Kadcoccinic Acids A–J, Triterpene Acids from ... - ACS Publications

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Kadcoccinic Acids A−J, Triterpene Acids from Kadsura coccinea Cheng-Qin Liang,†,‡,§ Yi-Ming Shi,† Wei-Guang Wang,† Zheng-Xi Hu,† Yan Li,† Yong-Tang Zheng,⊥ Xiao-Nian Li,† Xue Du,† Jian-Xin Pu,† Wei-Lie Xiao,† Hong-Bin Zhang,§ and Han-Dong Sun*,† †

State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, Yunnan, People’s Republic of China ‡ Guilin Medical University, Guilin 541004, Guangxi, People’s Republic of China § Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, School of Chemical Science and Technology, Yunnan University, Kunming 650091, Yunnan, People’s Republic of China ⊥ Key Laboratory of Animal Models and Human Disease Mechanisms of Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, People’s Republic of China S Supporting Information *

ABSTRACT: Eleven triterpene acids including 10 new compounds (kadcoccinic acids A−J, 1−10) were isolated from the stems of Kadsura coccinea. Except for 10, these compounds feature a rearranged lanostane skeleton with a 6/6/5/6 tetracyclic ring system, and compounds 1 and 2 are the first examples of 2,3-seco-6/6/5/6-fused tetracyclic triterpenoids. Their structures were established primarily by spectroscopic and spectrometric methods. Additionally, the absolute configuration of 3 was determined by single-crystal X-ray diffraction. Several of the compounds isolated were tested for their anti-HIV-1 and cytotoxic activities.

P

Ziyuan County of Guangxi Province, People’s Republic of China, resulted in the isolation of several new triterpenoids featuring intriguing structures based on rearranged lanostanetype skeletons.13,14 The main differences between these compounds have concerned the nature of the C and D rings and the extension of the side chain. For example, kadcoccitones A and B have an unprecedented carbon skeleton with a 6/6/5/ 5-fused tetracyclic ring system and a C9 unit side chain,13 kadcotrione A features a 12,14β-dimethyl-6/6/6-fused tricyclic skeleton, while kadcotriones B and C are characterized with a 6/6/5-ring system.14 In the course of a systematic search for chemically novel and biologically potent active compounds from K. coccinea, 10 new triterpene acids, named kadcoccinic acids A−J (1−10), together with a known analogue, seco-neokadsuranic acid A (11), were obtained. Except for compound 10, these triterpene acids feature a rearranged lanostane framework with a 6/6/5/6ring system, with only five triterpenoids possessing such a skeleton having been reported previously. The unusual rearrangements in the C and D rings occur primarily through the Wagner−Meerwein reaction.6 Additionally, compounds 1 and 2 are the first example of 2,3-secotriterpenoids with a 6/6/ 5/6-fused tetracyclic system. Due to the folkloric use of K.

lants of the family Schisandraceae, consisting of the genera Schisandra and Kadsura, have attracted much attention as a rich source of triterpenoids with novel complex polycyclic structures and diverse biological activities.1 Representative examples of this type of metabolite are henrischinins A and B, having cytotoxicity against the HL-60 cell line, obtained from S. henryi,2 and schilancitrilactone A,3 lancolide A,4 and lancifonins E, 5 with an antifeedant effect, antiplatelet aggregation activity, and protective activity against H2O2induced oxidative damage on Caco-2 cells, respectively, isolated also from S. lancifolia.3−5 In the past decade, several significant advances have been made in the field of Schisandraceae triterpenoids, such as a dramatic increase in the number of newly identified triterpenoids, the first complete synthesis of a schinortriterpenoid, and substantial progress in investigations of their biological activity and mechanism of action.1 Kadsura species are used as common folk medicines in mainland China for the treatment of blood disorders, numbness of the limbs, arthralgia, and irregular menstruation.6 Kadsura species are rich in dibenzocyclooctadiene lignans and triterpenes.7 Some of these compounds have been proved to be effective as antitumor,8 anti-HIV,9 antihepatitis B,10 and cytotoxic agents.11 Kadsura coccinea (Lem.) A. C. Smith, a climbing plant, is distributed widely in southern mainland China and has been used as a folk medicine for the treatment of cancer and dermatosis and as an anodyne to relieve pain.12 Previous chemical investigations of this species collected from © XXXX American Chemical Society and American Society of Pharmacognosy

Received: May 4, 2015

A

DOI: 10.1021/acs.jnatprod.5b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H NMR Spectroscopic Data for Kadcoccinic Acids A−E (1−5) (δ in ppm, J in Hz)

coccinea, some of the compounds isolated were tested for their anti-HIV-1 activity and/or evaluated for their cytotoxicity on cancer cell lines. Herein are described the isolation, structure elucidation, and biological evaluation of the isolates obtained.

1a

no.

RESULTS AND DISCUSSION Compound 1 was obtained as a yellow, amorphous solid. Its molecular formula, C30H44O6, was deduced from its HREIMS data ([M]+ m/z 500.3126, calcd 500.3138), with nine indices of hydrogen deficiency. The IR spectrum showed absorptions at 3433, 1712, and 1641 cm−1, indicating the presence of hydroxy groups and carbonyl and double-bond functionalities. The 1H NMR spectrum (Table 1) showed signals for two olefinic protons (δH 4.93, s; δH 6.04, t, J = 7.4 Hz), an exocyclic methylene (δH 4.65, 4.71, d, J = 2.8 Hz),15 a secondary methyl group (δH 0.70, d, J = 6.3 Hz), and five methyl groups (δH 0.95, 1.16, 1.19, 1.31, and 1.86). The 13C NMR and DEPT spectra (Table 2) exhibited 30 carbon signals, including six methyls, eight methylenes (one olefinic), seven methines (two olefinic), and nine quaternary carbons (three olefinic and three carbonyl). Apart from three double bonds and three carbonyl groups, the remaining elements of unsaturation in 1 were assumed to represent a tricyclic skeleton. These data were consistent with the empirical formula from the HREIMS and suggested that 1 is a tricyclic triterpenoid. Analysis of its 1D and 2D NMR spectra indicated that 1 has structural features closely similar to those of seco-neokadsuranic acid A (11),16 differing only in the A ring. There are a carboxylic acid group, a methyl, three methylenes, a methine, and two quaternary carbons in the A ring of seco-neokadsuranic acid A, and a 3,4-seco-ring is evident. However, in the A ring of 1, two carboxylic acid groups (δC 178.3, C-2; 186.7, C-3), two methyls (δH 1.16, δC 20.6, Me-29; δH 1.19, δC 29.8, Me-30), a

3a

c

1.72 1.99 2.75 2.83 1.56

m m m m mc mc mc mc m m

mc m m m m

1.86 1.86 1.98 1.80

mc mc mc mc

4.94 s

5.14 s

6.98 s

2.89 s 1.06 m 1.49 m 1.46 mc 1.55 mc 1.86 m 4.66 d (2.7) 4.77 d (2.7) 1.22 s 1.55 mc 0.82 d (6.4) 1.06 mc 1.58 mc 2.47 m 2.52 m 6.05 t (7.5) 1.89 s 0.95 s 1.46 s 1.40 s

2.51 1.23 1.49 1.75 1.88

2.24 m 2.45 d (12.7)

1.80 mc

3.04 s

6a 6b 7a 7b 8

1.47 m 1.67 mc 1.13 m 1.77 mc 2.04 dd (12.1, 3.9) 4.93 s

1.94 2.07 1.95 1.95

mc m mc mc

1.77 1.79 1.80 1.15 2.19

2.23 2.33 2.21 1.17 1.36 1.49 1.60 1.87 4.73

mc m m m m m mc m d (2.9)

s m m mc mc mc d (2.8)

18b

4.71 d (2.8)

4.79 d (2.9)

19 20 21

1.31 s 1.66 mc 0.70 d (6.3)

1.03 s 1.64 m 0.89 d (6.3)

22a 22b 23a 23b 24

1.04 1.53 2.29 2.55 6.04

m mc m m t (7.4)

27 28 29 30

1.86 0.95 1.16 1.19

s s s s

1.04 m 1.62 mc 2.03 m 2.89 m 5.87 dd (11.9, 5.5) 1.97 s 0.94 s 1.15 s 1.28 s

c

5a 1.97 2.23 2.52 2.71 1.49

2.64 d (16.2) 2.74 d (16.2)

2.91 1.01 1.57 1.53 1.82 1.79 4.65

4a 1.90 m 1.90 mc 1.38 mc 2.38 mc 1.27 dd (12.3, 2.8) 1.49 mc 1.62 mc 1.13 m 1.83 m 2.21 m

1a 1b 2a 2b 5

11a 11b 12 15a 15b 16a 16b 17 18a

■

2b

s m mc mc m

1.64 s

1.14 s 2.67 mc 0.92 d (6.8) 1.40 2.66 2.36 2.36 6.05

mc m mc mc t (7.4)

1.87 0.90 1.02 1.03

s s s s

1.45 1.77 1.57 1.89 2.62 9.98

m mc m mc m s

1.34 s 1.90 mc 0.89 d (6.9) 1.20 m 1.44 mc 2.35 m 2.47 m 5.97 t (7.6) 1.86 s 0.98 s 1.11 s 1.12 s 3.03 s

a c

Recorded in CDCl3 at 400 MHz. bRecorded in CDCl3 at 500 MHz. Overlapped signals.

methylene (δH 2.64, 2.74, d, J = 16.2 Hz, δC 42.5, C-1), a methine (δH 1.80, m, δC 55.5, C-5), and two quaternary carbons (δC 44.9, C-4; δC 41.6, C-10) were found to occur. These data, along with the HREIMS data, supported a secosubstructure in the A ring of 1. The HMBC correlations (Figure 1) of H2-1 with C-2, Me-19 with C-1, C-5, and C-10, Me-29 and Me-30 with C-3, C-4, and C-5, and H-5 with C-3 supported the assignment of 1 as a 2,3-seco-derivative. The relative configuration of 1 was proved to be the same as that of 11 by detailed analysis of its ROESY spectrum (Figure S6, Supporting Information), in which H-12 (δH 2.91, s) correlated to Me-28 (δH 0.95, s) and H-17 (δH 1.79, m), and Me-19 (δH 1.31, s) correlated to H-8 (δH 2.04, dd, J = 12.1, 3.9 Hz) and Me-29 (δH 1.16, s), indicating that H-12, Me-28, and H-17 are α-oriented, while H-8 and Me-19 are β-oriented. The geometry of the double bond between C-24 (δC 146.9) and C25 (δC 126.0) was assigned in the Z configuration, due to the ROESY correlation between H-24 (δH 6.04, t, J = 7.4 Hz) and B

DOI: 10.1021/acs.jnatprod.5b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 13C NMR Spectroscopic Data for Kadcoccinic Acids A−J (1−10) (δ in ppm)a no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 MeO a

1b 42.5 178.3 186.7 44.9 55.5 24.1 28.8 54.3 151.3 41.6 122.0 58.1 149.3 43.1 31.7 22.2 48.7 112.6 19.0 29.9 17.3 33.1 26.0 146.9 126.0 174.4 20.2 20.1 20.6 29.8

2c t s s s d t t d s s d d s s t t d t q d q t t d s s q q q q

45.1 179.0 187.5 45.0 40.9 19.5 19.7 143.0 137.1 38.6 35.8 53.6 149.8 49.1 28.2 23.9 49.0 113.1 19.3 31.5 19.0 34.1 27.5 143.5 127.7 174.8 20.5 22.7 21.1 28.5

3b t s s s d t t s s s t d s s t t d t q d q t t d s s q q q q

33.6 32.1 175.4 86.7 56.2 25.9 29.0 53.8 153.9 40.4 121.1 59.1 149.2 43.0 33.7 24.1 48.5 112.9 18.9 32.6 17.9 33.3 26.9 147.1 125.8 172.8 20.6 21.2 24.1 33.8

4b t t s s d t t d s s d d s s t t d t q d q t t d s s q q q q

35.2 34.8 216.9 47.9 54.9 22.9 29.1 52.5 153.1 37.4 118.1 58.1 126.4 39.8 33.9 19.8 132.8 18.1 18.5 34.5 18.7 34.6 28.3 147.5 125.7 173.6 20.5 20.9 21.6 25.8

5b t t s s d t t d s s d d s s t t s q q d q t t d s s q s q q

36.3 34.1 216.0 47.7 53.1 19.0 30.5 88.1 163.5 38.0 122.7 169.9 130.8 48.6 24.7 19.4 35.5 190.4 20.6 35.9 18.1 33.5 28.1 146.3 126.2 172.7 20.5 28.8 21.8 26.7 52.1

6b t t s s d t t s s s d s s s t t d d q d q t t d s s q q q q q

36.0 34.1 216.3 47.8 52.6 18.9 30.7 83.2 168.0 37.8 120.0 169.7 132.4 49.6 24.7 18.1 35.7 190.5 21.9 35.7 19.1 32.6 28.4 146.0 126.2 172.4 20.4 27.1 21.7 26.4

7b t t s s d t t s s s d s s s t t d d q d q t t d s s q q q q

34.3 30.3 180.1 76.1 54.7 27.1 29.0 54.7 153.0 42.7 123.1 59.2 150.1 43.6 33.5 24.6 49.2 112.6 21.2 32.9 18.1 33.9 27.4 146.3 126.8 172.6 21.0 21.8 27.8 33.5

8b t t s s d t t d s s d d s s t t d t q d q t t d s s q s q q

34.1 29.9 175.6 75.5 54.3 26.9 28.6 54.3 152.5 42.4 122.8 58.9 149.8 43.2 33.4 24.2 48.6 112.2 21.0 32.6 17.7 33.4 26.8 146.6 126.1 172.7 20.6 21.5 27.7 33.6 51.5

9b t t s s d t t d s s d d s s t t d t q d q t t d s s q q q q q

31.1 28.8 180.7 147.0 49.1 27.6 24.7 37.9 122.1 39.9 131.9 100.2 142.8 38.9 28.9 22.0 49.2 116.3 27.0 30.8 18.3 33.8 26.6 147.2 125.8 173.6 20.5 17.3 23.1 114.0 49.6

10d t t s s d t t d s s d s s s t t d t q d q t t d s s q q q t q

32.0 29.6 181.5 147.5 49.6 27.2 27.5 43.0 146.3 43.6 117.5 82.7 47.8 46.3 34.8 27.7 43.6 15.1 27.1 36.5 16.9 35.6 27.3 147.5 125.9 174.1 20.7 19.9 23.3 114.1 55.9

t t s s d t t d s s d d s s t t d q q d q t t d s s q q q t q

Recorded in CDCl3. bRecorded at 100 MHz. cRecorded at 125 MHz. dRecorded at 150 MHz.

Figure 1. Key 1H−1H COSY (bold ), HMBC (→), and ROESY (↔) correlations of 1.

COSY correlation of 2 from H2-11 (δH 2.23, m; 2.33, m) to H12 (δH 2.21, m), and the HMBC correlations from H2-11 to C8, C-9, and C-10, Me-19 (δH 1.03, s) to C-9 and C-10, and Me28 (δH 0.94, s) to C-8 and C-12, indicated the occurrence of a quaternary double bond between C-8 and C-9 and a methylene located at C-11. Comparison of its chemical shifts and analysis of the ROESY spectrum allowed the relative configuration of 2 to be determined as the same as that of 1. Consequently, compound 2 (kadcoccinic acid B) was elucidated as shown.

Me-27 (δH 1.86, s). Thus, the structure of 1 was established as shown, and this compound has been named kadcoccinic acid A. Compound 2 was determined to possess the same molecular formula as 1 from its HREIMS data ([M]+ m/z 500.3146, calcd 500.3138). Comparison of the NMR data of these two compounds (Tables 1 and 2) suggested them to be similar, except for the appearance of a quaternary double bond (δC 143.0, s; δC 137.1, s) in 2 rather than the trisubstituted double bond (δC 151.3, s; δC 122.0, d) in 1 and the replacement of a methine (δC 54.3) by a methylene (δC 35.8). The 1H−1H C

DOI: 10.1021/acs.jnatprod.5b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX

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six methines (including two olefinic carbons), and nine quaternary carbons (including four olefinic and two carboxylic groups). The three trisubstituted double bonds and two carboxylic groups accounted for five out of the nine degrees of unsaturation, and the four remaining thus required 4 to be tetracyclic. A careful analysis of the 2D NMR spectroscopic data of 4 and comparison with kadpolysperin A6 led to the conclusion that these two compounds possess the same planar structure. The main differences found between 4 and kadpolysperin A concerned the substituent group located at C-3 and the location of a trisubstituted double bond in their tetracyclic core. The acetyl group at C-3 in kadpolysperin A was substituted by a keto group (δC 216.9) in 4, which was confirmed by HMBC correlations from Me-29 (δH 1.02, s), Me-30 (δH 1.03, s), and H2-2 (δH 1.38, m; δH 2.38, m) to C-3 (δC 216.9). A trisubstituted double bond in kadpolysperin A was assigned to C-7 and C-8, but in 4 was located at C-9 (δC 153.1) and C-11 (δC 118.1), as supported by HMBC correlations from Me-19 (δH 1.14, s) to C-8 and C-9 and from H-11 (δH 5.14, s) to C-8, C-9, and C-12 and the 1H−1H COSY correlations of H-8/H2-7/H2-6/H-5. The ROESY correlation from Me-19 to H-8 was used to establish the βorientation of H-8. The overall relative configuration was confirmed to be the same as that of kadpolysperin A. Thus, the structure of 4 (kadcoccinic acid D) was elucidated as shown. Compounds 5 and 6 were obtained as amorphous solids. Comparison of the NMR data of 5 and 6 with those of compound 4 disclosed that they have the same skeleton. Compound 5 gave a molecular formula of C31H44O5 from its HREIMS data at m/z 496.3177 [M]+ (calcd 496.3189). Detailed comparison of the MS and NMR data of 5 with those of 4 revealed that the structural differences between them concerned the substituents at C-8, C-12, C-13, and C-17. A methoxy group (δH 3.03, s; δC 52.1) at C-8 (δC 88.1), an aldehyde group (δH 9.98, s; δC 190.4) at C-13 (δC 130.8), as well as a double bond between C-12 (δC 169.9) and C-13 (δC 130.8) were apparent for 5, while an olefinic signal at C-17 (δC 132.8) found in 4 was absent for 5. These were established in compound 5 by HMBC correlations of the methoxy group signal (δH 3.03, s) with C-8, of H-11 (δH 6.98, s) with C-8, C-9, C-12, and C-13, of Me-28 (δH 0.98, s) with C-8 and C-12, and of H-18 (δH 9.98, s) with C-12, C-13, and C-17. The molecular formula of 6, determined as C30H42O5 by HREIMS at m/z 482.3034 [M]+ (calcd 482.3030), is 14 mass units less than that of 5. The 1H and 13C NMR data of 6 were closely comparable to those of 5, except for the absence of a methoxy carbon signal at around δC 52. On the basis of this observation, it was reasonable to assume that 6 is a demethylation derivative at C-8 of 5, which was also supported by the downfield shift of C-8 (by Δ 4.9 ppm, from δC 88.1 to δC 83.2). HMBC correlations observed from the proton signals at H2-6 (δH 1.56, m; 1.95, m), H-11 (δH 6.76, s), and H-28 (δH 0.96, s) to C-8 corroborated this assumption. In the ROESY spectrum of 5, MeO-8 correlated to Me-19, while in that of 6, H2-7 correlated to Me-28, suggesting MeO-8 in 5 and OH-8 in 6 both to be β-oriented. The other stereocenters in 5 and 6 were assigned as being identical with those of 4 by comparison of their relevant NMR data (Tables 1 and 3) and by the analysis of their ROESY spectrum (Supporting Information). Thus, the structures of 5 (kadcoccinic acid E) and 6 (kadcoccinic acid F) were determined as shown.

Compound 3 was obtained as colorless crystals, and its molecular formula was determined as C30H44O4, with nine indices of hydrogen deficiency on the basis of its HREIMS data ([M]+ m/z 468.3245, calcd 468.3240). The 1H NMR spectrum showed signals for two olefinic protons (δH 6.05, t, J = 7.5 Hz and δH 4.94, s), an exocyclic methylene (δH 4.66, 4.77, d, J = 2.7 Hz), a secondary methyl group (δH 0.82, d, J = 6.4 Hz), and five tertiary methyl groups (δH 0.95, 1.22, 1.40, 1.46, and 1.89). The 13 C NMR and DEPT spectra exhibited 30 carbon signals, consisting of six methyls, nine methylenes (including one olefinic carbon), seven methines (including two olefinic carbons), and eight quaternary carbons (including one oxygenated, three olefinic, and two carboxylic groups). These data were consistent with the elemental formula obtained from the HREIMS and suggested 3 also to be a tetracyclic triterpene. On analysis of its 1H and 13C NMR spectra, compound 3 was found to show a close resemblance to 1. Differences between the two compounds were evident in the substituents of the A ring. The presence of a carboxylic acid group (δC 175.4, C-3) and an oxygenated carbon (δC 86.7, C-4) was apparent in 3. After considering the HREIMS data, the A ring of 3 was deduced as a seven-membered lactone ring, which could be confirmed by the HMBC correlations from H2-1 (δH 1,72, m; δH 1.99, m) to C-3, C-5, C-9, and C-10, from H2-2 (δH 2.75, m; δH 2.83, m) to C-3 and C-10, and from Me-29 (δH 1.46, s) and Me-30 (δH 1.40, s) to C-4 and C-5. The geometry of the double bond between C-24 and C-25 was in the Z configuration, which was supported by a ROESY correlation between H-24 (δH 6.05, t, J = 7.5 Hz) and Me-27 (δH 1.89, s). In addition, an X-ray crystallographic study (Figure 2) was performed to confirm unambiguously the structure and determine the absolute configuration of 3. Therefore, the structure of 3 (kadcoccinic acid C) was assigned as shown.

Figure 2. X-ray crystal structure of 3.

Compound 4 was isolated as an amorphous solid. The molecular formula was assigned by the positive HREIMS data (m/z 452.3284 [M]+) as C30H44O3. The 1H NMR spectrum of 4 showed signals for two olefinic protons (δH 6.05, t, J = 7.4 Hz and δH 5.14, s), a secondary methyl group (δH 0.92, d, J = 6.8 Hz), and five methyl groups (δH 0.90, 1.02, 1.03, 1.14, 1.64, and 1.87). Its 13C NMR and DEPT spectra (Table 2) resolved 30 carbon resonances comprising seven methyls, eight methylenes, D

DOI: 10.1021/acs.jnatprod.5b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 3. 1H NMR Spectroscopic Data for Kadcoccinic Acids F−J (6−10) (δ in ppm, J in Hz) 6a

no. 1a 1b 2a 2b 5 6a 6b 7a 7b 8 11 12 15a 15b 16a 16b 17 18a

c

1.89 m 2.18 m 2.45 m 2.66 m 1.45 dd (12.1, 2.1) 1.56 m 1.95 mc 1.78 mc 1.78 mc 6.76 s

1.30 1.91 1.67 1.83 2.57 9.87

mc mc m mc m s

18b

a c

19 20 21

1.37 s 1.92 mc 0.90 d (6.9)

22a 22b 23a 23b 24

1.08 1.30 2.29 2.29 5.87

m mc mc mc t (7.2)

27 28 29 30a 30b MeO

1.77 0.96 1.07 1.07

s s sc sc

7a

8a

9a c

10b c

2.14 2.30 2.30 2.43 1.28

m mc mc mc m

2.26 2.26 2.38 2.49 1.32

m mc m m m

1.82 1.95 2.48 2.59 2.03

m mc mc m m

1.87 2.13 2.49 2.58 2.07

1.41 1.62 0.94 1.64 1.97 4.90 2.81

mc mc mc mc m s s

1.46 1.65 0.99 1.70 2.02 4.95 2.86

mc mc mc mc m s s

1.56 1.73 1.07 1.65 2.37 6.04

m mc m mc m s

1.58 mc 1.80 mc 1.35 m 1.60 m 2.17 m 5.86 br s 3.36 d (4.6) 1.38 mc 1.38 mc 1.32 mc 1.94 mc 2.28 m 0.67 s

1.55 mc 0.95 mc 1.50 mc 1.50 mc 1.78 m 4.58 d (2.6) 4.68 d (2.6) 1.08 s 1.58 mc 0.76 d (6.2) 0.97 mc 1.55 mc 2.43 mc 2.43 mc 5.94 t (7.3) 1.82 s 0.86 s 1.18 sc 1.18 sc

1.61 mc 1.04 mc 1.54 mc 1.54 mc 1.84 m 4.64 d (2.8) 4.74 d (2.8) 1.14 s 1.65 mc 0.81 d (6.4) 0.99 mc 1.63 mc 2.45 mc 2.54 mc 6.05 t (7.5) 1.88 s 0.92 s 1.24 sc 1.24 sc 3.64 s

methoxy group was revealed to be located at C-3 by the HMBC correlation between OCH3 (δH 3.64, s) and C-3. Similar ROESY correlations showed that the relative configuration of 8 was the same as 7. Accordingly, the structure of 8 (kadcoccinic acid H) was proposed as shown. Compound 9 gave the molecular formula C31H46O5 from the HREIMS data at m/z 498.3347 ([M]+, calcd 498.3345). The 1 H and 13C NMR data (Tables 2 and 3) of 9 showed similarities with the structurally related compound 11.16 However, signals for a methoxy group (δH 2.96, s; δC 49.6) were evident, and a methine (C-12) in 11 was replaced by an oxygenated quaternary carbon (δC 100.2) in 9. The HMBC correlations from a methoxy group, Me-28, H2-18, and H-11 to C-12 revealed that C-12 is substituted by a methoxy group. The ROESY correlation from MeO-12 to Me-28 suggested that the MeO-12 group is α-oriented, and the remaining relative configuration was confirmed to be the same as that of 11. Therefore, the structure of 9 (kadcoccinic acid I) was assigned as shown. Compound 10 was obtained as a yellow, amorphous powder. It gave the molecular formula C31H48O5 according to its HREIMS data (m/z 500.3494 [M]+, calcd 500.3502). Analysis of the 1D and 2D NMR spectra indicated that the planar structure of 10 resembled that of 12-β-hydroxycoccinic acid17 in rings B−D and in the C-17 side chain, except that the A ring is opened, and a methoxy group (δC 55.9), a carboxyl group (δC 181.5), and a disubstituted double bond (δC 147.5, 114.1) are presant. The HMBC correlations from Me-19 (δH 1.10, s) to C-1, C-5, C-9, and C-10, H2-1 (δH 1.87, m; 2.13, m) to C-2 and C-3, and Me-29 (δH 1.80, s) and H2-30 (δH 4.76, s; 4.91, s) to C-4 and C-5, in combination with the 1H−1H COSY correlation of H2-1/H2-2, supported the structural assignments made in the opened A ring. Additionally, the HMBC correlations from MeO-12 (δH 3.30, s) to C-12 (δC 82.7) implied that this methoxy group is connected to C-12. The αorientation of MeO-12 was apparent from the ROESY correlations of H-12/Me-18. The geometry of the double bond between C-24 (δC 147.5) and C-25 (δC 125.9) was assigned in the Z configuration, due to the ROESY correlation between H-24 (δH 6.16, t, J = 7.5 Hz) and Me-27 (δH 1.94, s). The other relative configurations in 10 were also determined by the ROESY experiment. Hence, the structure of compound 10 was established as shown, and it was named kadcoccinic acid J. Identification of the known compound seco-neokadsuranic acid A (11) 16 was performed by comparison of its spectroscopic data with those in the literature.16 In previous studies on plants of the family Schisandraceae, many triterpenoids with anti-HIV activities have been found.1 To determine their anti-HIV-1 activities and compare with those of other triterpenic acids isolated from plants of this family, compounds 1, 2, 4, 5, 7−9, and 11 were tested using a microtiter syncytium formation infectivity assay, with AZT as a positive control (Table 4).18 Among them, compounds 4 and 7 demonstrated anti-HIV-1 activity with respective EC50 values of 28.0 and 28.5 μg/mL (AZT, EC50 = 0.005 μg/mL). Additionally, compounds 1, 3, 4, 8, 9, and 11 were also tested for cytotoxicity against the A-549, HL-60, MCF-7, SMMC7721, and SW480 human tumor cell lines by the MTT method.19 However, none of them showed inhibitory activity against any of the cell lines used (IC50 >10 μM).

1.39 mc 1.39 mc 1.60 mc 1.79 mc 1.98 mc 5.07 d (2.6) 5.35 d (2.6) 1.09 s 1.96 mc 0.86 d (6.0) 1.14 mc 1.61 mc 2.44 mc 2.55 mc 6.10 t (7.3) 1.91 s 0.77 s 1.75 s 4.73 s 4.87 s 2.96 s

m m m m m

1.10 s 1.41 m 0.94 d (6.5) 1.21 m 1.55 m 2.32 m 2.71 m 6.16 t (7.5) 1.94 s 0.86 s 1.80 s 4.76 s 4.91 s 3.30 s

b

Recorded in CDCl3 at 400 MHz. Recorded in CDCl3 at 600 MHz. Overlapped signals.

Compound 7 was isolated as an amorphous solid. The molecular formula, C30H46O5, was deduced from the HREIMS data ([M]+ m/z 486.3351, calcd 486.3345). A comparison of the NMR data of 7 with those of 3 indicated that these two compounds are closely related in structure. The only significant difference found was that the seven-membered lactone ring in 3 is opened and a carboxyl group (δC 180.1, C-3) and an oxygenated quaternary carbon (δC 76.1, C-4) occur in 7 (Table 2). These assignments were consistent with its elemental formula and degree of unsaturation that were obtained from the HREIMS data of 7. ROESY correlations suggested this compound to have the same relative configuration as that of 3. Thus, the structure of 7 (kadcoccinic acid G) was determined as shown. Compound 8 was assigned a molecular formula of C31H48O5 on the basis of its [M]+ ion peak at m/z 500.3497 (calcd 500.3502), being 14 mass units greater than that of 7. The NMR data of 8 differed from those of 7 (Tables 2 and 3) only in the presence of signals for a methoxy group (δC 51.5). This E

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(3 mL/min, eluent 60−100% CH3CN−H2O) to yield compounds 3 (5 mg), 5 (8 mg), 6 (13 mg), and 7 (15 mg). Using this same purification procedure, compounds 8 (17 mg), 9 (21 mg), and 10 (25 mg) were obtained from fraction 3Mc. Kadcoccinic acid A (1): amorphous solid; [α]21 D −29.7 (c 0.07, MeOH); UV (MeOH) λmax (log ε) 205 (3.4) nm; IR (KBr) νmax 3433, 2971, 2938, 2879, 1712, 1641, 1459, 1380, 1240 cm−1; 1H and 13C NMR data, see Tables 1 and 2; negative ESIMS m/z 499 [M − H]−; positive HREIMS m/z 500.3126 [M]+ (calcd for C30H44O6, 500.3138). Kadcoccinic acid B (2): amorphous solid; [α]22 D −63.0 (c 0.5, CHCl3); UV (CHCl3) λmax (log ε) 240 (2.9) nm; IR (KBr) νmax 3425, 2957, 2932, 1697, 1641, 1460, 1252 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 523 [M + Na]+; positive HREIMS m/z 500.3146 [M]+ (calcd for C30H44O6, 500.3138). Kadcoccinic acid C (3): colorless crystals (MeOH); [α]22 D −172.9 (c 0.6, CHCl3); UV (CHCl3) λmax (log ε) 239 (2.8); IR (KBr) νmax 3424, 2926, 1720, 1691, 1642, 1441, 1189, 891 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 491 [M + Na]+; positive HREIMS m/z 468.3245 [M]+ (calcd for C30H44O4, 468.3240). Kadcoccinic acid D (4): amorphous solid; [α]23 D −173.9 (c 0.8, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.0) nm; IR (KBr) νmax 3423, 2957, 2931, 2867, 1706, 1640, 1458, 1264 cm−1; 1H and 13C NMR data, see Tables 1 and 2; positive ESIMS m/z 475 [M + Na]+; positive HREIMS m/z 452.3284 [M]+ (calcd for C30H44O3, 452.3290). Kadcoccinic acid E (5): amorphous solid; [α]23 D −80.0 (c 0.7, CHCl3); UV (CHCl3) λmax (log ε) 336 (3.2), 239 (3.1), 223 (3.0) nm; IR (KBr) νmax 3432, 2963, 2932, 1706, 1641, 1457, 1203 cm−1; 1H and 13 C NMR data, see Tables 1 and 2; positive ESIMS m/z 519 [M + Na]+; positive HREIMS m/z 496.3177 [M]+ (calcd for C31H44O5, 496.3189). Kadcoccinic acid F (6): amorphous solid; [α]21 D −37.2 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 293 (3.1), 256 (3.2), 203 (3.4) nm; IR (KBr) νmax 3434, 2968, 2934, 2873, 1705, 1440, 1209, 1112 cm−1; 1H and 13C NMR data, see Tables 2 and 3; negative ESIMS m/z 481 [M − H]−; positive HRESIMS m/z 482.3034 [M]+ (calcd for C30H42O5, 482.3030). Kadcoccinic acid G (7): amorphous solid; [α]22 D −168.5 (c 0.4, CHCl3); UV (CHCl3) λmax (log ε) 239 (3.0) nm; IR (KBr) νmax 3435, 2959, 2928, 2869, 1698, 1638, 1458, 1377 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 509 [M + Na]+; positive HREIMS m/z 486.3351 [M]+ (calcd for C30H46O5, 486.3345). Kadcoccinic acid H (8): amorphous solid; [α]23 D −101.4 (c 0.8, CHCl3); UV (CHCl3) λmax (log ε) 239 (3.0) nm; IR (KBr) νmax 3441, 2956, 2929, 2870, 1720, 1692, 1460, 1377, 1170 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 523 [M + Na]+; positive HREIMS m/z 500.3497 [M]+ (calcd for C31H48O5, 500.3502). Kadcoccinic acid I (9): amorphous solid; [α]23 D −29.5 (c 1.1, CHCl3); UV (CHCl3) λmax (log ε) 239 (3.0) nm; IR (KBr) νmax 3441, 2934, 2869, 1701, 1638, 1453, 1257 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 521 [M + Na]+; positive HREIMS m/z 498.3347 [M]+ (calcd for C31H46O5, 498.3345). Kadcoccinic acid J (10): amorphous solid; [α]22 D +30.8 (c 0.4, CHCl3); UV (CHCl3) λmax (log ε) 240 (3.0) nm; IR (KBr) νmax 3440, 2955, 2931, 2870, 1698, 1638, 1461, 1378, 1264 cm−1; 1H and 13C NMR data, see Tables 2 and 3; positive ESIMS m/z 523 [M + Na]+; positive HREIMS m/z 500.3494 [M]+ (calcd for C31H48O5, 500.3502). X-ray Crystal Structure Analysis of Compound 3. A colorless crystal of 3 was obtained in MeOH. Intensity data were collected at 100 K on an Bruker APEX DUO diffractometer equipped with an APEX II CCD, using Cu Kα radiation. Cell refinement and data reduction were performed with Bruker SAINT. The structure was solved by direct methods using SHELXS-97.20 Refinements were performed with SHELXL-97 using full-matrix least-squares, with anisotropic displacement parameters for all the non-hydrogen atoms. The H atoms were placed in calculated positions and refined using a riding model. Molecular graphics were computed with PLATON. Crystallographic data (excluding structure factor tables) for the

Table 4. In Vitro Anti-HIV-1 Activities of Selected Isolated Compounds (μM)

a

compound

CC50

EC50

TIb

1 2 4 5 7 8 9 11 AZTa

>200 >200 116.1 >200 143.4 142.5 183.9 203.2 4690

68.7 120.1 62.0 116.2 58.7 81.2 70.0 88.2 0.02

>2.9 >1.7 1.9 >1.7 2.4 1.8 2.6 2.3 >230 000

Positive control. bTI = CC50/EC50.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with Horiba SEPA-300 and JASCO P-1020 polarimeters. UV spectra were recorded on a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectra. 1D and 2D NMR spectra were recorded on Bruker AM-400, DRX-500, and DRX-600 spectrometers using TMS as internal standard, with chemical shifts (δ) expressed in ppm and referenced to the solvent signals. HREIMS were performed on a Waters AutoSpec Premier P776 spectrometer. ESIMS were performed on a Xevo TQ-S spectrometer. Preparative HPLC was performed on a Shimadzu LC8A preparative liquid chromatograph with a Shimadzu PRC-ODS (K) column (34 mm × 15 cm). Semipreparative HPLC was performed on an Agilent 1100 or Agilent 1200 liquid chromatograph with a Zorbax SB-C18 (9.4 mm × 25 cm) column. Column chromatography (CC) was performed on silica gel (100−200 mesh and 200−300 mesh; Qingdao Marine Chemical Inc., Qingdao, People’s Republic of China), Lichroprep RP-18 gel (40−63 μm, Merck, Darmstadt, Germany), MCI gel (75−150 μm, Mitsubishi Chemical Corporation, Tokyo, Japan), and Sephadex LH-20 (Pharmacia). Fractions were monitored by thinlayer chromatography (TLC) (Qingdao Marine Chemical Inc.), and spots were visualized by heating the silica gel plates sprayed with 10% H2SO4 in EtOH (10:90, v/v). Plant Material. The stems of Kadsura coccinea were collected in Ziyuan, Guangxi, People’s Republic of China, in October 2010. The sample was identified by Prof. Xi-Wen Li, and a voucher specimen (KIB 20101018) has been 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 stems of K. coccinea (24.8 kg) were extracted three times (3 days each time) with 70% aqueous acetone (3 × 150 L) at room temperature and concentrated at reduced pressure to afford a crude extract, which was partitioned between H2O and EtOAc. The EtOAc fraction (1.2 kg) was chromatographed on a silica gel (4.5 kg, 100−200 mesh) column using CHCl3−acetone as elution solvents (from 1:0 to 0:1), to afford fractions 1−8. Fraction 2 (34 g) was chromatographed using silica gel CC (200−300 mesh), eluted with a petroleum ether−acetone gradient system (50:1−10:1), to obtain a major fraction, which was then purified by Sephadex LH-20 column chromatography with CHCl3−MeOH (1:1) as the mobile phase, to yield compound 11 (500 mg). Fraction 3 (50 g), obtained with CHCl3−acetone (9:1), was subjected to further column chromatographic separation, first with MCI eluted with 90% MeOH, then on RP-18 (eluent MeOH−H2O, from 20% to 100%), to afford subfractions 3A−3O. Subfraction 3K (3.5 g) was subjected to semipreparative HPLC on a reversed-phase column (3 mL/min, eluent 80−100% MeOH−H2O) to yield compound 4 (3 mg). Subfraction 3M (5.5 g) was subjected to preparative HPLC, eluted with MeOH−H2O (25 mL/min, 50%−100%), to afford three major fractions, 3Ma-3Mc. Fraction 3Ma was purified by semipreparative HPLC, with a solvent gradient of CH3CN in water (3 mL/min, 50− 70%), to afford compounds 1 (10 mg) and 2 (16 mg). Fraction 3Mb was subjected to semipreparative HPLC on a reversed-phase column F

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structure reported for 3 have been deposited with the Cambridge Crystallographic Data Center as supplementary publications no. CCDC 1059968. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: Int. +44(0) (1223) 336 033); e-mail: [email protected]]. Crystallographic data for kadcoccinic acid C (3): C30H44O4, Mw = 468.65, orthorhombic, a = 7.49900(10) Å, b = 8.9180(2) Å, c = 19.5887(4) Å, α = 90.00°, β = 95.46°, γ = 90.00°, V = 1304.08(4) Å3, T = 100(2) K, space group P21, Z = 2, μ(Cu Kα) = 0.605 mm−1, 10 568 reflections measured, 4222 independent reflections (Rint = 0.0387). The final R1 values were 0.0393 (I > 2σ(I)). The final wR(F2) values were 0.1045 (I > 2σ(I)). The final R1 values were 0.0393 (all data). The final wR(F2) values were 0.1046 (all data). The goodness of fit on F2 was 1.043. Flack parameter = 0.06(16). The Hooft parameter is 0.10(7) for 1661 Bijvoet pairs. Anti-HIV-1 Assay. Using an MTT method, the cytotoxicity assay against C8166 cells (CC50) was assessed, and the anti-HIV-1 activity was evaluated by the inhibition assay for the cytopathic effects of HIV1 (EC50).18 AZT (Sigma) was used as the positive control substance. Cytotoxicity Assays. The cytotoxicity assay was performed using an MTT method, and the following human tumor cell lines were used: A-549 (lung adenocarcinoma), HL-60 (promyelocytic leukemia), MCF-7 (breast), SMMC-7721 (liver carcinoma), and SW480 (colon). Each tumor cell line was exposed to each test compound at various concentrations in triplicate for 48 h, with cisplatin and paclitaxel (Sigma) used as positive control substances.19

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(9) Chen, D. F.; Zhang, S. X.; Xie, L.; Xie, J. X.; Chen, K.; Kashiwada, Y.; Zhou, B. N.; Wang, P.; Cosentino, L. M.; Lee, K. H. Bioorg. Med. Chem. 1997, 5, 1715−1723. (10) Kuo, Y. H.; Li, S. Y.; Wu, M. D.; Huang, R. L.; Yang Kuo, L. M.; Chen, C. F. Chem. Pharm. Bull. 1999, 47, 1047−1048. (11) Shen, Y. C.; Lin, Y. C.; Cheng, Y. B.; Kuo, Y. H.; Liaw, C. C. Org. Lett. 2005, 7, 5297−5300. (12) Anonymous. Chinese Medicinal Herbs; Shanghai Science and Technology Press: Shanghai, 1999; Vol. 2, pp 895−896. (13) Liang, C. Q.; Shi, Y. M.; Luo, R. H.; Li, X. Y.; Gao, Z. H.; Li, X. N.; Yang, L. M.; Shang, S. Z.; Li, Y.; Zheng, Y. T.; Zhang, H. B.; Xiao, W. L.; Sun, H. D. Org. Lett. 2012, 14, 6362−6365. (14) Liang, C. Q.; Shi, Y. M.; Li, X. Y.; Luo, R. H.; Li, Y.; Zheng, Y. T.; Zhang, H. B.; Xiao, W. L.; Sun, H. D. J. Nat. Prod. 2013, 76, 2350− 2354. (15) (a) Pu, J. X.; Li, R. T.; Xiao, W. L.; Gong, N. B.; Huang, S. X.; Lu, Y.; Zheng, Q. T.; Lou, L. G.; Sun, H. D. Tetrahedron 2006, 62, 6073−6081. (b) Pu, J. X.; Yang, L. M.; Xiao, W. L.; Li, R. T.; Lei, C.; Gao, X. M.; Huang, S. X.; Li, S. H.; Zheng, Y. T.; Huang, H.; Sun, H. D. Phytochemistry 2008, 69, 1266−1272. (16) Li, L. N.; Xue, H.; Ge, D. L.; Kangouri, K.; Miyoshi, T.; Omura, S. Planta Med. 1989, 3, 300−302. (17) Li, L. N.; Hong, X.; Kangouri, K.; Kawashima, A.; Omura, S. Planta Med. 1989, 6, 548−550. (18) Wang, R. R.; Yang, L. M.; Wang, Y. H.; Pang, W.; Tam, S. C.; Tien, P.; Zheng, Y. T. Biochem. Biophys. Res. Commun. 2009, 382, 540−544. (19) (a) Reed, L. J.; Muench, H. Am. J. Hyg. 1938, 27, 493−497. (b) Alley, M. C.; Scudiero, D. A.; Monks, A.; Hursey, M. L.; Czerwinski, M. J.; Fine, D. L.; Abbott, B. J.; Mayo, J. G.; Shoemaker, R. H.; Boyd, M. R. Cancer Res. 1988, 48, 589−601. (c) Monks, A.; Scudiero, D.; Skehan, P.; Shoemaker, R.; Paull, K.; Vistica, D.; Hose, C.; Langley, J.; Cronise, P.; Vaigro-Wolff, A. J. Natl. Cancer Inst. 1991, 83, 757−766. (20) Sheldrick, G. M.; Schneider, T. R. Methods Enzymol. 1997, 277, 319−343.

ASSOCIATED CONTENT

* Supporting Information S

Supplemetary data associated with this article (1D and 2D NMR, MS, IR, and UV spectra of compounds 1−10). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00392.

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AUTHOR INFORMATION

Corresponding Author

*Tel: (86) 871-65223616. Fax: (86) 871-65216343. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This project was supported financially by the National Natural Science Foundation of China (81373290, 81422046, and 21322204).

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REFERENCES

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DOI: 10.1021/acs.jnatprod.5b00392 J. Nat. Prod. XXXX, XXX, XXX−XXX