Citation

Shuai-Zhen Zhou, Chun-Ping Tang, Chang-Qiang Ke, Sheng Yao, Ge Lin, Yang Ye. Three new dimeric diterpenes from Rhododendron molle[J]. Chin. Chem. Lett., 2017, 28(6): 1205-1209  

Shuai-Zhen Zhou^a,b, Chun-Ping Tang^a,b, Chang-Qiang Ke^a,b, Sheng Yao^a,b, Ge Lin^b,c, Yang Ye^a,b,d    

Species of the Ericaceae family are world-widely utilized for ornamental as well as medicinal use. Some of those species are used as analgesic and insecticide in traditional medicine [1]. Previous research reported that grayanotoxins, a type of diterpenoids exclusive in Ericaceae family, are the bioactive constituents of Ericaceae plants [2, 3].

Rhododendron molle G. Don is one of the most well-known poisonous plants of the Ericaceae family. Distributing widely in China, its flowers have long been recorded as narcotic, anodyne and insecticidal agents in ancient monographs [4]. Phytochemical studies of R. molle revealed the existence of grayanotoxins, the characteristic diterpenoids of the Ericaceae family. Over fifty grayanane diterpenoids [5-9] and four novel modified skeletons [10-13] have been reported from this plant in recent years.

Diterpenoids are a class of natural products that has been wellknown for its highly diverse structures and strong bioactivities, such as antitumor, antiinflammatory and neurotoxicity [14-16]. With the passion of searching for more interesting diterpenoids with diversified structures, a detailed chemical investigation of R. molle was carried out, resulting in the isolation of three unprecedented diterpenoid dimers named birhodomolleins A-C (1-3, Fig. 1). Herein, we present the isolation and structural elucidation of compounds 1-3.

Figure 1

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Fig. 1. Chemical structures of birhodomolleins A–C (1–3).

The flowers of R. molle were air-dried, ground and then extracted with 95% ethanol at room temperature. The extract was partitioned with petroleum ether, CH₂Cl₂, and EtOAc, successively. The CH₂Cl₂ extract was separated with repeated column chromatography to afford compounds 1-3.

Compound 1 was obtained as amorphous powder. The quasimolecular ion peak at m/z 901 [M+HCOOH-H]^- and an isotopic ion at m/z 903 [M+2+HCOOH-H]^- with ca. 30% intensity suggested the existence of a chlorine atom. The HRESIMS of 1 recording the quasi-molecular ion peak at m/z 879.4271 [M+Na]⁺ (calcd. 879.4274), together with its ¹³C NMR data, established a molecular formula of C₄₄H₆₉O₁₄Cl, corresponding to ten degrees of unsaturation. The IR absorption band at 3442 cm^-1 indicated the presence of hydroxyl groups.

The ¹H NMR data of 1 (Table 1) displayed signals for eight singlet methyls (δ_H 0.98, 1.16, 1.16, 1.22, 1.28, 1.29, 1.41, 1.41), two acetyl methyls (δ_H 2.07, 2.11) and eight oxygenated or chlorinated methines (δ_H 3.72, 3.91, 4.18, 4.30, 4.49, 4.53, 5.25, 5.33). Apart from four carbons ascribed to two acetyl groups (δ_C 21.5, 21.7, 172.1, 172.8), 40 carbon resonances were observed in the ¹³C NMR (DEPT) data (Table 1), including eight methyls, eight methylenes, fourteen methines (seven oxygenated at δ_C 78.3, 78.8, 79.2, 85.6, 88.9, 89.1, 89.7) and ten quaternary carbons (six oxygenated at δ_C 77.9, 79.6, 80.5, 81.0, 84.7, 85.8). In comparison to a known grayanane diterpene rhodomollein XI³, which contained four methyls, four methylenes, seven methines (four oxygenated) and five quaternary carbons (three oxygenated), the above data suggested the presence of two grayanane diterpenoid moieties in the molecule.

Table 1

Table 1 1H and 13C NMR data for compounds 1–3, measured at 500 MHz (1H) and 125 MHz (13C) in methanol-d4.

Table 1 1H and 13C NMR data for compounds 1–3, measured at 500 MHz (1H) and 125 MHz (13C) in methanol-d4.

The ¹H-¹H COSY correlations of 1 (Fig. 2) revealed the presence of the following spin systems: Two --CH--CH--CH(OH)--, two --CH(OAc)--CH₂-, and two --CH--CH₂--CH₂--CH--, which were further identified as two sets of characteristic fragments of --C(1) H--C(2) H--C(3) H-, --C(6) H--C(7) H₂-and --C(9) H--C(11) H₂--C (12) H₂--C(13) H--in a common grayanane skeleton. These structural features supported the speculation of two grayanane moieties. These two moieties could be distinguished through the HMBC experiment (Fig. 2). Correlations from H-2 to C-4 and C-10, from H-3 to C-5, from H-6 to C-1 and the carbonyl carbon of one acetyl group (δ_C 172.8), from H-14 to C-9, C-12, C-15 and C-16, from H₃-18(19) to C-3, from H-1 to C-20, from H₃-20 to C-9, and from H₃-17 to C-13 identified moiety A as a grayanane fragment, as drawn. Correlations from H-2' to C-4' and C-10', from H-3' to C-5', from H-6' to C-1' and the other carbonyl carbon of one acetyl group (δ_C 172.1), from H-14' to C-9', C-12', C-15' and C-16', from H₃-18'(19') to C-3', from H-1' to C-20', from H₃-20' to C-9', and from H₃-17' to C-13' constructed the other fragment, moiety B, similar with moiety A. Thus, both moieties were elucidated. The two proposed structural segments contributed to 10° of unsaturation, which suggested that the linkage between two moieties should be a single bond.

Figure 2

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Fig. 2. Selected COSY (—), and HMBC correlations (H→C) of 1.

Further analyses revealed that the spectroscopic data of the respective moiety (Table 1) closely resembled those of rhodomollein XI³ except for three obvious differences. The chemical shift of C-2 of moiety A was observed at a higher field (δ_C 65.6, CH) compared with that of rhodomollein XI (δ_C 80.7, CH), suggesting C (2) H could be a chlorinated methine rather than an oxygenated one. In addition, C-14 of moiety A (δ_C 88.9, CH) and C-2' of moiety B (δ_C 89.1, CH) were found to resonate at lower field compared with those of rhodomollein XI (δ_C 80.7 and 78.9, respectively), revealing that the oxygenated C-14 and C-2' might be the connecting positions of the two moieties. HMBC correlations from H-14 to C-2' and from H-2' to C-14 further confirmed the existence of a C--O--C linkage between two moieties. Further HSQC, HMBC, and ROESY studies enabled full assignments of the ¹H NMR and ¹³C NMR spectra of 1 (see Fig. S4-S6 in Supporting information).

The relative configuration of 1 was inferred from the ROESY experiment. The cross-peaks of H-2/H₃-20, H-2'/H₃-20', H-6/H-14, H-1/H-14, H-6'/H-14' and H-1'/H-14' suggested the same configuration of two respective moiety as rhodomollein XI (Fig. 3). Accordingly, the structure of 1 was established as α-chlorine-14'β-hydroxy-14β, 2'α-oxo-di-(6β-acetoxyl-3β, 5β, 10α, 16α-tetrahydroxygrayanane) and named birhodomollein A (Fig. 1). Birhodomollein A represents the first dimeric grayanane type diterpenoid with a rare chloro-substitution.

Figure 3

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Fig. 3. Key ROESY correlations (H↔H) of 1.

Compound 2 was obtained as amorphous powder. The HRESIMS, combined with the ¹³C NMR spectrum of 2, gave a molecular formula of C₄₄H₇₀O₁₅ with 10° of unsaturation on the basis of the quasi-molecular ion at m/z 837.4644 [M-H]^- (calcd. 837.4636). The IR data indicated the presence of hydroxyl groups (3431 cm^-1).

Detailed analysis of 1D and 2D NMR data (Table 1) revealed a high similarity between the structures of 1 and 2 except for the difference observed for C-2. Compared with 1, the carbon resonance of C-2 low-field shifted to δ_C 80.3 from δ_C 65.6, which, combined with its molecular formula, suggested that an oxygen atom instead of a chlorine atom was substituted at C-2. The relative configuration of 2 was deduced to be the same as that of 1 due to the highly resembled NMR data. The configuration was further proven by the ROESY cross-peaks of H-2/H₃-20, H-2'/H₃-20', H-6/ H-1, H-1/H-14, H-6'/H-1' and H-1'/H-14' (see Fig. S13 in Supporting information). Thus, the structure of 2 was fully established as 2α, 14'β-dihydroxy-14β, 2'α-oxo-di-(6β-acetoxyl-3β, 5β, 10α, 16α-tetrahydroxygrayanane) and named birhodomollein B (Fig. 1).

The molecular formula of compound 3 was established as C₄₄H₆₈O₁₄ according to the quasi-molecular ion at m/z 843.4539 [M+Na]⁺ (cal. 843.4507) in the HRESIMS, indicating 11° of unsaturation. Compared with 2, the molecular formula of 3 was one more degree of unsaturation and a H₂O unit less.

A detailed analysis of the ¹H and ¹³C NMR data of 3 (Table 1) revealed a high similarity between the structures of 2 and 3, suggesting again a dimeric diterpene comprising two grayanane moieties. The major differences were observed for C-1, C-2 and C-3, which resonated at higher filed for 3 (δ_C 54.5, 60.4, 65.5) compared with those for 2 (δ_C 59.7, 80.3, 86.6). These data were very similar to those of rhodojaponin Ⅱ [17], suggesting that 3 could also contain a 2, 3-epoxy fragment. Similarly, the relative configuration of 3 was deduced by ROESY correlations of H-6/H-1, H-1/H-14, H-2'/H₃-20', H-6'/H-1'and H-1'/H-14' (see Fig. S20 in the Supporting information). Accordingly, the structure of 3 was established as 2β, 3β-epoxy-3'β, 14'β-dihydroxy-14β, 2'α-oxo-di-(6β-acetoxyl-5β, 10α, 16α-trihydroxygrayanane) and named birhodomollein C (Fig. 1).

Bihodomolleins A-C represent a novel class of compounds dimerized from two grayanane type units through an oxygen bridge. They are the first examples of dimeric diterpenes from the Ericaceae family although diversified monomeric diterpenoids have been reported from the ericaceous plants. Bihodomollein A features a chlorine-substitution which is rarely discovered from plants. These findings expand our knowledge of the chemical diversity in natural products.

The optical rotations were obtained on a Perkin-Elmer 341 polarimeter. IR spectra were recorded with a Nicolet Magna FTIR-750 spectrometer. Analytical HPLC and ESIMS spectra were performed on a Waters 2695 instrument with a 2998 PAD coupled with a Waters Acquity ELSD and a Waters 3100 SQDMS detector. All HPLC analyses were carried out on a Waters Sunfire^® RP C-18 column, 3.5 μm, 4.6 mm × 100 mm eluted with a gradient of CH₃CN-H₂O (5% to 95%) with 0.1% CH₃COOH. HRESIMS spectra were recorded on a Waters Q-Tof Ultima mass detector. ¹H, ¹³C, and 2D NMR spectra were recorded using an AVANCE Ⅲ 500 instrument. Chemical shifts are reported in ppm (δ) with solvent (methanol-d₄) signals used as internal standards and coupling constants (J) in hertz. Silica gel for flash chromatography was produced by Qingdao Marine Chemical Industrials. MCI gel CHP20P (75-150 μm, Mitsubishi Chemical Industries, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for column chromatography (CC). TLC was carried out on precoated silica gel 60 F₂₅₄ aluminium sheets (Merck, Germany) and the TLC spots were viewed at 254 nm and visualized by 5% sulfuric acid in alcohol containing 10 mg/mL vanillin.

The flowers of R. molle were collected in Jinxiu county, Guangxi province of China in 2010, and identified by Associate Professor JinGui Shen from Shanghai Institute of Materia Medica. A voucher specimen (No. 20100811) was deposited at the herbarium of Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

The air-dried flowers of R. molle (5 kg) were ground into powder and extracted with 95% ethanol (3 × 25 L) at room temperature for three times (72 h each). Evaporation of the combined percolates under reduced pressure yielded a dark brown crude extract (400 g). The extract was then suspended in water and partitioned with petroleum ether, CH₂Cl₂, and EtOAc, successively, yielding the fractions of PE (90 g), CH₂Cl₂ (90 g) and EtOAc (30 g). The CH₂Cl₂ extract was applied to an MCI gel column (eluted with MeOH in water 50%, 60%, 70%, 80% and 95%, successively) to afford 9 fractions (CA-CI) on the basis of TLC analysis. Fraction CD was subjected to a Sephadex LH-20 column (eluted with MeOH) to give subfractions CD1-CD3. CD1 was chromatographed on a silica gel column (300-400 mesh) eluted with petroleum ether-acetone 1:2 to afford 1 (13 mg) and 3 (9 mg). Fraction CF was subjected to a Sephadex LH-20 column (eluted with MeOH) to give subfractions CF1-CF4. CF1 was separated by a silica gel column (200-300 mesh, eluted with petroleum ether-acetone 3:1) to afford subfraction CF1a. Subfractoin CF1a was further purified by Sephadex LH-20 to yield 2 (13 mg).

Birhodomollein A (1): Amorphous powder, [α]_D²⁰ -21.8 (c 0.06, MeOH). IR (KBr) v_max 3442, 2922, 1714, 1631, 1261, 1099, 1043 cm^-1; ¹H NMR and ¹³C NMR data (in methanol-d₄), see Table 1; ESIMS m/z 901 [M+HCOOH-H]^-, HR-ESIMS m/z 879.4271 [M+Na]⁺ (calcd. for C₄₄H₆₉O₁₄ClNa 879.4274).

Birhodomollein B (2): Amorphous powder, [α]_D²⁰ -18.0 (c 0.05, MeOH). IR (KBr) v_max 3431, 2927, 1714, 1647, 1261, 1101, 1045 cm^-1; ¹H NMR and ¹³C NMR data (in methanol-d₄), see Table 1; ESIMS m/z 883 [M+HCOOH-H]^-, HR-ESIMS m/z 837.4644 [M-H]^- (calcd. for C₄₄H₆₉O₁₅ 837.4636).

Birhodomollein C (3): Amorphous powder, [α]_D²⁰ -20.4 (c 0.05, MeOH). IR (KBr) v_max 3446, 2922, 1712, 1623, 1376, 1263, 1043 cm^-1; ¹H NMR and ¹³C NMR data (in methanol-d₄), see Table 1; ESIMS m/z 865 [M+HCOOH-H]^-, HR-ESIMS m/z 843.4539 [M+Na]⁺ (calcd. for C₄₄H₆₈O₁₄Na 843.4507).

We are thankful to the financial support of the National Science & Technology Major Project "Key New Drug Creation and Manufacturing Program" (Nos. 2012ZX09301001-001, 2015ZX09103002). Our thanks are also given to the National Natural Science Fundation of China (Nos. 81302657, 81473112, 81573305).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2017.02.020.