Dioscorea nipponica Makino, a plant used in traditional medicine for various ailments. Nine compounds were isolated, including a new compound named as diosniposide E, which was elucidated by analyzing its 1H-NMR, 13C-NMR, DEPT, COSY, HMBC and MS data and comparing them with data available in literature. The other eight compounds were identified as known compounds. Theoretical calculations of energy and the generation of a molecular electrostatic potential surface map were employed to assess the antioxidant capacity of nine compounds, the calculation results exhibited that compounds 5 and 6 had strong antioxidant capacities. To further evaluate the antioxidant activities of the investigated compounds, the DPPH and ABTS assays were conducted. The results from the DPPH scavenging activity test revealed that compounds 4–6 exhibited enhanced scavenging activities compared to L-ascorbic acid, while displaying similar efficacy to trolox. Moreover, the ABTS scavenging activities of compounds 4–6 were found to surpass those of L-ascorbic acid and trolox. In terms of α-glucosidase inhibition, compounds 3 and 4 displayed remarkable inhibitory activities that surpassed the effects of acarbose. Additionally, compound 2 exhibited potent anticholinesterase activities, outperforming donepezil. This research provides insights into the potential bioactive compounds present in Dioscoreanipponica Makino and may contribute to its use in traditional medicine.This study reported the isolation and identification of bioactive compounds from The online version contains supplementary material available at 10.1186/s12906-023-04086-6. Oxygen is a highly reactive element that can cause damage to biological molecules such as proteins and fats, resulting in various diseases such as cancer, aging, and diabetes. Free radicals generated by oxidative stress are responsible for the damage caused to biological molecules , 2. AntiThe decline in the body's capacity to counteract free radicals with age has emerged as the primary factor contributing to human aging and is implicated in 85% of diverse pathological conditions . ScientiRhodiola rosea, Panax ginseng, and Panax notoginseng, both in in vitro and in vivo experiments conducted on murine and rodent models +. The 1H-NMR  spectrum of compound 2 showed the presence of seven aromatic protons at δH 7.52 , 7.44 , 7.39 , 6.28 , 6.25 , an oxygenated methine proton at 5.61 , a methylene proton at 3.25  and 3.11 ; 13C-NMR (125 MHz) δ 170.1 (14-COOH), 165.0 (C-11), 164.3 (C-13), 138.7 (C-1), 142.0 (C-9), 128.3 , 125.9 , 106.6 (C-10), 100.9 (C-12), 100.3 (C-14), 80.5 (C-7), 34.7 (C-8). The compound was identified as diosniponol C, by comparison of data to that of known compounds in literature [13C NMR spectrum , supporting the presumed molecular formula C16H16O6; The 1H-NMR  spectrum of compound 1 showed the presence of six aromatic protons at δH 7.52 , 7.44 , 7.39 , 6.37 . an oxygenated methine proton at 5.61 , 3.86 , a methylene proton at 3.25  and 3.11 ; 13C-NMR (125 MHz) δ 170.3 (14-COOH), 157.2 (C-11), 156.3 (C-13), 138.6 (C-1), 136.0 (C-9), 128.3 , 125.9 , 106.5 (C-10), 133.8 (C-12), 101.0 (C-14), 80.8 (C-7), 59.5 (12-OCH3), 34.2 (C-8). The 13C NMR spectrum , and the absolute configuration of the new compound C7 in comparison with known literature is in the S form [1 was named as diosniposide E.A strong correlation between H-7 and H-8, as well as between H-2,6 and H-3,5 can be clearly seen in the COSY spectrum, indicating that these hydrogens are connected. Furthermore, the HMBC spectrum confirms correlations between C-7 and H-2, C-7 and H-6, C-10 and H-8, C-14 and H-8, C-12 and H-10, C-12 and 12-OCHe S form , therefo3: Molecular Formula: C19H18O3, Molecular Weight: 294. Yellow solid, EI-MS m/z: 294. 1H-NMR  δ 7.35 , 7.28 , 6.98 , 6.95 , 6.80 , 6.63 , 6.62 , 6.18 , 2.82 , 2.73 . 13C-NMR (125 MHz) δ 198.7 (C-3), 158.7 (C-4′′), 155.4 (C-4′), 143.3 (C-5), 141.4 (C-7), 131.1 (C-1′), 128.9 , 128.8 , 127.8 (C-4), 127.0 (C-1′′), 123.6 (C-6), 115.6 , 41.6 (C-2), 29.0 (C-1). The data are consistent with the literature [Compound 13C-NMR 1 MHz δ 194: Molecular Formula: C16H14O4, Molecular Weight: 270. Yellow solid, EI-MS m/z: 270. 1H-NMR  δ 7.79 , 7.36, 7.27 , 7.06 , 6.70 , 6.63 , 3.99 , 3.93 ; 13C-NMR (125 MHz) δ 159.2 (C-2), 154.8 (C-4), 147.5 (C-8), 144.5 (C-9), 134.8 (C-14), 127.2 (C-11), 126.6 (C-13), 124.6 (C-12), 124.3 (C-6), 114.5 (C-5), 111.4 (C-10), 108.6 (C-1), 104.2 (C-7), 98.6 (C-3), 54.8 (8-OCH3), 54.7 (4-OCH3). The data were consistent with the literature [Compound 5: Molecular Formula: C7H6O5, Molecular Weight: 170. Yellow solid, EI-MS m/z: 170. 1H-NMR  δ 7.06 ; 13C-NMR (125 MHz) δ 170.8 (C-7), 146.9 , 139.6 (C-4), 122.4 (C-1), 110.5 . The data are consistent with the literature [Compound terature  and the 6: Molecular Formula: C8H8O5, Molecular Weight: 184. Yellow solid, EI-MS m/z: 184. 1H-NMR  δ 7.11 , 3.78 . 13C-NMR (125 MHz) δ 167.3 (C-7), 146.1 , 138.8 (C-4), 121.9 (C-1), 109.9 , 52.0 (7-OCH3). The data were consistent with the literature [Compound terature  and the 7: Molecular Formula: C8H8O4, Molecular Weight: 168. Yellow solid, EI-MS m/z: 168. 1H-NMR  δ 7.59 , 7.55 , 6.90 , 3.90 ; 13C-NMR (125 MHz) 167.7 (C-7), 152.2 (C-4), 148.2 (C-3), 124.9 (C-6), 123.0 (C-1), 115.6 (C-2), 113.5 (C-5), 56.4 (3-OCH3). The data were consistent with the literature [Compound terature  and the 8: Molecular Formula: C32H26O8, Molecular Weight: 538. Yellow solid, EI-MS m/z: 538. 1H-NMR  δ 9.24 , 7.28 , 7.12 , 7.00 , 6.91 , 4.23 , 4.09 ; 13C-NMR (125 MHz) δ 166.4 , 158.1 , 147.7 , 145.0 , 133.8 , 126.3 , 124.1 , 122.5 , 115.2 , 114.1 , 111.6 , 109.1 , 99.0 , 55.1 . The data were consistent with the literature [Compound terature  and the 9: Molecular Formula: C15H10O5, Molecular Weight: 270. Yellow solid, EI-MS m/z: 270. 1H-NMR  δ 12.95 , 7.91 , 6.90 , 6.75 , 6.47 , 6.18 ; 13C NMR (125 MHz) 181.8 (C-4), 164.3 (C-7), 163.8 (C-2), 161.5 (C-5), 161.2 (C-4'), 157.4 (C-9), 128.5 , 121.2 (C-1'), 116.0 , 103.7 (C-10), 102.8 (C-3), 98.9 (C-6), 94.0 (C-8). The data were consistent with the literature [Compound terature  and the 1–9. Through density functional theory simulations, it has been determined that these theoretical models align well with the initial trial configurations depicted in Fig. Figure 1–9 have been calculated to assess the strength of their redox reactions. A smaller energy difference between deoxidation and oxidation signifies a favorable reaction. Table 1–9 are more inclined towards reduction rather than oxidation. Notably, compounds 5–7 exhibit significantly stronger abilities to acquire electrons compared to losing electrons, suggesting their potential for enhanced antioxidant capacity. These findings imply that compounds 5–7 may possess stronger antioxidant properties due to their pronounced electron-acquiring abilities.The deoxidation and oxidation energies of compounds 5 and 6 exhibited more positive electrostatic potential than the other compounds, which is consistent with the energy calculation results.Figure 1–9 in comparison with L-ascorbic acid and trolox according to the DPPH assay. compounds 4–6 exhibited high scavenging activity, which is comparable to trolox and superior to L-ascorbic acid. In contrast, the remaining six compounds exhibit relatively weak scavenging activity in the assay. These findings emphasize the potential of compounds 4–6 as effective DPPH scavengers, suggesting their promising antioxidant capabilities.Table 4–6 exhibited strong scavenging activity at low concentrations, with their IC50 values being 6–7 times lower than that of the positive controls (L-ascorbic acid and trolox). In addition, compounds 1–3 and 7 also exhibited strong scavenging activity, similar to L-ascorbic acid. However, compounds 8 and 9 had weaker scavenging activity compared to the other compounds tested.Table 1–7 exhibit potential antioxidant properties. Consequently, these compounds warrant further research and development to explore their efficacy and potential applications as antioxidants.In the search for natural free radical scavengers, both DPPH and ABTS assays are considered to be fast, effective, and economical methods, due to their simplicity, low cost, and reproducibility. These methods can be used to evaluate the antioxidant activity of various compounds, and the results provide valuable information on the potential health benefits of these compounds. DPPH is a fat-soluble free radical and ABTS is a water-soluble free radical. The capacity to scavenge ABTS is considered a more representative measure of antioxidant performance within the human body, as it corresponds to the water-soluble environment. Based on this consideration, compounds 4, categorized as a phenanthrene derivative, aligns with previous reports on phenanthrene derivatives derived from Dioscorea communis, which have demonstrated notable scavenging activities against DPPH and ABTS [5 is a naturally occurring substance found in various plants, vegetables, nuts and fruits [5 has demonstrated non-toxicity when orally administered at a dose of 5000 mg/kg body weight [6 has exhibited strong antioxidant activity without inducing any adverse effects even at a dosage of 1000 mg/kg [7 has been found to effectively reduce reactive oxygen species production and scavenge free radicals [Compound and ABTS . The datd fruits . It has d fruits . Importay weight . Similar00 mg/kg . Furtherradicals . These o1–4 have not been previously reported, making this study the first to assess their antioxidant potential. However, further comprehensive and systematic evaluations are necessary before these compounds can be established as antioxidants. In future research, when additional antioxidant compounds are isolated from the ethanol extract of D. Nipponica, the extract itself can be considered for antioxidant investigations. By expanding the scope of research and identifying more antioxidant compounds, a more comprehensive understanding of the antioxidant capacity of D. Nipponica and its extracts can be achieved.The antioxidant activities of compounds 1–9 as evaluated using the α-glucosidase inhibition assay in comparison with acarbose are summarized in Table 3 and 4 demonstrated significantly higher α-glucosidase inhibitory activities compared to acarbose, which is a recognized α-glucosidase inhibitor. These two compounds show promising potential as α-glucosidase inhibitors. However, the remaining seven compounds exhibited weaker α-glucosidase inhibitory activities when compared to acarbose. Further investigations and structure–activity relationship studies are warranted to understand the variations in α-glucosidase inhibitory activities among these compounds.The results for the α-glucosidase inhibitory activities of compounds 2 demonstrated the highest inhibitory activity against both AChE and BChE hepta-4E,6E-dien-3-one, 3,5-dimethoxyphenanthrene-2,7-diol, gallic acid, methyl gallate, vanillic acid, 2,2',7,7'-tetrahydroxy-4,4',6,6'-tetramethoxy-1,1'-biphenanthrenes and apigenin) and one new compound (diosniposide E), which differed from the known compound diosniponol C, only by one methoxy substitution at C-12.A total of nine compounds were successfully isolated from the ethyl acetate layer of the ethanol extract of 1–9. The outcomes of these calculations revealed that the investigated compounds have a higher tendency towards reduction, with compounds 5 and 6 exhibiting notably stronger electron acquisition abilities compared to electron loss. This observation suggests that compounds 5 and 6 may possess enhanced antioxidant activities. Subsequently, a comprehensive screening was conducted to evaluate the in vitro antioxidant activity and enzyme inhibitory potential of the nine compounds. Compounds 4–6 showed robust scavenging activities against DPPH and ABTS radicals, surpassing the efficacy of L-ascorbic acid and demonstrating similar potency to trolox in the DPPH assay, and surpassing both L-ascorbic acid and trolox in the ABTS assay. Furthermore, compounds 3 and 4 exhibited significant inhibitory effects on α-glucosidase, surpassing the activity of the standard acarbose. Notably, compound 2 displayed noteworthy inhibitory activity against both AChE and BChE, outperforming the reference compound donepezil. These findings lay a solid foundation for further research and development of natural antioxidants, antidiabetic agents and anticholinesterase drugs derived from D. nipponica.Theoretical calculations of energy and MEP surface maps were performed to assess the redox properties of compounds Additional file 1: Figure S1. 1HNMR spectrum of compound 1. Figure S2. 13C NMR spectrum of compound 1. Figure S3. DEPT spectrum of compound 1. Figure S4. COSY spectrum of compound 1. Figure S5. HMBC spectrum of compound 1. Figure S6. HRESIMS spectrum of compound 1. Figure S7. 1H NMR spectrum of compound 2. Figure S8. 13C NMR spectrum of compound 2. Figure S9. HRESIMS spectrum of compound 2. Figure S10. 1HNMR spectrum of compound 3. Figure S11. 13C NMR spectrum of compound 3. Figure S12. EIMS spectrum of compound 3. Figure S13. 1H NMR spectrum of compound 4. Figure S14. 13C NMR spectrum of compound 4. Figure S15. EIMS spectrum of compound 4. Figure S16. 1H NMR spectrum of compound 5. Figure S17. 13C NMR spectrum of compound 5. Figure S18. 1H NMR spectrum of compound 6. Figure S19. 13C NMR spectrum of compound 6. Figure S20. EIMS spectrum of compound 6. Figure S21. 1H NMR spectrum of compound 7. Figure S22. 13C NMR spectrum of compound 7. Figure S23. EIMS spectrum of compound 7. Figure S24. 1H NMR spectrum of compound 8. Figure S25. 13C NMR spectrum of compound 8. Figure S26. EIMS spectrum of compound 8. Figure S27. 1H NMR spectrum of compound 9. Figure S28. 13C NMR spectrum of compound 9. Figure S29. EIMS spectrum of compound 9.


R)-6,8-dihydroxy-4,5-dimethyl-3-methylidene-3,4-dihydro-1H-2-benzopyran-1-one (1), methyl acetate (2), (R)-5, 7-dimethoxy-3-((S)-(1-hydroxyethyl)-3,4-dimethylisobenzofuran-1(3H)-one (4b), (S)-7-hydroxy-3-((S)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran 1(3H)-one (5), and a p-hydroxyphenyl-2-pyridone derivative, avellaneanone (6), were isolated together with the previously reported (R)-3-acetyl-7-hydroxy-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (3), (R)-7-hydroxy-3-((S)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (4a) and isosclerone (7), from the ethyl acetate extract of a culture of a marine sponge-derived fungus, Hamigera avellanea KUFA0732. The structures of the undescribed compounds were elucidated using 1D and 2D NMR, as well as high-resolution mass spectral analyses. The absolute configurations of the stereogenic carbons in 1, 4b, 5, and 6 were established by X-ray crystallographic analysis. The absolute configurations of C-3 and C-4 in 2 were determined by ROESY correlations and on the basis of their common biosynthetic origin with 1. The crude fungal extract and the isolated compounds 1, 3, 4b, 5, 6, and 7 were assayed for their growth inhibitory activity against various plant pathogenic fungi viz. Alternaria brassicicola, Bipolaris oryzae, Colletotrichum capsici, C. gloeosporiodes, Curvularia oryzae, Fusarium semitectum, Lasiodiplodia theobromae, Phytophthora palmivora, Pyricularia oryzae, Rhizoctonia oryzae and Sclerotium rolfsii.Five undescribed pentaketide derivatives, ( Hamigera  are widespread and common soil fungi, despite one species being associated with beetles -2-[(4-methoxyphenyl)methylene]-1,2-ethanediyl]bis-formamide from an ethanol extract of the mycelium of H. avellanea (CBS 501.94) methyl acetate (2), (R)-5,7-dimethoxy-3-((S)-(1-hydroxyethyl)-3,4-dimethylisobenzofuran-1(3H)-one (4b), (S)-7-hydroxy-3-((S)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran 1(3H)-one (5), and avellaneanone (6), together with the previously reported (R)-3-acetyl-7-hydroxy-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (3) +  (13C NMR spectrum (C 166.3), six non-protonated sp2 , one protonated sp2 (δC 101.0), one methylene sp2 (δC 96.2), one methine sp3 (δC 34.2) and two methyl (δC 22.3 and 10.2) carbons. The 1H NMR spectrum  and 4.73 (J = 1.6 Hz), one methine quartet at δH 4.02 (J = 7.2 Hz), one methyl singlet at δH 2.02 and one methyl doublet at δH 1.27 (J = 7.0 Hz). The type and chemical shift values of the carbon atoms revealed that 1 is a 3, 4, 5, 6, 8-pentasubstituted chromone. That the hydroxyl group at δH 10.70 was on C-8, the methyl group at δH 2.02, s  was on C-5, and another hydroxyl group was on C-6 was evidenced by the chemical shift value of the hydrogen-bonded OH-8 as well as by the HMBC correlations (3-11 to C-5 (δC 113.2), C-4a (δC 143.6), 164.1 (C-6), and H-7  to C-5, C-6, C-8 (δC 161.8) and C-8a (δC 97.7). The COSY correlation from the methyl doublet at δH 1.27  to the quartet at δH 4.02 (J = 7.2 Hz/δH 34.2) , and H-4 to C-4a, C-5, C-8a, and C-11. That the methylidene group was on C-3 was supported by HMBC correlations from the two doublets of the olefinic protons at δH 4.73 (J = 1.6 Hz/δC 96.2) and δH 4.75 (J = 1.6 Hz/δH 96.2) to C-3 and C-4  , requirispectrum  exhibitespectrum , can be spectrum  displayeelations  from H3-δH 34.2)  revealed and C-4 . Taking 1 has one stereogenic center (C-4), and it is, therefore, necessary to determine the absolute configuration of this carbon. Since 1 could be obtained as a suitable crystal for X-ray analysis using a diffractometer equipped with CuKα radiation, its stereostructure was obtained. The Ortep view of 1, shown in R. Therefore, the absolute structure of 1 was elucidated as (R)-6,8-dihydroxy-4,5-dimethyl-3-methylidene-3,4-dihydro-1H-2-benzopyran-1-one.Compound 1 has never been previously reported; however, a structurally similar compound, but with opposite stereochemistry at C-4, i.e. (S)-8-hydroxy-6-methoxy-4,5-dimethyl-3-methyleneisochromen-1-one, was reported by Tayone et al. +  (13C NMR spectrum (C 170.7), one conjugated ester carbonyl (δC 168.2), two oxygen-bearing sp2 (δC 164.9 and 163.1), four non-protonated sp2 , one pronated sp2 (δC 97.5), one oxymethylene sp3 (δC 65.9), one methoxy (δC 55.9), one methine sp3 (δC 35.9) and three methyl  carbons. The 1H NMR spectrum , two terminally coupled doublets at δH 4.25 (J = 11.8 Hz/δC 65.9) and 4.59 (J = 11.8 Hz/δC 65.9), one methoxyl singlet at δH 3.85 (δC 55.9), one quartet at δH 4.33 (J = 7.1 Hz/δC 35.9), two methyl singlets at δH 2.19 (δC 20.8) and 2.08 (δC 10.1) and one methyl doublet at δH 1.19 (J = 7.1 Hz/δC 16.2). The presence of a 4,5-dimethyl-8-hydroxy-6-methoxy-3,4-dihydro-1H-2-benzopyran-2-one was substantiated by HMBC correlations from the singlet at δH 11.14 (OH-8) to the carbons at δC 163.1 (C-8) and 97.5 (C-7), the singlet at δHz/6.37 (H-7) to C-8, the carbons at δC 115.5 (C-5) and 99.0 (C-8a), a methyl singlet at δH 2.08 (H3-11) to the carbon at δC 164.9 (C-6), 141.5 (C-4a) and 115.5 (C-5), and the methyl doublet at δH 1.19 (H3-10) to C-4a, the carbons at δC 102.2 (C-3) and 35.9 (C-4)  and 4.59, d, J = 11.8 Hz) to C-3 and the carbonyl of the acetyl group at δC 170.7 (C-12) and the methyl protons of the acetyl group (δH 2.19) to C-12 (Compound 11.1131) , indicatspectrum  displayespectrum , can be spectrum , in combspectrum , exhibit.9 (C-4) . The che to C-12 . 2 was elucidated as methyl acetate. Therefore, the planar structure of 2 has two stereogenic carbons (C-3 and C-4); therefore, it is necessary to establish their absolute configurations. Since 2 is a viscous mass and could not be obtained as a suitable crystal for X-ray diffraction, the ROESY spectrum was obtained. The ROESY spectrum  with four malonyl CoA (II) gives a pentaketide (III), which undergoes cyclization and is followed by enolization to give IV, and further dehydration of IV gives rise to V. Stereospecific methylation at C-3 and C-5 of V by S-adenosyl methionine (SAM) gives rise to 1. Epoxidation of the exocyclic double bond of 1 gives an epoxide in VI. Enzymatic hydrolysis of the epoxide and methylation of a phenolic hydroxyl group in VI produces VII, which undergoes acetylation by acetyl CoA to give 2. Consequently, the absolute configuration at C-4 in 2 must be the same as that of C-4 in 1, i.e., 4R.Biosynthetically, VII, except for the stereochemistry at C-4 which is opposite to that of VII, from the EtOAc extract of the culture broth of Leptosphaeria sp. KTC 727. These authors have detected the difference in the 1H and 13C NMR chemical shift values between the major component  and the minor component  in the mixture but were not able to isolate them individually. On the contrary, the 1H and 13C NMR spectra -7-hydroxy-3-((R)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one, isolated from the culture extract of a marine mangrove-associated fungus, Xylaria sp. BL321 +   on the aromatic ring and the lack of a broad signal of the phenolic hydroxyl group around 7 ppm. Thus, the structure of 4b was deduced as a methoxy derivative of 4a. The correlations observed in the COSY, HSQC, and HMBC spectra -3,4-dimethylisobenzofuran 1(3H)-one.Compound 67.1232) , requiri spectra  are quit spectra  allowed 4b is also levorotatory  similar to 4a , we concluded that the absolute configurations at C-3 and C-8 in 4b are 3R and 8S also. After many attempts, we were finally able to obtain 4b as a suitable crystal and an X-ray analysis was performed. The Ortep view of 4b . The (+)-HRESIMS spectrum showed the [M + H]+ peak at m/z 253.1077   , reveali spectra  of 5 res spectra  revealedH 1.40, J = 6.4 Hz) in 5 is higher than that of 4a  around 0.5 ppm while the proton chemical shift value of its Me-10 singlet (δH 1.67) was lower than that of 4a (δH 1.78) just around 0.1 ppm. The differences in chemical shift values of the methyl protons of Me-9 and Me-10 can be attributed to the anisotropic effect of the benzene ring on both Me-9 and Me-10. As the chemical shift value of Me-10 in 5 is higher than that of its counterpart in 4a, while Me-10 in 5 is lower than that of its counterpart in 4a, the absolute configuration at C-3 in 5 must be opposite to that at C-3 in 4a, i.e., 3S. Since 5 could be obtained in a suitable crystal, an X-ray analysis was performed. The Ortep view of 5, shown in S configuration but also determined the configuration at C-8 as 8S. Therefore, a complete structure of 5 is (S)-7-hydroxy-3-((S)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran 1(3H)-one, which is the enantiomer of (R)-7-hydroxy-3-((R)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran 1(3H)-one  +  (13C NMR spectrum (C 162.4), two oxygen-bearing sp2 (δC 160.2 and 156.6), three non-protonated sp2 , five protonated sp2 (δC 115.1 (2C), 130.6 (2C), 131.5), one oxyquaternary sp3 (δC 82.2), three methine sp3 , three methylene sp3  and three methyl  carbons. The 1H NMR spectrum  and 7.15 , one olefinic singlet at δH 6.98 (δC 131.5), a double doublet at δH 2.06 , a double of double doublet at δH 1.71 , a quartet at δH 1.55 (J = 7.1 Hz/δC 33.8), one methyl singlet at δH 1.11 (δC 18.6), one methyl doublet at δH 1.47 (J = 6.2 Hz/δC 25.1) and one methyl triplet at δH 0.85 (J = 7.3 Hz/δC 7.7), in addition to various overlapped multiplets. The presence of a 4-hydroxy phenyl moiety was evidenced by the COSY correlation from the doublet at δH 7.15  to the doublet at δH 6.73  (C 156.6) and C-22 (δC 130.6), H-22 to C-18 (δC 130.6), and C-20, H-19 to C-17 (δC 126.7), and C-21 (δC 115.1), and H-21 to C-17, and C-19  to C-2 (δC 162.4), C-4 (δC 160.2), and C-5 (δC 113.6). Since H-18 and H-22 showed HMBC cross peaks to C-5 while H-6 showed cross peaks to C-17, the p-hydroxyphenyl group was on C-5 of the 2-pyridone ring. That the last portion of the molecule was 1-ethyl-1,5-dimethyl-1,4a,5,6,7,7a-hexahydrocyclopenta[c]pyran was supported by the COSY correlations from H3-16  to H-15 , H3-12  to H-11 , and a coupling system from H3-12 through H-11/H-7 /H-8 /H-9  and H-10  (3-16 to C-13 (δC 82.2) and C-15, H3-14  to C-8, C-13, C-15, H3-12 to C-7, C-10 and C-11, H-7 to C-11 and C-12, H-8 to C-13 and C-14  , requirispectrum  displayespectrum , can be spectrum , in conjspectrum , showed 9, H-21)  as well and C-19 . That anδC 34.5) . This waand C-14 . That thand C-13 . Taking 6 possesses four stereogenic centers viz. C-7, C-8, C-11 and C-13. Therefore, it is challenging to determine the absolute configurations of these carbons. Since we were able to obtain a suitable crystal of 6, its X-ray analysis was carried out using an X-ray diffractometer equipped with CuKα radiation. The Ortep view of 6 is shown in Compound R, 8R, 12S, and 13S. Since 6 has never been reported, we named it avellaneanone.The Ortep view showed clearly that the absolute configuration at C-7, C-8, C-12, and C-13 are 7p-Hydroxy phenyl-2-pyridones with alkyl or alkenyl side chains on C-3 have been previously isolated from different fungal strains. Examples of these are tenellin + .White crystal. mp 217–218 °C. +  and 333.0948 [M + Na]+ .Pale yellow viscous mass. + .White crystals. mp 174–176 °C. + .White crystal. mp 116–118 °C. + .White crystal. mp 219–210 °C.  × 100, where x = colony radius of the plant pathogenic fungi in the negative control, and y = colony radius of the plant pathogenic fungi in the presence of the crude extract. Each treatment was performed with five replications and repeated three times independently. The mean inhibition levels and standard deviations were calculated from five replications and three repetitions. Data were subjected to analysis of variance, and subsequently, means were compared using Duncan’s multiple range test (p ˂ 0.05) in the SPSS version 19 statistical program .The in vitro growth inhibitory activity of the crude EtOAc extract of p et al. . The cru2O was added to prepare a stock solution of 1000 µg/L. Two-fold serial dilutions of the stock solution by potato dextrose broth mixed with a spore suspension of each pathogen at 106 spores/mL provided the tested concentrations at 500, 250, and 125 µg/L. Each treatment was loaded at 200 µL per well into the 96-well U-shaped untreated polystyrene plates with five wells per treatment, and then the plate was incubated for 7 days at 25 °C. The MIC was determined as the lowest concentration of the compound that inhibited visible growth. Mancozeb , at the same concentration, was used as a positive control.The minimum inhibitory concentrations (MICs) for the antifungal activity of the isolated compounds on the plant pathogenic fungi were determined according to CLSI recommendation . Each paHamigera avellanea KUFA0732, isolated from the marine sponge Mycale sp., which was collected from the Gulf of Thailand, was found to have the potential to inhibit the growth of various plant pathogenic fungi. Chemical study of the crude extract of the culture of this marine-derived fungus led to the isolation of five undescribed specialized metabolites, viz. (R)-6,8-dihydroxy-4,5-dimethyl-3-methylidene-3,4-dihydro-1H-2-benzopyran-1-one (1), -3,8-dihydroxy-6-methoxy-4,5-dimethyl-1-oxo-3,4-dihydro-1H-isochromen-3-yl]methyl acetate (2), (R)-5, 7-dimethoxy-3-((S)-(1-hydroxyethyl)-3,4-dimethylisobenzofuran-1(3H)-one (4b), (S)-7-hydroxy-3-((S)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran 1(3H)-one (5), and avellaneanone (6), as well as three previously reported (R)-3-acetyl-7-hydroxy-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (3), (R)-7-hydroxy-3-((S)-1-hydroxyethyl)-5-methoxy-3,4-dimethylisobenzofuran-1(3H)-one (4a) and isosclerone (7). The common biosynthetic pathways of the pentaketide derivatives 1 and 2, as well as of 3, 4a, 4b, and 5, were determined. The biosynthesis of the PKS-NRPS hybrid product, avellaneanone (6), was also proposed based on the previously proposed pathways for other analogs. Finally, it is important to point out that although the crude EtOAc extract of H. avellanea KUFA0732 was able to completely inhibit the growth of many plant pathogenic fungi tested, only 3 and 5 displayed strong inhibitory activity (MIC = 125 µg/mL) comparable to the positive control, mancozeb, against Curvularia oryzae, while 6 exhibited only moderate activity (MIC = 250 µg/mL). These results give a new perspective on the potential application of marine-derived fungi and their specialized metabolites as biocontrol agents in agriculture. The crude EtOAc extract of a solid-rice culture of a marine sponge-associated fungus,


In addition, protocols for selective mono- and di-halogenations of positions 3 and 4 were also developed, which could be useful for further exploration of this scaffold.The construction of duocarmycin-like compounds is often associated with lengthy synthetic routes. Presented herein is the development of a short and convenient synthesis of a type of duocarmycin prodrug. The 1,2,3,6-tetrahydropyrrolo[3,2- Sinectively ,17,18,19ectively  have beeectively ,17 , the generation of complex mixtures when performing the halogenation reaction on the aniline, and problems with controlling the mono-Boc protection of the aniline.The existing synthetic pathways are elaborative and/or give the wrong substitution pattern . Further12  with tBu-carbamate performed well, and compound 13 could be isolated in 78% yield (14) and 3-iodo (15) products could be isolated in 74% and 71% yields, respectively, using the two different halogen sources. We envisioned that the deprotonation of the Boc-protected aniline with NaH prior to the halogenation might render the aromatic ring sufficiently electron-rich to direct the halogenation to the right position 8% yield . Performtion see . Gratifyaccharin ), other 12 to give 3-iodo-4-bromo compound 16 went smoothly under acidic conditions (NIS/TsOH) in 71% yield. To conclude the synthesis towards the duocarmycin-type prodrug, compound 12 smoothly underwent allylation with 1,3-dichloropropene to give 17  in 82% yield, followed by a tris(trimethylsilyl)silane (TTMSS)/azaisobutyronitrile (AIBN)-induced radical 5-exo-trig cyclization according to published procedures to furnish compound 10 . HRMS was performed using a microTOF instrument with electrospray ionization (ESI), and sodium formate was used as a calibration chemical. Optical rotations were measured on a polarimeter at 589 nm (D line of sodium) and 20 °C. Chiral chromatography was performed on supercritical fluid chromatography equipment, using mixtures of MeOH and supercritical CO2 as eluents.General Methods: All solvents and reagents were used as received from commercial suppliers. tert-butyl 1-(chloromethyl)-1,2-dihydropyrroloindole-3,6-dicarboxylate (10): tert-Butyl-4-bromo-5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate 17  was dissolved in dry toluene (40 mL), and the solution was degassed for 1 h (by bubbling N2 gas through the solution under stirring). Azobisisobutyronitrile (AIBN)  and tris(trimethylsilyl)silane (TTMSS)  were added, and the reaction was heated to 90 °C (with a preheated oil bath) in a sealed tube for 5 h. The solvent was evaporated, and the crude material was dissolved in MeOH (12 mL) and stirred at rt for 10 min. The solvent was evaporated, and the crude product was purified by column chromatography on silica gel (hexanes:EtOAc 95:5) to give compound 10 as a colorless oil . The spectral data agreed with the published data + Calcd for C18H24N2O4Na 355.1634; Found 355.1633.tert-Butyl 3-bromo-5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate (14): tert-Butyl 5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate 13  was dissolved in DMF (2 mL), NBS  and TsOH·H2O  were added, and the reaction was stirred at rt for 10 min. The reaction mixture was poured onto saturated NaHCO3 (aq) and extracted with EtOAc. The organic phase was dried (Na2SO4), filtered, and concentrated. The crude material was purified by column chromatography on silica gel (hexanes:EtOAc 95:5) to give compound 14 as a colorless foam . 1H-NMR  δ 8.02 , 7.65 , 7.60 , 7.24 , 6.67 , 1.65 , 1.54 ; 13C-NMR  δ 153.1, 148.9, 134.5, 130.9, 130.0, 125.5, 117.5, 115.6, 109.2, 97.9, 84.4, 80.6, 28.5 (3C), 28.3 (3C); HRMS (ESI/TOF) m/z: [M + Na]+ Calcd for C18H23BrN2O4Na 433.0739; Found 433.0755.tert-Butyl 5-((tert-butoxycarbonyl)amino)-3-iodo-1H-indole-1-carboxylate (15): tert-Butyl 5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate 13  was dissolved in DMF (14 mL), NIS  and TsOH·H2O  were added, and the reaction was stirred at rt for 15 h. The reaction mixture was poured onto saturated NaHCO3 (aq) and extracted with EtOAc. The organic phase was washed with 10 wt% Na2S2O5 (aq), dried (Na2SO4), filtered, and concentrated. The crude material was purified by column chromatography on silica gel (hexanes:EtOAc 95:5) to give compound 15 as a colorless foam . 1H-NMR  δ 8.00 , 7.69 , 7.53–7.46 , 7.27 , 6.73 , 1.65 , 1.54 ; 13C-NMR  δ 153.1, 148.7, 134.6, 132.7, 131.1, 130.8, 117.5, 115.5, 111.3, 84.3, 80.6, 65.4, 28.5 (3C), 28.2 (3C); HRMS (ESI/TOF) m/z: [M + Na]+ Calcd for C18H23IN2O4Na 481.0601; Found 481.0595.tert-Butyl 4-bromo-5-((tert-butoxycarbonyl)amino)-3-iodo-1H-indole-1-carboxylate (16): tert-Butyl 4-bromo-5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate 12  was dissolved in DMF (1.4 mL), NIS  and TsOH·H2O  were added, and the reaction was stirred at rt for 16 h. The reaction mixture was poured onto saturated NaHCO3 (aq) and extracted with EtOAc. The organic phase was washed with 10 wt% Na2S2O5 (aq), dried (Na2SO4), filtered, and concentrated. The crude material was purified by column chromatography on silica gel (hexanes:EtOAc 95:5) to give compound 16 as a colorless foam . 1H-NMR  δ 8.11 , 7.77 , 7.08 , 1.65 , 1.54 ; 13C-NMR  δ 153.0, 148.2, 134.0, 132.7, 131.6, 126.5, 118.5, 114.5, 105.3, 85.0, 81.1, 61.2, 28.5 (3C), 28.2 (3C); HRMS (ESI/TOF) m/z: [M + Na]+ Calcd for C18H22BrIN2O4Na 558.9706; Found 558.9700.tert-Butyl-4-bromo-5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate (17): tert-Butyl 4-bromo-5-((tert-butoxycarbonyl)amino)-1H-indole-1-carboxylate 12  was dissolved in dry DMF (12 mL) and cooled to 0 °C, NaH  was added, and the reaction was stirred at 0 °C for 5 min. 1,3-Dichloropropene was added, the ice bath was removed, and the reaction was stirred at rt for 1 h. The reaction mixture was poured onto saturated NaHCO3 (aq) and extracted with EtOAc. The organic phase was dried (Na2SO4), filtered, and concentrated. The crude material was purified by column chromatography on silica gel (hexanes:EtOAc 95:5) to give compound 17 as a colorless oil . The spectral data agreed with the published data [hed data .e]indol-3(2H)-yl)(5-fluoro-1H-indol-2-yl)methanone (18): Di-tert-butyl 1-(chloromethyl)-1,2-dihydropyrroloindole-3,6-dicarboxylate 10  was dissolved in 4 M HCl in 1,4-dioxane , and the reaction was stirred at rt for 22 h. The solvent was evaporated, and the crude material was coevaporated from EtOAc two times. The crude material, together with 5-fluoro-1H-indole-2-carboxylic acid 19 , N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) , and NaHCO3 , were mixed in dry DMF (10 mL), and the reaction was stirred at rt for 5 h. The reaction mixture was poured onto saturated NaHCO3 (aq) and extracted with EtOAc. The organic phase was dried (Na2SO4), filtered, and concentrated. The crude material was purified by column chromatography on silica gel (hexanes:EtOAc 60:40 to 50:50) to give compound 18  as an off-white solid. The spectral data agreed with the published results D  +17 and (−)—18, [α]D  -17, both with ee ≥ 99% -1,6-dihydropyrroloindole core 10, utilizing an unconventional NaH promoted site-selective bromination of Boc-protected amino indole 13 as the key step. Additionally, 3-iodo-4-bromo indole 16 constitutes an interesting starting point for further diversification. Closely related 3-iodo-4-bromo-indoles have been used in Pd-catalyzed cross-couplings such as the Mizoroki-Heck [18 was separated with chiral supercritical fluid chromatography for further investigation of this interesting prodrug.In conclusion, we developed a four-step route to the desired chloromethyl-substituted 1,2,3,6-tetrahydropyrrolo[3,2-oki-Heck ,25,26, Noki-Heck , and Suzoki-Heck ,29 react


Isodon ternifolius (D.Don) Kudô is an important Asian herb used in traditional medicine against several diseases. Nineteen compounds were isolated from the dichloromethane–methanol (1 : 1) extract of I. ternifolius roots, including ten new α-pyrone derivatives, named ternifolipyrons A–J. The chemical structures of the isolates were determined by a combination of 1D and 2D NMR, along with LR- and HRMS spectroscopy. The absolute configurations of the α-pyrone derivatives were constructed based upon the X-ray signal crystal of the bromobenzoyl derivative of 1 as well as the electronic circular dichroism (ECD). All isolates (1–19) were investigated for their growth-inhibitory potential towards CCRF-CEM-leukemia cells at a fixed concentration of 30 μM. The compounds which exerted more than 50% inhibition at this concentration, compounds , were tested at a different concentration range to determine their IC50 values in CCRF-CEM leukemia, MDA-MB-231 triple-negative breast cancer, and MCF7 breast cancer cell lines. Ursolic acid (16) showed the most potent activity against the three cancer cell lines with IC50 values of 8.37, 18.04, and 18.93 μM, respectively. Isodon ternifolius (D.Don) Kudô is an important Asian herb used in traditional medicine against several diseases. Because of the plant's historic significance and documented chemical variety, several biological actions of plant extracts and/or metabolites involving anti-cancer activity have been reported6–8,10,11 and the inhibition of DNA topoisomerase IB (TOP1) and tyrosyl-DNA phosphodiesterase 1 (TDP1).8,9 Longikaurin A from I. ternifolius exerted anticancer activity against several cancer cell lines, specifically hepatocellular carcinoma cells.10I. ternifolius along with other known compounds, (ii) the absolute configuration of the isolated compounds by NMR, X-ray signal crystal, and electronic circular dichroism (ECD), and (iii) the growth inhibition of these compounds towards CCRF-CEM leukemia, MDA-MB-23 triple-negative breast cancer, and MCF7 breast cancer cell lines.The present investigation described (i) ten new α-pyrone derivatives isolated and identified from the roots of 2.2.1.I. ternifolius roots via different chromatographic and spectroscopic tools -6--5,6-dihydro-2H-pyran-2-one (1),12 synargentolide A (12),13 hyptolide (13),14,15 6R--5,6-dihydro-2H-pyran-2-one (14),16 oleanolic acid (15),17 ursolic acid (16),18,19 sodoponin (18),20 rabdosianin B (17),21,22 and acacetin (19).23Ten new α-pyrones (2–11) along with further nine well-known compounds were isolated and identified from the dichloromethane–methanol (1 : 1) extract of the ic tools . The kno12 the structure of 1 was confirmed as α-pyrone derivative, 6--5,6-dihydro-2H-pyran-2-one. The absolute configuration of 1 was constructed and confirmed by the X-ray single crystal diffraction of its bromobenzoate derivative (S1) (ε) 265.0 nm (+45.1). This detailed analysis allowed the unequivocal determination of 1 as -6--5,6-dihydro-2H-pyran-2-one that was isolated previously from Hyptis oblongifolia leaves.12Compound 1 was identified using mass spectroscopy with low and high resolution, as well as NMR analysis. Comparing with the reported data,ive (S1)  and the m/z at 315.1453 that revealed a molecular formula of C15H23O7  and five unsaturation indexes. The presented 1H NMR data in δH 4.44 dt , 3.57 dd , 3.72 dd , 4.05 t (J = 5.0 Hz), and 4.79 m, four olefinic methene protons at δH 5.88 dt , 6.97 dd , 5.66 dd , and 5.73 dd , one methylene at δH 2.45 m and one methyl protons at δH 1.11 d (J = 6.5 Hz). Also, two protons characteristic for two methyl groups were assigned in oxygenated systems, including methoxy and acetoxy groups, at respective δH 3.22 s and 1.93 s. Totally, 15 carbon resonances were characterized based on the 13C NMR data (δC 164.9 (carbonyl of δ-lactone moiety) and δC 171.0 (acetoxyl carbonyl group), four olefinic methenes at δC , five oxygenated methenes at δC , one aliphatic methylene at δC 24.7, one, and one methyl proton at δC 14.1, one methyl for acetoxy at δC 19.7, and one methyl of methoxy group at δC 55.6. All these data revealed that 2 has the same structure as 1  and the hydroxylated proton (H5′) at δH 4.05 t (J = 5.0 Hz), H-5′/H-6′ at δH 4.79 m, and H-6′ and methyl proton (H-7′) at δH 1.11 d (J = 6.5 Hz). The 3J HMBC correlations (δC 73.0), H-6′/Ac-CO (δC 171.0), H-7′/C-5′ (δC 73.2), H-5 (δH 2.45 m)/C-1′ (74.3), and H-3′)/C-1′ confirmed the localization of the acetoxyl group in C-6′ and the two hydroxyl groups in C-1′ and C-5′. Based upon these 1D and 2D NMR analyses, the structure of 2 was deduced as 6--5,6-dihydro-2H-pyran-2-one. The trans (E) geometry of the C-3′/C-4′ olefinic system was confirmed by the discernible coupling constants of both sets of olefinic protons at 15.7 Hz.12,13 Comparing with 1 and the literature,12 the absolute orientation of 2 was affirmed via the coupling constants of the chiral carbons  and the positive ECD cotton effect at (Δε) 259.2 nm (+109.9). Thus, 2 was elucidated as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron A).The positive mode HRCIMS of 2 exhibited a molecular ion peak NMR data  and claselations  of the oelations  between m/z at 315.1453, indicating a molecular formula of C15H23O7  with five unsaturation indexes. The assigned 1D NMR, including 1H /δC 77.7, the upfield shift of H-6′/C-5′ by 1.01/4.9 ppm at δH 3.78 dd /δC 68.1 and C-7′ by 3.0 ppm at δC 17.1 indicating the presence of the acetoxyl group in C-6′ and the hydroxyl group in C-5′. These localizations were confirmed via1H 1H COSY correlations )/H-5′ ), H-5′/H-6′ ), and H-6′/H-7′ (δH 1.06 d (J = 6.5 Hz)) along with the 3J HMBC (δC 170.7) and H-7′/C-5′ (δC 77.7). From above, 3 was constructed as 6--5,6-dihydro-2H-pyran-2-one. As above compounds, the large coupling constants of the C-3′/C-4′ olefinic system sets at 15.8 Hz allowed its unequivocal geometry as trans (E).12,13 The determination of the absolute configuration of 3 was carried out depending upon the comparing of the chiral carbons′ coupling constants with the literature16 along with the positive ECD cotton effect at (Δε) 261.8 nm (+83.4).16 So, 3 was predicted as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron B).The positive mode HRCIMS of 3 showed a molecular ion peak uding 1H  and 13C  and four unsaturation indexes. The overall data presented in 1D NMR /70.4, 4.03 t (J = 5.5 Hz)/73.4 and 4.78 m/73.1, (ii) the absence of the methoxyl group that deduced via the downfield shift of H-2′ by 0.46 at δH 4.18 dd  and upfield of C-2′ by 9.8 ppm at δC 70.4, (iii) the upfield shift of the methyl proton, H-7′, by 3.1 ppm at δH 13.9, indicating that the only acetoxyl group was located in another carbon except C-6′. The localization of the acetoxyl group was affirmed in C-1′ via the 1H 1H COSY correlations of H-6 (δH 4.46 m)/H-1′ , H-1′/H-2′  and H-2′/H-3′  along with the 3J HMBC correlations of H-1′/Ac-CO (δC 171.1), H-1′/C–H-5 (δC 24.9) and H-1′/C-3′ (δC 132.6)  geometry.13,16 Similar to above compounds, the absolute configuration of 4 was established based upon the positive ECD cotton effect at (Δε) 258.0 nm (+46.2).12 Thus, 4 was assigned as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron C).The HRCIMS of 4 in a positive mode exhibited a molecular ion peak n 1D NMR  and 2 deC 132.6) . Hence, m/z at 301.1269, indicating a molecular formula of C14H21O7  and four unsaturation indexes. By assignment of the 1H , upfield shift of C-2′ by 9.7 ppm at δC 70.4, and the absence of the proton and carbon resonances of the methyl of methoxyl group. The 1H 1H COSY correlations )/H-5′ ), H-5′/H-6′ ), H-6′/H-7′ (δH 1.06 d (J = 6.5 Hz)) along with the 3J HMBC (δC 170.7) and H-7′/C-5′ (δC 77.7) confirmed the hydroxylation of C-1′, C-2′, and C-6′ along with acetoxylation of C-5′. From these data, 5 was established as 6--5,6-dihydro-2H-pyran-2-one. As well, the trans (E) configuration of the C-3′/C-4′ olefinic system was constructed via the large coupling constants of both sets at 15.9 Hz.12,13 The construction of the absolute stereochemistry of 5 was performed by the positive ECD cotton effect at (Δε) 257.4 nm (+31.8).12 So, 5 was predicted as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron D).Based upon the positive mode HRCIMS of 5, which exhibited a molecular ion peak f the 1H  and 13C  1H  alongside of five unsaturation indexes. The 1H /δC 35.7, δH 1.55 m/δC 18.1, and δH 0.86 t (J = 7.4 Hz)/δC 12.5 in addition to its carbonyl at δC 173.1. The sequence of the butanoyl group was established by the 1H 1H COSY of the butanoyl protons, CH3 (δH 0.86 t (J = 7.4 Hz)/CH2 (δH 1.55 m), and CH2 (δH 1.55 m)/CH2 (δH 2.26 t (J = 7.4 Hz)), along with the 3J HMBC (2 (δH 1.55 m)/CO (δC 173.1), and CH3 (δH 0.86 (t (J = 7.4 Hz))/CH2 (δC 35.7). While the placement of butanoyl group in C-5′ was proven through 3J HMBC )/CO (δC 173.1). Based on these data, the structure of 6 was established as 6--5,6-dihydro-2H-pyran-2-one. The large coupling constants of the olefinic bond, C-3′/C-4′, at 15.8 Hz confirmed its trans (E) configuration.12,13 Furthermore, the absolute configuration verification of 6 was achieved by the positive ECD cotton effect at (Δε) 258.6 nm (+41.8).13 So, 6 was predicted as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron E).The positive mode HRCIMS molecular ion peak at . The 1H  and 13C  1H  from the HRCIMS molecular ion peak m/z at 371.1705, showing four unsaturation indexes. The structure of 7 was closely constructed as that of 5via the 1H /δC 70.4, along with the 3J HMBC correlation (δC 173.2). Subsequently, the structure of 7 was assigned as 6--5,6-dihydro-2H-pyran-2-one. As described for all above compounds, the orientation of the olefinic system, C-3′/C-4′, was concluded as trans (E) from the large coupling constants at 15.8 Hz.12,13 Also, the absolute stereochemistry of 7 was derived by means of ECD that exhibited a positive cotton effect at (Δε) 259.2 nm (+94.7).12 So, 7 was predicted as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron F).The molecular formula of 7 was predicted as Ca the 1H  and 13C  1H  and five unsaturation indexes. According to the 1H )/CO (δC 173.1) (H-pyran-2-one. The C-3′/C-4′ olefinic system's orientation was determined to be trans (E) given a large coupling constant at 15.8 Hz as indicated in all of the compounds above.12,13 The absolute stereochemistry of compound 8 was verified using the ECD, which showed a positive cotton effect at (Δε) 257.8 nm (+104.8), in comparison to data from compound 1 and published data.12 Thereby, the predicted structure of 8 was -6-[5′-acetyloxy-6′-butyroyl-1′-hydroxy-2′-methoxy-3-heptenyl]-5,6-dihydro-2H-pyran-2-one (ternifolipyron G).The HRTOFESI-MS of 8 exhibited a molecular ion peak at o the 1H  and 13C  1H (13C  NMR dataelations . MoreoveC 173.1) . After cm/z 267.1236 in the HRCIMS of 9, the chemical formula was determined to be C14H19O5 , which revealed five unsaturation indices. Based upon the analysis of the 1H and 13C /δC 127.1, δH 6.16 t (J = 11.0 Hz)/δC 131.9, δH 6.56 ddt /δC 125.9, and δH 5.76 dd /δC 135.5, and (ii) the presence of the only two functional groups in the heptanyl chain, including one hydroxyl at δH 4.10 ddd /δC 73.3 and one acetoxyl at δH 4.78 m/δC 73.0. In addition to the C-3′/C-4′ olefinic system in all above compounds, the other olefinic system was located in C-1′/C-2′ based upon the 1H 1H COSY correlations (δH 5.40 m)/H-1′ ), H-1′/H-2′ (δH 6.16 t (J = 11.0 Hz)), H-2′/H-3′ ), H-3′, H-4′ ). The site of C-1′/C-2′ olefinic system was assured by 3J HMBC correlations (δH 2.38 m)/C-1′ (δC 127.1), H-6/C-2′ (δC 131.9), H-1′/C-3′ (δC 125.9), and H-2′/C-4′ (δC 135.5). Also, the presence of the hydroxyl and acetoxyl groups in C-5′ and C-6′, respectively, was confirmed by the same described 1H 1H COSY and HMBC correlations in compound 2. After taking all aforementioned information into account, the structure of 9 was determined to be 6--5,6-dihydro-2H-pyran-2-one. According to what was previously stated, the significant coupling constant at 15.7 Hz validated the geometry of the C-3′/C-4′ olefinic system as trans (E).12,13 In contrast, the C-1′/C-2′ olefinic system's geometry was predicted to be cis (Z) according to the modest coupling constant at 11.0 Hz in both sites.13,14,24 The absolute configuration of 9 was ascertained by the ECD study, which showed a positive cotton effect at (Δε) 264.0 nm (+35.3) by comparison with compound 1, other compounds, and the literature.12 With the aforementioned information, 9 was ultimately determined as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron H).Based on the molecular ion peak at  and 13C  NMR dataelations  of H-6  and six unsaturation indices. The study of the 1H and 13C )/Ac-CO (δC 170.3) corroborated the placement of the acetoxyl group in C-5′. The butanoyl group was created, as mentioned for the aforementioned compounds by the 1H 1H COSY and HMBC correlations (3J HMBC correlations (δH 5.00 m)/But-CO (δC 173.2) showed that the butanoyl group existed in C-6′. The olefinic systems, C-1′/C-2′ and C-3′/C-4′, in 10 were confirmed by the same in 9. Based on the data discussed above, 10 was chemically created as 6--5,6-dihydro-2H-pyran-2-one. The cis (Z) and trans (E) geometries of the olefinic systems were confirmed by the coupling constants of C-1′/C-2′ and C-3′/C-4′, respectively, at 10.9 and 15.2 Hz.12–14,24 In a similar way, the absolute configuration of 10 was established using the ECD experimental data, which revealed a positive cotton effect at (Δε) 264.0 nm (+76.9).14 Based on the information above, 10 was eventually -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron I).Compound 10's TOFESIMS results showed a molecular ion peak at  and 13C  NMR datarelation  of H-5′ elations  of its pelations  of the Hm/z 397.1871, compound 11's molecular formula was inferred to be C20H29O8  coupled with six unsaturation indices. The 11's 1H and 13C /δC 65.9 along with one methylene carbon at δH 1.73 m, 1.89 m/δC 35.0. The olefinic system was demonstrated to be C-1′/C-2′ by the 1H 1H COSY correlations (δH 5.32 m)/H-1′ ), H-1′/H-2′ ) along with the 3J HMBC correlation (δC 29.4). Furthermore, the new acetoxylated carbon and methylene carbon were localized in C-3′ and C-4′, respectively, depending on the 1H 1H COSY correlations ), H-3′/H-4′ , and H-4′/H-5′ (δH 5.00 m) as well as the 3J HMBC correlation (δC 65.9), H-4′/C-2′ (δC 130.9), and H-3′/Ac-CO (δC 170.6). The other sections of this compound, including 5′-acetoxyl and 6′-butyroyl, were determined as described for 10. Thus, 11 was established as 6--5,6-dihydro-2H-pyran-2-one. The C-1′/C-2′ olefinic system's cis (Z) geometry was decided by the system's modest coupling constant at 10.6 Hz.13,14,24 Also, 11′ absolute configuration was defined depending upon the positive cotton effect at (Δε) 257.8 nm (+91.2) in the ECD experimental data.14 Therefore, 11 was determined as -6--5,6-dihydro-2H-pyran-2-one (ternifolipyron J).From the HRCIMS molecular ion peak at  and 13C  NMR dataelations  of the Hrelation  of H-1′/elations  of the Hrelation  of H-1′/2.2.−1 for three cancer cell lines . Ursolic acid (16) exhibited the strongest cytotoxic activity against three cancer cell lines as follows: the IC50 for the three cell lines were 8.37 μM, 18.04 μM, and 18.93 μM, respectively  . The comectively .3.3.1.1H, 13C, and DEPT-135, and the 2D NMR, including HSQC, HMBC, 1H 1H COSY, and NOESY. All NMR analyses were measured in deuterated methanol (CD3OD) at room temperature. The chemical shifts and coupling constants were given in delta  and Hertz (Hz), respectively. CD3OD was referenced at δH 4.77 and δH 3.31 ppm in 1H NMR, and at δC 49.14 in 13C NMR. The experimentally measured electronic circular dichroism (ECD) of the compounds were measured in CH3OH by a JASCO-810 spectrometer. The mass spectral data including, LR and HR-MS, were derived by a JEOL (JMS-700) instrument .Silica gel 60 (230–400 and 100–200 mesh), pre-coated Kieselgel-60 F-254-TLC plates silica gel , and Sephadex LH-20 (Pharmacia Co. Ltd) were used as packing materials for column chromatography. Isolera-one flash chromatography  was used for flash chromatographic analysis as well as the isolation and purification processes. The compounds were purified using HPLC with a Jasco-pump (PU-980) equipped with a Jasco UV-970 intelligent detector (UV/VIS) at 210 nm. A HPLC semi-preparative Supelco C18-RP-column  was also used. The optical rotation of the isolated compounds was measured by a JASCO (P-2300) polarimeter . A Bruker 500 Hz  spectrometer was used for recording the 1D NMR, including 3.2.I. ternifolius were collected in West Imphal, Manipur, India, 24.6637° N, 93.9063° E, during March/April 2020. The identification and authentication of the collected plant were kindly performed by Dr Biseshwori Thongam, Taxonomist, Bioresource and Sustainable Development Institute, IBSD Imphal, Manipur, India. A plant voucher (No.: IBSD/Z-ITF-1578) was stored at the Plant Bioresource Division Herbarium, IBSD Imphal, India.The roots of 3.3.I. ternifolius roots were carefully cleaned, left for one week in a dry and shady place at room temperature until complete dryness, and then crushed using a clean plant grindery into fine powder. The extraction of the powdered roots (1.3 kg) occurred by maceration in dichloromethane-methanol  at room temperature for successive 72 h and filtered. The filtrate was extracted with the same steps two more times. The total extract (84.5 g) was obtained as black gum by complete drying of the overall amount of liquid extract under vacuum at 45–50 °C.3.4.n-hexane/CH2CL2  followed by CH2CL2/MeOH  as elution systems afforded 46 fractions. These fractions were collected to 8 main fractions (designated YGS-1 to YGS-8) after examination with thin layer chromatography (TLC) with different solvent systems. Fraction YGS-4 (1.4 g) was fractionated over Isolera-one flash CC using a step gradient of CHCl3/MeOH that yielded compound 1 (178.7 mg) along with three sub-fractions . The further fractionation of YGS-4A (152.1 mg) with a mixture of CHCl3/MeOH (1 : 1) as an elution system over Sephadex LH-20 CC yielded 2 (7.9 mg), 3 (8.5 mg), 4 (10.2 mg), and 18 (17.6 mg). Fraction YGS-4B (131.2 mg) was carefully filtered, and then the clearly soluble portion was subjected to C18-RP-HPLC with an eluting system of MeOH–H2O (3 : 2) to yield 5 (13.4 mg), 6 (11.6 mg), and 7 (9.8 mg). With the same sequence, 9 (7.7 mg), 12 (56.8 mg), and 19 (143.2 mg) were purified from the fraction YGS-4C (94.3 mg). The filtrated methanol-soluble portion of fraction YGS-2 (1.0 g) was subjected to C18-RP-HPLC using MeOH/H2O (4 : 1) as eluting system and yielded 8 (14.3 mg), 15 (46.1 mg), and 16 (53.5 mg). The further fractionation of fraction YGS-3 (1.3 g) over silica gel CC using CHCl3/MeOH  yielded 17 (161.4 mg), 14 (128.2 mg), and sub-fraction YGS-3A. The eluting of the sub-fraction YGS-3A (68.7 mg) by MeOH–H2O (3 : 2) as the mobile phase using the C18-RP-HPLC led to the purification of 10 (12.4 mg), 11 (16.1 mg), and 13 (33.7 mg).The extract was subjected to rapid fractionation over silica gel (230–400 mesh) column chromatography (CC) starting with 3.5.p-bromobenzoyl chloride  was added to a solution of compound 1  in dried pyridine (0.1 mL). After stirring for 19 h, at room temperature, the resulting solution was diluted with ethyl acetate and washed with aqueous KHSO4 (1 M) and aqueous saturated NaHCO3  in quantitative yield as a colourless oil.The d NaHCO3 . The res3.6.F2 with 311 variables converged at R1 = 2.51%, for the observed data and wR2 = 5.92% for all data. The ORTEP plot was obtained by the program PLATON . Crystallographic data (excluding structure factors) for the structures of S1 have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication numbers CCDC 2246696.†Single crystals of bromobenzoate derivative of 1 (S1) were obtained by slow evaporation of a hexane and ethyl acetate solution, selected and fitted onto a glass fiber, and measured at −173 °C with a Bruker Apex II ultra diffractometer using MoKα radiation. Data correction and reduction were performed with the crystallographic package Apex II. The structure was solved and refined using the Bruker SHELXTL software package. All non-hydrogen atoms were refined anisotropically, and hydrogen atoms were positioned geometrically. The final anisotropic full-matrix least-squares refinement on 3.7.α]25D +16.1; c 0.1, MeOH); ECD  (Δε) + 45.1 (265.0 nm); the spectroscopic spectra including LREIMS & TOFESIMS, 1H (500 Hz), 13C NMR  : 1H NMR (300 MHz in CDCl3) δH 7.91 , 7.61 , 6.88 , 6.06 , 5.78 , 5.61 , 5.38 , 5.23 , 4.99 , 4.90 , 4.25 , 3.37 , 2.47–2.40 , 2.01 , 1.87 , 1.07 . Crystal data: C24H27BrO9, MW = 539.36, monoclinic, space group P21, Z = 2, a = 13.5903 (13) Å, b = 6.6575 (6) Å, c = 13.8833 (13) Å, β = 100.1930 (10)°, V = 1236.3 (2) Å3, Flack parameter = 0.020 (4), GOF = 0.937. The spectroscopic spectra including 1H NMR ; ECD  (Δε) + 109.9 (259.2 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 315 [M + 1]+ (22%), 297 (37%), 255 (21%), 233 (54%), 205 (42%), 181 (44%), 163 (34%), 127 (63%); 111 (83%); 99 (43%); 95 (100%); 43 (26%); 41 (59%); HRCIMS m/z 315.1453 . Unsaturation indexes: 5. The spectral data involving the LR & HRCIMS, 1D, 2D NMR ; ECD  (Δε) + 83.4 (261.8 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 315 [M + 1]+ (7%), 297 (22%), 255 (20%), 238 (21%), 223 (46%), 205 (26%), 181 (84%), 163 (41%), 155 (46%), 127 (64%); 111 (65%); 99 (43%); 95 (100%); 69 (26%); 45 (22%); 41 (43%); HRCIMS m/z 315.1453 . Unsaturation indexes: 5. The spectral data involving the LR & HRCIMS, 1D, 2D NMR ; ECD  (Δε) + 46.2 (258.0 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 301 [M + 1]+ (4%), 283 (31%), 223 (55%), 205 (47%), 181 (64%), 163 (44%), 141 (36%), 128 (68%), 113 (47%); 97 (92%); 95 (100%); 86 (46%); 81 (38%); 69 (48%); 43 (45%); 41 (48%); HRCIMS m/z 301.1265 . Unsaturation indexes: 4. The spectral data involving the LR & HRCIMS, 1D, 2D NMR ; ECD  (Δε) + 31.8 (257.4 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 301 [M + 1]+ (17%), 283 (56%), 223 (100%), 205 (45%), 198 (58%), 181 (67%), 127 (100%), 111 (71%), 97 (90%); 95 (97%); 86 (54%); 81 (51%); 69 (45%); 43 (53%); 41 (98%); HRCIMS m/z 301.1269 . Unsaturation indexes: 4. The spectral data involving the LR & HRCIMS, 1D, 2D NMR ; ECD  (Δε) + 41.8 (258.6 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 343 [M + 1]+ (18%), 325 (45%), 297 (27%), 255 (42%), 223 (62%), 183 (63%), 181 (100%), 169 (68%), 163 (49%), 127 (89%), 111 (85%), 99 (41%); 95 (86%); 81 (30%); 71 (64%); 43 (57%); 41 (28%); HRCIMS m/z 343.1755 . Unsaturation indexes: 5. The spectral data involving the LR & HRCIMS, 1D, 2D NMR ; ECD  (Δε) + 94.7 (259.2 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 371 [M + 1]+ (6%), 353 (17%), 311 (100%), 223 (51%), 205 (38%), 183 (43%), 155 (45%), 141 (38%), 128 (64%), 113 (46%), 97 (65%), 95 (100%); 81 (41%); 71 (95%); 43 (39%); 41 (67%); HRCIMS m/z 371.1705 . Unsaturation indexes: 3. The spectral data involving the LR & HRCIMS, ECD, 1D, 2D NMR ; ECD  (Δε) + 104.8 (257.8 nm); 1H NMR  and 13C NMR : presented in m/z 407.1678 . Unsaturation indexes: 5. The spectral data involving the TOFESIMS, ECD, 1D, 2D NMR ; ECD  (Δε) + 35.3 (264.0 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 267 [M]+ (5%), 249 (65%), 189 (100%), 180 (47%), 171 (36%), 162 (74%), 147 (42%), 133 (58%), 121 (20%), 105 (24%), 95 (25%), 81 (34%); 43 (66%); 41 (62%); HRCIMS m/z 267.1236 . Unsaturation indexes: 5. The spectral data involving the LR & HRCIMS, 1D, 2D NMR ; ECD  (Δε) + 76.9 (264.0 nm); 1H NMR  and 13C NMR : presented in m/z 359.1465 . Unsaturation indexes: 5. The spectral data involving the TOFESIMS, 1D, 2D NMR ; ECD  (Δε) + 91.1 (257.8 nm); 1H NMR  and 13C NMR : presented in m/z (rel. int.): 397 [M + 1]+ (10%), 337 (100%), 329 (24%), 277 (64%), 249 (19%), 239 (43%), 207 (57%), 189 (66%), 179 (18%), 171 (22%), 162 (24%), 151 (22%), 71 (26%), 45 (23%), 43 (19%); 41 (69%); HRCIMS m/z 397.1871 . Unsaturation indexes: 6. The spectral data involving the LR & HRCIMS, 1D, 2D NMR –streptomycin (100 μg mL−1), CCRF-CEM leukaemia cells were grown. The MDA-MB-231-pcDNA3 and MCF-7 human breast cancer cell lines were grown in DMEM media with 10% FBS and 1% penicillin/streptomycin supplementation. All cell lines were maintained at 37 °C in a humid environment with 5% CO2.In RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin (100 U mL3.9.25 Suspension cells (1 × 104 cells per well) and/or adherent cells  were seeded in 96-wells plate in a volume of 100 μL. Three cancer cell lines were screened using a single concentration (30 μm), and different concentrations of test substances were added to create a total volume of 200 μL for the creation of dose–response curves. After 72 h, 20 μL 0.01% w/v resazurin (Sigma-Aldrich) was added to each well. Cells were incubated for 4 h at 37 °C. Fluorescence at the excitation wavelength of 544 nm and emission at 590 nm was measured using Infinite M2000 Pro™ plate reader . Three independently performed assays for each set of six replicates were performed. Fifty percent inhibitory concentration (IC50) values were calculated using the concentration–response curve fit to the non-linear regression model using GraphPad Prism® v8.0 software . All IC50 values are expressed as mean ± standard deviation (SD). Previously, this protocol had been described.26,27Living cells convert the inactive dye resazurin into the fluorescent dye resorufin through a metabolic process.4.2Cl2–MeOH (1 : 1) extract of I. ternifolius roots. In addition to the ECD, X-ray signal crystal diffraction was used to determine the isolates' absolute stereochemistry. Ternifolipyron A, among of all isolated compounds, exhibited the most significant inhibitory effect on the growth of the MDA-MB-231 triple-negative breast cancer cell line, the MCF7 breast cancer cell line, and the CCRF-CEM leukaemia cell line.Ternifolipyrons A–J, ten new α-pyrone derivatives, and nine known metabolites have been isolated from the CHConceptualization, A. I. E., M.-E. F. H. and A. U.; formal analysis, A. I. E., T. A. M., H. I., M.-E. F. H. and A. U.; investigation, A. I. E., T. A. M., N. S., H. I., M.-E. F. H. and A. U.; writing—original draft preparation, A. I. E., T. A. M., H. I., M.-E. F. H. and Y. K.; writing—review and editing, A. I. E., T. A. M., N. S., Y. K., T. E., H. I., M.-E. F. H. and A. U.; funding acquisition, A. I. E. All authors have read and agreed to the published version of the manuscript.The authors declare no conflict of interest.RA-013-D3RA03146B-s001RA-013-D3RA03146B-s002


A1E3, associated with the fruit of Rhizophora mucronata, together with averufanin (3). The planar structures and absolute configurations of rhizoaspergillinol A (2) and averufanin (3) were established by extensive NMR investigations and quantum-chemical electronic circular dichroism (ECD) calculations. Most notably, the constitution and absolute configuration of rhizoaspergillin A (1) were unambiguously determined by single-crystal X-ray diffraction analysis of its tri-pivaloyl derivative 4, conducted with Cu Kα radiation, whereas those of averufanin (3) were first clarified by quantum-chemical ECD calculations. Rhizoaspergillin A is the first orsellinic acid–ribose–pyridazinone-N-oxide hybrid containing a unique β-oxo-2,3-dihydropyridazine 1-oxide moiety, whereas rhizoaspergillinol A (2) and averufanin (3) are sterigmatocystin and anthraquinone derivatives, respectively. From the perspective of biosynthesis, rhizoaspergillin A (1) could be originated from the combined assembly of three building blocks, viz., orsellinic acid, β-D-ribofuranose, and L-glutamine. It is an unprecedented alkaloid-N-oxide involving biosynthetic pathways of polyketides, pentose, and amino acids. In addition, rhizoaspergillinol A (2) exhibited potent antiproliferative activity against four cancer cell lines. It could dose-dependently induce G2/M phase arrest in HepG2 cells.Two new compounds, named rhizoaspergillin A ( N-oxides are photoactivatable O(3P) precursors for applications in organic synthesis and chemical biology  , 6.28 , 2.47 , 10.85 , 10.17 ; δC 106.3 , 162.6 , 100.9 , 162.1 , 111.2 , 142.4 , 169.1 , 23.1 ]  to the nonprotonated C-15 (δC 142.4) placed it at C-15. Most notably, the key HMBC cross-peak from H-7 to the carbonyl C-16 connected the β-D-ribofuranose unit and the orsellinic acid moiety through the C-7–O–C-16 bond ] , was corH-8/H2-9 a. The ex, C-17)] , was con-16 bond a.δH 11.40 , 5.78 , 7.96 ; δC 163.3 , 102.6 , 140.7 , 151.1 ] ] , was cor H-3/C-4 a. The Keas shown a. 1 was determined by NOE interactions ] . To the best of our knowledge, rhizoaspergillin A is the first reported orsellinic acid–ribose–pyridazinone-N-oxide hybrid.The relative configuration of ractions b. Those 2) was isolated as a light white amorphous powder. The molecular formula C32H26O9 was determined by the positive HR-ESIMS ion at m/z 555.1644 , indicating twenty degrees of unsaturation. According to 1H- and 13C-NMR spectroscopic data of 2 , a methylene group, thirteen methine groups (twelve olefinic and one oxygenated), and fifteen nonprotonated carbons . Rhizoaspergillinol A , 6.51 , 6.400 , 6.399 , 6.29 , 6.56 , 2.30 , 2.27 , 4.71 ; δC 157.6 , 112.0 , 140.9 , 114.0 , 157.8 , 158.2 , 111.4 , 141.0 , 111.1 , 156.5 , 103.3 , 105.1 , 21.5 (Me-30), 21.7 (Me-31)]. The existence of the diorcinol moiety was corroborated by HMBC correlations between H-19/C-18, H-19/C-21, H-19/C-29, H3-30/C-19, H3-30/C-20, H3-30/C-21, H-21/C-22, H-24/C-23, H-24/C-28, H3-31/C-24, H3-31/C-25, H3-31/C-26, 27-OH/C-26, 27-OH/C-27, and 27-OH/C-28. HMBC cross-peaks from protons of Me-30  to the nonprotonated C-20 (δC 140.9) and those from protons of Me-31  to the nonprotonated C-25 (δC 141.0) placed Me-30 at C-20 and Me-31 at C-25, respectively. In addition, the key HMBC correlation from H-17  to the nonprotonated C-18  connected the dihydrosterigmatocystin unit and the diorcinol moiety through the C-17–O–C-18 bond. Therefore, the constitution of 2 was elucidated as shown  calculations -2 showed good agreement with that of the experimental curve of 2 -2 exhibited mirrored Cotton effects. Therefore, the absolute configuration of 2, named rhizoaspergillinol A, was concluded to be .The relative configuration of ractions . Those bulations . The calrve of 2 , whereas3 was obtained as amorphous powder. The molecular formula C20H18O7 was established by the positive HR-ESIMS ion at m/z 371.1136 , indicating twelve degrees of unsaturation. According to the NMR spectroscopic data for 3 , 7.03 , 7.00 ; δC 188.5 , 108.4 , 165.5 , 108.0 , 164.3 , 109.0 , 134.7 , 181.0 , 133.1 , 109.0 , 161.5 , 119.8 , 162.9 , 108.2 ], was confirmed by HMBC correlations between H-4/C-2, H-4/C-5, H-10/C-1, H-10/C-8, H-10/C-12, and H-10/C-14. The presence of a 20-methyltetrahydropyran moiety was confirmed by 1H–1H COSY cross-peaks between H-15/H2-16, H2-16/H2-17, H2-17/H2-18, H2-18/H-19, and H-19/H3-20 and HMBC correlations between H-15/C-11, H-15/C-12, H-15/C-13, H-15/C-16, H-15/C-19, H3-20/C-18, and H3-20/C-19. HMBC correlations from H-15 to C-11, C-12, and C-13 connected the 3,5,11,13-tetrahydroxyanthraquinone core and the 20-methyltetrahydropyran moiety through the C-12–C-15 bond  and , were confused in the literature . Data/restraints/parameters: 5739/35/426. Goodness-of-fit on F2: 1.049. Final R indexes [I ≥ 2σ (I)]: R1 = 0.0415, wR2 = 0.1143. Final R indexes [all data]: R1 = 0.0439, wR2 = 0.1172. Largest diff. peak/hole: 0.73/−0.61 e Å−3. Flack parameter: −0.12(9) (CCDC 2291154).C1 (20.0 mg) was dissolved in pyridine (0.5 mL). DMAP (3.0 mg) was then added, along with 50.0 μL of pivaloyl chloride under ice bath and stirred for 10 min. After the completion of the reaction , the reaction solution was evaporated under reduced pressure. The resulting residue was dissolved in methanol, purified by semi-preparative HPLC, and eluted with the mixture of MeCN/H2O  to afford 4 (5.0 mg). Suitable crystals of 4 were obtained in MeOH after considerable effort.Compound 1–3 and MS-275 (positive control) against four cancer cell lines, including a human lung cancer cell line (HepG2), mouse Lewis lung carcinoma (LLC) cells, a mouse skin melanoma cell line (B16-F10), and a human breast cancer cell line (MCF-7). Fetal bovine serum  in RPMI-1640 medium was used to culture MCF-7 cell lines and Fetal bovine serum  in DMEM medium was used to culture B16-F10, HepG2 and LLC cells. Cells were seeded into 96-well plates at a density of 5000 cells/well and incubated at 37 °C with 5% CO2 overnight. The next day, the three compounds and MS-275 (InvivoChem) were dissolved in a complete medium to prepare different concentrations of solution, which were added to each well, followed by incubation at 37 °C for 48 h. Finally, 20 μL of MTT (5 mg/mL dissolved in PBS) was added to each well and incubated with cells at 37 °C for 4 h. Then, the complete medium was removed and the formazan crystals were dissolved in 100 μL of DMSO in each well. The absorbance was measured in a TECAN microplate reader at 490 nm. GraphPad Prism was utilized to calculate the IC50 values by a model of nonlinear regression to fit a normalized dose response. All of the experiments were performed independently three times.An MTT assay was used to determine the antiproliferative activities of compounds 5 cells/well followed by incubation at 37 °C overnight. Then, different concentrations of 1–3  were added to the plate and incubated for 48 h. The collected cells were washed once with PBS. Then the cells were added 1.0 mL of DNA staining solution and 10.0 μL of permeabilization solution followed by incubation in the dark for 30.0 min. The DNA content of the samples was analyzed by flow cytometry.HepG2 cells were seeded to a six-well plate at a density of 5 × 101) and rhizoaspergillinol A (2), were obtained from the rice culture of the mangrove endophytic fungus Aspergillus sp. A1E3, together with the known compound averufanin (3). The planar structures and absolute configurations of rhizoaspergillinol A (2) and averufanin (3) were established by extensive NMR investigations and quantum-chemical electronic circular dichroism (ECD) calculations, whereas the constitution and absolute configuration of rhizoaspergillin A (1) were unambiguously determined by single-crystal X-ray diffraction analysis of its tri-pivaloyl derivative 4, conducted with Cu Kα radiation. In addition, the absolute configuration of averufanin (3) was first clarified by ECD calculations. Most notably, rhizoaspergillin A (1) is the first reported orsellinic acid–ribose–pyridazinone-N-oxide hybrid containing a unique β-oxo-2,3-dihydropyridazine 1-oxide moiety, whereas rhizoaspergillinol A (2) is a sterigmatocystin derivative containing an additional diorcinol motif. From the perspective of biosynthesis, rhizoaspergillin A (1) could be originated from the combined assembly of three building blocks, viz., orsellinic acid, β-D-ribofuranose, and L-glutamine. It is an unprecedented alkaloid-N-oxide involving biosynthetic pathways of polyketides, pentose, and amino acids. Rhizoaspergillinol A (2) exhibited potent antiproliferative activity against a panel of cancer cell lines. Additionally, it could dose-dependently induce G2/M phase arrest in HepG2 cells. This work demonstrates that mangrove endophytic fungi of the genus Aspergillus harbor secondary metabolites with new structures.In summary, two new compounds, named rhizoaspergillin A (


Their structures were confirmed by spectroscopic data. Herein, compounds 1–10 were rhapontigenin, trans-resveratrol, 5,7,4′-trihydroxy-6,3′,5′-trimethoxy-isoflavone, irisflorentin, 6-hydroxybiochannin A, iridin S, pinoresinol, 31-norsysloartanol, isoiridogermanal, and iristectorene B, respectively. All compounds were evaluated for their antiproliferative effects against five tumor cell lines . Among them, compound 9  showed the highest activity against 4T1 and MDA-MB-468 cells. Further studies displayed that compound 9 inhibited cell metastasis, induced cells cycle arrest in the G1 phase, exhibited significant mitochondrial damage in 4T1 and MDA-MB-468 cells including excess reactive oxygen species, decreased mitochondrial membrane potential, and induced 4T1 and MDA-MB-468 cell apoptosis for the first time. In summary, these findings demonstrate that compound 9 exerts promising potential for triple-negative breast cancer treatment and deserves further evaluation.Four compounds ( Belamcanda chinensis (L.) DC is the sole species in the genus Belamcanda Adans. nom. conserv., native to Southeast Asia , and it is widely distributed in Hubei, Henan, Jiangsu, and Anhui in China [rhizome of Belamcanda chinensis (L.) DC was introduced as a medicine in the first Chinese monograph on herbal medicines, Shennong’s Classic of Materia Medica [in China . The rhia Medica . It is ka Medica ,4,5,6,7.Belamcandae chinensis rhizoma is rich in a variety of flavonoids, stilbenoids, and triterpenoids. Flavonoids mainly contain flavones  [O-decanoyl-16-O-acetyl-isoiridogermanal [Belamcandae chinensis rhizoma.To date, d so on) ,8,9 and d so on) ,11,12. Sd so on) . In addid so on) ,15, epiad so on) , seconord so on) ,18, 3-O-germanal , belamchgermanal , and belgermanal . Moreovegermanal  have beeBelamcandae chinensis rhizoma and isolated compounds have shown anti-tumor [Belamcandae chinensis rhizoma belong to isoflavonoids [Belamcandae chinensis rhizoma and their mode of action has not been provided with triple-negative breast cancer cells in any literature. Consequently, we aim to elucidate the mechanism of anti-triple-negative breast cancer of iridal-type triterpenoids isolated from Belamcandae chinensis rhizoma, so as to provide a theoretical basis for the further development and utilization of Belamcandae chinensis rhizoma.In recent decades, the pharmacological activities of ti-tumor ,23,24, ati-tumor ,27,28,29ti-tumor ,31,32, ati-tumor ,34,35,36ti-tumor ,38, antiti-tumor ,40,41,42ti-tumor , and antti-tumor , human oti-tumor , non-smati-tumor , gastricti-tumor , prostatti-tumor , malignati-tumor , and the1, 5, 7, and 8) first isolated from the genus Belamcanda Adans. nom. conserv. and six known compounds , and their structures were established by spectroscopic analyses. Herein, compounds 1–10 are rhapontigenin, trans-resveratrol, 5,7,4′-trihydroxy-6,3′,5′-trimethoxy- isoflavone, irisflorentin, 6-hydroxybiochannin A, iridin S, pinoresinol, 31-norsysloartanol, isoiridogermanal, and iristectorene B, respectively. Furthermore, all isolates were tested for antiproliferative effects against five kinds of tumor cell lines . Several compounds, including compound 9, exhibited significant cytotoxicity in five cell lines, especially for the 4T1 and MDA-MB-468 cell lines. Subsequently, compound 9  was examined in this study for the first time for its effect on proliferation, metastasis, cell cycle, apoptosis, mitochondrial membrane potential (MMP) collapse, and reactive oxygen species (ROS) formation in 4T1 and MDA-MB-468 cell lines.In this study, we discovered four compounds (Rhapontigenin (1): colorless crystallization, 1H NMR  δH: 7.11 , 6.99–6.96 , 6.95 , 6.83 , 6.78 , 6.48 , 6.18 , 3.91 .13C NMR  δc: 159.6 , 149.2(C-3), 147.6 (C-4), 141.3 (C-1′), 131.0 (C-1), 129.7 (C-α), 127.3 (C-β), 121.2 (C-6), 116.3 (C-5), 110.4 (C-2), 105.8 (C-2′ and C-6′), 102.7 (C-4′), 56.4 (OMe). All presented data of 1 were identical to rhapontigenin [1 was elucidated as rhapontigenin (ntigenin .Trans-Resveratrol (2): colorless powder, 1H-NMR  δH: 7.37–7.34 , 6.96 , 6.81 , 6.77 , 6.46 , 6.17 . 13C-NMR  δc: 159.6 , 158.4 (C-4′), 141.3 (C-1″), 130.4 (C-1′), 129.4 (C-1), 128.8 , 127.0 (C-2), 116.5 , 105.8 , 102.6 (C-4″). All NMR data of 2 were consistent with trans-resveratrol [2 was established as trans-resveratrol : yellowish crystallization, 1H-NMR  δH: 13.08 , 10.79 , 8.54 , 8.41 , 6.85 , 6.51 , 3.79 , 3.75 . 13C-NMR  δc:180.5 (C-4), 157.5 (C-7), 154.6 (C-2), 153.3 (C-5), 152.7 (C-9), 147.7 , 135.8 (C-4′), 131.4 (C-6), 121.9 (C-3), 120.7 (C-1′), 106.8 (C-2′), 104.9 (C-10), 93.9 (C-8), 60.0 (6-OCH3), 56.1 . The NMR signals of 3 were the same as 5,7,4′-trihydroxy-6,3′,5′-trimethoxyisoflavone [3 was identified as 5,7,4′-trihydroxy-6,3′,5′-trimethoxyisoflavone (oflavone .Irisflorentin (4): colorless powder, 1H-NMR  δH: 8.14 , 6.84 , 6.81 , 6.13 , 4.08 , 3.88 , 3.81 . 13C-NMR  δc: 173.7 (C-4), 153.9 (C-5), 152.7 (C-9), 152.5 , 152.1 (C-2), 140.5 (C-7), 137.2 (C-4′), 136.0 (C-6), 127.5 (C-1′), 124.1 (C-3), 113.2 (C-10), 106.8 (C-2′), 102.7 (-O-CH2-O-), 93.7 (C-8), 60.8 (5-OCH3), 60.1 (4′-OCH3), 56.0 . All NMR signals of 4 were similar to irisflorentin [4 was proposed as irisflorentin (lorentin .6-hydroxybiochannin A (5): colorless needle crystallization, 1H-NMR  δH: 8.03 , 7.36 , 6.84 , 6.42 , 3.86 .13C-NMR  δc: 182.5 (C-4), 158.9 (C-7), 158.8 (C-5), 155.0 (C-4′), 154.9 (C-2), 154.5 (C-9), 132.8 (C-6), 131.4 (C-2′), 124.2 (C-1′), 123.3 (C-3), 116.2 (C-3′), 106.6 (C-10), 95.0 (C-8), 60.9 (-OCH3). All presented data of 5 were in accordance with 6-hydroxybiochannin A [5 was determined as 6-hydroxybiochannin A (hannin A .Iridin S (6): colorless powder, 1H-NMR  δH: 12.88 , 8.56 , 6.92 ,6.90 , 5.46 , 5.16 , 5.11–4.63 , 3.81 , 3.77 , 3.70 .13C-NMR  δc: 180.5 (C-4), 156.7 (C-7), 155.7 (C-2), 152.9(C-5), 152.7(C-9), 152.4 , 137.6 (C-4′), 132.6 (C-6), 126.2 (C-3), 122.0 (C-1′), 106.7 , 106.5 (C-10), 100.1 (C-1″), 94.1 (C-8), 77.3 (C-3″), 76.7 (C-5″), 73.1 (C-2″), 69.7 (C-4″), 60.7 , 60.3 (4′-OCH3), 60.1 ), 56.0 (6-OCH3). The NMR signals of 6 were in accordance with iridin S [6 was assigned as iridin S (iridin S .Pinoresinol (7): colorless powder, 1H-NMR  δH: 6.89 , 6.74 , 4.60 , 4.15–4.07 , 3.76 , 3.72 , 3.03 . 13C-NMR  δc: 147.5 , 145.9 , 132.2 , 118.6 , 115.1 , 110.4 , 85.2 , 70.9 , 55.6 (2 × -OCH3), 53.6 . The NMR signals of 7 were the same as pinoresinol [7 was established as pinoresinol (oresinol . As a cooresinol .31-norsysloartanol (8): colorless flaky crystallization, 1H-NMR  δH: 3.21 , 2.01–1.93 , 1.70 –1.48 , 1.42–1.24 , 1.20–1.08 , 0.98–0.96 , 0.89–0.85 , 0.58 , 0.38 , 0.14 .13C-NMR  δc: 76.7 (C-3), 52.5 (C-17), 49.0 (C-14), 47.0 (C-8), 45.4 (C-13), 44.7 (C-4), 43.5 (C-5), 39.7 (C-24), 36.6 (C-22), 36.3 (C-20), 35.5 (C-12), 35.0 (C-2), 33.0 (C-15), 30.9 (C-1), 29.7 (C-10), 28.3 (C-25), 28.2 (C-7), 27.4 (C-19), 27.1 (C-16), 25.3 (C-11), 24.8 (C-6), 24.3 (C-23), 23.7 (C-9), 23.0 (C-27), 22.7 (C-25), 19.3 (C-28), 18.5 (C-21), 17.9 (C-18), 14.5 (C-29). The NMR signals of 8 were identical to 31-norsysloartanol [8 was elucidated as 31-norsysloartanol : yellow oil, 1H-NMR  δH: 10.21 , 5.20 , 5.09 , 5.00 , 3.86 , 3.51 , 3.37 , 2.72–2.62 , 2.56 , 2.20 , 2.08–2.01 , 1.95 , 1.82 , 1.68 , 1.61 , 1.58 , 1.52 , 1.36–1.17 , 1.12 .13C-NMR  δc: 192.1 (C-1), 166.5 (C-7), 137.8 (C-15), 137.4 (C-19), 134.0 (C-2), 132.1 (C-23), 127.4 (C-14), 125.4 (C-22), 121.7 (C-18), 78.7 (C-16), 75.2 (C-10), 63.2 (C-3), 46.1 (C-11), 45.0 (C-6), 40.9 (C-20), 38.2 (C-4), 37.9 (C-9), 34.5 (C-17), 33.7 (C-12), 28.0 (C-21), 27.8 (C-5), 26.0 (C-27), 25.9 (C-24), 25.0 (C-8), 22.8 (C-13), 18.4 (C-26), 17.8 (C-30), 16.4 (C-29), 11.3 (C-28), 10.9 (C-25). The NMR data of 9 were consistent with isoiridogermanal [9 was established as isoiridogermanal : yellow oil, 1H-NMR  δH: 10.14 , 5.32 , 5.04 , 3.99 , 3.90 , 3.27 , 2.52 , 2.25 , 2.04 .13C-NMR  δc: 190.1 (CHO), 174.2 (C=O), 162.9 (C-4), 138.8 (C-8′), 137.2 (C-4′), 133.5 (C-8), 131.8 (C-12′), 125.5 (C-3′), 124.3 (C-11′), 120.1 (C-7′), 76.9 (C-5′), 75.1 (C-1), 64.4 (C-3″), 44.9 (C-2), 43.5 (C-3), 40.0 (C-9′), 37.2 (C-6), 37.0 (C-1′), 34.6 (C-6′), 34.5 (C-5″), 32.1 (C-15″), 29.8 (C-11″), 29.8 (C-12″), 29.8 (C-13″), 29.6 (C-10″), 29.5 (C-9″), 29.5 (C-14″), 29.3 (C-8″), 28.8 (C-2″), 26.7 (C-10′), 26.4 (C-7), 25.9 (C-7), 25.2 (C-13′), 25.1 (C-6″), 24.0 (C-5), 22.8 (C-16″), 21.9 (C-2′), 18.1 (C-11), 17.9 (C-16′), 16.4 (C-15′), 14.3 (C-17″), 12.0 (C-14′), 11.1 (C-10). All presented NMR data of 10 were in accordance with iristectorene B [10 was determined as iristectorene B   C. To furer to G1 . We dete S phase D. In sum9 was able to suppress MDA-MB-468 and 4T1 cell metastasis. As shown in the wound-healing assay, treatment with compound 9 of 30 μM in the MDA-MB-468 cells compared with the control group could significantly increase the percentage wound closure (%) (p < 0.001), but treatment with compound 9 of 20 μM in the 4T1 cells could markedly increase the percentage wound closure (%) (p < 0.001). In addition, the higher the concentration or the longer the treatment time of compound 9, the higher the percentage of wound closure (%), which revealed that compound 9 could dose- and time-dependently inhibit migration in the MDA-MB-468 and 4T1 cells (9 of 10 μM could significantly decrease the number of invaded MDA-MB-468 and 4T1 cells (p < 0.001)  B, and coApoptosis, programmed cell death, regulates the balance between cell growth and death. Tumor cells that evade apoptosis could promote metastasis . Accordi9 of 10 μM to 30 μM, the percentage of MDA-MB-468 and 4T1 cells in PI- and Annexin-V-FITC-positive cells was significantly increased via flow cytometry, which demonstrated that the apoptotic rate of the MDA-MB-468 and 4T1 cells was significantly increased (p < 0.001)  A. The reentation B in the 9, we examined MMP by JC-1 staining in the MDA-MB-468 and 4T1 cells. Red fluorescence revealed JC-1 aggregation, which represented an intact MMP, while green fluorescence represented JC-1 monomers, indicating a dissipated MMP. The decrease in MMP could be easily detected by the change of JC-1 from red fluorescence to green fluorescence, and the change of JC-1 from red fluorescence to green fluorescence could also be used as a characteristic sign of cells’ early apoptosis.A decrease in mitochondrial membrane potential (MMP) levels is one of the characteristics of early apoptosis in tumor cells. To evaluate the role of mitochondria-induced cell death after treatment with compound 9 of 10 μM to 30 μM in the MDA-MB-468 and 4T1 cells, the red fluorescence became weaker, whereas, on the contrary, the green fluorescence became stronger  are a heterogeneous group of highly reactive ions and molecules that are produced by metabolic reactions in mitochondria. ROS maintain homeostasis through the redox pathway, and increasing ROS levels are able to promote tumor cell death and limit cancer progression . We inve9 in the MDA-MB-468 and 4T1 cells through flow cytometry, the fluorescence became stronger which indicated that the ROS levels were higher (9 of 30 μM in the MDA-MB-468 cells and 20 μM in the 4T1 cells significantly increased the ROS levels (p < 0.001) compared to the control group. In addition, the results from the fluorescence microscope were identical; DCF fluorescence revealed a significant increase in ROS levels in a dose-dependent manner  DC, including four compounds  first isolated from the genus Belamcanda Adans. nom. conserv. and six known compounds . Ten compounds had antiproliferative effects against five breast cancer cell lines . Among them, compound 9 had the best inhibitory effect against the 4T1 and MDA-MB-468 cells. In addition, antiproliferative effects, apoptosis induction, and potential molecular mechanisms of action were evaluated for the first time. It was demonstrated that compound 9 showed significant antiproliferative activity and inhibited metastasis in the MDA-MB-468 and 4T1 cells. Subsequently, Annexin V-FITC/PI showed an increase in apoptotic rates, and with Hoechst 33342 staining, significant changes in cell morphology were found after treatment with compound 9 of 10 μM to 30 μM in the MDA-MB-468 and 4T1 cells. As we know, depolarization of the mitochondrial membrane leads to the release of pro-apoptotic factors in mitochondria and the activation of apoptotic proteases and ultimately induces apoptosis. Excessive production of ROS leads to oxidative damage to important cellular molecules and structures, resulting in mitochondrial dysfunction and activation of endogenous apoptotic pathways and ultimately causing cell apoptosis [9 could induce apoptosis in MDA-MB-468 and 4T1 cells for the first time. In conclusion, all of the results revealed compound 9 may be a leading compound for triple-negative breast cancer to merit further study.In summary, ten compounds were isolated from the poptosis . As mentBelamcandae chinensis rhizoma was purchased from Heshun Tang Pharmacy in Shenzhen, China, in October 2020 and identified by Dr. Li Chenyang from Shenzhen University. Approximately 19 kg Belamcandae chinensis rhizoma was ground into a fine powder and extracted with 95% ethanol by refluxing extraction three times, each time for 2 h. The extraction solution was combined, the pressure was reduced, and it was evaporated to a certain volume, and then the crude extract was further fractionated through macroporous resin column chromatography and eluted successively with 45%, 75%, and 95% ethanol, which was brought to dryness under reduced pressure, yielding dried 45% (3.461 kg), 75% (350 g), and 95% (341 g) ethanol extract, respectively. The cytotoxicity assay of the three extracts (Belamcandae chinensis rhizoma was carried out.extracts  indicate8 (30.0 mg) was purified by crystallization from Fr. B with methanol. Compound 10 (81.3 mg) was purified by semi-preparative RP-HPLC  under the following gradient: MeOH−H2O (95:5 to 100:0 over 30 min) from Fr. C. Fr. D was separated by medium- and low-pressure ODS column chromatography, and gradient elution was performed with MeOH−H2O (50%−100%). The obtained compounds, 1 (194.0 mg), 2 (376.0 mg), 4, (39.7 mg), 7 (32.4 mg), and 3 (20.0 mg), were purified by crystallization with methanol. In addition, compound 5 (39.0 mg) was isolated with 45% methanol aqueous solution using semi-preparative RP-HPLC  from Fr. D. Subsequently, compound 9 (179.0 mg) was separated with 71% methanol aqueous solution by semi-preparative RP-HPLC (Phenomenex Luna C18 (2) 250 mm × 10 mm, flow rate: 3 mL/min) from Fr. E. Finally, compound 6 (4.0 mg) was purified by Sephadex LH−20  from Fr. F. Compounds 1–10 were confirmed by spectroscopic data, and they were rhapontigenin, trans-resveratrol, 5,7,4′-trihydroxy-6,3′,5′-trimethoxy-isoflavone, irisflorentin, 6-hydroxybiochannin A, iridin S, pinoresinol, 31-norsysloartanol, isoiridogermanal, and iristectorene B, respectively.The 75% ethanol extract was fractionated by silica gel column chromatography using petroleum ether: ethyl acetate  and methanol successively, and it produced seven sub-fractions (Fr. A−Fr. G). Compound 2 at 37 °C.Five breast cancer cell lines: MDA-MB-468, MDA-MB-231, BT549, MCF7, and 4T1 were obtained from the American Type Culture Collection . The MDA-MB-468 cells were routinely cultured in RPMI-1640 , and the 4T1 cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Corning), which was supplemented with 10% fetal bovine serum (FBS)  and penicillin/streptomycin  in 5% CO4 cells and 1 × 104 cells per well, incubated in a 37 °C incubator with 5% CO2 added overnight. The cells were treated with compounds at different concentrations for 48 h in 5% CO2 at 37 °C. The original medium was discarded, and each well was filled with 100 μL of culture medium containing 10% CCK-8 and incubated for 1 h at 37 °C in an incubator, and then the absorbance at 450 nm was detected with a microplate reader .The cells were seeded in 96-well plates with 3 × 103 cells/well) and cultured overnight in 5% CO2 at 37 °C for 7 days. When the colonies formed, they were treated with compounds at different concentrations, and then they were cultured in a 37 °C incubator with 5% CO2 for 10 days. The fresh complete culture medium was replaced every other day. When the colonies were cultured to the naked eye, the colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution.The cells were seeded in a 6-well plate  ethanol overnight at 4 °C. After that, the fixed cells were centrifuged and washed with cold phosphate-buffered saline (PBS), and then, they were further resuspended in a solution of 0.5 mL propidium iodide (PI) and RNAase staining buffer for 30 min at 37 °C in the dark. The cells were determined by an Agilent NovoCyte Flow Cytometer, and the data were analyzed by NovoExpress software (version 1.6.1).The cells were seeded in a 6-well plate and cultured overnight in 5% CO2, and after the cells reached confluency, a sterile pipette tip was used to draw parallel lines in each cell, and the cells were washed with PBS. In succession, a medium with 2% FBS was added with compounds at different concentrations, and a microscope was used to take pictures at 0 h, 24 h, and 48 h to analyze the results.The cells were seeded in a 6-well plate in a 37 °C incubator with 5% COThe cells were resuspended in 200 μL FBS-free medium into the upper chamber, and 600 μL complete medium with compounds at different concentrations was added into the lower chamber. After incubating for 48 h, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet solution. The cells were photographed and counted under the microscope.2 at 37 °C. Then, the cells were treated with drugs of different concentrations for 48 h in 5% CO2 at 37 °C. The original medium was discarded, and the cells were collected and washed twice with cold PBS. The collected cells were dispersed with 100 μL 1 × binding buffer, and then, they were incubated with 5 μL Annexin V-FITC and 10 μL PI staining solution for 15 min at room temperature in the dark. The stained cells were detected by an Agilent NovoCyte Flow Cytometer, and the data were analyzed by NovoExpress software (version 1.6.1).Apoptosis was detected by an Annexin V-FITC/PI staining kit. The cells were seeded in a 6-well plate and cultured overnight in 5% CO2 at 37 °C. After the cells were treated with compounds at different concentrations for 48 h in 5% CO2 at 37 °C, the cells were washed twice with PBS and then stained with Hoechst 33342 fluorescent dye for 30 min at 37 °C in the dark. Then, using a fluorescence microscope, morphological changes in the nucleus were examined.The cells were seeded in a 6-well plate and cultured overnight in 5% CO2 at 37 °C. After the cells were treated with compounds of different concentrations, the medium was removed and washed with sterile PBS, and then, the cells were stained with a JC-1 fluorescent probe for 30 min at 37 °C in the dark. Thereafter, the cells were washed with 1 × JC-1 buffer solution three times, and the cells were pictured via fluorescence microscopy.Mitochondrial membrane potential (MMP) was determined by an MMP assay kit. Briefly, the cells were seeded in a 6-well plate and cultured overnight in 5% CO2 at 37 °C. After the cells were treated with compounds at different concentrations, the medium was removed and washed with sterile PBS, and, on one hand, incubated with a DCFH-DA probe for 20 min at 37 °C in the dark, and then, the cells were washed with PBS three times, and the cells were pictured via fluorescence microscopy. On the other hand, the cells were collected, stained with a DCFH-DA probe, and ROS were determined by an Agilent NovoCyte Flow Cytometer, and the data were analyzed by NovoExpress software (version 1.6.1).The intracellular ROS levels were detected with an ROS assay kit. The cells were seeded in a 6-well plate and cultured overnight in 5% COt-test. p < 0.05 represents a statistical difference.All cell culture assays were performed in triplicate. The data were analyzed and processed by GraphPad Prism 9.0 software. The data were expressed in the form of the mean ± SD. Differences between the groups were compared with the statistical significance by a 1, 5, 7, and 8) were first isolated from the genus Belamcanda Adans. nom. conserv., and six known compounds  were isolated from the rhizome of Belamcanda chinensis (L.) DC. The antiproliferative effects against five tumor cell lines  suggested that compound 9 had the best inhibitory effect against the MDA-MB-468 and 4T1 cells. Subsequently, we found compound 9 could inhibit the proliferation and metastasis of MDA-MB-468 and 4T1 cells and induce cell cycle arrest in the G1 phase. Moreover, we found that compound 9 could downregulate mitochondrial membrane potential and produce excessive ROS to induce apoptosis of the MDA-MB-468 and 4T1 cells for the first time. In conclusion, our findings demonstrate that compound 9 may be a leading compound for triple-negative breast cancer to merit further study.In this study, four compounds (