Heterocyclic molecules for biomedical imaging and therapeutic applications

Abstract

Probes which target diffuse and fibrillar forms of amyloid beta (A) are described. These probes demonstrate high initial brain penetration and facile clearance from non-targeted regions. The agents can be used to image amyloid quantitatively for monitoring efficacy of A-modifying therapeutics and assist in premortem diagnosis of Alzheimer's disease (AD). Disclosed probes can bind A aggregates of preformed A.sub.1-42 fibrils in vitro and can be used to image fibrillar and diffuse plaques ex vivo in brain sections. Disclosed probes can be used to determine A burden in early stages of AD. These probes can be used for multimodality imaging of A. F-AI-187 (1 M) can detect A plaques in brain sections of APP/PS1 mice. F-AI-187 (10 M) can detect A plaques in the frontal lobe in a brain section of a patient with confirmed AD. Some probes can be used for fluorescence imaging of plaque.

Claims

1. A compound or a pharmaceutically acceptable salt thereof selected from the group consisting of ##STR00148## or a pharmaceutically acceptable salt thereof.

2. A gold nanoparticle conjugated to the compound according to claim 1.

3. A method of imaging distribution of amyloid beta in a sample or a subject, comprising: administering the compound or a pharmaceutically acceptable salt thereof according to claim 1 to the sample or subject wherein the compound or pharmaceutically acceptable salt thereof comprises a radionuclide; and subjecting the sample or subject to PET or SPECT scanning.

4. A method of imaging cardiac systemic amyloidosis in a subject, comprising administering an imaging effective amount of the compound or a pharmaceutically acceptable salt thereof according to claim 1 to the subject, and imaging systemic amyloidosis in the subject by PET or SPECT scanning.

5. A complex comprising: the compound or a pharmaceutically acceptable salt thereof according to claim 1; and a gold nanoparticle.

6. The compound or a pharmaceutically acceptable salt thereof according to claim 1, Wherein the compound is ##STR00149##

7. The compound or a pharmaceutically acceptable salt thereof according to claim 6, wherein the F is .sup.18F.

8. The compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein the F is .sup.18F.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) In drawings based on multi-color originals, gray-scale versions of each color channel (red, green and/or blue) are shown, as well as a composite gray scale that combines all 3 (RGB) color channels.

(2) FIG. 1 illustrates concentration dependent and saturable binding (binding constant, 597 nM) to preformed A1-42 fibrils of agent F-AI-182, of structure

(3) ##STR00123##

(4) FIG. 2 illustrates staining of both fibrillar and diffuse plaques ex vivo in the hippocampus and cortical region of brain sections in APPsw+//PS1 mice using agent F-AI-182.

(5) FIG. 3 illustrates a comparative analysis of pharmacokinetics in normal mice for .sup.18F-AI-182 of structure

(6) ##STR00124##
and .sup.18F-AV-45 of structure

(7) ##STR00125##

(8) FIG. 4 illustrates that .sup.18F-AI-182 is washed out from blood (25% faster than AV-45) in absence of targeted plaques and remains non-metabolized in human serum. .sup.18F-AI-182 shows facile penetration of the brain and clearance in normal mice.

(9) FIG. 5 illustrates that F-AI-182 can be used to detect both diffuse and compact AP plaques in the brain cross-sections of frontal lobe of a 90-year-old female with neuropathologically confirmed Alzheimer's disease.

(10) FIG. 6 illustrates real time imaging using F-AI-182.

(11) FIG. 7 illustrates assessment of binding sites of PIB, AV-45, and AI-182.

(12) FIG. 8 illustrates staining of brain tissue sections from APPsw+/ (24 months old) mice using F-AI-183 of structure

(13) ##STR00126##

(14) FIG. 9 illustrates detection of compact A plaques in the brain cross-sections of frontal lobe of an 88-year-old female with neuropathologically confirmed Alzheimer's disease using F-AI-183.

(15) FIG. 10 illustrates fluorescence imaging of tumor cells in vitro, labeled by uptake of fluorescent AI-182.

DETAILED DESCRIPTION

(16) Abbreviations

(17) A Amyloid beta

(18) AD Alzheimer's Disease

(19) ADME absorption, distribution, metabolism, and excretion

(20) APP amyloid precursor protein

(21) BBB blood-brain barrier

(22) BS1 binding site 1

(23) BS2 binding site 2

(24) BS3 binding site 3

(25) .sup.13C NMR carbon nuclear magnetic resonance

(26) CAA cerebrovascular amyloid angiopathy

(27) CERAD Consortium to Establish a Registry for Alzheimer's Disease

(28) DS Down Syndrome

(29) .sup.19F NMR fluorine nuclear magnetic resonance

(30) .sup.1H NMR proton nuclear magnetic resonance

(31) HPLC high-performance liquid chromatography

(32) HRMS high-resolution mass spectroscopy

(33) MIRD Medical Internal Radionuclide Dose Committee

(34) NIA-RI National Institute of Aging-Reagan Institute

(35) NFT neurofibrillary tangle

(36) NMR nuclear magnetic resonance

(37) PBS phosphate buffered saline

(38) PET positron emission tomography

(39) SAR Structure-Activity Relationships

(40) SP senile plaque

(41) SPECT single photon emission computed tomography

(42) WT wild type

(43) The present teachings disclose agents that can be used for imaging cancers and neurodegenerative diseases. Reagents described herein also have therapeutic use in neurodegenerative diseases and cardiovascular diseases.

(44) In various embodiments, a fluorine-18-based PET probe can be capable of targeting high prevalence sites of A and displaying faster kinetics compared to non-targeted regions, such as white matter. In various embodiments, a fluorine-18-based PET probe can be used for quantitative amyloid imaging for monitoring progress of A-modifying treatments in the presymptomatic and symptomatic stages of Alzheimer's disease (AD), and/or premortem diagnosis of AD.

(45) In various embodiments, the present teachings include heterocyclic molecules (exemplified as F-AI-182) that can bind to A aggregates in vitro with concentration dependent and saturable binding. For example and without limitation, binding constants to preformed A.sub.1-42 fibrils can be F-AI-182, 597 nM; F-AI-183, 17 nM; F-AI-187, 1.58 nM0.05 nM. In various configurations, these probes can stain both fibrillar and diffuse plaques ex vivo in the hippocampus and cortical region of brain sections in APPsw.sup.+//PS1 mice and human tissues. In some aspects, F-AI-182 can incorporate .sup.18F (t.sub.1/2=110 min), a radionuclide for medical PET imaging (Mahmood, A. & Jones, A. Technetium Radiopharmaceuticals. Handbook of Radiopharmaceuticals. 323-362 (2003); Eckelman, W. The Development of 99mTc Radiopharmaceuticals for Perfusion and Biochemistry: In Technetium and Rhenium in Chemistry and Nuclear Medicine 3. M. Nicolini, G. Bandoli and U. Mazzi (Eds.). Cortina Int., Verona, Italy. pp. 571-580. (1990); Narra, R., et al. A Neutral Tc-99m Complex for Myocardial Imaging. J. Nucl. Med. 30, 1830-1837 (1989); Stadalnik, R., Kudo, M., Eckelman, W. & Vera, D. In vivo functional imaging using receptor-binding radiopharmaceuticals: 99mTc-galactosyl-neoglycoalbumin (TcNGA). Investigative Radiology 28, 64-70 (1993)). In some aspects, F-AI-182 can be used for diagnostic assessment of A burden in earlier stages of AD prior to expression of clinical symptoms. In some aspects, a radiolabeled counterpart .sup.18F-AI-182 can demonstrate a high initial brain penetration (7.280.46% % ID/g) of FVB mice, followed by 25% faster clearance from the blood pool (compared with AV-45) in normal mice in the absence of targeted plaques. In some aspects, .sup.18F-AI-182 can remain non-metabolized until about 30 min (investigated highest time-point) in human serum. In some aspects, F-AI-182 can demonstrate characteristics that enhance overall signal to background ratios and assist image analysis including lack of metabolites and high first-pass extraction into brain of coupled with fast clearance from the blood pool.

(46) In some embodiments, a tracer of the present teachings can provide high target/background ratios. In some aspects, multiphoton microscopy can demonstrate that an unlabeled counterpart F-AI-182 of the radiolabeled PET agent can label brain parenchymal A plaques as well as tracked cerebrovascular amyloid angiopathy (CAA), indicating its ability to serve as a noninvasive probe for assessment of plaque burden in brain. In some aspects, these data can illustrate a platform technology for image analysis in biomedical PET imaging, using an F-18 labeled PET agent.

(47) In various embodiments, a functional probe of the present teachings can have hydrophobic characteristics to cross the blood-brain barrier (BBB) and not be retained in non-targeted regions of the brain. In various aspects, a fluorescent molecule of the present teachings can show enhanced fluorescence upon binding to fibrils, can stain both fibrillar and diffuse plaques in brain cross sections of APP/PS1 transgenic mice and human AD tissues, and can show high initial penetration in the normal brain followed by clearance in the absence of targeted plaques. In various aspects, the agent can clear rapidly from other organs, such as liver and kidney, remain non-metabolized in human serum, and display modest hydrophobicity (log P 1.2) for formulation in 2% ethanol and 98% saline for intravenous injections. The scaffold of F-AI-182 can be used for interrogating AD in a prodromal phase.

(48) In some embodiments, heterocyclic small organic molecules of the present teachings can also be used for multimodality imaging of A using PET/Optical imaging in preclinical applications.

(49) In some embodiments, an agent can exhibit enhanced brain penetration, A interaction, and the ability to interact with highly prevalent or more-dense binding sites on A. In some embodiments, an agent can be identified by either the lack of binding or reduced binding to the white matter for enhancing sensitivity of tracers for A detection in human tissues. In some embodiments, an agent of the present teachings can label A plaques in brain parenchyma <5 min post-intravenous administration.

(50) In some embodiments, the specificity of agents can be determined for A compared to other biomarkers prevalent in neurodegenerative disorders (with overlapping symptoms) such as, tan protein, neurofibrillary tangles (NFT) and Lewy body, including further optimization of targeting properties through SAR study. In some embodiments, an agent can exceed or mimic the pharmacokinetic profiles (brain uptake and blood clearance) of .sup.18F-AV-45, an FDA approved agent for imaging A in brain. In an embodiment, .sup.18F-AI-182 showed facile penetration of the blood-brain barrier (BBB) in in vitro targeting of A in a mouse model.

(51) In some embodiments, a compound of the present teachings can be used for noninvasive assessment of A in early stages of AD prior to clinical expression, and can allow therapeutic interventions for disease management. In some embodiments, a compound of the present teachings can be used for stratification of patients in early phases of AD to allow for therapeutic interventions.

(52) Embodiments of A-targeted agent can include functional components including but not limited to the following examples.

(53) An embodiment of an A-targeted agent can include a benzothiazole moiety without the methyl group on the heterocyclic nitrogen of thioflavin T. This can allow the removal of the positive charge to increase the affinity of the probe to A fibrils and enhance hydrophobicity to facilitate BBB penetration.

(54) An embodiment of an A-targeted agent can include modifications on the 6.sup.th position of the benzothiazole ring has been shown to impact affinity of probes for plaques.

(55) An embodiment of an A-targeted agent can include the introduction of an olefin bond between the benzothiazole moiety and the aromatic ring to increase electron density as well as flexibility of the molecule to promote interactions with other binding sites on A plaques.

(56) An embodiment of an A-targeted agent can include substituting a basic dimethylamino group into an aromatic ring at p-position to the olefinic carbon. In some configurations, this can allow an increase electron density on nitrogen.

(57) An embodiment of an A-targeted agent can include incorporation of a heteroatom, such as nitrogen in the aromatic ring ortho to the highly basic dimethyl-amino group. In some configurations, this can allow better resonance stabilization of the molecule for influencing Pi-Pi interactions and can allow targeting of highly dense and moderate affinity sites on A fibrils.

(58) Methods

(59) Methods and compositions described herein utilize laboratory techniques well-known to skilled artisans. Such technique guidance can be found in laboratory manuals and textbooks such as Spector, D. L. et al., Cells: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998; Hedrickson et al., Organic Chemistry 3rd edition, McGraw Hill, New York, 1970; Carruthers, W., and Coldham, I., Modern Methods of Organic Synthesis (4th Edition), Cambridge University Press, Cambridge, U.K., 2004; Curati, W. L, Imaging in Oncology, Cambridge University Press, Cambridge, U.K., 1998; Welch, M. J., and Redvanly, C. S., eds. Handbook of Radiopharmnaceuticals: Radiochemistry and Applications, J. Wiley, New York, 2003.

(60) In some embodiments of the present teachings, biochemical characterization of F-AI-182 and other molecules can by performed agents via multiple binding and competitive displacement assays using PIB, AV-45, AZD4694, and BAYER 94-9172 for evaluation of targeted sites on A, phosphorimaging studies in vitro. In vivo, ex vivo binding studies of AD brain homogenates and human AD brain sections can be performed, including specificity for A evaluated compared with other biomarker proteins (tau, prion, TDP43, and -synclein) prevalent in other neurodegenerative diseases to determine target selectivity, and perform metabolite studies.

(61) In some embodiments of the present teachings, the inventors have biochemically characterized and validated agents via multiple in vitro bioassays to evaluate target sensitivity and specificity. The inventors can evaluate an A-targeted agent of the present teachings to detect A plaques via MicroPET imaging with pharmacokinetic analysis in APP transgenic mice and their WT counterparts. Investigations of the present teachings include a focused Structure-Activity Relationships (SAR) study to discover A-targeted agents. The agents obtained from SAR can be biochemically characterized and evaluated through biodistribution and pharmacokinetic studies. The findings can be used to further characterize A-targeted probes such as .sup.18F-AI-182.

(62) In some embodiments of the present teachings, heterocyclic small organic molecules can be characterized and validated through various analytical steps. In various embodiments, molecules can also be radiolabeled, HPLC purified, and undergo a chemical characterization for developing as either radiopharmaceuticals or optical probes. In some aspects, he HPLC purified organic molecules and their radiolabeled counterparts can be tested for binding affinity. The compounds can be evaluated in animal models using either microPET imaging or multiphoton imaging. The agents that can detect A plaques in mice can also undergo metabolite analysis in vivo for interrogating their translational potential. The agents that remain non-metabolized in the targeted tissue (brain) can also be investigated via pharmacokinetic studies in age-matched APP transgenic and control mice for assessing preliminary signal-to-noise ratios.

(63) In some embodiments of the present teachings, binding assays to preformed A fibrils or AD brain homogenates are disclosed.

(64) In some configurations, the present teachings include preparation of A fibrils or AD brain homogenates. In vitro binding assays can be performed to evaluate interactions of radiolabeled peptides with fibrils of A.sub.1-40/42 or extracts of AD brain homogenates (Choi, S. R., et al. Preclinical properties of .sup.18F-AV-45: a PET agent for A plaques in the brain. J Nucl Med 50, 1887-1894 (2009)) in histopathological core of the Alzheimer's Disease Research Center (ADRC), using standard procedures described in the literature (Zhuang, Z., et al. Structure-activity relationships of imidazo[1,2-a]pyridines as ligands for detecting amyloid plaques in the brain. J Med Chem 46, 237-243 (2003)).

(65) In some embodiments of the present teachings, binding assays to preformed fibrils or AD brain homogenate extracts can be performed using literature procedures (Klunk, W., et al. Uncharged thioflavin-T derivatives bind to amyloid-beta protein with high affinity and readily enter the brain. Life Sci 69, 1471-1484 (2001); Zhen, W., et al. Synthesis and amyloid binding properties of rhenium complexes: preliminary progress towards a reagent for SPECT imaging of Alzheimer's disease brain. J Med Chem 42, 2805-2815 (1999)). Prior to binding assays, the stock solution (2 M) can be thawed. To aliquots of this stock solution, .sup.18F-AI-182 (also exemplified for either .sup.18F-AI-183 or .sup.18F-AI-187) can be added at various concentrations to a final concentration of 200 nM A fibrils or 200 L of AD brain extracts (20-25 g). The aggregate-bound .sup.18F-AI-182 (or other analogues) can be collected on Whatman GF filters using Brandon M-24R cell harvester, washed, and counted in a -counter (Perkin Elmer). Inhibition constants (K.sub.i) can be calculated as described previously (Han, H., Cho, C. & Lansbury, P. J. Technetium complexes for quantification of brain amyloid. J Am Chem Soc 118, 4506-4508 (1996)). Binding assay results can assist in evaluation of target specificity.

(66) Some embodiments of the present teachings include evaluation of binding sites. In some configurations, binding assays can be done as described above in at least Example 8 below. Fixed concentrations of A.sub.1-42 fibrils and .sup.18F-AI-182 can be incubated in the presence of increasing concentration of cold competitors [thioflavin T (BS1), PIB (BS3 & BS1), FDDNP (BS3 & BS1) and BSB (BS2)]. Cold PIB, BSB, and FDDNP can be synthesized using published procedures. Experiments can also be performed with AV-45, BAYER 94-9172, and AZD4694. Measurements can be performed in triplicate and processed as described in the Examples. Agents competing for sites targeted by .sup.18F-AI-182 can be expected to displace .sup.18F-AI-182. Agents competing for different sites can be expected to have minimal effects. This analysis can identify binding site specificity on A.

(67) Some embodiments of the present teachings include immunohistochemistry and phosphorimaging of labeled probes ex vivo and in vivo.

(68) In various configurations, staining experiments on mice (WT and APP transgenic) brain sections can be performed with either fluorescent A-targeted F-AI-182 (exemplified for other analogues) or highly specific A-targeted HJ3.4 mouse monoclonal antibody conjugated to Alexa 568 (DeMattos, R. B., et al., Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease. Proc Natl Acad Sci USA 99, 10843-10848 (2002)), and phosphorimaging can also be performed using .sup.18F-AI-182. For in vivo experiments, .sup.18F-AI-182 (exemplified for other analogues) can be intravenously injected. After 2 min and up to 2 h, mice (transgenic APP or APP/PS1 or WT) can be sacrificed, brains removed, dissected into two halves, processed and analyzed as described in this disclosure. Stained brain tissue sections of APPsw.sup.+/ can serve as positive controls and the non-stained tissue sections from brains of WT mice can provide negative controls. Radioactive brain tissues can be analyzed directly on a phosphorimager. .sup.18F-AI-182 (exemplified for other analogues) showing activity patterns consistent with the staining of unlabeled F-AI-182 or A-targeted HJ3.4 mouse monoclonal antibody-Alexa 568 can be further examined.

(69) In some embodiments, an A-targeted heterocyclic molecule can stain or label A plaques in cortical and hippocampal brain sections of APPsw.sup.+/ transgenic mice compared to none or minimal interaction in WT controls. Other embodiments can include labeled heterocyclic molecules that show a correlation between immunohistochemistry, phosphorimaging, and staining using A-targeted HJ3.4 mouse monoclonal antibody-Alexa 568.

(70) Some embodiments of the present teachings include evaluation of target specificity in human brain tissues using F-AI-182 or other A-targeted agents.

(71) In various configurations, specificity of F-AI-182 or other agents can be interrogated. Staining using F-AI-182 or immunohistochemistry can be performed using antibodies such as antibodies against A (10D5, Eli Lilly), phosphorylated tau (PHF-1, Albert Einstein Medical School, Bronx, N.Y.), ubiquitin (Dako, Glostrup, Denmark), -synuclein (LB-509, Zymed, CA), and TDP-43 (Proteintech, Inc., Chicago, Ill.) using established methods (e.g., Burack, M. A., et al. In vivo amyloid imaging in autopsy-confirmed Parkinson disease with dementia. Neurology 74, 77-84 (2010)). Sections can be processed and analyzed on a Zeiss LSM 5 PASCAL confocal system coupled to a Zeiss Axiovert 200 microscope. A targeted agents that demonstrate specificity for A in brain sections of diseased subjects consistent with the expected regional distribution of plaques compared to their healthy controls and lack of cross reactivity with histopathological markers (tau, TDP43; and -synuclein) can thus be investigated.

(72) In some embodiments of the present teachings, metabolic stability of .sup.18F-AI-182 and/or other agents can be evaluated.

(73) In various configurations, identified heterocyclic molecules can be assessed for metabolic stability for use in biomedical imaging applications both in vitro and in vivo using established procedures (e.g., Mathis, C., et al. Synthesis and evaluation of .sup.11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents. J. Med. Chem. 46, 2740-2754 (2003); Sharma, V. Radiopharmaceuticals for assessment of multidrug resistance P-glycoprotein-mediated transport activity. Bioconjug. Chem. 15, 1464-1474 (2004)). In some embodiments, .sup.18F-AI-182, another agent, or a combination thereof can be incubated in either serum or human serum albumin at time points corresponding to uptake in vivo (5 min to 2 h) and filtered through filters (30 kDa). Free and bound radiotracer can be calculated using previously describe methods (Bartholoma, M. D., et al. Effect of the prosthetic group on the pharmacologic properties of .sup.18F-labeled rhodamine B, a potential myocardial perfusion agent for positron emission tomography (PET). J Med Chem 55, 11004-11012 (2012)), and can be analyzed by radio-TLC scanner and radio-HPLC. For in vivo pharmacokinetics experiments, .sup.18F-AI-182 agent or other agents can be injected into mice via tail-vein, and mice can be sacrificed at the time points corresponding with data of our biodistribution studies (5 min to 2 h). Brain tissues, liver, and kidney can be removed (liver and kidney can be used to evaluate their metabolic stability in more stringent in vivo environments), sonicated, extracted and analyzed through radio-TLC and -HPLC. The .sup.18F-AI-182, other agents, or combinations thereof that demonstrate stability (>95%) at the targeted site through this analysis can be investigated further in nonhuman primates models.

(74) Various embodiments of the present teachings include biodistribution and pharmacokinetic studies of .sup.18F-AI-182 or other agents in normal and transgenic APPsw.sup.+//PS1 mice.

(75) In various configurations, pharmacokinetic analysis of unlabeled fluorescent small organic molecules, .sup.18F-heterocyclic molecules, and/or their .sup.18F-counterparts via biodistribution studies in age-matched APPsw.sup.+/ transgenic mice and WT mice can be performed to determine target-specificity, and measure the detection of in vivo A plaques, in APPsw.sup.+//PS1 transgenic mice versus control mice, using either multiphoton imaging or microPET/CT imaging system by .sup.18F-heterocyclic small organic molecules.

(76) In various configurations, agents can be evaluated in part by exploring the tissue distribution and kinetics of .sup.18F-AI-182 or other agents in normal mice and transgenic mice. Because these heterocyclic molecules can be labeled with .sup.18F using the methods described herein, biodistribution in normal mice can be determined. In such investigations, BL/6 (control mice; Taconic) or APPsw.sup.+//PS1 (transgenic, Taconic) mice can be anesthetized by isoflurane inhalation and injected with .sup.18F-AI-182 or other agents (20 Ci in 50-100 l saline) via bolus injection through a tail vein. Animals can be sacrificed by cervical dislocation at 2, 30, 60, and 120 min post-injection (n=2-4) and data can be quantified into % ID/g as described (Sivapackiam, J., et al. Synthesis, molecular structure, and validation of metalloprobes for assessment of MDR1 P-glycoprotein-mediated functional transport. Dalton Trans 39, 5842-5850 (2010)). The brains can be removed and dissected into cerebellums and remaining whole brain fractions prior to weighing and counting to evaluate regional differences in the location of radiotracer in comparison with transgenic mice.

(77) In some configurations, biodistribution and pharmacokinetic studies can assist in pharmacokinetic analysis, in general, and in evaluation of .sup.18F-AI-182 and/or other agents to permeate the BBB. In the absence of target, radiolabeled heterocyclic molecules can demonstrate uptake in brains of control mice, followed by washout of activity, resulting in low background signals. However, in the presence of plaques in transgenic APPsw.sup.+//PS1 mice, enhanced accumulation and retention in brains can allow noninvasive imaging of mice.

(78) Various embodiments of the present teachings include validation and correlation of MicroPET imaging with .sup.18F-AI-182 and/or other agents.

(79) In various configurations, validation and correlation of MicroPET imaging with .sup.18F-AI-182 and/or other agents can be performed in age-matched BL/6 (control) and APPsw.sup.+//PS1 mouse models on MicroPET/CT Focus 220 scanner. Twenty-six frames can be acquired over a 3 hour scan period with the following frame sequences: 51 min, 52 min, 55 min, 810 min, and 320 min. Frames of the original reconstructed PET data can be summed, and this summed image can be co-registered with CT. Regions of interest can be drawn and tissue-time activity curves (TAC) can be constructed by plotting the percent injected dose per c.c. tissue (% ID/cc).

(80) From control mice, peak activity in the brain can be detected within the first 5 minutes post bolus injection and rapid clearance can be detected over the subsequent 2 to 3 hours. For APPsw.sup.+//PS1 mice, the early peak can be comparable in magnitude and time, but tracer clearance can be significantly slower, reflecting binding of .sup.18F-AI-182 and/or other agents to A plaques. The differences between normal and APPsw.sup.+//PS1 mice can increase with time. This difference can be correlated with plaque load in a cohort of mice.

(81) Various embodiments of the present teachings include SAR studies to develop agents including but not limited to heterocyclic molecules capable of detecting A plaques in early stages of AD prior to clinical expression.

(82) In various configurations, candidate A-targeted imaging agents can include but are not limited to the following characteristics: a) specific binding to A plaques; b) specific binding to a prevalent binding site on A; c) high first-pass extraction into the brain and region specific binding consistent with pathological localization of A; d) minimal binding to the white matter for sensitivity to detect plaques at earlier stages of the disease to segregate pools of patients likely to benefit from therapeutics, and e) excretion from organs of the body over a time period for MIRD analysis. .sup.18F-AI-182 as an agent demonstrates the above listed characteristics. .sup.18F-AI-182 can offer a scaffold template for further SAR exploration to develop second generation A-targeted agents.

(83) Various embodiments of the present teachings include characterization of molecules via standard analytical tools.

(84) In various configurations, these embodiments can include docking studies that can utilize Glide and ADME calculations using QProp. Molecules can be chemically characterized via standard analytical tools. Binding affinities with other agents, such as PIB, AV-45, and AZD4694 can be compared. Molecules demonstrating different binding sites on A compared to these agents can be identified. Molecules demonstrating high first-pass extraction into brains of transgenic mice and low white matter binding to nonhuman primate or human tissues can be characterized in vivo through biochemical characterization via multiple binding and competitive displacement assays as well as through biodistribution and pharmacokinetic studies.

EXAMPLES

(85) The present teachings including descriptions provided in the Examples, are not intended to limit the scope of any claim or aspect. Unless specifically presented in the past tense, an example can be a prophetic or an actual example. The following non-limiting examples are provided to further illustrate the present teachings. Those skilled in the art, in light of the present disclosure, will appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present teachings.

Example 1

(86) This example illustrates an A targeted probe of the present teachings.

(87) Utilizing the agent F-AI-182, a heterocyclic molecule was synthesized via multiple steps, purified via chromatography, crystallized in methylene chloride and pentane mixture, and the single crystal structure was determined. F-AI-182 was further characterized via standard analytical tools, including .sup.1H NMR, proton-decoupled .sup.13C-NMR, .sup.19F NMR, high resolution mass spectroscopy (HRMS), and analyzed for uniformity using HPLC (Waters) equipped with a dual detector (2487) set to 280 and 364 nm on a semi-preparative C-18 column (Vydac).

Example 2

(88) This example illustrates .sup.18F-AI-182 synthesis and testing.

(89) For bioassays described in following sections, .sup.18F-AI-182 was synthesized via standard nucleophilic substitution, employing 2,2,2-kryptofix/.sup.18F and AI-182-tosylate analog, purified on a C-18 (Vydac) column employing a gradient eluent mixture of ethanol and water, using radio-HPLC system equipped with a radiodetector (Bioscans). The fraction at R.sub.t=15 min was collected, concentrated, and resuspended in PBS to 5% ethanol for all radiotracer bioassays. Furthermore, .sup.18F-AI-182 was also characterized by spiking with an analytically characterized sample of an unlabeled F-AI-182 counterpart, prior to injection on the radio-HPLC.

(90) The agent F-AI-182 shows concentration dependent and saturable binding (binding constant, 597 nM; FIG. 1) to preformed A1-42 fibrils, stains both fibrillar and diffuse plaques ex vivo in the hippocampus and cortical region of brain sections in APPsw.sup.+//PS1 mice (FIG. 2) and human tissues (FIG. 5), and incorporates F-18 (.sup.18F; t.sub.1/2=110 min), a radionuclide for medical PET imaging (Mahmood, A. & Jones, A. Technetium Radiopharmaceuticals. Handbook of Radiopharmaceuticals. 323-362 (2003); Eckelman, W. The Development of .sup.99mTc Radiopharmaceuticals for Perfusion and Biochemistry: In Technetium and Rhenium in Chemistry and Nuclear Medicine 3. M. Nicolini, G. Bandoli and U. Mazzi (Eds.). Cortina Int., Verona, Italy. pp. 571-580. (1990); Narra, R., et al. A Neutral Tc-99m Complex for Myocardial Imaging. J. Nucl. Med. 30, 1830-1837 (1989); Stadalnik, R., Kudo, M., Eckelman, W. & Vera, D. In vivo functional imaging using receptor-binding radiopharmaceuticals: 99mTc-galactosyl-neoglycoalbumin (TcNGA). Investigative Radiology 28, 64-70 (1993)). The radiolabeled counterpart .sup.18F-AI-182 showed a transient high uptake in brains (7.280.46% % ID/g) of FVB mice (FIG. 3), and followed by washout from blood (25% faster than AV-45; FIG. 4) in absence of targeted plaques and remains non-metabolized in human serum. The high first-pass extraction into brain coupled with faster clearance from the blood pool and lack of metabolites offer characteristics that could potentially enhance overall signal to background ratios and assist image analysis. Multi-photon microscopy in live APPsw.sup.+//PS1 (15 months old) mice demonstrated that F-AI-182 labels plaques in brain parenchyma and blood vessels (CAA), by 5-min post intravenous administration (FIG. 6). The F-AI-182 showed facile clearance from non-targeted regions and the plaques remain labeled for investigated time points.

(91) Binding assays of F-AI-182 with preformed A.sub.1-42 aggregates were performed in PBS. Following excitation at 410 nm, fluorescence spectrum of F-AI-182 recorded in PBS containing 1% ethanol showed a broad emission peak 540-610 nm with E.sub.max at 570 nm. Upon incubation with preformed of A(1-42) aggregates, the peak 570 nm showed remarkable enhancement in the fluorescence indicating binding to A aggregates, similar to enhancement in fluorescence of thioflavin T in PBS (a positive control; data not shown). Fluorescence was not observed using A aggregates alone in PBS upon excitation at 410 nm (a negative control). Binding assays of the F-AI-182 with preformed A.sub.1-42 aggregates indicated a nearly saturable binding with a K.sub.d=597 nM (FIG. 1). Comparative analyses can be performed with other compounds such as AV-45, BAYER 94-9172, and AZD4694. An agent can be interacting with either of the two modestly high affinity binding sites (BS1& BS2) or recognizing an entirely new site on A.sub.1-42. F-AI-182 can register different complementary binding sites in relation to A-pathophysiology compared to current agents.

Example 3

(92) This example illustrates ex vive staining studies.

(93) FIG. 2 illustrates ex vivo staining studies were performed on brain sections (50 m) of an APPsw.sup.+//PS1 mouse (24 months old) and a control WT mouse (BL/6; 24 months old) using well-established procedures. Briefly, tissue sections were immunostained with mouse monoclonal antibody and visualized by donkey-anti-mouse HJ3.4 A monoclonal antibody-Alexa 568 as a positive control. Brain sections of APPsw.sup.+/PS1.sup.+/mice showed abundant staining of A compared with minimal levels in WT mouse (FIG. 2). Using F-AI-182 (100 nM, 60 min), abundant staining of fibrillar and diffuse plaques in the hippocampus and cortical regions of brain sections in APPsw.sup.+/PS1.sup.+/mice was observedarrows indicate labeling of A plaques (arrows, diffuse; arrow head, fibrillar). By comparison, no staining in WT mice was seen either with F-AI-182 or the antibody indicating the targeting specificity of F-AI-182. The slides were analyzed on a Zeiss LSM 5 PASCAL confocal system coupled to a Zeiss Axiovert 200 microscope.

Example 4

(94) This example illustrates biodistribution studies of .sup.18F-AI-182.

(95) For in vivo imaging of A plaques, the basic pharmacokinetic model in an unaffected normal brain involves high initial penetration of the agent, followed by rapid clearance due to lack of a binding target. However, in AD brains, high initial penetration can be followed by regional retention as the agent binds to A thus leading to differential kinetics. To accomplish this objective, biodistribution studies of .sup.18F-AI-182 were performed in normal FVB mice for assessment of signal to noise ratios and clearance profiles. Brain uptake of .sup.18F-AI-182 was analyzed in terms of percent injected dose per gram of the brain tissue (% ID/g). Biodistribution studies with HPLC purified .sup.18F-AI-182 in normal mice revealed a transient brain uptake value of 7.280.46% ID/g and 1.540.06% ID/g, 5 min and 120 min post tail-vein injection, respectively, giving a 5 min/120 min clearance a ratio of 4.73, providing evidence for the ability of .sup.18F-AI-182 to cross the BBB and permeate into brain in vivo (FIG. 3). This initial brain uptake value (5 min) in normal mice is approximately 15-fold high compared with our A-targeted .sup.99mTc-Peptides (Harpstrite, S. E., Prior, J., Binz, K., Piwnica-Worms, D. & Sharma, V. .sup.99mTc-Peptide conjugates for imaging -amyloid in the brain. ACS Med Chem Lett (2013) under review). Additionally, compared to .sup.18F-AV-45 (Liver: 17.0 t 0.69 (2 min), 4.960.90 (120 min); Kidney: 14.192.34 (2 min), 2.190.36 (120 min), .sup.18F-AI-182 clears rapidly from non-targeted tissues, such as liver and kidney (Liver: 16.321.41 (5 min), 2.710.21 (120 min); Kidney: 6.761.57 (5 min), 1.570.08 (120 min) and these clearance profiles could translate into better MIRD analysis. For comparison, .sup.18F-AV-45 demonstrates brain uptake values of 7.331.54% ID/g and 1.800.07% ID/g at 2 min and 120 min post-injection (Choi, S. R., et al. Preclinical properties of 18F-AV-45: a PET agent for Abeta plaques in the brain. J Nucl Med 50, 1887-1894 (2009)) respectively, thus providing a 2 min/120 min clearance ratio of 4.07 in normal mice that lack target sites (FIG. 3). Net brain uptake of .sup.18F-AI-182 is 1.2-fold higher than that of .sup.18F-AV-45. The initial data point for .sup.18F-AI-182 is at 5 min compared with 2 min for .sup.18F-AV-45. Our data indicates a 5 min uptake compared with a 2 min data point reported for .sup.18F-AV-45 thus we do expect these 2 min/120 min ratios to be much superior, upon comparative analysis at the same time points. .sup.18F-AI-182 undergoes 25% faster blood clearance from 5 min to 120 min compared with the 18F-AV-45 (FIG. 4). Compared with .sup.11C-PIB (Mathis, C., et al. Synthesis and evaluation of .sup.11C-labeled 6-substituted 2-arylbenzothiazoles as amyloid imaging agents. J Med Chem 46, 2740-2754 (2003)) and .sup.18F-AV-45 (Choi, S. R., et al. Preclinical properties of .sup.18F-AV-45: a PET agent for Abeta plaques in the brain. J Nucl Med 50, 1887-1894 (2009)) that undergo facile metabolism in vivo, .sup.18F-AI-182 remains non-metabolized in human serum.

Example 5

(96) This example illustrates staining experiments with an F-AI-182 agent.

(97) Staining experiments were performed with human brain tissues. Tissue samples were obtained from the frontal lobe of clinically and neuropathologically well-characterized cases. The neuropathological diagnosis of AD was based on the criteria of the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) (Mirra, S., et al. The Consortium to Establish a Registry for Alzheimer's Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer's disease. Neurology 41, 479-486 (1991)) or the National Institute of Aging-Reagan Institute (NIA-RI) (Hyman, B. & Trojanowski, J. Consensus recommendations for the postmortem diagnosis of Alzheimer disease from the National Institute on Aging and the Reagan Institute Working Group on diagnostic criteria for the neuropathological assessment of Alzheimer disease. J Neuropathol Exp Neurol 56, 1095-1097. (1997)). For experiments, highly specific A-targeted antibody (10D5, Eli Lilly, a positive control, used in histopathological core of the ADRC post-mortem cases) confirmed the presence of A plaques. As shown in FIG. 5, A-targeted F-AI-182 showed abundant staining of A plaques in the hippocampus of a 90 year-old female with AD. In FIG. 5 on the left, the fluorescent probe (F-AI-182, 50 nM) labels both the compact fibrillar amyloid (arrow) and more diffuse beta-amyloid deposits (arrowhead); on the right of FIG. 5, A (10D5, Eli Lilly) immunohistochemistry reveals similar beta-amyloid plaques in a section from the same tissue block as in (b); bar=100 m. Additionally, F-AI-182 demonstrated labeling of both the fibrillar and the diffuse plaques. The ability of the F-AI-182 agent to detect diffuse plaques represents an advancement to enable PET imaging of mildly demented individuals (an earlier manifestation of AD) prior to clinical expression (Price, J. L., et al. Neuropathology of nondemented aging: presumptive evidence for preclinical Alzheimer disease. Neurobiology of aging 30, 1026-1036 (2009); Morris, J. C., et al. Cerebral amyloid deposition and diffuse plaques in normal aging: Evidence for presymptomatic and very mild Alzheimer's disease. Neurology 46, 707-719 (1996); Price, J. L. & Morris, J. C. Tangles and plaques in nondemented aging and preclinical Alzheimer's disease. Annals of neurology 45, 358-368 (1999); Schmitt, F. A., et al. Preclinical AD revisited: neuropathology of cognitively normal older adults. Neurology 55, 370-376 (2000)), thereby offering a window of opportunity for therapeutic interventions for better management of disease.

(98) To demonstrate ability of the agent to label plaques in vivo, multiphoton imaging was conducted in live APP/PS1 12 month old mice, post intravenous injection of F-AI-182. Prior to imaging, dextran-Texas Red was injected for mapping the blood vessels. Following labeling of blood vessels, F-AI-182 (2 mg/kg, dissolved in DMSO/PEG; 20:80) was intravenously injected. A z-stack image series was acquired from cortex surface to a depth of approx. 100 m using microscope LSM 510META NLO (Carl-Zeiss Inc). Multi-photon microscopy in live APPsw+//PS1 (15 months old) mice demonstrated that F-AI-182 can label plaques in brain parenchyma and blood vessels (CAA), less than 5-min post intravenous administration. The labeling of brain parenchymal plaques was visible within 10 min, indicating facile clearance from non-targeted regions and remained labeled for 30 min (FIG. 6). Multi-photon imaging can be done using .sup.18F-AI-182, other agents, or second generation agents (Bacskai, B., et al. Four-dimensional multiphoton imaging of brain entry, amyloid binding and clearance of an amyloid-3 ligand in transgenic mice. Proc Natl Acad Sci USA 100, 12462-12467 (2003)).

Example 6

(99) This example illustrates methods of assessment of binding sites.

(100) There is an NMR-deduced structure in the protein data bank for A.sub.1-42 (PDB ID: 2BEG). To assess binding sites of PIB, AV-45, and AI-182, using procedures described earlier in our laboratories (Sundaram, G. S. M, Harpstrite, S. E., Kao, J. L., Collins, S. D. & Sharma, V. A New Nucleoside Analogue with Potent Activity against Mutant sr39 Herpes Simplex Virus-1 (HSV-1) Thymidine Kinase (TK). Organic letters (2012)), we used sitemap to determine binding sites on A.sub.1-42, generated a grid, then docked PIB, AV-45, and AI-182 to determine rank order (AV-45>F-AI-182>PIB) based upon the Glide score. FIG. 7 depicts the post docking view of PIB (left), AV-45 (Middle), and F-AI-182 (Right). Ligand interaction diagram indicated that PIB 6-hydroxy substituent of the benzothiazole ring forms a hydrogen bond with Leu 17 and smallest surface of hydrophobic interactions with amino acid residues of A-42. While pyridine ring of AV-45 participated in - interactions with Phe 19 as well as highest surface of hydrophobic interactions, F-AI-182 retained - interactions but also shows intermediate hydrophobic interaction surface thus supporting the rank order of the glide score. Building on the principles that: a) electrons play a role in biochemical interactions (Kumpf, R. A. & Dougherty, D. A. A mechanism for ion selectivity in potassium channels: computational studies of cation-pi interactions. Science 261, 1708-1710 (1993)); b) A recognizes planner molecules; and c) extended conjugation systems are more likely to offer more flexibility for interaction with other sites, we can explore a focused SAR around two scaffolds: a) slight variations in position of the heteroatoms in the six membered pyridine ring or 5-membered ring of benzothiazole in addition to variation in number of ethylene glycol moieties on the 6-position of benzothiazole ring and b) modification of dialkyl amino group with other functional groups.

Example 7

(101) This example illustrates labeling of A plaques in APPsw+/ (24 months old) mice using F-AI-183.

(102) Examples of brain tissue section staining of APPsw+/ (24 months old) mice using F-AI-183 are shown in FIG. 8. Arrows indicate labeling of A plaques (arrows, fibrillar plaques). The slides were analyzed using a Nikon Ti-E PFS inverted microscope equipped with a Nikon 10x 0.3 NA Plan APO objective, Prior H117 ProScan flat top linear encoded stage, and Prior Lumen 200PRO illumination system with standard DAPI and FITC filter sets. The images were acquired using a Photometrics CoolSNAP HQ2 digital camera and MetaMorph microscopy automaton, and imaging software. Images were processed and analyzed using the Image J software package (NIH).

Example 8

(103) This example illustrates detection of compact A plaques in brain cross-sections of frontal lobe by F-AI-183.

(104) F-AI-183 detected compact A plaques in the brain cross-sections of frontal lobe of an 88-year-old female with neuropathologically confirmed Alzheimer's disease as shown in FIG. 9. The fluorescent probe (F-AI-183) labels fibrillar amyloid (arrow). The slides were analyzed on a using a Nikon Ti-E PFS inverted microscope equipped with a Nikon 100.3 NA Plan APO objective, Prior H117 ProScan flat top linear encoded stage, and Prior Lumen 200PRO illumination system with standard DAPI and FITC filter sets. The images were acquired using a Photometrics CoolSNAP HQ2 digital camera, and MetaMorph microscopy automaton, and imaging software. Images were processed and analyzed using the Image J software package (NIH).

Example 9

(105) This example illustrates NMR data for some compounds of the present teachings.

(106) ##STR00127##

(107) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.17 (s, 6H), 3.83 (s, 3H), 6.59 (d, J=8.4 Hz, 1H), 7.02 (d, J=9.2 Hz, 1H), 7.16 (d, J=16.0 Hz, 1H), 7.25 (t, J=14.0 Hz, 2H), 7.74 (d, J=8.4 Hz, 1H), 7.83 (d, J=8.4 Hz, 1H), 8.45 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 37.14, 55.77, 104.13, 106.05, 115.31, 118.33, 119.21, 123.02, 134.05, 134.22, 148.93, 159.15, 171.33

(108) ##STR00128##

(109) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.10 (s, 6H), 6.75 (d, J=8.8 Hz, 1H), 6.81 (d, J=8.4 Hz, 1H), 7.32 (d, J=16.4 Hz, 1H), 7.43 (d, J=16.4 Hz, 1H), 7.81 (d, J=8.0 Hz, 1H), 8.18 (d, J=8.4 Hz, 1H), 8.37 (s, 1H), 9.62 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 38.21, 107.08, 108.21, 109.62, 115.05, 116.25, 119.69, 121.37, 125.88, 126.44, 135.49, 136.17, 136.54, 157.05, 172.69.

(110) ##STR00129##

(111) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.15 (s, 6H), 4.28 (d, J=27.8 Hz, 2H), 4.77 (d, J=47.2 Hz, 2H), 6.56 (dd, J=8.8, 3.2 Hz, 1H), 6.96 (d, J=8.4 Hz, 1H), 7.14 (d, J=16.4 Hz, 1H), 7.27 (d, J=6.0 Hz, 1H), 7.52 (d, J=16.8 Hz, 1H), 7.72 (dd, J=8.6, 3.0 Hz, 2H), 8.31 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 38.12, 67.29, 67.50, 81.00, 82.70, 106.08, 107.81, 115.05, 119.31, 120.78, 124.98, 134.41, 134.80, 136.36, 149.22, 157.85; .sup.19F NMR (282 MHz, CFCl.sub.3): 224 ppm; HRMS (FAB) m/z calc. for C.sub.18H.sub.18FN.sub.3OSe: [M].sup.+ 391.0599. found: 391.0602.

(112) ##STR00130##

(113) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.24 (s, 6H), 3.87 (s, 3H), 6.93 (d, J=7.2 Hz, 1H), 7.16 (s, 2H), 7.50 (s, 1H), 7.70 (d, J=7.4 Hz, 1H), 8.50 (s, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): 37.27, 55.52, 107.18, 114.85, 117.07, 121.54, 124.81, 128.10, 132.81, 156.54, 159.16, 161.64, 172.49

(114) ##STR00131##

(115) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.17 (s, 6H), 6.83 (dd, J=8.0, 1.6 Hz, 1H), 7.30 (s, 1H) 7.40 (d, J=8.8 Hz, 2H), 7.82 (d, J=8.8 Hz, 1H), 8.75 (s, 2H), 9.63 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 39.28, 109.72, 115.23, 117.66, 121.91, 125.92, 126.55, 133.49, 156.95, 157.07, 157.49, 161.59, 172.49

(116) ##STR00132##

(117) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.24 (s, 6H), 4.28 (d, J=27.6 Hz, 2H), 4.80 (d, J=47.2 Hz, 2H), 6.97 (d, J=7.2 Hz, 1H), 7.17 (bs, 1H), 7.50 (s, 1H), 7.72 (d, J=7.2 Hz, 1H), 8.52 (s, 2H); .sup.13C NMR (100 MHz, CDCl.sub.3): 37.28, 67.31, 67.51, 80.97, 82.67, 107.95, 110.00, 115.36, 117.04, 121.46, 125.01, 128.78, 133.01, 156.47, 156.57, 157.93, 158.57, 161.66, 172.72; .sup.19F NMR (282 MHz, CFCl.sub.3): 224 ppm; HRMS (FAB) m/z calc. for C.sub.17H.sub.18FN.sub.4OSe: [M].sup.+ 392.0594. found: 392.0603.

(118) ##STR00133##

(119) .sup.1H NMR (400 MHz, CDCl.sub.3): 0.12 (s, 6H), 0.92 (s, 9H), 3.24 (s, 6H), 4.01-4.03 (m, 2H), 4.09-4.12 (m, 2H), 6.94 (dd, J=8.8, 2.4 Hz, 1H), 7.17 (d, J=1.2 Hz, 1H), 7.50 (d, J=2.4 Hz, 1H), 7.70 (d, J=8.4 Hz, 1H), 8.53 (s, 2H); .sup.11C NMR (100 MHz, CDCl.sub.3): 31.04, 42.24, 68.44, 76.22, 110.76, 112.22, 121.21, 124.34, 129.86, 140.02, 142.74, 149.96, 152.44, 162.68, 172.86.

(120) ##STR00134##

(121) .sup.1H NMR (400 MHz, CDCl.sub.3): 2.43 (s, 3H), 3.24 (s, 6H), 4.20 (bs, 2H), 4.41 (bs, 2H), 6.71 (d, J=9.0 Hz, 1H), 6.81 (d, J=9.0 Hz, 1H), 7.17-7.41 (m, 4H), 7.65-7.69 (m, 1H), 7.82 (d, J=7.6 Hz, 2H), 8.52 (s, 1H), 8.66 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 21.64, 37.23, 65.72, 68.06, 108.04, 108.25, 115.08, 115.50, 116.88, 121.41, 124.78, 124.96, 125.25, 128.00, 129.85, 131.04, 133.12, 144.95, 156.60, 157.35, 158.59, 162.68, 171.84

(122) ##STR00135##

(123) .sup.1H NMR (300 MHz, CD.sub.3CN): 8.25 (s, 1H), 8.08 (d, 1H), 7.63 (d, 1H), 7.27 (dd, 1H), 3.92 (s, 3H)

(124) ##STR00136##

(125) .sup.1H NMR (300 MHz, acetone-d.sub.6): 10.00-9.00 (br, s, 1H), 8.51 (s, 1H), 8.09 (d, 1H), 7.63 (d, 1H), 7.28 (dd, 1H)

(126) ##STR00137##

(127) .sup.1H NMR (300 MHz, acetone-d.sub.6): 8.55 (s, 1H), 8.16 (d, 1H), 7.85 (d, 1H), 7.39 (d, 1H), 4.93-4.29 (m, 4H).

(128) ##STR00138##

(129) .sup.1H NMR (300 MHz, dmso-d.sub.6): 8.59 (s, 1H), 8.25 (d, 2H), 8.08 (d, 2H), 8.00 (d, 1H), 7.77 (d, 1H), 7.18 (dd, 1H), 3.87 (s, 3H)

(130) ##STR00139##

(131) .sup.1H NMR (300 MHz, acetone-d.sub.6): 9.00 (br, s, 1H), 8.39 (s, 1H), 8.30 (d, 2H), 8.18 (d, 2H), 7.94 (d, 1H), 7.52 (d, 1H), 7.14 (dd, 1H). MS(LRESI) m/z=304.0547 (M+H.sup.+).

(132) ##STR00140##

(133) .sup.1H NMR (300 MHz, acetone-d.sub.6): 8.39 (s, 1H), 8.31 (d, 2H), 8.18 (d, 2H), 8.02 (d, 1H), 7.73 (d, 1H), 7.25 (dd, 1H), 4.94-4.36 (m, 4H). MS(LRESI) m/z=350.2 (M+H.sup.+).

(134) ##STR00141##

(135) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.15 (s, 6H), 3.88 (s, 3H), 6.55 (d, J=8.4 Hz, 1H), 7.04 (d, J=9.2 Hz, 1H), 7.14 (d, J=16.0 Hz, 1H), 7.29 (t, J=14.0 Hz, 2H), 7.71 (d, J=8.4 Hz, 1H), 7.83 (d, J=8.4 Hz, 1H), 8.29 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 38.14, 55.79, 104.13, 106.05, 115.31, 118.0, 119.41, 123.02, 134.08, 134.22, 148.93, 159.15, 165.33

(136) ##STR00142##

(137) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.03 (s, 6H), 6.65 (d, J=8.4 Hz, 1H), 6.88 (d, J=7.6 Hz, 1H), 7.24-7.32 (m, 2H), 7.66 (d, J=8.4 Hz, 2H), 7.90 (d, J=7.6 Hz, 1H), 8.29 (s, 1H), 9.82 (s, 1H); .sup.13C NMR (100 MHz, CDCl.sub.3): 38.04, 106.51, 107.08, 116.11, 118.03, 119.58, 123.17, 134.13, 134.96, 135.60, 147.47, 149.36, 155.90, 159.25, 163.97.

(138) ##STR00143##
AI-182

(139) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.14 (s, 6H), 4.28 (d, J=26.0 Hz, 2H), 6.65 (d, J=8.4 Hz, 1H), 4.77 (d, J=49.6 Hz, 2H), 6.55 (d, J=8.8 Hz, 1H), 7.08 (d, J=9.2 Hz, 1H), 7.13 (d, J=16.4 Hz, 1H), 7.26-7.33 (m, 2H), 7.70 (d, J=8.4 Hz, 1H), 7.84 (d, J=9.2 Hz, 1H), 8.29 (s, 1H), 9.82 (s, 1H); .sup.11C NMR (100 MHz, CDCl.sub.3): 38.10, 38.13, 67.63, 67.84, 81.01, 82.70, 105.32, 106.05, 115.67, 117.86, 119.34, 123.12, 134.23, 134.33, 148.98, 156.36, 159.17, 165.74; .sup.19F NMR (282 MHz, CFCl.sub.3): 224 ppm; HRMS (FAB) m/z calc. for C.sub.18H.sub.18FN.sub.3OS: [M].sup.+ 343.155. found: 343.1152.

(140) ##STR00144##

(141) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.17 (s, 6H), 3.96 (s, 2H), 4.20 (s, 2H), 6.56 (d, J=8.8 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 7.14 (d, J=16.4 Hz, 1H), 7.24-7.32 (m, 2H), 7.72 (d, J=8.8 Hz, 2H), 7.82 (d, J=8.8 Hz, 1H), 8.29 (s, 1H).

(142) ##STR00145##

(143) .sup.1H NMR (400 MHz, CDCl.sub.3): 2.42 (s, 3H), 3.16 (s, 6H), 4.22 (s, 2H), 4.41 (s, 2H), 6.56 (d, J=8.8 Hz, 1H), 7.06 (d, J=8.4 Hz, 1H), 7.14 (d, J=16.4 Hz, 1H), 7.24-7.32 (m, 2H), 7.72 (d, J=8.8 Hz, 2H), 7.82 (d, J=8.8 Hz, 1H), 8.22-8.43 (m, 3H), 8.51-8.92 (m, 2H).

(144) ##STR00146##

(145) .sup.1H NMR (400 MHz, CDCl.sub.3): 3.14 (s, 6H), 3.61-3.80 (m, 6H), 3.82 (s, 2H), 4.22 (s, 2H), 4.44 (d, J=49.4 Hz, 2H), 6.55 (d, J=8.8 Hz, 1H), 7.08 (d, J=9.2 Hz, 1H), 7.13 (d, J=16.4 Hz, 1H), 7.26-7.33 (m, 2H), 7.70 (d, J=8.4 Hz, 1H), 7.84 (d, J=9.2 Hz, 1H), 8.29 (d, J=9.2 Hz, 2H); .sup.19F NMR (282 MHz, CFCl.sub.3): 224 ppm; HRMS (FAB) m/z calc. for C.sub.23H.sub.18FN.sub.2O.sub.3S: [M].sup.+ 430.1726. found: 430.1780.

Example 10

(146) This example illustrates imaging of cancer cells using AI-182 as a fluorescent probe.

(147) In these experiments, human carcinoma cells were incubated with AI-182

(148) ##STR00147##

(149) (5 M) at 37 C. in the presence of 5% CO.sub.2 for 30 min, and examined using a Nikon Ti-E PFS inverted high resolution microscope equipped with a Nikon (Magnification: 20) Plan APO objective, Prior H117 ProScan flat top linear encoded stage, and Prior Lumen 200PRO illumination system with standard DAPI and FITC filter sets. Results are shown in FIG. 10. Top row: Live Cell Imaging of Human Glioblastoma (U87) Cells Using AI-182. Middle row: Live Cell Imaging of Human Pancreatic Cancer Cells (PANC1) Using AI-182. Bottom row: Live Cell Imaging of Human Pancreatic Cancer Cells (Mia PaCa-2) Using AI-182. Note accumulation of the probe within cells.

(150) All references cited herein are incorporated by reference, each in its entirety. Applicant reserves the right to challenge any conclusions presented by any of the authors of any reference.