Kinetic mechanism of dopamine transporter interaction with 1-(2-(bis-(4-fluorophenyl)methoxy)ethyl)-4-(3-phenylpropyl)piperazine (GBR 12909)

Interaction of piperazine-based dopamine transporter inhibitor GBR12909 with rat dopamine transporters has been studied by means of competition kinetics analysis, employing [(3)H]PE2I as the reporter ligand. It has been found that GBR12909 is capable of inducing so-called "slow isomerization step" upon binding to DAT, probably consisting of a conformational change in the transporter protein. The mechanism exhibited by GBR12909 appears to be similar to the mechanism of PE2I that has been reported earlier and also confirms previous observations for GBR12783 made by Do-Rego and co-workers using dopamine uptake data. It appears that the isomerization phenomenon previously described for PE2I is not limited to tropane-based DAT inhibitors, but is, in fact, a general property of dopamine transporter protein, similar to "isomerization" process reported previously for G-protein coupled receptors. The rapid first step of association of the GBR 12909 is characterized by the equilibrium constant K(L)=34+/-11nM and the second slow step by k(i)=0.033+/-0.005s(-1).     

Synthesis and dopamine transporter affinity of chiral 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(2-hydroxypropyl)piperazines as potential cocaine abuse therapeutic agents

A series of optically pure phenyl-and non-phenyl-substituted 1-[2-[bis(4-fluorophenyl)methoxy]ethyl]-4-(2-hydroxypropyl)piperazines was synthesized and their binding affinity for dopamine transporter (DAT) was investigated. The analogues with a hydroxyl group in the S configuration were more selective for the DAT over the serotonin transporter (SERT) than the corresponding R enantiomers. Compound (+)-11 showed high affinity and selectivity for DAT over the SERT and, therefore, is a potential candidate for the development of a long-acting cocaine abuse therapeutic agent.     

3-{2-[Bis-(4-fluorophenyl)methoxy]ethyl}-6-substituted-3,6-diazabicyclo[3.1.1]heptanes as novel potent dopamine uptake inhibitors

A series of analogues 2a-i related to 3-{2-[bis-(4-fluorophenyl)methoxy]ethyl}-8-(1H-indol-2-ylmethyl)-3,8-diazabicyclo[3.2.1]octane (1) in which the 3,8-diazabicyclo[3.2.1]octane core was replaced by 3,6-diazabicyclo[3.1.1]heptane ring system has been synthesized and evaluated for their ability to inhibit DA reuptake into striatal nerve endings (synaptosomes). Biological data showed that compound 2a, the closest analogue of lead 1, possessed an increased reuptake inhibition activity over 1 (2a, K(i)=5.5 nM). Replacement of the indole ring with bioisosteric aromatic rings--benzothiophene (2b), benzofurane (2c), or indene (2d)--resulted, with the exception of 2d, in a double digit nanomolar activity. Changing the indenyl moiety of 2d with simplified aryl groups led to compounds 2e-h which displayed a similar or slightly decreased activity with respect to the ground term. Naphthalene derivative (2i) demonstrated a weaker activity than aromatic analogues.     

Structure-activity relationships of substituted N-benzyl piperidines in the GBR series: Synthesis of 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(2-trifluoromethylbenzyl)piperidine, an allosteric modulator of the serotonin transporter

A series of 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-(substituted benzyl) piperidines with substituents at the ortho and meta positions in the aromatic ring of the N-benzyl side chain were synthesized and their affinities and selectivities for the dopamine transporter (DAT), serotonin transporter (SERT), and norepinephrine transporter (NET) were determined. One analogue, 4-(2-(bis(4-fluorophenyl)methoxy)ethyl)-1-(2-trifluoromethylbenzyl)piperidine (the C(2)-trifluoromethyl substituted compound), has been found to act as an allosteric modulator of hSERT binding and function. It had little affinity for any of the transporters. Several compounds showed affinity for the DAT in the low nanomolar range and displayed a broad range of SERT/DAT selectivity ratios and very little affinity for the NET. The pharmacological tools provided by the availability of compounds with varying transporter affinity and selectivity could be used to obtain additional information about the properties a compound should have to act as a useful pharmacotherapeutic agent for cocaine addiction and help unravel the pharmacological mechanisms relevant to stimulant abuse.     

Enantioselective synthesis of S-(+)-2beta-carboalkoxy-3alpha-[bis(4-fluorophenyl)methoxy]tropanes as novel probes for the dopamine transporter

Synthesis of a series of pure S-(+)-2beta-carboalkoxy-3alpha-[bis(4-fluorophenyl)methoxy]tropanes (>99% ee) was achieved by employing a chiral amine-induced asymmetric reaction of tropinone with methyl cyanoformate as the key step. In this series, all of the S-(+)-enantiomers were 2-fold more potent than their racemic mixtures and all displayed high-affinity binding for DAT (K(i)=13-40 nM). These data support previous findings of significant divergence in structural requirements for high-affinity DAT binding among tropane-based inhibitors. Furthermore, the 2-substituent in the 3alpha-[bis(4-fluorophenyl)methoxy]tropane series is well tolerated at the DAT but not at SERT (K(i)=690-2040 nM), or muscarinic M(1) receptors (K(i)=133-4380 nM) resulting in highly selective DAT ligands that may provide new leads toward a cocaine-abuse therapeutic.     

In vitro and in vivo pharmacological profile of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl] ethyl] piperidine (NRA0161)

Atypical antipsychotic properties of 4-(4-fluorobenzylidene)-1-[2-[5-(4-fluorophenyl)-1H-pyrazol-4-yl]ethyl] piperidine (NRA0161) were investigated by in vitro receptor affinities, in vivo receptor occupancies and findings were compared with those of risperidone and haloperidol in rodent behavioral studies. In in vitro receptor binding studies, NRA0161 has a high affinity for human cloned dopamine D(4) and 5-HT(2A) receptor with Ki values of 1.00 and 2.52 nM, respectively. NRA0161 had a relatively high affinity for the alpha(1) adrenoceptor (Ki; 10.44 nM) and a low affinity for the dopamine D(2) receptor (Ki; 95.80 nM). In in vivo receptor binding studies, NRA0161 highly occupied the 5-HT(2A) receptor in rat frontal cortex. In contrast, NRA0161 did not occupy the striatal D(2) receptor. In behavioral studies, NRA0161, risperidone and haloperidol antagonized the locomotor hyperactivity in mice, as induced by methamphetamine (MAP). At a higher dosage, NRA0161, risperidone and haloperidol dose-dependently antagonized the MAP-induced stereotyped behavior in mice and NRA0161 dose-dependently and significantly induced catalepsy in rats. The ED(50) value in inhibiting the MAP-induced locomotor hyperactivity was 30 times lower than that inhibiting the MAP-induced stereotyped behavior and 50 times lower than that which induced catalepsy.These findings suggest that NRA0161 may have atypical antipsychotic activities yet without producing extrapyramidal side effects.     

Design, synthesis, and characterization of a novel, 4-[2-(diphenylmethoxy)ethyl]-1-benzyl piperidine-based, dopamine transporter photoaffinity label

The dopamine transporter (DAT) has been implicated strongly in cocaine's reinforcing effects. Many derivatives of piperidine analogs of GBR 12909 have been developed and were found to be quite potent and selective for the DAT. In this regard, most of these derivatives were found to be much more selective for the DAT than conventional GBR compounds e.g. GBR 12909 when their selectivity was compared with the serotonin transporter (SERT). A brief structure-activity relationship (SAR) study has been carried out in the development of a novel photoaffinity ligand which illustrated the effect of the presence of a sterically bulky iodine atom next to the azido group in activity and selectivity for the DAT. This SAR study also led to the development of the compound 4 which is one of the most potent and selective blockers for the DAT known today. The photoaffinity ligand [125I]AD-96-129 was incorporated into the DAT molecule as was demonstrated by immunoprecipitation with serum 16 which is specific for DAT. This photolabeling was antagonized by DAT-specific blockers and was unaffected by specific SERT and norepinephrine transporter (NET) blockers indicating interaction of this novel ligand with the DAT.     

1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

Evidence for the Sequential Formation of Two Complexes Between an Uptake Inhibitor, GBR 12783 [1-[2-(Diphenylmethoxy)ethyl]-4-(3-Phenyl-2-Propenyl)piperazine], and the Neuronal Transporter of Dopamine

Abstract : Incubation of a crude synaptosomal fraction from rat striatum with GBR 12783 at 37°C produced an inhibition of the specific uptake of [³H]dopamine that increased with time. The inhibition increased when GBR 12783 was present during preincubation and incubation (IC₅₀ = 1.85 ± 0.1 nM) instead of incubation alone (IC₅₀ = 25 ± 3.5 nM). Time-course studies of uptake inhibition demonstrated that a first collision transporter-inhibitor complex (TI) was formed immediately after addition of GBR 12783 so that the initial uptake velocity (V_o) decreased for increasing concentrations of inhibitor (K_i≥ 20 nM). TI slowly isomerized to a more stable complex TI^* (K^*_i≤ 5 nM) with a value of t_1/2 = 20-270 s. Fits of data to model 2 in which the steady-state uptake (V_S) is set to zero were generally preferred, suggesting that formation of TI^* could tend to irreversibility, as a consequence of a very low reverse isomerization. As expected, k, V_o, and V_S tended to steady-state values in an asymptotic manner for high concentrations of GBR 12783. GBR 12783 at 2.5 nM produced a mixed inhibition of the uptake, with an increase in K_M and a decrease in V_max ; these effects were improved for 10 nM GBR 12783 and at 20°C. These results are discussed in relation to previous data concerning [³H]GBR 12783 binding. The present work gives the first experimental demonstration that dopamine uptake blockers can act according to a two-step mechanism of inhibition ; this is of great interest, because these inhibitors can oppose the effects of cocaine or amphetamine on the transporter according to a reaction that is partly nondependent on the concentration of the abused agent.     

Efficient asymmetric syntheses, determination of absolute configurations and biological activities of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine as a novel potent dopamine uptake inhibitor in the central nervous system

An efficient asymmetric synthesis of the chiral N-(3-chloro-2-hydroxypropyl)anilines (2a and 2b) was achieved through the regioselective ring-opening reaction of chiral epichlorohydrin with aniline. This was applied to an asymmetric synthesis of the enantiomers of 1-[4,4-bis(4-fluorophenyl)butyl]-4-[2-hydroxy-3-(phenylamino)propyl]piperazine 1 as a novel potent dopamine uptake inhibitor. Both enantiomers as trihydrochlorides, 4a.3HCl and 4b.3HCl, could be synthesized in good total yields and optical purities of 100% ee in three steps synthesis, respectively. The absolute configurations of 4a.3HCl and 4b.3HCl were determined using the modified Mosher's method with the related compounds, the intermediates (2a and 2b) and the free bases (4a and 4b). The analytical results indicated that 4a.3HCl and 4b.3HCl have the (S)- and (R)-configuration, respectively, and a series of reactions to provide them proceeded without the apparent influence on the stereochemistry at the chiral centers. In in vitro pharmacological evaluations, 4a.3HCl and 4b.3HCl showed potent dopamine transporter binding affinities, high dopamine, moderate serotonin, and weak norepinephrine uptake inhibitory activities, and 4a.3HCl exhibited a more potent and selective dopamine uptake inhibition over the serotonin or norepinephrine uptake inhibition as compared with 4b.3HCl. An ex vivo evaluation revealed that the oral administrations of both enantiomers at a dose of 30 mg/kg in rats displayed apparent dopamine uptake inhibitory activities and 4a.3HCl had a stronger tendency to inhibit dopamine uptake compared with 4b.3HCl.     

Antipsychotic profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562) in rats

The antipsychotic profile of 5-[2-[4-(6-fluoro-1H-indole-3-yl)piperidin-1-yl]ethyl]-4-(4-fluorophenyl)thiazole-2-carboxylic acid amide (NRA0562) was investigated using the conditioned avoidance test in rats. NRA0562 is a putative "atypical" antipsychotic agent with moderate to high affinities for dopamine D(1), D(2), D(4), 5-hydroxytryptamine(2A) receptors and alpha(1) adrenoceptor. NRA0562 (1 and 3 mg/kg, p.o.) dose-dependently and significantly impaired the conditioned avoidance response. Likewise other atypical antipsychotics such as risperidone (1 and 3 mg/kg, p.o.) and clozapine (100 mg/kg, p.o.) dose-dependently and significantly impaired the conditioned avoidance response in rats. In addition, typical antipsychotics, haloperidol (1 and 3 mg/kg, p.o.) potently impaired the conditioned avoidance response.These results suggest that antipsychotic profile of NRA0562 is consistent with profiles of clozapine or risperidone and may be considered an atypical antipsychotic agent.     

Identification ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid with a metabolic intermediate betweenS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine andS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid

Summary S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptopyruvic acid (I) was chemically synthesized in 15% yield by incubating a reaction mixture oftrans-urocanic acid and 3-fold excess of 3-mercaptopyruvic acid at 45°C for 6 days. The synthesized compound was characterized by fast-atom-bombardment mass spectrometry and high-voltage paper electrophoresis. CompoundI was identified with a product of an enzymatic reaction ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-l-cysteine (II) with rat liver homogenate in a phosphate buffer, pH 7.4. CompoundI was degraded toS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]-3-mercaptolactic acid (III), a compound previously found in human urine [Kinuta et al. (1994) Biochem J 297: 475–478], by incubation with rat liver homogenate. From these results, we suggest that compoundI is a metabolic intermediate for the formation of compoundIII from compoundII. The present pathway follows a formation of compoundII fromS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl] gluthathione [Kinuta et al. (1993) Biochim Biophys Acta 1157: 192–198], a proposed metabolite ofl-histidine.     

Synthesis and in vitro antimicrobial studies of medicinally important novel N-alkyl and N-sulfonyl derivatives of 1-[bis(4-fluorophenyl)-methyl]piperazine

A series of novel substituted 1-[bis(4-fluorophenyl)-methyl]piperazine derivatives (4a-g) and (5h-m) have been synthesized. The synthesized compounds were characterized by IR and 1H NMR. All the synthesized compounds were evaluated in vitro for their efficacy as antimicrobial agents against representative strains of Gram-positive (Staphylococcus aureus ATCC 25953, Streptococcus pneumoniae ATCC 49619, Bacillus cereus 11778, and Bacillus subtilis 6051) and Gram-negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 2853, Proteus vulgaris ATCC 2853, and Salmonella typhi ATCC 9484) by paper disc diffusion and microdilution methods. Among the newly synthesized compounds 4e, 5l, and 5m showed potent antimicrobial activities, when compared to the standard drug.     

Discovery of 6-[5-(4-fluorophenyl)-3-methyl-pyrazol-4-yl]-benzoxazin-3-one derivatives as novel selective nonsteroidal mineralocorticoid receptor antagonists

In the course of our study on selective nonsteroidal mineralocorticoid receptor (MR) antagonists, a series of novel benzoxazine derivatives possessing an azole ring as the core scaffold was designed for the purpose of attenuating the partial agonistic activity of the previously reported dihydropyrrol-2-one derivatives. Screening of alternative azole rings identified 1,3-dimethyl pyrazole 6a as a lead compound with reduced partial agonistic activity. Subsequent replacement of the 1-methyl group of the pyrazole ring with larger lipophilic side chains or polar side chains targeting Arg817 and Gln776 increased MR binding activity while maintaining the agonistic response at the lower level. Among these compounds, 6-[1-(2,2-difluoro-3-hydroxypropyl)-5-(4-fluorophenyl)-3-methyl-1H-pyrazol-4-yl]-2H-1,4-benzoxazin-3(4H)-one (37a) showed highly potent in vitro activity, high selectivity versus other steroid hormone receptors, and good pharmacokinetic profiles. Oral administration of 37a in deoxycorticosterone acetate-salt hypertensive rats showed a significant blood pressure-lowering effect with no signs of antiandrogenic effects.     

[3-cis-3,5-Dimethyl-(1-piperazinyl)alkyl]-bis-(4′-fluorophenyl)amine analogues as novel probes for the dopamine transporter

In a continuing effort to identify novel probes with which to study the dopamine transporter (DAT), we discovered that the σ receptor antagonist, rimcazole, binds with moderate affinity (K_i=224 nM) to the DAT. The results from previous SAR studies suggested that substitution of the carbazole ring system of rimcazole with bis-(4′-fluorophenyl)amine might improve binding affinity and selectivity for the DAT. Thus, a novel series of [3-cis-3,5-dimethyl-(1-piperazinyl)alkyl]bis-(4′-fluorophenyl)amines were synthesized. The most potent compound in this series (9b) displaced [³H]WIN 35,428 binding in rat caudate-putamen (K_i=17.6 nM) with comparable affinity to GBR 12909. Despite high-affinity binding at DAT, and structural similarity to GBR 12909, preliminary studies suggest 9b behaves more like rimcazole than GBR 12909 and does not demonstrate cocaine-like psychostimulant behavior in mice.     

(S,E)-N-[1-(3-heteroarylphenyl)ethyl]-3-(2-fluorophenyl)acrylamides: synthesis and KCNQ2 potassium channel opener activity

Replacement of the morpholinyl moiety in (S,E)-N-[1-(3-morpholinophenyl)ethyl]-3-phenylacrylamide (1) with heteroaryl groups led to the identification of (S,E)-N-1-[3-(6-fluoropyridin-3-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (5) as a potent KCNQ2 potassium channel opener. Among this series of heteroaryl substituted acrylamides, (S,E)-N-1-[3-(1H-pyrazol-1-yl)phenyl]ethyl-3-(2-fluorophenyl)acrylamide (9) exhibits balanced potency and efficacy. The syntheses and the KCNQ2 opener activity of this series of acrylamides are described.     

Rational design, synthesis and evaluation of (6aR( *),11bS( *))-1-(4-fluorophenyl)-4-{7-[4-(4-fluorophenyl)-4-oxobutyl]1,2,3,4,6,6a,7,11b,12,12a(RS)-decahydropyrazino[2',1':6,1]pyrido[3,4-b]indol-2-yl}-butan-1-one as a potential neuroleptic agent

In our pursuit to prepare a potent antipsychotic compound, a novel 1,2,3,4,6,6a,7,11b,12,12a-decahydropyrazino[2',1':6,1]pyrido[3,4-b]indole derivative was synthesized which incorporates the butyrophenone substructure twice. This molecule has shown D(1), D(2) and 5-HT(2A) receptor blocking activity where the ratio pK(i) (5-HT(2A)) to pK(i) (D(2)) is 1.42 better than risperidone (1.15). It blocks amphetamine induced hyperactivity/stereotypy and secondary conditioned avoidance responses in rodents at lower doses than those required for the neuroleptic drugs haloperidol and centbutindole (biriperone).     

6-[2-(4-Aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones: dual-acting 5-HT1 receptor antagonists and serotonin reuptake inhibitors

Investigation of a series 6-[2-(4-aryl-1-piperazinyl)ethyl]-2H-1,4-benzoxazin-3(4H)-ones has led to the discovery of potent 5-HT(1A/1B/1D) receptor antagonists with and without additional SerT affinity. Modulation of the different target activities gave compounds with a range of profiles suitable for further in vivo characterization.     

2'-O-[2-(methylthio)ethyl]-modified oligonucleotide: an analogue of 2'-O-[2-(methoxy)-ethyl]-modified oligonucleotide with improved protein binding properties and high binding affinity to target RNA

A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.     

Synthesis, cytostatic, and antiviral activity of novel 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, 6-[2-(alkylsulfanyl)ethyl]-, and 6-[2-(dialkylamino)vinyl]purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-[2-(dialkylamino)vinyl]-, 6-[2-(dialkylamino)ethyl]-, 6-(2-alkoxyethyl)-, and 6-[2-(alkylsulfanyl)ethyl]purine nucleosides (35 examples of both ribo- and 2'-deoxyribonucleosides) was developed. The key transformations involved conjugate nucleophilic additions of amines, alcoholates, or thiolates to Tol-protected 6-alkylylpurine or 6-vinylpurine nucleosides. 6-[(2-Dialkylamino)vinyl]- and some 6-[(2-dialkylamino)ethyl]purine ribonucleosides exerted significant cytostatic effects and some anti-HCV activity with low selectivity.