Structures of Polyacetoxybriaranes Extracted from the Sea Whip Octocoral Junceella fragilis

Abstract

In the present study, an algae-containing octocoral, Junceella fragilis, was subjected to chemical screening. The analysis resulted in the extraction of six polyacetoxybriaranes: a new compound, identified as fragilide Z (1), alongside previously identified analogs, which included 12-epi-fragilide G (2), fragilide P (3), junceellolide D (4), junceellonoid A (5), and juncin ZI (6). The structures of compounds 2–6 were investigated through single-crystal X-ray diffraction analysis, whereas that of 1 was examined through two-dimensional nuclear magnetic resonance analysis. Compounds 1–6 proved active in enhancing the growth of MG-63 human mesenchymal stem cells.

Introduction

Briarane-type natural products, which usually contain a γ-lactone moiety, refer to marine-derived diterpenoids with a bicyclo[8.4.0]tetradecane ring system. Since the discovery of briarein A in 1977,¹⁾ more than 900 briaranes have been identified in different marine organisms, with Junceella octocorals being among the most crucial briarane sources.²⁾ The waters surrounding the main island of Taiwan are at the confluence of three ocean currents: the Kuroshio Current, the South China Sea Warm Current, and the Cold China Coastal Current. Biodiversity is highly correlated with natural product diversity. Therefore, the present study collected samples of the octocoral Junceella fragilis (Ridley, 1884) (phylum: Cnidaria, subphylum: Anthozoa, class: Octocorallia, order: Scleralcyonacea, and family: Ellisellidae),³⁾, which has rich biota, to investigate its briarane-type natural products. This study isolated six polyacetoxybriaranes from J. fragilis: one new compound, namely, fragilide Z (1), and five known analogs, namely, 12-epi-fragilide G (2),⁴⁾ fragilide P (3),⁵⁾ junceellolide D (4),⁶⁾ junceellonoid A (5),⁷⁾ and juncin ZI (6)⁸⁾ (Fig. 1). The structure of 1 was investigated through two-dimensional (2D) NMR analysis, whereas those of 2–6 were examined through single-crystal (SC) X-ray diffraction (XRD) analysis.

Fig. 1. Structures of Fragilide Z (1), 12-epi-Fragilide G (2), Fragilide P (3), Junceellolide D (4), Junceellonoid A (5), Juncin ZI (6), and Robustolide H (7)

Results and Discussion

Compound 1 was identified as an amorphous powder ([α]_D²³ +132°, c = 0.02, CHCl₃), and it was found to have a chemical formula of C₂₉H₃₉ClO₁₂ (Ω = 10); this determination was performed via high-resolution electrospray ionization mass spectrometry (HR-ESI)-MS at a mass-charge ratio (m/z) of 637.20190 (Calcd for C₂₉H₃₉ClO₁₂Na, 637.20223). The IR spectrum of 1 exhibited major bands at 1735, 1792, and 3663 cm⁻¹, which indicated ester, γ-lactone, and hydroxy groups, respectively. According to analysis of the distortionless enhancement by polarization transfer (DEPT) and ¹³C-NMR signals of 1, this compound had an exocyclic double bond. This was inferred from the resonance signals of the carbon atoms, which presented carbon chemical shift (δ_C) values of 146.7 (C-5) and 121.3 (CH₂-16). We confirmed the aforementioned identification by observing olefin proton signals at hydrogen chemical shift (δ_H) values of 5.54 (1H, s, H-16a) and 5.82 (1H, s, H-16b) in the ¹H-NMR spectrum of 1 (Table 1).

Table 1. ¹H- and ¹³C-NMR Data for 1

C/H	δΗa) (J in Hz)	δΧ,b) type
1		47.9, C
2	5.99 d (8.8)	73.3, CH
3α/β	1.63 m; 2.73 m	28.2, CH2
4	2.45 m	33.6, CH2
5		146.7, Cc)
6	4.63 d (3.2)	52.7, CHd)
7	4.45 br s	81.2, CH
8		81.4, C
9	5.76 s	72.7, CH
10	3.70 s	35.5, CH
11		57.6, C
12	4.55 dd (3.2, 2.4)	73.5, CH
13α/β	2.26 m; 2.04 m	29.1, CH2
14	4.88 dd (2.4, 2.4)	73.3, CH
15	1.16 s	14.0, CH3
16a/b	5.54 s; 5.82 s	121.3, CH2
17	2.96 q (7.2)	51.5, CH
18	1.27 d (7.2)	5.9, CH3
19		174.6, C
20a/b	2.36 d (4.0); 2.83 dd (4.0, 1.2)	50.3, CH2
OCOEt-2	2.29 q (7.6) 1.10 t (7.6)	174.2, C 27.7, CH2 8.9, CH3
Acetate methyls	2.22 s 2.00 s 1.97 s	21.2, CH3 20.9, CH3 21.1, CH3
Acetate carbonyls		170.3, C 169.5, C 169.4, C
OH-8	3.45 s

a) Spectra recorded at 400 MHz in CDCl₃. b) Spectra recorded at 100 MHz in CDCl₃. c) Data assigned on the basis of HMBC spectra. d) Data assigned on the basis of HSQC spectra.

Furthermore, carbonyl resonances were observed in the compound’s ¹³C-NMR spectrum at δ_C values of 174.6 (C-19), 174.2 (C), 170.3 (C), 169.5 (C), and 169.4 (C), which confirmed that it contained γ-lactone and four additional ester groups. An n-propionoxy group (δ_H 2.29, 2H, q, J = 7.6 Hz; δ_H 1.10, 3H, t, J = 7.6 Hz) and three acetoxy groups (δ_H 2.22, 2.00, and 1.97; 3H × s for each signal) were revealed by the ¹H-NMR spectrum. The aforementioned results indicated that 1 is a tetracyclic compound.

The overall structure of 1 was determined via 2D-NMR spectroscopy analysis. The following spin systems were identified for 1 from the results of a ¹H–¹H correlation spectroscopy (COSY) experiment: H-2/H₂-3/H₂-4, H-6/H-7, H-12/H₂-13/H-14, and H-17/H₃-18. In addition, the ¹H–¹H COSY spectrum of 1 was correlated, indicating that H₂-4/H-16a and H-6/H-16a were allylically coupled, whereas H-20b and H-10 exhibited w-coupling. On the basis of these findings and the results of a heteronuclear multiple bond connectivity (HMBC) study (Fig. 2), the carbon skeleton structure of 1 was determined. According to the findings of the HMBC experiments, correlations between H₂-16 and C-4/C-5, H-6 and C-16, as well as H₂-4 and C-16. indicated the presence of an exocyclic double bond at C-5. Additionally, the HMBC results for H₂-20/C-11, C-12, and H-10/C-20 suggested the presence of an epoxy group at C-11/20. The correlations observed for H₃-15/C-1, C-2, C-10, and C-14, as well as between H-2/C-15 and H-10/C-15, indicated that a methyl group (Me-15) was attached to C-1. Moreover, in the HMBC spectrum, the oxymethine protons appearing at δ_H 5.99 (H-2) and δ_H 5.76 (H-9) were correlated with the ester carbonyls at δ_C 174.2 (n-propionate carbonyl) and δ_C 169.4 (acetate carbonyl). This indicated that the n-propionoxy group and the acetoxy group are positioned at C-2 and C-9, respectively. Notably, the H-12 and H-14 oxymethine protons were not correlated with the acetate carbonyls; the acetoxy groups at C-12 and C-14 were identified on the basis of their distinct NMR signals (δ_H 4.55/δ_C 73.5 for CH-12 and δ_H 4.88/δ_C 73.3 for CH-14). Finally, the presence of a hydroxy group at C-8 was confirmed by the HMBC correlation of a hydroxy proton (δ_H 3.45, 1H, s) with carbons C-7, C-8, and C-9.

Fig. 2. Key HMBC and COSY Correlations of 1

The prominent intensity ratio of 1 : 3 for the sodiated molecular isotope peak (M + 2 + Na)⁺ compared with (M + Na)⁺ in the ESI-MS spectrum strongly suggested that compound 1 contains a chlorine atom. In the heteronuclear single quantum coherence spectroscopy (HSQC) spectrum, the methine unit (δ_C 52.7) shifted more upfield than typical for an oxygenated carbon atom, and it was correlated with the methine proton at δ_H 4.63 (1H, d, J = 3.2 Hz, H-6). This proton demonstrated ³J- and ⁴J-correlations with H-7 and H-16a, respectively, in the ¹H–¹H COSY spectrum, confirming the presence of a chlorine atom attached at C-6.

The chemical shifts for exocyclic 11,20-epoxy groups in briarane derivatives have been documented, revealing that when the ¹³C-NMR signals for C-11 and C-20 occur at δ_C 55–61 and 47–52 ppm, the epoxy group takes on an α-orientation (11R*), and the cyclohexane ring assumes a chair conformation. Conversely, if the epoxy group exists in the 11S* configuration, the ¹³C-NMR data for C-11 and C-20 experience a downfield shift, appearing at δ_C 62–63 and 58–60 ppm, respectively, resulting in the cyclohexane rings adopting a twist-boat conformation.⁹⁾ The ¹³C-NMR chemical shifts of C-11 at 57.6 ppm and C-20 at 50.3 ppm for compound 1 resemble the previously noted values, suggesting that the epoxy group in 1 also adopts an 11R* configuration and confirming that the cyclohexane ring assumes a chair conformation.

Vicinal ¹H–¹H proton coupling constant and nuclear Overhauser effect spectroscopy (NOESY) correlation analyses were performed to investigate the relative stereochemistry of 1. The NOESY experiment (Fig. 3) revealed that H-10 was correlated with H-2, H-9, and H₃-18. Additionally, the presence of an NOE correlation between OH-8 and H-2 suggested that these protons are α-protons located on the same face. This conclusion was drawn because of the lack of correlation between H₃-15 and H-10 alongside the β orientation of the C-15 methyl group at C-1. The absence of coupling between H-9 and H-10 indicated that the protons have a dihedral angle close to 90°, confirming that H-9 is α-oriented. The interactions between the H-14 proton and H₃-15 indicated the β-orientation of the proton at C-14. Additionally, the NOE interactions observed between the C-13 methylene protons and both H-12 and H-14 implied that H-12 and H-14 have a β-orientation within the six-membered ring of 1. Moreover, the coupling constants observed for the C-13 methylene protons with H-14 (J = 2.4, 2.4 Hz) and with H-12 (J = 2.8, 2.4 Hz) reinforced the conclusion that H-14 and H-12 are situated in the β-equatorial orientation within the six-membered ring of 1. The identified correlations involved H-9 with H-17 and one of the C-20 oxymethylene protons, namely, H-20a (δ_H 2.36). Through modeling analysis, it was deduced that H-17 and H-20a are in close proximity to H-9, suggesting that H-9 is likely positioned on the α face of the 10-membered ring of 1, whereas H-17 is situated in the γ-lactone moiety with a β-orientation. We identified an interaction between H-17 and H-7, which was subsequently found to link to H-6, suggesting that H-6 and H-7 are positioned on the β face. The aforementioned information enabled the relative configuration of all the stereogenic centers of 1 to be identified (1S*, 2S*, 6S*, 7R*, 8R*, 9S*, 10S*, 11R*, 12R*, 14S*, 17R*). On the basis of the above analysis and an NMR-based comparison with the literature, we deduced that 1 shares a similar structure with robustolide H (7)¹⁰⁾ (Fig. 1). The distinction between these compounds lies in 7 possessing a 2β-acetoxy group, whereas 1 features an n-propionoxy group. Consequently, we identified 1 as the 2-O-deacetyl-n-propionyl derivative of 7 and named it fragilide Z.

Fig. 3. Computer-Modeled Stereoview of 1 and Calculated Interproton Distances (Å) for Key NOESY Correlations

On the basis of the spectroscopic data, five known briaranes were identified: 12-epi-fragilide G (2),⁴⁾ fragilide P (3),⁵⁾ junceellolide D (4),⁶⁾ junceellonoid A (5),⁷⁾ and juncin ZI (6).⁸⁾ We performed SC-XRD analysis to determine the absolute configurations of 2–6, as shown in Fig. 4. Table 2 provides detailed information on the crystal data and structure refinement of compounds 2–6. Because 1–6 all had the same origin (i.e., the same organism), we assumed that they had the same absolute configuration. As a result, we determined that the absolute configuration of 1 is 1S, 2S, 6S, 7R, 8R, 9S, 10S, 11R, 12R, 14S, 17R.

Fig. 4. Computer-Generated ORTEP Drawings of 12-epi-Fragilide G (2), Fragilide P (3), Junceellolide D (4), Junceellonoid A (5), and Juncin ZI (6)

Table 2. Details of the Crystal Data and Structure Refinement for 2–6

Crystal data	2	3	4	5	6
Empirical formula	C28H37ClO13	C29H39ClO13	C28H38O11	C28H37ClO11	C26H33ClO13
Formula weight	617.02	631.05	550.58	585.02	556.97
Temperature	200 (2) K	200 (2) K	200 (2) K	200 (2) K	200 (2) K
Wavelength	1.54178 Å	1.54178 Å	1.54178 Å	1.54178 Å	1.54178 Å
Crystal system	Hexagonal	Hexagonal	Tetragonal	Orthorhombic	Orthorhombic
Space group	P65 (# 170)	P65 (# 170)	P43 (# 78)	P212121 (# 19)	P212121 (# 19)
Unit cell dimensions	a = 24.8909 (10) Å α = 90° b = 24.8909 (10) Å β = 90° c = 10.2810 (8) Å γ = 120°	a = 24.9439 (7) Å α = 90° b = 24.9439 (7) Å β = 90° c = 10.2687 (3) Å γ = 120°	a = 10.23260 (14) Å α = 90° b = 10.23260 (14) Å β = 90° c = 27.3237 (5) Å γ = 90°	a = 10.4140 (2) Å α = 90° b = 13.6216 (3) Å β = 90° c = 20.8693 (4) Å γ = 90°	a = 10.2033 (4) Å α = 90° b = 12.7941 (6) Å β = 90° c = 42.1368 (17) Å γ = 90°
Volume	5516.3 (6) Å3	5533.2 (4) Å3	2860.96 (9) Å3	2960.42 (10) Å3	5500.6 (4) Å3
Z	6	6	4	4	8
Density (calculated)	1.114 Mg/m3	1.136 Mg/m3	1.278 Mg/m3	1.313 Mg/m3	1.345 Mg/m3
Absorption coefficient	1.385 mm−1	1.391 mm−1	0.823 mm−1	1.638 mm−1	1.737 mm−1
F(000)	1956	2004	1176	1240	2352
Crystal size	0.570 × 0.047 × 0.025 mm3	0.496 × 0.048 × 0.024 mm3	0.582 × 0.208 × 0.144 mm3	0.336 × 0.124 × 0.034 mm3	0.329 × 0.237 × 0.022 mm3
θ range for data collection	2.049 to 67.493°	2.045 to 67.498°	4.321 to 72.489°	3.875 to 74.564°	3.610 to 67.494°
Index ranges	−29 ≤ h ≤ 29 −29 ≤ k ≤ 29 −10 ≤ l ≤ 12	−27 ≤ h ≤ 29 −29 ≤ k ≤ 27 −10 ≤ l ≤ 12	−12 ≤ h ≤ 12 −12 ≤ k ≤ 12 −30 ≤ l ≤ 33	−13 ≤ h ≤ 11 −15 ≤ k ≤ 16 −25 ≤ l ≤ 26	−11 ≤ h ≤ 8 −15 ≤ k ≤ 15 −49 ≤ l ≤ 50
Reflection collected	30897	31998	38730	32480	50359
Independent reflections	6341 [R(int) = 0.1330]	6505 [R(int) = 0.1164]	5528 [R(int) = 0.0331]	6016 [R(int) = 0.0416]	9652 [R(int) = 0.2491]
Completeness to θ = 67.493°	100.0%
Completeness to θ = 67.498°		100.0%
Completeness to θ = 67.679°			99.8%
Completeness to θ = 67.679°				99.7%
Completeness to θ = 67.494°					98.3%
Absorption correction	Semiempirical from equivalents	Semiempirical from equivalents	Semiempirical from equivalents	Semiempirical from equivalents	Semiempirical from equivalents
Max. and min. transmission	0.9423 and 0.7240	0.9805 and 0.8681	0.9819 and 0.8248	0.9819 and 0.8510	0.9768 and 0.7826
Refinement method	Full-matrix least-squares on F2	Full-matrix least-squares on F2	Full-matrix least-squares on F2	Full-matrix least-squares on F2	Full-matrix least-squares on F2
Data/restraints/parameters	6341/22/393	6505/7/396	5528/2/366	6016/1/368	9652/6/698
Goodness-of-fit on F2	0.914	0.989	0.624	1.028	0.954
Final R indices [I > 2σ(I)]	R1 = 0.0713, wR2 = 0.1836	R1 = 0.0597, wR2 = 0.1434	R1 = 0.0367, wR2 = 0.1108	R1 = 0.0333, wR2 = 0.0879	R1 = 0.0644, wR2 = 0.1311
R indices (all data)	R1 = 0.1344, wR2 = 0.2365	R1 = 0.1217, wR2 = 0.1796	R1 = 0.0370, wR2 = 0.1126	R1 = 0.0345, wR2 = 0.0891	R1 = 0.2418, wR2 = 0.1950
Absolute structure parameter	0.06 (4)	0.04 (2)	0.04 (9)	0.004 (5)	0.033 (17)
Extinction coefficient	0.0037 (6)	0.0029 (4)	n/a	n/a	0.00179 (19)
Large diff. peak and hole	0.220 and −0.264 e.Å−3	0.272 and −0.268 e.Å−3	0.384 and −0.202 e.Å−3	0.534 and −0.524 e.Å−3	0.350 and −0.324 e.Å−3

Studies have shown that briaranes can be used to treat osteoclastogenic disease.^11,12) Using MG-63 human mesenchymal stem cells, we conducted an alkaline phosphatase (ALP) ELISA (Table 3); the results indicated that 1–6 did not increase ALP activity but promoted the growth of MG-63 cells at a concentration of 10 µM.

Table 3. ALP Activity and Viability of MG63 Cells after Treatment with 1–6 at a Concentration of 10 µM for 72 h

Compounds	ALP activity (%)	Cell viability (%)
Control	100.00 ± 1.12	100.00 ± 6.89
1	99.26 ± 7.75	148.22 ± 1.92***
2	86.47 ± 1.29	148.62 ± 5.09***
3	91.85 ± 4.35	146.82 ± 5.52***
4	93.56 ± 5.30	161.46 ± 11.16***
5	89.40 ± 3.98	166.32 ± 10.43***
6	80.16 ± 3.88	137.08 ± 8.14**
17β-Estradiola)	140.76 ± 3.94***	123.12 ± 2.80*

The data are expressed as the standard error of the mean (n = 3). Significance was determined via Student’s t test (* p < 0.05, ** p < 0.01, *** p < 0.001) and by comparing the results with those for untreated cells. a) The positive control was 10 µM 17β-estradiol.

Experimental

General

Optical rotation and infrared spectra measurements were performed with a polarimeter and spectrophotometer, respectively, as described in our previous study.¹³⁾ NMR spectra were collected on a JEOL ECZ spectrometer at 400 and 100 MHz for ¹H-NMR and ¹³C-NMR, respectively. In this study, the coupling constant (J) is measured in hertz, while the chemical shift (δ) is expressed in parts per million (ppm). For reference points, we utilized the residual peaks of the deuterated solvent CDCl₃, noted at δ_H 7.26 and δ_C 77.0. To conduct ESI-MS and HR-ESI-MS, we used the solariX FTMS system (with seven Tesla magnet; Bruker Corporation) which features an electrospray-ionization ion source. To isolate the extracted compounds, column chromatography (CC) and TLC were used under the conditions as described in our previous publication.¹³⁾ The separation process was conducted via a system that included an injection port (model 7725, RHEODYNE, U.S.A.) paired with a semipreparative normal-phase column (Supelco Ascentis Si, Catalog No.#: 581514-U, Sigma-Aldrich, U.S.A.) and a pump (model L-7110, HITACHI, Japan) for normal-phase (NP)-HPLC. Additionally, an alternative system was utilized for separation through reverse-phase HPLC (RP-HPLC) with a setting as described previously.¹³⁾

Animal Material

In 2012, J. fragilis samples were harvested from the southern coast of Taiwan via scuba diving techniques. One of these samples was presented to the National Museum of Marine Biology & Aquarium located in Pingtung County, Taiwan. To determine the species of the sample, its physical attributes and microscopy images of the coral sclerites were analyzed and compared with data from previous studies.^3,14–16)

Extraction and Isolation

We first freeze-dried the J. fragilis sample, with a wet to dry weight ratio of 6.01 to 2.39 kg, sliced it into pieces, and treated it with an equal blend of methanol (MeOH) and dichloromethane (CH₂Cl₂) at ambient temperature. This procedure resulted in a crude extract weighing 140.1 g. We then performed liquid–liquid partitioning to separate the mixture into ethyl acetate (EtOAc) and water layers. In the ethyl acetate phase, the product, weighing 19.2 g, was subsequently further purified by using silica gel column chromatography (Si CC). Initially, we utilized a gradient solvent system containing nonpolar n-hexane for elution. The subsequent elution was carried out by employing mixtures with progressively higher polarity: combinations of n-hexane with EtOAc, as well as pure acetone and then pure methanol. Overall, 13 fractions (A−M) were obtained through the elution process. Si CC was performed to separate fraction E, which was then eluted via an isocratic solvent system (ISS), namely, a 10 : 1 CH₂Cl₂ : acetone mixture. This process yielded eight fractions (E1–E8). We purified fraction E3 through NP-HPLC with another ISS, namely, a 2 : 1 mixture of n-hexane and EtOAc (flow rate = 3 mL/min), thus obtaining 4 (3.0 mg) and 5 (1.8 mg). Si CC was performed using an ISS comprising a 15 : 1 mixture of CH₂Cl₂ and acetone to separate fraction F. This process resulted in fractions F1–F8. We separated fraction F4 through the Si CC and then eluted it by using an ISS consisting of a 20 : 1 CH₂Cl₂ : EtOAc mixture; this process led to the formation of six fractions, namely, F4A–F4F. We purified fraction F4E through RP-HPLC with an ISS comprising a 70 : 30 mixture of MeOH and H₂O (flow rate = 3 mL/min), thereby obtaining 6 (0.8 mg). In addition, NP-HPLC was conducted using an ISS consisting of a 20 : 1 mixture of CH₂Cl₂ and acetone (flow rate = 4 mL/min) to separate fraction F6; this process led to the formation of five fractions, namely, F6A–F6E. We purified fraction F6C through RP-HPLC with an ISS comprising a 75 : 25 mixture of MeOH and H₂O (flow rate = 3 mL/min), thus obtaining 1 (1.0 mg), 2 (8.0 mg), and 3 (1.2 mg).

Fragilide Z (1): The form of compound 1 upon its production was an amorphous powder. Some relevant information regarding this compound is given as follows: [α]_D²³ +132 (c = 0.02, CHCl₃); IR (KBr) ν_max 3663, 1792, and 1735 cm⁻¹; ¹H-NMR (CDCl₃, 400 MHz) and ¹³C-NMR (CDCl₃, 100 MHz) data are listed in Table 1; ESI-MS m/z: 637 (M + Na)⁺, 639 (M +2 + Na)⁺; HR-ESI-MS m/z: 637.20190 (Calcd for C₂₉H₃₉ClO₁₂Na, 637.20223).

SC-XRD of 12-epi-Fragilide G (2), Fragilide P (3), Junceellolide D (4), Junceellonoid A (5), and Juncin ZI (6)

Crystals of prisms 2–6 were derived from a methanol solution. Data for diffraction were gathered via a Bruker D8 Venture diffractometer with CuKα radiation for compounds 2–6. The structures were determined via direct methods and refined via a full-matrix least-squares procedure.^17,18) The crystallographic data for briarane structures 2–6 have been submitted to the Cambridge-Crystallographic Data Center (CCDC) as supplementary publication numbers CCDC 2342713, 2331400, 2323850, 2353595, and 2353603, respectively.¹⁹⁾

ALP Activity Assay

In this study, we conducted assays to evaluate ALP activity. For each assay, MG63 cells were exposed to one of the 1–6 compounds at a 10 µM concentration for three days. After treatment, the MG63 cells in each well were washed with phosphate-buffered saline (PBS) twice, and then lysis buffer with 0.1% Triton X-100 was added. After sonication, the cell lysate samples were subjected to ALP activity measurement with p-nitrophenyl phosphate in 0.2 M Tris hydrochloride-magnesium chloride hexahydrate buffer (pH 9.5). The ALP activity of each sample was normalized by the protein levels determined with a protein assay kit (BCA kit; Thermo Fischer Scientific, Waltham, MA, U.S.A.). The enzymatic reaction was halted by adding 0.1 N NaOH to the solution as soon as a noticeable color change occurred, thereby ceasing the conversion of p-nitrophenylphosphate to p-nitrophenol. The absorbance at 450 and 562 nm was measured via a spectrophotometer, and the ALP activity was subsequently quantified by relating these absorbance measurements to a standard curve derived from known concentrations of p-nitrophenol.²⁰⁾

Cell Viability Assay

Cell viability assays were conducted by seeding 1 × 10³ cells into each well of a 96-well plate, and after 24 h, a mixture of culture medium supplemented with either 10.0 µM of a specific drug or 10.0 µM alendronate sodium hydrate was added to the wells. After incubation for 72 h at 37 °C, the cells in each well were rinsed and treated with a solution composed of 10 µL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5 mg/mL) and 90 µL of culture medium. After another incubation at 37 °C for 4 h, this process led to the formation of formazan crystals, which were subsequently dissolved by adding 100 µL of dimethyl sulfoxide (DMSO) to each well. Upon complete dissolution, the optical density of the solution was measured at a wavelength of 570 nm via an ELISA reader (Thermo Fischer Scientific).²¹⁾

Acknowledgments

The authors extend their gratitude to Hsiao-Ching Yu and Chao-Lien Ho from Center of the High-Value Instrument at National Sun Yat-sen University for their support in acquiring mass (MS−006500) and NMR (NMR−001100) spectra under grant NSTC 113−2740−M−110−002. They also wish to acknowledge the Instrumentation Center at National Taiwan University for offering X-ray facilities, supported by grants NSTC 113−2740−M−002−007 and XRD 000200. This research was funded primarily by Grants from the National Museum of Marine Biology & Aquarium, the National Science and Technology Council (NSTC 112−2320−B−29−002−MY3, 113−2320−B−291−001, and 112−2811−B−291−002), and the Kaohsiung Armed Forces General Hospital (KAFGH-D-113028) in Taiwan, awarded to C.-Y.H. and P.-J.S. Their generous support is deeply appreciated.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

References

• 1) Burks J. E., van der Helm D., Chang C. Y., Ciereszko L. S., Acta Crystallogr. B, 33, 704–709 (1977).
• 2) Chung H.-M., Wang Y.-C., Tseng C.-C., Chen N.-F., Wen Z.-H., Fang L.-S., Hwang T.-L., Wu Y.-C., Sung P.-J., Mar. Drugs, 16, 339 (2018).
• 3) McFadden C. S., van Ofwegen L. P., Quattrini A. M., Bull. Soc. Syst. Biol., 1, 8735 (2022).
• 4) Chang Y.-C., Hwang T.-L., Huang S.-K., Huang L.-W., Lin M.-R., Sung P.-J., Heterocycles, 81, 991–996 (2010).
• 5) Lin C.-C., Su J.-H., Chen W.-F., Wen Z.-H., Peng B.-R., Huang L.-C., Hwang T.-L., Sung P.-J., Molecules, 24, 2487 (2019).
• 6) Shin J., Park M., Fenical W., Tetrahedron, 45, 1633–1638 (1989).
• 7) Zhang W., Guo Y.-W., Mollo E., Cimino G., Helv. Chim. Acta, 87, 2341–2345 (2004).
• 8) Qi S.-H., Zhang S., Qian P.-Y., Xiao Z.-H., Li M.-Y., Tetrahedron, 62, 9123–9130 (2006).
• 9) Sheu J.-H., Chen Y.-P., Hwang T.-L., Chiang M. Y., Fang L.-S., Sung P.-J., J. Nat. Prod., 69, 269–273 (2006).
• 10) Sung P.-J., Tsai W.-T., Lin M.-R., Su Y.-D., Pai C.-H., Chung H.-M., Su J.-H., Chiang M. Y., Chem. Lett., 37, 88–89 (2008).
• 11) Lin Y.-Y., Jean Y.-H., Lee H.-P., Lin S.-C., Pan C.-Y., Chen W.-F., Wu S.-F., Su J.-H., Tsui K.-H., Sheu J.-H., Sung P.-J., Wen Z.-H., Mar. Drugs, 15, 9 (2017).
• 12) Qi X., Zhang X., Meng J., Wu J., Cheng W., Huang J., Lin W., Eur. J. Med. Chem., 246, 114948 (2023).
• 13) Liu C.-J., Wang C.-C., Chang C.-F., Zhang M. M., Tsou L. K., Wen Z.-H., Hwang T.-L., Chen Y.-Y., Sung P.-J., Phytochem. Lett., 57, 92–95 (2023).
• 14) Dai C.-F., Chin C.-H., “Octocoral Fauna of Kenting National Park,” Kenting National Park Headquarters, Kenting, Pingtung, 2019, pp. 492–493.
• 15) Dai C.-F., “Octocoral Fauna of Taiwan,” Ocean Center, National Taiwan University, Taipei, 2019, pp. 636–637.
• 16) Dai C.-F., “Corals of Taiwan, Vol.2: Octocorallia,” Owl Publishing House Co., LTD., Taipei, 2022, p. 381.
• 17) Sheldrick G. M., Acta Crystallogr. A Found. Adv., 71, 3–8 (2015).
• 18) Sheldrick G. M., Acta Crystallogr. C Struct. Chem., 71, 3–8 (2015).
• 19) CCDC homepage. ‹http://www.ccdc.cam.ac.uk/conts/retrieving.html.›
• 20) Ansari S., Ito K., Hofmann S., ACS Omega, 7, 12724–12733 (2022).
• 21) Kuo H.-M., Tseng C.-C., Chen N.-F., Tai M.-H., Hung H.-C., Feng C.-W., Cheng S.-Y., Huang S.-Y., Jean Y.-H., Wen Z.-H., Mar. Drugs, 16, 8 (2018).