A Novel Cytotoxic Oxetane ent-Kauranoid from Isodon japonicus

• Abstract
• Materials and Methods
• References

Abstract

A novel 11,20 : 1,20-diepoxy-ent-kaurane diterpenoid, maoyecrystal I (1), was isolated from Isodon japonicus, and its structure was elucidated by spectroscopic methods and comparison with another new ent-kauranoid, rubescensin W (2) from Isodon rubescens var. taihangensis. The structure of 2 was determined by single crystal X-ray diffraction analysis. A bioassay of their cytotoxity against K562 cells showed that the oxetane group of 1 might be a bioactive moiety.

More than 400 ent-kaurane diterpenoids have been reported from the plant genus Isodon, most of them showed inhibitory effects against K562 cells [1]. The cyclopentanone moiety conjugated with an exo-methylene group was regarded as the antitumor active center of this kind of diterpenoids [1], [2]. Our investigation of the bioactive substances of Isodon japonicus (Burman f.) Hara, a folk medicinal plant popularly used in China and Japan for its antibacterial, anti-inflammatory and antitumor activities, led to the isolation of a series of ent-kauranoids [3], [4], [5], as well as a novel compound maoyecrystal I (1) which was deduced to be a 1,20 : 11,20-diepoxy-ent-kauranoid. The oxetane moiety at C-1/C-20 was so rare in ent-kauranoids that this structure could not be identified, until the structure of its analogue rubescensin W (2), a new 11,20-epoxy-ent-kauranoid from Isodon rubescens var. taihangensis had been determined by the single crystal X-ray diffraction analysis. Finally, compound 1 was elucidated as 6β,15β-dihydroxy-1α,20 : 11α,20-diepoxy-ent-kaur-16(17)-en-7-one, by analysis of its spectroscopic data and comparison with those of 2. Compound 1, without the above-mentioned bioactive moiety, however, showed considerable cytotoxicities against K562 cells. This paper presents their structure elucidation and bioassay results.

The molecular formula of maoyecrystal I (1) was determined as C₂₀H₂₆O₅ by HREIMS and NMR spectra. Its UV spectrum indicated the absence of any conjugated system. Considering the ent-configuration of the diterpenoids previously isolated from this plant and the characteristic signals of three non-oxymethines (C-5, C-9 and C-13), three non-oxy-quaternary carbon (C-4, C-8 and C-10) and two tertiary methyls (C-18 and C-19) for the kaurane skeleton, compound 1 was presumed to be an ent-kauranoid. Furthermore, the HMBC correlations of an oxymethine proton (δ_H = 4.94, H-11) with an acetal carbon (δ_C = 104.1, C-20), a non-oxy-quaternary carbon (δ_C = 56.8, C-8) and a non-oxymethine carbon (δ_C = 37.0, C-13) revealed that compound 1 was an 11, 20-epoxy-ent-kauranoid.

Comparison of the 1D-NMR and HMBC spectra of 1 with those of rubescensin W (2, a new 11, 20-epoxy-ent-kauranoid from Isodon rubescens var. taihangensis, the structure of which was determined by X-ray analysis, Fig. [1]), suggested that compounds 1 and 2 were very similar and had only few minor differences at ring A (Table [1]). On the basis of the difference of their HREIMS data, it was supposed that compound 2 might be formed by the trans-acetalization of 1 in MeOH/CHCl₃. This deduction was confirmed by the key NOEs of H-2β/H-5β and H-1β/H-11β (Fig. [2]), indicating the unusual boat-configuration of ring A, which was caused by the formation of the oxetane moiety. Furthermore, the S configuration of C-20 was assigned by the NOEs between H-6 (δ_H = 4.41), H-19 (δ_H = 1.46, 3H), and H-20 (δ_H = 6.25). Therefore, compound 1 was established to be 6β,15β-dihydroxy-1α,20 : 11α,20-diepoxy-ent-kaur-16(17)-en-7-one, and named maoyecrystal I. All of the ¹H- and ¹³C-NMR data were unambiguously assigned by careful inspection of its 2D NMR spectra including ¹H-¹H COSY, HMQC, HMBC, and ROESY spectra. This is the first example of a naturally occurring ent-kauranoid containing an oxetane heterocycle from the genus Isodon plants [7].

Compounds 1 and 2 were tested for their cytotoxicity against K562 cells. Without any substructure of an α,β-unsaturated ketone, compound 1 exhibited a comparable inhibitory effect (IC₅₀ = 7.30 μg/mL) as cis-platinum (IC₅₀ = 1.14 μg/mL), while 2 was inactive (IC₅₀ = 38.96 μg/mL). The unusual oxetane group of 1 was the only difference between the structures of 1 and 2, and was therefore suggested to be a new bioactive moiety in this class of natural products.

General interest in naturally occurring oxetanes continues to be high for their variable bioactivities, such as the antiviral oxetanocin A, the antibacterial oxetin, an oxatricyclic norbornane as potent herbicide and plant growth regulator, and thromboxane A₂ (TXA₂) being highly active against aggregation of blood platelets [8]. Especially, the oxetane moiety is necessary for bioactivity of taxol and its analogues [9].

Materials and Methods

The spectroscopic instruments and the identification of plant materials were described in our previous paper [5]. Fractions I - X were obtained from 70 % Me₂CO extraction of the dried and powdered leaves (10.0 kg) of I. japonicus, as described previously [5]. The CHCl₃-Me₂CO (9 : 1) fraction (II, 100.6 g) was further separated on a silica gel CC (200 - 300 mesh, 1.0 kg, 6 × 80 cm) column into six parts by eluting with a gradient system of CHCl₃-MeOH (1 : 0, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 0 : 1, each 10.0 L). The part exhibiting a blue spot in TLC test was collected from the CHCl₃-MeOH (9 : 1) fraction, and purified by preparative TLC (developed three times in petroleum ether-Me₂CO, 4 : 1, Rf = 0.4) to yield 1 (4 mg). The yield of 1 was so low that it was too difficult to get a suitable single crystal for X-ray analysis, which could determine the novel structure of 1. Thus, the use of the structure of 2 seemed to be necessary. In the same way, the EtOAc extract (400 g) of the leaves of I. rubescens var. taihangensis (10 kg) was obtained and subjected to silica gel CC (200 - 300 mesh, 3.0 kg, 10 × 130 cm) using CHCl₃ (15 L), CHCl₃-Me₂CO (9 : 1, 8 : 2, 7 : 3, 6 : 4, each 20 L) and Me₂CO (5 L) as eluents. Similarly, the CHCl₃-Me₂CO (9 : 1) fraction (80.4 g) was further chromatographed on silica gel to give eight parts, eluting with CHCl₃-MeOH (1 : 0, 9 : 1, 8 : 2, 7 : 3, 6 : 4, 0 : 1, each 10.0 L). Separation of the CHCl₃-MeOH (9 : 1) part by the silica gel CC (eluting with petroleum ether-Me₂CO, 5 : 1) yielded a portion, which also presented a blue spot in its TLC test, and from this portion, compound 2 (16 mg) was obtained as colorless cubes by recrystallization from MeOH. Both compounds were bioassayed for their cytotoxicity against K562 cells, using a previously described method [6], in which the OD values were recorded in X ± S, and the IC₅₀ values were calculated by the software GWBASIC, with cis-platinum being the positive control.

Maoyecrystal I (1): a white amorphous powder, C₂₀H₂₆O₅; [α]_D ^25.2: -32.47 (c 0.15, MeOH); UV (MeOH): λmax no absorption; IR (KBr): ν_max = 3418, 2927, 1736, 1712, 1433, 1375, 1245, 1071, 1034, 905 cm^-1; EIMS: m/z (rel. intensity) = 346 (M⁺, 15), 328 (28), 318 (1), 310 (14), 300 (35), 282 (100), 272 (60), 253 (85), 226 (82), 197 (46), 170 (42), 128 (64), 105 (72), 91 (97); HREIMS: m/z = 346.1778 [M]⁺, calcd.: 346.1780; ¹H- and ¹³C-NMR: Table [1].

Rubescensin W (2): colorless cubes, C₂₁H₃₀O₆; m. p. 109 - 110 °C; [α]_D ^26.9: + 57.69 (c 0.34, MeOH); UV (MeOH): λmax no absorption; IR (KBr): ν_max = 3477, 2954, 2900, 1704, 1630, 1459, 1417, 1382, 1246, 1195, 1122, 1091, 1022, 1011, 990 cm^-1; EIMS: m/z (rel. intensity) = 378 (M⁺, 18), 360 (6), 346 (100), 328 (30), 318 (2), 300 (6); HREIMS m/z = 378.2024 [M]⁺, calcd.: 378.2042; ¹H- and ¹³C-NMR: Table [1].

Crystal data of 2: colorless cubes (MeOH), monoclinic space group P21, C₂₁H₃₀O₆·H₂O, M = 378.45, a = 10.1970(6), b = 10.2000(6), c = 10.7650(6) Å, β = 65.069(4)°, V = 1015.33(10) ų, Z = 2, d = 1.297 g/cm³. Rf = 0.065, Rw = 0.066 (w = 1/σ|F|²). Linear absorption coefficient μ = 1.0 cm^-1. A colorless cubic crystal of dimensions 0.40 × 0.50 × 0.60 mm was used for X-ray measurements on a MAC DIP-2030 diffractometer with graphite monochromated Mo Kα radiation, a maximum 2Θ value of 50.0° was set. The total of 1829 independent reflections was measured, all were considered to be observed (|F|² ≥ 8σ|F|²). Hydrogen atoms were fixed at calculated positions. The structure was solved by the direct method SHELX-86 and expanded using difference Fourier techniques, refined by the program and method NOMCSDP and full-matrix least-squares calculations. The X-ray data are deposited in Cambridge Crystallographic Data Centre (deposition number CCDC 232 710). 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 - 336 033 or e-mail: deposit@ccdc.cam.ac.uk).

References

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• 2 Node M. Research and development of new anticancer agent from Isodon japonica and I. trichocarpa .  Suzuken Memorial Foundation. 1984;  3 112-8
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• 6 Abbaskhan A, Choudhary M I, Tsuda Y, Parvez M, Rahman A, Shaheen F, Parween Z, Tareen R B, Aaidi M A. A new diepoxy-ent-kauranoid, rugosinin, from Isodon rugosus .  Planta Medica. 2003;  69 94-6
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• 7 Niu X M, Li S H, Li M L, Zhao Q S, Mei S X, Lin Z W, Sun H D. Cytotoxic ent-kaurane diterpenoids from Isodon eriocalyx var. laxiflora .  Planta Medica. 2002;  68 528-33
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  Search in Google Scholar

Prof. Dr. Han-Dong Sun

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

Chinese Academy of Sciences

Heilongtan

Kunming 650204

People’s Republic of China

Fax: +86-871-5216-343

Email: hdsun@mail.kib.ac.cn

References

• 1 Sun H D, Xu Y L, Jiang B. Diterpenoids from Isodon species. 1st Edition Beijing; Sciences Press 2001: pp 95-106
  Search in Google Scholar
• 2 Node M. Research and development of new anticancer agent from Isodon japonica and I. trichocarpa .  Suzuken Memorial Foundation. 1984;  3 112-8
  PubMedSearch in Google Scholar
• 3 Zhang J X, Han Q B, Zhao Q S, Li S H, Sun H D. Two new ent-kauranoids from Isodon japonica .  Chinese Chemical Letters. 2002;  13 1075-8
  PubMedSearch in Google Scholar
• 4 Zhang J X, Han Q B, Zhao A H, Sun H D. Diterpenoids from Isodon japonica .  Fitoterapia. 2003;  74 435-8
  CrossrefPubMedSearch in Google Scholar
• 5 Han Q B, Zhang J X, Shen Y H, Sun H D. Diterpenoids from Isodon japonicus .  Chinese Journal of Natural Medicines. 2003;  1 13-7
  PubMedSearch in Google Scholar
• 6 Abbaskhan A, Choudhary M I, Tsuda Y, Parvez M, Rahman A, Shaheen F, Parween Z, Tareen R B, Aaidi M A. A new diepoxy-ent-kauranoid, rugosinin, from Isodon rugosus .  Planta Medica. 2003;  69 94-6
  Thieme ConnectPubMedSearch in Google Scholar
• 7 Niu X M, Li S H, Li M L, Zhao Q S, Mei S X, Lin Z W, Sun H D. Cytotoxic ent-kaurane diterpenoids from Isodon eriocalyx var. laxiflora .  Planta Medica. 2002;  68 528-33
  Thieme ConnectPubMedSearch in Google Scholar
• 8 Proemmel S, Wartchow R, Hoffmann H. M. R. Synthesis and studies of marine natural products: the dictyoxetane core from 8-oxabicyclo[3.2.1]oct-6-en-3-ones.  Tetrahedron. 2002;  58 6199-206
  CrossrefPubMedSearch in Google Scholar
• 9 Suffness M. Overview of paclitaxel research: progress on many fronts. In: Georg GI, Chen TT, Ojima I, Vyas DM (editors) Taxane Anticancer Agents, A.C.S. Symposium Series 583. American Chemical Society Washington DC; 1995: pp 1-17
  Search in Google Scholar

Prof. Dr. Han-Dong Sun

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

Chinese Academy of Sciences

Heilongtan

Kunming 650204

People’s Republic of China

Fax: +86-871-5216-343

Email: hdsun@mail.kib.ac.cn