Phytochemistry 102 (2014) 205–210
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Hikiokoshins A–I, diterpenes from the leaves of Isodon japonicus Naonobu Tanaka a, Eri Tsuji a, Kanae Sakai b, Tohru Gonoi b, Jun’ichi Kobayashi a,⇑ a b
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Medicinal Mycology Research Center, Chiba University, Chiba 260-0856, Japan
a r t i c l e
i n f o
Article history: Received 25 November 2013 Received in revised form 12 February 2014 Available online 2 April 2014 Keywords: Diterpenes Isodon japonicus Lamiaceae Hikiokoshins A–I
a b s t r a c t Diterpenes, hikiokoshins A–I, and twelve known diterpenes were isolated from the leaves of Isodon japonicus (Burm. f.) H. Hara (Lamiaceae). The hikiokoshins A–I possess various skeletons such as ternifonane {hikiokoshin A}, ent-6,7:8,15-diseco-6,8-cyclokauran-7,20-olide {hikiokoshin B}, ent-6,7-secokauran-7,20-olide {hikiokoshin C}, and ent-7,20-epoxykaurane {hikiokoshins D–I}. Their structures were elucidated on the basis of spectroscopic analysis. Antimicrobial activities of hikiokoshins A and B were evaluated. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction The plants belonging to the genus Isodon (Lamiaceae), which consists of about 150 species of under-shrubs, sub-undershrubs, or perennial herbs, are distributed throughout the world mainly in tropical and subtropical Asia. A large number of diterpenes with various chemical structures were isolated from Isodon plants. These diterpenes were reviewed in 2006, and classified into 11 groups including five subgroups (Sun et al., 2006). After that review, some unique diterpenes with novel carbon skeletons, such as maoecrystal Z (Han et al., 2006), bisrubescensin A (Huang et al., 2006), nervonin A (Li et al., 2008), ternifolide A (Zou et al., 2012), and scopariusic acid (Zhou et al., 2013) were isolated from this genus. Diterpenes from Isodon spp. have attracted considerable attention as antibacterial, antitumor, anti-inflammatory, and anti-feeding agents (Sun et al., 2006). Adenanthin, an ent-kaurane diterpene isolated from Rabdosia (=Isodon) adenantha, has attracted attention as the lead natural compound for the development of peroxiredoxin I- and II-targeted therapeutic agents, which may represent a promising approach to induce differentiation of acute promyelocytic leukemia cells (Liu et al., 2012). In Japan, Isodon japonicus (Burm. f.) H. Hara (Hikiokoshi in Japanese) has been used as a folk medicine for treatment of gastrointestinal disorders. As a part of a program to discover new structurally unique and bioactive natural products from plants (Tanaka et al., 2009, 2010, 2011, 2012), the extracts of the leaves of I. japonicus was examined. Thus resulted in the isolation of nine new diterpenes, hikiokoshins
⇑ Corresponding author. Tel.: +81 11 706 3239; fax: +81 11 706 4989. E-mail address: [email protected] (J. Kobayashi). http://dx.doi.org/10.1016/j.phytochem.2014.03.001 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
A–I (1–9). In this paper, the isolation and structure elucidation of 1–9 are described. Results and discussion The leaves of I. japonicus (1.05 kg, dry) were extracted with MeOH, and the extracts were partitioned successively with n-hexane, EtOAc, and H2O. Chromatographic separation of the EtOAc soluble materials afforded nine new diterpenes, hikiokoshins A–I (1–9). During the purification process, twelve known diterpenes including two ent-6,7-secokauran-7,20-olide diterpenes, rabdolasional (Takeda et al., 1990) and isorubesin D (Gao et al., 2011), four ent-7,20-epoxykaurane diterpenes, odonicin (Wang et al., 1994), neorabdosin (Wang et al., 1994), effusanin A (Wang et al., 1994), and rabdolongin A (Takeda et al., 1988), and six ent-6,7-secokauran-7,1-olide diterpenes, nodosin (Takeda and Matsumoto, 1994), epinodosin (Chen et al., 1989), isodocarpin (Isobe et al., 1972), sculponeatin E (Jiang et al., 2002), serrin C (Zhao et al., 2004), and ent-6,7-seco-6,20-epoxy-6b-methoxykaur-16-ene-15a,20b-diol (Yan et al., 2007) were isolated and identified by comparison of their physicochemical data with the reported data. Several artefacts with the ent-7,20-epoxykaurane and ent-6,7-secokauran-7,1-olide skeletons were also isolated, arising from conjugate addition of methanol to the unsaturated ketone in ring D. Hikiokoshin A (1) was obtained as an optically active colorless amorphous solid {[a]D +24.2 (c 0.23, MeOH)}. The molecular formula of 1 was elucidated to be C20H26O5 (m/z 369.16736 [M+Na]+, D+0.11 mmu) by the HRESIMS. IR absorptions at 3484, 1708, and 1683 cm1 implied the presence of hydroxyl and carbonyl functionalities. Analysis of the 1H NMR spectrum
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established the existence of one exo-methylene {dH 5.48 and 5.20 (H2-17)}, one oxygenated sp3 methine {dH 3.97 (H-1)}, two oxygenated sp3 methylenes {dH 5.08 and 3.92 (H2-6); 5.09 and 4.86 (H2-20)}, and two tertiary methyls (Table 1). The 13C NMR spectrum displayed 20 carbon signals including two carboxyl groups, two olefins, one oxygenated methine, and two oxygenated methylenes. Interpretation of the 1H–1H COSY and HMBC spectra (Fig. 1) implied that 1 is structurally related to ternifolide A (Fig. S1), a ternifonane diterpene with a 10-membered lactone from the leaves of Isodon ternifolius (Zou et al., 2012). HMBC correlations from H2-11 to C-8 and C-9, H2-14 to C-8, and H2-20 to C-9 indicated the absence of a ketone group at C-11 and the presence of a double bond between C-8 and C-9 in 1. The presence of a hydroxyl group at C-1 was deduced by the chemical shifts of H-1 and C-1 and the molecular formula. The relative stereochemistry of 1 was assigned on the basis of NOESY analysis (Fig. 1). NOESY correlations for H-1/H-3b, H-3b/H-5, H-2a/H3-19, and H3-19/H-20a indicated the axial orientations of H-1, H-2a, H-3b, H-5, CH3-19, and CH2-20. Thus, the cyclohexane ring (C-1–C-5 and C-10) adopts a chair conformation. In addition, NOESY correlations for H-11b/H-1 and H-11b/H-5 disclosed that CH2-11 was located at the upper side of the cyclohexane ring. The presence of the 10-membered lactone ring restricted the orientation of H-13 to the a side. Therefore, the structure of hikiokoshin A (1) was assigned as shown in Scheme 1. Hikiokoshin B (2) was isolated as an optically active amorphous solid {[a]D 6.4 (c 0.24, MeOH)}. The molecular formula, C23H32O8, of 2 was assigned by the HRESIMS (m/z 459.19907 [M+Na]+, D0.13 mmu). Comparison of the 1H and 13C NMR spectroscopic data for 2 (Table 1) with the literature data for diterpenes from Isodon spp. implied that 2 has two hydroxyl, one acetoxyl, one
methoxyl, and one carboxyl groups, as well as the same skeleton as that of maoecrystal Z (Fig. S1) (Han et al., 2006). The substituent at C-13 in 2 was assigned as a methyl acrylate by HMBC correlations from the methoxyl group to C-15 and H2-17 to C-13, C-15, and C-16. The presence of the acetoxyl group at C-1 and the hydroxyl group at C-11 was deduced from the chemical shifts of CH-1 (dH 5.39, dC 76.0) and CH-11 (dH 3.99, dC 67.4). Analysis of the NOESY spectrum of 2 (Fig. S2) suggested that the tetracyclic core of 2 has the same relative configuration as that of maoecrystal Z. NOESY analysis also indicated the a-orientation for the 1-OAc and the b-orientations for the 6-OH, 11-OH, and the substituent at C-13. Thus, the structure of hikiokoshin B (2) was elucidated as shown in Scheme 1. Hikiokoshin C (3) was isolated as an optically active colorless amorphous solid {[a]D 7.4 (c 0.19, MeOH)}. The molecular fomula, C22H30O6, was determined by the HRESIMS (m/z 413.19327 [M+Na]+, D0.19 mmu). Resemblance of the 1H and 13C NMR spectra (Table 1) of 3 with those of rabdolasional (Fig. S1) (Takeda et al., 1990) implied that 3 is an ent-6,7-secokauran-7,20-olide diterpene. The signal of an sp3 methylene (dH 1.53 and 1.34) found in 3 in place of an oxygenated methine (CH-11) in rabdolasional suggested that 3 was an 11-deoxy derivative of rabdolasional. This was confirmed by analysis of the 1H–1H COSY cross-peaks of H-9/ H2-11 and H2-11/H2-12 (Fig. S3). The relative stereochemistry of the tetracyclic core and the orientations of substituents at C-1, C-5, and C-15 in 3 were assigned as the same as those of rabdolasional on the basis of NOESY analysis. Hikiokoshin D (4) was obtained as an optically active colorless amorphous solid {[a]D 60.1 (c 0.28, MeOH)}. The 13C NMR spectra (Table 2) displayed 22 signals including one exo-methylene and
Table 1 H and 13C NMR spectroscopic data for hikiokoshins A–C (1–3) in pyridine-d5.
1
Position
1 dC
1 2
72.4 27.4
3
39.9
4 5 6
32.1 48.7 62.6
7 8 9 10 11
164.2 123.0 161.4 46.9 22.3
12
26.3
13 14
38.4 29.1
15 16 17 18 19 20 1-OH 15-OH 15-OMe 1-OAc
a
170.6 148.2 120.6 32.9 22.0 67.1
Overlapped with signal of HOD.
2 dH (J in Hz) 3.97 (dd, 13.4, 2.2) 1.86 (brd, 13.4) 1.75 (dddd, 13.4, 13.4, 13.4, 2.4) 1.38 (ddd, 13.4, 13.4, 2.4) 1.31 (brd, 13.4) – 2.06 (d, 11.7) 5.08 (m) 3.92 (d, 11.7) – – – – 2.72 (dd, 16.0, 8.8) 2.17 (m) 2.01 (m), 1.93 (m) 2.90 3.12 1.98 – – 5.48 0.91 0.73 5.09 6.75
(m) (d, 14.8) (m)
(brs), 5.20 (brs) (3H, s) (3H, s) (d, 12.2), 4.86 (d, 12.2) (brs)
dC 76.0 26.3
3 dH (J in Hz)
32.0 64.9 74.0
5.39 (dd, 14.2, 4.6) 1.85 (m) 1.72 (dddd, 14.2, 14.2, 14.2, 3.1) 1.45 (ddd, 14.2, 3.1, 3.1) 1.32 (ddd, 14.2, 14.2, 3.1) – 1.91 (d, 6.4) 4.37 (d, 6.4)
174.5 57.1 57.2 47.1 67.4
– – 2.15 (d, 10.3) – 3.99 (ddd, 10.3, 10.3, 3.7)
39.8
43.1
5.00 (m)a 2.05 (ddd, 13.1, 13.1, 4.0) 1.90 (m)
39.9 34.0 62.7 203.5
1.43 (ddd, 13.1, 13.1, 4.0) 1.35 (m) – 2.49 (d, 5.2) 10.17 (d, 5.2)
175.6 52.6 38.0 42.9 16.4
– – 2.73 (dd, 12.8, 4.3) – 1.53 (m), 1.34 (m)
32.8
1.91 (m), 1.34 (m)
36.3 29.9
2.62 2.22 1.71 4.90 – 5.39 0.89 1.06 5.43
167.2 144.9 123.0 32.9 20.7 68.3
51.5 170.0
3.60 (3H, s) –
170.2
21.4
2.12 (3H, s)
21.3
(brs), 5.59 (brs) (3H, s) (3H, s) (2H, brs)
dH (J in Hz)
75.7 24.2
2.55 1.58 3.29 2.68 1.95 – – 6.16 1.18 1.09 4.88
35.1 32.4
(brd, 10.3) (q, 10.3) (m) (brd, 12.5) (dd, 12.5, 12.5)
dC
82.9 159.7 109.5 32.8 23.8 67.7
(m) (dd, 12.2, 5.0) (d, 12.2) (brs) (brs), 5.14 (brs) (3H, s) (3H, s) (d, 11.6), 5.37 (d, 11.6)
8.24 (d, 4.6) – 2.17 (3H, s)
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17 2
3 4
18 19 1H-1H
5 6
OH 1 10 20
12
11
13
9
14 8
O
7
COSY
HMBC
15
O O
13
11
16
O
1 10 2
20
6 5
3
1
15
8
9
17
18 19
NOESY
Fig. 1. Selected 2D NMR correlations and relative stereochemistry (protons of methyl groups are not shown) for hikiokoshin A (1).
Scheme 1. Structures of hikiokoshins A–I (1–9).
one acetoxyl group, as well as the characteristic resonance of an acetal group (dC 95.4). Comparison of the 1D NMR spectroscopic data for 4 with literature data suggested that 4 is an analog of maoecrystal X (Fig. S1), an ent-7,20-epoxykaurane diterpene (Shen et al., 2005). The molecular formula, C22H32O5, of 4, which was elucidated by HRESIMS (m/z 399.21398 [M+Na]+, D0.22 mmu), was smaller than that of maoecrystal X by 16 mass units. These observations implied that 4 is a deoxy derivative of maoecrystal X. HMBC correlations from H2-6 to C-7 and C-8, and an 1H–1H COSY cross-peak of H-5/H-6 indicated that 4 has an sp3 methylene in place of an oxymethine (CH-6) in maoecrystal X. Thus, the structure of hikiokoshin D (4) was assigned as shown in Scheme 1. The HRESIMS of hikiokoshin E (5) established that the molecular formula of 5 was the same as that of 4 (m/z 399.21448 [M+Na]+, D +0.28 mmu). The 1H and 13C NMR spectroscopic data for 5 (Table 2) were similar to those of 4, except for the signals from ring A (C-1–C-5 and C-10). The 1H–1H COSY cross-peaks of an oxygenated methine (H-2) to H2-1 and H2-3 disclosed that 5 has a hydroxyl group at C-2 (Fig. S5). The b-orientation of the hydroxyl group at C-2 was deduced by the NOESY correlations for H-2/H3-19 and H-2/H-20a (Fig. S5). Therefore, the structure of hikiokoshin E (5) was assigned as a 2-hydroxy analog of 4. The HRESIMS has the same molecular formula, C24H34O8, for hikiokoshins F (6) and G (7) (m/z 473.21503 [M+Na]+, D +0.44 mmu for 6; m/z 473.21429 [M+Na]+, D 0.30 mmu for 7). Interpretation of the 1H and 13C NMR spectra of 6 and 7 (Table 2) implied that 6 and 7 are diterpenes possessing an ent-7, 20-epoxykaurane skeleton with two hydroxyl and two acetoxyl groups. In 6, the presence of oxygen functions at C-1, C-2, C-6,
and C-15 was deduced by interpretation of the 1H–1H COSY and HMBC spectra (Fig. S6), taking their carbon chemical shifts into consideration. In addition, the chemical shifts of H-6 (dH 5.88) and H-15 (dH 6.25) were at lower field relative to those of H-1 (dH 3.89) and H-2 (dH 4.12). These observations indicated the presence of acetoxyl groups at C-6 and C-15, and of hydroxyl groups at C-1 and C-2 in 6. Analysis of the NOESY spectrum of 6 established the orientations of 1b-OH, 2b-OH, 6b-OAc, and 15b-OAc (Fig. S6). Similarly, the structure of hikiokoshin G (7) was elucidated to be an ent-7,20-epoxykaurane with 3b-OH, 6b-OAc, 14b-OH, and 15b-OAc groups (Scheme 1). Hikiokoshin H (8), C24H32O8, was isolated as an optically active colorless amorphous solid {[a]D 6.6 (c 0.50, MeOH)}. Its IR spectrum implied the existence of hydroxyl (3393 cm1), ester carbonyl (1742 cm1), and ketone (1699 cm1) functionalities. Analysis of the 1H and 13C NMR spectra (Table 3) suggested it to be an ent-7,20-epoxykaurane diterpene having one ketone, one hydroxymethyl, two acetoxyl, and one exo-methylene groups. The positions of these functionalities were assigned by 1H-1H COSY and HMBC analysis as shown in Scheme 1. NOESY analysis (Fig. S8) indicated the a-orientation of the hydroxymethyl group (C-19) and the b-orientations of the acetoxyl groups at C-6 and C-15. Accordingly, the structure of hikiokoshin H (8) was shown in Scheme 1. The 1H and 13C NMR spectra of hikiokoshin I (9) (Table 3), C24H32O8, were similar to those of 8, and also showed the resonances due to an sp3 oxymethine (dH 4.28, dC 74.8) and a tertiary methyl in place of the signals of an sp3 methylene (CH2-3) and hydroxymethyl (CH2-19) in 8. An 1H–1H COSY cross-peak of the oxymethine proton (H-3) with H2-2 and HMBC correlations from
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Table 2 H and 13C NMR spectroscopic data for hikiokoshins D–F (4–6) in pyridine-d5.
1
Position
4 dC
a
1 2 3
73.7 30.4 39.0
4 5 6
33.7 48.5 34.7
7 8 9 10 11 12 13 14
95.4 52.7 46.6 40.1 19.0 33.1 36.6 26.1
15 16 17 18 19 20 1-OH 2-OH 7-OH 6-OAc
76.6 158.8 108.2 31.8 20.7 63.7
15-OAc
170.6 21.1
5 dH (J in Hz) 3.70 1.84 1.41 1.28 – 1.69 2.14 2.04 – – 2.22 – 2.21 2.21 2.58 2.11 2.04 6.28 – 5.10 0.80 1.07 4.84 5.72
(m) (dd, 13.0, 12.0) (m)
dH (J in Hz)
1.89 (m), 1.24 (m) 4.02 (m) 2.07 (m), 1.43 (m)
69.9 66.8 42.9
35.1 48.4 33.9
– 1.65 2.12 2.00 – – 2.07 – 1.47 2.07 2.54 1.99 1.92 6.23 – 5.09 0.85 1.08 4.26
34.9 49.5 74.9
3.89 4.12 2.46 1.67 – 2.33 5.88
95.6 51.0 38.8 42.2 15.1 31.7 36.7 27.5
– – 3.29 – 2.02 2.13 2.57 2.09
75.1 159.6 108.6 33.1 24.4 65.8
6.25 – 5.22 1.05 1.31 4.22 5.92 6.21 8.32 – 2.11 – 2.16
76.6 158.4 108.7 32.7 22.1 67.0
(dd, 12.8, 7.7) (dd, 14.5, 12.8) (dd, 14.5, 7.7)
(m) (m), 1.24 (m) (m), 1.43 (m) (dd, 8.6, 4.5) (dd, 12.2, 4.5) (d, 12.2) (s) (brs), 5.08 (brs) (3H, s) (3H, s) (d, 9.5), 4.06 (d, 9.5)
6.06 (brs) 7.94 (brs)
7.88 (brs)
– 2.21 (3H, s)
dC
41.5 63.9 51.1
95.6 52.3 45.4 37.0 15.6 32.7 36.5 26.3
(m)
(2H, m)a (3H, s) (3H, s) (d, 9.4), 4.48 (d, 9.4) (brd, 3.7)
dH (J in Hz)
dC
(brs) (2H, m) (brd, 13.4) (m)
(m), 2.02 (m) (m), 1.52 (m) (dd, 8.2, 4.9) (d, 12.0) (m) (s)
6
170.7 21.2
– 2.18 (3H, s)
171.0 21.1 171.0 21.8
(brs) (brd, 12.4) (dd, 12.4, 12.4) (dd, 12.4, 3.4) (d, 7.1) (d, 7.1)
(dd, 12.5, 5.0) (m), 1.64 (m) (m), 1.58 (m) (brd, 10.3) (2H, m) (s) (brs), 5.09 (brs) (3H, s) (3H, s) (d, 9.4), 4.18 (d, 9.4) (brs) (brs) (s) (3H, s) (3H, s)
Overlapped with signal of HOD.
Table 3 H and 13C NMR spectroscopic data for hikiokoshins G–I (7–9) in pyridine-d5.
1
Position
7 dC
dH (J in Hz)
dC
dH (J in Hz)
212.2
–
210.8
–
24.3
2
25.8
2.04 (ddd, 13.2, 13.2, 6.1) 1.06 (brd, 13.2) 1.71 (2H, m)
3 4 5 6 7 8 9 10 11 12 13 14
74.5 38.2 48.1 73.5 98.0 52.4 46.6 35.6 15.0 32.0 45.5 75.6
3.65 – 2.53 5.98 – – 2.74 – 1.53 2.29 2.77 5.02
74.1 158.8 111.2 28.6 23.1 67.1
6.70 – 5.38 1.25 1.22 4.34 6.09 8.31 7.97
15-OAc
170.8 21.3 171.1 22.0
Overlapped with signal of HOD.
9
dC
1
15 16 17 18 19 20 3-OH 7-OH 14-OH 19-OH 6-OAc
a
8 dH (J in Hz)
(brs) (d, 7.9) (d, 7.9)
(m) (m), 1.22 (m) (m), 1.55 (m) (m) (m)a (s) (brs), 5.23 (brs) (3H, s) (3H, s) (d, 9.7), 3.99 (d, 9.7) (brs) (s) (brs)
– 2.14 (3H, s) – 2.24 (3H, s)
35.7
2.84 (m), 2.40 (m)
47.4
34.1 36.9 53.9 74.5 96.3 52.1 42.9 48.9 18.2 32.3 35.9 27.1
1.98 – 2.99 6.41 – – 2.80 – 1.86 2.07 2.55 2.15 2.07 6.20 – 5.18 1.19 3.80 4.75
74.8 39.1 51.7 73.5 96.4 52.2 42.7 48.9 17.8 32.3 35.8 27.0
75.3 158.8 109.7 25.1 66.6 64.7
(m), 1.85 (m) (d, 8.9) (d, 8.9)
(dd, 14.2, 6.1) (m), 1.19 (m) (m), 1.39 (m) (dd, 8.8, 4.9) (d, 12.8) (m) (brs) (brs), 5.14 (brs) (3H, s) (d, 11.0), 3.74 (d, 11.0) (d, 10.0), 4.73 (d, 10.0)
6.80 (brs) – 2.13 (3H, s) – 2.36 (3H, s)
(dd, 16.9, 9.2) (dd, 16.9, 4.6) (dd, 9.2, 4.6) (m) (d, 9.2)
(m) (m), 1.23 (m) (m), 1.37 (m) (dd, 8.3, 4.7) (m), 2.04 (m)
75.3 158.6 109.6 25.8 16.5 65.5
6.16 – 5.14 1.26 1.12 4.66 6.95 8.64
170.8 21.2 170.7 21.9
– 2.13 (3H, s) – 2.28 (3H, s)
8.48 (brs)
170.8 21.2 170.8 22.0
3.37 2.74 4.28 – 2.71 5.83 – – 2.71 – 1.84 2.07 2.55 2.12
(brs) (brs), 5.13 (brs) (3H, s) (3H, s) (d, 9.9), 4.23 (d, 9.9) (brs) (brs)
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Me-18 to C-3, C-4, C-5, and C-19 established that 9 had a geminal dimethyl group at C-4 and a hydroxyl group at C-3. A NOESY correlation from H-3 to H-5 indicated as orientation of the hydroxyl group at C-3. Thus, the structure of hikiokoshin I (9) was assigned as shown in Scheme 1. Antimicrobial activities of hikiokoshins A (1) and B (2) against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, Trichophyton mentagrophytes, Candida albicans, and Cryptococcus neoformans were evaluated. Hikiokoshin A (1) exhibited antifungal activity against A. niger and C. neoformans (IC50 16 lg/mL each), whereas hikiokoshin B (2) did not show such activity against any strains (IC50 > 32 lg/mL). Hikiokoshins A–I (1–9) showed no cytotoxicity (IC50 > 10 lg/mL) against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro. Conclusion Investigation for the extract of the leaves of I. japonicus (Burm. f.) H. Hara resulted in the isolation of nine new diterpenes, hikiokoshins A–I (1–9), together with twelve known diterpenes. Although a large number of diterpenes with various skeletons have been isolated from Isodon spp. to date (Sun et al., 2006), hikiokoshins A (1) and B (2) have the rare ternifonane (Zou et al., 2012) and ent-6,7:8,15-diseco-6,8-cyclokauran-7,20-olide skeletons (Han et al., 2006), respectively. Hikiokoshin C (3) possesses an ent-6,7-secokaurane skeleton, while hikiokoshins D–I (4–9) are ent-7,20-epoxykaurenes. Experimental section General procedures Optical rotations were recorded using a JASCO P-1030 digital polarimeter. IR spectra were recorded on a JASCO FT/IR-460 Plus spectrophotometer. NMR spectra were measured on a Bruker AMX-600 spectrometer. The 7.19 and 123.5 ppm resonances of residual pyridine were used as internal references for 1H and 13C chemical shifts, respectively. ESIMS spectra were obtained on a Thermo Scientific Exactive spectrometer. C18 HPLC column: column A, YMC-Pack ODS-AQ, YMC Co., Ltd., 20 250 mm; column B, Cosmosil 5C18-MS-II, Nacalai Tesque, Inc., 10 250 mm. Plant Material I. japonicus (Burm. f.) H. Hara was cultivated at the Experimental Station for Medicinal Plant Studies, Hokkaido University, and the aerial parts were collected at in October 2009. Herbarium specimens were deposited in Graduate School of Pharmaceutical Sciences, Hokkaido University (specimen number: HIJ200910). Extraction and isolation The dried leaves of I. japonicus (1.05 kg) were extracted with MeOH (18 L, 3 days 3) at room temperature to give the extract (211 g). A part (107 g) of the extract was partitioned successively with n-hexane (500 mL 4), EtOAc (500 mL 4), and H2O (500 mL), respectively. The EtOAc-soluble portion (27.2 g) was subjected to silica gel column chromotography (CC) (eluent CHCl3/MeOH, 100:0?0:100) to give seventeen fractions (frs. 1– 17). Fr. 10 was loaded on a Sephadex LH-20 column (MeOH/toluene, 50:50) to remove fatty acids, and then applied to a silica gel column (n-hexane/EtOAc, 70:30?0:100) to afford ten fractions (frs. 10.1–10). Fr. 10.5 was purified by silica gel CC (CHCl3/acetone, 97:3?0:100) and C18 HPLC (column A; flow rate 6.0 mL/min; UV
209
detection at 220 nm; MeOH/H2O, 60:40) to yield hikiokoshin C (3, 0.8 mg, 1.4 104%). Separation of fr. 10.6 on a silica gel column (CHCl3/acetone, 97:3?0:100) gave nine fractions (frs. 10.6.1–9). Fr. 10.6.4 was loaded on a C18 column (MeOH/H2O, 30:70?100:0) and C18 HPLC (column A; 5.0 mL/min; 220 nm; MeOH/H2O, 50:50) to isolate hikiokoshin B (2, 1.2 mg, 2.2 104%). Fr. 10.6.5 was subjected to silica gel CC (n-hexane/EtOAc, 50:50?0:100) and then purified by C18 HPLC (column A; 6.0 mL/min; 220 nm; MeOH/ H2O, 60:40) to give hikiokoshin I (9, 1.9 mg, 3.6 104%). Purification of fr. 10.6.6 on a C18 column (MeOH/H2O, 30:70?100:0) and C18 HPLC (column A; 4.0 mL/min; 220 nm; MeOH/H2O, 60:40) gave hikiokoshins D (4, 1.2 mg, 2.2 104%) and H (8, 2.3 mg, 4.2 104%). Fr. 10.8 was applied to a silica gel column (CHCl3/acetone, 97:3?0:100) to give seven fractions (frs. 10.8.1–7). Fr. 10.8.4 was purified by a C18 column (MeOH/H2O, 20:80?100:0) and C18 HPLC (column B; 3.0 mL/min; 220 nm; MeOH/H2O, 50:50) to afford hikiokoshin A (1, 1.2 mg, 2.2 104%). Fr. 10.8.5 was applied to a C18 column (MeOH/H2O, 40:60?100:0) and then purified by C18 HPLC (column A; 5.0 mL/min; 220 nm; MeOH/H2O, 50:50) to give hikiokoshins E (5, 1.4 mg, 2.6 104%), F (6, 0.5 mg, 0.9 104%), and G (7, 0.7 mg, 1.3 104%). During the purification process, twelve known diterpenes, rabdolasional (4.1 mg, 7.7 104%), isorubesin D (3.9 mg, 7.4 104%), odonicin (639 mg, 1.2 101%), neorabdosin (23.8 mg, 4.5 103%), effusanin A (0.7 mg, 1.3 104%), rabdolongin A (1.0 mg, 1.9 104%), nodosin (4.6 mg, 8.7 104%), epinodosin (2.4 mg, 4.5 104%), isodocarpin (1.3 mg, 2.5 104%), sculponeatin E (12.0 mg, 2.3 103%), serrin C (6.4 mg, 1.2 103%), and ent-6,7-seco6,20-epoxy-6b-methoxykaur-16-ene-15a,20b-diol (10.0 mg, 1.9 103%) were isolated. Hikiokoshin A (1) Colorless amorphous solid; ½a29 D +24.2 (c 0.23, MeOH); IR (KBr) mmax 3484, 1708, and 1683 cm1; for 1H and 13C NMR spectroscopic data see Table 1; HRESIMS m/z 369.16736 ([M+Na]+, calcd for C20 H26O5Na, 369.16725). Hikiokoshin B (2) Colorless amorphous solid; ½a28 D 6.4 (c 0.24, MeOH); IR (KBr) mmax 3440 and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 1; HRESIMS m/z 459.19907 ([M+Na]+, calcd for C23H32O8 Na, 459.19894). Hikiokoshin C (3) Colorless amorphous solid; ½a24 D 7.4 (c 0.19, MeOH); IR (KBr) mmax 3447, 1734, and 1716 cm1; for 1H and 13C NMR spectroscopic data see Table 1; HRESIMS m/z 413.19327 ([M+Na]+, calcd for C22 H30O6Na, 413.19346). Hikiokoshin D (4) Colorless amorphous solid; ½a29 D –60.1 (c 0.28, MeOH); IR (KBr) mmax 3446, 1739, and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS m/z 399.21398 ([M+Na]+, calcd for C22 H32O5Na, 399.21420). Hikiokoshin E (5) Colorless amorphous solid; ½a28 D 40.3 (c 0.23, MeOH); IR (KBr) mmax 3423, 1737, and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS m/z 399.21448 ([M+Na]+, calcd for C22 H32O5Na, 399.21420). Hikiokoshin F (6) Colorless amorphous solid; ½a25 D 41.2 (c 0.13, MeOH); IR (KBr) mmax 3446, 1734, and 1717 cm1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS m/z 473.21503 ([M+Na]+, calcd for C24 H34O8Na, 473.21459).
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Hikiokoshin G (7) Colorless amorphous solid; ½a25 D 68.1 (c 0.17, MeOH); IR (KBr) mmax 3358, 1735, and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 473.21429 ([M+Na]+, calcd for C24 H34O8Na, 473.21459). Hikiokoshin H (8) Colorless amorphous solid; ½a28 D 6.6 (c 0.50, MeOH); IR (KBr) mmax 3393, 1742, and 1699 cm1; for 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 471.19882 ([M+Na]+, calcd for C24 H32O8Na, 471.19894). Hikiokoshin I (9) Colorless amorphous solid; ½a29 D 23.9 (c 0.42, MeOH); IR (KBr) mmax 3447, 1734, and 1701 cm1; for 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 471.19850 ([M+Na]+, calcd for C24 H32O8Na, 471.19894). Antimicrobial testing Antimicrobial assays of hikiokoshins A (1) and B (2) were carried out as previously described (Nagai et al., 1993). Amphotericin B, micafungin, hygromycin B, and kanamycin were used as controls for antimicrobial activities against E. coli (MIC, >31.2, >62.5, 2.0, and 31.2, 0.5, 0.5, and 31.2, 0.5, 2.0, and 31.2, 7.8, 1.0, and 1.0 lg/mL, respectively), A. niger (IC50, 1.8,
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Hikiokoshins A–I, diterpenes from the leaves of Isodon japonicus Naonobu Tanaka a, Eri Tsuji a, Kanae Sakai b, Tohru Gonoi b, Jun’ichi Kobayashi a,⇑ a b
Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Medicinal Mycology Research Center, Chiba University, Chiba 260-0856, Japan
a r t i c l e
i n f o
Article history: Received 25 November 2013 Received in revised form 12 February 2014 Available online 2 April 2014 Keywords: Diterpenes Isodon japonicus Lamiaceae Hikiokoshins A–I
a b s t r a c t Diterpenes, hikiokoshins A–I, and twelve known diterpenes were isolated from the leaves of Isodon japonicus (Burm. f.) H. Hara (Lamiaceae). The hikiokoshins A–I possess various skeletons such as ternifonane {hikiokoshin A}, ent-6,7:8,15-diseco-6,8-cyclokauran-7,20-olide {hikiokoshin B}, ent-6,7-secokauran-7,20-olide {hikiokoshin C}, and ent-7,20-epoxykaurane {hikiokoshins D–I}. Their structures were elucidated on the basis of spectroscopic analysis. Antimicrobial activities of hikiokoshins A and B were evaluated. Ó 2014 Elsevier Ltd. All rights reserved.
Introduction The plants belonging to the genus Isodon (Lamiaceae), which consists of about 150 species of under-shrubs, sub-undershrubs, or perennial herbs, are distributed throughout the world mainly in tropical and subtropical Asia. A large number of diterpenes with various chemical structures were isolated from Isodon plants. These diterpenes were reviewed in 2006, and classified into 11 groups including five subgroups (Sun et al., 2006). After that review, some unique diterpenes with novel carbon skeletons, such as maoecrystal Z (Han et al., 2006), bisrubescensin A (Huang et al., 2006), nervonin A (Li et al., 2008), ternifolide A (Zou et al., 2012), and scopariusic acid (Zhou et al., 2013) were isolated from this genus. Diterpenes from Isodon spp. have attracted considerable attention as antibacterial, antitumor, anti-inflammatory, and anti-feeding agents (Sun et al., 2006). Adenanthin, an ent-kaurane diterpene isolated from Rabdosia (=Isodon) adenantha, has attracted attention as the lead natural compound for the development of peroxiredoxin I- and II-targeted therapeutic agents, which may represent a promising approach to induce differentiation of acute promyelocytic leukemia cells (Liu et al., 2012). In Japan, Isodon japonicus (Burm. f.) H. Hara (Hikiokoshi in Japanese) has been used as a folk medicine for treatment of gastrointestinal disorders. As a part of a program to discover new structurally unique and bioactive natural products from plants (Tanaka et al., 2009, 2010, 2011, 2012), the extracts of the leaves of I. japonicus was examined. Thus resulted in the isolation of nine new diterpenes, hikiokoshins
⇑ Corresponding author. Tel.: +81 11 706 3239; fax: +81 11 706 4989. E-mail address: [email protected] (J. Kobayashi). http://dx.doi.org/10.1016/j.phytochem.2014.03.001 0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
A–I (1–9). In this paper, the isolation and structure elucidation of 1–9 are described. Results and discussion The leaves of I. japonicus (1.05 kg, dry) were extracted with MeOH, and the extracts were partitioned successively with n-hexane, EtOAc, and H2O. Chromatographic separation of the EtOAc soluble materials afforded nine new diterpenes, hikiokoshins A–I (1–9). During the purification process, twelve known diterpenes including two ent-6,7-secokauran-7,20-olide diterpenes, rabdolasional (Takeda et al., 1990) and isorubesin D (Gao et al., 2011), four ent-7,20-epoxykaurane diterpenes, odonicin (Wang et al., 1994), neorabdosin (Wang et al., 1994), effusanin A (Wang et al., 1994), and rabdolongin A (Takeda et al., 1988), and six ent-6,7-secokauran-7,1-olide diterpenes, nodosin (Takeda and Matsumoto, 1994), epinodosin (Chen et al., 1989), isodocarpin (Isobe et al., 1972), sculponeatin E (Jiang et al., 2002), serrin C (Zhao et al., 2004), and ent-6,7-seco-6,20-epoxy-6b-methoxykaur-16-ene-15a,20b-diol (Yan et al., 2007) were isolated and identified by comparison of their physicochemical data with the reported data. Several artefacts with the ent-7,20-epoxykaurane and ent-6,7-secokauran-7,1-olide skeletons were also isolated, arising from conjugate addition of methanol to the unsaturated ketone in ring D. Hikiokoshin A (1) was obtained as an optically active colorless amorphous solid {[a]D +24.2 (c 0.23, MeOH)}. The molecular formula of 1 was elucidated to be C20H26O5 (m/z 369.16736 [M+Na]+, D+0.11 mmu) by the HRESIMS. IR absorptions at 3484, 1708, and 1683 cm1 implied the presence of hydroxyl and carbonyl functionalities. Analysis of the 1H NMR spectrum
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established the existence of one exo-methylene {dH 5.48 and 5.20 (H2-17)}, one oxygenated sp3 methine {dH 3.97 (H-1)}, two oxygenated sp3 methylenes {dH 5.08 and 3.92 (H2-6); 5.09 and 4.86 (H2-20)}, and two tertiary methyls (Table 1). The 13C NMR spectrum displayed 20 carbon signals including two carboxyl groups, two olefins, one oxygenated methine, and two oxygenated methylenes. Interpretation of the 1H–1H COSY and HMBC spectra (Fig. 1) implied that 1 is structurally related to ternifolide A (Fig. S1), a ternifonane diterpene with a 10-membered lactone from the leaves of Isodon ternifolius (Zou et al., 2012). HMBC correlations from H2-11 to C-8 and C-9, H2-14 to C-8, and H2-20 to C-9 indicated the absence of a ketone group at C-11 and the presence of a double bond between C-8 and C-9 in 1. The presence of a hydroxyl group at C-1 was deduced by the chemical shifts of H-1 and C-1 and the molecular formula. The relative stereochemistry of 1 was assigned on the basis of NOESY analysis (Fig. 1). NOESY correlations for H-1/H-3b, H-3b/H-5, H-2a/H3-19, and H3-19/H-20a indicated the axial orientations of H-1, H-2a, H-3b, H-5, CH3-19, and CH2-20. Thus, the cyclohexane ring (C-1–C-5 and C-10) adopts a chair conformation. In addition, NOESY correlations for H-11b/H-1 and H-11b/H-5 disclosed that CH2-11 was located at the upper side of the cyclohexane ring. The presence of the 10-membered lactone ring restricted the orientation of H-13 to the a side. Therefore, the structure of hikiokoshin A (1) was assigned as shown in Scheme 1. Hikiokoshin B (2) was isolated as an optically active amorphous solid {[a]D 6.4 (c 0.24, MeOH)}. The molecular formula, C23H32O8, of 2 was assigned by the HRESIMS (m/z 459.19907 [M+Na]+, D0.13 mmu). Comparison of the 1H and 13C NMR spectroscopic data for 2 (Table 1) with the literature data for diterpenes from Isodon spp. implied that 2 has two hydroxyl, one acetoxyl, one
methoxyl, and one carboxyl groups, as well as the same skeleton as that of maoecrystal Z (Fig. S1) (Han et al., 2006). The substituent at C-13 in 2 was assigned as a methyl acrylate by HMBC correlations from the methoxyl group to C-15 and H2-17 to C-13, C-15, and C-16. The presence of the acetoxyl group at C-1 and the hydroxyl group at C-11 was deduced from the chemical shifts of CH-1 (dH 5.39, dC 76.0) and CH-11 (dH 3.99, dC 67.4). Analysis of the NOESY spectrum of 2 (Fig. S2) suggested that the tetracyclic core of 2 has the same relative configuration as that of maoecrystal Z. NOESY analysis also indicated the a-orientation for the 1-OAc and the b-orientations for the 6-OH, 11-OH, and the substituent at C-13. Thus, the structure of hikiokoshin B (2) was elucidated as shown in Scheme 1. Hikiokoshin C (3) was isolated as an optically active colorless amorphous solid {[a]D 7.4 (c 0.19, MeOH)}. The molecular fomula, C22H30O6, was determined by the HRESIMS (m/z 413.19327 [M+Na]+, D0.19 mmu). Resemblance of the 1H and 13C NMR spectra (Table 1) of 3 with those of rabdolasional (Fig. S1) (Takeda et al., 1990) implied that 3 is an ent-6,7-secokauran-7,20-olide diterpene. The signal of an sp3 methylene (dH 1.53 and 1.34) found in 3 in place of an oxygenated methine (CH-11) in rabdolasional suggested that 3 was an 11-deoxy derivative of rabdolasional. This was confirmed by analysis of the 1H–1H COSY cross-peaks of H-9/ H2-11 and H2-11/H2-12 (Fig. S3). The relative stereochemistry of the tetracyclic core and the orientations of substituents at C-1, C-5, and C-15 in 3 were assigned as the same as those of rabdolasional on the basis of NOESY analysis. Hikiokoshin D (4) was obtained as an optically active colorless amorphous solid {[a]D 60.1 (c 0.28, MeOH)}. The 13C NMR spectra (Table 2) displayed 22 signals including one exo-methylene and
Table 1 H and 13C NMR spectroscopic data for hikiokoshins A–C (1–3) in pyridine-d5.
1
Position
1 dC
1 2
72.4 27.4
3
39.9
4 5 6
32.1 48.7 62.6
7 8 9 10 11
164.2 123.0 161.4 46.9 22.3
12
26.3
13 14
38.4 29.1
15 16 17 18 19 20 1-OH 15-OH 15-OMe 1-OAc
a
170.6 148.2 120.6 32.9 22.0 67.1
Overlapped with signal of HOD.
2 dH (J in Hz) 3.97 (dd, 13.4, 2.2) 1.86 (brd, 13.4) 1.75 (dddd, 13.4, 13.4, 13.4, 2.4) 1.38 (ddd, 13.4, 13.4, 2.4) 1.31 (brd, 13.4) – 2.06 (d, 11.7) 5.08 (m) 3.92 (d, 11.7) – – – – 2.72 (dd, 16.0, 8.8) 2.17 (m) 2.01 (m), 1.93 (m) 2.90 3.12 1.98 – – 5.48 0.91 0.73 5.09 6.75
(m) (d, 14.8) (m)
(brs), 5.20 (brs) (3H, s) (3H, s) (d, 12.2), 4.86 (d, 12.2) (brs)
dC 76.0 26.3
3 dH (J in Hz)
32.0 64.9 74.0
5.39 (dd, 14.2, 4.6) 1.85 (m) 1.72 (dddd, 14.2, 14.2, 14.2, 3.1) 1.45 (ddd, 14.2, 3.1, 3.1) 1.32 (ddd, 14.2, 14.2, 3.1) – 1.91 (d, 6.4) 4.37 (d, 6.4)
174.5 57.1 57.2 47.1 67.4
– – 2.15 (d, 10.3) – 3.99 (ddd, 10.3, 10.3, 3.7)
39.8
43.1
5.00 (m)a 2.05 (ddd, 13.1, 13.1, 4.0) 1.90 (m)
39.9 34.0 62.7 203.5
1.43 (ddd, 13.1, 13.1, 4.0) 1.35 (m) – 2.49 (d, 5.2) 10.17 (d, 5.2)
175.6 52.6 38.0 42.9 16.4
– – 2.73 (dd, 12.8, 4.3) – 1.53 (m), 1.34 (m)
32.8
1.91 (m), 1.34 (m)
36.3 29.9
2.62 2.22 1.71 4.90 – 5.39 0.89 1.06 5.43
167.2 144.9 123.0 32.9 20.7 68.3
51.5 170.0
3.60 (3H, s) –
170.2
21.4
2.12 (3H, s)
21.3
(brs), 5.59 (brs) (3H, s) (3H, s) (2H, brs)
dH (J in Hz)
75.7 24.2
2.55 1.58 3.29 2.68 1.95 – – 6.16 1.18 1.09 4.88
35.1 32.4
(brd, 10.3) (q, 10.3) (m) (brd, 12.5) (dd, 12.5, 12.5)
dC
82.9 159.7 109.5 32.8 23.8 67.7
(m) (dd, 12.2, 5.0) (d, 12.2) (brs) (brs), 5.14 (brs) (3H, s) (3H, s) (d, 11.6), 5.37 (d, 11.6)
8.24 (d, 4.6) – 2.17 (3H, s)
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17 2
3 4
18 19 1H-1H
5 6
OH 1 10 20
12
11
13
9
14 8
O
7
COSY
HMBC
15
O O
13
11
16
O
1 10 2
20
6 5
3
1
15
8
9
17
18 19
NOESY
Fig. 1. Selected 2D NMR correlations and relative stereochemistry (protons of methyl groups are not shown) for hikiokoshin A (1).
Scheme 1. Structures of hikiokoshins A–I (1–9).
one acetoxyl group, as well as the characteristic resonance of an acetal group (dC 95.4). Comparison of the 1D NMR spectroscopic data for 4 with literature data suggested that 4 is an analog of maoecrystal X (Fig. S1), an ent-7,20-epoxykaurane diterpene (Shen et al., 2005). The molecular formula, C22H32O5, of 4, which was elucidated by HRESIMS (m/z 399.21398 [M+Na]+, D0.22 mmu), was smaller than that of maoecrystal X by 16 mass units. These observations implied that 4 is a deoxy derivative of maoecrystal X. HMBC correlations from H2-6 to C-7 and C-8, and an 1H–1H COSY cross-peak of H-5/H-6 indicated that 4 has an sp3 methylene in place of an oxymethine (CH-6) in maoecrystal X. Thus, the structure of hikiokoshin D (4) was assigned as shown in Scheme 1. The HRESIMS of hikiokoshin E (5) established that the molecular formula of 5 was the same as that of 4 (m/z 399.21448 [M+Na]+, D +0.28 mmu). The 1H and 13C NMR spectroscopic data for 5 (Table 2) were similar to those of 4, except for the signals from ring A (C-1–C-5 and C-10). The 1H–1H COSY cross-peaks of an oxygenated methine (H-2) to H2-1 and H2-3 disclosed that 5 has a hydroxyl group at C-2 (Fig. S5). The b-orientation of the hydroxyl group at C-2 was deduced by the NOESY correlations for H-2/H3-19 and H-2/H-20a (Fig. S5). Therefore, the structure of hikiokoshin E (5) was assigned as a 2-hydroxy analog of 4. The HRESIMS has the same molecular formula, C24H34O8, for hikiokoshins F (6) and G (7) (m/z 473.21503 [M+Na]+, D +0.44 mmu for 6; m/z 473.21429 [M+Na]+, D 0.30 mmu for 7). Interpretation of the 1H and 13C NMR spectra of 6 and 7 (Table 2) implied that 6 and 7 are diterpenes possessing an ent-7, 20-epoxykaurane skeleton with two hydroxyl and two acetoxyl groups. In 6, the presence of oxygen functions at C-1, C-2, C-6,
and C-15 was deduced by interpretation of the 1H–1H COSY and HMBC spectra (Fig. S6), taking their carbon chemical shifts into consideration. In addition, the chemical shifts of H-6 (dH 5.88) and H-15 (dH 6.25) were at lower field relative to those of H-1 (dH 3.89) and H-2 (dH 4.12). These observations indicated the presence of acetoxyl groups at C-6 and C-15, and of hydroxyl groups at C-1 and C-2 in 6. Analysis of the NOESY spectrum of 6 established the orientations of 1b-OH, 2b-OH, 6b-OAc, and 15b-OAc (Fig. S6). Similarly, the structure of hikiokoshin G (7) was elucidated to be an ent-7,20-epoxykaurane with 3b-OH, 6b-OAc, 14b-OH, and 15b-OAc groups (Scheme 1). Hikiokoshin H (8), C24H32O8, was isolated as an optically active colorless amorphous solid {[a]D 6.6 (c 0.50, MeOH)}. Its IR spectrum implied the existence of hydroxyl (3393 cm1), ester carbonyl (1742 cm1), and ketone (1699 cm1) functionalities. Analysis of the 1H and 13C NMR spectra (Table 3) suggested it to be an ent-7,20-epoxykaurane diterpene having one ketone, one hydroxymethyl, two acetoxyl, and one exo-methylene groups. The positions of these functionalities were assigned by 1H-1H COSY and HMBC analysis as shown in Scheme 1. NOESY analysis (Fig. S8) indicated the a-orientation of the hydroxymethyl group (C-19) and the b-orientations of the acetoxyl groups at C-6 and C-15. Accordingly, the structure of hikiokoshin H (8) was shown in Scheme 1. The 1H and 13C NMR spectra of hikiokoshin I (9) (Table 3), C24H32O8, were similar to those of 8, and also showed the resonances due to an sp3 oxymethine (dH 4.28, dC 74.8) and a tertiary methyl in place of the signals of an sp3 methylene (CH2-3) and hydroxymethyl (CH2-19) in 8. An 1H–1H COSY cross-peak of the oxymethine proton (H-3) with H2-2 and HMBC correlations from
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Table 2 H and 13C NMR spectroscopic data for hikiokoshins D–F (4–6) in pyridine-d5.
1
Position
4 dC
a
1 2 3
73.7 30.4 39.0
4 5 6
33.7 48.5 34.7
7 8 9 10 11 12 13 14
95.4 52.7 46.6 40.1 19.0 33.1 36.6 26.1
15 16 17 18 19 20 1-OH 2-OH 7-OH 6-OAc
76.6 158.8 108.2 31.8 20.7 63.7
15-OAc
170.6 21.1
5 dH (J in Hz) 3.70 1.84 1.41 1.28 – 1.69 2.14 2.04 – – 2.22 – 2.21 2.21 2.58 2.11 2.04 6.28 – 5.10 0.80 1.07 4.84 5.72
(m) (dd, 13.0, 12.0) (m)
dH (J in Hz)
1.89 (m), 1.24 (m) 4.02 (m) 2.07 (m), 1.43 (m)
69.9 66.8 42.9
35.1 48.4 33.9
– 1.65 2.12 2.00 – – 2.07 – 1.47 2.07 2.54 1.99 1.92 6.23 – 5.09 0.85 1.08 4.26
34.9 49.5 74.9
3.89 4.12 2.46 1.67 – 2.33 5.88
95.6 51.0 38.8 42.2 15.1 31.7 36.7 27.5
– – 3.29 – 2.02 2.13 2.57 2.09
75.1 159.6 108.6 33.1 24.4 65.8
6.25 – 5.22 1.05 1.31 4.22 5.92 6.21 8.32 – 2.11 – 2.16
76.6 158.4 108.7 32.7 22.1 67.0
(dd, 12.8, 7.7) (dd, 14.5, 12.8) (dd, 14.5, 7.7)
(m) (m), 1.24 (m) (m), 1.43 (m) (dd, 8.6, 4.5) (dd, 12.2, 4.5) (d, 12.2) (s) (brs), 5.08 (brs) (3H, s) (3H, s) (d, 9.5), 4.06 (d, 9.5)
6.06 (brs) 7.94 (brs)
7.88 (brs)
– 2.21 (3H, s)
dC
41.5 63.9 51.1
95.6 52.3 45.4 37.0 15.6 32.7 36.5 26.3
(m)
(2H, m)a (3H, s) (3H, s) (d, 9.4), 4.48 (d, 9.4) (brd, 3.7)
dH (J in Hz)
dC
(brs) (2H, m) (brd, 13.4) (m)
(m), 2.02 (m) (m), 1.52 (m) (dd, 8.2, 4.9) (d, 12.0) (m) (s)
6
170.7 21.2
– 2.18 (3H, s)
171.0 21.1 171.0 21.8
(brs) (brd, 12.4) (dd, 12.4, 12.4) (dd, 12.4, 3.4) (d, 7.1) (d, 7.1)
(dd, 12.5, 5.0) (m), 1.64 (m) (m), 1.58 (m) (brd, 10.3) (2H, m) (s) (brs), 5.09 (brs) (3H, s) (3H, s) (d, 9.4), 4.18 (d, 9.4) (brs) (brs) (s) (3H, s) (3H, s)
Overlapped with signal of HOD.
Table 3 H and 13C NMR spectroscopic data for hikiokoshins G–I (7–9) in pyridine-d5.
1
Position
7 dC
dH (J in Hz)
dC
dH (J in Hz)
212.2
–
210.8
–
24.3
2
25.8
2.04 (ddd, 13.2, 13.2, 6.1) 1.06 (brd, 13.2) 1.71 (2H, m)
3 4 5 6 7 8 9 10 11 12 13 14
74.5 38.2 48.1 73.5 98.0 52.4 46.6 35.6 15.0 32.0 45.5 75.6
3.65 – 2.53 5.98 – – 2.74 – 1.53 2.29 2.77 5.02
74.1 158.8 111.2 28.6 23.1 67.1
6.70 – 5.38 1.25 1.22 4.34 6.09 8.31 7.97
15-OAc
170.8 21.3 171.1 22.0
Overlapped with signal of HOD.
9
dC
1
15 16 17 18 19 20 3-OH 7-OH 14-OH 19-OH 6-OAc
a
8 dH (J in Hz)
(brs) (d, 7.9) (d, 7.9)
(m) (m), 1.22 (m) (m), 1.55 (m) (m) (m)a (s) (brs), 5.23 (brs) (3H, s) (3H, s) (d, 9.7), 3.99 (d, 9.7) (brs) (s) (brs)
– 2.14 (3H, s) – 2.24 (3H, s)
35.7
2.84 (m), 2.40 (m)
47.4
34.1 36.9 53.9 74.5 96.3 52.1 42.9 48.9 18.2 32.3 35.9 27.1
1.98 – 2.99 6.41 – – 2.80 – 1.86 2.07 2.55 2.15 2.07 6.20 – 5.18 1.19 3.80 4.75
74.8 39.1 51.7 73.5 96.4 52.2 42.7 48.9 17.8 32.3 35.8 27.0
75.3 158.8 109.7 25.1 66.6 64.7
(m), 1.85 (m) (d, 8.9) (d, 8.9)
(dd, 14.2, 6.1) (m), 1.19 (m) (m), 1.39 (m) (dd, 8.8, 4.9) (d, 12.8) (m) (brs) (brs), 5.14 (brs) (3H, s) (d, 11.0), 3.74 (d, 11.0) (d, 10.0), 4.73 (d, 10.0)
6.80 (brs) – 2.13 (3H, s) – 2.36 (3H, s)
(dd, 16.9, 9.2) (dd, 16.9, 4.6) (dd, 9.2, 4.6) (m) (d, 9.2)
(m) (m), 1.23 (m) (m), 1.37 (m) (dd, 8.3, 4.7) (m), 2.04 (m)
75.3 158.6 109.6 25.8 16.5 65.5
6.16 – 5.14 1.26 1.12 4.66 6.95 8.64
170.8 21.2 170.7 21.9
– 2.13 (3H, s) – 2.28 (3H, s)
8.48 (brs)
170.8 21.2 170.8 22.0
3.37 2.74 4.28 – 2.71 5.83 – – 2.71 – 1.84 2.07 2.55 2.12
(brs) (brs), 5.13 (brs) (3H, s) (3H, s) (d, 9.9), 4.23 (d, 9.9) (brs) (brs)
N. Tanaka et al. / Phytochemistry 102 (2014) 205–210
Me-18 to C-3, C-4, C-5, and C-19 established that 9 had a geminal dimethyl group at C-4 and a hydroxyl group at C-3. A NOESY correlation from H-3 to H-5 indicated as orientation of the hydroxyl group at C-3. Thus, the structure of hikiokoshin I (9) was assigned as shown in Scheme 1. Antimicrobial activities of hikiokoshins A (1) and B (2) against Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Aspergillus niger, Trichophyton mentagrophytes, Candida albicans, and Cryptococcus neoformans were evaluated. Hikiokoshin A (1) exhibited antifungal activity against A. niger and C. neoformans (IC50 16 lg/mL each), whereas hikiokoshin B (2) did not show such activity against any strains (IC50 > 32 lg/mL). Hikiokoshins A–I (1–9) showed no cytotoxicity (IC50 > 10 lg/mL) against murine lymphoma L1210 and human epidermoid carcinoma KB cells in vitro. Conclusion Investigation for the extract of the leaves of I. japonicus (Burm. f.) H. Hara resulted in the isolation of nine new diterpenes, hikiokoshins A–I (1–9), together with twelve known diterpenes. Although a large number of diterpenes with various skeletons have been isolated from Isodon spp. to date (Sun et al., 2006), hikiokoshins A (1) and B (2) have the rare ternifonane (Zou et al., 2012) and ent-6,7:8,15-diseco-6,8-cyclokauran-7,20-olide skeletons (Han et al., 2006), respectively. Hikiokoshin C (3) possesses an ent-6,7-secokaurane skeleton, while hikiokoshins D–I (4–9) are ent-7,20-epoxykaurenes. Experimental section General procedures Optical rotations were recorded using a JASCO P-1030 digital polarimeter. IR spectra were recorded on a JASCO FT/IR-460 Plus spectrophotometer. NMR spectra were measured on a Bruker AMX-600 spectrometer. The 7.19 and 123.5 ppm resonances of residual pyridine were used as internal references for 1H and 13C chemical shifts, respectively. ESIMS spectra were obtained on a Thermo Scientific Exactive spectrometer. C18 HPLC column: column A, YMC-Pack ODS-AQ, YMC Co., Ltd., 20 250 mm; column B, Cosmosil 5C18-MS-II, Nacalai Tesque, Inc., 10 250 mm. Plant Material I. japonicus (Burm. f.) H. Hara was cultivated at the Experimental Station for Medicinal Plant Studies, Hokkaido University, and the aerial parts were collected at in October 2009. Herbarium specimens were deposited in Graduate School of Pharmaceutical Sciences, Hokkaido University (specimen number: HIJ200910). Extraction and isolation The dried leaves of I. japonicus (1.05 kg) were extracted with MeOH (18 L, 3 days 3) at room temperature to give the extract (211 g). A part (107 g) of the extract was partitioned successively with n-hexane (500 mL 4), EtOAc (500 mL 4), and H2O (500 mL), respectively. The EtOAc-soluble portion (27.2 g) was subjected to silica gel column chromotography (CC) (eluent CHCl3/MeOH, 100:0?0:100) to give seventeen fractions (frs. 1– 17). Fr. 10 was loaded on a Sephadex LH-20 column (MeOH/toluene, 50:50) to remove fatty acids, and then applied to a silica gel column (n-hexane/EtOAc, 70:30?0:100) to afford ten fractions (frs. 10.1–10). Fr. 10.5 was purified by silica gel CC (CHCl3/acetone, 97:3?0:100) and C18 HPLC (column A; flow rate 6.0 mL/min; UV
209
detection at 220 nm; MeOH/H2O, 60:40) to yield hikiokoshin C (3, 0.8 mg, 1.4 104%). Separation of fr. 10.6 on a silica gel column (CHCl3/acetone, 97:3?0:100) gave nine fractions (frs. 10.6.1–9). Fr. 10.6.4 was loaded on a C18 column (MeOH/H2O, 30:70?100:0) and C18 HPLC (column A; 5.0 mL/min; 220 nm; MeOH/H2O, 50:50) to isolate hikiokoshin B (2, 1.2 mg, 2.2 104%). Fr. 10.6.5 was subjected to silica gel CC (n-hexane/EtOAc, 50:50?0:100) and then purified by C18 HPLC (column A; 6.0 mL/min; 220 nm; MeOH/ H2O, 60:40) to give hikiokoshin I (9, 1.9 mg, 3.6 104%). Purification of fr. 10.6.6 on a C18 column (MeOH/H2O, 30:70?100:0) and C18 HPLC (column A; 4.0 mL/min; 220 nm; MeOH/H2O, 60:40) gave hikiokoshins D (4, 1.2 mg, 2.2 104%) and H (8, 2.3 mg, 4.2 104%). Fr. 10.8 was applied to a silica gel column (CHCl3/acetone, 97:3?0:100) to give seven fractions (frs. 10.8.1–7). Fr. 10.8.4 was purified by a C18 column (MeOH/H2O, 20:80?100:0) and C18 HPLC (column B; 3.0 mL/min; 220 nm; MeOH/H2O, 50:50) to afford hikiokoshin A (1, 1.2 mg, 2.2 104%). Fr. 10.8.5 was applied to a C18 column (MeOH/H2O, 40:60?100:0) and then purified by C18 HPLC (column A; 5.0 mL/min; 220 nm; MeOH/H2O, 50:50) to give hikiokoshins E (5, 1.4 mg, 2.6 104%), F (6, 0.5 mg, 0.9 104%), and G (7, 0.7 mg, 1.3 104%). During the purification process, twelve known diterpenes, rabdolasional (4.1 mg, 7.7 104%), isorubesin D (3.9 mg, 7.4 104%), odonicin (639 mg, 1.2 101%), neorabdosin (23.8 mg, 4.5 103%), effusanin A (0.7 mg, 1.3 104%), rabdolongin A (1.0 mg, 1.9 104%), nodosin (4.6 mg, 8.7 104%), epinodosin (2.4 mg, 4.5 104%), isodocarpin (1.3 mg, 2.5 104%), sculponeatin E (12.0 mg, 2.3 103%), serrin C (6.4 mg, 1.2 103%), and ent-6,7-seco6,20-epoxy-6b-methoxykaur-16-ene-15a,20b-diol (10.0 mg, 1.9 103%) were isolated. Hikiokoshin A (1) Colorless amorphous solid; ½a29 D +24.2 (c 0.23, MeOH); IR (KBr) mmax 3484, 1708, and 1683 cm1; for 1H and 13C NMR spectroscopic data see Table 1; HRESIMS m/z 369.16736 ([M+Na]+, calcd for C20 H26O5Na, 369.16725). Hikiokoshin B (2) Colorless amorphous solid; ½a28 D 6.4 (c 0.24, MeOH); IR (KBr) mmax 3440 and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 1; HRESIMS m/z 459.19907 ([M+Na]+, calcd for C23H32O8 Na, 459.19894). Hikiokoshin C (3) Colorless amorphous solid; ½a24 D 7.4 (c 0.19, MeOH); IR (KBr) mmax 3447, 1734, and 1716 cm1; for 1H and 13C NMR spectroscopic data see Table 1; HRESIMS m/z 413.19327 ([M+Na]+, calcd for C22 H30O6Na, 413.19346). Hikiokoshin D (4) Colorless amorphous solid; ½a29 D –60.1 (c 0.28, MeOH); IR (KBr) mmax 3446, 1739, and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS m/z 399.21398 ([M+Na]+, calcd for C22 H32O5Na, 399.21420). Hikiokoshin E (5) Colorless amorphous solid; ½a28 D 40.3 (c 0.23, MeOH); IR (KBr) mmax 3423, 1737, and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS m/z 399.21448 ([M+Na]+, calcd for C22 H32O5Na, 399.21420). Hikiokoshin F (6) Colorless amorphous solid; ½a25 D 41.2 (c 0.13, MeOH); IR (KBr) mmax 3446, 1734, and 1717 cm1; for 1H and 13C NMR spectroscopic data see Table 2; HRESIMS m/z 473.21503 ([M+Na]+, calcd for C24 H34O8Na, 473.21459).
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N. Tanaka et al. / Phytochemistry 102 (2014) 205–210
Hikiokoshin G (7) Colorless amorphous solid; ½a25 D 68.1 (c 0.17, MeOH); IR (KBr) mmax 3358, 1735, and 1719 cm1; for 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 473.21429 ([M+Na]+, calcd for C24 H34O8Na, 473.21459). Hikiokoshin H (8) Colorless amorphous solid; ½a28 D 6.6 (c 0.50, MeOH); IR (KBr) mmax 3393, 1742, and 1699 cm1; for 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 471.19882 ([M+Na]+, calcd for C24 H32O8Na, 471.19894). Hikiokoshin I (9) Colorless amorphous solid; ½a29 D 23.9 (c 0.42, MeOH); IR (KBr) mmax 3447, 1734, and 1701 cm1; for 1H and 13C NMR spectroscopic data see Table 3; HRESIMS m/z 471.19850 ([M+Na]+, calcd for C24 H32O8Na, 471.19894). Antimicrobial testing Antimicrobial assays of hikiokoshins A (1) and B (2) were carried out as previously described (Nagai et al., 1993). Amphotericin B, micafungin, hygromycin B, and kanamycin were used as controls for antimicrobial activities against E. coli (MIC, >31.2, >62.5, 2.0, and 31.2, 0.5, 0.5, and 31.2, 0.5, 2.0, and 31.2, 7.8, 1.0, and 1.0 lg/mL, respectively), A. niger (IC50, 1.8,
