Citation

Xue Zhou, Qing-Lan Guo, Cheng-Gen Zhu, Cheng-Bo Xu, Ya-Nan Wang, Jian-Gong Shi. Gastradefurphenol, a minor 9, 9'-neolignan with a new carbon skeleton substituted by two p-hydroxybenzyls from an aqueous extract of "tian ma"[J]. Chin. Chem. Lett., 2017, 28(6): 1185-1189  

Xue Zhou, Qing-Lan Guo, Cheng-Gen Zhu, Cheng-Bo Xu, Ya-Nan Wang, Jian-Gong Shi    

Gastrodia elata Blume (Orchidaceae) is a holomycotrophic perennial plant living on a symbiotic mycorrhizal fungus Armillaria mellea at a vegetative growth stage of its life cycle [1-3]. The steamed and dried rhizome of G. elata, known as "tian ma" in Chines e, is an important traditional Chinese herbal medicine that has been used for the treatment of various neuralgic and nervous disorders (such as vertigo, blackout, headaches, migraine, epilepsy, neuralgia, and paralysis) for over 2000 years [1-4]. This herbal medicine has health benefits enhancing strength and virility and improving memory and blood circulation [4]. The fresh and prepared rhizomes of G. elata are used also in Chinese cuisines. Although this plant has become a wildly endangered species in China, pharmaceutical and catering demands are completely fulfilled by agricultural cultivation, which is benefited from a great achievement of scientific researches on symbiotic associations with fungi engaging in seed germination and vegetative growth since the late of 1950 [1-3]. Previous pharmacological and chemical studies mainly focusing on ethanol or methanol extracts of the herbal medicine showed that p-hydroxybenzyl analogues were the main active constituents [5-10]. To be consistent with a traditional and practical application of the herbal medicines by decocting with water, we conducted investigation of an aqueous extract of G. elata rhizomes as part of a program to systematically study the chemical diversity of traditional Chinese medicines and their biological effects [11-31]. In our previous papers [32-38], 50 compounds were reported from polar fractions of the extract, along with preliminary bioassays of those isolates and pharmacological functions of a minor component N⁶-(p-hydroxybenzyl) adenosine (NHBA). Herein, reported is the isolation and structure elucidation of an unprecedented 9, 90-neolignan possessing a novel carbon skeleton modified by two p-hydroxybenzyl at C-7, named gastradefurphenol (1) (Fig. 1) from a less polar fraction of the extract. In addition, free rotational restriction of C-7 -C-7'' and C-7 -C-7''' bonds and transformational equilibrium between two molecular states of 1 in acetone-d₆ were observed and discussed by further analysis of the NMR spectroscopic data in CD₃OD.

Figure 1

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Fig. 1. Structure of 1.

Compound 1 was obtained as yellowish amorphous powder. Its IR spectrum showed absorptions due to hydroxyl (3373 cm^-1) and aromatic ring (1612 and 1514 cm^-1) functionalities. The molecular formula of 1 was determined as C₃₂H₂₈O₅ by HR-ESIMS at m/z 515.1831 (calcd. for C₃₂H₂₈O₅Na, 515.1829) and NMR spectroscopic data (Table 1). The ¹H NMR spectrum of 1 in acetone-d₆ showed resonances attributable to two inequivalent para-substituted phenyls at δ_H 6.70 (d, 2H, J = 8.4 Hz, H-3/5), 6.75 (d, 2H, J = 8.4 Hz, H-3'/5'), 6.93 (d, 2H, J = 8.4 Hz, H-2/6), and 7.07 (d, 2H, J = 8.4 Hz, H-2'/6'); two equivalent para-substituted phenyls at δ_H 6.49 (d, 4H, J = 8.4 Hz, H-2''/2'''/6''/6''') and 6.53 (d, 4H, J = 8.4 Hz, H-3''/3'''/5''/5'''); and a disubstituted furan ring at δ_H 5.98 (d, 1H, J = 3.6 Hz, H-9) and 5.94 (d, 1H, J = 3.6 Hz, H-9'). In addition, the spectrum showed resonances assignable to three isolated methylene at δ_H 3.82 (s, 2H, H-7'), 3.13 (d, 2H, J = 13.8 Hz, H-7''a/7'''a), and 3.10 (d, 2H, J = 13.8 Hz, H-7''b/7'''b), as well as signals due to four phenolic hydroxyls at δ_H 8.17 (s, 1H, OH-4), 8.15 (s, 1H, OH-4'), and 8.02 (s, 2H, OH-4''/4'''). The ¹³C NMR and DEPT spectra displayed 32 carbon resonances (Table 1) corresponding to the above units and an additional quaternary carbon at δ_C 50.3 (C-7). As compared with those of the previously isolated compounds from "tian ma" [32, 34, 35, 37], these spectroscopic data indicate that 1 possesses an unusual structure consisting of one furan ring and four p-hydroxybenzyl units, which was further constructed by 2D NMR data analysis.

Table 1

Table 1 NMR spectroscopic data for compound 1.a

Table 1 NMR spectroscopic data for compound 1.a

The proton and proton-bearing carbon resonances in the NMR spectra of 1 were unambiguously assigned by homonuclear vicinal coupling cross-peaks in the ¹H-¹H COSY spectrum, in combination with one-bond heteronuclear correlations in the HSQC spectrum. In the HMBC spectrum, the two-and three-bond heteronuclear correlations (Fig. 2) from H-2/6 to C-3/5, C-4, and C-7; from H-3/5 to C-1 and C-4, and from OH-4 to C-4 and C-3/5, in combination with their chemical shifts and quaternary nature of C-7 (Table 1), demonstrated that there was a 7, 7-disubstituted 4-hydroxybenzyl unit in 1. The HMBC correlations from H-2'/6' to C-3'/5', C-4', and C-7'; from H-3'/5' to C-1' and C-4'; from H₂-7' to C-1' and C-2'/6'; and from OH-4' to C-4' and C-3'/5' confirmed the presence of a 4'-hydroxybenzyl. Similarly, the HMBC correlations from H-2''/2'''/6''/ 6''' to C-3''/3'''/5''/5''', C-4''/4''', and C-7''/7'''; from H-3''/3'''/5''/5''' to C-1''/1''' and C-4''/4'''; from H₂-7''/7''' to C-1''/1''' and C-2''/2'''/ 6''/6'''; and from OH-4''/4''' to C-4''/4''' and C-3''/3'''/5''/5''' verified the occurrence of the two equivalent p-hydroxybenzyl (e.g. 4''-and 4'''-hydroxybenzyls). In addition, the HMBC correlations of both C-1 and C-7 with H₂-7''/7''' revealed that the two equivalent p-hydroxybenzyl located at the quaternary C-7 of the 4-hydroxybenzyl, which was further supported by the overlapped correlations of H-7''/C-7''' and H-7'''/C-7''. Meanwhile, the presence of the disubstituted furan ring was supported by the ¹H-¹H COSY cross-peaks of H-9/H-9' and the HMBC correlations of H-9/C-8, C-8', and C-9' in the HMBC spectrum, together with chemical shifts of these proton and carbon resonances and the coupling constant value ³J_{H-9, H-9'} (3.6 Hz). The HMBC correlations of C-8 with H₂-7''/ 7''' indicated that the two equivalent p-hydroxybenzyl connected via the quaternary C-7 to C-8 of the furan ring in 1. Moreover, the HMBC correlations of C-8' and C-9' with H₂-7' unequivocally located the 4'-hydroxybenzyl at C-8' of the furan ring. Therefore, the structure of compound 1 was determined as shown and named gastradefurphenol. According to the IUPAC suggested nomenclature of neolignans [39], this compound is systematically named 4, 4'-dihydroxy-7, 7-bis(p-hydroxybenzyl)-8, 8'-epoxy-9, 9'-neoligna-8, 8'-diene.

Figure 2

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Fig. 2. Main 1H-1H COSY (thick lines) and bond HMBC correlations (red arrows, from 1H to 13C) of 1.

Interestingly and notably, the ¹H NMR spectrum of 1 in acetone-d₆ showed a singlet for H₂-7', an overlapped AB coupling system for H₂-7''/7''', and vicinal coupling doublets for aromatic protons of the p-hydroxybenzyl (Table 1 and Figs. S7 and S8 in Supporting information), as assigned unambiguously by 2D NMR spectroscopic data analysis. In addition, in the ¹³C NMR spectrum, for the symmetric p-hydroxybenzyl the carbons attaching to and closing to the methylenes (C-1, C-1', C-1''/1''', C-2/6, C-2'/6', C-2''/6'', and C-2'''/6''') appeared as the normal singlets. However, the carbons attaching to and closing to the phenolic hydroxyls appeared as paired resonances with almost identical intensities and chemical shift differences in Δδ_C 0.10 (15 Hz) for C-4, C-4', and C-4''/4''' and 0.09 (13.5 Hz) for C-3/5, C-3'/5', C-3''/5'', and C-3'''/5''' (Table 1 and Figs. S9-S11 in Supporting information). Because 1 is achiral the AB coupling system of H₂-7''/7''' and the paired carbon resonances are abnormal. The AB coupling system of H₂-7''/7''' demonstrated that the germinal protons at C-7''/7''' were chemically inequivalent. This could be explained from that free rotation of the C-7-C-7'' and C-7-C-7''' bonds was restricted by intramolecular interaction of four bulky groups at C-7 of 1 in the acetone-d₆ solution, because there is no chiral carbon or double bond to induce inequivalence of the germinal protons at C-7''/C-7''' in the structure. However, the paired resonances of C-4, C-4', C-4''/4''', C-3/5, C-3'/5', C-3''/5'', and C-3'''/5''' must be due to the presence of two chemically inequivalent states of the molecule since these spin nuclei are away from the crowded center C-7 and toward outside of the molecule. Accordingly, we speculate that there is transformational equilibrium between two molecular states in the acetone-d₆ solution of 1. In order to explore a solvent effect on the chemical inequivalence of the germinal protons and the carbon nuclei in solution state, the NMR spectra of 1 in CD₃OD were acquired (Figs. S16-S20 in Supporting information). As compared, the AB system and the paired carbon resonances in acetone-d₆ were replaced by a singlet for the germinal protons and corresponding unpaired carbon resonances in CD₃OD, respectively. This revealed that free rotational restriction of the C-7-C-7'' and C-7-C-7''' bonds in 1 as well as transformational equilibrium between two molecular states were solvent-dependent and that the restriction and equilibrium only existed in the aprotic solvent acetone-d₆ instead in the protic solvent CD₃OD. Therefore, intermolecular interactions between the solute molecules and between the solute and solvent molecules play potent roles in both restriction of the bond rotations and equilibrium of the molecular states. Although details of the solvent-dependent equilibration were not further investigated due to limitation of the sample amount, in the acetone-d₆ the paired carbon resonances of C-4, C-4', C-4''/4''', C-3/ 5, C-3'/5', C-3''/5'', and C-3'''/5''' in the symmetric p-hydroxybenzyl demonstrated that the phenolic hydroxyls in 1 must involve in the intermolecular interactions.

Gastradefurphenol (1) represents the first 9, 90-neolignan with the furan ring motif formed between the C₃ units and with the novel carbon skeleton modified at C-7 by two p-hydroxybenzyl though the only natural product of 9, 9'-neolignan category, e.g. 4, 4'-dimethoxy-9, 9'-neoligna-7, 7'-diene (ocimin) was reported from an essential oil of Ocimum americanum seeds [40, 41]. Based on the unique structure feature consisting of two p-hydroxyphenylpropene (C₆C₃) units and two p-hydroxybenzyl, the biosynthetic precursors of 1 are traced to p-hydroxyphenylpropene derivatives, such as (+)-(2S)-2-hydroxy-3-(p-hydroxyphenyl)propyl aldehyde (2) and 1-(p-hydroxyphenyl)propan-1, 2-dione (3). A plausible biosynthetic pathway for 1 is postulated in Scheme 1. An enzyme-catalyzed Aldol addition of 2 and 3, followed by sequential or simultaneous intramolecular nucleophilic addition and dehydration, would generate a key intermediate 4. The intermediate undergoes condensation with two molecules of p-cresol (5) to afford 1. The plausible biosynthetic pathway was supported by concomitant isolation of the acid form of 2, as well as of compound 3 and the β-D-glucoside of 5 from the extract in this study. These biosynthetic precursors would be generated from biosynthetic and/or metabolic processes of L-tyrosine [42, 43], which is supported by the occurrence of L-tyrosine in the rhizome of G. elata [44].

Scheme 1

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Scheme 1. The plausible biosynthetic pathway of 1.

A novel neolignan, named gastradefurphenol (1) possessing an unprecedented 7, 7-bis(benzyl)-9, 9'-neolignane skeleton, was isolated as the minor constituent from the aqueous extract of the G. elata rhizomes. Although biological activity of 1 was not assayed due to limitation of the sample amount, the unique structure adds a new skeletal entity to the neolignan natural products, and provides a selectable framework for synthesis and biological evaluation in future. In particular, the plausible biosynthetic pathway provides a strategy for studies of biomimetic and total synthesis to obtain enough amount of the sample for biological evaluation, as well as for biosynthesis of the diverse benzylcontaining metabolites in this orchid plant. Solvent-dependent equilibrium of intermolecular interactions as indicated by the ¹³C NMR spectroscopic data of 1 also deserve detailed investigations. The result in this study continuously reveals that the chemical constituents of holomycotrophic G. elata have characteristic structure features modified by and derived from p-hydroxybenzyl, indicating that production of these metabolites is predominated by the metabolic process of L-tyrosine. However, a literature survey showed that the symbiotic mycorrhizal fungus Armillaria mellea mainly produced antimicrobial and cytotoxic aryl esters of protoilludane-type sesquiterpenoids without p-hydroxybenzyl [45-47]. This, together with identification of various p-hydroxybenzyl analogues and p-hydroxybenzyl-substituted metabolites, including p-hydroxybenzyl-substituted amino acids, glutathione derivatives, and nucleosides [32, 34, 35, 37], suggests the presence of independent and unique biosynthetic pathways and associated enzymes to accumulate the diverse p-hydroxybenzyl-substituted natural products in the holomycotrophic plant.

The UV spectrum was recorded on a V-650 spectrometer (JASCO). The IR spectrum was recorded on a Nicolet 5700 FT-IR Microscope spectrometer (FT-IR Microscope Transmission) (Thermo Electron Corporation, Madison, WI, USA). 1D-and 2DNMR spectra were obtained at 600 MHz for ¹H and 150 MHz for ¹³C, respectively, on a SYS 600 MHz (Varian Associates Inc., Palo Alto, CA, USA) spectrometer, with in acetone-d₆ as a solvent and solvent peaks as references. ESIMS and HR-ESIMS data were obtained on Agilent 1100 Series LC-MSD-Trap-SL and Agilent 6520 AccurateMass Q-TOFL CMS spectrometers (Agilent Technologies, Ltd., Santa Clara, CA, USA), respectively. Column chromatography (CC) was performed with macroporous adsorbent resin (HPD-100, Cangzhou Bon Absorber Technology Co. Ltd., Cangzhou, China), MCI gel (CHP 20P, 75-150 μm) (Mitsubishi Chemical Corporation, Tokyo, Japan), silica gel (200-300 mesh, Qingdao Marine Chemical Inc., China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). HPLC separation was performed on an instrument equipped with an Agilent ChemStation for LC system, an Agilent 1100 pump, and an Agilent 1100 single-wavelength absorbance detector (Agilent Technologies, Ltd.), using a Shiseido column (250 mm × 10 mm, i.d.) packed with C₈ reversed phase silica gel (5 μm) (Shiseido Co., Ltd, Tokyo, Japan). TLC was carried out with glass precoated silica gel GF₂₅₄ plates (Qingdao Marine Chemical Inc.). Spots were visualized under UV light or by spraying with 5% H₂SO₄ in EtOH followed by heating. Unless otherwise noted, all chemicals were purchased from commercially available sources and were used without further purification.

See Ref. [34].

For extraction and preliminary fractionation of the extract, see refs 34 and 35. Fractions C4 and C5 were merged together on the basis of TLC analysis to give a mixture C4 + 5 (6.8 g), which was subjected to CC over silica gel (200-300 mesh), eluted with a gradient of increasing acetone (0-100%) in petroleum ether, to yield C4 + 5-1-C4 + 5-18. Subfraction C4 + 5-13 (199 mg) was separated by CC over Sephadex LH-20 (petroleum ether-CH₂Cl₂-MeOH, 5:5:1, v/v/v) to yield C4 + 5-13-1-C4 + 5-13-3, of which C4 + 5-13-2 (60 mg) was further isolated by CC over silica gel, with elution of CH₂Cl₂-MeOH (30:1, v/v), to afford C4 + 5-13-2-1-C4 + 5-13-2-3. Purification of C4 + 5-13-2-2 (6.5 mg) by RP-HPLC (C₈ column, 70% MeOH in H₂O) obtained 1 (2.0 mg, t_R = 16.1 min, 0.000004%).

Gastradefurphenol (1): Yellowish amorphous powder; UV (MeOH) λ_max (log ε): 203 (2.85), 222 (2.61), 279 (1.94) nm; IR v_max 3373, 3027, 2925, 2854, 1679, 1612, 1514, 1446, 1370, 1209, 1149, 1107, 965, 834, 801, 726, 557, 537 cm^-1; ¹H NMR (acetone-d₆, 600 MHz) data, see Table 1; ¹³C NMR (acetone-d₆, 150 MHz) data, see Table 1; (+)-ESIMS m/z 515 [M + Na]⁺, 531 [M + K]⁺; (+)-HRESIMS m/z 515.1831 (calcd. for C₃₂H₂₈NaO₅, 515.1829).

Financial support from the National Natural Science Foundation of China (Nos. 81502942 and 81630094) is acknowledged.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.03.028.