Emestrin-type epipolythiodioxopiperazines from Aspergillus nidulans with cytotoxic activities by regulating PI3K/AKT and mitochondrial apoptotic pathways

1 Introduction

Fungal natural products are a crucial source for drug discovery, serving as a rich reservoir for novel bioactive compounds [1–3]. Their fascinating structural diversity and biological activities hold out the ideal choices for the development of novel agents, and they also help discover novel structures, providing effective agents for the treatment of various human diseases [2, 4–6]. Epipolythiodioxopiperazines (ETPs) represent a distinct class of fungal metabolites, characterized by a di- or polysulfide-bridged dioxopiperazine ring, and derived from two amino acids. These compounds are renowned for broad-spectrum of bioactivities, including cytotoxicity against tumor cells, antibacterial function, antiviral, immunologic suppression, and anti-inflammatory action [7–10]. Based on the structure of the core ETP moiety and the modifications, emestrins are a notable group of monomeric ETPs, distinguished by a dihydrooxepino[4,3-b]pyrrole core and 15-membered macrolide [11]. Emestrin, first obtained from Emericella striata in 1985, demonstrated antifungal activity and cytotoxicity via DNA fragmentation in HL-60 cells [12–15]. To date, numerous emestrin-type ETPs have been identified, with their structural uniqueness and promising bioactivities drawing increasing scientific interest [16–22]. For instance, secoemestrin C exhibits potent anti-cancer activity in GEM-resistant and GEM-sensitive pancreatic adenocarcinoma (PAAD) cells, inducing mitochondria-mediated apoptosis and causing severe endoplasmic reticulum damage [23, 24].

Previously, we identified four emestrin hybrid polymers, asperemestrins A–D, and a highly productive ETP, secoemestrin C, which exhibited notable immunosuppressive effect, from Aspergillus nidulans [19, 25]. Recently, we found that the first cysteine-retained emestrin, nidustrin A, features a unique sulfur-containing 18-membered macrocyclic lactone [26]. Building on this work, our ongoing efforts to explore bioactive ETPs have led to the discovery of prenylemestrins C−G (1 − 5). These compounds are distinguished by a 2,5-dithia-7,9-diazabicyclo[4.2.2]decane-8,10-dione core involving a hemiterpene moiety (Fig. 1). In this study, the isolation, structural elucidation, and biological activities of these compounds were described in detail.

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2 Results and discussion

Prenylemestrin C (1) was isolated as a white powder with the molecular formula C₃₂H₃₂N₂O₁₁S₂, based on the HRESIMS spectrum with an ion peak at m/z 707.1345 ([M + Na]⁺, calcd. for 707.1345), indicating 18 degrees of unsaturation. The IR spectrum revealed the presence of hydroxyl (3419 cm⁻¹), carbonyl (1691 and 1662 cm⁻¹), and aromatic ring (1513 cm⁻¹) functionalities. The ¹H NMR data of 1 recorded in CD₃OD (Table 1) showed signals corresponding to two 1,3,4-trisubstituted benzene rings protons at δ_H 7.98 (d, J = 2.0 Hz), 7.81 (dd, J = 8.6, 2.0 Hz), and 7.19 (d, J = 8.6 Hz), and δ_H 8.42 (d, J = 2.3 Hz), 7.28 (dd, J = 8.6, 2.3 Hz), and 6.90 (d, J = 8.6 Hz), seven methines including three olefinic [δ_H 6.92 (d, J = 2.4 Hz), 6.42 (dd, J = 8.1, 2.4 Hz), and 5.01 (dd, J = 8.1, 2.3 Hz)] and three oxymethines [δ_H 5.49 (dt, J = 8.3, 2.3 Hz), 5.12 (s), and 4.72 (s)], one methylene [δ_H 3.46 (dd, J = 16.5, 1.8 Hz) and 2.89 (dd, J = 16.5, 5.3 Hz)], a methoxy group (δ_H 3.99), an N-methyl (δ_H 3.36), and two singlet methyls (δ_H 0.80, 0.68). The ¹³C NMR and DEPT spectroscopic data of 1 recorded in CD₃OD displayed 32 carbon signals (Table 2), including three carbonyls (δ_C 167.2, 167.1, 161.5), 10 nonprotonated carbons (seven olefinic and three oxygenated sp³ at δ_C 78.0, 76.5, 73.8), 14 methines (nine olefinic and five sp³), a methylene (δ_C 35.3), and four methyls (δ_C 56.8, 29.0, 26.1, 28.4).

The planar structure of compound 1 was elucidated through 2D NMR spectral analysis. The independent spin system of H-5a/H-6/H-7/H-8, along with HMBC correlations of H-10/C-5a, C-8, and C-10a, confirmed the presence of a 6,7-dihydrooxepine ring. Further HMBC spectrum showed correlations from H-11 to C-5a, C-10a, C-11a, and C-1, as well as from H-5a to C-4 and from the N-methyl protons to C-1 and C-3. These findings established that the 6,7-dihydrooxepine ring was fused to a dioxopiperazine moiety via an 11-hydroxypyrrolidine ring. Additionally, a 3′-oxygen-4′-methoxy-benzoate fragment and a 3′′-oxygen-4′′-hydroxybenzyl unit were identified as being connected to the 6,7-dihydrooxepine ring at C-6 and the dioxopiperazine ring at C-3, respectively. This connectivity was supported by the HMBC correlations from H-6 to C-7′ and from H-7′′ to C-3, C-4, C-1′′, C-2′′, and C-6′′. Moreover, the ¹H−¹H COSY correlation of H₂-12/H-13, combined with the HMBC cross-peak from H₂-12 to C-11a, H-13 to C-3, H₃-15/H₃-16 to C-13 and C-14, confirmed the attachment of an isopentan-3-ol fragment to C-11a and C-3 through sulfur atoms. Consequently, compound 1 was deduced to feature an epipolythiodioxopiperazine skeleton with a thioethanothio bridge, closely resembling the planar structure with prenylemestrins A and B (6 and 7) [17]. A detailed comparison of their ¹H and ¹³C NMR data revealed that the key difference lies in the 2,5-dithia-7,9-diazabicyclo[4.2.2]decane-8,10-dione core. HMBC correlations (Fig. 2) from H₂-12 to C-11a and from H-13 to C-3 confirmed that C-12 and C-13 were linked to C-11a and C-3, respectively. With the hemiterpene moiety switched, 1 can also be interpreted as a migration of the 2-hydroxyisopropyl group.

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To determine the relative configuration of compound 1, NMR data were recorded in DMSO-d₆ (Tables 1 and 2). The trans-orientation of H-5a and H-6 was confirmed by the large coupling constant (J = 8.3 Hz) observed between those protons [21]. The NOESY correlation between H-5a and OH-11 indicated that H-11 and H-6 were on the same side, tentatively assigned as α orientation [17]. Furthermore, NOESY correlations (Fig. 3) of H-6/H-13, H-2′/Me-15/Me-16, H-2″/Me-15/Me-16, suggested that the configuration of C-13 should be R^*. Accordingly, the C-3 − S and C-11a − S bonds were deduced to be α-oriented. Additionally, NOESY interactions of N-Me/H-7″, H-7″/H-2″, and H-7″/H-6″, OH-7″/H-6″, and OH-7″/N-Me, supported the assignment of the configurations at C-7′′ as S^*. Based on these observations, the relative configuration of 1 was established.

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Prenylemestrin D (2) was determined to share the same molecular formula, C₃₂H₃₂N₂O₁₁S₂, as compound 1, based on HRESIMS and NMR spectra. A comparison of the 1D (Tables 1 and 2) and 2D NMR spectra (Fig. 3) revealed significant similarities between the two compounds, except for the slightly different chemical shifts of C-1 (2: δ_C 170.4; 1: δ_C 167.1), C-4 (2: δ_C 165.4; 1: δ_C 161.5), and C-11a (2: δ_C 75.3; 1: δ_C 78.0), which inferred that 2 could be the C-13 epimer of 1 (Fig. 1). This deduction was further supported by NOESY interactions of H-13/N-Me and H-6/H-12a (δ_H 2.76) in 2. Additionally, the experimental ECD spectra (Fig. 4) of compounds 1 and 2 closely matched those of 6 and 7, whose absolute configurations had been previously confirmed by X-ray diffraction (Fig. 5). These findings conclusively established the absolute configurations of 1 and 2 as depicted in Fig. 1.

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Prenylemestrin E (3) was assigned the molecular formula of C₃₂H₃₂N₂O₁₁S₂, as indicated by its HRESIMS spectrum, showing an ion peak at m/z 707.1372 ([M + Na]⁺, calcd. for C₃₂H₃₂N₂O₁₁S₂Na⁺, 707.1345), corresponding 18 degrees of unsaturation. The 1D NMR data of 3 closely resembled those of 1 and 2 (Tables 1 and 2), except for the appearance of distinctive signals for a conjugated aldehyde group (δ_C 190.9 and δ_H 9.75), and a methine (C-3, δ_H 4.96, s; δ_C 65.7), which replaced the nonprotonated carbon C-3 (δ_C 76.5) in 1. These differences suggested that 3 was structurally related to secoemestrin C, with cleavage of the 15-membered macrolactone ring between C-3 and C-7′′. HMBC correlations from H-7′′ to C-1′′, C-2′′, and C-6′′ confirmed the presence and position of an aldehyde group. Moreover, HMBC interactions from H-13 to C-3, and from H₂-12 to C-11a along with the ¹H − ¹H COSY correlations of H₂-12/H-13 further established the caged core structure of 3 as consistent with that of 1 and 2. The relative configuration of 3 was deduced from the NOESY spectrum and coupling constant analysis. The coupling constants (J_H‑5a/H‑6 = 8.0 Hz) indicated that H-5a was trans-oriented. The NOESY correlation of between H-11 and H-6 indicated that H-11 is α-oriented. The observed NOESY correlation (Fig. 3) of H-6/H-13 suggested that the configuration of C-13 was R^*, with the C-3−S and C-11a−S bonds being α-oriented, and H-3 being β-oriented. Furthermore, the theoretical ¹³C NMR calculations with DP4 + analysis of two isomers13R^*-3 and 13S^*-3 were conducted through the GIAO method at the mPW1PW91/6-311G(d, p) level in chloroform with the Gaussian 09 software. The results (Fig. S1) showed that 13R^*-3 had a better coefficient of determination (R² = 0.9983) between the experimental and calculated ¹³C NMR chemical shits than 13S^*-3 (R² = 0.9964). Additionally, the DP4 + analysis with a high probability of 100% permitted the relative configuration 13R^*-3. To further confirm the absolute configuration of 3, the ECD spectra of (3R^*,5aS^*,6S^*,11R^*,11aR^*,13R^*)-3 were calculated using time-dependent density functional theory (TDDFT) methods (Tables S3 and S4). The calculated spectra matched well with the experimental data (Fig. 6), leading to the assignment of the absolute configurations of 3 as 3R,5aS,6S,11R,11aR,13R.

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Prenylemestrin F (4) was determined to have the same molecular formula, C₃₂H₃₂N₂O₁₁S₂, as compound 3, as confirmed by HRESIMS and NMR data. The ¹H and ¹³C NMR data (Tables 1 and 2) of 4 closely resembled those of 3, indicating a similar structural framework. Further interpretation of the ¹H − ¹H COSY and HMBC spectra (Fig. 2) confirmed that compound 4 shares the same ETPs core as 3. Notable differences between 3 and 4 were observed in the chemical shifts of C-1 and C-4, which were significantly downfield-shifted by + 4.5 ppm and + 4.9 ppm, respectively, in 3. Detailed ¹H − ¹H COSY and HMBC analyses confirmed that both compounds possess the same planar structure. Therefore, 3 and 4 were concluded to be stereoisomers. The NOESY interaction of H-13/N-Me was observed for 4, as opposed to H-13/H-6 in 3, which, along with careful NOESY elucidation, suggested that 4 is the C-13 epimer of 3. Furthermore, the experimental ECD spectra (Fig. 6) of compound 4 closely matched those of 3, leading to the determination of the absolute configuration of 4 as 3R,5aS,6S,11R,11aR,13S.

Prenylemestrin G (5) was assigned the same molecular formula of C₃₂H₃₂N₂O₁₁S₂ as 3 and 4, based on HRESIMS analysis. The ¹H and ¹³C NMR data of 5 (Tables 1 and 2) closely resembled those of 3. HMBC correlations (Fig. 2) from H₂-12 to C-3, H-13 to C-11a confirmed that the connection between the hemiterpene moiety and the ETP core in 5 was consistent with prenylemestrins A and B (6 and 7) [17]. NOESY correlations of H-6/H-13 (Fig. 3) indicated that H-13 in 5 adopts an α-orientation. The absolute configuration of 5 was determined by comparison its experimental ECD spectrum with the calculated one. The calculated ECD spectrum for (3R,5aS,6S,11R,11aR,13S)-5 was in good agreement with the experimental data (Fig. 6), leading to the assignment of the absolute configuration as 5 as 3R,5aS,6S,11R,11aR,13S.

Based on previous biosynthetic studies of ETPs, hypothetic biogenetic pathways of 1−7 were proposed to explain their origins (Scheme S1) with two molecules of L-phenylalanine as precursors. The key intermediate Ⅰ was obtained via a series of peptide cyclization, ring-expansion, and esterification reactions. Then, intermediate Ⅰ underwent methylation and macrocyclization to form Ⅱ. Sulfurization of the intermediate Ⅱ at C-3 and C-11a produced the key dithiol intermediate Ⅲ, which was further decorated with dimethylallyl diphosphate via pathway a or b to form Ⅲ or Ⅴ, respectively. Subsequently, the followed epoxidation and nucleophilic attack of the sulfhydryl group led to the production of 1−2 and 6−7. The intermediate Ⅵ, with the C-3−C-7′′ bond cleavaged, could be derived from Ⅲ; ultimately, compounds 3−5 could be derived via pathways c and d, with dimethylallyl diphosphate and modified farnesyl diphosphate, respectively.

In our biological evaluation, compounds 1−7 exhibited no anti-inflammatory activity. Additionally, compounds 1−5 and 7 showed no cytotoxicity against the tested cell lines (A549, L1210, HL-60, SW-480, and Hep3B). Interestingly, 6 demonstrated moderate cytotoxicity against L1210 mouse leukemia cells. Using L1210 cells as a model, we further investigated the antitumor effect and the underlying mechanism of compound 6. The results revealed that 6 inhibited proliferation and induced G2/M cell cycle arrest in L1210 cells by regulating PI3K/AKT pathway and cell cycle-related proteins, such as p-Chk1, Cyclin B, Cdc2, p-Cdc2, and γh2ax (Fig. 7). Furthermore, 6 inhibited mitochondrial membrane potential (MMP), increased ROS levels, and induced apoptosis in L1210 cells (Fig. 8). These changes in MMP and ROS levels suggest that the antitumor effects of 6 are closely related to mitochondrial dysfunction. Western blot analysis further supported this hypothesis, showing that 6 altered the expression of mitochondria-related proteins, including Bak and Bcl-xl, indicating that apoptosis induced by 6 occurs predominantly via the mitochondrial pathway. The antitumor effects of compound 6 were also validated in other leukemia cell lines, with results consistent with those observed in L1210 cells (data not shown). These findings highlight the potential of 6 as a candidate for anti-leukemia research, warranting further in-depth studies on its mechanism and therapeutic potential.

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3 Conclusion

This study elucidates the structures of prenylemestrins C−G (1−5) and highlights the moderate cytotoxicity of compound 6, which is likely attributed to its distinct structural features. Compound 6 was found to induce G2/M cell cycle arrest and apoptosis in L1210 cells, primarily through the regulation of the signaling pathway and mitochondrial apoptotic mechanisms. These findings underscore its significant impact on cell growth and survival. The discovery of prenylemestrins C−G expands the structural diversity of epipolythiodioxopiperazine (ETP) compounds and provides a solid foundation for investigating their pharmacological and medicinal potential. Future research should focus on a detailed exploration of the mechanism of action of compound 6 and its potential therapeutic applications in cancer treatment. Additionally, the biological activities and pharmacological properties of other prenylemestrins, as well as their interactions with cellular signaling pathways, warrant further study.

In conclusion, this work not only uncovers the structures and bioactivities of novel ETP compounds but also paves the way for their exploration in synthetic and pharmacological research, fostering interest in their potential applications in drug discovery and development.

4 Experimental

4.1 General experimental procedures

Using Shanghai SGW^® X-4B microscopic melting point instrument (uncorrected, China) and hot stage melting point determination to measure melting points. HRESIMS was performed on a microOTOF-Q-Ⅱ acquisition parameter (Bruker, Germany). Measure the optical rotations of compounds in methanol or dichloromethane using a Perkin Elmer 341 polarimeter. The ultraviolet (UV) spectrum was obtained with the Lambda 35 UV-1 spectrometer (PerkinElmer, US). CD curve was obtained on the J-810 spectrometer (JASCO, Japan). IR spectrum was acquired on a Nicolet iS50R FT-IR spectrometer (Thermo Scientific, US). NMR spectrum was recorded on an AM-400 and an AVANCE NEO 600 spectrometer (Bruker, Germany), with chemical shifts are expressed in ppm relative to CD₃OD (δ_C 49.0, δ_H 3.31) and CDCl₃ (δ_C 77.16, δ_H 7.26). Silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Inc., Qingdao, China) was used for normal-phase column chromatography (CC). Reversed-phase CC was accomplished using YMC-Gel (50 μm, YMC, Japan), and Sephadex LH-20 (Pharmacia Biotech AB, Uppsala, Sweden) were used for CC performed with methanol/dichloromethane as the mobile phase. Thin-layer chromatography (TLC) analyses were carried out with precoated silica gel F₂₅₄ plates (Qingdao Marine Chemical Inc., Qingdao, China). The HPLC separations were conducted on an Agilent 1220 HPLC system equipped with a UV detector, accompanied by reversed-phase Ultimate XB-C₁₈ column (5 μm, 10 × 250 mm). 10% H₂SO₄ in EtOH was used to visualize spots in TLC.

4.2 Fungal material

The fungus Aspergillus nidulans, deposited at the Huazhong University of Science and Techbology (HUST, 414JZ-1), Hubei Province, China, was isolated from the Annelida Whitmania pigra Whitman. The ITS sequence data for this strain have been submitted to DDBJ/EMBL/GenBank under accession No. MK397763.

4.3 Fermentation, extraction, and isolation

The fungus A. nidulans was maintained as seeds on potato dextrose agar (PDA) medium at 28 ℃ for 6 days, which was carried out on rice medium at room temperature for three weeks. After incubation, extract the fermented rice substrate seven times with ethanol. After removing the solvent under vacuum, the entire extract was suspended in water and then extracted with ethyl acetate to obtain a dark solid (400.0 g). Based on the TLC analysis, dark solid was chromatographed over silica gel using petroleum ether/ethyl acetate (20:1, 10:1, 5:1, 3:1, 1:1, 0:1, v/v) and ethyl acetate/methanol gradient (60:1 to methanol, v/v) as eluting solvent, to yield six subfractions (Fr.1−Fr.6). Fr.3 (1.5 g) was further separated through Sephadex LH-20 by eluting with dichloromethane/methanol (1:1, v/v), to produce three major fractions (Fr.3.1–Fr.3.3), Fr.3.3 (210 mg) was further separated through a silica gel column eluted with dichloromethane/methanol (100:1, 80:1, 50:1, v/v) and semipreparative HPLC to yield 4 (acetonitrile/water, 65:35, 2.0 mL/min; t_R 14.0 min, 2 mg). The Fr.4 (400 mg) was fractionated on a silica gel column eluted with dichloromethane/methanol (200:1, 100:1, 80:1, 50:1, v/v) to yield six major fractions (Fr.4.1–Fr.4.6), Fr.4.2 (100 mg) was further separated via a Sephadex LH-20 CC in methanol and purified by HPLC to obtain compound 6 (acetonitrile/water, 46:54, 2.0 mL/min; 27 mg; t_R 39.3 min), Fr.4.3 (89.2 mg) was separated by a Sephadex LH-20 CC in dichloromethane/methanol (1:1, v/v) and further purified by HPLC to yield compounds 2 (acetonitrile/water, 40:60, 2.0 mL/min; 10.2 mg; t_R 35.2 min) and 5 (acetonitrile/water, 42:58, 2.0 mL/min; 11.5 mg; t_R 13.0 min). The Fr.5 (12 g) was subjected to an reverse phase C₁₈ silica gel eluted with a gradient methanol/water (20%, 40%, 60%, 80%, 100%, v/v) to yield eight fractions (Fr.5.1−Fr.5.8), Fr.5.3 (260 mg) was separated through Sephadex LH-20 eluted with dichloromethane/methanol (1:1, v/v), and followed by semipreparative HPLC to yield compounds 1 (acetonitrile/water, 40:60, 2.0 mL/min; t_R 36.0 min, 20 mg) and 3 (acetonitrile/water, 40:60, 2.0 mL/min; t_R 48.5 min, 1.9 mg). Fr.5.6 (2.3 g) was chromatographed on a silica gel column by eluting with dichloromethane/methanol (100:1, 80:1, 60:1, v/v) to afford three fractions Fr.5.6.1 − Fr.5.6.3. Fr.5.6.2 (146.0 mg) was further purified to semipreparative HPLC to yield 7 (acetonitrile/water, 30:70, 2.0 mL/min; t_R 36.0 min, 38.8 mg).

Prenylemestrin C (1): White powder, [α]_D²⁰ –99 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) = 203 (4.90), 263 (4.33) nm; IR (KBr) ν_max = 3419, 2942, 2838, 1691, 1662, 1513, and 1381 cm⁻¹; ECD (MeOH) λ_max (Δε) = 221 (–50.7), 275 (–1.4), 302 (–8.8) nm; ¹H and ¹³C NMR data see Tables S1 and S2; HRMS (ESI-TOF) m/z: [M + Na]⁺ 707.1345 (calcd for C₃₂H₃₂N₂O₁₁S₂Na, 707.1345).

Prenylemestrin D (2): White powder; [α]_D²⁰ –29 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) = 262 (4.08), 202 (4.62) nm; IR (KBr) ν_max = 3423, 2974, 2954, 2934, 1675, 1653, 1513, and 1383 cm⁻¹; ECD (MeOH) λ_max (Δε) = 225 (–34.3), 258 (+ 10.0), 303 (–5.9) nm; ¹H and ¹³C NMR data see Tables S1 and S2; HRMS (ESI-TOF) m/z: [M + Na]⁺ 707.1377 (calcd for C₃₂H₃₂N₂O₁₁S₂Na, 707.1345).

Prenylemestrin E (3): White powder; [α]_D²⁰ –15 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) = 264 (4.33), 228 (4.50), 203 (4.68) nm; IR (KBr) ν_max = 3430, 2920, 2850, 1681, 1606, 1512, and 1384 cm⁻¹; ECD (MeOH) λ_max (Δε) = 224 (–25.8), 284 (–11.8) nm; ¹H and ¹³C NMR data see Tables S1 and S2; HRMS (ESI-TOF) m/z: [M + Na]⁺ 707. 1372 (calcd for C₃₂H₃₂N₂O₁₁S₂Na, 707.1345).

Prenylemestrin F (4): White powder; [α]_D²⁰ –67 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) = 263 (4.38), 226 (4.56) nm; IR (KBr) ν_max = 3429, 2922, 2850, 1681, 1605, 1511, and 1277 cm⁻¹; ECD (MeOH) λ_max (Δε) = 215 (–55.8), 253 (+ 16.8), 304 (–7.6) nm; ¹H and ¹³C NMR data see Tables S1 and S2; HRMS (ESI-TOF) m/z: [M + Na]⁺ 707.1433 (calcd for C₃₂H₃₂N₂O₁₁S₂Na, 707.1345).

Prenylemestrin G (5): White powder; [α]_D²⁰ +51 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) = 263 (4.43), 204 (4.92) nm; IR (KBr) ν_max = 3363, 2920, 2850, 1683, 1662, 1604, 1511, and 1277 cm⁻¹; ECD (MeOH) λ_max (Δε) = 221 (–59.0), 256 (+ 4.27), 274 (–0.2), 281 (+ 1.2) nm; ¹H and ¹³C NMR data see Tables S1 and S2; HRMS (ESI-TOF) m/z: [M + Na]⁺ 707.1338 (calcd for C₃₂H₃₂N₂O₁₁S₂Na, 707.1345).

4.4 X-ray crystal structure analysis

After crystallization from methanol using the vapor diffusion method, colorless crystals of 6 and 7 were obtained at room temperature. Collect crystal data using Bruker D8 Quest (Bruker, Germany) with a graphite-monochromated Cu Kα radiation. The structure was solved with the SHELXTL refinement packages using least squares minimization. All crystallographic data were stored in the Cambridge Crystallographic Data Centre (2133579 for 6 and 2133560 for 7).

Crystal data of 6. C₃₂H₃₂N₂O₁₁S₂, M = 684.72, space group P212121, a = 9.416(0) Å, b = 10.339(0), c = 33.754(0), α = β = γ = 90°, V = 3286.0(3) ų, T = 293(2) K, Z = 4, μ(Cu Kα) = 2.012 mm⁻¹, 83,642 reflections measured, 6631 independent reflections (R_int = 0.0325). The final R₁ values = 0.0332 (I > 2σ(I)), wR(F²) values = 0.1116 (I > 2σ(I)), R₁ values = 0.0334 (all data), and wR(F²) values = 0.1118 (all data). The goodness of fit on F² = 1.044. Flack parameter = 0.023(4). m.p. 181 − 184 ℃.

Crystal data of 7. C₃₂H₃₂N₂O₁₁S₂•4(H₂O), M = 748.71, space group P43212, a = 18.8206(4) Å, b = 18.8206(4) Å, c = 20.6776(4) Å, α = β = γ = 90°, V = 7324.3(3) ų, T = 100(2) K, Z = 8, μ(Cu Kα) = 1.938 mm⁻¹, 108,024 reflections measured, 7232 independent reflections (R_int = 0.0743). The final R₁ values = 0.0807 (I > 2σ(I)), wR(F²) values = 0.2240 (I > 2σ(I)), R₁ values = 0.1109 (all data), and wR(F²) values = 0.2508 (all data). The goodness of fit on F² = 1.562. Flack parameter = 0.021(7). m.p. 188 − 190 ℃.

4.5 Computational section for electronic circular dichroism (ECD)

The methodology utilized for the theoretical computation of ECD spectra, as well as the production of corresponding calculated ECD spectra, remained consistent with the previously outlined procedure[19]. The details were described in the supplementary material.

Notes

Acknowledgements

The Analytical and Testing Center and Medical Subcenter at HUST is acknowledged for the NMR data and the ECD, UV, and IR spectra collection. We thank the HPC Platform of HUST for assistance in completing computation.

Author contributions

P.L. and Q.L. isolated and purified compounds, identified them, and wrote the original manuscript; A.F. provided assistance in processes such as extraction, isolation, and structural characterisation. Y.X. performed a portion of the pharmacological experiments. C.C. and H.Z. were responsible for the verification and optimization of the manuscript and the delivery of the manuscript. W.W. and Y.Z. guided the experiments. Y-H.Z. supervised, acquired the funding, and revised the manuscript. All authors above reviewed this manuscript.

Data availability

The datasets used or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing financial interests.