirmed that an acetyl group is attached to C-5 in the sesquiterpene unit. The correlations from H-6 to C-3/C-8/ C-10 and from H3-14 to C-1/C-5/C-9 allowed the establishment of the sesquiterpene moiety as an isofaurinone derivative featuring a bicyclic 5,6-fused ring system. The key correlations from H-7′ to C-1′/C-3′, from H-8′ to C-3′/C-5′, and from H9′ to C-1′/C-5′/C-6′ not only confirmed the presence of a 3,5diformyl-phloroglucinol unit but also established its attachment to the sesquiterpene moiety via a C-1−C-9′ bond, which was also consistent with the 1H−1H COSY correlation between H-1 and H-9′ (Figure 1). With the aid of 1H−1H COSY, HMBC, and HSQC spectra, assignments of the 1H and 13 C NMR data were achieved as shown in Tables 1 and 2. The assignment of the relative configuration for 1 was accomplished based on its ROESY spectrum. The ROESY 2639

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

Article

Table 1. 1H NMR (600 MHz, Methanol-d4) Spectroscopic Data of Meroterpenoids 1−4 (δ in ppm, J in Hz)

Table 2. 1H NMR (600 MHz) Spectroscopic Data for Meroterpenoids 5−8 (δ in ppm, J in Hz)

position

position

1 2a 2b 3a

1 3.03, ddd (11.0, 11.0, 8.3) 1.61, m 1.22, m 4.84, dd (10.0, 7.3)

3b 5a 5b 6

5.60, s

7 8a 8b 9a 9b 12 13 14a 14b 15 4′ 7′ 8′ 9′

2.44, m 2.17, brdd (14.3, 5.7) 1.89, brdd (14.3, 6.4) 1.77, ddd (13.9, 12.1, 6.0) 1.11, d (6.7) 1.11, d (6.7) 0.92, s 2.13, s

10′a

10.06, s 10.07, s 3.34, td (11.3, 4.2) 2.12, m

10′b

1.34, m

11′ 12′ 13′

1.46, m 0.82, d (6.5) 0.86, d (6.5)

2

2.09, m 1.68, m

3

2.21, m 2.15, overlap

1.57, td 2.34, m (13.4, 3.6) 1.48, m 1.76, m 1.45, m 1.34, brd (13.7) 2.08, m

2.17, d (6.2)

2.40, m 2.24, ddd (13.5, 10.8, 2.5) 2.04, td (13.4, 2.2) 1.67, ddd (13.4, 10.7, 9.7) 2.30, d (11.9)

0.47, m

2.68, m

0.52, dd (11.9, 9.0) 0.47, m

2.05, m

2a 2b

1.58, m 1.25, m

3a

1.83, overlap

3b

1.66, m

0.87, s 1.07, s 2.15, s 1.06, s 5.85, s 10.02, s

1.16, s 5.85, s 10.00, s

2.49, m

10.05, s 10.07, s 3.04, dd 3.41, dd (12.7, 4.2) (12.5, 4.1) 2.31, td 2.40, td (12.1, 1.7) (12.3, 3.1) 1.17, m 1.21, brdd (12.8, 4.1) 1.18, m 1.27, m 0.85, d (6.1) 0.80, d (6.4) 0.75, d (6.1) 0.86, d (6.4)

2.42, t (6.8)

1.58, m 1.25, m

1.91, m 1.74, m

1.50, m

1.64, dt (11.6, 8.1) 1.44, m

1.41, m 2.45, m

5.27, dd (15.9, 10.4)

8b 9a

0.73, m 2.08, brt (12.3) 1.83, overlap

6.00, d (15.9)

1.10, s 1.10, s 1.32, s

1.10, s 1.14, s 4.86, 2H, brs

1.15, s 1.17, s 1.15, s

1.34, s 10.13, s

1.35, s 10.15, s

1.17, d (7.4) 2.70 dd (13.4, 2.1) 2.37, t (12.2)

2.98, 2H brd (6.7)

3.00, 2H brd (5.5)

11′ 12′ 13′ OH-3′ OH-5′

1.65, m 0.96, d (6.3) 0.83, d (6.3) 13.42, s 13.22, s

10.00, s 3.06, ddd (9.7, 4.2, 1.7) 1.49, ddd (13.2, 10.5, 1.7) 1.27, ddd (13.2, 10.0, 3.5) 1.71, overlap 0.99, d (6.5) 0.86, d (6.5) 13.41, s 13.26, s

10.06, s

10′b

10.00, s 3.14, brd (8.5) 1.42, brt (11.8) 1.23, m

2.03, m 2.44, dd (12.6, 6.3) 2.02, brt (12.6) 1.10, s 1.00, s 4.74, brs 4.68, brs 0.91, s 2.72, d (14.3) 2.27, d (14.3) 10.10, s

2.23, m 0.98, d (6.6) 0.98, d (6.6)

2.26, m 0.99, d (6.7) 0.99, d (6.7) 15.46, s 14.38, s

1.07, overlap

8′ 9′

1.19, m 0.73, d (6.5) 0.83, d (6.5)

10′a

5.49, d (3.2)

2.06, ddd (8.9, 5.2, 3.2) 1.72, 2H m

1.80, m 1.67, m

7′b

a

1.45, m

1.77, m

12 13 14a 14b 15 7′a

trum, the observed correlations from H-6 to C-1/C-2/C-10, from H2-14 to C-1/C-9, from H3-12/H3-13 to C-7/C-11, and from H3-15 to C-3/C-5/C-9′ suggested a cadinene-type sesquiterpene moiety in 2, similar to that in eucalyptal B, which was previously obtained from the fruits of the same species.13 However, one less IHD and the replacement of an oxygenated methine by a methylene revealed that 2 is a macrocarpal rather than an euglobal meroterpenoid. Compound 2 and eucalyptal B share the same relative configuration as suggested by the ROESY correlations of H-6/H3-15, H-6/ H-2b, and H-6/H3-12. The calculated and experimental ECD spectra of 2 were compared (Figure S73, Supporting Information), and a (1S,4R,6S,7R,9′R) absolute configuration for 2 was subsequently established. Accordingly, the structure of eucalypglobulusal B was elucidated as depicted. The molecular formula, C27H38O6, of eucalypglobulusals C (3) and D (4) was the same, and was established by deprotonated molecular ions observed at m/z 457.2603- [M − H]− and 457.2605 [M − H]− in their HRESIMS spectra, respectively. The NMR spectra of 3 and 4 both showed a close resemblance to those of eucalyptone except for lacking a formyl group and having an aromatic methine in the

8a

8a

9b

3.14, dd (12.0, 4.2) 3.33, brt (12.0)

2.48, m 2.32, brdd (14.5, 6.3) 1.79, m 1.37, ddd (14.9, 10.6, 2.6) 2.07, ddd (14.9, 10.9, 3.2) 1.71, dt (15.5, 3.2)

7b

7

6b

2.63, brdd (9.4, 6.0) 2.48, brdd (9.4, 5.8) 1.01, s 1.08, s 2.15, s

6a

5.55, d 2.02, brdd (9.7, (15.5) 4.5) 5.46, dd 1.84, overlap (15.5, 10.3) 0.82, td (10.1, 3.2) 1.79, m 1.84, brt (10.6)

6a

1.88, m 1.50, m

5a

1a 1b

4 5

2.18, dt 0.45, m (13.2, 3.4) 1.67, m 1.85, m 1.41, m 1.36, m 2.50, td (13.6, 4.6) 2.19, brt (15.1) 1.13, s 1.04, s 4.95, s 4.90, s 1.04, s

4

1.20, t (10.5) 0.62, t (10.2)

0.77, dt (10.2, 6.7) 2.26, m

Measured in CDCl3. bMeasured in methanol-d4.

phloroglucinol portion (Tables 1 and 3). The HMBC correlations from H-4′ to C-6′/C-5′/C-3′/C-2′ indicated a 3-formyl-phloroglucinol moiety in 3 and 4. The different 13C NMR chemical shifts for C-9′ (δC 39.0 in 3 and δC 41.4 in 4) were consistent with 3 and 4 being epimers at C-9′. The other portions of 3 and 4 were identical to eucalyptone.11 The absolute configurations of 3 and 4 were established as (4S,5R,6R,7R,9′S) and (4S,5R,6R,7R,9′R), respectively, from their ECD spectra (Figure 4). Accordingly, the structures of eucalypglobulusals C and D were elucidated as depicted. Eucalypglobulusal E (5) exhibited a molecular formula of C28H40O7, as determined from the deprotonated molecular ion at m/z 487.2710 [M − H]− in the HRESIMS. Careful comparison of the NMR spectroscopic data (Tables 2 and 3) 2640

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products Table 3.

13

Article

C NMR (150 MHz) Spectroscopic Data for Meroterpenoids 1−8 (δ in ppm)

position

1a

2a

3a

4a

5b

6b

7a

8b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′ 11′ 12′ 13′

40.9, CH 37.2, CH2 73.3, CH 213.2, C 69.0, C 118.3, CH 147.6, C 23.6, CH2 32.6, CH2 45.9, C 37.1, CH 21.6, CH3 22.1, CH3 19.9, CH3 31.0, CH3 171.3, C 106.8, C 169.9, C 106.6, C 171.1, C 110.7, C 193.2, CH 193.2, CH 35.4, CH 41.5, CH2 28.1, CH 25.2, CH3 22.1, CH3

75.1, C 34.7, CH2 35.5, CH2 40.1, C 34.2, CH2 42.2, CH 46.0, CH 23.6, CH2 34.3, CH2 148.9, C 73.5, C 29.5, CH3 27.4, CH3 111.0, CH2 21.0, CH3 171.8, C 106.8, C 171.0, C 106.7, C 171.4, C 108.3, C 193.0, CH 193.1, CH 45.4, CH 35.9, CH2 28.1, CH 25.0, CH3 21.9, CH3

225.7, C 36.9, CH2 33.2, CH2 48.9, C 53.6, CH 28.2, CH 27.3, CH 21.9, CH2 45.3, CH2 212.9, C 19.2, C 16.8, CH3 29.4, CH3 29.9, CH3 22.9, CH3 165.6, C 105.9, C 163.3, C 95.4, CH 168.5, C 108.0, C 192.6, CH

226.2, C 36.9, CH2 32.8, CH2 48.7, C 55.4, CH 28.1, CH 27.4, CH 21.5, CH2 45.3, CH2 212.8, C 19.5, C 16.6, CH3 29.3, CH3 30.0, CH3 22.8, CH3 166.0, C 106.0, C 163.5, C 95.5, CH 168.5, C 107.6, C 192.7, CH

39.0, 37.1, 28.4, 22.3, 24.9,

41.4, 37.5, 28.1, 21.9, 24.9,

38.4, CH 22.5, CH2 39.0, CH2 73.0, C 136.2, CH 130.6, CH 57.1, CH 22.6, CH2 39.2, CH2 87.4, C 71.8, C 26.8, CH3 27.7, CH3 24.9, CH3 31.3, CH3 164.1, C 103.9, C 168.0, C 103.6, C 168.1, C 109.2, C 191.6, CH 192.0, CH 25.2, CH 41.7, CH2 26.2, CH 21.3, CH3 24.4, CH3

32.2, CH2 25.2, CH2 38.9, CH2 87.6, C 39.5, CH 22.8, CH2 57.0, CH 135.9, CH 131.8, CH 148.0, C 71.6, C 26.9, CH3 28.4, CH3 111.4, CH2 25.2, CH3 164.0, C 104.0, C 168.1, C 103.7, C 168.2, C 109.1, C 191.7, CH 192.0, CH 26.3, CH 41.8, CH2 26.5, CH 21.4, CH3 24.3, CH3

48.7, CH 21.5, CH2 34.5, CH2 41.2, CH 151.6, C 123.4, CH 47.0, CH 21.5, CH2 37.2, CH2 39.2, C 74.5, C 27.5, CH3 27.7, CH3 23.0, CH3 22.9, CH3 172.3, C 108.6, C 166.7, C 106.3, C 170.0, C 105.5, C 23.4, CH2 193.7, CH 207.6, C 54.1, CH2 26.5, CH 23.3, CH3 23.3, CH3

50.9, CH 26.0, CH2 40.1, CH2 47.0, C 53.7, CH 27.7, CH 27.5, CH 25.5, CH2 39.3, CH2 154.9, C 20.1, C 28.9, CH3 16.6, CH3 105.5 20.4, CH3 172.8, C 104.5, C 162.3, C 104.2, C 168.0, C 103.4, C 32.8, CH2 192.1, CH 206.6, C 52.9, CH2 25.0, CH 22.8, CH3 22.8, CH3

CH CH2 CH CH3 CH3

CH CH2 CH CH3 CH3

a

Measured in methanol-d4. bMeasured in CDCl3.

Figure 2. Calculated and experimental ECD spectra of eucalypglobulusal A (1).

The cross-peaks from H3-12/H3-13 to C-7, from H3-14 to C1/C-9, and from H3-15 to C-3/C-5 in the HMBC spectrum not only were used to construct a 10-membered monocyclic ring system but also confirmed the position of the double bond and the oxygenated quaternary carbon. The orientation of H-9′ was assigned as an α, by comparing the C-9′ chemical shift (δC 26.2) with that of eucalteretial C (δC 26.3),7 whereas the crosspeaks of H-11′ with H-1, H-1 with H-5/H3-14, and H-5 with H3-15/H3-13 in the ROESY spectrum supported the βorientations for these groups. The ROESY cross-peaks (Figure 1) of H-7 with H-6 and of H3-13 with H-5 enabled the proposal of an E configuration for the C-5/C-6 double bond. The absolute configuration of 5 was established as

Figure 1. 2D NMR correlations of 1, 5, and 6.

of 5 with those of euglobal-IIIa15 revealed that they possess the same 3,5-diformyl-phloroglucinol moiety but with the replacement of one trisubstituted double bond by a disubstituted one as well as the occurrence of an oxygenated quaternary carbon for the sesquiterpene unit of 5. The 1H−1H COSY cross-peaks (Figure 1) of 5 indicated two spin systems. 2641

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

Article

Figure 3. Proposed biosynthetic pathway for eucalypglobulusal A.

presence of a dihydropyran ring, which could be connected to the 3,5-diformyl-phloroglucinol and the sesquiterpene moieties. The ROESY correlations (Figure 1) of H3-15 with H-9/ H-10′a and H-9 with H-7 indicated that these groups are cofacial, and they were assigned randomly as having βconfigurations, while the correlation of H-8 with H-5 indicated that H-5 is α-oriented. An E configuration for the C-8/C-9 double bond was supported by the observed correlations of H9 with H3-15 and H-8 with H3-12. The experimental and calculated ECD curves matched for (4S,5R,7S,9′S)-6 (Figure 5). Accordingly, the structure of eucalypglobulusal F was elucidated as depicted.

Figure 4. Calculated and experimental ECD spectra of eucalypglobulusals C (3) and D (4).

Figure 5. Experimental and calculated ECD spectra of eucalypglobulusal F (6).

(1R,4R,7R,10R,9′S) as deduced from its ECD spectra (Figure S73, Supporting Information). Accordingly, the structure of eucalypglobulusal E was elucidated as depicted. Eucalypglobulusal F (6) was assigned a molecular formula of C28H38O6, based on a peak at m/z 469.2590 [M − H]− in the HRESIMS. The 13C NMR data demonstrated a total of 28 carbon resonances (Table 3), especially those for a 3,5diformyl-phloroglucinol moiety and two double bonds, which accounted for eight out of 10 IHDs, with the two remaining thus requiring the presence of a bicyclic framework. A 2D NMR analysis was carried out to elucidate the planar structure of 6 (Figure 1). Two spin systems were revealed based on the 1 H−1H COSY spectrum of 6. The HMBC correlations from H3-12/H3-13 to C-7 were used to place a 2-hyrdoxyisopropyl group at C-7. When combined with the aforementioned information, the observed HMBC correlations from H2-14 to C-1/C-9 and from H3-15 to C-3/C-5 allowed the proposal of a germacrane skeleton for the sesquiterpene moiety. The higher frequency shift for C-4 (δC 87.6) was consistent with the

The molecular formula, C28H40O6, of eucalypglobulusal G (7) was determined from the HRESIMS (m/z 471.2758, [M − H]−). Detailed inspection of NMR data indicated that 7 and macrocarpal Q16 share the same sesquiterpene moiety (Tables 2 and 3). The HMBC correlations from H2-7′ to C-1′/C-2′/C3′, from H-8′ to C-3′/C-4′/C-5′, from H3-12′/H3-13′ to C10′/C-11′, and from H 2 -10′ to C-9′ indicated the phloroglucinol moiety to be grandinol.17 The connectivity of the sesquiterpene with the phloroglucinol moiety was established via a C-1−C-7′ bond due to the 1H−1H COSY cross-peaks between H-1 and H2-7′ as well as the observed correlations from H2-7′ to C-1/C-10 in the HMBC spectrum. The ROESY correlations of H-7/H-9a, H-9a/H-1, and H-1/H4 supported these protons as being cofacial, and they were assigned randomly as α-configured. The H3-14 group was assigned to be a β-oriented from its ROESY cross-peak with H9b. Comparison of experimental ECD data with calculated values (Figure S73, Supporting Information) was used to 2642

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

Article

for a quaternary carbon (δC 67.1, C-4) and an oxygenated methine (δC 59.7, C-3).18 Three spin systems were determined from the 1H−1H COSY spectrum, while the correlations between H3-7 and C-2/C-6 as well as between H-3/H2-5/H-8 and C-4 in the HMBC experiment confirmed the monoterpene unit as being a γ-terpinene derivative.19 Based on the downfield chemical shift of C-1 (δC 81.9), the molecular formula information, and the HMBC correlation between H-9′ and C-1, a dihydropyran ring was constructed which connected the 3,5-diformyl-phloroglucinol and monoterpene moieties. The orientation of H-9′ was assigned as β, by comparing the C-9′ chemical shift (δC 35.7) with that of euglobal-IIIa (δC 36.2).15 The ROESY correlations of H-9′/H3-7, H3-7/H-6, and H-6/ H-2 revealed β-configurations for H3-7 and H-2, but the relative configuration of the epoxy group could not be established due to the lack of sufficient evidence from the ROESY spectrum. The absolute configuration of 9, (1S,2R,3S,4S,6R,9′S), was determined on the basis of comparing its computational and experimental ECD curves (Figure S73, Supporting Information) for the (1S,2R,3S,4S,6R,9′S) and (1S,2R,3R,4R,6R,9′S) alternatives. Accordingly, the structure of eucalypglobulusal I was elucidated as depicted. The molecular formula of eucalypglobulusal J (10) was determined as C23H30O6 from the HRESIMS data (m/z 401.1967 [M − H]−). Its 1D NMR spectra (Table 4) were highly similar to those of euglobal IIc,20 but with the occurrence of a C-4 hydroxy group due to correlations between H-2/H2-5/H-8 and C-4 in the HMBC spectrum. The ROESY cross-peaks of H-6 with H3-7/H-8 indicated a cofacial arrangement for these protons. The establishment of the absolute configuration of 10, (1S,4S,6S), was proved by comparing the calculated and experimental ECD values (Figure S73, Supporting Information). Accordingly, the structure of eucalypglobulusal J was elucidated as depicted. All of the compounds except 3, 8, and 9 (which were sample limited) were screened for their cytotoxic effects on five selected human tumor cell lines (HCT116, A549, DU145, Huh7, and CCRF-CEM). Among these compounds, 6, eucalrobusone C, eucarobustol C, and eucalyptin A showed discernible cytotoxic effects (Table 5) as compared to VP-16 and CPT. Furthermore, the topoisomerase I inhibitory activities of the isolated meroterpenoids were determined by a Top1-mediated relaxation assay (Table 5). Although compounds 1, macrocarpal A, macrocarpal B, and macrocarpal D did not show significant cytotoxic activities (IC50 > 10 μM), these isolates and eucarobustol C exhibited moderate to high Top1 inhibition (Figure 6). Based on the above results, compound 1 and macrocarpal A were selected for further evaluation in a Top1-mediated cleavage assay, which showed that the positive control (CPT) exhibited activity by stabilizing the topoisomerase I cleavage complex and resulted in increased nicked DNA (Figure 7). After treatment with 1 and macrocarpal A, the content of nicked DNA decreased in a concentration-dependent manner, which indicated that they inhibit the activity of Top1 to cleave DNA. Top1-mediated electrophoretic mobility shift assay (EMSA) demonstrated that 1 and macrocarpal A did not inhibit the binding of Top1 to DNA. An unwinding assay mediated by Top1 was performed with a positive control, ethidium bromide (a DNA intercalator), to investigate whether 1 and macrocarpal A initiate DNA unwinding due to excess

establish the absolute configuration of (1R,4R,7S,10R) for 7. Accordingly, the structure of eucalypglobulusal G was elucidated as depicted. Eucalypglobulusal H (8) was found to possess a molecular formula of C28H38O5, as deduced from its HRESIMS data (m/ z 453.2652 [M − H]−). The NMR spectroscopic data (Tables 2 and 3) indicated a similar basic skeleton of 8 and eucalyptin A,10 except for a double bond in the sesquiterpene portion of 8. A Δ10,14 double bond in 8 was established by HMBC correlations between H2-14 and C-1/C-9. Compound 8 and eucalyptin A were shown to have the same relative configuration, as determined from their ROESY data analysis. The calculated and experimental ECD data (Figure S73, Supporting Information) supported the absolute configuration of 8 as (1R,4R,5R,6R,7R). Accordingly, the structure of eucalypglobulusal H was elucidated as depicted. The molecular formula of eucalypglobulusal I (9) was determined to be C23H30O7 as deduced from its negative HRESIMS data (401.1967 [M − H]−), suggesting nine IHDs. The 13C NMR spectroscopic data of 9 (Table 4), in addition to 13 characteristic signals for the same 3,5-diformylphloroglucinol unit as that of 5 and 6, revealed 10 carbon signals that were attributed to the monoterpene unit. The presence of an epoxy ring was indicated by the chemical shift Table 4. 1H (600 MHz) and 13C (150 MHz) NMR Spectroscopic Data for Meroterpenoids 9 and 10 9a position 1 2 3 4 5a 5b 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 7′a 7′b 8′ 9′ 10′a 10′b 11′ 12′ 13′ OH-1′ OH-5′

δC 81.9, 72.2, 59.7, 67.1, 29.4,

C CH CH C CH2

δH (mult., J in Hz) 3.97, d (4.4) 3.21, d (4.4) 1.92, dd (15.3, 5.6) 1.47, dd (15.3, 10.6) 2.23, brdd (10.6, 5.6) 1.44, s 1.54, sept (6.9) 0.87, d (6.9) 0.94, d (6.9)

10b δC 75.3, C 132.2, CH 136.0, CH 72.9, C 32.8, CH2

33.1, CH 23.0, CH3 35.5, CH 18.5, CH3 18.0, CH3 171.2, C 105.5, C 168.9, C 105.7, C 163.6, C 106.6, C 193.8, CH 10.15, s

29.8, CH 24.6, CH3 37.7, CH 17.3, CH3 16.1, CH3 172.0, C 98.5, C 160.5, C 103.7, C 168.2, C 103.6, C 21.6, CH2

193.2, CH 10.05, s 35.7, CH 2.55, brd (8.0) 42.77, 1.77, td (13.6, 3.4) CH2 1.65, ddd (13.6, 9.2, 5.5) 28.1, CH 1.83, m 22.6, CH3 0.97, d (6.4) 23.9, CH3 0.94, d (6.4)

191.7, CH 206.4, C 52.7, CH2

25.0, CH 22.7, CH3 22.7, CH3

δH (mult., J in Hz) 5.88, brd (9.7) 5.81, d (9.7) 1.59, 1.41, 2.34, 1.41, 1.71, 0.82, 0.91,

t (13.5) overlap m s sept (6.6) d (6.6) d (6.6)

2.82, dd (17.1, 6.4) 2.51, brd (17.1) 10.00, s 2.97, 2H brd (6.6)

2.25, m 0.98, d (6.5) 0.98, d (6.5) 15.46, s 14.45, s

a

Measured in methanol-d4. bMeasured in CDCl3. 2643

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

Article

Table 5. Cytotoxic and Top1 Inhibitory Activities for 6, Eucalrobusone C, Eucarobustol C, and Eucalyptin Aa cytotoxicity [IC50 ± SD (μM)] compound

HCT116

CCRF-CEM

DU145

Huh7

A549

6 eucalrobusone C eucarobustol C eucalyptin A VP-16 CPT

>10 9.6 ± 2.4 >10 9.6 ± 0.3 >10 0.013 ± 0.01

3.3 ± 0.7 >10 6.9 ± 1.5 >10 1.1 ± 0.4 0.0020 ± 0.0003

>10 >10 >10 8.9 ± 2.0 >10 0.016 ± 0.004

>10 >10 >10 >10 7.4 ± 1.3 0.0023 ± 0.0004

>10 5.2 ± 0.4 5.3 ± 0.3 4.9 ± 0.6 >10 0.040 ± 0.001

Relaxation assay (50 μM): compound 6, 60−89%; eucalrobusone C, eucarobustol C, eucalyptin A, and CPT, >90%.

a

Figure 6. Top1-mediated relaxation assay.

Figure 7. (A) Top1-mediated DNA cleavage assay; (B) Top1-mediated EMSA; (C) Top1-mediated unwinding assay. and Sephadex LH-20 (GE Chemical Corporation) were used for column chromatography (CC). An Agilent 1260 instrument with a Zorbax SB-C18 column (5 μm, 9.4 × 250 mm) was used for semipreparative HPLC. Fractions were monitored based on TLC (GF254, Qingdao Marine Chemical Co., Ltd., People’s Republic of China) spots by spraying with 10% FeCl3 in EtOH. Plant Material. The fruits of E. globulus, authenticated by Dr. Rong Li, were collected at Kunming, People’s Republic of China, in July 2014. A voucher specimen (HY0027) was deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China (Kunming Institute of Botany, Chinese Academy of Sciences). Extraction and Isolation. The air-dried and powdered fruits (6.0 kg) of E. globulus were extracted with EtOAc three times at room temperature to afford a crude extract (550 g), which was eluted with petroleum ether−acetone (30:1 to 1:1, v/v) by Si gel (200−300 mesh) CC to give nine fractions (A−I). Fraction D (30 g) was subjected to a Sephadex LH-20 column (CHCl3−MeOH, 3:2 v/v) and then purified by RP-18 silica gel (MeOH−H2O, 60:40 → 100:0 v/v) to obtain five subfractions (D1−D5). Subfraction D4 (128 mg) was then subjected to semipreparative HPLC using CH3CN−0.01%

Top1. Ethidium bromide exhibited a clear unwinding effect with pBR322 DNA, both supercoiled (upper) or relaxed (bottom), as a substrate. In contrast, 1 and macrocarpal A did not exhibit an unwinding effect even at 25.0 μM. These findings indicated that they are excellent Top1 catalytic inhibitors.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured by a JASCO P-1020 polarimeter. UV spectra were recorded on a Shimadzu UV2401 PC spectrophotometer. An Applied Photophysics spectropolarimeter was used to record the ECD spectra. A Bruker FT-IR Tensor-27 infrared spectrophotometer was utilized for measuring the IR spectra (KBr disks). An Agilent 1290 UPLC/ 6540 Q-TOF mass spectrometer was employed for HRESIMS measurement. Bruker AM-400 and Avance III-600 spectrometers, with residual solvent signals as references, were used to record the NMR spectra. Si gel (200−300 mesh, Qingdao Marine Chemical Co., Ltd., People’s Republic of China), RP-18 (50 μm, Merck, Germany), 2644

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

Article

385 (3.40) nm; ECD (MeOH) 215 (Δε −10.41), 276 (Δε −1.96) nm; IR (KBr) νmax 3434, 2928, 1626, 1461, 1094 cm−1; 1H and 13C NMR, see Tables 2 and 3; (−)-HRESIMS m/z 471.2758 [M − H]− (calcd for C28H39O6, 471.2752). Eucalypglobulusal H (8): white, amorphous powder; [α]26D −42.3 (c 0.27, MeOH); UV (MeOH) λmax (log ε) 220 (4.11), 389 (4.27), 384 (3.67) nm; ECD (MeOH) 220 (Δε +12.66), 267 (Δε −3.44) nm; IR (KBr) νmax 3428, 2926, 1631, 1462, 1064 cm−1; 1H and 13C NMR, see Tables 2 and 3; (−)-HRESIMS m/z 453.2652 [M − H]− (calcd for C28H37O5, 453.2646). Eucalypglobulusal I (9): colorless gum; [α]20D +70.7 (c 0.21, MeOH); UV (MeOH) λmax (log ε) 205 (4.29), 294 (4.56), 367 (3.77) nm; ECD (MeOH) 211 (Δε +5.21), 279 (Δε +4.86), nm; IR (KBr) νmax 3440, 2929, 1628, 1435, 1057 cm−1; 1H and 13C NMR, see Table 4; (−)-HRESIMS m/z 401.1967 [M − H]− (calcd for C23H29O7, 401.1970). Eucalypglobulusal J (10): colorless gum; [α]20D −51.7 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 207 (4.35), 286 (4.50), 371 (4.00) nm; ECD (MeOH) 206 (Δε −4.22), 226 (Δε +3.30), 244 (Δε −0.17), 269 (Δε +6.00), 304 (Δε −2.18) nm; IR (KBr) νmax 3432, 2948, 1622, 1464, 1460, 1098 cm−1; 1H and 13C NMR, see Table 4; (−)-HRESIMS m/z 401.1967 [M − H]− (calcd for C23H29O6, 401.1970). ECD Calculations. The conformations of compounds 1−10 were obtained using the MM2 force field with ChemBio3D software due to their observed ROESY correlations. Gaussian 09 software was used for semiempirical PM3 quantum mechanical geometry optimizations and time-dependent density functional theory (TDDFT) ECD calculations at the B3LYP/6-31++G(2d,p) level.21 Cytotoxicity Assay. Cytotoxic activities were evaluated against the A549, HCT116, Huh7, DU145, and CCRF-CEM cancer cell lines by a previously reported method.5b Top1-Mediated Relaxation and Cleavage Assays. Top1mediated relaxation and cleavage assays were carried out with slight modifications as previously described.5b Top1-Mediated EMSA. A previously described procedure5b was used to carry out the Top1-mediated electrophoretic mobility shift assay. Top1-Mediated Unwinding Assay. An unwinding assay was performed following a previously described procedure with slight modifications.5b

trifluoroacetic acid (TFA) (90:10 → 99:1 v/v) to give 10 (20 mg), eucalrobusone C (8.2 mg), and eucarobustol C (6.5 mg). Fraction E (20 g) was purified on a Sephadex LH-20 column (CHCl3−MeOH, 3:2 v/v) and then on an RP-18 CC (MeOH−H2O, 60:40 → 100:0 v/ v) to afford six subfractions (E1−E6). Subfraction E3 (110 mg) was subjected to semipreparative HPLC (CH3CN−0.01% TFA, 95:10 → 99:1 v/v) to afford 7 (5.0 mg), 8 (2.2 mg), 9 (2.0 mg), and eucalyptal B (5.0 mg). Subfraction E4 (50 mg) was further purified by preparative TLC (petroleum ether−acetone−TFA, 10:1:0.01 v/v) to give 6 (15 mg) and eucalyptin A (13 mg). Fraction G (35 g) was separated by Si gel CC (petroleum ether−EtOAc, 20:1 → 1:1 v/v) to give five subfractions (G1−G5). Subfraction G3 (2.5 g) was purified by a Sephadex LH-20 CC (CHCl3−MeOH, 1:1 v/v) and subsequently by a recrystallization method in acetone to obtain macrocarpal D (32 mg). Subfraction G4 (4.1 g) was chromatographed on a Sephadex LH20 column (CHCl3−MeOH, 1:1 v/v) and subsequently purified by semipreparative HPLC (CH3CN−0.01% TFA, 70:30 → 80:20 v/v) to give 2 (15 mg), 3 (1.9 mg), 4 (4.5 mg), and macrocarpal E (11 mg). Fraction H (18 g) was separated using a Si gel CC (CHCl3− MeOH, 100:1 to 10:1 v/v) to yield four subfractions (H1−H4). Compound 5 (7.2 mg) and macrocarpal L (35 mg) were obtained from subfraction H2 (135 mg) by semipreparative HPLC (MeOH− 0.01% TFA, 90:10 → 100:0 v/v). Fraction I (40 g) gave six subfractions (I1−I6) on an RP-18 CC (MeOH−H2O, 50:50 → 95:5 v/v). Subfraction I4 (1.8 g) was subjected to passage over a Sephadex LH-20 column (CHCl3−MeOH, 1:1 v/v) and finally purified by semipreparative HPLC (CH3CN−0.01% TFA, 80:20 → 95:5 v/v) to give 1 (9.0 mg) and eucalyptone (167 mg). Macrocarpals A (3.7 g) and B (62 mg) were isolated from subfractions I5 (6.8 g) and I6 (320 mg), respectively, by a recrystallization method. Eucalypglobulusal A (1): white, amorphous powder; [α]26D +49.0 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 218 (4.23), 298 (4.39), 379 (3.82) nm; ECD (MeOH) 239 (Δε −14.32), 305 (Δε +12.64) nm; IR (KBr) νmax 3433, 2931, 1716, 1628, 1470, 1023 cm−1; 1H and 13 C NMR, see Tables 1 and 3; (−)-HRESIMS m/z 485.2558 [M − H]− (calcd for C28H37O7, 485.2545). Eucalypglobulusal B (2): white, amorphous powder; [α]26D −51.7 (c 0.10, MeOH); UV (MeOH) λmax (log ε) 219 (4.10), 296 (4.22), 382 (3.69) nm; ECD (MeOH) 276 (Δε +9.50), 302 (Δε −2.89) nm; IR (KBr) νmax 3430, 2938, 1625, 1448, 1061 cm−1; 1H and 13C NMR, see Tables 1 and 3; (−)-HRESIMS m/z 487.2712 [M − H]− (calcd for C28H39O7, 487.2701). Eucalypglobulusal C (3): white, amorphous powder; [α]20D +71.9 (c 0.16, MeOH); UV (MeOH) λmax (log ε) 211 (4.19), 298 (4.32), 338 (3.56) nm; ECD (MeOH) 243 (Δε −4.43), 271 (Δε +3.28), 396 (Δε −3.86), 329 (Δε +1.13) nm; IR (KBr) νmax 2428, 2952, 1712, 1623, 1459, 1073 cm−1; 1H and 13C NMR, see Tables 1 and 3; (−)-HRESIMS m/z 457.2603 [M − H]− (calcd for C27H37O6, 457.2596). Eucalypglobulusal D (4): white, amorphous powder; [α]20D +67.4 (c 0.14, MeOH); UV (MeOH) λmax (log ε) 211 (4.20), 298 (4.33), 338 (3.56) nm; ECD (MeOH) 263 (Δε −0.36), 293 (Δε +2.22), 342 (Δε −2.06) nm; IR (KBr) νmax 2430, 2950, 1711, 1622, 1461, 1068 cm−1; 1H and 13C NMR, see Tables 1 and 3; (−)-HRESIMS m/z 457.2605 [M − H]− (calcd for C27H37O6, 457.2596). Eucalypglobulusal E (5): white, amorphous powder; [α]26D +66.9 (c 0.12, MeOH); UV (MeOH) λmax (log ε) 296 (4.43), 375 (3.74) nm; ECD (MeOH) 236 (Δε −10.99), 281 (Δε +14.65), 321 (Δε −2.40), 350 (Δε +2.22) nm; IR (KBr) νmax 3448, 2940, 1627, 1439, 1109 cm−1; 1H and 13C NMR, see Tables 2 and 3; (−)-HRESIMS m/ z 487.2710 [M − H]− (calcd for C28H39O7, 487.2701). Eucalypglobulusal F (6): white, amorphous powder; [α]25D +74.5 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 215 (4.22), 296 (4.39), 373 (3.62) nm; ECD (MeOH) 228 (Δε −12.59), 280 (Δε +14.22), 319 (Δε −0.74), 347 (Δε +3.14) nm; IR (KBr) νmax 3440, 2939, 1629, 1442, 1176 cm−1; 1H and 13C NMR, see Tables 2 and 3; (−)-HRESIMS m/z 469.2590 [M − H]− (calcd for C28H37O6, 469.2596). Eucalypglobulusal G (7): white, amorphous powder; [α]26D −22.7 (c 0.11, MeOH); UV (MeOH) λmax (log ε) 219 (3.96), 282 (4.08),

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00430.

■

NMR and HRESIMS spectra and computational data of 1−10, experimental and calculated ECD data of 2, 5, and 7−10, as well as the structures and physicochemical data of known compounds (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (L.-K. An). *E-mail: [email protected] (H.-Y. Liu). ORCID

Xiao-Jiang Hao: 0000-0001-9496-2152 Lin-Kun An: 0000-0002-6088-1503 Hai-Yang Liu: 0000-0002-1050-6254 Author Contributions

# X.-J. Qin, L.-Y. Jin, and Q. Yu have equally contributed to this work.

Notes

The authors declare no competing financial interest. 2645

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646

Journal of Natural Products

■

Article

(18) Jin, A.; Wu, W. M.; Yu, H. Y.; Zhou, M.; Liu, Y.; Tian, T.; Ruan, H. L. J. Nat. Prod. 2015, 78, 2057−2066. (19) Singh, I. P.; Umehara, K.; Asai, T.; Etoh, H.; Takasaki, M.; Konoshima, T. Phytochemistry 1998, 47, 1157−1159. (20) Kokumai, M.; Konoshima, T.; Kozuka, M. J. Nat. Prod. 1991, 54, 1082−1086. (21) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Keith, T.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision E.01; Gaussian, Inc: Wallingford, CT, 2013.

ACKNOWLEDGMENTS The authors are grateful to the National Natural Science Foundation of China (Nos. 31570363, 31600283, 81703668, and 81373257), Scientific and Technological Projects of Yunnan Province (No. 2017FB128), Major Biomedical Project of Yunnan Province (2018ZF005), and Foundation of State Key Laboratory of Phytochemistry and Plant Resources in West China (Nos. P2017-KF06 and S2017-ZZ08) for the financial supported. We are also thankful to CNGrid (China National Grid, http://www.cngrid.org) for the ECD computational work.

■

REFERENCES

(1) (a) Pommier, Y. Chem. Rev. 2009, 109, 2894−2902. (b) Pommier, Y. ACS Chem. Biol. 2013, 8, 82−95. (2) (a) Akerman, K. J.; Fagenson, A. M.; Cyril, V.; Taylor, M.; Muller, M. T.; Akerman, M. P.; Munro, O. Q. J. Am. Chem. Soc. 2014, 136, 5670−5682. (b) Motiur-Rahman, A. F. M.; Park, S. E.; Kadi, A. A.; Kwon, Y. J. Med. Chem. 2014, 57, 9139−9151. (3) Lian, Q.; Xu, J.; Yan, S.; Huang, M.; Ding, H.; Sun, X.; Bi, A.; Ding, J.; Sun, B.; Geng, M. Cell Res. 2017, 27, 784−800. (4) (a) Wall, M. E.; Wani, M. C.; Cook, C. E.; Palmer, K. H.; McPhail, A. T.; Sim, G. A. J. Am. Chem. Soc. 1966, 88, 3888−3890. (b) Pommier, Y. Nat. Rev. Cancer 2006, 6, 789−802. (5) (a) Qin, X. J.; Yan, H.; Ni, W.; Yu, M. Y.; Khan, A.; Liu, H.; Zhang, H. X.; He, L.; Hao, X. J.; Di, Y. T.; Liu, H. Y. Sci. Rep. 2016, 6, 32748. (b) Qin, X. J.; Yu, Q.; Yan, H.; Khan, A.; Feng, M. Y.; Li, P. P.; Hao, X. J.; An, L. K.; Liu, H. Y. J. Agric. Food Chem. 2017, 65, 4993− 4999. (c) Qin, X. J.; Liu, H.; Yu, Q.; Yan, H.; Tang, J. F.; An, L. K.; Khan, A.; Chen, Q. R.; Hao, X. J.; Liu, H. Y. Tetrahedron 2017, 73, 1803−1811. (d) Qin, X. J.; Shu, T.; Yu, Q.; Yan, H.; Ni, W.; An, L. K.; Li, P. P.; Zhi, Y. E.; Khan, A.; Liu, H. Y. Nat. Prod. Bioprospect. 2017, 7, 315−321. (e) Qin, X. J.; Feng, M. Y.; Liu, H.; Ni, W.; Rauwoilf, T.; Porco, J. A., Jr.; Yan, H.; He, L.; Liu, H. Y. Org. Lett. 2018, 20, 5066−5070. (f) Liu, H.; Feng, M. Y.; Yu, Q.; Yan, H.; Zeng, Y.; Qin, X. J.; He, L.; Liu, H. Y. Tetrahedron 2018, 74, 1540−1545. (g) Qin, X. J.; Zhi, Y. E.; Yan, H.; Zhang, Y.; Liu, H.; Wang, S.; Zhao, Q.; He, L.; Ma, X.; An, L. K.; Liu, H. Y. Tetrahedron 2018, 74, 6658− 6666. (6) Shang, Z. C.; Yang, M. H.; Jian, K. L.; Wang, X. B.; Kong, L. Y. Chem. - Eur. J. 2016, 22, 11778−11784. (7) Yu, Y.; Gan, L. S.; Yang, S. P.; Sheng, L.; Liu, Q. F.; Chen, S. N.; Li, J.; Yue, J. M. J. Nat. Prod. 2016, 79, 1365−1372. (8) Murata, M.; Yamakoshi, Y.; Momma, S.; Aida, K.; Hori, K.; Ohashi, Y. Agric. Biol. Chem. 1990, 54, 3221−3226. (9) Yamakoshi, Y.; Murata, M.; Shimizu, A.; Homma, S. Biosci., Biotechnol., Biochem. 1992, 56, 1570−1576. (10) Yang, S. P.; Zhang, X. W.; Ai, J.; Gan, L. S.; Xu, J. B.; Wang, Y.; Su, Z. S.; Wang, L.; Ding, J.; Geng, M. Y.; Yue, J. M. J. Med. Chem. 2012, 55, 8183−8187. (11) Osawa, K.; Yasuda, H.; Morita, H.; Takeya, K.; Itokawa, H. Phytochemistry 1995, 40, 183−184. (12) Shibuya, Y.; Kusuoku, H.; Murphy, G. K.; Nishizawa, Y. Nat. Med. 2001, 55, 28−31. (13) Yin, S.; Xue, J. J.; Fan, C. Q.; Miao, Z. H.; Ding, J.; Yue, J. M. Org. Lett. 2007, 9, 5549−5552. (14) Tran, D. N.; Cramer, N. Chem. - Eur. J. 2014, 20, 10654− 10660. (15) Peng, L. Y.; He, J.; Xu, G.; Wu, X. D.; Dong, L. B.; Gao, X.; Cheng, X.; Su, J.; Li, Y.; Zhao, Q. S. Nat. Prod. Bioprospect. 2011, 1, 101−103. (16) Chenavas, S.; Fiorini-Puybaret, C.; Joulia, P.; Larrouquet, C.; Waton, H.; Martinez, A.; Casabianca, H.; Fabre, B. Phytochem. Lett. 2015, 11, 69−73. (17) Crow, W. D.; Osawa, T.; Paton, D. M.; Willing, R. R. Tetrahedron Lett. 1977, 12, 1073−1074. 2646

DOI: 10.1021/acs.jnatprod.8b00430 J. Nat. Prod. 2018, 81, 2638−2646