The Discovery of Citalopram and Its Refinement to Escitalopram

Abstract

List of Abbreviations Introduction Discovery of Talopram Discovery of Citalopram Synthesis and Production of Citalopram The Pharmacological Profile of Citalopram Clinical Efficacy of Citalopram Synthesis and Production of Escitalopram The Pharmacological Profile of the Citalopram Enantiomers R-Citalopram's Surprising Inhibition of Escitalopram Binding Site(s) for Escitalopram on the Serotonin Transporter Future Perspectives on the Molecular Basis for Escitalopram's Interaction with the SERT Clinical Efficacy of Escitalopram Conclusions References

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11
The Discovery of Citalopram and Its Refinement to Escitalopram
Klaus P. Bøgesø and Connie S
anchez
List of Abbreviations
CHO Chinese hamster ovary
DA dopamine
DAT dopamine transporter
DMPK drug metabolism and pharmacokinetics
DTTA ditoluoyl tartaric acid
gSERT chicken (gallus) serotonin transporter
5-HIAA 5-hydroxyindoleacetic acid
HPLC high-pressure liquid chromatography
hSERT human serotonin transporter
5-HT 5-hydroxytryptamin
5-HT1A 5-hydroxytryptamin 1A (receptor)
5-HTP 5-hydroxytryptophan
LeuT leucine transporter
MAOI monoamine oxidase inhibitor
NE norepinephrine
NET norepinephrine transporter
NMR nuclear magnetic resonance
NRI norepinephrine reuptake inhibitor
PET positron emission tomography
PKC protein kinase C
QSAR quantitative structure–activity relationship
SAR structure–activity relationship
SERT serotonin transporter
SIP serotonin interaction protein
SLC6 solute carrier 6
SMB simulated moving bed
SNRI serotonin norepinephrine reuptake inhibitor
WT SERT wild type serotonin transporter
Analogue-based Drug Discovery III, First Edition. Edited by J
anos Fischer, C. Robin Ganellin, and
David P. Rotella.
#2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.
j
269

SPECT single-photon emission computer tomography
SSRI selective serotonin reuptake inhibitor
TCA tricyclic antidepressants
11.1
Introduction
The serendipitous discoveries of the monoamine oxidase inhibitor (MAOI) ipronia-
zid and the tricyclic antidepressant (TCA) imipramine in the late 1950s were major
breakthroughs in the treatment of depression. The discovery of imipramine initi-
ated a search for new tricyclic antidepressants using analogue design. Among these
analogues were amitriptyline (1), nortriptyline (2), and melitracen (3) (Figure 11.1).
Lundbeck developed new patentable syntheses of these three drugs (at that time,
product patents were not obtainable, only process patents) and entered the market
with these products in the early 1960s. However, the use of TCAs is associated with
disturbing and serious side effects, such as dryness of the mouth, constipation,
confusion, dizziness, sedation, orthostatic hypotension, tachycardia, and/or
arrhythmia. Relatively narrow therapeutic indices limit the dose range in which
they can be used and pose a risk in the case of overdose.
As increasing knowledge was gained about the mechanism of action of the
TCAs and appropriate screening assays were developed, chemists started to
look for ways to improve them. TCAs either inhibit the reuptake of serotonin
1 Amitriptyline, R=CH3
2 Nortriptyline, R=H
3 Melitracen, R=H
4 R=CF3
5
N R
CH3
H
N CH3
H
CH3
OH
OH NCH3
R
CF3
CH3
6 Talopram, X=O, R=R1=H
7 X=O, R=CH3, R1=CF3
8 Talsupram, X=S, R=R1=H
X
NR
CH3
R1
Figure 11.1 Lundbeck’s tricyclic antidepressants and the selective NE uptake inhibitors talopram
and talsupram.
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

(5-hydroxytryptamine, 5-HT) and norepinephrine (NE) (imipramine, amitripty-
line), or predominantly NE (desipramine, nortriptyline). But they also block a
number of postsynaptic receptors, notably those for NE, acetylcholine, and his-
tamine. The latter effects were mainly associated with the side effects, whereas
inhibition of the monoamine transporters was associated with the therapeutic
effects. Two major hypothesis of depression emerged from this research, the 5-
HT and the NE hypotheses, where lowered 5-HT levels in the brain were associ-
ated with lowered mood, and lowered NE levels were associated with lowered
psychomotor drive. The discovery of the selective serotonin reuptake inhibitors
(SSRIs) has already been described broadly in a previous volume of this
series [1]. Thus, the following will focus on the discovery of citalopram and esci-
talopram [2–4].
11.2
Discovery of Talopram
The discoveries of both citalopram and escitalopram (S-citalopram) are good exam-
ples of how analogue design can lead to drugs with either totally different or greatly
improved therapeutic profiles compared with the starting structure.
The discovery of citalopram started with the discovery of talopram (6)
(Figure 11.1) in 1965. In an attempt to make a trifluoromethyl-substituted deriva-
tive of melitracen (4) using a precursor molecule (5) and reaction conditions (ring
closure in concentrated sulfuric acid) similar to those used in the production of
melitracen, a chemist at Lundbeck ended up with a different product, which after
meticulous structural elucidation proved to be the phthalane structure 7. This com-
pound proved to be a surprisingly selective NE uptake inhibitor. Analogue design
revealed that the unsubstituted N-des-methyl analogue 6(talopram, Lu 03-010) was
a highly selective and very potent NE uptake inhibitor. The sulfur analogue of talo-
pram, called talsupram (8, Lu 05-003), was likewise a very potent, selective inhibitor
of the NE transporter (NET). Both compounds were therefore major improvements
with regard to selectivity compared with the nonselective TCAs, desipramine and
nortriptyline.
Both drugs entered early clinical testing in the late 1960s, but were stopped in
phase II for various reasons, among which was a rather activating profile. This
observation was supportive of the hypothesis proposed by Professor Arvid Carlsson
that NE uptake inhibition would mainly increase psychomotor drive. Carlsson had
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 the psychomotor drive in the depressed patients [5].
Carlsson advocated the development of selective 5-HT uptake inhibitors to treat the
lowered mood state and avoid the potential suicide risk associated with an
increased psychomotor drive in a depressed patient. Carlsson presented his theory
at Lundbeck, and in 1971 it was decided to start a search for a selective serotonin
reuptake inhibitor.
11.2 Discovery of Talopram
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Although it may seem paradoxical, it was decided to use talopram as template for
designing an SSRI. However, the reason was that there were already a few ana-
logues of talopram available in-house that had dual 5-HT/NE uptake inhibition [2].
In particular, derivatives lacking the 3,3-dimethyl substituents of talopram, but with
a chlorine either at the 5-position or at the 40-position (see the structure in
Table 11.1), were more potent as 5-HT uptake inhibitors than NE uptake inhibitors.
Like their tricyclic dual-acting counterparts, these compounds also had a dimethy-
lamino group, instead of the monomethylamino group found in the selective NE
uptake inhibitors.
We, therefore, decided to use the structure at the top of Table 11.1 as the core
structure for a program investigating the structure–activity relationships (SARs) of
aromatic substitution in the two benzene rings.
11.3
Discovery of Citalopram
We published our initial SAR study (actually a quantitative structure–activity rela-
tionship (QSAR) study) in 1977 [7]. At that time, in vitro assays measuring inhibi-
tion of neuronal 5-HT and NE uptake were not available at Lundbeck, so 5-HT and
NE uptake inhibition was measured as inhibition of ½3H-5-HT uptake into rabbit
platelets and as inhibition of ½3H-NE uptake into the mouse heart ex vivo, respec-
tively. Although these models were not directly comparable, they were acceptable as
long as the goal was to develop selective compounds. Later on, assays based on
inhibition of ½3H-5-HT and ½3H-NE uptake into rat brain synaptosome prepara-
tions as well as recombinant cell-based assays expressing the cloned human sero-
tonin transporter (hSERT) or NET were developed. Table 11.1 shows a number of
key citalopram derivatives (9–16) with the original blood platelet results as well as
data for inhibition of ½3H-5-HT uptake in recombinant cells expressing the cloned
hSERT. All citalopram derivatives are highly selective serotonin transporter (SERT)
inhibitors compared to inhibition of the NET and the dopamine transporters
(DATs). Thus, data for NETand DAT inhibition are not shown.
Applying the observations mentioned above regarding monosubstituted
chloro derivatives led to the synthesis of the 5,40-dichloro derivative (10,
Table 11.1) that proved to be a very potent SSRI. SAR studies of inhibitors of
SERT, NET, and DAT very often show that optimal potency is found in 3,4-
dichlorophenyl derivatives [8]. This derivative (11)alsoprovedtobevery
potent, whereas the corresponding 30,50-dichloro derivative (12)had25–100
times weaker activity. Substitution with chlorine at the 6-, 4-, and 7-positions
(compounds 13,14,and15) was also allowed. The most potent derivatives
were generally found among the 40,5-disubstituted derivatives substituted with
F, Cl, Br, or CF
3
. Electron-donating groups (30-OCH
3
,4
0-OCH
3
,or4
0-isopropyl)
had very low activity [7].
At that time, a cyano group was not considered an obvious choice in system-
atic aromatic substitution SAR investigations. One reason was that a nitrile
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

Table 11.1 Inhibition of 5-HT uptake and allosteric effect in a series of selected citalopram derivatives.
Inhibition of uptake of ½3H5-HT
Rat blood pl
a
hSERT hSERT hSERT allosteric
b
Compound R1 R2 IC
50
(nM) IC
50
(nM) IC
50
(nM) IC
50
(mM)
95-CN (citalopram) 40-F 14 6.7 8.7
S-95-CN (escitalopram) 40-F 2.1 5.1
R-95-CN (R-citalopram) 40-F 170 25
10 5-Cl 40-Cl 20 7.4 12.1
11 H3
0,40-Cl
2
17 13 29.6
12 H3
0,50-Cl
2
1600 200 340 15
12a
c
H3
0,50-Cl
2
350 8.8
12b
c
H3
0,50-Cl
2
300 20
13 6-Cl 40-Cl 120 23
14 4-Cl 40-Cl 47 33 9.2 6.1
15 7-Cl 40-Cl 63 26
16 5-Br 40-F 22 13 30.2
17 5-(CH
3
)
2
N(CH
2
)
3
CO 40-F 1.6 714
18 5-CN 40-(4-F-C
6
H
4
-S) 200 12
(continued )
O
N
CH3
CH3
R1
R2
2′
3′
4'
5′
6′
4
5
6
7
11.3 Discovery of Citalopram
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273

Table 11.1 (Continued)
Inhibition of uptake of ½3H5-HT
Rat blood pl
a
hSERT hSERT hSERT allosteric
b
Compound R1 R2 IC
50
(nM) IC
50
(nM) IC
50
(nM) IC
50
(mM)
18a
c
5-CN 40-(4-F-C
6
H
4
-S) 310 8.3
18b
c
5-CN 40-(4-F-C
6
H
4
-S) 330 22
19 5-CN [2-Naphthyl] 34 68
19a
c
5-CN [2-Naphthyl] 10 41
19b
c
5-CN [2-Naphthyl] 150 55
20 5-CN [1-Naphthyl] 250 19
20a
c
5-CN [1-Naphthyl] 740 14
20b
c
5-CN [1-Naphthyl] 280 19
If not referred to in the text, data are unpublished data, H. Lundbeck A/S. Inhibition of ½3H-5-HTuptake at the hSERT was performed in Chinese hamster ovary (CHO) cells
stably expressing wild-type hSERT, as described by Ref. [6]. The two columns represent essentially similar assay conditions.
a) Data from Ref. [7].
b) Dissociation binding studies at the hSERT were conducted as described [6].
c) a and b refer to different enantiomeric forms.
Table 11.1 (Continued )
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

may be metabolically labile to be transformed to an amide or carboxylic acid.
However, one of the authors of this chapter (KPB) had previously worked on a
project in which nitriles were key intermediates and had used the relatively
new reaction where the aromatic halogen was reacted with cuprous cyanide to
produce the nitrile. In August 1972, having already synthesized the potent 5-
bromo-40-fluoro-derivative 16, he used this reaction directly on 16 and made
the first sample of 9(citalopram). The compound proved to be a very potent
SSRI. It was selected together with a few other potent SSRIs from the series
for further preclinical studies, proved overall to have the best safety profile,
and it was selected as a clinical development candidate. Interestingly, the
cyano group proved to be totally metabolically stable.
11.4
Synthesis and Production of Citalopram
The syntheses used for preparation of citalopram and derivatives are outlined
in Scheme 11.1. The starting materials were phthalides (I). These were either
made by methods published in the literature, or by improved or completely
new procedures. The majority of compounds could then be produced by a
“double Grignard”reaction, in which the phthalide was reacted with a substi-
tuted phenyl magnesium bromide to give the benzophenone intermediate II,
which was further reacted with 3-(dimethylamino)propyl magnesium chloride
to afford the “dicarbinol”III. The dicarbinols were then ring closed in strong
acid to the final product VI.
O
O
X
X
MgBr
Y
O
OH
Y
XOH
OH
Y
H3CNMgCl
CH3N
CH3
CH3
O
NCH3
CH3
2′
3′
4′
5′
6′
4
5
6
7
X
Y
H+
IIII
O
H
X
Y
XOH
OH
Y
H3CNCl
CH3
NaH
H+
VIVIV
LiAlH4
II
(CuCN)[A]
(CuCN)[A]
Scheme 11.1 The syntheses used for preparation of citalopram and derivatives. [A] represents
extra step exchanging Br with CN using cuprous cyanide in DMF if the end product was a cyano-
substituted derivative.
11.4 Synthesis and Production of Citalopram
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275

The 5-bromo derivative 16 was made by this synthesis (from 5-bromophtha-
lide) and as mentioned above, citalopram (X ¼5-CN, Y ¼40-F) was synthesized
for the first time by reacting the Br in 16 with cuprous cyanide. However, it
quickly turned out that this method was unsuited for the preparation of larger
quantities, so an alternative method was developed. Starting again with 5-bro-
mophthalide, the benzophenone II was reduced with LiAlH
4
to the intermedi-
ate IV, which was ring closed in strong acid to the phenylphthalane V(X ¼5-
Br, Y ¼40-F). By reaction with cuprous cyanide, a crystalline 5-cyano-1-(40-
fluorophenyl)phthalane was obtained. Citalopram was then obtained by metal-
lation with NaH in dimethyl sulfoxide and subsequent reaction with 3-(dime-
thylamino)propyl chloride. This method was used to produce the first 5 kg.
Unfortunately, this method was also unsuited for large-scale production. This
was a critical point in the development of citalopram, but we made a very sur-
prisingdiscovery.Itturnedoutthatthecyanogroupin5-cyanophthalidedid
not react with the Grignard reagents, contrary to expectation, and did not
hydrolyze in the strong acid used in the final ring closure. We also found that
the second “side chain”Grignard reagent could be added directly after the 4-
fluorophenyl magnesium bromide without isolating the intermediate benzo-
phenone. This was a major improvement, and this synthesis proved to be an
excellent production method.
11.5
The Pharmacological Profile of Citalopram
The pharmacological characterization of citalopram showed that it potently inhibited
the uptake of ½3H-5-HT in rabbit and rat thrombocytes and rat brain synaptosomes
with IC
50
values in the nanomolar range (14 and 1.7nM, respectively) [9, 10]. Fur-
thermore, citalopram turned out to be the most selective 5-HT uptake inhibitor
among the SSRIs in clinical use at that time, with a selectivity ratio of 3400 relative
to the inhibition of ½3H-NE uptake in rat brain synaptosomes compared to selectiv-
ity ratios of 840, 280, and 54 for sertraline, paroxetine, and fluoxetine, respectively,
and the selectivity relative to the inhibition of ½3H-dopamine (DA) uptake was even
greater, that is 22000 compared to selectivity ratios of 250, 18000, and 740 for ser-
traline, paroxetine, and fluoxetine [11]. Citalopram had very low affinity for the
receptors studied, the highest affinity being for the s
1
and histamine H1 receptors
(IC
50
200 and 350 nM, respectively) [11]. Interestingly, early binding dissociation
studies of ½3H-imipramine, ½3H-paroxetine, and ½3H-citalopram suggested that
these drugs bind to different areas of the SERT [12], even though the implications
of this were unclear at that time.
The available mechanistic in vivo assays only provided indirect measures of
uptake inhibition and selectivity. For example, citalopram inhibited the uptake of
the 5-HT depleting agent, H75/12, into neurons with an ED
50
of 0.80 mg/kg and
failed to inhibit NE depletion by the depleting agent H77/77 at doses as high as
160 mg/kg [9]. Similarly, studies of amine turnover showed that an acute dose of
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

citalopram reduced 5-HT turnover in the brain (i.e., [5-HT] was unchanged and the
metabolite, [5-hydroxyindoleacetic acid, 5-HIAA], was decreased) and had no effect
on NE synthesis. This suggested that citalopram was the most selective of the com-
pounds tested [13, 14]. Finally, simple behavioral screening assays, such as the
potentiation of 5-hydroxytryptophan (5-HTP)-induced 5-HT syndrome (5-HT
uptake inhibition), tetrabenazine-induced ptosis (NE uptake inhibition), and apo-
morphine-induced gnawing (DA uptake inhibition), supported a selective 5-HT
uptake inhibition in vivo [15]. Citalopram, like other SSRIs, had limited and variable
effects in validated behavioral models predictive of antidepressant effect, such as
the forced swim test, which had been validated with TCAs and MAOIs [16]. It was
not until after the development of citalopram had been completed that more
advanced in vivo assays, such as microdialysis, in vivo electrophysiology, and more
complex behavioral models, became available.
11.6
Clinical Efficacy of Citalopram
Citalopram was first launched for the treatment of major depressive disorder in
1989 in Denmark as Cipramil
1
and subsequently marketed worldwide, including
the United States in 1998 under the trade name Celexa
1
. Following the approval
for major depressive disorder, citalopram was also approved for the treatment
of panic disorders. After only a few years on the market, the drug attained block-
buster status.
A large number of clinical short- and long-term studies of patients with major
depressive disorder have been conducted with citalopram over the years, and differ-
ent subsets of the clinical data have been subjected to meta-analyses as well. In
general, citalopram was found to have an efficacy comparable to that of other SSRIs
and, in some studies, also like other SSRIs, to be slightly less efficacious than the
TCAs [17, 18]. Citalopram has also been shown to be efficacious in the treatment of
other conditions, such as social phobia, obsessive compulsive disorder, post-trau-
matic stress disorder, mixed anxiety and depression, and poststroke depres-
sion [17, 18]. Citalopram has a favorable pharmacokinetic profile with good
bioavailability and linear kinetics and a low potential for interactions with concomi-
tant medication [17]. It is generally well tolerated with a better tolerability than the
TCAs [19, 20]. These favorable properties made citalopram a good choice, particu-
larly for depressed patients who required continuation and long-term treatment, as
well as for elderly patients [17]. Overall, citalopram was on par with the other SSRIs
with respect to efficacy, but has more favorable drug metabolism and pharmaco-
kinetics (DMPK) properties (e.g., approximately 80% bioavailability and 80% pro-
tein binding, dose-proportional linear pharmacokinetics, an elimination half-life of
1.5 days, negligible pharmacological activity of metabolites, and low drug–drug
interaction potential), which are the likely reasons why citalopram became such a
commercial success, even though it was the fifth SSRI introduced onto the US
market.
11.6 Clinical Efficacy of Citalopram
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11.7
Synthesis and Production of Escitalopram
The preparation of the closest analogues of citalopram, its single enantiomers,
proved to be a major challenge. Direct resolution via diastereomeric salts failed
after many fruitless attempts. Different chiral acids, different solvents, and differ-
ent stoichiometric ratios of citalopram and acid were tried, but the major problem
was the lack of crystal formation in almost all cases. A crystalline (þ)-camphor sul-
fonate was obtained, but no separation of enantiomers could be accomplished.
Chiral high-pressure liquid chromatography (HPLC) was in its infancy, and the
available analytical columns were tried with negative results. We wanted to avoid
acid ring closure of resolved intermediates due to the risk of racemization. There-
fore, we spent some time on various asymmetric syntheses focusing on derivatives
with a partially finished side chain that after potential resolution could be trans-
formed to the citalopram enantiomers without risk of racemization. However,
these attempts were also unsuccessful.
Finally, we decided to try to resolve the intermediate diol III (Scheme 11.1),
although we did not have a strategy for a subsequent stereoselective ring closure
at hand. We made the diasteromeric esters with the enantiomers of a-methoxy-
a-trifluoromethyl acetic acid (Mosher’s acid, known as a shift reagent for nuclear
magnetic resonance (NMR)) and tried to separate them on preparative (nonchiral)
HPLC. By repeated peak shaving, we obtained small samples of the pure diastereo-
mers. Importantly, we had noticed a seemingly spontaneous slow ring closure to
citalopram of the mixture of diastereomeric esters (in the presence of triethyl-
amine) during their synthesis. This encouraged us to try a stronger base (potas-
sium tert-butoxide), and to our great surprise, this resulted in a stereoselective ring
closure of the pure diastereomers to afford the very first small sample of the pure
citalopram enantiomers. Later we found that the diol III also could be resolved by
diastereomeric salt formation with di-p-toluoyl tartaric acid (DTTA) and, in this
way, it became possible to produce larger quantities of the enantiomers (using a
basic ring closure with a mesylate of the diol and triethylamine).
This method was possibly also suited for production scale, but as chromato-
graphic separation of the diols using simulated moving bed (SMB) technology in
the late 1990s proved very effective, two SMB plants (a pilot and a full scale) were
built for escitalopram production. Later, production became even more cost-effec-
tive when development chemists discovered that acidic ring closure of the R-diol
led to a mixture of approximately 30% R-citalopram and 70% escitalopram, which
could subsequently be isolated as citalopram and pure escitalopram.
In recent years, many chemists outside Lundbeck have worked with alterna-
tive syntheses (for a recent review, see Ref. [21]) of citalopram and escitalo-
pram. Of special interest was a publication in 2007 by researchers from Dr.
Reddy’s Laboratories who published a direct resolution of citalopram using
DTTA [22]. At that point, we had completed a systematic study with a large
number of chiral acids (including DTTA) without finding a single one that
worked. So we were not surprised to find that in our hands the Dr. Reddy
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

method did not work. Through a series of experiments, we showed that resolu-
tion of citalopram was not possible by Dr. Reddy’s method [23]. In a subse-
quent response, Dr. Reddy researchers admitted that the method did not work
as described. They then claimed success with a highly modified procedure, but
in our hands that did not work either [24, 25].
In conclusion, the preparation and production of citalopram and escitalopram
have been major challenges, but we finally succeeded in developing highly innova-
tive and effective syntheses that are still the best and most cost-effective production
methods.
11.8
The Pharmacological Profile of the Citalopram Enantiomers
Shortly after the citalopram enantiomers had been successfully produced in the
laboratory in 1988, they were characterized in vitro in the rat brain synaptosome
assay of ½3H-5-HT uptake inhibition and in vivo in the mouse 5-HTP potentiation
assay. Completely unexpectedly, the 5-HT uptake inhibition of citalopram turned
out to reside in the S-enantiomer, and R-citalopram was found to be practically
devoid of this activity in both the in vitro and in vivo assays. These initial findings
were reproduced and substantiated by studies of NE and DA uptake inhibitory
activity and published in 1992 [26]. Based on these studies and the fact that racemic
citalopram is a highly selective inhibitor of the SERT, R-citalopram was thought to
be pharmacologically inactive. Only very few pharmacological studies were con-
ducted with escitalopram in the next few years. It was not until new production
methods (see above) made it feasible to produce escitalopram at an industrial scale
and it was decided in 1997 to develop escitalopram for major depressive disorder
that there was a renewed interest in pharmacological studies of escitalopram. Based
on the knowledge available then, the original expectation was that escitalopram
would be comparable to citalopram with respect to efficacy, and that the tolerability
would be improved by removing the presumably pharmacologically inactive R-
enantiomer and thereby minimizing the drug load in the body.
11.9
R-Citalopram’s Surprising Inhibition of Escitalopram
It was not until about a decade after the first characterization of the citalopram
enantiomers that observations from behavioral studies made us hypothesize
that R-citalopram counteracts the effect of escitalopram. A comparison of esci-
talopram and citalopram in a simple rat model predictive of anxiolytic activity,
inhibition of footshock-induced ultrasonic vocalization, revealed that escitalo-
pram dose dependently and completely abolished the vocalization, whereas cit-
alopram only partially reduced the vocalization and reached a plateau of about
60% inhibition when the dose was increased [27]. These findings made us
11.9 R-Citalopram’s Surprising Inhibition of Escitalopram
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279

hypothesize that R-citalopram might counteract the anxiolytic-like activity of
escitalopram in this model.
Knowing that the ultrasonic vocalization model is very sensitive to serotoner-
gic mechanisms, it was a logical next step to investigate the effect of R-citalo-
pram alone and in combination with escitalopram on the extracellular 5-HT
levels in relevant brain regions, such as the frontal cortex and the ventral hip-
pocampus. From pharmacokinetics studies in humans, we also knew that the
citalopram enantiomers are metabolized at a different rate, which meant that
the plasma level of R-citalopram is at least twofold higher than that of escitalo-
pram in individuals treated with racemic citalopram [28]. Thus, these first
behavioral observations were followed up by a series of microdialysis studies
in freely moving rats where the effects of escitalopram and citalopram as well
as ratios of escitalopram to R-citalopram of 1:1, 1:2, and 1:4 were studied.
The outcome of these studies was that escitalopram produced a significantly
higher increase in the level of extracellular 5-HT compared to equivalent doses
of citalopram, and that R-citalopram antagonized the effect of escitalopram in
a dose-dependent manner [29].
These observations triggered a series of experiments aimed at identifying the
mechanism by which R-citalopram counteracts the therapeutic effect of escitalo-
pram. Studies of brain exposure levels in rats treated with escitalopram alone ver-
sus combinations of escitalopram and R-citalopram quickly ruled out a
pharmacokinetic interaction as the explanation [29]. A very broad receptor screen-
ing including 144 targets did not reveal any obvious target through which R-citalo-
pram could exert its action [30]. A reverse dialysis study in rats dosed systemically
with escitalopram showed that R-citalopram could exert its inhibitory action when
administered at terminal areas of 5-HT neurons, such as the prefrontal cortex [29].
In vivo recordings of individual 5-HT neurons in the rat dorsal raphe nucleus dem-
onstrated that desensitization of somatodendritic 5-HT
1A
autoreceptors, a mecha-
nism believed to be associated with the delayed clinical response of SSRIs,
occurred after only 2 weeks’treatment with escitalopram, whereas 3 weeks’treat-
ment was required for an equivalent dose of citalopram [31]. In another study using
the same methodology, it was shown that R-citalopram delayed the recovery of 5-
HT neuronal firing and the 5-HT
1A
receptor desensitization produced by escitalo-
pram [31–33]. The superior effect of escitalopram compared to citalopram and the
inhibitory action of R-citalopram on the pharmacological activities of escitalopram
have been confirmed in numerous animal models using acute or repeated dos-
ing [34–36] (Table 11.2).
The mechanistic in vivo studies all pointed toward the SERT as the target for
the interaction between escitalopram and R-citalopram. This and the original
in vitro reports from Plenge et al. [12] that SSRIs could bind differently to the
SERT made us decide to investigate the in vitro binding kinetics of the individ-
ual citalopram enantiomers in further detail. Kinetic studies of ½3H-escitalo-
pram binding to the SERT demonstrated that cold escitalopram slows down
the dissociation rate of ½3H-escitalopram from the primary (inhibitory) site of
the SERT [6]. This is indicative of the existence of a secondary allosteric
280
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

Table 11.2 Superior effect of escitalopram over citalopram and inhibitory action of R-citalopram
on the pharmacological activities of escitalopram in nonclinical models after acute or prolonged
exposure.
Assay Outcome References
Mechanistic
Microdialysis
Extracellular 5-HT in
prefrontal cortex and ventral
hippocampus in rats
Escitalopram increases 5-HT more than
citalopram
[29, 32, 37, 38]
R-Citalopram antagonizes escitalopram-
induced increase of 5-HT
Voltammetry in rats Escitalopram is more potent than
citalopram at inhibiting 5-HT clearance
[39]
R-Citalopram counteracts escitalopram
inhibition of 5-HT clearance
Electrophysiology .
5-HT neuronal firing in
dorsal raphe nucleus in rats
Escitalopram is four times more potent
than citalopram, acute dosing
[31–33]
R-Citalopram antagonizes escitalopram-
induced suppression of firing, acute
dosing
Escitalopram normalizes DRN firing
faster than citalopram, chronic dosing
R-Citalopram delays effect of
escitalopram on firing
DA neuron firing in ventral
tegmental area in rats
Escitalopram increases firing rate and
burst firing of DA neurons, acute dosing
[40]
R-Citalopram antagonizes the effect of
escitalopram on DA neuron firing
NMDA-induced currents in
hippocampal pyramidal
neurons in rats
Escitalopram but not citalopram
potentiates NMDA-induced currents in
pyramidal neurons
[40]
R-Citalopram antagonizes the effect of
escitalopram
LTP in rats R-Citalopram counteracts escitalopram
effect on LTP
[41]
Neurogenesis in rats R-Citalopram counteracts escitalopram-
induced cell proliferation in the dentate
gyrus
[33]
HPA axis
Plasma corticosterone in rat R-Citalopram antagonizes escitalopram-
induced increase of plasma
corticosterone
[42]
Behavior
Potentiating of 5-HTP-
induced behavior, rat,
mouse
R-Citalopram antagonizes the
potentiating effect of escitalopram
[42, 43]
(continued )
11.9 R-Citalopram’s Surprising Inhibition of Escitalopram
j
281

binding site that modulates the binding properties of the drug at the primary
site. In addition, we found that R-citalopram attenuated the decreased dissocia-
tion rate of ½3H-escitalopram in the presence of cold escitalopram [6, 49].
Finally, we found that R-citalopram also slowed the association rate of
½3H-escitalopram to the SERT [32, 49]. These findings were supportive of the
notion that R-citalopram counteracts the effects of escitalopram at the SERT,
and that the interaction takes place at a secondary allosteric modulator site. As
described in further detail in the next section, species comparisons and site-
directed mutagenesis studies have led to identification of domains on the
SERT that are critical for the delayed dissociation produced by escitalopram
and the negative effects of R-citalopram on the binding of escitalopram to the
SERT [49, 50].
Using brain imaging techniques, positron emission tomography (PET), or sin-
gle-photon emission computer tomography (SPECT) and appropriate radiolabeled
ligands, it was demonstrated that the occupancy at the SERT in the human brain is
significantly higher with escitalopram 10 mg than with the corresponding dose of
citalopram (20 mg, containing 10mg of R-citalopram and 10 mg of S-citalopram) at
steady state. The mean occupancies at 6 and 54 h after last doses were 82 and 63%
for escitalopram, respectively, and 64 and 49% for citalopram, respectively [51]. The
results were confirmed in a pooled analysis, in which the authors concluded that
the buildup of the R-enantiomer after repeated citalopram dosing may lead to lower
SERT occupancy by the S-enantiomer [52].
Table 11.2 (Continued)
Assay Outcome References
Anxiolytic and antidepressant effects and cognitive function
Elevated plus maze, rat R-Citalopram antagonizes anxiolytic
effect of escitalopram
[44]
Vogel conflict test, rat R-Citalopram antagonizes anxiolytic
effect of escitalopram
[44]
Separation-induced
vocalization, mouse pup
R-Citalopram antagonizes anxiolytic
effect of escitalopram
[45]
Footshock-induced ultrasonic
vocalization, rat
R-Citalopram antagonizes anxiolytic
effect of escitalopram
[27]
Conditioned fear, rat R-Citalopram antagonizes anxiolytic
effect of escitalopram
[46]
Resident intruder, rat Escitalopram produces antidepressant
effect earlier than citalopram
[47]
R-Citalopram counteracts escitalopram-
induced antidepressant effect
Chronic mild stress, rat Escitalopram produces antidepressant
effect earlier than citalopram
[48]
R-Citalopram counteracts escitalopram-
induced antidepressant effect
Novel object recognition, rat Escitalopram but not citalopram
improved recognition memory
[40]
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

11.10
Binding Site(s) for Escitalopram on the Serotonin Transporter
Early on in the era of antidepressant drugs, medicinal chemists started to speculate
about the mode of binding of these drugs to their target, that is SERT and NET.
With respect to citalopram and escitalopram, as described above, we noticed that
relatively small changes in the structure could transform a selective NE uptake
inhibitor such as talopram into an SSRI such as citalopram. Eli Lilly made a similar
observation with the NRIs tomoxetine and nisoxetine, and the SSRI fluoxetine.
However, the chiral SSRIs differed with regard to stereoselectivity. While the
enantiomers of fluoxetine were equipotent as SSRIs, a high stereoselectivity was
observed with the enantiomers of citalopram and with paroxetine and its
enantiomers.
We worked in the 1980s with another structural class of antidepressants, the 3-
phenyl-1-indanamines where the amine could be a dimethyl- or monomethylamine
or a piperazine [8, 53]. We observed a moderate stereoselectivity of the enantiomers
of trans-isomers with the small amines in favor of the 1R,3Sconformation, but a
high stereoselectivity of the piperazine enantiomers with 1S,3Ras the active stereo-
isomer. We tried to rationalize this in a simple cartoon of a three-point binding
model [54]. However, this model was not useful for the prediction of the stereose-
lectivity of citalopram.
Smith presented a three-dimensional model that he interpreted as predicting a
high stereoselectivity of the R-enantiomer of citalopram versus the S-enantiomer
(this was obviously later proved wrong) [55]. However, the author did not take the
semisymmetrical structure of citalopram into account. If one does that, both enan-
tiomers fit his model equally well, and the model could just as well predict no
stereoselectivity.
Later on when we established computational chemistry (and citalopram had been
resolved into its individual enantiomers), we developed a pharmacophore model
that was used to rationalize both the structural elements determining SERT or NET
selectivity and the observed stereoselectivity of citalopram and several other
SSRIs [56]. When fitting R-andS-citalopram into the model, we took advantage
of the semisymmetrical nature of the compound mentioned above, that is, the
p-fluorophenyl ring of one enantiomer was superimposed onto the phthalane ring
of the other enantiomer. Interestingly, these different binding modes were also the
outcome of a subsequent, more comprehensive computational study of the binding
of the enantiomers [57]. In order to further study the structural elements in talo-
pram and citalopram that were important for SERT and NET selectivity, respec-
tively, 14 derivatives of the two compounds were either selected or synthesized [58]
and tested for 5-HT and NE uptake inhibition [59]. This study showed, in agree-
ment with our previous pharmacophore model study, that the phthalane 3,3-
dimethyl substituents and 5-cyano group are key determinants for inhibitory activ-
ity and selectivity toward NET and SERT, respectively.
However, the real breakthrough came with the publication of the X-ray struc-
ture of the leucine transporter (LeuT), a bacterial homologue to the
11.10 Binding Site(s) for Escitalopram on the Serotonin Transporter
j
283

mammalian solute carrier 6 (SLC6) transporters [60]. We developed a SERT
homology model based on the LeuT structure with escitalopram bound to a
site equivalent to the leucine binding site in LeuT [61, 62]. Subsequently, sev-
eral other homology models supported by mutational studies strongly sup-
ported this site as the escitalopram binding site, despite some differences in
the suggested escitalopram conformation and binding mode at the
site [57, 63, 64].
Interestingly, two X-ray structures of LeuT were then published with the
TCAs clomipramine and desipramine bound within the transporter [65, 66].
The TCAs bound (with low affinity) in a cavity (called the vestibule or the S2
site), located 15 A
above the S1 site (the substrate binding site). This site had
also been found in a study we made of the driving forces for migration of
leucine through LeuT [67]. Subsequently, X-ray structures of LeuT with the
SSRIs sertraline and R-andS-fluoxetine bound within the S2 site were pub-
lished [68]. The authors of this study speculated that the equivalent site in the
SERT might be the binding site for antidepressants. However, the mutational
studies mentioned above showed not only a strong influence on escitalopram
binding of mutations at the S1 site on escitalopram binding, but also that
mutations in the S2 pocket did not influence escitalopram binding [63, 64].
Moreover, we have recently shown that mutation of just five amino acids
within the S1 binding pocket of the NET to the corresponding residues in the
SERT conferred affinity equivalent to WT SERT for citalopram and its enan-
tiomers at the mutated NET. In contrast, the binding of talopram was almost
unaffected by all mutations at both the SERT and the NET. The binding mode
of talopram to NET is, therefore, not at the S1 site [59].
As mentioned above, escitalopram has an allosteric effect that we believe is
attained through a separate allosteric binding site on the SERT. Studies on
chicken SERT (gSERT) have demonstrated a low allosteric potency of escitalo-
pram in this species. However, mutation of nine residues in transmembrane
domains 10, 11, and 12 to their corresponding human residues restored the
allosteric effect [50]. The location of these nine residues did not seem to com-
prise a cavity for a putative allosteric binding site. It has recently been pro-
posed that the SERT allosteric site could be identical to the S2 site [57].
Accordingly, the nine residues might have an indirect effect on escitalopram
binding at the S2 site (Figure 11.2).
A clear indication of the different nature of the S1 and S2 sites is that escitalo-
pram derivatives show a different SAR toward to two sites. In Table 11.1, the effect
on the hSERT S1 site (inhibition of ½3H-5-HT uptake) is shown, as well as the allo-
steric effect (inhibition of ½3H-escitalopram dissociation) of a number of escitalo-
pram derivatives. While the 5-chloro (10), 5-bromo (16), and 30,40-dichloro (11)
derivatives retain their effect at both sites, the 30,50-dichloro analogues (12 and
enantiomers 12a and 12b), which lack an effect at the S1 site, are still potent alloste-
ric modulators. A study of naphthyl derivatives of citalopram was recently pub-
lished [69]. Here we found that while the 2-naphthyl derivative of citalopram (19
and enantiomers 19a and 19b) retained its effect at both sites, the 1-naphthyl
284
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

Figure 11.2 Escitalopram’s two binding sites
at SERT. (a) A schematic drawing of the
suggested positions of escitalopram’s primary
binding site (S1) and allosteric binding site
(S2). (b) The X-ray structure model of LeuT
taken from Ref. [68]. The leucine binding pocket
is encircled in green. The “vestibule”pocket is
encircled in red. The corresponding ligands
from the X-ray structure (leucine and sertraline,
respectively) are depicted in yellow.
Superimposed on these is the docked structure
of escitalopram, where the additional aromatic
ring of compound 20 has been sketched in.
11.10 Binding Site(s) for Escitalopram on the Serotonin Transporter
j
285

derivatives (20 and enantiomers 20a and 20b) were selective allosteric modulators
(Figure 11.3).
Compounds 17 and 18 were originally identified as impurities formed in escita-
lopram production. Amazingly, testing showed that 17 (citalopram with an extra
side chain at the 5-position) has a very high affinity at the S1 site, but is devoid of
an allosteric effect. Conversely, 18 (and enantiomers 18a and 18b) are selective allo-
steric modulators. The stereoselectivity at the S2 site is generally low as can be seen
from the data on the enantiomers of 9,12,18,19, and 20.
In conclusion, analogue design of citalopram has turned out to be potentially
much more complicated than originally anticipated. The insight gained in recent
years into the dual effect of escitalopram and the different SARs of derivatives for
the S1 and S2 sites underlines how fortunate it is that citalopram was originally
selectedfordevelopment.Inprinciple,therewasarisk,ofwhichwewerethen
unaware, of selecting a derivative such as 17, which has no allosteric effect.
11.11
Future Perspectives on the Molecular Basis for Escitalopram’s Interaction
with the SERT
It has been shown that SSRIs in addition to blocking the reuptake of 5-HT also
cause an internalization of the SERT [70], and that the potency ranking of SSRIs to
cause internalization and to block the uptake of 5-HT appear to differ [71]. Recent
research shows that R-citalopram antagonizes escitalopram’s effect on the expres-
sion of SERT in the cell membrane and that protein kinase C (PKC) appears to be
involved in this effect as the PKC inhibitor staurosporine abolished the effect of R-
citalopram [72]. A putative interaction between the allosteric mechanism of the
SERT and the so-called serotonin interaction proteins (SIPs), of which PKC is one,
18 R=
19 R=
20 R=
O
R
NCH3
CH3
NC
S
F
Figure 11.3 Citalopram derivatives with selective (S2 site) allosteric effect (18 and 20) and dual
(S1 and S2 sites) effect (19).
286
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11 The Discovery of Citalopram and Its Refinement to Escitalopram

may potentially open up a new area of research into the function of the SERT and
eventually lead to new drug targets [73].
11.12
Clinical Efficacy of Escitalopram
In 2002, escitalopram was launched as Cipralex
1
in Europe and as Lexapro
1
in the
United States. Escitalopram is approved for the treatment of major depressive dis-
order and social anxiety disorder in the United States, and major depressive dis-
order, panic disorder, social anxiety disorder, generalized anxiety disorder, and
obsessive compulsive disorder in Europe and elsewhere.
Four pivotal randomized placebo-controlled short-term studies were conducted
for the registration file. Three of these studies had a citalopram group included as
active reference. Interestingly, and surprisingly, there was a signal that escitalopram
had a faster onset of acting and/or was more efficacious than an equivalent dose of
citalopram [74, 75]. These studies were not designed or powered to show differ-
ences to an active reference, but subsequent studies with head-to-head comparisons
of citalopram and escitalopram confirmed the original observation that escitalopram
is superior to citalopram, particularly in patients with severe depression [76, 77]. In
a meta-analysis of pooled data at an individual patient level, escitalopram was statis-
tically significantly superior to citalopram, with response rates of 59.7 and 52.3%,
respectively, for the complete data set, and 61.2 and 49.9%, respectively, for the sub-
set of patients with severe depression [78]. In a meta-analysis of publicly available
data [79], statistically superior response rates of 72.3% for escitalopram versus
63.9% for citalopram were reported. The efficacy of escitalopram has been further
confirmed in a prospective, randomized, double-blind study against paroxetine [80],
and in a post-hoc analysis of the effect of anxiety symptoms on patients with severe
depression from the same study [81]. Data from this study and an analysis of data
from two studies comparing escitalopram with paroxetine [82] confirmed the supe-
rior efficacy of escitalopram over another SSRI. Further comparisons of pooled data
from patients with severe depression treated with escitalopram versus other antide-
pressants again confirm that escitalopram is superior to other comparators, includ-
ing the serotonin norepinephrine reuptake inhibitors (SNRIs) venlafaxine and
duloxetine [83–87]. In patients with generalized anxiety disorder, escitalopram has
also shown statistically significant superiority over the comparator, paroxe-
tine [88, 89]. Overall, the tolerability of escitalopram was very similar to that of cit-
alopram [34], whereas toxicity in relation to overdose has been reported to be more
serious for citalopram than escitalopram [90, 91]. Thus, the original expectation of
similar efficacy of escitalopram and citalopram and escitalopram being better toler-
ated proved to be wrong, at least at therapeutic doses. Escitalopram not only
resulted in being clinically superior to citalopram, but has also proven to be supe-
rior to other SSRIs and to SNRIs in several clinical studies. In 2010, it reached the
status of being the most prescribed branded antidepressant globally and, by mid-
2011, more than 265 million patients have been treated with the drug.
11.12 Clinical Efficacy of Escitalopram
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287

11.13
Conclusions
The development of citalopram, and subsequently of escitalopram, has not been
trivial and was full of surprises, where time after time predictions based on the
established rules and perceptions have been proven wrong as the data emerged.
The discovery of citalopram and escitalopram underlines the point that analogue
design is not trivial and can lead to drugs with either totally different or greatly
improved profiles compared with the starting structure. Similarly, the complexity
of the biological mechanisms we are dealing with calls for caution when making
predictions and for an open mind to recognize and follow through on serendipi-
tous findings.
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Klaus P. Bøgesø,B.Sc.,D.Sc.,hasbeenworkingatH.
Lundbeck A/S with drug discovery within the CNS field for
more than 40 years. Klaus P. Bøgesø started at Lundbeck as a
Research Chemist in 1971. In 1986, he became Director of
Medicinal Chemistry and in 1999 he became Vice President
of Medicinal Chemistry Research. In 2006, he became Vice
President of Research, Denmark. In 2008, he became Vice
President of External Affairs, Lundbeck Research Manage-
ment. In 2011, he became Vice President, External Scientific
Affairs and Patents.
The research activities of Klaus P. Bøgesø have focused on drug design and
development within psychiatric and neurological diseases, such as schizophrenia,
depression, anxiety, Alzheimer’s disease, Parkinson’s disease, and epilepsy. Pri-
mary targets have been serotonin, norepinephrine, and dopamine receptors and
transporters, muscarinic receptors, and glutamate receptors. Klaus P. Bøgesø is
inventor of the selective serotonin reuptake inhibitor citalopram (Cipramil
1
, Cel-
exa
1
) and co-inventor of its S-enantiomer, escitalopram (Cipralex
1
,Lexapro
1
).
Both drugs have attained blockbuster status. The differential action of stereoisom-
ers of chiral drugs has been a focus area in many of the research projects Klaus P.
Bøgesø has been involved in, and has been a major topic in several of his publica-
tions, including his doctoral thesis.
Connie S
anchez, currently at Lundbeck Research USA and
before that at H. Lundbeck A/S, in Copenhagen, Denmark,
has more than 25 years of drug discovery experience. She
graduated from the Pharmaceutical University of Denmark
and has acquired a D.Sc. degree in Pharmacology at the
same institution. She has a broad experience within drug
discovery and drug development for neuropsychiatric and
neurological diseases, including depression, anxiety, schizo-
phrenia, Alzheimer’s disease, Parkinson’s disease, epilepsy,
and insomnia, and she has contributed to bringing various drug candidates into
clinical development, some of which have been brought to the market. Most nota-
bly, she established and successfully led a research project aiming at uncovering
the mechanism of action of the antidepressant drug escitalopram (Cipralex
1
). She
has authored approximately 100 scientific papers and more than 20 patent
publications.
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