Nine New Antibacterial Diterpenes and Steroids from the South China Sea Soft Coral Lobophytum catalai Tixier-Durivault

Five new cembrane-type diterpenes, lobocalines A–E (1–5), and four new steroids, lobocaloids A–D (9–12), along with six known related compounds (6–8 and 13–15) were isolated from the Yalong Bay soft coral Lobophytum catalai Tixier-Durivault. The structures of the new compounds were elucidated by extensive spectroscopic analysis, NMR calculation with DP4+ analysis, time-dependent density functional theory–electronic circular dichroism (TDDFT-ECD) calculations, X-ray diffraction analyses and comparison with the reported spectroscopic data of known compounds. Further, with the aid of X-ray diffraction analysis, the structure of lobocrasol B (15) was firmly revised as 15a. In in vitro bioassays, compound 2 showed moderate antibacterial activities against fish pathogenic bacteria Streptococcus parauberis KSP28 and Phoyobacterium damselae FP2244 with minimum inhibitory concentration (MIC) values of 8.7 and 17.3 µg/mL, respectively. All the steroids exhibited antibacterial activities against the S. parauberis KSP28 with MIC values ranging from 12.3 to 53.6 µg/mL. Compounds 2, 7 and 14 have remarkable inhibitory effects on the hemolysin production of Staphylococcus aureus, while compounds 8–12 have medium inhibitory effects on the pyocyanin production in Pseudomonas aeruginosa.


Introduction
Aquaculture is a crucial component of agriculture, the foundational element of agricultural economic growth and a crucial source of food.With the rapid and intensive development of aquaculture, many problems related to aquatic animal diseases have gradually been exposed.The main pathogens of aquatic animals include bacteria, viruses, parasites, etc.It is estimated that economic loss of aquatic animals caused by numerous bacteria (such as Streptococcus parauberis and Aeromonas salmonicida) accounts for 58%, which is the most serious factor leading to the economic loss of aquaculture [1][2][3].As antibiotic resistance and drug residues in aquaculture have become global focus, the search for new and effective antibacterial compounds against fish pathogenic bacteria has become more important and urgent.In addition, bacteria Staphylococcus aureus and Pseudomonas aeruginosa could also cause a broad range of life-threatening diseases in humans.For example, S. aureus cause infectious disease by the production of virulence factors such as hemolysins [4].Pyocyanin, the secondary metabolite produced by P. aeruginosa, is considered to play an essential role of oxidative stress in the infection [5].
Marine invertebrates are important sources of natural products and have been recognized to be the rich source of bioactive secondary metabolites with structural diversity [6][7][8][9][10].Therefore, the marine invertebrates have enormous potential for exploring new marine Marine invertebrates are important sources of natural products and have been recognized to be the rich source of bioactive secondary metabolites with structural diversity [6][7][8][9][10].Therefore, the marine invertebrates have enormous potential for exploring new marine drugs.In particular, soft corals of the genus Lobophytum (order Alcyonacea, family Alcyoniidae) have become an intensive research subject, which produced varieties of structurally intriguing and biologically interesting molecules [11,12].More recently, two breakthrough studies have been reported toward the biosynthesis of terpenes in the soft corals and the identification of coral-encoded terpene cyclase genes enabled the enzyme that construct the coral-exclusive terpene scaffolds [13,14], which attract significant attentions to conduct the metabolic patterns of marine soft coral metabolites.In addition, a survey of the extensive literature revealed that abundant Lobophytum soft corals are inhabiting in the South China Sea, and the titled species L. catalai has been rarely studied.Until now, only a total of four studies have been reported on the chemical investigations of this species, leading to the discovery of several terpenes [15][16][17] and steroids [18] with antiinflammatory [17] and/or cytotoxic [17] activities.
In the course of our continuing effort to explore chemically fascinating and biologically active secondary metabolites of South China Sea marine invertebrates [12], we recently carried out a chemical investigation on the soft coral L. catalai collected off Yalong Bay, Hainan Province, China, resulting in the isolation and characterization of nine new compounds, namely lobocalines A-I (1-5 and 9-12), together with six known compounds (6-8 and 13-15) (Figure 1).The structural difference between the five new diterpenes 1-5 is reflected in the different positions of substituents.Moreover, the structural diversities of six steroids 9-14 are mainly attributed to the different configurations of C-5 and C-6 in ring B of steroidal nucleus and the variations in functional groups on the side chains.What is more is that the structural determinations of new compounds are also a challenging problem, and X-ray diffraction analysis as a powerful tool [19] was applied in this study.Herein, we report the detailed isolation, planar structural elucidation, stereochemistry determination, and antibacterial activity evaluation of these isolated compounds.
Compound 1, namely lobocaline A, was isolated as colorless crystals and gave the molecular formula of C 22 H 34 O 3 as established by HRESIMS from the protonated molecular ion peak observed at m/z 347.2580 [M + H] + (calcd.for C 22 H 35 O 3 , 347.2581), implying six degrees of unsaturation.The characteristic peak at ν max 1737 cm −1 in its IR spectrum showed the presence of ester carbonyl group, which was further confirmed by the diagnostic 13 C NMR chemical shift at δ C 170.6.The 1 H and 13 C NMR data (Tables 1 and 2) of 1 along with the assistance of DEPT spectrum revealed the presence of two trisubstituted double bonds [δ H 6.06 (1H, d, J = 10.8Hz, H-2), δ C 118.6 (CH, C-2) and 146.9 (qC, C-1); δ H 5.97 (1H, d, J = 10.9Hz, H-3), δ C 135. .The above-mentioned moieties accounted for five of the six degrees of unsaturation, indicating the monocyclic carbon framework for 1.A comparison of the NMR data of 1 with those of the co-occurring known compound sarcoboettgerol D (6), which had been recently isolated from soft coral Sarcophyton boettgeri and its absolute configuration was determined by the TDDFT-ECD calculation method [20], which revealed that they were structural analogues, with the only difference being that the hydroxyl group at C-7 in 6 was substituted by an acetyloxy group at C-7 in 1, in agreement with the 42 mass unit difference in their molecular weights.The position of the acetyloxy group at C-7, as evidenced by the observation of the upfield shift of C-6 from δ C 32.9 in 6 to δ C 30.0 in 1.This assignment was further confirmed by strong HMBC correlations (Figure 2) from H-7 (δ H 5.16) to C-19 (δ C 113.0), C-8 (δ C 146.8) and carbonyl carbon (δ C 170.6).Thus, the planar structure of 1 was determined as shown in Figure 2.   The relative configuration of 1 was established mainly by the analysis of NOESY correlations (Figure 2).The clear NOE correlations of H-2 (δH 6.06)/H3-16 (δH 1.10), H-2/H3-18 (δH 1.78) and H-3 (δH 5.97)/H-5α (δH 2.24) indicated the "1E, 3E" geometry of the two double bonds Δ 1,2 and Δ 3,4 .The highly similar NOE correlations of H-7/H-9β, H-9β/H-10β and H-10β/H3-20 and H-9α/H-11 between compounds 1 and 6 suggested that these two compounds shared the same stereochemistry.Furthermore, the suitable single crystals of 1 in MeOH were obtained.The X-ray crystallographic analysis using Cu Kα radiation (λ = 1.54178Å) firmly disclosed the absolute configuration of 1 to be 7R, 11S, 12S with the Flack parameter of 0.00 (10) (Figure 3, CCDC 2290865).What is more is the acetylation of 6 yielding 1 further confirmed the structure and stereo-configuration of 1.The chemical structure The relative configuration of 1 was established mainly by the analysis of NOESY correlations (Figure 2).The clear NOE correlations of H-2 (δ H 6.06)/H 3 -16 (δ H 1.10), H-2/H 3 -18 (δ H 1.78) and H-3 (δ H 5.97)/H-5α (δ H 2.24) indicated the "1E, 3E" geometry of the two double bonds ∆ 1,2 and ∆ 3,4 .The highly similar NOE correlations of H-7/H-9β, H-9β/H-10β and H-10β/H 3 -20 and H-9α/H-11 between compounds 1 and 6 suggested that these two compounds shared the same stereochemistry.Furthermore, the suitable single crystals of 1 in MeOH were obtained.The X-ray crystallographic analysis using Cu Kα radiation (λ = 1.54178Å) firmly disclosed the absolute configuration of 1 to be 7R, 11S, 12S with the Flack parameter of 0.00 (10) (Figure 3, CCDC 2290865).What is more is the acetylation of 6 yielding 1 further confirmed the structure and stereo-configuration of 1.The chemical structure of 1 was thus established as shown in Figure 1.
The relative configuration of 1 was established mainly by the analysis of NOESY correlations (Figure 2).The clear NOE correlations of H-2 (δH 6.06)/H3-16 (δH 1.10), H-2/H3-18 (δH 1.78) and H-3 (δH 5.97)/H-5α (δH 2.24) indicated the "1E, 3E" geometry of the two double bonds Δ 1,2 and Δ 3,4 .The highly similar NOE correlations of H-7/H-9β, H-9β/H-10β and H-10β/H3-20 and H-9α/H-11 between compounds 1 and 6 suggested that these two compounds shared the same stereochemistry.Furthermore, the suitable single crystals of 1 in MeOH were obtained.The X-ray crystallographic analysis using Cu Kα radiation (λ = 1.54178Å) firmly disclosed the absolute configuration of 1 to be 7R, 11S, 12S with the Flack parameter of 0.00 (10) (Figure 3, CCDC 2290865).What is more is the acetylation of 6 yielding 1 further confirmed the structure and stereo-configuration of 1.The chemical structure of 1 was thus established as shown in Figure 1.   1 and 2) were reminiscent of 1. Careful comparison of their NMR data revealed they possessed the same planar structure and only chemical shifts at C-7 (δ C 72.5 in 1 vs. δ C 75.9 in 2) were different, suggesting that 2 was simply the C-7 epi-isomer of 1, thus suggesting the assignment of 7S*, 11S*, 12S* configuration of 2. Further analysis of 2D NMR spectra and the obvious NOESY correlations of H-7/H-11 confirmed this hypothesis (Figure 2).Subsequently, the absolute configuration of 2 was then assigned to be 7S, 11S, 12S by the comparison of similar experimental ECD spectra of compounds 1 (Figure S1.9) and 2 (Figure S2.9).Accordingly, the structure of 2 was determined as depicted in Figure 1.
Lobocaline C (3) was isolated as a colorless oil, and its molecular formula was assigned as C 20 H 32 O 2 by HRESIMS ion peak at m/z 327.2295 [M + Na] + (calcd.for C 20 H 32 O 2 Na, 327.2295), indicating five degrees of unsaturation.The 1D NMR data of 3 (Tables 1 and 2) showed great similarities to those of lE,3E,7E,11:12-epoxy-l,3,7-cembratriene, a known cembranoid isolated from the South Andaman Coast soft coral Lobophytum sp.[26], with the only difference being the presence of an additional hydroxyl group at C-6 in 3, in agreement with the 16 mass unit difference between these two compounds.This assignment was further confirmed by the strong 1 H-1 H COSY correlations of H-6 (δ H 4.54)/H-7(δ H 5.30) (Figure 2).As mentioned above, the planar structure of 3 was determined as shown in Figure 2. The geometries of the double bonds at ∆ 1,2 , ∆ 3,4 and ∆ 7,8 were both assigned to be E by the NOESY correlations of H-2 (δ H 5.94)/H 3 -16 (δ H 1.05), H-2/H 3 -18 (δ H 1.78) and H-7 (δ H 5.30)/H-9α (δ H 2.30) (Figure 2), respectively.The relative configurations of C-11 and C-12 in 3 were proven to be the same as those of 1 due to the diagnostic NOESY correlations of H 3 -20 (δ H 1.25)/H-10b (δ H 1.46), H 3 -20/H-13b (δ H 2.00), H-11 (δ H 2.82)/H-3 (δ H 5.92) and H-11/H-14a (δ H 2.36).To figure out the relative configuration of 3, the QM-NMR calculation was performed to give the best match of more than 99% probability in DP4+ with the 6R*, 11S*, 12S* isomer (see the details in the Supplementary Materials).The absolute configuration of 3 was determined by time-dependent density functional theory (TDDFT) ECD calculation.The theoretical ECD spectrum of 3 was calculated by the DFT method at the B3LYP/6-311G(d,p) level.As a result, the Boltzmann-averaged ECD spectrum of (6R, 11S, 12S)-3 highly matched the experimental ECD spectrum of 3, while the calculated ECD spectrum of enantiomer showed a completely opposite curve (Figure 4).Consequently, the absolute configuration of 3 was established to be 6R, 11S, 12S.
ESY correlations of H3-20 (δH 1.25)/H-10b (δH 1.46), H3-20/H-13b (δH 2.00), H-11 (δH 2.82)/H-3 (δH 5.92) and H-11/H-14a (δH 2.36).To figure out the relative configuration of 3, the QM-NMR calculation was performed to give the best match of more than 99% probability in DP4+ with the 6R*, 11S*, 12S* isomer (see the details in the Supplementary Materials).The absolute configuration of 3 was determined by time-dependent density functional theory (TDDFT) ECD calculation.The theoretical ECD spectrum of 3 was calculated by the DFT method at the B3LYP/6-311G(d,p) level.As a result, the Boltzmann-averaged ECD spectrum of (6R, 11S, 12S)-3 highly matched the experimental ECD spectrum of 3, while the calculated ECD spectrum of enantiomer showed a completely opposite curve (Figure 4).Consequently, the absolute configuration of 3 was established to be 6R, 11S, 12S.Lobocaline D (4), a colorless oil, has the same molecular formula (C 20 H 32 O 2 ) as that of 3, implying they are isomers.Comparison of 1 H and 13 C chemical shifts of 4 and 3 (Tables 1 and 2) followed by a detailed analysis of 2D NMR data revealed that the structure of 4 was similar to that of 3 with a different substituted position of the hydroxyl group.This conclusion was further supported by HMBC correlations from H 3 -18 (δ H 1.74) to C-5 (δ C 78.1) and 1 H-1 H COSY correlations of H-5 (δ H 4.12)/H-6α (δ H 2.39)/H-7 (δ H 5.19).Thus, the planar structure of 4 was determined.The relative configurations of C-11 and C-12 in 4 were assigned to be the same as those of 1 due to the similar 13 C NMR data.Similarly, the relative configuration of 4 was also elucidated via QM-NMR calculations using the DP4+ protocol.The best match was observed for the 5R*, 11S*, 12S* relative configuration with a DP4+ probability over 99%.Moreover, the absolute configuration of 4 was further determined by the TDDFT-ECD approach.As shown in Figure 5, the Boltzmann-averaged ECD curve calculated for the (5S, 11R, 12R)-4 enantiomer matched very well with the experimental ECD spectrum of 4. Accordingly, the absolute configuration of 4 was determined to be 5S, 11R, 12R.Lobocaline D (4), a colorless oil, has the same molecular formula (C20H32O2) as that of 3, implying they are isomers.Comparison of 1 H and 13 C chemical shifts of 4 and 3 (Tables 1 and 2) followed by a detailed analysis of 2D NMR data revealed that the structure of 4 was similar to that of 3 with a different substituted position of the hydroxyl group.This conclusion was further supported by HMBC correlations from H3-18 (δH 1.74) to C-5 (δC 78.1) and 1 H-1 H COSY correlations of H-5 (δH 4.12)/H-6α (δH 2.39)/H-7 (δH 5.19).Thus, the planar structure of 4 was determined.The relative configurations of C-11 and C-12 in 4 were assigned to be the same as those of 1 due to the similar 13 C NMR data.Similarly, the relative configuration of 4 was also elucidated via QM-NMR calculations using the DP4+ protocol.The best match was observed for the 5R*, 11S*, 12S* relative configuration with a DP4+ probability over 99%.Moreover, the absolute configuration of 4 was further determined by the TDDFT-ECD approach.As shown in Figure 5, the Boltzmann-averaged ECD curve calculated for the (5S, 11R, 12R)-4 enantiomer matched very well with the experimental ECD spectrum of 4. Accordingly, the absolute configuration of 4 was determined to be 5S, 11R, 12R.Lobocaline E (5) was obtained as a colorless oil.Its molecular formula, C22H34O3, was deduced from the ion peak observed at m/z 369.2397 [M + Na] + (calcd.for C22H34O3Na, 369.2400) in its HRESIMS spectrum.The 1 H and 13 C NMR data (Tables 1and 2) of 5 were extremely similar to those of the co-occurring compound 4, indicating that they are structural analogues.In fact, the only difference between these two compounds is the replace- Lobocaline E (5) was obtained as a colorless oil.Its molecular formula, C 22 H 34 O 3 , was deduced from the ion peak observed at m/z 369.2397 [M + Na] + (calcd.for C 22 H 34 O 3 Na, 369.2400) in its HRESIMS spectrum.The 1 H and 13 C NMR data (Tables 1 and 2) of 5 were extremely similar to those of the co-occurring compound 4, indicating that they are structural analogues.In fact, the only difference between these two compounds is the replacement of the hydroxyl group at C-5 in 4 by the acetyl group at C-5 in 5, in agreement with the 42 mass unit difference between compounds 4 and 5. To determine the relative configuration of 5, the theoretical and experimental NMR data were correlated and their corresponding DP4+ probabilities were estimated.Consequently, the candidate structure 5a (Figure S11.3) showed the dominant probability of 99.52%, indicating the 5R*, 11S*, 12S* relative configuration of 5.In this case, the TDDFT-ECD calculation was also employed to determine the absolute stereochemistry of 5.As shown in Figure 6, the calculated ECD spectrum of (5S, 11R, 12R)-5 fairly matched with the experimental one, allowing us to assign the absolute configuration of 5 to be 5S, 11R, 12R.Moreover, the hydrolysis reaction of 5 was also performed, but unfortunately, no product was detected due to the low number of isolated compounds.The chemical structure of 5 was thus established as depicted in Figure 1.Lobocaloid A (9) was isolated as a white powder.Its molecular formula was determined as C29H50O6 by HRESIMS m/z 517.3504 [M + Na] + (calcd.for C29H50O6Na, 517.35), appropriate for five degrees of unsaturation.The 1 H NMR data of 9 (Table 3) showed the general characters of polyhydroxylated sterols, including an olefinic proton resonating at δH 5.70 (1H, dd, J = 3.5, 1.5 Hz, H-16), two oxymethines at δH 4.27(1H, s, H-3), 3.82 (1H, dd, J = 12.2, 4.9 Hz, H-6), a hydroperoxyl group signal at δH 9.20 (br s), and seven methyls at δH 0.94 (3H, s, Me-18), 0.95 (3H, s, Me-19), 1.30 (3H, s, Me-21), 1.20 (3H, s, Me-26), 1.19 (3H, s, Me-27), 0.86 (3H, d, J = 7.3 Hz, Me-28), 0.93 (3H, d, J = 7.1 Hz, Me-29).Moreover, the 13 C NMR data (Table 3) and DEPT spectrum of 9 indicated the presence of 29 signals, which comprised seven methyls, eight methylenes, eight methines (including one olefinic at δC 126.9, and seven sp 3 hybridized at δC 67.9, 72.0, 32.5, 43.5, 58.0, 30.2, 50.3) and six quaternary carbons (including an olefinic at δC 158.3 and five sp 3 hybridized at δC 78.2, 41.3, 47.5, 85.7, 75.6).These above data suggest the presence of a trisubstituted double bond, which accounted for one degree of unsaturation.The remaining four degrees of unsaturation were attributed to a tetracyclic system in the molecule.In the 1    13 C NMR data (Table 3) and DEPT spectrum of 9 indicated the presence of 29 signals, which comprised seven methyls, eight methylenes, eight methines (including one olefinic at δ C 126.9, and seven sp 3 hybridized at δ C 67.9, 72.0, 32.5, 43.5, 58.0, 30.2, 50.3) and six quaternary carbons (including an olefinic at δ C 158.3 and five sp 3 hybridized at δ C 78.2, 41.3, 47.5, 85.7, 75.6).These above data suggest the presence of a trisubstituted double bond, which accounted for one degree of unsaturation.The remaining four degrees of unsaturation were attributed to a tetracyclic system in the molecule.In the 1 H-1 H COSY spectrum, it was possible to identify three different structural units extending from C-1 to C-4; from C-6 to both C-12 and C-16 through C-8; and from C-22 to both C-28 and C-29 through C-23 (Figure 7).From the HMBC spectrum, the correlations from H 3 -19 to C-1, C-5, C-9 and C-10; from H 3 -18 to C-12, C-13, C-14 and C-17; from H-6 to C-4 and C-5; from H-16 to C-20; from H 3 -21 to C-17, C-20 and C-22; from both H 3 -26 and H 3 -27 to C-24; and from H 3 -28 to C-25 permitted the establishment of the carbon skeleton of a 23,24dimethycholestane (Figure 7).The hydroperoxyl group substituted at C-20 was confirmed by the HMBC correlation of the hydroperoxyl proton δ H 9.20 (br s) to the oxygenated carbon at δ C 86.1 (C-20).An extensive survey of the literature revealed that the structure of 9 closely resembled that of michosterols A ( 16), a polyoxygenated steroid isolated from the soft coral Lobophytum michaelae [27].A comparison of the NMR data of 9 with 16, revealed that they were structural analogues, and the only difference occurred at the position C-25, where the acetyloxy group in 16 was replaced by a hydroxyl group in 9, in agreement with the 42 mass unit difference between compounds 9 and 16.As mentioned above, the planar structure of 9 was thus established.The relative configuration of 9 was established mainly by analysis of the NOESY correlations.As depicted in Figure 7, it was found that the NOE interactions displayed by both H 3 -18/H-8, H 3 -19/H-8, H 3 -19/H-6 and H-6/H-8, assuming the β-orientation of H 3 -19, H-6, H-8 and H 3 -18.Moreover, the NOESY correlation of H-9/H-14 assigned the αorientation of H-9 and H-14.Further, the chemical shifts of C-20, C-21, C-22, C-23, C-28 and C-29 on the side chain are similar to that of 16, suggested the same relative configuration 2) with the model known compound 5β-cholestane-3β,5,6α-triol, which was previously isolated from the soft coral Sinularia sp.[28,29].Consequently, the relative configuration of 9 was determined.Furthermore, based on the biogenetic consideration of the biosynthetic pathway of steroids, Me-18 and Me-19 should be positioned on the β-orientation; thus, the absolute configuration of 9 was elucidated as shown in Figure 1.    3) of 10 closely resembled that of michosterols A ( 16), suggesting that they were also structural analogues.In fact, the only difference between 10 and 16 appeared at C-20, where the hydroperoxyl group in 16 was substituted by a hydroxyl group in 10, in agreement with a 16 mass unit difference between their molecular weights.This assignment was supported by not only the carbon chemical shift of C-20 significantly upfield shifted from δ C 85.6 ppm to δ C 75.7 ppm, but also the chemical shift of the distinctive olefinic proton H-16 shifted from δ H 5.70 (d, J = 2.0 Hz) to δ H 5.49 (dd, J = 3.4, 1.5 Hz).The relative stereochemistry of 10 was determined by the analysis of NOE correlations (Figure 7) and by comparison of NMR spectroscopic data with those of model molecule sarcophytosterol, a known steroid isolated from the Dongsha atoll soft coral Lobophytum sarcophytoides, whose structure has been unambiguously determined by X-ray diffraction analysis [30].On the basis of the above findings, the structure of compound 10 was determined as shown in Figure 1.
The molecular formula of lobocaloid C (11) was the same as michosterols A ( 16 The following detailed analysis of 1 H-1 H COSY and HMBC correlations assigned the planar structure of 11, the same as 16, which suggested that 11 was the isomer of 16.Literature checking revealed that the NMR data of rings A and B in 11 were strongly reminiscent of the known compound 23,24-dimethylcholest-16-ene-3β,5α,6β,11α,20(R)-pentol-3-monoacetate [31], indicating that the rings' structures were identical.Thus, the absolute stereochemistry of C-3, C-5, and C-6 in 11 was speculatively assigned as 3S, 5R, 6R.4), revealed that they were structural analogues, with the only difference being the presence of a hydroxyl group substituted at C-20 on the side chain of 12 instead of a hydroperoxyl group in 11, in agreement with the 16 mass unit difference between compounds 11 and 12.The hydroxyl group was substituted at C-20, as evidenced by the observation of the upfield shifting of C-20 from δ C 86.1 in 11 to δ C 75.8 in 12. Similarly, the relative configuration of 12 was determined by analyzing NOE correlations and comparing its NMR data with those of compounds 10 and 11.As mentioned above, the structure of 12 was assigned as shown in Figure 1.
Compound 15 was readily identified as lobophytrol B, a capnosane-type diterpenoid previously reported to be isolated from Lobophytum sp. by our group [23].By comparing the nuclear magnetic and optical rotation data, they proved to be exactly the same.In this study, the suitable crystals of 15 were eventually obtained, and they were then sent for X-ray crystallographic analysis using Cu Ka radiation (λ = 1.54178Å).Analysis of the X-ray data unambiguously determined the planar structure and absolute configuration of 15 with the Flack parameter of 0.12 (7) (CCDC 2290866).Surprisingly, the X-ray result disclosed that the correct structure of lobophytrol B is 15a, instead of 15 (Figure 8).Compared to the mass spectrum, the molecular weight is 18 more than before, probably due to the presence of different molecular fragmentation peaks.We carefully and analyzed in depth the data to determine why the structure of 15 was incorrectly assigned, realizing that Compound 15 was readily identified as lobophytrol B, a capnosane-type diterpenoid previously reported to be isolated from Lobophytum sp. by our group [23].By comparing the nuclear magnetic and optical rotation data, they proved to be exactly the same.In thi study, the suitable crystals of 15 were eventually obtained, and they were then sent for X ray crystallographic analysis using Cu Ka radiation (λ = 1.54178Å).Analysis of the X-ray data unambiguously determined the planar structure and absolute configuration of 1 with the Flack parameter of 0.12 (7) (CCDC 2290866).Surprisingly, the X-ray result dis closed that the correct structure of lobophytrol B is 15a, instead of 15 (Figure 8).Compared to the mass spectrum, the molecular weight is 18 more than before, probably due to th presence of different molecular fragmentation peaks.We carefully and analyzed in depth the data to determine why the structure of 15 was incorrectly assigned, realizing that th error was triggered by the chemical shift of carbon substituted by the hydroxyl group and the same oxygen ring established at positions C-  In the in vitro bioassay, the isolated compounds were tested for their antibacterial cytotoxic and anti-inflammatory effects.In the antibacterial bioassays (Table 5), com pound 2 showed moderate antibacterial activities against the fish pathogenic bacteri Streptococcus parauberis KSP28 (MIC 8.7 µg/mL) and Phoyobacterium damselae FP2244 (MIC 17.3 µg/mL).Compounds 7-9 showed weak antibacterial activities against S. parauberi KSP28 (MIC 30.4,32.2, 49.4 µg/mL) and P. damselae FP2244 (MIC 30.4,16.1, 49.4 µg/mL) All the steroids exhibited antibacterial activities against S. parauberis KSP28 with MIC val ues ranging from 12.3 to 53.6 µg/mL.Furthermore, compounds 10 and 13 exhibited sig nificant antibacterial activities against a variety of strains; compound 13 especially dis played potent inhibitory activity against P. damselae FP2244, S. parauberis SPOF3K and S agalactiae WR10 with MIC value of 6.2 µg/mL, 12.3 µg/mL and 12.3 µg/mL, respectively Furthermore, compound 13 showed the growth inhibitory activities against the vancomy cin-resistant Enterococcus faecium G1 and G8 with the MIC values of 12.3 µg/mL and 12. µg/mL, respectively, comparable with that of positive control levofloxacin hydrochlorid (MIC > 39.78 µg/mL), ampicillin sodium (MIC > 37.14 µg/mL) and vancomycin hydrochlo ride (MIC > 297 µg/mL and 74.25 µg/mL).In the in vitro bioassay, the isolated compounds were tested for their antibacterial, cytotoxic and anti-inflammatory effects.In the antibacterial bioassays (Table 5), compound 2 showed moderate antibacterial activities against the fish pathogenic bacteria Streptococcus parauberis KSP28 (MIC 8.7 µg/mL) and Phoyobacterium damselae FP2244 (MIC 17.3 µg/mL).Compounds 7-9 showed weak antibacterial activities against S. parauberis KSP28 (MIC 30.4,32.2, 49.4 µg/mL) and P. damselae FP2244 (MIC 30.4,16.1, 49.4 µg/mL).All the steroids exhibited antibacterial activities against S. parauberis KSP28 with MIC values ranging from 12.3 to 53.6 µg/mL.Furthermore, compounds 10 and 13 exhibited significant antibacterial activities against a variety of strains; compound 13 especially displayed potent inhibitory activity against P. damselae FP2244, S. parauberis SPOF3K and S. agalactiae WR10 with MIC value of 6.2 µg/mL, 12.3 µg/mL and 12.3 µg/mL, respectively.Furthermore, compound 13 showed the growth inhibitory activities against the vancomycin-resistant Enterococcus faecium G1 and G8 with the MIC values of 12.3 µg/mL and 12.3 µg/mL, respectively, comparable with that of positive control levofloxacin hydrochloride (MIC > 39.78 µg/mL), ampicillin sodium (MIC > 37.14 µg/mL) and vancomycin hydrochloride (MIC > 297 µg/mL and 74.25 µg/mL).a Tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OT), levofloxacin hydrochloride (LF), Ampicillin (AMP) and Vancomycin hydrochloride (VAN) were used as positive controls.b '-' indicated they were not subjected to the antibacterial rescreening experiments since their inhibition rates against these bacteria were <90% in the preliminary antibacterial bioassays.
In hemolytic activity, the results showed that the hemolytic activity of S. aureus treated with compounds 1-4, 6-8 and 13-15 were obviously reduced when compared with the control (without compound treatment).In particular, the hemolysis percentage of compound 2, 7 and 14 test groups reached about 35.7%, 42.2% and 39.1% (Figure 9).These results suggest that compounds 2, 7 and 14 have a strong inhibitory effect on the hemolysin production of S. aureus.Furthermore, in the pyocyanin quantitation assay experiment (Figure 10), compounds 8-12 have medium inhibition effects on the pyocyanin production in P. aeruginosa, compared with the control (without compound treatment).Tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OT), levofloxacin hydrochloride (LF), Ampicillin (AMP) and Vancomycin hydrochloride (VAN) were used as positive controls.b -' indicated they were not subjected to the antibacterial rescreening experiments since their inhibition rates against these bacteria were <90% in the preliminary antibacterial bioassays.
In hemolytic activity, the results showed that the hemolytic activity of S. aureus treated with compounds 1-4, 6-8 and 13-15 were obviously reduced when compared with the control (without compound treatment).In particular, the hemolysis percentage of compound 2, 7 and 14 test groups reached about 35.7%, 42.2% and 39.1% (Figure 9).These results suggest that compounds 2, 7 and 14 have a strong inhibitory effect on the hemolysin production of S. aureus.Furthermore, in the pyocyanin quantitation assay experiment (Figure 10), compounds 8-12 have medium inhibition effects on the pyocyanin production in P. aeruginosa, compared with the control (without compound treatment).

General Experimental Procedures
Melting points were measured on an X-4 digital micro melting point apparatus.Op-

Calculation Section
Conformational search was performed by using the torsional sampling (MCMM) approach and OPLS_2005 force field within an energy window of 21 kJ/mol.Conformers above 1% Boltzmann populations were re-optimized at the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for chloroform.Frequency analysis was also carried out to confirm that the re-optimized geometries were at the energy minima.Subsequently, NMR calculations were performed at the PCM/mPW1PW91/6-31G(d) level, as recommended for DP4+.NMR shielding constants were calculated using the GIAO method.Finally, shielding constants were averaged over the Boltzmann distribution obtained for each stereoisomer and correlated with the experimental data.ECD spectra were obtained by TDDFT calculations with the B3LYP/6-311G(d,p) level with the IEFPCM solvent model for CH 3 CN.At last, the Boltzmann-averaged ECD spectra of the compounds were obtained with SpecDis (Version 1.71).

Acetylation of Compound 6
Compound 6 (0.5 mg) was dissolved in dry pyridine (2.0 mL) and mixed with 50 mL of Ac 2 O.The mixtures were stirred at room temperature overnight, and the reaction was detected on the TLC by heating after spraying with vanillin H 2 SO 4 reagent.The crude acetylated product, after evaporating the solvent in vacuo, was purified by silica gel CC (petroleum ether/ether, 9:1) to afford a colorless crystal compound 1a (0.5 mg, 87% yield), which was identical to the natural sample of 1 in all respects (Figure S1.10).

Antibacterial Activity Bioassays
The strains Streptococcus parauberis KSP28, Streptococcus parauberis SPOF3K, Phoyoba cteriumdamselae FP2244, Aeromonas salmonicida AS42, Photobacterium halotolerans LMG 22194T, Enterococcus faecium 5270 MDR8 and Lactococcus garvieae FP MP5245 were provided by National Fisheries Research & Development Institute, Korea.The vancomycin-resistant Enterococcus faecium bacteria G1, G4, G7, G8 and G13 were provided by Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.The strain Streptococcus agalactiae WR10 and Edwardsiella piscicida TH1 were provided by Chinese Academy of Tropical Agricultural Sciences.The MIC values for all antimicrobial agents were measured by the 96-well micro-dilution method.Mueller-Hinton II broth (cation-adjusted, BD 212322) was used for MIC value determination.Generally, compounds were dissolved with DMSO to 20 mM as stock solutions.All samples were diluted with culture broth to 500 µM as the initial concentration.Further 1:2 serial dilutions were performed by addition of culture broth to reach concentrations ranging from 500 µM to 0.24 µM.A total of 100 µL of each dilution was distributed in 96-well plates, as well as sterile controls, growth controls (containing culture broth plus DMSO, without compounds) and positive controls (containing culture broth plus control antibiotics, such as tetracycline).Each test and growth control well was inoculated with 5 µL of an exponential-phase bacterial suspension (about 10 5 CFU/well).The 96-well plates were incubated at 37 °C for 24 h.MIC values of these compounds were defined as the lowest concentration to inhibit the bacterial growth completely.All MIC values were interpreted according to recommendations of the Clinical and Laboratory Standards Institute (CLSI).

Antihemolytic Activity Bioassays
Blood was collected from the eye sockets of SD rats and stood for 30 to 60 min.After centrifugation (800 rpm, 5 min), the red blood cells were cleaned twice with 0.9% normal saline and then added to create a 4% red blood cells suspension.S. aureus suspension was incubated with compounds (100 µM) in a centrifuge tube for 12 h at 37 °C.After centrifugation (6000 rpm, 15 min), 500 µL of the supernatant was taken from each tube, filtered by a 0.2 µm filter membrane and incubated with 500 µL of freshly red blood cells suspension at 37 °C for 2 h.The incubation of S. aureus and red blood cells suspension was used as the positive control, and the incubation of LB liquid medium and red blood cells suspension served as the negative control.After centrifugation (800 rpm, 5 min), the absorbance of supernatants at 540 nm was examined.The percentage of hemolysis value was calculated by comparing it with the positive control (100% hemolysis).

Pyocyanin Quantitation Assay
The P. aeruginosa strain was mixed with the compounds (100 µM) at 37 °C and 140 rpm/min for 24 h, and the supernatant was mixed with 1 mL chloroform.Then, the lower chloroform phase was mixed with 200 µL of 0.2 N HCl; after shaking and centrifugal (4500 rpm, 10 min), the color layer was removed and measured at 520 nm.Concentrations, expressed as micrograms of pyocyanin produced per milliliter of culture supernatant, were determined by multiplying the optical density at 520 nm (OD 520 ) by 17.072.

Conclusions
In summary, five new cembrane-type diterpenes lobocalines A-E (1-5) and four new steroids lobocaloids A-D (9-12), along with six known analogues (6-8 and 13-15), were isolated and characterized from the soft coral L. catalai collected off Yalong Bay of the South China Sea.Structurally, all the isolated new cembrane-type diterpenes have hydroxyl or acetoxy groups substituted at C-5, C-6 or C-7.All the steroids possessed the hydroxyl groups at C-3, C-5 and C-6, and the stereotypes of hydroxyl groups (3β, 5β, 6α or 3β, 5α, 6β) are significantly different in 9-14.It is worth noting that the structure of known compound 15 has been revised by the X-ray diffraction analysis in this study.

Figure 3 .
Figure 3. Perspective ORTEP drawing of the X-ray structure of 1 (Carbon atoms are represented by black ellipsoids, oxygen atoms are represented by red ellipsoids, and hydrogen atoms are represented by green spheres).

Figure 3 .
Figure 3. Perspective ORTEP drawing of the X-ray structure of 1 (Carbon atoms are represented by black ellipsoids, oxygen atoms are represented by red ellipsoids, and hydrogen atoms are represented by green spheres).Lobocaline B (2), a colorless oil, gave the same molecular formula as 1 on basis of its HRESIMS ion peak at m/z 347.2581 [M + H] + (calcd.for C 22 H 35 O 3 , 347.2581).Overall, the 1 H and 13 C NMR data of 2 (Tables1 and 2) were reminiscent of 1. Careful comparison of their NMR data revealed they possessed the same planar structure and only chemical shifts at C-7 (δ C 72.5 in 1 vs. δ C 75.9 in 2) were different, suggesting that 2 was simply the C-7 epi-isomer of 1, thus suggesting the assignment of 7S*, 11S*, 12S* configuration of 2. Further analysis of 2D NMR spectra and the obvious NOESY correlations of H-7/H-11 confirmed this hypothesis (Figure2).Subsequently, the absolute configuration of 2 was then assigned to be 7S, 11S, 12S by the comparison of similar experimental ECD spectra of compounds 1 (FigureS1.9)and 2 (FigureS2.9).Accordingly, the structure of 2 was determined as depicted in Figure1.Lobocaline C (3) was isolated as a colorless oil, and its molecular formula was assigned as C20 H 32 O 2 by HRESIMS ion peak at m/z 327.2295 [M + Na] + (calcd.for C 20 H 32 O 2 Na, 327.2295), indicating five degrees of unsaturation.The 1D NMR data of 3 (Tables1 and 2) showed great similarities to those of lE,3E,7E,11:12-epoxy-l,3,7-cembratriene, a known cembranoid isolated from the South Andaman Coast soft coral Lobophytum sp.[26], with the only difference being the presence of an additional hydroxyl group at C-6 in 3, in agreement with the 16 mass unit difference between these two compounds.This assignment was further confirmed by the strong 1 H-1 H COSY correlations of H-6 (δ H 4.54)/H-7(δ H 5.30) (Figure2).As mentioned above, the planar structure of 3 was determined as shown in Figure2.The geometries of the double bonds at ∆ 1,2 , ∆ 3,4 and ∆7,8 were both assigned to be E by the NOESY correlations of H-2 (δ H 5.94)/H 3 -16 (δ H 1.05), H-2/H 3 -18 (δ H 1.78) and H-7 (δ H 5.30)/H-9α (δ H 2.30) (Figure2), respectively.The relative configurations of C-11 and C-12 in 3 were proven to be the same as those of 1 due to the diagnostic NOESY correlations of H 3 -20 (δ H 1.25)/H-10b (δ H 1.46), H 3 -20/H-13b (δ H 2.00), H-11 (δ H 2.82)/H-3 (δ H 5.92) and H-11/H-14a (δ H 2.36).To figure out the relative configuration of 3, the QM-NMR calculation was performed to give the best match of more than 99% probability in DP4+ with the 6R*, 11S*, 12S* isomer (see the details in the Supplementary Materials).The absolute configuration of 3 was determined by time-dependent density functional theory (TDDFT) ECD calculation.The theoretical ECD spectrum of 3 was calculated by the DFT method at the B3LYP/6-311G(d,p) level.As a result, the Boltzmann-averaged ECD

Figure 7 .
Figure 7.The 1 H-1 H COSY, selected key HMBC and NOESY correlations of compounds 9-12.Lobocaloid B (10) was isolated as a white powder with the molecular formula of C 31 H 52 O 6 on the basis of the HRESIMS ion peak at m/z 543.3655 [M + Na] + (calcd.for C 31 H 52 O 6 Na, 543.3656), corresponding to six degrees of unsaturation.The 13 C NMR and DEPT spectra of 10 displayed 31 carbon signals, including eight methyls, eight methylenes, eight methines (including one olefinic at δ C 123.6) and seven quaternary carbons (including an olefinic at δ C 161.2 and one carbonyl at δ C 170.6), accounting for two out of six degrees of unsaturation, thus requiring four extra rings in the molecule structure of 10.The 13 C NMR data (Table3) of 10 closely resembled that of michosterols A (16), suggesting that they were also structural analogues.In fact, the only difference between 10 and 16 appeared at C-20, where the hydroperoxyl group in 16 was substituted by a hydroxyl group in 10, in agreement with a 16 mass unit difference between their molecular weights.This assignment was supported by not only the carbon chemical shift of C-20 significantly upfield shifted from δ C 85.6 ppm to δ C 75.7 ppm, but also the chemical shift of the distinctive olefinic proton H-16 shifted from δ H 5.70 (d, J = 2.0 Hz) to δ H 5.49 (dd, J = 3.4, 1.5 Hz).The relative stereochemistry of 10 was determined by the analysis of NOE correlations (Figure7) and by comparison of NMR spectroscopic data with those of model molecule sarcophytosterol, a known steroid isolated from the Dongsha atoll soft coral Lobophytum sarcophytoides, whose structure has been unambiguously determined by X-ray diffraction analysis[30].On the basis of the above findings, the structure of compound 10 was determined as shown in Figure1.The molecular formula of lobocaloid C (11) was the same as michosterols A(16), which was determined by HRESIMS ion peak at m/z 559.3609 [M + Na] + (calcd.for C 31 H 52 O 7 Na, 559.3605).The proton and carbon resonances of 11 showed a high degree of similarity to those of 16.By comparison of the 1 H and 13 C NMR data (Table 4) of 11 and 16, the differences were found in the chemical shifts of carbons in rings A and B [C-1 (δ C 32.3), C-2 (δ C 31.0), C-4 (δ C 40.8), C-5 (δ C 76.3) and C-6 (δ C 76.2) in 11 and C-1 (δ C 25.2), C-2 (δ C 27.7), C-4 (δ C 30.0), C-5 (δ C 78.1) and C-6 (δ C 71.8) in 16)].The following detailed analysis ), which was determined by HRESIMS ion peak at m/z 559.3609 [M + Na] + (calcd.for C 31 H 52 O 7 Na, 559.3605).The proton and carbon resonances of 11 showed a high degree of similarity to those of 16.By comparison of the 1 H and 13 C NMR data (Table 4) of 11 and 16, the differences were found in the chemical shifts of carbons in rings A and B [C-1 (δ C 32.3), C-2 (δ C 31.0), C-4 (δ C 40.8), C-5 (δ C 76.3) and C-6 (δ C 76.2) in 11 and C-1 (δ C 25.2), C-2 (δ C 27.7), C-4 (δ C 30.0), C-5 (δ C 78.1) and C-6 (δ C 71.8) in 16)].
the error was triggered by the chemical shift of carbon substituted by the hydroxyl group and the same oxygen ring established at positions C-8 and C-12, as well as the existence of various molecular fragment peaks in the mass spectrum.Careful re-examination of the HRESIMS spectrum revealed wrong identification of the molecular ion peak at m/z HREIMS 363.2517 [M + Na] + (calcd for C 20 H 36 O 4 Na, 363.2506), while the cluster of quasi-molecular ion peaks centered at m/z 323.2581 [M + H] + (calcd for C 20 H 35 O 3 , 323.2581) was overlooked (Figure S10.1).Mar.Drugs 2024, 22, x FOR PEER REVIEW 12 of 2 8 and C-12, as well as the existence o various molecular fragment peaks in the mass spectrum.Careful re-examination of th HRESIMS spectrum revealed wrong identification of the molecular ion peak at m/ HREIMS 363.2517 [M + Na] + (calcd for C20H36O4Na, 363.2506), while the cluster of quasi molecular ion peaks centered at m/z 323.2581 [M + H] + (calcd for C20H35O3, 323.2581) wa overlooked (Figure S10.1).

Figure 8 .
Figure 8. Revised structure (15a) and originally structure of lobophytrol B (15) (Carbon atoms are represented by black ellipsoids, oxygen atoms are represented by red ellipsoids, and hydrogen atoms are represented by green spheres).

Figure 8 .
Figure 8. Revised structure (15a) and originally structure of lobophytrol B (15) (Carbon atoms are represented by black ellipsoids, oxygen atoms are represented by red ellipsoids, and hydrogen atoms are represented by green spheres).

δ H Mult. (J in Hz) δ H Mult. (J in Hz) δ H Mult. (J in Hz) δ H Mult. (J in Hz) δ H Mult. (J in Hz)
a Recorded at 600 MHz; b Recorded at 800 MHz; c Overlapped.

Table 4 .
(12)600 MHz) and13C (150 MHz) NMR data of compounds 11 and 12 in CDCl 3 .Lobocaloid D(12), which was obtained as a white powder, gave the molecular formula C 31 H 52 O 6 on the basis of its HRESIMS ion peak at m/z 543.3653 [M + Na] + (calcd.for C 31 H 52 O 6 Na, 543.3656), 16 mass units less than that of 11.A comparison of the NMR data of 12 with 11 (Table