Basicity Enhancement by Multiple Intramolecular Hydrogen Bonding in Organic Superbase <i>N</i>,<i>N</i>′,<i>N</i>″,<i>N</i>‴‑Tetrakis(3-(dimethylamino)propyl)triaminophosphazene

With the synthesis of N,N′,N″,N‴-tetrakis­(3-(dimethylamino)­propyl)­triaminophosphazene (TDMPP, 1), we present the first phosphazene superbase with enhanced basicity through the effect of multiple intramolecular hydrogen bonding (IHB). Due to intramolecular solvation of four NH protons, the proton affinity is even higher than that of second-order phosphazene (dma)­P2-tBu. X-ray structural proof, NMR titration experiments, and computational investigations provide a more detailed quantitative description of the IHB influence on the superbasicity of 1 in solid-state, solution, and the gas-phase

Xenograft model of CEL with the human cell line EOL-1 in wild-type and E- and P-selectin knockout scid mice.

<p>Selectin deficiency dramatically increases survival of the animals after injection of 2×10⁶ EOL-1 cells and decreases the number of leukemia cells in the blood. <b>A</b>: Kaplan-Meyer survival curve for wild-type (wt, selectin competent, 8 animals, grey curve) and selectin knockout (k.o., E-and P-selectin deficient, 10 animals, black curve). The percentage of surviving animals on a given day is shown. The experiment was terminated after 53 days. Median survival after transplantation was: wt 32 days, k.o. not reached. The curves were significantly different, ***: P<0.0001 (Log-rank test). <b>B</b>: Human EOL-1 cells in the animals’ blood at the time of death as determined by quantitative real-time PCR. Selectin competent (wt, 8 animals) are compared with E- and P-selectin deficient mice (k.o., 9 animals). Given in the box plot are the median (line), highest and lowest number of EOL-1 cells per ml of the animals’ blood (whiskers) and upper and lower quartile (box). Median cell number per ml blood was 32950 for the wt group and 7.8 for the k.o. group. The difference between the groups was significantly different, ***: P = 0.0002 (Mann Whitney test).</p

Analysis for potential selectin ligands on the surface of the human CEL and CML cell lines EOL-1 and K562 by flow cytometry.

<p>Only EOL-1 cells are positive for sialyl Lewis x, both cell lines are positive for CD162 (PSGL-1). Cells were incubated with antibodies against CA19-9 (sialyl lewis a), CD15s (sialyl lewis x) or CD162 (PSGL-1) or the respective isotype controls followed by an APC-labelled secondary antibody. Given in the histograms are the fluorescence signals and event numbers, labeling with the antibodies against selectin ligands is represented by the filled curves, the respective isotype controls by the open curves. Only a small subpopulation (3.7%) of EOL-1 cells was positive for CA19-9. The cells were either completely negative or more than 95% positive for the other ligands. All experiments were repeated twice, representative results are shown.</p

Human EOL-1 cells and K562 cells bind to E- and P-selectin <i>in vitro</i>.

<p>Binding of human E- and P-selectin to human CEL and CML cells was analyzed by flow cytometry. Given in the histograms are the fluorescence signal (FL-1 for AlexaFluor488 or FL-2 for phycoerythrin) and event number, selectin binding is represented by the filled curves, controls by the open curves. Cell lines and selectins used were: EOL-1 and human E-selectin (<b>A</b>), EOL-1 and human P-selectin (<b>B</b>), K562 and human E-selectin (<b>C</b>) and K562 and human P-selectin (<b>D</b>). All experiments were repeated twice, representative results are shown.</p

Xenograft model of CML with the human cell line K562 in wild-type and E- and P-selectin knockout scid mice.

<p>Selectin deficiency dramatically increases survival of the animals after injection of 2×10⁶ K562 cells and decreases the number of leukemia cells in blood and bone marrow. <b>A</b>: Kaplan-Meyer survival curve for wild-type (wt, selectin competent, 10 animals, grey curve) and selectin knockout (k.o., E-and P-selectin deficient, 10 animals, black curve). The experiment was ended after 56 days. Median survival after transplantation was: wt 48.5 days, k.o. not reached. The curves were significantly different, **: P = 0.0085 (Log-rank test). <b>B</b>: Human K562 CML cells (given in cells/ml) in the animals’ blood at the time of death as determined by quantitative real-time PCR (qRT-PCR). Selectin competent (wt, n = 10) compared with E- and P-selectin deficient mice (k.o., n = 10). Given in the box plot are the median (line), highest and lowest number of K562 cells per ml of the animals’ blood (whiskers) and upper and lower quartile (box). Median cell number per ml blood was 268.8 for the wt group and 0.2 for the k.o. group. The difference between the groups was significantly different, **: P = 0.0068 (Mann Whitney test). <b>C</b>: Human K562 CML cells in the animals’ bone marrow at the time of death as determined by qRT-PCR (given in cells/60 ng template DNA). Given in the box plot are the median (line), highest and lowest number of K562 cells (whiskers) and upper and lower quartile (box). Median cells per 60 ng template DNA in the wt group were 243 compared with 0 cells in the k.o. group. This difference was significant, **: P = 0.0089 (Mann Whitney test).</p

Human CEL and CML cells can be clearly detected by immunohistochemistry in the tissues of the scid mice.

<p><b>A</b>: Tissue section of an EOL-1 chloroma of selectin wt scid mouse. Immunohistochemical staining for human HLA-DR in red. <b>B</b>: Tissue section of a K562 chloroma of a wt scid mouse. Immunohistochemical staining for human mitochondria in red.</p

Human EOL-1 cells and K562 cells cells show tethering and adhesion to E- and P-selectin in laminar flow assays.

<p>Interactions of human CEL and CML cells from culture under laminar flow (0.25 dyn/cm²) in selectin coated channels. The cell lines EOL-1 and K562 were tested for adherence (<b>A</b>) or tethering (<b>B</b>). Given are the numbers and corresponding standard deviations of adhering and tethering cells, respectively. Channels were coated with human E-selectin (E), human P-selectin (P), murine E-selectin (mE) or murine P-selectin (mP). No adhering or tethering cells were observed in Fc control coated channels. All experiments were done in triplicates.</p

Inhibition of selectin binding to human CEL and CML cells by monoclonal antibodies and neuraminidase treatment as determined by flow cytometry.

<p><b>A</b>: Blocking of P-selectin binding by (pre)incubation with monoclonal antibodies against CD15s and CD162. Filled curves in the histograms represent incubation of the leukemia cells with isotype control, open curves represent incubation with the respective specific antibody. The only observed inhibitory effect was caused by anti-CD162 on EOL-1, but not on K562 cells. <b>B</b>: Blocking of E-selectin binding by (pre)incubation with monoclonal antibodies against CA19-9. Filled curves in the histograms represent incubation of the cells with isotype control, open curves represent incubation with the specific antibody. The only observed inhibitory effect by anti-CA19-9 was observed on PaCa 5061 pancreatic adenocarcinoma cells (positive control). <b>C</b>: Binding of selectins to the leukemia cells after neuraminidase treatment. Filled curves in the histograms: selectin binding without neuraminidase incubation (positive control); open curves, black: selectin binding to neuraminidase treated cells; open curves, grey: negative controls. Neuraminidase treatment abolished E-selectin binding to both cell lines and reduced P-selectin binding to EOL-1 but not to K562 cells. All experiments were repeated twice, representative results are shown.</p

Development of total tumour volume as determined by MR imaging under nilotinib treatment.

<p>(A) Total tumour volume in mm³ for each animal from the therapy group on days 27 (Therapy d0), 31 (Therapy d4) and 34 (Therapy d7). (B) Total tumour volume in mm³ for each animal from the placebo group on days 27 (Placebo d0), 31 (Placebo d4) and 34 (Placebo d7). (C) Total tumour volume in mm³ for the therapy and placebo group before (d0) and after (d7) nilotinib-treatment. n.s.: non-significant, P = 0.0869; ***: highly significant, P = 0.0008, unpaired t-test.</p

MR imaging.

<p>MR images of one mouse each of the therapy (A, imatinib) and placebo group (B). Coronar (left panels) and sagittal (left panels) T2 images of EOL-1 bearing scid mice on days 27 (d0 of treatment), 30 (d3 of treatment) and 34 (d7 of treatment). In the selected plane, remission (therapy) or growth (placebo) of a large abdominal chloroma can be observed (arrows). Chloroma is in complete remission on day 34 under imatinib treatment (A, d7).</p