Fungi from the genus Pestalotiopsis are widely distributed in diverse substrata such as plants, soil, alga, polluted steam water, and extreme environments in temperate and tropical regions (Maharachchikumbura et al., 2011, 2014). They are famous not only as phytopathogens but also as endophytes living in a well-balanced equilibrium with their host plants (Hopkins and McQuilken, 2000). The discovery of new bioactive natural products from this genus has established it as a promising candidate for bioprospecting (Xu et al., 2010; Deshmukh et al., 2017). Examples of recently discovered new compounds from Pestalotiopsis include benzophenones from the marine-derived fungus P. neglecta, which were able to suppress cell proliferation and induce apoptosis of the pancreatic cancer cell line PANC-1 at a low micromolar dosage by targeting the MEK/ERK pathway (Wang et al., 2019). Cuautepestalorin, which was isolated from a bioactive extract of Pestalotiopsis sp., was found to be active when evaluated in vitro as an α-glucosidase inhibitor (Rivera-Chavez et al., 2019).

Caryophyllene is a sesquiterpene consisting of a four-membered ring and a flexible nine-membered ring with an E-configurated double bond. Caryophyllene is known as a constituent of many essential oils from plants (Rampogu et al., 2018), but it also occurs as a natural product of plant-associated fungi such as Cytospora spp. (Li et al., 2017), Seiridium spp. (Tayone et al., 2020), Trichoderma spp. (Yu et al., 2015), and Pestalotiopsis spp. (Liu S. et al., 2016; Liu Y. et al., 2016). Some caryophyllene derivatives have also been reported from marine sources. For instance, the caryophyllene-type sesquiterpenes punctaporonins H–M were obtained from the fermentation broth of the sponge-associated fungus Hansfordia sinuosae, while punctaporonin H was reported as a new natural product capable of reducing triglycerides and total cholesterol content (Wu et al., 2014). Pestalotiopsins A and B are highly oxygenated caryophyllene derivatives from Pestalotiopsis sp. isolated from Taxus brevifolia (Pulici et al., 1996). (+)-Pestalotiopsin A bears an unprecedented oxatricyclic core structure with seven stereogenic centers, and its absolute stereochemistry was established based on an enantioselective total synthesis of both enantiomers (Takao et al., 2008, 2009).

The fungus P. lespedezae was isolated from the underground part of the medicinal plant Youngia japonica (L.) DC (Asteraceae) gathered in China. In a previous study, 10 new polyketide-terpenes, including a new iodized compound, were obtained following the addition of halogen salts such as NaCl, NaBr, and NaI to the solid rice medium (Yu et al., 2021). In the present study, we report two new caryophyllene-type sesquiterpenes pestalotiopsins U and V (1 and 2) and three known compounds pestalotiopsin B (7), pestaloporinate B (8), and pestalotiopsin C (9) from the solid rice medium culture of P. lespedezae, whereas four additional new caryophyllene pestalotiopsins W–Z (3–6) were obtained from the same fungus when cultured on solid rice medium with the addition of 3.5% NaI. All compounds were evaluated for their cytotoxicity against the mouse lymphoma cell line L5178Y, with only 6 showing significant cytotoxicity with an IC₅₀ value of 2.4 μM (Figure 1).

The HRESIMS data of 1 showed the pseudomolecular ion peaks at m/z 341.1960 [M + H]⁺, 358.2225 [M + NH₄]⁺, and 363.1780 [M + Na]⁺, which indicated the molecular formula C₁₈H₂₈O₆ with five degrees of unsaturation, containing one additional oxygen atom by comparison with that of the co-isolated known compound pestalotiopsin B (7) (Pulici et al., 1996). In the ¹H NMR spectrum, compound 1 was observed as a mixture of two conformational isomers with a ratio of approximately 80:20, which was also found for pestalotiopsin B (7) and other previously reported caryophyllene-type sesquiterpenes such as pestalotiopsins C₁ (Yu et al., 2015), M, and N (Tayone et al., 2020). The ¹H and ¹³C NMR data of 1 (Table 1) resembled those of pestalotiopsin B (7). However, signals of the one methyl group were replaced by signals of an oxygenated methylene at δ_C 70.3, δ_H 4.28 and 4.17 (CH₂-12, major conformer) in 1. The HMBC correlations from Me-13 (δ_H 1.14) to C-1 (δ_C 82.1), C-10 (δ_C 29.2), C-11 (δ_C 44.5), and C-12, as well as the HMBC correlations from H₂-12 and protons of the acetoxy group (δ_H 2.10) to the carbonyl carbon (δ_C 170.8) of the acetoxy group, confirmed the location of the acetoxy group at C-12 in 1 rather than location at C-2 in pestalotiopsin B (7). The remaining structure of 1 was found to be identical to that of 7 after the detailed analysis of the 2D NMR spectra of 1 (Figure 2). 4E and 7E geometries were assigned by NOE correlations of H-5/H-3a, H-7/H₂-14 in both conformers of 1 (Figure 3). However, characteristic differences between the two conformers were found for the ROESY correlations associated with H-5 and Me-15. In the major conformer of 1, the ROESY correlations between Me-15/H-2, Me-15/H-6, Me-15/H-9, H-9/H-2, H-9/H-6, H-9/H-10b (δ_H 1.81), and between Me-13/H-10a (δ_H 2.00) established that H-2, H-6, H-9, and Me-15 were on the same face of the ring, whereas OH-1 and Me-13 were on the opposite side. In the minor conformer, the ROESY correlations from H-5 to H-2, H-6 and H-9, and between H-9/H-10b (δ_H 1.74), Me-13/H-10a (δ_H 2.09) indicated α-orientation of H-2, H-5, H-6, and H-9, whereas OH-1 and Me-13 were β-oriented. Thus, the relative configuration of 1 was assigned as shown, and the obvious difference in the two conformational isomers was the orientation of H-5 and Me-15 caused by coupled hindered rotations regarding the C-3/C-4 and C-5/C-6 bonds. Thus, compound 1 was elucidated as a new caryophyllene derivative, for which the trivial name pestalotiopsin U is proposed.

Compound 2 had the molecular formula C₁₈H₂₈O₆ as established by the sodiated-molecular peak at m/z 363.1782 in the HRESIMS data, containing one additional oxygen atom when compared to that of pestalotiopsin B (7) (Pulici et al., 1996). The ¹H and ¹³C NMR data (Table 1) of 2 were similar to those of 7, except for the fact that two olefinic carbons were replaced by two oxygenated carbons at δ_C 65.5 (C-5) and 56.4 (C-4) in 2. An epoxy substituent at C-4/C-5 was evident from the COSY correlations between H-5 (δ_H 2.93)/H-6 (δ_H 3.80)/H-7 (δ_H 5.96), together with the HMBC correlations from Me-15 (δ_H 1.62) to C-3 (δ_C 38.8), C-4, and C-5. Detailed analysis of the 2D NMR spectra for 2 revealed that the remaining planar structure of 2 was identical to that of pestalotiopsin B (7). The double bond of C-7/C-8 was E-configurated as determined by the NOE correlation between H-7 and H-14b (δ_H 4.03). Moreover, the ROESY correlations between H-2 (δ_H 5.29)/Me-15, Me-15/H-6, H-6/H-9 (δ_H 3.28), H-9/H-2, H-2/H-3a (δ_H 2.33), H-3a/Me-15, and Me-15/H-9 indicated the cofacial orientation of these protons, and thus the relative configuration of 2 was determined as shown.

Pestalotiopsin W (3) possessed the same molecular formula as 1, according to the sodiated-molecular peak at m/z 363.1780 in the HRESIMS data. The ¹H NMR spectrum of 3 (Table 2) suggested an approximately 56:44 mixture of two conformers. Comparison of the ¹H and ¹³C NMR data of 3 and 1 suggested that their structures were closely related. The major differences found were a downfield shifted oxygenated methine (H-2, δ_H 5.23 in major conf. and 5.26 in minor conf. in 3 compared to δ_H 4.19 in major conf. and 4.14 in minor conf. in 1) together with an upfield shifted oxygenated methylene (H_ab-12, δ_H 3.84, 3.42 in major conf. and 3.60, 3.51 in minor conf. in 3 compared to δ_H 4.28, 4.17 in major conf. and 4.37, 4.27 in minor conf. in 1). These findings suggested the attachment of the acetoxy group at C-2 in 3 rather than at C-12 in 1, which was further confirmed by the COSY correlation between H_ab-3 and the above-mentioned downfield-shifted oxygenated methine together with the HMBC correlation from the latter and a methyl group at δ_H 2.03 to the carbonyl carbon δ_C 172.0 or 171.9 in both conformers of 3. Detailed analysis of the 2D NMR spectra of 3 revealed that the remaining substructure and relative configuration of 3 were identical to those of 1.

Based on the HRESIMS data, the molecular formula of 4 was deduced as C₁₈H₂₈O₆, which was identical to that of 3. Two sets of signals with a ratio of approximately 73:27 were observed in the ¹H NMR spectrum of 4 (Table 2). Analysis of the ¹H and ¹³C NMR data (Table 3) revealed structural similarity between 4 and 3. However, the replacement of signals for a hydroxymethyl group by those of an additional methyl group (Me-12) at δ_H 1.02 and δ_C 17.9 (major conf.) or at δ_H 0.99 and δ_C 19.0 (minor conf.) in 4 indicated a different location of the hydroxy group in 4. The COSY correlation between H-10 (δ_H 4.18 and 4.28 for major and minor conformers, respectively) and H-9, together with the HMBC correlations from the additional methyl group Me-12 to C-1, C-10, C-11, and C-13, confirmed the location of a hydroxy group at C-10 in 4 rather than at C-12 in 3. The remaining structure of 4 was determined to be identical to that of 3 after the detailed analysis of the 2D NMR spectra of 4. The olefinic double bonds at C-4/C-5 and C-7/C-8 were shown to be E-configurated, as indicated by the NOE correlations between H-5/H-3a and H-7/H₂-14 in both conformers of 4, respectively. In the major conformer of 4, the NOE correlations between H-6/H-5, H-5/H-9, H-9/Me-12, Me-12/H-2, H-2/H-5, and between Me-13 and H-10 indicated α-orientation of H-2, H-5, H-6, H-9, and Me-12, whereas OH-1, Me-13, and H-10 were β-orientated. Meanwhile, in the minor conformer of 4, the NOE correlations between H-6/Me-15, Me-15/H-9, H-9/Me-12, Me-12/H-2, H-2/Me-15, and between Me-13 and H-10 indicated the α-orientation of H-2, H-6, H-9, Me-12, and Me-15, whereas OH-1, Me-13, and H-10 were β-orientated. Thus, the relative configuration at C-1, C-2, C-6, and C-9 of 4 was assigned to be identical as 1 and 3, while the additional OH-10 was α-oriented in 4. It is worth mentioning that in 1 and 3, H-5 and H-9 were aligned on the opposite and the same side of the cyclononadiene ring in the major and minor conformers, respectively, whereas in 4, H-5 and H-9 were aligned on the same and the opposite side of the cyclononadiene ring in the major and minor conformers, respectively.

Compound 5 had the molecular formula C₂₁H₃₄O₈ as deduced by HRESIMS, indicating 5 degrees of unsaturation. The ¹³C NMR data of 5 (Table 3) displayed a carbonyl carbon at δ_C 172.2 and carbon signals of a double bond at δ_C 138.2 and 125.9, accounting for two of the five degrees of unsaturation, thus implying the presence of a tricyclic ring system. The ¹H and ¹³C NMR data of 5 were almost identical to those of co-isolated known tricyclic caryophyllene pestalotiopsin C (9) (Pulici et al., 1997), except for the absence of signals for a methoxy group and appearance of two additional oxygenated methylene (CH₂-16 at δ_C 71.3, δ_H 3.90 and 3.60; CH₂-18 at δ_C 64.7, δ_H 3.62 and 3.53) and an additional oxygenated methine (CH-17 at δ_C 72.4, δ_H 3.83) in 5. The COSY correlations between H-5/H-6/H-7/H-8/H-14 and between H_ab-16/H-17/H_ab-18, together with the HMBC correlations from H_ab-16 to C-7, indicated the attachment of an additional 2,3-dihydroxypropoxy group at C-7. The remaining structure of 5 was determined to be identical to that of 9 after detailed analysis of the 2D NMR data of 5. In the ROESY spectrum (Figure 4), the cross-peaks between Me-13/H-10b (δ_H 1.56), H-10b/H-8, H-8/H-7, H-7/H-14, H-14/H-8, H-14/H-5, H-5/OMe-6, H-5/H-3b (δ_H 2.46), and between H-9/H-10a (δ_H 1.98), H-10a/Me-12, Me-12/H-2, H-2/Me-15, Me-15/H-6, H-6/H-9, H-9/Me-15, and Me-15/H-3a (δ_H 2.48) indicated the relative configuration of 5 as shown, which is identical to that of structurally related pestalotiopsins A (Pulici et al., 1996), C (9) (Pulici et al., 1997), E (Putra et al., 2019) and J (Xiao et al., 2017).

The molecular formula of pestalotiopsin Z (6) was determined to be identical to that of 5 based on the HRESIMS data. Comparison of their ¹H and ¹³C NMR data (Table 2) and analysis of the 2D NMR spectra indicated that both compounds shared the same tricyclic caryophyllene core in addition to the presence of a glycerol subunit. However, the HMBC correlation from the oxygenated methine at δ_H 3.72 (H-16) in 6 instead of an oxygenated methylene in 5 to C-7 confirmed that the substituent at C-7 was (1,3-dihydroxypropan-2-yl)oxy in 6 rather than 2,3-dihydroxypropoxy in 5. The relative configuration of 6 was assigned to be identical to that of 5 by the comparison of their coupling constants and NOE relationships.

Sequencing data of the fungal genomes indicated that a large part of secondary metabolic gene clusters are silent under standard laboratory culture conditions (Debbab et al., 2013; Rutledge and Challis, 2015). In our current research, more new analogs were isolated when 3.5% NaI was added. These results, together with other reported successful examples (Wang et al., 2015; Yamazaki et al., 2015), suggested that OSMAC could be an effective approach that may result in a steep accumulation of target compounds or enhancement of diverse secondary metabolites from fungi by activating their silent gene clusters (Wiemann and Keller, 2014).

Pestalotiopsins A-T have been reported from different fungi so far (Pulici et al., 1996, 1997; Baker et al., 2008; Xiao et al., 2017; Putra et al., 2019; Tayone et al., 2020; Kang et al., 2023); however, they seldom showed cytotoxicity. In our study, all compounds were tested for cytotoxicity against the mouse lymphoma cell line L5178Y, but only compound 6 exhibited potent cytotoxicity with an IC₅₀ value of 2.4 μM compared to the positive control of 4.3 μM. The other tested compounds did not show any inhibitory effect at a concentration of 10 μM. The presence of a (1,3-dihydroxypropan-2-yl)oxy moiety in 6 rather than 2,3-dihydroxypropoxy in 5 or a methoxy group in 9 might contribute to the cytotoxicity.

In summary, six new caryophyllene-type sesquiterpenes pestalotiopsins U–Z (1–6) and three known derivatives pestalotiopsin B (7), pestaloporinate B (8), and pestalotiopsin C (9) were obtained through the fermentation of the endophytic fungus P. lespedezae on solid rice medium. Pestalotiopsins W–Z (3–6) were not detected when the fungus was grown on rice medium without the addition of 3.5% NaI. Cytotoxicity against the mouse lymphoma cell line L5178Y of all isolated compounds was evaluated, but only pestalotiopsin Z (6) showed significant activity with an IC₅₀ value of 2.4 μM.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary material.

XY contributed to extraction, isolation, and manuscript preparation. WM carried out cytotoxicity assay. MF and YG contributed to part of fungal fermentation. ZG and KZ contributed to part of structure elucidation. PP and ZL supervised the research work and revised the manuscript. All authors contributed to the article and approved the submitted version.