Tomenphantadenine, an unprecedented germacranolide-adenine hybrid heterodimer from the medicinal plant Elephantopus tomentosus L.

Fitoterapia 125 (2018) 217–220

Contents lists available at ScienceDirect

Fitoterapia journal homepage: www.elsevier.com/locate/fitote

Tomenphantadenine, an unprecedented germacranolide-adenine hybrid heterodimer from the medicinal plant Elephantopus tomentosus L.

T

Zhi Kai Guoa,b,c,1, Bei Wanga,b,c,1, Cai Hong Caia,b,c, Sheng Zhuo Huanga,b,c, Jing Zhe Yuana,b,c, ⁎ ⁎ Wen Li Meia,b,c, , Hao Fu Daia,b,c, a

Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Haikou 571101, PR China Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, PR China c Hainan Key Laboratory for Research and Development of Natural Products, Li Folk Medicine, Haikou 571101, PR China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Elephantopus tomentosus Medicinal plant Sesquiterpene lactone Antibacterial activity

An unusual adenine-substituted germacrane sesquiterpene lactone, tomenphantadenine (1), has been isolated from the whole plant of Elephantopus tomentosus L. The structure of this compound was established by comprehensive spectroscopic analysis including high resolution (HR) ESI-MS, 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data. This compound features novel hybrid pattern of germacrane sesquiterpene with adenine through C-N linkage, and a possible biosynthetic pathway for it was proposed. Compound 1 showed potent antibacterial activity against the gram-positive Staphylococcus aureus and weak acetylcholinesterase (AChE) inhibitory activity.

1. Introduction Species of the genus Elephantopus (Asteraceae), mainly distributed in the regions of America, are sources of abundant sesquiterpene lactones with significant antileishmanial and cytotoxic activity [1–3]. In China, there are two species including Elephantopus scaber L. and Elephantopus tomentosus L. Only Elephantopus tomentosus have long been used as a folk traditional medicine in south of China and for the treatment of various diseases such as fever, bronchitis, hepatitis, and the cough associated with pneumonia, and arthralgia [4]. As part of a program to investigate the chemical and biological diversity of the ethnic Li folk medicinal plants in Hainan Province [5–9], we phytochemically investigated the ethanol extracts of the medicinal plant Elephantopus tomentosus. From the EtOAc portion of the ethanol extract of the whole plants, six triterpenes with antibacterial activity and five new sesquiterpene lactones tomenphantopin C–F, H with antibacterial and cytotoxic activities have been isolated from this species for the first time [10–13]. Subsequent investigation on the fractions of the EtOAc portion by 1H NMR and LC-MS revealed the presence of a sesquiterpene derivative with [M + H]+ ion at m/z 500, which did not match the previously isolated compounds, indicative of a new germacranolide derivative. Eventually it was purified, identified and designated as tomenphantadenine (1) (Fig. 1). To our knowledge, this is the first example of germacranolide derivatives coupled with adenine. Herein, we

⁎

1

report details of the isolation, structure determination, and hypothetical biosynthetic pathway of this novel compound, as well as its antibacterial and AChE inhibitory activity. 2. Results and discussion Tomenphantadenine (1) was obtained as a white powder. The molecular formula of 1 was found to be C24H29N5O7 derived from the HRESI-MS, giving an [M + H]+ ion at m/z 500.2141 (calculated 500.2140) and an [M − H]− ion at 498.2002 (calculated 498.1994). The IR spectrum displayed the presence of hydroxyl (3450 cm− 1) and carbonyl (1773, 1721 cm− 1). The complete assignments of all proton and carbon resonances, as shown in Table 1, were deduced by comprehensive analysis of various 2D experiments including HSQC, 1H-1H COSY, HMBC, and ROESY, confirmed the structure of 1 as shown in Fig. 1. The 1H NMR spectrum of 1 (acquired in DMSO-d6) showed signals of two methyls [δH 1.54 (3H, s, H-14) and 1.86 (3H, s, H-3″)], four olefinic protons [δH 5.13 (2H, m, H-15), 5.90 (1H, br s, Hα-4″), and 5.55 (1H, br s, Hβ-4″)], two aromatic protons [δH 8.00 (1H, s, H-2′) and 7.84 (1H, s, H-8′)], four methylenes [δH 2.44 (1H, d, J = 13.5 Hz, Hα-1) and 2.29 (1H, d, J = 13.5 Hz, Hβ-1); δH 2.57 (1H, d, J = 13.5 Hz, Hα-3) and 1.80 (1H, d, J = 13.5 Hz, Hβ-3); δH 2.57 (2H, m, overlap, H-9), and 4.37 (2H, br d, J = 6.5 Hz, H-13)], two aliphatic methines [δH 3.22 (2H, m, overlap, H-7 and H-11)], three oxygenated methines [δH 4.07

Corresponding authors at: Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Haikou 571101, PR China. E-mail addresses: [email protected] (W.L. Mei), [email protected] (H.F. Dai). These authors contributed equally to this work.

https://doi.org/10.1016/j.fitote.2017.11.022 Received 16 October 2017; Received in revised form 27 November 2017; Accepted 28 November 2017 Available online 29 November 2017 0367-326X/ © 2017 Elsevier B.V. All rights reserved.

Fitoterapia 125 (2018) 217–220

Z.K. Guo et al.

(1H, d, J = 6.5 Hz, H-5), 4.30 (1H, dd, J = 6.5, 3.6 Hz, H-6), and 5.20 (1H, m, H-8)], two hydroxyl groups [δH 6.05 (1H, br s, 2-OH) and 5.31 (1H, br s, 4-OH)], and one NH2 group [δH 7.15 (2H, br s, 6-NH2)]. The 13 C NMR and DEPT135 spectra of 1 (in DMSO-d6) exhibited twenty four carbons resonances, including two methyls [δC 31.9 (q, C-14) and 18.2 (q, C-3″)], two olefinic methylene carbons [δC 119.8 (t, C-15) and 125.7 (t, C-4″)], two olefinic methine carbons [δC 152.1 (d, C-2′), 141.4 (d, C8′)], and five non-protonated olefinic carbons [149.6 (s, C-4′), 118.8 (s, C-5′), 155.9 (s, C-6′), 135.8 (s, C-2″), and 141.1 (s, C-10)], four aliphatic methylene carbons [δC 50.9 (t, C-1), 49.1 (t, C-3), 33.9 (t, C-9), and 44.2 (t, C-13)], two aliphatic methine carbons [δC 43.4 (d, C-11) and 40.1 (d, C-7)], and seven oxygenated carbons [δC 105.9 (s, C-2), 77.6 (s, C-4), 83.9 (d, C-5), 79.3 (d, C-6), 76.9 (d, C-8), 175.4 (s, C-12) and 165.9 (s, C-1″)]. Comprehensive interpretation of 1H-1H COSY correlations from H-6 to H-5 and H-7, from H-7 to H-6, H-8, and H-11, from H-8 to H-7 and H2-9, and from H-11 to H-7 and H2-13 revealed the partial linkage of C-5-C-6-C-7(-C-11-C-13)-C-8-C-9. HMBC correlations from H2-1 and H2-9 to C-10 and C-15, and from H2-1 to C-9 assigned the connectivity of C-1-C-10(-C-15)-C-9, while the HMBC correlations from H3-14 to C-3, C-4, and C-5, from H2-3 to C-1 and C-2 revealed the fragment of C-1-C2-C-3-C-4(-C-14)-C-5, which was also supported by the HMBC correlations from OH-2 to C-2 and C-3 and from 4-OH to C-3, C-4 and C-5. The lactone fragment was deduced from the HMBC correlations from H-7 and H2-13 to C-12. Therefore the germacrane sesquiterpene skeleton was determined. The position of the methacrylate ester side chain was suggested by the analysis of HMBC correlations from H-3″ and H-4″ to C-1″ and C-2″, from H-3″ to C-4″, and from H-4″ to C-3″. The key HMBC correlations from H2-13 to C-4′ and C-8′ indicated that C-13 was linked to the N-9′ on the adenine, which was also secured by the observation of NOE correlations of H-8′ with H2-13 and H-11. The relative configuration of 1 was determined by ROESY and NOESY experiments in DMSO-d6 and CDCl3 + methanol-d4, respectively. Both of the spectra displayed correlations of H-8 with H-6 and H-11, of H-5 with H-6 and H3-14, and of H3-14 with OH-2, demonstrating that OH-2, H3-14, H-5, H-6, H-8 and H-11 were on the same plane, while the NOE correlation of H-7 with H2-13 proved them in the opposite plane. Thus the structure of compound 1 was assigned as shown in Fig. 2 and named as tomenphantadenine, representing a new type of germacrane sesquiterpene lactone coupled with adenine through C-N linkage in the germacranolide family. A plausible biosynthetic pathway for 1 is proposed in Scheme 1. The germacrane skeleton is formed from farnesyl diphosphate (FPP) and undergoes a series of cyclization, oxidation and esterification reactions to an known intermediate tomenphantopin D (4) [13], then two hydrogens could be transferred by a dehydrogenase, using FAD (flavin adenine dinucleotide) as an electron acceptor, to form a terminal double bond in the lactonic ring of a proposed intermediate 5. The construction of the unprecedented hybrid 1 involves the initial step of hydrogen atom abstraction from the 9-NH of a primary metabolite adenine by a key catalytic basic amino acid (here we showed a histidine

Fig. 1. Structure of tomenphantadenine (1).

Table 1 1 H (500 MHz) and

13

C NMR (125 MHz) spectroscopic data for tomenphantadenine (1).

Position

δHa

1

2.44, d (13.5, Hα); 2.29, d (13.5, Hβ)

2 2-OH 3 4 4-OH 5 6 7 8 9 10 11 12 13 14 15 2′ 4′ 5′ 6′ 6-NH2 8′ 1″ 2″ 3″ 4″

a b

(mult, J in Hz)

δCa

δHb (mult, J in Hz)

δCb

50.9, CH2

2.51, d (13.5, Hα); 2.41, d (13.5, Hβ)

51.1

105.9, C 6.05, br s 2.57, d (13.5, Hα); 1.80, d (13.5, Hβ)

49.1, CH2

106.7 2.51, d (13.7, Hα); 1.85, d (13.7, Hβ)

77.6, C 5.31, 4.07, 4.30, 3.22, 5.20, 2.57,

br s d (6.5) dd (6.5, 3.6) m (overlap) m m (overlap)

3.22, m (overlap) 4.37, 1.54, 5.13, 8.00,

br d (6.5) s m s

7.15, br s 7.84, s

1.86, s 5.90, br s; 5.55, br s

83.9, 79.3, 40.1, 76.9, 33.9,

CH CH CH CH CH2

141.1, C 43.4, CH 175.4, C 44.2, CH2 31.9, CH3 119.8, CH2 152.1, CH 149.6, C 118.8, C 155.9, C 141.4, CH 165.9, C 135.8, C 18.2, CH3 125.7, CH2

48.9 78.5

4.24, 4.28, 3.34, 5.23, 2.71, 2.65,

d (3.5) dd (7.0, 3.5) m (overlap) dt (11.2, 3.0) dd (16.0, 3.0, Hα); d (16.0, Hβ)

3.20, m 4.56, 1.62, 5.40, 8.15,

m s s-like; 5.19, br s s

7.90, s

2.02, s 6.29, s-like; 5.67, t-like (1.4)

84.6 80.8 40.4 78.4 34.9 140.7 45.5 176.9 44.3 31.6 121.4 152.8 150.6 119.0 155.8 142.5 167.4 136.7 18.7 127.0

Acquired in DMSO-d6. Acquired in CDCl3 + methanol-d4 (v/v, 1:1).

Fig. 2. The key 1H-1H COSY, HMBC and NOESY correlations supporting the structure of tomenphantadenine (1).

218

Fitoterapia 125 (2018) 217–220

Z.K. Guo et al.

Scheme 1. A proposed biosynthetic pathway for the formation of the hybrid adenine-germacrane sesquiterpene lactone (1).

3.3. Extraction and isolation

group) of an enzyme, then undergoes a Michael-type addition to the nitrogen-carbon bond of 6 [14–17]. In the end, the hydrogen proton abstracted by the histidine group could be transferred back to C11 of 6 and then the novel molecule 1 is built. Sesquiterpene lactones have been reported to inhibit the growth of bacteria [11–13]. In this paper, compound 1 was assessed in vitro for antibacterial activities against Staphylococcus aureus and Ralstonia solanacearum, and for the AChE inhibitory activity. As a result, it exhibited potent antibacterial activity against the gram-positive Staphylococcus aureus with the minimum inhibitory concentration (MIC) value of 9.6 μg/mL, as compared to the positive control kanamycin sulfate with an MIC value of 4.2 μg/mL. Whereas the compound was inactive against the gram-negative Ralstonia solanacearum. Also compound 1 showed weak inhibitory activity against AChE with the inhibition percentage of 24.3% at concentration of 20 mg/mL.

The whole plants of Elephantopus tomentosus (19.4 kg) were powered, and then extracted three times with 95% EtOH at room temperature. Then the combined solution (3 × 20 L) was concentrated in vacuo, and the residue was suspended in H2O (2 L) and partitioned with petroleum ether (1 L × 3), EtOAc (1 L × 3), and n-BuOH (1 L × 3), respectively. The EtOAc fraction (81.3 g) was first fractionated by silica gel (200–300 mesh, 2.5 kg) CC eluted with CHCl3-MeOH (v/v, 9:1, 3 L) to afford five fractions (Fr.1–Fr.5) according to TLC analysis. Fr.2 (7.6 g) was further subjected to chromatography over a silica gel column (200–300 mesh, 4 cm × 98 cm, 350 g) eluting with CHCl3MeOH (v/v, 1:0, 50:1, 30:1, 20:1, 15:1, 10:1, 8:1, 5:1, 2:1, 0:1, each 1 L) to give 10 subfractions (Fr.2-1–Fr.2-10). Fr.2-4 (1.1 g) was chromatographed on silica gel CC (200–300 mesh, 3 cm × 65 cm, 64 g) with a gradient of CHCl3-MeOH (v/v, 30:1, 20:1, 10:1, 5:1, each 800 mL) to yield six subfractions (Fr.2-4-1–Fr.2-4-6), and then Fr.2-4-4 (166.2 mg) was further separated on ODS C-18 column (40–70 μm, 3 cm × 20 cm) eluted with MeOH-H2O (gradient elution of 30, 40, 50, 60, 70, 80, and 90%, each 350 mL) to obtain subfractions Fr.2-4-41–Fr.2-4-4-7. Fraction Fr.2-4-4-4 was purified by Sephadex LH-20 CC (3 cm × 45 cm) using MeOH as eluent to afford compound 1 (2.8 mg).

3. Experimental 3.1. General experimental procedures Optical rotations were acquired on an AUTOPOL IV automatic polarimeter. UV spectra (Rudolph Research Analytical, Hackettstown, NJ, USA) were recorded on a NanoDrop 2000C spectrophotometer (Thermo Scientific, Wilmington, DE, USA). IR spectra were measured on a Spectrum One FT-IR spectrometer (PerkinElmer, Shelton, CT, USA). HR-ESI-MS data were obtained on an Agilent 1260 Infinity LC coupled to a 6230 TOF. 1D and 2D NMR spectra were acquired on a Bruker AvanceIII 500 MHz NMR spectrometer at 500 MHz for 1H NMR and 125 MHz for 13C NMR. The chemical shifts were given in δ (ppm) and referenced to the solvent signal (DMSO-d6, δH 2.50, δC 39.5) or TMS. Column chromatography (CC) was conducted on silica gel (200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, People's Republic of China), ODS (40–70 μm, Fuji Silysia Chemical Ltd., Nagoya, Japan) and Sephadex LH-20 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).

3.3.1. Tomenphantadenine (1) White powder; [α]D23 + 35 (c 0.02, MeOH); IR (neat) vmax 3450, 2970, 1773, 1721, 1626, 1520, 1398 cm− 1; UV (MeOH) λmax (log ε) 210 (4.05), 261 (4.45) nm; 1H and 13C NMR data see Table 1; HR-ESIMS m/z 500.2141 [M + H]+ (calcd for C24H30N5O7, 500.2140), m/z 498.2002 [M − H]− (calcd for C24H28N5O7, 498.1994). 3.4. Bioassay of AChE inhibitory activity The AChE inhibitory activities were tested by Ellman method as described previously [18]. In the assays, the known AChE inhibitor tacrine was used as the positive control and displayed an inhibition percentage of 57%.

3.2. Plant material

3.5. Antibacterial activity

In October 2010, the whole plant of Elephantopus tomentosus was collected in Changjiang County of Hainan Province, People's Republic of China. The plant was identified by Professor Zheng-Fu Dai at Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou, People's Republic of China, where a voucher specimen (No. ET201010) was deposited.

The antibacterial activity was evaluated against Staphylococcus aureus (CMCC(B) 26,003, obtained from Hainan Institute for Food and Drug Control) and Ralstonia solanacearum (Yunnan Key laboratory of biological resource conservation and utilization, Yunnan University) by the reported methods [19]. In the assays, the antibacterial activity was firstly tested using the agar diffusion method with 6 mm filter paper discs containing 20 mg/mL of compound 1, and 0.64 mg/mL of kanamycin sulfate (Staphylococcus aureus) and streptomycin sulfate 219

Fitoterapia 125 (2018) 217–220

Z.K. Guo et al.

(Ralstonia solanacearum) as the positive control. MIC values for the active compound was then determined in the 96-well plates. The effect on the bacterial growth was evaluated after incubation at 37 °C for 18 h.

[6]

[7]

Conflict of interest [8]

The authors declare no conflict of interest. [9]

Acknowledgement

[10]

This work was supported in part by the Natural Science Foundation of Hainan (2017CXTD020), and National Nonprofit Institute Research Grant of CATAS-ITBB (ITBB2015RC03, ITBB2015RC02, 1630052016008).

[11] [12]

Appendix A. Supplementary data [13]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2017.11.022.

[14]

References

[15] [16]

[1] H. Fuchino, T. Koide, M. Takahashi, S. Sekita, M. Satake, New sesquiterpene lactones from Elephantopus mollis and their leishmanicidal activities, Planta Med. 67 (2001) 647–653. [2] S.M. Kupchan, Y. Aynehchi, J.M. Cassady, H.K. Schnoes, A.L. Burlingame, Tumor inhibitors. XL. The isolation and structural elucidation of elephantin and elephantopin, two novel sesquiterpenoid tumor inhibitors from Elephantopus elatus, J. Org. Sci. 34 (1969) 3867–3875. [3] T.K. Tabopda, J.W. Liu, B.T. Ngadjui, B. Luu, Cytotoxic triterpene and sesquiterpene lactones from Elephantopus molis and induction of apoptosis in neuroblastoma cells, Planta Med. 73 (2007) 376–380. [4] Y.L. Chen, Flora Republicae Popularis Sinicae, Science Press, Beijing, 1982, p. 43. [5] H. Wang, H.M. Jiang, F.X. Li, H.Q. Chen, W.C. Liu, S.Z. Ren, W.L. Mei, H.F. Dai,

[17] [18]

[19]

220

Flavonoids from artificially induced dragon's blood of Dracaena cambodiana, Fitoterapia 121 (2017) 1–5. S.Z. Huang, Q.Y. Ma, F.D. Kong, Z.K. Guo, Q. Wang, H.F. Dai, Y.Q. Liu, J. Zhou, Y.X. Zhao, Daphnauranins A and B, two new antifeedants isolated from Daphne aurantiaca roots, Fitoterapia 122 (2017) 11–15. H. Shao, W.L. Mei, F.D. Kong, W.H. Dong, C.J. Gai, W. Li, G.P. Zhu, H.F. Dai, Sesquiterpenes of agarwood from Gyrinops salicifolia, Fitoterapia 113 (2016) 182–187. Z.K. Guo, T. Yang, C.H. Cai, W.H. Dong, C.J. Gai, J.Z. Yuan, W.L. Mei, H.F. Dai, A new apotirucallane-type triterpenoid from Atalantia buxifolia, J. Asian Nat. Prod. Res. 17 (2015) 1018–1023. W. Li, C.H. Cai, W.H. Dong, Z.K. Guo, H. Wang, W.L. Mei, H.F. Dai, 2-(2-phenylethyl)- chromone derivatives from Chinese agarwood induced by artificial holing, Fitoterapia 98 (2014) 117–123. B. Wang, W.L. Mei, W.J. Zuo, Y.B. Zeng, G.D. Liu, H.F. Dai, Antibacterial activity components from Elephantopus tomentosus, J. Trop. Subtrop. Bot. 20 (2012) 413–417. B. Wang, W.L. Mei, Y.B. Zeng, Z.K. Guo, G.D. Liu, H.F. Dai, A new sesquiterpene lactone from Elephantopus tomentosus, J. Asian Nat. Prod. Res. 14 (2012) 700–703. B. Wang, W.L. Mei, W.J. Zuo, Y.X. Zhao, W.H. Dong, G.D. Liu, H.F. Dai, Two new sesquiterpene lactones from Elephantopus tomentosus, Chin. J. Chem. 30 (2012) 1320–1322. W.L. Mei, B. Wang, W.J. Zuo, Y.X. Zhao, W.H. Dong, G.D. Liu, H.F. Dai, Two new germacranolides from Elephantopus tomentosus, Phytochem. Lett. 5 (2012) 800–803. S. Raoufmoghaddam, Recent advances in catalytic C-N bond formation: a comparison of cascade hydroaminomethylation and reductive amination reactions with the corresponding hydroamidomethylation and reductive amidation reactions, Org. Biomol. Chem. 12 (2014) 7179–7193. O. Torre, I. Alfonso, V. Gotor, Lipase catalyzed Michael addition of secondary amines to acrylonitrile, Chem. Commun. (2004) 1724–1725. J.L. Wang, J.M. Xu, Q. Wu, D.S. Lv, X.F. Lin, Promiscuous enzyme-catalyzed regioselective Michael addition of purine derivatives to α,β-unsaturated carbonyl compounds in organic solvent, Tetrahedron 65 (2009) 2531–2536. J. Bariwal, E. Van der Eycken, C-N bond forming cross-coupling reactions: an overview, Chem. Soc. Rev. 42 (2013) 9283–9303. G.L. Ellman, K.D. Courtney, V. Andres Jr., R.M. Featherstone, A new and rapid colorometric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7 (1961) 88–95. I. Wiegand, K. Hilpert, R.E.W. Hancock, Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances, Nat. Protoc. 3 (2008) 163–175.