Qi Luoab,
Lei Dia,
Xiao-Hua Yangac and
Yong-Xian Cheng*a
aState Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, People's Republic of China. E-mail: yxcheng@mail.kib.ac.cn; Fax: +86-871-65223048; Tel: +86-871-65223048
bUniversity of Chinese Academy of Sciences, Yuquan Road 19, Beijing 100049, People's Republic of China
cGuangdong Pharmaceutical University, Guangzhou 510006, People's Republic of China
First published on 26th April 2016
Applanatumols A (1) and B [(±)-2], two unique meroterpenoids with a novel spiro[benzofuran-2,2′-bicyclo[3.2.2]nonane] ring system and a naturally unusual dioxacyclopenta[cd]inden motif, respectively, were isolated from Ganoderma applanatum. Their structures were determined using spectroscopic data. In particular, the absolute configurations of 1 and 2 were assigned by X-ray diffraction analysis using the Flack parameter and ECD calculations, respectively. A plausible biosynthetic pathway for 1 and 2 is proposed. The biological activities of 1 and 2 against renal fibrosis were accessed in rat proximal tubular epithelial cells.
In recent years, the search for renoprotective substances from natural sources has become our research focus. Inspired by the traditional uses of Chinese medicine, we have conducted an extensive investigation of Ganoderma fungi, which led to the isolation of several novel meroterpenoids with profound kidney protection activities. For examples, lingzhiol was identified as a potent Smad3 phosphorylation inhibitor and Nrf2 activator from G. lucidum,3 and lingzhilactone B from G. lingzhi was found to be able to ameliorate adriamycin-induced nephropathy in mice via scavenging reactive oxygen species and activating Nrf2.4 These findings not only add new facets to Ganoderma chemistry but also prompt us to further explore the structural diversity of meroterpenoids in this genus and their biological potential to fight kidney diseases. G. applanatum is a medicinal mushroom and has been used to treat various chronic diseases in Chinese folk medicine. We have characterized applanatumin A from this species, which exhibits potent antifibrotic activity.5 In our follow-up study on this mushroom, two structurally novel compounds applanatumols A (1) and B (2) were isolated and their biological activities towards renal fibrosis were evaluated. Interestingly, 1 is a spiro compound with a novel 6/5/7/6 ring system, and 2 bears an unusual dioxacyclopenta[cd]inden motif making it look like a naturally hemisphere-shaped structure. A plausible biosynthetic pathway further confirmed the structural rationality of 1 and 2 (Fig. 1).
Applanatumol A (1) was obtained as an optically active yellow gum. Its molecular formula, C16H16O6, was determined by means of HRESIMS, 13C NMR, and DEPT spectra, having 9 degrees of unsaturation. The 1H NMR spectrum (Table 1) of 1 contains an ABX spin system [δH 6.98 (1H, d, J = 2.7 Hz, H-3), 7.29 (1H, dd, J = 8.9, 2.7 Hz, H-5), 7.06 (1H, d, J = 8.9 Hz, H-6)], suggesting the presence of a 1,2,4-trisubstituted benzene ring. The 13C NMR and DEPT spectra show 16 carbons ascribed to 4 methylene (one oxygenated), 6 methine (three sp2 and three sp3), and 6 quaternary carbons (one ketone, one carbonyl, three olefinic, including two oxygenated, and one aliphatic). The 1H-1H COSY spectrum (Fig. 2) of 1 shows the presence of H-5/H-6, H-3′/H-4′/H-5′/H-6′/H-7′/H-8′ and H-7′/H-9′ fragments. The planar structure of 1 was determined mainly through 2D NMR experiments. The HMBC correlations of H-3, H-3′, H-8′/C-1′ (δC 202.5), H-3′, H-4′, H-8′/C-2′ (δC 87.6), and H-8′/C-3′ (δC 44.9) and in consideration of their downfield shifts indicate that C-1 and C-2′ are linked via an oxygen bridge. Moreover, this suggests that rings B and C (Fig. 2) are present as part of the novel spiro[benzofuran-2,2′-bicyclo[3.2.2]nonane] ring system. In addition, except for rings A-C, one ketone, and one carbonyl, the remaining one degree of unsaturation is attributed to an additional ring in 1. HMBC correlations of H-3′, H-4′, H-6′ (δH 4.79)/C-10′ (δC 170.3) suggest that C-10′ and C-6′ are linked by formation of an ester bond (ring D). These findings also indicate the presence of two isopentyl moieties (Fig. 2, pink line and green line).
1 | 2 | ||||
---|---|---|---|---|---|
No. | δH | δC | No. | δH | δC |
a In acetone-d6.b In DMSO-d6 (600 MHz). | |||||
1 | 165.7 | 1 | 148.5 | ||
2 | 119.9 | 2 | 129.7 | ||
3 | 6.98 (d, 2.7) | 108.3 | 3 | 6.87 (d, 2.8) | 114.0 |
4 | 153.7 | 4 | 150.8 | ||
5 | 7.29 (dd, 8.9, 2.7) | 128.4 | 5 | 6.64 (d, 8.6, 2.8) | 117.6 |
6 | 7.06 (d, 8.9) | 115.2 | 6 | 6.67 (d, 8.6) | 118.5 |
1′ | 202.5 | 1′ | 108.9 | ||
2′ | 87.6 | 2′ | 3.14 (dd, 10.4, 8.6) | 41.9 | |
3′ | 2.76 (dd, 6.0, 2.6) | 44.9 | 3′ | 3.28 (t-like, 8.1) | 50.6 |
4′a | 2.56 (m) | 17.9 | 4′ | 181.3 | |
4′b | 1.92 (m) | 5′a | 2.18 (m) | 29.9 | |
5′ | 2.15 (m) | 20.1 | 5′b | 1.73 (m) | |
6′ | 4.79 (t-like, 5.9) | 78.0 | 6′a | 1.90 (m) | 32.5 |
7′ | 2.45 (m) | 43.9 | 6′b | 1.51 (m) | |
8′a | 2.07 (overlap) | 30.1 | 7′ | 2.28 (m) | 37.9 |
8′b | 1.66 (dd, 15.0, 4.1) | 8′ | 1.69 (m) | 38.5 | |
9′a | 3.58 (dd, 11.0, 5.2) | 64.0 | 9′a | 3.98 (dd, 12.1, 2.4) | 59.0 |
9′b | 3.48 (dd, 11.0, 8.0) | 9′b | 3.91 (dd, 12.1, 3.5) | ||
10′ | 170.3 | 10′a | 4.02 (dd, 10.7, 3.9) | 64.1 | |
4-OHa | 8.67 (s) | 10′b | 3.77 (dd, 10.7, 2.9) | ||
9′-OHa | 4.04 (s) | 1-OHb | 9.17 (s) | ||
4-OHb | 8.80 (s) | ||||
10′-OHb | 4.68 (s) |
There are four stereogenic centers in 1 and the conformationally immobile bicyclo[3.2.2]nonane ring system in 1 requires H-3′ and H-6′ to be at the same orientation. ROESY correlations of Hb-4′ (δH 1.92)/Ha-8′, Ha-8′/H-9′, H-9′/H-5′ (Fig. 3) show that all these protons are spatially adjacent, which naturally assign the relative configuration at C-7′. This conclusion was further confirmed by the observed ROESY correlation of H-6′/H-7′. Unfortunately, it is impossible to assign the relative configuration at the spiro center using ROESY observations. Finally, the absolute configuration of 1 was unambiguously assigned as 2′S,3′R,6′S,7′R by utilizing single-crystal X-ray diffraction data and anomalous scattering of CuKα radiation. As shown in Fig. 4, the formation of rings A–D made the structure of 1 cage-like.
(±)-Applanatumol B (2), obtained as a yellow gum, has a molecular formula C16H18O6 (8 degrees of unsaturation), based on its HREIMS, 13C NMR and DEPT spectra. The 13C NMR and DEPT spectra show that this substance contains 16 carbons, including four methylene (two oxygenated), seven methine (three olefinic and four aliphatic), and five quaternary carbons (one carbonyl, three olefinic including two oxygenated, and one oxygenated aliphatic). The 1H NMR spectrum (Table 1) of 2 contains a typical ABX spin system [δH 6.87 (1H, d, J = 2.8 Hz, H-3), 6.64 (1H, dd, J = 8.6, 2.8 Hz, H-5), 6.67 (1H, d, J = 8.6 Hz, H-6)]. The architecture of 2 was constructed mainly based on the 1H-1H COSY and HMBC spectra (Fig. 5). The 1H-1H COSY spectrum shows correlations of H-5/H-6, H-2′/H-3′/H-5′/H-6′/H-7′/H-8′/H-9′, H-2′/H-7′, and H-8′/H-10′. Moreover, the HMBC spectrum presents correlations between H-3′, H-7′, H-9′/C-1′, H-2′, H-3′, H-5′/C-4′. Considering the chemical shift of C-1′ (δC 108.9) and C-4′ (δC 181.3) and the degree of unsaturation of 2, the data indicates the existence of a naturally unusual dioxacyclopenta[cd]inden motif comprising rings A–C, which makes the architecture of 2 hemisphere-shaped. In addition, HMBC correlations of H-3, H-6/C-1′, H-2′/C-2 (δC 129.7) suggest that one phenyl unit (ring D) in 2 is linked to C-1′ through C-2. With this, the planar structure of 2 was deduced as shown.
Fig. 5 Key COSY and HMBC correlations of 2; pink and green in 2 represent two independent isopentyl moieties. |
The relative configuration of 2 was assigned by ROESY correlations (Fig. 6). In the ROESY spectrum, correlations between Hb-9′/Ha-10′, Hb-10′, Ha-9′/Hb-6′, and H-7′/Hb-10′ were observed, indicating the relative configurations at C-7′ and C-8′. Due to the ring tension, the formation of rings A and B naturally requires H-2′ and H-7′ to be at the same orientation. This was further confirmed by ROESY correlations of H-2′/H-7′ and H-2′/Ha-10′ (in DMSO-d6). In addition, ROESY correlation of H-2′/H-3′ along with weak cross peak between H-3′ and H-7′ suggests that these protons are spatially adjacent, allowing the stereochemistry at C-3′ to be assigned. As far as the relative configuration at C-1′, we measured ROESY correlations in DMSO-d6, in which interactions between 1-OH/H-2′, Ha-10′, H-3′ (weak), Hb-10′ (weak) were observed, and clearly indicate the orientation of the phenyl group. With this, the relative configuration of 2 was assigned. It can be noted that compound 2 was isolated as a racemic mixture and further purification by chiral-phase HPLC yielded (+)-2 and (−)-2 in a ratio of 1.2:1. Computational methods were relied on to determine the absolute configuration of (+)-2 (ESI†). As shown in Fig. 7, the calculated ECD spectrum of (+)-2 agrees well with those of the experimental one, suggesting 1′S,2′S,3′R,7′R,8′S-configuration for (+)-2.
Fig. 7 Comparison of calculated ECD spectrum for (1′S,2′S,3′R,7′R,8′S) with the experimental spectra of (+)-2 and (−)-2 in MeOH. σ = 0.27 eV; shift = +15 nm. |
Meroterpenoids in the genus Ganoderma feature a hydroquinone and a terpenoidal side chain. To date, several structurally diverse meroterpenoids have been characterized by us,4,6,7 providing structure targets for synthetic chemists. Lingzhiol from G. lucidum is such an example that has been successfully synthesized by Yang and co authors,8 demonstrating that Ganoderma could be a potential source for searching molecules of interest. The present finding of exceptional structures of 1 and 2 makes a convincing footnote for our abovementioned hypothesis. In general, the ketone group at the benzylic location is rather stable. However, the ketalation reaction occurred in 2 and the formation of C-2′-C-7′ led to the construction of rings A–C, representing, to the best of our knowledge, the first example of a new carbon skeleton meroterpenoid with a rare dioxacyclopenta[cd]inden group. Moreover, that rings A–C of 2 sharing a common carbon (C-2′) makes it a hemisphere-like structure, which might be a challenge for synthetic chemists to finish the total synthesis of 2.
One more thing that needs to be mentioned is that 2 was isolated as a racemic mixture. Similar phenomena have also been found in the other meroterpenoids from Ganoderma.4,6,7 We have tried to check whether the previously isolated racemic compounds are artificial products, but our efforts gave negative feedback for this question.7 The reasons for the racemic nature of enzyme-catalyzed natural products in nature remain unknown to date. One possibility is that these racemic meroterpenoids might only act as intermediates for fungi to synthesize their end products. However, this needs further exploration.
Analyses of the structures of 1 and 2 indicate that they are derived from the combination of the shikimic acid pathway and the mevalonic acid pathway (Scheme 1).9 First, chizhine D,10 a precursor, which has been isolated from G. lucidum, is produced from geranyl diphosphate and 4-hydroxybenzonic acid under geranyltransferase.9 Two intermediates, A and F, are generated from chizhine D by oxidation reaction. The monomer A could be cyclized via nucleophilic addition to generate B, which could be transformed to C by further oxidation. An interesting feature in the biosynthesis of 1 is the formation of a spiro structure by a stereospecific phenol radical coupling.5 Second, esterification and reduction of monomer D would yield 1. Finally, monomer G can be cyclized by conjugated addition of F and ring A in 2 is formed via esterification (Scheme 1).
Footnote |
† Electronic supplementary information (ESI) available: 1D, 2D NMR, and MS spectra, crystallographic data, MTT assay. CCDC 1439639. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra05148k |
This journal is © The Royal Society of Chemistry 2016 |