Diterpenoids and Sesquiterpenoids from the Roots of Illicium majus

Article pubs.acs.org/jnp

Diterpenoids and Sesquiterpenoids from the Roots of Illicium majus Ya-Dan Wang,† Gui-Jie Zhang,† Jing Qu,† Yu-Huan Li,‡ Jian-Dong Jiang,†,‡ Yun-Bao Liu,† Shuang-Gang Ma,† Yong Li,† Hai-Ning Lv,† and Shi-Shan Yu*,† †

State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China ‡ Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China S Supporting Information *

ABSTRACT: Five new diterpenoids (1−5), five new sesquiterpenoids (6−10), and three known compounds (11−13) were isolated from the roots of Illicium majus. Their structures were elucidated by extensive spectroscopic analysis. The absolute configuration of 1 was assigned by X-ray crystallography, whereas those of the 1,2-diol moieties in 3 and 4 were determined using Snatzke’s method. The abietane acids 1, 2, 11, 12, and 13 displayed antiviral activity against the Coxsackie B3 virus, with IC50 values of 3.3−51.7 μM/mL. Illicium is a genus of flowering plants that contains 42 species and is the sole genus in the family Illiciaceae.1 Most of the plants in the genus are distributed throughout the southern parts of China and are considered toxic. 2 Chemical investigations of members of this genus have resulted in the isolation of a series of prenylated C6−C3 compounds with cytotoxic, anti-inflammatory, and antioxidant activities,3 neoligans with anti-inflammatory and antioxidant activities,4 diterpenes with potent antiviral activity toward the Coxsackie B virus,5 sesquiterpene lactones that inhibit the release of βglucuronidase and promote neurite outgrowth,6 and phenylpropanoids that significantly attenuate SH-SY5Y cell damage induced by H2O2.4b,7 Thus, the Illicium species have been regarded as an attractive plant source for chemically and biologically intriguing secondary metabolites.8 Illicium majus Hook et Thoms (Illiciaceae), a poisonous arbor, is used in folk medicine for treating rheumatoid arthritis.9 Although many plants of the genus Illicium have been extensively investigated in recent years, studies of this specific species have been limited.10 Our previous studies on the twigs and leaves of I. majus have led to the isolation of four new abietane diterpenes, majusanic acids A−D, as well as two new sesquiterpenes, majusanols A and B. Among them, majusanic acid B showed potent activity against the release of βglucuronidase in rat polymorphonuclear leukocytes induced by platelet-activating factor in vitro, with an IC50 value of 0.26 ± 0.03 μM.11 Ethanol extracts of roots of I. majus have now been subjected to chemical analysis. New diterpenoids, majusanic acids E (1) and F (2) and majusanins A−C (3− 5), sesquiterpenoids, majusanside (6), majusanols C−E (7−9), and majusanol E-13-O-β-D-glucopyranoside (10), and the known compounds 4-epi-dehydroabietic acid (11), majusanic acid B (12), and majusanic acid D (13) were isolated. The abietane diterpenoids (1−5, 11−13) exhibit structural © 2013 American Chemical Society and American Society of Pharmacognosy

characteristics similar to those of reported diterpenoids isolated from Illicium jiadifengpi, which showed significant antiviral activity against Coxsackie virus.5 Therefore, in order to further elucidate their structure−activity relationships, the antiviral activity of these compounds against Coxsackie B3 virus was studied. In this paper, we describe the isolation and structure elucidation of these new compounds and their activity against Coxsackie B3 virus.

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RESULTS AND DISCUSSION Majusanic acid E (1) was assigned the molecular formula C20H28O3 (seven degrees of unsaturation) based on HRESIMS (m/z 339.1931 [M + Na]+, calcd 339.1931) and NMR data (Tables 1 and 2). The 1H NMR spectrum showed resonances Received: August 8, 2013 Published: September 26, 2013 1976

dx.doi.org/10.1021/np400638r | J. Nat. Prod. 2013, 76, 1976−1983

Journal of Natural Products

Article

for three methyl groups at δH 1.16 (d, J = 7.0 Hz, H3-17), 1.23 (s, H3-18), and 1.06 (s, H3-20); one oxygenated methylene group at δH 3.56 (dd, J = 11.0, 7.0 Hz, H-16a) and 3.46 (dd, J = 11.0, 7.0 Hz, H-16b); and three aromatic protons at δH 7.13 (d, J = 8.0 Hz, H-11), 6.89 (br d, J = 8.0 Hz, H-12), and 6.81 (1H, br s, H-14). The 13C NMR spectrum indicated the presence of a carboxyl carbon (δC 181.8), six aromatic carbons (δC 126.2, 126.9, 129.2, 136.6, 142.8, 147.7), an oxygenated methylene carbon (δC 69.5), and three methyl carbons (δC 18.7, 24.0, 29.6). Analysis of the 1H and 13C NMR data for compound 1 revealed structural features similar to those of 4-epidehydroabietic acid (11),12 a known compound also isolated from the crude extract, with the exception that the C-16 methyl group (δH/δC 1.24/24.2) in 11 was replaced by an oxygenated methylene (δH/δC 3.56, 3.46/69.5) in 1, as confirmed by HMBC correlations from H2-16 to C-13, C-15, and C-17 (Figure S1, Supporting Information). The relative configuration of 1 was determined by analysis of the NOE data (Figure S1). Upon irradiation of H3-18 in the NOE experiment, enhancements were observed for H-3α (δH 1.02), H-5, and H-6α (δH 2.13), suggesting that these protons were located on the same side of the molecule. Upon irradiation of H3-20, enhancements for H-2β (δH 1.98) and H-6β (δH 1.97) were observed, indicating their spatial proximity. Therefore, the relative configuration of 1 is consistent with that of 11.12 Finally, the absolute configuration of 1 was deduced to be 4S, 5R, 10S, 15S by single-crystal X-ray diffraction analysis based on the anomalous dispersion of Cu Kα radiation (Figure 1). Thus, compound 1 was determined to be (15S)-16-hydroxyabieta-8,11,13-trien-19-oic acid, and it was named majusanic acid E. Compound 2 was assigned the same molecular formula, C20H28O3, as 1 by HRESIMS and NMR data (Tables 1 and 2). The 1D and 2D NMR spectra for 2 were nearly identical to those for 1, revealing that they possessed the same planar structure. On the basis of the specific rotation values for 1 20 ([α]20 D +112.1) and 2 ([α]D +134.6), it was suggested that 1

Table 1. 1H NMR Data (500 MHz, CD3OD) for 1−5 δH (J in Hz) no.

1

2

3

4

5

1α

1.29, dd (13.0, 3.5) 2.24, br d (13.0) 1.52, m 1.98, m 1.02, dd (13.5, 4.0) 2.17, br d (13.5) 1.47, dd (12.0, 1.5) 2.13, m

1.28, dd (13.0, 3.5) 2.24, br d (13.0) 1.52, m 1.98, m 1.02, dd (13.5, 4.0) 2.17, br d (13.5) 1.47, br d (12.5) 2.13, m



1.97, m 2.80, dd (17.0, 4.5) 2.71, m 7.13, d (8.0) 6.89, br d (8.0) 6.81, br s 2.72, m 3.56, dd (11.0, 7.0) 3.46, dd (11.0, 7.0) 1.16, d (7.0, 3H) 1.23, s (3H)

1.97, m 2.80, dd (17.0, 4.5) 2.71, m 7.13, d (8.0) 6.89, br d (8.0) 6.81, br s 2.72, m 3.56, dd (10.5, 7.0) 3.46, dd (10.5, 7.0) 1.16, d (7.0, 3H) 1.23, s (3H)

1.65, m 2.85, dd (17.0, 6.0) 2.75, m 7.15, d (8.5) 7.11, br d (8.5) 7.05, br s

1.65, m 2.86, dd (17.0, 6.0) 2.75, m 7.15, d (8.0) 7.11, dd (8.5, 1.5) 7.05, br s

1.51, dd (13.0, 3.5) 2.39, br d (13.0) 1.62, m 1.75, m 1.00, dd (14.0, 4.0) 1.86, br d (14.0) 1.92, dd (13.0, 5.0) 2.73, dd (18.0, 13.0) 2.75, overlap

3.51, d (11.0) 3.48, d (11.0) 1.40, s (3H)

3.51, d (11.0) 3.48, d (11.0) 1.40, s (3H)

1.46, s (3H)

1.06, s (3H)

1.06, s (3H)

0.97, s (3H) 3.77, d (11.0) 3.37, d (11.0) 1.10, s (3H)

0.97, s (3H) 3.77, d (11.0) 3.37, d (11.0) 1.10, s (3H)

0.97, s 3.77, d (11.0) 3.55, d (11.0) 1.21, s (3H)

1β 2α 2β 3α 3β 5 6α

6β 7α 7β 11 12 14 15 16

17 18 19

20

7.38, d (8.5) 7.66, dd (8.5, 2.0) 8.00, d (2.0) 1.46, s (3H)

Table 2. 13C NMR Data (125 MHz, CD3OD) for 1−5 δC, mult. no.

1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

41.1, 21.5, 39.1, 45.2, 54.7, 22.6, 33.5, 136.6, 147.7, 39.8, 126.9, 126.2, 142.8, 129.2, 43.6, 69.5, 18.7, 29.6, 181.8, 24.0,

2 CH2 CH2 CH2 C CH CH2 CH2 C C C CH CH C CH CH CH2 CH3 CH3 C CH3

41.1, 21.5, 39.1, 45.2, 54.7, 22.7, 33.5, 136.6, 147.7, 39.8, 126.9, 126.2, 142.8, 129.2, 43.6, 69.5, 18.7, 29.9, 181.8, 24.0,

3 CH2 CH2 CH2 C CH CH2 CH2 C C C CH CH C CH CH CH2 CH3 CH3 C CH3

40.6, 20.3, 36.7, 40.2, 53.3, 20.6, 32.6, 135.8, 149.8, 39.0, 125.5, 124.3, 144.3, 127.2, 75.6, 72.2, 26.3, 27.9, 65.3, 26.5, 1977

4 CH2 CH2 CH2 C CH CH2 CH2 C C C CH CH C CH C CH2 CH3 CH3 CH2 CH3

40.6, 20.4, 36.7, 40.2, 53.3, 20.6, 32.6, 135.8, 149.8, 39.0, 125.5, 124.3, 144.3, 127.2, 75.7, 72.1, 26.3, 27.9, 65.3, 26.6,

5 CH2 CH2 CH2 C CH CH2 CH2 C C C CH CH C CH C CH2 CH3 CH3 CH2 CH3

39.7, 19.9, 36.8, 39.7, 51.7, 37.5, 201.9, 131.4, 156.4, 39.5, 125.5, 132.4, 149.4, 124.3, 72.9, 32.0, 32.0, 27.4, 65.4, 24.5,

CH2 CH2 CH2 C CH CH2 C C C C CH CH C CH C CH3 CH3 CH3 CH2 CH3



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Figure 1. X-ray crystal structure of compound 1.

and 2 could be diastereomers. In the NOE spectrum of 2, irradiation of H3-18 enhanced H-3α (δH 1.02), H-5, and H-6α (δH 2.13), while irradiation of H3-20 enhanced H-2β (δH 1.98) and H-6β (δH 1.97). These observations indicated that 2 adopted the same relative configuration as 1 for all of the stereocenters with the exception of C-15, whose relative configuration was inconclusive, suggesting that 2 is the 15epimer of 1. Therefore, compound 2 was determined to be (15R)-16-hydroxyabieta-8,11,13-trien-19-oic acid. Majusanins A (3) and B (4) gave pseudomolecular ions [M + Na]+ at m/z 341.2087 and 341.2090 (calcd 341.2087) by HRESIMS, respectively, consistent with the molecular formula C20H30O3 (six degrees of unsaturation). Interpretation of their 1D and 2D NMR spectroscopic data (Tables 1 and 2) established the same planar structure, which resembled that of majusnic acid B (12),11 with the exception that the carboxyl group (δC 180.3) in 12 was replaced by oxygenated methylene units (δH/δC 3.77, 3.37/65.3) in 3 and 4. This deduction was confirmed by HMBC correlations from H2-19 to C-3, C-5, and C-18. The relative configurations of 3 and 4 were determined to be the same as 12, although NOE experiments were unable to conclusively determine the configuration at C-15. The aforementioned data combined with the specific rotation values 20 of 3 ([α]20 D +81.3) and 4 ([α]D +58.4) indicated that they could be 15-epimers, which was supported by the in situ dimolybdenum CD method developed by Snatzke and Frelek.13 A metal complex of the 1,2-diol moiety and Mo2(OAc)4 was generated as an auxiliary chromophore after the addition of Mo2(OAc)4 to a DMSO solution of 3 or 4. The indicator of the Cotton effect at approximately 300 nm in the induced spectrum originates from the chirality of the vic-diol expressed by the O− C−C−O torsion angle. A positive Cotton effect observed at 316 nm in the induced CD spectrum of 3 permitted the assignment of the S configuration at C-15 based on the empirical rule, with the bulkier group pointed away from the remaining portion of the complex. A negative Cotton effect at 316 nm for 4 indicated that the absolute configuration at C-15 was opposite that of 3 (Figures 2 and 3). Hence, compounds 3 and 4 were deduced to be (15S)-15,16-dihydroxyabieta8,11,13-trien-19-ol and (15R)-15,16-dihydroxyabieta-8,11,13trien-19-ol, respectively. Majusanin C (5) was established as C20H28O3 (seven degrees of unsaturation) based on HRESIMS (m/z 339.1933 [M + Na]+, calcd 339.1931) and NMR data (Tables 1 and 2). The 1H and 13C NMR spectra of 5 revealed structural features nearly identical to those found in majusanic acid D (13),11 with the exception of an oxygenated methylene unit (δH/δC 3.77, 3.55/ 65.4) at C-19 in 5 rather than a carboxyl group, which was confirmed by HMBC correlations from H2-19 to C-3, C-5, and C-18. Compound 5 was determined to have the same relative configuration as 13 by analysis of the NOE data. Thus,

Figure 2. CD spectra of 3 and 4 in DMSO containing Mo2(OAc)4 with the inherent CD spectra subtracted.

Figure 3. Conformations of the Mo4+ complexes of 3 and 4.

compound 5 was determined to be 15-hydroxy-7-oxoabieta8,11,13-trien-19-ol. Majusanside (6) was determined to be C21H28O11 (eight degrees of unsaturation) based on HRESIMS and NMR data (Tables 3 and 4). The 1H NMR spectrum exhibited resonances for two methyl groups at δH 0.97 (s, H3-8) and 1.14 (s, H3-13); four methylene groups at δH [2.58 (dd, J = 19.0, 2.0 Hz, H-3α), 2.43 (dd, J = 19.0, 2.0 Hz, H-3β)], [3.29 (d, J = 19.0 Hz, H10a), 2.54 (d, J = 19.0 Hz, H-10b)], [3.99 (d, J = 10.0 Hz, H14a), 3.97 (d, J = 10.0 Hz, H-14b)], and [4.42 (dd, J = 13.5, 1.5 Hz, H-15a), 4.21 (dd, J = 13.5, 1.5 Hz, H-15b)]; an oxymethine at δH 4.32 (s, H-7); and a trisubstituted olefinic proton at δH 5.68 (m, H-2). The 13C NMR spectrum showed 21 carbons, including two γ-lactone carbonyls (δC 181.5, 178.9), two olefinic carbons (δC 145.7, 128.2), four oxygenated carbons (δC 103.4, 78.3, 74.1, 67.0), and two methyl carbons (δC 16.5, 10.8). In addition, the 1H and 13C NMR resonances observed in the spectra of 6 were indicative of a glucose moiety.14 The data suggested that 6 was a sesquiterpene glucoside. The 1H and 13C NMR spectra of the aglycone revealed structural fragments similar to those found in merrilactone B,15 with the exceptions that an oxymethine at C-1 and a methylene at C-2 in merrilactone B were replaced by an olefin moiety in 6. In addition, an oxygenated methylene was present at C-15 in 6 in place of the methyl group found in merrilactone B. A key HMBC correlation from the anomeric proton of glucose (H-1′; δH 4.26) to the oxygenated methylene carbon (C-15; δC 67.0) indicated that the glucose was attached at C-15 (Figure S2). The relative configuration of 6 was proposed based on NOE data (Figure S3). Upon irradiation of H-7, an enhancement of H2-15 was observed, suggesting that the OH at C-7 adopted an α-orientation.15 Irradiation of H3-8 enhanced H3-13 and H-10a (δH 3.29), indicating that these protons were cofacial. 1978



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monosubstituted benzene ring at δH [8.02 (2H, dd, J = 7.5, 1.5 Hz, H-3′, 7′), 7.49 (2H, t, J = 7.5 Hz, H-4′, 6′), and 7.62 (1H, t, J = 7.5 Hz, H-5′)]. The 13C NMR spectrum (Table 4) indicated the presence of two γ-lactone carbonyls (δC 181.0, 177.9); a benzoyl moiety (δC 166.2, 135.4, 131.1, 131.1, 130.4, 130.3, 130.3); four oxygenated carbons (δC 97.7, 89.6, 75.0, 74.0); and three methyl carbons (δC 20.9, 17.3, 11.8). Analysis of the 1H, 13C, and 2D NMR spectra of 7 revealed that 7 was a benzoate of the known sesquiterpene anislactone A,17 and the HMBC correlation from H-7 to C-1′ indicated that the benzoyl unit was connected to the sesquiterpene moiety at C-7 (Figure S2). The relative configuration of 7 was determined to be identical to that of anislactone A based on analysis of the NOE data (Figure S3). Irradiation of H-7 enhanced H3-15 and H-14a (δH 4.47), indicating that these protons were on the same face of ring B. Comparatively, H-10b (δH 3.02), H-14b (δH 3.97), and H3-13 were enhanced upon irradiation of H3-8, indicating that these protons were located on the opposite face of the ring.17 Therefore, 7 was determined to be 7-O-benzoylanislactone A. The elemental composition of majusanol D (8) was established as C22H28O7 (nine degrees of unsaturation) by HRESIMS. The 1H NMR data (Table 3) revealed four methyl groups at δH 0.92 (s, H3-13), 1.00 (s, H3-12), 1.13 (d, J = 7.5 Hz, H3-15), and 1.92 (t, J = 2.0 Hz, H3-7′); two isolated AB methylene groups at δH [2.47 (d, J = 17.5 Hz, H-8α), 2.22 (d, J = 17.5 Hz, H-8β)] and [4.05 (d, J = 8.5 Hz, H-14a), 3.77 (d, J = 8.5 Hz, H-14b)]; and an oxymethine at δH 4.13 (s, H-10). The 13 C NMR spectrum of 8 (Table 4) showed 22 carbons, including three carbonyl carbons (δC 213.5, 211.7, 165.9); two olefinic carbons (δC 155.3, 149.7); a ketal carbon (δC 113.8); two oxygenated carbons (δC 86.0, 76.6); and four methyl carbons (δC 14.7, 14.2, 10.4, 10.3). The aforementioned data indicated a structural similarity to tashironin A (11-Odebenzoyl-11α-O-2-methylcyclopent-1-enecarboxyltashironin),18 except that a methylene group at C-4′ (δH/δC 2.47/ 41.0) in tashironin A was replaced by a carbonyl group (δC 211.7) in 8. This structural difference was confirmed by the HMBC cross-peak of H3-7′ with C-4′ (Figure S2). In the NOE spectrum of 8 (Figure S3), irradiation of H-10 enhanced H3-15 and H-8β (δH 2.22), while irradiation of H3-12 enhanced H3-13 and H3-7′. The data suggested that 8 had the same relative configuration as tashironin A.18 Accordingly, compound 8 was deduced to be 4′-oxo-tashironin A. Majusanol E (9) gave a pseudomolecular ion [M + Na]+ at m/z 337.1257, consistent with the molecular formula C15H22O7 (five degrees of unsaturation). The 1H NMR spectrum (Table 3) revealed the presence of two methyl groups at δH 0.94 (d, J = 7.0 Hz, H3-15) and 1.29 (s, H3-12); three isolated AB methylene groups at δH [1.60 (d, J = 13.5 Hz, H-8α), 1.50 (d, J = 13.5 Hz, H-8β)], [3.84 (d, J = 11.0 Hz, H-13a), 3.20 (d, J = 11.0 Hz, H-13b)], and [5.04 (d, J = 13.5 Hz, H-14a), 4.38 (d, J = 13.5 Hz, H-14b)]; and an oxymethine at δH 4.43 (s, H-10). The 13C NMR spectrum of 9 (Table 4) revealed signals of a carbonyl carbon (δC 175.5); a hemiketal carbon (δC 110.1); five oxygenated carbons (δC 95.4, 81.5, 76.0, 67.6, 64.6); and two methyl carbons (δC 21.0, 13.8). The data above suggested that 9 was an analogue of the pseudoanistin-type sesquiterpene cycloparviflorolide.19 The major difference between that compound and 9 was that the methyl group (δH/δC 0.94/ 17.7) at C-13 in cycloparviflorolide was replaced by a hydroxymethyl moiety (δH/δC 3.84, 3.20/64.6) in 9, which

Table 3. 1H NMR Data (500 MHz, CD3OD) for 6−10 δH (J in Hz) no. 1 2α 2β 3α 3β 7 8α

6 5.68, m

7

9

10

2.23, dd (13.0, 8.0) 1.99, m 2.02, m

2.22, m 2.01, m

2.42, m 1.88, m

2.43, m 1.89, m

1.58, m 2.32, m

1.75, m

1.59, m

1.54, m 1.96, dt (13.0, 4.5) 1.69, m

1.54, m 2.00, dt (13.0, 4.5) 1.65, m

2.47, d (17.5) 2.22, d (17.5) 4.13, s

1.60, d (13.5) 1.50, d (13.5) 4.43, s

1.60, d (14.0) 1.50, d (14.0) 4.44, s

1.00, s (3H) 0.92, s (3H)

1.29, s (3H)

1.32, s (3H)

3.84, d (11.0) 3.20, d (11.0) 5.04, d (13.5) 4.38, d (13.5) 0.94, d (7.0, 3H)

3.75, d (9.5)

2.58, dd (19.0, 2.0) 2.43, dd (19.0, 2.0) 4.32, s 0.97, s (3H)

5.65, s 1.07, s (3H)

3.29, d (19.0) 2.54, d (19.0)

3.05, d (17.5) 3.02, d (17.5)

8β 10a 10b 12 13

14a

1.14, s (3H)

3′

3.99, d (10.0) 3.97, d (10.0) 4.42, dd (13.5, 1.5) 4.21, dd (13.5, 1.5) 4.26, d (8.0) 3.13, dd (9.0, 8.0) 3.29, t (9.0)

4′ 5′

3.21, overlap 3.20, overlap

6′

3.82, dd (11.5, 1.5) 3.59, dd (11.5, 5.5)

14b 15

1′ 2′

7′

1.12, s (3H)

4.47, d (10.0) 3.97, d (10.0) 1.30, s (3H)

8.02, dd (7.5, 1.5) 7.49, t (7.5) 7.62, t (7.5)

8

4.05, d (8.5) 3.77, d (8.5) 1.13, d (7.5, 3H)

3.72, d (9.5) 5.07, d (13.5) 4.45, d (13.5) 0.94, d (7.0, 3H)

4.20, d (8.0) 3.11, dd (9.0, 8.0) 3.28, t (9.0)

7.49, t (7.5)

2.40, t (4.5) 2.66, m

8.02, dd (7.5, 1.5)

1.92, t (2.0, 3H)

3.21, overlap 3.20, m 3.81, dd (12.0, 1.5) 3.60, dd (12.0, 5.5)

Meanwhile, H-14a (δH 3.99) was enhanced upon irradiation of H-3α (δH 2.58), indicating their spatial proximity. The glucose moiety was connected to the sesquiterpene aglycone via a β-linkage based on the 8.0 Hz coupling constant observed for the anomeric proton. Upon the acid hydrolysis of 6 with 2 N HCl, the liberated sugar was treated with L-cysteine methyl ester followed by trimethylsilylation. This treatment afforded a derivative matching that of D-glucose by GC analysis when compared with the standard D-glucose derivative.16 Thus, compound 6 was determined to be 7α-hydroxyl-Δ 1,2merrilactone B-15-O-β-D-glucopyranoside. Majusanol C (7) was assigned the molecular formula C22H24O7 (11 degrees of unsaturation). The 1H NMR spectrum of 7 (Table 3) displayed resonances that were attributed to three methyl groups at δH 1.07 (s, H3-8), 1.12 (s, H3-13), and 1.30 (s, H3-15); two isolated AB methylene groups at δH [3.05 (d, J = 17.5 Hz, H-10a), 3.02 (d, J = 17.5 Hz, H10b)] and [4.47 (d, J = 10.0 Hz, H-14a), 3.97 (d, J = 10.0 Hz, H-14b)]; an oxymethine at δH 5.65 (s, H-7); and a 1979



Article

Table 4. 13C NMR Data (125 MHz, CD3OD) for 6−10 δC, mult. no.

6

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

145.7, C 128.2, CH 40.8, CH2 103.4, C 56.5, C 58.8, C 78.3, CH 10.8, CH3 69.6, C 35.4, CH2 178.9, C 181.5, C 16.5, CH3 74.1, CH2 67.0, CH2 103.9, CH 75.3, CH 78.44, CH 71.9, CH 78.38, CH 63.0, CH2

7 97.7, 38.3, 36.9, 89.6, 57.1, 57.6, 74.0, 11.8, 68.2, 33.5, 177.9, 181.0, 17.3, 75.0, 20.9, 166.2, 130.4, 131.1, 130.3, 135.4, 130.3, 131.1,

8 C CH2 CH2 C C C CH CH3 C CH2 C C CH3 CH2 CH3 C C CH CH CH CH CH

40.1, 32.6, 33.9, 86.0, 57.2, 62.2, 213.5, 45.3, 54.5, 76.6, 113.8, 10.4, 14.7, 74.5, 14.2, 165.9, 149.7, 155.3, 211.7, 35.0, 27.6, 10.3,

9 CH CH2 CH2 C C C C CH2 C CH C CH3 CH3 CH2 CH3 C C C C CH2 CH2 CH3

46.1, 33.4, 27.1, 95.4, 56.6, 81.5, 110.1, 39.5, 56.6, 76.0, 175.5, 21.0, 64.6, 67.6, 13.8,

10 CH CH2 CH2 C C C C CH2 C CH C CH3 CH2 CH2 CH3

46.1, 33.4, 27.3, 95.1, 56.7, 80.7, 110.3, 39.0, 56.6, 75.9, 175.4, 21.1, 72.6, 67.5, 13.8, 105.4, 75.2, 78.4, 71.8, 78.5, 63.0,

CH CH2 CH2 C C C C CH2 C CH C CH3 CH2 CH2 CH3 CH CH CH CH CH CH2

In our previous studies using the roots of I. jiadifengpi, an array of abietane diterpenes with significant antiviral activity against Coxsackie virus were reported as well as their structure−activity relationships (SAR), which showed that an acetyl substituent at C-13 and an OH at C-7 or C-17 resulted in increased antiviral activity and decreased cytotoxity.5 On the basis of those studies, compounds 1−5 were tested for activity against the Coxsackie B3 virus in Vero cells by a cytopathogenic effect assay to supplement the SAR of the abietane diterpenes. As shown in Table 5, the diterpenes with carboxyl groups at C-

was supported by HMBC correlations from H2-13 to C-4, C-5, C-6, and C-14 (Figure S2). Compound 9 was assigned the same relative configuration as cycloparviflorolide based on analysis of the NOE data (Figure S3). Irradiation of H-10 enhanced H3-15 and H-8β (δH 1.50), while H-8β and H-14b (δ 4.38) were enhanced upon irradiation of H3-12, indicating that these protons were cofacial. Irradiation of H-8α (δH 1.60) enhanced H-1, which revealed that these two protons were also cofacial. Furthermore, upon irradiation of H-13a (δH 3.84), an enhancement of H-3β (δH 1.69) was observed indicating spatial proximity.19 Thus, it was concluded that 9 was 13-hydroxycycloparviflorolide. The molecular formula of compound 10 was determined to be C21H32O12 (six degrees of unsaturation). The 1H, 13C, and 2D NMR data (Tables 3 and 4) revealed structural features nearly identical to those found in 9, with the exception of the appearance of an additional glucose moiety. Furthermore, 10 differed from 9 in the chemical shift of C-13 (δH/δC 3.84, 3.20/ 64.6 in 9; 3.75, 3.72/72.6 in 10), indicating that the glucose unit was located at C-13 in 10. This assignment was confirmed by HMBC cross-peaks of H-1′ with C-13 and H2-13 with C-1′. The anomeric relative configuration of the glucose was deduced to be β based on the coupling constant of 8.0 Hz observed for H-1′. The sugar was determined to be D-glucose after acid hydrolysis, preparation of a chiral derivative, and GC analysis following the procedure described above for compound 6. Therefore, 10 was proposed to be majusanol E-13-O-β-Dglucopyranoside. The known compounds 11−13 isolated from the crude extract were identified as 4-epi-dehydroabietic acid (11),12 majusanic acid B (12),11 and majusanic acid D (13),11 respectively, by comparison of their NMR and MS data with reported values. The absolute configuration of C-15 in 12 was assigned by the in situ dimolybdenum CD method as described for compounds 3 and 4.

Table 5. Antiviral Activities of 1, 2, 6, 8, 9, and 11−13a against Coxsackie B3Virus in Vero Cells no.

TC50 (μM/mL)

IC50 (μM/mL)

SIb

1 2 6 8 9 11 12 13 RBVc

138.7 66.7 200.0 200.0 200.0 22.2 138.7 200.0 8.1

17.4 12.8 51.7 66.7 66.7 3.3 22.2 51.7 0.9

8.0 5.2 3.9 3.0 3.0 6.7 6.2 3.9 9.0

a

Compounds 3−5, 7, and 10 were inactive at their maximal nontoxic concentration. bSelectivity index value equaled TC50/IC50. cRibavirin (positive control).

19 (1, 2, 11, 12, and 13) displayed antiviral effects, with IC50 values of 3.3−51.7 μM/mL and SI values of 3.9−8.0 (the positive control ribavirin had an IC50 of 0.9 μM/mL and a SI of 9.0). Their C-19 alcohol analogues (3, 4, and 5) were inactive at their maximal nontoxic concentrations, suggesting that the carboxyl group at C-19 plays a significant role in the antiviral activity. The sesquiterpenoids 6−10 were also tested against Coxsackie B3 virus, but only 6, 8, and 9 exhibited weak antiviral 1980



Article

effects, with IC50 values of 51.7, 66.7, and 66.7 μM/mL, respectively.

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E1-3 (30 g, eluted with 30% EtOH) was purified by ODS CC eluting with MeOH/H2O (5:95 to 70:30) to yield 10 fractions (E1-3-1−E1-310). Fraction E1-3-3 (2.5 g) was separated on a Sephadex LH-20 column using MeOH/H2O (1:1) to give four subfractions (E1-3-3-1− E1-3-3-4). Fraction E1-3-3-2 (0.5 g) was further separated by RPHPLC (CH3CN/H2O/TFA, 15:85:0.03) to afford 10 (70 mg), 6 (10 mg), and 9 (48 mg). Majusanic acid E (1): colorless needles (CH3OH/H2O, 1:1); mp 152−153 °C; [α]20 D +112.1 (c 0.067, MeOH); UV (MeOH) λmax (log ε) 203 (4.29) nm; IR (KBr) νmax 3386, 2961, 2931, 2672, 1693, 1612, 1498, 1468, 1377, 1265, 1235, 1193, 1026, 974, 822 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 1 and 2; HRESIMS m/z 339.1931 [M + Na]+ (calcd for C20H28O3Na, 339.1931). Majusanic acid F (2): colorless needles (CH3OH/H2O, 1:1); mp 179−180 °C; [α]20 D +134.6 (c 0.067, MeOH); UV (MeOH) λmax (log ε) 203 (4.21) nm; IR (KBr) νmax 3367, 2985, 2932, 1694, 1613, 1569, 1498, 1468, 1454, 1377, 1264, 1235, 1192, 1074, 1026, 974, 822 cm−1; 1 H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 1 and 2; HRESIMS m/z 339.1930 [M + Na]+ (calcd for C20H28O3Na, 339.1931). Majusanin A (3): white powder; [α]20 D +81.3 (c 0.067, MeOH); UV (MeOH) λmax (log ε) 202 (4.39) nm; IR (KBr) νmax 3357, 2966, 2927, 2870, 1782, 1673, 1497, 1473, 1458, 1376, 1188, 1149, 1041, 974, 896, 826 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 1 and 2; HRESIMS m/z 341.2087 [M + Na]+ (calcd for C20H30O3Na, 341.2087). Majusanin B (4): white powder; [α]20 D +58.4 (c 0.067, MeOH); UV (MeOH) λmax (log ε) 202 (4.40) nm; IR (KBr) νmax 3376, 2966, 2930, 2856, 1783, 1675, 1498, 1454, 1408, 1376, 1206, 1172, 1040, 974, 826 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 1 and 2; HRESIMS m/z 341.2090 [M + Na]+ (calcd for C20H30O3Na, 341.2087). Majusanin C (5): white powder; [α]20 D +23.0 (c 0.050, MeOH); UV (MeOH) λmax (log ε) 209 (4.67), 252 (4.27), 299 (3.52) nm; IR (KBr) νmax 3463, 3358, 3197, 2969, 2923, 2851, 1667, 1633, 1603, 1489, 1468, 1408, 1406, 1377, 1042, 960, 845 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD) see Tables 1 and 2; HRESIMS m/z 339.1933 [M + Na]+ (calcd for C20H28O3Na, 339.1931). Majusanside (6): white powder; [α]20 D −100.4 (c 0.067, MeOH); IR (KBr) νmax 3420, 2989, 2921, 2873, 1769, 1676, 1450, 1419, 1368, 1311, 1207, 1177, 1081, 1015, 971, 930, 819 cm−1; 1H (500 MHz) and 13 C NMR (125 MHz) data (CD3OD), see Tables 3 and 4; HRESIMS m/z 479.1539 [M + Na]+ (calcd for C21H28O11Na, 479.1524). Majusanol C (7): colorless crystals (CH3OH); mp 262−263 °C; [α]20 D −13.6 (c 0.067, MeOH); UV (MeOH) λmax (log ε) 231 (4.18) nm; IR (KBr) νmax 3392, 2989, 2941, 1767, 1732, 1602, 1485, 1450, 1414, 1391, 1345, 1267, 1207, 1159, 1106, 1090, 1070, 1011, 981, 952, 941, 714 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 3 and 4; HRESIMS m/z 423.1418 [M + Na]+ (calcd for C22H14O7Na, 423.1414). Majusanol D (8): colorless needles (CH3OH); mp 230−232 °C; [α]20 D −31.5 (c 0.067, MeOH); UV (MeOH) λmax (log ε) 247 (4.20) nm; IR (KBr) νmax 3566, 3450, 2976, 2939, 2870, 1716, 1689, 1636, 1500, 1450, 1388, 1238, 1135, 1106, 1051, 1027, 996, 968, 883 cm−1; 1 H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 3 and 4; HRESIMS m/z 427.1744 [M + Na]+ (calcd for C22H28O7Na, 427.1727). Majusanol E (9): colorless crystals (CH3OH/H2O, 1:1); mp 222− 223 °C; [α]20 D +68.2 (c 0.067, MeOH); IR (KBr) νmax 3369, 2951, 2874, 1784, 1715, 1469, 1412, 1308, 1269, 1184, 1157, 1105, 1037, 999, 963, 897, 881 cm−1; 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD), see Tables 3 and 4; HRESIMS m/z 337.1257 [M + Na]+ (calcd for C15H22O7Na, 337.1258). Majusanol E-13-O-β-D-glucopyranoside (10): white powder; [α]20 D +26.4 (c 0.067, MeOH); IR (KBr) νmax 3418, 2949, 2877, 1782, 1717, −1 1 1469, 1410, 1308, 1266, 1183, 1080, 1047, 962, 894, 881 cm ; H (500 MHz) and 13C NMR (125 MHz) data (CD3OD) see Tables 3

EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were measured on an XT5B (Beijing Keyi Electric Light Instrument Co., Ltd.) melting instrument and are uncorrected. Optical rotations were measured on a JASCO P-2000 automatic polarimeter, and the UV data were recorded on a JASCO V-650 spectrophotometer. CD spectra were obtained on a JASCO J-815 spectropolarimeter. IR spectra were obtained with a Nicolet 5700 FT-IR microscope spectrophotometer. NMR spectra were acquired with an INOVA-500 spectrometer using solvent signals (acetone-d6: δH 2.05/δC 29.8, 206.1; CD3OD: δH 3.25/ δC 49.3) as references. ESIMS data were recorded on an Agilent 1100 Series LC/MSD Trap/SL (Turbo Ionspray source) spectrometer. HRESIMS data were obtained on an Agilent 6520 Accurate-Mass-QTOF LC/MS spectrometer. Preparative HPLC was performed on a Shimadzu LC-6AD instrument with an SPD-20A detector or an RID detector using a YMC-Pack ODS-A column (250 × 50 mm, 5 μm). Silica gel (200−300 mesh, Qingdao Marine Chemical Inc., China), Sephadex LH-20 (Amersham Pharmacia Biotech AB, Sweden), ODS (50 μm, YMC, Japan), polyamide (30−60 mesh, Jiangsu Linjiang Chemical Reagents Factory, China), and macroporous resin (D101 type, The Chemical Plant of NanKai University, China) were used for column chromatography (CC). TLC was conducted with glass precoated with silica gel GF254 (Qingdao Marine Chemical Inc., China). Plant Material. Roots of I. majus were collected from Guangxi Province, China, in October 2009 and identified by Prof. Song-Ji Wei (Guangxi Traditional Medical College). A voucher specimen (ID21975) has been deposited in the Herbarium of the Department of Medicinal Plants, Institute of Materia Medica, Chinese Academy of Medical Sciences, People’s Republic of China. Extraction and Isolation. The air-dried and powdered roots of I. majus (11.8 kg) were extracted three times with 95% EtOH (40 L × 2 h), and the solvent was evaporated to dryness under vacuum to afford the crude extract (590 g), which was absorbed in Kieselguhr followed by successive extraction with petroleum ether, CH2Cl2, EtOAc, and MeOH. The CH2Cl2 extract (58 g) was subjected to silica gel CC (200−300 mesh, 1 kg) eluting with a petroleum ether/EtOAc gradient, yielding five fractions, C1−C5. Fraction C2 (4.6 g) was separated by chromatography on Sephadex LH-20 eluting with CH2Cl2 to yield six major fractions (C2-1−C2-6). Fraction C2-3 (0.30 g) was separated by RP-HPLC (CH3CN/H2O/ TFA, 85:15:0.03) to afford 11 (30 mg). Fraction C3 (10.4 g) was separated via an ODS column eluting with MeOH/H2O (20:80 to 95:5) to give 10 fractions (C3-1−C3-10). Fraction C3-3 (1.35 g) was purified by CC with a Sephadex LH-20 column using CH2Cl2/MeOH (1:1) as the eluent to obtain six subfractions (C3-3-1−C3-3-6). Fraction C3-3-2 (0.25 g) was separated by RP-HPLC (CH3CN/H2O/TFA, 40:60:0.03) to afford 13 (15 mg) and 5 (5 mg). Fraction C3-5 (0.85 g) was further separated by Sephadex LH-20 and RP-HPLC (CH3OH/H2O/TFA, 65:35:0.03) to afford 3 (5 mg), 4 (5 mg), 1 (7 mg), and 2 (8 mg). Fraction C4 (8.6 g) was separated by ODS CC eluting with MeOH/H2O (5:95 to 90:10) to yield nine fractions (C4-1−C4-9). Fraction C4-5 (1.0 g) was subsequently separated over Sephadex LH20 using MeOH/H2O (50:50) to afford five subfractions (C4-5-1−C45-5). Compound 8 (10 mg) was obtained from fraction C4-5-2 (0.12 g) by preparative HPLC (CH3CN/H2O/TFA, 40:60:0.03). Compound 7 (8 mg) was obtained from fraction C4-5-3 (0.20 g) by preparative HPLC (CH3CN/H2O/TFA, 37:63:0.03), and compound 12 (5 mg) was obtained from fraction C4-5-4 (0.15 g) by preparative HPLC (CH3OH/H2O/TFA, 50:50:0.03). The EtOAc extract (110 g) was subjected to polymide resin CC eluting with an EtOH/H2O gradient, yielding three fractions (E1− E3). The water-eluted fraction, E1 (60 g), was subjected to chromatography over the macroporous resin D101 using an EtOH/ H2O gradient for elution to yield four fractions (E1-1−E1-4). Fraction 1981



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and 4; HRESIMS m/z 499.1797 [M + Na]+ (calcd for C21H32O12Na, 499.1786). 4-epi-Dehydroabietic acid (11): The 1H (500 MHz) and 13C NMR (125 MHz) data (Me2CO-d6) and MS data were consistent with literature values.12 Majusanic acid B (12): The 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD) and MS data were consistent with literature values.11 Majusanic acid D (13): The 1H (500 MHz) and 13C NMR (125 MHz) data (CD3OD) and MS data were consistent with literature values.11 X-ray Crystallographic Analysis of 1 (ref 20). Upon crystallization from MeOH/H2O (1:1) using the vapor diffusion method, colorless crystals were obtained for 1. A crystal (0.55 × 0.15 × 0.04 mm) was separated from the sample and mounted onto a glass fiber. Data were collected using a Rigaku MicroMax 002 CCD detector with a graphite monochromator and Cu Kα radiation, λ = 1.5418 Å, at 173(2) K. Crystal data: C20H28O3; M = 316.42; space group monoclinic, C2; unit cell dimensions a = 21.2772(9) Å, b = 10.1711(5) Å, and c = 8.2882(3) Å; V = 1790.51(14) Å3; Z = 4; Dcalcd = 1.174 mg/m3; F(000) = 688. The structure was solved by direct methods using the SHELXS-97 program and refined by using full-matrix least-squares difference Fourier techniques. The 5865 measurements yielded 3404 independent reflections after the equivalent data were averaged and Lorentz and polarization corrections were applied. The final refinement gave R1 = 0.0364, wR2 = 0.0941 [I > 2σ(I)], and a Flack parameter of 0.15(18). Absolute Configurations of the 1,2-Diol Functionalities in 3 and 4. HPLC grade DMSO was dried with 4 Å molecular sieves. According to the published procedure,14 mixtures of 1:1.2 diol/ Mo2(OAc)4 for 3 and 4 were subjected to CD measurements at concentrations of 0.125 mg/mL, respectively. The first CD spectrum was recorded immediately after mixing, and its time evolution was monitored until it was stationary (approximately 10 min after mixing). The inherent CD was subtracted. The observed signs of the diagnostic band at approximately 310 nm in the induced CD spectra were correlated to the absolute configuration of the 1,2-diol moiety. Determination of D-Glucose (ref 16). A sample of compound 6 (2 mg) was dissolved in 2 M HCl in H2O (3 mL) and heated at 70 °C for 12 h. After extraction with EtOAc (3 × 3 mL) to remove the aglycone, the aqueous layer was evaporated and cryodesiccated to afford a neutral residue. The residue was dissolved in anhydrous pyridine (1 mL), to which L-cysteine methyl ester hydrochloride (2 mg) was added. The mixture was stirred at 60 °C for 2 h, and then 0.2 mL of N-trimethylsilylimidazole was added. The mixture was then heated to dryness at 60 °C for another 2 h. The dried reactant was partitioned between n-hexane and H2O (2 mL of each), and the nhexane layer was directly subjected to GC analysis (column: HP-5, 30 m × 0.25 mm × 0.25 μm, Dikma; detector: FID; detector temperature: 280 °C; injector temperature: 250 °C; carrier: N2 gas). The sugar moiety in 6 was identified as D-glucose by comparing the retention times of its derivative with that of the authentic standard (tR 14.90 min). The absolute configuration of the sugar unit in 10 was determined using the same method as for 6. Antiviral Activity Assay for the Coxsackie Virus B3 (ref 5). Confluent Vero cells (African green monkey kidney cell line) grown in 96-well microplates were infected with 100 median tissue culture infective doses (100TCID50) of Cox B3 virus. After 1 h of adsorption at 37 °C, the monolayers were washed with phosphate-buffered saline and incubated at 37 °C in maintenance media containing 2% fetal bovine serum in the absence or presence of various concentrations of the test compounds. The viral cytopathic effect (CPE) was observed when the viral control group reached 4+, and the antiviral activities of the tested compounds were determined by Reed−Muench analyses. Cytotoxicity Assay (ref 5). Vero cells were seeded into a 96-well plate. After 24 h, the monolayer cells were incubated with different concentrations of the test compounds. After 48 h of culture at 37 °C and 5% CO2 in a carbon dioxide incubator, the cells were monitored by CPE. The median toxic concentration (TC50) was calculated by Reed−Muench analyses.

Article

ASSOCIATED CONTENT

S Supporting Information *

Key 1H−1H COSY, HMBC correlations, and NOEs for compounds 1 and 6−9 (Figures S1−S3); copies of IR, HRESIMS, and NMR spectra for compounds 1−10; UV spectra for compounds 1−5, 7, and 8. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +86-10-63165324. Fax: +86-10-63017757. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge financial support from Natural Science Foundation of China (No. 201072234, No. 21132009), the National Science and Technology Project of China (No. 2012ZX09301002-002, No. 2011ZX09307-002-01), and PCSIRT (No. IRT1007).

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REFERENCES

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Diterpenoids and Sesquiterpenoids from the Roots of Illicium majus

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