Benny Bang Andersen and Klaus P B0ges0

CONTENTS

18.1 Introduction 299

18.2 Receptor Ligands 300

18.2.1 Antipsychotic Drugs 300

18.2.1.1 Classical Antipsychotic Drugs 300

18.2.1.2 Atypical Antipsychotic Drugs 302

18.3 Transporter Ligands 304

18.3.1 Antidepressant Drugs 304

18.3.1.1 First Generation Drugs 305

18.3.1.2 The Selective Serotonin Reuptake Inhibitors 306

18.3.1.3 Discovery of Escitalopram—An Allosteric Serotonin

Reuptake Inhibitor 309

18.3.1.4 The SSRI Pharmacophore and SERT Homology Model 310

18.4 Concluding Remarks 312

Further Readings 312

18.1 INTRODUCTION

Dopamine (DA), serotonin (5-hydroxytryptamine, 5-HT), and norepinephrine (NE) are important neurotransmitters in the human brain. These neurotransmitters activate postsynaptic and presynap-tic receptors, and their concentration is regulated by active reuptake into presynaptic terminals by transporters.

DA and 5-HT receptors are found in multiple subtypes that are divided into subclasses based on structural and pharmacological similarities. The DA and 5-HT receptors are all putative seven transmembrane (TM) G protein-coupled receptors (GPCRs) except for the 5-HT3 receptor, which is a ligand-gated ion channel regulating the permeability of sodium and potassium ions. Five subtypes of DA receptors are known and grouped into the Dj-like receptors (Dj and D5) and the D2-like receptors (D2, D3, and D4), whereas 14 subtypes of 5-HT receptors are known and grouped into seven subclasses, namely 5-HT1 (5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F), 5-HT2 (5-HT2A, 5-HT2B, and 5-HT2C), 5-HT3, 5-HT4, 5-HT5 (5-HT5a and 5-HT5B), 5-HT6, and 5-HT7. In addition, a variety of polymorphic and splice variants (functional and nonfunctional) have been described for subtypes of both DA and 5-HT receptors.

Transporters for DA (DAT), 5-HT (SERT), and NE (NET) belong to the same family, the so-called solute carrier 6 (SLC6) gene family of ion-coupled plasma membrane cotransporters. These transporters are able to transport DA, 5-HT, and/or NE from the synapse and into the cell using the sodium gradient. They are not specific for their substrates, and NET is, for example, important for the transport/clearance of DA in the cortex. This also fits with the fact that the highest homology among the cloned human transporters is found between DAT and NET. Recently, a high-resolution crystal structure of a bacterial homolog (LeuTAa) of these transporters was published revealing a dimeric protein with each of the protomers being a 12 TM spanning protein in a unique fold. Thus, this x-ray structure has given a new insight into the structure of mammalian transporters, and a SERT homology model will be discussed as an example of the application of computational methods in drug design (Section 18.3.1.4). The structure and function of transporters are discussed in more detail in Chapter 14.

Selective ligands have been described for many of these receptor subtypes and transporters and in the following text, we have chosen to focus on ligands that have shown potential as antip-sychotic or antidepressant drugs or which have been important in the discovery of these ligands. Antipsychotic drugs that are used in the treatment of schizophrenia will be discussed as an example of ligands for DA and 5-HT receptors (Section 18.2), whereas antidepressant drugs that are used for the treatment of depression and anxiety will be discussed as examples of ligands for transporters (Section 18.3).

18.2 RECEPTOR LIGANDS 18.2.1 Antipsychotic Drugs

Antipsychotic drugs are primarily used to treat schizophrenia. Schizophrenia is distinguished from other psychotic disorders based on a characteristic cluster of symptoms, where the positive symptoms appear to reflect an excess or distortion of normal function (i.e., delusion, hallucinations, disorganized thinking, disorganized behavior, and catatonia), whereas the negative symptoms appear to reflect a diminution or loss of normal functions (i.e., affective flattening, poverty of speech, and an inability to initiate and persist in goal-directed activities). The cognitive symptoms (i.e., impairment of memory, executive function, and attention) have in recent years attracted more and more attention, and recently much research is directed toward understanding the role of these symptoms.

The antipsychotic drugs are divided into the classical and the atypical antipsychotic drugs. The classical antipsychotic drugs were discovered in the 1950s with chlorpromazine (18.1, Figure 18.1) as the first prominent example, whereas the atypical antipsychotic drugs were introduced into the treatment of schizophrenia during the 1990s. It is believed that the antipsychotic drugs exert their effect on positive symptoms by reducing DA hyperactivity in limbic areas of the brain.

The term classical antipsychotic drug is linked to compounds that show effect in the treatment of positive symptoms at similar doses that induce extrapyramidal symptoms (EPS, i.e., Parkinsonian symptoms, dystonia, akathisia, and tardive dyskinesia). It is believed that EPS is caused by the blockade of DA activity in striatal areas of the brain. The classical antipsychotic drugs are without effect on negative and cognitive symptoms, and these drugs may even worsen these symptoms. It has been argued that the deterioration of negative and cognitive symptoms by classical antipsychotic drugs may be a consequence of their EPS, and the separation of the antipsychotic effect and EPS is the foremost important property of the atypical antipsychotic drugs.

Thus, the term atypical antipsychotic drug is linked to a diverse group of drugs having antipsychotic effect at doses not giving EPS. However, all drugs from this group have their own compound specific limitations, such as a strong tendency to increase weight for some of the compounds, whereas others have a tendency to prolong the QT interval (total duration of cardiac ventricular electrical activity) in the surface electrocardiogram. In the following text, the classical as well as atypical antipsychotic drugs will be discussed with focus on their discovery, including structural considerations and pharmacological profile of key compounds.

18.2.1.1 Classical Antipsychotic Drugs

Chlorpromazine was discovered in the beginning of the 1950s, and the structure of chlorpromazine with its phenothiazine backbone was an excellent lead for medicinal chemists. Thus, the modification of chlorpromazine without changing the phenothiazine backbone led to a number of drugs such as perphenazine (18.2) and fluphenazine (18.3) (Figure 18.1). Medicinal chemists also replaced the phenothiazine backbone with other tricyclic structures, and these modifications led to other classes of classical antipsychotic drugs such as the thioxanthenes and the 6-7-6 tricyclics. Lundbeck in

Phenothiazines

Perphenazine (R = Cl) (18.2) Fluphenazine (R = CF3) (18.3)

6-7-6 tricyclics rA^B^

Loxapine (R = Cl, X = O, Y=N, unsaturated bond) (18.6) Octoclothepin (R = Cl, X = S, Y = CH2, saturated bond) (18.7) Isoclozapine (R = Cl, X = NH, Y= N, unsaturated bond) (18.8)

Perphenazine (R = Cl) (18.2) Fluphenazine (R = CF3) (18.3)

Zuclopenthixol (R = Cl) (18.4) (Z)-Flupentixol (R = CF3) (18.5)

Zuclopenthixol (R = Cl) (18.4) (Z)-Flupentixol (R = CF3) (18.5)

Butyrophenones

U Haloperidol (18.9)

U Haloperidol (18.9)

FIGURE 18.1 Classical antipsychotic drugs.

Denmark investigated in particular the thioxanthene backbone, and this work resulted in drugs such as zuclopenthixol (18.4) and (Z)-flupentixol (18.5) (Figure 18.1). The 6-7-6 tricyclic backbone has also led to a number of classical antipsychotic drugs such as loxapine (18.6), octoclothepin (18.7), and isoclozapine (18.8) (Figure 18.1). The R group found in all of these compounds is called the "neuroleptic substituent," and this substituent increases the D2 affinity/antagonism relative to unsub-stituted molecules and is essential for potent neuroleptic effect.

In the late 1950s, researchers at Janssen discovered an entirely new class of classical antipsychotic drugs without a tricyclic structure, namely the butyrophenones. Haloperidol (18.9, Figure 18.1) is the most prominent representative of this class of compounds, and today haloperidol is considered the archetypical classical antipsychotic drug for both preclinical experiments and clinical trials.

The classical antipsychotic drugs were all discovered using in vivo animal models, as the current knowledge about receptor multiplicity and in vitro receptor-binding techniques were unknown at that time. However, many of the in vivo models, which were used at that time as predictive for antipsychotic effect, is today considered more predictive of various side effects, e.g., EPS, and in hindsight it was difficult to find new antipsychotic drugs without the potential to induce EPS with the models available at that time. Thus, the development of new animal models modeling key properties of putative new antipsychotics is essential to the progress toward novel pharmacotherapies of schizophrenia. The ventral tegmental area (VTA) and the substantia nigra pars compacta (SNC) model is a good example of such a "model breakthrough" (see the following text).

An examination of the classical antipsychotic drugs by today's range of receptor-binding techniques and other more advanced biochemical methods has revealed that these drugs are postsynaptic D2 receptor antagonists, and it is believed that this accounts for both their antipsychotic effect as well as their potential to induce EPS. However, these drugs also target several other receptors, which may contribute to both their antipsychotic effect, and to their side effect profile.

18.2.1.2 Atypical Antipsychotic Drugs

Isoclozapine (18.8, Figure 18.1), which has the "neuroleptic chloro substituent" in benzene ring A, is a classical antipsychotic drug. On the contrary, clozapine (18.10, Figure 18.2), which has the chloro substituent in benzene ring C (Figure 18.1), has revolutionized the pharmacotherapy of schizophrenia. Thus, clozapine was the first antipsychotic drug that was effective in the treatment of positive symptoms of schizophrenia and free of EPS, but unfortunately clozapine can cause fatal agranulocytosis in a small percentage (1%-2%) of individuals, and much effort has been directed toward the identification of new antipsychotics with a clozapine-like clinical profile but without the risk of causing agranulocytosis.

This search has resulted in a number of atypical antipsychotics such as olanzapine (18.11), que-tiapine (18.12), risperidone (18.13), ziprasidone (18.14), aripiprazole (18.15), and sertindole (18.16) (Figure 18.2). The structure of these compounds reveals that olanzapine and quetiapine were obtained by structural modification of clozapine, whereas risperidone and ziprasidone were obtained from the butyrophenones. Aripiprazole and sertindole are quite different in chemical structure, and the discovery of sertindole will be discussed in more detail in the following text.

In vitro binding data for selected DA, 5-HT, and NE receptors as well as data from the catalepsy model (in vivo rat model predictive of EPS in humans) are shown for haloperidol and key atypical antipsychotics (Table 18.1). All the compounds display mixed receptor profiles with affinity for even more receptors and sites than included in the table (data not shown). The tendency is that classical antipsychotics display high affinity for D2 receptors relative to 5-HT2A receptors, whereas the atypical antipsychotics display an increased affinity for 5-HT2A receptors relative to D2. The relative ratio of D2 versus 5-HT2A receptor affinity has been suggested as a reason for atypicals not giving rise to EPS at therapeutic doses. A number of other factors may influence both the antipsychotic potential and the propensity to induce EPS, such as the affinity and efficacy at some of the other receptors, but also in vivo preference for limbic versus striatal regions of the brain might explain these differences. Interestingly, aripiprazole is a partial D2 receptor agonist, whereas the other antipsychotics are D2 receptor antagonist. Partial D2 receptor agonists are envisaged to stabilize a dysfunctioning

h3c o

Clozapine (18.10)

h3c"

Olanzapine (18.11)

"v

Quetiapine (18.12)

Risperidone (18.13) Ziprasidone (18.14) Aripiprazole (18.15)

FIGURE 18.2 Atypical antipsychotic drugs.

Sertindole (18.16)

TABLE 18.1

Receptor Profile and EPS Potential of Antipsychotic Drugs

Receptor Binding Ki (nM)

Compounds

Classical antipsychotic drug Haloperidol (18.9) 15

Atypical antipsychotic drugs

0.82

1500

In Vivo ED50 (mmol/kg)

Catalepsy Max., sca

0.34

Classical antipsychotic drug Haloperidol (18.9) 15

Atypical antipsychotic drugs

0.82

1500

Risperidone (18.13)

21

0.44

14

7.1

0.39

6.4

0.69

17

Olanzapine (18.11)

10

2.1

71

32

1.9

2.8

7.3

37

Quetiapine (18.12)

390

69

1100

2400

82

1500

4.5

>80

Ziprasidone (18.14)

9.5

2.8

n.t.

73

0.25

0.55

1.9

>48

Sertindole (18.16)

12

0.45

2.0

17

0.20

0.51

1.4

>91

Clozapine (18.10)

53

36

310

30

4.0

5.0

3.7

120

Sources: Data froma Arnt, J. and Skarsfeldt, T. Neuropsychopharmacology, 18, 63, 1998; Lundbeck Screening

Database, H. Lundbeck A/S, Valby, Denmark. Note: n.t; not tested.

Sources: Data froma Arnt, J. and Skarsfeldt, T. Neuropsychopharmacology, 18, 63, 1998; Lundbeck Screening

Database, H. Lundbeck A/S, Valby, Denmark. Note: n.t; not tested.

5-HT2Aa

DA system, inhibiting transmission in synapses with high tonus, and increasing function in those with low activity. This profile might explain why aripiprazole does not induce EPS.

18.2.1.2.1 Discovery of Sertindole

In the 1970s, Lundbeck had successfully marketed a number of classical antipsychotic drugs, and the medicinal chemistry program at Lundbeck was still aimed at finding new antipsychotic drugs based on the phenothiazine or thioxanthene template. However, in 1975, the first compounds were synthesized in a project directed toward the identification of nonsteroidal anti-inflammatory drugs (NSAIDs), and fortunately the compounds were also examined in in vivo models predictive of antipsychotic and antidepressant action. It was found that the two trans-1-piperazino-3-phenylindanes 18.17 and 18.18 (Figure 18.3) were relatively potent in the methyl phenidate-induced hyperactivity model (predictive of antipsychotic effect/mechanistic model for D2 antagonism), but at the same time about a factor of 10 weaker in the catalepsy model (predictive of EPS). Thus, these compounds were seen as prototypes of a new class of antipsychotic drugs with an improved side effect profile with respect to EPS.

In 1980, the trans-racemate tefludazine (18.19, Figure 18.3) was selected from this series as a development agent with potential antipsychotic effect. Tefludazine displayed a similar ratio in the methyl phenidate hyperactivity versus the catalepsy model as the two lead compounds, but

18.17

18.18

FIGURE 18.3 Selected trans-1-piperazino-3-phenylindanes.

f3c f3c

Tefludazine (18.19)

18.17

18.18

Tefludazine (18.19)

Irindalone (18.20)

FIGURE 18.3 Selected trans-1-piperazino-3-phenylindanes.

tefludazine was at least 100 times more potent in these in vivo models. It was also shown that tefludazine was an extremely potent and long-acting in vivo 5-HT2 receptor antagonist (quipazine model). Thus, tefludazine was a mixed D2 and 5-HT2 receptor compound.

During the 1980s, an electrophysiological in vivo model for the evaluation of limbic (linked to positive symptoms) versus striatal (linked to EPS) selectivity was introduced at Lundbeck. The model was a chronic one where rats were treated with a compound for 3 weeks before the number of active dop-amine neurons were counted in the VTA/SNC from where neurons project to limbic and striatal areas, respectively. In this so-called VTA/SNC model treatment with classical antipsychotic drugs such as chlorpromazine and haloperidol led to complete inhibition of neurons in both VTA and SNC by equal doses, whereas clozapine selectively inactivated the dopamine neurons in the VTA. These results corresponded to clinical data with regards to antipsychotic effect and EPS, and the model became of key importance at Lundbeck as it had the potential to predict the therapeutic window between antipsy-chotic effect and EPS of putative new antipsychotic drugs. It was subsequently shown that tefludazine displayed some selectivity in this model predicting tefludazine to be atypical, but unfortunately, the development of tefludazine was discontinued in Phase I due to toxicological findings in dogs.

It was also discovered that the removal of the "neuroleptic substituent" in the indane benzene ring (i.e., the trifluoromethyl group in 18.19), reduced the D2 receptor antagonism, whereas the 5-HT2 receptor antagonism was retained. Concurrent replacement of the hydroxyethyl side chain with the more bulky 1-ethyl-2-imidazolidinone side chain, resulted in irindalone (18.20, Figure 18.3), which was a very potent and selective 5-HT2 antagonist. Irindalone was developed as a potential antihyper-tensive drug, but the development was discontinued in Phase II in 1989 because of market considerations. Irindalone was, in contrast to tefludazine, developed as the pure (1R,3S)-enantiomer. This configuration of the 1-piperazino-3-phenylindanes is generally associated with receptor antagonistic properties, whereas the (1S,3R)- and the (1R,3R)-enantiomers are NET/DAT reuptake inhibitors.

The piperazinoindanes are chiral molecules, which at that time complicated many stages of drug discovery and development processes. Therefore, the corresponding piperazino-, tetrahydropyridino-, and piperidino-indoles were designed, and it was discovered that the piperidinoindole moiety bioisos-terically substituted for the irans-piperazinoindane with respect to D2 and 5-HT2 antagonism. One of the compounds synthesized in this series was sertindole (18.16, Figure 18.2), which incorporates structural elements from both tefludazine (neuroleptic substituent) and irindalone (imidazolidinone side chain). Despite high affinity for both D2 and 5-HT2 receptors, sertindole displayed an in vivo profile of a selective 5-HT2 receptor antagonist. Therefore, it was surprising that sertindole in the VTA/SNC model displayed a more than 100-fold selectivity for inhibition of dopamine neurons in the VTA as compared to the SNC. Sertindole was subsequently pushed through development and marketed in 1996 for the treatment of schizophrenia. Sertindole was temporarily withdrawn from the market in 1998 because of uncertainties regarding the relation between QT prolongation and the ability to induce potentially fatal cardiac arrhythmias in humans. However, the suspension of sertindole was lifted in 2002, and in January 2006, Estonia became the first country to reintroduce it. Sertindole is currently being introduced in several European, Asian, Latin American and the USA. Interestingly, recent preclinical evidence suggests sertindole being effective in the treatment of cognitive impairment in schizophrenia, with superiority over other antipsychotics such as risperidone, olanzapine, and clozapine. The effect has putatively been linked to potent 5-HT6 receptor antagonism combined with lack of antimuscarinic activity. It is currently being investigated whether the preclinical finding can be confirmed in clinical trials.

18.3 TRANSPORTER LIGANDS 18.3.1 Antidepressant Drugs

Antidepressant drugs represent ligands that target DAT, SERT, and NET to various degrees, and these include first generation antidepressants (i.e., tricyclic antidepressants, TCAs), selective serotonin reuptake inhibitors (SSRIs), combined serotonin and norepinephrine reuptake inhibitors (SNRIs), and the more recently introduced allosteric serotonin reuptake inhibitor (ASRI), escitalopram.

The SSRIs have been highly successful in the treatment of depression due to their high safety in use, and a number of new indications (e.g., panic disorder, obsessive compulsive disorder, and social phobia) have been registered for many of these drugs in addition to major depression. However, there are still major unmet needs in the treatment of depression, and since the inhibition of 5-HT reuptake ensures a certain degree of antidepressant activity, there has been a large interest in combining

5-HT reuptake inhibition with additional pharmacological effects. Some of these have resulted in marketed antidepressants, such as the SNRIs. In the following text, key events related to the discovery of antidepressants and in particular citalopram and escitalopram at Lundbeck will be discussed, including the use of pharmacophore and homology models.

18.3.1.1 First Generation Drugs

The pharmacotherapy of depression started in the late 1950s with the introduction of the two drugs iproniazid (18.21) and imipramine (18.23, Figure 18.4). Iproniazid was originally an antituberculosis drug, but it was noticed that the drug had an antidepressant effect. It was subsequently discovered that iproniazid was an unselective, irreversible inhibitor of the enzymes MAO-A and MAO-B, which deaminate the monoamines NE, DA, and 5-HT. Structural modifications of the tricyclic antipsychotic drugs with chlorpromazine (18.1, Figure 18.1) as a prototype led to the

6-7-6 tricyclic compound imipramine that was found to block the transporters for NE and 5-HT. These mechanisms led to an increase in the concentrations of NE and 5-HT in the synapse, which in turn led to the so-called amine hypothesis of depression, stating that there is a decreased availability of these neurotransmitters in depression.

Although the discovery of these two classes of drugs was of major therapeutic importance, it quickly turned out that both types had fatal side effects. Treatment with MAO inhibitors could induce a hypertensive crisis because of a fatal interaction with foodstuffs containing tyramine such as cheese. Dietary restrictions during treatment with MAO inhibitors were therefore required. Reversible MAO-A inhibitors (such as moclobemide (18.22)) have later been developed, but such drugs are still not completely devoid of the "cheese-effect" because the tyramine potentiation is inherent to blockade of MAO-A in the periphery. MAO inhibitors are therefore only used to a lesser extent in antidepressant therapy.

0"Lnh aOiK.

Iproniazid (18.21) Moclobemide (18.22)

Imipramine (R' = H; R = CH3) (18.23) Desipramine (R' = H; R= H) (18.24) Clomipramine (R' = Cl; R = CH3) (18.25)

Imipramine (R' = H; R = CH3) (18.23) Desipramine (R' = H; R= H) (18.24) Clomipramine (R' = Cl; R = CH3) (18.25)

Amitriptyline (R = CH3) (18.26) Nortriptyline (R = H) (18.27)

Melitracen (18.28)

Amitriptyline (R = CH3) (18.26) Nortriptyline (R = H) (18.27)

J,CH3

Melitracen (18.28)

FIGURE 18.4 Antidepressant drugs from MAO-inhibitor and tricyclic classes.

A major problem with the tricyclic antidepressants such as imipramine (18.23), desipramine (18.24), amitriptyline (18.26), nortriptyline (18.27), and melitracen (18.28) is that, due to their fundamental tricyclic structures, in addition to their blockade of SERT and/or NET, they also block a number of postsynaptic receptors notably for acetylcholine, histamine, and NE. Therefore, they may induce a number of anticholinergic, antihistaminergic, and cardiovascular side effects, such as dryness of the mouth, constipation, confusion, dizziness, sedation, orthostatic hypotension, tachycardia, and/or arrhythmia. Moreover, they are potentially lethal in overdose. So even if these drugs represented a major therapeutic breakthrough, it became clear that there was an inevitable need for better and safer drugs.

18.3.1.2 The Selective Serotonin Reuptake Inhibitors

Nortriptyline (18.27) is a relative selective NE reuptake inhibitor, while the corresponding dimethyl derivative, amitriptyline (18.26), is a mixed 5-HT/NE reuptake inhibitor with concomitant high affinity for the postsynaptic receptors mentioned earlier. The same is true for the corresponding pair desipramine (18.24)/imipramine (18.23). Swiss psychiatrist Paul Kielholz coupled these observations to the clinical profiles of these drugs, and Swedish scientist Arvid Carlsson noticed that the tertiary amine drugs, which were mixed 5-HT and NE reuptake inhibitors, were "mood elevating," while the secondary amines, being primarily NE reuptake inhibitors, increased more "drive" in the depressed patients. As the foremost quality of an antidepressant drug should be mood elevation (elevation of drive before mood could induce a suicidal event), Carlsson advocated for the development of selective 5-HT reuptake inhibitors. Consequently, a number of pharmaceutical companies initiated drug discovery programs aiming at design of such drugs in the early 1970s.

18.3.1.2.1 Discovery of Citalopram

In the mid-1960s, chemists at Lundbeck were looking for more potent derivatives of the tricyclic compounds amitriptyline, nortriptyline, and melitracen, which the company had developed and marketed previously. The trifluoromethyl group had in other in-house projects proved to increase potency in thioxanthene derivatives with antipsychotic activity (see Figure 18.1), and it was therefore decided to attempt to synthesize the 2-CF3 derivative (18.30) of melitracen (Figure 18.5). The precursor molecule 18.29 was readily synthesized, but attempts to ring-close it in a manner corresponding to the existing melitracen method, using concentrated sulfuric acid, failed. However, another product was formed, which through meticulous structural elucidation proved to be the bicyclic phthalane (or dihydroisobenzofuran) derivative 18.31. Fortunately, this compound was examined in models for antidepressant activity and was very surprisingly found to be a selective NET inhibitor. Some derivatives were synthesized, among them two compounds that later got the International Nonproprietary Name (INN) names talopram (18.32) and talsupram (18.33). These compounds are still among the most selective NE reuptake inhibitors (SNIs) ever synthesized (Figure 18.5 and Table 18.2).

Both talopram and talsupram were investigated for antidepressant effect in clinical trials but were stopped in Phase II for various reasons, among which an activating profile in accordance with their potent NE reuptake inhibition. A project was therefore started in the beginning of 1971 with the aim of discovering an SSRI from the talopram structure.

It may not be obvious to use an SNI as template structure for an SSRI. However, in the first series synthesized, two compounds (18.35 and 18.36, Table 18.2) without the dimethylation of the phthalane ring showed a tendency for increased 5-HT reuptake, and in accordance with the structure-activity relationship (SAR) studies mentioned earlier for tricyclics, the A,A-dimethyl derivative 18.36 was the more potent. Therefore, compound 18.36 became a template structure for further structural investigation.

In this phase of the project, test models for measuring neuronal reuptake were not available, so 5-HT reuptake inhibition was measured as inhibition of tritiated 5-HT into rabbit blood platelets, while inhibition of NE reuptake was measured ex vivo as inhibition of tritiated NE into the heart of the mouse (Table 18.2). Although these models were not directly comparable, they were acceptable for the discovery of selective compounds.

FIGURE 18.5 Discovery of phenylphthalane antidepressants.

TABLE 18.2

5-HT and NE Reuptake Inhibition of Selected Talopram Derivatives

.chs

5-HT Reuptake (In Vitro) NE Reuptake (In Vivo)

Rabbit Blood pl. IC50

Mouse Heart ED,

Compound

Ri

R2

X

Y

(nM)

(mmol/kg)

Talopram (18.32)

ch3

H

H

H

3,400

2.2

(18.34)

ch3

ch3

H

H

53,000

5

(18.35)

H

H

H

H

1,300

43

(18.36)

H

ch3

H

H

600

66

(18.37)

H

ch3

H

cl

110

170

(18.38)

H

ch3

cl

H

220

>200

(18.39)

H

ch3

cl

cl

24

>80

(18.40)

H

ch3

H

Br

310

n.t.

(18.41)

H

ch3

H

CN

54

23

(18.42)

H

ch3

CN

cl

10

>80

Citalopram (18.43)

H

ch3

CN

F

38

>40

Source: Data from Lundbeck Screening Database, H. Lundbeck, Valby, Denmark.

Source: Data from Lundbeck Screening Database, H. Lundbeck, Valby, Denmark.

The introduction of a chloro substituent into the template structure 18.36 further increased 5-HT reuptake and decreased NE reuptake inhibition (18.36 and 18.38), in accordance with observations by Carlsson that halogen substituents in both zimelidine (18.47, Figure 18.6) (see the following text) derivatives and in of imipramine (clomipramine, 18.25, Figure 18.4) increased 5-HT reuptake. Indeed, the dichloro derivative 18.39 proved to be a selective 5-HT reuptake inhibitor. So the goal of obtaining an SSRI from an SNI was achieved very fast (in 1971), when less than 50 compounds had been synthesized.

The SAR were further explored, and it was established that high activity was generally found in 5,4'-disubstituted compounds where both substituents were halogen or other electron-withdrawing groups. Cyano-substituted compounds were obtained by the reaction of the bromo precursors (e.g., 18.40) with CuCN. One of the cyano-substituted compounds was (18.43), later known as citalopram (INN name). The compound was synthesized for the first time in August 1972. The cyano group could be metabolically labile, but it was subsequently shown not to be the case neither in animals nor in humans. Citalopram displayed the best overall preclinical profile within this series and was consequently selected for development. The 5-cyano substituent in citalopram also proved to be chemically stable in a surprising manner; for example, it does not react with Grignard reagents, which has led to a new and patentable process for its production.

Citalopram was launched in Denmark in 1989, and it has since been registered worldwide. Citalopram is a racemate, having an asymmetric carbon at the 1-position. When it was synthesized in 1972, classical resolution via diastereomeric salts was the only realistic alternative for separation of the enantiomers. However, it is generally difficult to make salts of citalopram, and eventually direct resolution was given up. Finally, an intermediate was resolved in this way, and the resolved intermediate could then be transformed into the pure (5)- and (R)-enantiomers of citalopram. Subsequent testing showed that all the 5-HT reuptake inhibition resided in the (5)-enantiomer. The high stereospecificity was later rationalized in the SSRI pharmacophore model discussed in the following text. The (5)-enantiomer (INN name escitalopram) has subsequently emerged as a new drug defining a new group of antidepressants, namely the ASRIs. The discovery and its implications are discussed in the following text.

Citalopram (18.43) 14.1.1976

Fluoxetine (18.44) 10.1.1974

Paroxetine (18.45) 30.1.1973

Fluvoxamine (18.46) 20.3.1975

Citalopram (18.43) 14.1.1976

Fluoxetine (18.44) 10.1.1974

Paroxetine (18.45) 30.1.1973

Fluvoxamine (18.46) 20.3.1975

Zimelidine (18.47) 28.04.1971

Indalpine (18.48) 12.12.1975

Sertraline (18.49) 1.11.1979

FIGURE 18.6 Selective serotonin reuptake inhibitors (SSRIs).

Zimelidine (18.47) 28.04.1971

Indalpine (18.48) 12.12.1975

Sertraline (18.49) 1.11.1979

FIGURE 18.6 Selective serotonin reuptake inhibitors (SSRIs).

18.3.1.2.2 Other Selective Serotonin Reuptake Inhibitors

In Figure 18.6, the seven SSRIs that have reached the market, with the priority dates of the first patent application indicated are shown. However, the two first compounds on the market were both withdrawn due to serious, although rare, side effects. Zimelidine (18.47) was found to induce an influenza-like symptom in 1%-2% of the patients, which in rare cases (1/10,000) resulted in the so-called Guillain-Barre syndrome. The drug was withdrawn in 1983 after B4 years on the market. Indalpine (18.48) induced agranulocytosis in one of 20,000 patients and was withdrawn in 1984.

As it appears from Figure 18.6, all the marketed SSRIs (except sertraline) were discovered in the first half of the 1970s, meaning that the companies lacked sufficient information regarding the structural classes their competitors were developing. Accordingly, rather diverse structures were developed. However, they were all selective 5-HT reuptake inhibitors (Table 18.3), although their selectivity ratios vary significantly, citalopram/escitalopram being the most selective compounds. In general, the SSRIs have low affinity for receptors for DA, NE, 5-HT, and other neurotransmitters, although exceptions exist. With regard to interaction with cytochrome P450 enzymes there are vital differences, e.g., paroxetine and fluoxetine having significant affinity for CYP2D6.

TABLE 18.3

The Effect of SSRIs, Talopram, and Talsupram on the Inhibition of Reuptake of 5-HT, NE, and DA

Uptake Inhibition IC50 (nM) Ratio

TABLE 18.3

The Effect of SSRIs, Talopram, and Talsupram on the Inhibition of Reuptake of 5-HT, NE, and DA

Uptake Inhibition IC50 (nM) Ratio

Compound

5-HT

NE

DA

NE/5-HT

DA/5-HT

Citalopram (18.43)

3.9

6100

40,000

1560

10,300

Escitalopram (S) (18.43)

2.1

2500

65,000

1200

31,000

r-citalopram (R) (18.43)

275

6900

54,000

25

200

Indalpine (18.48)

2.1

2100

1,200

1000

570

Sertraline (18.49)

0.19

160

48

840

250

Paroxetine (18.45)

0.29

81

5,100

280

17,600

Fluvoxamine (18.46)

3.8

620

42,000

160

11,000

Zimeldine (18.47)

56

3100

26,000

55

460

Fluoxetine (18.44)

6.8

370

5,000

54

740

Talopram (18.32)

1400

2.5

44,000

0.0017

0.00006a

Talsupram (18.33)

770

0.79

9,300

0.0010

0.00008a

Sources: Data in italics are from Hyttel, J. Int. Clin. Psychopharmacol. 9(Suppl. 1), 19, 1994;

Remaining are from Lundbeck Screening Database, H. Lundbeck A/S, Valby, Denmark.

Sources: Data in italics are from Hyttel, J. Int. Clin. Psychopharmacol. 9(Suppl. 1), 19, 1994;

Remaining are from Lundbeck Screening Database, H. Lundbeck A/S, Valby, Denmark.

18.3.1.3 Discovery of Escitalopram—An Allosteric Serotonin Reuptake Inhibitor

The 5-HT reuptake inhibition of citalopram resides in the (S)-enantiomer (escitalopram) whereas the (^)-enantiomer is about 100 times less potent. Escitalopram was launched as a single-enantiomer drug in 2002 and is today an effective antidepressant with several advantages as compared to citalopram and other SSRIs. In preclinical studies escitalopram shows greater efficacy and faster onset of action than comparable doses of citalopram. This is attributed to the fact that a number of studies have shown that the (R)-enantiomer of citalopram counteracts the activity of the (S)-enantiomer. Further in randomized, controlled clinical studies escitalopram shows better efficacy than citalopram, with higher response and remission rates, and faster onset of action.

The existence of an allosteric-binding site on SERT and its possible relevance for the effect of antidepressants has been known since the early 1990s, and among the SSRIs only citalopram and to a lesser extent paroxetine exert an effect via this low-affinity binding site. The dual action on both the allosteric and the primary-binding site results in an increased dissociation half-life of escitalopram from its primary-binding site. The (^)-enantiomer has a three times weaker allosteric effect, and it significantly reduces the association rate for [3H] escitalopram binding to SERT in low (40-80 nM) concentrations. These effects of the (^)-enantiomer may be important in relation to its inhibition of the (S)-enantiomer in citalopram.

18.3.1.4 The SSRI Pharmacophore and SERT Homology Model

Despite very different molecular structures, the SSRIs all bind to SERT. As information about the 3D structure of the transporter is lacking, development of a pharmacophore model was of major interest in the early 1990s. Thus, a pharmacophore model of the SSRIs was developed at Lundbeck based on extensive conformational studies and superimpositions of SSRIs and other reuptake inhibitors (Figure 18.7a). The model operates with three fitting points, namely the centroids of the two aromatic rings and a site point positioned 2.8 A from the nitrogen atom in the direction of the lone pair. The nitrogen site point mimics a hypothetical hydrogen-binding atom on SERT, most likely the carboxy group of Asp 98 (see also discussion in the following text). The use of a nitrogen site point as fitting point in this model gave a very good superimposition of all key SSRIs (i.e., escitalopram, (S)- and (R)-fluoxetine, (1S,4^)-sertraline, and (3S,4^)-paroxetine), which was not possible when the basic nitrogen atoms were superimposed. Additionally, many SSRIs have aromatic substituents (cyano, trifluoromethyl, chloro, methylendioxo, etc.) that all, in this model, occupy the same volume marked in yellow. Hence, this volume of the transporter is allowed for SSRIs but not for NRIs. On the contrary, the volume marked in white defines a forbidden volume for SSRI ligands. Protrusion of ligands into this volume allows for design of NRIs (Figure 18.7a).

The pharmacophore model has been validated with a number of 5-HT and NE reuptake inhibitors in addition to the compounds in Figure 18.6. Importantly, the model explains the more than 100-fold stereoselectivity of the citalopram enantiomers. Thus, it is possible to find a conformation of the (^)-enantiomer that is superimposable with the proposed bioactive conformation of escitalopram, but the conformational energy penalty is 2.8 kcal/mol, which corresponds closely to a 100-fold affinity difference. The enantiomers of fluoxetine display no stereoselectivity at SERT, and they can be fitted to the model with no differences in conformational energy in accordance with their equipotency.

An x-ray crystal structure of a leucine transporter (LeuTAa), which is a bacterial homolog of SERT, from Aquifex aeolicus has offered an opportunity to build a more reliable homology model of SERT as compared to earlier transporter models based on more distantly related proteins. In Figure 18.7b, the secondary structure of the LeuTAa is illustrated with the primary-binding site for leucine marked with a triangle, and in Figure 18.7c the corresponding homology model of SERT built at Lundbeck is shown. The homology model of SERT is colored and oriented in a similar manner as LeuTAa in Figure 18.7a and with escitalopram docked into the proposed primary-binding site. In Figure 18.7d a "close up" of the primary-binding site with bound escitalopram is shown. The "close up" is taken from the intracellular side looking up into the transporter. From this illustration it is possible to see the putative interaction points between escitalopram and SERT, notably the hydrogen bond between the basic amine of escitalopram and Asp 98. It is also possible to see the putative hydrophobic interaction between the two aromatic rings of escitalopram and the hydrophobic amino acid Ile 172. Importantly, it has been shown in a number of mutation studies that Asp 98 and Ile 172 are essential for the binding of escitalopram to SERT giving validity to this model.

The nine amino acids (shown in white in Figure 18.7c) that have been shown to be involved in the allosteric binding of escitalopram on SERT are located in TM 10, 11, and 12 near the C-terminal. Very little is known about the interaction of escitalopram with this putative-binding site. However, it is highly likely that the allosteric effect is important for the superior effect of escitalopram as an antidepressant, especially in patients with severe depression.

FIGURE 18.7 (a) Pharmacophore model of the 5-HTreuptake site. Purple: Three fitting points; centroids of aromatic rings and site point for interaction with transporter amine. Green: Phenyl rings. Blue: Nitrogen atoms. Yellow: Allowed volume for SSRI suhstituents. White: Forbidden volume at SERT. allowed at NET. Red: Possible hydrogen bond acceptor site. (Courtesy of Klaus Gundertofte. H. Lundbeck A/S. Copenhagen-Valby. Denmark.) (b) The leucine transporter topology. The position of leucine is depicted as a yellow triangle. (From Yamashita. A. et al. Nature, 437.215.2005. With permission.) (c) The 5-HT transporter (SERT) homology model. Escitalopramis highlighted in green/blue space filling and dockedinto the primary-binding site. The transmembrane domains (TMs) involved in the primary-binding site are shown in the same colors as in (b) (TM1: red. TM3: orange. TM6: green, and TM8: blue), and the important amino acids are shown in yellow. The TMs important for allosteric effects are highlighted in purple (TM 10-12). and the corresponding amino acids are shown in white. (Courtesy of Anne Marie J0rgensen, H. Lundbeck A/S. Copenhagen-Valby. Denmark.) (d) A "close up" of the primary-binding site of Figure 18.7c with escitalopram docked into the site. The three fitting points from the pharmacophore model is illustrated with purple spheres, including the site point on Asp 98 involved in the hydrogen bond (yellow dotted line) with escitalopram. (Courtesy of Anne Marie J0rgensen, H. Lundbeck A/S. Copenhagen-Valby. Denmark.)

FIGURE 18.7 (a) Pharmacophore model of the 5-HTreuptake site. Purple: Three fitting points; centroids of aromatic rings and site point for interaction with transporter amine. Green: Phenyl rings. Blue: Nitrogen atoms. Yellow: Allowed volume for SSRI suhstituents. White: Forbidden volume at SERT. allowed at NET. Red: Possible hydrogen bond acceptor site. (Courtesy of Klaus Gundertofte. H. Lundbeck A/S. Copenhagen-Valby. Denmark.) (b) The leucine transporter topology. The position of leucine is depicted as a yellow triangle. (From Yamashita. A. et al. Nature, 437.215.2005. With permission.) (c) The 5-HT transporter (SERT) homology model. Escitalopramis highlighted in green/blue space filling and dockedinto the primary-binding site. The transmembrane domains (TMs) involved in the primary-binding site are shown in the same colors as in (b) (TM1: red. TM3: orange. TM6: green, and TM8: blue), and the important amino acids are shown in yellow. The TMs important for allosteric effects are highlighted in purple (TM 10-12). and the corresponding amino acids are shown in white. (Courtesy of Anne Marie J0rgensen, H. Lundbeck A/S. Copenhagen-Valby. Denmark.) (d) A "close up" of the primary-binding site of Figure 18.7c with escitalopram docked into the site. The three fitting points from the pharmacophore model is illustrated with purple spheres, including the site point on Asp 98 involved in the hydrogen bond (yellow dotted line) with escitalopram. (Courtesy of Anne Marie J0rgensen, H. Lundbeck A/S. Copenhagen-Valby. Denmark.)

18.4 CONCLUDING REMARKS

DA, 5-HT, and NE receptors and transporters have shown their relevance as drug targets, and although research toward the pharmacotherapy of schizophrenia and depression nowadays are targeting other targets as well, future antipsychotics and antidepressants may very well interact with these receptors and sites also. Recently, it has been shown that DA signals via several different second messenger systems and as discussed earlier an allosteric site has been identified on SERT. Only the future will tell us whether these new discoveries will result in new and effective pharma-cotherapies based on the DA and 5-HT systems.

FURTHER READINGS

Gether, U., Andersen, P. H., Larsson O. M., and Schousboe, A. (2006) Neurotransmitter transporters: Molecular function of important drug targets. Trends Pharmacol. Sci., 27, 375-383. Gray, J. A. and Roth, B. L. (2007) The pipeline and future of drug development in schizophrenia. Mol. Psychiatry, 12, 904-922.

Hyttel, J. (1994) Pharmacological characterisation of selective serotonin reuptake inhibitors (SSRIs). Int. Clin.

Psychopharmacol., 9(Suppl. 1), 19-26. J0rgensen, A. M., Tagmose, L., J0rgensen, A. M. M., Topiol, S., Sabio, M., Gundertofte, K., B0ges0, K. P., and Peters, G. H. (2007) Homology modeling of the serotonin transporter: Insight into the primary escitalopram-binding site. ChemMedChem, 2, 815-826. Moltzen, E. K. and Bang-Andersen, B. (2006) Serotonin reuptake inhibitors: The corner stone in treatment of depression for half a century—A medicinal chemistry survey. Curr. Top. Med. Chem., 6, 1801-1823. Sánchez, C., B0ges0, K. P., Ebert, B., Reines, E. H., and Br®strup, C. (2004) Escitalopram versus citalopram:

The surprising role of the R-enantiomer. Psychopharmacology, 174, 163-176. Werkman, T. R., Glennon, J. C., Wadman, W. J., and McCreary, A. C. (2006) Dopamine receptor pharmacology: Interactions with serotonin receptors and significance for the aetiology and treatment of schizophrenia. CNS Neurol. Disord. Drug Targets, 5, 3-23. Wood, M. and Reavill, C. (2007) Aripiprazole acts as a selective dopamine D2 receptor partial agonist. Expert

Opin. Invest. Drugs, 16, 771-775. Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., and Gouaux, E. (2005) Crystal structure of a bacterial homologue of Na+/Cl--dependent neurotransmitter transporters. Nature, 437, 215-223.

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