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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Osteoclastogenesis Regulation Metabolites from the CoralAssociated Fungus Pseudallescheria boydii TW-1024‑3 Da-Hua Liu,†,‡ Yi-Zhe Sun,†,‡ Tibor Kurtań ,§ Attila Mań di,§ Hua Tang,† Jiao Li,†,⊥ Li Su,† Chun-Lin Zhuang,† Zhi-Yong Liu,*,∥ and Wen Zhang*,† †

School of Pharmacy, Second Military Medical University, 325 Guo-He Road, Shanghai 200433, People’s Republic of China Department of Organic Chemistry, University of Debrecen, POB 400, H-4002 Debrecen, Hungary ⊥ School of Pharmacy, Zhejiang Chinese Medical University, 548 Bin-Wen Road, Hangzhou 310053, People’s Republic of China ∥ Department of Urology, Changhai Hospital, Second Military Medical University, 168 Chang-Hai Road, Shanghai 200433, People’s Republic of China

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§

S Supporting Information *

ABSTRACT: Three new compounds (9−11) were isolated together with eight known analogues from the fungus Pseudallescheria boydii associated with the South China Sea soft coral Sinularia sandensis. The structures of the new compounds were elucidated on the basis of the spectroscopic analysis, and the absolute configurations including the sulfur stereogenic center of a sulfoxide moiety were determined by comparison of experimental ECD spectra to TDDFT/ECD calculations. Epimeric chiral sulfoxides differing in the absolute configuration of the sulfur chirality center could be efficiently distinguished and assigned by comparing the experimental ECD to those of calculations for the sulfur epimers. In the in vitro biotests for osteoclastogenesis effects, compounds 1, 5, 7, and 10 exhibited a stimulatory activity, while compound 3 displayed an inhibitory activity. Pseudallescheria boydii is a ubiquitous filamentous fungus that is widely distributed in soil, decaying vegetation, and even polluted waters.1,2 The fungus has been found to be an opportunistic fungal pathogen that causes fatal invasive infections in both immunocompromised patients and immunocompetent individuals.3,4 Chemical investigations of the fungus may trace back to 1985, when pseurotin A and a pyrazine analogue, PB-4, were obtained from a soil-derived strain of P. boydii. The metabolites exhibited monoamine oxidase inhibitory activity.5 In the years that followed, the chemistry of this fungus extended to peptides,6 polyketides,7,8 terpenoids,9−11 and alkaloids,11 showing antibacterial, antifungal, antitumor, and immunosuppressive activities. Recently, two strains were reported from marine invertebrates. The strain associated with the starfish Acanthaster planci produced two cytotoxic isobenzofuranone derivatives, pseudaboydins A and B,12 while the strain associated with the soft coral Lobophytum crassum yielded 25 compounds, including terpenoids, polyketides, and alkaloids.13 Pseuboydone C, one of the new alkaloids with a diketopiperazine skeleton, displayed potential as a natural insecticidal agent.13 As part of our continuing search for bioactive metabolites from invertebrates and associated fungi,14−16 a fungal strain of P. boydii was obtained from the soft coral Sinularia sandensis collected from Dongsha Atoll. Chemical investigation of this fungus resulted in the isolation of 11 compounds having mixed © XXXX American Chemical Society and American Society of Pharmacognosy

polyketide and terpenoid origins. Their structures were elucidated on the basis of detailed spectroscopic analysis, and the absolute configurations were determined by comparison of experimental electronic circular dichroism (ECD) spectra to calculated ECD spectra. These compounds were evaluated for their osteoclastogenesis regulatory activity. Here, we report the isolation, structure elucidation, and bioactivity evaluation of these compounds.

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RESULTS AND DISCUSSION The fungus P. boydii was cultivated on both biomalt agar and rice media and then extracted with EtOAc, respectively. The organic extracts were subjected to repeated column chromatography on silica gel, Sephadex LH-20, and reversed phase high performance liquid chromatography (RP-HPLC) to yield compounds 1−11. Based on the detailed spectroscopic analysis in combination with comparison to reported data, the known compounds were determined as pseurotin A (1),17−20 (−)-ovalicin (2),21,22 chlovalicin (3),23 dihydroxybergamotene (4),24 AM6898B (5), 25 aspergiketone (6),26 and the (−)-ovalicin derivatives 7 and 8.27,28 Compounds 7 and 8 were previously reported via chemical conversions of (−)-ovalicin (2) when determining its absolute Received: December 12, 2018

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DOI: 10.1021/acs.jnatprod.8b01053 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Table 2. 13C NMR Data for Compounds 7−9 (150 MHz, in CDCl3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 2-OMe

configuration. The absolute configurations for 7 and 8 were effectively also established. However, the C-8 configuration in both compounds was not explicitly represented, and their 1H nuclear magnetic resonance (NMR) data were incomplete.27 A full assignment of the NMR data for both compounds was conducted (Tables 1 and 2) and the relative configuration at

7

8

9

80.2, C 86.5, CH 207.7, C 35.7, CH2 30.6, CH2 73.7, C 74.0, C 80.1, CH 27.6, CH2 121.4, CH 133.6, C 25.9, CH3 18.1, CH3 72.6, CH2 20.0, CH3 59.4, CH3

80.8, C 86.3, CH 206.6, C 35.3, CH2 36.2, CH2 74.6, C 78.8, C 65.6, CH 24.4, CH2 122.4, CH 132.9, C 26.0, CH3 17.9, CH3 58.7, CH2 21.8, CH3 59.4, CH3

78.3, C 78.9, CH 65.8, CH 26.2, CH2 31.1, CH2 75.4, C 79.2, C 65.9, CH 24.6, CH2 122.6, CH 132.7, C 25.9, CH3 17.9, CH3 59.6, CH2 21.4, CH3 55.6, CH3

Table 1. 1H NMR Data for Compounds 7−9 (600 MHz in CDCl3, J in Hz) 7 2 3 4α 4β 5α 5β 8 9α 9β 10 12 13 14α 14β 15 1-OH 2-OMe 3-OH 6-OH 7-OH

8

4.59, s

4.59, s

2.38, ddd (14.0, 5.5, 1.8) 2.71, ddd (14.0, 13.8, 7.4) 2.13, ddd (13.9, 13.8, 8.3) 1.59, ddd (13.9, 6.9, 1.8) 3.42, dd (10.4, 2.1) 2.33, ova 2.09, ova 5.22, tt (6.7, 1.4) 1.72, s 1.63, s 3.82, d (11.9) 3.48, d (11.9) 1.45, s 3.70, s 3.56, s

2.33, ova

2.42, s 3.37, s

2.70, ddd (14.6, 13.7, 7.0) 2.33, ova

9 3.82, d (3.5) 4.31, ova 1.90, ova 1.90, ova

Figure 1. Key NOE correlations for compound 7.

2.31, ddd (14.6, 10.9, 7.8) 1.82, ddd (12.9, 6.6, 1.33, ddd (10.2, 5.3, 2.3) 5.1) 3.08, t (5.3) 3.05, t (5.4) 2.63, dt (14.7, 6.6) 2.65, dt (14.4, 6.4) 2.84, dt (12.7, 6.0) 2.82, dt (13.0, 9.3.) 5.39, t (7.5) 5.42, t (7.3) 1.69, s 1.70, s 1.65, s 1.66, s 3.26, d (12.2) 3.34, d (12.5) 3.45, d (12.2) 3.19, d (12.5) 1.55, s 1.48, s 3.75, s 4.29, s 3.53, s 3.49, s 4.16, s 5.12, s 2.50, s 4.34, s 4.69, s

reported from a soil-derived Sporothrix sp. and depicted with a C-8R configuration without experimental support.28 Compound 8 had a similar planar structure to that of 7 based on the 2D NMR analysis (Figure 2). The observation of

Figure 2. Key HMBC (arrow) and COSY (bold) corrections for compound 8.

the shielded signals for the ether bridge groups in 8 (δC 58.7, CH2; 65.6, CH) with respect to those in 7 (δC 72.6, CH2; 80.1, CH) suggested the replacement of the oxygen atom in the pyran ring of 7 by a sulfinyl group in 8. The assignment of the sulfoxide group was supported by the reported 13C NMR shift values correlated to the sulfoxide moiety.29−32 The relative configurations for the stereogenic centers in 8 were determined to be the same as those in 7 (Figure 3). The obvious NOE of H-2 with H-4β and H3-15 indicated the same orientation of these protons, whereas the distinct NOE cross-peaks between 1-OH, 7-OH, and H-8 indicated these protons were oriented on the other side of the thiopyran ring. A β-orientation of 6OH was deduced from its NOE with H3-15 and was further

a

Overlapped signal.

C-8 was determined by nuclear Overhauser enhancement spectroscopy (NOESY) experiments. For compound 7, an αorientation of H-8 was indicated by its nuclear Overhauser effect (NOE) with 1-OH and H-14α and further supported by the NOEs of H3-15 with H-2, 6-OH, and H2-9 and of H-2 with H-4β (Figure 1). The absolute configuration for 7 was confirmed as (1R,2S,6S,7S,8S) by comparing the experimental ECD spectrum to those obtained from time-dependent density functional theory (TDDFT)/ECD calculations (Figures S62 and S63, Supporting Information). Compound 7 was once B



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supported by the pyridine-induced solvent shifts for H3-15 (Δδ 0.30) (Supporting Information).14,33 Besides the five tetrasubstituted carbon chirality centers, 8 had an asymmetric sulfoxide moiety, in which the sulfur atom was a center of chirality. The lack of duplication for NMR signals indicated that the sample contains a single epimer regarding the absolute configuration of the sulfur atom. The absolute configuration of the sulfoxide group was determined by comparison of experimental ECD spectra to those of TDDFT-ECD calculations performed on (1R,2S,6R,7R,8S,16SS)-8 and (1R,2S,6R,7R,8S,16RS)-8.34,35 Two diastereomers of 8 were selected for calculations because the sulfoxide group can have a major impact on the ECD spectrum36 and TDDFT-ECD calculations can be utilized for elucidation of the absolute configuration of sulfoxides. ECD measurements and TDDFT-ECD calculations have been efficiently used to determine the absolute configuration of even conformationally flexible chiral sulfoxides.37−39 The ECD spectrum of 8 in MeCN showed positive Cotton effects (CEs) at 291 and 205 nm with a shoulder at 221 nm. DFT reoptimization of the initial eight Merck molecular force field (MMFF) conformers of (1R,2S,6R,7R,8S,16SS)-8 and seven MMFF conformers of (1R,2S,6R,7R,8S,16RS)-8 resulted in six low-energy conformers over 1% population with both applied optimization methods for (1R,2S,6R,7R,8S,16SS)-8 (Figure S64, Supporting Information) and two for (1R,2S,6R,7R,8S,16RS)-8 (Figure S65, Supporting Information). The Boltzmann-weighted ECD spectra computed for (1R,2S,6R,7R,8S,16SS)-8 nicely resembled the experimental ECD spectrum at all of the applied combinations of levels, while those of the other diastereomer gave a mismatch with the experimental ECD spectrum (Figure 4). Thus, the absolute configuration of 8 could be determined unambiguously as (1R,2S,6R,7R,8S,16SS). The computed ECD spectra of both diastereomers had a positive CE for the 291 nm transition, which suggested that this transition is determined by the n → π* transition of the substituted cyclohexanone moiety. In contrast, the two diastereomers gave opposite computed CEs for the intense 205 nm ECD transition, which is mostly governed by the transitions of the sulfoxide moiety overlapping with other transitions. In this case, the epimeric chiral sulfoxides differing in the absolute configuration could be efficiently distinguished and assigned by ECD calculations. Compound 9 was obtained as a white solid. Its molecular formula of C16H28O6S was established from the high resolution electrospray ionization mass spectrometry (HRESIMS), being two mass units more than that of 8. The 1H and 13C NMR spectra of 9 showed similarities to those of 8, except that the keto group (δC 206.6) in 8 was replaced by a secondary alcohol in 9 (δH 4.31, ov, 1H; δC 65.8, CH). The secondary alcohol was readily assigned at C-3 due to the proton sequences from

Figure 4. Comparison of the experimental ECD of 8 measured in MeCN with the CAM-B3LYP/TZVP PCM/MeCN spectrum of (1R,2S,6R,7R,8S,16SS)-8 (average of six conformers, level of optimization: CAM-B3LYP/TZVP PCM/MeCN) and the PBE0/ TZVP PCM/MeCN spectrum of (1R,2S,6R,7R,8S,16RS)-8 (average of two conformers, level of optimization: CAM-B3LYP/TZVP PCM/ MeCN).

H-2 to H-5 as deduced from the correlation spectroscopy (COSY) spectrum (Figure 5). The assignment was further

Figure 5. Key HMBC (arrow) and COSY (bold) corrections for compound 9.

supported by the change in the multiplicity of H-2 from a singlet in 8 to a doublet in 9 in the 1H NMR spectrum. The βorientation of H-3 was deduced by the NOE effect between H2 with both H-4β and H-3. The relative configurations of 9 at C-1, C-2, C-6, C-7, and C-8 were shown to be the same as those of 8 by NOESY experiment (Figure 6). The configuration of the 16-sulfoxide group remained unsolved.


The absolute configuration of 9 was elucidated upon biosynthetic considerations and the partial similarity of its experimental ECD spectrum to that of 8 as (1R,2R,3R,6R,7R,8S,16SS). In the ECD spectrum of 9, the 291 nm ECD transition, associated with the n → π* transition C



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of 8, was missing and the 202 nm positive CE derived from the sulfoxide moiety as was found for compound 8. Compound 10 was obtained as a colorless oil. Its molecular formula C15H23ClO5 was determined by HRESIMS spectrum, possessing four double-bond equivalents. The presence of a chlorine atom in the molecule was supported by the observation of isotopic peaks at m/z 317/319 ([M − H]−) and 363/365 ([M + HCOO]−) with a ratio of 3:1. The IR spectrum showed the absorption of hydroxy (3407 cm−1) and lactone (1770 cm−1) functionalities. These observations were consistent with 13C NMR signals (Table 3) for one deshielded

Figure 7. Key HMBC (arrow) and COSY (bold) correlations of compound 10.

group was readily deduced from the characteristic 13C NMR shift values for C-6 and C-7 (δ 62.0, C; 58.3, CH), accounting for one double-bond equivalent. The remaining double-bond equivalent led to the assignment of a six-membered lactone from C-1 to C-5, rather than C-4, because the presence of OH4 was confirmed by its proton resonance (δ 3.09) and an HMBC correlation with C-4. The planar structure of 10 was then determined as a lactone derivative of 2. The relative configuration of 10 was established by a NOESY experiment (Figure 8). The NOE correlations

Table 3. 1H NMR (600 MHz) and 13C NMR (150 MHz) Data for 10 and 11 (in CDCl3) 10 δC, type

δH, mult (J in Hz,)

11 δC, type

1 2

176.6, C 28.6, CH2

3

26.0, CH2

4 5

87.7, C 76.1, C

105.5, C 35.7, CH2

6

62.0, C

71.6, CH

7 8

58.3, CH 27.0, CH2

α 2.55, ddd (19.0, 12.8, 5.0) β 2.73, ova α 2.30, m β 2.73, ova

9

3.17, 2.43, 2.18, 117.5, CH 5.16,

10 11 12 13

136.1, C 25.8, CH3 18.2, CH3 50.1, CH2

14

63.5, CH2

15 NH 4OH

14.6, CH3

1.75, 1.67, 3.80, 3.72, 4.01, 3.72, 1.46,

t (6.6) dt (14.0, 6.7) dt (14.8, 7.5) t (7.3)

s s d (11.8) ova d (11.8) ova s

172.7, C 33.8, CH2

52.3, CH

38.8, C 81.0, CH 21.5, CH2 11.5, CH3 22.1, CH3 11.5, CH3 170.6, C 23.3, CH3

δH, mult (J in Hz) α 2.46, dd (17.2, 8.3) β 2.88, dd (17.2, 8.3) 4.59, dt (10.8, 8.5) α 1.99, dd (13.6, 11.8) β 1.88, dd (13.7, 5.1) 3.75, dd (11.8, 5.1) 3.46, dd (10.6, 2.0) 1.61, 1.40, 0.92, 0.97, 0.81,


m m t (7.3) s s

between H2-14 and H-3β and 4-OH indicated the same orientation of these protons. The NOE of H3-15 with H2-8 suggested these protons were on the same side as the epoxy group. The absolute configuration of 10 is tentatively suggested as (4R,5R,6S,7R) upon biosynthetic considerations based on the absolute configuration of 3. Compound 11 was obtained as a colorless oil. The molecular formula of 11 was determined as C14H23NO5 by the HRESIMS, requiring four double-bond equivalents. The 13 C NMR and DEPT data (Table 3) displayed 14 carbon signals including two deshielded sp2 carbon atoms (2 × C O) and 12 shielded sp3 carbon atoms (1 × OC, 2 × OCH, 1 × C, 1 × CH, 3 × CH2, 4 × CH3), accounting for two doublebond equivalents. The remaining double-bond equivalents were attributed to the presence of two rings in the molecule. The planar structure of compound 11 was elucidated by 2D NMR (HSQC, heteronuclear single quantum coherence (HSQC), and COSY) (Figure 9). By interpretation of COSY correlations, it was possible to establish three partial structures

2.07, s

5.96, d (8.8) 3.09, s

a

Overlapped signal.

ester carbon and two double-bond carbons and 12 shielded sp3 carbons (3 × OC, 1 × OCH, 1 × OCH2, 4 × CH2, 3 × CH3), accounting for two double-bond equivalents. The remaining two double-bond equivalents were due to the presence of two rings in the molecule. The planar structure of 10 was deduced from 2D NMR spectra (Figure 7). The COSY spectrum gave proton sequences from H2-2 to H2-3 and from H-7 to H3-11 and H3-12. The key heteronuclear multiple bond correlation (HMBC) correlations from H2-13 to C-3, C-4, and C-5, H214 to C-4, C-5, and C-6, and H3-15 to C-5, C-6, and C-7 allowed the connection of the two proton sequences through the oxygenated tertiary carbons C-4, C-5, and C-6. The longrange correlations from both H2-2 and H2-3 to C-1 extended the above substructure to the carbon atom C-1. A 6,7-epoxy

Figure 9. Key HMBC (arrow) and COSY (bold) corrections for compound 11. D



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of consecutive proton systems: H-2/H-3/NH (a), H-8/H-9/ H-10 (b), and H-5/H-6 (c). HMBC correlations from both H3-11 and H3-12 to C-6, C-7, and C-8 led to the linkage of the two methyls to the fragments b and c by the quaternary carbon C-7. The HMBC correlations from H-3 to C-4, C-5, and C-13 and from H2-2 to C-1 gave the connections of fragment c to the N-acetyl group and the carbonyl group through fragment a. HMBC signals from H-8 to C-4 resulted in the formation of a pyran ring in the molecule, satisfying one double-bond equivalent. The remaining double-bond equivalent allowed a connection from C-1 to C-4 by an oxygen atom to form a fivemembered lactone ring where C-4 was a spiro atom (δ 105.5, C) of the two rings. This conclusion was further supported by the appearance of the NMR signal of 6-OH in DMSO-d6 (Supporting Information), ruling out the possibility of the lactone at C-6. The assignment of an α-substituent acetamide to a spiro carbon atom was supported by the reported 13C NMR data of synthetic analogues.40 The relative configuration of compound 11 was deduced from the NOESY spectrum (Figure 10). The NOE correlations

Figure 11. Comparison of the experimental ECD spectrum of 11 measured in MeCN with the PBE0/TZVP PCM/MeCN spectrum of (3S,4R,6S,8S)-11 (level of optimization: CAM-B3LYP/TZVP PCM/ MeCN). The bars represent the rotational strength values of the lowest energy conformer.

The isolated compounds 1−11 were tested in vitro for their regulatory effects on osteoclastogenesis at the concentrations of 0.1 and 1.0 μM, as they showed no significant cytotoxicity below 10 μM. Compounds 5 and 7 significantly increased the number of the osteoclasts at both 0.1 and 1 μM and enhanced the area of osteoclasts at 0.1 μM. Compound 1 increased the number of osteoclasts at 0.1 μM, while compound 10 elevated the numbers of osteoclasts at 1.0 μM. In contrast, compound 3 significantly decreased the number and reduced the area of the osteoclasts at both 0.1 and 1.0 μM (Figures 12 and 13). It is noteworthy that the title fungus produced different metabolites when cultivated with different media. It produced 1 and 2 in both media and yielded 5−7 in biomalt media and 3, 4, and 8−11 in rice media. It is said that systematic alteration of easily accessible cultivation parameters (such as media composition, pH value, temperature, addition of enzyme inhibitors, oxygen-supply level, and culture vessel) may influence the structure and number of secondary metabolites from a single microorganism. This is the so-called OSMAC (one strain many compounds) theory raised by Zeeck and coworkers.47 The isolation of compounds 1−11 gives insight into the productivity of this fungus. Compounds 2−10 are biogenetically correlated sesquiterpene derivatives. Their chemical diversities are attributed to various atom substitutions, the degree of oxidation, and the ring cleavage and rearrangement of the carbon skeleton. Compound 11 is a spiro polyketide with a pyran ring and a five-membered lactone having an acetamide substituent. The occurrence of the thiopyran-S-oxide moiety in 8 and 9 is unusual, generating an additional stereogenic center in these structures. Recently, two metabolites having this subunit were obtained by biotransformation of thiochroman-4-ones in the fungus of Trichoderma viride.48 Several metabolites with a sulfoxide-bearing fivemembered ring were isolated from the garlic plants Allium sativum49,50 and A. f istulosum,51,52 the shrubs Breynia of f icinalis,30 B. vitis-idaea,53 and B. f ruticosa,32 the herbs Nuphar lutea54 and N. pumilum,55 and a sponge-derived Penicillium sp. fungus.56 The relative configuration of the sulfoxide group in the metabolites was assigned mainly by chemical semisynthesis and NMR analysis including solventinduced shifts, with no report on the absolute configuration.


of H3-11 with H-6 and H-8 indicated these protons were on the same side as the pyran ring, while H-5β with H3-12 indicated these protons were on the other side of the pyran ring. H-3 and C-5 were oriented on the same side of the furan ring due to the NOE effect of H-3 with H2-5. In order to assign the relative configuration of C-3, DFTNMR calculations were performed on the (3R,4R,6S,8S)-11 and (3S,4R,6S,8S)-11 diastereomers (Table S4, Supporting Information).41,42 B3LYP/6-31+G(d,p) level reoptimization of the 29 and 21 MMFF conformers resulted in three low-energy conformers for each of the two stereoisomers. Boltzmannaveraged mPW1PW91/6-311+G(2d,p)43 level 13C NMR data corrected for chloroform were compared with the experimental 13 C NMR data measured in CDCl3.44 Both simple comparison and the DP4+ analysis45,46 indicated the (3S*,4R*,6S*,8S*) relative configuration. The latter gave 99.89% confidence for this diastereomer. The MMFF conformers of (3S,4R,6S,8S)-11 were also reoptimized at both the B3LYP/6-31G(d) and the CAMB3LYP/TZVP PCM/MeCN levels, resulting in three lowenergy conformers over 1% population (Figure S63, Supporting Information). Boltzmann-weighted ECD spectra computed at various levels for both sets of conformers reproduced well the experimental ECD spectrum, and all low-energy conformers exhibited similar ECD spectra (Figure 11), which allowed unambiguous elucidation of the absolute configuration as (3S,4R,6S,8S)-11. E



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Figure 12. Effects of compounds 1−11 on osteoclastogenesis. The numbers (A, B) and areas (C, D) of osteoclasts were measured. Data are presented as mean ± SD (*p < 0.05, **p < 0.01, vs control, n = 3).

of cancer, and arthritis, and if the function of osteoclasts is impaired or declined, it can cause osteosclerosis, bone deficiency, etc. This research may serve as a basis for further investigations of this cluster of metabolites on osteoclastogenesis regulation activity.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in MeOH with a Rudolph AUTOPOL VI polarimeter at the sodium D line (590 nm). UV spectra were determined by a Hitachi U-3010 spectrophotometer. ECD spectra were measured with a Jasco-715 spectropolarimeter. The IR spectra were recorded on a Nexus 470 FT-IR spectrophotometer (Nicolet). NMR data were acquired at 300 K on Bruker DRX 600 and DRX 500 NMR spectrometers. Chemical shifts are reported relative to the residual CDCl3 signal (δH 7.26; δC 77.0), C5D5N signal (δH 8.74, 7.58, 7.22; δC 150.3, 135.9, 123.9), and DMSO-d6 (δH 2.50; δC 39.6) as internal standards for 1H and 13C NMR spectra. The 1H and 13C NMR assignments were supported by COSY, HSQC, HMBC, and NOESY experiments. The mass spectra were acquired on a Waters Q-TOF Micro mass spectrometer and Thermo Scientific DFS Magnetic Sector GC-HRMS. HPLC was performed using Agilent Technology 1100 and 1200 systems with DAD/DIR detector and YMC Pack ODS-A (250 × 10 mm, 5 μm) and Agilent XDB-C18 (250 × 30 mm, 5 μm) columns, respectively. Commercial silica gel (Yantai) was used for column chromatography (CC). Precoated silica gel plates (HSGF254, Yantai) were used for analytical thin-layer chromatography (TLC). Spots were detected on TLC under UV or by heating after spraying with anisaldehyde sulfuric acid reagent. Commercial KBr and NaCl (Titan, AR) and KCl, MgCl2·6H2O, CaCl2·2H2O, SrCl2·6H2O, Na2SO4, NaHCO3, and H3BO3 (Sinopharm, AR) were used for preparation of artificial seawater (ASW).57 Fungal Material. The fungus was isolated from the internal tissues of the soft coral Sinularia sandensis collected by Dr. Yalan Chou from the Dongsha Atoll in the South China Sea at a depth of 15

Figure 13. Bone marrow macrophage cells (BMMs) were stimulated with RANKL and M-CSF at indicated concentrations of compounds and stained in a TRAP assay (magnification = 40×; scale bar = 200 μm).

The absolute configurations of the sulfoxide 8 and 9 were directly determined by comparing experimental ECD to TDDFT/ECD calculations, which may give a reference for the configurational assignment of the sulfoxide moiety. In the in vitro osteoclastogenesis assay, compounds 1, 5, 7, and 10 showed stimulatory activity, while compound 3 displayed inhibitory activity. It is known that drugs for bonerelated diseases mainly affect the absorption of bone by osteoclasts in three aspects: differentiation, function, and apoptosis. Hyperfunctionality of osteoclasts can lead to degenerative diseases such as osteoporosis, bone metastasis F



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1664, 1449, 1383, 1089, 1008, 899 cm−1; 1H and 13C NMR data, Tables 1 and 2; HRESIMS m/z 347.1531 [M − H]− (calcd for C16H27O6S, 347.1528). Compound 10: colorless oil (CH2Cl2); Rf 0.43 (CH2Cl2/MeOH, 15:1); [α]25D +5.3 (c 0.47, MeOH); UV (MeCN) λmax (Δε) 195 (2.139) nm; ECD (MeCN, c 3.1 × 10−3) λmax (Δε) 219 (+0.19) nm; IR (film) νmax 3407, 2977, 2930, 1770, 1647, 1386, 1252, 1182, 1054 cm−1; 1H and 13C NMR data, Table 3; HRESIMS m/z 317.1161 [M − H]− (calcd for C16H23ClO5, 317.1156). Compound 11: colorless oil (CH2Cl2); Rf 0.32 (CH2Cl2/MeOH, 20:1); [α]25D −89.4 (c 0.64, MeOH); UV (MeCN) λmax (Δε) 251 (0.090), 242 (0.086),

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