Polygala tenuifolia Willd. have pharmacological effects related to anti-inflammation. Therefore, the aim of this study is to investigate the chemical constituents from P. tenuifolia roots as anti-inflammatory drug candidates. In the present work, twenty-three compounds were isolated from P. tenuifolia roots, including three saponins (1–3), ten phenylpropanoid sucrose esters (4–12), one benzoic acid sugar ester derivative (13), four xanthones (14–17), two hydroxy benzophenone derivatives (18 and 19), two phenolic derivatives (20 and 21), and two ionones (22 and 23). All isolates were tested for their inhibitory effects of LPS-stimulated NO and PGE2 production in RAW 264.7 macrophages. Among these, 3-O-,6′-O-(p-methoxybenzoyl) sucrose ester  together with compounds 3 and 21 exhibited significant inhibitory effects on NO production, while TCMB and compounds 17, 19, and 21 showed strong inhibitory effects on PGE2 production. Specifically, TCMB (11) downregulated the protein levels of iNOS and COX-2 in LPS-induced RAW 264.7 macrophages. In addition, TCMB (11) dose-dependently diminished the relative mRNA expression levels of iNOS, PGE2, and proinflammatory cytokines . A molecular docking study showed that TCMB (11) has strong binding affinities with iNOS and COX-2.Increasing scientific evidence has demonstrated that the roots of  P4-7 was further subjected to silica gel CC  and generated 20 subfractions (P4-7-1~P4-7-20). P4-7-9 produced compounds 13 (50.4 mg) and 6 (259.0 mg) using silica gel CC . P4-12 produced 16 subfractions using silica gel CC . P4-12-4 was fractionated into 12 fractions (P4-12-4-1~P4-12-4-12) by medium-pressure liquid chromatography (MPLC) using Redi Sep-C18 cartridge  to obtain compounds 4 (29.9 mg), 5 (151.5 mg), and 7 (10.8 mg). Compound 18 (16.8 mg) was obtained from P4-12-9 by MPLC using Redi Sep-C18 cartridge . Compound 20 (17.9 mg) was purified from P4-16 using MPLC with Redi Sep-C18 cartridge , successively.P4 was fractionated using Sephadex LH-20 column chromatography  with MeOH:H2O = 8:1.8:0.2 to 5:4.5:0.5 v/v/v) to produce 13 fractions (P5-1~P5-13). Compound 14 (1.04 g) was purified by recrystallization with MeOH from P5-10. P5-2 was fractionated into P5-2-1~P5-2-9 by MPLC using Redi Sep-C18 cartridge . Compound 9 (30.3 mg) was purified from P5-2-6 using MPLC . P5-3 was fractionated into P5-3-1~P5-3-16 by GLAS RP-18 CC . Compound 8 (352.5 mg) was purified from P5-3-8 using MPLC with Redi Sep-silica . P5-4 was fractionated using Sephadex LH-20 CC (3.8 × 60.0 cm) with MeOH:H2O mixture (7:3 v/v) and produced 14 fractions (P5-4-1~P5-4-14). Compound 16 (9.4 mg) was purified by recrystallization with MeOH from P5-4-11.P5 was subjected to silica gel CC  and produced 11 fractions (P9-1~P9-11). P9-6 was further fractionated using silica gel CC  and produced 18 subfractions (P9-6-1~P9-6-18). Compounds 9 (100.0 mg) and 12 (92.4 mg) were obtained from P9-6-9 and P9-6-14, respectively. Compounds 22 (3.3 mg) and 23 (3.5 mg) were generated from P9-6-2 by performing prep HPLC with Gemini NX-C18 110A column . Compound 11 (48.0 mg) was isolated from P9-6-6 using MPLC with Redi Sep-C18 cartridge . P9-9 was chromatographed over silica gel  to afford 17 pooled fractions (P9-9-1~P9-9-17). Compound 15 (112.3 mg) was obtained by recrystallization with MeOH from P9-9-15.P9 was fractionated using Sephadex LH-20 (5.8 × 50.0 cm) with MeOH:H2O mixture (7:3 v/v) and produced 12 fractions (P10-1~P10-12). P10-9 was further fractionated using silica gel CC , producing compound 10 (34.3 mg).P10 was fractionated using Sephadex LH-20 CC (5.8 × 56.0 cm) with MeOH:H2Cl2:EtOH:H2O = 5:4.5:0.5 v/v/v) and produced 10 subfractions (P13-1~P13-10). P13-5 was further fractionated into P13-5-1~P13-5-16 by GLAS CC  to give yielding compounds 1 (75.8 mg), 2 (158.0 mg), and 3 (25.8 mg).P13 was fractionated using silica gel CC  and gave 16 fractions (P15-1~P15-16). Fraction P15-7 was fractionated into P15-7-1~P15-7-7 by MPLC  to purify compounds 19 (9.7 mg) and 21 (15.5 mg). Fraction P15-13 produced compound 17 (8.3 mg) after using MPLC .P15 was separated by Sephadex LH-20 CC (5.8 × 56.0 cm) with MeOH:H5/mL. After 24 h of incubation, cells were incubated with compounds at concentrations of 6.25, 12.5, 25, and 50 μM and then stimulated with LPS 100 ng/mL. After incubation for 24 h, the cell viability was estimated by MTT assay. A total of 20 μL of MTT solution (5 mg/mL) was added to each well and then incubated for 2 h. The supernatant was removed and remained formazan was dissolved with DMSO. The absorbance was determined by using microplate reader  at 540 nm.The RAW 264.7 macrophage cells were obtained from the Korea Cell Line Bank . These cells were cultured with Dulbecco’s modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin and seeded in 24-well plates at a density of 2 × 105/mL. After incubation for 24 h, cells were pretreated with compounds at concentrations of 6.25, 12.5, 25, and 50 μM for 1 h and then stimulated with LPS 100 ng/mL for 24 h. The cultured media were collected to estimate NO and PGE2 production. To observe the NO production, 100 μL of cultured media was transferred to 96 wells, and then Griess reagent was added to each well. L-NIL (40 μM) was used as a positive control for the NO assay. The estimation of PGE2 production was performed using PGE2 enzyme-linked immunosorbent assay (ELISA) Kit (Enzo Life Sciences). NS398 (10 nM) was treated as a positive control for the PGE2 assay. The absorbance was determined by using microplate reader  at 504 nm and 405 nm for NO production and PGE2 production, respectively.The RAW 264.7 macrophage cells were seeded in 24-well plates at a density of 2 × 105/mL. Cells were pretreated with 50 μM of TCMB (11) for 1 h and then stimulated with LPS 100 ng/mL for 6, 12, and 24 h. After incubation, the cultured media were removed and cells were washed with PBS. Whole protein was extracted by using PRO-PREP protein extraction solution . Extracted proteins were quantified by Bio-Rad protein assay using a reagent . The whole protein (25 μg) was separated by SDS-PAGE using 10% acrylamide gel and then transferred to the PVDF membrane. The blots were incubated with iNOS  and COX-2  antibodies diluted in 5% skim milk with 1:1000 and 1:5000 ratio, respectively. After incubation at 4 °C for 18 h, the blots were washed 3 times using TBS/T. The washed blots were then incubated for 2 h with horseradish peroxidase-conjugated secondary antibody diluted in 5% skim milk with 1:2000 ratio and washed 3 times using TBS/T. The washed blots were developed using an ECL chemiluminescence substrate . The developed blots were then visualized with Amersham Hyperfilm ECL .The RAW 264.7 macrophage cells were seeded in a culture dish at a density of 5 × 105/mL. After incubation for 6 h, cells were pretreated with 12.5, 25, and 50 μM of TCMB (11) for 1 h and then stimulated with LPS 100 ng/mL for 6, 12, and 24 h. The cultured media were removed and cells were washed with PBS. Total mRNA was extracted by using Easy Blue® kits  and synthesized to cDNA using 0.5 mg/mL random oligonucleotide primers  and TOPscriptTM RTDryMIX . PCR amplification was analyzed by using the incorporation of SYBR green  and primers for iNOS , COX-2 , TNF-α , IL-1β , and IL-6 . All primers were purchased from Bioneer .The RAW 264.7 macrophage cells were seeded in a culture dish at a density of 5 × 10p < 0.05 vs. non-LPS-induced control group; * p < 0.05, ** p < 0.01, and *** p < 0.001 as compared to the LPS-only-induced group. p-values below 0.05 are considered significant.All results are expressed as the mean ± SD. Statistical significance was analyzed by using one-way analysis of variance (ANOVA) and Dunnett’s post hoc test using GraphPad Prism software . # 11) was minimized with MMFF94 force field by Chem 3D pro 14.0. Proteins and ligands were prepared using AutoDock 4.2 software . The active sites were determined by previously published data [Molecular docking studies were performed by a previously described method . iNOS ,6′-O-(p-methoxybenzoyl) sucrose ester (TCMB) (11) exhibited significant inhibitory effects on both NO and PGE2 production. Western blot analysis revealed that TCMB (11) effectively downregulated the protein expression of iNOS and COX-2 in dose-dependent manner. Moreover, TCMB (11) showed inhibitory effect on the relative mRNA expression level of iNOS, COX-2, and cytokines  significantly. Taken together, our data imply that TCMB (11) has an anti-inflammatory effect by inhibiting proinflammatory mediators. A molecular docking study showed that TCMB (11) has strong binding affinities with iNOS and COX-2 protein.A total of 23 compounds were obtained from the roots of


Correction: BMC Medicine 20, 445 (2022)https://doi.org/10.1186/s12916-022-02625-5The original article  containe1) Abstract, lines 48 and 49:“Median TDI score on SST was 26.8 (IQR 23.6-29.3) in the placebo group and 28.8 (IQR 24.0-30.9) in the prednisolone group, with a median difference of 2.0 (95% CI 0.75 to 1.5).”to“Median TDI score on SST was 26.8 (IQR 23.6-29.3) in the placebo group and 28.8 (IQR 24.0-30.9) in the prednisolone group, with a median difference of - 1.5 (-3.0 to 0.25).”2) Outcome, line 233-235:“Median TDI score was 26.8 (IQR 23.6- 29.3) in the placebo group and 28.8 (IQR 24.0- 30.9) in the prednisolone group, with a median difference of 2.0 .”to“Median TDI score was 26.8 (IQR 23.6- 29.3) in the placebo group and 28.8 (IQR 24.0- 30.9) in the prednisolone group, with a median difference of - 1.5 (-3.0 to 0.25).”3) Line 237-238:"Self-reported smell function on VAS-score was 3.2 (IQR 1.8- 6.5) in the placebo group and 3.6 (IQR 1.0- 5.8) in the prednisolone group with a median difference of 0.45 ."to"Self-reported smell function on VAS-score was 3.2 (IQR 1.8- 6.5) in the placebo group and 3.6 (IQR 1.0- 5.8) in the prednisolone group with a median difference of 0.3 ."4) Line 240-242:“Self-reported taste function on VAS-score was 5.6 (IQR 2.3- 7.6) in the placebo group and 5.0 (IQR 2.0- 7.8) in the prednisolone group with a median difference of 0.25 .”to“Self-reported taste function on VAS-score was 5.6 (IQR 2.3- 7.6) in the placebo group and 5.0 (IQR 2.0- 7.8) in the prednisolone group with a median difference of 0.1 .”


Tripterygium wilfordii Hook. f. is a well-known traditional Chinese medicine used to treat autoimmune diseases. Sesquiterpene pyridine alkaloids (SPAs) are a major class of components found in this herb that have piqued the interest of researchers due to their complex and diverse structures as well as significant biological activities. In this study, ten new SPAs, wilfordatine A–J (1–10), were isolated from the roots of T. wilfordii, along with ten known analogues (11–20). Their structures were primarily elucidated by extensive 1D and 2D NMR spectroscopic analysis. To search for more immunosuppressive ingredients related to the clinical efficacy of T. wilfordii, the total alkaloids (TA) and compounds 4, 5, and 9–16 were tested for their inhibitory effects on nuclear factor-kappa B (NF-κB) pathway in Lipopolysaccharide (LPS) induced HEK293/NF-κB-Luc cells. Among them, TA, compounds 5, 11, and 16 showed potent immunosuppressive activity, with IC50 values of 7.25 μg/mL, 8.75 μM, 0.74 μM, and 15.66 μM, respectively, and no influence on the cell viability at a concentration of 100 μg/mL (TA) or 100 μM . Accordingly, TA, 5, 11, and 16, especially 11, were identified as promising candidates for further investigation into their potential use as immunosuppressive agents. Tripterygium wilfordii Hook. f., a member of the Celastraceae family, has been used medicinally in China for the treatment of rheumatoid arthritis and other autoimmune diseases for centuries  dicarbolactone dihydro-β-agarofuran and was named wilfordatine A.The relative configuration of 2) was isolated as white amorphous powder. Its molecular formula was deduced to be C34H43NO16 by the HRESIMS data . The 1H- and 13C-NMR data  compared to that (δH 5.37) in 1, indicating that the furanoyloxy group at C-2 in 1 is replaced by a hydroxy group in 2. The relative configuration of 2 was determined to be identical to that of 1 based on the ROESY correlations for H-8/H-1, H-14, H-12/H-3, H-5, and H-11 (2) was identified to be 1β,7β,8β,11-tetraacetoxy-2β,4α,5α-trihydroxy-3α,15-[2′-methyl-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine B (NMR data  of 2 werand H-11  as well 3) was isolated as white amorphous powder with the same molecular formula of compound 2 determined by HRESIMS data . The 1H- and 13C-NMR data of 3 , 3 showed a downfield shift for H-5 (δH 6.70) but an upfield shift for H-7 (δH 4.38), suggesting an acetoxy group at C-5 and a free hydroxy group at C-7, as corroborated by the key HMBC correlation for H-5/δC 169.9 (5-OAc) (3 was determined to be the same as those of 1 and 2 by the ROESY experiment (3) was identified to be 1β,5α,8β,11-tetraacetoxy-2β,4α,7β-trihydroxy-3α,15-[2′-methyl-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine C (ata of 3  were com (5-OAc) . The relperiment . Thus, t4) was isolated as white amorphous powder with a molecular formula of C39H45NO19 determined by HRESIMS data . A comparison of the 1H- and 13C-NMR data , whereas in 4, it was observed as a singlet. Furthermore, 4 showed a downfield shift for C-9′ (δC 78.0) relative to 1 (δC 38.1), indicating that C-9′ in 4 was oxygenated. By analyzing the HMBC spectrum, the four acetoxy groups and the 3-furanoyloxy group were assigned to C-1, C-7, C-8, C-11, and C-2, respectively (4) was identified to be 2β-furanoyloxy-1β,7β,8β,11-tetraacetoxy-4α,5α-dihydroxy-3α,15-[2′-methyl-2′-hydroxy-4′-(3′′-carboxy-2′′ -pyridyl) butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine D (NMR data  between ectively . Accordi5) was isolated as white amorphous powder with a molecular formula of C41H47NO18 determined by HRESIMS data . The 1H- and 13C-NMR data , 7.63 , and 7.50 . The benzoyloxy group was assigned to C-2 based on the HMBC correlations for H-2/δC 164.9 (2-OBz) (5) was identified to be 2β-benzoyloxy-1β,7β,8β,11- tetraacetoxy-4α,5α-dihydroxy-3α,15-[2′-methyl-2′-hydroxy-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine E (NMR data  of 5 wer (2-OBz) . Thus, t6) was isolated as white amorphous powder with a molecular formula of C36H45NO18 determined by HRESIMS data . The 1H- and 13C-NMR data  was identified to be 1β,2β,7β,8β,11-pentaacetoxy-4α,5α-dihydroxy-3α,15-[2′-methyl -2′-hydroxy-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β- agarofuran.Wilfordatine F (NMR data  of 6 wer7) was isolated as white amorphous powder with a molecular formula of C41H51NO19 determined by HRESIMS data . The 1H- and 13C-NMR data , 6.96 , and 1.87 . Furthermore, 7 showed a downfield shift for H-5 (δH 6.95) compared to 5 (δH 5.26), indicating that C-5 was attached with an ester group. Based on the corresponding HMBC correlations, the tigloyloxy group was allocated at C-2, and the extra acetyloxy group was allocated at C-5 (7) was identified to be 1β,5α,7β,8β,11-pentaacetoxy-2β-tigloyoxy-4α-droxy-3α,15-[2′-methyl-2′-hydroxy-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine G  was isolated as white amorphous powder with a molecular formula of C43H53NO20 determined by HRESIMS data . The 1H- and 13C-NMR data  and C-8′ (δC 21.9) were significantly different from those of 7 , indicating that the hydroxy group at C-9′ was esterified. Based on the corresponding HMBC correlations, the six acetoxy groups were allocated at C-1, C-2, C-5, C-7, C-8, and C-11 (8) was identified to be 1β,2β,5α,7β,8β,11-hexaacetoxy-4α -droxy-3α,15-[2′-methyl-2′-tigloyloxy-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine H (NMR data  of 8 werand C-11 . Accordi9) was isolated as white amorphous powder with a molecular formula of C41H45NO20 determined by HRESIMS data . The 1H- and 13C-NMR data  in 9, resulting in about 10 ppm downfield shifts of C-6 (δC 62.2) and C-8 (δC 79.4) in the 13C-NMR spectrum of 9. Additionally, the key HMBC corrections for H-6/C-7 and H-8/C-7 supported the assignment of the keto carbonyl at C-7 , 7.32 , and 6.59 ]. The acetoxy groups were assigned to C-1, C-2, C-8, and C-11 by the corresponding HMBC correlations (C 80.5) compared to that of 8 (δC 81.5). Thus, the structure of wilfordatine I (9) was identified to be 1β,2β,5α,8β,11-pentaacetoxy-4α-droxy-7-oxo-3α,15-[2′-methyl-2′-(3-furoyloxy)-4′-(3′′-carboxy-2′′-pyridyl) butanoic acid] dicarbolactone dihydro-β- agarofuran.Wilfordatine I (NMR data  of 9 werl at C-7 . In addielations . Accordi10) was isolated as white amorphous powder with a molecular formula of C43H47NO19 determined by HRESIMS data . The 1H- and 13C-NMR data , 7.35 , and 7.49 . The positions of the five acetoxy groups were determined by the HMBC experiment, and they were assigned to C-1, C-2, C-5, C-8, and C-11 (10) was identified to be 1β,2β,5α,8β,11- pentaacetoxy-4α-droxy-7-oxo-3α,15-[2′-methyl-2′-(3-benzoyloxy)-4′-(3′′-carboxy-2′′-pyridyl)butanoic acid] dicarbolactone dihydro-β-agarofuran.Wilfordatine J (NMR data  of 10 weand C-11 . AccordiT. wilfordii and identified as tripfordine A (11) + ; 1H- and 13C-NMR data, see Wilfordatine A (2): white amorphous powder; c 0.10, MeOH); UV ε): 224 (3.50), 269 (3.13) nm; HRESIMS m/z 722.2645 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine B (3): white amorphous powder; c 0.03, MeOH); UV ε): 224 (3.96), 269 (3.56) nm; IR (KBr) υmax: 3469, 2922, 2851, 2361, 2342, 1741, 1647, 1585, 1434 1372, 1239, 1162, 1134, 1046, 1006, 883, 604 cm−1; HRESIMS m/z 722.2670 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine C (4): white amorphous powder; c 0.06, MeOH); UV ε): 225 (4.03), 268 (3.63) nm; IR (KBr) υmax: 3408, 2988, 2361, 1747, 1572, 1508, 1442, 1372, 1307, 1257, 1232, 1158, 1136, 1081, 1051, 1002, 952, 917, 874, 760, 713, 603 cm−1; HRESIMS m/z 832.2690 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine D (5): white amorphous powder; c 0.07, MeOH); UV ε): 229 (4.23), 268 (3.63) nm; IR (KBr) υmax: 3411, 2988, 2360, 1747, 1585, 1571, 1451, 1371, 1315,1272, 1252, 1232, 1171, 1141, 1117, 1052, 1003, 913, 870, 745, 712, 600 cm−1; HRESIMS m/z 842.2870 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine E (6): white amorphous powder; c 0.05, MeOH); UV ε): 222 (3.81), 269 (3.52) nm; IR (KBr) υmax: 3422, 2989, 1748, 1584, 1437, 1373, 1261, 1235, 1142, 1047, 1002, 946, 914, 870, 833, 763, 621, 603 cm−1; HRESIMS m/z 780.2752 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine F (7): white amorphous powder; c 0.06, MeOH); UV ε): 221 (4.22), 269 (3.58) nm; IR (KBr) υmax: 3466, 2994, 2934, 1748, 1648, 1571, 1441, 1371, 1314, 1232, 1095, 1054, 1007, 939, 883, 765, 731, 601 cm−1; HRESIMS m/z 862.3159 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine G (8): white amorphous powder; c 0.05, MeOH); UV ε): 220 (3.52), 266 (4.21) nm; IR (KBr) υmax: 3556, 3477, 3153, 3006, 2963, 1758, 1586, 1572, 1509, 1434, 1372, 1318, 1219, 1177, 1159, 1083, 1042, 1008, 969, 915, 874, 764, 628, 609 cm−1; HRESIMS m/z 904.3234 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine H (9): white amorphous powder; c 0.06, MeOH); UV ε): 225 (3.96), 263 (3.60) nm; IR (KBr) υmax: 3547, 3484, 2998, 2963, 2361, 2343, 1758, 1724, 1572, 1508, 1436, 1373, 1319, 1219, 1177, 1143, 1083, 1042, 1008, 969, 874, 765, 629, 604 cm−1; HRESIMS m/z 872.2642 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine I (10): white amorphous powder; c 0.06, MeOH); UV ε): 229 (4.21), 268 (3.67) nm; IR (KBr) υmax: 3548, 3483, 2998, 2943, 1756, 1725, 1586, 1569, 1452, 1437, 1372, 1288, 1248, 1218, 1135, 1084, 1042, 1008, 942, 914, 868, 760, 716, 628, 592 cm−1; HRESIMS m/z 882.2830 [M + H]+ ; 1H- and 13C-NMR data, see Wilfordatine J  was added into each well and continuously incubated for 2 h at 37 °C. Absorbance at a wavelength of 450 nm was measured by a microplate reader, which was further used to calculate the cell viability. All the experiments were performed in triplicate.The viability of HEK293 cells was determined by a CCK-8 assay according to the established protocol . BrieflyThe NF-κB inhibitory activities were measured using the HEK293/NF-κB-Luc cells, which were generated as described previously . BrieflyT. wilfordii. Their structures were mainly elucidated by spectroscopic analysis. The total alkaloids and compounds 5, 11, and 16 were found to have potent NF-κB inhibitory effects with IC50 values at μg/mL or μM level and no effects on the cell viability. The results obtained in this study highlight the bioactive potential of SPAs, which can be used as promising compounds for future optimization and development of potential immunosuppressive agents.In summary, 20 wilfordate-type SPAs, including 10 new ones and 10 known ones, were obtained from the roots of


These promising analogues were further examined for their inhibitory assessment against EGFR, VEGFR-2 and CDK-2/cyclin A2 kinases. The coumarin-tetrazole 10 displayed broad superior inhibitory activity against all screened enzymes compared with the reference drugs, erlotinib, sorafenib and roscovitine, respectively. The impact of coumarin-tetrazole 10 upon cell cycle and apoptosis induction was determined to detect its mechanism of action. Additionally, it upregulated the levels of casp-3, casp-7 and cytochrome-c proteins and downregulated the PD-1 level. Finally, molecular docking study was simulated to afford better rationalization and gain insight into the binding affinity between the promising derivatives and their targeted enzymes, which could be used as an optimum lead for further modification in the anticancer field.This study represents the design and synthesis of a new set of triazole-coumarin-glycosyl hybrids and their tetrazole hybrid analogues possessing various sugar moieties and modified analogues. All the newly synthesized derivatives were screened for their cytotoxic activities against a panel of human cancer cell lines. The coumarin derivatives  Cancer is one of the most serious and complicated health disorders all over the world . Owing tSeveral kinases function as an on/off switch for cellular motility and proliferation. Thus, mutations of these kinases are responsible for cellular irregularities affording cancer initiation, metastasis or progression . MultiplOne of the most prominent oncogenic kinases is epidermal growth factor receptor (EGFR), which is a receptor tyrosine kinase and plays an essential mediating role in cell proliferation, angiogenesis, apoptosis and metastatic spread . It is oIt has been apparent that unresponsive cancer cases that require integration therapy  involvinI–IV have been well reported and characterized by their potent anticancer activity through inhibition of EGFR, VEGFR-2 or CDK-2 kinases  gave the title products 13–15, respectively.d-glucopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-. Yield: 71%; mp 131–132 °C; IR (KBr) cm−1, ν: 3070 (C-H), 2924 (C-H), 1720 (C=O), 1749 C=O); 1H NMR (DMSO-d6) δ/ppm: 1.98, 1.99, 2.03, 2.04 , 3.62–3.77 , 4.16- 4.25 , 4.49–4.54 , 5.23–5.29 , 5.23–5.26 , 5.55 , 6.12 , 6.19 , 7.09–7.17 , 7.32 , 7.50 , 7.68–7.78 . Anal. calcd. for C32H31N3O12: C, 59.17; H, 4.81; N, 6.47. Found: C, 59.02; H, 4.86; N, 6.41.d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-. Yield: 69%; m.p. 93–94 °C; IR (KBr) cm−1, ν: 3055 (C-H), 2922 (C-H), 1715 (C=O), 1747 (C=O); 1H NMR (DMSO-d6) δ/ppm: 1.92, 1.95, 1.97, 2.01 , 3.94–3.99 , 4.14- 4.20 , 4.87–4.98 , 5.28 , 5.32–5.34 , 5.43–5.48 , 5.58–5.64 , 6.19 , 6.30 , 7.09–7.16 , 7.32 , 7.51 , 7.66–7.78 . 13C NMR (DMSO-d6) δ/ppm: 19.2, 20.4, 20.9, 21.0 (4CH3), 62.1 (C-6′), 68.3 (C-4′), 67.8 (CH2), 70.9 (C-2′), 73.5 (C-3′), 79.7 (C-5′), 99.0 (C-1′), 102.6, 102.7, 112.0, 112.6, 113.5. 128.3, 128.9, 129.4, 130.2, 135.4, 143.1, 155.6, 155.9, 160.5 , 161.5 (coumarin-C2), 170.0, 170.4, 170.5 (4C=O). Anal. calcd. for C32H31N3O12: C, 59.17; H, 4.81; N, 6.47. Found: C, 59.29; H, 4.88; N, 6.38.d-xylopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-. Yield: 65%; mp 89–90 °C; IR (KBr) cm−1, ν: 3058 (C-H), 2923 (C-H), 1718 (C=O), 1745 (C=O); 1H NMR (DMSO-d6) δ/ppm: 1.96, 1.99, 2.03 , 3.74–3.76 , 3.78–3.80 , 4.01–4.11 , 5.25 , 5.48–5.55 , 5.81–5.95 , 6.38 , 7.02–7.12 , 7.33 , 7.50 , 7.67–7.92 . Anal. calcd. for C29H27N3O10: C, 60.31; H, 4.71; N, 7.28. Found: C, 60.42; H, 4.65; N, 7.39.d-glycopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-((1-(β- (16–18). The O-acetylated-1,2,3-triazole glycosidic compounds 13–15 (5 mmol) was added portion wise while stirring to dry methanol saturated with gaseous ammonia (20 mL) at 0 °C. The reaction mixture was stirred at room temperature for a further 7 h. After completion of the deacetylation process , the solvent was evaporated under reduced pressure at 40 °C to give a residue, which, upon treatment with cold diethyl ether (25 mL), afforded a solid product that was filtered and dried. d-glucopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-((1-(β- (16). Yield: 70%; mp 180–182 °C; IR (KBr) cm−1, ν: 3427–3400 (OH), 3070 (C-H), 2922 (C-H), 1716 (C=O); 1H NMR (DMSO-d6) δ/ppm: 3.50–3.55 , 3.60–3.66 , 3.72–3.75 , 4.09–4.12 , 5.06–5.17 , 5.22–5.28 , 5.77 , 6.21 , 7.10–7.18 , 7.37 , 7.52 , 7.81–7.98 . 13C NMR (DMSO-d6) δ/ppm: 62.1 (C-6′), 79.1 (CH2), 72.6 (C-4′), 77.5 (C-2′), 80.53 (C-3′), 88.1 (C-5′), 102.0 (C-1′), 112.1, 112.6, 113.4, 113.4, 124.7, 128.9, 129.4, 130.2, 135.4, 142.4, 155.6, 155.9, 160.5 , 161.7 (coumarin-C2) Anal. calcd. for C24H23N3O8: C, 59.87; H, 4.82; N, 8.73. Found: 59.71; H, 4.75; N, 8.82.d-galactopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-((1-(β- (17). Yield: 71%; mp 185–186 °C; IR (KBr) cm−1, ν: 3422–3390 (OH), 3055 (C-H), 2924 (C-H), 1713 (C=O); 1H NMR (DMSO-d6) δ/ppm: 3.52–3.58 , 3.63–3.69 , 3.73–378 , 4.08–4.11 , 5.04–5.22 , 5.25–5.31 , 5.79 , 6.21 , 7.15–7.22 , 7.39 , 7.51 , 7.73–7.90 .13C NMR (DMSO-d6) δ/ppm: 62.3 (C-6′), 69.5 (CH2), 74.3 (C-4′), 78.9 (C-2′), 79.4.7 (C-3′), 88.7 (C-5′), 102.6 (C-1′), 102.8, 103.2, 112.0, 112.2, 113.4, 128.3, 128.9,129.3, 130.1, 135.4, 135.7, 155.6, 155.9, 160.4 , 160.8 (coumarin-C2) Anal. calcd. for C24H23N3O8: C, 59.87; H, 4.82; N, 8.73. Found: 60.02; H, 4.89; N, 8.64.d-Xylopyranosyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one4-Phenyl-7-((1-(β- (18). Yield: 69%; mp 177–178 °C; IR (KBr) cm−1, ν: 3420–3395 (OH), 3048 (C-H), 2927 (C-H), 1711 (C=O); 1H NMR (DMSO-d6) δ/ppm: 3.74–3.79 , 3.93–3.98 , 4.08–4.12 , 4.40–4.48 , 4.93–4.95 , 5.18–5.31 , 5.92 , 6.22 , 7.11–7.19 , 7.37 , 7.55 , 7.74–7.91 . Anal. calcd. for C23H21N3O7: C, 61.19; H, 4.69; N, 9.31. Found: 61.27; H, 4.61; N, 9.42.Human melanoma (A-375), melanoma (Mel-501), colon carcinoma (Caco-2), prostate cancer (PC-3) and pancreatic cancer (Paca-2) were obtained from Karolinska Centre, Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden. 1–18 were assessed using the 3-[4,5- dimethyl-2-thiazolyl)-2,5-diphenyl-2 H-tetrazolium bromide (MTT) assay, following the previously reported method [The cytotoxic screening of all synthesized derivatives d method ,62,63. 3 cells per well in case of PC-3, 20 × 103 cells/well in a fresh complete growth medium in case of A-375, caco2, Paca-2 and Mel-501 at 37 °C for 24 h in water-jacketed carbon dioxide incubator. After 48 h incubation, the medium was aspirated and then 40 μL MTT salt (2.5 mg/mL) was added for a further 4 h. Then, 200 μL 10% sodium dodecyl sulphate (SDS) in deionized water was added to each well and incubated overnight at 37 °C. The absorbance was measured at 595 nm with reference 690 nm. Doxorubicin was used as the positive control and 0.5% DMSO was used as the negative control. The IC50 values were calculated, using a probit analysis, and by utilizing the SPSS computer program .Following culturing for 10 days, the cells were seeded at a concentration of 10 × 1010, 13 and 15 were evaluated for their in vitro inhibitory activity against EGFR, VEGFR-2 and CDK-2/cyclin A2 using erlotinib, sorafenib and roscovitine as references, respectively, following the previously mentioned procedure [The targets with promising cytotoxicity rocedure ,65. EGFR assay: The master mixture (6 μL 5X Kinase Buffer + 1 μL ATP (500 μM) + 1 μL 50 X PTK substrate + 17 μL water) was prepared, then 25 μL to every well was added along with 5 μL of inhibitor solution of each well labelled as “Test Inhibitor”. However, for the “Positive Control” and “Blank”, 5 μL of the same solution without inhibitor (Inhibitor buffer) was added. Then, 3 mL of 1X Kinase Buffer by mixing 600 μL of 5X Kinase Buffer with 2400 μL water was prepared. So, 3 mL of 1X Kinase Buffer became sufficient for 100 reactions. To the wells designated as “Blank”, 20 μL of 1X Kinase Buffer was added. EGFR enzyme on ice was thawed. Upon first thaw, briefly, the tube containing the enzyme was spun to recover the full content of the tube. The amount of EGFR required for the assay and dilute enzyme to 1 ng/μL with 1X Kinase Buffer was calculated. Moreover, the remaining undiluted enzyme in aliquots was stored at −80 °C. The reaction was initiated by adding 20 μL of diluted EGFR enzyme to the wells designated “Positive Control” and “Test Inhibitor Control”, after that it was incubated at 30 °C for 40 min. After the 40 min reaction, 50 μL of Kinase-Glo Max reagent was added to each well and the plate was covered with aluminium foil and incubated at room temperature for 15 min. Luminescence was measured using the microplate reader.1 and 3c on the level of VEGFR-2 in human breast cancer cell line MCF-7 was determined. The cells in culture medium were treated with 20 μL of IC50 values of the compounds dissolved in DMSO, then incubated for 24 h at 37 °C, in a humidified 5% CO2 atmosphere. The cells were harvested, and the homogenates were prepared in saline using a tight pestle homogenizer until complete cell disruption. The kit used a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) to determine the level of human VEGFR-2 in samples. A monoclonal antibody for VEGFR-2 was pre-coated onto 96-well plates. The test samples were added to the wells and a biotinylated detection polyclonal antibody from goat specific for VEGFR-2 was added, subsequently followed by washing with PBS buffer. Avidin–Biotin–Peroxidase complex was added, and the unbound conjugates were washed away with PBS buffer. HRP substrate TMB was used to visualize HRP enzymatic reaction. TMB was catalysed by HRP to produce a blue colour product that changed into yellow after the addition of acidic stop solution. The density of yellow colour is proportional to the human VEGFR-2 amount of the sample captured in the plate. The chroma of colour and the concentration of the human VEGFR-2 of the samples were positively correlated and the optical density was determined at 450 nm. The level of human VEGFR-2 in samples was calculated (pg/mL) as duplicate determinations from the standard curve. The % inhibition was calculated in comparison to control untreated cells.VEGFR-2 assay: Additionally, the effect of the most promising cytotoxic compounds Assay of CDK-2/cyclin A2 was achieved through ELISA using an affinity tag labelled capture antibody. Adding the samples and the standard to the wells was carried out after the addition of the antibody mix. After the incubation period, the unrestrained substance was discarded and the wells were washed. The added TMB  substrate was prompted by Horseradish peroxidase (HRP) and blue coloration appeared. This reaction was stopped with the addition of stop solution to complete the change in colour from blue to yellow. The created signals were equivalent to the quantity of bound analyte and Robonik P2000 ELISA reader was used to record the intensity at a certain wavelength (450 nm). The concentrations of the screened compounds were calculated through the plotted curve.4 and incubated at 37 °C in 5% CO2 overnight. After treatment with the tested compound 10 for 24 h, cell pellets were collected and centrifuged . For cell cycle analysis, cell pellets were fixed with 70% ethanol on ice for 15 min and collected again. The pellets were incubated with propidium iodide (PI) staining solution at room temperature for 1 h and analysed by a Gallios flow cytometer . Apoptosis detection was carried out by FITC AnnexinV/PI commercial kit  following the manufacturer’s protocol. The samples were analysed by fluorescence-activated cell sorting (FACS) with a Gallios flow cytometer  within 1 h after staining. Data were analysed using Kaluza v 1.2 .Apoptosis investigation and cell cycle analysis were elucidated by flow cytometry . Paca-2 The precoated micro-ELISA plate with an antibody specific to Caspase-3 was applied using the Human Caspase-3 ELISA Kit from Invitrogen INC. Catalog # KHO1091 (96 tests)  according to the manufacturer’s instructions .The micro-ELISA plate provided in this kit was pre-coated with a Caspase-7-specific antibody. A biotinylated Caspase-7 antibody and Avidin–horseradish peroxidase (HRP) conjugate was added. The excess components were removed. The substrate solution was added. Wells containing Caspase-7, biotinylated detection antibody, and Avidin-HRP conjugate appeared blue in colour. The colour turned yellow following the addition of sulphuric acid solution. The optical density (OD) was measured at a wavelength of 450 ± 2 nm.Cytochrome-c specific antibodies were precoated onto 96-well plates. Cytochrome-c were analysed by enzyme-linked immunosorbent assays (ELISAs) using commercial kits . The optimal dilutions were determined and ELISAs were performed according to the manufacturer’s instructions.Samples or standards were added to the wells Human PD-1 eliza kit , followed by the antibody mix. After incubation, the wells were washed to remove unbound material. TMB Development Solution was added and during incubation was catalysed by HRP, generating blue coloration. This reaction was then stopped by the addition of Stop Solution, completing any colour change from blue to yellow. The optical density (OD) was measured at a wavelength of 450 ± 2 nm.10 with the highest inhibitory activity were examined within the active sites of EGFR, VEGFR-2 and CDK-2/cyclin A2  [13 and 15 were studied against CDK-2/cyclin A2. Molecular docking is a powerful computational process to rationalize gained biological data. Interactions between the newly synthesized target ctively) ,71,72 usctively) ,69,70. F10, 13 and 15 were drawn through chem. Draw. The protonated 3D was employed using standard bond lengths and angles. Then, the geometry optimization and energy minimization were applied to obtain the Conf Search module in MOE, followed by saving of the MOE file for the upcoming docking process. The co-crystallized structures of EGFR, VEGFR-2 and CDK-2/cyclin A2 kinases with their ligands erlotinib, sorafenib and roscovitine were downloaded  from the protein data bank. All minimizations were performed using MOE until an RMSD gradient of 0.05 kcal∙mol−1Å−1 with MMFF94x force field and the partial charges was automatically calculated. The preparation of the enzyme structures was carried out for molecular docking using Protonate 3D protocol with the default options in MOE. London dG scoring function and the Triangle Matcher placement method were used in the docking protocol. At first, validation of the docking processes was established by docking the native ligands, followed by docking of the derivatives 10, 13 and 15 within the ATP-binding sites after the elimination of the co-crystallized ligands.The 2D structure of the newly synthesized derivatives 10, 13 and 15 derivatives against Paca-2, Mel-501, PC-3 and A-375 cancer cell lines were further assessed for their inhibitory activities against EGFR, VEGFR-2 and CDK-2/cyclin A2 kinases to detect their mechanism of action. The coumarin-tetrazole 10 illustrated excellent broad inhibitory activity in comparison with the reference drugs, erlotinib, sorafenib and roscovitine, respectively. The molecular docking simulation was carried out to confirm these promising enzymatic results. Moreover, mechanistic studies found that the coumarin-tetrazole 10 arrested the cell cycle at pre-G 1 and S phases and induced apoptosis through upregulating casp-3, casp-7 and cytochrome-c proteins and downregulating PD-1 levels, in comparison with the untreated MCF-7 cells. Finally, this study may effectively promote further achievements in the field of discovering anticancer drug candidates.The present work afforded the design and synthesis of a new set of triazole-coumarin-glycosyl hybrids and their tetrazole analogues. The antiproliferative activity of these derivatives were carried out against human Paca-2, Mel-501, PC-3, A-375 and Caco-2 cancer cell lines through colorimetric MTT assay and compared with the standard drug, doxorubicin. The promising coumarin derivatives


Inula britannica, and the structure was determined using spectroscopic and chemical methods. The antimelanogenic effects of inularin on B16F10 melanoma cells and zebrafish embryos were evaluated. Inularin dose-dependently reduced melanocyte-stimulating hormone-induced melanin production and L-DOPA oxidation in B16F10 cells. Zebrafish embryos were used to confirm the antimelanogenic activity. Inularin significantly decreased the pigmentation of embryos compared with untreated controls.In the search for novel, natural melanogenesis inhibitors, a new sesquiterpene, inularin, was isolated from the flowers of  Melanogenesis is a complex process by which melanin is produced by melanocytes in melanosomes . MelaninInula britannica Linnaeus (Asteraceae) is well known in China (“Xuan-Fu-Hua”), as its flowers are used in traditional Chinese medicine to treat digestive disorders, bronchitis, and inflammation + on high-resolution electron ionization mass spectrometry (HRESIMS), corresponding to a molecular formula of C17H26O6. The 1H-NMR spectrum displayed the characteristic signals of paired olefinic protons at δH 6.28  and 5.68 , two oxygenated methine protons at δH 5.29  and 3.28 , two singlet methyls at δH 1.17  and 1.03 , and an acetoxymethyl at δH 1.95   correlations of inularin established two segments: CH(1)-CH2(2)-CH2(3) and CH(5)-CH2(6)-CH(7)-CH(8)-CH2(9) (H 5.29 and 3.28 were assigned to H-8 and H-1 by the heteronuclear multiple bond correlation (HMBC) correlations of δH 5.29 with C-7 (δC 44.2), C-9 (δC 45.5), and C-10 (δC 40.4) and those of δH 3.28 with C-2 (δC 29.0), C-3 (δC 41.9), and C-10 (δC 40.4) (H 1.17 (H3-15) with C-4 (δC 72.3) and of δH 5.29 (H-8) with the acetoxy carbon (δC 172.2) indicated that the other hydroxy group was attached to C-4, and an acetoxy moiety was linked to C-8, respectively. The presence of an α-methylene-carboxylic acid was inferred by carbon signals at δC 170.0  and 143.2 and 126.1  and the HMBC correlation between paired olefinic protons at δH 6.28 (H-13a) and 5.68 (H-13b) and the carboxylic carbon at δC 170.0 (C-12). Attachment of an α-methylene-carboxylic acid moiety to C-7 was indicated by the HMBC cross-peaks of H-7 with C-11 and C-12, and H-13 with C-7. The relative configuration of inularin was determined based on nuclear Overhauser effect spectroscopy (NOESY) data and comparisons of coupling constants with data from the literature. The coupling constant of H-5  suggested a trans-fused eudesmane skeleton [J = 14.4, 3.2 Hz) and H-9β  indicated that H-8 placed in ring B in an equatorial geometry, indicating a β-configuration of the acetoxy group at C-8. The absolute configuration of inularin was considered to be 1R, 4R, 5R, 7R, 8R, 10R, as established for the co-occurring analogue eudesmolides of known absolute stereostructure [1R, 4R, 5R, 7R, 8R, 10R)-1β,4α-dihydroxy-8β-acetoxy-5αH-eudesma-11(13)-en-12-oic acid.An ethanol extract of inularin . The mat  . The 13Cspectrum , combine)-CH29) . Two oxyδC 40.4) . The HMB . Two oxWe used B16F10 cells to investigate the effect of inularin on melanogenesis. The MTT (cell viability) assay showed that inularin did not affect viability at concentrations up to 100 μM (data not shown). B16F10 cells were treated with inularin at concentrations of 10, 50, and 100 μM. As shown in The zebrafish serves as a useful model for phenotype-based screening of melanogenic inhibitors or stimulators . We explInula species. In the Inula species, most sesquiterpenes occur in a lactonized form and exert various biological properties, including anti-inflammatory, antibacterial, anticancer, and cytotoxic activities [O-acetylbritannilactone and britannilactone, isolated from I. britannica flowers have been reported to exhibit antimelanogenic activities in vitro [I. britannica flowers and inularin are promising therapeutics for melanogenesis-related diseases. The specific mechanisms involved require further study.Sesquiterpenes are the 15-carbon subgroup of terpenoids and characteristic of tivities , 13. It tivities , 15. Thuin vitro . These r


Moreover, it exhibits an interesting action on inflammation-related cytokine IL-6.Fourteen triazole-scaffold derivatives were synthetized and biologically evaluated as potential oncoimmunomodultator agents by targeting both PD-L1 and c-Myc. First, the antiproliferative activity of these molecules on the monocultures of several tumor cell lines  and on the non-tumor cell line HEK-293 was studied. Then, the effects on the mentioned biological targets were also evaluated. Finally, the effect on cancer cell viability when the molecules were co-cultured with immune cells (Jurkat T cells or THP-1) was also determined. Compounds bearing a bromoophenyl group were selected because of their excellent results, and their effect on IL-6 secretion was also studied. In conclusion, we found compounds that are capable of downregulating c-Myc, as well as influencing and altering the distribution of PD-L1 in tumor cells; the compounds are thus capable of influencing the behavior of defensive cells towards cancer cells.  Tumors are not just masses of isolated cancer cells, as they are able to interact with the surrounding cells and with the extracellular matrix, thus generating the tumor’s microenvironment (TME). In a tumor microenvironment, the extracellular matrix serves as a support on which cells can adhere and grow . FurtherFor the last ten years, our research has been focused on the development of potential anticancer agents for application in targeted therapies ,13,14. MUsing antagonist antibodies to block PD-1/PD-L1 interaction has been a breakthrough in cancer treatment, to such an extent that it has become the gold standard of treatment for some types of cancer . For exaTo date, the PD-1/PD-L1 pathway inhibitors available in clinical practice have all been mAbs. However, despite the amazing initial therapeutic response, a durable response has not yet proved to be as encouraging, and prolonged survival is not promising. In addition, monoclonal antibodies present clinical disadvantages, such as in their safety, low oral bioavailability, and difficult and expensive production, which limit their clinical use. Therefore, the development of small-molecule inhibitors is a very promising area of work, as these would allow for overcoming the above drawbacks. In this vein, small-molecule agents that modulate the PD-1/PD-L1 axis have already been described in the literature and are being evaluated in preclinical and clinical trials. For example, CA-170 is a small-molecule inhibitor of the PD-1/PD-L1 axis that is designed from a peptide derived from a region located at the interface of PD-1 and PD-L1. It was the first small-molecule inhibitor of PD-L1 to enter clinical trials in 2016 for the treatment of lymphoma and several advanced solid tumors . AnotherThe development of these molecules had its starting point in a study carried out by P. Holak’s group from Bristol-Meyers Squibb, which resulted in the elucidation of the crystal structures of the PD-L1 dimer complex with BMS small-molecule inhibitors , A-549 (pulmonary adenocarcinoma), and MCF-7 (breast adenocarcinoma), as well as towards the non-tumor cell line HEK-293 (human embryonic kidney cells). Neither of the compounds were active, and we found that all of them exhibited IC50 values above 300 µM in all tested cell lines.The effect of triazole derivatives ®647 and anti-c-Myc conjugated to fluorescein to determine the amount of both targets related to the negative control. The effect of these triazole derivates on c-Myc and PD-L1 was measured in three different cancer cell lines  by flow cytometry. Thus, cells were incubated for 24 h with the corresponding compound at a concentration of 100 μM. Dimethyl sulfoxide (DMSO)-treated cells were used as the negative control, and a PD-L1 inhibitor, BMS-8 , was use3, 5, 7) were the most active as dual inhibitors in the three cancer cell lines. In addition, the results clarify that compounds bearing the protecting group 1, 3, 5, 7, 9, 11 and 13 have a greater inhibitory activity than the deprotected derivatives.As can be seen from 3, 5 and 7, we found that m-MeO derivative 11 inhibited this target by half in HT-29 and in A-549 . In this last cell line, A-549, methoxy derivatives 9 and 13 were also quite active, with inhibition rates of 40 and 50%, respectively. As regards the action on total PD-L1, the protected compounds were the most active in the HT-29 and in MCF-7 cell lines, although the effect in this latter line was very weak (around 10%), whereas in HT-29, the inhibitory effect reached to 50–60%. On the other hand, the unprotected compounds were the most active ones in the A-549 cell line, with inhibitions ranging from 50–60%. Even so, the p-Br protected 3 was as active as the unprotected 4.As regards the action on c-Myc, in addition to 3, 5 and 7 on both targets in MCF-7 by immunofluorescence microscopy , 6 (m-Br), 8 (o-Br), 12 (m-MeO), and 14 (o-MeO), with inhibition rates around 30%. In addition, we observed that protected and unprotected p-Br derivatives 3 and 4 were both active, whereas protected and unprotected p-OMe derivatives 9 and 10 showed no activity. The remaining compounds had no effect on the A-549 cell line. From these observations, we can assume that the more polar the molecule (free hydroxy group), the greater the effect on A-549 cell viability when they are cultured together with Jurkat T cells. On the other hand, the effect on HT-29 cell viability when co-cultured with Jurkat T cells was higher than that on A-549. In this case, the protected derivatives, 5 (m-Br), 7 (o-Br), 9 (p- MeO), and 11 (m- MeO), were more active than the unprotected ones, showing inhibition rates on cell proliferation of around 50–40%. Contrary to that which we observed for A-549, in the HT-29 cell line, both protected and deprotected p-Br derivatives 3 and 4 were not active, whereas m-MeO derivatives 11 and 12 were both active, showing around 40% inhibition rates. The most active compound was derivative 7 (o-Br), showing around 50% inhibition of cell viability. Finally, on the MCF-7 cell line, all the compounds showed relatively high inhibition rates when co-cultured with Jurkat T cells. In general, protected derivatives were more active than deprotected ones, with inhibition rates of around 60% and 30%, respectively. The most active derivative was protected compound 5 (m-Br 5), with a 75% inhibition rate, followed by p- and o-Br derivatives 3 and 7, and both o-MeO 13 and 14, which inhibited almost 60% of cancer cell viability in the presence of Jurkat T cells. Derivative 11 (m-MeO) was also outstanding; it was able to decrease cell viability in half.The effect on A-549 cell viability when co-cultured with Jurkat T cells was moderate for the unprotected derivatives ®647. We took advantage of the versatility of flow cytometry to simultaneously measure the expression of membrane VEGFR-2 by using anti-VEGFR-2 conjugated to fluorescein. We determined by flow cytometry the effect of these compounds on the expression of membrane PD-L1 in cancer cells when they were co-cultured with immune cells. Thus, interferon γ-stimulated cancer cells were treated for 24 h with the compounds at 100 μM in presence of Jurkat T cells. Then, cells were fixed and treated with anti-PD-L1 conjugated to AlexaFluor4, 6 and 8, together with protected p-MeO 9 and both o-MeO derivatives 13 and 14, exerted a higher action, showing membrane PD-L1 inhibition rates of about 40–50%. As regards the effect on PD-L1 in MCF-7, protected bromo derivatives 3, 5 and 7, together with methoxy derivatives 9–13, exerted excellent inhibition rates, with values ranging from 60–70%. This could explain why MCF-7 was the most sensitive cell line to the effect of the synthetic compounds in enhancing T cells’ antiproliferative action towards this cancer cell line . Assays were performed by ELISA using cell media after treatments with selected compounds at 100 μM for 24 h.The results in Here, we describe the synthesis and biological evaluation of new entities bearing a triazole core as shown in As shown in the results description and in 3–14 on each of the targets and the viability of tumor cells in the presence of T cells for the three cell lines HT-29 (a), MCF-7 (b), and A-549 (c). As regards the effect on HT-29, we can say that in general, there is a good correlation between the inhibition of cancer cell viability and total c-Myc and total PD-L1, with protected derivatives being more active than deprotected ones.To establish correlations between the effect on the targets and the activation of the immune cells towards the tumor cells, in 3–8, bearing electron-withdrawing substituents, cell viability becomes lower; with compounds 5 or 7, so do total PD-L1 and c-Myc, with inhibition rates from moderate to good. Better results were observed on the MCF-7 cell line. In this case, the correlation between the effect on cancer cell viability by the promotion of T cells and the effect on the targets is clearer. Again, bromo derivatives fit better in this correlation than methoxy ones, and we can highlight compounds 3, 5, and 7 because of their higher effect on this cell line. Contrary to that observed for HT-29 and MCF-7, in the case of A-549, the correlation is unclear, and this could be attributed to the very mild effect of the compounds in this particular cell line.This correlation fits better for bromo than for methoxy compounds. That is, with derivatives 3, 5, and 7, are able to regulate not only PD-L1 but also c-Myc expression. This effect on the inhibition of both c-Myc and PD-L1 has been previously observed and described in the literature for other small molecules. For example, THZ1 +. Calc. for C18H19N3O 294.1606H-1,2,3-triazole 3:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl)-4-(4-bromophenyl)-1 yield 56%; colorless oil; 1H NMR  δ 8.54 , 7.72 , 7.59 , 7.26–7.11 , 6.83 , 4.64 , 3.72 , 3.19 , 3.10 , 2.77 ; 13C NMR  δ 157.95 (2xC), 145.05 (C), 137.43 (C), 135.86 (2xC), 135.24 (C), 131.80 (2xCH), 130.02 (C), 129.54 (4xCH), 129.09 (CH), 128.48 (CH), 127.72 (CH), 127.59 (CH), 127.00 (2xCH), 126.52 (CH), 121.60 (CH), 120.67 (C), 113.07 (8xCH), 85.37 (C), 64.35 (CH2), 59.72 (C), 54.98 (2xCH3), 50.68 (CH2), 35.49 (CH2), 35.13 (CH2); HR ESMS m/z 696.1835 [M-Na]+. Calc. for C39H36N3O3Br 696.1833.H-1,2,3-triazol-1-yl)ethyl)phenyl)ethan-1-ol 4:2-(4-(2-(4-(4-bromophenyl)-1 yield 52%; white solid; m.p. 135–140 °C; 1H NMR  δ 8.57 , 7.76 , 7.64 , 7.12 , 4.63 , 4.57  3.56 , 3.17 , 2.67 ; 13C NMR  δ 145.04 (C), 137.77 (C), 134,91 (C), 131.82 (2xCH), 130.05 (C), 128.91 (2xCH), 128.40 (2xCH), 127.01 (2xCH), 121.62 (CH), 120.67 (C), 62,07 (CH2), 50,68 (CH2), 35.10 (CH2), 30.68 (CH2); HR ESMS m/z 372.0710 [M-H]+. Calc. for C18H18N3OBr 372.0711.H-1,2,3-triazole 5:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl)-4-(3-bromophenyl)-1 yield 70%; colorless oil; 1H NMR  δ 8.53 , 7.99 , 7.78 , 7.51 , 7.37 , 7.26–7.09 , 6.84 , 4.64 , 3.72 , 3.19 , 3.10 , 2.77 ;13C NMR  δ 157.96 (2xC), 145.03 (C), 144.67 (C),137.43 (C), 135.86 (2xC), 135.22 (C), 133.13 (C), 131.10 (CH), 130.41 (CH), 129.56 (4xCH), 129.09 (CH), 128.49 (CH), 127.73 (CH), 127.60 (CH), 127.49 (CH), 126.52 (CH), 123.93 (CH), 122.24 (C), 121.99 (CH), 113.08 (8xCH), 85.38 (C), 64.37 (CH2), 54.99 (2xCH3), 50.74 (CH2), 35.50 (CH2), 35.07 (CH2); HR ESMS m/z 696.1835 [M-Na]+. Calc. for C39H36N3O3Br 696.1833.H-1,2,3-triazol-1-yl)ethyl)phenyl)ethan-1-ol 6:2-(4-(2-(4-(3-bromophenyl)-1 yield 64%; white solid; m.p. 129–131 °C; 1H NMR  δ 8.63 , 7.99 , 7.82 , 7.51 , 7.41 , 7.12 , 4.62 , 4.57  3.56 , 3.17 , 2.66 ; 13C NMR  δ 144.63 (C), 137.77 (C), 134,89 (C), 133.13 (CH), 131.12 (CH), 130.41 (C), 128.91 (2xCH), 128.40 (2xCH), 127.01 (CH), 123.91 (CH), 122.21 (CH), 121.98 (C), 62,07 (CH2), 50,74 (CH2), 38.59 (CH2), 35.05 (CH2); HR ESMS m/z 372.0708 [M-H]+. Calc. for C18H18N3OBr 372.0711.H-1,2,3-triazole 7:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl)-4-(2-bromophenyl)-1 yield 40%; colorless oil; 1H NMR  δ 8.49 , 7.87 , 7.70 , 7.46 , 7.30–7.20 , 6.83 , 4.68 , 3.72 , 3.18 , 3.11 , 2.77 ; 13C NMR  δ 157.95 (2xC), 145.03 (C), 143.85 (C),137.33 (C), 135.87 (2xC), 135.27 (C), 133.42 (CH), 131.38 (C), 130.32 (CH), 129.67 (CH), 129.56 (4xCH), 129.05 (CH), 128.58 (CH), 127.92 (CH), 127.73 (CH), 127.61 (CH), 126.53 (CH), 124.01 (CH), 120.58 (C), 113.09 (8xCH), 85.41 (C), 64.44 (CH2), 54.98 (2xCH3), 50.64 (CH2), 35.51 (CH2), 35.38 (CH2); HR ESMS m/z 696.1835 [M-Na]+. Calc. for C39H36N3O3Br 696.1833.H-1,2,3-triazol-1-yl)ethyl)phenyl)ethan-1-ol 8:2-(4-(2-(4-(2-bromophenyl)-1 yield 75%; white solid; m.p. 130–133 °C; 1H NMR  δ 8.51 , 7.89 , 7.72 , 7.48 , 7.30 , 7.12 , 4.68 , 4.50 , 3.56 , 3.17 , 2.67 ; 13C NMR  δ 143.87 (C), 137.74 (C), 134,99 (C), 133.46 (CH), 131.39 (C), 130.35 (C), 129.71 (CH), 128.94 (2xCH), 128.54 (2xCH), 127.97 (CH), 124.05 (CH), 120.59 (CH), 62.17 (CH2), 50.69 (CH2), 38.64 (CH2), 35.41 (CH2); HR ESMS m/z 372.0716 [M-H]+. Calc. for C18H18N3OBr 372.0711.H-1,2,3-triazole 9:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl)-4-(4-methoxyphenyl)-1 yield 54%; yellow oil; 1H NMR  δ 8.38 , 7.69 , 7.29–7.09 , 6.97 , 6.83 , 4.61 , 3.77 , 3.72 , 3.18 , 3.10 , 2.77 ; 13C NMR  δ 158.92 (C), 157.96 (2xC), 146.07 (C), 145.03 (C), 137.41 (C), 135.88 (2xC), 135.36 (C), 129.56 (6xCH), 129.09 (CH), 128.51 (CH), 127.74 (CH), 127.61 (CH), 126.40 (2xCH), 123.41 (CH), 120.28 (CH), 114.27 (2xCH), 113.10 (6xCH), 85.39 (C), 64.40 (CH2), 59.75 (C), 55.13 (CH3), 54.99 (2xCH3), 50.58 (CH2), 35.51 (CH2), 35.21 (CH2); HR ESMS m/z 648.2838 [M-Na]+. Calc. for C40H39N3O4 648.2835.H-1,2,3-triazol-1-yl)ethyl)phenyl)ethan-1-ol 10:2-(4-(2-(4-(4-methoxyphenyl)-1 yield 20%; white solid; m.p. 172–174 °C; 1H NMR  δ 8.40 , 7.72 , 7.12 , 7.00 , 4.62–4.57 , 3.78 , 3.56 , 3.17 , 2.67 ; 13C NMR  δ 158.93 (C), 146.04 (C), 137.75 (C), 135.03 (C), 128.92 (2xCH), 128.43 (2xCH), 126.40 (2xCH), 123.43 (C), 120.29 (CH), 114.29 (2xCH), 62.10 (CH2), 55.13 (CH3), 50.58 (CH2), 38.60 (CH2), 35.18 (CH2); HR ESMS m/z 324.1710 [M-H]+. Calc. for C19H21N3O2 324.1712.H-1,2,3-triazole 11:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl)-4-(3-methoxyphenyl)-1 yield 74%; colorless oil; 1H NMR  δ 8.53 , 7.38–7.07 , 6.9–6.82 , 4.63 , 3.79 , 3.71 , 3.19 , 3.11 , 2.77 ; 13C NMR  δ 159.69 (C), 157.99 (2xC), 146.08 (C), 145.05 (C), 137.44 (C), 135.91 (2xC), 135.32 (C), 132.16 (C), 130.03 (CH), 129.59 (4xCH), 129.11 (2xCH), 128.53 (2xCH), 127.76 (2xCH), 127.64 (2xCH), 126.56 (CH), 121.49 (CH), 117.45 (CH), 113.53 (CH), 113.11 (4xCH), 110.30 (CH), 85.43 (C), 64.43 (CH2), 55.10 (CH3), 55.00 (2xCH3), 50.69 (CH2), 35.54 (CH2), 35.20 (CH2); HR ESMS m/z 648.2838 [M-Na]+. Calc. for C40H39N3O4 648.2835.H-1,2,3-triazol-1-yl)ethyl)phenyl)ethan-1-ol 12:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy) 2-(4-(2-(4-(3-methoxyphenyl)-1 yield 55%; white solid; m.p. 90–93 °C; 1H NMR  δ 8.54 , 7.39–7.32 , 7.13 , 6.89 , 4.62 , 4.57 , 3.81 , 3.56 , 3.18 , 2.67 ; 13C NMR  δ 159.65 (C), 145.99 (C), 137.75 (C), 134.96 (C), 132.13 (C), 129.98 (CH), 128.91 (2xCH), 128.41 (2xCH), 121.47 (CH), 117.39 (CH), 113.50 (CH), 110.25 (CH), 62.07 (CH2), 55.07 (CH3), 50.64 (CH2), 38.59 (CH2), 35.13 (CH2); HR ESMS m/z 324.1716 [M-H]+. Calc. for C19H21N3O2 324.1712.H-1,2,3-triazole 13:1-(4-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)phenethyl)-4-(2-methoxyphenyl)-1 yield 51%; colorless oil; 1H NMR  δ 8.27 , 8.12 , 7.32–7.01 , 6.84 , 4.64 , 3.82 , 3.71 , 3.17 , 3.11 , 2.78 ; 13C NMR  δ 157.99 (2xC), 155.30 (C), 145.05 (C), 141.58 (C), 137.38 (C), 135.90 (2xC), 135.48 (C), 129.59 (4xCH), 129.08 (2xCH), 128.81 (CH), 128.60 (2xCH), 127.77 (2xCH), 127.63 (2xCH), 126.54 (2xCH), 123.85 (CH), 120.62 (CH), 119.22 (C), 113.12 (4xCH), 111.54 (CH), 85.44 (C), 64.46 (CH2), 55.37 (CH3), 55.01 (2xCH3), 50.46 (CH2), 35.54 (CH2), 30.68 (CH2); HR ESMS m/z 648.2838 [M-Na]+. Calc. for C40H39N3O4 648.2835.H-1,2,3-triazol-1-yl)ethyl)phenyl)ethan-1-ol 14:2-(4-(2-(4-(2-methoxyphenyl)-1 yield 37%; white solid; m.p. 76–80 °C; 1H NMR  δ 8.28 , 8.12 , 7.32 , 7.14 , 7.11 , 7.04 , 4.64 , 4.59 , 3.88 , 3.57 , 3.17 , 2.68 ; 13C NMR  δ 155.29 (C), 141.54 (C), 137.73 (C), 135.14 (C), 128.91 (2xCH), 128.78 (2xCH), 128.5 (CH), 126.51 (CH), 123.81 (CH), 120.60 (CH), 119.21 (C), 115.55 (CH), 62.12 (CH2), 55.41 (CH3), 50.43 (CH2), 38.62 (CH2), 35.38 (CH2); HR ESMS m/z 324.1711 [M-H]+. Calc. for C19H21N3O2 324.1712.Cell culture media were purchased from Gibco . Fetal bovine serum (FBS) was obtained from Harlan-Seralab . Supplements and other chemicals not listed in this section were obtained from Sigma Chemical Co. . Plastics for cell cultures were supplied by Thermo Scientific BioLite. All tested compounds were dissolved in DMSO at a concentration of 20 mM and stored at −20 °C until use.HT-29, A549, MCF-7, and HEK-293 cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (1 g/L), glutamine (2 mM), penicillin (50 μg/mL), streptomycin (50 μg/mL), and amphotericin B (1.25 μg/mL), supplemented with 10% FBS.3  cells per well were incubated with serial dilutions of the tested compounds in a total volume of 100 μL of their growth media. The 3--2,5-diphenyltetrazolium bromide  dye reduction assay in 96-well microplates was used. After 2 days of incubation , 10 μL of MTT  was added to each well, and the plate was incubated for a further 3 h (37 °C). After that, the supernatant was discarded and replaced by 100 µL of DMSO to dissolve formazan crystals. The absorbance was then read at 550 nm by spectrophotometry. For all concentrations of the compound, cell viability was expressed as the percentage of the ratio between the mean absorbance of treated cells and the mean absorbance of untreated cells. Three independent experiments were performed, and the IC50 values  were graphically determined using GraphPad Prism 4 software.In 96-well plates, 5 × 10To study the effect of the compounds on every biological target in cancer cell lines, the compounds were used at a 100 µM dose.5 cells per well were incubated for 24 h with the corresponding dose of the tested compound in a total volume of 500 μL of their growth media.For the assay, 10™ X-100 was performed, and finally, cells were stained with FITC Mouse monoclonal Anti-c-Myc (ab223913) and Alexa Fluor® 647 Rabbit monoclonal Anti-PD-L1 (ab215251).To detect total PD-L1 and c-Myc, after the cell treatments, they were collected and fixed with 4% in PBS paraformaldehyde. After fixation, a treatment with 0,5% in PBS Triton5 or 2 × 105 of the corresponding cancer cells line per well were seeded and incubated for 24 h, then the medium was changed by one cell culture medium supplemented with IFN-γ  and containing 5 × 105 or 105 Jurkat T cells or THP-1, respectively, per well and 100 µM of the corresponding compound or DMSO for the positive control. After 24 h/48 h of incubation, supernatants were collected to determine Jurkat T or THP-1 living cells, and on the other hand, corresponding cancer cells were collected with trypsin. Both types of suspension cells were fixed with 4% in PBS paraformaldehyde and counted by flow cytometry.To study the effect of the compounds on the cell viability in co-culture with Jurkat T cells or THP-1 cells, 10To study the effect of the compounds on every biological target in co-cultured cancer cell lines, the compounds were incubated for 24h as described before.® 647 Rabbit monoclonal Anti-PD-L1 (ab215251).To detect membrane PD-L1 and VEGFR-2, after the cell treatments, they were collected, fixed with 4% in PBS paraformaldehyde, and stained with FITC Mouse monoclonal Anti-Human VEGFR-2 (ab184903) and Alexa Fluor® 647 Rabbit monoclonal Anti-PD-L1 (ab215251). Then, cells were washed in PBS, and cover glasses were mounted with a drop of ProLong® antifade solution (Invitrogen). The cytoskeleton was imaged by a confocal laser scanning microscope (CLSM) Leica SP5 with a Leica inverted microscope, equipped with 20x objective. Each image was recorded with the CLSM’s spectral mode selecting specific domains of the emission spectrum. The FITC fluorophore was excited at 488 nm with an argon laser, and its fluorescence emission was collected between 496 nm and 535 nm.For immunofluorescence microscopy of the microtubule network, 106 cells were plated on cover glass and incubated with the selected compounds. Cells were then fixed in 4% formaldehyde (in PBS pH 7.4) for 20 min at room temperature and permeabilized with PBS-Triton X-100 0.5% for 1 min at room temperature. Direct immunostaining was carried out for 1 h at room temperature with FITC Mouse monoclonal Anti-c-Myc (ab223913) and Alexa Fluor™ TaqMan® probes. TaqMan® Gene Expression Master Mix Fast containing the appropriate buffer for the amplification conditions, dNTPs, thermostable DNA polymerase enzymes, and a passive reference probe was used. To amplify each of the genes, the predesigned primers sold by Life Technologies TaqMan® Gene Expression Assays, Hs99999903-m1 (β-actin), Hs00153408-m1 (c-Myc), and Hs00204257-m1, (PD-L1) were used.MCF-7 cells at 70–80% confluence were collected, and 1.5 × 105 cells were placed in a six-well plate in 1.5 mL of medium. After 24h, cells were incubated with the corresponding compounds for 48 h. Cells were collected, and the total cellular RNA from MCF-7 cells was isolated using the Ambion RNA extraction kit according to the manufacturer’s instructions. The cDNA was synthesized by MMLV-RT with 1–21 µg of extracted RNA and oligo(dT)15 according to the manufacturer’s instructions. Genes were amplified by a thermal cycler and StepOnePlus