Hypericum roeperianum (HRB) was evaluated towards a panel of 9 cancer cell lines. The mode of action of the HRB and trichadonic acid (1) was also studied. Column chromatography was applied to isolate the constituents of HRB. The cytotoxicity of botanicals and phytochemicals was evaluated by the resazurin reduction assay (RRA). Caspase-Glo assay was used to evaluate the activity of caspases, and reactive oxygen species (ROS) (H2DCFH-DA) were assessed by flow cytometry. Phytochemicals isolated from HRB were trichadonic acid (1), fridelan-3-one (2), 2-hydroxy-5-methoxyxanthone (3), norathyriol (4), 1,3,5,6-tetrahydroxyxanthone (5), betulinic acid (6), 3′-hydroxymethyl-2′--5′,6′:5,6--1′,4′-dioxane (7), and 3′-hydroxymethyl-2′--5′,6′:5,6-(xanthone)-1′,4′-dioxane (8). Botanicals HRB, HRBa, HRBa2-4, HRBb, and doxorubicin displayed cytotoxic effects towards the 9 tested cancer cell lines. The recorded IC50 values ranged from 11.43 µg/mL (against the P-glycoprotein (gp)-overexpressing CEM/ADR5000 leukemia cells) to 26.75 µg/mL (against HCT116 (p53+/+) colon adenocarcinoma cells) for the crude extract HRB. Compounds 1, 5, and doxorubicin displayed cytotoxic effects towards the 9 tested cancer cell lines with IC50 values varying from 14.44 µM (against CCRF-CEM leukemia cells) to 44.20 µM (against the resistant HCT116 (p53−/−) cells) for 1 and from 38.46 µM (against CEM/ADR5000 cells) to 112.27 µM (against the resistant HCT116 (p53−/−) cells) for 5. HRB and compound 1 induced apoptosis in CCRF-CEM cells. The apoptotic process was mediated by enhanced ROS production for HRB or via caspases activation and enhanced ROS production for compound 1. This study demonstrated that Hypericum roeperianum is a potential source of cytotoxic phytochemicals such as trichadonic acid and could be further exploited in cancer chemotherapy.The global cancer burden remains a serious concern with the alarming incidence of one in eight men and one in eleven women dying in developing countries. This situation is aggravated by the multidrug resistance (MDR) of cancer cells that hampers chemotherapy. In this study, the cytotoxicity of the methanol extract (HRB), fractions , and compounds from the bark of  Multidreollidin , licoagreollidin , 7-dihydaejasmin , and alkaejasmin , isotetraejasmin , and ungaejasmin . Howeverterility , as antiterility  and as aterility . Previouthe bark  and 10 onthone D . The cytµg/mL) in culture media to maintain the features of MDA-MB-231-BCRP, U87MG.ΔEGFR, and HCT116 (p53−/−), respectively, was obtained from Sigma-Aldrich and stored at 72.18 mM. Hydrogen peroxide (H2O2) and valinomycin (at 1 mg/mL) were provided by Sigma-Aldrich .Doxorubicin (98.0% purity) from Sigma-Aldrich  was obtained from the Johannes Gutenberg University Medical Center . Geneticin >98% (used at 800 ng/mL and 400 Hypericum roeperianum Schimp. p. ex A. Rich (Guttiferae) was collected in Bangang Wabane (South West Region of Cameroon) in October 2018. No permission was necessary for sample's collection. The identification of the plant was carried out by Dr. Tchiengue Barthelemy at the Cameroon National Herbarium (Yaoundé) where a voucher specimen was deposited under the number 24584/SRF/Cam. Dried barks of the plant (3.0 kg) were powdered and extracted with methanol  for 72 h at room temperature to afford a crude extract  after filtration with Whatman paper no.1 and evaporation in vacuum, under reduced pressure. A portion of the resulting extract (140.0 g) was, then, exhausted in ethyl acetate to yield 65.0 g of the ethyl acetate extract (EtOAc) (HRBa) and the residue .The bark of Part of the ethyl acetate extract  was submitted to silica gel flash chromatography using hexane-EtOAc and, then, EtOAc-MeOH mixtures of increasing polarities. Eighty fractions (frs) of 500 mL each were collected as follows: hexane 100% (sub-frs 1–3), hexane-EtOAc 90 : 10 (sub-frs 4–12), hexane-EtOAc 80 : 20 (sub-frs 13–18), hexane-EtOAc 70 : 30 (sub-frs 19–22), hexane-EtOAc 60 : 40 (sub-frs 23–27), hexane-EtOAc 50 : 50 (sub-frs 28–37), hexane-EtOAc 30 : 70(sub-frs 38–43), AcOEt 100% (sub-frs 44–52), EtOAc-MeOH 95 : 5 (sub-frs 53–57), EtOAc-MeOH 90 : 10 (sub-frs 58–62), EtOAc-MeOH 80 : 20 (sub-frs 63–69), and MeOH 100% (sub-frs 70–80). Based on their analytical thin-layer chromatography (TLC) profiles, these fractions were pooled into five fractions (frs) as follows: HRBa1 , HRBa2 , HRBa3 , HRBa4 , and HRBa5 .1 (15 mg). The filtrate was subjected to silica gel column chromatography using hexane-AcOEt mixtures of increasing polarities as elution solvents. Sixty-five sub-frs of 150 mL each were collected as follows: Hex 100% (1–3), Hex-AcOEt 90:10 (Sub-frs 4–9), Hex-AcOEt 80 : 20 (Sub-frs 10–15), Hex-AcOEt 70 : 30 (Sub-frs 16–19), Hex-AcOEt 60 : 40 (Sub-frs 20–35), Hex-AcOEt 50 : 50 (Sub-frs 36–42), Hex-AcOEt 40 : 60 (Sub-frs 43–50), AcOEt 100% (Sub-frs 51–55), AcOEt-MeOH 90 : 10 (Sub-frs 56–60), and MeOH 100% (Sub-frs 61–65). Sub-frs 6–9 yielded compound 2 (15.0 mg) as a white powder. Sub-frs EC23-29 yielded compound 3 (12.0 mg) as a yellow powder.Dry fraction HRBa2 (15.0 g) was dissolved in methanol affording a nonsoluble powder which was, then, filtered to give compound 4 (18.0 mg) as a green-yellowish powder. Sub-frs 29–35 yielded compound 5 as a yellow powder (15.0 mg).HRBa3 (9.5 g) was subjected to silica gel column chromatography using Hex-AcOEt mixtures of increasing polarities as elution solvents. Seventy-five sub-frs of 150 mL each were collected as follows: hexane 100% (sub-frs 1–4), Hex-AcOEt 90 : 10 (sub-frs 5–9), Hex-AcOEt 80 : 20 (sub-frs 9–16), Hex-AcOEt 70 : 30 (sub-frs 17–23), Hex-AcOEt 60 : 40 (sub-frs 24–35), Hex-AcOEt 50 : 50 (sub-frs 36–42), Hex-AcOEt 40 : 60 (sub-frs 43–47), AcOEt 100% (sub-frs 48–57), AcOEt-MeOH 90 : 10 (sub-frs 58–63), AcOEt-MeOH 80 : 20 (sub-frs 64–70), and MeOH 100% (sub-frs 71–75). Sub-frs 22–25 yielded compound 2Cl2-MeOH mixtures of increasing polarities as elution solvents. Fifty sub-frs of 150 mL each were collected as follows: CH2Cl2 100% (sub-frs 1–4), CH2Cl2-MeOH 95 : 5 (sub-frs 5–13), CH2Cl2-MeOH 90 : 10 (sub-frs 14–25), CH2Cl2-MeOH 85 : 15 (sub-frs 26–35), CH2Cl2-MeOH 80 : 20 (sub-frs 36–42), and MeOH 100% (subfrs 43–50). Sub-frs 6–11 yielded compound 6 (40.0 mg) as a white powder. Sub-frs 13–15 yielded compound 7 (12.0 mg) as a yellow powder. Sub-frs 19–23 yielded compound 8 (14.0 mg) as a yellow powder.HRBa4 (12.5 g) was subjected to silica gel column chromatography using CHEGFR subline, HCT116 (p53+/+) colon cancer cell line and its knockout clone HCT116 (p53−/−), and MDA-MB-231-pcDNA3 breast cancer cell line and its BCRP-transfected multidrug-resistant MDA-MB-231-BCRP clone 23 cell line [Cell lines used in this work included drug-sensitive and drug-resistant phenotypes of earlier reported origin. They were all provided by Prof. Dr. Thomas Efferth from his cell lines collection; they have being used in cytotoxicity screening by our team for a decade –21. Thesell line , as wellell line .1–5, 7, and 8), and doxorubicin on the cell growth as reported earlier [2 atmosphere at 37°C. Cells were further coloured with resazurin and incubated for 1–2 h; the fluorescence was further measured with an Infinite M2000 Pro™ plate reader  at 544 nm as the excitation wavelength and 590 nm as the emission wavelength. The IC50 values represented the concentrations of the sample required to inhibit 50% of cell proliferation and were calculated from a calibration curve by linear regression using Microsoft Excel 2007 [The RRA was applied to evaluate the cytotoxicity of botanicals, the isolated phytochemicals  were used to treat CCRF-CEM cells (1 × 106 cells). Cells were further incubated for 24 h in humidified 5% CO2 atmosphere at 37°C and analyzed using a BD Accury C6 Flow Cytometer  by measuring the propidium iodide fluorescence of individual nucleus, as described earlier [Various concentrations of botanical HRB, phytochemical ells 1 × 6 cells.  earlier . Experim6; 1 ml) were also treated with HRB, compound 1 and doxorubicin for 24 h (in humidified 5% CO2 atmosphere at 37°C), and apoptosis was further assessed by flow cytometry using the flouresceinisothiocynate- (FITC-) conjugated annexin V/PI assay kit , as previously published [µl binding buffer, and stained with 5 µl FITC-conjugated annexin V (10 mg/mL) and 10 µl PI (50 mg/ml). After 15 min incubation at room temperature (RT) in the dark, cells were analyzed using a BD Accury C6 Flow Cytometer (BD Biosciences). Cells stained with only annexin V were evaluated as being in early apoptosis. Cells stained with both annexin V and propidium iodide were evaluated as being in late apoptosis or in a necrotic stage.The CCRF-CEM cells 1 × 106; 1 ml wublished . Briefly1 were used to treat CCRF-CEM cells for 6 h. The activities of caspases were determined using Caspase-Glo 3/7, Caspase-Glo 8, and Caspase-Glo 9 Assay kits  by measuring the luminescence using an Infinite M2000 ProTM plate reader (Tecan), as reported previously [Different concentrations of HRB and compound eviously .1 were used to treat CCRF-CEM cells (1 × 106 cells); DMSO (solvent control); or hydrogen peroxide . After 24 h incubation in humidified 5% CO2 atmosphere at 37°C, the production of ROS was evaluated using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFH-DA) (Sigma-Aldrich) staining, as described earlier [Various concentrations of HRB and triterpenoid  earlier –32.p value was calculated to determine significant differences .Statistical analyses were performed with Graph pad prism 5 software. Representative data from three independent experiments are shown as mean value ± S.E.M. One-way Analysis Variance (ANOVA) followed by post hoc Tukey's test was used to determine the significance of the difference between mean values relative to the control. The 30H48O3  [30H50O  [14H01O4  [13H8O6  [13H8O6  [30H48O3  [24H20O8  [2420 O10  [The chemical structures of the isolated phytochemicals were determined by exploiting the physical, mass spectra, and NMR data, followed by direct comparison of these data with those of similar reported compounds in the literature. Compounds were identified as trichadonic acid Cm/z 456) , fridelam/z 426) , 2-hydrom/z 242) , 1,3,6,7m/z 260) , 1,3,5,6m/z 456) , 3′-hydrm/z 436) , 38, andm/z 468)   to 26.75 µg/mL (against HCT116 (p53+/+) colon adenocarcinoma cells) for the crude extract HRB, from 15.65 µg/mL (against CEM/ADR5000 leukemia cells) to 41.17 µg/mL (against HCT116 (p53+/+) cells) for HRBa, from 13.92 µg/mL (against U87MG.ΔEGFR glioblastoma cells) to 33.44 µg/mL (against HCT116 (p53+/+) cells) for HRBa2, from 16.13 µg/mL (against U87MG glioblastoma cells) to 33.63 µg/mL (against HCT116 (p53−/−) cells) for HRBa3, from 10.52 µg/mL (against U87MG.ΔEGFR cells) to 28.43 µg/mL (against HCT116 (p53+/+) cells) for HRBa4, and from 28.30 µg/mL (against U87MG.ΔEGFR cells) to 69.48 µg/mL (against HepG2 cells) for HRBb  to 44.20 µM (against the resistant HCT116 (p53−/−) cells) for 1, from 38.46 µM (against CEM/ADR5000 cells) to 112.27 µM (against HCT116 (p53−/−) cells) for 5, and from 0.02 µM (against CCRF-CEM cells) to 122.96 µM (against CEM/ADR5000 cells) for doxorubicin  cells to the mother botanical, HRB compared to their sensitive counterparts CCRF-CEM cells, MDA-MB-231 cells, and HCT116 (p53+/+) cells, respectively, was observed. Hypersensitivity of all resistant cell lines to fraction HRBa4 and HRBb compared to their sensitive parental cell lines was also recorded  cells compared to their sensitive counterparts HCT116 (p53+/+) was observed with a D.R. of 2.55; however, this value was lower than that obtained with doxorubicin (D.R. of 3.73)  had the best activity and was consequently selected, together with the crude extract, HRB, for further mechanistic studies.Collateral sensitivity (D.R. below 1) of CEM/ADR5000 cells, recorded . Collateobserved . Concernof 3.73) . Apart aof 3.73) . Compounatocytes . Regardi1, as well as doxorubicin, caused dose-dependent alteration of CCRF-CEM cells' cycle distribution after 24 h treatment (1) induced increase of cells in the sub-G0/G1 phase, varying from 1.68% (1/4 × IC50) to 24.30% (2 × IC50) for HRB and from 1.86% (1/4 × IC50) to 17.40% (2 × IC50) for compound 1, while doxorubicin induced increase in the range of 3.28% (1/4 × IC50) to 12.05% (2 × IC50). This suggests that HRB and compound 1 induced apoptosis in CCRF-CEM cells. Increase of cells in the Go/G1 phase also suggests that both HRB and trichadonic acid caused cycle arrest in this phase; meanwhile, doxorubicin induced S and G2/M phase arrest  induced late apoptosis (Q2-UR) while compound 1 significantly (p < 0.05) induced early apoptosis with, respectively, 14.6% annexin V (+)/PI (−) and 52.5% annexin V (+)/PI (+) cells (The crude extract (HRB) and triterpenoid reatment . Figure e arrest . A concestaining . At 2 × +) cells .1), respectively, for caspases 3/7, 8, and 9 , or DMSO was analyzed, and the results are depicted in p < 0.05) induced increase of ROS production in a range of 8.98% (1/4 × IC50) to 71.92% (2 × IC50); compound 1 also significantly (p < 0.05) induced increase of ROS production in a range of 12.35% (7.22 µM) to 68.12% (57.76 µM). The reference compound, H2O2, increased the ROS levels to 94.30% at 50 µM, while ROS production in nontreated cells was 0.6%.The production of reactive oxygen species (ROS) in CCRF-CEM cells treated with HRB, triterpenoid BCRP cells, HCT116 (p53−/−) cells, and U87.MGΔEGFR cells, were used, and their susceptibilities to isolated phytochemicals were compared with those of their parental sensitive counterparts, CCRF-CEM cells, MDA-MB-231 cells, HCT116 (p53+/+) cells, and U87.MG cells, respectively  cells to HRB was achieved, as well as the hypersensitivity of all resistant cell lines to fraction HRBa4 and HRBb compared to their sensitive parental cell lines  is a well-known cytotoxic compound and has previously been found active towards the cancer cell lines tested in the present work, with IC50 values ranging from 7.65 µM (in CEM-ADR5000 cells) to 44.17 µM (in HepG2 cells) [6 can be ranked amongst the best active principles of Hypericum roeperianum. Also, the cytotoxicity of norathyriol (4) in JB6 P+ mouse skin epidermal cells was reported [5) had low cytotoxic effects against K562 leukemia cells with 12.98 µg/mL (49.92 µM) [Taking into account the rapid development of resistance by cancer cell lines, the use of MDR phenotypes when screening phytochemicals is an interesting approach. Collateral or normal sensitivity  of MDR cells to phytochemicals combined to their good cytotoxicity could be better criteria to select substances for clinical studies. In the present work, four MDR cells lines, CEM/ADR5000 cells, MDA-MB-231-ll lines . Collateblastoma . This clectively , 40. Impll lines . It can,2 cells) . Althougreported . 1,3,5,69.92 µM) .1 induced apoptosis in CCRF-CEM cells with cell cycle arrest in the Go/G1 phase  in C-13  against all cancer cell lines tested in the present study [7 and 8 is the presence of two hydroxyl (-OH) groups in C-6 and C-7 (Compound 8) (8 was active against HCT116 (p53+/+) cells, with an IC50 value of 37.79 µM compared to the IC50 value above 91.74 µM obtained for compound 7  . This diell line .Hypericum roeperianum is a source of cytotoxic compounds. Triterpenoids such as trichadonic acid (1) and betulinic acid (6), xonthones (2-hydroxy-5-methoxyxanthone (3), norathyriol (4), and 1,3,5,6-tetrahydroxyxanthone (5)), and xantholignoids -5′,6′:5,6--1′,4′-dioxane (7) and 3′-hydroxymethyl-2′--5′,6′:5,6-(xanthone)-1′,4′-dioxane (8)) are amongst the active constituents of this plant. Trichadonic acid (1) induced apoptosis in CCRF-CEM leukemia cells, through caspases activation and enhancement of ROS production. The crude extract, HRB, also induced apoptosis in CCRF-CEM cells, mediated by enhancement of ROS production. These compounds can potentially be useful in the fight against recalcitrant cancers.The present work demonstrated that


UBE2L3 which drives E6-mediated proteasomal degradation of p53 in cervical cancer. Downregulation of UBE2L3 by overexpression of HP1γ suppressed UBE2L3-dependent p53 degradation-promoting apoptosis of cervical cancer cells. Our findings propose a useful strategy to overcome p53 degradation in cervical cancer through the blockage of nuclear export of HP1γ.E6 oncoprotein derived from high-risk human papillomavirus (HPV) drives the development of cervical cancer through p53 degradation. Because cervical cancer therapies to inactivate HPV or E6 protein are not available, alternative strategies are required. Here, we show that HPV-mediated nuclear export of human heterochromatin protein 1γ (HP1γ) reduces the stability of p53 through UBE2L3-mediated p53 polyubiquitination during cervical cancer progression. In general, HP1 plays a key role in heterochromatin formation and transcription in the nucleus. However, our immunostaining data showed that the majority of HP1γ is localized in the cytoplasm in HPV-mediated cervical cancer. We found that HPV E6 protein drives unusual nuclear export of HP1γ through the interaction between the NES sequence of HP1γ and exportin-1. The mutation of the NES sequence in HP1γ led to nuclear retention of HP1γ and reduced cervical cancer cell growth and tumor generation. We further discovered that HP1γ directly suppresses the expression of  Cervical carcinogenesis is induced by persistent high-risk human papillomavirus (HPV) infection, which is different from other cancers or malignant diseases . Among tOncogenic transformations by high-risk HPVs are mediated by the viral E6 and E7 oncogenes, which are regularly co-expressed . The E6 Heterochromatin protein 1 (HP1), a histone code reader, specifically recognizes and binds to methylated histone H3 lysine 9 (H3K9me) , 14. TheHere, we found that abnormal nuclear export of HP1γ, which is mediated by exportin-1, is responsible for E6-dependent p53 degradation. Inhibition of exportin-1 or expression of nuclear export signal (NES)-depleted HP1γ blocked the nuclear export of HP1γ and subsequently restored p53 signaling reducing the tumorigenic potential of cervical cancer cells. Genome-wide analysis identified UBE2L3, which plays a role in p53 degradation, as a target gene of HP1γ. Overexpression of HP1γ suppressed UBE2L3 and restored p53 stability, further inducing apoptosis of cervical cancer cells. These observations prompted us to take a closer look at the role of HP1γ in the cervical cancer and provide a pathogenic rationale to treat cervical cancer.The DNA constructs used in this study were pcDNA-EGFP-HP1β, pcDNA-EGFP-HP1γ, pcDNA-EGFP-HP1γ V32A, pcDNA-EGFP-HP1γ I165E, pcDNA-EGFP-HP1γ W174A, pcDNA-EGFP-HP1γ 150A, 152A, and pNCMV 16E6no*. The mutant constructs for HP1γ were generated using site-directed mutagenesis . Anti-HP1α , anti-HP1β , anti-HP1γ , anti-p53 , anti-UBE2L3 , anti-ubiquitin , anti-Exportin-1 , anti-Bax , anti-Noxa , anti-Puma , anti-Caspase 3 , anti-Flag , anti-GFP , anti-α-tubulin , anti-actin , and anti-Lamin A/C  antibodies were used in this study.2 at 37 °C. To inhibit exportin-1, cells were treated with 50 nM of leptomycin B (LMB) purchased from Sigma-Aldrich for 4 h or 100 nM of KPT-330 purchased from Selleckchem for 24 h.HeLa, SiHa, CaSki, and U2OS cells were purchased from the American Type Culture Collection (ATCC). The cells were cultured according to the instructions from ATCC and were maintained under a fully humidified atmosphere of 95% air and 5% COHP1γ inserted under the TRE3G promoter in SiHa cells according to the manufacturer’s protocol. After cloning HP1γ wild-type (WT) or AA mutant into the pLVX-TRE3G-IRES vector, we gathered lentiviral supernatants from 293T cells expressing both regulator vector (pLVX-EF1a-Tet3G) and response vector (pLVX-TRE3G-IRES-HP1γ WT or pLVX-TRE3G-IRES-HP1γ AA). Then, SiHa cells were coinfected with the two viruses following G418 selection (500 μg/ml). After incubation with or without doxycycline (1 μg/ml) for additional 48 h, the cells were harvested for analyses.The Tet-on 3G doxycycline-inducible expression system  was used to promote the expression of Cells were transfected with siRNA targeting HP1α, HP1β, HP1γ, UBE2L3, or HPV16 E6 using Lipofectamine 2000 reagent (Life Technologies) according to the manufacturer’s protocol. The sequences of siRNAs are as follow:HP1α sense, 5′-GUUCCAGUCCUCUCUCAAAGC-3′HP1α antisense, 5′-GCUUUGAGAGAGGACUGGAAC-3′;HP1β sense, 5′-GACUCCAGUGGAGAGCUCAUG-3′HP1β antisense, 5′-CAUGAGCUCUCCACUGGAGUC-3′;HP1γ sense, 5′-AUUCUUCAGGCUCUGCCUC-3′HP1γ antisense, 5′-GAGGCAGAGCCUGAAGAAU-3′;UBE2L3 sense, 5′-UUUCUUUGUAAACUCUUCA-3′UBE2L3 antisense, 5′-UGAAGAGUUUACAAAGAAA-3′;HPV16 E6 sense, 5′-CCACAGUUAUGCACAGAGC-3′HPV16 E6 antisense, 5′-GCUCUGUGCAUAACUGUGG-3′.Formalin-fixed and paraffin-embedded tissue sections were acquired from normal people and HPV-positive precancer, endocervical adenocarcinoma, and invasive squamous cell carcinoma patients. The 4-μm sections were deparaffinized in xylene and rehydrated through a graded alcohol series to distilled water. The antigen retrieval was performed by microwave irradiation, and the primary antibody against HP1γ  was then applied. The specific binding was detected with biotinylated anti-mouse immunoglobulin, followed by peroxidase-labeled streptavidin with 3,3′-diaminobenzidine chromogen as substrate. Slides were counterstained with Harris hematoxylin. All protocols and procedures with human cervical tissues were approved by the Yonsei University Institutional Review Board (4-2017-0898).3VO4, 10 mM β-glycerophosphate, 0.3% CHAPS, and protease inhibitors). The lysates were centrifuged for 20 min at 13,000 rpm at 4 °C. The specific antibodies were incubated with the supernatants overnight at 4 °C, followed by incubation with anti-rabbit Ig-IP beads (Trueblot) for 1 h at 4 °C. The beads were spun-down for 1 min at 2000 rpm and washed three times with IP wash buffer (IP lysis buffer without CHAPS). The proteins were eluted by boiling for 5 min in Laemmli buffer (Bio-Rad) and subjected to immunoblotting.For immunoblotting, each sample was subjected to SDS-polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes using the semi-dry transfer (Bio-Rad). The membranes were incubated overnight with the indicated primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h (Abcam). The signals were detected using chemiluminescence reagents (Intron). For immunoprecipitation, the cells were lysed with IP lysis buffer (HEPES 40 mM (pH 7.4) containing 120 mM NaCl, 1 mM EDTA, 50 mM NaF, 1.5 mM Na2, 10 mM KCl, 1 mM EDTA, 1 mM DTT, 0.5 μg/ml leupeptin, 1 mM PMSF, 1 μM pepstatin A, and 0.05% NP-40), and cytoplasmic extracts were separated by centrifugation at 4 °C at 3000 rpm for 10 min. The remained pellet was resuspended in Buffer B  and incubated on ice for 30 min. Nuclear extracts were separated by centrifugation at 4 °C at 13,000 rpm for 20 min.For the fractionation of cytoplasmic and nuclear extracts, cells were suspended in buffer A (10 mM HEPES containing 1.5 mM MgCl3 cells per well of a six-well plate and cultured with doxycycline (0.5 μg/ml) for 10–20 days. For transiently expressing cells, HeLa or Siha cells were seeded in 2 × 103 cells per well of a six-well plate. Five days after, the cells were transfected with siRNA or DNA plasmids and the cells were grown 3–5 days more. Cells were fixed with glutaraldehyde (6%) and stained with 0.5% crystal violet. The images of stained colonies were visualized using a digital scanner.For the stable cells, SiHa cells expressing doxycycline-inducible HP1γ WT or AA were seeded in 1 × 106 stable SiHa cells expressing pLVX-EF1a-Tet3G (control), pLVX-TRE3G-IRES-HP1γ WT, or pLVX-TRE3G-IRES-HP1γ AA into each flank under 100 µL of saline/zoletil/rompun (7:1:1) anesthesia. Mice were randomized into groups (n = 8 per group), and treatment was started 14 days after tumor implantation. Doxycycline  was administered to tumor-bearing animals orally, two times a week for 3 weeks at a dose of 50 mg/kg. From palpable tumor formation until termination, tumor sizes were measured every 3–4 days using calipers, and tumor volume were calculated with the following formula: length × width2 × 0.5236. Mice were sacrificed in a 7.5% CO2 chamber, and tumors were harvested for immunohistochemistry and other analyses.All animal experiments were approved by the Institutional Review Board of the Yonsei University College of Medicine and were performed in specific pathogen-free facilities according to the university’s guidelines for the Care and Use of Laboratory Animals (2018–0155). Female Balb/c nude mice (5–7 weeks old) were inoculated subcutaneously with 3 × 10p value of <0.05 was considered statistically significant and termed differentially expressed genes.RNA labeling and hybridization were performed by using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol . Briefly, 200 ng of total RNA from each sample was linearly amplified and labeled with Cy3-dCTP. The labeled cRNAs were purified by the RNAeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 (NanoDrop). A total of 600 ng of each labeled cRNA was fragmented by adding 5 μl 10× blocking agent and 1 μl of 25× fragmentation buffer, and then heated at 60 °C for 30 min. Finally, 25 μl of 2× GE hybridization buffer was added to dilute the labeled cRNA. Fifty microliters of hybridization solution was dispensed into the gasket slide and assembled to the SurePrint G3 Human Microarrays, 8 × 60 K . The slides were incubated for 17 h at 65 °C in an Agilent hybridization oven and then washed at room temperature by using the Agilent One-Color Microarray-Based Gene Expression Analysis protocol . The hybridized array was immediately scanned with an Agilent Microarray Scanner . The cDNA microarray experiments (one-channel method) were repeated twice and the average of two gene expression values for each gene was used for further analysis. Statistical significance of the expression data was determined using LPE  test and fold change in which the null hypothesis was that no difference exists among 2 groups. For statistical analysis, the LPE test was used for multiple group comparisons across all samples in the experiment by MeV (MultiExperiment Viewer) Software. A GAPDH mRNA for each reaction. The qPCR primer sequences used are as follow:Total RNA was extracted using Easy-Blue reagent . Then 1 μg of total RNA was reverse transcribed into cDNA using a Reverse Transcription kit . Quantitative real-time PCR was performed using KAPATM SYBR FAST qPCR (KAPABIOSYSTEMS) with a CFX96TM or Chromo4TM real-time PCR detector (Bio-Rad). Relative levels of mRNA were normalized to the values of GAPDH forward, 5′-GAGTCAACGGATTTGGTCGT-3′;GAPDH reverse, 5′-TTGATTTTGGAGGGATCTCG-3′;TP53 forward, 5′-GAGGGATGTTTGGGAGATGTAAGAAATG-3′;TP53 reverse, 5′-TTCACAGATATGGGCCTTGAAGTTAGAGAA-3′;UBE2L3 forward, 5′-TTGACCCTT TGTAGGATTGGAATT-3′;UBE2L3 reverse, 5′-CGACCCCAGACTGGTGCTT-3′;Bax forward, 5′-TCTACTTTGCCAGCAAACTGG-3′;Bax reverse, 5′-TGTCCAGCCCATGATG GTTCT-3′;Noxa forward, 5′-AGAGCTGGAAGTCGAGTGT-3′;Noxa reverse, 5′-GCACCT TCACATTCCTCTC-3′;Puma forward, 5′-GACCTCAACGCACAGTA-3′;Puma reverse, 5′-CTAATTGGGCTCCATCT-3′;TP53AIP1 forward, 5′-TCTTCCTCTGAGGCGAGCT-3′;TP53AIP1 reverse, 5′-AGGTGTGTGTGTCTGAGCCC-3′;14-3-3σ forward, 5′-TTTCCTCT CCAGACTGACAAACTGTT-3′;14-3-3σ reverse, 5′-TAGAACTGAGCTGCAGCTGTAAA -3′;Gadd45α forward, 5′-TGCGAGAACGACATCAACAT-3′;Gadd45α reverse, 5′-TCCCG GCAAAAACAAATAAG-3′;p21 forward, 5′-CACCGAGACACCACTGGAGG-3′;p21 reverse, 5′-GAGAAGATCAGCCGGCGTTT-3′;Tp53inp1 forward, 5′-TGTTGCAGCTCTT GCTGCTCA-3′;Tp53inp1 reverse, 5′-GCTGATGAACAACCCAGCCAT-3′;HPV16 E6 forward, 5′-GAGTCAACGGATTTGGTCGT-3′;HPV16 E6 reverse, 5′-CATAAATCCCGAAAAGCAAAG-3′.Total ChIP was performed as previously described . In brieBax forward, 5′-TAATCCCAGCGCTTTGGAAG-3′;Bax reverse, 5′-TGCAGAGACCTGGATCTAGCA-3′;Noxa forward, 5′-CCTGGCCCCACCCCACCCCAC-3′;Noxa reverse, 5′-TCAGGGCTATTTTACGCTCTC-3′;14-3-3σ forward, 5′-TTTCCTCTCCAGACTGACAAA-3′;14-3-3σ reverse, 5′-TAGAACTGAGCTGCAGCTGTA-3′;Gadd45α forward, 5′-AGCGGAAGAGATCCCTGTGA-3′;Gadd45α reverse, 5′-CGGGAGGCAGGCAGATG-3′.2) for 15 min. Afterward, PI  was added and incubated for another 15 min, then applied to a flow cytometer for data collection.Apoptosis of cervical cancer cells was assessed using Annexin V-PI double staining as previously described . Cells whttps://gdac.broadinstitute.org. A total of 61 patients with available clinical information was analyzed for overall survival and disease-free survival. Kaplan–Meier survival curves and log-rank statistics were employed to evaluate time to tumor recurrence and overall survival. Statistical significance was when p < 0.05.The normalized level 3 TCGA RNA sequencing data of cervical cancer were retrieved from t test (two-tailed) and Pearson’s correlation test. Statistical differences were determined based on a P value .Statistical significance was analyzed using Student’s CBX3 (a gene encoding HP1γ)  project. HPA analysis showed cervical cancer-specific cytoplasmic localization of HP1γ, whereas no distinctive cancer type-specific localization of HP1α and HP1β was observed  and -positive (SiHa) cells Fig. , implyinXPO1) is involved in high-risk HPV-mediated elevation of the nuclear export system [XPO1 in cervical cancer cell lines and cervical cancer tissues, compared with normal cervix epithelium tissues , whereas the expression of UBE2L3 and CBX1 (a gene encoding HP1β) showed a positive correlation  levels . Although E6 expression in U2OS cells lowered the level of HP1α and HP1β while upregulating HP1γ expression  to alanine -dependent p53 degradation through the transcriptional repression of UBE2L3 while increasing overall p53 protein levels and restoring p53 function (Fig. During the development of cervical cancer, oncogenic HPV E6 plays a central role by disabling the tumor-suppressing function of p53 . In thision Fig. . IntriguAnother important issue we found here is the novel role of high-risk HPV E6 in cervical cancer progression through the exportin-1-mediated nuclear export of HP1γ. Although a few papers have proposed the presence of HP1γ in the cytoplasm during maternal to embryonic transition  or in myFigure S1Figure S2Figure S3Figure S4Figure S5Figure S6Figure S7Figure S8Figure S9Figure S10Figure S11Figure S12Figure S13Figure S14Table S1-S7


Goniothalamus (Annonaceae), have demonstrated in vitro antitumor activity. However, the aqueous solubility of these compounds is poor. In this study, we identified the absolute configurations of the previously isolated compounds, which were first isolated in our laboratory, by single-crystal X-ray diffraction analysis using Cu Kα radiation. Subsequently, the antitumor activities of the compounds were evaluated by 3--2,5-diphenyl-tetrazolium bromide staining in four tumor cell lines. The induced apoptosis activity of leiocarpin E-7ʹ-Monoacetate was studied by an annexin V fluorescein isothiocyanate/propidium iodide double-staining experiment, and the caspase activity was tested in the SW1116 cell line. The results demonstrated that the antitumor activities of cheliensisin A and goniodiol-7-monoacetate were limited by their poor water solubility. To address this issue, hydroxypropyl-β-cyclodextrin (HP-β-CD) complexes of the compounds were synthesized by the saturated aqueous method. The complexes were then analyzed using a differential scanning calorimeter. The IC50 of cheliensisin A was reduced by 45% and 58% against SW1116 and SMMC-7721 cell lines, respectively. Similarly, the IC50 of goniodiol-7-monoacetate was reduced by 55% and 34% against the two tumor cell lines, respectively. To further evaluate whether the styryllactones and complexes possessed selectivity against cancer cell lines and normal cell lines, toxicity against human normal cell line (HEK293T) was evaluated. The results demonstrated that the HP-β-CD complexes displayed more cytotoxicity than the respective pristine compounds against the HEK293T cell line. However, there existed a therapeutic window when the complexes were applied against cancer cell lines. In summary, the synthesis of several styryllactone compounds complexed with HP-β-CD was reported for the first time. These complexes could significantly enhance the cytotoxic effects of styryllactone compounds.Styryllactones, a class of compounds obtained from the genus  Colon cancer is the third most common type of cancer worldwide in both men and women, and is associated with a high recurrence rate and increasing mortality rate . The exiGoniothalamus belonging to the Annonaceae family, which are mostly indigenous in southeast Asia. Styryllactones are classified based on different structural skeletons as follows  . Vendramc effect .BIRC5 gene at the RNA level, thus subjecting NCI-H460 cells to DNA damage -associated X protein (Bax)/Bcl-2, phosphorylate c-Jun N-terminal kinase (p-JNK1)/JNK1, and p-p38/p38 ratios, which led to cleavage of poly (ADP-ribose) polymerase (PARP) and, finally resulted in apoptosis of the HT-29 cells . The celA damage .Goniothalamus griffithii (Annonaceae) and Goniothalamus leiocarpus (Annonaceae)  . Their ronaceae) .β-CD on their antitumor activity. The styryllactones displayed enhanced antitumor activity when complexd with HP-β-CD.In this study, the absolute configurations of several styryllactone compounds, first isolated in our laboratory, were determined by single-crystal X-ray diffraction analysis using Cu Kα radiation. In addition, we evaluated the effect of complexation of sytryllactones with HP-β-cyclodextrin (HP-β-CD) was obtained from Nihon Shoukuhin Kako Co. Ltd. . Phosphate buffer saline (PBS), annexin V fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit, and caspase activity kit were purchased from Keygentec . The water used in the experiments was obtained from the Milli-Q Water Purification System .Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute 1640 (RPMI-1640) medium, minimum Eagle's medium (MEM), fetal bovine serum (FBS), and 0.25% trypsin–ethylenediaminetetraacetic acid (EDTA) were obtained from Life Technologies INC. . Trypan blue, penicillin, streptomycin, dimethyl sulfoxide (DMSO), VP-16 (Etoposide) and taxol were supplied by Sigma Chemical Co. . 3--2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Molecular Probes Inc. . Hydroxypopyl-via direct methods using SHELXS-97 . Non–hydrogen atoms were anisotropically refined with SHELXL-97 . Hydrogen atoms were located by geometry and positioned on the related atoms during refinements, with a temperature factor.Data for diffraction intensity was obtained using a Bruker APEX-IICCD X-ray diffractometer  and graphite-monochromated Cu Kα radiation (λ= 1.54178 Å). Cell refinement and data reduction were performed with Bruker SAINT . The absorption correction was determined semi-empirically from equivalent compounds. The structures were determined 2 at 37°C using a CO2 incubator . Cell count was assessed using the trypan blue dye exclusion method.The human colon cancer SW1116 cell line, the human hepatocellular carcinoma SMMC-7721 cell line, the human gastric cancer SGC-7901 cell line, and the human hepatocellular carcinoma HepG2 cell line were kindly provided by Xiao-li Xu from the Cancer Center, Fudan University. The human embryonic kidney 293T (HEK293T) cell line was kindly donated by Professor You-hua Xie from Fudan University. These human cancer cell lines were cultured in DMEM medium or RPMI-1640 medium, whereas the HEK293T cell line was cultured in MEM medium. All of the cell lines were supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 mg/ml) under a humidified atmosphere of 5% CO3 cells/well in 96-well plates . After attachment, the culture media were replaced with various concentrations of chemicals for 24 h. Then, the media in 96-well plates were incubated with MTT reagent (5 mg/ml) for 4 h at 37°C. Subsequently, the culture medium was discarded and 100 μl of DMSO was added to each well, to solubilize the formazan crystal formed. The absorbance of formazan crystal solution was determined at 570 nm on a Multiskan FC from Thermo Fisher Scientific Inc. .The antiproliferative effects of the treatments were evaluated using the MTT assay. Cells were seeded at a density of 5×102 at 37°C. After 36 h of incubation, the culture medium was discarded, and the cells were collected. For the apoptosis analysis, cells were suspended with 1×binding buffer (1×106 cells/ml) and then labeled with annexin V-FITC/PI, as per the manufacturer's instructions . The analysis of the samples was performed by flow cytometry , and the acquired data was analyzed by the CellQuest software .The growing cells were incubated in 24-well microplates  for 24 h. The cells were then treated with various concentrations of leiocarpin E-7′-monoacetate or taxol in humidified air with 5% CO2 at 37°C. After 8 h of incubation, the culture medium was discarded, the cells collected and washed twice with PBS. The mixture was then centrifuged at 2,000 rpm for 5 min. The PBS supernatant was discarded and the cells  were collected. To these cells, ice-cold lysis buffer (150~200 μl) was added. The mixture was placed on ice for 30 min, and then centrifuged  at 4°C. The supernatant, containing lysed protein, was carefully aspirated and transferred to a new tube. The protein concentration was then measured in 2 μl of the supernatant using the Bradford method. The caspase assays were then performed in 96-well microtiter plates  by incubating 10 μl of protein cell lysate per sample in 80 μl of reaction buffer , 137 mM NaCl, and 10% glycerol) containing 10 μl of caspase substrate (2 mM). Lysates were incubated at 37°C for 4 h. Measuremet was done at 405 nm on a Multiskan FC from Thermo Fisher Scientific Inc. . The detailed analysis procedure is described in the manufacturer's protocol .To evaluate the activity of caspases, cell lysates were prepared after their respective designated treatments. The incubation of the growing cells was carried out in 24-well microplates  for 24 h. The cells were then treated with different concentrations of leiocarpin E-7′-monoacetate under humidified air with 5% COβ-CD (molecular ratio of 1:2). The dispersion was equilibrated for 24 h at room temperature, under constant stirring. The supernatant was then lyophilized using a Christ Alpha1-2 Ld10 Freeze Dryer  to obtain the inclusion complex in a dry powder form. The content of styryllactones in the complex was determined using an ultraviolet (UV) spectrophotometer . When the complexes were used in the antitumor test, we first prepared a solution of the compound, and then performed full wavelength scanning. Subsequently, we selected the maximum absorption wavelength of the compound as the detection wavelength. A 20-mg quantity of the complex was accurately weighed and placed in a 10-ml volumetric flask. It was then dissolved in 0.1 mol/L hydrochloric acid-acetonitrile-water (1:1:2), and the volume was recorded. The absorbance was measured at the detection wavelength. We calculated the total amount of compound (W1) in the complex, according to the standard equation obtained with a compound solution prepared with 0.1 mol/L hydrochloric acid-acetonitrile-water (1:1:2). Another 20 mg of the same complex was accurately weighed, and dissolved in 0.1 mol/L hydrochloric acid-acetonitrile (1:1) and placed in an ultrasound machine for 10 min. Subsequently, it was filtered and the filtrate was used to measure the absorbance at the detection wavelength. We calculated the free compound content (W2), according to the standard equation obtained with a compound solution prepared with 0.1 mol/L hydrochloric acid-acetonitrile (1:1). The difference between W1 and W2 represented the quantity of the compound that formed HP-β-CD complex in a 20-mg inclusion compound sample.A fixed quantity of the compound was weighed and evenly dispersed in an aqueous solution of HP-β-CD, the physical mixtures, and the complexes were determined using a differential scanning calorimeter , equipped with a computerized data station . Samples were accurately weighed in a crimped aluminum pan and heated under an inert atmosphere of nitrogen. An empty pan sealed in the same manner, was used as a reference. The scanning rate was 10°C/min, and the scanning temperature ranged between 30°C and 400°C.The thermal characteristics of the raw material, HP-t-tests. Statistical analyses were performed using SPSS 16.0 . A value of p < 0.05 was considered statistically significant.All data were expressed as means ± standard error of the mean (SEM) and were analyzed using two-tailed Student's R,3S)-6-oxo-2--3-phenyloxiran-2-yl)-3,6-dihydro-2H-pyran-3-yl acetate, and the absolute configuration of cheliensisin A was defined as 5S, 6S, 7S, 8R. The structure of leiocarpin B was established as (S)-5-hydroxy-7--2-hydroxy-2-((R)-6-oxo-3,6-dihydro-2H-pyran-2-yl)-1-phenylethoxy)-2-phenylchroman-4-one, and the absolute configuration of leiocarpin B was defined as 2′S, 6R, 7S, 8R. The structure of leiocarpin E was established as (R)-6-((R)-hydroxy-3-((R)-6-oxo-3,6-dihydro-2H-pyran-2-yl)-4-phenylisochroman-1-yl)methyl)-5,6-dihydro-2H-pyran-2-one, and the absolute configuration of leiocarpa E was defined as 6R, 6′R, 7S, 7′S, 8S, 8′S. Both goniodiol and goniodiol-7-monoacetate were identified as 6R, 7R, 8R. The structure of goniodiol was established as (R)-6--1,2-dihydroxy-2-phenylethyl)-5,6-dihydro-2H-pyran-2-one. The absolute configuration of leiocarpin E-7′-monoacetate, which is the acetate of leiocarpin E, was consistent with that of leiocarpin E.Single-crystal X-ray diffraction analysis, using Cu Kα radiation, was used to identify the absolute configurations of styryllactone compounds (15H14O5) were as follows  Å, b = 11.0918 (10) Å, c = 17.5287 (3) Å; α= 90°, β= 90°, γ = 90°; V = 1366.75 (3) Å3, T = 123 (2) K; Z = 4; Dcalc = 1.333 g·cm−3; index range: −8 ≤ h ≤ 8, −13 ≤ k ≤ 13, −18 ≤ l ≤ 20; absorption coefficient = 0.842 mm−1; completeness: 99.9%; F(0 0 0) = 576; GOF (goodness of fit) = 1.068. A colorless prismatic crystal with approximate dimensions of 0.21 mm × 0.15 mm × 0.14 mm was chosen and mounted on a Bruker APEX-II CCD diffractometer. The θ range for data collection was 4.72°–67.48°. A total of 8,080 reflections were collected, of which 2,435 were unique (R(int) = 0.0300) and 2,418 were considered observed (I> 2σ(I)). The maximum and minimum transmissions were 0.7456 and 0.3475. The refinement method was full-matrix least squares on F2. Data/restraints/parameters were 2435/0/183. The final R values were R1 = 0.0272 and wR2 = 0.0670 for 2,418 observed reflections, and R1 = 0.0274 and wR2 = 0.0673 for all observations. A full list of crystallographic data was deposited at the Cambridge Crystallographic Data Center, CCDC 1007949.The crystal data of cheliensisin A (C28H24O7) were as follows  Å, b = 7.9013 (10) Å, c = 16.4209 (3) Å; α = 90°, β = 121.2160 (10)°, γ = 90°; V = 2335.75 (7) Å3, T = 140 (2) K; Z = 4; Dcalc = 1.344 g·cm−3; index range: −25 ≤ h ≤ 25, −9 ≤ k ≤ 9, −19 ≤ l ≤ 19; absorption coefficient = 0.798 mm−1; completeness: 98.2%; F(0 0 0) = 992; GOF (goodness of fit) = 1.079. A colorless block crystal, with approximate dimensions of 0.35 mm × 0.26 mm × 0.20 mm, was chosen and mounted on a Bruker APEX-II CCD diffractometer. The θ range for data collection was 3.15°–69.15°. A total of 6,301 reflections were collected, of which 3,373 were unique (R(int) = 0.0432) and 3,343 were considered observed (I> 2σ(I)). The maximum and minimum transmissions were 0.7532 and 0.4386. The refinement method was full-matrix least squares on F2. Data/restraints/parameters were 3373/1/319. The final R values were R1 = 0.0419 and wR2 = 0.1082 for 3,343 observed reflections, and R1 = 0.0421 and wR2 = 0.1089 for all observations. A full list of crystallographic data was deposited at the Cambridge Crystallographic Data Center, CCDC 1008036.The crystal data of leiocarpin B (C26H24O6) were as follows  Å, b = 11.1611 (2) Å, c = 20.3823 (3) Å; α = 90°, β = 90°, γ = 90°; V = 2215.29 (6) Å3, T = 123 (2) K; Z = 4; Dcalc = 1.297 g·cm−3; index range: −11 ≤ h ≤ 11, −13 ≤ k ≤ 13, −24 ≤ l ≤ 24; absorption coefficient = 0.754 mm−1; completeness: 99.5%; F(0 0 0) = 912; GOF (goodness of fit) = 1.058. A colorless prismatic crystal, with approximate dimensions of 0.19 mm × 0.15 mm × 0.12 mm, was chosen and mounted on a Bruker APEX-II CCD diffractometer. The θ range for data collection was 4.34°–65.50°. A total of 12,242 reflections were collected, of which 3,746 were unique (R(int) = 0.0432) and 3,700 were considered observed (I> 2σ(I)). The maximum and minimum transmissions were 0.9129 and 0.8706. The refinement method was full-matrix least squares on F2. Data/restraints/parameters were 3746/0/291. The final R values were R1 = 0.0426 and wR2 = 0.1191 for 3,700 observed reflections, and R1 = 0.0430 and wR2 = 0.1195 for all observations. A full list of crystallographic data was deposited at the Cambridge Crystallographic Data Center, CCDC 1007951.The crystal data of leiocarpin E (C13H14O4) were as follows  Å, b = 9.7650 (2) Å, c = 13.0267 (3) Å; α = 90°, β = 90°, γ = 90°; V = 1175.93 (4) Å3; T = 140 (2) K; Z = 4; Dcalc = 1.323 g·cm−3; index ranges: −10 ≤ h ≤ 10, −10 ≤k ≤ 11, −14 ≤ l ≤ 15; absorption coefficient = 0.814 mm−1; completeness: 98.1%; F(0 0 0) = 496; GOF (goodness of fit) = 1.141. A colorless block crystal, with approximate dimensions of 0.35 mm × 0.26 mm × 0.22 mm, was chosen and mounted on a Bruker APEX-II CCD diffractometer. The θ range for data collection was 5.66°–69.43°. A total of 5,322 reflections were collected, of which 2,070 were unique (R(int) = 0.0369) and 2,058 were considered observed (I> 2σ(I)). The maximum and minimum transmissions were 0.7532 and 0.5891. The refinement method was full-matrix least squares on F2. Data/restraints/parameters were 2070/0/157. The final R values were R1 = 0.0404 and wR2 = 0.1007 for 2,058 observed reflections, and R1 = 0.0405 and wR2 = 0.1008 for all observations. A full list of crystallographic data was deposited at the Cambridge Crystallographic Data Center, CCDC 1008511.The crystal data of goniodiol (C15H16O5) were as follows  Å, b = 8.8394 (7) Å, c = 15.3120 (13) Å; α = 94.379 (6)°, β = 91.949 (5)°, γ = 105.106 (6)°; V = 709.58 (11) Å3; T = 296 (2) K; Z = 2; Dcalc = 1.293 g·cm−3; index ranges: −6 ≤ h ≤ 6, −10 ≤ k ≤ 10, −18 ≤ l ≤ 15; absorption coefficient = 0.811 mm−1; completeness: 94.0%; F(0 0 0) = 292; GOF (goodness of fit) = 1.042. A colorless block crystal, with approximate dimensions of 0.2 mm × 0.12 mm × 0.05 mm, was chosen and mounted on a Bruker APEX-II CCD diffractometer. The θ range for data collection was 2.90°–69.66°. In total, 5,692 reflections were collected, of which 3,307 were unique (R(int) = 0.0419) and 3,046 were considered observed (I> 2σ(I)). The maximum and minimum transmissions were 0.7532 and 0.4727. The refinement method was full-matrix least squares on F2. Data/restraints/parameters were 3307/3/365. The final R values were R1 = 0.0468 and wR2 = 0.1251 for 3,046 observed reflections, and R1 = 0.0505 and wR2 = 0.1309 for all observations. A full list of crystallographic data was deposited at the Cambridge Crystallographic Data Center, CCDC 1442700.The crystal data of goniodiol-7-monoacetate  than the normal group (cell viability in 0 μM). The highest cytotoxic effect against SW1116 cells was demonstrated by leiocarpin E-7′-monoacetate, while cheliensisin A and goniodiol-7-monoacetate showed relatively lower inhibitory effect (The TT assay . The resin vitro cytotoxic effect of styryllactones (concentration ranging from 0 and 100 μM) against HEK293T cells  cell line is used as a normal human cell line in various biological experiments . In thisin vitro cytotoxicity experiments, SW1116 cells were found to be sensitive to leiocarpin E-7′-monoacetate. To further evaluate the apoptotic activity of leiocarpin E-7′-monoacetate, the cells were treated with various concentrations of leiocarpin E-7′-monoacetate and then analyzed by flow cytometry. Taxol was chosen as the positive control, because it could stabilize microtubules, and subsequently cause cell apoptosis by arresting the cell cycle at G2/M , while HP-β-CD did not show any inhibition of the growth of the two cell lines . For example, the IC50 of cheliensisin A complex and goniodiol-7-monoacetate complex were 98.46 ± 3.00 and 35.02 ± 0.63 μM, respectively  and Goniothalamus leiocarpus (Annonaceae) (Styryllactones are a series of secondary metabolites isolated from the genus one ring . In our onaceae) , and theonaceae) . In this50 values of 13.1, 23.7, and 60.2 μM, respectively , HepG2 (liver cancer), and MCF7 (breast carcinoma) cell lines with ICectively . Goniothectively , HL-60 cectively , HGC-27 ectively , HT-29 cectively . Among tectively , and casectively . Howeverectively , HepG2 cectively , SW1116 ectively  and SGC-ectively  were allIt was found that leiocarpin B, leiocarpin E, and leiocarpin E-7'-monoacetate had significant cytotoxic activities against human colon cancer SW1116 cell line. Further, the results of Annexin V-FITC/PI double staining showed that leiocarpin E-7'-monoacetate induced apoptosis of SW1116 cells in a concentration-dependent manner  is a suitable thermal analysis technique used in the pharmaceutical industry for determining the purity, polymorphic forms, and melting point of a sample . In addiobserved . HP-β-CDn routes . For exalexation . As a reβ-CD was significantly enhanced. From the experiments, the IC50 of the cheliensisin A complex was 51.44 ± 4.78 μM and 46.91 ± 1.60 μM against SW1116 cells and SMMC-7721 cells, respectively. The IC50 of goniodiol-7-monoacetate complex was 46.48 ± 3.25 μM and 75.76 ± 4.19 μM, whereas the IC50 of the cheliensisin A monomer was 93.18 ± 0.78 μM and 110.90 ± 2.41 μM against SW1116 cells and SMMC-7721 cells, respectively. The IC50 of the goniodiol-7-monoacetate monomer was 102.14 ± 4.15 μM and 115.54 ± 3.97 μM, respectively (β-CD had no inhibitory effect on the growth of the two cell lines (β-CD complexes showed greater cytotoxic activity than the styryllactone compounds against HEK293T cell line. This suggested that the complexes could also inhibit the growth of normal cell line. The IC50 of cheliensisin A complex and goniodiol-7-monoacetate complex were 98.46 ± 3.00 and 35.02 ± 0.63 μM, respectively (β-CD is known to increase the solubility of poorly water-soluble compounds (β-CD complex will be developed and cytotoxicity will be studied. Subsequently, we intend to investigate the anticancer effect in the animal model using the styryllactones inclusion complex, and conduct efficient test methods including radiolabeling test and permeability test, to illustrate the exact mechanism underlying the improved potency of complexes toward tumor cell lines.It was observed that the antitumor effect of styryllactones complexed with HP-ompounds . Thus, iβ-CD for the first time. The in vitro antitumor experiments showed that the inhibitory activity of these complexes was greater than the respective pristine form of the compounds. These results suggest that the water solubility of styryllactones can be improved by complexation of styryllactones with HP-β-CD, which in turn, can enhance the antitumor activity of these compounds.This paper described the identification of the absolute configurations of several styryllactones by single-crystal X-ray diffraction analysis using Cu Kα radiation. We synthesized styryllactones complexes with HP-All datasets generated for this study are included in the article/RM and QM contributed to the conception and design of the study. RM performed the experiments and wrote the first draft of the manuscript. J-TC assisted in the performance of experiments. X-LX donated the cells in the research. QM and X-YJ revised the manuscript. QM gave final approval of the version to be submitted. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Sepsis is a complex syndrome characterized by a dysregulated inflammatory response to systemic infection and leads to shock, multiple organ failure and death especially if not recognized early and treated promptly. Previous studies have suggested Maresin 1 (MAR1) can alleviate systemic inflammation in sepsis, but its mechanism has not been clarified.RAW 264.7 cells and human primary peripheral blood mononuclear cells (hPBMCs) were pretreated with LPS and MAR1. The mRNA expression and supernatant levels of pro-inflammatory cytokines, tumor necrosis factor (TNF-α), interleukin (IL)-1β and IL-6 were evaluated by RT-qPCR and ELISA, respectively. The expression levels of Sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), and Peroxisome proliferator-activated receptor gamma (PPAR-γ) were determined by RT-qPCR and Western blot analysis, respectively.Our results show that LPS-induced inflammation increased the expression and secretion of proinflammatory cytokines TNF-α, IL-1β and IL-6 and induced suppression of SIRT1, PGC-1α, and PPAR-γ expression, which could be reversed by MAR1. And the effect of MAR1 was eliminated by repression of SIRT1/PPAR-γ and enhanced by PGC-1α overexpression.MAR1 suppressed inflammatory response in LPS-induced RAW 264.7 macrophages and hPBMCs via the SIRT1/PGC-1α/PPAR-γ pathway. Severe sepsis with multiple organ dysfunctions is still the main cause of death in intensive care unit patients , 2. Durig sepsis , such asg sepsis , 10.Specialized pro-resolving mediators such as resolvins, protectins, and maresins, actively turn off the inflammatory response by acting on different G protein-coupled receptors expressed on immune cells that activate the dual anti-inflammatory process . Maresinin vitro sepsis models of LPS-induced RAW 264.7 cells and human primary peripheral blood mononuclear cells (hPBMCs).In our current study, the relationship between reduction of inflammatory response by MAR1 and SIRT1/PGC-1α/PPAR-γ signaling axis was systematically investigated and intended to explain the specific mechanism of MAR1 in 2 at 37 °C. For establishment of in vitro model, lipopolysaccharide  was used to treat the cells for 24 h. For treatment of MAR1, cells were treated with different concentrations  of MAR1  for 12 h before LPS treatment. For analysis of time effects, cells were treated with 10 nM MAR1 with different time duration .The mouse mononuclear macrophage leukemia cell line RAW264.7 was purchased from the American Type Culture Collection (ATCC), and primary human peripheral blood mononuclear cells (hPBMCs) was purchased from PriCells . Cells were cultured in DMEM containing 10 % fetal bovine serum  and 1 % penicillin (100 U/mL)/streptomycin (100 µg/mL)  in a 37℃ in an incubator containing 5 % CO−ΔΔCt method. All experiments were repeated in triplicate.Total RNA was extracted from cells using TRIzol reagent (Invitrogen). After total RNA was reverse transcribed into cDNA by using a Prime-Script™ One Step RT-qPCR kit , PCR reactions for SIRT1, PGC-1α, PPAR-γ, IL-1β, IL-6 and TNF-α mRNA were performed on a LightCycler 480  system with GAPDH used as an internal control. The light cycler DNA master SYBR green I kit  was then used for PCR experiments. The following primers were used: SIRT1 Forward: 5′-GGTGTTAAATACCAAACTGC-3′ and reverse: 5′-AGGAGTGATGTTCAAAATG-3′; PGC-1α Forward: 5’-AATTCACAATCACAGGATCAGAACA3’ and reverse: 5’-ACTTAAGGTGCGTTCAATAGTCTT-3’; PPAR-γ Forward: 5’-TTGGCCATATTTATAGCTGTCATTATT-3′ and reverse: 5’- TGTCCTCGATGGGCTTCA-3’; TNF-α Forward: 5’-GAGCTGTGGGGAGAACAAAAGGA-3′ and reverse: 5’- TTGGCCCTTGAAGAGGACCTG-3’; IL-1β Forward: 5’-GAC CTT CCA GGA TGA GGA CA-3′ and reverse: 5’-AGC TCATATGGGTCCGACAG-3’; IL-6 Forward: 5’-TCC AGT TGC CTTCTT GGG AC-3′ and reverse: 5’-GTGTAATTAAGCCTCCGACTTG-3’; GAPDH Forward: 5’-AGAAGGCTGGGGCTCATTTG-3’ and reverse: 5’-AGGGGCCATCCACAGTCTTC-3. The relative expression of the target genes was calculated using the 2The contents of IL-1β, IL-6 and TNF-α in the cell supernatant were determined using an ELISA assay. Centrifuge at 4000 r/min for 15 min at 4 °C to collect the supernatant. ELISA kits were obtained from Boster , and each sample was measured three times according to the manufacturer’s instructions.Cells were washed in phosphate-buffered saline (PBS), then in 10 mM TrisHCl (pH7.5), 100 mM NaCl, 1 % NP-40, 50 mM NaF, 2 mMEDTA (pH8.0), 1 mM PMSF, Cells were lysed in 10 µg/mL leupeptin, and 10 µg/mL aprotinin. The mixture was collected as a whole cell extract. The protein extract was separated by sodium lauryl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane . The membrane was blocked with Tris buffered saline and 3 % bovine serum albumin (BSA) in Tween 20  for 1.5 h, and then specific primary antibodies with SIRT1, PGC-1α and PPAR-γ treated at 4 °C overnight. GAPDH was used as a protein-loading control. All antibodies were purchased from Proteintech  The membrane was then treated with horseradish peroxidase (HRP) -conjugated secondary antibody  for 2 h. The bands were visualized using an enhanced chemiluminescence system  and LAS image software .5) were seeded into 6-well plates for cell transfection after cell confluence of 40–60 %. Replace with new medium and prepare cell transfection buffer. Incubate 5 µL/Wells of lipofectamine 2000  and 250 µL of Opti-MEM medium  for 5 min at room temperature and mix with PGC-1α, sh-Sirt1 vector, sh-PPAR-γ vector and negative control (5 µL/well) 250 µL of Opti-MEM medium for 20 min. Then, inoculate the cells into a 6-well plate and incubate in the incubator for 6 h. Then, the cells are incubated in 10 % FBS continuous culture in new DMEM medium .GenePharma  synthesized PGC-1α overexpression, sh-Sirt1 vector, sh-PPAR-γ vector and corresponding negative controls. Cells . The data were expressed as mean ± SD. with either unpaired two-tailed t-test or one-way ANOVA as appropriate. p < 0.001)  . The changes of inflammatory factors in LPS-treated RAW264.7 cells and hPBMCs at different intervention time  of MAR1 were analyzed by RT-qPCR. And we demonstrated that MAR1 inhibited the LPS-induced increase of the mRNA levels of proinflammatory factors in both RAW264.7 cells and hPBMCs with the treatment time  for 12 h after sh-PPAR-γ and control vectors were stalely transfected. The results of RT-qPCR showed that the mRNA expression of PPAR-γ was markedly suppressed following transfection with the shRNA-containing vector Fig. a. Both mP < 0.001), the effect of MAR1 can be counteracted by SIRT1 knockdown and further enhanced by PGC-1α overexpression , the effect of MAR1 can be abolished by SIRT1 knockdown and further augmented by PGC-1α overexpression , while MAR1 could restore their expression, and the effect of MAR1 can be counteracted by SIRT1 knockdown and further enhanced by PGC-1α overexpression (Fig. The expression of TNF-α, IL-1β, and IL-6 induced by LPS were significantly reduced after treatment with MAR1 (ion Fig. a. The chion Fig. b. Additiion Fig.  c and d.in vitro inflammation model can more closely study sepsis and LPS-treated macrophages were adopted as an in vitro model of endotoxin-induced inflammation during sepsis [More and more studies have proved that using LPS-stimulated cells to establish an g sepsis , 29, 30.g sepsis . HoweverSepsis is considered a serious disease with multiple organ damage caused by uncontrollable inflammation in severe infections, while MAR1 plays a role in reducing the bacterial burden and mitigating excessive inflammatory response and increases the survival rate of sepsis mice . The stuThere is evidence that increased expression of SIRT1 can reduce multiple organ damage, including lung, kidney, and liver, caused by sepsis , 37. Intin vitro study, thus the in vivo effects of MAR1 on sepsis as well as its clinical effects are not clear. More researches are still necessary to confirm the in vivo and potential clinical application of MAR1 in sepsis treatment.The present study also has some limitations. It is an in vitro sepsis model and provides clues for further exploring the mechanism of MAR1 in reducing inflammation in sepsis. Altogether, we propose that MAR1 could not only be a potential biomarker but also a novel therapeutic target for sepsis.In conclusion, this study demonstrates that the SIRT1/PGC-1α/PPAR-γ axis plays an important role in the process of MAR1 in reducing LPS-induced inflammation in


N-tosylated secondary vinylamine was obtained by alkaline treatment of 1,5-bis(1-phenoxy)-3-azapentane at moderate heating. Theoretical predictions suggest that the title compound could inhibit gluconate 2-dehydrogenase (85% probability), as well as to act as a mucomembranous protector (73%).The title  21H23NO4S, obtained by alkaline treatment of 1,5-bis(1-phenoxy)-3-azapentane at moderate heating, is a N-tosylated secondary vinylamine. An intramolecular S=O⋯H—C hydrogen bond generates a 13-membered ring. The benzalacetone moiety adopts a trans conformation with respect to the C=C double bond, which is slightly longer than usual due to the conjugation with a neighbouring acetyl group. Theoretical predictions of potential biological activities were performed, suggesting that the title compound can inhibit gluconate 2-dehydrogenase (85% probability), as well as to act as a mucomembranous protector (73%).The title compound, C The amine N atom is significantly flattened due to conjugation with a vinyl group, the C1—S1—N1—C13 torsion angle being 28.46 (13)°. The N1—C13 bond distance [1.4138 (13) Å] is slightly shorter than that of a standard C—N single bond in similar compounds  macrocycle in the crystal.The title compound is the product of an unexpected transformation starting from 1,5-bis(1-phenoxy)-3-azapentane. Its molecular structure is presented in Fig. 1d Table 1, leadinget al., 2019et al., 2020In the crystal, the molecules of the title enamine are linked by pairs of intermolecular C—H⋯O contacts into chains stretched along the [011] direction Fig. 2. A similet al., 2016N-tosylated vinylamine fragment. At the same time, the CSD revealed the existence of some examples of structurally similar vinyl ketones, viz. 1-(4-chlorophenyl)-3-prop-2-en-1-one -1-(pyridin-2-yl)-3-prop-2-en-1-one -1-(pyridin-2-yl)-3-prop-2-en-1-one -5-hydroxy-1,7-bis(2-methoxyphenyl)-1,4,6- heptatrien-3-one , δ, ppm: 9.79–9.81 , 7.76–7.81 , 7.53 , 7.29–7.34 , 6.99 , 6.82 , 6.70 , 4.10 , 3.41–3.44 , 2.41 ; 2.36 , 2.20 .Equimolar amounts of 1,5-bis(1-phenoxy)-3-azapentane  and guanidine hydrochloride  were stirred in an ethanol/sodium hydroxide mixture at 313–323 K in the presence of ammonium acetate . The reaction was monitored by TLC and completed after 48 h. The reaction mixture was allowed to cool to room temperature (298 K). Then, the product was extracted with dichloromethane (3 × 30 ml) and dried with NaUiso(H) = 1.2–1.5Ueq(C)].Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989020015194/yk2141sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989020015194/yk2141Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S2056989020015194/yk2141sup3.pdf1H NMR Spetrum of unexpected product. DOI: Click here for additional data file.10.1107/S2056989020015194/yk2141Isup4.cmlSupporting information file. DOI: 2044436CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


A database survey showed that whilst this coordination mode is unusual for Ge and Pb compounds, Sn displays a wider range of coordination modes to cyclopentadienyl ligands and their derivatives.Analysis of the coordination of the Sn to the indenyl ring shows that the Sn interacts in an η N:2(η2),κ2N,N′-phenyl]azanidyl}ethyl)[2-(1H-inden-1-yl)ethyl]azanido)lithiumtin, [LiSn(C8H16O4)(C25H31N2)], at 100 K has monoclinic (P21/n) symmetry. Analysis of the coordination of the Sn to the indenyl ring shows that the Sn interacts in an η2 fashion. A database survey showed that whilst this coordination mode is unusual for Ge and Pb compounds, Sn displays a wider range of coordination modes to cyclopentadienyl ligands and their derivatives.The structure of (μ-1κ With an unsaturated backbone, they have been found to be thermally unstable  Å], as well as to the 12-crown-4 tetradentate ether ligand  and 2.701 (3) Å] with Sn⋯C distances that are similar to those in stannocene [Sn(η5-Cp)2], Sn⋯C = 2.56 (2)–2.85 (3) Å  and 2.701 (3) Å] than the other three  in the five-membered ring of the indenyl moiety. The only two other crystallographically characterized Sn-indenyl complexes  and  . The results are shown in Fig. 2a–c for germanium, tin and lead, respectively. Germanium and lead are almost always projected near the centre of the Cp ring; however, tin shows a wide range of projection points. The datapoints for this structure are displayed in red in Fig. 2b, showing the distinct interaction with two carbon centres, a unique coordination mode for group 14 metals.By surveying the coordination of cyclopentadienyl ligands to main group atoms using the CSD C2H4NC2H4N}]2, 1Pr9H7)C2H4N(H)C2H4N(H) 2]2  dissolved in THF (2 ml) was added slowly at room temperature under nitrogen in a two-necked-flask in a glovebox. After 2 h, the solvent was removed by pipette and the precipitate was washed five times with 5 ml of petroleum ether by dispersing it and pipetting off the solvent after the residue had settled. Evaporation of the remaining solvent under high vacuum yielded the desired product as a light-yellow solid .To a solution of : δ = 7.5–6.9 , 6.28 (m), 3.67 (d), 3.48 (m), 3.38 (m), 3.00 (m), 2.88 (m), 2.79 (m), 1.19 (d); 119Sn ; δ = 79.7 ppm. Analysis calculated for C25H32N2Sn: C 62.65, H 6.73, N 5.85; Found: C 62.53, H 6.66, N 5.68Synthesis of indenide-tethered N-heterocyclic stannylene 21  in a glass vial under nitrogen in a glovebox was added Li[N(SiMe3)2]  in THF (0.5 mL) then 12-crown-4  in THF (0.2 ml). This vial was placed in a freezer, producing a small number of single crystals. Reactions on larger scales led to concentrations that were too high, leading to decomposition processes. The material that was produced was not soluble in d8-THF.To Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl).Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S205698902000047X/ff2165sup1.cifCrystal structure: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698902000047X/ff2165Isup3.cdxSupporting information file. DOI: 1946071CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal and molecular structure of [2-chloro-3-nitro-5-(trifluoromethyl)phenyl](piperidin-1-yl)methanone, a side product in the synthesis of an 8-nitro-1,3-benzothiazin-4-one, which belongs to a class of new anti-tuberculosis drug candidates, is reported. 13H12ClF3N2O3, occurs as a side product as a result of competitive reaction pathways in the nucleophilic attack during the synthesis of the BTZ 8-nitro-2-(piperidin-1-yl)-6-(trifluoromethyl)-1,3-benzothiazin-4-one, following the original synthetic route, whereby the corresponding benzoyl isothiocyanate is reacted with piperidine as secondary amine. In the title compound, the nitro group and the nearly planar amide group are significantly twisted out of the plane of the benzene ring. The piperidine ring adopts a chair conformation. The trifluoromethyl group exhibits slight rotational disorder with a refined ratio of occupancies of 0.972 (2):0.028 (2). There is structural evidence for intermolecular weak C—H⋯O hydrogen bonds.1,3-Benzothiazin-4-ones (BTZs) are a promising new class of anti-tuberculosis drug candidates, some of which have reached clinical trials. The title compound, the benzamide derivative [2-chloro-3-nitro-5-(trifluoromethyl)phenyl](piperidin-1-yl)methanone, C The latter step is favoured when electron-withdrawing substituents are present on the benzene ring.1,3-Benzothiazin-4-ones (BTZs) are promising anti-tuberculosis drug candidates, some of which have already reached clinical trials  with thionyl chloride and subsequently ammonium thiocyanate, the corresponding 2-chloro-3-nitro-5-(trifluoromethyl)benzoyl isothiocyanate (2) was reacted with piperidine. As illustrated, nucleophilic attack of the piperidine nitrogen atom at the isothiocyanate carbon atom leads to the anticipated 8-nitro-2-(piperidin-1-yl)-6-(trifluoromethyl)-1,3-benzothiazin-4-one (3). The alternative nucleophilic attack at the carbonyl carbon atom affords the side product (2-chloro-3-nitro-5-(trifluoromethyl)phenyl)(piperidin-1-yl)methanone (4), which was structurally characterized by X-ray crystallography in the present work. The ratio of 3 to 4 was found to vary depending on the reaction conditions. Temperatures at or below 283 K favour the formation of the anticipated 3, whereas substantial amounts of 4 form at elevated temperatures  = 4.0° indicate an almost planar amide group , the band at 1639 cm−1 can be assigned to the C=O stretching vibration of the amide group. The molecule is axially chiral, although the centrosymmetric crystal structure contains both enantiomers. The 13C NMR spectrum of 4 in methanol-d4 as well as chloroform-d at room temperature (see supporting information) displays five distinct signals in the aliphatic region, which are assigned to the piperidine carbon atoms, indicating that the rotation about the amide C—N bond is slow in solution under these conditions. The 13C NMR chemical shift of the α-carbon atom C13 syn to the carbonyl oxygen atom of the amide group is shielded compared with that of the anti α-carbon atom C9. In chloroform-d, the observed shielding magnitude of ΔδC = 5.0 ppm is within the range expected for a benzoylpiperidine . Complete assignments of 1H and 13C NMR data in chloroform-d by 13C,1H-HSQC and -HMBC NMR spectra can be found in the supporting information. Notably, the two separated methylene 1H NMR signals assigned to C10 in chloroform-d appear as one signal in methanol-d4.Fig. 24 adopts a low-energy chair conformation with some minor angular deviations from ideal tetrahedral values, resulting from planarity at N2 due to involvement in the amide linkage. The puckering parameters of the piperidine six-membered ring, as calculated with PLATON  Å, θ = 4.1 (2)° and φ = 161 (3)°. By way of comparison, the total puckering amplitude Q is 0.63 Å and the magnitude of distortion θ is 0° for an ideal cyclohexane chair . The corresponding geometric parameters . The corresponding geometric parameters (piperidin-1-yl)methanone phenyl moiety present in 4 led to only one structure, viz. 2-chloro-1,3-dinitro-5-(trifluoromethyl)benzene . Chemical shifts are referenced to the residual signals of methanol-d4  or chloroform-d .Chemicals were purchased and used as received. The synthesis of 2 were added to a stirred solution of 1  in toluene, and the mixture was heated to reflux for two h. The solvent was subsequently removed under reduced pressure, and the acid chloride thus obtained was used without purification. The residue was taken up in 6.5 mL of acetonitrile and a solution of 1.41 g (18.5 mmol) NH4SCN in 55 mL of acetonitrile was added dropwise with stirring to obtain 2in situ. After stirring for 5 min at 313 K, the resulting NH4Cl precipitate was filtered off, and 3.7 mL (37.0 mmol) of piperidine were added. The mixture was refluxed overnight, and then the solvent was removed under reduced pressure. Water was added to the residue and, after extraction with dichloromethane, the organic phase was washed with 10% aqueous NaHCO3 and dried over MgSO4. After removal of the solvent, the crude product was subjected to flash chromatography on silica gel, eluting with ethyl acetate/n-heptane (gradient 10–50% v/v), to isolate 1.09 g  of 3 and a minor amount of the side product 4. 1H and 13C NMR spectroscopic and mass spectrometric data of 3 were in agreement with those in the literature  by slow evaporation of the solvents at room temperature. NMR spectroscopic data for 4:2.7 mL (37.0 mmol) of SOCl1H NMR  δ 8.42 , 8.09 , 3.88–3.71 , 3.33–3.21 , 1.76 , 1.64  ppm; 13C NMR  δ 165.5, 150.7, 141.8, 132.3 , 129.2 , 128.1, 124.4 , 124.1 , 49.5, 44.3, 27.6, 26.7, 25.5 ppm.1H NMR  8.07 , 7.73 , 3.83–3.68 , 3.22 , 3.15 , 1.70 , 1.65–1.57 , 1.56–1.47  ppm; 13C NMR  163.5 , 148.9 (C3), 141.1 (C1), 131.2 , 127.8 , 127.6 (C2), 122.7 , 122.4 , 48.3 (C9), 43.3 (C13), 26.7 (C10), 25.7 (C12), 24.6 (C11) ppm.a—H = 0.95 Å and Cm—H = 0.99 Å (a = aromatic and m = methylene), and refined with the appropriate riding model and Uiso(H) = 1.2 Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S2056989020010658/vm2238sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989020010658/vm2238Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989020010658/vm2238Isup3.cdxSupporting information file. DOI: 10.1107/S2056989020010658/vm2238sup4.pdfIR and NMR spectra, assay protocol for anti-mycobacterial activity. DOI: Click here for additional data file.10.1107/S2056989020010658/vm2238Isup5.cmlSupporting information file. DOI: 2021003CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Garcinia mangostana peel, has been effectively used in wound healing. However, its poor solubility in aqueous solution causes low bioavailability for skin ulcers, hindering its application in wound healing. The aim of this study was to improve the solubility of α-M through complex formation with 2-hydroxypropyl-β-cyclodextrin (α-M/HP-β-CD CX) and to evaluate the healing activity of the complex. The α-M/HP-β-CD CX was incorporated in a sodium carboxymethylcellulose hydrogel (α-M/HP-β-CD CX HG), and the in vivo healing activity was examined in mice. Evaluation of α-M/HP-β-CD CX HG, including organoleptic evaluation, homogeneity, pH, spreadability, swelling ratio, consistency, scanning electron microscopy (SEM), and in vitro drug release, was carried out. The complex formation of α-M/HP-β-CD CX was confirmed by FTIR and PXRD analysis. The solubility of the α-M/HP-β-CD CX in water linearly increased about 11.7-fold compared to α-M alone, and by 3.5-fold compared to the α-M/HP-β-CD physical mixture (α-M/HP-β-CD CX PM). The α-M/HP-β-CD CX HG was homogenous, the pH was found to be in the neutral range, the spread area was 5 cm, and the consistency was stable until 14 days. SEM analysis showed that α-M/HP-β-CD CX HG surged due to the porous structure of the HG. In addition, in vitro release of α-M from α-M/HP-β-CD CX HG was considerably increased compared to α-M/HP-β-CD PM HG and α-M HG. Notably, in vivo evaluation in mice showed that α-M/HP-β-CD CX HG significantly accelerated the wound healing ability compared to other HGs. Thus, α-M/HP-β-CD CX HG has potential as a new formulation of α-M for wound healing therapy.α-Mangostin (α-M), one of the active compounds in  Garcinia mangostana peel, an Indonesian native plant containing more than 50 xanthone compounds. This plant contains α-Mangostin, which has been used for a long time in wound care [Several herbal plants have been developed for wound healing because of their safety and remarkable pharmacological activity . One of und care ,3,4.Wounds are a skin disruption caused by chemical, microbial, and physical trauma, as well as fire, etc. The wound healing process occurs in four phases, i.e., the inflammatory and hemostatic, migration, proliferation, and remodeling phases . α-M, onHowever, the poor water solubility of α-M hinders its application in several preparations. Various methods to increase the solubility have been developed, such as micronization, chemical modification, pH adjustment, and complexation . HydroxyWound healing therapies are classified into traditional healing agents, i.e., topical pharmaceutical formulations (liquid and semi-solid), traditional dressings (gauze and bandage), and modern dressings ). Gauze is a woven or non-woven fabric which can be combined with drugs in a suitable carrier, but limits microbial growth and leaves a second trauma when the gauze is removed from the ulcer. Foam provides a high surface area for water absorption, resulting in an insufficient wound treatment . The liqHG is one type of modern wound dressing that can be used in wound healing applications due to its biodegradability, biocompatibility, and favorable permeability. This form has the ability to minimize the irritation surrounding the tissue because of the smooth and moist surface of the HG ,15. SodiRecently, in vitro and in vivo studies of the α-Mangostin (α-M) and HP-β-CD complex have not been observed, especially in HG formulation for wound healing applications. In this study, the first solubility improvement of α-M was investigated by complex formation with HP-β-CD. The complex was incorporated into Na-CMC HG to increase residence time and bioavailability in the wound healing process.−1. The α-M spectra indicated the absorption band at 1033.83 cm−1 (C–O–C stretching vibration), 1373.32 cm−1 (C–O stretching vibration), and 2380.87 cm−1 (C–H stretching vibration). The HP-β-CD spectra indicated absorption at 1643.35 cm−1 (H–O–H stretching vibration), 2075.41 cm−1 (C–H bending), 2927.94 cm−1 (C-H stretching vibration), and 3394.72 cm−1 (O–H stretching vibration) [FTIR analysis was performed to determine the functional group and the interaction that probably occurred in α-M/HP-β-CD CX. The result of FTIR evaluation a showed bration) .To predict the interaction between α-M and HP-β-CD, molecular docking was performed. The PXRD patterns of α-M, HP-β-CD, α-M/HP-β-CD PM, and α-M/HP-β-CD CX can be seen in The solubility was studied by dissolving the samples in deionized water and then filtering. The degree of solubility  of α-M/Hα-M/HP-β-CD CX HG was successfully fabricated with Na-CMC as a gel base. An organoleptic evaluation was performed to determine the visual appearance of the preparation. The homogeneity of the preparations was observed using a microscope to observe whether or not the HG preparation showed a homogeneous appearance with no grains b. The α-SEM analysis was performed to determine the morphological properties of the HGs. The sample was coated with gold-palladium for 10 s during the analysis. The morphologies of α-M HG, α-M/HP-β-CD PM HG, and α-M/HP-β-CD CX HG can be seen in The HGs should have a pH in the acceptable pH skin range to avoid irritation on the skin a. The pHThe spreadability of the HGs at 1, 7, and 14 days can be seen in The swelling ratio of α-M/HP-β-CD CX HG was the greatest compared to the other HGs c. The swThe consistency of HGs was evaluated to determine their physical properties. Consistency evaluation was done through the centrifugal method d. The reThe results showed that α-M/HP-β-CD CX HG significantly increased the release of α-M from α-M/HP-β-CD CX HG compared to other HGs .The drug release from α-M/HP-β-CD CX HG was followed by a biphasic curve which showed the first release at the 1st hour (1.048 µg/mL), and until the 5th hour (6.397 µg/mL), with the regression parameters showing that the correlation coefficient (r) was 0.99 .Finally, to clarify the efficacy of α-M/HP-β-CD CX HG on wound healing ability, the in vivo study was performed on mice. Mice were divided into four treatment groups . The wou−1 and 3431.72 cm−1 disappeared, indicating a physical or chemical interaction occurred due to the complexation of α-M and HP-β-CD. In addition, molecular docking predicted the interactions between α-M and HP-β-CD, including five hydrogen bonds and one hydrophobic interaction in α-M/HP-β-CD CX.We successfully prepared α-M/HP-β-CD CX as a fine light-yellow powder. FTIR analysis was performed to determine the functional group and the interaction that probably occurred in α-M/HP-β-CD CX. The absorption at >3000 nm showed a sloping spectrum. The spectra of α-M/HP-β-CD CX showed that the fingerprint of α-M at the wavelengths 2924.09 cmThe complex formation between HP-β-CD CX with various drugs depends on the ability of drug molecules to enter the HP-β-CD cavity. The inclusion process did not break the covalent bonds, but some molecular interactions might have occurred during the complex formation, i.e., hydrophobic interaction, van der Waals interaction, hydrogen bond, and the release of high energy water from the HP-β-CD cavity ,21,22. TPXRD studies were done to identify the crystallinity of the α-M in complex α-M/HP-β-CD CX. The PXRD patterns of α-M, HP-β-CD, α-M/HP-β-CD PM, and α-M/HP-β-CD CX showed the successful complex formation between α-M and HP-β-CD CX. An amorphous pattern was seen in HP-β-CD and α-M/HP-β-CD CX. The diffractogram of α-M/HP-β-CD CX was more amorphous than α-M/HP-β-CD PM. The solubility of α-M/HP-β-CD CX significantly improved compared to α-M and α-M/HP-β-CD PM. The hydrophilic surface surrounding the outer HP-β-CD molecules was able to improve the solubility of the drug (α-M) entrapped within the cavity of HP-β-CD. Even though the increasing of solubility was achieved in α-M/HP-β-CD PM, there was no hydrogen bond formed between α-M and HP-β-CD PM. This suggested that the interaction between both molecules was due to the interaction between the hydroxypropyl group of the HP-β-CD molecule with α-M . Relatedα-M/HP-β-CD CX HG was incorporated in Na-CMC hydrogel, resulting in a yellowish gel. Na-CMC is a “smart” cellulose due to its sensitivity to various ionic strength and pH. The presence of sodium in the CMC structure provides electrostatic charges as a linker in HG formation and creates a double effect swelling ratio. Na-CMC plays an important role in the repulsion between the similar electric charge of long polymer chains that form a neutral network, leading to the enhancement of swelling capacity. In addition, the presence of counterions provides an increasing number of water molecules entering the network due to the Donnan effect . The homogeneity evaluation was done by applying the HG on a glass slide, covering it with another one, and then observing it under a microscope. The HG containing α-M/HP-β-CD CX had a more homogenous appearance compared to α-M/HP-β-CD PM, which showed the grains. Homogeneity was also affected by the water solubility of the active substance to be dispersed in the HG . In addiThe results of SEM strongly elaborated on previous results showing the homogeneity of α-M/HP-β-CD CX HG. The α-M particles appeared to disappear in α-M/HP-β-CD CX HG, suggesting that α-M/HP-β-CD CX was successfully prepared and dissolved within the Na-CMC gel base.The pH of the HGs was observed to confirm the safety of the preparation when the HG was applied to the skin. The pH of α-M HG was found to be relatively stable during storage. On the other hand, the pH of α-M/HP-β-CD PM HG and α-M/HP-β-CD CX HG decreased at 7 days. The factors affecting the pH are environment, temperature, and storage . The pre+ and replaced it with H+, and a greater cohesion force caused the lower spread area of α-M HG and α-M/HP-β-CD PM HG [Spreadability was investigated to determine the ability of the HGs to spread on the skin. When HGs spread and cover skin ulcers, proliferation will occur. The preparation containing α-M/HP-β-CD CX had the highest spreadability due to the solubility effect from the active substance. In an aqueous solution, Na-CMC released NaCD PM HG .The swelling ratio of the HGs revealed the swelling ability of the HGs to entrap water molecules in their polymer network. The presence of HP-β-CD in α-M/HP-β-CD PM HG and α-M/HP-β-CD CX HG increased the swelling ratio of the HG due to the ability of HP-β-CD to bind the water molecules. Swelling ratio is related to the ability of the HG to absorb the exudate of the skin ulcer, thus speeding up the healing process. Swelling ability accelerates the wound healing process by maintaining a moist environment around the wound .The separated materials at the bottom of the tube were insoluble material from α-M HG and α-M/HP-β-CD PM HG. The consistency caused by the concentration of Na-CMC in the range of 1–10% was able to maintain the consistency of the HG preparation . InteresTo determine the release of drug from the HGs, the in vitro release study of α-M from α-M/HP-β-CD CX HG was inspected. The drug release from α-M/HP-β-CD CX HG followed a biphasic curve, which showed the first release at the 1st hour (1.048 µg/mL) and until the 5th hour (6.397 µg/mL), with the regression parameters showing that the correlation coefficient was (r) 0.99 .The drug release followed Fickian diffusion, which illustrated that the drug release was not dependent on drug concentration . These rThe wound healing activity of α-M/HP-β-CD CX HG was almost 100% in 7 days, and the wound was completely closed in 14 days, while the α-M HG treated group still showed an open wound. The soluble complex in water caused faster drug release to the wound, resulting in a faster healing process than the α-M and α-M/HP-β-CD PM without the complex. The healing process occurred without the solubilization of the drug at the ulcer, but was directly distributed onto the ulcer to give the healing activity.O-methyl mangostin, 3,6-di-O-methyl mangostin, mangostin triacetate, 1-isomangostin, mangostin-3,6-di-O-(tetraacethyl)-glucoside, and mangostin-3,6-di-O-glucoside, significantly reduced inflammation in rats [2 release [The previous study stated that synthetic compounds from α-M derivatives, i.e., 3- in rats . In addi release . Moreove release . The pre release . Thus, t release . In this2, O2, and H2O, in which the HGs have the characteristics of 90% water and 10% polymer, with a high-water content. Thus, they are well fitted to treating dry and necrotic wounds. Moist environment in the HG could improve the process and facilitate the resolution of injury debridement effectively through the HG’s absorption ability [Considering the properties of the HGs in wound healing therapy, permeable HGs allow the exchange of CO ability .α-M was procured from Chengdu Biopurify Phytochemicals Ltd. . HP-β-CD (DS 4) was obtained from Nihon Shokuhin Kako . Na-CMC, glycerin, propylene glycol, and methylparaben were bought from Brataco . All the reagents were of analytical grade and used without further purification.The inclusion complex of α-M/HP-β-CD (α-M/HP-β-CD CX) was prepared by α-M and HP-β-CD in a ratio of 1:1 as follows: HP-β-CD powder (1205 g) was dissolved in 20 mL of deionized water. In a separate flask, α-M 0.410 g) was dissolved in 30 mL of methanol. The α-M solution was slowly added into the HP-β-CD solution, which was mixed and sonicated for 15 min, then mixed at 120 rpm for 48 h at room temperature. The solvent was evaporated using a rotary evaporator . Furthermore, the concentrated solution was dried in the oven at 50 °C for 12 h 0 g was d.−1 [The functional groups in α-M, HP-β-CD, α-M/HP-β-CD PM, and α-M/HP-β-CD CX were determined by FTIR (Shimadzu’s IRPrestige-21) analysis in accordance with the following procedures: each sample of α-M, 2-HP-β-CD, α-M/HP-β-CD PM, and α-M/HP-β-CD CX was crushed with KBr and compressed to form a pellet. Each pellet containing the sample was then scanned in the range of 400–4000 cm−1 .® 2020 Client system [The 3D structure of HP-β-CD (DS 4) was established through the 3D structure modeling process using a Chem3D Pro 12.0 from β-CD (PDB ID: 4RER) . The strt system . The intα radiation , scanning rate 2°/minute at 2θ, and a diffraction angle of 3° to 50° [PXRD  analysis was performed to determine the crystallinity of α-M, HP-β-CD, α-M/HP-β-CD PM, and α-M/HP-β-CD CX. Briefly, each sample (5 mg) of α-M, α-M/HP-β-CD PM, and α-M/HP-β-CD CX was placed on a sample holder, and the tests were conducted using the diffractometer with filtered Cu-K° to 50° .g for 30 min, and filtered with a syringe filter at 0.45 µm. The concentration of α-M was determined using UV spectrophotometry at 246 nm [Each sample (60 mg) of α-M, α-M/HP-β-CD PM, and α-M/HP-β-CD CX was dissolved in 25 mL of deionized water (Milli-Q), and stirred at 120 rpm at a temperature of 25 °C for 48 h. The mixtures were centrifuged (Hettich EBA 200) at 3500× t 246 nm .The HG was composed of Na-CMC as a base, and the excipients included glycerin, propylene glycol, and methylparaben . The HG Organoleptic evaluation was performed by human sense organs to study the visual appearance of the preparation. Samples (20 g) of α-M HG, α-M/HP-β-CD CX HG, and α-M/HP-β-CD PM HG were placed in a 25 mL glass beaker at room temperature and were visually analyzed by a human panel, consisting of 3 trained postgraduate students from the Faculty of Pharmacy, Universitas Padjadjaran ,49.The pH evaluation was carried out using a pH meter (Mettler Toledo S220). Briefly, 1% of each sample  was diluted with 20 mL of distilled water in a 25 mL glass beaker, and then the pH was measured using a pH meter. The pH should have been in the range of 4.5–6.5 ,31.To clarify the formulation process of α-M HG, α-M/HP-β-CD CX HG, and α-M/HP-β-CD PM HG preparations, the homogeneity test was performed using a microscope (Olympus CX23). A few samples were dispersed over a microscope glass slide and closed by a glass cover. Each sample was observed under the microscope (magnification of ×40). The samples were taken from 3 parts of the preparation, i.e., the upper, middle, and bottom of the preparation ,50.Samples (0.5 g) of α-M HG, α-M/HP-β-CD CX HG, and α-M/HP-β-CD PM HG were placed in the center of a Petri dish in an upside-down position to form a circle of 2 cm in diameter, which was signed as an initial diameter. The other Petri dish was employed in reverse position. A weight of 150 g was added above the upper dish for 5 min, and the final diameter was measured. The diameter of the spreadability area should have been in the range of 5–7 cm ,48,51.The morphological structure of the HG was studied by SEM (JEOL JSM 6510 LA). Briefly, 2 g of each sample  was lyophilized and then freeze-dried. The samples were attached to a sample holder and coated with gold-palladium for 10 s, then observed by SEM (magnification of ×300) ,52,53.Wd is the weight of the first HG, and Ws is the weight of the swollen HG at a certain time.Samples (0.5 g) of  were immersed in phosphate buffer saline (PBS) solution pH 7.4 for 15, 30, and 60 min at room temperature. The swelling ratio was determined by the following Equation (1) ,55.(1)Sg for 5 h [To study the consistency alteration of the HGs, the consistency test was carried out using a centrifuge (Eppendorf Centrifuge 5424 R). The HG was inserted into the centrifuge tube to as much as 2 mL. The α-M HG, α-M/HP-β-CD CX HG, and α-M/HP-β-CD PM HG were centrifuged at 1800×  for 5 h .To investigate the drug release mechanism, the in vitro release study was done by dissolving 0.2 g of samples  in 20 mL PBS pH 7.4 and mixed. Then, 5 mL of the release medium was taken at various time points and replaced with the same volume of fresh PBS. The α-M amount released from each sample was measured using UV spectrophotometry at 246 nm .The wound healing activity of the α-M HG, α-M/HP-β-CD CX HG, and α-M/HP-β-CD PM HG was examined in mice. The procedures were approved by the Ethical Committee of Medical Faculty Universitas Padjadjaran, with ethical approval number 1036/UN6.KEP/EC/2018. The anesthesia  was injected before the evaluation to minimize pain in the animal tests .Mus musculus) that were 2–3 months old and weighed 20–30 g. The number of mice used was 20, divided into four groups, with 5 mice in each group. The animals were placed at a constant temperature (26 ± 1 °C) before the evaluation [In vivo healing activity was performed in Swiss–Webster strain mice  below .(2)% ofIn this study, α-M/HP-β-CD CX HG was successfully prepared and confirmed by FTIR, PXRD, and solubility studies. α-M/HP-β-CD CX was an amorphous complex which significantly improved solubility by about 11.7 fold compared to α-M alone, and by 3.5 fold compared to α-M/HP-β-CD PM. The better solubility of α-M/HP-β-CD CX created improved properties of α-M/HP-β-CD CX HG compared to α-M HG and α-M/HP-β-CD PM HG. The pH was found to be in the neutral range, the spread area was 5 cm, and the consistency was stable until 14 days. In addition, in vitro and in vivo studies of α-M/HP-β-CD CX HG showed the linear correlation in enhancement of α-M wound healing ability by complexation with HP-β-CD in Na-CMC HG formulation. These results suggest that α-M/HP-β-CD CX HG has potential as a new formulation of α-M for wound healing therapy.


CFL5, an endophytic fungus of Cephalotaxus fortunei. The chemical structures of all new compounds were elucidated on the basis of extensive spectroscopic data analyses and electronic circular dichroism calculation, as well as optical rotation calculation. Biological activities of compounds 1, 4‒9 were evaluated, including cytotoxic, LAG3/MHC II binding inhibition and LAG3/FGL1 binding inhibition activities. Compounds 6 and 9 possessed cytotoxicity against AGS cells at 5 μM, with inhibition rates of 94% and 64%, respectively. In addition, all tested isolates, except compound 6, exhibited obvious inhibitory activity against the interaction of both LAG3/MHC II and LAG3/FGL1. Compounds 1, 5, 7, and 8 inhibited LAG3/MHC II with IC50 values ranging from 2.37 to 4.74 μM. Meanwhile, the IC50 values of compounds 1, 7, and 8 against LAG3/FGL1 were 11.78, 4.39, and 7.45 μM, respectively.Three previously undescribed cytochalasins, named xylariasins A‒C (The online version of this article (10.1007/s13659-020-00279-5) contains supplementary material, which is available to authorized users. Xylaria +m/z 476.2793 ; 1H and 13C NMR data, found in Tables Xylariasin A : white pentclass1pt{minima2): colorless oil; c 0.885); IR (KBr) νmax 3456, 3302, 2969, 2933, 1740, 1692, 1454, 1373, 1229, 738, 702 cm−1; ECD (MeOH) λmax (Δε), 212 (+ 6.70), 231 (− 2.44), 243 (− 0.50), 266 (+ 0.70), 298 (+ 4.70) nm; HRESIMS [M + H]+m/z 492.2741 ; 1H and 13C NMR data, found in Tables Xylariasin B mentclass2pt{minimpt{minim23): white powder; c 0.336); IR (KBr) νmax 3369, 2925, 1773, 1719, 1686, 1262, 1232, 1107, 1074, 1027, 740, 702 cm−1; ECD (MeOH) λmax (Δε), 203 (− 17.70), 222 (− 8.82) nm; HRESIMS [M + Na]+m/z 500.2768 ; 1H and 13C NMR data, found in Tables Xylariasin C mentclass2pt{minimpt{minim31, 4–9 were assessed for cytotoxicity against AGS, U251, and MDA-MB-231 by the CCK8 assay  were added into each well. The concentration of DMSO in each well was kept below 0.5% and the final volume in each well was 20 μL in the moment. Then the 96-well dishes were kept incubating for 1 h to overnight at room temperature. Subsequently, remove the plate sealer and read specific emission signal at 665 nm on an HTRF compatible reader. The positive control was yielded by replacing tested solution with 2 μL diluent. The negative control consisted of 4 μL Tag2-MHC II protein, 5 μL anti-Tag1-Tb3+ and 5 μL anti-Tag2-XL665, as well as 6 μL diluent. In blank control, 5 μL anti-Tag1-Tb3+, 10 μL diluent, and 5 μL detection buffer were existed.Utilizing HTRF (Homogeneous Time-resolved Fluorescence) technology , 35, thep), blank control (Fb), and experimental groups (Fe), the inhibition rate was calculated at each of the compound concentration levels. The inhibitory activity of compound against the interaction of LAG3 with MHC II was evaluated based on the formula as follow: Inhibition rate = (Fp − Fe)/(Fp − Fb) × 100%. IC50 values were calculated by linear regression of a dose-dependent inhibition rate curve.Using the following fluorescence intensity (F) measurements at 665 nm, such as the signals of positive control Below is the link to the electronic supplementary material.