Taichunins A–D, Norditerpenes from Aspergillus taichungensis (IBT

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Taichunins A−D, Norditerpenes from Aspergillus taichungensis (IBT 19404) Hikaru Kato,† Momona Sebe,† Mika Nagaki,† Keisuke Eguchi,† Ippei Kagiyama,† Yuki Hitora,† Jens C. Frisvad,‡ Robert M. Williams,§,⊥ and Sachiko Tsukamoto*,† †

Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan Section for Eukaryotic Biotechnology, Departments of System Biology, Technical University of Denmark, Building 221, 2800 Kongens Lyngby, Denmark § Department of Chemistry, Colorado State University, 301 West Pitkin Street, Fort Collins, Colorado 80523, United States ⊥ University of Colorado Cancer Center, Aurora, Colorado 80045, United States

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‡

S Supporting Information *

ABSTRACT: Four new norditerpenes, taichunins A−D (1− 4), were isolated from the fungus Aspergillus taichungensis (IBT 19404). Compound 1 has a new carbon framework. The absolute configurations were determined by the calculated ECD spectral method. Compound 1 was cytotoxic against HeLa cells with an IC50 value of 4.5 μM, whereas 2−4 were nontoxic at 50 μM.

T

Taichunin A (1) has the molecular formula C19H24O4, which was determined by HRESIMS. The 1H NMR spectrum showed two olefinic methines [δH 6.94 (s, H-11) and 5.82 (dd, J = 17.5, 10.6 Hz, H-14)], an exomethylene [δH 5.09 (dd, J = 17.5, 1.4 Hz, H-15b) and 4.89 (dd, J = 10.6, 1.4 Hz, H-15a)], and three methyls [δH 1.23 (s, H3-18), 1.22 (s, H3-16), and 0.92 (s, H317)] (Table 1). The 13C NMR and HSQC spectra indicated the presence of two ester carbonyl carbons [δC 174.2 (C-19) and 165.8 (C-13)], a disubstituted olefin carbon [δC 126.4 (C-12)], three protonated olefin carbons [δC 150.4 (C-11), 142.2 (C-14), and 114.6 (C-15)], two oxygen-bearing carbons [δC 90.8 (C-6) and 85.2 (C-7)], five methylenes, a methine, three methyls [δC 32.3 (C-18), 26.2 (C-16), and 23.4 (C-17)], and two quaternary carbons [39.1 (C-10) and 33.7 (C-4)] (Table 1). Comprehensive analyses of 2D NMR spectra revealed the presence of a gem-dimethyl cyclohexane with the ester carbonyl carbon (C19) bonded to C-5 (substructure A, Figure 1a). Another ester carbonyl carbon (C-13) was attached to C-12 of the 3-ethenyl-3methylcyclohexene (substructure B, Figure 1b). An HMBC correlation from H-5 to C-7 showed that substructures A and B were connected at C-6 and C-7, respectively (Figure 1c). The chemical shifts observed in the lower field for C-6 (δC 90.8) and C-7 (δC 85.2), together with the molecular formula, suggested that these carbons were oxygenated and disclosed the planar

he fungi of the genus Aspergillus are well known as a rich source of bioactive secondary metabolites, whereas the compounds reported from A. taichungensis are limited to indole alkaloids1−4 and polyhydroxy-p-terphenyls.5 Recently, we isolated prenylated indole alkaloids with unique skeletons, taichunamides, from the fungus A. taichungensis (IBT 19404).1 During isolation of these biosynthetically interesting alkaloids, we discovered several terpenes in the culture. In this work, we report the isolation, structure determination, and biological activities of four new norditerpenes, taichunins A−D (1−4), together with a known compound, sphaeropsidin B (5).6 The carbon framework of 1 is unprecedented, to the best of our knowledge.

Received: December 7, 2018 © XXXX American Chemical Society and American Society of Pharmacognosy

A

DOI: 10.1021/acs.jnatprod.8b01032 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 1. NMR Data of 1−3 (600 MHz, DMSO-d6) 1 no. 1α 1β 2α 2β 3α 3β 4 5 6 7 8α

δC, mult. 29.1, CH2 18.0, CH2 36.9, CH2 33.7, C 54.6, CH 90.8, C 85.2, C 24.3, CH2

8β 9α 9β 10 11

30.4, CH2 39.1, C 150.4, CH

12 13 14

126.4, C 165.8, C 142.2, CH

15a

114.6, CH2

15b 16 17 18 19 1′ 2′ 3′ 4′ 5′ 6′

26.2, CH3 23.4, CH3 32.3, CH3 174.2, C

2

δH, mult., (J in Hz)

HMBC

δC, mult.

1.67, m 1.87, m 1.37, m 1.63, m 1.49, m 1.37, m

2, 5, 6

5

32.3, CH2

2.75, s

6, 7, 17, 18, 19

32.4, C 57.4, CH

1.76, ddd (13.9, 13.9, 2.7) 2.10, ddd (13.9, 3.0, 3.0) 1.87, m 1.67, m 6.94, s

29.7, CH2 17.9, CH2

86.7, C 168.3, C 21.8, CH2 7, 9, 12 28.3, CH2

5.82, dd (10.6, 17.5)

9, 10, 11, 16

4.89, dd (10.6, 1.4)

10, 14

5.09, dd (17.5, 1.4)

10, 14

1.22, s 0.92, s 1.23, s

9, 10, 11, 14 3, 4, 5, 18 3, 4, 5, 17

HMBC

2.19, m 1.10, m 1.59, m 1.71, m 1.80, m 1.21, m

6

2.08, s

3, 4, 6, 17, 18, 19

δC, mult. 29.8, CH2 17.8, CH2 32.4, CH2

2.07, m

7, 12

40.0, C 64.7, CH

2.19, m 1.13, br d (13.5) 1.62, m 1.76, m 1.71, m 1.24, m

HMBC 5, 6

17 4, 5, 18

2.16, s

1, 3, 4, 6, 7, 17, 18, 19

86.5, C 168.9, C 21.7, CH2

1.91, m

7

2.44, m

1.50, m 1.71, m

10, 14, 16

3.89, s

7, 12

40.0, C 64.9, CH

28.3, CH2

9, 10, 11

127.9, C 170.0, C 143.7, CH

128.2, C 170.0, C 143.4, CH 5.64, dd (11.1, 17.6) 112.6, 4.90, dd (17.6, CH2 1.3) 4.92, dd (11.1, 1.3) 22.1, CH3 0.96, s 30.0, CH3 0.91, s 28.2, CH3 1.15, s 172.8, C

11OH 2′-OH 3′-OH 4′-OH 6′-OH

δH, mult., (J in Hz)

32.9, C 56.7, CH

2.20, m

7, 10, 14 7, 9, 10, 13, 14, 16

3

δH, mult., (J in Hz)

10, 14

112.3, CH2

10, 14 9, 10, 11, 14 3, 4, 5, 18 3, 4, 5, 17

22.0, CH3 29.9, CH3 28.1, CH3 170.1, C 94.3, CH 71.8, CH 76.4, CH 69.7, CH 77.8, CH 61.1, CH2

1.43, br d (13.0) 1.67, m

7, 8, 10, 11 7, 8, 10, 11, 14

3.88, d (6.1)

7, 9, 10, 12, 13, 14, 16

5.62, dd (11.0, 17.7) 4.87, dd (11.0, 0.7) 4.90, dd (17.7, 0.7) 0.95, s 0.90, s 1.18, s

9, 10, 11, 16

5.33, d (8.3) 3.11, m 3.21, m 3.08, m 3.24, m 3.42, dt (11.1, 6.2) 3.65, br d (11.1) 5.23, d (6.1)

19 1′, 3′ 2′ 5′

5.24, d (7.8) 5.10, d (5.1) 5.01, d (5.5) 4.34, dd (5.2)

1′, 2′, 3′ 2′, 3′, 4′ 3′, 4′, 5′ 5′, 6

10, 14 10, 14 9, 10, 11, 14 3, 4, 5, 18 3, 4, 5, 17

5′ 4′ 11, 12

H-14, and H2-15 were α-oriented, whereas the correlation of H5/18-Me disclosed β-orientations of these protons (Figure 2a). Because the skeleton of 1 appears to be unprecedented, the 13C NMR chemical shifts were calculated (Table S1) to support the structure. The standard deviation of differences between calculated and experimental data was 1.4, which supported the structure of 1 having a new norditerpene skeleton. The ECD spectrum of 1 was calculated with a configuration of 5S,6S,7S,10R, and the calculated and experimental spectra matched well with the negative Cotton effect around 190−220 nm (Figure 2b). Therefore, the absolute configuration of 1 was assigned as 5S,6S,7S,10R. Taichunin B (2) has the molecular formula C19H26O5, having one more H2O unit than 1. The 1H and 13C NMR data of 2

Figure 1. Substructures A (a) and B (b) and the planar structure of 1 (c).

structure of 1 as shown in Figure 1c. The NOE correlations, H2α/H-8α, H-2α/H-8β, and H-8β/17-Me, indicated that H-2α, H2-8, and 17-Me were located close together. The NOE correlations, H-8α/H-14 and H-8α/H2-15, indicated that H-8α, B

DOI: 10.1021/acs.jnatprod.8b01032 J. Nat. Prod. XXXX, XXX, XXX−XXX

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sp. YXf3.7 The C19 norditerpene 2 is an acid version of aspergiloid I and contains the opposite absolute configuration at the spiro carbon C-6. Taichunin C (3) has the molecular formula C25H36O10 determined by HRESIMS, having one more C6H10O5 unit than that of 2. The 1H and 13C NMR data (Table 1) were very similar to those of 2 and indicated the presence of a hexose moiety. Although hexose signals were overlapped in the 1H NMR spectrum in DMSO-d6, those in pyridine-d5 were separated and the axial−axial coupling constant (J = 8.2 Hz) was observed for H-1′/H-2′, H-2′/H-3′, H-3′/H-4′, and H-4′/ H-5′, which clearly indicated that the hexose was β-linked glucose. The HMBC correlation from H-1′ to C-19 suggested that the glucose moiety was attached to the C-19 position. The absolute configuration of glucose liberated from 2 by acid hydrolysis was determined to be of the D-series by HPLC analysis after derivatization.8 Thus, the structure of 3 was defined as the 19-O-β-D-glucopyranoside of 2. The molecular formula of taichunin D (4) was determined as C19H22O3 by HRFABMS. Although the 1H and 13C NMR spectra (Table 2) showed the presence of three methyl groups

Figure 2. (a) Key NOEs observed in a stable conformer of 1 and (b) experimental and calculated ECD spectra of 1.

Table 2. NMR Data of 4 (600 MHz, DMSO-d6)

could be superimposed on those of 1, indicating the presence of a similar carbon skeleton (Table 1). Analysis of 2D spectra (Figure 3a) showed that the olefinic methine (C-11) of 1 (δH

Figure 3. (a) COSY and key HMBCs of 2, (b) key NOEs observed in a stable conformer of 2, and (c) experimental and calculated ECD spectra of 2.

no.

δC, mult.

1 2 3 4 5 6 7 8 9 10 11 12 13 14α 14β 15 16

26.0, CH2 17.8, CH2 40.0, CH2 33.8, C 142.0, C 180.5, C 146.9, C 114.2, C 144.2, C 143.6, C 125.4, CH 201.2, C 48.3, C 33.0, CH2

17 18 19

22.6, CH3 27.95, CH3 28.00, CH3

141.0, CH 114.7, CH2

δH, mult. (J in Hz)

HMBC (J value of 8 Hz)

2.57 (2H), m 1.64 (2H), m 1.47 (2H), t (5.9)

2, 5, 10 4, 10 1, 4, 5, 18, 19

6.34, s

8, 10, 13

3.07, d (15.9) 2.66, d (15.9) 5.80, dd (17.5, 10.6) 4.99, d (17.5) 5.05, d (10.6) 1.17, s 1.26, s 1.29, s

7, 8, 9, 12, 13, 15, 17 7, 8, 9, 12, 13, 15, 17 12, 13, 14, 17 13, 15 13 12, 13, 14, 15 3, 4, 5, 19 3, 4, 5, 18

[δC 22.6 (C-17), 27.95 (C-18), and 28.00 (C-19)] and an ethenyl group [δH 5.80 (dd, J = 17.5, 10.6 Hz), δC 141.0 (C-15); δH 4.99 (d, J = 17.5 Hz), 5.05 (d, J = 10.6 Hz), δC 114.7 (C-16)], the resonances of two carbonyl carbons [δC 201.2 (C-12) and 180.5 (C-6)] and six olefinic carbons [δC 125.4 (C-11), 114.2 (C-8), 142.0 (C-5), 143.6 (C-10), 144.2 (C-9), and 146.9 (C7)] indicated that the carbon skeleton of 4 was different from those of 1−3. Analysis of COSY and HMBC spectra showed a 1,2-disubstituted-3,3-dimethylcyclohexene moiety as substructure A (Figure 4a) and a 3,4-disubstituted-6-methyl-6-vinylcyclohex-2-en-1-one moiety as substructure B (Figure 4b). The HMBC from H-11 to C-10 indicated that the remaining component of the formula, C2HO2 containing a carbonyl carbon [δC 180.5 (C-6)] and an olefinic carbon [δC 146.9 (C-7)], should be located between C-5 and C-8. The olefinic carbon (C7) had the HMBC correlation from H2-14, indicating the linkage

6.94; δC 150.4) was changed to an oxygen-bearing methine in 2 (δH 3.89; δC 64.7). The double bond C-11/C-12 in 1 was shifted to C-12 (δC 128.2)/C-7 (δC 168.3) in 2, and then the lactone linkage in 1 was cleaved to a carboxylic acid at C-5 in 2. The planar structure of 2 was determined as shown in Figure 3a. The relative configuration of 2 was identical to that of 1, as confirmed by NOE correlations (Figure 3b). The calculated ECD spectrum of (5S,6S,10R,11R)-2 showed positive Cotton effects around 220 nm and negative Cotton effects around 200 nm, similarly to the experimental ECD of 2 (Figure 3c). The absolute configuration of 2 was thereby confirmed as 5S,6S,10R,11R. A C18 norditerpenoid, aspergiloid I, was isolated from Apergillus C

DOI: 10.1021/acs.jnatprod.8b01032 J. Nat. Prod. XXXX, XXX, XXX−XXX

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wentii,11 4 is the first norditerpene lacking a methyl group at C10. The biological activities of 1−5 were tested, and 1 exhibited cytotoxicity against HeLa cells with an IC50 value of 4.5 μM, whereas 2−5 were nontoxic at 50 μM. None of the compounds showed antimicrobial activities, inhibitory activity of the cholesterol ester accumulation in macrophages, inhibitory activity of the RANKL-induced formation of multinuclear osteoclasts, or inhibitory activities of the ubiquitin−proteasome system [proteasome, E1, Ubc13 (E2)−Uev1A interaction, p53−Mdm2 (E3) interaction, and USP7].

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Figure 4. Substructures A (a) and B (b) and the planar structure of 4 (c).

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 polarimeter in MeOH. UV spectra were measured on a JASCO V-550 spectrophotometer in MeCN or MeOH. ECD spectra were measured on a JASCO J-820 spectropolarimeter in MeCN. IR spectra were recorded on a PerkinElmer Frontier FT-IR spectrophotometer. 1H and 13C NMR spectra were recorded on a JEOL JNM-ECX-400, Bruker AVANCE III 500, or Bruker AVANCE III 600 NMR spectrometer in DMSO-d6 or pyridined5. Chemical shifts were referenced to the residual solvent peaks (δH 2.49 and δC 39.5 for DMSO-d6; δH 7.55 and δC 135.5 for pyridine-d5). Mass spectra were measured on a Bruker BioTOFQ mass spectrometer, Waters Xevo G2-XS Qtof, or JEOL JMS-700 mass spectrometer. Fungal Strain and Culture Condition. The fungus Aspergillus taichungensis (IBT 19404) was obtained from the IBT Culture Collection of Fungi in Technical University of Denmark. It was isolated from soil in Taiwan. The fungus was cultured on rice medium. Rice (5 kg, dried weight) was autoclaved with water (6 L), and it was put into six plastic containers (W17 cm × D22 cm × H9 cm). The fungus suspended in sterilized water was added on rice medium and cultured at 25 °C for 30 days. Extraction and Isolation. The culture was extracted with n-BuOH, and the extract partitioned with H2O. The n-BuOH layer was concentrated and then partitioned between n-hexane and 90% MeOH−H2O. The aqueous MeOH fraction (50 g) was subjected to ODS chromatography with 40% and 75% (Fr. A) and 95% MeOH− H2O. Fr. A was subjected to SiO2 column chromatography with 95% and 80% (Fr. A1) and 50% n-hexane−EtOAc (Fr. A2), EtOAc (Fr. A3), 80% CH2Cl2−MeOH (Fr. A4), and CH2Cl2−MeOH−H2O (6:4:1). Fr. A1 was purified by HPLC [Asahipak GS-310P (21.5 × 500 mm), Asahi Chemical Industry Co., Ltd., MeOH; Develosil C30-UG-5 column (20 × 250 mm), Nomura Chemical Co., Ltd., 80% MeOH− H2O] to afford taichunin D (4, 0.55 mg). Fr. A2 was purified by ODS HPLC [Develosil C30-UG-5 column (20 × 250 mm)] eluted with 75% MeOH−H2O to afford taichunin A (1, 1.8 mg). Fr. A3 was purified by gel filtration HPLC [Asahipak GS-310P (21.5 × 500 mm)] eluted with MeOH to afford sphaeropsidin B (5, 640 mg). Fr. A4 was subjected to NH2 column chromatography with 90% and 80% (Fr. A5) and 50% CH3CN−H2O (Fr. A6). Fr. A5 was purified by SiO2 HPLC [COSMOSIL 5SL-II column (20 × 250 mm), Nacalai Tesque Inc.] eluted with n-hexane−CH2Cl2−MeOH (5:8:1) to afford taichunin C (3, 2.0 mg). Fr. A6 was purified by HPLC [Develosil C30-UG-5 column (20 × 250 mm)] eluted with 60% MeOH−H2O to afford taichunin B (2, 9.0 mg). Taichunin A (1): [α]23D −85 (c 1.5, MeOH); UV (MeCN) λmax (log ε) 208 (4.2) nm; ECD (400 μM, CH3CN) λmax (Δε) 212 (−8.6); IR (film) νmax 2931, 1773, 1685, 1243 cm−1; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 317.1735 [M + H]+ (calcd for C19H24O4, 317.1753). Taichunin B (2): [α]20D +70 (c 7.5, MeOH); UV (MeCN) λmax (log ε) 218 (4.0) nm; ECD (400 μM, CH3CN) λmax (Δε) 223 (5.0), 202 (−2.7) nm; IR (film) νmax 3382, 2932, 1731, 1391, 1258, 1165, 1023 cm−1; 1H and 13C NMR data, see Table 1; HRESITOFMS m/z 357.1657 [M + Na]+ (calcd for C19H26O5Na, 357.1678). Taichunin C (3): [α]20D +90 (c 1.7, MeOH); UV (MeOH) λmax (log ε) 216 (3.9) nm; IR (film) νmax 3352, 1738, 1639, 1024, 687 cm−1; 1H

of C-7−C-8. The carbonyl carbon (C-6) had no HMBC with the optimized J value of 8 Hz, whereas, with the optimized J value of 4 Hz, the HMBCs from H3-18, H3-19, and H-14 (δH 2.66) were observed to C-6 (Figure 4c). These data indicated that the carbonyl carbon existed between C-5 and C-7, and the carbon chemical shift (δC 146.9) of C-7 suggested the substitution of a hydroxy group to this olefinic carbon. Thus, the planar structure of 4 was determined and the 13S configuration was assigned based on biogenetic considerations (see Scheme 1). Scheme 1. Possible Formation of 1−5

Norditerpenoids taichunins A−D (1−4) and sphaeropsidin B (5) are possibly formed from geranylgeranyl diphosphate through isopimarane 6 (Scheme 1). Oxidation of 6 followed by lactonization may afford the known compound 5. Decarboxylation from 7 followed by oxidation may afford 4 via 8. Ring cleavage in 8 followed by lactonization affords 1−3, having new carbon skeletons. Although pimarane diterpenes with a carbonyl group at C-12 have been isolated from the plants Croton insularis9 and Jatropha divaricate10 and the fungus A. D

DOI: 10.1021/acs.jnatprod.8b01032 J. Nat. Prod. XXXX, XXX, XXX−XXX

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and 13C NMR data (DMSO-d6), see Table 1; 1H NMR (600 MHz, pyridine-d5) δ 1.10 (3H, s), 1.17 (3H, s), 1.18 (1H, m, H-3), 1.26 (1H, d, J = 13.7 Hz, H-1), 1.39 (3H, s), 1.55 (1H, d, J = 13.7 Hz, H-2), 1.75 (1H, m, H-9), 1.88 (1H, t, J = 13.7 Hz, H-2), 2.00 (1H, ddd, J = 13.7, 13.7, 3.4 Hz, H-3), 2.30 (1H, s, H-5), 2.31 (1H, m, H-8), 2.32 (1H, m, H-9), 2.48 (1H, ddd, J = 13.7, 13.7, 4.7 Hz, H-1), 3.12 (1H, d, J = 19.0 Hz, H-8), 4.07 (1H, m, H-5′), 4.10 (1H, t, J = 8.2 Hz, H-2′), 4.24 (2H, d, J = 8.2 Hz, H-3′ and H-4′), 4.33 (1H, dd, J = 11.4, 5.3 Hz, H-6′), 4.51 (1H, dd, J = 11.4, 2.3 Hz, H-6′), 4.59 (1H, s, H-11), 4.99 (1H, d, J = 10.9 Hz, H-15), 5.06 (1H, d, J = 17.8 Hz, H-15), 5.85 (1H, dd, J = 17.8, 10.9 Hz, H-14), 6.20 (1H, d, J = 8.2 Hz, H-1′); 13C NMR (150 MHz, pyridine-d5) δ 17.7 (CH2, C-2), 21.1 (CH3), 22.0 (CH2, C-8), 27.9 (CH3), 29.3 (CH2, C-9), 29.5 (CH2, C-1), 29.6 (CH3), 32.0 (CH2, C3), 56.7 (CH, C-5), 61.9 (CH2, C-6′), 65.6 (CH, C-11), 70.4 (CH, C4′), 72.8 (CH, C-2′), 77.9 (CH, C-3′), 78.4 (CH, C-5′), 95.0 (CH, C1′), 111.5 (CH2, C-15), 143.9 (CH, C-14); HRESITOFMS m/z 519.2241 [M + Na]+ (calcd for C25H36O10Na, 519.2206). Taichunin D (4): [α]20D +13 (c 0.43, MeOH); UV (MeOH) λmax (log ε) 322 (3.8), 334 (3.8) nm; ECD (800 μM, MeOH) λmax (Δε) 337 (−0.5), 266 (0.4), 219 (−1.3) nm; IR (film) νmax 3359, 2925, 2854, 1723, 1657, 1609, 1456, 1362, 1301, 1025 cm−1; 1H and 13C NMR data, see Table 2; HRFABMS m/z 299.1654 [M + H]+ (calcd for C19H23O3, 299.1647). Conformational Analyses and ECD Calculations for 1 and 2 and Chemical Shift Calculation for 1. These experiments were performed as previously described.1 ECD calculations for 1 and 2 were performed at the B3LYP/TZVP level, and no wavelength correction was conducted. The chemical shift calculation for 1 was performed at the ωB97X-D/6-31G* level. Absolute Configuration of Glucose. Compound 3 (0.1 mg) was hydrolyzed by 0.5 M HCl (0.1 mL) at 60 °C for 1 h. After evaporation, the residue was dissolved in pyridine (0.1 mL) and reacted with Lcysteine methyl ester (0.5 mg) at 60 °C. After 1 h, isothiocyanate (0.5 mg) was added to the solution, and the reaction mixture was kept at 60 °C for 1 h. A portion (2 μL) of the solution was subjected to analysis by HPLC: column, Cosmosil 5C18-AR-II (4.6 × 250 mm); solvent, CH3CN−0.2% tetrafluoroacetic acid in H2O (25:75); flow rate, 1 mL/ min; detector, UV (254 nm). The retention times of the standard D- and L-glucose were 13.5 and 12.4 min, respectively. Biological Assays. Cytotoxicity assay was performed using daunorubicin as a positive control. The details of the biological assays performed in this experiment were previously described.12

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REFERENCES

(1) Kagiyama, I.; Kato, H.; Nehira, T.; Frisvad, J. C.; Sherman, D. H.; Williams, R. M.; Tsukamoto, S. Angew. Chem., Int. Ed. 2016, 55, 1128− 1132. (2) Cai, S.; Sun, S.; Peng, J.; Kong, X.; Zhou, H.; Zhu, T.; Gu, Q.; Li, D. Tetrahedron 2015, 71, 3715−3719. (3) Cai, S.; Luan, Y.; Kong, X.; Zhu, T.; Gu, Q.; Li, D. Org. Lett. 2013, 15, 2168−2171. (4) Cai, S.; Kong, X.; Wang, W.; Zhou, H.; Zhu, T.; Li, D.; Gu, Q. Tetrahedron Lett. 2012, 53, 2615−2617. (5) Cai, S.; Sun, S.; Zhou, H.; Kong, X.; Zhu, T.; Li, D.; Gu, Q. J. Nat. Prod. 2011, 74, 1106−1110. (6) Antonio, E.; Lorenzo, S.; Olga, F.; Giovanni, B.; Federico, G.; Andrea, M. Phytochemistry 1997, 45, 705−713. (7) Guo, Z. K.; Wang, R.; Huang, W.; Li, X. N.; Jiang, R.; Tan, R. X.; Ge, H. M. Beilstein J. Org. Chem. 2014, 10, 2677−2682. (8) Tanaka, T.; Nakashima, T.; Ueda, T.; Tomii, K.; Kouno, I. Chem. Pharm. Bull. 2007, 55, 899−901. (9) Maslovskaya, L. A.; Savchenko, A. I.; Gordon, V. A.; Reddell, P. W.; Pierce, C. J.; Parsons, P. G.; Williams, C. M. Org. Lett. 2011, 13, 1032−1035. (10) Denton, R. W.; Harding, W. W.; Anderson, C. I.; Jacobs, H.; McLean, S.; Reynolds, W. F. J. Nat. Prod. 2001, 64, 29−31. (11) Li, X.; Li, X.-D.; Li, X.-M.; Xu, G.-M.; Liua, Y.; Wang, B.-G. RSC Adv. 2017, 7, 4387−4394. (12) Inhibition of p53−Mdm2 (E3) interaction: Tsukamoto, S.; Yoshida, T.; Hosono, H.; Ohta, T.; Yokosawa, H. Bioorg. Med. Chem. Lett. 2006, 16, 69−71. E1 inhibition: Yamanokuchi, R.; Imada, K.; Miyazaki, M.; Kato, H.; Watanabe, T.; Fujimuro, M.; Saeki, Y.; Yoshinaga, S.; Terasawa, H.; Iwasaki, N.; Rotinsulu, H.; Losung, F.; Mangindaan, R. E. P.; Namikoshi, M.; de Voogd, N. J.; Yokosawa, H.; Tsukamoto, S. Bioorg. Med. Chem. 2012, 20, 4437−4442. Inhibition of Ubc13 (E2)−Uev1A interaction: Ushiyama, S.; Umaoka, H.; Kato, H.; Suwa, Y.; Morioka, H.; Rotinsulu, H.; Losung, F.; Mangindaan, R. E. P.; de Voogd, N. J.; Yokosawa, H.; Tsukamoto, S. J. Nat. Prod. 2012, 75, 1495−1499. Inhibition of the cholesterol ester accumulation in macrophages: Eguchi, K.; Fujiwara, Y.; Hayashida, A.; Horlad, H.; Kato, H.; Rotinsulu, H.; Losung, F.; Mangindaan, R. E. P.; de Voogd, N. J.; Takeya, M.; Tsukamoto, S. Bioorg. Med. Chem. 2013, 21, 3831−3838. Antimicrobial activities: Gushiken, M.; Kagiyama, I.; Kato, H.; Kuwana, T.; Losung, F.; Mangindaan, R. E. P.; de Voogd, N. J.; Tsukamoto, S. J. Nat. Med. 2015, 69, 595−600. Cytotoxic activity: Afifi, A. H.; ElDesoky, A. H.; Kato, H.; Mangindaan, R. E. P.; de Voogd, N. J.; Ammar, N. M.; Hifnawy, M. S.; Tsukamoto, S. Tetrahedron Lett. 2016, 57, 1285−1288. USP7 inhibition: Tanokashira, N.; Kukita, S.; Kato, H.; Nehira, T.; Angkouw, E. D.; Mangindaan, R. E. P.; Voogd, N. J. D.; Tsukamoto, S. Tetrahedron 2016, 72, 5530−5540. Inhibition of the RANKL-induced formation of multinuclear osteoclasts: Kato, H.; Kai, A.; Kawabata, T.; Sunderhaus, J. D.; McAfoos, T. J.; Finefield, J. M.; Sugimoto, Y.; Williams, R. M.; Tsukamoto, S. Bioorg. Med. Chem. Lett. 2017, 27, 4975−4978. Proteasome inhibition: Kato, H.; El-Desoky, A. H.; Takeishi, Y.; Nehira, T.; Angkouw, E. D.; Mangindaan, R. E. P.; de Voogd, N. J.; Tsukamoto, S. Bioorg. Med. Chem. Lett. 2019, 29, 8−10.

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b01032. 1D and 2D NMR spectra of 1−4; experimental and calculated 13C NMR data based on ωB97X-D/6-31G for 1 (PDF)

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Note

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Sachiko Tsukamoto: 0000-0002-7993-381X Author Contributions

H.K. and M.S. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by JSPS KAKENHI Grant Numbers 17H03994 (S.T.), 18K14933 (Y.H.), and 18K06719 (H.K.) of Japan. E

DOI: 10.1021/acs.jnatprod.8b01032 J. Nat. Prod. XXXX, XXX, XXX−XXX