Novel Cadinane and Norcadinane Sesquiterpenes and a New Propanoate from Goldfussia psilostachys

• Abstract
• Materials and Methods
• References

Abstract

Three new compounds, goldfussins A (1) and B (2) and goldfussinol (3), and sixteen known compounds were isolated from the ethanolic extract of the whole plants of Goldfussia psilostachys. The structures of the new compounds were elucidated as a novel cadinane sesquiterpene, 1,8-dimethyl-5-hydroxymethyl-9-oxo-6,7,8,9-tetrahydronaphtho[2,1-b]furan (1), a novel norcadinane sesquiterpene, 4-acetyl-3-hydroxy-1-hydroxymethyl-6-methyl-5-oxo-5,6,7,8-tetrahydronaphthalene (2) and methyl 3-hydroxy-2-(3,5-dimethoxy-4-hydroxyphenyl)-propanoate (3) on the basis of spectral data.

The genus Goldfussia Nees (Acanthaceae), containing more than 30 species, is distributed in India, Malaysia and China [1]. About 9 species are distributed in the East and from South to Southwestern China [1]. Goldfussia psilostachys (C. B. Clarke ex W. W. Smith) Brem., ”Liuyueqing” in the Traditional Chinese Medicine [2], [3], is found in the Yunnan and Guangxi Provinces of China at an altitude of 750 - 1600 meters [4], [5], [6]. The whole plants were used for excreting toxins, eliminating blood block, and treating snake-bite [2], [3].

The ethanolic extract of G. psilostachys showed antiproliferative activity on K562 leukemia cells [7]. To the best of our best knowledge, no chemical study on this plant has been reported before. In this investigation, the petroleum ether-soluble fraction of the ethanolic extract of the whole plants of G. psilostachys was separated repeatedly by column chromatography with various materials. New compounds 1 - 3 along with 16 known compounds were isolated. Their structures were established on the basis of UV, IR, NMR, MS data and of optical rotations.

The molecular ion peak at m/z = 244.1087 in the HR-EI-MS of compound 1 afforded the molecular formula C₁₅H₁₆O₃, corresponding to eight degrees of unsaturation. The ¹H-NMR signals (Table [1]) at δ = 1.27 [3H, d, J = 6.4 Hz, Me(15)] and 2.39 [3H, s, Me(13)] suggested two methyl groups. The IR absorption peak at 1743 cm^-1 and the ¹³C-NMR (DEPT) signal (Table [1]) at δ = 201.9 [s, C(4)] showed the presence of a carbonyl group. Fifteen signals were observed in the ¹³C-NMR (DEPT) spectrum (Table [1]). Thus, a sesquiterpene was postulated. Moieties 1A and 1B were concluded from the cross signals in the HSQC and HMBC diagrams (Fig. [1] and Table [1]). The remaining quaternary carbon atom resonated at δ = 128.3 [s, C(5)] in the ¹³C-NMR (DEPT) spectrum and should be connected with C(4), C(6), and C(10) considering the degree of unsaturation. Thus, the structure of compound 1 was elucidated as 1,8-dimethyl-5-hydroxymethyl-9-oxo-5,6,7,8-tetrahydronaphtho[2,1-b]furan (goldfussin A, Fig. [1]).

Compound 2 was isolated as a yellowish powder. The molecular formula C₁₄H₁₆O₄ was calculated from the quasi-molecular ion peak at m/z = 271.0946 [M + Na]⁺ in its HR-ESI-MS (positive-ion mode). The ¹H-NMR signals (Table [1]) at δ = 1.17 [3H, d, J = 6.4 Hz, Me(15)] and 2.44 [3H, s, Me(13)] suggested two methyl groups. The IR absorption peaks at 1734 and 1697 cm^-1, and the ¹³C-NMR (DEPT) signals (Table [1]) at δ = 202.9 [s, C(4)] and 208.9 [s, C(11)] indicated the presence of two carbonyl groups. Fourteen ¹³C-NMR signals were observed. Compound 2 was probably a norsesquiterpene, which was elucidated as a norcadinane sesquiterpenoid, 4-acetyl-3-hydroxy-1-hydroxymethyl-6-methyl-5-oxo-5,6,7,8-tetrahydronaphthalene (goldfussin B, Fig. [1]) based on the HSQC and HMBC experiments (Fig. [1] and Table [1]).

Compound 3 was isolated as a white powder. Its HR-EI-MS gave the molecular ion peak at m/z = 256.0936 for C₁₂H₁₆O₆. The ¹H-NMR signals at δ = 3.84 (6H, s) and 3.72 (3H, s) suggested two kinds of methoxy groups. The fragment -CH₂-CH- was concluded from the ¹H-NMR signals at δ = 4.11 [1H, dd, J = 7.2, 7.1 Hz, H-C(2)], 4.17 [1H, dd, J = 10.8, 7.1 Hz, Ha-C(3)] and 4.23 [1H, d, J = 10.8, 7.2 Hz, Hb-C(3)]. The IR absorption band at 1725 cm^-1 and the ¹³C-NMR (DEPT) signal at δ = 175.4 (s) suggested the presence of a carbonyl group. One 1,3,4,5- or 1,2,4,6-tetrasubstituted phenyl was deduced from the ¹³C-NMR (DEPT) signals at δ = 106.4 (d), 125.7 (s), 150.0 (s) and 138.9 (s) and ¹H-NMR signal at δ = 6.58 [s, 2H, H-C (2′) and H-C(6′)]. No absorption peak for a carboxyl group was observed in the IR spectrum. From the evidence mentioned above and the NOESY correlations between H-C(2′) and H-C(3′-OMe), and between H-C(2) and H-C(2′), compound 3 was determined to be methyl 3-hydroxy-2-(3,5-dimethoxy-4-hydroxyphenyl)-propanoate (goldfussinol, Fig. [1]) which is structurally related with 2-(4-hydroxy-3,5-dimethoxyphenyl)- propane-1,3-diol, a product of degradation of wood lignin [8]. Thus, goldfussinol may be a product of degradation of lignin.

No reference compounds were found in order to elucidate the absolute configurations of 1 and 2 with circular dichroism. In an attempt to obtain suitable crystals for X-ray analysis, the preparation of their oxamazone derivatives from the optically active reagent 5-(α-phenethyl)semioxamazide was unsuccessful. Also, the derivative of 3 with chiral amino acids did not yield crystals suitable for X-ray analysis. The absolute configurations of compounds 1, 2, and 3 could thus only be determined by stereoselective synthesis, which was beyond the scope of this study.

Additionally, 2-ethenyl-2,6,6-trimethyl-3,6-dihydro-2H-pyran (4) was isolated as a natural product for the first time. Lupeol (5), betulin (6), betulinic aldehyde (7), betulinic acid (8), phytol (9), (22E,20S,24R)-5α,8α-epidioxyergosta-6,22-dien-3-β-ol (10), 5,7-dihydroxy-6,4′-dimethoxyflavone (11), pomolic acid (12), (E)-ferulaldehyde (13), hyptinin (14), 3,5-dimethoxy-4-hydroxybenzaldehyde (15), β-sitosterol (16), and 2,6-dimethoxy-p-benzoquinone (17) were isolated and identified based on their spectral data and optical rotations. p-Methoxybenzoic acid (18) and vanillin (19) were identified by co-TLC and co-melting point with authentic samples. For the references, see the Supporting Information.

All the three new compounds were subjected to biological testing including cytotoxicity [9], [10] and antiviral activity against herpes simplex virus types I and II (HSV-1 and HSV-2) [11], [12]. None of them was active. However, lupeol (5), isolated in large amounts from this plant, may be the active component considering its cytotoxicity [13], [14], induction of apoptosis [15], and cell differentiation [16].

Materials and Methods

General experimental procedures: Melting points were determined on an XRC-1 micro-melting point apparatus and are uncorrected. Optical rotations were measured on a Perkin-Elmer 341 automatic polarimeter. IR spectra were recorded on a Nicolet Protege 460 spectrometer using KBr disc and ν_max are given in cm^-1. UV spectra were measured on a GBC Cintra 20 spectrometer. NMR spectra were recorded on a Varian^unity Ionva-400 spectrometer (¹H-NMR: 400 MHz, ¹³C-NMR: 100 MHz) at room temperature. The chemical shifts (δ) are reported in ppm and the coupling constants (J) are given in Hz. HR-ESI-MS were obtained on an API QSTAR Pulsar i mass spectrometer. APCI-MS were recorded on an HP110-LC/MSD mass spectrometer. HR-EI-MS and EI-MS were obtained on a VG 7070E (70 eV) mass spectrometer. Column chromatography (CC) was performed on a self-packed open column with silica gel from Qingdao Ocean Chemical Engineering Company (QOCEC), or with high porous polymer MCI gel (CHP20P, 75 - 150 μ) from Mitsubishi Chemical Corporation, or reverse phase C₁₈ linked silica gel (40 - 63 μ) from Phenomenex Corporation. TLC analyses were carried out on plates precoated with 10 - 40 μ of silica gel G from QOCEC. Visualization on TLC was achieved by spraying with 8 % phosphomolybdic acid-ethanol solution (w/v) followed by heating. Fractions from all columns were generally collected by hand and by an auto-collecting apparatus according to TLC analyses.

Plant material: The whole plants of G. psilostachys were purchased from Xishuangbanna, Yunnan Province of China, in September 1999, and identified by Professor Jinyun, Cui from the Xishuangbanna Tropical Botanical Garden, the Chinese Academy of Sciences (CAS). A voucher specimen (Z-17) is deposited in the Herbarium of the Chengdu Institute of Biology, CAS.

Extraction and isolation: The dried and powdered whole plants of G. psilostachys were soaked with 95 % ethanol at room temperature. After removing ethanol under vacuum, the syrupy residue was suspended in water and successively extracted with petroleum ether (60 - 90 °C), ethyl acetate and n-butanol to afford the corresponding fractions P, E and B. The fraction P was divided into eighteen fractions P1 - P18 by flash chromatography. P13 (30 g, 0 - 2000 mL, solvent B) was subjected to CC over MCI gel (130 mL) eluted at a rate of 2 mL/min to give P13A [MeOH and H₂O (6 : 4) as eluent, 0 - 2000 mL], P13B [MeOH and H₂O (7 : 3) as eluent, 0 - 2000 mL] and P13C [MeOH and H₂O (8 : 2) as eluent, 8500 - 23 500 mL]. Compound 1 (19 mg) was crystallized from P13B. P18C3 (450 mg) was separated on CC with 50 g of C₁₈ silica gel eluted with methanol and water (6 : 4, 4 mL/10 min) to give P18C3A (80 mg, 200 - 290 mL) which was subjected to CC over silica gel (200 - 300 mesh, 6 g) eluted with chloroform and methanol (30 : 1, 0.5 mL/min) to furnish compound 2 (10 mg, 110 - 160 mL). Compound 3 (15 mg, 250 - 320 mL) was isolated from P18H1 (580 mg) by CC over silica gel (200 - 300 mesh, 50 g) and eluted with petroleum ether and acetone (3 : 1, 1 mL/3 min). For detailed extraction and isolation procedures, see Supporting Information.

Goldfussin A (1): White needle crystals (19 mg); m. p. 170 - 171 °C (MeOH/H₂O, 7 : 3); [α]_D ²⁵: + 19.2° (c 0.12, CHCl₃); UV (MeOH): λ_max (log ε) = 234 (4.39), 292 (3.93), 324 (3.80) nm; IR: ν_max = 3417, 2928, 1743, 1624, 1509, 1480, 1435, 1347, 1265, 1231, 1171, 1135, 1080, 1040, 772, 684 cm^-1; ¹H- and ¹³C-NMR, see Table [1]; APCI-MS (positive-ion mode): m/z = 267 [M + Na]⁺; HR-EI-MS: m/z = 244.1087 ([M]⁺, calcd. for C₁₅H₁₆O₃ : 244.1099).

Goldfussin B (2): Yellowish powder (10 mg); m. p. 204 - 206 °C; [α]_D ²⁵: -6.0° (c 0.1, MeOH); UV (MeOH): λ_max (log ε) = 215 (4.20), 257 (3.68), 328 (3.49) nm; IR: ν_max = 3422, 2924, 1734, 1697, 1677, 1637, 1598, 1415, 1377, 1294, 1243, 1169, 1125, 1064 cm^-1; ¹H- and ¹³C-NMR, see Table [1]; APCI-MS (positive-ion mode): m/z = 519 [2 M + Na]⁺, 271 [M + Na]⁺; HR-ESI-MS (positive-ion mode): m/z = 271.0946 ([M + Na]⁺, calcd. for C₁₄H₁₆O₄Na: 271.0946).

Goldfussinol (3): White powder (15 mg); [α]_D ²⁵: + 5.0° (c 0.1, MeOH); UV (MeOH): λ_max (log ε) = 272 (3.69) nm; IR: ν_max = 3415, 2937, 1725, 1614, 1519, 1461, 1427, 1330, 1221, 1116, 1039, 671 cm^-1; ¹H-NMR (CD₃OD): δ = 6.58 [2H, s, H-C(2′) and H-C(6′)], 4.23 [1H, dd, J = 10.8, 7.2 Hz, Ha-C(3)], 4.17 [1H, dd, J = 10.8, 7.1 Hz, Hb-C(3)], 4.11 [1H, dd, J = 7.2, 7.1 Hz, H-C(2)], 3.84 [6H, s, H-C(3′-OMe) and H-C(5′-OMe)], 3.72 [3H, s, H-(1-OMe)]; ¹³C-NMR (CD₃OD): δ = 175.4 [s, C(1)], 55.6 [d, C(2)], 65.3 [t, C(3)], 125.7 [s, C(1′)], 106.4 [d, C(2′) and C(6′)], 150.0 [s, C(3′) and C(5′)], 138.9 [s, C(4′)], 52.4 [q, C(1-OMe)], 56.7 [q, C(3′-OMe) and C(5′-OMe)]; NOESY: H-C(2)/H-C(1-OMe), H-C(2′) and H-C(6′)/H-C(3′-OMe) and H-C(5′-OMe); HR-EI-MS: m/z = 256.0936 ([M]⁺, calcd. for C₁₂H₁₆O₆ : 256.0947).

References

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  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Guolin Zhang

Chengdu Institute of Biology

Chinese Academy of Sciences

P. O. Box 416

Chengdu 610041

People’s Republic of China

Phone: +86-28-8522-5401

Fax: +86-28-8522-5401

Email: zhanggl@cib.ac.cn

References

• 1 How F C, Wu T L, Ko W C, Chen T C. A Dictionary of the Families and Genera of Chinese Seed Plants. 2nd edn South China Institute of Botany, Academic Sinica, Beijing; Science Press 1998: p 211
  Search in Google Scholar
• 2 Editors Committee for Dictionary of Chinese Traditional M edicine. Dictionary of Chinese Traditional Medicine. Beijing; Chinese Publishing House for Medical Science and Technology 1999: pp 1267-8
  Search in Google Scholar
• 3 Working Group of Collects of Chinese Medicines and H erbs. Collects of Chinese Medicines and Herbs, Section B. Beijing; The People’s Health Publishing House 1988: p 836
  Search in Google Scholar
• 4 Delectis Florae Reipublicae Popularis Sinicae Agendae Academiae Sinicae E dita. Flora Reipublicae Popularis Sinicae, Tomus 70. Beijing; Sciences Press 2002: pp 168-9
  Search in Google Scholar
• 5 Wu C Y, Yin W Q, Bao S Y, Tao D Y, Yuan S H, Deng X F. et al .Index Florae Yunnanensis, Tomus II. Institutum Botanicum Kunmingense Academiae Sinicae Edita. Yunnan; The People’s Publishing House 1984: pp 1675
  Search in Google Scholar
• 6 Yunnan Institute of Tropical B otany, Academia S inica. List of Plants in Xishuangbanna. Kunming; Yunnan Nationality Publishing House 1984: p 368
  Search in Google Scholar
• 7 Gao X P, Zhang G L, Zhou M, Luo D Y, Li B G. Antiproliferative activity of Goldfussia psilostachys ethanolic extract on K562 leukemia cells.  Fitoterapia. 2004;  75 639-44
  CrossrefPubMedSearch in Google Scholar
• 8 Namba H, Nakatsubo F, Higuchi T. Degradation of β-1 linked dilignols by Fusarium solani M-13 - 1.  Wood Res. 1983;  69 52-60
  PubMedSearch in Google Scholar
• 9 Skehan P, Storeng R, Scudiero D, Monk A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 10 Rubinstein L V, Shoemaker R H, Paull K D, Simon R M, Tosini S, Skehan P. et al . Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines.  J Natl Cancer Inst. 1990;  82 1113-8
  CrossrefPubMedSearch in Google Scholar
• 11 Chen Z J, Liu W G, Song Z J. Experimental studies of 9-(1,3-dihydroxy-2-propoxymethyl)-guanine (DHPG) against herpes simplex virus.  Yaoxue Xuebao (Acta Pharmac Sin). 1989;  24 331-4
  PubMedSearch in Google Scholar
• 12 Wang H Y, Zhang M H, Wang Y, Yang W S. Protection effect of the convalescent serum from HFRS patient on the HTNV infected neurons in vitro .  Zhonghua Weishengxue he Mianyixue Zazhi (Chin Microbiol Immunol). 1999;  10 105-8
  PubMedSearch in Google Scholar
• 13 Cordero C P, Gomez-Gonzalez S, Leon-Acosta C J, Morantes-Medina S J, Aristizabal F A. Cytotoxic activity of five compounds isolated from Colombian plants.  Fitoterapia. 2004;  75 225-7
  CrossrefPubMedSearch in Google Scholar
• 14 Saleem M, Afaq F, Adhami V M, Mukhtar H. Lupeol modulates NF-κB and PI3K/Art pathways and inhibits skin cancer in CD-1 mice.  Oncogene. 2004;  23 5203-14
  CrossrefPubMedSearch in Google Scholar
• 15 Aratanechemuge Y, Hibasami H, Sanpin K, Katsuzaki H, Imai K, Komiya T. Induction of apoptosis by lupeol isolated from mokumen (Gossampinus malabarica L. Merr) in human promyelotic leukemia HL-60 cells.  Oncol Rep. 2004;  11 289-92
  PubMedSearch in Google Scholar
• 16 Hata K, Hori K, Takahashi S. Role of p38 MAPK in lupeol-induced B16 2F2 mouse melanoma cell differentiation.  J Biochem. 2003;  134 441-5
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. Guolin Zhang

Chengdu Institute of Biology

Chinese Academy of Sciences

P. O. Box 416

Chengdu 610041

People’s Republic of China

Phone: +86-28-8522-5401

Fax: +86-28-8522-5401

Email: zhanggl@cib.ac.cn

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