**2. Results**

*2.1. Taxonomic Identification (Phenotypic and Genotypic Data) of Monascus purpureus wmd2424*

The sample WMD2424 is a filamentous fungal strain collected from the Chiayi mangrove wetland, inoculated in CYA medium, and cultured at 25 ◦C for 7 days. The diameter of the colony on the CYA plate is 15 mm, and the front color of the colony is reddish orange; the colony is velutinous, without radial grooves (sulcate), exudate (exudate), and soluble pigment (soluble pigment); the back of the colony is reddish orange. Observed under an optical microscope, the mycelium has a septate, and the thin wall is colorless; the conidia

are colorless, and the wall is smooth; conidia (conidia) grow on the top or lateral hyphae, with several clusters, pear-shaped, and a truncated bottom, 8.1–16.7 × 6.3–15.5 μm in size, with smooth walls that are colorless; the outer walls of the ascomata are light red but all immature; the same condition was found after 14 days of culture, and only one ascoma was found. The fruit contains ascospores, and the ascospores have a smooth, colorless outer wall and a broad oval shape, with a size of 4.3–5.6 × 3.8–4.8 μm. Partial sequence analysis of the β-tubulin gene fragment was carried out. The total length of the sequence was 1019 bp, compared with the GenBank database, and analyzed and judged with reference to the taxonomic literature. The results showed that the sequence similarity with Monascus purpureus wmd2424 was 99.88% (838/839). The strain was identified as Monascus purpureus according to the colony culture morphology, microstructural characteristics and partial sequence analysis of β-tubulin gene fragment.

#### *2.2. Structure Elucidation of Compounds*

Compound **1** was obtained as oil with an [α]26D: +34.2 (*c* 0.01, CHCl3). The molecular formula was established as C22H24O8 via HRESIMS, indicating 11 degrees of unsaturation. The UV spectrum showed maximum absorption at 268 and 360 nm. The IR spectrum showed absorptions at 3406, 1710, and 1680 cm<sup>−</sup>1, corresponding to the hydroxyl group and carbonyl groups. The 1H NMR spectroscopic data (Table 1) of **1** show three methyl groups, including one singlet at *δ*H 1.48 (3H, s, H-12), one triplet at *δ*H 1.28 (3H, t, *J* = 6.4 Hz, H-17), and one acetyl at *δ*H 2.60 (3H, s, H-11). It also showed two methylene groups [*δ*H 2.72 (1H, d, *J* = 16.2 Hz, H-15), 2.75 (1H, d, *J* = 16.2 Hz, H-15), 3.72 (1H, d-like, *J* = 17.0 Hz, CH2-13), 3.77 (1H, d-like, *J* = 17.0 Hz, CH2-13)], one oxymethine [*δ*H 4.25 (1H, m, H-16)], one *meta*-coupling aromatic ring at *δ*H 6.68 (1H, dd, *J* = 8.8 Hz, H-1) and 7.71 (1H, d, *J* = 8.8 Hz, H-2), two olefinic protons at *δ*H 5.94 (1H, s, H-6) and 7.53 (1H, s, H-9), one intramolecular hydrogen bond at *δ*H 13.4 (1H, s, OH-4), and two hydroxyl groups at *δ*H 3.50 (1H, s, OH-8 or OH-8b) and 4.15 (1H, s, OH-8b or OH-8). The 1H (Table 1), 2D-NMR (Figures 3 and 4), IR, and UV spectra showed that compound **1** was a xanthene derivative similar to xanthonoides as monasxanthone A [35]. The molecular weight of **1** is 30 units more than **1** and showed another proton peak at *δ*H 4.25 (1H, m, H-16) and 3.21 (3H, s, OCH3-16), suggesting the existence of a methoxy group in **1**. The NMR spectra of **1** represent a 4-methoxy-2-oxopentyl moiety at C-5 position in **1** instead of a pentan-2-one in monasxanthone A. Thus, the structure of **1** was elucidated as 7-acetyl-4,8-dihydroxy-1-(4-methoxy-2-oxopentyl)-4-methyl-4,4a-dihydro-3*H*-xanthen-3-one and named monascuspurin A. The relative configuration of **1** was deduced from the NOESY spectrum (Figure 4). The absolute configuration of **1** was further established as (8*S*,8b*R*,16*S*), for the experimental electronic circular dichroism (ECD) curve was in line with its theoretical curve, which was calculated by using the time-dependent density functional theory (TD-DFT) approach [36] (Figure 5).



**Table 1.** *Cont.*

**Figure 3.** Key COSY (1H–1H) and HMBC (1H→13C) correlations of compounds **1**–**5**.

Compound **2** was obtained as an optically active colorless oil. [α]26D: +54.2 (*c* 0.01, CHCl3). The molecular formula was determined as C15H22O5 (five degrees of unsaturation) via HR-ESI-MS (*m*/*z* 305.13598, ([M+Na]+, C21H30NaO<sup>+</sup>5 ; calcd. 305.13592)), which was in agreemen<sup>t</sup> with the 1H- and 13C-NMR data (Table 1). The UV spectrum absorption λmax (MeOH) at 242 nm, and a strong IR absorption at 1675 cm<sup>−</sup>1, as well as the observation of the featuring carbon resonances [δC 132.1 (C-8a), 146.2 (C-4a), and 195.1 (C-8)] in the 13C-NMR spectrum (Table 1), revealed the presence of an <sup>α</sup>,β-unsaturated carbonyl functionality in **1**. The remaining IR spectrum revealed the presence of the OH group (3410 cm<sup>−</sup>1), and ester (1715 cm<sup>−</sup>1), respectively. The 1H-NMR spectrum of **2** exhibited signals attributed to one allylic Me (δH 1.76 (3H, q, *J* = 1.2 Hz, Me-1), signals of *α*-methylene protons of one ketone [δH 3.33/3.48 (each 1H, d, J = 16.8 Hz, CH2-4), 2.45 (2H, t, *J* = 7.8 Hz, CH2-9)], one *β*-methylene signals of ketone (δH 1.61 (2H, sextet, *J* = 7.8 Hz, CH2-10)), one aliphatic CH2

proton (δH 2.49 (1H, ddd, J = 18.0, 10.7, 1.2 Hz, Hax-5) and 2.53 (1H, ddd, J = 18.0, 6.2, 1.2 Hz, Heq-5)]), one oxymethine [*δ*H 4.83 (1H, dd, *J* = 10.7, 6.2 Hz, H-6)], one acetoxy group [*δ*H 2.09 (3H, s, H-15)], and one terminal Me moiety (*δ*H 0.92 (3H, t, *J* = 7.8 Hz, H-11)). Fifteen C-atom signals (Table 2) corresponding to six quaternary C-atoms (including three carbonyl groups, one oxygenated quaternary carbon), one OCH, two olefinic carbons, four CH2, and four CH3 groups were observed in the 13C-NMR and DEPT spectra.

**Figure 4.** Key NOESY correlations (↔) of compounds **1**–**5**.

**Figure 5.** *Cont*.

**Figure 5.** *Cont*.

**Figure 5.** Experimental CD spectra (upper) and the calculated ECD (lower) spectra of compounds **1**–**5**.

The 1H- and 13C-NMR spectra of **2** (Tables 1 and 2) were similar to those of monaphilone C [31], except that an acetoxy moiety at C-6 of **2** replaced a 2-oxoheptyl moiety at C-6 of monaphilone C. The planner structure of **2** was confirmed using the COSY and HMBC experiments (Figure 3). The stereochemistry of **2** was proposed on the basis of the NOESY experiments (Figure 4). The H-6/CH3-12 has no correlation in the NOESY spectrum (Figure 4) represented acetoxy group and CH3-12 are *syn*-form. The physicochemical data and NMR spectra of compound **2** and the known compound monapurpureusone [31] are similar; the only difference is that the specific rotation of monapurpureusone is negative, while the specific rotation of **2** is positive, and it can be inferred that the two are stereoisomers. On comparing the reference to the (6*S*,7*<sup>R</sup>*)-configuration of FK17-P2b (α]26D: + 26.0 (c 0.1, MOH)) [37], the relative configuration of **2** can be assigned as *rel*-(6*S*,7*R*)- configuration, and named monascuspurin B. The absolute configuration of **2** was defined via a comparison of the experimental and calculated ECD data (Figure 5). Therefore, the absolute configuration of **2** was undoubtedly determined as (6*S*,7*R*).

Compound **3** was isolated as oil with [α]26D: +74.2 (*c* 0.01, CHCl3). Its molecular formula was determined to be C18H26O5 based on the HRESIMS [M+Na]+ peak at 345.16780 (Calcd.: C18H26O5, 345.16779), referring six degrees of unsaturation. The maximum absorption of an IR spectrum showed the presence of the hydroxyl group (3410 cm<sup>−</sup>1), γ-lactone (1770 cm<sup>−</sup>1), and <sup>α</sup>,β-unsaturated C=O (1715 cm<sup>−</sup>1). The UV spectrum exhibited the absorption band at 285 nm. The CD spectrum showed a positive Cotten effect at 250 nm and depict the negative Cotten effect at 225, 290, and 335 nm.


**Table 2.** 13C-NMR data for compounds **2**–**5** (*δ* in ppm, 150 MHz for 13C NMR in CDCl3).

The 1H-NMR spectrum (Table 1) displayed an oxononyl group at [*δ*H 0.90 (3H, t, *J* = 7.2 Hz, H-17), 1.20–1.35 (8H, m, H-13~H-16), 1.55–1.60 (2H, m, H-12), 2.44–2.46 (2H, m, H-11)], one methyl group [*δ*H 1.24 (3H, s, H-8)], signals of the *α*-methylene protons of one ketone [*δ*H 2.49–2.52 (1H, m, 1H of CH2-9), 3.03 (1H, dd, J = 18.0, 3.2 Hz, 1H of CH2-9), and 2.44–2.47 (2H, m, CH2-11)], one oxymethylene [*δ*H 4.89 (1H, dd, J = 18.0, 4.5 Hz, 1H of CH2-1), 5.05 (1H, dd, J = 18.0, 3.3 Hz, 1H of CH2-1)], one non-equivalent methylene proton at [*δ*H 2.10–2.12 (1H, m, 1 H of CH2-4), 2.95 (1H, ddd, J = 19.0, 4.5, 3.3 Hz, 1 H of CH2-4)], and one methine [*δ*H 2.80–2.82 (1H, m, H-5)]. Eighteen C-atom signals (Table 2) corresponding to six quaternary C-atoms, one CH, nine CH2, and two CH3 groups, were observed in the 13C-NMR and DEPT spectra. Since four out of six unsaturation equivalents were accounted for via the above-mentioned 13C-NMR data, **1** was inferred to have two rings (one as a six-membered and another as a five-membered ring). In addition, two rings

were further determined as a cyclohex-2-enone skeleton combined with one *γ*-lactone ring via the detail HMBC and COSY analyses.

The 1H- and 13C-NMR spectra of **3** (Table 1) were similar to those of monaphilone A [31]; the major difference was the presence of signals for an *γ*-lactone attached to C-3a and 7a in **3**, instead of signals for a 4*H*-pyran group in monaphilone A [31]. HMBC correlations between the H-atom signals at δH 4.89/5.05 (CH2(1)) and the C-atom signals at δC 198.3 (C-7) once indicated that the *γ*-lactone was located at C-3a and 7a of the cyclohex-2- enone ring. The relative configuration of **3** was derived using a NOESY spectrum (Figure 4) and a comparison with similar compounds [31], the relative configuration of which was based on a NOESY analyses. No NOEs for H-5/Meax-8 and Hax-4 indicated that Me-8 and Hax-4 were on the same side of the molecular plane, tentatively assumed as *α*-orientation.

The H-5 was occupied at axial *β*-oriented, which was further confirmed by the NOE H-5/Heq-4. The relative configuration at C-5 and 6 were determined to be (5*S\**,6*S\**) based on the correlation between the [α]D value and the known configuration at C-5/C-6 for monaphilone A type derivatives [31]. In order to determine the absolute configuration of **3**, the theoretical electronic circular dichroism (ECD) spectra of 4 possible stereoisomers were calculated using a time-dependent density-functional theory (TDDFT) calculation, and the calculated ECD curve of (5*S*,6*S*) revealed good agreemen<sup>t</sup> with the experimental spectrum of **2** (Figure 5). Therefore, the absolute configuration of **3** was assigned as (5*S*,6*S*) and named as monascuspurin C.

Compound **4** was obtained as colorless oil. The molecular formula was determined as C18H16N2O3 on the basis of the [M+Na]+ peak at *m*/*z* 331.10588 (calcd. 331.10586 for C18H16NaN2O3) in its HR-ESI-MS. The UV absorptions (λmax 220, 252, and 312 nm) confirmed the presence of a pyridine moiety [38]. IR absorption bands were assigned to amide (3400 cm<sup>−</sup>1), multiple carbonyls C=O (1712 and 1656 cm<sup>−</sup>1), and the pyridine ring (1589, 1535, and 1458 cm<sup>−</sup>1) functional groups. Twelve indices of hydrogen deficiency (IHD) were determined from the molecular formula, 13C-NMR (Table 2), and DEPT spectra. The CD spectrum showed positive Cotten effect at 240, 262, 319, and 333 nm, and negative Cotten effect at 365 nm.

Interpretation of the 1H-NMR spectrum of **4** (Table 1) exhibited the signals of one 2,4,5-trisubstituted pyridine ring [δH 9.03 (1H, *s*, H-1), 7.59 (*s*, H-4)], one trans-propenyl unit [δH 2.05 (3H, dd, *J* = 6.8, 1.8 Hz, H-11), 6.65 (1H dq, *J* = 15.6, 1.8 Hz, H-9), 7.13 (1H, dq, *J* = 15.6, 6.8 Hz, H-10)], one Me group [δH 1.85 (3H *s*, Me-12)], as well as one ABC system aromatic ring [*δ*H 7.70 (1H, t, *J* = 8.0 Hz, H-14), 7.90 (1H, dd, *J* = 8.0, 0.8 Hz, H-15), and 8.04 (1H, dd, *J* = 8.0, 0.8 Hz, H-13)]. The 13C and DEPT NMR spectra indicated (Table 2) that compound **4** is a pyridine derivative with signals for 18 C-atoms, which were classified as nine quaternary C-atoms comprising six olefinic C-atoms, one amide C-atom (δC 168.5 (C-17), one ketone groups (δC 192.8 (C-8)), one oxygenated quaternary carbon [*δ*C 84.9 (C-7)], one Me group (δC 27.3 (C-12), and one trans-propenyl unit [*δ*C 131.8 (C-9), 137.2 (C-10), 18.8 (C-11)].

The 1H- and 13C-NMR spectra of **4** (Table 1) were similar to those of monascopyridine C and D [38]; the major difference was the presence of signals for ABC system aromatic ring attached between C-5 and C-6 in **4**, instead of signals for an alkyl groups in monascopyridine C and D. HMBC correlations between the H-atom signals at δH 8.04 (H-13) and the C-atom signals at δC 151.0 (C-6), and 143.5 (C-4a) and δH 7.70 (H-14) and the C-atom signals at δC 126.7 (C-5), indicated that the ABC system aromatic ring was bounded at C-5 and 6. The other key correlations of HMBC were illustrated in Figure 3.

Furthermore, the attachment of the amide to C-17, the methyl group to C-7, and the trans-propenyl group located at C-3, were disclosed according to the HMBC cross-peaks of *δ*H 7.90 (H-15)/*δ*C 168.5 (C-17), *δ*H 1.85 (H-12) to C-6/C-7/C-8, and *δ*H 7.59 (H-4) to C-9.

On the basis of the evidence, the entire structure of **4** was confirmed and named monascuspurin D. The relative configuration at C-7 was determined to be 7*R* based on the correlation between the [α]26D:+ 15.9 (*c* 0.01, CHCl3) and the known configuration at C-7 for (*R*)-2-hydroxy-2-methylcyclohexanone derivatives [39]. The absolute configuration of

**4** was defined via a comparison of the experimental and calculated ECD data (Figure 5). Therefore, the absolute configuration of **4** was determined as 7 *R*.

Compound **5** was obtained as an optically active oil. [ α]26D: +56.7 (*c* 0.01, CHCl3). The molecular formula was determined as C23 H30 O5 on the basis of the [ *M*+H]+ peak at *m*/*z* 409.19912 (calcd. 409.19909 for C23 H30NaO5) in its HR-ESI-MS. The UV absorptions (λmax 235 and 285 nm) confirmed the presence of a benzenoid nucleus. The bands at 3400, 1780, 1695, and 1615/1577 cm<sup>−</sup><sup>1</sup> in the IR spectrum revealed the presence of a hydroxyl group, γ-lactone, and aromatic ring, respectively. Nine indices of hydrogen deficiency (IHD) were determined from the molecular formula, 13C-NMR (Table 1), and DEPT spectra. The 1H-NMR and 13C-NMR spectra (Table 2) of **5** were similar to those of ankaflavin [9], except that a 2-ethylphenol group of **5** replaced a (*E*)-6-(prop-1-en-1-yl)-2 *H*-pyran group at C-4a–C-8a of ankaflavin. Further confirmation using the HMBC correlations (Figure 3) of H-1/C-3, 4a, 2a, H-4/C-2, 3, 5, 8a, and H-2b/C-2, 2a, verified the junction of the 2- ethylphenol unit at C-4a and C-8a. The correlations of H-1/H-2a and H-4/CH2-5 were also observed in the NOESY experiment (Figure. 4) and further supported the position of each aromatic substitution. The 1H- and 13C-NMR, COSY (Figure 3), NOESY (Figure 4), HSQC, and HMBC (Figure 3) experiments confirmed the structure as 7-ethyl-3-hexanoyl-6- hydroxy-9a-methyl-3a,9adihydronaphtho[2,3-*b*]furan-2,9(3 *H*,4 *H*)-dione, and designated monascuspurin E.

The dextrorotatory optical activity of **5**, gathered from the NOESY spectrum (Figure 4), indicates that Hax-5 is correlated to H-12 and H-13, and H-6 has no NOE contacts with Hax-5, H-12, and H-13. It can be concluded that Hax-5, H-12, and H-13 are on the same side, and H-6, H-12, and H-13 are on the opposite side, and once again it indicated that the relative configuration of **5** is (6 *R*,7 *R*,13*S*), as in the case of ankaflavin [9]. In order to determine the absolute configuration of **5**, the theoretical ECD spectra of all possible stereoisomers were calculated using the TDDFT calculation, and the calculated ECD curve of the isomer (6 *R*,7 *R*,13*S*) revealed a good agreemen<sup>t</sup> with the experimental one (Figure 5). Therefore, the absolute configuration of **5** was assigned as (6 *R*,7 *R*,13*S*)-form and named as monascuspurin E.
