*Article* **Sesquiterpenoids and Xanthones from the Kiwifruit-Associated Fungus** *Bipolaris* **sp. and Their Anti-Pathogenic Microorganism Activity**

**Jun-Jie Yu , Ying-Xue Jin, Shan-Shan Huang and Juan He \***

National Demonstration Center for Experimental Ethnopharmacology Education, School of Pharmaceutical Sciences, South-Central University for Nationalities, Wuhan 430074, China; junjieyu98@outlook.com (J.-J.Y.); Yancy6020@163.com (Y.-X.J.); HuangSS1998@126.com (S.-S.H.)

**\*** Correspondence: 2015048@mail.scuec.edu.cn

**Abstract:** Nine previously undescribed sesquiterpenoids, bipolarisorokins A–I (**1**–**9**); two new xanthones, bipolarithones A and B (**10** and **11**); two novel sativene-xanthone adducts, bipolarithones C and D (**12** and **13**); as well as five known compounds (**14**–**18**) were characterized from the kiwifruitassociated fungus *Bipolaris* sp. Their structures were elucidated by extensive spectroscopic methods, electronic circular dichroism (ECD), <sup>13</sup>C NMR calculations, DP4+ probability analyses, and single crystal X-ray diffractions. Many compounds exhibited anti-pathogenic microorganism activity against the bacterium *Pseudomonas syringae* pv. *actinidiae* and four pathogenic microorganisms.

**Keywords:** *Bipolaris* sp.; kiwi-associated fungus; sesquiterpenoid; xanthone; anti-pathogenic microorganism activity

### **1. Introduction**

Kiwifruit (*Actinidia chinensis* Planch., Actinidiaceae) is an emerging, healthy, and economical fruit which has become increasingly popular worldwide owing to its flavor and nutritional properties [1]. It is an excellent source of vitamin C and provides balanced nutritional components of minerals, dietary fiber, folate, and health-promoting metabolites [2,3]. China is the leading kiwifruit producer in the world, followed by Italy and New Zealand. The cultivation area and annual output reached 243,000 hm<sup>2</sup> and 2,500,000 tons at the end of 2020 [4]. Nevertheless, as the cultivation of kiwifruit expands rapidly, many serious diseases such as bacterial canker, soft rot, bacterial blossom blight, brown spot, and root rot are a serious and ongoing threat to kiwifruit production [5–12]. Particularly, the destructive bacterial canker disease, which is associated with an infection by *P*. *syringae* pv. *actinidiae* (Psa), has led to reduced kiwifruit production and huge economic losses worldwide [13,14]. Although the application of copper-based chemicals and streptomycin have played a positive role in the prevention and treatment of bacterial canker, these chemical residues are extremely threatening to human health and the ecological environment [15,16]. Additionally, chemical fungicides easily induce pathogen resistance [17,18]. Thus, it is urgent to develop safer and more effective biological pesticides.

Endophytic microorganisms reside within different tissues of the host plant without causing any disease symptoms and produce various metabolites with different activities [19,20]. Therefore, the endophytic fungi have been proved to be valuable sources of important natural products [21,22]. Some natural products from endophytic fungi play important roles in plant defense systems. Therefore, we carried out the excavation of anti-Psa active substances from metabolites of kiwifruit endophytes and harvested a number of bioactive molecules. For instance, 3-decalinoyltetramic acids and cytochalasins from the kiwifruit endophytic fungus *Zopfiella* sp showed anti-Psa activity [23,24], while imidazole alkaloids ether were characterized as anti-Psa agents from *Fusarium tricinctum* [25]. These

**Citation:** Yu, J.-J.; Jin, Y.-X.; Huang, S.-S.; He, J. Sesquiterpenoids and Xanthones from the Kiwifruit-Associated Fungus *Bipolaris* sp. and Their Anti-Pathogenic Microorganism Activity. *J. Fungi* **2022**, *8*, 9. https://doi.org/10.3390/ jof8010009

Academic Editor: Frank Surup

Received: 13 December 2021 Accepted: 21 December 2021 Published: 23 December 2021

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**Copyright:** © 2021 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/).

discoveries prompted us to search for more novel and bioactive metabolites from kiwifruitassociated fungi. In the current study, a total of eighteen compounds have been isolated from the large-scale fermentation of the kiwifruit-associated fungus *Bipolaris* sp. (Figure 1), which included nine new sativene or longifolene sesquiterpenoids, bipolarisorokins A–I (**1**–**9**); two new xanthones, bipolarithones A and B (**10** and **11**); two novel sativene-xanthone adducts, bipolarithones C and D (**12** and **13**); as well as five known ones (**14**–**18**). Their structures were established by means of spectroscopic methods, namely, ECD and <sup>13</sup>C NMR calculations, DP4+ probability analyses, and single crystal X-ray diffractions. All compounds were evaluated for their inhibitory activities against Psa. Additionally, their inhibitory activity against four phytopathogens (*Phytophthora infestans*, *Alternaria solani*, *Rhizoctonia solani*, and *Fusarium oxysporum*) were assessed. Here, the details of isolation, structural elucidation, and bioactivity evaluations for **1**–**18** are reported. [25]*.* These discoveries prompted us to search for more novel and bioactive metabolites from kiwifruit-associated fungi. In the current study, a total of eighteen compounds have been isolated from the large-scale fermentation of the kiwifruit-associated fungus *Bipolaris*  sp. (Figure 1), which included nine new sativene or longifolene sesquiterpenoids, bipolarisorokins A–I (**1**–**9**); two new xanthones, bipolarithones A and B (**10** and **11**); two novel sativene-xanthone adducts, bipolarithones C and D (**12** and **13**); as well as five known ones (**14**–**18**). Their structures were established by means of spectroscopic methods, namely, ECD and 13C NMR calculations, DP4+ probability analyses, and single crystal X-ray diffractions. All compounds were evaluated for their inhibitory activities against Psa. Additionally, their inhibitory activity against four phytopathogens (*Phytophthora infestans*, *Alternaria solani*, *Rhizoctonia solani*, and *Fusarium oxysporum*) were assessed. Here, the details of isolation, structural elucidation, and bioactivity evaluations for **1**–**18** are reported.

of bioactive molecules. For instance, 3-decalinoyltetramic acids and cytochalasins from the kiwifruit endophytic fungus *Zopfiella* sp showed anti-Psa activity [23,24], while imidazole alkaloids ether were characterized as anti-Psa agents from *Fusarium tricinctum* 

*J. Fungi* **2021**, *7*, x FOR PEER REVIEW 2 of 18

**Figure 1.** Structures of compounds **1**–**18**. **Figure 1.** Structures of compounds **1**–**18**.

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

#### *2.1. General Experimental Procedures 2.1. General Experimental Procedures*

Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured with an Autopol IV polarimeter (Rudolph, Hackettstown, NJ, USA). UV spectra were obtained using a double beam spectrophotometer UH5300 (Hitachi High-Technologies, Tokyo, Japan). IR spectra were obtained by a Shimadzu IRTracer-100 spectrometer using KBr pellets. 1D and 2D NMR spectra were run on a Bruker Avance III 600 MHz spectrometer with TMS as an internal standard. Chemical Melting points were obtained on an X-4 micro melting point apparatus. Optical rotations were measured with an Autopol IV polarimeter (Rudolph, Hackettstown, NJ, USA). UV spectra were obtained using a double beam spectrophotometer UH5300 (Hitachi High-Technologies, Tokyo, Japan). IR spectra were obtained by a Shimadzu IRTracer-100 spectrometer using KBr pellets. 1D and 2D NMR spectra were run on a Bruker Avance III 600 MHz spectrometer with TMS as an internal standard. Chemical shifts (δ) were expressed in ppm with references to the solvent signals. High resolution electrospray ionization mass spectra (HR-ESIMS) were recorded on a LC-MS system consisting of a Q Exactive™ Orbitrap mass spectrometer with an HRESI ion source (ThermoFisher Scientific, Bremen, Germany) used in ultra-high-resolution mode (140,000 at m/z 200) and a UPLC system

(Dionex UltiMate 3000 RSLC, ThermoFisher Scientific, Bremen, Germany). Column chromatography (CC) was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., Qingdao, China), RP-18 gel (20–45 µm, Fuji Silysia Chemical Ltd., Kasugai, Japan), and Sephadex LH-20 (Pharmacia Fine Chemical Co. Ltd., Uppsala, Sweden). Medium-pressure liquid chromatography (MPLC) was performed on a Büchi Sepacore System equipped with a pump manager C-615, pump modules C-605, and a fraction collector C-660 (Büchi Labortechnik AG, Flawil, Switzerland). Preparative high-performance liquid chromatography (prep-HPLC) was performed on an Agilent 1260 liquid chromatography system equipped with Zorbax SB-C18 columns (5 µm, 9.4 mm × 150 mm, or 21.2 mm × 150 mm) and a DAD detector. Chiral resolution was achieved by HPLC equipped with a Daicel AD-H column. Fractions were monitored by TLC (GF 254, Qingdao Haiyang Chemical Co. Ltd. Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H2SO<sup>4</sup> in EtOH.

#### *2.2. Fermentation, Extraction, and Isolation*

The fungus *Bipolaris* sp. was isolated from fresh and healthy stems of kiwifruit plants (*Actinidia chinensis* Planch., Actinidiaceae), which were collected from the Cangxi county of the Sichuan Province (GPS: N 31◦120 , E 105◦760 ) in July 2018. Each fungus was obtained simultaneously from at least three different healthy tissues. The fungus was identified as one species of the genus *Bipolaris* by observing the morphological characteristics and analysis of the internal transcribed spacer (ITS) regions. A living culture (internal number HFG-20180727-HJ32) has been deposited at the School of Pharmaceutical Sciences, South-Central University for Nationalities, China.

This fungal strain was cultured on a potato dextrose agar (PDA) medium at 24 ◦C for 10 days. The agar plugs were inoculated in 500 mL Erlenmeyer flasks, each containing 100 mL potato dextrose media. Flask cultures were incubated at 28 ◦C on a rotary shaker at 160 rpm for two days as the seed culture. Four hundred 500 mL Erlenmeyer flasks, each containing 150 mL potato dextrose broth (PDB), were individually inoculated with 25 mL of seed culture and were incubated at 25 ◦C on a rotary shaker at 160 rpm for 25 days.

The cultures of *Bipolaris* sp. (60 L) were extracted four times by EtOAc to afford a crude extract (32.0 g) which was subjected to CC over silica gel eluted with a gradient of CHCl3-MeOH (a gradient from 1:0 to 0:1) to give six fractions, A–F. Fraction B (13.0 g) was fractionated by MPLC CC over RP-18 eluted with MeOH–H2O (from 10:90 to 100:0, *v*/*v*) to give twelve sub-fractions (B1–B12). Fraction B<sup>3</sup> (1.2 g) was applied to Sephadex LH-20 eluting with CHCl3–MeOH (1:1, *v*/*v*) and was further purified by preparative HPLC with MeCN–H2O (19:81, *v*/*v*, 4.0 mL/min) to obtain compounds **9** (18.6 mg, retention time (tR) = 40 min), **18** (22.6 mg, t<sup>R</sup> = 15.8 min), **2** (3.3 mg, t<sup>R</sup> = 32 min), and **1** (5.4 mg, t<sup>R</sup> = 36 min). Fraction B<sup>5</sup> (2.1 g) was separated by CC over silica gel with a gradient elution of the CHCl3–MeOH system (50:1→0:1) and was prepared by HPLC with MeCN–H2O (12:88, *v*/*v*, 4.0 mL/min) to obtain **3** (4.9 mg, t<sup>R</sup> = 36 min), **4** (14.4 mg, t<sup>R</sup> = 46 min), **17** (28.3 mg, t<sup>R</sup> = 43 min), and **5** (2.1 mg, t<sup>R</sup> = 40 min). Fraction B<sup>6</sup> (1.8 g) was purified over Sephadex LH-20 eluted with MeOH to give four subfractions (B6.1–B6.4). Fraction B6.2 (210 mg) was purified using semipreparative HPLC with MeOH-H2O (28:72, *v*/*v*, 3.0 mL/min) to afford **8** (8.8 mg, t<sup>R</sup> = 17.8 min) and **7** (9.6 mg, t<sup>R</sup> = 21.1 min). Fraction B6.3 (170 mg) was purified by preparative HPLC with MeCN–H2O (23:77, *v*/*v*, 4 mL/min) to yield **6** (4.3 mg, 26 min). Fraction C (4.3 g) was separated by CC over silica gel with a gradient elution of PE-acetone (50:1→0:1) to afford subfractions C1–C8. Fraction C<sup>2</sup> (340 mg) was purified by preparative HPLC with MeCN-H2O (55:45, *v*/*v*, 4 mL/min) to give **12** (10.3 mg, t<sup>R</sup> = 38 min), **13** (3.7 mg, t<sup>R</sup> = 39 min), **14** (3.1 mg, t<sup>R</sup> = 36 min) and **15** (3.4 mg, t<sup>R</sup> = 34 min). Fraction C<sup>5</sup> (230 mg) was isolated by CC over Sephadex LH-20 (MeOH) and was prepared by HPLC (32:68, *v*/*v*, 4 mL/min) to give **10** (3.7 mg, t<sup>R</sup> = 28 min), **11** (4.2 mg, t<sup>R</sup> = 29 min), and **16** (5.1 mg, t<sup>R</sup> = 24 min).

Bipolarisorokin A (**1**): colorless crystals; mp 145–148 ◦C; [*α*] 20 <sup>D</sup> + 67.8 (*c* 0.01, MeOH); UV (MeOH) *λ*max (log *ε*) 205 (3.30); IR (KBr) *ν*max 3360, 2947, 2833, 1651, 1454, 1114, 1031 cm−<sup>1</sup> ; <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 1; positive ion HRESIMS *m/z* 251.16624 [M–H]<sup>+</sup> , (calculated for C15H23O<sup>3</sup> − 251.16527).


**Table 1.** <sup>1</sup>H (600 MHz) and <sup>13</sup>C (150 MHz) NMR Spectroscopic Data for **1**–**3**.

*<sup>a</sup>* Measured in CDCl3; *<sup>b</sup>* Measured in methanol-*d*4.

Bipolarisorokin B (**2**): colorless oil; [*α*] 22 <sup>D</sup> − 100.1 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 210 (3.23); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 1; positive ion HRESIMS *m/z* 275.16166 [M+Na]<sup>+</sup> , (calculated for C15H24O3Na<sup>+</sup> 275.16177).

Bipolarisorokin C (**3**): colorless, needle-like crystals (MeOH); mp 135–138 ◦C; [*α*] 22 D − 21.8 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 265 (3.49) nm; <sup>1</sup>H NMR (600 MHz, methanol*d*4) and <sup>13</sup>C NMR (150 MHz, methanol-*d*4) data, see Table 1; positive ion HRESIMS *m/z* 253.17971 [M+H]<sup>+</sup> (calculated for C15H25O<sup>3</sup> <sup>+</sup> 253.17982).

*Bipolarisorokin* D (**4**): colorless oil; [*α*] 25 <sup>D</sup> + 32.0 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 255 (3.65); <sup>1</sup>H NMR (600 MHz, methanol-*d*4) and <sup>13</sup>C NMR (150 MHz, methanol-*d*4) data, see Table 2; positive ion HRESIMS *m/z* 275.16153 [M+Na]<sup>+</sup> (calculated for C15H24NaO<sup>3</sup> + 275.16177). *Bipolarisorokin E* (**5**): colorless oil; [*α*] 25 <sup>D</sup> − 22.7 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 210 (3.24); <sup>1</sup>H NMR (600 MHz, methanol-*d*4) and <sup>13</sup>C NMR (150 MHz, methanol-*d*4) data, see Table 2; positive ion HRESIMS *m/z* 221.15529 [M–H]− (calculated for C14H21O<sup>2</sup> − 221.15470).

*Bipolarisorokin F* (**6**): white powder; [*α*] 20 <sup>D</sup> − 3.3 (*c* 0.04, MeOH); UV (MeOH) *λ*max (log *ε*) 215 (3.72); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 2; positive ion HRESIMS *m/z* 225.18506 [M+H]<sup>+</sup> (calculated for C14H25O<sup>2</sup> <sup>+</sup> 225.18491).

*Bipolarisorokin G* (**7**): colorless oil; [*α*] 20 <sup>D</sup> + 17.2 (*c* 0.02, MeOH); UV (MeOH) *λ*max (log *ε*) 230 (3.21); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 3; positive ion HRESIMS *m/z* 275.20059 [M+H]<sup>+</sup> (calculated for C18H27O<sup>2</sup> <sup>+</sup> 275.20056).


**Table 2.** <sup>1</sup>H (600 MHz) and <sup>13</sup>C (150 MHz) NMR Spectroscopic Data for **4**–**6**.

*<sup>a</sup>* Measured in CDCl3; *<sup>b</sup>* Measured in methanol-*d*4.


*<sup>a</sup>* Measured in CDCl3; *<sup>b</sup>* Measured in methanol-*d*4.

*Bipolarisorokin H* (**8**): colorless oil; [*α*] 25 <sup>D</sup> − 136.9 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 225 (3.93); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 3; positive ion HRESIMS *m/z* 277.17984 [M+H]<sup>+</sup> (calculated for C17H25O<sup>3</sup> <sup>+</sup> 277.17982).

*Bipolarisorokin I* (**9**): colorless crystals; mp 191–194 ◦C; [*α*] 22 <sup>D</sup> + 8.8 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 210 (3.46); <sup>1</sup>H NMR (600 MHz, methanol-*d*4) and <sup>13</sup>C NMR (150 MHz, methanol-*d*4) data, see Table 3; positive ion HRESIMS *m/z* 251.16621 [M–H]−, (calculated for C21H23O<sup>3</sup> − 251.16527).

*Bipolarithone A* (**10**): colorless oil; [*α*] 23 <sup>D</sup> + 136.0 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 245 (3.30); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 4; positive ion HRESIMS *m/z* 349.09143 [M+H]<sup>+</sup> , (calculated for C17H17O<sup>8</sup> <sup>+</sup> 349.09179).


**Table 4.** <sup>1</sup>H (600 MHz) and <sup>13</sup>C (150 MHz) NMR Spectroscopic Data for **10** and **11**.

*<sup>a</sup>* Measured in CDCl3; *<sup>b</sup>* Measured in methanol-*d*4.

*Bipolarithone B* (**11**): colorless oil; [*α*] 23 <sup>D</sup> − 24.2 (*c* 0.05, MeOH); UV (MeOH) *λ*max (log *ε*) 245 (3.30); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 4; positive ion HRESIMS *m/z* 349.09157 [M+H]<sup>+</sup> , (calculated for C17H17O<sup>8</sup> <sup>+</sup> 349.09179).

*Bipolarithone C* (**12**): colorless oil; [*α*] 25 <sup>D</sup> + 52.9 (*c* 0.5, MeOH); UV (MeOH) *λ*max (log *ε*) 245 (4.06); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 5; positive ion HRESIMS *m/z* 541.24310 [M+H]<sup>+</sup> , (calculated for C30H37O<sup>9</sup> <sup>+</sup> 541.24321).

*Bipolarithone D* (**13**): colorless oil; [*α*] 25 <sup>D</sup> + 10.2 (*c* 0.5, MeOH); UV (MeOH) *λ*max (log *ε*) 245 (3.88); <sup>1</sup>H NMR (600 MHz, CDCl3) and <sup>13</sup>C NMR (150 MHz, CDCl3) data, see Table 5; positive ion HRESIMS *m/z* 541.24316 [M+H]<sup>+</sup> , (calculated for C30H37O<sup>9</sup> <sup>+</sup> 541.24321).

Crystal data for Cu\_**1**\_0m: C15H24O3, *M* = 252.34, a = 9.7038(6) Å, b = 13.7866(8) Å, c = 16.6333(10) Å, *α* = 95.329(3)◦ , *β* = 104.898(2)◦ , *γ* = 102.525(3)◦ , *V* = 2073.0(2) Å<sup>3</sup> , *T* = 100(2) K, space group P 1, Z = 6, *µ*(Cu K*α*) = 1.54178 mm−<sup>1</sup> , *F*(000) = 828, 82979 reflections measured, 16831 independent reflections (*Rint* = 0.0695). The final *R<sup>1</sup>* values were 0.0437 (*I* > 2*σ*(*I*)). The final *wR*(*F 2* ) values were 0.1047 (*I* > 2*σ*(*I*)). The final *R<sup>1</sup>* values were 0.0531 (all data). The final *wR*(*F 2* ) values were 0.1143 (all data). The goodness of fit on *F <sup>2</sup>* was 1.039. Flack parameter = <sup>−</sup>0.10(7). CCDC: 2124305. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu\_**3**\_0m: C15H24O3, *M* = 252.34, a = 7.0044(5) Å, b = 10.1468(8) Å, c = 20.1433(14) Å, *α* = 90.00◦ , *β* = 90.00◦ , *γ* = 90.00◦ , *V*= 1431.63(18) Å<sup>3</sup> , *T* = 295(2) K, space group P 21 21 21, with Z = 4, *µ*(Cu K*α*) = 1.54178 mm−<sup>1</sup> , *F*(000) = 552, 6263 reflections measured, 2527 independent reflections (*Rint* = 0.0500). The final *R<sup>1</sup>* values were 0.0519 (*I* > 2*σ*(*I*)). The final *wR*(*F 2* ) values were 0.1538 (*I* > 2*σ*(*I*)). The final *R<sup>1</sup>* values were 0.0719 (all data). The final *wR*(*F 2* ) values were 0.2087 (all data). The goodness of fit on *F <sup>2</sup>* was 1.117. Flack parameter = <sup>−</sup>0.40(17). CCDC: 2124306. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).


**Table 5.** <sup>1</sup>H (600 MHz) and <sup>13</sup>C (150 MHz) NMR Spectroscopic Data for **12** and **13**.

*<sup>a</sup>* Measured in CDCl3; *<sup>b</sup>* Measured in methanol-*d*4.

Crystal data for Cu\_**9**\_0m: C15H24O3, *M* = 252.34, a = 6.8634(2) Å, b = 15.0872(4) Å, c = 13.5156(3) Å, *α* = 90.00◦ , *β* = 90.4010(10)◦ , *γ* = 90.00◦ ,*V* = 1399.50(6) Å<sup>3</sup> , T = 295(2) K, space group P 1 21 1, with Z = 4, *µ*(Cu K*α*) = 1.54178 mm−<sup>1</sup> , *F*(000) = 552, 32232 reflections measured, 5982 independent reflections (*Rint* = 0.0279). The final *R<sup>1</sup>* values were 0.0300 (*I* > 2*σ*(*I*)). The final *wR*(*F 2* ) values were 0.0808 (*I* > 2*σ*(*I*)). The final *R<sup>1</sup>* values were 0.0304 (all data). The final *wR*(*F 2* ) values were 0.0812 (all data). The goodness of fit on *F <sup>2</sup>* was 1.057. Flack parameter = <sup>−</sup>0.01(3). CCDC: 2124307. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu\_**17**\_0m: C14H24O2, *M* = 224.33, a = 13.6388(2) Å, b = 13.6388(2) Å, c = 13.0174(2) Å, *α* = 90.00◦ , *β* = 90.00◦ , *γ* = 90.00◦ , *V* = 2097.04(7) Å<sup>3</sup> , *T* = 296(2) K, space group P 31 2 1, with Z = 6, *µ*(Cu Kα) = 1.54178 mm−<sup>1</sup> , *F*(000) = 744, 39026 reflections measured, 3033 independent reflections (*Rint* = 0.0459). The final *R<sup>1</sup>* values were 0.0353 (*I* > 2*σ*(*I*)). The final *wR*(*F 2* ) values were 0.0988 (*I* > 2*σ*(*I*)). The final *R<sup>1</sup>* values were 0.0366 (all data). The final *wR*(*F 2* ) values were 0.1003 (all data). The goodness of fit on *F <sup>2</sup>* was 1.047. Flack parameter =0.01(5). CCDC: 2126101. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

Crystal data for Cu\_**18**\_0m: C15H26O2, *M* = 238.36, a = 13.1977(2) Å, b = 13.1977(2) Å, c = 8.49040(10) Å, *α* = 90.00◦ , *β* = 90.00◦ , *γ* = 90.00◦ , *V* = 1478.85(5) Å<sup>3</sup> , *T* = 297(2) K, space group P 43, with Z = 4, *µ*(Cu K*α*) = 1.54178 mm−<sup>1</sup> , *F*(000) = 528, 14568 reflections measured, 3063 independent reflections (*Rint* = 0.0269). The final *R<sup>1</sup>* values were 0.0534 (*I* > 2*σ*(*I*)). The final *wR*(*F 2* ) values were 0.1525 (*I* > 2*σ*(*I*)). The final *R<sup>1</sup>* values were 0.0541 (all data). The final *wR*(*F 2* ) values were 0.1539 (all data). The goodness of fit on *F <sup>2</sup>* was 1.051. Flack parameter =0.12(7). CCDC: 2126105. Available online: https://www.ccdc.cam.ac.uk (accessed on 11 December 2021).

#### *2.3. ECD Calculations*

The ECD calculations were carried out using the Gaussian 16 software package [26]. Systematic conformational analyses were performed via SYBYL-X 2.1 using the MMFF94 molecular mechanics force field calculation with 10 kcal/mol of cutoff energy [27,28]. The optimization and frequency of conformers were calculated on the B3LYP/6-31G(d) level in the Gaussian 09 program package. The ECD (TDDFT) calculations were performed on the B3LYP/6-311G(d) level of theory with an IEFPCM solvent model (MeOH). The ECD curves were simulated in SpecDis V1.71 using a Gaussian function [29]. The calculated ECD data of all conformers were Boltzmann averaged by Gibbs free energy.

#### *2.4. NMR Calculations*

All the optimized conformers in an energy window of 5 kcal/mol (with no imaginary frequency) were subjected to gauge-independent atomic orbital (GIAO) calculations of their <sup>13</sup>C NMR chemical shifts, using density functional theory (DFT) at the mPW1PW91/6- 311+G (d,p) level with the PCM model. The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energy. The <sup>13</sup>C NMR chemical shifts for TMS were also calculated by the same procedures and used as the reference. After the calculation, the experimental and calculated data were evaluated by the improved probability DP4<sup>+</sup> method [30].

### *2.5. Antibacterial Activity Assay*

The bacterium *P*. *syringae* pv. *actinidiae* was donated by Dr. He Yan of Northwest A&F University, China. A sample of each culture was then diluted 1000-fold in fresh Luria-Bertani (LB) (Beijing Solarbio Science & Technology. Co. Ltd., Beijing, China) and incubated with shaking (160 rpm) at 27 ◦C for 10 h. The resultant mid-log phase cultures were diluted to a concentration of 5 <sup>×</sup> <sup>10</sup><sup>5</sup> CFU/mL, then 160 <sup>µ</sup>L was added to each well of the compound-containing plates. Subsequently, 1:1 serial dilutions with sterile PBS of each compound were performed, giving a final compound concentration range from 4 to256 µg/mL. The minimum inhibitory concentration (MIC, with an inhibition rate of ≥90%) was determined by using photometry at OD<sup>600</sup> nm after 24 h. Streptomycin was used as the positive control.

#### *2.6. Anti-Phytopathogens Assay*

Four phytopathogens (*Phytophthora infestane*, *Alternaria solani, Rhizoctonia solani*, and *Fusarium oxysporum*) were cultured in PDA with micro glass beads at 27 ◦C for a week, as well as shaking (160 rpm). Ninety microliters of PDA, together with a 10 µL volume of an aqueous test sample solution, was added into each well of the 96-well plate. The test solutions contained different concentrations of the sample being tested. Then, agar plugs (1 mm<sup>3</sup> ) with fresh phytopathogens were inoculated into each well. Subsequently, a two-fold serial dilution in the microplate wells was performed over a concentration range of 4 to 256 µg/mL. Plates were covered and incubated at 27 ◦C for 24 h. Finally, the minimum inhibitory concentration was determined by observing the plates, with no growth in the well taken as that value. Hygromycin B was used as the positive control.

#### **3. Results and Discussion**

Bipolarisorokin A (**1**) was isolated as colorless crystals. Its molecular formula of C15H24O<sup>3</sup> was determined on the basis of the HR-ESIMS data (measured at *m/z* 251.16624

[M–H]−, calculated for C15H23O<sup>3</sup> − 251.16527), corresponding to four degrees of unsaturation. The <sup>1</sup>H and <sup>13</sup>C NMR spectra, in association with the HSQC spectrum, revealed two methyls, four methenes, seven methines, and two quaternary carbons (Table 1). Of them, signals at *δ*<sup>C</sup> 66.9 (t, C-11), 69.6 (d, C-14), and 74.9 (d, C-15) were identified as the oxygenated methylene and methines. Two olefinic carbons at *δ*<sup>C</sup> 156.8 (s, C-2) and 103.5 (t, C-12) corresponded to a double bond, which suggested that **1** possessed a tricyclic system. Considering the 15 carbons in **1**, as well as those isolates from the same source, compound **1** was suggested to be a tricyclic sesquiterpenoid. In the <sup>1</sup>H–1H COSY spectrum, a fragment was revealed, as shown with bold lines in Figure 2. The HMBC correlations from to *δ*<sup>H</sup> 4.94 (H, s, H-12a) and 4.62 (H, s, H-12b), to *δ*<sup>C</sup> 156.8 (s, C-2), 54.3 (d, C-1) and 43.0 (s, C-3), established the connections between C-12, C-2, and C-1. Further analyses of <sup>1</sup>H–1H COSY, as well as HMBC correlations from *δ*<sup>H</sup> 0.92 (3H, d, *J* = 6.8 Hz, H-10) to *δ*<sup>C</sup> 37.6 (d, C-6), 40.5 (d, C-9) and 66.9 (t, C-11), indicated a hydroxy group at C-11. In addition, the connections of C-8/C-3, C-3/C-4, C-3/C-2, and C-3/C-13 were deduced from HMBC correlations from *δ*<sup>H</sup> 1.05 (3H, s, H-8) to *δ*<sup>C</sup> 43.0 (s, C-3), 39.9 (t, C-4), 156.8 (s, C-2), and 58.2 (d, C-13). Moreover, the proton of an oxygenated methine at *δ*<sup>H</sup> 4.02 (H, d, *J* = 5.9 Hz, H-14) showed key correlations to C-13, C-3, and *δ*<sup>C</sup> 42.2 (d, C-7), which indicated that *δ*<sup>C</sup> 69.6 (d, C-14) should be placed at C-13. The above 2D NMR data analysis suggested that compound **1** possessed a sativene type sesquiterpene backbone. A ROESY experiment was carried out to establish the relative configuration of **1** (Figure 3). The key correlations of H-13/H-8, H-13/H-6, H-8/H-14, and H-7/H-13 suggested that H-6, H-7, H-8, and H-13 were *β* oriented, while the correlation of H-1/H-9 indicated that H-1 and H-9 were *α*-oriented. Because of the rigid structure and the ROESY correlation of H-8/H-14, both H-14 and H-15 were assigned as an *α* orientation [31]. Finally, the single-crystal X-ray diffraction not only confirmed the planar structure, as elucidated above, but also established the absolute configuration of **1** (Flack parameter = −0.10(7), CCDC: 2124305; Figure 4). *J. Fungi* **2021**, *7*, x FOR PEER REVIEW 10 of 18

**Figure 2.** Key 1H–1H COSY and HMBC correlations for **1**, **3**, **5**, **6**, **7**, and **9**–**13**. **Figure 2.** Key <sup>1</sup>H–1H COSY and HMBC correlations for **1**, **3**, **5**, **6**, **7**, and **9**–**13**.

**Figure 3.** Key ROESY correlations for **1**, **3**, **5**, **6**, **7**, **9**, **10,** and **12**.

**Figure 2.** Key 1H–1H COSY and HMBC correlations for **1**, **3**, **5**, **6**, **7**, and **9**–**13**.

**Figure 3.** Key ROESY correlations for **1**, **3**, **5**, **6**, **7**, **9**, **10,** and **12**. **Figure 3.** Key ROESY correlations for **<sup>1</sup>**, **<sup>3</sup>**, **<sup>5</sup>**, **<sup>6</sup>**, **<sup>7</sup>**, **<sup>9</sup>**, **10,** and **<sup>12</sup>**.

**Figure 4**. ORTEP diagrams of **1**, **3,** and **9**. **Figure 4.** ORTEP diagrams of **1**, **3,** and **9**.

Bipolarisorokin C (**3**) was obtained as colorless needles. Its molecular formula of C15H24O3 was determined on the basis of the HR-ESIMS data (measured at *m/z* 253.17971 [M+H]+, calculated for C15H25O3+ 253.17982), corresponding to four degrees of unsaturation. The 1H NMR data (Table 1) showed characteristic signals, including three methyls at *δ*H 0.78 (3H, d, *J* = 6.4 Hz, H-10), 1.06 (3H, d, *J* = 6.4 Hz, H-11), and 2.13 (3H, s, H-12), and the proton of an aldehyde group at *δ*H 10.02 (H, s, H-15). The 1H and 13C NMR data, in association with the HSQC data, revealed three methyls, four methenes, five methines, and three nonprotonated carbons (Table 1). Preliminary analyses on the 1D NMR data revealed that **3** was likely to be a seco-sativene type sesquiterpenoid. Detailed analyses of the 2D NMR data indicated that the majority of the data of **3** was the same as those of helminthosporol [32], except for a hydroxy group at C-8 (t, *δ*C 64.6) in **3**, which was confirmed by the HMBC correlations from *δ*H 3.63 (H, d, *J* = 11.6 Hz, H-8a) and 3.71 (H, d, *J* = 11.6 Hz, H-8b) to *δ*C 58.0 (s, C-3), 29.6 (t, C-4), 167.0 (s, C-2), and 60.8 (d, C-13) (Figure 2). A ROESY experiment was carried out to establish the relative configuration of The molecular formula of bipolarisorokin B (**2**) was determined to be C15H24O<sup>3</sup> from the HRESIMS data (measured at *m/z* 275.16166 [M+Na]<sup>+</sup> , calculated for C15H24O3Na<sup>+</sup> 275.16177). Close similarities were observed in the 1D NMR data (Table 1) of compound **1**. However, signals for a methyl (*δ*<sup>H</sup> 0.94, d, *J* = 6.9 Hz, H-11; *δ*<sup>C</sup> 16.4, C-11) and an oxygenated quaternary carbon (*δ*<sup>C</sup> 73.7, C-6) in **2** was suggested to replace the oxymethylene (*δ*<sup>H</sup> 3.64, overlap, H-11; *δ*<sup>C</sup> 66.9, C-11) and the methine (*δ*<sup>H</sup> 1.65, m, H-6; *δ*<sup>C</sup> 37.6, C-6) in **1**. These observations indicated that the hydroxy group at C-10 in **1** migrated to C-6 in **2**. The observed <sup>1</sup>H−1H COSY cross-peak of H-10 (*δ*<sup>H</sup> 0.88, 3H, d, *<sup>J</sup>* = 6.9 Hz) and H-9 (*δ*<sup>H</sup> 1.57, 1H, m), and H-9/H-11, along with the HMBC correlations from H-10 to C-6, C-9, and C-11 confirmed the above deduction (Figure 2). Furthermore, ROESY correlations of H-13/H-8, H-8/H-14, H-7/H-13, and H-1/H-9 revealed that compounds **2** and **1** shared the same relative configuration. In consideration of its biosynthetic origin, the absolute configuration of compound **2** was identified the same as that of **1**.

**3** (Figure 3). The cross peaks of H-13/H-8a, H-13/H-4b, H-4b/H-6, and H-7/H-14b were observed, which indicated that H-6, H-7, H-8, and H-13 were *β* oriented. Furthermore,

0.40(17), CCDC: 2124306; Figure 4), and the absolute configuration of **3** was determined

Bipolarisorokin D (**4**) was isolated as a colorless oil. The molecular formula was determined to be C15H24O3 according to the HRESIMS spectra (measured at *m/z* 275.16153 [M+Na]+, calculated for C15H24NaO3+ 275.16177). Compound **4** had the same molecular formula and NMR spectral patterns to that of **3** (Table 2). The key difference was an oxygenated quaternary carbon (*δ*C 73.5, s) in **4** instead of the methine in **3** (*δ*C 46.4, d). The HMBC correlations from H-4a (*δ*H 1.38, m), H-5b (*δ*H 1.61, m), H-7 (*δ*H 3.16, br s), H-10 (*δ*<sup>H</sup> 1.02, d, *J* = 6.6 Hz), and H-11 (*δ*H 0.80, d, *J* = 6.6 Hz) to *δ*C 73.5 established the quaternary carbon to be C-6. In addition, a methyl (s, *δ*H 1.07, H-8; *δ*C 18.7, C-8) in **4** replaced the oxygenated methylene (*δ*C 64.6) of C-8 in **3**, which was verified by HMBC correlations from H-8 (*δ*H 1.07, s) to C-2 (*δ*C 170.4, s), C-3 (*δ*C 52.0, s), C-4 (*δ*C 32.4, t), and C-13 (*δ*C 55.3, d). Detailed analyses of 2D NMR (HSQC, HMBC, 1H-1H COSY and ROESY) data

confirmed that the other fragments of **4** were the same as those of **3**.

by ECD calculations, as shown in Figure 5.

Bipolarisorokin C (**3**) was obtained as colorless needles. Its molecular formula of C15H24O<sup>3</sup> was determined on the basis of the HR-ESIMS data (measured at *m/z* 253.17971 [M+H]<sup>+</sup> , calculated for C15H25O<sup>3</sup> <sup>+</sup> 253.17982), corresponding to four degrees of unsaturation. The <sup>1</sup>H NMR data (Table 1) showed characteristic signals, including three methyls at *δ*<sup>H</sup> 0.78 (3H, d, *J* = 6.4 Hz, H-10), 1.06 (3H, d, *J* = 6.4 Hz, H-11), and 2.13 (3H, s, H-12), and the proton of an aldehyde group at *δ*<sup>H</sup> 10.02 (H, s, H-15). The <sup>1</sup>H and <sup>13</sup>C NMR data, in association with the HSQC data, revealed three methyls, four methenes, five methines, and three nonprotonated carbons (Table 1). Preliminary analyses on the 1D NMR data revealed that **3** was likely to be a seco-sativene type sesquiterpenoid. Detailed analyses of the 2D NMR data indicated that the majority of the data of **3** was the same as those of helminthosporol [32], except for a hydroxy group at C-8 (t, *δ*<sup>C</sup> 64.6) in **3**, which was confirmed by the HMBC correlations from *δ*<sup>H</sup> 3.63 (H, d, *J* = 11.6 Hz, H-8a) and 3.71 (H, d, *J* = 11.6 Hz, H-8b) to *δ*<sup>C</sup> 58.0 (s, C-3), 29.6 (t, C-4), 167.0 (s, C-2), and 60.8 (d, C-13) (Figure 2). A ROESY experiment was carried out to establish the relative configuration of **3** (Figure 3). The cross peaks of H-13/H-8a, H-13/H-4b, H-4b/H-6, and H-7/H-14b were observed, which indicated that H-6, H-7, H-8, and H-13 were *β* oriented. Furthermore, single crystal X-ray diffraction established the relative configuration (Flack parameter = −0.40(17), CCDC: 2124306; Figure 4), and the absolute configuration of **3** was determined by ECD calculations, as shown in Figure 5.

Bipolarisorokin D (**4**) was isolated as a colorless oil. The molecular formula was determined to be C15H24O<sup>3</sup> according to the HRESIMS spectra (measured at *m/z* 275.16153 [M+Na]<sup>+</sup> , calculated for C15H24NaO<sup>3</sup> <sup>+</sup> 275.16177). Compound **4** had the same molecular formula and NMR spectral patterns to that of **3** (Table 2). The key difference was an oxygenated quaternary carbon (*δ*<sup>C</sup> 73.5, s) in **4** instead of the methine in **3** (*δ*<sup>C</sup> 46.4, d). The HMBC correlations from H-4a (*δ*<sup>H</sup> 1.38, m), H-5b (*δ*<sup>H</sup> 1.61, m), H-7 (*δ*<sup>H</sup> 3.16, br s), H-10 (*δ*<sup>H</sup> 1.02, d, *J* = 6.6 Hz), and H-11 (*δ*<sup>H</sup> 0.80, d, *J* = 6.6 Hz) to *δ*<sup>C</sup> 73.5 established the quaternary carbon to be C-6. In addition, a methyl (s, *δ*<sup>H</sup> 1.07, H-8; *δ*<sup>C</sup> 18.7, C-8) in **4** replaced the oxygenated methylene (*δ*<sup>C</sup> 64.6) of C-8 in **3**, which was verified by HMBC correlations from H-8 (*δ*<sup>H</sup> 1.07, s) to C-2 (*δ*<sup>C</sup> 170.4, s), C-3 (*δ*<sup>C</sup> 52.0, s), C-4 (*δ*<sup>C</sup> 32.4, t), and C-13 (*δ*<sup>C</sup> 55.3, d). Detailed analyses of 2D NMR (HSQC, HMBC, <sup>1</sup>H-1H COSY and ROESY) data confirmed that the other fragments of **4** were the same as those of **3**.

Bipolarisorokin E (**5**) was obtained as a colorless oil. Its molecular formula C14H22O<sup>2</sup> was characterized according to HRESIMS (measured at *m/z* 221.15529 [M–H]- , calculated for C14H21O<sup>2</sup> − 221.15470), implying four degrees of unsaturation. The general features of its NMR data closely resembled that of **3** (Table 2). Detailed analyses of 1D and 2D NMR data revealed the differences. At first, the loss of the aldehyde group at C-1 was revealed by the chemical shift of C-1 at *δ*<sup>C</sup> 124.2, along with the data from <sup>1</sup>H–1H COSY and HMBC spectra as shown in Figure 2. Second, the hydroxy migrated from C-8 to C-12 (*δ*<sup>C</sup> 59.8, t) as identified by the HMBC correlation from *δ*<sup>H</sup> 4.06 (2H, m, H-12) to *δ*<sup>C</sup> 124.2 (d, C-1), 147.2 (s, C-2), and 47.7 (s, C-3). Third, one double bond between C-9 and C-10 was built by HMBC correlations from *δ*<sup>H</sup> 4.69 (2H, d, *J* = 5.1 Hz, H-10) to *δ*<sup>C</sup> 22.7 (q, C-11) and 45.2 (d, C-6). The other parts of **5** were elucidated as the same as those of **3** by a detailed analysis of 2D NMR data.

Bipolarisorokin F (**6**) was purified as white powder, and its molecular formula C14H24O<sup>2</sup> was determinded according to HRESIMS (measured at *m/z* 225.18506 [M+H]<sup>+</sup> , calculated for C14H25O<sup>2</sup> <sup>+</sup> 225.18491). Analyses of the 1D and 2D NMR data (Table 2) suggested that **6** showed structural similarities to **3.** The distinction between the two compounds was that the *α*,*β*-unsaturated aldehyde group (*δ*<sup>C</sup> 140.0, C-1; *δ*<sup>C</sup> 167.0, C-2; *δ*<sup>C</sup> 190.0, C-15) in **3** was replaced by a carbonyl (*δ*<sup>C</sup> 212.0, C-1) and a methylene group (*δ*<sup>C</sup> 50.7, C-2) in **6**. It was supported by HMBC correlations from *δ*<sup>H</sup> 2.70 (H, br s, H-7), 0.96 (3H, q, *J* = 7.2 Hz, H-12), and 1.72 (H, dd, *J* = 7.9, 5.0 Hz, H-13) to *δ*<sup>C</sup> 212.0 (s, C-1), 50.7(d, C-2), and the COSY cross-peak of *δ*<sup>H</sup> 2.10 (1H, m, H-2) and H-12. The hydroxymethyl group (C-8) in **3** was replaced by a methyl group at C-8 (*δ*<sup>C</sup> 22.1, q) in **6**, as well as the HMBC correlations from *δ*<sup>H</sup> 1.09 (3H, s, H-8) to C-2, *δ*<sup>C</sup> 41.8 (s, C-3), *δ*<sup>C</sup> 36.1 (t, C-4), and *δ*<sup>C</sup> 54.9 (d, C-13). The key

ROESY cross-peak (Figure 3) of H-2/Ha-14 (H, dd, *J* = 10.7, 5.0 Hz, *δ*<sup>H</sup> 3.85) suggested that H-2 was *β* oriented. Other ROESY data revealed the same patterns to **3**. Finally, regarding the same origin of **6** and **3**, the absolute configuration of **6** was identified to be the same as that of **3,** as depicted. *J. Fungi* **2021**, *7*, x FOR PEER REVIEW 12 of 18

Bipolarisorokin E (**5**) was obtained as a colorless oil. Its molecular formula C14H22O2 was characterized according to HRESIMS (measured at *m/z* 221.15529 [M‒H]- , calculated for C14H21O2− 221.15470), implying four degrees of unsaturation. The general features of its NMR data closely resembled that of **3** (Table 2). Detailed analyses of 1D and 2D NMR data revealed the differences. At first, the loss of the aldehyde group at C-1 was revealed by the chemical shift of C-1 at *δ*C 124.2, along with the data from 1H–1H COSY and HMBC The molecular formula of bipolarisorokin G (**7**) was assigned as C18H26O<sup>2</sup> based on its HRESIMS spectrum (measured at *m/z* 275.20059 [M+H]<sup>+</sup> , calculated for C18H27O<sup>2</sup> + , 275.20056), which contained three more carbon atoms than **3**. The interpretation of the <sup>1</sup>H and <sup>13</sup>C NMR data of **7** (Table 3) indicated the same structure skeleton to that of **3**. Analyses of 2D NMR spectra revealed modifications in **7** (Figure 2). HMBC correlations from *δ*<sup>H</sup>

spectra as shown in Figure 2. Second, the hydroxy migrated from C-8 to C-12 (*δ*C 59.8, t) as identified by the HMBC correlation from *δ*H 4.06 (2H, m, H-12) to *δ*C 124.2 (d, C-1), 147.2

HMBC correlations from *δ*H 4.69 (2H, d, *J* = 5.1 Hz, H-10) to *δ*C 22.7 (q, C-11) and 45.2 (d,

0.97 (3H, s, H-8) to *δ*<sup>C</sup> 165.3 (s, C-2), 52.6 (s, C-3), 33.7 (t, C-4), and 63.6 (d, C-13) suggested that a hydroxy group was missing in **7**. In addition, an *α*,*β*-unsaturated ketone group was identified by the HMBC correlations from *δ*<sup>H</sup> 6.55 (H, dd, *J* = 15.9, 9.6 Hz, H-14), 6.08 (H, d, *<sup>J</sup>* = 15.9 Hz, H-16), and 2.20 (3H, s, H-18) to *<sup>δ</sup>*<sup>C</sup> 198.6 (s, C-17). In the <sup>1</sup>H−1H COSY spectrum, correlations from H-14 to *δ*<sup>H</sup> 2.22 (H, d, *J* = 9.6 Hz, H-13) and H-16 indicated that the *α*,*β*-unsaturated carboxyl moiety was located at C-13. Finally, the absolute configuration of **7** can be fully resolved by the ECD calculation, as shown in Figure 5.

Bipolarisorokin H (**8**) was obtained as a colorless oil. Its molecular formula, C17H24O3, was inferred from the pseudomolecular ion peak at m/z 277.17984 [M+H]<sup>+</sup> in the HRESIMS (calculated for C17H25O<sup>3</sup> <sup>+</sup> 277.17982). The NMR data of **8** (Table 3) resembled that of **7**, except for the presence of a carboxyl (*δ*<sup>C</sup> 171.1, C-17) in **8** instead of a carbonyl (*δ*<sup>C</sup> 198.6, C-17) in **7**, as well as the loss of a methyl group. This was supported by HMBC correlations from *δ*<sup>H</sup> 6.80 (H, dd, *J* = 15.4, 9.9 Hz, H-14) and 5.81 (H, d, *J* = 15.5 Hz, H-16) to *δ*<sup>C</sup> 171.1 (s, C-17). Detailed analyses of 2D NMR data suggested that the other data were the same as those of **7**.

Bipolarisorokin I (**9**) was isolated as colorless crystals. Its molecular formula was identified as C15H24O<sup>3</sup> by HRESIMS (measured at *m/z* 251.16621 [M–H]−, calculated for C21H23O<sup>3</sup> − 251.16527). All the spectroscopic data indicated similar patterns to those of longifolene [33]. Detailed analyses of 1D and 2D NMR data revealed the differences. Signals at *δ*<sup>C</sup> 67.0 (d, C-5), 70.5 (d, C-14), and 74.9 (d, C-15) were identified as the oxygenated methines. Therefore, three hydroxyls were suggested to be placed at C-5, C-14, and C-15, respectively, which were identified by the HMBC and <sup>1</sup>H–1H COSY correlations, as shown in Figure 2. Comprehensive analyses of other data suggested that the other parts of **9** were the same as those of longifolene. The relative configuration of **9** was revealed by a ROESY experiment, as shown in Figure 3. The ROESY correlations of Me-10/H-13, H-13/H-5, Me-8/H-13, Me-10/H-9, and Me-10/H-5 indicated these groups were cofacial (assigned as *β* orientation). In addition, the Me-11/H-1 interaction suggested that H-1 should be *α* oriented. Moreover, the coupling constant between H-14 and H-15 (*J*14,15 = 6.2 Hz), as well as the ROESY correlations of Me-8/H-14 and Me-8/H-15, suggested that H-14 and H-15 were *α* oriented. Finally, the single-crystal X-ray diffraction not only confirmed the planar structure but also established the absolute configuration of **9** (Flack parameter =0.01(3), CCDC: 2124307; Figure 4).

Bipolarithone A (**10**) was isolated as a yellow oil, and its molecular formula was determined to be C17H16O<sup>8</sup> by HRESIMS (measured at *m/z* 349.09143 [M+H]<sup>+</sup> , calculated for C17H17O<sup>8</sup> <sup>+</sup> 349.09179). The NMR data (Table 4) of **10** were similar to those of the dechlorinated methyl ester (**16**) isolated in this study [34]. The major difference was that **10** exhibited a dihydrofuran ring rather than a furan ring. HMBC correlations from H-8 (H, d, *J* = 3.9 Hz, *δ*<sup>H</sup> 5.64) to C-8a (*δ*<sup>C</sup> 114.7, s), C-7 (*δ*<sup>C</sup> 170.0, s), C-9 (*δ*<sup>C</sup> 178.3, s), and C-10a (*δ*<sup>C</sup> 167.7, s), together with H-5 (H, ddd, *J* = 6.6, 4.4, 3.9 Hz, *δ*<sup>H</sup> 5.73) to C-10a, C-8a, C-6 (*δ*<sup>C</sup> 37.7, t), and C-20 (*δ*<sup>C</sup> 169.5, s), supported the above assignment. The relative configuration of **10** was identified by the analysis of its ROESY data. The ROESY correlation between H-8 and H-5 indicated that H-8 had the same orientation as H-5 (assigned as an *α* orientation). The calculated ECD of **10** established the configuration of **10,** as shown in Figure 5. Therefore, the structure of **10** was characterized as depicted.

Bipolarithone B (**11**) was isolated as a yellow oil. The HRESIMS spectrum of **11** suggested a molecular formula of C17H16O<sup>8</sup> (measured at *m/z* 349.09157 [M+H]<sup>+</sup> , calculated for C17H17O<sup>8</sup> <sup>+</sup> 349.09179), the same as that of **10**. The planar structure of **11** was elucidated to be the same as that of **10** by the analysis of its 1D and 2D NMR data. The main difference was suggested as its stereochemistry at C-8 (*δ*<sup>C</sup> 79.8, d). Analyses of the <sup>1</sup>H NMR information showed that the coupling constants of H-8, H-5, and H-6 were significantly different from those of **11,** as shown in the Table 4. Furthermore, the ROESY correlation of H-8 (*δ*<sup>H</sup> 5.63, 1H, d, *J* = 1.7 Hz)/H-5 (*δ*<sup>H</sup> 5.62, 1H, ddd, *J* = 8.4, 3.8, 1.7 Hz) was not observed in **11**. These data suggested that **11** was an epimer of **10**. The ECD calculation for **11** was performed, and the results of **11** matched well with the experimental ECD curve (Figure 5). Hence, the absolute configuration of **11** can be fully assigned, as shown.

Bipolarithone C (**12**) was assigned a molecular formula of C30H36O<sup>9</sup> based on its HRESIMS data (measured at *m/z* 541.24310 [M+H]<sup>+</sup> , calculated for C30H37O<sup>9</sup> <sup>+</sup> 541.24321). The NMR data of **12** were very similar to those of bipolenin I (**14**) (Table 5), a novel sesquiterpenoid-xanthone adduct isolated from the fungus *Bipolaris eleusines* [35]. The significant differences were that there was an absence of an aldehyde group and two olefinic carbons, as well as the presence of an additional methine and carbonyl, in **12**. These data suggested that the *α*,*β*-unsaturated aldehyde moiety disappeared in **12**. This assignment was confirmed by the HMBC correlations of *δ*<sup>H</sup> 2.16 (H, m, H-2), 1.29 (H, m, H-6), 2.56 (1H, br s, H-7), 0.95 (3H, d, *J* = 7.2 Hz, C-12), and 1.90 (H, m, H-13) to *δ*<sup>C</sup> 50.6 (d, C-2) and 221.6 (s, C-1). The ROESY spectrum displayed similar patterns to those of **14**. Furthermore, a cross peak between H-2 and H-14a (*δ*<sup>H</sup> 4.05, 1H, dd, *J* = 11.3, 5.1 Hz) confirmed the relative configuration of C-2, as shown. The absolute configuration of **12** was elucidated by the quantum chemistry calculations. At first, the ECD calculations were conducted on the four possible conformers (**12**a–d), using time-dependent density functional theory (TDDFT) at the B3LYP/6-311G (d) level in methanol with the PCM model. The overall calculated ECD spectrum of each configuration was then generated according to the Boltzmann weighting of the conformers. As a result, the calculated ECD curves of **12**a and **12**d matched well with the experimental data (Figure 5). To determine its final structure, the theoretical NMR calculations and DP4+ probabilities were employed. The <sup>13</sup>C NMR chemical shifts of **12**a and **12**d were calculated at the mPW1PW91/6-311+G (d,p) level in the gas phase. According to the DP4+ probability analyses, **12**a was assigned with 100% probability (see data in the Supporting Information). Structurally, compound **12** comprised of a seco-sativene sesquiterpenoid unit and a xanthone unit, whose absolute configurations were in accord with compound **6** and compound **10**, respectively. Therefore, the structure of **12** was established as depicted.

Bipolarithone D (**13**) had the same molecular formula (C30H36O9) as that of **12**, according to their HRESIMS spectra (measured at *m/z* 541.24316 [M + H]<sup>+</sup> , calculated for C30H37O<sup>9</sup> <sup>+</sup> 541.24321). The NMR resonances for **13** (Table 5) resembled those of **12**, except that the resonances of C-60 (∆*δ*<sup>C</sup> + 1.5), H-60a (∆*δ*<sup>H</sup> + 0.08), and H-60b (∆*δ*<sup>H</sup> + 0.15) were shifted downfield, while the data H-50 (∆*δ*<sup>H</sup> − 0.08) were shifted upfield. A detailed comparison of the 1D and 2D NMR data of **13** with that of **12** indicated that the two compounds possessed the same planar structure. The main difference was the stereochemistry at C-80 . A key ROESY correlation of H-50/H-80 could be detected in **12** but not in **13.** In addition, the coupling constants of H-80 in **13** (*J* = 1.8 Hz) were different from that in **12** (*J* = 3.9 Hz). All the data suggested that compound **13** was a C-80 epimer of **12**. Finally, the absolute configuration of **13** was confirmed by ECD calculations (Figure 5).

Five known compounds were determined as bipolenins I and J (**14** and **15**), dechlorinated methyl ester (**16**), drechslerines A (**17**), and (+)-secolongifolene diol (**18**) by the comparison of their spectral data with that reported in the literature [32,34,35]. In this study, the absolute configurations of compounds **17** and **18** were confirmed by single crystal X-ray diffractions (Figure 6), which could support the absolute configurations of **1**–**9**, **12,** and **13** as depicted in the text, since they were obtained from the same source.

All compounds (**1**–**18**) were evaluated for their anti-Psa activity. As a result, compounds **10** and **15** showed significant inhibitory activity, with MICs of 64 and 16 µg/mL, respectively, while compounds **7**, **11**, **13**, and **16** showed moderate activity, with MICs of 128 µg/mL (Table 6).

that the resonances of C-6′ (*Δδ*C + 1.5), H-6′a (*Δδ*H + 0.08), and H-6′b (*Δδ*H + 0.15) were shifted downfield, while the data H-5′ (*Δδ*<sup>H</sup> − 0.08) were shifted upfield. A detailed comparison of the 1D and 2D NMR data of **13** with that of **12** indicated that the two compounds possessed the same planar structure. The main difference was the stereochemistry at C-8′. A key ROESY correlation of H-5′/H-8′ could be detected in **12** but not in **13.** In addition, the coupling constants of H-8′ in **13** (*J* = 1.8 Hz) were different from that in **12** (*J* = 3.9 Hz). All the data suggested that compound **13** was a C-8′ epimer of **12**. Finally, the absolute configuration of **13** was confirmed by ECD calculations (Figure 5). Five known compounds were determined as bipolenins I and J (**14** and **15**), dechlorinated methyl ester (**16**), drechslerines A (**17**), and (+)-secolongifolene diol (**18**) by the comparison of their spectral data with that reported in the literature [32,34,35]. In this study, the absolute configurations of compounds **17** and **18** were confirmed by single crystal X-ray diffractions (Figure 6), which could support the absolute configurations of **1**‒**9**, **12,** and **13** as depicted in the text, since they were obtained from the same source.

**Figure 6**. ORTEP diagrams of **17** and **18**. **Figure 6.** ORTEP diagrams of **17** and **18**.



<sup>a</sup> Compounds without any bioactivity are not listed; <sup>b</sup> Positive controls; <sup>c</sup> NA = no activity at 256 µg/mL.

In addition, our previous study on chemicals from *B. eleusines* suggested that sativenexanthone adducts have promising inhibitory activity against plant pathogenic microorganisms [35]. Therefore, all compounds were evaluated for their inhibitory activity against four plant pathogenic microorganisms, including *P*. *infestane*, *A*. *solani*, *R*. *solani*, and *F*. *oxysporum*. As a result, many compounds showed certain inhibitory activity, as given in Table 6.

A brief structure–activity relationship analysis suggested that the aldehyde-containing sativene sesquiterpenoids were more active than the others, while the xanthones or their derivatives showed better inhibitory activities than sativene sesquiterpenoids.

## **4. Conclusions**

A total of 18 compounds, including 13 new ones, were characterized from the kiwifruitassociated fungus *Bipolaris* sp. Their structures, with absolute configurations, were established by means of spectroscopic methods. Many compounds possessed anti-Psa activity and inhibitory activity against plant pathogens. It is concluded that *Bipolaris* sp. is rich in sativene sesquiterpenoids and xanthones, and both sativene sesquiterpenoids and xanthones possess potential antimicrobial application prospects. This study also suggested that it is an effective way to find natural anti-Psa agents from kiwifruit-associated fungi.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/jof8010009/s1, Section S1: Supplementary of NMR, HRESIMS and CD spectra for **1**–**13**, Section S2: Calculational details for **3**, **7**, **10**, **11**, **12** and **13**.

**Author Contributions:** Conceptualization, J.H.; methodology, J.-J.Y., Y.-X.J., S.-S.H. and J.H.; resources, J.H.; data curation, J.-J.Y.; writing—original draft preparation, J.-J.Y.; writing—review and editing, J.H.; project administration, J.H.; funding acquisition, J.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China (grant number 22177139).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** X-ray crystallographic data of **1**, **3**, **9**, and **18** (CIF) is available free of charge at https://www.ccdc.cam.ac.uk (accessed on 1 December 2021).

**Acknowledgments:** The authors thank the Analytical & Measuring Centre, South-Central University for Nationalities for the spectra measurements.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

