New Dihydrodibenzoxepins from Bulbophyllum kwangtungense

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

Three new dihydrodibenzoxepins 7,8-dihydro-5-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (1), 7,8-dihydro-4-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (2), and 7,8-dihydro-3-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (3), were isolated from Bulbophyllum kwangtungense Schlecht, along with three known compounds, cumulatin (4), densiflorol A (5) and plicatol B (6). Their structures were established by a combination of 1D and 2D NMR spectroscopic techniques. All new compounds (1 - 3) and the known compound densiflorol A (5) exhibited anti-tumor activities against HeLa and K562 human tumor cell lines.

The genus Bulbophyllum, a member of the Orchidaceae family, consists of over 1000 species found in Africa and Asia [1]. It is a rich source of aromatic compounds such as phenanthrenes and bibenzyls [2], [3], [4], [5]. Bulbophyllum kwangtungense Schlecht (Chinese name ”Shi dou-lan”) has long been used in traditional Chinese medicine as a Yin tonic to nourish the lung, promote the production of body liquid and reduce fever [6]. To the best of our knowledge, no phytochemical investigation on this plant has been reported. Our research on the chemistry and on biological activities of this species resulted in the isolation of three new dihydrodibenzoxepins, 7,8-dihydro-5-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (1), 7,8-dihydro-4-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (2), and 7,8-dihydro-3-hydroxy-12, 13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (3), along with three known compounds, cumulatin (4), densiflorol A (5) and plicatol B (6) from the EtOAc extract. The antitumor activities of these compounds also are reported.[]

Compound 1 was obtained as colorless needles from methanol. The high resolution FT-ICR-MS exhibited a molecular ion peak at m/z = 287.0909 [M + H]⁺ (calculated: 287.0914), corresponding to the molecular formula C₁₆H₁₄O₅. The IR spectrum showed hydroxy (3444 cm^-1) and aromatic ring (1617, 1496 and 1352 cm^-1) absorptions, and its UV spectrum showed absorption bands at 214, 253 and 272 nm. A positive reaction with FeCl₃ and the IR spectral data suggested the structural characteristics of phenolic compounds [7]. The ¹H-NMR spectrum of 1 in DMSO-d ₆ revealed the presence of three coupled aromatic protons and one isolated aromatic proton. Additional signals in the ¹H-NMR spectrum were typical for two methylenedioxy protons and three methoxy protons (Table [1]). The ¹³C-NMR spectrum of 1 (Table [1]) showed 16 carbon resonances, among which 12 resonances in the deshielded region were consistent with the presence of two benzene rings. Two benzene rings were accounted for by 8 units of unsaturation, indicating the existence of two additional rings from the molecular formula C₁₆H₁₄O₅. The structure of 1 was deduced from a detailed analysis of ¹H- and ¹³C-NMR data aided by 2D NMR including COSY, HMQC, and HMBC experiments (Fig. [1]). HMBC correlations completed the definition of all the functional groups in compound 1. The long-range correlations from the two coupled benzylic methylene groups (H-7 and H-8) to two oxygenated aromatic carbons at δ _C = 144.00 (s, C-6) and 134.99 (s, C-14) observed in the HMBC spectrum indicated a dihydrodibenz[b,f]oxepin skeleton [9], [10]. The long-range correlations from the hydroxyl proton at δ _H = 6.16 to the carbon signals at δ _C = 113.02 (d, C-4), 147.25 (s, C-5) and 144.00 (s, C-6) established the hydroxy group at C-5. The long-range correlation from the methoxy protons at δ _H = 3.86 to the carbon signal at δ _C = 140.01 (s, C-11) indicated the presence of a methoxy group at C-11. The methylenedioxy protons at δ _H = 6.02 (s, H-16) had long-range correlation to the aromatic carbons at δ _C = 134.29 (s, C-12) and 138.75 (s, C-13), revealing that the methylenedioxy group is attached to the aromatic ring at C-12 and C-13. Thus, the structure of 1 was established as 7,8-dihydro-5-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin. The occurrence of oxepin derivatives appears to be rare in nature [8]. 7,8-Dihydrodibenz[b,f]oxepins, which were characterized by a diphenyl ether probably formed by a coupling from 9,10-dihydrobibenzyl derivatives, are even more uncommon in nature, and only two compounds have been found from natural sources [9], [10]. This is the first time that a 7,8-dihydrodibenz[b,f]oxepin with a methylenedioxy group was isolated.

Compound 2 was obtained as colorless needles from methanol. The high resolution FT-ICR-MS exhibited a molecular ion peak at m/z = 287.0912 [M + H]⁺ (calculated: 287.0914), corresponding to the same molecular formula C₁₆H₁₄O₅ as that of compound 1. The IR spectrum showed hydroxy (3518 cm^-1) and aromatic ring (1624, 1459 and 1375 cm^-1) absorptions, and its UV spectrum showed absorption bands at 211, 255 and 270 nm. The ¹H- and ¹³C-NMR spectra of 2 showed similar chemical shifts and the same multiplicities for all carbon atoms as in 1 with minor differences, indicating a dihydrodibenz[b,f]oxepin skeleton with one methoxy, one hydroxy and one methylenedioxy group in the structure of 2. Extensive analysis on the proton couplings of 2 in the ¹H-NMR spectrum revealed that the structure of 2 had a 1,3,4-substituted benzene ring [δ _H = 6.56 (1H, d, J = 3.00 Hz, H-5), 6.58 (1H, dd, J = 8.18, 3.00 Hz, H-3), 7.03 (1H, d, J = 8.18 Hz, H-2)]. HMBC correlations completed the definition of all the functional groups in compound 2 (Fig. [1]). The long-range correlations from the proton at δ _H = 7.03 (H-2) to the carbon signals at δ _C = 30.82 (t, C-7) and 150.65 (s, C-6) as well as the long-range correlations from the proton at δ _H = 6.56 (H-5) to the carbon signals at δ _C = 132.73 (s, C-1) and 150.65 (s, C-6), together with the identification of a 1,3,4-substituted benzene ring pattern, indicated the presence of a hydroxy group attached at C-4. Thus the structure of 2 was established as 7,8-dihydro-4-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin.

Compound 3 was obtained as colorless needles from methanol. The high resolution FT-ICR-MS exhibited a molecular ion peak at m/z = 287.0913 [M + H]⁺ (calculated: 287.0914), corresponding to the same molecular formula C₁₆H₁₄O₅ as those of compounds 1 and 2. The UV and IR spectra of 3 exhibited the same patterns as those of 1 and 2. The ¹H- and ¹³C-NMR spectra of compound 3 (Table [1]) were also similar to those of 1 and 2, revealing a dihydrodibenz[b,f]oxepin skeleton with one methoxy, one hydroxy and one methylenedioxy group in the structure of 3. Extensive analysis on the proton couplings of 3 in ¹H-NMR spectrum revealed that the structure of 3 also had a 1,3,4-substituted benzene ring [δ _H = 6.55 (1H, d, J = 2.73 Hz, H-2), 6.52 (1H, dd, J = 8.51, 2.73 Hz, H-4), and 6.87 (1H, d, J = 8.51 Hz, H-5)]. HMBC correlations completed the definition of all the functional groups in compound 3 (Fig. [1]). The long-range correlations from the proton at δ _H = 6.55 (d, J = 2.73 Hz, H-2) to the carbon signals at δ _C = 150.09 (s, C-6) and 31.35 (t, C-7) in the HMBC spectrum revealed that the hydroxy group was attached to C-3. Thus, the structure of 3 was established as 7,8-dihydro-3-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin.

Furthermore, three known compounds, cumulatin (4), densiflorol A (5) and plicatol B (6) were identified by comparison of their spectroscopic data with those in the literature [11], [12], [13].

Compounds 1 - 6 were tested for anti-tumor activity in vitro against two human tumor cell lines using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] colorimetric method [14]. All new compounds (1 - 3) and the known compound densiflorol A (5) exhibited anti-tumor activities against Hela and K562 human tumor cell lines. IC₅₀ values of compounds 1, 2, 3 and 5 in HeLa cells were 140.9 μM, 78.3 μM, 61.2 μM and 79.4 μM, and the IC₅₀ values of compounds 1, 2, 3 and 5 in K562 cells were 138.8 μM, 88.5 μM, 64.7 μM and 67.6 μM, respectively. However, compounds 4 and 5 did not show antitumor activities.

Materials and Methods

The spectroscopic instruments and the antitumor assay method were described in our previous paper [15]. The leaves and stems of Bulbophyllum kwangtungense Schlecht were collected in the Zhejiang province, China, in September 2003 and identified by Dr. Hongxiang Sun (Zhejiang University, Hangzhou, China.). A voucher specimen (No. zju 3587) is kept in the College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China. The shade-dried, powdered leaves and stems (3 kg) of B. kwangtungense Schlecht were extracted at room temperature three times with methanol (3 × 5000 mL). The extracts were evaporated under vacuum to afford a gummy residue (321 g). This residue was partitioned in H₂O (3000 mL) and extracted with petroleum ether (4 × 3000 mL) and EtOAc (4 × 3000 mL), successively. The EtOAc extract (102 g) was adsorbed onto silica gel (100 g) and submitted to chromatography over a silica gel column (9 × 100 cm, 2 kg, 200 - 300 mesh) eluting with n-hexane/EtOAc gradient mixtures. TLC-guided combination of eluents resulted in eleven main fractions which were subsequently tested for antitumor activity. The viscous yellow oil (5.1 g) obtained from the fourth and fifth fractions showed high antitumor activity. The fourth fraction was subjected to preparative HPLC (Waters 600 HPLC, Shim-pack PREP-ODS 250 × 20 mm, flow rate 8 mL/min, UV detector 254 nm) using CH₃OH/H₂O (75 : 25) as eluent to afford compounds 1 (10.6 mg, t_R = 65.3 min) and 2 (13.5 mg, t_R = 80.9 min). The fifth fraction (2.3 g) was applied to a Sephadex LH-20 column (8 × 150 cm, 300 g) and eluted with 70 % MeOH at 15 °C for 2 days. Collected fractions were separated by HPLC using CH₃OH/H₂O (72 : 28) as eluent, to yield compounds 3 (13.5 mg), 4 (15.6 mg), 5 (14.6 mg) and 6 (10.7 mg).

7,8-Dihydro-5-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (1): Colorless needles (MeOH), C₁₆H₁₄O₅; UV (MeOH): λ_max = 214 (4.56), 253 (2.45), 272 nm (3.00); IR (KBr): ν_max = 3444, 2947, 1617, 1496 1454, 1459, 1352, 1330, 1195, 1133, 1044, 948, 901 cm^-1; ¹H-NMR and ¹³C-NMR: see Table [1]; ESI-MS: m/z = 309 [M + Na]⁺, 285 [M - H]^-; FT-ICR-MS: m/z = 287.0909 [M + H]⁺ (calculated: 287.0914).

7,8-Dihydro-4-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (2): Colorless needles (MeOH), C₁₆H₁₄O₅; UV (MeOH): λ_max = 211 (4.32), 255 (2.64), 270 nm (3.23); IR (KBr): ν_max = 3518, 2917, 1624, 1591, 1509, 1459, 1375, 1319, 1130, 1054, 964 cm^-1; ¹H-NMR and ¹³C-NMR, see Table [1]; ESI-MS: m/z = 309 [M + Na]⁺, 285 [M - H]^-; FT-ICR-MS: m/z = 287.0912 [M + H]⁺ (calculated: 287.0914).

7,8-Dihydro-3-hydroxy-12,13-methylenedioxy-11-methoxyldibenz[b,f]oxepin (3): Colorless needles (MeOH), C₁₆H₁₄O₅; UV (MeOH): λ_max = 212 (4.67), 253 (2.33), 272 nm (3.05); IR (KBr): ν_max = 3510, 2932, 1620, 1564, 1145, 1366, 1148, 1046, 958, 903 cm^-1; ¹H-NMR and ¹³C-NMR, see Table [1]; ESI-MS: m/z = 309 [M + Na]⁺, 285 [M - H]^-; FT-ICR-MS: m/z = 287.0913 [M + H]⁺ (calculated: 287.0914).

Acknowledgements

This work was supported by NSFC of China (20 472 073).

References

• 1 Leong Y -W, Kang C -C, Harrison L J, Powell A D. Phenanthrenes, dihydrophenanthrenes and bibenzyls from the Orchid Bulbophyllum vaginatum .  Phytochemistry. 1997;  44 157-65
  CrossrefPubMedSearch in Google Scholar
• 2 Leong Y -W, Harrison L J. A biphenanthrene and a phenanthro[4,3-b]furan from the orchid Bulbophyllum vaginatum .  J Nat Prod. 2004;  67 1601-3
  CrossrefPubMedSearch in Google Scholar
• 3 Majumder P L, Pal S, Majumder S. Dimeric phenanthrenes from the orchid Bulbophyllum reptans .  Phytochemistry. 1999;  50 891-7
  CrossrefPubMedSearch in Google Scholar
• 4 Majumder P L, Kar A, Shoolery J N. Bulbophyllathrin, a phenanthrene of the orchid Bulbophyllum leopardium .  Phytochemistry. 1985;  25 2083-7
  PubMedSearch in Google Scholar
• 5 Majumder P L, Sen R C. Bulbophyllanthrone, a phenanthraquinone from Bulbophyllum odoratissimum .  Phytochemistry. 1991;  30 2092-4
  CrossrefPubMedSearch in Google Scholar
• 6 Yi Y F, Xing F W, Huang X X, Chen H F. Medicinal plants of Bulbophyllum species in China.  J Trop Subtrop Bot. 2005;  13 65-9
  PubMedSearch in Google Scholar
• 7 Andrea S, Rudolf M, Floris E, Thawatchai S, Suttiporn C. Phenolic compounds from Anomianthus dulcis .  Phytochemistry. 1999;  50 1069-72
  CrossrefPubMedSearch in Google Scholar
• 8 Pettit G R, Numata A, Iwamoto C, Usami Y, Yamada T, Ohishi H. Antineoplastic gents. 551. Isolation and structures of bauhiniastatins 1 - 4 from Bauhinia purpurea .  J Nat Prod. 2006;  69 323-7
  CrossrefPubMedSearch in Google Scholar
• 9 Marina D G, Antonio F, Antonio M, Pietro M, Lucio P. A bioactive dihydrodibenzoxepin from Juncus effusus .  Phytochemistry. 1993;  34 1182-4
  CrossrefPubMedSearch in Google Scholar
• 10 Reddy M VB, Reddy M K, Gunasekar D, Caux C, Bodo B. A flavanone and a dihydrodibenzoxepin from Bauhinia variegata .  Phytochemistry. 2003;  64 879-82
  CrossrefPubMedSearch in Google Scholar
• 11 Majumder P L, Pal S. Cumulatin and tristin, two bibenzyl derivatives from the orchid Denrobium cumulatum and Bulbophyllum triste .  Phytochemistry. 1993;  32 1561-5
  CrossrefPubMedSearch in Google Scholar
• 12 Fan C Q, Wang W, Wang Y P, Qin G, Zhao W. Chemical constituents from Dendroium densiflorum .  Phytochemistry. 2001;  57 1255-8
  CrossrefPubMedSearch in Google Scholar
• 13 Chie H, Masae Y. Phenanthrenes from Dendrobium plicatile .  Phytochemistry. 2000;  53 987-90
  CrossrefPubMedSearch in Google Scholar
• 14 Tweentyman P R, Luscombe M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity.  Brit J Cancer. 1987;  56 279-85
  PubMedSearch in Google Scholar
• 15 Wang K W, Sun H X, Wu B, Pan Y J. Two novel olean triterpenoids from Celastrus hypoleucus .  Helv Chim Acta. 2005;  88 990-5
  CrossrefPubMedSearch in Google Scholar

Prof. Yuan-Jiang Pan

Department of Chemistry

Zhejiang University

Hangzhou 310027

People’s Republic of China

Phone: +86-571-879-51264

Fax: +86-571-879-51264

Email: panyuanjiang@zju.edu.cn

References

• 1 Leong Y -W, Kang C -C, Harrison L J, Powell A D. Phenanthrenes, dihydrophenanthrenes and bibenzyls from the Orchid Bulbophyllum vaginatum .  Phytochemistry. 1997;  44 157-65
  CrossrefPubMedSearch in Google Scholar
• 2 Leong Y -W, Harrison L J. A biphenanthrene and a phenanthro[4,3-b]furan from the orchid Bulbophyllum vaginatum .  J Nat Prod. 2004;  67 1601-3
  CrossrefPubMedSearch in Google Scholar
• 3 Majumder P L, Pal S, Majumder S. Dimeric phenanthrenes from the orchid Bulbophyllum reptans .  Phytochemistry. 1999;  50 891-7
  CrossrefPubMedSearch in Google Scholar
• 4 Majumder P L, Kar A, Shoolery J N. Bulbophyllathrin, a phenanthrene of the orchid Bulbophyllum leopardium .  Phytochemistry. 1985;  25 2083-7
  PubMedSearch in Google Scholar
• 5 Majumder P L, Sen R C. Bulbophyllanthrone, a phenanthraquinone from Bulbophyllum odoratissimum .  Phytochemistry. 1991;  30 2092-4
  CrossrefPubMedSearch in Google Scholar
• 6 Yi Y F, Xing F W, Huang X X, Chen H F. Medicinal plants of Bulbophyllum species in China.  J Trop Subtrop Bot. 2005;  13 65-9
  PubMedSearch in Google Scholar
• 7 Andrea S, Rudolf M, Floris E, Thawatchai S, Suttiporn C. Phenolic compounds from Anomianthus dulcis .  Phytochemistry. 1999;  50 1069-72
  CrossrefPubMedSearch in Google Scholar
• 8 Pettit G R, Numata A, Iwamoto C, Usami Y, Yamada T, Ohishi H. Antineoplastic gents. 551. Isolation and structures of bauhiniastatins 1 - 4 from Bauhinia purpurea .  J Nat Prod. 2006;  69 323-7
  CrossrefPubMedSearch in Google Scholar
• 9 Marina D G, Antonio F, Antonio M, Pietro M, Lucio P. A bioactive dihydrodibenzoxepin from Juncus effusus .  Phytochemistry. 1993;  34 1182-4
  CrossrefPubMedSearch in Google Scholar
• 10 Reddy M VB, Reddy M K, Gunasekar D, Caux C, Bodo B. A flavanone and a dihydrodibenzoxepin from Bauhinia variegata .  Phytochemistry. 2003;  64 879-82
  CrossrefPubMedSearch in Google Scholar
• 11 Majumder P L, Pal S. Cumulatin and tristin, two bibenzyl derivatives from the orchid Denrobium cumulatum and Bulbophyllum triste .  Phytochemistry. 1993;  32 1561-5
  CrossrefPubMedSearch in Google Scholar
• 12 Fan C Q, Wang W, Wang Y P, Qin G, Zhao W. Chemical constituents from Dendroium densiflorum .  Phytochemistry. 2001;  57 1255-8
  CrossrefPubMedSearch in Google Scholar
• 13 Chie H, Masae Y. Phenanthrenes from Dendrobium plicatile .  Phytochemistry. 2000;  53 987-90
  CrossrefPubMedSearch in Google Scholar
• 14 Tweentyman P R, Luscombe M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity.  Brit J Cancer. 1987;  56 279-85
  PubMedSearch in Google Scholar
• 15 Wang K W, Sun H X, Wu B, Pan Y J. Two novel olean triterpenoids from Celastrus hypoleucus .  Helv Chim Acta. 2005;  88 990-5
  CrossrefPubMedSearch in Google Scholar

Prof. Yuan-Jiang Pan

Department of Chemistry

Zhejiang University

Hangzhou 310027

People’s Republic of China

Phone: +86-571-879-51264

Fax: +86-571-879-51264

Email: panyuanjiang@zju.edu.cn