Editorial Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1799
Editorial Neurotransmitter transporters can be divided into two types: the vesicular transporters mediating the uptake and storage of neurotransmitters in vesicles in the presynaptic terminal and two families of membrane-bound transporters responsible for the transport of neurotransmitters from the synaptic cleft back into the presynaptic terminal or glia cells. One of these families, the Na+/Cl--dependent transporters, contains transporters mediating the synaptic reuptake of the monoamines norepinephrine, dopamine and serotonin and the major inhibitory neurotransmitters g-aminobutyric acid (GABA) and glycine, and other transporters in this family have creatine, taurine and proline as their substrates. The other transporter family consists of five Na+-dependent excitatory amino acid transporters responsible for the synaptic reuptake of glutamate, the major excitatory neurotransmitter in the mammalian CNS.
Being essential regulators of the synaptic activity in these important neurotransmitter systems, the membrane-bound neurotransmitter transporters constitute highly interesting targets for pharmacological intervention in a wide range of psychiatric and neurological disorders. Drugs targeting transporters are already being administered in the clinic for depression, attention-deficit hyperactivity disorder (ADHD), obesity and epilepsy and as smoking cessation aids. The present issue of Current Topics in Medicinal Chemistry is focused on medicinal chemistry and pharmacology aspects of ligands targeting membrane-bound neurotransmitter transporters and the possibilities and challenges in connection with the development of novel drugs acting at these transporters or the improvement of already existing ones.
The serotonin transporter (SERT) is the key molecular target in the treatment of depression and is thus by far the most successful drug target amongst the neurotransmitter transporters. Selective serotonin reuptake inhibitors (SSRIs) hold significant advantages to older classes of antidepressants, in particular when it comes to their relatively mild side effects. However, the obvious clinical benefits of SSRIs are hampered by their characteristic slow onset of action and the fact that a considerable fraction of depression patients are non-responders to SSRI therapy. In the first review of this issue Moltzen and Bang-Andersen outline the add-on and augmentation strategies currently being pursued in the industry to address these problems through the development of compounds combining SERT inhibition with activities at other monoamine transporters or at various different neurotransmitter receptors [1].
The dopamine transporter (DAT) is a potentially interesting target for a wide range of neurodegenerative and psychiatric disorders, and furthermore it is the key mediator of the psychostimulant effects of recreational drugs such as amphetamine and cocaine. Runyon and Carroll summarize the findings in recent structure-activity studies of 3-phenyltropane, 1,4- dialkylpiperazine, phenylpiperidine and benztropine analogs as well as other classes of DAT ligands [2]. Furthermore, the authors outline the results from studies of DAT inhibitors in non-human primate models of psychostimulant abuse, Parkinson’s Disease and ADHD.
In their review of monoamine transporter substrates Rothman and Baumann elucidate how the basic inhibitor/substrate properties and the NET/DAT/SERT selectivity profile of the psychostimulant drug determine its pharmacological effects, including its therapeutic potential and its abuse liability [3]. Since equipotent DAT/SERT substrates do not appear to induce the cardiovascular side-effects caused by ‘clean’ serotonin releasers or the psychostimulant side-effects and abuse liabilities characteristic for ‘clean’ dopamine releasers, the authors propose these ligands as candidate drugs for the treatment of cocaine abuse.
The GABA and glycine transporters have not attracted nearly as much medicinal chemistry attention as the monoamine transporters. In their review of the GABA transporter (GAT) field, Clausen and colleagues present the structure-activity relationships for different series of conformationally restricted GAT ligands and propose a pharmacophore model for the GAT1 subtype [4]. Several selective GAT1 inhibitors have been published, whereas no truly selective pharmacological tools have been identified for the other three GAT subtypes. Considering the clinical administration of the GAT1-inhibitor tiagabine in epilepsy and the well-established therapeutic potential in augmentation of GABAergic neurosignalling in other disorders like anxiety, convulsive states, pain and sleeping disorders these three subtypes could be interesting as drug targets as well. 1800 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Editorial
In addition to having glycine, the co-agonist of the NMDA receptors, as its substrate, the glycine transporter subtype 1 (GlyT1) is co-localized with these receptors in several CNS regions. In keeping with the ’glutamate/NMDA dysfunction’ hypothesis of schizophrenia, inhibition of GlyT1 and the resulting augmentation of NMDA receptor signaling have been proposed as an alternate way to treat this disorder. Kinney and coworkers present a selection of the sarcosine and non-sarcosine based GlyT1 inhibitors published to date and outline the performances of these ligands in in vivo models for schizophrenia [5]. The major excitatory neurotransmitter glutamate plays key roles in a wide range of processes in the CNS, and an exhaustive numbers of ligands acting at ionotropic and metabotropic glutamate receptors have been published over the years. In the last review of this issue, Dunlop and Butera outline the relatively limited medicinal chemistry work done in the field of excitatory amino acid transporters (EAATs) [6]. While the therapeutic potential in EAAT inhibitors may be limited, some b-lactam antibiotics have recently been shown able to increase expression levels of EAAT proteins and have displayed promising neuroprotective effects in various in vitro and in vivo models.
I would like to thank all the authors for their enthusiasm and dedication in contributing to this CTMC issue, and the insightful suggestions and constructive criticism of the reviewers are also thankfully acknowledged. I hope this issue will be an inspiring read for the scientist engaged in the neurotransmitter transporter area and that it also will serve as an introduction to this exciting field of research for the general reader.
REFERENCES [1] Moltzen, E. K.; Bang-Andersen, B. Serotonin reuptake inhibitors: The corner stone in treatment of depression for half a century – A medicinal chemistry survey. Curr. Topics Med. Chem. 2006, 6(17), 1801-1823. [2] Runyon, S. P.; Carroll, F. I. Dopamine transporter ligands: Recent developments and therapeutic potential. Curr. Topics Med. Chem. 2006, 6(17), 1825-1843. [3] Rothman, R. B.; Baumann, M. H. Therapeutic potential of monoamine transporter substrates. Curr. Topics Med. Chem. 2006, 6(17), 1845-1859. [4] Høg, S.; Greenwood, J. R.; Madsen, K. B.; Larsson, O. M.; Frølund, B.; Schousboe, A.; Krogsgaard-Larsen, P.; Clausen, R. P. Structure-activity relationships of selective GABA uptake inhibitors. Curr. Topics Med. Chem. 2006, 6(17), 1861-1882. [5] Lindsley, C. W.; Wolkenberg, S. E.; Kinney, G. G. Progress in the preparation and testing of glycine transporter type-1 (GlyT1) inhibitors. Curr. Topics Med. Chem. 2006, 6(17), 1883-1896. [6] Dunlop, J.; Butera, J. A. Ligands targeting the excitatory amino acid transporters (EAATs). Curr. Topics Med. Chem. 2006, 6(17), 1897-1906.
Anders A. Jensen, Ph.D. Department of Medicinal Chemistry The Danish University of Pharmaceutical Sciences Universitetsparken 2 DK-2100 Copenhagen Denmark Email: [email protected] Current Topics in Medicinal Chemistry, 2006, 6, 1801-1823 1801 Serotonin Reuptake Inhibitors: The Corner Stone in Treatment of Depression for Half a Century – A Medicinal Chemistry Survey
Ejner K. Moltzen* and Benny Bang-Andersen
Medicinal Chemistry Research, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby-Copenhagen, Denmark
Abstract: Inhibition of serotonin (5-HT) reuptake has been a central theme in the therapy of depression for half a century. Through the years these therapies have improved, particularly with regard to side effects, and today’s selective serotonin reuptake inhibitors (SSRIs) constitute a reasonably effective offer for the patients. However, there is still room for major improvement and considering that almost 20% of the population in the western world will experience a depressive period in their lifetime, there is a large need for improved therapies. A large spectrum of targets and strategies are currently being pursued, but so far none of these new approaches have been successful, mainly due to lack of a deeper understanding of the disease biology. Since inhibition of 5-HT reuptake ensures a certain degree of antidepressant efficacy, there has been a large interest in various combinations with serotonin reuptake inhibitors (SRIs) in order to improve on the shortcomings of treatment with SSRIs. Some of these approaches have resulted in marketed antidepressants, eg combinations of SRI with norepinephrine (NE) reuptake inhibition, whereas other approaches are still at an experimental stage. This review attempts to present the current status of these add-on/combination approaches with particular focus on the medicinal chemistry aspects.
Keywords: Serotonin, norepinephrine, dopamine, SERT, DAT, NET, SSRI, 5-HT1A, 5-HT2C, 5-HT2A, reuptake inhibition, combination, add-on, augmentation, ASRI.
INTRODUCTION not respond to treatment [5]. Apart from that, a number of other side effects, such as sleep and sexual disturbances, also Drug therapies for treatment of depression disorders have became apparent. been available since the late fifties, where the first tricyclic antidepressants (TCAs) and monoamine oxidase inhibitors During the past ten years, the demand from society for (MAOI) became available. Amitriptyline (1) was one of the better treatments of depression has increased substantially. most prominent members of the TCA class and among the Where success of treatment earlier was equated to reduction core pharmacological activities accounting for their antidep- in symptoms as measured by various rating scales (e.g. 50% ressant effect were potent serotonin transporter (SERT) reduction on the HAM-D scale), it is now being replaced by inhibition and norepinephrine transporter (NET) inhibition. demand for full remission and patient wellness. Combined However, this drug class was also associated with severe with the fact that today the total potential patient population side effects, eg dry mouth (see ref [1] for further references). is considered much larger than earlier anticipated, a renewed and intense research interest was triggered within the In the late sixties and early seventies, the importance of depression field in general. SERT in the regulation of mood became more obvious. This resulted in the formulation of the monoamine hypothesis of A number of drug targets have been pursued, including depression [2] and the first selective SERT inhibitors (the other monoamine and non-monoamine receptors and trans- SSRIs), that were discovered, were zimelidine (2) [3] and porters, various peptide receptors, hormone receptors, enzy- fluoxetine (3) [4]. In the following years, a large number of mes, and the immune system (see eg refs [6] and [7]). Lately, structures claiming SSRI activity were patented. the importance of neuroplasticity has also become subject of investigation [8]. However, with the exception of the The advent of the SSRIs meant a major breakthrough in serotonin noradrenaline reuptake inhibi-tors (SNRIs), none the treatment of depressive illnesses. Not so much in terms of these approaches have so far resulted in therapies that are of better efficacy, but more in terms of a highly improved just as efficacious as the SSRIs. The future will show side effect profile, which allowed a much larger patient whether some of them will be successful. population to be treated. Particularly the improved overdose safety of the SSRIs was a major improvement. During the Driven by the success of the SSRIs, it has been a popular eighties and nineties, a number of SSRIs came on the market strategy to pursue various augmentation princi-ples to and with them the shortcomings of these drugs also became serotonin (5-HT) reuptake inhibition, thereby hoping to clear: full antidepressant effect requires 3-6 weeks of improve on some of the shortcomings of the SSRIs. Fast treatment, and a significant number of the patients (30%) do onset of action, in particular, has been in focus [9]. This review will mainly focus on the medicinal chemistry aspects *Address correspondence to this author at the Medicinal Chemistry of these SRI augmentation strategies. For other aspects Research, H. Lundbeck A/S, Ottiliavej 9, DK-2500 Valby-Copenhagen, regarding SRIs, including more biological and Denmark; E-mail: [email protected] pharmacological related information, the reader is referred to
1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1802 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen a number of excellent reviews published in recent years [1, efficacious in severe depression than the TCAs [12], their 6, 7, 10, 11]. much improved side effect profile warrants their widespread use. The main problems encountered with SSRIs are SELECTIVE SEROTONIN REUPTAKE INHIBITORS CYP450 liver enzyme interactions, slow onset of action, (SSRIs) poor or no effect in at least 30% of the patients, sexual dys- Since the early seventies and until today, a large number function, insomnia, and nausea. Some withdrawal symptoms of SSRIs have been described and marketed. However, more have also been observed. However, these side effects are than 95% of the SSRI market is covered by 5 products: much more benign than the severe anticholinergic and Fluoxetine (Lilly: ProzacTM) 3, Escitalopram (Lundbeck/ cardiovascular problems seen with TCAs [1]. TM TM Forrest Labs: Lexapro , Cipralex ) 4, Sertraline (Pfizer: This benign profile has allowed the SSRIs to be explored ZoloftTM) 5, Paroxetine (GSK: PaxilTM) 6, and Fluvoxamine TM in a much wider range of indications, and clinical efficacy (Solvay: Fevarin ) 7. A few new SSRIs are still being has now also been demonstrated in panic disorder, general developed. However, they cannot be considered of impor- anxiety disorders, post traumatic stress disorder, social tance for two main reasons: the currently marketed SSRIs phobia, obsessive-compulsive disorder, eating disorders, pre- are all as efficacious as one can expect from this class of menstrual dysphoric disorder, and a further range of drugs and most of them will go generic within a few years, indications are still being explored [1]. thus making it very difficult for new me-too SSRIs to make it to the market. As will be outlined below, Escitalopram ALLOSTERIC SITE ON THE SEROTONIN TRANS- constitutes a special case. It was originally marketed as the PORTER racemate, but recent evidence has shown that the R- The pharmacological selectivity of marketed SSRIs is enantiomer inhibits the action of the S-enantiomer, thus relative rather than absolute, and these agents can have making the now marketed pure S-enantiomer a much more clinically significant properties that differentiates one drug in efficacious antidepressant with allosteric action on SERT. this class from another [13, 14]. It must also be emphasized As mentioned above, this class of drugs provided a major that Fluoxetine and Citalopram were first marketed as race- improvement compared to treatments with TCAs and mates, whereas Paroxetine and Sertraline were introduced as MAOIs. Although the SSRIs cannot be considered more single enantiomers [15], and that single enantiomers of both
Br N
H O N
F C N 3 N
Amitriptyline (1) Zimelidine (2) Fluoxetine (3)
NC
F O N Cl
Cl NH HN O O
F O
S-Citalopram (4) Sertraline (5) Paroxetine (6)
O N NH2 O Cl NH2 N O N Cl
O N N CF3 H H
Fluvoxamine (7) SoRI-6238 (8)
Scheme (1). Amitriptyline and Selective Serotonin Reuptake Inhibitors (SSRIs). Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1803
Citalopram and Fluoxetine have been pursued as second- degree of stereoselectivity at the allosteric site [37]. Thus, generation SSRIs [14]. In the case of Fluoxetine, the Escitalopram may be superior toward SSRIs because of its enantiomers are equipotent as SRIs, but the R-enantiomer is unique interaction with the allosteric binding site on SERT, an antagonist at human 5-HT 2A and 5-HT2C receptors leaving making this site especially interesting as a future target for the S-enantiomer as the more selective SSRI [16]. However, the design of improved antidepressants. The term ASRI due to cardiovascular side effects, R-Fluoxetine never made (Allosteric Serotonin Reuptake Inhibitors) [38] has been it to market. In the case of Citalopram, it has been shown assigned to these new types of dual acting compounds. that the SRI activity is attributable to the S-enantiomer [17, In order to assist the design of novel SSRIs and to under- 18], Escitalopram. The R-enantiomer is about a factor of 30- stand the structural organisation as well as the molecular 40 times less potent than Escitalopram at the human mechanisms responsible for the function of SERT, a number transporter [19, 20]. In 2002, Escitalopram was launced as a of homology models have been developed during the years new single-enantiomer drug (Lundbeck/Forrest Labs: TM TM based on non-crystallographic approaches [39], but also Lexapro , Cipralex ). based on X-ray structures of related transporters, such as the Today, Escitalopram has demonstrated improved clinical Escherichia coli Na+/H+ antiporter [40] and the lactose efficacy as antidepressant when compared to SSRIs and with permease symporter (lac permease) [41]. demonstrated efficacy in anxiety disorders [21, 22]. The Recently, an X-ray structure of a bacterial homologue of improved effect was first demonstrated in a human clinical SERT from Aquifex aeolicus was published [42]. As this study in patients with major depressive disorder, and in non- transporter belongs to the same family as SERT, this clinical studies using rat behavioural models of depression. structure may offer an opportunity to build a more reliable In both clinical and preclinical studies it was found that the computational model of SERT than hitherto possible. A onset of effect of Escitalopram versus placebo was faster preliminary insight into such a model and its limitations has than Citalopram versus placebo [23]. This was a surprising recently been described [43], but it is probably only a matter result at that time because the animals and patients in these of time before a more comprehensive account will be pub- two studies received equivalent amounts of the S-enan- lished. It may be envisaged that such a model will give better tiomer. However, the Citalopram-treated groups also insight into the primary binding site of SERT, but hopefully received R-Citalopram. Since then it has been shown in a also into the allosteric binding site, which has been shown to number of studies that the R-enantiomer in Citalopram actu- be independent of the primary binding site [36, 44] and ally counteracts the activity of the S-enantiomer in in vitro located at the C-terminal part of SERT containing the and in vivo mechanistic models [24-27] and in behavioural transmembrane domains 10 to 12 [45]. animal models [19, 23, 28-30]. For a more detailed discussion regarding Escitalopram and the surprising role of AUGMENTATION STRATEGIES the R-enantiomer of Citalopram, the reader is referred to a number of reviews published recently [15, 22, 31]. The concept of combining serotonin reuptake inhibition with other pharmacological activities within treatment of The mechanism behind the inhibitory action of R-Citalo- depression has attracted much research attention during the pram on Escitalopram may be related to its action on the past 15 years. Two different approaches have mainly been allosteric binding site on SERT [32]. The existence of an attempted: either combination of an SSRI with other allosteric binding site on SERT and its possible relevance for substances of varying pharmacological activity (add-on) or the effect of antidepressants has been known since the early preparation of molecules with build-in activities at multiple 1990s [33]. In vitro binding kinetics using brain neuronal targets. Although the terms combination and augmentation membranes and platelet membranes demonstrated the are sometimes used interchangeably [46], some distinction existence of this secondary, affinity-modulating binding site can be made. Thus, for add-on treatment the term combi- on SERT. It is a low-affinity binding site and only Escitalo- nation refers to the use of more than one disease-specific pram and to a lesser extent Paroxetine exert an allosteric treatment to treat a specific disease, ie pharmacotherapy of effect via this mechanism, which is not correlated to their treatment-resistant depression by the use of at least two affinity for the primary high-affinity binding site and their different antidepressants, whereas augmentation refers to the SRI potencies. Interestingly, SoRI-6238 (8) has been descri- addition of a non-antidepressant medication, ie addition of bed as an allosteric modulator without affinity for the lithium or an antipsychotic to an SSRI. In the case of single primary binding site [34]. molecules with build-in activities at multiple targets, the Recently, the allosteric binding site has attracted renewed augmentation term will be used to describe both the addition interested in connection with the improved antidepressant of targets known to treat related symptoms, eg 5-HT1A effect seen for Escitalopram, and it has been shown that agonism to treat anxiolytic symptoms, but also to describe Escitalopram and R-Citalopram via this site can modulate the improvement of the antidepressant potential, eg 5-HT1A association [35] and dissociation [36, 37] rates of Escitalo- antagonism to increase onset of antidepressant action. pram from the primary binding site. Furthermore, it has been The discovery by Artigas of the augmentative effect of 5- 3 shown that Escitalopram stabilises the binding of [ H]- HT1A antagonists on the antidepressant efficacy of SSRIs Escitalopram to hSERT, but not the binding of other SSRIs [47] triggered the development of these combination/ [37]. In comparison, Paroxetine also stabilises its own augmentation strategies. The main focus was – and still is - binding, but to a lesser extent, and it has no effect on the to obtain faster onset of action, which is one of the major binding of the other ligands. R-Citalopram is about a factor problems in depression, but other unmet needs have also of 3 times less potent than Escitalopram suggesting some been addressed, for example treatment resistance and sleep 1804 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen disturbances. In more recent years, a broader range of trials [60]. The interesting challenge for these compounds indications has been targeted, eg treatment of bipolar will be whether the DAT component is tolerable without in- disorder with a combination of SSRIs and antipsychotics. ducing abuse liability due to increased dopamine stimulation. In the following, a survey of the various medicinal In the patent literature, the interest in this area is also chemistry strategies reported in the literature on this subject reflected by the increasing number of patents being during the last 15 years will be presented. published, both on dual and triple active compounds. Some of the more recent ones are listed in Table (1). SERT/NET and SERT/NET/DAT Inhibitors SERT Inhibitors/5-HT Antagonists The SERT/NET uptake inhibitor class is relatively new 1A and so far there are only three on the market for depression During the early nineties, it was discovered that blockade TM (Scheme 2): Venlafaxine, 9, Duloxetine (Cymbalta ), 10, of 5-HT1A autoreceptors in raphé nuclei augments the and Milnacipran, 11. NET inhibition is a component of the serotonergic output in cortical and other brain areas follo- profiles of the old TCAs, and the antidepressant effect of wing application of SRIs as shown by both microdialysis and elevating the norepinephrine (NE) levels in the brain has electrophysiological studies [61]. The importance of this long been realized [48]. The NET effect is mainly associated discovery was emphasised by Artigas’ clinical study with improvement of anhedonic and melancholic features of combining Paroxetine with Pindolol (18) [47], which besides depression as well as higher remission rates [49, 50]. The being a beta blocker is also a 5-HT1A antagonist. A rapid antidepressant effect of selective NET uptake inhibition is improvement of the depressive symptoms in the participating supported by the NET-selective Reboxetine, 13, which is on patients was observed. This finding was rationalised by the the market and displays clinical efficacy, particularly in hypothesis that the 4-6 weeks of treatment with SRIs major depression [50]. normally required to obtain full antidepressant efficacy leads Venlafaxine has been on the market since 1994 and is at to desensitisation of 5-HT1A autoreceptors, thus allowing the serotonergic neurons to resume their normal function. Simul- relatively high doses (>225 mg) associated with faster onset taneous application of a 5-HT antagonist inhibits this of action than some of the SSRIs [52]. Duloxetine is a potent 1A feedback mechanism. This would in turn allow an immediate SERT/NET inhibitor and has recently entered the market and sufficient increase in extracellular concentrations of 5- where it has been positioned as an antidepressant, possibly HT to obtain antidepressant efficacy. with pain relieving properties [53, 54]. Milnacipran is a somewhat weaker SERT/NET inhibitor, but still shows clini- It is still being discussed whether this hypothesis holds cal efficacy [51]. Sibutramine, 12, is also a selective SERT/ [62]. A number of similar clinical trials have been performed, NET inhibitor, but has been marketed for treatment of and the results are very mixed, although a recent meta- obesity [55]. analysis concludes that Pindolol seems to hasten the The major issue with this type of profile concerns safety. response to SSRIs in depression [63]. It has also been Although the new generation is devoid of the anticholinergic questioned whether the applied 7.5 mg dose of Pindolol and antihistaminic activity of the old TCAs, the increased would be sufficient to occupy the 5-HT1A receptors, but PET NE tone still represents an increased risk of side effects, studies have now confirmed that this is indeed the case [64]. primarily blood pressure changes. This is also seen in It has also been suggested that stimulation of postsynaptic 5- clinical studies [52, 56], and for new derivatives of this kind HT1A receptors is important for antidepressant action, and 5- HT antagonism would be detrimental to this effect. This the challenge is to find the right balance between increased 1A efficacy, putatively greater response and remission rates and, issue is still not fully resolved. Finally, it should be on the other side, norepinephrine-related side effects. mentioned that genetic links between depression and SERT Duloxetine might be the first compound of this type on the and 5-HT1A receptors have recently been reviewed [65]. market with relatively modest effect on heart rate and blood A large number of groups have been pursuing the pressure. SERT/5-HT1A concept. Combinations of SSRIs with various 5-HT antagonists have appeared in the patent literature. The success of SERT/NET inhibitors has initiated an 1A This includes combinations with Pindolol and other beta increased activity to find new derivatives. The Lilly group blockers [66-69], with the silent 5-HT antagonist WAY- has published further SAR efforts around Duloxetine [54] 1A 100635 (19) [70], and with other types of 5-HT antagonists resulting in two related structures showing potent SERT/ 1 [71-75]. However, none of these approaches have so far NET inhibition: 14 (SERT: K 0.5 nM, NET: K 4.4 nM) [57] i i reached the market. and 15 (SERT: Ki 1.4 nM, NET: Ki 3.1 nM) [58]. The concept of combining the two effects in one In an older work, Servier presented S33005, 16, which molecule has attracted much more attention. The Eli Lilly displays potent SERT uptake inhibition (K 2.1 nM) and i group has recently published a series of papers on Pindolol more modest NET inhibition (K 1514 nM). However, the i derivatives where the isopropylamino group has been repla- compound is effective in increasing 5-HT and NE levels in ced by a 5-HT active biarylalkylamino moiety exemplified rat frontal cortex (500% for both 5-HT and NE at 10 mg/kg) 1A by 20 [76-79]. A comprehensive SAR study shows that in as measured by microdialysis [59]. vitro the series potently binds to 5-HT1A receptors and to Recently, triple compounds with uptake inhibiting acti- SERT. The series is in general antagonistic at 5-HT1A vity at SERT, NET, and the dopamine transporter (DAT) receptors according to testing in a [35S]GTPgS assay in cells have started to appear. The most prominent example is transfected with human 5-HT1A receptors. Compound 20 (5- DOV-21947, 17, which has been reported to be in clinical Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1805
O N H S O H N O OH 2
N N
Venlafaxine (9) Duloxetine (10) Milnacipran (11)
O S
Cl O N H O O
N S N H H
Sibutramin (12) Reboxetine (13) (14)
CF3 N Cl N O HO Cl
N N H H DOV-21947 (17) (15) S33005 (16)
Scheme (2). Combined SERT/NET and SERT/NET/DAT inhibitors.
Table 1. Recently Patented SERT/NET and SERT/NET/DAT Inhibitors
Structure Comment Reference
H N
N SERT, NET, DAT reuptake inhibitors. [147] S Lundbeck, 2006
HN
O SERT, NET reuptake inhibitors. Br [148] Baxter et al, 2006
N 1806 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen
(Table 1) Contd….
Structure Comment Reference
NH N SERT (Ki 27 nM), NET (Ki 62 nM) uptake inhibitor. [149] Pfizer, 2005
F3C
SERT, NET, DAT reuptake inhibitors. [150] N Lilly, 2004
HN CN
S SERT, NET, DAT reuptake inhibitors. [151] N Lilly, 2005
HN
SERT, NET N O reuptake inhibitors. [152] Lilly, 2004 N H
H SERT (<100 nM), NET (<100 nM)
H2N O reuptake inhibitors. [153] F Lilly, 2004
O O SERT, NET N reuptake inhibitors. [154] Lundbeck, 2003 Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1807
(Table 1) Contd….
Structure Comment Reference
SERT, NET, DAT reuptake inhibitors. [155] Dupont, 2001 N
SERT, NET, DAT reuptake inhibitors. S [156] BMS, 2006 N
HT1A: Ki 14 nM, SERT: Ki 0.86 nM) was selected based on a whereas the 5-HT1A part mainly circles around an aryloxy- potent effect in microdialysis with 700% increase in 5-HT alkyl motif. Some representative examples are shown in concentration as measured in rat hypothalamus at 10 mg/kg, Scheme 3, structures 23 and 24. The profiles of these two po. The effects on SERT and 5-HT1A receptors of 20 were derivatives demonstrate the main challenges in this work. also substantiated in vivo [80]. Derivative 23 is rather weak at both SERT and 5-HT1A ([3H]5-HT uptake: K 45 nM, 5-HT : K 78 nM) and is a The Lilly group has also reported on structures relating to i 1A i 35 g the selective 5-HT antagonist WAY100635 [81-85]. Data partial 5-HT1A agonist according to [ S]GTP- S assay 1A 24 3 were presented on LY433221, 21 [86-89], and they showed testing. Derivative is more potent ([ H]5-HT uptake: Ki 4.5 nM, 5-HT1A: Ki 10.7 nM) and shows no 5-HT1A agonism. potent affinity for cloned human 5-HT1A receptors (Ki 5.4 3 However, both 23 and 24 bind potently to a 1 adrenoceptors nM), inhibition of synaptosomal [ H]5-HT uptake (IC50 0.8 nM), and inhibition of 5-HT-stimulated [35S]GTP-gS binding (Ki 4.7 nM and 4.6 nM, respectively). Extensive SAR variations on both the aryloxy group as well as the linker (K 2.7 nM, E 12.4%). The compound also displays i min were performed, whereas some substituent variations on the affinity for 5-HT receptors (K 13.1 nM). A large number 2A i indole moiety were performed. However, the optimal profile of in vivo studies were reported, indicating antidepressant was not reached. This optimisation challenge: to obtain and anxiolytic potential, eg the compound increased extra- potent SERT inhibition and 5-HT antagonism without cellular 5-HT by 600% in prefrontal cortex (30 mg/kg, po). 1A affecting other monoamine receptors is a general problem Combination of the Pindolol/SSRI motives has also been within this field. addressed by the GSK group. Based on a functional high throughput screening using a [35S]GTP-gS binding assay The Lundbeck group faced similar challenges. An followed by optimisation resulted in compound 22 [90]. The approach similar to the Wyeth’s was used combining the 3- SAR study included variations of the indole moiety and the indolylalkylamine moiety as the SERT-active part and the ortho-alkyloxyarylpiperazine as the 5-HT part [97]. The linker, whereas the benzoxazinone part was kept. As in other 1A main result of this work was Lu 36-274, 25, which shows similar SAR studies, the main issue was to maintain high 3 affinity for and antagonism/low partial agonism at 5-HT potent SERT inhibition ([ H]5-HT uptake: Ki 5 nM) and 5- 1A HT antagonism (K 4.5 nM, no partiality). Selectivity receptors and potent SERT inhibition. This was 1A i towards a 1 adrenoceptors and D2 receptors was a main accomplished in 22 (5-HT1A: Ki 0.3 nM (intrinsic activity 3 problem, but it was solved by introducing the 8-cyano group 0.2), inhibition of synaptosomal [ H]5-HT uptake K 6.3 i in the benzodioxane ring. Another problem was to obtain 5- nM). The compound also showed affinity for 5-HT (pK 1B i HT antagonism with no partiality. This was solved by 8.1) and 5-HT (pK 8.7) receptors, but was otherwise 1A 1D i introducing the 6-chloro substituent in the indole ring. Lu selective. Oral bioavailability and brain penetration could 36-274 shows a 500% increase in extracellular 5-HT also be demonstrated. concentration in rat frontal cortex (16 mg/kg, sc), and it The Wyeth group has published a series of studies shows a potent and immediate effect in the schedule-induced towards compounds combining SERT-active with 5-HT1A- polydipsia model, which is an indicator of antidepressant active moieties, respectively [91-96]. For the SERT-active efficacy [98]. part the main focus is on the 3-indolyl-alkylamino scaffold Although the main focus has been on 5-HT antagonists, (known from eg the potent SERT inhibitor indalpine), 1A there has also been some interest in developing 5-HT1A 1808 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen agonists. Clinically, combination of Buspirone (BusparTM, a SSRIs, with atypical antipsychotics. Thus, Risperidone, 29 5-HT1A partial agonist) with various SSRIs has been [111, 112], Olanzapine, 30 [113-115] Ziprasidone, 31 [116] attempted and in a smaller study including thirty outpatients Aripiprazole, 32 [117, 118] and Quetiapine, 33 [119] have improved response- and remission rates were observed [99]. all shown superior efficacy in the treatment of treatment- However, these results have not been substantiated in larger resistant depression as add-on to various SSRIs. Further- trials, and compounds with this level of 5-HT1A efficacy more, Quetiapine in combination with ongoing SRI therapy might not be the optimal profile for combination with has been shown to be effective in treatment of treatment- serotonin reuptake inhibition. It shall be mentioned that refractory obsessive compulsory disorder [120] and in the anecdotal reports indicate that co-prescriptions of Buspirone treatment of co-morbid anxiety in depressed patients [121]. and SSRIs occur to some extent. Olanzapine in combination with Fluoxetine (SymbiaxTM) has VN2222, 26, is the result of an optimisation starting reached the US market for the treatment of bipolar depression [122], whereas Risperidone in combination with mainly from known selective 5-HT1A agonists and, some- what unexpectedly, building in SERT inhibition via a Paroxetine only showed modest effects when added to a mood stabilizer in bipolar depression [123]. Thus, combining benzothiophene moiety [100-102]. As expected by a 5-HT1A agonist, VN2222 acutely reduces extracellular 5-HT levels in an SSRI with an atypical antipsychotic drug cannot be seen rat striatum, but after 14 days of chronic treatment 5-HT as a common class of drugs that gives similar effects. This levels are unaffected by VN2222, indicating desensitization may not be too surprising, as the atypical antipsychotics all display slightly different multireceptorial pharmacologies of the 5-HT1A autoreceptors [103]. Effect in the learned helplessness test was demonstrated [104]. that probably account for these differences. The success of such a combination will furthermore depend on compound- The indole derivative Vilazodone (EMD68843), 27, is a specific limitations known for all of the atypical antipsy- very interesting compound. It was developed by Merck chotics, such as varying degrees of potential for inducing KGaA via systematic structural modification of indole- weight gain/metabolic disturbances, QTc prolongation, and alkylphenyl piperazines [105] and is now being developed elevation of prolactin [124]. by Genaissance Pharmaceuticals. It recently entered phase Mirtazepine (and Mianserin) is a potent antagonist at III clinical trials. Vilazodone is a potent SERT inhibitor 3 central a 2-adrenergic auto- and heteroreceptors, and an ([ H]5-HT uptake: IC50 0.5 nM), a 5-HT1A ligand (IC50 0.2 35 antagonist at 5-HT2 and 5-HT3 receptors [125]. It has been nM) and an agonist at 5-HT1A as measured in the [ S]GTP- shown that Mirtazepine is an effective antidepressant in its gS assay. However, in vivo the compound behaves as a 5- own right [126], but beneficial effects in combination with HT1A antagonist and induces a 2- to 3- fold increase in extra- SSRIs have also been demonstrated [127]. cellular 5-HT concentration in rat frontal cortex [106]. This discrepancy is apparently still not understood. The Merck The mechanism behind these combinations has not been group has further elaborated on the SAR around Vilazodone fully elucidated, and different groups have put forward 3 various explanations [119, 128]. It has been suggested that resulting in eg 28 (5-HT1A: IC50 2.5 nM (agonist), [ H]5-HT blockade of 5-HT2A receptors is of importance because a low uptake: IC50 0.2 nM), which increased 5-HT concentration 2- to 3- fold in rat frontal cortex [107]. dose of Risperidone supposed to block only 5-HT2A receptors are indeed effective as add-on [129]. It has been Besides the SERT/5-HT1A active compounds described suggested, that the ability of antipsychotics to augment the so far, a rather large number of further structures with similar therapeutic efficacy of SSRIs in major depression and profiles have been claimed in the patent literature. Since no treatment-resistant depression may be due, at least in part, to further biological data are available, only representative potentiation of SSRI-induced increases in cortical dopamine structures and references are given in Table (2). and norepinephrine levels [130, 131].
SERT Inhibition/Antipsychotics and SERT Inhibition/ SERT Inhibition/5-HT2A Antagonism Mirtazepine or Mianserin Multiple lines of evidence are consistent with the Antipsychotics show only modest efficacy as mono- hypothesis that blockade of 5-HT2A receptors might enhance therapy of depression, but they are effective in those patients the therapeutic effectiveness of SSRIs [128, 129, 132]. It has who also present psychotic symptoms [46]. However, an also been hypothesized that blockade of 5-HT2A receptors increasing body of evidence suggests that antipsychotics might reduce some of the side-effects related to SSRI may also augment the antidepressant action of SSRIs in non- treatment, eg sexual and sleep disturbances [133], making a psychotic patients and be useful in the treatment of treat- combined SERT inhibitor/5-HT2A antagonist an attractive ment-resistant depression [108] and bipolar disorder [109]. target for an improved antidepressant. Augmentation with typical antipsychotics has never been Trazodone (DesyrelTM) 34 and Nefazodone (SerzoneTM) widely used because of the risk of extrapyramidal symptoms 35 are antidepressants, which both show high affinity for 5- (EPS) and/or tardive dyskinesia [110]. The introduction HT2A receptors (34: Ki 11 nM and 35: Ki 32 nM) and during the 1990’s of the atypical antipsychotic drugs, which somewhat lower affinity for SERT (34: Ki 115 nM and 35: do not induce EPS to the same extent, spurred the interest for Ki 181 nM) as measured in rat cortex homogenate [133, combining SSRIs with an atypical antipsychotics in order to 134]. It has been shown in vivo in rat and mouse brain, that potentially augment the effects of SSRIs. Nefazodone is an antagonist at 5-HT2A receptors. The in vivo In the meantime, a growing number of clinical trials have SERT activity has been more difficult to verify in various been published on the combination of SRIs, in particular laboratories [133-135], and this may be due to the relatively weak in vitro affinity for SERT. It has also been shown that Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1809
N
N OH N N O NH H N O NH O N O OH
S O Pindolol (18) WAY-100635 (19) (20)
N O R O Ar N N HN O N O O N NH S (23): R = H, Ar = LY433221 (21) (22)
O O
CN Cl OH (24): R = F, Ar = N O Cl N O N O S N N NH
HN Lu 36-274 (25) VN2222 (26) O
O O O NC NC N NH2 N N N HN O
HN Vilazodone (27) HN (28)
Scheme (3). Combined SERT inhibitors and 5-HT1A antagonists.
Table 2. Combined SERT Inhibitors and 5-HT1A Antagonists Reported in the Patent Literature
Structure Comment Reference
H N Br 5-HT1A modulator and SERT inhibitor. N Sumitomo Pharmaceuticals [157] Cl 2003
N Cl N O 5-HT1A antagonist and SERT inhibitor. Cl OH O Welfide Corp. [158] N O 2000 1810 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen
(Table 2) Contd….
Structure Comment Reference
OH H O N O NH 5-HT1A modulator and SERT inhibitor. Pierre Fabre [159] F3C 1998
O F O 5-HT1A ligand and SERT inhibitor. N American Home Products [160] N 2000 H NH
F N N 5-HT1A antagonist and SERT inhibitor. American Home Products [161] 2000 O NH
HN N N 5-HT1A antagonist and SERT inhibitor. American Home Products [162] NH 2000
CN
O
HN N 5-HT1A ligand and SERT inhibitor. S American Home Products [163] 2000
CN
F
5-HT1A ligand and SERT inhibitor. O N American Home Products [164] H 2000 NH N
F
O 5-HT1A modulator and SERT inhibitor. N H American Home Products [165] NH 1999 O O Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1811
(Table 2) Contd….
Structure Comment Reference
NH
5-HT1A antagonist and SERT inhibitor. O American Home Products [166] N H F 1999 N H
F
5-HT1A antagonist and SERT inhibitor. O N American Home Products [167] H NH 1999 NH N NH
5-HT1A modulator and SERT inhibitor. O N American Home Products [168] H F 1999
HN
N 5-HT antagonist and SERT N N 1A/1B/1D N O inhibitor. [169] O N BASF 1999
O O S N N 5-HT1A/1B/1D antagonist and SERT N inhibitor. [170] BASF 1999
O
N N N 5-HT1A antagonist and SERT inhibitor. N GSK [171] O 2006 N O N 1812 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen
(Table 2) Contd….
Structure Comment Reference
N 5-HT1A ligand and O SERT inhibitor. [172] N Adir et Co 2001
F
S O H N S N N N
N N N N O N Cl N N O N H Risperidone (29) Olanzapine (30) Ziprasidone (31)
S H O N O N N N N
Cl N Cl HO 33 Aripiprazole (32) O Quetiapine ( ) Scheme (4). Atypical antipsychotics.
Nefazodone occupies 5-HT2A receptors in humans at starting LY367265, 37, has been shown to have high affinity for clinical doses [136]. Trazodone and Nefazodone also display SERT (Ki 2.3 nM) and 5-HT2A receptors (Ki 0.81 nM), and it affinity for a number of other receptors believed to be has been shown to be a potent inhibitor of [3H]5-HT uptake relevant at therapeutic doses, most notably 5-HT1A (partial into rat synaptosomes (IC50 3.1 nM) with selectivity towards agonist) and a 1 for Trazodone [133, 137], and NET and a 1 NET (IC50 > 1000 nM) [139]. This compound is no longer for Nefazodone [133]. Yamanouchi was developing a dual under development [10]. Lilly has described a number of acting SERT/5-HT2A compound, Lubazodone (YM-992/YM- further analogues of LY367265 in the patent literature, and 35995) 36, which is a more potent SERT inhibitor than also Pfizer, Lundbeck, Merck KGaA, and Baylor University Trazolone and Nefazodone. Lubazodone is also a more have filed patents claiming combined SERT inhibitors and 5- balanced SERT/5-HT2A compound (SERT: Ki 21 nM, 5- HT2A antagonists. Representative structures and references HT2A: Ki 86 nM) [138] displaying good selectivity toward a for these patents are given in Table (3). range of monoaminergic receptors of which the closest SERT Inhibition/5-HT2C Antagonism affinity was found for a 1 adrenoceptors (Ki 200 nM) and 5- HT2C receptors (K i 680 nM) [138]. Lubazodone has been in Selective 5-HT2C antagonists have mostly received inte- phase II clinical trials, but the development was discontinued rest as potential anxiolytics, but also as potential antidep- in 2001 due to erosion of the SSRI market in the US. The ressants, whereas 5-HT2C agonists have been related to anti- Lilly group has also reported on structures that are even depressant activity [140-145]. Furthermore, it was recently more balanced with regard to SERT versus 5-HT2A. demonstrated that blockade of 5-HT2C receptors potentiates Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1813
O O N N N N N N N N O N N
Cl Cl Trazodone (34) Nefazodone (35)
O O N S O O N N N N N N N H N O
F N F N Lubazodone (36) H LY-367265 (37) (38)
Scheme (5). Combined SERT inhibitors and 5-HT2A/5-HT2C antagonists.
Table 3. Combined SERT Inhibitors and 5-HT2A Antagonists Reported in the Patent Literature
Structure Comment Reference
N 5-HT2A antagonist and SERT inhibitor. N O [173] Baylor University, 2005
F Cl
Novel composition comprising an SSRI, particularly Sertraline, and a selective inhibitor for the binding of serotonin at the 5-
HT2A site for the treatment of depression and anxiety disorders [174] Pfizer, 2005
O
N N 5-HT2A antagonist and SERT inhibitor. N [175] O N Pfizer, 2002 H Cl Cl
O
N N 5-HT2A antagonist and SERT inhibitor. [176] O N Pfizer, 2001 H Cl 1814 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen
(Table 3) Contd….
Structure Comment Reference
O
N N
5-HT2A antagonist and SERT inhibitor. [177] Lilly, 1999
N H F
O O S N
N 5-HT2A antagonist and SERT inhibitor. [178, 179] Lilly, 1999
N F H
Cl
N
N O 5-HT2A antagonist and SERT inhibitor. O [180] Merck KGaA, 1998
N H
N
5-HT2A antagonist and SERT inhibitor. [181] HN O Lundbeck, 1995
Cl N H both the neurochemical and antidepressant-like behavioural support the combination of an SSRI with a 5-HT2C action of SSRIs [146]. In in vivo microdialysis the 5-HT2C antagonist in order to find an improved antidepressant. selective antagonists SB-242084 and RS-102221 substan- Compounds with this specific profile have been reported tially augmented the serotonergic response to Citalopram, as in a number of patent applications, but not specifically in the indicated by a 900% increase in extracellular 5-HT levels as literature. However, some of the compounds discussed above compared to baseline values. The two selective 5-HT2C targeting SERT/5-HT2A will probably also exert 5-HT2C antagonists did not alter the 5-HT level when given alone, affinity, as the SAR for 5-HT and 5-HT often follows a and Citalopram gave an increase of approximately 400-500% 2A 2C similar pattern in chemical series. R-Fluoxetine may to some relative to baseline [146]. Thus, several lines of evidence Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1815 extent be representative for such a compound (SERT: Ki 7.7 antagonists, respectively, most of these new suggestions nM and 5-HT2C: Ki 68 nM), although the increase in have only appeared in the patent literature so far. This is for extracellular 5-HT as measured by in vivo microdialysis, example the case with combinations with GABA-A/GABA- when compared to the SSRI S-Fluoxetine, does not match B ligands. The future will show whether these approaches the combinations described above [16]. The 5-HT2C antago- are viable. A summary is shown in Table (5). nism of R-Fluoxetine is probably too weak. CONCLUSIONS The Adir group has filed a patent in year 2000 on Serotonin reuptake inhibition has meant a major break- SERT/5-HT2C compounds and described in vitro data for a representative compound from this application, 38.230 through in the treatment of depression over the last 50 years. The drugs have been refined over the years, and with the Compound 38 exhibited a Ki value of 9.8 nM for SERT as measured by displacement of [3H]Paroxetine and an affinity advent of the SSRIs it seems that this type of treatment is close to have reached its full potential as stand-alone between 1 and 10 nM for 5-HT2C receptors as measured by displacement of [3H]mesulergine. Since no biological data treatment. Due to the relatively benign side effect profiles of are available for specific compounds in the other patents, the SSRIs, their use has spread out to a number of different representative structures and references for these are given in mood and anxiety disorders, and further uses will undoub- Table (4). tedly be found. However, SSRI treatment does not constitute the ideal depression treatment. The issues with slow onset of SERT Inhibitors/Other action, treatment resistance in almost 30% of the patients, A number of other SRI combinations have been descri- and various other side effects as mentioned above cannot be bed. While some degree of validation exists for a few of addressed by SSRIs alone. these possibilities, like combinations with 5-HT1B/1D and a 2
Table 4. Combined SERT Inhibitors and 5-HT2C Antagonists Reported in the Patent Literature
Structure Comment Reference
H N
5-HT2C antagonist and SERT inhibitor. [182] Lundbeck, 2004 S
F H N
5-HT2C antagonist and SERT inhibitor. [183] Lundbeck, 2004 S
Cl CF3
H N
5-HT2C antagonist and SERT inhibitor. CF N [184] 3 Lundbeck, 2003 S
Use of compounds and composition of compounds having SRI activity and 5-HT2C antagonistic, partial agonistic or inverse agonistic activity for the treatment of depression and other affective disorders [185] Lundbeck, 2001
Use of combination of Irindalone and SRIs or any other compounds, which causes elevation in the level of 5-HT, for the treatment of depression and other affective disorders [186] Lundbeck, 2001 1816 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Moltzen and Bang-Andersen
Table 5. Other SRI Combinations
Combination for treatment of depression Reference
Concurrent administration of an SRI, 5-HT, a peripheral decarboxylase inhibitor or tetrahydrobiopterin Duke University, 2005 [187]
Administration of a 5-HT3 antagonist with an SRI Lundbeck, 2005 [188]
Administration of a 5-HT3 antagonist with an SRI Pfizer, 2002 [189]
Administration of a 5-HT3 antagonist with an SRI Eli Lilly, 1999 [190]
Compounds with combined SRI activity and 5-HT1D antagonism Eli Lilly, 2004 [191]
Compounds with combined SRI activity and 5-HT1B/D antagonism Merck KgaA, 2000 [192]
Compounds with combined SRI activity and 5-HT1B antagonism or partial agonism Astra, 1999 [71]
Concurrent administration of an SRI and a 2 antagonist yohimbine Yale University, 2004 [193]
Compounds with combined SRI activity and a 2 antagonism Abbott, 1997 [194]
Compounds with combined SRI activity and NK1 BMS, 2006 [195]
Compounds with combined SRI activity and NK1 BMS, 2006 [196]
Compounds with combined SRI activity and NK1 Glaxo, 2005 [197]
Compounds with combined SRI activity and NK1 AstraZeneca, 2005 [198]
Compounds with combined SRI activity and NK1 AstraZeneca, 2005 [199]
Compounds with combined SRI activity and NK1 AstraZeneca, 2004 [200]
Compounds with combined SRI activity and NK1 AstraZeneca, 2004 [201]
Compounds with combined SRI activity and NK1 AstraZeneca, 2004 [202]
Compounds with combined SRI activity and NK1 Glaxo, 2004 [203]
Compounds with combined SRI activity and NK1 UCB, 2002 [204]
Compounds with combined SRI activity and NK1 UCB, 2002 [205]
Concurrent administration of an SRI and a GABA-B antagonist Lundbeck, 2004 [206]
Concurrent administration of an SRI and a GABA-A a 2/3 agonist Pfizer, 2002 [207]
Concurrent administration of an SRI and a GABA-A a 2/3 agonist MSD, 1999 [208]
Compound with combined SRI activity and MC4 receptors antagonism Taisho Pharmaceutical Co, 2005 [209]
Concurrent administration of an SRI and N-acetylcysteine Yale University, 2006 [210]
Concurrent administration of SRI and 5-hydroxytryptophan Duke University, 2005 [211]
Concurrent administration of an SRI and an imidazoline I2 agonist or compounds with combined activity Merck KgaA, 2005 [212]
Concurrent administration of an SRI and Mecamylamine Yale University, 2005 [213]
Concurrent administration of an SRI and Loxapine Lundbeck, 2005 [214]
Concurrent administration of an SRI and a glycine type 1 (GlyT-1) inhibitor Lundbeck, 2005 [215]
Concurrent administration of an SRI and Agomelatine Lundbeck, 2005 [216]
Concurrent administration of an SRI and Alverine Cerep, 2005 [217]
Concurrent administration of an SRI and an NMDA antagonist Forrest Labs, 2005 [218]
Concurrent administration of an SRI and Gaboxadol Lundbeck, 2004 [219]
Concurrent administration of an SRI and a nitric oxide synthase inhibitor National University of Ireland, 2004 [220]
Concurrent administration of an SRI and Modafinil Emory University, 2004 [221] Serotonin Reuptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1817
(Table 5) Contd….
Combination for treatment of depression Reference
Compounds with combined SRI activity and acetylcholinesterase inhibition Sankyo, 2002 [222]
Concurrent administration of an SRI and a sigma ligand Pfizer, 2002 [223]
Concurrent administration of an SRI and an estrogenic agent American Home Products, 2002 [224]
Concurrent administration of an SRI and Lithium University Prilly-Lausanne, 2001 [225]
Concurrent administration of an SRI and Lithium Free University of Berlin, 1995 [226]
Concurrent administration of an SRI and Buspirone University of Helsinki, 2001 [227]
Concurrent administration of an SRI, Buspirone and Pindolol Sanofi, 2000 [67]
Concurrent administration of an SRI and Buspirone University of Thessaloniki, 1998 [99]
Concurrent administration of an SRI and a betablocker SKB, 1999 [66]
Concurrent administration of an SRI and a 5-HT4 agonist McGill University, 2006 [228]
Concurrent administration of an SRI and Triiodothyronine Harvard University, 2005 [229]
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Scott P. Runyon and F. Ivy Carroll*
Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, North Carolina 27709, USA
Abstract: The dopamine transporter (DAT) is a target for the development of pharmacotherapies for a number of central disorders including Parkinson’s disease, Alzheimer’s disease, schizophrenia, Tourette’s syndrome, Lesch-Nyhan disease, attention deficit hyperactivity disorder (ADHD), obesity, depression, and stimulant abuse as well as normal aging. Considerable effort continues to be devoted to the development of new ligands for the DAT. In this review, we present some of the more interesting ligands developed during the last few years from the 3-phenytropane, 1,4-dialkylpiperazine, phenylpiperidine, and benztropine classes of DAT uptake inhibitors as well as a few less studied miscellaneous DAT uptake inhibitors. Studies related to the therapeutic potential of some of the more studied compounds are presented. A few of the compounds have been studied as pharmacotherapies for Parkinson’s disease, ADHD, and obesity. However, most of the drug discovery studies have been directed toward pharmacotherapies for stimulant abuse (mainly cocaine). A number of the compounds showed decreased cocaine maintained responding in rhesus monkeys trained to self-administer cocaine. One compound, GBR 12,909, was evaluated in a Phase 1 clinical trial. Keywords: Dopamine transporter, 3-phenyltropane, 1,4-dialkylpiperazine, phenylpiperidine, benztropine.
INTRODUCTION Extracellular dopamine (DA) for dopaminergic neuro- transmission is controlled largely by the dopamine trans- porter (DAT) [1]. In fact, the DAT is the primary mechanism by which extracellular DA is cleared from the synaptic space. The DAT regulates the concentration of DA available for binding to dopamine receptors, thus terminating synaptic transmission. Biochemical and pharmacological studies suggest that alteration in DAT function may play a part in Parkinson’s disease, Alzheimer’s disease, normal aging, schizophrenia, Tourette’s syndrome, Lesch-Nyhan disease, attention deficit hyperactivity disorder (ADHD), obesity, depression, and stimulant abuse [2-4]. Thus, DAT uptake inhibitors are a class of compounds that continue to be of high interest. The primary sequence of the DAT is divided into 12 hydrophobic regions that span the plasma membrane, Fig. (1). These putative transmembrane domains (TMs) are con- nected by hydrophilic loops. The crystal structure of a leucine transporter from Aquifex aeolicus, a bacterial homo- logue of Na+-dependent transporter like DAT, has been reported [5]. Both the leucine and the Na+ binding sites are within the TMs. The binding site for DA, Na +, and Cl- on the DAT may also be located on the TMs. It will be possible to generate a preliminary homology model of the DAT from this crystal structure, which may provide useful information Fig. (1). Schematic representation of the cloned DAT. for the design of potent and selective DAT uptake inhibitors. However, the new ligands developed over the last few years Numerous reports from several laboratories demonstrate have resulted from lead optimization of known DAT uptake the extensive variety of structural types of compounds inhibitors. capable of binding to the DAT [5, 6]. Reasons for this have not been established; nevertheless, many studies suggest that the DAT possesses a high degree of adaptability toward a *Address correspondence to this author at the Organic and Medicinal variety of ligand structures. This diversity of ligand Chemistry, Research Triangle Institute, P.O. Box 12194 / 3040 Cornwallis Road, Research Triangle Park, NC 27709, USA; Tel: 1 919 541-6679; Fax: structures complicates the problem of formulating a unique 1 919 541-8868; E-mail: [email protected] DAT binding site at the molecular level on which to base a
1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1826 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll complimentary relationship with its ligand. In the absence of and dependence liability [16, 17]. These efforts resulted in this information, structure-activity relationship (SAR) the development of WIN 35,065-2 (A2) which was five-fold studies are used to develop potent and selective DAT uptake more potent than cocaine and provided a new molecular inhibitors. SARs have been developed for several classes of template on which to further investigate the pharmacological compounds, and for the most part each class has its unique properties of compounds commonly referred to as 3- SAR requirements. In this review, we will present some of phenyltropanes [16, 17]. the more interesting compounds that have been developed Prior structure-activity studies with 3-phenyltropanes from SAR studies during the last few years (since 2003) indicate affinity for the DAT is highly dependent on the using inhibition of radioligand binding or inhibition of nature and position of substituents on the 3-phenyl ring [13, [3H]DA uptake. The compound classes will include analogs 18]. Early work incorporated 3'- or 4'-lipophilic substituents of 3-phenyltropane, 1,4-dialkylpiperazines, phenylpiperi- on the aryl ring in order to enhance affinity for the DAT dine, benztropine, and other miscellaneous classes. relative to WIN 35,065-2. In follow-on studies, our A. 3-PHENYLTROPANES laboratory synthesized 3',4'-disubstituted 3b-phenyltropanes as potential therapies for cocaine abuse. Recently, we Cocaine (A1) is one of the most commonly recognized reported that substitution of the 3b-phenyl group with tropane alkaloids due in part to its significant abuse and combinations of 3'- and 4'-chloro, -bromo, -iodo, or -methyl dependence liability. Thus, substituted derivatives of cocaine groups provides compounds with subnanomolar affinity at have received significant attention over the past 20 years as the DAT [19]. The 3'-chloro, 4'-bromo derivative A3 was potential pharmacotherapies for psychostimulant abuse. In non-selective among transporters, having an IC of 0.12 nM 1992, our laboratory summarized information strongly sug- 50 at the DAT, and had Kis of 0.23 and 0.65 nM at the 5-HTT gesting interaction of cocaine with the dopamine transporter and NET, respectively. Although 3'-halogens such as chloro, (DAT) as one of the molecular mechanisms responsible for iodo, and bromo are tolerated at the DAT, there appears to be the reinforcing effects of cocaine [7]. Since that time, a limited tolerance for 3'-hydroxy substituents. Meltzer and numerous tropane-based ligands have been synthesized for colleagues prepared catechol derivative A4 to determine if the purpose of selectively modulating the function of the the aryl group of 3-phenyltropanes interacted with the DAT dopamine transporter and ultimately providing pharmaco- in a manner similar to the 3,4-dihydroxyphenyl group of therapies for various disorders including depression, ADHD dopamine [20]. The monosubstituted 3-(4'-hydroxyphenyl)- [8, 9], Parkinson’s disease [10-12], and psychostimulant tropane A5 had an IC50 of 12.1 nM [21], while the 3',4'- abuse [7]. Fundamental research with tropane-based ligands dihydroxy analog A4 was 113-fold less potent at the DAT. has spawned several excellent reviews [6, 13-15] and This suggests that dopamine and the 3b-phenyltropanes do provided researchers many promising molecules. Because not occupy identical binding sites on the DAT. the study of tropane-based ligands has been extensively reviewed, our focus here will be on the most recent findings. The tolerance for 3'-position substitution on 3b-phenyl- tropanes has also been examined by Bois and colleagues Since no effective pharmacotherapy for cocaine abuse [22]. Introduction of 3'-(3-thiophene) (A6), 3'-(2-furanyl) has been discovered to date, the continued development of (A7), and 3'-phenyl (A8) provided compounds roughly tropane-based ligands has primarily focused on providing four–nine-fold more potent than cocaine. As an extension of high-affinity DAT ligands for the study of cocaine abuse. this work, Tamagnan and co-workers further probed the 4'- Clark and co-workers originally set out to separate the position of 3b-phenyltropanes with various heterocyles [23]. stimulant and depressant actions of cocaine from its toxicity
CH3 CH N 3 CH3 N N
CO2CH3 CO2CH3 CO2 CH3
O O Br
Cocaine WIN 35-065-2 Cl A1 A2 A3
CH3 H2N N CH3 N CO2CH3 CO2CH3 OH OH OH OH OH A4 A5 Dopamine Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1827
This work identified A10 and A11 as potent inhibitors of the [29, 30], and aryl groups [31]. Although the DAT has a DAT with Kis of 4.4 and 6.62 nM, respectively. The most broad tolerance for 2-position substitution, the nature of 2- interesting aspect of this study was selectivity of 4'- position substituents can impart selectivity for the DAT over heterocycles for the 5-HTT. Compound A9 had a Ki of 0.017 the NET, and the 5-HTT. A15 (RTI-177) was prepared nM at the 5-HTT and was 710-fold and 11,118-fold selective through bioisosteric replacement of the 2b-carbomethoxy for the 5-HTT over the DAT and NET, respectively. group with a 2b-isoxazole group [30]. A15 had an IC50 of Meltzer and colleagues have previously determined that 1.28 nM at the DAT and also had prominent DAT selectivity replacement of the 8-aza group on 3-phenyltropanes with 8- versus the NET (394-fold) and 5-HTT (1890-fold). Further b carba [24], 8-oxa [24], or 8-thia [25] groups did not exploration of this structural class led to numerous 2 -(1,2- isoxazoles) with high affinity and selectivity for the DAT significantly alter DAT affinity. Flattening the tropane ring through introduction of a 2,3-double bond was also shown to [32]. Substitution on the 1,2-isoxazole ring with alkyl or 4'- enhance DAT selectivity over the 5-HTT. Thus, Meltzer and substituted phenyl groups provided a series of selective co-workers [25] have extended their work to include 8- compounds with IC50s ranging from 0.50–26 nM at the DAT. In all cases compounds having a 3b-(4'-chlorophenyl) group thiabicyclo[3.2.1]oct-2-enes. Replacement of the 8-oxo b functionality with an 8-thia group provided a series of highly as opposed to a 3 -(4'-methylphenyl) group were more potent analogs. Affinity for the DAT was dependent on the potent at the DAT. size of the 4-position aryl substituent. Replacement of 4- Since 2-substituted isoxazoles possessed properties favor- chloro (A12a, IC50 = 13 nM) with 4-bromo (A12b, IC50 = able to cocaine abuse pharmacotherapies, locomotor stimula- 9.1 nM), 4-iodo (A12c, IC50 = 6.7 nM), 3,4-dichloro (A12d, tion, and gross observation were evaluated in mice and drug IC50 = 4.5 nM) and 3,4-naphthyl group (A12e, IC50 = 8.0 discrimination properties were evaluated in rats [32]. In nM) enhanced affinity at the DAT. particular, A16 (RTI-336), which had an IC50 of 4.09 nM at the DAT, an IC of 1710 nM at the NET, and an IC of In an attempt to expand the SAR for 3-aryl-4-substituted 50 50 (para position) heterocyclic derivatives, Torun and others 5741 nM at the 5-HTT possessed favorable in vivo proper- synthesized a series of 2b,3a- and 2b,3b-3-aryl-8-oxa- ties. It completely generalized to cocaine and had an ED50 of bicyclo[3.2.1]octanes with various 4-heterocycles [26]. The 3.9 mg/kg for locomotor stimulation with 70% efficacy relative to cocaine and a peak onset in hour 3. The slow 2b,3b-4-(2-furanyl) derivative (A13) was the most potent with an IC of 64 nM at the DAT and an IC of 30 nM at onset, long duration, selectivity profile, and cocaine genera- 50 50 A16 the 5-HTT. The 2b,3b-4-N-methylpyrrole derivative (A14) lization proper-ties indicate encompasses the attributes thought useful for potential cocaine pharmacotherapies. was the most selective for the 5-HTT having an IC50 of 566 nM and an IC50 at the DAT of 12,400. Although 2b-(1,2-isoxazole) compounds in general were potent and selective inhibitors of the DAT, A17 (RTI-370) Structural modification of the 2b-position on 3-phenyl- A18 tropanes has provided several interesting results. The broad and (RTI-371) displayed unusual properties in vivo. tolerance of the 2-position to structural modification is Both were potent and extremely selective ligands with IC50s of 8.74 and 13 nM at the DAT and had IC s of 6980 and evident through the diversity of analogs that have equal or 50 greater affinity than cocaine and WIN 35-065-2 at the DAT. 100,000 nM at the NET and IC50s of 100,000 nM at the 5- Examples include 2b-esters, amides [27, 28], heterocycles HTT. These compounds appear to have a unique
CH3 N R1 = O CO2CH3 S
A6 A7 A8
R1 CH 3 CH3 N N S
CO2CH3 CO2 CH3 CO2CH3 S
A9 A10, X = Cl S X A11, X = Br A12
O O A12a, R = Cl R A12b, R = Br CO CH CO CH CH 2 3 2 3 3 A12c, R = I O N A12d, R = 3,4-dichloro A12e, R = 3,4-naphthyl A13 A14 1828 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll
CH 3 CH3 N O N N O N
CH3
Cl Cl
A15 (RTI-177) A16 (RTI-336)
CH 3 CH3 N O N N O N CH3 Cl
CH3 CH3
A17 (RTI-370) A18 (RTI-371) pharmacological profile because neither compound produced NET and 5-HTT respectively. Addition of a 4'-CH3 (A21) or locomotor stimulation in mice. In addition, A17 weakly a 4'-OCH3 (A22) group to the 2-phenyl ring did not generalized to cocaine while A18 did not generalize at all. significantly alter DAT affinity or selectivity; however, This phenomenon has been observed for other classes of compounds with a 2-(3'-4'-dimethoxyphenyl) group (A23) DAT uptake inhibitors [33] and could potentially be caused decreased affinity at the DAT by 10-fold but enhanced by a lack of brain penetration or most likely interactions at selectivity over the 5-HTT and NET. alternate receptor sites. Clearly, compounds lacking The preparation of bivalent ligands with varied linkers locomotor stimulation but possess potent and selective DAT has led to the development of a number of compounds with affinity require further investigation. interesting biological activity. Taking advantage of the 2- The diarylmethoxy group has been shown to impart high position tolerance for substitution, Fandrick and colleagues DAT affinity when incorporated on the 3-position of the [36] synthesized a series of bivalent analogs of RTI-31 benztropine nucleus [6, 13, 34]. In order to further examine having amide linkers separated by spacers of 3 to 8 2-position structural requirements for 3-phenyltropanes, Xu methylene units (24a–d). The order of potency increased and colleagues synthesized a series of 2-diarylmethoxy with increasing spacer lengths. The most potent compound tropanes [35]. In general, the 2b-isomers displayed higher 24d had a Ki of 6.7 nM at the DAT with an uptake IC50 of affinity (Kis = 34–112 nM) than the 2a isomers (Kis = 871 nM. Although DAT uptake did not correlate with 60–690 nM), while the unsubstituted diarylmethoxy analog potency the apparent affinity of 24d demonstrates a unique A19 had the highest affinity at the DAT (Ki = 34 nM). mode of binding for bivalent tropane analogs. Based on prior studies with cocaine [37], it was recognized that the stereochemistry at the 2- and 3-positions may prove important for modulating the pharmacological properties of 3-phenyltropanes. A series of substituted 2- carbomethoxy-3-(4'-substituted)phenyltropanes encompas- CH3 N O sing all four 2- and 3-position stereoisomers were synthesi- zed and evaluated for DAT binding, locomotor activity, and drug discrimination properties [38]. Generally, the order of potency for DAT binding was 2b,3b>2b,3a >2a ,3a > CH3 2a ,3b. In all cases, substitution of the 4'-position with halogens or CH3 enhanced affinity of 3-(4'-substituted) A19 phenyltropanes at the DAT. Locomotor stimulation and drug discrimination paralleled that of binding and was most Modification of the 2-position through replacement of the prominent for 2b,3b derivatives followed by 2b,3a. One of 2-carbomethoxy group with substituted aromatic rings has the more interesting findings with this study was the fact that also provided compounds with high DAT affinity and 2a ,3a 4'-methylphenyl derivatives (A25) and 2a ,3a 4'- selectivity. Our laboratory has prepared a series of 2b,3b- chloro (A26) were roughly four- and eight-fold more potent and 2a ,3a -diaryltropanes, and in all cases the 2b,3b than cocaine at the DAT but displayed no locomotor derivatives had higher affinity at the DAT [31]. Compound stimulation and drug discrimination properties. These A20 having an unsubstituted 2-aryl group had an IC50 of 1.96 properties are comparable to that observed for A17 and A18. nM at the DAT and IC50s of 480 nM and 1100 nM at the Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1829
CH3 N
R1 R1 A20, R = H, R = H CH3 R 1 2 N 2 A21, R1 = H, R2 = CH3 R2 A22, R1 = H, R2 = OCH3 A23, R1 = OCH3, R2 = OCH3
CH3 2b,3b 2a ,3a CH3
CH3 CH3 N O O N (CH )n N 2 N H H
Cl Cl
A24 24a, n = 1 24b, n = 2 24c, n = 4 24d, n = 6
CH H H CH3 3 N N N N CO2CH3 CO2 CH3 A27 R = CH CO CH 1 3 CO2CH3 2 3 A28 R = Cl 1 A30 A29 R1 = F
F
R1 CH3 Cl CH3 B. 1,4-DIALKYLPIPERAZINES A25 A26 Van der Zee et al. was the first to report on the 1,4- Nortropane derivatives lacking an N-alkyl substituent dialkylpiperazine class of DAT uptake inhibitors [41]. Two display enhanced selectivity for the NET [39]. In addition, of the more interesting compounds developed were GBR the tropane ring conformations can be influenced to favor a 12,909 (B1) and GBR 12,935 (B2). Similar to cocaine and 3- boat or chair by altering the stereochemistry of the 2- and 3- phenyltropane analogs, the 1,4-dialkylpipera-zines showed position substituents. 2b,3b-Substitution favors the chair inhibition of radioligand binding to the DAT [42-45]. conformer while 2b,3a substituents favors a flattened boat However, in contrast to cocaine and 3-phenyltro-pane conformer. Our laboratory has synthesized a series of 2b,3a - analogs, which show a high and low affinity binding site, the nortropanes in order to determine structural features 1,4-dialkylpiperazines label only one DAT site [45]. The necessary for affinity and selectivity for the NET [40]. 1,4-dialkylpiperazines also bind to a piperazine-acceptor site Substitution of the 4'-position on 2b,3a -aryl nortropanes [45]. with methyl, fluoro, or chloro substituents (A27–A29) GBR 12,909 (B1) has an affinity for the DAT similar to provides relatively nonselective compounds with IC50s that of WIN 35,065-2 (A2). It was found that pretreatment ranging between 5 and 9 nM at the NET and 3–34 nM at the with B1 produced reduction of cocaine self-administration in DAT and 53–500 nM at the 5-HTT. The 2b,3a derivative rhesus monkeys [46, 47], and high levels of DAT occupancy A30 has the greatest affinity and selectivity for the NET. It were observed for doses of B1 that produced robust incorporates a 3'-fluoro, 4'-methylphenyl group and had a Ki decreases in cocaine self-administration [47]. Based on this of 0.43 nM at the NET and was 21-fold selective versus the and other preclinical development information, B1 was DAT and 55-fold selective versus the 5-HTT. This NET developed as a potential indirect DA agonist for treatment of selective compound now provides a tool capable of deter- stimulant abuse. Unfortunately, it was not possible to mining the importance of the NET regarding psychos- determine its potential as a pharmacotherapy, since clinical timulant abuse. studies were not continued beyond Phase 1 because of cardiovascular QTc problems [48]. 1830 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll
R R F F benzhydryl ether
O O ethylene ether
N N(CH2)3C6 H5 N proximal nitrogen
B1, R = F N distal nitrogen B2, R = H phenylpropyl (CH2)3 substitutent B3
Even though GBR 12,909 may not be a useful Compound (S)-B6 was also highly selective relative to the 5- pharmacotherapy, it has served as a highly useful structure HTT and NET [53]. Analogs (S)- and (R)-B7, which are the for lead optimization of the 1,2-dialkylpiperazine class of desfluoro analogs of (S)- and (R)-B6, were almost as potent DAT uptake inhibitors. Numerous analogs have been as (S)- and (R)-B6 at the DAT, with a Ki of 1.4 and 6.9 nM, synthesized and evaluated for inhibition of DAT binding. respectively. (S)-B7 was even more selective for the DAT The studies have involved modifications of the benzhydryl than (S)-B6. When the hydroxy group was moved to the C-3 ether, piperazine ring, the ethylene ether linker between the position in the difluoro or desfluoro analogs, the resulting benzhydryl moiety and the proximal piperazine nitrogen and compounds (S)- and (R)-B8 and (S)- and (R)-B9 retained the phenylpropyl substitutent on the distal nitrogen (see high affinity for the DAT (Ki of 0.8 to 2.1 nM) and were still structure B3). SARs resulting from earlier studies have been selective, particularly relative to the NET [53]. Exchanging summarized in previous reviews [13, 49]. Significant new the hydroxyl groups in (S)- and (R)-B6 by methyl groups had SAR findings will be presented in this review. opposite effects [53]. Compound (S)-B10 with the (S)- oriented methyl group had a K of 8 nM and is 20 times less Interesting New 1,4-Dialkylpiperazines Analogs i potent than (S)-B6. In contrast, (R)-B10 with a Ki of 0.9 nM One of the problems with GBR 12,909 (B1) is high is six times more potent than (R)-B6 [53]. Exchanging the C- lipophilicity. In order to overcome this problem, B1 analogs 3 position hydroxy groups in (S)- and (R)-B8 with methyl possessing additional heteroatoms have been studied. Along groups had little effect on DAT binding affinity. (R)- and this line, (R,S)-, (R)-, and (S)-B4, which has a hydroxy group (S)-B11 had Kis of 2.2 and 1.3 nM compared to Ki values of at the C-3 position on the phenylpropyl group, was deve- 0.9 and 0.8 for (S)- and (R)-B8 [53]. loped [50]. Racemic B4 as well as the (R)- and (S)-isomers Replacement of the N-phenylpropyl group of the GBR all possessed about the same affinity (Ki of 2.1 to 4.4 nM) 12,909-piperidine analog (proximal nitrogen replaced with a for the DAT [50]. In contrast, the C-2 position hydroxy methine group) with N-(4-substituted benzyl) groups, analog, (S)-(+)-B5 with an IC50 of 0.75 nM was 16 times compound B12, provided analogs that retained high DAT more potent than (R)-(-)-B5 [50]. affinity [54, 55]. Compound B12 with X = 4-F, 4-Cl, 4-Br, Earlier SAR studies had shown that removal of the 4-I, 4-CF3, 4-CN, and 4-NO2 had Ki values of 0.98 to 1.9 nM proximal nitrogen in 1,2-dialkylpiperazine analogs did not compared to 9.9 nM for the unsubstituted compound [55]. have a significant effect on DAT affinity [51, 52]. However, The 4-methyl and 4-methoxy analogs (B12, X = CH3 or this change reduces chances of binding to the piperazine site CH3O) had Ki values of 5.7 and 7.9 nM [53]. The most [52]. Compound B6 is a C-2 position hydroxy GBR 12,909 potent compound evaluated was the 2-naphthyl analog B14, analog in which the proximal nitrogen has also been replaced which had a Ki of 0.7 nM at the DAT [56]. All of the 4- by a methine group [53]. The analogs (S)- and (R)-B6 were substituted analogs showed greater than 100-fold selectivity highly potent at the DAT, Ki of 0.4 and 5.1 nM, respectively. for the DAT relative to the 5-HTT and NET. As expected,
F F F F
O O OH N N N N
OH B4 B5 Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1831
R R R R
O O
R1 R2 N N
R1 R2
(S)-B6, R = F, R1 = H, R2 = OH (S)-B8, R = F, R1 = H, R2 = OH (R)-B6, R = F, R1 = OH, R2 = H (R)-B8, R = F, R1 = OH, R2 = H (S)-B7, R = H, R1 = H, R2 = OH (S)-B9, R = H, R1 = H, R2 = OH (R)-B7, R = H, R1 = OH, R2 = H (R)-B9, R = H, R1 = OH, R2 = H
F F F F
O O
R1 R2 N N
R1 R2
(S)-B10, R1 = F, R1 = H, R2 = CH3 (S)-B11, R1 = H, R2 = CH3 (R)-B10, R1 = F, R1 = CH3, R2 = H (R)-B11, R1 = CH3, R2 = H
R R
O O
X
N CH2 N CH2 B12, R = F B14 B13, R = H
O O HO OH
N CH F N CH F B15 2 B16 2
the desfluoro analogs B13 (X = 4-F, 4-Cl, 4-Br, 4-I, 4-CF3, was separated into its (S,S)- and (R,R)-isomers. The (R,R)- 4-CN, 4-NO2) were slightly less potent than the corres- isomer with a Ki of 0.46 nM was 123 times more potent at ponding B12 analog, Ki values of 2.5–7.2 nM [54]. The 3- the DAT than the (S,S)-isomer. This analog was also highly substituted analogs B12 (X = 3-F, 3-Cl, 3-Br, 3-I, 3-CN, 3- selective for the DAT relative to the 5-HTT and NET. (R,R)- NO2) were all slightly less potent than the corresponding 4- and (S,S)-B16 were also evaluated for their ability to inhibit substituted analogs. The 3- and 4-methyl analogs of B12 (X synaptosomal uptake of DA, 5-HT, and NE and compared to = 3-CH 3 or 4-CH3) possessed about the same DAT affinity. values for GBR 12,909 (B1) [58]. (R,R)- and (S,S)- B16 have The 2-substituted analogs B12 (X = 2-F, 2-Cl, 2-Br, 2-I, 2- Ki values of 3.01 and 28.3 nM in DAT uptake compared to CN, 2-NO2) all had relatively low affinity for the DAT. 10.6 nM for GBR 12,909 (B1). However, (R,R)-B16 was 360- and 66-fold selective for DA uptake relative to 5-HT The addition of a hydroxy group to the piperidine ring of B1 B13 provides cis-B15 and trans-B16, which have K values and NE uptake. GBR 12,909 ( ) showed only 8.6- and 9.6- i fold selectivity for DA uptake over that of 5-HT and NE. of 11.2 and 4.1 nM, respectively [57]. The trans isomer B16 1832 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll
Compounds B17 and B18 can be viewed as confor- enantioselectivity was observed for the minus isomer of C1 mationally-constrained analogs of B1 [59]. B17 also has a (IC50 of 85.2 nM) versus the plus isomer of C1 (IC50 of 5090 hydroxy group attached to the constrained portion. Analogs nM) in DAT uptake. In addition, little correlation was B17 and B18 possessed Ki values of 1.3 and 6.4 nM at the observed between DAT uptake potency and relative DAT compared to B1 of 3.7 nM in the same study [59]. configuration of the 3- and 4-position substituents. The trans Compound B19, (S,S)-B20, and (R,R)-B20 are also confor- derivative C2 had an IC50 of 34.6 nM while the cis derivative mationally-constrained analogs [53]. All three compounds C1 had an IC50 of 85.2 nM. The relative potency is reversed possess high affinity for the DAT (Ki of 0.5 to 1.4 nM). in the case of 3-n-propyl derivatives. The trans derivative C6 C4 Replacement of the 4-fluorobenzhydryl group of B1 with ( ) had an IC50 of 23.3 nM while the cis isomer ( ) had an IC of 8.3 nM. Substitution of the 4-aryl group with 4'- a bis-(4-fluorophenyl)amine group and the ether oxygen with 50 C1 a methylene group gives B21, which retained moderate hydrophobic substituents such as chloro ( ) and 2-naphthyl C5, affinity for the DAT [60]; K of 18.1 nM compared to 11.9 [68] ( IC50 of 23 nM) enhance potency for the DAT i C3, nM for B1. An analog of B21 where the phenylpropyl group relative to hydrogen ( IC50 of 769 nM). Replacement of the 3-carbomethoxy group with 3-position alkyl groups also was replaced by phenylpropanol (B22) [60] or anilinopro- C4 panol B23 group [61] showed affinity at the DAT slightly enhance potency at the DAT: cis-(-)- is one of the more better than B21. The GBR 12,909 (B1)-like compounds B24, potent derivatives with an IC50 of 8.3 nM at the DAT. B25, and B26 also showed moderate affinity for the DAT A pharmacophore model has been proposed by Petukhov [61, 62]. The iodine-125 derivative of the 3'-iodo, 4'-azido and colleagues based on structure-activity studies with analog [125I]B27 was prepared and shown to photolabel the various 3-substituted piperidine derivatives [69]. A 10 Å DAT protein [63]. The 3,6-disubstituted piperidine B28 was conical region extending from the 3-positon has been designed as GBR 12,935 (B2) analogs [64]. The (S,S)-B28 hypothesized to encompass a sterically allowed region with a Ki of 11.3 nM showed the highest affinity for the between 0 and 5.5 Å and a sterically prohibited region DAT relative to GBR 12,909 (Ki of 10.6 nM). Bridging the between 5.5 and 10 Å from the piperidine ring. In order to two nitrogen with an ethylene group gave the 1,4-diaza- further explore this hypothesis, a series of derivatives having bicyclo[3.3.1]nonane analog B29 [65]. The 4-methoxybenzyl elongated 3-position spacers were prepared [69, 70]. In analog (S,S)-B29 with a Ki of 22.5 nM was the most potent general, compounds with greater 3-position flexibility were at the DAT. Replacement of the piperidine nitrogen in B28 more potent at the DAT. Racemic C2 (Ki of 233 nM) has the with an oxygen gives B30 (X = various substitutents), which carbomethoxy group directly attached to the piperidine ring show lower affinity for the DAT [66]. and is three-fold less potent than C8 (Ki of 79 nM) having a methylene spacer between the tropane ring and the ester C. PHENYLPIPERIDINE DERIVATIVES group. Extending the spacer to two methylenes provides C12 Kozikowski and colleagues investigated a series of with a Ki of 68 nM at the DAT. Another example of this piperidine derivatives that preserve the essential pharmaco- trend is seen by comparing compounds C7 and C10. phore features of 3-phenyltropanes yet enhance flexibility of Addition of a one carbon spacer significantly enhanced the tropane ring [67]. Removal of the ethylene bridge from potency for C10 (Ki of 44 nM) versus C7 (Ki of >3,000 nM). RTI-31 provided a series of piperidine derivatives with Two additional derivatives having greater flexibility at the 3- moderate efficacy at the DAT. In general, prior structure- position are C9 and C11. Carboxamide C9 has a Ki of 16 activity studies established several key piperidine features as nM, whereas phenylpropyl ester C11 has a Ki of 36 nM at important for efficacy at the DAT [67]. A marked the DAT. Substitution of a terminal hydroxyl or fluorine for
F F F F
O O HO
N N N N B17 B18
F F F F
O O
N N B19 (S,S)-B20 (R,R)-B20 Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1833
F F F F
N N
HO
N N N N N B21, R = H B23 H B22, R = OH R
F F F F
O HO HO
N N N N N N B24 H B25 H
F F F F
N I
N3 HO
N N N N N B26 H B27
HN N O
NHCH2 CN N CH2 OCH3 NHCH2 X B28 B29 B30 hydrogen on the 3-alkyl chain provided C13 and C14. Interestingly, the cis isomer C21 [72] was only two-fold less Fluoro substitution (C13, Ki = 43 nM) slightly enhanced potent at the DAT but was 26-fold less potent at the NET. potency but significantly improved selectivity for the DAT Oxidation of the sulfide C15 to the sulfoxide C16 had little and NET over the 5-HTT. Hydroxy derivative C14 had a Ki effect on potency (Ki of 12 nM) [70]. Methylation of the of 16 nM at the DAT, 2810 nM at the 5-HTT, and 564 nM at terminal -OH provided compound C17 with a Ki of 15 nM, the NET and was 175- and 35-fold selective for the DAT and whereas acetylation afforded C18 with a Ki of 9 nM. N- NET, respectively. In summary, it appears that flexibility of Methyl amide derivative C19 had a Ki of 13 nM while the the 3-position piperidine substituent is more important in cis isomer C22 was 300-fold less efficacious at the DAT determining potency than distance from the piperidine ring. [72]. N-Isopropyl carboxamide C20 was potent but nonselective among the transporters having a K of 1 nM at Since substitution at the 3-position of the piperidine ring i the DAT, NET, and 5-HTT. The cis isomer C23 also posse- was identified as an important component for altering transporter selectivity and efficacy, Zhou and colleagues ssed high affinity at the DAT and the NET with Kis of 13 and 16 nM but had reduced affinity at the 5-HTT (K of 154 prepared a series of piperidines, which possessed a i modafinil-like substituent at the 3-position [71]. Two series nM). were prepared having either a thioether or an oxidized D. BENZTROPINES sulfoxide linker. Modifications were then made at the terminal end with a variety of substituents. Compound C15, As the title indicates, benztropine (D1) has served as the having a terminal -OH, had a K of 16 nM at the DAT but lead structure for this class of DA uptake inhibitors. Even i though benztropine is better known as an anticholinergic was more potent at the NET (Ki of 0.94 nM) [71]. 1834 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll
H3 C N CO2 CH3 CO CH CO CH H3C N 2 3 H3 C N 2 3 Cl Cl R1
RTI-31 (-)-C1, R1 = Cl (+)-C1 (-)-C3, R1 = H (-)-C5, R1 = 2-naphthyl
H3C N CO2CH3 H C N H3C N 3 Cl Cl Cl
(+)-C2 (-)-C4 (+)-C6
R R2 3 H3 C N H3 C N R1 H3C N Cl Cl Cl
C2, CO2 CH3 C8, CO2C2H5 C12, CO2CH3
O C7, N C13, -CH2-F C9, CH3 O O N C14, -CH -OH N 2 O CH3 N C10, O O N O O C11, O
H3C N R1 = R1 R1 = Cl O
C15, C21, H2C S OH C18, H2C S O CH3
C15–C20 O O
CH3 C16, H2C S OH C19, C22, H2C S N R H O H3C N 1 Cl O CH3 C17, H2C S O C20, C23, H2C S N O C21–C23 H Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1835 agent, it has reasonable affinity for the DAT. Studies to aminopropyl group provided D3 and D4, which had Kis of improve DAT affinity have investigated the effects of 13.9 and 26 nM, respectively. The N-benzoyl (D5) and N- changes of the tropane ring and the benzhydryl ether group. nicotinoyl (D6) analogs of D3 had K is of 13.1 and 12.6 nM, These studies have been summarized in previous reviews [6, respectively. The reverse amide analog (D7) of D5 had a Ki 13, 34]. The addition of small halogens to the 3- or 4- of 27.6 nM. position on the phenyl rings improved DAT affinity comp- Recent studies of the previously reported N-butyl-3a - ared to benztropine. One of the best compounds developed [bis(4-fluorophenyl)methoxy]tropane (D8, JHW007) have D2 was 4',4'-difluorobenztropine ( ). Replacement of the N- shown that this relatively selective DAT uptake inhibitor D2 methyl group in with a 4-phenylbutyl group had little with a K of 24.6 nM [74] does not show cocaine-like effect on DAT affinity but improved DAT selectively i stimulant or subjective effects [75]; properties very similar to relative to the muscarinic receptor [34]. the 3-phenyltropane analog RTI-371 (A18), which was discussed in the 3-phenyltropane section. In vivo binding CH3 N studies with rats showed that D8 associates with the DAT slowly. The compound was found to antagonize the locomotor stimulant and subjective effects of cocaine [75]. X Compound D8 had some affinity for muscarinic receptors. However, a study on a number of muscarinic ligands showed that they did not antagonize cocaine locomotor effects [76]. O Benztropine analogs, where an a - or b-hydroxy or D1, X = H methoxy group has been added to the 6-position of the D2, X = F X tropane ring, have been synthesized and evaluated for inhibition to the DAT [77-79]. The compounds showing the
Recent SAR studies have continued to evaluate the effects highest DAT affinity were the (±)-6b-hydroxy and (±)-6a - of changes to both the tropane ring and the benzhydryl ether methoxyl-4',4'-difluorobenztropine [(±)-D9 and (±)-D10, group. In order to improve the pharmacological and pharma- respectively]. Compounds (±)-D9 and (±)-D10 possessed Kis cokinetic properties of the benztropine class of DAT uptake of 12 and 10 nM, respectively. 6a -Hydroxy-4',4'-difluoro- inhibitors, a series of N-substituted analogs possessing hetero benztropine (D11) with a Ki of 140 nM is 12 times less atoms were designed, synthesized, and evaluated for DAT potent than D9. In a separate study, (±)-D9 and (±)-D10 affinity and for selectivity relative to the 5-HTT, NET, and were reported to have Kis of 6.28 and 31.3 nM at the DAT muscarinic M1 receptor [73]. Exchanging the N-methyl [79]. The (1R)- and (1S)-6b-methoxy-4',4'-difluorobenztro- group in D2 (Ki of 11.8 nM) for an aminoethyl and pines [(1R)-D12 and (1S)-D12] were synthesized and their
H2N(CH2 )n N O H R N C N
F
O F O
D3, n = 2 D5, R = C6H5 D4, n = 3 F D6, R = N F
O
C6H5 N N C4H9 N H
F
O O
D7 D8 F 1836 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll
CH3 N
F HO
O
D9 (±)- F
CH3 N CH3 N
F F
CH3O HO O O
(±)-D10 F (±)-D11 F
CH 3 N CH3 N
CH3O F F CH3O
O O
(1S)-D12 (1R)-D12 F F
DAT affinity compared. The (1R)-isomer had a Ki of 25 nM Compound D15 can be viewed as a GBR 12,909 analog compared to 95 nM for the (1S)-isomer [77, 78]. or a 4',4'-difluorobenztropine analog with a two methylene The isosteric replacement of benzhydryl ether group with linker between the tropane ring and the benzhydryl ether a benzhydryl amino moiety led to analogs with affinity group [81]. This compound had a Ki of 52 nM. The N- D17 similar to their ether counterparts [80]. For example, the phenylpropyl analog possessed a Ki of 19 nM in one difluoro analog D13 has a K of 11.3 nM at the DAT report [81] and 7.4 nM in another report [82]. The N-benzyl i analog D16 had a K of 5.7 nM [82]. Compound D18, which compared to 4.11 nM for the ether analog D2 [80]. The N- i has a double bond methylene linker between the tropane ring ethylamino-4',4'-difluoro analog D14 had a Ki of 12.5 nM at the DAT compared to 5.59 nM for the ether analog D3 [80]. and the benzhydryl ether, has a Ki of 19 nM [81]. The N- benzyl and N-phenylpropyl analogs D19 and D20 had K s of This compound was also highly selective showing i 7.9 and 3.7 nM, respectively [82]. SERT/DAT, NET/DAT, and M1/DAT ratios of 872, 284, and 169, respectively. In earlier studies, Meltzer et al. developed some benztropine analogs that possessed a 2- carbomethoxy group [83]. (S)-2b-Carbomethoxy-3a -[bis(4'-fluorophenyl)me- R N thoxy]tropane (D21), with a Ki of 10.9 nM had a higher affinity for the DAT than the seven other possible isomers [83]. The ethyl, isopropyl benzyl, and phenylethyl ester F D22–D25, respectively, had Kis of 16.4, 30.3, 48.4, and 36.7 nM compared to 13.4 for D21 in the same study [84].
HN Compound D26 can be viewed as a flexible analog of D2. Compound D26 possessed an IC50 of 22 nM at the DAT compared to 420 nM for the standard DPP (D27) [85]. The F compound showing the highest DAT affinity in these flexible analogs was the N-butyl-4',4'-dichloro analog D28, D13, R = CH3 which had an IC of 12.5 nM [85]. D14, R = CH2CH2NH2 50 Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1837
R N R N CH3 N CO2R F
F F O O O
D21, R = CH3 F D15, R = CH 3 D22, R = CH2CH3 D16, R = C H CH D18, R = CH 6 5 2 F 3 F D23, R = CH(CH3 )2 D17, R = C H (CH ) D19, R = C H CH 6 5 2 3 6 5 2 D24, R = CH2Ph D20, R = C H (CH ) 6 5 2 3 D25, R = CH2CH2Ph
X Cl CH3 N C4 H9 N
O O
X D26, X = F D27, X = H D28 Cl
Using combinatorial methods, a library of benztropine 51 nM), -CH2OH (E6, Ki of 49 nM), and –Cº CCH3 (E7, K i analogs having structures D29 were synthesized where R and of 61 nM). A moderate enhancement in DAT affinity over R' were varied [51]. None of the analogs possessed DAT pyrovalerone was observed for 3,4-dichloro (E8, K i of 11.5 affinity higher than that of benztropine (D1). nM) and naphthyl derivatives (E9, K i of 20.1 nM). The two most selective compounds from this series were E4 and E10. E10 was very DAT selective against the 5-HTT having K s R' i N of 125 nM, >10 mM, and 1290 nM at the DAT, 5-HTT, and NET, respectively, while E4 was both potent and highly selective for the DAT having Kis of 30 nM, >10 mM, and 4000 nM at the DAT, 5-HTT, and NET, respectively. Addi- tion of a methyl group to the alkyl chain moderately enhanced affinity compared to pyrovalerone (E11, K of 14 O i R nM) while shortening the chain by one carbon slightly reduced affinity (E12, K i of 28.8 nM). Further evaluation of pyrovalerone analogs in drug discrimination assays may pro- vide interesting leads for the treatment of cocaine addiction. D29 Indatraline (E13) is a high affinity, non-selective monoamine transport inhibitor that has shown promise as a E. MISCELLANEOUS DAT UPTAKE INHIBITORS potential pharmacotherapy for methamphetamine abuse [88]. Indatraline has K s of 1.7 nM, 0.4 nM, and 6 nM at the DAT, Pyrovalerone (E1, K of 21 nM), a closely related analog i i 5-HTT, and NET, respectively [89]. Studies aimed at of bupropion (E2), was reported to inhibit the DAT and the developing long lasting depot derivatives of indatraline were NET by Lancelot and colleagues in 1992 [86]. Although undertaken by Yu and colleagues [90]. 6-Oxy analogs were clinical interest in pyrovalerone has emerged, little SAR has prepared to provide an attachment point for long chain been reported for this structural class. In order to further alkanoic acids. Addition of either a 6-methoxy (E14, K of investigate potential pharmacotherapies for cocaine addic- i 12 nM) or 6-hydroxy group (E15, K of 11 nM) afforded tion, Meltzer and co-workers evaluated a family of pyrovale- i compounds with high affinity at the DAT, although both rone analogs as monoamine transporter inhibitors [87]. compounds were slightly less potent than indatraline (E13, Resolution of pyrovalerone provided compounds that Ki of 1.7 nM). The 6-substituted analogs were also enantio- displayed significant enantioselectivity at the DAT. The 2S selective among all three monoamine transporters. isomer had a Ki of 18 nM at the DAT and was 74-fold more Resolution of the trans diastereomers provided (1R,3S)-E14 potent than the 2R isomer (Ki of 1330 nM). Minimal effects having a Ki of 9 nM while the (1S,3R)-E14 had a Ki of 600 on DAT potency were observed for removal of the 4-methyl nM at the DAT. 6-Hydroxy derivatives followed a similar group (E3, Ki of 34 nM) or replacement of the 4-methyl pattern. (1R,3S)-E15 had a Ki of 12 nM while (1S,3R)-E15 group with -NHCOCH3 (E4, K i of 30.2 nM), -Br (E5, Ki of had a Ki of 86 nM at the DAT. 1838 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll
O O O N H N N
H3C R1
E1 Cl E2 R2
E3, R1 = -H, R2 = H O E4, R = -NHCOCH , R = H O 1 3 2 N E5, R1 = -Br, R2 = H N E6, R1 = -CH2OH, R2 = H E7, R1 = -C CCH3, R2 = H E8, R1 = R2 = Cl E10, R1 = -OH, R2 = H
E11 E9
O N
Cl Cl
E12
Evaluation of E15 in monoamine reuptake and autoradio- most potent oxacyclic compound in the series was compound graphic assays provided interesting results. Although E17, which has the (2R,2’R) stereochemistry, IC50 = 17 nM (1R,3S)-E15 displayed roughly equal efficacy between DA at the DAT and >10,000 nM at the 5-HTT. Carbon analog (Ki of 3 nM) and 5-HT (2 nM) uptake inhibition and only a E18 also retained affinity and selectivity for the DAT having six-fold lower binding affinity at the DAT, (1R,3S)-E15 an IC50 of 47 nM at the DAT and 7000 nM at the 5-HTT. persistently occupied the 5-HTT in rat brain over the DAT. Many new structural classes have been examined as Clearly, more data are needed to better understand the inhibitors of the dopamine transporter since the subject was relationship between transporter occupancy and uptake last reviewed. In this review we have highlighted the most efficacy for indatraline-like inhibitors. potent and selective molecules from each structural class; however, several other unique structures have been identified HN CH3 that possess either moderate affinity and or moderate selectivity for the DAT. Rhoden and colleagues prepared R 1 meperidine derivatives (E19) having altered N-and ester substituents [94]. Krunic and co-workers studied a series of 3-aryl substituted trop-2-enes (E20) [95]. Kim and others prepared a series of 3-azabicyclo[3.2.1]octanes (isotropanes) with a variety of 8-substituents (E21) [96]. Airaksinen and Cl colleagues have investigated 6- and 7-methyl derivatives of 3-phenyltropanes (E22) [97]. Quirante and others have Cl evaluated 6-azabicyclo[3.2.1]octanes (E23) [98]. Dar and E13, R1 = H others have examined trihexylphenidyl analogs (E24) as E14, R1 = OCH3 DAT ligands [99]. Sakamuri and co-workers synthesized a E15, R1 = OH series of quinuclidine analogs (E25) [100], and Kozikowski and others have significantly expanded their work on Meltzer and colleagues have shown that replacement of tricyclic tropanes (E26) [101-103]. the 3-phenyltropane nitrogen with oxygen, carbon, or sulfur provides compounds that retain affinity for the DAT[24, 25]. F. THERAPEUTIC POTENTIAL FOR DAT LIGANDS A continuation of this work led to the discovery of methyl- The ability of compounds to selectively modulate DAT phenidate derivatives where the aryl ring was disubstitued function has provided pharmacotherapies that may poten- with 3,4-Cl, and nitrogen was replaced with oxygen and tially address a number of clinically significant disorders. carbon[91]. Methylphenidate (E16) binds to the DAT (17 nM) and has been used clinically for the treatment of Specifically, DAT pharmacotherapies have reached advan- ced stages of testing for psychostimulant abuse, Parkinson's attention deficit hyperactivity disorder (ADHD)[92, 93]. The disease, and ADHD. Even though only a clinical trial has Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1839
O O O
OCH OCH3 N OCH3 O 3 H
Cl Cl Cl Cl E16 E17 E18
Cl H3C N N H3C R1 N N R1 CO2CH3 Cl Ar H3C I O R2 Ar E19 E20 E21 E22
Cl Ar OH N R CO2CH3 1 R1 N R N Cl 2 N H3C
E23 E24 E25 E26 CH3 been conducted with DAT inhibitors, several DAT Tesofensine (NS2330, F2) a 3-phenyltropane from Neuro- pharmacotherapies have reached advanced stages of testing search has recently entered Phase II clinical trials for obesity. in non-human primates. In this section we highlight the most Tesofensine has also been identified as a potential pharma- advanced leads. cotherapy for Parkinson's disease, Alzheimer's disease, and Pharmacotherapies based on 3-phenyltropanes have ADHD [105]. Another DAT inhibitor, SPD 473 (BTS 74 F3 advanced through Phase II clinical trials for the treatment of 398, ) is currently in Phase 1 clinical trials for ADHD Parkinson's disease. Brasofensine (F1) has been reported to with the potential for treatment of Parkinson’s disease. SPD have excellent efficacy, with a favorable adverse effect 473 has shown preliminary efficacy in MPTP treated profile after oral administration in relevant animal models of common marmosets [106, 107]. Parkinson's disease [104]. Although initial testing appeared The development of new drugs for the treatment of promising, brasofensine was withdrawn from clinical trials cocaine addiction has focused largely on the dopaminergic due to in vivo cis-anti isomerization of the 2a -methyloxime. system. In the case of the DAT, efforts have been directed
CH3
H3C OCH3 H3 C N N N O
H
Cl Cl Brasofensine Tesofensine F1 Cl F2 Cl
O CH2CH3 S (CH2)3—N CH2CH3
Cl Cl F3 1840 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Runyon and Carroll toward development of compounds acting as indirect addition, A16 (LD50 180 mg/kg) had a higher LD50 than dopamine agonists. This approach is analogous to methadone RTI-177 (A15) (LD50 49 mg/kg) in acute rat toxicity maintenance treatment for heroin addiction or nicotine studies. Overall, A16 possesses characteristics believed replacement therapy for smoking cessation. Several classes important for a cocaine pharmacotherapy. of compounds (3-phenyltropanes, (+)CPCA, and GBR F6 (RTI-111) was evaluated to determine if the reinfor- 12,909) have been examined in non-human primates as cing and discriminative stimulus effects of amphetamine potential pharmacotherapies for psychostimulant abuse. could be blocked with pretreatment. F6 functioned as a Several 3-phenyltropanes have recently been evaluated as positive reinforcer under a FR 25 schedule in rhesus indirect agonists of the DAT for the treatment of cocaine monkeys and when given as a pretreatment to monkeys self- abuse. The analogs studied possess differing degrees of administering methamphetamine under a progressive-ratio affinity for the DAT, 5-HTT, or NET. Evaluation of these schedule, F6 shifted the methamphetamine dose response combinations has provided insight into the biochemical curve down and to the left [112]. When F6 was given to requirements for effective pharmacotherapies to reduce monkeys trained to discriminate amphetamine from saline, cocaine self-administration. F4 (RTI-113), a DAT selective full amphetamine responding was observed. inhibitor with a duration of action five-times longer than Piperidine derivative C2 [(+)CPCA] has roughly the cocaine in squirrel monkeys [108], reduced cocaine self- same degree of DAT uptake inhibition as cocaine and is administration in rhesus monkeys [109]. DAT occupancies moderately selective for the DAT and NET over the 5-HTT. of 72–78% were required for the pretreatment doses of F4 in C2 has been evaluated in rhesus monkeys under a order to reduce responding maintained by cocaine. In cont- progressive ratio of i.v. cocaine self-administration [113]. rast, F5 (RTI-112), a 3-phenyltropane with roughly equal Cocaine was found to be four-fold more potent than C2 as a reuptake efficacy at the DAT, 5-HTT, and NET did not reinforcer. In a separate study with rhesus monkeys, 100% of exhibit levels of DAT occupancy above the detection thre- the monkeys showed reinforcement at an equivalent dose of shold at doses that produced significant decreases in cocaine cocaine (30 mg/kg) [114]. Considering the preliminary self-administration in rhesus monkeys [110]. However, the results from studies in mice, rats, and non-human primates, F5 same dose of displayed high levels of 5-HTT occupancy C2 may have utility for the treatment of cocaine dependence. [47]. Interestingly, F5 did not function as a cocaine reinforcer in rhesus monkeys. Clearly, more data is needed GBR 12,909 (B1, vanoxerine) is a DAT uptake inhibitor to better understand the unique properties of F5. that was originally developed as an antidepressant. Based on
its behavioral pharmacology properties, particularly its ability A15 Evaluation of a DAT/NET selective analog (RTI- to reduce cocaine self-administration in rhesus monkey, B1 177) with more favorable drug-like properties demonstrated was evaluated in a Phase 1 clinical trial. Further studies were a pharmacological profile similar to RTI-113 (F3) in rhesus stopped due to cardiovascular QTc problems [48]. GBR A15 monkeys [110]. exhibited high levels of DAT occu- 12,909 analogs (R)- and (S)-B4 and (R)- and (S)-B5 were pancy (73%) at doses that reduced cocaine self- reported to dose dependently decrease cocaine maintained administration by 50%. In drug substitution studies, A15 also responding in rhesus monkeys [115, 116]. The (R)- and (S)- maintained drug self-administration [47]. B4 analogs showed approximately equal potency, whereas A16 (RTI-336) has been developed in our laboratory as a the (S)-B5 analog was more potent than the (R)-B5 in more DAT selective analog of A15 [32]. A16 is a highly decreasing cocaine maintained responding [115, 116]. potent and selective DAT uptake inhibitor that is being A few compounds F7–F10 that were part of our last pursued as a clinical candidate for the treatment of cocaine review [13] were reported to display potency comparable to addiction. A16 produced a dose-dependant reduction in B1 in decreasing cocaine-maintained responding [117]. cocaine self-administration in rhesus monkeys and is equally effective at 0.1 and 0.3 mg/kg/injection of cocaine [111]. In
CH3 CH3 N N
CO2 CO2CH3
Cl Cl
F4 (RTI-113) F5 (RTI-112) CH3
CH3 N CO2 CH3
Cl
F6 (RTI-111) Cl Dopamine Transporter Ligands Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1841
F F F F
O O
N N(CH )R N NCH CH=CHR F7, R = 2-thienyl 2 F9, R = 2-thienyl 2 F8, R = 3-pyridyl F10, R = 3-pyridyl
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Richard B. Rothman* and Michael H. Baumann
Clinical Psychopharmacology Section, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, Baltimore, Maryland, USA
Abstract: Monoamine transporter proteins are targets for many psychoactive compounds, including therapeutic and abused stimulant drugs. This paper reviews recent work from our laboratory investigating the interaction of stimulants with transporters in brain tissue. We illustrate how determining the precise mechanism of stimulant drug action (uptake inhibitor vs. substrate) can provide unique opportunities for medication discovery. An important lesson learned from this work is that drugs which display equipotent substrate activity at dopamine (DA) and serotonin (5-HT) transporters have minimal abuse liability and few stimulant side-effects, yet are able to suppress ongoing drug-seeking behavior. As a specific example, we describe the development of PAL-287 (a -methylnapthylethylamine), a dual DA/5-HT releasing agent that suppresses cocaine self-administration in rhesus monkeys, without the adverse effects associated with older phenylethylamine 5-HT releasers (e.g., fenfluramine) and DA releasers (e.g., amphetamine). Our findings demonstrate the feasibility of developing non-amphetamine releasing agents as potential treatments for substance abuse disorders and other psychiatric conditions. Keywords: Transporters, dopamine, serotonin, release, amphetamine, PAL-287.
PSYCHOMOTOR STIMULANTS Table 1. Representative Examples of Psychostimulants Psychomotor stimulants are drugs that produce a spect- rum of effects in humans characterized by cardiovascular Therapeutic Drugs Indication activation, increased energy and mood elevation. After high doses or extended periods of use, stimulants can induce a Methylphenidate Attention Deficit Disorder range of disordered thought processes, including severe psychotic episodes. In laboratory animals, stimulants produce Amphetamine Attention Deficit hyperactivity and support self-administration behavior, Disorder/Narcolepsy effects that are mediated by increases in extracellular dopamine (DA) in mesolimbic reward circuits (DA) [1]. The Pemoline Attention Deficit Disorder effects of stimulants are often described as “amphetamine- Phentermine Anorectic like” since amphetamine is the prototypical stimulant agent. Table 1 shows some examples of stimulants. Many of these Diethypropion Anorectic drugs are medications with long histories of efficacy and Phendimetrazine Anorectic safety, whereas others are highly addictive substances associated with significant morbidity and mortality. In some Abused Drugs cases, as with amphetamine itself, the same drug can be Cocaine therapeutic or abused depending upon the context in which the drug is administered. Indeed, the route of administration Methamphetamine can play a crucial role in determining the addictive potential stimulants, since a slower rate of drug action is associated 3,4-Methylenedioxymethamphetamine with diminished reinforcing strength [2, 3]. (MDMA) 1-Benzylpiperazine (BZP) Most stimulants exert their pharmacological effects by interacting with monoamine transporter proteins expressed on various cell types. Nerve cells that synthesize, store, expressed on NE neurons (i.e., NE transporters, NET), DA release and metabolize monoamine transmitters [DA, nore- neurons (i.e., DA transporters, DAT), and 5-HT neurons pinephrine (NE) and serotonin (5-HT)] are widely (i.e., 5-HT transporters, SERT). These proteins belong to a distributed in the mammalian CNS. These cells display superfamily of Na+/Cl- dependent transporters that share specialized membrane proteins that transport previously genetic, structural, and functional homologies [6, 7]. Under released transmitter from the extracellular space back into normal circumstances, the transporter-mediated uptake of the cytoplasm [4, 5]. Distinct transporter proteins are amine transmitters is the principal mechanism for inactiva- tion of monoamine signaling in the brain. Accordingly, *Address correspondence to this author at the Clinical Psychopharmacology stimulants that interact with monoamine transporters can Section, Intramural Research Program, NIDA, NIH, 5500 Nathan Shock have profound effects on normal neurotransmission [8]. It is Drive, Baltimore, MD 21224; Tel: 410-550-1487; Fax: 410-550-2997; E- important to note that certain stimulant drugs, especially mail: [email protected] substituted amphetamines, interact with other neuronal
1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1846 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Rothman and Baumann proteins such as monoamine oxidase (MAO) [9] and the mechanism of action. Both types of agents increase synaptic vesicular monoamine transporter type 2 (VMAT2) [10]. concentrations of transmitter via transporter-dependent Stimulants that target transporter proteins can be divided processes, and there are important differences in their modes into two classes based on their precise molecular mechanism of action at the cellular and organismic level. In particular, of action - reuptake inhibitors and substrate-type releasers. the activity of reuptake inhibitors requires ongoing release of Reuptake inhibitors (e.g., methylphenidate and cocaine) bind transmitters via exocytosis – a cellular mechanism directly to transporter proteins but are not transported. These drugs dependent upon electrical depolarization and extracellular elevate extracellular transmitter concentrations by blocking calcium. Thus, the ability of reuptake inhibitors to increase transporter-mediated recapture of transmitters from the synaptic transmitter levels is described as being impulse- and calcium-dependent. Substrates, on the other hand, increase synapse. Substrate-type releasers (e.g., amphetamines) also bind to transporter proteins, and these drugs are subsequently synaptic transmitter levels by a process that is largely transported into the neuronal cytoplasm where they evoke independent of ongoing cell firing and exocytotic transmitter non-exocytotic transmitter release. Releasers elevate release. Plasma membrane autoreceptors mediate negative feedback mechanisms that serve to dampen the ability of extracellular transmitter concentrations by a two-pronged mechanism: (1) they promote efflux of transmitter by reuptake inhibitors to elevate synaptic transmitter [26]. Such reversing the normal direction of transporter flux and (2) negative feedback effects exist for 5-HT [27-29], DA [30], they increase free cytoplasmic levels of transmitter by and NE [31] neuronal systems. While autoreceptor activation can abolish the ability of reuptake inhibitors to elevate interacting with VMAT2 to disrupt vesicular storage [11, 12]. The precise underpinnings of transporter-mediated release synaptic transmitter levels, autoreceptor mechanisms have involve complex processes that are the topic of intensive little or no effect on substrate-induced neurotransmitter investigation [13-15]. Because substrate-type releasing release [32-36]. Because of autoreceptor-mediated feedback agents must be transported into nerve terminals to promote inhibition, reuptake inhibitors tend to produce small transmitter release, reuptake inhibitors can block the effects increases in extracellular neurotransmitter whereas releasers of releasers. In some cases, drugs can act as allosteric tend to produce more robust increases [37]. The in vivo 1 modulators of transporter function [16-20]. microdialysis data in Fig. illustrate the modest and sustained elevation of extracellular 5-HT evoked by the 5- For more than a decade, we have carried out experiments HT reuptake inhibitor, fluoxetine, compared to the much investigating the interaction of psychostimulants with larger and transient effect of the 5-HT releaser, (+)- monoamine transporters in order to understand how these fenfluramine [33, 38, 39]. drugs affect the brain. It is anticipated that the knowledge Given the important distinctions between uptake inhibi- gained from these studies will aid in the development of pharmacotherapies for treating stimulant dependence and tors and releasers, we sought to develop assays which could other psychiatric disorders [21]. Specific aspects of our work easily determine if test drugs interacted with transporters as have been recently reviewed [22-25]. In the present paper, inhibitors or substrates. This work served to identify the mechanism of action of known stimulant-type agents [40-43] we illustrate how determining the mechanism of action of a wide range of transporter ligands (uptake inhibitor vs. and to guide the synthesis and evaluation of novel treatment substrate) can provide unique opportunities for medication agents for addictive disorders [21]. Traditionally, it has been development. difficult to use simple test tube assays to discriminate between drugs that are uptake inhibitors vs. those that are TRANSPORTER MECHANISMS substrate-type releasers [8, 11]. Thus we spent considerable time and effort in developing a rapid high-throughput As noted above, stimulants can be classified as either 3 method for measuring the release of preloaded [ H]DA, uptake inhibitors or releasers based on their molecular
Fig. (1). Effects of fluoxetine (a 5-HT reuptake inhibitor) and (+)-fenfluramine (a 5-HT releaser) on extracellular 5-HT in rat nucleus accumbens. Dialysis methods were carried out as described previously [22]. Drugs were administered i.v. at time zero minutes. Data are expressed as a percentage of the mean of three basal dialysate samples collected prior to drug treatment. Basal dialysate 5-HT level was 0.46 ± 0.17 nM. Values are mean ± SEM for N=5 rats/group. Therapeutic Potential of Monoamine Transporter Substrates Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1847
[3H]NE and [ 3H]5-HT from nervous tissue in vitro [44]. The basic strategy employed in the release assay is to first incubate synaptosomes with [3H]neurotransmitter for sufficient time to achieve steady state. At steady state, test drugs are added to synaptosomes and the reaction is terminated by rapid filtration after 5 min. Transmitter “release” is quantified by measuring the amount of tritium retained on the filter – decreases in retained tritium reflect increases in [3H]neurotransmitter released. A key requirement of our release assay is the inclusion of reserpine in the assay buffer; reserpine prevents accumulation of neurotransmitters into synaptic vesicles thereby maximizing the amount of preloaded [3H]neurotransmitter available for substrate-induced release. The activity of transporter substrates can be readily distinguished from that of transporter blockers using our in vitro release assay. As depicted in Fig. 2, the non-selective uptake inhibitor indatraline, which has high affinity for DAT, NET and SERT, displays very weak activity in release Fig. (3). Effects of indatraline (5 nM and 25 nM) on assays. By contrast, the transporter substrate, methamphet- methamphetamine-evoked release of [3H]dopamine. Each point is amine, causes dose-dependent release of [3H]DA, [3H]NE the mean±SD (n=3). Data are from [44]. and [3H]5-HT. Methamphetamine is much more potent at releasing DA and NE when compared to its effects on 5-HT, test drugs at all three monoamine transporters under similar consistent with the known pharmacology of this drug [40, experimental conditions. Table 2 reports the results obtained 44]. The data in Fig. 3 demonstrate that low concentrations with a wide range of pharmacological agents using these of indatraline antagonize methamphetamine-induced release assays. Although the results are discussed in detail in the of [3H]DA, shifting the methamphetamine release curve to individual papers cited in Table 2, we highlight important the right. Thus, indatraline blocks the DA-releasing activity findings here. of methamphetamine by binding to DAT sites in the tissue. APPETITE SUPPRESSANTS The apparent Ki value for indatraline (~2 nM) calculated from the shift of the methamphetamine release curve is Figure 4 shows that many clinically available appetite 3 similar to its Ki value for inhibition of [ H]DA uptake (1.9 suppressants are phenylethylamines structurally related to 3 nM). In more recent studies, we have employed [ H]1- amphetamine. Not surprisingly, these drugs share behavioral 3 + methyl-4-phenylpyridinium ([ H]MPP ) as a radiolabeled properties with abused stimulants but are typically less 3 + substrate for DAT and NET assays. [ H]MPP yields results potent and less addictive [45-48]. Preclinical studies have comparable to the endogenous substrates DA and NE, with shown that amphetamine-type appetite suppressants decrease an improved signal-to-noise ratio [43]. The collective cocaine and methamphetamine self-administration in various findings illustrate that our assay systems can readily animal species [49-53]. Thus, we believed that clinically discriminate releasers from uptake blockers. Moreover, the available appetite suppressants, such as phentermine, di- assays can be used to evaluate the pharmacological profile of ethylpropion, and phendimetrazine, might be logical candi-
Fig. (2). Effects of the reuptake blocker indatraline (left panel) and the transporter substrate methamphetamine (right panel) on the release of [3H]dopamine, [3H]norepinephrine and [3H]5-HT in synaptosomes. Each point is the mean±SD (n=3). Data are from [44]. 1848 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Rothman and Baumann
Table 2. Pharmacological Profile of Selected Agents in the Dopamine, Norepinephrine and 5-HT Release and Uptake Inhibition Assays
Test Drug Release NE Release DA Release 5-HT NETa Uptake DATa Uptake SERT Uptake
EC50 (nM ± SD) Ki (nM ± SD) EC50 (nM ± SD) Ki (nM ± SD) EC50 (nM ± SD) Ki (nM ± SD)
Appetite Suppressants and their Metabolites
Phentermine 39.4 ± 6.6 262 ± 21 3511 ± 253
(+)-Amphetamine 7.07 ± 0.95 24.8 ± 3.5 1765 ± 94
(-)-Ephedrine 43.1±4.0 236±9 >10,000 >50,000
(+)-Ephedrine 218±14 2104±68 inactive
Diethylpropion > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000
N-Ethylaminopropiophenone 99.3 ± 6.6 1014 ± 80 2118 ± 98
N,N- > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 > 10,000 Diethylnorpseudoephedrine
Phendimetrazine 8300 ± 445 >10,000 19,000±537 >10,000 >100,000 >100,000
(±)-Phenmetrazine 50.4±5.4 131±11 7765±610
Pseudophenmetrazine 514 ± 52 2630 ± 198 >10,000 >10,000
(-)-Pseudophenmetrazine 2511 ± 561 2691 ± 176 >10,000 >10,000
(+)-Pseudophenmetrazine 349 ± 28 1457 ± 138 >10,000 >10,000
Appetite suppressants removed from clinical use
(+)-Fenfluramine 302 ± 20 22000±1100 51.7 ± 6.1
(-)-Fenfluramine 7187±559 >10,000 >20,000 147±19
(±)-Fenfluramine 739 ± 57 23700±1300 79.3 ± 11.5
(±)-Norfenfluramine 168±17 1925±295 104±5
(+)-Norfenfluramine 72.7±5.4 924±112 59.3±2.4
(-)-Norfenfluramine 474±40 19194±1048 287±14
Aminorex 26.4 ± 2.8 49.4± 7.5 193 ± 23
Chlorphentermine 451 ± 66 2650 ± 273 30.9 ± 5.4
Abused Stimulants
(+)-Methamphetamine 12.3 ± 0.7 24.5 ± 2.1 736 ± 45
(-)-Methamphetamine 28.5 ± 2.5 416 ± 20 4640 ± 243
(±)-MDMA 110±10 278±7 72±3
(+)-MDMA 136±9 142±4 74±3
(-)-MDMA 560±4 3700±100 340±20
(±)-MDA 108±7 190±6 160±7
(+)-MDA 50±5 98±4 100±4
(-)-MDA 290±10 900±30 310±10
1-Benzylpiperazine (BZP) 62±6.5 175±13 6050±835 Therapeutic Potential of Monoamine Transporter Substrates Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1849
(Table 2) Contd….
Test Drug Release NE Release DA Release 5-HT NETa Uptake DATa Uptake SERT Uptake
EC50 (nM ± SD) Ki (nM ± SD) EC50 (nM ± SD) Ki (nM ± SD) EC50 (nM ± SD) Ki (nM ± SD)
Endogenous Substrates
Tyramine 40.6 ± 3.5 119 ± 11 2775 ± 234
Norepinephrine 164 ± 13 869 ± 51 >10,000 >50,000
Dopamine 66.2 ± 5.4 86.9 ± 9.7 6489 ± 200
Serotonin >10000 3013 ± 266 1960±147b 44.4 ± 5.3
Transporter Inhibitors
GBR12935 277 ± 23 4.90±0.30 289±29
GBR12909 79.2 ± 4.9 4.3±0.3 73.2 ± 1.51
Cocaine 779 ± 30 478 ± 25 304 ± 102
Mazindol 2.88 ± 0.17 25.9 ± 0.56 272 ± 11
Methylphenidateb 118±12 90.2±7.9 inactive
Desipramine 8.32 ± 1.19 5946 ± 193 350 ± 13
Fluoxetine 688 ± 39 >5,000 9.58 ± 0.88
Citalopram 4332 ± 295 20485 ± 923 2.40 ± 0.09
RTI-55 5.89 ± 0.53 0.83±0.09 1.00 ± 0.03
Indatraline 12.6 ± 0.5 1.90 ± 0.05 3.10 ± 0.09
Miscellaneous Agents
Nantenineb >10,000 >10,000 >10,000 >10,000 >10,000 >10,000
Tramadolb >10,000 2770±250 >10,000 >10,000 >10,000 1820±100
JDTicb >10,000 1756±100c >10,000 3620±230 >10,000
From: [21, 40, 41, 43, 74, 86, 93, 94]. Each value is the mean±SD of three experiments. For substrates, we report only the release EC50 values. For uptake inhibitors, we report the Ki for inhibition of [3H]neurotransmitter uptake. aSome studies used [3H]MPP+, with appropriate blocking agents, to measure release via DAT and NET. bUnpublished data. c 125 3 The Ki of JDTic at NET is 6.8 ± 0.9 nM (n=3) using [ I]RTI-55 and the cloned hNET, and is 126 ± 23 nM (n=2) at the rat brain NET labeled with [ H]nisoxetine. dates for the pharmacotherapy of stimulant dependence [54]. Unfortunately, to our knowledge, a dose-response compari- We have performed experiments to examine the molecular son of the NE- and DA-releasing effects of these stimulants mechanisms associated with anorectic drugs. has not been directly determined using in vivo microdialysis methods in rats. Phentermine is one of the more widely prescribed appetite suppressant medications. In vitro studies (Table 2) To help clarify this issue, a recent study was conducted in reveal that phentermine is a substrate at NET, DAT and baboons to compare the effects of phentermine, (±)- SERT, with its most potent action being NE release (EC50 = ephedrine and (+)-amphetamine in vivo [56]. Following 39 nM). While the administration of phentermine to rats administration of high (1.5 mg/kg) iv doses of the drugs to evokes DA release in the brain [55], the in vitro data suggest anesthetized baboons, both dopaminergic (central DA release that phentermine might increase NE release at much lower and plasma prolactin) and noradrenergic (plasma NE) doses than those required to release DA. The same holds true endpoints were measured. Central DA release was for (±)-ephedrine, which is at least 10-times more potent at determined via positron emission tomography using the releasing NE than DA. (+)-Amphetamine, in contrast, is only method of [11C]raclopride displacement. As shown in Figs. 5 3-times more potent at NE release than DA release. and 6, only (+)-amphetamine decreases plasma prolactin and 1850 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Rothman and Baumann
H NH2 N CH3
CH3 CH3 Amphetamine Methamphetamine
CH3 CH3 H NH2 NH2 F3C N CH2CH3 CH3 CH3 CH3 Cl Phentermine Chlorphentermine Fenfluramine
NH2 O O O CH2CH3 N N N CH2 CH3 CH3
CH3 CH3
Diethylpropion Phendimetrazine Aminorex Fig. (4). Structure of selected anorectic agents. From [25].
Fig. (5). Mean plasma levels of prolactin in two adult male Papio anubis baboons after the IV bolus administration of amphetamine, (±)- ephedrine, or phentermine. Mean % change = ([prolactin at indicated time] - [prolactin at time 0])*100/[prolactin at time 0]. From [56]. increases central DA release as indicated by reductions in the stimulant-induced increases in NE contribute to the acute binding potential of [11C]raclopride. In contrast, all three subjective effects of amphetamine-like agents. More studies stimulants increase plasma NE and DA (Fig. 7). Given that are needed to determine the role of NE in mediating effects plasma DA levels are about 10-fold less than plasma NE of psychomotor stimulants. levels, and DA is a precursor in NE biosynthesis, we believe Diethylpropion and phendimetrazine are clinically that these stimulants release both NE and DA from available anorectic agents that display minimal interactions peripheral noradrenergic nerves. The plasma levels of the with monoamine transporters in vitro (Table 2). On the other various drugs tested in baboons were at the high end of hand, these medications are known to be psychomotor typical therapeutic plasma levels observed in human subjects stimulants when administered in vivo as indicated by their (1-3 µM) [57, 58]. These data suggest, therefore, that typical shared properties with illicit drugs like cocaine. For example, clinical doses of phentermine and (±)-ephedrine may not diethylpropion and phendimetrazine are self-administered by release central DA in humans. Both phentermine [59] and animals [45, 62], and both drugs exhibit discriminative (±)-ephedrine [60, 61] produce amphetamine-like subjective stimulus properties that generalize to cocaine [47, 48]. One effects in humans, and the EC values of stimulants to 50 hypothesis consistent with the available data is that release NE, but not DA, correlate with the oral doses that diethylpropion and phendimetrazine are “prodrugs” which produce amphetamine-type subjective effects (Fig. 8) [40]. are converted to bioactive metabolites upon systemic Viewed collectively these data support the hypothesis that administration. In the case of diethylpropion, the N-deethyla- Therapeutic Potential of Monoamine Transporter Substrates Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1851
Fig. (6). Dopamine (DA) release as percent change in DA binding potential in two adult male Papio anubis baboons following the intravenous bolus injection of 1.5 mg/kg (+)-amphetamine, (±)-ephedrine, or phentermine. From [56].
Fig. (7). Mean plasma levels of NE and DA in two adult male Papio anubis baboons after the intravenous bolus administration of amphetamine, (±)-ephedrine, or phentermine. Mean % change = ([amine at indicated time] - [amine at time 0])*100/[amine at time 0]. From [56].
Fig. (8). Correlation of oral doses of stimulants which produce amphetamine-like subjective effects with their potency in releasing [3H]NE (Panel A) and [3H]DA (Panel B). From [40]. 1852 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Rothman and Baumann ted metabolite, N-ethylaminopropiophenone, appears to be Ephedrine has attracted unfavorable attention because of its the bioactive metabolite since this compound potently use as a synthetic precursor in the clandestine production of releases NE (EC50 = 99.3 nM) with less potent effects on 5- methamphetamine [64], and due to its potential for toxicity HT release (EC50 = 2118 nM). Interestingly, N-ethyl- (including death) [65]. The compound is obtained as an aminopropiophenone is not a DAT substrate, but instead herbal extract derived from plants of the genus Ephedra or as blocks DA reuptake (EC50 = 1014 nM) [63]. In the case of a synthetic chemical. All forms of ephedrine have been phendimetrazine, the N-demethylated metabolite phenmet- removed from the OTC market due to the occurrence of razine potently releases NE and DA (Table 2). Stereo- cardiovascular side-effects, but the synthetic chemical is still chemistry plays an important role in the transporter activity available as a prescription product. Current and past thera- of (±)-phenmetrazine. While the clinically available trans- peutic applications of ephedrine include treatment of asthma stereoisomer ((+)-phenmetrazine) is a potent NE and DA and use as a hypertensive agent, decongestant, central releaser, the cis-stereoisomer ((-)-phenmetrazine) is some- stimulant, and anorectic agent. what less active. A more intriguing finding is that enantio- Ephedrine has a complex stereochemistry due to the mers of (±)-pseudophenmetrazine display different molecu- presence of two chiral centers. Specifically, ephedrine- lar mechanisms at DAT. Specifically, (-)-pseudophenmetra- related phenylpropanolamines can exist as four distinct zine is a DA uptake inhibitor whereas (+)-pseudophenme- stereoisomers (see Table 3 for chemical structures). [1R,2S]- trazine is a DAT substrate. To our knowledge, this represents (- )-2-(Methylamino)-1-phenylpropan-1-ol is typically identi- the first example of two enantiomers exhibiting differential fied as (- )-ephedrine, while [1S,2R]-(+)-2-(methylamino)-1- transporter mechanisms [41]. phenylpropan-1-ol is typically identified as (+)-ephedrine. EPHEDRINE-RELATED COMPOUNDS [1R,2R]-(- )-2-(Methylamino)-1-phenylpropan-1-ol is (- )- pseudoephedrine, and [1S,2S]-(+)-2-(methylamino)-1-phe- Ephedrine is a familiar and widely-used stimulant that nylpropan-1-ol is (+)-pseudoephedrine. N-demethylation was once available in over-the-counter (OTC) preparations. also results in four optical isomers: [1R,2S]-(- )-2-(amino)-1-
Table 3. Structure-Activity Profile of Phenylpropanolamines at the Biogenic Amine Transporters EC50 nM (±SD)a
From [43]. aAll agents behaved as substrates except for (-)-pseudoephedrine which displayed activity in the DA uptake assay. bData reported previously [40]. These experiments used [3HDA and [3H]NE instead of [3H]MPP+. c(-)-Pseudoephedrine behaved as an uptake inhibitor. Each value is the mean±S.D. of three experiments. Therapeutic Potential of Monoamine Transporter Substrates Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1853 phenylpropan-1-ol, commonly termed (- )-norephedrine or established risks of prescribed NET and DAT substrates (e.g. (- )-phenylpropanolamine, [1R,2S]-(- )-2-(amino)-1-phenyl- amphetamine). More recently, adverse effects associated propan-1-ol, commonly referred to as (+)-norephedrine or with the use SERT substrates have been widely publicized; (+)-phenylpropanolamine, (- )-norpseudoephedrine or (- )- these serious side-effects include cardiac valve disease cathine, and (+)-norpseudoephedrine or (+)-cathine. Table 3 (CVD) and primary pulmonary hypertension (PPH). (±)- shows how the structures of the phenylpropanolamines can Fenfluramine, and its more potent enantiomer (+)-fenflur- be related to those of the phenylisopropylamines (i.e. amine, were once commonly prescribed anorectic agents. phenylethylamines) methamphetamine and amphetamine. These medications were removed from clinical use in 1997 The phenylpropanonamines methcathinone and cathinone due to the occurrence of CVD in some patients [67]. We differ from methamphetamine and amphetamine with respect reported that fenfluramine, aminorex, and other amphetamine- to stereochemistry and the oxidation state of the benzylic related drugs known to increase the risk for developing PPH position. Methamphetamine and amphetamine lack a (e.g., chlorphentermine) share the common feature of being benzylic substituent, and S(- )-methcathinone and S(- )- SERT substrates [68]. Our findings implicated SERT in the cathinone can be viewed as analogs of ephedrine and mechanism underlying fenfluramine-induced PPH. It is norephedrine where the benzylic hydroxyl group has been noteworthy that not all SERT substrates are associated with oxidized to the corresponding ketone. PPH, and the specific role of SERT proteins in this disease Ephedrine has been extensively studied for decades, but likely involves a complex array of factors that requires further study. the pharmacological activity of ephedrine-related phenyl- propanolamines across a wide array of CNS receptors and Experimental data from a mouse model of hypoxic transporters was not reported until our study published in pulmonary hypertension have suggested that 5-HT2B 2003 [43]. The most potent action of ephedrine-like receptors are involved in the pathogenesis of PPH [69]. phenylpropanolamines is substrate activity at NET. In Aminorex is a SERT substrate that caused an epidemic of contrast, methcathinone, cathinone, methamphetamine and PPH in the 1960s [70] and case reports implicate the related amphetamine, release both NE and DA. Importantly, the designer drug, 4-methylaminorex, as a potential cause of the ephedrine-like phenylpropanolamines have much lower or disease [71]. If 5-HT2B receptors are involved in the negligible affinity for adrenergic receptors, indicating that pathogenesis of aminorex-associated PPH, then we surmised the pharmacological effects of these agents most likely result that aminorex should display activity at 5-HT2B sites. As from the “indirect” NET-mediated release of NE. This latter reported in Table 4, aminorex activates the cloned human 5- finding is unexpected, since pharmacology text books teach HT2B receptor with moderate potency and high efficacy that the phenylpropanolamines stimulate the sympathetic (EMAX = 76%). However, the EC50 of aminorex for 5-HT2B nervous system by a variety of mechanisms that include receptor activation (870 nM) is 33-times higher than its EC50 direct agonist activity at adrenergic receptors and “indirect” for NET release (26.4 nM). Moreover, the activity of effects via carrier-mediated exchange with NE [66]. aminorex at 5-HT2B sites is nearly 50-fold less than the known 5-HT agonist (+)-norfenfluramine, the N- ADVERSE EFFECTS 2B deethylated metabolite of (+)-fenfluramine. Chlorphen- The clinical utility of amphetamine-type anorectic agents termine is a substituted amphetamine analog that causes PPH is limited by a number of adverse side-effects. Cardio- in animal models. As shown in Table 4, this drug has vascular complications and high abuse liability are negligible affinity for 5-HT2 receptor subtypes. Collectively,
Table 4. Functional Activity of Selected Anorectic Agents at the Cloned Human 5-HT2 Receptors
5-HT2A 5-HT2B 5-HT2C SERT NET
(EC50 nM) (EC50 nM) (EC50 nM) Release Release
[Emax] [Emax] [Emax] (EC50 nM) (EC50 nM)
5-HT 10.9±0.7 42.6±4.2 0.07±0.01 44.4±5.3c [100±2] [100±4] [100±3]
Aminorex 4365±108 870±27 525±17 193±3c 26.4±2.8c [47±2] [76±3] [71±2]
Chlorphentermine inactive 5370±288 6456±237 30.9±5.4c >10000 [50±5] [48±3]
Phentermine inactive inactive inactive 3511±253c 39.4±±6.6c
(+)-Norfenfluramine 630±141a 18.4±5.3a 13±2a 59.3±2.4b 88±5 [73±4] [100±6]
The data for 5-HT, aminorex and chlorphentermine are previously unpublished. The functional endpoint for these data, provided by the NIMH Psychoactive Drug Screening Program ++ a [73] b c (http://pdsp.cwru.edu/), is Ca mobilization. The functional endpoint of these data is phosphoinositide hydrolysis . From [42]. From [40]. The values in brackets are the EMAX values, with the effect of 5-HT being 100%. Each value is ±SD. 1854 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Rothman and Baumann the data argue against an important role for 5-HT2B receptors able to fenfluramine [76]. Other examples of SERT subst- in the pathogenesis of anorectic-associated PPH. On the rates that do not cause 5-HT neurotoxicity include the other hand, it seems feasible that a metabolite of aminorex or tetralin and indan analogues of 3,4-(methylenedioxy) chlorphentermine may act more potently at 5-HT2B recep- amphetamine (MDA) reported by Nichols et al. [77] and tors, and this possibility deserves to be examined. PAL-287, which is discussed in the next section. Like mCPP, PAL-287 produces elevations in extracellular 5-HT The evidence supporting a role for 5-HT2B receptors in the pathogenesis of fenfluramine-associated CVD is much but does not produce long-term 5-HT depletion, even after stronger. As noted above, the association of fenfluramine exposure to very high doses (i.e., 18 mg/kg, i.p. x 3). These with an increased prevalence of cardiac valvulopathy led to data indicate that SERT substrate activity is necessary, but not sufficient to produce long-term depletion of brain 5-HT. its withdrawal from the market place. Numerous investi- gations have demonstrated that the fenfluramine metabolite, PAL-287 AND RELATED AGENTS norfenfluramine, activates 5-HT2B receptors on heart valves to stimulate mitogensis, and this action may represent the Along with other researchers, we have advocated the use principal mechanism underlying fenfluramine-induced CVD of amphetamine-type monoamine releasers as agonist thera- [72-74]. Findings summarized by Setola et al. [74] pies for cocaine dependence [54, 78]. Similar to cocaine, emphasize that valvulopathic agents are “pro-drugs” in many these compounds target monoamine transporters to elevate instances, since it is the bioactive metabolites which serve to synaptic levels of NE, DA and 5-HT [8, 25]. Preclinical studies support the utility of amphetamines as agonist stimulate 5-HT2B receptor sites. Specific examples of this pro-drug phenomenon are shown in Fig. 9, where N- treatments [49, 79]. For example, Negus and Mello [80] demethylated metabolites of MDMA and methysergide are demonstrated that slow infusion of the DA releaser (+)- potent efficacious 5-HT agonists. amphetamine decreases cocaine self-administration behavior 2B in monkeys, with minimal effects on food-maintained H behavior. Perhaps more importantly, Grabowski et al. [81] F3 C N CH3 F3 C NH2 and Shearer et al. [82] showed that (+)-amphetamine is an effective treatment adjunct for reducing illicit cocaine use in CH CH 3 in vivo 3 cocaine-dependent human patients. Unfortunately, the use of Fenfluramine Norfenfluramine amphetamine as a medication is limited by at least two problems. First, DA releasers possess significant abuse H potential due to activation of mesolimbic DA neurons in O N O NH2 reward pathways [83]. Second, DA releasers will not correct CH3 CH 5-HT deficits that accompany long-term cocaine abuse [84]. O CH3 O 3 MDMA in vivo MDA We have suggested the possibility of designing dual DA/5-HT releasers that can overcome both of the afore- mentioned limitations [85]. For example, a growing body of CH3 CH3 H H evidence indicates that increases in 5-HT release can dampen O O N N the effects of concurrent DA release, suggesting that mixed DA/5-HT releasers would exhibit less stimulant side-effects. OH OH To test whether the balance between DA and 5-HT trans- N N mission can predict the occurrence of stimulant-like beha- CH3 CH3 viors, we examined the neurochemical and locomotor effects produced by a series of monoamine releasers that display in vivo varying degrees of potency as DAT and SERT substrates. N HN The chemical structures of the drugs tested are shown in Fig. H3C 10; it should be noted that PAL-313, PAL-314, PAL-303 and Methysergide Methylergonovine PAL-353 are ring-substituted amphetamines whereas PAL- 287 is a non-amphetamine compound [21, 86]. Fig. (9). Metabolism of MDMA, fenfluramine and methysergide results in the formation of norfenfluramine, MDA and Table 5 summarizes in vitro potencies of test drugs as 3 3 methylergonovine. These metabolites are 5-HT2B receptor agonists releasers of [ H]DA and [ H]5-HT. The DA/5-HT ratio for and are associated with cardiac valve disease. From [74]. each drug was calculated by dividing the EC50 value for DA release by the corresponding value for 5-HT release. With As comprehensively reviewed elsewhere [75], certain this method, ratios greater than 1 indicate increasing 5-HT SERT substrates, such as MDMA and fenfluramine, produce selectivity while ratios less than 1 indicate DA selectivity. 5-HT neurotoxicity. The term ‘5-HT neurotoxicity’, when All of the drugs are substrate-type releasers that cause efflux used in the present context, refers to the fact that high-dose of preloaded tritiated transmitter via transporter-mediated administration of 5-HT releasers often causes persistent mechanisms. The selectivity of drugs for DA and 5-HT depletion of brain tissue 5-HT and 5-HT transporters. A key transporters differs substantially. (+)-Amphetamine has observation is that not all SERT substrates deplete 5-HT. For much higher potency for DA release when compared to 5- example, repeated administration of the SERT substrate m- HT release (DA/5-HT ratio=0.005). The naphthalene chlorophenylpiperazine (mCPP) fails to deplete brain 5-HT, derivative PAL-287 has 500-fold greater potency for 5-HT despite producing elevations of extracellular 5-HT compar- release when compared to amphetamine, while effects on Therapeutic Potential of Monoamine Transporter Substrates Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1855
3 NH2 NH2 Table 5. Effects of Test Compounds on the Release of [ H]DA and [3H]5-HT From Rat Brain Synaptosomes
CH3 CH3 a -Methylphenylethylamine a -Methylnaphthylethylamine Test Drug [3H]DA [3H]5-HT DA/5-HT (amphetamine) (PAL-287) release release ratio
EC50 (nM) EC50 (nM) NH2 NH2 (+)-amphetamine 1 8.0 ± 0.4 1756 ± 94 0.005 CH CH3 3 1 H3C PAL-287 12.6 ± 0.4 3.4 ± 0.2 3.706 p-Methylamphetamine m-Methylamphetamine CH3 2 (PAL-313) (PAL-314) PAL-313 44.1 ± 2.6 53.4 ± 4.1 0.824 2 NH PAL-314 33.3 ± 1.3 218 ± 22 0.154 NH2 2 PAL-303 2 51.5 ± 1.7 939 ± 76 0.055 CH CH3 3 F PAL-353 2 24.2 ± 1.1 1937 ± 202 0.012 p-Fluoroamphetamine F m-Fluoroamphetamine 1 Data taken from [21]. 2 Data taken from [86]. Data are mean ± SD for N=3 (PAL-303) (PAL-353) experiments.
Fig. (10). Chemical structures of (+)-amphetamine, PAL-287, and amphetamine-treated rats, forward locomotion is markedly PAL amphetamines. stimulated and reaches a level of 6000 cm/20 min after the 1.0 mg/kg dose. Motor activation produced by (+)-amphe- DA are comparable (DA/5-HT ratio=3.706). The substituted tamine increases in parallel with elevations in dialysate DA, amphetamines, PAL-313, -314, -303 and -353, display suggesting a direct relationship between these two endpoints. roughly equivalent potency as NE and DA releasers, but The data in Fig. 12 illustrate that i.v. administration of PAL- these compounds have varying potencies as 5-HT releasers, 287 increases extracellular levels of DA and 5-HT in with PAL-313 exhibiting highest potency and PAL-353 prefrontal cortex, with effects on 5-HT being somewhat exhibiting lowest potency. greater. It is noteworthy that elevations in dialysate DA We first compared the in vivo neurochemical and induced by PAL-287 are similar to those induced by (+)- behavioral effects of (+)-amphetamine and PAL-287 in rats. amphetamine. Despite the large increase in extracellular DA Fig. 11 shows that i.v. administration of (+)-amphetamine produced by PAL-287, this drug produces very little forward increases extracellular DA in the prefrontal cortex, with locomotion (Fig. 12). Specifically, after a 3 mg/kg i.v. dose minimal effects on extracellular 5-HT. The microdialysis of PAL-287, dialysate DA levels are increased 8-fold above data are consistent with in vitro release data demonstrating baseline but ambulation distance is only 1000 cm/20 min. (+)-amphetamine is a potent DAT substrate (Table 5). In These data provide compelling evidence that stimulation of 5-HT release can antagonize the locomotor stimulation produced by DA release.
Fig. (11). Left panel. Effects of (+)-amphetamine on extracellular DA and 5-HT in rat prefrontal cortex as determined by in vivo microdialysis. Rats received i.v. injection of 0.3 mg/kg (+)-amphetamine at time zero, followed by 1 mg/kg 60 min later. Data are mean ± SEM for 7 rats/ group, expressed as % baseline. Baseline levels of DA and 5-HT were 0.38 ± 0.07 and 0.24 ± 0.06 pg/5 µl. * P<0.05 compared to pre-injection control at a given time point, Duncan’s post hoc test. Right panel. Effects of (+)-amphetamine on ambulation and stereotypy in rats undergoing microdialysis sampling. Rats received i.v. injections of 0.3 mg/kg (+)-amphetamine at time zero, followed by 1 mg/kg 60 min later. Data are mean ± SEM for 7 rats/ group, expressed as distance traveled in cm (ambulation) and number of repetitive movements (stereotypy). * P<0.05 compared to pre-injection control, Duncan’s post hoc test. From [21]. 1856 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Rothman and Baumann
Fig. (12). Left panel. Effects of PAL-287 on extracellular DA and 5-HT in rat prefrontal cortex as determined by in vivo microdialysis. Rats received i.v. injection of 1 mg/kg PAL-287 at time zero, followed by 3 mg/kg 60 min later. Data are mean ± SEM for 7 rats/ group, expressed as % baseline. Baseline levels of DA and 5-HT were 0.43 ± 0.07 and 0.27 ± 0.06 pg/5 µl. * P<0.05 compared to pre-injection control at a given time point, Duncan’s post hoc test. Right panel. Effects of PAL-287 on ambulation and stereotypy in rats undergoing microdialysis sampling. Rats received i.v. injections of 1 mg/kg PAL-287 at time zero, followed by 3 mg/kg 60 min later. Data are mean ± SEM for 7 rats/group, expressed as distance traveled in cm (ambulation) and number of repetitive movements (stereotypy). * P<0.05 compared to pre-injection control, Duncan’s post hoc test. From [21].
The reinforcing effects of PAL-287 have been evaluated blunt the reinforcing effects normally associated with DA in rhesus monkeys. Findings depicted in Fig. 13 demonstrate release. It should be noted that PAL-287 can readily that PAL-287 is not self-administered by monkeys trained to suppresses on-going cocaine self-administration, even self-inject cocaine. Similar results were obtained when a though PAL-287 drug lacks stimulating and reinforcing series of PAL amphetamines were tested in the monkey self- effects [21]. The findings support the feasibility of develo- administration assay. As shown in Fig. 14, PAL-313 displays ping non-amphetamine releasers with low abuse potential, by the lowest reinforcing efficacy in vivo, and this drug is the designing dual DA and 5-HT releasing activity into a single most potent SERT substrate in vitro (Table 5). The data molecule. Additionally, PAL-287 displays a more desirable suggest that serotonergic effects of PAL-287 and PAL-313 side-effects profile when compared to amphetamine-type SERT substrates. PAL-287 has reduced affinity for 5-HT2B receptors and does not persistently deplete brain 5-HT. Taken together, our findings support the possibility of developing dual DA/5-HT releasers that will not produce adverse effects associated with other SERT substrates, such as fenfluramine [22]. MISCELLANEOUS DRUGS We have used our assay procedures to screen a variety of compounds that might possess transporter activity, and previously unpublished data for a few specific agents are mentioned here. The alkaloid nantenine (9,10-methyl- enedioxy-1,2-dimethoxyaporphine) is a naturally-occurring compound from the fruit of Nandina domestica that bears structural similarity to MDMA. Nantenine is reported to antagonize many of the effects of MDMA in mice [87]. Because MDMA is a substrate of the monoamine transpor- ters, nantenine could block the effects of MDMA by acting as a monoamine reuptake inhibitor. As shown in Table 2, Fig. (13). Self-administration of cocaine and PAL 287 by rhesus nantenine (supplied by Dr. Fantegrossi) is inactive as a monkeys. Drugs were available under a FR 25 schedule of monoamine releaser and as an uptake inhibitor. Tramadol is reinforcement for two hours/day. Each point is the mean of two an analgesic drug sometimes linked to the development of sessions of access to each dose of the drugs. Data are mean ± SEM the 5-HT behavioral syndrome [88]. As reported in Table 2, for N=4 monkeys. Symbols without bars have variability smaller tramadol inhibits NET and SERT uptake with Ki values in than the points. * P<0.05 compared to saline-injected control, the low micromolar range. Our SERT results are similar to Newman-Keul’s post hoc test. From [21]. Therapeutic Potential of Monoamine Transporter Substrates Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1857
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Signe Høg†, Jeremy R. Greenwood†, Karsten B. Madsen‡, Orla M. Larsson‡, Bente Frølund†, Arne Schousboe‡, Povl Krogsgaard-Larsen† and Rasmus P. Clausen†,*
Departments of Medicinal Chemistry† and Pharmacology & Pharmacotherapy‡, The Danish University of Pharmaceutical Sciences (DUPS), DK-2100 Copenhagen, Denmark
Abstract: For more than four decades there has been a search for selective inhibitors of GABA transporters. This has led to potent and selective inhibitors of the cloned GABA transporter subtype GAT1, which is responsible for a majority of neuronal GABA transport. The only clinically approved compound with this mechanism of action is Tiagabine. Other GABA transporter subtypes have not been targeted with comparable selectivity and potency. We here review a comprehensive series of competitive inhibitors that provide information about the GABA recognition site and summarise the structure-activity relations in a ligand-based pharmacophore model that suggests how future compounds could be designed. Finally, some of the recent results on subtype-characterised competitive inhibitors and recent lipophilic aromatic GABA uptake inhibitors are reviewed.
1. INTRODUCTION: DEVELOPMENT OF relevance in other CNS-related disorders, where GABA SELECTIVE GABA UPTAKE INHIBITORS mediated neurotransmission is believed to be involved [10,11]. The initial isolation of g-aminobutyric acid (GABA, see Table 1) in brain tissue in 1950 was followed by studies in 2. GABA TRANSPORTER HETEROGENEITY the 1960s that revealed the vital role of this compound as the major inhibitory neurotransmitter in the central nervous So far, five different GABA transporters have been cloned, one vesicular GABA transporter (VGAT) [12] and system (CNS) [1]. The discovery of an active transport + system [2] for GABA in the late 60s initiated the search for four that are members of the Na -dependent transporter family, SLC-6 [13,14]. The nomenclature of the latter group compounds capable of inhibiting these transporters in a selective manner. This endeavour has continued over the is species-dependent and mGAT1-mGAT4 in mouse subsequent four decades, and the current review provides an correspond to rGAT-1, rBGT-1, rGAT-2 and rGAT-3 in rat, respectively. Rat nomenclature is used for human subtypes overview of the progress towards specific inhibitors and also but the human homologue of mGAT3 has not been cloned. describes a pharmacophore model for future design of such compounds. The review covers many of the small substrate- The mouse nomenclature will be used in this review. After the discovery of the GABA transport system, it became related GABA analogues developed at our institution and by apparent that there are pharmacological differences in the others, and we will focus on GABA analogues that are uptake systems in glial and neuronal cells. Thus, 2,4- conformationally restricted and provide information about diaminobutyric acid (DABA) and cis-3-aminocyclohexane- the GABA transporter recognition site. The developments in carboxylic acid (ACHC) displayed different substrate the group of lipophilic aromatic GABA uptake inhibitors are inhibitory effects compared with b-alanine in glial versus also updated in the end of the review since this group of neuronal cells [15,16]. When the first member of the group compounds are more likely to have a therapeutic potential as of GABA transporters was cloned from rat brain and termed they generally tend to penetrate the blood-brain barrier. GAT-1 [13], it was believed to be the neuronal transporter. However, they provide little information on the GABA However, GAT-1 has later been shown to be present also in binding site, which is the main focus of this review. astrocytes, and the distinction between neuronal and glial Very early on it was realised that inhibition of GABA transporters is not very clear. In general, the subtypes display uptake could provide a therapeutic strategy in epileptic varying distribution in different brain regions and at the disorders where GABA mediated inhibition was believed to cellular level [17]. be impaired [3-5] since inhibition of GABA uptake would Initially, research was focused on identifying compounds augment GABA neurotransmission. Later, the development that could effectively discriminate GABA uptake from of very potent inhibitors disclosed this strategy to be useful GABA receptor activity, and these efforts have indeed been ultimately leading to approval of the GABA transport successful. Later, the heterogeneity of transporters was inhibitor Tiagabine in the treatment of partial seizures [6,7]. targeted. The apparent existence of different glial and Although GABA uptake inhibitors have primarily been neuronal GABA transport systems prompted a parallel investigated in relation to epilepsy [8,9] more recent studies search for compounds that could discriminate between these indicate that this therapeutic intervention may be of transporters; however this task has not been solved convincingly. Compounds that effectively block glial uptake *Address correspondence to this author at the Department of Medicinal Chemistry, Danish University of Pharmaceutical Sciences, are believed to be useful since this transport leads to Universitetsparken 2, DK-2100 Copenhagen, Denmark; Tel: +45 35306114; degradation of GABA and thereby impairment of inhibitory E-mail: [email protected] neurotransmission. In contrast, GABA transported into
1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1862 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al. neurons is reused and therefore is crucial for maintaining corresponds to neuronal GABA transport, although synapto- neuronal GABA levels [18]. It has been evidenced that some preparations contain GFAP, a marker for astrocytes targeting the glial transport could be therapeutically favour- [28]. Cortical astrocytes are cultured essentially as described able and that the weak glial preference may be an important by Hertz et al. [29] providing astroglial cells with virtually property for the anticonvulsive action of Tiagabine [19]. no neurons remaining. Furthermore, because of the extensive Conversely, compounds like DABA with a neuronal biochemical and biophysical similarities between this preference display proconvulsive behaviour [20]. Since it is preparation and those of its in vivo counterparts, this not a single transporter subtype that accounts for the glial technique is ideal for studying astrocytes in vitro [30]. uptake it is at present unclear how compounds with a glial Primary cultures of cortical neurons (neu) are prepared preference can be developed. essentially as described by Hertz et al. [31] resulting in well With the cloning of four different GABA transporter differentiated GABAergic neurons without notable contami- subtypes, GAT1-GAT4, it became apparent that several of nation from astrocytes and non-neuronal cells. Considering the very potent GABA uptake inhibitors were GAT1- these preparation methods, it is likely that GAT1-mediated transport predominates. Thus most of the compounds tested selective, including Tiagabine [21]. Thus, most of the in vivo and in vitro investigations involving GABA uptake inhibi- for GABA transport inhibition in neuronal preparations may tors have been probing the role of GAT1 mediated GABA only have been investigated at the GAT1 subtype. These uptake. A GAT1 knock-out mouse was recently developed, aspects may explain the high abundance of GAT1-selective compounds and why so few selective compounds have been which enable further studies of the functional role of GAT1 [22]. found for GAT2-4. Subsequent efforts have been devoted to the development 4. STRUCTURE ACTIVITY RELATIONSHIPS. of subtype specific inhibitors, but very potent and specific An overview is presented of the compounds that have inhibitors of the remaining subtypes are, as yet, unavailable. been evaluated as GABA uptake inhibitors in neuronal and As an implication, little knowledge exists on both the role of glial uptake assay systems, starting with the small substrate- the remaining subtypes for GABA regulation in the CNS and related compounds. The focus will be on conformationally the therapeutic prospects of targeting these subtypes. restricted analogues that provide information about the Recently, we demonstrated that GAT2 could have an GABA transporter recognition site. Such data have been important functional role in the CNS. This was based on the used to construct a pharmacophore model that summarises synergistic action among a GAT1/GAT2 selective inhibitor the known structure-activity relationships (SARs). As stated in combination with GAT1-selective inhibitors like Tiaga- above, the assay methods employed in the characterisation of bine [23]. However, the GAT1 activity of the inhibitor most of the compounds imply that the pharmacophore precludes any firm conclusions. Thus, there continues to be a features elucidated belong to a neuronal “GAT1-like” model. need for potent and selective inhibitors for GAT2-GAT4 to The following sections will briefly deal with the GABA clarify the functional roles and the therapeutic potential of transporter subtype pharmacological profile of selected these subtypes. inhibitors, and finally an update will be given on inhibitors 3. GABA UPTAKE ASSAYS. containing aromatic lipophilic side-chains. When relevant, GABA receptor activity of the compounds will be discussed A number of different assays have been applied for since this activity limits the specificity of the compounds and determining the potency of GABA uptake inhibitors. Most thereby the use as a pharmacological tool. commonly an IC50 value is determined from measuring the uptake of radiolabelled GABA by incubating a tissue prepa- 4.1. Acyclic GABA Analogues ration in the presence of varying concentrations of inhibitor. GABA itself (Table 1) is a potent inhibitor of [3H]- Non-specific binding, which includes receptor bound GABA GABA uptake into both glial and neuronal cells [32-34] and is measured and subtracted. Although comparison of IC50 only slightly weaker than (R)-nipecotic acid (Table 5), which values can be problematic since it depends on the GABA is the most potent small substrate-related inhibitor known. concentration applied, the same concentrations are often The most potent inhibitors among the earliest tested acyclic used and normally a reference compound is included, compounds are the GABA analogues 2-fluoro-GABA and enabling an approximate comparison of the uptake data from trans-4-aminocrotonic acid [35] which had potency similar different laboratories and studies. In the following sections to GABA, whereas DABA and 3-hydroxy-GABA are we will describe compounds investigated in different assays. slightly weaker [36]. (S)-DABA is approximately twice as Compounds have been characterised using synaptosomes potent as (RS)-DABA. Studies of the enantiomers of 4- (syn), brain slices (sli), cultured neuronal cells (neu), or glial methyl-GABA and trans-4-aminopent-2-enoic acid revealed cells (astro). Both mini-slices and prisms are prepared from that these compounds display different stereoselectivity in the outer convexity of the cerebral cortex using the first slice neuronal GABA uptake and GABAA receptor binding [33]. 0.5 mm in depth [24] and the major difference between slices (R)-4-Methyl-GABA interacts more potently with neuronal and prisms is the subsequent chopping. The preparations are and glial GABA uptake systems than (S)-4-methyl-GABA, basically identical and believed to represent neuronal uptake whereas the two enantiomers are equipotent in GABAA [25]. The majority of compounds have been characterised in receptor binding. Likewise, (R)-trans-4-aminopent-2-enoic a synaptosome preparation assay in which nerve terminals acid is an inhibitor of GABA uptake systems, whereas the are pinched off during homogenisation and centrifugation (S)-enantiomer is inactive. (S)-Trans-4-aminopent-2-enoic and the synaptosomes isolated [26,27]. A major part of acid, however, display a much higher potency at GABAA synaptosomal GABA transport capability probably receptors than the (R)-enantiomer [33]. Shortening or Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1863
Table 1. Acyclic GABA Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes and Brain Slices and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R3 R1 R3
H2N H2N CO2H H N CO H CO H 2 2 n 2 R2 III Uptake inhibition I II Compound IC50 (mM)
R1 R2 R3 n Syn Sli Astro
I H H H GABA 2.6a 15#,b 35b 20c
I F H H 2-fluoro-GABA 35d
d I NH2 H H DABA 108
d I NH2 H H (S)-DABA 50
I H OH H 3-hydroxy-GABA 100d
I H H Me (R)-4-methyl-GABA 200#,b 120b
I H H Me (S)-4-methyl-GABA 750#,b 1000b
II H trans-4-aminocrotonic acid 26e
II Me (R)-trans-4-aminopent-2-enoic acid 160#,b 500b
II Me (S)-trans-4-aminopent-2-enoic acid >5000#,b >5000b
III 1 ß-alanine >5000#,b 1000b
III 3 5-amino-pentanoic acid 230d
The brain slices were prepared from rat brains unless otherwise specified. #Preparation from mice. a) Ref [32]. b) Ref [33]. c) Ref [34]. d) Ref [36]. e) Ref [35]. extending the GABA chain by one carbon atom to give ß- selectivity at GABAergic recognition sites [37]. 4,5-Dihy- alanine and 5-aminopentanoic acid, respectively, leads to dromuscimol is equipotent with muscimol at the GABAA more than an order of magnitude reduction in potency as receptor, and it is more potent as an inhibitor of neuronal compared to GABA (Table 1) [33,36]. ß-Alanine is inactive than glial GABA uptake systems [39]. The enantiomers of as an inhibitor of neuronal GABA uptake and is much less dihydromuscimol showed different pharmacology. The potent at GABAA receptor binding sites than GABA [33]. A uptake activity resides with the (R)-enantiomer, unlike the variety of other acyclic compounds have been tested for GABAA receptor binding affinity conferred by the (S)- activity as GABA uptake inhibitors. However, as these enantiomer. (R)-Dihydromuscimol displays a weak prefe- molecules are often very flexible, they do not provide much rence for neuronal uptake systems [27]. Both 5-amino- information about the conformation in which GABA methyl-3-isothiazolol (thio-muscimol) [37], and 5-amino- interacts with the transporter recognition site. methyl-3-hydroxyfuran-2(5H)-one [40] lack measurable activity as inhibitors of GABA uptake. 4.2. Muscimol Analogues 4.3. THPO Analogues 5-Aminomethyl-3-isoxazolol (muscimol, Table 2), a Incorporation of the amino sidechain of muscimol into a constituent of the fly agaric mushroom Amanita muscaria, ring led to the synthesis of the bicyclic GABA analogue possesses a bioisosteric substitute for the carboxylic acid 4,5,6,7-tetrahydroisothiazolo[5,4-c]pyridin-3-ol (THIP) and group of GABA, in the form of the 3-isoxazolol moiety. the isomeric ß-alanine analogue 4,5,6,7-tetrahydroisoxa- Accordingly, muscimol has a variety of GABAergic effects. zolo[4,5-c]pyridin-3-ol (THPO). While THIP is a potent Early studies revealed that it is a potent GABAA receptor agonist that also has effects on synaptosomal GABA uptake GABAA receptor agonist, THPO is a selective GABA uptake inhibitor [27,41], however it is almost two orders of sites albeit two orders of magnitude weaker than nipecotic magnitude weaker than nipecotic acid. THPO was previously acid. Furthermore, muscimol is a moderately potent GABAB receptor agonist and a substrate for GABA transaminase shown to be a selective inhibitor of glial GABA uptake 3 [37]. Unlike the zwitterionic acyclic GABA uptake systems (Table ) [27], but more recent studies indicate that inhibitors, muscimol is able to penetrate the blood-brain THPO displays very weak glia selectivity at best [42]. THPO displays competitive inhibition of GABA uptake [41,43], and barrier [38], and these properties made it an obvious lead compound for the development of compounds displaying is not a substrate for GABA transport [27,39]. THPO has been found to penetrate the blood-brain barrier (BBB) in 1864 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
Table 2. Muscimol Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes and Cultured Neurones and Inhibition of Glial GABA Uptake into Cultured Astrocytes
Uptake inhibition
IC50 (mM)
Compound Syn Neu Astro
OH
muscimol 320a 2500*,b 2000b N H2N O
a ,b b OH dihydromuscimol 130 1500* 4000 (R)-dihydromuscimol 70a 800c 2000c N a c c H2N O (S)-dihydromuscimol >500 >5000 >5000
OH
thiomuscimol n.s.d N H2N S
OH
5-aminomethyl-3-hydroxyfuran-2(5H)-one n.s.*,e O H2N O
*Rat Brain slices. n.s. = no significant inhibition. a) Ref [39]. b) Ref [33]. c) Ref [27]. d) Ref [37]. e) Ref [40]. newborn mice [27], but THPO only penetrates to a very exchange renders the 7-membered iso-THAO effectively limited extent into the brain after systemic administration in inactive. animals with a fully developed brain [44]. A number of 4.4. Exo-THPO Analogues analogues of THPO were designed and synthesised in an attempt to obtain glia selective GABA uptake inhibitors Moving of the amino group in THPO to an exo-cyclic more potent than THPO. The 7-membered ring analogue of position provides exo-THPO, which displays potency similar THPO, 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol to that of THPO at neuronal and glial GABA uptake sites (THAO), was found to have a similar pharmacological (Table 4) [19,44]. Monomethylation of the amino group of profile to that of THPO [27,42]. Replacing the oxygen atom exo-THPO to give the secondary N-methyl-exo-THPO leads of the isoxazole ring of THPO with sulphur led to the weak to slightly increased IC50 for neuronal uptake over glial neuronal GABA uptake inhibitor 4,5,6,7-tetrahydroiso- uptake, making it a glia-selective GABA uptake inhibitor. thiazolo[4,5-c]pyridin-3-ol (thio-THPO). Saturation of one By contrast, the dimethylated tertiary amine N,N-dimethyl- of the double bounds of THPO resulted in the inactive cis- exo-THPO is less potent than exo-THPO. The GABA uptake 3a,4,5,6,7,7a-hexahydroisoxazolo[4,5-c]pyridin-3-ol (cis- inhibitory activity of exo-THPO and N-methyl-exo-THPO dihydro-THPO) [27]. resides with the (R)-enantiomers. The 5-isoxazolol isomer of THPO, 4,5,6,7-tetrahydroiso- N-Ethyl-exo-THPO and N-2-hydroxyethyl-exo-THPO xazolo[4,3-c]pyridin-3-ol (iso-THPO) is equipotent with display similar potency to exo-THPO in both neuronal and THPO and THAO, whereas the seven-membered ring glial uptake. N-Allyl-exo-THPO and N-acetyloxyethyl-exo- analogues, 5,6,7,8-tetrahydro-4H-isoxazolo[4,3-c]azepin-3- THPO display similar potencies at neuronal GABA uptake ol (iso-THAO) and 5,6,7,8-tetrahydro-4H-pyrazolo[4,5- sites, but are both glia selective [19,43,44]. Substitution of c]azepin-3-ol (aza-THAO) show no effect on neuronal the methyl groups of N,N-dimethyl-exo-THPO with bulkier GABA uptake. Iso-THPO also displays weak binding to butyl groups does not change the potency significantly. The GABAA receptors, as well as strychnine-sensitive glycine 7-membered ring analogue of exo-THPO, 4-aminocyc- receptor antagonism [45]. The distribution of the negative lohepteno[1,2-d]isoxazol-3-ol (exo-THAO), lacks measur- charge of 3-isoxazolol and 5-isoxazolol moieties differs as a able activity as an inhibitor of neuronal GABA uptake [46], result of the interchange of the nitrogen and oxygen atoms and both the 3-isothiazolol analogue of exo-THPO, 4-amino- [45]. It is notable that this change in regioelectronic 3-hydroxy-4,5,6,7-tetrahydro-1,2-benz-isothiazole (exo-thio- distribution is tolerated in the recognition of the 6-membered THPO), and its N-methylated derivative N-methyl-exo-thio- ring analogues THPO and iso-THPO, whereas a similar Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1865
Table 3. THPO Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes and Cultured Neurones and Inhibition of Glial GABA Uptake into Cultured Astrocytes
Uptake inhibition
IC50 (mM)
Compound Syn Neu Astro
OH
THIP >300a >5000a >5000a N HN O
OH 5000a 300a HN THPO 160a N 501*,b 262*,b O
OH HN >5000a 500a THAO 180a N 487*,b 258*,b O
OH
HN thio-THPO 230a 5000a >5000a N S
OH
HN cis-dihydro-THPO >300a >5000a >5000a N O
OH
HN iso-THPO 130c O N
OH HN iso-THAO >300c O N
OH HN aza-THAO >200d NH N
The brain slices were prepared from rat brains. *Ki values (µM). a) Ref [27]. b) Ref [41]. c) Ref [45]. d) Unpublished data from DUPS.
THPO are rather weak inhibitors of neuronal and glial modification was the addition of lipophilic aromatic side- GABA uptake systems [44]. chains [32]; however, these derivatives will be discussed separately as they constitute a separate class of small 4.5. Nipecotic Acid and Related Compounds molecule derivatives. A number of these lipophilic aromatic derivatives display a noncompetitive type of inhibition. The discovery of the conformationally restricted ß- alanine analogue nipecotic acid [41] as a potent inhibitor of 4.5.1. Nipecotic Acid and Isomeric Analogues GABA uptake provided a very important lead structure for (R)-Nipecotic acid was found to be more potent than the the development of structurally related GABA uptake corresponding (S)-isomer as an inhibitor of neuronal and inhibitors. The pharmacologically most important structural glial GABA uptake systems [33,47]. (R)-Nipecotic acid has 1866 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
Table 4. Exo-THPO Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes, Brain Slices and Cultured Neurones, and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R1 R2 N OH
N X Uptake inhibition
Compound IC50 (mM)
R1 R2 X Syn Neu Astro
H H O exo-THPO 181a 780b 250b
H H O (R)-exo-THPO 118a 681a 281a
H H O (S)-exo-THPO >300a
Me H O N-methyl-exo-THPO 63a 405b 48b
Me H O (R)-N-methyl-exo-THPO 39a 510a 60a
Me H O (S)-N-methyl-exo-THPO >300a
Et H O N-ethyl-exo-THPO 105a 390b 301b
a c c CH2=CHCH2- H O N-allyl-exo-THPO 98 220 73
a c c HOCH2CH2- H O N-2-hydroxyethyl-exo-THPO 173 300 200
a c c AcOCH2CH2- H O N-acetyloxyethyl-exo-THPO 125 200 18
Me Me O N,N-dimethyl-exo-THPO 291a >1000c >1000c
Bu Bu O N,N-dibutyl-exo-THPO 201a
H H S exo-thio-THPO 290a ~3000d ~3000d
Me H S N-methyl-exo-thio-THPO 242a 2000d 1200d
NH2 OH
exo-THAO n.s.*,e N O n.s. = no significant inhibition. *Rat brain slices. a) Ref [44]. b) Ref [19]. c) Ref [43]. d) Unpublished data from DUPS. e) Ref [46]. been reported to be slightly glia selective, whereas (S)- tested in GABAA receptor binding assays have shown any nipecotic acid displays the opposite selectivity, being slightly activity [27]. The nipecotic acid side-chain homologue, more potent at neuronal than glial GABA uptake sites (Table piperidine-3-acetic acid, a GABA analogue, is a much 5) [27]. (R)-Nipecotic acid and THPO display competitive weaker inhibitor of neuronal GABA uptake systems than inhibition of glial as well as neuronal uptake systems [43], nipecotic acid [37,42]. Phosphinic acid has been used as a but unlike THPO, nipecotic acid is a substrate for neuronal bioisostere for carboxylic acid groups in the development of and glial GABA uptake systems [48]. Substitution of the ligands for GABAB and GABAC receptors. Accordingly, methylene group at the 2-position of the piperidine ring of piperidine-3-phosphinic acid was synthesised and found to nipecotic acid with an oxygen atom as in 1,2-oxazinane-6- inhibit GABA uptake into synaptosomes, although its carboxylic acid or with a nitrogen atom as in (R)- and (S)- potency is lower than that of nipecotic acid (Table 5) [51]. piperazic acid leads to a decrease in potency [49]. The The corresponding methyl phosphinic acid was found to be isomeric piperazine-2-carboxylic acid was found to be inactive. Piperidine-3-sulphonic acid displays no potency at inactive as well. The constitutional isomers of nipecotic acid, neuronal and glial GABA uptake sites [37]. The 7-membered piperidine-4-carboxylic acid (isonipecotic acid) [27,50] and ring analogue of nipecotic acid, perhydroazepine-3- piperidine-2-carboxylic acid (pipecolic acid) [42], are both carboxylic acid (homonipecotic acid), proved to be without measurable activity as inhibitors of neuronal GABA essentially inactive as an inhibitor of neuronal and glial uptake. The GABA analogue isonipecotic acid, however, is a GABA uptake systems [46,52]. It is notable that the potent GABAA receptor agonist [27]. None of the bioisosteric transformation from THPO to nipecotic acid isonipecotic analogues tested on GABA uptake systems have leads to a significant increase in potency, whereas the same been found active and none of the nipecotic acid analogues transformation from THAO to homonipecotic acid does not. Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1867
Table 5. Nipecotic Acid and Isomeric Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes and Brain Slices, and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R1 X CO H 1 2 R2 HN CO2 H HN HN
X2 R3 I II III Uptake inhibition
Compound IC50 (mM)
X1 X2 Syn Sli Astro
I H H nipecotic acid 4a 9b
I H H (R)-nipecotic acid 2a 5c 30d 70d
I H H (S)-nipecotic acid 10a 30c 1000d 500d
I O H 1,2-oxazinane-6-carboxylic acid 380e
I NH H (S)-piperazic acid 21e
I NH H (R)-piperazic acid >500e
I H NH piperazine-2-carboxylic acid >500e
R1 R2 R3
d c II H H CO2H isonipecotic acid >300 >5000
b II CO2H H H pipecolic acid >1000
b II H CH2CO2H H piperidine-3-acetic acid 372
f II H PO2H2 H piperidine-3-phosphinic acid 24
f II H P(Me)O2H H P-methylpiperidine-3-phosphinic acid >1000
#,g g II H SO3H H piperidine-3-sulfonic acid 5000 5000
III homonipecotic acid 3000#,h502i 1000#,h
The brain slices were prepared from rat brains unless otherwise specified. #Preparation from mice. n.s. = no significant inhibition. a) Ref [32]. b) Ref [42]. c) Ref [50]. d) Ref [27]. e) Ref [49]. f) Ref [51]. g) Ref [27,33]. h) Ref [34]. i) Ref [58].
4.5.2. Substituted Analogues of Nipecotic Acid acid are much weaker GABA uptake inhibitors than cis-4- hydroxynipecotic acid [53]. Methylation of the 5-position of A number of substituted analogues of nipecotic acid have cis-4-hydroxynipecotic acid results in a marked decrease in been synthesised and characterised for their GABA uptake potency at neuronal GABA uptake sites, (3RS,4SR,5SR)-4- inhibitory properties. In general, the substituents decrease the hydroxy-5-methylnipecotic acid being weakly active and the potency of nipecotic acid, but it is not known whether any (3RS,4SR,5RS)-stereoisomer inactive [27,33]. 3-Hydroxy- subtype-specific compounds might be found among these nipecotic acid was found to have no effect on neuronal analogues. These compounds will be summarised since they GABA uptake [25]. Cis- and trans-4-mercaptonipecotic acid give important information about the structural constraints of are almost inactive. 3-Amino-, cis- and trans-4-amino-, cis- the GABA binding site of the neuronal transporter. 5-amino-, cis-5-acetamido and cis-4-acetamidonipecotic acid Substitution at the 4-position of nipecotic acid with a show very weak or no inhibitory effect upon GABA uptake hydroxy group led to the potent GABA uptake inhibitor cis- into synaptosomes [27,37,53]. Substitution with an epoxy 4-hydroxynipecotic acid (Table 6). Whereas (R)-nipecotic function at the 3,4-position of nipecotic acid led to the very acid and cis-4-hydroxynipecotic acid are equipotent inhibi- weak inhibitor, 3,4-epoxynipecotic acid. tors of GABA uptake into synaptosomes, cis-4-hydroxyni- N-methylation of nipecotic acid (Table 7) results in a pecotic acid is apparently more glia selective than (R)- substantial decrease in potency at neuronal GABA uptake nipecotic acid [27,33]. Like nipecotic acid, cis-4-hydroxy- systems [33,54,55] and the N,N-dimethyl derivative was nipecotic acid is a substrate for glial GABA uptake systems. found to be even weaker [42]. Monomethylated derivatives Trans-4-hydroxynipecotic acid and cis-5-hydroxynipecotic of nipecotic acid have been synthesised at the 2-, 3-, 4-, 5- 1868 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
Table 6. Hydroxy-, Amino-, Mercapto- and Related Substitutions of Nipecotic Acid. Inhibition of Neuronal GABA Uptake into Synaptosomes And Brain Slices and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R1 CO2H HN
R2
R3 Uptake inhibition
Compound IC50 (mM)
R1 R2 R3 Syn Sli Astro
H OH H cis-4-hydroxy-nipecotic acid 5a 200b 10a
H OH H trans-4-hydroxy-nipecotic acid 203c
H H OH cis-5-hydroxy-nipecotic acid 173c 5000d >5000d
H OH Me (3RS,4SR,5SR)-4-hydroxy-5-methyl-nipecotic acid 300b >5000#,b
H OH Me (3RS,4SR,5RS)-4-hydroxy-5-methyl-nipecotic acid 3000b 4000#,b
OH H H 3-hydroxynipecotic acid >200e >5000d
H SH H cis-4-mercapto-nipecotic acid 160e >5000d
H SH H trans-4-mercapto-nipecotic acid >200e >5000d 5000d
c b,e #,b,e R1-R3=NH2 or amino- and acetamido- nipecotic acids >200 >5000 >5000
R2, R3= NHAc
CO2H HN e e O 3,4-epoxynipecotic acid 370 >5000
The brain slices were prepared from mouse brains. a) Ref [27]. b) Ref [33]. Ref [53]. d) Ref [25]. e) Unpublished data from DUPS. and 6-positions, as well as the phenyl derivatives at 2-, 4-, 5- 4.7. Ecgonine Analogues and 6-positions [57]. All of these congeners were reported to be inactive as inhibitors of GABA uptake into synaptosomes, (1R,2R,3S,5S)-3-hydroxytropane-2-carboxylic acid but since the compounds were only characterised at (ecgonine, Fig. 1), possessing the amino acid skeleton of concentrations up to 100 µM, some of them might be weak cocaine, is a structural analogue of cis-4-hydroxynipecotic inhibitors. acid. Ecgonine and (1R,2S,3S,5S)-3-hydroxytropane-2-car- boxylic acid (y -ecgonine), the corresponding analogue of 4.6. Guvacine Analogues trans-4-hydroxynipecotic acid, and their demethylated derivatives (1R,2R,3S,5S)-3-hydroxynortropane-2-carboxylic Guvacine and nipecotic acid are approximately equipo- acid (nor-ecgonine) and (1R,2S,3S,5S)-3-hydroxynortropane- tent inhibitors of GABA uptake (Table 8) [32,54]. Guvacine 2-carboxylic acid (nor-y -ecgonine) were all found to be was previously found to display weak glia selectivity [27], inactive as inhibitors of neuronal GABA uptake systems in but more recent studies indicate that guvacine displays slices from mouse or rat brain [33,58]. equipotent inhibition of glial and neuronal GABA uptake It has been demonstrated by 1H-NMR spectroscopy that [27]. Like nipecotic acid, but unlike THPO (Table 3), the preferred conformations of the piperidine rings of guvacine is a substrate for neuronal transport systems [37]. ecgonine, y -ecgonine, nor-ecgonine and nor-y -ecgonine N-Methylation of guvacine led to a large reduction in adopt slightly distorted chair conformations with an potency, and the N,N-dimethylated analogue was found to be equatorial orientation of the hydroxy groups [58]. In the inactive [56]. 5-Methylguvacine is a very weak inhibitor of preferred conformations of (1R,2R,5R)-nortropane-2-car- neuronal GABA uptake [58], whereas 6-methylguvacine boxylic acid and (1R,5S)-2-nortropene-2-carboxylic acid, the proved to be moderately potent [57]. Increasing the size of rings and the carboxylate groups adopt the same the N-substituent led to the inactive compounds 6-ethylgu- conformation as the preferred conformations of (R)-nipecotic vacine and 6-propylguvacine [59]. The ring homologue of guvacine, 2,5,6,7-tetrahydro-1H-azepine-3-carboxylic acid acid and guvacine, respectively. The preferred conformation of nipecotic acid is a chair conformation with the carboxylate (homoguvacine), was found to be inactive as an inhibitor of group in an equatorial position in both the crystalline state neuronal and glial GABA uptake systems, thus paralleling homonipecotic acid [58]. and in aqueous solution [60]. As (1R,2R,5R)-nortropane-2- Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1869
Table 7. Alkyl- and Phenylsubstitutions of Nipecotic Acid. Inhibition of Neuronal GABA Uptake into Synaptosomes and Brain Slices, and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R2 R3 R1 CO2 H N
R6 R4 Uptake inhibition R5 Compound IC50 (mM) R Syn Sli Astro
a R1=Me 122 N-methylnipecotic acid 70#,c 300#,b
R2-R6 = Me or >100d R1, R2 or R4-R6=Ph
CO2 H N N,N-dimethyl-nipecotic acid 48% †,#,e
CO2H HN 1,2,3,4-tetrahydro-isoquinoline-4-carboxylic acid >100d
The brain slices were prepared from rat brains unless otherwise specified. #Preparation from mice. † Percent inhibition at 10 mM. a) Ref [54]. b) Ref [33]. c) Ref [55]. d) Ref [57]. e) Ref [56].
enantiomer. (RS)-ß-proline and (S)-ß-proline apparently CO2H CO2 H CO2H N N HN display similar potency at neuronal GABA uptake sites. The observation that cis-4-hydroxynipecotic acid and nipecotic OH OH OH acid are equipotent as neuronal GABA uptake inhibitors, the former displaying some glial selectivity, prompted the synthesis of cis-4-hydroxy-ß-proline. However, cis-4- hydroxy-ß-proline proved to be essentially inactive as an CO2H CO2 H CO2H HN HN HN inhibitor of neuronal as well as glial GABA uptake [33]. Introduction of a hydroxy group at the 3-position of ß- OH proline or an amino group at the 3- or cis-4-position led to inactive compounds. The 5-membered ring analogue of the Fig. (1). Ecgonine and related structures are all inactive as weak GABA uptake inhibitor 1,2-oxazinane-6-carboxylic inhibitors of GABA uptake [33, 58]. acid (Table 5), isoxazolidine-5-carboxylic acid, was found to be essentially inactive [33]. By contrast with nipecotic acid carboxylic acid and (1R,5S)-2-nortropene-2-carboxylic acid and its side-chain homologue, piperidine-3-acetic acid, the are inactive as inhibitors of GABA uptake it has been latter being much less potent, the ß-proline homologue, concluded that the ethylene bridges in the ecgonine-type pyrrolidine-3-acetic acid (homo-ß-proline), proved to be a compounds are responsible for their lack of affinity for neuronal and glial GABA uptake inhibitor more potent than neuronal GABA transport systems. ß-proline and equipotent with (R)-nipecotic acid [25,37,61]. However, homo-ß-proline is rather unselective since it also 4.8. ß-Proline Analogues has the same affinity as isonipecotic acid at the GABAA receptor. Homo-ß-proline seems to be a substrate for both Pyrrolidine-3-carboxylic acid (ß-proline) was found to be neuronal and glial uptake systems [61]. The enantiomers of a much weaker GABA uptake inhibitor than the ring homo-ß-proline are approximately equipotent as inhibitors of homologue, nipecotic acid, although displaying greater glia GABA uptake into synaptosomes. The a -hydroxylated selectivity (Table 9) [33]. Furthermore, ß-proline displays homo-ß-proline analogue is inactive as an inhibitor of GABAA receptor binding activity as well, albeit with a GABA uptake [37]. Introduction of a double bond at the 3,4- considerably lower potency than isonipecotic acid. (RS)-ß- position of ß-proline led to a large reduction in potency for Proline displays a higher potency at glial GABA uptake glial GABA uptake systems. On the other hand, 3-pyrroline- systems than the (S)-enantiomer indicating that the affinity 3-carboxylic acid is more potent at GABAA receptor binding for glial GABA uptake systems resides with the (R)- 1870 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
Table 8. Guvacine Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes, Brain Slices and Cultured Neurons, and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R1 CO2H N
R3
R2 Uptake inhibition
Compound IC50 (mM)
R1 R2 R3 Syn Sli Neu Astro
H H H Guvacine 5a 8b 32c 29c
Me H H N-methyl-guvacine 122b
H Me H 5-methyl-guvacine 547d
H H Me 6-methyl-guvacine 40f
H H Et 6-ethylguvacine >100g
H H Pr 6-propyl-guvacine >100g
HN CO2H Homoguvacine >1000d 5000d >5000d
CO2H N N,N-dimethyl-guvacine 4%†,#,e
The brain slices were prepared from rat brains unless otherwise specified. #Preparation from mice. †Percent inhibition at 10 mM. a) Ref [32]. b) Ref [54]. c) Ref [44]. d) Ref [58]. e) Ref [56]. f) Ref [57]. g) Ref [59]. sites than ß-proline [25,33]. Imidazole-4-acetic acid displays 4.10. Aminocyclopentanoic Acid (ACPC) Analogues no affinity for neuronal or glial GABA uptake systems. The homologue of homo-ß-proline, 3-(pyrrolidine-3-yl)-propio- The cis- and trans-isomers of 3-aminocyclopentane- nic acid is inactive as an inhibitor of neuronal GABA uptake carboxylic acid (3-ACPC) are nearly equipotent as inhibitors [27,37]. of neuronal GABA uptake (Table 11) [68]. Pharmacological characterisation of the enantiomers of trans-3-ACPC has 4.9. Aminocyclohexanecarboxylic Acid (ACHC) Analo- revealed that the GABA uptake inhibitory activity of trans- gues 3-ACPC resides with the (1R,3R)-enantiomer, whereas the (1S,3S)-enantiomer displays specific GABAA receptor Cis-3-ACHC was found to be a potent inhibitor of activity [68]. The 2-ACPC isomers were reported to be of GABA uptake with selectivity for neuronal GABA uptake similar potency as cis-3-ACHC as GABA uptake inhibitors, (Table 10), the active enantiomer being (1S,3R)-3-ACHC with the cis-isomer being slightly more potent than the trans- [62,63]. The isomeric trans-3-ACHC was shown to be isomer [64]. The unsaturated 3-ACPC analogue 4- essentially inactive as an inhibitor of neuronal GABA uptake aminocyclopent-1-enoic acid shows similar potency to trans- [35,64]. Cis- and trans-2-ACHC were found to be essentially 3-ACPC at GABA uptake systems as well as GABAA inactive as inhibitors of neuronal and glial GABA uptake. receptor activation. GABA uptake activity of this compound Likewise, neither cis- nor trans-4-ACHC displays any resides with the (R)-enantiomer, whereas the GABAA activity towards GABA uptake systems [64]. Introduction of receptor activity resides with the (S)-enantiomer [68]. Still, a double bond at the 4,5-position of cis-3-ACHC to give cis- the compounds are somewhat unselective since the 5-aminocyclohex-3-enecarboxylic acid results in similar enantiomers of cis- and trans-3-ACPC are GABAC receptor potency to cis-3-ACHC [34]. Likewise, 5-aminocyclohexa- partial agonists, whereas (S)-4-aminocyclopent-1-enoic acid 1,3-dienecarboxylic acid (gabaculine) containing two ring is a potent GABAC receptor antagonist [68]. Unlike cis- and double bonds is equipotent with cis-3-ACHC as inhibitor of trans-3-ACPC, the trans-isomer of 4-aminocyclopent-2- neuronal GABA uptake [65]. Gabaculine is a naturally enoic acid is fairly potent at both neuronal GABA uptake occurring neurotoxin isolated from Streptomyces toyacaensis systems and at GABAA receptors, whereas the cis-isomer is and is a potent irreversible inhibitor of GABA-T [66,67]. less potent at both GABA uptake and GABAA receptors [68]. (1R,4R)-4-aminocyclopent-2-enoic acid displays no activity Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1871
Table 9. ß-Proline Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes and Brain Slices and, Inhibition of Glial GABA Uptake into Cultured Astrocytes
X CO2 H R3 HN R1 HN CO2 H R2
I II Uptake inhibition
Compound IC50 (mM)
I X R1 R2 R3 Syn Sli Astro
a a b I CH2 H H ß-proline 48 453 320 1200#,b
#,b b I CH2 H H (S)-ß-proline 1700 1900
a #,c c I CH2 OH H 3-hydroxy-ß-proline >200 >5000 >5000
a #,b b I CH2 H OH cis-4-hydroxy-ß-proline >200 2500 1800
a #,c c I CH2 NH2 H 3-amino-ß-proline >200 >5000 >5000
#,c c I CH2 NH2 H cis-4-amino-ß-proline >5000 >5000
I O H H isoxazolidine-5-carboxylic acid 1000#,b >5000#,b
II H homo-ß-proline 2.5d 75*,c 20c
II H (S)-homo-ß-proline 2.7d
II H (R)-homo-ß-proline 1.6d
II OH hydroxy-(pyrrolidine-3-yl)acetic acid n.s.e
CO2 H HN 3-pyrroline-3-carboxylic acid 160a 1800#,b 1200b
HN CO2H imidazole-4-acetic acid >5000#,b >5000b N
CO2H HN 3-(pyrrolidine-3-yl)-propionic acid n.s.e
The brain slices were prepared from rat brains unless otherwise specified. #Preparation from mice. *Cultured neurones. n.s. = no significant inhibition. a) Unpublished data DUPS. b) Ref [33]. c) Ref [25]. d) Ref [61]. e) Ref [37].
Table 10. Aminocyclohexanecarboxylic Acid Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes, Brain Slices and Cultured Neurones, and Inhibition of Glial GABA Uptake into Cultured Astrocytes
R1
H2N R2
Uptake inhibition R3
Compound IC50 (mM)
R1 R2 R3 Syn Sli Neu Astro
a c d d H CO2H H cis-3-ACHC 8 85 200 700 75b
e H CO2H H (1S,3R)-3-ACHC 25 1872 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
(Table 10) Contd…
R1
H2N R2
Uptake inhibition R3
Compound IC50 (mM)
R1 R2 R3 Syn Sli Neu Astro
e H CO2H H (1R,3S)-3-ACHC >500
b c H CO2H H trans-3-ACHC 1330 1400
b g g CO2H H H cis-2-ACHC >1000 >300 >300 22 %†,*,b
†, ,b g g CO2H H H trans-2-ACHC 19 % * >300 >300
b H H CO2H cis-4-ACHC 398 4 %†,*,b
†, ,b H H CO2H trans-4-ACHC 6 % *
H2N CO2 H cis-5-amino-cyclohex-3-ene- 131f carboxylic acid
H2N CO2H gabaculine 69h
The brain slices were prepared from rat brains. *Synaptic membrane vesicles. †Percent inhibition at 10 mM. a) Ref [27]. b) Ref [64]. c) Ref [35]. d) Ref [63]. e) Ref [62]. f) Ref [34]. g) Unpublished data from DUPS. h) Ref [65].
Table 11. Aminocyclopentanecarboxylic Acid Analogues. Inhibition of Neuronal GABA Uptake into Synaptosomes and Brain Slices
R2 R1 R R H2 N R2 1 3
Uptake inhibition I II R4 Compound IC50 (mM)
R1 R2 Syn Sli
a I H CO2H cis-3-ACPC 13
a I H CO2H trans-3-ACPC 29
a I H CO2H (1R,3R)-3-ACPC 11
a I H CO2H (1S,3S)-3-ACPC n.s.
b I CO2H H cis-2-ACPC 63
b I CO2H H trans-2-ACPC 119
R1 R2 R3 R4
a II H NH2 H CO2H 4-aminocyclopent-1-enecarboxylic acid 16
a II H NH2 H CO2H (R)-4-aminocyclopent-1-enecarboxylic acid 7.4
a II H NH2 H CO2H (S)-4-aminocyclo-pent-1-enecarboxylic acid n.s. Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1873
(Table 11) Contd…
R2 R1 R R H2 N R2 1 3
Uptake inhibition I II R4 Compound IC50 (mM)
R1 R2 Syn Sli
a II NH2 H CO2H H cis-4-aminocyclopent-2-enecarboxylic acid 85
a II NH2 H CO2H H trans-4-aminocyclopent-2-enecarboxylic acid 19
a II NH2 H CO2H H (1R,4R)-4-aminocyclo-pent-2-enecarboxylic acid n.s.
a II NH2 H H CO2H 3-aminocyclopent-1-enecarboxylic acid 15
CO H 2 †, ,b H2N cis-3-aminomethyl-cyclopentane-carboxylic acid 42% *
The brain slices were prepared from rat brains. *Synaptic membrane vesicles. †Concentration of test compound 10 mM. n.s. = no significant inhibition. Ref [68]. b) Ref [64].
at GABA transporters but activates GABAA receptors low energy structures based on the charge-bearing functional indicating that the GABA uptake activity of trans-4- groups. aminocyclopent-2-enoic acid resides with the (1S,4S)- A consistent alignment of selected low energy enantiomer [68]. 3-Aminocyclopent-1-enoic acid displays conformations of these inhibitors was possible, indicating a moderate activity with respect to both GABA uptake common binding mode to GAT1 of these compounds. By inhibition and GABAA receptor activation [68]. fitting known ligands to this initial pharmacophore align- 4.11. Neuronal GABA Transport Pharmacophore Model ment, information about the space available for ligands in the binding pocket of GAT1 (sterically allowed volume, Fig. 3), With the pharmacological activities and stereospecifici- as well as some areas of the binding pocket not available for ties of a large number of active and inactive substrate ligands (sterically disallowed volumes, Fig. 4) has been analogues available, 3D pharmacophore modelling can be obtained. A potential application for such a model would be used to describe the structural requirements of the neuronal a 3D database query for retrieving novel active structures, as GABA transport [69]. As stated earlier, a major part of has for example been performed for other GABAergic neuronal GABA transport is mediated by GAT1 and the targets [70]. The model can also be used and refined as activities determined in the available assays therefore follows to rationalise the structure activity data described primarily reflect GAT1 uptake. It therefore follows that the above, lending confidence that the model features accurately model can be considered to be a GAT1 model. An initial reflect the three dimensional characteristics of the transporter alignment was based upon a template set of seven binding site. structurally diverse, active, and reasonably conformationally restricted GABA uptake inhibitors (Fig. 2). THPO and its seven-membered ring analogue, THAO, are equipotent inhibitors of neuronal GABA uptake. The Conformational analyses were performed for the global energy minimum conformation of THAO fits well to structures in the template set, followed by superposition of the initial pharmacophore alignment and the extra volume
OH NH2 OH CO2H CO2H HN HN HN N N O O THPO (R)-exo-THPO (R)-nipecotic acid guvacine
H N CO H 2 2 H N CO H HN 2 2 CO2H
(R)-4-aminocyclopent- (R)-homo-b-proline (1S,3R)-3-ACHC 1-enecarboxylic acid Fig. (2). Structures of GABA uptake inhibitors chosen as templates for a neuronal GAT1-type pharmacophore model. 1874 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
Conversely, the 7-membered ring analogue of nipecotic acid, homonipecotic acid, is essentially inactive as an inhibitor of neuronal GABA uptake. The global energy minimum of (R)-homonipecotic acid fits well to the pharma- cophore alignment, and the inactivity of homonipecotic acid is thus probably due to steric repulsion between the 7- membered ring and the target (Fig. 6).
Fig. (6). Superposition of (R)-homonipecotic acid (green) with (R)- Fig. (3). Sterically allowed volume of the GABA transporter nipecotic acid (grey). Nitrogen atoms are blue, oxygen atoms red. GAT1, showing the position and conformation of (R)-nipecotic The displayed conformation of (R)-homonipecotic acid is the global acid. energy minimum.
Homonipecotic acid occupies a larger volume than the combined volume of the pharmacophore alignment, and this extra volume has been included in the sterically disallowed volumes of the pharmacophore model. This may explain why ring expansion for THPO is allowed, whereas for nipecotic acid it results in a large activity decrease. Racemic exo- THAO does not display any inhibitory activity at neuronal GABA uptake sites. The lowest energy conformer of exo- THAO that fits reasonably well with the pharmacophore alignment has a conformational energy penalty of 38 kJ/mol (Fig. 7). Thus, in the case of exo-THAO it seems that a
Fig. (4). Sterically disallowed volumes (four views). The structure of (R)-nipecotic acid is displayed. These volumes are the sterically allowed volume subtracted from the combined van der Waals volumes of the aligned conformations of (R)-homonipecotic acid, (R)-3,4-epoxynipecotic acid, (1R,2R,5R)-nortropane-2-carboxylic acid and (1R,5S)-2-nortropene-2-carboxylic acid. Fig. (7). Superposition of (R)-nipecotic acid and (R)-exo-THAO occupied by THAO compared to the volume occupied by (two views). Nitrogen atoms are blue, oxygen atoms red. The THPO is thus included in the sterically allowed volume of displayed conformer of (R)-exo-THAO has a conformational energy the pharmacophore alignment (Fig. 5). penalty of 38 kJ/mol.
Fig. (8). The initial pharmacophore model. Only the pharmacophore elements are displayed. Left: The distances between the nitrogen atom and the heteroatoms of the acid group Fig. (5). Superposition of (R)-nipecotic acid (grey) with THPO are 4.6 and 5.3 Å. Right: The dihedral angle between the N-H bond (green) and THAO (cyan). Nitrogen atoms are blue, oxygen atoms and the plane of the acid group is 61°. red. The displayed conformers of THPO and THAO are the global energy minimum conformations. Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1875 conformational energy barrier may explain why exo-THAO binding pocket “below” nipecotic acid as displayed in Fig. 4. is inactive as an inhibitor of GABA uptake. Additional steric By contrast, the space “above” nipecotic acid is relatively effects cannot be excluded. unexplored. As expected, there seems to be available space Cis-4-hydroxynipecotic acid is a potent inhibitor of in the equatorial direction from the nitrogen atom of neuronal GABA uptake systems. Only the racemic nipecotic acid. This is where the lipophilic sidechains of 9 compound has been tested, but the enantiomer with the same Tiagabine (Fig. ) and other potent lipophilic analogues are stereochemistry of the carbon atom bearing the carboxylate attached. group as in (R)-nipecotic acid is presumably the active The available structure-activity data suggest that whereas enantiomer. Since cis-4-hydroxynipecotic acid and nipecotic only one hydrogen bond donor is required for binding, the acid are equipotent inhibitors of neuronal GABA uptake presence of two hydrogen bond donors is optimal. Likewise, systems, the volume occupied by the axial 4-hydroxy group two hydrogen bond acceptors seem to be optimal for must be available to ligands, and (3S,4R)-4-hydroxynipecotic interaction with GAT1. As compounds with both shorter and acid was included in the sterically allowed volume of the longer distances between the hydrogen bond donor and the pharmacophore model. In addition to THAO, the volume of hydrogen bond acceptors have been tested and found less (3S,4R)-4-hydroxynipecotic acid was included in the potent, the optimal distance between the hydrogen bond sterically allowed volume of the model. donor and the hydrogen bond acceptors seems to be within Following the same line of reasoning as for (R)- the distances of the GABA uptake inhibitors in the template set. The ideal distances between the nitrogen atom and the homonipecotic acid, the extra volume occupied by the global hydrogen bond accepting heteroatoms appear to be around energy minimum conformation of (R)-3,4-epoxynipecotic 4.6Å and 5.3Å, with a dihedral angle between the N-H acid was included in the sterically disallowed volumes of the vector and the plane of the acid functionality of 61º (Fig. 8). model. This rather simple pharmacophore model encapsulates As described earlier, (1R,2R,5R)-nortropane-2-carboxylic the GABA uptake inhibitory activity of the known substrate acid and (1R,5S)-2-nortropene-2-carboxylic acid are inactive analogues described above. The conformational space of the as inhibitors of GABA uptake and therefore the ethylene GABA uptake inhibitors was searched employing MM3* or bridge in ecgonine type compounds must be responsible for MMFFs and the GB/SA solvation model as implemented in their lack of affinity. In line with this reasoning, the MacroModel 8.1 [71]. The MM3* force field applied has conformers of (1R,2R,5R)-nortropane-2-carboxylic acid and added parameters for 3-isoxazololes based on ab initio (1R,5S)-2-nortropene-2-carboxylic acid that give the best fit with the pharmacophore alignment are low energy confor- calculations. The alignments were based on superpositions of the nitrogen atom and a hydrogen atom of the amino group mers, and the inactivity of these compounds is most likely as well as the three atoms of the carboxylate or its due to steric conflict between the ethylene bridges and the protein. The extra volumes occupied by (1R,2R,5R)- equivalent. The structures have been superimposed using nortropane-2-carboxylic acid and (1R,5S)-2-nortropene-2- rigid conformations and atoms as fitting points. The superimpositions have been evaluated visually, and not carboxylic acid were included in the sterically disallowed volumes of the model. exclusively on the basis of RMS-values. The conformational energy penalties were calculated by subtracting the internal 2-, 3-, 4-, 5- and 6-methyl and phenyl substitution of energy of the global energy minimum conformation from the nipecotic acid leads to inactive or low potency GABA uptake internal energy of the bioactive conformation. These inhibitors. The results indicate that methyl and phenyl procedures follow the principles outlined by Liljefors and substitution of nipecotic acid is not favourable and that the Petterson [72]. space around the combined volume of the inhibitors in the Since most of the known GABA uptake inhibitors are ß- template set may be rather limited in these directions. However, the fact that cis-4-hydroxynipecotic acid and 6- alanine analogues, it seems surprising that ß-alanine is almost inactive at GAT1. The conformation of the ß-alanine methylguvacine display affinity for neuronal GABA moiety of the putative bioactive conformation of (R)- transporters means that some extra volume may be tolerated. nipecotic acid is identical to the conformation of the global Likewise, methylation of the 5-position of cis-4- energy minimum of ß-alanine according to MM3* / GB/SA. hydroxynipecotic acid as well as 5-substitution of nipecotic Thus, the model fails to explain the lack of GAT1 activity of acid with a hydroxy group or an acetamido group leads to ß-alanine. This small molecule does however only fill a decreased potency at neuronal GABA uptake sites. The chair fraction of the available sterically allowed volume, a SAR conformations of the methylated compounds with the characteristic which may not be expected to promote carboxylate group positioned equatorially are most likely activity. low-energy conformers, and the decrease in potency of these compounds is therefore unlikely to be the result of a Unfortunately, in the absence of sufficient selectivity data conformational energy penalty. The moderate potencies of for the compounds, such a model predicts very little about the requirements of the other GABA transporter subtypes. As these compounds indicates that the volume around the 5- position of nipecotic acid is not completely inaccessible by described below, only a few compounds have been ligands, although 5-substitution does not seem to be characterised at the GAT subtypes, and those that have been favourable. characterised are either not selective or without sufficient structural diversity to allow adaptation of the GAT1 model According to the sterically disallowed volumes, there directly to other subtypes. However, a new route forward has does not seem to be much space available for ligands in the recently been provided by the publication of the X-ray 1876 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
O O
N OH S N OH
S
SKF100330A Tiagabine
F3C O O O N OH O N N OH
CF3
NNC-711 CI-966
Fig. (9). Structures of GAT1-selective inhibitors. structure of a homologous leucine transporter [73]. By subtypes. Interestingly, the anticonvulsant properties of exo- combining the ligand-based model with mutation data and THPO analogues correlate with their glial activities, and not homology models of the subtypes, it may become possible the neuronal activities that follow the potencies at GAT1 for the first time to build comparative models to shed light [19]. THPO inhibits GABA uptake via GAT3 with the same on areas that can be targeted for the design of subtype- weak potency seen for GAT1, whereas THAO does not selective GABA uptake inhibitors. display any inhibitory activity of GAT3. Both THPO and THAO are essentially inactive as inhibitors of GABA uptake 4.13. Compounds Characterised at GABA Transporter via GAT2 and GAT4. Subtypes 4.14. Lipophilic Aromatic GABA Uptake Inhibitors As stated earlier, only potent and highly selective GABA uptake inhibitors for the GAT1 subtype have so far been N-Alkylation of the amino groups of nipecotic acid and discovered, not for the other individual GAT2-4 subtypes. guvacine with a 4,4-diphenyl-3-butenyl side-chain resulted The lipophilic aromatic inhibitors with few exceptions are not only in marked increases in the potency of these selective either GAT1-selective or unselective. A more diverse GABA uptake inhibitors. This also provides a general pharmacology is displayed by the small substrate-related strategy for enabling BBB penetration, and for making inhibitors (Table 12). Thus, GABA has a higher affinity for inhibitors that are not substrates, which is a characteristic for GAT1, GAT3 and GAT4 than for GAT2 [74,75]. most of the small substrate-related inhibitors. The discovery ß-Alanine is inactive as an inhibitor of GABA uptake is notable since the introduction of small alkyl substituents at nitrogen results in a reduction of the potency of nipecotic into both synaptosomes and neurones and it inhibits neither acid, suggesting that the larger substituents are able to reach GAT1, as expected, nor GAT2 [21,75]. However, it is a potent inhibitor of GABA uptake via GAT3 and GAT4. The or induce a novel hydrophobic zone. Subsequently, a large number of derivatives of nipecotic acid and guvacine N- unsaturated GABA analogue trans-4-aminocrotonic acid has substituted with certain lipophilic aromatic side-chains have a pharmacological profile similar to GABA, whereas the cis- been synthesised. Some of the most potent derivatives that form has a ß-alanine-like profile [76]. have been extensively pharmacologically characterised are Recently, 2-hydroxy-ß-alanine (isoserine) was reported to shown in Fig. 9. In general, the compounds contain a di- or have the same profile as ß-alanine [76]. Also the 2-amino tri-aromatic moiety separated by a flexible linker of 3-4 analogue displays similar profile. Interestingly, these authors atoms from the GABA resembling part of the molecule. also report that 3-guanidino-propionic acid is a potent The majority of the reported compounds have only been GABA uptake inhibitor. (RS)- and (R)-nipecotic acid inhibit characterised in synaptosome assays reflecting the neuronal all subtypes potently except for GAT2, whereas the (S)- uptake mediated by GAT1. The cloning of GABA enantiomer is less active [17,21]. Cis-4-hydroxy-nipecotic transporter subtypes has revealed that several of the most acid and guvacine have a similar profile to nipecotic acid, potent GABA uptake inhibitors are also highly GAT1- though they are less potent. Cis-3-ACHC seems to be rather selective, including the structures shown in Fig. 9. The SAR GAT1-selective, as are the exo-THPO analogues, albeit with of this class of lipophilic aromatic GABA uptake inhibitors reduced potency [17]. The latter is notable since the N- methylated analogue is glia selective and therefore high glial was recently reviewed [77]. However, several publications activity does not seem to be related to any of the other Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1877
Table 12. Compounds Tested on GABA Transporter Subtypes. Inhibition of GABA Uptake into Cell Lines Expressing Cloned GABA Transporters
Uptake inhibition
IC50 (mM)
Compound GAT1 GAT2 GAT3 GAT4
5*,a 36*,a 5#,a 7*,a H N CO H GABA 2 2 13b 97b 9b 12b
trans-4-amino-crotonic acid 3c 64c 7c 5c H2 N CO2H
cis-4-amino-crotonic acid 280c 280 c 9c 9c H2N CO2H
9000b 1000b 15b 30b H2N CO H ß-alanine 2 2920#,d 1100*,d 66#,d 110#,d
OH (RS)-isoserine 2500c 230c 4c 5c H2N CO2 H
NH c c c c (CH2 )3CO2H 3-guanidino-propionic acid 26 24 3 3 H2 N N H
nipecotic acid 8*,d 2370*,d 38#,d 159#,d CO2 H HN (R)-nipecotic acid 6*,d 2310*,d 19#,d 50#,e
(S)-nipecotic acid 116*,d 6410*,d 763#,d 2320#,e
CO2H HN cis-4-hydroxy-nipecotic acid 44#,e 3840*,e 567#,e 467#,e OH
CO2H HN guvacine 39#,e 1420*,e 228#,e 378*,e
H2 N CO2H cis-3-ACHC 132#,e 1070*,e 25900#,e 55100#,e
OH
HN THPO 1000f 3000f 800f 5000f N O
OH HN THAO 1000f 4500f >3000f >10000f N O
NH 2 OH
exo-THPO 1000g 3000g >3000g >3000g N O 1878 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
(Table 12) Contd….
Uptake inhibition
IC50 (mM)
Compound GAT1 GAT2 GAT3 GAT4
NH OH N-methyl-exo-THPO 450g >3000g >3000g >3000g N O
NH OH N-ethyl-exo-THPO 320g >1000g >1000g >1000g N O
AcO NH OH N-acetoxyethyl-exo-THPO 550h >1000h >1000h >1000h N O
The cloned transporters are obtained from mouse unless otherwise specified. *Human. #Rat. a) Ref [74]. b) Ref [75]. c) Ref [76]. d) Ref [21]. e) Ref [17]. f) Ref [41]. g) Ref [19]. h) Ref [43]. describing the synthesis and pharmacological characteri- be established by characterisation at the remaining GABA sation of new derivatives have since appeared. Three transporter subtypes. publications from Andersen et al. describe the extension of We recently reported the synthesis and characterisation the lipophilic aromatic series based on nipecotic acid and of a series of lipophilic aromatic analogues based on exo- guvacine in their search for second generation anticon- THPO [83]. In this series EF1502 (Fig. 11), containing the vulsants following the success of Tiagabine [78-80]. The side-chain of Tiagabine, turned out to have similar potency first publication describes variation of the electronic and at GAT1 and GAT2. Thus, the pharmacological profile of steric properties of the diaromatic substituents, as well as EF1502 is novel and distinct from Tiagabine [23]. This chain length of the flexible linker. It was concluded that an finding prompted an in vivo study of the anti-convulsant 9 ethyloxy linker, as in NNC-711 and CI-966 (Fig. ), confers properties of the compound that revealed a synergistic effect high potency. The authors speculate that the oxygen atom between EF1502 and GAT1-selective inhibitors, indicating a may participate in hydrogen bonding, but may also increase possible role for GAT2 as a therapeutic target [84]. The the flexibility of the chain. The second publication describes GAT1 activity was dependent on the stereochemistry of the a series of tricyclic diaromatic substituents at the end of the compound whereas GAT2 activity was not, and as a result ethyloxy linker whereas the third introduces more extensive (S)-EF1502 is a GAT2 selective compound [23,83,84]. variation that leads to several very potent and more Thomsen et al. [75] have previously reported a GAT2 structurally diverse compounds. This shows that some selective compound (NNC-05-2090, Fig. 11); however, this variation can be tolerated at the distal part of the ethyloxy compound also affects other transmitter systems. linker. Together with SNAP-5114 which is a GAT3/GAT4 Zheng et al. [81] recently reported a series based on the selective inhibitor, EF1502 and NNC-05-2090 are the only bis-thienylbutenyl side-chain from Tiagabine, adding known lipophilic aromatic compounds displaying a selective variation to both the aromatic and the GABA-like moieties subtype pharmacological profile different from that of the (Fig. 10). Interestingly, the compounds in Fig. 10 displayed GAT1-selective inhibitors. potencies similar to that of Tiagabine despite the significant increase in steric bulk attached to the thiophene moieties. CONCLUSION Recently Zhao et al. [82] reported some lipophilic Despite the large number of GABA uptake inhibitors aromatic derivatives based on pyrrolidine-2-acetic acid, synthesised and characterised, there continues to be a need proline and hydroxylated analogues. The compounds were for the development of further types of selective GABA characterised at GAT1 and GAT3, but it seems that the uptake inhibitors. Although GABA transporters have proven pharmacology is similar to the corresponding compounds to be therapeutically useful targets [6], the complete based on nipecotic acid and guvacine, although this needs to therapeutic potential of GABA transport inhibition seems not Structure-Activity Relationships of Selective GABA Uptake Inhibitors Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1879
OPh OPh O CO2H
S N OH S N
S S PhO PhO
Fig. (10). Tiagabine analogues that are potent GABA uptake inhibitors [81].
N OH S N OH
S N O O
NNC-05-2090 EF 1502
MeO
O O N OH
MeO
OMe SNAP-5114 Fig. (11). Selective GABA uptake inhibitors with activity at GABA transporter subtypes other than GAT1. to have been realised, not even for GAT1. Thus, several BBB penetrating properties. Most of these inhibitors have studies indicate that GABA uptake inhibitors could be been based on nipecotic acid and extensive variation has applied in the treatment of anxiety, pain, abuse, and sleep been introduced in the lipophilic aromatic side-chains. disorders, and clinical trials are investigating the application However, in order to target other subtypes a switch in of Tiagabine in these disorders [10,11]. This emphasises the strategy may be necessary. Our recent finding that continued need for specific GABA uptake inhibitors. substitution of the Tiagabine side-chain to another GABA To demonstrate that sufficient SARs are available for mimicking amino acid moiety than nipecotic acid leads to ligand-based design and virtual screening to assist in the the GAT2-active compound EF1502, shows that greater discovery process, we have presented a pharmacophore variation in the amino acid part of the inhibitors may lead to selective ligands for the other GABA transporter subtypes. model that summarises the known activities of small substrate-related GABA uptake inhibitors at neuronal uptake Although many companies have now left their GABA uptake mediated by GAT1. The recent publication of the X-ray programmes this field may gain renewed industrial attention crystal structure of a bacterial leucine transporter that is if new medicinal chemistry strategies prove successful. homologous to the GABA transporters [73] opens up the REFERENCES way to homology modelling and structure-based design at [1] Roberts, E. GABA: The road to neurotransmitter status. In GABA transporter subtypes, as well as a greater understan- Benzodiazepine/ GABA Receptor and Chloride Channels: ding of the mechanics of substrate transport and transporter Structural and Functional Properties. Olsen, R. W. and Venter, J. inhibition. Thus, a hybrid ligand-based and structure-based C. Eds.; Alan R. Liss: New York, 1986; pp 1-39. approach, integrated with synthetic exploration of the [2] Iversen, L. L.; Neal, M. J. The uptake of [3H]GABA by slices of rat cerebral cortex. J. Neurochem. 1968, 15, 1141-1149. subtype binding site volumes, appears most promising with a [3] Meldrum, B.S. Epilepsy and g-aminobutyric acid-mediated view to the discovery of future selective GABA uptake inhibition, Int. Rev. Neurobiol., 1975, 17, 1-36. inhibitors. In particular, homology models based on the [4] Meldrum, B.S. Pharmacology of GABA, Clin. Neuropharmacol., leucine transporter structure can disclose binding site 1982, 5, 293-316. differences between subtypes that can be exploited in the [5] Neurotransmitters, Seizures, and Epilepsy. Morselli, P.L.; Lloyd, K.G.; Löscher. W.; Meldrum, B. and Reynolds, E.H. Eds.; Raven design of new subtype selective inhibitors. Undoubtedly, Press: New York, 1981, pp. 361. these compounds will be important pharmacological tools. [6] Braestrup, C.; Nielsen, E. B.; Sonnewald, U.; Knutsen, L. J. S.; Andersen, K. E.; Jansen, J. A.; Frederiksen, K.; Andersen, P. H.; From a therapeutic point of view the lipophilic aromatic Mortensen, A.; Suzdak, P. D. (R)-N-[4,4-Bis(3-methyl-2- inhibitors has so far been the most promising due to their thienyl)but-3-en-1-yl]nipecotic acid binds with high-affinity to the 1880 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Clausen et al.
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Craig W. Lindsley†, Scott E. Wolkenberg† and Gene G. Kinney‡,*
†Medicinal Chemistry and ‡Neuroscience Drug Discovery, Merck Research Laboratories, West Point, PA 19486
Abstract: Clinically utilized antipsychotic agents share as a common mechanism the ability to antagonize dopamine D2 receptors and it is widely assumed that this activity contributes to their efficacy against the positive symptoms of schizophrenia. The efficacy of currently marketed antipsychotic agents on the negative and cognitive symptoms of this disease, however, is not optimal. One alternate hypothesis to the “dopamine hypothesis” of schizophrenia derives from the observation that antagonists of NMDA receptor activity better mimic the symptomatology of schizophrenia in its entirety than do dopamine agonists. Findings from this line of research have led to the NMDA receptor hypofunction (or glutamate dysfunction) hypothesis of schizophrenia, which complements existing research implicating dopamine dysfunction in the disease. According to the NMDA receptor hypofunction hypothesis, any treatment that enhances NMDA receptor activity may prove useful for the treatment of the complex symptoms that define schizophrenia. This idea is now supported by numerous clinical studies that have reported an efficacious response following treatment with activators of the NMDA receptor co-agonist glycineB site. One area of study, aimed at potentiating the NMDA receptor via activation of the glycineB site is small molecule blockade of the glycine reuptake transporter type 1 (GlyT1). Broadly, these efforts have focused on derivatives of the substrate inhibitor, sarcosine, and non-sarcosine based GlyT1 inhibitors. Accordingly, the following review discusses the development of both sarcosine and non-sarcosine based GlyT1 inhibitors and their current status as putative treatments for schizophrenia and other disorders associated with NMDA receptor hypoactivity.
INTRODUCTION hypofunction of NMDARs may be a critical component of schizophrenia. This idea is formally known as the NMDA Glutamate is a major excitatory neurotransmitter in the receptor hypofunction hypothesis. This hypothesis predicts mammalian central nervous system that exerts its actions that any drug capable of potentiating NMDAR activity in a through a variety of ionotropic and metabotropic receptors physiologically relevant manner may ameliorate schizo- (iGluR and mGluR, respectively), which are distributed both phrenic symptoms. pre and post-synaptically throughout neuronal populations in the brain. One class of iGluR is the N-methyl-D-aspartate Glycine is a simple amino acid inhibitory and excitatory receptor (NMDAR) complex. The NMDAR is distinguished neurotransmitter [4, 5]. As an inhibitory neurotransmitter, from other iGluRs by several unique properties. Among glycine acts at the ligand-gated, strychnine sensitive these is dependence on depolarization in order to relieve glycineA binding site in cerebellum, brainstem and spinal Mg2+ block of the channel, a complex subunit makeup that cord [4]. As an excitatory neurotransmitter, glycine interacts provides for functional heterogeneity and a requirement for with the glycineB site in forebrain areas like cortex, co-activation of a glutamate binding site and a strychnine hippocampus and thalamus [5-7], and activation of this insensitive glycine binding site (i.e., the glycineB site). The glycineB binding site on the NR1 subunit of the NMDAR is NMDAR was initially implicated in schizophrenic condi- required for subsequent glutamate binding to the NR2 tions based on the observation that use-dependent NMDAR subunit of the receptor complex. Interestingly, the affinity of antagonist treatment produced a variety of symptoms that glycine for the glycineB binding site can vary widely resemble those observed in the clinic [1, 2]. Although a depending on the NR2 isoform makeup of the NMDAR variety of psychoactive drugs are known to induce psycho- complex [see 7], whereas expression of an NR3 subunit sis, including psychomotor stimulants (cocaine, amphet- causes the NMDAR complex to act as an excitatory glycine amine), hallucinogens (LSD), and NMDA receptor antago- receptor that is insensitive to glutamate. Further, NR3 nists (PCP, ketamine), only NMDA-receptor antagonists containing NMDAR complexes are inhibited by D-serine, appear to reproduce the full spectrum of symptoms observed which normally acts as an agonist at the glycineB binding in schizophrenia [1, 2]. In addition to producing these site [8]. These findings suggest that glycine serves a symptoms in normal humans, NMDAR antagonists were modulatory role on NMDAR function whose sensitivity is also shown to exacerbate existing symptoms in schizophre- dependent on regional NMDAR subunit expression. The nics and trigger the re-emergence of symptoms in stable modulatory nature of this interaction has led several groups patients [3]. These clinical observations, combined with a to hypothesize that enhancing activity at the glycineB site growing body of preclinical literature, suggest that will allow for an increase in normal glutamatergic signaling at the NMDAR thereby decreasing the likelihood of toxicity *Address correspondence to this author at Merck Research Laboratories, associated with glutamate receptor agonism. WP44E-200, P. O. Box 4, West Point, PA 19486. Tel: (215)-652-5628; Fax: (215)-993-6240; E-mail: [email protected]
1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1884 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al.
Accordingly, several strategies have been employed as improved properties for therapeutic use [21-27]. None- therapeutic approaches to potentiate NMDAR function via theless, due to the activity of sarcosine and its structural activation of the glycineB binding site. Among these, bloc- similarity to glycine it served as a rational starting point in kade of transporters that are specific for glycine represents a the design of specific GlyT1 inhibitors. particularly useful approach. GlyT1 and GlyT2 have been Structurally, modification of sarcosine has taken many identified as two transporters that modulate glycine reuptake forms, and a unifying theme is the incorporation of large [9-13]. These transporters belong to the 12-transmembrane + - hydrophobic groups on the amine and retention of a domain Na /Cl dependent family of neurotransmitter carboxylic acid or ester (see Table 1). These modifications g transporters which includes taurine, -aminobutyric acid are exemplified in the earliest disclosures of specific GlyT1 (GABA), proline and monoamine transporters. Both forms inhibitors, a series of patents from Trophix (which later of transporter are known to exist in multiple isoforms became Allelix and then NPS). While no publication (GlyT1a-d and GlyT2a-c, respectively), which have describing the initial development of this series has differential expression and development [10, 14]. Although appeared, the structures disclosed can be viewed as hybrids GlyT1 and GlyT2 show high inter-species homology [15] between sarcosine (2) and fluoxetine (4), a serotonin they are only ~50% homologous to each other, display transporter inhibitor. This progression seems reasonable differential regional distribution and underlie differential since GlyT1 belongs to the same family of Na+/Cl-dependent functional activity. GlyT1 is found throughout the forebrain transporters as the serotonin transporter [24]. while GlyT2 is localized in the brain stem and cerebellum [13, 16, 17]. At the cellular level, GlyT2 is colocalized with The Trophix/Allelix/NPS patents cover a range of diaryl- glutamic acid decarboxylase (GAD) and expressed at linked sarcosine analogues (5, 6, 8–10, 14, Table 1) [28, 29, glycinergic nerve endings in the spinal cord, brainstem and 31-36, 40], and an important contribution to the field was the cerebellum [13, 16, 17]. In addition to an overlapping synthesis and detailed characterization of one example, distribution with GlyT2, GlyT1 is highly expressed by glial ALX-5407 (3) [45]. This compound, whose racemate is cells in areas of the cortex, hippocampus, septum and referred to in the literature as N-[3-(4’-fluorophenyl)-3-(4’- thalamus, and is found presynaptically in glutamatergic phenylphenoxy)propyl]sarcosine (NFPS), is a potent (IC50 nerve terminals in forebrain regions [13, 18]. 3–22 nM) and selective inhibitor of GlyT1 and has been an invaluable pharmacological tool in evaluating the neuro- Recent publications suggest that a great deal of physiological role of GlyT1. NFPS selectively increases pharmaceutical industry and academic interest exists in the extracellular concentrations of glycine in rat prefrontal identification and development of selective GlyT1 inhibitors. cortex (no effect on other amino acids, taurine, or glutamate) These efforts have led to the development of numerous and [45, 46]. In vitro experiments in rat hippocampal and structurally diverse GlyT1 inhibitors. Broadly these can be brainstem slices demonstrated that blockade of GlyT1 with classified as sarcosine-based and non-sarcosine-based. NFPS potentiates NMDA receptor-mediated excitatory post- Although the potency and efficacy of many of these novel synaptic currents [47, 48]. Subsequent experiments have GlyT1 inhibitors are not known in detail, the multiplicity of shown that NMDAR activity is enhanced following selective structures and number of pharmaceutical companies invol- blockade of GlyT1 in vivo [49, 50]. Chen et al. [49] ved in this area of research highlights the intense activity and demonstrated that NFPS both potentiated NMDA mediated interest in GlyT1 inhibitor research. Accordingly, the present responses and reversed a reduction of NMDA response in review focuses on progress in the synthesis and characte- prefrontal cortical neurons produced by local application of a rization of sarcosine and non-sarcosine based inhibitors of specific glycine site antagonist, (+)HA-966. Further, syste- GlyT1. mic NFPS administration potentiated long-term potentiation GLYT1 INHIBITOR CHEMISTRY (LTP) in the dentate gyrus of the hippocampus and increased c-fos expression in neurons of the nucleus accumbens, two 2.1. Sarcosine-Derived GlyT1 Inhibitors physiological responses known to depend upon activation of Following their cloning and expression, pharmacological NMDA receptors [50]. Additional evidence comes from investigations of glycine transporters distinguished the two preclinical behavioral studies using assay conditions (a) isoforms GlyT1 and GlyT2. Pharmacologically, sarcosine sensitive to NMDAR antagonists and (b) sensitive to (N-methyl glycine, 2) acts as a selective GlyT1 substrate antipsychotic drug treatment. Early studies using glycyldo- with functional antagonist activity [10, 11, 13, 19]. In a decylamide (GDA) (19, Fig. 2) demonstrated a selective recent 6-week double blind, placebo-controlled trial, stably reversal of PCP-induced locomotor activity in mice [51, 52]. treated schizophrenic patients (N=17) were treated with the More recent studies using NFPS demonstrated a potentiation GlyT1 inhibitor sarcosine (2 g/day). Supporting an effica- of prepulse inhibition (PPI) of the acoustic startle response in cious role for GlyT1 inhibition in the treatment of schizo- DBA/2J mice [50], a strain that demonstrates low basal phrenia, the results from this trial demonstrated a significant levels of PPI [53]. NFPS and the closely related sarcosine and efficacious response on negative, positive and cognitive derivative, Org 24461 (20, Fig. 2), have also inhibited PCP symptoms [20]. and amphetamine-induced locomotor activity in mice and PCP induced changes in rat EEG [54]. Glycine (1) and Org Like glycine itself, sarcosine lacks appropriate pharma- 24598 (21, Fig. 2) are effective in reversing amphetamine- ceutical properties (potency, pharmacokinetic profile, and induced locomotor activity and PPI deficits in neonatal blood brain barrier permeability) for use as a treatment, and ventral hippocampally lesioned rats [55], an animal model thus the favorable clinical trial results have motivated a resulting in a developmentally regulated psychotic-like significant effort in pursuit of GlyT1 inhibitors with phenotype [56]. Finally, NFPS partially reverses the Progress in the Preparation and Testing of Glycine Transporter Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1885
O O
H2 N HN OH OH 1 2
F O N NH OH O O
F3C
3 4
Fig. (1). Glycine (1), sarcosine (2), ALX-5407 (R-NFPS, 3), and fluoxetine (4).
Table 1. Structures of Sarcosine-Derived GlyT1 Inhibitors Disclosed by Various Companies in the Patent Literature
Compound Number Structure Patent Number Company Reference
O O N OH 5 WO199745115 Trophix [28]
F3C
O N O 6 WO199745423 Trophix [29]
F O N OH 7 WO200007978 AKZO NOBEL [30]
O
H2 N OH
8 F US006103743 Allelix [31]
F F F 1886 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al.
(Table 1) Contd….
Compound Number Structure Patent Number Company Reference
O
H2N OH
9 S US006166072 Allelix [32]
O
O 10 N US020169197 NPS Allelix [33-36] OH
F F
11 WO200136423 AKZO NOBEL [37]
O O N OH
F O N OH O 12 WO200200602 Pfizer [38]
O
Cl
O 13 N WO200208216 H. Lundbeck [39] OH
Cl Progress in the Preparation and Testing of Glycine Transporter Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1887
(Table 1) Contd….
Compound Number Structure Patent Number Company Reference
O O N 14 OH WO2002066456 NPS Allelix [40]
S
F O N OH O 15 WO2003000646 Pfizer [41]
O
F O N OH O 16 EP1284257 Pfizer [42]
F F
N O 17 WO2003053942 H. Lundbeck [43] S N OH
O
O O 18 WO2004096761 H. Lundbeck [44] S N OH
F 1888 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al.
F O O N N OH OH O O O H2N N H 10 CF 3 OCF3 19 20 21 Fig. (2). Glycyldodecylamide (19), Org 24461 (20) and Org 24598 (21). disruptive effects of PCP in a spatial memory task, sugges- (NPS-Allelix, Glaxo, F. Hoffmann-La Roche, Merck, ting that GlyT1 inhibition may provide a cognitive benefit as Sanofi-Aventis) and that a great diversity of chemical well. Therefore, NFPS provides strong support for the structures can inhibit GlyT1. The reader is referred to NMDA hypofunction hypothesis in animal models (where previous reviews for additional information on these series known antipsychotic agents afford similar, positive results) of GlyT1 inhibitors [22-24, 62, 63]. This portion of the and validated GlyT1 as a novel target for drug discovery [50, review will focus on the most advanced and characterized 51, 55]. series of non-sarcosine-derived GlyT1 inhibitors that have recently been disclosed by Roche and Sanofi. Simultaneous with the preparation and characterization of NFPS, numerous patents were published describing In late 2005, researchers from F. Hoffmann-La Roche similarly modified sarcosine-based structures (Table 1). published one of the first detailed accounts of structure- While little biological data is available, these publications activity-relationships (SAR) and drug metabolism/phar- suggest that changes, including the incorporation of rings macokinetics (DMPK) issues with a unique series of non- and unsaturation in the linker between sarcosine and the two sarcosine-derived GlyT1 inhibitors based on an N-(2-aryl- aryl groups, are tolerated. For example, a series of inhibitors cyclohexyl) substituted spiropiperidine scaffold 41-43 (Fig. described by a team at Lundbeck introduced a spiroindane 4) [79, 80]. (13, Table 1) and retained GlyT1 activity (IC <500 nM). In 50 Screening of the F. Hoffmann-La Roche compound addition, researchers at Pfizer have prepared modified repository led to the identification of racemic, cis-41, a analogues by substituting a benzophenone structure for the potent GlyT1 inhibitor with an IC of 26 nM. However, the NFPS biaryl (Fig. 3). The resulting inhibitors ((R)-NPTS 50 spiropiperidine motif is a privileged structure in a number of 22 23 ( ) and CP-802,079 ( )) retain high potency and CNS agents and selectivity within this series was found to be selectivity and were shown to significantly enhance NMDA an issue. As a result, 41 showed inhibitory activity against receptor-dependent LTP in area CA1 of the rat hippocampus GlyT2 (IC50 = 12 mM, 400-fold selective) as well as against [57, 58]. Recently, a series of potent (GlyT1 IC50 <1 nM) the m opioid receptor and the nociceptin/orphanin FQ and selective GlyT1 inhibitors based on a sarcosine-linked receptor (NOP). Selectivity against GlyT2 could be 24 indandione structure were reported by a group at Merck ( ) improved to almost 700-fold by the addition of a 4-fluoro [59]. moiety to afford 42; however, selectivity against the m opioid The GlyT1 inhibitors described here represent the first receptor and NOP receptor was poor. Further SAR studies generation of potent and selective agents. Furthermore, led to the development of 43, an N-(2-hydroxy-2-aryl- several members of this class were evaluated in vivo and cyclohexyl) substituted spiropiperidine. Incorporation of the demonstrated efficacy similar to clinically useful antipsy- key hydroxyl group provided complete selectivity versus chotics. Despite these promising characteristics, the GlyT2 and NOP while improving selectivity versus the sarcosine-derived GlyT1 inhibitors suffer from a range of m opioid receptor. In addition, 43 and related congeners deleterious side effects including ataxia, hypoactivity and displayed improved PK and a predicted maximal achievable decreased respiratory activity [22, 40]. It remains to be bioavailability (MAB) of 81% in mouse and 84% in human definitively determined whether these effects are due to microsomes [79, 80]. overstimulation of the inhibitory strychnine-sensitive glycine Sanofi-Aventis has published a number of patent applica- binding site or, alternatively, a chemotype-specific activity. tions claiming the series 44-46 and culminating in the most Nonetheless, because of this profile, the field has largely advanced non-sarcosine-derived inhibitor to date SSR504734, moved to the study of non-sarcosine derived inhibitors. 47 (Fig. 5) [81-87]. Sanofi has recently disclosed detailed 2.2. Non-sarcosine-Derived GlyT1 Inhibitors biological data in support of the NMDA receptor hypofunc- tion hypothesis with 47 which compliments the earlier data High throughput screening efforts have delivered a obtained with NFPS, rac-3 [46, 50, 87]. Historically, 47 and myriad of non-sarcosine-derived GlyT1 inhibitors 25-40 related congeners were developed based on the theme (Table 2) [22-24, 26, 62-78]. The majority of these GlyT1 introduced with NFPS, rac-3, where a known inhibitor from inhibitors have been disclosed only in the patent literature; the Na+/Cl--dependent neurotransmitter transporter family, in accordingly, there are no details available regarding this instance methylphenidate 48 (a dopamine transporter biological activity. However, it is clear that GlyT1 is a target inhibitor, sold as Ritalin), was employed as starting point for of considerable interest within the pharmaceutical industry the development of this GlyT1 inhibitor series [24, 87]. Progress in the Preparation and Testing of Glycine Transporter Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1889
Cl O O N N F OH OH Cl O O O Cl
O O MeO O OH N 22 N S 23 O 24
Fig. (3). NPTS (22), CP-802,079 (23), and indandione (24) GlyT1 inhibitors.
Table 2. Structures of Non-Sarcosine-Derived GlyT1 Inhibitors Disclosed by Various Companies in the Patent Literature
Compound Number Structure Patent Number Company Reference
F F
25 WO199944596 Janssen [64]
N N N N N O
Cl
N N 26 S WO200222581 Telik [65] N S Cl
N
OH H SmithKline N N 27 S WO2003055478 Beecham [66] O 2 PLC
Cl
OH H N 28 Cl S WO2004113280 Glaxo [67] O O N CF3 1890 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al.
(Table 2) Contd….
Compound Number Structure Patent Number Company Reference
O F. Hoffmann- 29 N WO2005023261 [68] La Roche N
SO2 F3C
R 1 R2
N R3 O N R4 F. Hoffmann- 30 US05040166 [69] N La Roche
OH
CO2Et
N N MERCK F DE 10149370 PATENT 31 [70] (2004) GMBH N Yamanouchi
O N NH
N MERCK N N DE10315570 PATENT 32 [71] F (2004) GMBH Yamanouchi Progress in the Preparation and Testing of Glycine Transporter Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1891
(Table 2) Contd….
Compound Number Structure Patent Number Company Reference
O O S MeO N
OMe 33 WO2001081308 NPS, Allelix [72] N
O Cl
N H Merck & Co., 34 WO2005046601 [73] Inc. N O S O
O Cl
N N H Merck & Co., 35 WO2005094514 [74] N Inc. O S O
O Cl
N N Merck & Co., 36 H WO2005110983 [75] Inc. N
O2 S
O F N
N 37 WO2005058885 Glaxo [76] N HN O
CF3 1892 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al.
(Table 2) Contd….
Compound Number Structure Patent Number Company Reference
O N
N N N 38 HN O WO2005058882 Glaxo [77]
O
O F N
N N 39 HN O WO2005058317 Glaxo [78]
O
N S H MERCK N N 40 WO2003087086 PATENT [91] N GMBH NC
F
H H N H O N O N O N
N N N N N HO N
41 42 43 cis, rac cis, rac cis, rac GlyT1 IC50 = 26 nM GlyT1 IC50 = 23 nM GlyT1 IC50 = 62 nM GlyT2 IC5 0 = >12 mM GlyT2 IC50 = >16 mM GlyT2 IC50 = >100 mM
Fig. (4). Representative F. Hoffman-La Roche GlyT1 inhibitors. One of very few reports describing SAR and DMPK issues for non- sarcosine-derived GlyT1 inhibitors. Progress in the Preparation and Testing of Glycine Transporter Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1893
O
O O N H R N 1 N N H R1 H R1 N N
44, Sanofi WO2004013100 45, Sanofi WO2005037783 46, Sanofi WO2005037785 WO2004013101 WO2005037782 WO2005037792
O Cl
CF3 N H CO2Me NH N
47, Sanofi SSR504734 48, Methylphenidate (Ritalin)
Fig. (5). Representative Sanofi-Aventis GlyT1 inhibitors. 47, SSR504734, the most advanced non-sarcosine-derived GlyT1 inhibitor that has validated the NMDA receptor hypofunction hypothesis in preclinical models of schizophrenia.
Interestingly, SAR studies have not yet been published for dopamine in rat prefrontal cortex (PFC) and displayed this series. efficacy in chronic mild stress models. In summary, SSR504734, 47, is the most advanced non-sarcosine-based SSR504734 (S,S) 47 is a selective and reversible inhibi- GlyT1 inhibitor, exhibiting significant activity in preclinical tor of human, rat and mouse GlyT1 (IC50 = 18, 15 and 38 nM, respectively) with its (R,R) enantiomer, SSR506204 10- models of schizophrenia, anxiety and depression, and fold weaker at 163 nM. In contrast to the selectivity issues therefore validating the NMDA receptor hypofunction model preclinically. Typical antipsychotics display similar effects encountered in the F. Hoffmann-La Roche series 41-43, 47 47 demonstrated no significant activity in a panel of over 120 suggesting that will afford antipsychotic activity in the 47 different receptors, ion channels, enzymes and transporters. clinic [87]. It remains to be seen if or some other GlyT1 Unlike rac-3 which blocks for periods in excess of 24 hours, inhibitor, by targeting hypoglutamergy, will be efficacious 47 inhibited glycine uptake ex vivo at 30 mg/kg p.o. with an against the positive, negative and cognitive symptoms of schizophrenia – clinical confirmation is eagerly awaited. ID50 of 4.6 mg/kg and maintained blockade for >7 hours, returning to basal levels within 24 hours. By microdialysis in GLYT1 MOUSE MODELS rats, 47 selectively increased glycine levels 2-fold over baseline when dosed at 10 mg/kg i.p., a result similar to that In addition to the pharmacological studies discussed obtained with rac-3 [46, 50, 87]. above, additional support for a role of GlyT1 in behaviors associated with schizophrenia come from the use of geneti- Having shown that 47 selectively increases CNS glycine cally engineered mice. Studies using GlyT1 heterozygotic levels, Sanofi-Aventis then evaluated 47 in in vitro knockout mice suggest that a 50% reduction of glycine electrophysiology experiments to determine its ability to reuptake is sufficient for an efficacious response in modulate NMDA currents. 47, at a concentration of 3 mM, preclinical assays and that this level of reuptake inhibition is significantly increased NMDA current in rat hippocampal generally well tolerated. While homozygotic knockout of slices and the effect was reversible, unlike that observed with GlyT1 in mice is lethal, heterozygotic GlyT1 knockout mice rac-3 [46, 50, 87]. display a ~50% reduction in glycine re-uptake and are In vivo, 47 was efficacious in a variety of preclinical devoid of any gross deleterious behavioral phenotype [88, models of schizophrenia, depression and anxiety. Compound 89]. This 50% reduction of glycine reuptake results in 47 prevented ketamine-induced metabolic activation in saturation of glycine at the NMDAR as demonstrated by an mouse limbic regions and reversed MK-801-induced hyper- inability of exogenous glycine application to enhance activity with minimal effective doses (MED) in the range of NMDAR currents at hippocampal synapses. These hetero- 10-30 mg/kg i.p.. In preclinical models of schizophrenia, 47 zygotic mice also showed improved performance during the normalized a PPI deficit in DBA/2J mice (MED: 15 mg/kg probe trial of a water maze task and a reversal or i.p.) and reversed amphetamine-induced hyperlocomotion augmentation of amphetamine and MK-801 induced deficits and PCP-induced attention deficits in adult rats (MED: 1-3 in PPI, respectively [89]. The differential effects of GlyT1 mg/kg i.p.). In addition, 47 was able to increase extracellular reduction on amphetamine versus MK-801 induced deficits 1894 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al. suggests that excess synaptic glycine in the heterozygotic [6] Betz, H. Ligand-gated ion channels in the brain: the amino acid mice increases the ability of NMDAR open-channel receptor superfamily. Neuron 1990, 5, 383-392. [7] Danysz, W.; Parsons, A. C. Glycine and N-methyl-D-aspartate blockers, such as MK-801, to access their site of action. A receptors: physiological significance and possible therapeutic recent report of a forebrain neuronal specific GlyT1 applications. Pharmacol. Rev. 1998, 50, 597-664. disrupted mouse further supports and extends the earlier [8] Chatterton, J. E.; Awouluyi, M.; Premkuman, L. S.; Takahashi, H.; findings [90]. These mice demonstrated enhancement of Talantova, M.; Shin, Y.; Cui, J.; Tu, S.; Sevarino, K. A.; Nakanishi, N.; Tong, G.; Lipton, S. A.; Zhang, D. Excitatory glycine receptors NMDAR, but not AMPA mediated, activity ex vivo. containing the NR3 family of NMDA receptor subunits. Nature Behavioral evaluation of these mice suggested both an 2002, 415, 793-798. antipsychotic and pro-cognitive phenotype. Thus, these mice [9] Borowsky, B.; Mezey, E.; Hoffmann, B. J. Two glycine transporter demonstrated a decreased sensitivity to the psychotomimetic variants with distinct localization in the CNS and peripheral tissues drugs, phencyclidine and amphetamine. Further, are encoded by a common gene. Neuron 1993, 10, 851-863. [10] Kim, K. M.; Kingsmore, S. F.; Han, H.; Yang-Feng, T. L.; enhancement of associational learning was noted in three Godinot, N.; Seldin, M. F.; Caron, M. G.; Giros, B. Cloning of the separate behavioral paradigms. 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Neurochem. 1998, 71, 1785-1803. encompassing explanation for the complex disease sympto- [16] Zafra, F.; Aragon, C.; Olivares, L.; Danbolt, N. C.; Gimenez, C.; matology and thereby lead to novel treatments for Storm-Mathisen, J. Glycine transporters are differentially expressed schizophrenic patients. Excellent progress has been made in among CNS cells. J. Neurosci. 1995, 15, 3952-3969. the past several years towards preclinical validation of the [17] Zafra, F.; Gomeza, J.; Olivares, L.; Aragon, C.; Gimenez, C. NMDA receptor hypofunction hypothesis of schizophrenia. Regional distribution and developmental variation of the glycine transporters GlyT1 and Glyt2 in the rat CNS. Eur. J. Neurosci. Researchers within both academia and the pharmaceutical 1995, 7, 1342-1352. industry have advanced several approaches by which NMDA [18] Cubelos, B.; Gimenez, C.; Zafra, F. 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M.; Levay, G. The glycine transporter-1 inhibitors NFPS and Org [82] Dachary, E.; Dargazanli, G.; Estenne-Bouhtou, G.; Veronique, C.: 24461: a pharmacological study. Pharmacol. Biochem. Behav. WO04013101 (2004). 2003, 74, 811-825. [83] Dargazanli, G.; Estenne-Bouhtou, G.; Veronique, C.: WO05037782 [55] Le Pen, G.; Kew, J.; Alberati, D.; Borroni, E.; Heitz, M. P.; (2005). Moreau, J. L. Prepulse inhibition deficits of the startle reflex in [84] Dargazanli, G.; Estenne-Bouhtou, G.; Magat, P.: WO05037792 neonatal ventral hippocampal-lesioned rats: reversal by glycine and (2005). a glycine transport inhibitor. Biol. Psychiatry 2003, 54, 1162-1170. [85] Dargazanli, G.; Estenne-Bouhtou, G.; Magat, P., Marabout, P.; [56] Lipska, B. K.; Weinerger, D. R. Delayed effects of neonatal Roger, P.: WO05037785 (2005). hippocampal damage on haloperidol-induced catalepsy and [86] Dargazanli, G.; Estenne-Bouhtou, G.; Medaisko, F.; Rakotoarisoa, apomorphine-induced stereotypic behaviors in the rat. Brain Res. N.: WO05037783 (2005). Dev. Brain Res. 1993, 75, 213-222. [87] Depoortere, R.; Daraganzanil, G.; Estenne-Bouhtou, G.; Coste, A.; [57] Lowe, J. A.; Drozda, S. E.; Fisher, K.; Strick, C.; Lebel, L.; Lanneau, C.; Desvignes, C.; Ponce;et, M.; Heaulme, M.; Santucci, Schmidt, C.; Hiller, D.; Zandi, K. S. [3H]-(R)-NPTS, a Radioligand V., Decobert, M.; Cudennec, A.; Voltz, C.; Boulay, D.; Terranova, 1896 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Kinney et al.
J.P.; Stemmelin, J.; Roger, P.; Marabout, B.; Sevrin, M.; Vige, X.; [89] Tsai, G.; Ralph-Williams, R. J.; Martina, M.; Bergeron, R.; Berger- Biton, B.; Steinberg, R.; Francon, D.; Alonso, R.; Avenet, P.; Oury- Sweeney, J.; Dunham, K. S.; Jiang, Z.; Caine, S. B.; Coyle, J. T. Donat, F.; Perrault, G.; Griebel, G.; George, P.; Soubrie, P.; Gene knockout of glycine transporter 1: characterization of the Scatton, B. Neurochemical, electrophysiological and pharma- behavioral phenotype. Proc. Natl. Acad. Sci. USA 2004, 101, 8485- cological profiles of the selective inhibitor of the glycine 8490. transporter-1 SSR504734, a potential new antipsychotic. [90] Yee, B. K.; Balic, E.; Singer, P.; Schwerdel C.; Grampp, T.; Neuropsychopharmacology 2005, 30, 1963-1985. Gabernet L.; Knuesel, I.; Benke, D.; Feldon, J.; Mohler, H.; [88] Gomeza, J.; Hulsmann, S.; Ohno, K.; Eulenburg, V.; Szoke, K.; Boison, D. Disruption of glycine transporter 1 restricted to Richter, D.; Betz, H. Inactivation of the glycine transporter 1 gene forebrain neurons is associated with a procognitive and discloses a vital role of glial glycine uptake in glycinergic antipsychotic phenotypic profile. J. Neurosci. 2006, 26, 3169-3181. inhibition. Neuron 2003, 40, 785-796. [91] Schadt, O.; Bottcher, H.; Leibrook, J.; Schiemann, K.; Heinrich, T.; Hozlemann, G.: WO03087086 (2003). Current Topics in Medicinal Chemistry, 2006, 6, 1897-1906 1897 Ligands Targeting the Excitatory Amino Acid Transporters (EAATs)
John Dunlop†,* and John A. Butera‡
Neuroscience Discovery Research† and Chemical and Screening Sciences‡; Wyeth Research, CN 8000, Princeton NJ 08543, USA
Abstract: This review provides an overview of ligands for the excitatory amino acid transporters (EAATs), a family of high-affinity glutamate transporters localized to the plasma membrane of neurons and astroglial cells. Ligand development from the perspective of identifying novel and more selective tools for elucidating transporter subtype function, and the potential of transporter ligands in a therapeutic setting are discussed. Acute pharmacological modulation of EAAT activity in the form of linear and conformationally restricted glutamate and aspartate analogs is presented, in addition to recent strategies aimed more toward modulating transporter expression levels, the latter of particular significance to the development of transporter based therapeutics. Keywords: Excitatory amino acid transporters, EAAT, glutamate.
INTRODUCTION glutamate uptake. The idea of blocking glutamate uptake via targeting a specific transporter subtype without concomitant Glutamate, the major excitatory transmitter in the mam- excitotoxicity, or the premise of positively upregulating malian central nervous system, signals via two major EAAT activity has resulted in efforts to develop subtype- receptor families, the ligand-gated ion channels, termed selective ligands for this important family of proteins. In ionotropic receptors, and the seven-transmembrane spanning addition, a number of studies have reported altered EAAT G-protein coupled receptors, termed metabotropic receptors. expression in various pathological conditions including The availability of glutamate to these receptors is regulated ischemia, traumatic brain injury, spinal cord injury, by a family of high-affinity glutamate transporters, the amyotrophic lateral sclerosis (ALS), Alzheimer’s disease excitatory amino acid transporters (EAATs), localized to the and schizophrenia. Such studies raise the possibility that plasma membrane of neurons and astroglial cells. In addition impaired or elevated glutamate transport activity might to providing a mechanism for clearing extracellular gluta- contribute to disease pathology and consequently agents mate, thereby regulating glutamate concentrations in the capable of stimulating or blocking glutamate transporter vicinity of glutamate receptors, the EAATs are also activity would be of interest to profile in animal models of recognized as critical to maintaining extracellular glutamate disease. For example, agents capable of stimulating concentrations below levels that would trigger glutamate- glutamate transport would be desirable in ischemia or ALS mediated cell death, termed excitotoxicity. These proteins where extracellular glutamate levels are raised or transporter catalyze the cellular uptake of glutamate, against a large expression levels diminished, respectively. Alternatively, concentration gradient, by a process driven largely by the agents capable of inhibiting glutamate transport activity in a transmembrane Na+-gradient. Translocation of glutamate + non-excitotoxic manner would be of interest to evaluate in into cells is stoichiometrically coupled to inward flux of Na the context of the glutamatergic deficit, or NMDA hypo- in an electrogenic process. In the early 1990s molecular function, observed in schizophrenia. cloning techniques revealed the existence of multiple EAAT subtypes, EAATs 1-5, and offered the possibility of develo- A very comprehensive overview of the glutamate trans- ping expression systems for each of the individual subtypes port system from pre- to post-cloning era has been published to further explore their function and pharmacology. [1], while several excellent reviews on the pharmacology [2- 4], function [5,6] or regulation of EAATs [7,8] and their While the ionotropic and metabotropic glutamate recep- potential role in pathological conditions [9,10] are available. tors have received considerable attention as targets for ligand This review provides an overview of the recent development and drug development, there has been much less attention of EAAT ligands for the pharmacological modulation of paid to the EAATs. This may in part be attributed to the EAAT subtypes both with respect to identifying tools to fundamental role the EAATs play in maintaining extra- further explore the function of individual EAAT subtypes cellular glutamate levels below a threshold that would trigger and in the pursuit of novel therapeutic agents. In addition, excitotoxicity, and consequently the concept of inhibiting efforts to modulate these proteins by means of manipulating this process, at first glance, would appear detrimental. transporter protein expression levels are also discussed. However, the cloning of multiple EAAT subtypes with distinct and overlapping distribution suggests a reappraisal LINEAR GLUTAMATE/ASPARTATE ANALOGS of this simplified concept of globally blocking cellular L-glutamic acid (1), the endogenous substrate with high affinity, defined for these targets in the 5-30 µM range, for *Address correspondence to this author at the Neuroscience Discovery the EAATs, is efficiently translocated by these proteins. It Research and Chemical and Screening Sciences; Wyeth Research, CN 8000, has been shown that the D-enantiomer of glutamate, while Princeton NJ 08543, USA; E-mail: [email protected]
1568-0266/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd. 1898 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Dunlop and Butera showing high affinity for other glutamate receptors, exhibits A novel chemoenzymatic strategy for the synthesis of L-2,4- poor affinity for the EAATs and is not efficiently transported syn-4-alkyl glutamate analogs has recently been developed [11]. Conversely, a second endogenous amino acid substrate which allows for expansion of the SAR within this class of for the EAATs, aspartic acid (2), is equally and efficiently ligands with respect to the methyl substituent. Starting from transported in both its D and L enantiomeric forms [11]. the corresponding substituted racemic ketoglutaric acids 5, a Consequently, this has afforded medicinal chemists two series of SYM2081 analogs 6 has been prepared where the related scaffolds, each amenable by different chemistries, methyl group was diversified to larger alkyl groups [16]. with which to initiate synthetic campaigns in search of Utilizing aspartate aminotransferase (AAT), the conversions selective ligands to use as pharmacological tools to dissect were achieved with high enantiomeric excess (>99% ee) to the function of the EAAT subtypes. These strategies have afford novel L-2,4-syn-4-alkyl glutamate analogs. The included (i) structural modifications of the distal carboxylic compounds were evaluated at human EAAT1-3 in HEK293 acid moiety, or (ii) appending lipophilic substituents along cell lines stably expressing the transporters as measured by a the glutamate / aspartate backbone either from the distal FLIPR membrane potential assay. All of the compounds position, directly on a chain carbon atom or via a heteroatom possessed inhibitory activity at all three EAAT subtypes with linker. These approaches have provided numerous potent Ki values in the mid to high micromolar range with no EAAT inhibitors possessing varying levels of selectivity appreciable selectivity for any of the three (with the across the 5 EAAT subtypes and other glutamate receptor exception of R = butyl, whose affinity for EAAT3 was ~10- targets, thus adding to our understanding of the function of fold less than EAAT1 and EAAT2). Of note was the these transporters. observed switch in the pharmacological profile of the ethyl Simply adding a methyl group a or b to the amino acid analog (R = Et) which was shown to be a non-substrate m m m chiral center of glutamate affords molecules with diverse inhibitor at EAATs 1-3 (Ki = 23 M, 14 M, and 38 M, 4 properties; threo-3-methylglutamic acid (T3MG, 3) and respectively); while the parent methyl analog ( ) is an EAAT1 substrate (K = 13 mM) and a non-substrate (2S,4R)-4-methylglutamic acid (SYM2081, 4), respectively. m inhibitor at EAAT2 (Ki = 13 mM) and EAAT3 (6.6 mM). Modeling data were presented suggesting two distinct binding conformations associated with these molecules, one O for substrates, and one for inhibitors. H NH2 H NH2 HO OH OH HO
O O O R O R NH2 1 2 AAT HO OH HO OH
O O O O NH2 CH3 NH2 5 6 HO OH HO OH Isosteric replacement of the distal carboxyl group with a O CH3 O O O sulphonic acid or hydroxamic acid affords L-serine-O- 3 4 sulphate (L-SOS, 7) and L-aspartate-b-hydroxamic acid (8), respectively. L-SOS has been shown to selectively inhibit EAAT1 (Ki = 107 mM) and EAAT3 (Ki = 150 mM) over T3MG has been shown to be a non-substrate inhibitor of EAAT2 (Ki = 1157 mM) and was determined to be a EAAT2 (Kb = 18 mM) without much effect on EAAT1 up to substrate at EAAT1 [2,17]. EC50 values for L-SOS transport concentrations of 300 mM [12]. More recently, T3MG was were found to be steeply voltage-dependent at EAAT2; studied in Xenopus oocytes expressing EAATs 1-4 and in whereas only modest voltage-dependence was observed for mammalian cells expressing EAATs 1-3 [13]. It was found transport by EAAT1 [18]. Hydroxamic acid 8 was relatively to inhibit EAAT2 (IC50 = 90 mM) selectively over EAAT1 unselective as a substrate inhibitor across EAAT1-3. and EAAT3. There was about a 50% inhibition at these sub- types at a concentration of 1 mM. T3MG produced substrate- like currents in EAAT4-injected oocytes and was found to NH2 O NH2 inhibit EAAT4-mediated currents with an IC50 value of 109 HO O OH HO OH mM. S N H O O O O The 4-methylated glutamate (4MG) analog SYM2081 7 8 was found to potently block EAAT2 (Kb = 3.4 mM) while acting as a substrate for EAAT1 (Km = 54 mM) [12]. Recently, [3H]4MG was shown to be a useful reagent for A prototype of an important set of potent ligands used binding studies in numerous preparations including astro- extensively as a scaffold to characterize functionality of the cytes [14]. These studies confirmed that SYM2081 is a EAATs has been L-threo-b-hydroxy-asparate (L-THA, 9). In substrate for EAAT1. SYM2081 was also found to potently its racemic form, 9 has been shown to be a substrate inhibitor and selectively bind to kainate receptors (inhibition of of EAAT1-3 with Km values in the low to mid-micromolar 3 [ H]kainic acid binding to rat forebrain IC50 = 32 nM) [15]. range [19] and a non-transportable blocker of EAAT5 [20]. Ligands Targeting the Excitatory Amino Acid Transporters (EAATs) Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1899
benzyloxyaspartate > L-erythro-benzyloxyaspartate > D-
O NH2 erythro-benzyloxyaspartate and the inhibitory IC50 values for 14 OH each isomer for inhibiting [ C]glutamate uptake in COS-1 HO cells expressing EAAT1-3 are shown in Table 1 [23]. OH O Table 1a 9
EAAT1 IC EAAT2 IC EAAT3 IC The hydroxyl functionality on C-3 of L-THA has 50 50 50 afforded a convenient handle to install a plethora of lipophilic substituents, which has allowed for the discovery L-TBOA 23 3.8 7.0 of a large number of potent and selective ligands that have D-TBOA 637 64 21 added substantially to the understanding of EAAT subtype function. DL-TAcOAsp (10) and DL-TpnOAsp (11) L-EBOA 817 164 134 represent two acetylated analogs which where determined to D-EBOA >1000 221 161 be competitive substrates at EAAT1 (bovine EAAT1 a Data in µM from reference 23 expressed in Xenopus oocytes) with Km values of 40 and 64 mM, respectively [21]. Bulkier aroyl groups such as benzoyl (TbzOAsp, 12) or naphthoyl (T1NpOAsp, 13 or T2NpOAsp, DL-TBOA is devoid of effects on either ionotropic or 14) gave compounds which were shown to be blocker type metabotropic glutamate receptors, as well as on the GABA, inhibitors (12: Ki = 17.2 mM; 13: Ki = 52.1 mM). histamine, and serotonin receptor families. Replacing the benzyl group of DL-TBOA with a naphthyl moiety has Due to the compromised aqueous stability associated provided two additional aspartate analogs, TNOA1 (16) and with the ester linkage in this class of molecules, the TNOA2 (17) with similar potencies and selectivity profiles. corresponding ethers where prepared to further expand the utility of these tools. DL-threo-b-benzyloxyaspartate (DL- Recently, it has been reported that excision of the ether TBOA, 15) was developed as a key pharmacological tool oxygen atom in L-TBOA to give L-b-threo-benzyl-aspartate which was shown to be a non-transportable inhibitor of (18), affords a molecule which exhibits EAAT3-prefering EAAT1-3 in COS-1 cells expressing these proteins. The inhibitory activity. Compound 18 was found to be a compound was found to be much more potent at inhibiting competitive inhibitor of EAAT1-3 showing about a 10-fold human EAAT2 (Ki = 5.7 mM) than human EAAT1 (Ki = 42 preference for the EAAT3 subtype. Ki values for blocking 3 mM) [22]. In Xenopus laevis oocytes expressing human uptake of [ H]D-aspartate in C17.2 cells expressing EAAT1- EAAT2, DL-TBOA potently inhibits glutamate-induced 3 were reported to be 8.7 mM, 10 mM, and 0.8 mM, currents with a Kb value of 0.116 mM. DL-TBOA has also respectively [24]. The corresponding L-threo-phenethyl- been found to act as a non-substrate inhibitor of EAAT4 and aspartate analog (19) has also been synthesized and has been EAAT5 (oocytes expressing human EAAT4 or EAAT5) shown to have a preference for blocking EAAT3 (IC50 = 15 with Ki values of 4.4 mM and 3.2 mM, respectively [20]. The mM) over EAAT1 (IC50 = 35 mM) [25]. four possible stereoisomers of DL-TBOA have been The [7-(carboxymethoxy)coumarin-4-yl]methyl ester synthesized and tested across EAAT1-3. The rank order of derivative of L-TBOA (a -CMCM-TBOA, 20) has recently potencies are L-threo-benzyloxyaspartate > D-threo- been synthesized [26] and has provided a useful caged aspar-
O NH2 O NH2 O NH2 OH OH OH HO HO HO O O O O O O
O O 10 O 11 12
O NH2 OH O NH2 HO OH HO O O O O O O
13 14 1900 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Dunlop and Butera
O NH2 O NH2 O NH2 OH HO OH OH O NH2 HO HO O O O NH2 OH O O O O OH HO HO O O
15 16 17 18 19 tate analog containing a photo-labile coumarin chromophore which, upon UV-radiation, unleashes L-TBOA. The comp- O NH2 ound exhibits low intrinsic inhibitory activity (IC50 = 124 OH mM), yet activity is restored in a time-dependent manner HO upon photo-activation. The coumarin group has also been O O installed onto the amine moiety of D-aspartic acid to give N- [(6-nitrocoumarin-7-yl)methyl]-D-aspartic acid (Ncm-D- aspartate, 21) [27]. Upon photolysis, the compound elicits a O transient inward current in astrocytes which was inhibited by N TBOA. Activation of NMDA receptors was also observed H upon release of D-aspartate. These caged analogs are useful tools to study the interaction between transporters and 22 CF3 NMDA receptors in the CNS.
glutamate in COS-1 cells transiently expressing EAAT1-3 with IC values of 22 nM, 17 nM, and 300 nM, respectively, O NH 50 2 making 22 one of the most potent EAAT inhibitors reported O to date. In MDCK cells stably expressing EAAT2 or HO 14 EAAT3, 22 inhibited the uptake of [ C]glutamate with IC50 O O values of 1.9 nM and 28 nM, respectively.
OH An alternative strategy of appending a lipophilic moiety O O O on the distal end of aspartic acid has lead to several novel O series of potent EAAT ligands with unique selectivity profiles. The diaminopropionic acid (DAPA) headpiece has 20 been attached to a urea scaffold, which has allowed for a special mapping of the lipophilic site putatively occupied by O the benzyl group of L-TBOA. The most potent compound in a series of DAPA-ureas was compound 23, which was O reported to inhibit glutamate uptake thru EAAT3 with an IC50 = 4 mM [29].
O2N Exchange of the DAPA-urea scaffold for aspartic acid gave compound 24 with improved potency as an EAAT3 O HN inhibitor (IC50 = 1.2 mM). With incorporation of a fluorene ring system, a further improvement in EAAT3 inhibitory OH HO potency was seen with (S)-4-(9H-fluoren-2-ylamino)-2- O amino-4-oxobutanoic acid (NBI-59159, 25). The compound 21 was shown to be EAAT3 preferring in two systems: (i) HEK cells expressing EAAT1-3 (IC50 = 1.03 mM, 1.4 mM, and 0.09 mM, respectively) and (ii) as a blocker of glutamate- In an attempt to further enhance the potency and modu- induced current in Xenopus oocytes expressing EAAT1-3 late the selectivity profile in the TBOA series, a number of (IC50 = 0.212 mM, 0.055 mM, and 0.023 mM, respectively). new TBOA analogs possessing substituents on the benzy- The compound did not elicit a current in oocytes, thus loxy group have recently been characterized [28]. A remark- suggesting it is a non-transportable inhibitor. NBI-59159 was able enhancement in potency was realized with (2S,3S)-3-{3- found to be devoid of activity at AMPA, NMDA, or [4-(trifluoromethyl)benzoylamino]benzyloxy}aspartate (TFB metabotropic glutamate receptors up to concentrations of 14 -TBOA, 22). The compound inhibited the uptake of [ C] 100 mM. Ligands Targeting the Excitatory Amino Acid Transporters (EAATs) Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1901
O NH2 H H N N OH O NH2 OH N O O H O O 23 24
O NH2 OH N H O 25
The SAR in this novel series of DAPA and aspartic acid analogs has recently been expanded to include a variety of CH3 CH3 lipophilic cores including substituted biphenyls, substituted OH OH fluorenes, and substituted biaryl ethers [30-33]. The H3C architecture of the lipophilic load was found to significantly O O affect the potency (from low micromolar to nanomolar) and OH OH N N selectivity profile (from non-subtype selective to very H H selective for EAAT2) of the compounds. Of note, was the O O discovery and characterization of (S)-2-amino-4-(4-(2-bromo- 27 28 4,5-difluorophenoxy)phenylamino)-4-oxobutanoic acid (WAY-213613, 26), a highly potent and selective EAAT2 inhibitor (inhibitory IC50 values in HEK cells transfected compounds exhibit selectivity for the EAAT2 subtype by with EAAT1-3 were reported to be 5 mM, 0.08 mM, and 3.8 measuring the inhibition of labeled glutamate uptake into mM, respectively). Compound 26 was also found to exhibit COS cells transiently transfected with each of the transporter high inhibitory activity in rat cortical synaptosomes (0.037 subtypes (Table 2), although the potency was rather low in mM) and in EAAT2 expressing oocytes (0.13 mM). The the 20-60 µM range [19]. In addition, the failure of 27 and compound was found to be a non-substrate inhibitor of 28 to elicit glutamate-induced currents in oocytes expressing EAAT2 and lacked activity at metabotropic and ionotropic EAAT2 provided evidence consistent with a non-substrate glutamate receptors. The molecule represents one of the most mode of action. potent and selective EAAT2 inhibitors identified to date. Table 2b
Br EAAT1 Ki EAAT2 Ki EAAT3 Ki O O NH2 Dihydrokainate > 3000 23 > 3000 OH F N H Kainate > 3000 59 > 3000 F O b Data in µM from reference 19 26 Despite their modest potency, these compounds have been frequently used as tools to study EAATs, with an CONFORMATIONALLY CONSTRAINED GLUTA- important caveat that while they exhibit selectivity within the MATE/ASPARTATE ANALOGS EAAT family they have overlapping activity on glutamate The approach of constraining the linear glutamate/ receptors, in particular kainate, the prototype ligand for the aspartate backbone in a cyclic template has been a common ionotropic kainate receptor. Consequently, the use of these strategy in the development of novel transporter ligands. agents is somewhat limited especially in native preparations This is perhaps best exemplified by kainate (27) and where receptor effects might be expected to influence dihydrokainate (28), two of the earliest compounds to be transporter-mediated effects. identified as exhibiting pharmacological blockade of the A significant number of other conformationally constrai- glutamate transport system in brain derived preparations [34- ned analogs with varying ring sizes have now been identified 36]. Subsequently, the cloning of the individual transporter including the carboxycyclopropylglycines, aminocyclobu- subtypes has allowed for the determination that these tane dicarboxylates, oxazole carboxylates, pyrrolidine dicar- boxylates, heptane dicarboxylates and octane dicarboxylates. 1902 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Dunlop and Butera
A family of 2-(carboxycyclopropyl)glycine analogs synthe- Two additional compounds exemplified by 3-hydroxy- sized and evaluated for both glutamate receptor and trans- 4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxy- porter activity resulted in the discovery of 2S,3S,4R-2- lic acid (HIP-A, 36) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH- (carboxycyclopropyl)glycine (L-CCG-III, 29) as an inhibitor pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B, 37) have of the glutamate transport system in cortical synaptosomal been disclosed. Compounds 36 and 37 inhibited the preparations, hippocampal astrocytes and glial plasmale- glutamate transport system in rat cortical synaptosomes with mmal vesicles (IC50 = 2-5 µM) [37]. Subsequent studies IC50 values of 18 and 17 µM, respectively and were reported using either cloned human EAAT2 expressed in mammalian to be non-substrate inhibitors [42]. However, the selectivity cells [38] or rat EAAT3 expressed in Xenopus oocytes [39] of these agents with respect to the cloned transporter demonstrated 29 to be non-selective across the EAAT subtypes or glutamate receptors has not been documented. subtypes. Other analogs in the series were found to have less activity on the glutamate transport system (L-CCG-IV, 30) O or significant glutamate receptor activity (L-CCG-I, 31 and OH LCCG-II, 32). L-CCG-III has consequently been used most frequently as a transporter tool, however its utility is some- O N what limited by the non-selective activity across the cloned O EAAT1-3 subtypes. OH
NH2 CO H HO2C CO H 2 H 2 HO2C 35 NH HO NH2 2 O H H H OH H HO H H H N NH 29 30 N NH O O H OH H CO2H H CO2H H H O
NH NH2 36 37 HO C 2 H 2 HO2C H H One very fruitful area in the discovery of novel 31 32 transporter inhibitors has been the pyrrolidine dicarboxylates such as L-trans-2,4-pyrrolidine dicarboxylate (L-trans-2.4- 38 In the case of the four member cyclic ring aminocyclobu- PDC, ) and L-anti-endo-3,4-methanopyrrolidine dicar- 39 38 tane dicarboxylates two compounds have been reported; the boxylate (L-anti-endo-3,4-MPDC, ). Compound was cis and trans analogs of 1-aminocyclobutane-1,3-dicar- synthesized as part of a series of conformationally restricted boxylate (ACBD) [40]. While the cis-ACBD analog (33) carboxyproline derivatives aimed to mimic distinct confor- 39 was found to inhibit the glutamate transport system in mations of glutamate and was subsequently described as a next generation molecule from that effort. Both compounds synaptosomes (IC50 ~ 10 µM), trans-ACBD (34) was found to have activity on glutamate receptors, but not the transport inhibit the glutamate transport system in synaptosomal 38 system. These compounds have not seen widespread utility. preparations; exhibited a Ki of 4.6 µM for the inhibition of L-[ 3H]-glutamate uptake into rat brain synaptosomes [43] and 39 had a Ki of 5 µM for the inhibition of D-[3H]- aspartate uptake into rat forebrain synaptosomes [44]. The compounds were also found not to have significant cross- HO C NH 2 2 H NH2 reactivity with glutamate receptors. However, as with many H CO2H CO H of these cyclic analogs 38 and 39 are non-selective EAAT HO C 2 2 inhibitors having approximately equivalent inhibitory 33 34 activity on each of the EAAT family members. For example, although 38 was shown to have some modest selectivity for EAAT2 (Ki = 8 µM) compared with EAAT1 (Ki = 79 µM) The oxazole carboxylates have been described more and EAAT3 (Ki = 61 µM), based on the inhibition of recently with 2-(2-amino-2-carboxy-ethyl)-4,5-dihydro-oxa- glutamate uptake in transiently transfected COS cells, the zole-4-carboxylic acid (35) being one of the first compounds compound was found to be a substrate for each of the to be reported [41]. Compound 35 is a weakly potent transporter subtypes with somewhat similar affinities (Km EAAT3 inhibitor with an affinity for the transporter itself as values of 28, 7 and 27 µM for EAAT1-3, respectively), as a substrate of 31 µM, but further characterization of its measured by the induction of transporter currents in oocytes pharmacology at other EAAT subtypes is lacking. [19]. The related PDC analog, L-trans-2,3-PDC (40) Interestingly, 35 exhibited negligible affinity for NMDA, inhibited the glutamate transport system in synaptosomal AMPA or kainate binding sites in rat cortical homogenates. Ligands Targeting the Excitatory Amino Acid Transporters (EAATs) Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1903
O O OH O
HO OH H H OH OH N OH H N O N H H O O 38 39 40 preparations (Ki = 33 mM), while in oocytes it was found to selectively inhibit EAAT2 (Ki = 12 mM) in a non-substrate O O HO manner while showing no appreciable inhibitory activity at HO EAAT1 or EAAT3 [45]. OH OH O Expansion of the ring size in the form of 3-amino- O NH 2.6 NH tricyclo[2.2.1.0 ]heptane-1,3-dicarboxylate (WAY-855, 41) H3C has resulted in some progress in this area [46]. Compound 41 42 43 exhibits preferential inhibition of the cloned EAAT2 subtype expressed in MDCK cells with IC50 values of 2.2, 24.5 and O O O HO HO OH >100 µM determined for the EAAT2, EAAT3 and EAAT1 H subtypes, respectively. Similarly, in oocytes expressing OH individual transporter subtypes 41 inhibited glutamate- O HN induced transporter currents with rank order of potency NH EAAT2 (IC50 = 1.3 µM) > EAAT3 (52.5 µM) > EAAT1 H (126 µM). The absence of transporter induced currents in EAAT2 expressing oocytes or heteroexchange of accumu- 44 45 lated D-[3H]-aspartate in cortical synaptosomes in the presence of 41 indicate a non-substrate mechanism. Finally, 41 was found to be selective over the metabotropic gluta- inhibitory activity on the cloned EAAT1-3 subtypes (Ki mate receptors and the NMDA and AMPA subtypes of values of 127, 52 and 46 µM, respectively for the inhibition ionotropic receptors. To date this is likely the most selective of EAAT 1-3) as determined using HEK cell lines express- agent of the cyclic analogs reported. ing each transporter and a membrane potential dye assay to measure the electrogenic uptake of glutamate. However, 45 exhibited more potent activity on the NMDA subtype of O OH ionotropic glutamate receptors with a Ki for the displace- ment of [3H]CGP39653 binding to NMDA receptor sites in H H rat brain membrane preparations of 2.9 µM. This latter O observation is likely to limit the utility of this first generation analog as a transporter tool. Clearly this question of non- HO selectivity within the EAAT family is a common theme H2N H among many of the conformationally constrained analogs, kainate (27) and dihydrokainate (28), and to some extent 41 WAY-855 (41), being the exceptions. There is much room for improvement in these molecules both with respect to A recently reported stereocontrolled synthesis of a selectivity and potency. Unlike the case for the linear substituted azetidine ring has allowed for characterization of analogs where a number of nanomolar potent compounds substituted analogs of trans-2-carboxyazetidine-3-acetic acid have been identified, none of the cyclic analogs have yet (t-CAA, 42) [47]. While the parent compound 42 exhibited improved beyond the low micromolar range, very modest no selectivity across EAAT1-3 (Ki = 5 mM), introduction of improvements on the prototypes kainate and dihydrokainate. an alkyl group with the S-configuration resulted in REGULATING EAAT EXPRESSION compounds with EAAT2 selectivity as seen with methyl analog 43 (IC50 values = >1000 mM, 32 uM, and 126 mM, Besides developing molecules aimed at the acute phar- respectively at EAAT1-3) and n-propyl analog 44 (IC50 macological modulation of EAATs, efforts have also been values = >1000 mM, 44 uM, and 375 mM, respectively at focused on the discovery of compounds capable of altering EAAT1-3). transporter expression, in particular within the context of therapeutics. Such a strategy appears to have been successful Lastly, the octane dicarboxylate (1R,4S,5R,6S)-3-azabi- in the screening of approved FDA drugs for their ability to cyclo[3.3.0]octane-4,6-dicarboxylic acid (45) has recently increase the expression of the rodent homolog of EAAT2, been reported [48]. Compound 45 has non-selective GLT-1, in organotypic spinal cord slice cultures [49-51]. 1904 Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 Dunlop and Butera
NH2 H H H H H H N N S S O N O N HO O OH O OH
46 O 47 O
CH3 O N H H H N S S CH3 N O N S N O NH H2N N O OH O 48 O
Using this approach several b-lactam antibiotics including specificity of the EAAT subtypes. Pharmacol. Therapeut. 2005, penicillin (46), amoxicillin (47) and ceftriaxone (48) were 107(3), 271-85. [4] Shigeri, Y.; Seal, R. P.; Shimamoto, K. Molecular pharmacology of found to increase GLT-1 protein expression levels. The glutamate transporters, EAATs and VGLUTs. Brain Res. Brain potential for this effect to translate into some therapeutic Res. Rev. 2004, 45(3), 250-65. benefit was supported by the demonstration that ceftriaxone [5] Bergles, D. E.; Diamond, J. S.; Jahr, C. E. Clearance of glutamate exhibited neuroprotective effects in an in vitro ischemia inside the synapse and beyond. Curr. Opin. Neurobiol. 1999, 9(3), 293-8. model and in the G39A SOD mouse model of amyotrophic [6] Fairman, W. A.; Amara, S. G. Functional diversity of excitatory lateral sclerosis. In a control study, CNS-penetrating amino acid transporters: ion channel and transport modes. Am. J. antibiotics shown not to affect GLT-1 protein expression Physiol. 1999, 277(4 Pt 2), F481-6. levels were not neuroprotective in the G39A SOD mouse [7] OShea, R. D. Roles and regulation of glutamate transporters in the model of amyotrophic lateral sclerosis. Clearly it will be central nervous system. Clin. Exper. Pharmacol. Physiol. 2002, 29(11), 1018-1023. very interesting to see if these preclinical observations can be [8] Gonzalez, M. I.; Robinson, M. B. Neurotransmitter transporters: translated into clinical benefit in neurodegenerative disease. why dance with so many partners? Curr. Opin. Pharmacol. 2004, 4(1), 30-5. CONCLUSION [9] Dunlop, J.; Zaleska, M. M.; Eliasof, S.; Moyer, J. A. Excitatory amino acid transporters as emerging targets for central nervous We have reviewed the evolution of ligands for the acute system therapeutics. Emer. Therapeut. Targets 1999, 3(4), 543- modulation of EAAT function, in addition to strategies 570. aimed at altering EAAT expression levels. Clearly, there [10] Maragakis, N. J.; Rothstein, J. D. Glutamate transporters: animal have been incremental improvements in the available ligands models to neurologic disease. Neurobiol. Dis. 2004, 15(3), 461-73. [11] Balcar, V. J.; Johnston, G. A. The structural specificity of the high for the family of EAATs and in particular for the linear affinity uptake of L-glutamate and L-aspartate by rat brain slices. J. analogs we now have compounds with nanomolar affinity, a Neurochem. 1972, 19(11), 2657-66. hurdle yet to be overcome with the conformationally cons- [12] Vandenberg, R. J.; Mitrovic, A. D.; Chebib, M.; Balcar, V. J.; trained glutamate/aspartate analogs. There is still significant Johnston, G. A. R. Contrasting modes of action of methylglutamate room for improvement in particular toward the discovery of derivatives on the excitatory amino acid transporters, EAAT1 and EAAT2. Mol. Pharmacol. 1997, 51(5), 809-815. compounds with greater selectivity for each of the individual [13] Eliasof, S.; McIlvain, H. B.; Petroski, R. E.; Foster, A. C.; Dunlop, transporter subtypes. The pursuit of such molecules is an J. Pharmacological characterization of threo-3-methylglutamic acid important area for future development with the ultimate goal with excitatory amino acid transporters in native and recombinant of using these agents to more clearly define the role of each systems. J. Neurochem. 2001, 77(2), 550-557. [14] Aprico, K.; Beart, P. M.; Crawford, D.; O'Shea, R. D. Binding and EAAT subtype and to assess their potential involvement in transport of [3H](2S,4R)- 4-methylglutamate, a new ligand for pathological conditions. glutamate transporters, demonstrate labeling of EAAT1 in cultured murine astrocytes. J. Neurosci. Res. 2004, 75(6), 751-9. REFERENCES [15] Zhou, L. M.; Gu, Z. Q.; Costa, A. M.; Yamada, K. A.; Mansson, P. [1] Danbolt, N. C. Glutamate uptake. Prog. Neurobiol. 2001, 65(1), 1- E.; Giordano, T.; Skolnick, P.; Jones, K. A. (2S,4R)-4- 105. Methylglutamic acid (SYM 2081): a selective, high-affinity ligand [2] Bridges, R. J.; Kavanaugh, M. P.; Chamberlin, A. R. A for kainate receptors. J. Pharmacol. Exp. Ther. 1997, 280(1), 422- pharmacological review of competitive inhibitors and substrates of 427. high-affinity, sodium-dependent glutamate transport in the central [16] Alaux, S.; Kusk, M.; Sagot, E.; Bolte, J.; Jensen, A. A.; Brauner- nervous system. Curr. Pharmaceut. Des. 1999, 5(5), 363-79. Osborne, H.; Gefflaut, T.; Bunch, L. Chemoenzymatic synthesis of [3] Bridges, R. J.; Esslinger, C. S. The excitatory amino acid a series of 4-substituted glutamate analogues and pharmacological transporters: pharmacological insights on substrate and inhibitor Ligands Targeting the Excitatory Amino Acid Transporters (EAATs) Current Topics in Medicinal Chemistry, 2006, Vol. 6, No. 17 1905
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