Two New Terpenoids from Talaromyces purpurogenus

A new sesquiterpenoid 9,10-diolhinokiic acid (1) and a new diterpenoid roussoellol C (2), together with 4 known compounds, were isolated from the extracts of laboratory cultures of marine-derived fungus Talaromyces purpurogenus. 9,10-diolhinokiic acid is the first thujopsene-type sesquiterpenoid containing a 9,10-diol moiety, and roussoellol C possesses a novel tetracyclic fusicoccane framework with an unexpected hydroxyl at C-4. These new structures were confirmed by spectroscopic data, chemical method, NMR data calculations and electronic circular dichroism (ECD) calculations. The selected compounds were evaluated for cytotoxicities against five human cancer cell lines, including SW480, HL-60, A549, MCF-7, and SMMC-7721 and the IC50 values of compound 2 against MCF-7 and 3 against HL-60 cells were 6.5 and 7.9 μM, respectively.


Introduction
Over the past forty years, more than 60% small molecule new drugs have been directly or indirectly derived from natural product source, which demonstrates that natural products continue to play a significant role in drug discovery and development process [1]. Fungi-derived natural products are rich sources of medicines due to their diverse chemical structures and bioactivities. For example, lovastatin, penicillin, echinocandin B, and cyclosporine A have been clinically used as effective medicines, illustrating the significance of fungi-derived metabolites in drug discovery [2].
Exhaustive analyses of the 2D NMR spectra of 1 revealed some similarity to (+)-thujopsene [17], a sesquiterpenoid derivative isolated from the liverwort Marchantia polymorpha. However, two oxygenated methines (δH 4.03, δC 72.8 and δH 3.23, δC 78.6) of 1 replaced the methylenes of (+)thujopsene and one methyl was oxidized to a carboxyl (δC 171.2) group. The presence of a 9,10-diol moiety was demonstrated by the 1 H-1 H COSY cross-peak of H-9 and H-10, and HMBC correlations from H-9 to C-7 and C-11 and from H-10 to C-8 and C-11. The carboxyl was located at C-4, which was substantiated by the HMBC cross-peaks from H-3 and H-5 to C-12 ( Figure 2). Therefore, the planar structure of 1 was identified as a 9, 10-diolhinokiic acid [18]. In the NOESY experiment ( Figure  2), the correlations of H-2/Me-15 and H-2/Me-13 suggested that these groups were co-facial and assigned as α-oriented, while, the interactions of H-3/Me-14 and Me-14/H-10 indicated that they were on the opposite face of the ring system and β-oriented. The hydroxyls at C-9 and C-10 were on the same side according to the coupling constant between H-9 and H-10 (J = 3.8 Hz). Thus, the relative configuration of 1 was determined. The absolute stereochemistry of the 9,10-diol moiety in 1 was verified by observing the induced electronic circular dichroism (IECD) spectrum after the addition of dimolybdenum tetraacetate in anhydrous DMSO [19,20]. The obvious negative Cotton effect at 310 nm in the IECD spectrum ( Figure 3) permitted the 9R,10S configuration assignment of 1. Combining with the relative configuration, the absolute stereochemistry of 1 was elucidated as 1R,3R,7R,9R,10S (Figure 1).
Exhaustive analyses of the 2D NMR spectra of 1 revealed some similarity to (+)-thujopsene [17], a sesquiterpenoid derivative isolated from the liverwort Marchantia polymorpha. However, two oxygenated methines (δ H 4.03, δ C 72.8 and δ H 3.23, δ C 78.6) of 1 replaced the methylenes of (+)-thujopsene and one methyl was oxidized to a carboxyl (δ C 171.2) group. The presence of a 9,10-diol moiety was demonstrated by the 1 H-1 H COSY cross-peak of H-9 and H-10, and HMBC correlations from H-9 to C-7 and C-11 and from H-10 to C-8 and C-11. The carboxyl was located at C-4, which was substantiated by the HMBC cross-peaks from H-3 and H-5 to C-12 ( Figure 2). Therefore, the planar structure of 1 was identified as a 9, 10-diolhinokiic acid [18]. In the NOESY experiment (Figure 2), the correlations of H-2/Me-15 and H-2/Me-13 suggested that these groups were co-facial and assigned as α-oriented, while, the interactions of H-3/Me-14 and Me-14/H-10 indicated that they were on the opposite face of the ring system and β-oriented. The hydroxyls at C-9 and C-10 were on the same side according to the coupling constant between H-9 and H-10 (J = 3.8 Hz). Thus, the relative configuration of 1 was determined. The absolute stereochemistry of the 9,10-diol moiety in 1 was verified by observing the induced electronic circular dichroism (IECD) spectrum after the addition of dimolybdenum tetraacetate in anhydrous DMSO [19,20]. The obvious negative Cotton effect at 310 nm in the IECD spectrum ( Figure 3) permitted the 9R,10S configuration assignment of 1. Combining with the relative configuration, the absolute stereochemistry of 1 was elucidated as 1R,3R,7R,9R,10S ( Figure 1).  Compound 2 was isolated as colorless oil that gave a [M + Na] + ion peak in the HRESIMS spectrum at m/z 371.1839 Δm (calcd. for C20H28O5Na, 371.1834) appropriate for a molecular formula of C20H28O5, corresponding to 7 degrees of unsaturation. The IR spectrum showed a hydroxyl (3430 cm −1 ) and an ester or lactone carbonyl (1742 cm −1 ). The 1 H NMR data (Table 1)  .0 (C-16), 24.9 (C-18), and 17.7 (C- 19)]. Consideration of these data and analyses of the 1 H-1 H COSY and HMBC spectra ( Figure 2) of 2 suggested existence of tetracyclic fusicoccane framework which was similar with that of roussoellol B [21]. Further analyses of the 2D NMR spectra indicated that the methylene at C-4 in roussoellos B was oxygenated (δC 80.0), which was confirmed by 1 H-1 H COSY cross-peak of H-3 and H-4 and the HBMC correlations from H-4 to C-3, C-5, C-6, and C-16. In addition, the Δ 10,14 -double bond in roussoello B shifted to C-13 and C-14 which was evidenced by the HBMC correlations from H-13 to C-10, C-11, and C-12. Moreover, HMBC correlations from H-15 and H-19 to the oxygenated methylene carbon at δC 67.0 (C-20) indicated the presence of a hydroxymethyl functionality. Hence, the planar structure of 2 was established as shown.
In the NOESY experiment (  Compound 2 was isolated as colorless oil that gave a [M + Na] + ion peak in the HRESIMS spectrum at m/z 371.1839 Δm (calcd. for C20H28O5Na, 371.1834) appropriate for a molecular formula of C20H28O5, corresponding to 7 degrees of unsaturation. The IR spectrum showed a hydroxyl (3430 cm −1 ) and an ester or lactone carbonyl (1742 cm −1 ). The 1 H NMR data (Table 1)  .0 (C-16), 24.9 (C-18), and 17.7 (C- 19)]. Consideration of these data and analyses of the 1 H-1 H COSY and HMBC spectra ( Figure 2) of 2 suggested existence of tetracyclic fusicoccane framework which was similar with that of roussoellol B [21]. Further analyses of the 2D NMR spectra indicated that the methylene at C-4 in roussoellos B was oxygenated (δC 80.0), which was confirmed by 1 H-1 H COSY cross-peak of H-3 and H-4 and the HBMC correlations from H-4 to C-3, C-5, C-6, and C-16. In addition, the Δ 10,14 -double bond in roussoello B shifted to C-13 and C-14 which was evidenced by the HBMC correlations from H-13 to C-10, C-11, and C-12. Moreover, HMBC correlations from H-15 and H-19 to the oxygenated methylene carbon at δC 67.0 (C-20) indicated the presence of a hydroxymethyl functionality. Hence, the planar structure of 2 was established as shown.
In the NOESY experiment ( Compound 2 was isolated as colorless oil that gave a [M + Na] + ion peak in the HRESIMS spectrum at m/z 371.1839 ∆m (calcd. for C 20 H 28 O 5 Na, 371.1834) appropriate for a molecular formula of C 20 H 28 O 5 , corresponding to 7 degrees of unsaturation. The IR spectrum showed a hydroxyl (3430 cm −1 ) and an ester or lactone carbonyl (1742 cm −1 ). The 1 H NMR data (Table 1)  ]. Consideration of these data and analyses of the 1 H-1 H COSY and HMBC spectra (Figure 2) of 2 suggested existence of tetracyclic fusicoccane framework which was similar with that of roussoellol B [21]. Further analyses of the 2D NMR spectra indicated that the methylene at C-4 in roussoellos B was oxygenated (δ C 80.0), which was confirmed by 1 H-1 H COSY cross-peak of H-3 and H-4 and the HBMC correlations from H-4 to C-3, C-5, C-6, and C-16. In addition, the ∆ 10,14 -double bond in roussoello B shifted to C-13 and C-14 which was evidenced by the HBMC correlations from H-13 to C-10, C-11, and C-12. Moreover, HMBC correlations from H-15 and H-19 to the oxygenated methylene carbon at δ C 67.0 (C-20) indicated the presence of a hydroxymethyl functionality. Hence, the planar structure of 2 was established as shown.
In the NOESY experiment (Figure 2 (Figure 4) were performed to semiempirical PM3 quantum mechanical geometry optimizations using Gaussian09 at the B3LYP/6-31G* level [22]. The experimental shifts were plotted against the calculated shifts, and least-squares fit lines was confirmed. The calculated shifts for 2a and 2b were corrected by the slope and intercept to get the corrected 13 C shifts (Table 1), and the differences between the corrected and experimental 13 C NMR chemical shifts were analyzed [23,24]. The result showed that the correlation coefficient R 2 of 2a (0.9966) was higher than that of 2b (0.9926) (Figures S21 and S22). Meanwhile, the MAE (mean absolute error) and MD (maximum deviation) of 2a (MAE = 2.28, MD = 6.5) were obviously lower than that of 2b (MAE = 2.74, MD = 14.3), suggesting that 2a was more consistent with the experimental values ( Figure 4). What's more, all of the reported fusicoccanes or ophiobolins with 5/8/5/5 ring system possess a cis-fused A/D ring [21,[25][26][27][28], and the 5-OH of 2 was finally assigned a β-orientation as 2a.
Mar. Drugs 2018, 16, x 4 of 10 semiempirical PM3 quantum mechanical geometry optimizations using Gaussian09 at the B3LYP/6-31G* level [22]. The experimental shifts were plotted against the calculated shifts, and least-squares fit lines was confirmed. The calculated shifts for 2a and 2b were corrected by the slope and intercept to get the corrected 13 C shifts (Table 1), and the differences between the corrected and experimental 13 C NMR chemical shifts were analyzed [23,24]. The result showed that the correlation coefficient R 2 of 2a (0.9966) was higher than that of 2b (0.9926) (Figures S21 and S22). Meanwhile, the MAE (mean absolute error) and MD (maximum deviation) of 2a (MAE = 2.28, MD = 6.5) were obviously lower than that of 2b (MAE = 2.74, MD = 14.3), suggesting that 2a was more consistent with the experimental values ( Figure 4). What's more, all of the reported fusicoccanes or ophiobolins with 5/8/5/5 ring system possess a cis-fused A/D ring [21,[25][26][27][28], and the 5-OH of 2 was finally assigned a β-orientation as 2a. To determine the absolute configuration of compound 2, the electronic circular dichroism (ECD) calculation was performed. The experimental and simulated spectra generated by BALLOON [29,30] were performed to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program ( Figures S2 and S3, Supplementary Materials) [31]. The ECD spectrum of each conformer was calculated using the TDDFT methodology at B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level. Comparison of the experimental and calculated spectra of 2 showed more agreement ( Figure  5) for the 2a configuration. The experimental ECD is consistent with the calculated ECD of 2 ( Figure  5), indicating a (2S,3R,4S,5S,6R,10R,11S)-configuration. Therefore, the structure of 2, namely, roussoellol C, was deduced as shown.   To determine the absolute configuration of compound 2, the electronic circular dichroism (ECD) calculation was performed. The experimental and simulated spectra generated by BALLOON [29,30] were performed to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program (Figures S2 and S3, Supplementary Materials) [31]. The ECD spectrum of each conformer was calculated using the TDDFT methodology at B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level. Comparison of the experimental and calculated spectra of 2 showed more agreement ( Figure 5) for the 2a configuration. The experimental ECD is consistent with the calculated ECD of 2 ( Figure 5), indicating a (2S,3R,4S,5S,6R,10R,11S)-configuration. Therefore, the structure of 2, namely, roussoellol C, was deduced as shown.
Mar. Drugs 2018, 16, x 4 of 10 semiempirical PM3 quantum mechanical geometry optimizations using Gaussian09 at the B3LYP/6-31G* level [22]. The experimental shifts were plotted against the calculated shifts, and least-squares fit lines was confirmed. The calculated shifts for 2a and 2b were corrected by the slope and intercept to get the corrected 13 C shifts (Table 1), and the differences between the corrected and experimental 13 C NMR chemical shifts were analyzed [23,24]. The result showed that the correlation coefficient R 2 of 2a (0.9966) was higher than that of 2b (0.9926) (Figures S21 and S22). Meanwhile, the MAE (mean absolute error) and MD (maximum deviation) of 2a (MAE = 2.28, MD = 6.5) were obviously lower than that of 2b (MAE = 2.74, MD = 14.3), suggesting that 2a was more consistent with the experimental values ( Figure 4). What's more, all of the reported fusicoccanes or ophiobolins with 5/8/5/5 ring system possess a cis-fused A/D ring [21,[25][26][27][28], and the 5-OH of 2 was finally assigned a β-orientation as 2a. To determine the absolute configuration of compound 2, the electronic circular dichroism (ECD) calculation was performed. The experimental and simulated spectra generated by BALLOON [29,30] were performed to semiempirical PM3 quantum mechanical geometry optimizations using the Gaussian 09 program ( Figures S2 and S3, Supplementary Materials) [31]. The ECD spectrum of each conformer was calculated using the TDDFT methodology at B3LYP/6-311++G(d,p)//B3LYP/6-31G(d) level. Comparison of the experimental and calculated spectra of 2 showed more agreement ( Figure  5) for the 2a configuration. The experimental ECD is consistent with the calculated ECD of 2 ( Figure  5), indicating a (2S,3R,4S,5S,6R,10R,11S)-configuration. Therefore, the structure of 2, namely, roussoellol C, was deduced as shown.

Cytotoxic Activities of Selected Compounds
The growth inhibitory effects of the selected compounds (1-3) against human colonic carcinoma cell line (SW480), human promyelocytic leukemia cells (HL-60), human non-small-cell lung cancer cells (A549), breast adenocarcinoma cell line (MCF-7), and human hepatocellular carcinoma cell line (SMMC-7721) were assayed by using MTT method [32], with adriamycin as the positive control. Compounds 1-3 exhibited moderate antiproliferative activities against these cells with IC 50 values ranging from 6.5 to 35.7 µM (Table 2). Normally, cytotoxic natural products display better activities against HL-60 than any other cancer cell lines because HL-60 cells are much sensitive in the assay. However, it is interesting that compound 2 showed significant selectivity toward MCF-7 cells with an IC 50 value of 6.5 µM but with an IC 50 value of 10.9 µM against HL-60. Although an IC 50 value of 6.5 µM does not indicate strong potency, the selectivity of 2 against MCF-7 still makes it a promising lead compound for further studies.

Fungal Material
The fungus PP-414 was isolated from a mud sample collected on the coastal beach in Qinghuangdao County, Hebei Province, China. The mud sample (5 g) was suspended in 50 mL sterile water with a concentration at 10 -1 g/mL and then every 0.5 mL mutterlauge was respectively diluted to 10 -2 , 10 -3 , 10 -4 g/mL with sterile water. Each sample was coated individually on potato dextrose agar (PDA) medium contained chloramphenicol, and incubated at 28 • C to get single colonies by routine microbiological methods. The internal transcribed spacer (ITS) region was amplified by PCR using primers ITS1 (5 -TCCGTAGGTGAACCTGCGG-3 ) and ITS4 (5 -TCCTCCGCTTATTGATATGC-3 ), then submitted to GenBank and identified as Talaromyces purpurogenus by ITS sequence homology (99% similarity with Talaromyces purpurogenus strain Q2, accession no. KX432212.1 (max score 974, query cover 96%, e value 0.0)) and physiological characteristics with accession no. MH120320. The voucher sample, PP-414, has been preserved in the culture collection center of Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China).

Fermentation and Isolation
The fungus PP-414 was incubated on potato dextrose agar (PDA) at 28 • C for 7 days, the agar cultures were cut into small pieces (approximately 0.5 × 0.5 × 0.5 cm 3 ) and then inoculated into 100 × 1 L Erlenmeyer flasks which containing 250 g rice and 250 mL distilled water. After incubating at 28 • C for 28 days, the solid rice medium was distilled with CH 3 CH 2 OH and then extracted three times with EtOAc. The EtOAc extract (80 g) was chromatographed on silica gel chromatography column (CC, 80-120 mesh) eluting with petroleum ether/EtOAc (100:0-0:1, v/v) to afford five fractions (Fr. A-Fr. E). Fr. C (4.

NMR Calculation
The 13 C NMR chemical shifts of each conformer were calculated at the B3LYP/6-311++G(d,p)// B3LYP/6-31G(d) level by the IEFPCM solvation model implemented using Gaussian 09 program with MeOH as solvent, which were then combined using Boltzmann weighting according to their population contributions. The detailed methods were the same as previously described [33].

ECD Calculation
The electronic circular dichroism (ECD) spectra of each conformer were calculated by the TDDFT methodology with MeOH as solvent. The detailed methods were the same as previously described [32] The ECD spectra of each conformer were simulated using a Gaussian function with a bandwidth σ of 0.4 eV. The spectra were combined after Boltzmann weighting according to their population contributions and UV correction was applied.

Cytotoxicity against Cancer Cell Lines
Cytotoxicity of the selected compounds against the five cancer cell lines (SW480, HL-60, A549, MCF-7, and SMMC-7721) was evaluated by the MTT method with adriamycin as positive control. All cells were cultured in RPMI.1640 medium contained 10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 • C in a humidified atmosphere with 5% CO 2 . Tumor cells were seeded in 96-well microtiter plates at 5000 cells/wel, and the test compounds at concentrations ranging from 1.56 to 50 µM were added to the wells 12 h later. After incubation for 48 h, the metabolic conversion of 20 µL of MTT (5 mg/mL) 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide was added and the incubation was continued for 4 h at 37 • C. The medium was exchanged with the medium containing 100 µL triplex solution of 10% SDS, 5% isopropyl alcohol and 12 mM HCl and then cultured 12-20 h at 37 • C. The results were obtained using a microplate spectrophotometer plate reader at 570 nm and the value of inhibition was calculated by formula: % inhibition = [(OD control − OD treated )/OD control ] × 100%. Selected compounds were tested at five concentrations (50, 25, 12.5, 6.25, 3.12 and 1.56 µM) in 100% DMSO with a final concentration of DMSO was 0.5% (v/v) in each well. The IC 50 values were calculated by the means ± SEM calculating by GraphPad Prism 5.

Conclusions
In conclusion, we have reported six metabolites, including two new structures from the culture extract of T. purpurogenus. Among them, 9,10-diolhinokiic acid (1) is the first reported thujopsene-type sesquiterpenoid containing a 9,10-diol moiety and roussoellol C (2) possesses a novel tetracyclic fusicoccane diterpenoid with an unexpected hydroxyl at C-4. This study further enriched the structure diversity of secondary metabolites of this species. Additionally, compound 2 showed significant selectivity aginst MF-7 with IC 50 values of 6.5 µM, which makes it a promising lead compound for further studies.