Talaroacids A–D and Talaromarane A, Diterpenoids with Anti-Inflammatory Activities from Mangrove Endophytic Fungus Talaromyces sp. JNQQJ-4

Five new diterpenes including four diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A–D (1–4) and an isopimarane diterpenoid talaromarane A (5) were isolated from the mangrove endophytic fungus Talaromyces sp. JNQQJ-4. Their structures and absolute configurations were determined by analysis of high-resolution electrospray ionization mass spectroscopy (HRESIMS), 1D/2D Nuclear Magnetic Resonance (NMR) spectra, single-crystal X-ray diffraction, quantum chemical calculation, and electronic circular dichroism (ECD). Talaromarane A (5) contains a rare 2-oxabicyclo [3.2.1] octan moiety in isopimarane diterpenoids. In bioassays, compounds 1, 2, 4, and 5 displayed significant anti-inflammatory activities with the IC50 value from 4.59 to 21.60 μM.

The key HMBC correlations (Figures 2 and S6) from H 2 -7 to C-8 and C-9; from H 3 -18/H 3 -19 to C-3, C-4, and C-5; from H 3 -20 to C-1, C-5, C-9, and C-10 together with the spin system of H 2 -1/H 2 -2/H 2 -3 and H-5/H 2 -6/H 2 -7 indicated the presence of a 5,5,9-trimethyl-∆ 1,2 -octalin moiety of 1.The HMBC correlations from H 2 -11 to C-8, C-9, C-10, and C-11 (δ C 178.0) revealed the branched chain of acetic acid located at C-9.Furthermore, the spin coupling system (Figure S5) of H-15/H 2 -16 and the HMBC correlations from H 3 -17 to C-13, C-14, and C-15; from H 2 -16 to C-13; from H 2 -13 to C-7, C-8, and C-9 indicated the fragment of 3-methylbut-2-en-1-ol was linked to C-8.Thus, the planar structure of 1 was established and shown.The NOESY correlations (Figures 3 and S7) between H-15 and H 3 -17 ensure the configuration of the ∆ 14 double bond as 14Z.Furthermore, the NOESY correlations of H 3 -18/H 3 -20 revealed they were positioned on the same face.In turn, the correlation of H-5/H 3 -19 suggested they were at the opposite orientation.Based on the above information, the relative configuration of 1 was assigned to 5S* and 10S*.Finally, the absolute configuration of 1 was determined as 5S, 10S, and 14Z based on a comparison of experimental and calculated ECD spectra (Figure 4).ensure the configuration of the Δ 14 double bond as 14Z.Furthermore, the NOESY correlations of H3-18/H3-20 revealed they were positioned on the same face.In turn, the correlation of H-5/H3-19 suggested they were at the opposite orientation.Based on the above information, the relative configuration of 1 was assigned to 5S* and 10S*.Finally, the absolute configuration of 1 was determined as 5S, 10S, and 14Z based on a comparison of experimental and calculated ECD spectra (Figure 4).ensure the configuration of the Δ 14 double bond as 14Z.Furthermore, the NOESY correlations of H3-18/H3-20 revealed they were positioned on the same face.In turn, the correlation of H-5/H3-19 suggested they were at the opposite orientation.Based on the above information, the relative configuration of 1 was assigned to 5S* and 10S*.Finally, the absolute configuration of 1 was determined as 5S, 10S, and 14Z based on a comparison of experimental and calculated ECD spectra (Figure 4).Talaroacid B (2) was obtained as a white powder.The HRESIMS data (Figure S8) suggested that 2 had the same molecular formula as that of 1.The NMR data (Tables 1 and 2; Figure S9, S10 and S11) closely resembled those of 1, except for the chemical shift at C-15 (Δδc −2.4).The 1 H-1 H COSY spectra and (Figure S12) and HMBC spectra (Figure S13) also indicate that compounds 2 and 1 have similar planar structures.The NOESY correlation (Figure 3 and S14) of H-15/H2-13 indicated that the configuration of Δ 14 double bond of 2 was 14E.Furthermore, the absolute configuration of 2 was determined as 5S, 10S, and 14E according to the NOESY correlations and ECD calculation (Figure 4).
Talaroacid D (4), a white powder, had a molecular formula of C20H34O3 and 4 degrees of unsaturation according to the HRESIMS data (Figure S22).The structure of 4 was similar to 2 by comparison of their NMR data (Tables 1 and 2; Figure S23, S24 and S25).The main difference was the Δ 14 double bond in 2 was reduced in 4. The deduction was further confirmed by the 1 H-1 H correlations (Figure 2   Talaroacid B (2) was obtained as a white powder.The HRESIMS data (Figure S8) suggested that 2 had the same molecular formula as that of 1.The NMR data (Tables 1  and 2; Figures S9-S11) closely resembled those of 1, except for the chemical shift at C-15 (∆δ c −2.4).The 1 H-1 H COSY spectra and (Figure S12) and HMBC spectra (Figure S13) also indicate that compounds 2 and 1 have similar planar structures.The NOESY correlation (Figures 3 and S14) of H-15/H 2 -13 indicated that the configuration of ∆ 14 double bond of 2 was 14E.Furthermore, the absolute configuration of 2 was determined as 5S, 10S, and 14E according to the NOESY correlations and ECD calculation (Figure 4).
Talaroacid D (4), a white powder, had a molecular formula of C 20 H 34 O 3 and 4 degrees of unsaturation according to the HRESIMS data (Figure S22).The structure of 4 was similar to 2 by comparison of their NMR data (Tables 1 and 2; Figures S23-S25).The main difference was the ∆ 14 double bond in 2 was reduced in 4. The deduction was further confirmed by the 1 H-1 H correlations (Figures 2 and S26   Talaromarane A ( 5) was obtained as a colorless crystal with the molecular formula of C22H30O8 and 8 degrees of unsaturation according to the HRESIMS data (Figure S29).The 1 H NMR spectrum (Table 1 and Figure 2 and S31) and HSQC data (Figure S32) to obtain 22 carbons including four methyls, five methylenes (one olefinic), four methines (two olefinic and an oxygenated), six non-hydrogenated carbons (two carbonyl carbons, three oxygenated and an olefinic), and three quaternary carbons.These data suggested that 5 belongs to an isopimarane diterpene [30].The 1 H-1 H COSY correlations (Figure 2  The NOESY correlations (Figure S35) of H-3/H-6/H-15/H3-18 indicated that these protons were in the same orientation.However, due to the absence of key NOE correlations for OH-5, OH-6, and OH-9 in the NOE spectrum (CDCl3), the relative configurations of 5 were difficult to determine.Luckily, the single crystal of 5 was successfully obtained by slow volatilization in MeOH.Finally, the absolute configuration of 5 was unambiguously determined as 3R, 5R, 6R, 7R, 9R, 10S, and 13R using single crystal X-ray diffraction analysis with a flack parameter of −0.22 (8) (Figure 6).In addition, ECD calculation also verifies the conclusion above (Figure 4).Talaromarane A (5) was obtained as a colorless crystal with the molecular formula of C 22 H 30 O 8 and 8 degrees of unsaturation according to the HRESIMS data (Figure S29).The 1 H NMR spectrum (Table 1 and Figure 2 and Figure S31) and HSQC data (Figure S32) to obtain 22 carbons including four methyls, five methylenes (one olefinic), four methines (two olefinic and an oxygenated), six non-hydrogenated carbons (two carbonyl carbons, three oxygenated and an olefinic), and three quaternary carbons.These data suggested that 5 belongs to an isopimarane diterpene [30].The 1 H-1 H COSY correlations (Figures 2 and S33 The NOESY correlations (Figure S35) of H-3/H-6/H-15/H 3 -18 indicated that these protons were in the same orientation.However, due to the absence of key NOE correlations for OH-5, OH-6, and OH-9 in the NOE spectrum (CDCl 3 ), the relative configurations of 5 were difficult to determine.Luckily, the single crystal of 5 was successfully obtained by slow volatilization in MeOH.Finally, the absolute configuration of 5 was unambiguously determined as 3R, 5R, 6R, 7R, 9R, 10S, and 13R using single crystal X-ray diffraction analysis with a flack parameter of −0.22 (8) (Figure 6).In addition, ECD calculation also verifies the conclusion above (Figure 4).

Anti-Inflammatory Activities
On RAW264.7 cells test all compound's cytotoxicity and anti-inflammatory activities (Table 3).The results indicated compound 2 had better anti-inflammatory activities than positive control quercetin (IC50 = 11.

Anti-Inflammatory Activities
On RAW264.7 cells test all compound's cytotoxicity and anti-inflammatory activities (Table 3).The results indicated compound 2 had better anti-inflammatory activities than positive control quercetin (IC 50 = 11.33 µM) with IC 50 values of 4.59 µM.Compounds 1, 4, and 5 showed moderate anti-inflammatory activities with IC 50 values of 15.78, 21.60, and 13.38 µM, respectively.None of the compounds were cytotoxic to RAW264.7 cells at the tested concentrations.

ECD and NMR Calculations
The ECD calculation was carried out using described previously [30].The conformers were subjected to geometric optimization at the level of B3LYP/6-31+G (d,p) and the optimized conformers were calculated on the TD-DFT method using the B3LYP/6-311+G (d,p).All NMR calculations were performed using the GIAO method at mPW1PW91-SCRF/6-311+G (d,p)/PCM (Chloroform) [34].

Plant and Fungal Material
The healthy leaves of Kandelia obovata were collected in Jinniu Island Mangrove Nature Reserve in Guangzhou province of China in July 2023.The plant was identified by Dr. Yayue Liu, Guangdong Ocean University, and voucher sp.(JNQJ202306) is stored at Sun Yat-sen University.The strain Talaromyces sp.JNQQJ-4 was isolated from the healthy leaves of Kandelia obovate.The specific separation process was as follows: the fresh leaf tissue of Kandelia candel was transferred to 3% sodium hypochlorite solution and 75% ethanol solution with sterilized tweezers, and washed with sterile water.The leaf tissue was cut into regular small pieces (about 0.2 × 0.6 cm) and cultured on an autoclaved Bengal Rose agar plate incubated at 28 • C for 3 days.After the colony appeared, the mycelia were picked and inoculated on PDA medium.Repeat the above steps until a pure single colony is obtained on PDA plate.Fungal species were identified using DNA amplification and ITS sequence analysis previously [35].The strain sequence data were reserved for the GenBank with accession number PP660349, and BLAST analysis revealed that it was 100% homologous to the sequence of Talaromyces sp.(MK450749.1).This strain was preserved at Sun Yat-sen University, China.

Crystallographic Data for Talaromarane A
The X-ray diffraction data of talaromarane A (5) were measured using a Rigaku XtaLAB Pro diffractometer with CuKα radiation (λ = 1.54184Å).The structure of 5 was resolved using SHELXT methods and refined by full-matrix least-squares difference Fourier techniques on an OLEX2 interface program.The crystallographic data of 5 were preserved at the Cambridge Crystallographic Data Centre.peak and hole = 0.24 and −0.18 eÅ −3 .Flack parameter = −0.22(8).Crystallographic data for the structure reported in this paper were deposited in the Cambridge Crystallographic Data Centre (Accession No. CCDC 2351536).

Anti-Inflammatory Assay
Standard Anti-inflammatory assays employing RAW264.7 cell lines were carried out as described previously [30].All compounds were tested for cytotoxic activity before antiinflammatory testing.The RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, NY, USA) at 37 • C with 5% CO 2 humidified incubator.Quercetin (Sigma, Burlington, VT, USA) or compound was dissolved in DMSO to prepare mother liquor (10 mm/mL).Cytotoxic activity was tested by MTT assay.The cells were pretreated with different concentrations of quercetin or compounds (5,10,20,30,40, and 50 µM) for 24 h, then 10 µL of MTT (0.5 mg/mL) was added to each well and cultured for 4 h to test the absorbance at 540 nm.The concentration of DMSO was 0.2% of the medium culture.The NO content was determined by the Griess method to evaluate the anti-inflammatory activity of the compounds.Firstly, 500 µL cells (3 × 10 6 cells/mL) were seeded in 24-well plates and cultured overnight.Different concentrations of quercetin or compounds (5,10,20,30,40, and 50 µM) pretreated with LPS were added and cultured for 24 h, and the absorbance of final products was measured at 540 nm.None compounds displayed cytotoxic on RAW264.7 cell at 50 µM.Quercetin was the positive control.

Solubility and the Stability
Compounds 1-5 were dissolved in chloroform, and no change in compounds 1-5 was found by TLC detection after overnight storage.It was shown that compounds 1-5 were stable under normal conditions.

Conclusions
In conclusion, four new diterpenes with 1,2,3,4,4a,5,6,8a-octalin skeleton talaroacids A-D (1-4) and a new isopimarane diterpenoid talaromarane A (5) were isolated from the mangrove endophytic fungus Talaromyces sp.JNQQJ-4.It is noteworthy that 5 contains a rare 2-oxabicyclo [3.2.1] octan moiety in isopimarane.Moreover, compound 2 exhibited promising NO inhibitory activity with IC 50 values of 4.59 µM.In addition, the better activity of compounds 1-2 than 3-4 indicated that the ∆ 14 double bond in the side chain makes a contribution to NO inhibitory activity.Nitric oxide (NO) is a signaling molecule produced by inducible nitric oxide synthase (iNOS), playing an important regulatory role in the occurrence and development of inflammation [36].It is closely related to many major inflammation-induced diseases, such as autoimmune diseases, arthritis, cardiovascular diseases, and diabetes [37].Inhibiting the production of NO can reduce inflammatory responses and prevent subsequent diseases [38].Therefore, NO inhibitors were considered a promising direction for anti-inflammatory drug research [39].Recently, several diterpenes with decalin skeleton have been reported to have significant NO inhibitory activity [40][41][42].Among diterpenes, tinopanoid M, a clerodane diterpenoid isolated from Tinospora crispa, exerts good anti-inflammatory effects by reducing the expression of various pro-inflammatory factors and modulating multiple inflammatory pathways [41].Thus, talaroacid B (2) might be worthy of further study as a potential anti-inflammatory lead compound.