Leishmania braziliensis, responsible for cutaneous leishmaniasis, is the most prevalent species in Brazil. Superoxide dismutase (SOD) belongs to the antioxidant pathway of the parasites and human host. Despite the differences between SOD of Leishmania braziliensis and human make this enzyme a promising target for drug development efforts. No medicinal chemistry effort has been made to identify LbSOD inhibitors. Herein, we show that thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) can be employed as primary and secondary screens to achieve this goal. Moreover, we show that thiazole derivatives bind to LbSOD with micromolar affinity.Leishmaniasis is considered as one of the major neglected tropical diseases due to its magnitude and wide geographic distribution.  Besides, MORENO-VIGURI et al. (2016) have reported that arylaminoketone derivatives inhibit FeSOD from trypanosomatids but have little effect over human CuZnSOD.The superoxide dismutase from L. braziliensis is a promising target for drug design efforts. However, no medicinal chemistry effort to target this protein has been carried out before. Aiming at overcoming this knowledge-gap, this work describes how thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) can be employed as primary and secondary HTS-friendly alternatives to indirect-kinetic assays that measure SOD catalytic activity. TSA relies on environment-sensitive fluorophores that reversibly binds to exposed hydrophobic regions when protein unfolds due to temperature increaseL. braziliensis FeSOD.These results suggest FeSOD from F254) with visualization by UV light 254 nm in the appropriated system for each compound. The Infrared spectra  were recorded on a Bruker IFS66 spectrophotometer , the wave numbers were given in cm−1 and are uncorrected. The 1H NMR and 13 C NMR spectra were recorded on a VARIAN VNMRS 400-MR , 400 MHz for 1H and 75.4 MHz for 13 C. The 1H spectra were recorded in DMSO-d6 while 13 C spectra were recorded in CDCl3 and DMSO using tetramethylsilane (TMS) as the internal standard. The peak and abbreviations were used to indicate multiplicity: s (singlet), d (doublet), dd (double doublet), ddd , t (triplet), and m (multiplet). The chemical shifts were reported in δ units and the coupling constants (J) were reported in Hertz. Mass spectra were recorded on a Varian MAT 711 spectrometer  70 eV electron impact.Chemical reagents and solvents were purchased from Sigma-Aldrich  with analytical grade purity. Melting points were determined in open capillaries on a Buchi apparatus and are uncorrected. Thin-layer chromatography (TLC) was carried out on aluminium-supported silica gel plates (Merck 60 −3 mol) of thiourea dissolved in 15 ml of methanol followed by the addition of 0.513 g (2.3 × 10−3 mol) of substituted acetophenone halide dissolved in 20 ml methanol, to form a white suspension. The reaction was left for 15 min at room temperature under magnetic stirring. After that, the mixture was dissolved in 0.6 ml of HCl (dropwise). The reaction was heated to 90 °C in an oil bath, the pH adjusted between 4 and 5. The reaction time was 2 h. The reaction was monitored by thin layer chromatography. The product was ice-cooled and filtered. The compound was purified by crystallization from methanol.The 2-aminothiazole derivatives were obtained from the mixture of equimolar amounts 0.175 g  MP 216–217 °C. Infrared: 3390–3317 (NH2), 3110 (C–H) 1620 (C = N), 1570 (C = C Ar),1185 (C–N), 1058(C–S–C), 815 (Ar) 739 (Ar–Br). 1HNMR: 6.39 (s 2H); 6,99 (s 1H); 7.77–7.74 (d 2H) J = 8.50 Hz; 7.76–7.71 (d 2H) J = 8.50 Hz. 13HNMR: 121.5, 129.5, 135.6, 129.5, 121.5, 123.4, 136.6, 124.8, 115.0, 125.6. +HRMS, calculated: 252.9561, found: 252.9558.Chemical formula: C9H7BrN2S, molecular weight: 255.1343, yield 92%, Rf = 0.47 . MP 202–204 °C. Infrared 3389–3520 (NH2), 3116 (C–H) 1614 (C = N), 1575 (C = C Ar),1180 (C–N), 1067 (C–S–C), 817 (Ar) 740 (Ar–Br). 1HNMR: 8.33 ; 8.10 ; 8.12 (s 1H CH); 7.76 (d 1H Ar J = 8.1); 7.63 ; 7.66 . 13HNMR: 148; 124; 126; 140; 178; 161; 130; 132; 129; 137; 163. +HRMS, calculated: 255.9493, found: 253.9513.Chemical formula: C−3 mol of 4–-2-aminothiazole and 2.3 × 1.0 −3 mol of substituted benzaldehydes. The substituted benzaldehydes were dissolved in 15 ml of MeOH under stirring at room temperature. After that, 0.08 ml of HCl (conc.) dissolved in MeOH was added dropwise. After 5 min. under cold stirring, the starting material -2-aminothiazole previously dissolved in 15 ml of MeOH was added dropwise. The pH was then adjusted between 4 and 5 and the reaction was warmed to 75 °C and refluxed in an oil bath for 2–24 h. The progress of the reaction was monitored by TLC. At the end, the solvent was reduced to one-half volume and ice-cooled. The precipitate was filtered off and recrystallized from the appropriate solvent.The 4–(4-bromophenyl)-thiazol-2-imine-arylidene-substituted and 4–(3 or 4-nitrophenyl)-thiazol-2-imine-arylidene-substituted derivatives were synthesized from the mixture of equimolar amounts 2.3 × 1.016H9Cl2N3O2S, MW 378.2326, Rdt 55%, Rf = 0.45 . MP 218.6–220.0 °C. Infrared: 3109 (C–H), 1610 (C = N), 1575 (C = C Ar),1189 (C–N), 1055(C–S–C), 810 (Ar), 730 (Ar–Cl). 1HNMR: 8.35 ; 8.10 , 8.11 (s 1H CH–S); 8,28 (s 1H CH = N); 8.27 (s 1H Ar); 7.63 ; 7.67  13CNMR: 124, 130, 135, 129, 121, 124, 111, 152, 173, 179, 133, 131, 137, 136, 130, 129. +HRMS, calculated: 376.9793, found: 378.9633.Chemical Formula: C16H10BrN3O2S, MW 388.2385, Rdt 45%, Rf = 0.54 : MP 262 °C. Infrared 3112 (C–H), 1613 (C = N), 1575 (C = C Ar), 1179 (C–N), 1061(C–S–C), 815 (Ar), 737 (Ar–Br). 1HNMR: 8.33 ; 8.10 , 8.11 (s 1H CH); 7.74 (d 1H Ar J = 8.1) 7.63 ; 7.67  13CNMR: 148; 126; 127; 140; 175; 3. 161; 129; 133; 129; 137; 162. +HRMS, calculated: 386.9677, found: 388.9557.Chemical Formula: C16H9Br2N3O2S, MW 467.1346, Rdt 70%, Rf = 0.53 : Melting Point = 273 °C. Infrared 3115 (C–H), 1615 (C = N), 1571 (C = C Ar), 1182 (C–N), 1064 (C–S–C), 818 (Ar), 739 (Ar–Br). 1HNMR: 8.33 ; 8.10 ; 8.11 (s 1H CH); 7.74 (d 1H Ar J = 8.1); 7.63 ; 7.67 . 13CNMR: 148; 126; 127; 140; 175; 161; 129; 133; 129; 137; 162. +HRMS, calculated: 464.8782, found: 466.8657.Chemical Formula: C17H13BrN2OS, Molecular Weight: 373.2669. Rdt 70%, Rf = 0.53 : MP 273–274 °C. Infrared: 3114 (C–H), 1615 (C = N), 1576 (C = C Ar), 1180 (C–N), 1063 (C–S–C), 815 (Ar), 740 (Ar–Br). 1HNMR: 8.34 ; 8.10 ; 8.10 (s 1H CH); 7.74 (d 1H Ar J = 8.1); 7.64 ; 7.64. 13CNMR: 147; 124; 126; 141; 175; 160; 1 133; 129; 138;162. +HRMS, calculated: 371.9933, found: 371.9630.Chemical Formula: C16H10BrN3O2S, Molecular Weight: 388.2385, yield 53%, Rf = 0.531 . MP 233–234 °C. Infrared 3115 (C–H), 1615 (C = N), 1575 (C = C Ar), 1181 (C–N), 1066 (C–S–C), 817 (Ar), 740 (Ar–Br). 1HNMR: 8.34 ; 8.11 ; 8.11 (s 1H CH); 7.74 (d 1H Ar J = 8.1); 7.63 ; 7.66 . 13CNMR: 147; 124; 127; 141; 177; 160; 130; 133; 129; 138;163. +HRMS, calculated: 385.9725., found: 386.9678.Chemical Formula: C16H10BrN3O2S, Molecular Weight: 388.2385, yield 55%, Rf = 0.55 . MP 155–156 °C. Infrared 3116 (C–H), 1614 (C = N), 1575 (C = C Ar), 1180 (C–N), 1067 (C–S–C), 817 (Ar), 740 (Ar–Br). 1HNMR: 8.33 ; 8.10 ; 8.12 (s 1H CH); 7.76 (d 1H Ar J = 8.1); 7.63 ; 7.66 . 13CNMR: 148; 124; 126; 140; 178; 161; 130; 132; 129; 137; 163. +HRMS, calculated: 387.9725, found: 385.9688.Chemical Formula: C16H10FN3O2S, Molecular Weight: 327.3329, yield 80%, Rf = 0.6 . MP 182–183 °C. Infrared: 3116 (C–H), 1614 (C = N), 1575 (C = C Ar), 1230 (Ar–F), 1180 (C–N), 1068 (C–S–C), 818 (Ar). 1HNMR: 8.29 ; 8.12 ; 8.11 (s 1H CH); 7.76 ; 7.64 ; 7.67 . 13CNMR: 149; 121; 124; 138; 179; 163; 132; 131; 130; 138; 164. +HRMS, calculated: 326.0525, found: 325.9786.Chemical Formula: C16H10FN3O2S, Molecular Weight: 327.3329, yield 63%, Rf = 0.53 . MP 118.4–119.5 °C. Infrared: 3117 (C–H), 1612 (C = N), 1571 (C = C Ar), 1241 (Ar–F), 1183 (C–N), 1069 (C–S–C), 820 (Ar). 1HNMR: 8.31 ; 8.11 ; 8.09 (s 1H CH); 7.73 (d 1H Ar J = 8.11); 7.64 ; 7.67 . 13CNMR: 149; 121; 124; 137; 179; 168; 133; 131; 129; 138; 162. +HRMS, calculated: 326.3448, found: 325.9581.Chemical Formula: C16H11N3O3S, Molecular Weight: 325.3418, yield 63%, Rf = 0.45 . MP 118.4–119.5 °C. Infrared: 3340 (O–H), 3118 (C–H), 1612 (C = N), 1572 (C = C Ar), 1182 (C–N), 1070 (C–S–C), 820 (Ar). 1HNMR: 8.30 ; 8.09 ; 8.08 (s 1H CH); 7.72 (d 1H Ar J = 8.11); 7.65 ; 7.68 . 13CNMR: 149; 120; 123; 137; 179; 166; 133; 132; 130; 138; 161. +HRMS, calculated: 325.0519, found: 326.0537.Chemical Formula: C16H11N3O3S, Molecular Weight: 325.3418, yield 59%, Rf = 0.46 (Hex/AcOEt 0.5:0.5). MP 187.5 °C. Infrared: 3338 (O–H), 3120 (C–H) 1613 (C = N), 1574 (C = C Ar), 1181 (C–N), 1072 (C–S–C), 821 (Ar). 1HNMR: 8.31 ; 8.08 ; 8.08 (s 1H CH); 7.72 (d 1H Ar J = 8.11); 7.65 ; 7.68 . 13CNMR: 149; 121; 123; 137; 180; 166; 131; 132; 130; 136; 160. +HRMS, calculated: 324.0569, found: 325.9581.Chemical Formula: C17H13N3O2S, Molecular Weight: 323.369, yield 61%, Rf = 0.56 (CH2Cl2/MeOH 0.98:0.02). MP 213–214 °C. Infrared: 3118 (C–H), 1613 (C = N), 1572 (C = C Ar), 1181 (C–N), 1070 (C–S–C), 821 (Ar). 1HNMR: 8.29 ; 8.11 ; 8.08 (s 1H CH); 7.71 (d 1H Ar J = 8.11); 7.64 ; 7.67 . 13CNMR: 149; 121; 123; 137; 179; 167; 133; 132; 131; 138; 160. +HRMS, calculated: 322.3810, found: 324.9580.Chemical Formula: CE. coli BL21 (DE3) cell carrying the pETM11-LbSOD plasmid were cultured at 37 °C/180 rpm in Luria Bertani medium supplemented with 30 μg/mL kanamycin until OD600 reached 0.6–0.9. At this moment, 1 mM  isopropyl-β-D-thiogalactoside (IPTG) was added to the culture and the temperature was reduced to 20 °C. After 16 h, the cells were harvested by centrifugation  and resuspended in lysis buffer  supplemented with 1 mM phenylmethanesulphonylfluoride (PMSF). Next, cells were incubated with Lysozyme 0.5 mg/mL, for 30 min and then disrupted by sonication . These steps were carried out in ice-bath. The soluble fraction was clarified by centrifugation  and then loaded onto a HisTrap HP column , pre-equilibrated with PBS 50 mM, NaCl 100 mM, 20 mM Imidazole pH 7.0.Amicon Ultra centrifugal filters , 4000 rpm at 4 °C.The column was washed with 20 column volumes of PBS 50 mM, 100 mM NaCl, 20 mM Imidazole pH 7.0 and then steps of increasing concentration of imidazole (50–500 mM) were used. Purification was monitored by UV absorbance measurement at 280 nm and the level of protein purity was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) 12%. The protein fraction was dialyzed in PBS 50 mM, NaCl 100 mM pH 7 with − 1 cm−1 at 280 nm calculated using ExPASy ((http://web.expasy.org/protparam/).Protein concentrations were determined spectrophotometrically using a theoretical extinction coefficient of 55775 M Tobacco Etch Virus) protease digestion (1 mg per 20 mg LbSOD), 4 °C overnight. After the proteolysis step, LbSOD was loaded again onto His-Trap column to separate cleaved and uncleaved His-tag LbSOD from TEV protease. The column was pre-equilibrated with PBS 50 mM, NaCl 100 mM pH 7 and then the elution of cleaved LbSOD was performed with 10 column volumes of this same buffer. The purification fractions were analyzed by UV measurement at 280 nm and gel electrophoresis (SDS-PAGE 12%) to confirm purification of the protein. The cleaved His-tag LbSOD was concentrated  to 10 mg/mL and stored in 30% glycerol at −80 °C.The enzyme was subjected to TEV , sealed with transparent capping strips . The plates were centrifuged for 2 min, 2000 rpm, 25 °C. After the plates were heated from 25 to 85 °C in increments of 1 °C per minute and fluorescence signal was monitored with SYPRO Orange® dye, with 492 (excitation) and 610 nm (emission) wavelengths.The assays were carried out in the RT-PCR Applied Biosystems 7500 , in triplicate, using 96-well PCR plate (PCR plates 96 well BioRadftp://ftp.sgc.ox.ac.uk/pub/biophysics).The results obtained by Applied Biosystems 7500 proprietary software (v2.0) were submitted to processing and analysis in Excel 2007 worksheet . Comparisons between Tm of different conditions (ΔTm) were considered as significantly different when p < .05, according to the Kruskal–Wallis test followed by Dunn's post-test for multiple comparisons.The melting temperature (Tm) values were calculated by non-linear fitting of the melting curves to a Boltzmann sigmoidal function, using GraphPad Prism version 5.0 for Windows  in the presence of SYPRO Orange®  (1:100 dilution) in ultrapure water qsp 20 µL. Then, the buffers  and pHs (4–9) were evaluated at a final concentration of 50 mM for each buffer, SYPRO Orange® (1: 100 dilution) and LbSOD. Finally, the influence of DMSO  on the thermal stability of the protein was investigated.Conditions were optimized to LbSOD was evaluated at a single concentration  and an equivalent volume of DMSO was employed as a control (ΔTm = 0.0). Briefly, LbSOD (5 μM) was diluted in 50 mM sodium phosphate buffer (pH 7.0) supplemented with 100 mM NaCl and SYPRO Orange® (1:100 dilution). The 96 well plates were heated from 25 to 85 °C in increments of 1 °C per minute. The results were analyzed as described in the previous section.The effect of the thiazoles derivatives over the melting temperature of LbSOD (2 mg/mL) was incubated with fluorescein-5-isothiocyanate (FITC) dye (10 mg/mL) for 2 h, at 25 °C, with continuous stirring. Then, the solution was loaded on a Hi-Trap HP desalting column , previously equilibrated with 50 mM sodium phosphate buffer (pH 8) and 1.5 column volumes of the buffer were injected. The absorbance of the collected fractions was monitored at 280 nm and 490 nm, so correct to the contribution of dye to Absorbance280nm could be calculated according to Aprotein stands for the protein absorbance after correction; A280 is the absorbance at 280 nm of the purified sample; Amax: maximal dye absorption at 280 nm or correction factor (CF) corresponding to 0.3 to fluorescein.LbSOD was diluted to 5 µM and the influence of putative inhibitors (50 µM) over its fluorescence was measured at 25 °C for 10 min on an Applied Biosystems 7500 RT-PCR machine. The experiments were carried out in triplicate using 96-well PCR plates (PCR plates 96 well BioRad®). Fluorescence data was recorded on the Applied Biosystems 7500 Software v2.0 and then analyzed on GraphPad Prism V5.0 software .Next, LbSOD (20 µM) diluted in sodium phosphate buffer 0.05 M pH 7 with DMSO (5%v/v). The injection syringe was loaded with ligand 400 µM (previously dissolved in the same solution as the protein). Control experiments were carried out with sodium phosphate buffer 0.05 M pH 7 and DMSO (5%v/v) in the sample cell to determine the dilution heat. Injections were started after baseline stability had been achieved. The ligands were titrated in the sample cell through 21 injections of 13 µL and the first injection of 10 µL. The solutions of the sample cell were stirred during the experiment at 200 rpm to ensure mixing.ITC experiments were carried out in a Microcal VP-ITC Model, at 298 K. All the solutions were thoroughly degassed prior to the titrations to avoid the formation of bubbles during the experiment. The reference cell contained distilled water. The sample cell was filled with The heat variation, following each injection of the ligand in the sample cell, were employed to build the titration isotherms, as available in Origin package supplied with the calorimeter.The thermodynamic parameters ΔG (Gibbs free energy), ΔS (entropy), and ΔH  were calculated using the L. infantum FeSOD than human CuZnSODLbSOD inhibitorsPrevious studies have shown that 1,3-thiazole derivatives have leishmanicidal activity1HNMR and 13CNMR absorption of CH = N between 4.92–4.04 and 171.76–168.25 ppm, respectively. The stereochemistry of CH = N bond is assumed to be E, as reported in literature for thiazole Schiff bases derivativesThe starting material 2-aminothiazole-4-substituted Ju-436 and Ju-533 were prepared, as described previously,E. coli Bl21 (DE3) cells were transformed with plasmid LbSOD-pET-M11 and LbSOD expression was performed with IPTG 1 mM at 20 °C for 16 h.6xhistidine tag, which allows the expression of the His-tag fused LbSOD and its purification by affinity chromatography. The purification was based on the interaction of histidine residues with nickel ions immobilized on the chromatographic column (Ni-sepharose) and LbSOD was eluted in a higher concentration of imidazole (500 mM)   . The oveLbSOD was obtained, the next step consisted the identification of potential inhibitors (LbSOD due to the absorption of light at the same wavelength of the assay (320 nm) (data not shown), the thermal shift assays (TSA) and fluorescent protein-labeled assays (FPLA) were employed to evaluate the potential LbSOD inhibitors.Once the pure hibitors . Conside,,LbSOD, buffer, pH, and organic solvent (DMSO), as described next.In general, TSA is used to identify the conditions and molecules that influence protein stabilityLbSOD concentrations  in this range  showed that Propionibacterium shermanha Fe-SOD activity decreases at pH <5 Plasmodium vickey also decreases at pH <6.0,Comparison of is range . Either LbSOD was also investigated. The assessment of different DMSO concentrations suggests that up to 5% (v/v) of DMSO does not significantly affect the thermal stability of LbSOD (p > .05)   and, forLbSOD hit identification campaigns. In order to increase the number of true-positives, it is common practice to employ a counter-screening method, whose end-point is different from the first screening assay. Accordingly, the first screening method described in this work relies on fluorescein-5-isothiocyanate (FITC) covalent binding to the target proteinLbSOD was labeled with FITC, any change in the protein conformation would lead to a change in the fluorescence. In case thiazole derivatives cause a conformational shift that exposes FITC-labeled residues, an increased fluorescence signal would be observed. On the other hand, if ligand binding hides the FITC-labeled residues, a decreased signal is expected. Then, the conformational modification due to thiazole derivatives binding to LbSOD was evaluated at a single concentration. This approach suggests that Ju-450 and Ju-480 are the most promising hits , the change in the fluorescence signal might be low and promising compounds would be discarded. An even more troublesome scenario would be a covalent modification of a residue that is crucial for binding. Considering all these shortcoming of protein covalent labeling, we resorted to thermal shift assays as a secondary counter-screening approach. Although this strategy also depends on a fluorescent label (Sypro-orange®), covalent binding is not an issue anymore.When protein labeling occurs far from the LbSOD (ΔTm ≅ +1.0 °C). However, only Ju-450, Ju-480 have a significant effect over LbSOD thermal stability (p < .05). Taking both results into consideration, Ju-450 and Ju-480 would be considered a true hit.TSA shows that Ju-514, Ju-517, and Ju-546 stabilize p-nitrophenyl moiety at the 4-position of the thiazole ring and increase the fluorescence signal in FLPA. On the other hand, Ju-514 has a p-bromophenyl moiety at the equivalent position and reduces the fluorescence signal in the FLPA. Given the overall similarity between these compounds, we decided to include Ju-514 in subsequent assays to make sure no true ligand was excluded from our study . Hence, structure-activity relationships based on those compounds would be of little help to design potent LbSOD inhibitors. Instead, we decided to focus our efforts on Ju-480, that shows low-micromolar affinity to the macromolecular target (Kd= 4.8 ± 12.5 μM) and employ a label-free method to study its thermodynamic signatureAlthough all evaluated compounds exhibited dose-response profile that are typical of true ligands, Ju-450 and Ju-514 have low affinity to ΔH= −4.22 ± 1.6970 kcal/mol) and entropic  contributions to affinity (LbSOD surface, as was observed in the optimization of HIV-1 protease inhibitors,Once ITC measures the heat released or absorbed upon the ligand-macromolecule interaction, it is possible to directly determine thermodynamic parameters ΔH and ΔSaffinity . The entLbSOD ligands. On the other hand, the combined use of TSA and FPLA assays lead to the identification of Ju-480, a thiazole derivative that has a low-micromolar affinity to L. braziliensis Superoxide dismutase. Label-free methods confirm that Ju-480 binds to LbSOD and suggests that structure rigidification might increase the ligand’s affinity to its macromolecular target.Although TSA is commonly employed for screening putative ligands of promising targets, the results presented in this work highlight that its use alone might lead to the selection of false-positive


Two complex molecules with similar bond lengths and angles are present in the asymmetric unit, each exhibiting a distorted octahedral N4O2 coordination environment around the CdII ion.The crystal structure of [Cd(C 13H18NO3)2(C12H8N2)]·2H2O, contains two crystallographically independent molecules that differ insignificantly in their geometrical parameters. In both, the CdII cation lies on a twofold rotation axis and is coordinated in a distorted octahedral fashion to two monodeprotonated residues of the l-leucine-derived ligand (S)-2-(2-hydroxybenzylamino)-4-methylpentanoic acid (L), as well as to a 1,10-phenanthroline ligand in a κ2N,N′ mode. The former coordinate in an N,O-chelating mode, exhibiting a trans-N,N′ mutual disposition. The phenolic oxygen donor groups remain protonated and do not coordinate to the cation but take part in intra- and intermolecular hydrogen bonds. In the crystal, O—H⋯O hydrogen bonding results in the formation of a three-dimensional network structure. The contribution to the electron density of two disordered water molecules was removed with the SQUEEZE procedure in PLATON . The studied crystal was refined as a two-component inversion twin. The title complex was also characterized by IR and 1H NMR spectroscopic methods.The asymmetric unit of the mononuclear mixed-ligand title complex, [Cd(CSpek 2015. Acta Cr The second molecule also exhibits similar geometrical parameters . All three sets of ligands form five-membered chelate rings. Unlike the essentially planar chelate rings formed by the phenanthroline ligands, the ones involving the l-leucine-derived ligands exhibit a λ-conformation in both complex molecules. The deviations of the carbon atoms from the planes defined by the central atom and donor atoms are 0.258 (6) Å for C7, 0.599 (7) Å for C8, −0.417 (7) Å for C26 and 0.632 (5) Å for C27. In the second molecule, the highest deviations are found to be 0.160 and 0.232 Å for O4 and N4, respectively. The N—Cd—O and N—Cd—N bite angles are 73.01 (13) and 71.2 (2)°, respectively, for the first molecule and 72.40 (14) and 70.8 (2)° for the second. The phenolic O—H group remains protonated and is non-coordinating, albeit participating in an extensive intermolecular hydrogen-bonding network. Intramolecular hydrogen bonds are also found to exist and take place between atoms H2A and O3 as well as between H4 and O6 of the l-leucine-derived ligands. To a minor extent, intramolecular C—H⋯O interactions are also present between a methylene group and O4  Å for the first molecule. The stacking interactions of the second molecule are between C33–C38 rings of two l-leucine-derived ligands L and the C23–C25/C23′–C25′ phenanthroline fragment with a centroid-to-centroid distance of 3.773 (4) Å.In the crystal structure, the complex molecules are linked s Table 1. π–π intet al., 2016II complex with a 2-hydroxybenzyl derivative of an amino acid (refcode WARLIL). In this mononuclear complex, the phenolic and β-carboxylic groups are deprotonated. The N-(2-hydroxybenzyl)-d,l-aspartic acid residue coordinates in an -tridentate mode including the phenolic O atom . These compounds have a Zn2O2 binuclear core, and the ligands also coordinate in an -tridentate manner, with an additional μ2-mode for the phenolic O atom -2-(2-hydroxybenzylamino)-4-methylpentanoic acid (L)l-leucine  and LiOH·H2O  in methanol (25 ml) was stirred for 10 min to dissolve. A methanolic solution of o-salicylaldehyde  was added dropwise to the above solution whereby the colour of the solution turned to yellow. Stirring was continued for 30 min before the solution was treated with NaBH4 , leading to a colourless solution. The solvent was evaporated under reduced pressure, and the resulting solid was dissolved in water. The clear solution was then acidified with diluted HCl (pH ∼5–7). The ligand precipitated as a white solid. The suspension was filtered, and the residue was washed thoroughly with water. The solid was dried in a vacuum desiccator . Because of its poor solubility, the 1H NMR spectrum for the ligand was recorded as the lithium salt of the ligand, prepared by adding 2 equiv. of LiOH·3H2O in CD3OD. 1H NMR Li2L : 0.76  , 0.81  , 1.36  , 1.41 , 1.67  , 3.07  , 3.65  , 3.94 , 6.35  , 6.45  , 6.94 . m/z ; calculated: 242.22, found 242.02. IR  ν(COO)asym 1600 (s), 1593 (s); ν (COO)sym 1393 (m), cm−1.A mixture of 2(phen)]·(H2O)2]Synthesis of ·2H2O. Yield: 60%. [Cd(L)2(phen)]·2H2O: IR  ν(COO)asym 1594, ν(COO)sym 1384, ν 1257. 1H NMR [Cd(L)2(phen)]·2H2O] : 0.6 , 0.7 , 1.3 , 1.5 , 2.7 , 2.9 , 6.6 , 6.4 , 6.6 , 6.1 , 8.0 , 8.1 , 8.7 , 9.1 . ESI–Mass (-ve) at 829.18 . Suitable needle-shaped crystals for X-ray data collection were obtained by slow evaporation of a methanol: DMF (2:1 v:v) solution within a week.A methanolic solution of Cd = 1.5Ueq(O). All C-bound H atoms were positioned geometrically and refined using a riding model with C—H = 0.93 Å and with Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 2PLATON , corresponding to 151 electrons  per unit cell, or 37.75 electrons per one complex molecule. This number agrees with two water molecules. Although not modelled in the refined structure, the two water molecules are included in the formula and other crystallographic data.After unsuccessful attempts to model disordered solvent molecules, their contributions to the diffraction data were removed by using the SQUEEZE routine in 10.1107/S2056989018013877/wm5464sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018013877/wm5464Isup2.hklStructure factors: contains datablock(s) I. DOI: 1534691CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


We assessed the efficacy and safety of avelumab, an anti-programmed death ligand 1 (PD-L1) antibody, in patients with previously treated metastatic adrenocortical carcinoma (mACC).In this phase 1b expansion cohort, patients with mACC and prior platinum-based therapy received avelumab at 10 mg/kg intravenously every 2 weeks. Continuation of mitotane was permitted; however, mitotane levels during the study were not recorded. Tumor response was assessed by Response Evaluation Criteria In Solid Tumors v1.1.n = 12) or PD-L1− (n = 30) tumors (≥5% tumor cell cutoff), ORR was 16.7% vs 3.3% (P = .192). Treatment-related adverse events (TRAEs) occurred in 82.0%; the most common were nausea (20.0%), fatigue (18.0%), hypothyroidism (14.0%), and pyrexia (14.0%). Grade 3 TRAEs occurred in 16.0%; no grade 4 to 5 TRAEs occurred. Twelve patients (24.0%) had an immune-related TRAE of any grade, which were grade 3 in 2 patients (4.0%): adrenal insufficiency (n = 1), and pneumonitis (n = 1).Fifty patients received avelumab and were followed for a median of 16.5 months. Prior treatment included ≥2 lines in 74.0%; mitotane was continued in 50.0%. The objective response rate (ORR) was 6.0% . Twenty-one patients (42.0%) had stable disease as best response . Median progression-free survival was 2.6 months , median overall survival (OS) was 10.6 months , and the 1-year OS rate was 43.4% . In evaluable patients with PD-L1+  contains supplementary material, which is available to authorized users. ZNRF3), telomerase reverse transcriptase (TERT), protein kinase cAMP-dependent type I regulatory subunit alpha (PRKAR1A), ribosomal protein L22 (RPL22), telomeric repeat binding factor 2 (TERF2), cyclin E1 (CCNE1), and neurofibromin 1 (NF1), may have a major role in the etiology of ACC ), AE (n = 5 [10.0%]), death (n = 3 [6.0%]), withdrawal of consent (n = 2 [4.0%]), protocol noncompliance (n = 1 [2.0%]), and other (n = 2 [4.0%]).Between September 9, 2014, and the data cutoff date of December 31, 2016, 50 patients were enrolled in 6 countries. Median age was 50 years  (Table n = 13), 2 (n = 18), or ≥ 3 (n = 19) prior lines of systemic therapy since diagnosis, ORR was 15.4%, 5.6%, and 0%, respectively. Response was ongoing in 1 patient, who had a duration of response of 19.4 months at data cutoff  Table . Of the off Fig. . One patoff Fig. .Table 2CMedian PFS was 2.6 months , and PFS rates at 6 months and 1 year were 20.9%  and 8.7% , respectively Fig. . Using in = 12) or PD-L1– (n = 30) tumors based on a 5% PD-L1 expression cutoff, ORR (95% CI) was 16.7% (2.1% to 48.4%) vs 3.3% (0.1% to 17.2%) (P = .192), median PFS was 5.5 (1.3 to 8.2) vs 1.7 (1.4 to 4.0) months , and median OS was 14.4 (7.4 to 14.4) vs 10.6  months  . Analyses of higher tumor cell PD-L1 cutoffs and PD-L1 expression on tumor-infiltrating immune cells were not informative because few patients had PD-L1+ tumors (0 to 5 patients).PD-L1 expression was evaluable in tumor samples from 42 patients (84%). In patients with PD-L1+ (9]) Fig. . Findingn = 10; 20.0%), fatigue , hypothyroidism , and pyrexia . Five patients (10%) had an infusion-related reaction; all were grade 1 or 2. Eight patients (16.0%) had a grade 3 TRAE and no patient had a grade 4 or 5 TRAE. Of patients with or without concomitant mitotane treatment during avelumab treatment (n = 25 in each group), 6 (24.0%) vs 2 (8.0%) patients had a grade 3 TRAE, respectively. Increase in alanine aminotransferase level (n = 2) was the only grade 3 TRAE that occurred in more than 1 patient; both patients received concomitant mitotane. Treatment-related grade 3 elevations in liver enzyme levels occurred only in patients who received concomitant mitotane. Twelve patients (24.0%) had an immune-related TRAE of any grade; those occurring in ≥1 patient were hypothyroidism (n = 7), adrenal insufficiency (n = 3), and pneumonitis (n = 2). Two patients (4.0%) had a grade 3 immune-related TRAE: adrenal insufficiency (n = 1) and pneumonitis (n = 1). Thirty-two patients (64.0%) had an SAE; in 4 patients (8.0%), the SAE was treatment related: pneumonitis (n = 1), adrenal insufficiency and transaminitis (n = 1), thyroiditis (n = 1), and pyrexia (n = 1). Six patients (12.0%) had AEs that led to death, but none were treatment related: disease progression (n = 4), failure to thrive (n = 1), and respiratory failure (n = 1).All 50 patients (100.0%) had an AE of any grade; 41 patients (82.0%) had a treatment-related AE (TRAE) Table . The mosn = 2) or 2 (n = 1) prior lines of treatment. This suggests that the activity of avelumab might be greatest in patients with limited pretreatment, although the small patient numbers in this study prevent any definitive conclusion. Reasons for improved response in patients with less pretreatment may include a smaller tumor burden, decreased proportion of treatment-resistant cells within the tumor, and reduced immunosuppression associated with multiple prior lines of chemotherapy. Although the ORR and median PFS were modest in this heavily pretreated population, the disease control rate was 48.0%, median OS was 10.6 months, and the 1-year OS rate was 43.0%. No association was seen between concomitant mitotane treatment and clinical activity of avelumab, although the absence of detailed patient data relating to ongoing mitotane treatment, including drug levels, is a limitation of the study.In this single-arm phase 1b study, which to our knowledge is the largest prospective trial of a checkpoint inhibitor in mACC, avelumab showed antitumor activity with an acceptable safety profile in a platinum-treated population. Three patients (6.0%) had an objective response, including patients with PD-L1+ and PD-L1− tumors, and all of whom had received only 1 , the disease-control rate was 58.3% vs 31.4% (P < .001), median PFS was 5.0 vs 2.1 months (P < .001), median OS was 14.8 vs 12.0 months (P = .07), and serious AEs occurred in 58.1% vs 41.6% of patients [n = 28), the ORR was 7.1%, disease control rate was 46.4%, median time to progression was 5.3 months, and median OS was 9.8 months; grade 3/4 AEs were leukopenia (21.4%), thrombocytopenia (3.5%), and mucositis (3.5%) [Current treatment options for patients with mACC are highly limited. In the first-line setting, response rates with mitotane monotherapy are estimated to be approximately 10% to 30%, although data from prospective trials are lacking . In a rapatients . In a phs (3.5%) . Thus, ts (3.5%) . This suP = .30), and median OS was 323 vs 356 days (P = .77) [Targeted molecular therapies have been explored in patients with previously treated advanced ACC, but efficacy has been poor or nonexistent. In a phase 3 trial of linsitinib (insulin-like growth factor receptor 1 and insulin receptor inhibitor) vs placebo in 139 patients with previously treated ACC, of whom 55% had received prior cisplatin, ORR was 3.3% vs 0%, disease control rate was 32.2% vs 34.7%, median PFS was 44 vs 46 days (P = .77) . TreatmeP = .77) . In otheP = .77)  had an oP = .77) . In a stIn patients with mACC previously treated with chemotherapy, avelumab had moderate clinical activity either as monotherapy or with concurrent mitotane (50% of patients), particularly in those with limited pretreatment or PD-L1+ tumors, and with an acceptable safety profile. These findings provide a rationale for studies of avelumab-based combination treatment in patients with advanced ACC.Additional file 1:Table S1. Confirmed best objective response based on modified immune-related response criteria. Table S2. Antitumor activity based on PD-L1 expression on tumor cells (1% and 5% cutoffs) in evaluable patients (n = 42). Figure S1. Computed tomography scans of a patient with adrenocortical carcinoma who experienced a long-term tumor response with avelumab treatment. The patient had a partial response documented at the second assessment (week 13) and remained on treatment without progression until last follow-up. Figure S2. (A) Progression-free survival (PFS) and (B) overall survival (OS) based on programmed death ligand 1 (PD-L1) expression on tumor cells (1% cutoff) in evaluable patients (n = 42). ACC, adrenocortical carcinoma; NE, nonevaluable. (DOCX 498 kb)


H-benzopyran-3-carbonitrile is reported. Hirshfeld surface analysis was performed on the molecule and frontier orbitals were investigated with density functional theory calculations.The crystal and molecular structures of the pyran derivative 2-amino-4-(2-chlorophenyl)-7-hydroxy-4 16H11ClN2O2, is planar [maximum deviation = 0.079 (2) Å] and is almost perpendicular to the chlorophenyl ring [dihedral angle = 86.85 (6)°]. In the crystal, N—H⋯O, O—H⋯N, C—H⋯O and C—H⋯Cl hydrogen bonds form inter- and intramolecular interactions. The DFT/B3LYP/6-311G method was used to determine the HOMO–LUMO energy levels. The molecular electrostatic potential surfaces were investigated by Hirshfeld surface analysis and two-dimensional fingerprint plots were used to analyse the intermolecular interactions in the molecule.The benzopyran ring of the title compound, C The pyran ring is a core unit in benzopyrans, chromones, flavanoids and coumarins. Numerous naturally-occurring compounds containing pyrans and benzopyrans show fascinating therapeutic activities, which include their use as antimicrobial -7-hydroxy-4P21/c. The benzopyran and chlorophenyl rings in the molecule are planar, as confirmed by the puckering parameters  Å, θ = 105.6 (11)° and φ = 349.9 (14)°  Å. The sum of the bond angles around atom N1 of the pyran ring is in accordance with the ii interaction leads to the formation of a C(10) chain running along the a axis. The molecules are also linked by pairs of intermolecular N1—H1A⋯O2i and O2—H2⋯N2ii hydrogen bonds, forming inversion dimers with et al., 1995i hydrogen bonds, forming b-axis direction, as shown in Fig. 3The packing of the molecules in the unit cell is stabilized by strong intermolecular C—H⋯O, O—H⋯N and N—H⋯O hydrogen bonds Table 1. The O2—fs Fig. 2  were calculated using the DFT/B3LYP/6-311G basis set implemented in the i.e. EHOMO and ELUMO, are −6.354 and −2.712 eV, respectively, and the HOMO–LUMO energy gap is 3.642 eV. The title compound has a small frontier orbital gap, hence the molecule has high chemical reactivity and low kinetic stability.The HOMO of the title molecule is localized on the entire molecule except for the chlorobenzene ring, while the LUMO is located on the whole molecule. However, the HOMO-1 is localized on the entire molecule, with the LUMO+1 confined to the chlorobenzene and benzopyran rings, except for the amino substituent. The DFT study shows that the FMO energies, et al., 2007CrystalExplorer17  surface resolution and are shown as blue and red regions around the atoms related with positive (hydrogen-bond donors) and negative (hydrogen-bond acceptors) electrostatic potentials, respectively.Hirshfeld surface analysis -7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile hemihydrate  in a 250 ml round-bottomed flask. The resulting solution was refluxed for about 2 h. The progress of the reaction was monitored by thin-layer chromatography using silica gel-G plates. After product formation, the reaction mixture was kept in a refrigerator overnight. The solid mass that settled was filtered off by suction and washed well with a mixture of methanol and water, and finally dried in air. The resulting crude solid was recrystalized from methanol giving a white solid. The purified sample was recrystallized from 1,4-dioxane using the slow-evaporation method (m.p. 250–255 °C).A mixture of 2-chlorobenzaldehyde , malononitrile  and resorcinol  in water (150 ml) was added to a 10% aqueous KUiso(H) = 1.5Ueq(C) for methyl H atoms and 1.2Ueq(C) otherwise.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019013537/sj5577sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989019013537/sj5577Isup2.hklStructure factors: contains datablock(s) I. DOI: 1873687, 1873687CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The title compound was synthesized by a one-pot Mannich condensation reaction. In the crystal, centrosymmetric dimers are linked into layers parallel to (011) by N—H⋯Cl and C—H⋯Cl hydrogen bonds. A Hirshfeld surface analysis indicates that O—H (20.5%) interactions make the largest contribution to the crystal packing. 31H46NO7+·Cl−, was synthesized by a one-pot Mannich condensation reaction. In the molecule, the piperidinone ring adopts a chair conformation, and the trimethoxy-substituted benzene rings and octyl chain are arranged equatorially. In the crystal, centrosymmetric dimers are linked into layers parallel to (011) by N—H⋯Cl and C—H⋯Cl hydrogen bonds. A Hirshfeld surface analysis indicates that the most important contributions for the crystal packing are O⋯H (20.5%) interactions followed by C⋯H (7.8%), Cl⋯H (5.5%), C⋯C (1.2%), C⋯O (0.5%) and Cl⋯O (0.4%) interactions.The title compound, C The octyl chain at C3, and the trimethoxy-substituted benzene rings attached at C2 and C6 are equatorially oriented. The trimethoxy benzene rings C7–C12 and C13–C18 form a dihedral angle of 73.91 (5)°, and are tilted with respect to the mean plane of the piperidone ring by 59.42 (4) and 78.54 (6)°, respectively. The C13—C2—C3—C22 and O4—C4—C3—C22 torsion angles are 56.36 (17) and −11.0 (2)°, respectively.In the molecule of the title compound Fig. 1, the hetIn the crystal, centrosymmetrically-related molecules are linked into dimers through pairs of N—H⋯O hydrogen bonds Table 1 forming ne Fig. 2.dnorm -3,5-dimethylpiperidin-4-one , dried and redissolved in ethanol. Crystals suitable for single-crystal X-ray diffraction analysis were obtained on slow evaporation of the solvent at room temperature.The title compound was synthesized according to the procedure given in literature  = 1.2Ueq(C) or 1.5Ueq(C) for methyl H atoms. A rotating model was used for the methyl groups. The N-bound hydrogen atoms were located in a difference-Fourier map and freely refined.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018008125/rz5236sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S2056989018008125/rz5236Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989018008125/rz5236Isup3.cmlSupporting information file. DOI: 1846705CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


AbstractPambolus (Phaenodus) shujaisp. nov., and Parachremylustrachysisp. nov., of braconid wasps are described as new to science. Parachremylustrachysisp. nov., is reared from larvae of the leaf miner Trachys sp.  on Corchorus sp. (Wild Jute Plant). A new species of Pambolus Haliday along with two known species is also recorded. A key to the Indian species of Pambolus is also provided. Diagnoses with morphological characters and illustrations are provided.Two new species viz.,  Hymenoptera, Braconidae) represent an important insect group of natural parasitoids which have an efficient role in biological control programs  and Phaenodus  (Phaenodus is often treated as a separate genus (see Pambolus is cosmopolitan in distribution and is particularly diverse in the Neotropics (P. (Phaenodus) ignarus Papp, P. (Ph.) topali Papp and P. (Ph.) ruficeps Belokobylskij are reported from India  . Phaenodenus see . The genotropics . Of the otropics  of whichom India . All speParachremylus Granger is disputed, either included in the subfamily Exothecinae  or more traditionally in the subfamily Hormiinae , P.seyrigi Granger and P.temporalis Belokobylskij .The systematic position of the genus ormiinae . The genobylskij . In the .litchi  were reaPambolus, and descriptions by Parachremylus species. We followed OOL – ocello-ocular line ; POL – post-ocellar line ; AOL – anterior-ocellar line ; ØOD – diameter of an ocellus; T1 – First metasomal tergite; F1 – First antennal flagellomere. All descriptions, measurements, and photographs of wings and body parts were made under a Zeiss Discovery V20 stereo zoom microscope while scanning electron microscope (SEM) photomicrographs were taken using a LEO 435VP SEM. The specimens have been deposited in the Insect Collection section of the Department of Zoology, Aligarh Muslim University, Aligarh, India (ZDAMU).The specimens were collected from northern Uttar Pradesh in order to study the biodiversity and conservation of parasitoid wasps in the northern region of India. The subfamily keys of Taxon classificationAnimaliaHymenopteraBraconidaePappF06045B2-20E0-5BCC-AF1A-D0FFE5C2B1ABPambolus (Phaenodus) ignarus Papp, 1996: 46; ZDAMU)”. 1♀, “INDIA: Uttar Pradesh, Aligarh, 09.X.01; Coll. Zubair Ahmad (ZDAMU)”.2 females, “INDIA: Uttar Pradesh, Aurriya, 23.IX.03; coll. M Shamim (Pambolus (Ph.) ignarus is known among all the Oriental species by its smaller size and almost absence of notauli, which is hardly impressed on the anterior part of mesoscutum and without any crenulation. A brief diagnosis is as follows: body length 1.8 mm long; forewing length 1.9 mm; ground color of head brownish yellow; meso- and metasoma rusty brown; antennae unicolor (yellowish brown); head in dorsal view less transverse 1.7 × as broad as long; eye 4.0 × as long as temple; antenna nearly 2.0 × as long as body and with 25 segments; F1 5.0 × as long as broad apically; face polished; notauli hardly distinct on disc of mesonotum; mesonotum finely granulose; propodeal spines short, as long as half of third tarsomere of hind tarsus; forewing vein r arising beyond middle of pterostigma; ovipositor sheath as long as hind basitarsus.Unknown.India: Karnataka, Uttar Pradesh .Taxon classificationAnimaliaHymenopteraBraconidaeAhmadsp. nov.F9F4D8BB-FE75-5BE0-BCC2-3CEB2F475469http://zoobank.org/D6EE6537-36D1-4F99-B972-71C77A6D9243Holotype, female, “INDIA: Uttar Pradesh, Etawah, 13.IV.02; coll. M Shamim (ZDAMU)”. Paratypes, 3 females with same data as holotype.Pambolusshujai Ahmad sp. nov., is closely related to Pambolusignarus Papp. However, it differs from P.ignarus in having (1) antennae with F17–F29 creamish-white (antennae yellowish brown in P.ignarus); (2) POL: ØOD: OOL = 2:1.5:6 (POL: ØOD: OOL = 2: 2: 5 in P.ignarus); (3) antennae as long as body (antennae 2.0 × as long as body length in P.ignarus); (4) propodeal spines present at the middle of propodeum, length of spine 0.4 × as long as fore basitarsus, 0.3 × the distance between them ; (5) hind basitarsus 1.2 × as long as tarsal segment 2–4 combined ; (6) forewings distinctly shorter than the body (forewings distinctly longer than the body P.ignarus). (7) pterostigma 3.0 × as long as wide (pterostigma 5 × as long as wide P.ignarus). The new species also differs from other Indian species, P.ruficeps Belokobylskij, by having (1) vertex finely granulates around ocelli, smooth elsewhere (rugulose in P.ruficeps); (2) forewing veins r, 2RS, 3RSa thickened ; (3) antennae as long as body (antennae 1.5–1.7 × as long as body length in P.ruficeps); (4) face smooth (face reticulate rugulose in P.ruficeps).The new species Female, body length: 2.8 mm; forewing: 2.3 mm.Head. Antennae 29 segmented, almost as long as body; scape 1.6 × as long as wide, F1 3.5–4.0 × as long as wide, head transverse, ca. 2.0 × as wide as long in dorsal view, temple smooth, distinctly widening ventrally, widest distance from eyes 2.0 × as long as eye dorsally; AOL: POL: ØOD: OOL =1:2:1.5:6; malar space 3.5 × basal width of mandible; face sparsely setose, smooth, 1.3 × as wide as long; clypeus smooth, distinctly separated from face, slightly convex, 3.5 × as wide as long; hypoclypeal depression elliptical almost 3.0 × as wide as long medially; frons strigose with few setae; vertex finely granulate around ocelli, rest smooth.Mesosoma. Mesosoma 1.8 × as long as wide in dorsal view, 2.0 × as long as high in lateral view; pronotum small and sparsely setose; median and lateral lobes of mesoscutum granulate, sparsely setose; notauli poorly developed anteriorly, distinct posteriorly with some longitudinal carinae; scutellar sulcus deep and broad with four crenulae, 0.6–0.7 × as long as scutellum, scutellum polished with fine setae laterally and posteriorly, side of scutellum concave with longitudinal striations, metanotum almost at the same level, longitudinally striated, sparsely setose; propodeum with strong areolation, areola elongate, with a pair of spines, present at the middle of propodeum; propodeal spine 0.4 × as long as fore basitarsus and 0.3 × the distance between them; propleuron anteriorly granulate, posteriorly rugose, sparsely setose; mesopleuron anteriorly rugose, otherwise smooth and polished; episternal scrobe deep and isolated, crenulate at the margins.Wings. Forewing 2.7 × as long as wide, 0.8–0.9 × as long as body, 2.7 × as long as hind tibia; pterostigma 3.0 × as long as wide, 0.8 × length of R1a, r arising from its middle; r about as long as width of pterostigma; second marginal cell of moderate size; 3RSa 0.9 × as long as r, 0.5 × 2RS, 0.25 × 3RSb; r-m 0.7 × 3RSa; CU1b arising from the middle of brachial cell; marginal cell somewhat short about 2.25 × as wide as high, 3RSb straight and falls much before the tip of wing; (RS+M)a slightly curved; hind wing 4.0 × as long as wide; M+CU 0.6 × 1M.Legs. Hind leg setose, hind femur 4.0 × as long as broad, hind tarsus 0.9 × as long as hind tibia, hind basitarsus 1.2 × as long as tarsal segment 2–4 combined.Metasoma. Metasoma as long as head and mesosoma combined in dorsal view, 2.0 × as long as wide; T1 longitudinally striated, strongly broadening posteriorly; 2 × as long as broad basally, spiracles present a little basally from middle; further tergites polished; ovipositor sheath in lateral view 1.1 × as long as tarsomere 2–4 combined; ovipositor short, straight and pointed.Color. Head brownish yellow with dark brown patches; eyes, stemmaticum, propleuron, mesopleuron, propodeum black; mesonotum, metanotum, legs brownish; metasoma reddish brown; ovipositor sheath brownish; mandibles yellowish brown; tip of mandible, claws, antennal segment F1–F16 dark brown; F17–F29 creamish-white; ocelli transparent; wings hyaline, pterostigma yellowish brown, veins yellowish brown and thickened.Unknown.Unknown.India (Uttar Pradesh).Braconidae.The species is named after Dr Shujauddin for his valuable contributions to the taxonomy of Indian Taxon classificationAnimaliaHymenopteraBraconidaeBelokobylskij34B0F990-7D5B-5D34-99B8-3A5961FDE82CPambolus (Phaenodus) ruficeps Belokobylskij, 1988: 27 (Taiwan); ZDAMU)”.2 females, “INDIA: Uttar Pradesh, Etawah, 13.IV.02; coll. M Shamim (Pambolus (Ph.) ruficeps is quite unlike all Oriental species of the genus Pambolus due to the presence of a heavily sculptured head. A brief diagnosis of P. (Ph.) ruficeps follows: body length 3.0–4.2 mm long, forewing length 3.0 mm long; eye 2.7–3.0 × as long as temple in dorsal view, latter receded; antenna with 33–40 segmented, 1.5–1.7 × as long as body; vertex, occiput coriaceous to rugulose; face reticulate rugulose; notauli distinct and crenulated; vein r, 2RS, 3RSa normal; propodeal spine as long as second or third tarsomere of hind tarsus; hind femur 3.5–3.8 × as long as broad medially; F1 1.1–1.3 × as broad behind as long medially.Unknown.India: Jammu and Kashmir, Orissa and Uttar Pradesh; Malaysia, Taiwan, Vietnam .Taxon classificationAnimaliaHymenopteraBraconidaeAhmadsp. nov.FA9BA224-6D9B-5717-B30D-45515B9C2143http://zoobank.org/6837B17F-77C7-48C1-A5A6-B11EAEFA9C69Holotype, female, “INDIA: Uttar Pradesh, Aligarh, 10.VIII.2005; ex Trachys sp. on Corchorus sp., Coll. Zubair Ahmad (ZDAMU)”. Paratypes, 3 females and 2 males; with same data as holotype.Parachremylus by Parachremylustrachysi sp. nov., runs near to another Indian species viz., P.oblongus (Papp). The new species shares similarities with P.oblongus in having the presence of longer temple; transverse diameter of eye 4.0 × as long as temple length in dorsal view; malar space 1.2 × basal width of mandible; mesopleuron smooth in upper half, striation partly present in subalar depression only; 1–4 segments of hind tarsomere with narrow and partly indistinct flanges and shallow field of hind half of mesonotum with a medio-longitudinal carinae running up to middle of mesoscutum. However, the new species can easily be distinguished from P.oblongus by having the head and mesosoma densely setose (head and mesosoma sparsely setose in P.oblongus); median longitudinal carina of metasoma reaching up to almost T3 ; pterostigma 3.5 × as long as high (pterostigma 5.0 × as long as high in P.oblongus); malar space 1.2 × as long as basal width of mandible ; scutellar sulcus with three crenulae (scutellar sulcus with five crenulae in P.oblongus); forewing veins 2RS and 3RSa 3.5 × as long r (forewing veins 2RS and 3RSa 2.7 × as long r in P.oblongus)Following the key to world species of Female, body length: 2.1 mm; forewing: 2.0 mm.Head. Antennae 27 segmented, about as long as body length, F1– F2 3–3.3 × as long as apical width, except the apical segment which is 4.5 × as long as wide; head transverse, 1.5–1.7 × as wide as long in dorsal view and 1.2 × as high as long in lateral view; eyes in lateral view 1.2 × as high as wide, 4.0 × as long as temple, inner margin of eyes not parallel; temple granulate, sparsely setose, its widest part behind eye 0.25 × as long as width of eye; occipital carina dorsally slightly curved towards ocelli, rather widely interrupted medially; occiput with uniform transverse striations; AOL: POL: OOL: ØOD = 2:1:3:2; face polished, laterally shagreened, sparsely setose, 1.5 × as wide as long medially; clypeus 1.6 × as wide as long; vertex granulate, sparsely setose; malar space 0.4 × as long as eye and 1.2 × as long as basal width of mandible;Mesosoma. Mesosoma 1.5 × as long as wide in dorsal view , 1.6 × as long as high in lateral view; pronotum small, indistinct; median and lateral lobes of mesoscutum granulate, sparsely setose; notauli broad anteriorly, shallow posteriorly, shallow field of hind half of mesonotum with a medio-longitudinal carinae running up to middle of mesoscutum; scutellar sulcus deep and 3.5 × as wide as long with three crenulae; scutellum indistinctly granulate, rather densely setose; propodeal areola with a median longitudinal carina anteriorly, two transverse carina inside, lateral areola with longitudinal striations anteriorly and inside with transverse ruguae; propleuron smooth; mesopleuron smooth and polished except few striation at subalar depression.Wings. Forewing 2.5 × as long as wide, as long as body; pterostigma 3.0 × as long as wide, issuing r from its middle; length of marginal cell along R1a about 1.2 × as long as pterostigma; 2RS and 3RSa 3.5 × as long r respectively; hind wing vein M+CU 0.8 × 1M; hind femur 3.4–3.6 × as long as broad medially; hind tibia 1.2 × as long as hind tarsus; hind basitarsus 0.6 × as long as tarsomere 2–4 combined.Metasoma. Metasoma dorsally sub-sclerotized, 1.0 × as long as head and mesosoma combined, T1 evenly and distinctly broadening posteriorly, T2 2.1 × as broad as long, 1. 5 × as long as T3; T3 3.0 × as broad as long; T4 4.0 ×, as wide as long medially; basal pair of carinae of T1 meeting at its basal one-third and continuing a medio-longitudinal carinae up to apical end of T3; all tergites sub- sclerotized; ovipositor sheaths setose and blunt, in lateral view 1.2 × as long as hind basitarsus.Color. Vertex yellow with brown marking, mandibles, scutellar sulcus, metanotum creamish; tip of mandible, mesoscutum, claws, dark brown; antennae brown; ocelli transparent; eyes, stemmaticum black; wings hyaline, pterostigma pale yellow, veins brown; scutellum, propodeum, legs yellow.Same as ♀ except body size (2 mm).Trachys sp. on Corchorus sp. (Wild Jute).India (Uttar Pradesh).The species name is derived from the name of the genus of the host insect.


Depression is one of the most common mental disorders characterized mainly by low mood and loss of interest or pleasure. About a third of patients with depression do not respond to classic antidepressant treatments. Recent evidence suggests that Mrp8/14 (myeloid-related protein 8/14) plays a crucial role in cognitive dysfunction and neuroinflammatory diseases, yet its role in mood regulation remains largely uninvestigated. In the present work, we explored the potential role of Mrp8/14 in the progression of depression.After 4 weeks of chronic unpredictable mild stress (CUMS), depressive-like symptoms and Mrp8/14 were determined. To verify the effects of Mrp8/14 on depressive-like behaviors, the inhibitor TAK-242 and recombinant Mrp8/14 were used. Furthermore, the molecular mechanisms in Mrp8/14-induced behavioral and biological changes were examined in vivo and ex vivo.Four-week CUMS contributed to the development of depressive symptoms. Mrp8 and Mrp14 were upregulated in the hippocampus and serum after exposure to CUMS. Pharmacological inhibition of Mrp14 attenuated CUMS-induced TLR4/NF-κB signaling activation and depressive-like behaviors. Furthermore, central administration of recombinant Mrp8, Mrp14, and Mrp8/14 resulted in neuroinflammation and depressive-like behaviors. Mrp8/14-provoked proinflammatory effects and depressive-like behaviors were improved by pretreatment with a TLR4 inhibitor. Moreover, pharmacological inhibition of TLR4 reduced the release of nitric oxide and reactive oxygen species in Mrp8/14-activated BV2 microglia.These data suggest that the hippocampal Mrp8/14-TLR4-mediated neuroinflammation contributes to the development of depressive-like behaviors. Targeting the Mrp8/14 may be a novel promising antidepressant approach.The online version of this article (10.1186/s12974-018-1296-0) contains supplementary material, which is available to authorized users. Depression is a common mental disorder with high rates of recurrence . Unfortu2+ binding S100 protein family. Mrp8 and Mrp14 have been identified as crucial endogenous DAMPs [Myeloid-related protein-8  and myeloid-related protein-14  are two important members of the Caus DAMPs , 12. Mrpus DAMPs , 14. Theus DAMPs . Other eus DAMPs , psoriasus DAMPs , and canus DAMPs , 19.In the central nervous systems (CNS), Mrp8 and Mrp14 have been found to be expressed in the microglia  and neurIn the present study, we investigated the levels of Mrp8 and Mrp14 expression in the hippocampus and serum after exposure to chronic unpredictable mild stress (CUMS). Further, the effects of Mrp14 inhibitor ABR-215757 on CUMS-induced neuroinflammation and depressive-like behaviors were assessed. Moreover, the recombinant proteins were injected into the cerebrospinal fluid (CSF) to determine the changes of depressive-like behaviors. Finally, the molecular mechanisms in Mrp8/14 heterodimer-induced behavioral and biological changes were examined in vivo and ex vivo.Male BALB/c mice  were purchased from the Animal Center . All animals were given 1–2 weeks to acclimate to colony conditions before the experiments began. The mice were maintained under standard conditions  with a 12-h light/dark cycle and ad libitum access to food and water unless otherwise stated. After acclimation, mice were randomly assigned to the control and experiment groups using random numbers generated in Microsoft Excel  and Mrp14  were purchased from Abcam . Recombinant mouse Mrp8/14 heterodimer  was obtained from R & D SYSTEMS . The endotoxin level is less than 0.10 EU per 1 μg in the recombinant proteins. TAK-242  was obtained from MedChem Express . Anti-Mrp8 goat polyclonal antibody (cat. no. sc-8113), anti-Mrp14 goat polyclonal antibody (cat. no. sc-8115), anti-TLR4 mouse monoclonal antibody (cat. no. sc-293072), and anti-RAGE mouse monoclonal antibody (cat. no. sc-365154) were purchased from Santa Cruz. Anti-MyD88 rabbit monoclonal antibody (cat. no. 4283) and Anti- NF-κB p65 rabbit monoclonal antibody (cat. no. 8242) were obtained from Cell Signaling Technology . Anti-Phospho-NF-κB p65 rabbit polyclonal antibody (cat. no. AB11011) was purchased from AbSci . Anti-iNOS rabbit monoclonal antibody (cat. no. ab205529) was purchased from Abcam. Anti-ACTB rabbit polyclonal antibody (cat. no. D110001–0100) was obtained from BBI Life Science. IRDye 800CW goat anti-mouse antibody (cat. no. 926–68070), goat anti-rabbit antibody (cat. no. 926–32211), and IRDye 680RD donkey anti-goat antibody (cat. no. 925–68074) were provided by LI-COR Biosciences.ABR-215757 (quinoline-3-carboxamide) was provided by Active Biotech AB . Recombinant mouse Mrp8 . Mice were acclimated to 1% sucrose for 48 h. The bottles were weighed both before and after the experiment, and consumption was quantified following overnight bottle choice. The sucrose preference was defined as the ratio of the weight of sucrose solution consumption to the total water intake, i.e., sucrose preference = sucrose consumption / (sucrose consumption + water consumption) × 100%. The test was performed in dark phase (6–10 p.m.).Sucrose preference test (SPT) was performed as described in a previous study . To deteTail suspension test (TST) was carried out according to our previous report . The mouAfter weighing and behavioral tests, mice under general anesthesia were fixed on a heated pad. Blood was collected from left ventricle. Then, mice were perfused transcardially with ice-cold saline for about 3 min. Hippocampi were isolated on ice, temporarily frozen in liquid nitrogen, and stored at − 80 °C. Blood samples were allowed to stand for 30 min at room temperature and centrifuged at 4000 rpm for 15 min. The supernatant serum was collected and stored at − 80 °C. Cell samples were prepared as stated below.Mouse serum Mrp8 and Mrp14 were determined using enzyme-linked immunosorbent assay kits  from CUSABIO and CusAb . All the assays were performed according to the manufacturer’s instructions. The detection range was 0.625–40 ng/mL for Mrp8, 0.45–30 ng/mL for Mrp14. The ELISA kits also have high precision, including high intra-assay precision (CV < 8%) and inter-assay precision (CV < 10%).The frozen mouse hippocampi and BV2 microglia were homogenized in ice-cold RIPA buffer  containing 1 mM protease inhibitor PMSF (Beyotime Institute of Biotechnology) and 10% PhosSTOP phosphatase inhibitor . Protein concentration in the lysate was determined with the BCA assay (Beyotime Institute of Biotechnology). Then, samples were mixed with 5× loading buffer and heated at 100 °C for 10 min. Protein samples were separated by 10–15% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis and transferred to polyvinylidene difluoride membranes . After two-hour blocking with 5% nonfat milk at room temperature, membranes were incubated with primary antibodies overnight at 4 °C. After washing, membranes were further incubated with fluorescent second antibodies for 1 h at room temperature. Bands were visualized using Odyssey Infrared Imaging System  and quantified with National Institutes of Health (NIH) Image J software.Intracerebroventricular (ICV) cannulation was operated for ICV injection as described previously . BrieflyMrp14 inhibitor ABR-215757 was dissolved and prepared as recommended by Active Biotech. Other drugs were dissolved or diluted using sterile endotoxin-free isotonic saline. ABR-215757 (10 mg/kg body weight ) was admThe levels of gene expression of inflammatory cytokines were determined by real-time RT-PCR. Brain tissue was stored in liquid nitrogen, and BV2 microglia were washed twice with ice cold PBS and then processed for RNA extraction. Total RNA was extracted from the hippocampus or BV2 microglia utilizing a standard method of TRIzol reagent . After quantification, 2 μg of total RNA was used for cDNA synthesis using PrimeScript™ RT Master Mix . Primer sequences were tested for sequence specificity using Primer-BLAST in NCBI. As tabulated in Additional file 2. For nitric oxide (NO) assay and Western Blot, cells were seeded onto 6-well culture plates. For real-time RT-PCR and reactive oxygen species (ROS) assay, cells were seeded onto 12-well culture plates. After treatment with TAK-242 (1 μM) for 30 min, cells were administrated with rMrp8/14 (0.5 μg/mL) for 24 h. Then, the supernatants were collected for NO assay, and cells were used for Western Blot, real-time RT-PCR, and ROS assay.A murine microglial cell line BV2 was obtained from American Type Culture Collection . Cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum  at 37 °C in an incubator with 95% air and 5% CONO of cell supernatant was detected using Griess Reagent  according to the manufacturer’s instructions. Samples and standards were added into a 96-well plate, and then Griess Reagent I and II were added successively. The absorbance was determined at a wavelength of 540 nm using a microplate reader. ROS was determined by a Reactive Oxygen Species Assay Kit (Beyotime Institute of Biotechnology). In brief, after washing, cells were incubated with fluorescent probe DCFH-DA  for 20 min at 37 °C in an incubator. Then, the extracellular DCFH-DA was cleared, and images were obtained using a fluorescence microscope (Carl Zeiss) at the 488 nm excitation wavelength and 525 nm emission wavelength.t test when comparing between two variables, and one-way or two-way ANOVA followed by LSD post hoc tests when comparing among multiple variables. Omnibus F values with degrees of freedom were reported for each ANOVA. Differences were considered statistically significant only when p < 0.05. All the analyses were performed with IBM SPSS 21.0 .All data were presented as mean ± standard error of the mean (SEM). Normality and homogeneity of variance were assessed before comparisons using Kolmogorov–Smirnov test and Levene’s test. Statistical analyses were carried out with Student’s t(27) = 3.63, p < 0.01, 29.18 ± 0.53 vs. 25.78 ± 0.41). CUMS significantly decreased the sucrose preference, indicating the impaired sensitivity to reward and anhedonia, which is a core symptom of major depression  = 2.51, p = 0.02, 81.65 ± 5.57 vs. 57.87 ± 7.66). The stressed mice also showed longer immobility duration in the TST  = 2.22, p = 0.04, 44.87 ± 3.05 vs. 57.98 ± 5.05). On the other hand, the results of ELISA showed that relative to the control group, serum Mrp8  = 2.15, p = 0.04, 4.93 ± 0.35 vs. 6.04 ± 0.37) and Mrp14  = 2.96, p < 0.01, 4.49 ± 0.08 vs. 4.77 ± 0.06) were slightly upregulated after CUMS intervention. Western blot revealed that hippocampal Mrp8  = 3.00, p = 0.01, 0.91 ± 0.26 vs. 2.14 ± 0.31) and Mrp14  = 5.41, p < 0.01, 1.10 ± 0.03 vs. 1.46 ± 0.08) were also increased. Collectively, these data demonstrate that CUMS elicited depressive-like behaviors and increased Mrp8 and Mrp14 protein in both serum and hippocampus.Behavioral tests and body weight were measured after CUMS period  = 3.18, p = 0.03, 1.00 ± 0.04 vs. 1.22 ± 0.06) but not MyD88  = 0.53, p = 0.62, 1.00 ± 0.11 vs. 1.06 ± 0.04) at the protein level. CUMS also activated the phosphorylation of NF-κB p65  = 3.74, p = 0.02, 1.00 ± 0.14 vs. 1.66 ± 0.11). Conversely, RAGE expression remained unaffected  = 0.92, p = 0.41, 1.00 ± 0.06 vs. 1.06 ± 0.03). Taken together, CUMS activated TLR4/NF-κB signaling pathway but did not affect the expression of RAGE.TLR4 signaling pathway has been shown to have a pivotal role in stress-induced neuroinflammation  and majoF = 4.54, p = 0.01; post hoc: vehicle vs. CUMS, p = 0.03, 28.44 ± 1.29 vs. 26.84 ± 1.78), while CUMS could not significantly decrease body weight of ABR-215757-treated mice . However, ABR-215757 did not significantly rescue body weight of stressed mice . Besides, ABR-215757 increased the sucrose preference  = 5.67, p = 0.02; post hoc: CUMS vs. CUMS + ABR-215757, p < 0.05, 57.28 ± 4.79 vs. 76.31 ± 4.26) and decreased the immobility duration in TST  = 6.91, p < 0.01; post hoc: CUMS vs. CUMS + ABR-215757, p < 0.001, 62.21 ± 6.70 vs. 32.12 ± 5.15). We also found that ABR-215757 alleviated TLR4 expression  = 7.85, p < 0.01; post hoc: CUMS vs. CUMS + ABR-215757, p = 0.02, 1.36 ± 0.05 vs. 1.14 ± 0.04) and p65 phosphorylation  = 9.68, p < 0.01; post hoc: CUMS vs. CUMS + ABR-215757, p = 0.03, 1.33 ± 0.06 vs. 1.13 ± 0.07) after CUMS exposure. The RAGE expression did not change significantly among these groups  = 0.72, p = 0.56, 0.91 ± 0.03 vs. 1.07 ± 0.09). Thus, ABR-215757 could alleviate CUMS-induced depressive-like behaviors and TLR4/NF-κB signaling activation.To further investigate the functional role of Mrp14 in the development of depressive symptoms, the inhibitor ABR-215757 was injected daily during the CUMS period  = 3.44, p = 0.03; post hoc: vehicle vs. Mrp8, Mrp14, and Mrp8/14, all p < 0.05, 83.17 ± 7.02 vs. 59.90 ± 6.83, 55.03 ± 7.08, 53.09 ± 8.82). All of the recombinant Mrp8, Mrp14, and Mrp8/14 treatment could result in a longer immobility duration  = 3.16, p = 0.05; post hoc: vehicle vs. Mrp8, p = 0.03; vehicle vs. Mrp14, p = 0.02; vehicle vs. Mrp8/14, p = 0.02, 45.02 ± 3.11 vs. 65.40 ± 6.08, 65.91 ± 5.45, 68.61 ± 7.92). Besides, we found that the mRNA levels of inflammatory cytokines  were elevated in the hippocampus after being challenged by any of these recombinant proteins  = 20.54, p < 0.001 for TNF-α, 1.00 ± 0.10 vs. 6.17 ± 0.47, 2.70 ± 0.31, 3.76 ± 0.71; F = 24.70, p < 0.001 for IL-1β, 1.00 ± 0.26 vs. 13.60 ± 0.97, 4.54 ± 1.14, 4.67 ± 1.93; F = 7.59, p < 0.01 for IL-6, 1.00 ± 0.22 vs. 1.13 ± 0.08, 1.72 ± 0.14, 2.09 ± 0.23). Microglia activation has been suggested to be mainly responsible for depression [F = 7.79, p < 0.01; post hoc: vehicle vs. Mrp8, p = 0.03; vehicle vs. Mrp14, p < 0.01; vehicle vs. Mrp8/14, p < 0.001, 1.00 ± 0.25 vs. 2.76 ± 0.31, 3.30 ± 0.57, 4.85 ± 0.79). After these recombinant proteins treatment, the hippocampal TLR4 level  = 9.13, p < 0.01; post hoc: vehicle vs. Mrp8, p = 0.02; vehicle vs. Mrp14, p < 0.01; vehicle vs. Mrp8/14, p < 0.001, 1.00 ± 0.04 vs. 1.23 ± 0.05, 1.34 ± 0.03, 1.41 ± 0.09) and p65 phosphorylation  = 10.29, p < 0.01; post hoc: vehicle vs. Mrp8, p = 0.03; vehicle vs. Mrp14, p < 0.01; vehicle vs. Mrp8/14, p < 0.001, 1.00 ± 0.03 vs. 1.60 ± 0.13, 1.90 ± 0.22, 2.34 ± 0.24) were elevated. No significant differences were found in RAGE expression among the four groups  = 1.51, p = 0.26, p < 0.001, 1.00 ± 0.09 vs. 1.12 ± 0.12, 1.25 ± 0.07, 1.01 ± 0.10). Collectively, central administration of Mrp8, Mrp14, and Mrp8/14 induced depressive-like behaviors, enhanced TLR4/NF-κB signaling pathway, and promoted the generation of proinflammatory cytokines.To determine whether Mrp8, Mrp14, and Mrp8/14 affected the development of depressive-like behaviors, recombinant Mrp8, Mrp14, Mrp8/14, and vehicle reagent were injected (ICV) to mice  = 4.99, p = 0.01; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.01, 53.30 ± 8.19 vs. 73.40 ± 6.48) and shorter immobility time in the tail suspension test  = 5.24, p = 0.01; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.01, 94.91 ± 8.56 vs. 66.88 ± 3.69). The NF-κB p65 phosphorylation evoked by recombinant Mrp8/14 was inhibited by TAK-242 treatment  = 18.21, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.001, 1.45 ± 0.09 vs. 0.95 ± 0.06). We further detected the changes of the expression of proinflammatory cytokines. The results showed that relative to the recombinant Mrp8/14 group, the mRNA levels of proinflammatory cytokines in the hippocampus of recombinant Mrp8/14 + TAK-242 were reduced significantly . Moreover, Mrp8/14-induced hippocampal IBA-1 overexpression was attenuated by TAK-242, which was shown by the RT-PCR assay of IBA-1  = 43.71, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.01, 3.42 ± 0.27 vs. 2.40 ± 0.19). Taken together, pretreatment of TAK-242 could inhibit the Mrp8/14-induced depressive-like behaviors and neuroinflammation.Since Mrp8/14 heterodimer is the most abundant form , 12, we F = 26.66, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.001, 1.60 ± 0.12 vs. 0.78 ± 0.04; Fig. F = 14.19, p < 0.001; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p < 0.001, 1.34 ± 0.13 vs. 0.63 ± 0.04). The production of NO was also significantly decreased after TAK-242 administration  = 17.81, p < 0.01; post hoc: Mrp8/14 vs. Mrp8/14 + TAK-242, p = 0.03, 5.17 ± 0.36 vs. 4.00 ± 0.30). Moreover, our data showed that TAK-242 reduced the generation of ROS induced by recombinant Mrp8/14 , such as TLR4 and RAGE . As repo−/− mice, Mrp8 is not expressed, but the transcription of Mrp8 is not altered [In Mrp14 altered . Thus, M altered . Our res altered , 48. As Furthermore, recombinant Mrp8, Mrp14, and Mrp8/14 heterodimer were administrated by ICV injection to detect their effects on behaviors and the underlying mechanism. The results showed that depressive-like behaviors and TLR4/NF-κB signaling activation were observed in all the three protein-administrated mice. However, we did not find any effects of these recombinant proteins on RAGE expression. These findings are in line with our previous results using a CUMS animal model. It should be noted that the recombinant proteins were used at high doses in this study. Central injection of these recombinants at lower doses may not induce neuroinflammation and depressive-like behaviors in mice.Mrp8/14 can induce the release of IL-6, IL-8, IL-1β, and TNF-α in monocytes or bone marrow cells , 49. As Next, we further verified whether the recombinant proteins-induced depressive-like behaviors were mediated by TLR4 signaling and microglia activation. Although all of Mrp8, Mrp14, and Mrp8/14 seem to be effective, we choose Mrp8/14 heterodimer for this issue. The main reason is that the heterodimer is the most abundant form , 12. BesAs two indicators of microglia activation, NO and ROS have been suggested to contribute to the development of depressive symptoms –58. iNOSIn conclusion, we identify a vital molecule contributing to the development of depressive symptoms and augment neuroinflammation. Our results validate that Mrp8/14 takes a critical role in CUMS-provoked neuroinflammation and depressive-like behaviors. The Mrp14 inhibitor ABR-215757 effectively ameliorates depressive symptoms and TLR4/NF-κB signaling activation. Central injection of bioactive recombinant protein confirms the role of Mrp8/14 in proinflammatory cytokines overexpression and the development of depressive-like behaviors. These Mrp8/14-induced cellular, biochemical, and behavioral changes depend on TLR4 signaling. Our results further reinforce the neuroinflammation hypothesis of depression. These findings also provide new sights into the underlying molecular mechanism of depression and raise a novel antidepressant approach by targeting the aberrant Mrp8/14 function.Additional file 1:Figure S1. The schematic diagrams showing the experimental designs. CUMS, chronic unpredictable mild stress; SPT, sucrose preference test; TST, tail suspension test; ICV cannulation, intracerebroventricular cannulation; IP, intraperitoneal. (TIF 1686 kb)Additional file 2:Table S1. Primer sequence used in this study. (DOCX 17 kb)


In the crystal, the trinuclear cationic units and dinuclear anionic units are assembled into infinite layers.In the pentanuclear title complex, [SmZn 2(C22H18N2O4)2(NCS)2(C3H7NO)2][Zn2(C22H18N2O4)(NCS)3]·C3H7NO·0.32H2O, namely bis{μ2-6,6′-dimethoxy-2,2′-diphenolato}-1κ4O,N,N′,O′:2κ3O,O′,O6;2κ3O,O′,O6:3κ4O,N,N′,O′-bis(dimethylformamide-2κO)dithiocyanato-1κN,3κN-2-samarium(III)-1,3-dizinc(II) {μ2-6,6′-dimethoxy-2,2′-diphenolato-1κ4O,N,N′,O′:2κ2O,O′}trithiocyanato-1κN;2κ2N,N-dizinc(II) dimethylformamide monosolvate 1.32-hydrate, a dinuclear unit and a trinuclear unit co-exist. One of the ZnII centers in the dinuclear unit as well as the two ZnII centers in the trinuclear unit are located in the inner N2O2 cavity of the ligand and are coordinated to the nitrogen atom of one thiocyanate moiety, giving rise to a square-pyramidal geometry. The second ZnII center in the dinuclear unit is coordinated to the two phenolate oxygen atoms of the ligand and to two thiocyanate groups via the nitrogen atom in a tetrahedral geometry. The SmIII ion is eight-coordinated by four phenolate O atoms from the two ligand molecules, two methoxy O atoms from the two ligand molecules and two O atoms from the DFM solvent molecule. In the dinuclear unit, the two methoxy oxygen atoms remain uncoordinated while in the trinuclear unit, for each ligand one methoxy oxygen is coordinated and the other one remains uncoordinated. In the crystal, the trinuclear cationic units and dinuclear anionic units are assembled into infinite layers. These layers are held together via electrostatic interactions, forming a three-dimensional structure. In the dinuclear unit, the C and S atoms of one of the thiocyanate groups are disordered over two sets of sites in a 0.680 (4)(4):0.320 (4) ratio.In the pentanuclear title complex, [SmZn The asymmetric unit 2(SCN)2(DMF)2]+, one dinuclear anionic unit, [Zn2(L)(SCN)3]−, one uncoordinated DMF solvent molecule and 0.32 of a water molecule.The title compound crystallizes in the triclinic system in the space group it Fig. 1 consists3O2 environment, which can be characterized by the Addison parameter τ  and Sm—O31 [2.6934 (13) Å]. The Sm—Ophenoxy distances are in the range 2.3348 (12)–2.4417 (12) Å and are comparable to those found for the Zn2Sm complex –2.0395 (12) Å. These values are comparable with the distances for the dinuclear complex [Zn(H2O)Sm(O2NO)3]  and cisoid angles [88.34 (6) and 88.94 (6)°], which are different from the ideal values of 180 and 90° for a square-planar geometry –119.03 (8)°, are indicative of a distorted tetrahedral environment. The geometry around the Zn62 atom is best described as a distorted square pyramidal, as indicated by the value of 0.105 for the Addison parameter τ. The apical position is occupied by the nitrogen atom N141 of the thiocyanate group with the basal plan occupied by atoms N51, N52, O62 and O63 from the ligand molecule. The angles between the N141 atom in the apical position and each of the four basal plane atoms fall in the range 106.67 (6)–111.37 (7)° and are far from the ideal value of 90°. The deformation of the basal plane around the Zn62 atom is indicated by the values of the y Table 1. The aniFig. 2et al., 20162O2 cage is occupied by a 3d metal and the larger, open O2O2 cage is occupied by one s-, p-, d- or f-block metal are well documented. Trinuclear complexes formed by two 3d metals with the above organic ligand in which the 3d metal atom is situated in the smaller N2O2 cage and one s-, d- or f-block metal atom is coordinated to the two larger O2O2 cages have also been reported: five entries corresponding to d–s [BIZBAO  ·(H2O)] was prepared according to a literature method  in 10 mL of acetonitrile. The resulting orange mixture was refluxed for 60 min, affording the organic H2L ligand. After cooling, a solution of Zn(CH3COO)2·2H2O  in 10 mL of acetonitrile was added. The mixture was heated under reflux for 60 min. On cooling, the orange precipitate was filtered off, washed with 3 × 10 mL of ether and dried in air, yielding a compound formulated as [(ZnL)·(H2O)] in 75% yield, m.p. 571–573 K. FT–IR : 3307 (OH) , 1609 (C=N) 1594 (C=C), 1586 (C=C), 1488 (C=C), 1439, 1234, 1187, 731. Analysis calculated for C22H20ZnN2O5: C, 57.72; H, 4.40; N, 6.12. Found: C, 57.68; H, 4.42; N, 6.07%. Λ (S cm2 mol−1): 5. The filtrate of a mixture of Sm(NO3)3·6H2O  and KSCN  in 20 mL of absolute ethanol was added to a DMF solution (5 mL) of [(ZnL)·(H2O)] . The resulting solution was heated under reflux for two h. After cooling, the solution was filtered and the filtrate was kept at 298 K. After four weeks, crystals suitable for X-ray diffraction were collected and formulated as [{Zn2(L)(SCN)3}]·[Sm{Zn(L)(SCN)}2(DMF)2]·(DMF)·0.18H2O. FT–IR : 2078 (S=C=N), 1654, 1607 (C=N), 1584 (C=C), 1545 (C=C), 1463 (C=C), 1440, 1238, 1191, 731. Analysis calculated for C80H75.36Zn4SmN14O15.18S5: C, 46.92; H, 3.71; N, 9.57; S, 7.83%. Found: C, C, 46.87; H, 3.68; N, 9.51; S, 7.85%. ΛM (S m2 mol−1): 28. μeff = 1.6 µB.The complex .Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989018016109/ex2013sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989018016109/ex2013Isup2.hklStructure factors: contains datablock(s) I. DOI: 1878960CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The crystal structure shows the molecules contain halogen bonds which are always between equivalent iodine atoms, either para–para or meta–meta.The title compound, C 15H10I2O, is a halogenated chalcone formed from two iodine substituted rings, one para-substituted and the other meta-substituted, linked through a prop-2-en-1-one spacer. In the molecule, the mean planes of the 3-iodophenyl and the 4-iodophenyl groups are twisted by 46.51 (15)°. The calculated electrostatic potential surfaces show the presence of σ-holes on both substituted iodines. In the crystal, the molecules are linked through type II halogen bonds, forming a sheet structure parallel to the bc plane. Between the sheets, weak intermolecular C—H⋯π interactions are observed. Hirshfeld surface analysis showed that the most significant contacts in the structure are C⋯H/H⋯C (31.9%), followed by H⋯H (21.4%), I⋯H/H⋯I (18.4%). I⋯I (14.5%) and O⋯H/H⋯O (8.1%).The title compound, C Substitutions on each of the benzene rings are currently being investigated in order to interrogate how the electronic properties of the crystal are altered. The iodo-substituted rings present in the title compound allows for the formation of iodine channels in the crystal, a conformation which may afford a change in the crystal’s electrical properties.Chalcones are aromatic ketones which have shown potential as antibacterial, antifungal and anti-inflammatory agents  and 3-iodophenyl (3-Ring), which are connected, respectively, to atoms C1 and C3 of the –CO—CH=CH– enone bridge Fig. 1. The baca)–(c)] show the presence of σ-holes on both substituted iodines, I1 and I2, which allow for halogen bonding of a bifurcated type II. Partial packing diagrams are shown in Fig. 3a)–(c). Interestingly, these halogen bonds form exclusively between equivalent iodine atoms, either para–para or meta–meta. The geometries of the halogen bonds are I1⋯I1iv = 4.0980 (9) Å, C7—I1⋯I1iv = 113.85 (13)°, I1⋯I1v =4.0980 (9) Å and C7—I⋯I1v = 154.47 (13)° for the para–para I⋯I bonds, and I2⋯I2vi = 3.9805 (8) Å, C12—I2⋯I2vi = 108.20 (13)°, I2⋯I2vii = 3.9805 (8) Å and C12—I2⋯I2vii = 157.30 (13)° for the meta–meta I⋯I bonds . A sheet structure is formed parallel to the bc plane. There are also three weak C—H⋯π interactions  and (c)] highlight the relationship between the contact distance and the van der Waals radii ] shows depressions on both 1-Ring and 3-Ring, which is indicative of C—H⋯π interactions. Two-dimensional fingerprint plots are used to illustrate the intermolecular contacts between molecules within the crystal structure. The fingerprint plots of all significant interactions are shown in Fig. 5a)–(f). C⋯H/H⋯C contacts [Fig. 5b)] make the largest contribution (31.9%) and show a pair of spikes at de + di = ∼2.8 Å, representative of intermolecular C—H⋯π interactions. The O⋯H/H⋯O plot also contains a pair of spikes at de + di = ∼2.7 Å [Fig. 5f)]. The negligible contributions from other contacts, not included in Fig. 5Hirshfeld surfaces, mapped over et al., 2016E)-1-(4-bromophenyl)-3-(4-fluorophenyl)prop-2-en-1-one -1-(4-bromophenyl)-3-(4-iodophenyl)prop-2-en-1-one -1-(4-bromophenyl)prop-2-en-1-one contains one meta-substituted ring and one para-substituted ring: each halogen bond exists between rings with the same substitution, either para–para or meta–meta, as seen in the title compound.A survey of the Cambridge Structural Database : δ (ppm) 8.37 , 8.0–7.94 , 7.88 , 7.81 , 7.68 , 7.26 . 13C NMR : δ (ppm) 188.44, 142.75, 139.04, 137.72, 136.95, 136.66, 136.61, 130.89, 130.38, 128.73, 122.81, 102.14, 95.57. Single crystals suitable for X-ray diffraction were obtained by the slow evaporation technique from an acetone solution at room temperature.4′-Iodoacetophenone , 3-iodobenzaldehyde , anhydrous zinc chloride  and absolute ethanol (1.5 ml) were added to a microwave vessel with a stir bar. Using a microwave reactor, the reaction mixture was heated to 468 K for 15 minutes. Upon cooling the reaction, yellowish solids were collected by vacuum filtration and washed with 95% ethanol. The resulting solid was recrystallized from 95% ethanol . Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2056989019016402/is5526sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2056989019016402/is5526Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2056989019016402/is5526Isup5.molSupporting information file. DOI: 10.1107/S2056989019016402/is5526sup3.pdf13C NMR of the synthesised product. DOI: 10.1107/S2056989019016402/is5526sup4.pdf1H NMR of the synthesised product. DOI: Click here for additional data file.10.1107/S2056989019016402/is5526Isup6.cmlSupporting information file. DOI: 1970266CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The cage cavities can accommodate different molecules such as anthranilamide and aromatic aldehyde through supramolecular interactions, allowing for a cascade condensation and cyclization to produce nonplanar 2,3-dihyroquinazolinones. The reaction is highly efficient with high rate enhancements  and multiple turnovers compared to the bulk reaction mixture. Control experiments and molecular simulations suggest that the acceleration is attributed to inherent strength of binding affinity for reactants and the release of products to establish catalytic turnover is due to the host−guest geometry discrepancy.The search for supramolecular reactors that contain no catalytically active sites but can promote chemical transformations has received significant attention, but it remains a synthetic challenge. Here we demonstrate a strategy of incorporating bulky and electro-rich aromatic linkers into metallocages to induce cascade reactions. Two hexahedral cages with a framework formula [(Zn Supramolecular containers are promising enzyme mimics, but they currently accommodate only a limited range of chemical transformations. Here, the authors describe coordination cages that catalyze two-component cascade reactions without relying on an external or encapsulated catalytic species. Of particular interest is the possibility of performing chemical transformations in confined spaces, in which the relativities and selectivities may be quite different from those in solution. In light of their well-defined yet tunable structures6, coordination cages provide an ideal platform for designing supramolecular catalysts for chemical transformations3. In this respect, pioneering works have been demonstrated by Fujita8 and Raymond10. Some representative reactions include Diels–Alder12, epoxidation13, the aza-Cope rearrangement14, Knoevenagel reaction15, Nazarov cyclization16, sigmatropic rearrangements17, and a few others23. Despite that some examples are known for efficient bimolecular reactions in containers without catalytically active sites, there is a great need for the design of new supramolecular capsules to promote more complicated reactions25. Moreover, the range of reaction types is narrow, limiting their use in practical organic synthesis. Cascade or sequential catalytic reactions, as sophisticatedly manipulated by nature, are of great value because such processes can guide the reactive intermediates to the targeted products via consecutive reactions27. However, it remains unexplored to rationally design catalytic cages for tandem reactions29. In artificial systems, product inhibition poses another challenge in establishing a catalytic cycle3. To address these issues, here we report a strategy of incorporating bulky and electro-rich aromatic linkers into metallocages to induce two-component cascade reactions and reduce product inhibition.Self-assembled supramolecular containers have attracted growing attention as enzyme mimetics for potential applications in molecular recognition and storage31. We envisioned that specific aromatic−aromatic and/or edge-to-face aromatic interactions may be used for hosts to concentrate aromatic reactants, regulate their orientations and even promote catalytic reactions to generate products that have weak host−guest interactions and can be expelled to allow catalytic turnover3. Tetraphenylethylene (TPE) and its derivatives have recently attracted much attention owing to their rich electrochemical and excited state properties31 and particularly have been employed for designing cages and metallacycles with aggregation-induced emission33. Considering their π-electron-rich and structural flexibility, in this study we select two TPE-derived tetraamines as ligands for subcomponent self-assembly of octanuclear Zn8L6 cages with tunable cavity sizes boronic acid pinacol ester and tetra(4-bromophenyl)ethylene and followed by reduction with Pd/C (OTf)16]·G (G = guest molecule) were obtained by heating Zn(OTf)2, L1 or L2 and 2-formylpyridine in CH2Cl2 and CH3CN at 70 °C with 64 and 35% yield, respectively. Single crystals of the cages were obtained by diffusion of a mixed solvents of Et2O and THF or 1,4-dioxane and THF  into a dilute and saturated CH3CN solution. The formulations were supported by the results of microanalysis, IR spectroscopy, 1H and 13C NMR, 1H COSY, NOESY, Quadrupole time-of-flight mass spectrometry (Q-TOF-MS), thermogravimetric analyses (TGA) and single-crystal X-ray diffraction.The ligand 1H and 13C NMR spectra of each cage displayed only one set of ligand resonances in solution, suggesting the formation of a discrete and highly symmetric assembly  for TPE-1  with a hydrodynamic radius of about 11.76 Å indicated the formation of single product. Similarly, 1H-DOSY of TPE-2 showed one band at log D = − 9.61 (m2 s−1) with a hydrodynamic radius of about 14.79 Å indicating a slightly larger size than TPE-1 6·xCH3CN·yTHF·zH2O·(16-n)OTf]n+ 6·xdioxane·yTHF·zCH3CN·(16-n)OTf]n+ zinc(II) vertices has an octahedral coordination geometry, with the Zn center chelated by three ligands. The Zn ions in one cage have the same Δ or Λ configuration. The Zn−N bond lengths range from 2.09 (2) to 2.27(2) Å, which are in good agreement with those reported for related Zn-pyridylimine complexes.Single-crystal X-ray diffraction study on TPE°, giving rise to the four-bladed propeller structure in the cage. This arrangement of metal ions and coordination ligands thus leads to a molecular cube with the eight corners occupied by the zinc ions and the six faces by six TPE ligands. The Zn−Zn separations along the cage edges are from 10.27(3) to 11.47(3) Å  , generating portals to allow the ingress/egress of guests  to 18.34(7) Å   and 4-fluorobenzaldehyde (4b) as substrates. After screening various reaction conditions including catalyst loading, reaction temperature, and solvent  can be used, but prolonged reaction time (45 h) was required. We measured the reaction rate under this condition by 1H NMR spectroscopy: the starting material transformed into intermediate 5b and product 6b nearly completely in 3 h and completed within 11 h to furnish the targeted 2,3-dihydroquinazolinone (6b) in 95% yield. Besides, the reactions of anthranilamide with other aromatic aldehydes such as benzaldehyde, 4-methylbenzaldehyde, and 4-methoxybenz aldehyde can also be catalyzed by TPE-1, which afforded 76−81% yields of the products  with little interference from the background reaction (kuncat), we assessed the model of Michaelis−Menten scheme 2, Sc(OTf)337 and Ga(OTf)338 or strong Brønsted acids such as sulfonic acid35 and phosphoric acid39 at 1.0 mol% catalyst loading  , the ligand L1  or a mononuclear tris(pyridylimine)zinc(II) complex (0.8 mol%) , but TPE-1 cannot promote the reaction at all  and 4b (6.0 × 8.5 Å2) with sizes smaller and even slightly larger than the portals can enter the cavities from windows41. However, the substrates are too large, so they cannot enter the cage cavities [1-formylnaphthalene (8.0 × 8.5 Å2) vs. TPE-1 and 9-anthracenealdehyde (8.0 × 10.9 Å2) vs. TPE-1 and TPE-2]. Taking together, the above results suggested that the sequential condensation and cyclization reaction was indeed associated with the substrates being bound in the cage cavity. We demonstrated that the catalytic reaction occurs with a lot of turnovers. To a solution of TPE-1 (0.1 mol% loading) in CH3CN and toluene, we added several successive portions of anthranilamide and 4-fluorobenzaldehyde, waiting until each aliquot had completely reacted before adding the next. We can see from Supplementary Fig. 1H NMR, TOF mass, and UV−Vis spectra  DMSO-d6/CD3CN, in which the diffusion coefficients were single set of resonances in the presence of substrates  were found to be 4.0×104 and 1.2×104 M−1 for TPE-1 and the analytes 3a and 4b, respectively, and 1.5×104 and 1.0×104 M−1 for TPE-2 and 3a and 4b, which are much higher than the Ka values of 1191 and 1754 M−1 observed for the two cages and the intermediate 5b, and 874 and 215 M−1 observed for the two cages and the product 6b  at ~3440 cm−1 and υ(C=O) at ~1697 cm−1 for 3a/4b@TPE-1/2  and hydrophobic CH···N and CH···O interactions (2.35-3.78 Å). Importantly, the cavity-confined microenvironment triggered the inherent strength of binding affinity between the two reactants via π···π and hydrophobic interactions (2.65~3.62 Å). For the substrates 3a + 4b, 3a + 4e and 3a + 4f in TPE-1, the binding energies were calculated to be −34.98, 44.39, and 52.67 kcal mol−1, respectively . Nevertheless, the bulky reactants 3a + 4f cannot fit into the cavity of TPE-2 and their condensation reaction is impeded.In order to provide microscopic insight into the cavity-confined effect, molecular dynamics (MD) simulations with the universal force fields 2.35-3.8 Å. Impos 2.35-3.8 Å. Impo3a + 4b with TPE-1 and TPE-2. In experiments, the solvent was present and, to a large extent, screened the binding, thus leading to a small difference in the associate constants obtained from UV−Vis titration. Nevertheless, these calculated interaction energies are consistent with the experimental observations of cavity-confined effect in the two cages. We believe that rate acceleration of this reaction is associated with the enhanced binding of the reactants in the confined cavity.It should be noted that the binding energies from simulations were based on an implicit solvent model. If the solvent molecules were included, the simulations would be quite time-consuming. Therefore, there is a large difference in the binding energies of 3a (higher Ka than reactant 4b) within their hydrophobic cavities (Step I). Second, the reactant 4b enter cavity and stack with encapsulated 3a face to face in the most stable configuration. The model of the host−guest binding system was conducted by molecular dynamic simulations revealing the enough negative binding energy . Third, the reaction of a subsequent intramolecular nucleophilic attack of the amide nitrogen on the activated imine group is followed by a 1,5-proton transfer to yield the final product in cages (Step III). The rich electronic and conjugate TPE faces of the cages may facilitate the deprotonating of the amide nitrogen via supramolecular interactions to render it more nucleophilic in the cyclization43. This is similar to the same cyclization reaction catalyzed by Lewis acids40 or Brønsted acids37 , 2-formylpyridine , and Zn(OTf)2  was dissolved in 30 mL CH2Cl2 and 30 mL CH3CN and then was heated at 70 °C for 8 h to give a clear solution. Single crystals suitable for X-ray diffraction analysis were obtained by slow diffusion of a mixture of Et2O and THF or 1,4-dioxane and THF (2:1 v/v) into the solution at r.t. for 7 days. The product can be best formulated as [Zn8(L1)6]16OTf·2THF·3CH3CN for TPE-1 and [Zn8(L2)6]16OTf·4THF·8dioxane for TPE-2 on the basis of microanalysis, TGA, Q-TOF-MS, and IR.A mixture of -1. Yield: 200 mg, about 64%. Anal (%). Calcd for C336H252N52Zn8S16O51F48: C, 59.16; H, 3.72; N, 10.68. Found: C, 59.23; H, 3.81; N, 10.86. 1H NMR  δ: 8.73 , 8.65 , 8.12 , 8.11 , 7.62 , 7.20 , 7.07 . 13C NMR  δ: 160.6, 150.1, 132.3, 125.9, 122.9, 121.8, 119.7, 116.4, 113.8. IR : 1598 (m), 1513 (m), 1445 (w), 1267 (vs), 1162 (s), 1031 (s), 914 (w), 839 (w), 777 (w), 639 (m), 574 (s), 518 (m). Q-TOF-MS: m/z: 797.84 [Zn8(L1)6·8OTf·THF·2CH3CN]8+, 829.24 [Zn8(L1)6·8OTf·2THF·6CH3CN·H2O]8+, 963.19 [Zn8(L1)6·9OTf·4THF·CH3CN·2H2O]7+, 1083.96 [Zn8(L1)6·10OTf]6+, 1331.55 [Zn8(L1)6·11OTf]5+, 1700.41 [Zn8(L1)6·12OTf]4+.TPE-2. Yield: 140 mg, about 35%. Anal (%). Calcd for C508H471N48Zn8S16O62F48: C, 59.95; H, 4.61; N, 6.61. Found: C, 59.89; H, 4.75; N, 6.76. 1H NMR  δ: 8.74 , 8.15 , 7.77 , 7.68−7.19 . 13C NMR  δ: 160.8, 150.0, 148.9, 141.1, 139.0, 132.1, 131.8, 127.6, 127.4, 126.4, 125, 123.0, 119.6, 114.7. IR : 1599 (m), 1494 (s), 1445 (w), 1267 (vs), 1164 (m), 1031 (s), 1004 (w), 823 (m), 803 (m), 776 (w), 639 (m), 574 (s), 517 (w). Q-TOF-MS: m/z: 559.11 [Zn8(L2)6·3OTf]13+, 650.41 [Zn8(L2)6·4OTf·4·THF·CH3CN]12+, 741.06 [Zn8(L2)6·5OTf·5·THF·CH3CN]11+, 795.89 [Zn8(L2)6·6OTf·2·CH3CN]10+, 916.64 [Zn8(L2)6·7OTf·3·THF·CH3CN]9+, 1267.19 [Zn8(L2)6·9OTf·7·THF]9+.TPE3CN and toluene , anthranilamide , aldehyde (0.055 mmol), and TPE-1  or TPE-2  were added. The resultant solution was stirred at 40 °C for 14 h. The reaction mixture was cooled down to r.t. and extracted with ethyl acetate. The ethyl acetate solution was washed with water and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the crude products were separated by the column on silica gel (EtOAc/petroleum ether) to get the isolated yield.To a solvent mixture of CH-1  or TPE-2  in the mixed solvents of CH3CN (1 mL) and toluene (2 mL), we added ten times successive portions of 3a (0.05 mmol) and 4b (0.055 mmol), waiting until each aliquot had completely reacted before adding the next.To a solution of the TPE−3 mol L−1) to a solution of TPE-1 or TPE-2 (1.0×10−5 mol L−1) in 3.0 mL CH3CN every 5 min. The absorption was measured at room temperature.The titration experiments were carried out by adding 30.0 μL solution of substrates (1.0×101 and TPE-2 were collected on a Bruker D8 VENTURE CMOS photon 100 diffractometer with helios mx multilayer monochromator Cu Kα radiation (λ = 1.54178 Å) at 173 K. The empirical absorption correction was applied by using the SADABS program . The structure was solved by direct methods with SHELXS-2014 and refined with SHELXL-2014 using OLEX2-1.2. In both cages, all non-H atoms were subjected to anisotropic refinement by full matrix program. Contributions to scattering due to these highly disordered solvent molecules were removed using the SQUEEZE routine of PLATON. Structures were then refined again using the data generated. Crystal data and details of the data collection are given in Supplementary Table Single-crystal XRD data for TPE-n λ = 1.5178 Å at 3a and 4  in TPE-1 and TPE-2, respectively. The cages and reactants were described by the Lennard−Jones (LJ) and electrostatic potentials. The LJ potential parameters as listed in Supplementary Table 44, and the atomic charges of the cages and reactants were estimated using the Qeq method45. To calculate the interaction energy for 3a + 4 in each cage, first, 3a and 4 together were inserted into TPE-1 or TPE-2, and followed by optimization using Materials Studio ; then, MD simulation was performed at 313 K for 15 ns. The cage structure was assumed to be rigid during MD simulation, but all the reactant molecules were flexible and free to move. The LJ interactions were evaluated with a cutoff of 12 Å, and the electrostatic interactions were estimated using the Ewald summation method with an accuracy of 10−3 kcal mol−1.Molecular simulations were conducted to estimate the interaction energies for Supplementary Information