HETEROCYCLES

Paper | Regular issue | Vol. 78, No. 6, 2009, pp. 1453-1462
Received, 11th December, 2008, Accepted, 29th January, 2009, Published online, 30th January, 2009.
DOI: 10.3987/COM-08-11624

■ New Cytotoxic Nor- and Bisnorditerpene Dilactones, Makilactones A-D, from Podocarpus macrophyllus D. Don

Kimihiko Sato, Keito Sugawara, Hirono Takeuchi, Hyun-Sun Park, Toshiyuki Akiyama, Tetsuo Koyama, Haruhiko Fukaya, Yutaka Aoyagi, and Koichi Takeya*

School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan

Abstract

Four new nor- and bisnorditerpene dilactones, makilactones A-D, were isolated from a MeOH extract of bark and root of Podocarpus macrophyllus D. Don (Podocarpaceae) with three known diterpene dilactones. The structures of the new nor- and bisnorditerpene dilactones having a 7:8, 9:11-dienolide moiety were determined by the spectroscopic studies, including 1D and 2D NMR spectral analysis, and single crystal X-Ray crystallographic analysis. Two of the presently isolated compounds were shown to be very strongly cytotoxic.

INTRODUCTION
Podocarpus macrophyllus D. Don (Podocarpaceae) (Japanese name: Inumaki) is a dioecious evergreen tree distributed in the subtropical areas of south eastern China, Taiwan, and Japan. From this plant, flavonoids, bisflavonoids, ecdysons, norditerpenoids,1-4 and antibiotic diterpenes have been reported.5 From a related Podocarpus plant, Podocarpus macrophyllus D. Don var. maki, nor- and bisnorditerpenoids having cytotoxic activities against P388 murine leukemia cells have also been reported by us.6-8
In the present study, from the bark and root of Podocarpus macrophyllus D. Don, we further isolated four new nor- and bisnorditerpene dilactones, makilactones A-D, along with three known norditerpene dilactones, nagilctone F, 2α-hydroxynagilactone F and 3β-hydroxynagilactone F, and determined the structures of the new diterpene dilactones mainly on the basis of the spectroscopic studies including X-ray crystallography. All the present diterpenoids were shown to have a 7:8, 9:11-dienolide moiety, which is considered to be associated with cytotoxic activity.

Makilactone A (1) was isolated as colorless needles, mp 236-239℃. The molecular formula was determined to be C18H20O4 from the [M++H] ion peak at m/z 301.1444 in HRESIMS. IR absorption bands at 1702 cm-1 and 1769 cm-1 indicated the presence of γ-lactone carbonyl and δ-lactone carbonyl groups. The profiles of the NMR spectra of 1 and nagilactone F, a known norditerpene dilactone also isolated in the present study, were generally quite similar to each other, as shown in Table 1. In the HMQC spectrum, the two methyl carbon signals at δC 24.0 and δC 24.3 correlated with the proton signals at δH 1.23 and δH

1.07, respectively, and were assigned to C-18 methyl and C-20 methyl, respectively. The fact implied that they had the same basic carbon skeleton structure with a 7:8, 9:11-dienolide unit (hereafter, the carbon skeleton structure without the C-14 side chain will be referred to as nagilactone F unit). The difference between 1 and nagilactone F was only in the C-14 side chain structure. The carbon signals in the lower magnetic field at δC 132.8 and δC 122.0 were assigned to C-15 and C-16, forming a vinyl group in the side chain. Thus, the structure of 1 was determined to be as shown in Figure 1. The ORTEP representation derived from the X-ray crystallographic analysis

is shown in Figure 2.
Makilactone B (2) was isolated as colorless needles, mp 218 ℃. The molecular formula was determined to be C19H23O7Cl from the [M++Na] ion peak at m/z 421.1017 in HRESIMS. The presence of a chlorine atom in 2, was implied by the ESIMS, by the [M＋Na]+ ion peak at 421.1017 and the chlorine isotope (37Cl) peak at 423.1029. IR absorptions bands at 1774 cm-1, 1693 cm-1 and 3311 cm-1 showed the presence of γ-lactone carbonyl, δ-lactone carbonyl and hydroxyl groups, respectively. The 13C and 1H NMR spectra of 2 showed that it had three methyls and three hydroxyls, and that it had the nagilactone F unit as 1, in which two of the three methyl carbon signals at δC 23.1 and δC 26.8 correlating with the singlet methyl proton signals at δH 1.66 and δH 1.99, respectively in the HMQC spectrum were assigned to C-18 methyl and C-20 methyl, respectively. The other methyl carbon signal at δC 14.7 correlating with the doublet methyl proton signal at δH 1.30 in the HMQC spectrum was assigned to C-16 methyl of the side chain. In the COSY spectrum, the methylene proton signals H-17 (δH 3.89 and δH 4.05) were both

correlated with the hydroxyl group at δH 6.34, which implied that a hydroxyl methyl group was connected to C-15. In the COSY spectrum, the proton signals of H-2 (δH 4.71) and of H-3 (δH 4.40) correlated with the hydroxyl protons at δH 8.17 and at δH 5.95, respectively, to show that the remaining two hydroxyls were at C-2 and C-3. The HMBC correlations were observed between H-14 and the carbons C-8, C-7, and C-16, which indicated the presence of 2-hydroxy-1-methylethan-2-yl substituent at C-14 (Figure 3a). In the NOESY spectrum, the proton signal H-1 (δH 5.01) was correlated with the methyl proton signal H-20 (δH 1.99), the proton signal H-2 (δH 4.71) with the proton signal H-18 (δH 1.66), and the proton signal H-3 (δH 4.40) with the proton signals H-18 (δH 1.66) and of H-5 (δH 2.72) to suggest that H-1 was β-oriented, and H-2 and H-3 were α-oriented (Figure 3b). Because of the presence of chlorine as a heavy atom, the single crystal X-ray crystallographic analysis established the absolute configuration as follows: the hydroxyl groups at C-2

and C-3 were both of β-orientation, the chlorine at C-1 was of α-orientation and the configuration at C-15 was R, as shown in Figure 4. Accordingly, the structure of 2 was determined to be as shown in Figure 1.
Makilactone C (3) was isolated as colorless needles, mp 233℃. The molecular formula was determined to be C19H22O6 from the [M++Na] ion peak at m/z 369.1294 in HRESIMS. IR absorptions bands for γ-lactone carbonyl, δ-lactone carbonyl and hydroxyl groups were seen at 1770 cm-1, 1702 cm-1 and 3430 cm-1, respectively. The 13C and 1H NMR spectra of 3 were generally similar to those of 2, implying that 3 had the nagilactone F unit with one double bond and one hydroxyl group. One methyl carbon signal at δc 15.4 was correlated with the doublet methyl proton signal at δH 1.42 to assign this methyl group to C-16 of the side chain. In the COSY spectrum, the methylene proton signals H-17 (δH 3.95 and δH 4.20) was correlated with the hydroxyl proton at δH 6.28,

which implied the presence of a hydroxyl methyl group at C-15. These facts revealed that the side chain in 3 was the same in the structure as that in 2. In the COSY spectrum, the proton signal of H-1 (δH 4.66) was correlated with the hydroxyl proton (δH 7.21) to show that the hydroxyl was at C-1. In 13C NMR, the carbon signals of C-2 (δC 135.2) and of C-3 (δC 127.6) were in the lower magnetic field, implying the presence of a double bond between C-2 and C-3. The OH-1 proton signal (δH 7.21) was correlated with H-20 (δH 1.45) in the NOESY spectrum, suggesting that OH-1 was β-oriented. The single crystal X-ray crystallographic analysis demonstrated that the hydroxyl group at C-1 was β oriented and that the configuration at C-15 was R* as shown in Figure 5. Accordingly, the structure of 3 was determined to be as shown in Figure 1.
Makilactone D (4) was isolated as colorless needles, mp 236℃. The molecular formula was determined to be C19H24O6 from the [M++Na] ion peak at m/z 371.1444 in HRESIMS. IR absorption bands for γ-lactone carbonyl, δ-lactone carbonyl and hydroxyl groups were observed at 1742 cm-1, 1697 cm-1 and 3413 cm-1, respectively. The 13C and 1H NMR spectra of 4 showing the presence of three methyl and two hydroxyl groups were generally quite similar to those of 3, implying that 4 had the nagilactone F unit. One of the three methyl carbon signals, the one at δc 15.3 was correlated with the doublet methyl proton signal at δH 1.40, so that this methyl group was

assigned to C-16 in the side chain. The methylene proton signals of H-17 (δH 3.94 and δH 4.18) were correlated with the hydroxyl proton (δH 6.29) in the COSY spectrum, implying the presence of a hydroxyl methyl group connected to C-15. These facts revealed that the side chain in 4 was the same in the structure as that in 2 and 3. In the COSY spectrum, the proton signal of H-1 (δH 4.10) was correlated with the hydroxyl proton at δH 6.70 to show the presence of OH at C-1. The hydroxy proton signal of OH-1 (δH 6.70) was correlated with the methyl proton signal of H-20 at δH 1.45 and the proton signal of H-1 at δH 4.10 was correlated with the proton signal of H-5 (δH 1.78) in the NOESY spectrum to suggest that H-1 was α-oriented. The single crystal X-ray crystallographic analysis confirmed that hydroxyl group at C-1 was of β-orientation and that the configuration at C-15 was R* as shown in Figure 6. Accordingly, the structure of 4 was determined as shown in Figure 1.
The absolute configuration of chlorine-containing makilactone B (2) was established by X-ray crystallographic analysis. Since the CD spectra of all four compounds (1-4) showed negative Cotton effect, the absolute configurations of 1-4 were concluded to be the same, as shown Figure 1. All these diterpenes isolated in the present study were tested for their cytotoxic activities on P388 murine leukemia cells and the results are given in Table 3. The IC50 values of makilactone A (1) (0.050 μg/mL) and nagilactone F (0.090 μg/mL) were almost the same as that of the positive control used, camptothecin (0.045 μg/mL). Makilactones C (3) and D (4), 2α-hydroxynagolactone F11 and 3β-hydroxynagilactone F12 were slightly less active. The chlorinated dilactone, makilactone B (2) was almost inactive.

These norditerpenoid compounds which are characterized by having a γ-lactone unit between C-4 and C-6 and a δ-lactone unit between C-12 and C-14, are classified into three subgroups according to their B/C ring structure:9 Subgroup A: having α-pyrone unit, Subgroup B: having 7α:8α-epoxy-9:11-enolide unit and Subgroup C: having 7:8, 9:11-dienolide unit. So far, not many norditerpenoids of Subgroup C have been reported. The presence of 7:8, 9:11-dienolide moiety as seen in Subgroup C is reported to be necessary for norditerpenoids to show a potent cytotoxic activity.10 The compounds isolated in the present study all belong to Subgroup C and and all were shown to be active in the cytotoxicity assay (Table 3.). The present results may support the proposed relationship between the activity and the 7:8, 9:11-dienolide structure unit.

EXPERIMENTAL
General Experimental Procedures
Optical rotations were measured on a JASCO DIP-360 automatic digital polarimeter, IR spectra on a JASCO FT/IR 620 spectrophotometer, and mass spectra on VG AutoSpec E and Micromass LCT (Manchester, UK) spectrometers. NMR spectra were obtained on a Brucker DRX-500 and on an AV-600 spectrometer at 300K in C5D5N. The chemical shifts (δ) of proton signals are given in ppm relative to the resonances of residual C5D4HN at 7.19 ppm and those of carbon signals in ppm relative to the resonance at 135.5 ppm for C5D5N. Silica gel (Merck Kiesel gel 60, 70-230 nm, Kanto silica gel N 60, 63-210 µm) and Diaion® HP-20 (Mitsubishi Chemical Co. Ltd.) were used for column chromatography and precoated Kieselgel 60 F254 (0.25 mm thick, Merck Co. Ltd.), RP–18 F254S (0.25 mm thick, Merck) plates for TLC, in which the spots were visualized by spraying of 10% H2SO4 solution, and subsequent heating. Preparative HPLC was carried out on a JASCO PU-986 equipped with a UV-970 UV detector (λ 220nm) and Inertsil PREP-ODS column (10 µm, 20 x 250 mm), by using MeOH/H2O or MeCN/H2O at a flow rate of 10mL/min. X-ray single crystallographic analysis was taken on a Mac Science DIP diffractometer with Mo Kα radiation (λ = 0.71073 Å).

Plant Material
Bark and root of Podocarpus macrophyllus D. Don collected in Kochi, Japan, in Nobember 2004 were air-dried. The botanical identification was made by K. Takeya, Professor of Plant Chemistry of Tokyo University of Pharmacy and Life Science. A voucher specimen (08JCP18) has been deposited in the herbarium of Tokyo University of Pharmacy and Life Science.

Extraction and isolation
The air dried bark of Podocarpus macrophyllus D. Don (3.2 kg) was extracted with hot MeOH (3 x 45 L). The combined MeOH extract was concentrated and the residue (284 g) was subjected to Diaion HP-20 resin column chromatography (10 cm x 40 cm) eluting sequentially with H2O/MeOH (1:1, 2:8), MeOH, and acetone (each 5 L) to give four fractions. The second fraction Fb (H2O/MeOH 2:8) was concentrated to give a residue (10.4 g), which was subjected to silica gel column chromatography eluting with CHCl3/MeOH (19:1) to give fractions Fb1-Fb12. Fb12 (145 mg) was subjected to ODS-HPLC eluting with H2O/MeOH (54:46) to give 1 (3.1 mg), nagilactone F (2.5 mg) and 3β-hydroxylnagilactone F (3.0 mg).
The air dried root of Podocarpus macrophyllus D. Don (22.66 kg) was extracted with MeOH (45 L x 3) at room temperature. As described above for the bark, the MeOH extract was concentrated and the residue (614 g) was subjected to Diaion HP-20 resin column chromatography (10 cm x 40 cm) eluting with H2O, H2O/MeOH (1:1, 2:8), MeOH and acetone (each 5 L). Fractions Er (H2O/MeOH 1:1) and Fr (H2O/MeOH 2:8) were treated as follows. Fraction Er was concentrated to give a residue (71.6 g), which was subjected to silica gel chromatography eluting with CHCl3/H2O (19:1) to give six fractions, Er1~Er6. Er2 (8.85 g) was further subjected to ODS-HPLC eluting with H2O/MeCN (93.5:6.5) to give eleven fractions. The 10th fraction gave 3 (6.1 mg).
Fraction Fr was concentrated to give a residue (82.4 g), which was subjected to silica gel column chromatography eluting sequentially with CHCl3/H2O (1:0, 19:1, 9:1, 2:1, 0:1) to give five fractions Fr1~Fr5. Fraction Fr2 (12.6 g) was subjected to silica gel chromatography eluting with CHCl3/H2O (30:1) to give fourteen fractions Fr2-1~Fr2-14. ODS-HPLC of Fr2-3 with H2O/MeOH (60:40) gave 2α- hydroxynagilactone F (4.2 mg), that of Fr2-7 with H2O/MeOH (77:23) gave 4 (5.7 mg), and that of Fr2-8 with H2O/MeOH (75:25) gave 2 (19.6 mg).

Single crystal X-ray Crystallographic Analysis
For X-ray analysis, samples were recrystallized from EtOAc-MeCN-MeOH. Crystallographic data for 1-4 reported in this paper (Figure 2-5) have been deposited at the Cambridge Crystallographic Data Centre, under the reference numbers CCDC 696338, 711930, 711929, and 711931, 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: +44-(0)-1223-336033 or e-mail: deposit@ccdc.cam.ac.UK)

Assay of Cytotoxic Activity
The cytotoxic assay was performed by using the MTT assay method. The murine P388 leukemia cells were cultured in RPMI 1640 medium (Nissui Co. Ltd.) supplemented with 5% heat-inactivated fetal bovine serum (FBS) and kanamycin (5.3 mL/L) in a humidified atmosphere of 95% air and 5% CO2 at 37 ℃. The 100 µL of the cell suspension was added to each well (3 x 103 cells/well) of a 96 microwell plate (Iwaki, flat bottomed, treated polystyrene) and the plate was incubated for 24 h. Test compounds were dissolved in DMSO in various concentrations (100, 30, 10, 3, 1, 0.3, 0.1 µg/mL) and 10 µl of the test solution or DMSO (control) was added to each well. The plate was kept in an incubator at 37 ℃ for 48 h. After termination of the cell culture by adding 20 µL MTT (5% in PBS) to each well, the plate was further incubated for 4 h. To each well was added 100 µL of 10 % SDS in 0.01 N HCl. The plate was read on a microplate reader (MPR A4i, Tosoh) at 550 nm. A dose-response curve was plotted for each compound, and the concentrations giving 50 % inhibition of the cell growth (IC50) were recorded.

Makilactone A (1) : Colorless needles, mp 236-239℃ (MeOH). [α]D24-81.4°(c 0.16, MeOH); CD (c 0.000173, MeOH) λ (Δε) 265 (-13.6) nm. IR (film) 1702, 1769 (C=O) cm-1, HRESIMS m/z:301.1444 [Calcd for C18H21O4 301.1440 (M++H)]. 1H- and 13C- NMR, see Tables 1 and 2.

Makilactone B (2) : Colorless needles, mp 218℃ (MeOH). [α]D24+101.5°(c 0.19, MeOH); CD (c 0.000113, MeOH) λ (Δε) 285 (-1.90) nm. IR (film) 1774, 1693 (C=O) cm-1, 3311 (OH) cm-1, HRESIMS m/z:421.1017 [Calcd for C19H23O7ClNa 421.1030 (M++Na)]. 1H- and 13C NMR, see Tables 1 and 2.

Makilactone C (3) : Colorless needles, mp 233℃ (MeOH). [α]D24-219.9 (c 0.06, MeOH); CD (c 0.000172, MeOH) λ (Δε) 268 (-12.6) nm. IR (film) 1770, 1702 (C=O) cm-1, 3430 (OH) cm-1, HRESIMS m/z: 369.1294 [Calcd for C19H22O6Na 369.1314 (M++Na)]. 1H- and 13C NMR, see Tables 1 and 2.

Makilactone D (4) : Colorless needles, mp 236℃ (MeOH). [α]D24-185.3 (c 0.08, MeOH); CD (c 0.000147, MeOH) λ (Δε) 267 (-11.0) nm. IR (film) 1742, 1697 (C=O) cm-1, 3413 (OH) cm-1, HRESIMS m/z: 371.1444 [Calcd for C19H24O6Na 371.1471 (M++Na)]. 1H- and 13C NMR, see Tables 1 and 2.

ACKOWLEDGEMENTS
This work was supported by a Grant-in-aid for Scientific Research (C) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

References

1. I. Saeki, M. Sumitomo, and T. Kondo, Holzforschung, 1970, 24, 83.
2. T. Hayashi, H. Kakisawa, S. Ito, Y. P. Chen, and H.-Y. Hsu, Tetrahedron Lett., 1972, 3385. CrossRef
3. M. Kodama, C. Kabuto, M. Sunagawa, and S. Ito, Tetrahedron Lett., 1977, 2909. CrossRef
4. T. Omoto and O. Yoshida, Chem. Pharm. Bull., 1980, 28, 1894.
5. K. Sato, K. Sugawara, H. Takeuchi, H. Park, T. Akiyama, T. Koyama, Y. Aoyagi, K. Takeya, and T. Tsugane, Chem. Pharm. Bull., 2008, 56, 1691. CrossRef
6. H. Park, Y. Takahashi, H. Fukaya, Y. Aoyagi, and K. Takeya, J. Nat. Prod., 2003, 66, 282. CrossRef
7. H. Park, N. Yoda, H. Fukaya, Y. Aoyagi, and K. Takeya, Tetrahedron, 2004, 60, 171. CrossRef
8. H. Park, N. Kai, H. Fukaya, Y. Aoyagi, and K. Takeya, Heterocycles, 2004, 63, 347. CrossRef
9. S. Ito and M. Kodama, Heterocycles, 1976, 4, 595. CrossRef
10. A. F. Barrero and S. Arseniyadis, J. Org. Chem., 2002, 67, 2501. CrossRef
11. I. Kubo, M. Himejima, and B. Ying, Phytochemistry, 1991, 30, 1467. CrossRef
12. Y. Hayashi and T. Matsumoto, Heterocycles, 1978, 10, 123. CrossRef