Introduction

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Several new tropane monoamine uptake inhibitors have been developed in an effort to further characterize the cocaine binding sites in the brain and identify the structural requirements that contribute to the specificity and potency of the respective transport processes (Davies et al., 1993, 1994). These monoamine uptake inhibitors were prepared using a unique synthetic strategy that uses the reaction of vinylcarbenoids with pyrroles to yield novel 2-methyl ketone and 2-ethyl ketone tropane compounds (Davies et al., 1991, 1993). These compounds are more resistant to esterase degradation and have been shown to have a longer duration of action in vivo (Hemby et al., 1995). Three compounds prepared using this strategy are WF-31, WF-50 and WF-11 (PTT) (fig. 1). As reported previously, WF-11 binds with high affinity to the dopamine transporter and with lower affinity for the serotonin and norepinephrine transporters (table 1; Davies et al., 1996). In contrast, WF-31 binds with higher affinity to the serotonin transporter than to the dopamine or norepinephrine transporters (Bennett et al., 1995; Davies et al., 1996). Demethylation of WF-31 yields the compound WF-50, which binds with even higher affinity to the serotonin transporter while binding with low affinity to the dopamine and norepinephrine transporters. The presence of an isopropyl vs. a methyl constituent at the para position on the 3-aryl ring results in a 50-fold decrease in potency at the dopamine transporter and a 5-fold increase in potency at the serotonin transporter (WF-31 vs. WF-11). The elimination of the ester group between the aryl group at the 3 position of WF-31, WF-50 and WF-11 and the tropane structure result theoretically in greater metabolic stability than related tropanes (i.e., cocaine), as evidenced by the increased duration of behavioral and neurochemical actions (Hemby et al., 1995).

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structures of WF-31 (A), WF-50 (B), fluoxetine (C), WF-11 (D) and GBR 12909 (E). Note the single structural difference between WF-31 and WF-11 is the substitution of the isopropyl for the methyl constituent at the para position on the phenyl ring, significantly increasing serotonin transporter selectivity, and the single structural difference between WF-31 and WF-50 is the N-demethylation at the 8 position.

View this table: [in this window] [in a new window]   TABLE 1 Ligand affinities for dopamine, serotonin and norepinephrine transporters Potencies of all tropane compounds in displacing [125I]RTI-55 binding in rat striatal membranes are expressed as IC50 values due to the multiphasic nature of tropane binding to striatal membranes. The potencies of these compounds in displacing [3H]paroxetine in rat frontal cortex membranes and [3H]nisoxetine in rat whole brain (minus brainstem and cerebellum) are expressed as Ki. [Binding data have been presented previously (Davies et al., 1996)].

Dopamine and serotonin are thought to be involved in a variety of psychiatric illnesses, including substance abuse (Dackis and Gold, 1985; for a review, see Hemby et al., 1997) and depression (Asberg and Martensson, 1993; Brown and Gershon, 1993). Sites on the dopamine and serotonin transporters have become targets for the development of new compounds to treat these disorders. Due to the high affinity and selectivity of the aforementioned novel tropanes at the dopamine and serotonin transporters, the efficacy of these compounds was assessed as potential antidepressant agents in the FST in the present study.

The FST, developed by Porsolt et al. (1978a) is one of the more widely used behavioral screens for antidepressant effects (for a review, see Porsolt and Lenegre, 1992). This procedure measures the development of behavioral immobility after a rodent has been placed in a tank of water for a 5-min test session. The development of immobility is facilitated by a 15-min pretest session 24 hr earlier. Antidepressant drugs administered between the pretest and test sessions decrease the duration of behavioral immobility in the FST, the principal dependent measure. The FST is sensitive to all major classes of antidepressant drugs, including tricyclic antidepressants, monoamine oxidase inhibitors and atypical antidepressants (for a review, see Borsini and Meli, 1988), and can distinguish antidepressants from compounds of other therapeutic classes. Recently, a scoring system for the FST was introduced that uses a behavior sampling procedure to quantify the frequency of behaviors (swimming and climbing) and immobility during the test session (Detke et al., 1995). This scoring system was shown to be sensitive for detecting a behavioral pattern for serotonin uptake inhibitors in the FST, which decreased immobility and increased swimming. The system also distinguished serotonin from norepinephrine uptake inhibitors, which decreased immobility and increased climbing but not swimming.

The present study examined the potential "antidepressant-like" effects of WF-11, WF-31 and WF-50 in the FST because of their affinity for monoamine transporter sites. These novel tropane compounds were compared with fluoxetine and GBR 12909 because of their selective inhibition of the neuronal uptake of serotonin and dopamine, respectively. The effects of these compounds on motor activity were also assessed to determine whether drugs that decreased immobility in the FST produced corresponding increases in motor activity.

	Materials and Methods

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Subjects

Adult male Sprague-Dawley rats (90-150 days old; 275-350 g; Sasco, Inc., Lincoln, NE) were housed two per cage and were maintained on a 12-hr reversed light/dark cycle (lights off, 7:00 a.m.) with food and water available ad libitum in the home cage. Rats were tested in a separate room under dim lighting during the dark phase of the cycle. This strain is more sensitive than some others to the effects of antidepressants in the FST (Porsolt et al., 1978b). All research was conducted according to the Guide for the Care and Use of Laboratory Animals as promulgated by the National Institutes of Health and approved by the Bowman Gray Animal Care and Use Committee.

FST Methods and Apparatus

Rats were handled for 10 min each for 2 days before initiation of experimentation. Swim sessions were conducted by placing rats in a 13.2-liter Pyrex cylindrical tank (22 × 46 cm; Fisher Scientific) containing water (at ~25°C) for 15 min. The water depth was 35 cm from the bottom, a depth at which rats could not touch the bottom with their tails. The initial 15-min swim pretest was followed 24 hr later by a 5-min test. Rats were removed from the tanks after each swim session, towel dried, placed in a heated cage for 15 min and then returned to their home cages. After the pretest session, rats were assigned randomly to groups to receive either saline (n = 26), propylene glycol/saline vehicle (n = 10), WF-31 (0.1, 0.3, 3.0 and 10.0 mg/kg; n = 10/dose), WF-50 (0.3, 3.0 and 10.0 mg/kg; n = 8, 10 and 10/dose, respectively), fluoxetine (0.3, 3.0 and 10.0 mg/kg; n = 10/dose), WF-11 (0.3, 1.0 and 3.0 mg/kg; n = 10/dose) or GBR 12909 (3.0, 10.0 and 30.0 mg/kg; n = 10/dose).

Rats received three injections between the pretest and the test swim. At 15 min after the initial swim, rats were injected with saline, vehicle or a dose of one of the five compounds before being returned to the home cage. The following day, rats were injected with saline, vehicle or drug 5 hr and 1 hr before the 5-min test swim. Thus, each rat received three injections before the swim test on the second day.

Scoring of the FST

For scoring purposes, the test session was divided into 60 five-sec bins, and each bin was assigned a score of immobility, swimming or climbing, according to the scale of Detke et al. (1995). The test sessions were videotaped and the tapes were later scored by an observer who was unaware of the experimental conditions.

Locomotor Activity and Stereotypy Rating

Several reports suggest that some compounds that potentiate monoaminergic transmission may yield false-positive effects in the FST (for a review, see Borsini and Meli, 1988). These potential false-positives can be detected by evaluating their effects on locomotor activity. For this reason, the effects of these compounds on locomotor activity were evaluated after an identical treatment regimen used in the FST. Subjects were randomly assigned to groups and were administered intraperitoneal saline (n = 8), propylene glycol/saline vehicle (n = 8), WF-31 (10.0 mg/kg; n = 8), WF-50 (10.0 mg/kg; n = 8), fluoxetine (10.0 mg/kg; n = 8), WF-11 (1.0 and 3.0 mg/kg; n = 8/dose) or GBR 12909 (3.0 and 30.0 mg/kg; n = 8/dose). For each compound, the dose that produced the largest effect in the FST was selected initially. If those doses stimulated motor activity, then the lowest effective dose in the FST was evaluated for a particular compound.

The locomotor chambers were clear, square Plexiglas boxes (33.2 × 33.2 × 29.9 cm) placed inside an activity monitor (Med Associates, St. Albans, VT). Each apparatus was equipped with 16 optical sensors spaced 2.5 cm apart on two perpendicular sides located 3.8 cm above the floor. A second set of optical sensors was located 17 cm above the floor and was used to record vertical activity. Sequential beam disruptions were required to register locomotor activity or vertical activity. Successive breaks of the same beam were recorded as repetitive activity. Activity was recorded by an IBM-compatible computer with a data collection program (Med Associates). On the first day, rats were placed in locomotor chambers for 15 min, analogous to the pretest in the FST experiment. The drug treatment regimen was identical to the one used for the FST. On the following day, subjects were placed in the locomotor chambers and activity was recorded for 5 min, analogous to the test day in the FST experiment. Stereotypic behaviors were rated immediately after the test session by an observer who was unaware of the experimental conditions. The observer rated the behavior of each subject in 1-min intervals for 5 min for stereotypy according to the scale of Ellinwood and Balster (1974).

Drugs

WF-11, WF-31 and WF-50 were prepared according to established procedures (Davies et al., 1994, 1996). The fumarate salts were prepared for the in vivo studies in the following manner.

WF-11 fumarate salt (1:1 ratio). Fumaric acid (1.12 g, 9.65 mmol) was added to a solution of the tropane (2.624 g, 9.67 mmol) in 2-propanol (50 ml). All solids were dissolved by heating the mixture carefully. The solvent was removed thoroughly in vacuo. The residue was triturated with dry ether, and the solid thus obtained was collected by filtration (3.064 g, 81% yield), m.p. 142° to 144°C: ¹H NMR (D₂O)  7.23 (d, J = 8.1 Hz, 2 H), 7.15 (d, J = 8.1 Hz, 2 H), 6.67 (s, 2 H), 4.07 (br. s, 1 H), 4.04 (br. s, 1 H), 3.56 (ddd, J = 13.6, 5.9, 5.2 Hz, 1 H), 3.46 (dd, J = 5.3, 1.5 Hz, 1 H), 2.82 (s, 3H), 2.65 (br. dd, J = 13.9, 13.8 Hz, 1 H), 2.49-2.13 (m, 5 H), 2.3 (s, 3H), 1.97 (ddd, J = 15.0, 3.8, 3.8 Hz, 1 H), 1.43 (dq, J = 19.7, 7.0 Hz, 1 H), 0.59 (t, J = 7.0 Hz, 3H). Analysis calculated for C₂₀H₂₅NO₃·C₂H₄O₂ 0.75H₂O: C, 65.90; H, 7.67; N, 3.49. Found: C, 66.03; H, 7.68; N, 3.47.

WF-31 fumarate salt (1:1 ratio). Fumaric acid (0.3541 g, 3.05 mmol) was added to a solution of WF-31 (0.9122 g, 3.05 mmol) in 2-propanol (25 ml). All solids were dissolved by heating the mixture carefully. The solvent was removed thoroughly in vacuo. The residue was triturated with dry ether, and the solid thus obtained was collected by filtration (1.192 g, 94% yield), m.p. 130° to 132°C: ¹H NMR (D₂O)  7.31 (d, J = 8.2 Hz, 2 H), 7.19 (d, J = 8.2 Hz, 2 H), 6.74 (s, 2 H), 4.07 (br. m, 1 H), 4.04 (br. m, 1 H), 3.57 (ddd, J = 13.3, 5.5, 5.5 Hz, 1 H), 3.46 (dd, J = 5.3, 1.2 Hz, 1 H), 2.87 (septet, J =6.9 Hz, 1 H), 2.67 (br. dd, J =13.5, 13.5 Hz, 1 H), 2.45-1.60 (m, 6 H), 1.40 (dq, J = 19.7, 7.0 Hz, 1 H), 1.19 (d, J = 6.9 Hz, 6 H), 0.58 (t, J = 7.0 Hz, 3 H). Analysis calculated for C₂₀H₂₈NO₃·C₂H₄O₂ 0.25H₂O: C, 68.63; H, 8.03; N, 3.33. Found: C, 68.53; H, 8.01; N, 3.29.

WF-50 fumarate salt (1:1 ratio). Fumaric acid (0.227 g, 1.96 mmol, 1.0 equiv.) was added to a solution of WF-50 (0.559 g, 1.96 mmol) in 2-propanol (30 ml). All solids were dissolved by heating the mixture carefully. The solvent was removed thoroughly in vacuo. The residue was triturated with dry ether, and the solid thus obtained was collected by filtration and washed with dry ether (0.577 g, 86% yield), m.p. 179° to 180°C: ¹H NMR (D₂O)  7.18 (d, J = 7.4 Hz, 2 H), 7.02 (d, J = 7.4 Hz, 2 H), 6.52 (s, 1 H), 4.10 (br. s, 1 H), 4.02 (br. s, 1 H), 3.40 (ddd, J = 13.2, 6.1, 5.4 Hz, 1 H), 3.21 (dd, J = 6.2, 1.8 Hz, 1 H), 2.70 (septet, J = 6.5 Hz, 1 H), 2.40 (ddd, J = 13.9, 13.9, 2.6 Hz, 1 H), 2.19-1.95 (m, 5 H), 1.79 (br. dd, J = 14.7, 14.7 Hz, 1 H), 1.21 (dq, J = 19.6, 7.0 Hz, 1 H), 1.02 (d, J = 7.0 Hz, 6 H), 0.70 (t, J = 7.0 Hz, 3 H).

Statistical Analysis

FST data were analyzed using a one-way ANOVA with dose as the main effect. Motor activity data were analyzed using Student's t test for WF-31, WF-50 and fluoxetine and by one-way ANOVA for WF-11 and GBR 12909. Drug effects were compared with saline (fluoxetine, WF-11, WF-31 and WF-50) or propylene glycol/saline (GBR 12909). Post hoc analyses were conducted using Dunnett's test (Neter et al., 1985), and results were considered statistically significant when P < .05.

	Results

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FST. All five compounds tested decreased immobility in the FST compared with vehicle controls. WF-31 produced a dose-dependent decrease in immobility [F(4,49) = 14.32, P < .0001) and a corresponding dose-dependent increase in swimming [F(4,49) = 25.40, P < .0001] without significantly affecting the frequency of climbing [F(4,49) = .44, P = NS; fig. 2]. WF-50, the demethylated analog of WF-31, decreased immobility [F(4,51) = 3.86, P < .0086] and increased swimming [F(4,51) = 4.60, P < .0032] without significantly affecting climbing [F(4,51) = .81, P = NS; fig. 3]. Fluoxetine administration produced a pattern similar to that of WF-31 (fig. 4): a dose-dependent decrease in immobility [F(3, 45) = 6.46, P < .0011] with a corresponding increase in swimming [F(3, 45) = 10.16, P < .0001]. Climbing was not significantly affected by fluoxetine administration [F(3, 45) = .66, P = NS].

View larger version (46K): [in this window] [in a new window]   Fig. 2.   The effects of treatment with WF-31 in the FST. Bars represent the mean ± S.E.M. counts for the test session. ANOVA revealed a significant effect of dose on immobility [F(4,49) = 14.32, P < .0001] and swimming [F(4,49) = 25.40, P < .0001]. *P < .05 from saline, #P < .05 from preceding dose.

View larger version (45K): [in this window] [in a new window]   Fig. 3.   The effects of treatment with WF-50 in the FST. Bars represent the mean ± S.E.M. counts for the test session. ANOVA revealed a significant effect of dose on immobility [F(4,51) = 3.86, P < .0086] and swimming [F(4,51) = 4.60, P < .0032]. *P < .05 from saline, #P < .05 from preceding dose.

View larger version (34K): [in this window] [in a new window]   Fig. 4.   The effects of treatment with fluoxetine in the FST. Bars represent the mean ± S.E.M. (n = 10/dose). ANOVA revealed a significant effect of dose on immobility [F(3,45) = 6.46, P < .0001] and swimming [F(3,45) = 10.16, P < .0001]. *P < .05 from saline, #P < .05 from preceding dose.

View larger version (35K): [in this window] [in a new window]   Fig. 5.   The effects of treatment with WF-11 in the FST. Bars represent the mean ± S.E.M. (n = 10/dose). ANOVA revealed a significant effect of dose on immobility [F(3, 45) = 57.38, P < .0001], swimming [F(3, 45) = 14.03, P < .0001] and climbing [F(3,45) = 23.32, P < .0001]. *P < .05 from saline, #P < .05 from preceding dose.

View larger version (37K): [in this window] [in a new window]   Fig. 6.   The effects of treatment with GBR 12909 in the FST. Bars represent the mean ± S.E.M. (n = 10/dose). ANOVA revealed a significant effect of dose on immobility [F(3,39) = 71.85, P < .0001] and climbing [F(3,39) = 11.76, P < .0001]. *P < .05 from saline, #P < .05 from preceding dose.

Locomotor activity and stereotypy rating. Several doses that were effective in decreasing immobility were assessed for their ability to stimulate locomotor activity to determine whether decreases in immobility in the FST corresponded with increases in general activity (table 2). At the doses tested, neither WF-31, WF-50 nor fluoxetine increased ambulatory or vertical activity or stereotypic rating. Fluoxetine decreased locomotor activity (t = 2.46, df = 14, P = .027) and vertical activity (t = 2.30, df = 14, P = .037) compared with saline. As previously demonstrated, WF-11 increased locomotor activity [F(2,23) = 5.03, P = .016] and ratings of stereotypic behavior [F(2,23) = 38.83, P < .0001; Hemby et al., 1995]. Post hoc analysis revealed 1.0 mg/kg significantly stimulated locomotor activity compared with saline, whereas 3.0 mg/kg significantly increased repetitive activity [F(2,23) = 9.16, P = .0014]. Both doses of WF-11 increased stereotypy rating. After the administration of GBR 12909, neither locomotor activity nor vertical activity was significantly different from propylene glycol/saline controls at either of the doses tested. However, there was a significant effect of drug on repetitive activity [F(2,23) = 44.82, P < .0001] and stereotypy rating [F(2,23) = 67.76, P < .0001]. Post hoc analysis revealed 30.0 mg/kg GBR 12909 significantly increased repetitive activity, whereas both 3.0 and 30.0 mg/kg increased stereotypy rating scores.

	Discussion

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The effects of the novel tropanes selective for the serotonin (WF-31 and WF-50) and dopamine (WF-11) transporters were compared with fluoxetine and GBR 12909 in the FST. All of the compounds tested decreased immobility in the FST, albeit to varying degrees. Moreover, administration of the selective serotonin uptake inhibitors WF-31, WF-50 and fluoxetine produced a behavioral pattern qualitatively different from the pattern observed after administration of the dopamine uptake inhibitors WF-11 and GBR 12909. WF-31, WF-50 and fluoxetine increased swimming while not affecting climbing, whereas WF-11 increased climbing and swimming and GBR 12909 increased climbing while not affecting swimming. In addition, the selective dopamine uptake inhibitors WF-11 and GBR 12909 administration increased indices of motor activity and stereotypy, an effect not observed for the selective serotonin uptake inhibitors. The effect of the latter may have been due to increased general activity observed after identical treatment regimens. The possibility remains that the doses selected for the locomotor activity tests may not have included the dose(s) produced the greatest amount of stimulation for each drug.

WF-31 and WF-50 induced patterns of behavioral effects similar to fluoxetine in the FST (decreased immobility, increased swimming and no change in climbing). This pattern has been reported previously after subcutaneous administration of the serotonin uptake inhibitors fluoxetine, paroxetine and sertraline (Detke et al., 1995). It should be noted that others have reported that fluoxetine is a false-negative in the FST (Borsini, 1995). The discrepancy may be due in part to the sensitivity of the behavioral scoring system and/or the specific parameters for the FST (cylinder diameter, water depth, etc.) used in the present study. Comparison of the serotonin uptake inhibitors WF-31, WF-50 and fluoxetine revealed both similarities and subtle differences among the compounds. WF-31 and fluoxetine were equipotent at decreasing immobility and increasing swimming, although the least effective dose for WF-31 (0.3 mg/kg) produced effects comparable with 3.0 mg/kg fluoxetine. These findings are interesting for two reasons. WF-31 is less potent than fluoxetine in inhibiting [³H]serotonin uptake in vitro and is less selective for the serotonin transporter, being ~20 times less potent at the serotonin vs. the dopamine transporters (Davies et al., 1996). These data suggest fluoxetine should have been more efficacious than WF-31 in the FST. Second, WF-50 is more potent than WF-31 in inhibiting [³H]serotonin uptake in vitro³ and is more selective for the serotonin transporter, being ~6 times more potent in binding to the serotonin vs. the dopamine transporter. However, WF-50 is 30 times less efficacious than WF-31 in the FST. Differences in in vivo potencies may be due to the duration of action of the parent compound or the metabolites of WF-31 and WF-50 inasmuch as they are less susceptible metabolism from plasma esterases due to the lack of ester linkage between the tropane and phenyl structures of these compounds. Experiments have been initiated to determine the duration of action of WF-31 on in vivo extracellular serotonin concentrations. In addition, metabolites of WF-31 and fluoxetine may potentiate serotonergic transmission and participate in the behavioral effects of these drugs. For example, fluoxetine is metabolized to norfluoxetine, an effective serotonin uptake inhibitor, which has a long half-life in rats (14 hr; Caccia et al., 1990). The relative efficacies of WF-31, WF-50 and their respective metabolites compared with fluoxetine and norfluoxetine warrant further study.

Apart from the similarity of effects in the FST, fluoxetine and WF-31 differ in other behavioral effects. At the doses tested, fluoxetine decreased locomotor and vertical activity, whereas neither WF-31 and WF-50 was significantly different from controls. Decreases in motor activity in laboratory animals after the administration of several serotonin uptake inhibitors have been reported (Detke et al., 1995) and appear to be a common feature of currently available compounds with this action. The doses of fluoxetine that were effective in the FST have been shown to decrease food maintained responding, whereas doses of WF-31 effective in the FST do not.⁴ These data suggest that although the potencies of WF-31, WF-50 and fluoxetine are similar in the FST, important differences exist between these compounds in their actions, especially for behaviors unrelated to their antidepressant effects. The lack of effect of WF-31 on both unconditioned and conditioned behaviors is a unique feature of this compound and may prove to be a desirable characteristic in the development of future antidepressant medications.

WF-11 and GBR 12909 differed from fluoxetine, the reference antidepressant, in several ways. Unlike fluoxetine, the decrease in immobility induced by GBR 12909 and WF-11 was accompanied by an increase in climbing for GBR 12909 and by an increase in climbing and swimming for WF-11. Increased climbing has been reported after the administration of desipramine and maprotiline, antidepressants that are selective inhibitors of noradrenergic uptake (Detke et al., 1995). However, amphetamine, a psychomotor stimulant and false-positive on many antidepressant tests, also increased climbing in the FST, and a secondary test of locomotor activity was necessary to distinguish amphetamine from the tricyclic antidepressants (Detke et al., 1995). Both WF-11 and GBR 12909 increased motor activity at the doses tested, whereas fluoxetine decreased motor activity. These findings lead to the consideration that WF-11 and GBR 12909 are false-positives in the FST because the decreases in immobility induced by WF-11 and GBR 12909 administration may be attributable to the motor-stimulating effects of these compounds and not a reflection of their potential antidepressant effects. However, WF-11 differed from GBR 12909 in that increases in both swimming and climbing behavior were observed. This dual-response pattern in the FST has also been observed after venlafaxine, an antidepressant drug that potently inhibits both norepinephrine and serotonin uptake.⁵ It is reasonable to speculate that potent effects of WF-11 at norepinephrine and dopamine uptake sites contribute to producing increases in climbing behavior and locomotor activity and that potent effects at serotonin uptake sites contribute to increased swimming behavior. Thus, the description of behaviors in the FST lends supports the suggestion that WF-11 produces antidepressant-like effects in addition to motor stimulation.

Over the past 20 years, several atypical antidepressant compounds have been developed. Some of the main advantages of these compounds in comparison to the tricyclics are that they are less toxic, exert fewer adverse side effects and are also efficacious in other clinical indications (Baldessarini, 1996). For example, fluoxetine is an equiefficacious antidepressant compared with imipramine but lacks the antimuscarinic side effects of that tricyclic (e.g., decreased gastrointestinal motility, tachycardia, dry mouth, and so on) and has proved to be useful in the treatment of panic disorder, obsessive-compulsive disorder and eating disorders. The development of tropanes using the synthesis strategy developed by Davies et al. (1991) may yield compounds with high affinity and selectivity for the respective monoamine transporter sites not previously available with existing synthesis strategies. One of the clinical uses of such compounds may be their activity as antidepressants. In addition, structural modifications may enhance or prolong the pharmacological action of these compounds (i.e., absence of the ester group). The development of selective serotonin uptake inhibitors using this synthesis strategy will also provide a new class of compounds for studying the functional roles of the serotonin in the brain.