Synthesis, binding affinity, radiolabeling, and microPET evaluation of 4-(2-substituted-4-substituted)-8-(dialkylamino)-6-methyl-1-substituted-3,4-dihydropyrido[2,3-b]pyrazin-2(1H)-ones as ligands for brain corticotropin-releasing factor type-1 (CRF1) receptors.

Compounds 1-14 were synthesized in a search for high-affinity CRF1 receptor ligands that could be radiolabeled with (11)C or (18)F for use as positron emission tomography (PET) radiotracers. Derivatives of 2 were developed which contained amide N-fluoroalkyl substituents. Compounds [(18)F]24 and [(18)F]25 were found to have appropriate lipophilicities of logP7.4=2.2 but microPET imaging with [(18)F]25 demonstrated limited brain uptake.

Synthesis, F-18 radiolabeling, and microPET evaluation of 3-(2,4-dichlorophenyl)-N-alkyl-N-fluoroalkyl-2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-amines as ligands of the corticotropin-releasing factor type-1 (CRF1) receptor.

A series of 3-(2,4-dichlorophenyl)-N-alkyl-N-fluoroalkyl-2,5-dimethylpyrazolo[1,5-a]pyrimidin-7-amines were synthesized and evaluated as potential positron emission tomography (PET) tracers for the corticotropin-releasing factor type-1 (CRF1) receptor. Compounds 27, 28, 29, and 30 all displayed high binding affinity (⩽1.2 nM) to the CRF1 receptor when assessed by in vitro competition binding assays at 23 °C, whereas a decrease in affinity (⩾10-fold) was observed with compound 26. The logP7.4 values of [(18)F]26-[(18)F]29 were in the range of ∼2.2-2.8 and microPET evaluation of [(18)F]26-[(18)F]29 in an anesthetized male cynomolgus monkey demonstrated brain penetrance, but specific binding was not sufficient enough to differentiate regions of high CRF1 receptor density from regions of low CRF1 receptor density. Radioactivity uptake in the skull, and sphenoid bone and/or sphenoid sinus during studies with [(18)F]28, [(18)F]28-d8, and [(18)F]29 was attributed to a combination of [(18)F]fluoride generated by metabolic defluorination of the radiotracer and binding of intact radiotracer to CRF1 receptors expressed on mast cells in the bone marrow. Uptake of [(18)F]26 and [(18)F]27 in the skull and sphenoid region was rapid but then steadily washed out which suggests that this behavior was the result of binding to CRF1 receptors expressed on mast cells in the bone marrow with no contribution from [(18)F]fluoride.

Preparation of 1-Tosyloxy-4-Substituted-2-butenes Using Ag(I) Salts.

The trans-fluoro-2-butenyl group has been previously utilized as an N-substituent on several nortropanes for imaging the dopamine transporter with positron emission tomography. We report here a simplified and shorter synthesis of trans-1-tosyloxy-4-substituted-2-butenes using Ag(I) salts. This methodology was also applied to the synthesis of the cis-isomers. Furthermore, these procedures allow for the recovery of the majority of the Ag(I) ions.

Ligand-based design of [18F]OXD-2314 for PET imaging in non-Alzheimer's disease tauopathies.

Positron emission tomography (PET) imaging of tau aggregation in Alzheimer's disease (AD) is helping to map and quantify the in vivo progression of AD pathology. To date, no high-affinity tau-PET radiopharmaceutical has been optimized for imaging non-AD tauopathies. Here we show the properties of analogues of a first-in-class 4R-tau lead, [18F]OXD-2115, using ligand-based design. Over 150 analogues of OXD-2115 were synthesized and screened in post-mortem brain tissue for tau affinity against [3H]OXD-2115, and in silico models were used to predict brain uptake. [18F]OXD-2314 was identified as a selective, high-affinity non-AD tau PET radiotracer with favorable brain uptake, dosimetry, and radiometabolite profiles in rats and non-human primate and is being translated for first-in-human PET studies.

Decreased brainstem and putamen SERT binding potential in depressed suicide attempters using [11C]-zient PET imaging.

BACKGROUND: Deficits in serotonergic neurotransmission have been implicated in the pathogenesis of depression and suicidality. The present study utilized a novel positron-emission tomography (PET) ligand to quantitate and compare brain regional serotonin transporter (SERT) binding potential in depressed patients with a past history of suicide attempts to that of healthy comparison subjects. METHOD: We used [(11) C]-ZIENT PET to label SERT in the serotonergic cell body rich brainstem, and forebrain projection fields. Quantitative PET emission data from 21 adults (10 healthy controls and 11 drug-free patients with major depression) was used for group comparison. SERT binding potential (BPND ) in eight MRI-based brain regions of interest (ROI) were compared in high-resolution PET images. RESULTS: SERT binding potential was significantly decreased in the midbrain/pons (P = .029) and putamen (P = .04) of depressed patients with a past suicide attempt relative to comparison subjects. Forebrain SERT binding was also reduced in the patient sample, though these region effects did not survive a multiple comparison correction. CONCLUSION: These results suggest that decreased availability of the brainstem and basal ganglia SERT represents a biomarker of depression and thus confirm and extend the role of dysregulation of brain serotonergic neurotransmission in the pathophysiology of depression and suicide.

(11) C and (18) F PET radioligands for the serotonin transporter (SERT).

The serotonin transporter (SERT) has been implicated in a variety of neuropsychiatric disorders including depression, anxiety, and suicide, and is the target of the selective serotonin reuptake inhibitor (SSRI) class of antidepressants. The availability of SERT-specific positron emission tomography (PET) radioligands will allow the SERT to be studied noninvasively in living subjects through PET imaging of the SERT and occupancy studies of SSRIs. Numerous diaryl sulfide and tropane derivatives have been developed and radiolabeled with (11) C or (18) F for imaging the SERT with PET.

In Silico Discovery and Subsequent Characterization of Potent 4R-Tauopathy Positron Emission Tomography Radiotracers.

A chemical fingerprint search identified Z3777013540 (1-(5-(6-fluoro-1H-indol-2-yl)pyrimidin-2-yl)piperidin-4-ol; 1) as a potential 4R-tau binding ligand. Binding assays in post-mortem Alzheimer's disease (AD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) brain with [3H]1 provided KD (nM) values in AD = 4.0, PSP = 5.1, and CBD = 4.5. In vivo positron emission tomography (PET) imaging in rats with [18F]1 demonstrated good brain penetration and rapid clearance from normal brain tissues. A subsequent molecular similarity search using 1 as the query revealed an additional promising compound, Z4169252340 (4-(5-(6-fluoro-1H-indol-2-yl)pyrimidin-2-yl)morpholine; 21). Binding assays with [3H]21 provided KD (nM) values in AD = 1.2, PSP = 1.6, and CBD = 1.7 and lower affinities for binding aggregated α-synuclein and amyloid-beta. PET imaging in rats with [18F]21 demonstrated a higher brain penetration than [18F]1 and rapid clearance from normal brain tissues. We anticipate that 1 and 21 will be useful for the identification of other potent novel 4R-tau radiotracers.

Biodistribution and dosimetry of ¹²³I-mZIENT: a novel ligand for imaging serotonin transporters.

PURPOSE: 123I-labelled mZIENT (2ß-carbomethoxy-3ß-(3'-((Z)-2-iodoethenyl)phenyl)nortropane) has been developed as a radioligand for the serotonin transporter. The aim of this preliminary study was to assess its whole-body biodistribution in humans and estimate dosimetry. METHODS: Three healthy controls and three patients receiving selective serotonin reuptake inhibitor (SSRI) therapy for depression were included (two men, four women, age range 41-56 years). Whole-body imaging, brain SPECT imaging and blood and urine sampling were performed. Whole-body images were analysed using regions of interest (ROIs), time-activity curves were derived using compartmental analysis and dosimetry estimated using OLINDA software. Brain ROI analysis was performed to obtain specific-to-nonspecific binding ratios in the midbrain, thalamus and striatum. RESULTS: Initial high uptake in the lungs decreased in later images. Lower uptake was seen in the brain, liver and intestines. Excretion was primarily through the urinary system. The effective dose was estimated to be of the order of 0.03 mSv/MBq. The organ receiving the highest absorbed dose was the lower large intestine wall. Uptake in the brain was consistent with the known SERT distribution with higher specific-to-nonspecific binding in the midbrain, thalamus and striatum in healthy controls compared with patients receiving SSRI therapy. CONCLUSION: ¹²³I-mZIENT may be a promising radioligand for imaging the serotonin transporters in humans with acceptable dosimetry.

Development of a PET radioligand selective for cerebral amyloid angiopathy.

INTRODUCTION: Positron emission tomography (PET) using radiolabeled amyloid-binding compounds has advanced the field of Alzheimer's disease (AD) by enabling detection and longitudinal tracking of fibrillar amyloid-ß (Aß) deposits in living people. However, this technique cannot distinguish between Aß deposits in brain parenchyma (amyloid plaques) from those in blood vessels (cerebral amyloid angiopathy, CAA). Development of a PET radioligand capable of selectively detecting CAA would help clarify its contribution to global brain amyloidosis and clinical symptoms in AD and would help to characterize side-effects of anti-Aß immunotherapies in AD patients, such as CAA. METHODS: A candidate CAA-selective compound (1) from a panel of analogues of the amyloid-binding dye Congo red was synthesized. The binding affinity to Aß fibrils and lipophilicity of compound 1 were determined and selectivity for CAA versus parenchymal plaque deposits was assessed ex-vivo and in-vivo in transgenic APP/PS1 mice and in postmortem human brain affected with AD pathology. RESULTS: Compound 1 displays characteristics of Aß binding dyes, such as thioflavin-S, in that it labels both parenchymal Aß plaques and CAA when applied to histological sections from both a transgenic APP/PS1 mouse model of Aß amyloidosis and AD brain. Thus, compound 1 lacks molecular selectivity to distinguish Aß deposits in CAA from those in plaques. However, when administered to living APP/PS1 mice intravenously, compound 1 preferentially labels CAA when assessed using in-vivo two-photon microscopy and ex-vivo histology and autoradiography. CONCLUSION: We hypothesize that selectivity of compound 1 for CAA is attributable to its limited penetration of the blood-brain barrier due to the highly polar nature of the carboxylate moiety, thereby limiting access to parenchymal plaques and promoting selective in-vivo labeling of Aß deposits in the vascular wall (i.e., "delivery selectivity").

Radiosynthesis, In Vitro and In Vivo Evaluation of [18F]CBD-2115 as a First-in-Class Radiotracer for Imaging 4R-Tauopathies.

CBD-2115 was selected from a library of 148 compounds based on a pyridinyl-indole scaffold as a first-in-class 4R-tau radiotracer. In vitro binding assays showed [3H]CBD-2115 had a KD value of 6.9 nM and a nominal Bmax of 500 nM in 4R-tau expressing P301L transgenic mouse tissue. In binding assays with human brain tissue homogenates, [3H]CBD-2115 has a higher affinity (4.9 nM) for progressive supranuclear palsy specific 4R-tau deposits than [3H]flortaucipir (45 nM) or [3H]MK-6240 (>50 nM). [18F]CBD-2115 was reliably synthesized (3-11% radiochemical yield with molar activity of 27-111 GBq/µmol and >97% radiochemical purity). Dynamic PET imaging was conducted in mice, rats, and nonhuman primates, and all species showed initial brain uptake of 0.5-0.65 standardized uptake value with fast clearance from normal tissues. [3H]CBD-2115 could be a useful lead radioligand for further research in 4R-tauopathies, and PET radiotracer development will focus on improving brain uptake and binding affinity.

Synthesis, in vitro characterization, and radiolabeling of reboxetine analogs as potential PET radioligands for imaging the norepinephrine transporter.

Six new (S,S)-enantiomers of reboxetine derivatives were synthesized and their binding affinities were determined via competition binding assays in cells expressing the human norepinephrine transporter (NET), serotonin transporter (SERT) or dopamine transporter (DAT). All six compounds prepared exhibit high affinity for the NET (K(i)

Synthesis and in vivo evaluation of fluorine-18 and iodine-123 labeled 2beta-carbo(2-fluoroethoxy)-3beta-(4'-((Z)-2-iodoethenyl)phenyl)nortropane as a candidate serotonin transporter imaging agent.

2Beta-carbo(2-fluoroethoxy)-3beta-(4'-((Z)-2-iodoethenyl)phenyl)nortropane (betaFEpZIENT, 1) was synthesized as a serotonin transporter (SERT) imaging agent for both positron emission tomography (PET) and single photon emission computerized tomography (SPECT). The binding affinity of 1 to human monoamine transporters showed a high affinity for the SERT (Ki = 0.08 nM) with respect to the dopamine transporter (DAT) (Ki = 13 nM) and the norepinephrine transporter (NET) (Ki = 28 nM). In vivo biodistribution and blocking studies performed in male rats demonstrated that [123I]1 was selective and specific for SERT. In vivo microPET brain imaging studies in an anesthetized monkey with [18F]1 showed high uptake in the diencephalon and brainstem with peak uptake achieved at 120 min. A chase study with (R,S)-citalopram.HBr displaced [18F]1 radioactivity from all SERT-rich brain regions. A chase study with the DAT ligand 2beta-carbophenoxy-3beta-(4-chlorophenyl)tropane (9, RTI-113) failed to displace [18F]1, indicating that [18F]1 is specific to the SERT. The in vivo evaluation of [18F]1 indicates that this radiotracer is a good candidate for mapping and quantifying CNS SERT.

Synthesis, radiosynthesis, and biological evaluation of carbon-11 labeled 2beta-carbomethoxy-3beta-(3'-((Z)-2-haloethenyl)phenyl)nortropanes: candidate radioligands for in vivo imaging of the serotonin transporter with positron emission tomography.

2beta-carbomethoxy-3beta-(3'-((Z)-2-iodoethenyl)phenyl)nortropane (mZIENT, 1) and 2beta-carbomethoxy-3beta-(3'-((Z)-2-bromoethenyl)phenyl)nortropane (mZBrENT, 2) were synthesized and evaluated for binding to the human serotonin, dopamine, and norepinephrine transporters (SERT, DAT, and NET, respectively) using transfected cells. Both 1 and 2 have a high affinity for the SERT (Ki=0.2 nM) and are approximately 160 times more selective for the SERT than the DAT. Compound 2 has a significantly higher affinity for the NET than 1, and this may be a result of the different size and electronegativity of the halogen atoms. MicroPET imaging in nonhuman primates with [11C]1 and [11C]2 demonstrated that both tracers behave similarly in vivo with high uptake being observed in the SERT-rich brain regions and peak uptake being achieved in about 55 min postinjection. Chase studies with citalopram and methylphenidate demonstrated that this uptake is the result of preferential binding to the SERT.

Synthesis, radiosynthesis, and biological evaluation of carbon-11 and fluorine-18 (N-fluoroalkyl) labeled 2beta-carbomethoxy-3beta-(4'-(3-furyl)phenyl)tropanes and -nortropanes: candidate radioligands for in vivo imaging of the serotonin transporter with positron emission tomography.

2beta-Carbomethoxy-3beta-(4'-(3-furyl)phenyl)nortropane (1) was synthesized along with the N-methyl (2), N-fluoroethyl (3), N-fluoropropyl (4), and N-fluorobutyl (5) derivatives. The binding affinity for each compound to the human serotonin, dopamine, and norepinephrine transporters was determined using transfected HEK-293 cells. Radiolabeling and microPET brain imaging studies were performed with [(11)C]1, [(11)C]2, and [(18)F]3 to determine their utility as in vivo imaging agents.

Fluorinated tropanes.

The development of tropane-derivatives as cocaine-addiction therapeutics identified numerous compounds with affinities for the dopamine, serotonin, and norepinephrine transporters. Many of these compounds were then adapted for use as imaging radiotracers. Incorporation of fluorine into the molecule often improved the physiochemical properties of the compound and also provided a position for radiolabeling with fluorine-18. This review provides an overview of the efforts devoted to developing fluorine-18 radiolabeled tropanes for imaging use with positron emission tomography.

In vivo evaluation of [¹²³I]mZIENT as a SPECT radioligand for the serotonin transporter.

INTRODUCTION: In vivo imaging of the serotonin transporter continues to be a valuable tool in drug development and in monitoring diseases that alter serotonergic function. The purposes of this study were to: 1) evaluate the test/retest reproducibility of [¹²³I] 2ß-Carbomethoxy-3ß-(3'-((Z)-2-iodoethenyl)phenyl)nortropane ([¹²³I]mZIENT); and 2) to assess displacement of [¹²³I]mZIENT following administration of SERT specific drugs. METHODS: Six female baboons (Papio anubis) were scanned following i.v. administration of [¹²³I]mZIENT. The regional binding potential (BP(nd)) was determined using a simplified reference tissue model, with the cerebellum used as a reference region. The test/retest reproducibility of BP(nd) was determined following repeated injection of [¹²³I]mZIENT on a different day. To assess the displacement of [¹²³I]mZIENT from SERT, citalopram (0.01-5mg/kg) or sertraline (0.01-0.5mg/kg) was given as iv bolus at ~4h following administration of [¹²³I]mZIENT. RESULTS: The test/retest variability of BP(nd) was less than 10% for all SERT-rich brain regions. Estimates of ED50 for displacement of [¹²³I]mZIENT in SERT-rich regions were consistent with previous reports for the [¹¹C] analog of [¹²³I]mZIENT. Both citalopram and sertraline displaced [¹²³I]mZIENT from SERT in a dose-dependent manner, with maximal observed displacements of greater than 80% in the diencephalon and greater than 75% in brainstem for both citalopram and sertraline. CONCLUSIONS: [¹²³I] mZIENT demonstrates good test-retest reproducibility; and initial displacement studies suggest that this compound is highly selective for SERT. Overall, this radioligand has favorable characteristics for use in drug development studies and/or longitudinal studies interrogating SERT.

Neurobiological changes mediating the effects of chronic fluoxetine on cocaine use.

Acute SSRI (selective serotonin reuptake inhibitor) treatment has been shown to attenuate the abuse-related effects of cocaine; however, SSRIs have had limited success in clinical trials for cocaine abuse, possibly due to neurobiological changes that occur during chronic administration. In order to better understand the role of serotonin (5HT) in cocaine abuse and treatment, we examined the effects of chronic treatment with the SSRI fluoxetine at clinically relevant serum concentrations on cocaine-related neurobiology and behavior. Rhesus macaques self-administering cocaine underwent a 6-week dosing regimen with fluoxetine designed to approximate serum concentrations observed in humans. Self-administration and reinstatement were monitored throughout the treatment and washout period. In vivo microdiaylsis was used to assess changes in dopaminergic and serotonergic neurochemistry. Positron emission tomography was used to assess changes in the 5HT transporter and 2A receptor binding potential (BP). Functional output of the 5HT system was assessed using prolactin levels. Cocaine-primed reinstatement and cocaine-elicited dopamine overflow were significantly suppressed following chronic fluoxetine treatment. 5HT2A receptor BP was increased in the frontal cortex following treatment while prolactin release was blunted, suggesting desensitization of the 5HT2A receptor. These effects persisted after a 6-week washout period. Measures of pre-synaptic serotonergic function and cocaine self-administration were unaffected. These data demonstrate that acute and chronic fluoxetine treatments exert different effects on cocaine-related behavior. Furthermore, chronic fluoxetine treatment causes alterations in 5HT2A receptors in the frontal cortex that may selectively disrupt cocaine-primed reinstatement. Fluoxetine may not be useful for treatment of ongoing cocaine abuse but may be useful in relapse prevention.

Synthesis, fluorine-18 radiolabeling, and biological evaluation of N-((E)-4-fluorobut-2-en-1-yl)-2beta-carbomethoxy-3beta-(4'-halophenyl)nortropanes: candidate radioligands for in vivo imaging of the brain dopamine transporter with positron emission tomography.

The N-(E)-fluorobutenyl-3beta-(para-halo-phenyl)nortropanes 9-12 were synthesized as ligands of the dopamine transporter (DAT) for use as (18)F-labeled positron emission tomography (PET) imaging agents. In vitro competition binding assays demonstrated that compounds 9-12 have a high affinity for the DAT and are selective for the DAT compared to the serotonin and norepinephrine transporters. MicroPET imaging with [(18)F]9-[(18)F]11 in anesthetized cynomolgus monkeys showed high uptake in the putamen with lesser uptake in the caudate, but significant washout of the radiotracer was only observed for [(18)F]9. PET imaging with [(18)F]9 in an awake rhesus monkey showed high and nearly equal uptake in both the putamen and caudate with peak uptake achieved after 20 min followed by a leveling-off for about 10 min and then a steady washout and attainment of a quasi-equilibrium. During the time period 40-80 min postinjection of [(18)F]9, the ratio of uptake in the putamen and caudate vs cerebellum uptake was > or = 4.

Fluorine-18 Radiolabeled PET Tracers for Imaging Monoamine Transporters: Dopamine, Serotonin, and Norepinephrine.

This review focuses on the development of fluorine-18 radiolabeled PET tracers for imaging the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET). All successful DAT PET tracers reported to date are members of the 3ß-phenyl tropane class and are synthesized from cocaine. Currently available carbon-11 SERT PET tracers come from both the diphenylsulfide and 3ß-phenyl nortropane class, but so far only the nortropanes have found success with fluorine-18 derivatives. NET imaging has so far employed carbon-11 and fluorine-18 derivatives of reboxetine but due to defluorination of the fluorine-18 derivatives further research is still necessary.