Discovery of novel trisubstituted asymmetric derivatives of (2S,4R,5R)-2-benzhydryl-5-benzylaminotetrahydropyran-4-ol, exhibiting high affinity for serotonin and norepinephrine transporters in a stereospecific manner

In our structure-activity relationship study on 3,6-disubstituted pyran derivatives, we have carried out asymmetric synthesis and biological characterization of trisubstituted (2S,4R,5R)-2-benzhydryl-5-benzylaminotetrahydropyran-4-ol and (3S,4R,6S)-6-benzhydryl-4-benzylaminotetrahydropyran-3-ol derivatives and their enantiomers. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]-5-HT, and [(3)H]NE, respectively. Compounds were also tested for their binding affinity at the DAT by their inhibition of [(3)H]WIN 35,428. Biological results indicated that regioselectivity and stereoselectivity played important roles in determining activity for monoamine transporters as only (-)-isomers of 2-benzhydryl-5-benzylaminotetrahydropyran-4-ol derivatives exhibited appreciable potency for the monoamine transporters, in particular for the SERT and NET. Among the active analogues, (-)-9d exhibited potent and selective affinity at the NET (K(i), [(3)H]NE = 4.92 nM; DAT/NET = 91 and SERT/NET = 140). One of the derivatives with p-methoxybenzyl substitution, (-)-9a, was potent at both SERT and NET (K(i), [(3)H]-5-HT = 25.9 and [(3)H]NE = 15.8 nM, respectively). In the active analogue series ((-)-9a-(-)-9e), a cis-relationship between the biphenyl and the amino moiety was maintained for the SERT and NET interactions, as was observed with our earlier 3,6-disubstituted pyran compounds for the DAT interaction. To the best of our knowledge, this current series of compounds represents a novel class of pyran derivatives as blockers for monoamine transporters.     

Design and synthesis of promiscuous high-affinity monoamine transporter ligands: unraveling transporter selectivity

A series of 4-[2-[bis(4-fluorophenyl)methoxy]ethyl]-piperidines and 4-[2-[(bisphenyl)methoxy]ethyl]-piperidines with different types of substituents in the phenylpropyl side-chain were synthesized and examined for their ability to bind to the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). All of the compounds showed high binding affinities for the DAT in the low to subnanomolar range. Their ability to bind to the SERT and the NET, while maintaining their high affinity for the DAT, could be altered by substitution in positions C2 and C3 of the phenylpropyl side-chain. This approach gave rise to a new set of compounds with selectivity for the DAT, the DAT and the SERT, or the DAT and the NET. Six compounds (7, 9, 11, 12, 14, and 20) with relatively low SERT/DAT ratios were selected for additional study in biogenic amine uptake inhibition assays based on the biogenic amine transporter binding results. Some of the new ligands can serve as pharmacological tools to block DAT or DAT and another transporter simultaneously.     

Further structural exploration of trisubstituted asymmetric pyran derivatives (2S,4R,5R)-2-benzhydryl-5-benzylamino-tetrahydropyran-4-ol and their corresponding disubstituted (3S,6S) pyran derivatives: a proposed pharmacophore model for high-affinity interaction with the dopamine, serotonin, and norepinephrine transporters

In our previous report, we described a novel series of asymmetric pyran derivatives (2S,4R,5R)-2-benzhydryl-5-benzylamino-tetrahydropyran-4-ol and their enantiomers as blockers of monoamine transporters in the brain. In this report, we describe the further exploration of this series of molecules by incorporating functional groups in the molecular template, which should promote the formation of H bonds with the transporters. In addition, a new synthetic scheme for the asymmetric synthesis of disubstituted cis-(6-benzhydryl-tetrahydro-pyran-3-yl)-benzylamine analogues and their biological characterization is reported. All synthesized derivatives were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. The compounds were also tested for their binding potency at the DAT by their ability to inhibit binding of [(3)H]WIN 35, 428. The results indicated that the presence of functional groups, such as -OH, -NH(2), and the bioisosteric 5-substituted indole moiety in both di and trisubstituted compounds, significantly increased their potencies for the SERT and NET, especially for the NET. Among the trisubstituted compounds, (-)-4b exhibited the highest potency for the NET and the SERT (K(i) of 2.13 and 15.3 nM, respectively) and was a serotonin norepinephrine reuptake inhibitor (SNRI). Compound (-)-4a exhibited the highest selectivity for the NET. Among the disubstituted compounds, a number of compounds, such as (-)-9a, (+)-9b, (-)-9b, and (+)-9d, exhibited significant low-nanomolar potencies for the SERT and the NET. Interestingly, compound (-)-9d exhibited appreciable potencies at all three transporters. On the basis of our present and past findings, we propose a qualitative model for the interaction of these compounds with monoamine transporters, which will be refined further in the future.     

Syntheses and binding studies of new [(aryl)(aryloxy)methyl]piperidine derivatives and related compounds as potential antidepressant drugs with high affinity for serotonin (5-HT) and norepinephrine (NE) transporters

In a wide search program toward new, efficient, and fast-acting antidepressant drugs, we have prepared series of new compounds having an (aryl)(aryloxy)methyl moiety linked directly or through a methylene chain to different substituted and unsubstituted cycles (isoquinoline, piperazine, piperidine, tetrahydropyran, or cyclopentane). These compounds have been evaluated for their affinities for serotonin (5-HT) transporter (SERT) and 5-HT(1A) and 5-HT(2A) receptors. Racemic mixtures of 4-[(aryl)(aryloxy)methyl]piperidine derivatives showed much higher affinity values for SERT than fluoxetine and resulted in lack of affinity for 5-HT(1A) and 5-HT(2A) receptors. Some of these racemic mixtures were resolved to their enantiomers and tested for binding to norepinephrine (NE) transporter (NET), dopamine (DA) transporter (DAT), and alpha(2) receptor. Several of these enantiomers [(-)-15b, (-)-15j, (-)-15t, (+)-15u] displayed a dual binding profile with affinities for SERT and NET with K(i) < 25 nM and a NET/SERT ratio <10. Compound (-)-15j (coded as F-98214-TA for development studies) showed a dual binding profile with very high affinity values for SERT and NET (K(i) = 1.9 and 13.5 nM, respectively), and further pharmacological characterization is in progress for its evaluation as a antidepressant.     

Synthesis and in vivo evaluation of halogenated N,N-dimethyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine derivatives as PET serotonin transporter ligands

N, N-dimethyl-2-(2'-amino-4'-hydroxymethylphenylthio)benzylamine (38), substituted on ring A, was reported to display high binding affinity and selectivity to the human brain serotonin transporter (SERT). In an attempt to explore the potential of compounds substituted on ring B of the phenylthiophenyl core structure, three derivatives of 38 were synthesized: N, N-dimethyl-2-(2'-amino-4'-hydroxymethyl-phenylthio)-5-fluorobenzylamine (35), N, N-dimethyl-2-(2'-amino-4'-hydroxymethyl-phenylthio)-5-bromobenzylamine (36), and N, N-dimethyl-2-(2'-amino-4'-hydroxymethyl-phenylthio)-5-iodobenzylamine (37). The in vitro binding studies in cells transfected with human SERT, norepinephrine transporter (NET), and dopamine transporter (DAT) showed that 35, 36, and 37 exhibited high SERT affinity with K is (SERT) = 1.26, 0.29, and 0.31 nM (vs [(3)H]citalopram), respectively. [(11)C]-(35), [(11)C]-(36), and [(11)C]-( 37) were prepared by methylation of their monomethyl precursors 16, 17, and 18, with [(11)C]iodomethane in 28, 11, and 14% radiochemical yields, respectively. The microPET images of [(11)C]-(35), [(11)C]-(36), and [(11)C]-(37) showed high uptake in the monkey brain regions rich in SERT with peak midbrain to cerebellum ratios of 3.41, 3.24, and 3.00 at 85 min post-injection, respectively. In vivo bindings of [(11)C]-(35), [(11)C]-(36), and [(11)C]-(37) were shown to be specific to the SERT as displacement with citalopram (a potent SERT ligand) reduced radioactivity in SERT-rich regions to the cerebellum level. These results suggest that [(11)C]-(35), [(11)C]-(36), and [(11)C]-(37) could be potential agents for mapping human SERT by PET and radiolabeling 37 with iodine-123, which could afford the first SPECT SERT imaging agent exhibiting fast kinetics.     

MDMA-induced impairment in primates: antagonism by a selective norepinephrine or serotonin, but not by a dopamine/norepinephrine transport inhibitor

Human MDMA (R,S-3,4-methylenedioxymethamphetamine) users display selective cognitive deficits after acute MDMA exposure, frequently attributed to serotonin deficits. We postulated that MDMA will compromise executive function in primates and that an inhibitor of the serotonin transporter (SERT) and the norepinephrine transporter (NET) but not the dopamine (DAT) transporter, will prevent impairment. The potencies of DAT/NET, NET and SERT inhibitors to block transport of [(3)H]MDMA and [(3)H]monoamines were compared in vitro. Subsequently, cynomolgus monkeys (Macaca fasicularis) were trained to stable performance in a reversal learning task. Effects of once-weekly oral or i.m. dose of MDMA (1.5 mg/kg, n = 4) on performance were monitored, alone or after pretreatment with inhibitors of the SERT, DAT or NET (prior to i.m. MDMA). 1) Drug potencies for blocking [(3)H]MDMA or [(3)H]monoamine transport were not consistent; 2) Oral MDMA increased error rates in a cognitive task for up to three days following exposure, whereas intramuscular MDMA prevented subjects from performing the cognitive task on the day of administration, but not on subsequent days; 3) The SERT inhibitor citalopram and the NET inhibitor desipramine, but not the DAT/NET inhibitor methylphenidate, reversed the effects of MDMA on task performance and mandibular movements induced by i.m. MDMA and 4) MDMA altered sleep latency. Oral MDMA impairs executive function in monkeys for several days, a finding of potential relevance to MDMA consumption by humans. Reversal of impaired executive function by a NET inhibitor implicates the NET and norepinephrine in MDMA-induced cognitive impairment and may be relevant to therapeutic strategies.     

Synthesis, monoamine transporter binding, properties, and functional monoamine uptake activity of 3beta-[4-methylphenyl and 4-chlorophenyl]-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes

Synthetic methods were developed for the synthesis of the 3beta-(4-substituted phenyl)-2 beta-[5-(substituted phenyl)thiazol-2-yl]tropanes (4a-s). The compounds were evaluated for their monoamine transporter binding and monoamine uptake inhibition properties using both rat brain tissue and cloned transporter assays. In general, the compounds showed higher dopamine transporter (DAT) affinity relative to the serotonin and norepinephrine transporters (SERT and NET, respectively) and greater [3H]dopamine uptake inhibition potency relative to [3H]serotonin and [3H]norepinephrine uptake inhibition. Several compounds were DAT selective relative to the SERT and NET in the monoamine transporter binding assays. The most potent and selective analog in the functional monoamine uptake inhibition test was 3beta-(4-methylphenyl-2 beta-[5-(3-nitrophenyl)thiazol-2-yl]tropane (4p).     

Synthesis and characterization of iodine-123 labeled 2beta-carbomethoxy-3beta-(4'-((Z)-2-iodoethenyl)phenyl)nortropane. A ligand for in vivo imaging of serotonin transporters by single-photon-emission tomography

2beta-Carbomethoxy-3beta-(4'-((Z)-2-iodoethenyl)phenyl)nortropane (ZIENT) (6) and 2beta-carbomethoxy-3beta-(4'-((E)-2-iodoethenyl)phenyl)nortropane (EIENT) (10) were prepared and evaluated in vitro and in vivo for serotonin transporter (SERT) selectivity and specificity. High specific activity [(123)I]ZIENT and [(123)I]EIENT were synthesized in 45% (n = 5) and 42% (n = 4) radiochemical yield (decay-corrected to end of bombardment (EOB)), respectively, by preparation of the precursor carbomethoxy-3beta-(4'-((Z)-2-trimethylstannylethenyl)phenyl)nortropane (7) and 2beta-carbomethoxy-3beta-(4'-((E)-2-tributylstannylethenyl)phenyl)nortropane (9), respectively, followed by treatment with no carrier-added sodium [(123)I]iodide and hydrogen peroxide in ethanolic HCl. Competition binding in cells stably expressing the transfected human SERT, dopamine transporter (DAT), and norepinephrine transporter (NET) using [(3)H]citalopram, [(3)H]WIN 35,428, and [(3)H]nisoxetine, respectively, demonstrated the following order of SERT affinity (K(i) in nM): ZIENT (0.05) > nor-CIT (0.12) > EIENT (1.15) > fluvoxamine (1.46). The affinity of ZIENT and EIENT for DAT was 69 and 1.6-fold lower, respectively, than for SERT. In vivo biodistribution and blocking studies were performed in male rats and demonstrated that the brain uptake of [(123)I]ZIENT was selective and specific for SERT-rich regions (hypothalamus, striatum, pons, and prefrontal cortex). SPECT brain imaging studies in monkeys demonstrated high [(123)I]ZIENT uptake in the diencephalon, which resulted in diencephalon-to-cerebellum ratios of 2.12 at 190 min. [(123)I]ZIENT uptake in the diencephalon achieved transient equilibrium at 157 min. In a displacement experiment of [(123)I]ZIENT in a cynomolgus monkey, radioactivity was reduced by 39% in the diencephalon at 101 min following injection of citalopram. The high specific activity one-step radiolabeling preparation and high selectivity of [(123)I]ZIENT for SERT support its candidacy as a radioligand for mapping brain SERT sites.     

Structure-activity relationship studies of highly selective inhibitors of the dopamine transporter: N-benzylpiperidine analogues of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine

A series of 4-[2-[bis(4-fluorophenyl)methoxy]ethyl-1-benzylpiperidines were examined for their ability to bind to the dopamine transporter (DAT), the serotonin transporter (SERT), and the norepinephrine transporter (NET). Binding results indicated that the presence of an electron-withdrawing group in the C(4)-position of the N-benzyl group is beneficial for binding to the DAT. Several analogues have been identified with high affinity for the DAT, up to 500-fold selectivity over the SERT and about 170-fold selectivity over the NET in binding and uptake inhibition assays.     

Design and synthesis of a novel photoaffinity ligand for the dopamine and serotonin transporters based on 2beta-carbomethoxy-3beta-biphenyltropane

Tropane-based photoaffinity ligands covalently bind to discrete points of attachment on the dopamine transporter (DAT). To further explore structure-activity relations, a ligand in which the photoactivated group was extended from the 3-position of the tropane ring was synthesized from cocaine via a Stille or Suzuki coupling strategy. 3-(4'-Azido-3'-iodo-biphenyl-4-yl)-8-methyl-8-aza-bicyclo[3.2.1]octane-2-carboxylic acid methyl ester (11; K(i) = 15.1 +/- 2.2 nM) demonstrated high binding affinity for the DAT. Moreover, this compound showed moderate binding affinity for the serotonin transporter (SERT, K(i) = 109 +/- 14 nM), suggesting the potential utility of [(125)I]11 in both DAT and SERT protein structure studies.     

Further structurally constrained analogues of cis-(6-benzhydrylpiperidin-3-yl)benzylamine with elucidation of bioactive conformation: discovery of 1,4-diazabicyclo[3.3.1]nonane derivatives and evaluation of their biological properties for the monoamine transporters

Our structure-activity relationship (SAR) study on piperidine analogues for monoamine transporters led to the development of a series of 3,6-disubstituted piperidine derivatives, structurally constrained versions of flexible piperidine analogues, with preferential affinity for the dopamine transporter (DAT). In our attempt to further rigidify this structure to study influence of rigidity on binding and in vivo activity, we have developed a series of 4,8-disubstituted 1,4-diazabicyclo[3.3.1]nonane derivatives. All synthesized derivatives were tested for their affinity at the DAT, serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in competing for the binding of [(3)H]WIN 35, 428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also tested for their ability to inhibit uptake of [(3)H]DA. The SAR study led to the discovery of a potent lead compound (-)-S,S-10c which exhibited high affinity and selectivity for the DAT (IC(50) = 22.5 nM; SERT/DAT = 384 and NET/DAT > 444). It is interesting to note that both (-)-10c and the lead compound from the 3,6-disubstituted series (-)-2 exhibited highest activity in their (S,S) isomer indicating similar requirement of regiospecificity for maximum interaction. Overall, our current SAR results corresponded well with the results from less constrained 3,6-disubstituted versions of these molecules albeit the former class exhibited more stringent requirement in molecular structure for activity. However, the potent compounds in the current series exhibited greater selectivity for the DAT compared to their corresponding lesser constrained 3,6-disubstituted versions indicating an effect of rigidity in selective interaction with the transporter proteins. In an effort to elucidate the bioactive conformational structure of the lead molecules in the current and the 3,6-disubstituted series, a preliminary molecular modeling study was carried out where the most rigid derivative (-)-10c was used as a template structure. Compounds (-)-2 and (-)-10c exhibited stimulant activity in locomotor tests in mice in which (-)-2 exhibited a slower onset and longer duration of action compared to (-)-10c. Both compounds occasioned complete cocaine-like responding in mice trained to discriminate 10 mg/kg ip cocaine from vehicle.     

Further structure-activity relationship studies on 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol: identification of compounds with triple uptake inhibitory activity as potential antidepressant agents

To investigate structural alterations of the lead triple uptake inhibitor molecule, disubstituted 4-((((3S,6S)-6-benzhydryltetrahydro-2H-pyran-3-yl)amino)methyl)phenol, we have carried out structure-activity relationship (SAR) studies to investigate the effect of alteration of aromatic substitutions and introduction of heterocyclic aromatic moieties on this molecular template. The novel compounds were tested for their affinities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) in the brain by measuring their potency in inhibiting the uptake of [(3)H]DA, [(3)H]5-HT, and [(3)H]NE, respectively. SAR results indicate dopamine norepinephrine reuptake inhibitory (DNRI) type activity in thiophene (10g) and pyrrole (10i) derivatives. On the other hand, 3-hydroxyphenyl derivative 10f and 4-methoxyphenyl derivative 10j exhibited a triple reuptake inhibitory (TUI) activity profile, as these molecules exhibited potent uptake inhibition for all the monoamine transporters (K(i) of 31.3, 40, 38.5 and K(i) of 15.9, 12.9, 29.3 for DAT, SERT, and NET for 10f and 10g, respectively). Compound 10f was further evaluated in the rat forced swim test to evaluate its potential antidepressant effect. The results show significant reduction of immobility by TUI 10f at 10 mg/kg dose, indicating potential antidepressant activity.     

Design and synthesis of 2- and 3-substituted-3-phenylpropyl analogs of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine and 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine: role of amino, fluoro, hydroxyl, methoxyl, methyl, methylene, and oxo substituents on affinity for the dopamine and serotonin transporters

Novel derivatives of 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(3-phenylpropyl)piperazine (GBR 12909, 1) and 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935, 2) with various substituents in positions C2 and C3 of the phenylpropyl side chain were synthesized and evaluated for their ability to bind to the dopamine transporter (DAT) and the serotonin transporter (SERT). In the C2 series, the substituent in the S-configuration, with a lone-pair of electrons, significantly enhanced the affinity for DAT, whereas the steric effect of the substituent was detrimental to DAT binding affinity. In the C3 series, neither the lone electron pair nor the steric effect of the substituent seemed to affect DAT binding affinity, while sp (2) hybridized substituents had a detrimental effect on affinity for DAT. In the series, the 2-fluoro-substituted (S)-10 had the highest DAT binding affinity and good DAT selectivity, while the 2-amino-substituted (R)-8 showed essentially the same affinity for DAT and SERT. The oxygenated 16 and 18 possessed the best selectivity for DAT.     

Benzo- and cyclohexanomazindol analogues as potential inhibitors of the cocaine binding site at the dopamine transporter

A series of mazindol (1), homomazindol (2), and bishomomazindol (3) derivatives with a benzo or cyclohexano ring fused at various sites were prepared as part of an SAR study to determine the effect of increased aliphatic and aromatic lipophilicity on selected in vitro assays used to identify potential cocaine-like and cocaine antagonism activity. Very good (IC(50) = 2-3 nM) inhibition of [(3)H] WIN 35,428 and [(125)I] RTI-55 binding on rat or guinea pig striatal membranes and HEK cells expressing cDNA for the human dopamine transporter (HEK-hDAT) was shown by the 8,9-benzomazindol 25 and 9,10-benzohomomazindol 28. All new compounds were weaker inhibitors of [(3)H] DA uptake in HEK-hDAT cells than 1 and 2. No improvement in the binding selectivity ratio (SERT/DAT and NET/DAT) was found when compared to 2. Compounds 25and 28 showed a considerable increase versus 1 in uptake/binding discrimination ratios at the DAT (311.0 and 182.1 vs 0.9), SERT (33.6 and 127.3 vs 1.9), and NET (7.3 and 10.0 vs 0.3).     

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.     

New PET imaging agent for the serotonin transporter: [(18)F]ACF (2-[(2-amino-4-chloro-5-fluorophenyl)thio]-N,N-dimethyl-benzenmethanamine)

A new F-18-labeled phenylthiophenyl derivative specific for imaging of serotonin transporters (SERT) in the brain by positron emission tomography (PET) is described. Fluorinated phenylthiophenyl derivative, ACF, 2-[(2-amino-4-chloro-5-fluorophenyl)thio]-N,N-dimethyl-benzenmethanamine, was prepared by first coupling 2,5-dichloro-4-nitroaniline with 2-mercapto-N,N-dimethylbenzamide. The amino group of the coupled adduct was converted to a fluoro group through a Schiemann reaction. Subsequently, a one pot reduction of both nitro and amide groups by BH(3)-tetrahydrofuran yielded the nonradioactive ACF (yield 25%). In vitro binding assays using cell membrane homogenates of LLC cells expressing SERT, dopamine transporters (DAT), or norepinephrine transporters (NET) showed excellent binding affinity and selectivity for SERT (K(i) = 0.05, 3020, and 650 nM for SERT, DAT, and NET, respectively). For preparation of the [(18)F]ACF, the NH(2) group of the initially coupled adduct was converted to the trimethylammonium salt, which was replaced by [(18)F]fluoride in the presence of Kryptofix 222 and potassium carbonate. The final product, [(18)F]ACF, was obtained after a borane and stannous chloride reduction reaction. The combined two step reaction gave a radiochemical yield of 10-15% (EOB) and a radiochemical purity of >99%. Synthesis of the novel PET tracer, [(18)F]ACF, as a probe for binding to SERT in the brain was successfully achieved. The new tracer [(18)F]ACF showed excellent brain penetration and selective localization after an iv injection in rats (brain uptake at 2, 30, 60, 120, and 240 min was 3.27, 1.28, 0.69, 0.21, and 0.06% dose/organ, respectively). The hypothalamus/cerebellum ratio at 60 min post iv injection was 3.55. This specific localization in the hypothalamus was blocked by pretreatment of (+)McN5652. This novel ligand is a potential PET tracer for in vivo evaluation of SERT in the brain.     

Synthesis and transporter binding properties of 3beta-[4'-(phenylalkyl, -phenylalkenyl,and -phenylalkynyl)phenyl]tropane-2beta-carboxylic acid methyl esters: evidence of a remote phenyl binding domain on the dopamine transporter

A series of 4'-substituted 3beta-phenyltropane-2beta-carboxylic acid methyl esters were synthesized and evaluated for binding at the dopamine transporter (DAT) in order to better define the pharmacophore for the cocaine binding site on the DAT. Results from the study of 3beta-[(4'-phenylalkyl)phenyl]tropane-2beta-carboxylic acid methyl esters (5a-c and 6a,b) revealed strong evidence of a previously unknown remote binding domain. The 3beta-[(4'-phenylethyl)phenyl]tropane-2beta-carboxylic acid methyl ester (5a), which has a two methylene linker between the 3beta-phenyl group and the remote phenyl group, has an IC(50) value of 5.14 nM at the DAT. The 3beta-[4'-(benzyl)phenyl] and 3beta-[4'-(phenylpropyl)phenyl] analogues 6b and 5b, respectively, are 102- and 68-fold less potent than 5a at the DAT. Compound 5a also has good affinity for the serotonin and norepinephrine transporters (K(i) = 21 and 6.5 nM, respectively) and is thus a nonselective monoamine uptake inhibitor. Electrostatic effects make a significant contribution to the DAT binding affinity of the 3beta-[(4'-phenylalkenyl)phenyl]tropane-2beta-carboxylic methyl esters (6c, 7a,b, and 8) and 3beta-[(4'-phenylalkynl)phenyl]tropane-2beta-carboxylic acid methyl esters (4a-e). However, the results from the DAT binding on these compounds suggest that there may be another binding domain even further remote from the 4'-position on the 3beta-phenyl group. In both cases, steric barriers have to be overcome before potent binding to the DAT is observed. 3beta-(4'(3-Phenyl-1-propynyl)phenyl)tropane-2beta-carboxylic acid methyl ester (4b), with an IC(50) value of 1.82 nM, was the most potent compound studied. This compound possessed K(i) values of 1.19 and 16.5 nM for the serotonin and norepinephrine transporter and is thus a nonselective monoamine uptake inhibitor.     

Synthesis, biodistribution, and primate imaging of fluorine-18 labeled 2beta-carbo-1'-fluoro-2-propoxy-3beta-(4-chlorophenyl)tr opanes. Ligands for the imaging of dopamine transporters by positron emission tomography

2beta-(R)-Carbo-1-fluoro-2-propoxy-3beta-(4-chlorophenyl) tro pane ((R)-FIPCT, R-6) and 2beta-(S)-carbo-1-fluoro-2-propoxy-3beta-(4-chlorophenyl) tro pane ((S)-FIPCT, S-6) were prepared and evaluated in vitro and in vivo for dopamine transporter (DAT) selectivity and specificity. High specific activity [(18)F](R)-FIPCT and [(18)F](S)-FIPCT were synthesized in 5% radiochemical yield (decay-corrected to end of bombardment (EOB)) by preparation of the precursors 2beta-carbo-R-1-mesyloxy-2-propoxy-3beta-(4-chlorop hen yl)tropane (R-12) and 2beta-carbo-S-1-mesyloxy-2-propoxy-3beta-(4-chlorop hen yl)tropane (S-12) followed by treatment with no carrier-added potassium[(18)F]fluoride and kyrptofix K222 in acetonitrile. Competition binding in cells stably expressing the transfected human DAT and serotonin transporter (SERT) labeled by [(3)H]WIN 35428 and [(3)H]citalopram, respectively, demonstrated the following order of DAT affinity (K(i) in nM): GBR 12909 (0.36) > CIT (0.48) > (S)-FIPCT (0.67) > (R)-FIPCT (3.2). The affinity of (S)-FIPCT and (R)-FIPCT for SERT was 127- and 20-fold lower, respectively, than for DAT. In vivo biodistribution studies were performed in male rats and demonstrated that the brain uptake of [(18)F](R)-FIPCT and [(18)F](S)-FIPCT were selective and specific for DAT rich regions (caudate and putamen). PET brain imaging studies in monkeys demonstrated high [(18)F](R)-FIPCT and [(18)F](S)-FIPCT uptake in the caudate and putamen which resulted in caudate-to-cerebellum and putamen-to-cerebellum ratios of 2.5-3.5 at 115 min. [(18)F](R)-FIPCT uptake in the caudate/putamen achieved transient equilibrium at 75 min. In an imaging experiment with [(18)F](S)-FIPCT in a rhesus monkey with its left hemisphere lesioned with MPTP, radioactivity was reduced to background in the caudate and putamen of the lesioned hemisphere. The high specific activity one-step radiolabeling preparation and high specificity and selectivity of [(18)F](R)-FIPCT and [(18)F](S)-FIPCT for DAT indicate [(18)F](R)-FIPCT and [(18)F](S)-FIPCT are potential radioligands for mapping brain DAT in humans using PET.     

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC(50) values of HD-205 were 4.1, 14 and 280nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC(50) values of 191, 230 and 840nM at DAT, SERT and NET, respectively. Saturation binding studies with [(125)I]RTI-55 in membranes pretreated with 100nM HD-205 showed that HD-205 significantly decreased the B(max) but not K(D) of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC(50) of HD-244 at DAT was 20nM. [(125)I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [(125)I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.     

Expansion of structure-activity studies of piperidine analogues of 1-[2-(diphenylmethoxy)ethyl]-4-(3-phenylpropyl)piperazine (GBR 12935) compounds by altering substitutions in the N-benzyl moiety and behavioral pharmacology of selected molecules.

A series of substituted N-benzyl analogues of the dopamine transporter (DAT) specific compound, 4-[2-(diphenylmethoxy)ethyl]-1-benzylpiperidine were synthesized and biologically characterized. Different 4'-alkyl, 4'-alkenyl, and 4'-alkynyl substituents were introduced in the phenyl ring of the benzyl moiety along with the replacement of the same phenyl ring by the isomeric alpha- and beta-naphthyl groups. Different polar substitutions at the 3'- and 4'-position were also introduced. Novel compounds were tested for their binding affinity at the dopamine, serotonin, and norepinephrine transporter systems in the brain by competing for [(3)H]WIN 35 428, [(3)H]citalopram, and [(3)H]nisoxetine, respectively. Selected compounds were also evaluated for their activity in inhibiting the uptake of [(3)H]dopamine. Binding results demonstrated that alkenyl and alkynyl substitutions at the 4'-position produced potent compounds in which compound 6 with a vinyl substitution was the most potent. In vivo evaluation of three selected compounds indicated that despite their high potency at the DAT, these compounds stimulated locomotor activity (LMA) less than cocaine when tested across similar dose ranges. In a drug discrimination study procedure, none of these three compounds generalized from cocaine in mice trained to discriminate 10 mg/kg cocaine from vehicle. In a 4 h time course LMA experiment, one of our previous lead piperidine derivatives (1a) showed considerable prolonged action. Thus, in this report, we describe a structure-activity relationship study of novel piperidine analogues assessed by both in vitro transporter assays and in vivo behavioral activity measurements.