A New Cytotoxic Amide from the Stem Wood of Hibiscus tiliaceus

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

A new coumarin, hibiscusin, and a new amide, hibiscusamide, together with eleven known compounds including vanillic acid, p-hydroxybenzoic acid, syringic acid, p-hydroxybenzaldehyde, scopoletin, N-trans-feruloyltyramine, N-cis-feruloyltyramine, a mixture of β-sitosterol and stigmasterol, a mixture of β-sitostenone and stigmasta-4,22-dien-3-one were isolated from the stem wood of Hibiscus tiliaceus. The structures of these new compounds were determined through spectral analyses. Among the isolates, three compounds exhibited cytotoxicity (IC₅₀ values < 4 μg/mL) against P-388 and/or HT-29 cell lines in vitro.

Hibiscus tiliaceus L. (Malvaceae) is an evergreen shrub or tree distributed in tropical and subtropical regions [1]. The root of this plant has been used as an antifebrile and emetic, and the leaves and bark have been used for the treatment of cough and bronchitis in Chinese folk medicine [2]. Investigation of the EtOAc-soluble fraction of the stem wood of Hibiscus tiliaceus has led to the isolation of a new coumarin, hibiscusin (1) and a new amide, hibiscusamide (2), together with eleven known compounds, vanillic acid (3), p-hydroxybenzoic acid (4), syringic acid (5), p-hydroxybenzaldehyde (6), scopoletin (7), N-trans-feruloyltyramine (8), N-cis-feruloyltyramine (9), a mixture of β-sitosterol (10) and stigmasterol (11), and a mixture of β-sitostenone (12) and stigmasta-4,22-dien-3-one (13). The structural elucidation of 1 and 2 and the cytotoxic properties of the isolates are described herein.[]

Hibiscusin (1) was isolated as a yellowish oil. The EI-MS afforded the positive ion [M]⁺ at m/z = 262, implying a molecular formula of C₁₄H₁₄O₅, which was confirmed by HR-EI-MS. The UV absorptions at 206, 227, 251, 296, and 344 nm were similar to those of scopoletin [3], and suggested the presence of a 6,7-dioxygenated coumarin nucleus. The presence of a carbonyl group in the molecule was revealed by a band at 1707 cm^-1 in the IR spectrum. The presence of hydroxy groups in the molecule was revealed by a band at 3289 cm^-1 in the IR spectrum and was confirmed by the signals at δ = 2.35 and 6.16 (each 1H, each br.s, disappeared on addition of D₂O) in the ¹H-NMR spectrum. The ¹H-NMR spectrum of 1 was very similar to that of scopoletin [3] except that the methoxy group (C-6) of scopoletin was replaced by a 2-hydroxy-3-methyl-3-butenyloxy group in 1. The presence of a 2-hydroxy-3-methyl-3-butenyloxy group was clearly demonstrated from a pair of double doublets at δ = 4.29 (J = 10.0, 7.2 Hz) and 4.37 (J = 10.0, 2.8 Hz) corresponding to the AB part of an ABX system in an -O-CH₂-CH(OH)- moiety, and a multiplet at δ = 4.75 showing the X portion of the same system. In addition, the ¹H-NMR spectrum exhibited two broad singlet signals at δ = 5.08 and 5.19, typical of terminal vinylic protons, and a singlet signal at δ = 1.86 (3H) characteristic of a methyl group, suggesting the presence of a CH₂=C(CH₃)- group. Because the presence of NOESY correlations (Fig. [1]) could be observed between H-5 [δ = 6.87 (1H, s)] and H-4 [δ = 7.59 (1H, d, J = 9.4 Hz)] and OCH ₂CH(OH)C(Me)=CH₂ [δ = 4.29, 4.37 (each 1H, dd)], the 2-hydroxy-3-methyl-3-butenyloxy group was reasonably assigned to C-6. On the basis of the above results and the COSY and NOESY (Fig. [1]) experiments, the structure of 1 was elucidated as 7-hydroxy-6-(2-hydroxy-3-methylbut-3-enyloxy)-2H-chromen-2-one, named hibiscusin.

Hibiscusamide (2) was isolated as colorless needles. The EI-MS afforded the positive ion [M]⁺ at m/z = 359, implying a molecular formula of C₂₀H₂₃O₆, which was confirmed by HR-EI-MS. The UV absorptions at 220, 288, and 316 nm were similar to those of N-trans-feruloyltyramine [4]. The presence of OH and NH groups in the molecule was revealed by a band at 3323 cm^-1 in the IR spectrum. The IR spectrum also showed an amide carbonyl group at 1654 cm^-1. The ¹H-NMR spectrum of 2 was very similar to that of N-trans-feruloyltyramine [4] except that N-(4-hydroxyphenethyl) group of N-trans-feruloyltyramine was replaced by N-(4-hydroxy-3,5-dimethoxyphenethyl) group in 2. The presence of a N-(4-hydroxy-3,5-dimethoxyphenethyl) group was clearly demonstrated from two symmetrical methoxy groups at δ = 3.85 (6H, s, OMe-5 and OMe-7), two symmetrical aromatic protons at δ = 6.45 (2H, s, H-4 and H-8), and four methylene protons at δ = 2.76, 3.47 (each 2H, each t, J = 7.6 Hz, H₂ - 2 and H₂ - 1). On the basis of the above results and the COSY and NOESY (Fig. [2]) experiments, the structure of 2 was elucidated as (E)-N-(4-hydroxy-3,5-dimethoxyphenethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide, named hibiscusamide.

The known isolates, including four benzenoids, vanillic acid (3) [5], p-hydroxybenzoic acid (4) [6], syringic acid (5) [7], p-hydroxybenzaldehyde (6) [6], a coumarin, scopoletin (7) [3], two amides, N-trans-feruloyltyramine (8) [4], N-cis-feruloyltyramine (9) [4], and four steroids, a mixture of β-sitosterol (10) [8] and stigmasterol (11) [8], and a mixture of β-sitostenone (12) [9] and stigmasta-4,22-dien-3-one (13) [9], were easily identified by comparison of physical and spectroscopic data (UV, IR, ¹H-NMR, [α]_D, and mass spectrometry data) with those of corresponding authentic samples or literature values.

The cytotoxic effects of the isolates from the stem wood of Formosan H. tiliaceus were tested in vitro against P-388 and HT-29 cell lines. The cytotoxicity data are shown in Table [1]. The clinically applied anticancer agent, mithramycin, was used as the reference compound. A value of IC₅₀ ≤ 4.0 μg/mL is considered to be indicative of significant cytotoxicity [10]. Hibiscusamide (2), N-trans-feruloyltyramine (8), and N-cis-feruloyltyramine (9) exhibited cytotoxicity with IC₅₀ values < 4 μg/mL against P-388 and/or HT-29 cell lines. The amide compounds (2, 8, and 9) were more effective against the P-388 cell line than the HT-29 cell line. Compounds 2 was the most cytotoxic, with IC₅₀ values = 1.7 ± 0.3 and 3.8 ± 0.8 μg/mL, respectively, against the P-388 and HT-29 cell lines.

Materials and Methods

Plant material: The stem wood of H. tiliaceus was collected from Kaohsiung City, Taiwan, in August, 2004, and identified by Dr. J. J. Chen. A voucher specimen (Chen 5003) was deposited in the herbarium of the Department of Pharmacy, Tajen University, Pingtung, Taiwan.

Extraction and isolation: The dried stem wood (12.7 kg) was extracted with cold MeOH, and the extract concentrated under reduced pressure. The MeOH extract (220 g), when partitioned between H₂O-EtOAc (1 : 1), afforded an EtOAc-soluble fraction (fr. A, 57.7 g). Fr. A (57.7 g) was chromatographed on silica gel (70 - 230 mesh, 2.3 kg), eluting with n-hexane and gradually increasing the polarity with EtOAc and MeOH to give 15 frs: fr. A1 (5 L, n-hexane), fr. A2 (3 L, n-hexane-EtOAc, 100 : 1), A3 (3 L, n-hexane-EtOAc, 90 : 1), A4 (3 L, n-hexane-EtOAc, 80 : 1), A5 (4 L, n-hexane-EtOAc, 50 : 1), A6 (4 L, n-hexane-EtOAc, 10 : 1), A7 (4 L, n-hexane-EtOAc, 5 : 1), A8 (4 L, n-hexane-EtOAc, 3 : 1), A9 (4 L, n-hexane-EtOAc, 2 : 1), A10 - A12 (4 L, n-hexane-EtOAc, 1 : 1), A13 (3 L, n-hexane-EtOAc, 1 : 2), A14 (3 L, EtOAc), A15 (3 L, MeOH). Fr. A8 (2.95 g) was chromatographed further on silica gel (230 - 400 mesh, 110 g) eluting with n-hexane-EtOAc (6 : 1) to give 14 frs (each 1.2 L, fr. A8 - 1 - fr. A8 - 14). Fr. A8 - 7 (192 mg) was purified further by preparative TLC (CH₂Cl₂-EtOAc, 35 : 1) to yield a mixture of 12 and 13 (18.5 mg) (R_f = 0.59). Fr. A9 (3.1 g) was chromatographed further on silica gel (230 - 400 mesh, 120 g) eluting with n-hexane-EtOAc (5 : 1) to give 12 frs (each 1.4 L, fr. A9 - 1 - fr. A9 - 12). Fr. A9 - 5 (192 mg) was purified further by preparative TLC (n-hexane-acetone, 3 : 1) to yield a mixture of 10 and 11 (13.8 mg) (R_f = 0.52). Fr. A10 (2.7 g) was chromatographed further on silica gel (230 - 400 mesh, 105 g) eluting with CH₂Cl₂-MeOH (30 : 1) to give 13 frs (each 2.0 L, fr. A10 - 1 - fr. A10 - 13). Fr. A10 - 2 (178 mg) was purified further by preparative TLC (n-hexane-EtOAc, 1 : 1) to obtain 7 (3.5 mg) (R_f = 0.31). Fr. A10 - 3 (178 mg) was purified further by preparative TLC (n-hexane-acetone, 2 : 1) to obtain 6 (3.2 mg) (R_f = 0.67). Fr. A10 - 10 (152 mg) was purified further by preparative TLC (n-hexane-EtOAc, 1 : 1) to obtain 1 (1.2 mg) (R_f = 0.14). Fr. A11 (2.8 g) was chromatographed further on silica gel (230 - 400 mesh, 125 g) eluting with CH₂Cl₂-MeOH (10 : 1) to give 11 frs (each 1.5 L, fr. A11 - 1 - fr. A11 - 11). Fr. A11 - 7 (206 mg) was purified further by preparative TLC (CH₂Cl₂-EtOAc, 1 : 1) to obtain 4 (2.8 mg) (R_f = 0.41). Fr. A12 (2.55 g) was chromatographed further on silica gel (230 - 400 mesh, 98 g) eluting with CH₂Cl₂-MeOH (20 : 1) to give 10 frs (each 1.8 L, fr. A12 - 1 - fr. A12 - 10). Fr. A12 - 4 (146 mg) was purified further by preparative TLC (CHCl₃-MeOH, 8 : 1) to obtain 2 (1.1 mg) (R_f = 0.28). Fr. A12 - 5 (206 mg) was purified further by preparative TLC (CHCl₃-MeOH, 10 : 1) to obtain 8 (3.5 mg) (R_f = 0.18). Fr. A12 - 6 (187 mg) was purified further by preparative TLC (CHCl₃-MeOH, 10 : 1) to obtain 9 (2.5 mg) (R_f = 0.19). Fr. A13 (3.4 g) was chromatographed further on silica gel (230 - 400 mesh, 135 g) eluting with CH₂Cl₂-MeOH (10 : 1) to give 12 frs (each 1.8 L, fr. A13 - 1 - fr. A13 - 12). Fr. A13 - 7 (206 mg) was purified further by preparative TLC (n-hexane-acetone, 1 : 1) to obtain 3 (2.8 mg) (R_f = 0.60) and 5 (3.7 mg) (R_f = 0.47).

Hibiscusin [7-hydroxy-6-(2-hydroxy-3-methylbut-3-enyloxy)-2H-chromen-2-one] (1): Yellowish oil; UV (MeOH): λ_max (log ε) = 206 (4.46), 227 (4.27), 251 (3.83), 296 (3.82), 344 nm (4.17); IR: ν_max = 3289 (OH), 1707 (C = O), 1607, 1562, 1509, 1453 cm^-1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 262 (M⁺, 21), 244 (25), 177 (100), 164 (19), 149 (54), 121 (35), 69 (46), 51 (22); HR-EI-MS: m/z = 262.0845, calcd. for C₁₄H₁₄O₅ [M]⁺: 262.0841; ¹H-NMR (CDCl₃, 400 MHz): δ = 1.86 (3H, s, Me-3′), 2.35 (1H, br s, OH-2′, exchangeable with D₂O), 4.29 (1H, dd, J = 10.0, 7.2 Hz, H-1′), 4.37 (1H, dd, J = 10.0, 2.8 Hz, H-1′), 4.75 (1H, m, H-2′), 5.08 (1H, s, H-4′), 5.19 (1H, s, H-4′), 6.16 (1H, br s, OH-7, exchangeable with D₂O), 6.27 (1H, d, J = 9.4 Hz, H-3), 6.85 (1H, s, H-8), 6.87 (1H, s, H-5), 7.59 (1H, d, J = 9.4 Hz, H-4).

Hibiscusamide [(E)-N-(4-hydroxy-3,5-dimethoxyphenethyl)-3-(4-hydroxy-3-methoxyphenyl)acrylamide] (2): Colorless needles from MeOH, m. p. 126 - 128 °C; UV (MeOH): λ_max (log ε) = 220 (3.87), 288 (3.76), 316 nm (3.79); IR: ν_max = 3323 (br, OH and NH), 1654 (C = O), 1595, 1513, 1453 cm^-1 (aromatic ring C = C stretch); EI-MS: m/z (rel. int.) = 359 (M⁺, 22), 193 (61), 192 (100), 181 (13), 180 (55), 177 (78), 176 (39), 165 (23); HR-EI-MS: m/z = 359.1492, calcd. for C₂₀H₂₃O₆ [M]⁺: 359.1495; ¹H-NMR (CDCl₃, 400 MHz): δ = 2.76 (2H, t, J = 7.6 Hz, H-2), 3.47 (2H, t, J = 7.6 Hz, H-1), 3.85 (6H, s, OMe-5 and OMe-7), 3.88 (3H, s, OMe-6′), 6.41 (1H, d, J = 15.8 Hz, H-2′), 6.45 (2H, s, H-4 and H-8), 6.79 (1H, d, J = 8.0 Hz, H-8′), 7.01 (1H, dd, J = 8.0, 2.0 Hz, H-9′), 7.11 (1H, d, J = 2.0 Hz, H-5′), 7.44 (1H, d, J = 15.8 Hz, H-3′).

Cytotoxic assays were carried out in accordance with methods discussed in the literatures [11], [12].

Acknowledgements

This work was supported by grants from the Council of Agriculture and the National Science Council of the Republic of China.

References

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Dr. J. J. Chen

Graduate Institute of Pharmaceutical Technology

Tajen University

Pingtung 907

Taiwan

Republic of China

Phone: +886-8-762-4002 ext. 332

Fax: +886-8-762-5308

Email: jjchen@mail.tajen.edu.tw

References

• 1 Chang C E. Malvaceae in the Flora of Taiwan. Vol. 3, 2nd edition Taipei; Editorial Committee of the Flora of Taiwan 1993: p 737-53
  Search in Google Scholar
• 2 Kan W S. Malvaceae in pharmaceutical botany. 9th edition Taipei; National Research Institute of Chinese Medicine 1997: p 333-9
  Search in Google Scholar
• 3 Wu T S, Shi L S, Wang J J, Iou S C, Chang H C, Chen Y P. et al . Cytotoxic and antiplatelet aggregation principles of Ruta graveolens .  J Chin Chem Soc. 2003;  50 171-8
  PubMedSearch in Google Scholar
• 4 Muñoz O, Piovano M, Garbarino J, Hellwing V, Breitmaier E. Tropane alkaloids from Schizanthus litoralis .  Phytochemistry. 1996;  43 709-13
  CrossrefPubMedSearch in Google Scholar
• 5 Chen J J, Duh C Y, Chen I S. Cytotoxic chromenes from Myriactis humilis .  Planta Med. 2005;  71 370-2
  Thieme ConnectPubMedSearch in Google Scholar
• 6 Chen C Y, Chang F R, Teng C M, Wu Y C. Cheritamine, a new N-fatty acyl tryptamine and other constituents from the stems of Annona cherimola .  J Chin Chem Soc. 1999;  46 77-86
  PubMedSearch in Google Scholar
• 7 Chen J J, Lin R W, Duh C Y, Huang H Y, Chen I S. Flavones and cytotoxic constituents from the stem bark of Muntingia calabura .  J Chin Chem Soc. 2004;  51 665-70
  PubMedSearch in Google Scholar
• 8 Chen J J, Duh C Y, Chen J F. New cytotoxic biflavonoids from Selaginella delicatula .  Planta Med. 2005;  71 659-65
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Chen J J, Duh C Y, Huang H Y, Chen I S. Cytotoxic constituents of Piper sintenense .  Helv Chim Acta. 2003;  86 2058-64
  CrossrefPubMedSearch in Google Scholar
• 10 Geran R I, Greenberg N H, Macdonald M M, Schumacher A M, Abbott B J. Protocols for screening chemical agents and natural products against animal tumors and other biological systems.  Cancer Chemother Rep 3. 1972;  3 1-103
  PubMedSearch in Google Scholar
• 11 Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays.  J Immunol Methods. 1983;  65 55-63
  CrossrefPubMedSearch in Google Scholar
• 12 Chen J J, Chou E T, Duh C Y, Yang S Z, Chen I S. New cytotoxic tetrahydrofuran- and dihydrofuran-type lignans from the stem of Beilschmiedia tsangii .  Planta Med. 2006;  72 351-7
  Thieme ConnectPubMedSearch in Google Scholar

Dr. J. J. Chen

Graduate Institute of Pharmaceutical Technology

Tajen University

Pingtung 907

Taiwan

Republic of China

Phone: +886-8-762-4002 ext. 332

Fax: +886-8-762-5308

Email: jjchen@mail.tajen.edu.tw

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