Rare dimeric guaianes from Xylopia vielana and their multidrug resistance reversal activity

Phytochemistry 158 (2019) 26–34

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Rare dimeric guaianes from Xylopia vielana and their multidrug resistance reversal activity

T

Ya-Long Zhang, Qi-Qi Xu, Xu-Wei Zhou, Lin Wu, Xiao-Bing Wang, Ming-Hua Yang, Jun Luo, Jian-Guang Luo∗∗, Ling-Yi Kong∗ Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 210009, People's Republic of China

A R T I C LE I N FO

A B S T R A C T

Keywords: Xylopia vielana Pierre Annonaceae Sesquiterpenoid dimers Guaiane

Thirteen undescribed dimeric guaianes were isolated from the leaves of Xylopia vielana Pierre. Their structures were elucidated by NMR spectroscopy and mass spectrometry, and the absolute configurations of vielanins G-Q were determined by a combination of the circular dichroism (CD) exciton chirality method, chemical conversion, and electronic CD (ECD) spectroscopy analysis. Vielaninors A and B are the first examples of trinor-guaianedimers. Multidrug resistance reversal activity assay of the isolates was evaluated in doxorubicin-resistant human breast cancer cells. Vielanins H, K-M, P, and Q were noncytotoxic and enhanced the cytotoxicity of doxorubicin by 2.1–41.6-fold at 10 μM.

1. Introduction Natural occurring dimeric sesquiterpenoids (DSs) have attracted great attention in recent years for their diverse structures and biological activities (Zhan et al., 2011; Liao and Yue, 2016; Li et al., 2010; Ohtsuki et al., 2008). Guaiane-type sesquiterpenoid dimers which are predominantly composed of dimeric guaianolides are one of the main classes of DSs, however, dimeric guaianes have rarely been isolated (Zhan et al., 2011; Liao and Yue, 2016). To date, all the dimeric guaianes from the plant kingdom have been reported to occur in the genus Xylopia from the family Annonaceae (Martins et al., 1998; Kamperdick et al., 2001, 2003; Zhang et al., 2017a). In our previous work, four dimeric guaianes, including xylopiana A with an unprecedented pentacyclo[5.2.1.01,2.04,5′.05,4′]decane-3,2′-dione core, and vielanins A, E, and F, had been isolated from the leaves of Xylopia vielana Pierre (Zhang et al., 2017a). Their fantastic structures are hypothesized to be formed via [4+2] or [2+2] cycloadditions, and vielanin E exhibited a potentiation effect on doxorubicin susceptibility in MCF-7/DOX cells. Multidrug resistance (MDR) is characterized by which neoplastic cells display resistance to many chemotherapeutic drugs that are chemically dissimilar with different cytotoxic targets (Daniel et al., 2013). Since MDR negates the efficacy of cancer chemotherapy, it has been a great challenge in the cancer treatment. As part of an ongoing search

∗

for novel MDR reversal agents from natural resources (Zhang et al., 2017a, 2017b), further investigation of the leave extract of X. vielana resulted in isolation of thirteen undescribed dimeric guaianes (1–13) (Fig. 1). Compounds 1–13 were presumably constructed through endoDiels-Alder cycloaddition of two monomeric guaiane units. Among these dimers, vielaninors A and B (1 and 2) are two unique trinorguaiane-dimers. MDR reversal activity assay of the isolates showed that compounds 4, 7–9, 12, and 13 exerted a 2.1–41.6-fold potentiation effect on doxorubicin susceptibility in MCF-7/DOX cells at 10 μM. In this paper, the isolation, structural elucidation, and MDR reversal activities of these undescribed dimeric guaianes are described. 2. Results and discussion Vielaninor A (1), yellowish powder, possessed a molecular formula of C29H36O6 based on a sodium adduct HRESIMS ion at m/z 503.2403 (calcd 503.2404), corresponding to 12 indices of hydrogen deficiency. The formation of a fragment ion at m/z 421.2376 revealed the presence of an acetoxy group, which was further confirmed by the corresponding signals in its NMR spectra [δH 2.02 (3H, s); δC 170.4 and 21.4]. Except for the acetoxy group, the remaining 27 carbon signals in its 13C NMR spectrum (Table 1) were assigned to be three ketonic, one oxygenated secondary, six olefinic, two quaternary, six methinic, three methylenic, and six methylic carbons with the aid of HSQC spectrum. The presence

Corresponding author. Corresponding author. E-mail addresses: [email protected] (J.-G. Luo), [email protected] (L.-Y. Kong).

∗∗

https://doi.org/10.1016/j.phytochem.2018.11.004 Received 15 May 2018; Received in revised form 17 October 2018; Accepted 6 November 2018 0031-9422/ © 2018 Published by Elsevier Ltd.

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Fig. 1. Structures of compounds 1–13 from Xylopia vielana.

of a α,β-unsaturated cyclopentenone (δC 145.4, 203.9, 56.3, 53.2, and 164.5), two doublet methyls [δH 0.88 (3H, d, J = 7.0 Hz) and 1.20 (3H, d, J = 6.5 Hz); δC 13.4 and 20.8], and only one isopropyl group [δH 1.92 (1H, m), 0.85 (3H, d, J = 7.0 Hz), and 0.78 (3H, d, J = 6.5 Hz); δC 27.6, 20.4, and 21.2] suggested that compound 1 might be a trinorguaiane-dimer. Two monomeric guaiane units (A and B) in the structure of 1 were assigned by a comprehensive analysis of its 2D NMR spectra (Fig. 2). Unit A was deduced to be a 11,12,13-trinor-guaiane moiety by the key HMBC cross-peaks from H-3 to C-1/C-2/C-4/C-5, from H-6 to C-1/C-4/ C-8, from H2-9 to C-1/C-7/C-8, from H3-14 to C-1/C-9/C-10, and from H3-15 to C-3/C-4/C-5. The key HMBC cross-peaks from H-2′ to C-4′/C5′/CH3eCOeO-2′, from H-3′ to C-1′/C-5′, from H2-6′ to C-1′/C-5′/C-7′/ C-8′/C-11′, from H2-9′ to C-1′/C-7′, from H3-12′/H3-13′ to C-7′, from H3-14′ to C-1′/C-9′/C-10′, and from H3-15′ to C-3′/C-4′/C-5′ enabled the construction of the structure of unit B, which was also found in the structure of vielanin A (Kamperdick et al., 2001; Zhang et al., 2017a). The connection between units A and B through C-3/C-3′ and C-4/C-1′ constructing a bicyclo[2.2.1]heptane moiety was deduced by the key HMBC cross-peaks from H-3 to C-3′/C-4′, from H3-15 to C-1′, from H-2′ to C-3/C-4, and from H-3′ to C-4. Therefore, the 2D structure of 1 was established as depicted. In the ROESY spectrum of 1 (Fig. 2), the key correlation between H6 and H-7′ revealed that the norbornene system is endo-orientated to the fused cyclopentenone moiety and H-7′ could be arbitrarily assigned as β-orientation. The ROESY correlation of H-7′/H-10′ indicated that H3-14′ is α-oriented, while the ROESY correlations of H-9b/H-6′ and H-

9a/H3-14 combined with the unobserved correlation of H-9b/H3-14 suggested that H3-14 is β-oriented. The relative configuration of C-2′ was assigned to be S* by the observed ROESY correlations between H-2′ and H-3/H3-15/H3-14′. Thus, compound 1 was elucidated as the first example of a trinor-guaiane-dimer. Vielaninor B (2) was assigned the same molecular formula as 1 by its HRESIMS ion at m/z 503.2399 (calcd 503.2404). The 1D NMR data of 2 showed high similarity with those of 1 (Table 1), suggesting that 2 is also a trinor-guaiane-dimer. According to the key HMBC cross-peaks from H-2 to C-1′/C-10′ and from H-3′ to C-2/C-10, two guaiane units (A and B) in 2 were connected through the C-1 to C-3′ and C-2 to C-1′ bonds (Fig. 3). The HMBC cross-peaks from H3-15 to C-3/C-4/C-5 and from H-2 to C-5/C-10 indicated that the conjugated carbonyl group of the cyclopentenone in unit A is located at C-3 instead of C-2 like in 1. The relative configuration of 2 was established by a ROESY experiment (Fig. 3). The key ROESY correlations from H-2′ to H-2/H-10 revealed that the norbornene system is endo-orientated to the fused cyclopentenone moiety and H-2′ as well as H3-14 is β-oriented. The ROESY correlations of H-7′/H-10′ and H-2′/H3-14′ indicated that H-7′ and H10′ are both β-oriented. Vielanin G (3) had the molecular formula of C32H42O6 on the basis of HRESIMS data. Except for three extra carbon signals (δC 73.5, 29.9, and 29.5), the 13C NMR data of 3 was similar to that of 2 (Table 1), indicating that 3 is a dimeric guaiane. The HMBC cross-peaks from H312 and H3-13 to C-11 (δC 73.5) and C-7 revealed that monomeric unit A in 3 is a complete guaiane skeleton and a hydroxyl group is located at C11 (Fig. 3). The 2D structure of 3 was thus established as depicted, 27

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Table 1 1 H (500 MHz) and No.

13

1 δH (J in Hz)

1 2 3 4 5 6 7 8 9a

2.58 d (4.0)

6.47 s

2.91 dd (15.5, 5.0) 2.55 dd (15.5, 3.0) 2.97a

9b 10 11 12 13 14 15 1′ 2′ 3′

0.88 d (7.0) 1.54 s 5.01 s 3.22 brd (4.0)

4′ 5′ 6′a 6′b 7′ 8′ 9′a 9′b 10′ 11′ 12′ 13′ 14′ 15′ CH3eCOeO CH3eCOeO a

which was further confirmed by analysis of the HMBC spectrum. In the ROESY spectrum of 3 (Fig. 3), the correlations from H-2′ to H-2/H-10/ H3-14′, from H-2 to H-10′/H3-14, and from H-7′ to H-10′ suggested that the relative configuration of 3 is identical with that of 2. In order to determine the absolute configuration of 3, the chemical conversion between 3 and biosynthetically related vielanin E (Fig. 4) was conducted (Kamperdick et al., 2003). Successful conversion of vielanin E to 3 under triphenylphosphine (PPh3) assigned the absolute configuration of 3 as 1R,2R,10R,1′S,2′S,3′S,7′S,10′R (Frimer et al., 2000). The molecular formula of vielanin H (4) was assigned as C30H36O6 based on the [M+Na]+ ion at m/z 515.2401 (calcd 515.2404). The 1D NMR data recorded in DMSO‑d6 (Table 2) of 4 showed resonances reminiscent of two guaiane unit (A and B). One OH signal at δH 6.37 and three carbon signals at δC 158.1, 102.2, and 84.9 revealed the presence of a five-membered peroxide ring. The peroxide ring was fused at C-7′ and C-8′ of unit B based on the HMBC cross-peaks from H-6′ to C-7′/C8′/C-11′, from H3-12′/H3-13′ to C-7′/C-11′, and from 8′-OH to C-7′/C8′/C-10′ (Fig. 3), which was similar to the corresponding moiety of vielanin D (Kamperdick et al., 2003). The carbonyl group in the fivemembered ring of unit B was located at C-3′, supported by the HMBC cross-peaks from H-2′/H3-15′ to C-3′ (δC 201.8). Unit A was deduced to be a disubstituted guaia-4,7(11)-dien-3,8-dione by comparison of the NMR data of 4 with those of vielanin F, which was further confirmed by the HMBC cross-peaks (Zhang et al., 2017a). Units A and B were linked through C-1 to C-4′ and C-2 to C-2′ bonds based on the key HMBC crosspeaks from H-2 to C-1′/C-2′, from H-2′ to C-2, and from H3-15′ to C-1. In the ROESY spectrum of 4 recorded in DMSO‑d6 (Fig. 3), 8′-OH showed the correlation with H3-15, indicating that two guaiane units are endo-orientated. The relative configurations of C-1, C-2, C-2′, C-4′, and C-8′ were thus assigned as S*, S*, R*, S*, and S*, respectively. The ROESY correlations of 8′-OH/H-10′, H-10/H3-15′, and H-2/H3-14 revealed that the relative configurations of C-10 and C-10′ are both R*. Therefore, the structure of 4 was established as depicted. Vielanin I (5) had the molecular formula of C30H38O6 based on HRESIMS (m/z 517.2562 [M+Na]+) and NMR data, which is 2 mass units more than that of 4. Comparison of the 13C NMR data (Table 2) for 5 with those for 4 indicated that 5 is a hydrogenation product of 4. This deduction was further confirmed by the upfield-shifted C-8 at δC 209.3 and the HMBC cross-peaks from H3-12/H3-13 to C-11/C-7. In the ROESY spectrum of 5 recorded in DMSO‑d6, the key correlations from 8′-OH to H3-15/H-10′, from H-10 to H3-15′/H-7, and from H-2 to H3-14 revealed that the relative configurations of C-1, C-2, C-7, C-10, C-2′, C4′, C-8′, and C-10′ is S*, S*, S*, R*, R*, S*, S*, and R*, respectively. Vielanin J (6) was deduced to have the molecular formula C32H40O7 by HRESIMS spectrum, which is 44 mass units more than that of 4. Comparison of the 1H and 13C NMR data of 6 (Table 2) with those of 4 indicated that the carbonyl group at C-3′ in 4 was replaced by an acetoxy group [δH 2.06 (3H, s); δC 170.0 and 21.0], which was further confirmed by the HMBC cross-peak from H-3′ to CH3eCOeO-3′. The relative configuration of 6 was determined by the key ROESY correlations from 8′-OH to H3-15/H-10′, from H-10 to H3-15′, and from H-2 to H-3′. Vielanin K (7) had the same molecular formula of C30H36O6 as 4 as indicated by its HRESIMS spectrum. Comparison of the 1D NMR data of 7 (Table 3) with those of 4 (Table 2) indicated that unit B in 7 is the same as that of 4, while unit A in 7 was deduced as a disubstituted guaia-1,7(11)-dien-3,8-dione by analysis of its HMBC spectrum. The HMBC cross-peaks from H2-6 to C-2′, from H3-15 to C-4′, and from H2′/H3-15′ to C-4 revealed that unit A is directly connected with unit B through the C-4-C-4′ and C-5-C-2′ bonds. The relative configuration of 7 was assigned as depicted by the key ROESY correlations (Fig. 5) from 8′-OH to H-2/H-10′, from H-10 to H-2′, and from H-2 to H3-14. The HRESIMS data of vielanin L (8) indicated a molecular formula of C30H36O6. A comparison of the NMR data of 8 (Table 3) with those of 4 (Table 2) revealed that the difference in their structures is the connection of units A and B. Units A and B in 8 were deduced to be linked

C (125 MHz) NMR data for compounds 1 and 2 in CDCl3.

2.42 dd (15.0, 6.0) 1.71 brd (15.0) 2.15 m 2.99 t (12.0) 2.05 d (12.0)a 2.50 m 1.92 m 0.85 d (7.0) 0.78 d (6.5) 1.20 d (6.5) 1.57 s 2.02 s

2 δC 145.4 203.9 56.3 53.2 164.5 107.1 153.7 194.7 44.1

22.4

13.4 23.0 66.2 86.1 52.1 136.1 132.8 26.5

3

δH (J in Hz)

2.36 s

6.73 s

3.48 dd (19.5, 5.0) 3.00 dd (19.5, 3.0) 2.41 m

0.94 d (6.5) 1.72 s

δC 56.5 55.2 205.4 143.1 159.4 109.4 150.7 195.6 43.8

32.5

4.86 s 3.04 s

16.9 8.5 64.0 85.9 55.3

2.37a

136.2 133.3 25.7

1.88 m 58.6 214.3 49.3

30.8 27.6 20.4 21.2 20.8 14.7 170.4 21.4

a

2.37

2.79 t (13.0) 2.17 brd (13.0) 2.87 m 1.97 m 0.91 d (6.5) 0.81 d (6.5) 1.09 d (6.5) 1.46 s 2.00 s

δH (J in Hz)

2.48 s

7.07 s

3.26 dd (14.5, 6.5) 2.61 dd (14.5, 4.5) 2.46 m

δC 58.2 54.3 206.1 145.6 161.1 126.2 153.0 206.3 51.5

4.88 brs 2.90 brs

34.3 73.5 29.9 29.5 18.8 8.8 64.5 86.0 56.9

2.30 m

136.4 132.6 25.6

1.49 s 1.46 s 0.95 d (6.5) 1.74 s

1.80 m 57.9 214.2 48.0

28.7 28.0 20.3 21.3a 17.5 14.3 170.9 21.3a

2.33 m 2.71 t (13.0) 2.12 dd (13.0, 1.0) 2.83 m 1.95 m 0.89 d (6.5) 0.79 d (6.5) 1.08 d (7.0) 1.43 s 1.98 s

57.7 214.0 48.0

28.6 27.9 20.3 21.2 17.3 14.3 170.6 21.3

Overlapped signals.

Fig. 2. Key HMBC and ROESY correlations of 1. 28

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Fig. 3. Key HMBC and ROESY correlations of 2–4.

Fig. 4. Conversion of vielanin E to compound 3.

spectrum to those of 8 defined the structure and absolute configuration of 9 as shown. The molecular formula of vielanin N (10) was assigned as C30H36O4, two oxygen atoms less than that of 8, based on HRESIMS (m/z 483.2503 [M+Na]+) and NMR data. The NMR data of 10 (Table 4) were similar to those of 8 (Table 3) except that the signals for the fivemembered peroxide ring were unobserved in 10, indicating that 10 is a deperoxidation product of 8. This was further confirmed by its HMBC experiment. The relative configuration of 10 was assigned by the key ROESY correlations from H-2 to H3-14/H3-13′ and from H-10 to H-10′. Since the ECD spectrum of 10 was similar to that of 7, the absolute configuration of 10 was determined as 4S,5S,10R,2′R,4′S,10′R. Vielanin O (11) was found to have the molecular formula of C30H36O4 by HRESIMS and NMR spectra. Similar to 10, compound 11 was assigned as a deperoxidation product of 8 by analysis of the HMBC spectrum of 11 and comparison of the NMR data of 8 and 11 (Tables 3 and 4). In the ROESY spectrum of 11, the correlations of H-2/H3-14, H10/H3-15′, and H-2′/H3-14′ revealed that H3-14 was β-oriented while H3-14′ was α-oriented. The ECD curve of 11 showed Cotton effects similar to those of 4. Therefore, the absolute configuration of 11 was assigned as 1R,2S,10R,2′S,4′S,10′R. The molecular formula of vielanin P (12) was established as C32H44O5 by HRESIMS and NMR spectra. Comparison of the NMR data of 12 (Table 4) with those of vielanin A revealed that unit B in 12 is the same as that of vielanin A (Zhang et al., 2017a). The HMBC cross-peaks from H3-15 to C-3/C-4/C-5, from H-3 to C-1/C-2/C-4/C-5, and from H7/H-11 to C-12/C-13 assigned unit A as a disubstituted guaia-1(5)-en2,8-dione moiety. The connection of these two guaiane moieties via C-

via the C-3-C-4′ and C-4-C-2′ bonds by the HMBC cross-peaks from H-3 to C-4′/C-5′, from H3-15 to C-2′, from H-2′ to C-3/C-15, and from H3-15′ to C-3, suggesting that the conjugated carbonyl group of the cyclopentenone in unit A is located at C-2. In the ROESY spectrum of 8 recorded in DMSO‑d6 (Fig. 5), the key correlations from 8′-OH to H-9a/H10/H-10′ defined the relative configurations of 8 as depicted. The absolute configurations of compounds 4–8 were determined by applying the CD exciton chirality method (Fig. 6). The ECD spectra of 4–8 recorded in MeOH showed intense negative first Cotton effects around 270 nm and positive second Cotton effects around 240 nm, indicating their negative chirality. Though there are more than two chromophores in their structures, their negative chirality should arise from the transition interaction between the trans-enone chromophore in unit A and the cis-diene chromophore in unit B by comparison of the ECD spectra of 4–6. Therefore, according to the exciton chirality rule, a counterclockwise screw sense between these two chromophores was assigned to compounds 4–8, eliciting their absolute configurations as depicted. Vielanin M (9) gave a molecular formula of C30H36O7, one more oxygen atom than that of 8, as deduced by its HRESIMS spectrum. The NMR data of 9 (Table 3) were comparable to those of 8 except for one OH signal [δH 5.13 (1H, s); δC 71.7] and one olefinic proton [δH 6.80 (1H, s)] in 9, suggesting that unit A in 9 is similar with that of 3. This deduction was supported by the HMBC cross-peaks from 11-OH (δH 5.13) to C-7/C-12/C-13 and from H-6 (δH 6.80) to C-1/C-4/C-8/C-11. The 2D structure of 9 was thus established as depicted. Similar to 8, the key correlations from 8′-OH to H-9a/H-10/H-10′ were also observed in the ROESY spectrum of 9. The similar ROESY correlations and ECD 29

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Table 2 1 H and 13C NMR data for compounds 4–6 in DMSO‑d6. No.

4a δH (J in Hz)

1 2 3 4 5 6a

3.15 d (15.5)

6b

3.07 d (15.5)

2.88 d (5.0)

7 8 9a

2.30 d (17.0)

9b 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′a 9′b 10′ 11′ 12′ 13′ 14′ 15′ 8′-OH CH3eCOeO CH3eCOeO a b c

1.93 dd (18.0, 12.0) 2.59 m 1.69 s 1.78 s 0.79 d (7.0) 1.36 s 3.37 d (5.0)

5.62 s

1.82 dd (13.5, 5.0) 1.49 d (13.0) 2.44 m 1.41 s 1.30 s 1.05 d (7.0) 1.46 s 6.37 s

5b δC 57.8 45.7 204.9 143.5 170.3 28.2

132.4 204.7 49.0

29.4 136.6 20.6 21.8 16.2 7.9 142.4 51.8 201.8 57.6 132.1 108.9 158.1 102.2 38.0

δH (J in Hz)

2.86 d (4.0)

2.45 t (10.5) 2.20 dd (10.5, 7.0) 3.12 m 2.17 d (15.0) 1.76 m 2.99 m 1.85 m 0.92 d (5.5) 0.78 d (5.0) 0.81 d (5.5) 1.29 sc 3.35 d (4.0)

5.57 s

1.80 m

6b δC 57.7 45.3 205.0 143.4 171.2 28.5

53.8 209.3 50.2

29.5 28.9 19.0 21.4 16.3 8.0 142.3 51.9 202.0 57.6 132.1 109.1 157.9 102.2 38.0

1.46 m 31.3 84.9 24.1 27.7 18.0 9.3

2.40 m 1.36 s 1.29 sc 1.03 d (5.5) 1.48 s 6.34 s

Table 3 1 H and 13C NMR data for compounds 7–9 in DMSO‑d6.

31.3 84.9 24.1 27.6 18.0 9.4

δH (J in Hz)

3.03c

3.45 d (12.0) 3.03c

3.35c 2.13 dd (10.0, 5.5) 2.87 m 1.92 s 1.98 s 0.71 d (5.5) 1.35 s 3.02c 4.32 s

5.69 s

1.78 dd (11.0, 4.0) 1.37 d (11.0) 2.18 m 1.50 s 1.30 s 1.01 d (6.0) 1.57 s 6.61 s 2.06 s

No. δC

7a

8a

δH (J in Hz)

61.5 47.8 206.9 141.4 171.6 30.2

1 2 3 4 5 6a 6b 7 8 9a

129.5 201.9 47.7

6.00 s

2.43 d (12.5)c 2.37 d (12.5)

2.63 dd (13.5, 9.5) 2.43 d (13.5)c

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

32.4 143.3 23.1 22.7 16.5 7.7 143.5 55.4 89.2 56.5 132.7 110.1 155.9 102.5 37.3

9′b 10′ 11′ 12′ 13′ 14′ 15′ 8′-OH 11-OH

30.5 84.9 23.6 28.1 18.8 13.6

a b

170.0 21.0

c

500 MHz for 1H and 125 MHz for 13C. 600 MHz for 1H and 150 MHz for 13C. Overlapped signals.

2.47 m

c

1.89 s 1.96 s 1.22 d (5.5) 0.93 s 3.11 s

5.38 s

1.85 dd (11.0, 3.0) 1.47 t (11.0) 2.47 mc 1.34 s 1.26 s 1.04 d (6.0) 1.19 s 6.59 s

δC 182.6 131.1 206.2 53.1 56.6 30.3 131.3 204.1 49.0

31.6 144.7 22.7 23.2 19.3 13.0 142.8 54.5 200.4 59.2 130.7 109.8 157.2 102.1 37.9

31.1 84.9 23.7 27.9 18.0 8.0

9b

δH (J in Hz)

2.12 s

3.62 d (14.0) 3.02 d (14.0)

3.25 d (12.5) 2.63 dd (12.5, 3.5) 2.64 m 1.86 s 1.91 s 0.77 d (6.0) 1.37 s 3.23 s

5.38 s

1.88 m 1.50 t (11.0) 2.71 m 1.33 s 1.27 s 1.13 d (6.0) 1.35 s 6.53 s

600 MHz for 1H and 150 MHz for 500 MHz for 1H and 125 MHz for Overlapped signals.

13

δC 149.2 203.0 56.0 49.4 169.9 27.6 132.0 204.4 46.3

26.1 142.9 22.3 22.9 18.1 21.1 143.4 57.3 201.1 57.4 129.2 109.5 156.9 102.3 37.8

31.1 84.9 24.0 27.8 18.2 11.3

δH (J in Hz)

2.17 s

6.80 s

3.08 dd (11.0, 5.0) 2.21 dd (11.0, 4.5) 2.87 m 1.28 sc 1.49 s 0.76 d (7.0) 1.28 sc 3.19 s

5.40 s

1.81 dd (13.0, 5.0) 1.44 t (13.0) 2.53 m 1.35 s 1.26 s 1.11 d (7.5) 1.37 s 6.60 s 5.13 s

δC 149.6 203.1 55.4 49.6 159.3 120.6 158.5 202.8 44.8

25.9 71.7 28.7 30.9 17.8 21.1 143.6 57.4 200.9 57.7 129.2 109.3 157.0 102.2 37.2

31.0 84.8 23.9 27.9 18.3 11.3

C. C.

13

determined by comparison of its ECD spectrum with those of 12 and vielanin A (Fig. 7). The presence of similar Cotton effects in the spectra of 12, 13, and vielanin A defined the absolute configuration of 13 as 1R,2R,7S,10R,1′S,2′S,3′S,7′S,10′R. Biosynthetically, compounds 1–13 are proposed to arise from the Diels-Alder reactions between two 10R-guaiane monomers on the basis of their structures. The different double bonds of cyclopentadienone in unit A approached the diene in unit B at C-1′/C-3′ (type A) or C-2′/C-4′ (type B), respectively, leading to the diverse skeleton types of these dimers (Fig. 8). Despite the different cycloaddition types, these dimers are all endo-adducts on account of the stereoselectivity of the [4+2] Diels-Alder reaction. The MDR reversal activity of compounds 1–5 and 7–13 in MCF-7/ DOX cells was evaluated encouraged by the previous results (Zhang et al., 2017a). The cytotoxicity assay showed that all compounds were noncytotoxic against the drug-sensitive and multidrug-resistant cells (IC50 > 30 μM), while only compounds 4, 7–9, 12, and 13 potentiated doxorubicin cytotoxicity by 2.1–41.6-fold when incorporated at 10 μM (Table 5). Compound 8 exhibited the highest potentiation effect of doxorubicin susceptibility with an enhancement of 41.6-fold at 10 μM. Compounds 10 and 11, the respective deperoxidation products of 7 and 4, were inactive, revealing that the peroxide ring may affect the activity. Compound 4 enhanced the cytotoxicity of doxorubicin by 5.5fold, while compound 5 was inactive, indicating that the exocyclic α,β-

3-C-1′ and C-4-C-3′ bonds was determined by the HMBC cross-peaks from H-3 to C-1′/C-10′, from H3-15 to C-3′, and from H-2′/H-3′ to C-3/ C-4. The relative configuration of 12 was assigned as depicted by the key ROESY correlations (Fig. 5) from H3-14 to H3-15′, from H-7 to H315, and from H-2′ to H-3/H3-15/H3-14′. The ECD spectrum of 12 showed similarity to that of vielanin A (Fig. 7). Thus, the absolute configuration of 12 was assigned as 3R,4R,7S,10R,1′S,2′S,3′S,7′S,10′R. Vielanin Q (13) had the molecular formula of C32H44O5 by HRESIMS spectrum, which is 2 mass units more than that of vielanin A (Zhang et al., 2017a). Comparison of the 13C NMR data (Table 4) for 13 with those for vielanin A indicated that 13 is a hydrogenation product of vielanin A, which was further confirmed by the HMBC experiment. The relative configurations of C-1, C-2, C-10, C-1′, C-2′, C-3′, C-7′, and C-10′ were assigned as R*, R*, S*, R*, S*, S*, S*, S*, and R*, respectively, by the key ROESY correlations from H3-15′ to H-6a, from H-2′ to H-2/H-10/H3-14′, and from H-10′ to H-7′. Owing to lack of useful ROESY correlations, the relative configuration of C-7 could not be determined through the ROESY spectrum. Since the absolute configuration of C-7 or C-7′ in compounds 3, 5, 12 and vielanin A was S, the absolute configuration of C-7 in 13 was tentatively assigned as S. The absolute configurations of the remaining stereogenic centers in 13 were 30

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Fig. 5. Key ROESY correlations of 7, 8, and 12.

unsaturated ketone group of unit A in 4 is essential for its MDR activity. Furthermore, comparing the activities of 7 and 8, 12 and 13, respectively, suggested that their 3D structures may have an influence on their activities. These findings may provide some guidances for structural modification to enhance the activity of this class of dimers. Consequently, dimeric guaianes isolated from the title plant are a new class of promising MDR reversal agents and their mechanism of action awaits further investigation.

collected from Jiangping Town, Dongxing City, Guangxi province of China in April 2016, and then were sun-dried. The material was identified by Prof. Mian Zhang in China Pharmaceutical University. A voucher specimen (No. XV201604) is deposited in the Department of Natural Medicinal Chemistry, China Pharmaceutical University. 3.3. Extraction and isolation The air-dried and powdered leaves of X. vielana (3.5 kg) were exhaustively extracted with 95% EtOH at room temperature (4 × 4 d). The EtOH extract was concentrated under reduced pressure. The crude extract (600 g) was suspended in H2O (2.0 L) and successively partitioned with CH2Cl2 and EtOAc. The CH2Cl2 (300 g) fraction was subjected to silica gel CC, eluted with a gradient of PE-EtOAc (15:1, 1:1, 1:5, = v/v), to afford four fractions (Fr. 1–4) based on TLC analysis. Fr. 3 (150 g) was further subjected to silica gel CC, eluted with a gradient of PE-EtOAc (10:1, 5:1), to obtain four subfractions (Fr. 3.1–Fr. 3.4). Fr. 3.2 (16 g) was applied to an MCI gel column (MeOHeH2O, 50:50–100:0) to yield two subfractions (Fr. 3.2.1–Fr. 3.2.2). Fr. 3.2.2 (4.5 g) was rechromatographed on an ODS column with MeOHeH2O (50:50–100:0) to afford four subfractions (Fr. 3.2.2.1–Fr. 3.2.2.4). Fr. 3.2.2.2 (200 mg) was further purified by preparative HPLC (CH3CNeH2O, 50:50) to obtain 12 (35 mg) and 13 (4 mg). Fr.3.3 (28 g) was subjected to an MCI gel column (MeOHeH2O, 50:50 to 100:0) to yield two subfractions (Fr. 3.3.1–Fr. 3.3.2). Fr. 3.3.1 (14 g) was rechromatographed on an ODS column with MeOHeH2O (40:60–100:0) to produce six subfractions (Fr. 3.3.1.1–Fr. 3.3.1.6). Fr. 3.3.1.2 (1 g) was subjected to silica gel CC (PE-EtOAc, 3:1) to afford four subfractions (Fr. 3.3.1.2.1–Fr. 3.3.1.2.4). Fr. 3.3.1.2.2 (350 mg) was further separated by preparative HPLC (CH3CNeH2O, 50:50, and then MeOHeH2O, 62:38) to yield 1 (18 mg), 9 (10 mg), and 11 (40 mg). Fr. 3.3.1.2.4 (280 mg) was further purified by preparative HPLC (MeOHeH2O, 60:40) to yield 4 (25 mg), 7 (8 mg), and 10 (10 mg). Fr. 3.3.1.3 (600 mg) was subjected to silica gel CC (PE-EtOAc, 3:1) and further purified by preparative HPLC (CH3CNeH2O, 50:50) to obtain 5 (15 mg). Fr. 3.3.1.4 (750 mg) was subjected to silica gel CC (PE-EtOAc,

3. Experimental 3.1. General experimental procedures Optical rotations were recorded on a JASCO P-1020 polarimeter. UV data were collected on a Shimadzu UV-2450 spectrophotometer. ECD spectra were recorded on a JASCO 810 spectropolarimeter in MeOH. IR spectra were recorded in KBr-disc on a Bruker Tensor 27 spectrometer. NMR spectra were acquired on Bruker Avance-500 or Ascend-600 NMR instruments in CDCl3 or DMSO‑d6 and chemical shift values were presented as δ values with TMS as the internal standard. HRESIMS data were recorded on an Agilent 6520B Q-TOF mass instrument. Column chromatography (CC) was done with silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China), MCI gel (Mitsubishi Chemical Industries Ltd., Japan), and ODS (40–63 μm, Fuji, Japan). Preparative HPLC was carried out using a Shimadzu LC-6AD series instrument with a Shim-park RP-C18 column (20 × 200 mm) and a Shimadzu SPD-20A detector. Analytical HPLC was performed on an Agilent 1200 Series instrument with a DAD detector using a Shim-pack VP–ODS column (4.6 × 250 mm). All the solvents used for CC were of analytical grade (Nanjing Chemical Reagents Co., Ltd., Jiangsu, China), and the solvents used for HPLC were of HPLC grade (Hanbon Science & Technology Co., Ltd., Jiangsu, China). 3.2. Plant material The fresh leaves of Xylopia vielana Pierre (Annonaceae) were

Fig. 6. ECD spectra of 4–8 in MeOH and the stereoview of 4 and 8. 31

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Table 4 1 H (500 MHz) and No.

13

C (125 MHz) NMR data for compounds 10–13 in CDCl3.

10 δH (J in Hz)

1 2 3 4 5 6a 6b 7 8 9a 9b 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ 6′a 6′b 7′ 8′ 9′a 9′b 10′ 11′ 12′ 13′ 14′ 15′ CH3eCOeO CH3eCOeO a

5.96 s

2.58 d (15.0) 2.19 d (15.0)

2.53 m 2.49a 1.97 s 2.08 s 1.22 d (6.0) 1.03 s 2.93 s

3.19 d (17.0) 2.99 d (17.0)

2.76 dd (12.0, 10.0) 2.48 dd (12.0, 4.5) 2.24 m 1.85 s 2.04 s 1.07 d (6.5) 1.29 s

11 δC 183.3 131.5 207.5 54.6 56.9 31.6 130.7 204.0 49.8 32.6 147.9 23.5 24.0 19.7 13.5 140.2 54.4 200.2 60.9 137.7 25.9 132.9 202.2 48.2 34.1 145.3 23.3 23.6 19.6 8.0

12

δH (J in Hz)

δC 58.9 45.8 205.0 144.8 170.5 29.2

2.68 d (5.0)

3.21a 3.03 d (15.0)

132.3 204.9 49.9

2.19 dd (18.5, 2.5) 2.07 dd (18.5, 12.0) 2.74 m

29.8 138.3 22.3 21.2 17.2 7.9 141.2 51.6 200.5 59.6 137.4 26.0

1.79 s 1.82 s 0.91 d (7.0) 1.53 s 3.24 d (5.0)

2.82 d (17.0) 3.21a

133.4 202.5 47.8

2.61 dd (12.5, 8.0) 2.44 dd (12.5, 4.5) 2.49 m

33.8 141.9 22.8 23.1 18.9 9.6

1.84 s 1.98 s 1.05 d (6.5) 1.49 s

13

δH (J in Hz)

2.20 s

2.46 m 2.14 m 2.70 m 2.39 dd (11.0, 6.5) 2.81 m 1.96a 0.99 d (7.0) 0.94 d (6.5) 0.97 d (7.0) 1.46 s 4.89 d (1.5) 2.73 brs

2.33 m 1.87 m 2.32a 2.77 m 2.12 m 2.78 m 1.97a 0.91 d (7.0) 0.79 d (6.5) 1.07 d (6.0) 1.60 brs 2.00 s

δC 146.2 205.5 55.4 51.1 173.1 26.9 60.2 211.9 46.1 28.1 30.8 19.6 21.1 20.0 21.0 65.2 87.1 58.0 135.9 131.4 25.9 58.1 214.3 47.9 28.9 27.9 20.4 21.2 17.4 14.5 170.6 21.3

δH (J in Hz)

2.40 s

2.67 m 2.11 m 2.43a 3.19 dd (11.0, 4.0) 2.44a 2.47 m 1.98 m 0.92 d (6.5)a 0.81 d (6.5) 0.73 d (6.5) 1.54 s 4.89 d (1.5) 3.41 brs

2.32 dd (15.0, 7.0) 1.86 m 2.65 m 2.73 t (13.5) 2.18 dd (13.5. 2.5) 2.93 m 2.07 m 0.97 d (7.0) 0.92 d (6.5)a 1.09 d (7.0) 1.60 d (1.0) 2.04 s

Overlapped signals.

Fig. 7. The ECD spectra of 12, 13, and vielanin A.

3:1) and further purified by preparative HPLC (CH3CNeH2O, 50:50, and then MeOHeH2O, 60:40) to obtain 2 (5 mg), 3 (30 mg), 6 (2 mg), and 8 (3 mg). Vielaninor A (1): yellowish powder; [α]25 D −259 (c 0.2, MeOH); UV (MeOH) λmax (log ɛ) 207 (3.92), 244 (3.78), 274 (3.44), 366 (3.75) nm; ECD (MeOH) λmax (mdeg) 203 (11.9), 216 (5.5), 243 (35.3), 284 (0.3), 315 (4.4), 382 (−20.3) nm; IR (KBr) νmax 3393, 2965, 2923, 2876,

Fig. 8. Cycloaddition types of 1, 2, 4, 7, 8, and 12.

32

δC 59.4 54.9 205.4 142.6 169.8 31.3 57.0 212.1 49.7 34.5 28.1 20.2 21.4 17.2a 8.4 64.5 87.1 56.3 135.5 133.0 26.0 55.1 214.1 48.5 28.5 28.8 19.8 21.8 17.2a 13.9 171.0 21.3

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(mdeg) 225 (53.8), 251 (−42.4), 302 (−4.8) nm; IR (KBr) νmax 2971, 2929, 2876, 1774, 1678, 1606, 1459, 1382, 1315, 1271 cm−1; 1H and 13 C NMR data, see Table 3; HRESIMS m/z 483.2503 [M+Na]+ (calcd for C30H36NaO4, 483.2506). Vielanin O (11): white powder; [α]25 D −155 (c 0.2, MeOH); UV (MeOH) λmax (log ɛ) 205 (3.62), 247 (3.84) nm; ECD (MeOH) λmax (mdeg) 226 (−6.5), 243 (13.0), 267 (−48.3), 329 (6.5) nm; IR (KBr) νmax 2965, 2928, 1776, 1694, 1680, 1634, 1456, 1382, 1338,1294, 1248 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 483.2502 [M+Na]+ (calcd for C30H36NaO4, 483.2506). Vielanin P (12): white powder; [α]25 D −65 (c 0.3, MeOH); UV (MeOH) λmax (log ɛ) 207 (3.92), 242 (3.81) nm; ECD (MeOH) λmax (mdeg) 204 (35.0), 240 (−26.5), 265 (−4.9), 294 (−7.8), 335 (2.4) nm; IR (KBr) νmax 2963, 2933, 2874, 1744, 1701, 1689, 1632, 1461, 1377, 1237 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 531.3080 [M+Na]+ (calcd for C32H44NaO5, 531.3081). Vielanin Q (13): white powder; [α]25 D −57 (c 0.2, MeOH); UV (MeOH) λmax (log ɛ) 206 (4.03), 246 (3.96) nm; ECD (MeOH) λmax (mdeg) 204 (33.0), 242 (−31.1), 328 (3.3) nm; IR (KBr) νmax 2964, 2933, 2876, 1746, 1694, 1628, 1462, 1384, 1241 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 531.3079 [M+Na]+ (calcd for C32H44NaO5, 531.3081).

Table 5 MDR reversal effects of 4, 7–9, 12, and 13 (10 μM) on MCF-7/DOX cells. Sample DOX DOX DOX DOX DOX DOX DOX DOX a

+ + + + + + +

4 7 8 9 12 13 Vera

IC50 (μM)

RF value

29.92 ± 10.22 5.41 ± 0.85 7.42 ± 1.01 0.72 ± 0.24 10.25 ± 3.50 4.03 ± 0.64 14.33 ± 2.04 2.98 ± 0.46

5.5 4.0 41.6 2.9 7.4 2.1 10.0

Verapamil was used as positive control at 10 μM.

1744, 1706, 1661, 1581, 1456, 1396, 1226, 1195, 1178 cm−1; 1H and 13 C NMR data, see Table 1; HRESIMS m/z 503.2403 [M+Na]+ (calcd for C29H36NaO6, 503.2404). Vielaninor B (2): yellowish powder; [α]25 D −6 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 206 (4.11), 342 (3.76) nm; ECD (MeOH) λmax (mdeg) 210 (9.9), 241 (−15.2), 270 (−1.0), 304 (−4.5), 367 (4.3) nm; IR (KBr) νmax 3436, 2962, 2928, 2876, 1744, 1701, 1460, 1381, 1237 cm−1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 503.2399 [M+Na]+ (calcd for C29H36NaO6, 503.2404). Vielanin G (3): white powder; [α]25 D +20 (c 0.2, MeOH); UV (MeOH) λmax (log ɛ) 207 (3.85), 298 (4.04) nm; IR (KBr) νmax 2965, 2934, 2875, 1746, 1683, 1615, 1455, 1383, 1338, 1237, 1174 cm−1; 1 H and 13C NMR data, see Table 1; HRESIMS m/z 545.2871 [M+Na]+ (calcd for C32H42NaO6, 545.2874). Vielanin H (4): white powder; [α]25 D −228 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 205 (3.90), 249 (4.19) nm; ECD (MeOH) λmax (mdeg) 201 sh (30.2), 241 (33.3), 273 (−52.8), 329 (4.1) nm; IR (KBr) νmax 3402, 2974, 2933, 2883, 1778, 1683, 1634, 1451, 1384, 1347, 1298, 1248 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 515.2401 [M+Na]+ (calcd for C30H36NaO6, 515.2404). Vielanin I (5): white powder; [α]25 D −152 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 206 (3.73), 249 (4.10) nm; ECD (MeOH) λmax (mdeg) 201 sh (19.8), 245 (38.6), 277 (−42.3), 330 (3.3) nm; IR (KBr) νmax 3440, 2970, 2931, 1779, 1712, 1697, 1674, 1624, 1454, 1416, 1384, 1159 cm−1; 1H and 13C NMR data, see Table 2; HRESIMS m/z 517.2562 [M+Na]+ (calcd for C30H38NaO6, 517.2561). Vielanin J (6): white powder; [α]25 D −94 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 206 (4.18), 255 (4.30) nm; ECD (MeOH) λmax (mdeg) 245 (43.5), 270 (−36.1) nm; IR (KBr) νmax 3456, 2973, 2929, 1738, 1671, 1626, 1463, 1383, 1342, 1237, 1159 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 559.2670 [M+Na]+ (calcd for C32H40NaO7, 559.2666). Vielanin K (7): light yellowish powder; [α]25 D −202 (c 0.2, MeOH); UV (MeOH) λmax (log ɛ) 242 (3.99) nm; ECD (MeOH) λmax (mdeg) 230 (38.8), 263 (−26.2) nm; IR (KBr) νmax 3296, 2971, 2930, 2877, 1775, 1696, 1656, 1586, 1461, 1381, 1364 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 515.2401 [M+Na]+ (calcd for C30H36NaO6, 515.2404). Vielanin L (8): white powder; [α]25 D −13 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 208 (3.94), 245 (4.21) nm; ECD (MeOH) λmax (mdeg) 237 (75.5), 278 (−45.5) nm; IR (KBr) νmax 3441, 2965, 2928, 2873, 1775, 1694, 1682, 1632, 1455, 1381 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 515.2399 [M+Na]+ (calcd for C30H36NaO6, 515.2404). Vielanin M (9): light yellowish powder; [α]25 D −100 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 207 (3.91), 229 (3.92), 273 (4.04), 310 (3.89) nm; ECD (MeOH) λmax (mdeg) 202 sh (−7.8), nm; IR (KBr) νmax 3400, 2987, 2972, 2926, 2867, 1780, 1685, 1658, 1593, 1456, 1409, 1382, 1359, 1326, 1277 cm−1; 1H and 13C NMR data, see Table 3; HRESIMS m/z 531.2347 [M+Na]+ (calcd for C30H36NaO7, 531.2353). Vielanin N (10): white powder; [α]25 D −110 (c 0.1, MeOH); UV (MeOH) λmax (log ɛ) 206 (3.94), 242 (4.04) nm; ECD (MeOH) λmax

3.4. Conversion of vielanin E to vielanin G (3) To a solution of vielanin E (5.0 mg, 0.009 mmol) in CH3CN (5 mL) was added triphenylphosphine (10.0 mg, 0.038 mmol) for 5 h at room temperature. The reaction mixture was concentrated in vacuo, followed by the addition of brine (10 mL). The resulting mixture was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over anhydrous Na2SO4, concentrated in vacuo and purified by preparative HPLC using MeOHeH2O (65:35) to afford 3 (3.4 mg, 70%).

3.5. Cytotoxicity assay MCF-7/DOX cells were cultured in RPMI 1640 containing 10% fetal bovine serum, harvested with trypsin, and resuspended in a final concentration of 5.0 × 104 cells/mL. Aliquots (0.1 mL) of cell suspension were seeded evenly into 96-well culture multiplates and incubated in a 37 °C incubator containing 5% CO2 for 24 h. A series of concentrations for the isolates in DMSO were added to designated wells. After 48 h, an MTT assay was performed as described previously (El-Readi et al., 2013; Xia et al., 2015).

3.6. MDR reversal assay The MDR reversal assay was performed as previously reported with slight modifications (Yuan et al., 2016; Zhang et al., 2017b). MCF-7/ DOX cells were distributed into 96-well culture plates at 5.0 × 103 cells per well. A full range of concentrations of DOX with or without 10 μM samples or 10 μM verapamil (positive control) were added to the cells. After 48 h, 20 μL MTT solution was then added to each well followed incubation for an additional 4 h. Finally, the purple formazan crystals formed were dissolved in 150 μL of DMSO by gently shaking for 10 min. The absorbance was evaluated at a test wavelength of 570 nm, and a reference wavelength of 630 nm by ELISA reader (Spectra Max Plus384; Molecular Devices, Sunnyvale, CA). IC50 values were calculated by using GraphPad Prism 5.0. The IC50 values of DOX were calculated from plotted results using untreated cells as 100%. The reversal fold, in terms of potency of reversal, was calculated using the following formula: reversal fold (RF) = IC50 (MCF-7/DOX cells)/IC50 (MCF-7/DOX cells combined with sample treatment). All assays were performed in triplicate and the IC50 values were shown as the mean ± SD. 33

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Acknowledgments

vielana. Phytochemistry 56, 335–340. Kamperdick, C., Phuong, N.M., Adam, G., Sung, T.V., 2003. Guaiane dimers from Xylopia vielana. Phytochemistry 64, 811–816. Li, C., Yu, X.L., Lei, X.G., 2010. A biomimetic total synthesis of (+)-ainsliadimer A. Org. Lett. 12, 4284–4287. Liao, S.G., Yue, J.M., 2016. Dimeric sesquiterpenoids. In: Kinghorn, A.D., Falk, H., Gibbons, S., Kobayashi, J. (Eds.), Progress in the Chemistry of Organic Natural Products. Springer-Verlag, Vienna, pp. 1–112. Martins, D., Osshiro, E., Roque, N.F., Marks, V., Gottlieb, H.E., 1998. A sesquiterpene dimer from Xylopia aromatica. Phytochemistry 48, 677–680. Ohtsuki, T., Tamaki, M., Toume, K., Ishibashi, M., 2008. A novel sesquiterpenoid dimer parviflorene F induces apoptosis by up-regulating the expression of TRAIL-R2 and a caspase-dependent mechanism. Bioorg. Med. Chem. 16, 1756–1763. Xia, Y.Z., Yang, L., Wang, Z.D., Guo, C., Zhang, C., Geng, Y.D., Kong, L.Y., 2015. Schisandrin A enhances the cytotoxicity of doxorubicin by the inhibition of nuclear factor-kappa B signaling in a doxorubicin-resistant human osteosarcoma cell line. RSC Adv. 5, 13972–13984. Yuan, W.Q., Zhang, R.R., Wang, J., Ma, Y., Li, W.X., Jiang, R.W., Cai, S.H., 2016. Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression. Oncotarget 7, 31466–31483. Zhan, Z.J., Ying, Y.M., Ma, L.F., Shan, W.G., 2011. Natural disesquiterpenoids. Nat. Prod. Rep. 28, 594–629. Zhang, Y.L., Zhou, X.W., Wang, X.B., Wu, L., Yang, M.H., Luo, J., Yin, Y., Luo, J.G., Kong, L.Y., 2017a. Xylopiana A, a dimeric guaiane with a case-shaped core from Xylopia vielana: structural elucidation and biomimetic conversion. Org. Lett. 19, 3013–3016. Zhang, Y.L., Zhou, X.W., Wu, L., Wang, X.B., Yang, M.H., Luo, J., Luo, J.G., Kong, L.Y., 2017b. Isolation, structure elucidation, and absolute configuration of syncarpic acidconjugated terpenoids from Rhodomyrtus tomentosa. J. Nat. Prod. 80, 989–998.

This research was financially supported by the 111 Project from Ministry of Education of China and the State Administration of Foreign Export Affairs of China (No: B18056), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Program for Changjiang Scholars and Innovative Research Team in University (IRT_15R63). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.phytochem.2018.11.004. References Daniel, C., Bell, C., Burton, C., Harguindey, S., Reshkin, S.J., Rauch, C., 2013. The role of proton dynamics in the development and maintenance of multidrug resistance in cancer. BBA – Mol. Basis Dis. 1832, 606–617. El-Readi, M.Z., Eid, S., Ashour, M.L., Tahrani, A., Wink, M., 2013. Modulation of multidrug resistance in cancer cells by chelidonine and Chelidonium majus alkaloids. Phytomedicine 20, 282–294. Frimer, A.A., Afri, M., Baumel, S.D., Gilinsky-Sharon, P., Rosenthal, Z., Gottlieb, H.E., 2000. Thermolysis and photosensitized oxygenation of tetrasubstituted cyclopropenes. J. Org. Chem. 65, 1807–1817. Kamperdick, C., Phuong, N.M., Sung, T.V., Adam, G., 2001. Guaiane dimers from Xylopia

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