Ligand-Free Pd-Catalyzed Double Carbonylation of Aryl Iodides with Amines to α‑Ketoamides under Atmospheric Pressure of Carbon Monoxide and at Room Temperature

A general Pd-catalyzed double carbonylation of aryl iodides with secondary or primary amines to produce α-ketoamides at atmospheric CO pressure has been developed. This transformation proceeds successfully even at room temperature and in the absence of any ligand and additive. A wide range of aryl iodides and amines can be coupled to the desired α-ketoamides in high yields with excellent chemoselectivities. Importantly, the current methodology has been demonstrated to be applied in the synthesis of bioactive molecules and chiral α-ketoamides

Preparation, Biological Evaluation, and First-in-Human Single-Photon Emission Computed Tomography (SPECT) Study of ^99mTc-Labeled Prostate-Specific Membrane Antigen (PSMA)-Targeted Radiotracers Containing Triazole with Reduced Kidneys Accumulation

Prostate-specific membrane antigen (PSMA), a well-established biological marker for prostate cancer (PCa) imaging and therapy, is overexpressed on the surface of prostate cancer lesions. In this study, a triazole ring was introduced into the linker by click chemistry to generate a HYNIC-derived ligand (T), which exhibited good PSMA affinity (Ki = 2.23 nM). Eight stable 99mTc-labeled complexes, [99mTc]Tc-T-Mn (n = 1–8), with hydrophilic properties were synthesized by incorporating different coligands at high radiochemical yields and purities without purification. The radioligands were concentrated in the kidneys of healthy Kunming male mice and were significantly blocked by the PSMA inhibitor ZJ-43. The uptake of the optimized complex [99mTc]Tc-T-M2 was correlated with PSMA, and it had good PSMA affinity (Kd = 5.42 nM). [99mTc]Tc-T-M2 accumulated on LNCaP (PSMA++) tumors and was significantly blocked by ZJ-43 at 2 h p.i., indicating high PSMA specificity. Relatively suitable kidney uptake was beneficial for reducing kidneys exposure in patients. SPECT/CT imaging of [99mTc]Tc-T-M2 in LNCaP (PSMA++) or 22Rv1 (PSMA+) tumor-bearing mice revealed high tumor uptake, low background uptake (especially low kidney uptake (49.06 ± 9.20 %ID/g) at 2 h p.i.), and obvious inhibition by ZJ-43, whereas PC-3 (PSMA−) tumors were undetectable. A freeze-dried [99mTc]Tc-T-M2 kit was successfully developed (T-M2 kit). Preliminary clinical trials showed that [99mTc]Tc-T-M2 clearly identified small prostate cancer lesions and has potential for clinical application

Preparation, Biological Evaluation, and First-in-Human Single-Photon Emission Computed Tomography (SPECT) Study of ^99mTc-Labeled Prostate-Specific Membrane Antigen (PSMA)-Targeted Radiotracers Containing Triazole with Reduced Kidneys Accumulation

Prostate-specific membrane antigen (PSMA), a well-established biological marker for prostate cancer (PCa) imaging and therapy, is overexpressed on the surface of prostate cancer lesions. In this study, a triazole ring was introduced into the linker by click chemistry to generate a HYNIC-derived ligand (T), which exhibited good PSMA affinity (Ki = 2.23 nM). Eight stable 99mTc-labeled complexes, [99mTc]Tc-T-Mn (n = 1–8), with hydrophilic properties were synthesized by incorporating different coligands at high radiochemical yields and purities without purification. The radioligands were concentrated in the kidneys of healthy Kunming male mice and were significantly blocked by the PSMA inhibitor ZJ-43. The uptake of the optimized complex [99mTc]Tc-T-M2 was correlated with PSMA, and it had good PSMA affinity (Kd = 5.42 nM). [99mTc]Tc-T-M2 accumulated on LNCaP (PSMA++) tumors and was significantly blocked by ZJ-43 at 2 h p.i., indicating high PSMA specificity. Relatively suitable kidney uptake was beneficial for reducing kidneys exposure in patients. SPECT/CT imaging of [99mTc]Tc-T-M2 in LNCaP (PSMA++) or 22Rv1 (PSMA+) tumor-bearing mice revealed high tumor uptake, low background uptake (especially low kidney uptake (49.06 ± 9.20 %ID/g) at 2 h p.i.), and obvious inhibition by ZJ-43, whereas PC-3 (PSMA−) tumors were undetectable. A freeze-dried [99mTc]Tc-T-M2 kit was successfully developed (T-M2 kit). Preliminary clinical trials showed that [99mTc]Tc-T-M2 clearly identified small prostate cancer lesions and has potential for clinical application

PEDF prevents VEGF-induced dissociation of endothelial AJs and TJs in the retinal vasculature of mice.

<p>(<b>a</b>) Representative confocal images of retinal vessels in flat mount preparations from unstimulated and animals, treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0021164#pone-0021164-g004" target="_blank">Figure 4</a>, immunostained with VE-cadherin or claudin-5 (red) and FITC-conjugated agglutinin (green) to visualize retinal vessels. Merged images are shown with colocalization of VE-cadherin and claudin-5 appearing as yellow. Scale bar = 50 µM. The lower panel (<b>b</b>) shows representative merged higher power images of retinal vessels stained for VE-cadherin (i–iii) or claudin-5 (iv–vi) (red) and FITC-conjugated agglutinin (green). <i>i, iv = unstimulated; ii, v = VEGF treatment; iii, vi = PEDF 6 hr after VEGF.</i> Scale bar = 10 µM.</p

Hypoxia increases vascular permeability.

<p>Temporal changes in transendothelial resistance across a microvascular endothelial monolayer grown on a Transwell insert treated with vehicle (unstimulated), VEGFA alone, PEDF alone, simultaneous VEGFA+PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γ-SI) under either (<b>a</b>) hypoxia or (<b>b</b>) retinal normoxia. (n = 4 independent experiments). VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. In the case of PEDF 6 hours post VEGF, Transwell inserts were transferred to new wells containing basal medium without fluorescent dextran. Data are respresented as means± SEM.</p

PEDF inhibits VEGF-induced phosphorylation of membrane-bound AJ proteins and promotes association of presenilin and nicastrin with VE-cadherin.

<p>Confluent retinal endothelial cells were either unstimulated (1) treated with VEGF alone (2), PEDF alone (3), VEGF followed by PEDF 6 hours later without (4) and with (5) γ-secretase inhibitor over 24 hours. VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. Cells were separated into membrane and cytosolic fractions followed by immunoprecipitation. (<b>a</b>) Total VE-cadherin levels were determined by immunoprecipitation of the membrane fraction with anti-PY20 and Western blots for VE-cadherin. A panel of representative Western blots is shown on left and band intensities of replicate experiments (n = 4) on the right. (<b>b</b>) Western blot analysis to assess the effect of the different treatments on tyrosine phosphorylation of VE-cadherin at sites Y658 and Y731 (n = 4 independent experiments). (<b>c</b>) Total phosphorylation of β-catenin was assessed by immunoprecipitation of the membrane fraction with anti-PY20 and Western blots for β-catenin. (n = 4) (<b>d</b>) The association of presenilin-1 (PS1) and nicastrin (NTC) were confirmed by immunoprecipitation with anti-VE-cadherin, followed by Western blot analysis using anti-presenilin-1 and nicastrin. (<b>e</b>) To further confirm a role for γ-secretase in regulating vascular permeability presenilin-1 or nicastrin were knocked down with by transfection with AAV 2 expressing PS1 and NTC siRNAs. Scrambled siRNA acted as a control. Paracellular macromolecular permeability to 40 kDa Dextran-FITC was determined in cells treated with VEGF, PEDF, VEGF+PEDF and compared to unstimulated control. Densitometry analyses in (a), (c) and (d) are shown as the relative ratio of phosphorylated pVE-cadherin, pβ-catenin, presenilin-1 and nicastrin to heavy IgG chains. Densitometry in b is shown as the relative ratio to α-tubulin. The data are mean ± SEM. *p<0.05, **p<0.01 versus unstimulated group.</p

The temporal response of AJs and TJs to VEGF or VEGF plus PEDF.

<p>Representative pictures of confluent cultures of microvascular endothelial cells treated with VEGFA alone or VEGFA followed by PEDF 6 hours later triple stained for VE-cadherin (red), claudin-5 (green) and nuclei (DAPI) and assessed at different times over 24 hours using confocal microscopy. VEGFA and PEDF were used at 100 ng/ml. Scale bar = 100 µm.</p

Hypoxia increases the expression of VEGF receptors and γ-secretase along with VEGFR dissociation from AJ proteins.

<p>(<b>a</b>) Confluent retinal microvascular endothelial cells were exposed to hypoxia for up to 24 hours. Cell lysated were examined by Western blot for VEGFR1, VEGFR2, presenilin-1 (PS-1), nicastrin (NCT), VE-cadherin and β-catenin. α-tubulin acted as the house keeping control. A representative Western blot is shown together with densitometric analysis from three separate experiments. (<b>b</b>) Confluent retinal microvascular endothelial cells were maintained under standard incubator conditions, retinal normoxia and hypoxia for 24 hours. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. A panel of representative Western blots is shown. Band intensities of replicate experiments (n = 4) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.</p

PEDF blocks VEGF-induced retinal vascular permeability in mice.

<p>C57BL/6 mice received intravitreal injections of vehicle (unstimulated), VEGFA alone, PEDF alone, simultaneous VEGFA+PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γSI) (n = 18 animals per treatment). Test compounds (1 µl per eye were given at the following concentrations, VEGF 80 ng/µl; PEDF (80 ng/µl); L685485 24 ng/µl; DAPT 16 ng/µl. 46 hours post the first injection mice received tail vein injections of FITC-labeled albumin. Uninjected animals acted as the baseline control. (<b>a</b>) 46 hours post the first injection mice received tail vein injections of FITC-labeled albumin and retinas were taken for analysis 2 hours later (n = 10–20 per group). Leakage of systemic FITC-labeled albumin into the retina was assessed by measuring total fluorescence in homogenized retina using a fluorescence plate reader. Control is the baseline fluorescence in untreated animals. Data are represented as means ± SEM. (<b>b</b>) Representative confocal microscopy showing dilated vessels and leakage of FITC-labeled albumin in the retinas of mice treated with vehicle (unstimulated), VEGF, PEDF, PEDF 6 hours post VEGF and PEDF 6 hours post VEGF+γ-secretase inhibitor (γ-SI). Scale bar = 50 µM.</p

PEDF maintains the association between AJ proteins and VEGF receptors in VEGF treated cells.

<p>Confluent retinal endothelial cells were either unstimulated (1) treated with VEGF alone (2), PEDF alone (3), VEGF followed by PEDF 6 hours later without (4) and with (5) γ-secretase inhibitor for 24 hours. VEGFA and PEDF were used at 100 ng/ml and γ-secretase inhibitor at 1 nM. Total cell lysates were split into four equal portions and immunoprecipitated with anti-VE-cadherin, anti-β-catenin, anti-VEGFR1-C-terminus and anti-VEGFR2-C-terminus, and then subsequent Western blot analyzed using these four antibodies, respectively. (<b>a</b>) A panel of representative Western blots. (<b>b</b>) Band intensities of replicate experiments (n = 4 independent experiments) were quantified as described in the Methods and regression analysis undertaken to assess the association of these proteins.</p