Fluselenamyl: A Novel Benzoselenazole Derivative for PET Detection of Amyloid Plaques (Aβ) in Alzheimer’s Disease

Fluselenamyl (5), a novel planar benzoselenazole shows traits desirable of enabling noninvasive imaging of Aβ pathophysiology in vivo; labeling of both diffuse (an earlier manifestation of neuritic plaques) and fibrillar plaques in Alzheimer’s disease (AD) brain sections, and remarkable specificity for mapping Aβ compared with biomarker proteins of other neurodegenerative diseases. Employing AD homogenates, [18F]-9, a PET tracer demonstrates superior (2–10 fold higher) binding affinity than approved FDA tracers, while also indicating binding to high affinity site on Aβ plaques. Pharmacokinetic studies indicate high initial influx of [18F]-9 in normal mice brains accompanied by rapid clearance in the absence of targeted plaques. Following incubation in human serum, [18F]-9 indicates presence of parental compound up to 3h thus indicating its stability. Furthermore, in vitro autoradiography studies of [18F]-9 with AD brain tissue sections and ex vivo autoradiography studies in transgenic mouse brain sections show cortical Aβ binding, and a fair correlation with Aβ immunostaining. Finally, multiphoton- and microPET/CT imaging indicate its ability to penetrate brain and label parenchymal plaques in transgenic mice. Following further validation of its performance in other AD rodent models and nonhuman primates, Fluselenamyl could offer a platform technology for monitoring earliest stages of Aβ pathophysiology in vivo.

binding to AD homogenates also indicate multiple binding sites on Aβ 24 thus mandating development of new tracers to study Aβ pathophysiology. To further supplement the existing armamentarium of FDA approved Aβ imaging agents, earlier we have shown that a heterocyclic fluorescent molecule is capable of traversing the BBB to label Aβ plaques in brains of APP +/− /PS1 +/− mice and also indicates sensitivity for detecting diffuse plaques in autopsy confirmed AD human tissues 25,26 . Although the PET counterpart showed high first pass extraction into the brains of normal mice and Aβ labeling in brain frontal cortex of APP +/− /PS1 +/− mice, its binding affinity to AD homogenates was 5-10 fold inferior compared with two FDA approved agents 27 . Importantly, this template scaffold of our first generation agent comprised a benzothiazole moiety, a pharmacologically active constituent with widespread medicinal chemistry applications. Compared with benzothiazole derivatives, investigations on biological activity of benzoselenazoles have not gained much attention primarily due to a lack of convenient and cost-efficient chemical methodologies 28 . To our knowledge, benzoselenazoles derivatives represent an entirely novel class of Aβ diagnostic agents. Herein, we report synthesis, characterization and crystal structure of (Z)-5-(2-(5-(2-fluoroethoxy)benzo[d] [1,3]selenazol-2-yl)vinyl)-N,N-dimethylpyrimidin-2-amine (Fluselenamyl: 5), F-18 labeled radiotracer ([ 18 F]-9), and perform its preclinical validation to evaluate its potential to serve as an Aβ -targeted PET radiopharmaceutical for monitoring plaque burden in AD. Fluselenamyl demonstrates potent binding to Aβ fibrils, autopsy confirmed AD homogenates, traverses the blood brain barrier (BBB) to detect Aβ plaques in a transgenic mice model, and is highly specific for probing Aβ plaques in AD.

Materials and Methods
Material details, organic synthesis procedures, analytical characterization of all intermediates including the final Fluselenamyl (1, 2, 3, 4, 5, 6, 7, and 8) and radiochemistry for synthesis of the PET tracer [ 18 F]-9 are described in supporting information. Additionally, X-ray crystallographic details for 5; Aβ binding assays (Fibrils and AD homogenates) using 5 and [ 18 F]-9; histochemical staining of 5 with transgenic mice brain cross sections, and AD brain sections; human serum stability of [ 18 F]-9; autoradiography of [ 18 F]-9 with AD human brain sections and ex vivo brain sections of APP/PS1 and WT mice biodistribution studies of [ 18 F]-9 in FVB mice; multiphoton imaging using 5 in transgenic mice, and finally microPET/CT imaging details of [ 18 F]-9 in age-matched transgenic mice and their WT counterparts are also included in the supporting information. While postmortem brain tissues from autopsy-confirmed AD patients and their approximate age-matched healthy controls were obtained through the Knight Alzheimer's Disease Research Center (ADRC) Washington University School of Medicine, and processed according to a protocol approved by institutional ADRC executive committees, the animal procedures were approved by the Washington University Animal Studies Committee (Protocols #20150147, PI: Sharma; protocol #20140182, PI: Jin-Moo Lee). All methods were carried out on animals and human tissues in "accordance" with the approved guidelines.

Results and Discussion
Our strategic design for obtaining a second generation Aβ -targeted agent involved five functional components described earlier 25 . Incorporating those characteristic features into a template scaffold, the Aβ targeted probe 5 was synthesized as shown ( Fig. 1; synthetic chemistry details are in supporting information). For synthesis, 5-methoxy-2-methylbenzo[d] [1,3]selenazole 2 was obtained using literature procedures from N-(acetyl) benzoyl-2-iodoaniline 1, using Woolins reagent under microwave conditions 29 and condensed with 2-(dimethylamino)pyrimidine-5-carbaldehyde in an aqueous potassium hydroxide (50%) solution dissolved in DMSO to obtain 3. Following purification, 3 was demethylated in the presence of BBr 3 to yield the phenolic derivative 4. Finally, 4 was alkylated with 2-fluoroethyl-4-methylbenzene sulfonate (prepared using literature procedure 30 ) in the presence of cesium carbonate to obtain 5. Alternatively, 4 was treated with (2-bromoethoxy)(tert-butyl) dimethylsilane in the presence of cesium carbonate to obtain (Z)-5-(2-(5-(2-((tert-butyldimethylsilyl)oxy)ethoxy)benzo[d] [1,3]selenazol-2-yl)vinyl)-N,N-dimethylpyrimidin-2-amine (6). Upon treatment with TBAF, its corresponding deprotected alcohol 7 was obtained. Following treatment of 7 with tosyl-chloride, 8 the precursor ligand for synthesis of the PET tracer was obtained. Finally, 8 was also treated with TBAF to obtain 5 via nucleophilic displacement. All intermediates 1, 2, 3, 4, 6, 7, 8 and the final compound 5 were characterized via standard analytical methods. Additionally, 5 was also analyzed for uniformity and purity, on a semi-preparative C-18 column (Phenomenex), using an HPLC system. Compound 5 eluted as a single chemical entity, with a retention time of 10.5 minutes thus indicating purity of the molecule. For determination of the solid-state structure, crystals suitable for X-ray analysis were obtained via a vapor diffusion method, involving slow diffusion of methanol into a DCM solution containing 1% ACN of 5. The benzoselenazole derivative 5 crystallized in a monoclinic space group P 2 1 /n. The ORTEP drawing showing the crystallographic numbering scheme for 5 is illustrated in Fig. 2. The crystal structure of 5 shows a planar molecule with the F atom displaced from the mean plane by 1.005 Å (mean plane deviation for the molecule except F = 0.048 Å). The heterocyclic molecule 5 indicates presence of both inter and intra-molecular hydrogen bonding (SI, Fig. 1). Noticeably, H13 forms intra-molecular H bond to N1 at 2.22 Å thus comprising a bond angle of C13-H13-N1 = 147°. Additionally, two intermolecular hydrogen bonds involve the N atoms N2 from the ring and the solvent N, N1s [C3-H3… .N2 (1.5-x, y-0.5, z-0.5) = 2.61 Å and the bond angle around H3 = 164.5°; C15-H15a… N1s (1+ x, y, z) = 2.68 Å and the angle around H15a = 170.5°]. While search of the Cambridge Crystallographic database does not show organic scaffolds sharing a similar arrangement of atoms around Se in the molecule, the closest chemical structure for comparative analysis remains the first generation Aβ targeted molecule reported earlier 25 . Importantly, the chemical structures of 5 and its first generation benzothiazole derivative counterpart (C 18 H 18 FN 3 OS) are identical except that Se is swapped with S (while the six membered aromatic ring of 5 possesses 2Ns, the aromatic ring of the benzothiazole derivative contains only one N atom). Therefore, Se-C interatomic distances (1.868(7) and 1.917(7) Å) in 5 are larger compared to the S-C distance (1.728(2) and 1.762(2) Å) in earlier reported benzothiazole derivative thus consistent with a larger size of the selenium atom. Furthermore, the bond angle around the Se atom of 5 is also significantly narrower than that of benzothiazole derivative (84.6° vs 89.1°). Overall, comparative analysis indicate both 5 and its first generation counterpart 25 share planar geometry with the F atoms displaying out of plane deviation. Finally, NMR spectral data of 5 was also consistent with the crystal structure thus indicating the presence of identical structures both in solid and solution state (J H-H = 7.4 Hz; alkene protons indicating the presence of Z isomer) and the results are in accord with the first generation Aβ agent 25 .
For assessing the ability of 5 to bind Aβ plaques; preliminary binding assays with preformed Aβ 1-42 fibrils were performed in PBS. Following excitation at 392 nm, fluorescence spectrum of 5 recorded in PBS containing 1% ethanol showed a broad emission peak at 450-540 nm with E max at 490 nm. Upon incubation with preformed Aβ (1-42) aggregates, the peak (490 nm) showed remarkable enhancement in fluorescence indicating binding to Aβ aggregates, similar to enhancement in fluorescence of thioflavin T in PBS (a positive control; data not shown). Additionally, it is also noteworthy that no fluorescence was observed using Aβ aggregates alone in PBS following excitation at 392 nm (a negative control). Preliminary binding assays of 5 with preformed Aβ 1-42 aggregates (using a single site binding model) indicated saturable binding with a K d = 1.58 ± 0.05 nM (SI, Fig. 2).
To further assess ability of the 5 to label Aβ plaques in human brain, staining experiments were also performed with postmortem tissues from clinically-characterized AD patients 35,36 As a positive control, a highly specific anti-Aβ antibody (10D5, Eli Lilly, Indianapolis, IN) was used to ascertain the presence of Aβ plaques (Fig. 4A), using well-established procedures for assessment of Aβ plaques in postmortem brain 37 . Importantly, 5 (2 μ M) demonstrated strikingly distinct labeling of Aβ plaques in the tissue sections of frontal lobe of a 90-year-old male with AD ( Fig. 4B) and absence of Aβ plaques in normal controls (SI, Fig. 4A,D), therefore indicating target specificity. Noticeably, 5 also indicated proficient and distinct labeling of plaque and leptomeningeal vessels (cerebral amyloid angiopathy; CAA) (Fig. 4B). These data are consistent with other unlabeled counterparts of FDA approved PET agents 38 . Furthermore, thioflavin S, an amyloid staining dye showed staining of amyloid in the blood vessel (CAA) and indicated only weak staining of diffuse plaques (Fig. 4C). Importantly, 5 demonstrated  labeling of numerous diffuse Aβ plaques (Fig. 4B) and specificity for Aβ plaques in AD compared with biomarkers of other neurodegenerative diseases (SI, Fig. 4A,B). This high specificity of 5 for Aβ could be attributed to presence of a benzoselenazole ring within Fluselenamyl. Overall, the ability and sensitivity of 5 to detect diffuse plaques and specificity for AD could represent an important advancement to enable PET imaging of mildly demented individuals, prior to onset of symptoms 6 .
For performing additional bioassays and correlating Aβ binding data of 5 obtained via fluorescence assay, the PET counterpart [ 18 F]-9 was synthesized via standard nucleophilic substitution, employing 2,2,2-kryptofix/ 18 F and tosylate analog 8. Following reaction and Sep-Pak treatment to separate free fluoride, the crude mixture was purified on a C-18 column, using a radio-HPLC system, with an overall radio-chemical yield of 35% (radiochemical purity > 99%; specific activity (1700-2000 Ci/mmol). Furthermore, [ 18 F]-9 was also characterized by spiking with an analytically characterized sample of an unlabeled counterpart 5 (SI Fig. 5), prior to injection on the radio-HPLC. The fraction eluting at R t = 10.5 min was collected, concentrated, and resuspended in PBS/ethanol (95/5) for all radiotracer bioassays.
For assessing the ability of [ 18 F]-9 to bind Aβ plaques; binding assays with either AD homogenates or preformed Aβ 1-42 aggregates were performed in 30 mM Tris, pH 7.4 buffer supplemented with 0.1% BSA 39 . Nonspecific binding was determined in the presence of 5 (1 μ M) as a competitor. Overall, the binding assay of [ 18 F]-9 with AD homogenates and Aβ 1-42 fibrils (Fig. 5A-D), indicates a saturable specific binding with K d = 1.7 nM (B max = 546 pmol/g wet wt.) and 1.6 nM (B max = 1.3 pmol/nmol), respectively (Fig. 5A,B). Scatchard plots of the binding data indicate that [ 18 F]-9 binds to a single high affinity site on AD homogenates and Aβ 1-42 fibrils (Fig. 5C,D). Importantly, the binding affinity of [ 18 F]-9 with autopsy confirmed AD homogenates is significantly superior to that of other FDA approved Aβ -targeted probes ( 42 . Previously, the incorporation of pyrimidine ring into benzothiazole has been shown to decrease binding affinity of the molecule to Aβ fibrils 43 . However, the design of Fluselenamyl includes an incorporation of pyrimidine ring into benzoselenazole, wherein the atomic radius of selenium (115 pm) is much larger than that of sulfur atom (100 pm) present within benzothiazole 43 , resulting in longer bond lengths involving selenium with neighboring atoms of the 5-membered ring (as evident from crystal structure data of 5), and the presence of an additional double bond between two ring systems (benzoselenazole and pyrimidine ring) thus generating a relatively better flow of electrons consistent with planarity of the molecule Additionally, the incorporation of dimethylamino group into the pyridine ring has been shown to promote selectivity for Aβ 44 . Therefore, the higher binding affinity of 5 to Aβ plaques could be attributed to these combined variations in the scaffold.
Literature precedents indicate the presence of at least three different binding sites, characterized as BS1, BS2 and BS3 on the Aβ fibrils in vitro 24 and extracts from AD homogenates 23 . A recent binding study using homogenates from AD confirmed human tissues and their control counterparts also indicates the presence of multiple binding site models for the amyloid tracers 23 , wherein [ 18 F]-AV-45 and [ 11 C]-PiB have been shown to bind to two different binding sites, a high-affinity site (visualized by PET) and a low affinity site. Additionally, while BF-227 shows binding to BS3, the FDDNP has been postulated to bind only to BS2 23 . Overall, these investigations indicate that different PET tracers may be beneficial to better understand Aβ pathophysiology in vivo. To assess the binding site targeted by [ 18 F]-9 on AD homogenates, binding assays were also performed in the presence of unlabeled analytically characterized samples of PiB, IMPY, and Chrysamine G. While no displacement was observed with Chrysamine G, significant displacement of [ 18 F]-9 specific binding was observed in the presence of unlabeled PiB and IMPY, suggesting that radiotracer [ 18 F]-9 binds to the same high affinity site on AD homogenates. This is further supported by the observation that the K i values obtained from analysis of the competition assays (K i = 4.9 nM for PiB, K i = 6.3 nM for IMPY) are also consistent with previously reported K d values obtained in assays measuring direct binding of these tracers to AD homogenates. Further, to evaluate ability of [ 18 F]-9 for labeling Aβ in autopsy confirmed human brain sections, autoradiography and immunohistochemical correlations were also performed. Following incubation of AD frontal cortex sections (12 μ m) with [ 18 F]-9 (2 nM) for 60 min, the agent showed labeling of cortical Aβ plaques and the binding was inhibited upon incubation in the presence of 5 (1 μ M) (Fig. 6A,C). These data indicate sensitivity and specificity of the [ 18 F]-9. Additionally, immunohistochemical staining of these sections using anti-Aβ -antibody indicated the presence of Aβ plaques in the cortex of these sections (Fig. 6B,D) thus demonstrating excellent correlation of immunohistochemical staining data with that of autoradiography data.
For biomedical imaging applications, the signal is a net function of target/background ratio. In PET imaging, this signal results from detection of γ -photons arising from annihilation events of positrons and imaging resolution is also dependent upon energy associated with a given radionuclide. Resultant radioactive metabolites could also contribute to nonspecific binding or compete with the parental tracer for binding to the target. Therefore, it is also important to investigate metabolic stability of the radiotracer to explicitly confirm whether or not biochemical targeting profiles of a given tracer are driven by a parental molecular imaging probe. Importantly, both [ 11 C]-PiB and [ 18 F]-AV-45 have demonstrated low biological half-lives in serum; while metabolites of [ 11 C]-PiB have been shown to be polar and thus postulated to not penetrate the brain 45 , two metabolites of [ 18 F]-AV-45 (desmethylated, 4.5% ID/g; acetylated analogue, 3.3% ID/g at 2 min in normal mice) 10 have been shown to permeate the brain and thus could potentially contribute nonspecific interaction and complicate image analysis. To perform preliminary evaluation of biological half-life, [ 18 F]-9 was also incubated at 37 °C in human serum as a function of time, and aliquots were analyzed on radio-HPLC. The presence of a single radio-peak indicated presence of parental tracer up to 3 h (SI Fig. 6).
To further assess whether or not PET counterpart [ 18 F]-9 administered at tracer concentrations relevant for nuclear imaging demonstrates optimal kinetics (signal/noise ratios) to enable brain imaging in vivo, quantitative biodistribution studies in normal mice were performed. Uptake in brain and other critical organs was analyzed in terms of percent injected dose per gram of the tissue (%ID/g) SI Table 1. For in vivo imaging of Aβ plaques, the basic pharmacokinetic model in normal brains involves a high initial penetration of the agent, accompanied by facile clearance due to lack of a binding target. Preliminary biodistribution studies (SI, Table 1) with HPLC purified [ 18 F]-9 in normal mice show transient brain uptake values of 8.86 ± 0.32% ID/g and 1.66 ± 0.01% ID/g, at 2 min and 120 min post tail-vein injection, respectively, thus providing a 2 min/120 min clearance a ratio of 5.33.  46 , respectively. Therefore, the brain uptake clearance ratio of [ 18 F]-9 (2 min/120 min) is 1.3-fold superior to [ 18 F]-AV-45 and is comparable to that of [ 18 F]-Florbetaben in healthy mice. For an agent to be able to serve as an Aβ -imaging agent, literature precedents indicate that brain uptake ratio (%ID/g; the earliest time-point; 2-5 min to that of the latest time point; typically for carbon-11; 30-60 min; and F-18, 2 h) of 3.5 or above could be considered as a benchmark for ability of a given agent to cross the blood-brain barrier 38 . Additionally, the brain uptake of a given imaging agent is also a net function of several components, such as cerebral regional blood flow, BBB permeability, plasma radiotracer concentration, and free fractions of the radiotracer in plasma and in the brain. Furthermore, the lipophilicity of a given compound also reflects a critical physicochemical trait for neuroimaging radiotracers due to its direct relationship to membrane permeability, solubility in water, and entropic contribution to binding. Literature precedents indicate that lipophilic drugs readily cross the BBB, although other chemical characteristics, including the number of hydrogen bonds, molecular weight, polar surface area and molecular size are also known to be critical traits for passive transport. Lipophilicity measured via (log P OCT ), the octanol/water partition coefficient for non-ionized molecules serves as a good indicator of a molecule to permeate brain and molecules possessing log P values of 0.9 and 3.0 have been shown to cross the BBB 47 . Conversely, radiotracers that are too lipophilic can also bind plasma proteins, undergo fast metabolism, while also contributing to high nonspecific binding, such as white matter. [ 18 F]-9 demonstrates a log P value of 1.28 which is similar to that of [ 11 C]-PiB (1.3) but considerably lower than that of [ 18 F]-AV-45 (2.4), and [ 18 F]-Florbetaben (3.22). While the clearance ratio of 5.33 (%ID/g; 2 min/120 min; SI Table 1) provides evidence for the ability of [ 18 F]-9 to traverse the BBB in vivo, the log P value of 1.28 could also lead to a low nonspecific interaction with white matter. Additionally, the agent [ 18 F]-9 excreted from other critical organs over 2 h (SI, Table 1); a critical factor likely to result in favorable dosimetry; although slight defluorination as a function of time is also evident from bone accumulation; yet consistent with pharmacokinetic profiles of other FDA approved agents 10,17,18 . For assessing directly the ability of radiotracer to traverse the BBB, [ 18 F]-9 (170 μ Ci) was injected into a 24 months old APP/PS1 transgenic mouse and its age-matched WT counterpart, and 30 min post tail-vein injection, the brains were removed, frozen, and sectioned for autoradiography ( Fig. 7A-E). Autoradiograms of the brain sections showed labeling of plaques in cortical regions, in addition to probable off-target binding in white matter (Fig. 7A). The labeling of plaques was confirmed by staining with anti-Aβ monoclonal antibody (mHJ3.4 conjugated to Alexa Fluor 568, Fig. 7B) and data show fair correlation with a caveat of some off-target binding attributed to differences between targeting profiles of a small organic molecule versus the large highly specific monoclonal antibody (Fig. 7A,B). For ROI analysis of the PET signal in brain section autoradiograms of transgenic mouse (Fig. 7A) and its WT counterpart (Fig. 7D), we used measured count densities in cortex and amygdala target regions and compared them with that of hypothalamus as reference region with a low plaque density as determined by Aβ immunostaining (Fig. 7B). The target/reference ratio (mean ± SD) in the transgenic mouse was 1.88 ± 0.22 compared to 0.96 ± 0.12 in the WT control mouse (Fig. 7E). Since brain stem was also devoid of plaques, we also analyzed the above target regions using brain stem as a reference region, and observed target/reference value in the transgenic to be 0.89 ± 0.1 compared to 0.45 ± 0.07 in the WT mouse.
For assessing viability of molecules as imaging probes in vivo 48 , various imaging modalities, such as nuclear imaging (PET/SPECT), optical imaging, and MRI have been used to investigate simultaneously distribution kinetics and target-receptor specificity. While the resolution of PET and MRI allow in vivo imaging at relatively moderate resolution, the multiphoton microscopy enables evaluation of kinetics at a sub-micrometer resolution 49 . Therefore, this technique enables characterization of probes in small animal models at a significantly high spatial and temporal resolution 49 . The generation of various transgenic (tg) animals overexpressing mutant human APP and/or PS1 and PS2, tg APP mice expressing ApoE isoforms including those for Tau offer attractive models for unravelling biochemical pathways prevalent in pathophysiology of AD [50][51][52][53][54] . Of note, compared to single transgenic animal models (APP or PS1), co-expression of PS1 with APP exhibits robust deposition of Aβ months earlier than APP tg mice alone. Literature precedents indicate Aβ deposition by 2 months of age, with gradual quantitative increase at six months 55 . Therefore APP/PS1 mice offers interesting models for evaluation of therapeutics and validation of imaging probes in vivo 25 . For assessing the ability of 5 to penetrate the BBB, label Aβ parenchymal plaques, and simultaneously interrogate the pharmacokinetic profiles from nearby brain regions, direct real-time imaging was performed in transgenic APP +/− /PS1 +/− mice. Prior to imaging, dextran-Texas Red conjugate (33 mg/kg; dissolved in PBS to mark the blood vessels) and 5 (2 mg/kg; dissolved in 20% DMSO in propylene glycol 56 ) were intravenously administered to anesthetized APP +/− /PS1 +/− mice (with cranial windows; supporting information). Following injection, 3D volumes were acquired (by collecting a stack of x-y sections from surface of the thinned skull to 100 μ m deep into the cortex). Compared with barely detectable auto-fluorescence levels prior to imaging transgenic APP +/− /PS1 +/− mice (Fig. 8, first panel), bright fluorescence appeared almost instantaneously following administration of 5. Fluorescence first appeared in large and small blood vessels within the brain. Within minutes (10 min), the brain parenchyma was uniformly bright in fluorescence, and Aβ deposits were labeled. While the complete labeling of CAA occurred instantaneously following injection, the labeling of parenchymal plaques peaked at approximately 10 min. Overall, these data demonstrate in real time that 5 permeates the brain rapidly, and visualizes parenchymal Aβ plaques (Fig. 8).
To directly access the potential of [ 18 F]-9 to bind Aβ plaques in vivo, we performed microPET/CT imaging in age-matched APP +/− /PS1 +/− mice (n = 3) compared their WT counterparts, 5 min-2 h post tail-vein injection. For any given agent to serve as an Aβ -targeted agent, a pharmacokinetic model would involve an initial high and equal influx of the tracer into the brains of transgenic and WT mice, followed by clearance of the radiotracer from brains of WT mice thus demonstrating differential retention in regions of brains, as the agent binds to Aβ plaques in transgenic mice. Indeed, the [ 18 F]-9 demonstrates a 1.2-folds (p < 0.05) higher retention in transgenic mice brains consistent with its binding to Aβ plaques, while exhibiting clearance of the unbound tracer from brains of WT counterparts (Fig. 9). Time activity curves (TAC) also indicated higher retention of activity within transgenic mice brains compared with their WT counterparts (Fig. 10). Additionally, ROI (cortical regions) were also analyzed using linearized method with a reference region (cerebellum) via Reference Logan plot (RefLogan), and distribution volume ratios (DVR) were obtained. The analysis indicated a statistically significant DVR value of 1.18 for transgenic mice brain compared with that of 1.03 for WT brains. These smaller differences in DVR values of targeted regions in transgenic mice compared with that of WT counterparts could also be attributed to a lack of true negative reference region in aged PS1/APP mice 57   did not show differences consistent with spatial localization of Aβ in brains of PS1/APP mice models compared with controls, [ 18 F]-9 shows statistically significant differences between PS1/APP transgenic mice and age-matched WT brains. The differences between these tracers in this rodent model could be attributed to ability of [ 18 F]-9 for targeting both diffuse and fibrillary plaques. To further evaluate, whether or not [ 18 F]-9 offers significant advantage over other approved tracers, the agent would need to be further investigated for its performance in other AD rodent models (APP23 and Tg2576). Nevertheless, the preclinical data indicate that [ 18 F]-9 could provide a novel benzoselenazole based PET tracer, worthy of further evaluation to enable interrogation of Aβ load within the brain.  Conclusions A novel heterocyclic benzoselenazole derivative 5 was synthesized and structurally characterized. Crystal structure revealed a planar geometry 25 with fluorine atoms deviating out of the plane, and the presence of intermolecular and intramolecular hydrogen bonding. The agent demonstrates potent binding affinity to autopsy confirmed AD homogenates and the binding affinity constant is superior to that of [ 18 F]-AV-45 41 ]-9 labels Aβ plaques in cortex of autopsy confirmed AD brain sections, while also demonstrating a close immunohistochemical correlation with anti Aβ antibody-conjugate thereby indicating specificity for mapping the biomarker protein. The fluorescent molecule 5 detects Aβ plaques in brain hippocampus sections of transgenic mice, while also visualizing CAA. Noticeably, 5 labels both diffuse and fibrillar plaques in brain sections of AD patients, while lacking interaction with biomarker proteins of other neurodegenerative diseases thereby indicating specificity for detecting Aβ in AD. The ability of 5 to detect diffuse plaques (the precursor to fibrillar neuritic plaques) in AD brain tissue sections could be beneficial in stratification of subjects with a risk factor for development of AD, while also enabling quantitative assessment of overall efficacy of anti-amyloid therapeutics. Finally, Fluselenamyl demonstrates favorable biological half-life in human serum, facile brain penetration, and ability to detect parenchymal plaques in transgenic mice, minutes post-intravenous injection. Overall, these data provide a provocative platform for further development of PET tracers comprising benzoselenazoles moiety in their organic scaffold to enable noninvasive assessment of Aβ plaques in vivo. Further investigations evaluating ability of 5 to map Aβ burden in various AD rodent models as a function of aging are under progress.