Geranylgeraniol-Type Diterpenoids, Boletinins AâJ, from Boletinus

958

J. Nat. Prod. 2004, 67, 958-963

Geranylgeraniol-Type Diterpenoids, Boletinins A-J, from Boletinus cavipes as Inhibitors of Superoxide Anion Generation in Macrophage Cells Tsunashi Kamo,* Kazuya Sato, Kikuo Sen, Hisao Shibata, and Mitsuru Hirota Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano 399-4598, Japan Received December 18, 2003

In addition to16-hydroxygeranylgeraniol (1) and cavipetin B (2), 10 new geranylgeraniol-type diterpenoids, named boletinins A-J (3-12), were isolated from the fruiting bodies of Boletinus cavipes. Compounds 1-9 and 11 exhibited inhibitory activities of less than 10% at 25-125 µM in the xanthine oxidase test. A bioassay on superoxide anion (O2•-) generation in macrophage cells revealed that 1 and 4-12 suppressed the generation by more than 20% at 25 µM. Compounds 4 and 5 showed inhibitory activities against O2•generation of more than 50% at 50 µM and exhibited no or low cytotoxicities against macrophage cells at 25-50 µM, suggesting that 4 and 5 are the most promising candidates for O2•- generation inhibitors. O-Acyl geranylgeraniol derivatives, 2 and 7-12, showed cytotoxicities at 25 µM. Generation of superoxide anion (O2•-), one of the active oxygens, is essential for the defense of living bodies and for signal transfer in living cells. The overgeneration of O2•-, however, causes oxidative stress-related diseases such as inflammation, hypertension, aging, and cancer.1 It would therefore be beneficial in maintaining a healthy body to scavenge overgenerated O2•- or to depress O2•- generation.2,3 A large number of natural products have already been isolated and recognized as radical scavengers;4,5 therefore, we have focused on O2•- generation inhibitors as candidates for prevention of oxidative stress-related diseases. An African legume, Cassia spectabilis, is one of the potent plants from which a new piperidine alkaloid, spectamine A, has been isolated as a possible inhibitor of O2•- generation.6 We also conducted screening tests of the extracts from various species of mushrooms, finding in some species remarkable inhibitory activities against O2•generation in macrophages. In the present paper, we describe the isolation and characterization of 10 new geranylgeraniol-type diterpenoids from Boletinus cavipes (Opat.) Kalchbr (Boletaceae). The inhibitory activities of O2•- generation of the isolated compounds were evaluated using macrophage cells induced by a tumor promoter, 12O-tetradecanoylphorbol-13-acetate (TPA). The cell viabilities and xanthine oxidase (XOD) tests were also carried out to distinguish cytotoxic compounds and O2•- scavengers, respectively, from inhibitors of O2•- generation. Results and Discussion Bioassay-guided purification from a methanolic extract of B. cavipes led to the isolation of 16-hydroxygeranylgeraniol (1).7 The notable inhibitory activity of 1 against O2•- generation urged us to examine the extract for related compounds, resulting in isolation of cavipetin B (2) and 10 new geranylgeraniol-type diterpenoids (3-12).8 The 1H NMR spectrum of 3 was similar to that of 1, except for the presence of trans-CHdCH (each 1H, δ 5.59 and 5.52, J ) 15.6 Hz), dC-CH2-Cd (2H, δ 2.69), and C-C-CH2-C-C (2H, δ 1.60) signals (Table 1).7 In the 13C NMR spectrum of 3 (Table 2), an additional signal assignable to an oxygen-bearing carbon was observed other than those at δ 68.9 (CH2) and 59.4 (CH2). The additional signal * To whom correspondence should be addressed. Tel: +81-265-77-1602. Fax: +81-265-77-1629. E-mail: [email protected].

10.1021/np030535g CCC: $27.50

(δ 72.9) is quaternary; therefore the position of oxygenation must be C-3, C-7, C-11, or C-15. These observations led to the partial structure (Figure 1a), from which the structure of 3 was elucidated using 2D NMR spectra. The H-H COSY spectrum of 3 demonstrated the correlations between H2-1 and H-2 and between H-2 and CH3-20, excluding C-3 from the possible positions of the oxygenated carbon. The correlations between H-14 and H2-16 and

© 2004 American Chemical Society and American Society of Pharmacognosy Published on Web 05/14/2004

Geranylgeraniol-Type Diterpenoids

Journal of Natural Products, 2004, Vol. 67, No. 6 959

Table 1. 1H NMR Data for 3-6 (500 MHz, CDCl3) position

3

4

5

1 2 4 5

4.14 (2H, d, 6.8 Hz) 5.39 (1H, t, 6.8 Hz) 2.21 (2H, m) 2.14 (2H, m)

6 8 9

5.13 (1H, t, 6.3 Hz) 2.69 (2H, d, 6.4 Hz) 5.59 (1H, dt, 15.6 and 6.4 Hz)

4.15 (2H, d, 6.9 Hz) 5.46 (1H, t, 6.9 Hz) 2.09 (2H, m) 1.64 (1H, m) 1.53 (1H, m) 4.05 (1H, d, 7.4 and 5.2 Hz) 2.14 (2H, m) 2.19 (2H, m)

10 12 13

5.52 (1H, d, 15.6 Hz) 1.60 (2H, m) 2.13 (2H, m)

14 16

6

4.12 (2H, d, 6.7 Hz) 5.39 (1H, t, 6.7 Hz) 2.02 (2H, m) 2.15 (2H, m)

4.15 (2H, d, 6.8 Hz) 5.41 (1H, t, 6.8 Hz) 1.99 (2H, m) 2.00 (2H, m) 5.15 (1H, t, 6.7 Hz) 1.99 (2H, m) 2.00 (2H, m)

5.14 (1H, t, 6.2 Hz) 2.06 (2H, m) 2.15 (2H, m)

5.17 (1H, t, 6.8 Hz) 2.02 (2H, m) 1.68 (1H, m) 1.58 (1H, m) 4.06 (1H, dd, 8.1 and 4.3 Hz) 2.21 (2H, m) 2.23 (2H, m)

5.42 (1H, t, 7.3 Hz) 3.99 (2H, s)

5.35 (1H, t, 6.9 Hz) 3.98 (2H, s)

5.42 (1H, t, 6.5 Hz) 3.99 (2H, s)

17

1.67 (3H, s)

1.66 (3H, s)

1.68 (3H, s)

18

1.29 (3H, s)

1.61 (3H, s)

19

1.67 (3H, s)

20

1.59 (3H, s)

5.05 (1H, s) 4.89 (1H, s) 1.69 (3H, s)

5.05 (1H, s) 4.87 (1H, s) 1.63 (3H, s)

Table 2.

13C

5.16 (1H, t, 6.7 Hz) 2.09 (2H, m) 1.76 (1H, m) 1.72 (1H, m) 4.23 (1H, t, 6.6 Hz) 4.30 (1H, d, 13.1 Hz) 4.16 (1H, d, 13.1 Hz) 5.13 (1H, s) 5.10 (1H, s) 1.62 (3H, s) 1.60 (3H, s)

1.66 (3H, s)

1.68 (3H, s)

NMR Data for 3-12 (125 MHz, CDCl3)

position

3

4

5

6

7

8

9

10

11

12

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

59.4 (CH2) 123.8 (CH) 139.2 (C) 39.3 (CH2) 26.0 (CH2) 124.8 (CH) 134.1 (C) 42.4 (CH2) 126.1 (CH) 138.0 (CH) 72.9 (C) 42.3 (CH2) 22.5 (CH2) 126.3 (CH) 135.0 (C) 68.9 (CH2) 13.7 (CH3) 28.2 (CH3) 16.2 (CH3) 16.2 (CH3)

59.4 (CH2) 123.7 (CH) 139.4 (C) 35.6 (CH2) 33.4 (CH2) 75.1 (CH) 151.4 (C) 31.3 (CH2) 26.5 (CH2) 124.3 (CH) 135.0 (C) 39.2 (CH2) 25.8 (CH2) 125.7 (CH) 135.2 (C) 68.9 (CH2) 13.7 (CH3) 16.0 (CH3) 110.1 (CH2) 16.3 (CH3)



59.5 (CH2) 123.0 (CH) 140.1 (C) 39.7a (CH2) 26.7b (CH2) 125.0c (CH) 135.4d (C) 39.4a (CH2) 26.2b (CH2) 123.8c (CH) 134.1d (C) 38.9a (CH2) 26.1b (CH2) 129.9 (CH) 129.2d (C) 71.0 (CH2) 14.0 (CH3) 16.0e (CH3) 15.9e (CH3) 16.3 (CH3) 164.7 (C) 132.9 (CH) 135.0 (CH) 167.3 (C)

62.3 (CH2) 117.7 (CH) 143.3 (C) 39.7a (CH2) 26.7b (CH2) 124.6c (CH) 135.6d (C) 39.4a (CH2) 26.2b (CH2) 123.5c (CH) 134.6d (C) 39.2a (CH2) 26.0b (CH2) 126.4 (CH) 134.4d (C) 69.0 (CH2) 13.7 (CH3) 16.1e (CH3) 16.0e (CH3) 16.5 (CH3) 164.8 (C) 132.8 (CH) 135.4 (CH) 168.4 (C)

62.4 (CH2) 117.6 (CH) 143.3 (C) 39.6 (CH2) 26.2 (CH2) 123.7 (CH) 135.4 (C) 39.5 (CH2) 26.6 (CH2) 125.6 (CH) 133.4 (C) 38.0 (CH2) 27.5 (CH2) 154.6 (CH) 139.4 (C) 195.4 (CH) 9.2 (CH3) 15.9 (CH3) 16.0 (CH3) 16.6 (CH3) 164.7 (C) 132.4 (CH) 135.7 (CH) 168.5 (C)

62.4 (CH2) 117.8 (CH) 143.2 (C) 39.5 (CH2) 26.0 (CH2) 123.6 (CH) 135.6 (C) 39.5 (CH2) 26.6 (CH2) 125.2 (CH) 133.7 (C) 38.0 (CH2) 27.5 (CH2) 145.3 (CH) 126.8 (C) 173.3 (C) 12.0 (CH3) 16.0 (CH3) 15.9 (CH3) 16.5 (CH3) 164.7 (C) 132.8 (CH) 135.4 (CH) 169.7 (C)

62.6 (CH2) 117.8 (CH) 143.1 (C) 39.5 (CH2) 26.2 (CH2) 123.7 (CH) 135.4 (C) 39.6 (CH2) 26.6 (CH2) 125.6 (CH) 133.4 (C) 38.0 (CH2) 27.5 (CH2) 154.6 (CH) 139.4 (C) 195.4 (CH) 9.2 (CH3) 15.9 (CH3) 16.0 (CH3) 16.6 (CH3) 166.9 (C) 146.3 (C) 125.6 (CH) 169.9 (C) 14.6 (CH3)

62.6 (CH2) 117.9 (CH) 143.0 (C) 39.5 (CH2) 26.0 (CH2) 123.6 (CH) 135.4 (C) 39.6 (CH2) 26.6 (CH2) 125.2 (CH) 133.7 (C) 38.0 (CH2) 27.6 (CH2) 145.2 (CH) 126.9 (C) 173.3 (C) 12.0 (CH3) 16.1 (CH3) 15.9 (CH3) 16.5 (CH3) 167.0 (C) 146.1 (C) 125.8 (CH) 170.8 (C) 14.6 (CH3)

a-e

Values with the same symbol may be interchanged.

Figure 1. Partial structures of (a) 3, (b) 4-6, and (c) 11 and 12.

between H-14 and CH3-17 ruled out C-15. The oxygenated carbon position turned out to be C-11 and not C-7, as proven by the H-H COSY and the HMBC correlations. The HREIMS of 3 showed an ion at m/z 286.2274 (C20H30O, [M - 2H2O]+), which corresponded to the molecular formula of an oxygenated form of 1 (C20H34O2). Thus, the structure

of 3 was established, and this new diterpenoid was named boletinin A (3). Compound 4 showed an [M]+ ion at m/z 322.2518 in the HREIMS, suggesting a molecular formula of C20H34O3. The 1H NMR spectrum of 4 exhibits signals similar to those of 1 (Table 1). A couple of signals assignable to one of the four -CHdC(CH3)- groups are absent, while signals due to CdCH2 (each 1H, δ 5.05 and 4.89) and -CH(OH)- (1H, δ 4.05) were observed in the spectrum of 4. With these data, the signals at δ 110.1 (CdCH2) and δ 75.1 [-CH(OH)-] in the 13C NMR spectrum suggested the partial structure shown in Figure 1b (Table 2). The signal assignable to CH320 was determined using the H-H COSY spectrum. The correlations from C-4 to CH3-20 and from C-4 to H-6 were observed in the HMBC spectrum of 4. All these spectral data revealed that the oxygenated position of 4 was located at C-6. We named this new diterpenoid boletinin B (4). The 1H and 13C NMR spectra of 5 were almost identical to those of 4 except for slight differences in the δ values

960

Journal of Natural Products, 2004, Vol. 67, No. 6

Kamo et al.

Table 3. 1H NMR Data for 7-10 (500 MHz, CDCl3) position

7

8

9

10

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

4.18 (2H, d, 6.9 Hz) 5.41 (1H, t, 6.9 Hz) 1.96 (2H, m) 2.01 (2H, m) 5.11 (1H, t, 6.9 Hz) 1.96 (2H, m) 2.01 (2H, m) 5.11 (1H, t, 6.9 Hz) 1.96 (2H, m) 2.01 (2H, m) 5.47 (1H, t, 7.0 Hz) 4.58 (2H, s) 1.67 (3H, s) 1.60 (3H, s) 1.60 (3H, s) 1.68 (3H, s) 6.85 (1H, d, 15.8 Hz) 6.92 (1H, d, 15.8 Hz)

4.72 (2H, d, 7.2 Hz) 5.37 (1H, t, 7.2 Hz) 1.96 (2H, m) 1.96 (2H, m) 5.08 (1H, t, 6.7 Hz) 1.96 (2H, m) 1.96 (2H, m) 5.11 (1H, t, 6.5 Hz) 1.96 (2H, m) 1.96 (2H, m) 5.39 (1H, t, 7.6 Hz) 4.00 (2H, s) 1.66 (3H, s) 1.60 (3H, s) 1.60 (3H, s) 1.73 (3H, s) 6.84 (1H, d, 15.8 Hz) 6.91 (1H, d, 15.8 Hz)

4.73 (2H, d, 7.0 Hz) 5.38 (1H, t, 7.0 Hz) 2.06 (2H, m) 2.06 (2H, m) 5.09 (1H, t, 6.4 Hz) 1.98 (2H, t, 8.0 Hz) 2.06 (2H, m) 5.15 (1H, t, 6.7 Hz) 2.16 (2H, t, 7.3 Hz) 2.45 (2H, q, 7.3 Hz) 6.47 (1H, t, 7.3 Hz) 9.38 (1H, s) 1.74 (3H, s) 1.63 (3H, s) 1.60 (3H, s) 1.73 (3H, s) 6.84 (1H, d, 15.8 Hz) 6.93 (1H, d, 15.8 Hz)

4.73 (2H, d, 7.2 Hz) 5.37 (1H, t, 6.9 Hz) 1.96 (2H, m) 1.96 (2H, m) 5.09 (1H, t, 6.3 Hz) 1.96 (2H, m) 1.96 (2H, m) 5.14 (1H, t, 6.4 Hz) 2.05 (2H, t, 7.4 Hz) 2.29 (2H, q, 7.4 Hz) 6.89 (1H, t, 7.4 Hz)

(Tables 1 and 2), suggesting that the partial structure shown in Figure 1b is located at a different position in the molecule. A peak at m/z 323.2597 (C20H35O3, [M + H]+) in the HRFABMS of 5 supported this presumption. The H-H COSY spectrum of 5 showed the correlation between H-14 and H2-16. In the HMBC spectrum, correlations from C-12 to H-18 and from C-12 to H-14 were observed. These spectral data showed that the oxygenation site is at C-10. Compound 5, named boletinin C, is a regioisomer of 4. Compound 6 is also a regioisomer of 4 on the basis of the 1H and 13C NMR spectra and the HRFABMS (Tables 1 and 2). The δ values (δ 4.23, 5.13, and 5.10) of -CH(OH)C(dCH2)- shifted downfield, compared with those of 4 (δ 4.05, 5.05, and 4.89) and 5 (δ 4.06, 5.05, and 4.87), probably due to the presence of the adjacent -CH2OH group at C-16. The HMBC spectrum showed the correlation from C-16 to H-17, elucidating the structure of 6, named boletinin D. The absolute configurations of 3-6 could not be determined because of their small amounts. The UV spectrum of 7 showed λmax at 198 nm, while those of 3-6 exhibited end absorption only (

Geranylgeraniol-Type Diterpenoids, Boletinins AâJ, from Boletinus

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Geranylgeraniol-Type Diterpenoids, Boletinins AâJ, from Boletinus