Hydroquinones from the Marine Sponge

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Sesquiterpene Quinones/Hydroquinones from the Marine Sponge Spongia pertusa Esper Jing Li,†,‡,⊥ Bin-Bin Gu,†,⊥ Fan Sun,† Jian-Rong Xu,§ Wei-Hua Jiao,† Hao-Bing Yu,† Bing-Nan Han,† Fan Yang,*,† Xi-Chun Zhang,*,∥ and Hou-Wen Lin*,† †

Research Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, 200127, China ‡ College of Pharmacy, Jinan University, Guangzhou 510632, China § Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai 20025, China ∥ Institute of Pharmaceutical & Food Engineering, Shanxi University of Traditional Chinese Medicine, Taiyuan 030024, China S Supporting Information *

ABSTRACT: Nine new sesquiterpene quinones/hydroquinones (1− 7, 10, and 12), three solvent-generated artifacts (8, 9, and 11), and three known compounds, 5-epi-smenospongine (13), smenospongine (14), and smenospongiadine (15), were isolated from the marine sponge Spongia pertusa Esper. The planar structures of the new compounds were elucidated on the basis of spectroscopic analyses. Their absolute configurations were determined by comparison between the calculated and experimental ECD spectra. In the cytotoxicity bioassay, compounds 13−15 exhibited activities against the human cancer cell lines U937, HeLa, and HepG2, with most potent cytotoxicities to U937 cells with IC50 values of 2.8, 1.5, and 0.6 μM, respectively. In addition, compound 6 displayed CDK-2 affinity with a Kd value of 4.8 μM in a surface plasmon resonance assay.

T

effectiveness and fewer adverse effects.22 As part of our ongoing bioactive marine natural product discovery project in the South China Sea, we focused our efforts on the extracts of the marine sponge Spongia pertusa Esper. Chemical investigation led to the isolation of nine new sesquiterpene quinones/hydroquinones (1−7, 10, and 12), three solvent-generated artifacts (8, 9, and 11), and three known compounds, 5-epi-smenospongine (13), smenospongine (14), and smenospongiadine (15). Details of the isolation, structure elucidation, and biological evaluation of these compounds are presented herein.

he marine sponges of the Spongia genus have been reported to produce many types of metabolites such as macrolides,1 steroids,2,3 diterpenoids,2,4−6 C-21 β-substituted furan terpenoids,7−9 sesterterpenoids,10−12 and sesquiterpene quinones/hydroquinones.13−15 These compounds have shown a large spectrum of biological properties, including cytotoxicity,13,15−17 inhibition of DNA polymerase β lyase activity,14,18 aromatase inhibition,6 human phospholipase A2 inhibition,4 and antagonism at the farnesoid X-activated receptor.12,19 Among the various compounds from the Spongia sponges, sesquiterpene quinones/hydroquinones are the most representative and attractive due to their structural diversity and pharmaceutical potential. Cyclin-dependent kinases (CDKs) are an important target for many disease treatments, as they play crucial roles in cell cycle progression, apoptosis, neuronal functions, and transcription in mammalian cells.20 CDKs belong to the serine/ threonine protein kinase family. There are 16 subtypes (CDKs 1−16), which can be combined with cell cycle proteins to regulate the cell cycle.21 Clinical applications of first-generation nonselective CDK inhibitors, designed to inhibit uncontrolled cell proliferation, were originally hampered by the high risk of toxicity and lack of efficacy. Selective CDK-2 inhibitors have enabled tumor types in which CDK-2 has a pivotal role in the G1-to-S phase cell-cycle transition to be treated with improved © 2017 American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION

Compound 1 was obtained as a yellow powder. Its molecular formula was determined as C24H33NO5 by the HRESIMS ion peak at m/z 414.2283 [M − H]−, which corresponded to nine degrees of unsaturation. Analyses of the 13C NMR and DEPT spectra of 1 displayed the presence of 24 carbons, including five quaternary carbons, four additional nonprotonated carbon, four methines (one formamido), seven methylenes, and four methyls (one methoxy). The 1H NMR spectrum of 1 indicated the presence of two phenolic hydroxy protons (δH 11.45 and 10.54), a formamide group (NH δH 8.00; CHO δH 8.23), one Received: November 29, 2016 Published: April 11, 2017 1436

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Chart 1

Figure 1. Key COSY and HMBC correlations of compounds 1−4, 6, 8, 9, and 12.

the other phenolic hydroxy 19-OH (δH 11.45) to C-18 (δC 113.5), C-19 (δC 151.8), and C-20 (δC 103.4) suggested the formamide group was tethered at C-18 and the hydroxy groups were located at C-17 and C-19, respectively (Figure 1). Futhermore, the observation of the HMBC cross-peaks of H215 (δH 2.66 and 2.61) with C-8 (δC 36.3), C-9 (δC 42.0), C-10 (δC 48.1), C-14 (δC 17.56), C-16 (δC 120.5), and C-17 (δC 154.1) and the aromatic proton H-21 (δC 151.8) with C-15 (δC 36.6), C-17 (δC 154.1), C-19 (δC 151.8), and C-23 (δC 13.8) indicated the hydroquinone ring was attached at C-15. The relative configuration of 1 was established by the correlations observed in the NOESY spectrum (Figure 2). The trans fusion of the rings A/B was deduced from the large coupling constant of H-10 (J = 13.0 Hz) and the chemical shift

methoxy (δH 3.91), and three methyls (δH 1.07, 1.02, and 0.85). An extensive inspection of the 1H and 13C NMR spectra allowed the establishment of a sesquiterpene hydroquinone system, which was similar to dictyoceratin B.23 In the HMBC spectrum, correlations from H2-11 (δH 4.42 and 4.38) to C-5, from H3-12 (δH 1.07) to C-4 (δC 160.4), C-5 (δC 40.3), C-6 (δC 36.7), and C-10 (δC 48.1), from H3-13 (δH 1.02) to C-7 (δC 27.7), C-8 (δC 36.3), and C-9 (δC 42.0), and from H3-14 (δH 0.85) to C-8 (δC 36.3), C-9 (δC 42.0), C-10 (δC 48.1), and C-15 (δC 36.6) confirmed the avarane Δ4(11) skeleton in compound 1 (Figure 1).24 In the hydroquinone ring, HMBC correlations from the formyl proton H-22 (δH 8.23) to C-18 (δC 113.5), from phenolic hydroxy 17-OH (δH 10.5) to C-16 (δC 120.5), C-17 (δC 154.1), and C-18 (δC 113.5), and from 1437

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Figure 2. Key NOESY correlations of compounds 1, 3, 5−7, 9, 11, and 12.

of H3-12 (δC 20.6).25 Additionally, NOESY cross-peaks of H1α (δH 1.54)/H3-12, H-1α/H3-14, H-3α (δH 2.35)/H3-12, H313/H3-14, and H-6α (δH 1.47)/H3-12 indicated these methyl groups and protons are α-oriented, whereas the correlations of H-10 (δH 0.92)/H-2β (δH 1.34), H-6β (δH 1.22)/H-8 (δH 1.26), and H-10/H-8 revealed that these protons are βoriented. The absolute configuration was determined via experimental and calculated electronic circular dichroism (ECD) data.26,27 Density functional theory (DFT) calculations performed at the B3LYP/6-31G(d) level were used to generate ECD spectra for the two lowest-energy conformers of 1. The resulting ECD spectra were combined by Boltzmann weighting to give a composite spectrum for each enantiomer. Comparison of the experimental and calculated ECD spectra for 1 showed excellent agreement with the 5R,8R,9S,10R-1 enantiomer (Figure 3). The similar Cotton effects (CEs) finally enabled assignment of the absolute configuration of 1 as 5R,8R,9S,10R. Compound 2 was obtained as a yellow powder. Its molecular formula was determined as C25H35NO6 by the HRESIMS sodium adduct ion peak at m/z 468.2725 [M + Na]+, which corresponded to nine degrees of unsaturation. The NMR data of 2 were nearly identical to those of 1, except for the presence of resonances for a hydroxymethyl amide group at δC 171.1 (C22), δC/δH 62.4/4.36 (CH2-25), and δH 9.12 (22-NH), as shown in Table 1. This group was tethered at C-18, which was deduced from an HMBC correlation from H2-25 to C-22 (δH 171.1) and the shielded chemical shift of the aromatic nonprotonated carbon C-18 (δC 113.5) similar to 1. Detailed NOESY analysis suggested that 2 possessed the same relative configurations at C-5 (δC 40.2), C-8 (δC 36.2), C-9 (δC 41.9), and C-10 as those of 1. The similar Cotton effects in the ECD spectra of 1 and 2 (Figure 3) and similar specific rotations (1

Figure 3. Experimental and calculated ECD spectra of 1, 2, 4, and 5.

[α]24D +5.3 in CHCl3; 2 [α]24D +6.3 in CHCl3) suggested that 2 shared the same absolute configuration as that of 1. Compound 3 was obtained as a yellow powder. Its molecular formula was established as C23H30O3 with nine degrees of unsaturation by HRESIMS. In the 1H NMR spectrum, three aromatic protons were observed at δH 7.80 (1H, s, H-21), 7.79 (1H, d, J = 8.4 Hz, H-19), and 6.76 (1H, d, J = 8.4 Hz, H-18), implying a 1,2,4-trisubstituted benzene moiety. Two olefinic protons at δH 6.07 (1H, d, J = 8.0 Hz, H-3) and 5.72 (1H, m, H-2), two exomethylene protons at δH 4.85 (1H, s, H-11a) and 4.79 (1H, s, H-11b), and one methoxy group at δH 3.89 (3H, s, CH3-23) were also observed in the 1H NMR spectrum. Analyses of the 13C NMR and HSQC data revealed the presence of 23 carbons, including three aliphatic and two aromatic quaternary carbons, one ester carbonyl carbon and one nonprotonated aromatic carbon, seven methines, four methylenes, and four methyls (one methoxy). Comparison of 1438

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Table 1. 1H (500 MHz) and 13C NMR (125 MHz) Data for 1−4 in CDCl3 1 position

δC

1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19 20 21 22 23 24 25 18-NH 22-NH 17-OH 19-OH

23.2, CH2 27.9, CH2 33.1, CH2 160.4, C 40.3, C 36.7, CH2 27.7, CH2 36.3, 42.0, 48.1, 102.6,

CH C CH CH2

20.6, CH3 17.6, CH3 17.56, CH3 36.6, CH2 120.5, 154.1, 113.5, 151.8, 103.4, 130.5, 160.1, 170.6, 52.2,

C C C C C CH CH C CH3

2 δH (J in Hz)

1.54, 2.09, 1.92, 1.34, 2.35, 2.09,

td br m dt td br

(13.0, 3.5) d (13.0)

1.26, m d s s s d s d d

23.2, CH2 27.9, CH2

(12.5, 4.5) (13.5, 5.0) d (13.0)

1.47, dt (12.5, 3.0) 1.22, m 1.40, m

0.92, 4.42, 4.38, 1.07, 1.02, 0.85, 2.66, 2.61,

δC

(13.0)

(6.5) (14.5) (14.5)

7.41, s 8.23, d (2.0) 3.91, s

33.1, CH2 160.4, C 40.2, C 36.7, CH2 27.7, CH2 36.2, 41.9, 48.0, 102.6,

CH C CH CH2

20.6, CH3 17.6, CH3 17.57, CH3 36.5, CH2 120.6, 154.7, 113.5, 152.6, 103.4, 130.3, 171.1, 170.7, 52.2, 62.4,

C C C C C CH C C CH3 CH2

3 δH (J in Hz)

1.55, 2.10, 1.91, 1.35, 2.34, 2.10,

dd (11.0, 3.0) m m td (11.0, 3.5) td (11.5, 7.0) m

1.47, 1.23, 1.41, 1.42, 1.26,

td (10.5, 3.0) m d (2.5) td (11.5, 3.0) m

0.92, 4.42, 4.38, 1.06, 1.02, 0.85, 2.63,

dd (9.5, 1.5) s s s d (6.6) s m

7.40, s

δC 25.4, CH2

4 δH (J in Hz)

δC

δH (J in Hz)

127.8, CH

2.61, m 2.49, m 5.72, m

28.7, CH2

130.0, CH

6.07, d, 8.0

33.0, CH2

149.2, C 37.7, C 36.1, CH2 27.3, CH2 37.6, 42.6, 44.6, 108.2,

CH C CH CH2

33.4, 17.8, 16.6, 36.5,

CH3 CH3 CH3 CH2

125.0, 158.6, 115.3, 129.3, 122.2, 135.0, 167.1, 51.9,

C C CH CH C CH C CH3

1.98, m 1.47, m 1.25, m 1.42, m 1.28, 4.85, 4.79, 0.96, 1.00, 0.76, 2.72, 2.57,

m s s s d (6.0) s d (14.4) d (14.4)

6.76, d (8.4) 7.79, d (8.4) 7.80, s

23.2, CH2

160.5, C 40.4, C 36.7, CH2 28.0, CH2 37.9, 42.9, 49.9, 102.5,

CH C CH CH2

20.6, 17.9, 17.3, 32.5,

CH3 CH3 CH3 CH2

113.6, 157.2, 178.2, 91.5, 151.3, 182.8, 29.4,

C C C CH C C CH3

1.42, 2.11, 1.85, 1.16, 2.33, 2.08,

m m m m td (14.0, 5.5) m

1.52, m 1.36, m 1.39, m 1.18, m 0.78, 4.44, 4.43, 1.04, 0.96, 0.83, 2.49, 2.40,

dd (11.5, 2.0) s s s d (6.5) s d (13.5) d (14.0)

5.36, s

2.91, s

3.89, s

4.36, m 3.92, s

8.00, br s 10.54, br s 11.45, br s

9.12, br s 10.92, s 11.38, br s

the NMR spectra of 3 (Table 1) with those of dictyoceratin C revealed that 3 possessed a similar planar structure.28 The chemical shift of the methyl CH3-12 (δC 33.4) implied the cis fusion of rings A/B,25 which was supported by the NOESY correlations between H-10 (δH 1.28) and H3-12 (δH 0.96), as depicted in Figure 2. NOESY correlations of H-10/H3-12 and H-10/H-8 (δH 1.42) indicated that protons H-8, H-10, and methyl CH3-12 are α-oriented, whereas the correlations of H313 (δH 1.02)/H3-14 (δH 0.85) revealed that the two methyls are β-oriented. The absolute configuration of 3 was determined as 5R,8S,9R,10S by comparison of the experimental and calculated ECD spectra as shown in Figure 4. Compound 4 was obtained as a purple-red powder. Its molecular formula was deduced as C22H30NO3 by HRESIMS (m/z 356.2226 [M − H]−). Two characteristic conjugated enone carbonyl resonances at δC 182.8 and 178.2 in the 13C NMR spectrum corroborated the presence of a 1,4benzoquinone.29 The molecular weight of 4 was 14 mass units more than that of smenospongine, a sesquiterpene aminoquinone isolated from a marine sponge. 30 The spectroscopic data of 4 were also similar to those of smenospongine, except for an additional methyl (δH 2.91/δC 29.4, CH3-22) attached to the nitrogen, which was supported

Figure 4. Experimental and calculated ECD spectra of 3.

by the COSY correlation of 20-NH (δH 6.46)/H3-22 and the HMBC correlation from H3-22 to C-20 (δC 151.3). The relative configuration of 4 was deduced from the NOESY spectrum. The NOESY correlations observed for H-1α (δH 1.42)/H3-12 (δH 1.02), H-1α (δH 1.42)/H3-14 (δH 0.83), H-3α (δH 2.33)/ H3-12 (δH 1.04), H3-13 (δH 0.96)/H3-14 (δH 0.83), and H-6α (δH 1.52)/H3-12 indicated these methyl groups and protons are α-oriented, whereas the correlations of H-10 (δH 0.78)/H-2β 1439

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Compound 7 exhibited the same molecular formula of C22H29NO3 as that of 6 by HRESIMS data. Analyses of the 1H and 13C NMR data revealed that 7 was a C-5 epimer of 6 based on the difference of chemical shifts of CH3-12 between 7 (δC 32.3/δ H 1.10) and 6 (δ C 21.1/δ H 1.10).The relative configuration of 7 was established based on the NOESY correlations of H-10 (δH 1.22)/H3-12 (δH 1.10) and H3-13 (δH 1.21)/H3-14 (δH 1.11), indicating that CH3-12 and H-10 were α-oriented and CH3-13 and CH3-14 were β-oriented (Figure 2); thus, the absolute configuration of 7 was determined as 5R,8R,9S,10R by comparison of the experimental and calculated ECD spectra of 7 (Figure 6).

(δH 1.16) and H-6β (δH 1.36)/H-8 (δH 1.18) revealed these protons are β-oriented. The two six-membered rings A and B were trans fused, which was also confirmed by the large coupling constant of H-10 (J = 11.5 Hz) and the chemical shift of CH3-12 (δC 20.6).25 The NOESY correlations and the similarity of their ECD spectra between 1 and 4 indicated 4 shared the same absolute configuration with 1 (Figure 3). Compound 5 was isolated as an isomer of 4 with a molecular formula of C22H30NO3 by its HRESIMS data. The 1H and 13C NMR spectra of 5 were quite similar to those of 4, except for the resonances of C-4 (δC 153.6) and CH3-12 (δC 33.2). Therefore, compound 5 appeared to have a cis ring junction. The cis fusion of rings A/B was supported by the NOESY correlation between H-10 (δH 1.19) and H3-12 (δH 1.05). In addition, the NOESY correlation of H-10 (δH 1.19)/H-8 (δH 1.18) indicated that H-8, H-10, and CH3-12 are α-oriented, whereas the correlation of H3-13 (δH 0.91)/H3-14 (δH 0.85) comfirmed the two methyls are β-oriented (Figure 2). For these molecules, the configuration at C-5 adjacent to the exocyclic double bond appeared to have the dominant effect on the ECD spectrum near 200 nm. Since the experimental ECD spectrum for 5 was similar to that for 4 and 13, the 5R configuration can be assigned. Moreover, the specific rotation value of 5 ([α]24D +73.4 in CHCl3) was similar to that of 13 ([α]24D +82.6 in CHCl3). Therefore, the absolute configuration of 5 was determined as 5R,8S,9R,10S. Compound 6 was obtained as a purple-red powder. Its molecular formula was determined as C22H29NO3, based on HRESIMS data, which corresponded to nine degrees of unsaturation. Further comparison of the NMR data between compound 6 and the reported dactyloquinone D31 showed that their NMR data were similar, with the only differences being the substituent attached to C-20. The substituent was deduced to be methylamine, which was supported by HMBC correlation from H3-22 (δH 2.84) to C-20 (δC 148.7). Hence, the planar structure of 6 was identified. The relative configuration for 6 was deduced from NOESY correlations shown in Figure 2. The large vicinal coupling constant of H-10 (J = 14.5 Hz) indicated the trans fusion of rings A/B, which was supported by the chemical shift of the methyl CH3-12 (δC 20.9).25 NOESY crosspeaks of H-6α (δH 2.00)/H-10 (δH 1.02), H-3β (δH 2.25)/H312 (δH 1.10), H-1β (δH 1.49)/H3-12/H3-14 (δH 1.07), and H313 (δH 1.25)/H3-14 indicated that H-6α and H-10 were αoriented and H-1β, H-3β, CH3-12, CH3-13, and CH3-14 were β-oriented (Figure 2). The absolute configuration of 6 was determined as 5S,8R,9S,10R by comparison of the experimental and calculated ECD spectrum of 6 (Figure 5).

Figure 6. Experimental and calculated ECD spectra of 7.

Compoud 8 exhibited the molecular formula C23H30O4 from the HRESIMS data. The 1H and 13C NMR spectra of 8 were similar to those of dactyloquinone D32 except for the presence of an O-ethyl group. The COSY cross-peaks of H3-23 (δH 1.46)/H2-22 (δH 3.98) and the HMBC correlations from H2-22 (δH 3.98) to C-20 (δC 159.4) demonstrated that an O-ethyl group rather than a methoxy was attached at C-20. Furthermore, 8 shared consistent key NOESY correlations as well as a similar ECD curve with 6. Therefore, the absolute configuration of 8 was determined as 5S,8R,9S,10R. Because EtOH was used as the extraction solvent, it is highly probable that compound 8 is an artifact. Compound 9 displayed the molecular formula C23H30O4 by HRESIMS. The 1H and 13C NMR data of 9 were similar to those of dactyloquinone B except that an additional oxymethylene quartet was observed at δH 3.98.32 The COSY crosspeaks of H3-23 (δH 1.46)/H2-22 (δH 3.98) demonstrated an Oethyl group in 9. The placement of the O-ethyl group at C-20 was deduced from the HMBC correlations from the oxymethylene H2-22 (δH 3.98) to the oxygenated olefinic carbon C-20 (δC 158.6). Detailed NOESY analysis revealed that 9 possessed the same relative configuration as that of dactyloquinone B (Figure 2). The absolute configuration of 9 was determined as 5S,8S,9R,10R by comparison of the experimental and calculated ECD spectra of 9 (Figure 7). Compound 9, however, could possibly be an artifact from the extraction process with EtOH as a solvent. Compound 10 has the molecular formula C22H29NO3 as determined by the HRESIMS, which corresponded to nine degrees of unsaturation. The 1H and 13C NMR data of 10 were similar to those of 9, except for the resonances for a N-methyl group instead of an O-ethyl group (Table 2). The placement of the N-methyl at C-20 was deduced from the COSY correlation

Figure 5. Experimental and calculated ECD spectra of 6 and 8. 1440

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22 (δH 3.98) to C-20 (δC 158.8) and COSY correlation crosspeaks of H2-22 (δH 3.98)/H3-23 (δH 1.46) then indicated the O-ethyl group was attached to C-20. The relative configuration of 11 was established by the correlations observed in the NOESY spectrum (Figure 2). NOESY correlations of H-5 (δH 0.97)/H-9 (δH 0.88) and H3-11 (δH 0.90)/H-5 (δH 0.97) revealed that these protons are α-oriented, while the correlations of H3-12 (δH 0.83)/H3-14 (δH 0.88) and H3-13 (δH 1.22)/H3-14 (δH 0.88) indicated that these methyl groups are β-oriented. The absolute configuration was determined as 5S,8R,9R,10S by comparison of the experimental and calculated ECD data, as shown in Figure 8. However, it is possible that compound 11 is an artifact formed during the extraction procedure with EtOH. Compound 12 has the molecular formula C21H32O2, which was deduced from the HRESIMS ion peak at m/z 339.2297 [M + Na]+, corresponding to six degrees of unsaturation. The IR spectrum exhibited absorptions for hydroxy groups at 3357 cm−1 and a benzene moiety at 1610, 1511, and 1419 cm−1. The 1 H NMR spectrum of 12 showed four aromatic protons at δH 7.31 (2H, d, J = 10.5 Hz, H-17, H-21) and 6.73 (2H, d, J = 10.5 Hz, H-18, H-20), implying a 1,4-disubstituted benzene moiety, which was confirmed by COSY correlations of H-17 (δH 7.31)/ H-18 (δH 6.73) and H-20 (δH 6.73)/H-21 (δH 7.31). Three singlet methyls at δH 0.99, 0.85, and 0.81 and a doublet methyl at δH 0.93 were also observed in the 1H NMR spectrum.

Figure 7. Experimental and calculated ECD spectra of 9 and 10.

of NH-20 (δH 5.94)/H3-22 (δH 2.84) and HMBC correlations from H3-22 (δH 2.84) to C-20 (δC 148.5). Identical key NOESY correlations and Cotton effects in the ECD spectra revealed that 10 shared the same absolute configuration as 9, as shown in Figure 7. Compound 11 has the molecular formula C23H33O4 deduced from the HRESIMS data. The 1H and 13C NMR data of 11 were similar to those of cyclospongiaquinone-1 except for an oxymethylene (δH 3.98/δC 65.3) and its coupled methyl resonances (δH 1.46/δC 13.8), which indicated the appearance of an O-ethyl group (Table 3).33 HMBC correlations from H2-

Table 2. 1H (500 MHz) and 13C NMR (125 MHz) Data for 5−8 in CDCl3 5 position

δC

1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19 20 21 22 23 20-NH

22.5, CH2 25.0, CH2 32.0, CH2 153.6, C 39.5, C 37.9, CH2 27.9, CH2 39.3, 44.4, 48.1, 105.6,

CH C CH CH2

33.2, 18.3, 18.6, 32.7,

CH3 CH3 CH3 CH2

113.6, 157.2, 178.2, 91.4, 151.3, 182.8, 29.4,

C C C CH C C CH3

6 δH (J in Hz) 2.12, 1.78, 1.49, 1.64, 2.09, 2.40,

m m m m m m

1.98, 1.07, 1.16, 1.46, 1.18,

m m m m m

1.19, 4.70, 4.67, 1.05, 0.91, 0.85, 2.52, 2.41,

m s s s d (8.0) s d (16.5) d (16.5)

5.38, s

2.92, s

δC 22.2, CH2 28.4, CH2 32.8, CH2 158.6, C 39.5, C 31.4, CH2 30.6, CH2 85.1, 37.1, 44.8, 103.8,

C C CH CH2

21.1, 23.3, 21.3, 27.8,

CH3 CH3 CH3 CH2

111.4, 155.7, 179.4, 95.2, 148.7, 182.1, 29.2,

C C C CH C C CH3

7 δH (J in Hz)

1.54, 1.49, 1.83, 1.14, 2.08, 2.25,

2.00, 1.43, 2.04, 1.28,

1.02, 4.52, 4.48, 1.10, 1.25, 1.07, 1.87, 2.76,

m m m m m td (17.0, 6.5)

δC 21.3, CH2 24.4, CH2 31.6, CH2 155.6, C 38.6, C 31.4, CH2

s m m m

30.4, CH2

dd (14.5, 4.0) s s s s s d (18.6) d (18.6)

5.29, s

2.84, d (5.4) 5.96, d (5.4)

85.7, 38.2, 42.9, 106.4,

C C CH CH2

32.3, 23.3, 22.8, 28.0,

CH3 CH3 CH3 CH2

111.9, 153.1, 179.5, 95.2, 148.7, 182.4, 29.2,

C C C CH C C CH3

8 δH (J in Hz) 2.15, 1.85, 1.81, 1.68, 2.46, 2.16,

2.05, 1.11, 1.50, 1.21,

1.22, 4.78, 4.77, 1.10, 1.21, 1.11, 1.87, 2.71,

m m m m m m

s m m m

m s s s s s d (18.6) d (18.6)

5.31, s

2.84, d (5.4)

δC

δH (J in Hz)

22.0, CH2 28.3, CH2 32.6, CH2 158.6, C 39.4, C 31.2, CH2 30.4, CH2 84.7, 37.1, 44.8, 103.5,

C C CH CH2

20.9, 23.1, 21.1, 27.8,

CH3 CH3 CH3 CH2

113.9, 152.4, 181.9, 105.1, 159.4, 181.2, 65.3, 13.8,

C C C CH C C CH2 CH3

1.54, 1.47, 1.84, 1.14, 2.08, 2.26,

m m m m m td (17.0, 6.5)

2.01, 1.42, 2.04, 1.28,

s m m m

1.02, 4.51, 4.48, 1.11, 1.23, 1.06, 1.90, 2.83,

dd (14.5, 4.0) s s s s s d (23.5) d (23.5)

5.73, s

3.98, q (8.5) 1.46, t (8.5)

5.96, d (5.4) 1441

DOI: 10.1021/acs.jnatprod.6b01105 J. Nat. Prod. 2017, 80, 1436−1445

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Table 3. 1H (500M Hz) and 13C NMR (125 MHz) Data for 9−12 in CDCl3 9 position

δC

1α 1β 2α 2β 3α 3β 4 5 6α 6β 7α 7β 8 9 10 11a 11b 12 13 14 15a 15b 16 17 18 19 20 21 22 23 20-NH

29.2, CH2 23.3, CH2 30.0, CH2 152.9, C 44.0, C 32.6, CH2 26.9, CH2 33.8, 38.8, 87.9, 107.4,

CH C C CH2

27.6, 16.1, 19.9, 28.1,

CH3 CH3 CH3 CH2

114.0, 152.4, 181.8, 105.0, 158.6, 181.2, 65.2, 13.8,

C C C CH C C CH2 CH3

10 δH (J in Hz)

1.91, 1.88, 1.86, 1.73, 2.20, 2.58,

1.95, 1.76, 1.62, 1.28, 1.42,

4.89, 4.79, 1.37, 0.75, 1.12, 2.10, 2.63,

m dd (11.5, 5.0) m m dd (11.5, 4.0) m

dt (12.0, 3.0) dd (12.0, 3.5) dd (11.5, 3.0) dq (11.5, 3.0) m

s s s d (6.5) s d (19.2) d (19.2)

5.69, s

3.98, q (6.0) 1.46, t (6.0)

δC 29.12, CH2 23.2, CH2 30.0, CH2 153.1, C 44.5, C 32.6, CH2 26.9, CH2 33.7, 38.8, 88.2, 107.4,

CH C C CH2

27.7, 16.2, 20.0, 27.9,

CH3 CH3 CH3 CH2

111.3, C 155.6, C 179.3, C 95.0, CH 148.5, C 182.0, C 29.05, CH3

11 δH (J in Hz)

1.94, 1.88, 1.86, 1.73, 2.20, 2.61,

1.81, 1.96, 1.61, 1.29, 1.47,

4.90, 4.80, 1.40, 0.75, 1.12, 2.57, 2.08,

m m m m dd (15.0, 5.4) m

td (14.4, 4.2) m qd, (16.8, 3.6) dq (13.2, 4.2) m

br s t (1.8) s d (6.6) s d (18.6) d (18.0)

5.25, br s

2.84, d (6.0)

δC

12 δH (J in Hz)

δC

δH (J in Hz)

39.3, CH2

1.75, m

25.5, CH2

18.4, CH2

1.63, 1.45, 1.39, 1.16,

m m m m

21.7, CH2

0.97, 1.75, 1.32, 2.23, 1.75,

m m m m m

41.7, CH2 33.2, C 56.0, CH 19.7, CH2 40.2, CH2 81.7, 51.3, 37.1, 33.4,

C CH C CH3

21.5, 20.5, 15.0, 16.1,

CH3 CH3 CH3 CH2

116.4, 152.4, 182.4, 105.0, 158.8, 181.5, 65.3, 13.8,

C C C CH C C CH2 CH3

37.9, CH2 39.4, C 77.8, C 29.0, CH2 27.4, CH2

0.90, s

36.5, 39.3, 40.0, 24.8,

CH C CH CH3

0.83, 1.22, 0.88, 0.85,

22.5, 16.1, 27.6, 38.8,

CH3 CH3 CH3 CH2

106.3, 133.0, 114.2, 153.3, 114.2, 133.0,

C CH CH C CH CH

0.88, m

s s s m

5.73, s

1.25, m 1.46, m 1.38, m 1.38, m 1.23, m

1.86, 1.44, 1.38, 1.73, 1.68,

td (16.0, 4.5) m m m m

1.74, dd (16.0, 3.5) 0.85, s 0.99, 0.93, 0.81, 3.57, 2.28,

s d (8.0) s d (16.0) d (16.0)

7.31, d (10.5) 6.73, d (10.5) 6.73, d (10.5) 7.31, d (10.5)

3.98, q (8.5) 1.46, t (8.5)

5.94, br s

(δC 27.4), C-8 (δC 36.5), and C-9 (δC 39.3) and from H3-14 (δH 0.81) to C-8, C-9, and C-10 (δC 40.0) revealed the two methyls were attached to C-8 and C-9, respectively. Furthermore, HMBC cross-peaks from H-10 (δH 1.74) to C1 (δC 25.5), C-5, and C-8 and from H-1 (δH 1.25) to C-2 (δC 21.7), C-5, and C-9 determined the decalin moiety with four methyl groups (CH3-11, CH3-12, CH3-13, and CH3-14). Moreover, HMBC correlations from H2-15 (δH 3.57 and 2.28) to C-8, C-9, C-10, and C-14 (δC 27.6) and to aromatic carbons C-16 (δC 106.3) and C-17 (δC 133.0) allowed for the linkage of the two moieties by the methylene CH2-15. Additionally, considering the chemical shift of C-5 (δC 77.8) and C-19 (δC 153.3), the two hydroxy groups were attached at C-5 and C-19, respectively. The relative configuration of 12 was established by the NOESY spectrum (Figure 2). NOESY correlations of H-8 (δH 1.68)/H-10 (δH 1.74) and H-10/H-15α (δH 3.57) revealed that these protons are α-oriented, while the correlations of H3-13 (δH 0.93)/H3-14 (δH 0.81), H3-13/H-15β (δH 2.28), and H314/H-1β (δH 1.46) indicated that these protons are β-oriented. Besides, the NOESY spectrum was remeasured in DMSO-d6, and the correlations of H3-11/5-OH and H3-12/H-10 confirmed the trans fusion of rings A/B and the β-orientation of 5-OH. In addition, the similar specific rotations for 12 ([α]24D −13.6) and the structurally related ambliol B ([α]24D −3.4) suggested the same absolute configuration34 (NMR data

Figure 8. Experimental and calculated ECD spectra of 11.

Analysis of the 13C NMR and HSQC data revealed the presence of 21 carbons, including four aromatic methines, one quaternary aromatic carbon, two aliphatic quaternary carbons, two nonprotonated carbons, two aliphatic methines, six methylenes, and four methyl groups (Table 3). Interpretation of the COSY and HSQC spectra of 12 led to the assignment of two isolated spin systems: (a) C-10−C-1−C-2−C-3 and (b) C6−C-7−C-8−C-13, as shown in Figure 1. HMBC correlations from H3-11 (δH 0.99) and H3-12 (δH 0.85) to C-3 (δC 37.9), C4 (δC 39.4), and C-5 (δC 77.8) located both of the methyls at C-4. Further HMBC correlations from H3-13 (δH 0.93) to C-7 1442

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from GE Healthcare Bio-Sciences. The machine was purged with running buffer prior to analysis. All solutions were prepared using ultrapure water obtained from a Milli-Q ultrapure H2O system, and all buffer and solutions used for SPR assay were filtered through a 0.22 μm filter. Animal Material. The sample of Spongia pertusa Esper was collected off the coast of Yongxing Island in April 2013. The sponge was identified by Prof. Jin-He Li (Institute of Oceanology, Chinese Academy of Sciences) and was frozen shortly after collection and transported frozen to the laboratory. A voucher sample (No. 1316) is maintained at Research Center for Marine Drugs, Department of Pharmacy, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China. Extraction and Isolation. The sponge (1.75 kg, dry weight) was extracted with 95% EtOH repeatedly to give an EtOH extract (75 g). The extract was dissolved in 1 L of H2O and partitioned with the same volume of CH2Cl2 three times to yield a CH2Cl2 solvent extract (56 g), which was partitioned between 90% aqueous MeOH and petroleum ether to afford a petroleum ether fraction (10 g) and an aqueous MeOH fraction (36 g). Addition of H2O to the aqueous MeOH fraction afforded a 60% aqueous MeOH solution, which was partitioned with CH2Cl2 three times to yield the CH2Cl2-soluble fraction (18 g). The CH2Cl2-soluble fraction was subjected to vacuum liquid chromatography (VLC) on silica gel by gradient elution using petroleum ether/EtOAc (100:1, 80:1, 60:1, 40:1, 20:1, 10:1, 8:1, 5:1, 2:1, 1:1, 0:1, v/v) as the solvent to give 12 fractions (D1−D12). Fraction D5 (251 mg) was passed through an ODS chromatography column eluted with a gradient of aqueous MeOH (from 50% to 100%) to give eight fractions, D5A−D5G. Fraction D5E was purified by reversed-phase HPLC (YMC-Park Pro C18, 10 × 250 mm, 2 mL/ min) with 68% CH3CN to give 4 (3.8 mg, tR = 33 min) and 5 (1.2 mg, tR = 34 min), while the reversed-phase HPLC purification of D5F by 60% CH3CN resulted in the isolation of 10 (5.2 mg, tR = 25 min), 9 (4 mg, tR = 40 min), and 15 (15 mg, tR = 35 min). Fraction D6 (2.2 g) was separated by ODS column chromatography, eluting with a gradient of aqueous MeOH (from 20% to 100%), to give eight fractions, D6A−D6G. Fractions D6A and D6C were further purified by reversed-phase HPLC with 60% CH3CN to give 1 (1.2 mg, tR = 45 min), 12 (2.3 mg, tR = 50 min), and 3 (1.8 mg, tR = 27 min). Fraction D8 (1.8 g) was subjected to column chromatography on Sephadex LH-20 using CHCl2/MeOH (1:1) as the solvent to give 10 fractions (D8A−D8J). Fractions D8H and D8J were further purified by reversed-phase HPLC with 60% CH3CN, to give 2 (2.1 mg, tR = 42 min) and 6 (5.9 mg, tR = 35 min). Fractions D8E, D8F, and D8H were combined and further purified by reversed-phase HPLC with 55% CH3CN to give 7 (1 mg, tR = 48 min), 8 (1 mg, tR = 64 min), 11 (1.5 mg, tR = 70 min), 13 (3.2 mg, tR = 19 min), and 14 (3.1 mg, tR = 19 min). 18-Deoxy-18-formamidodictyoceratin B (1): yellow powder; [α]24D +5.3 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 205 (4.19), 230 (4.16) nm; ECD (c 0.3 mM, MeOH), λmax (Δε) 205 (−14.1), 252 (1.28) nm; IR (KBr) νmax 3282, 2926, 2857, 1665, 1598, 1460, 1439, 1365, 1275, 1198, 1096, 795 cm−1; 1H NMR (500 MHz, CDCl3) and 13 C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS m/z 414.2283 [M − H]− (calcd for C24H32NO5, 414.2280). 18-Deoxy-18-(2-hydroxyacetyl)aminodictyoceratin B (2): yellow powder; [α]24D +6.3 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 204 (4.14), 233 (4.21), 309 (3.48) nm; ECD (c 0.3 mM, MeOH), λmax (Δε) 204 (−12.4), 252 (1.88) nm; IR (KBr) νmax 3319, 2924, 2854, 1664, 1551, 1439, 1354, 1261, 1211, 1095, 1030, 798 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS m/z 468.2725 [M + Na]+ (calcd for C25H35NO6Na, 468.2726). Dictyoceratin D (3): yellow powder; [α]24D −7.8 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 206 (4.07), 259 (3.65), 320 (3.23) nm; ECD (c 0.3 mM, MeOH), λmax (Δε) 205 (−0.86), 215 (−4.70), 233 (5.83) nm; IR (KBr) νmax 3260, 2925, 2855, 1714, 1602, 1514, 1461, 1282, 1096, 1037, 803 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS m/z 377.2096 [M + Na]+ (calcd for C23H30O3Na, 377.2093).

of compound 12 in DMSO-d6, Supporting Information Table S1). Besides the nine new compounds and three solventgenerated artifacts, three known compounds, 5-epi-smenospongine (13), smenospongine (14), and smenospongiadine (15), were also obtained. Their structures were determined by comparison of their MS, NMR, ECD, and specific rotation data with the reported literature values.35−37 The cytotoxicities of all 15 compounds were evaluated against four human cancer cell lines, U937, A549, HeLa, and HepG2, by the CCK-8 method.38 There were differences in the sensitivity of the above cell lines to these sesquiterpene quinones/hydroquinones. Among them, none were cytotoxic to the A549 cancer cells, but compounds 13−15 exhibited cytotoxicities to the U937, HeLa, and HepG2 cancer cells with IC50 values from 0.6 to 8.6 μM (Table 4). Especially, these Table 4. Cytotoxicities of Compounds 13−15 IC50 (μM) compound

U937

A549

HeLa

HepG2

13 14 15 doxorubicin

2.8 1.5 0.60 0.10

>10 >10 >10 0.10

>10 8.6 5.4 0.10

>10 6.7 3.5 0.10

three sesquiterpene quinones displayed potent activity against the U937 cell line with IC50 values of 2.8, 1.5, and 0.6 μM, respectively. A previous study showed compound 13 had no growth inhibition on the proliferation of human prostate cancer DU145 cells,39 but we found that U937 cells were sensitive to 13. Compound 14 had been reported to have activity against the K562, HL60, and U937 cell lines, and we confirmed its cytotoxicity to the U937 cells.17 In addition, all of the compounds were tested for their affinities to CDK-2 in a surface plasmon resonance (SPR) assay. The new compound 6 displayed CDK-2 affinity with a Kd value of 4.8 μM (Figure S139). Compound 6 is the first example of a marine sesquiterpene quinone with CDK-2 affinity. A future functional study will be needed for the further exploration of the effect that this new sesquiterpene quinone has on CDK-2.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotation measurements were conducted using an Autopol I polarimeter (No. 30575, Rudolph Research Analytical) with a 10 cm length cell at room temperature. UV and IR (KBr) spectra were recorded on a Hitachi U3010 spectrophotometer and Jasco FTIR-400 spectrometer, respectively. ECD spectra were obtained on a Jasco J-715 spectropolarimeter. 1 H, 13C, DEPT135, COSY, HSQC, HMBC, and NOESY NMR spectra were obtained using a Bruker Avance DRX-500 MHz NMR spectrometer with CDCl3 as the solvent and internal standard. Spectra were referenced to residual solvent signals with resonances at δH/δC 7.26/77.0 for CDCl3 and 2.49/39.5 for DMSO-d6. HRESIMS data were acquired using a Waters Q-Tof micro YA019 mass spctrometer. Column chromatography was conducted using silica gel 60 (200−300 mesh; Yantai), Sephadex LH-20 (18−110 μm, Pharmacia Co.), and ODS (50 μm, YMC Co.) Analytical thin-layer chromatography (TLC) was performed on silica gel 60 F254 plates. Reversed-phase HPLC was performed on YMC-Pack Pro C18 RS (5 μm) columns using a Waters 1525 separation module equipped with a Waters 2998 photodiode array detector. All chemicals were of analytical grade, solvents for open column chromatography and MPLC were also analytical grade, whereas solvents for HPLC were chromatography grade. The SPR biosensor (Biacore T200) and SPR sensor chips (CM5) were obtained 1443

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Smenospongine (14): red powder; [α]24D −3.5 (c 0.1, CHCl3); ECD (c 0.15 mM, MeOH), λmax (Δε) 312 (−1.32), 200 (7.40) nm; ESIMS m/z 344.0 [M + H]+. Smenospongiadine (15): purple-red powder; [α]24D −16.5 (c 0.25, CHCl3); ECD (c 0.15 mM, MeOH), λmax (Δε) 376 (−0.56), 200 (7.15) nm; ESIMS 448.2 [M + H]+. Energy Minimization and ECD Calculations. The initial conformations of 1−12 were optimized using the MMFF94 method in MarvinSketch 5.8.1 and then the HF/6-31G(d) method in Gaussian 09. Further optimization at the B3P86/6-31G(d) level led to the final dihedral angles. The optimized conformations were used for the ECD calculations, which were performed with Gaussian 09 (B3P86/6-311+ +G(2d,p)). The solvent effects were taken into account by the conductor-like polarizable calculation model (MeOH as the solvent). Cytotoxicity Bioassay. The CCK-8 method was used for in vitro evaluation of the cytotoxicity potential of all these 15 compounds against the human cancer cell lines U937, A549, HeLa, and HepG2. The cell lines were cultured in RPMI-1640 or DMEM medium (Hyclone), supplemented with 10% fetal bovine serum (Hyclone) in 5% CO2 at 37 °C. The cytotoxicity assay was performed in 96-well microplates. Briefly, adherent cells (100 μL) were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition. Suspended cells were seeded at an initial density of 1 × 108 cells/mL. Each cancer cell line was exposed to the tested compound at various concentrations in triplicate for 72 h with doxorubicin as the positive control. The cells in each well were then solubilized with DMSO (100 μL for each well), and the optical density (OD) was measured at 450 nm in a 96-well microtiter plate reader (Spectra MAX340). IC50 values were derived from the mean OD values of the triplicate tests versus drug concentration curves. Surface Plasmon Resonance Assay. SPR measurements were performed using a Biacore T200 instrument with four flow channels and a CM5 sensor chip with a dextran matrix. According to the amine coupling protocol, CDK-2 (50 μg/mL in 10 mM acetate buffer at pH 4.5) was immobilized on the second flow cell of the CM5 chip, while bovine serum albumin (BSA) (50 μg/mL in 10 mM acetate buffer at pH 3.8) was immobilized on the first flow cell as a reference. The immobilization level of CDK-2 was about 5500 RU, and it was about 6000 RU for BSA. For the binding assay, the reaction temperature was controlled at 25 ± 0.1 °C, and the flow rate was set at 30 μL/min. The response obtained from the detection channel (Fc 2) was normalized by subtracting the signal simultaneously acquired from the control channel (Fc 1), which could eliminate nonspecific binding and bufferinduced bulk refractive index changes. PBS buffer (10 mM phosphate buffer, 2.7 mM KCl, and 137 mM NaCl, pH 7.4) was used as the running buffer. Different concentrations of analytes were prepared and measured in the SPR assay, and a solvent correction was performed to adjust the solvent response. The sample solutions were injected onto the chip surface for 120 s. After each binding reaction, a further dissociation time of 120 s was applied after each injection. In addition, the analytes were regenerated from the chip surface with glycine-HCl buffer at pH 2.1 for 30 s. The binding affinity between CDK-2 and the analytes was fitted in the affinity model with Biacore T200 Evaluation Software 1.0.

N-Methyl-ent-smenospongine (4): purple-red powder; [α]24D +16.2 (c 0.2, CHCl3); UV (MeOH) λmax (log ε) 204 (3.84), 317 (3.46) nm; ECD (c 0.15 mM, MeOH), λmax (Δε) 205 (−14.7) nm; IR (KBr) νmax 3279, 2924, 2854, 1656, 1582, 1513, 1378, 1205 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 1; HRESIMS m/z 356.2224 [M − H]− (calcd for C22H30NO3, 356.2226). N-Methyl-5-epi-smenospongine (5): purple-red powder; [α]24D +73.4 (c 0.1, CHCl3); UV (MeOH) λmax (log ε) 203 (3.60), 319 (3.12) nm; ECD (c 0.3 mM, MeOH), λmax (Δε) 206 (−7.79), 217 (0.25) nm; IR (KBr) νmax 3275, 3083, 2956, 2921, 2852, 1731, 1651, 1580, 1514, 1379, 1260, 1208, 1094, 1030, 799 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS m/z 356.2223 [M − H]− (calcd for C22H30NO3, 356.2226). 20-Demethoxy-20-methylaminodactyloquinone D (6): purple-red powder; [α]24D +60.6 (c 0.05, CHCl3); UV (MeOH) λmax (log ε) 203 (3.81), 317 (3.20) nm; ECD (c 0.15 mM, MeOH), λmax (Δε) 203 (−7.02), 314 (4.43) nm; IR (KBr) νmax 3333, 2954, 2926, 2855, 1707, 1656, 1593, 1514, 1460, 1225, 1206 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS m/z 378.2047 [M + Na]+ (calcd for C22H29NO3Na, 378.2045). 20-Demethoxy-20-methylamino-5-epi-dactyloquinone D (7): purple-red powder; [α]24D +31.6 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 204 (3.81), 318 (3.39) nm; ECD (c 0.15 mM, MeOH), λmax (Δε) 214 (−0.61), 225 (−1.24), 315 (2.72) nm; IR (KBr) νmax 3342, 2927, 2856, 1654, 1593, 1514, 1455, 1387, 1260, 1202, 1075, 801 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS m/z 378.2044 [M + Na]+ (calcd for C22H29NO3Na, 378.2045). 20-Demethoxy-20-ethoxydactyloquinone E (8): yellow powder; [α]24D −1.6 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 206 (4.05), 291 (4.02) nm; ECD (c 0.25 mM, MeOH), λmax (Δε) 206 (2.48), 249 (−2.65), 219 (2.33) nm; IR (KBr) νmax 2934, 2862, 1663, 1641, 1600, 1447, 1224, 1202, 1052 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 2; HRESIMS m/z 393.2309 [M + Na]+ (calcd for C23H30O4Na, 393.2042). 20-Demethoxy-20-ethoxydactyloquinone B (9): yellow powder; [α]24D −23.0 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 204 (3.92), 291 (3.76) nm; ECD (c 0.25 mM, MeOH), λmax (Δε) 218 (3.81), 293 (−4.13) nm; IR (KBr) νmax 3295, 2931, 2859, 1663, 1644, 1600, 1221, 1045 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 3; HRESIMS m/z 371.2226 [M + H]+ (calcd for C23H31O4, 371.2217). 20-Demethoxy-20-methylaminodactyloquinone B (10): purplered powder; [α]24D −62.2 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 206 (4.12), 292 (4.04) nm; ECD (c 0.15 mM, MeOH), λmax (Δε) 209 (−5.80), 231 (2.59), 314 (−5.93) nm; IR (KBr) νmax 3299, 3084, 2933, 1732, 1662, 1645, 1599, 1465, 1348, 1269, 1221, 1095, 1046, 799, 736 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 3; HRESIMS m/z 356.2219 [M + H]+ (calcd for C22H30NO3, 356.2220). 20-Demethoxy-20-ethoxycyclospongiaquinone-1 (11): yellow powder; [α]24D +18.5 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 204 (4.01), 288 (3.60) nm; ECD (c 0.15 mM, MeOH), λmax (Δε) 249 (−4.41), 288 (4.34) nm; IR (KBr) νmax 3361, 2926, 2856, 1735, 1661, 1635, 1598, 1460, 1381, 1261, 1228, 1087, 1036, 803 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 3; HRESIMS m/z 373.2377 [M + H]+ (calcd for C23H33O4, 373.2273). Yahazunol B (12): yellow powder; [α]24D −13.6 (c 1.12, CHCl3); UV (MeOH) λmax (log ε) 204 (4.02), 224 (3.95), 277 (3.37) nm; ECD (c 0.15 mM, MeOH), λmax (Δε) 200(9.55), 228 (−4.03) nm; IR (KBr) νmax 3357, 2960, 2928, 2862, 1694, 1610, 1511, 1449, 1370, 1258, 1241, 1092, 1029, 824, 804 cm−1; 1H NMR (500 MHz, CDCl3) and 13C NMR (125 MHz, CDCl3) data, Table 3; HRESIMS m/z 339.2297 [M + Na]+ (calcd for C21H32O2Na, 339.2300). 5-epi-Smenospongine (13): red powder; [α]24D +82.6 (c 0.2, CHCl3); ECD (c 0.15 mM, MeOH), λmax (Δε) 336 (−0.62), 253 (1.24), 200 (−9.33) nm; HRESIMS m/z 344.2226 [M + H]+.

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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.6b01105. Copies of 1D and 2D NMR, HRESIMS, UV, IR, and ECD spectra for 1−12 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-21-68383346. Fax: +86-21-58732594. E-mail: [email protected] (F. Yang). 1444

DOI: 10.1021/acs.jnatprod.6b01105 J. Nat. Prod. 2017, 80, 1436−1445

Journal of Natural Products

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*E-mail: [email protected] (X. C. Zhang). *E-mail: [email protected] (H. W. Lin).

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ORCID

Hou-Wen Lin: 0000-0002-7097-0876 Author Contributions ⊥

J. Li and B. B. Gu contributed equally to this paper.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Natural Science Fund for Distinguished Young Scholars of China (81225023), the National Natural Science Fund of China (Nos. U1605221, 41476121, 81373321), the Innovation Program of Shanghai Municipal Education Commission (No. 14YZ037), National High Technology Research and Development Program of China (863 Projects, No. 2013AA092902), Beijing Medical Award Foundation (No. YJHYXKYJJ-125), and the Fund of the Science and Technology Commission of Shanghai Municipality (Grant No. 15431900900).

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DOI: 10.1021/acs.jnatprod.6b01105 J. Nat. Prod. 2017, 80, 1436−1445