Cytotoxic Ceanothane- and Lupane-Type Triterpenoids from the Roots

Article pubs.acs.org/jnp

Cytotoxic Ceanothane- and Lupane-Type Triterpenoids from the Roots of Ziziphus jujuba Kyo Bin Kang, Jung Wha Kim, Won Keun Oh, Jinwoong Kim, and Sang Hyun Sung* College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea S Supporting Information *

ABSTRACT: Ziziphus jujuba, a plant in the family Rhamnaceae, is used in several Asian countries as a food and traditional medicine. Fifteen new ceanothane-type (1−15) and three new lupane-type triterpenoids (16−18) were isolated from the roots of Z. jujuba, as well as 12 previously known triterpenoids (19−30). Their structures were elucidated by 1D and 2D NMR spectroscopic and HR mass spectrometric data analysis. Compounds 12 and 13 were found to possess a rare E-ring γ-lactone structure, and 14 was assigned as the first 2,28-dinorlupane derivative isolated as a natural product. Twenty-five of the isolates were examined for cytotoxicity against human hepatocellular carcinoma HepG2 cells, and compounds 6−8, 14, 17, 23, 25, 29, and 30 showed cytotoxicity with IC50 values ranging from 1.9 to 5.9 μM.

T

Together with the isolation and chemical elucidation of the compounds, the evaluation of their cytotoxicity against human hepatocellular carcinoma HepG2 cells is also described in this report.

riterpenoids have been found in plant species as both free acid and aglycone forms of triterpenoid saponins.1 Betulinic acid, which is one of the most abundant lupanetype triterpenoids in the plant kingdom, has attracted much interest for its biological and pharmaceutical potential, including its anti-inflammatory activity, antiviral effects against human immunodeficiency virus (HIV)-1, and antitumor activity against several cancer cell lines.2−4 Therefore, its natural and synthetic derivatives have also been investigated as potential drug candidates for the chemotherapeutic treatment of HIV-1 and cancer. Ziziphus jujuba Mill. (Rhamnaceae) is a 5−8 m tall deciduous tree, and its fruits are used widely as a food and as an ingredient of traditional medicine formulations in East Asia.5,6 More than 15 triterpenoids have been isolated from Z. jujuba, and most of them are lupane-type triterpenoids and their rearranged derivatives possessing a five-membered A-ring, which are called ceanothane-type triterpenoids.7 According to Grishko et al., only 27 natural ceanothane-type triterpenoids have been discovered previously, and most of these have been found from species in the Rhamnaceae.8 The unusual structural characteristics of ceanothane-type triterpenoids have generated much interest by the scientific community, and natural derivatives such as zizymauritic acid A and epiceanothic acid exhibit antitumor and anti-HIV-1 activities, respectively.9,10 After prior research on the isolation of cyclopeptide alkaloids from the roots of Z. jujuba,11 it was recognized that the roots of Z. jujuba also contain large amounts of ceanothane- and lupanetype triterpenoids, but their chemical constituents have been investigated rarely, when compared to the leaves, fruits, or seeds of this species. Using a series of chromatographic steps, 15 new ceanothane-type triterpenoids (1−15) and three new lupane-type triterpenoids (16−18), as well as 12 known triterpenoids (19−30), were isolated from the roots of Z. jujuba. © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION The roots of Z. jujuba were extracted in an ultrasonicator with MeOH to yield a crude extract. The water-suspended crude extract was fractionated sequentially with CHCl3, EtOAc, and n-BuOH. The triterpenoid-enriched CHCl3 and EtOAc fractions were subjected to a series of column chromatographic separations and then purified by preparative HPLC to afford 30 triterpenoids (1−30). The known compounds identified by comparison of their spectroscopic data with reference values were ceanothic acid (19),12 epiceanothic acid (20),13 3-Oprotocatechuoylceanothic acid (21),14 3-O-vanilloylceanothic acid (22),15 zizyberenalic acid (23),14 24-hydroxyceanothic acid (24),16 betulinic acid (25),17 alphitolic acid (26),18 2-Oprotocatechuoylalphitolic acid (27),14 2-O-benzoylalphitolic acid (28),19 2-O-trans-p-coumaroylalphitolic acid (29),20 and 2-O-cis-p-coumaroylalphitolic acid (30).20 The full assignments of their 1H and 13C NMR spectroscopic data were also made, which are listed in Tables S1−S3 (Supporting Information). Compound 1 gave a molecular formula of C30H44O6, which was indicated by the HRESIMS deprotonated ion peak at m/z 499.3062 ([M − H]−, calcd for C30H43O6, 499.3060). The 1H NMR spectrum of 1 (Table 1) showed five tertiary methyl groups [δH 1.87, 1.24, 1.23, 0.98, and 0.94 (each 3H, s)] and two vinyl protons at δH 5.03 (1H, br s, H-29a) and 4.70 (1H, br s, H-29b). The 13C NMR spectrum (Table 2) exhibited 30 Received: June 8, 2016

A

DOI: 10.1021/acs.jnatprod.6b00525 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Chart 1

carbon resonances including three carboxylic acid carbons at δC 179.8, 179.1, and 179.0. The multiplicity-edited HSQC (meHSQC) NMR spectrum was used to classify the rest of the carbons as five methyls, 10 methylenes, six methines, an olefinic quaternary carbon, and five quaternary carbons, in agreement with the molecular formula of 1. The two sp2 carbons at δC 151.6 (C-20) and 110.7 (C-29) were consistent with the Δ20(29) double bond characteristic of lupane- and ceanothane-type skeletons, which was confirmed by the HMBC correlations of the downfield-shifted methyl group signal at δH 1.87 (C-30) with C-29 and the methine carbon at δC 48.3 (C19) (Figure 1). From the HMBC correlations of two germinal methyls at δH 1.24 (H-23) and 0.94 (H-24) with the quaternary carbon at δC 38.8 (C-4) and the methylene carbon at δC 43.2 (C-3), it was revealed that 1 does not possess an oxygenated functional group at C-3, which is observed commonly in most of the triterpenoids because of their biosynthetic pathway from 2,3-oxidosqualene. The HMBC correlation of H-25 (δH 0.98) with the methine carbon at δC 56.3 (C-1) and 1H−1H COSY correlations between H-1 (δH 2.88) and H-3s (δH 2.06 and 1.88) suggested that 1 possesses a cyclopentanyl A-ring system of a ceanothane-type triterpenoid. This was confirmed by the HMBC correlations of H-1 and H-3 with the carboxylic acid carbon resonance at δC 179.1 (C-2). The HMBC couplings between H-25 and C-9 (δC 46.7), between H-26 (δH 1.23) and C-9, and between H-26 and quaternary C-8 (δC 41.9) implied the attachment of H-26 at C-8. The H-26 signal also correlated with that of a downfield quaternary carbon at δC 60.8 (C-14),

for which the chemical shift suggested that C-27 is substituted as a carboxylic acid group. This was confirmed by the HMBC correlations of H-13 (δH 3.01) and H-15 (δH 2.54 and 1.95) with the carboxylic acid carbon at δC 179.0 (C-27). The HMBC correlation between H-18 (δH 2.21) and C-28 (δC 179.8) also confirmed the position of the carboxylic acid group. A ROESY experiment showed a NOE correlation between H-1 and H-25, which suggested an α-orientation of the C-2 carboxylic acid group.21 This was also supported by the downfield chemical shift of H-5 (δH 2.06) and H-23. Among the isolated known compounds, ceanothic acid (19), which has an α-oriented C-2 carboxylic acid group, showed its H-5 signal at δH 2.20 and H23 at δH 1.40, while its β-epimer, epiceanothic acid (20), exhibited H-5 at δH 1.08, and H-23 at δH 1.12 (Table S1, Supporting Information). This downfield shift of the α-oriented protons in 19 was due to the deshielding effect of the αoriented C-2 carboxylic acid group. Taken together, spectroscopic data analysis was used to establish the structure of 1 as 2α-carboxy-A(1)-norlup-20(29)-en-27,28-dioic acid. This is the same as the 3-dehydroxy form of ceanothetric acid;12 so compound 1 was assigned the trivial name 3-dehydroxyceanothetric acid. Compound 2 exhibited a deprotonated molecular ion at m/z 513.3213 ([M − H]−, calcd for C31H45O6, 513.3216) in the HRESIMS, suggesting a molecular formula of C31H46O6. The 1 H and 13C NMR spectra of 2 (Tables 1 and 2) were similar to those of 1, but the major difference was the presence of a methyl ester group signal (δH 3.78/δC 51.7). This suggested 2 B


C

a

b

3.78, s

3.74, s

5.05, s 4.80, s 1.90, s

3.62, s

m m m m m m m m m m m dt (4.2, 10.2) m m m m s s s s s s s s

1.62, 1.75, 1.63, 1.98, 1.28, 2.77, 1.92, 1.25, 2.64, 1.52, 1.73, 3.53, 2.25, 1.52, 2.26, 1.59, 1.11, 1.16, 1.62, 1.12, 1.06, 4.93, 4.75, 1.78,

m m m m m m m dt (4.7, 12.7) m m m m m dt (3.5, 10.5) m m m m s s s s

m m m m

1.04, 1.56, 1.37, 1.40,

1.93, m 1.44, m 2.04, 1.83, 2.17, 1.90, 1.60, 2.59, 2.03, 2.98, 2.51, 1.91, 2.88, 1.91, 2.17, 3.68, 2.26, 1.52, 2.28, 1.51, 1.22, 1.19, 1.37, 1.21,

4.57, d (7.5)

2.77, d (7.5)

4a

4.64, s

3.06, s

3b

m m m m m dd (3.3, 12.8) m m m m dt (3.5, 13.0) m m m m t (11.2) m m m m m s s s s s s s s

3.30, s

1.32, 1.31, 1.28, 1.36, 1.32, 1.75, 1.39, 1.32, 1.89, 1.23, 2.68, 1.85, 1.19, 2.63, 1.54, 1.69, 3.50, 2.24, 1.52, 2.25, 1.57, 1.06, 1.00, 0.94, 0.97, 1.00, 4.94, 4.78, 1.78,

2.53, dd (4.0, 8.7) 10.12, d (4.0) 4.03, d (8.7)

5a

Recorded at 600 MHz. Recorded at 500 MHz. Recorded at 400 MHz. Spectra were measured in pyridine-d5.

5.03, s 4.70, s 1.87, s

c

m m m m m m m dt (4.8, 12.9) m m m m m m m m m m s s s s

1.99, 1.81, 2.17, 1.89, 1.55, 2.62, 2.05, 2.98, 2.50, 1.91, 2.91, 1.95, 2.18, 3.71, 2.28, 1.53, 2.29, 1.53, 1.17, 0.89, 0.89, 1.16,

5.08, s 4.82, s 1.92, s

m m m m

1.85, 1.78, 1.80, 1.31,

2.06, 1.88, 2.06, 1.35, 1.31, 2.10, 1.86, 2.65, 2.26, 1.65, 2.83, 2.09, 3.01, 2.54, 1.95, 2.92, 1.97, 2.21, 3.70, 2.26, 1.52, 2.29, 1.53, 1.24, 0.94, 0.98, 1.23,

m m m m m m m dd (2.2, 12.2) m m m m m m m m m dd (10.9, 11.2) dt (4.7, 10.9) m m m m s s s s

2.69, d (7.6)

2.88, d (7.3)

1 2 3a 3b 5 6a 6b 7a 7b 9 11a 11b 12a 12b 13 15a 15b 16a 16b 18 19 21a 21b 22a 22b 23 24 25 26 27 29a 29b 30 2′ 3′ 5′ 6′ −OMe −OMe′

2a

1a

position

m m m m m dt (3.4, 12.7) m m m m m dt (4.8, 10.7) m m m m s s s s s s s s d (1.8)

m m m m

6c

7.32, d (8.3) 7.90, dd (1.8, 8.3) 3.72, s

2.08, 2.14, 1.58, 1.90, 1.30, 2.52, 1.89, 1.27, 2.33, 1.38, 1.62, 3.27, 2.01, 1.42, 1.96, 1.42, 1.52, 1.17, 1.07, 1.07, 0.97, 4.80, 4.63, 1.62, 8.10,

2.15, 1.41, 1.15, 1.40,

5.93, s

3.11, s

Table 1. 1H NMR Spectroscopic Data (δ in ppm and J Values in (Hz) in Parentheses) of Compounds 1−9

m m m m m m m m m m m dt (4.2, 10.9) m m m m s s s s s s s s br s

m m m m

3.70, s

7.10, d (8.2) 8.02, d (8.2)

1.76, 1.93, 1.86, 1.97, 1.28, 2.79, 1.94, 1.27, 2.63, 1.57, 1.73, 3.50, 2.22, 1.48, 2.25, 1.57, 1.36, 1.03, 1.67, 1.15, 1.08, 4.89, 4.73, 1.77, 7.92,

1.21, 1.55, 1.37, 1.43,

5.95, d (7.7)

3.13, d (7.7)

7a

7.31, 7.95, 3.74, 3.80,

1.48, 1.41, 2.14, 2.17, 1.59, 1.91, 1.30, 2.54, 1.58, 1.15, 2.34, 1.40, 1.63, 3.29, 2.01, 1.43, 1.97, 1.43, 1.58, 1.17, 1.30, 1.10, 1.01, 4.85, 4.65, 1.64, 7.91,

d (8.2) dd (8.2, 1.9) s s

m m m m m m m dt (3.5, 12.7) m m m m m dt (4.2, 10.9) m m m m s s s s s s s s d (1.9)

2.20, m 1.48, m

5.99, s

3.20, s

8a

1.49, 1.41, 2.13, 2.18, 1.61, 2.00, 1.35, 2.80, 1.90, 1.21, 2.61, 1.49, 1.70, 3.52, 2.25, 1.49, 2.24, 1.54, 1.57, 1.13, 1.26, 1.14, 1.06, 4.87, 4.67, 1.68, 8.27, 7.25, 7.25, 8.27,

m m m m m m m dt (3.4, 12.7) m m m m m dt (4.8, 10.7) m m m m s s s s s s s s d (8.7) d (8.7) d (8.7) d (8.7)

2.20, m 1.44, m

5.98, s

3.18, s

9a

Journal of Natural Products Article


D

56.3 179.1 43.2 38.8 56.7 19.4 38.3 41.9 46.7 51.6 24.6 27.2 41.0 60.8 29.4 35.9 57.1 52.8 48.3 151.6 31.7 38.2 32.1 27.5 20.4 18.5 179.0 179.8 110.7 20.0

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 1′ 2′ 3′ 4′ 5′ 6′ 7′ −OMe −OMe′

b

51.7

55.7 176.8 42.6 38.6 56.5 19.1 38.1 41.7 46.5 51.6 24.4 27.2 40.7 60.8 29.3 35.8 57.0 52.7 48.3 151.6 31.6 38.1 31.9 27.0 19.8 18.5 178.9 179.8 110.7 19.8

2a

51.3

66.3 175.7 84.0 43.6 57.0 18.9 37.6 41.4 46.0 50.3 24.1 26.8 40.3 60.5 28.7 35.3 56.5 52.3 47.8 151.1 31.1 37.6 31.2 20.2 18.8 18.1 178.4 179.3 110.1 19.3

3b

c

51.2

63.3 173.6 83.5 43.4 63.0 18.8 35.1 43.5 51.2 48.4 25.1 26.2 38.9 42.3 30.9 33.4 57.0 50.2 48.3 151.7 31.6 38.1 32.5 19.9 15.1 17.2 15.4 179.3 110.5 20.3

4a

60.0

72.4 205.6 91.4 41.2 63.4 18.6 34.9 42.7 50.7 49.2 25.5 26.0 38.9 43.5 30.8 33.3 56.8 50.1 48.2 151.7 31.6 38.1 25.8 26.9 15.4 17.4 15.2 179.4 110.5 19.9

5a 64.1 176.5 85.8 43.7 56.8 18.7 34.5 43.4 45.2 49.5 24.1 26.1 39.0 42.0 30.3 32.4 56.8 49.7 47.3 150.7 31.0 37.0 30.6 20.2 18.4 16.8 14.9 176.5 109.9 19.6 122.2 117.6 152.5 147.1 116.4 123.5 166.5 51.4

6c

d

55.6

61.4 174.0 85.3 43.1 62.6 18.1 34.7 41.9 51.0 47.6 24.7 25.8 38.5 42.9 30.5 32.9 56.5 49.8 47.8 151.2 31.2 37.6 31.0 19.5 14.4 16.8 15.0 178.8 110.0 19.5 121.8 113.6 148.3 153.2 116.2 124.7 166.6

7a 64.8 177.3 86.6 44.3 57.5 19.3 35.1 42.6 45.8 50.3 24.7 26.7 39.6 43.9 30.9 32.9 57.4 50.3 47.9 151.2 31.6 37.6 31.2 20.8 19.1 17.4 15.5 177.0 110.5 20.2 122.5 114.1 149.2 153.9 116.9 125.1 166.9 51.9 56.4

8a 64.7 177.3 86.4 44.2 57.4 19.2 35.1 42.6 45.8 50.1 24.7 26.7 39.5 44.0 31.1 33.4 57.1 50.1 48.1 151.6 31.8 38.0 31.1 20.7 19.1 17.5 15.5 179.4 110.3 20.2 122.2 132.9 116.8 164.2 116.8 132.9 166.7

9a 60.5 174.5 83.4 42.4 56.3 19.6 35.0 42.6 44.5 48.2 24.4 26.5 39.3 44.0 31.0 33.4 57.1 50.2 48.0 151.6 31.7 38.0 25.6 26.3 19.8 17.5 15.7 179.3 110.3 19.9 122.2 133.0 116.5 164.1 116.5 133.0 166.9

10b 58.7 65.1 85.4 43.5 58.5 19.0 34.5 42.4 43.1 46.9 24.0 25.6 38.9 43.3 30.3 32.9 56.8 49.7 47.8 151.5 31.0 37.5 32.8 20.4 18.9 17.2 14.8 179.4 110.1 19.3 123.3 117.9 152.9 147.5 116.1 123.0 167.7

11a,d 56.2 178.8 43.2 38.8 56.5 19.6 29.7 42.0 46.6 51.4 23.9 26.2 38.4 55.1 28.0 25.5 55.1 58.3 92.9 142.3 35.2 37.7 32.1 27.2 20.2 17.8 177.9 179.3 112.4 19.3

12a 64.8 177.3 86.2 44.0 57.2 19.0 35.2 42.5 45.7 49.7 23.8 26.4 36.1 42.3 28.8 34.7 54.5 55.9 92.7 141.8 24.2 29.6 30.9 20.6 19.1 16.7 14.2 179.1 112.7 19.6 123.4 118.1 153.2 147.5 116.8 123.5 167.0

13a

110.9 21.3

137.7 52.3 63.7 19.2 39.2 42.5 49.5 51.6 24.3 27.1 49.1 59.5 31.5 27.9 146.1 51.5 54.3 150.7 39.7 120.1 25.3 67.1 21.8 18.7 178.5

143.4

14a 55.3 176.4 42.9 38.3 52.8 25.6 80.9 46.6 46.7 51.6 24.2 27.0 41.2 61.3 31.5 36.0 56.6 52.2 48.6 151.8 31.6 37.9 31.8 27.0 19.3 13.5 178.7 179.8 110.7 19.8 123.4 114.0 148.8 153.5 116.8 123.5 165.9 51.9 56.1

15a

56.2

45.5 74.6 80.1 41.0 56.2 19.2 35.1 41.5 51.3 39.2 21.7 26.5 39.0 43.3 30.7 33.3 57.0 50.1 48.2 151.8 31.6 38.1 29.6 17.9 17.9 16.7 15.3 179.3 110.5 19.8 123.2 113.9 148.8 153.5 116.6 125.2 167.2

16a 45.5 74.5 80.2 41.0 56.1 19.2 35.1 41.5 51.4 43.3 21.7 26.5 39.0 43.3 30.7 33.3 57.0 50.1 48.2 151.8 31.6 38.1 29.5 17.8 17.9 15.3 16.7 179.3 110.5 19.8 123.0 132.9 116.5 163.9 116.5 132.9 167.2

17a

49.3 67.0 85.6 40.5 56.0 18.9 35.0 41.5 51.2 38.9 21.7 26.4 39.0 43.1 30.5 33.2 57.0 50.1 48.2 151.7 31.6 38.0 29.2 18.5 16.8 17.9 15.3 179.4 110.8 19.8 123.5 118.3 152.7 147.5 116.7 123.5 167.8

18a,d

Recorded at 150 MHz. Recorded at 125 MHz. Recorded at 100 MHz. The assignments were based on the HSQC and HMBC spectra, because of their scarce quantities. Spectra were measured in pyridine-d5.

a

1a

position

Table 2. 13C NMR Spectroscopic Data (δ in ppm) of Compounds 1−18




Article

Figure 1. 1H−1H COSY and selected HMBC correlations of 1, 5, 11, 12, 14, and 15.

1−3, 4 was suggested to be a ceanothane-type triterpenoid derivative possessing a hydroxy, a methyl ester, and a carboxylic acid group. The presence of the C-3 hydroxy group was confirmed by the HMBC correlations of H-3 with two methyl carbons, C-23 (δC 32.5) and C-24 (δC 19.9). The 1H−1H COSY correlation between H-1 and H-3 and the HMBC correlations of H-1, H-3, and the methyl ester protons with the carboxyl C-2 (δC 173.6) confirmed the five-membered A-ring system of 4. The HMBC spectrum also exhibited correlations of H-18 (δH 1.73) and H-22a (δH 2.26) with C-28 (δC 179.3), which substantiated the carboxylic acid substitution of C-28. In contrast to 3, H-1 and H-3 exhibited a doublet correlation with J = 7.5 Hz. The β,β cis-orientation of the C-2 and C-3 hydroxy group was confirmed by comparing this J value with reference data.13,22 The upfield chemical shift of the α-oriented H-5 (δH 1.04) and the downfield chemical shift of the β-oriented H-25 (δH 1.62) also supported the β-orientation of the C-2 methyl ester group at C-1. Thus, spectroscopic data analysis was used to establish the structure of 4 as epiceanothic acid 2-methyl ester. The HRESIMS of compound 5 exhibited a deprotonated molecular ion at m/z 483.3484 ([M − H]−, calcd for C31H47O4, 483.3474) in the HRESIMS, suggesting a molecular formula of C31H48O4. The 1H NMR spectrum (Table 1) showed six tertiary methyl groups [δH 1.78, 1.06, 1.00, 1.00, 0.97, and 0.94 (each 3H, s)], two vinyl protons at δH 4.94 (1H, br s, H-29a) and 4.78 (1H, br s, H-29b), a hydroxylated methine proton at δH 4.03 (1H, d, J = 8.7 Hz, H-3), a methoxy group at δH 3.30 (3H, s), a methine proton at δH 2.53 (1H, dd, J = 8.7, 4.0 Hz, H-1), and an aldehyde proton at δH 10.12 (1H, d, J = 4.0 Hz, H-2). The vinyl protons and the downfield-shifted methyl group at δH 1.78 (C-30) suggested the presence of a characteristic isopropenyl group of a ceanothane- or lupanetype triterpenoid. Vicinal proton couplings between H-1 and H-2 and between H-1 and H-3 were estimated from their J values and were confirmed by the 1H−1H COSY spectrum

is a methyl ester derivative of 1, as supported by the molecular formula. An HMBC experiment verified this suggestion, showing a correlation between the methyl ester protons and C-2 (δC 176.8). Thus, the structure of 2 was assigned as 3dehydroxyceanothetric acid 2-methyl ester. The HRESIMS of 3 showed a deprotonated molecular ion peak at m/z 529.3165 ([M − H]−, calcd for C31H45O7, 529.3165), corresponding to the molecular formula C31H46O7. Compound 3 exhibited 1H and 13C NMR spectra (Tables 1 and 2) similar to those of 2, including three carboxylic acid carbons and a methyl ester group. However, the 1H NMR spectrum of 3 also showed an oxygenated methine proton at δH 4.64 (1H, s, H-3) and a methine proton at δH 3.06 (1H, s, H-1), which were correlated to carbons at δC 84.0 (C-3) and 66.3 (C-1), respectively, in the HSQC spectrum. These differences in the NMR spectra and the molecular formula suggested that 3 is a hydroxylated derivative of 2. This inference was confirmed by an HMBC experiment, which exhibited correlations of H-23 (δH 1.22) and H-24 (δH 1.19) with C-3 and of H-1 and H-3 with C-2 (δC 175.7), as well as of the methyl ester protons (δH 3.74) with C-2. The trans relationship between H-1β and H-3α was deduced from their singlet shapes, by comparing with the reference data.22 The chemical shift of H-5 (δH 1.93) also supported the α-orientation of the carbonyl C-2 at C-1. Accordingly, compound 3 was determined to be ceanothetric acid 2-methyl ester. Compound 4 gave the molecular formula C31H48O5, as indicated by the deprotonated molecular ion at m/z 499.3420 ([M − H]−, calcd for C31H47O5, 499.3423) in the HRESIMS. The 1H NMR spectrum of 4 (Table 1) exhibited six tertiary methyl groups [δH 1.78, 1.62, 1.16, 1.12, 1.11, and 1.06 (each 3H, s)], two vinyl protons at δH 4.93 (1H, br s, H-29a) and 4.75 (1H, br s, H-29b), a hydroxylated methine proton at δH 4.57 (1H, d, J = 7.5 Hz, H-3), a methyl ester group at δH 3.62 (3H, s), and a methine proton at δH 2.77 (1H, d, J = 7.5 Hz, H1). When compared to the spectroscopic data of compounds E



Article

Figure 2. Key ROESY correlations of 1, 5, 11, 12, 14, and 15.

observed, which suggested 6 is a derivative of ceanothic acid 2methyl ester,26 the epimeric compound of 4. The downfield chemical shift of H-3 suggested that the protocatechuoyl moiety is connected to C-3, and this was confirmed by the HMBC correlation of H-3 and C-7′ (δC 166.5). The HMBC spectrum also showed the coupling of the methoxy group and C-2 (δC 176.5). On the basis of its spectroscopic data analysis, compound 6 was determined to be 3-O-protocatechuoylceanothic acid 2-methyl ester. The HRESIMS of 7 exhibited a deprotonated molecular ion at m/z 635.3594 ([M − H]−, calcd for C38H51O8, 635.3584), indicating that its molecular formula is identical with that of 6, namely, C38H52O8. However, the prominent fragment ion of 7 at m/z 167.0347 (calcd for C8H5O4, 167.0344) suggested the presence of a vanilloyl moiety, instead of the protocatechuoyl unit of 6. The 1H and 13C NMR spectra of 7 (Tables 1 and 2) were similar to those of 6, but the chemical shifts of the 3,4disubstituted benzoyl moiety [δH 8.02 (1H, d, J = 8.2 Hz, H6′), 7.92 (1H, br s, H-2′), and 7.10 (1H, d, J = 8.2 Hz, H-5′)] were slightly different. Downfield-shifted H-2′ and H-6′ (ortho and para to C-3′) and upfield-shifted H-5′ (meta to C-3′) signals supported the hydroxy function at C-3′ as being substituted as a methoxy group. The vanilloyl moiety was confirmed by the HMBC spectrum, in which the methoxy proton (δH 3.70) showed a correlation with C-3′ (δC 148.3). The doublet coupling of H-1 (δH 3.13) and H-3 (δH 5.95) with J = 7.7 Hz indicated β,β-orientations of the C-2 carboxylic acid group at C-1 and the vanilloyl moiety at C-3, respectively. Therefore, the structure of compound 7 was elucidated as 3-Ovanilloylepiceanothic acid. The molecular formula of 8 was determined as C39H54O8, as indicated by the deprotonated molecular ion peak at m/z 649.3737 ([M − H]−, calcd for C39H53O8, 649.3740) in the HRESIMS. The 1H and 13C NMR spectra of 8 (Tables 1 and 2) were quite similar to those of 7, except for the presence of an additional methyl ester group at δH 3.80 and the singlet-shaped coupling of H-1 (δH 3.20) and H-3 (δH 5.99). The αorientation of C-2 at C-1 and β-orientation of the vanilloyl moiety at C-3 were determined by the singlet signals. The HMBC correlation of the methyl ester protons with C-2 (δC 177.3) revealed a methyl ester substitution at the C-2 carboxyl group. Accordingly, the structure of 8 was defined as being 3-Ovanilloylceanothic acid 2-methyl ester.

(Figure 1). The HMBC spectrum showed correlations of H-23 (δH 1.06) and H-24 (δH 1.00) with C-3 (δC 91.4), of H-25 (δH 0.94) with C-1 (δC 72.4), and of H-3 with C-2 (δH 205.6), which confirmed the five-membered A-ring system of 5. The methoxy proton group exhibited an HMBC correlation with C3, suggesting its location at C-3. For the configuration at C-1 and C-3, at first the vicinal doublet-shaped coupling of H-1 and H-3 was thought to be caused by the α,α cis-orientation of C-2 and the methoxy group, as in compound 4. However, the J1,3 value of 5 was slightly larger than in compoound 4, so a ROESY experiment was performed to confirm the configuration (Figure 2). Interestingly, H-1 showed spatial correlations with the αoriented protons of H-5 (δH 1.32), H-9 (δH 1.75), and H-23, while H-3 showed a correlation with the β-oriented H-25. These NOE correlations suggested the β,α-orientation of C-2 and the methoxy group at C-1 and C-3, respectively, different from the estimation using the J value alone. From the literature, it was found that the J value between H-1α and H-3β has been calculated and observed around 9.0 Hz.22 Additionally, previously isolated ceanothane-type triterpenoids with a β,αorientation also showed similar J values: 8.9 Hz for zizyberanalic acid,23 8.7 Hz for isoceanothic acid,24 and 8.6 Hz for ceanothanolic acid.25 Consequently, the structure of compound 5 was assigned as 2β-aldehydo-3α-methoxy-A(1)norlup-20(29)-en-28-oic acid and as the 3-O-methyl derivative of the previously isolated compound zizyberanalic acid.23 The HRESIMS of 6 exhibited a deprotonated molecular ion at m/z 635.3580 ([M − H]−, calcd for C38H51O8, 635.3584), indicating a molecular formula of C38H52O8. Compound 6 yielded a prominent fragment ion at m/z 153.0192 (calcd for C7H5O4, 153.0188) in the HRESIMS and also showed UV absorption bands at λmax 262 and 295 nm, suggesting the presence of a protocatechuoyl group moiety. The latter was confirmed by the 1H NMR spectrum (Table 1), in which an ABX aromatic system [δH 8.10 (1H, d, J = 1.8 Hz, H-2′), 7.90 (1H, dd, J = 1.8, 8.3 Hz, H-6′), and 7.32 (1H, d, J = 8.3 Hz, H5′)] of a 3,4-disubstituted benzoyl moiety was observed. In the 1 H NMR spectrum (Table 1), an isopropenyl group [δH 4.80 (1H, br s, H-29a), 4.63 (1H, br s, H-29b), and 1.62 (3H, s, H30)], five additional quaternary methyl groups [δH 1.52, 1.17, 1.07, 1.07, and 0.97 (each 3H, s)], an oxygenated methine proton at δH 5.93 (1H, s, H-3), a methoxy group at δH 3.72 (3H, s), and a methine proton at δH 3.11 (1H, s, H-1) were also F



Article

Compound 12 gave a molecular formula of C30H42O6, as indicated by the deprotonated molecular ion peak at m/z 497.2897 ([M − H]−, calcd for C30H41O6, 497.2903) in the HRESIMS. The 1H and 13C NMR spectra of 12 (Tables 2 and 3) were similar to those of 1, but several uncommon characteristics were also observed. The vinyl proton resonances of the isopropenyl group were shifted significantly downfield [δH 5.54 (1H, br s, H-29a) and 5.02 (1H, br s, H-29b)] when compared to other ceanothane- and lupane-type triterpenoids, and the peak shapes of H-29a and H-30 (δH 1.72) were broadened (Figure S60, Supporting Information). Additionally, the characteristic doublet of triplet proton signal corresponding to H-19 was absent in the 1H NMR spectrum. This β-oriented proton generally is observed at δH 3.20−3.70 in triterpenoids possessing a 28-oic acid substituent and occurs downfield from the spatial effect of the β-oriented C-28 carboxylic acid group (Tables 1 and 3). In the 13C NMR spectrum of 12, an unusual quaternary carbon resonance was observed at δC 92.9 (C-19). From these characteristic NMR data and the IR absorption band at 1780 cm−1, the presence of an E-ring γ-lactone structure was deduced. The HMBC experiment exhibited correlations of H-29b and H-30 with the quaternary carbon at δC 92.9, which confirmed the presence of the lactone bridge at C-19 (Figure 1). The downfield effect on H-29 protons was estimated to be a result of anisotropic effects generated by the carbonyl function of the lactone system. The α-orientation of carboxy C-2 at C-1 was determined by the ROESY spectrum, in which a NOE correlation between H-1 (δH 2.87) and H-25 (δH 0.94) was observed (Figure 2). The downfield chemical shift of the α-oriented H-5 (δH 2.05) also supported the configuration at C-1, as in 1. Consequently, compound 12 was identified as 3dehydroxyceanothan-27α-carboxy-28β,19β-olide. To the best of our knowledge, a ceanothane-type triterpenoid with an E-ring γ-lactone was reported only once before, namely, alphitexolide (3-O-p-methoxybenzoylceanothan-28β,19β-olide) from Alphitonia exelsa.28 The 1H NMR spectroscopic characteristics, including the downfield-shifted H-29s signal and the broadened H-29a and H-30 signals in compound 12, were consistent with those in alphitexolide.28 The HRESIMS of 13 exhibited a deprotonated molecular ion peak at m/z 619.3275 ([M − H]−, calcd for C37H47O8, 619.3271), indicating a molecular formula of C37H48O8, along with a fragment ion at m/z 153.0188 (calcd for C7H5O4, 153.0188), corresponding to a protocatechuoyl moiety. The 1H and 13C NMR spectra of 13 (Tables 2 and 3) also showed characteristic features of an E-ring γ-lactone structure, which indicated 13 to be a protocatechuoyl derivative of 12. An oxygenated methine singlet at δH 5.97 (1H, s, H-3) suggested the presence of the β-O-protocatechuoyl moiety at C-3. This was confirmed by the HMBC correlations of H-23 (δH 1.53) and H-24 (δH 1.08) with C-3 (δC 86.2) and of H-3 with C-7′ (δC 167.0). Accordingly, compound 13 was proposed as 3-Oprotocatechuoylceanothan-28β,19β-olide. The molecular formula of 14 was suggested to be C28H40O3 by the HRESIMS deprotonated molecular ion peak at m/z 423.2901 ([M − H]−, calcd for C28H39O3, 423.2889) and was supported by the 13C NMR spectrum (Table 2), in which 28 carbon resonances were observed. Compound 14 was deduced to be a dinorlupane-type triterpenoid derivative, from its molecular formula and the isoproprenyl group signals [δH 5.04 (1H, br s, H-29a), 4.84 (1H, br s, H-29b), and 1.79 (3H, s, H30)] in the 1H NMR spectrum (Table 3). Signals for three additional methyl groups [δH 1.31, 1.16, and 1.16 (each 3H, s)],

Compounds 9 and 10 showed HRESIMS deprotonated molecular ion peaks at m/z 605.3472 ([M − H]−, calcd for C37H49O7, 605.3468), corresponding to the same molecular formula of C37H50O7. Both of these compounds exhibited major fragment ions at m/z 137.0236 (calcd for C7H5O3, 137.0239) and UV absorption bands at λmax 255 nm, suggesting the presence of a p-hydroxybenzoyl group moiety. This suggestion was confirmed by the AA′XX′ aromatic systems in their 1H NMR spectra (Tables 1 and 3). The 1H and 13C NMR spectra (Table 2) of 9 and 10 also showed the characteristics of ceanothane-type triterpenoid derivatives. By comparing the coupling constants of H-1 and H-3, the configurations at C-1 and C-3 were determined to be α,β and β,β for 9 and 10, respectively. Consequently, compound 9 was determined to be 3-O-p-hydroxybenzoylceanothic acid, while 10 was 3-O-phydroxybenzoylepiceanothic acid. The molecular formula of compound 11 was determined as C37H52O7 based on its deprotonated molecular ion peak at m/z 607.3639 ([M − H]−, calcd for C37H51O7, 607.3635) in the HRESIMS. An aromatic ABX system [δH 8.22 (1H, d, J = 1.9 Hz, H-2′), 7.90 (1H, dd, J = 8.3, 1.9 Hz, H-6′), and 7.28 (1H, d, J = 8.3 Hz, H-5′)], six tertiary methyl groups [δH 1.74, 1.31, 1.21, 1.16, 1.10, and 1.09 (each 3H, s)], two vinyl protons at δH 4.93 (1H, br s, H-29a) and 4.79 (1H, br s, H-29b), an oxygenated methylene group [δH 4.73 (1H, dd, J = 10.0, 5.5 Hz, H-2a) and 4.46 (1H, dd, J = 10.0, 8.8 Hz, H-2b)], a hydroxylated methine proton at δH 4.37 (1H, s, H-3), and a methine proton at δH 2.45 (1H, dd, J = 8.8, 5.5 Hz, H-1) were observed in the 1H NMR spectrum of 11 (Table 3). The 1 H−1H COSY spectrum revealed vicinal proton couplings between H-2s and H-1 and between H-1 and H-3, which suggested 11 is a ceanothane-type triterpenoid derivative with a methylene group at C-2 (Figure 1). This inference was confirmed by the HMBC correlations of H-25 (δH 1.31) with C-1 (δC 58.7) and of H-23 (δH 1.16) and H-24 (δH 1.21) with C-3 (δC 85.4). The aromatic ABX system of the 3,4disubstituted benzoyl moiety indicated the presence of a protocatechuoyl moiety, supported by the HRESIMS fragment ion at m/z 153.0183 (calcd for C7H5O4, 153.0188). The chemical shift of H-3 was not as far downfield as in compounds 6−10, indicating that a protocatechuoyl group was not connected to C-3. The HMBC correlation of H-2b and C-7′ (δC 167.7) revealed the presence of the protocatechuoyl moiety at C-2. The singlet shape of H-3 suggested the α,β-orientation of C-2 at C-1 and the hydroxy group at C-3 as in compounds 3, 6, 8, and 9. The ROESY experiment confirmed the configuration of the A-ring, showing NOE correlations between H-1 and the β-oriented H-25 and between H-3 and the αoriented H-23 and H-5 (δH 1.55) (Figure 2). Correlations of H-2b and H-5 and of H-2b and H-9 (δH 1.84) were also observed, which supported the α,β-orientation at C-1 and C-3. Therefore, the structure of 11 was assigned as 2α-Oprotocatechuoylmethyl-3β-hydroxy-A(1)-norlup-20(29)-en-28oic acid. To the best of our knowledge, only four ceanothanetype triterpenoids with a C-2 methylene unit have been isolated before: ceanothanolic acid, 25 2-O-trans-p-coumaroylceanothanolic acid,25 gouanic acid A,27 and epigouanic acid A.21 Among them, ceanothanolic acid is a compound with a hydroxy group at C-3 and a C-27 methyl as in compound 11, but it exhibited a β,α-orientation at C-1 and C-3. Therefore, following the naming rule for isoceanothic acid,24 11 was assigned as 2-O-protocatechuoylisoceanothanolic acid. G


a

H

s s s s s s d d d d

0.99, 1.10, 1.13, 4.87, 4.66, 1.65, 8.32, 7.01, 7.01, 8.32,

(8.7) (8.7) (8.7) (8.7)

s s s s s s d (1.9)

7.28, d (8.3) 7.90, dd (1.9, 8.3)

1.31, 1.10, 1.09, 4.93, 4.79, 1.74, 8.22,

m m m m m m m m m m m m m s s

1.92, 1.29, 2.75, 1.88, 1.19, 2.59, 1.51, 1.71, 3.52, 2.22, 1.48, 2.23, 1.54, 1.16, 1.21, m m m m s s

2.00, 1.56, 2.16, 1.77, 1.24, 0.95,

5.54, s 5.02, s 1.72, s

0.94, s 1.01, s

m m m m m m m m m m m m m m m m m d (10.8)

2.05, 1.89, 2.05, 1.37, 1.30, 1.56, 1.44, 2.47, 2.15, 1.56, 2.70, 2.13, 1.94, 2.30, 1.76, 2.38, 1.96, 2.46,

2.87, d (7.9)

12a

m m m m m m m m m m m

1.15, 0.91, 0.88, 5.49, 4.99, 1.62, 8.14, 7.40, 7.92,

s s s s s s br s d (8.3) d (8.3)

1.53, s 1.08, s

1.58, m

1.55, m

2.11, 2.13, 1.55, 1.94, 1.25, 1.73, 1.74, 1.17, 2.07, 1.37, 1.77,

1.58, m

2.16, m 1.39, m

5.97, s

3.14, s

13a

5.04, s 4.84, s 1.79, s

s d (10.6) d (10.6) s s

t (8.6) dd (8.4, 16.6) m m s

3.01, 2.77, 2.58, 2.22, 5.33, 1.31, 4.03, 3.84, 1.16, 1.16,

m m m m m m

m m m m m m m

2.55, 1.97, 1.60, 2.40, 1.48, 2.56,

1.66, 1.80, 1.66, 2.31, 1.88, 2.39, 1.66,

5.85, d (5.7)

6.24, d (5.7)

14a

m m m m m m m m m m m dt (4.5, 11.0) m m m m s s

m m m m m dd (4.4, 10.3)

s s s d (1.1) 7.20c 8.01, dd (8.2, 1.1) 3.78, s 3.76, s

5.09, 4.84, 1.95, 7.97,

0.99, s 1.63, s

2.21, 1.95, 1.74, 2.62, 2.10, 3.06, 3.00, 2.30, 2.76, 1.79, 2.29, 3.71, 2.24, 1.48, 2.20, 1.42, 1.18, 0.90,

1.87, 1.83, 2.12, 2.28, 1.55, 6.02,

2.71, d (7.6)

15a

Recorded at 500 MHz. bRecorded at 600 MHz. cOverlapped with the solvent signal. Spectra were measured in pyridine-d5.

m m m m m m m m m m m m m m m m m m s s

1.57, 1.42, 1.29, 1.83, 1.53, 1.94, 1.31, 2.74, 1.91, 1.23, 2.59, 1.23, 1.67, 3.49, 2.24, 1.48, 2.23, 1.52, 1.40, 1.26,

m m m m m m m

5.86, d (7.8)

1.55, 1.44, 1.36, 1.41, 1.37, 1.84, 1.48,

4.73, dd (5.5, 10.0) 4.46, dd (8.8, 10.0) 4.37, s

2.79, m 1.44, m

2.45, dd (5.5, 8.8)

3.51, d (7.8)

1a 1b 2a 2b 3a 3b 5 6a 6b 7a 7b 9 11a 11b 12a 12b 13 15a 15b 16a 16b 18 19 21a 21b 22a 22b 23 24a 24b 25 26 27 29a 29b 30 2′ 3′ 5′ 6′ −OMe −OMe′

11a

10b

position

Table 3. 1H NMR Spectroscopic Data (δ in ppm and J Values in (Hz) in Parentheses) of Compounds 10−18

s s s s s s d (1.9)

m m m m m m m m m m m m m m m m dt (4.8, 10.7) m m m m s s

3.72, s

7.28, d (8.3) 8.06, dd (1.9, 8.3)

1.02, 1.04, 1.09, 4.97, 4.82, 1.82, 7.99,

1.01, 1.56, 1.39, 1.46, 1.39, 1.43, 1.27, 1.18, 1.92, 1.16, 2.74, 1.87, 1.26, 2.64, 1.56, 1.78, 3.56, 2.26, 1.54, 2.26, 1.59, 1.31, 1.13,

3.72, d (9.8)

2.36, dd (4.7, 12.3) 1.20, m 5.78, dt (4.7, 10.7)

16a

1.12, 1.09, 1.04, 4.97, 4.82, 1.82, 8.34, 7.21, 7.21, 8.34,

1.01, 1.55, 1.40, 1.46, 1.39, 1.44, 1.26, 1.17, 1.92, 1.16, 2.74, 1.90, 1.26, 2.26, 1.57, 1.77, 3.56, 2.27, 1.54, 2.28, 1.59, 1.31, 1.02, s s s s s s d d d d

(8.6) (8.6) (8.6) (8.6)

m m m m m m m m m m m m m m m m dt (4.8, 10.8) m m m m s s

3.71, d (9.9)

2.35, m 1.17, m 5.74, dt (4.7, 11.1)

17a

s s s s s s d (1.8)

m m m m m dt (3.5, 12.7) m m m m m dt (4.3, 9.9) m m m m s s

m m m m

7.31, d (8.2) 7.97, dd (1.9, 8.2)

1.05, 0.92, 1.07, 4.95, 4.78, 1.79, 8.21,

1.47, 1.50, 1.23, 1.94, 1.21, 2.75, 1.86, 1.26, 2.65, 1.56, 1.76, 3.56, 2.26, 1.53, 2.27, 1.59, 1.00, 0.99,

1.04, 1.43, 1.35, 1.35,

5.34, d (9.8)

2.39, dt (4.1, 12.8) 1.35, m 4.31, m

18a




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three olefinic protons [δH 6.24 (1H, d, J = 5.7, H-1), 5.85 (d, J = 5.7 Hz, H-3), and 5.33 (s, H-22)], and a hydroxylated methylene group [δH 4.03 (1H, d, J = 10.6 Hz, H-24a) and 3.84 (1H, d, J = 10.6 Hz, H-24b)] were also observed in the 1H NMR spectrum. The detailed structure of 14 was elucidated by 1 H−1H COSY, HMBC (Figure 1), and ROESY (Figure 2) experiments. Initially, the cyclopentenyl 2-nor A-ring system was confirmed by HMBC correlations of H-25 (δH 1.16) with C-1 (δC 143.4) and of H-23 (δH 1.31) with C-3 (δC 137.7) and supported by the COSY coupling between two olefinic protons of H-1 and H-3. The hydroxylated methylene group exhibited HMBC correlations with C-3, C-4 (δC 52.3), C-5 (δC 63.7), and C-23 (δC 25.3), suggesting its position as H-24. The relative configuration at C-4 was confirmed by the ROESY spectrum, in which NOE correlations between H-24s and H-25 and between H-23 and H-5 (δH 1.66) were observed. The elimination of any carboxylic acid substituent at C-28 was deduced by the upfield chemical shift of the β-oriented H-19 signal (δH 2.77), when compared to compounds 1−11, possessing a β-oriented C-28 carboxylic acid group. The olefinic H-22 correlated with C-17 (δC 146.1), C-18 (δC 51.5), C-19 (δC 54.3), and C-21 (δC 39.7) in the HMBC spectrum. These correlations confirmed a cyclopentenyl E-ring structure, which was generated by elimination of C-28. The carboxylation at C-28 was deduced from the downfield-shifted resonance of H-18 (δH 3.01) and was confirmed by the HMBC correlations of H-13 (δH 1.60) and H-15b (δH 1.48) with C-27 (δC 178.5). On the basis of the above spectroscopic information, the structure of 14 was elucidated as 2,28-dinor-24-hydroxylup1,17(22)-dien-27-oic acid. Only a small number of 2-norlupane triterpenoids have been reported before, such as ceanothenic acid,29,30 gouanic acid B,27 and zizymauritic acids A−C.31 28Norlupane derivatives were also reported,32 but to the best of our knowledge, compound 14 is the first 2,28-dinorlupane molecule isolated from Nature. The molecular formula of 15 was determined to be C39H52O10 from the deprotonated molecular ion peak at m/z 679.3474 ([M − H]−, calcd for C39H51O10, 679.3482) in the HRESIMS. Compound 15 also exhibited a prominent fragment ion at m/z 167.0345 (calcd for C 8 H 5 O 4 , 167.0344), corresponding to the presence of a vanilloyl moiety. The 1H NMR spectrum of 15 exhibited signals for five quaternary methyl groups [δH 1.95, 1.63, 1.18, 0.99, and 0.90 (each 3H, s)], an ABX aromatic system [δH 8.01 (dd, J = 8.2, 1.1 Hz, H6′), 7.97 (d, J = 1.1 Hz, H-2′), and 7.20 (overlapped with solvent signal, H-5′)], an oxygenated methine proton at δH 6.02 (dd, J = 4.4, 10.3 Hz, H-7), two vinyl protons at δH 5.09 (1H, s, H-29a) and 4.84 (1H, s, H-29b), a methyl ester group (δH 3.78), and a methoxy group (δH 3.76) (Table 3). Three carboxyl carbons at 179.8 (C-28), 178.7 (C-27), and 176.4 (C2) were observed in the 13C NMR spectrum (Table 2). These data suggested that compound 15 is a derivative of 2 possessing a vanilloyl moiety. However, the downfield chemical shift of the methyl group at δH 1.63 (H-26) suggested that the vanilloyl moiety is substituted not at C-3 but at another position near this methyl group. In the HMBC spectrum of 15 (Figure 1), H26 exhibited a correlation with the oxygenated carbon at δC 80.9 (C-7), and H-7 correlated with C-7′ (δC 165.9), which confirmed the O-vanilloyl substitution at C-7. A ROESY experiment was performed to determine the relative configuration at C-1 and C-7 (Figure 2). The H-7 signal exhibited NOE correlations with the α-oriented H-5 (δH 2.12) and H-9 (δH 2.21), indicating the β-orientation of the O-vanilloyl moiety

at C-7. A NOE cross-peak between H-1 (δH 2.71) and H-25 (δH 0.99) was also observed, which indicated the α-orientation of the methyl ester C-2 at C-1. Therefore, compound 15 was assigned as 7β-O-vanilloyl-3-dehydroxyceanothetric acid 2methyl ester. Compound 16 showed a deprotonated molecular ion peak at m/z 621.3787 ([M − H]−, calcd for C38H53O7, 621.3791) in the HRESIMS, indicating its molecular formula to be C38H54O7. In the 1H NMR spectrum of 16, six quaternary methyl groups [δH 1.82, 1.31, 1.13, 1.09, 1.04, and 1.02 (each 3H, s)], an aromatic ABX system [δH 8.06 (1H, dd, J = 1.9, 8.3 Hz, H-6′), 7.99 (1H, d, J = 1.9 Hz, H-2′), and 7.28 (1H, d, J = 8.3 Hz, H-5′)], two vinyl protons [δH 4.97 (1H, s, H-29a) and 4.82 (1H, s, H-29b)], a methoxy group (δH 3.72), and two oxygenated methine protons [δH 5.78 (1H, dt, J = 10.7, 4.7 Hz, H-2) and 3.72 (1H, d, J = 9.8 Hz, H-3)] were observed (Table 3). The six-membered A-ring system was determined by 1 H−1H correlations between H-3 and H-2 and between H-2 and H-1 (δH 2.36 and 1.20). The relative configuration at C-2 and C-3 was determined to be identical to 26, by comparing the J values of H-2 and H-3 with the literature.18 The downfield chemical shift of H-2 suggested that the hydroxy group at C-2 was substituted in the aromatic ester, and this was confirmed by the HMBC correlation of H-2 and C-7′ (δC 167.2). The HMBC spectrum of 16 also showed the correlation of the methoxy group and C-3′ (δC 148.8), which revealed the 3,4disubstituted benzoyl moiety to be a vanilloyl moiety. Therefore, the structure of compound 16 was established as 2-O-vanilloylalphitolic acid. The HRESIMS of 17 exhibited the deprotonated molecular ion peak at m/z 591.3678 ([M − H]−, calcd for C37H51O6, 591.3686), indicating its molecular formula as C37H52O6. The 1 H and 13C NMR spectra of 17 (Tables 2 and 3) also showed that 17 is a 2-O-aromatic ester derivative of alphitolic acid, similar to compound 16. An aromatic AA′XX′ system at δH 8.34 and 7.21 Hz observed in the 1H NMR spectrum suggested the presence of a p-hydroxybenzoyl moiety, for which the position was confirmed by the HMBC correlation of H-2 (δH 5.74) and C-7′ (δC 167.2). Thus, compound 17 was assigned as 2-O-p-hydroxybenzoylalphitolic acid. Compound 18 gave a molecular formula of C37H52O7, as indicated by the HRESIMS (m/z 607.3630 [M − H]−, calcd for C37H51O7, 607.3635). The 1H NMR spectrum of 18 suggested it to be a protocatechuoyl derivative of 26 (Table 3). However, in the 1H NMR spectrum, H-2 was observed in a relatively upfield position (δH 4.31), while H-3 was observed downfield (δH 5.34) instead. This suggested that the protocatechuoyl moiety is substituted at C-3, instead of C-2. The HMBC experiment confirmed this suggestion, exhibiting the correlation of H-3 and C-7′ (δH 167.8). Consequently, the structure of 18 was elucidated as 3-O-protocatechuoylalphitolic acid. Compounds 2−4, 6, 8, and 15 contain methyl ester moieties, and therefore it was determined whether these are actual natural products or experimental artifacts, because MeOH was used for the extraction and isolation. An EtOH extract of Z. jujuba roots was prepared without using any MeOH and was analyzed by LC-MS. The methyl ester compounds were detected in the EtOH extract, which showed that they are naturally occurring constituents of Z. jujuba and not artifacts. Detailed information on this LC-MS analysis are described in the Supporting Information. The cytotoxicity of the isolated triterpenoid compounds against the HepG2 cell line was evaluated. Compounds 5, 11, I



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eluted with a mixture of CH2Cl2−MeOH (3:1). Compounds 16 (1.2 mg) and 22 (1.6 mg) were isolated from subfraction C3e5 by ODS silica gel HPLC eluted with 83% aqueous acetonitrile. Subfraction C3f was subjected to silica gel CC eluted with mixtures of CHCl3−MeOH (200:1, 100:1, 50:1, 20:1, and 10:1) to yield six subfractions (C3f1−C 3f6). Compound 29 (124.6 mg) was isolated from subfraction C3f3 by recrystallization (MeOH). Before being subjected to further isolation, the MeOH-insoluble precipitate of fraction C5 was collected and purified as compound 25 (12.5 g), by washing with MeOH. The rest of the fraction was subjected to silica gel CC eluted with CHCl3− MeOH mixtures of increasing polarity (100:1, 50:1, 25:1, 15:1, and 10:1) to give 10 subfractions (C5a−C5j). Subfraction C5e was separated into seven further subfractions (C5e1−C5e7) by silica gel CC with mixtures of CHCl3−MeOH of increasing polarity (100:1, 50:1, 25:1, 15:1, and 10:1). White pellets of subfraction C5e2, which were insoluble in MeOH, were filtered and purified by recrystallization with MeOH to yield compound 2 (45.8 mg). The MeOH-soluble part of subfraction C5e2 was subjected to ODS silica gel HPLC (acetonitrile−H2O, 78:22), yielding compounds 5 (0.7 mg), 8 (10.8 mg), 23 (9.0 mg), and 28 (11.3 mg). Subfraction C5g was chromatographed by ODS silica gel HPLC with 65% aqueous acetonitrile to yield six subfractions (C5g1−C5g6). Compounds 9 (8.5 mg), 10 (2.8 mg), 14 (5.6 mg), 15 (3.3 mg), and 26 (30.3 mg) were isolated from subfraction C5g3 by ODS silica gel HPLC eluted with 60% aqueous acetonitrile. Subfraction C5g5 was further purified with ODS silica gel HPLC with 80% aqueous acetonitrile and yielded compounds 7 (6.4 mg) and 17 (4.2 mg). Compound 1 (45.8 mg) was isolated from the MeOH-insoluble pellets of subfraction C5i by recrystallization with MeOH. The rest of subfraction C5i was further separated on Sephadex LH-20 eluted with a 1:1 mixture of CH2Cl2− MeOH to give four subfractions (C5i1−C5i4). Compound 12 (6.5 mg) was purified from subfraction C5i4 using ODS silica gel HPLC eluted with 70% aqueous acetonitrile. Fraction C8 was separated into five subfractions (C8a−C8e) by passage over Sephadex LH20, eluted with a 1:1 mixture of CH2Cl2−MeOH. Compounds 4 (12.1 mg), 13 (2.3 mg), 21 (5.8 mg), and 27 (2.8 mg) were isolated from subfraction C8c using ODS silica gel HPLC with 62% aqueous acetonitrile. Compounds 11 (0.8 mg) and 18 (1.0 mg) were purified from subfraction C8d by ODS silica gel HPLC with 70% aqueous acetonitrile. The EtOAc fraction was subjected to silica gel CC eluted with mixtures of CHCl3−MeOH (50:1, 25:1, 15:1, 10:1, 7:1, 5:1, 3:1, and 1:1) to yield 14 fractions (E1−E14). Three major triterpenoid acids, compounds 25 (18.7 g), 19 (14.5 g), and 20 (276.4 mg), were isolated from fractions E4, E7, and E8 by recrystallization with MeOH, respectively. Compound 3 (15.6 mg) was isolated from fraction E6 using ODS silica gel HPLCs eluted with 65% aqueous acetonitrile. Fraction E9 was further separated into four subfractions (E9a−E9d) using Sephadex LH-20 with a 1:1 mixture of CH2Cl2−MeOH. Compound 24 (39.6 mg) was isolated from subfraction E9c using ODS silica gel HPLC eluted with 50% aqueous acetonitrile. Fraction E11 was subjected to silica gel CC eluted with mixtures of CHCl3− MeOH (50:1, 25:1, 15:1, 10:1, 5:1, and 3:1) to yield seven subfractions (E11a−E11e). Compound 6 (56.0 mg) was purified from subfraction E11d using ODS silica gel HPLC eluted with acetonitrile−H2O mixtures of decreasing polarity (4:6 to 9:1). 3-Dehydroxyceanothetric acid (1): white, amorphous powder; mp 288−290 °C; [α]20 D +58.6 (c 0.10, MeOH); IR νmax 2966, 2869, 2361, 2322, 1698, 1456, 1054, 1033, 1014 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 499.3062 [M − H]− (calcd for C30H43O6, 499.3060). 3-Dehydroxyceanothetric acid 2-methyl ester (2): white, amorphous powder; mp 296−298 °C; [α]20 D +73.4 (c 0.10, MeOH); IR νmax 3704, 2950, 2869, 2361, 2327, 1687, 1054, 1033, 1013 cm−1; 1 H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 513.3213 [M − H]− (calcd for C31H45O6, 513.3216). Ceanothetric acid 2-methyl ester (3): white, amorphous powder; mp 288−290 °C; [α]20 D +44.6 (c 0.10, MeOH); IR νmax 2966, 2873, 2360, 2337, 1717, 1684, 1456, 1054, 1033, 1011 cm−1; 1H and 13C

13, 16, and 18 were excluded from such biological testing due to the scarce amounts obtained. Among the 25 tested triterpenoids, compounds with an IC50 lower than 10.0 μM are shown in Table 4 along with their IC50 values and 95% Table 4. Cytotoxicity of the Isolated Triterpenoids against HepG2 Cellsa compound

IC50 (μM)

95% confidence interval

6 7 8 14 17 23 25 29 30 docetaxelb

2.8 3.9 1.9 4.9 2.9 2.0 5.9 2.0 2.1 8.3

1.5−5.4 3.2−4.7 1.5−2.5 2.9−8.3 2.1−4.1 1.4−2.7 4.1−8.3 1.5−2.7 1.6−2.8 4.2−16.5

a

Compounds 1−4, 9, 10, 12, 19−22, 24, and 26−28 showed IC50 values of >10.0 μM. bDocetaxel was used as a positive control.

confidence intervals. As shown in the table, compounds 8, 23, and 29 were the most potently cytotoxic against HepG2 cells, with IC50 values of 1.9, 2.0, and 2.0 μM, respectively.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on a Buchi B-545 apparatus (Büchi Labortechnik AG, Postfach, Switzerland). Optical rotations were measured on a JASCO P-2000 polarimeter using a 1 cm cell. UV spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics, Surrey, UK), and IR spectra were acquired using a JASCO FT/IR-4200 spectrometer. 1D and 2D NMR spectra were obtained with a JEOL JMN-LA300 spectrometer (JEOL Ltd., Tokyo, Japan) and Bruker GPX 400, AMX 500, and AVANCE 600 spectrometers (Bruker, Billerica, MA, USA). HRESIMS was performed using a Waters Xevo G2 QTOF mass spectrometer (Waters MS Technologies, Manchester, UK), which was equipped with an electrospray interface (ESI). Column chromatography (CC) was performed with Kieselgel 60 silica gel (40−60 μm, 230−400 mesh, Merck) and Sephadex LH-20 (25−100 μm, Pharmacia, Piscataway, NJ, USA). Preparative HPLC was performed with a system consisting of a Gilson 321 pump and a UV/vis-151 detector (Gilson Inc., Middleton, WI, USA), equipped with a YMC Triart C18 column (YMC Co. Ltd., Kyoto, Japan). Extra-pure grade solvents for extraction, fractionation, and isolation were purchased from Dae Jung Pure Chemical Engineering Co. Ltd., Korea. Deuterated pyridine for NMR analyses was purchased from Merck (Darmstadt, Germany). Plant Materials. The roots of Z. jujuba were collected in April 2012 in Jinju, Korea, and authenticated by Prof. Dr. Eun Ju Jeong (Gyeongnam National University of Science and Technology, Jinju, Korea). A voucher specimen (SUPH-1204-01) was deposited in the Herbarium of the Medicinal Plant Garden, College of Pharmacy, Seoul National University, Koyang, Korea. Extraction and Isolation. Pulverized, air-dried roots of Z. jujuba (7.5 kg) were extracted with MeOH (2 × 30 L, for 3 h each) with ultrasonication at room temperature and then concentrated in vacuo. The crude extract (630.4 g) was suspended in H2O and partitioned successively into CHCl3 (103.5 g), EtOAc (75.0 g), and BuOH fractions (127.3 g), respectively. The CHCl3 fraction was subjected to silica gel CC eluted with mixtures of CHCl3−MeOH (100:1, 50:1, 25:1, 15:1, 10:1, 7:1, 5:1, and 3:1) to yield 10 fractions (C1−C10). Fraction C3 was further separated into six subfractions (C1a−C1f) by silica gel CC, eluted with mixtures of CHCl3−MeOH (200:1, 100:1, 70:1, 50:1, 30:1, and 20:1). Subfraction C3e was further divided into five subfractions (C3e1−C3e5) by passage over Sephadex LH-20, J



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NMR, see Tables 1 and 2; HRESIMS m/z 529.3165 [M − H]− (calcd for C31H45O7, 529.3165). Epiceanothic acid 2-methyl ester (4): white, amorphous powder; [α]20 D +10.0 (c 0.10, MeOH); IR νmax 3649, 2950, 2869, 2360, 2337, 1698, 1054, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 499.3420 [M − H]− (calcd for C31H47O5, 499.3423). 3-O-Methylzizyberanalic acid (5): white, amorphous powder; [α]20 D −13.7 (c 0.10, MeOH); IR νmax 2928, 2869, 2366, 2326, 1713, 1600, 1456, 1360 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 483.3484 [M − H]− (calcd for C31H47O4, 483.3474). 3-O-Protocatechuoylceanothic acid 2-methyl ester (6): pale yellowish powder; [α]20 D −4.2 (c 0.10, MeOH); UV λmax (log ε) 295 (3.56), 262 (3.71) nm; IR νmax 3648, 2972, 2866, 2360, 2327, 1698, 1507, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 635.3580 [M − H]− (calcd for C38H51O8, 635.3584). 3-O-Vanilloylepiceanothic acid (7): yellow, amorphous powder; mp 250−252 °C; [α]20 D −31.2 (c 0.10, MeOH); UV λmax (log ε) 292 (3.88), 262 (4.00) nm; IR νmax 3649, 2972, 2866, 2360, 2322, 1698, 1507, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 635.3594 [M − H]− (calcd for C38H51O8, 635.3584). 3-O-Vanilloylceanothic acid 2-methyl ester (8): yellowish, amorphous powder; [α]20 D +7.0 (c 0.10, MeOH); UV λmax (log ε) 292 (3.76), 262 (4.06) nm; IR νmax 3649, 2972, 2866, 2360, 2327, 1698, 1507, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 649.3737 [M − H]− (calcd for C39H53O8, 649.3740). 3-O-p-Hydroxybenzoylceanothic acid (9): white, amorphous powder; [α]20 D −2.1 (c 0.10, MeOH); UV λmax (log ε) 257 (4.13) nm; IR νmax 3647, 2972, 2866, 2322, 1698, 1507, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 1 and 2; HRESIMS m/z 605.3472 [M − H]− (calcd for C37H49O7, 605.3468). 3-O-p-Hydroxybenzoylepiceanothic acid (10): pinkish, amorphous powder; [α]20 D +20.0 (c 0.10, MeOH); UV λmax (log ε) 255 (3.67) nm; IR νmax 3649, 2971, 2864, 2317, 1684, 1507, 1055, 1033, 1012 cm−1; 1 H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 605.3472 [M − H]− (calcd for C37H51O7, 605.3468). 2-O-Protocatechuoylisoceanothanolic acid (11): white, amorphous powder; [α]20 D +34.9 (c 0.10, MeOH); UV λmax (log ε) 298 (3.74), 262 (3.91) nm; IR νmax 3649, 2966, 2865, 2321, 1684, 1507, 1055, 1032, 1012 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 607.3639 [M − H]− (calcd for C37H51O7, 607.3635). 3-Dehydroxyceanothan-27α-carboxy-28β,19β-olide (12): white, amorphous powder; mp 246−248 °C; [α]20 D +87.2 (c 0.10, MeOH); IR νmax 2949, 2876, 2361, 2307, 1780, 1708, 1456, 1223, 1179, 1032 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 497.2897 [M − H]− (calcd for C30H41O6, 497.2903). 3-O-Protocatechuoylceanothan-28β,19β-olide (13): white, amorphous powder; [α]20 D −1.4 (c 0.10, MeOH); UV λmax (log ε) 297 (3.91), 262 (4.12) nm; IR νmax 3853, 2972, 2866, 2844, 2310, 1698, 1507, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 619.3275 [M − H]− (calcd for C37H47O8, 619.3271). 2,28-Dinor-24-hydroxylup-1,17(22)-dien-27-oic acid (14): colorless solid; mp 116−120 °C; [α]20 D +0.61 (c 0.10, MeOH); IR νmax 3393, 2938, 2867, 2322, 1689, 1455, 1054, 1033, 1014 cm−1; 1H and 13 C NMR, see Tables 2 and 3; HRESIMS m/z 423.2901 [M − H]− (calcd for C28H39O3, 423.2889). 7β-O-Vanilloyl-3-dehydroxyceanothetric acid 2-methyl ester (15): white, amorphous powder; [α]20 D +79.9 (c 0.10, MeOH); UV λmax (log ε) 289 (3.91), 261 (4.30) nm; IR νmax 3649, 2972, 2866, 2360, 2332, 1684, 1507, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 679.3474 [M − H]− (calcd for C39H51O10, 679.3482). 2-O-Vanilloylalphitolic acid (16): white, amorphous powder; [α]20 D +29.5 (c 0.10, MeOH); UV λmax (log ε) 292 (3.61), 262 (3.80) nm; IR νmax 3811, 2939, 2866, 2360, 1682, 1054, 1032, 1013 cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 621.3787 [M − H]− (calcd for C38H53O7, 621.3791). 2-O-p-Hydroxybenzoylalphitolic acid (17): white, amorphous powder; [α]20 D −16.5 (c 0.10, MeOH); UV λmax (log ε) 257 (4.12) nm; IR νmax 3649, 2939, 2865, 2360, 2326, 1684, 1055, 1033, 1013

cm−1; 1H and 13C NMR, see Tables 2 and 3; HRESIMS m/z 591.3678 [M − H]− (calcd for C37H51O6, 591.3686). 3-O-Protocatechuoylalphitolic acid (18): white, amorphous powder; [α]20 D +19.8 (c 0.10, MeOH); UV λmax (log ε) 296 (3.63), 256 (3.86) nm; IR νmax 3631, 2971, 2864, 2360, 1510, 1055, 1033, 1013 cm−1; 1H and 13C NMR, see Tables 2 and 3; ESI-qTOF-MS m/z 607.3630 [M − H]− (calcd for C37H51O7, 607.3635). Cytotoxicity Assay Using HepG2 Cells. HepG2 cells (human hepatocellular carcinoma cell line; ATCC HB-8065) were purchased from the Korean Cell Line Bank (KCLB, Korea). HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone), 100 U/mL penicillin (Sigma-Aldrich, St. Louis, MO, USA), and 100 μg/ mL streptomycin (Sigma-Aldrich) in a humidified atmosphere environment with 5% CO2 at 37 °C. Each cytotoxicity assay was conducted according to the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5diphenyl-2H-tetrazolium bromide] method in 96-well microplates.33 Briefly, 200 μL of adherent cells was seeded into 96-well microculture plates and allowed to adhere for 12 h with an initial density of 5 × 104 cells/mL in 200 μL of medium. HepG2 cells were then exposed to the compounds dissolved in DMSO at four concentrations, 1, 5, 10, and 25 μM, with docetaxel (Korea United Pharm Inc., Seoul, Korea) as a positive control. After incubation for 48 h under growth conditions, MTT (Sigma-Aldrich) was added, and the incubation of cells continued for another 4 h. After removing the supernatant, DMSO (100 μL) was added to solubilize the formazan crystals. Consequently, the absorbance was measured at 550 nm. The percentage cell viability is the absorbance in the experiment well compared to that in the control wells, and compound toxicity is the percentage cell viability. IC50 and 95% confidence interval were calculated using the SigmaPlot Statistical Analysis software (Systat Software Inc., San Jose, CA, USA), based on regression analysis between the logarithmic concentration versus the percentage cell viability. The experiments were performed for three independent replicates.

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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.6b00525. Description of LC-MS analysis for determining if methyl ester compounds 2−5, 8, and 15 are actual natural products; spectroscopic data of known compounds 19− 30 including fully assigned 1 H and 13 C NMR spectroscopic data; raw 1D and 2D NMR data of compounds 1−18 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel (S. H. Sung): (+82)-2-880-7859. E-mail: [email protected]. kr. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), which was funded by the Ministry of Science, ICT and Future Planning (NRF-2015M3A9A5030733). We would like to thank Mr. B. G. Jeong and Prof. E. J. Jeong (Gyeongnam National University of Science and Technology, JinJu, Republic of Korea) for kindly providing the plant material. We also would like to thank Dr. Y.-J. Ko of the National Centre for Interuniversity Research Facilities for her efforts and kindness in the NMR experiments. K



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Article

(32) Abdel Bar, F. M.; Zaghloul, A. M.; Bachawal, S. V.; Sylvester, P. W.; Ahmad, K. F.; El Sayed, K. A. J. Nat. Prod. 2008, 71, 1787−1790. (33) Mosmann, T. J. Immunol. Methods 1983, 65, 55−63.

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