Immunosuppressive Ergosterol−emodin Hybrids from Penicillium sp. XB152

Immunosuppressive Ergosterol−emodin Hybrids from Penicillium sp. XB152 | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 November 2025 V1 Latest version Share on Immunosuppressive Ergosterol−emodin Hybrids from Penicillium sp. XB152 Authors : Qianxi Ouyang , Chunlun Qin , Jinling Chang , Jiao Pei , Yinhui Zhou , Huifang Pi , An Jin , and Han-Li Ruan 0000-0003-0882-1009 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176300128.81685363/v1 153 views 76 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Text：Ergostemodins A−D ( 1 − 4 ), the first examples of ergosterol−emodin carbon skeletons formed by Diels−Alder reactions between ergosterol and emodin moieties, were separated from Penicillium sp. XB152, alongside two previously unreported ergosterols ( 5 and 6 ). Their architectures were established via spectroscopic investigations and single-crystal X-ray diffraction (SC-XRD). Compounds 1 − 4 feature a distinctive 6/6/6/5/6/6/5/6 octacyclic ring system. Compounds 1 − 6 were immunosuppressive against concanavalin A (ConA)-induced T cell proliferation and Lipopolysaccharide (LPS)-induced B cell proliferation with EC 50 values ranging from 5.80 to 27.97 μ M and from 5.44 to 25.66 μ M, respectively. Compound 1 exhibited inhibitory activity against HT29, MCF-7, HeLa, HePG2, and A549 human cancer cell line with the IC 50 values of 17.09, 21.70, 12.71, 11.53, and 24.74 μ M. Compound 5 exhibited inhibitory activity against HT29, HeLa, and A549 human cancer cell line with the IC 50 values of 25.63, 18.96, and 16.82 μ M. Cite this paper: Chin. J. Chem. 2025 , 43 , XXX—XXX. DOI: 10.1002/cjoc.70XXX Immunosuppressive Ergosterol−emodin Hybrids from Penicillium sp. XB152 Qianxi Ouyang, a,b,1 Chunlun Qin, a,1 Jinling Chang, a Jiao Pei, a Yinhui Zhou, a Huifang Pi,* , a An Jin,* , c and Hanli Ruan* , a a School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Hubei Key Laboratory of Natural Medicinal Chemistry and Resource Evaluation, Wuhan 430030, People’s Republic of China b Department of Pharmacy, The Central Hospital of Wuhan, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People’s Republic of China c School of Pharmaceutical Sciences, Hunan University of Medicine, Huaihua, 418000, People’s Republic of China ergostemodins A−D | ergosterol−emodin heterodimers | Penicillium sp. XB152 | immunosuppressive activity | Diels-Alder reactions Comprehensive Summary Text：Ergostemodins A−D ( 1 − 4 ), the first examples of ergosterol−emodin carbon skeletons formed by Diels−Alder reactions between ergosterol and emodin moieties, were separated from Penicillium sp. XB152, alongside two previously unreported ergosterols ( 5 and 6 ). Their architectures were established via spectroscopic investigations and single-crystal X-ray diffraction (SC-XRD). Compounds 1 − 4 feature a distinctive 6/6/6/5/6/6/5/6 octacyclic ring system. Compounds 1 − 6 were immunosuppressive against concanavalin A (ConA)-induced T cell proliferation and Lipopolysaccharide (LPS)-induced B cell proliferation with EC 50 values ranging from 5.80 to 27.97 μ M and from 5.44 to 25.66 μ M, respectively. Compound 1 exhibited inhibitory activity against HT29, MCF-7, HeLa, HePG2, and A549 human cancer cell line with the IC 50 values of 17.09, 21.70, 12.71, 11.53, and 24.74 μ M. Compound 5 exhibited inhibitory activity against HT29, HeLa, and A549 human cancer cell line with the IC 50 values of 25.63, 18.96, and 16.82 μ M. Background and Originality Content Figure 1 . Structures of 1 − 6 . Steroids are amphipathic compounds characterized by the presence of the 1,2-cyclopentanoperhydrophenanthrene ring skeleton. [1] They play a crucial role as components of membrane lipids, influencing membrane fluidity, stability, and permeability. Additionally, steroids play a key regulatory role in most eukaryotes, governing cell cycle progression and functions of membrane-associated enzymes. [2] Steroidal drugs represent the second largest class of drugs and are routinely applied for autoimmune and inflammatory diseases. [3-5] Furthermore, some steroidal drugs, including chenodeoxycholic acid, obeticholic acid and abiraterone, have demonstrated promising anti-cancer efficacy. [6] This points to the prospect of steroids in anticancer medication, attracting interest from pharmacists. [7] In the ongoing investigation of natural steroids, a series of steroids with unique backbones has been reported. [8-10] Furthermore, steroid monomers undergoing Diels-Alder reactions with cytochalasins, [11-12] alkaloids, [13-15] and polyketides, [16-21] have led to the formation of steroid heterodimers featuring complex ring systems and novel backbones with diverse activities, attracting continuous attention. Among the seventeen reported heterodimers, certain compounds have demonstrated antibacterial, [19] immunosuppressive, [11-12] cytotoxic, [11, 18] antiviral [11] and anti-inflammatory [17] activity, as well as inhibitory effect against MptpB. [17] To discover more biologically active secondary metabolites with unique structures from fungi, Penicillium sp. XB152 was studied, which was isolated from the stem of Rhododendron lapponicum . As a result, four unprecedented naturally occurring Diels-Alder cycloadducts of ergosterol and emodin derivatives, ergostemodins A−D ( 1 − 4 ), and two undescribed ergosterols, penicillisterenes A and B ( 5 and 6 ), were purified and characterized. Hence, we present the separation, structural clarification and biological assessment of 1 − 6 , along with the hypothesized biosynthesis of compounds 1 − 4 . Results and Discussion Ergostemodin A ( 1 ) was designated the empirical formula C 45 H 56 O 8 from HRESIMS data ( m/z 747.3860 [M + Na] + , and calcd 747.3873), suggesting an index of hydrogen deficiency (IHD) of 18. Its 1 H NMR ( table 1 ) demonstrated seven methyl groups, two methoxy groups, four olefinic protons, and two aromatic protons. Its 13 C NMR data ( table 1 ) revealed 45 carbons attributed to 7 methyls, 2 methoxys, 6 methylenes, 15 methines (1 oxygenated, 4 olefinic and 2 aromatic), and 15 unprotonated carbons (3 carbonyls). The above analyses indicated that 1 was an ergosterol heterodimer composed of an ergosterol (unit A) and a polyketide (unit B). [16-17] The planar architecture and key connectivity of 1 were established through interpretation of the NMR data. The characteristic 1,2-cyclopentanoperhydrophenanthrene ring skeleton was confirmed through the COSY ( Figure 2 ) correlations of H 2 -1/H 2 -2/H-3/H 2 -4, H-6/H-7, H 2 -12/H 2 -11/H-9, and H-15/H 2 -16/H-17 and the crucial HMBC interactions ( Figure 2 ) of H 2 -12/C-9; H-6/C-4, C-8, C-10; H 2 -16/C-13, C-14; H 3 -18/C-12, C-14, C-17; and H 3 -19/C-1, C-5, C-9, C-10. The side chain of ergosterol was determined by COSY couplings of H-20/H 3 -21; H-20/H-22/H-23/H-24/H-25; H-24/H 3 -28; and H 3 -26/H-25/H 3 -27. The above analyses indicated that the unit A’s planar structure was likely an ergosterol moiety. [22] Figure 2 . The HMBC (blue arrow) and COSY (red bold) associations of 1 − 6 . The NMR signals remaining from unit B indicated that it had a structure similar to 2′-hydroxy bisdechlorogeodin, [23] which is a polyketide belonging to the emodin family. The structural divergence between unit B and 2′-hydroxy bisdechlorogeodin lies in the absence of the OH at C-2′′ and a Δ 1′′,2′′ double bond in unit B, which could be unambiguously determined through the essential HMBC associations of H-2′′/C-2′, C-4′′, C-7′′ and the higher field δ values of C-1′′ and C-2′′ in unit B. Thus, the unit B’s planar structure was constructed. Table 1 1 H NMR (400 MHz) and 13 C NMR (100 MHz) data for compounds 1 and 2. No. δ H ( J in Hz) δ C , type δ H ( J in Hz) δ C , type 1 1.78, m 37.3, CH 2 1.75, m 1.24, m 36.1, CH 2 2 1.86, m 1.45, m 32.5, CH 2 1.89, m 1.48, m 31.9, CH 2 3 3.50, m 71.0, CH 3.64, m 70.5, CH 4 2.47, m 2.19, m 42.5, CH 2 2.56, d (14.2) 2.20, t (12.8) 41.9, CH 2 5 144.3, C 142.9, C 6 5.55, d (2.6) 124.0, CH 5.64, s 122.8, CH 7 2.69, m 38.6, CH 2.67, brs 37.4, CH 8 130.2, C 129.1, C 9 1.87, m 48.7, CH 1.89, m 47.6, CH 10 37.1, C 36.0, C 11 1.68, m 20.8, CH 2 1.62, m 19.9, CH 2 12 2.07, m 1.36, m 39.9, CH 2 2.05, m 1.32, m 38.7, CH 2 13 43.3, C 42.2, C 14 145.3, C 143.7, C 15 2.87, d (8.2) 42.3, CH 2.83, d (8.8) 41.0, CH 16 2.60, dd (15.3, 7.3) 1.85, m 36.0, CH 2 2.42, dd (15.1, 7.3) 1.84, m 34.5, CH 2 17 0.97, m 55.5, CH 0.90, m 55.1, CH 18 0.96, s 21.5, CH 3 0.94, s 20.8, CH 3 19 0.91, s 17.7, CH 3 0.89, s 17.6, CH 3 20 2.03, m 40.9, CH 2.32, m 34.1, CH 21 0.99, d (6.4) 21.1, CH 3 0.90, d (6.4) 20.7, CH 3 22 5.14, dd (15.2, 8.4) 137.0, CH 4.79, s 131.7, CH 23 5.28, dd (15.2, 8.0) 133.3, CH 135.9, C 24 1.85, m 44.6, CH 1.62, m 50.8, CH 25 1.48, m 34.4, CH 1.49, m 30.9, CH 26 0.88, d (6.8) 20.6, CH 3 0.75, d (6.5) 21.9, CH 3 27 0.86, d (6.8) 20.4, CH 3 0.85, d (6.5) 20.6, CH 3 28 1.01, d (6.6) 18.7, CH 3 0.99, d (6.8) 17.7, CH 3 29 1.52, s 12.7, CH 3 2′ 92.2, C 91.4, C 3′ 197.1, C 198.3, C 4′ 174.3, C 171.4, C 5′ 6.35, s 104.3, CH 6.32, s 109.7, CH 6′ 153.7, C 153.4, C 7′ 6.17, s 110.6, CH 6.42, s 104.3, CH 3′a 107.4, C 106.2, C 7′a 157.8, C 156.3, C 1′′ 57.4, C 56.6, C 2′′ 3.15, d (8.6) 55.0, CH 3.12, d (8.9) 53.8, CH 3′′ 199.4, C 196.3, C 4′′ 5.60, s 103.6, CH 5.53, s 103.2, CH 5′′ 169.7, C 166.7, C 6′′ 2.32, s 22.8, CH 3 2.35, s 23.4, CH 3 7′′ 173.3, C 171.4, C 5′′-OCH 3 3.64, s 57.9, CH 3 3.63, s 57.2, CH 3 7′′-OCH 3 3.36, s 52.5, CH 3 3.40, s 52.2, CH 3 a measured in CD 3 OD; b measured in CDCl 3 ; With an IHD of 17 accounted for by the ergosterol (unit A) and the polytide (unit B) moieties, compound 1 demanded an additional unsaturation site. This was rationalized by a bridging ring system (ring E) which unit A linked to unit B via C-2′′−C-7 and C-1′′−C-15 bonds, as evidenced by the crucial COSY association of H-7/H-2′′, combined with the vital HMBC associations of H-15/C-2′′, H 2 -16/C-1′′. Hence, its planar architecture was elucidated as an [4+2] cycloaddition of 14-dehydroergosterol and bisdechlorogeodin, featuring an unprecedented 6/6/6/6/5/6/5/6 octacyclic scaffold. Table 2 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data in CDCl 3 for compounds 3 and 4. No. δ H ( J in Hz) δ C , type δ H ( J in Hz) δ C , type 1 1.79, m 1.12, m 36.5, CH 2 1.35, m 33.4, CH 2 2 1.85, m 1.46, m 31.6, CH 2 2.08, m 1.66, m 29.0, CH 2 3 3.54, m 71.1, CH 4.33, m 68.4, CH 4 2.40, m 2.19, m 42.4, CH 2 5.58, d (2.3) 130.7, CH 5 143.6, C 144.0, C 6 5.46, t (2.6) 121.4, CH 6.71, s 135.6, CH 7 2.92, t (2.8) 39.0, CH 130.5, C 8 131.3, C 62.7, C 9 2.00, m 45.7, CH 1.56, m 47.2, CH 10 36.7, C 35.7, C 11 1.30, m 22.8, CH 2 1.68, m 1.35, m 33.4, CH 2 12 1.97, m 1.34, m 37.3, CH 2 1.69, m 1.35, m 38.6, CH 2 13 41.8, C 41.8, C 14 148.3, C 74.4, C 15 2.59, m 41.6, CH 2.42, m 33.8, CH 16 2.00, m 1.69, m 35.2, CH 2 2.01, m 1.56, m 32.2, CH 2 17 1.30, m 57.4, CH 2.42, m 55.2, CH 18 0.96, s 19.1, CH 3 0.91, s 16.7, CH 3 19 0.94, s 17.7, CH 3 1.08, s 19.4, CH 3 20 2.19, m 38.6, CH 2.08, m 40.4, CH 21 1.02, d (6.6) 21.3, CH 3 1.01, d (6.6) 21.1, CH 3 22 5.57, m 135.5, CH 5.24, m 135.4, CH 23 5.57, m 132.6, CH 5.24, m 132.5, CH 24 1.87, m 43.1, CH 1.86, m 43.0, CH 25 1.49, m 33.3, CH 1.47, m 33.3, CH 26 0.85, d (6.5) 20.1, CH 3 0.84, d (6.8) 20.2, CH 3 27 0.85, d (6.5) 19.9, CH 3 0.83, d (6.8) 19.8, CH 3 28 0.96, d (6.6) 21.2, CH 3 0.92, d (6.9) 18.0, CH 3 29 2′ 90.9, C 89.5, C 3′ 196.6, C 196.4, C 4′ 170.0, C 170.6, C 5′ 6.43, s 104.4, CH 6.53, s 104.6, CH 6′ 153.8, C 154.3, C 7′ 6.35, s 109.7 CH 6.35, s 110.0, CH 3′a 107.9, C 105.4, C 7′a 156.4, C 156.4, C 1′′ 59.8, C 59.9, C 2′′ 3.01, d (10.1) 55.6, CH 4.28, d (5.7) 46.9, CH 3′′ 196.6, C 196.2, C 4′′ 5.50, s 103.4, CH 5.31, s 104.1, CH 5′′ 170.4, C 166.5, C 6′′ 2.37, s 23.3, CH 3 2.47, s 23.3, CH 3 7′′ 171.4, C 168.9, C 5′′-OCH 3 3.68, s 57.2, CH 3 3.63, s 57.6, CH 3 7′′-OCH 3 3.40, s 57.4, CH 3 3.30, s 52.7, CH 3 The essential NOESY couplings ( Figure 3 ) of H-15/H-7, H 3 -18; H-7/H 3 -19; and H-20/H 3 -18 demonstrated that H-7 and H-15 were oriented β , and H-17 was oriented α . The association between H-2′′ and H-7 ( J 7,2′′ = 8.6 Hz) proved that H-2′′ oriented α . The NOESY associations of H-1a/H-3, H-9 implied that H-3 and H-9 oriented α . Nevertheless, the relative configurations at C-1′′, C-2′, C-20, and C-24 remained unassigned owing to the absence of detectable NOESY linkages and the free rotation of the side chain. Ultimately, the absolute stereochemistry of 1 was unequivocally established as 3 S , 7 S , 9 R , 10 R , 13 R , 15 S , 17 R , 20 R , 24 R , 2′ R , 1′′ S , 2′′ S by X-ray crystallographic diffraction [Flack parameter 0.01(7) ( Figure 4 )]. Figure 3. NOESY (pink dashed arrows) couplings of 1 − 6 . The NMR signals remaining from unit B indicated that it had a structure similar to 2′-hydroxy bisdechlorogeodin, [23] which is a polyketide belonging to the emodin family. The structural divergence between unit B and 2′-hydroxy bisdechlorogeodin lies in the absence of the OH at C-2′′ and a Δ 1′′,2′′ double bond in unit B, which could be unambiguously determined through the essential HMBC associations of H-2′′/C-2′, C-4′′, C-7′′ and the higher field δ values of C-1′′ and C-2′′ in unit B. Thus, the unit B’s planar structure was constructed. With an IHD of 17 accounted for by the ergosterol (unit A) and the polytide (unit B) moieties, compound 1 demanded an additional unsaturation site. This was rationalized by a bridging ring system (ring E) which unit A linked to unit B via C-2′′−C-7 and C-1′′−C-15 bonds, as evidenced by the crucial COSY association of H-7/H-2′′, combined with the vital HMBC associations of H-15/C-2′′, H 2 -16/C-1′′. Hence, its planar architecture was elucidated as an [4+2] cycloaddition of 14-dehydroergosterol and bisdechlorogeodin, featuring an unprecedented 6/6/6/6/5/6/5/6 octacyclic scaffold. The essential NOESY couplings ( Figure 3 ) of H-15/H-7, H 3 -18; H-7/H 3 -19; and H-20/H 3 -18 demonstrated that H-7 and H-15 were oriented β , and H-17 was oriented α . The association between H-2′′ and H-7 ( J 7,2′′ = 8.6 Hz) proved that H-2′′ oriented α . The NOESY associations of H-1a/H-3, H-9 implied that H-3 and H-9 oriented α . Nevertheless, the relative configurations at C-1′′, C-2′, C-20, and C-24 remained unassigned owing to the absence of detectable NOESY linkages and the free rotation of the side chain. Ultimately, the absolute stereochemistry of 1 was unequivocally established as 3 S , 7 S , 9 R , 10 R , 13 R , 15 S , 17 R , 20 R , 24 R , 2′ R , 1′′ S , 2′′ S by X-ray crystallographic diffraction [Flack parameter 0.01(7) ( Figure 4 )]. Ergostemodin B ( 2 ) was designated the empirical formula of C 46 H 58 O 8 from the HRESIMS data ( m/z 761.4027 [M + Na] + , and calcd 761.4029). The planar structure of 2 was almost identical to that of 1 , with the only notable difference being the replacement of the olefinic proton of H-23 in 1 by a methyl in 2. This substitution could be demonstrated by a crucial COSY association ( Figure 2 ) of H-20/H-22, together with the vital HMBC associations of H 3 -29/C-22, C-23, and C-24. The stereochemistry of 2 was elucidated based on the NOESY associations depicted in Figure 3 . The H-15 oriented β and H-17 oriented α were evidenced by the couplings of H 3 -18/H-15, H-20. Additionally, the NOESY coupling of H-7/H 3 -19 demonstrated H-7 oriented β . Meanwhile, the J 2′′, H-7 (8.9 Hz), similar to that in 1, determined H-2′′ oriented α . Combining the above analysis with the biosynthetic pathways of ergosterols [24] and polyketides [25] , and the close resemblance of ECD curves between 1 and 2 ( Figure S1 ), the absolute stereochemistry of 2 was confirmed as 3 S , 7 S , 9 R , 10 R , 13 R , 15 S , 17 R , 20 R , 24 R , 2′ R , 1′′ S , 2′′ S . Figure 4. Crystallographic structures of 1 and 5 . Table 3 1 H NMR (400 MHz) and 13 C NMR (100 MHz) data for compounds 5−6. No. δ H ( J in Hz) δ C , type δ H ( J in Hz) δ C , type 1 1.78, m 31.1, CH 2 1.80, m 1.92, m 31.1, CH 2 2 1.86, m 1.45, m 30.8, CH 2 1.72, m 2.01, m 30.8, CH 2 3 3.50, m 68.7, CH 3.99, dt (11.3, 6.2) 68.7, CH 4 2.47, m 2.19, m 38.5, CH 2 2.30, m 1.56, m 38.5, CH 2 5 64.7, C 64.7, C 6 5.55, d (2.6) 62.6, CH 3.42, s 62.6, CH 7 2.69, m 195.6, C 195.6, C 8 124.6, C 124.5, C 9 1.87, m 156.8, C 156.8, C 10 40.7, C 40.7, C 11 1.68, m 24.0, CH 2 2.31, m 23.8, CH 2 12 2.07, m 1.36, m 35.7, CH 2 1.51, m 2.06, m 35.7, CH 2 13 44.9, C 44.9, C 14 141.1, C 141.0, C 15 2.87, d (8.2) 126.4, CH 6.03, t (2.7) 126.3, CH 16 2.60, dd (15.1, 7.3) 1.85, m 37.0, CH 2 2.26, m 2.16, m 37.5, CH 2 17 0.97, m 56.7, CH 1.51, m 56.1, CH 18 0.96, s 15.7, CH 3 0.80, s 15.6, CH 3 19 0.91, s 24.6, CH 3 1.23, s 24.6, CH 3 20 2.03, m 33.4, CH 2.24, m 38.9, CH 21 0.99, d (6.4) 21.0, CH 3 1.04, d (6.6) 21.2, CH 3 22 5.14, dd (15.2, 8.4) 131.0, CH 5.19, dd (15.3, 8.0) 135.2, CH 23 5.28, dd (15.2, 8.0) 136.4, C 5.27, dd (15.3, 7.4) 132.6, CH 24 1.85, m 50.4, CH 1.86, m 43.0, CH 25 1.48, m 30.9, CH 1.48, m 33.2, CH 26 0.88, d (6.8) 20.3, CH 3 0.88, d (6.8) 20.6, CH 3 27 0.86, d (6.8) 21.9, CH 3 0.86, d (6.8) 20.4, CH 3 28 1.01, d (6.6) 17.2, CH 3 0.92, d (6.8) 17.7, CH 3 29 3.36, s 13.3, CH 3 Ergostemodin C ( 3 ) and 1 share the identical planar architecture. The interpretation of 1D NMR data indicated that 3 should be an isomer of 1, with the δ values and the coupling constants altered at H-7 and H-2′′. Upon examination of the 2D NMR data, it was pointed out that units A and B of 3 are connected via C-2′′−C-15 and C-1′′−C-7 bonds, as indicated by the critical COSY correlation of H-2′′/H-15, along with the crucial HMBC associations ( Figure 2 ) of H-2′′/C-16, C-14. Hence, the gross structure of 3 was identified. The relative stereochemistry of 3 was assigned through detailed coupling constants and the NOESY associations ( Figure 3 ). The key NOESY couplings of H 3 -19/7′′-OCH 3 and H-2′′/H 3 -18, H-16b indicated that 7′′-OCH 3 and H-2′′ oriented β . Furthermore, the observed large trans-diaxial J 15,2′′ = 10.1 Hz suggested H-15 oriented α . Meanwhile, the significant couplings of H-7/H-15, H-9 demonstrated that H-7 and H-9 oriented α. Considering the biosynthetic pathways of ergosterols [24] and polyketides, [25] the absolute stereochemistry of 3 was confirmed as 3 S , 7 S , 9 R , 10 R , 13 R , 15 S , 17 R , 20 R , 24 R , 2′ R , 1′′ S , 2′′ S . Ergostemodin D ( 4 ) was designated the empirical formula C 45 H 54 O 9 from HRESIMS ( m/z 761.3667 [M + Na] + , suggesting an IHD of 19. Its 1D NMR spectra pointed out that 4’s planar structure resembled that of 3 . The main differences were that 4 featured two double bonds at Δ 4,5 and Δ 6,7 , as judged by the H-3/H-4 association in the crucial COSY correlation ( Figure 2 ), together with essential HMBC associations ( Figure 2 ) of H-4/C-10, and H-6/C-4, C-8, C-10, C-1′′. Additionally, an extra epoxy moiety at C-8, C-14 was identified, supported by δ C 62.7 and 74.4, together with the vital HMBC associations of H 3 -18, H-2′′/C-14, and H-6/C-8. Moreover, significant shifts in the δ value and coupling constant of H-2′′ suggested a possible reversal in the chirality of C-15, C-1′′, and C-2′′. The NOESY association of H-15/H 3 -18 in Figure 3 indicated that H-15 was oriented β . Additionally, the NOESY association of H-2′′/H-16a confirmed that H-2′′ was α -oriented. Since 7′′-OCH 3 and H-2′′ are in the same orientation, governed by the Woodward-Hoffmann rules, it could be concluded that 7′′-OCH 3 is α -orientated. The configuration reversal at C-2′′ and C-15 was definitively established by the observed small coupling constant between H-2′′ and H-15. Following a comprehensive analysis, C-8 and C-14 remained the only carbons with undetermined configurations. It was reported that the δ value of C-14 ranged from 70 to 75 ppm [26-28] when configured as 8 R , 14 R , and 85 ppm [29] when configured as 8 S , 14 S . The δ values observed for C-14 ( δ C 74.4) in compound 4 were more consistent with the configuration of 8 R , 14 R , thus establishing the configuration of the epoxide group. Consequently, the absolute stereochemistry of 4 was appointed as 3 S , 8 R , 9 R , 10 R , 13 R , 14 R , 15 R , 17 R , 20 R , 24 R , 2′ R , 1′′ S , 2′′ R . Penicillisterene A (5) was designated the empirical formula C 29 H 42 O 3 from HRESIMS data ( m/z 461.3070 [M + Na] + , and calcd 461.3032). The spectral information (Table 3) of 5 matched those reported for gargalol C 27 , differing only in the absence of a hydroxyl and an olefinic proton, alongside the existence of a methyl and an additional olefin unit. The COSY association of H-15/H 2 -16, complemented by the vital HMBC associations (Figure 2) of H-15/C-13, C-17, H 3 -18/C-14, and H 3 -29/C-22, C-23, C-24, revealed that the olefin unit was located at Δ 14,15 and the olefinic proton at C-23 was replaced by a singlet methyl. Consequently, the planar architecture of 5 was resolved. Table 4 Immunosuppressive effects of 1−6 on murine lymphocyte proliferation induced by ConA (5 μ g/mL) or LPS (10 μ g/mL) EC 50 ( μ M) SI EC 50 ( μ M) SI CC 50 ( μ M) 1 5.80 >8.62 7.94 >6.29 >50 2 14.79 >3.38 12.13 >4.12 >50 3 13.63 >3.67 15.17 >3.29 >50 4 27.97 >1.79 25.56 >1.96 >50 5 18.83 >2.65 6.74 >7.41 >50 6 9.48 >5.27 5.44 >9.19 >50 CsA 0.17 – – – – MMF – – 1.05 – – The crystals of 5 were obtained from a methanol solution, establishing its relative stereochemistry as 3 S * , 5 R * , 6 R * , 10 R * , 13 R * , 17 R * , 20 R * , 24 R * [Figure 4, Flack parameter 0.0(2)]. Combined with the biosynthetic pathways of ergosterols, [24] the absolute stereochemistry of 5 was determined as 3 S , 5 R , 6 R , 10 R , 13 R , 17 R , 20 R , 24 R . Table 5 Cytotoxicity of 1−6 against HT-29, MCF-7, HeLa, HepG2, and A549 HT29 MCF-7 HeLa HepG2 A549 1 17.09 21.70 12.71 11.53 24.74 2 46.66 >50 49.82 45.26 >50 3 42.17 46.53 48.29 43.80 >50 4 >50 >50 >50 >50 >50 5 25.63 >50 18.96 >50 16.82 6 32.48 37.77 27.50 49.76 36.24 adriamycin 0.62 0.17 1.01 0.80 1.83 Penicillisterene B (6) was designated the empirical formula C 28 H 40 O 3 based on its HRESIMS data ( m/z 447.2869 [M + Na] + , calcd for C 28 H 40 O 3 Na, 447.2875). The 1D NMR data of 6 were consistent with those of 5 (Table 3), indicating structural similarities, with the only distinction being the absence of C-23 methyl, corroborated by the crucial COSY associations (Figure 2) of H-22/H-23/H-24. The β configurations of H 3 -18, H 3 -19, and H-6, and the α configurations of H-3 and H-17 were designated according to the NOESY associations (Figure 3) of H-6/H 3 -19, H-3/H-1a, and H 3 -18/H-20. The absolute stereochemistry of C-20 and C-24 was verified by comparing the δ values for H 3 -21 and H 3 -28 with documented data: H 3 -21 ( δ H 1.28 for 20 S , δ H 1.13 for 20 R ) and H 3 -28 ( δ H 0.912 for 24 R , δ H 0.819 for 24 S ). [30] Therefore, 6 was designated as 20 R , 24 R . Combined with the similar ECD spectra between 6 and 5 (Figure S2), the absolute stereochemistry of 6 was confirmed as 3 S , 5 R , 6 R , 10 R , 13 R , 17 R , 20 R , 24 R. Compounds 1 − 4 represent novel heterodimers biosynthetically formed via Diels-Alder reactions of an ergosterol compound, 14-dehydroergosterol, and an emodin compound, bisdechlorogeodin ( Figure 5 ). These compounds vary in configuration, ergosterol unit, and subsequent modifications. Compounds 1 , 3 , and intermediate a are isomers arising from the regioselectivity of the Diels-Alder reaction. Compound 4 is presumed to be derived from intermediate a through two consecutive oxidation reactions. Compound 2 is the Diels-Alder cycloadduct of intermediate b and bisdechlorogeodin. Figure 5. Proposed biosynthetic pathway for compounds 1 − 4 . The immunosuppressive activities of 1 − 6 were assessed toward ConA-activated T and LPS-activated B lymphocyte cells in vitro . As a result ( Table 4 ), 1 − 6 inhibited ConA-activated T and LPS-activated B cell proliferation, exhibiting EC 50 values of 5.80−27.97 μ M and 5.44−25.56 μ M, respectively. Additionally, the cytotoxicity of 1 − 6 against HepG2, HT-29, HeLa, MCF-7, and A549 cells was assessed using the MTT assay ( Table 5 ), and compound 1 displayed cytotoxicity toward HepG2 cells, exhibiting an IC 50 value of 11.53 ± 0.43 μ M. Conclusions In summary, four unreported Diels–Alder [4 + 2] cycloadducts derived from ergosterol and an emodin derivative, ergostemodins A−D ( 1 − 4 ), along with two previously unreported ergosterols ( 5 and 6 ), were isolated from Penicillium sp. XB152. Compounds 1 − 4 feature a distinctive 6/6/6/5/6/6/5/6 octacyclic ring system. Compounds 1 , 5 , and 6 exhibited potential immunosuppressive activities. Notably, 1 also displayed significant cytotoxicity against HepG2 cells. These discoveries expand the structural diversity of steroidal Diels–Alder cycloadducts and provide a foundation for the targeted discovery of steroid-based immunosuppressive agents. Experimental General experimental protocols Optical rotation was assessed using a PerkinElmer 341 instrument. HRESIMS was conducted utilizing the Bruker micro TOF II spectrometer. The relevant NMR experiments were performed on AVANCE NEO 600 and AM-400, manufactured by the Bruker company . SC-XRD experiments utilized a Bruker Photon III diffractometer employing Ga K α radiation. ECD, IR, and UV were separately conducted on the JASCO J-1700 or J-810, Thermo Scientific Nicolet iS50R FT-IR microscope, and SolidSpec-3700 UV−vis spectrophotometer. Column chromatography (CC) was carried out using the following stationary phases: silica gel (100−200 mesh and 200−300 mesh), Sephadex LH-20 gel, and octadecylsilyl (ODS) gel (S-50 µ m). Semi-preparative HPLC separation employed a SilGreen HPLC column and a Daicel CHIRALPAK ® IG chiral column as stationary fractionation and was achieved through the Agilent 1260 and 1100 systems. Fermentation and isolation Penicillium sp. XB152 was purified from Rhododendron lapponicum gathered from Sun Mountain, Xianning County, Hubei Province of China, in January 2021 and determined based on ITS rDNA sequence (Accession Number for GenBank: PP859464). P. sp. XB152 was propagated on PDA under axenic conditions at room temperature for a week to obtain primary inoculum. Subsequently, agar plugs were transferred to 150 Erlenmeyer flasks (1 L) containing a solid-state fermentation medium (rice substrate 250 g, L-leucine 1.5g, and water 250 mL). The cultures were incubated for 4 weeks. Following incubation, four extractions were made using EtOAc (EA). After distilling on a rotary evaporator, the crude extract (403 g) was processed through 100−200 mesh silica gel CC and eluted by applying a gradient elution of PE−EtOAc (v/v, 10:1 to 0:1), affording 7 main fractions (Fr. A−G). Fr. C (13 g) was subdivided on Sephadex LH-20 (eluent: CH 2 Cl 2 −CH 3 OH, v/v, 1:1) to provide 3 sub-fractions (Fr. C1−C3). Fr. C2 was directly exposed to ODS CC with CH 3 OH−H 2 O gradient (v/v, 40:60 to 100:0) to result in 17 sub-fractions (Fr. C2.1−C2.17). Fr. C2.15 was exposed to column chromatography on silica gel and subsequently was prepared using semipreparative HPLC (eluted: CH 3 OH−H 2 O, v/v, 80:10) to furnish 5 (8.1 mg). Fr. D (71.4 g) was directly exposed to Sephadex LH-20 chromatography (eluted: CH 2 Cl 2 −CH 3 OH, v/v, 1:1), yielding 3 sub-fractions (Fr. D1−D3). Fr. D2 underwent ODS CC utilizing CH 3 OH−H 2 O mixtures (v/v, 40:60 to 100:0), resulting in 30 sub-fractions (Fr. D2.1−2.30). Fr. D2.24 underwent silica gel column and was subsequently purified via semi-prep HPLC (CH 3 CN−H 2 O, v/v, 90:10), yielding 6 (9.6 mg). Fr. E (46.4 g) was processed using Sephadex LH-20 (eluted: CH 2 Cl 2 −CH 3 OH, v/v, 1:1) to afford 7 sub-fractions (Fr. E1−E7). Fr. E3 underwent ODS CC utilizing CH 3 OH−H 2 O mixtures (v/v, 40:60 to 100:0), resulting in 34 sub-fractions (Fr. E3.1−E3.34). Fr. E3.31 was applied to a silica gel column and then was prepared using semi-prep HPLC (eluted: CH 3 CN−H 2 O, v/v, 90:10), yielding 1 (24.5 mg), 2 (5.3 mg). Fr. E3.31.8.4 was refined utilizing HPLC (ODS, CH 3 CN−H 2 O, v/v, 85/15) to furnish 3 (2.0 mg). Fr. E3.30 was employed in column chromatography on silica gel and then was prepared using semi-prep HPLC (eluted: CH 3 CN−H 2 O, v/v, 90:10; chiral HPLC columns, CH 3 CN−H 2 O, v/v, 62:38) to yield 4 (0.8 mg). ergostemodin A (1) Colorless crystals (in CH 3 OH); mp 129 ℃; [ α ] −236 (CH 3 OH , c 0.1 mg/mL); Its ECD (CH 3 OH, c 0.5 mg/mL) displayed λ max ( Δε ) values at 212 (−17.77), 259 (2.86), 319 (−13.64) nm; Its UV spectrum (CH 3 OH) displayed λ max (log ε ) at 207 (5.02), 276 (4.70), 345 (3.99) nm; Its IR absorptions (film) included ν max 3440, 2956, 1720, 1628, 1458, 1219, 1045 cm −1 ; Its HRMS (ESI) displayed [M + Na] + at m/z 747.3860 (calcd C 45 H 56 O 8 Na 747.3873); 1D NMR is presented in Table 1. ergostemodin B (2) White solid; [ α ] −166 ( c 0.1, CH 3 OH); Its ECD (CH 3 OH , c 0.5 mg/mL) displayed λ max ( Δε ) values at 215 (−9.49), 256 (0.29), 284 (−5.53), 295 (−4.84), 317 (−7.10) nm; Its UV specturm (CH 3 OH) displayed λ max (log ε ) at 205 (4.40), 266 (4.09), 342 (3.45) nm; Its IR absorptions (film) included ν max 3431, 2929, 1728, 1629, 1459, 1222, 1031 cm −1 ; Its HRMS (ESI) displayed [M + Na] + at m/z 761.4027 (calcd C 46 H 58 O 8 Na 761.4029); 1D NMR is presented in Table 1. ergostemodin C (3) White powder; [ α ] −92 ( c 0.1, CH 3 OH); Its ECD (CH 3 OH , c 0.5 mg/mL) displayed λ max ( Δε ) values at 208 (3.00), 230 (−0.46), 262 (1.41), 320 (−3.67) nm; Its UV spectrum (CH 3 OH) displayed λ max (log ε ) at 204 (4.46), 274 (4.16), 343 (3.46) nm; Its IR absorptions (film) included ν max 3432, 2927, 1731, 1629, 1459, 1223 cm −1 ; Its HRMS (ESI) displayed [M + Na] + at m/z 747.3826 (calcd C 45 H 56 O 8 Na 747.3873); 1D NMR is presented in Table 2. ergostemodin D (4) White solid; [ α ] −104 ( c 0.1, CH 3 OH); Its ECD (CH 3 OH , c 0.5 mg/mL) displayed λ max ( Δε ) values at 216 (5.08), 233 (1.33), 245 (2.07), 278 (−8.68) nm; Its UV specturm (CH 3 OH) displayed λ max (log ε ) at 204 (3.94), 250 (3.80), 273 (3.77), 335 (3.08) nm; Its IR absorptions (film) included ν max 3432, 2927, 1729, 1665, 1629, 1459, 1222 cm −1 ; Its HRMS (ESI) displayed [M + Na] + at m/z 761.3667 (calcd C 45 H 54 O 9 Na 761.3666); 1D NMR is presented in Table 2. penicillisterene A (5) White solid; [ α ] +34 ( c 0.1, CH 3 OH); Its ECD (CH 3 OH , c 0.25 mg/mL) displayed λ max ( Δε ) values at 206 (−14.86), 233 (1.33), 248 (−2.77), 265 (−0.33), 283 (−0.67), 332 (3.97) nm; Its UV specturm (CH 3 OH) displayed λ max (log ε ) at 201 (4.22), 227 (4.22), 306 (3.51) nm; Its IR absorptions (film) included ν max 3408, 2956, 2929, 1669, 1458, 1385, 1280 cm −1 ; Its HRMS (ESI) displayed [M + Na] + at m/z 461.3070 (calcd C 29 H 42 O 3 Na 461.3032); 1D NMR is presented in Table 3. penicillisterene B (6) White powder; [ α ] +21 ( c 0.1, CH 3 OH); Its ECD (CH 3 OH, c 0.25 mg/mL) displayed λ max ( Δε ) values at 200 (−11.03), 234 (0.30), 247 (−2.65), 266 (−0.08), 282 (−0.28), 331 (4.17) nm; Its UV specturm (CH 3 OH) displayed λ max (log ε ) at 228 (4.15), 3.07 (3.48) nm; Its IR absorptions (film) included ν max 3443, 2958, 2928, 1666, 1458 cm −1 ; Its HRMS (ESI) displayed [M + Na] + at m/z 447.2869 (calcd C 28 H 40 O 3 Na 461.3032); 1D NMR is presented in Table 3. X-ray crystallography Single crystals were produced via the methanol vapor diffusion. The crystal data was acquired on D8 Venture diffractometer equipped with monochromatized Ga Kα radiation. Crystallographic information was stored in the CCDC. The data are freely accessible at www.ccdc.cam.ac.uk/deposit Compound 1 (CCDC 2361726). C 48 H 68 O 11 , M = 821.02 g/mol, P1, triclinic, a = 8.3746(11) Å, b = 10.9209(14) Å, c = 13.7627(18) Å, V = 1168.8(3) Å 3 , Z = 1, T = 200(2) K, α = 101.645(5)°, β = 91.309(5)°, γ = 107.819(5)°, μ (Ga K α ) = 0.420 mm –1 , Flack parameter = 0.01(7). Compound 5 (CCDC 2361727). C 58 H 86 O 7 , M = 895.26 g/mol, P2 1 2 1 2 1 , orthorhombic, a = 7.0961(11) Å, b = 10.3397(16) Å, c = 71.568(10) Å, Z = 4, T = 100.00 K, α = β = γ = 90°, μ (Ga K α ) = 0.359 mm –1 , Flack parameter = 0.0(2). Immunosuppressive activity assay Splenic cells were collected from male BALB/ c mice (8 weeks). Following processing, the resulting lymphocytes were obtained and resuspended in Gibco 1640 medium. For the immunosuppressive assay, cells were inoculated at 1 × 10 6 cells/well in 96-well microplates and stimulated with 2.5 μ g/mL ConA (T cells) or 10 μ g/mL LPS (B cells), respectively. The cells were co-treated with serial dilutions of the test compounds or controls and incubated for 48 h at 37 °C under 5% CO₂. The cyclosporin A (CsA) and mycophenolate mofetil (MMF) were positive controls for T and B cell proliferation, respectively. Cell viability was then assessed by adding CCK-8 reagent for 4 h, and the absorbance at 450 nm was measured with a Bio-Tek Synergy 2 microplate reader. EC₅₀ and CC₅₀ values were generated using GraphPad Prism 8. The selectivity index (SI) was expressed as CC 50 /EC 50 . Cytotoxicity assay A549, HT-29, HeLa, MCF-7, HepG2 cells were exposed to an incubator at 37 °C with 5% CO 2 , using DMEM medium. For cytotoxicity assays, Cells were added to 96-well microplates and incubated with serial dilutions of test samples and adriamycin for 24 h. Adriamycin was the positive control. Subsequently, cells were treated with MTT solution (15 μ L/well) and incubated for 4 h. Formazan solubilization was achieved with DMSO after aspiration of the supernatant. Absorbance readings at 490 and 570 nm were detected. Calculation of IC 50 values was performed with GraphPad Prism 8. Supporting Information The supporting information for this article is available on the WWW under https://doi.org/10.1002/cjoc.70XXX. Acknowledgement We sincerely thank the National Natural Science Foundation of China (No. 22477035 ) for financial support. Special thanks are extended to Mrs. Chaomei Xiong and Mr. Wei Wang at the Analytical and Testing Center of Huazhong University of Science and Technology for their assistance with NMR data acquisition. 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Manuscript received: XXXX, 2024 Manuscript revised: XXXX, 2024 Manuscript accepted: XXXX, 2024 Version of record online: XXXX, 2024 Entry for the Table of Contents Immunosuppressive Ergosterol−emodin Hybrids from Penicillium sp. XB152 Qianxi Ouyang, a,b,1 Chunlun Qin, a,1 Jinling Chang, a Jiao Pei, a Yinhui Zhou, a Huifang Pi,* , a An Jin,* , c and Hanli Ruan* , a Chin. J. Chem. 2025 , 43 , XXX—XXX. DOI: 10.1002/cjoc.70XXX Ergostemodins A−D ( 1 − 4 ), the first examples of ergosterol−emodin carbon skeletons formed by Diels−Alder reactions between ergosterol and emodin moieties, were separated from Penicillium sp. XB152. Compounds 1 − 4 feature a distinctive 6/6/6/5/6/6/5/6 octacyclic ring system. Compounds 1 − 4 inhibite ConA-T cell and LPS-B cell proliferation with EC 50 values ranging from 5.80 to 27.97 μ M and from 5.44 to 25.66 μ M, respectively. Information & Authors Information Version history V1 Version 1 13 November 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords diels-alder reactions ergostemodins a−d ergosterol−emodin heterodimers immunosuppressive activity penicillium sp. xb152 Authors Affiliations Qianxi Ouyang Huazhong University of Science and Technology Tongji Medical College View all articles by this author Chunlun Qin Huazhong University of Science and Technology Tongji Medical College View all articles by this author Jinling Chang Huazhong University of Science and Technology Tongji Medical College View all articles by this author Jiao Pei Huazhong University of Science and Technology Tongji Medical College View all articles by this author Yinhui Zhou Huazhong University of Science and Technology Tongji Medical College View all articles by this author Huifang Pi Huazhong University of Science and Technology Tongji Medical College View all articles by this author An Jin Hunan University of Medicine School of Pharmaceutical Science View all articles by this author Han-Li Ruan 0000-0003-0882-1009 [email protected] Huazhong University of Science and Technology Tongji Medical College View all articles by this author Metrics & Citations Metrics Article Usage 153 views 76 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Qianxi Ouyang, Chunlun Qin, Jinling Chang, et al. Immunosuppressive Ergosterol−emodin Hybrids from Penicillium sp. XB152. Authorea . 13 November 2025. DOI: https://doi.org/10.22541/au.176300128.81685363/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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