The precancerous disease of breast cancer is an inevitable stage in the tumorigenesis and development of breast neoplasms. Quercetin (Que) has shown great potential in breast cancer treatment by inhibiting cell proliferation and regulating T cell function. γδ T cells are a class of nontraditional T cells that have long attracted attention due to their potential in immunotherapy. In this study, we revealed the immunomodulatory function of Que through regulation of the JAK/STAT1 signaling pathway, which was followed by the synergistic killing of breast cancer cells.In the experimental design, we first screened target genes with or without Que treatment, and we intersected the Que target with the disease target by functional enrichment analysis. Second, MCF-10A, MCF-10AT, MCF-7 and MDA-MB-231 breast cancer cell lines were treated with Que for 0 h, 24 h and 48 h. Then, we observed the expression of its subsets by coculturing Que and γδ T cells and coculturing Que and γδ T cells with breast tumor cells to investigate their synergistic killing effect on tumor cells. Finally, Western blotting was used to reveal the changes in proteins related to the JAK/STAT1 signaling pathway after Que treatment in MCF-10AT and MCF-7 cells for 48 h.P < 0.05). Most importantly, Que promoted the differentiation of γδ T cells into the Vδ2 T cell subpopulation. The best ratio of effector cells to target cells (E/T) was 10:1, the killing percentages of γδ T cells against MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 were 61.44 ± 4.70, 55.52 ± 3.10, 53.94 ± 2.74, and 53.28 ± 1.73 , and the strongest killing effect on precancerous breast cancer cells and breast cancer cells was found when the Que concentration was 5 μM and the E/T ratio was 10:1 . In addition, our results showed that Que increased the protein levels of IFNγ-R, p-JAK2 and p-STAT1 while decreasing the protein levels of PD-L1 (P < 0.0001).The pathway affected by Que treatment was the JAK/STAT1 signaling pathway and was associated with precancerous breast cancer, as shown by network pharmacology analysis. Que induced apoptosis of MCF-10AT, MCF-7 and MDA-MB-231 cells in a time- and concentration-dependent manner (In conclusion, Que plays a synergistic role in killing breast cancer cells and promoting apoptosis by regulating the expression of IFNγ-R, p-JAK2, p-STAT1 and PD-L1 in the JAK/STAT1 signaling pathway and promoting the regulation of γδ T cells. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of breast cancer. Precancerous lesions of breast cancer refer to some breast diseases that are not malignant tumors but have an increased possibility of developing into malignant tumors. The World Health Organization (WHO) defines such lesions as precancerous because they are more than 20% likely to transform from nonneoplastic lesions to tumors . The nexThe study and development of food with anticancer effects is an important link in the field of antitumor therapy. Quercetin  is a flavonoid that is widely found in wild fields, mainly in the form of rutin, quercetin, hypericin and so on. Que can be obtained by an acid hydrolysis reaction . Therefoγδ T cells are innate immune cells, accounting for 0.5 ~ 5% of all peripheral T lymphocytes. γδ T cells are mainly densely distributed in the mucosal epithelium and belong to a special class of immune cells involved in acquired immunity and natural immunity. Its subgroups are usually divided into Vδ1 T cells and Vδ2 T cells . γδ T ceThe JAK-STAT1 signaling pathway is involved in the process of tumor cell recognition and tumor-driven immune escape . STAT1  with “precancerous lesions” as the key word to obtain disease target information.Genes previously associated with breast cancer lesions were searched in the GeneCards database  analysis and GO and KEGG enrichment analysis were performed using the STRING database. PPI data were imported into Cytoscape 3.6.1 for visualization.After obtaining the consent of participants, peripheral blood samples were obtained from healthy controls (HCs), and peripheral blood mononuclear cells (PBMCs) were separated by Ficoll-Hyperaque gradient centrifugation. All the above procedures were carried out according to the guiding principles of the Medical Ethics Committee of Guangdong Provincial Health Bureau, and the ethics involved were examined and approved by the Ethics Committee of the First Affiliated Hospital of Jinan University in Guangdong Province.2 plastic culture flasks) in F12/DMEM and DMEM (1x) medium with 10% fetal bovine serum (Gibco South America) and Donor Equine Serum , Penstrep at 37 °C in an incubator with an atmosphere containing 5% CO2. The cells were cultured every 2 days.MCF-10A , MCF-10AT (breast cancer precancerous cells) and MCF-7 and MDA-MB-231 (human breast cancer cells) cells  were maintained as monolayer cultures  was added. Que working solution with concentrations of 0, 2.5, 5 and 10 μM was added to the culture medium (constant volume to 1 mL system). The culture was again placed at 37 ℃ and 5% CO2. γδ T cells were cultured for 16 days, Que was cultured for 13 days, and γδ T cell subsets were detected.Peripheral blood of sterile anticoagulant normal subjects was collected and separated with lymphocyte separation solution to obtain peripheral blood mononuclear cells. Then, the cells were resuspended in RPMI 1640 complete medium  and coated in a 24-well culture plate with 1 μL (1 μg/mL) solid phase anti-human TCR γδ monoclonal antibody. Stimulated at 37 ℃ and 5%CO50 value of Que was detected by the MTT method. In short, MCF-10AT, MCF-7 and MDA-MB-231 cells were seeded in 96-well plates for 48 h. MTT solution (Sigma) was incubated at 37 ℃ for 4 h. A total of 140 μL of dimethyl sulfoxide (Sigma) working solution was added to each well and shaken on a low-speed shaker for 10 min to completely dissolve the crystal. The IC50 value of Que was determined by absorbance at 490 nm .The IC5/mL, the cells were washed and centrifuged with PBS, and the supernatant was discarded. Then, 195 μL of binding buffer with 5 μL of annexin V-FITC  was incubated for 15 min at room temperature. The cells were resuspended with 190 μL binding buffer and 10 μL of PI (20 µg/mL) were added to the machine and then the cells were immediately placed in the machine  to detect cell apoptosis, and data were analyzed by Flowjo software  , and necrotic cells (annexin −/PI +).The apoptosis rates were verified by Annexin V staining. As negative controls, untreated cells were used, and cells treated with staurosporine , which was used to induce apoptosis, were used as positive controls. Cells were collected, the number of cells was approximately 3*10PBMCs were isolated from HC samples and then incubated with the following antibodies: CD3-FITC (clone SK7), TCR γδ-PE (clone IP26), Vδ1- PE-Cy7 (clone TS8.2), Vδ2-PerCP (clone B6), PE-Cy7 isotype control (clone P3.6.2.8.1), PE isotype control (clone eBM2a), FITC isotype control (clone MOPC-21), and PerCP isotype control (clone MOPC-21) . PBMCs were stained with different combinations of anti-human TCR γδ, Vδ1, Vδ2, and CD3 antibodies at room temperature in a dark room for 20 min. A total of 30,000–50,000 CD3\+ T cells were collected with a Cytoflex S flow cytometer , and data were analyzed by FlowJo software .6 cells/mL. One milliliter of resuspended related cell lines was removed and placed in a 1.5 mL centrifuge tube, and 0.4 μL CFSE (concentration of 5 mM) was added to a final sample concentration of 2 μM. Then, the trypsinized cells were preincubated with γδ T cells diluted in RPMI 1640 medium with 10% fetal bovine serum  and Penstrep  at 37 °C for 4–5 h in a humidified atmosphere containing 5% CO2. After this period of coculture with γδ T cells, the cells were removed and stained with 0.5 μL PI, and the killing efficiency of γδ T cells was immediately detected by Cytoflex S .Subconfluent (80%) monolayers of MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 cells were trypsinized  and adjusted to 2 × 10Total protein was extracted according to the protein extraction kit instructions. Protein concentration  was determined by the BCA method. After electrophoresis, the proteins were transferred to PVDF membranes  and blocked with 5% skimmed milk powder for 2 h at room temperature. The membrane was incubated overnight with primary antibody (diluted according to manufacturer’s instructions) at 4 °C. On the second day, the horseradish peroxidase-conjugated secondary antibody diluted at 1:5000 was incubated at room temperature for 1 h. After washing with TBST, the PVDF membrane was detected by a gel automatic exposure imaging system. Image-Pro Plus software was used for gray value analysis of Western blotting.SPSS 13.0 statistical software was used to analyze the data, the mean ± standard deviation was used to represent the data, and the Wilcoxon test method for comparison of two independent samples of the original data was used to analyze whether the data were significantly different. One-Way ANOVA was used for comparison between groups. The inspection standard shall be as per P < 0.05 indicates a significant difference, * indicates P < 0.05, ** indicates P ≤ 0.01, *** indicates P ≤ 0.001. The experimental data and related statistical data were analyzed by GraphPad Prism 5 software.The chemical structure of Que was obtained from the PubChem database, as shown in Fig. https://www.genecards.org/) when the keywords "precancerous lesions" were used. Intersecting 482 potential target genes of Que with 1602 disease targets resulted in 314 genes, as shown in Fig. Information on a total of 1602 disease target genes associated with breast cancer lesions were obtained in the GeneCards database  values of Que on MCF-10AT, MCF-7 and MDA-MB-231 cells were 52.39 µM, 53.76 µM and 64.23 μM, respectively.The effects of Que on the proliferation of MCF-10AT, MCF-7 and MDA-MB-231 cells were determined by an MTT assay, and the results are shown in Fig. P = 0.006, P = 0.109, and P = 0.004, respectively), and the difference was statistically significant. MCF-10AT cells were compared with MCF-10A, MCF-7, MDA-MB-231 cell lines treated with 20 μM Que. The percentage of the apoptotic cell population was 7.60 ± 0.21% and 3.31 ± 0.12%, 12.76 ± 0.37%, and 20.17 ± 2.90% . There was a significant statistical difference. MCF-10AT cells were compared with MCF-10A, MCF-7, and MDA-MB-231 cells treated with 80 μM Que. The percentage of the apoptotic cell population was 35.63 ± 0.60% and 1.64 ± 0.05%, 25.34 ± 5.06%, 29.80 ± 0.98% , which showed statistical significance. MCF-10AT cells were compared with MCF-10A, MCF-7 and MDA-MB-231 cell lines treated with 120 μM Que. The percentage of the apoptotic cell population was 40.25 ± 0.71% and 3.40 ± 0.07%, 29.38 ± 2.50%, and 29.40 ± 2.51% , with statistical significance. Next, MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 5 μM concentration of Que, the percentage of apoptotic cell population was determined as 1.75 ± 0.33% and 1.34 ± 0.15%, 9.63 ± 1.70%, 9.58 ± 0.32% . MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 20 μM concentration of Que and percentage of the apoptotic cell population was determined as 8.44 ± 1.54% and 0.84 ± 0.10%, 11.12 ± 1.38%, 14.23 ± 1.62%  with significant statistical differences. MCF-10AT was compared with MCF-10A, MCF-7 and MDA-MB-231 cell lines at 80 μM Que, and the percentage of apoptotic cell population was determined to be 72.36 ± 1.77% and 3.52 ± 0.14%, 61.95 ± 1.22%, 74.73 ± 0.77%  with statistical significance. MCF-10AT was compared with MCF-10A, MCF-7, MDA-MB-231 cell lines at 120 μM concentration of Que, the percentage of apoptotic cell population was determined to be 79.19 ± 1.76% and 5.52 ± 0.30%, 61.95 ± 1.22%, 74.37 ± 0.80%  with significant statistical differences    ratio was 10:1, the killing rates of γδ T cells against MCF-10A, MCF-10AT, MCF-7, and MDA-MB-231 cells were 61.44 ± 4.70, 55.52 ± 3.10, 53.94 ± 2.74, and 53.28 ± 1.73 . The cell killing rates of MCF-7 at E/T  were 13.23 ± 2.68, 24.39 ± 3.13, 55.59 ± 5.98, compared with MCF-10AT, the killing rate of MCF-7 versus MCF-10AT was statistically significant at 5:1 . The cell killing rates of MDA-MB-231 cells at E:T ratios of 1:1, 5:1, and 10:1 were 12.77 ± 3.64, 22.7 ± 1.39, and 59.04 ± 5.67, respectively. Thus, compared with MDA-MB-231 cells, MCF-10AT cells were also statistically significant at a 5:1 ratio . In addition, with the increase in effector cell proportion, the Que concentration was still 5 μM, and MCF-10A > MCF-10AT > MCF-7 > MDA-MB-231, 1:1 < 5:1 < 10:1  and investigate the specific killing effect. We found that the cell killing rates of MCF-10A cells in E/T  were 24.12 ± 4.34, 51.93 ± 6.47, 64.94 ± 3.61. The cytotoxicity rates of MCF-10AT in E/T  were 19.38 ± 5.30, 33.45 ± 5.49, 64.96 ± 5.45, and MCF-10A > MCF-10AT (P < 0.0001), while PD-L1 protein levels were decreased   by producing IL-2 and IFN-γ when intracellular bacteria are infected. When extracellular parasites and other infections occur, interleukin-10 (IL-10) and interleukin-4 (IL-4) are produced and play a role in Th2 (type 2 helper T lymphocytes) regulation . With fuThe JAK/STAT1 pathway is involved in almost the entire immune regulation process, including immune surveillance and immune escape. STAT1 promotes cell apoptosis, inhibits cell growth and differentiation and plays an important role in inhibiting tumorigenesis and tumor development by regulating the interferon (IFN) system . STAT1-dWe demonstrated for the first time the proapoptotic effect of Que on different breast cancer cells and explored the mechanisms by which Que influences immunity by affecting the JAK/STAT1 signaling pathway. Notably, Que promotes the proliferation of Vδ2 T subsets of γδ T cells, thus enhancing the killing effect on breast cancer cells. This finding suggests that Que and γδ T cells play a synergistic role in killing tumor cells and cellular immune regulation. Que, as a natural flavonoid, should be widely used in clinical practice due to its antitumor and immune regulation functions. Que may be a potential drug for the prevention of precancerous breast cancer and adjuvant treatment of breast cancer.


Protonation of synthetic μ-1,2-peroxo model complexes occurred at the μ-oxo and not at the μ-1,2-peroxo bridge. Here we report a stable μ-1,2-peroxo complex {FeIII(μ-O)FeIII} using a dinucleating ligand and study its reactivity. The reversible oxidation and protonation of the μ-1,2-peroxo-diferric complex provide μ-1,2-peroxo FeIVFeIII and μ-1,2-hydroperoxo-diferric species, respectively. Neither the oxidation nor the protonation induces a strong electrophilic reactivity. Hence, the observed intramolecular C-H hydroxylation of preorganized methyl groups of the parent μ-1,2-peroxo-diferric complex should occur via conversion to a more electrophilic high-valent species. The thorough characterization of these species provides structure-spectroscopy correlations allowing insights into the formation and reactivities of hydroperoxo intermediates in diiron enzymes and their conversion to activated P′ or high-valent intermediates. IIIFeIII and a μ-1,2-peroxo FeIVFeIII, and study their reactivity in C-H activation.Iron coordination complexes can be used to gain insight on biologically relevant iron-oxygen compounds generated in iron metalloenzymes. Here, the authors characterise a μ-1,2-hydroperoxo Fe Their catalytic cycles generally employ a diferrous form that reacts with dioxygen to a peroxo-diferric intermediate FeIII} core afforded μ-hydroxo-bridged {FeIII(μ-OH)FeIII} species36 questioning the principle accessibility of hydroperoxo-diferric species.Syntheses and detailed spectroscopy and reactivity studies of μ-1,2-peroxo complex [(susan6-Me){FeIII(μ-O)FeIII}](ClO4)2 using the dinucleating ligand susan6-Me {FeIII(μ-O)(μ-O2)FeIII}]2+ is reversibly oxidized to the high-valent μ-1,2-peroxo complex [(susan6-Me){FeIV(μ-O)FeIII}]3+ and reversibly protonated to the μ-1,2-hydroperoxo complex [(susan6-Me){FeIII(μ-O)FeIII}]3+. The study of the electrophilic reactivity for oxygen-atom transfer (OAT) using PPh3 and hydrogen-atom transfer (HAT) using DHA and TEMPOH provides not only a low electrophilic character of the parent μ-1,2-peroxo-FeIIIFeIII complex but also for the oxidized μ-1,2-peroxo-FeIVFeIII and protonated μ-1,2-hydroperoxo-FeIIIFeIII species. Only the oxidized μ-1,2-peroxo-FeIVFeIII species reacts with the relatively weak substrate TEMPOH. The determination of the pKa = 9.5 ± 0.1 and the bond dissociation free energy −1 of the protonated μ-1,2-hydroperoxo-FeIIIFeIII species quantifies the low electrophilic character even of the oxidized μ-1,2-peroxo-FeIVFeIII species. Therefore, the intramolecular C–H activation of preorganized 6-methyl pyridine groups to benzylalcoholato and carboxylato donors in the parent 1,2-peroxo-FeIIIFeIII complex should not occur via the 1,2-peroxo-ligand but via conversion to a more reactive but fluent high-valent species. The low electrophilic character and the spectroscopic signatures of this μ-1,2-hydroperoxo-diferric model are discussed in relation to assignments of reactive intermediates postulated for diiron enzymes.Here, we present the synthesis, characterization, and reactivity of the rationally stabilized -Me Fig. 37–39. Th6-Me and Fe(ClO4)2·6H2O provided [(susan6-Me){FeII(μ-OH)2FeII}](ClO4)2 {FeIII(μ-O)FeIII}](ClO4)2  and with O2 trans to a pyridine (N44). This results in a shorter Fe1–O1peroxo and a longer Fe2–O2peroxo bond. The resulting different charge donation is compensated by a longer Fe1–O3oxo and a shorter Fe2–O3oxo bond. The O1–O2 bond length of 1.432(2) Å is the longest yet established for a peroxo-diferric complex (1.396–1.426 Å)26.The reaction of susan4)2 Fig.  and subs4)2 Fig. . Single-δ = 0.53 mm s−1 and quadrupole splitting |ΔEQ| = 1.69 mm s−1 {FeIIIF(μ−O)FeIIIF}]2+  providing an exact value for a μ-1,2-peroxo,μ-oxo-diferric complex. This antiferromagnetic coupling is significantly stronger than −100 ± 20 cm−1 usually found for μ-oxo-diferric complexes43 including those with our dinucleating ligands38. This applies also to μ-oxo,μ-carboxylato-diferric complexes which exhibit decreased ∢(FeIII-(μ-O)-FeIII) angles as the μ-1,2-peroxo,μ-oxo-diferric complex. This comparison indicates a significant contribution of the FeIII-μ-1,2-peroxo-FeIII exchange pathway due to shorter (1.88 and 1.93 Å) and hence more covalent FeIII-μ-Operoxo bonds44 compared to the longer FeIII-μ-carboxylato bonds {FeIII(μ-O)(μ-OAc)FeIII}]3+)45 that are considered not to contribute significantly to the exchange coupling.Despite this structural asymmetry, the Mössbauer spectrum exhibits one quadrupole doublet with isomer shift s−1 Fig. . This isnts Fig.  revealed−1 (ε = 1180 M−1 cm−1), 15,400 cm−1 (ε = 1000 M−1 cm−1), and 11,800 cm−1 (ε = 190 M−1 cm−1) that are characteristic for {FeIII(μ-O)FeIII} complexes29 with the 19,300 cm−1 band assigned to the μ-oxo→FeIII LMCT and the 15,400 cm−1 band to the μ-1,2-peroxo→FeIII LMCT involving π*π → t2g transitions11. This low LMCT energy demonstrates strong covalency of the FeIII–Operoxo bond representing the origin of the strong antiferromagnetic coupling.The UV-Vis-NIR spectrum Fig.  exhibits18O/18O2-isotopomers as microcrystalline solids. The resonance Raman (rR) and FTIR spectra FeIII} core  stretch by isotopic labeling (Δ(16O2−18O2) = 46 cm−1 consistent with a Hooke’s law calculation for a harmonic O–O vibration of 48 cm−1).We synthesized all four possible tra Fig.  both rev3CN Fig. . The 831ν(O–O) stretch of the crystallographically characterized [(6Me2BPP){FeIII(μ-O)FeIII}(6Me2BPP)] (A) appears at higher energy at 847 cm−1 (Table 22. Higher ν(O–O) stretches were attributed22 to increasing ∢(Fe–O–O) angles11. However, ∢(Fe–O–O) is slightly smaller in A than in our complex (115° vs 117°). Interestingly, the lower ν(O–O) stretch correlates with a longer O–O distance (1.432(2) Å vs 1.411 Å) indicating a stronger dependence of ν(O–O) on d(O–O) than on ∢(Fe–O–O).The ized [6MeBPP{FeIII6-Me){FeIII(μ-O)FeIII}]2+ shows no indication of decay for hours in CH3CN at −40 °C {FeIII(μ-O)(μ-O2)FeIII}]2+ can be reversibly oxidized at E1/2ox = 0.55 V and irreversibly reduced at Epred = −1.28 V vs Fc+/Fc {FeIII(μ-O)FeIII}]2+ at 0.68 V vs Fc+/Fc  in CH3CN at −40 °C resulted in slight changes of the absorption features  restored the initial UV-Vis spectrum to ~95%  generated the UV-Vis spectrum of [(susan6-Me){Fe(μ-O)2(μ-O2)Fe}]3+ ClO]3+ Fig.  within 16-Me){Fe(μ-O)Fe}]3+ . Mössbauer spectroscopy (vide infra) demonstrates that the iron ions remain high-spin ruling out an interpretation as FeIII l.s. species. The severe deviation of gav = 2.15 from 2.0023 and the large g-anisotropy demonstrate a strong contribution of orbital angular momentum and rule out a ligand-centered oxidation to a μ-oxo,μ-1,2-superoxo-FeIIIFeIII complex, disclosing a metal-centered oxidation to a μ-oxo,μ-1,2-peroxo-FeIVFeIII complex with antiferromagnetically coupled FeIV(S1 = 2) and FeIII(S2 = 5/2) ions. The FeIV h.s. S1 = 2 configuration results in g1 < 2.0 by spin-orbit coupling, while the FeIII h.s. is close to isotropic g2 ≈ 2.0046. Projection of the local spins onto the antiferromagnetic St = 1/2 ground state47 results in gav > 2.00 for the St = 1/2 as observed experimentally39. Despite significant efforts, we were not able to obtain resonance-enhanced Raman features of [(susan6-Me){Fe(μ-O)Fe}]3+ by excitation at 647 , 568 , and 514 nm  corresponding to the three absorption features of this oxidized complex.The EPR spectrum of [{Fe(μ-O)Fe}]3+ {Fe(μ-O)Fe}]3+ restores the Mössbauer spectrum of the starting complex [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+  while class III stands for quantum-mechanically delocalized states. In class II systems, two different states exist that correspond roughly to the excess electron localized on the one or the other metal ion. Between these two states is an energy barrier and there can be temperature-dependent and light-induced mechanisms to transfer the excess electron from the reduced to the oxidized metal ion. In symmetric cases, the two states are energetically degenerate while the asymmetry observed here results in an energy difference between these two states . To differentiate between these two possibilities, we recorded Mössbauer spectra at lower temperatures {FeIII(μ-O)FeIII}]2+ with HClO4 at −60 °C resulted in the loss of the 15,400 cm−1 band while the 11,800 and 19,300 cm−1 bands are only slightly affected . Neither were resonance-enhanced vibrations observed by excitation at 568  and 514 nm  close to the μ-oxo→FeIII LMCT {FeIII(μ-O)FeIII}]2+ exhibits only one quadrupole doublet despite the significant differences in its two coordination sites (vide supra) requires a different source of asymmetry for the protonated species to explain its two strongly differing quadrupole doublets. This rules out a μ-1,1-hydroperoxo-bridge, while a μ-1,2-hydroperoxo-bridge provides the different source of asymmetry as the protonated site becomes much less charge-donating. Although a terminally bound hydroperoxo-ligand would be in-line with the absence of a μ-1,2-peroxo→FeIII LMCT and two quadrupole doublets, the persistence of the μ-oxo → FeIII LMCTs around 19,000 and 11,800 cm−1 strongly favors a doubly-bridged structure of almost the same ∢(FeIII-(μ-O)-FeIII) angle hence ruling out an almost linear {FeIII(OOH)(μ-O)FeIIIX} core40 that is also inaccessible with the ligand susan6-Me (vide infra).The Mössbauer spectrum provided two quadrupole doublets with s−1 Fig. . Since tμ-1,2-hydroperoxo-bridged complex is supported by DFT calculations (μ-peroxo-O1 being 1200 cm−1 higher in energy. Although the μ-hydroxo species (protonation of the μ-oxo-O3) seems energetically feasible, its isomer shifts are even higher than for the starting peroxo complex {FeIII(μ-O)(μ-O2)FeIII}]2+ with HClO4 demonstrates the nucleophilic character of the μ-1,2-peroxo ligand. On the other hand, the reaction of [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ with 2-phenylproponal as a typical substrate to evaluate the nucleophilic character of peroxo ligands52 is slow in CH3CN at −5 °C {FeIII(μ-O)(μ-O2)FeIII}]2+ but also for the oxidized μ-1,2-peroxo-FeIVFeIII and the protonated μ-1,2-hydroperoxo-diferric species as both oxidation and protonation should increase the electrophilic character. This is already reflected in the different stabilities of these species {FeIII(μ-O)FeIII}]2+ shows no indication of decay for hours in CH3CN at −40 °C, oxidized [(susan6-Me){FeIV(μ-O)(μ-O2)FeIII}]3+ and protonated [(susan6-Me){FeIII(μ-O)FeIII}]3+ decay with half-lives of τ1/2 ≈ 90 min τ1/2 ≈ 11 min, respectively. Therefore, the clean characterization of the latter two species required lower temperatures of −60 °C and hence addition of a certain amount of CH2Cl2.The electrophilic character was not only evaluated for [{FeIII(μ-O)(μ-O2)FeIII}]2+ were performed in CH3CN at −40 °C, while that with [(susan6-Me){FeIV(μ-O)(μ-O2)FeIII}]3+ and [(susan6-Me){FeIII(μ-O)FeIII}]3+at −60 °C in CH3CN/CH2Cl2 mixtures (vide supra). The parent [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ showed no reactivity towards DHA and PPh3 {FeIV(μ-O)(μ-O2)FeIII}]3+ also showed no reactivity towards DHA {FeIII(μ-O)(μ-O2)FeIII}]2+ {FeIV(μ-O)(μ-O2)FeIII}]3+ and PPh3 but suggests an oxidation of PPh353 by [(susan6-Me){FeIV(μ-O)(μ-O2)FeIII}]3+. Analogous observations were made with the protonated [(susan6-Me){FeIII(μ-O)FeIII}]3+ that showed no reactivity with DHA {FeIII(μ-O)(μ-O2)FeIII}]2+ {FeIII(μ-O)FeIII}]3+ and PPh3. The partial recovery of [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ indicates a protonation equilibrium between PPh354 and [(susan6-Me){FeIII(μ-O)FeIII}]3+.The electrophilic character of the three complexes were initially investigated using 9,10-dihydroanthracene (DHA) and PPhh3 Figs. , S21. ThDHA Fig. , while t]2+ Fig. . The refDHA Fig.  and with]2+ Fig. . Again, 55 that [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ exhibits no electrophilic reactivity against DHA, the non-reactivity of both oxidized [(susan6-Me){FeIV(μ-O)2(μ-O2)FeIII}]3+ and protonated [(susan6-Me){FeIII(μ-O)FeIII}]3+ is quite surprising. To further understand this non-reactivity, we determined the 6-Me){FeIII(μ-O)FeIII}]3+. In this respect, we determined the pKa of [(susan6-Me){FeIII(μ-O)FeIII}]3+ in CH3CN that provided 9.5 ± 0.1 {FeIV(μ-O)(μ-O2)FeIII}]3+ should be capable as oxidant for HAT for substrates with a lower BDFE(X-H)−159. However, the intrinsic difference between BDFE and BDE57, the temperature-dependence of BDFE especially for transition metal complexes, and the experimental error explain the non-reactivity of [(susan6-Me){FeIV(μ-O)(μ-O2)FeIII}]3+ with DHA. In this respect, TEMPOH should be a suitable HAT substrate with BDFE(O–H)−1 and BDE(O–H) = 70.6 kcal mol−157 especially for [(susan6-Me){FeIV(μ-O)(μ-O2)FeIII}]3+.As it is not surprising0.1 Fig. . Using t0.1 Fig.  and the 6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ showed no reactivity with TEMPOH {FeIII(μ-O)(μ-O2)FeIII}]2+ should have a driving force for HAT significantly lower than 78 kcal mol−1 of [(susan6-Me){FeIV(μ-O)2(μ-O2)FeIII}]3+. The reaction of protonated [(susan6-Me){FeIII(μ-O)FeIII}]3+ with TEMPOH resulted in the reoccurence of the UV-Vis signature of the parent [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ {FeIII(μ-O)FeIII}]3+ and TEMPOH and suggests a protonation of TEMPOH60. The reaction of [(susan6-Me){FeIV(μ-O)(μ-O2)FeIII}]3+ with TEMPOH resulted in the reoccurence of the UV-Vis signature of the parent [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ {FeIV(μ-O)(μ-O2)FeIII}]3+ resulting in [(susan6-Me){FeIII(μ-O)FeIII}]3+ that reacts again by protonation of excess TEMPOH to [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+.The parent [{FeIV(μ-O)(μ-O2)FeIII}]3+ is capable for HAT from TEMPOH corroborated by the BDFE while the parent [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ and the protonated [(susan6-Me){FeIII(μ-O)FeIII}]3+ does not exhibit enough electrophilic character. However, [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}]2+ that shows no decay at −40 °C exhibits a change in the UV-Vis spectra at room temperature with the formation of the typical signature of complexes with a {FeIIIX(μ-O)FeIIIX} core38 without the observation of intermediates accompanied by deposition of an inhomogenous solid and a few single-crystals. The crystallographic analysis provided the structure of the decay product {FeIII(OH)(μ-O)FeIII(OH)}]2+61 with a disorder of the coordinated OH- ligands, which could be resolved to coordination of oxidized 6-methyl groups. At Fe1, only 20% is OH- while 80% is a carboxylate while at Fe2, 65% is OH− and 35% consists of a benzylalcoholato donor. NMR spectroscopy of the demetalated bulk decay product shows the formation of more than one product but a significant signal at 173.4 ppm for a benzoic acid group in the 13C NMR spectrum indicates that the hydroxylation of the 6-methyl group is not only a minor reaction path.Thus, only the oxidized 2+, catalyzes the oxidation of CH3OH with H2O2 to HCHO62. We could observe the μ-1,2-peroxo intermediate [(susan){FeIII(μ-O)FeIII}]2+. However, the temperature-dependencies ruled out this μ-1,2-peroxo intermediate to be the active species indicating the conversion to a high-valent active species. This conversion is faster in the presence of a proton suggesting a transient μ-1,2-hydroperoxo species.To mimic dinuclear active sites of metalloenzymes, we have developed a dinucleating ligand system with varying terminal donorsμ-oxo-diferric complexes with susan6-Me was not possible under identical aerobic conditions. Only the small ligand F- with H2O2 as oxidant allowed the isolation of [(susan6-Me){FeIIIF(μ-O)FeIIIF}]2+ 40. Its comparison to [(susan){FeIIIF(μ-O)FeIIIF}]2+ showed a steric repulsion between the 6-methyl group and the terminal F− ligand in cis-position, which explains the inaccessibility of susan6-Me complexes with larger terminal ligands as Cl− or OAc− that are easily accessible with susan. Thus, the μ-oxo-brigded core {FeIIIX(μ-O)FeIIIX} is not the thermodynamic sink for susan6-Me as it is for susan. Moreover, the steric repulsion of the 6-methyl group enforces longer Fe–N6-Me-py than Fe–Npy bonds68, and hence a lower electron donation. This results in an anodic shift of +250 mV making FeIV less accessible with susan6-Me than with susan.In contrast, formation of 6-Me should be able to stabilize a μ-1,2-peroxo complex {FeIII(μ-O)FeIII} that is with susan only a reactive intermediate decaying via a high-valent FeIV species to its thermodynamic sink {FeIIIX(μ-O)FeIIIX}. In constrast, with susan6-Me not only this thermodynamic driving force is absent but FeIV is also less accessible. Indeed, we could present here the synthesis and characterization of the stable μ-1,2-peroxo complex [(susan6-Me){FeIII(μ-O)FeIII}]2+. Inspection of the space-filling model {FeIV(μ-O)FeIII}]3+, which is stabilized by the additional highly covalent μ-1,2-peroxo ligand. It is interesting to note, that this high-valent μ-1,2-peroxo species stores one oxidation-equivalent more than intermediate Q of sMMO. Comparing the FeIVFeIII/FeIIIFeIII redox potential of E1/2 = 0.55 V to 0.41 V vs Fc+/Fc for the analogous redox couple of [(tpa6-Me){FeIII(μ-O)2FeIII}(tpa6-Me)]2+ 69 shows only a slightly lower electron-donating character of the μ-1,2-peroxo-bridge than a μ-oxo-bridge.Although susanμ-1,2-hydroperoxo-diferric complex [(susan6-Me){FeIII(μ-O)FeIII}]3+. Generally, protonation of a Fe-coordinated peroxo ligand is regarded to enhance its reactivity, e. g. protonation of the cis-μ-1,2-peroxo intermediate P of sMMO was proposed to promote the conversion to intermediate Q11. The relatively high stability of the μ-1,2-hydroperoxo complex [(susan6-Me){FeIII(μ-O)FeIII}]3+  is thus remarkable and must owe its origin to a low stabilization of the FeIV conversion product by susan6-Me.We could further demonstrate the reversible protonation to the 6-Me){FeIII(μ-O)FeIII}]2+, the μ-1,2-peroxo complex A is protonated at the μ-oxo-bridge forming a {FeIII(μ-OH)FeIII} species22 indicating different nucleophilicities. The nucleophilic character of a ligand should increase with less electron donation to the FeIII ions, i.e. less covalent, longer bonds. But for A, the FeIII-μ-Ooxo bonds are shorter than for [(susan6-Me){FeIII(μ-O)FeIII}]2+ (1.72/1.74 Å vs 1.82/1.89 Å), whereas the situation is reversed for the FeIII-μ-Operoxo bonds (2.07/2.10 Å vs 1.88/1.93 Å). This structural argumentation is in contrast to the experimentally determined protonation sites. However, the disorder of the μ-oxo/μ-1,2-peroxo ligands in A questions the significance of this comparison. Moreover, as protonation should occur at a pπ orbital and a Fe–O bond consists of σ- and π-bonding, a pure structural analysis does not need to provide the answer for the reactivity.In contrast to [{FeIII(μ-O)FeIII}]2+ is more intense (ε = 1180 M−1 cm−1) than the μ-peroxo → FeIII LMCT (ε = 1000 M−1 cm−1). Note that also the integrated absorption intensity is smaller in the susan6-Me complex than in A for the μ-1,2-peroxo→FeIII LMCT indicating - without the intention of a quantitative analysis - less charge-donation and hence more nucleophilic character of the μ-1,2-peroxo ligand. This UV-Vis spectroscopic argumentation is supported by a comparison of the vibrational signature in the rR spectra. Interestingly, the strongest difference is observed for the νs(Fe–O2–Fe), which are at 465 and 448 cm−1 for A and [(susan6-Me){FeIII(μ-O)FeIII}]2+, respectively, indicative for less covalent FeIII-μ-Operoxo bonds and hence a higher nucleophilicity of the μ-1,2-peroxo ligand in [(susan6-Me){FeIII(μ-O)FeIII}]2+.Thus, spectroscopic markers might provide a better correlation to the nucleophilic character of the peroxo group than structural parameters. Solomon and coworkers proposed to extract the donor strength of a given ligand from the integrated absorption intensities of all CT transitions associated with this ligandμ-1,2-peroxo-FeIIIFeIII complex that is not unexpected for such complexes55. Interestingly, also protonation to the μ-1,2-hydroperoxo-FeIIIFeIII species turned out to be not sufficient to increase the electrophilic character for HAT with substrates of weak to modest BDE (TEMPOH and DHA). Only the oxidized μ-1,2-peroxo-FeIVFeIII species reacts with the relatively weak substrate TEMPOH. The determination of the pKa = 9.5 ± 0.1 and the bond dissociation free energy BDFE(O–H)−1 of the protonated μ-1,2-hydroperoxo-FeIIIFeIII species quantifies this low electrophilic character even of the oxidized μ-1,2-peroxo-FeIVFeIII species. In this respect, the intramolecular C–H activation of preorganized 6-methyl pyridine groups to benzylalcoholato and carboxylato donors by the parent μ-1,2-peroxo-FeIIIFeIII complex is remarkable. Considering that this complex does not react with TEMPOH (BDE(O–H) = 70.6 kcal mol−1) and using the BDE(C–H) = 90 kcal mol−159 of the methyl group of toluene as an approximation for the BDE(C–H) of the 6-methyl groups of the coordinated pyridines, HAT should not occur via the bridging μ-1,2-peroxo-ligand. This indicates that this intramolecular reaction requires the conversion of the μ-1,2-peroxo-diferric core to a more reactive high-valent species as already postulated for the CH3OH oxidation62 of the analogous μ-1,2-peroxo-diferric complex of susan (vide supra).The study of the electrophilic reactivity demonstrated only a low electrophilic character of the parent 6-Me){FeIII(μ-O)FeIII}]3+ is an μ-1,2-hydroperoxo model complex and provides spectroscopic signatures for the assignment of postulated hydroperoxo intermediates in diiron enzymes: Upon protonation, the prominent μ-1,2-peroxo→FeIII LMCT around 14,000–16,000 cm−1 disappears and the isomer shift decreases. The presence of an μ-oxo-bridge is indicated by the typical μ-oxo→FeIII LMCTs around 12,000 and 19,000 cm−1 and large values of | ΔEQ | ≥ 1.3 mm s−1. The best signature to differentiate between a μ-1,1-hydroperoxo and a μ-1,2-hydroperoxo is the appearance of two strongly differing quadrupole doublets for the latter due to the strongly differing donation abilities of the two μ-1,2-hydroperoxo-oxygen atoms.[(susan6-Me){FeIII(μ-O)FeIII}]2+ to [(susan6-Me){FeIII(μ-O)FeIII}]3+ reflect the UV-Vis-NIR and Mössbauer spectroscopic differences between P and the more reactive P′ intermediates in non-heme diiron enzymes20. For the latter, a μ-1,2-hydroperoxo structure has been suggested, which is thus strongly supported by the results of this study.The protonation of (ClO4)2·MeOH precipitated at 0 °C, which were filtered off, washed three times with water, a small amount of cold MeOH, three times with Et2O, and dried under reduced pressure. Yield: 704 mg . IR (KBr): −1 = 3642 w, 3083 w, 2976 w, 2856 m, 1603 s, 1576 m, 1462 s, 1429 m, 1093 vs, 1009 m, 956 m, 847 m, 780 s, 723 w, 623 s, 490 m. ESI-MS (+) (CH2Cl2): m/z = 361.2 [(susan6-Me){Fe(μ-O)Fe}]2+. Anal. Found: C 44.90, H 5.76, N 11.51. Calcd. for [(susan6-Me){Fe(μ-OH)2Fe}](ClO4)2·H2O C36H54N8Cl2Fe2O11: C 45.16, H 5.68, N 11.70.A solution of susan6-Me){FeII(μ-OH)2FeII}](ClO4)2  in CH2Cl2 (2 mL) was added to a solution of 25% aqueous ammonia  in MeOH (6 mL) at −15 °C. Pouring an O2 stream of approximate 0.1 L min−1 through this yellow solution for 1.5 h at −15 °C results in immediately color change to dark green followed by precipitation of a black microcrystalline solid, which was filtered off, washed three times with cold water, a small amount of cold MeOH, three times with Et2O, and dried under reduced pressure. Yield: 52 mg . Anal. Found: C 43.34, H 5.20, N 11.02. Calcd. for [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}](ClO4)2·2H2O C36H54N8Cl2Fe2O13: C 43.30, H 5.55, N 11.22. For single-crystal X-ray diffraction and magnetic measurements, the sample was recrystallized by slow evaporation of a filtered (0.2 μm pores size PTFE filter) solution of [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}](ClO4)2·2H2O  in MeCN (20 mL) at −30 °C. The resulting black crystals of [(susan6-Me){FeIII(μ-O)(μ-O2)FeIII}](ClO4)2·0.85 MeCN·0.7 H2O were filtered off, washed three times with cold water, three times with cold MeOH, three times with Et2O, and dried under reduced pressure. Yield: 52 mg . IR (KBr): −1 = 3079 w, 2999 w, 2918 w, 2881 w, 2811w, 1605s, 1573 w, 1466 s, 1454 s, 1090 vs, 1002 m, 955 m, 933 w, 842 m, 832 w, 788 s, 686 m, 624 s, 500 w, 447 w. ESI-MS (+) (CH2Cl2/MeCN): m/z = 377.1 [(susan6-Me){Fe(μ-O)(μ-O2)Fe}]2+. Anal. Found: C 44.66, H 5.21, N 12.27. Calcd. for [(susan6-Me){Fe(μ-O)(μ-O2)Fe}](ClO4)2·0.8MeCN·H2O C37.6H54.4N8.8Cl2Fe2O12: C 44.97, H 5.46, N 12.27.A solution of [(susanSupplementary InformationPeer Review File


Lung cancer is the most common malignancy and leads to around one-quarter of all cancer deaths. Great advances have been achieved in the treatment of lung cancer with novel anticancer agents and improved technology. However, morbidity and mortality rates remain extremely high, calling for an urgent need to develop novel anti–lung cancer agents. 1,2,3-Triazole could be readily interact with diverse enzymes and receptors in organisms through weak interaction. 1,2,3-Triazole can not only be acted as a linker to tether different pharmacophores but also serve as a pharmacophore. This review aims to summarize the recent advances in 1,2,3-triazole–containing compounds with anti–lung cancer potential, and their structure–activity relationship (SAR) together with mechanisms of action is also discussed to pave the way for the further rational development of novel anti–lung cancer candidates. Lung cancer has high morbidity and represents the leading cause of cancer-related deaths  . With th1,2,3-Triazole moiety , a majorIn recent years, a variety of 1,2,3-triazole–containing compounds are designed, synthesized, and evaluated for their anti–lung cancer activity, and some of them have the potential to act on dual-/multi-targets simultaneously . This reChromene (benzopyran) and coumarin (chromen-2-one) moieties are present in a large number of biologically active compounds, and their derivatives are potent inhibitors of a variety of proteins, such as EGFR, tyrosine kinase, ERK1/2, PI3K, HSP 90, Bax, STAT proteins, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and telomerase associated with lung cancer . Therefo1  revealed that the fluoro atom on the phenyl ring contributes to the activity. However, 1,2,3-triazole is not crucial for the activity and replacement of 1,2,3-triazole by 1,2,4-triazole is also tolerated  against A549 cells , the most active against A549 cells, are less potent than the reference Chrysin (IC50: 8.80 μm).1,2,3-Triazole–containing chromene derivatives 1  possess olerated . In addi49 cells . Among t3  exhibit excellent activity against A549, taxol-resistant A549 (A549/taxol), and cisplatin-resistant A549 (A549/cisplatin) cancer cell lines, and an SAR study proved that the morpholino group at the R position could enhance the activity  is highly potent against A549, A549/taxol and A549/cisplatin lung cancer cell lines, and the resistance index (RI: ICresistant cells/ICsensitive cells) is approximately 1, demonstrating its potential for the treatment of drug-sensitive and drug-resistant lung cancers. The activity of DDO-6318 is at the same level as those of DDO-6106 (IC50: 0.46 μm) and gambogic acid (IC50: 0.29 μm) against A549/taxol cells but is around eight times superior to those of DDO-6106 (IC50: 0.46 μm) and gambogic acid (IC50: 0.29 μm) against A549 and A549/cisplatin cells. In the mouse model xenografted with A549, DDO-6318  displays significant inhibitory effect on the growth of inoculated A549 in mice without causing vascular irritation or weight loss, and its activity is higher than those of DDO-6101 (34.56% inhibition at 20 mg/kg) and 5-fluorouracil (64.71% inhibition at 20 mg/kg). Similar results were also observed when oral administraion of DDO-6318 . Thus, DDO-6318 can be considered as a new and orally active natural product–like anti–lung cancer candidate for further clinical studies.1,2,3-Triazole–containing chromene derivatives activity . In part4  are active against various cancer cell lines, and an antiproliferative SAR study against A549 cells illustrates that the methyl group at the R1 position could result in the improved activity  are more potent than cisplatin (IC50: 24.15 μm) against A549 cells. Moreover, compound 4a (IC50: 49.07 μm) shows low cytotoxicity toward mouse embryonic fibroblast cells NIH/3T3, and the selective index (SI: IC50(NIH-3T3)/IC50(A549)) is 16.5. A mechanistic study elucidated that compound 4a could induce sub-G1 phase arrest, increase apoptosis, decrease the mitochondrial membrane potential, and promote reactive oxygen species (ROS) production. 1,2,3-Triazole–containing coumarin derivatives 5  show weak to moderate activity against a panel of cancer cell lines, including A549 cells, and a strong correlation exists between lipophilicity profile and antiproliferative activity (5a (IC50: 8.87 μm) displays the highest inhibitory activity against A549 cells, but it is toxic against normal fibroblasts WI38 (IC50: 13.96 μm).Some 1,2,3-triazole–containing coumarin derivatives activity . Among tactivity . Compoun6 , show certain activity against A549 cells , histone deacetylase (HDAC), matrix metalloproteinase (MMP), NF-κB, tubulin, vascular endothelial growth factor, and vascular endothelial growth factor receptor 2 (VEGFR-2) kinase; thus, chalcones are endowed with broad-spectrum antiproliferative activity against drug-susceptible and drug-resistant cancers, even MDR cancers . Accordi7  show potential activity against A549 cells. An SAR study illustrated that the bromo group is essential for the activity, and replacement by heterocycles results in loss of activity  with single digital micromolar inhibitory activity are comparable to doxorubicin (IC50: 3.24 μm). Compounds 8  are active against A549 cells, and a mechanistic study revealed that these compounds could exert antiproliferative activity by inducing apoptosis and G2/S arrest as well as triggering mitochondrial potential loss  is considerably inferior to that of combretastatin-A4 (GI50: 0.08 μm) against A549 cells  show comparable activity to doxorubicin (IC50: 39.86 μm) against A549 cells  are more potent than doxorubicin (IC50: 69.33 μm) against A549 cells, demonstrating their potential to fight against lung cancer  against A549 cells reveals that the electron-withdrawing group at the R position could boost up the activity, and the representative compound 12a (IC50: 5.01–7.31 μm) not only exhibits the highest activity against the four tested cancer cell lines but also displays low cytotoxicity (IC50: 39.21 μm) toward normal NIH/3T3 cells  shows 85.4% tumor growth inhibition (TGI) after treatment for 16 days without causing obvious toxicities. Accordingly, compound 12a could be considered as a promising candidate for the chemotherapy of lung cancer.The antiproliferative SAR of 1,2,3-triazole–tethered chalcone-matrine hybrids T3 cells . A mechaIndoles, which could inhibit various enzymes and receptors, such as HDAC, proviral insertion site in moloney murine leukemia virus (Pim), and tubulin in cancer cells, are scaffolds to avail for developing novel anticancer agents . Thus, c13  against A549 cells revealed that the phenyl ring is crucial for the activity, and replacement by the naphthyl group results in loss of activity  display comparable activity to doxorubicin (IC50: 3.30 μm). Thus, these compounds can serve as promising lead candidates for further investigations. 1,2,3-Triazole–containing indole derivatives 14  and 15  show considerable activity against A549 cells, and the most potent compound 14a (IC50: 9.07 μm) is not inferior to doxorubicin (IC50: 4.39 μm)  .16  are active against A549 cells, and an SAR study implied that the para bromobenzyl group is more favorable than the alkyl group at the N-1 position of oxindole moiety (16a (IC50: 1.87 μm) is comparable to that of doxorubicin (IC50: 1.98 μm) but 3.8-fold superior to that of 5-fluorouracil (IC50: 7.24 μm). Further investigations demonstrated that compound 16a could arrest the A549 cells at the G2/M phase of the cell cycle and induce apoptosis in A549 cells through collapse of the mitochondrial membrane potential as well as elevation of intracellular ROS levels. Overall, compound 16a has the potential to act as a lead compound for the development of novel anticancer agents against lung adenocarcinoma A549 cancer cells.The majority of spirooxindole-derived morpholine-fused 1,2,3-triazoles e moiety . In part17  against NCI-H23 lung cancer cells disclosed that substituents at the C-3 and C-5 positions have great influence on the activity  is around 2 and 13 times superior to doxorubicin (IC50: 1.12 μm) and 5-fluorouracil (IC50: 8.84 μm), respectively. Some other 1,2,3-triazole–containing isatin compounds also hold certain activity against A549 cells, and the representative compounds 18a,b  demonstrate the highest activity  are highly potent against A549 cells, and compounds 19a-c (IC50: 21.1–29.4 nM) are comparable to podophyllotoxin (IC50: 27.1 nM)  . Mechani20  exhibit considerable activity against A549 cells, and an SAR study implies that introduction of a carbon spacer between 1,2,3-triazole and phenyl ring reduces the activity (20a-e (IC50: 0.97–1.96 μm) possess higher activity than the references podophyllotoxin (IC50: 4.60 μm) and etoposide (IC50: 1.97 μm), and the cytotoxicity (IC50: 53.86–89.04 μm) toward NIH/3T3 cells is much lower. A mechanistic study demonstrates that these hybrids could inhibit topoisomerase II, arrest G2/M phase of cell cycle, and induce apoptosis.1,2,3-Triazole–containing epipodophyllotoxin derivatives activity . Compoun21  show higher antiproliferative activity than their analogues 22 (IC50: 19.6->100 μm) against A549 cells, revealing that the amino linker to couple 1,2,3-triazole with coumarin moieties is more favorable than ether linker  are found much more potent than the reference etoposide (IC50: 25.6 μm) against A549 cells, and hybrid 21b could disrupt microtubules and induce cell cycle arrest at the G1 phase by regulating P21 and cyclin D1.1,2,3-Triazole–tethered podophyllotoxin-coumarin hybrids r linker . In addi23a possesses potent broad-spectrum antiproliferative activity, and the activity against A549 cells  is 2-fold higher than that of cisplatin (IC50: 9.24 μm) and etoposide (IC50: 11.92 μm) (23b (IC50: 1.52 μm) is six times superior to cisplatin (IC50: 9.24 μm) and etoposide (IC50: 11.92 μm) against A549 cells  . An SAR 49 cells .bis-glucose hybrids  are devoid of activity against A549 cells, but hybrid 24 (IC50: 3.84 μm) demonstrates higher activity than the references cisplatin (IC50: 6.15 μm) and etoposide (IC50: 11.92 μm) . Only hybrid 25 (IC50: 0.89 μm) displays excellent inhibitory activity against A549 cells, and the activity is 6.9- and 13.3-folds superior to those of cisplatin (IC50: 6.15 μm) and etoposide (IC50: 11.92 μm) (50: 15.38 μm) also shows relatively low cytotoxicity against normal BEAS-2B cells, and the SI is 17.2. Accordingly, hybrid 25 could serve as a lead compound for further investigations.The majority of 1,2,3-triazole–tethered epipodophyllotoxin-1.92 μm) . Similar1.92 μm) . This hyβ/PDGFR-β and VEGFR2, and some quinoline-/quinolone-based agents, such as anlotinib and lenvatinib, have already been approved for lung cancer therapy  are active against H460 and HCC827 human lung cancer cell lines, and compound 26  is more potent than chloroquinoline (IC50: 52 and 76 μm)  demonstrates promising activity against a series of cancer cell lines, including eight NSCLC (GI50: 0.25–1.91 μm), and a mechanistic study reveals that this hybrid could induce apoptosis  display potential activity against A549 cells, and an SAR study elucidates that the piperidinyl group at the R1 position enhances the activity (28a-c (IC50: 0.07–0.14 μm) are not inferior to foretinib (IC50: 0.11 μm). In particular, the most active compound 28a with an IC50 value of 2.27 nM against c-Met is identified as a multitargeted receptor tyrosine kinase inhibitor. Thus, this compound is worthy of further investigation. Some 1,2,3-triazole–containing 2-chloroquinoline derivatives, such as compound 29 , also display certain antiproliferative activity against lung cancer cells, but most of them are less potent than the references  is comparable to doxorubicin (IC50: 1.83 μm) against A549 cells  . A mechapoptosis . Compounactivity . Among tferences . Further49 cells .31a,b  is comparable to that of cisplatin (IC50: 3.5 μm) against A549 cells  shows promising activity against drug-sensitive NCI-H460 and MDR NCI-H460/R; probably due to that this compound could increase ROS production and induce mitochondrial damage in MDR cancer cells, demonstrating its potential against MDR lung carcinoma  exhibit excellent activity against A549 cells and low cytotoxicity (IC50: 25.24 and 16.05 μm) toward mouse mesangial cells  is more potent than vorinostat (IC50: 76.3 μm), revealing that these types of hybrids could be taken for further investigations  against A549 cells indicates that the methoxy group on the 2-arylpyridine moiety (R1 position) and the 3-phenoxy group on the benzyl group are favorable for the activity (35a-c (IC50: 1.023–1.148 μm) exhibit higher activity than the reference E7010 (IC50: 1.622 μm), and mechanistic studies reveals that these compounds could inhibit the microtubule assembly, arrest the cell cycle at the G2/M phase, and induce cell death by apoptosis. BD7  shows potent antiproliferative activity against various cancer cell lines, and its activity is identical to that of sorafenib (IC50: 0.08–0.30 μm)  is not inferior to doxorubicin (IC50: 38.2 μm) against A549 cells, and BTPT  with the thiophene moiety at the para position of the pyridine moiety holds enhanced activity against A549 cells  . Biologi49 cells .38 against A549 cells demonstrates that the methyl group on the 1,2,3-triazole motif and the fluoro on the phenyl ring are advantageous to the activity  are more potent than golvatoinib (IC50: 8.14 μm), and the most active compound 38b could block cells in the G0/G1 phase. 1,2,3-Triazole–containing isoxazole–thiazole–pyridine hybrids 39  has promising activity against A549 cells, and an SAR study implies that electron-withdrawing groups, especially the nitro group on the phenyl ring, could greatly increase the activity (39b (IC50: 0.01 μm) is 308-fold more potent than etoposide (IC50: 3.08 μm) against A549 cells, so it could serve as a lead compound for further development of novel anti–lung cancer candidates.An antiproliferative SAR study of 1,2,3-triazole–containing pyridine derivatives activity . The moractivity . The mos40  exhibit excellent activity against A549 cells, and an SAR study demonstrates that the fluoro at the R1 position and the trifluoromethyl group at the R2 position are beneficial for the activity  is higher than that of foretinib (IC50: 0.49 μm), and compound 40a (IC50: 1.68–4.57 nM) shows excellent inhibitory activity against c-Met, Flt-3, and PDGFR-β. Further study disclosed that incorporation of oxime into the pyrrolopyridine moiety  is permitted, and the most active compound 41 (IC50: 0.12 μm) could intercalate into calf thymus DNA efficiently to form a 41-DNA complex that might block DNA replication to exert its antiproliferative activity pyridines activity . The actactivity .42  show considerable activity against A549 cells, and an SAR study illustrated that a substitution at the R2 position, regardless if the substituent is an electron-donating or electron-withdrawing group, could reduce the activity as compared with hydrogen  are more potent than nocodazole (IC50: 1.47 μm) against A549 cells. Flow cytometry reveals that these compounds result in A549 cell cycle arrest at the G2/M phase, and further studies indicates that these compounds could inhibit tubulin and induce cell death by apoptosis.1,2,3-Triazole–containing imidazopyridines hydrogen . In partPyrimidines, quinazolinones, and nucleosides could inhibit various MDR proteins, such as P-gp and MDR-associated protein-1, and several pyrimidine-/quinazolinone-/nucleoside-based agents, such as azacitidine and gemcitabine, demonstrate high efficacy for a broad spectrum of cancers . Therefo43  is 3.3 folds more potent than 5-flurouracil (IC50: 7.86 μm) against NCI-H1650 cells, and mechanistic studies disclose that this compound can inhibit the migration of cancer cells and induce apoptosis  hold potential activity against A549 and the NSCLC cell line H2228 expressing EML4-ALK, and both of them display superior activity to crizotinib (IC50: > 1 μm) and ceritinib (IC50: > 1 μm) against A549 cells  is 5.2 times more potent than foretinib (IC50: 4.7 μm) against A549 cells  display inhibitory activity against A549 cells, and three compounds 46a-c (IC50: 0.03–0.14 μm) are 35–163.3 folds more potent than 5-fluorouracil (IC50: 4.9 μm), revealing the potential of these compounds as novel anti–lung cancer agents pyrimidine poptosis . 1,2,3-T49 cells . Further49 cells . A mechat manner . The majr agents .47a  and its derivative 47b (GI50: 15 and 17 μm) show considerable activity against SW1573 and A549 lung cancer cell lines  shows moderate activity against A549 cells  are more potent than pemetrexed (IC50: 3.29 μm) against A549 cells, and an SAR study illustrates that the chloro on the phenyl rings enhance the activity pyrimidinone-6-carboxylate 50  shows slightly lower potency than the reference doxorubicin (IC50: 0.14 μm) against A549 cells, demonstrating its potential as a lead compound for further development of new therapeutic drug candidates  are active against A549 cells, and the most active compound 51a (GI50: 0.03 μm) is more potent than the reference nocodazole (GI50: 0.08 μm)  to 5-fluorouracil (IC50: 2.80 μm) is also observed, but both the compounds 52a,b (IC50: 2.22 and 0.04 μm) display high cytotoxicity toward normal NIH 3T3 cells  also exhibits considerable activity against A549 cells, but it is less potent than combretastatin-A4 (IC50: 0.11 μm)  . SimilarT3 cells . Compoun0.11 μm) .54  possess potential activity against SK-Lu-1 NSCLC, and the activity is superior to that of erlotinib (IC50: 99.76 μm)  possess comparable activity to ellipticine (IC50: 1.38 μm), and molecular docking studies indicates that these compounds could exert antiproliferative activity through targeting EGFR.Most of 1,2,3-triazole–containing quinazoline derivatives 9.76 μm) . An SAR H)-one derivatives also demonstrate considerable activity against lung cancer cell lines, and an antiproliferative SAR study of compounds 55  against NCI-H23 lung cancer cells elucidates that introduction of substituents into the quinazolin-4(3H)-one motif, especially at the C-7 position, is unfavorable to the activity (55a-c (IC50: 2.01–2.69 μm) compared with doxorubicin (IC50: 1.29 μm) and SAHA (IC50: 1.44 μm), and compounds 55a-c are around six folds more potent than 5-fluorouracil (IC50: 13.45 μm). The most active compound 55a could induce early apoptosis and arrest at the G2/M phase. Thus, it could serve as a new lead for the design and development of potent anticancer agents.Some 1,2,3-triazole–containing quinazolin-4 are devoid of activity against SK-Lu-1 NSCLC, whereas the activity of hybrid 56  is at the same level as that of ellipticine (IC50: 1.30 μm), and 3.4 times higher than that of erlotinib hydrochloride (IC50: 31.15 μm)  also display certain activity against A549 cells, but the activity is considerably inferior to that of combretastatin-A4 (IC50: 0.11 μm)  . The 1,20.11 μm) .Steroids that are ubiquitous in natural resources are involved in several physiological functions and exhibit low cytotoxicity, high bioavailability, and excellent efficiency against diverse cancers, including lung cancers . Thus, c58  show promising cytotoxic potential against A549 cells, and the activity is higher than that of the parent betulinic acid (IC50: 23.0 μm)  against A549 cells indicates that introduction of electron-donating or electron-withdrawing groups into the phenyl ring is detrimental to the activity (59a-d (IC50: 5.54–7.23 μm) are comparable to BEZ-235 (IC50: 6.52 μm) but more potent than diosgenin (IC50: 13.3 μm). Most 1,2,3-triazole–containing dehydroepiandrosterone derivatives 60  are active against A549 cells, and these compounds exert antiproliferative effects by arresting cells in the G2 phase of the cell cycle and inducing apoptosis  . An SAR activity . Among tpoptosis .61  show promising activity against A549, NCI-H460, and NCI-H460/DOX lung cancer cell lines, and an SAR study reveals that the electron-withdrawing group at the ortho position of the phenyl ring is favorable to the activity (61a (IC50: 2.67–4.84 μm) is the most active against the three tested lung cancer cell lines, and the RI value is 1.08, suggesting its potential to fight against drug-resistant lung cancer. Moreover, compound 61a (IC50: > 50 μm) is nontoxic toward normal HL-7702 cells, and the SI values are > 10.3. Mechanistic studies illustrate that compound 61a is a potential NF-κB inhibitor and could induce apoptosis and suppress cell migration. Accordingly, rational design of 1,2,3-triazole–containing asiatic acid derivatives may offer a new class of NF-κB inhibitors with the ability to suppress cancer cell migration and induce apoptosis.1,2,3-Triazole–containing asiatic acid derivatives activity . The rep62a,b  show broad-spectrum antiproliferative activity, and compound 62a (IC50: 3.53 μm) is comparable to celastrol (IC50: 3.02 μm) against A549 cells  display comparable activity to doxorubicin (IC50: 6.36 μm), but it is more potent than dihydroartemisinin (IC50: 43.43 μm) against A549 cells, and this hybrid (IC50: > 100 μm) is nontoxic toward human erythrocyte and normal kidney cells (HEK-293)  . An SAR 64  show considerable activity against A549 cells, and an SAR study demonstrated that the alkyl or alkyloxy group at the para position of the phenyl ring has advantages to the activity (64b (IC50: 9.19 μm) is slightly more active than tamoxifen (IC50: 10.87 μm), and it could arrest the cell cycle at the G0/G1 phase and induce apoptotic cell death. Accordingly, compound 64b could be taken as a lead molecule for further development of potent anti–lung cancer therapeutic agents. In LL2  and Hepa1-6–xenografted mouse models, compound 65  demonstrates moderate in vivo anticancer efficacy with TGI rates of 62.5% and 80%, and the potency is higher than that of epacdostat (TGI: 50.0%)  . Moreove66  not only displays comparable activity to 5-fluorouracil (IC50: 37.9 μm) against A549 cells but also exhibits synergistic effect with 5-fluorouracil (IC50: 16.6 μm) (66 (IC50: 32 nM) is a potential aminopeptidase N inhibitor, and it is nontoxic toward normal HUVECs (IC50: > 2000 μm). In addition, in the H22-xenografted mouse model, compound 66 alone or in combination with 5-fluorouracil could effectively inhibit tumor growth without causing loss of body weight. 1,2,3-Triazole–tethered dihydropyridin-2-one-hydroxamic acid hybrid 67 (IC50: 35 and 49 nM) is a potential HDAC1 and HDAC6 inhibitor, and it  is as potent as SAHA (IC50: 8.9 μm) against A549 cells (67 (IC50: > 50 μm) is nontoxic toward normal RWPE-1 and VERO cells, and SI > 5.6. Compound 68 reveals outstanding potency (IC50: 1.1–10.3 nM) against EGFR, HDAC1, and HDAC6 and promising activity  against A549 and NCI-H1975 lung cancer cell lines, and its antiproliferative activity is also superior to that of vorinostat (IC50: 2.67 and 23.76 μm) (69 (IC50: 23.9 and 2.85 nM) demonstrates excellent inhibitory activity against HDAC1 and HDAC6, and the activity of this hybrid  is around five-fold higher than that of SAHA (IC50: 5.00 and 3.27 μm) against A549 cells  . Further49 cells . Moreove3.76 μm) . Thus, t49 cells .D-ribofuranose-pyridine hybrid 70  is 3.4-fold more potent than cisplatin (IC50: 17.7 μm) against A549 cells  are more active than 5-fluorouracil (IC50: 69.07 μm)  are highly potent against A549 cells, and mechanistic investigations demonstrate that these hybrids could inhibit the proliferation by inducing apoptosis and arresting the cell cycle at the G1 and S phases.Some 1,2,3-triazole–containing sugar derivatives also possess certain antiproliferative activity against lung cancer cell lines , and 1,249 cells , whereas9.07 μm) . An SAR 72  shows remarkable cytotoxic efficacy against A549 cells, and the activity is 3.7-fold higher than that of paclitaxel (IC50: 13.2 nM), revealing the potential of 1,2,3-triazole–containing colchicine derivatives as novel anti–lung cancer candidates  show higher activity than the parent rapamycin (IC50: 18.1 μm) against A549 cells, and compound 73 (50: 12.8 μm) could induce apoptosis and cell cycle arrest in A549 cells  are endowed with considerable activity against A549 cells, and an SAR study demonstrates that the trifluoromethyl group on the phenyl ring is beneficial for the activity , which is comparable to doxorubicin (IC50: 1.98 μm), demonstrates potent activity. Cell cycle analysis show that compound 74a could induce G2/M phase arrest and lead to the accumulation of cyclin B1 protein. Cell-based tubulin polymerization assays and docking studies implies that compound 74a disrupts tubulin assembly by occupying the colchicine binding pocket of tubulin.1,2,3-Triazole–containing bergenin derivatives activity . In part75  has excellent inhibitory activity to A549 cells, which is higher than those of pemetrexed (IC50: 3.39 μm), rucaparib (IC50: 4.91 μm), and harringtonine (IC50: 10.55 μm)  show enhanced activity against A549 cells, and the activity is ∼5 times superior to that of rucaparib (IC50: 4.69 μm)  . 1,2,3-T4.69 μm) . Flow cy77  are active against A549 cells, whereas the parent genipin (IC50: > 20.0 μm) is devoid of activity  are the most active against A549 cells. Thus, both of them merit further investigations. 1,2,3-Triazole–containing bavachinin derivative 78  is more potent than bavachinin (IC50: 30.5 μm) against A549 cells, and a mechanistic study reveals that this compound could induce apoptotic cell death through loss of MMP and PARP cleavage  show considerable activity against A549 cells, and most of them are more potent than the parent melampomagnolide B (IC50: 9.94 μm) (79d (IC50: 1.35 μm) shows significant efficacy by inducing apoptosis, inhibiting the proliferation and migration of cancer cells. Thus, this compound might be considered as a promising anticancer drug candidate for further evaluation. 1,2,3-Triazole–tethered Jiyuan Oridonin A–thiazolin-dione hybrids 80a,b  demonstrates higher activity than Jiyuan Oridonin A (IC50: 16.4 μm) against A549 cells, and further mechanistic studies reveals that hybrid 80a has inhibition on the proliferation of cancer cells by inducing apoptosis and arresting the cell cycle at the G1 phase  . The repG1 phase .81  possesses excellent antiproliferative activity against A549 cells, whereas the parent ʟ-ascorbic acid (IC50: > 100 μm) is inactive, suggesting that 1,2,3-triazole–containing ʟ-ascorbic acid derivatives may be promising for the discovery of novel anti–lung cancer agents  show potential activity against A549 cells, and the activity is superior to that of doxorubicin (IC50: 4.4 μm), revealing that these compounds warrant further investigations  against A549 cells is much higher than that of 5-fluorouracil (IC50: 3.47 μm), and this hybrid could induce the G2/M phase of cell cycle arrest and apoptosis, indicating that hybrid 83 could be used for further studies as anti–lung cancer candidates phenazine/paeonol/naphthalimide/sesquiterpene/sulfonate ester/sapinofuranone/triterpene/thiourea/benzothiazinone/benzoxazinone/furan/nimesulide  also posLung cancer, as a cancer with the highest morbidity, is the leading cause of cancer-related deaths and has already posed heavy burden on the world health system, which makes an urgent need to develop novel anti–lung cancer agents. 1,2,3-Triazole could readily interact with diverse enzymes and receptors in organisms through weak interaction and has been considered as a privileged structure in medicinal chemistry. 1,2,3-Triazole–containing agent CAI synergizes with sorafenib to combat NSCLC through the inhibition of NANOG and aggravation of apoptosis, indicating that 1,2,3-triazole–containing derivatives are useful scaffolds to develop novel anti–lung cancer agents.The azide-alkyne cycloaddition reaction is the major strategy to synthesize 1,2,3-triazoles. The thermal Huisgen 1,3-dipolar cycloaddition usually results in poor regioselectivity, but the Huisgen cycloaddition catalyzed by metal catalyst could give excellent regioselectivity. For example, the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) usually affords 1,4-disubstituted 1,2,3-triazoles, whereas ruthenium(II)-catalyzed azide-alkyne cycloaddition often provides 1,5-disubstituted triazoles. However, azides are energy-rich and potentially explosive substances, so synthesis of azides should be careful. Even though various 1,2,3-triazoles showed poor solubility, combination of the 1,2,3-triazole moiety with the other pharmocophores demonstrated good soluability, and may solve the issue of solubility.in vitro and in vivo efficacy. Overall, rational design of 1,2,3-triazole–containing derivatives may provide novel anticancer agents with excellent potency against various lung cancers, including drug-resistant forms.This review summarizes the current developments, mechanisms of action and SAR of a series of 1,2,3-triazole–containing derivatives , and some of them suggest great


Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines.1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h.In this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production.The 1-OH-PHZ production of The online version contains supplementary material available at 10.1186/s12934-021-01731-y. As tely used , 6. Althely used , the conely used . Most naely used , 2-hydroused [2) , and pheused [2)  have beePseudomonas aeruginosa, Pseudomonas monteilii, Nocardiopsis dassonvillei (1.0 mg extracted from 96 L fermentation medium), and Streptomyces sp. (30 mg extracted from 20 L fermentation medium) [Pseudomonas chlororaphis H18 in the natural plant rhizosphere, which was confirmed to produce phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ). The titers of both compounds are higher than most of the known wild type Pseudomonas strains. Through genome sequencing and protein sequence alignment, the biosynthetic gene cluster of PCA in P. chlororaphis H18 was located. The core loci phzABCDEFGO is responsible for PCA and 2-OH-PHZ biosynthesis  is a phenazine family compound separated from  medium) –13. Prevhis GP72  , 2-OH-PHZ (2), and 2-hydroxy-phenazine-1-carboxylic acid (3). Using genome-mining and sequence alignment, their biosynthetic gene cluster was confirmed  generation was detected and the production of 2-OH-PHZ (2) and 2-hydroxy-phenazine-1-carboxylic acid (3) was abolished  and verified its activity by whole-cell catalysis  to catalyze PCA , which limited its industrial application. To address this issue, the promotion of the 1-OH-PHZ conversion rate is an essential job. We know that enzymes are the most critical factors in the biosynthesis process of natural products, especially those rate-limiting step enzymes. In the 1-OH-PHZ biosynthetic pathway, the flavin-dependent hydroxylase PhzS catalyzes the crucial step of decarboxylation and hydroxylation at PCA 1-position. We believe that optimizing PhzS will be an effective way to improve pathway efficiency. Semi-rational design and molecular remodeling are feasible methods for promoting protein catalytic performance. First, PCA was docked into the crystal structure of PhzS (PDB: 2RGJ) as a ligand to predict the PCA-binding hydrophobic pocket or activity site dominated by N48, L76, M205, V207, R215, V217, Y219, M238, P317, M318, G319 [ mg/L, whM318A/V207I gene to replace PhzS in PH18-1, the constructed strain PH18-1–3 produced 202.4 mg/L 1-OH-PHZ and more PCA is consumed.The PH18-1–2 fermentation result showed that there is still a certain amount of precursor PCA in the fermentation broth. Afterward, we try to enhance the conversion of PCA to 1-OH-PHZ by adding PhzS copy numbers. When we inserted two copies of the PhzS P. chlororaphis H18. Chorismate, a shikimate pathway end product is the key phenazine derivative precursor. Several enzymes such as phosphoenolpyruvate (PEP) synthetase PpsA, transketolase TktA, quinate/shikimate dehydrogenase AroE, and dehydroquinic acid synthase AroB have been studied to enhance the shikimate pathway and chorismate [P. chlororaphis H18 genome. Hence, we planned to introduce one more copy of ppsA, tktA, aroB, and aroE genes into P. chlororaphis H18. Besides, RpeA, RsmE, PsrA, and Lon have been reported as negative regulators in Pseudomonas chlororaphis strains [aroE, tktA, aroB, and ppsA genes from P. chlororaphis H18 to replace the possible negative regulatory genes repA, rsmE, psrA, and lon to obtain strains PH18-1-4, PH18-1-5, PH18-1-6, PH18-1-7 respectively  semi-rational design remodeling of critical enzyme PhzS, (2) blocking 2-hydroxyphenazine synthesis to reduce the consumption of intermediate PCA, (3) enhancing the precursor pool by adding copy numbers of shikimate pathway genes or directly feeding shikimic acid into the fermentation system, (4) engineering regulatory genes, etc. The 1-OH-PHZ concentration of the final engineered strain PH18-1-8 reached 938.5 mg/L in 50 mL shake flasks, which is 12 times more than the original strain PH18-1. Finally, through fed-batch optimization, the titer of 1-hydroxyphenazine reached 3.6 g/L in a 5 L fermenter in 54 h, which is the highest reported to date. This research paved a way for the further combinatorial biosynthesis and metabolic engineering of 1-hydroxyphenazine and its derivatives.In this study, we screened a PCA and 2-OH-PHZ producing strain Escherichia coli DH5α, BL21 (DE3), Rosetta (DE3) and S17-1 were cultured at 37 °C in a Luria–Bertani (LB) medium. P. chlororaphis H18 and mutants were grown in King’s B (KB) media. For fermentation, one colony of P. chlororaphis H18 was picked from the KB plate and inoculated into 3 mL of the KB liquid medium as a seed culture at 30 °C with 200 rpm of shaking overnight. Then, the seed culture (1 mL inoculum) was inoculated into 50 mL KB liquid medium in 250 mL flasks at 30 °C with 200 rpm for 24–72 h. When required, chloromycetin  or kanamycin  was used in the media. The production of 1-hydroxyphenazine was tested with different media , PPM medium contained 22 g/L tryptone, 20 g/L glucose, 5 g/L KNO3, pH 7.5; PCM medium contained 30.0 g/L soybean meal, 7.5 g/L corn steep liquor, 10.0 g/L glucose, 10.0 g/L tryptone, 1.0 g/L MgSO4, 2.5 g/L KNO3, pH 7.5; PPN medium contained 22 g/L Tryptone, 20 g/L glucose, and 15 g/L KNO3, pH 7.5; SCM medium contained 65.02 g/L soybean meal, 15.36 g/L corn steep liquor, 12 g/L glucose, 21.70 mL/L ethanol, and 1 g/L MgSO4, pH 7.5. The DNA sequencing and Polymerase chain reaction (PCR) primers were performed by Tsingke Biotechnology Co.,Ltd. The genes were synthesized by Beijing Liuhe BGI Co.,Ltd. DNA extraction and kits for plasmid preparation were purchased from Omega.The strains and Plasmids used in this work are indicated in Additional file P. chlororaphis H18 was obtained by amplification of primers 16 S rRNA (27F) and 16 S rRNA (1492R), the PCR product of which was sequenced by Tsingke BiotechnologyCo.,Ltd. The whole genome sequencing of P. chlororaphis H18 was performed by GENEWIZ Biotechnology Co., Ltd.The 16 S rRNA sequence of sacB vector. Primers F/R were used to amplify the fragment of overexpressed gene and its promotor using synthetic gene or total DNA of P. chlororaphis H18 as a template. Meanwhile, primers LF/LR and RF/RR were used to amplify the fragment of target gene upstream and downstream, respectively. Since the success rate of four-fragment ligation is too low, the plasmids were constructed step by step. Utilizing pK18mobsacB as a vector, it is connected to the downstream fragment of target gene and overexpressed gene through T4 ligase , and then connected to the upstream fragment of the target gene. The correctly sequenced plasmid was introduced into S17-1 (λ pir) and transferred into P. chlororaphis H18 through conjugative transfer, and the target gene was replaced with an overexpressed gene by double crossover. Eventually, the successful replacement was verified by PCR.Homologous recombination was used for gene replacement. The construction of plasmid included three fragments and pK18mobPhzS mutants were constructed by the splicing-by-overlap-extension method. The sequences upstream and downstream of the mutation site were amplified first individually from pET30a-PhzS. The upstream of primer pairs were F1/R1 and the downstream of primer pairs were F2/R2. The PCR products of the above primer pairs were used as templates for another round of PCR using primers F1 and R2. The PCR products of the second round were digested with restriction enzymes BamHI/XhoI and ligated into the BamHI/XhoI sites of pET-30a containing an N-terminal 6xHis-tag. The mutations were verified by DNA sequencing.3OH and evaporated to dryness under vacuum at 40 °C with 3 g silica gel  and applied uniformly onto the top of the column. Gradient elution of the column was carried out with mixtures of Petroleum ether, dichloromethane, and ethyl acetate in increasing order of polarity. Fractions of 150 mL each were collected and a total of 36 fractions were collected and pooled based on their TLC profile into 8 fractions. It was further purified by preparative thin-layer chromatography (PTLC) on pre-coated Silica Gel plates . The PTLC plates were developed in CH2Cl2. The desired band was scratched out of the PTLC plate after air-drying visualized under UV light (254 nm) and the metabolite was extracted with MeOH.Fermentation was performed in 500 mL flasks containing 100 mL of the PPM medium and incubated for 3 days at 30 °C with 200 rpm for a total of 1 L. The broth was extracted with ethyl acetate three times and the extract was evaporated to dryness under vacuum at 40 °C. Chromatography column (5 × 20 cm) was packed with silica gel (50 g). The crude extract was dissolved in 50 mL of CHg for 10 min, the organic phase was taken into the bottle and detected with HPLC at 250 nm. The mobile phase detected by HPLC was acetonitrile and water containing 0.1% TFA. 3 μL of extracted phenazine compounds sample was taken for HPLC analysis (Nexera XR) under the following conditions: C18 reversed-phase column (Agilent 5 TC-C18 (2) (250 × 4.6 mm)) eluted with acetonitrile and water containing 0.1% TFA. 1-OH-PHZ production was quantified using peak area (A) in HPLC elute according to the following formula: 1-OH-PHZ (mg/L) = A/21,033.7–9.45, which was derived from a dose-peak area plot using purified 1-OH-PHZ with a correlation co-efficiency (R2) of 0.999. Liquid chromatography high-resolution mass spectrometry (LC-HRMS) analysis was performed on an Agilent Technologies 6520 Accurate-Mass Q-TOF LC–MS instrument with an Agilent Eclipse Plus C18 column (4.6 × 100 mm). LC detection procedure: 15% CH3CN in H2O from 0 to 5 min, 15% to 95% CH3CN in H2O from 5 to 12 min, 95% CH3CN in H2O from 12 to 15 min, 95% to 15% CH3CN in H2O from 15 to 18 min, 15% CH3CN in H2O from 18 to 20 min (H2O and CH3CN containing 0.1% formic acid (vol/vol)). The flow rate of 0.2 mL/min was used. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker Biospin 600 MHz spectrometer.In order to extract the phenazine compounds, 5 mL of fermentation culture was mixed with 250 μL of diluted HCl and extracted three times using an equal volume of ethyl acetate with vigorous shaking. After centrifugation at 13,000×3OH. After centrifugation at 13,000×g for 10 min, the mixture was taken into the bottle and detected with HPLC at 250 nm. HPLC detection procedure: 15% to 30% CH3CN in H2O from 0 to 5 min, 30% to 60% CH3CN in H2O from 5 to 10 min, 60% to 80% CH3CN in H2O from 10 to 15 min, 80% CH3CN in H2O from 15 to 18 min, 80% to 15% CH3CN in H2O from 18 to 20 min, 15% CH3CN in H2O from 20 to 28 min (H2O and CH3CN containing 0.1% TFA). The flow rate of 1 mL/min was used.The enzymatic activity of PhzS mutants was measured and the reaction mixture (100 μL) contained substrate (0.5 mM), NADH (50 μM), PhzS mutants whole-cell in 50 mM HEPES (pH 7.5) at 30 °C for 1 h. The reaction was quenched by an equal volume of CHBipolaris maydis, Alternaria solani, Fusarium graminearum, Phytophthora parasitica, Aspergillus flavus. Paper discs of 5 mm diameter were autoclaved at 121 °C for 20 min. On this paper discs, 1-hydroxyphenazine and phenazine-1-carboxylic acid (dissolved CH2Cl2) was slowly applied in sterile conditions and the concentrations was 40 μg/disc. Paper discs with CH2Cl2 alone were used as control. The plates were incubated at 30 °C. These discs were then used to check the antibacterial activity of 1-hydroxyphenazine and phenazine-1-carboxylic acid.The antibacterial activity 1-hydroxyphenazine and phenazine-1-carboxylic acid was tested by paper-disc diffusion assay against five plant pathogenic fungi, Using KB medium for seed culture, and then inoculating 500 μL of seed into 50 mL of KB medium in a 250 mL triplicate baffled Erlenmeyer flasks at 30 °C with 200 rpm for 12 h. The 50 mL seed culture was added into a 5 L fermenter containing 2 L PPM medium at 30 °C with an airflow rate of 3 L/min. The dissolved oxygen was kept at 30% by adjusting the agitation speed from 300 to 600 rpm. When the glucose is exhausted, the solution containing 500 g/L glucose was added into the fermenter at 2 mL/h and maintained 0.5–1 g/L of glucose.Additional file 1.  Tables of list of strains, plasmids (Table S1), primers (Table S2), comparison of P. chlororaphis H18 with GP72 (Table S3) and DNA sequences of phzS (Table S4). Antagonistic activity of phenazine-1-carboxylic acid and 1-hydroxyphenazine against plant pathogenic fungi (Table S5). And figures of PhzS mutation , Generation of mutant for P. chlororaphis H18  and schematic of engineering strains of P. chlororaphis H18 for 1-OH-PHZ production . 1 H NMR and 13C NMR spectra of 1-hydroxyphenazine .


Terminalia chebula var. tomentella (Combretaceae) led to the isolation of two new lignan glucosides with a furofuran skeleton, termitomenins F (1) and G (2). In addition, 19 known compounds including five lignan glucosides (3–7), six hydrolyzable tannins (8–13) and eight simple phenolics (14–21) were also identified. Their structures were determined by comprehensive spectroscopic analyses. It is noted that 8 and 9 were C-glycosidic hydrolyzable tannins with one hexahydroxydiphenoyl and one gallagyl group linked to an open-chain glucosyl C-1/O-2/O-3 and O-4/O-6, respectively, which were rarely found in plants. Nine known compounds, 6–9, 13, and 18–21, were procured from the titled plant for the first time, while 3–5, 10–12 and 14–17 were also found in the fruits. Notably, the known hydrolyzable tannins 8–13 exhibited stronger α-glucosidase inhibitory activities with IC50 values ranging from 0.10 to 3.12 μM, than the positive control, quercetin (IC50 = 9.38 ± 0.33 μM).The extensive chemical investigation on the branches and leaves of The online version contains supplementary material available at 10.1007/s13659-021-00314-z. Terminalia Linn, the second largest genus in the family Combretaceae, is distributed globally in the tropical and subtropical areas. Among which, some species, such as T. catappa Linn, T. bellirica Roxb, and T. chebula Retz, are widely used medicinal plants. Particularly, T. chebula is a famous and commonly used medicinal plant in Ayurveda, Tibetan, and traditional Chinese medicinal systems. So far, 39 Terminalia species have been chemically and pharmacologically studied, from which 368 compounds, including terpenoids, hydrolyzable tannins, flavonoids, lignans, phenols and glycosides with a wide range of bioactivities, e.g., liver and kidney protection, antibacterial, anti-inflammatory, anticancer, immune regulation, anti-diabetes, and wound healing, were reported  asarininone (5) ,pyranose  − , indicated a molecular formula of C27H32O12, corresponding to 12 unsaturation degrees. In the 13C NMR and DEPT spectra of 1 (Table δC 100–160) arising from two benzene rings, one methylenedioxy (δC 102.7), four aliphatic methines  with two oxygen-bearing ones , two oxymethylenes , and two methoxys  were observed, in addition to a glucosyl moiety . The 1H NMR spectrum showed the presence of one trisubstituted  and one 1,2,3,4-tetrasubstituted  benzene rings. These 1H and 13C NMR features were closely related to those of termitomenin D (3), a furofuran lignan glucoside isolated from the fruits of the titled plant , and six carbon signals  from a glucosyl moiety. The 1H NMR spectrum of 2 showed characteristic signals of two ortho-coupled aromatic protons at δH 6.53, 6.88 , one two-proton singlet at δH 6.68 , one anomeric proton at δH 5.39 , and a set of signals from δH 2.99 to 5.18 due to a furofuran skeleton, a glucosyl and a methylenedioxy moieties. The 13C and 1H NMR features of 2 were quite similar to those of 1, except that 2 had one more methoxy group (δC 61.2) than 1. Compound 2 was also a furofuran lignan glucoside. Instead of the 1,3,4-trisubsituted benzene ring in 1, a symmetric 1,3,4,5-tetrasubstituted benzene ring appeared in 2. These were further confirmed by the 2D NMR experiment. In the HMBC spectrum of 2, δH 3.84  and 3.74  showed respectively correlations with δC 154.8  and 138.6 (C-4′), indicating the three methoxy groups were at C-3′, C-4′ and C-5′  with H-6 (δH 6.88) / H-8′ (δH 2.99), and H-8′ with H-2′ (δH 6.68) / H-6′ indicated that H-7 and H-8, and H-7′ and H-8′ were at the opposite orientations, respectively. This was confirmed by the ROESY correlations of H-7 with H-9β, H-8 with H-9 α, H-7′ with H-9′β, and H-8′ with H-9′α. Eventually, the ECD calculation of -2 matched well with the experimental ECD curve of 2, revealed the absolute configuration of 2 as 7S,8R,7′S,8′R , while the other compounds did not show obvious inhibitory activity at a concentration of 50 μM. Among them, compound 12 with five galloyl group in molecular showed the strongest effect on α-glucosidase, followed with 10 > 9 > 8 > 11 > 13 by the order of activity strength. When the open-chain glucosyl moiety was fixed by a C-glycosidic bond with a HHDP group (8 and 9), it displayed less activity than that one (10) with a free glucosyl anomeric center. Moreover, compound 11 with nine phenol OHs arising from three galloyl groups showed a little bit stronger activity than 13 with the same nine phenol OHs from one galloyl and one HHDP groups. The results suggested that galloyl and HHDP groups may play a vital role for the α-glucosidase inhibitory activity of these compounds (8–13), much stronger than those lignan glucosides and phenolic compounds.Compounds controls . As showd4 or D2O with Bruker Ascend 600 and AV-800 spectrometers. Coupling constants were expressed in hertz (Hz), and chemical shifts were recorded in a δ  scale with TMS  as an internal standard. ESI mass spectra were measured on a VG Auto Spec300 spectrometer. High-resolution electrospray ionization mass (HRESIMS) spectra were measured on an API QSTAR Pular-1 spectrometer. Optical rotations were taken on JASCO P-1020 digital polarimeter. IR spectra were recorded on Bio-Rad FTS 135 series spectrometer with KBr pellets. UV spectra were given on and UV-2410PC Shimadzu spectrometer. Column chromatography (CC) was performed on 25–100 μm Sephadex LH-20 , 75–100 μm MCI-gel CHP20P , and 100–150 mesh silica gel . Thin-layer chromatography (TLC) was performed on precoated 0.20–0.25 mm thick silica gel GF254 plates , Agilent series 1260 (Agilent Technologies) were used for semi-preparative HPLC with an Agilent ZORBAX SB-C18 column . Acetonitrile (chromatographic grade) were purchased from Merck . Water was purified in a Milli-Q apparatus (Millipore). 4-Nitrophenyl-α-D-glucopyranoside (PNPG) was procured from America Sigma Chemical Co. α-Glucosidase and quercetin were purchased from Sigma Chemical . Potassium phosphate buffer solution (PPBS) was purchased from Shanghai Xilong Biochemical Technology Co. Ltd. 96-well plates was obtained from Qingdao Haiyang Chemical Co., Ltd.One- and two-dimensional NMR spectra were determined on methanol-Terminalia chebula var. tomentella (Kurz) C. B. Clarke (Combretaceae), identified by Dr. En-De Liu from Kunming Institute of Botany, Chinese Academy of Sciences, were collected in Lincang County, Yunnan Province, P. R. China, on November 2017. A standard sample (KIB-1-Z-20171102) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resource in West China of Kunming Institute of Botany.The branches and leaves of T. chebula var. tomentella (10 kg) were crushed and extracted by 60% aqueous acetone solution at room temperature. After removal of organic solvent by rotary evaporator at 45 °C under reduced pressure, the crude aqueous extract (3 L) was applied to a Diaion HP-20 column chromatography (water–methanol 1:0–0:1) to obtain three fractions, Fr. I—III. Fr. I (355.9 g) was applied to repeated CC over Sephadex LH-20, MCI-gel CHP20P, Toyopearl HW-40F, and MCI-gel CHP20P, eluting with water–methanol (1:0–0:1), to furnish compounds 7 (5 mg), 8 (3 mg), 9 (3 mg), 10 (55 mg), and 11 (15 mg). Fr. II (163.9 g) was applied to repeated CC over Sephadex LH-20, MCI-gel CHP20P, Toyopearl HW-40F, and Rp18, eluting with water–methanol (1:0–0:1), to obtain compounds 3 (5 mg), 4 (2 mg), 5 (2 mg), and 14 (3 mg). Fr. III (993.4 g) was separately chromatographed over Sephadex LH-20, MCI-gel CHP20P, Toyopearl HW-40F water–methanol (1:0 − 3:7), and silica gel CC eluting with CHCl3/MeOH 15:1–1:1, to yield compounds 1 (5 mg), 2 (5 mg), 6 (20 mg), 12 (13 mg), 13 (10 mg), 15 (16 mg), 16 (4 mg), 17 (3 mg), 18 (4 mg), 19 (5 mg), 20 (0.9 mg), and 21 (6 mg).The air-dried branches and leaves of α]22D ‒ 13.2 . UV (MeOH) λmax (log ε) 205 (3.65) nm. IR (KBr) νmax 3399, 2923, 2875, 1467, 1260 cm−1. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table m/z 593.1885 [M + 45 (COOH)]− .Colorless oil, [α]22D ‒ 4.1 . UV (MeOH) λmax (log ε) 205 (3.71) nm. IR (KBr) νmax 3409, 2925, 1630, 1465 cm−1. 1H (600 MHz) and 13C (150 MHz) NMR (in methanol-d4) data, see Table m/z 613.1700 [M + Cl]− .Colorless oil, [−1. The results indicated six lowest energy conformers for compounds 1 and 2. Then, the conformers were re-optimized using DFT at the PBE0-D3(BJ)/def2-SVP level in Methanol using the polarizable conductor calculation model by the ORCA-4.2.1 program [σ = 0.30 for all) [1 and 2 were gained by weighing the Boltzmann distribution ratio of each geometric conformation.Conformational analyzes were conducted by random searching in the Sybyl Software X 2.0 using the MMFF-94S force field with an energy shortoff of 2.0 kcal mol program . The enefor all) . Eventuad-glucopyranoside (PNPG) was used as an enzyme inhibitor screening model. α-glucosidase solution (0.025 U·mL−1), PNPG (0.1 M), phosphate buffer (pH 6.8) and test samples (50 μM) were incubated in 96-well plates at 37 °C for 1 h. A microplate reader was recorded by the absorbance at 405 nm. No enzyme as the blank readings were subtracted from each well and compared to the control. In this assay, all reactions were performed in triplicate. The α-glucosidase inhibitory activity was presented as inhibition ratio. The formula to calculate the inhibition rate is as follows: inhibition rate (%) =  × 100%, and IC50 values were calculated according to the Reed and Muench method [.The α-glucosidase inhibitor screening assay was conducted as reported previously and slightly modified . Querceth method .1–21) including seven lignan glucosides (1–7), six hydrolyzable tannins (8–13) and eight simple phenolics (14–21) were isolated and determined from the branches and leaves of T. chebula var. tomentella. Compounds 1 and 2 were two new lignan glucosides with a furofuran skeleton, while 8 and 9 were C-glycosidic hydrolyzable tannins with one HHDP and one gallagyl groups linked to an open-chain glucosyl C-1/O-2/O-3 and O-4/O-6, respectively, rarely found to exist in plants. Compounds 1–2, 6–9, 13, and 18–21 were obtained from the titled plant for the first time, while 3–5, 10–12, and 14–17 were also find in the fruits. The hydrolyzable tannins 8–13 exhibited strong α-glucosidase inhibitory activities.In conclusion, 21 phenolic compounds (Supplementary file1 (DOCX 4492 kb)Below is the link to the electronic supplementary material.