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Quaternary α-(hetero)arylnitriles are desirable biologically relevant products, however the existing methods for their synthesis can be unselective or require the use of undesirable reagents, such as cyanide salts. Herein we report a one-pot method for transnitrilation-mediated decyanation-metalation of disubstituted malononitriles, followed by treatment with (hetero)aryl electrophiles to access quaternary α-(hetero)arylnitrile products. A number of products were prepared using this method (34 examples, 27-99% yield). This method highlights the usefulness of malononitriles as precursors for alkylnitrile-containing compounds.

DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of

Herein, we report a Ni-catalyzed reductive coupling for the synthesis of benzonitriles from aryl (pseudo)halides and an electrophilic cyanating reagent, 2-methyl-2-phenyl malononitrile (MPMN). MPMN is a bench-stable, carbon-bound electrophilic CN reagent that does not release cyanide under the reaction conditions. A variety of medicinally relevant benzonitriles can be made in good yields. Addition of NaBr to the reaction mixture allows for the use of more challenging aryl electrophiles such as aryl chlorides, tosylates, and triflates. Mechanistic investigations suggest that NaBr plays a role in facilitating oxidative addition with these substrates.

α-Aryl amines are prevalent in pharmaceutically active compounds and natural products. Herein, we describe a Ni-catalyzed protocol for their synthesis from readily available α-aryl ethers. While α-aryl ethers have been used as electrophiles in Ni-catalyzed C–C bond formations, their use in C–heteroatom bond formation is much less prevalent. Preliminary mechanistic insight suggests that oxidative addition is facilitated by an anionic ligand and that reductive elimination is a reversible process.

The efficient preparation of nitrile-containing building blocks is of interest due to their utility as synthetic intermediates and their prevalence in pharmaceuticals. As a result, significant efforts have been made to develop methods to access these motifs which rely on safer and non-toxic sources of CN. Herein, we report that 2-methyl-2-phenylpropanenitrile is an efficient, non-toxic, electrophilic CN source for the synthesis of nitrile-bearing quaternary centers by a thermodynamic transnitrilation and anion-relay strategy. This one-pot process leads to nitrile products resulting from the gem-difunctionalization of alkyl lithium reagents.

BACKGROUND: High-level expression of the Fcγ receptor, CD32(hi), on CD4(+) T cells was associated with enhanced human immunodeficiency virus (HIV) infection of the latent reservoir in a study of adults receiving antiretroviral therapy. We tested the hypothesis that CD32 was the preferential marker of the latent HIV reservoir in virally suppressed, perinatally HIV-infected adolescents. METHODS: The frequency of CD32(hi)CD4(+) T cells was determined by flow cytometry (N = 5) and the inducible HIV reservoir in both CD32(hi) and CD32(−)CD4(+) T cells was quantified (N = 4) with a quantitative viral outgrowth assay. Viral outgrowth was measured by the standard p24 enzyme-linked immunosorbent assay and an ultrasensitive p24 assay (Simoa; Quanterix) with lower limits of quantitation. RESULTS: We found a 59.55-fold enrichment in the absolute number of infectious cells in the CD32(−) population compared with CD32(hi) cells. Exponential HIV replication occurred exclusively in CD32(−)CD4(+) T cells (mean change, 17.46 pg/mL; P = .04). Induced provirus in CD32(hi)CD4(+) T cells replicated to substantially lower levels, which did not increase significantly over time (mean change, 0.026 pg/mL; P = .23) and were detected only with the Simoa assay. CONCLUSIONS: Our data suggests that the latent HIV reservoir resides mainly in CD32(−)CD4(+) T cells in virally suppressed, perinatally HIV-infected adolescents, which has implications for reservoir elimination strategies.

The efficient preparation of nitrile-containing building blocks is of interest due to their utility as synthetic intermediates and their prevalence in pharmaceuticals. As a result, significant efforts have been made to develop methods to access these motifs which rely on safer and non-toxic sources of CN. Herein, we report that 2-methyl-2-phenylpropanenitrile is an efficient, non-toxic, electrophilic CN source for the synthesis of nitrile-bearing quaternary centers via a thermodynamic transnitrilation and anion-relay strategy. This one-pot process leads to nitrile products resulting from the gem-difunctionalization of alkyl lithium reagents.

Background: Current thrombolytic therapies utilize exogenous plasminogen activators (PAs) to effectively lyse clots, restoring blood flow, and preventing tissue and organ death. These PAs may also impair normal hemostasis, leading to life-threatening bleeding, including intracerebral hemorrhage. Aims: This study aims to develop new thrombus-targeted fibrinolytic agents that harness the multifunctional theranostic capabilities of nanomaterials, potentially allowing for the generation of efficacious thrombolytics while minimizing deleterious side effects. Materials & methods: A thrombus-targeted nano-fibrinolytic agent was synthesized using a magnetofluorescent crosslinked dextran-coated iron oxide nanoparticle platform that was conjugated to recombinant tissue PA (tPA). Thrombus-targeting was achieved by derivatizing the nanoparticle with an activated factor XIII (FXIIIa)-sensitive peptide. Human plasma clot binding ability of the targeted and control agents was assessed by fluorescence reflectance imaging. Next, the in vitro enzymatic activity of the agents was assessed by S2288-based amidolytic activity, and an ELISA D-dimer assay for fibrinolysis. In vivo targeting of the nanoagent was next examined by intravital fluorescence microscopy of murine arterial and venous thrombosis. The fibrinolytic activity of the targeted nanoagent compared to free tPA was then evaluated in vivo in murine pulmonary embolism. Results: In vitro, the targeted thrombolytic nanoagent demonstrated superior binding to fresh-frozen plasma clots compared to control nanoagents (analysis of variance, p < 0.05). When normalized by S2288-based amidolytic activity, targeted, control and free tPA samples demonstrated equivalent in vitro fibrinolytic activity against human plasma clots, as determined by ELISA D-dimer assays. The FXIIIa targeted fibrinolytic nanoagent efficiently bound the margin of intravascular thrombi as detected by intravital fluorescence microscopy. In in vivo fibrinolysis studies the FXIIIa-targeted agent lysed pulmonary emboli with similar efficacy as free tPA (p > 0.05). Conclusion: The applicability of a FXIIIa-targeted thrombolytic nanoagent in the treatment of thromboembolism was demonstrated in vitro and in vivo. Future studies are planned to investigate the safety profile and overall efficacy of this class of nanoagents. Original submitted: 8 July 2011; Revised submitted: 16 November 2011; Published online 21 February 2012

Digital ELISA (Enzyme Linked Immunosorbent Assay) based on single molecule arrays (Simoa) has improved sensitivity of traditional ELISA from picomolar (10−12M) to femtomolar (10−15M), increasing the quality and quantity of biomarkers that can be measured for health and disease. Digital ELISA counts signal generated from single immunocomplexes formed on superparamagnetic beads confined in arrays of femtoliter-sized wells in which fluorescent molecules are highly concentrated. Quanterix has developed digital ELISA assays in a fully-automated instrument (Simoa HD-1 Analyzer), ideal for use in pharmaceutical companies, drug discovery, clinical research and other areas necessitating full automation and high throughput. Recent advancements in the Simoa technology and workflow have been integrated into the new SR-X benchtop reader, with a smaller footprint and more flexible workflow. Operators prepare assays in microtiter plates at the bench in a semi-automated format similar to traditional ELISA, with the notable exception that plates are preserved by drying after assay completion, and can be read immediately or the next day. We present results comparing performance of the following Simoa assays on SR-X to HD-1: PSA; HIV p24; Tau; Neurofilament-light; PD-L1; TNFa; IL-10; IL-17A; IL-6 and a neurology multiplex panel consisting of NF-L, tau, GFAP and UCH-L1. Measured sample levels correlated with R2 values from 0.96 to 1.00, with average LOD and LLOQ within 1.4 and 1.5 fold of HD-1, respectively. Inter-assay precision ranged from 4.0 to 11.2% CV across assays. Operators tested full-plates from start to finish within 1 – 2 hours (1/2 hour hands on time) and a read time of 2 hours (5 minutes hands on time).

Thrombosis underlies numerous life-threatening cardiovascular syndromes. Development of thrombosis-specific molecular imaging agents to detect and monitor thrombogenesis and fibrinolysis in vivo could improve the diagnosis, risk stratification, and treatment of thrombosis syndromes. To this end, we have synthesized efficient multimodal nanoagents targeted to two different constituents of thrombi, namely, fibrin and activated factor XIII. These agents are targeted via the conjugation of peptide-targeting ligands to the surface of fluorescently labeled magnetic nanoparticles. As demonstrated by in vitro and in vivo studies, both nanoagents possess high affinities for thrombi, and enable mutimodal fluorescence and magnetic resonance imaging.

Patient: Female, 72 Final Diagnosis: Renal cell carcinoma Symptoms: — Medication: — Clinical Procedure: — Specialty: Oncology Objective: Challenging differential diagnosis Background: Lithium salts are widely used in the treatment of affective disorders of the bipolar type. Lithium is a nephrotoxic substance which can cause both acute and chronic renal disease, including cyst formation. Cysts appear to predispose the kidney to renal cell carcinoma. Case Report: A case of renal cell carcinoma in a background of acquired cystic disease due to chronic lithium toxicity is described. Conclusions: Kidneys with multiple cysts are at risk of renal cell carcinoma. Although it is difficult to determine if long term Lithium use renal cell carcinoma, patients leads to the development of on long-term lithium therapy should undergo regular renal function and imaging tests.

Background : Activated factor XIII (FXIIIa) is a blood transglutaminase that mediates fibrinolytic resistance and is a hallmark of acute thrombi. Noninvasive molecular imaging of FXIIIa may offer a novel approach to identify acute thrombi and to gauge fibrinolytic resistance in vivo. Here we developed and validated a FXIIIa thrombosis imaging strategy using noninvasive integrated SPECT/CT. Methods: A FXIIIa-targeted peptide agent (F13) was synthesized using NQEQVSPLTLLK chelated to DOTA and then labeled with 111 InCl 3 . A control agent (C13, 111 In-NAEQVSPLTLLK) was analogously synthesized. In vitro validation of the F13 agent was performed in human plasma clots. Next, the in vivo blood-half life of F13 was determined in mice (n=4). In vivo thrombosis studies (n=15 mice) were then performed using 10% ferric chloride jugular venous thrombi aged 1 hour or 16 hours. Mice were intravenously injected with 200 μCi of F13 or C13. After 4 hours, mice underwent integrated CT angiography (72 μm isotropic resolution) and SPECT imaging (32 minute acquisition). In situ thrombi were then resected for radioactivity and weight measurements. Results : Human plasma clots incubated with F13 showed 280–740% greater counts per minute (CPM) than controls (p90% inhibited by pretreatment with iodoacetamide, an alkylating agent. The blood half-life of F13 was calculated to be 16 minutes. In one hour thrombi, in vivo SPECT/CT imaging revealed strong focal F13 SPECT signal in the co-registered ipsilateral venous thrombi but not the contralateral normal jugular vein. One hour thrombi in the F13 group had 15-fold greater radioactivity than the C13 group (4.6±3.6% vs. 0.3±0.2% injected dose per gram tissue, IDGT, p Conclusions : Blood transglutaminase FXIIIa can be noninvasively detected using a FXIIIa-sensitive and specific imaging agent for integrated SPECT/CT. The current in vivo results further validate that activated factor XIII is a hallmark of acute thrombi and declines in activity over time. This clinically translatable imaging strategy could permit visualization of FXIIIa in patients with thrombotic syndromes.