**1. Introduction**

Fungi have proven to be a valuable source of new secondary metabolites with a wide spectrum of biological activities [1]. Natural products from Polar fungi remain the nonnegligible sources of pharmacologically active compounds [1]. Cytosporins are a family of hexahydrobenzopyran metabolites derived from fungi with a distinct heptene side chain residue [2]. Initially isolated from endophytic *Cytospora* sp. in 1996, cytosporins were recognized as inhibitors of angiotensin II binding inhibitors [2]. To date, nearly 30 natural cytosporins of this structural class have been predominantly isolated from four genera of fungi: *Cytospora* sp. [2], *Pestalotiopsis* sp. [3], *Eutypella* sp. [4,5], and *Pseudopestalotiopsis* sp. [6]. The cytosporin family exhibits diverse bioactive effects, including cytotoxic, antibacterial, and antagonistic activity [2,4]. Besides cytosporins, *Eutypella* species have been extensively investigated as a rich source of various bioactive compounds, pimarane diterpenes, γlactones, benzopyrans, *ent*-eudesmanes, cytochalasins, and dipeptides, which display a spectrum of bioactivities [7–9].

The OSMAC approach has emerged as a powerful tool in the field of natural product biodiscovery, stimulating the production of a wider range of new metabolites [10]. During

**Citation:** Yu, H.-B.; Ning, Z.; Hu, B.; Zhu, Y.-P.; Lu, X.-L.; He, Y.; Jiao, B.-H.; Liu, X.-Y. Cytosporin Derivatives from Arctic-Derived Fungus *Eutypella* sp. D-1 via the OSMAC Approach. *Mar. Drugs* **2023**, *21*, 382. https://doi.org/ 10.3390/md21070382

Academic Editor: Dehai Li

Received: 14 June 2023 Revised: 26 June 2023 Accepted: 27 June 2023 Published: 28 June 2023

**Copyright:** © 2023 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/).

our exploration of structurally diverse bioactive natural products from polar fungi, we discovered a series of terpenoids with unique skeleton characteristics from the talented Arctic fungus strain *Eutypella* sp. D-1 [5,7,9]. This strain has proven to be a prolific source of metabolites with diverse biological activities [7,9]. To enhance the chemical diversity of *Eutypella* sp. D-1, we employed the one strain many compounds (OSMAC) strategy, utilizing different culture conditions. Through high-performance liquid chromatography (HPLC) analysis, some structural analogs during fermentation on two distinct media, solid rice medium and defined solid medium, were dramatically discovered. Subsequent chemical investigation led to the isolation of 12 cytosporin derivatives, including seven new cytosporins—eutypelleudesmane A (**1**), cytosporin Y (**2**), cytosporin Z (**3**), cytosporin Y1 (**4**), cytosporin Y2 (**5**), cytosporin Y3 (**6**), and cytosporin E1 (**7**)—together with five known biogenetic-related analogs—cytosporin X (**8**), cytosporin E (**9**), cytosporin L (**10**), and cytosporins D and F (**11**–**12**) (Figure 1). Herein, we present the detailed purification, structure elucidation, and bioactive evaluation of these compounds.

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

#### **2. Results**

Eutypelleudesmane A (**1**) was isolated as a light-brown oil. The molecular formula was determined as C36H56O7 from HRESIMS and NMR data (Table 1), indicating the presence of nine degrees of unsaturation. The IR spectra confirmed the presence of hydroxy (3357 cm−1) and carbonyl (1741 cm−1) groups [3–5]. Additionally, the 13C NMR analysis revealed one ester carbonyl signal (δ<sup>C</sup> 171.0) and six double-bond carbon signals (δ<sup>C</sup> 121.2, 124.7, 125.1, 134.1, 135.6, and 136.4), accounting for four degrees of unsaturation. The remaining five degrees of unsaturation were attributed to the pentacyclic ring structure present in the molecule.


**Table 1.** 1H (500 MHz) and 13C NMR (125 MHz) spectroscopic data of **1** in CDCl3.

Upon comparing the 1D NMR data of compound **1** and cytosporin F (**12**), it was observed that one set of signals was similar to compound **12**, while the remaining signals resembled a derivative of eudesmane-type sesquiterpene, dihydroalanto glycol. By utilizing 2D NMR correlations (Figure 2), these two structural fragments, labeled as A and B, were deduced. The COSY spectrum revealed the presence of seven continuous spin systems: (a) C-3−C-4, (b) C-6−C-7, (c) C-14–C-15–C-16–C-17–C-18–C-19–C-20, (d) C-23–C-24–C-25, (e) C-27–C-28–C-29–C-30–C-31, (f) C-29–C-33–C-34, and (g) C-33–C-35 (Figure 2). Fragment A, comprising C-2 to C-22, exhibited similarity to compound **12** based on a comparison of their 1D NMR spectra. HMBC correlations from H-4α to C-2, C-5, C-6, and C-10; from H-6 to C-8; from H-7 to C-5, C-8, and C-9; from H-10 to C-5, C-6, C-8, and C-9; from H3-11 and H3-12 to C-2 and C-3; and from H2-13 to C-8, C-9, and C-10 were detected. These correlations, along with the chemical shift of C-2 (δ<sup>C</sup> 76.6) and C-10 (δ<sup>C</sup> 67.5), indicated the formation of two six-membered rings by connecting C-5 (δ<sup>C</sup> 55.7) with C-10 and C-2 with C-10 via an *O*-atom, as well as the location of the two methyl groups CH3-11 and CH3-12 both at C-2 and one methylene group CH2-13 at C-9. The presence of an oxirane resulting from the conjugation of C-5 and C-6 via *O*-atom was supported by the downfield shift of C-5 and C-6 (δ<sup>C</sup> 59.7) [3,4]. Furthermore, the direct linkage between C-8 and C-14 was established by HMBC correlations from H-14 to C-7, C-8, and C-9. An additional acetyl group was identified to be connected to C-13 based on the HMBC correlations from H-13 and H-22 to C-21 and the chemical shift of C-13 (δ<sup>C</sup> 61.5). Fragment B, spanning from C-23 to C-37, exhibited characteristics of a eudesmane-type sesquiterpene moiety, as deduced from the analysis of the remaining 1H and 13C NMR data. HMBC correlations from H-31α and H-31β to C-27 and C-32; from H2-23 to C-27 and C-32; and from H3-36 to C-23, C-27, C-31, and C-32 confirmed the presence of a linkage of C-23, C-27, and C-31 via the quaternary carbon C-32, and placed the methyl group CH3-36 at C-32 as well. The methyl group CH3-37 was demonstrated to be connected to C-25 and C-27 via C-26 by the HMBC correlations from H3-37 to C-25, C-26, and C-27. The linkage of fragments A with B through C-3 (δ<sup>C</sup> 73.8) and C-30 (δ<sup>C</sup> 66.7) via an *O*-atom was supported by the downfield resonance of C-3 and C-30, along with HMBC correlations from H-30 to C-3. Additionally, the connection of two hydroxyl groups with a downfield carbon shift at C-7 (δ<sup>C</sup> 64.6) and C-34 (δ<sup>C</sup> 67.5) were determined to satisfy the molecular formula. Consequently, the planar structure of **1** was established as depicted.

**Figure 2.** Key COSY and HMBC correlations of **1**–**7**.

The relative configuration of **1** was established by analyzing coupling constants and NOESY experiments [9]. The *trans* configuration of the conjugated C-14/C-15 double bond was inferred based on the large coupling constant (16.0 Hz) and the NOESY correlations of H-14/H2-16. The observed similarity in the NMR chemical shift values and NOESY correlations of H-7/H-10, H-10/H3-12, H3-12/H-4β, H-4α/H-6, and H-3/H3-11 indicated that the relative configurations of fragment A in **1** were identical to those of **12** [3–5]. Additional NOESY correlations of H-30/H3-35, H-30/H-36β, and H-31β/H-36β and those of H-27/H-29, H-27/H-31α, and H-27/H-33 indicated the β-orientation and α-orientation of these protons in fragment B, respectively (Figure 3). Furthermore, the absence of a NOESY correlation between H-3 and H-30 supported the *trans* relationship between these two protons [5]. Thus, the relative structure of **1** was determined. Furthermore, the characteristic positive Cotton effect at 242 nm in the CD spectrum of **1** was virtually identical to that of cytosporins D and F (**11**–**12**) (Figure 4) [5], which suggested the absolute configuration of **1** was assigned as 3*S*,5*R*,6*S*,7*R*,10*S*,27*S*,29*S*,30*R*,32*S*,33*R*.

Cytosporin X (**2**) was obtained as a light-brown oil and determined to have a molecular formula of C19H30O4 based on HRESIMS and NMR data, corresponding to an unsaturation index of 5. The presence of hydroxy functionality was indicated by IR absorption bands at 3359 cm<sup>−</sup>1. The 13C NMR (Table 2) and DEPT spectra revealed the presence of 19 carbons, including six double-bond carbon signals (δ<sup>C</sup> 117.3, 124.6, 131.4, 131.6, 135.4, and 135.9) and five oxygenated carbon signals (δ<sup>C</sup> 57.4, 59.3, 62.2, 64.3, and 69.5). The COSY spectrum of **2** showed three distinct spin systems: C-2/C-3, C-7/C-8/C-9/C-10/C-11/C-12/C-13, and C-15/C-16 (Figure 2). HMBC correlations from H-2 to C-4 and C-6; from H-3 to C-1 and C-4; from H-6 to C-1, C-2, and C-4; and from H2-14 to C-4, C-5, and C-6, along with the comparison of the chemical shifts of C-1 (δ<sup>C</sup> 59.3) and C-2 (δ<sup>C</sup> 57.5) to those of cytosporins D and F [3,4], determined the oxirane-fused cyclohexene moiety with one methylene group (CH2-14) attached at C-5. The isoamylene group was connected to C-1 based on the HMBC correlations from H2-15 to C-1, C-2, and C-6, as well as from H3-18 and H3-19 to C-16 and C-17. Further HMBC correlations from H-7 to C-3, C-4, and C-5 established the connectivity of C-4 and C-7. With this assignment secured, each of the three oxygenated carbon at C-3 (δ<sup>C</sup> 64.3), C-6 (δ<sup>C</sup> 69.5), and C-14 (δ<sup>C</sup> 62.2) had to be substituted with a hydroxy group to satisfy the molecular formula. The relative stereocenter of 2 was determined from NOESY correlations and coupling constants in comparison with those

of **11** and **12** [3,4]. The conjugated C-7/C-8 double bond was assigned as *trans* upon its large coupling constant (16.0 Hz). The NOESY correlations of H-2/H2-15 and H-6/H2-15 in CDCl3 and 3-OH/H-6 in DMSO-*d*<sup>6</sup> (Figure S16), combined with the similarity between the calculated and the experimental ECD spectra, confirmed the absolute configurations of **2** as 1*R*,2*S*,3*R*,6*R* (Figure 4).


**Table 2.** 1H (500 MHz) and 13C NMR (125 MHz) spectroscopic data of **2** and **3** in CDCl3.

Cytosporin Y (**3**) exhibited a negative HRESIMS with a pseudomolecular ion at *m/z* 319.1912 [M − H]−, consistent with the molecular formula of C19H28O4. The similarity of the 1H and 13C NMR data between **3** and **11** indicated that compound **3** was the derivative of **11**. The presence of a pentasubstituted benzene moiety (δ<sup>H</sup> 6.61 (1H, s); δ<sup>C</sup> 115.1 (CH), 118.8 (C), 123.6 (CH), 126.8 (C), 144.8 (C), and 146.7 (C)) instead of the oxirane-fused cyclohexene moiety in **11** was suggested by the 1H and 13C NMR spectra. This was further confirmed by the further HMBC correlations from H-4β to C-5, C-6, and C-10; from H-6 to C-7, C-8, and C-10; from H2-13 to C-8, C-9, and C-10; and from H-14 to C-7, C-8, and C-9 (Figure 2). Additionally, one hydroxy group was attached to C-7, as evidenced by its chemical shift (δ<sup>C</sup> 146.7) and the molecular formula. The conjugated C-14/C-15 double bond in **3** was assigned as *trans* based on the similar 1H NMR chemical shift values and coupling constants (16.5 Hz) observed in **3** and **2**. To determine the absolute configuration at C-3 in compound **3**, the specific rotation ([*α*] 25 *<sup>D</sup>* +12.3, MeOH, *c* 0.1 and [*α*] 25 *<sup>D</sup>* +1.9, CDCl3, *c* 0.1) was measured. The configuration of C-3 could be assigned as *S* by comparison to the literature data for synthetic (*R*)-2,2-dimethylchromane-3,7-diol ([*α*] 20 *<sup>D</sup>* −1.2, MeOH, *c* 0.03) [11] and (*S*)-2,2-dimethylchromane-3,7-diol ([*α*] 23 *<sup>D</sup>* +11.5, CHCl3, *c* 1.0) [12] (differences in measured versus literature values likely stem from the different concentration and solvent).

**Figure 3.** Key NOESY correlations of **1**, **2**, and **4**–**7**.

**Figure 4.** ECD spectra of **1**, **8**, and **10**–**12** and calculated and experimental ECD spectra of **2**.

Cytosporin Y1 (**4**), in the form of a light-yellow oil, had a molecular formula of C19H32O5 based on HRESIMS (*m/z* 363.2139 [M + Na]+), which is larger than that of cytosporin Y (**2**) by 18 amu. The NMR data of **4** (Table 3) were nearly identical to those of **2**, indicating the same carbon skeleton. Considering the degrees of unsaturation of **4**, the observed downfield shift of one quaternary carbon (δ<sup>C</sup> 59.3) and one methine (δH/δ<sup>C</sup> 3.29/57.5) in **2** to δ<sup>C</sup> 74.3 and δH/δ<sup>C</sup> 3.76/75.0 in **4**, respectively, suggested that **4** was the oxirane ring-opening product of **2**. This hypothesis was further supported by further COSY and key HMBC correlations, as shown in Figure 2. The *E*-geometry of the Δ7,8 double bond was deduced from a NOESY correlation between H-7 and H2-9, as well as the coupling constants (16.5 Hz). Additional NOESY correlations of H-2/H2-15, H-2/H-6, H-3/H-6, and H-6/H2-15 indicated the same orientation of these protons. Furthermore, the comparison of the calculated and the experimental ECD spectra confirmed the absolute configurations of **4** as 1*S*,2*R*,3*R*,6*S* (Figure 5).


**Table 3.** 1H (500 MHz) and 13C NMR (125 MHz) spectroscopic data of **4** and **5** in MeOD-*d*4.

**Figure 5.** Calculated and experimental ECD spectra of **4** and **5**.

Cytosporin Y2 (**5**) was obtained as a light-yellow oil. Extensive NMR analyses and HRESIMS data (*m/z* 389.1928 [M + Na]+) led to the determination of its molecular formula as C20H30O6. The overall NMR data of **5** indicated a structure similar to **4**, with the notable difference of an additional quaternary carbon. This carbon was identified as a carbonate moiety based on the strong IR absorption at 1647 cm−<sup>1</sup> and the diagnostic 13C NMR signal at δ<sup>C</sup> 154.8 [4]. Another significant difference was observed for C-1 and C-2, resonating at δ<sup>C</sup> 74.3 and 75.0 in compound **4**, whereas in compound **5**, these signals resonated at δ<sup>C</sup> 84.4 and 71.3, respectively (Table 3). This observation, along with the key HMBC correlations from H-2 to C-20, led to the linkage of the carbonyl to both oxygen atoms at C-1 and C-2 to form a cyclic carbonate moiety. The relative configurations of **5** were determined via a detailed analysis of the NOESY correlations of H-2/H-15a, H-3/H-15b, H-6/H-15a, H-6/H-15b, and H-7/H2-9, as well as the coupling constants (16.5 Hz) of H-7/H2-9. Furthermore, the calculated ECD spectrum of **5** exhibited a close resemblance to the experimental one, confirming the absolute configuration as 1*R*,2*R*,3*R*,6*S* (Figure 5).

Cytosporin Y3 (**6**) was isolated as a light-yellow oil. Its molecular formula was determined to be C20H30O6, the same as that of **5**, based on HRESIMS data. A comparison of the IR, UV, and NMR data (Table 4) of **6** with those of **5** suggested that **6** was an isomer of **5**. Further analysis of the 13C NMR chemical shift of C-2 (δ<sup>C</sup> 79.2) and C-3 (δ<sup>C</sup> 75.2), along with the unambiguous HMBC correlations from H-2 and H-3 to C-20 of **2**, revealed that the cyclic carbonate moiety was fused with C-2-C-3 in **6**. The relative configuration and *E*-geometry of the Δ7,8 double bond in **6** were determined from the NOESY correlations of H-2/H-3, H-2/H-6, H-3/H-6, H-2/H-15b, H-6/H-15a, H-6/H-15b, and H-7/H2-9. The absolute configuration of **6** was subsequently determined to be 1*R*,2*S*,3*S*,6*R* based on the opposite CD spectra (Figure 6) and a comparison of the specific rotation ([*α*] 20 *<sup>D</sup>* +37.8, MeOH, *c* 0.1) with that of **4** ([*α*] 20 *<sup>D</sup>* −15.8, MeOH, *c* 0.1) and **5** ([*α*] 20 *<sup>D</sup>* −60.1, MeOH, *c* 0.1) (Figure 6).


**Table 4.** 1H (500 MHz) and 13C NMR (125 MHz) spectroscopic data of **6** and **7** in CDCl3.

**Figure 6.** Calculated and experimental ECD spectra of **6** and **7**.

Cytosporin E1 (**7**) was also purified as a light-yellow oil and exhibited a HRESIMS ion peak at *m/z* 407.2034 [M + Na]+, consistent with the molecular formula C20H32O7 with five degrees of unsaturation. The 1H and 13C NMR data of **7** (Table 4) closely resembled those of the known compound cytosporin E (**9**), except for two additional methylenes (δC/δ<sup>H</sup> 31.0/2.26 and 30.0/1.49) in **7** and the absence of two olefinic methines (δC/δ<sup>H</sup> 123.5/6.39 and 137.1/6.03) in **9**. These observations indicated that C-8 in **7** was substituted by a heptane subunit instead of the 1-heptene part in **9**, which was also confirmed by COSY correlations of H2-14 (δ<sup>H</sup> 2.26)/H2-15 (δ<sup>H</sup> 1.49), H2-15/H2-16 (δ<sup>H</sup> 2.25), H2-16/H2-17 (δ<sup>H</sup> 1.35), H2- 17/H2-18 (δ<sup>H</sup> 1.31), H2-18/H2-19 (δ<sup>H</sup> 1.32), and H2-19/H3-20 (δ<sup>H</sup> 0.90), as well as HMBC correlations from H2-14 to C-7 (δ<sup>C</sup> 78.5) and C-8 (δ<sup>C</sup> 133.3). The relative configuration of **7** was inferred to be different from that of compound **9** through a comparison of the 13C NMR data between **7** (C-6 δ<sup>C</sup> 81.3 and C-7 δ<sup>C</sup> 78.5) and **9** (C-6 δ<sup>C</sup> 81.0 and C-7 δ<sup>C</sup> 75.3), as well as the analysis of NOESY correlations of H-3/H3-11, H-4α/H-6, H-4α/H-7, H-4β/H-10, H-4β/H3-12, and H-10/H3-12 in MeOD-*d*<sup>4</sup> and 3-OH/H-10 and 5-OH/H-10 in DMSO-*d*<sup>6</sup> (Figure S75). The absolute configurations of **7** were subsequently assigned as 3*S*,5*R*,6*S*,7*S*,10*S* based on the similarity of its calculated and the experimental ECD spectra (Figure 6).

In addition to the seven new compounds **1**–**7**, the five known cytosporins—cytosporin X (**8**) [13], cytosporin E (**9**) [4], cytosporin L (**10**) [14], cytosporin D (**11**) [4], and cytosporin F (**12**) [3]—were also isolated and identified through a comparison of its NMR spectroscopic data with reported values in the literature.

Structurally, considering the close relationship in biosynthesis among compounds **1**–**12**, a biosynthetic pathway different from the previous literature for these compounds is proposed (Figure 7) [3]. The possible precursor originated from phenylmethanol [3]. The subsequent addition of an isoprenyl unit, followed by hydroxylation and the addition of an aliphatic chain, would give the intermediate i. The hydroxylation of the C-1/C-6 double bond in i gave rise to the key intermediate **4**. Compound **2** was derived from **4** via a dehydration cyclization reaction. Compound **3** was generated from i via the epoxidation of the C-16/C-17 double bond and a cyclization reaction. Compounds **5** and **6** were derived from the dehydration reaction of compound **4** with carbonic acid by different attack directions and substitution positions, respectively. Compounds **7**, **10**, and **11** were obtained from **6**, **4**, and **2** via the same cyclization reaction as **3**, respectively. Compound **9** was derived from **10** via the carbonic acid substitution, while compound **8** was formed through the hydrogenation of **11**. The cyclization of compound **2**, followed by an acetylation reaction, resulted in the formation of compound **12**. Another possible precursor, the eudesmane-type sesquiterpene dihydroalanto glycol, was generated from farnesyl pyrophosphate with two steps of cyclization, dehydrogenation, and hydroxylation reaction [15]. Then, **1** was formed from the above two precursors, ii and **12**, via a condensation reaction.

All the isolated compounds **1**–**12** were evaluated for their cytotoxicity against four human cancer cell lines, including DU145, SW1990, Huh7, and PANC-1, and antibacterial activity against *Staphylococcus aureus*, *Escherichia coli*, and *Bacillus subtilis*. Unfortunately, all compounds were not active during the above test, with IC50 values higher than 50 μM or MIC values higher than 128 μg/mL. Additional immunosuppressive activity against ConA-induced T cell proliferation for **1**–**12** was also tested. However, only compounds **3**, **6**, **8**, and **10**–**11** displayed immunosuppressive activity, demonstrating inhibitory rates of 62.9%, 59.5%, 67.8%, 55.8%, and 68.7%, respectively, at a concentration of 5 μg/mL.

**Figure 7.** Proposed biogenesis pathway of **1**–**12**.

### **3. Materials and Methods**

#### *3.1. General Experimental Procedures*

Specific rotations and IR (KBr) data were measured on a PerkinElmer model 341 polarimeter (Perkin-Elmer Inc., Waltham, MA, USA) and Jasco FTIR400 spectrometer (Jasco Inc., Tokyo, Japan), respectively. CD and UV spectra were obtained on a Jasco J-715 spectropolarimeter (Jasco Inc., Tokyo, Japan) and UV-8000 spectrophotometer (Shanghai Metash instruments Co., Shanghai, China) in MeOH, respectively. 1D and 2D NMR spectra were acquired using a Bruker AMX-500 instrument (500 MHz for 1H NMR, 125 MHz for 13C NMR) (Bruker Biospin Corp., Billerica, MA, USA) at room temperature. HRESIMS data were measured on an Agilent 6210 LC/MSD TOF mass spectrometer (Agilent Technologies Inc. Lake Forest, CA, USA). HPLC separation was performed using a YMC-Pack Pro C18 (5 μm) column (YMC Co. Ltd., Kyoto, Japan) using a Waters 1525 separation module equipped with a Waters 996 Photodiode Array (PDA) detector (Waters Corp., Milford, MA, USA). Column chromatographic purifications were performed on silica gel 60 (200–300 mesh, Qingdao Ocean Chemical Co., Qingdao, China), ODS (50 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (Pharmacia Co., Piscataway, NJ, USA).
