Phragmalin Limonoids from Chukrasia tabularis var. velutina

• Abstract
• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Two new C-15-acyl phragmalin limonoids, velutinalides A (2) and B (3), featuring a C-16/C-30 δ-lactone ring, and a new structurally related natural product, R310B8 (1), were isolated from the leaves of Chukrasia tabularis var. velutina. Their structures were elucidated on the basis of extensive spectroscopic data analyses and by comparison of their NMR data with those of related known compounds.

Phragmalins are a type of rings B,D-seco limonoids with characteristic tricycle [3.3.1^2,10.1^1,4]decane or tricycle [4.2.1^10,30.1^1,4]decane (rearranged phragmalin) A and B ring systems, most of which bear orthoester groups at positions 1,8,9 or 8,9,14 or 8,9,30 or 8,9,11, and a C-16/C-17 δ-lactone ring (ring D) [1], [2], [3], [4]. In a few cases, the ring D was cleaved, producing C-16/C-17 δ-lactone-seco [5], C-16/C-8 γ-lactone [6], and C-16/C-30 δ-lactone derivatives [7]. Many phragmalins have shown a potential biological significance especially in relation to their insect antifeeding activity [1], [8], [9]. Phragmalins have so far only been found in two tribes (Swietenieae and Xylocarpeae) of the family Meliaceae, such as in the genera Chukrasia [1], Swietenia [5], and Neobeguea [10] in the Swietenieae tribe and Xylocarpus [3] in the Xylocarpeae tribe.

Chukrasia tabularis var. velutina (Meliaceae), a timber tree, is widely distributed in tropical areas of Asia such as India and southern mainland China [11]. Its stem bark has been used traditionally as an astringent, antidiarrheal, and anti-influenza agent in China [12]. Previous chemical investigation of the stem barks, twigs, and leaves of the title plant collected from Xishuangbanna, Yunnan Province, China, has led to the isolation of a series of phragmalin limonoids with interesting carbon skeletons, such as 16-norphragmalins [13], phragmalins incorporating a cyclopropany ring (C-13, C-14, and C-18) [14] and C-15-acyl phragmalins with a C-16/C-30 δ-lactone ring [7]. In our continuing search for structurally and biologically interesting metabolites from Chinese flora resources [15], [16], [17], [18], the leaves of C. tabularis var. velutina were collected from Zhanjiang, Guangdong Province, China. A chemical investigation of the EtOAc-soluble fraction of the methanolic extract led to the isolation of two new C-15-acyl phragmalin limonoids, named velutinalides A (2) and B (3), and a new structurally related natural product, R310B8 (1) ([Fig. 1]). Although the structure of 1 was already reported previously as a deacetylation derivative of R310B (4) in a published US patent WO/145996/2008 [10], there was no NMR data of 1 either disclosed in the above-mentioned literature or published in other academic journals. In the present paper, we report the isolation and structural determination of these three compounds.

The chipped leaves of C. tabularis var. velutina were extracted exhaustively with MeOH. The MeOH extract was partitioned consecutively between EtOAc and H₂O, n-BuOH and H₂O, respectively. The EtOAc-soluble portion was subjected to repeated column chromatography (silica gel, Sephadex LH-20, and reversed phase HPLC) to afford compounds 1–3.

R310B8 (1), obtained as an optically active white powder, [α]_D ²⁰ − 122 (c 0.05, CHCl₃), gave the molecular formula C₃₅H₄₄O₁₃ as determined by HR-ESI-MS at m/z = 695.2665 [M + Na]⁺ (calcd.: 695.2680), which was supported by a pseudomolecular ion peak at m/z = 695.3 [M + Na]⁺ in the positive ESI-MS. The IR (KBr) absorption bands implied the presence of hydroxyl (3466 cm⁻¹), ester (1748 cm⁻¹), and β-dicarbonyl (1636 and 1610 cm⁻¹) groups. Its UV spectrum λ _max (MeOH) = 206 (3.17) and 269 (3.36) nm was characteristic of a typical furanyl and a β-dicarbonyl chromophore. The ¹³C-NMR spectrum displayed 35 carbon resonances including eight methyls (one methoxyl), four methylenes, nine methines (three olefinic and three oxygenated), and 14 quaternary carbons (three olefinic, three ester carbonyl, and four oxygenated). Furthermore, a combined analysis of its ¹H- and ¹³C-NMR data ([Tables 1] and [2]) revealed the presence of an orthoacetate group, a β-furyl ring, an acetyl group, and an isopropyl moiety. Two proton signals at δ _H = 13.91, 2.74 (br s, each ca. 1H), which disappeared in D₂O, were assigned to exchangeable OH protons (one enolic). The molecule of 1 had 14 degrees of unsaturation, of which seven were occupied by three ester carbonyls, one double bond, and the β-furyl ring, and the remaining seven required 1 to be heptacyclic at the central core. The aforementioned data suggested that 1 is a phragmalin-type limonoid orthoester.

A comparison of overall ¹H- and ¹³C-NMR data ([Tables 1] and [2]) revealed great similarities between the model compound R310B (4), previously isolated from Neobeguea mahafalensis, and compound 1. In fact, the only significant difference is the ¹H-NMR chemical shift of H-3 (δ _H 3.68 for 1 and δ _H 4.84 for 4). This difference could be easily rationalized due to the deacetylation at C-3 in agreement with the molecular weight of 1 that was 42 mass units less than that of 4. Thus, the structure of 1 was unambiguously elucidated to be 3-deacetyl derivative of 4. The complete NMR data assignments were made by detailed analysis of 1D- and 2D-NMR (HMBC, HSQC, ROESY) spectra of 1 ([Tables 1] and [2]).

Velutinalide A (2), a white amorphous powder, gave the molecular formula C₃₇H₄₈O₁₃ as determined by HR-ESI-MS (m/z = 723.2974 [M + Na]⁺, calcd.: 723.2993), 28 mass units more than that of 1. The data from ¹H- and ¹³C-NMR and the subsequent 2D NMR studies (HMBC and HSQC) suggested that 2 was also a phragmalin limonoid with the same basic skeleton as 1. In fact, the only difference between 2 and 1 was that the acetoxyl group at C-17 of 1 was replaced by an isobutyryloxyl group [δ _H = 2.46–2.56 (1H, m), 1.08 (3H, t, J = 7.8 Hz), 1.08 (3H, t, J = 7.8 Hz); δ _C = 175.2 (s), 33.6 (d), 19.1 (q), 17.8 (q)] in 2. The HMBC correlation ([Fig. 2]) from H-17 (δ _H = 5.77) to the isobutyryloxyl carbonyl (δ _C = 175.2) further confirmed the assignment. Its relative configuration was determined to be the same as 1 by the ROESY experiment ([Fig. 3]) and biogenetic consideration.

Velutinalide B (3) had the molecular formula C₃₆H₄₆O₁₃ as determined by HR-ESI-MS (m/z 709.2826 [M + Na]⁺, calcd.: 709.2836), 14 mass units less than that of 2. Comparison of its spectral data [UV (log ε): 206 (3.17), 268 (3.31) nm; IR (KBr): λ _max = 3466, 1745, 1635, 1601 cm⁻¹; ¹H- and ¹³C-NMR data ([Tables 1] and [2])] with those of 2 clearly indicated that it differs from 2 only in the substitutent at C-17, where the isobutyryloxyl group at C-17 of 2 was replaced by a propionyloxyl group [δ _H = 2.27–2.30 (2H, m), 1.06 (3H, t, J = 7.2 Hz); δ _C = 172.8 (s), 27.3 (t), 8.8 (q)] in 3. The rest of the structure of 3 is the same as in compound 2.

Although many phragmalin limonoids continue to be isolated from the plants of the family Meliaceae, C-15-acyl limonoids featuring a C-16/C-30 δ-lactone ring are very rare. To the best of our knowledge, until now, only seven such compounds, chukvelutilides A–F [7] and R310B (4) [10], have been discovered from Chukrasia tabularis var. velutina and Neobeguea mahafalensis, respectively. The discovery of compounds 1–3 has widened the knowledge on these fascinating phragmalin limonoids that are rapidly expanding. Moreover, this is the first report to isolate these intriguing compounds from the title plant collected in Zhanjiang, Guangdong Province, China.

In order to explore other pharmacological potentials of these new molecules, a series of bioassays, such as cytoxicity, enzyme inhibition, and antimicrobial assays, were also carried out. Firstly, the cytotoxic activities of compounds 1–3 against the growth of tumor cell lines [A549 (human lung adenocarcinoma) and HL-60 (human lymphocytic leukemia)] were evaluated. Unfortunately, the results indicated that compounds 1–3 were inactive against the above cancer cells at concentrations up to 20 µg/mL (IC₅₀ > 10 µM). Furthermore, 1–3 were also tested for their inhibitory activities towards several enzymes, such as hPTP1B (human protein tyrosine phosphatase 1B) [19], a key target for the treatment of type II diabetes and obesity, CDC25B dual specificity phosphatase [20], a key enzyme for cell cycle progression and observed in a variety of cancers with a striking association with tumor aggressiveness and poor prognosis, and pancreatic lipase [21], a primary lipase that hydrolyzes dietary fat molecules in the human digestive system, converting triglyceride substrates found in ingested oils to monoglycerides and free fatty acids. Once again, all tested compounds did not show inhibitory activities. Very recently, these molecules were also subjected to antimicrobial bioassay against the bacteria Staphylococcus aureus and Pseudomonas aeruginosa. The screening results obtained also showed no activity.

Materials and Methods

Plant material: The leaves of Chukrasia tabularis var. velutina were collected from Zhanjiang, Guangdong Province, China in July 2009 and identified by Associate Professor J.-G. Shen of Shanghai Institute of Material Medica, Chinese Academy of Sciences. A voucher sample (NO. 09-P-49) is available for inspection at the Herbarium of SIMM-CAS.

Extraction and isolation: The chipped leaves of C. tabularis var. velutina (1 kg) were extracted exhaustively with MeOH (5 L × 3) at room temperature. The MeOH extract was concentrated in vacuo to give a residue, which was suspended in H₂O and partitioned successively with EtOAc and n-BuOH. The EtOAc-soluble extract was evaporated in vacuo to give a residue (27 g), which was subjected to Sephadex LH-20 column (3 × 120 cm), eluted with CHCl₃-MeOH (1 : 1) to afford three fractions (Fr. A–C). Fr. B (8 g) was chromatographed on a column of silica gel (4.5 × 40 cm, 200 g) successively eluted with a gradient of petroleum ether-EtOAc (90 : 10, 70 : 30, 50 : 50, each 1.5 L) and followed by a gradient of CHCl₃-MeOH (90 : 10, 80 : 20, 70 : 30, 60 : 40, each 1.5 L) to give five subfractions (Fr. B1–B5). Fr. B3 (1.6 g) was subjected to a column of silica gel (3 × 30 cm, 100 g) eluted with a gradient of CHCl₃-MeOH (99 : 1, 98 : 2, 95 : 5, each 500 mL) to give four subfractions (Fr. B3a–B3d), then Fr. B3d was separated by semipreparative HPLC using CH₃CN-H₂O (80 : 20, 2.0 mL/min) as the mobile phase, yielding pure compounds 1 (> 98 %, 1.5 mg, t _R 14.3 min), 2 (> 98 %, 40.0 mg, t _R 20.9 min), and 3 (> 98 %, 3.4 mg, t _R 17.3 min), respectively.

R310B8 (1): White amorphous powder; [α]_D ²⁰: − 122 (c 0.05, CHCl₃); UV (log ε): 206 (3.17), 269 (3.36) nm; IR (KBr): λ _max = 3466, 2970, 2867, 1748, 1636, 1610, 1400, 1250, 1144, 1035, 882 cm⁻¹; ¹H- and ¹³C-NMR spectral data: see [Tables 1] and [2]; ESI-MS: m/z = 695.3 [M + H]⁺; HR-ESI-MS: m/z = 695.2665 [M + Na]⁺ (calcd. for C₃₅H₄₄O₁₃Na: 695.2680).

Velutinalide A (2): White amorphous powder; [α]_D ¹⁸: − 67.8 (c 0.85, CHCl₃); UV (log ε): 206 (3.17), 270 (3.36) nm; IR (KBr): λ _max = 3466, 2970, 2877, 1740, 1635, 1606, 1402, 1242, 1144, 1028, 876 cm⁻¹; ¹H- and ¹³C-NMR spectral data: see [Tables 1] and [2]; ESI-MS: m/z = 701.4 [M + H]⁺; HR-ESI-MS: m/z = 723.2974 [M + Na]⁺ (calcd. for C₃₇H₄₈O₁₃Na: 723.2993).

Velutinalide B (3): White amorphous powder; [α]_D ¹⁸: − 63.3 (c 0.45, CHCl₃); UV (log ε): 206 (3.17), 268 (3.31) nm; IR (KBr): λ _max = 3466, 2973, 2870, 1745, 1635, 1601, 1379, 1242, 1140, 1025, 865 cm⁻¹; ¹H- and ¹³C-NMR spectral data: see [Tables 1] and [2]; ESI-MS: m/z = 709.3 [M + Na]⁺; HR-ESI-MS: m/z = 709.2826 [M + Na]⁺ (calcd. for C₃₆H₄₆O₁₃Na: 709.2836).

Supporting information

Original spectra for compounds 1–3 and details regarding the experimental protocols are available as Supporting Information.

Acknowledgements

This research work was financially supported by the Natural Science Foundation of China (Nos. 31070310, 40976048, 30730108, and 21021063) and National Marine “863′′ Project (No. 2011AA09070102), and was partially funded by the EU 7th Framework Programme-IRSES Project (2010–2014), STCSM Project (No. 10540702900), NSFC-TRF International Cooperation Project (No. 20911140471), Hungarian-Chinese Intergovernmental S & T Cooperation Programme (2009–2011), SKLDR/SIMM Projects (Nos. SIMM1105KF-04 and SIMM1106KF-11), as well as by a CAS grant (KSCX2-YW-R-18). We are indebted to Miss Feifei Chen and Professor Lefu Lan of the SIMM-CAS for the antimicrobial bioassay.

Conflict of Interest

The authors have no conflict of interest to declare.

References

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  Search in Google Scholar
• 18 Liu H L, Huang X Y, Dong M L, Xin G R, Guo Y W. Piperidine alkaloids from Chinese mangrove Sonneratia hainanensis.  Planta Med. 2010;  76 920-922
  Search in Google Scholar
• 19 Byon J C H, Kusari A B, Kuseti J. Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction.  Mol Cell Biochem. 1998;  182 101-108
  Search in Google Scholar
• 20 Lammer C, Wagerer S, Saffrich R, Mertens D, Ansorge W, Hoffmann I. The cdc25B phorsphatase is essential for the G2/M phase transition in human cell.  J Cell Sci. 1998;  111 2445-2453
  Search in Google Scholar
• 21 Wignot T M, Stewart R P, Schray K J, Das S, Sipos T. In vitro studies of the effects of HAART drugs and excipients on activity of digestive enzymes.  Pharm Res. 2004;  21 420-427
  Search in Google Scholar

Prof. Dr. Yue-Wei Guo

State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica
Chinese Academy of Sciences

Zu Chong Zhi Rd. 555

Zhangjiang, Hi-Tech Park

Shanghai 201203

People's Republic of China

Phone: +86 21 50 80 58 13

Fax: +86 21 50 80 58 13

Email: ywguo@mail.shcnc.ac.cn

References

• 1 Nakatani M, Abdelgaleil S A M, Saad M M G, Huang R C, Doe M, Iwagawa T. Phragmalin limonoids from Chukrasia tabularis.  Phytochemistry. 2004;  65 2833-2841
  Search in Google Scholar
• 2 Baxter R L, Dijksma F J J, Gould R O, Parsons S. β-dihydroentandrophragmin-ethyl acetate (1/0.355).  Acta Crystallogr Sect C Cryst Struct Commun. 1998;  54 1182
  Search in Google Scholar
• 3 Wu J, Xiao Q, Huang J S, Xiao Z H, Qi S H, Li Q X, Zhang S. 8,9,30-phragmalin ortho esters from Xylocarpus granatum.  Org Lett. 2004;  6 1841-1844
  Search in Google Scholar
• 4 Fan C Q, Wang X N, Yin S, Zhang C R, Wang F D, Yue J M. Tabularisins A–D, phragmalin ortho esters with new skeleton isolated from the seeds of Chukrasia tabularis.  Tetrahedron. 2007;  63 6741-6747
  Search in Google Scholar
• 5 Saad M M G, Iwagawa T, Doe M, Nakatani M. Swietenialides, novel ring D opened phragmalin limonoid orthoesters from Swietenia mahogani JACQ.  Tetrahedron. 2003;  59 8027-8033
  Search in Google Scholar
• 6 Chen Y Y, Wang X N, Fan C Q, Yin S, Yue J M. Swiemahogins A and B, two novel limoids from Swietenia mahogani.  Tetrahedron Lett. 2007;  48 7480-7484
  Search in Google Scholar
• 7 Luo J, Wang J S, Huang X F, Luo J G, Kong L Y. Chukvelutilides A–F, phragmalin limonoids from the stem barks of Chukrasia tabularis var. velutina.  Tetrahedron. 2009;  65 3425-3431
  Search in Google Scholar
• 8 Wu J, Xiao Q, Zhang S, Li X, Xiao Z H, Ding H X, Li Q X. Xyloccensins Q–V, six new 8,9,30-phragmalin ortho ester antifeedants from the Chinese mangrove Xylocarpus granatum.  Tetrahedron. 2005;  61 8382-8389
  Search in Google Scholar
• 9 Abdelgaleil S A M, Doe M, Morimoto Y, Nakatani M. Rings B, D-seco limonoids from the leaves of Swietenia mahogany.  Phytochemistry. 2006;  67 452-458
  Search in Google Scholar
• 10 Wikberg J E S, Rasoanaivo P, Benoit R, Razafimahefa A S. Novel compounds and pharmaceutical preparations from Neobeguea mahafalensis extracts and their use for treatment of sexual dysfunction. US Patent 145996A2 2008
  Search in Google Scholar
• 11 Chen P Y. Meliaceae. In: Chen S K, editor Flora reipublicae popularis sinicae (Zhongguo Zhiwu Zhi). Volume 13 Beijing: Science Press; 1997: 47-48
  Search in Google Scholar
• 12 Editorial Committee of the Administration Bureau of Traditional Chinese Medicine .Chinese materia medica (Zhonghua Bencao). Volume 5 Shanghai: Shanghai Science and Technology Press; 1999: 31-32
  Search in Google Scholar
• 13 Luo J, Wang J S, Luo J G, Wang X B, Kong L Y. Chukvelutins A–C, 16-norphragmalin limonoids with unprecedented skeletons from Chukrasia tabularis var. velutina.  Org Lett. 2009;  11 2281-2284
  Search in Google Scholar
• 14 Zhang C R, Yang S P, Zhu Q, Liao S G, Wu Y, Yue J M. Nortriterpenoids from Chukrasia tabularis var. velutina.  J Nat Prod. 2007;  70 1616-1619
  Search in Google Scholar
• 15 Sun Y Q, Guo Y W. Gymonorrhizol, an unusual macrocyclic polydisulfide from the Chinese mangrove Bruguiera gymnorrhiza.  Tetrahedron Lett. 2004;  45 5533-5535
  Search in Google Scholar
• 16 Wang J D, Guo Y W. Agallochaols A and B, two new diterpenes from the Chinese mangrove Excoecaria agallocha L.  Helv Chim Acta. 2004;  87 2829-2833
  Search in Google Scholar
• 17 Wang J D, Zhang W, Li Z Y, Xiang W S, Guo Y W, Krohn K. Elucidation of excogallochaols A–D, four unusual diterpenoids from the Chinese mangrove Excoecaria agallocha.  Phytochemistry. 2007;  68 2426-2431
  Search in Google Scholar
• 18 Liu H L, Huang X Y, Dong M L, Xin G R, Guo Y W. Piperidine alkaloids from Chinese mangrove Sonneratia hainanensis.  Planta Med. 2010;  76 920-922
  Search in Google Scholar
• 19 Byon J C H, Kusari A B, Kuseti J. Protein-tyrosine phosphatase-1B acts as a negative regulator of insulin signal transduction.  Mol Cell Biochem. 1998;  182 101-108
  Search in Google Scholar
• 20 Lammer C, Wagerer S, Saffrich R, Mertens D, Ansorge W, Hoffmann I. The cdc25B phorsphatase is essential for the G2/M phase transition in human cell.  J Cell Sci. 1998;  111 2445-2453
  Search in Google Scholar
• 21 Wignot T M, Stewart R P, Schray K J, Das S, Sipos T. In vitro studies of the effects of HAART drugs and excipients on activity of digestive enzymes.  Pharm Res. 2004;  21 420-427
  Search in Google Scholar

Prof. Dr. Yue-Wei Guo

State Key Laboratory of Drug Research
Shanghai Institute of Materia Medica
Chinese Academy of Sciences

Zu Chong Zhi Rd. 555

Zhangjiang, Hi-Tech Park

Shanghai 201203

People's Republic of China

Phone: +86 21 50 80 58 13

Fax: +86 21 50 80 58 13

Email: ywguo@mail.shcnc.ac.cn

• Supplementary Material