*Biological Studies*

Culture broth from *M*. *purpureus* wmd2424 was tested for antifungal activity against the following fungi: *Aspergillus niger* (BCRC-31512), *Penicillium italicum* (BCRC-30567), *Candida albicans* (BCRC-21538), and *Saccharomyces cerevisiae* (BCRC-20822). The antifungal data are shown in Table 3 and the clinically used antifungal drug ketoconazole was employed as a positive control.


**Table 3.** Antifungal activity of five sufficient compounds isolated from the culture broth of *A*. *punica* 04107M (diameter of the zone of growth-inhibitory fungicidal zone is given in mm, including the diameter of the disk, which is 8 mm).

Inhibitory zone diameter (mm); ± inhibitory zone; positive control (STD): ketoconazole. Each value represents the mean ± SD.

Our results indicate that compounds **3**–**5** have moderate antifungal activity compared to ketoconazole, with **1** being weaker. From the results of the antifungal tests, the following conclusions can be drawn about these isolates: (a) within the novel strain, the 2,3-dimethylcyclohex-2-en-1-one (compound **2**) and γ-lactone (compound **3**) showed antifungal activities with inhibition zones of 29, 28, 27, and 30 mm, and 29, 29, 36, and 21 mm against *Aspergillus niger* (BCRC-31512), *Penicillium italicum* (BCRC-30567), *Candida albicans* (BCRC-21538), and *Saccharomyces cerevisiae* (BCRC-20822), respectively. (b) The xanthonoids (compound **1**) exhibited weak antifungal activities against the *Aspergillus niger* (BCRC-31512), *Penicillium italicum* (BCRC-30567), *Candida albicans* (BCRC-21538), and *Saccharomyces cerevisiae* (BCRC-20822) strains. (c) The other type of isoquinoline, Monascuspurin D (compound **4**), indicated effective inhibition zones of 32, 28, 31, and 28 mm against *Aspergillus niger* (BCRC-31512), *Penicillium italicum* (BCRC-30567), *Candida albicans* (BCRC-21538), and *Saccharomyces cerevisiae* (BCRC-20822), respectively. (d) The azaphilone compound **5** exhibited moderate antifungal activities against the *Aspergillus niger* (BCRC-31512) and *Candida albicans* (BCRC-21538) strains (Table 3).

The inhibitory activity of compounds **3**–**5** against *A*. *niger*, *P*. *italicum*, *C*. *albicans*, and *S*. *cerevisiae* was further tested using the method described in the experimental section (Table 4). Compound **2** has inhibitory activity against *S*. *cerevisiae*, with MIC values of 43.45 μg/mL. Compound **3** has inhibitory activity against *C*. *albicans*, with an MIC value of 32.87 μg/mL. Compound **4** was found to have moderate inhibitory activity against the *A*. *niger*, and *C*. *albican* strains with MIC values ranging from 29.65 and to 58.43 μg/mL. They were less biologically active than the reference compound, ketoconazole, which had MIC values of 4.10, 5.34, 10.88, and 3.57 μg/mL against *A*. *niger*, *P*. *italia*, *C*. *albicans*, and *S*. *cerevisiae*, respectively. In this bioassay, no antifungal activity (MIC > 100) was observed for compound **5** at concentrations below 100 μg/mL.


**Table 4.** MIC values of compounds **2**–**5** in μg/mL against four fungi strains.

a Each value represents the mean ± SD.

## **4. Materials and Methods**

## *4.1. General Experimental Procedures*

For the TLC, we used silica gel 60 F254-precoated plates (Merck); for column chromatography (CC), we used silica gel 60 (70–230 or 230–400 mesh, Merck) and Spherical C18 100A Reversed Phase Silica Gel (RP-18) (particle size: 20–40 μm) (Silicycle). For the HPLC analysis, we used a spherical C18 column (250 mm × 10 mm, 5 μm) (Waters) and LDC-Analytical-III apparatus. For the UV spectra, we used a Jasco UV-240 spectrophotometer, with λmax (log ε) in nm. For optical rotation, we used a Jasco DIP-370 polarimeter, in CHCl3. For the IR spectra, we used a Perkin-Elmer-2000 FT-IR spectrophotometer, with ν in cm<sup>−</sup>1. For the 1H-, 13C-, and 2D-NMR spectra, we used Varian-VNMRS-600 and Varian-Unity-Plus-400 spectrometers; δ in ppm relative to Me4Si, J in Hz. For the ESI and HRESIMS, we used a Bruker APEX-II mass spectrometer, in *<sup>m</sup>*/*<sup>z</sup>*.

#### *4.2. Microorganism, Cultivation, and Preparation of the Strain*

This WMD2424 strain was isolated from the mangrove wetland collected in Chiayi County, Taiwan, using HV agar and cultured at 28 ◦C for 3 weeks. A voucher specimen was immersed in 15% glycerol–water solution at −80 ◦C and deposited at the Bioresource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI). Analysis of the ITS rDNA using the BLAST database screening provided a 99.9% match with *Monascus purpureus*, whose sequence has been submitted to GenBank.

To each 500-mL flask containing 150 mL of liquid RGY medium (3% rice starch, 7% glycerol, 1.5% polypeptone, 3% soybean powder, 0.2% MgSO4, and 0.2% NaNO3) were added 10 mL of fungal inocula and incubated at 25◦ for 2 weeks on a rotary shaker at the speed of 100 circles/min without illumination. A total of 14.0 L of fungal fermented broth was harvested and then filtered to remove fungal mycelium.

#### *4.3. Isolation and Characterization of Secondary Metabolites*

Liquid fermentate of M. purpureus (14.0 L) was extracted with BuOH to yield a BuOH extract (16.9 g), which was partitioned in EtOAc–H2O (1:1; 2 L × 3) to produce an EtOAc-soluble fraction (8.9 g) and an H2O-soluble fraction. The active EtOAc-soluble fraction (8.9 g) was subjected to silica gel column chromatography (CC) using CH2Cl2– MeOH (100:1) as the primary eluent, gradually increasing the eluent polarity with MeOH to produce 10 fractions (Frs. 1–Frs. 10). Fr. 2 was subjected to RP-18 silica gel CC using H2O–acetone (2:1) as the eluent to produce 5 fractions (Frs. 2-1–2-5), Fr. 2-5 (432 mg) was subjected to silica gel CC using CH2Cl2–EtOAc (3:1) as the eluent to produce 4 fractions (Frs. 2-5-1–Frs. 2-5-4), Fr. 2-5-3 was further subjected to silica gel CC using CH2Cl2–EtOAc (2:1) as the eluent to give **1** (1.2 mg) and **2** (3.0 mg). Fr. 3 was subjected to RP-18 silica gel CC using H2O–acetone (1:1) as the eluent to obtain 8 fractions (Frs. 3-1–3-8), Fr.3-8 was further subjected to silica gel CC using CH2Cl2–acetone (1:1) as the eluent to give 11 fractions (Frs. 3-8-1–Frs. 3-8-11), Fr. 3-8-10 was purified with prep. TLC (CH2Cl2/EtOAc 6:1) to obtain **4** (1.8 mg). Fr. 5 (1132 mg) was subjected to RP-18 silica gel CC using H2O–acetone (1:1) as the eluent to give **3** (1.2 mg) and **5** (3.3 mg).

Monascuspurin A (compound **1**): Oil. [ α]26D: +34.2 (*c* 0.01, CHCl3). UV (MeOH) λmax (log *ε*) 268 (4.11), 360 (3.89) nm. IR *ν*max (neat) 3406 (OH), 1710, 1680 (C=O), 1615, 1450, 1406 (aromatic ring) cm<sup>−</sup>1. CD (MeOH) λext 215 ( Δε –10.9), 232 ( Δε –4.2), 251 ( Δε –7.9), 273 ( Δε +5.2), 296 ( Δε –2.3), 342 ( Δε +7.3), 400 ( Δε –6.7) nm. ESI-MS *m*/*z* 439 [M+Na]+. 1H NMR (600 MHz, CDCl3): see Table 1. HRESI-MS *m*/*z*: 439.13640 [M+Na]+ (calculated for C22 H24 O8Na, 439.13636).

Monascuspurin B (compound **2**): Oil. [ α]26D: + 54.2 (*c* 0.01, CHCl3). UV (MeOH): 242 (3.98) nm. IR (neat): 3410 (OH), 1715 (C=O), 1675 (C=O) cm<sup>−</sup>1. CD (MeOH) λext 225 ( Δε –1.9), 241 ( Δε +0.9), 282 ( Δε –0.3) nm. 1H NMR (600 MHz, CDCl3): see Table 1; 13C NMR (150 MHz, CDCl3): see Table 2. ESI-MS *m*/*z* 305 [M+Na]+. HRESI-MS *m*/*z*: 305.13598 [M+Na]+, (calculated for C15 H22 O5Na, 305.13592).

Monascuspurin C (compound **3**): Oil. [ α]26D: +74.2 (*c* 0.01, CHCl3). UV (MeOH): 285 (3.26) nm. IR (Neat): 3410 (OH), 1770, 1715 (C=O) cm<sup>−</sup>1. CD (MeOH) λext (Δε): 225 ( Δε –1.89), 250 ( Δε +1.79), 290 ( Δε –1.08), 335 ( Δε –1.69) nm. 1H-NMR (600 MHz, CDCl3): see Table 1; 13C-NMR (150 MHz, CDCl3): see Table 2. ESI-MS *m*/*z* 345 [M+Na]+. HRESI-MS *m*/*z*: 345.16780 [M+Na]+, C18 H26 O5 (calculated for C15 H13O, 345.16779.

Monascuspurin D (compound **4**): oil; [ α]26D: + 15.9 (*c* 0.01, CHCl3); UV (MeOH): 220 (4.01), 252 (4.22), 312 (3.89) nm; IR (neat): 3400 (OH), 1712, 1656 (C=O), 1589, 1535, 1458 (pyridine) cm<sup>−</sup>1; CD (MeOH) λext (Δε) 240 ( Δε +13.19), 262 ( Δε +5.13), 319 ( Δε +1.98), 333 ( Δε +2.01), 365 ( Δε −2.81) nm. 1H-NMR (600 MHz, CDCl3): see Table 1; 13CNMR (150 MHz, CDCl3): see Table 2; ESI-MS *m*/*z* 331 [M+Na]+; HRESIMS *m*/*z* 331.10588 [M+Na]+ (calculated for C18 H16NO4, 331.10586).

Monascuspurin E (compound **5**): oil; [ α]26D: +56.7 (*c* 0.01, CHCl3); UV (MeOH): 235 (4.22), 285 (3.89) nm; IR (neat): 3400 (OH), 1780, 1695 (C=O), 1615, 1577 (aromatic ring) cm<sup>−</sup>1; 1H-NMR (600 MHz, CDCl3): see Table 1; 13C-NMR (150 MHz, CDCl3): see Table 2; ESI-MS *m*/*z* 409 [M+Na]+; HRESIMS *m*/*z* 409.19912 [M+Na]+ (calculated for C23 H30 O5Na, 409.19909).
