Tricalysiolides A–F, new rearranged ent-kaurane diterpenes from Tricalysia dubia

Tetrahedron 62 (2006) 1512–1519

Tricalysiolides A–F, new rearranged ent-kaurane diterpenes from Tricalysia dubia Koichi Nishimura,a Yukio Hitotsuyanagi,a Noriko Sugeta,a Kei-ichi Sakakura,a Kazuya Fujita,a Haruhiko Fukaya,a Yutaka Aoyagi,a Tomoyo Hasuda,a Takeshi Kinoshita,b Dong-Hui He,c Hideaki Otsuka,c Yoshio Takedad and Koichi Takeyaa,* a

b

School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan Faculty of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko-machi, Tsukui-gun, Kanagawa 199-0195, Japan c Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan d Faculty of Integrated Arts and Sciences, The University of Tokushima, 1-1 Minamijosanjima-cho, Tokushima 770-8502, Japan Received 29 September 2005; accepted 4 November 2005 Available online 15 December 2005

Abstract—Six rearranged ent-kaurane diterpenes, tricalysiolides A–F, having the cafestol-type carbon framework were isolated from the wood of Tricalysia dubia (Rubiaceae). Their absolute structures were determined on the basis of the 2D NMR spectroscopy, X-ray crystallographic analysis, and chemical methods. q 2005 Elsevier Ltd. All rights reserved.

1. Introduction

13

11 20

Tricalysia dubia (Lindl.) Ohwi (Rubiaceae) is an evergreen shrub or tree that is wildly distributed in Taiwan and the southern parts of China and Japan. From the leaves of this plant, unique rearranged ent-kaurane glycosides, tricalysiosides A–G, have been isolated.1 In the present study, from the wood of this plant, we isolated six new rearranged entkaurane diterpenes, tricalysiolides A–F (1–6), and determined their structures.

C

1

9

A

HB

14

17

16

2

R1

4

O

OR

OH

R

2

OH H

15 7

H 19

OH

O

6

H

18

O tricalysiolide A (1) tricalysiolide B (2) tricalysiolide C (3) tricalysiolide D (4)

R1

R2

H OH OMe H

H H H Me

O

R tricalysiolide E (5) tricalysiolide F (6)

H OH

Figure 1. Structures of tricalysiolides A–F (1–6).

2. Results and discussion By a series of column chromatography on highly porous synthetic resin (Diaion HP-20), silica gel, aminopropylbonded silica gel, and ODS HPLC, a hot MeOH extract of air-dried wood of T. dubia afforded six diterpenes named tricalysiolides A–F (1–6) (Fig. 1). Tricalysiolide A (1) was isolated as an amorphous solid, whose molecular formula was determined to be C20H28O4 from the [MCH]C peak at m/z 333.2074 (calcd for C20H29O4, 333.2066) in the HRESIMS. The IR spectrum indicated that 1 possessed hydroxyl Keywords: Tricalysiolide; Diterpene; Tricalysia dubia. * Corresponding author. Tel.: C81 426 76 3007; fax: C81 426 77 1436; e-mail: [email protected] 0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tet.2005.11.024

(3365 cmK1) and lactone (1742 cmK1) groups. The 1H NMR spectrum showed the presence of one tertiary methyl (d 0.67) and one olefinic proton (d 5.73) (Table 1). The 13C NMR spectrum suggested that 1 was a diterpenoid derivative with a total of 20 carbons, consisting of one methyl, nine methylenes, four methines, one trisubstituted double bond giving highly shielded (d 111.4) and deshielded (d 175.1) signals, one carbonyl, and three quaternary carbons (Table 2). On the basis of detailed analysis of the 1H–1H COSY, HMQC, and HMBC spectra, 1 was shown to be a cafestol-type rearranged kaurane diterpenoid with two hydroxyls at C-16 and C-17. In the HMBC spectrum, H-18 was correlated with C-3, C-4, C-5, and C-19, which implied that 1 had an a,b-unsaturated-g-lactone group attached to ring A (Fig. 2). From the NOESY correlations between

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519

1513

Table 1. 1H NMR data for tricalysiolides A–F (1–6) in C5D5Na Proton

1b

2b

3b

4b

5c

6b

1a 1b 2a 2b 3 5 6a 6b 7a 7b 9 11a 11b 12a 12b 13 14a 14b 15a 15b 17a 17b 18 20 OMe-3 OMe-16 OH-1 OH-3 OH-16 OH-17

1.67 (m) 0.97 (td, 13.8, 3.7) 2.21 (m) 1.52 (m) 4.75 (t, 8.9) 1.90 (m) 1.50 (m) 1.41 (m) 1.61 (m) 1.49 (m) 1.13 (d, 8.8) 1.69 (m) 1.47 (m) 1.86 (m) 1.42 (m) 2.43 (s-like) 2.02 (dd, 11.3, 4.1) 1.92 (d, 11.3) 1.82 (d, 14.2) 1.72 (d, 14.2) 4.11 (dd, 10.8, 4.6) 4.05 (dd, 10.8, 4.6) 5.73 (s) 0.67 (s, 3H)

1.78 (m) 1.52 (m) 2.52 (d, 13.7) 2.00 (m)

1.68 (m) 1.27 (m) 2.40 (ddd, 14.0, 4.1, 2.3) 1.87 (dd, 14.0, 4.7)

2.32 (dd, 17.6, 6.7) 1.80 (m) 5.62 (dt, 6.7, 2.1)

4.40 (t, 6.1) 6.09 (dd, 6.4, 1.6)

2.57 (d, 9.7) 1.59 (m) 1.38 (m) 1.65 (m) 1.56 (m) 1.29 (d, 8.6) 1.76 (m) 1.54 (m) 1.91 (m) 1.50 (m) 2.47 (s-like) 2.06 (dd, 11.1, 4.7) 1.97 (d, 11.1) 1.86 (d, 14.3) 1.77 (d, 14.3) 4.14 (dd, 10.8, 5.0) 4.06 (dd, 10.8, 5.0) 5.81 (s) 0.80 (s, 3H)

2.12 (m) 1.56 (m) 1.48 (m) 1.67 (m) 1.58 (m) 1.26 (d, 9.0) 1.76 (m) 1.52 (m) 1.92 (m) 1.50 (m) 2.50 (s-like) 2.10 (m) 1.98 (d, 11.4) 1.89 (d, 14.2) 1.80 (dd, 14.2, 1.5) 4.18 (dd, 10.9, 4.7) 4.09 (dd, 10.9, 4.7) 6.00 (d, 1.7) 0.79 (s, 3H) 3.16 (s, 3H)

1.66 (m) 0.94 (td, 13.8, 3.6) 2.20 (m) 1.49 (m) 4.75 (t, 9.3) 1.87 (m) 1.44 (m) 1.40 (m) 1.52 (m) 1.45 (m) 1.13 (d, 8.8) 1.69 (m) 1.46 (m) 1.85 (m) 1.45 (m) 2.48 (s-like) 1.82 (d, 11.0) 1.69 (m) 1.74 (d, 14.5) 1.48 (d, 14.5) 4.08 (dd, 12.3, 4.7) 4.00 (dd, 12.3, 4.7) 5.73 (s) 0.66 (s, 3H)

2.17 (dd, 11.7, 2.4) 1.64 (m) 1.43 (m) 1.65 (m) 1.53 (m) 1.21 (d, 8.7) 1.48 (m) 1.41 (m) 1.88 (m) 1.42 (m) 2.46 (s-like) 2.04 (dd, 11.3, 4.4) 1.88 (d, 11.3) 1.84 (d, 14.1) 1.71 (dd, 14.1, 1.6) 4.14 (dd, 10.8, 4.0) 4.05 (dd, 10.8, 4.0) 5.91 (s-like) 0.84 (s, 3H)

3.05 (dt, 11.8, 2.5) 1.78 (m) 1.56 (m) 1.68 (m) 1.62 (m) 2.40 (d, 8.5) 1.92 (m) 1.72 (m) 1.95 (m) 1.59 (m) 2.51 (s-like) 2.10 (dd, 11.4, 4.4) 2.01 (d, 11.4) 1.88 (d, 14.2) 1.83 (d, 14.2) 4.14 (dd, 10.8, 5.2) 4.05 (dd, 10.8, 5.2) 5.97 (dd, 2.5, 1.6) 0.94 (s, 3H)

3.31 (s, 3H) 6.87 (d, 5.8) 9.49 (s) 5.22 (s) 6.16 (br s)

5.24 (s) 6.16 (t, 6.2)

5.22 (s) 6.16 (t, 5.1)

5.24 (s) 6.16 (br s)

5.72 (br s)

5.21 (s) 6.12 (t, 5.3)

a

Assignments based on 1H–1H COSY, HMBC, and HMQC experiments. Multiplicity and J-values in Hz are given in parentheses. Recorded at 500 MHz. c Recorded at 400 MHz. b

H-1a/H3-20, H-1b/H-9, H-3/H-5, H-5/H-9, H-9/H-15b, H-11a/H-15b, H-11a/H-17a, H-11a/H-17b, H-12a/H-17a, H-12a/H-17b, and H-14b/H3-20 (Fig. 3), its stereostructure was determined to be as shown in Figure 1. Compound 1 corresponds to the aglycone moiety of Table 2.

C NMR data for tricalysiolides A–F (1–6) in C5D5N

OH

1a

2a

3a

4a

5b

6a

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

35.8 29.9 81.4 175.1 48.7 22.0 40.2 44.5 53.5 42.9 19.4 26.3 45.8 38.0 53.8 81.5 66.4 111.4 173.7 14.7

36.1 35.0 105.7 173.7 47.4 22.1 40.3 44.6 53.7 43.8 19.5 26.4 45.9 38.1 53.8 81.5 66.4 112.6 171.5 14.4

35.6 33.6 107.3 171.2 47.5 21.8 40.1 44.5 53.6 43.6 19.5 26.3 45.9 38.0 53.7 81.5 66.3 115.2 170.5 14.5 49.9

35.9 29.9 81.4 175.0 48.7 22.0 40.2 44.2 53.5 42.9 19.5 26.0 42.0 37.7 49.8 87.3 60.9 111.4 173.7 14.7

39.6 107.4 149.7 159.9 45.5 21.8 39.9 44.3 52.8 41.9 18.8 26.3 45.9 37.6 53.5 81.5 66.3 110.5 170.6 15.3

69.0 109.3 151.4 160.3 39.2 21.8 40.0 44.3 44.4 44.3 18.4 26.4 46.0 37.6 53.9 81.5 66.4 111.7 170.3 16.1

b

The absolute configuration of 1 was determined by the X-ray crystallographic analysis of its p-bromobenzoate 7

13

Carbon

a

tricalysiosides B–E obtained previously from the leaves of the same plant source.1

Recorded at 125 MHz. Recorded at 100 MHz.

OH H O O

H HMBC H C

Figure 2. Selected HMBC correlations for 1.

49.3 Figure 3. Selected NOESY correlations for 1.

4

OMe

H

H

O

O

h

H

Br

O

H O

O

H

8

H OH

O

O

H

5

H OH

O

O

H

6

H

OH OH O

O

MeO

H

3

H

OH

c

O

O

a

HO

H

2

H

b

OH

OH

O

H

H

d

O

O

g OH

OTs

1

H

f

OH H

9

H H

O

O

e

H

H

H

7

O

OH

O

O OH OH OH

Scheme 1. Reagents and conditions: (a) LiOH, THF–H2O, 48 h, 87%; (b) (trimethylsilyl)diazomethane, CHCl3–MeOH, 1 h, 15%; (c) triethylsilane, BF3$OEt2, CH2Cl2, 4 h, 36%; (d) NaBH4, EtOH, 5 min, 36%; (e) p-bromobenzoyl chloride, DMAP, pyridine–CH2Cl2, 4 h, 94%; (f) TsCl, pyridine, 18 h, 75%; (g) Na2CO3, EtOH, 48 h, 74%; (h) BF3$OEt2, MeOH, 20 min, 31%.

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519

OH

1514

(Scheme 1, Fig. 4). The value of the final refined Flack parameter 0.012(7) confirmed that the absolute configuration of 1 was as depicted in Figure 1. Tricalysiolide B (2) was isolated as an amorphous powder. Its molecular formula was determined to be C20H28O5 from the [MCH]C peak at m/z 349.2033 (calcd for C20H29O5, 349.2015) in the HRESIMS. The 1H and 13C NMR spectra of 2 were similar to those of 1. The differences noted between 1 and 2 were that the C-3 signal, observed as a methine carbon signal (d 81.4) in 1, was observed as a quaternary carbon signal (d 105.7) in 2. The C-3 signal was correlated with the hydroxyl proton (d 9.49) in the HMBC spectrum in 2, showing the location of the hydroxyl group was at C-3. The NOESY correlation detected between H-5 and OH-3 indicated that the hydroxyl group at C-3 was of b-orientation. The structure and absolute stereochemistry of 2 were determined to be as shown in Figure 1, by the preparation of 1 from 2 (Scheme 1). Reduction of 2 with sodium borohydride afforded a product, which was shown to be identical to natural 1 by comparison of their spectral data and optical rotations. Tricalysiolide C (3) was isolated as colorless prisms. Its molecular formula was determined to be C21H30O5 from the [MCH]C peak at m/z 363.2182 (calcd for C21H31O5, 363.2171) in the HRESIMS. The 1H and 13C NMR spectra of 3 were very similar to those of 2, implying that 2 and 3 had the same basic structure. The only difference noted between 2 and 3 was that the NMR spectra of 3 displayed signals due to a methoxyl group (dH 3.16, dC 49.9), whose methoxyl protons were correlated with the C-3 signal in the HMBC spectrum, thus demonstrating the location of the methoxyl group being at C-3 in 3. A NOESY correlation detected between H-5 and OCH3-3 indicated that the methoxyl group was of b-orientation. Thus, compound 3 was determined to be the 3-O-methyl analogue of 2. This structure was confirmed by the X-ray analysis (Fig. 5). Alkaline hydrolysis and subsequent acid treatment of 3 afforded 2 (Scheme 1), establishing that the absolute structure of 3 was as shown in Figure 1. Tricalysiolide D (4) was isolated as colorless prisms. Its molecular formula, C21H30O4, determined from the [MC H]C peak at m/z 347.2232 (calcd for C21H31O4, 347.2222) in the HRESIMS showed that it was higher than that of 1 by one methylene unit. The 1H and 13C NMR spectra of 4 were similar to those of 1, except that a singlet methoxyl proton signal (d 3.31) and one carbon signal (d 49.3), which were not observed in the spectra of 1, were observed in the spectra of 4 and that the C-16 signal (d 87.3) of 4 was in a lower field than the corresponding signal of 1 (d 81.5). The HMBC correlation between the methoxyl protons and C-16 indicated that the location of the methoxyl group was at C-16 to show that 4 was the 16-O-methyl ether of 1. The NOESY correlation between H-14b and OCH3-16 indicated that the methoxyl group at C-16 was of a-orientation. This structure was confirmed by the X-ray analysis (Fig. 6). Its absolute configuration was established by the preparation of 4 from 1 (Scheme 1). Tosylate 8, produced by treatment of 1 with p-toluenesulfonyl chloride, gave, on treatment with sodium carbonate, epoxide 9. Methanolysis of 9 in the presence of boron trifluoride diethyl etherate afforded a

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519

1515

Figure 4. ORTEP representation of 7 as determined by single-crystal X-ray analysis.

product, which was shown to be identical to natural 4 by comparison of their spectral data and optical rotations. Thus, the absolute structure of 4 was determined to be as shown in Figure 1. Tricalysiolide E (5) was obtained as an amorphous powder. Its molecular formula was determined to be C20H26O4 from the [MCH]C peak at m/z 331.1901 (calcd for C20H27O4, 331.1909) in the HRESIMS, which

was less than that of 1 by two hydrogen atoms. The 1H and 13C NMR spectra of 5 were generally similar to those of 1, except for the C-2 and C-3 signals. The C-2 methylene (d 29.9) and C-3 methine (d 81.4) signals in 1 were of olefinic methine (d 107.4) and quaternary (d 149.7) carbons, respectively, in 5. When 2 was treated with (trimethylsilyl)diazomethane2 in chloroform– methanol (10/1), the product was shown to be identical to natural 5 by their spectral data and optical rotations.

Figure 5. ORTEP representation of tricalysiolide C (3) as determined by single-crystal X-ray analysis.

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K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519

Figure 6. ORTEP representation of tricalysiolide D (4) as determined by single-crystal X-ray analysis.

Thus, the structure of 5 was determined to be as shown in Figure 1. Tricalysiolide F (6) was isolated as an amorphous powder. Its molecular formula was determined to be C20H26O5 from the [MCH]C peak at m/z 347.1882 (calcd for C20H27O5, 347.1858) in the HRESIMS, which was higher than that of 5 by one oxygen atom. The 1H and 13C NMR spectra of 6 resembled those of 5, demonstrating that they were of the same basic structure. The differences noted between them were that the C-1 methylene signal (d 39.6) in 5 was observed as an oxymethine carbon signal (d 69.0) in 6. The NOESY correlation between H-1 and H3-20 indicated that the hydroxyl group at C-1 was of b-orientation. Treatment of 6 with triethylsilane and boron trifluoride diethyl etherate3 afforded a reduction product, which was shown to be identical to natural 5 by comparison of their spectral data and optical rotations. Accordingly, the absolute structure of 6 was determined to be as shown in Figure 1. Tricalysiolides A–F (1–6) and tricalysiosides A–G1 are unusual compounds in which one of the geminal methyls is rearranged to the other methyl to form an ethyl unit at C-4 of the ent-kaurane skeleton. So far, the only known compounds with this unusual carbon framework are cafestol4,5 and several related furokauranes6–10 from Coffea plants (Rubiaceae). Tricalysiolides A (1), D (4) and tricalysiosides A–G are characterized by an a,b-unsaturated-g-lactone ring fused to the ring A, and tricalysiolides B (2), C (3), E (5), and F (6) by a further oxidized ring A structure.

Tricalysiolides A–F (1–6) showed a weak cytotoxic activity on P-388 murine leukemia cells with IC50 values of 40, 40, 50, 40, 17, and 30 mg/mL, respectively. 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a JASCO P1030 digital polarimeter. IR spectra were recorded on a JASCO FT/IR 620 spectrophotometer. UV spectra were obtained using a JASCO V-530 spectrophotometer. NMR spectra were measured on Bruker DRX-500 and DPX-400 spectrometers at 300 K. The 1 H chemical shifts in C5D5N were referenced to the residual C5D4HN resonance at 7.21 ppm, and the 13C chemical shifts to the solvent resonance at 135.5 ppm. Mass spectra were obtained using a Micromass LCT spectrometer. Preparative HPLC was carried out on a JASCO PU-986 pump unit equipped with a UV-970 UV detector (l 220 nm) and an Inertsil PREP-ODS column (10 mm, 20!250 mm), by using a MeOH–H2O or a MeCN–H2O solvent system at a flow rate of 10 mL/min. X-ray single crystal analysis was taken on a Mac Science DIP diffractometer with Mo Ka radiation ˚ ). (lZ0.71073 A 3.2. Plant material Wood of T. dubia was collected in Iriomote Island, Okinawa, in March 2003, and the plant origin was identified by Dr. T. Kinoshita (Teikyo University, Japan). A voucher specimen has been deposited at the Herbarium of Teikyo University.

K. Nishimura et al. / Tetrahedron 62 (2006) 1512–1519

3.3. Extraction and isolation Cut and air-dried wood (1.44 kg) of T. dubia was extracted with hot MeOH (3!6 L). After removal of MeOH under reduced pressure, the residue (104 g) was placed on a column of HP-20 (DIAION, 600 g) and eluted with H2O, H2O–MeOH (1/1), H2O–MeOH (1/4), MeOH, and acetone (each 3 L) sequentially to give five fractions. After removal of the solvent, the residue of the H2O–MeOH (1/4) fraction (17 g) was subjected to silica gel (Merck Kieselgel 60, 230–400 mesh, 200 g) column chromatography eluting sequentially with EtOAc, CHCl3–MeOH (10/1), CHCl3–MeOH (3/1), and MeOH (each 500 mL). After evaporation the CHCl3–MeOH (10/1) fraction (1.2 g) was subjected to aminopropyl-bonded silica gel (Chromatorex, 200–350 mesh, 40 g) column chromatography eluting sequentially with CHCl3–MeOH (50/1, then 30/1, then 10/1), and MeOH (each 100 mL) to give fractions 1–5. Fraction 2 (16.2 mg) was further applied to ODS HPLC eluting with MeOH–H2O (43/57) to afford 4 (5.6 mg). Fraction 3 (465.0 mg) was further separated by ODS HPLC eluting with MeOH–H2O (44/56) to afford 1 (141.5 mg), 3 (50.8 mg), and 5 (6.8 mg). The CHCl3–MeOH (3/1) fraction (7.15 g) was subjected to aminopropyl-bonded silica gel (35 g) column chromatography eluting sequentially with CHCl3–MeOH (30/1, then 10/1, then 5/1, then 3/1), and MeOH (each 100 mL) to give fractions A–G. Fraction B (276.7 mg) was further purified by repeated ODS HPLC using MeOH–H2O (40/60) and then MeCN–H2O (18/82) to afford 6 (14.8 mg). Fraction E (551.7 mg) was further purified by repeated ODS HPLC using MeOH–H2O (40/60) and then MeCN–H2O (17/83) to afford 2 (7.7 mg). 3.4. Characteristics of each terpenoid 3.4.1. Tricalysiolide A (1). Amorphous solid; [a]25 D K215 (c 0.11, CHCl3); UV (MeOH) lmax nm (log 3) 217 (4.29), 276 (1.25); IR (film) nmax 3365, 2932, 2865, 1775, 1742, 1645, 1044, 1021 cmK1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 333.2074 [MCH]C (calcd for C20H29O4, 333.2066). 3.4.2. Tricalysiolide B (2). Amorphous powder; [a]25 D K160 (c 0.33, pyridine); UV (MeOH) lmax nm (log 3) 215 (3.93); IR (film) nmax 3394, 2929, 2866, 1754, 1735, 1657, 1451, 1220, 1038 cmK1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 349.2033 [MCH]C (calcd for C20H29O5, 349.2015). 3.4.3. Tricalysiolide C (3). Colorless prisms (CHCl3– MeOH); mp 243–246 8C; [a]25 D K243 (c 0.11, CHCl3); UV (MeOH) lmax nm (log 3) 217 (4.46); IR (film) nmax 3419, 2933, 2865, 1762, 1656, 1452, 1194, 1170, 1053 cmK1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 363.2182 [MCH]C (calcd for C21H31O5, 363.2171). 3.4.4. Tricalysiolide D (4). Colorless prisms (CHCl3– MeOH); mp 251–253 8C; [a]25 D K198 (c 0.25, pyridine); UV (MeOH) lmax nm (log 3) 217 (4.13); IR (film) nmax 3398, 2937, 2864, 1750, 1646, 1448, 1159, 1069, 1047, 1014 cmK1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 347.2232 [MCH]C (calcd for C21H31O4, 347.2222).

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3.4.5. Tricalysiolide E (5). Amorphous powder; [a]25 D K203 (c 0.26, pyridine); UV (MeOH) lmax nm (log 3) 276 (3.99); IR (film) nmax 3389, 2925, 2852, 1746, 1669, 1605, 1464, 1455, 1445, 1259, 1213, 1055, 1021 cmK1; 1H and 13 C NMR data, Tables 1 and 2; HRESIMS m/z 331.1901 [MCH]C (calcd for C20H27O4, 331.1909). 3.4.6. Tricalysiolide F (6). Amorphous powder; [a]25 D K218 (c 0.14, pyridine); UV (MeOH) lmax nm (log 3) 270 (3.89); IR (film) nmax 3419, 2927, 2867, 1781, 1747, 1672, 1608, 1454, 1395, 1209, 1017 cmK1; 1H and 13 C NMR data, Tables 1 and 2; HRESIMS m/z 347.1882 [MCH]C (calcd for C20H27O5, 347.1858). 3.5. Identification of structural relations between the terpenoids by synthetic procedures (Scheme 1) 3.5.1. Preparation of compound 7. To a solution of 1 (20.0 mg, 0.060 mmol) in pyridine–CH2Cl2 (1/1, 1 mL) were added p-bromobenzoyl chloride (66.0 mg, 0.301 mmol) and 4-(dimethylamino)pyridine (8.0 mg, 0.065 mmol) in one portion. After stirring at room temperature for 4 h, the mixture was diluted with EtOAc. The extract was washed successively with 5% aqueous HCl and brine, dried (MgSO4), filtered, and concentrated to give a residue, which was then purified by silica gel column chromatography with CHCl3–MeOH (50/1) to afford p-bromobenzoyl ester 7 (29.0 mg, 94%) as colorless prisms (pyridine); mp 230–232 8C; [a]25 D K92 (c 0.11, CHCl3); IR (film) nmax 3442, 2924, 2852, 1721, 1679, 1647, 1452, 1396, 1266, 1013 cmK1; 1H NMR (500 MHz, pyridine-d5) d 8.07 (d, 2H, JZ8.4 Hz), 7.61 (d, 2H, JZ 8.4 Hz), 5.75 (s, 1H), 4.93 (d, 1H, JZ11.3 Hz), 4.76 (t, 1H, JZ9.3 Hz), 4.71 (d, 1H, JZ11.3 Hz), 2.49 (br s, 1H), 2.12 (m, 1H), 2.06 (m, 1H), 1.97–1.92 (m, 2H), 1.89 (d, 1H, JZ 14.4 Hz), 1.84 (d, 1H, JZ14.4 Hz), 1.71–1.63 (m, 4H), 1.59–1.43 (m, 6H), 1.16 (d, 1H, JZ7.9 Hz), 0.98 (td, 1H, JZ13.8, 3.3 Hz), 0.69 (s, 3H); 13C NMR (125 MHz, pyridine-d5) d 174.9, 173.7, 166.2, 132.0 (2C), 131.7 (2C), 130.1, 128.1, 111.5, 81.4, 79.3, 70.1, 53.8, 53.4, 48.7, 46.4, 44.7, 42.9, 40.0, 37.9, 35.8, 29.9, 26.2, 22.0, 19.3, 14.7; HRESIMS m/z 515.1410 [MCH]C (calcd for C27H32O5Br, 515.1433). 3.5.2. Reduction of compound 2. Sodium borohydride (2.5 mg, 0.066 mmol) was added to a solution of 2 (5.0 mg, 0.014 mmol) in EtOH (1.5 mL), and then the mixture was stirred at room temperature for 5 min. After evaporation of the solvent, the residue was purified by ODS HPLC with MeCN–H2O (23/77) to give a compound [1.7 mg, 36%, [a]25 D K186 (c 0.09, CHCl3)], which was shown to be identified to the natural product 1, by comparison of their 1H NMR and mass spectra, and optical rotations. 3.5.3. Treatment of compound 3 with lithium hydroxide. To a solution of 3 (5.0 mg, 0.014 mmol) in THF–H2O (10/1, 1.5 mL) was added lithium hydroxide monohydrate (3.0 mg, 0.071 mmol), and the mixture was stirred at room temperature for 48 h. After addition of acetic acid (1 mL) to the mixture, it was evaporated to dryness. The residue was subjected to ODS HPLC with MeCN–H2O (23/77) to give a compound [4.2 mg, 87%, [a]25 D K173 (c 0.11, pyridine)],

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which was shown to be identical to natural 2 by comparison of their 1H NMR and mass spectra, and optical rotations. 3.5.4. Tosylation of compound 1. To a solution of 1 (6.4 mg, 0.019 mmol) in pyridine (1 mL) was added p-toluenesulfonyl chloride (20 mg, 0.10 mmol) at 0 8C. The mixture was kept at room temperature for 18 h, and then was diluted with EtOAc. The solution was washed successively with aqueous Na2CO3 and brine, dried (MgSO4), filtered, and concentrated to give a residue, which, by ODS HPLC with MeCN–H2O (50/50), gave compound 8 (7.0 mg, 75%) as an amorphous solid: [a]25 D K152 (c 0.17, CHCl3); IR (film) nmax 3445, 2930, 1746, 1644, 1357, 1189, 1175 cmK1; 1H NMR (500 MHz, pyridine-d5) d 8.06 (d, 2H, JZ8.0 Hz), 7.27 (d, 2H, JZ 8.0 Hz), 5.72 (s, 1H), 4.74 (t, 1H, JZ9.0 Hz), 4.58 (d, 1H, JZ9.6 Hz), 4.55 (d, 1H, JZ9.6 Hz), 2.40 (br s, 1H), 2.22 (s, 3H), 2.19 (m, 1H), 1.98 (m, 1H), 1.86–1.84 (m, 2H), 1.79 (d, 1H, JZ14.3 Hz), 1.71 (d, 1H, JZ14.3 Hz), 1.64–1.55 (m, 3H), 1.51–1.32 (m, 7H), 1.07 (d, 1H, JZ8.5 Hz), 0.95 (td, 1H, JZ13.6, 3.6 Hz), 0.62 (s, 3H); 13C NMR (125 MHz, pyridine-d5) d 174.8, 173.6, 145.1, 133.6, 130.3 (2C), 128.4 (2C), 111.5, 81.3, 78.8, 75.5, 53.3, 53.2, 48.6, 45.9, 44.5, 42.8, 39.9, 37.8, 35.8, 29.8, 25.8, 21.9, 21.3, 19.1, 14.6; HRESIMS m/z 487.2130 [MCH]C (calcd for C27H35O6S, 487.2154). 3.5.5. Epoxidation of compound 8. A solution of 8 (6.1 mg, 0.013 mmol) in EtOH (3 mL) was stirred with Na2CO3 (15 mg, 0.14 mmol) at room temperature for 48 h. The mixture was filtered and the filtrate was evaporated. By ODS HPLC with MeCN–H2O (50/50), the residue gave compound 9 (2.9 mg, 74%) as an amorphous powder: [a]25 D K223 (c 0.06, CHCl3); IR (film) nmax 2929, 2861, 1778, 1749, 1645, 1456, 1258, 1180, 1160, 1127, 1048, 1037, 1022 cmK1; 1H NMR (500 MHz, pyridine-d5) d 5.75 (s, 1H), 4.75 (t, 1H, JZ8.7 Hz), 2.89 (d, 1H, JZ5.0 Hz), 2.82 (d, 1H, JZ5.0 Hz), 2.21 (m, 1H), 1.92–1.83 (m, 4H), 1.71–1.56 (m, 4H), 1.55–1.30 (m, 8H), 1.11 (d, 1H, JZ 8.0 Hz), 0.95 (td, 1H, JZ13.8, 3.7 Hz), 0.64 (s, 3H); 13C NMR (125 MHz, pyridine-d5) d 174.8, 173.6, 111.7, 81.3, 65.7, 52.2, 49.9, 49.0, 48.7, 45.1, 42.7, 42.6, 39.2, 38.9, 35.9, 29.8, 28.7, 21.6, 20.3, 14.9; HRESIMS m/z 315.1949 [MCH]C (calcd for C20H27O3, 315.1960). 3.5.6. Methanolysis of compound 9. To a solution of 9 (2.6 mg, 0.0083 mmol) in MeOH (1.5 mL) was added BF3$OEt2 (3 mL, 0.024 mmol). The mixture was stirred at room temperature for 20 min. After neutralization with sodium acetate (5 mg), the solvent was evaporated under reduced pressure. By ODS HPLC with MeCN–H2O (40/60) the residue gave a compound [0.9 mg, 31%, [a]25 D K241 (c 0.05, pyridine)], which was shown to be identical to the natural product 4 by comparison of their 1H NMR spectra and optical rotations. 3.5.7. Treatment of compound 2 with (trimethylsilyl)diazomethane. To a solution of 2 (11.0 mg, 0.032 mmol) in CHCl3–MeOH (10/1, 3 mL) was added (trimethylsilyl)diazomethane (2.0 M in hexanes, 1 mL, 2.0 mmol). The mixture was stirred at room temperature for 1 h. After removal of the solvent and subsequent ODS HPLC of the residue with MeCN–H2O (22/78), along with the starting

material 2 (1.0 mg, 9%) and 3 (3.8 mg, 33%), another compound [1.6 mg, 15%, [a]25 D K136 (c 0.06, pyridine)] was obtained. On the basis of the 1H NMR and mass spectra, and optical rotations, the third compound was shown to be identical to the natural product 5. 3.5.8. Reduction of compound 6. Triethylsilane (0.5 mL, 6.1 mmol) and BF3$OEt2 (5 mL, 0.039 mmol) were added to a solution of compound 6 (5.0 mg, 0.014 mmol) in CH2Cl2 (2 mL). The mixture was stirred at room temperature for 4 h. After neutralization with sodium acetate (10 mg), the solvent was removed by evaporation and the residue was separated by ODS HPLC with MeCN–H2O (28/72) to give a compound [1.7 mg, 36%, [a]25 D K101 (c 0.05, pyridine)], which was shown to be identical to the natural product 5, by comparison of their 1H NMR and mass spectra, and optical rotations. 3.6. X-ray single crystallographic analysis The structures of compounds 3, 4, and 7 were determined by the direct method using the maXus crystallographic software package11 and the refinement was carried out by the program SHELXL-97.12 Crystal data for 3. C21H30O5; MZ362.45; orthorhombic; ˚ ; bZ13.087(3) A ˚ ; cZ space group P212121; aZ11.155(3) A ˚ ˚ 12.476(3) A; VZ1821.31(8) A; ZZ4; DXZ1.322 Mg mK3; m(Mo Ka)Z0.093 mmK1; RZ0.0349; RwZ0.0974. Crystal data for 4. C21H30O4; MZ346.45; orthorhombic; ˚ ; bZ11.322(5) A ˚ ; cZ space group P212121; aZ6.899(10) A ˚; ˚; 22.828(10) A VZ1783.11(11) A ZZ4; DXZ 1.291 Mg m K3; m(Mo Ka)Z0.088 mmK1; RZ0.0403; RwZ0.1064. Crystal data for 7. C27H31BrO5$C5H5N; MZ594.52; ˚ ; bZ orthorhombic; space group P212121; aZ6.259(10) A ˚ ; cZ22.348(10) A ˚ ; VZ2796.12(19) A ˚ ; ZZ4; 19.99(10) A D X Z1.412 Mg mK3; m(Mo Ka)Z1.512 mm K1; RZ 0.0501; RwZ0.067; Flack parameterZ0.012(7). Crystallographic data for 3, 4, and 7 reported in this paper have been deposited at the Cambridge Crystallographic Data Centre, under the reference numbers CCDC 280148, 280149, and 280147, respectively. Copies of the data can be obtained, free of charge, on application to the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: C44 1223 336033 or e-mail: [email protected]).

References and notes 1. He, D.-H.; Otsuka, H.; Hirata, E.; Shinzato, T.; Bando, M.; Takeda, Y. J. Nat. Prod. 2002, 65, 685–688. 2. Hashimoto, N.; Aoyama, T.; Shioiri, T. Chem. Pharm. Bull. 1981, 29, 1475–1478. 3. Adlington, M. G.; Orfanopoulos, M.; Fry, J. L. Tetrahedron Lett. 1976, 2955–2958. 4. Wettstein, A.; Hunziker, F.; Miescher, K. Helv. Chim. Acta 1943, 26, 1197–1218.

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5. Djerassi, C.; Cais, M.; Mitscher, L. A. J. Am. Chem. Soc. 1959, 81, 2386–2398. 6. Kaufmann, H. P.; Sen Gupta, A. K. Chem. Ber. 1963, 96, 2489–2498. 7. De Rostolan, J.; Poisson, J. Cafe´, Cacao, The´ 1970, 14, 47–49. 8. Ducruix, A.; Pascard-Billy, C.; Hamonnie`re, M.; Poisson, J. J. Chem. Soc., Chem. Commun. 1975, 396–397. 9. Richter, H.; Spiteller, G. Chem. Ber. 1979, 112, 1088–1092.

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10. Prewo, R.; Guggisberg, A.; Lorenzi-Riatsch, A.; Baumann, T. W.; Wettstein-Ba¨ttig, M. Phytochemistry 1990, 29, 990–992. 11. Mackay, S.; Gilmore, C. J.; Edwards, C.; Stewart, N.; Shankland, K. maXus Computer Program for the Solution and Refinement of Crystal Structures; MacScience, Japan and The University of Glasgow: Bruker Nonius, The Netherlands, 1999. 12. Sheldrick, G. M. SHELXL-97: Program for the Refinement of Crystal Structures; University of Go¨ttingen: Go¨ttingen, Germany, 1997.