Ceylonamides A–F, Nitrogenous Spongian Diterpenes That Inhibit

Aug 15, 2016 - ... Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands ... Maria Camila A. Ramirez , David E. Williams , Juliana R. Gu...
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Ceylonamides A−F, Nitrogenous Spongian Diterpenes That Inhibit RANKL-Induced Osteoclastogenesis, from the Marine Sponge Spongia ceylonensis Ahmed H. El-Desoky,† Hikaru Kato,† Esther D. Angkouw,‡ Remy E. P. Mangindaan,‡ Nicole J. de Voogd,§ and Sachiko Tsukamoto*,† †

Graduate School of Pharmaceutical Sciences, Kumamoto University, Oe-honmachi 5-1, Kumamoto 862-0973, Japan Faculty of Fisheries and Marine Science, Sam Ratulangi University, Kampus Bahu, Manado 95115, Indonesia § Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA Leiden, The Netherlands ‡

S Supporting Information *

ABSTRACT: Seven new spongian diterpenes, ceylonamides A−F (1−6) and 15α,16-dimethoxyspongi-13-en-19-oic acid (7), were isolated from the Indonesian marine sponge Spongia ceylonensis along with eight known spongian diterpenes, 8−15. Compounds 1−6 were determined to be nitrogenous spongian diterpenes. The isolated compounds were examined for the inhibition of RANKLinduced osteoclastogenesis in RAW264 macrophages. Ceylonamide A (1) exhibited the most potent inhibitory activity with an IC50 value of 13 μM, followed by ceylonamide B (2) (IC50, 18 μM). An examination of the structure−activity relationships of the isolated compounds revealed that the position of the carbonyl group of the γ-lactam ring and bulkiness of the substituent at its nitrogen atom were important for inhibitory activity.

B

sponges and marine-derived fungi. The EtOAc-soluble fraction of the extract derived from the marine sponge Spongia ceylonensis exhibited inhibitory activity. The sponge was collected in Indonesia in 2007 and extracted with EtOH. After evaporation, the extract was partitioned between EtOAc and H2O, and the EtOAc layer was subjected to silica gel column chromatography followed by HPLC to afford seven new compounds (1−7) and eight known compounds (8−15). Ceylonamide A (1) showed a protonated molecule by HRESIMS corresponding to a molecular formula of C28H37NO3. 1H and 13C NMR spectra displayed characteristic signals reminiscent of terpenes including three methyl signals [δH 1.05 (s), δC 22.2 (C-17); δH 1.22 (s), δC 29.6 (C-18); δH 0.80 (s), δC 14.0 (C-20)], two carbonyl carbons [δC 172.0 (C16) and δC 182.7 (C-19)], two olefinic carbons [δC 129.1 (C13) and δC 160.2 (C-14)], and signals of a phenyl group (Table 1). An analysis of 2D NMR spectra including COSY, HSQC, and HMBC (Figure 1) and molecular formulas revealed that 1 was a structural isomer of haumanamide (8).7 Marked differences in the carbon chemical shifts of these compounds were observed at δC 22.2 (C-12), 129.1 (C-13), and 160.2 (C14) (CDCl3) for 1 and at δC 26.3 (C-12), 147.7 (C-13), and 141.1 (C-14) (CDCl3−MeOH-d4) for 8,7 which indicated that the γ-lactam ring of 1 possessed the opposite orientation of that

one density is maintained by the balance between bone formation by osteoblasts and bone resorption by osteoclasts.1 The stimulation of a monocyte/macrophage lineage with receptor activator of nuclear factor-κB ligand (RANKL) was shown to induce its differentiation into multinuclear osteoclasts.1−3 RANKL stimuli activate several downstream signaling pathways such as the NF-κB and MAPK signaling pathways, which, in turn, up-regulate the expression of osteoclast-specific genes including those encoding tartrateresistant acid phosphatase (TRAP) and enzymes involved in cell fusion. Several diseases are associated with malfunctions in osteoclasts such as osteoporosis and bone metastasis, which has attracted increasing attention to compounds that affect osteoclastogenesis and the functions of osteoclasts for the treatment of osteoclast-related diseases.4,5 In our continuing efforts to search for inhibitors of the osteoclastogenic differentiation of murine RAW264 cells from natural sources,6 we performed screening in which the RANKLinduced up-regulation of TRAP activity was measured in RAW264 cells. We herein report the isolation and structure elucidation of seven new spongian diterpenes (1−7) along with eight known terpenes (8−15) and their inhibition of TRAP activity.

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RESULTS AND DISCUSSION Screening was performed with the EtOAc- and water-soluble fractions prepared from the EtOH extracts of 250 marine © XXXX American Chemical Society and American Society of Pharmacognosy

Received: February 23, 2016

A

DOI: 10.1021/acs.jnatprod.6b00158 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 1. 1H and 13C NMR Data (500/125 MHz, CDCl3) for Ceylonamide A (1) δC, type

δH, mult (J in Hz)

1

39.9, CH2

2

18.9, CH2

3

37.9, CH2

0.86, 1.80, 1.43, 1.85, 1.01, 2.14,

m m m m td (13.2, 3.8) br d (13.2)

4 5 6

44.0, C 57.1, CH 19.7, CH2

7

38.1, CH2

1.10, 1.88, 2.01, 1.21, 1.65,

br d (13.2) m m m dt (12.5, 2.8)

8 9 10 11

36.3, 55.5, 38.0, 17.4,

12

22.2, CH2

no.

13 14 15 16 17 18 19 20 21 22 23 24, 28 25, 27 26

of 8. Hence, carbons at C-12 and C-13 were deshielded, which was confirmed by the HMBC correlation from H-12 (δH 2.36) to C-16 (δC 172.0) and from H3-17 (δH 1.05) to C-14 (δC 160.2) (Figure 1a). NOE correlations showed that the ring junctions A/B and B/C were trans and a methyl group at C-4 was equatorially oriented. Ceylonamides B (2) and D (4) had the same molecular formula of C25H39NO3, as established by HRESITOFMS. Their 1 H and 13C NMR spectra (Table 2) were similar to those of 1. Marked differences were the absence of aromatic signals and presence of two equivalent methyl groups [δH 0.90 (6H, d, J = 6.5 Hz)/δC 22.6 (2C) for 2 and δH 0.90 (6H, d, J = 6.6 Hz)/δC 22.6 (2C) for 4] at C-24 and C-25. An inspection of 2D NMR spectra showed the replacement of the phenyl group in 1 by an isopropyl group (C-23−C-25) in 2 and 4 (Figure 2). The carbon chemical shifts of C-12 (δC 22.2/26.0, 2/4), C-13 (δC 129.3/147.2, 2/4), and C-14 (δC 159.8/141.2, 2/4) indicated the orientations of the γ-lactam ring of 2 and 4, which were the same as those of 1 and 8, respectively. Ceylonamides C (3) and E (5) had the same molecular formula of C24H37NO3, which is one methylene unit less than 2 and 4, and their NMR data (Table 3) were similar to those of 2 and 4. HMBC correlations [H2-21 (δH 3.19 (2H))/C-15 (δC 49.2) and C-16 (δC 172.3) for 3; H2-21 (δH 3.22 and 3.09)/C15 (δC 171.0) and C-16 (δC 52.8) for 5] (Figure 2) revealed that an isobutyl group was attached to a nitrogen atom in the γlactam ring. The orientations of the γ-lactam ring of 3 and 5 were indicated to be the same as those of 2 and 4, respectively. The molecular formula C20H29NO3 and NMR data (Table 4) of ceylonamide F (6) suggested that it was a simple congener of 1−5. HMBC correlations from NH (δH 7.65) to C-13 (δC 151.2), C-14 (δC 140.2), C-15 (δC 173.3), and C-16 (δC 47.3)

129.1, 160.2, 49.1, 172.0, 22.2, 29.6, 182.7, 14.0, 43.7, 35.2, 139.1, 128.5, 128.7, 126.4,

C CH C CH2

C C CH2 C CH3 CH3 C CH3 CH2 CH2 C CH CH CH

1.08, br d (12.0) 1.41, 1.79, 2.07, 2.36,

m m m dd (17.5, 5.0)

3.54, br s 1.05, s 1.22, s 0.80, s 3.62, m 2.85, t (7.1) 7.18, m 7.26, m 7.19, m

Figure 1. (a) COSY and key HMBC correlations of 1. (b) Key NOE correlations in the energy-minimized conformation of 1, calculated using Spartan’14 (Wavefunction, Inc.).

(Figure 2) showed that 6 contained a nonsubstituted γ-lactam ring as depicted. The specific rotations of 1−6 showed negative signs (−26, −29, −26, −38, −23, and −110, respectively), which were similar to those of spongolactams A, B, and C (−37, −12, and −30, respectively).8 The absolute stereostructures of the spongolactams were established by semisyntheses8 from B

DOI: 10.1021/acs.jnatprod.6b00158 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 2. 1H and 13C NMR Data (500/125 MHz, CDCl3) for Ceylonamides B (2) and D (4) 2

Table 3. 1H and 13C NMR Data (500/125 MHz, CDCl3) for Ceylonamides C (3) and E (5)

4

3

5

no.

δC, type

δH, mult (J in Hz)

δC, type

δH, mult (J in Hz)

no.

δC, type

δH, mult (J in Hz)

δC, type

1

39.9, CH2

40.1, CH2

39.9, CH2

2

18.9, CH2

3

37.6, CH2

3

37.9, CH2

4 5 6

43.7, C 57.1, CH 19.0, CH2

0.88, m 1.79, m 1.44, m 1.90, m 1.01, td (13.5, 3.8) 2.14, br d (13.5)

40.1, CH2

18.9, CH2

0.86, br d (6.7) 1.78, m 1.42, m 1.93, m 0.99, td (13.2, 4.2) 2.12, br d (13.2)

1

2

0.85, m 1.79, m 1.44, m 1.90, m 1.01, td (13.2, 4.0) 2.13, br d (13.2)

4 5 6

43.7, C 57.1, CH 19.8, CH2

7

38.1, CH2

1.09, m 1.82, m 1.94, m 2.83, dt (13.4, 2.9) 1.17, m

7

38.3, CH2

1.08, m

8 9 10 11

36.4, 55.5, 38.1, 17.5,

12

22.3, CH2

8 9 10 11 12

36.4, 55.5, 37.9, 17.5,

C CH C CH2

22.2, CH2

1.10, 1.87, 2.04, 1.32, 1.75,

m m br d (12.2) br t (12.2) m

1.10, m 1.41, m 1.82, m 2.07, m 2.36, dd (17.9, 4.3)

13 14 15

129.3, C 159.8, C 48.4, CH2

16

172.0, C

17 18 19 20 21

22.6, 28.9, 182.6, 14.1, 40.5,

CH3 CH3 C CH3 CH2

1.11, s 1.22, s

22 23 24 25

37.6, 25.9, 22.6, 22.6,

CH2 CH CH3 CH3

1.40, 1.54, 0.90, 0.90,

3.68, d (18.6) 3.75, d (18.6)

19.7, CH2 38.1, CH2 43.7, C 57.3, CH 19.0, CH2 35.9, CH2 36.3, 56.4, 38.0, 17.7,

C CH C CH2

26.0, CH2

m m d (6.5) d (6.5)

21.0, 28.8, 182.6, 14.0, 40.2,

CH3 CH3 C CH3 CH2

37.6, 26.0, 22.6, 22.6,

CH2 CH CH3 CH3

m m br d (13.4) br d (13.4) br d (12.6)

1.11, m 1.44, m 1.82, m 2.06, m

38.0, CH2

3.56, 3.66, 1.16, 1.22,

d (18.6) d (18.6) s s

36.3, CH2 35.8, 56.4, 38.0, 17.6,

C CH C CH2

26.4, CH2

0.80, s 3.30, td (13.8, 7.5) 3.42, td (13.8, 7.5) 1.39, q (7.5) 1.54, m 0.90, d (6.6) 0.90, d (6.6)

13 14 15

129.1, C 159.9, C 49.2, CH2

16

172.3, C

17 18 19 20 21

22.3, CH3 28.9, CH3 182.1, C 14.1, CH3 49.8, CH2

1.11, s 1.22, s 0.81, s 3.19, d (7.4)

21.0, CH3 28.8, CH3 181.7, C 14.0, CH3 49.6, CH2

22 23 24

28.0, CH 20.2, CH3 20.2, CH3

1.88, m 0.87, d (6.5) 0.87, d (6.5)

28.0, CH 20.1, CH3 20.2, CH3

3.69, d (18.7) 3.77, d (18.7)

m m m m td (13.0, 4.3)

2.13, br d (13.0) 43.6, C 57.3, CH 19.0, CH2

2.37, dd (17.8, 3.8)

147.2, C 141.2, C 170.7, C 52.0, CH2

0.82, s 3.41, m

1.43, m 1.79, m 2.20, m 2.29, dd (18.1, 5.4)

C CH C CH2

1.13, 1.86, 2.02, 1.32, 1.77,

19.7, CH2

δH, mult (J in Hz) 0.86, 1.78, 1.44, 1.96, 1.00,

1.11, 1.84, 1.98, 1.15, 2.84,

br d, (13.1) m br d (13.1) m br d (13.1)

1.08, m 1.43, m 1.80, m 2.21, ddd (18.2, 11.0, 6.5) 2.28, dd (18.2, 6.5)

147.1, C 141.2, C 171.0, C 52.8, CH2

3.58, 3.68, 1.16, 1.22,

d (18.6) d (18.6) s s

0.81, 3.09, 3.22, 1.86, 0.86, 0.87,

s dd (13.6, 7.6) dd (13.6, 7.6) m d (6.6) d (6.6)

and B (11), indicated by analyses of NMR data,10 except for the presence of two methoxy groups [δH 3.42/δC 55.0 (C-21) and δH 3.32/δC 53.0 (C-22)] at C-15 and C-16 instead of a carbonyl and hemiacetal carbons in their lactone rings (Table 5). The positions of the methoxy groups were determined to be C-15 and C-16 by HMBC correlations, δH 5.41 (H-15)/δC 55.0 (C-21) and δH 5.29 (H-16)/δC 53.0 (C-22). NOE correlations readily established the A/B trans and B/C trans ring junctions (Figure 3a). The key NOE cross-peaks, H-7β/H-15, H-7α/H15, and H-12α/H-16 (Figure 3b), excluded the 15R,16R-, 15S,16R-, and 15S,16S-configurations and matched the 15R,16S-configuration (Figure 3a). Thus, 7 was determined to be 15R,16S-dimethoxyspongi-13-en-19-oic acid.11 All isolated compounds were tested for their potential to inhibit the RANKL-induced up-regulation of TRAP activity in RAW264 cells, whereas they were not cytotoxic against RAW264 cells. Ceylonamide A (1) exhibited the most significant inhibitory activity, with an IC50 value of 13 μM followed by ceylonamide B (2) (IC50, 18 μM) (Figure 4A), and the IC50 values of other compounds (3−15) were more than 50 μM (Table 6). The RANKL stimulation induced the differ-

Figure 2. Key COSY and HMBC correlations of 2−6.

spongia-13(16),14-dien-19-oic acid (13),9 the absolute stereostructure of which is known. The specific rotation of 13 isolated in this study was +22 (c 0.50, CHCl3) (lit.9 +15; lit.8 +10 (c 0.50, CHCl3)). These results showed the absolute stereostructures of 1−6 as shown. Compound 7 had the molecular formula C22H34O5 and contained the same ring system as spongiabutenolides A (12) C

DOI: 10.1021/acs.jnatprod.6b00158 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Table 4. 1H and 13C NMR Data (600/150 MHz, CDCl3) for Ceylonamide F (6) no.

δC, type

1

40.0, CH

2

19.2, CH2

3

38.2, CH2

4 5 6

43.4, C 56.8, CH 19.9, CH2

7

36.7, CH2

8 9 10 11

35.7, 56.4, 38.0, 17.7,

12

26.5, CH2

C CH C CH2

13 14 15 16

151.2, 140.2, 173.3, 47.3,

C C C CH2

17 18 19 20 NH

21.2, 29.5, 178.9, 14.4,

CH3 CH3 C CH3

Table 5. 1H and 13C NMR Data (600/150 MHz, CDCl3) for 15R,16S-Dimethoxyspongi-13-en-19-oic acid (7)

δH, mult (J in Hz) 0.86, 1.73, 1.36, 1.82, 0.94, 1.99,

m m m dt (13.6, 3.1) td (13.3, 3.1) br d (13.3)

1.05, 1.72, 1.91, 1.03, 2.68,

m m dd (27.2, 14.3) m dt (12.8, 2.9)

1.02, m 1.34, 1.72, 2.18, 2.29,

3.59, 3.63, 1.05, 1.09,

m m ddd (18.1, 11.4, 6.6) dd (18.1, 5.4)

d (19.1) d (19.1) s s

0.75, s 7.65, s

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entiation of RAW264 cells and formation of TRAP-positive multinuclear osteoclasts. However, the presence of 1 or 2 decreased the formation of these cells (Figure 4B), which resulted from the inhibition of RANKL-induced osteoclastogenesis by 1 and 2 (Figure 4C). Examinations of structure− activity relationships indicated that the compounds containing the amide carbonyl at C-16 (1 and 2) exhibited significantly stronger inhibitory activity than those containing the amide carbonyl at C-15 (8 and 4). Because inhibitory potencies were in the order of 1, 2, and 3, the presence of a bulkier substituent bonded at the amide nitrogen may result in more potent inhibitory activity. Tetracyclic diterpenes of the spongian type, containing a furan ring, such as spongia-13(16),14-dien-19-oic acid (13),7,9,10 spongiadiol (14),12 and isospongiadiol (15),12 are metabolites in sponges of the genus Spongia. In addition, diterpenes in which the furan is replaced with an unsaturated γlactone, such as 15-oxospongi-13-en-19-oic acid (9)13 and 16oxospongi-13-en-19-oic acid (10),13 or with a γ-hydroxybutenolide, such as spongiabutenolides A (12) and B (11),10 were reported from Spongia species. In contrast, only three studies have been published on nitrogenous spongian diterpenes with a γ-lactam, namely, haumanamide (8),7 spongolactams A−C,8 and oxeatamides A−G.14 We herein report new nitrogenous spongian diterpenes, ceylonamides A−F (1−6), as inhibitors of RANKL-induced osteoclastogenesis in RAW264 macrophages. These nitrogenous diterpenes may be biosynthesized from spongian diterpenes and decarboxylated amino acids.

no.

δC, type

1

40.1, CH2

2

18.9, CH2

3

37.9, CH2

4 5 6

43.8, C 57.3, CH 19.5, CH2

7

37.6, CH2

8 9 10 11

35.4, 56.1, 38.0, 17.5,

12

22.9, CH2

13 14 15 16 17 18 19 20 21 22

133.7, 145.9, 107.1, 107.1, 20.9, 28.8, 183.5, 14.0, 55.0, 53.0,

C CH C CH2

C C CH CH CH3 CH3 C CH3 CH3 CH3

δH, mult (J in Hz) 0.86, 1.80, 1.45, 1.91, 1.00, 2.14,

td m dt m td br

(13.0, 3.9)

1.10, 1.84, 2.01, 1.26, 1.90,

dd (12.3, 2.4) m ddd (26.3, 13.5, 2.6) ddd (26.3, 12.9, 3.3) m

(14.9, 3.0) (13.6, 4.3) d (13.6)

0.98, br d (10.7) 1.52, 1.76, 1.92, 2.18,

dtd (12.6, 12.6, 6.5) m m dd (18.1, 6.5)

5.41, 5.29, 1.17, 1.23,

d (2.0) s s s

0.81, s 3.42, s 3.32, s

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO DIP-1000 polarimeter in MeOH. UV absorptions were measured on a JASCO V-550 spectrophotometer in MeOH. The IR spectra were recorded on a JEOL JIR-6500W spectrophotometer. 1H and 13C NMR spectra were recorded on a Bruker Avance III 500 or Bruker Avance III 600 NMR spectrometer in CDCl3. Chemical shifts were referenced to the residual solvent peaks (δH 7.24 and δC 77.0 for CDCl3). ESIMS spectra were measured on a Bruker BioTOFQ mass spectrometer. A preparative HPLC system was composed of an Waters 515 HPLC pump, Waters 2489 UV/visible detector, and Pantos Unicorder U-228. Animal Material, Extraction, and Isolation. The sponge, Spongia ceylonensis, was collected by scuba at a depth of 10 m in Tiwoho, North Sulawesi, Indonesia, in 2007 and soaked in EtOH immediately. Voucher specimens (RMNH POR 8676) of the sponge have been deposited in the Naturalis Biodiversity Center. The sponge (wet weight 270 g) was extracted with EtOH. After evaporation, the residual aqueous solution was extracted with EtOAc. The EtOAc fraction (2.3 g) was subjected to SiO2 column chromatography with a stepwise gradient elution using n-hexane−EtOAc, EtOAc, and MeOH to yield 12 fractions (Frs. 1−12). Fr. 2 that eluted with n-hexane− EtOAc (9:1) afforded spongia-13(16),14-dien-19-oic acid (13) (150 mg). Fr. 5 (40 mg) that eluted with n-hexane−EtOAc (8:2) was purified by C30 reversed-phase HPLC (Develosil C30-UG-5 column, Nomura Chemical Co., Ltd., 20 × 250 mm) with 70% MeOH−H2O (0−30 min), 85% MeOH−H2O (30−60 min), and 93% MeOH−H2O (60−80 min) (6 mL/min) to yield 9 (0.68 mg), 10 (1.1 mg), and 7 (3.0 mg). Fr. 8 (24 mg) that eluted with n-hexane−EtOAc (1:1) was further purified by gel filtration HPLC (Asahipack GS-310P column, Asahi Chemical Industry Co., Ltd., 21.5 × 500 mm) with CH2Cl2− MeOH−H2O to yield four fractions: Fr. 8-1 (5.4 mg), Fr. 8-2 (23 mg), D

DOI: 10.1021/acs.jnatprod.6b00158 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Figure 3. (a) Energy-minimized 15R,16S-, 15R,16R-, 15S,16R-, and 15S,16S-7 obtained from calculations with B3LYP/6-31G*. Blue dashed arrows are NOE correlations for 15R,16S-7. Green solid curves for 15R,16R-, 15S,16R-, and 15S,16S-7 are calculated distances between two selected hydrogens. (b) Observed key cross-peaks in the NOESY spectrum of 7.

Figure 4. Inhibitory effects of 1 and 2 on RANKL-induced osteoclastogenesis. (a) RAW264 cells were treated with RANKL (50 ng/mL) in the presence or absence of 1 or 2 at the indicated concentrations and allowed to differentiate for 4 days. TRAP activity was measured as absorbance at 405 nm. (b) RAW264 cells were allowed to differentiate by treatment with RANKL (50 ng/mL) in the presence or absence of 1 (10 μM) and 2 (20 μM) for 4 days and were then stained with TRAP-staining solution. TRAP-positive cells stained red. (c) TRAP-positive multinuclear cells (nuclei ≥3) in the presence of 1 and 2 were counted. Fr. 8-3 (36 mg), and Fr. 8-4 (7.7 mg). Fr. 8-2 was purified by C30 reversed-phase HPLC with 80% MeOH−H2O (0−40 min) and 90% MeOH−H2O (40−80 min) (5 mL/min) to yield 5 (1.0 mg), 8 (3.7 mg), and 4 (2.4 mg). Fr. 8-4 was purified by C30 reversed-phase HPLC with 75% MeOH−H2O (0−100 min) (5 mL/min) to yield 12 (1.9 mg), 11 (0.65 mg), 14 (2.0 mg), and 15 (0.74 mg). Fr. 9 (82 mg) that eluted with n-hexane−EtOAc (1:1) was purified by C30 reversed-

phase HPLC with 70% MeOH−H2O (0−60 min), 85% MeOH−H2O (60−120 min), and 93% MeOH−H2O (120−140 min) (6 mL/min) to yield 12 (2.3 mg), 11 (3.5 mg), 3 (1.6 mg), 1 (0.56 mg), and 2 (1.5 mg). Fr. 12 (340 mg) was subjected to C18 open column chromatography with 75% MeOH−H2O followed by purification by C18 HPLC (COSMOSIL 5C18-AR-II, Nacalai Tesque Inc., 20 × 250 E

DOI: 10.1021/acs.jnatprod.6b00158 J. Nat. Prod. XXXX, XXX, XXX−XXX

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conformational searches with Spartan’14 using MMFF16 as the force field, in which the number of initial conformers was set to 10 000. The most stable 100 conformers obtained were further optimized by the Hartree−Fock (HF) method with 3-21G. The resultant conformers of >1% were finally optimized by the density functional theory (DFT) method with B3LYP/6-31G*, giving stable conformers for further simulations. TRAP Activity. TRAP activity was measured as previously described.6 Osteoclastogenesis Assay. The osteoclastogenesis assay was performed as previously described.6

Table 6. Biological Data of 1−15 compound

RANKL-induced TRAP activity, IC50 (μM)

cell survival ratio (%) at 5 μM

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

13 18 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50 >50

100 87 100 100 100 100 98 97 100 95 100 100 100 97 96

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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.6b00158. 1D and 2D NMR spectra of 1−7 (PDF)

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mm; COSMOSIL 5C18-MS-II, Nacalai Tesque Inc., 20 × 250 mm), gel filtration HPLC, and C30 HPLC to yield 6 (0.24 mg). Ceylonamide A (1): white, amorphous solid; [α]21D −26 (c 1.8, MeOH); UV (MeOH) λmax (log ε) 210 (4.64) nm; IR (film) νmax 2950, 2925, 2853, 1722, 1619, and 1459 cm−1; 1H and 13C NMR data, Table 1; HRESITOFMS m/z 436.2812 [M + H]+ (calcd for C28H38NO3, 436.2846). Ceylonamide B (2): white, amorphous solid; [α]21D −29 (c 0.91, MeOH); UV (MeOH) λmax (log ε) 212 (4.33) nm; IR (film) νmax 3287, 2950, 2922, 2853, 1707, and 1464 cm−1; 1H and 13C NMR data, Table 2; HRESITOFMS m/z 424.2803 [M + Na]+ (calcd for C25H39NO3Na, 424.2822). Ceylonamide C (3): white, amorphous solid; [α]21D −26 (c 0.34, MeOH); UV (MeOH) λmax (log ε) 208 (4.20) nm; IR (film) νmax 3284, 2950, 2923, 2853, 1709, and 1459 cm−1; 1H and 13C NMR data, Table 3; HRESITOFMS m/z 388.2826 [M + H]+ (calcd for C24H38NO3, 388.2846). Ceylonamide D (4): white, amorphous solid; [α]21D −38 (c 1.5, MeOH); UV (MeOH) λmax (log ε) 206 (4.96) nm; IR (film) νmax 3365, 2950, 2923, 2853, 1709, and 1547 cm−1; 1H and 13C NMR data, Table 2; HRESITOFMS m/z 402.2980 [M + H]+ (calcd for C25H40NO3, 402.3003). Ceylonamide E (5): white, amorphous solid; [α]21D −23 (c 0.57, MeOH); UV (MeOH) λmax (log ε) 206 (4.96) nm; IR (film) νmax 3356, 2950, 2923, 2853, 1709, and 1647 cm−1; 1H and 13C NMR data, Table 3; HRESITOFMS m/z 388.2827 [M + H]+ (calcd for C24H38NO3, 388.2846). Ceylonamide F (6): white, amorphous solid; [α]21D −110 (c 0.15, MeOH); UV (MeOH) λmax (log ε) 210 (3.91) nm; IR (film) νmax 3453, 2950, 2922, 2852, 1578, and 1095 cm−1; 1H and 13C NMR data, Table 4; HRESITOFMS m/z 332.2179 [M + H]+ (calcd for C20H30NO3, 332.2220). 15α,16-Dimethoxyspongi-13-en-19-oic acid (7): white, amorphous solid; [α]21D −17 (c 1.5, MeOH); UV (MeOH) λmax (log ε) 206 (4.46) and 218 (4.22, sh) nm; IR (film) νmax 3350, 2950, 2922, 2852, 1725, and 1710 cm−1; 1H and 13C NMR data, Table 5; HRESITOFMS m/z 401.2311 [M + Na]+ (calcd for C22H34O5Na, 401.2298). Conformational Analyses of 15R,16S-, 15R,16R-, 15S,16R-, and 15S,16S-7. Conformational searches were performed with Spartan’14 (ver. 1.1.8 by Wavefunction Inc.) using a commercially available PC (operating system: Windows 7 Professional SP1 64-bit, CPU: QuadCore Core i7-3770 processor 3.40 GHz, RAM 8 GB), and DFT calculations were conducted with Gaussian09 (revivion D.01 by Gaussian)15 using a PC (operating system: CentOS a Linux, CPU: Intel Xeon E5-2603 v3 processors 1.60 GHz, RAM 32 GB). The input structures were constructed on a graphical user interface considering the absolute configurations of interest and were subjected to

AUTHOR INFORMATION

Corresponding Author

*E-mail (S. Tsukamoto): [email protected]. Tel/fax: +81-96-371-4380. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. M. Namikoshi and Dr. K. Ukai of Tohoku Pharmaceutical University, Dr. H. Kobayashi of the University of Tokyo, and Dr. H. Rotinsulu of Universitas Pembangunan for collecting the sponges and F. Losung of Sam Ratulangi University for technical assistance. This work was supported by Grants-in-Aid for Scientific Research (Nos. 18406002, 25293025, and 26305005 to S.T.) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan.

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