Their physicochemical properties can be modulated by an appropriate choice of N‐substituents and counteranion. Tested against a panel of human cancer cell lines, the complexes were shown to possess promising antiproliferative activity and to circumvent multidrug resistance. Interestingly, most derivatives also retained a significant cytotoxic activity against human cancer 3D cell cultures. Among them, the complex with R=4‐C6H4OMe and R’=Me emerged as the best performer of the series, being on average about six times more active against cancer cells than a noncancerous cell line, and displayed IC50 values comparable to those of cisplatin in 3D cell cultures. Mechanistic studies revealed the ability of the complexes to release carbon monoxide and to act as oxidative stress inducers in cancer cells.A straightforward two‐step procedure via single CO removal allows the conversion of commercial [Fe A class of cationic diiron complexes containing a variable bridging iminium ligand displays promising antiproliferative activity towards a panel of 2D and 3D cancer cell lines, and a selectivity not correlated to a different degree of uptake in cancerous and noncancerous cells. The mode of action appears to be mainly related to the interference with cell redox processes , initiated by intracellular CO release. Remarkably, 3D cell culture studies constitute an excellent base for more advanced in vivo cancer research, surpassing the traditional assays on monolayer cultures;To the best of our knowledge, the present 3D investigation is one of the first ones ever reported on iron compounds. The proposed diiron complexes combine within a cationic structure two Cp rings, three carbonyls and a variable bridging aminocarbyne ligand, the latter tuning the water solubility, the lipophilicity and the activity. Several experiments aimed to clarify the mechanism of action of the complexes will be discussed.1A–H were prepared by the thermal substitution reaction of the corresponding isocyanides with a slight molar excess of [Fe2Cp2(CO)4] in acetonitrile.2–5]+ 2} core, connected to a further CO and an aminocarbyne group occupying the two bridging sites. Along this series of complexes, the C(4)‐N(1) distance does not significantly vary, indicating a substantial double‐bond character which accounts for the complementary description of the bridging ligand as iminium (see below).New compounds were fully characterized by elemental analysis, IR and multinuclear NMR spectroscopy. In addition, the structures of ACF3SO3, 2C]+ was calculated and compared to that of the parent compound cis‐1C with a bridging cyclohexyl‐isocyanide ligand (CNCy). DFT‐optimized structures and relevant bonding parameters are reported in Table S1 and Figure S1. In 1C, the (μ‐C)−N Mayer bond order (b.o.)iPr instead of CNCy].2 C]+. The nature of the nitrogen substituents has a negligible influence, and for instance the corresponding b.o. is 1.54 in the case of [2A]+, the related bond length being consistent with the crystallographic characterization of [2A]CF3SO3 + as an almost perfect aminocarbyne‐iminium hybrid structure } ligands in dimetal complexes is not clearly defined in the literature, therefore we performed a DFT study to shed light on this point. The representative structure of C+ was ca2Cp2Cl(CO)(μ‐CO){μ‐CNMe(Cy)}], 6 . Based on a comparison of the 1H NMR and IR spectra with data available in the literature for similar compounds,6 exists in CDCl3 solution as a nearly equimolar mixture of two isomers, maintaining the cis‐geometry of the Cp ligands  and differing in the orientation of the N‐substituents with respect to the chloride (E/Z isomers). This feature suggests that the (μ‐C)−N bond in the neutral 6 still holds a substantial double bond character. The X‐ray structure of the E isomer was determined and is shown in Figure S2, although the low quality of the crystals prevents a detailed discussion of bonding parameters.Then, in order to clarify the possible effect of the net ionic charge of the complex, we also synthesized and characterized the novel neutral derivative +, with only a slight decrease of the (μ‐C)−N bond order (from 1.58 to 1.52). It is interesting to note that the bond length of the bridging carbonyl in 6 is very close to that in 1C (1.187 vs. 1.188 Å), highlighting that the isocyanide/CO and aminocarbyne/Cl combinations supply very similar electron densities for back‐donation to the diiron framework.In agreement with the NMR evidence, DFT outcomes indicate that 2Cl2 solution), the vibrations due to the terminal carbonyl ligands occur in the ranges 2018–2028 (anti‐symmetric stretching) and 1985–1996 cm−1 (symmetric stretching), while the band related to the bridging carbonyl is found at 1833–1853 cm−1. Moreover, the absorption attributed to the carbyne‐N bond falls in between 1530 to 1602 cm−1, the higher values being associated to the N‐substituents with higher electron‐donor ability. The 1H and 13C NMR spectra  display one set of resonances, ascribable to a single isomeric form featuring the Cp ligands in mutual cis position, as found in the X‐ray structures and confirmed by 1H NOE experiments in the case of [4G]+ . Two resonances for the Cp and terminal CO ligands are distinguishable in the unsymmetrical complexes, due to hampered rotation around the partially double C−N bond (see above); accordingly, such resonances coincide in the complexes [2F]+ and [5H]+, displaying two identical N groups. In the 13C NMR spectra, the bridging {CN} unit is detected at low fields , as it is typical for bridging aminocarbynes.[2B]+ features a 31P NMR resonance at 17 ppm for the phosphonato group, and the 13C signal for the carbyne appears as a doublet (3JCP ca. 6 Hz).Salient spectroscopic features of all complexes are summarized in Table S2, and NMR and IR spectra are supplied in Figures S3–S32. In the IR spectra (CH2C]CF3SO3 and [2C]Cl in chlorinated solvents are ascribable to the presence of ion pairs (or higher aggregates);1H NMR spectra in protic polar solvents (D2O and CD3OD), where ion pairs are practically absent.Small differences between the spectroscopic data of Cl. The octanol/water partition coefficients (log Pow) were measured by a UV/Vis method and fall in between 0 and −1, reflecting an amphiphilic or slightly hydrophilic character. An exception is represented by [2B]BF4, wherein the marked hydrophilicity imparted by the diethylphosphonato does not appear sufficiently balanced (log Pow<−1.5). Conversely, those complexes with aromatic groups  are less soluble in water and display increased affinity for n‐octanol (log Pow≈0). The diiron compounds are inert in aqueous solution at room temperature, and undergo a slow degradation at 37 °C. Under such conditions, a variable amount  of the starting material was detected by 1H NMR in D2O or D2O/CD3OD mixtures after 72 h.2C]CF3SO3 solution) was separated and identified as γ‐Fe2O3 by Raman analysis. In addition, headspace GC analyses performed on 5 % MeOH aqueous solutions of the compounds maintained at 37 °C allowed the identification and quantitation of the carbon monoxide released over a 48 h period (Table S4). These facts indicate that the thermal degradation process in aqueous solution leads to the progressive disassembly of the organometallic scaffold.II ions was previously associated to their cytotoxicity,A detailed study on the behaviour of a selection of the diiron complexes in aqueous media was undertaken. Experimental procedures are provided in the Experimental Section and in the Supporting Information, with data collected in Tables 1H NMR. Notably, a considerable fraction  of the starting material was detected at the end of the experiment, with a limited formation of an orange‐brown precipitate analogous to that described above.Next, the stability of the diiron compounds in a cell culture medium (DMEM) solution at 37 °C, over a shorter time range (24 h), was assessed by 2–3]+ was the methylated derivative, which was also checked by HPLC separation . The expected diiron counterparts, that is, 1A–H, could not be detected possibly due to their water insolubility. The [lysozyme+Me+] to [lysozyme] ratios, calculated on extracted ion chromatograms after protein peak reconstruction, are reported in Table N‐methyl groups  did not react with lysozyme. Methylation is a common post‐translational modification of protein/enzymes (methyltransferase enzymes) but it has also been observed as a result of the interaction of proteins with various electrophiles.To gain insights into the reactivity of the diiron compounds with possible biomolecular targets, we selected lysozyme, a small model protein often used to test interactions with metal compounds. Figure S1. The ex2A]+, [2C–E]+, [3F–G]+, [5H]+  and [2B]+, [4G]+  were tested for their cytotoxic potential by means of the MTT assay, as reported in the Experimental Section. The most convenient counter anion in terms of preparation and handling/storage was chosen in each case; in particular, [2C]CF3SO3 was included in the study, while [2C]Cl was excluded, being highly moisture sensitive and thus affecting the accuracy of the weighting operation. The in‐house human cancer cell line panel contains examples of ovarian (2008), colon (HCT‐15), pancreatic (PSN‐1), and breast (MCF‐7) cancers as well as of melanoma (A375). Cisplatin was used as a reference and tested under the same experimental conditions. The cytotoxicity parameters, expressed in terms of IC50 obtained after 72 h of exposure, are listed in Table 2B]BF4 and [3F]CF3SO3 did not impact cell viability , whereas the other compounds elicited IC50 values in the micromolar range. Despite all cytotoxic complexes showed an average in vitro antitumor potential lower than that of cisplatin, [2C]CF3SO3 and [2D]CF3SO3 proved to be the most effective derivatives, with average IC50 values of 17.4 and 16.6 μM , and showed a similar pattern of response over the five tested cell lines. In particular, [2D]CF3SO3 exhibits a comparable activity with respect to cisplatin against HCT‐15, PSN‐1 and MCF‐7 cells.The diiron complexes [[3F–G]+, H+ . Overall, it appears that slight structural variations on the iminium group may have a notable impact on the activity of the cations, although not obvious: for instance, [2D]CF3SO3 and [2E]CF3SO3 differ from each other in the nature and the position of peripheral arene substituents and display almost identical log Pow values, notwithstanding the former is significantly more cytotoxic and selective than the latter.As one of the main drawbacks of chemotherapeutics is the toxic effect toward noncancerous cells, we measured the cytotoxicity of tested complexes against a noncancerous cell line (HEK293) and calculated the selectivity index . All complexes exhibited activity levels very similar on both sensitive (MCF‐7) and multi‐drug resistant (MCF‐7ADR) cell lines; interestingly, the resistance factor (RF) values Table  range fr2D]CF3SO3 (log Pow=−0.25) together with the less active/selective [2E]CF3SO3 (log Pow=−0.27) and [3G]CF3SO3 (log Pow=+0.29), to be assessed for their accumulation in human pancreatic PSN‐1 cancer cells and in untransformed HEK293 cells. Thus, cells were treated for 24 h with equimolar concentrations (50 μM) of the tested compounds. The cellular iron levels were quantified by means of GF‐AAS analysis, and the results, expressed as μg Fe per 106 cells, are shown in Figure 3G]CF3SO3 was ineffective in entering human cells, while both [2D]CF3SO3 and [2E]CF3SO3 were able to induce cellular iron accumulation, and no significant differences in the ability to enter cells were evidenced for the two complexes. Considering the log Pow values, the obtained outcomes highlight that the hydrophilic complexes [2D]CF3SO3 and [2E]CF3SO3 are significantly more effective in entering cells compared to the more lipophilic one [3G]CF3SO3. In addition, both [2D]CF3SO3 and [2E]CF3SO3 led to major iron accumulation in untransformed cells with respect to pancreatic cancer cells. Intriguingly, these findings attest that their selectivity towards cancer cells with respect to untransformed ones cannot be attributable to their different ability to cross cellular plasmalemma and to enter human cells, and the reason of their preferential activity against cancer cells might be dependent on their ability to interfere with a cancer specific target.In order to correlate the cytotoxic potential to the ability of the newly developed complexes to enter cells, we performed uptake experiments. To this purpose, we selected the most promising compound CF3SO3 and [3F]CF3SO3 were ineffective against cancer cells spheroids, whereas [2D]CF3SO3 was the most effective complex among the series, thus indicating its ability to better penetrate the spheroid and reach the hypoxic core. It is noteworthy that [2D]CF3SO3 exhibited a level of activity either comparable (PSN‐1 cells) or even superior (2008 cells) to that of cisplatin.The remarkable cell‐killing effect observed in 2D cultured cells prompted us to evaluate the in vitro antitumor activity of the complexes on 3D cell cultures. Differently from 2D monolayer culture, 3D spheroid cell culture systems comprise cancer cells in various cell growth stages. Consequently, the multicellular cancer spheroid model is recognized to better reflect the tumour mass in vivo regarding drug permeation, cell interactions, gene expression, hypoxia and nutrient gradients with respect to monolayer cell cultures, making 3D models more predictive than conventional 2D monolayer cultures in screening antitumor drugs.50 ratio related to 3D and 2D experiments ranging from 2 to 5 for both cell lines; in particular, this ratio is approximately 2 for the cytotoxic diiron complexes against the 2008 cell line, while the corresponding value for cisplatin is 26. Note that a substantial drop of antiproliferative activity has been often recognized for potential anticancer metal drugs in 3D cell cultures compared to the corresponding 2D ones, highlighting the need for a combination of both models for screening of compounds.An important aspect of the collected data is that only a limited decrease of activity is observed with the cytotoxic compounds, the IC2C]CF3SO3 and [2D]CF3SO3 to release carbon monoxide inside PSN‐1 pancreatic cancer cells and HEK293 noncancer cells. A fluorometric test was conducted over a short time interval (30 min) by means of a CO sensing dye (L)(μ‐CO){μ‐CNR(Me)}]0/+.CF3SO3 and [2D]CF3SO3 determined a substantial increase in cellular ROS production, in a time‐dependent manner  oxides has been recognized from water solution, see above). Otherwise, based on previous voltammetric measurements on [2E–F]CF3SO3,2–3]+, some contribution to ROS might be provided by the partial generation of the neutral products 1A–H upon CH3+ elimination (see above); in principle, the absence of a net positive charge in 1A–H might favour the oxidation of these complexes or their possible derivatives in the cells.Iron is a redox active metal that can generate ROS in cells via the Fenton reaction, thus inducing cellular damage and ultimately leading to cell death.r Figure A. Remark3 Figure B. The ti2C]CF3SO3 and [2D]CF3SO3 to target TrxR both in cell‐free experiments and in cells. The two complexes showed a similar pattern of response, being completely ineffective in hampering TrxR in cell‐free experiment (data not shown) but elicited a substantial inhibition of the selenoenzyme in human pancreatic PSN‐1 cancer cells . On the other hand, TrxR inhibition might also be consistent with the capacity of the complexes to methylate specific substrates, as we have assessed for lysozyme. Actually, it was previously described that several alkylating agents are able to selectively modify the redox active Cys or Sec of TrxR, thus causing irreversible inhibition.It has recently emerged that the redox stress induced by some iron complexes might also be attributed to their ability to inhibit the selenoenzyme thioredoxin reductase (TrxR). In particular, Rigobello and co‐workers reported the capacity of a ferrocenyl diphenol and of a Tamoxifen‐like ferrocifen to target and hamper TrxR activity.s Figure . In part2Cp2(CO)4]. They have ideal properties for a potential anticancer drug, such as the presence of a nontoxic metal element, appreciable water solubility and amphiphilicity, and stability in aqueous media.2D]CF3SO3, with a 4‐methoxy phenyl substituent on the bridging hydrocarbyl ligand, was revealed to be the most promising and exhibited a noteworthy selectivity toward cancer cell lines compared to noncancerous cells. According to cellular‐uptake experiments, such selectivity is not correlated to a different ability to enter cancerous or noncancerous cells. Remarkably, the cytotoxicity profile of the complexes does not substantially decrease in 3D cell cultures, in contrast to what has often been observed in the literature upon comparison of 2D/3D studies on various transition metal compounds. Interestingly, the activity exhibited by [2D]CF3SO3 in the ovarian cancer cell 3D model was even superior to that of cisplatin. Experiments on selected complexes outlined their power to behave as carbon monoxide‐releasing molecules (CORMs) inside cells, to unbalance cellular redox homeostasis and to alkylate biological targets. The preferential CO release in cancer cells with respect to untransformed cells could, at least in part, be consistent with the ability to target TrxR. Actually, it is well known that the Trx system is overexpressed in cancer cells, and has been recognized as a tumour‐specific target for the development of selective anticancer agents. In summary, we suggest that a simple diiron platform might offer an arsenal of tools not available for widely investigated ferrocenes, paving the way for the development of iron‐based drugs with optimized features. Further biochemical and biophysical studies will be performed to clarify in more detail the mechanism of action of this new class of organometallic anticancer candidates.We have described a synthetic strategy to obtain a family of diiron carbonyl complexes containing a bridging aminocarbyne/iminium hybrid ligand from the readily available [FeGeneral experimental details: [Fe2Cp2(CO)4] (99 %) was purchased from Strem Chemicals, other reactants and solvents were obtained from Alfa Aesar, Merck, Apollo Scientific or TCI Chemicals and were of the highest purity available. Isocyanides were stored at 4 °C or −20 °C and used as received; contaminated labware was treated with bleach. Methyl triflate was stored under N2 at 4 °C; allyl iodide and triethyloxonium tetrafluoroborate (1 M solution in CH2Cl2) were stored under N2 at −20 °C. Compounds [1A–H]2D–F]CF3SO3X , [2C]Cl, [5H]CF3SO3 (hygroscopic) and 6 were stored under N2; all other Fe compounds being air‐ and moisture‐stable in the solid state. NMR spectra were recorded at 25 °C on a Bruker Avance II DRX400 instrument equipped with a BBFO broadband probe. Chemical shifts (expressed in parts per million) are referenced to the residual solvent peaks1H, 13C) or to external standards46 . In [D6]DMSO/D2O mixtures, 1H chemical shifts are referenced to the [D5]DMSO signal as in pure [D6]DMSO (δ/ppm=2.50); in D2O/CD3OD mixtures, 1H chemical shifts are referenced to the CD3OD residual peak as CH3OH in pure D2O (δ/ppm=3.34). 1H and 13C spectra were assigned with the assistance of 1H{31P}, 13C DEPT 135, 1H,1H COSY, 1H‐13C gs‐HSQC and 1H‐13C gs‐HMBC experiments.3 stored in the dark over Na2CO3 was used for NMR analysis. IR spectra of solid samples (650–4000 cm−1) were recorded on a Perkin Elmer Spectrum One FTIR spectrometer, equipped with a UATR sampling accessory; IR spectra of solutions were recorded using a CaF2 liquid transmission cell (2300–1500 cm−1) on a Perkin Elmer Spectrum 100 FTIR spectrometer. UV/Vis spectra (250–800 nm) were recorded on a Ultraspec 2100 Pro spectrophotometer using PMMA cuvettes (1 cm path length). IR and UV/Vis spectra were processed with Spectragryph software.2Cp2(CO)3(CNR)], [1A[Fe–H].General procedure. In a 250 mL round‐bottom flask equipped with a reflux condenser under N2, the selected isocyanide, or its solution in MeCN , was added dropwise to a suspension of [Fe2Cp2(CO)4] (1.6 equiv) in anhydrous MeCN (80–100 mL). Alkyl isocyanides: the mixture was heated at reflux (T≥100 °C) for 8 h, then stirred at room temperature for additional 14 h. Aryl isocyanides: the mixture was stirred at room temperature for 14 h then heated at reflux (T≥100 °C) for 1–3 h. Next, conversion was checked by IR and the dark red‐brown suspension was dried under vacuum (40 °C). The resulting solid, consisting of a mixture of [Fe2Cp2(CO)4] and [Fe2Cp2(CO)3(CNR)], was directly used in the following alkylation procedure without any purification.2Cp2(CO)2(μ‐CO){μ‐CNMe(1H‐indol‐6‐yl)}]CF3SO3, [2A]CF3SO3CF3SO3 is soluble in MeCN, THF, MeOH, CH2Cl2, less soluble in CH2Cl2, CHCl3, insoluble in Et2O, toluene and water. X‐ray quality crystals of [2A]CF3SO3 were obtained from an acetone solution layered with Et2O and settled aside at −20 °C. Anal. calcd. for C25H21F3Fe2N2O6S: C 46.47, H 3.28, N 4.34; found: C 46.09, H 3.28, N 4.34. IR (solid state): ν˜/cm−1=3330w‐br (νNH), 3114w, 2004 s (νCO), 1985 s (νCO), 1833 s (νμ‐CO), 1583w‐sh, 1555 m, 1540 m (νCN), 1475w, 1458w, 1433w, 1420w, 1397 m, 1361w, 1345w, 1323w, 1290–1276 m, 1244 s (νSO3), 1223 s (νSO3), 1154 s (νSO3), 1107 m‐sh, 1067w, 1028 s, 1016 s‐sh, 1002 m‐sh, 942w, 931w, 893w, 875w, 860 m, 845 m, 817w, 797 m, 772 s, 756w‐sh, 743 m‐sh, 728 m. IR (CH2Cl2): ν˜/cm−1=2022 s (νCO), 1992 m‐sh (νCO), 1836 s (νμ‐CO), 1554 m, 1542 m (νCN). IR (MeCN): ν˜/cm−1=2023 s (νCO), 1991 m‐sh (νCO), 1836 s (νμ‐CO), 1554 m, 1542 m‐sh (νCN). 1H NMR (CD3CN): δ/ppm=9.78 , 7.85 , 7.70 , 7.48 , 7.37 , 6.67−6.62 ; 5.39, 4.65 , 4.53 . 13C{1H} NMR (CD3CN): δ/ppm=324.9 (CN), 255.9 (μ‐CO); 209.8, 209.2 (CO); 144.6 (C2), 136.3 (C5), 129.1 (C8), 128.8 (C6), 118.8 (C3), 117.3 (C9), 113.8 (C4), 103.5 (C7); 91.2, 91.0 (Cp); 58.5 (C1). 19F{1H} NMR (CD3CN): δ/ppm=−79.3. 1H NMR (CDCl3): δ/ppm=9.57 , 8.0–7.0 , , 7.42–7.39 , 6.60 ; 5.41, 4.63 ; 4.56 .In a 150 mL Schlenk tube under N3SO3[2B]CF. In a 150 mL Schlenk tube under N2, CF3SO3Me  was added dropwise to a dark red solution of 1B (1.38 mmol) in anhydrous CH2Cl2 (40 mL) under stirring. Therefore, the mixture was stirred at room temperature for 7 h and conversion was checked by IR (CH2Cl2). Next, the solution was moved on top of an alumina column . Impurities were eluted with neat CH2Cl2, THF and MeCN, then a red band containing the title product was eluted with MeCN/MeOH . Volatiles were removed under vacuum (40 °C), affording a red, highly hygroscopic solid. Yield: quantitative. IR (CH2Cl2): ν˜/cm−1=2028 s (νCO), 1997w‐sh (νCO), 1836 m (νμ‐CO). 1H NMR (CDCl3): δ/ppm=5.32 , 5.23 , 4.93 , 4.38 , 4.23–4.10 , 1.30 . 19F{1H} NMR (CDCl3): δ/ppm=−78.2. 31P{1H} NMR (CDCl3): δ/ppm=17.3.4[2B]BF. In a 500 mL round bottom flask, [2B]CF3SO3 was suspended in water (100 mL) with vigorous stirring until completely dissolved then treated with NaBF4 (500 mg). The red solution was extracted with CH2Cl2 (3×20 mL) and the combined organic fractions were dried under vacuum. The residue was suspended in water and the ion exchange procedure with NaBF4 was repeated (×3); 19F{1H} NMR analysis of the final CH2Cl2 solution indicated the complete removal of CF3SO3− anions. Therefore, volatiles were removed under vacuum; the residue was dissolved in CH2Cl2 and filtered over celite. A red foamy hygroscopic solid, obtained upon volatiles removal without heating, was dried under vacuum and stored under N2. Yield: 751 mg, 90 % (with respect to 1B). Compound [2B]BF4 is soluble in MeOH, MeCN, CH2Cl2, CHCl3, water, insoluble in Et2O and hexane. Anal. calcd. for C20H25BF4Fe2NO6P: C 39.71, H 4.17, N 2.31; found: C 39.20, H 4.25, N 2.26. IR (solid state): ν˜/cm−1=3117w, 2987w, 2938w, 2913w, 2875w, 2012 s (νCO), 1986 s‐sh (νCO), 1823 s (νμ‐CO), 1568 m (νCN), 1479w, 1446w, 1435w, 1421w, 1399 m, 1370w‐sh, 1297–1286w, 1254 m, 1225 m‐sh, 1180w, 1163w, 1075 m‐sh, 1033 s‐sh (νBF4), 1008 s, 973 s, 951 s‐sh, 904 m, 864 m‐sh, 851 m, 837 m‐sh, 780s, 745 s, 710 m‐sh. IR (CH2Cl2): ν˜/cm−1=2028 s (νCO), 1996w (νCO), 1836 m (νμ‐CO), 1574w (νCN). IR (MeCN): ν˜/cm−1=2026 s (νCO), 1994w (νCO), 1838 m (νμ‐CO), 1569w (νCN). 1H NMR (CDCl3): δ/ppm=5.29 , 5.15 , 4.93 , 4.35 , 4.24–4.10 , 1.30 . No changes were observed in the 1H spectrum after 14 h at room temperature. 13C{1H} NMR (CDCl3): δ/ppm=323.7 , 254.7 (μ‐CO); 207.4, 207.1 (CO); 90.3, 90.1 (Cp); 63.3 (C3), 62.5 , 53.2 (C1), 16.4 . 19F{1H} NMR (CDCl3): δ/ppm=‐151.9 (10BF4), −152.0 (11BF4). 31P{1H} NMR (CDCl3): δ/ppm=17.3.3SO3[2C]CF. In a 500 mL round bottom Schlenk flask under N2, CF3SO3Me  was added dropwise to a dark red suspension of 1C (22.5 mmol) in anhydrous CH2Cl2 (100 mL) under stirring. Therefore, the mixture was stirred at room temperature for 4 h and the conversion was checked by IR (CH2Cl2). Next, the dark red solution was moved on top of an alumina column . Impurities were eluted with neat CH2Cl2 and THF, then a red band containing [2C]CF3SO3 was eluted with MeCN/MeOH . A red band, containing a minor fraction of [2C]Cl, was collected using MeOH as eluent. Volatiles were removed under vacuum from the MeCN/MeOH solution and residue was triturated in Et2O. The suspension was stirred at room temperature for a few h then filtered. The resulting red solid was washed with toluene, Et2O and dried under vacuum (40 °C). Yield: 11.44 g, 85 %. Compound [2C]CF3SO3 is soluble in MeCN, MeOH, CH2Cl2, CHCl3, less soluble in water, poorly soluble in toluene, insoluble in Et2O, hexane. X‐ray‐quality crystals of [2C]CF3SO3 were obtained from a CH2Cl2 solution layered with heptane and settled aside at −20 °C. Anal. calcd. for C22H24F3Fe2NO6S: C 44.10, H 4.04, N, 2.34; found: C 44.47, H 4.04, N, 2.34. IR (solid state): ν˜/cm−1=3102w, 2937w, 2862w, 2185w, 2146w, 2006 s (νCO), 1982 s‐sh (νCO), 1822 s (νμ‐CO), 1565 m (νCN), 1544 m‐sh, 1452w, 1435w, 1421w, 1402w, 1366w, 1352w, 1321w, 1272 s‐sh, 1257 s (νSO3), 1223 s‐sh (νSO3), 1150s (νSO3), 1056 m, 1029 s, 990 m, 864 m, 854 m, 796 s, 746 s. IR (CH2Cl2): ν˜/cm−1=2020s (νCO), 1988w‐sh (νCO), 1835 m (νμ‐CO), 1567w (νCN). IR (MeCN): ν˜/cm−1=2021 s (νCO), 1988w‐sh (νCO), 1835 m (νμ‐CO), 1568w (νCN). 1H NMR (CDCl3): δ/ppm=5.36, 5.27 ; 4.68 , 4.06 , 2.81 , 2.15 , 2.03–1.94 , 1.86–1.72 , 1.53–1.35 , 1.34–1.20 . No changes were observed in the 1H spectrum after 14 h at room temperature. 13C{1H} NMR (CDCl3): δ/ppm=316.4 (CN), 255.3 (μ‐CO); 208.5, 207.6 (CO); 90.3, 90.1 (Cp); 79.8 (C2), 46.8 (C1), 31.2 (C5), 30.7 (C4), 26.1 (C3), 26.0 (C6), 25.1 (C7). 19F{1H} NMR (CDCl3): δ/ppm=−78.2. 1H NMR (CD3OD): δ/ppm=5.40, 5.36 , 4.07 , 2.41 , 2.17–2.07, 2.03–1.91, 1.85–1.50, 1.42–1.32 .[2C]Cl. Compound [2C]CF3SO3  was dissolved in MeOH (2 mL) then moved on top of an Amberlyst 15 column . NaCF3SO3 was eluted with neat MeOH then a red band containing [2C]+ was eluted with NaCl‐saturated MeOH. Volatiles were removed under vacuum and the residue was suspended in MeCN. The suspension was filtered over celite; the filtrate was checked for absence of CF3SO3− by 19F NMR then dried under vacuum (40 °C). The resulting red, hygroscopic solid was stored under N2. Yield: 83 mg, 93 %. Compound [2C]Cl shows the same solubility pattern as the triflate salt. X‐ray quality crystals of [2C]Cl ⋅ 2.34H2O were obtained from a CH2Cl2 solution layered with hexane and settled aside at −20 °C. Anal. calcd. for C21H24ClFe2NO3: C 51.94, H 4.98, N 2.88; found: C 51.99, H 5.02, N 2.83. IR (solid state): ν˜/cm−1=3376 m‐br* (νOH), 3098–3059w, 2933 m, 2858w, 2180–2160w, 1997 s (νCO), 1969 s‐sh (νCO), 1811 s (νμ‐CO), 1662w, 1631w, 1565 s (νCN), 1543 m, 1450 m, 1434w, 1419 m, 1400 m, 1350w, 1319w, 1263w‐sh, 1249w, 1173w, 1154w, 1054 m, 1026w, 1015w, 990 m, 924w‐sh, 893w‐sh, 852 m, 795 s, 746 s, 729 s‐sh. *Due to moisture. IR (CH2Cl2): ν˜/cm−1=2018 s (νCO), 1985w‐sh (νCO), 1833 m (νμ‐CO), 1569w (νCN). IR (MeCN): ν˜/cm−1=2020s (νCO), 1987w‐sh (νCO), 1834 m (νμ‐CO), 1568w (νCN). 1H NMR (CDCl3): δ/ppm=5.51, 5.39 ; 4.75–4.66 , 4.14 , 3.16 , 2.15 , 2.03–1.93 , 1.84–1.72 , 1.51–1.35 , 1.33–1.23 . No changes were observed in the 1H spectrum after 24 h at room temperature. 13C{1H} NMR (CDCl3): δ/ppm=316.2 (CN), 255.8 (μ‐CO); 208.7, 207.8 (CO); 90.7, 90.3 (Cp); 79.7 (C2), 47.1 (C1), 31.3 (C5), 31.0 (C4), 26.1 (C3), 26.0 (C6), 25.2 (C7). 35Cl NMR (CDCl3): δ/ppm=7.61 (Δν1/2=266 Hz).2, CF3SO3Me  was added dropwise to a dark red solution of 1B (4.63 mmol) in anhydrous CH2Cl2 (40 mL) under stirring. The mixture was stirred at room temperature for 5.5 h then moved on top of an alumina column . Impurities were eluted with CH2Cl2 and CH2Cl2/THF , then a red band containing [2D]CF3SO3 was eluted with neat MeCN. Volatiles were removed under vacuum (40 °C) to afford a red foamy solid. Yield: 2.446 g, 85 %. Compound [2D]CF3SO3 is soluble in MeCN, MeOH, CH2Cl2, CHCl3, water, insoluble in Et2O and hexane. X‐ray quality crystals of [2D]CF3SO3 were obtained from a CHCl3 solution layered with hexane and settled aside at −20 °C. Anal. calcd. for C23H20F3Fe2NO7S: C 44.33, H 3.24, N 2.25; found: C 43.92, H 3.31, N 2.17. IR (solid state): ν˜/cm−1=3113w, 3091w, 2946w, 2849w, 2011 s (νCO), 1978 s (νCO), 1821 s (νμ‐CO), 1608w, 1563 m, 1539 m (νCN), 1506 s, 1468w, 1449w, 1433w, 1420w, 1395 m, 1304w‐sh, 1278 s, 1251 s (νSO3), 1224 s‐sh (νSO3), 1176 m‐sh, 1146 s (νSO3), 1117 m‐sh, 1108 m, 1067w, 1028 s, 890w, 855 s, 825w‐sh, 773 s, 756 m‐sh, 736w, 702 s. IR (CH2Cl2): ν˜/cm−1=2021 s (νCO), 1989w (νCO), 1836 m (νμ‐CO), 1607w, 1562w, 1540w (νCN), 1507 m. IR (MeCN): ν˜/cm−1=2024 s (νCO), 1991w (νCO), 1837 m (νμ‐CO), 1610w, 1563w, 1539w (νCN), 1508w. 1H NMR (CDCl3): δ/ppm=8.3–7.6 , 7.10 ; 5.43, 4.78 ; 4.54 , 3.90 . 19F{1H} NMR (CDCl3): δ/ppm=−78.1.The title compound was prepared with minor modifications of the literature procedure.[3F]I. The preparation of the title compound was optimized with respect to the literature .2, allyl iodide  was added dropwise to a dark red solution of 1F (1.54 mmol) in anhydrous MeCN (30 mL) under vigorous stirring. The mixture was stirred for 4.5 h under protection from the light and conversion was checked by IR (MeCN). Next, the dark red solution was moved on top of an alumina column . Impurities were eluted with neat Et2O, THF and MeCN, then a red band containing the title product was eluted with MeCN/MeOH . Volatiles were removed under vacuum (40 °C) and the residue was triturated in Et2O. The suspension was stirred at room temperature then filtered. The resulting red solid was washed with Et2O and dried under vacuum (40 °C). Yield: 646 mg, 78 %. Compound [3F]I is soluble in MeOH, CH2Cl2, CHCl3, poorly soluble in water, insoluble in Et2O, hexane. IR (CH2Cl2): ν˜/cm−1=2021 s (νCO), 1989w (νCO), 1835 m (νμ‐CO), 1585w (νCN). 1H NMR (CDCl3): δ/ppm=6.09–5.97 , 5.53–5.46 ; 5.45, 5.53 ; 5.17–5.11 , 4.25 .3SO3[3F]CF. In a 50 mL round bottom flask, a solution of Ag(CF3SO3)  in MeCN (5 mL) was added to a dark red suspension of [3F]I  in MeCN (6 mL) at 0 °C. The mixture stirred at 0 °C for 2.5 h while maintaining the system under protection from the light. The resulting suspension (dark red solution+yellow AgI precipitate) was filtered over celite. Volatiles were removed under vacuum from the filtrate solution and the residue was triturated in a Et2O/toluene mixture. The suspension was stirred at room temperature for a few h, then filtered. The resulting red solid was washed with toluene, Et2O and dried under vacuum (40 °C). Yield: 928 mg, 94 %. Compound [3F]CF3SO3 is soluble in MeCN, acetone, MeOH, water, less soluble in CH2Cl2 > CHCl3, poorly soluble in toluene, insoluble in Et2O. Anal. calcd. for C19H18F3Fe2NO6S: C 40.96, H, 3.26, N, 2.51; found: C 41.20, H, 3.20, N, 2.72. IR (solid state): ν˜/cm−1=3106w, 2213w, 2178w, 2012 s (νCO), 1988 s (νCO), 1813 s (νμ‐CO), 1644w, 1581 m (νCN), 1435w, 1416w, 1395w, 1261 s (νSO3), 1243 s‐sh, 1222 m‐sh (νSO3), 1169 s (νSO3), 1050 m, 1028 s, 989w‐sh, 931 m, 888 m, 854 m, 758 s, 666 m. IR (CH2Cl2): ν˜/cm−1=2022 s (νCO), 1990w (νCO), 1836 m (νμ‐CO), 1584 m (νCN). IR (MeCN): ν˜/cm−1=2023 s (νCO), 1990w (νCO), 1836 m (νμ‐CO), 1582 m (νCN). 1H NMR (CDCl3): δ/ppm=5.98 , 5.50–5.45 ; 5.34, 5.32 ; 5.30–5.23 , 5.15–5.08 , 4.17 . No changes were observed in the 1H spectrum after 14 h at room temperature. 13C{1H} NMR (CDCl3): δ/ppm=318.1 (CN), 255.3 (μ‐CO); 208.1, 207.8 (CO), 130.2 (C3), 122.2 (C4), 121.0 ; 90.20, 90.16 (Cp); 70.6 (C2), 51.3 (C1). 19F{1H} NMR (CDCl3): δ/ppm=−78.2. 1H NMR ([D6]acetone): δ/ppm=6.33–6.19 , 5.62 ; 5.58, 5.54 ; 5.41–5.16 , 4.28 .2, methyl iodide  was added to a dark red solution of 1G (0.48 mmol) in anhydrous MeCN (8 mL). The mixture was stirred for 2.5 h and conversion was checked by IR (MeCN). Next, the dark red solution was moved on top of an alumina column . Impurities were eluted with neat CH2Cl2 and THF, then a red band containing the title product was eluted with THF/MeOH . Next, volatiles were removed under vacuum and the residue was dissolved in CH2Cl2. The suspension was filtered over celite and the filtrate was taken to dryness under vacuum (40 °C), affording a dark red solid. Yield: 208 mg, 69 %. A considerable decrease in yield and purity was observed for reactions carried out in refluxing CHCl3 or in MeCN at lower temperatures. Anal. calcd. for C25H20Fe2INO3: C 48.35, H 3.25, N 2.56; found: C 48.20, H 3.15, N 2.49. Compound [3G]I is soluble in MeOH, CH2Cl2, CHCl3, sparingly soluble in water, insoluble in hexane. IR (CH2Cl2): ν˜/cm−1=2021 s (νCO), 1989 m‐sh (νCO), 1836 m (νμ‐CO), 1564w, 1545w, 1538w (νCN). 1H NMR (CDCl3): δ/ppm=8.10 , 7.98−7.95 , 7.94−7.77 , 7.66−7.52 , 7.55−7.47  (C10H7); 5.62, 4.82 ; 4.75 .In a 25 mL Schlenk tube under N2, [Et3O]BF4 , was added dropwise to a dark red solution of 1G (0.724 mmol) in anhydrous MeCN (30 mL) at 0 °C under vigorous stirring. Therefore, the mixture was stirred for 3 h, while allowing to reach room temperature. Next, conversion was checked by IR (MeCN) then volatiles were removed under vacuum. The residue was dissolved in a small volume of THF then moved on top of an alumina column . Impurities were eluted with neat Et2O and THF, then a red band containing the title product was eluted with MeCN/MeOH . Volatiles were removed under vacuum (40 °C) and the residue was triturated in Et2O. The suspension was stirred at room temperature for a few h then filtered. The resulting dark red solid was washed with Et2O and dried under vacuum (40 °C). Yield: 284 mg, 66 %. A considerable decrease in yield and purity was observed for reactions carried out in CH2Cl2 or in MeCN at room temperature. Compound [4G]BF4 is soluble in MeOH, MeCN, CH2Cl2, CHCl3, poorly soluble in Et2O, insoluble in water. Anal. calcd. for C26H22BF4Fe2NO3: C 52.49, H 3.72, N 2.35; found: C 52.11, H 3.67, N 2.30. IR (solid state): ν˜/cm−1=3116w, 3060w, 2979w, 2937w, 2160w‐br, 2007 s (νCO), 1937 s‐sh (νCO), 1836 s (νμ‐CO), 1596w, 1532 s (νCN), 1506 m‐sh, 1458w, 1434w, 1421w, 1381w, 1360w, 1341w, 1275w, 1247w, 1211w, 1179w, 1134w, 1075 s‐sh, 1049 s‐br, 1033 s (νBF4), 1014 s‐sh, 963w, 935w, 865w, 850w, 825w, 808 m, 745 s, 707 m, 657 m. IR (CH2Cl2): ν˜/cm−1=2021 s (νCO), 1989w (νCO), 1836 m (νμ‐CO), 1538w (νCN). IR (MeCN): ν˜/cm−1=2023 s (νCO), 1990w (νCO), 1837 m (νμ‐CO), 1537w (νCN). 1H NMR ([D6]acetone): δ/ppm=8.7–8.3 , 8.30 , 8.16–8.09 , 8.1–7.7 , 7.75–7.70 , 5.68 , 5.19 , 4.87 , 1.51 . No changes were observed in the 1H spectrum after 14 h at room temperature. 13C{1H} ([D6]acetone): δ/ppm=325.2 (CN), 255.2 (μ‐CO); 210.1, 209.8 (CO); 146.1 (C3); 134.3, 133.9 (C6+C11); 131.4 (C5); 129.5, 129.0 (C7+C10); 128.8, 128.6 (C8+C9); 126.3, 125.1 (C4+C12); 91.7, 91.2 (Cp); 66.8 (C1), 14.4 (C2). 19F{1H} NMR ([D6]acetone): δ/ppm=−151.0 (10BF4), −151.1 (11BF4). 1H NMR (CDCl3): δ/ppm=8.5–7.8 , 7.70–7.60 , 7.60–7.48 , 5.44 , 5.20–5.05 , 4.95 , 4.65 , 1.44 . 13C{1H} NMR (CDCl3): δ/ppm=323.2 (br), 254.9, 209.2 (br), 208.4, ca. 145 (br); 133 (br), 132.9 (br); 130.1 (m), 128.1, 127.9, 126–125 (m‐br), 124.0, 120.5, 117.1, 90.6, 90.3, 66 (br), 14.0. 19F{1H} NMR (CDCl3): δ/ppm=−150.9, −151.0. The cis orientation of the Cp ligands in [4G]BF4 was ascertained by 1H NOE experiment in CDCl3. Thus, irradiation of one Cp resonance (δH 5.44 or 4.65 ppm) evidenced a NOE interaction with the other.In a 150 mL Schlenk tube under N[5H]Br. In a 100 mL Schlenk tube under N2, benzyl bromide  was added dropwise to a dark red solution of 1H (1.58 mmol) in anhydrous MeCN (50 mL) under vigorous stirring at 60 °C. The mixture was stirred at reflux for 1.5 h and conversion was checked by IR (MeCN). Next, the dark red solution was moved on top of an alumina column . Impurities were eluted with neat CH2Cl2, THF and MeCN, then a red band containing the title product was eluted with MeCN/MeOH . Volatiles were removed under vacuum (40 °C), affording a red solid. Yield: 892 mg, 92 %. Compound [5H]Br is soluble in CH2Cl2, CHCl3, THF, iPrOH, poorly soluble in toluene, EtOAc, insoluble in Et2O. IR (CH2Cl2): ν˜/cm−1=2020s (νCO), 1988w (νCO), 1837 m (νμ‐CO), 1550w (νCN), 1534w. 1H NMR (CDCl3): δ/ppm=7.48 , 7.40 , 7.30–7.26 , 5.69 , 5.56 , 5.52 .3SO3[5H]CF. In a 50 mL round bottom flask, a solution of Ag(CF3SO3)  in MeCN (5 mL) was added to a dark red solution of [5H]I  in MeCN (40 mL) at 0 °C. The mixture was stirred at 0 °C for 1.5 h while maintaining the system under protection from the light. The resulting suspension  was filtered over celite and the filtrate solution was dried under vacuum. The residue was treated with hexane (10 mL) then Et2O (50 mL) and settled aside for 30 min. Therefore, the solution was discarded and the washing procedure was repeated (x 2). The resulting red solid was dried under vacuum (40 °C) and stored under N2 (slightly hygroscopic). Yield: 1076 mg, 93 %. Compound [5H]CF3SO3 is soluble in MeCN, acetone, MeOH, CH2Cl2, poorly soluble in Et2O, toluene, water and insoluble in hexane. Anal. calcd. for C29H24F3Fe2NO6S: C 50.98, H, 3.54, N, 2.05; found: C 50.59, H, 3.58, N, 2.26. IR (solid state): ν˜/cm−1=3106w, 3070–3230w, 2166w, 2013 s (νCO), 1985 s‐sh (νCO), 1827 s (νμ‐CO), 1588w, 1549 m (νCN), 1533 m, 1497w, 1454w, 1433w, 1420w, 1352w, 1260s (νSO3), 1223 s‐sh (νSO3), 1153 s (νSO3), 1096w, 1079w, 1029 s, 858 m, 767 s, 737 m‐sh, 698 s. IR (CH2Cl2): ν˜/cm−1=2023 s (νCO), 1991 m (νCO), 1840 m (νμ‐CO), 1550w (νCN), 1534w. IR (MeCN): ν˜/cm−1=2023 s (νCO), 1990w (νCO), 1839 m (νμ‐CO), 1552w (νCN), 1538w. 1H NMR (CDCl3): δ/ppm=7.50–7.44 , 7.44–7.39 , 7.20 , 5.66 , 5.50 , 5.40 . No changes were observed in the 1H spectrum after 2 days at room temperature. 13C{1H} NMR (CDCl3): δ/ppm=324.4 (CN), 253.7 (μ‐CO), 208.2 (CO), 132.1 (C2), 129.9 (C4), 129.4 (C5), 127.8 (C3), 121.1 , 90.6 (Cp), 68.9 (C1). 19F{1H} NMR (CDCl3): δ/ppm=−78.1 ppm.2, a red suspension of [2C]CF3SO3 , Me3NO ⋅ 2H2O  and LiCl  in acetone (8 mL) was stirred at reflux for 5 h. Conversion was checked by IR (acetone) then volatiles were removed under vacuum. The brown residue was suspended in CH2Cl2 and moved on top of an alumina column , under N2. Impurities were eluted with CH2Cl2 then a brown band containing the title product was eluted with THF. The eluate was dried under vacuum and the residue was suspended hexane. The suspension was filtered; the resulting brown solid was washed with hexane and dried under vacuum (40 °C). Yield: 55 mg, 70 %. A related one‐pot reaction with [2C]CF3SO3/Me3NO ⋅ 2H2O (1.6 equiv)/NaCl (3.6 equiv) in deaerated MeOH (8 mL) at 40 °C overnight gave 6 in 43 % yield. A two‐step procedure comprising the [2C]CF3SO3/Me3NO ⋅ 2H2O (1 equiv) reaction in MeCN at room temperature followed by LiCl (3.0 equiv) in refluxing acetone for 3 h gave 6 in 54 % yield. Compound 6 is soluble in CH2Cl2, CHCl3, scarcely soluble in MeOH, insoluble in Et2O and hydrocarbons. Crystals of 6 for X‐ray analysis were obtained from a CHCl3 solution layered with pentane and settled aside at −20 °C. IR (CH2Cl2): ν˜/cm−1=1977 s (νCO), 1798 s (νμ‐CO), 1537 m (νCN). IR (MeCN): ν˜/cm−1=1971 s (νCO), 1795 m (νμ‐CO), 1539w (νCN). IR (solid state): ν˜/cm−1=3104w, 3070w, 1939 s (νCO), 1782 s (νμ‐CO), 1558w‐sh, 1536 s (νCN), 1456w, 1447w, 1419w, 1394 m, 1360w, 1346w, 1317w, 1251w, 1182w, 1153w, 1116w, 1062 m, 1016w, 1001w, 993 m, 857w‐sh, 838 m, 814 m, 800s, 757 s. 1H/13C NMR: mixture of isomers in 3 : 2 ratio (1H CDCl3). Signals attributable to the minor isomer are italicized. 1H NMR (CDCl3): δ/ppm=6.09, 5.01 ; 4.72, 4.70 ; 4.64, 4.62 ; 4.53, 4.05 ; 2.59, 2.37 ; 2.28–2.06, 1.98–1.69, 1.42–1.24 . No change in the 1H NMR spectrum was observed after 14 h at room temperature. 13C{1H} NMR (CDCl3): δ/ppm=334.8, 334.5 (CN); 268.1, 267.5 (μ‐CO); 212.5, 211.9 (CO); 86.8, 86.6 (Cp); 86.4, 86.3 (Cp’); 76.6, 75.4 (C2‐H); 44.4, 43.6 (C1‐H), 32.5, 32.1 (C3‐H); 32.3, 30.6, 26.3, 26.2, 26.0, 25.6, 25.6, 25.5 (C3’‐H+C4‐H+C5‐H).In a 25 mL Schlenk tube under N2A]CF3SO3, [2C]Cl ⋅ 2.34H2O, [2C]CF3SO3, [2D]CF3SO3 and 6 are reported in Table Kα radiation. The structures were solved by direct methods and refined by full‐matrix least‐squares based on all data using F2.2A]CF3SO3 which was located in the Fourier map and refined isotropically using the 1.2‐fold isoU of the parent N(2) atom. The H2O molecules of [2C]Cl ⋅ 2.34H2O are disordered and it was not possible to locate the hydrogen atoms. All non‐hydrogen atoms were refined with anisotropic displacement parameters. The crystals of 6 display a low quality, allowing the full determination of the overall connectivity and geometry of the complex, while the bonding parameters cannot be discussed in detail.Crystal data and collection details for BF4, [2C]Cl and [3F]CF3SO3 an inverse procedure was followed, starting from a solution of the compound in octanol‐saturated water. The partition coefficient was calculated as Pow=(A0aq−Afaq)/Afaqwhere A0aqand Afaqare the absorbance in the aqueous phase before and after partition with the organic phase, respectively. The wavelength of the maximum absorption of each compound (320–390 nm range) was used for UV/Vis quantitation. The procedure was repeated three times for each sample (from the same stock solution); results are given as mean ± standard deviation  and cell culture mediumStability in water  in MeOH (0.50 mL) then diluting with H2O up to 10.0 mL total volume (5 % v/v MeOH). Next, 3.20 mL of the solution (nFe ca. 6 ⋅ 10−3 mmol) and a 7x2 mm stir bar were transferred into a 4‐mL screw top vial (1.72 mL neat gas phase volume). The mixture was sealed with a screw cap with a PTFE/silicone septum and heated at 37 °C for 24 h under stirring. Afterwards, the headspace was sampled with a gas tight microsyringe (250 mL) and analysed by GC‐TCD. Therefore, the vial was vented, sealed and heated for further 24 h, and GC‐TCD analysis was repeated. The amount of released CO  was calculated based on a calibration curve obtained from analyses of known air/CO mixtures (1–10 % v/v), and the number of equivalents released over a 24 h period refers to the initial amount of compound (eqCO=nCO/nFe). The residual amount of starting diiron complex after 48 h was calculated by assuming the release of 3 equivalents of CO per mole and the total amount of CO released. The procedure was repeated three times for each sample (from the same stock solution); results are given as mean ± standard deviation (Table S4).4OAc (1.25×10−2 M)cFe2=1×10−3 M) were prepared by dissolving the selected compound in DMSO (0.25 mL) or MeOH , then diluting with HPLC water up to 5.0 mL total volume. Next, aliquots of the Fe (0.50 mL) and lysozyme (2.0 mL) stock solutions were mixed and the final solution  was kept at 37 °C for 24 h. The resulting suspensions were centrifuged  to separate the orange‐brown precipitate. A blank solution (lysozyme only) was prepared and treated by the same procedure. Next, samples were diluted 1 : 20 (v/v) with a water/MeCN  solution containing 1 % formic acid and analysed by HPLC‐MS and flow injection MS. For HPLC analyses, elution was conducted with a MeCN/water linear gradient (90 : 10 to 5 : 95 v/v over 60 min), containing 0.05 % trifluoroacetic acid. The following compounds were identified by their MS pattern: [2A]+ (tR=18.0 min), [2B]+ (tR=13.8 min), [2C]+ (tR=18.8 min), [2D]+ (tR=17.0 min), [2E]+ (tR=18.3 min), [2F]+ (tR=9.7 min), [3F]+ (tR=13.1 min), [4G]+ (tR=23.0 min), [5H]+ (tR=24.3 min), lysozyme‐CH3 (tR=21.0 min), lysozyme (tR=21.6 min). In all cases, the starting organometallic cation was identified as the major Fe‐containing compound in solution. Protein peaks pattern were obtained following peak reconstruction. Peak integrals for lysozyme and methyl lysozyme were calculated from the extracted ion chromatograms from the flow injection MS analysis  was stored at 4 °C as received. A stock solution was prepared by dissolving the powder protein (125 μM) and NH2B]BF4, [2C]CF3SO3, [2D]CF3SO3 and [3F]CF3SO3 were dissolved in water while the other compounds were dissolved in the minimum DMSO amount prior to cell culture testing. A calculated amount of the stock drug DMSO solution was added to the cell culture media to reach a final maximum DMSO concentration of 0.5 %, which had no effects on cell viability.Compounds , cisplatin and ImmunoPure Cell cultures. Human colon (HCT‐15), pancreatic (PSN‐1) and breast (MCF‐7) carcinoma cell lines along with human melanoma cells (A375) were obtained from American Type Culture Collection . Embryonic kidney HEK293 cells were obtained from the European Collection of Cell Cultures . Human ovarian 2008 cancer cells were kindly provided by Prof. G. Marverti . MCF‐7 ADR cells were kindly provided by Prof. N. Colabufo . Cell lines were maintained in the logarithmic phase at 37 °C in a 5 % CO2 atmosphere using the following culture media containing 10 % fetal calf serum , antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin) and 2 mM l‐glutamine: i) RPMI‐1640 medium  for PSN‐1, 2008, HCT‐15, MCF‐7 and MCF‐7 ADR cells; ii) DMEM for A375 and HEK293 cells.Spheroid cultures. Spheroid cultures were obtained by seeding 2.5×103 cells/well in round bottom non‐tissue culture treated 96 well‐plate  in phenol red free RPMI‐1640 medium , containing 10 % FCS and supplemented with 20 % methyl cellulose stock solution.MTT assay. The growth inhibitory effect towards tumour cells was evaluated by means of MTT assay as previously described.3 cells/well, dependent upon the growth characteristics of the cell line, were seeded in 96‐well microplates in growth medium (100 μL). After 24 h, the medium was removed and replaced with a fresh one containing the compound to be studied at the appropriate concentration. Triplicate cultures were established for each treatment. After 24 or 72 h, each well was treated with 10 μL of a 5 mg/mL MTT saline solution, and following 5 h of incubation, 100 μL of a sodium dodecyl sulfate (SDS) solution in HCl 0.01 M were added. After an overnight incubation, cell growth inhibition was detected by measuring the absorbance of each well at 570 nm using a Bio‐Rad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control untreated well absorbance and plotted against drug concentration. IC50 values, the drug concentrations that reduce the mean absorbance at 570 nm to 50 % of those in the untreated control wells, were calculated by the four‐parameter logistic (4‐PL) model. Evaluation was based on means from at least four independent experiments.Acid phosphatase (APH) assay. An APH modified assay was used for determining cell viability in 3D spheroids, as previously described.50 values were calculated with a four‐parameter logistic (4‐PL) model. All the values are the means ± SD of not less than four independent experiments.Mitochondrial membrane potential (ΔΨ). The ΔΨ was assayed using the Mito‐ID® Membrane Potential Kit according to the manufacturer's instructions  as previously described.3 per well) were seeded in 96‐well plates; after 24 h, cells were washed with PBS and loaded with Mito‐ID detection reagent for 30 min at 37 °C in the dark. Afterwards, cells were incubated with increasing concentrations of tested complexes. Fluorescence intensity was estimated using a VICTOR X3  plate reader at λex=490 and λem=590 nm. Antimycin (3 μM) was used as positive control.ROS production. The production of ROS was measured in PSN‐1 cells (104 per well) grown for 24 h in a 96‐well plate in RPMI medium without phenol red (Merck). Cells were then washed with PBS and loaded with 10 μM 5‐(and‐6)‐chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate acetyl ester  for 25 min, in the dark. Afterwards, cells were washed with PBS and incubated with increasing concentrations of tested compounds. Fluorescence increase was estimated by using λex=485 nm and λem=527 nm in a VICTOR X3 (PerkinElmer) plate reader. Antimycin , a potent inhibitor of Complex III in the electron transport chain, was used as positive control.Intracellular CO release. PSN‐1 cell (1×104) were seeded in 96‐well plates. After 24 h, cells were treated with 20 μM of tested complexes or CORM‐401 (Merck) for 15 min at 37 °C and followed by 30 min incubation with a fluorescent probe BioTracker Carbon Monoxide Probe 1 Live Cell Dye® (Merck). The intracellular fluorescence, indicative of CO accumulation in cells, was quantified using a VICTOR X3 (PerkinElmer) plate reader.Cellular uptake. PSN‐1 and HEK293 cells (3×106) were seeded in 75 cm2 flasks in growth medium (20 mL). After overnight incubation, the medium was replaced and the cells were treated with tested compounds for 24 h. Cell monolayers were washed twice with cold PBS, harvested and counted. Samples were than subjected to three freezing/thawing cycles at −80 °C, and then vigorously vortexed. The samples were treated with highly pure nitric acid  and transferred into a microwave Teflon vessel. Subsequently, samples were submitted to standard procedures using a speed wave MWS‐3 Berghof instrument . After cooling, each mineralized sample was analysed for iron using a Varian AA Duo graphite furnace atomic absorption spectrometer (Varian) at the wavelength of 248.3 nm. The calibration curve was obtained using known concentrations of standard solutions purchased from Sigma Chemical Co.Statistical analysis. All values are the means ± SD of no less than three measurements starting from three different cell cultures. Multiple comparisons were made by ANOVA followed by the Tukey‐Kramer multiple comparison test , using GraphPad software.50 values after 24 h of incubation; MS spectra from lysozyme interaction experiments. Deposition numbers 2A]CF3SO3), 2045778 (for [2C]CF3SO3), 2045777 (for [2C]Cl), 2045780 (for [2D]CF3SO3) and 2045781 (for 6)2045779  should be addressed to the authors.SupplementaryClick here for additional data file.


The molecular and crystal structure of a ferrocenyl derivative with an undecyl-1,11-diol chain on one cyclopentadienyl ring is reported: O—H⋯O, C—H⋯O and C—H⋯π(ring) contacts occur in the extended structure. 5H5)(C16H27O2)], comprises an α,ω-diol-substituted undecyl chain with a ferrocenyl substituent at at one terminus. The alkane chain is inclined to the substituted ring of the ferrocene grouping by 84.22 (13)°. The ferrocene rings are almost eclipsed and parallel. The crystal structure features O—H⋯O and C—H⋯O hydrogen bonds and C—H⋯π contacts that stack the molecules along the c-axis direction. A Hirshfeld surface analysis reveals that H⋯H interactions (83.2%) dominate the surface contacts.The racemic title compound, [Fe(C It was synthesized to provide a ferrocenyl-substituted diol for the preparation of polyesters with regular pendant electroactive groups. Similar ferrocenyl neo-pentyl diol-derived terephthalate polymers have been shown to display interesting electrochemical properties  using LiAlH4. Enantiomeric selection of the individual chiral forms should be possible using more complex synthetic methodology (C16H27O2)], comprises a ferrocene unit that carries a well-ordered undecane chain (atoms C11–C21) with hydroxyl substituents at the 1 and 11 positions along the chain ° between them. The C11 undecyl chain in 1 is conformationally extended with the typical antiperiplanar  Å. The C1–C5 and C6–C10 cyclopentadienyl rings of the ferrocenyl group are approximately 3° from being eclipsed and are almost coplanar with a dihedral angle of 1.7 (2)° between them; the separation of the ring centroids is is 3.298 (2) Å.The title compound, , 1.7–1.3 . 13C NMR (CDCl3): 94.7 (Fc ipso), 69.7 (–CHOH–), 68.3 (Cp), 67.9, 67.7, 67.3, 65.2 (Fc—Cα & β), 63.2 (–CH2OH), 38.3, 32.9, 29.6, 29.6, 29.5, 29.5, 26.1, 25.8 (–CH2–). UV–vis (CH2Cl2): 325 (90), 440 (110) nm (ɛ).The title compound iso(H) = 1.5 Ueq(O). All H-atoms bound to C were refined using a riding model with C—H = 0.95–1.00 Å and Uiso(H) = 1.2Ueq(C). Despite repeated attempts to grow crystals of better quality, the crystals obtained were weakly diffracting and the extent of diffraction observed is poor with sin (θmax)/λ = 0.544 (2θmax = 44.5°). Despite this, the structure solved and refined adequately.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698902101358X/hb8007sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S205698902101358X/hb8007Isup2.hklStructure factors: contains datablock(s) I. DOI: 2130725CCDC reference:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


In the crystal, dimeric C—H⋯O hydrogen bonds connect pairs of cations, producing two  30H28NO2+·CF3O3S−, the four tetrahydrofuran rings adopt envelope conformations. In the crystal, pairs of cations are linked by dimeric C—H⋯O hydrogen bonds, forming two R22(6) ring motifs parallel to the (001) plane. The cations and anions are connected by further C—H⋯O hydrogen bonds, forming a three-dimensional network structure. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯H (47.6%), C⋯H/H⋯C (20.6%), O⋯H/H⋯O (18.0%) and F⋯H/H⋯F (9.9%) interactions.In the cation of the title salt, C The molecular conformation of the cation is stabilized by weak intramolecular C21—H21B⋯O12 and C21—H21B⋯O13 contacts (Table 1B/C11C/C3A/C3) in the cation exhibits a chair conformation . The benzene ring (C7A/C8–C11/C11A) fused with the central tetrahydrofuran ring makes dihedral angles of 53.43 (5) and 58.64 (5)°, respectively, with the C22–C27 and C32–C37 phenyl rings of the benzyl groups attached to the N atom. These phenyl rings make a dihedral angle of 73.81 (5)° with each other.In the cation  −x + 1, −y + 1, −z + 1], forming two et al., 1995A⋯O1i, C6—H6A⋯O12ii, C6A—H6AA⋯O3ii, C7—H7A⋯O13ii, C31—H31A⋯O2i and C9—H9A⋯Cg10iii hydrogen bonds, forming a three-dimensional network  interactions constitute the primary factor in the crystal packing, with C⋯H/H⋯C , O⋯H/H⋯O  and F⋯H/H⋯F  interactions constituting the next stronger contributions. Numerical values of these interactions together with other percentage contributions of weaker interactions are compiled in Table 3The intermolecular interactions Table 2 were qu⋯C Fig. 8c, O⋯H/H⋯O Fig. 8d and F⋯⋯F Fig. 8e interet al., 2016viz. IQOTOA  and ac plane. Additionally, C—H⋯O hydrogen bonds form a C(6) chain, linking the molecules along the b-axis direction. In the crystal of ERIVIL, molecules are connected into b axis. In MIGTIG, the molecules are linked only by weak van der Waals interactions.IQOTOA, OMUTAU and OMEMAX each crystallize with two molecules in the asymmetric unit. In the crystal, molecule pairs generate centrosymmetric rings with -2,2-Dibenzyl-2,3,6a,11c-tetrahydro-1H,6H,7H-3a,6:7,11b-diepoxydibenzoisoquinolin-2-ium trifluoromethanesulfonate (1)3CN (20 ml). Then an equivalent of 2-(trimethylsilyl)phenyl trifluoromethanesulfonate  was added to the solution under an argon atmosphere. The mixture was refluxed for 4 h . After one more portion of 2-(trimethylsilyl)phenyl trifluoromethanesulfonate  and CsF  had been added to the mixture, all procedures were repeated. After the mixture was cooled to room temperature, the CsF was filtered off through a thin layer of SiO2, and the resulting solution was concentrated under reduced pressure. The residue (yellow oil) turned out to be a multicomponent mixture. It was separated using column chromatography on silica gel. The least mobile fraction represented the target product, 1. In addition, two by-products 2 (12%) and 3 (17%) were isolated. Single crystals of 1 were obtained by slow crystallization from ethyl acetate.Cesium fluoride (CsF)  was added to benzylbis(furan-2-ylmethyl)amine (0.0022 mol) dissolved in dry CHUiso(H) = 1.2Ueq(C). Five reflections  I. DOI: 10.1107/S2056989021010173/wm5617Isup2.hklStructure factors: contains datablock(s) I. DOI:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


In the crystal, the title molecules are linked by intermolecular C—H⋯N, C—H⋯Cl, C—H⋯π contacts and π–π stacking interactions. A Hirshfeld surface analysis was undertaken to quantify the intermolecular interactions. 24H20ClNO2, the mean planes of 4-chlorophenyl, 2-methylphenyl and phenylene rings make dihedral angles of 62.8 (2), 65.1 (3) and 15.1 (2)°, respectively, with the 5-methyl-1,2-oxazole ring. In the crystal, molecules are linked by intermolecular C—H⋯N, C—H⋯Cl, C—H⋯π contacts and π–π stacking interactions between the phenylene groups. Hirshfeld surface analysis indicates that the most important contributions to the crystal packing are from H⋯H (48.7%), H⋯C/C⋯H (22.2%), Cl⋯H/H⋯Cl (8.8%), H⋯O/O⋯H (8.2%) and H⋯N/N⋯H (5.1%) interactions.In the title compound, C SCXD has thus become an essential tool for drug development to unambiguously determine the three-dimensional structures of molecules, which eventually paves the way for rapid development of new molecules  = 3.958 (2) Å]  to 1.5787 Å (blue)  Fig. 4.dnorm values on the surface, correspond to the C—H⋯N (C17—H17A⋯N1), C—H⋯Cl (C8—Cl1⋯H1C—C1) and C—H⋯π (C6—H6⋯phenylene) interactions  shows the total two-dimensional fingerprint plot providing information on the major and minor percentage contributions of the interatomic contacts to the Hirshfeld surface of the title compound. The blue colour refers to the frequency of occurrence of the  pair and the grey colour is the outline of the full fingerprint  show that the H⋯H contacts clearly make the most significant contribution to the Hirshfeld surface (48.7%). The H⋯C/C⋯H, Cl⋯H/H⋯Cl, H⋯O/O⋯H and H⋯N/N⋯H contacts contribute 22.2, 8.8, 8.2 and 5.1%, respectively . The remaining weaker contacts are listed in Table 3Fig. 6ly Fig. 6c–f. Theet al., 2015The large number of H⋯H, H⋯C/C⋯H, Cl⋯H/H⋯Cl, H⋯O/O⋯H and H⋯N/N⋯H interactions suggest that van der Waals interactions play the major roles in the crystal packing (Hathwar E)-2- (dimethylamino)ethenyl]-1,2-oxazole-4- carboxylate , whereas in molecule B, it points back towards the benzene ring [C—C—C=O = 17.9 (4)°]. The dihedral angles between the oxazole and benzene rings are also somewhat different . Each molecule features an intramolecular C—H⋯O interaction, which closes an S(6) ring. In the crystal, the B molecules are linked into C(12) chains along the c-axis direction by weak C—H⋯Cl interactions. In the crystal of (II), the components are linked by O—H⋯N and N—H⋯O hydrogen bonds, where the water molecule acts as both an H-atom donor and an acceptor, into a tape along the a-axis direction with an In compound (I)N-hydroxy-4-[(2-methylbenzyl)oxy]benzimidoyl chloride  in diethyl ether (6 ml) was added Et3N . The resulting mixture was stirred for 2 h in an ice bath, and the precipitate formed was filtered off. The filtrate was evaporated under vacuum to obtain the arylnitriloxide intermediate.Step 1: To a solution of 2SO4, filtered and evaporated to dryness. The crude product was purified by automated-flash chromatography on silica gel (12 g) eluting with a gradient of 0 to 40% EtOAc in hexane. The obtained pure product was recrystallized from methanol. Crystals for structural study were obtained by slow cooling of the solution, yield 77%, m.p. 387.2–388.6 K.Step 2: To a solution of NaH  in dry THF (4 ml), 4-chlorophenylacetone  was added dropwise, and stirred for 1 h under a nitrogen atmosphere in an ice bath. At the end of the period, the arylnitriloxide intermediate was dissolved in dry THF (4 ml), and was added to the reaction mixture, then stirred at room temperature overnight. Upon completion of the reaction, aqueous ammonium chloride solution was added, and the product was extracted with EtOAc (2 × 50 mL). The combined organic extracts were dried over anhydrous Na1H NMR : δ 2.29 , 2.39 , 5.07 , 7.05 , 7.15–7.25 , 7.27 , 7.38 , 7.47 .13C NMR : δ 11.21, 18.42, 67.98, 113.96, 114.97, 120.84, 125.77, 128.15, 128.59, 128.86, 129.43, 130.12, 131.44, 132.57, 134.58, 136.64, 159.49, 160.09, 166.93. HRMS (m/z): [M + H]+ calculated for C24H21ClNO2: 390.1261; found: 390.1263.Uiso(H) = 1.2Ueq(C) or 1.5Ueq(C-methyl). In the final refinement, three outliers  I. DOI: 10.1107/S2056989021002383/yk2147Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021002383/yk2147Isup3.cmlSupporting information file. DOI: 2067449CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


This study was designed to identify the fluid spaces that are most changed during ultrafiltration (UF) according to intradialytic blood pressure (BP) difference. BP data were collected five times . Intradialytic BP difference was calculated as the highest minus lowest of these BP measurements. Intradialytic systolic BP (SBP) difference over 20 mm Hg and diastolic BP (DBP) difference over 10 mm Hg were defined as wide intradialytic SBP difference (SYS-W) and DBP difference (DIA-W), respectively. We measured the various fluid spaces before HD and 1–4 h of HD, and 30 min after HD using a portable, whole-body bioimpedance spectroscopy (BIS). In this study, 85 prevalent patients aged over 18 years with a fixed dry weight , undergoing HD had participated. 1) Mean relative reduction of extracellular water (ECW) was significantly higher in SYS-W than in narrow intradialytic SBP difference (SYS-N) patients from 1 h to 30 min after HD. 2) Mean relative reduction of intracellular water (ICW) was significantly lower in DIA-W than in narrow intradialytic DBP difference (DIA-N) patients from 1 h to 30 min after HD. 3) ECW of patients with SYS-W was significantly lower than that of patients with SYS-N. Patients with SYS-W have the characteristics of fluid shifts in which reduction of ECW was steeper than patients with SYS-N whereas fluid shifts of ICW were lower in patients with DIA-W than patients with DIA-N. A recent large, retrospective cohort study observed first-year mortality to be higher in HD patients with BP variability than in those with stable BP, independent of the absolute BP level, suggesting that BP variability is associated with worse outcomes at all levels of BP . The majvs. intracellular [IC]) source of fluid shifts during the UF procedure and the relative ratio of these compartments during conventional renal replacement therapy in patients with asymptomatic intradialytic BP variability. An authoritative journal accepted the definition of BP variability by the difference between the highest and lowest BP during each session , patients with a history of ischemic or hemorrhagic stroke, or heart disease including atrial fibrillation  < 45% by 2-D echocardiography); those with peripheral arterial occlusive disease, such as gangrene, and amputees; and those with infection or hemiplegia were excluded. Some of the patient’s characteristics are given in 2 hollow fiber poly-sulfone membranes, blood flow rates of 250–280 mL/min, and a dialysate flow rate of 500 mL/min, using the Fresenius 5008S machine . The mean UF volume was 2457.65 ± 947.73 mL, and treatment time was 4 h. The dialysis fluid contained 138 mEq/L sodium, 2 mEq/L potassium, 2.5 mEq/L calcium, 1.0 mEq/L magnesium, 108.5 mEq/L chloride, 35 mEq/L bicarbonate, and 99.1 mg/dL glucose, and the temperature was maintained at 36 °C.All patients underwent high-flux HD thrice weekly with a bicarbonate buffer for 4 h, using 1.0–1.4-mBP data were collected five times (before HD and 1–4 h of HD), and the highest and lowest measurements were identified. Intradialytic BP difference was calculated as the highest minus lowest BP measurements of each dialysis session . Intradiurea) in liters (L). The BCM determines resistance and reactance at 50 discrete frequencies from 3 to 1000 kHz. ECW and ICW resistance are obtained on the basis of a Cole model [BCM measurement has been recommended that subjects should be resting in a supine position for at least 5 min, before the BCM-measurement is started, such that fluid volume equilibration has taken place . We measle model . Using tle model .Because of the small number of patients, normal distribution was tested using a single sample Kolmogorov–Smirnov analysis. Continuous variables are expressed as mean ± SD or median (range) according to normal distribution of data. Categorical variables are expressed as percentage (%). Between-group differences were assessed for significance using a Student t-test for normal distribution, Mann–Whitney U tests for skewed distribution, or Chi-Square test for categorical variables. Statistical analyses were performed using SPSS software version 14.0 .The study included a total of 85 patients undergoing HD (vintage of 70.98 ± 59.47 months), 46 (54.12%) men with a mean age of 65.38 ± 12.45 years. Here, 44 (51.76%) patients had diabetes mellitus and 50 (58.82%) patients had hypertension. The anti-hypertensive medications administered by the patients included angiotensin-converting enzyme inhibitor by nine patients (10.59%), angiotensin II receptor blocker by 18 patients (21.18%), calcium channel blocker by 30 patients (35.29%), alpha blocker by three patients (3.53%), beta-blocker by 21 patients (24.71%), diuretic by seven patients (8.24%), and other anti-hypertensive medications by 2 patient (2.35%). In 62 (72.94%) or 35 (41.17%) of the patients on HD, phosphorus binders or active vitamin D metabolites  were administered, respectively. Other variables are summarized in vs. 0.984 ± 0.027; 2 h, 0.988 ± 0.047 vs. 0.975 ± 0.034; 3 h, 0.970 ± 0.032 vs. 0.958 ± 0.032; 4 h, 0.960 ± 0.035 vs. 0.948 ± 0.035, 30 min after HD; 0.955 ± 0.029 vs. 0.941 ± 0.032, respectively)  .vs. 0.977 ± 0.019*; 2 h, 0.966 ± 0.027 vs. 0.952 ± 0.026*; 3 h, 0.942 ± 0.032 vs. 0.924 ± 0.033*; 4 h, 0.923 ± 0.037 vs. 0.901 ± 0.038*, 30 min after HD; 0.916 ± 0.038 vs. 0.895 ± 0.042*, respectively, *p < .05)  .vs. 0.991 ± 0.052; 2 h, 1.008 ± 0.081 vs. 0.996 ± 0.064; 3 h, 0.996 ± 0.047 vs. 0.989 ± 0.054; 4 h, 0.997 ± 0.044 vs. 0.991 ± 0.058, 30 min after HD; 0.992 ± 0.038 vs. 0.987 ± 0.049, respectively)  .vs. 0.986 ± 0.024; 2 h, 0.971 ± 0.025 vs. 0.982 ± 0.040; 3 h, 0.955 ± 0.027 vs. 0.964 ± 0.036; 4 h, 0.945 ± 0.034 vs. 0.953 ± 0.036, 30 min after HD; 0.938 ± 0.026 vs. 0.947 ± 0.036, respectively)  .vs. 0.975 ± 0.021; 2 h, 0.959 ± 0.021 vs. 0.953 ± 0.030; 3 h, 0.932 ± 0.027 vs. 0.926 ± 0.038; 4 h, 0.910 ± 0.035 vs. 0.905 ± 0.042, 30 min after HD; 0.901 ± 0.036 vs. 0.898 ± 0.046, respectively)  .vs. 0.997 ± 0.049*; 2 h, 0.982 ± 0.043 vs. 1.009 ± 0.073*; 3 h, 0.977 ± 0.045 vs. 1.001 ± 0.053*; 4 h, 0.980 ± 0.056 vs. 0.999 ± 0.050*, 30 min after HD; 0.975 ± 0.040 vs. 0.995 ± 0.050, respectively, *p < .05)  .vs. 0.993 ± 0.021; 2 h, 0.977 ± 0.021 vs. 0.977 ± 0.016; 3 h, 0.965 ± 0.024 vs. 0.966 ± 0.029; 4 h, 0.953 ± 0.020 vs. 0.953 ± 0.025, 30 min after HD; 0.950 ± 0.021 vs. 0.950 ± 0.030, respectively)  .vs. 0.977 ± 0.019*; 2 h, 0.969 ± 0.014 vs. 0.949 ± 0.026*; 3 h, 0.946 ± 0.018 vs. 0.923 ± 0.034*; 4 h, 0.924 ± 0.018 vs. 0.904 ± 0.038*, 30 min after HD; 0.922 ± 0.019 vs. 0.885 ± 0.043*, respectively, *p < .05)  .vs. 1.014 ± 0.050*; 2 h, 0.983 ± 0.039 vs. 1.010 ± 0.034*; 3 h, 0.982 ± 0.042 vs. 1.015 ± 0.037*; 4 h, 0.982 ± 0.034 vs. 1.012 ± 0.038*, 30 min after HD; 0.984 ± 0.036 vs. 1.010 ± 0.039*, respectively, *p < .05)  .urea, TBW, ICW, and E/I ratio were no significant difference between the groups  weight, UFV, Ve groups .The main findings of our study are 1) changes in the ECW of patients with wide intradialytic SBP difference were significantly steeper than those of patients with narrow intradialytic SBP difference from 1 h to 30 min after HD. 2) there were no significant fluid shifts in the ICW of patients with wide intradialytic DBP difference, whereas that of patients with narrow intradialytic DBP difference was significantly decreased from 1 h to 30 min after HD. 3) ECW of patients with wide intradialytic SBP difference was significantly lower than that of patients with narrow intradialytic SBP difference.The results of the study suggest that maintenance of intradialytic SBP stability is dependent on the amount of ECW reduction during HD and that DBP stability is associated with the shifts of ICW. That is because there was no difference in the changes of TBW, whereas the difference in ECW or ICW changes could be observed between patients with narrow SBP difference and wide SBP difference, between patients with narrow DBP difference and wide DBP difference during HD, respectively. This phenomenon has been confirmed from patients with narrow intradialytic SBP/DBP difference, once again. In particular, ICW in patients with narrow intradialytic SBP/DBP difference showed a significant decrease, whereas that in patients with wide intradialytic SBP/DBP difference demonstrated no significant reduction of ICW from 1 h of HD compared to the start of HD. The above changes were continued until 30 min after HD without further reduction or increase. This indicates that the amount of shifts of ICW to ECW may affect the maintenance of SBP and DBP stability in situations where ECW is continuously reduced after HD. Thus, in contrast to a previous report , fluid rA recently introduced equipment called a BCM utilizes a well-established technical solution to assess the absolute volume of the body fluid distribution  using 50 multiple, discrete frequencies from 3 to 1000 kHz . DespiteThe clinical dilemmas and prognostic uncertainties exist in a patient with asymptomatic intradialytic BP falls, elevations, and fluctuations compared to overt intradialytic BP abnormalities such as IDH or IH. This absence of associated symptoms contributes to the tendency to regard asymptomatic BP fluctuations as ‘normal’ BP. However, there is a possibility that aberrant, asymptomatic intradialytic BP changes induce harmful effects to HD patients. Thus, our study was designed to evaluate factors that may influence the fluid shifts in asymptomatic patents with intradialytic BP falls. But, there were no significant differences between the hydration status, presence of DM, age, sex, HT medication, HD vintage, and fluid distribution index. Only we found that ECW of patients with wide intradialytic SBP difference was significantly lower than that of patients with narrow intradialytic SBP difference. Unfortunately, it would be difficult to explain the exact meaning of our results. Nevertheless, further study should be needed to elucidate the changes of IVF and ISF absolute values and relevant factors in asymptomatic patients with wide intradialytic BP difference.per se and a relation between a larger decline in ECW and BP is to be expected. If we repeated the analysis with additional BP variability metrics such as the BP residual in a random-effects model [This study had some limitations. First, we used intradialytic BP difference as an index of BP fluctuation instead of other intradialytic BP variability index, such as standard deviation, absolute value of the difference between successive BP measurements and BP residual. However, this reflects more the BP drop during HD than the variability ts model , we thouPatients with wide intradialytic SBP difference have the characteristics of fluid shifts in which reduction of ECW was steeper than patients with narrow intradialytic SBP difference whereas fluid shifts of ICW were lower in patients with wide intradialytic DBP difference than patients with narrow intradialytic DBP difference. This phenomenon has been confirmed from patients with narrow intradialytic SBP/DBP difference.


Scientific Reports 10.1038/s41598-021-85129-1, published online 10 March 2021Correction to: The original version of this Article contained errors.Author Fatih Semerci was incorrectly given as Faith Semerci.In addition, in the Introduction,“The NSC-6 Ab, which produced the most robust staining of NPCs, corresponded to Brain-Associated Signal Protein 1 (BASP1), a protein not previously described in NPCs.”now reads:“The NSC-6 Ab, which produced the most robust staining of NPCs, corresponded to Brain-Abundant, membrane-attached Signal Protein 1 (BASP1), a protein not previously described in NPCs.”In the Results section, under subheading “NSC-6 stains mouse and human NPCs”,“In addition, we derived human neuroprogenitor cells (hNPCs) from inducible pluripotent stem cells (iPSCs) obtained from a healthy adult and stained them with NSC-6.”now reads:“In addition, we derived human neuroprogenitor cells (hNPCs) from induced pluripotent stem cells (iPSCs) obtained from a healthy adult and stained them with NSC-6.”Under the subheading “NSC-6-labeled BASP1 is regulated temporally in the mammalian brain”,“Consistent with the data obtained in mice, our results show high BASP1 expression in the hippocampus, the brainstem, and the spinal cord .”now reads:“Consistent with the data obtained in mice, our results show high BASP1 expression in the human hippocampus, the brainstem, and the spinal cord .”In the Materials and methods section, under subheading “Embryonic neurosphere culture and hybridoma production”,5 cells/10 ml of proliferation media containing Neurocult Basal Media (STEM CELL TECHNOLOGIES), 10% Proliferation Supplement (STEM CELL TECHNOLOGIES), 20 ng/ml EGF and FGF (SIGMA) and 1% antibiotic–antimycotic (GIBCO).”“Whole brains from C57/BL6 embryonic day 12 (E12) mice were dissected, digested with collagenase  for 2 h at 37 °C, filtered twice through 0.4 μm filters and plated at 3 × 10now reads:5 cells/10 ml of proliferation media containing Neurocult Basal Media (STEM CELL TECHNOLOGIES), 10% Proliferation Supplement (STEM CELL TECHNOLOGIES), 20 ng/ml EGF and FGF (SIGMA) and 1% antibiotic–antimycotic (GIBCO).”“Whole brains from C57/BL6 embryonic day 12 (E12) mice were dissected, digested with collagenase  for 2 h at 37 °C, filtered twice through 40 μm filters and plated at 3 × 10Under subheading “Two-dimensional electrophoresis”,50.”“Briefly, IPG strips  were passively equilibrated under mineral oil for 18 h at 23 °C with 90 µg of solubilized protein in 200 µl STnow reads:50.”“Briefly, IPG strips  were passively equilibrated under mineral oil for 18 h at 23 °C with 90 µg of solubilized protein in 200 µl STUnder the subheading “Neurosphere culture from adult brain SVZ”,“3 ml of Trypsin inhibitor (from glycine max) was added and gently mixed and the tissue suspension was passed through 0.7 μm pore size filter followed by centrifuge at 700 rpm for 5 min. The supernatant was removed and the pellet was resuspended in the NSC culture media .”now reads:“3 ml of Trypsin inhibitor (from glycine max) was added and gently mixed and the tissue suspension was passed through 70 μm pore size filter followed by centrifuge at 700 rpm for 5 min. The supernatant was removed and the pellet was resuspended in the NSC culture media .”Under the subheading, “Human brain organoids and hNPCs”,“Specifically, iPSCs colonies were first dissociated into single cells (D0) with Accutase (SIGMA-ALDRICH) and 1.5 million cells seeded per well of an AggreWell00 plate (STEM CELL TECHNOLOGIES) in a medium containing KnockOut DMEM, 20% KnockOut Serum Replacement, 1% penicillin and streptomycin (P/S) solution, 0.5X GlutaMAX, 1× MEM Non-Essential Amino Acids , 5 µM Dorsomorphin (PEPROTECH), 10 µM SB431542 (PEPROTECH), 100 µM 2-Mercaptoethanol (SIGMA), and 10 µM Y-27632 (TOCRIS).”now reads:“Specifically, iPSCs colonies were first dissociated into single cells (D0) with Accutase (SIGMA-ALDRICH) and 1.5 million cells seeded per well of an AggreWell800 plate (STEM CELL TECHNOLOGIES) in a medium containing KnockOut DMEM, 20% KnockOut Serum Replacement, 1% penicillin and streptomycin (P/S) solution, 0.5X GlutaMAX, 1× MEM Non-Essential Amino Acids , 5 µM Dorsomorphin (PEPROTECH), 10 µM SB431542 (PEPROTECH), 100 µM 2-Mercaptoethanol (SIGMA), and 10 µM Y-27632 (TOCRIS).”Lastly, Reference 11 was incorrectly given as:Front. Biol. (Beijing) 11, 151–167 (2016).Semerci, F. & Maleic-Savatic, M. Transgenic mouse models for studying adult neurogenesis. The correct reference is listed below:Front. Biol. (Beijing) 11, 151–167 (2016).Semerci, F. & Maletic-Savatic, M. Transgenic mouse models for studying adult neurogenesis. The original Article has been corrected.


The title molecule adopts a conformation with the two phenyl substituents disposed on opposite sides of the mean plane of the isoquinoline unit. In the crystal, corrugated layers of molecules are formed by N—H⋯O, C—H⋯N and C—H⋯S hydrogen bonds together with C—H⋯π(ring) interactions. These layers are connected by C—H⋯O contacts. 29H29N3O4S, adopts a conformation with the two phenyl substituents disposed on opposite sides of the mean plane of the isoquinoline unit. In the crystal, corrugated layers of molecules are formed by N—H⋯O, C—H⋯N and C—H⋯S hydrogen bonds together with C—H⋯π(ring) interactions. These layers are connected by C—H⋯O contacts. The Hirshfeld surface analysis of the crystal structure indicates that the most important contributions for the crystal packing are from H⋯H (45.2%), C⋯H/H⋯C (20.2%), O⋯H/H⋯O (15.8%) and N⋯H/H⋯N (11.0%) interactions.The title molecule, C In AKIVUO, a layer structure with the layers parallel to . In POPYEB, molecules are packed in a herringbone manner parallel to (103) and (10via weak C—H⋯O and C—H⋯π(ring) interactions. In ENOCIU, various C—H⋯π and C—H⋯O bonds link the molecules together. In NIWPAL, the molecules are linked by N—H⋯O intermolecular hydrogen bonds involving the sulfonamide function to form an infinite two-dimensional network parallel to (001).In the crystal of NAQRIJ, dimers are formed through complementary sets of inversion-related O—H⋯O and C—H⋯O hydrogen bonds, which are further connected into zigzag chains by pairwise C—H⋯N interactions that also form inversion dimers. In KUGLIK, the heterocyclic amines are alternately connected by hydrogen bonds thus forming syndiotactic polymeric chains. The hydrogen-bonding network of water molecules forms planes parallel to (100). In the crystal of DUSVIZ, molecules are linked H)-thione (10 mmol), N-(phenyl)-2-chloroacetamide (10 mmol) and sodium acetate trihydrate  in ethanol (100 ml) was heated under reflux for 1 h. The reaction mixture was allowed to stand at room temperature overnight. The precipitate that formed was collected and recrystallized from ethanol giving colourless crystals of the title compound, m.p.: 508–510 K, yield 84%. Its IR spectrum showed characteristic absorption bands at 3474 cm−1 (OH); 3311 cm−1 (NH); 3023 cm−1 (C-H aromatic); 2910, 2956 cm−1 ; 1800, 1900 cm−1 (overtones of phenyl group); 2220 cm−1 (C≡N) and 1694 cm−1 (C=O). Its 1H NMR  spectrum exhibited the following signals: δ 10.21 , 7.48–7.49 ; 7.22–7.25 ; 6.97–7.00 ; 6.89–6.91 ; 6.75–6.77 ; 4.84 ; 4.41–4.43 ; 4.04–4.11 ; 3.66 ; 3.20–3.24 ; 2.83–2.85 ; 2.81–2.84 ; 2.08 ; 1.86  and 1.21 .A mixture of 7-acetyl-4-cyano-1,6-dimethyl-6-hydroxy-8-(4-methoxyphenyl)-5,6,7,8-tetrahydroisoquinoline-3 I, global. DOI: 10.1107/S2056989021005430/yk2151Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989021005430/yk2151Isup3.cmlSupporting information file. DOI: 2085564CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Three crystal structures from the reaction of 2,6-dimethylphenyl isocyanide with bis(anthracene)ferrate(−1) are presented. 2]1+ (thf is tetrahydrofuran) salt of bis(anthracene)ferrate(−1), or [Fe(C14H10)2]−, with 2,6-dimethylphenyl isocyanide (CNXyl) in thf resulted in the formation of two new iron isocyanide complexes, namely, [-anthracene]trisiron, [Fe(C14H10)(C9H9N)3] or [Fe(CNXyl)3], and {5,6-bis-3--1,2,7-tris[imino]-3-azoniahept-3-ene-1,4,7-triido}trisiron tetrahydrofuran disolvate, [Fe(C54H56N6)(C9H9N)3]·2C4H8O or [Fe(C54H56N6)(CNXyl)3]·2C4H8O, which were characterized by single-crystal X-ray diffraction. The former is likely an intermediate along the path to the known homoleptic [Fe(CNXyl)5], while the latter contains a tridentate ligand that is formed from the ‘coupling’ of six CNXyl ligands. A third crystal structure from this reaction, (7-methylindol-1-ido-κN)potassium, [K(C9H8N)(C12H24O6)] or [K(C9H8N)(18-crown-6)], contains a 7-methylindol-1-ide anion, in which one CNXyl ligand has shed a proton during its reductive cyclization.The reaction of the [K(18-crown-6)(thf) On this basis, the corresponding reaction with CNXyl in tetrahydrofuran, thf, was examined to determine whether the unknown [Fe2(CNXyl)8]2− could be accessed. Bis(anthracene)ferrate(−1) was also reacted with excess CNXyl in the presence of one equivalent of a reducing agent to see whether the previously reported monometallic [Fe(CNXyl)4]2− 5] cobaltate(−1) has been shown to be an excellent source of spin-paired atomic Co(−1) anions in substitution reactions in which both anthracene ferrate(−1) with excess CNXyl in THF. First an aliquot was taken from the reaction mixture early on and placed in a 243 K freezer until orange blocks were observed. A single crystal X-ray diffraction experiment revealed these to be [Fe(C14H10)(CNXyl)3] 1 iron(0). However, the related carbonyl, [Fe(C14H10)(CO)3], has been known for more than 50 years (CNXyl)3] 2 6X]+ (many variations on R and X) by Zn in the presence of water generated a bisacetylene ligand with protonated nitrogen atoms (C9H8N)] 3 ]. The hydrogen atom lost during the reduction and cyclization that forms e.g., four), intractable tars resulted. It should be noted, however, that this equation is only speculative and requires further investigation for confirmation.Interestingly, in support of this equation, when less than eight equivalents of CNXyl were employed (Equation (1)2][Fe(C14H10)2] + 8 CNXyl → 0.5 [Fe(CNXyl)5] + 0.5 [Fe(C54H56N6)(CNXyl)3] + [K(18-crown-6)(C9H8N)] portion of a trinuclear molecule, 97.5° 3], 97.7° 3], 97.9°  bond lengths  π–π interactions  π–π interaction between phenyl rings C56–C61 and C65–C70 [Fe(C14H10)2] and CNXyl were prepared according to previously reported procedures 2]  in thf  was added CNXyl  in thf . The reaction mixture was warmed slowly to room temperature. A solution IR spectrum showed no anionic species, but a broad peak with shoulders that matched the well-known [Fe(CNXyl)5]  were identified as 2 by X-ray diffraction. IR spectroscopy on the crystals (Nujol mull) gave νCN = 2110w and 2055vs cm−1. The filter cake  was redissolved in THF and layered with pentane, which resulted in crystals of 3 as determined by a single crystal X-ray experiment. No characterization beyond what is presented above was performed.To a deep-orange solution of . The 18-crown-6 macrocycle is also disordered in a similarly lopsided component ratio; the eight largest residual peaks are the two peaks near the K atom and those for six O atoms of the minor component of disorder. However, the data-to-parameter ratio drops below eight if this disorder is modeled. Thus only the anion disorder was modeled.In 2, bond lengths were restrained toward ideal values and anisotropic displacement parameters were additionally restrained toward the expected motion relative to bond direction.To model the various disordered species, analogous bond lengths and angles were restrained to be similar and anisotropic displacement parameters for proximal atoms were restrained to be similar. For the THF solvent molecules in 1 were refined freely to confirm their nature and better describe their true positions. In 2, H7 was also refined freely. All other H atoms were placed geometrically and treated as riding atoms. For 1 and 3 (173 K), methylene, C—H = 0.99 Å, aromatic/sp2, C—H = 0.95 Å, with Uiso(H) = 1.2Ueq(C), and methyl, C—H = 0.98 Å, with Uiso(H) = 1.5Ueq(C). For 2 (293 K), methylene, C—H = 0.97 Å, aromatic/sp2, C—H = 0.93 Å, N—H = 0.86 Å, with Uiso(H) = 1.2Ueq(C), and methyl, C—H = 0.96 Å, with Uiso(H) = 1.5Ueq(C).The H atoms on the metal-coordinating carbon atoms (C1–C4) of 1 the maximum residual peak of 0.36 e− Å−3 and the deepest hole of −0.35 e− Å−3 are found 0.97 and 0.53 Å from atoms C2 and Fe1, respectively.For 2 the maximum residual peak of 0.38 e− Å−3 and the deepest hole of −0.18 e− Å−3 are found 0.81 and 0.39 Å from atoms H15 and C14, respectively.For 3 the maximum residual peak of 0.58 e− Å−3 and the deepest hole of −0.23 e− Å−3 are found 1.15 and 1.25 Å from atoms C15 and K1, respectively. The peak is part of the minor component of disorder of the 18-crown-6 ring, which was not modeled (see above).For 10.1107/S205698902101313X/yz2014sup1.cifCrystal structure: contains datablock(s) 1, 2, 3, global. DOI: 10.1107/S205698902101313X/yz20141sup2.hklStructure factors: contains datablock(s) 1. DOI: 10.1107/S205698902101313X/yz20142sup3.hklStructure factors: contains datablock(s) 2. DOI: 10.1107/S205698902101313X/yz20143sup4.hklStructure factors: contains datablock(s) 3. DOI: 2127598, 2127597, 2127596CCDC references:  crystallographicinformation; 3D view; checkCIF reportAdditional supporting information:


Scientific Reports 10.1038/s41598-021-85972-2, published online 22 March 2021Correction to: The original version of this Article contained an error in Figure 3 where the labels in panel D were incorrect. As a result,“mg/ml”now reads:“mg/kg”Additionally,“μg/ml”now reads:“mg/kg”Furthermore, in the legend of Figure 3,A) 20 μm; in (B) 50 μm”“Scale bar in (now reads:B) 20 μm; in (D) 50 μm”“Scale bar in (The original Figure


Intracerebral hemorrhage (ICH) can induce intensively oxidative stress, neuroinflammation, and brain cell apoptosis. However, currently, there is no highly effective treatment available. Puerarin (PUE) possesses excellent neuroprotective effects by suppressing the NF‐κB pathway and activating the PI3K/Akt signal, but its role and related mechanisms in ICH‐induced early brain injury (EBI) remain unclear. In this study, we intended to observe the effects of PUE and molecular mechanisms on ICH‐induced EBI. ICH was induced in rats by collagenase IV injection. PUE was intraperitoneally administrated alone or with simultaneously intracerebroventricular injection of LY294002 . Neurological deficiency, histological impairment, brain edema, hematoma volume, blood–brain barrier destruction, and brain cell apoptosis were evaluated. Western blot, immunohistochemistry staining, reactive oxygen species (ROS) measurement, and enzyme‐linked immunosorbent assay were performed. PUE administration at 50 mg/kg and 100 mg/kg could significantly reduce ICH‐induced neurological deficits and EBI. Moreover, PUE could notably restrain ICH‐induced upregulation of the NF‐κB pathway, pro‐inflammatory cytokines, ROS level, and apoptotic pathway and activate the PI3K/Akt signal. However, LY294002 delivery could efficaciously weaken these neuroprotective effects of PUE. Overall, PUE could attenuate ICH‐induced behavioral defects and EBI possibly by PI3K/Akt signal stimulation‐mediated inhibition of the NF‐κB pathway, and this made PUE a potential candidate as a promising therapeutic option for ICH‐induced EBI. The research protocol and animal care were authorized by the Southern Medical University Ethics Committee and were performed according to the National Institutes of Health guidelines on care and use of animals.2.2Supplementary materials, respectively   solution of PUE  (100 mg/ml) was prepared. The rats were injected intraperitoneally with either PUE at 50 mg/kg and 100 mg/kg, or just DMSO solution 30 min before modeling and at the time points of 30 min, 6 h, 12 h, 24 h, and 48 h after ICH. We selected the dosages and administration route based on previous studies, and intraperitoneal injection of PUE at 50 mg/kg and 100 mg/kg could both produce significantly neuroprotective effects.2.5The modified neurological severity score (mNSS) scale was applied to estimate the neurobehavioral deficiency at 24 h and 72 h following ICH, which was executed by two experienced researchers who were blinded to animal groups.2.6Paraffin sections of rat brains were made, as previously described.2.7H&E staining was performed as our previous method.2.8V = T1*S1 + T2*S2 + … + Tn*Sn .Hematoma volume was assessed as previously reported with some modifications.2.9M1−M2)]/M1*100% .Brain water content (BWC) was measured by the wet/dry weight method, as previously described.2.10Evans blue (EB) dye  was applied to assess the disruption of the blood–brain barrier (BBB), as reported previously.2.11+ cells were counted in a blinded manner.At 24 h and 72 h after ICH, the brain samples were obtained and paraffin sections were prepared. TUNEL staining was performed with an In Situ Cell Death Detection Kit  as our previous method.2.122O2 for 10 min and antigen blocking with 5% bovine serum albumin for 20 min, the samples underwent overnighted co‐incubation at 4°C with the following primary antibodies: cleaved caspase‐3 , Ser536p‐NF‐κB p65 , NF‐κB p65  , 3‐nitrotyrosine (3‐NT) , and 8‐hydroxyguanosine (8‐OHdG) . Then, the brain slices were orderly incubated with corresponding secondary antibodies and horseradish peroxidase–streptavidin for 20 min. After further reaction with 3, 3‐diaminobenzidine and being counterstained with hematoxylin, representative IHC images were obtained using a microscope .IHC staining was performed as previously reported.2.13Ser536p‐NF‐κB p65 (Cat. No.: #3033), NF‐κB p65 (Cat. No.: #8242), Ser473p‐Akt (Cat. No.: #4060), Akt (Cat. No.: #9272), Bax (Cat. No.: #2772), cleaved caspase‐3 (Cat. No.: #9664) , Bcl‐2 , Tyr607p‐PI3K , and PI3K . β‐actin  and Lamin A  were employed as the internal reference. WB protein bands were quantified by ImageJ software . Protein expression levels were indicated by the ratio of interest protein bands to that of β‐actin or Lamin A bands.Western blot (WB) was carried out as our previous method.2.14At 24 h after ICH, the brain samples were collected and used to detect pro‐inflammatory cytokine levels using a Rat TNF‐α ELISA Kit (Cat. No.: SEA133Ra), Rat IL‐1β ELISA Kit (Cat. No.: SEA563Ra), and Rat IL‐6 ELISA Kit (Cat. No.: SEA079Ra)  as our previous method.2.15The reactive oxygen species (ROS) level was detected using a ROS assay kit  at 24 h after ICH.2.16P‐value was statistically significant when <0.05.All data were expressed as means ± standard deviation (SD). Data analyses were conducted with SPSS 19.0 , and related diagrams were prepared with GraphPad Prism 5 . All data were analyzed using the Shapiro–Wilk and Levene tests. If data satisfy normal distribution and homogeneity of variance, one‐way analysis of variance (ANOVA) was considered, and then the least significant difference (LSD) test was applied to compare the difference among multiple experimental groups; conversely, for unsatisfied data, Dunnett's T3 test was adopted. The 33.1P < .001) and 72 h (P < .001)  and 100 mg/kg , yet the beneficial effects of two treatment dosages were not significantly different  . The schedule of research, including ICH induction of rats, treatments with different agents , and corresponding experimental assessments, is shown Fig. . The mNS) Figure  after IC) Figure . Analogo) Figure  and 72 h) Figure  followin3.2P < .001, 24 h; P < .01, 72 h)  and 100 mg/kg  could significantly reduce an ICH‐induced increase in BWC   . Besides, PUE (100 mg/kg) also could notably repress ICH‐induced EB dye extravasation    Figure . Extrava) Figure , and PUE) Figure . Similar) Figure .3.3P <.001, 24 h and 72 h)  and 100 mg/kg  could markedly reduce the ICH‐induced increase in TUNEL+ cells. However, the effects of PUE at 50 mg/kg and 100 mg/kg had no statistical difference    after ICH   Figure . Treatme) Figure . Typical) Figure . PUE cou) Figure . BesidesH Figure . Consist) Figure .3.4P < .001), p‐NF‐κB p65 (P < .05), and nuclear NF‐κB p65 (P < .05)       Figure ,E,G,H, a) Figure  at 24 h ) Figure . Similar) Figure , p‐NF‐κB) Figure , and nuc) Figure  and sign) Figure .3.5P < .001)  at 24 h after ICH   at 24 h following ICH as well   could significantly upregulate the expression levels of PI3K  Figure  and p‐AkH Figure . Similar) Figure  and p‐Akl Figure . However) Figure .3.6P <.001) and 100 mg/kg (P < .001) could significantly increase the expression level of Bcl‐2 at 24 h after ICH  and that of cleaved caspase‐3  at 24 h following ICH  are shown Figure . The resH Figure . MoreoveH Figure . TypicalH Figure .3.7+ and 8‐OHdG+ cells are shown        Figure  and 8‐OH) Figure . Moreove) Figure , and PUE) Figure  and 100 ) Figure , yet the) Figure .3.8P < .001)         notably increased  Figure ,H, and P) Figure , IL‐1β  Figure , and IL‐) Figure . Similar) Figure , IL‐1β  Figure , and IL‐) Figure  were all) Figure ,H.3.9P < .05)   was used to ulteriorly explore the protective mechanisms of PUE in ICH‐induced EBI, and the acquired results indicated that LY294002 could markedly weaken the beneficial effects of PUE on ICH‐induced behavioral deficiency indicated by increasing the mNSS scores  Figure  and BBB ) Figure  at 24 h 3.10P < .05)      Figure , and p‐A) Figure  proteins) Figure  and p‐NF) Figure  at 24 h 4Increasing evidence has shown that OS and neuroinflammation are incredibly crucial for ICH‐induced EBI, which is characterized by massive brain cell apoptosis.Research studies have confirmed that activation of NFthe ‐κB signal pathway widely exists in various kinds of CNS damages, including ischemic stroke,Stimulation of the PI3K/Akt signal has also been reported by different groups that it could notably mitigate acute CNS disorder‐induced brain injury, including ICH 21H20O9], chemical structure ‐7‐hydroxy‐3‐(4‐hydroxyphenyl)‐4H‐1‐benzopyran‐4‐one, Ce Figure , belongsPuerarin is also well‐confirmed to produce various beneficial effects by activation of the PI3K/Akt signal pathway. A recent report has exhibited that PUE could ameliorate OS‐induced neurodegeneration by stimulating the PI3K/Akt signal after TBI, and the specific inhibitor of the PI3K/Akt signaling pathway LY294002 could markedly reduce PUE’s protective effects.Research studies have suggested that the PI3K/Akt signal is the powerful upstream regulator of the NF‐κB pathway.Some potential limitations deserve special attention in our study. First, PUE can generate multiple beneficial effects by manipulation of different signal pathways, but we primarily focused on PI3K/Akt signal activation‐mediated suppression of the NF‐κB pathway. Hence, PUE may exert neuroprotective effects by other signal pathways such as the Nrf2 signaling pathway Overall, our results have indicated that PUE could notably improve ICH‐induced EBI and neurological deficiency, and related mechanisms might be involved in the suppression of NF‐κB signal pathway activation‐induced brain injury partly by the triggering of PI3K/Akt signal pathway‐mediated neuroprotection. Our findings might provide a promising therapeutic selection for ICH‐induced EBI.All authors listed in this manuscript declared that no any conflicts of interest existed.Jun Zeng: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing‐original draft . Shizhong Zheng: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Software ; Validation ; Visualization ; Writing‐original draft . Yizhao Chen: Conceptualization ; Data curation ; Funding acquisition (lead); Project administration (lead); Resources (lead); Supervision (lead); Writing‐review & editing (lead). Yaoming Qu: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Jiayu Xie: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Enhui Hong: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Hongzhu Lv: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Rui Ding: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Liang Feng: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting). Zhichong Xie: Formal analysis (supporting); Investigation (supporting); Methodology (supporting); Software (supporting); Visualization (supporting); Writing‐review & editing (supporting).Fig. S1.Click here for additional data file.Fig. S2.Click here for additional data file.Fig. S3.Click here for additional data file.Fig. S4.Click here for additional data file.Fig. S5.Click here for additional data file.Fig. S6.Click here for additional data file.Supplementary MaterialClick here for additional data file.