ent-Kaurane Diterpenoids from the Leaves of Isodon xerophilus

• Abstract
• Materials and Methods
• References

Abstract

From the ethanolic extract of the leaves of Isodon xerophilus, three new ent-kaurane diterpenoids named xerophilusins L-N (1 - 3), together with three known ones designated as rabdoternin A (4), longikaurin F (5) and ponicidin (6), were isolated and structurally elucidated. Compound 6 demonstrated most potent cytotoxic activity against K562 and T24 human tumor cell lines with IC₅₀ = 0.09 and 0.32 μg/ml, respectively.

Recent phytochemical studies on some Isodon species (Labiatae) have brought the discovery of some interesting novel ent-kaurane diterpenoids [1], [2]. Our previous investigation on the EtOAc extracts of Isodon xerophilus has led to the discovery of a series of new and known ent-kauranoids [3], [4], [5]. The ongoing work resulted in the isolation of another six ent-kauranoids including three new ones named xerophilusins L-N (1 - 3) and three known ones identified as rabdoternin A (4) [6], longikaurin F (5) [7], and ponicidin (6) [8].

Xerophilusin L (1) was obtained as colorless block crystals. EIMS showed the [M]⁺ ion peak at m/z 380, consistent with a molecular formula of C₂₀H₂₈O₇, as supported by HREIMS (found: m/z 380.1825, calcd. 380.1835). Signals observed in the NMR spectra (Tables [1] and 2) of 1, were very similar to those of rabdoternin A (4), suggesting that 1 possessed the same skeleton and equivalent oxygenation pattern with those for 4. A detailed comparison between the NMR data of these two compounds revealed that 1 differed from 4 only by the existence of an additional hydroxy group. The oxymethine signal at δ _H 4.21 (H-11) showed ¹H-¹H COSY interaction with H-9 and HMBC couplings with C-9, C-10, C12 and C-13, indicating that the extra hydroxy group was assignable to C-11. Relative stereochemistry of the 11-OH was established as β-orientation due to the observation of 2D ROESY correlations of H-11 with H-1α and H-14α (Fig. [1]). Finally xerophilusin L was concluded as ent-7β, 20-epoxy-kaur-16-en-6α,7α,11α,14α,15α-pentaol-20-one.

Xerophilusin M (2) was isolated as colorless block crystals. Its NMR data (Tables [1] and 2) coupled with a [M]⁺ ion peak at m/z 422.1943 in HREIMS established a molecular formula of C₂₂H₃₀O₈ (calcd. 422.1941). The NMR spectra of 2 were very similar with those of 1 except for the presence of an additional acetyl group, which we propose to be attached to 11-OH considering the obviously downfield shifted H-11/C-11 signals [H-11: δ _H 4.21 for 1 and 5.17 for 2; C-11: δ _C 62.4 for 1 and 65.9 for 2]. The long-range ¹H-¹³C correlation from H-11 to the acetyl carbonyl carbon in the HMBC spectrum confirmed the above speculation. Xerophilusin M was accordingly deduced as ent-7β,20-epoxy-11α-acetoxy-kaur-16-en-6α,7α,14α,15α-tetraol-20-one.

Xerophilusin N (3) was purified as colorless needles. HREIMS (found: m/z 362.1729, calcd. 362.1729) provided a molecular formula of C₂₀H₂₆O₆. The NMR spectra (Tables [1] and 2) of 3 closely resembled those of 4, indicating that 3 was also an ent-kauranoid closely related to 4. Compound 3 differed from 4 mainly by the replacement of a hydroxy group by a keto group, which presumably occurred at C-15 considering the δ _H/C changes of H₂ - 17, C-17, C-16 and C-9 signals. Xerophilusin N was proved to be the expected ent-7β,20-epoxy-kaur-16-en-6α,7α,14α-triol-15,20-dione by extensive 2D NMR (including ¹H-¹H COSY, HMQC, HMBC and ROESY) analysis.

Compounds 1 - 3 and 6 were evaluated for their in vitro cytotoxic activity against two human tumor cell lines (K562 and T24) using the same bioassay methods as previously described [9]. Compound 6 exhibited most significant cytotoxic activities, the potency of inhibition was approximately 22 fold (against K562 cell, IC₅₀ = 0.09 μg/mL) and 3.5-fold (against T24 cell, IC₅₀ = 0.32 μg/mL) higher than cis-platin (IC₅₀ = 2.02 and 1.16 μg/mL, respectively). In contrast, compound 3 only showed very weak inhibitory effects (IC₅₀ &γ> 15.6 μg/mL). The cytotoxic activities of compounds 1 and 2, however, were undeterminable in the same conditions. Although nearly 500 ent-kaurane diterpenoids have been discovered from different Isodon species [10], those 7,20-epoxy diterpenoids possessing a carbonyl group at C-20 are still rare, with only 2 such compounds including rabdoternin A (4) were reported [6]. Our present study implied that phytochemical changes in I. xerophilus are possible due to seasonal conditions.

Materials and Methods

General: Previously described experimental procedures were adopted [3]. Fractions were monitored by silica gel TLC (CHCl₃-MeOH 19 : 1, 14 : 1, 9 : 1, 7 : 1 or petroleum ether-EtOAc, 3 : 1, 2 : 1, 1 : 1, 1 : 2) and spots were visualized by heating plates sprayed with 10 % H₂SO₄ in EtOH.

Plant material: The leaves of I. xerophilus were collected and identified in September 1999 as previously described [3]. A voucher specimen (No. KIB 99-9-28 Lin) has also been deposited in the same Herbarium.

Extraction and isolation: The air-dried leaves of I. xerophilus (7 kg) were pulverized and extracted with EtOH 95 % EtOH (30 L) at 50 °C for 3 × 24 hrs. The extract was filtered and concentrated, in vacuo, and suitably diluted with water, then partitioned with EtOAc (4 × 10 L). The organic layer was evaporated, in vacuo, to dryness to afford 400 g of a residue. 390 g of this extract was absorbed on 1.3 kg of silica gel and chromatographed on a pre-packed silica gel column (2.0 kg), eluting with a gradient system of CHCl₃-Me₂CO (1 : 0/20 L, 9 : 1/25 L, 8 : 2/25 L, 7 : 3/20 L, 6 : 4/15 L, 0 : 1/10 L) to give six fractions (Frs. 1 - 6). Compound 6 (298 mg) was crystallized from Fr. 3 in acetone directly. Fr. 2 (54 g) was rechromatographed on silica gel (300 g) developing with petroleum ether-EtOAc (2 : 1, 5 L) and on RP₁₈ gel (100 g) using CH₃OH-H₂O (70 : 30, 3 L) as eluents to provide compound 3 (405 mg). The remaining Fr. 3 (37 g) was further subjected CC over silica gel (200 g) eluting with petroleum ether-EtOAc (1 : 1, 3.5 L) and n-hexane-i-PrOH (5 : 1, 3 L) and over RP₁₈ gel (80 g) with eluting system of CH₃OH-H₂O (65 : 35, 2 L) to yield compounds 2 (238 mg), 4 (323 mg) and 5 (10 mg). Compound 1 (112 mg) was purified from Fr. 4 (107 g) by sequential CC on silica gel (600 g) using a mixture of cyclohexane-CHCl₃-i-PrOH (3 : 2:1, 9 L) as eluting solvents and on RP₁₈ gel (200 g) with the eluents of aqueous CH₃OH (55 %, 5 L).

Xerophilusin L (1): C₂₀H₂₈O₇, colorless needles, m. p. 272 °C; [α]^23.1 _D: -7.6° (c 0.33, CH₃OH); UV (MeOH): λ_max (log ε) = 204.5 nm (3.82); IR (KBr): ν_max = 3393, 2938, 1726, 1390, 1360, 1256, 1222, 1193, 1163, 1115, 1081, 1060, 1032, 985, 957, 932 cm^-1; ¹H-NMR data, see Table [1]; ¹³C-NMR data, see Table [2]; EIMS: m/z = 380 [M]⁺ (4), 364 (2), 344 (2), 328 (6), 316 (3), 300 (15), 284 (10), 269 (15), 255 (16), 239 (7), 227 (10), 213 (13), 199 (8), 185 (10), 165 (12), 149 (16), 137 (12), 129 (16), 115 (23), 105 (31), 91 (100), 77 (41), 69 (37), 55 (63); HREIMS: m/z = 380.1825 [M]⁺, calcd. 380.1835.

Xerophilusin M (2): C₂₂H₃₀O₈, colorless needles, m. p. 267 °C; [α]^22.9 _D: + 16.2° (c 0.39, CH₃OH); UV (MeOH): λ_max (log ε) = 203 nm (3.86); IR (KBr): ν_max = 3527, 3436, 2956, 1740, 1717, 1447, 1371, 1321, 1243, 1220, 1195, 1159, 1111, 1062, 1026, 957, 906, 856 cm^-1; ¹H-NMR data, see Table [1]; ¹³C-NMR data, see Table [2]; EIMS: m/z = 422 [M]⁺ (81), 404 (5), 378 (30), 362 (5), 344 (31), 326 (24), 316 (16), 300 (42), 282 (27), 271 (36), 253 (27), 243 (2), 239 (30), 229 (28), 213 (36), 199 (22), 190 (45), 185 (25), 183 (25), 171 (21), 161 (27), 159 (28), 157 (30), 152 (41), 133 (43), 109 (32), 91 (85), 77 (59), 69 (67), 55 (100); HREIMS: m/z = 422.1943 [M]⁺, calcd. 422.1941.

Xerophilusin N (3): C₂₀H₂₆O₆, colorless needles, m. p. 225 °C; [α]^23.2 _D: -82.8° (c 0.46, CH₃OH); UV (MeOH): λ_max (log ε) = 235.5 nm (4.15); IR (KBr): ν_max = 3595, 3288, 2948, 1752, 1711, 1641, 1457, 1393, 1372, 1305, 1261, 1218, 1194, 1158, 1141, 1095, 1058, 949, 598, 578 cm^-1; ¹H-NMR data, see Table [1]; ¹³C-NMR data, see Table [2]; EIMS: m/z = 362 [M]⁺ (17), 344 (19), 333 (10), 326 (6), 318 (26), 300 (19), 288 (7), 271 (15), 255 (8), 243 (5), 229 (8), 213 (8), 199 (7), 190 (8), 177 (11), 167 (12), 161 (15), 149 (22), 135 (22), 121 (20), 115 (21), 105 (44), 91 (84), 77 (61), 69 (62), 55 (100); HREIMS: m/z = 362.1729 [M]⁺, calcd. 362.1729.

Rabdoternin A (4): C₂₀H₂₈O₆, colorless crystals; [α]²⁴ _D: -45.6° (c 0.19, CH₃OH) [6]; Longikaurin F (5): C₂₄H₃₂O₈, colorless crystals; [α]²⁵ _D: -120.4° (c 0.11, C₅H₅N) [7]; Ponicidin (6): C₂₀H₂₆O₆, colorless crystals; [α]¹⁷ _D: -118.0° (c 0.10, C₅H₅N) [8] Copies of the original spectra are obtainable from the author of correspondence.

References

• 1 Sun H D, Qiu S X, Lin L Z, Zhang R P, Zhou Y, Zheng Q T, Johnson M E, Fong H HS, Farnsworth N R, Cordell G A. Crystal structure of a 1 : 1 complex of natural diterpenoid: absolute configurations and unambiguous NMR spectral assignments of neoangustifolin and epinodosinol.  J Nat Prod. 1997;  60 203-6
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• 4 Hou A J, Zhao Q S, Jiang B, Lin Z W, Sun H D, Zhou Y P, Lu Y, Zheng Q T. Cytotoxic 7,20-epoxy-ent-kauranoids from Isodon xerophilus .  Phytochemistry. 2001;  58 179-83
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• 5 Hou A J, Yang H, Liu Y Z, Zhao Q S, Lin Z W, Sun H D. Novel ent-kaurane diterpenoids from Isodon xerophilus .  Chin J Chem. 2001;  19 365-70
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  PubMedSearch in Google Scholar
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• 8 Fujita E, Taoka M, Shibuya M, Fujita T, Shingu T. Terpenoids. XXVII. Structure and stereochemistry of ponicidin, a diterpenoid of Isodon japonicus . J Chem Soc Perkin Trans I 1973: 2277-81
  Search in Google Scholar
• 9 Niu X M, Li S H, Li M L, Zhao Q S, Mei S X, Na Z, Wang S J, 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
• 10 Sun H D, Xu Y L, Jiang B. Diterpenoids from Isodon species. Science Press Beijing, China; October 2001 P5
  Search in Google Scholar

Prof. Dr. Han-Dong Sun

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

The Chinese Academy of Sciences

Heilongtan

Kunming 650204

Yunnan Province

People’s Republic of China

Email: hdsun@mail.kib.ac.cn

Fax: +86 871 5216343

References

• 1 Sun H D, Qiu S X, Lin L Z, Zhang R P, Zhou Y, Zheng Q T, Johnson M E, Fong H HS, Farnsworth N R, Cordell G A. Crystal structure of a 1 : 1 complex of natural diterpenoid: absolute configurations and unambiguous NMR spectral assignments of neoangustifolin and epinodosinol.  J Nat Prod. 1997;  60 203-6
  CrossrefPubMedSearch in Google Scholar
• 2 Sun H D, Qiu S X, Lobkovsky E B, Lin L Z, Farnsworth N R, Clardy J, Fong H HS. Crystal and molecular structures of a natural equimolecular mixture of two epimeric diterpenes.  Tetrahedron. 2001;  57 65-70
  CrossrefPubMedSearch in Google Scholar
• 3 Hou A J, Li M L, Jiang B, Lin Z W, Ji S Y, Zhou Y P, Sun H D. New 7,20 : 14,20-diepoxy-ent-kauranoids from Isodon xerophilus .  J Nat Prod. 2000 ;  63 599-601
  CrossrefPubMedSearch in Google Scholar
• 4 Hou A J, Zhao Q S, Jiang B, Lin Z W, Sun H D, Zhou Y P, Lu Y, Zheng Q T. Cytotoxic 7,20-epoxy-ent-kauranoids from Isodon xerophilus .  Phytochemistry. 2001;  58 179-83
  CrossrefPubMedSearch in Google Scholar
• 5 Hou A J, Yang H, Liu Y Z, Zhao Q S, Lin Z W, Sun H D. Novel ent-kaurane diterpenoids from Isodon xerophilus .  Chin J Chem. 2001;  19 365-70
  PubMedSearch in Google Scholar
• 6 Takeda Y, Takeda K, Fujita T, Sun H D, Minami Y. Studies on the diterpenoid constituents of Rabdosia ternifolia: structural elucidation of new diterpenoids, rabdoternins A, B and C.  Chem Pharm Bull. 1990;  38 439-42
  PubMedSearch in Google Scholar
• 7 Fujita T, Takeda Y, Shingu T. Longikaurin C, D, E and F; new anti- bacterial diterpenoids from Radosia longituba .  Heterocycles. 1981;  16 227-30
  PubMedSearch in Google Scholar
• 8 Fujita E, Taoka M, Shibuya M, Fujita T, Shingu T. Terpenoids. XXVII. Structure and stereochemistry of ponicidin, a diterpenoid of Isodon japonicus . J Chem Soc Perkin Trans I 1973: 2277-81
  Search in Google Scholar
• 9 Niu X M, Li S H, Li M L, Zhao Q S, Mei S X, Na Z, Wang S J, 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
• 10 Sun H D, Xu Y L, Jiang B. Diterpenoids from Isodon species. Science Press Beijing, China; October 2001 P5
  Search in Google Scholar

Prof. Dr. Han-Dong Sun

State Key Laboratory of Phytochemistry and Plant Resources in West China

Kunming Institute of Botany

The Chinese Academy of Sciences

Heilongtan

Kunming 650204

Yunnan Province

People’s Republic of China

Email: hdsun@mail.kib.ac.cn

Fax: +86 871 5216343