Garcinia schomburgkiana is an edible tree widely distributed in the southern region of Vietnam. Little is known about the alpha-glucosidase inhibition of the Vietnamese Garcinia schomburgkiana. The aim of the current study was to explore the anti-diabetic potential of G. schomburgkiana fruits. All the fractions of G. schomburgkiana were evaluated for alpha-glucosidase inhibition, followed by bioassay-guided isolation. A new compound, epi-guttiferone Q (1), together with ten known compounds, guttiferones I–K (2–3), hypersampsone I (4), sampsonione D (5), sampsonione H (6), β-mangostin (7), α-mangostin (8), 9-hydroxycalabaxanthone (9), and fuscaxanthone (10), were isolated and structurally elucidated. The structure of the new metabolite 1 was confirmed through 1D and 2D NMR spectroscopy, and MS analysis. To the best of our knowledge, the metabolites (except 3) have not been isolated from this plant previously. All isolated compounds were evaluated for their alpha-glucosidase inhibition. Compounds 1–6 showed potent activity with IC50 values ranging from 16.2 to 130.6 μM. Compound 2 was further selected for a kinetic study. The result indicated that it was a competitive type. Additionally, in silico docking was employed to predict the binding mechanism of 1–2 and 4–6 in the active site of alpha-glucosidase, suggesting their potential as promising anti-diabetic compounds. Molecular dynamic simulation was also applied to 1 to better understand its inhibitory mechanism. Garcinia schomburgkiana is an edible tree widely distributed in the southern region of Vietnam. To date, there have been two reports on the phytochemical constituents of G. schomburgkiana fruits, uncovering 14 phloroglucinols and xanthones.8,10 In 2016, Le and co-workers reported the presence of 12 new phloroglucinols, showing strong cytotoxicity against several cancer cell lines.8 In 2021, Nguyen and co-workers reported two potent alpha-glucosidase inhibitors, namely schomburgkianone I and guttiferone K, isolated from Thai fruits.10 Only a report regarding alpha-glucosidase inhibition of G. schomburgkiana fruits was published by Jaisamut and Vongsak from the Thai bio-source, indicating the strong activity with an IC50 value of 8.31 μg mL−1.11 In this paper, preliminary screening of alpha-glucosidase inhibition was conducted to find the most active fractions. Then, these fractions were selected for further analysis. A new compound, epi-guttiferone Q (1), together with ten known compounds, guttiferones I–K (2–3), hypersampsone I (4), sampsonione D (5), sampsonione H (6), β-mangostin (7), α-mangostin (8), 9-hydroxycalabaxanthone (9), and fuscaxanthone (10), were isolated and structurally elucidated. The structures of the compounds were confirmed using 1D and 2D NMR spectroscopy, as well as MS analysis. All isolated compounds were evaluated for their alpha-glucosidase inhibition. A kinetic study, molecular docking, and molecular dynamic simulation were also performed to gain a deeper understanding of the inhibitory mechanism.2.Garcinia schomburgkiana based on alpha-glucosidase inhibition. All extracts and fractions were screened to identify the bioactive fractions, ultimately selecting the fraction EA6 as the most active (epi-guttiferone Q (1), together with ten known compounds, guttiferones I–K (2–3), hypersampsone I (4), sampsonione D (5), sampsonione H (6), β-mangostin (7), α-mangostin (8), 9-hydroxycalabaxanthone (9), and fuscaxanthone (10). To the best of our knowledge, these metabolites (except 3) have been reported for the first time in this plant.Bioactive-guided isolation was performed on the fruits of t active  for furt33H42O4 by HRESIMS and from the NMR spectra , 7.51 , and 7.43 ], three olefinic protons , a downfiled methine at δH 3.41 , and six downfield methyls  characteristic of three isoprenyl groups, one methyl at δH 1.00 and other methylene and methine protons in the range δH 1.45–1.70 ppm and δH 2.02–2.49 ppm. The 13C NMR in accordance with the HSQC spectrum indicated the presence of 33 carbon signals, including three ketone carbons , five aromatic methines , three olefinic methines , and six sp2 substituted carbons , two quaternary carbons (δC 65.1 and 48.8), two carbon methines , five carbon methylenes , and seven carbon methyls . The full analysis of both 1D (1H and 13C) and 2D  NMR spectra suggested that 1 contained a benzoylphloroglucinol skeleton, and its NMR spectra were also close to those of guttiferones I–K (2–3), major constituents of this plant material,10,12 except for the absence of the isoprenyl group at C-4. Comparison of NMR data of 1 and 2 , C-5 (δC 48.8), C-6 (δC 41.8), and C-18 (δC 39.3), supporting the position of H-4.Compound 1 was obtained as a colorless gum, and its molecular formula was determined to be C Table S1. The 1H 3-17 gave a key NOE correlation with H-6, indicating their syn-facial while the NOE correlations of H2-18/H2-24, H2-29/H-4, and H2-29/H2-24 indicated their same orientation. The downfield chemical shift of H3-17 in 1 compared to that of 2 [δH 1.00 in 1vs. 0.83 in 2–3] indicated the reversion of the configuration of C-5. The characteristic methyl group at δH 0.80–0.83 that could be found in 2–3 and previously reported benzoylphloroglucinols8 in the same bio-source ,14 sampsonione D (5),14,15 sampsonione H (6),16 β-mangostin (7),17 α-mangostin (8),17 9-hydroxycalabaxanthone (9),18 and fuscaxanthone (10)19 were identified in this plant for the first time. They were classified into three types of skeletons: benzoylphloroglucinols (1–3), homo-adamantanes (4–6), and xanthones (7–11). Guttiferone I (2) was reported in G. cowa by Phakhatmuen and co-workers, showing a consistent result regarding alpha-glucosidase inhibition (IC50 13.1 μM).12 The inhibition of guttiferone K (3) was isolated from Thai G. schomburgkiana sources in our previous reports.10,20 Molecular docking of 3 was reported previously.10 This compound was also reported their strong cytotoxicity against several cancer cell lines: HT-29, SW620, KATO-III, HepG2, CHAGO, and HCT11.21–23The metabolites guttiferone I (2),Garcinia plants, i.e., G. mangostana,24–26G. fusca,27G. cylindrocarpa,28G. planchonii.29 These compounds showed strong cytotoxicity against various cancer cell lines and potent alpha-glucosidase inhibition.24–29Xanthones 7–11 are well-known in the Hypericum plants: H. sampsonii, H. cohaerens, and H. hookerianum.14–16,30 Interestingly, this is the first report of these compounds in the genus Garcinia. Ye and co-workers provided a comprehensive review on NMR assignments and the stereochemistry for over 100 homo-adamantanes.16 Based on this review, the chemical shifts of the methyl groups H3-22 and H3-23 were reassigned  was graphed against the reciprocal of substrate concentrations (1/[S]). This approach allowed monitoring the effect of the inhibitor on both Km and Vmax. As shown in V vs. 1/[S] of 2 gave a group of straight lines that all intersected at the y-axis. The data revealed that with an increasing concentration of 2, Vmax remained constant, while Km increased, indicating that 2 is a competitive inhibitor of alpha-glucosidase. The results suggested that 2 bound to the active site of the enzyme, competed with the substrate and retarded the conversion of substrate into product. The inhibition constant for the inhibitor binding with free enzyme (Ki) was determined from a plot of the slope (Km/Vm) versus the inhibitor concentration (Ki) of 2 was determined as 69.25 μM. In previous work, the kinetic analysis of the positive control, acarbose, showed that it acts as a competitive inhibitor with a Ki of 162.49 μM. The smaller Ki of 2 means the greater binding affinity.32The results of alpha-glucosidase inhibition isolated compounds obtained from rk plots , where tntration . The inhG. cowa by Phakhatmuen and co-workers, showing a consistent result regarding alpha-glucosidase inhibition (IC50 13.1 μM).12 The inhibition of guttiferone K (3) and schomburgkianone I isolated from Thai G. schomburgkiana sources showed potent alpha-glucosidase inhibition with IC50 values of 34.8 and 21.2 μM, respectively.10,20 Guttiferone Q was thought to be a moderate inhibitor with an IC50 value of 117.5 μM.29 To date, there have been no reports on the alpha-glucosidase inhibition of homo-adamantanes 4–6. The activity of xanthones 7–11 was reported by Trinh and co-workers29 with IC50 values ranging from 31.3 to 128.9 μM. However, these compounds showed weak activity in our assay. The skeleton might play an important role in the inhibition. The inhibitory order is as follows: polyprenyl benzoylphloroglucinols (1–3) > homo-adamantanes (4–6) > xanthones (7–11).Up to now, very few polyprenyl benzoylphloroglucinols have been reported their alpha-glucosidase. Guttiferone I (2) was reported in 2.2The significant docking results for 1–7, obtained using AutoDock software, are presented in 2.2.1.4J5T enzyme: PDB with the values of free Gibbs energy and inhibition constant of −12.43 Kcal mol−1 and 0.08 μM, respectively, and it formed four hydrogen bondings with residual amino acids on 4J5T enzyme. Among them, hydrogen bonding, pose 304:H-A:Leu 432:O was the strongest bonding due to the shortest bonding length, as seen in 4J5T. Pose 304 has been considered well in ligand interaction model due to three parts of pose 304 has been identified via interactions such as capping group (aromatic ring or heterocyclic ring on pose), linker , and functional group (hydrogen bonding).33 The capping group of pose 304 has been discovered by pi–pi T shaped and pi–alkyl interactions from Phe 342 and Ala 436 to pi electrons of aromatic ring-hydrophobic interactions. The linker of this pose has been detected by pi-alkyl and alkyl interactions–hydrophobic interactions from Ile 36 Val 205 to alkenyl group, from Pro 372 and Gly 433 to methyl group, and from Gly 433 to carbon of alkenyl group on pose. The functional groups of this pose have been detected by hydrogen bondings from Leu 432 to oxygen atom of pose, from Tyr 343 to carbon atom of carbonyl group, Glu 435 to carbon atom of alcohol group, Leu 432 to oxygen atom of hydroxyl atom of alcohol group, and Arg 26 to oxygen atom of ketone group on this pose. Pose 304 has been considered good ligand interactions with 4J5T and proved its great inhibition on 4J5T in in silico docking model. As seen in 4J5T. The overlap interactions between atoms on pose and 4J5T enzyme have been depicted by violet circles on atom of pose 304. The bigger the diameters of violet circles, the stronger interactions are between pose 304 and 4J5T.The best docking pose of 1 or 304 among 500 confirmations (the number of models of 500). As indicated in 2.2.2.4J5T with the values of the free Gibbs energy and inhibition constant of −10.53 Kcal mol−1 and 0.027, respectively as seen in 4J5T and pose 149 has interacted well to 4J5T enzyme because 3 parts of pose 149 have shown full interactions. The capping group of pose 149 has detected via one pi–sigma interaction forming from Phe 385 to pi electrons of phenyl group. Linker part or connecting unit (CU) has identified by hydrophobic interactions via pi–sima interactions from Phe 395 and Thr 789 to methyl groups on pose, and pi-alkyl or alkyl from Phe 786 to methyl group on pose. The functional groups of this pose have been discovered by hydrogen bondings from Arg 428 to oxygen atom of ketone group, and Glu 771 to oxygen atom of hydroxyl alcohol on pose. In 4J5T. There are many hydrogen bondings: Arg 428, and Glu 771 and steric interactions: Arg 387, Arg 428, Glu 429, Asp 568, Tyr 709, and Glu 771. The overlap interactions between atoms on pose 304 and residual amino acids are manifested by violet circles. As the size of violet circles get bigger, the interactions between this pose and 4J5T get stronger.The best docking pose 149 among 500 conformation has anchored to active center of 2.34J5T in dynamic environment with parameters such as temperature, simulation, atoms, water, and charge of 300 K, 100 ns, 83 681 atoms, 23 630 water molecules, and 0 charge are presented in profile results, simulation interactions diagram report. The information of pose 304 has shown by 37 heavy atom/79 atoms, 0 charge, 10 rotation bonds, and counter ion/salt information is ion Na of 31.547 mM and 41 positives. As seen in 4J5T enzyme is 27.56% helix, 13.79% strand, and 41.35% SSE total (secondary structure elements) that were observed in whole simulation time. The protein secondary structure elements (SSE) contained the alpha-helices (red color) and beta-strands (light blue color). The plot above reported SSE distribution by residue index throughout the enzyme structure. As indicated in 4J5T and pose 304 are made around by water solvent. As seen in 4J5T enzyme have been depicted, and the values of RMSDs of the enzyme such as alpha-carbon and side chain that are varied the range of 1 to 3 Å in permission ranges. ‘The lig fit lig’ means that the values of RMSD of atoms of pose 304 are changed from 0.5 to 1.5 Å  whole simulation course from 0 to 100 ns. ‘Lig fit Prot’ exposed the RMSD of a pose 304 when the pose 304-4J5T complex is first aligned on the enzyme backbone of the reference, and then the RMSD of the ligand heavy atoms (37 atoms) is calculated. The values observed are lower than that of the RMSD of the 4J5T-alpha-carbon or side chain curves, then it is likely that the ligand has formed one equivalent from its initial binding site. As shown in 4J5T and their entropic role in the binding event. The ‘Fit Ligand on Protein’ curve demonstrated the fluctuations of pose 304 with respect to the 4J5T enzyme. The pose 304-4J5T complex has been first aligned on the backbone of 4J5T and then the RMSF of this pose has been measured on the ligand heavy atoms. As seen in 4J5T and pose 304 interactions have been monitored throughout the simulation. They have been categorized by type and summarized as hydrogen bonds, hydrophobic, ionic, and water bridges. The stacked bar charts are normalized over the course of the trajectory; for example, Leu 432 interacted hydrogen with active atom on pose 304 in 100% simulation time (100 ns). The Tyr 343 forming hydrogen with active atom on pose 304 is possible, as some residue amino acids may make multiple contacts of same hydrogen bonding with the pose 304. The Glu 435 has made hydrogen bonding with active atom on this pose via water bridge interaction that maintained at 40% simulation time. The Arg 26 has linked hydrogen bonding via water bridge that is maintained at 30% simulation time. The Phe 432 and Ala 436 have formed hydrophobic interactions with active atom on pose 304 and it keep at 70 and 50% simulation time, respectively. Other hydrophobic interactions appeared and keep less than 20% simulation time such as Ile 36, Val 205, Trp 206, and Pro 372. As shown in 4J5T contacts are presented in a detailed via water bridge that are stable at 68% simulation time. Leu 432 has formed one hydrogen bonding and has kept at 98% simulation time. Finally, Phe 342 has linked one pi–pi stacking interaction to pi electron system of phenyl group and made stable at 70% simulation time. As seen in 2, 120 to 180 Å2, and 100 to 120 Å2, respectively. As demonstrated in 4J5T has been demonstrated by 2D diagram. The pharmacokinetics of pose 304 has exposed 2 areas including hydrophobic (green light) and polar (light blue). The hydrogen bondings have formed from the hydrogen atom of hydroxyl alcohol to Leu 432 (hydrophobic), Tyr 343 (hydrophobic amino acid) to oxygen atom of the ketone group, and water bridge interactions from water molecules to 2 atom oxygen of two ketone groups on pose 304. Pharmacophore is relative to hydrophobic interactions: Ile 430, Ile 431, Leu 432, Ala 436, Pro 372, Tyr 343, and Phe 342, polar interactions: Asn 434 and Asn 345, negative charged interaction: Glu 435, and positive charged interaction: Arg 387.As seen in 3.3.11H-NMR and 125 MHz for 13C-NMR). The HR-ESI-MS was recorded using an HR-ESI-MS MicrOTOF-Q mass spectrometer on an LC-Agilent 1100 LC-MSD Trap spectrometer. Thin layer chromatography (TLC) was carried out on precoated silica gel 60 F254 or silica gel 60 RP-18 F254S (Merck). Spots were visualized by spraying with 10% H2SO4 solution followed by heating. Gravity-column chromatography was performed on silica gel 60 .The NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 3.2Garcinia schomburgkiana were collected in Tay Ninh Province (Vietnam) from July to August 2022. The scientific name of the material was identified as Garcinia schomburgkiana by Dr Dang Van Son, Institute of Tropical Biology, Vietnam Academy of Science and Technology. The voucher specimen (No UE-P010) was deposited in Ho Chi Minh University of Education.Fruits of 3.3Garcinia schomburgkiana (5 kg) were extracted with methanol (10 L × 3) at room temperature then the filtrated solution was evaporated to give a crude extract (695.5 g). This extract was partitioned into n-hexane and EtOAc to afford the corresponding extracts H (46.7 g) and EA (245.2 g), respectively. The water-soluble solution was evaporated to obtain the extract R (358.7 g). The EA extract was applied to silica gel column chromatography (CC) using the mobile phase of n-hexane : EtOAc  to afford 16 fractions (EA1–EA16): EA1 (41.20 g), EA2 (13.30 g), EA3 (2.42 g), EA4 (5.1 g), EA5 (6.92 g), EA6 (31.01 g), EA7 (4.48 g), EA8 (14.87 g), EA9 (4.63 g), EA10 (11.19 g), EA11 (4.23 g), EA12 (31.01 g), EA13 (4.05 g), EA14 (5.59 g), EA15 (4.69 g), and EA16 (75.12 g). Extracts and fractions EA1–EA16 were evaluated for the alpha-glucosidase inhibition to choose the fraction for further analysis. The isolation of 1–11 was described in Dried and ground fruits of 3.3.1.38H50O5: [α]D25 + 283 . IR νmax (neat): 1706, 1522, 1368, 1229, 1045 cm−1. 1H-NMR : 7.57 , 7.51 , 7.51 , 7.43 , 7.43 , 5.23 , 5.14 , 4.99 , 3.41 , 2.49 , 2.42 , 2.34 , 2.32 , 2.20 , 2.02 , 2.01 , 1.74 , 1.70 , 1.68 , 1.67 , 1.65 , 1.63 , 1.63 , 1.62 , 1.56 , 1.47 , 1.00 . 13C-NMR : 208.2 (C-9), 198.7 (C-10), 196.7 (C-1), 188.8 (C-3), 138.5 (C-11), 134.7 (C-31), 133.7 (C-26), 133.1 (C-14), 132.0 (C-21), 129.5 (C-12), 129.5 (C-16), 128.6 (C-13), 128.6 (C-15), 125.2 (C-20), 123.4 (C-25), 121.0 (C-30), 118.5 (C-2), 65.6 (C-4), 65.1 (C-8), 48.8 (C-5), 43.1 (C-7), 41.8 (C-6), 39.3 (C-18), 31.1 (C-29), 28.8 (C-24), 26.2 (C-33), 25.9 (C-23), 25.9 (C-28), 22.7 (C-19), 18.2 (C-17), 18.1 (C-32), 18.0 (C-27), 17.8 (C-22). HR-ESI-MS m/z: 543.3059 [M + H2O + Na]+ , m/z: 503.3166 [M + H]+ .Colorless oil C3.3.2.35H44O4: 1H-NMR : 7.47 , 7.36 , 7.36 , 7.18 , 7.18 , 5.16 , 5.08 , 2.63 , 2.63 , 2.53 , 2.52 , 2.40 , 2.32 , 2.32 , 2.09 , 2.07 , 2.07 , 1.99 , 1.82 , 1.80 , 1.68 , 1.64 , 1.58 , 1.48 , 1.44 , 1.03 , 0.92 . 13C-NMR : 206.4 (C-12), 206.1 (C-2), 206.1 (C-14), 193.4 (C-15), 138.2 (C-26), 134.7 (C-16), 132.7 (C-19), 132.0 (C-31), 129.8 (C-17), 129.8 (C-21), 128.7 (C-18), 128.7 (C-20), 124.9 (C-30), 120.5 (C-25), 81.3 (C-1), 70.8 (C-3), 69.3 (C-11), 58.3 (C-7), 51.2 (C-13), 45.2 (C-10), 44.6 (C-6), 43.9 (C-9), 42.2 (C-5), 40.6 (C-27), 30.4 (C-24), 30.3 (C-23), 29.3 (C-8), 29.2 (C-4), 27.2 (C-29), 25.4 (C-34), 25.4 (C-32), 22.8 (C-35), 22.0 (C-22), 17.7 (C-33), 16.5 (C-28).Colorless oil C3.3.3.38H48O4: 1H-NMR : 7.39 , 7.26 , 7.26 , 7.12 , 7.12 , 5.11 , 5.05 , 4.91 , 4.84 , 3.15 , 2.64 , 2.52 , 2.29 , 2.10–2.12 , 2.06 , 2.03 , 2.03 , 1.95 , 1.89 , 1.80 , 1.71 , 1.66 , 1.65 , 1.57 , 1.46 , 1.41 , 0.93 , 0.87 . 13C-NMR : 206.2 (C-14), 204.7 (C-12), 203.8 (C-2), 192.4 (C-15), 145.2 (C-22), 138.2 (C-29), 134.8 (C-16), 131.9 (C-19), 131.2 (C-34), 128.8 (C-17), 128.8 (C-21), 127.9 (C-18), 127.9 (C-20), 124.0 (C-33), 118.9 (C-28), 111.7 (C-23), 80.8 (C-1), 73.9 (C-3), 68.8 (C-11), 57.1 (C-7), 54.9 (C-5), 50.6 (C-13), 44.2 (C-6), 43.8 (C-9), 42.5 (C-10), 39.8 (C-30), 34.3 (C-4), 29.3 (C-27), 28.8 (C-8), 27.0 (C-26), 26.7 (C-25), 26.5 (C-32), 25.6 (C-35), 25.1 (C-38), 23.7 (C-24), 22.7 (C-37), 17.5 (C-36), 16.3 (C-31).Colorless oil C3.3.4.35H44O4: 1H-NMR : 7.38 , 7.26 , 7.26 , 7.06 , 7.06 , 5.30 , 5.06 , 2.61 , 2.61 , 2.50 , 2.43 , 2.37 , 2.21 , 2.10 , 2.07 , 2.07 , 1.95 , 1.92 , 1.75 , 1.75 , 1.75 , 1.66 , 1.66 , 1.59 , 1.42 , 1.39 , 1.01 , 0.94 . 13C-NMR : 207.5 (C-12), 203.8 (C-2), 203.8 (C-14), 193.4 (C-15), 139.1 (C-26), 135.2 (C-16), 132.3 (C-19), 131.8 (C-31), 128.7 (C-17), 128.7 (C-21), 128.4 (C-18), 128.4 (C-20), 124.4 (C-30), 119.2 (C-25), 79.7 (C-1), 75.1 (C-3), 67.7 (C-11), 55.6 (C-7), 47.8 (C-13), 44.5 (C-6), 42.7 (C-5), 42.5 (C-9), 40.2 (C-27), 35.3 (C-10), 29.1 (C-24), 28.6 (C-22), 28.2 (C-4), 26.7 (C-29), 25.9 (C-32), 25.2 (C-34), 23.8 (C-8), 22.6 (C-35), 20.7 (C-23), 17.8 (C-33), 16.5 (C-28).Colorless oil C3.434Evaluation of the inhibitory activity of 1–11 against yeast alpha-glucosidase followed a previous procedure.3.5p-NPG substrate in the absence or presence of tested compound at different concentrations . The Lineweaver–Burk plots were used to determine the inhibition type. In addition, the inhibition constant Ki is an indication of the potency of an inhibitor and was determined from secondary plots of 1/V vs. [I].35 The Lineweaver–Burk double-reciprocal plot (1/V vs. 1/[S]) was employed to discern the Vmax, Kmax, and mode of inhibition  of , coordinators of active center of (−18.418–20.917 8.099). MD simulation has been performed on 4–6,36,374.G. schomburgkiana fruits, a bioassay-guided isolation was applied, resulting in the presence of 11 compounds. They were structurally elucidated epi-guttiferone Q (1), guttiferones I–K (2–3), hypersampsone I (4), sampsonione D (5), sampsonione H (6), β-mangostin (7), α-mangostin (8), 9-hydroxycalabaxanthone (9), and fuscaxanthone (10). To the best of our knowledge, compounds 1–2 and 4–11 were reported for the first time from this plant. Notably, compounds 1–6 displayed potent activity. Compound 2 was further selected for a kinetic study, indicating that it was a competitive type. In silico docking and molecular dynamic simulation were employed to predict well the most stable binding mechanism of a new compound 1 or pose 304 to 4J5T enzyme.From No potential conflict of interest was reported by the authors.RA-013-D3RA06760B-s001


In order to expand the structure–activity relationship (SAR) knowledge, a new series of 7-analogues was also designed and synthetized. All the compounds were evaluated for their antitumor activity against melanoma (A375-C5), breast adenocarcinoma (MCF-7), and colorectal carcinoma (HCT116) cell lines, as well as non-tumor HPAEpiC cells. Three of the newly synthesized compounds  exhibited potent antiproliferative activity, mainly on colorectal tumor cells (GI50 = 2.66–3.26 μM), showing hybrid 7 selectivity for tumor cells. We performed molecular mechanism studies to evaluate the potential interference of compounds with the p53 pathway, namely, p53–MDM2 interaction and mitosis in HCT116 cells. The antiproliferative activities of compounds were shown to be p53-independent. Compound 7 emerged as an antimitotic agent by inducing the mitotic arrest of colorectal tumor cells, and subsequently, cell death.In this work, the design and synthesis of a new chalcone-trimethoxycinnamide hybrid ( CM-M345  of colchicine methanaminium hexafluorophosphate (COMU) to obtain chalcone-trimethoxycinnamide hybrid 7 with 20% yield.The synthetic approach planned for the synthesis of chalcone-trimethoxycinnamide hybrid acid (4) A. These ee steps B. In the8 , was obtained using the same synthetic approach as for 7 , resulting from the isosteric substitution of the amide linker by a retro-amide, was conducted using a three-steps pathway (9) was synthetized via a nucleophilic substitution reaction of 1-ethan-1-one with methyl bromoacetate in the presence of K2CO3 and anhydrous acetone as a solvent with 45% yield. The compound dimethyl 2,2’-bis(oxy))diacetate (10) was also obtained as a by-product of the reaction in 25% yield. This reaction was followed by an aldol condensation between each synthesized acetophenone (9 or 10) with 3,4,5-trimethoxybenzaldehyde. In these cases, the aldol condensation reaction in basic medium also allowed for the hydrolysis of the ester group to obtain (E)-2-acryloyl)phenoxy)acetic acid (11) and (E)-2,2’-acryloyl)-1,3-phenylene)bis(oxy))diacetic acid (12), respectively, with moderate yields . Lastly, chalcone 11 gave rise to hybrid 13 through the reaction with 3,4,5-trimethoxyaniline in the presence of the coupling reagent COMU, with 30% yield.The synthesis of another chalcone-trimethoxyphenylamide hybrid,  pathway C. Firstl7 analogues with a flavone scaffold 15 and 16  was firstly planned using chalcone 6  or 3,4,5-trimethoxybenzoic acid, respectively. However, the expected products were not obtained. Therefore, an alternative synthetic pathway was carried out . For comparison purposes, the synthesis of 17 was also conducted in a two-step pathway  when compared to Method A. The overall results allowed us to conclude that the synthesis of flavone hybrids was most favorable if the cyclization of chalcone hybrids was the last step of the synthetic pathway.Considering that the synthesis of 3, 4, and 9 was established using 1H and 13C nuclear magnetic resonance (NMR) techniques (5–8 and 10–18 was established on the basis of NMR (13C NMR assignments were determined using 2D heteronuclear single quantum correlation (HSQC) and heteronuclear multiple bond correlation (HMBC) experiments.The structure elucidation of compounds chniques , and dats of NMR  and highs of NMR  techniqu50), was calculated. Among all of the tested compounds, three  showed a high growth arrest in all cell lines, and 7 displayed potent growth inhibitory activity against the colorectal tumor HCT116 cell line , 7 showed high selectivity for tumor cells, and even higher than that obtained for two previously reported hit compounds, CM-345 (1) and BP-M345 (2). The overall results prompt us to further investigate the mechanism behind the cytotoxic activities of compounds 6, 7, and 13 in colorectal tumor cells. Moreover, in silico studies were performed, aiming to screen for drug-likeness.A sulforhodamine B (SRB) assay was performed to evaluate the in vitro growth inhibitory activity of all synthesized hybrids, as well as intermediates in the human tumor cell lines of melanoma (A375-C5), breast adenocarcinoma (MCF-7), and colorectal carcinoma (HCT116), as well as on the non-tumor Human Pulmonary Alveolar Epithelial cell line (HPAEpiC). The concentration inducing 50% cell growth inhibition (GIell line . Interes7) by a 3,4,5-trimethoxyphenyl group (8) is associated with a reduction in activity, suggesting that the α,β-unsaturated ketone moiety on the linker is an important feature in the enhancement of the antiproliferative activity (7: GI50 = 3.26 µM vs. 8: GI50 = 13.49 µM). Nevertheless, this effect is not observed for the flavone derivatives 15 and 16. Concerning the isosteric substitution of the amide linker by a retro-amide, for chalcone derivatives (8 vs. 13), the results suggested that the presence of the carbonyl of the amide group adjacent to the 3,4,5-trimethoxyphenyl ring was less favorable for the activity (8: GI50 = 13.49 µM vs. 13: GI50 = 2.66 µM), but for flavone derivatives, this isosteric substitution has no effect on the activity. Interestingly, the cyclization of chalcones, resulting in flavone-hybrids  led to the loss of antiproliferative activity, compared with chalcone-hybrids . Additionally, with the screening of the synthetic intermediates, some conclusions can also be inferred (11 and 12 with CM-M345 (1), it seems that the substitution of the hydroxyl group by a carboxylic acid side chain in the 4′ position of the chalcone scaffold (11) is associated with a remarkable decrease in the growth inhibitory effect (a GI50 between 26.87 and 58.34 µM), compared with the compound CM-M345 , and the substitution of both hydroxyl groups at 2′ and 4′ is associated with a complete loss of activity . However, the presence of a BOC-protected amine in the 4′ position of the chalcone scaffold is tolerated .Considering the antiproliferative effects on tumor cells of all the studied compounds , some SAinferred . When coCM-M345 (1) was associated with a p53–MDM2 inhibitory effect, and compounds 6, 7, and 13 displayed a preferential antiproliferative effect on HCT116 p53+/+ cells, they were also tested in HCT116 cells, in which p53 has been knocked out (HCT116 p53−/−), in order to confirm the contribution of the p53 pathway to the antiproliferative activity  has been previously reported as a p53-MDM2 inhibitor by our research group [6, 7, and 13 on the p53–MDM2 interaction was analyzed using yeast cell-based assays. The results obtained showed that none of the tested compounds significantly reversed the negative inhibitory growth effect of MDM2 on p53 (data not shown).Compounds ch group , the eff6, 7, and 13 generate antiproliferative activity against colorectal carcinoma cells. For that, HCT116 cells were exposed to each compound for 48 h. As shown in Double labeling with annexin V-FITC and propidium iodide dyes was used to detect cells going through apoptotic events, using a flow cytometer in order to obtain insights into the mechanism by which compounds BP-M345 (2) has been previously reported [6, 7, and 13 were tested for their capability to induce cell cycle arrest. HCT116 cells were exposed for 24 h to the compounds and then analyzed using phase contrast microscopy for morphological changes. Untreated and dimethyl sulfoxide -treated cells were used as negative controls. Cell exposure to nocodazole, an antimitotic agent, was used as a positive control. We found an accumulation of bright and rounded cells, reminiscent of mitotic cells, under 7 treatment, which was not observed under 6 or 13 treatment  compared to the untreated (12.68 ± 1.98%) or DMSO-treated (12.81 ± 1.45%)  induced by the parent compound BP-M345 (5) used at 2×GI50 [Disruption of cell cycle progression is considered one of the most common cytotoxic mechanisms of anticancer drugs . Moreovereported . Thereforeatment a. DAPI somosomes b, while ± 1.45%) c. Interet 2×GI50 .7 exerts its antiproliferative activity on colorectal tumor cells by inducing apoptotic cell death, probably by acting as an antimitotic agent.These results suggest that compound 7, we treated HCT116 cells with the GI50 of the compound. Twenty-four hours later, we performed an immunofluorescence assay using an anti-α-tubulin antibody to visualize the spindle morphology. Under fluorescence microscopy, we observed that most compound 7-treated cells showed collapsed mitotic spindles (91.41 ± 4.87%), contrasting with untreated or DMSO-treated cells  (7-treated cells (6.57 ± 2.13%) was significantly lower than in untreated or DMSO-treated cells  (7 induces mitotic delay by interfering with mitotic spindle assembly by generating monopolar-like spindles.To understand the mechanism behind the induction of mitotic arrest after treatment with the compound ctively) . Likewisctively) . This fihttp://www.swissadme.ch/, (accessed on 12 May 2023)). For each compound, the above-mentioned molecular descriptors/physicochemical properties were calculated  were higher than the recommended range of values according to the drug-likeness guidelines (150 < MW < 500), as well as the mean MW value (313 g mol−1) reported for orally bioavailable cancer drugs [The most promising compounds  were screened for drug-likeness properties using in silico models. The drug-likeness was evaluated according to the following chemical features: (i) flexibility, inferred by the number of rotatable bonds (RBs); (ii) unsaturation, inferred by the fraction of carbon sp3 (Fsp3); (iii) solubility, inferred by log S; (iv) polarity, inferred by the polar surface area (PSA); and (v) lipophilicity, inferred by the log P, using the SwissADME web server , and the fraction of aromatic heavy atoms (Far = AHA/HA) are frequently used to infer molecular flexibility. Analyzing these molecular descriptors for all compounds, it was observed that while the number of Fsp3 respects the values preconized by the drug-likeness guidelines, the RB value obtained did not .6, 7, and 13, the values of HBD and PSA were in agreement with the literature for synthetic drugs  [The molecular polarity can be determined by the number of HBA, the number of HBD, and the PSA values. Regarding these values , it was  125 Å2) .6, 7, and 13 have a higher mean Log P value  than anticancer drugs . Considering the range of values of Log S preconized by the drug-likeness guidelines (Log S > −4) [Lipophilicity and solubility were inferred by log P and Log S, respectively, using different methods . Conside S > −4) , all com6, 7, and 13 should also consider the improvement of their drug-likeness profile, namely through nanotechnology [Therefore, taking into account the overall results, in the near future, the optimization of compounds chnology . In factchnology  and docechnology , and som1H and 13C NMR spectra were taken in CDCl3, DMSO, or THF at room temperature on Bruker Avance 300 and 400 instruments . Chemical shifts are expressed in δ (ppm) values relative to tetramethylsilane (TMS), used as an internal reference; 13C NMR assignments were made using 2D (HSQC and HMBC) NMR experiments (long-range 13C-1H coupling constants were optimized to 7 Hz). The spectral treatment was executed using the MestReNova v6.0.2-5475 software. The HRMS mass spectra of compounds were performed on an LTQ Orbitrap 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 following materials were synthesized and purified using the described procedures. The 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 of a 100 µL loop, and a Thermo Scientific SpectraSystem UV8000 DAD detector (USA). 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 . 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 an isocratic mode for 30 min. The peak purity index was determined using a total peak UV-Vis spectra of between 210 and 650 nm, with a step of 4 nm.All reactions were monitored using thin-layer chromatography (TLC). The purification of compounds via flash column chromatography (CC) and preparative TLC was performed using Macherey-Nagel silica gel 60 (0.04–0.063 mm) and Macherey-Nagel silica gel 60 (GF254) plates, respectively. Melting points were obtained under a Köfler microscope and are uncorrected. E)-3- acrylate (3) and 3,4,5-trimetoxycinnamic acid (4) were synthesized  and characterized according to the described procedure [3 and 4 is presented in the Methyl  and 2-(Boc-amino)-ethyl bromide  were added. The reaction proceeded for 24 h at 70 °C with gentle stirring. After cooling, the reaction mixture was poured onto ice and neutralized with HCl. The obtained solid was filtrated, washed with water, and dried. A white solid (90% yield) corresponding to tert-butyl (2-(4-acetyl-3-hydroxy-2-propylphenoxy)ethyl)carbamate (5) was obtained.1-ethan-1-one  was dissolved in 15 mL of anhydrous acetone. Then, Ktert-butyl (2-(4-acetyl-3-hydroxy-2-propylphenoxy)ethyl)carbamate (5): 99.3% purity; mp 175–177 °C (acetone); 1H NMR  δ: 12.74 , 7.58 , 6.42 , 4.91 , 4.09 , 3.56 , 2.63 , 1.58–1.50 , 2.56 , 1.45 , 0.94  ppm. 13C NMR (CDCl3. 75.47 MHz) δ: 203.2 (1-COCH3), 162.5 (C-4), 162.3 (C-2), 156.0 (C-4″), 130.2 (C-6), 118.5 (C-3), 114.6 (C-1), 102.9 (C-5), 79.8 (C-5″), 67.6 (C-1″), 40.2 (C-2″), 28.5 (C-6″), 26.5 (1-COC3H), 24.5 (C-1′), 22.1 (C-2′), 14.3 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C18H27NO5 (M+Na+) 360.17814., found 360.17821.tert-butyl (2-(4-acetyl-3-hydroxy-2-propylphenoxy)ethyl)carbamate  in methanol was added an aqueous solution of sodium hydroxide (40%) to pH 13–14. Then, a solution of 3,4,5-trimethoxybenzaldehyde  in methanol was slowly added to the reaction mixture. The reaction was refluxed for 4 days. After cooling, the reaction was poured onto crushed ice, and the pH was adjusted to 6–8 with a 5 M HCl solution. The obtained solid was filtrated, washed with water, and purified via flash CC . A yellow solid (20% yield) corresponding to tert-butyl(E)-acryloyl)phenoxy)ethyl)carbamate (6) was obtained.To a solution of tert-butyl(E)-acryloyl)phenoxy)ethyl)carbamate (6): 98.9% purity; mp 196–198 °C (ethyl acetate); 1H NMR  δ: 13.68 , 8.27 , 7.98 , 7.79 , 7.26 , 7.00 , 6.69 , 4.10 , 3.87 , 3.72 , 3.35 , 2.59 , 1.52–1.45 , 1.39 , 0.89  ppm; 13C NMR  δ: 192.4 (C=O), 162.7 (C-2), 162.6 (C-4), 155.7 (C-4″), 153.1 , 144.7 (C-β), 140.0 (C-4‴), 130.6 (C-6), 130.1 (C-1‴), 120.2 (C-α), 117.1 (C-3), 113.9 (C-1), 106.9 , 103.3 (C-5), 77.7 (C-5″), 67.0 (C-1″), 60.2 (4‴-OCH3), 56.2 , 40.0 (C-2″), 28.2 (C-6″), 24.3 (C-1′), 21.9 (C-2′), 14.0 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C28H37NO8 (M+Na+) 538.24114., found 538.24191.Tert-butyl(E)-acryloyl)phenoxy)ethyl)carbamate  was dissolved in 5 mL of dichloromethane, and then 5 mL of TFA was added to the mixture. The reaction proceeded for 26 h with gentle stirring. After completion, the reaction mixture was diluted with dichloromethane, and then a saturated sodium hydrogen carbonate solution was added. The solution was then extracted with ethyl acetate (3 × 50 mL). The organic layers were collected, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude obtained was reserved. Then, in a round-bottomed flask, (E)-3-acrylic acid  was dissolved in 5 mL of anhydrous dichloromethane and N-ethyl-N-isopropylpropan-2-amine  was added. Then, 50% of the crude material was added to the mixture. After 10 min, COMU  was also added to the mixture. The reaction proceeded for 20 h at room temperature with stirring. After completion, the reaction mixture was poured into ice. The solution was subsequently washed with dichloromethane (3 × 80 mL), with a 1 M HCl solution and saturated NaHCO3 solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and solvent evaporated under reduced pressure. The reaction mixture obtained was dissolved in chloroform and purified using TLC . A yellow solid (20% yield) corresponding to (E)-N-(2-(3-hydroxy-2-propyl-4-((E)-3-acryloyl)phenoxy)ethyl)-3-acrylamide (7) was obtained.E)-N-(2-(3-hydroxy-2-propyl-4-((E)-3-acryloyl)phenoxy)ethyl)-3-acrylamide (7): 98.9% purity; mp 205–207 °C (chloroform); 1H NMR  δ: 13.68 , 8.32–8.27 , 7.99 , 7.79 , 7.28 , 6.89 , 6.87 , 6.62 , 6.73 , 4.21 , 3.87 , 3.80 , 3.72 , 3.78 , 3.67 , 2.60 , 1.54–1.44 , 0.88  ppm; 13C NMR  δ: 192.5 (C=O), 165.4 (C-4″), 162.7 (C-2), 162.6 (C-4), 153.2 , 153.1 , 144.8 (C-β), 140.0 (C-4‴), 139.1 (C-4″), 130.5 (C-6), 130.5 (C-1‴), 130.1 (C-5″), 121.3 (C-α’), 120.4 (C-α), 117.2 (C-3), 114.0 (C-1), 106.9 , 104.9 , 103.6 (C-β’), 103.4 (C-5), 66.9 (C-1″), 60.2 (4‴-OCH3), 60.1 (8″-OCH3), 56.2 , 56.0 , 38.7 (C-2″), 24.0 (C-1′), 21.6 (C-2′), 14.1 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C35H41NO10 (M+Na+) 658.26227., found 658.26343.(Tert-butyl(E)-acryloyl)phenoxy)ethyl)carbamate  was dissolved in 5 mL of dichloromethane. Then, 5 mL of TFA was added to the mixture. The reaction proceeded for 26 h with gentle stirring. After completion, the reaction mixture was diluted with dichloromethane, and then a saturated sodium hydrogen carbonate solution was added. The solution was then extracted with ethyl acetate (3 × 50 mL). The organic layers were collected, washed with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude obtained was reserved. Then, in a round-bottomed flask, 3,4,5-trimethoxybenzoic acid  was dissolved in 5 mL of anhydrous dichloromethane and N-ethyl-N-isopropylpropan-2-amine  was added. Then, 50% of the crude material was added to the mixture. After 10 min, COMU  was also added to the mixture. The reaction proceeded for 3 h at room temperature with stirring. After completion, the reaction mixture was poured onto ice. The solution was subsequently washed with 1M HCl solution and saturated NaHCO3 solution. The organic phase was dried over anhydrous sodium sulfate, filtered, and solvent evaporated under reduced pressure. The reaction mixture obtained was purified via TLC . A light-yellow solid (20% yield) corresponding to (E)-N-acryloyl)phenoxy)ethyl)-3,4,5-trimethoxybenzamide (8) was obtained.E)-N-acryloyl)phenoxy)ethyl)-3,4,5-trimethoxybenzamide( (8): 97.8% purity; mp 201–203 °C (chloroform); 1H NMR  δ: 13.67 , 8.63 , 8.28 , 7.98 , 7.29 , 7.26 , 7.20 , 6.76 , 4.28 , 3.87 , 3.82 , 3.72 , 3.69–3.66 , 3.68 , 2.58 , 1.49–1.40 , 0.82  ppm; 13C NMR  δ: 192.4 (C=O), 166.0 (C-4″), 162.7 (C-2), 162.6 (C-4), 153.1 , 152.5 , 144.7 (C-β), 140.0 (C-4‴), 140.0 (C-4″), 130.6 (C-6), 130.6 (C-1‴), 129.5 (C-5″), 120.2 (C-α), 117.1 (C-3), 113.9 (C-1), 106.9 , 104.8 , 103.4 (C-5), 66.5 (C-1″), 60.2 (4‴-OCH3), 60.1 (8″-OCH3), 56.2 , 56.0 , 38.9 (C-2″), 23.9 (C-1′), 21.0 (C-2′), 14.0 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C33H39NO10 (M+Na+) 632.24662, found 632.24594.2CO3  and methyl 2-bromoacetate  were added. The reaction proceeded for 24 h at 70 °C with gentle stirring. After cooling, the reaction mixture was poured onto ice and neutralized with HCl. The obtained solid was filtrated, washed with water, and dried. A white solid (45% yield) corresponding to methyl 2-(4-acetyl-3-hydroxy-2-propylphenoxy)acetate (9) was obtained. The aqueous phase was then extracted with dichloromethane (3 × 50 mL). The organic layers were collected, washed with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified using flash CC . A gum (25% yield) corresponding to dimethyl 2,2’-bis(oxy))diacetate (10) was obtained.1-ethan-1-one  was dissolved in 5 mL of anhydrous acetone. Then, K9 is presented in the The experimental description of compound Dimethyl 2,2’-bis(oxy))diacetate (10): 99.7% purity; mp N.D. (gum); 1H NMR  δ: 7.50 , 6.53 , 4.69 , 4.47 , 3.81 , 3.79 , 2.69 , 1.63–1.53 , 2.56 , 0.97  ppm; 13C NMR (CDCl3. 75.47 MHz) δ: 198.9 (1-COCH3), 169.2 (C-2‴), 168.9 (C-2″), 160.1 (C-4), 156.8 (C-2), 129.2 (C-6), 126.8 (C-1), 126.4 (C-3), 107.1 (C-5), 71.8 (C-1‴), 65.3 (C-1″), 52.4 (C-3‴), 52.2 (C-3″), 30.0 (1-COCH3), 26.0 (C-1′), 22.9 (C-2′), 14.5 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C17H22O7 (M+Na+) 361.12577, found 361.12594.9, 200 mg, 1 Eq, 751 µmol) in methanol was added an aqueous solution of 40% sodium hydroxide to pH 13–14. Then, a solution of 3,4,5-trimethoxybenzaldehyde  in methanol was slowly added to the reaction mixture. The reaction was refluxed for 2 days. After cooling, the reaction was poured into crushed ice and neutralized with a 5 M HCl solution. The solution was then extracted with ethyl acetate (3 × 50 mL). The organic layers were collected, washed with water, and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness, and the obtained reaction mixture was purified via flash CC . A yellow solid (30% yield) corresponding to (E)-2-acryloyl)phenoxy)acetic acid (11) was obtained.To a solution of methyl 2-(4-acetyl-3-hydroxy-2-propylphenoxy)acetate (E)-2-acryloyl)phenoxy)acetic acid( (11): 98.7% purity; mp 200–202 °C (chloroform); 1H NMR  δ: 13.39 , 12.73 , 7.79 , 7.75 , 7.56 , 7.45 , 6.86 , 4.72 , 3.92 , 3.90 , 2.73–2.66 , 1.63–1.53 , 0.94  ppm; 13C NMR  δ: 192.4 (C=O), 172.8 (C-2″), 163.7 (C-2), 162.5 (C-4), 153.7 , 145.0 (C-β), 140.8 (C-4‴), 130.1 (C-5), 130.4 (C-1‴), 129.0 (C-6), 119.7 (C-α), 119.4 (C-3), 115.3 (C-1), 106.1 , 65.1 (C-1″), 61.2 (4‴-OCH3), 56.4 , 24.7 (C-1′), 22.0 (C-2′), 14.4 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C23H26O10 (M+H+) 431.17004, found 431.16961.10, 200 mg, 1 Eq, 751 µmol) in methanol was added an aqueous solution of 40% sodium hydroxide to pH 13–14. Then, a solution of 3,4,5-trimethoxybenzaldehyde  in methanol was slowly added to the reaction mixture. The reaction was refluxed for 4 days. After cooling, the reaction was poured onto crushed ice and the pH was adjusted to 6–8, with a 5 M HCl solution. The obtained solid was filtrated, washed with water, dried, and purified via flash CC . A yellow solid (35% yield) corresponding to (E)-2,2’-acryloyl)-1,3-phenylene)bis(oxy))diacetic acid (12) was obtained.To a solution of methyl 2-(4-acetyl-3-hydroxy-2-propylphenoxy)acetate (E)-2,2’-acryloyl)-1,3-phenylene)bis(oxy))diacetic acid( (12): 98.7% purity; mp 212–214 °C (chloroform); 1H NMR  δ: 7.55–7.52 , 7.10 , 6.79 , 4.80 , 4.35 , 3.84 , 3.70 , 2.69 , 1.60–1.51 , 0.93  ppm; 13C NMR  δ: 190.3 (C=O), 170.0 (C-2″), 169.5 (C-2‴), 159.6 (C-4), 156.3 (C-2), 153.1 , 144.0 (C-β), 139.6 (C-4‴’), 130.1 (C-1‴’), 129.0 (C-6), 125.4 (C-α), 125.1 (C-1), 124.5 (C-3), 107.4 (C-5), 106.2 , 71.4 (C-1‴), 65.0 (C-1″), 60.1 (4‴’-OCH3), 56.0 , 25.4 (C-1′), 22.3 (C-2′), 14.3 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C25H28O10 (M+Na+) 511.15747, found 511.15885.E)-2-acryloyl)phenoxy)acetic acid  was dissolved in 5 mL of anhydrous dichloromethane and N-ethyl-N-isopropylpropan-2-amine  was added. Then, 3,4,5-trimethoxyaniline  was added to the mixture. After 10 min, COMU  was also added to the mixture. The reaction proceeded for 3 h at room temperature with stirring. After completion, the reaction mixture was poured onto ice. The solution was subsequently washed with a 1 M HCl solution and a saturated NaHCO3 solution, and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness, and the obtained reaction mixture was purified via TLC . A yellow solid (30% yield) corresponding to (E)-2-acryloyl)phenoxy)-N-acetamide (13) was obtained.(E)-2-acryloyl)phenoxy)-N-acetamide( (13): 95.9% purity; mp 199–201 °C (chloroform); 1H NMR  δ: 13.66 , 10.18 , 8.27 , 7.95 , 7.80 , 7.25 , 7.01 , 6.61 , 4.90 , 3.86 , 3.74 , 3.72 , 3.62 , 2.66 , 1.58–1.51 , 0.94  ppm; 13C NMR  δ: 192.4 (C=O), 165.8 (C-2″), 162.6 (C-2), 162.1 (C-4), 153.1 , 152.8 , 145.0 (C-β), 140.0 (C-4‴), 134.6 (C-4″), 133.7 (C-7″), 130.5 (C-6), 130.5 (C-1‴), 120.2 (C-α), 117.4 (C-3), 114.2 (C-1), 106.8 , 103.4 (C-5), 97.0 , 67.2 (C-1″), 60.1 , 56.2 , 55.7 , 24.2 (C-1′), 21.4 (C-2′), 14.2 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C32H37NO10 (M+Na+) 618.23097, found 618.23023.tert-butyl (E)-acryloyl)phenoxy)ethyl)carbamate  in DMSO , I2  was added. The mixture was heated at reflux for 24 h. Then, the mixture was poured into ice and a sodium thiosulfate solution (10%) was added. The obtained mixture was extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The solvent was evaporated to dryness, and the obtained reaction mixture was purified via TLC . A white solid (30% yield) corresponding to tert-butyl -4H-chromen-7-yl)oxy)ethyl)carbamate (14) was obtained.To a solution of tert-butyl -4H-chromen-7-yl)oxy)ethyl)carbamate (14): 98.9% purity; mp 191–193 °C (ethyl acetate); 1H NMR  δ: 7.88 , 7.34 , 7.20 , 7.07 , 7.02 , 4.18 , 3.91 , 3.90–3.67 , 3.76 , 2.74 , 1.68–1.61 , 1.38 , 0.97  ppm; 13C NMR  δ: 177.0 (C=O), 161.8 (C-2), 160.6 (C-7), 155.7 (C-4″), 154.4 (C-8a), 153.3 , 140.4 (C-4‴), 126.8 (C-1‴), 123.8 (C-5), 118.2 (C-4a), 117.2 (C-8), 110.4 (C-6), 106.1 (C-3), 103.5 , 77.7 (C-5″), 67.6 (C-1″), 60.2 (4‴-OCH3), 56.3 (C-2″), 56.1 , 28.2 (6″-CH3), 24.8 (C-1′), 22.1 (C-2′), 14.2 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C28H35NO8 (M+H+) 514.24354, found 514.24311.E)-N-(2-(3-hydroxy-2-propyl-4-((E)-3-acryloyl)phenoxy)ethyl)-3-acrylamide  in DMSO , I2  was added. The mixture was heated at reflux for 2 h. Then, the mixture was poured into ice and a sodium thiosulfate solution (10%) was added. The obtained mixture was extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The solvent was evaporated to dryness, and the obtained reaction mixture was purified via TLC . A white solid (35% yield) corresponding to (E)-N--4H-chromen-7-yl)oxy)ethyl)-3-acrylamide (15) was obtained.To a solution of (E)-N--4H-chromen-7-yl)oxy)ethyl)-3-acrylamide( (15): 98.4% purity; mp 198–199 °C (chloroform); 1H NMR  δ: 8.09 , 7.59 , 7.15 , 7.02 , 6.73 , 6.72 , 6.32 , 5.97 , 4.28 , 3.95 , 3.94 , 3.88 , 3.87 , 3.95–3.90 , 2.98 , 1.78–1.72 , 1.06  ppm; 13C NMR  δ: 178.9 (C=O), 166.2 (C-4″), 162.9 (C-2), 160.6 (C-7), 155.4 (C-8a), 153.8 , 153.6 , 142.1 (C-β), 140.0 (C-4‴), 139.2 (C-8″), 130.4 (C-5″), 127.4 (C-1‴), 124.7 (C-5), 119.4 (C-4a), 119.3 (C-α), 118.7 (C-8), 109.9 (C-6), 106.6 (C-3), 105.2 , 103.5 , 67.8 (C-1″), 61.2 (4‴-OCH3), 61.1 (4″-OCH3), 56.4 , 56.3 , 39.5 (C-2″), 25.7 (C-1′), 22.8 (C-2′), 14.7 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C35H39NO10 (M+CH3OHH+) 666.29089, found 666.29022.E)-N-acryloyl)phenoxy)ethyl)-3,4,5-trimethoxybenzamide  in DMSO , I2  was added. The mixture was heated at reflux for 2 h. Then, the mixture was poured into ice and a sodium thiosulfate solution (10%) was added. The obtained mixture was extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The solvent was evaporated to dryness and the reaction mixture was purified via TLC . A white solid (33% yield) corresponding to 3,4,5-trimethoxy-N--4H-chromen-7-yl)oxy)ethyl)benzamide (16) was obtained.To a solution of -4H-chromen-7-yl)oxy)ethyl)benzamide3,4,5-trimethoxy- (16): 95.8% purity; mp 196–199 °C (chloroform); 1H NMR  δ: 8.64 , 7.90 , 7.33 , 7.27 , 7.07 , 7.07 , 4.02 , 3.90 , 3.81 , 3.75 , 3.69 , 3.51–3.46 , 2.89 , 1.68–1.59 , 0.89  ppm; 13C NMR  δ: 177.4 (C=O), 166.1 (C-4″), 162.5 (C-2), 160.2 (C-7), 155.9 (C-8a), 154.5 , 154.4 , 140.4 (C-4‴), 140.2 (C-8″), 129.8 (C-5″), 126.8 (C-1‴), 124.3 (C-5), 119.8 (C-4a), 117.3 (C-8), 109.7 (C-6), 106.0 (C-3), 104.7 , 103.5 , 67.6 (C-1″), 61.4 (4‴-OCH3), 60.0 (8″-OCH3), 56.1 , 55.9 , 38.9 (C-2″), 30.7 (C-1′), 22.1 (C-2′), 14.1 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C33H37NO10 (M+H+) 608.24902, found 608.24725.Method A: To a solution of (E)-2-acryloyl)phenoxy)-N-acetamide  in DMSO , I2  was added. The mixture was heated at reflux for 24 h. Then, the mixture was poured into ice and a sodium thiosulfate solution (10%) was added. The obtained mixture was extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The solvent was evaporated to dryness and the obtained mixture was purified via TLC . A yellow solid (30% yield) corresponding to -4H-chromen-7-yl)oxy)-N-acetamide (17) was obtained.Method B: 2--4H-chromen-7-yl)oxy)acetic acid  was dissolved in 5 mL of anhydrous dichloromethane and N-ethyl-N-isopropylpropan-2-amine , and 3,4,5-trimethoxyaniline  was added to the mixture. After 10 min, COMU  was also added to the mixture. The reaction proceeded for 5 h at room temperature with stirring. After completion, the reaction mixture was poured onto ice. The solution was subsequently washed with 1M HCl solution and saturated NaHCO3 solution, and dried over anhydrous sodium sulfate. The solvent was evaporated to dryness and the obtained reaction mixture was purified via TLC . A yellow solid (17.5% yield) corresponding to -4H-chromen-7-yl)oxy)-N-acetamide (17) was obtained.H-chromen-7-yl)oxy)-N-acetamide (17)-4: 96.4% purity; mp 195–197 °C (chloroform); 1H NMR  δ: 10.15 , 7.90 , 7.36 , 7.00 , 7.14 , 7.10 , 4.94 , 3.92 , 3.76 , 3.76 , 3.62 , 3.07 , 1.71–1.67 , 1.01  ppm; 13C NMR  δ: 177.0 (C=O), 166.9 (C-2″), 162.9 (C-2), 160.1 (C-7), 154.6 (C-8a), 153.0 , 152.8 , 140.0 (C-4‴), 134.6 (C-7″), 133.8 (C-4″), 126.7 (C-1‴), 123.7 (C-5), 119.5 (C-8), 118.5 (C-4a), 110.4 (C-6), 106.1 (C-3), 103.8 , 97.0 , 67.9 (C-1″), 60.3 (4‴-OCH3), 60.1 (7″-OCH3), 56.1 , 55.7 , 24.1 (C-1′), 22.3 (C-2′), 14.5 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C33H35NO10 (M+K+) 632.18925, found 632.18769.E)-2-acryloyl)phenoxy)acetic acid  in DMSO , I2  was added. The mixture was heated at reflux for 24 h. Then, the mixture was poured into ice and a sodium thiosulfate solution (10%) was added. The obtained mixture was extracted with ethyl acetate, washed with water, and then dried over anhydrous sodium sulfate. The solvent was evaporated to dryness, and the obtained oil was purified via TLC . A white solid (35% yield) corresponding to 2--4H-chromen-7-yl)oxy)acetic acid (18) was obtained.To a solution of (H-chromen-7-yl)oxy)acetic acid2-((4-oxo-8-propyl-2-(3.4.5-trimethoxyphenyl)-4 (18): 95.9% purity; mp 199–201 °C (chloroform); 1H NMR  δ: 7.92 , 7.38 , 7.10 , 7.01 , 4.85 , 3.94 , 3.79 , 3.04 , 1.73–1.72 , 1.04  ppm; 13C NMR  δ: 178.1 (C=O), 170.8 (C-2″), 163.2 (C-2), 161.1 (C-7), 156.1 (C-8a), 154.9 , 142.2 (C-4‴), 128.3 (C-1‴), 124.9 (C-5), 119.8 (C-8), 119.2 (C-4a), 111.1 (C-6), 107.3 (C-3), 104.7 , 66.4 (C-1″), 61.0 (4‴-OCH3), 57.0 , 26.4 (C-1′), 23.5 (C-2′), 15.1 (C-3′) ppm; HRMS (ESI+): m/z Anal. Calc. for C23H24O8 (M+H+) 429.15439, found 429.15658.2, and cell viability was regularly monitored using the exclusion trypan blue dye.A375-C5 (melanoma) and MCF-7 (breast adenocarcinoma) cell lines were obtained from the European Collection of Cell Culture, UK, and were grown in RPMI-1640 medium  with 5% inactivated FBS . HCT-116 (colon adenocarcinoma) cells were grown in DMEM medium  with 5% inactivated FBS. The non-tumor human pulmonary alveolar epithelial cells (HPAEpiC) were obtained from ScienCell Research Laboratories and grown in DMEM medium supplemented with 10% FBS and 1% non-essential amino acids . All cell lines were maintained in a humidified incubator  at 37 °C and with 5% COAll tested compounds were dissolved to 60 µM in sterile DMSO . To preserve the activities of compounds, several aliquots were prepared and stored at −20 °C. Immediately prior to use, compounds were diluted in fresh culture medium at the required concentrations.4 A375-C5, MCF-7, and HCT-116 cells, or 6.0 × 104 HPAEpiC cells were seeded in 96-well plates and incubated at 37 °C and 5% CO2 to allow for cell attachment. After 24 h, cells were treated with two-fold serial dilutions of test compounds (0–150 µM), or with DMSO as the compound solvent. In parallel, cells in a time-zero (T0) plate, without treatment, were fixed with 50% (m/v) trichloroacetic acid , for 1 h at 4 °C, and then washed with distilled water and left to dry. Forty-eight hours after compound treatment, cells were fixed with 50% TCA and stained with SRB  for 30 min at room temperature, followed by 5 rounds of washing with 1% (v/v) acetic acid . SRB–protein complexes were then solubilized with 10 µM Tris buffer  for 30 min, and the absorbance was measured at 515 nm in a microplate reader . A dose-response curve was obtained for each cell line, and the concentration that caused 50% cell growth inhibition (GI50) was calculated.A total of 5.0 × 10Saccharomyces cerevisiae (strain CG379) expressing human wt p53 alone and combined with human MDM2 were obtained in previous works [w/w) galactose, 1% (w/w) raffinose, 0.7% (w/w) yeast nitrogen base without amino acids from Difco , and all the amino acids required for yeast growth (50 g/mL) except leucine and tryptophan. Yeast cells were incubated at 30 °C under continuous orbital shaking (200 rpm) with 10 µM compounds or 0.1% DMSO only, for approximately 42 h . Yeast growth was analyzed by counting the number of colony-forming units (CFU) after 2 days of incubation at 30 °C on Sabouraud Dextrose Agar from Liofilchem .us works ,33. For 5 cells/well were seeded into 6-well plates, and 24 h later, cells were treated with compounds 6, 7, and 13. After 48 h, floating and adherent cells were collected, pelleted via centrifugation , and suspended in binding buffer. Annexin V-FITC was added and incubated for 10 min, protected from light. Cells were washed and resuspended in binding buffer, and 20 μg/mL of propidium iodide (PI) was added. Fluorescence was assessed using the BD Accuri™ C6 Plus flow cytometer , and the data were analyzed with the BD Accuri TM C6 Plus software . At least 20,000 events per sample were collected.To evaluate apoptotic cell death, the “Annexin V-FITC Apoptosis Detection Kit”  was used according to the manufacturer’s instructions. Briefly, 1.2 × 105 cells were grown on poly-L-lysine-coated coverslips for 24 h. Then, cells were treated with 6, 7, and 13, and fixed with methanol  for 10 min at −20 °C and washed 3 times with PBS for 5 min. DNA was stained with 2 µg/mL 40,6-diamidino-2-phenylindole  diluted in Vectashield mounting medium .A total of 1.2 × 105 HCT-116 cells were grown in six-well dishes and treated for 24 h with 3.26 µM of compound 7. Cells treated with 1 µM nocodazole were used as a positive control for antimitotic activity. Untreated cells and cells treated with DMSO were included to exclude the cytotoxicity of the compound solvent. Mitotic index, and the percentage of mitotic cells over the total cell population were determined via cell-rounding under phase-contrast microscopy from at least ten random microscope fields.A total of 1.2 × 10Cells were treated as for DAPI staining. After methanol fixation, cells were blocked with 10% FBS in PBST (0.05% Tween-20 in PBS) for 30 min at room temperature, followed by 1 h incubation with a primary antibody  diluted in 5% FBS in PBST. Then, cells were washed three times in PBST and incubated with an Alexa Fluor 488 conjugated secondary antibody . DNA was stained with DAPI.For phase contrast microscopy images, a Nikon TE 2000-U microscope  with a 10× objective was used, coupled to a DXM1200F digital camera with Nikon ACT-1 software . For fluorescence images, an Axio Observer Z.1 SD microscope  was used, coupled to an AxioCam MR3, with the Plan Apochromatic 63×/NA 1.4 objective. The images were processed using ImageJ, version 1.51.p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 were considered as statistically significant.All assays were performed in triplicate with at least three independent experiments. Data were expressed as mean ± standard deviation (SD), statistical analysis was carried out in GraphPad Prism Software Inc. v6 using an unpaired t-test, and values of * http://www.swissadme.ch/, (accessed on 12 May 2023).The selected compounds were further investigated for drug-likeness properties, namely, molecular descriptors/physicochemical properties, by SwissADME (CM-M345 (1) and BP-M345 (2), a new series of chalcone-trimethoxycinnamide/trimethoxyphenylamide hybrids have been designed, synthesized, and evaluated for their antitumor activity against melanoma (A375-C5), breast adenocarcinoma (MCF-7), and colorectal carcinoma (HCT116) cell lines, as well as non-tumor HPAEpiC cells. Among these, compounds 6, 7, and 13 demonstrated potent antiproliferative activity in the studied human tumor cell lines, with this effect being particularly intensified against colorectal tumor cells, with a relevant degree of selectivity when compared to non-tumor cells for compound 7. The three referred compounds exert antitumor activity against colorectal tumor cells through apoptosis. Mechanistically, compound 7 emerged as an antimitotic agent that, through its antimicrotubule activity, induces mitotic delay in colorectal tumor cells, and consequently, cell death. Notably, compound 7 affected colorectal tumor cells more severely than the non-tumor cells, indicating a high degree of selectivity, as suggested by the 17-fold higher GI50 value of the compound on non-tumor cells. This highlights the potential of 7 as a hit compound, since the discovery of compounds with selective cytotoxicities that are capable of distinguishing between normal and tumor cells is a major challenge in tumor therapy. Considering the results obtained through in silico drug-likeness prediction, in the near future, the optimization of this compound should also consider the improvement of its drug-likeness profile, namely through nanotechnology.Inspired by


Beauveria bassiana KCH J1.5, Isaria fumosorosea KCH J2, and Isaria farinosa KCH J2.6 strains on six flavonoid compounds obtained in chemical synthesis. As a result of the biotransformation of 6-methyl-8-nitroflavanone using the strain I. fumosorosea KCH J2, two products were obtained: 6-methyl-8-nitro-2-phenylchromane 4-O-β-D-(4″-O-methyl)-glucopyranoside and 8-nitroflavan-4-ol 6-methylene-O-β-D-(4″-O-methyl)-glucopyranoside. 8-Bromo-6-chloroflavanone was transformed by this strain to 8-bromo-6-chloroflavan-4-ol 4′-O-β-D-(4″-O-methyl)-glucopyranoside. As a result of microbial transformation by I. farinosa KCH J2.6 effectively biotransformed only 8-bromo-6-chloroflavone into 8-bromo-6-chloroflavone 4′-O-β-D-(4″-O-methyl)-glucopyranoside. B. bassiana KCH J1.5 was able to transform 6-methyl-8-nitroflavone to 6-methyl-8-nitroflavone 4′-O-β-D-(4″-O-methyl)-glucopyranoside, and 3′-bromo-5′-chloro-2′-hydroxychalcone to 8-bromo-6-chloroflavanone 3′-O-β-D-(4″-O-methyl)-glucopyranoside. None of the filamentous fungi used transformed 2′-hydroxy-5′-methyl-3′-nitrochalcone effectively. Obtained flavonoid derivatives could be used to fight against antibiotic-resistant bacteria. To the best of our knowledge, all the substrates and products presented in this work are new compounds and are described for the first time.Combining chemical and microbiological methods using entomopathogenic filamentous fungi makes obtaining flavonoid glycosides possible. In the presented study, biotransformations were carried out in cultures of  The microbial transformation process is a well-known method of obtaining modified organic compounds like glycosides or hydroxylic derivatives using enzymes and whole cells ,2,3. ThiStenotrophomonas maltophilia KB2 can carry out demethylation of 7-methoxyflavanones, and open the heterocyclic C ring in various methoxyflavanones, 7-methoxyflavanone, 3′-methoxyflavanone, and 5,7-dimethoxyflavanone D = −32.311 (0.38 w/v % in acetone); 1H NMR  δ (ppm): 7.69 , 7.59 , 7.56–7.54 , 7.43 , 7.37–7.33 , 5.55 , 5.06 , 4.50 , 4.43 , 4.25 , 3.91 , 3.86 , 3.72 , 3.54 , 3.50 , 3.36 , 3.30 , 3.12 , 2.60 , 2.37 , 2.19–2.13 ; 13C NMR (acetone-d6) δ (ppm): 147.27 (C-8a), 141.48 (C-1′), 137.48 (C-5), 130.03 (C-6), 129.33 , 128.78 (C-4′), 126.83 , 126.53 (C-7), 125.43 (C-8), 123.87 (C-4a), 101.49 (C-1″), 80.59 (C-4″), 78.11 (C-3″), 77.10 (C-5″), 75.29 (C-2″), 75.12 (C-2), 69.42 (C-4), 62.54 (C-6″), 60.52 (C4″-O-CH3), 36.72 (C-3), 20.09 (-CH3).6-Methyl-8-nitro-2-phenylchromane 4-O-β-D-(4″-O-methyl)-glucopyranoside (5b). C23H27NO10, mp: 177 °C; HPLC Rt = 11.6 min; [α]D = −37.348 (1.1 w/v % in acetone); 1H NMR  δ (ppm): 7.87 , 7.71 , 7.54 , 7.45–7.42 , 7.36 , 5.49 , 4.90 , 4.90 , 4.68 , 4.41 , 4.29 , 3.83 , 3.76–3.70 , 3.65 , 3.53 , 3.51 , 3.31–3.25 , 3.13–3.08 , 2.39–2.36 , 2.23–2.18 ; 13C NMR (acetone-d6) δ (ppm): 148.21 (C-8a), 141.39 (C-1′), 139.96 (C-8), 137.40 (C-4a), 135.41 (C-5), 131.11 (C-6), 129.40 , 128.84 (C-4′), 126.78 , 124.95 (C-7), 103.29 (C-1″), 80.50 (C-4″), 78.09 (C-3″), 76.96 (C-5″), 75.24 (C-2), 75.15 (C-2″), 69.79 (6-CH2-O-), 63.10 (C-4), 62.44 (C-6″), 60.49 (4″-O-CH3), 38.83 (C-3).8-Nitroflavan-4-ol 6-methylene-O-β-D-(4″-O-methyl)-glucopyranoside (6a). C23H23NO10, mp: 140–143 °C; HPLC Rt = 6.7 min; [α]D = −12.707 (0.35 w/v % in acetone); 1H NMR  δ (ppm): 8.34 , 8.24 , 8.11–8.08 , 7.29–7.26 , 6.93 , 5.12 , 4.75 , 4.46 , 4.16–4.13 , 3.87 , 3.74–3.70 , 3.68–3.66 , 3.58–3.57 , 3.54–3.50 , 3.27–3.23 , 2.59 ; 13C NMR (acetone-d6) δ (ppm): 179.09 (C=O), 163.80 (C-2), 161.81 (C-4′), 153.12 (C-8a), 139.88 (C-8), 136.11 (C-6), 131.58 (C-5), 131.36 (C-7), 129.17 , 125.75 (C-4a), 124.92 (C-1′), 117.77 , 106.73 (C-3), 101.14 (C-1″), 80.02 (C-4″), 77.97 (C-3″), 77.20 (C-5″), 74.86 (C-2″), 62.02 (C-6″), 60.58 (4″-O-CH3), 20.58 (-CH3).6-Methyl-8-nitroflavone 4′-O-β-D-(4″-O-methyl)-glucopyranoside (7a). C22H22BrClO8, mp: 97 °C; HPLC Rt = 13.6 min; [α]D = −60.024 (0.41 w/v % in acetone) 1H NMR  δ (ppm): 7.91 , 7.77 , 7.38 , 7.32–7.29 , 7.25 , 7.09 , 5.81 , 5.01 , 4.64 , 4.39 , 3.83 , 3.73 , 3.70–3.66 , 3.65–3.60 , 3.56 , 3.50–3.46 , 3.26 , 3.24–3.20 , 3.03 ; 13C NMR (acetone-d6) δ (ppm): 190.26 (C=O), 158.93 (C-3′), 157.58 (C-8a), 140.87 (C-1′), 138.83 (C-7), 130.68 (C-5′), 127.35 (C-6), 126.12 (C-5), 123.68 (C-4a), 120.72 (C-6′), 117.43 (C-4′), 115.23 (C-2′), 113.60 (C-8), 101.35 (C-1″), 80.84 (C-2), 80.07 (C-4″), 78.08 (C-3″), 77.06 (C-2″), 74.94 (C-5″), 62.05 (C-6″), 60.55 (4″-O-CH3), 43.85 (C-3). 8-Bromo-6-chloroflavanone 3′-O-β-D-(4″-O-methyl)-glucopyranoside (8a). C22H24BrClO8, mp: 117 °C; HPLC Rt = 11.0 min; [α]D = −8.725 (0.4 w/v % in acetone); 1H NMR  δ (ppm): 7.54 , 7.45 , 7.40 , 7.11 , 5.37 , 4.98 , 4.85–4.81 , 4.63 , 4.38 , 3.85 , 3.76 , 3.69 , 3.66–3.61 , 3.57 , 3.51–3.45 , 3.24–3.20 , 2.29–2.25 , 2.19–2.13 ; 13C NMR (acetone-d6) δ (ppm): 158.63 (C-4′), 151.36 (C-8a), 135.03 (C-1′), 132.58 (C-7), 130.45 (C-5), 128.78 (C-4a), 128.25 , 125.45 (C-6), 117.36 , 111.84 (C-8), 101.64 (C-1″), 80.12 (C-4″), 78.04 (C-3″), 77.06 (C-5″), 74.98 (C-2″), 74.91 (C-2), 63.44 (C-4), 62.11 (C-6″), 60.55 (4″-O-CH3), 38.57 (C-3).8-Bromo-6-chloroflavan-4-ol 4′-O-β-D-(4″-O-methyl)-glucopyranoside (9a). C22H20BrClO8, mp: 160 °C; HPLC Rt = 11.1 min; [α]D = −17.181 (1.39 w/v % in acetone) 1H NMR  δ (ppm): 8.12 , 8.01 , 7.27 , 6.90 , 5.12 , 4.75 , 4.47 , 3.87 , 3.84–3.82 , 3.73–3.70 , 3.66 , 3.58 , 3.56 , 3.54–3.50 , 3.24 ; 13C NMR (acetone-d6) δ (ppm): 176.14 (C=O), 164.06 (C-2), 161.82 (C-4′), 152.40 (C-8a), 137.19 (C-7), 131.44 (C-6), 129.11 , 126.65 (C-4a), 125.38 (C-1′), 124.88 (C-5), 117.81 , 113.75 (C-8), 106.48 (C-3), 101.16 (C-1″), 80.05 (C-4″), 77.96 (C-3″), 77.22 (C-5″), 74.86 (C-2″), 62.05 (C-6″), 60.59 (4″-O-CH3).8-Bromo-6-chloroflavone 4′-4) was subjected to small-scale biotransformation by B. bassiana KCH J1.5, I. fumosorosea KCH J2, and I. farinosa KCH J2.6. A High-Pressure Liquid Chromatography (HPLC) analysis showed no flavonoid derivative products in the obtained extracts. The compound 2′-hydroxy-5′-methyl-3′-nitrochalcone (5) was transformed microbiologically in the culture of I. fumosorosea KCH J2 (O-β-D-(4″-O-methyl)-glucopyranoside (5a) with 5.3% yield (4.3 mg) and 8-nitroflavan-4-ol 6-methylene-O-β-D-(4″-O-methyl)-glucopyranoside (5b) with 14.8% yield (12.5 mg). The biotransformation took 10 days.6-Methyl-8-nitroflavanone (a KCH J2 . The pro5a) and (5b) were determined by NMR spectroscopy using 1H NMR, 13C NMR, HMBC, and HSQC spectra and mass confirmation by LC-MS analysis (O-β-D-(4″-O-methyl)-glucopyranoside (5a), the presence of glucose in position C-4 is confirmed by the lack of a characteristic signal from carbonyl carbon in the 13C NMR spectrum as it was in a substrate (5). The NMR spectrum shows the signal of one proton at C-4, which proves the reduction of the carbonyl group at this position (δ = 5.06 ppm 1H NMR) and carbon δ = 69.42 ppm (13C NMR)  and the presence of one signal from carbon at δ = 60.52 ppm (13C NMR) shows that glucose is O-methylated at the C-4″ position  . There i13C NMR) . The chaposition . There aspectrum  Moreoverspectrum . Proton 5b), the presence of the one proton attached to carbon C-2 at δ = 5.49 ppm in the 1H NMR spectrum (carbon at δ = 75.24 ppm 13C NMR) and the two protons: H-3ax at δ = 2.37 ppm and H-3eq at δ = 2.20 ppm (carbon C-3 at δ = 38.83 ppm 13C NMR) indicates the structure of the flavanone. Moreover, the correlation of these protons is visible in the COSY spectrum (marked with an arrow—1H NMR), there is a signal from a single proton suggesting a reduction of the carbonyl group at the C-4 position. There is also a dehydrogenation of the -CH3 group at the C-6 position to a -CH2- group (two characteristic proton doublets at δ = 4.90 ppm and 4.68 ppm)  (In the case of product (.68 ppm) . In the position . In the ylation) . Proton 6) in the culture of B. bassiana KCH J1.5, the 6-methyl-8-nitroflavone 4′-O-β-D-(4″-O-methyl)-glucopyranoside (6a) was formed (6a) was obtained (5%).As a result of the biotransformation of the 6-methyl-8-nitroflavone (s formed . After sO-β-D-(4″-O-methyl)-glucopyranoside (6a) was determined based on NMR spectroscopy and LC-MS analysis (13C NMR spectrum with the characteristic attached proton doublet H-1” in the 1H NMR spectrum at δ = 5.12 ppm having the coupling constant J = 7.8 Hz indicates the β-configuration of the attached glucose molecule (O-methylated in position C-4″ (3 group located at δ = 3.57 ppm (1H NMR) correlates with the carbon C-4″  in the HMBC spectrum  is correlated with the one proton at C-3 carbon in the HMBC spectrum  was biotransformed by entomopathogenic fungi B. bassiana KCH J1.5 into 8-bromo-6-chloroflavanone 3′-O-β-D-(4″-O-methyl)-glucopyranoside (7a) with 6.24% yield (4.9 mg)  . The pro7) was converted to the flavanone structure by the cyclization of the C ring and glycosylated at C-3′ position. The structure of the product was confirmed by NMR spectroscopy and LC-MS analysis (1H NMR) and the carbon C-3′ δ = 158.93 ppm (13C NMR) at the HMBC contour map (J = 2.6 Hz) and H-5  evidence the presence of substitution in positions C-6 and C-8 (13C NMR) and protons 3-axial and 3-equatorial  at HMCB   . Other p8) in the culture of I. fumosorosea KCH J2 allowed receiving 8-bromo-6-chloroflavan-4-ol 4′-O-β-D-(4″-O-methyl)-glucopyranoside (8a)   with 2.68a). The 1H NMR spectrum shows two doublets coming from protons H-7  and H-5 , evidencing the substitution at C-8 and C-6 positions, as in the case of the substrate (8). The correlation is visible in the HMBC spectrum (J = 8.6 Hz), and protons from C-3′ and C-5′  suggesting additional substitution at the C-4′ position  which proves the reduction of the carbonyl group  and the presence of one carbon at δ = 60.55 ppm (13C NMR) shows that glucose is O-methylated at C-4″ position  was biotransformed by enthomopathogenic fungi I. farinosa KCH J2.6 into 8-bromo-6-chloroflavone 4′-O-β-D-(4″-O-methyl)-glucopyranoside (9a) (The 8-bromo-6-chloroflavone (ide (9a)  with 19.9a) was an O-methylglycosylated 8-bromo-6-chloroflavone derivative. The accurate NMR and LC-MS analysis confirming the structure and mass of the obtained product is presented in 1H NMR and 13C NMR spectra (9) was confirmed by the presence of a proton doublet from the proton H-1″ at the anomeric carbon C-1″ at δ = 5.12 ppm in the 1H NMR spectrum with the characteristic coupling constant J = 7.7 Hz . Other proton–proton correlations COSY and proton–carbon correlations HMBC are shown in The obtained product , was observed for 6-methylflavanone D = 0.108 (0.5 w/v % in acetone); 1H NMR  δ (ppm): 8.02 , 7.96–7.95 , 7.61 , 7.47 , 7.41 , 5.87 , 3.30 , 3.07 , 2.44 ; 13C NMR (acetone-d6) δ (ppm): 190.29 (C=O), 152.95 (C-6), 140.62 (C-8), 139.39 (C-1′), 132.30 (C-5), 132.09 (C-7), 131.87 (C-8a), 129.57 , 129.53 (C-4′), 127.10 , 123.97 (4a), 81.36 (C-2), 44.35 (C-3), 20.06 (-CH3)6-Methyl-8-nitroflavanone (6). C16H11NO4, mp: 219 °C; HPLC Rt = 17.1 min; 1H NMR  δ (ppm): 8.35 , 8.24 , 8.15 , 7.65–7.61 , 7.01 , 2.59 ; 13C NMR (acetone-d6) δ (ppm): 176.23 (C=O), 163.90 (C-2), 147.56 (C-8a), 139.61 (C-8), 136.24 (C-6), 133.00 (C-4′), 131.92 (C-1′), 131.57 (C-5), 131.53 (C-7), 130.11 , 127.45 , 126.31 (C-4a), 107.97 (C-3), 20.60 (-CH3)6-Methyl-8-nitroflavone (7). C15H10BrClO2, mp: 133–135 °C; HPLC Rt = 19.9 min; 1H NMR  δ (ppm): 13.63 , 8.40 , 8.14 , 8.03 , 7.94 , 7.90 , 7.54–7.48 ; 13C NMR (acetone-d6) δ (ppm): 194.21 (C=O), 159.79 (C-1′), 148.25 (C-β), 139.40 (C-4′), 135.44 (C-1), 132.33 (C-4), 130.34 , 130.13 (C-6′), 129.94 , 124.46 (C-5′), 122.09 (C-2′), 120.75 (C-α), 113.07 (C-3′)3′-Bromo-5′-chloro-2′-hydroxychalcone (8). C15H10BrClO2, mp: 117–119 °C; HPLC Rt = 18.6 min; [α]D = -5.331 (0.325 w/v % in acetone); 1H NMR  δ (ppm): 7.91 , 7.77 , 7.64–7.61 , 7.50–7.46 , 7.44–7.40 , 5.84 , 3.27 , 3.03 ; 13C NMR (acetone-d6) δ (ppm): 190.27 (C=O), 157.66 (C-8a), 139.43 (C-1′), 138.81 (C-7), 129.61 , 127.32 (C-6), 127.22 , 126.14 (C-5), 123.70 (C-4a), 113.58 (C-8),81.05 (C-2), 43.87 (C-3)8-Bromo-6-chloroflavanone (9). C15H8BrClO2, mp: 186–189 °C; HPLC Rt = 19.2 min; 1H NMR  δ (ppm): 8.19–8.17 , 8.14 , 8.03 , 7.66–7.62 , 7.00 ; 13C NMR (acetone-d6) δ (ppm): 176.30 (C=O), 164.21 (C-2), 152.51 (C-8a), 137.38 (C-7), 133.05 (C-4′), 132.06 (C-1′), 131.56 (C-6), 130.16 , 127.43 , 126.69 (C-4a), 124.90 (C-5), 113.90 (C-8), 107.72 (C-3) 8-Bromo-6-chloroflavone : Methanol  in a ratio of 9:1 v/v was used to monitor the course of biotransformation. In both cases, the plates were observed under a UV lamp using a wavelength of λ = 254 nm and λ = 365 nm. Preparative TLC was used to separate the product mixture on scale-up biotransformation. For this purpose, preparative TLC Silica plates   were used with the eluent consisting of chloroform  and methanol  in the ratio (9:1 v/v). The products were observed under a UV lamp using a wavelength of λ = 254 nm and λ = 365 nm. Then, products were extracted thrice with 15 mL of ethyl acetate . The extracts were filtered and evaporated in a vacuum evaporator.Analytical and preparative TLC (Thin Layer Chromatography) was used to assess the course of synthesis as well as biotransformation and product isolation. Analytical TLC was used to monitor the course of chemical syntheses and the progress of biotransformation. For this purpose, TLC Silica gel 60/Kieselguhr F254 (0.2 mm thick) aluminum sheets 20 cm × 20 cm  were used. The eluent consisted of cyclohexane : Ethyl acetate  in the ratio 9:1 and 4:1 v/v (A) and acetonitrile (B). The gradient program was as follows: initial conditions—32.5% B in A, 4 min—40% B in A, 8 min—40% B in A, 10 min—45% B in A, 15 min—95% B in A, 18 min—95% B in A, 19 min—32.5% B in A, and 23 min—32.5% B in A. The flow rate was 1 mL/min, the injection volume was 5 µL, and the detection wavelength was 280 nm [HPLC chromatography was performed to check the biotransformation’s progress and determine the retention times of their substrates and products. Dionex Ultimate 3000 instrument  with a DAD-3000 diode array detector using an analytical octadecylsilica (ODS) 2 column  and pre-column. The eluent was a mixture of 0.1% aqueous acid formic acid s 280 nm . Data we1H NMR, 13C NMR, COSY, HSQC, and HMBC spectra were performed using the DRX AvanceTM 600 MHz NMR spectrometer . All samples were dissolved in 0.7 mL of deuterated acetone.For the NMR analysis of substrates and biotransformation products, v/v (A) and acetonitrile (B). The flow rate was 0.4 mL/min, and the injection volume was 5 µL. The gradient program was as follows: Initial conditions—80% B in A, 6.5 min—100% B, 7 min—80% B in A. The principal operating parameters for the LC-MS were set as follows: nebulizing gas flow: 3 L/min, heating gas flow: 10 L/min, interface temperature: 300 °C, drying gas flow: 10 L/min, data acquisition range, m/z 100–1000 Da, positive ionization mode. Data were collected with LabSolutions version 5.97  software.The mass of the obtained biotransformation substrates and products was confirmed using LC-MS analysis, LC-MS 8045 SHIMADZU Triple Quadrupole Liquid Chromatograph Mass Spectrometer with electrospray ionization (ESI) source . Analyses were conducted using method “product ion scan”. Only a specific ion with a known molecular mass  was searched in each sample with a pure compound. The separation was achieved on the Kinetex column  operated at 30 °C. The mobile phase was a mixture of 0.1% aqueous formic acid Optical rotation was measured using digital polarimeter P-2000-Na .B. bassiana KCH J1.5, I. fumosorosea KCH J2, and I. farinosa KCH J2.6 were used for small-scale biotransformation. Each of the six substrates underwent biotransformation by all the microorganisms above-mentioned. These fungi have been chosen for their ability to biotransform flavonoid compounds (production of glycoside derivatives) based on previous screening studies conducted by our team in the Department of Food Chemistry and Biocatalysis [4), (5), (6), (7), (8), and (9) for further scale-up studies.Three entomopathogenic filamentous fungi atalysis ,54. The 4), filtered, and concentrated in a vacuum evaporator. Samples for HPLC analysis from obtained extracts were prepared. The dried extracts were suspended in 1 mL of acetonitrile and analyzed by HPLC. The results of this analysis made it possible to select the appropriate biotransformation time and strain for the increased scale of the experiment so that the process would run as efficiently as possible.Small-scale biotransformation was carried out in 300 mL flat-bottomed conical flasks (Erlenmeyer flasks) containing 100 mL of modified Sabouraud medium . The flasks were inoculated with about 1 mL of a culture of entomopathogenic filamentous fungi and then shaken for 72 h at 140 rpm and 25 °C. Next, 10 mg of the substrate was added to each flask and shaken again at 140 rpm at 25 °C. Samples were collected after 3, 7, and 10 days of the experiment. After that, samples were extracted with ethyl acetate in the ratio of 1:1 ethyl acetate: medium, and collected in a separate flask. Extracts were dried with magnesium sulfate . The scale-up biotransformation was used to obtain more products for further analysis. 4, filtered, and evaporated to dryness. The obtained samples were separated using preparative TLC plates. For this purpose, the extract was dissolved in approx. two mL of THF and applied to the plate. In a chromatography chamber, the products were separated using chloroform: methanol mixture  as the eluent. TLC plates were visualized under a UV lamp (254 nm and 365 nm), and separated fractions were removed from the plate. Fractions were extracted three times with 15 mL ethyl acetate for 30 min. The extracts were filtered and evaporated to dryness in a vacuum evaporator. Then, the fractions prepared this way were dissolved in 0.7 mL of deuterated acetone and subjected to NMR analysis.To the flask with a prepared sterile medium 1 mL of preincubation culture of entomopathogenic filamentous fungi was added. Then, the flask was incubated for 72 h at 25 °C with shaking at 145 rpm. After this time, 50 mg of substrate dissolved in 2 mL of DMSO was added to the flask. The time of scale-up biotransformation was based on previous small-scale studies for a given microorganism and substrate. The next step was to extract the obtained products three times using 350 mL of ethyl acetate. The combined extracts were then dried with MgSONMR analyses are presented in 4), (7), flavanones (5), (8), and flavones (6), (9) using entomopathogenic filamentous fungi. In this paper, we present the microbial transformations of chalcones . B. bassiana KCH J1.5 strain was able to transform 3′-bromo-5′-chloro-2′-hydroxychalcone (7). In this case, the chalcone was cyclized to flavanone, and the glycosidic unit was attached at the C-3’ position creating an 8-bromo-6-chloroflavanone 3′-O-β-D-(4″-O-methyl)-glucopyranoside (7a). 5) and (8) were only effectively biotransformed by entomopathogenic filamentous fungi I. fumosorosea KCH J2. The carbonyl groups of both substrates were reduced. During the biotransformation of 6-methyl-8-nitroflavanone (5), the carbonyl group was reduced with the attachment of a glycosidic unit at the C-4 position (6-methyl-8-nitro-2-phenylchromane 4-O-β-D-(4″-O-methyl)-glucopyranoside (5a)), and in the product (5b) the sugar unit has become attached to the -CH3 group at the C-6 position (8-nitroflavan-4-ol 6-methylene-O-β-D-(4″-O-methyl)-glucopyranoside (5b)). Moreover, flavanones (6), the most efficient reaction was carried out by B. bassiana KCH J1.5, and for compound (9) by I. farinosa KCH J2.6.In the case of the transformation of flavanones, glucose was attached at the C-4′ position. For compound (The presented research results show the glycosylation capacity of flavonoids with various substituents by entomopathogenic filamentous fungi. All biotransformation products have not been previously described in the scientific literature. We aim at further studies to assess their biological properties and bioavailability.