Valeriadimers A–C, three sesquiterpenoid dimers from valeriana officinalis var. latifolia

Abstract

Valeriadimer A (1), formed by a C–C (4-1′) bond between a bicyclogermacrane nor-sesquiterpene moiety and a maaliane nor-sesquiterpene moiety, together with two further sesquiterpenoid dimers, valeriadimers B and C (2, 3) were isolated from the root of Valeriana officinalis var. latifolia. Their structures were elucidated by extensive analysis of spectroscopic data and confirmed by Cu-Kα X-ray diffraction experiment.

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The genus Valeriana of the Valerianaceae family is widely distributed throughout the world. Previous investigations on Valeriana species led to a large number of structurally diverse compounds and showed multiple bioactivities, such as anxiolytic, antidepressant, antispasmodic, sedative, and anti-HIV.¹ Valeriana officinalis var. latifolia is commonly used in mainland China as an alternative species for V. officinalis. In recent years, phytochemical studies on this plant, including our work, have afforded sesquiterpenoids and iridoids.² As part of our investigation on structurally novel compounds from natural sources, especially on sesquiterpenoid oligomers,³ a nor-sesquiterpenoid dimer with an unprecedented bicyclogermacrane–maaliane skeleton (1), together with two sesquiterpenoid dimers (2 and 3) (Fig. 1), were isolated from the titled plant. In this work, we describe the isolation and structural elucidation of compounds 1–3 and their angiogenesis activity in HUVECs.

Fig. 1 Chemical structures of 1, 2, and 3.

Valeriadimer A (1) ([α]²⁵_D +2.36, c 0.12, CH₃OH) was obtained as colourless orthorhombic crystals (acetone). Its molecular formula was assigned as C₂₈H₄₂O₂ by positive HRESIMS at 433.3113 [M + Na]⁺ (calcd 433.3083), which required 8 degrees of unsaturation. The IR spectrum showed the presence of carbonyl group (1711 cm⁻¹). In the ¹H NMR spectrum, 6 singlet methyls [δ_H 0.73 (3H, s), 0.95 (3H, s), 1.01 (3H, s), 1.03 (3H, s), 1.08 (3H, s), and 1.12 (3H, s)], one olefinic proton [5.03 (1H, d, J = 8.3 Hz)], and one oxymethine [3.10 (1H, dd, J = 11.0, 2.0 Hz)] signals were observed. The ¹³C NMR and DEPT spectroscopic data displayed 28 carbon signals (Table S1, ESI†), sorted into 6 methyls, 8 methylenes, 8 methines, and 6 quaternary carbons. The ¹³C NMR spectrum also indicated the presence of a carbonyl (δ_C 215.7), a double bond (δ_C 142.2 and 126.2), and an epoxide ring (δ_C 64.5 and 61.9).

Detailed analysis of 1D and 2D NMR spectra of compound 1, two dimethylcyclopropane rings were inferred by the characteristic methyl and quaternary carbon signals at upfield in ¹³C NMR spectrum [δ_C 16.3 (C-12′, –CH₃), 16.6 (C-13, –CH₃), 29.9 (C-13′, –CH₃), 30.0 (C-12, –CH₃), 19.5 (C-11′, qC), and 21.3 (C-11, qC)],⁴ which was further confirmed by the HMBC correlations from H-12 to C-11, C-13, C-6 (δ_C 28.7), and C-7 (δ_C 35.3); correlations from H-13 to C-11, C-12, C-6, and C-7, correlations from H-12′ to C-11′, C-13′, C-6′ (δ_C 23.6), and C-7′ (δ_C 18.6), and correlations from H-13′ to C-11′, C-12′, C-6′, and C-7′. The ¹H–¹H COSY spectrum of compound 1 gave four fragments [a: –CH(1)–CH₂(2)–CH₂(3)–; b: –CH(5)–CH(6)–CH(7)–CH₂(8)–CH₂(9)–; c: –CH(1′)–CH₂(2′)–CH₂(3′)–; d: –CH(5′)–CH(6′)–CH(7′)–CH₂(8′)–CH₂(9′)–] according to its protons spin systems (Fig. 2). By combination with HMBC correlations from H-14 to C-1, C-9, and C-10, and correlations from H-3 to C-5, the fragments a and b, together with one of the above dimethylcyclopropane moieties, constructed a bicyclogermacrane-type nor-sesquiterpene. The remanent section of 1, including the fragments c and d, was just in accordance to a maaliane nor-sesquiterpene skeleton, which was certified by the HMBC signals of H-2′, H-3′, and H-6′ to C-4; and signals of H-14′ to C-1′, C-5′, C-9′, and C-10′. The key HMBC correlations of H-1′/C-5 and H-5/C-1′ disclosed without doubt these two sesquiterpene parts was linked each other by C–C (4-1′) single bond (Fig. 2).

Fig. 2 Key ¹H–¹H COSY (), HMBC (), and NOESY () correlations of 1.

The relative configuration of 1 came from the analysis of its NOESY spectrum. In the bicyclogermacrane sesquiterpoid segment, H-1, H-6, and H-7 were validated on the same side by the cross peak of H-1/H-7, and H-7/H-6, while Me-14 was on the opposite side due to the correlations of Me-14/H-9a and H-7/H-9b. In addition, the correlations of H-5/H-12 and H-13 illustrated the geometric configuration of C=C double bond between C-4 and C-5 belonged to E-type. In the maaliane sesquiterpene segment, the NOESY correlations of H-7′/H-6′, H-6′/H-1′, H-1′/H-2′b, and Me-14′/H-2′a suggested that H-1′, H-6′, and H-7′ was assumed to possess the same orientation and Me-14′ to be in the adverse orientation (Fig. 2). The absolute configurations of 1 were finally determined to be 1R, 6S, 7R, 10R, 1′S, 5′S, 6′R, 7′R, 10′S (Fig. 3) by Cu-Kα X-ray diffraction experiment.

Fig. 3 Single X-ray crystallographic structure of 1.

Compound 2 ([α]²⁵_D +41.7, c 0.20, CH₃OH) showed the [M + H]⁺ peak at m/z 455.3154 by HRESIMS (calcd 455.3156), consistent with the molecular C₂₉H₄₂O₄. Its 1D NMR spectra (Table S2, ESI†) exhibited the presence of two dimethylcyclopropane rings, one epoxide ring, double bond, and one carbonyl, which revealed its structural similarity to valeriadimer A (1), except that the signal of C-1′ in 1 was replaced by an oxygenated methine at δ_C 71.2, and an extra acyloxy signal (δ_C 167.7, qC, C-15) appeared in the ¹³C NMR spectrum of 2. Further detailed analysis of 2D NMR spectra of 2 disclosed the presence of a bicyclogermacrane-type sesquiterpenoid part, identical to madolin B.⁵ The other fragment was deduced to be identical with the maaliane-type nor-sesquiterpenoid moiety of 1 apart from oxidation at C-1′. The key HMBC correlation of H-1′/C-15 verified that the above two moieties was linked through an ester bond (Fig 4). The NOESY correlations observed in 2 (Fig 4) suggested the same relative configuration as 1. Thus, the structure of 2 was elucidated and named valeriadimer B.

Fig. 4 Key ¹H–¹H COSY (), HMBC (), and NOESY () correlations of 2.

Valeriadimer C (3) ([α]²⁵_D −30.0, c 0.35, CH₃OH) was isolated as colourless crystals (70% MeOH–H₂O) and exhibited the molecular formula C₃₀H₄₈O₆ as evidenced by the positive HRESIMS at m/z 527.3318 [M + Na]⁺ (calcd 527.3343). The ¹H NMR spectrum of 3 displayed 6 singlet methyls [δ_H 1.03 (3H, s, H-13), 1.04 (3H, s, H-13′), 1.05 (3H, s, H-12′), 1.06 (3H, s, H-12), 1.13 (3H, s, H-14), 1.14 (3H, s, H-14′)] and 2 typical acetal or hemiacetal protons [δ_H 5.12 (s, H-15), δ_H 5.40 (s, H-15′)]. In the ¹³C NMR spectrum 30 carbon signals were observed, which were sorted into 6 methyls, 8 methylenes, 10 methines, and 6 quaternary carbons due to the DEPT spectrum. (Table S3, ESI†) Among them, two quaternary carbon signals at upfield [δ_C 20.5 (qC, C-11), δ_H 20.7 (qC, C-11′)], together with four methyl singlets [δ_C 16.3 (CH₃, C-13), δ_C 16.4 (CH₃, C-13′), δ_C 28.4 (CH₃, C-12′), δ_C 28.7 (CH₃, C-12)] suggested the presence of two dimethylcyclopropane fragments. In addition, two methines at δ_C 98.2 (CH, C-15) and δ_C 103.0 (CH, C-15′) indicated the existence of a hemiacetal and an acetal group, respectively.

Compound 3 appeared to be a dimeric sesquiterpenoid because its characteristic pair of signals with close chemical shifts in the ¹H and ¹³C NMR spectra was reminiscent of the presence of two sesquiterpenoid moieties. By further analysis of 2D NMR spectra of 3, two aromadendrane-type sesquiterpenoid parts were deduced according to the two spin systems (H-2/H-1/H-10/H-4/H-5/H-6/H-7/H-8; H-2′/H-1′/H-10′/H-4′/H-5′/H-6′/H-7′/H-8′) combined with the HMBC correlations (H-14/C-8, C-9, C-10; H-14′/C-8′, C-9′, C-10′; H-15/C-2, C-3, C-4; H-15′/C-2′, C-3′, C-4′; H-13/C-5, C-11, C-12; H-13′/C-5′, C-11′, C-12′) (Fig. 5). These two sesquiterpenoid units should be connected via a five-membered ring possessing an acetal and a hemiacetal functionalities, which was inferred by the key HMBC correlations from H-15 to C-15′ and C-3′, and H-15′ to C-15, combined with the molecular formula and one residual degree of unsaturation.

Fig. 5 Key ¹H–¹H COSY (), HMBC (), and NOESY () correlations of 3.

In the NOESY spectrum of 3, Me-14 was determined to be β-oriented by the key correlation of H-4/H-14, and the α-orientations of H-5, H-6, and H-10 by the correlations of H-5/H-6 and H-6/H-10 (Fig. 5). The same NOE correlations could be found in the other sesquiterpenoid moiety (H-4′/H-14′, H-5′/H-6′, H-6′/H-10′).

However, the configuration of 3 could not be clarified merely on the basis of NOESY spectrum because the chirality of four carbons (C-3, C-3′, C-15 and C-15′) could not be determined in this experiment. Therefore, further evidence, such as X-ray diffraction, was necessary. Fortunately, orthorhombic crystals of 3 were obtained in 70% MeOH–H₂O and the X-ray crystallographic analysis was realized (Fig. 6). Thus, the absolute configuration of 3 was established unambiguously to be 3S, 4S, 5R, 6R, 9R, 10R, 15S, 3′S, 4′S, 5′R, 6′R, 9′R, 10′R, and 15′R.

Fig. 6 Single X-ray crystallographic structure of 3.

Compounds 1, 2, and 3 were tested in vitro to investigate their stimulation of angiogenesis in cultured human umbilical vein endothelial cells (HUVECs).⁶ 1 and 2 exhibited moderate tube proliferation effects at 20 μM with viability rate of 146.8% and 140.7%, respectively.

Conclusions

Valeriadimer A (1) is a rare nor-sesquiterpenoid dimer formed by a C–C (4-1′) bond between a bicyclogermacrane nor-sesquiterpene moiety and a maaliane nor-sesquiterpene moiety. We propose as a plausible biogenetic pathway that 1 could be derived from a maaliane type sesquiterpenoid and the bicyclogermacrene type sesquiterpenoid madolin B. Compound 2 should be a key intermediate which then forms 1 via decarboxylative alkylation reaction. Compound 3 possessing a unique five-member ring system connecting two aromadendrane-type sesquiterpenoid units could be formed via the hemiacetal reaction of two aldehyde groups, similar to the first dimer volvalerelactone B from the same plant.^2a

Acknowledgements

The work was supported by program NCET Foundation, NSFC (81230090, 81102335, 81473109), Shanghai Leading Academic Discipline Project (B906), Key laboratory of drug research for special environments, PLA, Shanghai Engineering Research Center for the Preparation of Bioactive Natural Products (10DZ2251300), the Scientific Foundation of Shanghai China (12401900801, 13401900101), National Major Project of China (2011ZX09307-002-03) and the National Key Technology R&D Program of China (2012BAI29B06).

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: The extraction scheme, compound characterization, spectroscopic data, and CIF files of 1 and 3 are included herein. CCDC 928251 and 931555. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c4ra11439f

‡ Zhu-zhen Han and Ji Ye contributed equally to this work.

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