Cell-based therapies using genetically engineered lymphocytes expressing antigen-specific T cell receptors (TCRs) hold promise for the treatment of several types of cancers. Almost all studies using this modality have focused on transfer of TCR from CD8 cytotoxic T lymphocytes (CTLs). The transfer of TCR from innate lymphocytes to other lymphocytes has not been studied. In the current study, innate and adaptive lymphocytes were transfected with the human NKT cell-derived TCRα and β chain mRNA (the Vα24 and Vβ11 TCR chains). When primary T cells transfected with NKT cell-derived TCR were subsequently stimulated with the NKT ligand, α-galactosylceramide , they secreted IFN-γ in a ligand-specific manner. Furthermore when γδT cells were transfected with NKT cell-derived TCR mRNA, they demonstrated enhanced proliferation, IFN-γ production and antitumor effects after α-GalCer stimulation as compared to parental γδT cells. Importantly, NKT cell TCR-transfected γδT cells responded to both NKT cell and γδT cell ligands, rendering them bi-potential innate lymphocytes. Because NKT cell receptors are unique and universal invariant receptors in humans, the TCR chains do not yield mispaired receptors with endogenous TCR α and β chains after the transfection. The transfection of NKT cell TCR has the potential to be a new approach to tumor immunotherapy in patients with various types of cancer. All participants gave written informed consent. Jurkat and K562 cell lines were purchased from RIKEN BRC. HEK293 cell line was purchased from ATCC.IL-2 was purchased from Shionogi & Co., LTD. Human recombinant IL-7 and IL-15 were purchased from Pepro TECH, Inc. α-GalCer and Zoendronic acid (Zol) were purchased from Funakoshi and Novartis Oncology respectively. The following monoclonal antibodies (mAbs) were purchased: anti-human CD11c (B-ly6), CD86 (2311), human invariant NKT Cell (6B11) from BD, anti-human Vα24 (C15), Vβ11 (C21), Vγ9 (IMMU360) from Beckman Coulter, anti-human CD3 (UCHT1) from e-Bioscience and anti-human CD1d (51.1) from Biolegend. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) rabbit monoclonal antibody from Cell Signaling and ERK1/2 rabbit polyclonal antibody from Promega were used. HRP-conjugated goat-anti-rabbit IgG was purchased from R&D systems. Laemmli Sample buffer, 2-mercaptoethanol, 10 x Tris/Glycine buffer, 10 x Tris/Glycine/SDS buffer, Any KD Mini-PROTEAN TGX Gel, Immun-Blot PVDF membrane were purchased form BIO-RAD. Immobilon Western Chemiluminescent HRP Substrate was purchased from Millipore. Tris Buffered Saline with Tween20 Tablets, pH 7.6 was purchased from TAKARA. PMA and Ionomycin were purchased from Sigma. The following ELISA kits were purchased: human IFN-γ and IL-4 from BD and human TNF-α from e-Bioscience.To prepare NKT cell lines, PBMCs were cultured in RPMI 1640 containing 10% FCS the presence of α-GalCer (100 ng/ml) and IL-2 (100 U/ml). After 10–14 days, human NKT cells were sorted using anti-Vα24 mAb and were maintained as NKT cell lines in the presence of IL-2 (100 U/ml), IL-7 (5 ng/ml), and IL-15 (10 ng/ml) in complete medium and restimulated with irradiated PBMCs pulsed with α-GalCer for at least one month as previously described [To prepare Vγ9Vδ2T cell lines, PBMCs were cultured in the presence of Zol (5 μM) and IL-2 (300 U/ml). After sorting using anti-Vγ9 mAb, Vγ9Vδ2T cell cells were maintained as γδT cell line in the presence of IL-2 (300 U/ml).5’-ATGAAAAAGCATCTGACGACCTTC-3’ and 5’-TCAGCTGGACCACAGCCGCAG-3’ for TCRα; 5’-ATGACTATCAGGCTCCTCTGC-3’ and 5’-TCAGAAATCCTTTCTCTTGACC-3’ for TCRβ. By sequencing analysis, we chose a cDNA clone for each of the TCRs that had a functional open reading frame, and subcloned them into EcoRI and BamHI sites of pGEM-3Z vector (Promega). Human NKT-TCR used in this study is as follows, α-chain: TRAV10*01/TRAJ18*01/TRAC*01 (GenBank Accession # DQ341448); β-chain: TRBV25-1*01/TRBD1*01/TRBJ1-4*01/TRBC1*01, with CDR3β sequence being CASEQRGGVDEKL , J and C regions; Genbank #BC073930).Total RNA was isolated from human NKT cells using RNeasy Mini Kit (QIAGEN Sciences). Complementary DNAs (cDNAs) for NKT-specific invariant TCRα and β chains were amplified by RT-PCR using OneStep RT-PCR Kit (QIAGEN GmbH) and cloned into a bacterial plasmid using QIAGEN PCR Cloning Kit (QIAGEN GmbH). The primer pairs used were as follows: BamHI as templates. Briefly, IVT was carried out using mMESSAGE mMACHINE T7 Ultra Kit (Ambion). This kit utilizes E. coli poly(A) polymerase to polyadenylate RNA transcripts at their 3’ ends to ensure longer half-life in the cells. The RNA was purified using RNeasy Midi Kit (QIAGEN Sciences) and its integrity was verified by gel electrophoresis.RNA was synthesized as described previously  with aboin vitro at 106 cells/ml with 50 ng/ml anti-CD3 mAb OKT3  and 300 U/ml IL-2. Three days later, T cells were washed and resuspended in OptiMEM. γδT cells were established as previously described and resuspended in OptiMEM. In transfection by electroporation, 10 μg each RNA was pulsed in square-wave pulse, 500V, 3 msec with an ECM 830 square were electroporation system (BTX). Immediately after electroporation, the cells were cultured with 300 U/ml IL-2 containing culture medium until use in the assay.RNA electroporation of T cells was performed as described previously . In brie5 effector cells for 12 h. After subtracting the background control value, cytotoxicity values (%) were calculated as previously described [The cytotoxic activity against adherent cells was analyzed using an LDH assay kit according to the manufacturer’s instructions (Takara Bio Company). CD1d gene-transfected HEK293 (CD1d-HEK293) were treated with or without 500 ng/ml α-GalCer or 5 μM Zol for 24 h to use them as target cells. Ten thousands target cells were cultured with 1x10escribed .in vitro data were analyzed using the Student T-test. P<0.05 was considered statistically significant.Differences in +Vβ11+NKT cells were cloned using RT-PCR. The resultant NKT-TCR α and β pairs, i.e., Vα24 and Vβ11 chains were separately inserted into a pGEM-3Z vector ) produced high amounts of IFN-γ in an α-GalCer-dependent manner (-Vβ11- TCR non-transfected cells (denoted as NKT-TCR(-)) did not produce IFN-γ  and IFN-γ secretion in the supernatants was measured. The Vα24t manner  bottom. +T cells: 0.036±0.035 vs 0.98±1.46). When PBMCs were stimulated with either Zol or α-GalCer, Vγ9Vδ2T cells proliferated more than NKT cells (+Vβ11+ NKT-TCR mRNA-transfected Vγ9Vδ2T cells (denoted as γδT-NKT-TCR (+)), these cells were stimulated using Zol and α-GalCer. It is well known that Vγ9Vδ2T cells in PBMC can be expanded after culturing with Zol. However, there are few reports about secondary expansion of γδT cells. It was previously reported that the proliferative response of γδT cells is transient and also that repeated injection of BrHPP and IL-2 induced activation induced cell death of Vγ9Vδ2T cell and an exhaustion of the response [ex vivo expansion from PBMCs using Zol, we assessed the number and function of γδT cells in secondary challenge with antigen. Vγ9Vδ2T cells were cultured again in the presence of soluble Zol to serve as a control for the subsequent groups  are a highly sensitive γδT cell target due to the expression of phosphoantigen while K562 cells (myelogenous leukemia cell line) are a weakly sensitive γδT cell target . We initThe current study evaluates the effects of transfecting human NKT cell-derived TCR α and β chain mRNA into either innate or adaptive lymphocytes. NKT-TCR transfected T cells and γδT cells secreted IFN-γ in a ligand-specific manner. These γδT cells demonstrated cytotoxicity against K562 tumor cells and proliferated in response to NKT cell and γδT cell ligands. This study demonstrated that γδT cells and primary adaptive lymphocytes transfected with the NKT-TCR retain parental T cell qualities while acquiring NKT cell functionality, thus creating a bi-potential T cell.NKT cell receptors are universal in humans and their specificity is not HLA restricted. Therefore, the approach of NKT-TCR mRNA transfection into primary lymphocytes and γδT cells has a number of unique features that makes it attractive for use as an immunotherapeutic approach to cancer treatment. NKT-TCR mRNA is not stably integrated into the genome , 6 and iA potential disadvantage of the TCR gene transfer approach is the possible formation of mixed TCR dimers. It has been reported that chains of the introduced TCR can pair with endogenous TCR chains, resulting in a dimer composed of both endogenous TCR and introduced TCR, thus altering TCR specificity . HoweverNKT cells in naïve mice have the potential to produce both IFN-γ and IL-4 in response to α-GalCer, therefore modulating whether the immune response is Th1 or Th2 . HoweverIn the current study, NKT-TCR-transfected lymphocytes responded to NKT cell ligand with the secretion of IFN-γ, but not IL-4. We found that the transfer of mRNA encoding NKT cell receptors drives the other lymphocytes toward IFN-γ producing NKT like cells, which might be useful against tumor cells. One of the reasons is that the transfected lymphocytes might be already polarized or skewed toward Th1 type in cytokine production. Previous studies showed that PBMCs stimulated by anti-CD3 plus IL-2 led to T-bet via TCR signaling pathway for IFN-γ in most of conventional T cells . T-bet iex vivo expanded Vγ9Vδ2T cells showed some clinical efficacy [Aminobisphosphonates, such as palindronate- or zoledronic acid-activated Vγ9Vδ2T cells to produce IFN-γ and exhibit cytotoxicity against some tumor cell types, e.g. Daudi, K562, AML cells, lymphoma cells and myeloma cells –32. Dielefficacy , 34. TheThe current study demonstrated that the transfer of NKT-TCR αβ chains enhances the function of both innate and adaptive lymphocytes and this approach may hold the promise of a new anti-cancer immunotherapy.


The title compounds, (I) and (II), are dispiro-indole-pyrrolidine-benzothiophene derivatives, with (II) having a chlorine substituent on the oxoindole unit. As a result, the conformation of the two molecules differs in the angle of inclination of the indole moiety with respect to the benzothiophene ring system, with a dihedral angle of 71.59 (5) in (I) and 82.27 (7)° in (II). 22H20N2O4S, (I), and C22H19ClN2O4S, (II), the pyrrolidine rings have twist conformations on the spiro–spiro C—C bonds. In (I), the five-membered ring of the oxindole moiety has an envelope conformation with the spiro C atom as the flap, while in (II) this ring is flat (r.m.s. deviation = 0.042 Å). The mean planes of the pyrrolidine rings are inclined to the mean planes of the indole units  and the benzothiophene ring systems (r.m.s. deviations = 0.019 and 0.034 Å for (I) and (II), respectively) by 79.57 (8) and 88.61 (7)° for (I), and by 81.99 (10) and 88.79 (10)° for (II). In both compounds, the ethoxycarbonyl group occupies an equatorial position with an extended conformation. The overall conformation of the two molecules differs in the angle of inclination of the indole unit with respect to the benzothiophene ring system, with a dihedral angle between the planes of 71.59 (5) in (I) and 82.27 (7)° in (II). In the crystal of (I), molecules are linked via pairs of N—H⋯O hydrogen bonds, forming inversion dimers enclosing R22(14) loops. The dimers are linked via C—H⋯O and bifurcated C—H⋯O(O) hydrogen bonds, forming sheets lying parallel to (100). In the crystal of (II), molecules are again linked via pairs of N—H⋯O hydrogen bonds, forming inversion dimers but enclosing smaller R22(8) loops. Here, the dimers are linked by C—H⋯O hydrogen bonds, forming ribbons propagating along [010].In the title compounds, C The dimvia pairs of N—-H⋯O hydrogen bonds, forming inversion dimers but enclosing smaller In the crystal of (II)s Table 2. Here thp-methoxyphenyl)-1′-methyl-1H-indole-3-spiro-2′-pyrrolidine-3′-spiro-1′′-cyclopentane-2(3H),2′′-dione -ethyl 2-(3-oxobenzo[b]thiophen-2(3H)-ylidene) acetate (1.0 mmol) and the relevant isatin together with sarcosine (1.1 mmol) was refluxed in methanol (20 ml) until completion of the reaction, as evidenced by TLC analysis. After completion of the reaction, the solvent was evaporated under reduced pressure. The crude reaction mixture was dissolved in dichloromethane (2 × 50 ml) and washed with water followed by brine solution. The organic layer was separated and dried over sodium sulfate. After filtration, the solvent was evaporation under reduced pressure. The product was separated by column chromatography using hexane and ethyl acetate (9:1) as eluent to give a white solid. This was dissolved in chloroform (3 ml) and heated for 2 min. The resulting solutions were allowed to evaporate slowly at room temperature and yielded colourless block-like crystals of compounds (I)The two compounds were prepared in a similar manner using isatin (1.1 mmol) for (I)Uiso(H) = 1.5Ueq(C-methyl) and = 1.2Ueq(C) for other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S1600536814015426/su2728sup1.cifCrystal structure: contains datablock(s) global, I, II. DOI: 10.1107/S1600536814015426/su2728Isup2.hklStructure factors: contains datablock(s) I. DOI: 10.1107/S1600536814015426/su2728IIsup3.hklStructure factors: contains datablock(s) II. DOI: 1011623, 1011624CCDC references:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The locations and arrangements of carotenoids at the subcellular level areresponsible for their designated functions, which reinforces the necessity ofdeveloping methods for constructing carotenoid-based suprastructures beyond themolecular level. Because carotenoids lack the binding sites necessary for controlledinteractions, functional structures based on carotenoids are not easily obtained.Here, we show that carotene-based suprastructures were formed via the induction ofpillararene through a phase-transfer-mediated host–guest interaction. Moreimportantly, similar to the main component in natural photosynthesis, complexescould be synthesized after chlorophyll was introduced into the carotene-basedsuprastructure assembly process. Remarkably, compared with molecular carotene orchlorophyll, this synthesized suprastructure exhibits some photocatalytic activitywhen exposed to light, which can be exploited for photocatalytic reaction studies ofenergy capture and solar conversion in living organisms. Carotenoids are photoactive organic pigments found in many plants.Here, the authors report the self-assembly of carotenoid suprastructures by forming apillararene host-guest complex, and show that these structures display functionalitiesincluding stimuli responsiveness and photocatalytic activity. Carotenoids are a diverse group of structurally related compoundsinvivo is now essentialRecently, in the carotenoid-related fields, this problem has been partially solvedthrough the use of hydrophilic vectorsFortunately, supramolecular chemistry provides a new framework to tackle this difficultquestion. Thus far, supramolecular macrocycles, such as crown ethers24252830-carotene (β-CAR) was selected as a type ofcarotenoid to represent the carotenoid family. A synthetic water-solublecarboxyl-modified pillararene (WP5) was also chosen because its cavity dimensions areappropriate for the accommodation of β-CAR, which could further induce theconstruction of a β-CAR-based substructure. We envisioned that β-CARmight bind WP5 in water to achieve WP5⊃β-CAR complexation (WCC) via thehydrophobic effect. Then, the resulting amphiphilic WCC might be able to form aβ-CAR-containing suprastructure in water via self-assembly. On the basis of thefascinating properties exhibited by natural chlorophyll/carotenoid complexes inphotosynthesis, chlorophyll-b (Chl-b) was selected as the co-assembly factorparticipating in the preparation process with WCC, and a similar suprastructure ofChl-b-containing LHC was synthesized. These hydrophilic complexes possess a suite ofunusual properties, including spontaneous growth, fusion, pH stimulus responsiveness andeven some photocatalytic activity.In this article, β1H-NMR spectroscopy after being dissolved in D2O.According to the results of Gaussian 09 calculations, the internal diameter ofthe cavity is 0.5 nm  and methyl protons(Ha and He) of the bulky terpene were observed(Δδ(Hb)=0.228 p.p.m.,Δδ(Hc)=0.259 p.p.m.,Δδ(Hd)=0.354 p.p.m.,Δδ(Ha)=0.160 p.p.m. andΔδ(He)=0.150p.p.m.) on theaddition of WP5 due to the shielding effect of the electron-rich cavities of WP5toward β-CAR, which clearly demonstrated the inclusion of the terpenesection of β-CAR into the hydrophobic WP5 cavity. Moreover, the signalsderived from protons Hg(Δδ(Hg)=−0.027 p.p.m.)and methyl protons (Hf) on linear unsaturated carbon chain shiftedfrom 6.1–6.8 p.p.m. to downfield6.6–6.3 p.p.m.(Δδ(Hf)=−0.5 p.p.m.).A similar phenomenon was also observed for the inclusion complexation betweenwater-soluble pillar[5]arene and guest in previousreports39a, Hb,Hc, Hd and He in the hydrophobic WP5 cavityand that the other protons on a linear unsaturated carbon chain  protruded out of the cavity. The binding affinity for suchhost–guest inclusion might be mainly driven by the hydrophobicinteractions. The generation of β-CAR-based WCC was then supported byRaman spectroscopy  blue-shifted(5 cm−1) from 1,521 to1,526 cm−1, which not only providedfurther direct evidence of host–guest interactions between WP5 andβ-CAR, but also further confirmed that the bulky groups at the ends ofthe β-CAR units were inserted into the cavityThe pillar[5]arene carboxyl derivative WP5 was preparedaccording to published procedures nm ref.  is prima nm ref. , the len nm ref. . Chl-bm nm ref. is likeltroscopy . The feaKa) ofWP5⊃β-CAR was calculated to be (2.34±2.06)× 105 M−1 using anon-linear curve-fitting method (– of WP5and the CH3– of β-CAR). As shown in −1 is attributed to anon-hydrogen-bonded –COOH group, which implies the presence of freecarboxyl groups−1 disappeared after thecomplexation. These results show that strong and orderly hydrogen bonds wereformed in the WCC.The encapsulation of a guest (G) in the hydrophobic cavity might also influencethe polarizability of the host (H) cavityg method . Aside fg method 44. To gag method  and lead⊃β-CAR-based HMS had an average diameter of1,855 nm . After the as-prepared solutions wereaged for 7 days, orange aggregates appeared in the solutions. As shown in 1,855 nm . Autoflu1,855 nm  and is a1,855 nm . The ele1,855 nm , across 1,855 nm  by SEM. 1,855 nm 46, indic1,855 nm ,providiq values (0.05 and0.1 Å−1) were observed in theprofile, indicating that there are multiple orders of structure with aninterlayer spacing of ∼3.1 nm in the HMS. However, thesepeaks are weak, possibly because of the partially inhomogeneous lamellarstructure within the HMS. In addition, considering the SAXS results, weconjectured that an interlayer spacing centred at 3.1 nm may exist inthe HMS. Furthermore, the zeta-potential of the HMS decreased from∼0 mV (pure β-CAR) to −31.6 mV,which provides further evidence that the hydrophilic WP5 is located on theexterior surface of the HMS , possibly due to the conformational change or partialcurling of the unsaturated carbon chain the HMS .On the basis of the aforementioned results, we proposed the following mechanismfor the formation of the WCC-based HMSs : Thehyd513 of the methylene moieties on bothrims were split into quartets from singlets, likely because of the inclusion ofChl-b in the cavity of WP5, which affected the swinging of the constituent unitsand led to the loss of the internal symmetry of the Hc protons.However, different from the remarkably chemical shift (β-CAR) beforeand after the complexation, the chemical shift of Chl-b changed slightly (Ka of WP5⊃Chl-b (WCB) wascalculated to be (3.46±0.14) ×103 M−1 . As in the LHCs containing Chl-b synthesis method, the β-CAR andChl-b powders were first dissolved in ethanol, and then, 2 ml of theβ-CAR and 2 ml of the Chl-b solutions were added to4 ml of an aqueous solution of WP5 using a pipette, without stirring.After the solution was aged for 7 days, pale-orange aggregates appeared.Meanwhile, a clear Tyndall effect .As a result, it is difficult to obtain a convincing data by Elementary Analysisor Inductive Coupled Plasma Emission Spectrometer (proportion of Chl-b in eachLHCs-b is different). Fortunately, EDX spectra provide a fine-testing method toinvestigate the β-CAR/Chl-b ratio in individual LHC-b. Thus, as shownin The route to synthesize the light-harvesting antenna is proposed in l effect  could bel effect . OMobsel effect . It is wl effect  demonstrl effect . The ultl effect .53 In adl effect , whichp2. The catalytic activity of LHCs-b was evaluated for the reduction of the pollutant4-nitrophenol (4-NP) after the addition of NaNOThese findings reveal that β-CAR suprastructures also exhibitedcatalytic activity. Reasonably, as the irradiation time was prolonged , a peak 612PtCl4, ascorbicacid (as an electron donor) and LHCs-b was irradiated for 2 h, LHCmicrospheres decorated with platinum nanoparticles were observed . Raman spectrawere measured in solution using an In Renishaw Via Raman spectrometer(λexc=532 nm). For Ramanmeasurements, aqueous dispersions were dried onto glass slides; the focus wasthen centred on an individual HMS for detection. Fluorescence spectra wererecorded on an F-4500 spectrometer; the spectra were corrected against aphotomultiplier and against the lamp intensity. The slit width of bothmonochromators was 5.0 nm. ultraviolet–visible absorptionspectra of the photocatalytic experiment  were recorded on a UV-2550 spectrophotometer.Ultraviolet–visible absorption spectra for assembly characterizationand photocatalysis experiments (0–120 min) were recorded ona UV-2501 spectrophotometer. FT-IR spectra were collected using potassiumbromide pellets on a TENSOR 27 spectrometer. Circular dichroism spectra weremeasured in a Jasco-815 spectropolarimeter. To compare the shift of each peakmore clearly, the intensity of the 649 nm peak in the LHCs containingChl-b was adjusted to the same intensity as the base of Chl-b. Dynamic lightscattering and zeta-potential measurements were carried out on a MalvernNanosizer S instrument at room temperature. OM and polarized OM images wereobtained using a Leica Microsystems DM LM/P instrument. Fluorescence OM imageswere acquired on an Olympus IX 73 W. D. 27 mm with an invertedconfiguration. For OM, polarized OM and fluorescence OM, aqueous dispersions oforange aggregates were dried onto glass slides for observation. The TEM imageswere obtained using a Philips TECNAI-12 instrument with an accelerating voltageof 120 kV. For TEM, aqueous dispersions of HMSs and Chl-b-containingLHCs were dried onto carbon-coated copper support grids. AHitachi S-4800 field-emission scanning electron microscope was used toinvestigate the HMS. For SEM, aqueous dispersions of HMSs were dried ontosilicon wafers. HRTEM and elemental mapping images were obtained using a TecnaiG2 F30 S-TWIN instrument. SAXS experiments were performed at room temperature ona NanoSTAR, Bruker-AXS, 30 W. For SAXS, dispersions of WCC-based HMSsand Chl-b-containing LHCs were loaded into special glass X-ray capillary tubeswith an internal diameter of 1.5 mm.All chemical reagents were purchased from Aladdin and used without furtherpurification. 1H-NMR spectroscopy in D2O. 1H-NMR: δ 6.7 ,4.28 , 3.90 .Carboxyl-modified pillar[5]arene WP5 was prepared by a modifiedliterature procedure1H-NMR spectroscopy in D2O.β-CAR (purity≥97%) and Chl-b(purity≥95%) were purchased from EKEAR (Shanghai). WP5 wasprepared by a modified literature procedure and was identified by1H-NMR characterization, β-CAR was dissolved inethanol-d6 to produce a 150 μM solution(curve a in 6). WP5 was dissolved inD2O to produce a 300 μM aqueous solution,and then, an equal volume of ethanol-d6 was added to the WP5solution to produce a 150 μM binary solvent solution(curve c in 6: D2O=1:1).The D2O aqueous solution of WP5 added to theethanol-d6 solution to produce a 150 μMWP5 and β-CAR complexation solution (curve b in 6: D2O=1:1). Clear1H-NMR spectra could not be achieved when β-CARethanol-d6 solution was added by an equal volume ofD2O due to the poor solubility and low concentration ofβ-CAR in water in the absence of WP5. Thus, to obtain clear spectra,ethanol-d6 instead of binary solvents(ethanol-d6: D2O=1:1) were used for theβ-CAR 1H-NMR characterization and for furthercomparison. In addition, the comparison between β-CAR(ethanol-d6) and WCC (ethanol-d6:D2O=1:1) is closer to the solvent environment changesin the assembly process conditions. For fluorescence characterization, variousconcentrations of β-CAR in ethanol solution were added to an aqueoussolution of WP5 to produce β-CAR/WP5 solutions with differentmolar ratios of 0, 0.2, 0.3, 0.6, 0.8, 1.2, 1.6, 2.0 and 2.5. The emissionintensities of the WP5 solutions at various molar ratios were monitored byfluorescence spectroscopy(λex=268 nm). Using anon-linear curve-fitting method, the Ka ofWP5⊃β-CAR was estimated.The non-linear curve fitting was basedon the equationΔF=(ΔF∞/[H]0)(0.5[G]0+0.5([H]0+1/Ka)−(0.5([G]02+(2[G]0(1/Ka−[H]0))+(1/Ka+[H]0)2) 0.5)) (1)  refs , 66. On 1H-NMR characterization, Chl-b was dissolved inethanol-d6 to produce a 150 μM solution(curve a in 6). WP5 wasdissolved in D2O to produce a 300 μM aqueoussolution, and then, an equal volume of ethanol-d6 was added toWP5 solution to produce a 150 μM binary solvent solution(curve c in 6:D2O=1:1). The D2O aqueous solution of WP5 addedto the ethanol-d6 solution to produce a150 μM WP5 and Chl-b complexation solution (curve b in6:D2O=1:1). In accordance with the method used in previousexperiments (WCC 1H-NMR characterization),ethanol-d6 was used for the Chl-b 1H-NMRcharacterization and for further comparison instead of binary solvents(ethanol-d6: D2O=1:1).For Ka for the complexation between WP5 and Chl-b,fluorescence titration experiments were carried out in solutions that had aconstant concentration of Chl-b and varying concentrations of WP5. Variousconcentrations of WP5 solutions were added to an ethanol solution of Chl-b atthe concentration of 100 μM to produce WP5/Chl-b solutions withdifferent molar ratios . In a typical experiment, 1,000 μl of WP5 at theconcentration of 20 μM were added to the Chl-b solution toproduce WP5/Chl-b solutions with a molar fraction of 0.2. Then,1,000 μl of WP5 at the concentration of40 μM were added to the Chl-b solution to produceWP5/Chl-b solutions with a molar fraction of 0.4. Subsequently,1,000 μl of WP5 at the concentration of60 μM were added to the Chl-b solution to produceWP5/Chl-b solutions with a molar fraction of 0.6. The solutions withthe other molar ratios  were preparedby a similar method. The emission intensities of the WP5 solutions at variousmolar ratios were monitored by fluorescence spectroscopy(λex=268 nm). Thenon-linear curve fitting was based on the equationΔF=(ΔF∞/[G]0)(0.5[H]0+0.5([G]0+1/Ka)−(0.5([H]02+(2[H]0(1/Ka−[G]0))+(1/Ka+[G]0)2) 0.5)) (2)  refs , 66, whiWP5 was first basified with sodium hydroxide to produce an aqueous solution.Then, β-CAR was dissolved in ethanol to produce an ethanol solution ofβ-CAR. Subsequently, β-CAR-based HMSs were prepared bypipetting an ethanol solution of β-CAR into an aqueous solution of WP5and then aging. The final concentration of host and guest could be controlled byadjusting their initial concentration. In a typical experiment, HMSs wereprepared by pipetting 2 ml of an ethanol solution of β-CAR(150 μM) into 2 ml of an aqueous solution ofWP5 (150 μM) and then aging the solution for 7 days atroom temperature.WP5 was first basified with sodium hydroxide to produce an aqueous solution.Then, β-CAR was dissolved in ethanol to produce an ethanol solution ofβ-CAR. Chl-b was dissolved in ethanol to produce an ethanol solution ofChl-b. Simlar to the synthesis method of HMSs, LHCs-b were prepared at roomtemperature by pipetting an ethanol solution of β-CAR and Chl-b into anaqueous solution of WP5. The final concentration of host and guest could becontrolled by adjusting their initial concentration. In a typical experiment,LHCs-b were prepared at room temperature by pipetting 2 ml of anethanol solution of β-CAR (150 μM) and2 ml of an ethanol solution of Chl-b (150 μM)into 4 ml of an aqueous solution of WP5. Mixing the solution resultedin a pale-orange turbid suspension that was subsequently aged for 7 days.2PtCl4(10 mM) and 25 μl of aqueous ascorbic acid(0.2 M) were sequentially added to a 2-ml glass vial and thenirradiated for 2 h with visible light from a 300-W Xe lamp. Pd and Agnanoparticles were prepared using similar methods.In a typical experiment, 0.50 ml of a suspension of LHCs-b,20 μl of aqueous KExperimental group. Blank sample: 1 ml of 4-NP stock solution(2 mM), 100 μl of sodium nitrite stock solution(0.1 M) and 300 μl of distilled water weremixed and homogenized in a glass vial and then irradiated with visible lightfrom a 500-W mercury lamp. β-CAR sample: One millilitre of 4-NP stocksolution (2 mM), 100 μl of sodium nitrite stocksolution (0.1 M) and 300 μl of a dispersion ofβ-CAR (75 μM) were mixed and homogenized in aglass vial and then irradiated with visible light from a 500-W mercury lamp.Chl-b sample: 1 ml of 4-NP stock solution (2 mM),100 μl of sodium nitrite stock solution (0.1 M)and 300 μl of a dispersion of Chl-b(75 μM) were mixed and homogenized in a glass vial andthen irradiated with visible light from a 500-W mercury lamp. HMS sample:1 ml of 4-NP stock solution (2 mM),100 μl of sodium nitrite stock solution (0.1 M)and 300 μl of a dispersion of HMS(75 μM) were mixed and homogenized in a glass vial andthen irradiated with visible light from a 500-W mercury lamp. LHCs-b sample:1 ml of 4-NP stock solution (2 mM),100 μl of sodium nitrite stock solution (0.1 M)and 300 μl of a dispersion of LHCs-b(75 μM) were mixed and homogenized in a glass vial andthen irradiated with visible light from a 500-W mercury lamp.Untreated control group. Blank sample: 1 ml of 4-NP stocksolution (2 mM), 100 μl sodium nitrite stocksolution (0.1 M) and 300 μl of distilled waterwere mixed and homogenized in a glass vial and then stored in the dark.β-CAR sample: 1 ml of 4-NP stock solution (2 mM),100 μl sodium nitrite stock solution (0.1 M)and 300 μl of a dispersion of β-CAR(75 μM) were mixed and homogenized in a glass vial andthen stored in the dark. Chl-b sample: 1 ml of 4-NP stock solution(2 mM), 100 μl sodium nitrite stock solution(0.1 M) and 300 μl of a dispersion of Chl-b(75 μM) were mixed and homogenized in a glass vial andthen stored in the dark. HMS sample: 1 ml of 4-NP stock solution(2 mM), 100 μl sodium nitrite stock solution(0.1 M) and 300 μl of a dispersion of HMS(75 μM) were mixed and homogenized in a glass vial andthen stored in the dark. LHCs-b: 1 ml of 4-NP stock solution(2 mM), 100 μl sodium nitrite stock solution(0.1 M) and 300 μl of a dispersion of LHCs-b(75 μM) were mixed and homogenized in a glass vial andthen stored in the dark. Changes in the absorption peaks over time associatedwith the photocatalytic reduction of 4-NP to 4-AP  were monitored by ultraviolet–visiblespectroscopy (UV-2501).For the photocatalytic activity investigation of the LHCs-b sample using a longerduration of irradiation, the procedure was as follows: A 4-NP stock solution(2 mM), a sodium nitrite stock solution (0.1 M) and adispersion of LHCs-b were mixed and homogenized in a glass vial and thenirradiated with visible light from a 300-W Xe lamp. Changes in the absorptionpeaks over time associated with the photocatalytic reduction of 4-NP to 4-AP(0–120 min) were monitored by ultraviolet–visiblespectroscopy (UV-2501).The authors declare that the data supporting the findings of this study areavailable within the article and its How to cite this article: Sun, Y. et al. Stimulus-responsivelight-harvesting complexes based on the pillararene-induced co-assembly ofβ-carotene and chlorophyll. Nat. Commun. 7:12042 doi:10.1038/ncomms12042 (2016).Supplementary Figures 1-27, Supplementary Discussion and SupplementaryReferences


H-pyrazoloquinoline are reported.The crystal structure and supramolecular features of 3-chloro-2-(4-methylphenyl)-2 H-pyrazoloquinoline, C17H12ClN3, (I), the dihedral angle between the planes of the pyrazole ring and the methylated phenyl ring is 54.25 (9)°. The bond distances in the fused tricyclic system provide evidence for 10-π delocalization in the pyrazolopyridine portion of the molecule, with diene character in the fused carbocyclic ring. In the crystal, molecules of (I) are linked by two independent C—H⋯N hydrogen bonds, forming sheets containing centrosymmetric R22(16) and R64(28) rings, and these sheets are all linked together by π–π stacking interactions with a ring-centroid separation of 3.5891 (9) Å.In the molecule of 3-chloro-2-(4-methylphenyl)-2 The ring-centroid separation is 3.5891 (9) Å, corresponding to a ring-centroid offset of ca 1.202 Å −1 rather than the usual kJ mol−1 b]quinolines carrying a substituent at the N2 position but not at N1, is sparse. In a series of pyrazoloquinolin-5-ones, each carrying a substituent at N2, the central heterocyclic ring is in reduced form, carrying H atoms at positions 4 and 8 .A sample of the title compound was prepared using the recently published procedure (Sowmya Uiso(H) = kUeq(C), where k = 1.5 for the methyl group, which was permitted to rotate but not to tilt, and 1.2 for all other H atoms.Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S205698901500818X/sj5454sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901500818X/sj5454Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S205698901500818X/sj5454Isup3.cmlSupporting information file. DOI: 1012361CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


SW/PS1dE9/APOE ε2-TR(APP/E2) and APPSW/PS1dE9/APOEε4-TR (APP/E4) mice, with targeted replacement (TR) of the murine Apoe forhuman ε2 or ε4 alleles, and used these models to investigate effects ofpharmacological inhibition of the apoE/Aβ interaction on Aβ deposition andneuritic degeneration. APP/E2 and APP/E4 mice replicate differential effectof human apoE isoforms on Aβ pathology with APP/E4 mice showing aseveral-fold greater load of Aβ plaques, insoluble brain Aβ levels, Aβoligomers, and density of neuritic plaques than APP/E2 mice. Furthermore,APP/E4 mice, but not APP/E2 mice, exhibit memory impairment on objectrecognition and radial arm maze tests. Between the age of 6 and 10 monthsAPP/E2 and APP/E4 mice received treatment with Aβ12-28P, a non-toxic,synthetic peptide homologous to the apoE binding motif within the Aβsequence, which competitively blocks the apoE/Aβ interaction. In both lines,the treatment significantly reduced brain Aβ accumulation, co-accumulationof apoE within Aβ plaques, and neuritic degeneration, and prevented memorydeficit in APP/E4 mice. These results indicate that both apoE2 and apoE4isoforms contribute to Aβ deposition and future therapies targeting theapoE/Aβ interaction could produce favorable outcome in APOE ε2 and ε4 allelecarriers.Accumulation of β-amyloid (Aβ) in the brain is essential to Alzheimer’sdisease (AD) pathogenesis. Carriers of the apolipoprotein E (APOE) ε4 alleledemonstrate greatly increased AD risk and enhanced brain Aβ deposition. Incontrast, APOE ε2 allele carries show reduced AD risk, later age of diseaseonset, and lesser Aβ accumulation. However, it remains elusive whether theapoE2 isoform exerts truly protective effect against Aβ pathology or apoE2plays deleterious role albeit less pronounced than the apoE4 isoform. Here,we characterizedAPPThe online version of this article (doi:10.1186/s40478-014-0075-0) contains supplementary material, which is available toauthorized users. To pharmacologically disrupt the apoE/Aβinteraction, we used a previously characterized non-toxic, synthetic peptideAβ12-28P, which is homologous to the apoE binding motif within the Aβsequence [I = 12.9 nM and neutralizesthe promoting effect of apoE on Aβ fibrillization invitro, but has no effect on self-assembly of Aβ in the absenceof apoE [Alzheimer’s disease (AD) is a familial and sporadic neurodegenerativedisease. Its neuropathological hallmarks include parenchymal plaques andvascular deposits of β-amyloid (Aβ), intraneuronal paired helical filamentscomposed of hyperphosphorylated tau and ubiquitin, widespread loss ofsynapses and neurons, and the appearance of reactive astrocytes andmicroglia. Converging lines of evidence derived from genetic,neuropathological, biomarker, and transgenic animal studies implicatedisturbance of Aβ homeostasis leading to its progressive accumulation in thebrain as a driving mechanism of AD pathogenesis . Aβ ieAβ1-42 ). ApoE ieAβ1-42  and apoEeAβ1-42 -8. ConveeAβ1-42 ,10. To peAβ1-42 . HowevereAβ1-42 ,12. In tpromoter . As prevque load  but do nine apoE . Since tsequence ,15. In A1-40, and Aβ1-42peptides were custom synthesized and purified in the WM Keck ProteomicFacility of Yale University  in the laboratory of Dr.James I. Elliott as previously described [Unless stated otherwise all reagents and antibodies were purchasedfrom Sigma-Aldrich . Aβ12-28P,Aβescribed .SW/PS1dE9 mice[SW/PS1dE9 transgene were used as anapoE isoform background control during the behavioral and serologicaltesting. After concluding the experiment, all animals were killed with anoverdose of sodium pentobarbital (150 mg/kg). The blood was collectedthrough a cardiac puncture and the serum was separated by centrifugation.Animals were transcardially perfused and their brains and internal organscollected for histopathological analysis as previously described[All mouse care and experimental procedures were approved byInstitutional Animal Care and Use Committees of the New York UniversitySchool of Medicine and the Washington University School of Medicine.APP/E2 and APP/E4 mice were generated by crossing-breeding ofAPPdE9 mice with APOdE9 mice,17 asprdE9 mice.APOE ε2dE9 mice andtherdE9 mice. Mice wedE9 mice,19,20. Abarbital 0 mg/kg. Series of 40 μm-thick serial brain sections evenly spaced every 400 μmalong the entire rostro-caudal brain axis were stained with Th-S tovisualize fibrillar Aβ deposits, with Gallyas silver stain to identifyplaque-associated neuritic dystrophy , or immux−40 and Aβx−42,respectively; as coating antibodies (1 μg/well) [x-40 and Aβx-42. ELISAreadouts were converted to actual peptide concentrations based onreadouts from standard curves prepared from FA-treated syntheticAβ1-40 and Aβ1-42peptides and multiplied by all dilutions made during DEA and FAextractions. Final values of brain concentrations of Aβ peptides werereported in reference to the total brain protein concentration, thelatter determined in a neat brain homogenate using the bicinchoninic acidassay . The concentration of Aβoligomers was determined in the neat homogenate using a human aggregatedβ-amyloid ELISA kit  [Cortical mantle and hippocampi were dissected out, homogenized, andsubjected to diethylamine (DEA) and FA extractions as previouslydescribed .Concentμg/well)  and biotμg/well) ,23.Sincbad, CA) .Sandwich ELISA for apoE was performed using mAb 3D12  as thecapture antibody and biotinylated goat anti-human apoE polyclonalantibody   as thedetection antibody. ApoE concentrations were quantified in DEA and FAbrain extracts. ELISA readouts were converted to apoE concentrationsusing standard curves prepared from recombinant human apoE2 or apoE4 and multiplied by alldilutions made during DEA or FA extractions. The final brain apoEconcentrations were reported in reference to the total brain proteinconcentration.t test with Welch’s correction following confirmation ofnormal data distribution by the Kolmogorov-Smirnov and Shapiro-Wilktests. All statistical analyses were performed using GraphPad Prism v5.04.RAM data were analyzed using one-way analysis of variance (ANOVA)followed by the Newman-Keuls post hoc test. All other data sets werecompared using the unpaired Student’s Female APP/E2 and APP/E4 mice received Aβ12-28P or vehicle throughintraperitoneal injections from the age of 6 to 10 months. The treatmentcommenced at the start of Aβ plaque formation in these Tg lines.Veterinary staff closely monitored the animals undergoing treatment forany signs of toxicity. Monitoring parameters included body weight,physical appearance, changes in coat appearance, occurrence of unprovokedbehavior, and blunted or exaggerated responses to external stimuli.APP/E2 and APP/E4 mice receiving vehicle and Aβ12-28P showed nodifferences in respect to those parameters compared to their WT/E2 andWT/E4 age and sex-matched littermates, respectively. Measurements of theimmune response against Aβ in post-treatment sera of APP/E2 and APP/E4mice showed no evidence of increased titer of anti-Aβ antibodies in IgGand IgM classes associated with Aβ12-28P treatment  , which tests animals’ long-term recognitionmemory, followed by the radial arm maze (RAM), which is a test ofanimals’ spatial working memory. Behavioral studies were conducted duringthe last 3 weeks of the treatment experiment and during the testing micecontinued to receive vehicle or Aβ12-28P injections. During theacquisition session of the ORT, when the animals are allowed to explorefreely the two identical objects presented in the testing arena, WT/E2,and vehicle- and Aβ12-28P-treated APP/E2 mice, equally interacted withboth objects as expected of normal mice (data not shown). Following a 3-hinterval, which the mice spent in their home cages, one of the twofamiliar objects was replaced with a novel one, and the behavior of themice was then observed during the retention session. All three testedmice groups spent significantly more time exploring the novel object thanthe familiar one during the retention session  andAβ12-28P-treated APP/E4 mice (p < 0.001), confirming the presence of memory impairmentin APP/E4 mice at the age of 10 months , and 34.4% (p < 0.05), respectively, while in APP/E4mice the reduction was 23.0% (p <0.01) and 21.7% (p < 0.05),respectively. As Th-S reveals only fibrillar structure of Aβ plaques, wealso stained serial brain sections with mAb HJ3.4 directed against theN-terminus of Aβ [The load of Th-S positive Aβ plaques  was quantified inthe neocortex and the hippocampus on serial coronal brain sections. Invehicle-treated APP/E4 mice, the load of Th-S-stained plaques in theneocortex and the hippocampus was 2.6- and 2.9-fold higher than that inAPP/E2 mice , respectively; while insolubleAβx-40 and Aβx-42 levelswere 5.6-fold (p < 0.001) and9.2-fold (p < 0.01) higher thanthose in APP/E2 mice, respectively , while in theAPP/E4 mice the opposite relation was observed, with the level of solubleAβx-42 being 1.4-fold higher than that ofsoluble Aβx-40 (p < 0.05). In APP/E2 mice, levels of insolubleAβx-40 and Aβx-42 weresimilar, while in APP/E4 mice the level of insolubleAβx-42 was 1.8-fold higher than that ofAβx-40 (p < 0.05). In both APP/E2 and APP/E4 mice, insolubleAβx-40 and Aβx-42 levelswere over 15-fold higher than those of soluble peptides (p < 0.001).Brain Aβy Figure A-D. Furtx-40 andAβx-42, which in APP/E2 mice were reduced by30.1% (p < 0.001) and 35.8%(p < 0.0001), respectively;while in APP/E4 mice by 32.1% (p < 0.05) and 38.2% (p < 0.01), respectively ; while inAβ12-28P-treated APP/E4 mice by 30.6% and 33.8%, respectively (p < 0.05) . Aβ12-28P treatment reduced the level of Aβoligomers by 46.1% and 23.5% in APP/E2 and APP/E4 mice, respectively(p < 0.001)   reduction in thesoluble apoE level in APP/E2 mice and a 45.7% reduction in the level inAPP/E4 mice (p < 0.0001). InsolubleapoE levels were reduced with Aβ12-28P treatment by 41.4% in APP/E2 mice(p < 0.0001) and by 54.6% inAPP/E4 mice (p < 0.0001).Levels of soluble and insoluble human apoE were measured in brainhomogenate extracted with DEA and FA, respectively. There was nosignificant difference in the level of soluble apoE betweenvehicle-treated APP/E2 and APP/E4 mice  and 4.2-fold higher in the hippocampus(p < 0.01) in vehicle-treatedAPP/E4 mice than in the corresponding structures of vehicle-treatedAPP/E2 mice  and a 37.5% reduction in the hippocampus(p < 0.05) in APP/E2 mice, andwith a 49.9% (p < 0.001) and 48.5%(p < 0.05) reduction in thecorresponding structures in APP/E4 mice, respectively.Furthermore, we quantified the load of apoE-positive plaque load,which also bound the amyloid dye Th-S. The load of apoE plaques was2.9-fold higher in the neocortex (p < 0.001) , while in APP/E4 mice by 48.8% (p < 0.001) and 43.6% (p < 0.01), respectively.Neuritic dystrophy associated with Aβ plaques was revealed using theGallyas silver stain  Figure A and B. SW/PS1dE9 AD Tg modelmice reproduces the differential effect of encoded by these alleles apoEisoforms on various aspects of Aβ pathology and associatedneurodegeneration. Ten-month-old APP/E2 mice showed no behavioral deficitcompared to WT/E2 mice, despite a modest Aβ plaque load and Aβ oligomerlevel. In contrast, age and sex matched APP/E4 mice revealed a pronouncedmemory deficit along with a several-fold higher load of Aβ plaques andinsoluble Aβ level, and nearly an 80-fold higher level of Aβ oligomerscompared to APP/E2 mice. Oligomeric Aβ species have been shown to causememory deficits in rodents [SW/PS1dE9 mice. APP/E4mice had significantly higher levels of soluble Aβ peptides than APP/E2mice. In particular, the level of soluble Aβx-42,which is considered more toxic and aggregation prone thanAβx-40, was 22.5-fold higher in APP/E4 mice thanin APP/E2 mice. Furthermore, in APP/E2 mice, the solubleAβx-40 level was 2.1-fold higher than that ofAβx-42, while in APP/E4 the ratio was reversed,with the level of soluble Aβx-42 being 1.4-foldhigher than that of Aβx-40. These strikingdifferences in the brain levels of soluble Aβx-40 andAβx-42 peptides, and the reversed ratio, betweenAPP/E2 and APP/E4 mice are likely related to the well-establisheddifferential effect of apoE2 and apoE4 isoforms on Aβ brain clearance[x-42 associated withthe apoE4 isoform, produce a background promoting greater Aβ deposition andenhanced neurodegeneration. In fact, the load of Th-S positive Aβ plaquesand the brain concentration of insoluble Aβ, which are associated withfibrillar Aβ deposits, were several-fold higher in APP/E4 mice than inAPP/E2 mice. Enhanced Aβ deposition was, in turn, associated with a greaterdegree of neurodegeneration, as demonstrated by a several-fold higherdensity of neuritic plaques in APP/E4 animals as compared to APP/E2 animals.APOE ε4 targeted replacement was also associated with significantly greaterco-deposition of apoE in Aβ plaques, as shown by the comparison ofapoE-positive plaque load and the level of insoluble apoE between APP/E4 andAPP/E2 mice. It has been previously demonstrated that co-deposition of apoEin Aβ plaques is a prerequisite for the occurrence of focal neuriticdystrophy [ApoE isoforms differentially modulate Aβ metabolism, Aβ accumulation, andAD morbidity in human subjects. We demonstrated here that APOE ε2 and APOEε4 targeted replacement inAPP rodents and are  rodents ,27, whiclearance. Increasystrophy .in vivoevidence that both apoE2 and apoE4 isoforms are involved in the process ofAβ aggregation and deposition and are associated with neurodegeneration,although the effect of apoE4 appears to be much stronger than that of apoE2.Our findings remain consistent with the effects of apoE knock-out noted inseveral AD Tg mouse models, where lack of apoE was associated with absenceof fibrillar Aβ deposits and prevented neurodegeneration [x-42 level, which is associated with theapoE4 background, there is experimental evidence to indicate that directinteraction between apoE and Aβ facilitates Aβ oligomeric assembly and thatall three isoforms promote this process in the rank order E4 > E3 > E2[SW/PS1dE9 mice canbe effectively reduced through targeting brain apoE with systemic anti-apoEpassive immunization [Epidemiological evidence and evidence from neuropathological assessmentof the impact of apoE isoforms on the brain Aβ load, have suggested thatapoE2 exerts a protective effect against Aβ pathology and AD morbidity,9,10, whneration ,29,31,32neration ,34.Alth E3 > E2. Reduced E3 > E2,23. It h E3 > E2,29,35. Rnization .x-40 andAβx-42 peptides were significantly reduced in bothAβ12-28P-treated APP/E2 and APP/E4 mice providing evidence that the apoE/Aβbinding antagonist may enhance soluble Aβ clearance.Clearance of soluble Aβ from the brain is critical in preventing itsaccumulation and there is evidence this process can be differentiallyregulated by apoE isoforms . SeveralThe accumulation of Aβ in the brain is considered to be a prime target ofdisease-modifying therapies for AD . Althoug


The title molecule crystallizes with two half-molecules in the asymmetric unit, the whole molecules being generated by twofold rotation symmetry. In the crystal, there are two interpenetrating three-dimensional networks involving the individual molecules that are linked by C—H⋯Br and Br⋯Br interactions. 8H8Br4N2, crystallizes in the enantiomorphic-defining space group P41212 and has a refined Flack x parameter of 0.04 (4). In the asymmetric unit, there are two half-molecules; the whole molecules (A and B) are generated by twofold rotation symmetry. In molecule A, the twofold axis is normal to the pyrazine ring passing through the centre of the ring, while in molecule B, the twofold rotation axis lies in the plane of the pyrazine ring bisecting the C—C aromatic bonds. The two molecules are pseudo-mirror images of one another, and the best fit of the two molecules was obtained for inverted molecule B on molecule A, with an r.m.s. deviation of 0.1048 Å and a maximum deviation of any two equivalent atoms of 0.2246 Å. In the crystal, the A molecules are linked by weak C—H⋯Br hydrogen bonds and Br⋯Br interactions [3.524 (3) Å], forming a three-dimensional framework. The B molecules are also linked by weak C—H⋯Br hydrogen bonds and Br⋯Br interactions [3.548 (3) Å], forming a three-dimensional network that interpenetrates the network of A molecules.The title compound, C In molecule A, the twofold axis is normal to the pyrazine ring passing through the centre of the ring. In molecule B, the twofold rotation axis lies in the plane of the pyrazine ring bisecting the C6—6ii and C7—C7ii bonds . Placed side by side, it can be seen that the two molecules are almost perfect mirror images of each other  ° in molecule B . The other torsion angles involving the Br—C—Car—Car (ar = aromatic) arms do not differ significantly (Table 2The main difference appears for the torsion angles Br1—C1—C2—C3 = −92.6 (15) ° in molecule y Table 2.A molecules, linked by weak C—H⋯Br hydrogen bonds and Br1⋯Br2iii interactions  ZnCl2], and a quasi-linear one-dimensional coordination polymer, {Mn(tampyz)Cl2·2H2O}n  was added well-ground N-bromosuccinimide . The mixture was stirred vigorously and heated to reflux. As soon as the reflux set in, the mixture was irradiated for 5 h with two 200 W lamps fitted at least 10 cm at opposite sides of the flask. After the mixture was then cooled firstly to room temperature and the floating succinimide filtered off. The orange filtrate was cooled overnight to 278 K to crystallize the remaining traces of succinimide, which was filtered off. The filtrate was evaporated and the residual orange oil dissolved in 50 ml of diethyl ether. This solution was maintained at 278 K for at least one week, whereupon a white crystalline material deposited. The solid was filtered off, then recrystallized in ethanol to give colourless rod-like crystals of the title compound: Yield 7.87 g (8%); m.p. 401–405 K; RF 0.54 . Analysis for C8H8Br4N2 (rM = 451.78 g/mol); Calculated (%): C 21.27; H 1.79; N 6.20. Found (%): C 21.41; H 1.72; N 6.10. Spectroscopic data: 1H-RMN : δ = 4.69  p.p.m.; 13C-RMN : δ = 150.41, 28.75 p.p.m. MS , m/z (%): 452 , 371 (100), 292 (13.2), 211 (20.7), 131 (32.7), 92 (20.4), 65 (18.8); IR : 3030 w, 2977 w, 1438 s, 1405 s, 1220 s, 1096 m, 923 w, 787 s, 731 m, 629 m, 596 w, 543 m, 445 m.The title compound was prepared by a modification of the procedure described by Ferigo  al. 1994. To 2,3,Uiso(H) = 1.2Ueq(C).Crystal data, data collection and structure refinement details are summarized in Table 310.1107/S1600536814011337/hb0005sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S1600536814011337/hb0005Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814011337/hb0005Isup3.cmlSupporting information file. DOI: 1004263CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


In comparison with the previous determination from photographic film data [Corbridge & Cox  and 2.2883 (11) compared to 2.25 (1) and 2.27 (1) Å]. In the title molecule, the ZnII atom is five-coordinated in a distorted square-pyramidal mode by two Cl atoms and by the three N atoms from the 2,2′:6′,2′′-terpyridine ligand. The latter is not planar and shows dihedral angles between the least-squares planes of the central pyridine ring and the terminal rings of 3.18 (8) and 6.36 (9)°. The molecules in the crystal structure pack with π–π interactions [centroid–centroid distance = 3.655 (2) Å] between pyridine rings of neighbouring terpyridine moieties. These, together with intermolecular C—H⋯Cl interactions, stablize the three-dimensional structure.The crystal structure of the title compound, [ZnCl Cox 1956. J. Chemfold 1966. Acta Cr R value = 0.24) and was later re-refined by Einstein & Penfold (1966R = 0.14). In both reports, the setting in P21/a of space group No. 14 was used. For background to terpyridine-based materials, see: Fermi et al.  crystallizing in space group No. 15, and the second polymorph (type II) crystallizing in space group No. 14 ] = 0.020wR(F2) = 0.056S = 1.052564 reflections190 parametersH-atom parameters constrainedmax = 0.24 e Å−3Δρmin = −0.24 e Å−3ΔρSMART  used to solve structure: SHELXS97  I, Global. DOI: 10.1107/S1600536814023605/wm5082Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S1600536814023605/wm5082fig1.tif. DOI: The molecular structure of (I), with the atom-numbering scheme. Displacement ellipsoids are drawn at the 50% probability level.Click here for additional data file.10.1107/S1600536814023605/wm5082fig2.tif. DOI: The arrangement of the molecules in the crystal structure of (I), showing π—π interactions (dashed red lines) and C—H⋯Cl hydrogen bonds (dashed green and turquoise lines).Click here for additional data file.10.1107/S1600536814023605/wm5082fig3.tif. DOI: Packing diagram of (I). All H atoms have been omitted for clarity.1029855CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


The geometry of oligomer metal complexes was carried out by electronic spectral analysis and magnetic measurement studies. Polymeric properties have also been carried out. Ligand was synthesized using pyromellitic dianhydride and 4-bromoaniline. It was duly characterized. All novel synthesized compounds 1 and 2a–f were evaluated for their antibacterial and antifungal activity. The results showed significantly higher antibacterial and antifungal activity of oligomer metal complexes compared to the ligand.Novel oligomer metal complexes ( The design and construction of polymeric metal complexes have received great attention –6. The s2 vapors, and visualized by spraying with Dragendorff's reagent. Infrared spectra (FT-IR) were obtained from KBr pellets in the range of 4000–400 cm−1 with a Perkin Elmer spectrum GX spectrophotometer (FT-IR) instrument. 1H NMR and 13C NMR spectra were acquired at 400 MHz on a Bruker NMR spectrometer using DMSO-d6  as a solvent as well as TMS an internal reference standard. Microanalytical  data was obtained by using a Perkin Elmer 2400 CHN elemental analyzer. The solid diffuse electronic spectra were recorded on a Beckman DK-2A spectrophotometer with a solid reflectance attachment. MgO was employed as a reference. Magnetic moments [4] as calibrant (Xg = 1644 × 10−6 cgs units at 20°C), by Citizen Balance (at room temperature). Molar susceptibilities were corrected using Pascal's constant n shows two broad bands around 15,949 cm−1 and 22,746 cm−1 due to the 2T2g → 2Eg transition while the second may be due to charge transfer, respectively. This suggests a distorted octahedral structure for the [Cu(L)(H2O)2]n polymer which was further confirmed by its μeff value 1.99 B.M. The [Ni(L)(H2O)2]n coordination polymer shows two absorption bands at 15,570 cm−1, 22,934 cm−1, and 9,851 cm−1 due to 3A2g → 3T1g(F) and 3A2g → 3T1g(P) and 3A2g → 3T2g, respectively. The [Co(L)(H2O)2]n polymer shows that two absorption bands, at 22,948 cm−1, 15,553 cm−1, and 9,845 cm−1 corresponding to 4T1g(F) →4T1g(P), 4T1g(F) →4A2g(F), and 4T1g(F) →4T2g(F) transitions, respectively, indicated an octahedral configuration for the [Ni(L)(H2O)2]n and [Co(L)(H2O)2]n polymers [μeff values 2.87 B.M. and 4.25 B.M. The spectrum of [Fe(L)(H2O)2]n shows bands at 36,062 cm−1 and 19,011 cm−1 assigned to the transitions 5T2g(F) →3T1g and 5T2g(F) →3Eg and its μeff 4.98 B.M. suggesting octahedral configuration. The spectrum of [Mn(L)(H2O)2]n shows weak bands at 16,468, 17,769, and 23,140 cm−1 assigned to the transitions 6A1g → 4T1g(4G), 6A1g → 4T2g(4G), and 6A1g → 4A1g, 4Eg, respectively, suggesting an octahedral structure for the [Mn(L)(H2O)2]n polymer [μeff value 5.54 B.M. As the spectrum of the [Zn(L)(H2O)2]n polymer is not well interpreted, its μeff value shows that it is diamagnetic as expected. Magnetic moments μeff of all oligomer metal complexes revealed that all oligomers except Zn(II) metal ion polymer are paramagnetic while Zn(II) metal ion oligomer is diamagnetic.The information regarding geometry of the oligomeric metal complexes were obtained from their electronic spectral data and magnetic moment values . The difpolymers . This co polymer . This co−1 in the temperature range 50–700°C under nitrogen which provides much information about the coordination compounds. In all the oligomer metal complexes, decomposition occurred in two steps  exhibited antibacterial activity against both Gram-positive and Gram-negative bacterial strains with zones of inhibition (ZOI) ranging from 20 mm to 37 mm  = 36 mm, ZOI(SA) = 35 mm, ZOI(ST) = 37 mm, and ZOI(EC) = 36 mm) was identified as a potent antibacterial agent against all Gram-positive and Gram-negative bacterial strains. Compound 2b (ZOI(BS) = 33 mm, ZOI(SA) = 32 mm, ZOI(ST) = 33 mm, and ZOI(EC) = 32 mm) also had good antibacterial activity against bacterial strains. Compounds 2a, 2c, 2d, and 2f exhibited moderate antibacterial activity. Compounds 1 and 2a–f exhibited less antibacterial activity as compared to standard antibiotic drug, ciprofloxacin (ZOI(BS) = 45 mm, ZOI(SA) = 46 mm, ZOI(ST) = 45 mm, and ZOI(EC) = 47 mm).Schiff base 2,5-bis((4-bromophenyl) carbamoyl)terephthalic acid was found less active than its metal complexes. Among the analogsπ-electron delocalization within the whole chelate ring system thus formed during coordination. This process of chelation thus increases the lipophilic nature of the central metal atom, which in turn favors its permeation through the lipoid layer of the membrane. This in turn is responsible for increasing the hydrophobic character and liposolubility of the molecule in crossing cell membrane of the microorganism and hence enhances the biological utilization ratio and activity of the testing drug/compound. The biological activity of compounds also depends on the nature of the ligand, concentration, lipophilicity, nature of metal ion, coordinating sites, and geometry of the complex.Comparative study of the growth inhibition zone values of Schiff base and its oligomer metal complexes indicated that the oligomer metal complexes exhibited higher antibacterial activity than free Schiff base . Such in1 and 2a–f) exhibited antifungal activity against different fungal strains ( 2e (ZOI(PE) = 35 mm, ZOI(BT) = 33 mm, ZOI(NS) = 36 mm, and ZOI(TS) = 34 mm) was identified as more active against all fungal strains. Compound 2b (ZOI(PE) = 33 mm, ZOI(BT) = 30 mm, ZOI(NS) = 32 mm, and ZOI(TS) = 31 mm) also had good antifungal activity against fungal strains. Compounds 2a, 2c, 2d, and 2f exhibited moderate antifungal activity. Compounds 1 and 2a–f exhibited less antifungal activity as compared to standard antibiotic drug, ketoconazole (ZOI(PE) = 43 mm, ZOI(BT) = 42 mm, ZOI(NS) = 45 mm, and ZOI(TS) = 41 mm).Based on the screening data from the antifungal studies, the following observations can be made. All compounds ( strains . Schiff Ligand and its oligomer metal complexes have been synthesized and were duly characterized by various spectroscopic techniques. The geometry of a central metal ion was confirmed by electronic spectra and magnetic susceptibility measurements. Antimicrobial activity of ligand and its oligomer metal complexes suggests that the complexes are more potent than the ligand.


The most promising compounds in this series were 1,1′-)bis)bis(methylene))bis(4-(dimethylamino)pyridinium) bromide (10a) and 1,1′-)bis)bis(methylene))-bis(7-chloro-4-(pyrrolidin-1-yl)quinolinium) bromide (10l), which inhibit human choline kinase (ChoKα1) with IC50 of 1.0 and 0.92 μM, respectively, in a range similar to that of the previously reported biscationic compounds MN58b and RSM932A. Our compounds show greater antiproliferative activities than do the reference compounds, with unprecedented values of GI50 in the nanomolar range for several of the cancer-cell lines assayed, and more importantly they present low toxicity in non-tumoral cell lines, suggesting a cancer-cell-selective antiproliferative activity. Docking studies predict that the compounds interact with the choline-binding site in agreement with the binding mode of most previously reported biscationic compounds. Moreover, the crystal structure of ChoKα1 with compound 10a reveals that this compound binds to the choline-binding site and mimics HC-3 binding mode as never before.A novel family of compounds derivative of 1,1′-)bis)bis(methylene))-bispyridinium or –bisquinolinium bromide ( Cancer is a worldwide health threat and the second leading cause of mortality in developed countries1et al.Protein kinases have emerged as one of the most important types of targets in cancer-drug discovery due to their major roles in regulating cell growth and survival and many other cell functions42+ to yield phosphocholine (PCho) and ADPCholine kinase (ChoK) EC 2.7.1.32) catalyzes the phosphorylation of choline by ATP in the presence of Mg.1.32 catchk-α) by alternative splicing, while ChoKβ (395 amino acids) is the product of a different gene (chk-β). The amino acid sequence identity is 56% between ChoKα and ChoKβ, and both chk-α and chk-β mRNAs, as well as their encoded protein isoforms, are ubiquitously expressed in diverse tissuesMammalian ChoK exists as three isoforms, encoded by two separate genes11Choline kinase is overexpressed in many tumors such as breast, lung, bladder, colon, prostate, ovary, and liver carcinomas1415Caenorhabditis elegans and human, in which two monomers were dimerized in each asymmetric unit1921HC-3, the first inhibitor of choline kinase described, MN58b and RSM923A, which belong to the first generation of Chok inhibitors2324252627RSM932A  has evidenced a low-toxicity profile with improved tolerability in micehttp://clinicaltrials.gov/ct2/show/NCT01215864).The determination of the crystal structures of ChoK proteins from 1 and 2, we identified the adenine and 1-benzyl-4-(dimethylamino)pyridinium as the most efficient fragments of these molecules by the deconvolution approach based on the ChoKα1/1 (PDB ID: 3ZM9)2 (PDB ID: 4BR3)HC-3.For compounds 3 and 4), asymmetrical bispyridinium compounds, proved to be good inhibitors and provide the discovery of a new inhibitory binding site on ChoKα1 Compound 4 3 (with biphenyl group as a linker) adopts a binding mode similar to the one observed for compound 2.The second generation of inhibitors )bis)bis(methylene))-bis[4-pyridinium or quinolinium] bromide derivatives with general structures 10a-l  of twelve 1,2-bis(p-methylphenoxy)ethane derivatives 10a-l, substituted in the methylphenoxy group with different cationic heads as moieties. These compounds can be also considered as more polar analogues of choline kinase inhibitor derivatives than those previously synthesized.Microwave-assisted reactions present several advantages, such as a remarkable reduction in reaction times compared to those of the conventionally heated reactions and often lead to improved yields4210a-l is shown in p-methylphenoy)ethane (7)444 also under microwave irradiation , to give the 1,2-bis(4-bromomethylphenoy)ethane (8)4545The syntheses of compounds 7 gave the best yields, while more quantity of 7 led to diminished yields. This result was due to the volume of the reactor, which allows only 5 mL of the mixture, while more than 100 mg of 7 derivative would need more solvent to dissolve it.We conducted different experiments to achieve these successful results with the MW . AlthougN2 reaction in acetonitrile under argon atmosphere for 72 h at reflux of 1,2-bis(4-bromomethylphenoxy)ethane (8) and the 4-substituted pyridine derivative (9a-c), quinuclidine derivative (9d-e) or 4-substituted quinoline or 7-Chloro-4-substituted quinoline (9f-l) to afford 10a-l with moderate or good yieldsFinally, the last step is the introduction of cationic heads (previously synthesized using the procedure reported3235363738392 (PDB ID: 4BR3)4 (PDB ID: 4CG8)2 and 4.Docking studies were made in order to design the new ChoK inhibitors. The crystal structures of greatest interest for docking studies are those of ChoKα1 isoenzyme in complex with compounds 2 (carbon atoms in yellow color) inserted into the Cho binding site, being stabilized by cation-π interactions with Tyr333, Tyr354, Tyr440, Trp420, Trp423, and Phe435 (carbon atoms in cyan color). In particular, the biphenyl group shows optimal parallel hydrophobic stacking interactions with Tyr354, and the 4-(dimethylamino)pyridinium moiety interacts through parallel cation-π interaction with Trp420. The orientation of this compound inside the Cho binding site was accommodated by a conformational change of Tyr333, which moved back to create an extra space30304 (carbon atoms in orange color) inserted into the ChoKα1 crystal structure. This compound adopts a new different binding mode, inducing a conformational change in some amino acids. Tyr256, Tyr333, and Trp420 are the residues that undergo the major changes, and the rotation of these side chains is critical to allow the insertion of the 4-chloro-N-methylaniline fragment into an additional binding site (carbon atoms in magenta color), being stabilized by hydrophobic interactions with Trp248, Tyr256, Tyr333, Leu419, Trp420, and Trp423. The rest of the molecule is located inside the Cho binding site (carbon atoms in cyan color), the pyridinium moieties being stabilized through cation-π interactions with Tyr333, Tyr354, Trp420, and Tyr4404 is more inserted into the Cho binding site in comparison to compound 2  has two 4-(dimethylamino)pyridinium cationic heads, similarly to compound 2 (2: i) the cationic head is situated close to Trp420, being stabilized by π-cation interactions with Trp420, Tyr333, and Trp423; and: ii) the benzyl fragment is also optimized by hydrophobic stacking interactions with Tyr354. The linker of compound 10a is extremely long, and the second 1-benzyl-4-(dimethylamino)pyridinium fragment is situated outside of the enzyme, an additional hydrophobic interaction occurring between the second phenyl fragment and Ile433. Compound 10l (carbon atoms in orange color) has two 7-chloro-4-(pyrrolidin-1-yl)quinolinium) cationic heads, one of which is inserted very similarly to compound 4 (methyl)amino)pyridinium cationic heads. One cationic head is also inserted into the additional binding site and into the choline binding site, as in compound 4, and the second cationic head is also inserted into the enzyme, but with a different orientation. Nevertheless, the most noteworthy effect is that in the resulting pose of these compounds the 1,2-dioxoethane fragment also adopted a synclinal conformation and, for this reason, all these compounds should be completely inserted into the enzyme and probably will show good ChoKα1 inhibition.ound 10l , one of It has been reported that a potent anticancer effect inducing maximal apoptosis is achieved only when ChoKα1 expression is specifically knocked down, without affecting ChoKβ levels10a for pyridinium compounds and 10f, 10g, 10k, and 10l for quinolinium compounds) and, since selectivity may be explained by a reduced flexibility of Trp353 in ChoKβ compared to its homologue Trp420 in ChoKα1, as was proposed for HC-34, the results of the spectroscopy analysis, depicted in Kd values of these compounds for ChoKα1 were in the low μM range (0.241–0.700 μM), indicating high affinity for the enzyme. These results agree with the first experimental validation of the docking studies described above.We selected the most representative compounds of each family,  and the inhibitory effect on purified human ChoKα1 activity.10a-b and 10j-l, offer the best results in terms of enzyme inhibition and antiproliferative assays.Of all tested compounds, the ones that present an alkylamine or a cycloalkylamine substituted at position 4 of the pyridinium or quinolinium system, RSM932A and MN58b. The docking studies indicated that all compounds could be inserted into the choline binding site and could be grouped into two families on the basis of their different insertion modes. Compounds 10a, 10b, 10d, and 10e could be inserted similarly to compound 2. Compound 10a showed good HsChoKα1 inhibition (IC50 = 1.0 μM), thanks to the presence of the 1-benzyl-4-(dimethylamino)pyridinium fragment, which performs a strong π-cation interaction with the Cho binding site (10b showed a reduced ChoKα1 inhibition (IC50 = 9.56 μM) attributable to the volume of the 4-(pyrrolidin-1-yl)pyridinium cationic head, causing a decrease in the π-cation interaction (10d showed very poor ChoKα1 inhibition (IC50 = 37.54 μM). This result may be due to the presence of the quinuclidine and the consequently low lipophilicity (clog P = −1.01) cationic head that probably prevented the interactions with Tyr333, Tyr354, Trp420, and Tyr440 (10e had a 3-hydroxyquinuclidine cationic head and low lipophilicity (clog P = −2.42), it showed better ChoKα1 inhibition (IC50 = 9.51 μM) than could be explained by the establishment of two additional H-bonds with the enzyme , low lipophilicity values are sufficient to achieve good inhibition of the enzyme, the pydidinic ring being the most appropriate moiety, and the quinuclidine ring decreases the activity unless the lack of aromatic ring is offset by the formation of a H bond provided by the hydroxyl group.In summary, when compounds bind to the choline site, such as compound 4. Compounds 10c 10j-l showed a good ChoKα1 inhibition , while compounds 10f-i showed a slightly reduced ChoKα1 inhibition . All these compounds had a 4-substituted and 7-substituted quinolinium cationic head, 10f-l, except 10c, which had the rest of the N-methylanilino at the 4 position of the pyridinium cationic head. The structure of the 4-substituted fragment conditions the inhibitory activity of these molecules. In fact, compounds 10j-l had a 4-cycloalkylamino fragment, while compounds 10c and 10f-i had a 4-N-methylanilino substituent. The resulting IC50 values indicated that the 4-cycloalkylamino favored the ChoKα1 inhibition, probably promoting a more effective insertion into the additional binding site. In this group the chloro atom increased the volume of molecule and the lipophilicity. Thus, the compound 10l offered the right balance between volume and lipophilicity. The perhydroazepine group (10i) provided the volume to be inserted into the choline binding site properly and the addition of a choro atom slightly depressed the activity (10j).The second group interacted with the choline binding site as compound N-methylanilino system at the 4 position of the pyridinium or quinolinium cationic heads showed lower IC50 than did those in the alkylamine system. In these compounds, the chloro atom in para position of 4-(methyl(phenyl)amino)quinolinium or pyridinium fragment  or in position 7 of the quinolinium ring (10h and 10i) seemed to play an essential role in the enzyme inhibition. In fact, the presence of the chloro atom allowed the cationic head to be accommodated at the choline-binding site, likely by an increase in the lipophilicity provided by the halogen atom, regardless of where the halogen was located . A direct correlation between volume-lipophilic activity was found in these compounds , so that the less bulky and lipophilic compounds offered the best values  while adding a second aromatic ring slightly diminished the activity of 10f-h. On the other hand, the two chloro atoms present in compound 10i made the molecule too bulky to bind to the choline site, lowering its inhibition activity , and raising its lipophilicity (clog P = 4.23). However, the absence of chloro atom in 10f also considerably decreased ChoKα1 inhibition (IC50 = 6.85 μM), highlighting the important role of the chloro atom.Conversely, the compounds with an 10a, 10j, 10k, and 10l , These results reveal that the volume of the cationic head probably fits properly into the choline binding site. The only exception was 10b (IC50 = 9.56 μM), and this was a consequence of the higher volume of the 4-(pyrrolidin-1-yl)pyridinium cationic head that diminished the π-cation interaction . Compared to quinolinium derivatives, in general the pyridinium moiety provided better results in all tested cell lines.10a-b). The switching of dimethylamine to pyrrolidine caused a curious decline in activity for the majority of cell lines . The replacement of this tertiary amine by a conjugate aromatic system (4-(4-chloro-N-methylanilino)pyridinium, 10c) resulted in lower activity (GI50 = 2.1 μM to 7.3 μM). It bears noting that despite of their low lipophilicity , the compounds 10a-b provided better results than did compound 10c (clog P = 1.8). This finding can be explained by the different mode to bind to enzymes of these compounds, as mentioned above.In the pyridinium family, the best results were found when the substitution was an alkyl or cycloalkylamine  and to the low IC50 value.Replacing the pyridinium system with quinuclidine as cationic head, such as compounds 10f-l), all compounds had GI50 in the range of submicromolar values, 10l being the best compound (0.007 μM for Jurkat cells). No differences in the activity were detected between the different substituents in the 4 position upon the quinolinium system, since 10f and 10l  offer the best antiproliferative activity over nearly all the cell lines. However, the presence or not of a chloro atom over the quinolinium system, seems to play a crucial role in inhibiting cell growth. The chloro atom provided not only a higher lipophilicity to these compounds, improving the passage through the plasma membrane, but also a larger volume that impaired the insertion in the choline binding site. In fact, compound 10f, which has an N-methylanilino group at the 4 position without any chloro atom, exhibited better antiproliferative activity than did compound 10h or 10g, which have a chloro atom in the quinolinium.Regarding the quinolinium family  than 10g and 10h  with chloro in para position of the N-methylanilino system (10g) or in 7 position of quinolinium fragment (10h). The chloro atom provided lipophilicity and good results of enzyme inhibition but this lipophilicity can allow binding to other targets in the cancer cells. On the other hand, the presence of two chloro atoms make compound 10i too bulky to inhibit the enzyme (IC50 = 16.22 μM), strongly reducing the antiproliferative activity, and it is also more lipophilic (clog P = 4.23), and thus does not bind to other targets. In conclusion, among the quinolinium family, the compounds with an N-methylanilino system upon the 4 position (10f-i), 10f offers the best antiproliferative values but lower inhibition of the enzyme (6.85 μM). This could be due to a greater affinity of 10f by the enzyme in a whole cancer cell while the higher lipophicility of 10g-h could help them to be more suitable for binding to other targets. This highlights the need for a balance between lipophilicity, inhibitory activity of the enzyme isolated (affinity and selectivity), and antiproliferative activity for achieving successful results.However, 10k improves the activity. This suggests that although the enzymatic inhibitory activity is almost the same for these compounds (IC50 from 0.92 to 2.02 μM), the chloro leads to an appreciable increase in the antiproliferative activity (10j vs. 10k) in nearly all the cell lines.The insertion of chloro in position 7 as in compound 10a and 10l were the compounds with the best GI50 values in almost all the cell lines. The two belong to different subfamilies. The first one, 10a, has a residue of dimethylaminopyridinium as a cationic head which could offer the best volume to fit in a choline binding site, while the compound 10l has a residue of 7-Chloro-4-pyrrolidinequinolinium which provides more lipophilicity to the quinolinium moiety, allowing the compound to cross the plasma membrane more easily.Finally, 10a was also evaluated by the trypan blue exclusion assay. The results depicted in 10a significantly inhibited cell growth in three cell lines  in non-tumoral cells e.g. human lymphocytes and human umbilical-vein endothelial cells (HUVEC) from healthy donors. As shown in 10a had lower toxicity compared to tumor cells. Instead, in lymphocytes stimulated with a mitogen , the compound had cytotoxicity comparable to that seen in tumor cells, indicating a certain preference only towards proliferating cells. In this context, it bears noting that other choline kinase inhibitors such as MN58b or RSM932A presented low or reduced cytotoxicity in oncogene-transformed cells and in tumor cells, in agreement with previously published data44We investigated the effect of the most active compound  was too hydrophobic pyridinium fragment adopted .This moiety was completely superimposed in the two poses, indicating the accuracy of the theoretical predictions. Nevertheless, the second 4-(dimethylamino)pyridinium moiety was flipped almost 90° towards the residue Phe361 but not towards the residue Ile433, as the docking had predicted. The reason for this difference is that the pyridinium ring set π-π interactions with Phe361 in the crystal pose, increasing the stability of the ligand-protein complex.2 (PDB ID:4BR3) and HC-3 (PDB ID: 3G15) pyridinium fragment was positioned at the choline binding site in the same place as one of the previously reported ChoKα1 inhibitors, such as compound D: 3G15) . This innic head .10a induced a G1 arrest of the cell cycle, which occurred in a concentration-dependent manner in the three cell lines tested . Together with the G1 increase, a concomitant reduction was found in the S phase  higher than that used in the present study.Compound  S phase . Notably10a causes cell death, we conducted a biparametric cytofluorimetric analysis using PI, which stains DNA and enters only dead cells, together with fluorescent immunolabeling of the protein annexin-V, which binds to PS in a highly selective manner. Dual staining for annexin-V and with PI enables the discrimination between live cells (annexin-V−/PI−), early apoptotic cells (annexin-V+/PI−), late apoptotic cells (annexin-V+/PI+), and necrotic cells (annexin-V−/PI+). For a positive control, we used two well-known anticancer drugs such as Cis-Pt and Etoposide that in all cell lines tested induce significant apoptosis.To evaluate whether 10a after 72 h of incubation induced a modest increment in apoptotic cells only in Jurkat cells while both in MCF-7 and MDA-MB-231 the increase appeared not to be significantly different from that of the untreated cells.As depicted in 10a-l) and their evaluation as inhibitors of choline kinase against a panel of cancer-cell lines are described. These compounds were efficiently synthesized in three steps, starting from the building block 6. The chemistry used was appropriate to obtain the designed target compounds, and both the yield and the time of reaction were improved considerably with microwave irradiation.In conclusion, the synthesis of a novel family of 1,1′-)bis)bis(methylene))-bispyridinium or –bisquinolinium bromide (2 (PDB ID: 4BR3) and (ChoKα1/4 PDB ID: 4CG8), and the analysis of the resulting poses, indicated that these compounds (10a-l) could adopt similar behaviour to that of compound 2 or to compound 4 thanks to the synclinal conformation of the linker that allowed the insertion of these molecules inside the Cho binding site and consequently enhanced the antiproliferative effect. The first experimental validation of the docking studies is shown with the results of tryptophan assays for these compounds, which offer very good Kd values. Among all the compounds, those belonging to the families of pyridinium and quinolinium offered similar or better IC50 ChoKα1 than did the lead compounds MN58b and RSM932A. In fact, the best inhibitors were 10a and 10l, and the crystal structure of ChoKα1/10a showed that the compound binds to the choline binding site, indicating the accuracy of the theoretical predictions, wherein the first one-benzyl- 4- (dimethylamino) pyridinium represents the appropriate fragment to inhibit the enzymePreliminary docking studies performed on both crystal structures, ChoKα1/RSM932A and MN58b in a panel of human tumor-cell lines. More importantly, our compounds presented lower or reduced toxicity in some non-tumor-cell lines in comparison to transformed cells. Indeed our results agree with previous observations indicating increased ChoKα1 activity in cell cultures treated with growth factors or insulin2349Also, we have shown these compounds to have an excellent antiproliferative profile, better than that of the two lead compounds 10a in fact presented higher activity only in rapidly proliferating cells such as mitogen-stimulated lymphocytes in comparison to quiescent cells.In this context it is important to note that our results indeed confirm these previous findings, and compound 10a significantly arrested the cell cycle in G1 together with a sharp reduction of the S phase, confirming their ability to inhibit cell growth. Curiously, despite their ability to block cell proliferation, 10a induced a low proportion of cell death, as reflected by a low level of Annexin-V positive cells , which can have an off-target effect.Another important finding is that ve cells . Notably10a is a highly promising new Chokα1 inhibitor and is worthy of further preclinical evaluation as a potential anticancer drug.This intriguing aspect is under active investigation by our group. Nevertheless, our data demonstrate that 8) in dry CH3CN was added drop by drop to a solution of 9a-l (2 eq) in dry CH3CN under argon conditions. The mixture was heated under reflux for a 3 additional days and, after cooling down to room temperature, washed with diethyl ether and hexane, filtered, and dried under vacuum to afford 10a-l as a solid product.A solution of 1eq 1,2-bis(4-bromomethylphenoy)ethane  δ: 8.20 , 7.35 , 7.02 , 6.99 , 5.30 , 4.33 , 3.24 . 13C RMN  δ: 161.80 × 2, 158.87 × 2, 143.76 × 4, 131.98 × 4, 129.20 × 2, 117.30 × 4, 109.93 × 4, 68.86 × 2, 62.22 × 2, 41.20 × 4. HRMS (m/z): [M]2+ calcd for C30H36N4O2: 242.1419, found: 242.1409.Following general procedure C furnished 10b as a brown solid, yield 48%, mp: 129–130 °C. 1H NMR  δ: 8.17 , 7.34 , 7.01 , 6.84 , 5.28 , 4.32 , 3.54 , 2.11 , 13C NMR  δ: 161.79 × 2, 156.05 × 2, 143.68 × 4, 131.92 × 4, 129.31 × 2, 117.29 × 4, 110.51 × 4, 68.86 × 2, 62.26 × 2, 50.28 × 4, 26.98 × 4. HRMS (m/z): [M]2+ calcd for C34H40N4O2: 268.1576, found: 268.1568.Following general procedure C furnished 10c as a white solid, yield 30%, mp: >300 °C. 1H NMR  δ: 8.28 , 7.58 , 7.37 , 7.02 , 6.92 , 5.35 , 4.33 , 3.53 . 13C NMR  δ: 160.85 × 2, 158. 31 × 2 143.52 × 4, 135.52 × 2, 131.94 × 4, 131.13 × 4, 129.28 × 4, 127.95 × 2, 116.27 × 8, 110.27 × 2, 67.81 × 2, 61.64 × 2, 30.55 × 2. HRMS (m/z): [M]2+ Calcd for C20H19N2OCl: 338.1186, found: 338.1194.Following general procedure C furnished 10d as a white solid, yield 56%, mp: >300 °C. 1H NMR  δ: 7.47 , 7.13 , 4.43 , 4.35 , 3.48 , 2.18 , 2.01 . 13C NMR  δ: 162.80 × 2, 136.48 × 4, 121.54 × 2, 117.14 × 4, 69.52 × 2, 68.87 × 2, 56.39 × 6, 25.81 × 6, 22.33 × 2. HRMS (m/z) [M]2+ calcd for C30H42N2O2: 231.1623, found: 231.1628.Following general procedure C furnished 10e as a white solid, yield 63%, mp: 268–270 °C. 1H NMR  δ: 7.48 , 7.13 , 4.43 , 4.41 , 4.08, 3.64, 3.35, 3.04, 2.27, 2.10 , 13C NMR  δ: 159.52 × 4, 134.43 × 8, 119.63 × 4, 114.77 × 8, 65.61 × 4, 62.26 66.37, 63.27, 53.46, 52.52, 26.83, 20.87, 17.29 × 4. HRMS (m/z) [M]2+ calcd for C30H42N2O4: 247.1572, found: 247.1565.Following general procedure C furnished 10f as a yellow solid, yield 64%, mp: 169–170 °C. 1H NMR  δ: 8.86 , 8.13 . 7.81 , 7.62 , 7.53 , 7.46 , 7.40–7.29 , 7.03 , 5.89 , 4.33 , 3.84 . 13C NMR  δ: 161.47 × 2, 160.80 × 2, 150.14 × 2, 148.54 × 2, 141.68 × 2, 135.51 × 2, 132.75 × 4, 130.63 × 4, 130.28 × 2, 130.15 × 2, 128.64 × 2, 127.85 × 2, 127.73 × 4, 122.29 × 2, 120.97 × 2, 117.30 × 4, 107.73 × 2, 68.85 × 2, 59.94 × 2, 46.75 × 2. HRMS (m/z) [M]2+ calcd for C48H44N4O2: 354.1732, found: 354.1736.Following general procedure C furnished 10g as a yellow solid, yield 70%, mp: 178–180 °C. 1H NMR  δ: 8.90 , 8.16 , 7.84 , 7.67 , 7.52 , 7.41–7.37 , 7.33 , 7.03 , 5.91 , 4.33 . 3.82 , 13C NMR  δ: 161.48 × 2, 160.92 × 2, 148.81 × 2, 148.77 × 2, 141.68 × 2, 135.68 × 2, 135.58 × 2, 132.72 × 4, 130.69 × 4, 130.00 × 2, 129.28 × 4, 128.52 × 2, 128.20 × 2, 122.41 × 2, 121.13 × 2, 117.30 × 4, 108.36 × 2, 68.84 × 2, 60.09 × 2, 46.53 × 2. HRMS (m/z) [M]2+ calcd for C48H42N4O2Cl2: 388.1342, found: 388.1338.Following general procedure C furnished 2Cl2: MeOH (9:1 v/v) as eluent to obtain 10h as a yellow-green solid, yield 61%, mp: 181–183 °C. 1H NMR  δ: 8.82 , 8.15 , 7.56–7.53 , 7.47 , 7.41–7.40 , 7.34–7.31 , 7.29 , 7.06–7.03 , 5.85 , 4.34 , 3.82 . 13C NMR  δ: 161.58 × 2, 160.51 × 2, 149.76 × 2, 148.95 × 2, 142.51 × 2, 141.90 × 2, 132.92 × 4, 131.80 × 2, 130.77 × 4, 130.59 × 2, 128.35 × 2, 128.24 × 2, 127.72 × 4, 128.24 × 2, 120.68 × 2, 120.45 × 2, 117.41 × 4, 107.93 × 2, 68.86 × 2, 59.91 × 2, 46.87 × 2. HRMS (m/z): [M]2+ calcd for C48H42N4O2Cl2: 388.1342, found: 388.1331.Following general procedure C furnished the crude residue which was purified by flash chromatography using CH2Cl2: MeOH (9:1 v/v) as eluent to obtain 10i as a yellow solid, yield 39%, mp: 185–186 °C, 1H NMR  δ: 8.87 , 8.20 , 7.63 , 7.55 , 7.42 , 7.40 , 7.38 , 7.35 , 7.06 , 5.88 , 4.35 , 3.82 . 13C NMR  δ: 161.59 × 2, 160.65 × 2, 149.22 × 2, 148.45 × 2, 142.50 × 2, 142.06 × 2, 135.92 × 2, 132.88 × 4, 131.68 × 2, 130.85 × 4, 129.33 × 4, 128.71 × 2, 128.15 × 2, 120.82 × 2, 120.61 × 2, 117.40 × 4, 108.57 × 2, 68.85 × 2, 60.05 × 2, 46.69 × 2. HRMS (m/z) [M]2+ calcd for C48H40N4O2Cl4: 422.0953, found: 422.0952.Following general procedure C furnished the crude residue which was purified by flash chromatography using CH10j as a yellow solid, yield 67%, mp: 75–77 °C. 1H RMN  δ: 8.54 , 8.42 , 8.05 , 7.90 , 7.66 , 7.27 , 7.10 , 7.00 , 5.74 , 4.30 , 4.09 , 2.09 , 1.75 , 13C NMR  δ: 161.91 × 2, 161.34 × 2, 146.48 × 2, 142.10 × 2, 135.52 × 2, 130.48 × 4, 130.44 × 2, 128.98 × 2, 127.04 × 2, 121.41 × 2, 120.33 × 2, 117.22 × 4, 104.55 × 2, 68.84 × 2, 58.99 × 2, 56.06 × 4, 29.37 × 4, 29.23 × 4. HRMS (m/z) [M]2+ calcd for C46H52N4O2: 346.6700, found 346.2039.Following general procedure C furnished 2Cl2: MeOH (8:1 v/v) as eluent to obtain 10k as a white solid, yield 41%, mp: 87–88 °C. 1H RMN  δ: 8.51 , 8.40 , 8.07 , 7.29 , 7.65 , 7.11 , 7.03 , 5.72 , 4.33 , 4.08 , 2.08 , 1.74 . 13C NMR  δ :161.56 × 2, 161.44 × 2, 146.77 × 2, 142.95 × 2, 141.82 × 2, 132.31 × 2, 130.59 × 4, 128.57 × 2, 127.45 × 2, 119.88 × 2, 119.78 × 2, 117.32 × 4, 105.00 × 2, 68.85 × 2, 59.00 × 2, 56.12 × 4, 29.28 × 4, 29.16 × 4. HRMS (m/z) [M-Br]+ calcd for C46H50N4O2Cl2Br : 839.24949, found: 839.2494.Following general procedure C furnished the crude residue which was purified by flash chromatography using CH2Cl2: MeOH (9:1 v/v) as eluent to obtain 10l as a white solid, yield 48%, mp: 118–120 °C. 1H RMN  δ: 8.63 , 8.51 , 8.04 , 7.66 , 7.27 , 7.01 , 6.90 , 5.71 , 4.32 , 4.02 , 2.20 . 13C NMR  δ: 161.37 × 2, 157.92 × 2, 146.91 × 2, 142.45 × 2, 141.76 × 2, 131.76 × 2, 130.45 × 4, 128.68 × 2, 127.71 × 2, 120.16 × 2, 119.73 × 2, 117.29 × 4, 104.64 × 2, 68.83 × 2, 59.06 × 2, 55.43 × 4, 24.64 × 4. HRMS (m/z) [M]2+ calcd for C42H42N4O2Cl2: 352.1337, found: 352.1353.Following general procedure C furnished the crude residue which was purified by flash chromatography using CH2 (PDB entry 4BR3) and 4 (PDB entry 4CG8) were used for docking studies. In both cases, using the Structure Preparation Tool module of Sybyl refined protein structure. Missing side chains of those residues situated far away from the binding sites were added and protein N-terminal and C-terminal were fixed with ACE and NME, respectively. Hydrogens and charges were also added and protonation type of Glu, Asp, Gln and Asp was analyzed and fixed. Hydrogen orientations were also checked in order to maintain intramolecular hydrogen bonds within the protein. Finally, the molecules of compounds 2 and 4 inserted into the ATP binding site were carefully checked to assure the correction of these molecules. Structures of compounds 10a-l were constructed from standard fragments of the Libraries of the Sybyl program, and used as ligands for docking studies. As previously describedMolecular-modeling studies were performed by using Sybyl program2 or 4 inserted into the ChoK binding site, with a threshold value of 0.5 and a Bloat of 0 A. Surflex-Dock GeomX (SFXC) protocol was used, the search grid was expanded in 5 Å, 50 additional starting conformations were used for each molecule and 30 conformations per fragment. The results were analyzed using the Sybyl program and the most stable pose for each molecule was chosen as the preferred one inside the ChoK enzyme. Figures were built using the PyMOL programThe Surflex-Dock14C]choline chloride (55 mCi/mmol) was supplied by Perkin Elmer . Fetal bovine serum (FBS) was from The Cell Culture Company . Minimal essential medium (MEM) was from Sigma-Aldrich . Cell proliferation reagent WST-1 was from Roche Applied Sciences . Thin-layer chromatography (TLC) plates of Silica Gel 60 A were acquired from Analtech . Microwell plates and culture dishes were obtained from NuncTM . All other reagents were of analytical grade.[methyl-14C from [methyl-14C]choline into phosphocholine both in the presence or absence of different inhibitor concentrations. Briefly, the final reaction mixture contained 100 mM Tris (pH 8.5), 10 mM MgCl2, and 10 mM ATP, and 20 ng of purified ChoKα1. After the samples were preincubated at 37 °C for 5 min, the reaction was initiated with 1 mM [methyl-14C]choline (4500 dpm/nmol) and incubated at 37 °C for 10 min, the final volume being 55 μl. The assay was stopped by immersing the reaction tubes in boiling water for 3 min. Aliquots of the reaction mixture were applied to the origin of Silica Gel plates in the presence of phosphocholine (0.1 mg) and choline (0.1 mg) as carriers. The chromatography was developed in methanol/0.6% NaCl/28% NH4OH in water  as solvent. Phosphocholine was visualized under exposure to iodine vapor and the corresponding spot was scraped and transferred to scintillation vials for measurement of radioactivity by a Beckman 6000-TA  liquid-scintillation counter. At least three experiments were performed in all assays. The 50% inhibitory concentrations  were determined from the % activity of the enzymes at different concentrations of synthetic inhibitors by using a sigmoidal dose-response curve (the ED50plus v1.0 software).The effect of the different inhibitors on human choline kinase (ChoK) was assayed in ChoKα1 purified as previously describedChoKα1 have been previously reportedDetails on cloning and purification of human Kds against human ChoKα1 were measured by monitoring the quenching of tryptophan fluorescence. All experiments were performed in a Cary Eclipse spectrofluorometer (Varian) at 25 °C with the enzymes at 1 μM, and concentrations of compounds varying from 0.1 to 5 μM for ChoKα1 in 25 mM Tris, 150 mM NaCl, pH 7.5. Fluorescence emission spectra were recorded in the 300–400 nm range with an excitation wavelength of 280 nm, with slit width of 5 nm. Controls were determined by incubating the enzymes with equivalent amounts of DMSO. As indicated previously, data analysis was performed in Prism (GraphPad software) considering a model with a single binding site (F0 is the intrinsic fluorescence of the enzyme in the absence of quencher (Q), F1 is the observed fluorescence at a given quencher concentration, fa is the fractional degree of fluorescence, and Kd is the dissociation constant.All compounds were prepared in 100% DMSO. Their ing site , where F10a, 10g, 10h, 10k, and 10l in DMSO  for two days. Only crystals soaked with compound 10a contained the compound. The crystals used in this study were cryoprotected in mother-liquor solutions containing 20% ethylenglycol and frozen in a nitrogen gas stream cooled to 100 K.ChoKα1 at 20 mg/ml in buffer 25 mM Tris/HCl, 150 mM NaCl pH 7.5 was used as the protein solution. The sitting-drop vapor-diffusion method was used to produce apo-crystals by mixing 0.5 μl of the protein solution and an equal volume of mother liquor . Tetragonal crystals (space group P43212) grew within in 3–4 days and were soaked in 2 μL of the mother liquor with 0.2 μL of a dilution 500 mM of compounds Diffraction data of the binary complexes were collected at beamline I04-1 . The data was processed and scaled using the XDS package and CCP4 software, relevant statistics are given in Table S2 of Supplementary informationThe structure of the binary complex was solved by molecular replacement using PDB ID 3G15 as a template. Initial phases were further improved by cycles of manual model building in Coot and refinement with REFMAC5. The final model was validated with PROCHECK, model statistics are given in 3 cells. The plates were incubated at 37 °C in a humidified 5% CO2 incubator for 18 h prior to the experiments. After medium removal, 100 μL of fresh medium containing the test compound at different concentrations was added to each well and incubated at 37 °C for 72 h. The percentage of DMSO in the medium in no case exceeded 0.25%. Cell viability was assayed by the -2,5-diphenyl tetrazolium bromide test as previously described50 was defined as the compound concentration required to inhibit cell proliferation by 50%, in comparison with cells treated with the maximum amount of DMSO (0.25%) and considered as 100% viability. In additional experiments, cell viability was also determined by calculating the values of trypan blue positive cells (dead cells) and trypan blue negative (live cells) from a mixture of the cell suspension and 0.4% trypan blue solution.Human T-cell leukemia (Jurkat), human B-cell leukemia  and human promyelocytic leukemia (HL-60) cells were grown in RPMI-1640 medium . Breast adenocarcinoma (MCF-7 and MDA-MB-231), human non-small cell lung carcinoma (A549), human cervix carcinoma (HeLa), human prostate adenocarcinoma (PC-3), and human colon adenocarcinoma (HT-29) cells were grown in DMEM medium . Both media were supplemented with 115 units/mL of penicillin G , 115 μg/mL of streptomycin  and 10% fetal bovine serum . Stock solutions (10 mM) of the different compounds were obtained by dissolving them in DMSO. Individual wells of 96-well tissue-culture microtiter plates were inoculated with 100 μL of complete medium containing 8 × 106 cells/mL) in RPMI-1640 with 10% fetal bovine serum and incubated overnight. For cytotoxicity evaluations in proliferating PBL cultures, non-adherent cells were resuspended at 5 × 105 cells/mL in growth medium, containing 2.5 μg/mL PHA (Irvine Scientific). Different concentrations of the test compounds were added, and viability was determined 72 h later by the MTT test. For cytotoxicity evaluations in resting PBL cultures, non-adherent cells were resuspended (5 × 105 cells/mL) and treated for 72 h with the test compounds, as described above.Peripheral mononuclear cells (PBMC) from healthy donors were obtained by separation on Lymphoprep (Fresenius KABI Norge AS) gradient. After extensive washing, cells were resuspended , were prepared from human umbilical-cord veins, as previously describedHuman fibroblasts from foreskin were isolated as previously described5 HeLa or Jurkat cells in exponential growth were treated with different concentrations of the test compounds for 24 and 48 h. After the incubation period, the cells were collected, centrifuged, and fixed with ice-cold ethanol (70%). The cells were then treated with lysis buffer containing RNAse A and 0.1% Triton X-100, and then stained with propidium iodide (PI). Samples were analyzed in a Cytomics FC500 flow cytometer (Beckman Coulter). DNA histograms were analyzed using MultiCycle for Windows (Phoenix Flow Systems).For flow-cytometric analysis of the DNA content, 5 × 10Surface exposure of PS on apoptotic cells was measured by flow cytometry with Cytomics FC500 (Beckman Coulter) by adding simultaneously annexin-V conjugated to fluorescein isothiocyanate (FITC) and PI to cells according to the manufacturer′s instructions .How to cite this article: Schiaffino-Ortega, S. et al. Design, synthesis, crystallization and biological evaluation of new symmetrical biscationic compounds as selective inhibitors of human Choline Kinase α1 (ChoKα1). Sci. Rep.6, 23793; doi: 10.1038/srep23793 (2016).