Cercis glabra is a plant belonging to the legume family, whose flowers and barks are commonly used as food and traditional Chinese medicines. However, its leaves are usually disposed of as wastes. This research comprehensively investigated the bioactive constituents of C. glabra leaves, and two new phenolic, ceroffesters A-B (1–2) and thirteen known compounds (3–15) were isolated. Their structures were elucidated by spectroscopic methods such as nuclear magnetic resonance (1D NMR and 2D NMR), high-resolution electrospray ionization mass spectra (HR-ESI-MS), optical rotatory dispersion (ORD) and electronic circular dichroism (ECD). All of them were assessed for their antioxidant activities through ABTS, DPPH and PTIO methodologies, and evaluated for inhibitory activities against two enzymes (mushroom tyrosinase and acetylcholinesterase). As a result, compounds 3–6, 10 and 13 exhibited evident antioxidant activities. Meanwhile, compounds 5, 10 and 13 showed the most potent tyrosinase inhibitory activities, with IC50 of 0.64, 0.65 and 0.59 mM, and compared with the positive control of 0.63 mM (kojic acid). In the initial concentration of 1 mg/mL, compounds 3, 5 and 6 demonstrated moderate inhibitory activities against acetylcholinesterase with 85.27 ± 0.06%, 83.65 ± 0.48% and 82.21 ± 0.09%, respectively, compared with the positive control of 91.17 ± 0.23% (donepezil). These bioactive components could be promising antioxidants, tyrosinase and acetylcholinesterase inhibitors. Antioxidation, whitening and Alzheimer’s disease (AD) have always been the focus of attention in the fields of health and medicine. Antioxidants have been widely used as food additives to reduce or avoid the degradation of food and improve its palatability. In addition, antioxidants are important in preventing diseases . The humAcetylcholinesterase (AChE) promotes the degradation of acetylcholine at synapses and neuromuscular junctions, leading to the termination of nerve impulses. Decreased acetylcholine levels are one of the major causes of cognitive impairment in Alzheimer’s disease ,8. The iHuman health has become the focus of food development. Functional ingredients and functional food are investigated and have become a trend for food research worldwide . IngrediCercis is one genera of the legume family and contains about eight species, namely C. glabra, C. chinensis, C. canadensi, C. siliquastrum, C. griffithii, C. chuniana, C. chingii and C. racemose. These plants are widely grown in Western and Central Asia, Southern Europe and North America [Cercis flowers are rich in volatile oil, amino acids [Cercis flowers’ pigment is often produced as a natural edible red pigment in the food industry [Cercis plants also have a variety of medicinal values. The flowers, barks and fruits from Cercis plants have been used as traditional Chinese medicines to treat various diseases, such as wind-dampness, cold-arthralgia, carbuncle and swelling. Phytochemical studies of the Cercis genus have found the isolation of stilbenes, flavonoids, lignans, phenolic acids, cyanogenic glycosides and others [Cercis plant are very scarce. More attention should be paid to it for the further development and utilization of Cercis resources. America . Cercis no acids , and havindustry . In addid others , while pd others ,17,18. HC. glabra is a megaphanerophyte whose flowers are highly ornamental and are often planted in courtyards and roadsides [C. glabra leaves has strong inhibitory activity. A further phytochemical investigation on the ethanolic extract led to two new esters of ceroffesters A-B (1–2), eight flavonoids (3–10) and five others (11–15) which were isolated and identified. Herein, details of the isolation, structure elucidation and biological activities of these isolated compounds are described.oadsides . In receoadsides . In the oadsides ,21, the 1–2) and thirteen known ones (3–15) were isolated from the leaves of C. glabra , ceroffester B (2), myricetin (3) [4) [5) [6) [7) [8) [O-rutinoside (9) [O-rhamnoside-(C7-I-O-C7-II)-myricetin-3-O-rhamnoside (10) [11) [O-β-d-glucopyranoside (12) [13) [β-d-glucopyranosyl tuberonate (14) [15) [1–4, 9–10 and 12–15 were reported from the C. glabra leaves for the first time, and compounds 1–2 and 10 were first reported from the Cercis leaves.By spectroscopic data analysis, they were determined as ceroffester A (etin (3) , isorham (3) [4) , quercet [4) [5) , kaempfe [5) [6) , afzelin [6) [7) , quercit [7) [8) , kaempfeside (9) , myricetide (10) , caroten10) [11) , 4-(4′-Hide (12) , gallic 12) [13) , methyl-ate (14) , hovetri14) [15) , by comp1 was obtained as a white powder. The IR absorption band at 3424 cm−1 suggested the presence of hydroxyl groups. The IR absorption bands at 1729, 1708 and 1695 cm−1 suggested the presence of three carbonyl groups  in the HR-ESI-MS spectrum , 6.82 , a trans-double bond , 6.36 ), suggesting the presence of a trans-coumaroyl group in 1. Additionally, the 1H-NMR spectrum displayed two doublets at δH 5.57  and 4.81 , corresponding to protons of two oxygenated methines, and two methoxyl groups δH 3.79  and 3.74 . In the 13C-NMR and DEPT spectra (trans-coumaroyl group (δC 167.8 (C-9), 161.7 (C-4), 148.3 (C-7), 131.6 (C-2/C-6), 127.1 (C-1), 117.0 (C-3/C-5), 113.8 (C-8)), another six signals were observed at δC 172.4 (C-4′), 169.3 (C-1′), 74.8 (C-2′), 72.0 (C-3′), 53.3 (1′-OCH3) and 53.2 (4′-OCH3), which were attributed to a tartaric acid dimethyl ester. The 1H-1H COSY correlations -trans-4-coumaroyltartaric acid dimethyl ester (ceroffester D) [R, 3′R), while compound 1 was negative optical rotation, and its absolute and relative stereochemistry was determined as 2′S and 3′S. Finally, the structure of compound 1 was identified as -trans-4-coumaroyltartaric acid dimethyl ester, namely ceroffester A.Compound l groups . The molspectrum , with eispectrum  revealed spectra , aside felations  between elations  from 1′-yl ester . The dow2 was obtained as a white powder. The IR absorption bands at 3434, 1739 and 1698 cm−1 suggested the presence of hydroxyl groups and carbonyl groups  in the HR-ESI-MS spectrum -2 and its three isomers, -2, -2 and -2, were calculated by the time-dependent density functional theory (TD-DFT) calculations -2, and is opposite to that of its enantiomer -2 -trans-4-coumaroyl-4′-methoxyl-tartaric acid, namely ceroffester B.Compound l groups . The molspectrum , with ei spectra  of 2 wer-1H COSY  and HMBC-1H COSY . The HMB-1H COSY  from 4′- to C-4′ . Therefoulations . The mea, 3′S)-2 A,D. Ther3). Yellow powder. 1H-NMR  δ: 7.76 , 6.38 , 6.18 ; 13C-NMR  δ: 177.4 (C-4), 165.7 (C-7), 162.6 (C-5), 158.3 (C-9), 148.1 (C-2), 146.9 (C-3′/5′), 137.5 (C-3), 137.1 (C-4′), 123.2 (C-1′), 108.7 (C-2′/6′), 104.3 (C-10), 99.5 (C-6), 94.5 (C-8) [Myricetin (.5 (C-8) .4). Yellow powder. 1H-NMR  δ: 12.47 , 10.81 , 9.77 , 9.45 , 7.75 , 7.69 , 6.94 , 6.48 , 6.19 , 3.84 ; 13C-NMR  δ: 176.8 (C-4), 164.8 (C-7), 161.6 (C-5), 157.1 (C-9), 149.7 (C-3′), 148.3 (C-4′), 147.5 (C-2), 136.7 (C-3), 122.9 (C-1′), 122.6 (C-6′), 116.5 (C-5′), 112.7 (C-2′), 103.9 (C-10), 99.1 (C-6), 94.5 (C-8), 56.7 (3′-OCH3) [Isorhamnetin (3′-OCH3) .5). Yellow powder. 1H-NMR  δ: 7.74 , 7.63 , 6.88 , 6.39 , 6.18 ; 13C-NMR  δ: 177.5 (C-4), 165.7 (C-7), 162.6 (C-5), 158.4 (C-9), 148.9 (C-4′), 148.2 (C-2), 146.4 (C-3′), 137.4 (C-3), 124.3 (C-1′), 121.8 (C-6′), 116.4 (C-5′), 116.2 (C-2′), 104.7 (C-10), 99.4 (C-6), 94.6 (C-8) [Quercetin (6). Yellow crystal. 1H-NMR  δ: 8.14 , 7.00 , 6.52 , 6.23 ; 13C-NMR  δ: 177.6 (C-4), 166.0 (C-7), 163.3 (C-5), 161.2 (C-4′), 158.8 (C-9), 148.1 (C-2), 137.7 (C-3), 131.5 (C-2′/6′), 124.4 (C-1′), 117.4 (C-3′/5′), 105.2 (C-10), 100.2 (C-6), 95.6 (C-8) [Kaempferol (7). Yellow powder. 1H-NMR  δ: 7.65 , 6.93 , 6.37 , 6.19 , 5.38 , 4.23–3.32 , 0.92 ; 13C-NMR  δ: 179.8 (C-4), 166.0 (C-7), 163.4 (C-4′), 161.7 (C-5), 159.4 (C-9), 158.7 (C-2), 136.4 (C-3), 132.1 (C-2′/6′), 122.8 (C-1′), 116.7 (C-3′/5′), 106.1 (C-10), 103.7 (C-1′′), 100.0 (C-6), 94.9 (C-8), 73.4 (C-4′′), 72.3 (C-3′′), 72.2 (C-2′′), 72.1 (C-5′′), 17.8 (C-6′′) [Afzelin ( (C-6′′) .8). Yellow powder. 1H-NMR  δ: 7.34 , 7.31 , 6.91 , 6.37 , 6.20 , 5.35 , 4.22 , 3.75 , 3.42 , 3.35 , 0.94 ; 13C-NMR  δ: 179.8 (C-4), 166.0(C-7), 163.4 (C-5), 159.5 (C-9), 158.7 (C-2), 149.9 (C-4′), 146.6 (C-3′), 136.4 (C-3), 123.1 (C-1′), 123.0 (C-6′), 117.1 (C-5′), 116.5 (C-2′), 106.1 (C-10), 103.7 (C-1′′), 100.0 (C-6), 94.9 (C-8), 73.4 (C-4′′), 72.3 (C-3′′), 72.2 (C-2′′), 72.1 (C-5′′), 17.8 (C-6′′) [Quercitrin ( (C-6′′) .O-rutinoside (9). Yellow powder. 1H-NMR  δ: 8.06 , 6.88 , 6.36 , 6.18 , 5.10 , 4.52 , 3.81 , 3.64 , 1.13 ; 13C-NMR  δ: 179.3 (C-4), 167.6 (C-7), 163.0 (C-5), 161.6 (C-4′), 159.4 (C-9), 158.8 (C-2), 135.6 (C-3), 132.5 (C-2′/6′), 122.9 (C-1′), 116.3 (C-3′/5′), 105.4 (C-10), 104.9 (C-1′′), 102.6 (C-1′′′), 100.6 (C-6), 95.4 (C-8), 78.3 (C-3′′), 77.3 (C-5′′), 75.9 (C-2′′), 74.1 (C-4′′′), 72.4 (C-3′′′), 72.2 (C-2′′′), 71.6 (C-4′′), 69.9 (C-5′′′), 68.7 (C-6′′), 18.1 (C-6′′′) [Kaempferol-3-(C-6′′′) .O-rhamnoside-(C7-I-O-C7-II)-myricetin-3-O-rhamnoside (10). Yellow powder. 1H-NMR  δ: 6.92 , 6.35 , 6.16 , 5.29 , 5.28 , 4.20 , 3.77 , 3.49 , 3.28 , 0.94 , 0.92 ; 13C-NMR  δ: 179.8 , 165.9 , 163.3 , 159.5 , 158.6 , 146.9 , 138.0 , 136.4 , 122.0 , 109.7 , 106.0 , 103.7 , 99.9 , 94.8 , 73.4 , 72.2 , 72.0 , 17.8  [Myricetin-3-11). White powder. 1H-NMR  δ: 5.32 , 4.43 , 4.22 , 3.56 , 3.05 , 2.91 , 0.96 , 0.90 , 0.85–0.79 , 0.65 ; 13C-NMR  δ: 141.4 (C-5), 122.1 (C-6), 101.8 (C-1′), 77.9 (C-3′), 77.7 (C-5′), 77.7 (C-3), 74.4 (C-2′), 71.0 (C-4′), 62.0 (C-6′), 57.1 (C-17), 56.4 (C-14), 50.4 (C-9), 46.1 (C-24), 42.8 (C-13), 40.2 (C-12), 39.3 (C-4), 37.8 (C-1), 37.1 (C-10), 36.4 (C-20), 34.3 (C-22), 32.4 (C-8), 32.3 (C-7), 30.2 (C-2), 29.6 (C-25), 28.7 (C-16), 26.4 (C-23), 24.8 (C-15), 23.5 (C-28), 21.5 (C-11), 20.6 (C-26), 20.0 (C-19), 19.9 (C-27), 19.5 (C-21), 12.7 (C-29), 12.6 (C-18) [Carotene . Colorless syrup. 1H-NMR  δ: 7.12 , 7.01 , 4.86 , 3.31–3.90 , 2.78 , 2.11 ; 13C-NMR  δ: 211.2 (C-2), 157.6 (C-8), 136.5 (C-5), 130.4 (C-6/10), 117.9 (C-7/9), 102.6 (C-1′), 78.2 (C-3′), 78.1 (C-5′), 75.0 (C-2′), 71.5 (C-4′), 62.6 (C-6′), 46.1 (C-4), 30.1 (C-1/3) [4-(4′-Hydroxyphenyl)-2-butanone-4′-13). White crystal. 1H-NMR  δ: 7.10 ; 13C-NMR  δ: 170.9 (C-7), 146.3 (C-3/5), 139.5 (C-4), 122.2 (C-1), 110.5 (C-2/6) [Gallic acid ( (C-2/6) .d-glucopyranosyl tuberonate (14). colorless oil. 1H-NMR . δ: 5.49 , 5.29 , 4.36 , 3.87 , 3.72 , 3.57–3.28 , 2.72 , 2.41 , 1.29–2.26 ; 13C-NMR  δ: 219.6 (C-3), 172.9 (C-1′′), 128.1 (C-3′), 127.9 (C-2′), 103.0 (Glc C-1), 76.6 (Glc C-3), 75.8 (Glc C-5), 73.6 (Glc C-2), 69.8 (Glc C-4), 69.5 (C-5′), 61.8 (Glc C-6), 54.1 (C-2), 52.0 (1′′-OCH3), 38.9 (C-4), 38.0 (C-1/2′′), 28.1 (C-4′), 27.4 (C-5), 25.6 (C-1′) [Methyl-β-6 (C-1′) .15). colorless syrup. 1H-NMR  δ: 6.98 , 6.56 , 6.04 , 5.94 , 4.87 , 3.86–3.38 , 3.08 ; 13C-NMR  δ: 197.1/197.0 (C-3), 174.7 (2C-8), 171.6 (2C-6), 158.6/158.4 (C-4), 157.4 (2C-4′), 132.7 (2C-2′/6′), 125.7 (2C-1′), 115.9 (2C-3′/5′), 107.8/107.7 (C-2), 103.8/103.6 (C-9), 101.8 (2C-1′′), 97.7/97.4 (C-5), 93.4/93.3 (C-7), 78.5 (2C-5′′), 77.5/77.4 (C-3′′), 74.2/74.1 (C-2′′), 71.3 (2C-4′′), 62.5 (2C-6′′), 42.3/42.1 (C-10) [Hovetrichoside C (1 (C-10) .C. glabra leaves were explored with l-ascorbic acid as the positive control  from  control .3–6, 10 and 13. In particular, compounds 3 (myricetin), 5 (quercetin), 6 (kaempferol) and 10 (myricetin-3-O-rhamnoside-(C7-I-O-C7-II)-myricetin-3-O-rhamnoside) displayed higher IC50 values than l-ascorbic acid. Five compounds  showed similar IC50 values to l-ascorbic acid for DPPH radical scavenging assay. Moreover, three compounds  showed similar IC50 values to l-ascorbic acid in PTIO radical scavenging assay.Six compounds showed ABTS radical scavenging rates of >86% at 1 mg/mL, including 13) are flavonoids. Therefore, the phenolic hydroxyl groups may be important for the radical scavenging activity. The ABTS, DPPH and PTIO methods are commonly used to evaluate the scavenging ability of free radicals. Thus, their reaction may be regarded as a direct antioxidant process. However, their scavenging mechanisms are in fact rather different. For example, DPPH and ABTS are nitrogen-centered radicals, while PTIO is an oxygen-centered radical. The ABTS radical is scavenged mainly involving one-electron transfer (ET), while DPPH and PTIO scavenging have been demonstrated to be involved in H+ transfer (HAT) [3, 5, 6 and 10 could effectively scavenge three types of free radicals through ET and HAT pathways, indicating that they could be used as novel effective antioxidants.Of these bioactive components, all except gallic acid (er (HAT) . As can 1–2 showed moderate tyrosinase inhibitory activities, while compounds 5, 10 and 13 showed the most potent tyrosinase inhibitory activities, with IC50 of 0.64, 0.65 and 0.59 mM, respectively, while the positive control was 0.63 mM (kojic acid). Tyrosinase inhibitors, characterized by reducing melanin production and improving skin elasticity, are widely used in pharmaceutical and skincare industries [5, 10 and 13 are of great pharmaceutical value both in cosmetics and pharmaceuticals, due to their potent biological properties.In dustries . Howeverdustries . Therefo3, 5 and 6 exhibited moderate acetylcholinesterase inhibitory activities, with a percentage inhibition value of 85.27 ± 0.06%, 83.65 ± 0.48% and 82.21 ± 0.09%, respectively, with donepezil used as the positive control (91.17 ± 0.23%). The inhibition of AChE serves as a strategy for the treatment of neurological disorders, including myasthenia gravis, glaucoma, Parkinson’s disease, senile dementia and ataxia [C. glabra leaves may be a potential therapeutic agent for the treatment of Alzheimer’s disease, due to its phytochemical components.The initial concentration was 1 mg/mL, and compounds d ataxia . The fin3) and kaempferol (6) are two flavonoids widely distributed in natural plants [3 and 6 were generally consistent with the previous research. Quercetin (5) spread widely in fruits and vegetables [10 has been tested to be a potential natural antioxidant [13 was found to show antioxidant and tyrosinase inhibition activities [Myricetin , and the spots were detected by UV illumination (365 and 254 nm) and by spraying 10% H2SO4 followed by heating. UV, FT-IR and NMR spectra were recorded on Puxi TU-1950, FTIR-650 and Bruker AM-400  instruments, respectively. HR-ESI-MS dates were obtained from a Bruker micrOTOF II spectrometer. The optical rotatory dispersion (ORD) was obtained on JASCO P-2200 polarimeter and ECD spectra were obtained on a JASCO J-810 spectrometer. The absorbance was gained from Multiskan FC microplate reader .Silica gel  was used for column chromatography, along with Sephadex LH-20 , ODS  and Polyamide . TLC was conducted with silica gel 60 FC. glabra were collected from Yanling Zhonglin Garden Engineering Co., Ltd., Xuchang, China, in May 2021. The species was identified by Prof. Lin Yang at Lanzhou Technology University. A specimen (No. SPH2021B) was stored in Xuchang University, China.The fresh leaves of C. glabra leaves (6.2 kg) were crushed and extracted three times with 50 L EtOH (95%) and soaked for 3 days at room temperature. The concentrated EtOH crude extract (418.1 g) was dissolved in warm H2O and partitioned with small polar solvent  and ethyl acetate (EtOAc). The EtOAc soluble fraction (81.4 g) was separated by chromatography on silica gel CC eluted with CH2Cl2-MeOH (100:0~2:1 v/v) to obtain nine fractions (F1–F9). The fraction F1 was separated by Sephadex LH-20 CC (CH2Cl2-MeOH 1:1 v/v) to yield compounds 1 (26.6 mg) and 4 (21.2 mg). The fraction F3 was fractionated on silica gel CC divided into multiple subfractions (F3-1–F3-3) by gradient elution with CH2Cl2-MeOH (100:1~15:1 v/v). Compounds 6 (27.6 mg) and 11 (18.6 mg) were purified from subfraction F3-2 on a silica gel CC eluted with gradient of CH2Cl2-MeOH (40:1~20:1 v/v). Subfraction F3-3 was chromatographed on Sephadex LH-20 CC eluted with MeOH to generate compound 5 (8.7 mg). Three subfractions (F5-1–F5-3) were obtained by ODS CC eluted with MeOH-H2O (40:60~90:10 v/v) from F5. Further purification gave compounds 2 (105.9 mg), 3 (3.5 mg) and 13 (95.0 mg) from subfractions F5-2 and F5-3. The fraction F7 was segmented into multiple subfractions (F7-1–F7-3) by ODS CC eluted with MeOH-H2O (30:70~90:10 v/v). Compounds 12 (25.4 mg), 7 (15.3 mg), 8 (10.3 mg) and 10 (27.1 mg) were obtained by the further separation of subfraction F7-1 utilizing Polyamide CC, Sephadex LH-20 CC and silica gel CC. Similarly, compound 14 (18.6 mg) was gained from subfraction F7-3. Fraction F9 was further isolated into compounds 15 (13.5 mg) and 9 (23.0 mg), which were applied to Sephadex LH-20 CC and silica gel CC.According to Ceroffester A (1). White powder. νmax 3424, 2962, 1729, 1708, 1695, 1635, 1608, 1513, 1442, 1355, 1309, 1284, 1253, 1224, 1164, 1068, 981, 835 cm–1. UV λmax (MeOH) nm (log ε): 315 (5.07), 228 (4.82) .8 (4.82) . 1H-NMR Ceroffester B (2). White powder. νmax 3434, 2960, 1739, 1698, 1625, 1606, 1515, 1434, 1349, 1315, 1286, 1243, 1224, 1199, 1157, 1074, 981, 825 cm–1. UV λmax (MeOH) nm (log ε): 313 (5.17), 228 (4.89) .8 (4.89) . 1H-NMR 3–15 can be found in Experimental data of compounds 2 and its isomers. Rotatory strengths for a total of 30 excited states were calculated. ECD spectra were generated using the program SpecDis 1.6  and GraphPad Prism 5  from dipole-length rotational strengths by applying Gaussian band shapes with sigma = 0.3 eV.Monte Carlo conformational searches were carried out by means of the Spartan’s 14 software using Merck Molecular Force Field (MMFF). The conformers with Boltzmann population of over 5% were chosen for ECD calculations, and then the conformers were initially optimized at B3LYP/6–31g level in gas. The theoretical calculation of ECD was conducted in MeOH using the time-dependent density functional theory (TD-DFT) at the B3LYP/6–31+g  level for all conformers of compound l-ascorbic acid was the positive control. After incubation at 37 °C for 10 min, the absorbance was tested at 745 nm using a microplate reader. Scavenging rate was calculated according to Equation (1).C and AS are the absorbance of the blank control and the compounds to be tested, respectively.The ABTS radical scavenging assay was modified according to the method with slight modifications . Brieflyl-ascorbic acid was the positive control. The absorbance at 517 nm was measured using a microplate reader after the solution had stood for 30 min at 37 °C under dark conditions. The calculation formula of DPPH radical scavenging activity is consistent with the formula of ABTS radical scavenging activity.The scavenging ability on DPPH radical was conducted based on the method with some modifications . The samS is the absorbance of the compounds to be tested and AC is the absorbance of the untreated control.The PTIO radical scavenging activity was assayed by referring to relevant methods . Brieflyl-tyrosine (3 mM) were added separately in 0.05 M potassium phosphate buffer (pH 6.5). A total of 80 µL of potassium phosphate buffer (pH 6.5), 80 µL of l-tyrosine (3 mM) solution, 20 µL of sample solution and 20 µL of mushroom tyrosinase (400 U/mL) were added to a 96-well microplate. The mixture reacted for 1 h at 37 °C. Kojic acid was selected as the positive control. The absorbance was measured at 490 nm using a microplate reader and Equation (3) was used to calculate the inhibition rate.The mushroom tyrosinase inhibitory activity was partially improved on the basis of reports ,54. MushC is the absorbance of the untreated control.Equation (3) is the absorbance of the test compound and A3. A total of 120 µL of 0.1 M phosphate buffer (pH 8.0), 20 µL of 3 mM DTNB solution, 20 µL of sample solution and 20 µL of AChE (0.2 U/mL) were sequentially added to the 96-well microplate, and the mixture reacted for 10 min at 37 °C. The reaction was started by adding 20 µL of 3 mM ATCI and the mixture was incubated at 37 °C for 20 min. Donepezil was chosen as the positive control. The absorbance was tested at 412 nm and the inhibition rate was calculated based on Equation (4).s and Aj are the absorbance of the compound to be tested and tested compound blanks, respectively, and AC is the absorbance of the untreated control.The experiment of acetylcholinesterase inhibition activity was slightly modified according to the literature . Acetylc1–2) and thirteen compounds (3–15) have been reported and isolated from C. glabra leaves. Their structures were identified mainly by NMR, UV, IR, HR-ESI-MS, ORD and ECD. Biologically, compounds 3–6, 10 and 13 exhibited obvious antioxidant activities, and compounds 5, 10 and 13 showed significant tyrosinase inhibitory activities. At an initial concentration of 1 mg/mL, compounds 3, 5 and 6 demonstrated moderate inhibitory activities against acetylcholinesterase. These results indicate that C. glabra is a potent source of natural antioxidants that could be used in managing diseases involved with the overexpression of tyrosinase and acetylcholinesterase.In conclusion, two new phenolic of ceroffesters A-B (


However, other D3 metabolites, such as 20-hydroxyvitamin D3 and lactone D3, also show interesting biological activities and stable isotope-labeled derivatives are required for LC-MS/MS analysis of their concentrations in serum. Here, we describe a versatile synthesis of deuterium-labeled D3 metabolites using A-ring synthons containing three deuterium atoms. Deuterium-labeled 25(OH)D3 (2), 25(OH)D3-23,26-lactone (6), and 1,25(OH)2D3-23,26-lactone (7) were synthesized, and successfully applied as internal standards for the measurement of these compounds in pooled human serum. This is the first quantification of 1,25(OH)2D3-23,26-lactone (7) in human serum.Blood levels of the vitamin D Among them, 25-hydroxyvitamin D3 (25(OH)D3) (2) is generated from D3 (1) by CYP2R1 and/or CYP27A1-mediated hydroxylation at C25 in the liver, and the resulting 25(OH)D3 (2) is further metabolized to the active form of D3, 1α,25-dihydroxyvitamin D3 2D3) (3), by CYP27B1-mediated oxidation at C1α in the kidneys (2D3 (3) plays a key role in the regulation of bone metabolism in vivo . Th. Th13-d3 content . In thison steps .16 bearing a hydroxyl group at C1α was synthesized 4 and L-(+)-DET [15 in a 79% yield (3 steps) [15 was 10:1, and the undesired C1β diastereomer was removed by kinetic resolution, using acylation with isopropyl acid anhydride in the presence of -HyperBTM [15 in an 82% yield as a single diastereomer. The undesired diastereomer at C3 was also removed via silica gel column purification. The deuterium-labeled enyne 16, in which the secondary alcohol was protected as the TBS ether, was obtained in an 82% yield.Next, the deuterium-labeled enyne thesized . The alc from 12 ,32. The -(+)-DET , and the3 steps) ,32. The HyperBTM , to give13-d3 and bromoolefin 17 followed by deprotection of the silyl groups provided 25(OH)D3-d3 (2-d3) in a 36% yield [3-23,26-lactone3-d (6-d3) and 1,25(OH)2D3-23,26-lactone-d3 (7-d3) were similarly synthesized by reacting bromoolefin 18 and enynes 13-d3 and 16-d3, respectively [2-d3 and 6-d3, the undesired diastereomers at C3α were separated by an HPLC -1,2,4-triazoline-3,5-dione-phenyl anthracene) [With the deuterium-labeled Dhracene) , and the2 and 2-d3 (tR: 5.60 min). Similarly, 6 and 6-d3 showed the same tR of 3.50 min, and 7 and 7-d3 showed the same tR of 2.15 min, indicating that the deuterium-labeled compounds are suitable as internal standards for the isotope dilution method. We also observed small peaks at the retention times of 5.20 min  was mixed with the internal standards solution (200 μL). Each sample was loaded onto a supported liquid extraction column  and eluted three times with 600 mL hexane/ethyl acetate  using a PRESSURE+48 positive pressure manifold . The combined eluates were evaporated to dryness in a centrifugal evaporator. The ion peaks of the metabolites matched well with those of the corresponding internal standards in the pretreated samples D3 (2), 25(OH)D3-23, 26-lactone (6), and 1,25(OH)2D3-23, 26-lactone (7). These deuterium-labeled D3 metabolites were successfully applied as internal standards for the quantification of the metabolites in pooled human serum by LC-MS/MS using the isotope dilution method. This is the first quantification of 1,25(OH)2D3-23, 26-lactone (7) in human serum.We synthesized deuterium-labeled A-ring-


Their structures were established based on spectroscopic methods. The absolute configurations of acrucipentyn A was assigned by the modified Mosher’s method and ROESY data analysis. Acrucipentyns A–E were identified to be the very first examples of chlorine-containing asperpentyn-like compounds. The cytotoxic and antimicrobial activities of the isolated compounds were examined. Acrucipentyns A–F were found as antimicrobial agents, which inhibited sortase A enzyme activity, bacterial growth and biofilm formation of Staphylococcus aureus and decreased LDH release from human keratinocytes HaCaT in S. aureus skin infection in an in vitro model.Six new polyketides acrucipentyns A–F ( Penicillium chrysogenum, isolated from marine sediments, was reported to be a producer of the unique dimeric nitrophenyl trans-epoxyamides, chrysamides A–C D20 – 36.0 (c 0.09 CH3OH); CD , λmax (∆ε) 210 (−0.97), 266 (−0.14) nm, see 3OH) λmax (log ε) 271 (2.54), 256 (2.50) and 224 (3.77) nm, see 1H and 13C NMR data, see m/z 229.0625 [M – H]– , 253.0599 [M + Na]+ .Acrucipentyn A (2): amorphous solids; [α]D20 +46.2 (c 0.09 CH3OH); CD , λmax (∆ε) 223 (+0.87) nm, see 3OH) λmax (log ε) 224 (3.93), 200 (3.41) and 194 (3.51) nm, see 1H and 13C NMR data, see m/z 229.0634 [M – H]– , 253.0601 [M + Na]+ .Acrucipentyn B (3): amorphous solids; [α]D20 –127.9 (c 0.07 CH3OH); CD , λmax (∆ε) 223 (−0.89), 297 (−0.11), 305 (−0.12) nm, see 3OH) λmax (log ε) 270 (3.06), 251 (2.63) and 223 (3.70) nm, see 1H and 13C NMR data, see m/z 229.0631 [M – H]– , 253.0595 [M + Na]+ .Acrucipentyn C (4): amorphous solids; [α]D20 – 94.3 (c 0.06 CH3OH); CD , λmax (∆ε) 210 (−3.73), 227 (−1.44), 238 (−1.17) nm, see 3OH) λmax (log ε) 259 (3.90), 226 (3.56) and 198 (3.80) nm, see 1H and 13C NMR data, see m/z 227.0471 [M – H]– , 251.0441 [M + Na]+ .Acrucipentyn D (5): amorphous solids; [α]D20 –60.5 (c 0.09 CH3OH); CD , λmax (∆ε) 199 (−5.11), 226 (−1.19), 237 (−0.83) nm, see 3OH) λmax (log ε) 260 (4.22), 226 (3.77) and 198 (4.10) nm, see 1H and 13C NMR data, see m/z 227.0470 [M – H]– , 251.0441 [M + Na]+ .Acrucipentyn E (6): amorphous solids; [α]D20 +40.3 (c 0.11 CH3OH); CD , λmax (∆ε) 199 (−4.56), 226 (−0.72), 259 (+1.13) nm, see 3OH) λmax (log ε) 259 (4.05), 226 (3.59) and 201 (3.88) nm, see 1H and 13C NMR data, see m/z 191.0704 [M – H]– , 215.0677 [M + Na]+ .Acrucipentyn F (1 (4.0 mg) 2,2-dimethoxypropane (0.5 mL) and catalyst p-toluenesulfonic acid (0.8 mg) at room temperature were added and the solution was stirred for 24 h. After the evaporation of the solvent, the product was dissolved in MeOH and purified by reversed-phase HPLC (YMC ODS-A column) eluted with CH3CN-H2O (50:50) to yield the acetonide product 1a (1.5 mg). Compound 4 (4.0 mg) was treated similarly and yielded the acetonide product 4a (1.4 mg).To a DMFA solution of 1a): amorphous solids; 1H NMR  δ: 5.20 , 5.19 , 4.37 , 4.20 , 4.11 , 4.00 , 3.50 , 2.03 , 1.97 , 1.85 , 1.47 , 1.34 ; 13C NMR  δ: 128.9 (C-3′), 122.0 (C-4′), 110.3 (C-1″), 90.3 (C-1′), 84.1 (C-2′), 80.5 (C-2), 77.4 (C-1), 70.7 (C-4), 67.1 (C-3), 35.0 (C-6), 29.3 (C-3″), 27.2 (C-2″), 26.5 (C-5), 24.6 (C-5′), see m/z 293.0912 [M + Na]+ .Acetonide of acrucipentyn A (4a): amorphous solids; 1H NMR  δ: 5.33 , 5.31 , 6.11 , 4.63 , 4.58 , 4.57 , 4.38 , 1.91 , 1.37 , 1.33 ; 13C NMR  δ: 137.7 (C-5), 130.0 (C-6), 129.3 (C-3′), 122.8 (C-4′), 110.6 (C-1″), 91.3 (C-2′), 87.9 (C-1′), 77.1 (C-2), 74.6 (C-1), 66.0 (C-4), 62.8 (C-3), 27.8 (C-2″/C-3″), 26.0 (C-2″/C-3″), 23.4 (C-5′), see m/z 291.0758 [M + Na]+ .Acetonide of acrucipentyn D (1a (0.7 mg) 4-dimethylaminopyridine  and (S)-MTPA-Cl (10 μL) at room temperature were added, and the solution was stirred for 24 h. After the evaporation of the solvent, the residue was purified by RP HPLC on a YMC ODS-AM column eluted with CH3CN-H2O (70:30) to afford the (R)-MTPA ester (1a-1). The (S)-MTPA ester (1a-2) was prepared in a similar manner using (R)-MTPACl. 1H NMR and COSY data, see To a pyridine solution of R)-MTPA ester of 1a: 1H NMR  δ: 5.56 , 5.22 , 5.20 , 4.32 , 4.30 , 4.09 , 2.80 , 2.24 , 2.09 , 1.84 , 1.50 , 1.33 . HRESIMS m/z 509.1310 [M + Na]+ (S)-MTPA ester of 1a: 1H NMR  δ: 5.65 , 5.24 , 5.21 , 4.29 , 4.44 , 4.06 , 3.27 , 2.33 , 2.22 , 1.85 , 1.50 , 1.34 . HRESIMS m/z 509.1307 [M + Na]+ .(6 (12.0 mg) at room temperature, and the solution was stirred for 12 h. After the evaporation of the solvent, the product was dissolved in MeOH and purified by reversed-phase HPLC (Phenomenex Hydro-RP column) eluted with CH3OH-H2O (50:50) to yield compound 4 (3.7 mg) and compound 5 (6.4 mg).Next, 2,2-dimethylpropanoyl chloride (0.5 mL) was added to an aqueous solution of The human normal cell lines HEK 293T and MRC-9 cell lines were purchased from ECACC . Human normal cell lines PNT2 and RWPE-1 as well as human cancer cell lines PC-3, DU145, 22Rv1, VCaP and LNCaP, as well as a human normal prostate line were purchased from ATCC . Human normal cell line HUVEC (passage 11) was kindly donated by Prof. Sonja Loges . The human keratinocytes cell line HaCaT was kindly provided by Prof. N. Fusenig . All the cells had a passage number ≤ 30.2 at 37 °C. The cells were continuously kept in a culture for 3 months maximum and regularly checked for mycoplasma infection using MycoAlert™ PLUS Mycoplasma Detection Kit  and stable phenotype using light microscopy – at m/z 229.0634 and m/z 229.0631, respectively. These data, coupled with 13C NMR spectral data (DEPT), established the molecular formulas of 2 and 3 as C11H15ClO3 for both. A close inspection of the 1H and 13C NMR data /H-4 (δH 4.22) and H-5β (δH 1.91)/H-1 (δH 3.92). Due to the small amount of compound 2, the absolute configuration establishing by Mosher’s method was impossible. Compound 2 was named acrucipentyn B.The main elations  indicateonstants  and ROESonstants  correlat1H–1H COSY correlations of H-1(OH)/H-2/H-3(OH)/H-4(OH)/H2-5/H-6 together with the 1H-13C HMBC correlations (H 4.38)/C-1 (δC 74.2), C-2 (δC 68.9) and C-6 (δC 35.0); OH-3 (δH 4.50)/C-2, C-3 (δC 79.5) and C-4 (δC 71.1), and OH-4 (δH 4.04)/C-3, C-4 and C-5 (δC 34.2) indicated the location of the hydroxy groups at C-1, C-3, C-4 and a chlorine atom at C-2 in a penta-substituted cyclohexane ring of 3. The structure of the 3-methyl-3-buten-1-ynyl side chain and its position at C-6 in 3 were determined by HMBC correlations (1).The elations  OH-1 , established the molecular formula of 4 as C11H13ClO3. The 1H and 13C NMR , one methyl group , one olefinic methylene  and five methines , including three oxygen-bearing methines and one olefinic methine, two 2sp quaternary carbons and one triple bond (δC 88.2 and 89.9).The HRESIMS of  13C NMR , DEPT anH 4.25) to C-1 (δC 67.9), C-2 (δC 68.6), C-4 (δC 65.1) and C-5 (δC 136.0); from H-5 (δH 5.92) to C-1, C-4, C-6 (δC 123.5) and C-1′ (δC 88.2), and from OH-4 (δH 5.25) to C-4, C-5, together with 1H–1H COSY correlations of H-1(OH)/H-2(OH)/H-3/H-4(OH)/H-5, indicated the presence of a penta-substituted cyclohexene ring with ∆5,6 double bond, the location of the hydroxy groups at C-1, C-2, C-4 and a chlorine atom at C-3 in 4. The structure of the 3-methyl-3-buten-1-ynyl side chain and its position at C-6 in 4 were determined by HMBC correlations (1).The HMBC correlations  from H-3elations , as for 4 was assigned based on 1H-1H coupling constants (H 3.87)/OH-4 (δH 5.25). The acetonide derivative (4a) of compound 4  and dissimilar magnetic environment of acetonide methyls (Δ = 0.01 ppm) (erythro configuration of the diol group at C-1 and C-2 – at m/z 227.0468. These data, coupled with 13C NMR spectral data (DEPT), suggested the molecular formula of 5 as C11H13ClO3. The 1H and 13C NMR data .The HRESIMS of NMR data  of 5 revelations , as for 5 was assigned based on ROESY correlations (H 3.69)/H-4 (δH 3.97), OH-1 (δH 5.73), OH-3 (δH 5.48); H-3 (δH 3.31)/H-1 (δH 4.05), OH-4 (δH 5.31) and 1H-1H coupling constants (5 were unsuccessful. Etherification of compound 5 with (S)- and (R)-MTPA-Cl resulted in the formation of esters at three hydroxyl groups, which made it impossible to establish the absolute configuration by the modified Mosher’s method. Compound 5 was named acrucipentyn E.The relative configuration of elations  H-2  and NMR data (1H and 13C NMR data for this compound were similar to those obtained for (+)-asperpentyn [6 with acrucipentyns D (4) and E (5), led us to suggest a configuration of asymmetric centers for it, different from the known asperpentyns.The molecular formula of compound NMR data . The 1H erpentyn ,25, with1H-1H coupling constants were useless to establish the relative stereochemistry of 6 unambiguously. Therefore, an epoxy ring-opening reaction was carried out in 6. The reaction of compound 6 with 2,2-dimethylpropanoyl chloride in an aqueous medium  were identical to acrucipentyns D (4) and E (5). The presence of only two reaction products confirmed the SN2 mechanism of the epoxy ring opening, which corresponded to the literature data [6. Compound 6 was named acrucipentyn F.The ROESY spectrum data and s medium  yielded ure data . The oriTruncatella angustata [Beauveria felina KMM 4639 [It should be noted that acrucipentyn F is a stereoisomer of well-known fungal metabolites (−)-asperpentyn ,28 and , their inhibitory effects on bacterial growth and biofilm formation of Staphylococcus aureus were investigated  release from human keratinocytes HaCaT, co-cultivated with . aureus .S. aureus caused a significant increase in the LDH release from keratinocytes during co-cultivation. The addition of compounds 1–6 at a concentration of 10 μM reduced the LDH release by 30–50%. The greatest effect was registered for compounds 1, 2, 4, and 5.In normal conditions, LDH weakly releases from cells to culture media. 2 and 4, which showed significant activity in co-cultivation HaCaT cells with S. aureus, did not show high antibacterial and anti-biofilm-forming activity, in contrast to substances 1 and 5. We assume that the cytoprotective effect of substances 1–6 in the in vitro infectious skin lesions could be due not only to their anti-S. aureus effects, but also to their anti-inflammatory and other cytoprotective effects. Recently, we observed similar dual effects during an investigation on flavuside B, an inhibitor of sortase A enzymatic activity derived from fungi. Flavuside B was able to inhibit S. aureus growth and biofilm formation, as well as protect HaCaT keratinocytes against S. aureus infection in a co-culture model via an anti-inflammatory pathway [Interestingly, compounds  pathway .Our work is the very first detailed investigation of anti-Staphylococcal activity of isoprenylated cyclohexanols. To the best of our knowledge, earlier, only oxirapentyns A and D were found as antimicrobial agents among related compounds . Thus, t1–6) were isolated from the alga-derived fungus Asteromyces cruciatus KMM 4696. The absolute configuration of acrucipentyn A was assigned by the modified Mosher’s method and ROESY data. Acrucipentyns A–E are the very first members of chlorine-contained monocyclic cyclohexanols containing a 3-methylbutenynyl unit. The compounds have shown inhibitory activity against sortase A from Staphylococcus aureus, as well as inhibition of S. aureus growth and biofilm formation, while no cytotoxicty to mammalian cells was observed. Moreover, acrucipentyns A–F (1–6) protected human keratinocytes HaCaT from S. aureus toxicity in skin infection in an in vitro model. Thus, the isolated compounds hold a good potential as antimicrobal agents and should be further investigated.Six new isoprenylated cyclohexanols acrucipentyns A–F (


Mammea siamensis belongs to the Calophyllaceae family, which is a medicinal plant that is used in traditional Thai preparations. The hexane and dichloromethane extracts of this plant were found to have potent antimalarial activity. Therefore, this study aimed to isolate active compounds from M. siamensis flowers and evaluate their antimalarial potential and their interactions with Plasmodium falciparum lactate dehydrogenase (PfLDH).The emergence of antimalarial drug resistance encourages the search for new antimalarial agents. M. siamensis flowers were isolated by chromatographic techniques and evaluated for their antimalarial activity against chloroquine (CQ)-resistant P. falciparum (K1) strains using a parasite lactate dehydrogenase (pLDH) assay. Interactions between the isolated compounds and the PfLDH enzyme were investigated using a molecular docking method.The compounds from 1) and a mixture of β-sitosterol and stigmasterol (5); two mammea coumarins, mammea A/AA cyclo D (6) and mammea A/AA cyclo F (7); and nine xanthones, 4,5-dihydroxy-3-methoxyxanthone (2), 4-hydroxyxanthone (3), 1,7-dihydroxyxanthone (4), 1,6-dihydroxyxanthone (8), 1-hydroxy-5,6,7-trimethoxyxanthone (9), 3,4,5-trihydroxyxanthone (10), 5-hydroxy-1-methoxyxanthone (11), 2-hydroxyxanthone (12), and 1,5-dihydroxy-6-methoxyxanthone (13). Compound 9 exhibited the most potent antimalarial activity with an IC50 value of 9.57 µM, followed by 10, 1, 2 and 13 with IC50 values of 15.48, 18.78, 20.96 and 22.27 µM, respectively. The molecular docking results indicated that 9, which exhibited the most potent activity, also had the best binding affinity to the PfLDH enzyme in terms of its low binding energy  and formed interactions with ARG109, ASN140, and ARG171.The isolation produced the following thirteen compounds: two terpenoids, lupeol (M. siamensis flowers exhibited antimalarial activity. The result suggests that 1-hydroxy-5,6,7-trimethoxyxanthone is a possible lead structure as a potent inhibitor of the PfLDH enzyme.These findings revealed that isolated compounds from  Plasmodium falciparum is the most virulent parasite, as it results in a high mortality and morbidity rate caused approximately 229 million cases and 409,000 deaths worldwide in 2019 [P. falciparum has now developed mechanisms to resist most antimalarial drugs, including current ACTs [P. falciparum (PfLDH) has been considered an important molecule and an essential drug target for malaria treatments. This enzyme catalyzes the interconversion of pyruvate to lactate in the final step of glycolysis, which is required for energy production in living cells [Malaria remains one of the most important tropical parasitic diseases, as it results in 1–3 million deaths annually [ in 2019 . Artemis in 2019 . ACTs co in 2019 . Howeverent ACTs . The emeent ACTs . As currng cells .Mammea siamensis T. Anders. is a Thai medicinal plant that is locally known as Saraphi and belongs to the family Calophyllaceae. This plant is widely distributed in Thailand, Myanmar, Laos, Cambodia, and Vietnam [In the continued search for new molecular targets for drug design, herbal medicine offers an interesting choice for the research and development of antimalarial drugs because plants are an excellent source of many pharmaceutical compounds . Mammea  Vietnam . Its flo Vietnam . This pl Vietnam , aromata Vietnam  and the  Vietnam . HoweverM. siamensis possessed potent antimalarial properties, with IC50 values of 5.32 and 5.67 µg/ml, respectively. Therefore, the compounds from this plant were isolated and investigated for antimalarial activity. Moreover, the mechanism of the isolated compound with the PfLDH enzyme was also evaluated using a molecular docking technique.In our study, the hexane and dichloromethane extracts of M. siamensis flowers were collected from Surat Thani Province, Thailand, in 2020. The collection of plant materials was performed in accordance with the relevant guidelines and regulations of the Plant Varieties Protection, Department of Agriculture, Ministry of Agriculture and Cooperatives, Thailand. The plant was identified by a botanist from the School of Pharmacy at Walailak University. The voucher specimen (SMD 122006002) was deposited in the School of Medicine, Walailak University.The 3 on an Avance NEO, a Bruker Spectrometer operating at 500 MHz for 1 H and 125 MHz for 13 C. Column chromatography was carried out using silica gel . All reagents were purchased from Sigma, USA. All solvents were analytical reagent grade and purchased from Labscan, Thailand.The nuclear magnetic resonance (NMR) spectra were recorded in Acetone-d6 and CDClM. siamensis flowers (3.5 kg) were ground and macerated successively with three solvents of increasing polarity. The plant material was initially extracted with hexane (2 × 20 L) for one week at room temperature. The solvent extraction was filtered through Whatman No. 1 filter paper and concentrated under reduced pressure at 50 °C to obtain a yellowish viscous crude hexane extract (21.3 g). The residue was further macerated with dichloromethane and ethanol under the same conditions mentioned above to afford a brownish viscous crude containing dichloromethane (29.7 g) and ethanol (16.2 g) extracts.The dried 50 = 5.32 and 5.67 µg/ml, respectively), were selected to further isolate active compounds by chromatographic techniques. The hexane extract (18.00 g) was separated by quick column chromatography (QCC) over silica gel and eluted by step-gradient elution, which starting with hexane and then increased in polarity with EtOAc to afford seven major fractions (F1-F7) based on thin-layer chromatography (TLC) profiles. Fraction 2 (3.64 g) was purified by column chromatography (CC) using 5% EtOAc in hexane as the eluent to produce compound 1 as a white crystal solid (470.10 mg) and compound 2 as a yellow oil (170.10 mg). Fraction 4 (2.13 g) was purified by CC using 10% EtOAc in hexane as an eluent to obtain six subfractions (H1-H6). Subfraction H3 (0.57 g) was purified by CC with 10% acetone in hexane to produce compound 3 as a yellow crystal (60.50 mg). Subfraction H6 (2.17 g) was purified by CC using 15% EtOAc in hexane to give five subfractions (H6A-H6E). Subfraction H6C (0.37 g) was further purified by CC using 15% acetone in hexane followed by preparative thin layer chromatography (PTLC) to produce compound 4 as a yellow oil (24.20 mg).A bioassay-guided isolation of antimalarial active compounds was performed. Hexane and dichloromethane, which exhibited high activities (IC5 as a white powder (45.30 mg) and compound 6 as a yellow powder (22.40 mg). Fraction 5 (2.87 g) was purified by CC to produce seven subfractions (5 A-5G). Subtraction 5B (0.48 g) was purified by CC with 15% EtOAc in hexane to produce compound 7 as a yellow powder (5.80 mg). Subtraction 5D was separated by CC with 15% EtOAc in hexane to afford compound 8 as a yellow powder (5.70 mg) and compound 9 as a yellow powder (5.30 mg).The dichloromethane extract (25.00 g) was subjected to QCC over silica gel by step-gradient elution starting from dichloromethane, and then the polarity was increased with EtOAc and acetone to produce 10 fractions (F1-F10) based on TLC profiles. Fraction 3 (4.17 g) was purified by QCC to produce six subfractions (C1-C6). Subtraction C3 (0.71 g) was further subjected to CC with 10% EtOAc in hexane to afford compound 10 as a yellow powder (41.50 mg) and compound 11 as a yellow powder (22.50 mg). Fraction 8 (0.22 g) was purified by CC to give eight subfractions (8 A-8 H). Subtraction 8 C was purified by CC with 25% EtOAc in hexane to afford compound 12 as a yellow powder (57.40 mg) and compound 13 as a yellow powder (25.30 mg).Subfraction 5 F (0.32 g) was subjected to CC eluting with 20% EtOAc in hexane to afford three subtractions (5F1-5F3). Subtraction 5F3 (0.08 g) was further purified by CC eluting with 20% EtOAc/hexane to obtain compound 1–13 were elucidated by NMR analysis, and were confirmed by comparison with previously reported data in the literature.The structures of compounds P. falciparum (K1) strain was obtained from Dr. Rapatbhorn Patrapuvich, Department of Drug Research Unit for Malaria, Faculty of Tropical Medicine, Mahidol University, Thailand. P. falciparum was cultured according to the method of Trager and Jensen with some modifications [2 incubator. The culture medium was changed, and Giemsa-stained slides were prepared daily to monitor parasitemia.The chloroquine-resistant ications . The parications . All theP. falciparum using the Plasmodium lactate dehydrogenase (pLDH) assay described by Makler with some modifications [2 incubator. At the end of incubation, the plates were subjected to three freeze/thaw cycles (frozen at -20 °C and thawed at 37 °C) for complete hemolysis. The lysed cells were transferred to a new 96-well plate that contained a mixture of 100 µl of Malstat reagent and 20 µl of nitroblue tetrazolium/phenazine ethosulfate solution  and were incubated for 1 h in the dark. The experiments were performed in triplicate. These solutions were used to determine the activity of the lactate dehydrogenase (LDH) enzyme in the cultures. When LDH was present, a purple product was formed, and the optical density was measured using a microplate reader at a wavelength of 650 nm. The concentrations at which 50% inhibition of parasite growth (IC50) was calculated using a nonlinear regression curve contained in GraphPad PRISM version 6 .The in vitro antimalarial activity of the isolated compounds was assessed on cultured ications . Briefly4 cell/ml) and incubated for 24 h at 37 °C with 5% CO2. Cells were then treated with various concentrations of the tested compounds ranging from 5 to 80 µM, and the final concentration of DMSO in the tested dilutions was not higher than 1% for 48 h at 37 °C with 5% CO2. Subsequently, MTT solution was added to each well, and the plate was incubated for 2 h in a CO2 incubator. The medium was then removed, and 100 µl of DMSO was added to each well. The negative control was performed using growth medium alone instead of the tested compounds, while doxorubicin  was used as the positive toxic control. The assay was performed in triplicates. Finally, the optical density was determined using a microplate reader at a wavelength of 590 nm. The 50% cytotoxic concentration (CC50) was calculated using a nonlinear regression curve contained in GraphPad PRISM version 6 .The toxicity of isolated compounds was assessed by a 3--2,5-diphenyl tetrazolium bromide (MTT) assay according to a previous method . BrieflyPfLDL structure preparation.PfLDH in complex with β-nicotinamide adenine dinucleotide phosphate disodium salt (NADH) and oxamate was downloaded from the Protein Data Bank with PDB code 1LDG. The PfLDH structure was prepared using AutoDock Tools. The missing residues were incorporated. All water molecules and the oxamate were removed so that a new ligand could enter the active site [The crystal structure of ive site .M. siamensis were used as ligands for the docking study. The 3D structures of compounds, including artesunate and chloroquine, were generated using the HyperChem Professional 8.0 program. . Each structure was geometrically optimized using the semiempirical PM3 method. Subsequently, Gasteiger charges were assigned to the ligands using AutoDock Tools to model the appropriate structures for docking calculations.Fourteen compounds from M. siamensis and PfLDH were determined by molecular docking using the AutoDock 4.2 program  according to a previous method [PfLDH active site was selected as the ligand binding site. A grid box was constructed of 60 × 60 × 60 Å3 with a grid spacing of 0.375 Å and centered on 32, 30 and 32 Å for x, y, and z, respectively. All calculations were performed using the Lamarckian genetic algorithm (LGA) method with protein-fixed and ligand-flexible molecules. The resulting docked poses with root mean-square deviations (RMSDs) less than 2.0 Å were clustered together. The lowest energy-minimized conformation of the most populated cluster was used for further analysis of the hydrogen bond interactions [The binding mode and interaction of compounds from s method . The PfLractions . The 3D ractions .M. siamensis flower extracts, hexane and dichloromethane extracts exhibited potent antimalarial activity with IC50 values of 5.32 and 5.67 µg/ml, respectively. Therefore, these two extracts were selected for the isolation of active compounds. The extracts were fractionated and isolated by chromatographic techniques, and thirteen known compounds were obtained  were isolated from the hexane extract, and nine compounds (5–13) were obtained from the dichloromethane extract. These included two terpenoids, lupeol (1) [5) [6) [7) [2) [3) [4) [8) [9) [10) [11), 2-hydroxyxanthone (12) [13) [From the bioassay-guided isolation of the ned Fig. . Four copeol (1)  and a mi (1) [5) ; two mam [5) [6)  and mamm [6) [7) ; and nin [7) [2) , 4-hydro [2) [3) , 1,7-dih [3) [4) , 1,6-dih [4) [8) , 1-hydro [8) [9) , 3,4,5-t[9) [10) , 5-hydro12) [13) .Lupeol (1): white crystal solid. 1 H NMR : δ 4.59 , 4.70 , 3.23 , 1.00 , 0.98 , 0.88 , 0.86 , 0.84 , 0.79 , 1.72 . 13 C NMR : δ 14.6 (C-27), 16.1 (C-24), 16.1 (C-26), 16.2 (C-25), 18.1 (C-28), 18.4 (C-6), 19.4 (C-30), 21.1 (C-11), 27.7 (C-2), 25.3 (C-12), 27.6 (C-15), 28.1 (C-23), 29.9 (C-21), 34.5 (C-7), 35.7 (C-16), 37.4 (C-10), 38.1 (C-1), 38.9 (C-13), 39.0 (C-4), 40.1 (C-22), 41.0 (C-8), 42.9 (C-14), 43.1 (C-17), 47.9 (C-19), 48.4 (C-18), 50.6 (C-9), 55.4 (C-5), 79.2 (C-3), 109.4 (C-29), 151.0 (C-20).4,5-Dihydroxy-3-methoxyxanthone (2): yellow oil. 1 H-NMR : δ 7.21 , 7.30 , 7.31 , 7.41 , 7.68 , 3.93 . 13 C-NMR : δ 61.2 (3-OCH3), 113.5 (C-2), 116.1 (C-8), 116.1 (C-9a), 119.3 (C-6), 122.8 (C-8a), 123.1 (C-1), 123.3 (C-7), 144.7 (C-10a), 145.1 (C-3), 146.0 (C-5), 146.6 (C-4), 150.3 (C-4a), 175.4 (C-9).4-Hydroxyxanthone (3): yellow crystal. 1 H-NMR : δ 7.26 , 7.36 , 7.45 , 7.62 , 7.71 , 7.84 , 8.24 . 13 C-NMR : δ 115.9 (C-1), 118.1 (C-5), 120.1 (C-3), 121.5 (C-8a), 122.6 (C-9a), 123.8 (C-2), 124.0 (C-7), 126.1 (C-8), 134.9 (C-6), 145.4 (C-4a), 146.6 (C-4), 155.8 (C-10a), 176.2 (C-9).1,7-Dihydroxyxanthone (4): yellow oil. 1 H-NMR : δ 6.77 , 6.90 , 7.34 , 7.39 , 7.54 , 7.60 , 12.66 . 13 C-NMR : δ 105.3 (C-8), 106.6 (C-4), 108.7 (C-9a), 110.0 (C-2), 119.0 (C-5), 120.8 (C-8a), 125.3 (C-6), 136.2 (C-3), 151.0 (C-10a), 156.0 (C-4a), 156.1 (C-7), 161.8 (C-1), 181.7 (C-9).Mixture of β-sitosterol and stigmasterol (2:1) (5): white powder.β-sitosterol; 1 H NMR : δ 5.35 , 3.52 , 1.00 , 0.92 , 0.83 , 0.82 , 0.80 , 0.67 . 13 C-NMR : δ 12.0 (C-24), 12.4 (C-29), 19.1 (C-28), 19.4 (C-19), 19.7 (C-27), 20.3 (C-26), 21.5 (C-11), 23.5 (C-23), 26.5 (C-15), 26.5 (C-21), 28.6 (C-16), 29.6 (C-25), 32.1 (C-2), 32.2 (C-8), 32.3 (C-7), 34.3 (C-20), 36.5 (C-10), 36.8 (C-18), 37.4 (C-1), 40.1 (C-12), 42.6 (C-4), 42.8 (C-13), 46.4 (C-22), 50.4 (C-9), 56.5 (C-17), 57.1 (C-14), 72.1 (C-3), 121.9 (C-6), 141.1 (C-5).Stigmasterol ; 1 H NMR : δ 5.31 , 3.51 , 5.14 , 4.97, 1.02 , 0.91 , 0.82 , 0.81 , 0.80 , 0.70 . 13 C-NMR : δ 12.2 (C-24), 12.4 (C-29), 19.0 (C-28), 19.8 (C-27), 20.4 (C-26), 21.7 (C-11), 21.9 (C-19), 24.6 (C-15), 25.5 (C-23), 29.4 (C-16), 29.7 (C-25), 31.9 (C-8), 32.1 (C-2), 32.3 (C-7), 36.7 (C-10), 37.6 (C-1), 40.0 (C-12), 40.7 (C-18), 42.3 (C-13), 42.6 (C-4), 50.2 (C-9), 56.4 (C-17), 56.9 (C-14), 72.3 (C-3), 121.7 (C-6), 129.8 (C-21), 138.9 (C-20), 141.3 (C-5).Mammea A/AA cyclo D (6): yellow powder. 1 H-NMR : δ 0.96 , 0.96 , 1.57 , 1.57 , 2.20 , 2.94 , 5.61 , 5.96 , 6.87 , 7.27 , 7.30 , 7.30 , 7.38 , 7.41 , 14.77 . 13 C-NMR : δ 22.4 (C-9ʹ), 22.4 (C-10ʹ), 24.9 (C-8ʹ), 28.1 (C-4ʹ), 28.1 (C-5ʹ), 53.3 (C-7ʹ), 79.6 (C-3ʹ), 101.3 (C-8), 102.0 (C-4a), 107.0 (C-6), 112.5 (C-3), 115.3 (C-1ʹ), 126.1 (C-2ʹ), 126.9 (C-4ʹʹ), 127.4 (C-2ʹʹ), 127.4 (C-6ʹʹ), 128.0 (C-5ʹʹ), 128.1 (C-3ʹʹ), 139.1 (C-1ʹʹ), 154.6 (C-8a), 156.1 (C-4), 157.9(C-7), 159.4 (C-2), 164.2 (C-5), 206.5 (C-6ʹ).Mammea A/AA cyclo F (7): yellow powder. 1 H-NMR : δ 0.97 , 0.97 , 1.31 , 1.43 , 2.23 , 2.97 , 3.32 , 4.90 , 5.94 , 7.32 , 7.32 , 7.32 , 7.42 , 7.42 . 13 C-NMR : δ 22.6 (C-9ʹ), 22.6 (C-10ʹ), 24.8 (C-5ʹ), 25.0 (C-8ʹ), 26.1 (C-4ʹ), 26.7 (C-1ʹ), 51.8 (C-7ʹ), 71.5 (C-3ʹ), 92.7 (C-2ʹ), 102.4 (C-4a), 103.2 (C-6), 104.9 (C-8), 112.0 (C-3), 127.0 (C-6ʹʹ), 127.1 (C-2ʹʹ), 127.4 (C-5ʹʹ), 127.5 (C-3ʹʹ), 128.1 (C-4ʹʹ), 138.9 (C-1ʹʹ), 155.5 (C-8a), 156.4 (C-4), 159.5 (C-2), 164.0 (C-5), 164.3 (C-7), 204.9 (C-6ʹ).1,6-Dihydroxyxanthone (8): yellow powder. 1 H-NMR : δ 6.77 , 7.00 , 7.43 , 7.52 , 7.61 , 7.70 . 13 C-NMR : δ 106.7 (C-4), 108.3 (C-5), 109.2 (C-9a), 109.3 (C-2), 119.2 (C-8), 120.8 (C-8a), 124.3 (C-7), 136.8 (C-3), 150.1 (C-6), 154.1 (C-10a), 156.4 (C-4a), 162.3 (C-1), 181.9 (C-9).1-Hydroxy-5,6,7-trimethoxyxanthone (9): yellow powder. 1 H-NMR : δ 6.95 , 7.13 , 7.20 , 7.68 , 12.70 , 3.96 , 3.93 , 3.95 . 13 C-NMR : δ 54.3 (5-OCH3), 55.4 (7-OCH3), 60.1 (6-OCH3), 96.3 (C-8), 105.5 (C-4), 109.5 (C-2), 111.6 (C-9a), 118.4 (C-8a), 134.3 (C-3), 139.0 (C-5), 140.1 (C-10a), 141.3 (C-6), 150.0 (C-7), 157.8 (C-4a), 160.6 (C-1), 174.1 (C-9).3,4,5-Trihydroxyxanthone (10): yellow powder. 1 H-NMR : δ 7.22 , 7.29 , 7.30 , 7.42 , 7.69 . 13 C-NMR : δ 113.7 (C-2), 116.0 (C-8), 116.2 (C-9a), 119.3 (C-6), 122.9 (C-8a), 123.0 (C-1), 123.3 (C-7), 144.7 (C-10a), 145.2 (C-3), 146.0 (C-5), 146.6 (C-4), 150.2 (C-4a), 175.4 (C-9).5-Hydroxy-1-methoxyxanthone (11): yellow powder. 1 H-NMR : δ 6.94 , 7.12 , 7.19 , 7.26 , 7.62 , 7.70 , 3.94 , 9.24 . 13 C-NMR : δ 55.5 (1-OCH3), 105.8 (C-4), 109.5 (C-2), 112.0 (C-9a), 115.8 (C-8), 119.1 (C-6), 123.3 (C-7), 123.9 (C-8a), 135.1 (C-3), 144.1 (C-10a), 145.7 (C-5), 157.5 (C-4a), 160.6 (C-1), 174.7 (C-9).2-Hydroxyxanthone (12): yellow powder. 1 H-NMR : δ 7.36 , 7.43 , 7.51 , 7.54 , 7.61 , 7.81 , 8.23 , 8.90 . 13 C-NMR : δ 114.2 (C-1), 123.2 (C-5), 124.5 (C-4), 126.2 (C-8a), 127.5 (C-9a), 128.8 (C-7), 129.3 (C-3), 131.3 (C-8), 139.9 (C-6), 155.2 (C-4a), 159.1 (C-2), 161.3 (C-10a), 181.1 (C-9).1,5-Dihydroxy-6-methoxyxanthone (13): yellow powder, 1 H-NMR : δ 6.53 , 6.79 , 6.81 , 7.45 , 7.61 , 12.62 , 3.79 . 13 C-NMR : δ 60.8 (6-OCH3), 106.6 (C-4), 107.8 (C-9a), 110.1 (C-2), 113.7 (C-7), 114.2 (C-8a), 121.1 (C-8), 134.5 (C-5), 136.3 (C-3), 150.9 (C-10a), 155.9 (C-4a), 156.7 (C-6), 161.8 (C-1), 181.1(C-9).1–13 was determined against the P. falciparum K1 strain by the pLDH assay and was compared with chloroquine and artesunate, a positive control (Table 9) exhibited the highest effect with good activity (IC50 = 9.57 µM), followed by 3,4,5-trihydroxyxanthone  and lupeol , while 4,5-dihydroxy-3-methoxyxanthone (2), 1,5-dihydroxy-6-methoxyxanthone (13), and 5-hydroxy-1-methoxyxanthone (11) also exhibited antimalarial effects with moderate activity with IC50 values of 20.96, 22.27 and 29.32 µM, respectively  with PfLDH were created, as shown in Fig. 9), which possessed the most potent antimalarial property against P. falciparum (IC50 = 9.57 µM), exhibited the best binding affinity to PfLDH in terms of its low binding energy of -7.35 kcal/mol, which was the highest observed affinity to an enzyme; however, its affinity was lower than that of artesunate, which had a binding energy of -8.57 kcal/mol. Four hydrogen bonds between the amino group of ARG109, ASN140, and ARG171 interacted with compound 9. An oxygen atom of the methoxy group at C5, C6, and C7 were responsible for these polar interactions , which also possessed good activity (IC50 = 15.48 µM), showed a remarkable binding affinity to PfLDH with a binding energy of -7.25 kcal/mol. It strongly interacted with PHE100, ASN140 and SER245. All three hydroxyl groups in this compound interacted with those residues, and double hydrogen bonds were observed for PHE100 and ASN140  interacted with amino acids lower than artesunate, which formed six hydrogen bonds, and interacted with GLY29, ILE31, GLY32, ILE54, THR97, and GLY99 of the PfLDH active site , a terpenoid compound, showed a remarkable binding affinity to PfLDH with a binding energy of -7.21 kcal/mol. It does not interact with any enzyme residue but forms hydrophobic interactions with residues ILE54, VAL55, PHE100, and ILE119. 4,5-Dihydroxy-3-methoxyxanthone (2), a moderately active compound, interacted with PHE100, ASN140 and SER245. A double hydrogen bond between ASN140 and the oxygen at O10 and the methoxy group at C3 was observed for this compound. This compound also forms hydrophobic interactions with VAL138, LEU167, ALA236, PRO246 and PRO250. 1,5-Dihydroxy-6-methoxyxanthone (13) had a lower binding energy  and formed three hydrogen bonds with ILE54, TYR85 and ILE199. It formed additional hydrophobic interactions with VAL26, PHE52, ASP53, ILE54, ALA98, ILE119, and ILE123. 5-Hydroxy-1-methoxyxanthone (11) showed a binding affinity to PfLDH with a binding energy of -7.01 kcal/mol and formed two hydrogen bonds with ILE54 and TYR85. It also forms hydrophobic interactions with residues VAL26, PHE52, ILE54, ALA98, ILE119 and ILE123.Lupeol , which were the most commonly found compounds in this study, are naturally secondary metabolite compounds that can be found in diverse terrestrial and marine plants, fungi, and lichen [Isolating d lichen . Xanthond lichen . Xanthond lichen . The biod lichen .2, 3, 4, 8, 9, 10, 11, 12, 13), the results showed that they possessed a wide range of antimalarial effects that ranged from 9.57 to 74.97 µM. 1-Hydroxy-5,6,7 trimethoxyxanthone (9), which contains one hydroxyl group and three methoxy groups, showed the highest activity (IC50 = 9.57 µM). 3,4,5-Trihydroxyxanthone (10), containing three hydroxyl groups, also possessed good activity (IC50 = 15.48 µM), whereas 4-hydroxyxanthone (3) and 2-hydroxyxanthone (12), which contain only one hydroxyl group, possessed weak activity . Remarkably, 1,7-dihydroxyxanthone (4) and 1,6-dihydroxyxanthone (8), which contain two hydroxyl groups, appeared to have comparable activities. The results clearly revealed that the increase in the hydroxyl group becomes more frequent with 10 compared with 3 and 12. Interestingly, the presence of a methoxy group also increased the antimalarial effect, as observed from the comparison of 5-hydroxy-1-methoxyxanthone  and 4-hydroxyxanthone  and as clearly observed in 1-hydroxy-5,6,7-trimethoxyxanthone , the strongest active compound bearing three methoxy groups, when compared with 11, which bears only one hydroxyl and methoxy group.Among nine xanthone compounds in the present study  and formed many interactions. Methoxy and hydroxyl groups of these compounds play an important role in forming hydrogen bonds. For compound 9, all three methoxy groups interacted with ARG109, ASN140, and ARG171. For compound 10, all three hydroxyl groups extensively formed five hydrogen bonds with PHE100, ASN140, and SER245.The docking results in accordance with the in vitro study showed that compounds PfLDH were weaker interactions than that of polyhydroxylated compounds in terms of binding energy. An interesting result was observed with compound 9, which contains one hydroxyl group but has three methoxy groups, and it showed relatively strong affinity with the enzyme in terms of lower binding energy.In compounds that consist of only one hydroxyl group, which result in a weak antimalarial effect based on the in vitro results, the predicted hydrogen bond interactions with 6) and mammea A/AA cyclo F (7), the docking results were also in accordance with the in vitro results. These compounds interacted with PfLDH with a low binding energy and formed only one and two hydrogen bonds for 6 and 7, respectively.In the case of mammea coumarins, mammea A/AA cyclo D  and PfATP6, the SERCA-type Ca2+-ATPase enzyme present in the malarial parasite have been proven to be major molecular drug targets of antimalarial drugs [Regarding other al drugs . For DHFal drugs . The actal drugs .M. siamensis flowers exhibited antimalarial activity. Xanthones are potent compounds that are responsible for the antimalarial activity of this plant. The findings suggest that 1-hydroxy-5,6,7-trimethoxyxanthone could be a promising lead structure as a potent inhibitor of the PfLDH enzyme. Further in vivo studies are necessary to determine the efficacy of the compounds and their toxicities.These findings revealed that isolated compounds from


Based on that information, we converted cyclic peptides to small molecules using PepMetics® scaffolds that can mimic the α-helix or β-turn of the peptide. Finally, we succeeded in generating small-molecule compounds with good IC50  against CTLA-4. This strategy is expected to be a useful approach for small-molecule design targeting PPIs, even without having structural information such as that associated with X-ray crystal structures.Currently, various pharmaceutical modalities are being developed rapidly. Targeting protein–protein interactions (PPIs) is an important objective in such development. Cyclic peptides, because they have good specificity and activity, have been attracting much attention as an alternative to antibody drugs. However, cyclic peptides involve some difficulties, such as oral availability and cell permeability. Therefore, while small-molecule drugs still present many benefits, the screening of functional small-molecule compounds targeting PPIs requires a great deal of time and effort, including structural analysis of targets and hits. In this study, we investigated a rational two-step strategy to design small-molecule compounds targeting PPIs. First, we obtained inhibitory cyclic peptides that bind to cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) by ribosomal display using PURE Protein–protein interactions (PPIs) regulate various cellular processes, some of which are responsible for severe diseases such as cancer. Therefore, disease-related PPIs have recently attracted attention as drug-discovery targets. As pharmaceutical modalities have become increasingly diversified, many of their characteristics have been clarified. Small-molecule drugs are suitable for targets with deep, narrow, and rigid binding surfaces, such as receptors and enzymes, but they are generally inadequate for targets with flat, wide, and flexible binding surfaces, as is often the case in PPIs. By contrast, peptide drugs are currently being actively developed, along with in vitro screening technologies, as the next generation of drugs able to inhibit PPIs while maintaining the same level of specificity as antibodies despite their low molecular weight (0.5–5 kDa). Unfortunately, even though peptide drugs can be synthesized chemically, the manufacturing cost is higher compared to small-molecule drugs. Moreover, they are not easily administered orally and have difficulty penetrating into cells. The development of small-molecule drugs targeting PPIs remains an important challenge. The current mainstream method of small-molecule drug discovery targeting PPIs demands first that the structural information of target proteins complexed with small molecules be identified using various structural analysis methods, such as cryogenic electron microscopy, X-ray crystallography, and NMR analysis. None of these is an easy task ,2,3. TheWe chose cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) as a target protein. CTLA-4 is expressed on the surface of T cells, activated by the antigen presentation from dendritic cells, and binds the related ligands B7-1 and B7-2 with higher affinity than T cell activator CD28, thereby suppressing T cell activation ,6. In otfrex®RD  inhibitor screening assay. The biotinylated C4m-3127 peptide was optimized to a concentration of 30 μM in an earlier experiment. CTLA-4 immobilized on a 384-well plate was reacted with PGF00506 in 11-point two-fold serial dilution from 1000 to 0.98 μM by assay buffer. Then, the biotinylated peptides were added to wells for competitive reaction. After washing, the residual amounts of biotinylated peptides were detected using streptavidin-HRP. A decrease in luminescence values with increasing concentration of PGF00506 was observed and an ICfrex®RD. PUREfrex®RD is a much simpler in vitro display technology than phage/mRNA/cDNA display, and it can fully explore a highly diverse library easily and effectively. C4m-3127 was obtained as the representative cyclic peptide in this process. Peptides that inhibit the interaction between CTLA-4 and its ligand B7-1 have already been reported -dimethyl-ammonium;hexafluorophosphateDMT-MM: 4--4-methylmorpholin-4-ium chlorideDBU: 1,8-diazabicyclo [5.4.0]-7-undeceneN,N-diisopropylethylamineDIEA: Trt: triphenylmethylPbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonylFmoc: 9-fluorenylmethyloxycarbonylThe synthetic routes and conditions are shown in S)-2-amino-N--N-phenethyl-3-(1-trityl-1H-imidazol-4-yl)propanamide (4).(N-phenethyl-1-amine (1) , Nα-(((9H-fluoren-9-yl)methoxy)carbonyl)-Nτ-trityl-L-histidine (2) , HATU , and DIEA  in THF (30 mL) was stirred at RT for 14 h. The mixture was added to water (20 mL), with extraction by EtOAc (20 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified using column chromatography  to give crude (9H-fluoren-9-yl)methyl (S)-(phenethyl)amino)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2-yl)carbamate (3) (8.01 g) as a white amorphous powder. This material was dissolved in THF (30 mL), and added to DBU  at 0 °C. The reaction mixture was warmed gradually to RT, and was stirred at RT for 17 h. The reaction mixture was diluted with EtOAc (10 mL) and washed with diluted aq. HCl solution. The aqueous phase was extracted with EtOAc–MeOH . The combined organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo. The residue was purified via column chromatography  to give 4  as a brown oil.A mixture of 2,2-diethoxy-m/z 617.4 [M + H]+.LC/MS R)-1-(((S)-1-(phenethyl)amino)-1-oxo-3-(1-trityl-1H-imidazol-4-yl)propan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)oxy)carbamate (6).Benzyl , (R)-2-((((benzyloxy)carbonyl)amino)oxy)-3-methylbutanoic acid (5) , 4-methylmorpholine , and DMT-MM  in THF (8 mL) was stirred at RT for 20 h. To the mixture was added saturated aqueous NaHCO3 (5 mL), with extraction by EtOAc (10 mL). The organic phase was washed with brine (10 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The residue was purified using column chromatography  to give 6  as a colorless oil.A mixture of m/z 867.3 [M + H]+.LC/MS R,6S,9aS)-6-((1H-imidazol-4-yl)methyl)-3-isopropyl-4,7-dioxo-8-phenethylhexahydropyrazino oxadiazine-1(6H)-carboxylate (PGF00432).Benzyl  and formic acid (1.5 mL) was left to stand at RT for 6 days. The mixture was concentrated in vacuo, and was azeotroped with CH2Cl2. The residue was purified using column chromatography . The obtained material was purified by preparative HPLC . After the collected fraction was concentrated in vacuo, the resulting aqueous solution was freeze-dried to give PGF00432  as a white powder.A mixture of 1H NMR  δ 7.44–7.33 , 7.25–7.12 , 6.77 , 5.33–5.14 , 5.02–4.99 , 4.38 , 3.65–3.60 , 3.49 , 3.14–3.12 , 2.99 , 2.94–2.78 , 2.42–2.32 , 1.00 , 0.62 .13C NMR  δ 166.11, 165.08, 153.70, 138.22, 135.20, 134.96, 128.80, 128.75, 128.72, 128.66, 128.42, 128.74, 84.90, 77.25, 68.89, 62.20, 55.40, 50.65, 49.64, 33.33, 28.74, 18.50, 15.87.m/z: calcd. for C29H33N5O5 [M + H]+, 532.2560; found, 532.2537.HRMS (EI) S)-2-amino-N--5-sulfonyl)guanidino)-N-phenethylpentanamide (9)., N2-((benzyloxy)carbonyl)-Nω-sulfonyl)-L-arginine (7) , and DIEA  in DCM (400 mL) was added HATU in portions at 0 °C. The reaction mixture was stirred at 15 °C for 18 h, then was washed with water (3 × 200 mL), dried over anhydrous Na2SO4, filtered, and concentrated in vacuo. The residue was purified using column chromatography  to give benzyl (S)-(phenethyl)amino)-1-oxo-5-sulfonyl)guanidino)pentan-2-yl)carbamate (8)  as a yellow oil. A part of this material  was dissolved in EtOH (150 mL), to which was added Pd/C  under nitrogen atmosphere. The suspension was degassed under vacuum and purged with hydrogen several times. The mixture was stirred under hydrogen (15 psi) at 25 °C for 72 h. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to give 9  as a yellow solid.To a stirring mixture of m/z 646.3 [M + H]+.LC/MS R,6S,9aS)-3-isopropyl-4,7-dioxo-6-sulfonyl)guanidino)propyl)-8-phenethylhexahydropyrazinooxadiazine-1(6H)-carboxylate (11).Benzyl , 5 , HATU , and DIEA  in DMF (1 mL) was stirred at RT for 4 days. To the mixture was added water (5 mL), with extraction by hexane-EtOAc . The organic phase was dried over anhydrous Na2SO4, and concentrated in vacuo to give benzyl (((R)-1-(((S)-1-(phenethyl)amino)-1-oxo-5-sulfonyl)guanidino)pentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)oxy)carbamate (10) as a crude product. This material was dissolved in formic acid (1 mL) and left to stand at RT for 21 h. The mixture was concentrated in vacuo. The residue was diluted with EtOAc and washed with saturated aqueous NaHCO3. The organic phase was dried over anhydrous Na2SO4 and concentrated in vacuo to give 11  as a white powder.A mixture of m/z 803.4 [M + H]+.LC/MS R,6S,9aS)-6-(3-guanidinopropyl)-3-isopropyl-4,7-dioxo-8-phenethylhexahydropyrazino oxadiazine-1(6H)-carboxylate (PGF00452).Benzyl  in TFA (1.0 mL) was stirred at RT for 21 h. The mixture was concentrated in vacuo. The residue was purified using preparative HPLC . The collected fraction was concentrated in vacuo. The resulting aqueous solution was freeze-dried to give PGF00452  as a white powder.A mixture of 1H NMR  δ 7.43–7.36 , 7.23–7.14 , 5.75–5.70 , 5.28 , 4.89–4.84 , 4.42–4.41 , 3.77–3.55 , 3.33–3.31 , 3.26–3.13 , 2.90 , 2.92–2.67 , 2.04–1.97 , 1.82–1.76 , 1.59–1.52 , 1.04 , 0.87 .13C NMR  δ 165.97, 157.48, 153.95, 137.94, 134.61, 128.95, 128.76, 128.66, 128.38, 126.85, 84.82, 77.24, 69.32, 60.87, 53.74, 50.11, 49.30, 40.20, 33.11, 28.83, 28.32, 24.81, 18.37, 15.88.m/z: calcd. for C29H38N6O5 [M + H]+, 551.2982; found, 551.2962.HRMS (EI) R)-3-cyclohexyl-1-(((S)-1-(phenethyl)amino)-1-oxo-5-sulfonyl)guanidino)pentan-2-yl)amino)-1-oxopropan-2-yl)oxy)carbamate (13).Benzyl (((R)-2-((((benzyloxy)carbonyl)amino)oxy)-3-cyclohexylpropanoic acid 12 , 9 , and DMT-MM  in MeOH (2.0 mL) was stirred at RT for 20 h. The mixture was loaded in an inject column and was purified using column chromatography  to give 13  as a colorless oil.A mixture of -3-(cyclohexylmethyl)-4,7-dioxo-6-sulfonyl)guanidino)propyl)-8-phenethylhexahydropyrazino oxadiazine-1(6H)-carboxylate (14).Benzyl  in formic acid (1.5 mL) was left to stand at RT for 20 h. The mixture was concentrated in vacuo. The residue was purified by column chromatography  to give 14  as a colorless amorphous substance.A solution of m/z 858.5 [M + H]+.LC/MS R,6S,9aS)-3-(cyclohexylmethyl)-6-(3-guanidinopropyl)-4,7-dioxo-8-phenethylhexahydropyrazino oxadiazine-1(6H)-carboxylate (PGF00478).Benzyl  in TFA (1.0 mL) and water (0.1 mL) was left to stand at RT for 2 h. The mixture was concentrated in vacuo. The residue was purified by preparative HPLC . The collected fraction was concentrated in vacuo. The resulting aqueous solution was freeze-dried to give PGF00478  as a white solid.A solution of 1H NMR  δ 7.42–7.38 , 7.29–7.16 , 5.73 , 5.31–5.22 , 4.90–4.84 , 4.57–4.54 , 3.67–3.60 , 3.32–3.14 , 2.93–2.87 , 2.02–1.42 , 1.18–1.11 , 0.94–0.83 .13C NMR  δ 179.45, 166.62, 166.01, 157.85, 153.84, 138.04, 134.42, 128.99, 128.81, 128.74, 128.64, 126.82, 79.58, 77.24, 69.49, 60.85, 53.93, 49.71, 49.33, 40.10, 36.78, 33.79, 33.74, 33.08, 31.77, 28.39, 26.28, 26.16, 25.94, 25.07, 24.82.m/z: calcd. for C33H44N6O5 [M + H]+, 605.3451; found, 605.3428.HRMS (EI) S)-2-amino-N--N--5-sulfonyl)guanidino)pentanamide (17).-3,3-diphenylpropan-1-amine (15) , 7 , and DIEA  in DCM (400 mL) was added HATU  in portions at 0 °C. The reaction mixture was stirred at 15 °C for 18 h. The reaction mixture was washed with water (3 × 200 mL), dried over anhydrous Na2SO4, filtered and concentrated in vacuo. The residue was purified by column chromatography  to give benzyl (S)-amino)-1-oxo-5-sulfonyl)guanidino)pentan-2-yl)carbamate (16)  as a yellow oil. A part of this material  was dissolved in EtOH (150 mL), and Pd/C  was added under nitrogen atmosphere. The suspension was degassed under vacuum and was purged with hydrogen several times. The mixture was stirred under hydrogen (15 psi) at 25 °C for 72 h. The reaction mixture was filtered. The filtrate was concentrated under reduced pressure to give 17  as a yellow solid.To a stirring mixture of m/z 736.3 [M + H]+.LC/MS R)-3-cyclohexyl-1-(((S)-1-amino)-1-oxo-5-sulfonyl)guanidino)pentan-2-yl)amino)-1-oxopropan-2-yl)oxy)carbamate (18).Benzyl , 17 , DIEA , and HATU  in DMF (2.0 mL) was stirred at RT for 0.5 h. The mixture was loaded in an inject column and was purified using column chromatography  to give 18  as a colorless oil.A mixture of m/z 1040.6 [M + H]+.LC/MS R,6S,9aS)-3-(cyclohexylmethyl)-8--4,7-dioxo-6-sulfonyl)guanidino)propyl)hexahydropyrazino oxadiazine-1(6H)-carboxylate (19).Benzyl  and formic acid (2.0 mL) was left to stand at RT for 20 h, and then at 60 °C for 3 h. After cooling to RT, the mixture was concentrated in vacuo to give 19  as a pale brown oil. This material was used in the next step without purification.A mixture of m/z 947.5 [M + H]+.LC/MS R,6S,9aS)-3-(cyclohexylmethyl)-8--6-(3-guanidinopropyl)-4,7-dioxohexahydropyrazino oxadiazine-1(6H)-carboxylate (PGF00506).Benzyl  in TFA (1.0 mL) and water (0.1 mL) was stirred at RT for 2 h, and then at 60 °C for 2 h. To the mixture was added TFA (2.0 mL). The mixture was stirred at 60 °C for 1.5 h. After cooling to RT, the mixture was concentrated in vacuo. The residue was purified by preparative HPLC . The collected fraction was concentrated in vacuo. The aqueous solution was freeze-dried to give PGF00506  as a white solid.A solution of 1H NMR  δ 7.43–7.36 , 7.31–7.11 , 5.74 , 5.33–5.25 , 4.86–4.81 , 4.57 , 3.93 , 3.78 , 3.43–3.38 , 3.24–3.16 , 2.39–2.36 , 1.95–1.86 , 1.77–1.42 , 1.16–1.10 , 0.93–0.75 .13C NMR  δ 179.29, 166.56, 165.87, 157.91, 153.89, 143.87, 143.85, 134.42, 129.01, 128.82, 128.69, 128.66, 127.53, 126.57, 79.76, 77.23, 70.56, 69.51, 61.01, 54.03, 49.26, 49.10, 47.19, 40.07, 36.72, 33.84, 33.73, 32.37, 31.82, 28.49, 26.28, 26.18, 25.96, 25.17, 24.75.m/z: calcd. for C40H50N6O5 [M + H]+, 695.3921; found, 695.3896.HRMS (EI) To examine the binding interactions of PGF00432 and PGF00506 against CTLA-4, molecular docking was performed using AutoDock Vina , as implfrex®RD and sequence mimic by a PepMetics® scaffold is anticipated to be a powerful approach for drug discovery targeting PPI inhibition by small molecules.In this study, we succeeded in obtaining peptidemimetic small-molecule compounds that inhibit PPI between CTLA-4 and B7-1 without relying on structural analysis such as X-ray crystallography. The combination of PURE


Perenniporia centrali-africana (originating from Kenya) and Cerrena sp. nov. (originating from Thailand). A new pereniporin A derivative (1), a new drimane-type sesquiterpene lactam (2), and the new 6,7-Dehydro-isodrimenediol (3) were isolated from P. centrali-africana. In parallel, the two new drimane-type sesquiterpene lactams 5 and 6 were isolated together with known isodrimenediol (4) from Cerrena sp. This is the first report of drimane-type sesquiterpene lactams from basidiomycetes. The structures were elucidated based on 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data, in combination with high-resolution electrospray mass spectrometric (HR-ESIMS) data. The compounds were devoid of significant antimicrobial and cytotoxic activities.Five new drimane-type sesquiterpenoids were isolated from cultures of the tropical basidiomycetes,  Mushroom-forming fungi (mostly belonging to the division Basidiomycota) have been investigated regarding their abilities as prolific producers of structurally diverse bioactive compounds, leading to the discovery of numerous unique highly functionalized secondary metabolites ,2. AlthoThe current study deals with the production, isolation, and characterization of further new sesquiterpenoid derivatives from two fungal species originating from Kenya and Thailand, respectively, which were examined in our ongoing projects aiming at the discovery of novel antibiotics and other beneficial molecules from Basidiomycota. These strains were selected for intensified evaluation because preliminary studies had revealed weak to moderate antimicrobial effects in the crude extracts, along with the presence of potentially undescribed molecules, as deduced from HPLC–DAD/MS (high performance liquid chromatography coupled with diode array detection and mass spectrometry) data. Since the molecules turned out to be structurally similar, we decided to report their isolation and structure elucidation in one paper and include data on the taxonomy of the producer strain.P. centrali-africana is a common white rot, wood decay basidiomycete belonging to the family, Polyporaceae. This fungus has a dimitic or trimitic hyphal structure with clamp connections on generative hyphae. It produces ellipsoid to distinctly truncate soft and thick-walled basidiospores + , indicating four degrees of unsaturation. 1H NMR and HSQC data , 0.99 , and 1.23 , three methylenes at δH 1.44 , 1.70 , 1.67 , 4.18 , and 4.46 , two methines at δH 1.31  and 2.25 , three oxymethines at δH 3.22 , 4.34 , and 5.17 , and one olefinic methine at δH 5.53 . The 13C and HMBC NMR data  and one methine ; three oxymethines ; two methines ; three methylene carbons ; and three methyl carbons . One degree of unsaturation is given by the two sp2-hybridized carbons, suggesting three rings in the scaffold of 3β-Hydroxy-9-dehydroxy-pereniporin A (1). Analysis of the 1H-1H COSY data gave the first spin system due to correlations between H2-1, H2-2, and H-3. Correlations between H-5, H-6, and H-7, as well as between H-9 and H-11, led to the identification of the second and third spin system. HMBC correlations from H3-13/H3-14 to C-3/C-4/C-5, H3-15 to C-1/C-5/C-9/C-10, and H-5 to C-3/C-4/C-5/C-6/C-7/C-8/C-9/C-10/C-15 revealed the presence of two six-membered rings that were fused across C-5 and C-10, carrying a hydroxyl function at C-3 and C-6, as well as one methyl group at C-10 and two methyl groups at C-4. Further HMBC correlations from H-11 to C-8/C-9/C-12 and from H2-12 to C-7/C-8/C-9/C-11 led to the identification of a tetrahydrofuran-11-ol substructure that was fused to the molecule across C-8 and C-9. Relative configuration could be obtained by evaluation of ROE data. Due to key correlations among H-1α/H-3α/H-5α/H-9α/H-12α/H3-14, these protons were arbitrarily assigned to the α face of the molecule. Correlations between H-1β/H-6β/H-11β/H-12β/H3-13/H3-15 indicated a β orientation of these protons. Finally, the absolute configuration of 1 was determined as 3S,5R,6S,9R,10S,11R, after comparison of its ECD data to the ones of related drimane derivatives + in the HRESIMS spectrum, indicating the presence of a nitrogen atom. One- and two-dimensional NMR spectra revealed the absence of the hydroxyl function at C-3, which was replaced by a methylene . 1H NMR, 13C, and HSQC data indicated the presence of another oxymethine  at C-7, which was confirmed by HMBC correlations from H-7α to C-5/C-6/C-8/C-9/C-12. Furthermore, a lactam functionality was placed at C-11 (δC 173.4), which was supported by the absence of H-11, the chemical shift of the methylene CH2–12 , and the HMBC correlations of H2-12 to C-11. Metabolite 2 is the nitrogen derivative of deacetylugandensolide. Since 2 and deacetylugandensolide have very similar coupling constants and chemical shifts for H-5, H-6 and H-7, we propose a common relative configuration +, 267.09 [M − H]−; HRESIMS m/z 291.1565 [M + Na]+ ; tR = 2.3 min.6,7-Hydroxy-12-deoxy-dysidealactam (2) white solid; max (logε) 206 (3.72) nm ; ECD : λmax (Δε) 235 (−0.8), 211 (+2.4) nm; NMR data  see m/z 266.10 [M + H]+, 265.01 [M − H]−; HRESIMS m/z 266.1750 [M + H]+ ; tR = 4.6 min.6,7-Dehydro-isodrimenediol (3) white solid; max (logε) 235 (3.70), 201 (3.50) nm ; ECD : λmax (Δε) 234 (+1.3) nm; NMR data  see m/z 219.09 [M − H2O + H]+; HRESIMS m/z 219.1738 [M − H2O + H]+ ; tR = 6.1 min.Isodrimeniol (4) yellow oil; max (log ε) 201 (0.46) nm ; ECD : λmax (Δε) 190 (+0.9) nm; NMR data  see + m/z 219.1740, calcd. 219.1743 for C15H23O, [2M + H]+ m/z 437.3412, calcd. 437.3414 for C30H45O2, [2M + Na]+ m/z 581.2830, calcd. 581.2833 for C30H42N2NaO8, tR = 7.09 min, data are in good agreement with the literature + m/z 374.1933, calcd. 374.1938 for C19H29NNaO5, [2M + H]+ m/z 725.3976, calcd. 725.4008 for C40H57N2O10, [2M + Na]+ m/z 747.3802, calcd. 747.3827 for C40H56N2NaO10, tR = 6.04 min.4-Aminobutyl derivative of ugandensolide (6) light yellow oil; max (log ε) 211.5 (0.681) nm ; ECD : λmax (Δε) 205 (+4.9) nm; NMR data  see + m/z 366.1915, calcd. 366.1911 for C19H28NO6, [M + Na]+ m/z 388.1727, calcd. 388.1731 for C19H27NNaO6, [2M + H]+ m/z 731.3747, calcd. 731.3750 for C38H55N2O12, [2M + Na]+ m/z 755.3627, calcd. 755.3608 for C52H48N2NaO2, tR = 7.64 min.50) was determined as previously described [5 was tested against further cell lines including epidermoid carcinoma cells (A431), adenocarcinomic human alveolar basal epithelial cells (A549), breast cancer cells (MCF-7), prostate cancer cells (PC-3), and human ovary adenocarcinoma (SK-OV-3). All cell lines were purchased from the DSMZ collection . Cell lines L929, A549, and KB-3.1 were cultured in Dulbecco’s modified Eagle’s medium ; MCF-7 and A431 cells were cultured in RPMI-1640 medium (Gibco) and PC-3 cells in F12K (Gibco) medium, all supplemented with 10% fetal bovine serum  and incubated under 5% CO2 at 37 °C. The tested compound was dissolved in methanol to yield a concentration of 1 mg/mL. A 60 μL amount of 1:1 serial dilutions of the test compounds was added to 120 μL aliquots of a cell suspension (50/mL) in 96-well microplates. After 5 days of incubation, an MTT -2,5-diphenyltetrazolium bromide) assay was performed. IC50 was reported at the concentration of the compounds that inhibited the growth of cells at 50% of the control. Methanol was used as a negative control.In vitro cytotoxicity (ICescribed . In brieThe minimum inhibitory concentrations (MICs) were determined according to previous reports . BrieflyPerenniporia centrali-africana, and Cerrena sp. nov.), denoted as one new derivative of pereniporin A, namely 3β-hydroxy-9-dehydroxy-pereniporin A, as well as three new drimane sesquiterpene lactams including 6,7-dihydroxy-12-deoxy-dysidealactam, glycinyl of deacetylugandensolide, and 4-aminobutyl derivative of ugandensolide, and 6,7-dehydro-isodrimenediol were isolated in this study. This is the first report of the isolation of sesquiterpene lactams from tropical Basidiomycota from two different countries . Although both basidiomycetes belong to different families, they produce similar types of compounds, some of which were never isolated previously. None of the sesquiterpene lactams showed biological activities in this study. Studies of the metabolites on other biological activities are presently underway.Five new drimane sesquiterpenoids from cultures of two tropical basidiomycetes (i.e.,


Their structures were determined by spectroscopic analysis and comparison with literature-reported data. These compounds were tested for their in vitro antibacterial activity against four Gram-(+) bacteria: Staphyloccocus aureus (SA), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (BC), Bacillus subtilis (BS), and the Gram-(−) bacterium Escherichia coli. Compounds 1, 2, 5 and 8 showed antibacterial activity toward SA, BC and BS with MIC values ranging from 7.8 to 62.5 µg/mL, but they were inactive to MRSA. Compound 4 not only showed the best antibacterial activity against SA, BC and BS, but it further displayed significant antibacterial activity against MRSA (MIC 1.95 µg/mL) even stronger than vancomycin (MIC 3.9 µg/mL). No compounds showed inhibitory activity toward E. coli. Further bioassay indicated that compounds 1, 4, 5, 6, 8 and 9 showed in vitro α-glucosidase inhibitory activity, among which compound 9 displayed the best α-glucosidase inhibitory activity with IC50 value (0.026 mM) about 15-fold stronger than the reference compound acarbose (IC50 0.408 mM). These results suggested that compounds 4, 8 and 9 were potentially highly valuable compounds worthy of consideration to be further developed as an effective anti-MRSA agent or effective α-glucosidase inhibitors, respectively. In addition, the obtained data also supported that S. latifolia was rich in structurally diverse bioactive compounds worthy of further investigation, at least in searching for potential antibiotics and α-glucosidase inhibitors.Three new polycyclic phenol derivatives, 2-acetyl-4-hydroxy-6H-furo chromen-6-one ( Spermacoce genus in the Rubiaceae family comprises 250–300 plant species that are widely distributed in tropical and subtropical zones around the world, including America, Europe, Africa, Australia and Asia − , which requires 10 degrees of unsaturation. The 1H NMR spectrum of 1 , two aromatic protons at δH 7.07  and 7.96 , and two cis-oriented vicinal olefinic protons at δH 6.42  and 7.96 , respectively. The 13C NMR and DEPT spectra , 1 carbonyl carbon (δC 188.4), 1 carboxyl carbon (δC 161.2), 4 olefinic methines and 6 olefinic quaternary carbons. These above findings supported 1 to be a highly conjugated compound bearing a basic coumarin skeleton. Careful analysis of the NMR data  to δC 153.4 (C-2) and 188.4 (C-1′), and from δH 7.96 (H-3) to δC 188.4 (C-1′), 147.2 (C-9a) and 142.5 (C-4) were observed, which supported the direct linkages of C-2 with C-1′ and C-3, of C-2′ with C-1′, and of C-3a with C-3, C-9a and C-4. The HMBC correlations from δH 7.07 (H-9) to δC 118.0 (C-3a), 140.7 (C-4a) and 144.9 (C-8) supported the connection of C-9a with C-9, and of C-8a with C-8, C-4a and C-9. The linkages of C-7 with C-6 and C-8 were supported by HMBC correlations of δH 7.96 (H-8) to δC 110.7 (C-9) and 140.7 (C-4a). In addition, the coupling constant of H-7  further evidenced the cis- configuration of the double bond between C-7 and C-8. Therefore, compound 1 was determined as 2-acetyl-4-hydroxy-6H-furo chromen-6-one.Compound rum of 1  showed s spectra , coupledNMR data  of 1 witarbon δC 8.4, 1 caspectrum , signifi2 was obtained as a purple amorphous powder. Its HR-ESI-MS (negative mode) showed a pseudo-molecular ion peak at m/z 275.0561 [M − H]−, corresponding to a molecular formula of C14H12O6 . The 1H NMR spectrum of 2 , a hydroxymethyl group at δH 3.81 , two singlet aromatic protons at δH 6.99  and 6.80 , and two vicinal cis-positioned olefinic protons at δH 6.32  and 7.92 . The 13C NMR and DEPT spectra , a hydroxymethyl group at δC 69.5 (C-2′), an oxygenated 3sp quaternary carbon at δC 73.1 (C-1′), 4 olefinic methine carbons at δC 119.9 (C-3), 114.1 (C-7), 147.1 (C-8), 108.7 (C-9), an ester bond carboxyl carbon at δC 163.8 (C-6) and 6 olefinic quaternary carbons. These spectral data closely resembled the NMR data of 1, which supported 2 to be also a coumarin derivative bearing a similar molecular skeleton to that of 1. Comparison of the 1H and 13C NMR spectral data of 2 with those of 1 indicated the major difference that the signals for the acetyl group at C-2 in 1 were absent in 2. Instead, proton and carbon signals for a propylene glycol group  were exhibited in the spectra of 2. This observation supported that the structure of 2, as shown in 1 with the only difference of the acetyl group at C-2 in 1 being replaced by a propylene glycol group in 2. This deduction was in accordance with the molecular formula of 2, and further well-supported by 2D-NMR analysis. In the HMBC spectrum (δH 3.81 (H2-2′) to δC 100.9 (C-2) and 23.8 (C-1′), and from δH 6.99 (H-3) to δC 73.1 (C-1′) were observed, which supported the direct linkages of C-1′ with C-2, C-2′, C-3′, and of C-2 with C-3. The HMBC correlations from δH 6.99 (H-3) to δC 143.9 (C-4) and 148.9 (C-9a) supported the linkages of C-3a with C-3, C-4 and C-9a. The observation of HMBC correlations from δH 6.80 (H-9) to δC 119.9 (C-3a), 142.0 (C-4a) and 147.1 (C-8) supported the direct linkages of C-9 with C-9a, and of C-8a with C-4a, C-8 and C-9. The exhibition of significant HMBC correlations from 7.92 (H-8) to δC 143.9 (C-4a) and 163.8 (C-6) supported C-8 linked with C-8a, and C-7 linked with C-8 and C-6. In addition, the coupling constant between H-7 and H-8  supported the cis-configuration of the double bond between C-7 and C-8. Thus, compound 2 was elucidated as 2--4-hydroxy-6H-furo [1] benzopyran-6-one.Compound rum of 2  showed s spectra  of 2, cospectrum , signifi8, obtained as a yellowish powder, was determined to have a molecular formula C15H12O7 on the basis of HR-ESI-MS data , which required 10 unsaturated degrees. The 1H NMR spectrum of 1 showed proton signals for two oxygenated methyl groups at δH 3.94  and 3.97 , two mutually coupled ortho aromatic hydrogen atoms at δH 8.64  and 6.82 , and a singlet aromatic hydrogen atom at δH 7.38 . The 13C-NMR spectra, coupled with HSQC analysis, showed the resonances for 15 carbons, including 1 carboxyl carbon, 2 methoxy carbons, 3 2sp methine carbons and 9 2sp quaternary C-atoms  and 3.97 ] in 1, respectively. These above findings supported preliminarily establishing the molecular structure of 1 as shown in J1,2 = 9.1 Hz) supported the pentasubstituted and the ortho tetrasubstituted benzene rings, respectively. Second, in the HMBC spectrum, the observation of 1H–13C long-range correlations from δH 7.38  to δC 163.2 (C-6), 142.9 (C-9) and 117.9 (C-10a), and from δH 3.97  to δC 142.9 (C-9) supported the location of a methoxy group at C-9, the linkages of C-8 with C-7 and C-9, and the linkages of C-6a with C-7, C-10a and C-6a (δH 8.64 (H-1) to δC 117.9 (C-10a) and 145.4 (C-4a), and from δH 6.82 (H-2) to 112.9 (C-10b) supported the linkages of C-10b with C-1, C-10a and C-4a. The HMBC correlations from H-1 to δC 151.3 (C-3), from H-2 to δC 136.0 (C-4) and from δH 3.94  to 136.0 (C-4) supported the location of a methoxy group at C-4 and further evinced the direct linkage of C-3 with C-2 and C-4, respectively. Therefore, the structure of compound 8 was unambiguously elucidated as 3,8,10-trihydroxy-4,9-dimethoxy-6H-benzo[c]chromen-6-one.Compound  C-atoms . Taking an-6-one , except and C-6a . The HMBH-furo benzopyra,2-g]chromen-6-one (1). Yellow amorphous powder; +; ESI-MS (−) m/z 243 [M − H]−, HR-ESI-MS (−) m/z 243.0300 [M − H]− . 1H NMR (500 MHz) and 13C NMR (125 MHz) in CD3OD, see 2--4-hydroxy-6H-furo +; ESI-MS (−) m/z 275 [M − H]−, HR-ESI-MS (−) m/z 275.0561 [M − H]− . 1H NMR (500 MHz) and 13C NMR (125 MHz) in CD3OD, see 3,8,10-trihydroxy-4,9-dimethoxy-6H-benzo[c]chromen-6-one (8). [α]c 0.21, MeOH); Yellow powder; HR-ESI-MS (pos.) m/z 327.0467 ; ESI-MS (pos.) m/z 305 [M + H]+, 327 [M + Na]+; ESI-MS (neg.) m/z 303 [M − H]–; For 1H NMR  and 13C NMR  spectroscopic data, see 6 cfu/mL, OD600 = 0.07) followed by transferring  to growth control wells (12th column) and all test wells (1–10th column). Then, each of 100 μL of test compounds (1 mg/mL) in beef extract peptone medium, along with the positive control solutions (prepared by adding kanamycin sulfate and vancomycin instead of the samples) and the negative control solution (3% DMSO of beef extract peptone medium), were applied to the wells in the 1st column of the plates. Once all samples and controls were properly applied to the 1st column of wells on the plate, half of the homogenized content (100 μL solution) was then parallel-transferred from the 1st column wells to the 2nd column wells, and each subsequent well was treated similarly (doubling dilution) up to the 10th column, followed by discarding the last 100 μL aliquot. Finally, the plates were incubated at 37 °C for 5–6 h until the color of growth control change to pink. The lowest concentration for each test compound at which color change occurred was recorded as the MIC value of the test compound. In the bioassay, Gram-(+) bacterial strains of Staphyloccocus aureus (CMCC26003), methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus (CMCC63302), Bacillus subtilis (CMCC63501), and Gram-(−) bacterial strain of Escherichia coli (CMCC44102) were used for the test, and they were all purchased from the Guangdong Institute of Microbiology .The antibacterial assay was monitored in 96-well plates by using a method as described previously ,41. Briea-glucosidase inhibitory activity of the ten isolated compounds was determined in 96-well microtiter plates following the method as described previously /(ODnegative blank − ODblank) × 100%. IC50 values were calculated and expressed as means ± standard deviations (SD) and SPSS 23.0 software was used for the analysis of variances.The eviously ,42. In b1, 2 and 8) along with seven known ones  were isolated from the leaves of S. latifolia. Their structures were determined by extensive spectroscopic analysis and comparison with literature-reported data. All the compounds were obtained from S. latifolia for the first time. Bioassays indicated that compounds 1, 2, 5 and 8 were active to show antibacterial activity toward three test Gram-(+) bacteria SA, BC and BS, but they were inactive to MRSA. Compound 4 was revealed to display the best antibacterial activity against all the four tested Gram-(+) bacteria (including MRSA) with MIC values comparable to reference compounds of kanamycin and vancomycin, and 4 in particular showed antibacterial activity against MRSA even stronger than vancomycin. No compounds showed inhibitory active toward the Gram-(−) bacteria E. coli. Further bioassay indicated that compounds 1, 4, 5, 6, 8 and 9 showed in vitro α-glucosidase inhibitory activity with IC50 values close to or more potent than acarbose (IC50 0.408 mM), especially for compound 9 which displayed α-glucosidase inhibitory activity (IC50 0.026 mM) about 15-fold stronger than the reference compound. The current research provided new data to support that S. latifolia is a plant rich in structurally diverse chemicals worthy of further development in medicinal or healthcare applications. In particular, compounds 4, 8 and 9 were discovered to be the three most highly valuable compounds worthy to be developed as an effective anti-MRSA agent or effective α-glucosidase inhibitor, respectively.Three new polycyclic phenol derivatives (