New Polyenes from the Marine-Derived Fungus Talaromyces cyanescens with Anti-Neuroinflammatory and Cytotoxic Activities

Three new polyene compounds, talacyanols A–C (1–3), along with two known compounds, ramulosin (4) and eurothiocin A (5), were isolated from the marine fungus Talaromyces cyanescens derived from a seaweed Caulerpa sp. Structures of 1–5 were established by one-dimensional and two-dimensional (1D/2D) NMR, HR-ESIMS, and the modified Mosher’s methods, as well as comparison with previously reported literature data. All the compounds (1–5) were tested for their in vitro cytotoxic and anti-neuroinflammatory activities. Among them, 1 showed moderate cytotoxic activity against a panel of cancer cell lines (HCT-15, NUGC-3, NCI-H23, ACHN, PC-3, and MDA-MB-231) with GI50 values ranging from 44.4 to 91.6 μM, whereas compounds 2 and 5 exhibited anti-neuroinflammatory effect without cytotoxicity against all the tested cell lines.


Introduction
Despite developing new therapeutic agents is a long, intricate, and costly process, the discovery and development of the new drugs are urgently needed due to the increase in the annual number of deaths caused by cancer, cardiovascular, respiratory, and neurodegenerative diseases, as well as the emergence and rapid growth of multidrug resistant pathogenic microbes [1,2].
Cancer and Alzheimer's disease are the top leading causes of death worldwide, and neuro-inflammation plays a crucial role in the pathogenesis of Alzheimer's disease [14,15]. Therefore, there is an urgent and continuous need to find new classes of anticancer and anti-neuroinflammatory drugs. As a part of our continuing studies for novel marine fungal agents with potent cytotoxic and anti-neuroinflammatory effects, we isolated three new compounds possessing a polyene skeleton, talacyanols A-C (1-3), and two known compounds ramulosin (4) and eurothiocin A (5) from the marine-derived fungus Talaromyces sp. 168ST-51.1 (Figure 1). In this paper, we describe the isolation and structure identification of the secondary metabolites 1-5 and their in vitro cytotoxic and anti-neuroinflammatory activities.
Molecules 2021, 26, x FOR PEER REVIEW 2 of 9 Cancer and Alzheimer's disease are the top leading causes of death worldwide, and neuro-inflammation plays a crucial role in the pathogenesis of Alzheimer's disease [14,15]. Therefore, there is an urgent and continuous need to find new classes of anticancer and anti-neuroinflammatory drugs. As a part of our continuing studies for novel marine fungal agents with potent cytotoxic and anti-neuroinflammatory effects, we isolated three new compounds possessing a polyene skeleton, talacyanols A−C (1-3), and two known compounds ramulosin (4) and eurothiocin A (5) from the marine-derived fungus Talaromyces sp. 168ST-51.1 (Figure 1). In this paper, we describe the isolation and structure identification of the secondary metabolites 1−5 and their in vitro cytotoxic and anti-neuroinflammatory activities.
Detailed analysis of 1 H-1 H COSY correlations determined the partial structures of 1 including two distinct spin systems from H-3 (δ H 7.13) to H 3 -8 (δ H 1.14), and from H-9 (δ H 5.98) to H 3 -11 (δ H 1.54). Furthermore, the linkages between the partial structures were identified on the basis of the HMBC spectrum. The HMBC correlations of H-1 (δ H 9.46), H-4 (δ H 6.66), and H-10 (δ H 5.92) to C-2 (δ C 139.1), and those of H-3, H-9 to C-1 (δ C 196.0) indicated that three parts of the compound were assembled via C-C bonds from C-2 to C-1, C-3, and C-9. Thus, the planar structure of 1 was determined as shown in Figure 1 The absolute configurations of four possible stereoisomers of the secondary 1,2-diols could be unambiguously determined using the modified Mosher's method ( Figure 3A) by comparing the 1 H NMR data (∆δ S-R ) of their corresponding bis-(S)-and -(R)-MTPA (α-methoxy-α-trifluoromethylphenylacetic acid) esters ( Figure 3B) [16,17]. Therefore, compound 1 was treated with Rand S-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride (MTPA-Cl) to give bis-S-and -R-MTPA esters (1a and 1b), respectively. As a result, the ∆δ S−R values of 1a and 1b were interpreted, which were consistent with syn-1,2-diols ( Figure 3A) and the absolute configurations of the stereogenic centers were determined as 6R and 7R. Thus, the structure of 1 was determined as (2E,4E,6R,7R)-6,7-dihydroxy-2-((Z)prop-1-en-1-yl)octa-2,4-dienal and named talacyanol A. Compound 2 was isolated as a colorless oil with the same molecular formula of C11H16O3 to that of 1, which was established from an HRESIMS ion peak at m/z 219.1003 [M + Na] + (calcd. for C11H16O3Na + , 219.0992). The one-dimensionial (1D) and two-dimensional (2D) NMR data of 2 were almost identical to those of 1. By detailed analysis of NMR data, the planar structure of 2 was determined to be the same as that of 1 including the geometries of the double bounds ( Figure 1). The only difference between 1 and 2 was the absolute stereochemistry of the secondary 1,2-diols at C-6 and C-7 according to their 1 H NMR data in CDCl3 (δH-6 4.28 and δH-7 3.96 for 1; δH-6 4.04 and δH-7 3.72 for 2, Figure S23  Compound 3 was obtained as a colorless oil with a molecular formula of C11H18O3 based on its HRESIMS data m/z 221.1155 [M + Na] + (calcd for 221.1148, C11H18O3Na + ). The 1 H NMR spectrum of 3 was quite similar to that of 2, the only difference lies in the chemical shift of the singlet proton H-1 (δH 9.46 in 2, δH 4.05 in 3), indicating that the aldehyde group at C-1 in 2 was replaced by a hydroxy group in 3. Thus, the planar structure of 3 was elucidated as shown in Figure 1 based on its 1D and 2D NMR data.
A literature search revealed that the planar structure of 3 was similar to pinophol A, which was isolated from a plant endophytic fungus Talaromyces pinophilus by Zhao et al. [18]. However, the absolute configurations at C-6 and C-7 of pinophol A had not been  Figure S22) suggested that they could be a pair of diastereomers. By analyzing 1 H NMR data of tri-S-and -R-MTPA esters of 3, the absolute configurations of C-6 and C-7 were determined as 6S and 7R ( Figure 3B). Thus, the structure of 3 was determined as a new derivative of pinophol A, (2E,4E,6S,7R)-2-((Z)-prop-1-en-1-yl)octa-2,4-diene-1,6,7-triol, and named talacyanol C.
Aliphatic aldehydes are easily reduced to the corresponding alcohols in high yield by heterogeneous catalytic hydrogenation. To verify whether compound 3 is a true natural product or an artifact arising from compound 2 by reduction of aldehyde during the extraction process with ethyl acetate, we cultured the strain again and the culture broth was extracted successively with dichloromethane and n-butanol. Compound 3 was found in the butanol extract with a detectable concentration (Figures S31 and S32). Therefore, it could be concluded that 3 is a true natural substance.
The structures of the known compounds were identified as ramulosin (4), and eurothiocin A (5) by comparison of their spectroscopic data with those reported in the literature [19][20][21].

Bioactivities
Over the past few decades, emerging evidence has shown that many marine natural products, such as cytarabine, eribulin mesylate, brentuximab vedotin, and trabectidine exhibit beneficial effects in the prevention and treatment of cancer [22]. Furthermore, acyclic polyene polyols are a wide group of polyketides, and many of them display cytotoxicity against various cancer cell lines [17,23]. Therefore, compounds 1−5 were screened for in vitro cytotoxicity against six different cancer cell lines (stomach NUGC-3, colon HCT-15, lung NCI-H23, breast MDA-MB-231, prostate PC-3, and renal ACHN), the most common cancers in Korea [24]. Notably, compound 1 displayed moderate cytotoxicity against all the cancer cell lines with GI50 values ranging from 44.4 to 91.8 μM (Table 2). Compound 2 was isolated as a colorless oil with the same molecular formula of C 11 H 16 O 3 to that of 1, which was established from an HRESIMS ion peak at m/z 219.1003 [M + Na] + (calcd. for C 11 H 16 O 3 Na + , 219.0992). The one-dimensionial (1D) and twodimensional (2D) NMR data of 2 were almost identical to those of 1. By detailed analysis of NMR data, the planar structure of 2 was determined to be the same as that of 1 including the geometries of the double bounds ( Figure 1). The only difference between 1 and 2 was the absolute stereochemistry of the secondary 1,2-diols at C-6 and C-7 according to their 1 H NMR data in CDCl 3 (δ H-6 4.28 and δ H-7 3.96 for 1; δ H-6 4.04 and δ H-7 3.72 for 2, Figure S23) and optical rotation values [α] 25 D +76.6 (c 0.2, MeOH) for 1; [α] 25 D −15 (c 0.2, MeOH) for 2). By comparing 1 H NMR data of bis-S-and -R-MTPA esters of 2, the absolute configurations at C-6 and C-7 were assigned as 6S and 7R ( Figure 3B), and the structure of 2 was determined as (2E,4E,6S,7R)-6,7-dihydroxy-2-((Z)-prop-1-en-1-yl)octa-2,4-dienal and named talacyanol B.
Compound 3 was obtained as a colorless oil with a molecular formula of C 11 H 18 O 3 based on its HRESIMS data m/z 221.1155 [M + Na] + (calcd for 221.1148, C 11 H 18 O 3 Na + ). The 1 H NMR spectrum of 3 was quite similar to that of 2, the only difference lies in the chemical shift of the singlet proton H-1 (δ H 9.46 in 2, δ H 4.05 in 3), indicating that the aldehyde group at C-1 in 2 was replaced by a hydroxy group in 3. Thus, the planar structure of 3 was elucidated as shown in Figure 1 based on its 1D and 2D NMR data.
A literature search revealed that the planar structure of 3 was similar to pinophol A, which was isolated from a plant endophytic fungus Talaromyces pinophilus by Zhao et al. [18]. However, the absolute configurations at C-6 and C-7 of pinophol A had not been  Figure S22) suggested that they could be a pair of diastereomers. By analyzing 1 H NMR data of tri-Sand -R-MTPA esters of 3, the absolute configurations of C-6 and C-7 were determined as 6S and 7R ( Figure 3B). Thus, the structure of 3 was determined as a new derivative of pinophol A, (2E,4E,6S,7R)-2-((Z)-prop-1-en-1-yl)octa-2,4-diene-1,6,7-triol, and named talacyanol C.
Aliphatic aldehydes are easily reduced to the corresponding alcohols in high yield by heterogeneous catalytic hydrogenation. To verify whether compound 3 is a true natural product or an artifact arising from compound 2 by reduction of aldehyde during the extraction process with ethyl acetate, we cultured the strain again and the culture broth was extracted successively with dichloromethane and n-butanol. Compound 3 was found in the butanol extract with a detectable concentration (Figures S31 and S32). Therefore, it could be concluded that 3 is a true natural substance.
The structures of the known compounds were identified as ramulosin (4), and eurothiocin A (5) by comparison of their spectroscopic data with those reported in the literature [19][20][21].

Bioactivities
Over the past few decades, emerging evidence has shown that many marine natural products, such as cytarabine, eribulin mesylate, brentuximab vedotin, and trabectidine exhibit beneficial effects in the prevention and treatment of cancer [22]. Furthermore, acyclic polyene polyols are a wide group of polyketides, and many of them display cytotoxicity against various cancer cell lines [17,23]. Therefore, compounds 1-5 were screened for in vitro cytotoxicity against six different cancer cell lines (stomach NUGC-3, colon HCT-15, lung NCI-H23, breast MDA-MB-231, prostate PC-3, and renal ACHN), the most common cancers in Korea [24]. Notably, compound 1 displayed moderate cytotoxicity against all the cancer cell lines with GI 50 values ranging from 44.4 to 91.8 µM (Table 2). Compounds 1-5 were also tested for their inhibitory effects on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. The cells were initially treated with a high concentration (200 µM) of each compound and LPS (200 ng/mL) to screen their inhibitory effect on NO production. All the compounds showed weak or strong inhibitory effects on NO production ( Figure S33), and compounds 2 and 5 showed the most potent anti-inflammatory activity. Therefore, talacyanol B (2) and eurothiocin A (5) were selected for further studies to investigate NO production in BV-2 cells and LPS-induced expression levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins by Western blot analysis. As shown in Figure 4A,B, the NO production and expression levels of COX-2 and iNOS proteins were suppressed by both 2 and 5 in a dose-related fashion at the concentrations of 50, 100, and 200 µM. Compounds 1−5 were also tested for their inhibitory effects on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. The cells were initially treated with a high concentration (200 μM) of each compound and LPS (200 ng/mL) to screen their inhibitory effect on NO production. All the compounds showed weak or strong inhibitory effects on NO production ( Figure S33), and compounds 2 and 5 showed the most potent anti-inflammatory activity. Therefore, talacyanol B (2) and eurothiocin A (5) were selected for further studies to investigate NO production in BV-2 cells and LPS-induced expression levels of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins by Western blot analysis. As shown in Figure 4A,B, the NO production and expression levels of COX-2 and iNOS proteins were suppressed by both 2 and 5 in a dose-related fashion at the concentrations of 50, 100, and 200 μM.   Figure S30). Cell viability was tested using the MTT (3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyltetrazol bromide) assay. Results are shown as the percentage of control samples; (B) Inhibition of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein and mRNA expression by compounds 2 and 5 in lipopolysaccharide (LPS)-stimulated BV-2 cells. The data (B) is expressed as the relative signal intensity for two independent experiments. Values are the mean ± standard error. ### p < 0.001, vs. control group and *** p < 0.001 vs. LPS-treated group.

Fungal Material and Fermentation
The fungal strain 168ST-51.1 was isolated from the seaweed Caulerpa sp. The seed and mass cultures were performed in Bennett's medium (1% glucose, 0.2% tryptone, 0.1% yeast extract, 0.1% beef extract, 0.5% glycerol, sea salts 32 g/L, and agar 17 g/L for agar medium). The fungus was initially cultured on Bennett's agar medium in a Petri dish for 7 days. The actively grown mycelium was transferred aseptically into a 500 mL conical flask containing 300 mL of Bennett's broth medium and incubated on a rotary shaker (140 rpm) at 28 • C for 4 days. An aliquot (0.1% v/v) from the seed culture was inoculated into twenty 2 L flasks each containing 1 L of the medium and grown under the same conditions as described for the seed culture for 7 days, and then harvested.