Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer’s disease (AD). Thus, modulating the neuroinflammatory response represents a potential therapeutic strategy for treating neurodegenerative diseases. Several recent studies have shown that dopamine (DA) and its receptors are expressed in immune cells and are involved in the neuroinflammatory response. Thus, we recently developed and synthesized a non-self-polymerizing analog of DA (CA140) and examined the effect of CA140 on neuroinflammation.To determine the effects of CA140 on the neuroinflammatory response, BV2 microglial cells were pretreated with lipopolysaccharide , followed by treatment with CA140 (10 μM) and analysis by reverse transcription-polymerase chain reaction (RT-PCR). To examine whether CA140 alters the neuroinflammatory response in vivo, wild-type mice were injected with both LPS ) and CA140 , and immunohistochemistry was performed. In addition, familial AD (5xFAD) mice were injected with CA140 or vehicle daily for 2 weeks and examined for microglial and astrocyte activation.Pre- or post-treatment with CA140 differentially regulated proinflammatory responses in LPS-stimulated microglia and astrocytes. Interestingly, CA140 regulated D1R levels to alter LPS-induced proinflammatory responses. CA140 significantly downregulated LPS-induced phosphorylation of ERK and STAT3 in BV2 microglia cells. In addition, CA140-injected wild-type mice exhibited significantly decreased LPS-induced microglial and astrocyte activation. Moreover, CA140-injected 5xFAD mice exhibited significantly reduced microglial and astrocyte activation.CA140 may be beneficial for preventing and treating neuroinflammatory-related diseases, including AD.The online version of this article (10.1186/s12974-018-1321-3) contains supplementary material, which is available to authorized users. Increasing evidence indicates a critical role of the immune system in neurodegenerative diseases such as Alzheimer’s disease (AD) . AbnormaLipopolysaccharide (LPS) is a well-established stimulator that induces the activation of microglial cells and is widely used both in vivo and in vitro to induce neuroinflammation in animal models , 6. The . We discovered that CA140 reduced proinflammatory responses in LPS-stimulated BV2 microglial cells, primary microglial cells, and primary astrocytes. In addition, CA140 inhibited LPS-induced neuroinflammatory responses by inhibiting the dopamine D1 receptor (D1R)/ERK/STAT3 signaling pathways. Moreover, CA140 significantly decreased the activation of microglia and astrocytes in wild-type mice as well as a mouse model of AD. Taken together, our results indicate that CA140 is a potential therapeutic agent for treating and/or preventing neuroinflammation-related diseases, including AD.In the present study, we examined whether CA140 regulates the neuroinflammatory response in vitro and in vivo2 incubator.BV2 microglial cells (a generous gift of Dr. Kyung-Ho Suk) or HEK cells (a generous gift of Dr. Hyung-Jun Kim) were maintained in high-glucose DMEM  with 5 or 10% fetal bovine serum  in a 5% COMouse primary microglial and astrocyte cultures were prepared from mixed glial cultures as previously described . Briefly5 cells per well. The remaining cells in the flask were harvested using 0.1% trypsin to obtain rat primary astrocytes. These rat primary astrocytes and primary microglial cells were cultured in 12-well plates (35 mm) pre-coated with poly-D-lysine (Sigma).Rat primary mixed glial cells were cultured from the cerebral cortices of 1-day-old Sprague Dawley rats. Briefly, the cortices were triturated into single cells in high-glucose DMEM containing 10% FBS/penicillin-streptomycin solution  and plated into 75 T culture flasks (0.5 hemisphere/flask) for 2 weeks. To harvest rat primary microglial cells, the plate was shaken continuously at 120 rpm for 2 h to facilitate microglial detachment from the plate. The fluid medium was subsequently collected and centrifuged at 1500 rpm for 15 min, and the cell pellets were resuspended to plate 1 × 10Escherichia coli, 10 mg/kg, i.p.) or PBS. After 3 h, immunostaining was performed with anti-IbaI or anti-GFAP antibodies. To examine whether post-treatment with CA140 regulates LPS-induced neuroinflammatory responses, wild-type mice were injected with LPS  or PBS, followed 30 min later by injection with CA140  or vehicle . Immunohistochemistry was then performed with anti-Iba-1 and anti-GFAP antibodies.All experiments were performed in accordance with the approved animal protocols and guidelines established by the Korea Brain Research Institute (IACUC-2016-0013). C57BL6/N mice were purchased from Orient-Bio Company. Male C57BL6/N mice  were housed in a pathogen-free facility with 12 h of light and dark per day at an ambient temperature of 22 °C. To determine if pretreatment with CA140 alters LPS-induced neuroinflammation, wild-type mice were intraperitoneally (i.p.) administered CA140 (30 mg/kg) or vehicle (10% DMSO) daily for 3 days and subsequently injected with LPS  were purchased from The Jackson Laboratory. 5xFAD mice overexpress two mutant human proteins: APP (695) with KM670/671NL (Swedish), I716V (Florida), and V717I (London) FAD mutations and PS1 with M146 L and L286 V FAD mutations. To examine the effects of CA140 on the neuroinflammatory response in a mouse model of AD, 5xFAD mice were injected with CA140  or vehicle  daily for 2 weeks, and immunohistochemistry was conducted with anti-Iba-1 or anti-GFAP antibodies. The animal groups were randomized for all experiments. Data were analyzed in a semi-automated manner using ImageJ software and confirmed by an independent researcher who did not participate in the current experiments. Only male mice were used for this study because the pathology of 5xFAD female mice is more severe than that of male mice, leading to huge variations in in vivo experiments.N-Methylisatoic anhydride  was added to 28.1 mg of 3,4-dimethoxyphenethylamine  and 25 μL of triethylamine  in dichloromethane (DCM). The reaction mixture was stirred for 2 h at room temperature and overnight at 20 °C. The mixture was warmed to room temperature, and a vacuum was subsequently applied to remove volatile organics. The amide product was purified by column chromatography  to provide 31.5 mg of N--2-(methylamino)benzamide as a white solid (91% isolated yield). 1H-NMR  δ ppm = 7.30 , 7.20 , 6.82 , 6.74 , 6.55 , 3.86 , 3.83 , 3.63 , 3.61 , 2.84 , 2.84 . 13C-NMR  δ ppm = 169.6, 150.0, 148.9, 147.6, 132.7, 131.4, 127.0, 120.6, 115.4, 114.8, 111.9, 111.4, 111.3, 55.8, 55.7, 40.9, 35.1, 29.8.N--2-(methylamino)benzamide  was dissolved in 0.5 mL of anhydrous DCM, and 120 μL of 1 M BBr3 in DCM  was added. The reaction was stirred overnight under an inert atmosphere. Excess methanol was added to the mixture, and volatile organics were removed in vacuo. The addition and removal of methanol was repeated at least three times to remove boric acid as methyl borate. The target compound, N--2-(methylamino)benzamide (CA140), was obtained in quantitative yield. 1H-NMR  δ ppm = 7.91 , 7.89 , 7.74 , 6.67 , 6.56 , 3.60 , 3.01 , 2.79 . LC-MS ESI positive mode m/z [M + H]+ = 287.11 .n = 3) were dosed with CA140 dissolved in DMSO/Tween-80/saline (10:5:85%) via a single intravenous administration (10 mg/kg). Blood was collected by cardiac puncture at 5 min and then centrifuged to isolate plasma. The brain was collected at 5 min and homogenized in PBS after washing with fresh PBS. The concentrations of CA140 in the plasma and brain were determined by LC-MS/MS. The LC-MS/MS system comprised a Nexera XR HPLC system  coupled to a TSQ Vantage triple quadrupole mass spectrometer equipped with Xcalibur version 1.1.1 .ICR mice  of either dopamine  or CA140 by dilution in 1 mL of preheated PBS buffer to yield final concentrations of 500 μM. The solutions were incubated at 37 °C in a thermoblock, and the concentration of CA140 or DA was followed over time in triplicate. Aliquots (50 μL) were taken at 0, 1, 2, 4, 6, 8, and 22 h and added to 200 μL of acetonitrile. The samples were mixed by vortexing for 30 s and then centrifuged at 4 °C for 15 min at 14,000 rpm. The clear supernatants were diluted in PBS (2-fold) and analyzed by HPLC at 254 and 280 nm for CA140 and DA, respectively.The following primary antibodies were used throughout this study: rat anti-mouse CD11b , rabbit anti-F-actin , rabbit anti-COX-2 , rabbit anti-IL-1β , rabbit anti-GFAP , rabbit anti-Iba-1 , goat anti-Iba-1 , rabbit anti-AKT , rabbit anti-p-AKT  , rabbit anti-ERK , rabbit anti-p-ERK (Thr42/44) , rabbit anti-STAT3 , rabbit anti-p-STAT3 , mouse anti-PCNA , rabbit anti-D2R , and rabbit anti-D1R  antibodies. We used the following small molecules: D1R antagonists , D1R agonist , D2R antagonist , a STAT3 inhibitor , and an ERK inhibitor .phenyltetrazolium bromide (MTT) assay. BV2 microglial cells were seeded in 96-well plates and treated with various concentrations of CA140 (1–50 μM) or vehicle (1% DMSO) for 24 h in the absence of FBS. The cells were subsequently treated with 0.5 mg/mL MTT and incubated for 3 h at 37 °C in a 5% CO2 incubator. The absorbance was read at 580 nm.BV2 microglial cell viability was assessed using the 3--2,5-diTotal RNA was extracted from cells using TRIzol (Invitrogen) following the manufacturer’s instructions. Total RNA was reverse transcribed into cDNA using a Superscript cDNA Premix Kit II with oligoDT , and RT-PCR was performed using Prime Taq Premix . RT-PCR products were separated by electrophoresis on 1.5% agarose gels with Eco Dye  and photographed. Images were analyzed using ImageJ (NIH) and Fusion (Korea).BV2 microglial cells were fixed in ice-cold methanol for 8 min, washed three times with 1 × PBS, and incubated with CD11b and COX-2 or CD11b and IL-1β antibodies in GDB buffer  overnight at 4 °C. The next day, the cells were washed three times with 1 × PBS and incubated with the following secondary antibodies for 1 h at room temperature: Alexa Fluor 488 and Alexa Fluor 555 . Images were obtained on a single plane using a confocal microscope  and analyzed using ImageJ software.Animals were perfused and fixed with 4% paraformaldehyde (PFA) solution, and brain tissues were flash-frozen and dissected using a cryostat (35-mm-thick sections). Each brain section was processed for immunofluorescence or immunohistochemical staining. For immunofluorescence staining, sections were rinsed in PBS and incubated with rabbit anti-Iba-1  for microglia or rabbit anti-GFAP  for astrocytes. Antibodies were diluted in 0.5% bovine serum albumin (BSA) and incubated at 4 °C overnight. The following day, tissues were rinsed with 0.5% BSA and incubated with Alexa Fluor 555-conjugated anti-rabbit IgG  for 1 h at room temperature. The tissues were subsequently mounted on a gelatin-coated cover glass and covered with DAPI-containing mounting solution (Vector Laboratories). Images of the stained tissues were captured using confocal microscopy .2O2. The sections were rinsed in 0.1 M PB and mounted on gelatin-coated slides, and images were obtained under a bright-field microscope (Leica).For immunohistochemistry, sections were permeabilized for 1 h in PBS with 0.2% Triton X-100 and 1% BSA at room temperature. The sections were then incubated with primary antibodies at 4 °C overnight. The next day, the tissues were washed three times with 0.5% BSA and incubated with biotin-conjugated anti-rabbit antibody  for 1 h at room temperature. After rinsing with 0.5% BSA, the sections were incubated for 1 h at room temperature in avidin-biotin complex solution , followed by rinsing three times in 0.1 M phosphate buffer (PB). The signal was detected by incubating the sections in 0.5 mg/mL 3,3′-diaminobenzidine  in 0.1 M PB containing 0.003% HTo measure the effects of pre- or post-treatment with CA140 on IL-1β, an enzyme-linked immunosorbent assay (ELISA) was performed. Briefly, BV2 microglial cells were treated with LPS (100 ng/mL) or PBS for 30 min, followed by treatment with CA140 (10 μM) or vehicle (1% DMSO). IL-1β ELISA was then performed using the conditioned medium. Mouse IL-1β ELISA kits  were used according to the manufacturer’s recommendations. Recombinant mouse IL-1β protein (R&D Systems) was used as a standard. The absorbance of the samples was measured at 450 nm using a microplate reader .N-(1-naphthyl)ethylenediamine dihydrochloride and 1% sulfanilamide in 2% phosphoric acid) in 96-well plates and incubated at room temperature for 5 min. The absorbance was measured at 540 nm using a microplate reader, and the level of nitrite was analyzed against a standard curve of sodium nitrite.To examine the effects of CA140 on nitrite (NO) production, the Griess assay was performed. BV2 microglial cells were incubated with CA140 (10 μM) or vehicle (1% DMSO) for 30 min, followed by treatment with LPS (100 ng/mL) or PBS for 23.5 h. The conditioned medium was mixed with Griess reagent . Western blot analysis was performed as previously described . Images 2, 10 mM KCl, 0.5 mM DTT, 300 mM sucrose, 0.1% NP-40, and 0.5 mM PMSF). After 5 min, the cell lysates were centrifuged at 10,000 rpm at 4 °C for 1 min, and the supernatant was stored as the cytosolic fraction. The pellet was lysed in nuclear fractionation buffer  on ice for 15 min, followed by centrifugation at 10,000 rpm at 4 °C for 15 min. The cytosolic and nuclear fractions were analyzed by western blot as previously described [BV2 microglial cells were lysed in cytosolic fractionation buffer .All data were analyzed using a two-tailed N-Methylisatoic anhydride  with To examine the effects of CA140 on the LPS-induced neuroinflammatory response, we initially assessed the cytotoxicity of CA140 in BV2 microglial cells. BV2 microglial cells were treated with vehicle (1% DMSO) or CA140  for 24 h, and MTT assays were performed. CA140 did not affect cell viability up to a concentration of 25 μM; however, CA140 exhibited some toxicity at 50 μM in BV2 microglial cells  or PBS for 30 min and treated with vehicle (1% DMSO) or CA140 (10 μM) for 5.5 h. After 6 h, BV2 microglial cells were fixed and immunostained with anti-CD11b and anti-F-actin antibodies. LPS treatment produced aberrant cell morphology of BV2 microglial cells, such as thin fibroblast-like processes from the cell body  or PBS for 30 min, followed by treatment with CA140 (5 or 10 μM) or vehicle (1% DMSO) for 5.5 h. Proinflammatory cytokine levels were then measured by RT-PCR. Interestingly, we observed that 5 μM CA140 only significantly decreased LPS-induced IL-1β mRNA levels  or PBS for 30 min, followed by treatment with CA140 (10 μM) or vehicle (1% DMSO) for 11.5 or 23.5 h. Proinflammatory cytokine levels were then measured by RT-PCR. Interestingly, post-treatment with CA140 significantly suppressed LPS-induced proinflammatory cytokine levels in a time-dependent manner  or vehicle (1% DMSO) for 30 min, followed by LPS (1 μg/mL) or PBS for 5.5 h. Proinflammatory cytokine levels were evaluated by RT-PCR. Pretreatment with 5 μM CA140 followed by LPS treatment did not decrease proinflammatory cytokine levels  or vehicle (1% DMSO) for 30 min, followed by LPS (1 μg/mL) or PBS for 11.5 or 23.5 h. Longer pretreatment with CA140 further reduced LPS-induced mRNA levels of the proinflammatory cytokines COX-2, IL-1β, and iNOS compared with pretreatment for 6 h  or vehicle (1% DMSO) for 30 min, followed by treatment with LPS (1 μg/mL) or PBS for 5.5 h. Proinflammatory cytokine levels were then measured by RT-PCR. Interestingly, pretreatment with CA140 reduced LPS-induced COX-2, IL-1β, iNOS, and TNF-α mRNA levels in rat primary microglial cells  to alter the neuroinflammatory response. To test this idea, we initially investigated whether BV2 microglial cells present endogenous dopamine D1 receptor (D1R) or dopamine D2 receptor (D2R). For the initial experiment, BV2 microglial cells were pretreated with LPS (1 μg/mL) or PBS for 30 min, followed by treatment with CA140 (10 μM) or vehicle (1% DMSO) for 5.5 h, and the mRNA levels of D1R were measured by RT-PCR. Interestingly, LPS treatment significantly increased D1R mRNA levels, whereas treatment with LPS followed by CA140 significantly downregulated D1R mRNA levels  or PBS for 30 min, followed by treatment with CA140 (10 μM) or vehicle (1% DMSO) for 5.5 h and immunocytochemistry with anti-CD11b and anti-D2R antibodies. Interestingly, LPS treatment significantly increased D2R levels in BV2 microglial cells  or PBS for 30 min, followed by treatment with a D1R antagonist  or vehicle (1% DMSO) for 5.5 h. Proinflammatory cytokine levels were then measured by RT-PCR. LE300 treatment significantly reduced LPS-stimulated COX-2 and IL-1β mRNA levels Fig. a–f, whicNext, to investigate whether CA140 further regulates the neuroinflammatory response in the presence of a D1R antagonist, BV2 microglial cells were pretreated with LPS (1 μg/mL) or PBS for 30 min, followed by treatment with LE300  or vehicle (1% DMSO) for 30 min and finally CA140 (10 μM) or vehicle (1% DMSO) for 5 h. Subsequent RT-PCR analysis revealed that treatment with LE300, CA140, and LPS further inhibited IL-1β mRNA levels compared with treatment with LE300 and LPS or CA140 and LPS  or PBS for 30 min, followed by another D1R antagonist  or vehicle (1% DMSO) for 5.5 h, and proinflammatory cytokine levels were measured by RT-PCR. SCH23390 treatment significantly decreased LPS-induced COX-2 and IL-1β mRNA levels Fig. i–n. In aNext, to examine whether D1R agonist treatment alters neuroinflammatory responses, BV2 microglial cells were treated with LPS (1 μg/mL) or PBS for 30 min, followed by a D1R agonist  or PBS for 5.5 h and measurement of proinflammatory cytokine levels by RT-PCR. A77636 treatment did not decrease any LPS-induced proinflammatory cytokine levels  or PBS for 30 min, followed by treatment with A77636  or PBS for 30 min and finally CA140 (10 μM) or vehicle (1% DMSO) for 5 h; we subsequently performed RT-PCR. Consistent with our findings above, post-treatment with CA140 dramatically reduced LPS-induced IL-1β mRNA levels  or PBS for 30 min, followed by treatment with PD98059  or vehicle (1% DMSO) for 30 min and finally vehicle (1% DMSO) or CA140 (10 μM) for 5 h. The mRNA levels of COX-2 and IL-1β were measured by RT-PCR. Consistent with our previously described findings, post-treatment with CA140 significantly decreased the mRNA levels of COX-2 and IL-1β Fig. g–i. In aWe then examined whether pretreatment with CA140 alters ERK and AKT signaling. BV2 cells were pretreated with vehicle (1% DMSO) or CA140 (10 μM) for 45 min, followed by LPS (1 μg/mL) or PBS treatment for 45 min and western blotting with anti-p-ERK/ERK or anti-p-AKT/AKT antibodies. Interestingly, pretreatment with CA140 significantly suppressed the phosphorylation of ERK and AKT in LPS-treated BV2 microglial cells  or CA140 (10 μM) for 30 min, followed by treatment with LPS (1 μg/mL) or PBS for 5.5 h and subcellular fractionation. Pretreatment with CA140 also significantly decreased LPS-induced cytosolic and nuclear p-STAT3 (Ser727) levels  or PBS for 30 min, followed by treatment with S31–201  or vehicle (1% DMSO) for 30 min and finally vehicle (1% DMSO) or CA140 (10 μM) for 5 h. The mRNA levels of COX-2 and IL-1β were then measured by RT-PCR. Compared with treatment with LPS and S3I-201 or with LPS and CA140, treatment with LPS, S3I-201, and CA140 further decreased IL-1β and COX-2 mRNA levels Fig. g–i. ThesNumerous studies have shown that activated microglia and astrocytes are involved in neuroinflammatory responses . To examWe subsequently examined whether post-treatment with CA140 alters LPS-stimulated proinflammatory cytokine levels in wild-type mice. For this experiment, wild-type mice were injected with LPS  or PBS, followed 30 min later by injection with CA140  or vehicle . Immunohistochemistry was performed with anti-IL-1β and anti-COX-2 antibodies. Post-treatment with CA140 significantly downregulated LPS-stimulated IL-1β Fig. a–e and CTo further examine whether pretreatment with CA140 attenuates microglial and astrocyte activation, wild-type mice were injected with CA140  or vehicle  daily for 3 days, followed by injection with LPS  or PBS. Three hours later, immunohistochemistry was conducted with anti-Iba-1 and anti-GFAP antibodies. We observed that pretreatment with CA140 also significantly decreased microglial and astrocyte immunoreactivity  , 40. DA With respect to the effects of D2R on neuroinflammation, the D2R agonist pramipexole increases nitrites in cultured primary microglia . In addiActivation of TLR receptors via LPS turns on downstream signaling cascades, such as MAP kinases, including ERK and AKT signaling in microglia and astrocytes , 50, 51.STAT3, a member of the STAT family, is a transcription factor that plays a critical role in regulating microglial activation and inflammatory responses , 57. STASystemic injection of LPS in wild-type mice significantly induces astrocyte and microglial activation and proinflammatory cytokine expression –62. MoreIn the present study, we have demonstrated that CA140 exhibits novel anti-inflammatory effects and provided initial evidence for its mechanism of action. We discovered that CA140 suppresses LPS-induced proinflammatory cytokine levels in BV2 microglial cells, primary microglia, and primary astrocytes. In addition, CA140 modulates D1R to regulate LPS-induced neuroinflammation in BV2 microglial cells. Moreover, CA140 affects ERK/STAT3 signaling to alter the LPS-induced neuroinflammatory response. In in vivo experiments, CA140 significantly reduced microglial and astrocyte activation in LPS-injected wild-type mice and 5xFAD mice. This study reveals that CA140 is a promising compound with anti-inflammatory effects and subsequent attenuation of neurotoxins such as LPS.Additional file 1:Figure S1. Post-treatment with CA140 at 5 μM only significantly reduced LPS-induced IL-1β mRNA levels. Figure S2. Post-treatment with CA140 significantly reduced LPS-induced proinflammatory cytokine levels in a longer treatment. Figure S3. Pretreatment with CA140 significantly decreased LPS-induced COX-2, IL-1β, and iNOS mRNA levels in BV2 microglial cells. Figure S4. Pretreatment with CA140 significantly decreased LPS-induced COX-2, IL-1β, and iNOS mRNA levels in a longer treatment. Figure S5. Post-treatment with CA140 significantly decreased LPS-mediated proinflammatory cytokine levels in rat primary microglial cells. Figure S6. Pretreatment with CA140 decreased LPS-induced proinflammatory cytokine levels in rat primary microglial cells and primary astrocytes. Figure S7. Pretreatment with CA140 decreased LPS-mediated proinflammatory cytokine levels in mouse primary microglial cells and primary astrocytes. Figure S8. Post-treatment with CA140 downregulated LPS-induced dopamine D1 receptor (D1R) levels in BV2 microglial cells. Figure S9. Inhibition of dopamine D2 receptor (D2R) did not reduce LPS-stimulated proinflammatory cytokine levels in BV2 microglial cells. Figure S10 Pretreatment with CA140 significantly decreased phosphorylation of ERK and AKT in LPS-stimulated BV2 microglial cells. Figure S11. Pretreatment with CA140 significantly decreased cytosolic and nuclear p-STAT3 levels in LPS-induced BV2 microglial cells. Figure S12. Pretreatment with CA140 significantly reduced microglia and astrocyte activation in wild-type mice. (DOCX 22915 kb)


Two independent N—H⋯O hydrogen bonds link all of the molecules into a continuous three-dimensional framework structure. The quinazoline ring adopts an envelope conformation with the 2,5-dimethoxyphenylunit occupying a quasi-axial site. 61H15ClN2O3, the heterocyclic ring adopts an envelope conformation, folded across the N⋯N line, with the 2,5-dimethoxyphenyl unit occupying a quasi-axial site. There are two N—H⋯O hydrogen bonds in the structure: one hydrogen bond links molecules related by a 41 screw axis to form a C(6) chain, and the other links inversion-related pairs of molecules to form an R22(8) ring. The ring motif links all of the chains into a continuous three-dimensional framework structure. Comparisons are made with the structures of some related compounds.In the title compound, C H)-one is a privileged scaffold in drug design -7-chloro-2--2,3-dihydroquinazolin-4(1H)-one (I)et al., 2014Quinazoline-4-one and its derivatives constitute an important class of fused heterocycles, which are found in more than two hundred naturally occurring alkaloids. In addition, 2,3-dihydroquinazolin-4(1R configuration at this atom: the centrosymmetric space group confirms that compound (I)A,C8A) in the R-enantiomer are Q = 0.258 (2) Å, θ = 121.8 (4)° and φ = 219.3 (6)°. For the ideal envelope form, the puckering angles take the values θ = 54.7°  and φ = (60k)°, where k represents an integer  Å for atom N3. However, atom C2 is displaced from this plane by 0.355 (3) Å. The 2,5-dimethoxyphenyl substituent occupies the quasi-axial site at atom C2. Within this unit, the two methoxy C atoms are almost coplanar with the aryl ring: the deviations from the mean plane of this ring are 0.020 (5) Å for atom C221 and 0.101 (5) Å for atom C251. Associated with this planarity, the two exocyclic C—C—O angles at atoms C22 and C25 are significantly different, by 11.9° at C22 and by 8.2° at atoms C25, as previously observed in planar or near-planar alkoxyarenes 1 screw axis along  into a C(6) chain  chain around the 41 screw axis (z) with four similar chains around the screw axes along (z), (−z), (z) and (z)  chains, so linking all of the molecules into a very elegant three-dimensional structure generated by only two hydrogen bonds.The structure of compound (I)if Fig. 3. This inz) Fig. 4. PropagaRS)-2-(2-chlorophenyl)-2,3-dihydroquinazolin-4(1H)-one  alternate with n + n represents an integer in each case se Fig. 5.H)-one , RS)-2-methyl-4-phenyl-3,4-dihydroquinazoline, and here molecules which are related by a 31 screw axis are linked by an N-–H⋯N hydrogen bond to form a C(5) chain -ones in which there is a substituent at atom N3  chain. However, when the substituent at atom N3 is an arylmethylamino group, the heterocyclic ring adopts a screw–boat conformation  was added to a suspension of 2-(dibromomethyl)-1,4-dimethoxybenzene (3.3 mmol) and 2-amino-4-chlorobenzamide (3.5 mmol) in a pyridine-dimethylformamide mixture . The resulting mixture was heated at 313 K for 4 h, with TLC monitoring. When the reaction was judged to be complete, an excess of water was added, followed by extraction with ethyl acetate (2 × 20 ml). The combined organic extract was washed with brine and then dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography using silica gel mesh 60–120, with 30% ethyl acetate in hexane as eluent, to give the product (I)A sample of compound (I)I41/a, No. 88, employed here the origin is located at a centre of inversion. All H atoms were located in difference maps. The H atoms bonded to C atoms were then treated as riding atoms in geometrically idealized position with C—H 0.93 Å (aromatic), 0.96 Å (CH3) or 0.98 Å , and with Uiso(H) = kUeq(C), where k = 1.5 for the methyl groups, which were permitted to rotate but not to tilt, and 1.2 for all other H atoms bonded to C atoms. For the H atoms bonded to N atoms, the atomic coordinates were refined with Uiso(H) = 1.2Ueq(N), giving the N—H distances shown in Table 1Crystal data, data collection and structure refinement details are given in Table 210.1107/S2056989019007023/zl2756sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989019007023/zl2756Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019007023/zl2756Isup3.cmlSupporting information file. DOI: 1875494CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


N-oxide and 3-nitroprop-1-ene. For the first time, the regiochemistry of this reaction was unambiguously proven by X-ray structural analysis. The quantum-chemical calculation performed at the M06-2X/6-31G(d) (PCM) theoretical level affords a basis for explaining the course of reaction as well as the nature of transition states. Next, further DFT calculations together with spectral data shed light on structural aspects of the product.5-(Nitromethyl)-3-phenyl-4,5-dihydroisoxazole was obtained as a product of a high-yielding [3 + 2] cycloaddition reaction of in situ-generated benzonitrile The online version of this article (10.1007/s00706-018-2227-6) contains supplementary material, which is available to authorized users. They are used as parasiticidal agents in veterinary medicine  cycloaddition (32CA) between nitrile s (TACs) , 12 and s (TACs) –15. At ts (TACs)  to date.N-oxide (2) and 3-nitroprop-1-ene (3)  in a reaction between 3-bromoprop-1-ene and silver nitrite. The second component for the 32CA reaction, benzonitrile N-oxide (2), was generated in situ from phenylhydraxamoyl chloride. Next, we performed cycloaddition between 2 and 3. The reaction was monitored by HPLC. It was found that the reaction was completed after 20 h. HPLC analysis of the crude reaction product confirmed the presence of unreacted residual nitroalkene and a single adduct formed. The product was isolated with sufficient purity for a full set of structural analyses using LC–MS IT-TOF, IR, and NMR techniques .In the first stage, we prepared 3-nitroprop-1-ene -3-phenyl-4,5-dihydroisoxazole  cycloaddition between benzonitrile The results obtained provide a valuable background for understanding of the chemistry of nitromethyl-4,5-dihydroisoxazoles. They are also useful for better understanding of other 32CA processes involving nitroallylic systems.1H NMR (500 MHz) and 13C NMR (125 MHz) spectra were taken on a Bruker Avance 500 MHz spectrometer calibrated to residual solvent peaks at 7.27 and 77.00 ppm for 1H and 13C in CDCl3. The chemical shifts are reported in ppm, and the following abbreviations are used in reporting the NMR data: d (doublet), dd (doublet of doublets), and m (multiplet). Coupling constants (J) are given in Hz. Spectra are reported as follows: chemical shift , multiplicity, integration, and coupling constants (Hz). Liquid chromatography (HPLC) was done using a Knauer apparatus equipped with a UV–Vis detector. For monitoring of the reaction progress and postreaction mixtures, a LiChrospher 18-RP 10 μm column (4 × 240 mm) and 70% methanol as the eluent at a flow rate of 1.3 cm3 min−1 were used. MS spectra were recorded on a Shimadzu LCMS IT-TOF spectrometer equipped with a Kinetex® 2.6 µm C18 100 Å, LC column 100 × 2.1 mm, eluted with 50% aqueous acetonitrile at a flow rate of 0.3 cm3 min−1.Melting points were determined on a Boetius apparatus. Elemental analysis was performed on a Perkin-Elmer PE-2400 CHN apparatus. The IR spectrum was recorded on an FTS Nicolet IS 10 apparatus. 3 of anhydrous diethyl ether at − 5 °C. Next, 35 g of 3-bromoprop-1-ene was slowly added dropwise, while the temperature was maintained at − 5 °C. The mixture was then left at 0–5 °C for 24 h, and next for 48 h at room temperature. The reaction mixture was filtered and the residue was washed with the diethyl ether. The filtrate and the ethereal extract were combined and the solvent was evaporated on a rotary evaporator. The liquid residue was distilled under reduced pressure. 7.2 g (29%) of 3-nitroprop-1-ene were obtained as a yellow liquid with a characteristic odor  was suspended in 100 cm/27 Torr ).3 of dry Et2O was stirred and cooled down to 0 °C. 20 mmol of Et3N in 10 cm3 of Et2O was added dropwise and the mixture was stirred at r.t. for 20 h. The organic layer was separated from the postreaction mixture, dissolved in DCM, washed with water, and dried over MgSO4. The solvent was evaporated to dryness and residues were crystallized from EtOH. Yield 80%; white crystals; m.p.: 94–95 °C; 1H NMR : δ = 7.59 , 7.37 , 5.33 , 4.62 , 4.43 , 3.58 , 3.20  ppm; 13C NMR : δ = 156.9 (C3), 130.8 (ArC4′), 128.9 (ArC3′+C5′), 128.4 (ArC1′), 126.9 (ArC2′+C6′), 76.8(C5) 76.1 (–CH2–), 38.7 (C4) ppm; IR (KBr): −1; HRMS (ESI): m/z = 207.0758, calc. for [C10H10N2O3 + H]+ 207.0764, diff. = 2.90 ppm.A mixture of 40 mmol of 3-nitroprop-1-ene and 20 mmol of phenylhydraxamoyl chloride  in 100 4 were obtained by recrystallization from ethanol. The crystals were twinned; however, the diffraction data produced good quality electron density maps that allowed us to unambiguously determine the structural model of the crystal (Table α radiation (λ = 1.54184 Å). Using Olex2 [Crystals of al Table . The difng Olex2 , the strng Olex2  structurhttp://www.ccdc.cam.ac.uk/conts/retrieving.html .CCDC 1576601 contains supplementary crystallographic data for this paper. These data can be obtained free of charge via The quantum-chemical calculations reported in this paper were performed on “Zeus” and “Prometheus” supercomputers in the “Cyfronet” regional computer center in Cracow. The M06-2X  and B3LYSupplementary material 1 (DOCX 3330 kb)Below is the link to the electronic supplementary material.


Seven distinct hydrogen-bonding interactions link the idelalisib, t-BuOH and water molecules into a complex chain structure with the topology of a 2,3,4,5-connected 4-nodal net having the point symbol (3.4.52.62)(3.4.52.64.72)(3.5.6)(5).In the title structure, 5-fluoro-3-phenyl-2-[(1 This compound was developed for the oral treatment of chronic lymphocytic leukemia and is currently marketed under the trade name Zydelig by Gilead Sciences, Inc. Carra  al. 2013 reportedhkin 2005.i.e. a molecule each of idelalisib and of t-BuOH as well as two water molecules, denoted as w1 (O37) and w2 (O38) 8) Fig. 1. The cont-BuOH molecule, via N25—H25⋯O36. Additionally, the secondary amino function attached to the pyrimidine ring of the purine fragment donates a hydrogen bond to a w2 water molecule, via N19—H19⋯O38. In turn, the idelalisib molecule accepts three hydrogen bonds. Its quinazoline group is linked to the w1 water molecule via an O37—H37A⋯N5 bond, and additionally each of N23 and N27 of the purine group is hydrogen-bonded to a water molecule of type w2 [O38—H38A⋯N23] or w1 [O37—H37B⋯N27]. Moreover, the water molecule w1 is an acceptor for two H-bonds, O36—H36⋯O37 from a t-BuOH molecule and O38—H38B⋯O37 from a w2-type water molecule. There are no hydrogen bonds between neighbouring idelalisib molecules. Overall, the seven classical hydrogen-bonding interactions listed in Table 1b axis , whilst w2 serves as a three-connected node . The hydrogen-bonded chain of (I)2.62)(3.4.52.64.72)(3.5.6)(5). The topology of the hydrogen-bonded structure was determined and classified with the programs ADS and IsoTest of the TOPOS package  of the Cambridge Structural Database (Groom H)-one -one -one adenine  at 296 K. The amorphous material was dissolved. Precipitation of solid material was observed after 5 min of stirring of the solution. The suspension was then stirred at 296 K for five days, which was followed by centrifugation and separation of the precipitate. Subsequent drying of the solid material yielded the title compound (I)The preparation of idelalisib was carried out according to the scheme displayed in Fig. 4 al. 2013. To amorUiso(H) = 1.5Ueq(C) of the parent carbon atom. All other hydrogen atoms bonded to carbon atoms were positioned geometrically (C—H = 0.95 Å) and refined with Uiso(H) = 1.5Ueq(C) of the parent carbon atom. Hydrogen atoms of OH and NH groups were refined with restrained distances  and their Uiso parameters were refined freely. The absolute structure was established by anomalous-dispersion effects  I. DOI: 10.1107/S2056989019002743/wm5487Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019002743/wm5487Isup3.cmlSupporting information file. DOI: 1898812CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In vitro, we showed that 89Zr-labeled Vγ9Vδ2 T cells retained their viability, proliferative capacity, and anti-cancer cytotoxicity with minimal DNA damage for amounts of 89Zr ≤20 mBq/cell. Using a mouse xenograft model of human breast cancer, 89Zr-labeled γδ-T cells were tracked by PET imaging over 1 week. To increase tumor antigen expression, the mice were pre-treated with PEGylated liposomal alendronate. Liposomal alendronate, but not placebo liposomes or non-liposomal alendronate, significantly increased the 89Zr signal in the tumors, suggesting increased homing of γδ-T cells to the tumors. γδ-T cell trafficking to tumors occurred within 48 hr of administration. The presence of γδ-T cells in tumors, liver, and spleen was confirmed by histology. Our results demonstrate the suitability of [89Zr]Zr(oxinate)4 as a cell-labeling agent for therapeutic T cells and the potential benefits of liposomal bisphosphonate treatment before γδ-T cell administration.Gammadelta T (γδ-T) cells are strong candidates for adoptive immunotherapy in oncology due to their cytotoxicity, ease of expansion, and favorable safety profile. The development of γδ-T cell therapies would benefit from non-invasive cell-tracking methods and increased targeting to tumor sites. Here we report the use of [  89Zr]Zr(oxinate)4 allows in vivo PET imaging of therapeutic γδ-T cells over 1 week. Furthermore, the trafficking of γδ-T cells to the tumor is increased by treatment with a liposomal aminobisphosphonate drug. This radiotracer is potentially translatable for clinical trials of therapeutic T cells.Man et al. demonstrate that [ Whole-body imaging is highly useful in this context by enabling in vivo tracking of administered cells. Many techniques exist for non-invasive cell tracking;Clinical studies of γδ-T cell immunotherapy have shown a good safety profile and efficacy comparable to second-line anticancer therapies, but they have also highlighted the need for improvements.111In]In(oxinate)3 and [99mTc]Tc-exametazime.89Zr .PET tracking of T cells has been performed with radiolabeled antibodies, antibody fragments, or lipophilic small molecules89Zr]Zr(oxinate)4 for in vitro radiolabeling and in vivo tracking of human γδ-T cells, including the effects of radiolabeling on γδ-T cell functionality, proliferation, and DNA integrity. We applied this strategy in a xenograft model of breast cancer with an engineered cancer cell line that allows multimodal imaging to track tumor cells. A liposomal aminobisphosphonate was administered to increase T cell trafficking to the tumor.Here we report the first use of [89Zr]Zr(oxinate)4 was obtained by mixing neutralized [89Zr]Zr4 with 8-hydroxyquinoline dissolved in chloroform (89Zr]Zr(oxinate)4  was significantly higher than with [89Zr]Zr4 4 (6−600 mBq/cell) for 10, 20, or 30 min at 4°C, room temperature (RT), or 37°C. We found no significant difference between incubation times and temperatures  were cultured at 0.83 × 10k N = 3; C.in vitro-expanded γδ-T cells plateaued 13−15 days post-isolation  and liver  in all groups, followed by lung and bone tissue (89Zr in tumors from PLA-treated groups (2.1% ± 0.8% ID/g) was significantly higher than in non-PLA groups  was significantly lower than in other groups . Uptake in kidneys was significantly higher with killed γδ-T cells than in other treatment groups 4 synthesis has been reported previously by our group89Zr]Zr(oxinate)4 suggests this is a passive process, in line with results from Sato et al.89Zr over 1 week in vitro, which we believe does not interfere with in vivo imaging within this time frame. Uptake of 89Zr in the bone can be used to estimate the amount of tracer that leaked from the cells.89Zr]Zr(oxinate)4 is dependent on cell type, and our results are comparable to those observed with dendritic, bone marrow, and chimeric antigen receptor (CAR)-T cells.111In]In(oxinate)3,[89Zr]Zr(oxinate)4 on γδ-T cell survival, proliferation capacity, and DNA damage were kept minimal for doses up to 20 mBq/cell but were significant at doses ≥50 mBq/cell. The cytotoxicity of radiolabeled γδ-T cells against the same tumor cells used for in vivo experiments was not affected by amounts of [89Zr]Zr(oxinate)4 of up to 600 mBq/cell, at least within 48 hr of radiolabeling. Cancer cell death was due to the combination of bisphosphonate treatment and γδ-T cells and not to the presence of 89Zr. Preserved cytotoxicity after radiolabeling, also recently observed in CAR-T cells by Weist et al.,89Zr]Zr(oxinate)4 as a T cell-tracking agent. However, the therapeutic efficacy of γδ-T cells presumably also relies on their in vivo proliferation ability; hence, we suggest that radiolabeling γδ-T cells with [89Zr]Zr(oxinate)4 should ideally not exceed 20 mBq/cell. This could lead to sensitivity issues on conventional PET scanners. Indeed, our experiments show that ex vivo gamma-counting tumors could reveal amounts of 89Zr indistinguishable from background in our PET imaging system at day 7. Assuming that a human cell-tracking study would require 37 MBq 89Zr9 γδ-T cells,89Zr activity per cell (by a factor of 40) and overcome these sensitivity issues.A radiotracer for cell tracking must not significantly alter the phenotype, survival, proliferation capacity, and functionality of labeled cells. We demonstrated that the effects of Zr(oxinate)4 (30−300 mBq/cell) by PET 1 hr, 48 hr, and 7 days after intravenous injection. We simultaneously used 99mTcO4− to visualize hNIS-expressing tumors by SPECT. The in vivo distribution of 89Zr-labeled γδ-T cells over time was similar to that observed in studies of adoptively transferred γδ-T89Zr uptake was significantly increased in PLA-treated tumors, suggesting that PLA increases homing of these cells to the tumor site. Accumulation of γδ-T cells at the tumor site 48 hr after administration was also observed by others.ex vivo bio-distribution. This can be explained by the small size of this organ and significant partial volume effect (spleen) and the liquid or necrotic tumor core that leaked upon dissection.For 89Zr than recommended above. However, the distinctly different distribution pattern observed with killed γδ-T cells suggests that radiolabeling with up to 300 mBq/cell, which preserved cytotoxic functionality in vitro over 48 hr, did not impair γδ-T cell trafficking and allowed us to track live cells. Furthermore, previous studies have shown that [89Zr]Zr4,89Zr]Zr(oxinate)4, and lysates from [89Zr]Zr(oxinate)4-labeled cells89Zr]Zr(oxinate)4. Cell concentrations during labeling and in vitro assays were in the range of 1−5 × 106/mL. In comparison, using in vitro89Zr retention values, cell concentrations extrapolated from PET-computed tomography (CT) images in the organs showing the strongest 89Zr signal  would be in the range of 0.5−5 × 106 cells/mL. We therefore expect the DNA damage sustained by γδ-T cells, due to both self-irradiation and crossfire, after in vivo administration to be comparable to that observed in vitro. Considering the strong affinity of the 89Zr4+ ion for bone,89Zr indicates limited efflux of weakly chelated 89Zr, and it suggests that 89Zr is mostly retained by γδ-T cells after injection. The lower accumulation of 89Zr in the bones of PLA-treated animals compared to other groups also suggests reduced efflux of 89Zr from γδ-T cells after PLA treatment.For instrument sensitivity reasons, some imaging studies were performed with higher doses of 89Zr in tumors, which would be challenging to observe by histology. Combined with the non-invasive nature of PET imaging, this further highlights the value of using PET tracers such as [89Zr]Zr(oxinate)4 for cell tracking. The high uptake of 89Zr in the liver and spleen was mirrored by the large numbers of human CD3+ cells observed in these tissues, consistent with the bio-distribution of radiolabeled γδ-T cells. In contrast, the apparent absence of CD3+ cells in the kidneys, despite higher 89Zr uptake than in the tumor, and the fact that the kidney uptake of 89Zr was significantly higher in animals administered killed γδ-T cells than in other groups both suggest that the radioactivity detected in the kidneys corresponds to 89Zr progressively released from γδ-T cells in other organs. A limitation of directly labeling cells is that the radionuclide can leak out over time and be taken up by adjacent tissue. Although immunohistochemistry demonstrates the presence of the administered γδ-T cells in the tumors, this technique cannot determine whether the 89Zr signal originates from the γδ-T cells or from in situ-labeled bystander cells.Histology confirmed the presence of γδ-T cells in the tumors, spleen, and liver, using the CD3 marker.89Zr reaching the tumor within 48 hr of radiolabeled γδ-T cell administration. Our results suggest that γδ-T cells home to the tumor within 2 days and remain there for at least 5 days. This was not observed in any other treatment group, demonstrating the importance of encapsulating the aminobisphosphonate in a tumor-targeting vehicle.A critical aspect of this type of cellular immunotherapy is that the therapeutic cells must be activated at the target site and reach the tumor in sufficient numbers. γδ-T cell toxicity toward cancer cells is greatly amplified by bisphosphonates, suggesting a role for γδ-T cells in the anti-cancer properties of bisphosphonates.111In]In(oxinate)3 have been performed by radiolabeling only a fraction of the total administered T cells,89Zr]Zr(oxinate)4 might be the preferable option to avoid imaging excessively damaged cells. In two notable studies, γ-scintigraphy revealed T cell uptake in tumors using only 1−3 mBq 111In per cell.89Zr]Zr(oxinate)4 is a credible prospect.Clinical imaging studies of therapeutic T cells with [89Zr]Zr(oxinate)4 as a PET tracer to track γδ-T cells in vivo, while previous work has shown the therapeutic efficacy of γδ-T cells in combination with PLA.89Zr]Zr(oxinate)4 can now be applied to answer fundamental questions in the preclinical and clinical development of γδ-T cell therapies, e.g., whether the accumulation of γδ-T cells at the tumor site or their distribution within the tumor correlates with therapeutic efficacy. Due to numerous molecular and cellular differences, the distribution of human γδ-T cells in an immunocompromised mouse model cannot fully predict their behavior in a human host. However, the results of this proof-of-principle study can be used to design a clinical trial that will answer the question of the distribution of γδ-T cells in humans after adoptive transfer.This study demonstrates the suitability of Zr(oxinate)4 is clinically translatable without significant methodological modifications, and the high similarity of [89Zr]Zr(oxinate)4 to the well-established [111In]In(oxinate)3 should facilitate regulatory approval. Our results support that T cell labeling with [89Zr]Zr(oxinate)4 is a realistic option for human studies and will benefit the development of cellular immunotherapy.Our results have implications for clinical translation, and they suggest using liposomal aminobisphosphonates as adjuncts to γδ-T cell therapy. In the context of clinical protocols involving repeated infusions of γδ-T cells,Animals experiments were approved by the UK Home Office under The Animals (Scientific Procedures) Act (1986), PPL reference 7008879 (Protocol 6), with local approval from King’s College London Research Ethics Committee (KCL-REC). Experiments using human T cells received approval from KCL-REC (Study Reference HR-16/17-3746). All donors provided written, informed consent.scidLystbg-J/Crl) and Nod scid gamma (NSG) (NOD.Cg-Prkdcscid Il2rgtm1WjI/SzJ) mice  were obtained from Charles River (UK). γδ-T cells were obtained as described previously,http://flowingsoftware.btk.fi). Only batches with ≥80% γδ-positive CD3+ cells were used for further experiments (≥95% for in vivo experiments). MDA-MB-231.hNIS-GFP cellsl-glutamine (2 mM), and they were tested for mycoplasma contamination .Unless otherwise indicated, reagents were purchased from Sigma-Aldrich and Merck. Female SCID/beige (CB17.Cg-Prkdc89Zr]Zr(oxinate)4 was synthesized as previously described.[in vitro2+/Mg2+ free) and re-suspended at 5 × 106/mL in PBS at RT. [89Zr]Zr(oxinate)4 (6−600 mBq/cell) in aqueous DMSO was added to the cell suspension, keeping DMSO concentrations ≤0.7%. Neutralized [89Zr]Zr4 with an equivalent amount of DMSO was used as a control. After 10−30 min of incubation, cells were pelleted and the supernatants kept aside. The cells were washed with PBS, centrifuged, and the washings combined with the previous supernatants. The cells were suspended in growth medium or PBS for further experiments. Viability was assessed using the trypan blue dye exclusion method. Radioactivity in re-suspended cells and combined supernatants was measured in a gamma-counter. Cell-labeling efficiency (LE[%]) was calculated as follows.γδ-T cells expanded 4 cells/well and incubated overnight were treated with 3 μM zoledronate or vehicle for 24 hr. The cells were washed and the medium was replaced with γδ-T cells in growth medium. As a control for radiolabeled γδ-T cells, an equal amount of 89Zr in medium was added to some wells. After 48 hr, γδ-T cells were removed by washing with PBS, and cancer cell viability was evaluated using the alamarBlue assay (Thermo Scientific), reading plates in a GloMax (Promega) reader (530 nm excitation and 590 nm emission filters).MDA-MB-231.hNIS-GFP cells seeded in a 96-well plate at 10l-lysine-coated coverslips and incubated for 1 hr. After centrifugation and gentle rinsing with PBS, the cells were fixed and permeabilized with 3.7% formalin, 0.5% Triton X-100, and 0.5% IGEPAL CA-630 in PBS, then blocked with 2% BSA and 1% goat serum. γH2AX foci were detected with an anti-γH2AX (Ser139) mouse monoclonal antibody (mAb)  and goat anti-mouse AF488-immunoglobulin G (IgG) . Nuclei were detected with Hoechst 33342. Images were acquired on a TCS SP5 II confocal microscope (Leica) with a 100×/1.40 HCX PL Apochromat objective (Leica) and Leica Application Suite Advanced Fluorescence (LAS-AF) control software. Ten sections (0.4-μm thickness) were imaged. At least 30 nuclei/slide were imaged (2 slides/treatment). Maximal intensity projections of z stacks were made using ImageJ v.1.51p (https://imagej.nih.gov/ij/). Nuclei and γH2AX foci were counted using CellProfiler v.2.2.0 (http://cellprofiler.org), calculating average numbers of γH2AX foci per nucleus in each image. Full details are provided in the Radiolabeled γδ-T cells in medium were seeded onto poly-6 MDA-MB-231.hNIS-GFP cells were injected subcutaneously in the mammary fat pad between the fourth and fifth nipples in the left flank; tumors were grown over 3 weeks. Animals were randomly assigned to experimental groups, and investigators were not blinded to cohort allocation when assessing outcomes. Cohort sizes were chosen based on prior experience,Approximately 1.5 × 1089Zr-radiolabeled γδ-T cells  were injected i.v. at t = 0 hr and imaged by PET/CT within 30 min. PET/CT imaging was performed for 30−240 min (as indicated) on a nanoScan PET-CT scanner (Mediso). For tumor imaging, 100 μL 99mTcO4− (15−25 MBq) in saline was injected i.v., and SPECT-CT was performed 40 min thereafter in a NanoSPECT/CT scanner . PET-CT and SPECT-CT were repeated at t = 48 and 168 hr. For PLA imaging by SPECT-CT, PLA was radiolabeled with [111In]In(oxinate)3 and administered i.v. (7 MBq 111In/mouse) to NSG mice. PET- and SPECT-CT datasets were reconstructed using a Monte Carlo-based full-3D iterative algorithm . Images were co-registered and analyzed using VivoQuant v.2.50 (Invicro). Regions of interest (ROIs) were delineated for PET activity quantification in specific organs. Uptake in each ROI was expressed as a percentage of injected dose per volume (% ID/mL).Mice from imaging studies were used for bio-distribution studies on day 2 or 7. After culling, organs were dissected, weighed, and γ-counted together with standards prepared from a sample of injected material. The percentage of injected dose per gram (% ID/g) of tissue was calculated. Organs were cryopreserved in optimal cutting temperature (OCT) compound (VWR) for autoradiography and/or formalin fixed and paraffin embedded (FFPE) for histologic analysis.l-lysine-coated slides (VWR), fixed in 4% paraformaldehyde (PFA), mounted in Mowiol, and exposed to a storage phosphor screen for 20 min at 3 hr post-dissection to obtain the 99mTc signal, then for 48 hr at 4 days post-dissection to obtain the 89Zr signal. The storage phosphor screen was read using a Cyclone Plus imager (PerkinElmer), and images were processed with ImageJ.Cryopreserved tissues were cut (50 μm), mounted on poly-Briefly, FFPE organ blocks were sliced and stained using a Discovery XT system  using the DAB Map detection kit (Ventana 760-124). For pre-treatment, CC1 (Ventana 950-124) was used. Sections were stained with anti-GFP  or anti-CD3  primary antibodies, followed by biotinylated anti-rabbit or anti-mouse IgG  secondary antibodies, as appropriate. Full details are provided in the http://invivostat.co.uk/), as indicated. Dunnett’s post hoc test was applied for comparisons back to a control group, or Bonferroni correction for multiple pairwise comparisons, unless otherwise specified. Exact significance values are reported in each figure.Independent experiments were performed on different days with γδ-T cell batches from different donors. Data were plotted using Prism v.7.01 (GraphPad). Differences between 2 groups were evaluated by Student’s two-tailed t test. To account for repeated measurements in a same animal or cell batch and multiple treatments tested on a same cell batch, analysis was performed using 2-way repeated-measures ANOVA in GraphPad Prism or a repeated-measures Mixed Model (MM)Conceptualization, F.M., L.L., G.O.F., and R.T.M.d.R.; Methodology, F.M., L.L., A.G., G.O.F., and R.T.M.d.R.; Investigation, F.M., L.L., A.V., B.D., A.C.P.-P., and R.T.M.d.R.; Writing – Original Draft, F.M.; Writing – Review and Editing, F.M., L.L., A.G., J.M., P.J.B., G.O.F., and R.T.M.d.R.; Funding Acquisition, G.O.F., R.T.M.d.R., P.J.B., and J.M.; Resources, F.M., L.L., A.G., H.S., and R.T.M.d.R.; Supervision, R.T.M.d.R., G.O.F., and P.J.B.J.M. is chief scientific officer of Leucid Bio, a company dedicated to the commercial development of CAR-T cells for solid tumors. The authors declare no other potential conflicts of interest.


H)-one derivative, crystallizes with two independent molecules in the asymmetric unit. The two molecules differ essentially in the orientation of the benzyl ring with respect to the central pyridazine ring; this dihedral angle being 3.70 (9) ° in one molecule and 10.47 (8) ° in the other.The title pyridazin-3(2 H)-one derivative, C25H19FN2O2, crystallizes with two independent molecules (A and B) in the asymmetric unit. In molecule A, the 4-fluorophenyl ring, the benzyl ring and the phenyl ring are inclined to the central pyridazine ring by 86.54 (11), 3.70 (9) and 84.857 (13)°, respectively. In molecule B, the corresponding dihedral angles are 86.80 (9), 10.47 (8) and 82.01 (10)°, respectively. In the crystal, the A molecules are linked by pairs of C—H⋯F hydrogen bonds, forming inversion dimers with an R22(28) ring motif. The dimers are linked by C—H⋯O hydrogen bonds and a C—H⋯π interaction, forming columns stacking along the a-axis direction. The B molecules are linked to each other in a similar manner and form columns separating the columns of A molecules.The title pyridazin-3(2 H)-ones are pyridazine derivatives, being constructed about a six-membered ring that contains two adjacent nitrogen atoms, at positions one and two, and with a carbonyl group at position three. The interest in these nitrogen-rich heterocyclic derivatives arises from the fact that they exhibit a number of promising pharmacological and biological activities. These include anti-oxidant -one derivative has been undertaken, along with a DFT study, in order to gain further insight into the molecular structure.Pyridazin-3(2A and B) in the asymmetric unit  is planar in both molecules; r.m.s. deviations are 0.029 Å for molecule A and 0.009 Å for molecule B. In molecule A, the 4-fluorophenyl ring , the benzyl ring  and the phenyl ring  are inclined to the central pyridazine ring  by 86.54 (11), 3.70 (9) and 84.87 (13)°, respectively. In molecule B, the corresponding dihedral angles are 86.80 (9), 10.47 (8) and 82.01 (10)°, respectively. Hence, the conformation of the two molecules differs essentially in the orientation of the benzyl ring (C) with respect to the central pyridazine ring (B); 3.70 (9)° in molecule A compared to 10.47 (8)° in molecule B. The two molecules have an r.m.s. deviation of 0.683 Å for the 30 non-hydrogen atoms  level  and the lowest-lying unoccupied molecular orbitals (LUMOs) are named as frontier molecular orbitals (FMOs). The FMOs play an important role in the optical and electric properties, as well as in quantum chemistry and UV–vis spectra. As a result of the interaction between the HOMO and LUMO orbitals of a structure, a transition state of the viz. atoms O1A and O2A of molecule A and O1B and O2B of molecule B). The positive regions are over hydrogen atoms, indicating that these sites are the most likely to be involved in nucleophilic processes.The molecular electrostatic potential (MEP) is a technique of mapping electrostatic potential onto the iso-electron density surface. The MEP surface provides information about the reactive sites. The colour scheme is as follows: red for electron rich, partial negative charge; blue for electron-deficient, partial positive charge; light blue for a slightly electron deficient region; yellow for a slightly electron-rich region; green for neutral -ones gave 297 hits, while a search for 6-phenyl-pyridazin-3(2H)-ones gave 40 hits, including 6-phenyl-pyridazin-3(2H)-one itself -ones gave only three hits, for example 4-(4-bromobenzyl)-6-phenylpyridazin-3(2H)-one -ones with an oxoethyl group in position-2 on the pyridazine ring gave eight hits, mostly esters. Four of these structures also have a phenyl substituent in position-6 on the pyridazine ring, as in the title compound. They include, for example 2-acetic acid -one , K2CO3  and 2-chloro-1-(4-fluorophenyl)ethan-1-one  in acetone (40 ml), was refluxed overnight. The solution was then filtered by suction and the solvent removed under reduced pressure. The residue was purified by recrystallization from ethanol to afford the title compound as colourless prismatic crystals (yield 68%).A mixture of 4-benzyl-6-phenylpyridazin-3(2Uiso(H) = 1.2Ueq(C). The image plate disc in the diffractometer used for the data collection was unfortunately distorted at the outer edges, hence the maximum 2θ value available was limited to 48.8°.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019008557/su5499sup1.cifCrystal structure: contains datablock(s) I, Global. DOI: 10.1107/S2056989019008557/su5499Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019008557/su5499Isup3.cmlSupporting information file. DOI: 1923216CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Three new complexes, {[RuCl(H2biim)(η6-p-cymene)]PF6}2·H2O (1), [(η5-Cp)RhCl(H2biim)]PF6 (2), and [(η5-Cp)IrCl(H2biim)]PF6 (3), were fully characterized by CHN, X-ray diffraction analysis, UV–Vis, FTIR, and 1H, 13C and 15N NMR spectroscopies. The complexes exhibit a typical pseudooctahedral piano-stool geometry, in which the aromatic arene ring (p-cymene or Cp) forms the seat, while the bidentate 2,2′-biimidazole and chloride ion form the three legs of the piano stool. Moreover, the cytotoxic activities of the compounds were examined in the LoVo, HL-60, MV-4-11, MCF-7 human cancer cell lines and BALB/3T3 normal mouse fibroblasts. Notably, the investigated complexes showed no cytotoxic effects towards the normal BALB/3T3 cell line compared to cisplatin, which has an IC50 value of 2.20 µg. Importantly, 1 displayed the highest activity against HL-60 (IC50 4.35 µg). To predict a binding mode, we explored the potential interactions of the metal complexes with CT-DNA and protein using UV absorption and circular dichroism. The obtained data suggest that the complexes could interact with CT-DNA via an outside binding mode. Moreover, binding of the complexes with the GSH via UV–Vis and ESI mass spectra was determined. Comparative studies have shown that the rhodium complex (2) is the most GSH reactive, which is probably responsible for its deactivation towards LoVo and MCF-7 tumour cells. The influence of the metal ion on the biological activity of isostructural Rh(III) and Ir(III) complexes was an important goal of the presented investigation.In this paper, we discussed the similarities and differences in dThe online version of this article (10.1007/s00775-019-01665-2) contains supplementary material, which is available to authorized users. With the aim of gaining deeper insights into the mechanisms of action, we investigated the interactions with DNA, HSA via UV–Vis and CD methods. Moreover, binding of the complexes with the GSH via UV–Vis and ESI mass spectra was determined.By screening the literature connected to the effort of scientists attempting to resolve the problems associated with cancer treatment, it should be noted that organometallic, bioinorganic and bioorganic chemistry are important branches of this subject. Contemporary medicine, including chemotherapy, is mainly based on Pt-anticancer drugs to overcome the following important problems: 1) drug resistance, 2) toxic side effects, and 3) lack of activity against several types of cancer PF6  and Rh(Ioxylate) , as welloxylate) , we focu6-p-Cymene)Ru(µ-Cl)Cl]2 (97%), [CpRh(μ-Cl)Cl]2 (99%), [CpIr(μ-Cl)Cl]2 (99%), 2,2′-biimidazole (H2biim), NH4PF6 (99%), cisplatin, 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB) (99%), human serum albumin (HSA) , l-glutathione reduced form (GSH)  were purchased from Sigma-Aldrich. Analytical grade solvents were purchased from Chempur. Tris(hydroxymethyl)aminomethane-HCl  and PBS  buffer were prepared using MiliQ water.2 , ligand 2,2′-biimidazole  and NH4PF6  was dissolved in a 20 ml mixed solution  with one drop of H2O. The reaction mixture was refluxed for 12 h (~ 65 °C). After 2 weeks, the resulting orange crystals suitable for single-crystal X-ray diffraction were filtered and dried in a vacuum box. The crystals were collected at 75% yield. Anal. Calc. (%) for C32H42Cl2F12N8OP2Ru2: C, 33.95; H, 3.67; N, 10.19. Found: C, 33.42; H, 3.50; N, 10.22; 1H NMR : 1.00 ; 2.61 ; 6.00 ; 5.80 ; 2.07 ; 7.84 ; 7.50 ; 12.91 . 13C NMR : 22.1 ; 30.9 (C27); 102.6 (C24); 83.3 ; 81.7 ; 99.8 (C21); 18.7 (C30); 138.2 [C2/2′(H2biim)]; 131.8 [C4/4′(H2biim)]; 121.1 [C5/5′(H2biim)]. 15N NMR : − 218.5 (N1/1′H); − 181.7 (N3/3′). IR (cm−1): 3260(br), 3145(w), 1527(ms), 1468(ms), 1321(ms), 1188(ms), 1093(ms), 849(vs), 754(ms), 679(ms), 557(s).A solution of the precursor compound ; 7.56 ; 7.54 ; 12.90 . 13C NMR : 9.3 (Cp–C11–15H3); 95.4 (Cp–C6–10) (J = 8.2 Hz); 138.2 [C2/2′(H2biim)]; 128.2 [C4/4′(H2biim)]; 121.4 [C5/5′(H2biim)]. 15N NMR : − 218.7 (N1/1′H); − 173.8 (N3/3′) (J = 18.6 Hz). IR (cm−1): 3169(w), 1531(ms), 1433(ms), 1179(ms), 1095(ms), 1021(ms), 836(vs), 746(ms), 608(ms), 557(s).A mixture of the precursor compound [CpRh(μ-Cl)Cl]3 was prepared similar to complex (2) using [CpIr(μ-Cl)Cl]2  as the metal source. The yellow crystals appeared after a week and were collected at 49% yield. Anal. Calc. (%) for C16H21ClF6N4PIr: C, 29.93; H, 3.29; N, 8.73. Found: C, 30.17; H, 3.03; N, 8.78; 1H NMR : 1.69 ; 7.56 ; 7.54 ; 13.15 . 13C NMR : 9.1 (Cp–C11–15H3); 87.1 (Cp–C6–10); 139.9 [C2/2′(H2biim)]; 127.8 [C4/4′(H2biim)]; 121.8 [C5/5′(H2biim)]. 15N NMR : − 218.1 (N1/1′H); − 189.7 (N3/3′). IR (cm−1): 3172(w), 1533(ms), 1458(ms), 1181(ms), 1093(ms), 1025(ms), 835(vs), 740(ms), 652(ms), 565(s).Complex 1–3) was determined by microculture with sulforhodamine B SRB  and MTT -2,5-diphenyltetrazoliumbromide)]) [for cells in suspension (leukaemia cells)] assays PF6}2·H2O (1), [(η5-Cp)RhCl(H2biim)]PF6 (2) and [(η5-Cp)IrCl(H2biim)]PF6 (3) with polymeric CT-DNA was evaluated via UV–Vis absorption titrations and CD spectroscopy. The stock solutions of the complexes and CT-DNA were prepared using 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2. The DNA concentration per nucleotide was determined by UV absorption measurements using the molar absorption coefficient ε258 = 6 600 M−1 cm−1 /(εa − εf) versus [DNA] gave a slope and an intercept equal to 1/(εb − εf) and 1/Kb/(εb − εf), respectively. The intrinsic binding constant Kb is the ratio of the slope to the intercept.Binding of {[RuCl(HM−1 cm−1 . Stock s2biim)(η6-p-cymene)]PF6}2·H2O (1), [(η5-Cp)RhCl (H2biim)]PF6 (2) and [(η5-Cp)IrCl(H2biim)]PF6 (3) with HSA was studied by UV–Vis absorption titrations at 210–320 nm wavelengths; λmax was recorded at 278 nm (1 cm cuvette). The CD spectroscopy was performed from 220 to 300 nm (0.2 cm cuvette) in 5 mM Tris–HCl/50 mM NaCl buffer at pH 7.2. Solid HSA was dissolved in Tris–HCl/NaCl buffer (5/50 mM at pH 7.2) and the resulting stock HSA solution was kept at 4 °C for a maximum of 1 day. All the experiments involved 16.5 µM HSA (UV–Vis spectroscopy) or 10 µM HSA (CD spectroscopy) and increasing amounts of the complexes dissolved in Tris–HCl/NaCl buffer (pH = 7.2). The CD spectra of these solutions were measured after 24 h of incubation at 37 °C, while the UV–Vis spectra were measured after 40 min and 24 h of incubation at room temperature. The concentrations of the stock solution of complexes 1, 2, and 3 were 1.5 × 10−4 M (UV–Vis spectroscopy) and 3 × 10−4 M (CD spectroscopy). The HSA concentration was determined spectrophotometrically using an extinction coefficient ε280 = 36 600 M−1 cm−1 [εa − εf)  versus [complex] is a linear curve, and the binding constant (Kb) can be obtained from the ratio of the intercept to the slope. The results of HSA interaction with complexes via CD spectroscopy were expressed as mean residue ellipticity (MRE) in deg cm2 dmol−1. The value of MRE can be obtained using the equation MRE = [Θ]/(nlC × 10), where [Θ] is the CD in millidegrees obtained from the spectra (at 208 nm), n is the number of amino acid residues (585 for HSA), l is the path length of the cell (0.2 cm), and C is the mol fraction of the protein. Then, the helical content of HSA can be calculated from the [Θ] value at 208 nm according to the equation  % helix = {(− MRE208 − 4000)/(33000 − 4000)} × 100 as described by Liu et al. 2, pentamethylcyclopentadienylrhodium(III) dimer [CpRh(μ-Cl)Cl]2, pentamethylcyclopentadienyliridium(III) dimer [CpIr(μ-Cl)Cl]2, with 2,2-biimidazole ligand (H2biim) with appropriate ratio in dry mixture of methanol/dichloromethane resulted in the formation of d6 low-spin half-sandwich ruthenium, rhodium and iridium complexes: {[RuCl(H2biim)(η6-p-cymene)]PF6}2·H2O (1), [(η5-Cp)RhCl(H2biim)]PF6 (2) and [(η5-Cp)IrCl(H2biim)]PF6 (3)  and Ru(2) is built up by an η6-p-cymene, a chloride and an N∩N-κ2N,N′ 2,2′-biimidazole ligand. The distance from ruthenium(II) to the centroid of the p-cymene ring is similar to that observed in the literature + cations are held together via π···π interactions between the parallel-displaced imidazole rings [3.7835(2) Å] and an N–H···Cl interaction  M(H2biim)Cl][PF6], in which M = Rh(III) (2) or Ir(III) (3) and H2biim represents 2,2′-biimidazole, display the standard “three-leg piano-stool” geometry  consists of a π-bonded η5-pentamethylcyclopentadienyl ligand, a doubly σ-bonded 2,2′-biimidazole, as well as a chloride ligand. The Rh–C bond distances in complex 2 [ranging from 2.1311(32) to 2.1632(22) Å] are consistent with previous reports [3) shows slightly longer distances between the metal ion and the Cp ring [2.1445(47)–2.1794(44) Å]. Accordingly, replacement of the rhodium with iridium in compound 3 surprisingly compresses the M–N bond lengths of the chelating κ2N,N′–H2biim ligand   and H2bi reports , 38. Theompounds , 38.Fig.N,N-H2biim bonds are similar in complexes 2 and 3; however, careful inspection of Table ∩N-2,2′-biimidazole ligand compared to the stronger interactions between the rhodium(III) ion and the Cp ring. Additionally, the M–Cl bond distance in both complexes 2 and 3  is relatively longer than that in previously reported complexes .The above-mentioned data pointed out that the presence of N–H/C–H hydrogen bond donors in complexes favours multiple noncovalent interactions ; the ensuing data are summarized in the experimental section, and some of the data are presented in Table Formation of 13C and 15N NMR are interesting to compare the strength of the coordination (by Lewis acids) of the rhodium and iridium central metal ions in the obtained complexes . Both the metal ions are coordinated by the same η5-pentamethylcyclopentadienyl ligand (Cp). Considering the δCp–C, we noticed that the π-donating effect is much more effective in the case of the rhodium complex (shift δ = 95.4 ppm) compared to the iridium complex (shift δ = 87.1 ppm). This was confirmed by the X-ray data . Consequently, the effective positive charge of the metal ion is decreasing, which is reflected in the Rh–N and Ir–N bond lengths with H2biim . This weaker coordination interaction of H2biim with the rhodium ion compared to the iridium ion was confirmed by the 15N NMR results (shift δRh = − 173.8 ppm and shift δIr = − 189.7 ppm). The chelating ligand H2biim is the same in both complexes; thus, Δcoord. = δcomplex − δligand can be estimated according to the δRh,Ir shift (the same is correct for the Cp ligand shift). It should be noted that the ligand H2biim is a tautomer under the experimental conditions, which effectively makes it impossible to measure the nitrogen spectrum and to determine the NH shift in the proton spectrum. Interestingly, by analysing the 13C NMR spectra of the rhodium complex, we observed a doublet around δ 95.4 ppm (JRh–C = 8.2 Hz). Moreover, the 15N NMR spectra of the same complex display a doublet centred ca. − 173.8 ppm, with a JRh–N value of 18.6 Hz, which is in the range typical for related compounds.1–3 and H2biim (ligand) were determined in 1 × 10−4 M dry methanol at room temperature, and these spectra are presented in Figure S9. The Ru(II), Rh(III) and Ir(III) complexes are d6 low-spin metal complexes containing proper geometry-filled orbitals at the metal centres, which can interact with the ligand’s low lying π* orbitals, possibly resulting in metal-to-ligand charge transfer (MLCT) transitions (Table S3). The low energy absorption bands observed in the range from 330 to 410 nm are assigned to metal-to-ligand charge transition (MLCT) dπ(M) to π*(L) transitions, while the high energy absorption bands observed in the range from 208 to 285 nm may be attributed to ligand-centre π–π*/n–π* transitions  which influenced compounds’ stability in solution significantly.Following the characterization of the complexes using standard spectroscopic methods before performing the biological studies, the stability of obtained complexes tra Fig. , which iP value for 1 at +0.22 ± 0.01 and 2 at − 0.54 ± 0.02, whereas for 3, the log P = + 1.55 ± 0.04, indicating that [(η5-Cp)RhCl(H2biim)]+ is more hydrophilic than {[RuCl(H2biim)(η6-p-cymene)]+ and [(η5-Cp)IrCl(H2biim)]+. Interestingly, in this case, the replacement of rhodium with a heavier homolog from the same group, iridium, in the obtained isostructural complex (2 and 3) led to significant differences in the lipophilicity  accumulation probably occurs mainly in the nuclei and lysosomes, whereas a log P > 0 suggests that the ruthenium (1) and iridium (3) compounds will specifically accumulate in the mitochondria and endoplasmic reticulum, but this requires further more detailed research. Taking this into consideration, the obtained complexes should possess different cytotoxic activities, which are mainly influenced by the type of metal centre ion in 1, 2, and 3 and the types of DNA/HSA interactions. The obtained unexpected results for the lipophilicity of the rhodium and iridium isostructural complexes were confirmed by multiple repetitions of the experiment.Lipophilicity is a very important physicochemical feature related to the pharmacokinetic behaviour of drug-like compounds . Using t(H2biim)η-p-cymene1–3 was determined using MTT and SRB assays after a 96 h incubation period against a panel of four human tumour cell lines, including LoVo, HL-60, MV-4-11, and MCF-7 cells, as well as the BALB/3T3 healthy mouse cell line (Table 50 = 2.20 µg). Importantly, {[RuCl(H2biim)(η6-p-cymene)]PF6}2·H2O (1) displayed the highest activity against HL-60 cells (IC50 4.35 µg), while the IC50 values of Rh(III) and Ir(III) were 36.2 µg and 30.86 µg, respectively. According to the log P values (log P > 0), the ruthenium and iridium complexes are active against the same cancer cell lines , whereas the rhodium complex (log P < 0) was only active against HL-60 and MV-4-11. All the compounds were not only inactive against the normal cell line at the tested concentration range but also against the MCF-7 cell line. These results suggest the cytotoxic selectivity of the obtained organometallic complexes. It should be underlined that the biological potencies of two isostructural half-sandwich rhodium and iridium complexes  in the same overall ligand set are not the same. Studying similarities and differences in biological activity of investigated analogues, it should be pointed out that the rhodium complex is not active against MCF-7 and LoVo cells, while the iridium compound is only not active against the MCF-7 cell line. Moreover, the rhodium complex is approximately twofold more active against MV-4-11 but slightly less active against the HL-60 cell line compared with the iridium complex. The differences in the biological activity of potential drugs, such as discussed complexes, are most likely derived from their absorption and distribution into different cell components, depending on their lipophilicity . Similar differences in anticancer activities in vitro and in vivo have been discussed by Sadler et al. PF6}2·H2O at 401 nm and [(η5-Cp)RhCl(H2biim)]PF6 at 398 nm; moreover, a hyperchromic effect with a red shift of approximately 1 nm was observed upon the addition of CT-DNA. The [(η5-Cp)IrCl(H2biim)]PF6 complex exhibited hyperchromism at 401 nm but with a blue shift of approximately 3 nm. These spectral characteristics (hyperchromic effect) suggest that the complexes interact with CT-DNA through non-intercalative binding modes such as outside binding. This may be due to electrostatic interactions between the positively charged [RuCl(H2biim)(η6-p-cymene)]+, [(η5-Cp)IrCl(H2biim)]+ and [(η5-Cp)IrCl(H2biim)]+ complex units and the negatively charged phosphate backbone at the periphery of the double-helix CT-DNA , the binding constants were calculated as 1.5 × 104 (1), 5 × 104 (2), and 1 × 104 (3). These values are within the range of 104 to 106 M−1, as expected for good HSA carrier activity in vivo  significantly influenced compounds’ stability in solution. Similar to the osmium and ruthenium complex analogues investigated by Sadler’s group, the biological potencies of the two obtained isostructural half-sandwich rhodium and iridium complexes  are not the same. The differences in complexes 2 and 3 are potentially derived from their absorption and distribution into different cell components, which is based on their lipophilicity . A comparison of the cytotoxicity showed that the rhodium complex is not active against MCF-7 and LoVo cells, while the iridium compound is only not active against the MCF-7 cell line. Moreover, the rhodium complex is approximately twofold more active against MV-4-11 cells but slightly less active against the HL-60 cell line compared with the iridium complex. Importantly, {[RuCl(H2biim)(η6-p-cymene)]PF6}2·H2O (1) displayed the highest activity against HL-60 cells (IC50 4.35 µg). Additionally, it should be emphasized that the successfully obtained complexes were inactive against the normal cell line. UV–Vis and CD data suggest that the compounds can efficiently bind to protein targets (HSA) and interact with calf thymus DNA. The spectral characteristics (hyperchromic effect) suggest that the complexes interact with CT-DNA through electrostatic interactions between the positively charged [RuCl(H2biim)(η6-p-cymene)]+, [(η5-Cp)IrCl(H2biim)]+ and [(η5-Cp)IrCl(H2biim)]+ complex units and the negatively charged phosphate backbone at the periphery of the DNA double-helix. Moreover, we studied the interactions of the analysed complexes with GSH. The ESI mass spectra results confirmed that 1, 2 and 3 obtained complexes bound to GSH and formed adducts with GSH, with fragments of GSH or GSSG. Additionally, the UV–Vis results demonstrated that ruthenium complex 1 is considerably less active in the reaction with GSH than the rhodium (2) and iridium (3) complexes. Simultaneously, the ruthenium(II) dimer displays better cytotoxicity compared to complexes 2 and 3.In conclusion, new dSupplementary material 1 (PDF 1779 kb)Supplementary material 2 (PDF 143 kb)Supplementary material 3 (PDF 139 kb)Supplementary material 4 (PDF 141 kb)Below is the link to the electronic supplementary material.


The crystal structures of two bulky triarylphosphines of emerging interest in coordination chemistry and catalysis have been determined, and the syntheses and crystal structures of their corresponding oxides are reported. Each oxide is isostructural to its corresponding phosphine. 24H27P), (I), trisphosphine oxide (C24H27OP), (II), trisphosphine (C27H33O3P), (III), and trisphosphine oxide (C27H33O4P), (IV), are reported. The strucure of (III) has been described before [Romain et al.  and (II) crystallize isostructurally in P21/c. Similarly, (III) and (IV) crystallize isostructurally in Pbca. The conformations of (I) and (II) in the solid state deviate strongly from helical, whereas those of (III) and (IV) are found to be closer to an ideal threefold rotational symmetry. The pyramidality indices, ∑(C—P—C), are 305.35 (16), 317.23 (15), 307.2 (4) and 318.67 (18)° for (I), (II), (III) and (IV), respectively. Each is found to be more pyramidal than Ph3P or Ph3PO. Hybrid DFT calculations incorporating terms for dispersion provide evidence that the causes of the increased pyramidality, despite the 3,5-dimethyl group substitution, include dispersion interactions. The calculated ∑(C—P—C) values are 304.8° for both (I) and (III) and 317.4° for both (II) and (IV), with no difference arising from the substitution at ring position 4.The crystal structures of trisphosphine (C al. 2000. Organom By contrast, phosphines with 2,6-disubstitution patterns have greatly reduced pyramidality. For example, ∑(C—P—C) = 335.6 (3)° in Dipp3P, 3PO reported in the CSD, the average value with s.u. of ∑(C—P—C) is 319.3 (3)°. Thus, for both the title phosphines and their oxides, the pyramidality index for the title compounds is lower than in the corresponding Ph3P or Ph3PO.Phosphine (I)al Fig. 1. It has more pyramidal than corresponding C6H5– derivatives is at first surprising. A plausible explanation for this is that the substitution induces greater intramolecular dispersion interactions, i.e. between the methyl groups and the π-clouds of adjacent rings. To find evidence for this, hybrid density functional theory (DFT) calculations [with Becke’s non-local three parameter exchange and the Lee–Yang–Parr correlation functional (B3LYP) and also incorporating Grimme’s D3 empirical dispersion corrections] with the 6-31G basis set, as implemented in the Gaussian16 program package 3O. This indicates that intermolecular interactions in the extended structures involving the methoxy groups affect the observed structures compared to that predicted by computation.That all these 3,5-dimethyl-substituted compounds should be 3 (0.7%) for the whole unit cell, or 3.5 Å3 per oxygen atom, whereas for (III)3 (2.2%) or 13.3 Å3 per oxygen atom. The van der Waals volume of an oxygen atom is 14.7 Å3. In the extended structure, the oxygen atoms in (II)ii separations in the crystal increase from 5.148 (2) Å along the b-axis direction in (III)x, −y, z]. As a consequence, the a:b lattice parameter ratio changes from 12.30:10.27 in (III)In the isostructural pairs, the volumes of the unit cells are larger due to oxygen incorporation. For (I)3P structures along the b-axis direction . There are some short intermolecular C—H⋯O interactions in structures (II)–(IV), as listed in Tables 1By contrast, the molecules of (I) Å Fig. 3b [closo-1-H-CB11Me11] bis(μ2-chloro)-dichlorotetrakis[trisphosphine]diruthenium [trisphosphine]molybdenum(II) -1,2-diphenylethylenediamine)bis[trisphosphine]ruthenium(II) [trisphosphine]hexacarbonyl-bis(η5-cyclopentadienyl)diiridiumdimolybdenum No crystal structures of (II)1H), CDCl3 at 77.23 (13C) and 85% H3PO4 at 0 ppm .Crystals of trisphosphine [69227-47-0], (I)fR = 0.32 in 1:9 ethyl acetate/hexanes), heating to the boil, and adding 3.0 mL of 4% aqueous H2O2 dropwise. After gentle reflux for 1.5 h, the mixture was checked again by TLC (Rf = 0) indicating reaction completion. Removal of all volatiles, dissolving in 10 ml CH2Cl2 and drying overnight with Na2SO4, filtering and evaporating, left a dry solid. Recrystallization from mixed solvents of 5 ml heptane and 2 ml CH2Cl2 at the boil produced colourless blocks on cooling, recovered by slow evaporation to afford 0.06 g (II)b). 1H NMR (CDCl3): δ 2.312 ; 7.144 ; 7.282 . 13C NMR (CDCl3): δ 21.47 ; 129.74 ; 132.67 ; 133.67 ; 138.16 . 31P NMR (CDCl3): δ +29.73, s (satellites: 1JPC = 102.6 Hz).Trisphosphine oxide [381212-20-0], (II)fR = 0.38 in 1:9 ethyl acetate/hexanes) and 3.0 ml of 4% aqueous H2O2. 1.5 h gentle reflux also sufficed for reaction completion (TLC: fR = 0). A similar workup and recrystallization procedure afforded colourless plates by slow evaporation, 0.08 g (II)b). 1H NMR (CDCl3): δ 2.282 ; 3.747 ; 7.311 . 13C NMR (CDCl3): δ 16.37 ; 59.75 ; 127.84 ; 131.41 ; 132.81 ; 160.09 . 31P NMR (CDCl3): δ +28.49, s (satellites: 1JPC = 105.8 Hz).Trisphosphine oxide [540743-36-0], (IV)Uiso(H) = 1.5Ueq(C) for methyl and C—H = 0.95 Å and Uiso(H) = 1.2Ueq(C) for aromatic H atoms.Crystal data, data collection and structure refinement details are summarized in Table 410.1107/S2056989018007831/hb7751sup1.cifCrystal structure: contains datablock(s) I, II, III, IV. DOI: 10.1107/S2056989018007831/hb7751Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751Isup6.molSupporting information file. DOI: 10.1107/S2056989018007831/hb7751IIsup3.hklStructure factors: contains datablock(s) II. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751IIsup7.molSupporting information file. DOI: 10.1107/S2056989018007831/hb7751IIIsup4.hklStructure factors: contains datablock(s) III. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751IIIsup8.molSupporting information file. DOI: 10.1107/S2056989018007831/hb7751IVsup5.hklStructure factors: contains datablock(s) IV. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751IVsup9.molSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751Isup10.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751IIsup11.cmlSupporting information file. DOI: Click here for additional data file.10.1107/S2056989018007831/hb7751IIIsup12.cmlSupporting information file. DOI: 10.1107/S2056989018007831/hb7751sup13.pdfNMR data  for compounds (II) and (IV). DOI: 1845429, 1845428, 1845427, 1845426CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Fe-1 has been thoroughly characterized in the solid state by single-crystal X-ray diffraction (SXRD), powder X-ray diffraction (PXRD), IR spectroscopy, and elemental analysis as well as in solution by UV–Vis spectroscopy. The crystal structure of Fe-1 reveals π–π-interactions between the aromatic systems of the unconventional 2,3-DAP counter cation. POM-protein interaction studies using SDS-PAGE revealed a non-proteolytic behavior of Fe-1 towards Human Serum Albumin (HSA) as a model protein.The novel iron-substituted Krebs-type polyoxotungstate (CThe online version of this article (10.1007/s00706-019-2381-5) contains supplementary material, which is available to authorized users. Polyoxometalates (POMs)  represenAmong the POM family, transition metal substituted POMs (TMSPs) represent the largest group, mainly counting the subgroup of Sandwich-type POMs. Sandwich-type POMs, which are generally composed of two lacunary building blocks linked by a belt of heteroatoms, can be further divided into various subgroups, among them the Krebs-archetype.9O33]9− [2(H2O)6(WO2)2(β-SbW9O33)2](14 − 2n)−  were reported by Krebs and co-workers in 1997 9− . The fir in 1997 , exhibit in 1997 . As Kreb in 1997 .12N4H11)4Na2H5[(Fe(H2O)3)2((FeO2)0.5(WO2)0.5)2(β-SbW9O33)2] (Fe-1) has been prepared. Herein, we report on the synthesis and thorough characterization of the novel Fe-substituted Krebs-type Sandwich tungstoantimonate Fe-1. Regarding the scarce number of studies on the POM-protein interactions of the Krebs-POM archetype  pH 5.5 to ensure a stable more accessible acidic conformation of the model protein  was prepared according to the literature procedure reported by Bösing et al.  (0.05 mmol) in 20 cm3 aqueous sodium acetate buffer , 81 mg FeCl3·6 H2O (0.3 mmol) was added. The resulting orange reaction mixture was stirred at 70 °C for 10 min. ortho-Phenylenediamine  was added to the reaction solution and the mixture was stirred for further 60 min at 85 °C. A color change from orange to dark red over the time period of 60 min was noticed. Dark red crystal plates of Fe-1 were obtained upon cooling the filtered reaction mixture to room temperature and further evaporation at 18 °C gave a total yield of 60% based on W after 3 days. IR (ATR): −1.To a stirred solution of 215 mg NaSupplementary material 1 (DOCX 80 kb)Supplementary material 2 (PDF 232 kb)Below is the link to the electronic supplementary material.


Dear Editor,2/FiO2) by re-expanding the collapsed lung with consequent benefits from mechanical ventilation interruption [We read with great interest the work by Razazi et al.  recentlyrruption . Howeverrruption  and proprruption  is validrruption . This merruption , 5 overerruption , other arruption  have fourruption  is up to1,2,3, MD, Armand Mekontso Dessap1,2,3, MD, PhDKeyvan Razazi1 AP-HP, Hôpitaux universitaires Henri Mondor, DHU A-TVB, Service de Réanimation Médicale, Créteil, 94010 France;2 Université Paris Est Créteil, Faculté de Médecine de Créteil, IMRB, GRC CARMAS, Créteil, 94010, France.3 Unité U955 , INSERM, Créteil, France.n = 41, 51%) or right side . We fully agree with the need for an urgent standardization of the method to assess pleural effusion volume. This point was not mentioned in the international recommendations of lung ultrasound [We thank Dr. Vetrugno and Dr. Bove for their interest and positive appreciations of our study “Pleural effusion during weaning from mechanical ventilation: a prospective observational multicenter study” . Vetrugntrasound .


Correction to: Antimicrob Resist Infect Controlhttps://doi.org/10.1186/s13756-018-0305-5Safety analysis sub-section of the results regarding the reported mean creatinine level. The correct mean creatinine level was 42.09 ± 18.54 μmol/L at 48 hours after polymyxin B injection instead of the reported 41.09 ± 11.46 μmol/L.The original article  contains


In the crystal, molecules are linked by C—H⋯π interactions between the bromophenyl and fluorophenyl rings of molecules, resulting in a two-dimensional layered structure parallel to the ab plane.In the title compound, the 3-bromophenyl and 4-fluorophenyl rings, linked  15H10BrFO, the molecular structure consists of a 3-bromophenyl ring and a 4-fluorophenyl ring linked via a prop-2-en-1-one spacer. The 3-bromophenyl and 4-fluorophenyl rings make a dihedral angle of 48.90 (15)°. The molecule has an E configuration about the C=C bond and the carbonyl group is syn with respect to the C=C bond. In the crystal, molecules are linked by C—H⋯π interactions between the bromophenyl and fluorophenyl rings of molecules, resulting in a two-dimensional layered structure parallel to the ab plane. The molecular packing is stabilized by weak Br⋯H and F⋯H contacts, one of which is on the one side of each layer, and the second is on the other. The intermolecular interactions in the crystal packing were further analysed using Hirshfeld surface analysis, which indicates that the most significant contacts are Cl⋯H/H⋯Cl (20.8%), followed by C⋯H/H⋯C (31.1%), H⋯H (21.7%), Br⋯H/H⋯Br (14.2%), F⋯H/H⋯F (9.8%), O⋯H/H⋯O (9.7%).In the title compound, C The bond lengths and angles are comparable with those found in the related compounds (2E)-3-(3-chlorophenyl)-1--prop-2-en-1-one -3-(3-bromo-4-fluorophenyl)-1-prop-2-en-1-one -3--1-prop-2-en-1-one -1-(3-bromophenyl)-3-prop-2-en-1-one -3-(2-bromophenyl)-1-prop-2-en-1-one  and exterior (de) atom and the van der Waals radii of the atoms . H⋯H contacts are disfavoured when the number of H atoms on the molecular surface is large. The Br⋯H/H⋯Br and F⋯H/H⋯F contacts  = 1.2Ueq(C). Owing to poor agreement between observed and calculated intensities, thirteen outliers  I. DOI: 10.1107/S2056989018018418/xu5955Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018018418/xu5955Isup3.cmlSupporting information file. DOI: 1036740CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information: