Lobosteroids A–F: Six New Highly Oxidized Steroids from the Chinese Soft Coral Lobophytum sp.

To explore the steroidal constituents of the soft coral Lobophytum sp. at the coast of Xuwen County, Guangdong Province, China, a chemical investigation of the above-mentioned soft coral was carried out. After repeated column chromatography over silica gel, Sephadex LH-20, and reversed-phase HPLC, six new steroids, namely lobosteroids A–F (1–6), along with four known compounds 7–10, were obtained. Their structures were determined by extensive spectroscopic analysis and comparison with the spectral data reported in the literature. Among them, the absolute configuration of 1 was determined by X-ray diffraction analysis using Cu Kα radiation. These steroids were characterized by either the presence of an α,β-α′,β′-unsaturated carbonyl, or an α,β-unsaturated carbonyl moiety in ring A, or the existence of a 5α,8α-epidioxy system in ring B, as well as diverse oxidation of side chains. The antibacterial bioassays showed that all isolated steroids exhibited significant inhibitory activities against the fish pathogenic bacteria Streptococcus parauberis FP KSP28, Phoyobacterium damselae FP2244, and Streptococcus parauberis SPOF3K, with IC90 values ranging from 0.1 to 11.0 µM. Meanwhile, compounds 2 and 6–10 displayed potent inhibitory effects against the vancomycin-resistant Enterococcus faecium bacterium G7 with IC90 values ranging from 4.4 to 18.3 µM. Therefore, ten highly oxidized steroids with strong antibacterial activities were isolated from the Chinese soft coral Lobophytum sp., which could be developed as new chemotypes of antibacterial drug leads.

As part of our ongoing research aimed at discovering bioactive substances from marine invertebrates in China [29], we recently collected Lobophytum sp. at the coast of Xuwen County, Guangdong Province, China.In our recent study, we have reported the isolation and structural elucidation of anti-tumor cembrane diterpenoids from the Hainan specimens of Lobophytum sp.[30].While our current investigation on the Guangdong collection of Lobophytum sp. has now resulted in the isolation of six new steroids, lobosteroids A-F (1-6), together with four known analogs 7-10 (Figure 1).The structural difference of six new steroids 1-6 is mainly attributed to the different degrees of oxidation in rings A and B of the steroidal nucleus and the variations of functional groups on the side chains.This paper describes the isolation, structural elucidation, and bioactivity of these compounds.
Compound 1, colorless crystals, had the molecular formula of C28H42O3 as established by HRESIMS (Figure S7) from the protonated molecular ion peak observed at m/z 427.3210
No. 12.4 (q) 12.2 (q) 12.5 (q) 12.4 (q) 12.6 (q) 12.7 (q) 19 18.8 (q) 18.8 (q) 18.8 (q) 13.1 (q) 13.1 (q) 18.3 (q) Compound 2 was obtained as a white amorphous powder and it displayed a protonated molecular ion peak at m/z 411.3255 ([M + H] + ; calcd.411.3258) in the HRESIMS spectrum (Figure S15), consistent with a molecular formula of C 28 H 42 O 2 .Inspection of the NMR data of compound 2 (Tables 1 and 2, Figures S9 and S10 [36], further confirming the established structure of the side chain.Due to the isomerization of a single isolated chiral center C-24 in a linear chain would not result in significant shifts of 1 H or 13 C NMR data, the configuration of C-24 was undetermined.Similar NOE correlations as those of 1 were observed in the NOESY spectrum of 2 (Figures 4 and S14), suggesting they had the same relative configurations for the chiral centers of the parent nucleus.Thus, the structure of 2 was depicted as shown in Figure 1.
the hydroxyl group attached to C-24 (δC 74.0 vs. δC 53.0) in 1 was lost in 2, which was consistent with their 16 mass units difference.The characteristic 1 H-1 H COSY correlations from H-17 (δH 1.13) through H-20 (δH 2.04) to H2-22 (δH 2.20, 2.44) and from H3-28 (δH 0.98) through H-24 (δH 2.29) and H-25 (δH 1.92) to H3-26 (δH 0.84)/H3-27 (δH 0.90), together with the diagnostic HMBC correlations from H2-22 to C-20 (δC 32.0) and C-23, from H3-28 to C-23, C-24, and C-25 (δC 30.2) (Figures 4, S12 and S13), supported the above-mentioned structure of the side chain.The literature surveys revealed that the NMR data of the side chain of 2 were almost identical to those of the synthetic steroid 3β-hydroxyergost-5,7-diene-23one [36], further confirming the established structure of the side chain.Due to the isomerization of a single isolate chiral center C-24 in a linear chain would not result in significant shifts of 1 H or 13 C NMR data, the configuration of C-24 was undetermined.Similar NOE correlations as those of 1 were observed in the NOESY spectrum of 2 (Figures 4 and S14), suggesting they had the same relative configurations for the chiral centers of the parent nucleus.Thus, the structure of 2 was depicted as shown in Figure 1.Compound 3, a white amorphous powder, had the molecular formula of C30H44O4 as established by HRESIMS (Figure S23) from the protonated molecular ion peak observed at m/z 469.3318 [M + H] + (calcd.469.3312).Detailed analysis of NMR data of 3 (Tables 1  and 2, Figures S17 and S18) disclosed that 3 and 1 possessed the same steroidal nucleus but differed in the side chain.The presence of an acetyl group in 3 was recognized by the characteristic NMR signals at δH 2.00 (s, H3-30) and δC 170.7 (s, C-29), and 21.0 (q, C-30) (Figure S19).The location of the acetyl group at C-21 was straightforward from the significant down-field shifted NMR signals at  S20).Moreover, the chemical shift of C-24 (δC 38.7) shifted significantly upfield, which indicated that the hydroxyl group attached to C-24 in 1 was lost in 3. Based on the analysis of the NOE correlations, as depicted in Figures 5 and S22, the structure of 3 was determined, as shown in Figure 1.However, the configuration of C-24 could not be assigned herein.Compound 3, a white amorphous powder, had the molecular formula of C 30 H 44 O 4 as established by HRESIMS (Figure S23) from the protonated molecular ion peak observed at m/z 469.3318 [M + H] + (calcd.469.3312).Detailed analysis of NMR data of 3 (Tables 1 and 2, Figures S17 and S18) disclosed that 3 and 1 possessed the same steroidal nucleus but differed in the side chain.The presence of an acetyl group in 3 was recognized by the characteristic NMR signals at δ H 2.00 (s, H 3 -30) and δ C 170.7 (s, C-29), and 21.0 (q, C-30) (Figure S19).The location of the acetyl group at C-21 was straightforward from the significant down-field shifted NMR signals at δ Compound 4 was obtained as a white amorphous powder.Its molecular form C29H46O3, was deduced from its protonated molecular ion peak observed at m/z 443.3 ([M + H] + ; calcd.443.3520) in the HRESIMS spectrum (Figure S31).Careful analysis o 1 H and 13 C NMR data (Tables 1 and 2, Figures S25 and S26) revealed the presence o α,β-unsaturated carbonyl group [δH 7.13 (d, J = 10.2Hz, H-1), 5.86 (dd, J = 10.2, 1.0 Hz 2), and δC 158.7 (d, C-1), 127.5 (d, C-2), 200.4 (s, C-3)] and a methyl ester functionality 3.67 (s, H3-29) and δC 176.9 (s, C-21), 51.2 (s, C-29)] in the molecule.Searching in our c pound library, it was found that the 13 C NMR data of C-1-C-21 were nearly identica those of methyl spongoate, a steroid previously reported from the soft coral Spongodes by our group [37], suggesting they had the same steroidal nucleus and a methoxycarbo group at C-21 of the side chain.The only difference between them was at the methyl a 24 in 4, which was deduced from the 1 H-1 H COSY correlations from H3-28 (δH 0 Compound 4 was obtained as a white amorphous powder.Its molecular formula, C 29 H 46 O 3 , was deduced from its protonated molecular ion peak observed at m/z 443.3520 ([M + H] + ; calcd.443.3520) in the HRESIMS spectrum (Figure S31).Careful analysis of its 1 H and 13 C NMR data (Tables 1 and 2, Figures S25 and S26) revealed the presence of an α,β-unsaturated carbonyl group [δ H 7.11 (d, J = 10.2Hz, H-1), 5.84 (dd, J = 10.2, 1.0 Hz, H-2), and δ C 158.7 (d, C-1), 127.5 (d, C-2), 200.4 (s, C-3)] and a methyl ester functionality [δ H 3.65 (s, H 3 -29) and δ C 176.9 (s, C-21), 51.2 (s, C-29)] in the molecule.Searching in our compound library, it was found that the 13 C NMR data of C-1-C-21 were nearly identical to those of methyl spongoate, a steroid previously reported from the soft coral Spongodes sp. by our group [37], suggesting they had the same steroidal nucleus and a methoxycarbonyl group at C-21 of the side chain.The only difference between them was at the methyl at C-24 in 4, which was deduced from the 1 H-1 H COSY correlations from H 3 -28 (δ H 0.76) through H-24 (δ H 1.24) and H-25 (δ H 1.55) to H 3 -26 (δ H 0.75)/H 3 -27 (δ H 0.84) as well as the HMBC correlations from H 3 -28 to C-23 (δ C 21.2), C-24 (δ C 38.7), and C-25 (δ C 31.4) (Figures 6, S28 and S29).The established structure of the side chain was further verified in agreement with the 13 C NMR data of those of (24S)-3β-acetoxyergost-5-en-21-oic acid, a secondary metabolite previously reported from the soft coral Cladiella australis [38].Similar NOE correlations as those of methyl spongoate were observed in the ROESY spectrum of 4 (Figures 6 and S30), suggesting they had the same relative configurations for the chiral centers of the parent nucleus.Therefore, compound 4 was established as a 24-methyl derivative of methyl spongoate, as shown in Figure 1, with the configuration of C-24 remaining unknown.
Compound 4 was obtained as a white amorphous powder.Its molecular formula, C29H46O3, was deduced from its protonated molecular ion peak observed at m/z 443.3520 ([M + H] + ; calcd.443.3520) in the HRESIMS spectrum (Figure S31).Careful analysis of its 1 H and 13 C NMR data (Tables 1 and 2, Figures S25 and S26) revealed the presence of an α,β-unsaturated carbonyl group [δH 7.13 (d, J = 10.2Hz, H-1), 5.86 (dd, J = 10.2, 1.0 Hz, H-2), and δC 158.7 (d, C-1), 127.5 (d, C-2), 200.4 (s, C-3)] and a methyl ester functionality [δH 3.67 (s, H3-29) and δC 176.9 (s, C-21), 51.2 (s, C-29)] in the molecule.Searching in our compound library, it was found that the 13 C NMR data of C-1-C-21 were nearly identical to those of methyl spongoate, a steroid previously reported from the soft coral Spongodes sp. by our group [37], suggesting they had the same steroidal nucleus and a methoxycarbonyl group at C-21 of the side chain.The only difference between them was at the methyl at C-24 in 4, which was deduced from the 1 H-1 H COSY correlations from H3-28 (δH 0.76) through H-24 (δH 1.24) and H-25 (δH 1.55) to H3-26 (δH 0.74)/H3-27 (δH 0.84) as well as the HMBC correlations from H3-28 to C-23 (δC 30.1),C-24 (δC 38.7), and C-25 (δC 31.4)(Figures 6, S28 and S29).The established structure of the side chain was further verified in agreement with the 13 C NMR data of those of (24S)-3β-acetoxyergost-5-en-21-oic acid, a secondary metabolite previously reported from the soft coral Cladiella australis [38].The correlations were observed in the ROESY spectrum of 4 (Figures 6 and S30), suggesting the relative configurations for the chiral centers of the parent nucleus.Therefore, compound 4 was established as a 24-methyl derivative of methyl spongoate, as shown in Figure 1, with the configuration of C-24 remaining unknown.Compound 5 was obtained as a white amorphous powder, and its molecular formula was established as C28H44O2 according to the protonated molecular ion at m/z 413.3411 ([M + H] + ; calcd.413.3414) in the HRESIMS spectrum (Figure S39).A comparison of overall 1 H and 13 C NMR data (Tables 1 and 2, Figures S33 and S34) revealed that 5 shared the identical steroidal nucleus with 4 but differed at the side chain, where the presence of a ketone at C-22 and the disappearance of a methoxycarbonyl group at C-21 were observed.These Compound 5 was obtained as a white amorphous powder, and its molecular formula was established as C 28 H 44 O 2 according to the protonated molecular ion at m/z 413.3411 ([M + H] + ; calcd.413.3414) in the HRESIMS spectrum (Figure S39).A comparison of overall 1 H and 13 C NMR data (Tables 1 and 2, Figures S33 and S34) revealed that 5 shared the identical steroidal nucleus with 4 but differed at the side chain, where the presence of a ketone at C-22 and the disappearance of a methoxycarbonyl group at C-21 were observed.These differences were evident by the NMR signals at δ H 1.09 (d, J = 6.9 Hz, H 3 -21)/δ C 16.7 (q, C-21) and δ C 214.8 (s, C-22) (Figure S35).The 1 H-  6, S36 and S37) supported the speculation.Furthermore, the coincident 13 C NMR data from C-20 to C-25 and C-28 for 5 and the synthetic steroid 3β-hydroxyergost-5,7-diene-22-one [36] confirmed they shared the same side chain.Based on the analysis of the ROESY correlations, as depicted in Figures 7 and S38, the structure of 5 was determined with the unknown configuration of C-24, as shown in Figure 1.
Mar. Drugs 2023, 21, 457 8 of 14 differences were evident by the NMR signals at δH 1.08 (d, J = 6.9 Hz, H3-21)/δC 16.7 (q, C-21) and δC 214.8 (s, C-22) (Figure S35).The 1 H- ) supported the speculation.Furthermore, the coincident 13 C NMR data from C-20 to C-25 and C-28 for 5 and the synthetic steroid 3β-hydroxyergost-5,7-diene-22-one [36] confirmed they shared the same side chain.Based on the analysis of the ROESY correlations, as depicted in Figures 7 and S38, the structure of 5 was determined with the unknown configuration of C-24, as shown in Figure 1.Compound 6 was obtained as a white amorphous powder.Its molecular formula C29H46O3 was determined by the HREIMS ion peak at m/z 424.3325 [M − H2O] + (calcd.424.3336, Figure S47), corresponding to seven degrees of unsaturation.Two vicinal coupled olefinic protons at δH 6.24 (d, J = 8.5 Hz, H-6) and 6.50 (d, J = 8.6 Hz, H-7) and an Compound 6 was obtained as a white amorphous powder.Its molecular formula C 29 H 46 O 3 was determined by the HREIMS ion peak at m/z 424.3325 [M − H 2 O] + (calcd.424.3336, Figure S47), corresponding to seven degrees of unsaturation.Two vicinal coupled olefinic protons at δ H 6.24 (d, J = 8.5 Hz, H-6) and 6.50 (d, J = 8.6 Hz, H-7) and an oxygenated methine at δ H 3.97 (tt, J = 11.2, 5.1 Hz, H-3) were characteristic of a 3β-hydroxy-6-en-5α,8αepidioxysterol nucleus, which was also recognized by the 13 C NMR signals at δ C 66.6 (d, C-3), 82.3 (s, C-5), 135.6 (d, C-6), 130.9 (d, C-7), and 79.6 (s, C-8) (Figure S43).These spectral data of 6 (Tables 1 and 2, Figures S41 and S42) were reminiscent of yalongsterol A, a sterol previously reported from the soft coral Sinularia sp. by our group [11].Detailed comparison of the full 1 H and 13 C NMR data of 6 with yalongsterol A, showing great similarity between them, clearly allowed the assignment of 3β-hydroxy-6-en-5α,8α-epidioxy-cholesta nucleus to 6, which was further justified by the extensive analyses of 2D NMR spectra involving 1   8 and S44) implied the location of germinal methyls at C-24 of the side chain of 6.With the established structure of the side chain in hand, the structure of 6 was depicted as shown in Figure 1.Compound 6 was obtained as a white amorphous powder.Its molecular formula C29H46O3 was determined by the HREIMS ion peak at m/z 424.3325 [M − H2O] + (calcd.424.3336, Figure S47), corresponding to seven degrees of unsaturation.Two vicinal coupled olefinic protons at δH 6.24 (d, J = 8.5 Hz, H-6) and 6.50 (d, J = 8.6 Hz, H-7) and an oxygenated methine at δH 3.98 (tt, J = 11.2, 5.1 Hz, H-3) were characteristic of a 3β-hydroxy-6-en-5α,8α-epidioxysterol nucleus, which was also recognized by the 13 C NMR signals at δC 66.6 (d, C-3), 82.3 (s, C-5), 135.6 (d, C-6), 130.9 (d, C-7), and 79.6 (s, C-8) (Figure S43).These spectral data of 6 (Tables 1 and 2, Figures S41 and S42) were reminiscent of yalongsterol A, a sterol previously reported from the soft coral Sinularia sp. by our group [11].Detailed comparison of the full 1 H and 13 C NMR data of 6 with yalongsterol A, showing great similarity between them, clearly allowed the assignment of 3β-hydroxy-6-en-5α,8αepidioxy-cholesta nucleus to 6, which was further justified by the extensive analyses of 2D NMR spectra involving 1 H-1 H COSY, HSQC, and HMBC (Figures 8 and S43-S45).However, they differed at the side chain.The NMR signals at 3) (Figures 8 and S44) implied the location of germinal methyls at C-24 of the side chain of 6.With the established structure of the side chain in hand, the structure of 6 was depicted as shown in Figure 1.In in vitro bioassays, all the isolates were tested for antibacterial, neuroprotective, and anti-inflammatory effects.In the antibacterial bioassays (Table 3), all the steroids exhibited significant antibacterial activities against the fish pathogenic bacteria Streptococcus parauberis FP KSP28, Phoyobacterium damselae FP2244, and Streptococcus parauberis SPOF3K with IC 90 values ranging from 0.1 to 11.0 µM.As observed in Table 3, the steroids possessing the unsaturated carbonyl moiety in ring A were favored for the inhibition against Streptococcus parauberis FP KSP28.Moreover, it seemed that a vinyl-type side chain in the steroid could lead to a small increase in the antibacterial activities against Phoyobacterium damselae FP2244 and Streptococcus parauberis SPOF3K, as indicated in Table 3.Only compound 7 displayed potent inhibitory activity against the fish pathogenic bacterium Aeromonas salmonicida AS42 with an IC 90 value of 8.8 µM.This might imply that the combination of an α,βα ,β -unsaturated carbonyl moiety and a vinyl-type side chain played a key role in the antibacterial activity against Aeromonas salmonicida AS42.The above-mentioned results indicated that these isolated steroids could be used as antibacterial agents in fish farming.
Meanwhile, compounds 2 and 6-10 also displayed potent inhibitory effects against the vancomycin-resistant Enterococcus faecium bacterium G7 with IC 90 values ranging from 4.4 to 18.3 µM (Table 3).Among them, steroid 10 also displayed antibacterial effects against the vancomycin-resistant Enterococcus faecium bacteria G1, G4, and G8 with IC 90 values of 8.0, 4.0, and 8.0 µM, respectively.The preliminary analysis of the structure-activity relationship for compounds 6-10 revealed that the higher degrees of unsaturation of ring A Mar. Drugs 2023, 21, 457 9 of 14 in the steroids could keep efficacy against more individuals of the vancomycin-resistant Enterococcus faecium bacteria.The above-mentioned results implied that these isolates could be developed as new chemotypes of antibacterial leads against drug-resistant bacteria.a Tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OT), levofloxacin hydrochloride (LF) 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.
Moreover, all the isolated steroids except 5, 6, and 9 showed strong inhibitory activities against Streptococcus agalactiae WR10 with IC 90 values ranging from 4.0 to 22.0 µM (Table 3).However, in the neuroprotective bioassays, none of these steroids displayed significant neuroprotective effects against the corticosterone-induced cellular injuries in human neuroblastoma SH-SY5Y cells at the concentration of 10 µM.In the evaluations of the anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells, all the isolates were judged as inactive at 10 µM, neither.

Animal Material
The soft coral Lobophytum sp. was collected in October 2021 in Xuwen Country, Guangdong Province, China.This specimen was identified by Prof. X.-B.Li from Hainan University.A voucher specimen (No.S-21-XW-6553) is available for inspection at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences.

Antibacterial Bioassays
The marine strains Streptococcus parauberis FP KSP28, Streptococcus parauberis SPOF3K, Phoyoba cteriumdamselae FP2244, and Aeromonas salmonicida AS42 were provided by National Fisheries Research & Development Institute, Korea.The strain Streptococcus agalactiae WR10 was provided by the Chinese Academy of Tropical Agricultural Sciences.The vancomycinresistant Enterococcus faecium bacteria G1, G4, G7, and G8 were provided by Ruijin Hospital, Shanghai Jiao Tong University School of Medicine.The minimum inhibitory concentration for 90% (MIC 90 ) values for all antimicrobial agents was measured by the 96-well microdilution method.Mueller-Hinton II broth (cation-adjusted, BD 212322) was used for MIC 90 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 the addition of culture broth to reach concentrations ranging from 500 µM to 0.24 µM. 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 90 values of these compounds were defined as the lowest concentration to inhibit bacterial growth completely.All MIC 90 values were interpreted according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI).Tetracycline hydrochloride (TC), oxytetracycline hydrochloride (OT), and levofloxacin hydrochloride (LF) were used as positive controls (Table 3).

Conclusions
In summary, six new steroids, lobosteroids A-F (1-6), together with four known compounds 7-10, were isolated from the Chinese soft coral Lobophytum sp.The chemical diversity of new steroids was mainly attributed to the high oxidation, which was characterized by the conjugated enone or dienone system of the nucleus and diverse oxidation of side chains.Although many steroids were reported from soft corals, those with an α,β-α ,β -unsaturated carbonyl or an α,β-unsaturated carbonyl moiety in ring A, or the existence of a 5α,8α-epidioxy system in ring B were rarely found from the genus Lobophytum.The discovery of steroids 1-6 expanded the diverse and complex array of steroids, which is still a research hotspot of marine natural products.In the bioassays, all of the isolates displayed significant antibacterial activities against the fish pathogenic bacteria Streptococcus parauberis FP KSP28, Phoyobacterium damselae FP2244, and Streptococcus parauberis SPOF3K with IC 90 values ranging from 0.1 to 11.0 µM.Meanwhile, compounds 2 and 6-10 exhibited excellent antibacterial activities against the vancomycin-resistant Enterococcus faecium bacterium G7 with IC 90 values ranging from 4.4 to 18.3 µM.These new findings implied that these isolated steroids could be developed as new chemotypes of antibacterial leads.