Non-amines, drugs without an amine nitrogen, potently block serotonin transport: novel antidepressant candidates?

The serotonin transporter (SERT) is a principal site of action of therapeutic antidepressants in the brain. Without exception, these inhibitors of serotonin transport contain an amine nitrogen in their structure. We previously demonstrated that novel compounds without an amine nitrogen in their structure (non-amines), blocked dopamine transport in cells transfected with the human dopamine transporter. The present study investigated whether, in the absence of an amine nitrogen, certain non-amines bind selectively to the SERT and block the transport of serotonin. At 10 microM concentration, select non-amines displayed no, or little, affinity for 9 serotonin, 5 dopamine, 7 adrenergic, 5 muscarinic cholinergic, 3 opiate and histamine receptors. The affinities of non-amines for [(3)H]citalopram binding sites on the SERT and their potencies for blocking [(3)H]serotonin transport were measured in cloned human SERT stably or transiently expressed in HEK-293. Whether oxa- or carba-based, non-amines bound to [(3)H]citalopram-labeled sites and blocked [(3)H]serotonin transport in the low nanomolar range, at values equal to or higher than those of some conventional antidepressants. A non-amine, O-1809, was 99-fold more selective for the serotonin over the dopamine transporter. As substituents on the aromatic ring of non-amines confer high affinity for the SERT, we investigated the hypothesis that aromatic-aromatic interactions may contribute significantly to non-amine/transporter association. A SERT mutant was produced in which a highly conserved aromatic amino acid, phenylalanine 548, was replaced by an alanine (F548A). Although the affinities of several non-amines were unchanged in the mutant SERT, the affinity of imipramine was decreased, revealing possible differences in amine and non-amine binding domains on the SERT. The similar affinities of non-amines and conventional antidepressant drugs for the SERT support the view that an amine nitrogen is not essential for drugs to block serotonin transport with high affinity. Non-amines open avenues for developing a new generation of antidepressants.

Synthesis of 6- and 7- hydroxy-8-azabicyclo[3.2.1]octanes and their binding affinity for the dopamine and serotonin transporters.

Cocaine is a potent stimulant of the central nervous system. Its reinforcing and stimulant effects are related to its ability to inhibit the membrane bound dopamine transporter (DAT). Inhibition of the DAT causes an increase of dopamine in the synapse with a resultant activation of postsynaptic receptors. The rapid onset and short duration of action of cocaine contribute to its high addictive potential. Consequently, the design of tropane analogues of cocaine that display longer onset times on the DAT and extended duration of action is driven by the need to develop cocaine medication. This study extends the exploration of bridge hydroxylated azabicyclo[3.2.1]octanes (tropanes). A series of 6- and 7-hydroxylated tropanes was prepared and evaluated biologically. Structure activity relationships lead to the following conclusions. Bridge hydroxylated tropanes retain biological enantioselectivity but display higher DAT versus SERT selectivity, particularly for the 3 alpha-aryl compounds as compared with the 3beta-aryl compounds, than the bridge unsubstituted analogues. The 7-hydroxyl compounds are more potent at the DAT than their 6-hydroxyl counterparts. The general SAR of the tropanes is maintained and the rank order of potencies based on substitution at the C3 position remains 3,4-dichloro > 2-naphthyl > 4-fluoro > phenyl.

[(11)C, (127)I] Altropane: a highly selective ligand for PET imaging of dopamine transporter sites.

The E isomer of (123)I-2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(1-iodoprop-1-en-3-yl)nortropane (Altropane(R)) shows high affinity (IC(50) = 6.62 +/- 0.78 nmol) and selectivity (DA/5-HT = 25) for DAT sites in the striatum. Recently, dynamic SPECT studies in healthy volunteers and patients with Parkinson disease demonstrated that the kinetics of striatal accumulation followed a pattern that is characteristic of a reversible tracer with maximal accumulation within 30 min after injection. These findings suggested that radiolabeling Altropane with [(11)C] might provide an equivalent and complementary tracer for PET studies. [(127)I] Altropane was treated with HCl to hydrolyze the methyl ester bond and yield a precursor for [(11)C] labeling. Introduction of an [(11)C] methyl ester group was achieved by treatment with [(11)C] CH(3)I followed by HPLC purification. Five healthy rhesus monkeys were injected with approximately 10 mCi of [(127)I,(11)C] Altropane and dynamic PET images were acquired over 90 min. Arterial blood samples were collected in parallel with imaging and metabolite analysis was performed by HPLC. The PET and metabolite corrected arterial blood data were to calculate k(3)/k(4) by two methods: 1) nonlinear least-squares fitting, and 2) a linear graphical method for reversible ligands. The synthetic procedure yielded high specific activity tracer, >1,000 mCi/micro mole, with radiochemical purity >95%. Synthesis time was approximately 30 min. The PET images revealed excellent striatal definition, with clear separation of caudate nucleus and putamen and minimal accumulation in brain regions with high 5HT transporter density. Metabolite analysis demonstrated that at 60 min after injection, approximately 80% of circulating tracer was intact [(127)I,(11)C] Altropane and the remainder was converted to polar metabolites. Values for k(3)/k(4) calculated by two analysis methods were remarkably similar: Method 1, 3.48 +/- 0.41; Method 2, 3.77 +/- 0.45 (mean +/- SEM, t = 2.31, df = 8, P = 0.64). These results establish that Altropane has the important characteristics of: 1) rapid and specific striatal binding; 2) high selectivity for DA vs. 5-HT transporter sites; 3) reversible binding kinetics; 4) potential for multiple injection studies; 5) high efficiency labeling with either [(11)C] or [(123)I]; 6) applicability for both PET and SPECT. These properties make Altropane an important DAT ligand for both research and clinical applications.

2-Carbomethoxy-3-aryl-8-bicyclo[3.2.1]octanes: potent non-nitrogen inhibitors of monoamine transporters.

Cocaine is a potent central nervous system stimulant with severe addiction liability. Its reinforcing and stimulant properties derive from inhibition of monoamine transport systems, in particular the dopamine transporter (DAT). This inhibition results in an increase in synaptic dopamine with subsequent stimulation of postsynaptic dopamine receptors. A wide variety of ligands manifest potent inhibition of the DAT, and these ligands include 3-aryltropane as well as 8-oxa-3-aryltropane analogues of cocaine. There has been considerable effort to determine structure-activity relationships of cocaine and congeners, and it is becoming clear that these inhibitors do not all interact with the DAT in the same manner. The functional role of the 8-heteroatom is the focus of this study. We describe the preparation and biology of a series of 2-carbomethoxy-3-arylbicyclo[3.2.1]octane analogues. Results show that methylene substitution of the amine or ether function of the 8-hetero-2-carbomethoxy-3-arylbicyclo[3.2.1]octanes yields potent inhibitors of monoamine transport. Therefore neither nitrogen nor oxygen are prerequisites for binding of tropane-like ligands to monoamine transporters.

3-Aryl-2-carbomethoxybicyclo[3.2.1]oct-2-enes inhibit WIN 35,428 binding potently and selectively at the dopamine transporter.

The search for medications for cocaine abuse has focused upon the design of potential cocaine antagonists or cocaine substitutes which interact at the dopamine transporter of mammalian systems. This manuscript describes the synthesis and biological evaluation of 8-substituted 2-carbomethoxy-3-arylbicyclo[3.2.1]oct-2-enes. These compounds prove potent and selective inhibitors of the dopamine transporter. Their selectivity results primarily from a reduced inhibitory potency toward the serotonin transporter. This work supports the notion that the orientation of the 3-aryl ring in the bicyclo[3.2.1]octane system affects the interaction of these molecules with the serotonin transporter far more markedly than it affects the interaction with the dopamine transporter.

Molecular and regional targets of cocaine in primate brain: liberation from prosaic views.

Abstract The neurochemical processes underlying initial exposure to and reinforcing effects of cocaine are not fully understood. An enduring hypothesis of cocaine addiction is based on an underlying premise that dopamine is the acute mediator of the rewarding effects of cocaine and this nefarious role extends through each phase of addiction. Cocaine is an effective inhibitor of the dopamine transporter, thereby increasing extracellular dopamine levels. Euphoria is attributed to the cocaine-induced inundation of extracellular dopamine and the withdrawal and craving for cocaine after cessation of drug use are attributed to neuroadaptive processes to dampen dopaminergic transmission. Nevertheless, our understanding of the role of dopamine transporter blockade in cocaine addiction is not fully understood. The objectives of this laboratory are to investigate the primary targets of cocaine in the brain, those associated with the initial phase of cocaine use and that can provide leads for investigating neuroadaptive processes that may trigger addiction. Two prosaic views of the neurobiology of cocaine addiction are examined in this review. The first is based on the assumption that the dopamine transporter contributes significantly to the stimulant and reinforcing effects of cocaine, and focuses on how stimulant drugs of abuse such as cocaine bind to the dopamine transporter. We present evidence that the widespread assumption that dopamine transporter blockers require an amine nitrogen in their structure is incorrect as non-amines are effective blockers of transporters. The second prosaic view, based on the assumption that the dopamine transporter fulfills a paramount role in cocaine addiction, is assessed in view of mounting evidence that the transporter may not account for the full spectrum of cocaine's effects. Other targets of cocaine, which may be relevant to the acute and chronic effects of cocaine, are presented.

Non-amine dopamine transporter probe [(3)H]tropoxene distributes to dopamine-rich regions of monkey brain.

Drug development in psychopharmacology has adhered to the unwritten precept that compounds targeting monoamine transporters must contain an amine nitrogen in the molecular structure. A series of non-amine-bearing aryloxatropanes that are potent inhibitors of the dopamine transporter (DAT) challenged this precept. In the present study, we investigated the brain distribution of a selective, high-affinity DAT non-amine, [(3)H]tropoxene (2-carbomethoxy-3, 4dichloro-3-aryl-8-oxabicyclo[3.2.1] octene), which binds to the DAT in monkey striatum. The autoradiographic distribution of [(3)H]tropoxene was conducted in tissue sections of rhesus (Macaca mulatta) monkey brain. Highest accumulation of the radioligand was detected in the putamen and caudate nucleus, with significant levels also observed in the nucleus accumbens and substantia nigra. Moderate to low levels of [(3)H]tropoxene binding were noted in the hypothalamus, amygdala, ventral tegmental area, and thalamus. The distribution of [(3)H]tropoxene was restricted to brain regions previously identified as expressing DAT, and the relative densities of [(3)H]tropoxene binding sites in various brain regions corresponded to those observed with other selective monoamine radioligands for the DAT. This is the first report to demonstrate that transporter-selective compounds that bear no amine nitrogen in their structure bind selectively to brain regions rich in the transporter. The results support our conclusion that an amine nitrogen is not necessary for compounds to bind to monoamine transporters and distribute in brain according to the known distribution of transporters. The findings provide further incentives to investigate the pharmacological potential of transport inhibitors lacking an amine nitrogen in the molecular structure.

Bicyclo[3.2.1]octanes: synthesis and inhibition of binding at the dopamine and serotonin transporters.

Herein we report the synthesis of a series of bicyclo[3.2.1]octanes and their binding characteristics at the dopamine and serotonin transporters. The data confirm that a heteroatom at position 8 of the tropane nucleus is not a prerequisite for binding since the bicyclo[3.2.1]octanes prove potent inhibitors of both transporters. Therefore the three-dimensional topology of the ligand may be more important than specific functionality with respect to stereospecific binding at the acceptor site.

Altropane, a SPECT or PET imaging probe for dopamine neurons: I. Dopamine transporter binding in primate brain.

Increasing evidence suggests that the dopamine transporter is an important marker for physiological and pathological changes in dopamine neurons. Potent dopamine transport inhibitors of the phenyltropane series (e.g., WIN 35,428 or CFT) are particularly suitable for PET (positron emission tomography) or SPECT (single photon emission computed tomography) imaging of the dopamine transporter in living brain. We investigated whether altropane, an N-iodoallyl analog of WIN 35,428 (IACFT:E-N-iodoallyl-2 -carbomethoxy-3beta-(4-fluorophenyl)tropane), displayed in vitro properties suitable for evaluation as a SPECT imaging agent. In brain striatum of cynomolgus monkey (Macaca fascicularis), the unlabeled E-isomer (IC50: 6.62 +/- 0.78 nM) was more potent than the Z-isomer (IC50: 52.6 +/- 0.3 nM) and displayed a relatively high dopamine:serotonin transporter selectivity (28-fold). In radiolabeled form, [125I]altropane bound to sites in the striatum with a single high affinity (KD: 5.33 +/- 0.55 nM) and with a site density (BMAX: 301 pmol/g original wet tissue weight) that was within the density range reported previously for the dopamine transporter in striatum. Drugs inhibited [125I]altropane binding with a rank order of potency that corresponded closely to their potencies for inhibiting [3H]WIN 35,428 binding (r2: 0.99; P < 0.0001) to the blocking dopamine transport. The favorable binding properties of altropane, together with its rapid entry into primate brain and highly localized distribution in dopamine-rich brain regions, suggest it is a suitable iodinated probe for monitoring the dopamine transporter in vitro and in vivo by SPECT or PET imaging.

Altropane, a SPECT or PET imaging probe for dopamine neurons: II. Distribution to dopamine-rich regions of primate brain.

The dopamine transporter in brain, localized almost exclusively on dopamine neurons, is an effective window on dopamine neurons. SPECT or PET imaging of the transporter in brain requires selective imaging agents that display appropriate pharmacokinetic properties. We previously reported that [125I]altropane ([125I]IACFT,2beta-carbomethoxy-3beta-(4-fluorophenyl)-n-(1- iodoprop-1-en-3-yl)nortropane) bound with high affinity (Kd: 5.33 nM) to a single site on the dopamine transporter and was selective for dopamine over the serotonin transporter in homogenates of monkey striatum. To determine whether the selective binding of [125I]altropane is reflected in its brain distribution, the in vitro and ex vivo distribution of [125I]altropane in squirrel monkey (Saimiri sciureus) brain was determined by quantitative autoradiography of coronal brain sections. In vitro, [125I]altropane (2 nM) distribution was discrete and was detectable primarily in the dopamine-rich putamen, caudate nucleus, and nucleus accumbens. The resulting putamen:cerebellum ratio exceeded 120:1 (n = 3). The selective in vitro binding of [125I]altropane to the dopamine transporter, at concentrations approaching its Kd value (Kd: 5.33 nM, a single high affinity site), highlight its suitability for investigating the density of the dopamine transporter in various brain regions in vitro. Ex vivo autoradiography was conducted in monkeys to determine whether the brain distribution of [125I]altropane in vitro was predictive of its brain distribution pattern after intravenous administration. Thirty minutes after intravenous injection, highest levels of [125I]altropane (0.3 nmol/kg) were detected in the caudate-putamen and nucleus accumbens and lowest levels in the cerebellum and cortex. The putamen or caudate:cerebellum ratio was 7. SPECT imaging of the brain within 30 min of i.v. injection confirmed the rapid and selective accumulation of [123I]altropane to the striatum. The selective binding of altropane to the dopamine-rich striatum within 30 min of i.v. administration indicates that it is uniquely suited for SPECT or PET imaging of the dopamine transporter and associated dopamine neurons.

Altropane, a SPECT or PET imaging probe for dopamine neurons: III. Human dopamine transporter in postmortem normal and Parkinson's diseased brain.

Increasing evidence suggests that the dopamine transporter is situated almost exclusively on dopamine neurons. Accordingly, it is an valuable marker for Parkinson's disease and other pathological states of dopamine neurons. We previously demonstrated that the potent dopamine transport inhibitor [125I]altropane (IACFT:E-N-iodoallyl-2beta-carbomethoxy-3beta-(4-fluor ophenyl)tropane) is a high affinity selective probe for the dopamine transporter in monkey brain and an effective SPECT imaging agent in nonhuman primate brain. We now report the binding properties of [125I]altropane in postmortem tissue of normal human brain and compare the findings to Parkinson's diseased brain. In homogenates of human brain putamen, [125I]altropane bound with high affinity (KD: 4.96 +/- 0.38 nM, n = 4) and site density (BMAX: 212 +/- 41.1 pmol/g original wet tissue weight) well within the density range reported previously for the dopamine transporter in this brain region. Drugs inhibited [125I]altropane binding with a rank order of potency that corresponded closely to their rank order for blocking dopamine transport (r 0.98, P < 0.001). In postmortem Parkinson's diseased brain, bound [125I]altropane (1 nM) was markedly reduced (89%, 99% in putamen, depending on measures of nonspecific binding) compared with normal aged-matched controls (normal putamen: 49.2 +/- 8.1 pmol/g; Parkinson's diseased putamen: 0.48 +/- 0.33 pmol/g; n = 4). In vitro autoradiography, conducted in tissue sections at a single plane of the basal ganglia, revealed high levels of [125I]altropane binding the caudate nucleus and putamen, but lower levels (73% of the caudate-putamen) in the nucleus accumbens (n = 7). In Parkinson's diseased brains (n = 4), [125I]altropane binding was 13% of the levels detected in normal putamen, 17% of normal values in the caudate nucleus, and 25% of normal levels in nucleus accumbens. The association of [125I]altropane to the dopamine transporter in human postmortem tissue, the marked reduction of [125I]altropane binding in Parkinson's diseased brains, its rapid entry into brain and highly localized distribution in dopamine-rich brain regions, support its use as a probe for monitoring the dopamine transporter in vitro and in vivo by SPECT imaging.

Rapid detection of Parkinson's disease by SPECT with altropane: a selective ligand for dopamine transporters.

Increasing evidence indicates that dopamine (DA) transporter density declines in Parkinson's disease (PD). 2Beta-carbomethoxy-3beta-(4-fluorophenyl)-n-(1-iodoprop-1-en -3-yl) nortropane (IACFT, Altropane) is a cocaine analog with high affinity and selectivity for dopamine transporter (DAT) sites in the striatum. In this study, single photon emission computed tomography (SPECT) with [123I]altropane was used to measure DAT density in seven healthy volunteers (five males, age 37-75, and two females, ages 26 and 39) and eight male patients with Parkinson's disease (age 14-79, Hoehn and Yahr stage: 1.5-3 (n = 5) and 4-5 (n = 3)). Dynamic SPECT images and arterial blood samples were acquired over 1.5-2 hr and plasma radioactivity was analyzed chromatographically to obtain metabolite corrected arterial input functions. Binding potential (BP, B'max/KD) for striatal (Str) DAT sites was calculated by two methods using occipital cortex (Occ) as a reference. In the first method, tissue time-activity curves (TAC) and metabolite corrected arterial input functions were analyzed by a linear graphical method developed for reversible receptor ligands. In the second method, the expression (Str(TAC) - Occ(TAC)) was fitted to a gamma variate function and the maximum divided by Occ(TAC) at the same time was used to estimate BP. In five of the PD patients, the SPECT data were compared with the results of PET with [18F] 6-fluoro DOPA (FD-PET). Plasma analysis indicated that [123I]altropane is rapidly converted to polar metabolites. SPECT images in healthy volunteers showed that [123I] altropane accumulated rapidly and selectively in the striatum and yielded excellent quality images within 1 h after injection. Both methods of analysis revealed a 7.6%/decade reduction in BP and average striatal values (corrected to age 25) were 1.83 +/- 0.22 and 2.09 +/- 0.20 by methods 1 and 2. In all the PD patients, striatal accumulation was markedly reduced and the pattern of loss was similar to that reported for DA; most profound in the posterior putamen with relative sparing of the caudate nuclei. A comparable pattern was observed with FD-PET. For total striatum, age-corrected BP was significantly (P < 0.001) reduced; 0.83 +/- 0.06 (method 1), 0.84 +/- 0.07 (method 2). BPs measured by the two methods were remarkably similar and highly correlated r2 = 0.88, (P < 0.001). These results indicate that [123I]altropane is an excellent SPECT ligand for imaging the DAT/DA neurons in human brain. The high selectivity and rapid striatal accumulation of the ligand allows for accurate quantitation of DAT sites in less than 2 hr. The results further demonstrate that [123I]altropane is an effective marker for PD.

2-Carbomethoxy-3-aryl-8-oxabicyclo[3.2.1]octanes: potent non-nitrogen inhibitors of monoamine transporters.

Cocaine is a potent stimulant of the mammalian central nervous system. Its reinforcing and stimulant properties have been associated with its propensity to bind to monoamine transporter systems. It has generally been assumed that the amino function on monoamines is a requirement for binding to monoamine transporters. In particular, the 8-amino function on the tropane skeleton of cocaine and cocaine analogs has been assumed to provide an ionic bond to the aspartic acid residue on the dopamine transporter (DAT). We have prepared the first 8-oxa analogs of the 3-aryltropanes (WIN compounds) and have found that the 3 beta-(3,4-dichlorophenyl) (6g) and 3 alpha-(3,4-dichlorophenyl) (7g) analogs are particularly potent (IC50 = 3.27 and 2.34 nM, respectively) inhibitors of the dopamine transporter. We now describe the synthesis and biology of the family of 2-carbomethoxy-3-aryl-8-oxabicyclo[3.2.1]octanes and demonstrate that an amino nitrogen is not required for binding to the DAT.

A technetium-99m SPECT imaging agent which targets the dopamine transporter in primate brain.

The dopamine transporter (DAT), located presynaptically on dopamine neurons, provides a marker for certain neurological diseases. In particular, the DAT is depleted in Parkinson's disease, and the extent of depletion correlates with the loss of dopamine. Herein we describe the design, synthesis, and biological evaluation of technepine, the first 99mTc-labeled SPECT imaging agent which targets the dopamine transporter in striatum. We have demonstrated that the DAT can accommodate a chelating unit attached to the 8-amine function of a tropane skeleton. Further, we have demonstrated for the first time that a molecule can be designed to carry the radionuclide 99mTc across the blood-brain barrier in sufficient quantity to obtain in vivo images of the striatum in monkeys. This advance will undoubtedly lead to the design of new receptor and transporter-mediated 99mTc agents which can label specific transporter and receptor targets in the central nervous system.

Nitrogen-based drugs are not essential for blockade of monoamine transporters.

In brain, monoamine transporters are principal targets of widely used therapeutic drugs including antidepressants, methylphenidate (Ritalin), and the addictive drug cocaine. Without exception, these transport blocking agents contain an amine nitrogen. A prevalent view and untested premise is that an amine nitrogen is needed to bind to the same counterion on the transporter as does the amine nitrogen of the monoamine neurotransmitter. We report that several compounds without nitrogen (8-oxa-bicyclo-3-aryl-[3.2.1] octanes, or aryloxatropanes) are active at monoamine transporters. One of these, tropoxane (0-914), bound with high affinity to the dopamine (IC50: 3.35 +/- 0.39 nM), serotonin (IC50: 6.52 +/- 2.05 nM), and norepinephrine (IC50: 20.0 +/- 0.3 nM) transporters in monkey brain, the human striatal dopamine transporter (IC50: 5.01 +/- 1.74 nM), and blocked dopamine transport (IC50: 7.2 +/- 3.0 nM) in COS-7 cells transfected with the human dopamine transporter. These unique compounds require a revision of current concepts of the drug binding domains on monoamine transporters, open avenues for discovery of a new generation of drugs and raise the issue of whether mammalian transporters and receptors may respond to, as yet, undiscovered non-amine bearing neurotransmitters or drugs.

Cocaine congeners as PET imaging probes for dopamine terminals.

UNLABELLED: The PET imaging properties of three phenyltropane drugs with differing affinities and selectivities for the dopamine over serotonin transporter, were compared. METHODS: Carbon-11-CFT (WIN 35,428, 2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane), 11C-CCT (RTI-131, 2 beta-carbomethoxy-3 beta-(4-monochlorophenyl)tropane), and 11C-CDCT (dichloropane, 2 beta-carbomethoxy-3 beta-(3,4-dichlorophenyl)tropane) were evaluated as imaging probes for dopamine neurons in five normal and in two MPTP-treated cynomolgus monkeys (macaca fascicularis) using a high-resolution PET imaging system (PCR-I). RESULTS: For 11C-CFT, the specific binding ratio (as defined by the ratio of radioactivity levels in striatum versus cerebellum) was 4.2 +/- 0.8 in caudate and 4.9 +/- 1.2 in putamen at 60 min and 4.9 +/- 1.2 and 5.5 +/- 1.1 at 90 min in control animals. In MPTP-treated monkeys the corresponding ratios were 1.4 +/- 0.1 in caudate and 1.5 +/- 0.1 in putamen at 60 min and 1.3 +/- 0.1 in caudate and 1.4 +/- 0.3 in putamen at 90 min. For the monochloro analog of CFT, 11C-CCT, the ratios in control caudate and putamen were 2.7 +/- 0.4 and 3.4 +/- 0.3, respectively, at 60 min and 3.7 +/- 0.5 and 4.4 +/- 0.6, respectively, at 90 min. In MPTP-treated animals, corresponding ratios were 1.4 +/- 0.4 and 1.5 +/- 0.3 at 60 min and 1.4 +/- 0.4 and 1.6 +/- 0.4 at 90 min. The dichloro analog of CFT, CDCT, which has the highest affinity for the dopamine transporter, generated the lowest ratios in control brains, 2.3 +/- 0.4 in caudate and 2.4 +/- 0.5 in putamen at 60 min. In one MPTP-treated monkey, the corresponding ratios were 1.6 +/- 0.4 and 1.8 +/- 0.3. In comparison with 11C-CFT, both 11C-CCT and 11C-CDCT were less selective and had high uptake in the thalamus. CONCLUSION: The present results clearly indicate that 11C-CFT is a useful ligand for monitoring dopamine neuronal degeneration.

Preparation and biological evaluation of iodine-125-IACFT: a selective SPECT agent for imaging dopamine transporter sites.

UNLABELLED: Parkinson's disease is a progressive neurodegenerative disorder that is associated with the loss of nerve terminals from specific brain areas, particularly in the caudate and putamen, which contains the highest concentrations of dopamine transporter sites. Previously, we synthesized and evaluated a series of 11C-labeled 2 beta-carbomethoxy -3 beta-aryltropane (WIN 35,428; CFT) derivatives as markers for the dopamine transporter system. These ligands have high affinity and specificity for dopamine transporter sites in vitro and in vivo in laboratory animals. The goal of this study was the preparation and preliminary biological characterization of two new ligands based on the structure of WIN 35,428, the E and Z isomers of N-iodoallyl-2 beta -carbomethoxy-3 beta-(4-fluorophenyl)tropane (E and A IACFT). METHODS: E and Z IACFT were synthesized and radiolabeled with 125I. The ligands were characterized by in vitro assays of binding to dopamine and serotonin transporters and by autoradiography. RESULTS: Iodine-125-IACFT was prepared in > 60% radiochemical yield, and > 98% radiochemical purity. Specific activity was 1500 Ci/mmole. In vitro, E-IACFT showed higher affinity for dopamine transporter sites than WIN 35,428 (6.6 versus 11 nM) and better selectivity than RTI-55. The Z isomer was found to have much lower affinity. One hour after an intravenous injection of 125I IACFT in monkeys, ex vivo autoradiographs of the brain revealed high concentrations of tracer in dopamine rich regions such as the caudateputamen. The striatum-to-cerebellum, striatum-to-cortex and striatum-to-thalamus ratios were 10.8, 7.2 and 8.3. CONCLUSION: These result suggest that radiolabeled E-IACFT may be a useful radioligand for SPECT imaging of dopamine transporter sites. IACFT could prove to be extremely useful for the noninvasive evaluation of patients with early Parkinson's disease.

2-Carbomethoxy-3-(diarylmethoxy)-1 alpha H, 5 alpha H-tropane analogs: synthesis and inhibition of binding at the dopamine transporter and comparison with piperazines of the GBR series.

We recently reported a new class of tropanes, based on benztropine, that bind uniquely, in the S-configuration, to the dopamine transporter. We have now extended this series to evaluate the effects of substituents on the nitrogen and the diarylmethoxy group. Herein we have described the synthesis and biological evaluation of a series of 2-carbomethoxy-3-(diaryl-methoxy)-1 alpha H, 5 alpha H-tropane (2-carbomethoxybenztropine) analogs. Examination of the binding data obtained for these compounds shows that while the 4,4'-difluoro compound is potent and selective for the dopamine transporter, introduction of larger groups such as 4,4'-dichloro, 4,4'- dibromo, 4,4'-diiodo, or 4,4'dimethyl on the 3-diphenylmethoxy moiety reduces this potency. However, although introduction of only one group (e.g., 4-chloro, 4-bromo, 4-iodo, or 4-methyl) leads to a similar reduction of binding affinity, these monosubstituted 2-carbomethoxybenztropines are significantly more potent than the related disubstituted compounds. Finally, from the data for the N-substituted 2-carbomethoxybenztropine analogs, it is evident that steric bulk can be tolerated at the nitrogen site. A comparison of structure-activity relationship data for the tropanes, GBR analogs, and these benztropines indicates that the 2-carbomethoxybenztropine analogs may be more like the GBR analogs in their mode of binding to the dopamine transporter than like the tropanes. This conclusion supports the notion that the binding site for (-)-cocaine [and the (1R)-tropanes] may differ from of the 2-carbomethoxybenztropine analogs.