Abstract
Peniadenisteroid A, also known as striasteroid C, is a unique marine-derived natural steroidal alkaloid with a characteristic adenine–steroid hybrid scaffold, in which an adenine moiety is bound to the C-3 position of the steroid skeleton. This natural metabolite displays evident antiviral activity through neuraminidase inhibition, with an IC50 value of 6.15 μM. In this work, the C-3 epimer, (3S)-peniadenisteroid A, was successfully synthesized in eight steps with an overall yield of 3.9%. Biological screening results revealed that the intermediate (3S)-P6 exhibited prominent inhibitory activity against cellular nitric oxide (NO) production, while both (3S)-peniadenisteroid A and peniadenisteroid A presented weak anti-inflammatory effects. Further mechanistic studies indicated that (3S)-P6 exerts significant anti-inflammatory bioactivity by negatively regulating the STAT3/NF-κB and HIF-1α signaling pathways. Moreover, network pharmacology combined with molecular docking predicted Heat Shock Protein 90α (HSP90α) as the putative binding target of (3S)-P6. Overall, this study completes the synthesis and biological evaluation of (3S)-peniadenisteroid A, highlighting that intermediate (3S)-P6 is a valuable lead compound for further anti-inflammatory drug discovery.
Keywords: peniadenisteroid A, striasteroid C, (3S)-peniadenisteroid A, steroidal alkaloid, synthesis, anti-inflammatory, network pharmacology
1. Introduction
Steroidal alkaloids are an important class of secondary metabolites, predominantly distributed in plant families including Solanaceae, Buxaceae, Apocynaceae, and Liliaceae [1,2]. Structurally, they possess a fundamental steroidal skeleton integrated with a nitrogen atom, which is embedded either in the ring system or the side chain (Figure 1) [1]. Steroidal alkaloids and their derivatives, exemplified by cyclopamine, solasodine, and tomatidine (Figure 1), exhibit diverse pharmacological properties, including potent anticancer, anti-inflammatory, and antibacterial activities [1,2,3,4]. Given their unique structural features and versatile biological activities, steroidal alkaloids have garnered substantial research interest, especially in the exploration of marine natural products.
Figure 1.
Chemical structures of representative steroidal alkaloid natural products. * Denotes a chiral center.
The first reported marine-derived steroidal alkaloids, plakinamine A and plakinamine B (Figure 1), were isolated from the marine sponge Plakina sp. by Rosser et al. in 1984 [5]. Subsequent biological tests demonstrated that plakinamine A suppressed the growth of Staphylococcus aureus and Candida albicans at doses of 25 μg/disk and 10 μg/disk, respectively. Meanwhile, the hydrochloride derivative of plakinamine B exerted inhibitory effects on the same pathogens at lower doses of 10 μg/disk and 2 μg/disk [5]. Furthermore, cortistatins represent the most extensively investigated subclass of marine-derived steroidal alkaloids. These compounds are characterized by a highly functionalized steroidal core with multiple chiral centers, which is conjugated to nitrogen-containing heterocyclic moieties such as isoquinoline. As illustrated in Figure 1, cortistatin A was first isolated from the marine sponge Corticium simplex by Aoki et al. in 2006 and it was reported with an anti-angiogenic activity against human umbilical vein endothelial cells (HUVECs), with an IC50 value of 1.8 nM [6]. In addition, Nicolaou et al. reported in 2009 that cortistatin A serves as a high-affinity ligand for the protein kinases Rho-associated coiled-coil containing protein kinase (ROCK), cyclin-dependent kinase 8 (CDK8), and cyclin-dependent kinase 11 (CDK11) [7]. Cephalostatin 1 (Figure 1), another representative steroidal alkaloid isolated from the marine worm Cephalodiscus gilchristi, possesses a unique bissteroidal skeleton and represents a structurally and biologically significant member of this compound class [8]. It exhibits exceptionally potent antitumor activity, with an ED50 value of less than 1 pM against P388 leukemia cells and potent cytotoxic activity against all 60 human tumor cell lines in the National Cancer Institute (NCI) screening panel [8,9,10].
Inspired by the novel chemical structures, diverse and excellent pharmacological activities, as well as high drug-likeness of steroidal alkaloids, we carried out research on the discovery and bioactivity evaluation of steroidal alkaloids from marine-derived microorganisms. Subsequently, a novel steroidal alkaloid, named peniadenisteroid A (Figure 1), was isolated and characterized from a deep-sea-sediment-derived fungus Penicillium janthinellum SH0301, and its structure was established by NMR, HRMS, and ECD analyses. Structurally, peniadenisteroid A consists of an ergostane-type steroid and a purine moiety, which are linked via a C-N bond between C-3 position of the steroid and N-9 position of the purine. The absolute configuration at C-3 was proposed to be R-configuration. Unfortunately, cytotoxic and anti-inflammatory activity screening revealed that it only exhibited weak inhibitory activity against NO production in LPS-induced RAW264.7 macrophages, with an inhibition rate of 43% at 20 μM. In 2021, Dong et al. isolated peniadenisteroid A from the mangrove sediment-derived fungus Penicillium brefeldianum ABC190807 [11]. Most recently, Zhou et al. reported the isolation of peniadenisteroid A from the marine-derived fungus Striaticonidium cinctum SCSIO41432 and named it striasteroid C. Notably, they unambiguously determined its absolute configuration for the first time via X-ray diffraction analysis, revealing an R-configuration at the C-3 stereo center. Meanwhile, they reported that peniadenisteroid A exhibited potent neuraminidase-inhibitory activity with an IC50 value of 6.15 μM [12].
Further investigations on peniadenisteroid A are hindered by its limited natural availability, due to its low abundance and complex extraction process. Furthermore, the effect of the C-3 chiral center on the biological activity of this compound remains to be elucidated, and no synthetic strategy for peniadenisteroid A or its diastereomers has been reported to date. Therefore, this study aims to explore the synthesis of peniadenisteroid A and its C-3 diastereomer, preliminarily compare the activity differences between analogs with opposite configurations at the C-3 position of the steroid nucleus, and to perform systematic bioactivity screening of structurally novel synthetic intermediates in order to identify lead candidates with therapeutic potential.
2. Results
2.1. Chemistry
Herein, a rational retrosynthetic analysis was performed for peniadenisteroid A and its C-3 epimer, (3S)-peniadenisteroid A (Figure 2). Both target compounds could be constructed via the Traube purine synthesis, in which the key chiral 4,5-diaminopyrimidine intermediate undergoes cyclization with trimethoxymethane. This chiral diaminopyrimidine intermediate would be available through a nucleophilic substitution reaction between 5-amino-4,6-dichloropyrimidine and a chiral steroidal amine precursor. The chiral steroidal amine could be synthesized from (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one by employing Ellman’s tert-butanesulfinamide-mediated asymmetric amine synthesis. (22E,24R)-ergosta-4,6,8(14),22-tetraen-3-one, in turn, could be prepared via DDQ oxidation of (22E,24R)-ergosta-4,7,22-trien-3-one, which would be ultimately derived from the oxidation of ergosterol.
Figure 2.
Retrosynthetic analysis of compound peniadenisteroid A and (3S)-peniadenisteroid A. * Denotes a chiral center.
Guided by reasonable retrosynthetic analysis, we performed synthetic studies of peniadenisteroid A and (3S)-peniadenisteroid A and the synthetic routes were summarized in Scheme 1. As shown in Scheme 1, compound (22E,24R)-ergosta-4,7,22-trien-3-one (P2) was efficiently prepared by oxidation of commercially available ergosterol (P1) in 70% yield via Oppenauer oxidation [13]. However, the subsequent oxidation of P2 to P3 turned out to be a challenging transformation. Initial attempts employing Jones reagent as the oxidant in THF at room temperature failed to obtain the desired product P3 (Table 1, entry 1) [14,15]. Optimizations regarding reaction temperature, solvent, and oxidant equivalents were then conducted, yet none of these modifications enhanced the reaction efficiency (Table 1, entries 2–4). Further screening of alternative oxidants, including PCC, PDC, IBX, and DMP, also afforded no improvement in oxidation yields (Table 1, entries 5–12) [16,17,18,19]. Gratifyingly, the adoption of DDQ as the oxidant in 1,4-dioxane at 40 °C moderately promoted the transformation, furnishing P3 in 19% yield, which was higher than that obtained under the room-temperature conditions (Table 1, entries 13–15). Graduated elevation of the reaction temperature to 110 °C combined with a shortened reaction duration of 4 h further improved the outcome, delivering P3 with a yield of 39% (Table 1, entry 16). In contrast, prolonged reaction time led to a slight reduction in product yield (Table 1, entry 17). Ultimately, the optimal reaction conditions for the synthesis of P3 are: DDQ as the oxidant, 1,4-dioxane as the solvent at 110 °C for 4 h. To our delight, when the reaction was scaled up to the 10 g scale, no decrease in yield was observed.
Scheme 1.
Synthesis of compound P3. Reagents and conditions: (a) aluminium isopropoxide, cyclohexanone, toluene, rt-115 °C, 3 h, 70%; (b) DDQ, 1,4-dioxane, 110 °C, 4 h, 39%.
Table 1.
Optimization of reaction conditions for the synthesis of compound P3.
| ||||||
| Entry | Reagent | Equivalent | Temperature | Time | Solvent | Yield |
| 1 | Jones | 4 | 25 °C | 12 h | THF | trace |
| 2 | Jones | 4 | 40 °C | 12 h | Toluene | 7% |
| 3 | Jones | 4 | 40 °C | 12 h | 1,4-dioxane | 11% |
| 4 | Jones | 8 | 40 °C | 12 h | 1,4-dioxane | 8% |
| 5 | PCC | 4 | 25 °C | 12 h | CH2Cl2 | 8% |
| 6 | PCC | 4 | 40 °C | 12 h | 1,4-dioxane | trace |
| 7 | PCC | 8 | 40 °C | 12 h | 1,4-dioxane | trace |
| 8 | PDC | 4 | 25 °C | 12 h | CH2Cl2 | trace |
| 9 | IBX | 4 | 25 °C | 12 h | CH2Cl2 | trace |
| 10 | IBX | 4 | 40 °C | 12 h | 1,4-dioxane | trace |
| 11 | DMP | 4 | 25 °C | 12 h | CH2Cl2 | trace |
| 12 | DMP | 4 | 40 °C | 12 h | 1,4-dioxane | trace |
| 13 | DDQ | 4 | 25 °C | 12 h | CH2Cl2 | 6% |
| 14 | DDQ | 4 | 25 °C | 36 h | CH2Cl2 | 10% |
| 15 | DDQ | 4 | 40 °C | 12 h | 1,4-dioxane | 19% |
| 16 | DDQ | 4 | 110 °C | 4 h | 1,4-dioxane | 39% |
| 17 | DDQ | 4 | 110 °C | 6 h | 1,4-dioxane | 30% |
As depicted in Scheme 2, following the successful preparation of sufficient P3 intermediate, we proceeded to construct the C-3 stereo center on the steroid scaffold. On the basis of the aforementioned retrosynthetic analysis, we sought to install the C-3 stereo center through chiral induction using Ellman’s tert-butanesulfinamide of different chirality to afford the target chiral steroidal amines [20,21]. Subsequently, intermediate P3 was reacted with the (S)- and (R)-configured tert-butylsulfinimine to afford the corresponding imine intermediates P4a and P4b, respectively. Reduction of compound P4a with DIBAL-H, followed by deprotection of the tert-butanesulfinyl group, afforded the C-3 S-configured steroidal amine (3S)-P6. However, following the same procedure that was used to prepare compound (3S)-P6 from compound P4b failed to obtain intermediate (3R)-P6. By comparing the reaction product of P5a, (3S)-P6, with that of P5b, we conclude that the reduction-deprotection product of P4b is (3S)-P6 rather than (3R)-P6 (Scheme 2 and Figure 3D). We speculate that the C-3 R-configured steroidal amine intermediate was not successfully synthesized. Accordingly, we performed a systematic analysis of the reduction product P5a/b derived from intermediate P4a/b using mass spectrometry, NMR, HPLC and ECD spectroscopy.
Scheme 2.
Synthesis of compound (3S)-P6. Reagents and conditions: (a) (S)-2-methylpropane-2-sulfinamide for P4a and (R)-2-methylpropane-2-sulfinamide for P4b, titanium tetraisopropanolate, THF, 70 °C, 24 h; (b) DIBAL-H, THF, −78 °C-rt, 5 h, 52% in two steps for P5a and 62% in two steps for P5b; (c) 2M HCl in 1,4-dioxane, rt, 4 h, 80%. Solid arrows: successful reactions; dotted arrows: planned but unattempted routes; crossed arrows: failed reactions.
Figure 3.
Absolute configuration studies of P5a, P5b, product from P4a and product from P4b. (A) Chiral HPLC profiles of compounds P5a and P5b. (B) Chiral HPLC analysis: P5a co-injected with P5b. (C) ECD spectral comparison of P5a and P5b for absolute configuration. (D) ECD spectral comparison of product from P4a and product from P4b for absolute configuration.
As shown in Figures S5–S8, S20 and S21, compounds P5a and P5b showed identical molecular weights (m/z (ESI) [M + H]+ = 498) and nearly identical NMR data. The HPLC analysis revealed that compounds P5a and P5b exhibited similar retention times (Figure 3A,B). Additionally, if the two compounds indeed vary at two stereogenic centers, their ECD profiles should not exhibit such minor differences; nevertheless, the measured ECD differences were considerably smaller than expected. These unexpectedly small ECD differences led us to suspect that the two compounds may in fact differ at only a single stereogenic center. (Figure 3C). Further 2D NOESY experiments on P5a and P5b showed no NOE cross-peak between H-3 and the C-19 angular methyl protons in either compound, suggesting that both share the same C-3 stereochemistry and are likely of the S configuration (Figures S22 and S23). Taken together, we concluded that reduction of P4b with DIBAL-H led to compound P5b rather than expected compound P5c (Scheme 2). The DIBAL-H reduction of the imine appears to be governed primarily by the pre-existing stereochemistry of the steroid skeleton rather than by the sulfur configuration of the Ellman auxiliary; this accounts for both P4a and P4b furnishing the same (3S) amine. Herein, although we failed to obtain (3R)-P6, (3S)-P6 was successfully synthesized, enabling us to proceed with the synthesis of (3S)-peniadenisteroid A following the aforementioned retrosynthetic analysis.
Subsequently, we initiated the synthesis of (3S)-peniadenisteroid A, and the results are illustrated in Scheme 3. Compound (3S)-P6 was reacted with NaHCO3, followed by nucleophilic substitution with 5-amino-4,6-dichloropyrimidine to furnish intermediate (3S)-P7 in 66% yield. Acid-catalyzed intramolecular cyclization of (3S)-P7 using trimethoxymethane then afforded the cyclized intermediate (3S)-P8 [22]. Finally, treatment of (3S)-P8 with 7 M ammonia in methanol delivered the target product (3S)-peniadenisteroid A in 69% yield.
Scheme 3.
Semi-synthesis of (3S)-peniadenisteroid A. Reagents and conditions: (a) 5-amino-4,6-dichloropyrimidine, NaHCO3, n-butanol, 118 °C, 24 h, 66%; (b) trimethoxymethane, conc. HCl, rt, 6 h, 64%; (c) ammonia (7M in MeOH), THF, 70 °C, 30 h, 69%. * Denotes a chiral center.
2.2. Characteristic Analysis
The high-resolution electron spray ionization mass (HRMS-ESI) of compound (3S)-peniadenisteroid A exhibited [M + H]+ peak at m/z 512.3751 (C33H46N5, calcd for 512.3748) (Figure S19) corresponding to the reported molecular formula of compound striasteroid C [12]. Whereas the 1H and 13C NMR data of natural striasteroid C and our isolated peniadenisteroid A were in close agreement, the synthesized epimer (3S)-peniadenisteroid A exhibited substantial discrepancies from both. As shown in Table 2, the 1H NMR spectrum of peniadenisteroid A recorded in CDCl3 exhibited the C-3 methine proton at δH 5.31 (t, J = 5.1 Hz) and the 13C NMR signal at δC 49.0 ppm, which were essentially identical to the values reported for striasteroid C (δH 5.32, t, J = 5.0 Hz; δC 49.1 ppm). However, (3S)-peniadenisteroid A showed different chemical shifts around the C-3 stereocenter, with H-3 observed at δH 5.37 (t, J = 5.0 Hz) and C-3 at δC 52.0 ppm. The neighboring H-4 signal also shifted upfield to δH 5.44 (d, J = 4.8 Hz), compared with δH 5.56 (d, J = 4.9 Hz) for peniadenisteroid A, and the C-4 carbon signal moved downfield from δC 119.9 to δC 116.8 ppm. Divergences were further noted at C-1 (δC 33.2 vs. 29.6), C-2 (δC 27.9 vs. 25.7). (Table 2, Figures S15–S18). These differences in the C-3/H-3 region and adjacent carbons are suggestive of an epimeric relationship between the synthesized compound and the natural product, an assignment that will be further substantiated by ECD, chiral-phase HPLC, and single-crystal X-ray analyses.
Table 2.
1H NMR and 13C NMR data of compound peniadenisteroid A and (3S)-peniadenisteroid A.
| Position | Striasteroid C (CDCl3, δ in ppm) a |
Peniadenisteroid A (CDCl3, δ in ppm) b | (3S)-Peniadenisteroid A (CDCl3, δ in ppm) c | |||
|---|---|---|---|---|---|---|
| 1H NMR | 13C NMR | 1H NMR | 13C NMR | 1H NMR | 13C NMR | |
| 1 | 1.62 d 1.31 d |
29.5 | 1.62 (m) 1.30 (m) |
29.6 | 1.79 (m) 1.45 (m) |
33.2 |
| 2 | 2.03 (m) 2.23 (m) |
25.6 | 2.03 (m) 2.23 (m) |
25.7 | 1.93 (m) 2.33 (m) |
27.9 |
| 3 | 5.32 (t, 5.0) | 49.1 | 5.31 (t, 5.1) | 49.0 | 5.37 (t, 5.0) | 52.0 |
| 4 | 5.56 (d, 5.0) | 116.7 | 5.56 (d, 4.9) | 116.8 | 5.44 (d, 4.8) | 119.9 |
| 5 | - | 150.4 | - | 150.4 | - | 150.0 |
| 6 | 6.00 (d, 9.5) | 124.8 | 6.00 (d, 9.7) | 124.8 | 5.92 (d, 9.7) | 124.9 |
| 7 | 6.35 (d, 9.5) | 128.3 | 6.35 (d, 9.6) | 128.3 | 6.28 (d, 9.6) | 127.4 |
| 8 | - | 124.4 | - | 124.4 | - | 124.5 |
| 9 | 2.10 (m) | 45.4 | 2.10 (m) | 45.5 | 2.10 (m) | 45.3 |
| 10 | - | 36.4 | - | 36.1 | - | 35.8 |
| 11 | 1.62 d 1.56 (m) |
19.6 | 1.63 (m) 1.56 (m) |
19.6 | 1.63 (m) 1.57 (m) |
19.3 |
| 12 | 2.05 (m) 1.31d |
36.1 | 2.04 (m) 1.33 (m) |
36.4 | 2.04 (m) 1.31 (m) |
36.3 |
| 13 | - | 43.9 | - | 43.9 | - | 43.8 |
| 14 | - | 151.9 | - | 151.8 | - | 151.3 |
| 15 | 2.36 (m) 2.46 (m) |
25.3 | 2.36 (m) 2.46 (m) |
25.3 | 2.38 (m) 2.47 (m) |
25.2 |
| 16 | 1.81 (m) 1.48 d |
28.0 | 1.80 (m) 1.48 (m) |
28.0 | 1.84 (m) 1.50 (m) |
28.0 |
| 17 | 1.25 (m) | 56.0 | 1.24 (m) | 56.0 | 1.24 (m) | 56.0 |
| 18 | 0.94 (s) | 19.3 | 0.94 (s) | 19.3 | 0.95 (s) | 19.3 |
| 19 | 0.95 (s) | 17.8 | 0.95 (s) | 17.8 | 1.01 (s) | 18.7 |
| 20 | 2.15 (m) | 39.5 | 2.14 (m) | 39.5 | 2.15 (m) | 39.5 |
| 21 | 1.05 (d, 7.0) | 21.4 | 1.05 (d, 6.7) | 21.4 | 1.06 (d, 6.7) | 21.4 |
| 22 | 5.21 (dd, 15.0, 8.0) | 135.3 | 5.20 (dd, 15.3, 7.8) | 135.3 | 5.21 (m) | 135.4 |
| 23 | 5.26 (dd, 15.0, 7.0) | 132.4 | 5.25 (dd, 15.2, 7.1) | 132.5 | 5.24 (m) | 132.4 |
| 24 | 1.88 (m) | 43.0 | 1.87 (m) | 43.0 | 1.88 (m) | 43.0 |
| 25 | 0.93 (d, 7.0) | 17.8 | 0.93 (d, 7.8) | 17.8 | 0.93 (d, 6.8) | 17.8 |
| 26 | 1.48 d | 33.2 | 1.46 (m) | 33.2 | 1.68 (m) | 34.0 |
| 27 | 0.83 (d, 7.0) | 20.1 | 0.83 (d, 6.8) | 20.1 | 0.83 (d, 6.6) | 20.1 |
| 28 | 0.85 (d, 7.0) | 19.8 | 0.85 (d, 6.7) | 19.8 | 0.85 (d, 6.6) | 19.8 |
| 29 | 7.79 (s) | 140.3 | 7.78 (s) | 140.3 | 7.88 (s) | 138.8 |
| 30 | - | 120.2 | - | 120.4 | - | 119.9 |
| 31 | - | 155.5 | - | 155.6 | - | 155.6 |
| 32 | 8.36 (s) | 152.7 | 8.38 (s) | 152.9 | 8.36 (s) | 152.9 |
| 33 | - | 149.5 | - | 149.6 | - | 148.4 |
| NH2 | 5.83 (br s) | - | 5.77 (br s) | - | 5.71 (br s) | - |
Analysis of the ECD spectra indicated that peniadenisteroid A exhibited a negative Cotton effect at ~250 nm and a positive one at ~300 nm (Figure 4A). By comparison, (3S)-peniadenisteroid A displayed only weak positive Cotton effect signals at both wavelengths, with an opposite sign at 250 nm and a substantially diminished intensity at 300 nm. This clearly distinguishes the synthetic epimer from the natural product and is consistent with an inverted C-3 configuration. Meanwhile, chiral HPLC analysis showed that peniadenisteroid A displayed a retention time of 7.8 min, whereas (3S)-peniadenisteroid A eluted at 10.8 min (Figure 4B). Furthermore, the structure of (3S)-peniadenisteroid A was unambiguously confirmed by single-crystal X-ray diffraction analysis (Figure 4C). In summary, we have accomplished the synthesis of (3S)-peniadenisteroid A via an 8-step sequence with an overall yield of 3.9%. While the natural product itself remains synthetically inaccessible, its C-3 epimer, (3S)-peniadenisteroid A, was successfully prepared, offering useful guidance for the future synthesis of the natural product.
Figure 4.
Absolute configuration studies of (3S)-peniadenisteroid A. (A) ECD spectral comparison for absolute configuration. (B) Chiral HPLC analysis: (3S)-peniadenisteroid A co-injected with peniadenisteroid A. (C) Crystal structure of (3S)-peniadenisteroid A determined by X-ray diffraction (CCDC number: 2526090).
2.3. Biological Evaluation
(3S)-Peniadenisteroid A and its synthetic intermediates were evaluated for their anti-inflammatory activity by measuring NO production in LPS-stimulated RAW264.7 macrophages. As listed in Table 3, both (3S)-peniadenisteroid A and peniadenisteroid A showed weak inhibitory activity against LPS-induced NO production. Notably, intermediates P3 and (3S)-P6 demonstrated promising NO production inhibitory activities, with inhibition rates reaching 59.7% and 86.1% at a concentration of 10 μM, respectively. To further elucidate the anti-inflammatory mechanism and molecular target of (3S)-P6, a high-throughput screening assay was performed to evaluate its regulatory effects on multiple signaling pathways, including STAT3/NF-κB, HIF-1α, WNT/β-catenin, and p53. The results revealed that (3S)-P6 exerted prominent inhibitory activity against two core transcription factors, STAT3/NF-κB and HIF-1α (Table 4).
Table 3.
Anti-inflammatory activity against LPS-induced NO production in RAW264.7 macrophages of compounds a.
| Compounds | Inhibition Rate at 10 µM (%) |
|---|---|
| P2 | 20.5 ± 6.9 |
| P3 | 59.7 ± 8.5 |
| P5a | 28.5 ± 12.4 |
| (3S)-P6 | 86.1 ± 5.6 |
| (3S)-P7 | 19.3 ± 11.8 |
| (3S)-P8 | 21.5 ± 8.8 |
| (3S)-peniadenisteroid A | 39.1 ± 10.3 |
| peniadenisteroid A | 12.2 ± 3.4 |
| Dexamethasone | 35.1 ± 8.6 |
a The values are the mean ± SEM of at least three independent experiments.
Table 4.
Effects of (3S)-P6 on STAT3/NF-kB and HIF-1α signaling pathways.
| Compounds | STAT3/NF-Kb Logratio a | HIF-1α Logratio a |
|---|---|---|
| (3S)-P6 c | −11.74 | −9.19 |
| KC7F2 c | NT b | −3.09 |
| Liangshanin A d | −4.15 | NT b |
a Compared with the luciferase in the untreated control group, the fold change of luciferase in the drug-treated group was measured as log2FoldChange (log2FC). b NT means not tested. c Concentration for compound is 20 μM. d Concentration for compound is 7.5 μM.
Given the anti-inflammatory potential of compound (3S)-P6 and its inhibitory effects on the HIF-1α and STAT3/NF-κB signaling pathways, we conducted a preliminary exploration of its possible targets under the guidance of network pharmacology. First, using “inflammation” as a key term, we searched the GeneCards database for disease-related therapeutic targets and obtained 12,121 candidate targets. Subsequently, based on the chemical structure of (3S)-P6, we queried the SuperPRED database to predict its potential binding targets. A total of 149 potential binding targets were compiled. These two sets, the disease-related targets (12,121) and the compound-predicted targets (149), were then intersected using a Venn diagram, yielding 141 overlapping targets (Figure S24). Following this initial screening, a protein–protein interaction (PPI) network was constructed using the STRING database. The resulting data were processed and visualized with Cytoscape software (version 3.10.0), and nodes were ranked according to their degree centrality. PPI network analysis revealed 10 proteins that may be closely associated with the anti-inflammatory activity of (3S)-P6 (Figure 5A). It is noteworthy that HIF-1α, STAT3 and NF-κB were among the 10 most relevant targets identified, which had already been identified in our earlier high-throughput screening assay (Table 4). This convergence provides mutual validation for both the screening assay results and the network pharmacology predictions.
Figure 5.
Network pharmacology and molecular docking analysis of compound (3S)-P6. (A) Prediction of potential targets for (3S)-P6 via Network Pharmacology. GeneCards database (https://www.genecards.org, accessed on 31 January 2026); SuperPRED (https://prediction.charite.de, accessed on 31 January 2026); STRING (https://cn.string-db.org, accessed on 31 January 2026). Red font denotes signaling pathway results consistent with Table 4 (Section 2.3). (B) Docking-predicted binding model for (3S)-P6 within HSP90α (PDB code: 1UY6).
Prioritizing among the targets identified by network pharmacology analysis, we focused on HSP90, which plays a critical regulatory role in both inflammatory processes and tumorigenesis [23,24,25,26]. Molecular docking of compound (3S)-P6 with the HSP90α protein revealed that (3S)-P6 fits well into the ligand-binding pocket of HSP90α (Figure 5B). The amino group at its C-3 position forms crucial hydrogen bonds with residues Asp93 and Thr184, explaining why introducing a bulky group at this site leads to a loss of anti-inflammatory activity (comparing (3S)-P6 to (3S)-P7, Table 3). Furthermore, an important C-H-π interaction was observed between the angular methyl group at C-19 of the steroidal core and Phe138. Additionally, the double bond in the C-17 side chain contributed positively to enhancing the compound’s affinity for the protein. Collectively, it is presumed that compound (3S)-P6 may interact with HSP90α. By interfering with the chaperone activity of HSP90α, compound (3S)-P6 compromises the folding and stability of its downstream client transcription factors (STAT3, NF-κB, and HIF-1α), which ultimately contributes to its anti-inflammatory action.
3. Materials and Methods
3.1. Materials and General Synthetic Procedures
All reagents and solvents were purchased from commercial suppliers and employed without further purification. Reactions were conducted under a nitrogen atmosphere and monitored by thin-layer chromatography (TLC) on silica gel GF254 plates (Yantai Jiangyou Silica Gel Development Co., Ltd., Yantai, Shandong, China). Products were routinely purified by silica gel column chromatography (Qingdao Marine Chemical Factory, Qingdao, Shandong, China) unless otherwise noted. NMR spectra were recorded on BRUKER AVANCE NEO (Bruker BioSpin, Ettlingen, Germany) with 400 MHz or 600 MHz for proton (1H NMR), and 100 MHz or 150 MHz for carbon (13C NMR). Chemical shifts (δ) are reported in parts per million (ppm) relative to TMS as an internal standard; coupling constants (J) are given in hertz (Hz). Splitting patterns are abbreviated as s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublet), and m (multiplet). High-resolution mass spectra (HRMS) were acquired on a Waters Micromass Q-TOF Premier mass spectrometer (Waters, Milford, MA, USA), and LRMS were measured with a Waters ACQUITY SQD2 Detector (Waters, Milford, MA, USA). Single-crystal X-ray diffraction data were collected using a Bruker D8 VENTURE diffractometer equipped with Cu Kα (λ = 1.54178 Å) radiation. (Bruker, Karlsruhe, Germany). ECD spectra were measured on a JASCO J-815 circular dichroism spectrometer (JASCO Corporation, Tokyo, Japan). The high-performance liquid chromatography (HPLC) analysis was conducted on an Agilent 1260 Infinity II Prime LC system (Agilent, Santa Clara, CA, USA), which was carried out on a 5-AmyCoat (4.6 × 250 mm) (Kromasil, Bohus, Sweden) with an isocratic mobile phase of 40% isopropanol in n-hexane at a flow rate of 1.0 mL/min. Detection was carried out by UV at 287 nm. Melting ––points of compounds were measured using an MP4 series video melting point apparatus (Hanon Advanced Technology Group Co., Ltd., Jinan, Shandong, China).
3.1.1. Synthesis of (22E,24R)-Ergosta-4,7,22-trien-3-one (P2)
To a solution of ergosterol (P1) (1.19 g, 3 mmol) in dry toluene (30 mL) was added cyclohexanone (1.47 g, 15 mmol), and the reaction was stirred at room temperature for 2 h. Then the aluminium isopropoxide (0.62 g, 3 mmol) was added rapidly to the solution, and the reaction was refluxed for 1 h under nitrogen atmosphere. After completion (TLC), the solution was cooled to room temperature. The solution was quenched with addition of 2 N HCl (30 mL) at 0 °C. Then the solution was extracted with ethyl acetate (20 mL × 3). The combined organic phases were washed with water, saturated aqueous sodium bicarbonate solution, saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography to give the product as a white solid, mp. 98–100 °C. Yield: 0.83 g, 2.1 mmol, 70%. Compound P2 is a known derivative, and our NMR data are in good agreement with those reported in the literature [13,16]. 1H NMR (400 MHz, Chloroform-d) δ 5.78 (s, 1H), 5.25–5.12 (m, 3H), 3.18–3.07 (m, 1H), 2.–2.58 (m, 1H), 2.38–2.28 (m, 2H), 2.20 (t, J = 8.2 Hz, 1H), 0–2.01 (m, 2H), 1–1.90 (m, 2H), 9–1.80 (m, 3H), 1.73 (d, J = 3.7 Hz, 1H), 1.63 (dt, J = 10.3, 5.1 Hz, 2H), 1.54–1.40 (m, 3H), 1.31–1.26 (m, 2H), 1.16 (s, 3H), 1.02 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.8 Hz, 3H), 0.82 (d, J = 6.4 Hz, 3H), 0.80 (s, 3H), 0.59 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 199.5, 169.2, 139.6, 135.5, 132.2, 122.8, 115.7, 55.9, 55.1, 45.9, 43.5, 42.9, 40.6, 39.1, 38.2, 34.3, 33.2, 33.2, 33.0, 28.2, 23.0, 22.1, 21.4, 21.2, 20.1, 19.8, 17.7, 12.3.
3.1.2. Synthesis of (22E,24R)-Ergosta-4,6,8(14),22-tetraen-3-one (P3)
To a solution of P2 (1.58 g, 4 mmol) in dry 1,4-dioxane (20 mL) was added DDQ (3.63 g, 16 mmol). The reaction was stirred at 110 °C for 4 h under nitrogen atmosphere. After cooling to room temperature, the mixture was filtered and the filtrate was concentrated under reduced pressure. The crude residue was purified by silica gel chromatography to give the product as a white solid, mp. 99–100 °C. Yield: 0.62 g, 1.56 mmol, 39%. Compound P3 is a known derivative, and our NMR data are in good agreement with those reported in the literature [27]. 1H NMR (400 MHz, Chloroform-d) δ 6.61 (d, J = 9.5 Hz, 1H), 6.03 (d, J = 9.5 Hz, 1H), 5.73 (s, 1H), 5.31–5.17 (m, 2H), 2.60–2.33 (m, 4H), 2.18–1.99 (m, 4H), 1.91–1.77 (m, 3H), 1.75–1.61 (m, 2H), 1.53–1.43 (m, 2H), 1.31 (dd, J = 12.8, 2.9 Hz, 2H), 1.06 (d, J = 6.7 Hz, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.85 (t, J = 6.4 Hz, 3H), 0.82 (s, 3H). 13C NMR (100 MHz, Chloroform-d) δ 199.6, 164.5, 156.2, 135.1, 134.1, 132.7, 124.6, 124.6, 123.1, 55.9, 44.5, 44.1, 43.0, 39.4, 36.9, 35.7, 34.3, 34.3, 33.2, 27.9, 25.5, 21.4, 20.1, 19.8, 19.1, 19.1, 17.8, 16.8.
3.1.3. Synthesis of (3S,22E,24R)-3-[(S)-tert-Butylsulfinamido]ergosta-4,6,8(14),22-tetraene (P5a)
To a solution of P3 (0.98 g, 2.5 mmol) and (S)-(−)-2-methyl-2-propanesulfinamide (1.82 g, 15 mmol) in tetrahydrofuran (20 mL) was added titanium tetraisopropanolate (5.68 g, 20 mmol) and the reaction was refluxed for 24 h. DIBAL-H (10 mmol, 1 M in THF) was then added drop wise to the solution under nitrogen at −78 °C. After completion of the addition, the mixture was stirred at room temperature for 5 h. After the reaction was completed (detected by TLC), saturated aqueous solution of ammonium chloride was then added slowly to the reaction mixture at −78 °C and stirred for 5 min. The aqueous layer was washed with ethyl acetate (30 mL × 3). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography to give the product as a pale yellow solid, mp. 142–144 °C. Yield: 0.65 g, 1.3 mmol, 52%. 1H NMR (400 MHz, Chloroform-d) δ 6.17 (d, J = 9.6 Hz, 1H), 5.87 (d, J = 9.7 Hz, 1H), 5.46 (s, 1H), 5.26–5.13 (m, 2H), 3.99–3.84 (m, 1H), 3.21 (d, J = 6.9 Hz, 1H), 2–2.35 (m, 1H), 2.34–2.24 (m, 1H), 2.16–2.09 (m, 1H), 2.09–1.94 (m, 3H), 1.90–1.72 (m, 3H), 1.67–1.5–m, 3H), 1.47–1.37 (m, 3H), 1.26 (s, 2H), 1.22 (s, 9H), 1.04 (d, J = 6.7 Hz, 3H), 0.97–0.90 (m, 6H), 0.89 (s, 3H), 0.86 –0.80 (m, 6H). 13C NMR (100 MHz, Chloroform-d) δ 150.0, 145.8, 135.4, 132.3, 126.2, 125.7, 124.9, 124.9, 56.0, 55.7, 54.2, 45.5, 43.7, 43.0, 39.6, 36.5, 35.7, 34.1, 33.2, 28.6, 28.0, 25.1, 22.8 (3C), 21.3, 20.1, 19.8, 19.3, 19.3, 18.5, 17.8. LRMS (ESI): m/z 497.980 [M + H]+.
3.1.4. Synthesis of (3S,22E,24R)-3-[(R)-tert-Butylsulfinamido]ergosta-4,6,8(14),22-tetraene (P5b)
P5b was synthesized following the same procedure of P5a using (R)-(−)-2-methyl-2-propanesulfinamide as a pale-yellow solid, mp. 145–148 °C. Yield: 0.77 g, 1.55 mmol, 62%. 1H NMR (400 MHz, Chloroform-d) δ 6.17 (d, J = 9.6 Hz, 1H), 5.85 (d, J = 9.7 Hz, 1H), 5.33 (s, 1H), 5.27–5.15 (m, 2H), 3.97–3.84 (m, 1H), 3.09 (d, J = 9.1 Hz, 1H), 2.46–2.35 (m, 1H), 2.35–2.23 (m, 2H), 2.17–2.06 (m, 1H), 2.05–1.92 (m, 2H), 1.86 (td, J = 6.9, 5.8 Hz, 1H), 1.81–1.71 (m, 2H), 1.60 (ddt, J = 16.0, 9.2, 3.1 Hz, 2H), 1.54–1.37 (m, 4H), 1.30–1.25 (m, 1H), 1.22 (s, 9H), 1.20–1.16 (m, 1H), 1.04 (d, J = 6.7 Hz, 3H), 0.95–0.89 (m, 6H), 0.87 (s, 3H), 0.85–0.80 (m, 6H). 13C NMR (100 MHz, Chloroform-d) δ 150.2, 145.9, 135.5, 132.3, 126.3, 125.6, 124.9, 124.6, 56.0, 55.9, 55.0, 45.5, 43.7, 43.0, 39.5, 36.5, 35.6, 34.3, 33.2, 29.9, 28.0, 25.2, 22.8 (3C), 21.4, 20.1, 19.8, 19.3, 19.3, 18.6, 17.8. LRMS (ESI): m/z 498.520 [M + H]+.
3.1.5. Synthesis of (3S,22E,24R)-Ergosta-4,6,8(14),22-tetraen-3-amine hydrochloride ((3S)-P6)
To a solution of P5a or P5b (200 mg, 0.4 mmol) in 1,4-dioxane (2 mL) was added 2 M HCl in 1,4-dioxane (3 mL). The resulting mixture was stirred at room temperature for 4 h. The solution was concentrated and then diluted with MeOH (10 mL) and filtrated. The solid was washed with petroleum ether and then dried overnight to give (3S)-P6 as a white solid, mp. 112–114 °C. Yield: 149 mg, 0.32 mmol, 80%. 1H NMR (400 MHz, Chloroform-d) δ 8.41 (br s, 2H), 6.21 (d, J = 9.5 Hz, 1H), 5.88 (d, J = 9.7 Hz, 1H), 5.50 (s, 1H), 5.29–5.14 (m, 2H), 4.13–3.87 (m, 1H), 2.47–2.36 (m, 1H), 2.34–2.21 (m, 2H), 2.16–2.08 (m, 1H), 2.06–1.90 (m, 3H), 1.89–1.83 (m, 1H), 1.80–1.74 (m, 2H), 1.60–1.56 (m, 1H), 1.54–1.34 (m, 4H), 1.25–1.15 (m, 2H), 1.04 (d, J = 6.6 Hz, 3H), 0.95–0.88 (m, 9H), 0.86–0.80 (m, 6H). 13C NMR (100 MHz, Chloroform-d) δ 150.9, 148.6, 135.4, 132.4, 127.3, 125.3, 124.6, 117.8, 77.4, 56.0, 49.4, 45.1, 43.8, 43.1, 39.6, 36.4, 35.8, 33.4, 33.3, 28.0, 25.2, 21.4, 20.1, 19.8, 19.3, 19.3, 18.5, 17.8. LRMS (ESI): m/z 394.37 [M + H]+.
3.1.6. Synthesis of N4-[(3S,22E,24R)-Ergosta-4,6,8(14),22-tetraen-3-yl]-6-chloropyrimidine-4,5-diamine ((3S)-P7)
A solution of (3S)-P6 (93 mg, 0.2 mmol) and 5-amino-4,6-dichloropyrimidine (36 mg, 0.22 mmol) in n-butanol (5 mL) was treated with sodium bicarbonate (50 mg, 0.6 mmol) and refluxed for 24 h. After the reaction was completed, the solution was quenched with H2O. Then the solution was extracted with ethyl acetate (15 mL × 3). The organic layers were combined and washed with water, saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude residue was purified by silica gel chromatography to give the product as a pale yellow solid, mp. 128–130 °C. Yield: 68 mg, 0.13 mmol, 66%. 1H NMR (400 MHz, Chloroform-d) δ 8.07 (s, 1H), 6.18 (d, J = 9.6 Hz, 1H), 5.84 (d, J = 9.7 Hz, 1H), 5.41–5.34 (m, 1H), 5.30–5.15 (m, 2H), 4.84 (br s, 2H), 3.48–3.27 (m, 2H), 2.48–2.36 (m, 1H), 2.35–2.22 (m, 2H), 2.16–2.08 (m, 1H), 2.07–1.97 (m, 2H), 1.90–1.84 (m, 1H), 1.77 (tt, J = 9.5, 3.5 Hz, 2H), 1.63 (td, J = 6.4, 2.9 Hz, 1H), 1.57–1.41 (m, 5H), 1.34–1.28 (m, 1H), 1.23–1.18 (m, 1H), 1.05 (d, J = 6.7 Hz, 3H), 0.97–0.88 (m, 9H), 0.86–0.81 (m, 6H). 13C NMR (100 MHz, Chloroform-d) δ 154.9, 150.2, 150.0, 146.3, 143.4, 135.4, 132.4, 126.3, 125.6, 124.9, 123.1, 121.7, 56.0, 48.8, 45.5, 43.7, 43.0, 39.5, 36.5, 35.8, 34.1, 33.2, 28.0, 26.7, 25.2, 21.4, 20.1, 19.8, 19.4, 19.3, 18.6, 17.8. LRMS (ESI): m/z 521.32 [M + H]+.
3.1.7. Synthesis of 6-Chloro-9-[(3S,22E,24R)-ergosta-4,6,8(14),22-tetraen-3-yl]-9H-purine ((3S)-P8)
Conc. HCl (30 μL) was added to a solution of (3S)-P7 (52 mg, 0.1 mmol) in trimethoxymethane (2 mL). The reaction was stirred for 6 h at room temperature and then concentrated in vacuo. The crude residue was purified by silica gel chromatography to give the product as a white solid, mp. 166–167 °C. Yield: 34 mg, 0.064 mmol, 64%. 1H NMR (400 MHz, Chloroform-d) δ 8.74 (s, 1H), 8.19 (s, 1H), 6.31 (d, J = 9.6 Hz, 1H), 5.92 (d, J = 9.7 Hz, 1H), 5.49–5.43 (m, 1H), 5.42–5.39 (m, 1H), 5.32–5.16 (m, 2H), 2.49–2.32 (m, 3H), 2.18–2.03 (m, 3H), 1.97–1.75 (m, 4H), 1.72–1.68 (m, 1H), 1.64–1.58 (m, 2H), 1.56–1.42 (m, 3H), 1.24–1.19 (m, 1H), 1.06 (d, J = 6.6 Hz, 3H), 1.03 (s, 3H), 0.96 (s, 3H), 0.93 (d, J = 6.8 Hz, 3H), 0.86–0.82 (m, 6H). 13C NMR (100 MHz, Chloroform-d) δ 151.9, 151.8, 151.1, 149.1, 146.1, 143.7, 135.3, 132.5, 132.0, 127.9, 124.6, 124.4, 118.7, 56.0, 53.0, 45.3, 43.8, 43.0, 39.5, 36.3, 35.9, 33.9, 33.2, 28.0, 27.6, 25.3, 21.4, 20.1, 19.8, 19.3, 19.3, 18.7, 17.8. LRMS (ESI): m/z 531.39 [M + H]+.
3.1.8. Synthesis of 9-[(3S,22E,24R)-Ergosta-4,6,8(14),22-tetraen-3-yl]-9H-purin-6-amine ((3S)-peniadenisteroid A)
To a solution of (3S)-P8 (27 mg, 0.05 mmol) in THF (1 mL) was added ammonia (7 M in MeOH) and saturated with ammonia in a sealed tube. The resulting solution was then heated to 70 °C and stirred for 30 h. Upon reaching room temperature, the reaction mixture was unsealed, monitored for completion, and then concentrated under reduced pressure. The residue was purified by silica gel chromatography to give the product as a white solid, mp. 238–240 °C. Yield: 18 mg, 0.035 mmol, 69%. NMR data were shown in Table 2. HRMS (ESI) for C33H46N5 [M + H]+ calcd 512.3748, found 512.3751.
3.2. General Procedures of Biological Studies
3.2.1. Nitric Oxide (NO) Production Inhibitory Activity
Nitric oxide (NO) production inhibitory activity was evaluated using the Griess assay as previously described [28]. Briefly, RAW264.7 cells (kindly provided by Prof. Chang-Lun Shao, Ocean University of China) were seeded into 96-well plates at a density of 1.5 × 105 cells/mL (100 μL per well) and incubated overnight at 37 °C in a 5% CO2 atmosphere. The cells were then treated with test compounds or dexamethasone (Macklin, Shanghai, China, positive control) at 10 μM in DMEM (Biosharp, Beijing, China) supplemented with 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel) for 24 h. Subsequently, the cells were stimulated with 1 μg/mL lipopolysaccharide (LPS, from E. coli, Promega (Beijing), Beijing, China) to induce NO production. The concentration of nitrite in the culture supernatant, reflecting NO levels, was determined colorimetrically based on the Griess reaction. In each experiment, NO release in the LPS-stimulated group was defined as 100%, and that in the unstimulated group was defined as 0%. All experiments were performed in triplicate and repeated three times independently.
3.2.2. Pathway Reporter Assay
SKA cells [29] were plated in white 96-well plates at a density of 104 cells per well and cultured overnight under standard conditions (37 °C, 5% CO2). Following attachment, the cells were incubated for 24 h with the test compounds: (3S)-P6 (at 20 μM) and KC7F2 (Selleck (Shanghai, China), at 20 μM), and Liangshanin A [30] (at 7.5 μM). Subsequently, luciferase activity was quantified using a commercial detection kit (Promega (Beijing), Beijing, China, product #E1483) and measured on a SpectraMax® L microplate reader (Molecular Devices, San Jose, CA, USA).
3.3. Molecular Docking Study
Molecular docking simulations were conducted in Molecular Operating Environment (MOE, version 2020.09) to explore the potential binding interactions between compound (3S)-P6 and its target proteins. The three-dimensional structure of each protein in complex with a reference ligand was obtained from the RCSB Protein Data Bank. Prior to docking, all water molecules and ions were deleted from the protein structure. Protein preparation was carried out using the QuickPrep module, which assigned protonation states, optimized hydrogen-bonding networks, and performed energy minimization. The binding site was defined based on the coordinates of the co-crystallized ligand. During the docking procedure, poses were initially generated and ranked using the London dG scoring function, then refined and rescored with the more accurate GBVI/WSA dG scoring method. The conformation exhibiting the most favorable binding free energy was selected as the predicted binding mode. The resulting PDB file from docking was imported into PyMOL (version 2.6.0) for processing. Key intermolecular interactions were visualized using the “find contacts” function, which identifies contacts between the ligand and the protein.
4. Conclusions
In this study, we accomplished the semi-synthesis of (3S)-peniadenisteroid A-the C-3 epimer of the natural product peniadenisteroid A-from ergosterol via an eight -step sequence in 3.9% overall yield. Although the total synthesis of the natural 3R product remains incomplete, this work provides a foundational step and a valuable reference for its eventual realization. Among the synthesized compounds, (3S)-P6 exhibited excellent inhibitory activity against NO production (86.1% at 10 μM), markedly outperforming the positive control dexamethasone (35.1% at 10 μM). Further high-throughput signaling pathway screening revealed that (3S)-P6 significantly suppressed both the STAT3/NF-κB and HIF-1α pathways. Integrated network pharmacology analysis of 141 intersecting targets identified 10 highly correlated candidates. Ultimately, by integrating network pharmacology and molecular docking, we postulate that HSP90α may serve as a putative target of compound (3S)-P6. We propose that the anti-inflammatory mechanism of (3S)-P6 may involve inhibition of HSP90α, which disrupts the chaperone-mediated maturation of key client transcription factors (STAT3, NF-κB, and HIF-1α), resulting in a synergistic multi-pathway effect [23,24,25,26].
In summary, our exploration of the synthetic routes toward the natural product and its diastereomer offers key guidance for the synthesis and structural diversification of this class of compounds. These findings provide a solid chemical and mechanistic foundation for the development of novel small-molecule anti-inflammatory agents based on marine natural product structures, and point the way toward designing multi-pathway modulators targeting the STAT3/NF-κB, HIF-1α network.
Supplementary Materials
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/md24100352/s1, Figures S1–S18: 1H and 13C NMR spectra of relevant compounds; Figure S19: HRMS-ESI spectra of compound (3S)-Peniadenisteroid A; Figures S20 and S21: LRMS-ESI spectra of compounds P5a and P5b; Figures S22 and S23: 2D NOESY spectra of compounds P5a and P5b; Figure S24: Venn diagram of overlapping targets for (3S)-P6.
Author Contributions
Conceptualization and design, P.W. and Z.L.; methodology, X.Z. (Xinxin Zhang), X.W., J.W., M.M., P.W. and Z.L.; acquisition of data X.Z. (Xuetao Zhang), X.W., M.M., D.G., J.Y. and L.Y.; data analysis, all authors; writing—original draft preparation, X.Z. (Xuetao Zhang) and P.W.; writing—review and editing, X.Z. (Xuetao Zhang), Z.L. and P.W.; supervision, P.W., Z.L. and C.-Y.W.; funding acquisition, P.W., Z.L., X.Z. (Xinxin Zhang) and C.-Y.W. All authors have read and agreed to the published version of the manuscript.
Institutional Review Board Statement
Not applicable.
Data Availability Statement
The data presented in this study are available in the manuscript and in the Supplementary Materials.
Conflicts of Interest
The authors declare no conflicts of interest.
Funding Statement
This research was funded by the National Natural Science Foundation of China (Nos. 42476106; 82473839; 32470764), National Natural Science Foundation of China for Young Scholar Project (82404586), Fundamental Research Funds for the Central Universities (Nos. 202212019), Taishan Scholars Program (tsqn202211050; tsqn202211060), China, Taishan Industry Leading Talent Project (No. tscx202408138), and Shandong Provincial Natural Science Foundation (ZR2026MS1180). We are also grateful for the excellent support from the core Facility of IEMB at OUC.
Footnotes
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Supplementary Materials
Data Availability Statement
The data presented in this study are available in the manuscript and in the Supplementary Materials.