Dongtingnoids A–G: Fusicoccane Diterpenoids from a Penicillium

Article Cite This: J. Nat. Prod. 2019, 82, 80−86

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Dongtingnoids A−G: Fusicoccane Diterpenoids from a Penicillium Species Qiong Bie,†,§ Chunmei Chen,†,§ Muyuan Yu,† Jieru Guo,‡ Jianping Wang,† Junjun Liu,† Yuan Zhou,† Hucheng Zhu,*,† and Yonghui Zhang*,† †

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Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China ‡ Department of Pharmacy, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China S Supporting Information *

ABSTRACT: Five new diterpenoid glycosides, dongtingnoids A−E (1−5), two new diterpenoid aglycones, dongtingnoids F and G (6 and 7), and two known analogues, cotylenins E and J (8 and 9), belonging to the fusicoccane family, were isolated from the fungus Penicillium sp. DT10, which was derived from wetland soil from Dongting Lake. Their structures and absolute configurations were elucidated based on spectroscopic analyses, acid hydrolysis, ECD calculations, and X-ray crystallography. Dongtingnoid C (3) is the first 16-nor-fusicoccane diterpenoid glycoside reported and is proposed to form by oxidative demethylation. Compounds 1, 4, and 5 showed comparable seed-germination-promoting activities to that previously reported for the growth regulator cotylenin E (8).

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RESULTS AND DISCUSSION Dongtingnoid A (1) was obtained as colorless crystals. The molecular formula of 1 was determined to be C29H48O9 by HRESIMS, requiring an index of hydrogen deficiency of six. The 1 H NMR data of 1 (Table 1) along with its HSQC spectrum displayed characteristic resonances for four methyl groups [δH 0.80 (3H, d, J = 7.0 Hz), 0.97 (3H, d, J = 6.8 Hz), 1.07 (3H, d, J = 6.5 Hz), and 1.23 (3H, s)], three methoxyl groups [δH 3.07 (3H, s) and 3.37 (6H, s)], and an olefinic proton signal at δH 5.35 (1H, br s). In addition, the characteristic signal of an anomeric proton at δH 4.98 (1H, d, J = 3.3 Hz), along with six other protons ranging from δH 3.47 to 3.79, suggested the presence of a sugar residue. The 13C NMR (Table 2) and DEPT data revealed the presence of 29 carbons, including seven methyls/ methoxyls, six methylenes (two oxygenated), 10 methines (seven oxygenated ones), two sp3 nonprotonated carbons (C-3 and C-11), and four olefinic carbons. Among these carbons, seven including one methoxyl are associated with the sugar moiety. The three indices of hydrogen deficiency are attributed to the two double bonds and the sugar, and the remaining indices of hydrogen deficiency required three additional rings. Comparison of the NMR data of 1 with those of cotylenin E (8)12 revealed that compound 1 may be a fusicoccane glycoside possessing the same 5−8−5 carbon skeleton as seen in 8.

usicoccanes, terpenoids characterized by 5−8−5 or 5−9−5 fused carbocyclic ring systems, include the diterpenoid families fusicoccins, cotylenins, and brassicicenes and the sesterterpenoid families ophiobolins and ceroplastols.1 Fusicoccin A and cotylenin A, two representative compounds of the fusicoccane family, exhibit significant phytohormone-like activities resulting from their interactions with plant 14−3−3 regulatory proteins,2,3 which are known to interact with hundreds of protein partners in eukaryotes.4 In addition, cotylenin A shows differentiation-inducing and antitumor activities against human acute myeloid leukemia in both cell and mouse models5,6 and proliferation-inhibiting, apoptosisinducing, and PAX6 mRNA transcription activities in retinoblastoma cell lines.7 Cotylenin E, an analogue of cotylenin A, was found to be a stimulator of germination of Monochoria vaginalis seeds and tubers of Sagittaria trifolia.8,9 In our previous study on fusicoccanes, structures of the brassicicene subclass were revised from 5−8−5 fused carbocyclic ring system to 5−9−5 systems containing an unusual bridgehead double bond.10 In our continuing search for bioactive fusicoccanes from fungi, five new diterpenoid glycosides, dongtingnoids A−E (1−5), two new diterpenoid aglycones, dongtingnoids F and G (6 and 7), and two known analogues, cotylenins E and J (8 and 9),11 were isolated from an extract of Penicillium sp. DT10, a fungus derived from wetland soil of Dongting Lake. Herein, we report the isolation, structural elucidation, and bioactive evaluation of these metabolites. © 2019 American Chemical Society and American Society of Pharmacognosy

Received: August 16, 2018 Published: January 11, 2019 80

DOI: 10.1021/acs.jnatprod.8b00694 J. Nat. Prod. 2019, 82, 80−86

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Table 1. 1H NMR Data of Compounds 1−7 in CDCl3 (J in Hz) 1a

no.

2a

3b

4a

5a

6a

7a 2.60 d (12.2); 2.31 d (12.2) 2.80 m 3.76 d (3.0)

1

5.35 br s

5.18 br s

4.1 d (8.9)

3.92 s

5.34 s

2.44 br d (16.6); 2.36 d (16.6)

3 4 5

1.67 m; 1.79 m 1.28 m; 1.94 m

1.48 m; 1.90 m 1.55 m; 1.87 m

2.45 m 2.58 d (17.6); 2.76 d (17.6)

2.25 m 1.89 m2.17 m

4.22 s

6 7 8 9

2.77 m 1.91 m 3.96 dd (9.9, 3.3) 3.88 d (9.9)

2.79 m 2.01 m 4.00 dd (10.0, 4.0) 3.92 d (10.0)

2.82 m 4.09 d (6.1) 3.31 d (8.4)

2.67 m 1.88 m 3.90 br d (9.5) 3.98 d (9.5)

5.86 s 1.98 d (17.1)2.83 dd (17.1, 7.9) 3.08 br d (8.0) 1.94 m 4.03 m 3.86 d (10.0)

12

1.69 m; 1.82 m

1.70 m

1.41 m; 2.55 m

1.72 m; 2.04 m

1.68 m; 1.76 m

13 15 16

2.10 m 2.26 m 3.21 m 3.14 m 3.10 d (10.1); 3.54 d (10.3) 0.85 d (7.0) 1.25 d (4.6)

2.23 m 3.10 m 4.40 d (11.5); 3.86 d (11.5) 1.07 d (7.0)

2.20 t (7.0) 3.20 m 4.02 overlapped

17

2.10 m 3.21 m 3.14 d (10.5); 3.28 d (10.5) 0.80 d (7.0)

18 19 20

1.23 s 0.97 d (6.8) 1.07 d (6.5)

1.28 s 0.96 d (6.7) 1.07 d (6.4)

1.02 s 0.97 d (5.2) 1.06 d (5.8)

1.11 s 0.97 d (6.4) 0.91 d (7.4)

1.17 s 1.00 d (6.9) 1.02 d (6.7)

1′ 2′ 3′ 4′ 5′ 6′

4.98 d (3.3) 3.58 dd (9.2, 3.3) 3.79 m 3.52 m 3.77 m 3.47 overlapped; 3.66 dd (10.2, 3.3)

5.02 d (3.6) 3.62 dd (10.9, 3.5) 3.82 m 3.55 m 3.79 m 3.48 overlapped; 3.64 dd (10.5, 3.3)

4.90 br s 3.57 br d (8.0) 3.77 m 3.53 m 3.73 m 3.44 dd (6.7, 4.0); 3.62 d (6.7)

4.90 br s 3.46 br d (9.6) 3.66 m 3.43 m 3.65 m 3.45 overlapped; 3.61 overlapped

4.95 d (3.5) 3.57 m 3.77 t (8.8) 3.55 m 3.70 m 3.43 dd (10.9, 2.8); 3.67 d (10.9)

3.34 s

3.35 s

3.33 s

3.36 s

33.07 s OMe 163.37 s OMe 6′3.37 s OMe 1-OH a

0.77 d (6.9)

3.47 m 2.52 m 1.55 m1.67 m 2.56 m2.15 m 1.42 dd (11.9, 7.5); 1.55 dd (11.9, 7.5) 2.18 m 2.81 m 1.64 s 3.27 dd (10.1, 7.8); 4.11 t (7.8) 1.00 s 0.85 d (6.9) 3.55 br d (7.1)

2.66 m 1.77 m1.91 m 2.25 m1.57 dt (13.5, 6.0) 1.73 m; 1.82 m 2.24 m 2.78 m 1.32 d (7.2) 3.81 dd (10.9, 5.5); 3.88 dd (10.9, 4.9) 1.06 s 0.94 d (6.8) 3.51 dd (10.5, 8.8); 3.58 dd (10.5, 6.2)

3.13 s 3.38 s 3.38 s

3.37 s 5.89 d (8.9)

b

Measured at 400 MHz. Measured at 800 MHz.

Natural products with a 6-O-methylglucose moiety are rare, with only a few examples reported.13,14 Due to the lack of 6-Omethyl-D-glucose and 6-O-methyl-L-glucose standards, the GC and HPLC analyses that are normally used to determine the absolute configuration of sugars are not available, and the absolute configurations of the 6-O-methylglucose moieties of the majority of these natural products were not definitively determined.15,16 A thin crystal of compound 1 was obtained from a methanol−water solvent mixture, and X-ray diffraction analysis was performed with Cu Kα radiation (Figure 2, CCDC 1843253). Thus, the sugar unit of 1 was unambiguously identified as 6-O-methyl-α-D-glucose, and the absolute configuration of the diterpenoid moiety was also assigned. To the best of our knowledge, this is the first time the absolute configuration of a natural product with a 6-O-methylglucose moiety has been determined by X-ray diffraction analysis. Dongtingnoid B (2) has the same molecular formula (C29H48O9) as that of 1 according to their HRESIMS spectra. The 1H and 13C NMR data of 2 (Tables 1 and 2) are very similar to those of 1, except that the resonances of C-2 (ΔδC +0.8), C-4 (ΔδC +6.8), and C-5 (ΔδC +0.7) are shifted downfield, while those of C-3 (ΔδC −2.0) and C-16 (ΔδC −1.9) are shifted upfield. These analyses suggested that the configuration of C-3

Further analysis of the HMBC spectrum of 1 (Figure 1) suggested that the main difference between 1 and 8 was that the hydroxy at C-16 in 8 was replaced by a methoxyl group in 1, which was established by the HMBC correlation from 16-OMe to C-16. In addition, the structure of the sugar residue was determined by analysis of the HMBC spectrum as well as comparing the NMR data of 1 with those of 8. The HMBC correlation from 6′-OMe to C-6′ and the relatively small coupling constant of H-1′ [δH 4.98, d (J = 3.3 Hz)] indicated that the sugar is a 6-O-methyl-α-glucose. Finally, the sugar residue was located at C-9 by the HMBC correlations from H-9 to C-1′ and H-1′ to C-9. The relative configuration of the diterpenoid part of 1 was revealed by a NOESY experiment (Figure 1) as well as by comparing the NMR data of 1 with those of 8. The NOESY correlations of Me-18/H-9, H-9/Me-17, and Me-17/H-16 indicated these groups are cofacial, and these groups were assigned as β-oriented. In addition, the Me-17/H-5 interaction suggested that H-6 should be α-oriented. Moreover, the coupling constant between H-8 and H-9 (J8,9 = 9.9 Hz) and NOESY correlation of H-8/H-6 revealed the α-orientation of H8. Thus, the relative configuration of the diterpenoid fragment was determined. 81

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HRESIMS data. The 1H and 13C NMR data of 4 (Tables 1 and 2) are also similar to those of compound 1. The significant differences between 4 and 1 were the presence of an oxygenated methine (δC 77.2 and δH 3.92) and the absence of a methoxyl and a nonprotonated sp3 carbon in 4. In addition, a tetrasubstituted double bond (δC 137.0 and 143.5) was found instead of a trisubstituted double bond (δC 135.3 and 134.5). The HMBC correlations from H-16 to C-2 and C-3 suggested that the tetrasubstituted double bond was located between C-2 and C-3 and that the 3-OMe group present in 1 was missing. Moreover, the HMBC correlations from H-1 to C-2, C-3, and C11 revealed the presence of a hydroxy group at C-1. Detailed analyses of the 2D NMR (HSQC, HMBC, and NOESY) data confirmed that the other fragments of 4 were the same as those of 1. Finally, the hydroxy group at C-1 was determined to be βoriented based on NOESY correlations of H-1/H-6 and H-1/H7. The HRESIMS data of 5 established a molecular formula of C28H44O8, implying seven degrees of unsaturation. The general features of its NMR data closely resembled those of 4. However, the signals for C-1 and C-4 were replaced by signals for two olefinic methines (δH 5.34 and 5.86; δC 128.8 and 132.3). These observations indicated the dehydration of the hydroxy group at C-1 in 4, leading to the migration of the double bond. The observed 1H−1H COSY cross-peak of H-4/H-5 along with the HMBC correlations from H-1 and H-4 to C-2 and C-3 and from H-16 to C-2, C-3, and C-4 confirmed the above deduction. The comprehensive analysis of the 2D NMR data of 5 established the overall structure. To determine the absolute configurations of the diterpenoid fragments of compounds 3−5, their Boltzmann-weighted ECD spectra were calculated using time-dependent density functional theory (TDDFT) with their sugar-removed structural models. The calculated electronic circular dichroism (ECD) spectra of 3−5 were in good agreement with the experimental ECD curves (Figure S1), revealing the absolute configurations of the diterpenoid fragments of 3−5 as 1R,7R,8R,9R,11S, 1R,6S,7R,8R,9R,11S, and 6S,7R,8R,9R,11R, respectively. To determine the absolute configuration of the 6-O-methyl-αglucose moieties of compounds 2−5, acid hydrolysis and trimethylsilyl-L-cysteine derivatization were performed for 1−5. Finally, GC analyses (Figure S2) revealed that the sugar moieties of 2−5 are identical to that of 1 (tR = 16.16 min) determined to be 6-O-methyl-α-D-glucose by X-ray diffraction analysis as described above. Dongtingnoid F (6) was assigned a molecular formula of C20H30O4 based on its HRESIMS data. The 1H and 13C NMR data of 6 (Tables 1 and 2) revealed that 6 is a diterpenoid similar to the aglycone part of 4. However, some significant differences were observed: hydroxy groups at C-8 and C-9 and the methoxyl at C-16 seen in 4 were missing, and C-4, C-17, and C-19 had been oxidized based on the HMBC correlations from H-4 (δH 4.22) to C-2 and C-5; from H-17 (δH 3.27) to C-6, C-7, and C-8; and from H-20 (δH 3.55) to C-14 and C-15 (Figure 3). Moreover, a furan was formed between C-5 (δC 112.3, a hemiacetal carbon) and C-17 based on the HMBC correlation from H-17 to C-5. The NOESY interaction between H-6 and H7 suggested they were cofacial, and the NOESY correlation between H-6 and H-12 indicated that Me-18 was on the opposite face (Figure 3). The remaining chiral centers, including C-4, C-5, and C-15, could not be determined by the NOESY data because of the absence of key interactions. The relative and absolute configuration of 6 was finally confirmed by single-

Table 2. 13C NMR Data of Compounds 1−7 in CDCl3 no.

1a

2a

3b

4a

5a

6a

7a

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1′ 2′ 3′ 4′ 5′ 6′ 3′OMe 16OMe 6′OMe

135.3 134.5 88.7 26.6 31.8 41.2 41.4 78.4 77.8 135.2 52.1 43.2 27.0 149.7 28.1 76.8 8.4 25.9 21.0 21.5 101.8 72.2 74.2 71.1 70.4 72.3 49.5

134.9 135.3 86.7 33.4 32.5 40.8 42.6 78.3 77.8 135.3 52.4 42.8 26.9 149.7 28.2 74.9 8.7 26.9 20.7 21.6 101.6 72.4 74.2 71.8 70.0 72.6 49.7

74.5 138.0 213.2 35.1 28.5 178.5 36.1 79.4 83.3 151.9 57.0 34.4 26.6 134.8 28.1

77.2 137.0 143.5 34.9 24.8 55.3 33.1 79.7 81.3 134.9 56.5 35.7 27.4 148.9 27.8 71.0 18.4 19.5 21.2 21.6 101.9 70.5 74.3 72.5 70.5 71.7

128.8 141.8 142.7 132.3 42.6 39.3 43.2 77.7 77.0 138.2 52.3 41.5 27.4 147.9 27.9 69.0 6.6 26.8 20.6 21.7 101.3 72.1 74.2 70.9 70.4 72.0

39.2 134.1 136.6 83.3 112.3 57.0 41.8 26.5 23.8 143.0 51.0 35.2 27.7 138.5 35.3 11.5 74.6 26.6 15.4 66.3

39.7 177.9 46.4 79.8 208.3 139.1 37.3 29.7 21.3 142.0 54.3 40.6 27.0 140.9 35.6 16.5 63.9 25.8 15.3 66.4

59.5

59.6

58.6

58.4

59.6

59.7

59.5

59.7

14.1 19.3 21.3 21.7 101.9 72.6 74.3 72.3 69.8 72.8

59.8

a

Measured at 100 MHz. bMeasured at 200 MHz.

of 2 might differ from that of 1. Further analysis of the 2D NMR spectra confirmed the planar structure of 2, and the relative configuration of C-3 was assigned according to the NOESY correlation between Me-17 and 3-OMe. Based on the similarity between the ECD curves of 2 and 1 (Figure S1), compound 2 was determined to be the C-3 epimer of 1. The molecular formula of 3 was assigned as C26H40O9 based on its HRESIMS spectrum, which contains three fewer carbon atoms than 1. Interpretation of the 1H and 13C NMR data of compound 3 (Tables 1 and 2) indicated that the sugar part of this compound is the same as that in 1, but the diterpenoid fragment was substantially different. Further analysis of the 2D NMR spectra revealed that the changes are in rings A and B. An α,β-unsaturated ketone was identified by the HMBC correlations from H-4 and H-5 to C-2, C-3, and C-6, which revealed the absence of C-16 and 3-OMe located at C-3 in 1. In addition, the HMBC correlations from H-1 to C-2, C-3, C-6, and C-11 together with the chemical shift of C-1 suggested that a hydroxy group was located at C-1. The relative configuration of C-1 was established by the NOESY correlation of H-1 and H-7, which suggested the α-orientation of H-1 and β-orientation of the hydroxy group. Thus, the structure of compound 3 was determined, and it represents the first 16-nor-fusicoccane diterpenoid glycoside, which was proposed to form by oxidative demethylation. Dongtingnoid D (4) has a molecular formula of C28H46O9, 14 mass units (CH2) less than that of 1, as deduced from its 82

DOI: 10.1021/acs.jnatprod.8b00694 J. Nat. Prod. 2019, 82, 80−86

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Figure 1. Key 2D NMR correlations of 1.

Figure 2. ORTEP drawing of compound 1.

83

DOI: 10.1021/acs.jnatprod.8b00694 J. Nat. Prod. 2019, 82, 80−86

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Figure 3. Key 2D NMR correlations of 6.

crystal X-ray diffraction analysis with Cu Kα radiation (Figure 4, CCDC 1846054). Figure 5. Germination of Monochoria vaginalis seeds in water and in water solutions of the test compounds (50 ppm) in the dark under nonflooded conditions for 13 days. Water and cotylenin E (8) were used as blank and positive controls, respectively.

showed no cytotoxicity (IC50 > 40 μM) to cell lines HL60, U87MG, MDA-MB-231, A549, HEP-3B, and SW480.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were obtained using an X-5 microscopic melting point apparatus (Beijing Tech, China), and optical rotations were recorded on a PerkinElmer 341 polarimeter (PerkinElmer Inc., Fremont, CA, USA). UV spectra were obtained in MeOH on a Lambda 35 instrument (PerkinElmer Inc.). ECD spectra were measured on a JASCO-810 spectrometer (JASCO, Tokyo, Japan). IR spectra were measured with a Vertex 70 FT-IR spectrophotometer (Bruker, Karlsruhe, Germany). The NMR spectra were measured on a Bruker AM-400 spectrometer or an Ascend 800 M spectrometer, and the chemical shifts were referenced to the signals of the residual CDCl3 peaks (δC 77.16 and δH 7.26). HRESIMS data were obtained with a Bruker micrOTOF II spectrometer (Bruker, Karlsruhe, Germany). Semipreparative HPLC separations were performed on a Dionex HPLC system using a reversed-phase (RP) column (5 μm, 10 × 250 mm. Welch Ultimate XB-C18). Column chromatography (CC) was performed with ODS (50 μm, YMC Co. Ltd., Japan), silica gel (200−300, 100−200, and 80−120 mesh; Qingdao Marine Chemical Inc., China), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden). Thin-layer chromatography (TLC) separations were performed with silica gel 60 F254 (Yantai Chemical Industry Research Institute) and RP-C18 F254 plates (Merck, Germany). Gas chromatography (GC) spectra were recorded on an Agilent 7820A GC system (column 30 m × 0.25 mm × 0.5 μm, Welch WM-1). Fungal Material. The fungus Penicillium sp. DT10 was isolated from wetland soil collected from Dongting Lake, Hunan Province, People’s Republic of China, in October 2016. The sequence data for this strain have been submitted to the DDBJ/EMBL/GenBank under accession No. MH458525. A voucher sample (BQ20161001) was preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology. Fermentation and Isolation. To prepare the seed culture, the strain was cultured on potato dextrose agar (PDA) at 28 °C for 7 days. The agar plugs were cut into small pieces and then inoculated into 150 Erlenmeyer flasks (1 L) containing 200 g of rice and 200 mL of distilled water (30 kg of rice in total), which were previously sterilized by autoclaving. All flasks were incubated at 28 °C for 21 days. The culture broths were extracted with ethanol, and the ethanol was removed under reduced pressure to yield a crude extract. The crude extract was partitioned between ethyl acetate and water to obtain the ethyl acetatesoluble fraction (400 g). This part was separated by column chromatography on silica gel (CC, 900 g, 15 × 100 cm, petroleum ether−ethyl acetate (30:1−0:1) and ethyl acetate−methanol (20:1− 0:1)) to furnish six fractions (Fr. 1−Fr. 6). Fr. 4 (10 g) was further separated on an ODS column (MeOH−H2O, 30−100%) to yield eight fractions (Fr. 4.1−Fr. 4.8). Fr. 4.3 was further separated using silica gel

Figure 4. ORTEP drawing of compound 6.

The molecular formula of dongtingnoid G (7) was determined to be the same as that of 6 based on its HRESIMS spectrum. Careful examination of its 1H and 13C NMR spectra indicated that compound 7 has an additional carbonyl carbon (δC 208.3), which formed an α,β-unsaturated ketone in ring A as disclosed by 1H−1H COSY correlations of Me-16/H-3/H-4 and H2-17/H-7 and by the HMBC correlations from H-4 to C-2 and C-5 and from H-7 to C-2, C-5, and C-6. The full structure of 7 was determined by further elucidation of its 2D NMR spectra. The relative configuration of 7 was resolved by a NOESY experiment. The NOESY correlation of Me-18/H-3 revealed they were cofacial, and they were assigned as β oriented. Accordingly, the NOESY correlation of Me-16/H-4 indicated H-4 was α oriented. Furthermore, based on the interactions of Me-18/H-1β (δH 2.31) and H-1α (δH 2.60)/H-7, H-7 was determined to be α-oriented. The absolute configuration of 7 was confirmed by ECD calculations (Figure S1). Compounds 1−5 are fusicoccane diterpenoids structurally related to cotylenin E (8), which was reported to be a plant regulator that showed significant seed-germination-promoting activity.12 Furthermore, it had been proposed that C-3-OH and sugar moieties are important for the seed-germinationpromoting activities of these compounds.8,11 Thus, considering the above-mentioned structural features, compounds 1−5 were examined for their seed-germination-promoting activities with water as the blank control and cotylenin E (8) as the positive control (Figure 5) using the previously reported method.8 As shown in Figure 5, compounds 1, 4, and 5 showed comparable seed-germination-promoting activities to that of cotylenin E (8) in the dark under nonflooded conditions, which suggested that the methylation or dehydration of the C-3-OH does not affect their seed-germination-promoting activities. These compounds 84

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V = 1849.09(2) Å3, Z = 2, Dcalcd = 1.2337 Mg/m3; absorption coefficient 0.689 mm−1; F(000) = 750.4, theta range for data collection 6.16° to 147.98°; index ranges −9 ≤ h ≤ 8, −35 ≤ k ≤ 35, −10 ≤ l ≤ 11; Reflections collected 40 459, independent reflections 7310[R(int) = 0.0295]; completeness to theta = 73.991°, 98%; largest diff peak and hole 0.15 and −0.15 e·Å−3; refinement method full-matrix least-squares on F2, with goodness-of-fit on F2 = 1.047; data/restraints/parameters 7310/0/459; final R indices [I > 2σ(I)], R1 = 0.0244, wR2 = N/A; R indices (all data) R1 = 0.0245, wR2 = 0.0638; Flack parameter 0.04(6) (CCDC 1846054). Acid Hydrolysis and GC Analyses to Determination the Absolute Configurations of the Sugars. Compounds 1−5 and 8 (1.5 mg of each) were hydrolyzed with 1 N CF3COOH (2.0 mL) at 100 °C for 3 h. Each reaction mixture was diluted with H2O and extracted with EtOAc (3 × 10 mL), and the aqueous layer was concentrated to dryness under reduced pressure. A portion of the residue was dissolved in dry pyridine (0.2 mL) containing (L)-cysteine methyl ester hydrochloride (0.2 mg) and kept at 65 °C for 1.5 h, Ntrimethylsilylimidazole (100 μL) was then added, and the mixture was kept for another 1.5 h. Then, the residue was partitioned between nhexane and H2O (1.0 mL each). The n-hexane fraction was subjected to GC under the following conditions: carrier gas, N2; constant flow, 1.0 mL/min; injector temperature, 250 °C; injected volume, 0.1 μL; split ratio, 1:10. The GC oven was set at 220 °C for 5 min and increased to 280 °C for 7 min at a rate of 2 °C/min. As the absolute configuration of the sugar of 1 was determined by X-ray diffraction analysis, it was used as a standard sample to determine the absolute configurations of the sugars of 2−5. Seed Germination. Ten Monochoria vaginalis seeds were placed on 8 cm glass Petri dishes and immersed in water with 50 ppm solutions of each compound. All the seeds were incubated at 25 °C for 10 to 13 days in the dark under nonflooded conditions. After incubation, the germination of the seeds was examined. The experiments were repeated three times.

column chromatography (dichloromethane−methanol, 50:1−0:1) to obtain six fractions (Fr. 4.3.1−Fr. 4.3.6). Fr. 4.3.4 was further separated using silica gel column chromatography (dichloromethane−methanol, 20:1−0:1) to obtain a mixture of compounds 1−5. The mixture was purified by repeated semipreparative HPLC (MeCN−H2O, 30%) separations to yield 1 (16.5 mg), 2 (3.0 mg), 3 (1.5 mg), 4 (30.5 mg), and 5 (15.0 mg). Fr. 4.3.2 was purified by semipreparative HPLC (MeCN−H2O, 50%) to yield a mixture of compounds 6 and 7. Purifying the mixture repeatedly by semipreparative HPLC (MeCN− H2O, 45%) afforded 6 (16.5 mg) and 7 (4.5 mg). Fr. 4.3.5 was further purified by repeated semipreparative HPLC (MeCN−H2O, 25%) separations to yield 8 (65.5 mg) and 9 (3.3 mg). Dongtingnoid A (1): colorless crystals, mp 168−169 °C; [α]23 D +9.0 (c 0.1, MeOH); IR νmax = 3437 and 1631 cm−1; UV (MeOH) λmax (log ε) = 204 (4.23) nm; ECD (MeOH) λ (Δε) 210 (−14.7) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 563.3187 (calcd for C29H48O9Na, 563.3196). Dongtingnoid B (2): colorless oil, [α]21 D +15.0 (c 0.1, MeOH); IR νmax = 3432 and 1631 cm−1; UV (MeOH) λmax (log ε) = 202 (4.08) nm; ECD (MeOH) λ (Δε) 210 (−9.2) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 563.3178 (calcd for C29H48O9Na, 563.3196). Dongtingnoid C (3): colorless oil, [α]22 D +27.0 (c 0.1, MeOH); IR νmax = 3433 and 1625 cm−1; UV (MeOH) λmax (log ε) = 205 (4.05) and 242 (3.93) nm; ECD (MeOH) λ (Δε) 204 (−15.3), 229 (−0.7), 305 (+3.4), nm; 1H NMR (800 MHz) and 13C NMR (200 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 519.2555 (calcd for C26H40O9Na, 519.2570). Dongtingnoid D (4): colorless oil, [α]26 D +17.0 (c 0.1, MeOH); IR νmax = 3429 and 1631 cm−1; UV (MeOH) λmax (log ε) = 201 (3.92) nm; ECD (MeOH) λ (Δε) 201 (−7.95) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 549.3045 (calcd for C28H46O9Na, 549.3040). Dongtingnoid E (5): colorless oil, [α]23 D +8.0 (c 0.1, MeOH); IR νmax = 3431 and 1632 cm−1; UV (MeOH) λmax (log ε) = 203 (4.09) and 245 (4.06) nm; ECD (MeOH) λ (Δε) 242 (−12.9) nm; for 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 531.2921 (calcd for C28H44O8Na, 531.2934). Dongtingnoid F (6): colorless crystals, mp 118−119 °C; [α]22 D + 4.0 (c 0.1, MeOH); IR νmax = 3436 and 1631 cm−1; UV (MeOH) λmax (log ε) = 204 (3.59) nm; ECD (MeOH) λ (Δε) 208 (−2.18) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 357.2038 (calcd for C20H30O4Na, 357.2042). Dongtingnoid G (7): colorless oil, [α]18 D −28.0 (c 0.1, MeOH); IR νmax = 3430 and 1628 cm−1; UV (MeOH) λmax (log ε) = 202 (3.93) and 246 (3.79) nm; ECD (MeOH) λ (Δε) 215 (+1.2), 220 (+1.4), 259 (−4.8) nm; 1H NMR (400 MHz) and 13C NMR (100 MHz) data see Tables 1 and 2; HRESIMS [M + Na]+ m/z 357.2049 (calcd for C20H30O4Na, 357.2042). Crystal data for compound 1: empirical formula C58H96O25; fw 1193.34; temp 296(2) K; wavelength 1.54178 Å; monoclinic, crystal size 0.100 × 0.040 × 0.020 mm3; unit cell dimensions: a = 17.3169(8) Å, b = 10.1910(5) Å, c = 19.8401(8) Å, α = 90°, β = 98.595(2)°, γ = 90°; V = 3462.0(3) Å3; Z = 2; Dcalcd = 1.145 Mg/m3; absorption coefficient 0.744 mm−1; F(000) = 1288; theta range for data collection 2.252° to 57.916°; index ranges −18 ≤ h ≤ 18, −10 ≤ k ≤ 10, −21 ≤ l ≤ 21; reflections collected 37 471, independent reflections 8999 [R(int) = 0.0772]; completeness to theta = 57.916°, 97.2%; largest diff peak and hole 0.999 and −0.328 e·Å−3; refinement method full-matrix leastsquares on F2, with goodness-of-fit on F2 = 1.069; data/restraints/ parameters 8999/119/838; final R indices [I > 2σ(I)]: R1 = 0.0753, wR2 = 0.2029, R indices (all data): R1 = 0.0839, wR2 = 0.2166; Flack parameter 0.10(12); extinction coefficient 0.0085(11) (CCDC 1843253). Crystal data for compound 6: empirical formula C40H62O9; fw 686.93; temp 100.01(10) K; wavelength 1.54184 Å; monoclinic, crystal size 0.5 × 0.15 × 0.05 mm3; unit cell dimensions: a = 7.22937(4) Å, b = 28.66155(14) Å, c = 9.57923(7) Å, α = 90°, β = 111.3149(7)°, γ = 90°;

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00694. Original HRESIMS, IR, UV, 1D and 2D NMR data for 1− 7; GC analyses for 1−5 (PDF) X-ray crystallographic data of 1 (CIF) X-ray crystallographic data of 6 (CIF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: 86-27-83692892. E-mail: [email protected]. *E-mail: [email protected]. ORCID

Junjun Liu: 0000-0001-9953-8633 Yonghui Zhang: 0000-0002-7222-2142 Author Contributions §

Q. Bie and C. Chen contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the Program for Changjiang Scholars of Ministry of Education of the People’s Republic of China (No. T2016088); the National Natural Science Foundation for Distinguished Young Scholars (No. 81725021); the National Science and Technology Project of China (2018ZX09201001-001-003); Innovative Research Groups of the National Natural Science Foundation of China 85

DOI: 10.1021/acs.jnatprod.8b00694 J. Nat. Prod. 2019, 82, 80−86

Journal of Natural Products

Article

(81721005); the Academic Frontier Youth Team of HUST; and the Integrated Innovative Team for Major Human Diseases Program of Tongji Medical College (HUST). We thank the Analytical and Testing Center at Huazhong University of Science and Technology for assistance in the acquisition of the ECD, UV, and IR spectra.

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REFERENCES

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DOI: 10.1021/acs.jnatprod.8b00694 J. Nat. Prod. 2019, 82, 80−86