CM-M345 (1) and diarylpentanoid BP-C4 (2) induced p53-dependent growth inhibitory activity in human cancer cells. Herein, CM-M345 (1) and BP-C4 (2) analogues were designed and synthesized in order to obtain more potent and selective compounds. Compounds 16, 17, 19, 20, and 22–24 caused pronounced in vitro growth inhibitory activity in HCT116 cells (0.09 < GI50 < 3.10 μM). Chemical optimization of CM-M345 (1) led to the identification of compound 36 with increased selectivity for HCT116 cells expressing wild-type p53 compared to its p53-null isogenic derivative and low toxicity to non-tumor HFF-1 cells. The molecular modification of BP-C4 (2) resulted in the discovery of compound 16 with more pronounced antiproliferative activity and being selective for HCT116 cells with p53, as well as 17 with enhanced antiproliferative activity against HCT116 cells and low toxicity to non-tumor cells. Compound 16 behaved as an inhibitor of p53–MDM2 interaction, and compound 17 was shown to induce apoptosis, associated with an increase in cleaved PARP and decreased levels of the anti-apoptotic protein Bcl-2. In silico studies allowed us to predict the druglikeness and ADMET properties for 16 and 17. Docking and molecular dynamics studies predicted that 16 could bind stably to the MDM2 binding pocket.We previously reported that chalcone  TP53, encoding the tumor suppressor protein p53, which is also known as the guardian of the genome . Th. Th−1) w 125 Å2) . The logP values , and comhttp://www.swissadme.ch/ (accessed on 1 January 2023)), several druglikeness rules were evaluated, namely Ghose, Veber, Egan, and Muegge rules), as well as bioavailability score and synthetic accessibility ) for featured prediction of other druglikeness properties, including CMC-like, Lead-like, WDI-like, and MDDR-like rules ) and the PreADMET web server ). The calculated ADMET properties of 16 and 17 included the parameters of oral absorption, namely gastrointestinal (GI) absorption (High = good absorption), the percentage of human oral absorption (%HIA > 70% = well absorbed), Caco-2 cell permeability in nm·s−1 (Caco-2 > 70 nm·s−1 = high permeability), parameters of distribution, namely blood–brain barrier (BBB) permeation (No = is not able to permeate the BBB), P-gp substrate (No = is not able to be subtract of P-gp), plasma protein binding , parameters of metabolism, namely the ability to inhibit one of nine major isoforms of CYP450 , parameters of toxicity, namely carcinogenic activity in a mouse or rat , capability of blocking hERG K+ channels, and properties of skin permeation (Log Kp). The calculated values of ADMET properties of compounds 16 and 17 are presented in The ADMET properties of the compounds were calculated using the SwissADME web server  and presented middle permeability in Caco-2 cells (56.90–57.50 nm·s−1). Interestingly, both compounds have a high probability of not being subtracts of the P-gp efflux pump. P-gp is found in a variety of organs, including the BBB and the brush border membrane of the small intestine [16 and 17 for GI absorption and P-gp, we can speculate that these compounds may have an interesting bioavailability. Analyzing the remaining distribution parameters, 16 and 17 have a good probability of being BBB permeates as well as a good probability of establishing chemicals strongly bound with plasma protein (PPB = 100%). Interestingly, using the Brain Or IntestinaL EstimateD permeation method (BOILED-Egg)   , which ad point) .16 and 17 were predicted to inhibit at least five of the nine major isoforms. Lastly, compound 17 has no evidence of carcinogenic activity, and both compounds have a medium risk of blocking hERG K+ channels.Considering the ability to inhibit isoforms of CYP450, A well-defined hydrophobic surface pocket in MDM2 mediates p53–MDM2 interactions, and at least three hydrophobic residues of the α-helix in p53  play a crucial role in the interaction ,48. The 16 on MDM2 (PDB id: 4HG7) was carried out to better characterize the activity profile of compound 16. AutoDock Vina was selected to predict docking conformations and scores since it has been reported as the best software for predicting crystallographic p53-MDM2 inhibitor poses (RMSD< 1.0 Å) by redocking tests [16, which was identified as a p53–MDM2 interaction inhibitor in the yeast assay, presented a similar docking score to nutlin-3A , a known p53-MDM2 inhibitor [BP-C4 (1) .A docking study of compound ng tests . The resnhibitor , as well16 in comparison to BP-C4 (2), docking poses and residues involved in the potential p53–MDM2 interaction were also examined (BP-C4 into MDM2 as suggested by the docking protocol are presented in BP-C4 (2), 16 was predicted to bind stably to the MDM2 binding pocket, establishing one hydrogen interaction with Val-93  and residues Ile-61, His-96, and Val-93 , Fisher’s value (F), and the correlation coefficient (R2) were used to assess the reliability of the regression equation [The squared standard error :2) of 0.7217, a Fisher value of 9.85, and an S2 of 0.1029, which suggests that the model has statistical stability and validity. R2 is a measure of how well a regression equation fits the data. As a high R2 was obtained (R2 = 0.7217) for the test set data, it was ensured that the model fits the data well [2 = 0.1029)[2 (Q2 = 0.6650) from the LOO internal validation process is higher than 0.5 and smaller than the overall R2, as expected, and the difference between R2 and Q2 is lower than 0.3, indicating that the model is not overfitted [Despite the modest number of molecules utilized to develop the model, the best QSAR model presented a squared correlation coefficient (Rata well . The F tata well . The mod= 0.1029). Two dis= 0.1029). With an= 0.1029). Additioerfitted .It is feasible to gain some understanding of the structural properties that are probably important for the growth inhibitory activity of the examined compounds by analyzing the descriptors in the regression model. The potency of the compounds was shown to be significantly influenced by five variables. Descriptors which appear in this model are topological (average bonding information content of order 2), constitutional (relative number of sulfur atoms), quantum–chemical (WPSA-2 weighted PPSA), geometrical (XY shadow/XY rectangle), and electrostatic .Four of the descriptors—relative number of sulfur atoms, XY shadow/XY rectangle, WPSA-2 weighted PPSA and minimal partial charge for a oxygen atom—have positive regression coefficients, which means that an increase in these descriptors will originate in an increase in growth inhibitory activity. As opposed to that, the average bonding information content (order 2) descriptor has a negative regression coefficient, demonstrating that the growth inhibitory activity of the molecules will decrease as the values of these descriptor rises.The presence of sulfur, which is less electronegative and 60% larger than oxygen, contributes positively to the activity. Also, the activity increases with increasing the value of XY-Shadow/XY-Rectangle, which is a geometrical descriptor related to the size and shape of the molecule, meaning that a higher area of molecular shadow in the enclosing rectangle will benefit the activity. WPSA-2 weighted PPSA is a surface-weighted charged partial-positive charged surface area, and it is defined as (PPSA2 × TMSA)/1000, where PPSA2 is the total charge weighted by the partial positive surface area and TMSA is the total molecular surface area. Minimal partial charge for an oxygen atom, an electrostatic descriptor related to charge distribution, also positively contributes to activity . A graphAll of the aforementioned information points to the QSAR model’s applicability for growth inhibitory activity analysis, which raises the possibility that the model may be able to predict the activity of additional hits.1H and 13C NMR spectra were taken in CDCl3 at room temperature on Bruker Avance 300 and 500 instruments (300.13 or 400.14 MHz for 1H and 75.47 or 100.63 MHz for 13C). Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS) used as an internal reference; 13C NMR assignments were made by HSQC and HMBC experiments (the long-range 13C-1H coupling constants were optimized to 7 Hz). The spectral treatment was executed using MestReNova v6.0.2-5475 software. High-resolution mass spectrometry (HRMS) was performed on an LTQ OrbitrapTM XL hybrid mass spectrometer  controlled by LTQ Tune Plus 2.5.5 and Xcalibur 2.1.0. at CEMUP—University of Porto, Portugal. Ions were generated using a Combi MALDI electrospray ionization (ESI) source. All reagents were purchased from Sigma Aldrich . The high performance liquid chromatography (HPLC) system consisted of a Thermo Scientific SpectraSystem P4000 pump, equipped with a degasser, a Thermo Scientific SpectraSystem AS3000 autosampler fitted with a maximum volume 100 µL loop, and a Thermo Scientific SpectraSystem UV8000 DAD detector . Data acquisition was performed using ChromQuest 5.0 software, version 3.2.1. The column used in this study was ACE-C18  manufactured by Advanced Chromatography Technologies Ltd. . The mobile phase composition was acetonitrile and water  or acetonitrile and water ; all were HPLC grade solvents obtained from Merck Life Science S.L.U. . The flow rate was 1.0 mL/min and the UV detection wavelength was set at 254 nm. Analyses were performed at room temperature in isocratic mode in a 30 min run. Peak purity index was determined by total peak UV-Vis spectra between 210–650 nm with a step of 4 nm.All reactions were monitored by thin-layer chromatography (TLC). Flash column chromatography (CC) and preparative TLC using Macherey–Nagel silica gel 60 (0.04–0.063 mm), and Macherey–Nagel silica gel 60 (GF254) plates, respectively, were used in the purification of the synthesized compounds. Melting points were obtained in a Köfler microscope and are uncorrected. 11 and 15 after the addition of crushed ice, the solution was extracted with chloroform (3 × 50 mL), and the organic layers were collected, washed with water, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified as indicated below for the referred compounds. For the synthesis of compounds 3–10, 12–14, and 16, the obtained solid was filtrated, washed with water, dried, and purified as indicated below for the referred compounds.An aqueous solution of 40% sodium hydroxide was added to a solution of appropriate ketone  in methanol until pH 13–14. Then, a solution of 4-chloro or 3,4,5-trimethoxybenzaldehyde  in methanol was slowly added to the reaction mixture. The reaction was left at room temperature for 22 h–7 days and was monitored by TLC. After, crushed ice was added to the reaction mixture and neutralized with 5 M HCl solution. For the synthesis of compounds 3, 5, 6, 8, 9, 11, and 13–16 is presented in the 4, 7, 10, 12, and 14 are described here for the first time, as indicated below.An experimental description of compounds E)-2--4,5-dimethoxy-2,3-dihydro-1H-inden-1-one( (4): Purified by crystallization from methanol. Yield: 86% as a white solid; 99.8% purity; mp 165–168 °C (methanol); 1H NMR  δ: 7.68 , 7.54 , 7.02 , 6.91 , 3.99 , 3.99 , 3.98 , 3.94 , 3.91  ppm; 13C NMR  δ: 192.9 (C=O), 157.7 (C-5), 153.5 , 145.3 (C-4), 142.3 (C-9), 139.8 (C-4′), 134.3 (C-2), 133.6 (C-1′’), 133.1 (C-1′), 132.2 (C-8), 121.2 (C-7), 112.7 (C-6), 61.1 (4′-OCH3), 60.7 (4-OCH3), 56.5 , 56.4 (5-OCH3), 29.1 (C-3) ppm; HRMS (ESI+): m/z Anal. Calc. for C21H22O6 (M + H+) 371.14892, found 371.14859.E)-2-(4-chlorobenzylidene)-4,5-dimethoxy-2,3-dihydro-1H-inden-1-one( (7): Purified by crystallization from methanol. Yield: 65% as a white solid; 99.9% purity; mp 206- 209 °C (methanol); 1H NMR  δ: 7.68 , 7.61 , 7.56 , 7.43 , 7.03 , 3.98 , 3.97 , 3.96  ppm; 13C NMR  δ: 192.8 (C=O), 157.9 (C-5), 145.3 (C-4), 142.5 (C-9), 135.8 (C-4′), 135.7 (C-2), 134.0 (C-1′), 132.1 (C-8), 132.0 (C-1′’), 131.9 , 129.3 , 121.4 (C-7), 112.7 (C-6), 61.5 (4-OCH3), 57.4 (5-OCH3), 28.6 (C-3) ppm; HRMS (ESI+): m/z Anal. Calc. for C18H16ClO3 (M + H+) 315.07825, found 315.07798.E)-2--6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one( (10): Purified by crystallization from methanol. Yield: 35% as a light yellow solid; 99.9% purity; mp 145–147 °C (methanol); 1H NMR  δ: 7.75 , 7.62 , 6.68 , 6.66 , 3.95 , 3.89 , 3.88 , 3.15 , 2.91  ppm; 13C NMR  δ: 186.7 (C=O), 153.7 (C-7), 153.2 , 148.4 (C-6), 138.6 (C-10), 138.2 (C-4′), 136.3 (C-1′′), 135.0 (C-2), 131.7 (C-1′), 126.7 (C-9), 110.0 (C-8), 109.7 (C-5), 107.3 , 61.1 (4′-OCH3), 55.3 , 55.2 , 28.7 (C-4), 27.7 (C-3) ppm. HRMS (ESI+): m/z Anal. Calc. for C22H25O6 (M + H+) 385.16156. found 385.16442.Z)-3-thiochroman-4-one( (12): Purified by crystallization with methanol. Yield: 81% as light orange; 99.8% purity; mp 115–118 °C (methanol); 1H NMR  δ: 8.60 , 7.61–7.57 . 7.56–7.52 , 7.42 , 6.49 , 3.94 , 3.84  ppm; 13C NMR  δ: 179.2 (C=O), 153.5 , 137.4 (C-10), 136.7 (C-4′), 134.6 (C-2), 134.4 (C-1′′), 131.7 (C-9), 131.2 (C-6), 129.2 (C-8), 127.7 (C-5), 126.6 (C-7), 125.2 (C-1′), 106.6 , 61.0 (4′-OCH3), 56.3 , 38.1 (C-3) ppm. HRMS (ESI+): m/z Anal. Calc. for C19H19O4S (M + H+) 343.09986, found 343.09779.E)-2-(4-chlorobenzylidene)-6,7-dimethoxy-3,4-dihydronaphthalen-1(2H)-one( (14): Purified by crystallization from methanol. Yield: 73% as a light yellow solid; 99.8% purity; mp 124–125 °C (methanol); 1H NMR  δ: 7.75 , 7.62 , 7.40–7.33 , 6.68 , 3.95 , 3.08 , 2.89  ppm; 13C NMR  δ: 186.8 (C=O), 153.8 (C-6), 148.4 (C-7), 138.3 (C-10), 136.1 (C-2), 134.7 (C-1′′), 134.6 (C-4′), 134.4 (C-1′), 131.2 , 128.8 , 126.6 (C-9), 110.9 (C-5), 109.7 (C-8), 58.1 , 28.3 (C-4), 27.2 (C-3) ppm. HRMS (ESI+): m/z Anal. Calc. for C19H17ClO3 (M + H+) 329.09390. found 329.09352.25)Synthesis of 1-(2-hydroxy-4-methoxy-3-propylphenyl) ethan-1-one (25) was synthesized (90% yield) and characterized according to the described procedure [25 is presented in the 1-(2-hydroxy-4-methoxy-3-propylphenyl) ethan-1-one (rocedure . An expeE)-3--1-(2-hydroxy-4-methoxy-3-propylphenyl)prop-2-en-1-one (1)Synthesis of (E)-3--1-(2-hydroxy-4-methoxy-3-propylphenyl)prop-2-en-1-one (1) was synthesized (10%) and characterized according to the described procedure [1 is presented in the (rocedure . An expeE)-1-(4-methoxy-2-((3-methylbut-2-en-1-yl)oxy)-3-propylphenyl)-3-prop-2-en-1-one (26)Synthesis of (E)-1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-prop-2-en-1-one  and tetrabutylammonium hydroxide 30-hydrate  in chloroform/ toluene 10:7 (17 mL), 1-bromo-3-methylbut-2-ene  was added, and the reaction was allowed to proceed at room temperature for 1.5 h under gentle stirring. The reaction mixture was then poured into a mixture of water and ice and extracted with chloroform (3x50 mL). The combined organic layers were washed with brine solution, rinsed with water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The resulting crude product was purified by TLC chromatography . A light yellow solid (66% yield) corresponding to (E)-1-(4-methoxy-2-((3-methylbut-2-en-1-yl)oxy)-3-propylphenyl)-3-prop-2-en-1-one (26) was obtained.To a solution of chalcone (E)-1-(4-methoxy-2-((3-methylbut-2-en-1-yl)oxy)-3-propylphenyl)-3-prop-2-en-1-one( (26): mp 100–103 °C (dichloromethane); 99.9% purity; 1H NMR  δ: 7.64 , 7.62 , 7.50 , 6.84 , 6.73 , 5.46–5.40 , 4.28 , 3.90 , 3.89 , 3.88 , 2.66  1.59–1.54 , 1.62 , 1.50 , 1.00  ppm; 13C NMR  δ: 191.5 (C=O), 162.0 (C-4), 157.9 (C-2), 153.4 , 142.8 (C-β), 140.1 (C-4′), 138.3 (C-4‴), 131.0 (C-1′), 129.8 (C-6), 126.7 (C-1), 126.1 (C-α), 125.4 (C-3), 120.1 (C-2‴), 106.4 (C-5), 105.6 , 73.1 (C-1‴), 61.1 (4′-OCH3), 56.3 , 55.8 (4-OCH3), 26.1 (C-1′′), 25.8 (C-4‴), 23.0 (C-2′′), 18.0 (C-5‴), 14.7 (C-3′′) ppm; HRMS (ESI+): m/z Anal. Calc. for C27H34O6K (M + K+) 493.19870, found 493.19794.17–24 is presented in the An experimental description and the NMR data of compounds Synthesis of 7-methoxy-8-propyl-2--4H-chromen-4-one (27)E)-1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-prop-2-en-1-one  in DMSO , I2  was added. The mixture was heated at reflux for 24 h and then the mixture was poured into ice and 10% sodium thiosulfate solution was added. The obtained mixture was extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to dryness and the obtained oil was purified by TLC ). A light yellow solid (60% yield) corresponding to 7-methoxy-8-propyl-2--4H-chromen-4-one (27) was obtained.To a solution of -4 (27): mp 153–155 °C (dichloromethane); 99.8% purity; 1H NMR  δ: 8.09 , 7.16 , 7.02 , 6.72 , 3.96 , 4.95 , 3.93 , 2.95 , 1.75–1.67 , 1.03  ppm; 13C NMR  δ: 178.7 (C=O), 162.7 (C-2), 161.6 (C-7), 155.3 (C-9), 153.7 , 141.1 (C-4′), 127.5 (C-1′), 124.6 (C-5), 118.7 (C-8), 117.9 (C-10), 109.0 (C-6), 106.5 (C-3), 103.4 , 61.2 (4′-OCH3), 56.3 , 56.2 (4-OCH3), 25.6 (C-1′′), 22.7 (C-2′′), 14.6 (C-3′′) ppm; HRMS (ESI+): m/z Anal. Calc. for C22H25O6 (M + H+) 385.16456, found 385.16371.30)Synthesis of (E)-6-allyl)-3-methoxy-2-propylphenol (28) and 3,5-bis((E)-4-chlorobenzylidene)tetrahydro-2H-pyran-4-ol (E)-1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-prop-2-en-1-one  or (E)-3-(4-chlorobenzyl)-5-(4-chlorobenzylidene)tetrahydro-4H-pyran-4-one  in methanol (10 mL). Mild sonication was used initially to disperse the substrate. Solid NaBH4  was then added to the magnetically stirred solution at 30 °C for 2–20 h and the reaction was monitored by TLC. After cooling, the reaction was poured into crushed ice and neutralized with 5M HCl solution. For the synthesis of compound 28 after the addition of crushed ice, the solution was extracted with chloroform (3 × 50 mL), and the organic layers were collected, washed with water, dried over with anhydrous sodium sulfate, and concentrated under reduced pressure. The obtained residue was purified as indicated below for the referred compound. For the synthesis of compound 30, the obtained solid was filtrated, washed with water, dried, and purified as indicated below for the referred compound.A reaction was carried out in 50 mL round-bottom flasks with (E)-4-chlorobenzylidene)tetrahydro-2H-pyran-4-ol3,5-bis(( (30): Purified by TLC . Yield: 88% as a white solid; 98.9% purity; mp 219–222 °C (methanol); 1H NMR  δ: 7.32 , 7.10 , 6.62 , 4.91 , 4.69 , 4.51  ppm; 13C NMR  δ: 139.4 (C-2), 134.6 (C-1′), 133.3 (C-4′), 130.2 , 128.8 , 124.3 (C-1′’), 75.5 (C-1), 66.8  ppm. HRMS (ESI+): m/z Anal. Calc. for C19H16Cl2O2 (M + k+) 385.01598. found 385.01545.29) and 3,5-bis(4-chlorobenzyl)tetrahydro-4H-pyran-4-one (31)Synthesis of 1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-propan-1-one (E)-1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-prop-2-en-1-one  or 3,5-bis((E)-4-chlorobenzylidene)tetrahydro-4H-pyran-4-one  and 4 mL of toluene. The reaction mixture was left to stir in a H2 atmosphere at room temperature for 2–3.5 h. The final product was filtered through celite, and recovery with ethyl acetate was carried out to remove the Pd/C catalyst from the reaction mixture. The solvent was evaporated under reduced pressure. The obtained residues were purified as indicated below for the referred compounds.A 10 mL round-bottom flask with stir bar was charged with 0.2 equivalents of 10% wt Pd/C, (1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-propan-1-one (29): Purified by TLC .Yield: 85% as a yellow gum; 99.8% purity; mp N.D. (gum); 1H NMR  δ: 12.78 , 7.60 , 6.45 , 6.43 , 3.87 , 3.84 , 3.82 , 3.24 , 3.00 , 2.63 , 1.58–1.48 , 0.94  ppm; 13C NMR (CDCl3. 75.47 MHz) δ: 204.2 (C=O), 163.6 (C-4), 162.3 (C-2), 153.4 , 137.0 (C-1′), 136.5 (C-4′), 129.4 (C-6),118.6 (C-3), 113.9 (C-1), 105.5 , 102.1 (C-5), 61.0 (4′-OCH3), 56.2 , 55.8 (4-OCH3), 40.0 (C-1‴), 31.2 (C-2‴), 24.5 (C-1′′), 22.0 (C-2′′), 14.3 (C-3′′) ppm; HRMS (ESI+): m/z Anal. Calc. for C22H29O6 (M+H+) 389.19587, found 389.19580.H-pyran-4-one3,5-bis(4-chlorobenzyl)tetrahydro-4 (31): Purified by crystallization from methanol. Yield: 50% as a white solid; 98.2% purity; mp 145–146 °C (methanol); 1H NMR  δ: 7.25 , 7.09 , 4.14 , 3.34 , 2.94–2.83 , 3.17 , 2.36  ppm; 13C NMR (CDCl3. 75.47 MHz) δ: 208.0 (C=O), 137.7 (C-1′), 132.3 (C-4′), 130.3 , 52.9 , 128.7 , 73.7 , 30.5 (C-1′) ppm. HRMS (ESI+): m/z Anal. Calc. for C19H18Cl2O2 (M+k+) 387.03154. found 387.03260.An aqueous solution of 40% sodium hydroxide was added to a solution of 1-(2-hydroxy-4-methoxy-3-propylphenyl)ethan-1-one  in methanol until pH 13–14. Then, a solution of appropriate benzaldehyde  in methanol was slowly added to the reaction mixture. The reaction was left at reflux for 2 days and was monitored by TLC. After cooling, the reaction was poured into crushed ice and neutralized with 5M HCl solution. The organic layer was rinsed with brine and water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained residue was purified as indicated below for the referred compounds.32 is presented in the An experimental description of compound E)-3-(4-fluorophenyl)-1-(2-hydroxy-4-methoxy-3-propylphenyl)prop-2-en-1-one( (33): Purified by TLC . Yield: 12% as a yellow solid; 96.4% purity; mp 115–117 °C (ethyl acetate); H NMR  δ: 13.33 , 7.84 , 7.78 , 7.67–7.62 , 7.53 , 7.16–7.08 , 6.50 , 3.90 , 2.66 , 1.62–1.49 , 0.96  ppm; 13C NMR  δ: 192.2 (C=O), 163.7 , 163.8 (C-4), 163.4 (C-2), 142.9 (C-β), 130.5 , 131.3 , 129.2 (C-6), 120.5 (C-α), 118.8 (C-3), 114.5 (C-1), 102.2 (C-5), 116.3 , 56.9 (4-OCH3), 24.6 (C-1′′), 22.0 (C-2′′), 14.4 (C-3′′) ppm; HRMS (ESI+): m/z Anal. Calc. for C19H19FO3 (M + H+) 315.13910, found 315.13906.E)-3-(4-bromophenyl)-1-(2-hydroxy-4-methoxy-3-propylphenyl)prop-2-en-1-one( (34): Purified by TLC . Yield: 10% as an orange solid; 96.2% purity; mp 115–116 °C (dichloromethane); 1H NMR  δ: 13.29 , 7.80 , 7.77 , 7.59 , 7.57–7.54 , 7.52–7.49 , 6.50 , 3.90 , 2.66 , 1.59–1.49 , 0.96  ppm; 13C NMR  δ: 192.1 (C=O), 163.9 (C-4), 163.5 (C-2), 142.7 (C-β), 134.0 (C-1′), 132.4 , 130.0 , 129.2 (C-6), 125.0 (C-4′), 121.4 (C-α), 118.8 (C-3), 114.5 (C-1), 102.2 (C-5), 56.4 (4-OCH3), 24.6 (C-1″), 22.0 (C-2″), 14.4 (C-3″) ppm; HRMS (ESI+): m/z Anal. Calc. for C19H20BrO3 (M + H+) 375.05903, found 375.05889.An aqueous solution of 40% sodium hydroxide was added to a solution of 1-(2-hydroxy-4-methoxy-3-propylphenyl)ethan-1-one  in methanol until pH 13–14. Then, a solution of appropriate benzaldehyde  in methanol was slowly added to the reaction mixture. The reaction was left at reflux for 2 days and was monitored by TLC. After cooling, the reaction was poured into crushed ice and neutralized with 5M HCl solution. The organic layer was rinsed with brine and water, dried over anhydrous sodium sulfate, and evaporated under reduced pressure. The obtained residue was purified as indicated below for the referred compounds.E)-3-(4-(dimethylamino)phenyl)-1-(2-hydroxy-4-methoxy-3-propylphenyl)prop-2-en-1-one( (35): Purified by flash column chromatography . Yield: 15% as an orange solid; 98.7% purity; mp 126–128 °C (ethyl acetate); 1H NMR  δ: 13.71 , 7.87 , 7.80 , 7.56 , 7.41 , 6.70 , 6.48 , 3.89 , 3.05 , 2.66 , 1.62–1.50 , 0.97  ppm; 13C NMR  δ: 192.5 (C=O), 163.3 (C-2), 163.2 (C-4), 152.2 (C-4′), 145.2 (C-β), 130.7 , 128.8 (C-6), 122.8 (C-1′), 118.5 (C-3), 115.1 (C-α), 114.8 (C-1), 111.9 , 101.8 (C-5), 55.8 (4-OCH3), 40.3 (4′-NCH3), 24.6 (C-1″), 22.1 (C-2″), 14.4 (C-3″) ppm; HRMS (ESI+): m/z Anal. Calc. for C21H26NO3 (M+H+) 340.19072, found 340.19093.E)-1-(2-hydroxy-4-methoxy-3-propylphenyl)-3-(4-morpholinophenyl)prop-2-en-1-one( (36): Purified by flash column chromatography . Yield: 15% as an orange solid; 99.1% purity; mp 129–131 °C (ethyl acetate); 1H NMR  δ: 13.56 , 7.85 , 7.80 , 7.58 , 7.47 , 6.90 , 6.49 , 3.90 , 3.87 , 3.28 , 2.66 , 1.59–1.52 , 0.96  ppm; 13C NMR (CDCl3. 75.47 MHz) δ: 192.5 (C=O), 163.5 (C-4), 163.4 (C-2), 152.9 (C-4′), 144.4 (C-β), 130.3 , 129.0 (C-6), 126.0 (C-1′), 118.6 (C-3), 117.1 (C-α), 114.8 , 102.0 (C-5), 66.8 (C-8′), 55.8 (4-OCH3), 48.1 (C-7′), 24.6 (C-1″), 22.1 (C-2″), 14.4 (C-3″) ppm. HRMS (ESI+): m/z Anal. Calc. for C23H28NO4 (M + H+) 382.20128, found 382.20104.37) and 4-((3-methylbut-2-en-1-yl)oxy)benzaldehyde (39) were synthesized  and characterized according to the described procedures [37 and 39 are presented in the 2-((3-methylbut-2-en-1-yl)oxy)benzaldehyde  in methanol an aqueous solution of 40% sodium hydroxide was added until pH 13–14. Then, a solution of 2-((3-methylbut-2-en-1-yl)oxy)benzaldehyde  or 4-((3-methylbut-2-en-1-yl)oxy)benzaldehyde ) in methanol was slowly added to the reaction mixture. The reaction was left at room temperature for 26 h–2 days and was monitored by TLC. After completion, the reaction mixture was poured into ice and neutralized with diluted HCl. For the synthesis of compound 38 after the addition of crushed ice, the solution was extracted with chloroform (3 × 50 mL), and the organic layers were collected, washed with water, dried over with anhydrous sodium sulfate, and concentrated under reduced pressure. The crude material was purified as below for the referred compound. For the synthesis of compound 40, the obtained solid was filtrated, washed with water, and purified as indicated below for the referred compound. To a solution of tetrahydro-4E)-2-((3-methylbut-2-en-1-yl)oxy)benzylidene)tetrahydro-4H-pyran-4-one3,5-bis(( (38): Purified by TLC . Yield: 50% as an orange gum; 99.9% purity; mp N.D. (gum); 1H NMR  δ: 8.10 , 7.32 , 7.06 , 6.97–6.91 , 5.51–5.47 , 4.80 , 4.57 , 1.79 , 1.74  ppm; 13C NMR  δ: 185.9 (C=O), 157.9 (C-2′), 137.8 (C-3‴), 133.2 , 132.7 (C-1′’), 130.9 (C-4′), 130.8 (C-6′), 124.5 (C-1′), 119.8 (C-2′’’), 120.1 (C-5′), 112.4 (C-3′), 69.1 , 65.6 (C-1‴), 25.9 (C-4‴), 18.4 (C-5‴) ppm; HRMS (ESI+): m/z Anal. Calc. for C29H31O4 (M-H+) 443.22278, found 443.22146.E)-4-((3-methylbut-2-en-1-yl)oxy)benzylidene)tetrahydro-4H-pyran-4-one3,5-bis(( (40): Purified by crystallization from methanol. Yield: 25% as a yellow solid; 99.9% purity; mp 145–147 °C (methanol); 1H NMR  δ: 7.79 , 7.28 , 6.95 , 5.52–5.47 , 4.93 , 4.55 , 1.89 , 1.76  ppm; 13C NMR  δ: 185.6 (C=O), 160.0 (C-4′), 138.9 (C-3‴), 136.1 (C-1′′), 132.6 , 131.3 , 127.6 (C-1′), 119.3 (C-2‴), 115.0 , 68.8 , 65.5 (C-1‴), 26.0 (C-4‴), 18.4 (C-5‴) ppm; HRMS (ESI+): m/z Anal. Calc. for C29H33O4 (M + H+) 445.23734, found 445.23760.+/+) and its p53-null isogenic derivative (HCT116 p53−/−) were given by Prof. Vogelstein ; the normal human fibroblasts HFF-1 cell lines were from ATCC . Human cells were cultured in RPMI-1640 with UltraGlutamine  with 10% FBS  at 37 °C with 5% CO2. The SH-SY5Y cells were cultured in DMEM/F-12 (Lonza) and 10% FBS and 2mM L-glutamine (Gibco). The cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. Mycoplasma was routinely checked using the MycoAlert™ PLUS kit (Lonza).The colorectal adenocarcinoma HCT116 cells  cells/wells for 24 h, and the GI50  values of compounds were obtained as described [Saccharomyces cerevisiae (strain CG379) expressing human wt p53 alone and combined with human MDM2 were used as described [w/w) galactose, 1% (w/w) raffinose, 0.7% (w/w) yeast nitrogen base without amino acids from Difco , and all the amino acids necessary for yeast growth (50 g/mL) except leucine and tryptophan. The yeast cells were incubated at 30 °C under continuous orbital shaking (200 rpm) with 0.1–50 M compounds or 0.1% DMSO only for over 42 h. Yeast growth was analyzed by counting the number of colony-forming units (CFUs) after 2 days of incubation at 30 °C on Sabouraud Dextrose Agar from Liofilchem .In the sulforhodamine B (SRB) assay, the cells were seeded in 96-well plates at 5.0 × 10escribed . In the escribed . For the5 HCT116 p53+/+ cells/well were seeded in 6-well plates and then treated with 5 µM 17 for 48 h. For the apoptosis analysis, an Annexin V-FITC Apoptosis Detection Kit I  was used following the manufacturer’s instructions. An AccuriTM C6 flow cytometer and the BD Accuri C6 software were used for data acquisition.The 1.5 × 105 cells (per well) of HCT116 p53+/+ cells were seeded in six-well plates and allowed to adhere for 24 h, followed by treatment with 5 μM 17 for 48 h. Protein extracts obtained from the human cancer cells were quantified using the Pierce™ BCA Protein Assay Kit , according to the manufacturer’s instructions. The proteins were then run in SDS-PAGE and transferred to a Whatman nitrocellulose membrane from Protan . Proteins were detected using the primary antibodies anti-p53 , anti-BCL-2 , anti-PARP , and anti-GAPDH , followed by HRP-conjugated secondary antibodies anti-mouse  and anti-rabbit . GAPDH was used as a loading control. The signal was detected with an ECL Amersham kit from GE Healthcare (VWR) and the ChemiDoc™ MP Imaging System .For Western blotting, 1.5 × 10p values < 0.05: statistically significant.Data analyzed using GraphPad Prism 7.0. Statistical tests were used according to the dataset; http://www.swissadme.ch/ (accessed on 1 January 2023)). Medicinal chemistry rules ) and PreADMET (https://preadmet.bmdrc.kr/ (accessed on 1 January 2023)) web servers. They provide a quick and detailed method for the calculation of a wide range of principal molecular descriptors that are useful in the prediction of physicochemical properties.The selected compounds were further investigated for druglikeness properties, namely molecular descriptors/physicochemical properties by SwissADME (http://www.swissadme.ch/ (accessed on 1 January 2023 and 8 September 2023)) [https://preadmet.bmdrc.kr/ (accessed on 1 January 2023)) web servers. SwissADME provides prediction of absorption (GI absorption), distribution (BBB permeant and P-gp substrate), and metabolism (ability to inhibit isoforms of CYP450). PreADMET provides a prediction of human intestinal absorption [The pharmacokinetic properties were evaluated using the SwissADME (r 2023))  and preAsorption , membransorption , the abi.A−1mol−1; the maximum number of geometry steps were set to 1000. The calculation was run until the maximum steps were reached, or the convergence criteria was met, whichever came first. The 3D structure of MDM2 was obtained from the Protein Data Bank (PDB id (MDM2): 4HG7) and prepared for docking using AutoDockTools 1.5.7. Docking simulations between the MDM2 and small molecules were undertaken in PyRx 0.8 using AutoDock Vina [The structures of compounds were drawn using ChemDraw 17.0. The structure of the known p53-MDM2 inhibitor nutlin-3A was obtained from Pubchem . The throck Vina . Dockingock Vina . Nine coock Vina .16 docked in MDM2. The X:MDM2 complex was then minimized using the MMFF94x force field until the RMS gradient < 0.1 kcal.Å−1.mol−1. The complex was then subjected to MD simulation using MOE-dynamic implemented in MOE 2014.09 . The MD simulation was performed using an MMFF94x force field and NVT  ensemble and the Nosé–Poincaré–Andersen (NPA) algorithm, with a 0.002 ps time step and sampling every 0.5 ps. The system was heated from 0 K to 300 K in 100 ps (heat stage), followed by a 5000 ps production stage at 300 K; the system was then cooled back to 0K in 100 ps (cooling stage). A potential energy analysis was used to keep track of the system’s behavior during the simulation. The intermolecular interactions between X and MDM2 were analyzed at the end of the simulation using Discovery Studio Visualizer v16 .A 12Å spherical droplet containing water molecules was placed surrounding compound 50 on HCT116 p53+/+ cell line) were utilized to develop a QSAR model using thirty chalcones and diarylpentanoid derivatives. The QSAR study used pGI50 as a dependent variable. A training set (25 molecules) and a test set (5 molecules) were created from the 30 molecules at random. Constitutional, topological, geometrical, electrostatic, quantum–chemical, and thermodynamical molecular descriptors were computed using CODESSA software . The optimal multilinear correlations with a wide range of descriptors were thoroughly searched by using heuristic multilinear regression methodology. The 2D-QSAR model with the best square of the correlation coefficient (R2), F-test (F), and squared standard error (S2) was selected. Utilizing the test set and internal leave-one-out (LOO) validation, the resulting model was further validated.The experimental data from the in vitro investigations (pGICM-M345 (1) and BP-C4 (2) and SAR studies, 33 compounds were planned, synthesized, and evaluated for their antiproliferative activity in human colorectal cancer HCT116 cells expressing wt p53 and its p53-null isogenic derivative HCT116 p53−/− cells, as well as in human normal fibroblasts cells to assess their selectivity towards cancer cells. The majority of the compounds displayed GI50 values lower than 10 μM in HCT116 p53+/+ cells, showing diarylpentanoids 16, 17, 19, and 20 ) and diarylpentanoids 22–24 ) lower values of GI50 than CM-M345 (1) and BP-C4 (2). When comparing the antiproliferative effect, in HCT116 p53+/+ cells, of CM-M345 (1) and BP-C4 (2) analogues, we may conclude that molecular rigidification and extension by prenylation were not favorable for antiproliferative activity. In general, most of the diarylpentanoids presented higher antiproliferative effects than chalcones. Additionally, the enone moiety seemed to be essential for a potent cytotoxic effect, with the reduction of this moiety being associated with a decrease in this activity. Moreover, the substitution of the 3,4,5-trimethoxyphenyl B ring by para-halogenated or para-aminated phenyl groups resulted in a decreased antiproliferative effect, with these results being in accordance with the results previously reported by our group [21.Aiming to perform the structural optimization of 16 and 36 were selective for p53+/+ HCT116 cells over p53-null cells, showing a selective index higher than CM-M345 (1) and BP-C4 (2), respectively. Notably, chalcone 16, possessing a chlorine group at the para position on B aromatic ring, showed nine-fold higher antiproliferative activity as well as seven-fold increased selectivity for p53, compared to BP-C4 (2), with this compound being identified as a p53–MDM2 interaction inhibitor by yeast cell-based assays.Chalcones 17, with a GI50 value of 2.8 μM in colorectal cancer cells, and 12-fold lower cytotoxicity towards normal cells. The growth-inhibitory effect of diarylpentanoid 17 was associated with the induction of p53-independent apoptotic cell death mediated by caspases and a mitochondrial pathway. Interestingly, in silico analysis allowed to predict that chalcone 16 and diarylpentanoid 17 have adequate pharmacokinetic and druglikeness profiles. Moreover, from docking studies, it was predicted that compound 16 binds stably to the MDM2 binding pocket. After MD simulation, it was found that chalcone 16 entered more profoundly into the MDM2 binding groove, establishing π-stacking, π-alkyl, and alkyl interactions with residues already described as important in the binding of MDM2 to p53. Moreover, a QSAR model allowed us to conclude that topological, constitutional, quantum–chemical, geometrical, and electrostatic descriptors influence the growth inhibitory activity of the test compounds on the HCT116 p53+/+ cell line.Additionally, optimization of the hit compounds led to the development of diarylpentanoid 16 and 17 as potential antitumor agents. The overall results obtained from this study will be valuable for the rational design of new and more selective and potent chalcones and diarylpentanoids with antiproliferative activity.Further studies are required to explore the molecular mechanism of action of


Allium cristophii and A. macleanii. The chemical constituents of ‘Globemaster’ bulbs have not yet been examined; thus, a systematic phytochemical investigation was undertaken herein. A series of chromatographic separations of the MeOH extract of ‘Globemaster’ bulbs afforded 27 steroidal glycosides (1–27), which are classified into 23 spirostanol glycosides (1–8 and 11–25), two furostanol glycosides (9 and 26), a pregnane glycoside (10), and a cholestane glycoside (27). The structures of the hitherto undescribed compounds (1–10) were determined from the two-dimensional NMR spectroscopic data and hydrolysis. The cytotoxicity of the isolated compounds (1–27) toward HL-60 human promyelocytic leukemia cells, A549 human adenocarcinoma lung cancer cells, and SBC-3 human small-cell lung cancer cells was evaluated. Compounds 8, 22, 23, 24, and 26 exhibited cytotoxicity toward all cell lines in a dose-dependent manner, with IC50 values in the 1.3–49 µM range.‘Globemaster’ is an ornamental hybrid cultivar whose parent plants are  Allium, belonging to the family Amaryllidaceae, is distributed throughout the Northern Hemisphere and comprises more than 950 species -3β,5α,6β-trihydroxyspirostan-2α-yl β-d-glucopyranoside (20) -3β,5α,6β-trihydroxyspirostan-2α-yl O-β-d-glucopyranoside (21) [R)-5α-spirostan-3β-yl O-α-l-rhamnopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl]-(1→4)-β-d-galactopyranoside (22) [R)-6β-hydroxy-5α-spirostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (23) -O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (24) -O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (25) [R)-26-[(β-d-glucopyranosyl)oxy]-2α,6β,22α-trihydroxy-5α-furostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside (26) [S)-16β-[(O-α-l-rhamnopyranosyl-(1→3)-β-d-glucopyranosyl)oxy]-3β-hydroxycholestan-5-en-1β-yl α-l-rhamnopyranoside (27) , two secondary methyl groups , five oxygenated methine groups , an oxygenated methylene group , an acetal carbon [δC 111.5 (C-22)], an oxygenated quaternary carbon [δC 75.6 (C-5)], and two anomeric protons/carbons . Enzymatic hydrolysis of 1 with β-d-glucosidase at 28 °C for nine days yielded 19 and a sugar fraction. The sugar fraction was subjected to direct HPLC analysis, which allowed the identification of d-glucose. The heteronuclear multiple bond correlation (HMBC) spectrum of 1 shows long-range correlations between H-1″ of β-d-glucopyranosyl [Glc (II): δH 5.37 ] and C-2′ of Glc (I) (δC 83.7), and between H-1′ of β-d-glucopyranosyl [Glc (I): δH 4.90 ] and C-24 of the aglycone (δC 81.7) -2α,3β,5α,6β-tetrahydroxyspirostan-24-yl O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside.Compound δC 81.7) . The β-a1H and 13C NMR spectra of 2 (C52H78O23) were similar to those of 21 (C45H74O22), 2 was larger than 21 because of the presence of C7H4O in the molecular formula of the former. The 1H and 13C NMR spectra of 2 displayed signals arising from a 1-substituted aromatic group  and a carbonyl group [δC 166.8 (C-7″″)]. Analysis of the 1H-1H correlation spectroscopy (COSY) and heteronuclear single quantum coherence (HSQC) spectra of 2 revealed that 2 has a benzoyl (Bz) moiety. The HMBC spectrum of 2 shows a 3JC,H correlation from H-3 of the aglycone [δH 6.29 ] to C-7″″ of Bz -3β-benzoyloxy-2α-[(β-d-glucopyranosyl)oxy]-5α,6β-dihydroxyspirostan-24-yl O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranoside.Although the ″″ of Bz . Accordi1H and 13C NMR spectra revealed that 3 (C50H82O25) is similar to 24, except for the F-ring part of the aglycone. The 1H-1H COSY spectrum of 3 shows spin-coupling correlations between the methine proton [δH 1.81 (m)] attributable to H-25 and the methyl protons [δH 1.07 ], oxygenated methylene protons [δH 3.70  and δH 3.58 ], and oxygenated methine proton [δH 3.99 ]. These correlations indicate the presence of a hydroxy group at C-24 of the aglycone. The configuration of C-24 was determined to be S based on the nuclear Overhauser effect (NOE) correlation between H-24 and H-26ax in the nuclear Overhauser enhancement spectroscopy (NOESY) spectrum of 3  and 3JH-23eq,H-24 (4.8 Hz) are consistent with the 24S configuration. Therefore, 3 was determined to be -2α,6β,24-trihydroxy-5α-spirostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.Analysis of the rum of 3 . The spi1H and 13C NMR spectra of 4 (C56H92O30) were closely related to those of 3; however, an additional anomeric proton signal [Glc (III): δH 4.90 ] and six carbon signals  corresponding to a terminal β-d-glucopyranosyl unit were detected in the 1H and 13C NMR spectra of 4. The HMBC spectrum of 4 shows a long-range correlation between H-1″″′ of Glc (III) and C-24 of the aglycone (δC 81.3) -24-[(β-d-glucopyranosyl)oxy]-2α,6β-dihydroxy-5α-spirostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The δC 81.3) . Thus, 45 (C62H102O35) was larger than that of 4 (C56H92O30) by C6H10O5. Comparison of the 1H and 13C NMR spectra of 5 with those of 4 indicated that one more β-d-glucopyranosyl unit  was present in 5, and the signal of C-2 of the β-d-glucopyranosyl unit  attached to C-24 of the aglycone was shifted downfield by 8.0 ppm. The HMBC spectrum of 5 showed long-range correlations between H-1″″″ of Glc (IV) and C-2″″′ of Glc (III) (δC 83.6) and between H-1″″′ of Glc (III) and C-24 of the aglycone (δC 81.7) -24-[(O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl)oxy]-2α,6β-dihydroxy-5α-spirostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The molecular formula of δC 81.7) . Accordi6 (C29H46O7) was obtained as an amorphous solid. Although the 1H and 13C NMR spectra of 6 were similar to those of 12, the signals of the acetyl group  were detected in the spectra of 6. A 3JC,H correlation was observed between H-3 of the aglycone [δH 6.08 ] and the carbonyl carbon of the acetyl group (δC 171.0) in the HMBC spectrum of 6 (6 was determined to be (25R)-3β-acetoxyspirostan-2α,5α,6β-triol.Compound rum of 6 . Therefo1H and 13C NMR spectral data of 7 (C56H92O28) were similar to those of (25R)-5α-spirostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside   was observed in the 1H and 13C NMR spectra of 7. The HMBC spectrum of 7 indicated a long-range correlation between H-1″″′ of Glc (III) and C-24 of the aglycone. The configuration of C-24 was determined to be S based on the NOE correlations observed between H-24 [δH 4.03 (m)] and H-26ax [δH 3.57 ]. The spin-coupling constants of 3JH-23ax,H-24 (13.2 Hz) and 3JH-23eq,H-24 (4.8 Hz) supported the 24S configuration. Thus, 7 was determined to be -24-[(β-d-glucopyranosyl)oxy]-5α-spirostan-3β-yl O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranoside.The tigonin) , which w1H and 13C NMR spectra of 8 with those of 23 showed that 8 and 23 shared the same aglycone. Analysis of 1H-1H COSY and HSQC spectra of 8 indicated the presence of a 2-substituted β-d-galactopyranosyl unit  and a terminal α-l-rhamnopyranosyl unit . The HMBC spectrum of 8 showed long-range correlations between H-1″ of Rha and C-2′ of Gal, and between H-1′ of Gal and C-3 of the aglycone (δC 77.9). Accordingly, 8 was determined to be (25R)-6β-hydroxy-5α-spirostan-3β-yl O-α-l-rhamnopyranosyl-(1→2)-β-d-galactopyranoside.Comparison of the 9 (C33H56O12) was obtained as an amorphous solid. The 1H and 13C NMR spectra of 9 displayed signals of the following groups: four steroidal methyl groups , four oxygenated methine groups , an oxygenated methylene group , an oxygenated quaternary carbon [δC 75.6 (C-5)], a hemiacetal carbon [δC 110.6 (C-22)], and an anomeric proton/carbon . The above data and positive Ehrlich’s test results suggest that 9 is a furostanol glycoside. Compound 9 was treated with β-d-glucosidase to afford the corresponding spirostanol glycoside (12) and d-glucose. The HMBC spectrum of 9 showed a long-range correlation from H-1′ of the β-d-glucopyranosyl (δH 4.81) to C-26 of the aglycone (δC 75.2). In the NOESY spectrum of 9, NOE correlations were detected between H-20 [δH 2.23 (m)] and Me-18/H2-23 [δH 2.04 and 1.98 (each m)], indicating the C-20α configuration. Therefore, 9 was determined to be (25R)-26-[(β-d-glucopyranosyl)oxy]-furostan-2α,3β,5α,6β,22α-pentol.Compound 10 (C44H70O23) was obtained as an amorphous solid, the 1H and 13C NMR spectra of which showed signals of the following groups: three methyl groups , three oxygenated methine groups , and four anomeric protons/carbons . The existence of an α,β-unsaturated carbonyl group was verified from the infrared (IR) (1700 cm−1), ultraviolet (UV) [255 (log ε 3.89) and 204 (log ε 3.83) nm], and 13C NMR  spectra. The HMBC spectrum of 10 indicated a 2JC,H correlation between the methyl singlet signal assignable to Me-21 (δH 2.22) and the carbonyl carbon [δC 196.3 (C-20)] and a 3JC,H correlation between Me-21 and the olefinic carbon [δC 155.3 (C-17)]. Furthermore, the olefinic proton [δH 6.57 ] exhibited a 3JC,H correlation with the quaternary carbon [δC 46.6 (C-13)] bearing an angular methyl group. These spectral data and comparison with those of previously reported compounds -O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranosyl. Accordingly, 10 was determined to be 3β-[(O-β-d-glucopyranosyl-(1→2)-O-[β-d-xylopyranosyl-(1→3)]-O-β-d-glucopyranosyl-(1→4)-β-d-galactopyranosyl)oxy]-2α,6β-dihydroxy-5α-pregn-16-en-20-one.Compound 0 C44H70O was obta1–27 towards HL-60, A549, and SBC-3 cells was evaluated using a modified 3--2,5-diphenyltetrazolium bromide (MTT) assay. Compounds 8, 22–24, and 26 exhibited cytotoxicity toward all cell lines in a dose-dependent manner , DPX-600  spectrometer , or JNM-ECZ600R/M1  spectrometer  at 300 K. Chemical shifts are read as δ values. The data of HRESITOFMS were obtained using a Waters Micromass LCT mass spectrometer . Diaion HP-20 porous polymer polystyrene resin , silica gel Chromatorex BW-300 , and ODS silica gel COSMOSIL 75C18-OPN  were applied for column chromatography (CC). Thin-layer chromatography (TLC) analysis was performed by precoated silica gel 60F254 or RP18 F254S plates . The sample spots were detected by spraying the TLC plate with H2SO4/H2O (1:9) and then heating. The preparative reverse-phase HPLC system was established from an LC-20 AD pump , an RID-10A detector (Shimadzu), a Rheodyne injection port , and a TSKgel ODS-100Z column . The following reagents and materials were adopted for the cell culture and cytotoxic assay: HL-60 cells (JCRB0085), A549 cells (JCRB0076), and SBC-3 cells (JCRB0818) ; Roswell Park Memorial Institute (RPMI)-1640 medium, minimum-essential medium (MEM), and cisplatin ; 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution ; fetal bovine serum ; MTT ; penicillin G sodium salt and streptomycin sulfate, and paraformaldehyde and phosphate-buffered saline (PBS) ; MCO-170AIC-PJ CO2 incubator ; Countess II FL automated cell counter (Thermo Fisher Scientific); SH-1300 Lab microplate reader ; 96-well flat-bottomed plates .Optical rotations were measured with a P-1030 automatic digital polarimeter . IR and UV spectral data were recorded on a Fourier transform infrared (FT-IR) 620 spectrometer (JASCO) and a V-630 UV-vis spectrophotometer (JASCO), respectively. NMR spectral data were collected using a DRX-500  in 2014. A voucher specimen was kept at the herbarium of the Tokyo University of Pharmacy and Life Sciences (KS-2014-009).2O , MeOH-H2O , MeOH (9 L), EtOH (6 L), and EtOAc (6 L). The MeOH-H2O (1:1) eluted fraction (10 g) was separated by ODS silica gel CC eluted with MeCN-H2O (3:7) to yield five subfractions (Fractions B1–B5). Fraction B1 (3.0 g) was subjected to ODS silica gel CC eluted with MeOH-H2O  and MeCN-H2O , and preparative reverse-phase HPLC using MeOH-H2O (9:11) and MeCN-H2O (1:3) to collect 2 , 9 , 20 , and 21 . Fraction B2 (740 mg) was divided by ODS silica gel CC eluted with MeOH-H2O  and MeCN-H2O , and preparative reverse-phase HPLC using MeOH-H2O (1:1) and MeCN-H2O (1:3) to obtain 1 , 4 , 5 , 10 , 18 , 19 , 26 , and 27 . The MeOH eluted portion (15 g) was chromatographed on a silica gel column eluted with a stepwise gradient mixture of CHCl3-MeOH-H2O  to afford five subfractions (Fractions C1–C5). Fraction C1 (5.0 g) was separated by silica gel CC eluted with CHCl3-MeOH-H2O (90:10:1) and EtOAc-MeOH-H2O , and ODS silica gel CC eluted with MeOH-H2O (4:1) and MeCN-H2O  to obtain 6 , 11 , 12 , 13 , and 15 . Fraction C2 (895 mg) was purified by ODS silica gel CC eluted with MeOH-H2O  and MeCN-H2O (2:3) to furnish 16 . Fraction C3 (4.1 g) was separated by silica gel CC eluted with CHCl3-MeOH-H2O (30:10:1) and ODS silica gel CC eluted with MeOH-H2O (4:1) and MeCN-H2O (2:3) to afford 8 , 17 , and 23 . Fraction C4 (8.0 g) was divided by silica gel CC eluted with CHCl3-MeOH-H2O (30:10:1) and ODS silica gel CC eluted with MeOH-H2O  and MeCN-H2O  to collect 3 , 7 , 14 , 22 , 24 , and 25 .The bulbs of ‘Globemaster’  were extracted with MeOH  for 2 h. Then, the solution was evaporated under reduced pressure to obtain MeOH extract (460 g). The MeOH extract was loaded on a Diaion HP-20 porous polymer polystyrene resin column, and successively eluted with MeOH-Hc 0.05); IR (film) νmax: 3376 (OH), 2930 (CH); For 1H NMR spectral data of the aglycone, see 1H NMR spectral data of the sugar moieties, see 13C NMR spectral data, see m/z: 827.4035 [M + Na]+ . For NMR spectra, see Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; + .Amorphous solid; [α1 (15 mg) and 9 (5.0 mg) were independently treated with β-d-glucosidase (EC 232-589-7) in AcOH-AcONa  at 28 °C for nine days (1) and 70 h (9). The crude hydrolysate of 1 was separated by silica gel CC eluted with EtOAc-MeOH-H2O (40:10:1) to obtain 19 (6.2 mg) and the sugar fraction (2.2 mg). The reaction mixture of 9 was purified by silica gel CC eluted with CHCl3-MeOH-MeOH (90:10:1) to yield 12 (0.94 mg) and the sugar fraction (0.20 mg). Each sugar fraction was analyzed by HPLC under the following conditions: pump, DP-8020 (Tosoh); detector, Shodex OR2 ; column, Capcell Pak NH2 ; solvent, MeCN-H2O (17:3); and flow rate, 1.0 mL/min. d-Glucose was identified by comparing the retention time (tR) and optical rotation with those of the authentic sample .Compounds l-glutamine, respectively. All cell lines were incubated at 37 °C in a 5% CO2/air atmosphere. HL-60, A549, and SBC-3 cells were cultured in a 96-well flat-bottomed plate with cell concentrations of 4 × 104, 1 × 104, and 2 × 104 cells/mL, respectively. After preincubation for 24 h, 4 μL of EtOH-H2O (1:1) solution, including each screened sample, was added and incubated for 72 h. The control cells were treated with 4 μL of EtOH-H2O (1:1) solution. The cell viability was examined using a modified MTT assay. After 72 h, 10 μL of MTT solution dissolved in PBS at a concentration of 5 mg/mL was added to each well, and the 96-well flat-bottomed plate was further incubated. After 4 h, MTT formazan was dissolved in dimethyl sulfoxide (DMSO), and the absorbance was measured at 550 nm. A dose–response curve was diagramed for 8, 11, 15, 22–24, and 26, which inhibited cell growth by more than 50% at a sample concentration of 50 μM, and the concentrations at which 50% inhibition (IC50) of cell growth occurred were calculated.HL-60 cells were maintained in RPMI-1640 medium, and A549 and SBC-3 cells were preserved in MEM containing 10% heat-inactivated FBS supplemented with 100 unit/mL penicillin G sodium salt, 100 μg/mL streptomycin sulfate, and


Curatella americana L. (Dilleniaceae) is a common shrub in the Brazilian Cerrado, whose ethanolic extract showed significant in vitro anti–Zika virus activity by the MTT colorimetric method. Currently, there is no drug in clinical use specifically for the treatment of this virus; therefore, in this work, the antiviral and cytotoxic properties of the ethanolic extract, fractions, and compounds were evaluated. The ethanolic extract of the leaves showed no cytotoxicity for the human MRC-5 cell and was moderately cytotoxic for the Vero cell (CC50 161.5 ± 2.01 µg/mL). This extract inhibited the Zika virus multiplication cycle with an EC50 of 85.2 ± 1.65 µg/mL. This extract was fractionated using the liquid–liquid partition technique, and the ethyl acetate fraction showed significant activity against the Zika virus with an EC50 of 40.7 ± 2.33 µg/mL. From the ethyl acetate fraction, the flavonoids quercetin-3-O-hexosylgallate (1), quercetin-3-O-glucoside (2), and quercetin (5) were isolated, and in addition to these compounds, a mixture of quercetin-3-O-rhamnoside (3) and quercetin-3-O-arabinoside (4) was also obtained. The isolated compounds quercetin and quercetin-3-O-hexosylgallate inhibited the viral cytopathic effect at an EC50 of 18.6 ± 2.8 and 152.8 ± 2.0, respectively. Additionally, analyses by liquid chromatography coupled to a mass spectrometer allowed the identification of another 24 minor phenolic constituents present in the ethanolic extract and in the ethyl acetate fraction of this species.The ethnomedicinal plant  Flavivirus, and it is transmitted by mosquitoes of the genus Aedes. The virus was first isolated in 1947 in Uganda, Africa, and by 2007, very few human cases were described, all of which were associated with mild clinical symptoms + 617.22, HRMS [M+H]+ 617.1137, cald. for C28H25O16 617.1142 Da; 1H-NMR  6.18 , 6.37 , 7.78 , 6.81 , 7.55 , 5.11 , 3.85 , 3.80 , 3.87 , 3.59 , 4.09 , 6.88 ; 13C-NMR  158.3 (C-2), 135.7 (C-3), 179.5 (C-4), 166.0 (C-5), 99.9 (C-6), 168.0 (C-7), 94.8 (C-8), 162.8 (C-9), 105.4 (C-10), 123.6 (C-1′), 117.7 (C-2′), 145.8 (C-3′), 149.9 (C-4′), 116.1 (C-5′), 123.0 (C-6′), 105.5 (C-1′′), 73.0 (C-2′′), 74.5 (C-3′′), 70.0 (C-4′′), 74.9 (C-5′′), 61.6 (C-6′′), 121.0 (C-1′′′), 109.8 , 146.3 (C-3′′′), 145.8 (C-5′′′), 135.7 (C-4′′′), 173.1 (C-7′′′).Quercetin-3-O-glucoside (compound 2): Yellowish powder, UV λmax 255, 354 nm; ESI-MS m/z: [M+H]+ 465.13, HRMS [M+H]+ 465.1030, cald. for C21H21O12 465.1033; 1H NMR  6.26 , 6.08 , 7.59 , 6.74 , 7.46 , 5.14 , 3.36 , 3.34 , 3.31 , 3.11 , 3.58 , 3.61 ; 13C NMR  158.6 (C-2), 135.7 (C-3), 179.6 (C-4), 163.1 (C-5), 100.0 (C-6), 166.1 (C-7), 94.8 (C-8), 159.1 (C-9), 105.8 (C-10), 123.3 (C1′), 117.7 (C-2′), 146.0 (C-3′), 149.9 (C-4′), 116.1 (C-5′), 123.2 (C-6′), 104.4 (C-1′′), 75.8 (C-2′′), 78.2 (C-3′′), 71.3 (C-4′′), 78.5 (C-5′′), 62.6 (C-6′′).Quercetin-3-O-rhamnoside (compound 3): Yellowish powder, UV λmax 256 and 349 nm; ESI–MS m/z: [M+H]+ 449.13, HRMS [M+H]+ 449.1080, cald. for C21H21O11 449.1083 Da; 1H NMR  6.09 , 6.27 , 6.82 , 7.24 , 7.22 , 5.25 , 3.80 , 4.12 , 3.32 , 3.66 , 0.83 ; 13C NMR , 159.4 (C-2), 136.4 (C-3), 179.6 (C-4), 163.2 (C-5), 100.0 (C-6), 166.2 (C-7), 94.8 (C-8), 158.5 (C-9), 105.7 (C-10), 123.1 (C1′), 116.5 (C-2′), 146.6 (C-3′), 149.9 (C-4′), 117.0 (C-5′), 123.0 (C-6′), 103.7 (C-1′′), 73.0 (C-2′′), 72.0 (C-3′′), 72.2 (C-4′′), 72.1 (C-5′′), 17.8 (C-6′′).Quercetin-3-O-arabinoside (compound 4): Yellowish powder, UV λmax 256 and 354 nm; ESI–MS m/z: [M+H]+ 435.15, HRMS [M+H]+ 435.0922, cald. for C20H19O11 435.0927 Da; 1H NMR  6.10 , 6.29 , 6.79 , 7.65 , 7.48 , 5.05 , 3.25 , 3.55 , 3.71 , 3.72/3.35 ; 13C NMR , 158.8 (C-2), 135.8 (C-3), 179.8 (C-4), 163.3 (C-5), 99.9 (C-6), 166.3 (C-7), 94.8 (C-8), 158.7 (C-9), 106.0 (C-10), 123.1 (C1′), 116.3 (C-2′), 146.1 (C-3′), 150.1(C-4′), 117.6 (C-5′), 123.1 (C-6′), 104.8 (C-1′′), 73.4 (C-2′′), 74.3 (C-3′′), 69.3 (C-4′′), 67.1 (C-5′′).Quercetin-3-max 274 and 369 nm; ESI–MS m/z: [M+H]+ 303.06, HRMS [M+H]+ 303.0498, cald. for C15H11O7 303.0504 Da; 1H NMR  6.01 , 6.21 , 7.56 , 6.71 , 7.47 ; 13C NMR , 148.9 (C-2), 137.4 (C-3), 177.4 (C-4), 162.6 (C-5), 99.4 (C-6), 165.7 (C-7), 94.5 (C-8), 158.3 (C-9), 104.6 (C-10), 124.3 (C1′), 116.1 (C-2′), 146.3 (C-3′), 148.1(C-4′), 116.4 (C-5′), 121.8 (C-6′).Quercetin (compound 5): Yellowish powder, UV λ®  ion trap mass spectrometer according to the method developed by Fonseca et al.  × 100, where A and B are the optical densities at 490 nm (OD490) from the wells where untreated (A) and treated (B) cells are present, respectively.Cell suspensions (Vero and MRC-5) were distributed in 96-well microplates containing 6.0 × 10h 1% SFB ,49. Subsh 1% SFB ,64. Seveh 1% SFB ,64. FormZika virus (ZIKV) was picked on C6/36 cells to generate working stocks for 5 days at 28 °C. The infected cell culture supernatant was harvested after 5 days and clarified by centrifugation at 4000 RPM and refrigerated for 10 min at 4 °C, and then aliquoted and stored at −70 °C as virus stock until use.4 cells per well), maintaining three uninfected columns for cell control. The plates were then incubated at 37 °C (5% CO2), and the cytopathic effect (ECP) was monitored. ECP was recorded every 24 h for 72 h, observing cell control.After the incubation period (24 h), for the formation of the Vero cell monolayer, the viral suspension was infected, containing in each well 100.0 μL of each dilution in 1% SFB DMEM medium. Viral stocks were diluted (10-1 to 10-18) and inoculated into Vero cell monolayers implanted in approximately 85% confluence 96-well plates (6 × 104 cells per well). After 24 h of incubation in 5% CO2 at 37 °C, the culture medium (DMEM 5% SFB) was removed, and 100.0 μL of sample dilutions were added to each well along with 100.0 μL of the viral suspension. Then, the cultures were incubated in a humid atmosphere containing 5% CO2 at 37 °C for 72 h. Cell viability was assessed by the MTT colorimetric technique  × 100, where A, B, and C are the OD490 of the wells, in which are present treated and infected (A), untreated and infected (B), and untreated and non-infected cells (C), respectively.The effective concentration that has 50% antiviral effect, or 50% effective concentration , corresponding to the therapeutic index, is defined as the ratio of CCon cycle .6 cells per well) was infected by viral suspensions with titers of 1.0 × 107 TCID50/mL, (MOI = 1), Zika virus, during 1 h for the viral adsorption. Afterwards, the viral suspension was removed and the wells were washed with PBS. Then, the wells were treated with the active concentration of the compound (20 μg/mL) and ethanolic extract of leaves of C. americana (85 μg/mL). The plates were incubated at 37 °C in a humidified 5% CO2 atmosphere and photographed 48 h after infection [In order to confirm the anti-ZIKV activity observed in the antiviral MTT assay, the Vero cell monolayer .Data from cytotoxicity and antiviral assays were evaluated according to their means and standard deviations. The cytotoxic concentration at 50% and the effective concentration at 50% were determined compared with the control obtained from nonlinear regression. These analyses were performed using the GraphPad Prism 5.04 statistical packageCuratella americana leaves are rich in phenolic compounds, mainly flavonoids. The bioguided phytochemical investigation of the ethanolic extract of C. americana leaves revealed that medium polarity constituents may be responsible for the observed anti-ZIKV activity, considering that only the ethyl acetate fraction obtained from the liquid–liquid partition of the ethanolic extract was active against ZIKV. From this fraction, three flavonoids were isolated: quercetin-3-O-hexosylgallate, quercetin-3-O-glucoside, and quercetin, in addition to a mixture (quercetin-3-O-rhamnoside and quercetin-3-O-arabinoside). Flavonoids can be considered primarily responsible for the antiviral activity observed, since the active substances obtained, quercetin-3-O-hexosylgallate and quercetin, showed significant biological activity, exhibiting selective and promising anti-ZIKV activity in vitro.


Annona purpurea seeds with a sequence of cyclo-Gly-Phe-Ile-Gly-Ser-Pro-Val-Pro. In our previous study, the cyclisation of linear cyclopurpuracin was problematic; however, the reversed version was successfully cyclised even though the NMR spectra revealed the presence of a mixture of conformers. Herein, we report the successful synthesis of cyclopurpuracin using a combination of solid- and solution-phase synthetic methods. Initially, two precursors of cyclopurpuracin were prepared, precursor linear A (NH2-Gly-Phe-Ile-Gly-Ser(t-Bu)-Pro-Val-Pro-OH) and precursor linear B (NH-Pro-Gly-Phe-Ile-Gly-Ser(t-Bu)-Pro-Val-OH, and various coupling reagents and solvents were trialled to achieve successful synthesis. The final product was obtained when precursors A and B were cyclised using the PyBOP/NaCl method, resulting in a cyclic product with overall yields of 3.2% and 3.6%, respectively. The synthetic products were characterised by HR-ToF-MS, 1H-NMR, and 13C-NMR, showing similar NMR profiles to the isolated product from nature and no conformer mixture. The antimicrobial activity of cyclopurpuracin was also evaluated for the first time against S. aureus, E. coli, and C. albicans, showing weak activity with MIC values of 1000 µg/mL for both synthetic products, whereas the reversed cyclopurpuracin was more effective with an MIC of 500 µg/mL.Cyclopurpuracin is a cyclooctapeptide isolated from the methanol extract of  Cyclic peptides isolated from animals, plants, bacteria, or fungi have been of interest due to their potential biological properties . The uni1)  is of interest. The initial effort to synthesise cyclopurpuracin by our group was unsuccessful as the cyclisation step was problematic. Fortunately, reversed cyclopurpuracin (2) (A new cyclic peptide of interest is cyclopurpuracin (1) , which wnfigured  9,10]. ,10. 1) (acin (2)  was succA combination of solid- and solution-phase methods is commonly used for the synthesis of cyclic peptides. In this strategy, a linear precursor is prepared through solid-phase synthesis, while cyclisation is performed in a solution. This method was applied by Muhajir et al. in 2021 for the synthesis of nocardiotide A , Kurnia t-Bu-based strategy. Fmoc is the protecting group of the α amino group, while t-Bu is the protecting group of the side chain for the serine residue. The Fmoc group is base labile and the peptides are released from 2-chlorotrityl resin along with t-Bu deprotection using acid. Therefore, this orthogonal strategy ensures the peptide and all side-chain protecting groups remain on the resin during the elongation step  for 45 min. The chromatogram of linear precursor A showed a peak of the target compound at a retention time of 33.2 min, whereas the peak was at a retention time of 34.2 min for linear precursor B. All the fractions were collected and concentrated using a rotary evaporator to produce white solids (6.1 mg for linear precursor A and 12.2 mg for linear precursor B). The purified products were then characterised by HR-ToF-MS and the MS spectra showed correct molecular ion peaks of the desired linear octapeptide A and B at m/z [M + H]+ 829.4820  and m/z [M + H]+ 829.4822 , respectively (1H-NMR and 13C-NMR (spectra can be found in The crude linear precursors were purified by semi-preparative RP-HPLC [Hectively . Furtherv/v DIPEA at room temperature [n-hexane:ethyl acetate (6:4)] until there was no purple colour in the TLC plate when it was sprayed with ninhydrin solution. The reaction took seven days to yield the protected cyclopurpuracin and the remaining solvent was concentrated by a rotary evaporator. The crude peptide was then added by a deprotecting reagent (TFA:H2O:TIPS = 95:2.5:2.5) to eliminate the t-Bu protecting group. The cyclopurpuracin was successfully obtained and confirmed by HR-ToF-MS, showing a molecular ion peak at m/z [M+Na]+ 777.3809  . The third trial cyclisation was performed using PyBOP but the desired product was still not formed after seven days with only one molecular ion peak of the linear precursor at m/z 829.4214 . The failure of the cyclisation of linear precursor A may be due to the high flexibility of the linear octapeptide because of the presence of two glycine and one serine residues + 777.3915  for cyclopurpuracin B, representing the correct molecular ion peak of the desired 1 , benzotriazole-1-yloxytri(pyrrollidino)phosphonium hexafluorophosphate (PyBOP), N-hydroxybenzotriazole (HOBt), n-hexane N,N-diisopropyletilamine (DIPEA), piperidine, ethyl acetate, sodium chloride, 2,2,2-trifluoroethanol, acetic acid, triisopropylsilane, acetonitrile, sodium sulphate, and trifluoroacetic acid. All of the amino acid residues, Fmoc-L-Proline, Fmoc-L-Valine, Fmoc-L-Isoleucine, Fmoc-L-Serine(t-Bu)-OH, Fmoc-L-Glycine, Fmoc-L-Phenylalanine-OH, and 2-chlorotrityl chloride, were purchased from GL-biochem Ltd., Shanghai, China.The chemicals used in this research were 2-chlorotrityl chloride resin, dimethylformamide (DMF), dichloromethane (DCM), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), O-(7-azabenzotriazole-1-yl)-1H-NMR and 13C-NMR on an Agilent NMR 500 MHz (1H) and 125 MHz (13C) using a deuterated solvent. Mass spectrometry spectra were recorded on the Waters HR-ToF-MS Lockspray. The loading resin absorbance was measured on the TECAN Infinite pro 200 UV-Vis Spectrophotometer.Analysis of the linear and cyclic octapeptide was achieved on the Waters 2998 Photodiode Array Detector (PDA) and LiChrospher 100 C-18 5 µm column RP-HPLC with a detection wavelength of 210 and 240 nm. The mobile phase consisted of acetonitrile (A) and deionised water (B) using gradient elution. The flow rate was 1.0 mL/min, and the column temperature was maintained at 25 °C over 40 min. The peptides were characterised by 1-OH) (1 eq.) was loaded onto the resin in dichloromethane (4 mL) and basic DIPEA (2 eq.). For loading resin absorbance measurement, 20% piperidine in DMF (3 mL) was added to 0.6 mg of Fmoc-L-AA1 resin in an Eppendorf tube and left for 30 min followed by sonification for 5 min. The loading resin was measured by a UV-Vis spectrophotometer at 290 nm. For the next step, the capping of the resin was carried out by adding MeOH:DCM:DIPEA 15:80:5 (10 mL) twice for 15 min before the Fmoc group in Fmoc-L-AA1 resin was removed using 20% piperidine in DMF (5 mL) for 2 × 5 min to yield free amino groups. The free amino group was coupled with the second Fmoc-protected amino acid (Fmoc-AA2-OH) in the presence of HBTU (3 eq.) and HOBt (3 eq.) as a coupling agent and N, N-diisopropylethylamine (DIPEA) (6 eq.) as an activating reagent in DMF (4 mL) for 4 h at room temperature. The Fmoc group was removed using 20% piperidine in DMF (5 mL) for 2 × 5 min to afford the resin Fmoc-AA1-AA2-NH2. This cycle of coupling and deprotection was repeated with subsequent Fmoc-protected amino acids to afford the resin-coupled octapeptide. Finally, the resin was cleaved using AcOH:TFE:DCM (2:2:6) for 2 × 2 h. After the filtration of the resin and subsequent evaporation, the crude peptides were washed with dichloromethane repeatedly and dried under a vacuum. The linear peptide was analysed using analytical RP-HPLC , and the crude peptide was purified by semi-preparative RP-HPLC using 5–80% ACN eluent for 40 min. The target fractions  were collected and characterised by ToF-ESI-MS, 1H-NMR , and 13C-NMR .Chlorotrityl chloride resin was used as the solid support. First, 2-chlorotrityl chloride resin  was swollen in dichloromethane (10 mL) over 30 min at room temperature. The first amino acid . 1H-NMR  4.29 , 2.18; 2.12 , 2.02; 1.90 , 3.74; 3.54 , 4.50 , 3.10 , 0.87 , 0.87 , 4.54 , 2.28; 2.12 , 2.02; 1.90 , 3.74; 3.58 , 4.78 4 Hα), 3.99; 3.38 4 Hβ), 1.16 4 CH3), 4.13 , 4.40 , 2.89 , 1.11 , 1.54 , 0.93 , 4.95 , 3.63; 3.18 , 7.22 , 7.24 , 3.83 . 13C-NMR  176.46 (Pro1 C=O), 62.1 (Pro1 Cα), 28.4 (Pro1 Cβ), 24.7 (Pro1 Cγ), 54.6 (Pro1 Cδ), 172.0 , 59.3 , 30.8 , 18.5 , 176.45 (Pro3 C=O), 63.7 (Pro3 Cα), 29.3 (Pro3 Cβ), 24.5 (Pro3 Cγ), 52.0 (Pro3 Cδ), 167.6 (Ser4 C=O), 56.1 (Ser4 Cα), 61.6 (Ser4 Cβ), 28.2 (Ser4 t-Bu), 81.4 (Ser4 Cq-tBu), 170.3 (Gly5 C=O), 42.0 (Gly5 Cα), 171.4 (Ile6 C=O), 60.4 (Ile6 Cα), 36.4 (Ile6 Cβ), 14.5 (Ile6 Cβ-CH3), 24.8 (Ile6 Cγ), 10.1 (Ile6 Cγ-CH3), 172.3 (Phe7 C=O), 58.5 (Phe7 Cα), 37.7 (Phe7 Cβ), 136.7 (Phe7 Cq), 128.2 , 129.1 , 126.5 (Phe7-4′), 165.0 (Gly8 C=O), 46.6 (Gly8 Cα).Precursor linear A: white solid; 6.1 mg, yield: 15.2%; HR-TOF-MS m/z [M+H]+ 829.4822 . 1H-NMR  4.25 , 1.75 , 0.78 , 0.78 , 7.97 , 4.37 , 2.74; 2.13 , 2.02; 1.88 , 3.60; 3.38 , 4.59 3 Hα), 4.16; 3.91 3 Hβ), 1.05 3 CH3), 8.12 3 NH), 4.08 , 8.38 , 4.25 , 2.78 , 0.78 , 1.55 , 0.78 , 7.50 , 4.67 , 3.44; 3.19 , 7.18 , 7.22 , 8.17 , 3.95 , 8.56 , 3.68 , 1.88; 1.84 , 1.70; 1.20 , 3.04; 3.02 . Precursor linear B: white solid; 12.2 mg, yield: 20.3%; HR-TOF-MS v/v). The mixture was stirred over 7 days at room temperature (monitored by TLC). The reaction mixture was concentrated to obtain a dark-yellow oil, which was deprotected with TFA:TIPS:H2O (95:2.5:2.5). The linear octapeptide was dissolved in dichloromethane in 1.25 mM before the addition of the coupling reagent  (3 equiv.) and DIPEA . The cyclooctapeptide was analysed using analytical RP-HPLC  and characterised by ToF-ESI-MS, 1H-NMR  and, 13C-NMR .The linear octapeptide was dissolved in DMF to 2.0 mM before the addition of PyBOP (1.14 equiv.) and DIPEA (1.65 equiv.). The mixture was stirred over 2 days at room temperature (monitored by TLC). The reaction mixture was extracted using a brine solution and ethyl acetate. The organic phase was added by NaS. aureus, E. coli, C. albicans). The peptides were dissolved in 2% DMSO to a concentration of 1000 ppm. Each solution was dissolved gradually: 1000; 500; 250; 125; 62.5; 31.25; 15.62; 7.81; 3.90; 1.95; 0.97; and 0.48 ppm. The sample solutions, ciprofloxacin, vancomycin, nystatin, and 2% DMSO, were incubated in a 96-well microplate at 37 °C for 18 h. The plates were read in a spectrophotometer at a wavelength of 600 nm and the MIC was calculated as the percentage of microbial inhibition.The cyclopurpuracin and the reversed cyclopurpuracin  were tes1H-NMR  and 13C-NMR  data can be found in m/z [M + H]+ 755.4110 . Cyclopurpuracin from precursor linear A: white solid; 3.8 mg, 20.8% yield; 1H-NMR  and 13C-NMR  data can be found in m/z [M + Na]+ 777.3915 . Cyclopurpuracin from precursor linear B: white solid; 3.3 mg, 18.1% yield; 2-Gly-Phe-Ile-Gly-Ser(t-Bu)-Pro-Val-Pro-OH) and linear precursor B (NH-Pro-Gly-Phe-Ile-Gly-Ser(t-Bu)-Pro-Val-OH) in overall yields of 3.2% and 3.6%, respectively. The NMR spectra of cyclopurpuracin showed a single conformer and high similarity with natural cyclopurpuracin. The antimicrobial activity of cyclopurpuracin A and B was categorised as weak with MIC values of 1000 µg/mL and slightly lower than the antimicrobial properties of the reversed cyclopurpuracin with MIC values of 500 µg/mL.Cyclopurpuracin  was synthesised through a combination of solid- and solution-phase methods using PyBOP with the addition of NaCl salt with linear precursor A (NH


Eupatorium heterophyllum leaves were collected at different locations in Yunnan and Sichuan provinces in China, and their chemical constituents were investigated. Thirteen previously undescribed sesquiterpene lactones—seven germacranolides, three eudesmanolides, two guaianolides, and a 2-norelemanolide—were isolated, and their structures were elucidated based on extensive spectroscopic analyses. The major constituents in the six samples from northwestern Yunnan and Sichuan are hiyodorilactones A and B, whereas that in the two samples from the region near Kunming, Yunnan is eupatoriopicrin. These results and previously reported results suggest the presence of locality-dependent intra-specific diversity in the chemical constituents of E. heterophyllum leaves.Eight samples of  The Hengduan Mountains and surrounding areas consist of many mountain ranges and deep river valleys with the elevation range from approximately 1500 to 6000 m, which separate these areas into various subdivisions. These areas are also climatically diverse and rich in plant resources, providing us with good plant materials for the study of diversity in secondary metabolites.Eupatorium heterophyllum DC. (Asteraceae) is an endemic species in China. It is widely distributed in grasslands and forest areas at altitudes of 1700–3000 m in Southwest China  and has not been artificially cultivated + at m/z 453.1764 in its HRFABMS, which suggests a molecular formula of C22H28O10. The 1H and 13C NMR spectra of 17 (16) + peak at m/z 433.1241 in HRFABMS, confirming its molecular formula as C20H26O8. The IR absorptions at 3380, 1745, and 1715 cm−1 suggested the presence of a hydroxy group, γ-lactone, and ester group, respectively. The 1D and 2D NMR spectra of 36 were recorded at 233 K because its 1H NMR spectrum exhibited broad signals at room temperature, suggesting conformational flexibility. The 1H and 13C NMR spectra of 36 (33)  and an exomethylene group , respectively, implying that 36 was a C-1 hydroperoxy analog of deacetyleupaserrin (33). This was confirmed by the COSY correlations of H-1/H-2/H2-3, H-5/H-6/H-7, H-8/H2-9, and H-3′/H-4′, along with the HMBC correlations from H3-15 to C-3, C-4, and C-5; from H2-14 to C-1; from H-1 to C-9; from H2-13 to C-7 and C-12; and from H3-4′ to C-2′ germacra-4,10(14),11(13)-trien--olide.Compound ra of 36  were sim to C-2′ . Therefo40 showed a [M + Na]+ peak at m/z 459.1632 to establish a molecular formula of C22H28O9 with nine degrees of unsaturation. The IR absorptions at 3400 cm−1 corresponded to a hydroxy group and those at 1743, 1735, and 1715 cm−1 are attributed to carbonyl groups. Similar to the case of 36, the 1H NMR spectrum of 40 also afforded broad signals at room temperature. Even at 233 K, the quality of the 13C NMR spectrum remained insufficient owing to the small amount of 40 obtained; however, the 1H NMR spectrum clearly showed pairs of signals (in a ratio of 2:3 based on the integration), suggesting the coexistence of two conformers. A careful analysis of the 1H NMR (1H-1H COSY spectra (40 with 4E-deacetyl chromolaenide-4′-O-acetate (39) [H 8.56 (major) and 8.43 (minor)] and an additional exomethylene [δH 5.44/5.13 (major) and 5.35/5.02 (minor)]. The differences in the 1H NMR spectrum of 40 with that of 39 were attributable to a 1β-hydroperoxy-10(14)-ene structure, as is the case with 36 and 33. Therefore, 40 was identified as 8β-(4′-acetoxytigloyloxy)-1β-hydroperoxy-3β-hydroxygermacra-4,10(14),11(13)-trien--olide.The HRESIMS spectrum of e 1H NMR  and the  spectra  of both ate (39)  as well 45 showed a quasimolecular ion [M + H]+ at m/z 363.1808 in its HRFABMS, which suggests a molecular formula of C20H26O6. The 1H NMR data suggested a structural similarity of 45 to that of the known eudesmanolide 44 [H 6.40 ] and H3-4′ [δH 2.04 ] and the NOESY correlation between H-3′ and H2-5′ indicated that the 4′,5′-dihydroxytigloyl group in 44 was replaced with a 5′-hydroxyangeloyl group in 45 (45 was identified as 1β-hydroxy-8β-(5′-hydroxyangeloyloxy)eudesma-4(15),11(13)-dien--olide.Compound olide 44  /H-2 (δH 3.62)/H2-3 (δH 2.66 and 2.11) and the NOE correlation between H-2β and H3-14 suggested the presence of another hydroxy group at C-2α in 50 compared to that in 45 eudesma-4(15),11(13)-dien--olide.The at in 45 . Thus, 551 was determined to be C24H30O10 using HRFABMS. Its 1H and 13C NMR data  and H-8 (δH 5.83) in the 1H NMR spectrum as well as the COSY and HMBC correlations shown in 51.The molecular formula of NMR data  were clo53 showed a [M + Na]+ peak at m/z 415.1368, which suggests the molecular formula C20H24O8 with nine degrees of unsaturation. The 1H and 13C NMR spectra revealed the presence of one methyl, two oxymethylenes, three oxymethines, two exocyclic double bonds, one trisubstituted double bond, one tetrasubstituted double bond, and two carbonyls  as well as the NOESY correlation between H-13b and H-8 suggested the presence of a 4′,5′-dihydroxytigloyloxy moiety at C-8 and a γ-lactone between C-12 and C-6. Moreover, the molecular formula of 53 and the chemical shift of C-3 (δ 94.2) suggested the presence of a hydroperoxy group at this position. The elucidated planar structure of 53 is shown in J6,7 = 10.5 Hz). Therefore, 53 was identified as 8β--3α-hydroperoxyguaia-4,10(14),11(13)-trien--olide.The HRESIMS spectrum of compound arbonyls . The abo54 revealed that it is also a guaianolide with the same molecular formula as that of 53  and C-3  indicated a disubstituted double bond. The chemical shift of C-4 observed at δC 95.2 in the 13C NMR spectrum and NOE correlation between H3-15 and H-6 indicated the presence of a hydroperoxy group at C-4α. Thus, 54 was concluded to be a 4α-hydroperoxy-2-ene isomer of 53.HRFABMS and 1D/2D NMR spectra of at of 53 . A COSY 62 was isolated as a colorless oil. A [M + Na]+ peak was observed at m/z 371.1472 in its HRESIMS, corresponding to the molecular formula of C19H24O6 with eight degrees of unsaturation. The IR spectrum suggested the presence of hydroxy (3501 cm−1), γ-lactone (1769 cm−1), aldehyde (1726 cm−1), and α,β-unsaturated carbonyl (1715 cm−1) groups. The 1H and 13C NMR spectra showed the characteristic signals for a 5′-hydroxyangeloyl moiety  as well as the NOE between H-8 and H-13b (δH 5.57) as shown in 62 is similar to that of 45 based on NOE correlations and coupling constants. Thus, 62 was identified as 8β-(5′-hydroxyangeloyloxy)-1-oxo-2-norelema-3,11(13)-dien--olide.Compound l moiety , which i to H2-9 . Further17, 18, 24, 45, 50, 51, 54, and 62 showed a similar trend to that of 10, especially the negative Cotton effect around 210 nm mainly owing to the α-methylene-γ-lactone moiety. This indicated that the absolute configurations at C-6 and C-7 of these compounds are the same as those of 10 while the other chromophore might have a weaker contribution to their experimental ECD spectra [31, 36, 40, and 53, would have the same stereochemistry.The experimental ECD spectra of E. heterophyllum collected in Yunnan and Sichuan provinces (1–40), eudesmanolides (41–52), guaianolides (53–56), and elemanolides (62). Most of the new compounds would be produced from major constituents vir oxidative metabolism, which often involve the introduction of a hydroperoxy group. The chemical composition of each sample is summarized in 1) and B (3) are the major constituents in samples 1–6, confirming that hiyodorilactone-type samples are predominant in Sichuan and northwestern Yunnan regions. In contrast, eupatoriopicrin (26), which is not detected in samples 1–6, is the major constituent in samples 7 and 8 from the region near Kunming. A variety of -germacranolides and eudesmanolides are also contained in these samples. Therefore, samples 7 and 8 should be classified as another chemotype, eupatoriopicrin-type. Considering these results and previously obtained results [E. heterophyllum.In this study, 63 compounds, including 13 new compounds, were isolated from 8 leaf samples of rovinces . Among t26) showed higher cytotoxicity against HL-60 cells than hyodorilactones A (1) and B (3) [E. heterophyllum [E. heterophyllum is related to its genetic background. These observations can provide us a new insight into a chemical aspect of adaptation and differentiation of Eupatorium plants. We plan to conduct further chemical studies on E. heterophyllum sampled from other regions to obtain secondary metabolites produced by limited populations within a species and to understand the relationship between chemical diversity, genetic diversity, and geographical distribution.Eupatoriopicrin (nd B (3) . Moreoveophyllum . Notablyplus 500 spectrometer  or JEOL JNM-AL 400 spectrometer . The coupling constants (J) are expressed in Hertz, and the chemical shifts (δ) are reported in ppm with the residual solvent signal used as a reference (CDCl3: TMS). Mass spectra, including high-resolution spectra, were recorded on a JEOL JMS-700 MStation. Column chromatography was performed on silica gel 60 . TLC was with silica gel 60 F254 plates . Preparative HPLC was performed on a JASCO chromatograph  equipped with a JASCO PU-2086 pump, a JASCO UV-970 detector, a JASCO RI-2031 detector, and various columns: COSMOSIL 5SL-II , COSMOSIL 5SL-II , COSMOSIL 5SL-II , YMC-Pack Diol-120-NP , Inertsil CN-3 , Inertsil Diol , TSK gel silica 60 , and TSK gel G1000HHR .Optical rotations were measured with a JASCO P1020NK digital polarimeter. IR spectra were recorded using a JASCO FT/IR-410 spectrophotometer with the diffuse reflectance method. The 1D and 2D NMR spectra were measured using a Varian Unity Samples were collected in August of 2014 (samples 1 and 2) and 2015 (samples 3–8) at the several geographically isolated locations shown in v/v; eluent B ), were delivered at a flow rate of 0.25 mL/min using a linear gradient program. The linear elution gradient program was set as follows: 0 min (95:5)–14.75 min (2:98)–17.00 min (2:98)–17.20 min (95:5)–20.00 min (95:5).Parts of the fresh leaves (a few grams) of each sample were extracted with ethanol immediately after harvesting, and the extracted ethanol solutions were filtered and then separated using a Waters Acquity™ UPLC I-Class system coupled with a Waters ACQUITY UPLC BEH C18 column . The temperature of the column was held at 45 °C. The mobile phases, which consisted of two solvent systems  to afford ten subfractions (Fr. 1–10). Compound 1 (573.7 mg) was obtained as Fr. 7. Fr. 4 (165.1 mg) was purified with semipreparative HPLC  to give 7 (11.0 mg) and 9 (36.4 mg). Fr. 5 (141.7 mg) was suspended in n-hexane–EtOAc (7:3) and then filtered to obtain 58 (6.3 mg) as a precipitate. The filtrate was separated by COSMOSIL 5SL-II  to afford 58 (7.8 mg), Fr. 5-4 (9.5 mg), and 14 (9.5 mg). Fr. 5-4 (9.5 mg) was purified on YMC-Pack Diol-120-NP  to yield 60 (0.7 mg), a mixture (4:3) of 12 and 13 (2.1 mg), and 15 (0.4 mg). Fr. 6 (670.1 mg) was separated by COSMOSIL 5SL-II  to give five fractions: Fr. 6-0–6-4. Fr. 6-4 (47.6 mg) was identified as 38. Fr. 6-2 (251.5 mg) was purified on COSMOSIL 5SL-II  and gave 11 (83.9 mg) and 3 (112.5 mg). Fr. 6-3 (90.3 mg) was purified on YMC-Actus Triart Diol-HILIC  and Inertsil CN-3  to obtain 22 (2.1 mg) and 24 (1.9 mg).The dried leaves of sample 1 (39.1 g) were cut into small pieces and extracted with MeOH two times at room temperature. After removal of the solvent under reduced pressure not exceeding 30 °C, a concentrated and combined MeOH extract (7.5 g) was obtained, which was then separated using a silica gel column , 2 (64.3 mg), 3 (85.4 mg), 4 (18.8 mg), 7 (9.6 mg), 8 (3.2 mg), 9 (38.1 mg), 10 (17.9 mg), 11 (20.0 mg), 14 (7.9 mg), 15 (10.8 mg), 17 (11.5 mg), 20 (16.6 mg), 21 (21.0 mg), 22 (4.2 mg), 24 (2.8 mg), 37 (28.1 mg), 51 (7.4 mg), 57 (26.5 mg), and 58 (4.3 mg).The extract of sample 2 (5.4 g from 31.2 g of leaves) was similarly treated to afford 1 (11.2 mg), 3 (68.6 mg), 4 (2.4 mg), 18 (1.5 mg), and 60 (1.7 mg).The extract of sample 3 (2.3 g from 14.7 g of leaves) was similarly treated to afford 1 (305.1 mg), 2 (129.6 mg), 3 (161.0 mg), 4 (15.5 mg), 5 (0.5 mg), 7 (6.9 mg), 16 (20.4 mg), 17 (6.4 mg), 19 (0.8 mg), 20 (1.9 mg), 40 (0.9 mg), 53 (0.5 mg), 54 (6.8 mg), and 61 (2.1 mg).The extract of sample 4 (7.8 g from 46.2 g of leaves) was similarly treated to afford 1 (101.7 mg), 2 (37.6 mg), 3 (12.9 mg), 4 (2.2 mg), 7 (0.9 mg), 9 (0.9 mg), 11 (4.8 mg), 14 (0.4 mg), 16 (4.8 mg), 17 (2.2 mg), 38 (6.8 mg), 57 (3.5 mg), and 60 (0.5 mg).The extract of sample 5 (1.0 g from 5.2 g of leaves) was similarly treated to give 1 (303.8 mg), 3 (34.6 mg), 4 (6.1 mg), 5 (0.6 mg), 6 (5.2 mg), 7 (1.4 mg), 9 (1.2 mg), 11 (8.1 mg), 12 (0.5 mg), 13 (0.3 mg), 14 (3.0 mg), 15 (0.5 mg), 37 (1.6 mg), 38 (16.1 mg), 39 (0.9 mg), 56 (0.7 mg), 57 (5.4 mg), 58 (1.7 mg), and 61 (1.3 mg).The extract of sample 6 (3.1 g from 24.8 g of leaves) was similarly treated to afford 3 (2.1 mg), 23 (0.6 mg), 25 (22.5 mg), 26 (514.5 mg), 27 (27.1 mg), 28 (73.0 mg), 29 (11.0 mg), 30 (70.4 mg), 31 (23.6 mg), 32 (1.0 mg), 34 (3.1 mg), 35 (4.7 mg), 42 (4.3 mg), 47 (2.6 mg), 57 (16.5 mg), 61 (1.8 mg), and 63 (21.3 mg).The extract of sample 7 (4.0 g from 25.9 g of leaves) was similarly treated to afford 1 (7.8 mg), 2 (16.3 mg), 3 (8.8 mg), 4 (1.1 mg), 11 (1.0 mg), 16 (0.2 mg), 26 (50.3 mg), 28 (10.0 mg), 29 (1.2 mg), 32 (7.9 mg), 33 (5.6 mg), 36 (2.0 mg), 38 (8.4 mg), 39 (0.4 mg), 41 (6.9 mg), 43 (1.9 mg), 44 (20.0 mg), 45 (4.6 mg), 46 (1.0 mg), 48 (1.9 mg), 49 (0.2 mg), 50 (1.4 mg), 52 (2.3 mg), 55 (0.2 mg), 57 (23.7 mg), 58 (3.4 mg), 59 (68.1 mg), 60 (7.2 mg), 61 (1.0 mg), and 62 (0.4 mg).The extract of sample 8 (3.3 g from 26.2 g of leaves) was similarly treated to afford A conformational search was performed using the Monte Carlo method and the MMFF94 force field with Spartan ′20 . The obtained low-energy conformers within 6 kcal/mol were optimized at the B3LYP/6-31G level in MeOH (PCM). The vibrational frequencies were also calculated at the same level to confirm their stability, and no imaginary frequencies were found. The energies, oscillator strengths, and rotational strengths of the low-energy conformers were calculated using TDDFT at the CAM-B3LYP/6-31G in MeOH (PCM) level and weight-averaged. The ECD spectra were simulated using GaussView  with the10, Colorless oil; [αc = 0.10, MeOH); FT-IR cm−1: 3448, 1765, 1743; MS (EI) m/z: 400 (M)+; HRMS (EI) m/z: 400.1518 . UV (CH3OH) λmax (log ε) 204 (0.84) nm; ECD  λmax (Δε): 208 (−22.74) nm. 1H and 13C NMR: see Compound 17, Colorless oil; [αc = 0.59, MeOH); FT-IR cm−1: 3411, 1740, 1715; MS (FAB) m/z: 453 (M + H)+; HRMS (FAB) m/z: 453.1764 . UV (CH3OH) λmax (log ε) 204 (1.03) nm; ECD  λmax (Δε): 233 (+1.39), 215 (−6.48) nm. 1H and 13C NMR: see Compound 18, Colorless oil; [αc = 0.12, MeOH); FT-IR cm−1: 3390, 1739, 1714; MS (FAB) m/z: 475 (M + K)+; HRMS (FAB) m/z: 475.1383 . UV (CH3OH) λmax (log ε) 203 (0.63) nm; ECD  λmax (Δε): 302 (+0.32), 255 (−0.25), 236 (+0.25), 214 (−4.86) nm. 1H and 13C NMR: see Compound 24, Colorless oil; [αc = 0.20, MeOH); FT-IR cm−1: 3419, 1760, 1730; MS (FAB) m/z: 457 (M + Na)+; HRMS (FAB) m/z: 475.1475 . UV (CH3OH) λmax (log ε) 203 (0.87) nm; ECD  λmax (Δε): 296 (+0.23), 263 (−0.16), 238 (+0.67), 214 (−10.21) nm. 1H and 13C NMR: see Compound 31, Colorless oil; [αc = 0.54, CHCl3); FT-IR cm−1: 1769, 1731; MS (CI) m/z: 333 (M + H)+; HRMS (CI) m/z: 333.2059 . 1H and 13C NMR: see Compound 36, Colorless oil; [αc = 0.15, MeOH); FT-IR cm−1: 3380, 1745, 1715; MS (FAB) m/z: 433 (M + K)+; HRMS (FAB) m/z: 433.1241 . 1H and 13C NMR: see Compound 40, Colorless oil; [αc = 0.12, MeOH); FT-IR cm−1: 3400, 1743, 1735, 1715; MS (ESI) m/z: 459 (M + Na)+; HRMS (ESI) m/z: 459.1632 . 1H and 13C NMR: see Compound 45, Colorless oil; [αc = 0.33, CHCl3); FT-IR cm−1: 3444, 1769, 1715; MS (FAB) m/z: 363 (M + H)+; HRMS (FAB) m/z: 363.1808 . UV (CH3OH) λmax (log ε) 208 (1.25) nm; ECD  λmax (Δε): 258 (−0.79), 238 (+0.02), 216 (−3.22) nm. 1H and 13C NMR: see Compound 50, Colorless oil; [αc = 0.16, MeOH); FT-IR cm−1: 3390, 1767, 1714; MS (FAB) m/z: 379 (M + H)+; HRMS (FAB) m/z: 379.1749 . UV (CH3OH) λmax (log ε) 203 (1.02) nm; ECD  λmax (Δε): 251 (−0.95), 212 (−3.26) nm. 1H and 13C NMR: see Compound 51, Colorless oil; [αc = 0.75, MeOH); FT-IR cm−1: 3459, 1765, 1745, 1721; MS (FAB) m/z: 501 (M + Na)+; HRMS (FAB) m/z: 501.1737 . UV (CH3OH) λmax (log ε) 204 (1.02) nm; ECD  λmax (Δε): 255 (−1.31), 216 (−5.00) nm. 1H and 13C NMR: see Compound 53, Colorless oil; [αc = 0.07, MeOH); FT-IR cm−1: 3384, 1759, 1714; MS (ESI) m/z: 415 (M + Na)+; HRMS (ESI) m/z: 415.1368 . 1H and 13C NMR: see Compound 54, Colorless oil; [αc = 0.63, CHCl3 + MeOH); FT-IR cm−1: 3398, 1748, 1714; MS (FAB) m/z: 415 (M + Na)+; HRMS (FAB) m/z: 415.1369 . UV (CH3OH) λmax (log ε) 203 (1.12) nm; ECD  λmax (Δε): 258 (−1.18), 216 (−1.42) nm. 1H and 13C NMR: see Compound 62, Colorless oil; [αc = 0.03, CHCl3); FT-IR cm−1: 3501, 1769, 1715; MS (ESI) m/z: 371 (M + Na) +; HRMS (ESI) m/z: 371.1472 . UV (CH3OH) λmax (log ε) 203 (0.62) nm; ECD  λmax (Δε): 255 (−1.00), 210 (−1.09) nm. 1H and 13C NMR: see Compound


Bioprospecting of actinobacteria isolated from Kubuqi desert, China for antibacterial, antifungal and cytotoxic metabolites production and their structure elucidation.Streptomyces as the predominant genus comprising 37 different species, along with the rare actinobacterial genus Lentzea. The methanolic extracts of 60.8% of strains exhibited potent antimicrobial activities against Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Escherichia coli, Salmonella enterica, Saccharomyces cerevisiae and high to mild in vitro cytotoxicity against PC3 (prostate cancer) and A549 (lung carcinoma) cell lines. The metabolomics analysis by TLC, HPLC-UV/vis, HPLC-MS and NMR showed the presence of compounds with molecular weights ranging from 100 to 1000 Da. The scale-up fermentation of the prioritized anti-Gram-negative strain PU-KB10–4 (Streptomyces griseoviridis), yielded three pure compounds including; griseoviridin  with 20 fold increased production as compared to previous reports and its crystal structure as monohydrate form is herein reported for the first time, mitomycin C  and a new bacterial metabolite 4-hydroxycinnamide .A total of 100 actinobacteria strains were selectively isolated from Kubuqi desert, Inner Mongolia, China. The taxonomic characterization revealed 3) is first time isolated from a bacterial source. This study demonstrated that actinobacteria from Kubuqi desert are a potential source of novel bioactive natural products. Underexplored harsh environments like the Kubuqi desert may harbor a wider diversity of actinobacteria, particularly Streptomyces, which produce unique metabolites and are an intriguing source to develop medicinally valuable natural products.This is the first report of the bioprospecting and exploration of actinobacteria from Kubuqi desert and the metabolite 4-hydroxycinnamide (The online version contains supplementary material available at 10.1186/s12866-023-02770-8. As the twenty-first century unfolds, the threat of antimicrobial resistance (AMR) to human health gets more pervasive. In 2019, an estimated 4.95 million deaths were attributed to bacterial AMR . AntibioStreptomyces is the largest bacterial genus in Actinomycetota, a dominating bacterial phylum  .Structure elucidation of purified fractions was performed through nuclear magnetic resonance (NMR) spectroscopy. All NMR spectra were recorded using a Varian Inova NMR spectrometers (1 were obtained in CH3OH/H2O (3:1), . X-ray diffraction data was collected at 90.0 (2) K on a Bruker D8 Venture dual-source diffractometer using graded-multilayer focused MoKa X-rays. Raw data was integrated, scaled, merged, and corrected for Lorentz-polarization effects using the APEX3 package . Corrections for absorption were applied using SADABS [R-tensor [Colorless bulk crystals of compound g SADABS . The strg SADABS  and refig SADABS . HydrogeR-tensor  and PlatR-tensor .The % cell viability in cytotoxicity assays and % growth inhibition in antimicrobial assays was calculated on Excel, each experiment was performed in triplicate and standard error was calculated.Additional file 1: Fig. S1. Morphological appearance of selected actinobacterial strains (A) strain PU-KB2–2 (Streptomyces griseoviridis) (B) strain PU-KB6–10 (Streptomyces mutabilis) (C) strain PU-KB8–2 (Streptomyces djakartensis) (D) strain PU-KB10–4 (Streptomyces griseoviridis) (E) strain PU-KB10–10 (Streptomyces luteogriseus) (F) strain PU-KB12–15 (Streptomyces atrovirens).Additional file 2: Table S1. Biochemical characterization of actinobacterial strains isolated from Kubuqi Desert, China.Additional file 3: Table S2. 16S rRNA gene sequence analysis and % similarity of the Kubuqi desert strains with various actinobacterial type strains.Additional file 4: Table S3. Microtiter plate assay for antimicrobial activity of all actinobacterial strains against bacteria (Gram-positive and Gram-negative) and yeast along with percentage cytotoxicity against cancer cell lines.Additional file 5: Fig. S2. TLC (CH2Cl2/10%MeOH) screening of the extracts produced PU-KB strains.Additional file 6: Fig. S3. HPLC/UV analyses of the generated extract produced by Streptomyces sp. PU-KB5-11 (A-medium). HPLC-conditions: Detection wavelength 280 and 320 nm; solvent A: H2O/0.1% TFA; solvent B: acetonitrile; flow rate: 1.0 mL min-1; 0-30 min, 95-0% A (linear gradient); 30-35 min 0% A; 35-36 min 0-95% A (linear gradient); 36-40 min 95% A. UV-vis inset of full wavelength scan (190-600 nm).Additional file 7: Fig. S4. HPLC/UV analyses of the generated extract produced by Streptomyces sp. PU-KB10-4 (A-medium). HPLC-conditions: Detection wavelength 320 and 280 nm;; solvent A: H2O/0.1% TFA; solvent B: acetonitrile; flow rate: 1.0 mL min-1; 0-30 min, 95-0% A (linear gradient); 30-35 min 0% A; 35-36 min 0-95% A (linear gradient); 36-40 min 95% A. UV-vis inset of full wavelength scan (190-600 nm).Additional file 8: Fig. S5. HPLC-MS analysis of PU-KB8-2 crude extract. HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm.Additional file 9: Fig. S6. HPLC-MS analysis of PU-KB9-11 crude extract. HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm.Additional file 10: Fig. S7. HPLC-MS analysis of PU-KB10-4 crude extract. HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm.Additional file 11: Fig. S8. HPLC-MS analysis of PU-KB12-3 crude extract. HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm.Additional file 12: Figs. S9. Fermentation cultures of actinomycin D producing strains . The dark brown culture of strain PU-KB6-7 is due to high concentration of metal-complex siderophores (Fe – complex), which was consistent with the detected peak in HPLC/UV analysis figure at 18.8 min, and the low concentration of actinomycin D at 25.3 min.Additional file 13: Fig. S10. HPLC/UV analyses of the generated extract produced by actinomycin D producing strains (A-medium).Additional file 14: Fig. S11. HPLC-MS analysis of one selected actinomycin D producer crude extract (PU-KB7-8).Additional file 15: Fig. S12. (+)-HRMS of analysis of actinomycin D.Additional file 16: Fig. S13. HPLC analysis of griseoviridin (1). HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm. UV-vis inset of full wavelength scan (190-600 nm). (+) and (–)-ESI-MS spectra of griseoviridin (1).Additional file 17: Fig. S14.1H NMR spectrum  of griseoviridin (1).Additional file 18: Fig. S15.13C NMR spectrum  of griseoviridin (1).Additional file 19: Fig. S16. APT NMR spectrum  of griseoviridin (1).Additional file 20: Fig. S17.1H (400 MHz) and 13C (100 MHz) NMR spectra of griseoviridin (1) in CD3OD.Additional file 21: Fig. S18. NOESY spectrum  of griseoviridin (1).Additional file 22: Fig. S19.1H,1H-COSY spectrum  of griseoviridin (1).Additional file 23: Fig. S20. HSQC spectrum  of griseoviridin (1).Additional file 24: Fig. S21. HMBC spectrum  of griseoviridin (1).Additional file 25: Fig. S22. TOCSY spectrum  of griseoviridin (1).Additional file 26: Fig. S23. HPLC analysis of mitomycin C (2). HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm. UV-vis inset of full wavelength scan (190-600 nm). (+) and (–)-ESI-MS spectra of mitomycin C (2).Additional file 27: Fig. S24.1H NMR spectrum  of mitomycin C (2).Additional file 28: Fig. S25.13C NMR spectrum  of mitomycin C (2).Additional file 29: Fig. S26.1H,1H-COSY spectrum  of mitomycin C (2).Additional file 30: Fig. S27.1H (400 MHz) and 13C (100 MHz) NMR spectra of mitomycin C (2) in CD3OD.Additional file 31 Fig. S28. HPLC analysis of 4-hydroxycinnamide (3). HPLC-conditions: solvent A: H2O/0.1% FA; solvent B: CH3CN; flow rate: 0.5 mL min-1; 0-30 min, 5-100% B; 30-35 min, 100% B; 35-36 min, 100-5% B; 36-40 min, 5% B; Phenomenex NX-C18 column ; 254 nm. UV-vis inset of full wavelength scan (190-600 nm). (+) and (–)-ESI-MS spectra of 4-hydroxycinnamide (3).Additional file 32: Fig. S29.1H NMR spectrum  of 4-hydroxycinnamide (3).Additional file 33: Fig. S30.13C NMR spectrum  of 4-hydroxycinnamide (3).Additional file 34: Fig. S31.1H (500 MHz) and 13C (125 MHz) NMR spectra of 4-hydroxycinnamide (3) in CD3OD.Additional file 35: Fig. S32.1H NMR spectrum  of 4-hydroxycinnamide (3).Additional file 36: Fig. S33.13C NMR spectrum  of 4-hydroxycinnamide (3).Additional file 37: Fig. S34.1H (400 MHz) and 13C (100 MHz) NMR spectra of 4-hydroxycinnamide (3) in DMSO-d6.Additional file 38: Fig. S35.1H,1H-COSY spectrum  of 4-hydroxycinnamide (3).Additional file 39. pdf: Fig. S36. HSQC spectrum  of 4-hydroxycinnamide (3).Additional file 40: Fig. S37. HMBC spectrum  of 4-hydroxycinnamide (3).Additional file 41.. Physiochemical properties of compounds 1-3.Additional file 42: Fig. S38. (A) Streptomyces sp. PU-KB10-4 grown on a M2-medium agar plate. (B) HPLC/UV analyses of the generated extracts produced by Streptomyces sp. PU-KB10-4 using three different media . SG medium was selected for scale-up as 10 L based on the high production of the major metabolite (Compound 1).Additional file 43: Fig. S39. (A) HPLC/UV analyses of the mycelium and XAD-extracts produced by Streptomyces sp. PU-KB10-4. Compound 1 was the major compound of the strain extract (42 mg/L). (B) Antimicrobial activity of crude extracts of PU-KB10–4 grown in three different media: the crude extracts of PU-KB10–4 obtained from A-media, M2- media and SG media showed less activity against Gram-negative bacteria (Escherichia coli) and huge zones of inhibition against Gram-positive bacteria (Staphylococcus aureus).Additional file 44: Fig. S40. (A and B) Cytotoxicity and antimicrobial analysis of PU-KB10-4 fractions obtained after RP-18 column chromatography (C and D) Cytotoxicity and antimicrobial analysis of PU-KB10-4 after combining the initial column fractions.