Anti-Inflammatory and Proangiogenic Metabolites from the Hadal Trench-Derived Fungus Acremonium dichromosporum YP-213

Four new compounds, including two ascochlorin-type meroterpenoids acremocholrins A (1) and B (2), one pyridone alkaloid acremopyridone A (7), and one cyclopentenone derivative acremoketene A (12), together with eight known compounds (3–6 and 8–11), were isolated and identified from the hadal trench-derived fungus Acremonium dichromosporum YP-213. Their structures were determined with a detailed spectroscopic analysis of NMR and MS data, NOE analysis, octant rule and quantum chemical calculations of ECD, and NMR (with DP4+ probability analysis). Among the compounds, 7 represent a novel scaffold derived from a pyridone alkaloid by cleavage of the C-16-C-17 bond following oxidation to give a ketone. Compounds 9, 11, and 12 showed potent in vivo anti-inflammatory activity in transgenic zebrafish, while compound 8 exhibited significant proangiogenic activity in transgenic zebrafish.


Introduction
The hadal trench, consisting of deep-sea trenches deeper than 6000 m, represents one of the most unique habitats in the deep sea, characterized by extreme high pressure, low temperature, geological isolation, complex topography, and high seismic activity [1].The steep slopes of the trenches, formed by the funnel effect of the V-shaped narrow troughs created by plate subduction, transport organic particles from the upper layers downwards, leading to the accumulation of benthic elements in the hadal zone.The hadal trench harbors a diverse range of large benthic organisms, as well as a rich microbial community with unique biodiversity and species specificity [2].Under extreme conditions, hadal fungi have gradually evolved physiological adaptations, genetic mechanisms, and metabolic systems that allow them to produce and accumulate secondary metabolites distinct from those of terrestrial and shallow-sea microorganisms.Currently, research on natural products from hadal microorganisms is still scarce, and the exploration and utilization of hadal microbial metabolites are lagging behind.However, the potential for discovering novel drug leads from hadal microorganisms is enormous.

Structure Elucidation
Acremocholrin A (1) was obtained as a pale yellow amorphous powder.The molecular formula of 1 was determined to be C 23 H 27 ClO 5 using HRESIMS (Figure S2), indicating ten degrees of unsaturation.Specifically, the existence of a chlorine group was further deduced using the isotopic peaks at m/z 441 and 443 with a ratio of 3:1.The 1 H NMR data (Table 1) and HSQC spectrum displayed signals for an aldehyde proton (H-8), six aromatic/olefinic protons (H-4 ′ , H-5 ′ , H-8 ′ , H-9 ′ , and H 2 -12 ′ ), an oxygenated methine proton (H-2 ′ ), and four methyls (H 3 -7, H 3 -13 ′ , H 3 -14 ′ , H 3 -15 ′ ).The 13 C NMR data (Table 1) revealed the presence of 23 carbon signals, sorted using DEPT into four methyls, two methylenes (including one olefinic), seven methines (including four olefinic), and nine quarternary carbons (including seven aromatic/olefinic and one conjugated keto).The general features of the 1 H and 13 C NMR data of 1 resembled 8 ′ ,9 ′ -dehydroascochlorin [5], a previously reported analogue isolated from the cultural mycelium of Verticillium sp.FO-2787.The major difference was that the signals of the olefinic methine (CH-2 ′ ) resonating at δ H/C 5.55/128.0 and of the methyl (3 ′ -Me) resonating at δ H/C 1.93/12.7 in the NMR spectra of 8 ′ ,9 ′ -dehydroascochlorin were replaced by an oxygenated sp 3 methine resonating at δ H/C 4.61/69.9and an olefinic methylene resonating at δ H/C 5.25, 5.12/148.2 in those of 1, respectively.The above observation suggested that, in comparison to 8 ′ ,9 ′ -dehydroascochlorin, compound 1 has undergone a hydroxylation of C-2 ′ and a dehydrogenation of C-12 ′ , which led to the rearrangement of ∆ 2 ′ double bond to ∆ 3 ′ .This deduction was further verified using the key HMBC from H-2 ′ to C-4 ′ , from H-4 ′ to C-3 ′ , and from H-12 ′ to C-2 ′ and C-4 ′ .The structure of 1 was fully defined using the HMBC correlations, as shown in Figure 2.  The relative configuration of compound 1 was proposed after an analysis of NOE difference spectroscopy (Figure 3).NOE correlations from H-13′ to H-14′ and H-15′ revealed that these groups are on the same side, while correlations from H-7′ to H-5′ and H-11′ suggested these groups were on the other side of the molecule.The absolute configurations of C-6′, C-7′, and C-11′ were established using the octant rule for cyclohexenones [9].The negative Cotton effect at 332 nm (Δεmax −0.2) for n→π* indicated the 6′S, 7′R, 11′R configuration (Figure 4).To further determine the whole absolute configuration of 1, timedependent, density functional (TDDFT)-ECD calculations at the BH&HLYP/TZVP level The relative configuration of compound 1 was proposed after an analysis of NOE difference spectroscopy (Figure 3).NOE correlations from H-13 ′ to H-14 ′ and H-15 ′ revealed that these groups are on the same side, while correlations from H-7 ′ to H-5 ′ and H-11 ′ suggested these groups were on the other side of the molecule.The absolute configurations of C-6 ′ , C-7 ′ , and C-11 ′ were established using the octant rule for cyclohexenones [9].The negative Cotton effect at 332 nm (∆ε max −0.2) for n→π* indicated the 6 ′ S, 7 ′ R, 11 ′ R configuration (Figure 4).To further determine the whole absolute configuration of 1, timedependent, density functional (TDDFT)-ECD calculations at the BH&HLYP/TZVP level were performed.The calculated ECD spectrum for the (2 ′ R, 6 ′ S, 7 ′ R, 11 ′ R)-1 matched well with that of the experimental curve (Figure 5A), allowing the establishment of the absolute configuration of 1 as 2 ′ R, 6 ′ S, 7 ′ R, 11 ′ R (Figure 1).were performed.The calculated ECD spectrum for the (2′R, 6′S, 7′R, 11′R)-1 matched well with that of the experimental curve (Figure 5A), allowing the establishment of the absolute configuration of 1 as 2′R, 6′S, 7′R, 11′R (Figure 1).Acremocholrin B (2) was obtained as a pale yellow amorphous powder.Its molecular formula of C23H27ClO5 was determined using HRESIMS (Figure S9), which was the same were performed.The calculated ECD spectrum for the (2′R, 6′S, 7′R, 11′R)-1 match with that of the experimental curve (Figure 5A), allowing the establishment of the a configuration of 1 as 2′R, 6′S, 7′R, 11′R (Figure 1).Acremocholrin B (2) was obtained as a pale yellow amorphous powder.Its mo formula of C23H27ClO5 was determined using HRESIMS (Figure S9), which was th were performed.The calculated ECD spectrum for the (2′R, 6′S, 7′R, 11′R)-1 matched well with that of the experimental curve (Figure 5A), allowing the establishment of the absolute configuration of 1 as 2′R, 6′S, 7′R, 11′R (Figure 1).Acremocholrin B (2) was obtained as a pale yellow amorphous powder.Its molecular formula of C23H27ClO5 was determined using HRESIMS (Figure S9), which was the same Acremocholrin B (2) was obtained as a pale yellow amorphous powder.Its molecular formula of C 23 H 27 ClO 5 was determined using HRESIMS (Figure S9), which was the same as that of 1.The NMR spectra of 2 was very similar to that of 1, with some minor differences on the chemical shifts for H 3 -14 ′ and H 3 -15 ′ .Inspection of the 1D NMR (Table 1) and NOESY data suggested that 2 is a diastereomer of 1, epimeric at C-2 ′ , which was further evidenced by the ECD calculations.The experimental ECD spectrum of 2 showed excellent accordance with that of (2 ′ S, 6 ′ S, 7 ′ R, 11 ′ R)-2 (Figure 5A).Both experimental and calculated data showed positive CEs near 245 and 280 nm and negative CEs near 230 and 330 nm.These close similarities allowed assignment of the absolute configuration for 2 as shown (Figure 1).
Acremopyridone A ( 7) was obtained as a white amorphous powder.Its molecular formula, C 27 H 29 NO 5 (Figure S16), was determined using HRESIMS data, indicating the presence of 14 degrees of unsaturation.In the 1 H NMR spectrum of 7, the signals indicative of a 1,4-disubstituted benzene ring system at δ H 7.29 (d, J = 8.5 Hz, H-2 ′ /6 ′ ) and δ H 6.82 (d, J = 8.5 Hz, H-3 ′ /5 ′ ) and of a trans-double bond at δ H 5.66 (dd, J = 14.9, 10.7 Hz, H-20) and δ H 5.37 (dd, J = 14.9, 6.6 Hz, H-21) were observed.The 13 C NMR data, HSQC, and HMBC spectra (Table 2) indicated the presence of 26 carbon signals, which were sorted using DEPT and HSQC spectra into three methyls, three methylenes, twelve methines (including eight aromatic/olefinic), and nine quaternary carbons (including three carbonyls).The NMR data of 7 (Table 2) are very similar to those of campyridone C [7], a pyridine alkaloid isolated from a mangrove endophytic fungus, Campylocarpon sp.HDN13-307.However, signals for the methine group at C-8, the oxygenated quaternary carbon at C-16, and the oxygenated methine group at C-17 of campyridone C were absent in the NMR spectra of 7. Instead, resonances for a trisubstituted-double bond at δ C 129.0 (C-8) and δ H 7.91/δ C 151.7 (CH-17) and for a ketone group at δ C 212.2 (C-16) were observed in the 1D and 2D NMR spectra of 7. The above observation suggested that the C-16-C-17 bond in the structure of campyridone C was cleaved in that of 7.Moreover, the formation of ∆ 8 and oxidation of C-16 were also observed in 7.This deduction was further verified by the key HMBC correlations (Figure 2) from H-15 to C-10, C-14, and C-16, from H-17 to C-4, C-7, and C-8, and from H 3 -18 to C-15 and C-16.The structure of 7 was fully defined using the HMBC correlations, as shown in Figure 3.The relative configuration of 7 was partially assigned with an analysis of NOESY data (Figure 1).The key NOE correlations from H-10 to H-12, H-14β, and H 3 -18 suggested the co-facial orientation of these groups, while NOESY correlations from H-11α to H- 15 and H 3 -19 and from H-14α to H 3 -19 placed these groups on the opposite face.The relative configuration of C-9 was determined with a comparison of the observed NMR data with those of computed values for two possible isomers (7a and 7b, Figure 6) using DFT calculations through DP4+ probability analysis [10].The experimental NMR data of 7 correspond to the computed NMR data for isomer 7a with DP4+ probabilities of 100%, which led to the determination of the α-orientation of H-9.The absolute configuration of 7 was also determined using TDDFT-ECD calculation (Figure 5B).The experimental ECD spectrum of 7 matched well with that calculated for (9R, 10R, 12S, 15S)-7 (Figure 5B).The structure and absolute configuration of 7 were thus assigned as shown in Figure 1.
Mar. Drugs 2024, 22, x FOR PEER REVIEW H3-19 and from H-14α to H3-19 placed these groups on the opposite face.The configuration of C-9 was determined with a comparison of the observed NMR da those of computed values for two possible isomers (7a and 7b, Figure 6) using DF lations through DP4+ probability analysis [10].The experimental NMR data of spond to the computed NMR data for isomer 7a with DP4+ probabilities of 100% led to the determination of the α-orientation of H-9.The absolute configuration o also determined using TDDFT-ECD calculation (Figure 5B).The experimental EC trum of 7 matched well with that calculated for (9R, 10R, 12S, 15S)-7 (Figure 5 structure and absolute configuration of 7 were thus assigned as shown in Figure 1  Acremoketene A (12), obtained as a pale yellow amorphous powder, had a m formula C20H16O4 as determined with HRESIMS data (Figure S23), indicating 13 of unsaturation.The signals in the 1 H NMR spectrum (Table 3) of 12 for a para-sub benzene ring (H-11/13 and H-10/14) and for a mono-substituted benzene ring (thro 16 to H-20) were observed.Meanwhile, protons for a trans-double bond (δH 6.63, d Hz, H-7; δH 7.75, d, J = 15.9Hz,H-8) and for two methylenes (H2-4 and H2-5) w found in the 1 H NMR spectrum.The 13 C NMR and DEPT data (Table 3) revealed t ence of two methylenes, eleven aromatic/olefinic methines, and seven quaternary (including one ketone and one ester).The key HMBC correlations (Figure 2) from C-1 and C-2 and from H2-5 to C-1 and C-3, together with 1 H-1 H COSY correlatio H2-4 to H2-5, established the cyclopent-2-en-1-one moiety of 12.The HMBC corr from H-16/20 to C-3 and from H2-4 to C-15 indicated that the phenyl was attache cyclopent-2-en-1-one portion via C-3.The remaining part of the structure of 12 wa mined as a cinnamoyl group connected to C-2 with an ester bond with the 1 H-1 H correlations from H-7 to H-8 and from H-10/14 to H-11/13 along with the HMBC tions from H-7 to C-6 and C-9, from H-8 to C-6 and C-10/14, from H-11/13 to C-9 12, and from H-10/14 to C-9.Based on the above data, the structure of compound assigned as shown in Figure 1.Acremoketene A (12), obtained as a pale yellow amorphous powder, had a molecular formula C 20 H 16 O 4 as determined with HRESIMS data (Figure S23), indicating 13 degrees of unsaturation.The signals in the 1 H NMR spectrum (Table 3) of 12 for a para-substituted benzene ring (H-11/13 and H-10/14) and for a mono-substituted benzene ring (through H-16 to H-20) were observed.Meanwhile, protons for a trans-double bond (δ H 6.63, d, J = 15.9Hz, H-7; δ H 7.75, d, J = 15.9Hz, H-8) and for two methylenes (H 2 -4 and H 2 -5) were also found in the 1 H NMR spectrum.The 13 C NMR and DEPT data (Table 3) revealed the presence of two methylenes, eleven aromatic/olefinic methines, and seven quaternary carbons (including one ketone and one ester).The key HMBC correlations (Figure 2) from H 2 -4 to C-1 and C-2 and from H 2 -5 to C-1 and C-3, together with 1 H-1 H COSY correlations from H 2 -4 to H 2 -5, established the cyclopent-2-en-1-one moiety of 12.The HMBC correlations from H-16/20 to C-3 and from H 2 -4 to C-15 indicated that the phenyl was attached to the cyclopent-2-en-1-one portion via C-3.The remaining part of the structure of 12 was determined as a cinnamoyl group connected to C-2 with an ester bond with the 1 H-1 H COSY correlations from H-7 to H-8 and from H-10/14 to H-11/13 along with the HMBC correlations from H-7 to C-6 and C-9, from H-8 to C-6 and C-10/14, from H-11/13 to C-9 and C-12, and from H-10/14 to C-9.Based on the above data, the structure of compound 12 was assigned as shown in Figure 1.

Bioactivity
The isolated compounds were evaluated for in vivo anti-inflammatory activity in a transgenic zebrafish Tg (zlyz-EGFP) model and in vivo proangiogenic activity in a transgenic zebrafish Tg (vegfr2:GFP) model.In the anti-inflammatory activity assay, zebrafish were treated with CuSO 4 , which induced a strong acute inflammatory response, including breaking down the neuromast and mechanosensory cells of the lateral line system in the zebrafish, as well as causing the infiltration and migration of macrophages in the zebrafish.Compared with the model control group, the number of migrating macrophages with fluorescence of zebrafish treated with compounds 11 and 12 was reduced significantly (p < 0.01) at concentrations of 20, 40, and 80 µM (Figure 7).In addition, compound 9 also significantly reduced (p < 0.01) the migration of zebrafish macrophages at concentrations of 10 and 20 µM.The results indicated compounds 9, 11, and 12 had potent anti-inflammatory activity.In addition, blastocolysis or death were observed after exposure to compounds 1-5 for 24 h, which showed potential cytotoxicity of these compounds, as well as in other subsequent activity experiments.
Mar. Drugs 2024, 22, x FOR PEER REVIEW transgenic zebrafish Tg (vegfr2:GFP) model.In the anti-inflammatory activity zebrafish were treated with CuSO4, which induced a strong acute inflammatory res including breaking down the neuromast and mechanosensory cells of the lateral li tem in the zebrafish, as well as causing the infiltration and migration of macroph the zebrafish.Compared with the model control group, the number of migrating phages with fluorescence of zebrafish treated with compounds 11 and 12 was r significantly (p < 0.01) at concentrations of 20, 40, and 80 μM (Figure 7).In addition pound 9 also significantly reduced (p < 0.01) the migration of zebrafish macroph concentrations of 10 and 20 μM.The results indicated compounds 9, 11, and 12 had anti-inflammatory activity.In addition, blastocolysis or death were observed afte sure to compounds 1-5 for 24 h, which showed potential cytotoxicity of these comp as well as in other subsequent activity experiments.In the proangiogenic activity assay, the intersegmental blood vessels (IS zebrafish in the model group were inhibited by PTK787.Compared to the model compound 8 remarkably increased the number of ISVs in model zebrafish in a d pendent manner at concentrations of 20, 40, and 80 μM (Figure 8), indicating that 8 ited significant proangiogenic activity.In the proangiogenic activity assay, the intersegmental blood vessels (ISVs) of zebrafish in the model group were inhibited by PTK787.Compared to the model group, compound 8 remarkably increased the number of ISVs in model zebrafish in a dose-dependent manner at concentrations of 20, 40, and 80 µM (Figure 8), indicating that 8 exhibited significant proangiogenic activity.

Fungal Material
The fungal strain was collected from a seawater sample from the Yap Trench Pacific Ocean (depth 6215 m, collected in 2017).The strain was identified as A. dichro rum based on ITS sequencing (GenBank No. KF022040) with 99% similarity.The se data of YP-213 have been deposited in GenBank with the accession No. OR85704 strain is preserved at the Shandong Provincial Key Laboratory of Applied Myc School of Life Sciences, Qingdao Agricultural University, Qingdao, China.

Fermentation, Extraction, and Isolation
The fungus A. dichromosporum YP-213 was transferred aseptically and grown a

Fungal Material
The fungal strain was collected from a seawater sample from the Yap Trench in the Pacific Ocean (depth 6215 m, collected in 2017).The strain was identified as A. dichromosporum based on ITS sequencing (GenBank No. KF022040) with 99% similarity.The sequence data of YP-213 have been deposited in GenBank with the accession No. OR857042.The strain is preserved at the Shandong Provincial Key Laboratory of Applied Mycology, School of Life Sciences, Qingdao Agricultural University, Qingdao, China.

Fermentation, Extraction, and Isolation
The fungus A. dichromosporum YP-213 was transferred aseptically and grown at 28 • C under static conditions.It was cultured in fermentation bags, each containing 80 g rice, 3.96 g sea salt, and 120 mL distilled water.
After the completion of the fermentation period, the whole fermentation broth (60 L) was filtered, and the broth media was extracted with ethyl acetate (EtOAc).The extracts

Figure 4 .
Figure 4.The octant rule for the cyclohexenone.

Figure 4 .
Figure 4.The octant rule for the cyclohexenone.

Figure 4 .
Figure 4.The octant rule for the cyclohexenone.

Figure 4 .
Figure 4.The octant rule for the cyclohexenone.

Figure 6 .
Figure 6.Structures of possible isomers for DP4+ probability analysis of compound 7.

Figure 6 .
Figure 6.Structures of possible isomers for DP4+ probability analysis of compound 7.

Figure 7 .
Figure 7.The anti-inflammatory activity of isolated compounds in Tg (zlyz-EGFP) zebrafi macrophages that migrated above the caudal notochord were numbered using an Olymp microscope.(A) Typical images of migratory fluorescent macrophages in transgenic zebrafis ibuprofen as a positive control.(B) Quantitative analysis of fluorescent macrophages in tra zebrafish (n = 10, mean ± SEM).## p < 0.01, compared to the control group.** p < 0.01 comp the model group.

Figure 7 .
Figure 7.The anti-inflammatory activity of isolated compounds in Tg (zlyz-EGFP) zebrafish.The macrophages that migrated above the caudal notochord were numbered using an Olympus IX53 microscope.(A) Typical images of migratory fluorescent macrophages in transgenic zebrafish, using ibuprofen as a positive control.(B) Quantitative analysis of fluorescent macrophages in transgenic zebrafish (n = 10, mean ± SEM).## p < 0.01, compared to the control group.** p < 0.01 compared to the model group.

Figure 8 .
Figure 8.In vivo proangiogenic activities of isolated compounds in fluorescent transgenic ze Tg(vegfr2:GFP) embryos.(A) Lateral view of the zebrafish larval trunk in all groups show (intersegmental blood vessel, red arrows) growth under a fluorescence microscope 24 h aft ment.(B) Statistic analysis of the number of ISVs in all groups.Data were derived from 1 pendent experiments and represented as mean ± SD; ## p < 0.01 compared to the control gro < 0.01 compared to the model group (PTK787).

Figure 8 .
Figure 8.In vivo proangiogenic activities of isolated compounds in fluorescent transgenic zebrafish Tg(vegfr2:GFP) embryos.(A) Lateral view of the zebrafish larval trunk in all groups showing ISV (intersegmental blood vessel, red arrows) growth under a fluorescence microscope 24 h after treatment.(B) Statistic analysis of the number of ISVs in all groups.Data were derived from 11 independent experiments and represented as mean ± SD; ## p < 0.01 compared to the control group, ** p < 0.01 compared to the model group (PTK787).

Table 1 .
H and C NMR data for compounds 1 and 2 (measured in DMSO-d6).
a Measured at 150 MHz; b measured at 600 MHz.

. δ C , Type a δ H (J in Hz) b No. δ C , Type a δ H (J in Hz) b
a Measured at 150 MHz; b measured at MHz; c not detected.
a Measured at 150 MHz; b measured at 600 MHz.
a Measured at 150 MHz; b measured at 600 MHz.