Germacrane-Type Sesquiterpenoids with Antiproliferative Activities

Article Cite This: J. Nat. Prod. 2018, 81, 85−91

pubs.acs.org/jnp

Germacrane-Type Sesquiterpenoids with Antiproliferative Activities from Eupatorium chinense Xiaoqin Yu,†,‡ Qingqing Zhang,† Li Tian,† Zhiyong Guo,† Chengxiong Liu,† Jianfeng Chen,† Weaam Ebrahim,‡,§ Zhen Liu,*,‡ Peter Proksch,*,‡ and Kun Zou*,† †

Hubei Key Laboratory of Natural Products Research and Development, College of Biological and Pharmaceutical Sciences, China Three Gorges University, Yichang 443002, People’s Republic of China ‡ Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University Duesseldorf, 40225 Duesseldorf, Germany § Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt S Supporting Information *

ABSTRACT: Ten new germacrane-type sesquiterpenoids (1−10) were isolated from a whole plant extract of Eupatorium chinense. The structures were elucidated by analysis of their NMR and MS data as well as by comparison with literature values. The absolute configuration of eupachinsin A (1) was determined by single-crystal X-ray diffraction analysis. Compounds 3 and 4 exhibited cytotoxicity against a human breast cancer cell line (MDA-MB-231), with IC50 values of 0.8 and 3.4 μM, respectively. In addition, compounds 3−5 showed cytotoxicity against the human hepatocellular carcinoma cell line (HepG2), with IC50 values ranging from 3.6 to 7.6 μM.

T

Chart 1

he Asteraceae is one of the largest plant families of the angiosperms. The main chemical constituents of plants of this family are flavonoids and terpenoids, with the latter comprising mainly sesquiterpene lactones.1 Plants from the Asteraceae are used in folk medicine to treat various diseases due to their anti-inflammatory, antimicrobial, antineoplastic, antisyphilitic, and antigonorrheal properties.2 Artemisinin, isolated from Artemisia annua, and its semisynthetic derivatives are sesquiterpene lactones used to treat malarial infections caused by multiresistant Plasmodium strains.3 The genus Eupatorium s.l. (tribe Eupatorieae, subtribe Eupatoriinae), which is one of the largest genera of the Asteraceae, contains around 1200 species and is distributed throughout the world.4 Eupatorium chinense L. is used as a traditional Chinese medicine by Tujia and Miao minorities in the People’s Republic of China for its varied effects such as analgesic, antiinflammatory,5 antiviral,6 and cytotoxic activities.7 Its dry root “Tu Niuxi” has been in usage for the treatment of pharyngitis in Guangdong Province in mainland China since 1979.8 In a previous study, various flavonoids as well as a series of terpenoids had been isolated from E. chinense.9−11 Among the latter, sesquiterpenoids attracted our attention not only due to their structural diversity but also due to their potential antitumor activities.12−14 In continuation of a search for new bioactive metabolites from folk medicines of the Tujia minority, 10 new germacrane-type sesquiterpenoids derivatives (1−10) were isolated from an ethanol extract of E. chinense. In this report, the isolation, structural elucidation, and cytotoxic activity of these compounds are presented. © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Compound 1 was obtained as colorless crystals. The HRESIMS of 1 showed a pseudomolecular ion at m/z 443.1674 [M + Na]+, indicating a molecular formula of C22H28O8 with nine degrees of unsaturation. The 13C NMR spectrum of 1 (Table 1) displayed three carbonyl carbons at δC Received: August 11, 2017 Published: December 27, 2017 85

DOI: 10.1021/acs.jnatprod.7b00693 J. Nat. Prod. 2018, 81, 85−91

Journal of Natural Products 13

Table 1.

Article

13a), 5.81 (d, J = 2.0 Hz, H-13b), 5.31 (d, J = 10.8 Hz, H-5), and 5.16 (d, J = 8.2 Hz, H-1). The above spectroscopic data accounted for seven degrees of unsaturation, and the remaining two degrees of unsaturation were represented by the presence of a bicyclic carbon skeleton in compound 1. The COSY correlations between H-1/H-2 (δH 4.85, dd), H-2/H-3 (δH 5.29, d), H-5/H-6 (δH 5.79, dd), H-6/H-7 (δH 3.04, m), H-7/ H-8 (δH 5.30, m), H-8/H-9a (δH 2.81, dd), and H-8/H-9b (δH 2.52, dd), together with the HMBC correlations from Me-14 to C-1, C-9 (δC 43.5), and C-10, from Me-15 to C-3 (δC 79.7), C-4, and C-5, and from the methyl of OAc-3 and H-3 (δH 5.29, d) to the carbonyl carbon of OAc-3, indicated the occurrence of a 10-membered ring from C-1 to C-10 with two methyls at C-4 and C-10 in addition to a hydroxy group and an acetoxy group at C-2 and C-3, respectively (Figure 1). The presence of

C NMR Data of Compounds 1−5

position

1a

2a

3b

4a

5,a

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

130.8, CH 70.9, CH 79.7, CH 135.6, C 127.0, CH 75.9, CH 48.5, CH 78.3, CH 43.5, CH2 134.5, C 137.0, C 169.9, C 125.2, CH2 20.0, CH3 25.5, CH3 165.7, C 130.9, C 142.8, CH 15.7, CH3 64.4, CH2

125.4, CH 29.4, CH2 76.8, CH 136.3, C 126.5, CH 75.8, CH 48.5, CH 78.7, CH 43.5, CH2 135.4, C 137.3, C 169.8, C 124.8, CH2 19.2, CH3 23.1, CH3 165.8, C 131.1, C 142.5, CH 15.7, CH3 64.5, CH2



OAc-3

169.3, C 20.9, CH3

127.1, CH 72.4, CH 77.2, CH 135.1, C 127.8, CH 75.5, CH 48.4, CH 78.1, CH 43.5, CH2 136.2, C 136.9, C 169.7, C 125.2, CH2 20.0, CH3 24.8, CH3 165.7, C 130.9, C 143.0, CH 15.7, CH3 64.6, CH2 169.3, C 20.7, CH3 168.7, C 21.0, CH3

169.6, C 21.1, CH3

170.1, C 21.1, CH3

170.3, C 20.9, CH3

Figure 1. Key COSY and HMBC correlations of compound 1.

a

Recorded at 100 MHz in CDCl3. bRecorded at 150 MHz in CDCl3.

a fused unsaturated lactone ring at C-6 and C-7 was established by the HMBC correlations from H-13a and H13b to C-7 (δC 48.5), C-11, and C-12 and from H-6 to C-12. Furthermore, the COSY correlation between H-3′ and Me-4′ along with the HMBC correlations from H-5′a (δH 4.22, d) and H-5′b (δH 4.15, d) to C-1′, C-2′, and C-3′ and from H-8 to C-1′ suggested a 2-hydroxymethyl-2-butenoyl group to be attached at the C-8 position through an ester bond. Thus, the

169.9 (C-12), 169.3 (OAc-3), and 165.7 (C-1′) and eight olefinic carbons at δC 130.8 (C-1), 134.5 (C-10), 135.6 (C-4), 127.0 (C-5), 137.0 (C-11), 125.2 (C-13), 130.9 (C-2′), and 142.8 (C-3′). The 1H NMR spectrum (Table 2) exhibited four methyl groups at δH 2.09 (s, OAc-3), 2.06 (d, J = 7.3 Hz, Me4′), 1.97 (s, Me-15), and 1.84 (s, Me-14) and five olefinic protons at δH 6.42 (q, J = 7.3 Hz, H-3′), 6.40 (d, J = 2.0 Hz, HTable 2. 1H NMR Data of Compounds 1−5 1a

2a

1 2

5.16, d (8.2) 4.85, dd (8.2, 3.0)

5.11, d (8.5) 5.79, dd (8.5, 2.8)

3 5 6 7 8 9

5.29, 5.31, 5.79, 3.04, 5.30, 2.81, 2.52, 6.40, 5.81, 1.84, 1.97,

d (3.0) d (10.8) dd (10.8, 2.2) m m dd (14.1, 3.8) dd (14.1, 2.7) d (2.0) d (2.0) s s

5.23, 5.32, 5.77, 3.02, 5.30, 2.82, 2.50, 6.40, 5.81, 1.92, 1.93,

d (2.8) d (10.9) dd (10.9, 2.2) m m dd (14.2, 3.8) dd (14.2, 2.6) d (2.0) d (2.0) s s

6.42, 2.06, 4.22, 4.15,

q d d d

6.43, 2.06, 4.21, 4.15, 2.08, 2.10,

q d d d s s

position

13 14 15 3′ 4′ 5′ OAc-2 OAc-3

(7.3) (7.3) (12.7) (12.7)

2.09, s

(7.3) (7.3) (12.7) (12.7)

3a

4a

5.19, 2.71, 2.29, 5.26, 5.20, 5.87, 2.99, 5.30, 2.80, 2.47, 6.38, 5.78, 1.80, 1.82,

m m m dd (3.4, 3.0) d (10.8) br d (10.8) m m br d (14.0) br d (14.0) d (2.1) d (2.1) s s

5.08, 2.75, 2.08, 5.57, 5.21, 5.24, 2.98, 5.30, 2.77, 2.41, 6.39, 5.78, 1.91, 1.79,

t (7.5) m m dd (11.6, 5.0) d (10.7) br d (10.7) m m dd (14.2, 3.0) dd (14.2, 3.0) d (2.0) d (2.0) s s

6.41, 2.06, 4.23, 4.16,

q d d d

6.39, 2.03, 4.27, 4.18,

q d d d

2.08, s

(7.3) (7.3) (12.7) (12.7)

2.10, s

(7.3) (7.3) (12.7) (12.7)

5a 5.19, 2.80, 2.33, 5.40, 5.48, 5.83, 3.04, 5.31, 2.81, 2.48, 6.40, 5.81, 1.84, 4.16, 4.10, 6.43, 2.07, 4.23, 4.17,

m m m dd (3.9, 2.7) d (10.8) br d (10.8) m m br d (12.8) br d (12.8) d (2.0) d (2.0) s d (13.4) d (13.4) q (7.3) d (7.3) d (12.7) d (12.7)

2.09, s

a

Recorded at 600 MHz in CDCl3. 86


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The molecular formula of compound 2 was established as C24H30O9 by HRESIMS. Its NMR data (Tables 1 and 2) were similar to those of compound 1, suggesting that 2 is an analogue of 1. The presence of an additional acetyl group in 2 was deduced from its molecular formula and from the NMR signals (δC 169.3, 20.7 and δH 2.08, s, OAc-2; δC 168.7, 21.0 and δH 2.10, s, OAc-3), indicative of two acetyl groups. The HMBC correlations from H-2 (δH 5.79, dd) to the carbonyl carbon of OAc-2 and from H-3 (δH 5.23, d) to the carbonyl carbon of OAc-3 suggested the two acetyl groups to be located at the C-2 and C-3 positions, respectively. Detailed analysis of the 2D NMR spectra of 2 revealed that its remaining structural units are identical to those of 1. The relative configuration of 2 was determined by the similarity of the ROESY correlations and the coupling constants compared to 1. Based on the similar ECD spectra and close biogenetic relationship of 1 and 2, the absolute configuration of 2 was assumed to be the same as that of 1. Eupachinsin B (3) gave the molecular formula C22H28O7, as established by HRESIMS, lacking one oxygen atom when compared to compound 1. Comparison of the 1H and 13C NMR data of 3 with those of 1 showed an additional methylene signal at δC 29.4 (C-2) and δH 2.71 (H-2a), 2.29 (H-2a) as well as the absence of an oxygenated methine signal in compound 3. The position of the additional methylene group was determined by the COSY correlations between H-1 (δH 5.19)/H-2a and H-1/H-2b. The remaining structure of 3 was identical to that of 1, as evident from detailed analysis of the 2D NMR data of 3. Based on the similar NOE correlations as shown in Figure 4, the relative configuration of 3 was proposed as being the same as determined for 1. Analysis of the HRESIMS and 2D NMR spectra revealed that compound 4 shares the same planar structure as compound 3. However, the chemical shift of H-3 shifted to δH 5.57 in 4 when compared to the corresponding chemical shift of H-3 at δH 5.26 in 3. Meanwhile, the coupling constants of H-3 (11.7 and 5.0 Hz) in 4 were different from those in 3 (3.3 and 3.3 Hz). The above evidence suggested an αorientation of the acetoxy substituent at C-3, which was confirmed by the ROESY correlations from H-3 to Me-14 (δH 1.91), H-2a (δH 2.75), and H-6 (δH 5.24) in 4 (Figure 4). Compound 5 was isolated as a yellow jelly-like compound. A pseudomolecular ion peak appeared at m/z 443.1678 [M + Na]+ in the HRESIMS, indicating the molecular formula, C22H28O8. Its NMR data (Tables 1 and 2) were similar to those of 3 except for the C-15 methyl group, which was replaced by an additional oxygenated methylene group resonating at δC 65.7 (C-15) and δH 4.16 (d, H-15a), 4.10

planar structure of 1 was elucidated as shown, representing a new germacrane-type sesquiterpenoid derivative, for which the name eupachinsin A is proposed. The ROESY correlations between H-1/H-9b, H-5/Me-15, H-3′/H-5′a, and H-3′/H-5′b inferred E-geometry for the C-1/ C-10 double bond and Z-geometry for the C-4/C-5 and C-2′/ C-3′ double bonds. The ROESY correlations between Me-14/ H-2, Me-14/H-6, Me-14/H-9a, H-6/3-OAc and between H-1/ H-7, H-7/H-9b, H-9b/H-1, H-8/H-7, H-7/H-5, H-5/Me-15, Me-15/H-3 suggested that H-2 and H-6 occur on the same side of the ring, while H-3, H-7, and H-8 are on the other side (Figure 2). Moreover, the absolute configuration of eupachinsin A (1) was determined as 2R, 3R, 6R, 7R, and 8R by singlecrystal X-ray diffraction analysis (Figure 3).

Figure 2. Key NOE correlations of compound 1.

Figure 3. Molecular structure of 1 by single-crystal X-ray diffraction.

Figure 4. Key NOE correlations of compound 3 (left) and 4 (right). 87



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and the carbonyl carbon of OAc-15 (δC 170.4) as well as the correlation from the methyl group of OAc-15 (δH 2.09, s) to the carbonyl carbon of OAc-15 revealed that an acetoxy group is attached to C-15. The presence of a hydroxy group at C-4′ (δC 59.7) was supported by the COSY correlation between H24′ (δH 4.33, t) and H-3′ (δH 6.80, d) in addition to the HMBC correlations from H2-4′ to C-2′ (δC 127.5) and C-3′ (δC 142.2). Thus, the structure of 7 was elucidated as shown. The molecular formula of eupachinsin D (8) was determined to be C22H28O8 by HRESIMS. The 1H and 13C NMR data of 8 (Tables 3 and 4) were similar to those of 3 except for the replacement of an olefinic proton and a methyl group by additional signals of an oxygenated methine at δC 71.0 (C-1) and δH 4.06 (H-1) and signals of a terminal double bond at δH 5.42 (br s, H-14a) and 5.21 (br s, H-14b). The HMBC correlations from H-14a and H-14b to C-1, C-9 (δC 36.8), and C-10 (δC 143.4) indicated a terminal double bond at C-10/C-14 and the attachment of a hydroxy group to C-1. The similarity of the ROESY correlations between 8 and 3 suggested that they share the same relative configuration regarding the double bonds at C-4/C-5 and C-2′/C-3′ as well as the chiral centers at C-3, C-6, C-7, and C-8 (Figure 5). Furthermore, NOE correlations between H-1/H-7, H-7/H-9b, H-9b/H-1, H-1/H-2b, H-2b/Me-15, Me-15/H-5, and H-5/H7 were observed, which indicated an α-orientation of H-1. Compound 9 gave the molecular formula C22H28O9, as established by HRESIMS, with one more oxygen atom when compared to 8. Comparison of the 1H NMR data of 9 with those of 8 revealed the presence of one additional oxygenated methylene group (CH2-15, δC 65.1, δH 4.11 and 4.07) and the disappearance of a methyl group signal. The location of this additional oxygenated methylene group at C-15 was confirmed by the HMBC correlations from H-15a and H-15b to C-3 (δC 72.4), C-4 (δC 142.8), and C-5 (δC 126.4). The remaining part of the structure of 9 was found to be identical to that of 8 by detailed analysis of the 2D NMR spectroscopic data. The relative configuration of 9 was determined to be identical to that of 8 on the basis of similar NOE correlations and coupling constants. Compound 10 was found to share the same planar structure as 8 as deduced from the 2D NMR and the HRESIMS data. However, the chemical shift and coupling constants of H-3 (δH 5.74, dd, J = 12.1, 4.1 Hz) in 10 were different from those in 8. The α-orientation of the acetoxy group at C-3 was determined by the ROESY correlations from H-3 to H-14a (δH 5.61), H-2a (δH 2.34), and H-6 (δH 5.65) and other similar ROESY correlations to those found in 8 (Figure 5). The remaining part of the structure of 10 was determined to be identical to that of 8, as confirmed by detailed interpretation of the 2D NMR data. Thus, the structure of 10 was elucidated as shown. The electronic circular dichroism (ECD) spectra of compounds 1−10 were found to be very similar to a weak positive Cotton effect around 250 nm and an intense negative one around 210 nm. Each ECD was mainly represented by the α,β-unsaturated lactone chromophore containing an exocyclic double bond, while the unsaturated ester chromophore and isolated double bonds would be expected to have a weaker contribution to the experimental ECD obtained. The similar ECD spectra of these compounds indicated that compounds 2−10 have the same absolute configuration at C-6 and C-7 as compound 1, for which the absolute configuration was determined by X-ray diffraction analysis.

(d, H-15b). These data indicated that 5 is a derivative of 3, differing from the latter by the presence of a hydroxymethylene group at C-15, which was supported by the HMBC correlations from H-15a and H-15b to C-3 (δC 74.0), C-4 (δC 139.2), and C-5 (δC 126.8). Detailed analysis of the 2D NMR spectra of 5 revealed that its remaining structural units and relative configuration are identical to those of 3. The molecular formula of eupachinsin C (6) was determined as C22H28O7 by HRESIMS. The NMR data of 6 (Tables 3 and 4) were closely related to those of 3. However, Table 3. position 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1′ 2′ 3′ 4′ 5′ OAc-3 OAc-15

13

C NMR Data of Compounds 6−10 6a

7a

8a

9b

10c

125.1, CH 30.3, CH2 74.2, CH 139.0, C 127.1, CH 75.4, CH 48.2, CH 78.3, CH 43.4, CH2 136.0, C 137.2, C 169.7, C 124.9, CH2 19.2, CH3

125.1, CH 30.8, CH2 74.0, CH 134.8, C 129.7, CH 75.1, CH 48.2, CH 78.6, CH 43.4, CH2 136.0, C 137.0, C 169.7, C 125.2, CH2 19.3, CH3

65.8, CH2 166.6, C 128.1, C 138.6, CH 14.5, CH3 11.9, CH3 169.9, C 21.0, CH3

67.1, CH2 166.0, C 127.5, C 142.2, CH 59.7, CH2 12.6, CH3 169.5, C 21.1, CH3 170.4, C 20.9, CH3

71.0, CH 34.9, CH2 72.6, CH 138.1, C 126.0, CH 73.4, CH 47.0, CH 75.6, CH 36.8, CH2 143.4, C 136.4, C 169.3, C 124.9, CH2 117.4, CH2 22.8, CH3 165.3, C 130.7, C 143.2, CH 15.5, CH3 64.4, CH2 169.7, C 20.8, CH3



a

Recorded at 100 MHz in CDCl3. bRecorded at 100 MHz in CD3OD. cRecorded at 150 MHz in CD3OD.

the HMBC correlations from H-15a (δH 4.15, d, J = 13.4 Hz) and H-15b (δH 4.09, d, J = 13.4 Hz) to C-3 (δC 74.2), C-4 (δC 139.0), and C-5 (δC 127.1) implied the presence of a hydroxy group attached to C-15. Furthermore, the HMBC correlations from Me-5′ (δH 1.79, s) to C-1′ (δC 166.6), C-2′ (δC 128.1), and C-3′ (δC 138.6) indicated that a methyl group is located at C-2′. The geometry of the double bond of the esterified acyl substituent was determined as E by comparison with (E)-2,3dimethylacrylic acid (δC 11.6, C-5; δC 14.6, C-4) and (Z)-2,3dimethylacrylic acid (δC 16.0, C-5; δC 20.3 C-4). The remaining substructures and relative configuration of 6 were found to be identical to 3. Compound 7 was obtained as a yellow oil. Its HRESIMS established a molecular formula of C24H30O9 with 10 degrees of unsaturation. The NMR data of 7 were similar to those of 6 except for an additional acetyl group, an additional oxygenated methylene group (δC 59.7 and δH 4.33), and the lack of a methyl group. In the HMBC spectrum, the correlations from H-15a (δH 4.60, d, J = 13.3 Hz) and H-15b (δH 4.57, d, J = 13.3 Hz) to C-3 (δC 74.0), C-4 (δC 134.8), C-5 (δC 129.7), 88



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Table 4. 1H NMR Data of Compounds 6−10 6a

position 1 2 3 5 6 7 8 9 13 14 15 3′ 4′ 5′ OAc-3 OAc-15

7a

5.18, 2.79, 2.31, 5.39, 5.48, 5.87, 3.00, 5.22, 2.75, 2.42, 6.35, 5.77, 1.80,

m m m dd (3.4, 2.9) d (10.9) br d (10.9) m m br d (14.3) br d (14.3) d (2.1) d (2.1) s

5.17, 2.79, 2.29, 5.37, 5.49, 5.92, 3.02, 5.25, 2.75, 2.46, 6.38, 5.80, 1.81,

m m m m d (10.8) br d (10.8) m m br d (14.0) br d (14.0) d (2.0) d (2.0) s

4.15, 4.09, 6.84, 1.77, 1.79,

d d q d s

4.60, 4.57, 6.80, 4.33, 1.80,

d (13.3) d (13.3) t (5.5) d (5.5) s

(13.4) (13.4) (7.2) (7.2)

2.08, s

2.11, s 2.09, s

8b

9c

4.06, 2.43, 2.21, 5.35, 5.32, 6.01, 3.15, 5.30, 2.94, 2.66, 6.38, 5.82, 5.42, 5.21, 1.84,

dd (11.6, 2.8) ddd (15.5, 11.6, 2.1) ddd (15.5, 5.5, 2.8) dd (5.5, 2.1) d (9.8) br d (9.8) m m dd (14.9, 5.0) dd (14.9, 3.2) d (2.0) d (2.0) s s s

6.44, 2.07, 4.20, 4.17, 2.06,

q d d d s

(7.3) (7.3) (13.8) (13.8)

4.04, 2.57, 2.13, 5.39, 5.58, 6.15, 3.34, 5.31, 2.90, 2.64, 6.31, 5.97, 5.44, 5.21, 4.11, 4.07, 6.45, 2.04, 4.22, 4.14, 2.04,

10c

dd (11.6, 2.8) ddd (15.6, 11.6, 2.1) ddd (15.6, 5.4, 2.8) dd (5.4, 2.1) d (10.3) br d (10.3) m m dd (14.8, 4.9) dd (14.8, 3.1) d (2.0) d (2.0) s s d (14.5) d (14.5) q (7.3) d (7.3) d (13.9) d (13.9) s

3.71, 2.34, 2.04, 5.74, 5.39, 5.65, 3.24, 5.29, 2.94, 2.55, 6.31, 5.96, 5.61, 5.28, 1.84,

dd (11.9, 3.4) ddd (12.7, 11.9, 4.1) ddd (12.7, 12.1, 3.4) dd (12.1, 4.1) d (10.7) dd (10.7, 2.4) m m dd (14.9, 4.3) dd (14.9, 2.7) d (2.0) d (2.0) s s s

6.42, 1.99, 4.22, 4.16, 2.09,

q d d d s

(7.3) (7.3) (13.4) (13.4)

a

Recorded at 400 MHz in CDCl3. bRecorded at 600 MHz in CDCl3. cRecorded at 600 MHz in CD3OD.

Table 5. Cytotoxicity (IC50, μM) of Compounds 1−9a

All compounds isolated except 10, which was obtained only in a limited amount, were assayed for their cytotoxicity against the Caski human cervical cancer cell line, the MDA-MB-231 human breast cancer cell line, the CNE2 human nasopharyngeal carcinoma cell line, and the HepG 2 human hepatocellular carcinoma cell line using an MTT assay (Table 5). Among them, compounds 3 and 4 exhibited cytotoxicity against the MDA-MB-231 cell line with IC50 values of 0.8 and 3.3 μM, respectively. Moreover, compounds 3−5 showed cytotoxicity against the HepG2 cell line with IC50 values ranging from 3.6 to 7.6 μM, respectively.

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compound

Caski

MDA-MB-231

CNE2

HepG2

3 4 5 mitomycin C

>10 >10 >10 >10

0.8 3.3 >10 4.4

>10 >10 >10 >10

3.6 6.3 7.6 >10

Compounds 1, 2, and 6−9 were not cytotoxic (IC50 > 10 μM) for any of the cell lines used. a

employed was prefilled with Eurosphere-10 C18 (Knauer, Germany), and the following gradient was used (MeOH, 0.1% formic acid in H2O): 0 min (10% MeOH); 5 min (10% MeOH); 35 min (100% MeOH); 45 min (100% MeOH). Semipreparative HPLC was performed using a Merck Hitachi HPLC System (UV detector L7400; pump L-7100; Eurosphere-100 C18, 300 × 8 mm), with mixtures of MeOH−H2O as mobile phases. Column chromatography utilized Merck MN silica gel 60 M (0.04−0.063 mm). TLC plates precoated with silica gel F254 (Merck) were used to monitor and collect fractions; detection was under 254 and 366 nm or by spraying the plates with anisaldehyde reagent. Distilled and spectroscopic grade solvents were used for column chromatography and spectroscopic measurements, respectively.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-2000 polarimeter. NMR spectra were recorded on Bruker ARX 400 or 600 NMR spectrometers. Chemical shifts were referenced to the solvent residual peaks. Mass spectra (ESI) were recorded with an LC-MS HP1100 Agilent Finnigan LCQ Deca XP Thermoquestman spectrometer, and HRESIMS were recorded on a UHR-QTOF maXis 4G (Bruker Daltonics) mass spectrometer. HPLC analysis was performed with a Dionex UltiMate3400SD system coupled to an LPG-3400SD pump and a photodiode array detector (DAD 300RS). The analytical column (125 × 4 mm)

Figure 5. Key NOE correlations of compounds 8 (left) and 10 (right). 89



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(0.04), 213 (−0.86) nm; 1H and 13C NMR data, Tables 3 and 4; HRESIMS m/z 437.1808 [M + H]+ (calcd for C22H29O9, 437.1806). 3-Epi-eupachinisin D (10): colorless oil; [α]20 D +15 (c 0.48, MeOH); UV (MeOH) λmax 203 nm; ECD (MeOH) λmax (Δε) 239 (0.13), 215 (−1.11) nm; 1H and 13C NMR data, Tables 3 and 4; HRESIMS m/z 438.2126 [M + NH4]+ (calcd for C22H32NO8, 438.2122), m/z 443.1675 [M + Na]+ (calcd for C22H28O8Na, 443.1676). X-ray Crystallographic Analysis of Compound 1. Crystallization conditions: X-ray quality crystals of 1 were obtained by slow evaporation of a MeOH solution containing compound 1. A suitable single crystal was carefully selected under a polarizing microscope. Data collection: SuperNova diffractometer (equipped with an Atlas detector) with Cu Kα radiation (λ = 1.541 84 Å). Structure analysis and refinement: The structure was solved by direct methods using SHELXS-2014; refinement was done by full-matrix least-squares on F2 using the SHELXL-2015 program suite.15 Crystal Data of 1. C22H28O8, M = 420.44, orthorhombic system, space group P212121, a = 10.6433(2) Å, b = 13.6676(3) Å, c = 14.8419(3) Å, V = 2159.04(8) Å3, Z = 4, Dcalc = 1.293 g/cm3, crystal size 0.25 × 0.2 × 0.16 mm3, μ(Cu Kα) = 0.820 mm−1, 4.398° < θ < 73.707°, Nt = 9822, N = 4248 (Rint = 0.0218), R1 = 0.0309, wR2 = 0.0777, S = 1.066, Flack parameter 0.07(7) determined using 1701 quotients. The structural data have been deposited in the Cambridge Crystallographic Data Center (CCDC No. 1542973). Cytotoxicity Assay. Cytotoxicity against the Caski human cervical cancer cell line, the MDA-MB-231 human breast cancer cell line, the CNE2 human nasopharyngeal carcinoma cell line, and the HepG2 human hepatocellular carcinoma cell line was evaluated using the MTT method with mitomycin C as positive control and medium with 0.1% DMSO used as negative control, as described previously.16

Plant Material. In August 2015, the aerial parts of Eupatorium chinense were collected at Changyang, Hubei Province, People’s Republic of China, and identified by Prof. Yu-Bin Wang. A voucher specimen (No. 201507162) has been deposited at the Herbarium of the Department of Medicinal Plants, College of Biological and Pharmaceutical Sciences, Three Gorges University. Extraction and Isolation. The dried powder (4.4 kg) of the whole plant of E. chinense was percolated with 95% EtOH. After removal of the solvent under reduced pressure, a dark green residue (135 g) was obtained, which was then suspended in water (3.6 L) and extracted with petroleum ether, EtOAc, and n-BuOH, respectively. The ethyl acetate-soluble fraction (67 g) was then subjected to silica gel column chromatography using a gradient solvent system of chloroform−methanol (100:1 to 0:100; v/v), to obtain six major fractions (Fr. A−F). Fr. B (7.2 g) was subjected to passage over a Sephadex LH-20 column (60 × 3 cm) with MeOH as mobile phase to remove pigments, then purified by semipreparative HPLC (MeOH− H2O: 0−5 min, 45%; 5−18 min, from 45% to 68%; 19−25 min, 100%) to give 1 (2.1 mg), 2 (2.4 mg), and 3 (2.9 mg). Following the same protocol, 4 (2.0 mg) and 5 (2.5 mg) were obtained from Fr. C (6.2 g) (HPLC sequence, MeOH−H2O: 0−10 min, from 20% to 40%; 10−22 min, from 40% to 60%; 22−30 min, 100%), while 6 (2.4 mg) and 7 (3.2 mg) were obtained from Fr. D (6.5 g) (HPLC sequence, MeOH−H2O: 0−5 min, from 20% to 30%; 5−20 min, from 30% to 60%; 20−25 min, 100%). Fr. F (22.5 g) was separated by Sephadex LH-20 column chromatography, with MeOH as mobile phase to give eight subfractions (Fr. F1−F8). Compounds 8 (3.5 mg), 9 (4.2 mg), and 10 (1.1 mg) were obtained from Fr. F3 (185.1 mg) by semipreparative HPLC using MeOH−H2O (HPLC sequence: 0−5 min, from 30% to 45%; 5−25 min, from 45% to70%; 25−30 min, 100%). Eupachinsin A (1): colorless crystals; [α]20 D −70 (c 0.22, MeOH); UV (MeOH) λmax 211 nm; ECD (MeOH) λmax (Δε) 253 (0.08), 214 (−3.28) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 443.1674 [M + Na]+ (calcd for C22H28O8Na, 443.1676). Eupachinisin A 2-acetate (2): colorless oil; [α]20 D −87 (c 0.15, MeOH); UV (MeOH) λmax 203 nm; ECD (MeOH) λmax (Δε) 254 (0.05), 213 (−1.59) nm; 1H and 13C NMR data, Tables 1and 2; HRESIMS m/z 463.1968 [M + H]+ (calcd for C24H31O9, 463.1963). Eupachinsin B (3): colorless oil; [α]20 D −61 (c 0.40, MeOH); UV (MeOH) λmax 205 nm; ECD (MeOH) λmax (Δε) 263 (0.05), 211 (−2.61) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 422.2176 [M+NH4]+ (calcd for C22H32NO7, 422.2173), m/z 427.1727 [M + Na]+ (calcd for C22H28O7Na, 427.1727). 3-Epi-eupachinisin B (4): colorless oil; [α]20 D −19 (c 0.30, MeOH); UV (MeOH) λmax 202 nm; ECD (MeOH) λmax (Δε) 246 (0.04), 210 (−2.13) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 422.2176 [M+NH4]+ (calcd for C22H32NO7, 422.2173). 15-Hydroxyeupachinisin B (5): colorless oil; [α]20 D −84 (c 0.30, MeOH); UV (MeOH) λmax 209 nm; ECD (MeOH) λmax (Δε) 263 (0.04), 211 (−2.57) nm; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 443.1678 [M + Na]+ (calcd for C22H28O8Na, 443.1676). Eupachinsin C (6): colorless oil; [α]20 D −54 (c 0.30, MeOH); UV (MeCN) λmax 209 nm; ECD (MeOH) λmax (Δε) 261 (0.05), 211 (−2.81) nm; 1H and 13C NMR data, Tables 3 and 4; HRESIMS m/z 422.2171 [M + NH4]+ (calcd for C22H32NO7, 422.2173), m/z 427.1724 [M + Na]+ (calcd for C22H28O7Na, 427.1727). 4′-Hydroxyeupachinisin C 15-acetate (7): colorless oil; [α]20 D −72 (c 0.30, MeOH); UV (MeCN) λmax 207 nm; ECD (MeOH) λmax (Δε) 260 (0.23), 212 (−7.20) nm; 1H and 13C NMR data, Tables 3 and 4; HRESIMS m/z 480.2225 [M + NH4]+ (calcd for C24H34NO9, 480.2228). Eupachinsin D (8): colorless oil; [α]20 D −7 (c 0.48, MeOH); UV (MeOH) λmax 207 nm; ECD (MeOH) λmax (Δε) 231 (0.16), 212 (−0.23) nm; 1H and 13C NMR data, Tables 3 and 4; HRESIMS m/z 443.1684 [M + Na]+ (calcd for C22H28O8Na, 443.1676). 15-Hydroxyeupachinisin D (9): colorless oil; [α]20 D −8 (c 0.20, MeOH); UV (MeOH) λmax 210 nm; ECD (MeOH) λmax (Δε) 245

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00693.

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HRESIMS, UV, ECD, and NMR spectra of compounds 1−10 (PDF) Crystallographic data (CIF)

AUTHOR INFORMATION

Corresponding Authors

*Tel: +49 211 81 15979. Fax: +49 211 81 11923. E-mail: [email protected] (Z. Liu). *Tel: +49 211 81 14163. Fax: +49 211 81 11923. E-mail: [email protected] (P. Proksch). *E-mail: [email protected] (K. Zou). ORCID

Zhen Liu: 0000-0003-3314-7853 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported by DFG (GRK 2158) and by the Manchot Foundation. It was also financially supported by grants from the National Natural Science Foundations of China (No. 21272136).

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REFERENCES

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Germacrane-Type Sesquiterpenoids with Antiproliferative Activities

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