**1. Introduction**

Fungi have attracted much attention of chemists and biologists due to their potential in producing bioactive secondary metabolites with diverse chemical skeletons [1,2]. Verrucosidins produced by *Penicillium* strains belong to a family of highly reducing fungal polyketides that are characterized with 2*H*-pyran-2-one and dicyclic fused 3,6 dioxabicyclo[3.1.0]hexane moieties interlinked by a polyene chain [3–6]. They have been reported to display important bioactivities, such as antitumor [7,8], antivirus [9], antibacterial [3,10], and neurological activities [11]. In order to explore in depth this kind of compounds with unique chemical structure and diverse biological activities, we explored *Penicillium* strains collected in our lab searching for verrucosidin analogues.

Molecular networking analyses include acquisition and similarity comparison of mass spectral fragment data, cluster grouping and visualization [12,13]. More recently, the MS/MS-based molecular networking has been demonstrated to be powerful in dereplicating known natural products from a targeted extract and searching for new analogues with

**Citation:** Han, J.; Chen, B.; Zhang, R.; Zhang, J.; Dai, H.; Wang, T.; Sun, J.; Zhu, G.; Li, W.; Li, E.; et al. Exploring Verrucosidin Derivatives with Glucose-Uptake-Stimulatory Activity from *Penicillium cellarum* Using MS/MS-Based Molecular Networking. *J. Fungi* **2022**, *8*, 143. https://doi.org/10.3390/jof8020143

Academic Editors: Tao Feng and Frank Surup

Received: 3 January 2022 Accepted: 29 January 2022 Published: 30 January 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 specific skeleton.Examples included thermoactinoamide A with moderate antiproliferative activity from *Thermoactinomyces vulgaris* DSM 43016 [14], suffranidines A-C with significant neuritogenic activity from *Flueggea suffruticosa* [15], and trilliumoside D with strong cytotoxicity against MOLT-4 cell lines from *Trillium tschonoskii* maxim [16]. To explore new reducing fungal polyketides from fungi, we applied the LC-MS/MS-based molecular networking for new verrucosidins from *Penicillium* strains using deoxyverrucosidin that was deposited in our compound library as the probing agent. with the specific skeleton.Examples included thermoactinoamide A with moderate antiproliferative activity from *Thermoactinomyces vulgaris* DSM 43016 [14], suffranidines A-C with significant neuritogenic activity from *Flueggea suffruticosa* [15], and trilliumoside D with strong cytotoxicity against MOLT-4 cell lines from *Trillium tschonoskii* maxim [16]. To explore new reducing fungal polyketides from fungi, we applied the LC-MS/MS-based molecular networking for new verrucosidins from *Penicillium* strains using deoxyverrucosidin that was deposited in our compound library as the probing agent.

*J. Fungi* **2022**, *8*, x FOR PEER REVIEW 2 of 17

The EtOAc extracts of *Penicillium* strains fermented on solid culture were first analyzed by high performance liquid chromatography (HPLC) with UV diode array detection (DAD) to find fungi potentially producing verrucosidin derivatives (Figure S1). In this work, the target isolation was further conducted on the selected fungus *P. cellarum* YM1 under the guidance of LC-MS/MS-based molecular networking (Figure S2). As a result, seven new verrucosidins, penicicellarusins A-G (**3–9**), as well as five known verrucosidins (compounds **1**, **2** and **10**–**12**) were identified from the culture of *P. cellarum* YM1 (Figure 1). The isolated compounds were evaluated for anti-bacterial effect, cytotoxicity, and glucose uptake-stimulating activities. This work described the details of the isolation, structure elucidation, and biological activities of the isolated secondary metabolites from *P. cellarum* YM1. The EtOAc extracts of *Penicillium* strains fermented on solid culture were first analyzed by high performance liquid chromatography (HPLC) with UV diode array detection (DAD) to find fungi potentially producing verrucosidin derivatives (Figure S1). In this work, the target isolation was further conducted on the selected fungus *P. cellarum* YM1 under the guidance of LC-MS/MS-based molecular networking (Figure S2). As a result, seven new verrucosidins, penicicellarusins A-G (**3–9**), as well as five known verrucosidins (compounds **1**, **2** and **10**–**12**) were identified from the culture of *P. cellarum* YM1 (Figure 1). The isolated compounds were evaluated for anti-bacterial effect, cytotoxicity, and glucose uptake-stimulating activities. This work described the details of the isolation, structure elucidation, and biological activities of the isolated secondary metabolites from *P. cellarum* YM1.

**Figure 1.** Structures of compounds **1**–**12**. **Figure 1.** Structures of compounds **1**–**12**.

#### **2. Materials and Methods 2. Materials and Methods**

#### *2.1. General*

*2.1. General*  NMR spectral data were obtained with an AVANCE-500 spectrometer (Bruker, Bremen, Germany) (CDCl3*, δ*H 7.26/*δ*C 77.16, and CD3OD, *δ*H 3.30/*δ*C 49.9). High-resolution electrospray ionization mass spectrometry (HRESIMS) data and LC-MS/MS measurements were procured on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a LC-30AD series UPLC (Shimadzu, Kyoto, Japan) equipped with an ACQUITY BEH C18 column (Waters, MA, USA; 2.1 × 100 mm, 1.7 µm). UV data, optical rotation, and IR data, were recorded on Genesys-10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), MCP 200 Automatic Polarimeter (Anton Paar, Graz, Austria) and IS5 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) respectively. The CD spectra were measured by a J-815 spectropolarimeter (JASCO, Tsukuba, Japan). Silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200–300 mesh), Sephadex LH-20 (GE NMR spectral data were obtained with an AVANCE-500 spectrometer (Bruker, Bremen, Germany) (CDCl3*, δ*<sup>H</sup> 7.26/*δ*<sup>C</sup> 77.16, and CD3OD, *δ*<sup>H</sup> 3.30/*δ*<sup>C</sup> 49.9). High-resolution electrospray ionization mass spectrometry (HRESIMS) data and LC-MS/MS measurements were procured on a Q Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) coupled with a LC-30AD series UPLC (Shimadzu, Kyoto, Japan) equipped with an ACQUITY BEH C18 column (Waters, MA, USA; 2.1 × 100 mm, 1.7 µm). UV data, optical rotation, and IR data, were recorded on Genesys-10S UV-Vis spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), MCP 200 Automatic Polarimeter (Anton Paar, Graz, Austria) and IS5 FT-IR spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) respectively. The CD spectra were measured by a J-815 spectropolarimeter (JASCO, Tsukuba, Japan). Silica gel (Qingdao Haiyang Chemical Co., Ltd., Qingdao, China, 200–300 mesh), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden), and ODS (50 µm, YMC Co., Ltd., Kyoto, Japan) were used for column chromatography. Semi-preparative

HPLC was performed on an Agilent 1200 HPLC system equipped with a DAD UV−vis spectrometric detector (Agilent Technologies Inc., CA, USA) using a reversed-phase Eclipse XDB-C8 column (5 µm, 9.4 × 250 mm, Agilent) with a flow rate of 2.0 mL/min and a CHIRALPAK IC column (5 µm, 4.6 × 250 mm, Daicel, Osaka, Japan) with a flow rate of 0.8 mL/min. For gas chromatography-mass spectrometry (GC-MS) a Shimadzu GCMS-QP2010 Ultra system (Shimadzu, Kyoto, Japan) was used.

### *2.2. Fungal Material*

The strain *Penicillium* sp. YM1 used in this work was isolated from mildewed corn, collected in China, in September 2017. The sequences of RPB2 (MT898427), Ben A (MT898428), and CaM (MT898429) of our fungus were deposited in GenBank and employed for phylogenetic analysis. The fungus is similar to *P. cellarum* in forming hyaline, roughened stipes with bearing terminal terverticillate penicillii; and producing typically two rami per stipe, which are usually hyaline, roughened, appressed or only narrowly divergent; and having four to five metulae typically per ramus, which are usually hyaline, roughened, appressed or only narrowly divergent as well; and producing typically six to eight per metula phialides, which are usually hyaline, smooth, ampulliform, slender; and with pale green conidia that were typically smooth, globose to sometimes subglobose [17,18]. The phylogenetic analyses based on a combined dataset of RPB2, Ben A, and CaM was conducted by using PhyML v.3.0, with 1000 bootstrap replicates presented that our taxon grouped with the other taxa of *P. cellarum* with strongly maximum likelihood bootstrap proportions value (Figure S3). In consideration of the morphological features and phylogeny, this fungus was identified as *P. cellarum* YM1.

### *2.3. Fermentation and Extraction*

*P. cellarum* was cultured on slant of PDA at 28 ◦C for 10 days. To prepare inoculum, the spores of the strain on the plate were collected with 0.01% sterile solution of Tween 80 (BTL, Warsaw, Poland) and adjusted to 1 <sup>×</sup> <sup>10</sup><sup>6</sup> CFU/mL. A large-scale fermentation was done in 40 × 500 mL Fernbach culture flasks containing 80 g of rice in 110 mL of distilled water (each with 0.5 mL of spore suspension) and incubated at 28 ◦C for 3 weeks. The fermented rice substrates were extracted with EtOAc (3 × 4 L) with the aid of ultrasonication, and the organic solvent was filtered and evaporated to dryness under vacuum to afford the crude extract (33.7 g).

#### *2.4. LC-MS/MS and Molecular Metworking Analysis*

LC-MS/MS (MS/MS scan 100−1500 Da) was performed with a Waters ACQUITY BEH C<sup>18</sup> column (2.1 × 100 mm, 1.7 µm particles) eluted by MeCN−H2O (0.005% TFA) (0.01−8 min 5−80% 8−12 min 80−99%, 12−15 min 99%) in a gradient manner. All the MS/MS data files were converted to ".mzML" format files using MSConver software and uploaded on the GNPS Web platform (http://gnps.ucsd.edu (accessed on 6 October 2021)) for MN analysis using Classic mode. For the network creation, a parent mass tolerance of 0.02 Da and a fragment ion tolerance of 0.05 Da were applied. The generated molecular network was visualized in Cytoscape 3.8.2 (www.cytoscape.org (accessed on 6 October 2021)) and guided the isolation of **1–12**. The MS/MS molecular network can be browsed and downloaded on the GNPS Web site with the following link: https: //gnps.ucsd.edu/ProteoSAFe/status.jsp?task=8716192add914a1fb3bd8f469f7d2d81 (accessed on 6 October 2021).

#### *2.5. Isolation and Characterization Data*

The EtOAc fraction was subjected to a silica gel column chromatography (CC) eluting witH-N-hexane/ether-ethyl acetate (*v*/*v*, 100:0, 100:1, 100:2, 100:4, 100:10) and dichloromethane/ methanol (*v*/*v*, 100:0, 100:1, 100:2, 100:4, 100:8, 100:12, 100:20, 0:100) to give 13 fractions (PC.1–PC.13). Fractions PC.6, PC.8, and PC.12 containing secondary metabolites with similar UV spectra were selected for further purification.

Fraction PC.6 (1.5 g) was further separated on silica gel column by a gradient elution with methanol-dichloromethane to give 25 fractions (PC.6-1–PC.6-25). PC.6–8 (60 mg) was purified finally by RP-HPLC with acetonitrile-water (63:37) to give **1** (13.5 mg, *t*<sup>R</sup> 42.3 min) and **2** (6.6 mg, *t*<sup>R</sup> 31.5 min). Compound **12** (44.5 mg) was obtained from subfractions PC.6–11 (65 mg) by Sephadex LH-20 chromatography eluting with methanol. Compounds **7** (8.6 mg, *t*<sup>R</sup> 31.1 min), **8** (9.5 mg, *t*<sup>R</sup> 40.5 min), and **9** (1.6 mg, *t*<sup>R</sup> 42.5 min) were obtained from PC.6–20 (75 mg) by RP-HPLC using 86% acetonitrile in acidic water (0.005% TFA).

Fraction PC.8 (3.5 g) was separated on an ODS column using a gradient elution with methanol (35%, 55%, 70%, and 100%) in acidic water (0.005%TFA) to afford 15 subfractions (PC.8-1–PC.8–15). Compounds **5** (14.2 mg) and **6** (12.8 mg) were obtained from subfractions PC.8–9 and PC.8–10 by Sephadex LH-20 chromatography eluting with methanol, respectively.

Fraction PC.12 (4.3 g) eluted with CH2Cl2-acetone (*v*/*v* 20:1) was first separated by ODS using a gradient of increasing methanol (35%, 55%, 70%, and 100%) in water to afford 21 subfractions (PC.12-1–PC.12-21). Compounds **3** (2.5 mg, *t*<sup>R</sup> 62.5 min) and **4** (3.5 mg, *t*<sup>R</sup> 30.5 min) were yielded from PC.12-15 (35 mg) by RP-HPLC using 23% acetonitrile in acidic water (0.005% TFA). Subfractions PC.12-6 (60 mg) was purified by RP-HPLC using 27% acetonitrile in water to afford a mixture of **10** and **11** (35.0 mg, *t*<sup>R</sup> 28.3 min). Enantioseparation of the mixture was carried out on CHIRALPAK IC using isopropanol/*n*-hexane (15:85) as mobile phase to afford **10** (10.0 mg, *t*<sup>R</sup> 21.5 min) and **11** (11.5 mg, *t*<sup>R</sup> 23.5 min).

*Penicicellarusin A (3).* light yellow oil, [α] 25 *D* + 55.6 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 240 (3.11), 302 (1.24) nm; IR (neat) *v*max 3429, 2972, 2931, 1703, 1574, 1450, 1378, 1210, 1042, 811 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 455.2041 [M+Na]<sup>+</sup> (calcd. for C24H32O7Na, 455.2040). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 1.


**Table 1.** <sup>1</sup>H and <sup>13</sup>C-NMR Data for compounds **3–4** in CD3OD.

*Penicicellarusin B (4)*. light yellow oil, [α] 25 *D* + 51.0 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 241 (3.35), 297 (1.50) nm; IR (neat) *v*max 3420, 2971, 2931, 1700, 1572, 1450, 1377, 1209, 1054, 812 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 441.1890 [M+Na]<sup>+</sup> (calcd. for C23H30O7Na, 441.1884). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 1.

*Penicicellarusin C (5).* light yellow oil, [α] 25 *D* + 45.0 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 236 (3.56), 303 (2.57) nm; IR (neat) *v*max 3410, 2974, 2930, 1685, 1560, 1450, 1378, 1224, 1089, 1043, 812 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 443.2047 [M+Na]<sup>+</sup> (calcd. for C23H32O7Na, 443.2040). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 2.


**Table 2.** <sup>1</sup>H and <sup>13</sup>C-NMR Data for compounds **5–6**.

<sup>a</sup> NMR data were measured in CD3Cl; <sup>b</sup> NMR data were measured in CD3OD; <sup>c</sup> NMR data reported in literature.

*Penicicellarusin D (6).* light yellow oil, [α] 25 *D* + 32.9 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 235 (3.56), 299 (2.54) nm; IR (neat) *v*max 3412, 2974, 2932, 1683, 1559, 1450, 1378, 1225, 1089, 1043, 812 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 457.2190 [M+Na]<sup>+</sup> (calcd. for C24H34O7Na, 457.2197). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 2.

*Penicicellarusin E (7).* light yellow oil, [α] 25 *D* +7 8.5 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 235 (3.65), 299 (2.58) nm; IR (neat) *v*max 2975, 2933, 1695, 1555, 1450, 1378, 1224, 1086, 1043, 812 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 695.4490 [M+Na]<sup>+</sup> (calcd. for C40H64O8Na, 695.4493). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 3.




**Table 3.** *Cont.*

"m" means multiplet or overlapped with other signals.

*Penicicellarusin F (8).* light yellow oil, [α] 25 *D* + 81.0 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 236 (3.38), 290 (2.54) nm; IR (neat) *v*max 2972, 2932, 1689, 1557, 1450, 1378, 1225, 1088, 1045, 811 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 721.4658 [M+Na]<sup>+</sup> (calcd. for C42H66O8Na, 721.4650). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 3.

*Penicicellarusin G (9).* light yellow oil, [α] 25 *D* + 76.5 (c 0.1 MeOH); UV (MeOH) *λ*max (log *ε*) 236 (3.38), 290 (2.54) nm; IR (neat) *v*max 2974, 2932, 1720, 1570, 1455, 1378, 1209, 1054, 812 cm−<sup>1</sup> ; Positive HRESIMS: *m/z* 719.4500 [M+Na]<sup>+</sup> (calcd. for C42H64O8Na, 719.4493). <sup>1</sup>H-NMR and <sup>13</sup>C-NMR, see Table 3.
