*2.2. Structural Elucidation*

Compound **1** was obtained as a white powder. Its molecular formula was determined to be C15H17O6 from the HRESIMS (*m/z* 293.1024 [M − H]<sup>−</sup> calcd for C15H17O6, 293.1031), indicating 7 degrees of unsaturation. The IR spectrum of **1** at 3696, 1646, and 1585 cm−<sup>1</sup> suggested the presence of hydroxyl, carbonyl, and aromatic ring groups. The 13C NMR data in combination with HMQC spectra (Table 1) displayed one methyl carbon at *δ*<sup>C</sup> 20.5, one methoxyl carbon at *δ*<sup>C</sup> 55.2, three methylene carbons at *δ*<sup>C</sup> 39.1, 38.9, and 38.5, four methine carbons at *δ*<sup>C</sup> 106.8, 99.3, 63.4, 63.0, and six nonprotonated carbons at *δ*<sup>C</sup> 169.2, 166.7, 164.4, 140.2, 104.6, 101.0. Analysis of the 1H NMR spectrum of **1** revealed two aromatic protons at *δ*<sup>H</sup> 6.35 (d, *J* = 2.1), 6.34 (s), indicating the presence of a tetrasubstituted phenyl. Two oxygenated methine protons signal at *δ*<sup>H</sup> 4.39 (m) and 4.17 (m), three aliphatic methylenes at *δ*<sup>H</sup> 1.53 (m), 1.78 (m), 1.87 (dd, *J* = 4.08, 15 Hz), 2.21 (dt, *J* = 15, 2.2 Hz), 2.95 (d, *J* = 16.5 Hz), and 3.17 (d, *J* = 16.5 Hz), one methoxyl at *δ*<sup>H</sup> 3.82 (s), and one methyl at *δ*<sup>H</sup> 1.07 (d, *J* = 6.3 Hz) were also recorded in this spectrum. The above spectroscopic features indicated that **1** belonged to the isocoumarin class. Further analysis of HMBC spectrum (Figure 2), the correlations from H-7 to C-2 (*δ*<sup>C</sup> 101.0)/C-5 (*δ*<sup>C</sup> 106.8)/C-6 (*δ*<sup>C</sup> 166.7)/C-8 (*δ*<sup>C</sup> 164.4), from H-5 to C-2/C-6, from H-4 to C-2/C-3 (*δ*<sup>C</sup> 140.2)/C-6' (*δ*<sup>C</sup> 104.6), from H-9 to C-6 suggested that **1** was an isocoumarin derivative with a hydroxyl group at C-8 and a methoxy group at C-6. The 1H-1H COSY correlations between H-2', H-3', H-4', H-5', and H-10 combined with the HMBC correlations from H-5' to C-3' (*δ*<sup>C</sup> 38.5)/C-4' (*δ*<sup>C</sup> 63.4), from H-3' to C-2' (*δ*<sup>C</sup> 63.0)/C-10 (*δ*<sup>C</sup> 20.5), from H-10 to C-2'/C-3' suggested an aliphatic fragment of -CH2-CH-CH2-CH-CH3. Furthermore, the key HMBC correlations from H-5' to C-1/C-4/C-6', from H-4' to C-6', and from H-10 to C-6', together with the

unsaturation of compound **1** and the chemical shift of C6' (*δ*<sup>C</sup> 104.6) indicated the presence of the spiroketal ring C. Therefore, the planar structure of **1** was shown in Figure 1. The absolute configuration of **1** was further verified by the X-ray diffraction analysis of a single crystal using Cu K*α* as 6'*S*, 2'*S*, 4'*S*-**1** (Figure 3). Hence, the structure of compound **1** was identified as 6 *S*, 2'*S*, 4 *S*-**1** and named penicillol A.


**Table 1.** 1H (600 MHz) and 13C NMR (150 MHz) data for compounds **1** and **2**.

<sup>a</sup> Measure in MeOD-d4. <sup>b</sup> measure in CDCl3.

**Figure 2.** Key HMBC and 1H-1H COSY correlations of **1**–**4**.

**Figure 3.** ORTEP representation of crystal structure of **1**.

Compound **2,** white powder, possesses the molecular formula C15H15O6, as assigned by the HRESIMS ion at *m/z* 291.08727 [M − H]<sup>−</sup> (calcd for C15H15O6, 291.08741), showing eight degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3680 cm<sup>−</sup>1), aromatic ring (1580 cm−1), and carbonyl (1736 cm−1, 1671 cm−1). Analysis of the 13C NMR and HMQC (Table 1) data indicated the presence of 15 carbon atoms consisting of one methoxy group, one methyl, three methylenes, three methines (including two aromatic methines), 7 nonprotonated carbons (including one ester carbon, one carbonyl, four olefinic carbons). The 1H NMR spectrum of **2** displayed two aromatic protons at *δ*<sup>H</sup> 6.38 (d, *J* = 2.3 Hz), 6.29 (s), indicating the presence of a tetrasubstituted phenyl. One oxygenated methine proton signal at *δ*<sup>H</sup> 4.41 (m), three aliphatic methylenes at *δ*<sup>H</sup> 3.14 (d, *J* = 16.4), 3.20 d, *J* = 16.4 Hz), 2.29 (dd, *J* = 11.5, 15.1 Hz), 2.53 (m), 2.59 (d, *J* = 15.4 Hz), and 2.85 (dd, *J* = 1.6, 15.3 Hz), one methoxyl at *δ*<sup>H</sup> 3.84 (s), one methyl at *δ*<sup>H</sup> 1.24 (d, *J* = 6.2 Hz), and one active proton at *δ*<sup>H</sup> 11.04 (s) were also recorded in this spectrum. Cumulative analyses of the 1H and 13C NMR spectra of compound **2** revealed that it possessed the similar planar structure as that of **1**. The main difference was the change of hydroxylated methine in **1** (*δ*<sup>C</sup> 63.4) to a carbonyl in **2** (*δ*<sup>C</sup> 202.5), indicating that compound **2** was an analog of **1**. Furthermore, the planar structure of **2** was further verified by the key HMBC correlation from H-5' to C-4' (*δ*<sup>C</sup> 202.5), and H-3' to C-4' (Figure 2). The absolute configuration of **2** was determined by comparison of experimental and calculated ECD. Experimental data showed that the compounds **1**, **2**, and **6** had extremely similar ECD spectra, inferring that the cotton effect of compound **2** might be only affected by the chiral centers of C-6' (*δ*<sup>C</sup> 104.7) and C-2' (*δ*<sup>C</sup> 67.9) (Figure 4A). Then, the ECD spectra of the four possible configurations (6'*S*, 2'*S*-**2**; 6'*S*, 2'*R*-**2**; 6'*R*, 2'*S*-**2**; 6'*R*, 2'*R*-**2**) were calculated. The results disclosed that 6'*S*, 2'*S*-**2** and 6'*S*, 2'*R*-**2** displayed similar ECD spectra with the experimental one, showing a negative cotton effect (CE) at about 266 nm and a positive CE at about 285 nm (Figure 4B). Therefore, it was rationally speculated that the cotton effect of compound **2** was only affected by the chiral center of C-6', and the configuration of C-6' was determined to be 6'S as same as that of compound **1**. Based on the biosynthetic point of view, the absolute configuration at C- 2 should be the same as that of compound **1**. Therefore, the structure of compound **2** was identified as 6'*S*, 2'*S*-**2** and named as penicillol B.

**Figure 4.** (**A**) Experimental ECD spectra of compounds **1**, **2,** and **6**. (**B**) Comparison between the experimental and calculated ECD spectra of **2**.

Compound **3** was obtained as pale-yellow oil with the molecular formula C23H30O8 established from HRESIMS at *m/z* 415.17607 [M − H3O]<sup>−</sup> (calcd for 415.17623), showing 9 degrees of unsaturation. The IR spectrum at νmax 3685, 1725, 1693, and 1612 cm−1, corresponded to a hydroxy, ester carbonyl, carbonyl and double bond group, respectively. Analysis of the 13C NMR and HMQC spectra revealed 23 carbon resonances, corresponding to one ketone carbonyl (*δ*<sup>C</sup> 204.7), one ester carbonyls (*δ*<sup>C</sup> 163.9), three sp<sup>2</sup> nonprotonated carbons (*δ*<sup>C</sup> 108.2, 154.5 and 170.8), three sp<sup>3</sup> nonprotonated carbon (*δ*<sup>C</sup> 86.3, 84.9 and 82.3), seven sp<sup>2</sup> methine carbons (*δ*<sup>C</sup> 88.9, −139.1), two sp<sup>3</sup> methine carbons (*δ*<sup>C</sup> 80.2, 78.1), and five methyl carbons (*δ*<sup>C</sup> 31.4, 16.9, 13.1, 12.5 and 9.0), one methoxyl (*δ*<sup>C</sup> 56.3). Analysis of 1H NMR spectrum of **<sup>3</sup>** exhibited a set of olefinic protons resonances at *<sup>δ</sup>*<sup>H</sup> 5.49 (s), 6.34 (d, *<sup>J</sup>* = 15.0 Hz), 6.27 (dd, *J* = 15.5, 10.5 Hz), 6.36 (dd, *J* = 15.0, 10.5 Hz), 7.16 (dd, *J* = 15.0, 10.5 Hz), 6.43 (dd, *J* = 15.0, 10.5 Hz), 6.05 (d, *J* = 15.5 Hz,). Five methyls at *δ*<sup>H</sup> 1.96 (s), 1.39 (s), 1.23 (d, *J* = 6.5 Hz), 1.35 (s), 1.46 (s), one methoxyl at *δ*<sup>H</sup> 3.82 (s), and two oxygenated methine protons signal at *δ*<sup>H</sup> 3.94 (s), 4.24 (dd, *J* = 6.5, 12.9 Hz) were also recorded. All the above data were indicative that compound **3** possessed a similar carbon skeleton as that of citreoviridin which was a polyketide derivative from a *Penicillium pulvillorum* [20]. The HMBC correlations from H-20 to C-2 (*δ*<sup>C</sup> 80.2)/C-3 (*δ*<sup>C</sup> 86.3), from H-2 to C-3, from H-21 to C-3/C-4 (*δ*<sup>C</sup> 78.1), from H-4 to C-2, and from H-22 to C-4/C-5 (*δ*<sup>C</sup> 84.9) suggested the presence of **3**, 4-dihydroxy-2, 3, 5-trimethyl-tetrahydrofuran moiety (Figure 2). The 1H-1H COSY correlation of H-2/ H3-20 and H-8/ H-9/ H-10/ H-11/ H-12/ H-13 indicated two hydrocarbon fragments of -CH-CH3 and -CH=CH-CH=CH-CH=CH-. Through the analysis of the 1H NMR spectra of **3** to those of citreoviridin, combined with key HMBC correlation from H-17 to C-16/C-18/C-19, from H-24 to C-19, and from H-25 to C-17/C-18, suggested the presence of 4-methyl-5-methoxy-3, 4, 5-trisubstituted-α-pyrone moiety. Moreover, the HMBC correlation from H-13 to C-14 (*δ*<sup>C</sup> 154.5)/C-19 (*δ*<sup>C</sup> 108.2), indicated the unsaturated hydrocarbon fragment was lactated at C-14 of α-pyrone moiety. The main difference was the replacement of two double bond carbons in citreoviridin by a carbonyl carbon C-6 (*δ*<sup>C</sup> 204.7) and an oxygenated quaternary carbon C-7 (*δ*<sup>C</sup> 82.3) in **3**. This deduction was supported by the key HMBC correlations from H-23 to C-7/C-6 (*δ*<sup>C</sup> 204.7)/C-8 (*δ*<sup>C</sup> 139.1), from H-8 to C-7 (*δ*<sup>C</sup> 82.3), and from H-22 to C-5/C-6. So, the planar structure of **3** was elucidated, as shown in Figure 1. The NOE correlations from H-2 to Me-21, from Me-21 to H-2/H-4, from Me-22 to H-4 revealed the relative configuration of the five-membered ring as 2*R\**, 3*S\**, 4*S\**, 5*R\** (Figure 5). The relative configuration of C-7 was subjected to DP4+ analysis. Therefore, a DP4+ analysis of two candidate structures (2*R\**, 3*S\**, 4*S\**, 5*R\**, 7*S\**-**3**/2*R\**, 3*S\**, 4*S\**, 5*R\**, 7*R\**-**3**) was performed by calculating for their theoretical 1D NMR chemical shifts. The result showed that the final score of the configuration 2*R\**, 3*S\*,* 4*S\**, 5*R\**, 7*S\**-**3** (100%) was the most probable (Figures S29 and S30). Thus, the structure of compound **3** was identified as 2*R\**, 3*S\**, 4*S\**, 5*R\**, 7*S\**-**3**. Coupling constants between protons H-8 and H-9, H-10 and H-11, H-12 and H-13 (3*J*H-8, H-9 = 15.5 Hz; <sup>3</sup>*J*H-10, H-11 = 15.0 Hz; <sup>3</sup>*J*H-12,

H-13 = 15.0 Hz) inferred that the conformations of these three double bonds are all *trans* conformations, and compound **3** is named citreoviridin H.

**Figure 5.** Key NOESY correlations of **1**, **3**, **4,** and **6**.

Compound **4** was isolated as pale-yellow oil, and its molecular formula was determined as C23H32O8 on the basis of the pseudomolecular ion peak observed at *m/z* 417.19163 [M − H3O]<sup>−</sup> (Calcd for 417.19188) in the HRESIMS spectrum, indicating 8 degrees of unsaturation. The IR spectrum showed absorption bands of hydroxyl, carbonyl, and double bond at νmax 3696, 1694, and 1623 cm−1. The 13C NMR and HMQC spectra (Table 2) displayed 23 carbon signals, consisting of six methyls (including one methoxyl), ten methines (including seven olefinic carbons), and seven quaternary carbons (including one ester carbon). The 1H NMR spectrum of **4** revealed a set of olefinic protons resonances at *δ*<sup>H</sup> 5.64 (s), 6.59 (d, *J* = 15.0 Hz), 6.43 (dd, *J* = 11.0, 15.4 Hz), 6.5 (dd, *J* = 11.0, 15.0 Hz), 7.16 (dd, *J* = 11.0, 15.0 Hz), 6.65 (dd, *J* = 11.0, 15.0 Hz), 6.01 (d, *J* = 15.4 Hz), five methyls at *δ*<sup>H</sup> 2.02 (s), 1.32 (s), 1.20 (d, *J* = 6.4 Hz), 1.22 (s), 1.30 (s), one methoxyl at *δ*<sup>H</sup> 3.92 (s), and three oxygenated methine protons signal at *δ*<sup>H</sup> 3.79 (s), 4.08 (m), 3.69 (s). All the above data were indicative that compound **4** possessed a similar carbon skeleton of **3**. The main difference was the replacement of a carbonyl group in **3** (*δ*<sup>C</sup> 204.7) by a hydroxymethine group in **4** (*δ*<sup>C</sup> 91.2), indicating that compound **4** was a homologue of **3**. This inference was further confirmed by the key HMBC correlation from H-22 to C-5 (*δ*<sup>C</sup> 86.3)/C-6 (*δ*<sup>C</sup> 91.2), from

H-6 to C-5/C-7 (*δ*<sup>C</sup> 73.3)/C-8 (*δ*<sup>C</sup> 140.9), and from H-23 to C-7/C-6 (Figure 2). Therefore, the planar structure of **4** was established as shown in Figure 1. Furthermore, the relative configuration of compound **4** was partly determined by NOESY spectrum. The NOE correlations from H-2 to Me-21, from Me-21 to H-2/H-4, from Me-22 to H-4 indicated the relative configuration of the tetrahydrofuran ring to be 2*R\**, 3*S\**, 4*S\**, 5*S\** (Figure 5). In order to determine the relative configuration of C-6 and C-7, the DP4+ analysis of four candidate structures (2*R\**, 3*S\**, 4*S\**, 5*S\*,* 6*S\**, 7*S\**-**4**/2*R\**, 3*S\**, 4*S\**, 5*S\**, 6*R\**, 7*S\**-**4**/2*R\**, 3*S\**, 4*S\**, 5*S\**, 6*R\**, 7*R\**-**4**/2*R\**, 3*S\**, 4*S\**, 5*S\**, 6*S\**, 7*R\**-**4**) was performed. The analysis results showed that the configuration of 2*R\**, 3*S\**, 4*S\**, 5*S\**, 6*S\**, 7*S\**-**4** was the correct structure with a 100% probability (Figures S31 and S32). Hence, the structure of compound **4** was identified as 2*R\**, 3*S\**, 4*S\**, 5*S\**, 6*S\**, 7*S\**-**4**. Coupling constants between protons H-8 and H-9, H-10 and H-11, H-12 and H-13 (3*J*H-8, H-9 = 15.4 Hz; <sup>3</sup>*J*H-10, H-11= 15.0 Hz; <sup>3</sup>*J*H-12, H-13 = 15.0 Hz) inferred that the conformations of these three double bonds are all *trans* conformations, and compound **4** is named citreoviridin I.

**Table 2.** 1H (600 MHz) and 13C NMR (150 MHz) data for compounds **3** and **4**.


<sup>a</sup> Measure in MeOD-d4; <sup>b</sup> measure in CDCl3.

Four known compounds including dichlorodiaportal (**5**) [21], citreoviranol (**6**) [22], citreopyrone D (**7**) [23], citreoviral (**8**) [24], were isolated and identified from this fungus. Their structures were determined by comparing their NMR and MS data with those reported in the literature. Moreover, the absolute configuration of compound **6** was further determined to be 6'*S*, 2'*S*, 4'*R*-**6** by comparing its NOE correlations and ECD spectrum with compound **1.**

A plausible typical fungal polyketide synthetase (PKS) involved biosynthetic pathway for compounds **1**–**4** was proposed as shown in Figure S33. The condensation of one mole of acetyl coenzyme A and six moles of malonyl coenzyme A gives a mole linear polyketide chain. Subsequent keto-reduction, cyclization, methylation, and hydroxylation furnish compounds **1** and **2.** Similarly, one mole acetyl coenzyme A and eight moles malonyl coenzyme A condensed to form a mole linear polyketide chain, then keto-reduction, dehydration, cyclization, methylation, and hydroxylation form compounds **3** and **4** [25,26].

### *2.3. Anti-Inflammatory Activity*

New compounds **1**–**4** were evaluated for anti-inflammatory activity in LPS-stimulated RAW 264.7 macrophages. Especially, Compound **2** significantly inhibited nitric oxide production with an IC50 value of 12 μM (Table 3).


**Table 3.** Inhibitory activities against LPS-induced NO production.

<sup>a</sup> Indometacin was used as positive control for the test.
