**1. Introduction**

Marine fungi, particularly the mangrove-derived fungi, have proven to be a prolific source of structurally novel and biologically active secondary metabolites, which increasingly attracted the attention of both pharmaceutical and natural product chemists [1–3]. Up to now, more than 1400 new secondary metabolites produced by mangrove-derived fungi have been reported, including polyketides, alkaloids, terpenes and so on, and more than 40% of secondary metabolites displayed cytotoxic, antibacterial and insecticidal activities etc. [3–8]. Among them, isocoumarins are lactone-type derivatives derived from the polyketide pathway [9,10], and they possess a wide range of pharmacological activities such as antibacterial 7-hydroxyoospolactone and parapholactone [11], anti-inflammatory (±)-prunomarin A [12], cytotoxic lunatinin [13], insecticidal peniciisocoumarins A and B, antiplasmodial monocerin [14], antioxidant and *α*-glucosidase inhibitory penicimarin N [15], brine shrimp (*Artemia salina*) lethal and broad-spectrum antimicrobial penicimarins G–K [16,17], penicoffrazins B and C [18].

**Citation:** Cai, J.; Zhu, X.-C.; Zeng, W.-N.; Wang, B.; Luo, Y.-P.; Liu, J.; Chen, M.-J.; Li, G.-Y.; Huang, G.-L.; Chen, G.-Y.; et al. Talaromarins A–F: Six New Isocoumarins from Mangrove-Derived Fungus *Talaromyces flavus* TGGP35. *Mar. Drugs* **2022**, *20*, 361. https://doi.org/ 10.3390/md20060361

Academic Editor: Hee Jae Shin

Received: 22 April 2022 Accepted: 24 May 2022 Published: 27 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The genus of *Talaromyces* has been studied and applied as a biocontrol agent, as a rich source of chitinolytic enzymes and producer of secondary metabolites [19–21]. Different classes of bioactive secondary metabolites have been found from the genus of *Talaromyces* [19–21], including some bioactive isocoumarins, such as selective antimigratory talarolactone A [22], *α*-glucosidase inhibitory sescandelin B and 3,4-dimethyl-6,8- dihydroxyisocoumarin [23], antibacterial tratenopyrone [24], antibacteria and antifungi (-)-8-hydroxy-3-(4-hydroxypentyl)-3,4-dihydroisocoumarin [25]. These results indicated that the genus of *Talaromyces* can be used for the control of pathogenic bacteria, phytopathogenic microorganisms, diabetes control agents and so on [19,26].

As part of our continuing exploration of structurally novel and biologically interesting secondary metabolites from mangrove-derived fungi [27–33], a fungus *Talaromyces flavus* TGGP35 obtained from the medicinal mangrove plant *Acanthus ilicifolius* attracted our attention, because of the EtOAc extract of *T. flavus* TGGP35 showed potent antioxidant activity. A chemical investigation of this fungus led to the identification of six new isocoumarins talaromarins A-F (**1**–**6**) and 17 known analogues (**7**–**23**) (Figure 1). Herein, we report the isolation, structure identification, and bioactivities of these compounds.

**Figure 1.** The structures of compounds **1**–**23**.

#### **2. Results and Discussion**

Compound **1** was isolated as a colorless oil. The molecular formula of **1** was established as C17H22O6 (seven degrees of unsaturation) by its HR-ESI-MS spectrum at *m*/*z* 323.1475 [M + H]+ (calcd for C17H23O6, 323.1472). The IR spectrum showed the presence of a hydroxyl group (3432 cm<sup>−</sup>1) and an aromatic ring (1743, 1618 and 1388 cm<sup>−</sup>1) in **1**. The 1H-NMR data (Table 1) displayed a pair of ortho coupled aromatic protons at *δ*H 7.00 (d, *J* = 8.8 Hz) and 6.78 (d, *J* = 8.8 Hz), one methoxyl group at *δ*H 3.88 (s), two oxygenated methine groups at *δ*H 4.90 (m) and 4.34 (m), four methylene groups at *δ*H [3.08 (m) and 2.64 (m)], 1.85 (m), 1.57 (m) and 1.52 (m), one methyl group at *δ*H 1.22 (d, *J* = 6.4 Hz). The combination of 13C NMR and DEPT data (Table 2) exhibited 17 carbon resonances, including two ester carbonyls at *δ*C (171.1 and 163.1), six aromatic carbons at *δ*C (155.5, 145.3, 128.4, 121.2, 114.6 and 111.5), one methoxyl group at *δ*C (56.6), two oxygenated methine groups at *δ*C (77.4 and 70.9), four methylene groups at *δ*C (35.7, 34.7, 28.0 and 21.0), two methyl groups at *δ*C (21.6 and 20.1). The above 1D NMR spectroscopic data indicated that **1** had an isocoumarin skeleton structure, and **1** is similar to penicimarin G (**12**) [16]. The major difference was the presence of an acetoxy group at [(*δ*C 171.1 (C), 21.6 (CH3) and *δ*H 2.04, (s)] in **1**, indicating that the hydroxyl group in **12** was replaced by an acetoxy group in **1**. The 1H-1H COSY correlations of CH2(2-)–CH2(3-)−CH(4-)−CH3(5-), combined with the HMBC correlations from H-5- to C-4-/C-3- (Figure 2), confirmed the acetoxy group connected at C-4- in **1**, and the planar structure of **1** was determined (Figure 1).

**Table 1.** 1H NMR spectroscopic data (400/600 MHz) (*δ* in ppm, *J* in Hz) for **1**–**6** in CDCl3.


**Table 2.** 13C NMR spectroscopic data (100/150 MHz) for **1**–**6** in CDCl3.


**Figure 2.** 1H-1H COSY and key HMBC correlations for compounds **1**–**6**.

The absolute configurations of C-3 and C-4- in **1** were determined by chemical hydrolysis, modified Mosher's method and a comparison of CD spectra with dihydroisocoumarins described in the literature [28,34]. The major hydrolysis product (**1a**) of **1** was obtained with K2CO3 and anhydrous ethanol at 28 ◦C for 1.5 h (Figure 3), and **1a** showed the same planar structure with **12** [16]. The modified Mosher's method was used to determine the configuration of C-4- for **1a**. The differences in 1H NMR chemical shifts of **1a** between (*S*)- and (*R*)-MTPA esters (Δ*δ* = *δS* − *δR*) were calculated to assign the absolute configuration of C-4- to be *R* (Figure 4), the same as **12** [16]. The negative cotton effect at 266 nm suggested the *R* configuration at C-3 (Figure 5), by comparison with data for dihydroisocoumarins described in the literature [34]. Thus, the structure of **1** was determined and named talaromarin A.

**Figure 3.** Reaction route of hydrolysis for compound **1**.

**Figure 4.** Δ*δ* (=*δS*−*δR*) values for (*S*)- and (*R*)-MTPA esters of compounds **1**–**3**.

Compound **2** was isolated as a white powder. The molecular formula was deduced to be C15H20O5 on the basis of HR-ESI-MS spectrum, implying six degrees of unsaturation. According to the IR spectrum, the hydroxyl group (3414 cm<sup>−</sup>1) and aromatic rings (1668, 1619, 1586, 1502 and 1442 cm<sup>−</sup>1) were observed. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) revealed that **2** also belonged to the isocoumarin class and had a similar structural relationship to penicimarin M [17], except for the presence of one oxygenated methine at [*δ*H 3.80 (m), *δ*C 67.9 (CH)] for C-4-, and the absence of a carbonyl group at *δ*C 208.4 (C) in **2**. The above results suggested a carbonyl group in penicimarin M was

replaced by an oxygenated methine group in **2**. Furthermore, the 1H-1H COSY correlations of H-5- to H-4- and H-3- to H-2-/H-4-, and the HMBC correlations from the methyl H-5- to C-3-/C-4- established the oxygenated methine at C-4- (Figure 2). The absolute configuration of C-4- was determined as *R* by Mosher's method [28] (Figure 4). The negative cotton effect at 265 nm suggested the *R* configuration at C-3 (Figure 5), by comparison with data for dihydroisocoumarins described in the literature [34]. Thus, the absolute configuration of **2** was established as 3*R*,4-*R* and named talaromarin B.

**Figure 5.** The experimental CD spectra of compounds **1**–**6**.

Compound **3** was isolated as a yellow oil, with the molecular formula C16H22O6 (six degrees of unsaturation) by the HR-ESI-MS spectrum. The IR spectrum indicated that **3** had hydroxyl group (3415 cm<sup>−</sup>1) and aromatic ring (1638, 1618 and 1384 cm<sup>−</sup>1). The 1H, 13C NMR data (Tables 1 and 2) and HR-ESI-MS data revealed that **3** closely resembled those of **2**, the main differences were the presence of a methoxyl group at [*δ*H 3.89 (s), *δ*C 61.8 (CH3)] in **3**, and an aromatic proton signal at *δ*H 6.62 (d, *J* = 8.0 Hz) was absented in **3**.Moreover, the chemical shift of C-5 at *δ*C (117.1) in **2** was downfield-shifted to *δ*C (147.7) in **3**. The HMBC correlations from 8-OCH3 to C-8, 7-OCH3 to C-7 and H-6 to C-8/C-7/C-4a (Figure 2), indicated the additional methoxyl group was attached to C-8 and the hydroxyl group was connected to C-5 (Figure 2). The absolute configurations of C-3 and C-4- were determined to be the same *R* by comparison with CD data described in the literature [34] and Mosher's method [28] (Figures 4 and 5). Thus, the structure of **3** was determined and named talaromarin C.

Compound **4** was isolated as a yellow oil, and the molecular formula was established as C16H22O5 (six degrees of unsaturation) on the basis of its HR-ESI-MS spectrum. The IR spectrum of **4** displayed absorption bands for hydroxyl (3475 cm<sup>−</sup>1), carbonyl (1706 cm<sup>−</sup>1) and aromatic (1637 and 1617 cm<sup>−</sup>1) groups. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) suggested that **4** was very similar to those of **2**, the only difference was the presence of a methoxyl group at [*δ*H 3.96 (s) and *δ*C 61.7 (CH3)] in **4**. The location of the methoxyl groups at C-7 and C-8 were established by HMBC correlations from 7-OCH3 to C-7, 8-OCH3 to C-8, H-6 to C-8/C-4a and H-5 to C-7/C-8a/C-4 (Figure 2). The 1H-1H COSY, HMQC, and HMBC spectra established the complete assignment for **4** (Figure 2). Mosher's method was tried to determine the absolute configuration of C-4- in **4** [28]; unfortunately, the reaction failed. The negative cotton effect at 259 nm suggested the *R* configuration at C-3 (Figure 5) [34]. Thus, the structure of **4** was determined and named talaromarin D.

Compound **5** was isolated as a colorless oil and had the molecular formula of C16H20O5 as determined by HR-ESI-MS and NMR data, requiring seven degrees of unsaturation. The presence of an aromatic ring (1638, 1617 cm<sup>−</sup>1) was observed in the IR spectrum. The 1H

and 13C NMR data (Tables 1 and 2) of **5** were structurally similar to those of **4**, except for the presence of a ketone carbonyl carbon at *δ*C 208.7 (C) and the absence of one oxygenated methine carbon at [*δ*C 68.1 (CH), *δ*H 3.83 (m)] at C-4- in **5**, indicating that the oxygenated methine group in **4** was replaced by a carbonyl group for C-4- in **5**. The HMBC correlations from H-3- to C-1-, H-5- to C-4-/C-3- further confirmed **5** with a carbonyl unit at C-4- (Figure 2). The whole structure was further determined by the 2D NMR spectra (Figure 2). The absolute configuration of C-3 was determined as *R* by CD spectra (Figure 5) [34], and **5** was named talaromarin E.

Compound **6** was isolated as a yellow oil. The molecular formula of **6** was established as C14H16O5 (seven degrees of unsaturation) on the basis of its HR-ESI-MS data. The IR spectrum of **6** showed the hydroxyl group at 3461 and 3407 cm<sup>−</sup><sup>1</sup> and the aromatic rings at 1736 and 1671 cm<sup>−</sup>1. The 1H and 13C NMR spectroscopic data (Tables 1 and 2) revealed that **6** was an isocoumarin derivative, with a similar structural relationship to peniciisocoumarin D (**18**), the obvious difference was that **6** lacked a methoxy group at C-8. The methoxy group (8-OMe) in **18** was replaced by a hydroxy group (8-OH) in **6**, which was supported by the appearance of a hydrogen-bonded hydroxyl group at *δ*H 11.00 (s). The HMBC correlations from the hydroxyl group 8-OH to C-8a/C-8/C-7 further confirmed the 8-OH was connected at C-8 (*δ*C 149.1) (Figure 2). The 1H-1H COSY, HMQC, and HMBC spectra determined the complete assignment for **6** (Figure 2). The absolute configuration of C-3 was determined as *R* by CD spectroscopy (Figure 5) [34] and **6** was named talaromarin F.

By comparing physical and spectroscopic data with the literature, the 17 known homologues were identified as (*R*)-3-(3-hydroxypropyl)-8-hydroxy-3,4-dihydroisocoumarin (**7**) [35], peniciisocoumarin C (**8**) [28], 7-hydroxy-3-(3-hydroxypropyl)-8-methoxyisochroman-1-one (**9**) [34], 5,6-dihydroxy-3-(4-hydroxypentyl)-isochroman-1-one (**10**) [23], peniciisocoumarin F (**11**) [28], penicimarin G (**12**) [16], penicimarin C (**13**) [36], peniciisocoumarin A (**14**) [28], peniciisocoumarin B (**15**) [28], aspergillumarin A (**16**) [37], penicimarin H (**17**) [16], peniciisocoumarin D (**18**) [28], peniciisocoumarin G (**19**) [28], peniciisocoumarin E (**20**) [28], penicimarin N (**21**) [15], 6,8-dihydroxy-3-(2-hydroxypropyl)-7-methyl-1*H*-isochromen-1- one (**22**) [38] and pestalotiorin (**23**) [39].

The plausible biosynthetic pathways of compounds **1**–**23** were also proposed (Scheme 1).Isocoumarins were originated from the acetate-malonate or the polyketide synthase (PKS) pathway [10]. Peniciisocoumarin C (**8**), penicimarin C (**13**) and peniciisocoumarin A (**14**) would be biosynthesized from malonyl-CoA and acetyl-CoA and can be considered as intermediates which would be transformed to other isolated compounds from the fungus TGGP35. Compound **8** would be transformed to **7**, **9** and **20**–**23** by condensation, aromatization, esterification, dihydroxylation, methoxylation reaction and so on. Compound **13** would be transformed to **10** by dehydroxylation, demethoxylation and dehydroxylation reaction. Compounds **1**–**6**, **10**–**15** and **17**–**19** would be deduced from **14** with dihydroxylation, methoxylation, methylation, hydroxylation, esterification, acetylation, Baeyer–Viliger oxidation reaction, etc.

The antioxidant activities of compounds **1**–**23** were evaluated. Compounds **6**–**11**, **17**–**19** and **22** exhibited potent antioxidant activity with the IC50 values ranging from 0.009 to 0.27 mM, while the positive control trolox was IC50 = 0.29 mM (Table 3).


**Table 3.** Antioxidant activity for compounds **2**, **6**–**11**, **17**–**19, 21** and **22**.

Trolox was used as a positive control.

**Scheme 1.** Plausible biosynthetic pathways of compounds **1**–**23**.

The preliminary structure–activity relationship of the isolated isocoumarins was discussed. The substitution site, orientation of hydroxyl and methoxy groups on the skeleton of isocoumarins, and the substitution of different groups by side chain C-4- can affect their antioxidant activity. Compound **2** which possesses a hydroxyl group on C-8, showed better antioxidant activity than that of **3** and **4**, indicating that the chelated hydroxyl group at C-8 is important in enhancing antioxidant activity. Compound **6** possesses two hydroxyl groups at C-7 and C-8, which showed higher antioxidant activity than **16** (only one hydroxyl group on C-8), indicating that the hydroxyl group at C-7 is an important antioxidant activity site. Compounds **17** and **18** possess a ketone group at C-4-, which showed higher antioxidant activity than **12** and **13,** which have an oxygenated methine at C-4-, suggesting that the substitution of different groups by side chain at C-4- can affect antioxidant activity. Compound **22** possesses a hydroxyl group at C-8, showed higher antioxidant activity than **23**, suggesting that the chelated hydroxyl group at C-8 is important in enhancing antioxidant activity. Furthermore, compounds **7**, **8**, **19** and **21** show antioxidant activities, which may be due to the existence of a chelated hydroxyl group.

Compounds **10**, **18**, **21**, and **23** showed strong inhibitory activity against *α*-glucosidase with the IC50 values of 0.10, 0.38, 0.62 and 0.54 mM, respectively (the positive control acarbose with the IC50 value of 0.5 mM).

All compounds were tested for their antibacterial activities against *Staphylococcus. aureus*, *Escherichia coli*, *S. epidermidis and Pseudomonas aeruginosa*; however, all compounds showed no antibacterial activity at the concentration of 50 μg/mL. All compounds showed no biological activity against five phytopathogens (*Colletotrichum asianum*, *C. acutatum*, *Fusarium oxysporum*, *Pyricularia oryzae* and *Curvularia australiensis*) at the concentration of 1 mg/mL.

#### **3. Materials and Methods**

## *3.1. General Experimental Procedures*

Optical rotations were measured on a JASCO P-1020 digital polarimeter (JASCO, Tokyo, Japan). IR spectra were recorded on a Thermo Nicolet 6700 (using KBr disks) spectrophotometer. UV spectra were measured on a PERSEE TU-1990 spectrophotometer. CD spectra were recorded on a Mos-450 spectrometer and 1D and 2D NMR spectra were recorded on a Bruker AV spectrometer (400 MHZ for 1H and 100 MHZ for 13C) and a JNM-ECZS spectrometer (600 HMZ for 1H and 125 MHZ for 13C). HR-ESI-MS spectra were obtained on a Q-TOF Ultima Global GAA076 LC mass spectrometer. ESI-MS spectra were recorded on a MAT-95-MS mass spectrometer. HPLC were used for the Agilent 1100 prep-HPLC system with an Agilent C18 analytical (9.4 × 250 mm, 5 μm) HPLC column. Silical gel (200 −300 mesh, Qingdao Marine Chemical Factory, Qingdao, China) were used for column chromatography (CC). Sephadex LH-20 gel column (GE Healthcare, Bio-Sciences Corp, Piscataway, NJ, USA) were used for CC. Biological activities were tested on an ultraclean workbench (Suzhou Sujing Company, Suzhou, China) and the biological activities' results were tested with a full wavelength multifunctional microplate reader (Bio-Tek Instruments, Winooski, VT, USA). Methanol, ethyl acetate, petroleum ether, chloroform, dimethyl sulfoxide and other conventional chemical reagents were used in the experimental operation (Guangzhou Xilong Chemical Reagent Factory, Guangzhou, China).
